key: cord-281086-fmftr5jn authors: morand, a.; roquelaure, b.; colson, p.; amrane, s.; bosdure, e.; raoult, d.; lagier, j-c; fabre, a title: child with liver transplant recovers from covid-19 infection. a case report date: 2020-05-06 journal: arch pediatr doi: 10.1016/j.arcped.2020.05.004 sha: doc_id: 281086 cord_uid: fmftr5jn abstract we present the case of a 55-month-old girl who recovered from coronavirus disease 2019 (covid-19) infection 5 months after undergoing liver transplantation; she had a co-infection with epstein–barr virus (ebv). to the best of our knowledge, this is the first case report of a covid-19 infection in a pediatric patient with liver transplantation. additionally, this is also the first report of confirmed co-infection between covid-19 and ebv. on the basis of this case, we suggest that liver transplantation is not associated with covid-19 symptom severity and development. moreover, covid-19 and ebv co-infections do not seem to aggravate the clinical outcome. j o u r n a l p r e -p r o o f 3 we herein present the case of a 55-month-old girl who was infected with coronavirus disease 2019 (covid-19) 5 months after undergoing liver transplantation. after an uneventful birth at term, she was diagnosed with congenital cholestasis due to biliary atresia and underwent kasai portoenterostomy (kpe) at 53 days of age. kpe was partially successful, but in the following years she developed portal hypertension with refractory ascites and angiocholitis. liver transplantation (from her father) was performed without major complications at the age of 50 months. the patient was discharged 20 days after the procedure on tacrolimus immunosuppression therapy and with no immunization against epstein-barr virus (ebv) before the transplantation. she had mildly elevated aspartate aminotransferase (ast) and alanine aminotransferase (alt) (approx. 1. a few days before the child's presentation, the mother, a 29-year-old woman with no medical history, presented with rhinopharyngitis. she felt progressively tired and had a fever, cough, polypnea, thoracic pain, and headache. she was referred to the méditerranée infection university hospital institute where she was diagnosed with covid-19 by rt-pcr of a nasopharyngeal swab [1] . she was hospitalized in the contagious infections diseases department. low-dose computed tomography showed bilateral, asymmetrical, peripheral frosted-glass images and two alveolar condensation foci in two different segments of the right lung. this was compatible with covid-19 pneumonia. the mother was treated with oral hydroxychloroquine and oral azithromycin [2] . on the fourth day of treatment, the results of her nasopharyngeal samples were negative and within 8 days she was discharged from hospital and followed up as an outpatient. the young liver transplant girl had rhinitis starting from mid-march, 2020, shortly after the onset of her mother's symptoms. two days later, she suffered from fever, cough, and polypnea, and 3 days later she was referred to the méditerranée infection university hospital institute at the same time as her mother, where she was also diagnosed with covid-19 after a nasopharyngeal swab test. to the best of our knowledge, this is the first case report of a liver transplantation patient with covid-19. although children seem to have a less severe reaction to covid-19 than j o u r n a l p r e -p r o o f 5 adults [3] , even when they have significant health complications [4] , there have been some doubts regarding immunosuppression therapy and the risk of severe infections as they are usually negatively correlated. an immunocompromised state has been associated with increased risk of severe lower respiratory tract disease in patients with coronavirus [5] . contrary to other viruses, during the covid-19 infection, the host's innate immune response seems to be the main cause of lung tissue damage [6] . it should be noted that the most important factors in adult patients are age, sex, and a history of hypertension [7] . a report of the successful recovery of 52-year-old renal transplantation patient was recently published [8] . additionally, this is also the first report of confirmed co-infection between covid-19 and ebv. two studies reported patients having a positive serology result for ebv at the same time as a covid-19 infection, but ebv dna was not assessed [9, 10] . a single study conducted in china aimed to assess the role of ebv co-infection during covid-19. in all, 35 out of 62 covid-19 patients (56.5%) were seropositive for ebv viral capsid antigen (vca) igm antibody, which reflects an acute ebv infection or reactivation [10] . ebv was reported to be correlated with a more severe course of covid-19. the authors noted that ebv-positive covid-19 patients had a 3.64-fold higher risk of presenting with fever (95% ci: 1.26-10.5; p=0.02), higher crp levels (p=0.01), and a lower proportion of cd8+ and cd4/cd8 lymphocytes (p=0.048 and =0.046, respectively) than ebv-negative patients. the median recovery time for ebv-seropositive covid-19 patients was higher than for ebv-negative patients, without being statistically significant (p=0.07) [10] . these authors hypothesized that ebv infection and covid-19 share some pathogenic characteristics such as an overactivation of t cells, which can lead to severe immune injury [11] . in fact, even if ebv infection is often asymptomatic [12] , some people present with fever, asthenia, myalgia, sore throat, anorexia, skin rash, and the disease can be fatal, especially in immunocompromised individuals [13] . therefore, the clinical case of our young immunocompromised patient recovering from ebv infection at the same time as covid-19 is particularly interesting. the hepatic anicteric cholestasis and cytolysis of our patient was probably due to her transplant state, as no hepatitis during covid-19 infection was described in the literature [14] . on the basis of this case, we suggest that liver transplantation is not associated with covid-19 symptom severity and development, even when there is a high level of immunosuppression under tacrolimus treatment. moreover, covid-19 and ebv co-infections do not seem to aggravate the clinical outcome. rapid viral diagnosis and ambulatory management of suspected covid-19 cases presenting at the infectious diseases referral hospital hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial sars-cov-2 infection in children epidemiology of covid-19 among children in china characteristics and outcomes of coronavirus infection in children: the role of viral factors and an immunocompromised state coronaviruses and immunosuppressed patients. the facts during the third epidemic host susceptibility to severe covid-19 and establishment of a host risk score: findings of 487 cases outside wuhan successful recovery of covid-19 pneumonia in a renal transplant recipient with long-term immunosuppression clinical outcome of 55 asymptomatic cases at the time of hospital admission infected with sars-coronavirus-2 in shenzhen positive epstein-barr virus detection in corona virus disease 2019 (covid-19) patients pathological findings of covid-19 associated with acute respiratory distress syndrome epstein-barr virus in healthy individuals from portugal positive epstein-barr virus detection and mortality in respiratory failure patients admitted to the intensive care unit clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study we thank the family for allowing us to publish this case report. key: cord-348388-nkosag8m authors: nirenberg, michael s.; herrera, maría del mar ruiz title: foot manifestations in a patient with covid-19 and epstein-barr virus: a case study date: 2020-06-22 journal: foot (edinb) doi: 10.1016/j.foot.2020.101707 sha: doc_id: 348388 cord_uid: nkosag8m sars-cov-2 (covid-19) is highly-contagious. it can lead to respiratory distress—and some cases—death. recent reports and observations have identified an association between covid-19 and manifestations in the feet. however, there are very few reports that describe the course of these foot manifestations in any detail. the authors present a case study chronicling the progression of foot issues in a covid-19 positive patient who also was positive for the epstein-barr virus. or necrosis. most reports of skin manifestations have not detailed the progression of the cutaneous issues, or whether there were other foot-related findings. one case study differed in not only describing the progression of the skin changes in the foot, but also documenting gait changes and pain symptoms [8] . most prior reports have also focused on patients who were often only presumed or proven positive for covid-19. borghetti at al. caution practitioners that during the covid-19 pandemic, it is important not to overlook other viral pathogens that mimic the symptoms of covid-19 and may be the sole cause of a patient's problems or may be present concomitantly with covid-19 or another disease [16] . this case study provides a description of the progression of foot manifestations in a patient in spain who tested positively for covid-19 and the epstein-barr virus (ebv), and may have also contracted parvovirus b19 (b19). the authors believe this may be the first detailed report of foot manifestations in a patient who had covid-19, ebv and possibly b19. this case concerns a 16-year-old female in madrid, spain. as a brief background, spain reported its first confirmed covid-19 case on january 31, 2020, and a state of emergency was declared march 14, 2020 when the government implemented a quarantine of its citizens [17] . the patient's history was negative for trauma, exposure to cold temperatures, or any health issues. she had no allergies, was not taking any medication, and denied cigarette use or vaping. however, she resided in a home with her 20-year-old sister, who on march 3, 2020, tested positive for active ebv and j o u r n a l p r e -p r o o f was also diagnosed with covid-19. at the time, tests for covid-19 were unavailable in the area, and the sister's diagnosis was based solely on her history and symptoms. on february 25, 2020, the patient's sister developed a sore throat and then two days later, a fever. her doctor prescribed augmentin (amoxicillin and clavulanic acid). the next day, a rash appeared on her chest and abdomen, which the doctor thought was due to an allergic reaction to the augmentin. the use of augmentin was discontinued. from march 3 to the 8, the sister's rash spread to all areas of her body, including her hands, feet, and face. when she was found to be positive for ebv, she was diagnosed with active mononucleosis and covid-19. the rash was treated with topical corticosteroids, which were of little help. the patient's sister's symptoms gradually resolved over the next 15 days. subsequently, the patient, residing with her sister, experienced a sore throat on march 6, soon followed by headache, diarrhea, and back pain. she was also prescribed augmentin, and within three days, by march 9, all her symptoms resolved. however, the next day, a rash appeared on the patient's chest, abdomen, and face, which was mildly itchy. this irritation gradually worsened, and by april 4, she was experiencing severe itching. the rash then spread to her toes, where it was also extremely irritating (figs. 1 and 2). on april 12, the patient and her mother consulted dr. del mar ruiz herrera, a podiatrist, via telemedicine. dr. del mar ruiz herrera advised the patient to be tested for covid-19; however, no tests were available at that time. the podiatrist also prescribed the patient hydrocortisone cream 1% for her skin issues. the cream was beneficial, and the skin manifestations began to improve. two days later, a pruritic rash appeared on some of the patient's fingers (fig. 3) . on april 17, the patient was tested covid-19, ebv, hepatitis b and c, mycoplasma pneumonia, syphilis, chlamydia, cytomegalovirus, and parvovirus b19. she was positive for ebv and parvovirus igg, but all other tests, including covid-19, were negative. on april 28, the patient tests positive for covid-19 antibodies. her final diagnosis was a co-infection of mononucleosis and covid-19. it is unclear when she had b19, as it can be asymptomatic and may have occurred at an earlier time. in early may 2020, the patient's rash completely resolves, with the cutaneous lesions on the feet disappearing last. the patient has had no further medical issues. this case study is one of the first to document the progression of the foot manifestations in a patient with covid-19, ebv, and who also possibly had b19. [19] . in the present case study, the timing of active ebv and covid-19 in relation to each other is uncertain. though reports have suggested that covid-19 causes skin manifestations in the feet, further research may refute this supposition. also, since numerous diseases are known to cause cutaneous manifestations, including some that can affect the feet [21, 22] , it is not known if skin issues observed in covid-19 patients are due to covid-19 or the result of a co-infection, perhaps unrecognized. if other infectious agent are found to be present with covid-19 they may be shown to be solely or in combination responsible for the foot issues. in the case presented, both ebv and b19 are known to cause cutaneous manifestations, and as such, either one of these may have been the cause of the patient's foot issues, alone or in combination with covid-19. further, this case study is of an adolescent, and at this time, research that has included younger patients suggests that in children, adolescents, and young people, covid-19 may have a propensity for presenting in the feet and hands [2] [3] [4] [5] [6] . one of the larger observational studies, done by recalcati, found that the skin manifestations mainly involved the trunk [23] . unlike other studies cited herein, which involved young people and children with foot involvement, recalcati's study was comprised entirely of adults and did not include any children. 1 perhaps, further research will show an age association between covid-19 and foot involvement. limitations of this case study include a lack of a biopsy, further laboratory investigations, vascular testing, and radiographs of the feet to determine if bone changes had occurred. there is the possibility that this patient's foot issues occurred at the time she was positive for covid-19 by coincidence, and there could be another reason for presenting in her feet. further study is needed to better understand the effect of covid-19 on the feet, including a consideration of how the foot practitioner should best 1 sebastiano recalcati (personal written communication, april 11, 2020) j o u r n a l p r e -p r o o f treat these patients. some focus should also be given to plotting the timing of foot manifestations in relation to covid-19's more severe systemic effects, such as fever, to guide patients on what preparations, if any, should be implemented. there is much work to be done to fully understand the effect of covid-19 on the feet. the foot practitioner can play a key role in adding to the body of research on this subject, but conclusions should be cautiously drawn. this case highlights the progression of foot manifestations in a patient co-infected with covid-19 and ebv, and possibly b19. however, for concrete conclusions and treatments to be established, further case studies and research is needed. 2020 who director-general's opening remarks at the media briefing on covid-19 acute acro-ischemia in the child at the time of covid-19 characterization of acute acro-ischemic lesions in non-hospitalized patients: a case series of 132 patients during the covid-19 outbreak chilblain-like lesions on feet and hands during the covid-19 pandemic a case report with histopathologic findings. jaad case reports lesions pernióticas y acrales en españa durante el confinamiento por covid: análisis retrospectivo de 12 casos. actas dermo-sifiliográficas chilblain-like lesions during covid-19 epidemic: a preliminary study on 63 patients foot manifestations in a covid-19 positive patient: a case study classification of the cutaneous manifestations of covid-19: a rapid prospective nationwide consensus study in spain with 375 cases viral exanthem with "pin and needles sensation" on extremities of covid-19 patient silent covid-19: what your skin can reveal. the lancet infectious diseases acral cutaneous lesions in the time of covid-19 journal of the european academy of dermatology and venereology silent covid-19: what your skin can reveal. the lancet infectious diseases clinical and coagulation characteristics of 7 patients with critical covid-2019 pneumonia and acro-ischemia. zhonghua xue ye xue za zhi= zhonghua xueyexue zazhi diagnosis does not rule out other concomitant diseases coronavirus: spain to quarantine 47m people as entire country put into lockdown. the independent coronavirus death counts prompt anger, confusion positive epstein-barr virus detection in corona virus disease 2019 (covid-19) patients. available at ssrn 3555268 child with liver transplant recovers from covid-19 infection. a case report. archives de pédiatrie viral exanthems in childhood-infectious (direct) part 2: other viral exanthems cutaneous manifestations in covid-19: a first perspective the authors whose names are listed immediately below certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patentlicensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. key: cord-297222-2danzbqt authors: quadri, syed p; jain, nitesh k; brandon, brooke l; modi, harshit; bawaadam, hasnain title: an intriguing presentation of epstein-barr virus-associated hemophagocytic lymphohistiocytosis date: 2020-08-05 journal: cureus doi: 10.7759/cureus.9561 sha: doc_id: 297222 cord_uid: 2danzbqt hemophagocytic lymphohistiocytosis (hlh) is an immune related clinical syndrome with protean manifestations, varying presentation, clinically complex, with diverse causes, and is an under-recognized entity which carries high morbidity and mortality. it is precipitated by an immunological trigger in a susceptible host resulting in immune activation and dysregulation leading to disruption of immune homeostasis, cytokine storm and multi-organ failure. we describe a case of epstein-barr virus (ebv) associated hlh with its typical diagnostic challenges and associated high mortality rate. certain diagnostic criteria and online tools may help to arrive at an earlier presumptive diagnosis which, in turn, may expedite treatment and lead to better clinical outcomes. hemophagocytic lymphohistiocytosis (hlh) is an extremely rare and potentially lifethreatening hematological disorder, characterized by clinical features of extreme inflammation and an unregulated immune system. it is precipitated by an immunological trigger in a susceptible host resulting in immune activation and dysregulation leading to disruption of immune homeostasis, cytokine storm and multi-organ failure. we describe a case of epstein-barr virus (ebv)-associated hlh with its typical diagnostic challenges and associated high mortality rate, but an early prompt diagnosis with appropriate treatment can lead to better outcomes. the objective of this clinical case report is to highlight the need for awareness and the complexity surrounding the diagnosis in an effort to lower subsequent morbidity and mortality. a 43-year-old caucasian male with no significant past medical history initially presented with severe fatigue, upper extremity joint pain, jaw pain, and new onset daily headaches. he was treated with a 10-day course of oral steroids for suspected temporomandibular joint dysfunction; this temporarily improved his symptoms. a few weeks later, his symptoms returned and he also developed daily high fevers with drenching night sweats. during this time, the patient was seen in the emergency room twice with dyspnea and cough and was treated for "presumed pneumonia" with broad spectrum oral antibiotics. he was eventually admitted to our hospital for failure of outpatient treatment of pneumonia. computed tomography scan of the thorax showed bilateral ground glass opacities, with no evidence of mediastinal lymphadenopathy ( figure 1) . he was noted to have pancytopenia, cervical lymphadenopathy and parotid gland swelling. ebv polymerase chain reaction (pcr) was elevated to 47,400 copies per ml. ferritin was markedly elevated at 54,212 mcg/l; lactate dehydrogenase (ldh) was elevated to 2,125 u/l. he underwent parotid gland biopsy which was ebv-negative and demonstrated acute sialadenitis with necrosis. an extensive autoimmune, infectious and hematologic workup was equivocal. he was treated with broad spectrum iv antibiotics and high dose steroids. he remained afebrile for four days, and was ultimately discharged home on a two-week course of doxycycline, valacyclovir and fluconazole. his symptoms returned the following month with fevers, headaches and night sweats. the outpatient evaluation revealed a new non-tender, non-pruritic erythematous rash on his chest. bone marrow biopsy was obtained which revealed a hypocellular marrow with trilineage hematopoiesis (figure 2 ), negative for dysplasia; moderate number of pelger-huet type neutrophils; single lymphoid aggregate with an unremarkable flow cytometric profile. subsequent positron emission tomography (pet)-scan demonstrated diffuse hypermetabolic lymphadenopathy in the mediastinum, upper abdomen and retroperitoneum, as well as pleural thickening and pulmonary hypermetabolic opacities. he was advised to obtain outpatient biopsy of the abdominal lymphadenopathy with interventional radiology, but he was unable to schedule it. the patient was readmitted to the hospital few days later due to continued weight loss and seizure-like activity at home. ct head was unremarkable. he was found to have elevated liver enzymes with aspartate aminotransferase (ast) of 1,329 u/l, alanine aminotransferase (alt) of 725 u/l and alkaline phosphatase (alp) of 328 u/l. liver biopsy revealed scattered acidophil bodies which can be seen in infections or drug-induced liver injury, rare ebv-positive cells but no evidence of fibrosis or lymphoproliferative disorder. he was once again found to have elevated ferritin of 35,732 mcg/l. high dose dexamethasone 40 mg/day was started for treatment of presumed hlh, based on labs and clinical presentation ( table 1) . chest x-ray showed right mid-lung opacity. the patient developed acute respiratory failure requiring intubation and mechanical ventilation. bronchoscopy with lavage revealed aspergillus species and he was started on appropriate anti-fungal treatment immediately. repeat bone marrow biopsy was performed due to pancytopenia and demonstrated changes consistent with hlh, with definitive evidence of hemophagocytosis. despite treatment, the patient continued to decline and developed rapid multi-organ failure. he was transitioned to comfort care and expired the same day. the major finding in the patient's autopsy was hemophagocytic histiocytosis with rare ebvpositivity noted in the bone marrow analysis, suggesting that the hlh was due to ebv infection. there was no evidence of any lymphoproliferative disorder. lung examination revealed acute aspergillus spp. pneumonia with left upper lobe abscess formation, likely related to his immunocompromised state in the setting of hlh. also discovered was multiorgan vasculitis, the significance of which is unclear. although there have been rare reported cases of hlh in the setting of vasculitis, the laboratory evidence of ebv infection and finding of rare ebv-positive cells in the bone marrow on autopsy and in the ante-mortem liver suggest that the hlh was related to ebv infection. the official cause of death in the patient was determined to be pulmonary aspergillosis with organizing pneumonia in the setting of hemophagocytic lymphohistiocytosis, likely secondary to epstein-barr virus infection. hlh is a rare life-threatening condition characterized by hyperinflammation and immune system dysregulation [1] . it can occur in both familial and acquired forms [2] . acquired hlh is associated with various infections including bacterial, viral, fungal and parasitic; autoimmune diseases including vasculitis, rheumatological disorders, malignancy, and acquired immune deficiency states including aids, and with drug use [3, 4] . the basic pathophysiology in hlh is that the natural killer (nk) cells and cytotoxic tlymphocytes, which represent the innate and acquired immunity respectively, have altered cytotoxic function. this is coupled with excessive macrophage activation. the nk cells and lymphocytes are not able to eliminate the macrophages by perforin-mediated cytotoxicity. eventually there is a huge amount of cytokine release by all these above-mentioned immune cells resulting in excessive tissue damage across multiple organ systems [1, 2] . toll like receptor activation of the immune system can also be another potential mechanism of hlh [5] . it usually presents with fevers, hepatosplenomegaly, pancytopenia along with elevated liver enzymes, significant ferritin elevation and hypertriglyceridemia [6] . ebv is the most common infection associated with acquired hlh [7] . other viruses such as adenovirus and cytomegalovirus can also trigger hlh. infectious triggers cause immune activation. alternatively, immune deficiency can also trigger hlh such as hiv disease, malignancy, and certain rheumatological diseases [1] . diagnosis of hlh is challenging and requires a high index of suspicion. it is not uncommon for patients to have had a prolonged or multiple hospitalizations associated with clinical decline in health status before the diagnosis is finally made. diagnostic criteria have been formulated as per the hlh-2004 protocol which includes molecular, clinical and laboratory criteria [6] . genetic testing should be performed in all suspected cases. the hlh-2004 protocol has not been validated in adults in secondary hlh. also, it has practical difficulty in application as the various tests required for completion of the diagnostic protocol take time and are not rapidly available. in one study of 369 patients, the clinical findings included in hlh-2004 protocol were found to variably distributed in the patient population [8] . the prevalence of low or absent nk cell function was 71%. prolonged fever was seen in 95% of the population, whereas depleted cell lines were seen in up to 92% cases. splenomegaly was seen in 89% of the cases. hypertriglyceridemia or hypofibrinogenemia were noted in 90% cases. elevated ferritin greater than 500 mcg/l was found in 94% of the patients. in contrast, evidence of hemophagocytosis on bone marrow exam was only seen in about 82% of the study subjects, the least prevalent of the markers. markers of increased t cell activation such as elevated cd25 counts were seen in up to 97% of the patients. it is therefore to be noted that the characteristic finding of "hemophagocytosis" in the liver, lymph nodes or bone marrow is not necessary for diagnosis, however, its presence can aid in diagnosis. flow cytometry in a specialized laboratory can be very helpful with immunological markers [8] . the h-score is a much more pragmatic and easier tool to apply by the patients' bed side and can help with rapid diagnosis and therefore help with treatment considerations, as timely intervention can help improve prognosis. an online calculator is also available for calculating the h-score [9, 10] . hlh should be considered in the differential diagnosis of any patient presenting with unexplained fever, pancytopenia, hepatosplenomegaly, rash, lymphadenopathy, neurologic symptoms, liver function abnormalities and high serum ferritin. elevated ferritin due to macrophage activation is helpful in children but much less specific in adults. however, a very high elevation of ferritin and scd25 are very helpful in making the diagnosis. treatment should be initiated in a timely manner and consists of supportive care, treating the underlying triggers like infection, immunosuppressive agents, monoclonal antibodies, cytotoxic therapy and biological agents [11] [12] [13] [14] . hematopoietic stem cell transplant may be an option for definitive treatment of hlh in selected patients [15] . delay in diagnosis is associated with poor prognosis due to multi-organ involvement. mortality rate remains high despite treatment, ranging from 18-24% in ebv-associated hlh [16] . given the current day pandemic, severe cases of covid-19 can act as hlh mimic, as the former manifests as severe sepsis with cytokine storm syndrome. this needs further elucidation and some therapies like il-6 inhibitor tocilizumab, which have been used for hlh/cytokine storm syndrome previously, are being tried as therapy for covid-19 [17] . several academic institutions have employed the h-score mentioned above to guide therapeutic decisions on ilinhibitors. an h-score greater than 250 confers a 99% probability of hlh and a score of less than 90 confers a less than 1% probability of hlh [18, 19] . our case highlights the importance of having a high index of suspicion for hlh in unusual presentations of febrile illness with pancytopenia, even in otherwise healthy individuals. it is possible that our patient could have benefited from earlier treatment with steroids or other appropriate agents, rather than later in the course of the disease. further research is needed to better understand the pathophysiology of hlh at a molecular level, which could lead to modification of existing treatment protocols and improve patient survival. human subjects: consent was obtained by all participants in this study. in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. hemophagocytic lymphohistiocytosis: when the immune system runs amok familial hemophagocytic lymphohistiocytosis reactive hemophagocytic syndrome in adult systemic disease: report of twenty-six cases and literature review hemophagocytic syndromes and infection pattern recognition receptors and inflammation hlh-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. pediatr blood cancer clinical features and treatment strategies of epstein-barr virus-associated hemophagocytic lymphohistiocytosis hemophagocytic lymphohistiocytosis: a review haemophagocytic lymphohistiocytosis in adult critical care chemoimmunotherapy for hemophagocytic lymphohistiocytosis: long-term results of the hlh-94 treatment protocol treatment of familial hemophagocytic lymphohistiocytosis with antithymocyte globulins, steroids, and cyclosporin a immunotherapy of familial hemophagocytic lymphohistiocytosis with antithymocyte globulins: a single-center retrospective report of 38 patients requirement for etoposide in the treatment of epstein-barr virus-associated hemophagocytic lymphohistiocytosis haematopoietic stem cell transplantation in haemophagocytic lymphohistiocytosis poor outcomes of chronic active epstein-barr virus infection and hemophagocytic lymphohistiocytosis in non-japanese adult patients a study to evaluate the safety and efficacy of tocilizumab in patients with severe covid-19 pneumonia (covacta) development and validation of the hscore, a score for the diagnosis of reactive hemophagocytic syndrome performances of the h-score for diagnosis of hemophagocytic lymphohistiocytosis in adult and pediatric patients key: cord-328647-0dut550o authors: riachy, m.; baaklini, c.; ibrahim, i.; azar, h.; yaghi, c.; dabar, g.; bazarbachi, t.; nasnas, r.; karam-sarkis, d.; germanos, m.; maacaron, n.; khayat, g.; choucair, j. title: sdra par pneumonie à ebv date: 2007-05-31 journal: revue des maladies respiratoires doi: 10.1016/s0761-8425(07)91134-1 sha: doc_id: 328647 cord_uid: 0dut550o résumé introduction chez l’adulte immunocompétent, l’epstein barr (ebv) entraîne une maladie autolimitée spontanément résolutive. observation un syndrome de détresse respiratoire aigu (sdra) compliquant une pneumonie grave à ebv est rapporté avec le recours à une ventilation artificielle prolongée. le diagnostic était confirmé par l’usage des sérologies spécifiques et la recherche de la charge d’adn virale par pcr. à part la stratégie protectrice de la ventilation mécanique, le traitement médical a compris l’utilisation de l’acyclovir et les immunoglobulines polyclonales dans la phase précoce ainsi que des corticoïdes systémiques dans la phase tardive. la guérison était progressive et complète. conclusion la pneumonie à ebv compliquée d’un sdra chez les immunocompétents existe. sa prise en charge est un défi diagnostique et thérapeutique. summary introduction in the immuno-competent adult ebstein-barr virus (ebv) infection is a self-limiting disease that resolves spontaneously. case report we report a case of acute respiratory distress syndrome (ards) complicating severe ebv pneumonia and requiring prolonged artificial ventilation. the diagnosis was confirmed by specific serology and estimation of the viral load by pcr. apart from supportive treatment with artificial ventilation the medical treatment included the use of acyclovir and polyclonal immunoglobulins in the early phase and corticosteroids in the late phase. recovery was progressive and complete. conclusion ards can complicate ebv pneumonia in an immuno-competent subject. its management represents a diagnostic and therapeutic challenge. introduction chez l'adulte immunocompétent, l'epstein barr (ebv) entraîne une maladie autolimitée spontanément résolutive. observation un syndrome de détresse respiratoire aigu (sdra) compliquant une pneumonie grave à ebv est rapporté avec le recours à une ventilation artificielle prolongée. le diagnostic était confirmé par l'usage des sérologies spécifiques et la recherche de la charge d'adn virale par pcr. à part la stratégie protectrice de la ventilation mécanique, le traitement médical a compris l'utilisation de l'acyclovir et les immunoglobulines polyclonales dans la phase précoce ainsi que des corticoïdes systémiques dans la phase tardive. la guérison était progressive et complète. la pneumonie à ebv compliquée d'un sdra chez les immunocompétents existe. sa prise en charge est un défi diagnostique et thérapeutique. la mononucléose est une pathologie infectieuse fréquente secondaire au virus epstein barr (ebv), aussi responsable d'un grand nombre de néoplasies. il appartient à la famille des gammas herpès virus, infectant environ 90 % de la population mondiale [1]. chez l'adulte immunocompétent, il entraîne en général un tableau autolimité et spontanément résolutif associant fièvre, douleur de gorge, pétéchies au niveau du voile du palais et adénopathies [2] . une évolution plus grave nécessitant une hospitalisation, avec atteinte de plusieurs organes systémiques dont le coeur, le cerveau, le foie, ou le poumon est retrouvée dans moins de 5 % des cas. les décès sont le plus souvent secondaires à une hépatite fulminante, une encéphalite, un syndrome d'hémophagocytose ou une rupture de rate [3] . plusieurs cas d'atteinte pulmonaire sévère par ebv sont rapportés dans la littérature chez des personnes immunocompétentes [4] [5] [6] . toutefois le développement d'un syndrome de détresse respiratoire de l'adulte (sdra) et le recours à une ventilation artificielle sont exceptionnels. une patiente âgée de 29 ans, sans aucun antécédent notable, est hospitalisée à la suite d'un voyage au canada (en dehors de l'épidémie du sars) pour une toux irritative associée à une fièvre traînante depuis 10 jours sans réponse au levofloxacine. à l'admission, l'examen révélait une fièvre à 39°c, des adénopathies douloureuses de la chaîne cervicale postérieure et des amygdales pultacées. le bilan biologique sanguin retrouvait des leucocytes à 6 100/mm3 avec 52 % de lymphocytes atypiques sur le frottis, une cytolyse hépatique (asat 342 ui/ml, alat 320 ui/ml, γgt 263 ui/ml, phosphatase alcaline = 221 ui/ml) et une crp à 90 mg/l. les gaz du sang à l'air libre montraient une hypoxémie légère (ph 7,43 ; po 2 71 mm hg ; pco 2 37 mm hg). le reste du bilan était sans particularité. la radiographie du thorax révélait un épaississement bronchique aux deux bases avec un foyer alvéolaire basal droit. la bronchoscopie objectivait la présence de sécrétions muqueuses peu abondantes avec une inflammation sévère diffuse des parois bronchiques. le lavage bronchoalvéolaire était stérile, mettant en évidence des cellules géantes multinucléées, faisant suspecter un syndrome mononucléosique. les hémocultures, le monospot, les sérologies de l'hépatite a et b, du vih et du mycoplasme, l'antigène urinaire pour légionella pneumophila et l'intradermoréaction à la tuberculine étaient négatifs. la sérologie pour le cytomégalovirus (cmv) était équivoque (igm 1,16 et igg 138 ua/ml pour un index de positivité à 0,5 et 15 respectivement) mais l'antigénémie précoce (pp65) négative. la sérologie pour le virus d'epstein barr (ebv) en igm vca était en densité optique de 1,93 (seuil à 0,30). un traitement symptomatique est débuté avec une antibiothérapie probabiliste par azithromycine. à j3, l'état respiratoire s'était franchement détérioré. la radiographie thoracique retrouvait une extension de la pneumonie basale droite et l'apparition d'images alvéolaires bilatérales ( fig. 1) . ce tableau clinico-radiologique associé aux résultats de la sérologie à cmv a fait retenir comme possible une pneumopathie à cmv. compte tenu de la gravité un traitement par ganciclovir iv (480 mg/jour) a été débuté. le diagnostic de syndrome mononucléosique se base sur un cortège de signes cliniques et biologiques simples [2] . les infections sévères avec une atteinte systémique compromettant la vie du sujet restent rares chez les immunocompétents et sont souvent secondaires au cytomégalovirus [3] . l'infection submortelle à ebv est exceptionnelle et l'atteinte pulmonaire si elle existe reste autolimitée [4] [5] [6] . chez notre malade d'âge jeune, la pneumonie étendue à ebv était compliquée d'un sdra sévère ayant nécessité le recours à une ventilation artificielle difficile durant 7 jours. plusieurs examens paracliniques évoquent le diagnostic d'une infection à ebv. la présence de lymphocytes atypiques dans le sang périphérique à l'examen sur lame n'est pas spécifique d'une infection à ebv. la culture virale n'est pas réalisée en routine mais dans des laboratoires spécialisés ou de référence. la recherche d'anticorps hétérophiles et d'anticorps spécifiques contre les antigènes du virus d'epstein barr est utile pour le diagnostic d'une infection aiguë surtout en présence d'un haut index de suspicion clinique [7] . toutefois une possible réaction croisée avec la sérologie du cmv nécessite une interprétation avec précaution [8] . chez notre malade, l'évolution temporelle de ces sérologies pouvait aider à discerner entre les deux virus. une infection aiguë est suggérée par la positivité des anticorps igm dirigés contre la capside virale (vca), avec positivité des anticorps dirigés contre la partie d des antigènes précoces (ea-d). le taux des igg anti-vca augmente lentement au cours d'une infection aiguë, mais persiste tout le long de la vie. durant la phase aiguë de l'infection, l'adn viral peut être détecté dans le sang par une méthode pcr avec une sensibilité de 94,9 % et une spécificité de 97,4 % lors d'une primoinfection [9] . il disparaît après 22 jours de l'apparition des symptômes [9] . ainsi la mesure de la charge virale est utile dans le diagnostic et le suivi des maladies associées à l'ebv [10] . le traitement de l'infection non compliquée à ebv est généralement symptomatique. le repos relatif pour 2 à 3 semaines est recommandé surtout en présence d'une splénomégalie. le traitement des infections virales à ebv par les corticoïdes est controversé. ils sont utilisés à la dose de 1 mg/kg/j en cas d'obstruction des voies aériennes, d'anémie hémolytique, de thrombopénie sévère et parfois dans les atteintes systémiques du cerveau et du coeur [11] . par ailleurs, les données concernant l'utilisation des corticoïdes chez l'homme pour traiter les pneumonies virales sont limitées. le bénéfice des corticoïdes dans le traitement des détresses respiratoires secondaires au coronavirus dans l'épidémie du sars n'est pas prouvé. dans une étude rétrospective menée chez des malades atteints de pneumonie sévère varicelleuse, les corticoïdes ont réduit le séjour hospitalier [12] . d'autre part l'utilisation des corticoïdes systémiques dans le cadre d'un sdra est discutable. l'effet néfaste d'une corticothérapie systémique empirique dans le sepsis et le choc septique est démontré [13] . une méta analyse plus récente [14] montre le bénéfice probable des corticoïdes sur la mortalité dans les phases tardives du sdra, se basant sur une seule étude prospective randomisée en double insu [15] . toutefois les résultats préliminaires de l'ardsnet sponsorisé par le « national institute of health » montre l'efficacité potentielle des corticoïdes dans la phase tardive du sdra à condition d'être débutés avant 14 jours d'évolution [16] . la corticothérapie chez notre malade dans la phase tardive était accompagnée d'une amélioration de l'atteinte pulmonaire et une extubation rapide. l'utilisation des antiviraux dans les infections aiguës à ebv reste peu étudiée. l'acide phosphonoacétique, l'adénine arabinoside, l'acyclovir, le desciclovir, le ganciclovir, le moribavir (1263w94) l'interféron-α et l'interféron-γ inhibent la réplication de l'ebv in vitro [17, 18] . cependant, les études cliniques de la thérapie antivirale restent insuffisantes pour établir des recommandations. l'acyclovir et le descyclovir ont tous les deux montré une action thérapeutique temporaire sur la leucoplasie chevelue orale chez des patients infectés par le vih [18] . l'acyclovir a induit une rémission temporaire d'une lymphoprolifération à cellules b chez un transplanté rénal et chez deux patients atteints de pneumonie interstitielle à l'ebv [19, 20] . de plus l'acyclovir a été rapporté seul ou en combinaison efficace dans le traitement des pneumonies à ebv [21] . le phosphonoformate trisodique (foscavir) inhibe la multiplication virale de l'ebv in vitro à la dose de 80 à 100 µmol/l [22] . la vaccination contre l'ebv reste décevante avec le risque oncogène potentiel de l'ebv.98. une étude utilisant la gp350 virale pour vaccination semble prometteuse [23] . les malades immunodéprimés et transplantés d'organes peuvent être traités par des immunoglobulines polyclonales lors des pneumonies virales sévères [24] . plusieurs cas dans la littérature rapportent l'utilisation des immunoglobulines dans des pneumonies sévères chez des immunocompétents, liés au virus de la varicelle [25] , de l'adénovirus [26] et du virus respiratoire syncytial, mais pas dans l'ebv. toutefois, les préparations commerciales d'immunoglobulines polyclonales, contenant une forte activité contre l'hsv1,2,6,7, le vzv, l'ebv, les virus de la rubéole, des oreillons et du parvovirus b19 en plus de certains types spécifiques de bactéries [27] , nous ont incité à les utiliser chez notre malade. la ventilation mécanique dans le sdra est bien standardisée et suit une stratégie protectrice à petits volumes avec une pep élevée [28] . des manoeuvres de recrutement alvéolaires sont utilisés parfois en pratique courante. néanmoins leur bénéfice sur la mortalité n'est pas prouvé. ce cas clinique rapporte une pathologie peu commune de sdra par pneumonie à ebv chez un immunocompétent. le diagnostic suspecté cliniquement doit être confirmé par l'usage des sérologies spécifiques et la pcr dans les meilleurs délais. le traitement médical n'est pas standardisé. l'utilisation de l'acyclovir et éventuellement les immunoglobulines polyclonales peuvent être envisagé dans la phase précoce de la maladie et les corticoïdes dans la phase tardive. infectious mononucleosis and epstein-barr virus epstein-barr virus infectious mononucleosis infectious mononucleosis; review of complications in hospitalized series infectious mononucleosis, diffuse pneumonia and acute respiratory failure in an elderly woman diffuse pneumonia and acute respiratory failure due to infectious mononucleosis in a middle-aged adult severe respiratory insufficiency complicating epstein-barr virus infection: case report and review diagnostic virology antigenic cross-reactions among herpes simplex virus types 1 and 2, epstein-barr virus, and cytomegalovirus puchhammer-stockl e : serum epstein-barr virus dna load in primary 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of desciclovir in the treatment of epstein-barr virus infection in oral hairy leukoplakia treatment of life-threatening epstein-barr virus infection with acyclovir chronic epstein-barr virus infection associated with fever and interstitial pneumonitis. clinical and serologic features and response to antiviral chemotherapy interferon gamma (ifn-gamma) deficiency in generalized epstein-barr virus infection with interstitial lymphoid and granulomatous pneumonia, focal cerebral lesions, and genital ulcers: remission following ifn-gamma substitution therapy sensitivity of kaposi's sarcoma-associated herpesvirus replication to antiviral drugs. implications for potential therapy sensitivity of the transforming and replicative functions of epstein--barr virus to inhibition by phosphonoacetate intravenous immunoglobulin in adult varicella pneumonia complicated by acute respiratory distress syndrome treatment of adenoviral pneumonitis with intravenous ribavirin and immunoglobulin in vitro antiviral and antibacterial activity of commercial intravenous immunoglobulin preparations -a potential role for adjuvant intravenous immunoglobulin therapy in infectious diseases ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. the acute respiratory distress syndrome network key: cord-005435-onghg1o8 authors: zhang, j; wilson, a; alber, s; ma, z; tang, z-l; satoh, e; mazda, o; watkins, s; huang, l; pitt, b; li, s title: prolonged gene expression in mouse lung endothelial cells following transfection with epstein–barr virus-based episomal plasmid date: 2003-04-17 journal: gene ther doi: 10.1038/sj.gt.3301958 sha: doc_id: 5435 cord_uid: onghg1o8 the development of a strategy to deliver a gene to pulmonary endothelium will be useful for gene function study and for pulmonary gene therapy. cationic lipidic vectors are efficient in gene transfer to pulmonary endothelium via the vascular route; however, gene expression is transient and lasts for only a few days. in this study, we show that pulmonary gene transfer via cationic lipidic vectors can be significantly improved using an epstein–barr virus (ebv)-based expression plasmid. systemic administration of cationic liposomes followed by the ebv-based plasmid led to gene expression in the lung that lasted for more than 3 weeks. prolonged and high levels of gene expression can also be obtained in primary mouse lung endothelial cells (mlec) following lipofection with an ebv-based plasmid. these results suggest the utility of this gene transfer protocol in studying the expression of cloned genes in lung endothelial cells and in pulmonary gene therapy. somatic gene transfer to the pulmonary endothelium may be a useful strategy for modifying the phenotype of endothelium and/or vascular smooth muscle in disorders such as primary pulmonary hypertension, ards, or pulmonary metastatic disease. it may also provide a useful research tool to study the function of cloned genes in pulmonary endothelial cells in vitro and in vivo. among the viral vectors employed for such potential use, 1,2 adenoviral vectors have proved to be limited with respect to efficiency in part because of difficulty in assuring significant residence time in the lung and/or paucity of receptors for adenovirus on endothelium. also, adenoviral vectors are only moderately effective in infecting primary mouse lung endothelial cells in vitro. first-generation cationic lipidic vectors also produce modest degrees of gene transfer to lung after i.v. administration. 3, 4 modification of cationic lipid composition and dna:lipid ratios led to considerably higher gene transfer efficiency in the lung with the endothelial cells being the major cell type transfected. [5] [6] [7] [8] [9] [10] however, gene expression in the lung via this gene transfer protocol is transient and lasts for only a few days. in the current study, we investigate whether in vitro and in vivo gene transfer to mouse lung endothelial cells (mlec) can be significantly improved using an ebvbased expression plasmid. ebv plasmid is a replicating episomal vector that has been developed to overcome the problem of rapid elimination of intracellularly delivered plasmid dna in nonviral vector-mediated gene transfer. the viral elements required for episomal replication and nuclear retention are the cis-acting replication origin (orip) of the ebv gene and the ebv nuclear antigen-1 (ebna-1), which interacts with the orip region. [11] [12] [13] plasmids containing the orip and ebna-1 sequences are maintained as low-copy number dna episomes in the cell nucleus and replicate once per cell cycle in primate cells. most studies suggest that long-term replication of ebvderived plasmids occurs only in primate cells but not in rodent cells. 11, 14 nonetheless, a more persistent gene expression was found in a number of rodent tissues following nonviral method-mediated delivery of ebv plasmid compared to non-ebv plasmid possibly because of the nuclear retention function and the enhancer activity of transgene expression induced by ebna-1. [15] [16] [17] [18] we hypothesize that lipofection of mlec can also be significantly improved both in vitro and in vivo using the ebv-based plasmid. figure 1 shows the maps of three different plasmids used in this study. plasmids pgeg.gl3 and pg.gl3 contain the firefly luciferase gene under the control of a cag promoter. pgeg.gl3 also contains an ebv ebna-1 gene driven by a cag promoter. in addition, pgeg.gl3 contains the ebv orip. plasmid pngvl3-luc contains the firefly luciferase gene under the control of a cytomegalovirus (cmv) promoter. in the initial experiment, we compared the level of gene expression in the lung with pgeg.gl3 using two different gene transfer protocols, that is, complex injection and sequential injection. with a non-ebv plasmid, we have shown recently that sequential injection of cationic liposomes and plasmid is more efficient in pulmonary gene transfer than injection of cationic liposome/dna complexes. 19 furthermore, sequential injection is associated with a significantly reduced proinflammatory cytokine response. 19 a similar result was observed in this study with an ebv-based plasmid. as shown in figure 2a , serum levels of tnf-a in sequential injection group were only 30% of those in complex injection group. furthermore, sequential injection of dotap:cholesterol liposomes and pgeg.gl3 led to a significantly higher level of gene expression in the lung than complex injection (figure 2b ). we and others have shown that proinflammatory cytokines can significantly inhibit transgene expression in either viral or nonviral vector-mediated gene transfer. [20] [21] [22] [23] the improved pulmonary gene transfer via sequential injection protocol is probably because of a decreased inhibitory effect of cytokines on transgene expression. thus, sequential injection protocol was used in subsequent studies. figure 3 shows the luciferase expression in the lungs over time following sequential injection of dotap:cholesterol liposomes (+/à charge ratio of 6:1) followed by figure 2 tnf-a cytokine response (a) and luciferase gene expression in mouse lungs (b) following i.v. injection of cationic liposome/dna complexes or cationic liposomes followed by plasmid dna. dotap:cholesterol liposome/pgeg.gl3 complexes were prepared at a +/à charge ratio of 6/1 as described 19 and injected into female cd-1 mice (charles river laboratories, wilmington, ma, usa; eight mice in each group) via tail vein at a dose of 35 mg dna per mouse. in a different group, mice received tail vein injection of dotap:cholesterol liposomes (1.3 mmol lipid/mouse) followed by pgeg.gl3 (35 mg/mouse). at 2 h following injection, mice were bled from the retro-orbital sinuses under anesthesia. the blood was allowed to stay at 41c for 4 h and then centrifuged at 14 000 g for 10 min at 41c. serum levels of tnf-a were determined with the specific cytokine immunoassay kit (r&d systems, minneapolis, mn, usa). in a separate experiment, groups of eight mice received i.v. injection of dotap: cholesterol liposome/pgeg.gl3 complexes or sequential injection of dotap:cholesterol liposome followed by pgeg.gl3 as described above. at days 1 and 3 following injection, mice were killed and lungs were removed and homogenized in 1 ml of ice-cold lysis buffer (0.05% triton x-100, 2 mm edta, and 0.1 m tris, ph 7.8) with a tissue tearer for 20 s at high speed. the homogenates were then centrifuged at 14 000 g for 10 min at 41c. of the supernatant 10 ml was analyzed with the luciferase assay system (promega, madison, wi, usa) using an automated lb953 luminometer (berthod, bad wildbad, germany). the protein content of the supernatant was measured with the bio-rad protein assay system (bio-rad, hercules, ca, usa). luciferase activity was expressed as relative lights units (rlu) per milligram of tissue protein. data were expressed as means7s.d. and analyzed by the two-tailed unpaired student's t-test using the prism software program (graphpad software, san diego, ca, usa). *po0.05; **po0.01 (versus complex injection). gene transfer to mlec via ebv-based plasmid j zhang et al pgeg.gl3, pg.gl3, or pngvl3-luc (35 mg/mouse). peak expression of luciferase occurred on day 1 for all plasmids. however, of the three plasmids, gene expression in pngvl3-luc group declined most rapidly with time and was not different from background by 21 days. gene expression in the pgeg.gl3 group declined during the first week following sequential injection, but then there was a rebound in gene expression over the subsequent 2 weeks. a similar phenomenon was observed in a study with complex injection. 24 the rapid decline in gene expression in the first few days following injection is probably largely because of the inhibitory effect of cytokines. despite a decreased cytokine response in sequential injection (figure 2a) , the level of tnf-a in the serum might be sufficient to cause significant inhibition on transgene expression. nevertheless, the levels of gene expression in pgeg.gl3 group were significantly higher than those in pg.gl3 or pngvl3-luc group at all time points examined. of note is that even after 3 weeks, there is a detectable level of gene expression in lungs of mice injected with pgeg.gl3. having characterized pulmonary gene transfer with pgeg.gl3, the sequential injection protocol was further evaluated using pgeg.egfp as a reporter gene. no green cells were observed in the lungs of mice treated with a control plasmid (pgeg.gl3) (data not shown). in contrast, there was localized gene expression throughout the distal lung of mice that received pgeg.egfp ( figure 4a ). the cell type of transfected cells was further analyzed by anti-platelet endothelial cell adhesion molecule-1 (pecam-1) immunofluorescence staining of the lung sections. constitutive expression of pecam-1 is a fundamental characteristic of endothelial cells. 25 as shown in figure 4a and b, egfp signal was substantially colocalized with pecam labeling, confirming that endothelial cells were the major cell type transfected. we then examined whether primary mlec can also be efficiently transfected via ebv-based plasmid. primary mlec were prepared by modification of an immunobead protocol. 26 briefly, mouse lungs were finely minced and digested in collagenase (type i, 100 mg/ml). cell suspensions were incubated with a monoclonal antibody (rat anti-mouse) to pecam-1 (bd pharmingen, san diego, ca, usa) for 30 min at 41c. pecam-1 has been used extensively as a reliable marker to isolate endothelial cells. 26 the cells were washed twice with buffer to remove unbound antibody, and resuspended in binding buffer containing the appropriate number of washed magnetic beads coated with sheep anti-rat igg (dynal, oslo, norway). attached cells were washed four to five times in cell culture medium, and then were digested with trypsin/edta to detach the beads. bead-free cells were centrifuged and resuspended for culture. at approximately passage 2, cells were incubated with fluorescent-labeled di-acetylated ldl (dii-ldl), which is taken up only by endothelial cells and macrophages, and sorted to homogeneity by facs. the enriched pecam and dii-ldl population were subcultured in dmem/f-12, 20% fbs, 6% plasma-derived human serum, 2 mm glutamine, and 30 mg/ml endothelialderived growth factor. figure 5 shows gene expression in mlec following transfection with pgeg.gl3, pg.gl3, or pngvl3-luc. more persistent and significantly higher level gene expression was achieved with pgeg.gl3. it should be noted that transfection of mlec in this study was performed using a nonoptimized reagent (dotap:cholesterol liposomes). gene expression in mlec should be further improved via optimization of transfection condition. this will greatly facilitate the use of this gene transfer protocol as a research tool to study the functions of cloned genes in mlec in vitro. ebv plasmid coupled with cationic liposome and hvjliposome has also been shown to efficiently transfect human primary cells in vitro. 27 one of the potential concerns over the use of ebv plasmid is the oncogenicity of ebna-1 protein. transgenic mice harboring the ebna-1 gene driven by b lymphocyte-specific enhancer developed b-cell lymphoma. 28 however, several studies have shown that ebna-1 mice received tail vein injection of dotap:cholesterol liposomes followed by pgeg.egfp. after 1 day, mice were killed and lungs were perfused intravascularly with pbs followed by 2% paraformaldehyde in pbs, and inflated with this fixative to near total lung capacity. the lungs were rinsed with cold pbs and immersed in 30% sucrose in pbs at 41c overnight. the lungs were then quickly frozen in oct with dry ice. lung cryo-sections (5 mm) were then cut. following three washes in pbs containing 0.5% bovine serum albumin and 0.15% glycine (pbg buffer), sections were incubated in a 1:100 dilution of rat anti-mouse pecam (pharmingen, san diego, usa) for 1 h at rt, washed with pbg three times, and labeled with cy3-labeled goat anti-rat igg (jackson immunoresearch laboratories) for 1 h at rt. the sections were mounted in gelvatol (monsanto, st louis) and the images were collected using a leica tcs nt confocal microscope at 1024 â 1024 pixel resolution. gene transfer to mlec via ebv-based plasmid j zhang et al itself is insufficient for b lymphocyte transformation in vitro in the absence of the latent viral properties ebna-2, 29 ebna-3a, 30 ebna-3c, 30 and lmp-1. 31 furthermore, 293 cells stably transfected with ebna-1 did not grow in soft-agar plates and were sensitive to serum depletion. 32 it is unlikely that the current gene expression system will induce tumor in the host. further studies are required to address the safety of long-term gene therapy using ebv plasmid. in summary, we have shown that systemic administration of cationic liposomes followed by an ebv plasmid led to a prolonged gene expression in the lung, which lasted for more than 3 weeks. an efficient transfection of primary mlec can also be achieved using ebv plasmid. these results suggest the utility of this gene transfer protocol in studying the expression of cloned genes in lung endothelial cells and in pulmonary gene therapy. figure 5 prolonged gene expression in primary lung endothelial cells following lipofection with an ebv-based plasmid. primary mlec were prepared as described in the text. cells of density 1 â 10 5 cells/well in a 48well plate were transfected with 1.5 mg of plasmid (pgeg.gl3, pg.gl3, or pngvl3-luc) complexed to dotap:cholesterol liposomes at a +/à charge ratio of 2:1 in serum-free medium. at 4 h later, the transfection medium was removed and replaced with complete medium. at days 1, 4, 7, and 11 following transfection, the cells were lysed using ice-cold lysis buffer (0.05% triton x-100, 2 mm edta, and 0.1 m tris, ph 7.8), the homogenate centrifuged at 14 000 g for 10 min at 41c and the supernatant analyzed for luciferase expression. *po0.05 (versus pg.gl3). gene transfer to mlec via ebv-based plasmid j zhang et al in vivo adenovirus-mediated gene transfer to lungs via pulmonary artery in vivo gene delivery to the pulmonary circulation in rats: transgene distribution and vascular inflammatory response systemic gene expression after intravenous dna delivery into adult mice aerosol and intravenous transfection of human alpha 1-antitrypsin gene to lungs of rabbits factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery factors controlling efficiency of cationic lipid-mediated transfection in vivo via intravenous administration 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vectors in mdx mice non-replicating epstein-barr virus-based plasmids extend gene expression and can improve gene therapy in vivo direct intra-cardiomuscular transfer of beta2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters highly efficient gene transfer into murine liver achieved by intravenous administration of naked epstein-barr virus (ebv)-based plasmid vectors sequential injection of cationic liposome and plasmid dna effectively transfects the lung with minimal inflammatory toxicity repression of retrovirus-mediated transgene expression by interferons: implications for gene therapy promoter attenuation in gene therapy: interferongamma and tumor necrosis factor-alpha inhibit transgene expression effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors the inhibitory role of cpg immunostimulatory motifs in cationic lipid vector-mediated transgene expression in vivo high and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter the biology of pecam-1 a general strategy for isolation of endothelial cells from murine tissues: characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants efficient gene transduction by epstein-barr-virusbased vectors coupled with cationic liposome and hvjliposome expression of epstein-barr virus nuclear antigen-1 induces b cell neoplasia in transgenic mice genetic analysis of immortalizing functions of epstein-barr virus in human b lymphocytes epstein-barr virus nuclear proteins ebna-3a and ebna-3c are essential for b-lymphocyte growth transformation epstein-barr virus latent membrane protein 1 is essential for b-lymphocyte growth transformation sustained transgene expression in vitro and in vivo using an epstein-barr virus replicon vector system combined with hvj liposomes gene transfer to mlec via ebv-based plasmid j zhang et al this work was supported by nih grants hl ro1 63080 (to s li), ai ro1 48851 (to l huang), and hl ro1 32154 (to b pitt). key: cord-277096-zvb7n9wo authors: bond, david a.; dotson, emily; awan, farrukh t.; baiocchi, robert a.; blum, kristie a.; maddocks, kami title: febrile hypotensive reactions following abvd chemotherapy in patients with ebv-associated classical hodgkin lymphoma date: 2018-11-29 journal: clin lymphoma myeloma leuk doi: 10.1016/j.clml.2018.11.020 sha: doc_id: 277096 cord_uid: zvb7n9wo nan classical hodgkin lymphoma (chl) comprises approximately 10% of all cases of lymphoma worldwide and is curable with multi-agent chemotherapy in the majority of cases, including in patients with advanced stage disease. the abvd regimen (doxorubicin, bleomycin, vinblastine, and dacarbazine) is currently the most widely used front-line treatment for patients with early and advanced stage chl, with no alternative regimen to date showing superior overall survival (os). [1] [2] [3] patients with human immunodeficiency virus (hiv) infection have an 11-fold increased risk for chl, 4 and chl is driven by the epstein-barr virus (ebv) in approximately 40% of cases, including nearly all cases associated with hiv infection. 5, 6 multiple prognostic risk factors have been identified, and the international prognostic score (ips) is widely used to provide riskstratification based upon clinical risk factors in patients with advanced stage disease. 7 chl is characterized by a relatively small proportion of pathologic reed-sternberg (rs) cells within a reactive inflammatory milieu and is associated with a state of increased clinical practice points abvd (doxorubicin, bleomycin, vinblastine, and dacarbazine) is a widely used front-line regimen for the treatment of early and advanced stage classical hodgkin lymphoma (chl). a fulminant syndrome characterized by pyrexia and shock was observed in early trials of bleomycin, occurring more frequently in patients with lymphoma. in the past 3 decades, only 1 case of a similar fulminant reaction following abvd had been reported, and thus there is limited literature regarding the risk factors, clinical course, and management for this life-threatening syndrome. we identified 3 patients experiencing febrile hypotensive reactions following abvd chemotherapy at our institution with shared baseline clinical features, including stage ivb disease, high risk disease by international prognostic score, male gender, and epstein-barr virus-positive chl. all 3 patients experienced fever, rigors, tachycardia, shortness of breath or hypoxia, and an elevated venous lactate with onset less than 2 hours after completing the first abvd infusion. all patients received intravenous fluid resuscitations and corticosteroids, 2 patients required vasopressors owing to refractory hypotension, and 1 patient required mechanical ventilation for respiratory failure. symptoms resolved within 24 hours in all cases. two patients received bleomycin with subsequent cycles, and 1 patient was treated with avd (doxorubicin, vinblastine, and dacarbazine); fulminant reactions were not observed with subsequent cycles. clinicians should be aware that fulminant febrile, hypotensive reactions can be seen following abvd treatment for chl. management with intravenous corticosteroids and intensive supportive care was associated with resolution within 24 hours of onset in the present series. cytokine production, including increased tumor necrosis factor-a, interleukin (il)-6, and il-8. 8, 9 cd68þ macrophages are a prominent component of the tumor microenvironment. a higher proportion of cd68þ cells in the tumor microenvironment are seen in ebv-positive chl in comparison to ebv-negative cases, and a high proportion of tumor-associated cd68þ macrophages have been associated with inferior outcomes. 10, 11 bleomycin is an antibody complex derived from streptomyces sp. with anti-neoplastic properties owing in part to inhibition of dna synthesis. 12 in early clinical investigation, bleomycin demonstrated single-agent activity in multiple solid organ malignancies, but the highest single-agent response rates were in hodgkin lymphoma. 13 fever was reported in 20% to 50% of patients following singleagent bleomycin, typically occurring after the first dose, and was observed more frequently in patients with chl compared with solid organ malignancies. 14 fever was typically self-limited, but in a review of the first 1174 patients treated with single-agent bleomycin, 4 cases of fulminant fever associated with hypotension and cardiorespiratory collapse leading to death were reported, all in patients with lymphoma. in a preclinical study, bleomycin was shown to provoke fever in a dose-dependent manner in rabbits with onset 1 to 2 hours after administration, and supernatant from cultures of human and rabbit leukocytes with bleomycin induced a febrile response with shorter latency, suggesting a cytokinemediated effect. 15 subsequent cases of fatal or life-threatening febrile, hypotensive reactions in patients with lymphoma treated with bleomycin as part of multi-agent therapy have been reported, [16] [17] [18] [19] including a patient with a febrile, hypotensive reaction following treatment with abvd associated with tumor lysis syndrome (tls) and markedly elevated serum il-6. 19 here we present 3 cases of patients with chl experiencing fulminant febrile, hypotensive reactions shortly after their first dose of abvd with similar baseline clinical features, including ebv-positive disease, and describe their clinical courses. cases were identified after surveying the lymphoma faculty at our institution. institutional review board approval was obtained. case series patient 1. patient 1 was 51 years old at initial presentation, with progressively worsening left inguinal lymph node enlargement, fever, malaise, night sweats, and weight loss. he had no significant past medical history. physical examination was significant for temperature of 103.4 f, palpable spleen tip 2 cm below the mid-costal margin, and a large left inguinal lymph node measuring 10 â 4 cm. computed tomography (ct) imaging revealed splenomegaly, and enlarged periaortic, celiac, retroperitoneal, and left inguinal lymph nodes. blood and urine cultures were collected and remained sterile; ebv polymerase chain reaction (pcr) was positive at 1,600,000 copies/ml. other laboratory values are summarized in table 1 . core needle biopsy of the left inguinal lymph node showed chl, with rs cells positive for epstein-barr encoding region (eber) by in situ hybridization (ish), and cd68 staining showed a predominance of macrophages comprising over 50% of the tumor background. bone marrow biopsy was negative for disease involvement. positron emission tomography (pet) scan showed diffuse hypermetabolic activity in lymph nodes above and below the diaphragm as well as focal sites of increased osseous uptake, consistent with ann arbor stage ivb disease, with ips risk score of 6. intravenous ganciclovir was initiated for treatment of ebv viremia prior to starting chemotherapy. the decision was made to begin treatment with abvd while inpatient, with doxorubicin dose reduced by 50% (12.5 mg/m 2 ) given bilirubin elevation (3.5 on date of treatment) and risk for reduced clearance, and vinblastine, bleomycin, and dacarbazine given at standard dosing. vital signs at initiation of treatment were significant for temperature (t) of 97.9 f, heart rate (hr) of 87, weight 112 kg, body max index (bmi) of 33.5 kg/m 2 , and body table 1 . vasopressors were weaned off within 12 hours, and the patient was extubated within 24 hours. bleomycin was omitted from further cycles without subsequent complications. after 6 cycles of treatment, pet imaging was consistent with complete response (cr). the patient remains in follow-up with no evidence of relapse over 3 years after completion of therapy. patient 2. patient 2 was 28 years old at diagnosis with a history of hiv infection treated with anti-retroviral therapy; baseline cd4 count was 287 cells/mm 3 with an undetectable hiv viral load. he presented with fever, shortness of breath, and a dry cough. physical examination was significant for dry mucous membranes, diminished breath sounds bilaterally, and an enlarged right axillary lymph node. baseline laboratory values are summarized in table 1 . ct scan of the chest revealed bilateral airspace consolidation and an enlarged right axillary lymph node. bone marrow biopsy was performed and was interpreted as nondiagnostic with necrotizing granulomas noted. the patient was intubated on hospital day 3 for worsening respiratory status and diagnosed with acute respiratory distress syndrome. bronchiolar lavage was consistent with pneumocystis pneumonia, and treatment with methylprednisolone and trimethoprim/sulfamethoxazole was initiated. on hospital day 14, the patient was extubated, and the following day, an excisional right axillary lymph node biopsy was performed that was consistent with chl, and the patient was transferred to our hospital for further care. the prior bone marrow biopsy was reviewed by hematopathology at our institution and read as consistent with chl with extensive necrosis with scattered fibro-histiocytic infiltrates containing typical rs cells, positive for eber by ish, and numerous cd68þ macrophages present (percentage not enumerated). pet scan showed mildly hypermetabolic left hilar and right peri-bronchial lymph nodes and hypermetabolic postoperative changes in the right axilla. the patient was staged as ann arbor stage ivb given the presence of bone marrow involvement, with ips risk score of 5. oral valgancyclovir was initiated for treatment of cmv viremia. therapy was initiated with abvd given at standard dosing. vital signs prior to start of treatment were significant for t 99. 8 table 1 . blood cultures were collected and remained sterile, and cefepime was initiated empirically. intravenous saline and 50 mg hydrocortisone were administered as well as hydromorphone for rigors. symptoms rapidly improved, with normalization of lactate and resolution of fever within 6 hours. antibiotics were discontinued after 24 hours of negative cultures. abvd was continued with dexamethasone 20 mg premedication with subsequent infusions, and no further reactions were observed. pet scan was performed after 3 cycles to evaluate response and showed progressive disease. platinum-based salvage therapy was initiated; the patient is currently alive receiving salvage therapy for refractory chl. patient 3. patient 3 was 62 years old at initial presentation, with a prior history of chronic lymphocytic leukemia (cll) treated with 6 cycles of obinutuzumab and otlertuzumab in an investigational protocol completed 3 months prior to presentation. he was diagnosed with igvh unmutated cll after presenting with bulky lymphadenopathy; baseline cytogenetics were positive for trisomy 12 as the sole abnormality. during treatment with obinutuzumab and otlertuzumab, he experienced clinical resolution of lymphadenopathy by physical exam. at his end of therapy evaluation 3 months after his sixth cycle of treatment, he noted daily fevers as high as 103 f. physical exam showed no evidence of lymphadenopathy. he was hospitalized for further evaluation with serum ebv pcr positive at 84,523 copies/ml; other laboratory values are summarized in table 1 . blood and urine cultures were collected and remained sterile, and other infectious workup was negative. bone marrow biopsy revealed a hypercellular marrow with absent cll cells by morphology or by flow cytometry, but classical rs cells were present, positive for eber by ish, with increased surrounding cd68þ macrophages. pet scan showed hypermetabolic activity in cervical, mediastinal, abdominal, and pelvic lymph nodes as well as hypermetabolic liver lesions and diffuse increased uptake throughout the bone marrow. the patient was staged as ann arbor stage ivb richter transformation to chl with an ips risk score of 5. oral valacyclovir was initiated, and the patient was discharged from the hospital. upon outpatient evaluation, the decision was made to initiate treatment with abvd, given at standard dosing. vital signs prior to treatment were significant for t 99.5 f, hr 130, weight 92.5 kg, bsa 2.17, and bmi 26.9. at 16:20, bleomycin was administered, and dacarbazine was completed at 16:40. at 17:17, rigors were noted with temperature of 103.5 f, and inpatient admission was requested. by 18:15, confusion and lethargy were noted with t 106 f, hr 144, bp 92/53, and rr 27. hydrocortisone was administered intravenously (50 mg) as well as oral acetaminophen and normal saline hydration. the patient developed progressive hypoxia with requirement of 13l oxygen by high-flow nasal canula; chest radiograph demonstrated a pulmonary edema pattern bilaterally. blood and urine cultures were collected and remained sterile, and an extended respiratory viral panel returned positive for coronavirus. other laboratory values are summarized in table 1 . despite fluid resuscitation, the patient developed progressive hypotension; norepinephrine infusion was initiated to maintain a mean arterial bp of 65, and 20 mg intravenous dexamethasone was administered. hypotension quickly improved, and norepinephrine was titrated off within 12 hours of onset. intravenous antibiotics were discontinued after blood cultures remained sterile for 48 hours, oxygen was weaned off within 24 hours, and the patient was discharged on hospital day 3. for the remaining cycles of abvd, dexamethasone 6 mg was given for 2 days prior to treatment in addition to 12 mg on the day of treatment. the patient experienced rigors at home after cycle 1 day 15 and took 6 mg of oral dexamethasone with resolution of symptoms. no subsequent episodes were noted, and the patient completed 6 cycles of abvd. end of therapy pet was consistent with cr, and end of therapy bone marrow biopsy was negative for chl or cll. within 6 months, relapse of cll was diagnosed by bone marrow biopsy, and the patient opted to undergo allogeneic hematopoietic cell transplant (hct) with reduced-intensity conditioning from a haplo-identical related donor for definitive treatment of cll and to mitigate the risk of relapse of richter transformation to chl. he remains alive in active follow-up with no evidence of relapse of cll or chl over 12 months after hct. given the high probability of cure in chl with front-line multiagent chemotherapy, short-and long-term toxicities from treatment are of particular importance, and awareness of potential therapyrelated toxicities and supportive management is essential. in this case series, we highlight a rare, life-threatening early complication of therapy with abvd. all 3 cases occurred during cycle 1 day 1 of abvd therapy, and the patients shared multiple baseline clinical characteristics including advanced stage disease, b symptoms, viremia, high risk ips score, and eber-positive disease (baseline characteristics summarized in table 2 ). the constellation of marked pyrexia, hypoperfusion, and respiratory failure in the absence of identified bacterial infection was reported as a rare complication in early clinical trials of single-agent bleomycin, 14 and at least 4 cases with a similar constellation of symptoms leading to fatal cardiorespiratory collapse in patients with lymphoma treated with bleomycin containing regimens were reported in the 1970s and 1980s. [16] [17] [18] 20 in a review performed in 2005 regarding the need to perform test dosing of bleomycin, the author postulated that the lack of reports of similar episodes in the 1990s or beyond may be owing to routine anti-emetic corticosteroid premedication with current bleomycin-containing regimens or that prior episodes were because of an unidentified contaminant that is no longer present in current bleomycin formulations. 21 since that time, there has been 1 subsequent case report of a febrile, hypotensive reaction with associated tls within the first hour of the first abvd infusion for chl. 19 this report and our series demonstrate that febrile hypotensive reactions can occur with modern bleomycin formulations and corticosteroid premedication. tls was not evident in the 3 cases from our institution and does not appear to be prerequisite for such reactions. in our series, the time to onset was 60 to 90 minutes after completion of bleomycin infusion, which coincides with the onset of pyrexia in animal studies of single-agent bleomycin. 15 the time course and the similarity to prior case reports following singleagent bleomycin treatment point to bleomycin as the causative agent, but it remains possible that other agents in abvd are contributory. although fulminant febrile episodes were fatal in many early case reports, [16] [17] [18] 20 all 3 patients in the present series survived with resolution of symptoms within 24 hours of onset. in addition to intensive supportive care, all patients received intravenous corticosteroid treatment after onset (management summarized in table 3 ). as this syndrome is clinically indistinguishable from bacterial sepsis, all patients received empiric antibiotics while cultures were pending. the high fevers, cardiorespiratory collapse, and neurologic symptoms seen in this series and prior reports are similar to the cytokine release syndrome (crs) seen following bispecific antibody and chimeric antigen receptor t cell therapy. [22] [23] [24] multiple cytokines have been implicated in crs, including interferon-g, il-10, and il-6, and monoclonal antibodies targeting the il-6/il-6 receptor axis such as tocilizumab are used for crs treatment, 24, 25 marked elevation of serum il-6 was noted in the case report by suzuki et al, 19 but to our knowledge, tocilizumab has not been used in the context of bleomycin-associated febrile, hypotensive reactions, and in all cases in the present series, symptoms resolved rapidly following corticosteroid treatment. given the rarity of this syndrome, the safety of bleomycin rechallenge is not well-defined. bleomycin was omitted from subsequent cycles for patient 1, but patient 2 and 3 tolerated bleomycin in subsequent cycles with increased steroid premedication. although bleomycin does contribute to the activity of abvd, it is the least active agent in the combination regimen 26 and can be safely omitted from cycles 3 to 6 of therapy for patients with advanced stage disease with negative interim pet imaging. 27 the substitution of brentuximab vedotin for bleomycin (a-avd) was compared with abvd in patients with stage iii/iv chl in the phase iii eche-lon-1 study, and resulted in a statistically significant improvement in the primary endpoint of modified progression-free survival at 2 years with no difference in os at the time of analysis. 2 a-avd is approved by the united states food and drug administration for front-line treatment of advanced stage chl and would be an alternative consideration to bleomycin rechallenge or avd therapy for advanced stage patients experiencing fulminant hyperpyrexia during cycle 1 of abvd. the shared clinical characteristics of the patients in our series may shed light on the pathophysiology of this rare syndrome. as in prior reports, all patients in the present series were febrile prior to the start of therapy and presented with advanced stage disease. as bleomycin can induce fever presumably through cytokine-mediated stimulation, 15 one possible explanation is that febrile patients have increased baseline levels of key cytokines, and the additive effect of cytokine stimulation by bleomycin crosses a threshold, triggering a fulminant reaction. 17 other common features of all 3 patients in this series include ebvþ disease and increased cd68þ tumor associated macrophages, and to our knowledge, this represents a novel observation not noted in prior case reports. the majority of prior reports occurred before the development of standard of care ish testing for eber 28 or routine cd68 staining, and thus it is unknown whether prior cases shared these common features. in comparison to ebv-negative chl, ebv-positive disease has been associated with a gene expression signature of macrophage response including upregulation of cxcl10, ccl5, cd14, cd68, 29,30 regulatory t cell type 1 recruitment including upregulation of ccl20 31 and lag3, 32 and altered cytokine signaling with upregulation of il10, tgfb-1, ifng, and tnf. 32 the febrile, hypotensive syndrome following bleomycin treatment is clinically similar to crs, a syndrome in which macrophage activating cytokines il-10 and il-6 are elevated, and macrophage activation appears to be central to pathogenesis. 33 the mechanism of fulminant febrile hypotensive reactions following bleomycin requires further elucidation, including determining the role of macrophage activation. fulminant febrile, hypotensive reactions following abvd therapy for chl are a rare but life-threatening complication of treatment. associated tls is not prerequisite for such reactions. with intensive supportive care and corticosteroid treatment, all patients in the present series survived these reactions with resolution of symptoms within 24 hours of onset. clinician awareness of this rare syndrome is essential for successful management. the authors have stated that they have no conflicts of interest. randomized phase iii trial of abvd versus stanford v with or without radiation therapy in locally extensive and advancedstage hodgkin lymphoma: an intergroup study coordinated by the eastern cooperative oncology group (e2496) brentuximab vedotin with chemotherapy for stage iii or iv hodgkin's lymphoma eight cycles of abvd versus four cycles of beacoppescalated plus four cycles of beacoppbaseline in stage iii to iv, international prognostic score >/¼ 3, high-risk hodgkin lymphoma: first results of the phase iii eortc 20012 intergroup trial incidence of cancers in people with hiv/aids compared with immunosuppressed transplant recipients: a meta-analysis the biology of hodgkin's lymphoma the impact of ebv and hiv infection on the microenvironmental niche underlying hodgkin lymphoma pathogenesis a prognostic score for advanced hodgkin's disease. international prognostic factors project on advanced hodgkin's disease expression of interleukin-6 and interleukin-6 receptor in hodgkin's disease production of multiple cytokines by hodgkin's disease 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single-arm, phase 2 study chimeric antigen receptormodified t cells in chronic lymphoid leukemia immunopharmacologic response of patients with b-lineage acute lymphoblastic leukemia to continuous infusion of t cell-engaging cd19/cd3-bispecific bite antibody blinatumomab chimeric antigen receptor-modified t cells for acute lymphoid leukemia omission of dacarbazine or bleomycin, or both, from the abvd regimen in treatment of early-stage favourable hodgkin's lymphoma (ghsg hd13): an open-label, randomised, non-inferiority trial adapted treatment guided by interim pet-ct scan in advanced hodgkin's lymphoma detection of epstein-barr viral genomes in reed-sternberg cells of hodgkin's disease molecular profiling of classical hodgkin lymphoma tissues uncovers variations in the tumor microenvironment and correlations with ebv infection and outcome expression of the interferoninducible chemokine ip-10 (cxcl10), a chemokine with proposed antineoplastic functions, in hodgkin lymphoma and nasopharyngeal carcinoma expression of the epstein-barr virus-encoded epstein-barr virus nuclear antigen 1 in hodgkin's lymphoma cells mediates up-regulation of ccl20 and the migration of regulatory t cells epstein-barr virus infection induces an increase of t regulatory type 1 cells in hodgkin lymphoma patients managing cytokine release syndrome associated with novel t cell-engaging therapies febrile hypotensive reactions following abvd e128 -clinical lymphoma, myeloma & leukemia key: cord-341106-algs6573 authors: min, hye-jin; cho, il-rae; srisuttee, ratakorn; park, eun-hee; cho, dae ho; ahn, jin-hyun; lee, im-soon; johnston, randal n.; oh, sangtaek; chung, young-hwa title: hexachlorophene suppresses β-catenin expression by up-regulation of siah-1 in ebv-infected b lymphoma cells date: 2009-04-18 journal: cancer lett doi: 10.1016/j.canlet.2008.10.041 sha: doc_id: 341106 cord_uid: algs6573 many studies have shown that the activation of β-catenin signaling can promote oncogenesis, and it is therefore of interest to find agents that modulate this pathway. recent work has shown using b lymphoma cells that infection by epstein–barr virus (ebv) and expression of its latent membrane protein (lmp)-1, cause increases in the expression of β-catenin and cellular transformation. conversely, results from cell-based small molecule screening studies have shown that the antibiotic hexachlorophene can down-regulate β-catenin in colon cancer cells. here we report that hexachlorophene also counteracts the elevated β-catenin levels in ebv-infected b lymphomas. this is associated with restoration in levels of siah-1 (an e3 ubiquitin ligase that is active in β-catenin regulation) which had been diminished by lmp-1. our results suggest that siah-1 is targeted by both lmp-1 and hexachlorophene with opposite effects. the hexachlorophene modulation of siah-1 and β-catenin is independent of p53 and results in reduced expression of cyclin-d1 and c-myc (target genes of β-catenin), leading to the growth arrest of b lymphoma cells. from these results we propose that hexachlorophene may provide a novel therapeutic strategy for ebv-infected b lymphoma cells by reducing β-catenin levels via the restoration of siah-1. many studies have shown that the wnt/b-catenin signaling pathway plays an important role in cell proliferation, differentiation, and oncogenesis [1, 2] . in particular, the abnormal up-regulation of wnt/b-catenin activity has been frequently detected as an early event in many cancers [3] . the level of b-catenin is normally regulated in part through its targeted decay as mediated by a glycogen synthase kinase-3b (gsk-3b)-dependent pathway [4] and alternatively by a siah-1-dependent pathway [5] . in the gsk-3b-depen-dent pathway, b-catenin is phosphorylated by a multi-protein complex composed of apc, axin, and gsk-3b [6, 7] , leading to the degradation of b-catenin through a ubiquitin-dependent mechanism [8] . conversely, in the siah-1dependent pathway, siah-1 interacts with the carboxyl terminus of apc, recruits the ubiquitination complex, and promotes the degradation of b-catenin through a pathway independent of both gsk-3b and b-trcp, an f-box protein in the e3 ubiquitin ligase complex [5] . in this report we have focused on the behavior of b cell lymphoma. it is known that infection by epstein-barr virus (ebv), a member of the human herpes virus family, enhances transformation of normal resting b cells into indefinitely proliferating lymphoblastoid cell lines. earlier work 0304-3835/$ -see front matter ó 2008 elsevier ireland ltd. all rights reserved. doi:10.1016/j.canlet.2008. 10 .041 has shown that latent membrane protein (lmp)-1 of ebv can transform rodent fibroblasts in vitro [9] and induces lymphomas in transgenic mice [10] . thus, lmp-1 has been considered as an important and multifunctional oncoprotein, causing the dysregulation of cell signaling pathways and inducing a variety of cellular genes that enhance cell survival and adhesive, invasive, and angiogenic potential [11] . for example, lmp-1 oncoprotein appears to mimic cellular cd40 molecules, leading to interaction with traf proteins and culminating in the activation of nf-jb and jnk [12, 13] . other studies have shown that lmp-1 also activates stat molecules through jak interactions [14] , and lmp-1 has recently been reported to elevate b-catenin protein levels through down-regulation of siah-1 e3 ubiquitin ligase [15] . screening of chemical libraries has been undertaken to identify small molecule inhibitors of b-catenin activity, yielding as one of the potential candidates hexachlorophene, which is an antimicrobial compound used in disinfectants and surgical scrubs [16] . hexachlorophene inhibits the activity of enoyl-acyl carrier protein reductase, which is the last enzyme in the fatty acid elongation cycle and a target for antibacterial agents [17] . hexachlorophene is also an inhibitor of the 3cl protease of sars-cov [18] , and is reported to suppress b-catenin levels through upregulation of siah-1 in colon cancer cells [19] . thus, we wanted to combine these disparate observations and ask whether hexachlorophene might be effective in diminishing the elevated levels of b-catenin achieved by lmp-1 activity in ebv-infected b lymphoma, to establish by what mechanism this might occur, and to observe any consequences in cell behavior. here we report that hexachlorophene does indeed suppress the level of lmp-1-induced b-catenin in ebv-infected b lymphoma, apparently through the rescue of siah-1 e3 ubiquitin ligase. our results thus raise the possibility that hexachlorophene or similar compounds may ultimately be useful in the treatment of ebv-mediated b lymphoma. bjab, im9, ramos, and raji b lymphocytes were cultured in rpmi 1640 supplemented with 10% fbs and 1% penicillin and streptomycin. bjab or im9 b cells were electroporated with 10 lg of dna at 260 v and 975 lf (bio-rad, hercules, ca). cells were harvested 48 h after electroporation and the expression of proteins was determined by immunoblotting. hexachlorophene and licl were from sigma (st. louis, mo) and mg132 and cyclohexamide were provided from calbiochem (san diego, ca). anti-cyclin-d1, anti-b-tubulin, anti-sp1, anti-c-myc, anti-p53, and hrp-conjugated secondary antibodies were purchased from santa cruz (santa cruz, ca). anti-b-catenin antibody was obtained from bd biosciences (san jose, ca). for the detection of siah-1 and lmp-1 proteins, anti-siah-1 (cosmobio, tokyo, japan) and anti-lmp-1 (dako, carpinteria, ca) antibodies were used, respectively. the ptopflash and pfopflash reporter plasmids were obtained from upstate biotechnology (lake placid, ny). total rna was extracted from cells using trizol reagent (invitrogen, carlsbad, ca) in accordance with the manufacturer's instructions. three micrograms of total rna was converted to cdna using superscript ii reverse transcriptase (invitrogen), and pcr was performed using specific primers as described elsewhere [15, 20] . the cdnas of each sample were diluted, and pcr was run at the optimized cycle number. b-actin mrna was measured as an internal standard. after amplification, the products were subjected to electrophoresis on 1.5% agarose and detected by ethidium bromide staining. bjab or im-9 b cells (1 â 10 7 cells) were electroporated with topflash-, fopflash-, or p53-luciferase reporter vectors. to normalize transfection efficiency, a pgk-bgal vector that expresses b-galactosidase from a phosphoglucokinase promoter was included in the transfection mixture. at 48 h post-transfection, cells were washed with cold pbs and lysed in lysis solution (25 mm tris [ph 7.8], 2 mm edta, 2 mm dtt, 10% glycerol, and 1% triton x-100). luciferase activity was measured with a luminometer by using a luciferase kit (promega, madison, wi). cells were harvested and lysed with lysis buffer (0.15 m nacl, 1% nonidet p-40, 50 mm tris [ph 7.5]) containing 0.1 mm na 2 vo 3 , 1 mm naf, and protease inhibitors (sigma). for immunoblotting, proteins from whole cell lysates were resolved by 10% or 12% sds-page and transferred to nitrocellulose membranes. primary antibodies were used at 1:1000 or 1:2000 dilutions, and secondary hrp-conjugated antibodies (santa cruz) were used at 1:2000 dilution in 6% nonfat dry milk. after final washing, nitrocellulose membranes were exposed using chemiluminescence assays (ge-amersham, piscataway, nj). as described elsewhere [21] , cells cultured in 100 mm plates were washed and harvested with ice-cold pbs and cell pellets were lysed with 800 ll of ttn buffer (20 mm tris-hcl [ph 7.4], 0.05% triton x-100, 150 mm nacl, 1 mm edta, 1 mm dtt, 10% glycerol, 0.5 mm pmsf, and 1â cocktail protease inhibitor) on ice for 20 min followed by centrifugation at 10,000g for 15 min. the supernatant was taken as the soluble fraction, and the pellets as insoluble fractions were subsequently solubilized in 800 ll of ripa buffer (50 mm tris-hcl [ph 7.4], 150 mm nacl, 1 mm edta, 1 mm dtt, 1% np-40, 0.5% deoxycholic acid, 0.1% sds, 10% glycerol, 0.5 mm pmsf, and 1â cocktail protease inhibitor) on ice for 30 min and were centrifuged at 12,000g for 15 min. thereafter, the supernatants were used for the nuclear extracts. to explore whether the infection of b cells with ebv induces up-regulation of b-catenin, we examined im9 and raji b lymphoma cells with a persistent infection of ebv, plus bjab and ramos lymphoma cells that are free of ebv. the infection with ebv was confirmed by the expression of lmp-1, a primary oncogenic protein in im9 and raji b cells (fig. 1a ). this figure also shows that the protein level of siah-1, an e3 ubiquitin ligase, was decreased whereas the b-catenin protein level was enhanced in the im9 and raji cells. by comparison, the siah-1 level was higher while the b-catenin level was lower in both bjab and ramos b cells. to test whether lmp-1 (instead of other viral proteins) is specifically linked to the up-regulation of b-catenin and the down-regulation of siah-1, we expressed lmp-1 protein in bjab cells using electroporated pcdna3-lmp-1 vector. we found that lmp-1 expression alone is sufficient to reduce siah-1 protein levels, leading to up-regulation of b-catenin (fig. 1b) . in addition, to determine whether the increased b-catenin due to the introduction of lmp-1 is transcriptionally active, we transfected cells with a reporter plasmid containing wild-type (topflash) or mutant (fopflash) binding sites for tcf [22] in the presence or absence of lmp-1. as seen in fig. 1c , lmp-1 expression specifically induced tcf reporter activity, indicating that at least some of the lmp-1-induced b-catenin is transcriptionally active. to examine whether lmp-1 also down-regulates siah-1 mrna levels, we measured siah-1 mrna from bjab cells in the presence and absence of lmp-1 using rt-pcr. as seen in fig. 1d , the presence of lmp-1 down-regulated siah-1 mrna levels in a dose-dependent manner. the results suggest that lmp-1 reduces siah-1 both protein and mrna levels. taken together, we propose that lmp-1 expression induces up-regulation of b-catenin through down-regulation of siah-1. hexachlorophene has been identified (by screening from small molecule libraries) as an inhibitor of b-catenin, and is possibly useful for the treatment of colon cancers [19] . we therefore examined whether hexachlorophene can also down-regulate the high levels of b-catenin observed in ebv-infected b lymphoma. as shown in fig. 2a and b, hexachlorophene significantly down-regulates protein as well as mrna levels of b-catenin level in a dose-dependent manner. furthermore, hexachlorophene treatment reduced lmp-1-induced tcf transcriptional activity in a dose dependent manner (fig. 2c) . next, to examine whether proteosomes are involved in the down-regulation of b-catenin following treatment with hexachlorophene, we administered mg132 (a proteosome inhibitor) into hexachlorophene-treated cells and then analyzed the b-catenin level. we found that b-catenin levels were restored by mg132 when compared with cells treated only with hexachlorophene (fig. 2d) , suggesting that down-regulation of b-catenin mediated by hexachlorophene is achieved via proteosome activation. of interest, the introduction of cycloheximide, an inhibitor of protein synthesis, also partly restored the b-catenin level from hexachlorophene-treated cells (fig. 2d) , suggesting that some aspect of protein synthesis is required for the activity of the b-catenin degradation pathway. since we observed that hexachlorophene administration caused the decrease in protein levels of b-catenin (fig. 2) , we next investigated the mechanism by which this down-regulation might be achieved. as seen in fig. 3a , hexachlorophene also induced up-regulation of siah-1 levels, associated with the decrease of b-catenin in ebv-infected b cells (1st and 2nd lanes). to confirm that this feature is mediated by lmp-1 expression, we also introduced the lmp-1 expression vector into bjab cells and treated them with hexachlorophene. we found that hexachlorophene treatment rescues the decreased siah-1 level mediated by lmp-1, resulting once again in up-regulation of b-catenin (fig. 3b) . next, since the phosphorylation of b-catenin by gsk-3b, followed by association with b-trcp, can lead independently to b-catenin degradation [8] , we tested whether gsk-3b and siah-1 might cooperate to regulate b-catenin. however, we found that gsk-3b inhibition using licl caused an increase in b-catenin protein levels without affecting siah-1 levels in im9 b cells (fig. 3a, 3rd lane) , suggesting that gsk-3b protein is not directly involved in the siah-1 signaling pathway. however, co-administration of licl and hexachlorophene caused the protein level of b-catenin to be still elevated (fig. 3a , 4th lane) in im9 b cells. this suggests that the effect of licl on the induction of b-catenin through gsk-3b inhibition overrides that of hexachlorophene through siah-1 in ebv-infected b lymphoma. it has been suggested that the expression of siah-1 can be activated by the tumor suppressor p53, leading to the induction of apoptosis and cell-cycle arrest in mammalian cells [5] . in addition, some genotoxic reagents, such as doxorubicin, may suppress the b-catenin response through the p53-inducible transcription pathway involving siah-1 [5] . to further explore this issue, we investigated whether hexachlorophene treatment might influence p53 transcription in im9 cells, which contain wild-type p53. as seen in fig. 4a , neither hexachlorophene nor doxorubicin treatment affected p53 transcript abundance [23, 24] . furthermore, we examined p53 protein levels after the treatment with hexachlorophene and found again no change in p53 protein level, even at 30 lm hexachlorophene, whereas doxorubicin induced up-regulation of p53 abundance (fig. 4b ). in addition, to determine whether the increased siah-1 due to treatment with hexachlorophene enhances p53 transcriptional activation of target genes, we transfected cells with a luciferase reporter vector containing a target sequence for binding of p53 protein. as seen in fig. 4c , hexachlorophene did not increase p53 transcriptional activity while doxorubicin drastically enhanced p53 transcriptional activation of the target. these results suggest that hexachlorophene induces the expression of siah-1 independently of p53 pathway activation. we first examined the expression of b-catenin target genes such as cyclin-d1 and c-myc in nuclear extracts because several lines of evidence have shown that nuclear localization of cyclind1 or c-myc is preferentially detected in proliferating and tumorigenic cells [25] [26] [27] [28] . as seen in fig. 5a , the expression levels of cyclin-d1 and c-myc were diminished in nuclear extracts when compared with sp-1 expression, in a hexachlorophene dose-dependent manner at 12 h post-treatment. as other groups have reported that shrna or chemical inhibition of b-catenin induces retardation of tumor cell growth [29] [30] [31] , we explored the potential biological consequence of the reduced b-catenin levels achieved by treatment with hexachlorophene. when we examined viable cells of ebv positive b lymphoma, we observed a striking dose-dependent decrease in viable cell numbers of im9 b lymphoma cells 48 h after treatment with hexachlorophene (fig. 5b) . these results suggest that hexachlorophene decreases the expression of b-catenin-dependent genes, leading to the growth arrest of b lymphoma cells. excessive accumulation of b-catenin is frequently detected in various types of cancer, such as colorectal, ovarian, endometrial, and prostate cancers [32, 33] . primary effusion lymphoma cells expressing kaposi's sarcoma associated herpes virus (kshv) also show elevated b-catenin levels. in particular, the lana protein of kshv plays a crucial role in up-regulation of b-catenin, by sequestration and downregulation of gsk-3b [34, 35] , which might be distinct from the action of lmp-1. here we show that lmp-1, a primary oncoprotein of ebv also induces up-regulation of b-catenin in b lymphocytes by the decrease of siah-1 protein and mrna levels. as it is known that siah-1 itself is a target for ubiquitination and proteosomal degradation [36] , lmp-1 may affect siah-1 protein destabilization. in addition, lmp-1 down-regulates siah-1 mrna levels, which indicates that lmp-1 might inhibit transcription of the siah-1 gene. although the promoter region of siah-1 has been recently identified [37] , more work is required to establish in detail how lmp-1 inhibits transcription of siah-1. the consequent over-expression of b-catenin target genes, including cyclin-d1, c-myc, matrix metalloproteinase-7, and peroxisome proliferators-activated receptor-d, can all play important roles in tumorigenesis [38] . the constitutive activation of b-catenin signaling is therefore a potential target for chemoprevention and treatment of various cancers, including b cell lymphomas. furthermore, we report that hexachlorophene drastically suppresses b-catenin levels through a siah-1-mediated pathway (rather than the gsk-3b-dependent pathway) in ebv-infected b lymphoma. hexachlorophene specifically up-regulates siah-1 mrna and protein in b lymphoma, counteracting its down-regulation by lmp-1. furthermore, prolonged exposure of ebv-infected b lymphoma cells to hexachlorophene also induces apoptotic cell death and down-regulation of b-catenin targeted gene expression. however, the up-regulation of b-catenin by licl through gsk-3b inhibition overrides that of hexachlorophene through siah-1 in ebv-infected b lymphoma (fig. 3) . since previous work showed that hexachlorophene can inhibit the expression of b-catenin induced by licl in hek293 cells [19] , this difference in response in the two cell types is currently under investigation. of interest, we observed that cycloheximide restores b-catenin levels in hexachlorophene-treated cells (fig. 2d) . as we suspect that the stability of proteins that are involved in the regulation of b-catenin expression is also a critical factor, siah-1 degradation by cycloheximide treatment may contribute to the recovery of b-catenin; this point will be the subject of further studies by our group. it has been proposed that siah-1 can be induced by p53 tumor suppressor activation, contributing to cell-cycle arrest, tumor suppression and apoptosis [5] . however, hexachlorophene does not affect p53 at either the transcriptional and translational level in our study: this suggests the existence of another hexachlorophene-responsive and p53independent pathway for the regulation of siah-1. a recent report showing that wnt5a also promotes the degradation of b-catenin through the p53-independent up-regulation of siah-1 level is consistent with our suggestion [39] . since hexachlorophene can suppress wnt/b-catenin signaling in a p53-independent manner, it is possible that this potential therapeutic strategy may be effective against cancer cells with mutant p53, which is commonly found in many cancers that are resistant to genotoxic chemotherapeutics. previous searches for small molecule inhibitors of b-catenin activity have revealed candidates that block the interaction between b-catenin and camp response element-binding protein [40] or that inhibit the association of b-catenin and the tcf4 complex [41] . in contrast, hexachlorophene specifically reduces the amount of free b-catenin protein through an independent pathway by siah-1-mediated degradation. from our results, we propose that hexachlorophene could ultimately provide a potential therapeutic strategy for the treatment of ebv-infected b lymphoma. wnt signaling in oncogenesis and embryogenesis -a look outside the nucleus new aspects of wnt signaling pathways in higher vertebrates caught up in a wnt storm: wnt signaling in cancer casein kinase 1: a wnt'er of disconnect siah-1 mediates a novel beta-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein downregulation of beta-catenin by human axin and its association with the apc tumor suppressor, beta-catenin and gsk3 beta axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin beta-catenin is a target for the ubiquitin-proteasome pathway all three domains of the epstein-barr virus-encoded latent membrane protein lmp-1 are required for transformation of rat-1 fibroblasts expression of the epstein-barr virus latent membrane protein 1 induces b cell lymphoma in transgenic mice epstein-barr virus latent membrane protein 1 induces the matrix metalloproteinase-1 promoter via an ets binding site formed by a single nucleotide polymorphism: enhanced susceptibility to nasopharyngeal carcinoma mimicry of cd40 signals by epstein-barr virus lmp1 in b lymphocyte responses epstein-barr virus latent membrane protein 1 activation of nf-kappab through irak1 and traf6 latent membrane protein 1 of epstein-barr virus interacts with jak3 and activates stat proteins up-regulation of betacatenin by a viral oncogene correlates with inhibition of the seven in absentia homolog 1 in b lymphoma cells soap bacteriostats inhibition of the staphylococcus aureus nadph-dependent enoyl-acyl carrier protein reductase by triclosan and hexachlorophene evaluation of metal-conjugated compounds as inhibitors of 3cl protease of sars-cov hexachlorophene inhibits wnt/beta-catenin pathway by promoting siah-mediated beta-catenin degradation novel cationic solid lipid nanoparticles enhanced p53 gene transfer to lung cancer cells ubiquitination and translocation of traf2 is required for activation of jnk but not of p38 or nf-kappab identification and cloning of tcf-1, a t lymphocyte-specific transcription factor containing a sequence-specific hmg box phosphorylation of p53 on thr55 by erk2 is necessary for doxorubicin-induced p53 activation and cell death regulation of p53 expression in response to 5-fluorouracil in human cancer rko cells pten induces cell cycle arrest by decreasing the level and nuclear localization of cyclin d1 role of cyclin d1 cytoplasmic sequestration in the survival of postmitotic neurons phosphorylation by glycogen synthase kinase-3 controls c-myc proteolysis and subnuclear localization critical role of cyclin d1 nuclear import in cardiomyocyte proliferation shrnamediated gene silencing of beta-catenin inhibits growth of human colon cancer cells silencing of wnt-1 by sirna induces apoptosis of mcf-7 human breast cancer cells beta-catenin/tcf-4 inhibition after progastrin targeting reduces growth and drives differentiation of intestinal tumors the abc of apc the significance of the wnt pathway in the pathology of human cancers a novel viral mechanism for dysregulation of beta-catenin in kaposi's sarcoma-associated herpesvirus latency regulation of the interaction between glycogen synthase kinase 3 and the kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen siah-1 n-terminal ring domain is required for proteolysis function, and c-terminal sequences regulate oligomerization and binding to target proteins siah-1b is a direct transcriptional target of p53: identification of the functional p53 responsive element in the siah-1b promoter identification of membrane-type matrix metalloproteinase-1 as a target of the beta-catenin/tcf4 complex in human colorectal cancers wnt-5a inhibits the canonical wnt pathway by promoting gsk-3-independent beta-catenin degradation a small molecule inhibitor of beta-catenin/creb-binding protein transcription small-molecule antagonists of the oncogenic tcf/beta-catenin protein complex this work was supported by the korea institute of science & technology evaluation and planning (kistep) and by the ministry of science & technology (most), korean government, through its national nuclear technology program (s. oh). key: cord-284235-ae37re3f authors: al-salam, suhail; dhaheri, shamma al; awwad, aktham; daoud, sayel; shams, ahmed; ashari, mouied al title: prevalence of epstein–barr virus in tonsils and adenoids of united arab emirates nationals date: 2011-07-12 journal: int j pediatr otorhinolaryngol doi: 10.1016/j.ijporl.2011.06.012 sha: doc_id: 284235 cord_uid: ae37re3f objective: given that epstein–barr virus (ebv) often inhabits human tonsils and adenoids, it remains to be distinctively determined its prevalence and in which cell and microenvironment the virus is present. methods: to determine the prevalence of ebv in the tonsils and adenoids of the united arab emirates (uae) nationals and to provide a basis for understanding the origin and biology of ebv-infected cells, the immunophenotype of all ebv-infected cells in 46 tonsils and 46 adenoids was determined by eber in situ hybridization and immunohistochemistry with monoclonal antibodies to t cells (cd3), b cells (cd20), and epithelial cells (cytokeratin ae1/ae3), as well as immunostaining with antibodies to ebv latent membrane protein-1 (lmp-1). results: ebv was found in 43% of tonsillectomy specimens and 15% of adenoidectomy specimens. all ebv-infected cells were found to be b lymphocytes. about 90% of the infected b cells are found in the interfollicular regions of tonsils and adenoids and the remaining 10% are found within the follicles. there is no significant association between ebv infection, age (p = 0.324) and gender (p = 0.442). conclusion: ebv is associated with tonsillar hypertrophy and is prevalent in 43% of our cases. ebv is only detected in b lymphocytes and we believe that b lymphocytes are sites of primary infection and latency. in situ hybridization is the gold standard for the detection of ebv in tissue. tonsils and adenoids, the common sites for recurrent inflammation in the pediatric population, are continually exposed to antigens, hence hyperplasia of their lymphoid component accounts for the increase in their size [1] . some authors have demonstrated a strong relation between viral infection [2] [3] [4] and recurrent pharyngotonsillitis. there are many viruses involved in the pathogenesis of pharyngotonsillitis including adenoviruses, parainfluenza viruses, rhinoviruses, herpes simplex viruses, respiratory syncytial viruses, epstein-barr viruses (ebv), influenza viruses, coxsackie a viruses, corona viruses, and cytomegaloviruses [2, 3, 5, 6] . in spite of high prevalence of viral infection in adenotonsillar tissue, the methods to detect viruses make this approach difficult in routine practice [2, 3, 5, 6] . commonly causing infectious mononucleosis, ebv is a member of g1-herpesvirus and has a genome comprised approximately 172 nucleotide base pairs [7] . ebv has a linear genome which is characterized by a distinctive sequence reiteration. at the termini there are 20 copies of a 500 bp repeated sequence that is complementary and therefore permits circularization of the linear genome to form the ebv episome. ebv episome is the molecular basis for latent infection. it is a circular intracellular form of genome that maintains a persistent relationship within the cell, like an autonomous piece of dna situated in the chromatin [7, 8] . ebv is b-lymphotropic and has the ability to transform memory bcells into blast cells, with permanent proliferation, leading to tonsillar enlargement [9] . ebv is associated not only with infectious mononucleosis but also with benign diseases, such as oral hairy leukoplakia, and malignancies, such as hodgkin's lymphoma, non-hodgkin's lymphomas, nasopharyngeal carcinoma, gastric carcinoma and breast carcinoma. it is recently being associated with autoimmune diseases, such as lupus erythematosus and multiple sclerosis [7, 10, 11] . for these reasons, our study is designed to identify the magnitude of ebv infection and types and pattern of distribution of ebv-infected cells in tonsils and adenoids among uae population which will be of great help in future planning to prevent this infection. to determine the prevalence of ebv in the tonsils and adenoids of the united arab emirates (uae) nationals and to provide a basis for understanding the origin and biology of ebv-infected cells, the immunophenotype of all ebv-infected cells in 46 tonsils and 46 adenoids was determined by eber in situ hybridization and immunohistochemistry with monoclonal antibodies to t cells (cd3), b cells (cd20), and epithelial cells (cytokeratin ae1/ae3), as well as immunostaining with antibodies to ebv latent membrane protein-1 (lmp-1). results: ebv was found in 43% of tonsillectomy specimens and 15% of adenoidectomy specimens. all ebv-infected cells were found to be b lymphocytes. about 90% of the infected b cells are found in the interfollicular regions of tonsils and adenoids and the remaining 10% are found within the follicles. there is no significant association between ebv infection, age (p = 0.324) and gender (p = 0.442). conclusion: ebv is associated with tonsillar hypertrophy and is prevalent in 43% of our cases. ebv is only detected in b lymphocytes and we believe that b lymphocytes are sites of primary infection and latency. in situ hybridization is the gold standard for the detection of ebv in tissue. ß 2011 elsevier ireland ltd. all rights reserved. in total, 46 cases of tonsillectomy with adenoidectomy, which were performed due to tonsillar and adenoidal enlargement, were randomly selected from surgical pathology archive in the department of pathology at tawam hospital in al ain city for the period june 2004 through may 2005. forty-six paraffin blocks of tonsillectomy specimens and another 46 paraffin blocks of adenoidectomy specimens were available for this study. the available hematoxylin and eosin (h&e) and immunohistochemical stained sections were reviewed. the age, sex, and clinical presentation were obtained by reviewing all the histopathologic reports and request forms of all cases. for the detection of ebv, two methods were used to increase the specificity and sensitivity of detection, the streptavidin-biotin immunohistochemical immunoperoxidase method to detect epstein-barr virus latent membrane antigen type i (ebv-lmp-1) and in situ hybridization (ish) for ebv encoded rna (eber). immunohistochemical (ihc) staining was performed by standard streptavidin-biotin immunoperoxidase technique [12] using the following mouse antihuman monoclonal antibodies (dako cytomation, glostrup, denmark); ebv-latent membrane protein-1(lmp1)(clone cs 1-4), cd3 (clone pc3/188a), cd20 (clone l26), cytokeratin (clone ae1/ae3), all diluted to 1:100, and visualized by a commercially available detection kit (dako envision plus-hrp, dako, glostrup, denmark) and 3-3 0 -diaminobenzidine (dako, glostrup, denmark) as a chromogen substrate to obtain a brown end-product. lymph node sections were used as positive controls for cd20, cd3. skin epidermis was used as a positive control for cytokeratin ae1/ae3. hodgkin lymphoma (hl) lmp-1-positive sections were used as positive controls for lmp-1. for negative controls, primary antibody was replaced with normal mouse serum. in situ hybridization (ish) was performed by standard techniques using a specific oligonucleotide probe (novocastra-lebv-k, uk) which hybridizes to ebv encoded rna (eber) transcripts concentrated in the nuclei of latently infected cells. with each batch of cases studied, positive and negative control slides were also run. the positive control slide was a known case of ebv positive hl to which a specific eber oligonucleotide probe was added. the negative control slide was another section of the same case of known ebv positive hl to which a random probe consisting of fluorescein labeled oligonucleotide cocktail was added. in addition, for each case studied two sections were used; the eber oligonucleotide probe was added to one section, and the random probe was added to the other. using this random probe as a negative background control alongside the ebv probe contributes to the validation of staining obtained by the ebv probe. if this negative control slide showed significant background staining in a particular case, the slide having the eber probe was considered non-interpretable and the test was repeated for that particular case. five-micrometer sections were stained for eber using in situ hybridization protocol described earlier. after nuclear visualization, mouse antihuman monoclonal antibodies for cd20 (dako cytomation, glostrup, denmark) were added and visualized by a commercially available detection kit (dako envision plus-hrp, dako, glostrup, denmark) and 3-3 0 -diaminobenzidine (dako, glostrup, denmark) as a chromogen substrate to obtain a brown end-product. subsequently, mouse antihuman monoclonal antibodies for cd3 (dako cytomation, glostrup, denmark) were added and visualized using the envision plus-alkaline phosphatase kit (dako, glostrup, denmark), and new fuchsin (merck, darmstadt, germany) as a second substrate to yield a red endproduct. finally, sections were mounted by water soluble mounting media. the statistical analysis was performed using spss for windows version 18 (spss inc, chicago, usa) and analyze it (analyze-it software ltd., leeds, uk). student's t-test was used to compare continuous variables. quantitative variables were analyzed with the x 2 -test and correlations of ordinal variables using the spearman rank correlation coefficient. p value <0.05 was considered significant. the project has been approved by al ain district human research ethical committee (protocol no. 07-145). in total, 46 cases of tonsillectomy and adenoidectomy, due to tonsillar and adenoid hypertrophy, were selected. all cases were in the 1st and 2nd decade of life with predominant clustering (63%) in the 1st decade. twenty-seven cases were females and 19 cases were males. although ebv-positive cases were more (n = 14) in the 1st decade than in the 2nd decade (n = 6), cross tab and logistic regression show no significant association between ebv expression and the age (p = 0.324) and gender (p = 0.442) distributions (table 1) . in total, 20 (43%) specimens were positive by in situ hybridization method for eber and show the black nuclear stain. eber-positive cells are seen within follicular ( fig. 1 ) and interfollicular (fig. 2) regions. the frequency of ebv is shown in table 2 . only 5 (11%) specimens of tonsillectomy show cytoplasmic and membranous positivity for lmp-1 as shown in fig. 3 . lmp-1positive large cells are seen within the mantle zone of lymphoid follicle and within the interfollicular area, however, lmp-1 expression is seen also in small lymphocytes in the interfollicular area. in total, 7 (15%) specimens were positive by in situ hybridization method for eber and show the black nuclear stain. eberpositive cells are seen within follicular (fig. 1) and interfollicular (fig. 2) regions. the frequency of ebv is shown in table 2 . all those cases were also ebv positive in their tonsillectomy specimens. lmp-1 staining was negative in all 46 case adenoid specimens. nearly 90% of the eber-positive cells were found within the tcell-rich interfollicular regions (fig. 2) while 10% were present within the b-cell-rich germinal centers of secondary follicles (fig. 1) . all eber-positive cells were b cells. the eber-positive cells show immunoreactivity to cd20 only (fig. 4) . there was no immunoreactivity to cd3 (fig. 4) and cytokeratin ( fig. 2a) . individual eber positive cells appeared to be randomly distributed within the interfollicular regions (fig. 3) . the distribution of eberpostive cells within lymphoid follicles is variable. some follicles have few positive cells within the germinal center (fig. 1) . some of them were atypical large cells (fig. 1d) . other follicles show their presence within the mantle zone layer (fig. 1c and d) . there is a third pattern where eber-positive cells form a circumferential distribution at the interphase between the germinal center cells and the mantle zone cells (fig. 2c and d) . enlargement of tonsils and adenoids is a common finding in children but the etiology remains controversial. in children, there is no correlation between hypertrophic tonsils and the body mass index [13] . the weight of normal tonsils in children is not well documented in anatomy literature; however, the mean weight of the tonsillectomy specimens from pathologic examination was shown to be 7.3 g in children between the ages of 2 and 12 years with 63% of it falls between 5 and 8 g [14] . tonsillectomy is a common surgical procedure in children, and the most common indication is tonsillar hypertrophy, which leads to obstructive symptoms in the upper airway. there is a poor correlation between enlarged tonsils and the associated symptoms [14] . recurrent bacterial tonsillitis is considered to be the main reason for the enlargement of the tonsils; however, the bacteriological examinations of the tonsils showed that the relationship is not very clear [15] . misuse of antibiotic therapy in acute tonsillitis may lead to change in tonsillar microflora and predisposing to recurrent tonsillitis and subsequent colonization by various bacteria and viruses [16] . the role of actinomycosis is considered in the etiology of recurrent tonsillitis, not as an active infection but as a factor in the development of lymphoid hyperplasia and hypertrophy [17] . many viruses are involved in the pathogenesis of pharyngotonsillitis [2, 3, 5, 6] . colonization of the tonsils by ebv shows no correlation between ebv-dna quantity and viral core antigen-igg quantity in the autologous sera [18] . ebv infection is very common, with a seroprevalence rates in excess of 90% worldwide. [19] . nearly all infections are acquired by oral contact with persons carrying ebv in saliva. although the exact details of ebv transmission in the oral cavity remain unknown, it is likely that initial infection of either oral epithelial cells or tonsillar b cells is followed by a brief period of replication and lifelong persistence in b lymphocytes [20] . the current study involves tonsils and adenoids of uae nationals only. cases were randomly selected from the surgical archive of the pathology department at tawam hospital for a period june 2004 to may 2005. the study includes patients from different parts of the country. tawam hospital is the main hospital that provides medical services for uae nationals. hence our samples are almost representative of the uae population. tonsils and adenoids were chosen to identify the prevalence of ebv in tissue since they are the initial sites of infection and may reflect the magnitude of ebv infection in the community. in addition, it may help in identifying the correlation between ebv infection and the prevalence of ebv-associated diseases in the community, which might help in the setting of plans to prevent these diseases. in this study we show that ebv is prevalent in 43% of tonsils and 15% of adenoids. it is interesting to see all our cases are children, a finding seen by endo et al. too [21] , indicating that tonsillar and adenoidal hypertrophy occur mainly during this age group. it is well known that ebv has a tropism for the oral and nasopharyngeal tissues, and leads to lymphocytic proliferation [21] . the role of oropharyngeal epithelial cells as a reservoir of ebv was already suggested [21] . we show ebv only inhabits b lymphocytes in tonsils and adenoids, but not t lymphocytes or epithelial cells indicating that b lymphocytes are the main reservoir for ebv. hudnall et al. [22] , showed ebv to be rarely found within epithelial cells and t lymphocytes in ebv-infected tonsils. following primary infection in the oropharynx, ebv persists in numerous anatomical sites including pharyngeal tonsils, adenoids, lymph nodes, and peripheral blood. in peripheral blood, the virus is present in small resting memory b cells with latent gene expression limited to eber, lmp2a, and perhaps ebna1 [19] . it is well known that the interleukin 10 (il-10) coding sequence is highly homologous to the ebv open reading frame bcrf1 [23] . bcrf1 protein, also termed viral il-10 (vil-10), inhibits the synthesis of t-helper 1 cytokines [24] and cytotoxic t lymphocytes (ctl) activity [25] . therefore, ebv-associated antigen-specific ctl activity might be down-regulated by vil-10 in ebv-infected areas of the tonsil. hence, ebv is much more likely to survive in the face of immune surveillance in the tonsils, suggesting that the immune response to ebv in tonsils may be different from that in peripheral blood [25] . the prevalence of ebv infection in tonsils varies according to the detection method. studies using the ish for detecting eber found 26%, [26] 29%, [3] and 65% [27] association of the ebv with tonsillitis. while in our study the prevalence of ebv infection in tonsils is 43%, which is intermediate between these studies and reflects a possible geographical difference in the prevalence of ebv. it is noteworthy to mention here that the prevalence of ebv (43%) in our specimens is close to its prevalence in hodgkin lymphoma among uae national (38%) [28] , which may reflect a causal relationship between ebv and hodgkin lymphoma. identification of a high prevalence (43%) of ebv-eber in tonsillectomy specimens in children suggests that the tonsils are the main reservoir for the ebv, and that this virus may be involved in tonsillar enlargement. on the other hand, the prevalence of ebv in adenoids (15%) is lower than in tonsils (43%), which indicate a lower association between adenoid hypertrophy and ebv infection. this finding is different from that found by endo et al. [21] which found the prevalence of ebv to be higher in adenoids (57%) than tonsils (29%) by using similar method for detection (ish), suggesting sampling and geographical differences. in this study, the immunohistochemical staining method was used to detect lmp-1 in tonsils and adenoids with the aim of strengthening our results in combination with eber-ish. the results are not encouraging since we detect ebv in only 5 cases (11%) and in few cells within the interfollicular area. this finding is less than that reported by dias et al. [16] , indicating a lesser expression of lmp-1 in ebv infected cells. in addition, it indicates that using eber-ish is the gold standard for detecting ebv in tissue. moreover, the use of eber-ish in the detection of ebv is more suitable since we can use it on paraffin-embedded tissue, which allows us to test archive material when required. this makes ish superior to immunohistochemistry in the detection of ebv in tissue. all detected ebv-infected cells in this study were cd20-positive b-cells and were located predominantly in the interfollicular regions. nearly 90% of the eber-positive cells were located within the t-cell-rich interfollicular regions of the tonsils, while a significant number (10%) were located within the lymphocyterich follicular region. these results are consistent with previous studies [22, 29] . almost 10% of eber-positive cells, all of which stained with cd20 b-cells, were located within the b-cell-rich germinal centers of secondary follicles. few follicles contained numerous eberpositive cells. these results are consistent with previous reports of eber-positive germinal center b-cells in tonsils [30] . it has been suggested that germinal center centroblasts may be a site of ebv persistence [31] . the presence of ebv-positive b-cells in germinal centers is reportedly more common in tonsils from areas endemic for ebv related lymphomas; leading to the suggestion that ebvinfected germinal center b-cells may be more prone to malignant transformation, perhaps due to somatic hypermutation [30] . in this study eber-positive cells were found within germinal centers of tonsils and adenoids, and some of these cells were atypical and large and might be a precursor for malignant transformation. although the pattern of distribution of eber-positive b lymphocytes in the interfollicular regions is random, they have 3 distinct patterns within lymphoid follicles; either within germinal center cells, or mantle zone cells or in the interphase between them. the interphase pattern is very interesting and involves some follicles which have one or more eber -positive atypical cell within the germinal center. the interphase eber-positive cells are distributed circumferentially in a round shape at the interphase between germinal center cells and mantle zone cells ( fig. 1c and d) as if they were generated from cell division of ebv-infected germinal center cells and moving outside the lymphoid follicle. whether this pattern has any role in ebv-persistent infection in tonsils and adenoids or carrying any risk for future malignant transformation needs to be determined in future studies. on the other hand, eber was not identified within tlymphocytes when we use triple staining for eber, cd20 and cd3 (fig. 4) . hudnall et al. [22] , shows that ebv is rarely found in t lymphocytes in ebv-infected tonsils. there is some controversy regarding the role of oropharyngeal epithelial cells in ebv infection. it has been suggested that primary and persistent ebv infection might be mediated through oropharyngeal epithelial cells [32] . the support for this perception has come mainly from infection detected in desquamated oropharyngeal epithelial cells from patients with infectious mononucleosis by in situ hybridization [33] . however, there is now increasing evidence pointing to b lymphocytes as the likely site of persistent ebv infection, and also as the possible target of primary ebv infection [34] . several studies have demonstrated ebv replication within the upper epithelial cell layers in oral hairy leukoplakia and are not accompanied by detectable latent infection of basal epithelial cells [35, 36] . thus, these results do not support the idea that ebv persists in epithelial cells. in our study we do not identify ebv in epithelial cells of tonsils and adenoids whereas it is only detected in b lymphocytes. we think that ebv infection might be mediated through oropharyngeal epithelium at early stages of acute infection but later on, the virus might have left epithelial cells or the infected epithelial cells died as a result of host antiviral immunological reaction, hence we do not have ebv persistence and latency within epithelial cells in our samples. on the other hand, we believe that b lymphocytes are sites of primary infection and latency in tonsils and adenoids. in conclusion, ebv is associated with tonsillar hypertrophy and is prevalent in 43% of our cases. ebv is only detected in b lymphocytes and we believe that b lymphocytes are sites of primary infection and latency. in situ hybridization is the gold standard for the detection of ebv in tissue. viral infection associated with recurrent tonsillitis detection of epstein-barr virus in tonsillar tissue of children and the relationship with recurrent tonsillitis immunology of the tonsils pitkä ranta, viral upper respiratory tract infections in young children with emphasis on acute otitis media acute tonsillitis in children: microbial pathogens in relation to age epstein-barr: 40 years on ebv persistence in memory cells detection of lymphocytes productively infected with epstein-barr virus in nonneoplastic tonsils primary infection with the epstein-barr virus and risk of multiple sclerosis epstein-barr virus-associated infectious mononucleosis and risk of systemic lupus erythematosus application of immunohistochemistry in the diagnosis of non-hodgkin and hodgkin lymphoma relation of isolated tonsillar hypertrophy with body mass index the significance of the size of the palatine tonsil level of streptococcous pyogenes in patients with recurrent tonsillitis and tonsillar hypertrophy, scan detection of epstein-barr virus in recurrent tonsillitis actinomycosis in the etiology of recurrent tonsillitis and obstructive tonsillar hypertrophy. answers from histopathologic point of view the correlation between ebv viral load in the palatine tonsils of patients with recurrent tonsillitis and concurrent serum titers of vca-igg identification of the site of epstein-barr virus persistence in vivo as a resting b cell tumour viruses-could they be an achilles' heel of cancer? the ebv action in tonsils and adenoid distribution and phenotype of epstein-barr virus-infected cells in human pharyngeal tonsils homology of cytokine synthesis inhibitory factor (il-10) to the epstein-barr virus gene bcrfi interleukin 10 is a potent growth and differentiation factor for activated human b il-10) and viral il-10 strongly reduce antigenspecific 1 human t cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class ii major histocompatibility complex expression detection of herpes simplex and epstein-barr viruses in patient with acute tonsillitis is there a relationship between the detection of human herpesvirus 8 and epstein-barr virus in waldeyers ring tissues? expression of epstein-barr virus in hodgkin lymphoma in a population of united arab emirates nationals ebv-infected b cells in infectious mononucleosis: viral strategies for spreading in the b cell compartment and establishing latency frequent expansion of epstein-barr virus (ebv) infected cells in germinal centres of tonsils from an area with a high incidence of ebv-associated lymphoma epstein-barr virus dna is present both in cd10/cd77 positive and negative subsets of human tonsillar lymphocytes role of epithelium in ebv persistence and pathogenesis of b-cell tumours replication of ebv in epithelial cells during infectious mononucleosis epstein-barr virus persistence and virus-associated tumours epstein-barr virus gene expression and epithelial cell differentiation in oral hairy leukoplakia morphology, immunophenotype, and distribution of latently and/or productively epstein-barr virus-infected cells in acute infectious mononucleosis: implications for the interindividual infection route of epstein-barr virus key: cord-257271-jzmwy4yi authors: lin, jung-chung; cherng, jaw-ming; hung, man-shan; baltina, lidia a.; baltina, lia; kondratenko, rimma title: inhibitory effects of some derivatives of glycyrrhizic acid against epstein-barr virus infection: structure–activity relationships date: 2008-03-31 journal: antiviral res doi: 10.1016/j.antiviral.2008.01.160 sha: doc_id: 257271 cord_uid: jzmwy4yi glycyrrhizic acid (18β-gl or gl) is a herbal drug with a broad spectrum of antiviral activities and pharmacological effects and multiple sites of action. previously we showed that gl inhibits epstein-barr virus (ebv) infection in vitro by interfering with an early step of the ebv replication cycle (possibly attachment/penetration). here we tested the effects of 15 gl derivatives against ebv infection by scoring the numbers of cell expressing viral antigens and quantifying ebv dna copy numbers in superinfected raji cells. the derivatives were made either by transformation of gl on carboxyl and hydroxyl groups or by conjugation of amino acid residues into the carbohydrate part. we identified seven compounds active against ebv and all showed dose-dependent inhibition as determined by both assays. among these active compounds, the introduction of amino acid residues into the gl carbohydrate part enhanced the antiviral activity in three of the seven active compounds. however, when glu(oh)-ome was substituted by glu(ome)-ome, its antiviral activity was completely abolished. introduction of potassium or ammonium salt to gl reduced the antiviral activity with no significant effect on cytotoxicity. the α-isomer (18α-gl) of 18β-gl was as potent as the β-form, but its sodium salt lost antiviral activity. the metabolic product of gl, 18β-glycyrrhetinic acid (18β-ga or ga), was 7.5-fold more active against ebv than its parental compound gl but, concomitantly, exhibited increased cytotoxicity resulting in a decreased therapeutic index. glycyrrhizic acid (18␤-gl or gl) is one of the bioactive compounds of licorice roots (glycyrrhiza radix) and is composed of one molecule of glycyrrhetinic acid (18␤-ga or ga), which has a steroid-like structure, and two molecules of glucuronic acid. after oral administration or intravenous injection, gl is hydrolyzed by glucuronidase in intestinal bacteria to its active principle aglycone, 18␤-ga, which is then absorbed into the blood (takeda et al., 1996) . gl and ga have been shown to possess several beneficial pharmacological activities (dirnagl et al., 1999; shibata, 2000; abe et al., 2003; armanini et al., 2002; cherng et al., 2006; cinatl et al., 2005; fiore et al., 2004) , which include an anti-ulcerative effect, anti-inflammatory activity (fujisawa et al., 2000) , interferon (ifn)-␥ induction, anti-hepatotoxic effect (chan et al., 2003; gumpricht et al., 2005; zheng and lou, 2003) , anti-tumor activity (hibasami et al., 2006) , and it is active against a range of viruses (lin, 2003 ; * corresponding author. tel.: +886 3 856 5301x7209; fax: +886 3 856 6724. e-mail address: jclin@mail.tcu.edu.tw (j.-c. lin). cinatl et al., 2003; crance et al., 2003; briolant et al., 2004; cherng et al., 2004; hoever et al., 2005) . clinically, gl has been used to treat patients with chronic active hepatitis (van rossum et al., 1999; bean, 2002; coon and ernst, 2004) . in previous years, we have shown that many nucleoside analogs selectively inhibit replication of epstein-barr virus (ebv) in vitro (lin et al., 1983 (lin et al., , 1984 (lin et al., , 1985 (lin et al., , 1987 (lin et al., , 1991 (lin et al., , 1992 lin and machida, 1988; mar et al., 1995; for a review see gershburg and pagano, 2005; lin, 2006) . the molecular target of all nucleoside analogs is the virus-encoded dna polymerase. recently, we have reported that gl is active in a dose-dependent fashion against ebv replication in superinfected raji cells (lin, 2003) . our results also indicated that gl interferes with an early step of the ebv replication cycle (possibly adsorption and/or penetration) (lin, 2003) . thus, gl represents a new class of anti-ebv compounds with a mode of action different from that of the nucleoside analogs that inhibit the viral dna polymerase. in the present studies we extend our previous findings after a search for more potent compounds against ebv. we tested the antiviral activity of 15 gl derivatives synthesized in the laboratory. here we report the identification of seven active compounds against ebv infection in vitro and elucidate their structure-activity relationships. glycyrrhizic acid (glycyrrhizin, 18␤-gl or gl) and 18␤glycyrrhetinic acid (18␤-ga or ga) were purchased from sigma-aldrich chemie gmbh, germany. the synthesis of gl derivatives was reported previously (baltina, 2003; baltina et al., 2006; kondratenko et al., 2006) . l-aminoacids were used for the synthesis of gl glycopeptides. unless otherwise specified, all drugs were dissolved in phosphate-buffered saline, ph 7.5. the chemical structures are shown in chart 1. a latently ebv-infected non-virus producing cell line (raji) was maintained at between 4 × 10 5 and 6 × 10 5 cells/ml in rpmi 1640 medium containing 10% fetal calf serum (fcs) supplemented with 100 iu penicillin/ml and 100 g streptomycin/ml, as described previously (lin et al., 1984) . a highly productive virus-producer cell line, p3hr-1 (ls), derived by low-serum cloning (lin, 2000) , was maintained in the same culture medium as for raji cells, except that the serum concentration was reduced to 2%. viral stocks were prepared from cultures of p3hr-1 (ls) that had been treated with 12-o-tetradecanoyl-phorbol-12-acetate (tpa) at a concentration of 30 ng/ml (lin, 2000) . briefly, the p3hr-1 (ls) cells were induced with tpa for 14 days without additional medium. the cells were removed by centrifugation at 1200 × g for 10 min, virus was then pelleted from the cell-free supernatant fluids by centrifugation at 13,000 × g for 90 min in a gs3 rotor (ivan sorvall, inc.). the centrifuged bottles were swabbed to remove residual medium chart 1. and the virus pellets were suspended in rpmi 1640 medium. the viral suspension was clarified to remove cellular debris by filtering through 1.2-, 0.8-, and 0.45-m-pore-size filters and stored at −80 • c. as an assay system for drug effects we used raji cells superinfected with p3hr-1 (ls) virus, which results in reactivation of the latent ebv infection and replication of virus. raji cells were first propagated in medium containing a sub-effective dose of compound (1 m). for superinfection (lin, 2003) , 10 6 raji cells growing exponentially were pelleted and resuspended in 0.5 ml of rpmi 1640 medium containing 2% fcs and appropriate concentrations of drug as specified. then 10 units of ebv early antigen (ea)-inducing virus were added to the cells. one unit of virus is defined as the amount of virus capable of inducing 10% of infected raji cells to express ea. after 2 h adsorption at 37 • c in a co 2 incubator, the cells were pelleted and suspended in 1 ml of the medium and incubation continued for 24 h. the levels of ebv early antigen (ea) and viral capsid antigen (vca) were monitored by indirect immunofluorescent assay (ifa) at 24 h after infection with ebv-positive sera from npc patients. the specificity of sera for ebv ea/vca was confirmed by western blots as reported previously (lin and pagano, 1986) . the detailed protocol for ifa was described previously (lin, 2000) . briefly, superinfected raji cells were smeared on slides and fixed in methanol for 15 min at room temperature. the fixed cells were first reacted with npc patients' serum, followed by fluorescein-conjugated goat anti-human igg (h + l) (santa cruz biotechnology, inc.). after counterstaining with 4 ,6 -diamidino-2-phenylindole (dapi) (0.1 g/ml in pbs) for 5 min in the dark, the slides were analyzed under the fluorescent microscope. the drug effects on ebv dna in superinfected raji cells were determined using a real-time quantitative pcr system toward the bamhi-w fragment region of the ebv genome as described (lo et al., 1999) . real-time quantitative pcr is based on the continuous optical monitoring of the progress of a fluorogenic pcr reaction. in this system, the amplification primers used in conventional pcr toward the bamhi-w were: w-44f (5 -cccaacactccaccacacc-3 ) and w-119r (5 -tcttaggagctgtccgaggg-3 ). the duallabeled fluorogenic hybridization probe was w-67t[5 -(fam)ca-cacactacacacacccacccgtctc(tamra)-3 ]. the fluorescent probes contained a 3 -blocking phosphate group to prevent probe extension during pcr. the effect of gl derivatives on raji cell cytotoxicity was carried out by mtt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue) cell-proliferative kit i (roche, mannheim, germany). raji cells were seeded in wells of a 96-well plates and treated with various concentrations of drug for 3 days. then 10 l of sterile mtt dye were added, and the cells were incubated for 6 h at 37 • c, followed by adding 100 l of acidic isopropanol (0.04 m hcl in isopropanol). spectrometric absorbance at 595 nm (for formazan dye) was measured with the absorbance at 655 nm for reference. to determine the dose-dependent effect of gl derivatives, raji cells were superinfected with p3hr-1 (ls) virus in the presence of various concentrations of compounds. the expression of ea and vca were monitored by ifa at 24 h after superinfection. a representative result from compound (xv) is shown in fig. 1 . without drug treatment, approximately 95% of infected cells became positive for viral antigens (fig. 1, panel a) . in the presence of drugs, a dose-dependent inhibition of the expression of viral antigens was observed (fig. 1, panels b, c, d, and e) . the concentration of drug required to inhibit ebv antigen expression by 50% (ec 50 ) was determined from the plot of drug concentrations against % of viral antigen-positive cells, assuming no-drug control as 100. fig. 2a shows graphical quantitation for compound (xv). the ec 50 determined from the graph was approximately 42 m. as shown in table 1 , seven gl derivatives were active against ebv infection with ec 50 ranging from 42 to 175 m. all these active compounds exhibited a dose-dependent inhibition manner. the parental compound gl was used as a positive drug control. in addition, ga, the metabolite of gl was also tested in parallel with the derivatives. it is interesting to note that ga was the most effective compound to block ebv infection with an ec 50 of 5 m, which was 7.6-fold lower than for gl (38 m). however, ga was approximately 60-fold more toxic (cc 50 = 75 m) than gl (cc 50 = 4500 m). the cytotoxicity of the gl derivatives was determined in 96-well microtiter plates by the mtt assay and the results are expressed as the concentration of the compound that decreased cell viability to 50% (cc 50 ) of the no drug control (table 1) . these compounds exhibited variable cytotoxicity, the cc 50 ranging from 1000 to 8000 m. to more precisely determine the drug effects, ebv dna copy numbers were measured using a real-time quantitative pcr system that amplified a dna segment in the bamhi-w fragment region of the ebv genome (lo et al., 1999) . a dose-dependent inhibition of viral genome copy numbers by compound (xv) was observed (fig. 2b) . the ec 50 obtained by this method is approximately 25 m, in contrast to 42 m determined by ifa (table 1) . data obtained by pcr and ifa are shown side by side in table 1 . it should be noted that the ec 50 values obtained by real-time quantitative pcr is approximately 20-40% lower than that obtained by ifa. however, the inhibitory profile of these compounds remains unchanged, indicating that ifa is as reliable as the quantitative pcr method for initial drug screening. evaluation of antiviral agents effective against ebv has been hampered by the lack of a permissive cell system for the replication of this virus. superinfection of raji cells with p3hr-1 virus results in shut-down of host cell dna synthesis and stimulation of viral dna replication and lytic antigen synthesis and production of virus. we have previously shown that the percentage of ea-and vca-positive cells is directly proportional to the amount of ebv dna in the cells (lin, 2003) . furthermore, we have also demonstrated that gl blocks ebv replication at an early step of the viral replication cycle. however, gl has no effects on ebv dna in the spontaneously virus-producing cell line p3hr-1 and the latently infected nonvirus-producing raji cells (lin, 2003) . the lack of effect of gl in p3hr-1 and raji cells, which are already infected, is consistent with our proposed mechanism of gl action, i.e., inhibition of virus attachment/penetration. thus, superinfection of raji cells represents a useful system for evaluation of compounds that block early steps in the ebv replication cycle. our results indicate that gl and several of its derivatives are highly selective in their antiviral action in that they inhibit ebv infection at concentrations far below the cytotoxic concentration. as demonstrated previously (lin, 2003) , pretreatment of cells with sub-effective dose of gl markedly reduced cytotoxic effects while decreasing the amount of drug (approximately 10-fold reduction) needed to reach the same effective level. the marked reduction of ec 50 obtained by pretreatment of cells with sub-effective doses of the compounds has clinical implication with regard to prophylactic and therapeutic effects. among seven active compounds (table 1) , three of them contain amino acid residues in the gl carbohydrate part. the ala-ome containing glycopeptide (xii), the glu(oh)-ome containing glycopeptide (xiii), and leu containing glycopeptides (xv) were active against ebv. however, compounds (xii) (ec 50 = 30 m) and (xv) (ec 50 = 25 m) were approximately 5-fold more active against ebv than compound (xiii) (ec 50 = 135 m). the cc 50 values of glycopeptides (xii), (xiii), and (xv) were, respectively, 2000, 4000, and 5000 m, resulting in a therapeutic index of 67, 30, and 200. in contrast, two other glycopeptide containing compounds such as (i) and (ix) did not exhibit activity against ebv at concentrations up to 1 mm. interestingly, when glu(oh)-ome was substituted by glu(ome)-ome as in compound (xiv), its antiviral activity was completely abolished. introduction of a hydroxyl or methoxyl group at the r position of the aglycon moiety of gl, such as in compounds (i), (ii), and (iii) did not show any antiviral activity. compounds (ix) and (xii) both contain a hydroxyl group at the r position of ga molecule, but with different substitution at the r position of the carbohydrate part of gl. compound (xii) with an ala-ome group at the r position showed good antiviral activity, whereas substitution of compound (ix) with an ala-obu group at the same position abolished the antiviral activity. interestingly, other substitutions at the r position of ga, such as lys(z)-oh in compound (v) and l-glu(ome)-ome in compound (xiv), did not confer any antiviral activity. thus, it appears that the modification of the carbohydrate moiety of gl rather than the r position of aglycon may affect the antiviral activity. these findings may provide a lead for further development of more active antiviral compounds. the two commercially available compounds gl and ga were tested in parallel with semisynthetic gl derivatives. unexpectedly, ga, the metabolic product of gl, was 7.5-fold more active (ec 50 = 4 m) against ebv than its parental compound (ec 50 = 30 m), but was 60-fold more toxic (cc 50 = 75 m) than gl (cc 50 = 4500 m), resulting in a therapeutic index of 19 and 150, respectively. introduction of potassium [compound (vi)] or ammonium salt [compound (vii)] to gl reduced the antiviral activity with a significant effect on cytotoxicity. the ␣-isomer [18␣-gl, compound (viii)] derived from 18␤-gl was as potent as the ␤-form, with ec 50 of 30 and cc 50 of 4800, resulting in a therapeutic index of 160. in contrast, the sodium salt of ␣-isomer of 18␤-gl [compound (xi)] lost antiviral activity as compared to its ␤-form [compound (x)]. previous study indicated that the sugar moiety of gl is essential for anti-sars-cov activity . the present study clearly demonstrated that compounds (vi), (vii), (viii), (x), (xii), (xiii), and (xv) are active inhibitors of ebv infection and all contain a sugar moiety. in summary, among the newly identified analogs four showed better or equal activity than the parent gl. recently we have demonstrated that gl interferes with an early step of ebv replication cycle (possibly attachment/penetration) (lin, 2003) . however, other possible mechanism of gl may also involve inhibition of ebv replication. both gl and ga have been shown to elicit a dose-dependent increase in no production and the level of inos mrna in macrophages (jeong and kim, 2002) . as a host defense molecule, no has an antimicrobial activity against a variety of pathogens including viruses. previous studies indicated that no is involved in maintaining ebv latency through inhibition of ebv reactivation (mannick et al., 1994; gao et al., 1999) . the molecular mechanism underlying the gl inhibition of viral attachment is not well understood. a complement protein called c3d is a breakdown product of complement c3. receptors for c3d, called type 2 complement receptors (cr2 or cd21), are expressed on mature b lymphocytes. when b lymphocytes recognize an antigen by their antigen receptors and simultaneously recognize c3d bound to the antigen by the complement receptor, the b cells are activated. the major ebv outer envelope glycoprotein gp350/220 is the cd21 ligand (nemerow et al., 1987; tanner et al., 1987) . cd21 is the only b-lymphocyte surface protein that binds to gp350/220 (nemerow et al., 1989) , which mediates virus attachment to the ebv/c3dg receptor (cr2) of human b lymphocytes. a recent study indicated that complement c3 is a gl-binding protein (kawakami et al., 2003) . synthetic peptides corresponding to two regions in gp350/220, which have a similar amino acid sequence with the complement c3dg protein, were used to identify a receptor binding epitope. a peptide corresponding to the n terminus of gp350/220, edpgffnvei, bound to purified cr2 and to cr2 positive but not cr2 negative b and t lymphoblastoid cell lines (nemerow et al., 1989) . this peptide sequence edpgffnvei is similar to the peptide sequence edpgkqlynvea, through which the c3d component of complement binds to cd21 (nemerow et al., 1989) . synthetic peptides containing the lynvea c3d sequence block c3d binding to cd21 (lambris et al., 1985) , whereas peptides containing the edpgffnvea sequence block ebv infection (nemerow et al., 1989 (nemerow et al., , 1990 . taken together these studies open the possibility that the inhibitory effect of gl could be in part due to the blockade of ebv receptor. the anti-ebv activity of glycopeptides (xii), (xiii), and (xv) could be attributed to the similarity of their amino acid residues with the peptide sequence edpgkqlynva of c3d component of complement that binds to cd21. ascertaining this possibility would require further study and is currently under investigation. from the target perspective, the drugs that might be candidates for treatment of ebv infection fall into two groups (for reviews see refs. gershburg and pagano, 2005; lin, 2006) . the first group targeting viral dna polymerase includes acyclic nucleoside analogues (acyclovir, ganciclovir, penciclovir, valaciclovir, valganciclovir and famciclovir); acyclic nucleotide analogues (cidofovir and adefovir); pyrophosphate analogues (phosphonoformic acid and phosphonoacetic acid); possibly 4-oxo-dihydroquinolines (pnu-182171 and pnu-183792). the second group contains compounds of mixed nature that have a distinct structure such as maribavir, ␤-l-5-iododioxolane uracil and indolocarbazole nigc-i. mechanisms of action of these drugs are still under study, but they do not involve inhibition of the viral dna polymerase. the molecular target in herpesviruses of all nucleoside analogs are the virus-encoded dna polymerases from which the mode of action of gl and its derivatives differs. the therapeutic and prophylactic effects of gl on chronic active viral hepatitis (bean, 2002; coon and ernst, 2004; shibata, 2000; van rossum et al., 1999) and the inhibitory effect on ebv replication observed in the present study together with its relative lack of toxicity at the cellular level all suggest that gl and selected derivatives may be worth further evaluating for their efficacy and safety in the treatment of active ebv infection. glycyrrhizin enhances interleukin-10 production by liver dendritic cells in mice with hepatitis history of the endocrine effects of licorice chemical 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ribavirin, 6-azauridine and glycyrrhizin: antiviral compounds active against pathogenic flaviviruses pathobiology of ischaemic stroke: an integrated view on the mechanism of mitochondrial permeability transition induction by glycyrrhetinic acid glycyrrhizin inhibits the lytic pathway of complement -possible mechanism of its antiinflammatory effect on liver cells in viral hepatitis nitric oxide down-regulates epstein-barr virus reactivation in epithelial cell lines epstein-barr virus infections: prospects for treatment licorice compounds glycyrrhizin and 18␤-glycyrrhetinic acid are potent modulators of bile acid-induced cytotoxicity in rat hepatocytes glycyrrhetic acid (a metabolic substance and aglycon of glycyrrhizin) induces apoptosis in human hepatoma, promyelotic leukemia and stomach cancer cells antiviral activity of glycyrrhizic acid derivatives against sars-coronavirus induction of inducible nitric oxide synthase expression by 18␤-glycyrrhetinic acid in macrophages characterization of complement c3 as a glycyrrhizin (gl)-binding protein and the phosphorylation of c3␣ by ck-2, which is potently inhibited by gl and glycyrrhetinic acid in vitro synthesis and immunomodulating activity of new glycopeptides of glycyrrhizic acid containing residues of l-glutamic acid mapping of the c3d receptor (cr2) binding site and a neoantigenic site in the c3d domain of the third component of complement strategies for evaluation of antiviral agents against epstein-barr virus in culture mechanism of action of glycyrrhizic acid in inhibition of epstein-barr virus replication in vitro pathogenesis and therapy of epstein-barr virus infection: novel therapeutic approaches comparison of two bromovinyl nucleoside analogs, 1-␤-d-arabinofuranosyl-e-5-(2-bromovinyl)uracil and e-5-(2-bromovinyl)-2 -deoxyuridine, with acyclovir in inhibition of epstein-barr virus replication sequential detection of different antigens induced by epstein-barr virus and herpes simplex virus in the same western blot by using dual antibody probes epstein-barr virus: inhibition of replication by three new drugs prolonged inhibitory effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine against replication of epstein-barr virus comparative efficacy and selectivity of some nucleoside analogs against epstein-barr virus hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human lymphoblastoid cell lines infected with epstein-barr virus novel acyclic adenosine analogs inhibit epstein-barr virus replication inhibitory effects of acyclic nucleoside phosphonate analogs, including (s)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, on epstein-barr virus replication structure-activity relationship between (e)-5-(2-bromovinyl)-and 5-vinyl-1-␤-d-arabinofuranosyluracil(bv-arau, v-arau) in inhibition of epstein-barr virus replication quantitative analysis of cell-free epstein-barr virus dna in plasma of patients with nasopharyngeal carcinoma nitric oxide produced by human b lymphocytes inhibits apoptosis and epstein-barr virus reactivation some nucleoside analogs with anti-human immunodeficiency virus activity inhibit replication of epstein-barr virus identification of gp350 as the viral glycoprotein mediating attachment of epstein-barr virus (ebv) to the ebv/c3d receptor of b cells: sequence homology of gp 350 and c3 complement fragment c3d identification of an epitope in the major envelope protein of epstein-barr virus that mediates viral binding to the b lymphocytes epstein-barr virus receptor (cr2) soluble recombinant cr2 (cd21) inhibits epstein-barr virus infection a drug over the millennia: pharmacognosy, chemistry, and pharmacology of licorice bioavailability study of glycyrrhetic acid after oral administration of glycyrrhizin in rats; relevance to the intestinal bacterial hydrolysis epstein-barr virus gp350/220 binding to the b lymphocyte c3d receptor mediates adsorption, capping, and endocytosis intravenous glycyrrhizin for the treatment of chronic hepatitis c: a doubleblind, randomized, placebo-controlled phase i/ii trial pathologic characteristics of immunologic injury in primary cultured rat hepatocytes and protective effect of glycyrrhizin in vitro we thank joseph pagano for his critical reading and comments on this manuscript. this work was supported in part by grants from the national science council of the republic of china (nsc94-2320-b-320-011; nsc95-2320-b-320-011) and by an institutional grant (tcirp 95002-05y1) of tzu chi university. russian authors thank the federal agency on science and innovation (russia) for financial support (contract 02.434.11.7060). key: cord-266218-r6xg9zts authors: law, arjun datt; salas, maria queralt; lam, wilson; michelis, fotios v.; thyagu, santhosh; kim, dennis (dong hwan); lipton, jeffrey howard; kumar, rajat; messner, hans; viswabandya, auro title: reduced-intensity conditioning and dual t lymphocyte suppression with antithymocyte globulin and post-transplant cyclophosphamide as graft-versus-host disease prophylaxis in haploidentical hematopoietic stem cell transplants for hematological malignancies date: 2018-08-07 journal: biol blood marrow transplant doi: 10.1016/j.bbmt.2018.07.008 sha: doc_id: 266218 cord_uid: r6xg9zts haploidentical hematopoietic stem cell transplantation (haplohsct) with conditioning regimens using post-transplant cyclophosphamide (ptcy) for peripheral blood stem cell (pbsc) grafts is limited by comparably higher rates of acute and chronic graft-versus-host disease (gvhd). antithymocyte globulin (atg) may mitigate this risk. we evaluated haplohsct after reduced-intensity conditioning (ric) with atg, ptcy, and cyclosporine to prevent rejection and gvhd. fifty adults underwent haplohsct from august 2016 to february 2018. ric included fludarabine (30 mg/m(2)/day on days –5 to –2), busulfan (3.2 mg/m(2)/day on days –3 and –2), and total body irradiation (200 cgy) on day –1. unmanipulated pbscs were infused on day 0. gvhd prophylaxis included atg (4.5 mg/kg over days –3 to –1), ptcy (50 mg/kg/day on days +3 and +4), and cyclosporine from day +5. median age was 56 years (range, 22 to 70 years); 25 (73.5%) patients were in first complete remission (cr1), 5 (14.7%) were in second complete remission (cr2), and 8 (23.5%) had active disease. median time to neutrophil engraftment was 16 days (range, 8 to 43 days). at day +100, the cumulative incidence of acute gvhd of any grade, and grades iii to iv was 38.3% and 5.2%, respectively. mild chronic gvhd was seen in 15.5%. cytomegalovirus (cmv) reactivation occurred in 37 (74%) cases and cmv disease occurred in 4 (11.5%) cases. epstein-barr virus (ebv) reactivation occurred in 21 (61.8%) patients. the incidence of histologically confirmed post-transplantation lymphoproliferative disorder (ptld) was 5.8%. four patients received rituximab. there were no cmv, ebv, or ptld-related deaths. six-month and 1-year overall survival (os), cumulative incidence of relapse (cir), and nonrelapse mortality (nrm) were 73.9%, 10.2%, and 19.4%, respectively, and 48.1%, 16% and 38.2%, respectively. infection was the most common cause of death (18%). unmanipulated haploidentical pbsc transplantation following ric with atg, ptcy, and cyclosporine as a gvhd prevention strategy results in low rates of acute and chronic gvhd. allogeneic hematopoietic stem cell transplantation (hsct) is a potentially curative therapy for patients with high-risk or advanced hematological disorders [1] . an hlamatched sibling donor is considered the first choice. approximately 70% of patients do not have a suitably matched sibling donor (msd) available for transplantation. alternatives such as matched unrelated donors (muds) can be identified for only 50% to 60% of patients, with the donor search and procurement process requiring a median of 4 months [2] . for patients who do not have suitable msds or muds, alternative stem cell sources include unrelated single or double umbilical cord blood transplants, hla-mismatched unrelated donors (mmuds), and hla haploidentical family members. despite the expansion of mud registries and cord blood banks, donor availability can often be uncertain particularly for under-represented ethnic minorities, which may be subject to prolonged and unproductive registry searches [3] . the use of haploidentical donors reduces this uncertainty to a large degree, as almost all patients have an immediately available related donor with whom they share a single hla haplotype. this not only facilitates a reduced time to transplantation, but also provides a more reliable source of donor there remains considerable variation between centers in conditioning regimens, graft sources and choice of graft-versus-host disease (gvhd) prophylaxis to achieve lower nonrelapse mortality (nrm) and optimum long-term outcomes in haploidentical hsct (haplohsct). the combination of a nonmyeloablative conditioning regimen with antithymocyte globulin (atg) and post-transplant high-dose cyclophosphamide (ptcy) has only been described in the setting of haplohsct for sickle cell disease [4] . in our institution, we established a reduced-intensity conditioning (ric) regimen consisting of fludarabine (flu), reduced-dose busulfan (bu), and total body irradiation (tbi) (total dose 200 cgy) combined with atg and followed by ptcy and cyclosporine for the prevention of graft rejection and gvhd for haplohsct recipients. we previously reported the use of this protocol in mud transplants wherein lower rates of severe acute gvhd were demonstrated compared with nonàatg-based regimens [5] . in the present study, we report the use of this protocol in 50 consecutive patients who underwent haplohsct at our center. from august 2016 to february 2018, 50 adult patients with hematological malignancies underwent haplohsct at the princess margaret cancer centre, toronto, canada. all patients were 18 years old with no upper age limit, had a karnofsky performance score (kps) >60%, and did not have active/ untreated infection or human immunodeficiency virus seropositivity. criteria for pretransplant organ function included left ventricular ejection fraction 45% without significant preexisting cardiac disease, pulmonary function testing demonstrating forced expiratory volume >50% predicted and diffusing capacity of carbon monoxide >50, normal/stable kidney function on biochemistry, and liver functions tests showing total bilirubin <2.5 times normal with transaminases <3 times the upper limit of normal. informed consent was obtained from all patients. the use of this protocol was approved by the institutional board of ethics. all patients with positive donor specific antibodies were excluded. high-resolution molecular typing for hla class i (a, b, c) and class ii (dr, dq) was performed for recipients and donors when patients were referred to the allogeneic blood and marrow transplantation program. haploidentical related donors were selected in all cases where msd or 9/10 mud could not be identified. peripheral blood stem cells (pbsc) were collected after granulocyte colony-stimulating factor (g-csf) mobilization. a minimum dose of 6 £ 10 8 cd34 + cells/kg recipient body weight was requested in all cases. cd3 + cell enumeration is not routinely performed in our institute, as unmanipulated stem cells are used in all cases. all donors provided informed consent. patients received conditioning therapy with fludarabine 30 mg/m 2 /day i. v. on days -5 to à2, busulfan 3.2 mg/m 2 /day i.v. days à3 and à2, and 200 cgy of tbi on day à1. rabbit atg (thymoglobulin; genzyme-sanofi, lyon, france) was administered in doses of .5 mg/kg on day à3, 2 mg/kg on day à2, and 2 mg/kg on day à1 (total 4.5 mg/kg). t cellàreplete pbsc grafts were infused on day 0. all patients received ptcy (cyclophosphamide 50 mg/kg/day i.v.) on days +3 and +4, with the first dose starting 72 hours after the start of allograft infusion. four doses of mesna were administered on days +3 and +4 at a total daily dose of 80% of the cyclophosphamide dose. cyclosporine was initiated at a dose of 2.5 mg/kg i.v. on day +5 and adjusted to achieve a therapeutic level of 200 to 400 mg/l ( figure 1 ). all patients received daily filgrastim 300 mg subcutaneously, starting on day +7 until neutrophils were >1.5 £ 10 9 /l. ursodeoxycholic acid 500 mg oral twice daily with meals (starting on the first day of conditioning) was used for prevention of sinusoidal obstruction syndrome (sos) in all patients. prophylactic antimicrobial therapy included posaconazole 300 mg daily, acyclovir 400 mg twice daily, ciprofloxacin 500 mg twice daily, and inhaled pentamidine 300 mg monthly. postengraftment, cytomegalovirus (cmv) titer was monitored twice per week and epstein-barr virus (ebv) was monitored weekly. cyclosporine tapering started around days +45 to 60 for all patients without gvhd. the schematic for the conditioning protocol and post-transplant immunosuppressive regimen is shown in figure 1 . neutrophil engraftment was defined as the first of 3 consecutive days with an absolute neutrophil count .5 £ 10 9 /l and platelet recovery was defined as a sustained and transfusion-independent platelet count >20 £ 10 9 /l (first of 3 days) without transfusion times 7 days. donor total chimerism was assessed in whole blood by pcr for variable number tandem repeat polymorphisms at days +30 and +60 in all patients. all patients underwent bone marrow examination at day +60 for marrow cellularity and disease monitoring. clinical features, post-transplant outcome, and side effects were collected through retrospective chart review. last follow-up was updated in february 2018. the study was conducted in accordance with the declaration of helsinki, and was reviewed and approved by the ethics committee at the princess margaret cancer centre, toronto, canada. patient and disease characteristics were reported using descriptive statistics (count and percentage). the time to event was calculated from the date of transplant to the date of event or last date of patients known to be alive. the main outcome variables of interest included overall survival (os), relapse-free survival (rfs), and nonrelapse mortality (nrm). the kaplan-meier method was used to estimate os and rfs. the cumulative incidences of acute and chronic gvhd were calculated accounting for death and relapse as competing risks. the incidence rate of acute gvhd was estimated at day +100 and chronic gvhd at day +180. nrm was estimated using the cumulative incidence method accounting for relapse as a competing risk (fine-and-gray analysis). survival rates were calculated 6 months and 1 year after hap-lohsct. analyses were performed using spss version 16.0 for windows (spss inc, chicago, il), and ezr [6] . table 1 shows baseline patient characteristics. table 2 shows donor characteristics, graft parameters, and gvhd characteristics. median follow-up for survivors was 168 days (range, 22 to 536 days), or 5 months (range, 0 to 17 months). nearly 80% of donor-recipient pairs were mismatched at 5 hla loci. sixteen patients and donors were cmv mismatched. while cryopreserved grafts were used in the majority, 10 patients received fresh product. the hematopoietic cell transplantation comorbidity index score was 3 in almost 40% cases. median time to neutrophil engraftment was 17 days (range, 8 to 43 days). median time to platelet engraftment was 22 days (range, 7 to 217 days). engraftment syndrome (characterized by fever, fluid retention/weight gain, hypotension, and hypoxemia in the absence of documented infection, and temporally correlated with count recovery) occurred in 3 (6%) patients, and all cases were successfully treated with steroids. ninetyfour percent of patients achieved donor chimerism >95% at day +30. four (8%) patients failed to engraft by day +30. one patient developed secondary graft failure 3 months post-transplant. this included 1 patient with primary myelofibrosis, 1 with myelodysplastic syndrome, and 2 with acute myelogenous leukemia. median of 9.94 £ 10 6 cd34 + /kg recipient body weight (range, 8.2 to 11.7 £ 10 6 cd34 + /kg recipient body weight) were infused. no donor-specific antibodies were documented. all patients had developed cmv reactivation, and 1 patient with secondary graft failure had biopsy-proven cmv colitis. ebv reactivation was documented in 2 cases, and 1 developed asymptomatic bk viruria. median length of hospitalization was 32 days (range, 13 to 83 days). post-transplant clinical course, organ toxicity, and infections are summarized in supplementary tables 1 and 2 . three (6%) cases developed low-grade cytokine release syndrome following stem cell infusion. this occurred on day 0 in all cases and resolved after cyclophosphamide administration in all cases by day +4. mild mucositis was seen in all patients, while 9 (18%) patients developed grade 3 to 4 mucositis requiring short-term total parenteral nutrition. mild-to-moderate sos was documented in 7 (17.4%) patients, with grade 3 sos in 1 case. all cases resolved with fluid restriction and diuretics. ten patients developed features of fluid overload, and 3 patients developed cardiogenic pulmonary edema secondary to fluid retention and hypoalbuminemia during the first 30 days during the post-transplant course. all cases responded to aggressive diuresis and fluid optimization, and no patient needed intensive care support. no ischemic events occurred. three patients developed pericardial complications (2 cases with pericarditis and 1 myopericarditis) during follow-up. all effusions were mild, resolved spontaneously, and did not show features of cardiac tamponade. diagnostic or therapeutic pericardiocentesis was not performed. one patient demonstrated an appreciable decline in left ventricular ejection fraction on follow-up echocardiography at day+60 but remained asymptomatic. one patient with a history of anthracycline-associated cardiomyopathy with induction chemotherapy sustained a fatal ventricular arrhythmia while undergoing treatment for cmv reactivation and gvhd. transient elevation of renal parameters without oliguria was seen in 13 (26%) patients. creatinine returned to baseline in all cases with conservative management. neutropenic fever occurred in 42 (84%) of the patients with positive blood cultures in nineteen cases (38%). no proven invasive fungal infections were encountered during hospitalization for the transplant. laboratory evidence of cmv reactivation by peripheral blood pcr was observed in 37 (74%) patients, with a median time to reactivation of 26 days (range, 13 to 61 days) post-transplant. cmv was also identified in bronchoalveolar lavage specimens in 3 cases and on intestinal biopsy in 1 case. all patients received intravenous ganciclovir or valgancyclovir until viremia clearance. two patients required foscarnet therapy due to persistence of cmv viremia despite appropriate doses of ganciclovir. twenty-seven (64%) patients developed ebv reactivation after transplantation with a median time of 56 days (range, 20 to 233 days). four patients (8%) developed biopsy proven post-transplant lymphoproliferative disease (ptld). ptld was managed by tapering immunosuppression and weekly rituximab for a maximum of 4 doses. rates of acute and chronic gvhd were 44% and 10%, respectively. acute gvhd was graded according to the keystone criteria and chronic gvhd was graded in accordance with the national institutes of health chronic graft-versus-host disease consensus [7, 8] . acute gvhd of any grade was identified in 20 (44%) patients; however, grade iii to iv acute gvhd occurred in only 2 cases (4%). the cumulative incidence of acute gvhd of any grade, grade ii to iv, and grade iii to iv at day +100 was 38.3%, 20.3%, and 5.2%, respectively ( table 2) . skin (36%) was the most commonly affected site, followed by liver (14%) and gut (10%). first-line therapy of clinically significant acute gvhd consisted of topical or systemic steroids based on site and severity. the cumulative incidence of chronic gvhd at 3 months was 15.5%. no systemic immunosuppression was required for patients with chronic gvhd. no patients developed moderate or severe chronic gvhd (table 3) . nineteen (38%) patients died during follow-up. table 4 lists the causes of death among transplanted patients. five (10%) patients relapsed during the follow-up period with a median time to relapse of 4 months (range, 1 to 9 months). six-month os, rfs, cir, and nrm were 73.9%, 57%, 10.2%, and 19.4%, respectively. no significant differences were found in os (p = .411) and progression-free survival (p = .563) between patients in cr1 versus cr2 versus active disease. one-year os, rfs, cir, and nrm were 48.1%, 35.7%, 16%, and 38.2%, respectively ( table 2 ). causes of death were relapse in 4 (8%) cases, graft failure in 2 (4%) patients, infection in 9 (18%) patients, multiorgan failure in 2 (4%) cases, acute gvhd in 1 case, and cardiac arrhythmia in 1 case (figures 2à4). haplohsct was initially described in the late 1950s. this was historically associated with high rates of graft rejection, severe acute gvhd, and worse outcomes when compared with msd transplants [9] . an important milestone in haplohsct was the use of t cellàreplete grafts in combination with posttransplantation high-dose cyclophosphamide [10] . in the setting of haplohsct, ptcy decreases the risk of gvhd and graft rejection by targeting and inducing cell apoptosis of alloreactive t cells rapidly proliferating early after transplant, sparing regulatory t cells and preserving nondividing hematopoietic stem and progenitor cells [10à13]. ptcy may be associated with high rates of severe acute gvhd if used alone, with 4 of 5 patients receiving single-agent ptcy succumbing to fatal, steroid-refractory gvhd in 1 single-center report [14] . therefore, post-transplant immunosuppression commonly includes additional agents such as calcineurin inhibitors, sirolimus, and/or mycophenolate mofetil [13, 14] . haplohsct using unmanipulated t cellàreplete pbsc grafts with ptcy, calcineurin inhibitors, and mycophenolate mofetil as gvhd prophylaxis have been associated with increased rates of acute and chronic gvhd and have shown commensurate outcomes with acceptable nrm when compared with other donor sources [12, 15, 16] . atg has also been extensively studied as a method to prevent graft rejection, acute gvhd, and chronic gvhd in msd and mud transplants [17à19]. however, its effectiveness in modulating severe acute and chronic gvhd is offset by a higher rate of infectious complications, including cmv and ebv reactivation [20à22] . ptcy induces selective destruction of proliferating alloanti-genàstimulated t cells. this effect is invaluable for haplohsct where there is an intense t cell proliferation due to major hla mismatch. donor memory t cells are relatively protected from ptcy and provide donor immunity in addition to immune reconstitution post-transplant [13] . reduced-intensity conditioning with fludarabine, reduceddose busulfan, and tbi (200 cgy) is a low-toxicity regimen that induces sufficient immunosuppression to permit donor hematopoietic cells to consistently engraft despite the hlahaplotype barrier. the feasibility of ric in haplohsct in adult patients with hematological malignancies is well described [23à25] . the combination of fludarabine and busulfan with ptcy may potentiate the antineoplastic activity of the conditioning regimen which may lead to resulting in further disease reduction. data in haplohsct using ric and pbsc as the stem cell source is limited. additionally, centers using pbsc in haploidentical transplants have shown rates of extensive chronic gvhd as high as 38% [16] . the combination of atg and low dose ptcy was found to increase regulatory t cell reconstitution in an animal model leading to lower rates of acute gvhd in a murine experiment [26] . our own experience with this combination in mud transplants led to its use in the haplohct setting [5] . we had also encountered a high incidence of severe acute and chronic gvhd with ric for msd and mud transplants [27] . this led to a concerted effort to design a conditioning regimen that would reduce both acute and chronic gvhd. ric with low-dose busulfan and fludarabine and atg + cyclosporineàbased gvhd prophylaxis showed outcomes comparable with mud with lower rates of cgvhd [18] . the inclusion of atg in conditioning regimens results in faster achievement of donor chimerism, especially when using alternative donors [17, 18] . however, the use of atg is also associated with delayed immune reconstitution and increased infective complications, particularly viral reactivations with cmv or ebv [19à21] . due to the higher degree of immunosuppression, hap-lohsct recipients have an increased risk of infectious complications, particularly viral infections including cmv reactivation. high incidence of cmv reactivation was noted in our study; however cmv-related mortality was very low. t cellàreplete haplohsct recipients have a lower incidence of infections than t cellàdepleted haplohsct patients do because of improved immune reconstitution; the incidence of cmv reactivation has been reported to be 60% [12] . the best approach to prevent cmv reactivation in the setting of hap-lohsct is not well established. the use of novel agents such as letermovir as prophylaxis may be potentially useful avenues of research [28] . ebv-related ptld after allogeneic hsct is associated with mortality rates as high as 50% to 90% [29, 30] . based on european data, the incidence of ptld after allogeneic hsct was 3.2%, varying from 1.2% in muds to 2.8% in mismatched related donors (including haploidentical and mismatched related donors). in the context of atg-based conditioning, the incidence of ptld has been reported to be 9.7% [31] . the risk of developing ebv-ptld is predominantly related to the degree of t cell depletion or impairment, as ebv-infected b cells are held in check by cytotoxic t cell lymphocytes. this loss of this mechanism leads to uncontrolled b cell proliferation and the development of ptld [32] . although the incidence of ebv reactivation and ptld in our patients was high, all patients responded well to rituximab monotherapy and no ptldrelated deaths occurred. the observed high rate of ebv reactivation and ptld may be attributed to t cell impairment between donor and recipient secondary to hla mismatch, the use of dual t cell depletion with ptcy and atg, and the high rate of cmv reactivation. patients were monitored closely with weekly pcr-based testing for ebv. this may have led to a higher rate of detection of ebv reactivation. ebv seromismatch has been described as a risk factor for ptld development, and the selection of an ebv-matched donor may affect rates of reactivation in a manner similar to cmv [31] . there are no clear guidelines for the monitoring and preemptive treatment of ebv reactivation or ptld in haplohsct. our study's major limitation is the short duration of followup. the focus of this report is to demonstrate the applicability of this novel conditioning regimen in a broad selection of patients. while a longer follow-up would certainly be more informative for outcomes such as chronic gvhd and longerterm complications, the lower incidence of acute gvhd grade ii to iv was encouraging. the incidence of nrm was of considerable concern and has been attributed to infective complications, particularly after viral infections. we have implemented a reduction in the atg dose in an attempt to mitigate the infection risk. finally, the proportion of older (35% over 65 years of age) patients may explain lower tolerance to infectious and other post-transplant complications. future modifications to this protocol may also include more aggressive antiviral prophylaxis and therapy. to summarize, our experience indicates that t cellàreplete haplohsct after ric with low-dose busulfan, fludarabine, tbi (200 cgy) combined with atg, ptcy, and cyclosporine is a feasible and effective transplant regimen. this approach produced consistent donor cell engraftment with low rates of acute gvhd; however, this was tempered by high rates of viral reactivation and consequently, nrm. haplohsct may be used when a suitable hlamatched donor is not available or when allogeneic hsct is needed urgently. prospective studies are needed to compare the outcomes of different conditioning regimens in haplohsct and the outcome of allogeneic hsct using alternative graft sources. financial disclosure: the authors have nothing to disclose. conflict of interest statement: there are no conflicts of interest to report. authorship statement: a.l. and q.s. collected and analyzed the data and contributed equally to the writing of this manuscript. w.l., s.t., j.l., f.m., r.k., d.k., h.m., and a.v. provided valuable input into the study design, analysis, and interpretation and reviewed the manuscript. supplementary data related to this article can be found online at doi:10.1016/j.bbmt.2018.07.008. hematopoietic stem-cell transplantation hla match likelihoods for hematopoietic stem-cell grafts in the u.s. registry on modeling human leukocyte antigen-identical sibling match probability for allogeneic hematopoietic cell transplantation: estimating the need for an unrelated donor source hla-haploidentical bone marrow transplantation with posttransplant cyclophosphamide expands the donor pool for patients with sickle cell disease reduction of severe acute graft-versus-host disease using a combination of pre transplant anti-thymocyte globulin and post-transplant cyclophosphamide in matched unrelated donor transplantation investigation of the freely available easy-to-use software 'ezr' for medical statistics consensus conference on acute gvhd grading measuring therapeutic response in chronic graft-versus-host disease: national institutes of health consensus development project on criteria for clinical trials in chronic graft-versushost disease: iv. response criteria working group report clonable t lymphocytes in t cell-depleted bone marrow transplants correlate with development of graft-v-host disease hla-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide durable engraftment of major histocompatibility complex-incompatible cells after nonmyeloablative conditioning with fludarabine, low-dose total body irradiation, and posttransplantation cyclophosphamide improved early outcomes using a t cell replete graft compared with t cell depleted haploidentical hematopoietic stem cell transplantation post-transplant high-dose cyclophosphamide for the prevention of graft-versus-host disease single-agent high-dose cyclophosphamide for graft-versus-host disease prophylaxis in human leukocyte antigenàmatched reduced-intensity peripheral blood stem cell transplantation results in an unacceptably high rate of severe acute graft-versushost disease alternative donor transplantation after reduced intensity conditioning: results of parallel phase 2 trials using partially hla-mismatched related bone marrow or unrelated double umbilical cord blood grafts haploidentical transplantation using t cell replete peripheral blood stem cells and myeloablative conditioning in patients with high-risk hematologic malignancies who lack conventional donors is well tolerated and produces excellent relapse-free survival: results of a prospective phase ii trial role of thymoglobulin in matched sibling allogeneic hematopoietic stem cell transplantation with busulfan and fludarabine conditioning in myeloid malignancies reduced-intensity conditioning with busulfan, fludarabine, and antithymocyte globulin for hematopoietic cell transplantation from unrelated or haploidentical family donors in patients with acute myeloid leukemia in remission antilymphocyte globulin for prevention of chronic graft-versus-host disease impact of donor and recipient cytomegalovirus serostatus on outcomes of antithymocyte globulin-conditioned hematopoietic cell transplantation optimal dose of rabbit thymoglobulin in conditioning regimens for unmanipulated, haploidentical, hematopoietic stem cell transplantation: long-term outcomes of a prospective randomized trial epstein-barr virus-related post-transplantation lymphoproliferative disorder after unmanipulated human leukocyte antigen haploidentical hematopoietic stem cell transplantation: incidence, risk factors, treatment, and clinical outcomes haploidentical transplant with posttransplant cyclophosphamide vs matched unrelated donor transplant for acute myeloid leukemia haploidentical transplantation for older patients with acute myeloid leukemia and myelodysplastic syndrome reduced-intensity transplantation for lymphomas using haploidentical related donors vs hlamatched unrelated donors low-dose post-transplant cyclophosphamide can mitigate gvhd and enhance the g-csf/atg induced gvhd protective activity and improve haploidentical transplant outcomes myeloablative versus reduced-intensity conditioning in patients with myeloid malignancies: a propensity score-matched analysis letermovir prophylaxis for cytomegalovirus in hematopoietic-cell transplantation strategies to prevent ebv reactivation and posttransplant lymphoproliferative disorders (ptld) after allogeneic stem cell transplantation in high-risk patients response to rituximab-based therapy and risk factor analysis in epstein barr virus-related lymphoproliferative disorder after hematopoietic stem cell transplant in children and adults: a study from the infectious diseases working party of the european group for blood and marrow transplantation risk factors for post-transplant lymphoproliferative disorder after thymoglobulin-conditioned hematopoietic cell transplantation post-transplantation lymphoproliferative disorders in adults key: cord-259194-9zllvfqb authors: cupples, sandra a. title: transplant infectious disease: implications for critical care nurses date: 2011-11-02 journal: crit care nurs clin north am doi: 10.1016/j.ccell.2011.08.001 sha: doc_id: 259194 cord_uid: 9zllvfqb infection is an important issue for critical care nurses as they care for patients throughout all phases of the transplant continuum: potential organ donors, transplant candidates, and transplant recipients. this article has reviewed salient issues relative to infections in each of these patient populations, including patients with vads, and has highlighted key points pertaining to bacterial, viral, and fungal infections. while they are on the transplant waiting list. urinary tract infections are common in kidney and pancreas transplant candidates. kidney transplant candidates are also at risk for infections in the native kidneys and occult abscesses. liver transplant candidates may have intra-abdominal infections or aspiration pneumonia. pneumonia is also common in the heart and lung candidate populations. hospitalized candidates are at risk for catheter-or device-related infections, such as those associated with dialysis access devices or ventricular assist devices (vads). as of august 2011, there were more than 3100 candidates on the heart transplant waiting list in the united states. 2 to date, only 949 heart transplant procedures have been performed in the united states in 2011. thus the demand for donor hearts far exceeds the supply. 3 vads were developed to augment circulation in patients with end-stage heart disease. these devices have been approved by the food and drug administration for three purposes: to bridge patients to heart transplantation, to bridge patients to recovery of their native myocardial function, and to provide permanent support for patients who are not deemed to be suitable heart transplant candidates ("life-time" or "destination" therapy). 4 vads can support the right or left ventricle or both. they stabilize the patient's condition by increasing cardiac output, improving perfusion to vital organs, and restoring mobility. 5, 6 these devices are typically implanted through a median sternotomy incision and placed in a pre-peritoneal or intra-abdominal pocket. the major components of a vad are inflow and outflow cannulae, unidirectional valves, a polyurethane chamber (for pulsatile devices), and a pump or rotor. the device is connected to an external power source through a driveline that exits through the abdominal wall (fig. 1) . 7, 8 vads contain biomaterials and, unfortunately, none of these materials are biologically inert. 9 therefore, events that occur at the host-implant interface can trigger aberrant immune activation. when the patient's blood comes in contact with the foreign vad surface, t cells can become activated and initiate a defective proliferative response and subsequent activation-induced cell death. as a result, the patient's immune system is impaired and the patient may be more susceptible to infection. 4, 5, 7, 9 infection is a common complication of vad therapy. 6 vad-related infections may delay or prevent transplantation altogether and they are a major cause of morbidity and mortality in lifetime therapy patients. 10 -12 the most recent international society for heart and lung/mechanical circulatory support device registry data indicate that infection occurred in 32.5% of the 655 vad patients enrolled in this database and that patients with vad infections had a 7.9% mortality rate. 13 device-related infection rates reported in the literature have ranged between 13% and 80%. 5 potential infection sites include the surgical site or any component of the vad (driveline, device pocket, or pump membrane). driveline infections are the most common; however, more than half of all infections involve several device sites simultaneously. 7 vad infections may remain localized or become systemic. if the infection spreads to multiple sites, serious complications such as bloodstream infections, bacteremia, sepsis, and endocarditis can ensue. 7 device-, patient-, and mechanical-related factors can contribute to vad infections. device-related factors include the exposure of percutaneous drivelines to pathogens and the vad cavities and pockets that can harbor pathogens. these microorganisms can cause blood flow through the pump to become turbulent; this in turn enables the pathogens to adhere to the surface of the device. 4 patient-related risk factors for infection include older age, poor nutritional status, indwelling catheters, prolonged intubation, postoperative bleeding, blood transfusions, multiorgan dysfunction, comorbidities such as diabetes mellitus and obesity, prolonged hospitalization before vad implantation, and surgical reexploration. 4, 6, 7 mechanical trauma to the driveline exit site is frequently associated with late-onset (ͼ30 days after implantation) infections. driveline trauma occurs when, for example, the controller or battery pack is dropped or when the driveline is snared on an object. these accidents result in shearing or torsion injuries that can lead to infection. 12 device-related infections can occur at any time; however, the majority develop between 2 weeks and 2 months of implantation. 7 gram-positive pathogens, particularly staphylococcus species, cause most infections. 14 these organisms are able to form a protective biofilm that blunts the host immune response and enables them to attach to and grow on inanimate surfaces. 8 fungal and gram-negative bacilli, such as pseudomonas aeruginosa and the enterobacter and klebsiella species, are other causative agents; these particular pathogens are associated with poorer outcomes. 4, 7 the administration of broad-spectrum antibiotics often leads to the development of fungal infections. 7 the clinical manifestations of vad-related infections are varied. presentation may be subtle or acute. if a device-related infection is suspected, the patient must have a thorough evaluation that includes a comprehensive physical examination and extensive work-up including blood cultures with gram stains. if possible, cultures should be obtained before initiation of antimicrobial therapy. 7 other sources of infection, such as pneumonia, urinary tract or catheter-related infections, must be investigated appropriately. additional diagnostic tests are site specific. for example, ultrasound is used to evaluate suspected pump pocket infections; transesophageal echocardiograms are useful in the setting of vad-related endocarditis. table 1 lists the typical signs and symptoms of devicerelated infections and potential treatment options. the evidence regarding the impact of device-related infection and posttransplant outcomes is mixed. some studies have indicated that these infections do not reduce 1-year 15 or overall 7, 16 survival. other studies have found that serious device-related infections can persist into the posttransplant period 7, 17 and are associated with decreased early 11 and long-term 17 posttransplant survival. although assist devices are often associated with infection, the benefits of this life-saving therapy are thought to outweigh the infection risk. 7 the major clinical implications for pre-and postoperative nursing care are listed in table 2 . infections can be transmitted via the allograft itself. 18 a donor-derived disease transmission is defined as "any disease present in the organ donor that is or has the potential to be transmitted to at least one of the recipients." 19(p234) donor-derived infectious diseases are rare. unexpected transmissions, that is, those that were either unrecognized in the donor or for which the donor was not screened, occur in fewer than 1% of all solid organ transplantation procedures. 19 although rare, these infections cause significant morbidity and mortality. factors that promote infection in potential organ donors include the use of medical devices and the treatment of patients in certain units that have high rates of bacterial contamination. 20 it is important to note that treatment of donor infections itself can further increase the potential donor's risk of iatrogenic infection, for example, via the insertion of intravascular catheters for antimicrobial therapy, the administration of immunomodulating medications such as corticosteroids, and prolonged hospitalization. 21 for a number of reasons, infections in potential organ donors may be difficult to diagnose: • the donor may not have the clinical manifestations of infection due to insufficient numbers or virulence of pathogens. • hemorrhage or aggressive fluid resuscitation may dilute both organisms and serologic infection markers such that they are undetectable by conventional laboratory tests. • the donor may not mount a fever response because brain death causes loss of temperature control and poikilothermia (a phenomenon whereby body temperature decreases to that of the environment). • the donor's white blood cell count may be already elevated due to trauma, tissue inflammation, or medications such as corticosteroids. 21 as a consequence, the diagnosis of infection may rely on culture and urinalysis reports, polymerase chain reaction (pcr) and nucleic acid testing results, characteristics of sputum, and changes in chest radiographs and computed tomography (ct) scans. 21 potential organ donors undergo a rigorous infectious disease evaluation. organ procurement and transplantation network (optn) policies mandate that potential donors must be screened for the following pathogens: human immunodeficiency 22 potential heart donors are screened for toxoplasmosis. many donors are also screened for nosocomial infections such as methicillinresistant staphylcoccus aureus or vancomycin-resistant enterocci. because infection can be transmitted via transfusions, serologic testing is typically performed both before and after a potential donor receives blood products. in addition, family members are questioned about the potential donor's infection risk, including prior infection exposure, history, and immunizations; travel to endemic areas; and risky behaviors such as intravenous drug abuse. table 3 displays donor organ acceptance and exclusion criteria based on the results of infectious disease screening. the acceptance of organs from donors with known infections with or exposure to hiv, hepatitis, or other viruses remains controversial. 21 given that the number of transplant candidates on the waiting list far exceeds the number of available organs, strategies to expand the donor pool include accepting donors with certain infections, higher-risk serological profiles, and social histories suggestive of prior exposure to bloodborne infections as well as donors who may be more at risk for transmitting infections (eg, older donors and donors with long icu stays). 20 informed consent effective treatment of bacterial infections in potential organ donors can result in successful transplantation. 21 box 1 displays important principles of antibiotic selection and administration for potential organ donors. the organ procurement organization's (opo's) coordinator has major responsibilities regarding the prevention and treatment of infections and reporting known infections to transplant centers that could potentially receive organs from infected donors. infections that must be reported to the transplant center are listed in box 2. moreover, all antimicrobial agents that are given to the potential donor must be documented and reported to each transplant center that receives an organ from that donor. 21 when a transplant center is informed that one of its organ recipients is confirmed positive for or has died from a potential donor-derived transmissible disease, that center must notify, within one working day, the opo that procured that organ. the opo must then notify the optn. these reports are forwarded to unos and uploaded to the disease transmission advisory committee's (dtac's) secure website. 1. select a bactericidal antibiotic over a bacteriostatic antibiotic. 2. use medication that will most directly target the identified bacteria to prevent the removal of harmless bacteria, the promotion of selective overgrowth of fungi, resistant organisms, or abnormal bacterial strains (eg, clostridium difficile), and the development of gene mutations and highly resistant organisms. 3. substitute directed antibiotic for broad-spectrum agent once sensitivities are available. 9. consider antibiotics specifically effective against anaerobic bacteria in the setting of facial injuries, pulmonary aspiration, or contaminated wounds from an injury scene. 10. administer antibiotics intravenously to maximize bioavailability. 11. adjust antibiotic doses in the setting of renal failure, hepatic failure, and older donor age. dtac data indicate that, between 2005 and 2007, there were 80 donors with reported possible donor-derived infectious disease transmission, 30 recipients with confirmed (proven, probable, or possible) donor-derived infections, and 14 recipient deaths attributed to donor-derived infections. these deaths were due to hepatitis c, tuberculosis, hiv, chagas disease, bacteremias, candidemia, strongyloides, and lymphocytic choriomeningitis virus. 18 there are three major factors that determine a transplant recipient's risk of infection. these include the patient's epidemiological exposure, either in the box 2 infections that must be reported to the transplant center 35 known conditions that may be transmitted by the donor organ must be communicated to the transplant center. these may include, but are not limited to, the following: hospital or in the community; the patient's current antimicrobial regimen, if any; and the patient's net state of immunosuppression, which is defined as "the combined effect of all of the factors that determine the patient's susceptibility to infection." 24(p138), 25 the net state of immunosuppression includes the patient's current immunosuppressive regimen (number and strength of antirejection agents), as well as any of the following concurrent factors: infection with an immunomodulating virus (eg, cmv or ebv); metabolic or autoimmune disorders (eg, diabetes mellitus); neutropenia or lymphopenia; disruption of mucocutaneous barriers; and surgical sequelae (eg, fluid collections). 25 approximately 80% of all transplant recipients have at least one significant infection during the first posttransplant year. 26 the three major groups of posttransplant pathogens are bacteria, viruses, and fungi (fig. 2) . bacterial infections are the most common, 26 followed by viral and fungal infections. bacterial infections frequently occur at the transplant site. bacterial pneumonias are common among all types of solid organ transplant recipients. nosocomial pathogens of particular concern include clostridium difficile, vancomycin-resistant enterococcus, methicillin-resistant staphylococcus aureus, and extended-spectrum ␤-lactamase gram-negative bacilli. common organ-specific bacterial infections and associated risk factors are listed in table 4 . most posttransplant viral infections are caused by two groups of pathogens: the herpes viruses (cmv, ebv, hsv 1 and 2, and varicella zoster) and the hepatitis viruses. viral infections are particularly deleterious because they have both direct and indirect effects. the direct effect is the clinical syndrome caused by the virus itself, such as cmv pneumonia or hepatitis. indirect effects include potential injury to the allograft, rejection, oncogenesis, and the virus's ability to alter the net state of immunosuppression, thereby increasing the patient's susceptibility to other opportunistic infections. the herpes viruses are characterized by latency. this means that once the virus is present, the patient will harbor the viral genome for life. immunosuppression, particularly augmented immunotherapy in the setting of rejection, can trigger replication of latent herpes viruses. 24 cytomegalovirus is the most important pathogen that affects transplant recipients. there is a bidirectional relationship between cmv and rejection. cmv can trigger rejection and the inflammatory effects of rejection and rejection therapy can increase cmv viral replication. the allograft is more likely to be affected by a cmv infection than a native organ. thus, liver transplant recipients with cmv infections are prone to develop vanishing bile duct syndrome, heart transplants recipients are at risk for coronary artery vasculopathy, lung transplant recipients are at risk for bronchiolitis obliterans, and so forth. the most common types of cmv disease are hepatitis, pneumonitis, and gastroenteritis. with regard to cmv serostatus, the risk of developing a posttransplant cmv infection is highest in cmv-seronegative recipients who receive an allograft from a cmv-seropositive donor and lowest in cmv-seronegative recipients who receive an allograft from a cmv-seronegative donor. recipients who receive potent antirejection therapy such as antithymocyte globulin are also at increased risk for developing a cmv infection. a concurrent critical illness can lead to the reactivation of a latent cmv infection; this is thought to be associated with proinflammatory cytokines and subsequent downregulation of the immune system. agents used to prevent or treat cmv infection include ganciclovir, valganciclovir, acyclovir, and cmv immune globulin. foscarnet is often used to treat ganciclovirresistant organisms. because cmv can be transmitted through blood transfusions, cmv-seronegative transplant candidates and recipients should receive cmv-negative, leukocyte-poor, or filtered blood products. 1, 24 given that most adults are ebv-seropositive, most posttransplant ebv infections in adults are reactivated from latent pretransplant infections. however, ebv-seronegative recipients can acquire an ebv infection through blood transfusions or community exposure. the incidence of posttransplant ebv infections is highest in multiorgan and intestinal transplant recipients followed, in decreasing order, by kidney-pancreas, lung, heart, liver, and kidney recipients. intravenous ganciclovir has been used as preemptive therapy for patients at high risk for ebv infections, for example, patients receiving antilymphocyte antibody therapy for rejection. the clinical sequelae of ebv infection range from a relatively mild mononucleosis-like syndrome to posttransplant lymphoproliferative disease (ptld). treatment options for mononucleosis include acyclovir. ptld is a set of syndromes that ranges from a benign, self-limiting polyclonal proliferation of b cells to an aggressive, malignant, monoclonal lymphoma. risk factors for ptld include pretransplant ebv-negative serostatus, primary ebv infection, high ebv viral load, cmv serostatus mismatch (recipient is cmv negative and donor is cmv positive), cmv disease, potent rejection treatment, and type of allograft. the incidence of ptld is highest in intestinal transplant recipients. treatment options for ptld range from antiviral agents (acyclovir, ganciclovir) and decreased immunosuppression for the benign polyclonal form to chemotherapy, radiation, resection, and decreased immunosuppression for malignant monoclonal lymphoma. although invasive fungal infections have the lowest incidence of all infections, they are associated with the highest morbidity and mortality rates. 27 risk factors for fungal infections include the use of high-dose corticosteroids and broad-spectrum antibiotics, rejection that requires increased immunosuppression, allograft dysfunction, and a simultaneous infection with an immunomodulating virus such as cmv. 24 two genera, aspergillus and candida, cause the vast majority of posttransplant fungal infections. together, these two pathogens account for more than 80% of invasive fungal infections. these infections typically present during the first month posttransplant, 27 but they can occur at any time. the most common fungal infection that involves the respiratory tract is invasive aspergillosis, which may affect approximately 30% of solid organ transplant recipients. 28 other portals of entry include the gastrointestinal tract and the skin. the risk of disseminated candidiasis is highest in neutropenic patients with central venous catheters who have received broadspectrum antibiotics and who have had prolonged icu stays. 29 liver transplant recipients are at highest risk for invasive candidiasis, followed, in decreasing order, by pancreas, lung, heart-lung, kidney, and heart transplant recipients. 29 pediatric transplant recipients are often at higher risk for posttransplant infections for a number of reasons, including: • lack of immunity to common pathogens such as cmv and ebv • incomplete immunizations • increased technical difficulty and prolonged transplant operative time due to pretransplant palliative surgeries • inability to close the abdomen or chest due to placement of a large allograft into a small child • social behavior of children in densely populated day care and school settings. 30 acute mediastinitis can develop after the implantation of mechanical circulatory assist devices or after heart, lung, and heart-lung transplantation. the risk of posttransplant mediastinitis is higher if the patient had a mechanical circulatory assist device or a total artificial heart as a bridge to transplantation. there are preoperative, intraoperative, and postoperative risk factors for mediastinitis. examples of preoperative risk factors include diabetes mellitus, prior sternotomy, renal failure requiring dialysis, prolonged hospitalization before the transplant surgery, and obesity. the risk of developing mediastinitis is more than double in patients with a body mass index greater than 30. intraoperative risk factors include blood transfusions and prolonged cardiopulmonary bypass, aortic cross-clamp, and operative times. examples of postoperative risk factors include surgical reexploration, prolonged icu stay, prolonged mechanical ventilation (ͼ24 -48 hours), having a tracheostomy, cardiopulmonary resuscitation, poor perioperative and postoperative glucose control, and low posttransplant cardiac output. 31 the major etiologic pathogens associated with mediastinitis include, in decreasing order, gram-positive cocci (staphylococcus aureus, staphylococcus epidermidis, enterococcus spp., streptococcus spp.), gram-negative bacilli (escherichia coli, enterobacter spp., klebsiella spp., proteus spp., other enterobacteriaceae, and pseudomonas spp.), and fungi (candida albicans). 31 the initial clinical manifestations of mediastinitis may be subtle: mild chest pain, and edema or erythema along the sternal incision. 24 the most common presenting symptom is fever; it may be associated with localized infection, erythema, cellulitis, purulent drainage, pleuritic-like pain, and sternal instability. diagnostic studies include ct scans, cultures, and laboratory tests. laboratory findings include elevations in the white blood cell count, c reactive protein, and procalcitonin. the latter test is particularly useful in distinguishing between rejection and infection. once mediastinitis is diagnosed, treatment should be initiated promptly. therapeutic options include surgical drainage/débridement, wound irrigation, tailored parenteral antimicrobial agents, and nutritional support. 31 in transplant recipients, central nervous system (cns) infections are among the most deleterious because they can be difficult to diagnose and treat. diagnosis is often challenging because presenting symptoms, such as mental status changes, seizures, focal neurologic deficits, and headache, may be blunted by immunosuppressive therapy. moreover, the neurotoxic effects of antibiotics, antiviral agents, and immunosuppressants themselves may make diagnosis even more complicated. 32, 33 the first step in diagnosing a suspected cns infection is a neuroimaging study to establish the presence, location, potential etiology, and characteristics of any lesion(s). magnetic resonance imaging studies of the brain or spinal cord or both are typically preferred to ct scans. neuroimaging studies are useful in determining if the infection is focal or nonfocal and if it involves the meninges. other diagnostic tests include cerebrospinal fluid analyses, electroencephalograms, viral polymerase chain reaction tests, cultures, and serologic tests. brain biopsies are rarely done, except in the setting of posttransplant lymphoproliferative disease and brain abscesses. the posttransplant interval, patient-specific risk factors, and the timing and evolution of clinical manifestations also help to inform the diagnosis. 32 cns infections may be caused by fungi, viruses, or bacteria. fungal infections carry the highest mortality rate-90% or higher-of all pathogens. 32 most brain abscesses are associated with aspergillus. these abscesses tend to occur early in the posttransplant period, particularly in recipients who have multiple risk factors for infection such as surgical reexploration, dependence on mechanical ventilation or dialysis, or retransplantation. 33 unlike meningitis in immunocompetent patients, posttransplant meningitis is typically caused by fungi. in this setting, the patient often develops a systemic infection that subsequently spreads to the cns. viral cns infections may be associated with the reactivation of a latent virus, such as jc virus-induced progressive multifocal leukoencephalopathy, or they may be caused by a new exposure to a pathogen such as west nile virus. other pathogens commonly associated with posttransplant encephalitis include the herpes simplex virus, varicella zoster virus, ebv, and cmv. bacterial cns infections are more frequently caused by listeria and nocardia rather than more common bacterial pathogens. 32 due to the severity of cns infections in transplant recipients, an infectious disease consult, coupled with prompt diagnosis and treatment, is imperative. empiric, broad-spectrum antimicrobial agents are typically administered until the causative organism is identified. 32 immunosuppressive agents blunt the inflammatory response to infection; however, in most cases, transplant recipients with infections will have an increase in temperature. some infections, however, tend to occur in the absence of fever. these include pneumocystis pneumonia, focal fungal lung infections, and cryptococcal meningitis. 29 patients with a persistent fever greater than 38°c or acute pulmonary infiltrates or both are typically hospitalized for an infection workup. fevers of unknown origin are most commonly associated with cmv or ebv viral syndromes. it is important to note that fevers in transplant recipients may also be caused by drug reactions (particularly antilymphocyte therapy), pulmonary emboli, deep vein thrombosis, and rejection. rejection-induced fever typically occurs in lung transplant recipients. it occurs less commonly in kidney and liver transplant recipients and rarely in heart recipients. 24, 29 given that other human beings are the most frequent source of infection in the patient's environment, it is essential that nurses prevent the nosocomial transmission of respiratory viruses and the transmission of organisms through contaminated hands or inanimate objects. 29 in addition, it is important for critical care nurses to: • follow standard precautions • use aseptic techniques with vascular and urinary catheters • ensure that ventilator circuits, catheters, and dressings are changed per protocol • inspect all percutaneous catheter sites for signs of infection • maintain closed systems for urinary and suction catheters • keep the head of the bed elevated to decrease the risk of aspiration. • restrict access to patients by visitors and staff with colds or other contagious illnesses • avoid transporting transplant recipients through areas of hospital construction • promptly recognize and report the clinical manifestations of infections • obtain and report diagnostic test results in a timely manner • administer and document antimicrobial agents in a timely manner • assess and report the vaccination status of transplant candidates and recipients • know the cmv, ebv, and other pertinent serostatuses of the donor and recipient. 21, 29 infection is an important issue for critical care nurses as they care for patients throughout all phases of the transplant continuum: potential organ donors, transplant candidates, and transplant recipients. this article has reviewed salient issues relative to infections in each of these patient populations, including patients with vads, and has highlighted key points pertaining to bacterial, viral, and fungal infections. approach to the immunocompromised host with infection in the intensive care unit organ procurement and transplantation network organ procurement and transplantation network. transplants by donor type complications in patients with ventricular assist devices nonvalvular cardiovascular device-related infections ventricular assist devices in the adult ventricular assist device-related infections device-related infections: a review immunobiologic consequences of assist devices for the randomized evaluation of mechanical assistance for the treatment of congestive heart failure (rematch) study group. long-term mechanical left ventricular assistance for end-stage heart failure left ventricular assist device-related infection: treatment and outcome late-onset driveline infections: the achilles' heel of prolonged left ventricular assist device support mechanical circulatory support device database of the international society for heart and lung transplantation: third annual report -2005 infection in ventricular assist devices: prevention and treatment effect of left ventricular assist device infection on post-transplant outcomes infections during left ventricular assist device support do not affect posttransplant outcomes lvad bloodstream infections: therapeutic rationale for transplantation after lvad infection donor-derived disease transmission events in the united states: data reviewed by the optn/unos disease transmission advisory committee the epidemiology and prevention of donor-derived infections donor infection and transmission to recipient of a solid allograft bacterial infection during adult donor care available at: http:// optn.transplant.hrsa.gov/policiesandbylaws2 the ast infection disease community of practice. screening of donor and recipient prior to organ transplantation transplant complications: infectious diseases infection in solid organ transplant recipients bacterial infections in the early period after liver transplantation: etiological agents and their susceptibility clinical aspects of invasive candidiasis in solid organ transplant recipients new therapies for fungal pneumonia risk factors and approaches to infections in transplant recipients intestine transplantation mandell, douglas and bennett's principles and practice of infectious diseases neurologic manifestations of transplant complications infections of the central nervous system in transplant recipients drive-line exit site infection in a patient with axial flow pump support: successful management using vacuum-assisted therapy lung and heart-lung transplantation liver transplantation liver transplantation pancreas and kidney-pancreas transplantation pancreas and simultaneous pancreas-kidney transplantation key: cord-275903-sfrpfy5g authors: yu, fenggang; tan, wei jian; lu, yanan; macary, paul a.; loh, kwok seng title: the other side of the coin: leveraging epstein–barr virus in research and therapy date: 2016-07-21 journal: oral oncol doi: 10.1016/j.oraloncology.2016.07.010 sha: doc_id: 275903 cord_uid: sfrpfy5g epstein-barr virus is (ebv) a ubiquitous virus prevalent in 90% of the human population. transmitted through infected saliva, ebv is the causative agent of infectious mononucleosis (im) and is further implicated in malignancies of lymphoid and epithelial origins. in the past few decades, research efforts primarily focused on dissecting the mechanism of ebv-induced oncogenesis. here, we present an alternate facet of the oncovirus ebv, on its applications in research and therapy. finally, discussions on the prospective utilization of ebv in nasopharyngeal carcinoma (npc) diagnosis and therapy will also be presented. epstein-barr virus (ebv) is the first human tumor virus discovered 52 years ago based on the electron microscopy observation of virus particles from burkitt's lymphoma (bl) biopsies [1] . it belongs to human herpesvirus family and infects more than 90% of the world population [2] . ebv exhibits dual tropism, infecting both b lymphocytes and epithelial cells. while infection with the virus during childhood is usually asymptomatic, ebv infection in an adolescent can manifest as infectious mononucleosis (im) [3, 4] . in immunocompetent individuals, the replication of ebv-infected b cells is kept in check by t cell immunity, driving the virus into dormancy. nonetheless, the virus is capable of persisting in the human population by restricting expression of viral products to ebv-encoded small rnas (ebers) and viral micrornas (mirnas), establishing latency 0 profile in the memory b cell pool [5] . as an opportunistic human pathogen, ebv was proposed to be involved in b cell malignancies observed in post-transplant lymphoma and aids-related lymphoma when the delicate balance between the host immunity and the ebv-infected b cells is perturbed [6] [7] [8] . unlike ebv-associated lymphoid malignancies, the link between ebv and epithelial malignancies is less clear. although the virus transforms and immortalizes b cell upon infection, the virus does not readily infect epithelial cells, putting forth the contribution of host genetics and environmental factors in ebv-associated npc. therefore, it is believed that ebv establishes latency aberrantly in epithelial cells that have already undergone pre-malignant genetic changes [9] as an essential initiation step in the development of npc. in this article, we will discuss the utility of targeting ebv gene products, the viral episome, and whole virus for research, screening, diagnostic, and future treatment of npc ( fig. 1 ). ebv gene product-zebra ebv zebra protein (product of gene bzlf1) is a basic leucine zipper transcriptional activator required for latent to lytic reactivation [10, 11] . apart from its intrinsic function in activating ebv lytic cycle, zebra was shown recently to cross the cell membrane and accumulate in the nucleus of lymphocytes. specifically, a minimal domain (md11) consisting of 43 amino acids peptide within the zebra protein was found to be efficient in delivering high molecular weight proteins across the lipid bilayer of the cell membrane via direct, non-endocytosis-dependent translocation [12] . as demonstrated by marchione et al., the utilization of md11 permitted high-efficiency (70-100%) delivery of fully biological active cargo proteins within two hours with almost no toxicity. indeed, using md11, apoptosis of melanoma and colorectal tumor cells can be observed upon the successful delivery of eif3f (f subunit of the eukaryotic initiation factor 3) across the cell membrane [13] . taken together, such ebv peptide-based delivery system presented here could represent a potentially powerful tool in cancer treatment, by facilitating the delivery of coupled therapeutics into cancer cells. ebv episomal vectors were first described by yates et al. in 1985 [14] . it is comprised of a latent origin of plasmid replication (orip) and its trans-activating protein ebv nuclear antigen-1 (ebna-1). structurally, orip consists of the family of repeats (fr) element and the dyad symmetry (ds) element, both containing multiple consensus sequences for ebna-1 binding. while ds serves as the site for the initiation of replication, fr functions as an anchor point for ebna-1 binding to ensure equal partitioning of viral episome into daughter cells. as a result, episomal vectors replicate once per cell cycle in synchrony with the host chromosomes as extrachromosomal entities [15] . given the non-integrative characteristic of the ebv episome and its ability to accommodate large transgene insertion, the use of ebv episomal vector had been extended to genetic studies, protein production, gene therapy, and ipscs generation. prior to the establishment of next-generation sequencing, the study of the human genome was technically challenging as the human genome is inherently large. to overcome this problem, the ebv episome was utilized as the shuttle vector to house large fragments of human genome sequences with size ranging from 60 to 330 kb [16] . the high insert capacity of ebv episomes allows the delivery of intact genomic dna loci to achieve physiological levels of transgene expression. these constructs known as human artificial episomal chromosomes permit the preservation of the human sequences from deletion, recombination, rearrangement, and facilitated the transfer and recovery between expression systems of different organism. the use of ebv episomes had greatly expedited the process of physical mapping and functional identification of human genes. in the production of recombinant protein, a stable cell line capable of chromosomal expression of the transgene is ideal but rare [17] . while the use of viral vectors such as retrovirus, adenovirus, and baculovirus can ensure stable expression, these viruses have to be modified to prevent uncontrolled replication in case of accidental release. apart from the laborious and technical challenges in generating these recombinant viruses, random integration of the transgene into the host chromosome with the use of these recombinant viruses can confound transgene expression and impact recombinant protein production. in this regard, the use of ebv episome can circumvent the erratic transgene expression and recombinant protein production as the result of random integration. episomal vectors persist in multiple copies per cell, resulting in amplification of transgene and higher protein expression in a relatively short period of time. indeed, rapid and high transgene expression had been achieved with the use of ebv episomes in the absence of drug selection [18] [19] [20] . coherent with the excellent capacity in accommodating large transgene, the expression of 185 kb human b-globing transgene had been reported with the stable and sustainable expression for 3 months [20] . even though high transgene expression can also be achieved with the use of plasmid-based vectors with constitutive promoters such as sv40, cmv, and ef1 promoters, sustainability of the plasmid is always a concern as plasmid often gets diluted upon cell division. leveraging on the binding interaction between ebna-1 protein and ebv episome, further improvements to achieve fast and high yield recombinant protein had been attempted. the enhancing transgene expression in ebv episomal vector is probably attributed to the binding of ebna-1 to orip to activate transcription of the transgene, and the orip enabling nuclear import of transgene [21] . with the establishment of the mammalian hek293-ebna-1 cell line (hek293e), where parental hek293 cells stably express ebna-1, the episomal expression of the transgene resulted in a threefold increase of protein yield [22] [23] [24] . as reported by backliwal et al., the utilization of hek293e combined with high-efficient polyethyleneimine-based transfection resulted in a remarkable efficiency of exceeding 1 g/l of recombinant protein [25] . notebly, production of recombinant protein at the scale of 100 l with 293e adapted suspension culture had been reported by philippe et al. [26, 27] appealing to industry production for biotechnology. gene therapy is a therapeutic approach to complement a deficient or to correct a defective gene via the introduction of a corrective cdna or dna. systems currently available utilizing viral vectors derived from retroviruses, lentiviruses, and adenoviruses pose safety concerns due to random insertion and accompanying cytopathic effects. therefore the possibility of activating an oncogene should not be disregarded [28] . in one of most the apprehensive incident of gene therapy, the use of retrovirus to correct severe combined immune deficiency had led to the development of leukemia-like condition [29] . with its non-integrating property, ebv episomal vector provides a safer alternative to the use of viral vector. indeed, the utility of ebv episome had been extended to the treatment of malignant and congenital disorders, accentuating its potential clinical application [30, 31] . however, efficient gene delivery remains a problem. the transfection procedures for ebv episomal vectors usually include electroporation and intramuscular injection [31] . the transfection efficiency can be improved when used in combination with cationic liposomes and polyamidodamine (pamam) dendrimer [32] . another approach to tackle the issue is to develop hybrid vectors, in which retroviral, adenoviral or herpes viral elements are utilized to deliver ebv-based replicon owing to high robust infectivity. regenerative medicine is a branch of applied research which involves the replacement of defective cells, tissues or organs to reinstate its functionality. with the advent of induced ipscs technology in 2006, embryonic stem-like cells can be directly reprogrammed from somatic cells by forcing the expression of four transcription factors (oct-4, sox-2, klf-4, and c-myc) [33] . in the early days of ipscs research, the yamanaka factors were efficiently delivered by engineered retroviruses or lentiviruses. akin to the drawback as seen with the use of viral vectors in gene therapy, random viral integration warrant safety consideration, making virallyderived ipscs unsafe for clinical application. as such, the use of integration-free ebv episome for ipscs reprogramming is preferred and gaining popularity [34] [35] [36] . actually, ipscs used for the first historic human trial for macular degeneration were reprogrammed using non-integrative ebv episomal vectors [37]. the oncogenic potential of ebv is best illustrated by its ability to infect b cells in vitro and transform them into b lymphoblastoid cell line (blcl) capable of indefinite proliferation. given the ability of ebv to immortalize b cells, it became apparent that the generation of these b cell lines would preserve the genetic information of an individual. as blcls can be cultured continuously, they provide an unlimited source of biological materials for genetic, epidemiologic and pedigree studies [38] . in addition, these cells can be thawed and frozen at users' convenience, enabling experimental observations to be made with more reliability, and at the same time minimizing intra-sample variability. apart from the use of ebv episome for the ipscs generation as mentioned earlier, the other aspect of ebv involvement in ipscs generation is the use of ebv-blcl as starting cell type. as reported by rajesh et al., ipscs can be successfully reprogrammed from blcl with the additional of episomal vectors containing oct3/4, sox2, nanog and sv40-t. intriguingly, with merely 0.5 ml of peripheral blood, blcl can be generated and reprogrammed into ipscs capable of differentiating into hematopoietic, neural, cardiac, and hepatocyte-like lineages. more importantly, ebv genes were not detectable at the transcriptional and protein levels in the resultant blcl-ipscs after 25 passages [36] . using this methodology, virusfree ipscs can be obtained, making an appeal to future clinical applications. the inherent plasticity of b cells, their receptivity to orip/ebna-1 plasmids, ease of generating blcls, and availability of banked blcl collections make ebv-blcl the ideal sources of somatic cells for ipsc generation. post-transplant lymphoproliferative disorder (ptld) is a condition whereby the immunosuppression of graft recipient resulted in the inability of t cells to control the proliferation of the ebvinfected b cells. as blcl expresses latency iii profile, they are the in vitro counterparts of the ebv-infected b cells in ptld patients. [39] . specifically, ebv-blcl is injected into immunodeficient mice to simulate ptld patient with b cell lymphoproliferation, and such mouse xenograft is classically used as a model for the assessment of potential therapeutics. the other utility of ebv-blcl in the treatment of ebv-associated malignancies entails its use in stimulating ebv-specific cytotoxic t lymphocytes (ctls). in this treatment modality, allogeneic ebv-specific ctls are harvested from donors and incubated with irradiated blcl in vitro to stimulate the expansion of ebv-specific ctls. these ebv-specific ctls are cultured and expanded in vitro prior to their administration into ptld and npc patients as a form of adoptive cell transfer therapy [40] [41] [42] . with the advent of using adoptive ebv-specific ctl therapy to treat ptld, blcl is also used to stimulate the proliferation of these ebv-specific ctls prior to injection into the patient [43] . taken together, the use of blcl is relevant for the modeling and treatment of ptld. antibody is the second largest class of drugs following vaccine. the use of ebv-blcls had also been extended to the sphere of antibody discovery and engineering. as therapeutic antibodies entering the clinical trials mostly are either from phage display or humanized in mice [44] , there is a risk of immunogenicity. therefore, ideal monoclonal antibodies (mabs) for clinical applications should be the natural antibodies produced in the human body as a result of the in vivo immune response. in this regards, a fully human-derived antibody can be obtained from the screening of individuals ('responder') who are naturally exposed and making specific humoral responses to the disease, with subsequent selection of the antigen-specific b cells followed by immortalization with ebv. using this approach, many fully human mabs have been isolated thus far targeting dengue virus, sars coronavirus, h5n1 influenza, hcmv and hiv-1 and plasmodia [45] [46] [47] [48] [49] [50] . an innovative fully human antibody platform is involved with the screening of individuals who naturally exposed from endemic regions for antigen-specific reactivity, targeted selection of antigen-specific b cells (e.g., by flow cytometric sorting) followed by subsequent ebv transformation, rescue of the antibody v region sequences and recombinant protein production in cho cells or plant cells. the key technology involving is to take the advantage of ebv transforming ability to immortalize immune antibodysecreting human b-lymphocytes. the poor ebv infectivity and cloning efficiency used to be a bottleneck, but now can be improved by combining cpg activation of toll-like receptor-9 (tlr9) and irradiated allogeneic mononuclear cells as feeder cells [51] . histopathological examination of nasopharyngeal biopsies and fine needle aspiration (fnac) of neck lumps are the gold standard for npc diagnosis. the detection of ebv dna (by pcr) and rna (eber-ish) can help when histopathological examination fails in clinically doubtful cases such as occult primaries, submucosal disease and fnac due to low accuracy. radiotherapy has been proved to be the most effective modality of treatment if the disease is diagnosed at an early stage, with a 5-year survival probability up to 90%. unfortunately, 75-90% of patients with npc already have developed local or regional spread upon diagnosis [52] . therefore, screening for early disease in asymptomatic individuals potentially may improve npc treatment outcomes. in endemic regions, the presence of ebv will highly suggest npc. since the high association with ebv infection, antibodies specific for ebv capsid antigen (iga/vca), early antigen (iga/ea) and plasma dna have been extensively studied for the screening of npc. data from both endemic and non-endemic regions demonstrate plasma dna can be used for screening, diagnosis, staging, follow-up and prognostication [53] [54] [55] [56] . chan and wong [53] recently studied 1318 healthy volunteers and demonstrated that plasma ebv dna analysis is useful for early detection of npc before it is clinically evident. shao et al. [57] proved that plasma ebv dna detection is a more sensitive and specific marker than the serum iga/vca titer for the diagnosis and monitoring of patients with npc. besides plasma antibody and dna, a circulating ebv mir-bart7 and mir-bart13 [58] were shown to serve as potentially new serological biomarkers for diagnosis and prediction of treatment efficacy of npc. in a very recent study, zheng et al. demonstrated ebvencoded mir-bart1-5p detection via a less invasive nasopharyngeal brush sampling can diagnose early-stage npc with 93.5% sensitivity and 100% specificity [59] . however, all these mirna detections need large patient cohorts across multiple centers for confirmation. successful strategies for targeting tumor cells depend on clear differences between tumor cells and normal cells. the consistent presence of ebv in every cell of undifferentiated npc provides a unique opportunity to target the virus itself. in endemic region, ebv is exclusively found in npc tumor cells but not in normal cells [60] . ebv-targeted therapy and immunotherapy have been recently reviewed in detail by many researchers [61, 62] mainly including (1) inhibit the ebv transforming proteins (ebna-1, lmp1 and lmp2) using antisense rna, small interfering rna (sirna) and ribozyme; (2) induce loss of the ebv episome, e.g. using hydroxyurea; (3) trigger the lytic form of ebv replication in tumor cells by expressing the ie proteins (zta or rta) or using radiation, chemo drugs, demethylation and histone acetylation agents, such as 5-azacytidine, gemcitabine, hdac inhibitors, phorbol esters and sodium butyrate. as ebv lytic replication promotes viral antigen exposure and provokes a strong host immune response, the use in conjunction with antiviral prodrug such as ganciclovir further enhances the killing of virus and tumor cells; (4) adopt t cell immune therapy by infusion ex vivo expanded of autologous ebv-specific ctls from peripheral blood mononuclear cells (pbmcs) [40, 41, 63, 64] , (5) active immunotherapy by vaccinating with lmp2 pulsed autologous monocyte-derived dendritic cells or viral vector loading with ebv peptides against lmp1, lmp2 and ebna-1 [65, 66] . ebv-targeted therapy and immunotherapy represent novel therapies that have currently become a center of interest in research and development of npc treatment. ebv-targeting therapies have so far no clinical breakthrough yet, although cytolytic virus activation (clva) therapy in 3 patients with end-stage npc showed biological effect, tolerability and moderate safety [67] . currently, immune-based strategies represent options with the most clinical benefit [68] . while these treatments advancing, knowledge to develop new therapies is emerging. in a genomewide mutation profiling, we find that npc has a relatively low level of genomic alteration as compared to other cancers. therese findings suggest that epigenetic alteration possibly is an oncogenic feature of ebv infection in neoplasia [69] . hence targeting dysregulated epigenetic regulators might be candidate targets for npc cancer therapy [70, 71] . recently the prokaryotic type ii crispr/cas9 system has been adapted for genetic engineering in mammalian cells, allowing researchers to edit the genomes with unprecedented speed and precision. this highly precise and versatile system has been used in cancer modeling, cancer genomics functional analyses and cancer drug targets screening [72] [73] [74] . in a proof of concept study, kanda et al. found that crispr/cas9-mediated cleavage of ebv episomal dna enabled cloning of disease-associated viral strains with unprecedented efficiency. two gastric cancer cell-derived novel ebv strains were cloned [75] . the same technology can accelerate the discovery new ebv strain variants associated with npc, which will provide important clues about the mechanisms of ebv-mediated epithelial carcinogenesis. in a study of bl patient-derived cells, the clearance of latent ebv genomes using crispr-cas9 leads to proliferation arrest and apoptosis in ebv-infected cells, with no observed cytotoxicity to uninfected cells [76] . the feasibility of crispr/cas9mediated editing of the ebv genome (ebna-1, ebna-3c, lmp1, and bart) has also been demonstrated in cultured npc cell lines [77] . the dormant viral genome during latent infection provides few therapeutic targets other than itself for antiviral drug development. thus, this strategy may lead to a generalized approach in curing latent viral infections, although many hurdles remain before this approach could be used in the clinic, such as methods of safe and efficient delivery. the rapid expansion of knowledge in the field of ebv biology in the past decades of research had empowered the utility of ebv in domains of gene therapy, regenerative medicine (generation of clinical-grade ipscs) and antibody discovery. with growing evidence on ebv-induced oncogenesis in both lymphoid and epithelial malignancies, the role which ebv plays is not merely 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authors have no other funding, financial relationships, or conflicts of interest to disclose. key: cord-256130-zhlvvuj4 authors: nordén, rickard; magnusson, jesper; lundin, anna; tang, ka-wei; nilsson, staffan; lindh, magnus; andersson, lars-magnus; riise, gerdt c; westin, johan title: quantification of torque teno virus and epstein-barr virus is of limited value for predicting the net state of immunosuppression after lung transplantation date: 2018-03-06 journal: open forum infect dis doi: 10.1093/ofid/ofy050 sha: doc_id: 256130 cord_uid: zhlvvuj4 background: major hurdles for survival after lung transplantation are rejections and infectious complications. adequate methods for monitoring immune suppression status are lacking. here, we evaluated quantification of torque teno virus (ttv) and epstein-barr virus (ebv) as biomarkers for defining the net state of immunosuppression in lung-transplanted patients. methods: this prospective single-center study included 98 patients followed for 2 years after transplantation. bacterial infections, fungal infections, viral respiratory infections (vrti), cytomegalovirus (cmv) viremia, and acute rejections, as well as ttv and ebv levels, were monitored. results: the levels of torque teno virus dna increased rapidly after transplantation, likely due to immunosuppressive treatment. a modest increase in levels of epstein-barr virus dna was also observed after transplantation. there were no associations between either ttv or ebv and infectious events or acute rejection, respectively, during follow-up. when tacrolimus was the main immunosuppressive treatment, ttv dna levels were significantly elevated 6–24 months after transplantation as compared with cyclosporine treatment. conclusions: although replication of ttv, but not ebv, appears to reflect the functionality of the immune system, depending on the type of immunosuppressive treatment, quantification of ttv or ebv as biomarkers has limited potential for defining the net state of immune suppression. for patients with end-stage lung disease, limited life expectancy, and otherwise acceptable physical condition, lung transplantation may be a life-saving therapeutic option. the 5-year survival rate is only approximately 50% in most centers, and 2 major limiting factors for the outcome of lung transplantation are the frequent occurrence of infectious complications and rejection of the transplanted organ [1] . standard immunosuppression, to prevent graft rejection, consists of a combination of 3 immunosuppressive drugs that subvert b and t cells [2] [3] [4] . currently, there is a lack of an easy-to-use marker that accurately predicts the net effect of the combined immunosuppression. such a biomarker would be an important tool in the management of patients with compromised immune function to identify overor underimmunosuppression and evaluate the risk of infectious complications and acute rejections. viruses are often only viewed as pathogenic entities, but there are several examples of viruses that are frequently found in healthy individuals without causing obvious disease [5, 6] . torque teno virus (ttv) is a human dna virus that causes asymptomatic viremia with a high prevalence in the general population [7] [8] [9] [10] [11] [12] [13] . cell-mediated immunity is considered important in controlling ttv infection [14] [15] [16] [17] . accordingly, serum levels of ttv-dna increase dramatically in patients who have undergone solid organ transplantation, presumably as a result of immunosuppression [16, 18, 19] . epstein-barr virus (ebv) is another dna virus, commonly acquired early in life, with a high prevalence in us adults [20] , that establishes latent infection in memory b cells [21, 22] . the levels of ebv often increase in blood after immunosuppressive treatment, and ebv can cause post-transplant lymphoproliferative disorder (ptld) when the immune system is repressed. hence, both ttv and ebv have previously been suggested as surrogate markers of the net state of immunosuppression [16, 23, 24] . the aim of the present study was to evaluate levels of ttv and ebv in relation to the frequency of infectious events and acute rejections over time in a prospective manner in a single-center cohort of lung-transplanted patients. all patients over the age of 18 years who received a single or double lung transplant, who survived the initial postoperative intensive care period and remained in gothenburg during the follow-up period, between january 1, 2009, and april 3, 2012, at the sahlgrenska university hospital transplant centre, gothenburg, sweden, were asked to participate in this prospective single-center cohort study. all included subjects underwent follow-up (fu) visits at 1, 2, 3, 4.5, 6, 9, 12, 18, and 24 months after transplantation. at every fu visit, whole blood, serum, and nasopharyngeal samples (nph) were collected. protocol bronchoscopies were performed at 1, 3, and 12 months post-transplantation and when indicated based on clinical presentation. bronchoalveolar lavage (bal) samples were collected at every bronchoscopy. all bal samples underwent direct microscopy for early detection of aspergillus hyphae and were cultured for bacteria and fungi and tested for legionella pneumophila, pneumocystis jirovecii, and human cytomegalovirus (cmv) using real-time polymerase chain reaction (pcr). bal samples from patients with cystic fibrosis were routinely cultured for mycobacterium tuberculosis and atypical mycobacteria. bacterial and fungal culture testing from blood, urine, and sputum was performed upon suspicion of infection. nph and bal samples from every fu visit, as well as additional nph samples obtained upon suspicion of airway infection between scheduled fu visits, were analyzed by multiplex real-time pcr for detection of airway pathogens. serum samples obtained at pretransplant evaluation were used for ttv-dna and ebv-dna quantification. for quantification of ttv and ebv during fu, serum and whole blood samples were used, respectively. induction therapy consisted of rabbit antithymocyte globulin, which was given for 1-3 consecutive days together with methylprednisolone intravenously. post-transplantation immunosuppression included prednisone 0.3 mg/kg/d and mycophenolate mofetil (mmf) 2 g/d. the patients then received either oral cyclosporine (csa; 1-2 mg/kg), adjusted to maintain a serum level of 300-350 ng/ml, or tacrolimus (tac; 0.075 mg/kg) given orally divided in 2 doses daily, adjusted to maintain a serum level of 14-16 ng/ml. the choice between csa and tac was made based on clinical presentation. patients previously treated with tac and patients with cystic fibrosis were given tac. there was also a preference for tac if the recipient was younger. the dosage of immunosuppression was gradually lowered during fu. further changes in immunosuppressive therapy were based on clinical presentation. four patients, who either underwent retransplantation or had previous immunosuppressive treatment due to autoimmune conditions, were given nonstandard immunosuppression therapy based on previous exposure to immunosuppressive drugs. all patients received a combination of 80 mg of sulfametoxazol and 400 mg of trimetoprim thrice weekly to prevent pneumocystis jirovecii infection. patients seropositive for cmv at the pretransplant evaluation were given valganciclovir at a dose of 900 mg daily for 3 months. patients seronegative for cmv pretransplant while the organ donor tested positive (ie, cmv mismatches) were given the same treatment for 6 months. bacterial infection was defined as a positive bacterial culture in conjunction with symptoms of clinical infection or, in the absence of positive culture, symptoms consistent with bacterial infection that was treated with antibiotics. fungal infection was defined as significant presence of fungi in culture from a sterile location in conjunction with symptoms of infection according to the european organization for research and treatment of cancer (eortc) criteria [25] . vrti or mycoplasma pneumoniae infection was defined as detection of a pathogen by multiplex real-time pcr in nph or bal. cmv viremia was defined as elevated levels of cmv-dna that prompted antiviral therapy. cmv-seronegative recipients were considered to have viremia if cmv-dna was detected, whereas recipients seropositive for cmv prior to transplantation where considered to have cmv viremia only if the level was above 3.0 log10 (1000) copies/ml. to distinguish milder infections from more severe infectious events, a subgroup of events requiring initial intravenous antimicrobial (antibacterial, antiviral, or antifungal) therapy were defined as severe. acute rejection was defined as either a lung biopsy showing rejection of ishlt grade 1a or higher [26] or, in the absence of a biopsy, physical (increased need for oxygen) and radiological findings (progressive infiltrate without signs of infection) consistent with acute rejection followed by a prompt response to high-dose (1 g/d for 3 consecutive days) corticosteroid therapy (methylprednisolone). nucleic acid isolation was performed with a magna pure lc total nucleic acid or dna isolation kit for serum (ttv) or whole blood (ebv and cmv) using a standardized protocol, according to the instructions (roche diagnostics, mannheim, germany). input/output volume was set to 200/100 µl, and the sample was eluted in extraction buffer. the ttv-dna levels were determined using a 7300 real-time pcr system (applied biosystems, foster city, ca). each pcr reaction contained 2 µl of extracted total nucleic acid, 10 µl of 2x universal master mix (applied biosystems, foster city, ca), 0.5 µl and 20 µm of forward and reverse primer, respectively, 0.3 µl and 20 µm of bhq hydrolysis probe, and 2 µl of rnase-free h2o. the reaction conditions were 50°c for 2 minutes and 95°c for 10 minutes prior to 45 cycles at 95°c for 15 seconds and 60°c for 60 seconds. the assay range was determined by serial dilution of plasmids with an insert of a synthesized sequence matching the ttv pcr product, and quantification was obtained from a plot of ct values. cmv-and ebv-dna levels were determined using a method described previously with minor modifications [27] . all primer and probe sequences are listed in supplementary table 1 . for ebv and cmv quantification, a control sample consisting of unrelated phocine herpesvirus 1 (phhv-1) was included prior to nucleic acid extraction. ttv load was quantified on the qx200 droplet digital pcr system using the ddpcr supermix for probes (no dutp; bio-rad, hercules, ca). primer and probe concentrations in the 20-µl final reaction were 300 and 200 nm, respectively. the pcr conditions were 50°c for 2 minutes followed by 95°c for 10 minutes, then 30 seconds at 95°c and 1 minute at 58°c for a total of 40 cycles, and finally a step at 98°c for 10 minutes. after overnight incubation at 4°c, the droplet fluorescence signal was determined. data analysis was done using bio-rad quanta soft analysis software. isolated dna and rna were analyzed using a multiplex real-time pcr system designed to detect adenovirus, bocavirus, chlamydophila pneumoniae, human coronavirus (nl63, hku1, oc43 and 229e), human enterovirus, human metapneumovirus, human rhinovirus, influenza a virus, influenza b virus, mycoplasma pneumoniae, parainfluenzavirus (1, 2, and 3), and respiratory syncytial virus. the pcr settings and a list of primers and probes have been described previously [28] . comparisons on group level for numerical variables were performed using the mann-whitney u test. pearson's correlation and linear regression were used to evaluate the relationship between the real-time pcr and ddpcr measurements. cox regression with ttv, ebv, and both as time-dependent covariates was used to analyze the relationship between the 2 biomarkers and the outcomes vrti, fungal infection, bacterial infection, cmv viremia, or any infectious event. each patient was treated as a cluster to handle multiple infections occurring in a single individual. univariable logistic regression was used in each of 4 periods, q1 (1-3 months), q2 (3-6), q3 (6) (7) (8) (9) (10) (11) (12) , and q4 (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) , with the outcomes vrti, fungal infection, bacterial infection, cmv viremia infection, or acute rejection and the predictors age, treatment, cmv mismatch, log ttv (beginning of period), or log ebv (beginning of the period). a p value <.05 was considered significant. r software, version 3.3.1 (r core team, 2016, r foundation for statistical computing, vienna, austria; http://www.r-project.org/), was used for all statistical analyses except for pearson's correlation analysis, which was performed using graphpad prism 6 software (graphpad software inc., san diego, ca). the study was approved by the regional ethical review board in gothenburg (dnr: 791-08), and all subjects provided written and oral consent to participate. in total, 98 lung transplant recipients were included in the study, of which 79 (80%) were still alive 24 months after transplantation. detailed patient description is outlined in table 1 . blood-, bal-, and nasopharyngeal samples (nph) were collected longitudinally at follow-up (fu) visits until 24 months post-lung transplantation (ltx; n = 837), including pretransplant samples. the total nucleic acid content was isolated from serum or whole blood samples and analyzed for ttv-, ebv-, and cmv-dna load by real-time pcr. the total nucleic acid content was isolated from the nph and bal samples and analyzed for respiratory pathogens using multiplex real-time pcr. a majority of the patients had detectable ttv-dna levels before transplantation (pre-ltx), and the levels subsequently increased until a peak was reached at 3 months post-ltx, after which the levels gradually decreased (figure 1 ). the mean ttv-dna levels for the respective time periods were 6.7 log10 1-3 months post-ltx, 6.4 log10 3-6 months post-ltx, 5.8 log10 6-12 months post-ltx, and 5.2 log10 12-24 months post-ltx, respectively. conversely, a majority of the patients had no detectable ebv-dna prior to transplantation as determined in serum. at 1 month post-ltx, a peak in mean ebv-dna levels was observed, which decreased already 2 months post-ltx and thereafter remained at a relatively constant level, determined in whole blood (figure 1 ). the mean ebv-dna levels for the respective time periods were 2.7 log10 1-3 months post-ltx, 2.6 log10 3-6 months post-ltx, 2.6 log10 6-12 months post-ltx, and 2.7 log10 12-24 months post-ltx, respectively. overall, fewer patients had detectable levels of ebv-dna compared with ttv-dna ( figure 1 ). although the individual ttv-dna levels varied, a majority of the patients had detectable levels across the entire fu period. in comparison, only a minority of the patients had detectable levels of ebv-dna over a longer consecutive period, and a large fraction had ebv-dna levels below the level of quantification (loq) at any given time point during fu (figure 1 ). three patients developed suspected ptld. no case was biopsy-verified, and all were reversed upon modification of immunosuppression. detailed individual ttvand ebv-dna level kinetics for each patient, including information regarding all infectious events and acute rejections, are displayed in supplementary figure 1 . comparison of ttv-and ebv-dna levels in lung transplant recipients who received either tacrolimus-or cyclosporinebased therapy revealed that cyclosporine-treated patients had significantly lower ttv-dna levels in serum at month 6 post-ltx and onwards, compared with the tacrolimustreated patients (figure 1 ). there was no significant difference in ebv-dna levels in whole blood between tacrolimus-and cyclosporine-treated patients at any time point during fu (figure 1 ). the fu was divided into 4 periods, denoted q1 through q4, defined as 1-3 (q1), 3-6 (q2), 6-12 (q3), and 12-24 (q4) months after transplantation. the periodic intervals were chosen to distinguish the initial phase of intensive immunosuppression from the later periods when immunosuppressive therapy was gradually lowered. viral infections (vrti) were more common during the first 6 months, while bacterial and fungal infections were evenly distributed throughout the period and cmv viremia occurred at a higher frequency 3-12 months after ltx after discontinuation of prophylactic ganciclovir treatment ( table 2) . events of acute rejections were most common during the first postoperative months and then decreased over time ( figure 1 ). all but 3 episodes of acute rejections were biopsy-verified. the frequency of infectious events ( figure 1 and table 2 ) was compared with the mean ttv-dna or ebv-dna levels during each period (q1-q4) and the during the total fu period, respectively. no statistically significant association was found. with logistic regression, log ttv-dna or log ebv-dna levels in the beginning of the period did not predict any infectious event in any of the periods (supplementary table 2 ). next, we performed a cox regression analysis to establish if ttv-or ebv-dna levels were associated with either viral table 3 ). no significant associations were found using this criterion. when comparing other factors relevant to infections and acute rejection, using logistic regression, we found that age, cmv mismatch, and dominant immunosuppressive treatment did not predict outcome in any of the 4 periods denoted q1 through q4 (supplementary table 4 ). most acute rejection events (ars) occurred within the first 6 months post-ltx (figure 1 ). logistic regression with initial ttv-dna or ebv-dna for each period, respectively, did not predict whether an acute rejection event would occur during that period (data not shown). previous studies [15, 24] have described higher mean ttv-dna levels in lung transplant patients than reported here. in order to verify the real-time pcr results, ttv-dna levels of patients with mean ttv-dna levels above 8 log10 in period q3 were assessed by digital droplet pcr (ddpcr). the ttv-dna level was underestimated by more than 1 log10 by real-time pcr in 2 samples, and overall the levels obtained by realtime pcr correlated well with the results obtained by ddpcr (pearson r = 0.67, p = .009) (supplementary table 5 ). ttv and ebv has been proposed as biomarkers reflecting the functionality of the immune system after ltx. in this prospective study, evaluating serial samples for ttv and ebv and carefully recording infectious and rejection events during an fu period of 24 months, we found no associations between the biomarkers and the risk of complications. we found that ttv-dna levels increased rapidly; 1 month after ltx, the levels were already markedly elevated, presumably due to ablation of the functional effector cells of the immune system that normally control the ttv infection [14, 15] . at 3 months post-ltx, the mean ttv-dna level peaked and then gradually declined, reflecting the gradual moderation of immunosuppressive therapy. interestingly, the ttv-dna level was lower in patients who received cyclosporine treatment, possibly mirroring a different immune modulatory mechanism for cyclosporine compared with tacrolimus. görzer et al. [16] previously suggested that this might be due to cyclosporine being less efficient as an immunosuppressant. however, we found no association between type of immunosuppressive regimen and acute rejection or other events that would indicate a difference in net immunosuppressive effect. recently, ttv-dna has been associated with chronic lung allograft dysfunction after lung transplantation [29] , but in our study this was not included as an outcome. it is possible that the observed higher mean ttv-dna levels in other studies could be explained by differences in induction therapy and distribution of respective immunosuppressive regimen. for example, in the study by görzer et al. [16] , the type of induction therapy was not defined and only 26% of the patients were treated with cyclosporine. there is some evidence that ttv replication is dependent on the type of induction therapy that appears to reflect changes in lymphocyte concentration; however, the duration is brief, after which the levels of ttv recover [30] . in the present study, ttv viral load was determined in serum samples, whereas plasma was used in previous studies [16, 31] . however, to our knowledge, there are no apparent reasons for variations in ttv-dna load, depending on whether plasma or serum is being used for analysis. to compare results regarding ttv levels between different transplantation centers, it is vital to reliably quantify the concentrations of ttv-dna in a standardized manner. here it was shown that the real-time pcr method was adequate as compared with the ddpcr method, which is considered a more reliable method for accurate quantification [32] . nonetheless, real-time pcr is prone to interlaboratory differences, and utilization of ddpcr for quantification has been suggested to circumvent this problem, facilitating direct comparison of results between centers [33, 34] . digital droplet pcr depends on partitioning of each master mix followed by end point pcr. quantitation is determined using poisson statistics to generate a result that is not dependent on a relation to a standard curve and should therefore exhibit lower variability between laboratories. however, the ddpcr assays still depend on amplification of a specific target sequence, and depending on the design of the primer and probe, the targeted sequence can vary. recent work has shown that viral dna in plasma samples is to a large extent free and not associated with viral particles and is also subjected to various degrees of degradation, influencing the results, depending on the amplicon length determined by the pcr-assay design [35] . thus, standardization of the entire assay including primer and probe design, use of international standards, and assessment of the commutability of reference material is needed before a direct comparison of interlaboratory results can be made with absolute confidence, even with the use of ddpcr [36, 37] . it remains to be clarified in which cell types ttv replication occurs, but cd4+ t cells and cd8 + 57+ t cells appear to be important for controlling the infection, whereas ebv resides within the b-cell pool and is controlled by cd4+ and cd8+ t cells [14, 15, 17, 21, 22, [38] [39] [40] [41] . in this work, we show that the choice of calcineurin inhibitor affects the ttv-but not the ebv-dna levels, possibly reflecting separate mechanisms for viral replication rather than merely ablation of the t-cell pool. as the choice of immunosuppressive regimen was made at the discretion of the treating physician, these findings should be interpreted with caution but warrant further investigation. in this study, infectious events were defined as symptoms prompting antimicrobial therapy also in cases without a positive culture (bacterial and fungal infections) or simply detection of a potential pathogen by pcr (vrti, cmv). attempting to focus on more severe infectious events, subgroup analysis of events requiring intravenous therapy was made. also, this analysis failed to reveal any significant association between ttv-or ebv-dna levels and infectious events. however, no proper validated scoring system for grading of the severity of infectious complications was used in this study, and we cannot exclude that our definitions cause underestimation of severe complications. further studies using proper grading of events are needed. ebv has previously been suggested as a surrogate marker for immunosuppression as an association between elevated ebv-dna levels and reduced incidence of acute rejection of lung transplants was observed [23] . in addition, ebv-dna was safely used as a trigger for reduction of immunosuppression late after lung transplantation and was found to be associated with a shorter time to development of infectious complications or solid tumors, but not ptld [24, 42] . we found no association between ebv-dna and acute rejection at any time during fu. a direct comparison could, however, be hampered by the fact that ebv-dna was analyzed in this study, and ebv-encoded rna in an earlier study [23] . in the present study, mean ebv-dna levels did not increase as markedly as ttv-dna levels after transplantation, even though ebv-specific t cells most likely are subverted by immunosuppressive therapy [43] . the seroprevalence pre-ltx and number of patients positive for ebv-dna at any time during fu were comparable to previous studies [24, 42, 44] . one possible caveat when determining ebv dna load is the use of valgangciclovir for preventing reactivation or primary infection of cmv. all patients received 3 months of valgangciclovir treatment initially after ltx; there are a few studies showing that valgangciclovir treatment reduces ebv load, which may have affected the results in this study [45, 46] . in conclusion, ttv-but not ebv-dna load appears to reflect the function of the immune system after lung transplantation, depending on the type of immunosuppressive treatment. however, we found no association between either ttv-or ebv-dna load and infectious events or acute rejections, which suggests a limited clinical applicability as biomarkers predicting short-term outcomes related to the net state of immunosuppression. further studies are warranted to define the effect of immunomodulation on ttv and ebv replication in relation to various immunosuppressive regimens. supplementary materials are available at open forum infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. international society for heart and lung transplantation. the registry of the international society for heart and lung transplantation: thirty-fourth adult lung and heart-lung transplantation report-2017; 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national institute of allergy and infectious diseases mycoses study group (eortc/ msg) consensus group. revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection comparison of serum and whole blood levels of cytomegalovirus and epstein-barr virus dna comparison of the filmarray assay and in-house real-time pcr for detection of respiratory infection association between plasma torque teno virus level and chronic lung allograft dysfunction after lung transplantation short-term kinetics of torque teno virus viraemia after induction immunosuppression confirm t lymphocytes as the main replication-competent cells pre-transplant plasma torque teno virus load and increase dynamics after lung transplantation quantification of hev rna by droplet digital pcr a miqe-compliant real-time pcr assay for aspergillus detection absolute quantification by droplet digital pcr versus analog real-time pcr determination of the biological form of human cytomegalovirus dna in the plasma of solid-organ transplant recipients quantitative assessment of commutability for clinical viral load testing using a digital pcr-based reference standard are we there yet? impact of the first international standard for cytomegalovirus dna on the harmonization of results reported on plasma samples dendritic cells cross-present latency gene products from epstein-barr virus-transformed b cells and expand tumor-reactive cd8(+) killer t cells human cd4(+) t lymphocytes consistently respond to the latent epstein-barr virus nuclear antigen ebna1 cd57+ t lymphocytes and functional immune deficiency changes in cd8 + 57+ t lymphocyte expansions after autologous hematopoietic stem cell transplantation correlate with changes in torquetenovirus viremia epstein-barr virus dnaemia is an early surrogate marker of the net state of immunosuppresion in solid organ transplant recipients human cytotoxic t lymphocyte responses to epstein-barr virus infection detection of epstein-barr virus dna in peripheral blood is associated with the development of bronchiolitis obliterans syndrome after lung transplantation efficacy and safety of valganciclovir in liver-transplanted children infected with epstein-barr virus valganciclovir for the suppression of epstein-barr virus replication financial support. this project was supported by funding from stiftelsen professor lars-erik gelins minnesfond, fou laboratoriemedicin, sahlgrenska university hospital, the swedish heart-lung foundation, the region västra götaland research funds (vgfoureg-228341, vgfoureg-82811), regional alf funds (alfgbg-217671, alfgbg-439391), and region halland. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-350807-qdq96723 authors: reckziegel, maria; weber-osel, claudia; egerer, renate; gruhn, bernd; kubek, florian; walther, mario; wilhelm, stefanie; zell, roland; krumbholz, andi title: viruses and atypical bacteria in the respiratory tract of immunocompromised and immunocompetent patients with airway infection date: 2020-05-27 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-020-03878-9 sha: doc_id: 350807 cord_uid: qdq96723 respiratory tract infections (rti) can take a serious course under immunosuppression. data on the impact of the underlying pathogens are still controversial. samples from the upper (n = 322) and lower rt (n = 169) were collected from 136 children and 355 adults; 225 among them have been immunocompromised patients. exclusion criteria were presence of relevant cultivable microorganisms, c-reactive protein > 20 mg/dl, or procalcitonin > 2.0 ng/ml. samples were tested by pcr for the presence of herpesviruses (hsv-1/-2; vzv; cmv; hhv6; ebv), adenoviruses, bocaviruses, entero-/rhinoviruses (hrv), parechoviruses, coronaviruses, influenza viruses (iv), parainfluenza viruses as well as for pneumoviruses (hmpv and rsv), and atypical bacteria (mycoplasma pneumoniae, m.p.; chlamydia pneumoniae, c.p.). viral/bacterial genome equivalents were detected in more than two-thirds of specimens. under immunosuppression, herpesviruses (ebv 30.9%/14.6%, p < 0.001; cmv 19.6%/7.9%, p < 0.001; hsv-1: 14.2%/7.1%, p = 0.012) were frequently observed, mainly through their reactivation in adults. immunocompromised adults tended to present a higher rsv prevalence (6.4%/2.4%, p = 0.078). immunocompetent patients were more frequently tested positive for iv (15.0%/5.8%, p = 0.001) and m.p. (6.4%/0.4%, p < 0.001), probably biased due to the influenza pandemic of 2009 and an m.p. epidemic in 2011. about 41.8% of samples were positive for a single pathogen, and among them ebv (19.9%) was most prevalent followed by hrv (18.2%) and iv (16.6%). hsv-2 and c.p. were not found. marked seasonal effects were observed for hrv, iv, and rsv. differences in pathogen prevalence were demonstrated between immunocompetent and immunocompromised patients. the exact contribution of some herpesviruses to the development of rti remains unclear. electronic supplementary material: the online version of this article (10.1007/s10096-020-03878-9) contains supplementary material, which is available to authorized users. infections of the upper respiratory tract (urti) are among the most frequent infections worldwide. these are mainly caused by rna viruses. among them, influenza viruses (iv), pneumoviruses (respiratory syncytial virus, rsv; human metapneumovirus, hmpv), but also parainfluenza viruses (piv), coronaviruses (cov), and rhinoviruses (hrv) are considered by world health organization a global health burden [1] . serious rti through respiratory viruses are frequently observed under immunosuppression, for example, in solid organ transplant recipients [2] . the current breakthroughs of immunomodulating therapies in medicine contribute to the continuous increase of patients being under iatrogenic immunosuppression and being at risk for pulmonary infections [3] . in general, suppression of t cell function is associated with a higher susceptibility for infection or reactivation of various viruses [4, 5] . impairment of th1cell activity but also of humoral immunity, both, facilitates the development of viral rti. in immunocompromised patients, higher morbidity and sometimes also mortality rates through infections, for example, with adenoviruses (adv), iv, piv, rsv, hmpv, but also of secondary complications like bacterial pneumonia have been observed [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . furthermore, particularly transplant patients are at risk for reactivation of diverse herpesviruses (herpes simplex virus-1/-2, hsv-1/-2; varicella zoster virus, vzv; cytomegalovirus, cmv; human herpesvirus 6, hhv-6; epstein-barr virus, ebv) [12, 15, [17] [18] [19] [20] . multiple viral infections/reactivations can occur [21] as well as indirect interactions of viruses with bacteria [22] [23] [24] . these aspects may challenge interpretation of diagnostic findings. the frequency of viral infections/reactivations is also influenced by factors like the underlying disease, therapeutic regimes, as well as the type of transplant and hla mismatches [12, 19] . thus, fast and efficient diagnostic methods that cover a broad spectrum of viruses, bacteria, but also fungi and parasites are in urgent need to deal with the aforementioned challenges. the availability of such methods is of particular importance in stem cell transplant recipients where clinical symptoms of rti are variable or may be mimicked by graft-versus-host disease [25, 26] . early diagnosis enables limited antiviral interventions [16, 27] and may prevent further cross-transmission [28] . however, detection of viral genome equivalents does not necessarily mean a causative role of this virus, and particularly immunocompromised patients can shed viruses over a prolonged time period [13, 29, 30] . furthermore, there is increasing evidence of the existence of a respiratory virome which is defined by the presence of common viral pathogens, rare viruses, and viruses of unknown pathogenicity [31] . thus, the exact contribution of a single virus to the development of rti is still controversial. we tried to consider most of these aspects by performing an observational study addressing the spectrum and impact of respiratory viruses but also of herpesviruses and the atypical bacteria mycoplasma pneumoniae (m.p.) and chlamydia pneumoniae (c.p.) in patients with respiratory symptoms. for this, an underlying infection through relevant cultivable microorganisms was largely ruled out. data were analyzed with respect to patients' immune status and age. this study included 322 samples from the upper (nasal or throat swabs and washings), and 169 samples from the lower (broncheoalveolar/tracheal washings, induced sputum) respiratory tract (urt/lrt) collected over a period of 40 months beginning in september 2009 to december 2012 from 266 healthy and 225 immunocompromised patients with symptoms of a rti (i.e., common cold, cough with/without sputum, dyspnea, and fever). this setting included samples from patients with respiratory symptoms under neutropenia or lungtransplant recipients with a recently observed decrease in forced expiratory volume in one second (fev1). a compromised immune status was defined (i) for solid organ or stem cell transplant recipients under iatrogenic immunosuppression, (ii) in patients with autoimmune disorders under immunosuppressing therapy but also (iii) in cancer patients under chemotherapy/radiation, and (iv) in patients with primary or secondary causes of immunodeficiency including hiv infection. samples found to contain relevant cultivable bacteria (streptococcus pneumoniae, staphylococcus aureus, haemophilus influenzae, various enterobacterales and nonfermenting gram-negative bacteria, mycobacterium tuberculosis) and fungi were excluded. furthermore, specimens obtained from patients with a present bacteremia/ sepsis were excluded as well as samples from patients with rti through pneumocystis jirovecii or legionella pneumophila. the oropharyngeal and tracheopulmonal flora was considered if data were available from routine diagnostics. in addition, samples obtained from the lrt of patients with more than 100,000 colonies/ml of oropharyngeal or tracheopulmonal flora, or more than 10,000 colonies/ml of enterococcus spp. or candida spp., were also excluded since such high concentrations may represent an infection rather than colonization. in addition, the presence of procalcitonin (pct) > 2 ng/ml and/or of c-reactive protein (crp) > 20 mg/ dl in serum leads to exclusion of the rt sample. the remaining samples were obtained from 99 immunocompetent (median age 1.0 years; 56 males/43 females) and 37 immunocompromised (median age 4.6 years; 18 males/19 females) children and adolescents as well as 167 immunocompetent (median age 46.2 years; 73 males/94 females) and 188 (median age: 57.2 years; 101 males/87 females) immunocompromised adults. among the immunocompromised cohort, most samples were obtained from patients with hemato-oncological malignancies (28.4%), followed by samples from patients after organ (24.9%) and stem cell (16.4%) transplantation or with autoimmune disorders (13.8%). about 9.8% of samples were included from patients with other conditions of immunosuppression or from solid tumor patients (6.7%). specimens were immediately deep frozen (− 80°c) until nucleic acid extraction. the extraction was done manually with the qiaamp minelute virus spin kit or automatically with the ez1 virus mini kit (both qiagen, hilden, germany). the nucleic acids were stored at − 20°c and used for synthesis of copy dna (cdna) applying the revertaid h minus first strand cdna synthesis kit (thermofisher scientific, langen, germany) with random hexamers. integrity of cdna was demonstrated by amplification of ß-actin or glyceraldehyde 3-phosphate dehydrogenase (gapdh) dna [32, 33] with dreamtaq dna polymerase (thermofisher scientific). presence of herpesviral dna (hsv-1/-2, vzv, cmv, and hhv-6) was demonstrated by applying conventional pcr [34] [35] [36] [37] [38] [39] [40] [41] together with the hotstartaq dna polymerase and qsolution (qiagen). quantitative detection of ebv dna in samples from the lrt was done in accordance with krumbholz et al. (2006) [42] , but sybr-green (quantitect sybr green pcr kit; qiagen) was used instead of hybridization probes. these diagnostic pcrs were continuously approved by successful participation in the external quality assurance service (eqas) program of instand e.v. (düsseldorf, germany). for detection of diverse respiratory viruses, all cdnas were tested with the seeplex rv5 ace (covers iv-a; iv-b; rsv-a/-b; adv; piv1-3; bocavirus, bov; hmpv; hrv-a/ b; cov 229e/nl63/oc43/hku1) and rv12 (covers the same spectrum as rv5, but is not able to detect bov) ace detection kits. the seeplex rv15 ace detection kit (includes also piv-4, hrv-c, and enterovirus detection but is not able to detect cov hku1) was used from november 2011, since the distribution of rv5 and rv12 versions was abandoned (all kits seegene, eschborn, germany). all three multiplex assays have been established in the laboratory using defined eqas samples from instand e.v. before testing of study samples. the rv5 kit is a screening kit and neither allows discrimination between adv, piv, and bov nor between hmpv, hrv, and cov. to overcome this problem, amplicons were purified after agarose gel-electrophoresis applying the qiaquick gel extraction kit (qiagen). purified dna was ligated into the pdrive cloning vector included in the qiagen pcr cloning kit, and used for transformation of competent escherichia coli cells. then, colonies were screened for inserts by pcr applying the dreamtaq dna polymerase and oligonucleotides specific for pdrive. amplicons with inserts were purified and sequenced using the dtcs quick start master mix. sequence analysis was done on a beckman ceq 8000 genetic analyzer (all beckman coulter, krefeld, germany). for detection of human parechovirus (hpev) genome equivalents, a semiquantitative real-time pcr was established using cdna and oligonucleotides [43] together with the quantitect sybr green pcr kit (qiagen) on a lightcycler 1.5 (roche, mannheim, germany). positive controls for hpev-pcr were kindly provided by dr. corinna pietsch and prof. dr. uwe gerd liebert (institute of virology, university of leipzig, germany). since enteroviruses were not covered by rv5 and rv12 assays, nearly all samples were screened by a nested pcr protocol detecting a conserved sequence of the 5′nontranslated region (5′-ntr) [44] . then, rough-typing was done by sequence analysis of purified pcr products. detection of hrv was performed by nested amplification of the vp4/2-encoding region [45] . parallel testing for m.p. and c.p. was done by applying the diagenode mycoplasma pneumoniae and chlamydophila pneumoniae kit (r-diamcpn, diagenode s. a., liège, belgium) on an abi7500 real-time pcr system (thermofisher scientific). sequence data were analyzed using mega 6.0 [46] . all other data were analyzed applying the two-sided fisher's exact test implemented in ibm® spss® statistics 20. a p value < 0.05 was considered statistically significant. this study included 491 samples from immunocompromised or immunocompetent patients with symptoms of rti collected over a period of 40 months. all samples tested positive for the presence of gapdh and/or ß-actin. thus, quality of sampling and nucleic acid extraction was demonstrated (data not shown). among the overall study population, genome equivalents of ebv were most frequently detected (22.3%, 84/377), followed by hhv-6 (20.3%, 32/158), hrv (14.1%, 69/ 491), cmv (13.2%, 65/491), rsv (11.2%, 55/491), and iv (10.8%, 53/491). genome equivalents of hsv-2 and c.p. were generally not detected (table 1) . with respect to patients' immune status, dna of ebv (30.9% vs. 14.6%), cmv (19.6% vs. 7.9%), and hsv-1 (14.2% vs. 7.1%) was significantly more prevalent in immunocompromised patients while genome equivalents of iv (5.8% vs. 15.0%) or m.p. (0.4% vs. 6.4%) were more frequently observed in their immunocompetent counterpart. the higher prevalence of cmv and ebv was only observed (supplementary fig. 1 ). in 26.3% (129/491) of samples found to be pathogen-positive, multiple agents were detected. among them were samples with two (20.4%), three (5.1%), four (0.6%), or even five (0.2%) different viruses/bacteria (supplementary fig. 1 ). the combination of two herpesviruses (hhv-6/ebv 13.2%, 5/38; cmv/ebv 9.6%, 8/83; cmv/hsv-1 8.0%, 8/100) but also of ebv and iv (6.0%, 5/83) or m.p. (6.0%, 5/83) as well as of rsv and hrv (5.0%, 5/100) was frequently observed. bocaviral dna was found together with other viruses/m.p. supplementary fig. 1 ). significant seasonal effects were recorded in the immunocompetent group for hrv with a high prevalence in autumn and for iv with an increased prevalence in winter. seasonal effect was significant in both patient groups for rsv with increased prevalence in winter and spring (supplementary table 2 ). interestingly, slight seasonality was also observed for hhv-6 in the immunocompetent group. in addition, some interannual variation was found for m.p. and iv (data not shown), which was most likely associated to the 2009influenza pandemic and an m.p. epidemic in 2011, respectively. in this monocentric study, genome equivalents of viruses and m.p. were frequently detected in immunocompromised (66.7%) and immunocompetent (69.2%) patients with respiratory symptoms (table 1) . since a contribution of relevant cultivable microorganisms to patient symptoms was largely excluded, a causative role of the pathogens detected in this study has to be considered. previously, a comparable approach was used to identify viral causes of severe rti in children [47] . the stringent exclusion criteria may account for the low number of patient samples included in this study and may have neglected possible additive or synergistic effects between bacteria, fungi, and viruses. in particular, we found a high prevalence of herpesviruses in immunocompromised adults with respiratory infections. nearly one-third of them was tested positive for ebv and every fourth patient presented cmv in his respiratory tract. in children, herpesviral dna was rarely detected which reflects the generally increasing infestation rate observed over life-time [48] [49] [50] [51] [52] and indicates viral reactivation as a major cause for pathogen detection. the higher prevalence of ebv, cmv, and hsv-1 in the airways of adults was associated with the state of immunosuppression. this is in line with the fact that herpesviral reactivation is facilitated by the impaired immune system [53] . it is still controversial whether this reactivation contributes to respiratory pathology or just represents an indicator of excessive immunosuppression. for cmv, however, there is no doubt that this betaherpesvirus is responsible for lrti in immunocompromised patients [17, 20] . cmv pneumonia is considered as likely when viral dna has been detected in bal of symptomatic patients [17] . thus, in our study, a remarkable proportion of immunocompromised adults revealed signs of suspected cmv pneumonia (table 1 ) and may benefit from antiviral prophylaxis or therapy. while hsv-1 dna was slightly more prevalent in the urt of immunocompromised patients, we found nearly comparable detection rates in the lrt of both patients groups (table 1 ). this is in line with a previous study [54] . interestingly, the same authors found that higher hsv-1 concentrations were associated with a poor patient outcome [54] . as for cmv, definitive diagnosis of hsv-1 pneumonitis depends on the presence of viral antigen within the lrt tissues [17] . in contrast to a recent report [55] , we found two-times higher ebv dna prevalence in immunocompromised patients compared to their immunocompetent counterparts (table 1) . previously, ebv dna was frequently detected in patients with pneumonia, respiratory insufficiency, and other bronchopneumopathies, but its presence was not associated with increased 28-day mortality [55] . in addition, the same authors reported no difference in ebv concentration between immunocompromised and immunocompetent patients [55] , which is in contrast to our findings (fig. 1, supplementary table 1 ). nevertheless, the contribution of ebv to the development of respiratory symptoms is still controversially discussed in the literature and remains unclear so far [55] [56] [57] . there is, however, some evidence that ebv reactivation-like that of other herpesviruses-may trigger fig. 1 comparison of ebv-dna copies/ml in respiratory specimens from immunocompromised and immunocompetent patients. data are presented in a logarithmic scale. the median ebv concentration is significantly higher in immunocompromised patients (p = 0.030, mann-whitney u test) inflammation which is associated to transplant rejection or interstitial lung disease [20, [58] [59] [60] . hhv-6 dna was found at similar high frequencies of ca. 20% in both patient collectives. interpretation of our results, however, is limited since our pcr protocol may have also detected chromosomally integrated viral dna [61] and cannot differentiate between hhv-6a and hhv-6b. the latter variant is more commonly implicated in human disease [62] . moreover, hhv-6 was frequently observed in combination with other herpesviruses ( table 2) as also seen by others [62] . thus, the contribution of hhv-6 to rti remains unclear. other herpesviruses (vzv, hsv-2) were found to be negligible in this study (table 1) which is in line with the literature [17, 20, 54, 63] . most of our results were obtained by end-point pcr. the consideration of viral concentration-as it is exemplarily shown here for ebv-may be useful in order to better unravel the contribution of herpesviruses to the development of lung pathology [20] . the observed frequencies of respiratory viruses were comparable to data from the german laboratory network (https://clinical-virology.net/en/charts/chart/ctype/ count/network/resp/section/viruses) and to another study from germany [64] . genome equivalents of rsv and hrv were prevalent in children while hrv and iv were frequent in adults. interestingly, immunocompromised adults tended to have a higher prevalence of rsv (table 1) . this supports previous data on the contribution of rsv to morbidity and mortality in this patient group [65] . there were various examples of single detections, which are probably indicative for infection, but also of co-presence of two or more pathogens ( table 2 , supplementary fig. 1 ). bocaviral dna, for instance, was frequently found in combination together with further viral genomes as also reported by others [66] . in children of 2 years and younger, however, this parvovirus was detected solely. previously, isolated bov infection was shown to be a likely cause of severe acute rti in children [67] . in a german study, bov dna was demonstrated in 10.3% of nasal swabs obtained from children with respiratory symptoms [68] . this prevalence is largely comparable to our results. same authors indicated a mean age of 1.8 years table 2 detection of multiple pathogens in the respiratory tract of the overall study population (a) as well as of immunocompromised (b) and immunocompetent (c) patients. the gray boxes indicate frequent co-infections. note that due to multiple detection (i.e., more than two pathogens), the sum of the frequencies given in these boxes may be higher the total frequency given in the black box. see also suppl. figure 1 a) overall study populaɵon adv bov cov ev hmpv hpev hrv iv-a iv-b piv rsv-a rsv-b cmv ebv hhv-6 hsv-1 for bov detection. in 39.1%, bocaviral dna was detected together with other pathogens [68] . interestingly, analysis of the ev 5′-ntr sequences gave some evidence for the presence of ev-d68 in the airways of three adults and one toddler. ev-d68 infection is associated with the development of acute flaccid myelitis and severe respiratory illness [69] . parechoviral rna was found only in a 2-year-old immunocompetent child with ia-v infection. human parechoviruses can cause mild gastrointestinal and respiratory disease but also sepsis-like illness and meningitis in infants [70] . the general low prevalence of hpev in this study is in line with a previous report [64] . the possible etiology of rti was not clarified in 45.9% of immunocompromised children (table 1) . under these conditions, application of broad diagnostic technologies like nextgeneration sequencing could be useful in identification of the underlying pathogen [71] . moreover, cmv detection rate in samples from the urt of immunocompetent children was surprisingly high (table 1) . seasonal effects were evident for several respiratory viruses (supplementary table 2 ). slight seasonality was also observed for hhv-6 in immunocompetent patients. interpretation of this finding, however, is unclear. the high prevalence of m.p. in 2011 may be explained by an epidemic observed in germany [72] . in line with this, the 2009 pandemic caused by a/h1n1pdm09 may account for a further study bias. dna of c.p. was generally not detected in our study. this is in line with the low prevalence of 0.2% reported recently [73] , but also with data from the respiratory viruses network (https://clinical-virology.net/en/charts/chart/ctype/count/ network/resp/section/bacteria). it is hypothesized that the prevalence of c.p. was overestimated in previous reports, most likely due to 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authors would like to thank all patients and their families for study support. furthermore, the great contribution of attended physicians is kindly acknowledged. the authors thank fabian cundano maltez, rosemarie carius, and martina müller for their excellent technical service. in addition, we are grateful for the continuous support given authors' contributions ak and rz conceived the study, helped with interpretation of data, and wrote the manuscript together with mw who performed statistical analyses. mr and cwo tested all respiratory tract samples by pcr and analyzed available patient data in frame of their m.d. theses. testing of nucleic acids for the presence of ev, hrv, and hpev was partially done by fk and sw as part of their bachelor theses. bg and re continuously supported the collection and analysis of samples. all authors read and approved the final version of this manuscript. the study was approved by the ethics committee of the jena university hospital (2612-07/09). the authors declare that they have no conflict of interest. key: cord-291481-ov1gkgpc authors: bonizzoli, manuela; arvia, rosaria; di valvasone, simona; liotta, francesco; zakrzewska, krystyna; azzi, alberta; peris, adriano title: human herpesviruses respiratory infections in patients with acute respiratory distress (ards) date: 2016-05-02 journal: med microbiol immunol doi: 10.1007/s00430-016-0456-z sha: doc_id: 291481 cord_uid: ov1gkgpc acute respiratory distress syndrome (ards) is today a leading cause of hospitalization in intensive care unit (icu). ards and pneumonia are closely related to critically ill patients; however, the etiologic agent is not always identified. the presence of human herpes simplex virus 1, human cytomegalovirus and epstein–barr virus in respiratory samples of critically ill patients is increasingly reported even without canonical immunosuppression. the main aim of this study was to better understand the significance of herpesviruses finding in lower respiratory tract of ards patients hospitalized in icu. the presence of this group of herpesviruses, in addition to the research of influenza viruses and other common respiratory viruses, was investigated in respiratory samples from 54 patients hospitalized in icu, without a known microbiological causative agent. moreover, the immunophenotype of each patient was analyzed. herpesviruses dna presence in the lower respiratory tract seemed not attributable to an impaired immunophenotype, whereas a significant correlation was observed between herpesviruses positivity and influenza virus infection. a higher icu mortality was significantly related to the presence of herpesvirus infection in the lower respiratory tract as well as to impaired immunophenotype, as patients with poor outcome showed severe lymphopenia, affecting in particular t (cd3+) cells, since the first days of icu hospitalization. in conclusion, these results indicate that herpesviruses lower respiratory tract infection, which occurs more frequently following influenza virus infection, can be a negative prognostic marker. an independent risk factor for icu patients with ards is an impaired immunophenotype. human herpes simplex virus 1 (hsv1), human cytomegalovirus (hcmv) and epstein-barr virus (ebv) are wellknown members of the herpesviridae family, which are highly prevalent and ubiquitous. primary infection takes place in the majority of cases early in the life and is followed by a lifelong latent infection, from which reactivation may occur with viral shedding at least in the saliva. the outcome of reactivation strongly depends from the host immunological status. in immunodepressed patients, all these three viruses may cause severe diseases, which may be different depending on the virus and on other factors, including host defences. mostly, hcmv and also hsv1 may cause severe respiratory diseases, whereas the role of ebv in pneumonia is debated [1] . in addition to a direct involvement of these viruses in respiratory diseases, their detection has been associated with other clinical aspects, which may promote viral reactivation or which outcome may be influenced by viral reactivation. an increasing number of papers report the presence of hsv1, hcmv and ebv in respiratory samples of critically ill patients even without canonical immunosuppression [1] [2] [3] [4] [5] . in patients requiring mechanical ventilation, herpesviruses, mainly hsv1 and hcmv, may be frequently detected from either upper or lower respiratory tract abstract acute respiratory distress syndrome (ards) is today a leading cause of hospitalization in intensive care unit (icu). ards and pneumonia are closely related to critically ill patients; however, the etiologic agent is not always identified. the presence of human herpes simplex virus 1, human cytomegalovirus and epstein-barr virus in respiratory samples of critically ill patients is increasingly reported even without canonical immunosuppression. the main aim of this study was to better understand the significance of herpesviruses finding in lower respiratory tract of ards patients hospitalized in icu. the presence of this group of herpesviruses, in addition to the research of influenza viruses and other common respiratory viruses, was investigated in respiratory samples from 54 patients hospitalized in icu, without a known microbiological causative agent. moreover, the immunophenotype of each patient was analyzed. herpesviruses dna presence in the lower respiratory tract seemed not attributable to an impaired immunophenotype, whereas a significant correlation was observed between herpesviruses positivity and influenza virus infection. a higher icu mortality was significantly related to the presence of herpesvirus infection in the lower respiratory tract as well as to impaired immunophenotype, as patients with poor outcome showed severe lymphopenia, affecting in particular t (cd3+) cells, since the first days of icu hospitalization. in conclusion, these results indicate that herpesviruses lower respiratory tract infection, which occurs samples [6, 7] . it has been suggested that the presence of hsv1 in the respiratory samples of icu patients correlates with the duration of tracheal intubation [5] . the detection of hsv1 in the lower respiratory tract of icu patients is reported with a variable frequency, from 5 to 64 % depending on the population and the diagnostic method used [2, 8, 9] . moreover, it is not always clear whether the demonstration of hsv1 dna in lower respiratory tract samples of non-immunocompromised ventilated patients is the consequence of a contamination from mouth or throat or is the result of local viral reactivation [4, 6, 7, 10] . some studies showed that there was a significant association between an hsv1 viral load >100.000 copies/ml of bal and admission to the icu (p < 0.0001), mechanical ventilation (p < 0.001) and death (p < 0.01) [5, 11, 12] . active hcmv infection, either restricted to the lower respiratory tract or involving both the lower respiratory airways and the systemic compartment, has been shown to occur frequently during critical illness in adult hcmv-seropositive patients [13] , and has been associated with prolonged icu hospitalization, extended periods of mechanical ventilation, higher rates of nosocomial infection and overall mortality [11, [14] [15] [16] [17] . the role of ebv presence in respiratory tract of icu patients is not clear. high degree of variability concerning the prevalence of ebv in bal samples from patients admitted in icu is reported in the literature [1, [18] [19] [20] [21] . ards is today a leading cause of hospitalization in icu. ards and pneumonia are closely related to critically ill patients [22] ; however, it is not always identified the etiologic agent. in most cases, bacterial infections are the main causative agent of pulmonary infections that evolve into framework of ards; more recently, viral infections, mainly related to influenza viruses, represent a new category of emerging cause of ards, and also viruses belonging to other families, in association or not to bacterial infections, may be involved. in still other cases, the causative agent remains unrecognized [23] . furthermore, the critically ill patients develop a state of immunosuppression, which can promote the onset and exacerbation of viral infections [24] . the aim of this study was to better understand the significance of herpesviruses finding in lower respiratory tract of patients hospitalized in icu and to assess the diagnostic and prognostic value of these findings. patients' characteristics, with attention to their immunological setting, were analyzed together with the virological data. institutional internal committee approval was waived for this study as it involved retrospective analysis of anonymous, routinely collected, group data. during the period september 2011-may 2014, 164 patients with diagnosis of ards were admitted to icu (intensive care unit of emergency department-careggi teaching hospital, florence-italy), from different clinical setting. for 54 out of these 164 patients, the causative agent of ards was unknown. the following samples were collected for microbiological analysis: • throat swab (ts) and bronchoalveolar lavage (bal) sent to general laboratory for research of common germs; • ts and bal sent to virology laboratory for the detection of influenza virus and other respiratory viruses like adenovirus (adv), parainfluenza viruses 1-4 (piv 1-4), enterovirus/rhinovirus (ev/rhv), respiratory syncytial virus (rsv), human coronaviruses (hcov) group i and group ii, human metapneumovirus (hmpv) and herpetic viruses. for each patient, the following data were collected: • anamnestic data: age, sex, body mass index (bmi), charlson comorbidity index (cci) adjusted for age; • data and severity scores at icu admission: saps ii at admission, sofa at admission, gcs at admission, provenience, length of stay before icu admission; • data related to respiratory samples: sampling timing, positivity for influenza viruses rna as well as for other respiratory viruses genome sequences, hsv1/hcmv/ ebv dna; hsv1/hcmv/ebv viral load in bal; • immunophenotyping analysis at icu admission; • data related to icu stay: treatment with antiviral, steroid; need for extracorporeal membrane oxygenation (ecmo) support and duration of treatment with ecmo; • outcome data: saps ii at discharge, gcs at discharge, ventilation length of stay (los), icu los, icu mortality, post-icu los, post-icu mortality. in the study period, 108 samples were analyzed with the aim to look for the presence of herpesviruses in 54 patients. all clinical samples were collected using standard techniques [25] . the throat swab was obtained with a nylon fiber tip (copan eswab™ system) inserted and rotated into the throat of patient. the bal samples were taken with sterile flexible bronchoscope through the oro-tracheal tube or the tracheal cannula; after the assessment of the tracheal-bronchial tree, 30 ml of sterile saline solution was instilled and picked up in a specimen trap (covidien argyle™). the detection and typing of influenza viruses were achieved as already described, using primers and probe sequence as indicated by the us centers for disease control (cdc) [26] . for the detection of other respiratory viruses, duplex real-time pcr, already described, was used [27] . the detection of hsv1 dna, hcmv dna and ebv dna was performed by in-house assays. the in-house assays here described were already used in the laboratory of virology and had shown a performance comparable with commercial assays, at a lower cost. any way the results here reported were confirmed by comparison with commercial, validated assays (realtime q-pcr kit, elitech molecular diagnostics). extraction of viral dnas from clinical samples was carried out using a commercially available kit (hp pcr template preparation kit, roche diagnostics, milan, italy). to detect hcmv, hsv1 and ebv dna in ts and bal samples, three real-time pcrs were developed, using primers listed in table 1 . the real-time pcrs were performed using 2 x hrm pcr master mix (qiagen, valencia, ca, usa). the reaction volume for each amplification was 25 μl (12.5 μl of master mix, 1.75 μl of each primer [10 μm], 5 μl of dna and h 2 o to reach the final volume). after initial activation step, 40 cycles of amplification [95 °c for 10 s, 55 °c for 30 s, 72 °c for 10 s (acquiring green)] were performed. for melting analysis, ramp from 78 to 92 °c was used, rising by 0.1 °c each step. the reaction was performed on rotor gene 6000 (qiagen, valencia, ca, usa). all herpesvirus-positive bal samples were quantified by quantitative real-time pcrs. to perform the calibration curves, serial dilutions of dna calibrator for each virus were used. these calibrators consisted of dna sequences obtained by the cloning the product of the pcr of viral dna of each virus in the pgem-t easy vector system (promega, madison, wisconsin, usa). the plasmid dna was purified by qiaprep spin miniprep kit (qiagen, valencia, ca, usa). the analytical sensitivity of all pcrs was determined using serial dilutions of cloned calibrators, quantified by nanodrop 1000 spectrophotometer (thermoscientific, wilmington, de, usa). the real-time pcr for ebv was able to detect 300 copies number/ml. the sensitivity of real-time pcr for hcmv and hsv1 was 100 copies number/ml. as the volumes and other characteristics of bal samples can vary, each bal sample was quantitatively analyzed also for the β-globin gene, as described below. then, the results obtained for each sample were normalized according to the ratio [sample target ct value × sample β-globin ct value/mean β-globin ct value] [28] . the detection of β-globin gene was performed using the primers described in the literature [29] . the sequence of primers was pf gh20 5′-caadttcatccacgttcacc-3′ and pr pc04 5′-gaagagccaaggacaggtac-3′. the real-time pcr was performed using 2 x hrm pcr master mix (qiagen, valencia, ca, usa). the reaction volume was 25 μl (12.5 μl of master mix, 1.75 μl of each primer [10 μm], 5 μl of dna and h 2 o to reach the final volume). after initial activation step, 40 cycles of amplification [95 °c for 10 s, 55 °c for 30 s, 72 °c for 10 s (acquiring green)] were performed. for melting analysis, ramp from 80 to 90 °c was used, rising by 0.1 °c each step. the reaction was performed on rotor gene 6000 (qiagen, valencia, ca, usa). peripheral blood samples (50 µl) were incubated with the appropriate fluorochrome-conjugated mabs (anti-cd3, cd4, cd8, cd19, cd16, cd56 and hla-dr) at room temperature for 15 min; red blood cells were then lysed by an appropriate lysing solution (500 µl, bd biosciences) and acquired with a bdlsr ii flow cytometer according to manufacturer's instructions (bd biosciences). at least 50.000 cells were acquired and analyzed by using the facs diva software (bd biosciences) [30] [31] [32] . the descriptive analysis is presented as mean and percentage frequencies. the mean values of the groups were compared using the student's t test for numeric values and chi-square test for ordinary variables. the analysis of variance (anova) was used for comparison of the four groups divided according to positivity for viral infections. we created a logistic model to search for variables predictors of death and a receiver operating characteristic (roc) curve to identify the cutoff of saps ii and cd3+ that discriminate for mortality. a p value <0.05 is considered statistically significant. for statistical analysis and graphic representation of data were used software microsoft excel 2007 © , graph pad prism 6.1 © and pasw 17.0 © for windows (ibm corporation, armonk, ny, usa). this study includes 54 patients who, since september 2011 to may 2014, were admitted to icu, from different clinical settings (other icus in 68.5 %, ward in 11.1 % and emergency department in 20.3 %; mean hospital stay pre-icu admission was 4.55 ± 6.65 days). this group represents 33.9 % of all patients admitted in icu with diagnosis of ards, without a known microbiological causative agent; within 48 h after icu admission, clinical samples from these patients were sent to the laboratory for the detection both bacterial and viral infections and for immunophenotyping analysis to assess the immunological status of patients. the descriptive analysis of the entire sample of patients is illustrated in table 2 . in 48.1 % of cases, patients required extracorporeal membrane oxygenation (ecmo) for severe ards, with hypoxia and/or hypercapnia unresponsive to conventional treatment. the ecmo los was on average 15.83 ± 13.13 days. one hundred and eight clinical samples from upper and lower respiratory tract from the 54 icu patients were analyzed to detect influenza and other respiratory viruses and a group of herpesviruses (ebv, hcmv and hsv1). a total of 35 patients (65 %) were positive for one or more herpesviruses in at least one respiratory sample (18 tf only, 7 bal only, 10 both samples). thus, altogether, herpesviruses were present in bal from 17 patients (31 %) and in ts from 28 patients (52 %). ebv was detected in 23 out of 54 patients (43 %), either as a single infection or as mixed infection. in only 5 patients (9 %), ebv dna was demonstrated in bal samples. in 3 cases, it was present as a single infection and in the two other as a mixed infection. hcmv was detected in 15 patients (28 %), either as single (in 2 patients) or mixed infection (in 13 patients). in seven patients (13 %), hcmv dna was demonstrated in bal samples. as regards hsv1, viral dna was detected in 15 patients (28 %). in 9 of these (17 %), it was present in bal. in addition, as bal represents a sample more suggestive of lower respiratory tract infection and/or of more invasive infection/reactivation, to understand better the significance of herpesviruses presence in this site, herpesviruses dna load in bal samples was assessed by quantitative realtime pcrs. ebv dna viral load in bal samples varied between traces (not quantifiable) in one sample to 1 × 10 9 copies number/ml in another sample with a median value of 10 3 copies/ml. altogether, ebv dna load (mean ± sd) was 21,254 ± 44,023. the range of hcmv dna load varied between traces (not quantifiable) in one sample to 1 × 10 5 copies number/ml with a median value of 10 3 copies/ml also in this case. altogether, hcmv dna load (mean ± sd) was 4240 ± 2619. the load of hsv1 in bal samples varied between 10 copies number/ml in one sample only and 10 9 copies number/ml with a median value of 10 7 . altogether, hsv1 dna load (mean ± sd) was 142,629,452 ± 422,767,741. according to herpetic viral infection positivity, patients were divided into 4 groups: group of hcmv-positive patients (n = 7); group of ebv-positive patients (n = 5); group of hsv1-positive patients (n = 9); and group of herpesvirus-negative patients (n = 37). patients positive for more than 1 herpesvirus have been included in more than one group. the analysis of the 4 groups is shown in table 3 . there were no statistically significant differences in the medical history data, the severity score values at icu admission and the provenience data. no statistically significant difference in corticosteroid treatment and in the need for extracorporeal treatment was observed. outcome data showed no statistically significant differences, except for a higher mortality in icu in patients with herpetic viral infection (hcmv group: 71.4 %, hsv1 group: 55.5 %, ebv group: 40.0 %, herpesvirus-negative group: 21.6 %; p < 0.05). a significant correlation emerged between influenza virus infection and herpetic viruses coinfection (p < 0.05). all patients with influenza positivity were treated with oseltamivir. in patients with persistent influenza infection, zanamivir was added. dividing patients into two groups, based on the positivity for influenza virus, no correlation emerged between influenza infection and icu mortality. the statistically significant data observed are reported in table 4 : patients with influenza infection showed higher incidence of herpesviruses coinfection in comparison with patients without influenza (52.6 vs 20.0 %; p = 0.01); saps ii demission score was 11 ± 6.78 for influenza-positive patients, whereas it was 27 ± 11.75 for patients without influenza. in addition, the cd3+ percentage was 68.23 ± 7.24 for influenzapositive patients and 60.37 ± 16.28 for influenza-negative patients. this observation is in agreement with the presence of lymphocytosis as a risk factor for icu admission in laboratory confirmed influenza patients [33] . an additional analysis was performed by dividing patients into two groups on the basis of icu mortality: the group of survivors included 37 patients discharged from the icu; the group of non-survivors included 17 patients died in icu. the statistically significant data are shown in table 5 . icu mortality was significantly associated with herpesviruses infection in the lower respiratory tract. in fact, 53 % of herpesviruses infected patients died in icu only few patients with laboratory confirmed herpesviruses infection were treated with acyclovir/ganciclovir and despite the treatment they died; however, the small number of observations and the lack of virological monitoring does not allow us to tray any conclusion. several immunological parameters were significantly impaired in the group of patients icu died. in particular, a clear reduction in circulating lymphocytes was evident in this group when compared to the group of patients discharged from icu. the cell reduction involves all lymphocytes populations: t (cd3+), b (cd19+) and nk cells (cd3cd16/56+). these data can account for both extravasation of cells that are recruited in inflamed organs and for cell apoptosis that typically affects hyper-activated lymphocytes. to evaluate the influence on icu mortality of viral coinfections, patients were divided into 4 groups depending on the presence of influenza and/or herpetic infection. anova analysis showed no statistically significant difference in icu mortality. in the group (n = 10) with presence of both infections (herpes and influenza), icu mortality was 40 %; in patients with only influenza positivity (n = 9), icu mortality was 22.3 %, while in patients with only herpetic positivity (n = 7), it was 71.4 %; icu mortality was 21.5 % in patients with no one viral positivity (n = 28) (p = 0.30). even if these differences are not significant, these data add further evidence to the association of icu mortality with herpesviruses infection, whereas icu mortality in patients with only influenza infection is similar to that of patients negative for both viruses. to better investigate the variables most associated with mortality, we built a logistic model with saps ii, herpesviruses positivity and total cd3 value. candidate variables were chosen as those statistically significant and/or clinically relevant to the outcome. table 6 shows that the only variable significantly associated with mortality is herpetic infection; this indicates that herpesviruses positivity is an independent predictor of death. moreover, we researched a cutoff value for saps ii and cd3+ (table 7 ; figs. 1, 2). the logistic regression is slightly over-fitted, but the hosmer-lemenshow test is not significant (p = 0.149), suggesting a good calibration of the model. ards is a relevant disease today, affecting patients of all ages that may require admission to intensive care unit. the mortality for this pathology is still high, despite the implementation of specific therapies in recent years. in patients with ards, bacterial infections are prevalent; however, there are no enough studies that highlight the presence of viral etiology. among respiratory viruses, influenza a viruses, above all of the subtype (h1n1)pdm09, may be associated with ards, as it became evident during and after the last influenza pandemic. some studies report the frequent presence of herpesviruses in respiratory samples of patients with ards [1] [2] [3] [4] [5] [6] [7] 34] . however, the significance of this positivity is still debated. this report concerns a group of 54 patients admitted to icu because of ards with unknown causative agent; 19 of them were infected by influenza virus, as demonstrated by the detection of viral rna in both upper and lower respiratory tract samples. instead, two other patients, influenza negative, were positive in the bal for rhv and adv, respectively. this study confirms that influenza viruses, mainly the h1n1 pandemic subtype, are frequently related to ards requiring icu hospitalization, whereas other common respiratory viruses showed to be involved only sporadically. in 10 of 19 influenza-positive patients, also the dna of one or more herpesvirus was present in the bal, whereas no coinfection with herpesviruses was found in the patients with rhv or adv infection. thus, in bal of 7 patients, dna of herpesviruses alone was found. these data indicate that in 28/54 patients viral infections seem to be involved in ards. however, the number of respiratory virus-positive patients could be underestimated because of the time elapsed between the onset of symptoms and the icu hospitalization. in addition, this study concerned the common respiratory viruses, whereas others like bocavirus and mimivirus were not included. moreover, it is possible that other already unknown viruses exist. data on other causative agents as bacteria or fungi have not been considered in this study. in addition to the detection of a direct viral cause of ards, this study highlights the existence of some interaction among different viruses and also among viruses, immune status and outcome of ards. in fact, a significant correlation was observed between influenza infection and herpesviruses reactivation, demonstrated by the detection of the viral dna in the bal. this observation could suggest that the respiratory mucosa damage caused by influenza virus replication can trigger herpesviruses reactivation. as regards each herpesvirus searched in the respiratory tract of the patients included in this study, ebv was the more frequently detected (43 %), whereas both hcmv and hsv1 were present in the respiratory tract of 28 % of patients. however, in bal ebv dna was found in 5 patients only (9 %), and hcmv dna and hsv1 dna were found in 7 (13 %) and 9 (17 %) patients, respectively. the frequency and dna load of hsv1 in bal samples were higher than that of hcmv and ebv, and in 6 patients, it was higher than 100,000 copies/ml, a value that is reported in the literature [5] as related to higher mortality. these results are in agreement with those of tachikawa [35] who reported that reactivation of hsv1 was predominantly observed in intubated patients regardless of their immune status, whereas reactivation of hcmv and ebv was rare in immunocompetent patients. herpesviruses reactivation, as could be inferred by the detection of viral dna in bal, was not significantly associated with impaired immunophenotype, whereas it showed to be related to icu mortality. in particular, the highest icu mortality was observed among patients with hcmv reactivation, followed by those with hsv1 reactivation and then by those with ebv reaction. as regards the role of each herpesvirus here considered, the small number of data for each virus does not allow to draw a definitive conclusion. altogether, it seems that ebv may be involved in ards like the two other herpesviruses, with a slightly lower frequency. furthermore, the data analyzed in this study indicate that icu mortality was significantly related to an impaired immunophenotype as patients with poor outcome showed severe lymphopenia, affecting in particular t (cd3+) cells, since the first days of icu hospitalization. in the present study, for the first time, as far as we know, several factors, like respiratory viral infections, respiratory infection/reactivation by some herpesviruses and immune status of the patients, have been considered and analyzed together. the results obtained, even if on a small number of patients, suggest that in a situation such complex as ards and in its outcome these factors may act at same time and synergistically: among these, viral respiratory infection, mainly by influenza a(h1n1)pdm09, herpesviruses reactivation (more frequently hsv1, hcmv and also ebv), which may be triggered by the influenza infection, and immune factors (as impaired immunophenotype). this study has several limitations which are in part related to its observational nature and the scanty samples number. it emphasizes the importance of bal analysis, whereas the analysis of viremia was performed only in few patients so that we were not able to afford a systematic analysis of these data, which must be implemented in future studies. in addition, it lacks dynamic data on herpesviruses infection, like resolution or persistence of viral infections. in addition, the usefulness of acyclovir/ganciclovir administration needs to be better studied. the data obtained imply that in ards icu patientsinfluenza virus laboratory diagnosis should be performed more frequently and as soon as possible; herpesviruses lower respiratory tract infection should monitored, together with the immunological evaluation. this could allow for a timely anti-influenza treatment which could decrease the influenza virus damage on the respiratory mucosa and eventually decrease the probability of herpesviruses reactivation. data deriving from the study of the immunological setting suggest that the evaluation of the immunophenotype is essential in order to improve the risk stratification in patients affected by systemic virus infection. detection of herpesvirus ebv dna in the lower respiratory tract of icu patients: a marker of infection of the lower respiratory tract? herpes simplex virus lung infection in patients undergoing prolonged mechanical ventilation detection of herpes viruses in respiratory secretions of patients undergoing artificial ventilation monitoring of herpes simplex virus in the lower respiratory tract of critically ill patients using real-time pcr: a prospective study clinical impact of hsv-1 detection in the lower respiratory tract from hospitalized adult patients herpes simplex virus the most frequently isolated pathogen in the lungs of patients with severe respiratory distress herpes simplex virus from the lower respiratory tract in adult respiratory distress syndrome herpes simplex virus in the respiratory tract of critical care patients: a prospective study comment on: 'nosocomial viral ventilatorassociated pneumonia in the intensive care unit' by daubin et al herpes simplex virus pneumonia: clinical, virologic, and pathologic features in 20 patients active cytomegalovirus infection is common in mechanically ventilated medical intensive care unit patients the clinical interest of hsv1 semi-quantification in bronchoalveolar lavage immunological insights into the pathogenesis of active cmv infection in non-immunosuppressed critically ill patients prevalence and mortality associated with cytomegalovirus infection in non-immunosuppressed patients in the intensive care unit cmv in critically ill patients: pathogen or bystander increased mortality in long-term intensive care patients with active cytomegalovirus infection active cytomegalovirus infection in patients with septic shock epstein-barr virus:40 years on quantitative detection of herpes simplex virus dna in the lower respiratory tract quantitative detection of epstein-barr virus in bronchoalveolar lavage from transplant and nontransplant patients herpesviruses detection by quantitative real-time polymerase chain reaction in bronchoalveolar lavage and transbronchial biopsy in lung transplant: viral infections and histopathological correlation acute respiratory distress syndrome and pneumonia: a comprehensive review of clinical data respiratory microbiology patterns within the first 24 h of ards diagnosis: influence on outcome assessment of immunological status in the critically ill a fiberoptic bronchoscopy technique to obtain uncontaminated lower airway secretions for bacterial culture the who collaborating centre for influenza at cdc atlanta, united states of america. cdc protocol of real time rt pcr for swine influenza a(h1n1) detection of 12 respiratory viruses by duplex real time pcr assays in respiratory samples pandemic a(h1n1) 2009 influenza virus detection by real time rt-pcr:is viral quantification useful? genital human papillomavirus infection in female university students as determined by a pcr-based method mortality prediction to hospitalized patients with influenza pneumonia:po 2 /fio 2 combined lymphocyte count is the answer persistent lymphopenia after diagnosis of sepsis predicts mortality lymphopenia associated with highly virulent h5n1 virus infection due to plasmacytoid dendritic cell-mediated apoptosis of t cells subjects hospitalized with the 2009 pandemic influenza a (h1n1) virus in a respiratory infection unit: clinical factors correlating with icu admission clinical relevance of herpes simplex virus viremia in intensive care unit patients detection of herpes viruses by multiplex and real-time polymerase chain reaction in bronchoalveolar lavage fluid of patients with acute lung injury or acute respiratory distress syndrome key: cord-261251-ylvqxpba authors: ansuini, valentina; rigante, donato; esposito, susanna title: debate around infection-dependent hemophagocytic syndrome in paediatrics date: 2013-01-16 journal: bmc infect dis doi: 10.1186/1471-2334-13-15 sha: doc_id: 261251 cord_uid: ylvqxpba background: hemophagocytic syndrome (hps) is clinically defined as a combination of fever, liver dysfunction, coagulation abnormalities, pancytopenia, progressive macrophage proliferation throughout the reticuloendothelial system, and cytokine over-production, and may be primary or secondary to infectious, auto-immune, and tumoral diseases. the most consistent association is with viral infections but, as it is still debated whether any micro-organisms are involved in its pathogenesis, we critically appraised the literature concerning hps and its relationship with infections. discussion: infection-dependent hps has been widely observed, but there are no data concerning its incidence in children. a better understanding of the pathophysiology of hps may clarify the interactions between the immune system and the variously implicated potential infectious agents. epstein-barr virus (ebv) infection has been prominently associated with hps, with clonal proliferation and the hyperactivation of ebv-infected t cells. however, a number of other viral, bacterial, fungal, and parasitic infections have been reported in association with hps. in the case of low-risk hps, corticosteroids and/or intravenous immunoglobulin or cyclosporine a may be sufficient to control the biological process, but etoposide is recommended as a means of reversing infection-dependent lymphohistiocytic dysregulation in high-risk cases. summary: hps is a potential complication of various infections. a polymerase chain reaction search for infectious agents including ebv, cytomegalovirus and leishmania is recommended in clinical settings characterised by non-remitting fever, organomegaly, cytopenia and hyperferritinemia. summary: hps is a potential complication of various infections. a polymerase chain reaction search for infectious agents including ebv, cytomegalovirus and leishmania is recommended in clinical settings characterised by non-remitting fever, organomegaly, cytopenia and hyperferritinemia. hemophagocytic syndrome (hps) is a potentially fatal condition due to dysregulated lymphocyte activation and proliferation, mainly characterised by impaired or inactive natural killer (nk) cells and cytotoxic t cells, which leads to macrophage hyperactivation and over-expression of cytokines [1] . the result of this process is uncontrolled and ineffective immune activation, multi-organ dysfunction, and hemophagocytosis throughout the reticuloendothelial system [2] . the pathognomonic characteristic of hps is the activation of well-differentiated macrophages, phagocyting erythrocytes, leukocytes and platelets in bone marrow, lymph nodes, spleen, liver and other organs, which can infiltrate almost anybody district and may account for many of its systemic features [3] . hsp is still often under-diagnosed and sub-optimally managed in children [4] , but the epidemiological data are fragmentary. the syndrome was first described in 1939 as poorlycontrolled histiocyte proliferation, but has since also been called hemophagocytic histiocytosis and macrophage activation syndrome [5] [6] [7] . it can be divided into a primary genetic form and a secondary reactive form (table 1) , a distinction that has historically been used to differentiate cases of often fatal infantile hps from those caused by other etiologies that appear later in life and have a better prognosis. this difference may be artificially scholastic because primary forms can occur at any age (not only during infancy or early childhood) [8] , and both primary and secondary forms can be precipitated by infections with a substantial risk of mortality [9] . even secondary hps occurs as an imbalance between insufficient host defense, obstinate hyperinflammation, and a heterogeneous triggering event, which can be of infectious, rheumatic or neoplastic nature: therefore, the clinical disease results as the signature of a dysregulated immune activation, leading to macrophage proliferation and widespread hemophagocytosis in the reticuloendothelial system. the aim of this review is to make a critical appraisal of the literature concerning infectionrelated hps in paediatrics. genetic hps is heterogeneous and arises from defects in the critical regulatory pathways responsible for the natural termination of immune responses that lead to the failure of the homeostatic removal of cells that are superfluous or dangerous to the host. since 1999, various genetic loci related to the activity of perforin and granzyme granules have been associated with genetic autosomal recessive hps, thus explaining the impaired or absent function of nk cells and cytotoxic t cells [2, 6] . the familial form, which was first described in 1952 [10] , is an autosomal recessive disorder that is estimated to occur in 1/30,000-50,000 births, and usually appears during the first year of life [11, 12] . sporadic cases of hps associated with multiple genetic mutations have also been described [13] . the different primary forms of hps are associated with immune deficiencies, including chediak-higashi syndrome, griscelli syndrome, x-linked lymphoproliferative syndrome, wiskott-aldrich syndrome, severe combined immunodeficiency, lysinuric protein intolerance, and hermansky-pudlak syndrome. acquired hps has also been associated with a variety of viral, bacterial and mycobacterial, fungal, and parasitic infections [14] , autoimmune diseases [15] , auto-inflammatory syndromes [16] , and tumours, particularly t cell malignancies [17] . the constellation of signs and symptoms of hps is not specific and none of the biochemical abnormalities is distinctive. the dramatic presentation of the syndrome includes unremitting fever, visceromegaly, thrombocytopenia, lethargy, seizures, skin rash, pulmonary failure, and cardiac and/or renal involvement, and the mortality rate is 8-22% [18] . the most common laboratory findings are due to liver dysfunction, and include low fibrinogen, and high serum triglycerides and ferritin levels [18] . two highly diagnostic clues are increased plasma concentrations of the alpha chain of interleukin-2 receptors (also known as scd25) and impaired nk cell activity. as treatment can be life-saving and some of the clinical criteria occur late during the course of the disease, it is not necessary to satisfy all of the criteria before beginning therapy. the hallmark of hps is the phagocytosis of blood cells and their precursors: bone marrow aspiration typically reveals the normal maturity of all cell lineages, and infiltration by activated macrophages "stuffed" with other blood cells [19] . criteria for a diagnosis of hps on the basis of clinical, laboratory, and histopatological findings are the following [3] : (1) genetic diagnosis: genes known to cause the syndrome (prf1, unc13d, stx11, stxbp2, rab27a) (2) signs and symptoms (at least five of the following criteria): a) fever b) splenomegaly c) cytopenias (minimum 2 cell lines reduced) d) hypertriglyceridemia (≥ 265 mg/dl) and/or hypofibrinogenemia (≤ 150 g/dl) e) hemophagocytosis in any involved organ f ) very little or no nk cell activity g) increased ferritin ≥ 500 mg/l h) increased soluble cd25 (serum interleukin-2 receptor alpha) ≥ 2.400 u/ml the main pathophysiological abnormality in hps is cytokine dysfunction, which leads to the uncontrolled accumulation and ectopic migration of activated t lymphocytes, antigen-presenting cells and histiocytes, and multi-system inflammation [20] . the pathophysiology of acquired hps has not been fully defined, but deficient cytolytic activity leads to the persistent activation of lymphocytes and histiocytes, followed by the hypersecretion of pro-inflamatory cytokines and high soluble interleukin-2 receptor levels that correlate with the prognosis [21] . the association between hps and infections has been widely documented and both familial or sporadic cases are often precipitated by acute infections. it must also be pointed out that every form of hps can mimic infectious diseases or overwhelming bacterial sepsis, thus hindering the diagnosis of a precipitating and treatable infectious illness. virus-associated hps was first described in 1979 by risdall et al., whose series consisted of 19 patients, most of whom were immunocompromised but without any confirmed genetic or acquired immunodeficiency, and all of whom showed serological signs of viral infection [22, 23] . since then, there have been reports of hps associated with a host of infections [22] . epstein-barr virus (ebv) is the most commonly reported trigger of hps [24] . the epidemiology of ebv-related hps is not well known, although a higher incidence has been observed in asian countries, where it has been theorised there may be a more pathogenic viral strain that is genetically similar to the strains observed in nasopharyngeal carcinoma cell lines [25] . two forms of ebv-related hps have been described: the first occurring during primary infection and the second during a reactivation process [26] . during primary infection, ebv typically infects and replicates in b cells, whereas ebv-specific cytotoxic t cells are required to produce memory cells. in rare cases, ebv may infect t and nk cells and induce persistent ebv infection, which may lead to chronic active ebv infection, lymphoproliferative disorders and fulminant ebv-related hps [27] [28] [29] . serological testing can help determine whether ebv-associated hps has occurred in the setting of acute infection or is the result of a reactivation process. in addition, the real-time polymerase chain reaction (pcr) detection and quantification of ebv nucleic acid is an important laboratory means of adequately reflecting viral replication and assessing ebv load in patients with ebvrelated hps [30] . the quantitative analysis of cell-free ebv genome copy numbers after four months of treatment can assess therapeutic responses and is prognostically significant [31] . the clonal expansion of ebv-infected t lymphocytes has been demonstrated in ebv-related hps [32] and ebvpositive t cell lymphoma [33] on the basis of the presence of homogeneous viral terminal repeat sequences. the clonality of infected t lymphocytes is further suggested by the finding of monoclonal rearrangements of the t cell receptor-alpha gene in ebv-related hps [34] . the distinction between the monoclonal proliferation of t lymphocytes seen in ebv-related hps and ebv-positive t cell lymphomas may describe the extremes of a spectrum of disordered t lymphocyte proliferation following ebv infection. the inflammatory cytokine over-production seen in patients with ebv-related hps tends to be much more pronounced than that observed in patients with other forms of hps [35] . of all of the infections associated with hps, ebv infection has the worst prognosis in the presence of underlying hereditary disorders, diffuse intravascular coagulation, neutropenia, or central nervous system involvement [36] . treatment strategies vary significantly depending on the clinical features of the infection: mild cases of ebvrelated hps are treated conservatively as spontaneous regression has been reported, and antiviral therapy with acyclovir, ganciclovir or cidofovir has led to disappointing results [37] . in the case of severe ebv-related hps, the introduction of immuno-chemotherapy and, if necessary, allogenic stem cell transplantation has radically changed the history and prognosis of the disease: in such cases, the optimal treatment strategy can be centred on immunosuppressive medications that inhibit overactive t and nk cell responses (i.e. corticosteroids, cyclosporine a, intravenous immunoglobulin, anti-thymocyte globulins, etoposide, rituximab, and plasma or blood exchange transfusions) [38, 39] . hematopoietic stem cell transplantation is the last treatment resort for refractory forms of ebvrelated hps, and in the case of ebv infection occurring in genetic forms of hps [40] . the most frequent herpes viruses associated with hps other than ebv are cytomegalovirus (cmv) and human herpes virus 8 (hhv8). cmv up-regulates tumour necrosis factor gene expression and has been associated with hps in otherwise healthy patients, patients with inflammatory bowel disease, rheumatological diseases and cancer, and transplant recipients [41] . hps was observed in seven of a series of 171 patients undergoing hematopoietic stem cell transplantation, and was triggered by cmv in three cases [42] . younger age may be associated with a worse prognosis [43] . a recent study has shown that the use of specific anti-cmv therapy, such as cmv immunoglobulin, foscarnet or ganciclovir, may be therapeutic [44] . hhv8 has been associated with hps in 13 patients: most of these cases occurred in patients with a lymphoproliferative disorder [45] or immunocompromised patients [46] , and rarely in immunocompetent hosts [47] . treatment based on etoposide, ganciclovir, foscarnet or rituximab has led o successful results [48] . hps can be associated with human immunodeficiency virus (hiv) infection, alone or with a wide variety of underlying disorders. it is likely that this condition is underestimated as hiv infection and hps have many clinical and laboratory similarities. about 10% of bone marrow biopsies taken from hiv patients before the start of highly active antiretroviral therapy show active signs of hemophagocytosis [49] . hiv-related hps can be observed in cases of acute or late hiv infection, and in conjunction with immune reconstitution inflammatory syndrome, opportunistic infections, or malignancies [50] . hps may even be the initial presentation of hiv infection [51] , and it has been suggested that hiv itself may play a direct role in triggering the syndrome [52] . other common viral triggers of hps in hiv patients are ebv, cmv and hhv8, and ebv-related hps seems to be more frequent in hiv-infected children [53] . influenza-related hps has been rarely reported in immunocompromised and otherwise healthy children [54] [55] [56] [57] . one fatal case of hps was observed among 32 children hospitalised with seasonal influenza in a prospective pediatric study [58] , but reactive hps has also been associated with avian and swine (non-pandemic) influenza [59, 60] . in particular, patients with severe h5n1 (avian) influenza infection have symptoms and laboratory findings that are similar to those observed in patients with hps, mainly encephalitis, organ dysfunction with hemophagocytosis, bone marrow failure, and pro-inflammatory cytokine over-production [61] . clinical studies have found that mutations in some viral genes (ns1, pb2, ha and na) are significantly related to cytokine release, and it has been demonstrated that recombinant hemagglutinin (h5) from h5n1 virus can suppress perforin expression and reduce the cytotoxicity of t cells, including their ability to kill h5-bearing cells [62] . some authors have suggested treatment with a shorter course of etoposide and dexamethasone [63] . hps has been reported in 28 cases of parvovirus b19 infection, most of whom had hereditary spherocytosis as the underlying disease: fewer than half were children [64] [65] [66] [67] [68] . of these patients, 16 did not receive any treatment and 22 survived, thus suggesting that the prognosis of parvovirus-associated hps is better than that of the other viral-mediated forms of hps. fulminant viral hepatitis may mimic and even cause hps, with hepatitis a virus being more frequently associated with hps than the other hepatotrope viruses. fifteen cases (including children) have been described, mainly in asia: three of these patients also had a concurrent rheumatological disease (systemic juvenile idiopathic arthritis or still's disease) and two also had hepatitis c. their treatment consisted of corticosteroids, variously combined with intravenous immunoglobulin, but four patients received no specific treatment and 11 of the 15 experienced a favourable outcome [69] [70] [71] . enterovirus-related hps has been described in 12 pediatric cases: five occurred in infants aged <1 year, and the oldest patient was 18 years old. an underlying disease was found in four patients who experienced a fatal outcome (lymphoid neoplasms, lymphoblastic leukemia and juvenile idiopathic arthritis). ten patients received intravenous immunoglobulin (six in combination with corticosteroids), but only seven patients survived [72] . other viruses associated with hps include adenovirus, paramyxovirus (leading to measles and mumps), rubella virus, human parainfluenza viruses, flavivirus (leading to dengue fever) and hantavirus (leading to hemorrhagic fever and severe acute respiratory syndrome), all of which have been treated with varying courses of corticosteroids and intravenous immunoglobulin. reactive hps has frequently been associated with intracellular pathogens. the pathophysiology of hps associated with non-viral agents may be related to the production of high levels of activating cytokines by host lymphocytes and monocytes. although the pathophysiological response of the host immune system to the infectious agent is not fully understood, it is hypothesised that functional deficiencies in nk and cytoxic t cells may occur during the illness [73] . hps can be associated with disseminated mycobacterium tuberculosis infection. thirty-six cases (including infants and children) have so far been reported, approximately half of which were accompanied by comorbidities: eight patients had end-stage renal disease and were receiving hemodialysis or had undergone renal transplantation, four had a history of a malignancy, two had aids, and one had sarcoidosis. fever was the most frequent clinical feature upon presentation, combined with visceromegaly and pancytopenia, and all of the patients underwent bone marrow aspirations that confirmed hemophagocytosis. evidence of extra-pulmonary tuberculosis was found in 83% of cases. the concluding remarks of the report stated that tuberculosis-related hps has a poor prognosis, with a mortality rate of approximately 50%, although anti-tuberculous and immunomodulatory therapy (consisting of high-dose corticosteroids, intravenous immunoglobulin, anti-thymocyte globulin, cyclosporine a, epipodophyllotoxin or plasma exchange) may lead to a better outcome [74] . early diagnostic confirmation and the timely administration of anti-tuberculous medication seem to be crucial in these patients. one reported case of hps occurred after childhood vaccination with the bacillus calmette-guérin [75] . hps has also been described in association with brucellosis, with brucella melitensis being the most frequently isolated organism [76] . leptospirosis can cause life-threatening hps as a result of an insufficient or misdirected immunological response to leptospira itself: antibiotic treatment alone is not sufficient in such cases, and treatment with corticosteroids, intravenous immunoglobulin or etoposide is required [77] . rickettsial diseases, transmitted to humans by arthropod bites and usually controlled at an intracellular level by nitric oxide synthesis, hydrogen peroxide production, and tryptophan degradation have also been related to hps: overall, 15 cases of rickettsial disease confirmed serologically and complicated by hps have been published in the period 1990-2010, with only 3 cases occurring in patients less than 15 years and a prognosis influenced by the specific rickettsia species, patient's immunologic equipment, and delay in antibiotic therapy or corticosteroid therapy [78] . in 2009, sepsis caused by multidrugresistant acinetobacter baumannii following urinary tract infection was reported for the first time in a previously healthy 3-year-old child, who recovered after multiple doses of granulocyte colony stimulating factor and red blood cell/platelet transfusions without any cytotoxic treatment or immunotherapy [79] . hps can be associated with leishmania donovani and leishmania infantum infections, but leishmaniasis may also mimic the syndrome, as it is characteristically associated with organomegaly and pancytopenia. this is particularly important in non-endemic areas, where visceral leishmaniasis is unlikely to be included in the differential diagnosis, and repeated bone marrow smears are often required to identify leishmania species by means of pcr with species-specific probes [80] . specific anti-leishmania treatment with amphotericin b is usually sufficient to control hps. unfortunately, sporadic cases of undiagnosed leishmaniasis have been treated as hps with fatal consequences [81] . malaria (caused by plasmodium falciparum and plasmodium vivax), toxoplasmosis, babesiosis, and strongyloidiasis have been rarely identified in association with hps: a history of travel from endemic countries may help to identify these triggering agents [82] . yeast (candida spp., cryptococcus spp. and pneumocystis spp.) and moulds (histoplasma spp., aspergillus spp. and fusarium spp.) have been associated with the occurrence of hps, most commonly during hiv infection, neoplastic diseases, protracted corticosteroid administration, and transplantation [83] [84] [85] . disseminated penicillium marneffei infection is common among hiv-infected patients in many regions in southeast asia: the first case of hps associated with penicilliosis in a thai hiv-infected child was reported in 2001, with complete recovery after antifungal and intravenous immunoglobulin therapy [86] . many immunological, neoplastic and genetic disorders may underlie hps, but infectious causes are the most prevalent and most frequently reported in association with this syndrome. the specific clinical and laboratory tests for microbiological identification of hps are the following: ( (7) serum cryptococcal antigen and serum galactomannans as a fatal outcome may occur when infection-related hps is only treated supportively, a multidisciplinary approach by experienced clinicians and infectious disease specialists is required in order to ensure the appropriate management of the syndrome itself, and the precipitating or underlying infection. pediatricians should be alert and aware of the risk of the syndrome, because an early diagnosis can change its natural history and it has been shown that prompt treatment improves the overall prognosis. a combination of high fever unresponsive to broad-spectrum antibiotics, hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, cytopenia, organomegaly and characteristic histological findings in the setting of an infectious process (particularly ebv infection, but also other viral and bacterial, parasitic and fungal infections) is the key diagnostic clue. a better understanding of the pathophysiology of hps should clarify the interactions between immune system pathways and infections. specific antimicrobial therapy can be beneficial in selected cases, whereas antiviral drugs do not seem to be curative. severe cases of infection-dependent hps require immunosuppressants or chemotherapeutic agents, while bone marrow transplantation is the ultimate choice for persistent refractory cases. hemophagocytic syndrome (hps) can occur as a rare complication of various infections in children. clonal proliferation of t lymphocytes with an excessive activation of macrophages can be triggered by different infectious agents, thus indicating that infection per se is involved in the pathogenetic mechanism of the process. a number of studies have demonstrated that hps is frequently triggered by one of many different viral, bacterial, parasitic or fungal infections, with large differences in terms of treatment responses and overall outcomes. all patients meeting the criteria for hps should undergo initial tests to diagnose the underlying infecting organism, which should be guided by epidemiological data and the patient's medical history. a polymerase chain reaction search for infectious agents, including ebv, cmv and leishmania, is recommended in a clinical scenario characterised by unremitting fever, organomegaly, cytopenia and hyperferritinemia. as hps may be associated with many infectious diseases and immunological, neoplastic or genetic disorders, the close cooperation of pediatricians and infectious disease specialists is crucial in order to define any precipitating or underlying condition. the authors declare that they have no competing interests. authors' contributions va and dr drafted the manuscript and contributed equally to its preparation; se revised the draft and made a substantial scientific contribution. all of the authors read and approved the final version of the manuscript. viral infection associated with haemophagocytic syndrome macrophage activation syndrome infections associated with the hemophagocytic syndrome nationwide survey of hemophagocytic lymphohistiocytosis in japan histiocytic medullary reticulosis diagnostic guidelines for hemophagocytic lymphohistiocytosis. the fhl study group of the histiocyte society familial hemophagocytic lymphohistiocytosis. clinical review based on the findings in seven children familial hemophagocytic 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of brucellosis and pancytopenia in a large series: hematological malignancies, the unusual cause of pancytopenia in patients with brucellosis myelodysplastic syndrome (mds) as a late stage of subclinical hemophagocytic lymphohistiocytosis (hlh): a putative role for leptospira infection. a hypothesis haemophagocytic syndrome and rickettsial diseases infection associated with hemophagocytic lymphohistiocytosis triggered by nosocomial infection secondary hemophagocytic lymphohistiocytosis in zoonoses. a systematic review hemophagocytic syndrome: a misleading complication of visceral leishmaniasis in children -a series of 12 cases infection-associated haemophagocytosis: the tropical spectrum a child case of haemophagocytic syndrome associated with cryptococcal meningoencephalitis reactive hemophagocytic syndrome: a new presentation of disseminated histoplasmosis in patients with aids reconstitution inflammatory syndrome related to histoplasmosis, with a hemophagocytic syndrome in hiv infection penicilliosis-associated hemophagocytic syndrome in a human immunodeficiency virus-infected child: the first case report in children submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution key: cord-268207-4dabwcfi authors: maakaroun, nadine rouphael; moanna, abeer; jacob, jesse t; albrecht, helmut title: viral infections associated with haemophagocytic syndrome date: 2010-02-01 journal: rev med virol doi: 10.1002/rmv.638 sha: doc_id: 268207 cord_uid: 4dabwcfi haemophagocytic syndrome (hps) or haemophagocytic lymphohistiocytosis (hlh) is a rare disease caused by a dysfunction of cytotoxic t cells and nk cells. this t cell/nk cell dysregulation causes an aberrant cytokine release, resulting in proliferation/activation of histiocytes with subsequent haemophagocytosis. histiocytic infiltration of the reticuloendothelial system results in hepatomegaly, splenomegaly, lymphadenopathy and pancytopenia ultimately leading to multiple organ dysfunctions. common clinical features include high fevers despite broad spectrum antimicrobials, maculopapular rash, neurological symptoms, coagulopathy and abnormal liver function tests. haemophagocytic syndrome can be either primary, i.e. due to an underlying genetic defect or secondary, associated with malignancies, autoimmune diseases (also called macrophage activation syndrome) or infections. infectious triggers are most commonly due to viral infections mainly of the herpes group, with ebv being the most common cause. hps can be fatal if untreated. early recognition of the clinical presentation and laboratory abnormalities associated with hps and prompt initiation of treatment can be life saving. hps triggered by viral infections generally does not respond to specific antiviral therapy but may be treated with immunosuppressive/immunomodulatory agents and, in refractory cases, with bone marrow transplantation. copyright © 2010 john wiley & sons, ltd. haemophagocytic syndrome (hps), also called haemophagocytic lymphohistiocytosis (hlh) [1] , is characterised by impaired or absent activity of nk cells and cytotoxic t-cells leading to cytokine dysregulation with proliferation and activation of histiocytes. hps results in multiorgan dysfunction and haemophagocytosis within the reticuloendothelial system characterised by pancytopenia and organomegaly [1] . if untreated, hps can be fatal. hps was first described by scott and robb-smith in 1939 [2] who termed it 'histiocytic medullary reticulosis'. virus-associated hps (vahs) was first characterised in 1979 by risdall in a case series of 19 patients [3] . the incidence of hps is difficult to estimate since the syndrome is certainly underdiagnosed. a nationwide survey of hps in japan, where a large number of hps studies have been done, reported [4] an annual incidence of 1 in 800 000. hps can be divided into primary or genetic hps and secondary or reactive hps (table 1) . primary hps usually occurs early in life and is associated with a higher mortality rate, while secondary hps occurs later in life and generally carries a better prognosis. this distinction is not categorical as primary hps can occur later in life [5] , may be triggered by infections, [6] and may not be associated (up to 50-60% [7] ) with a 'typical' genetic mutation. the familial form of hlh (fhlh) was first described in 1952 [8] by farquhar and claireaux. fhlh is an autosomal recessive disorder estimated to occur in 1 out of 30 000 to 50 000 births [9, 10] and in 70-80% of cases manifests in the first year of life. other forms of genetic hps are associated with immune deficiency syndromes including chediak-higashi syndrome, griscelli syndrome, x-linked lymphoproliferative syndrome (xlp), wiskott-aldrich syndrome, severe combined immunodeficiency, lysinuric protein intolerance and hermansky-pudlak syndrome. the true incidence of reactive hps is difficult to define. reactive hps can be divided into three categories: 1. macrophage activation syndrome [11] associated with autoimmune diseases. 2. malignancy associated hlh [12] (especially lymphoma-associated haemophagocytic syndrome). 3. infection associated hlh [13] (iahs). iahs are most commonly associated with viral infections, which are the focus of this review. iahs can also be caused by bacterial infections including mycobacteria and spirochetes as well as fungal and parasitic pathogens [1] . a review of the published cases in 219 children diagnosed with iahs before 1996 [12] found that more than half were from the far east. ebv was the triggering virus in 74% of the children in whom an infectious agent was identified. overall mortality was 52% but was higher (73%) in patients with ebv-hps. haemophagocytic disorders arise from defects in critical regulatory pathways responsible for the natural termination of immune and inflammatory responses with a subsequent failure of a homeostatic removal of cells that are superfluous or dangerous to the host organism. the role of granule (perforin/granzymes)-mediated cytotoxicity is important in both the killing of infected cells and the termination of the immune response [14] . genetic hps can be attributed to a defect in the mechanism of granule (perforin/granzymes)mediated cytotoxicity. several genetic loci related to the activity of perforin/granzymes granules have been implicated in the pathophysiology of genetic hps [15] . a defective triggering of apoptosis in fhlh has also been described in reference [16] . specific genes or mutations associated with primary hps include the prf1 gene (perforin), the munc 13-4 family of genes, the syntaxin 11 gene and the sh2-domain containing gene 1a. the pathophysiology of acquired hps is not fully understood. it certainly involves the interaction between t cells and macrophages but this does not exclude a genetic predisposition in affected cases. the persistent activation of lymphocytes is the result of an uncontrolled immune response causing a hypersecretion of pro-inflammatory cytokines such as ifn [17] , tnf [18] , il-6 [19] , il-8, il-10 [18] , il-16 [20] , il-18 [21] and macrophage-colony-stimulating factor (m-csf). il-18 appears to play a crucial role particularly in autoimmune related hps. in addition, higher levels of ifn and il-10 are associated with a worse prognosis in childhood hps [22] . the activation of t lymphocytes also results in an elevation of soluble il-2 receptor [23] which is one of the diagnostic criteria of hps and correlates with prognosis [24] . furthermore, the presence of a specific tnf polymorphism has been associated with an increased susceptibly to reactive hps in certain asian populations [25] . the proliferation of histiocytes is characterised by an up-regulation of adhesion and mhc class i and ii molecules [26] on monocytes/macrophages and expansion of inflammatory monocytes (increase in cd14/cd16 expression [27] ). the persistent activation of lymphocytes [28] and proliferation of histiocytes leads to organ infiltration and hemophagocytosis of various blood cells by macrophages present in bone marrow, lymph nodes and spleen. diagnosis of hps relies on specific clinical, laboratory, and histopathological findings proposed by the histiocyte society in 1991 [29] and updated in 2004 [30] ( table 2 ). the main symptoms and signs of hps are prolonged high fever, splenomegaly and cytopenias involving more than two cell lines. less frequently observed clinical findings include jaundice, hepatomegaly, lymphadenopathy, rash and neurological signs (table 3) . common laboratory findings result from prominent liver dysfunction [31] : low fibrinogen levels, high bilirubin levels, elevated serum transaminases, elevated prothrombin and partial thromboplastin times. high triglyceride levels are common in hps and are therefore included in the diagnostic criteria. in a study of 28 patients with secondary hps, 68% had hypertrigyceridemia at diagnosis or at some time during the disease process and triglyceride levels subsequently decreased with successful treatment of hps [32] . an often striking elevation of ferritin [33] levels is characteristic though it can be found in other diseases. a study from texas found that a ferritin level above 10 000 mg/l was 90% sensitive and 96% specific for hps [34] . two other main diagnostic parameters are an increased plasma concentration of the alpha chain of the il-2 receptor (scd25) and impaired nk cell activity. typical histopathologic findings include activated macrophages with engulfed leukocytes, erythrocytes, platelets and their precursor cells. the haemophagocytosis can be seen in any organ but is particularly common in bone marrow, lymph nodes, liver and spleen eventually resulting in organomegaly and organ failure. at presentation, hemophagocytosis is only found in a minority of cases, but can be more easily detected as the syndrome progresses. therefore, if haemophagocytosis is absent in initial biopsy specimens, the biopsy may need to be repeated in cases with high suspicion. the diagnosis of hps is established if one or two of the following criteria are fulfilled: (1) a molecular diagnosis consistent with hps (i.e., prf mutations, sap mutations, munc13-4 mutations) (2) five out of eight of the following criteria clinical ¼ fever splenomegaly laboratory ¼ cytopenia (affecting > 2 cell lineages, hemoglobin 9 g/dl, platelets < 100 000/ml, neutrophils < 1000/ml) elevated triglyceride levels ( 5 265 mg/dl) and/or low fibrinogen levels ( 150 mg/dl) elevated ferritin levels (( ! 500 ng/ml)* histopathology ¼ hemophagocytosis without evidence of malignancy biologic markers ¼ low or absent nk cell cytotoxicity* elevated soluble cd25 (il-2r chain; ! 2400u/ml)* *criteria added to the previous hlh diagnostic guidelines. virus associated haemophagocytic syndromes virus associated haemophagocytic syndromes 95 95 once a diagnosis of hps is established, a search for an underlying disease (genetic, rheumatologic or malignant disease) and a possible infectious trigger should be performed. treatment hps is frequently fatal if not treated promptly. as early therapy for hps can be life saving and some of the diagnostic criteria may only become apparent late in disease it is not necessary to fulfil all diagnostic criteria before initiating therapy. treatment is based on the control of the cytokine storm and the cellular proliferation. in iahs, treatment of the underlying infectious trigger is not sufficient to control the disease especially if hps is associated with ebv. immunochemotherapy as proposed by the histiocyte society (with the hlh-94 protocol [35, 36] and subsequently the hlh-04 protocol [30] ) consists of combination therapy with etoposide, dexamethasone and cyclosporine a as well as, in selected patients, intrathecal therapy with methotrexate and corticosteroids. the hlh-94 protocol was found to be highly effective in an early series of 17 cases of ebv-hps [37] . the efficacy of hlh-94 was later confirmed by a large series including 47 patients of ebv-hps [38] where none of the 33 patients receiving more than four doses of etoposide died. the prompt use of etoposide (less than 4 weeks after diagnosis) greatly improves the outcome in both pediatric [38] and adult patients [39] . the use of cyclosporine in treating hps was first suggested by oyama et al., in 1989 [40] when it was found to reduce the rate of fatal infections associated with neutropenia. although these immunemodulating chemotherapies are effective, they are commonly associated with side effects, most of which are more pronounced following prolonged courses of treatment. cyclosporine is associated with severe headaches, hypertension, seizures and renal impairment. corticosteroids have many well-described adverse effects, including hyperglycemia, fluid retention, hypertension and weight gain. etoposide is associated with myelosuppression and development of secondary leukemia. intravenous gamma immunoglobulins (ivig) [41] used with or without corticosteroids to treat hps are usually not effective as monotherapy. seventeen out of 21 patients treated with ivig ae corticosteroids had to be switched to an etoposide-based regimen to better control the dis-ease [38] . the 4 year survival was 68% for the ivig(corticosteroids group, compared to 86% for the group that received an etoposide containing regimen as first line therapy. the use of growth factors such as granulocyte (g)-csf or granulocyte monocyte (gm)-csf is generally not recommended as they can exacerbate hps [42] . haematopoietic stem cell transplantation (hsct) is recommended for patients with genetic forms and for patients with recurrent, persistent or refractory severe disease. hsct has greatly improved the survival of fhlh. a retrospective review of fhlh 25 years ago [43] described a mean survival of less than a month after onset of symptoms and a 1 year overall survival of less than 5%. currently, definitive treatment and potential cure of fhlh is only achieved by hsct with projected 5 year survival rates [44] ranging from 50% to 70%. in 1996, arico et al. [45] reviewed 122 patients with fhlh. bone marrow transplantation (bmt) recipients had a significantly superior outcome in comparison with nontransplanted patients with a 66% versus 10.1% survival rate. combining both, chemotherapy and bmt treated patients; the probability of survival at 3 years was 55% for all cases. ebv epidemiology epstein-barr virus (ebv) is a ubiquitous herpesvirus associated with most cases of infection associated hps [46] . the epidemiology of ebv associated hps/hlh (ebv-hps or ebv-hlh) is not well known. ebv-hps has a higher incidence in asian countries where each year in japan, 25 cases of ebv hps are diagnosed with female predominance and a peak incidence between 1 and 2 years of age [47] . the higher incidence rates in asian countries are theorised to be due to a more pathogenic viral strain of ebv [48] genetically similar to viral strains obtained from nasopharyngeal carcinoma cell lines. ebv-hps has been described in western countries and the prevalence in non-asian populations may be underestimated [49] [50] [51] . information on the epidemiology of ebv-hps in adults is lacking. primary ebv infection is usually asymptomatic expect for infectious mononucleosis [52] (im), seen commonly in children and young adults. during primary infection, ebv typically infects and replicates in b cells. after primary infection, ebv-specific t cells (cytotoxic t cells or ctls) are usually acquired to regulate ebv-infected b cells and induce memory b cells. while ebv has a well-described tropism for b cells and nasopharyngeal epithelial cells, the invasion of non-b cell populations plays an important role in the pathogenesis of several severe ebv related diseases, with disease manifestations generally depending on the type of ebv-infected cell [53] and the state of the host immunity. in rare cases, for instance, refractory and severe ebv disease results from either (i) chronic active ebv infection [54] (ii) acute fulminant ebv hps [55] (iii) lymphoproliferative disorders (t/nk cell leukemia/lymphoma [56] or lymphoma associated haemophagocytic syndrome [57] ). in chronic ebv infection, primarily cd4+ t cells or nk cells are affected. a dysfunction in t/nk cells results in a decrease in ebvspecific ctls and subsequent ebv-associated t/ nk cell lymphoproliferative disease. in ebv-hps, ebv infects primarily cd8þ cells [58] and results in a cytokine storm with the release of proinflammatory and th1-type cytokines [59] including tnf and ifn, resulting in the secondary activation of histiocytes and macrophages [53] . the cytokine response seen in ebv-hps tends to be much more pronounced than the one observed in non-ebv-hps [60] . although ebv-hps probably develops as a result of some element of immune dysfunction, the precise characteristics of host vulnerability to ebv are largely unknown, and the majority of ebv-hps cases occur in apparently immunocompetent and previously healthy children, adolescents and young adults. cases of ebv-hps can also be seen in the setting of established immune deficiencies such as fhlh [43] and xlp [61] . serological testing can help to determine if the ebv-hps occurred in the setting of primary infection or is the result of a reactivation process. in a recent epidemiological study from japan, the vast majority of ebv-hps cases occurred in children with primary infection. reactivation was considered a risk factor for worse outcome [4] . however, serologic methods carry some limitations. realtime pcr allows the quantitative measurement of ebv viral load. even though the vast majority of ebv in peripheral blood is found within leukocytes, ebv pcr of whole blood and serum are considered to adequately reflect the ebv load in terms of viral replication [63] . ebv pcr levels in ebv-hps are usually higher than those seen in ebv-im [64] . ebv viral load is considered a prognostic factor and a measure of the efficacy of treatment [65] in ebv-hps. various cell sorting techniques can be used to determine if t cells or nk cells are primarily involved and to confirm the diagnosis [50] . clonality should be determined by cytogenetic analysis, t-cell receptor gene rearrangements, or southern blot analysis for fused termini of the ebv genome, utilising blood, bone marrow or other lymphoid tissues. genetic testing for inherited conditions linked to hemophagocytosis, such as familial hlh and xlp should be considered, particularly in young (< 1 year of age) or male patients with ebv-hps, when there is consanguinity between parents, when hps is present in another sibling, or when hps is relapsing or refractory. mild cases of ebv-hps are treated conservatively since spontaneous regression of ebv-hps has been described in reference [66] . antiviral therapy with acyclovir, ganciclovir or cidofovir is generally ineffective in im and has provided disappointing results in ebv-hps. the optimal treatment strategy [67] for ebv-hps consists of: (i) control of the cytokine storm resulting in multiorgan failure and coagulopathy. antithymocyte globulins [68] , splenectomy [69] and plasma exchange or blood exchange transfusion [70] have been used in the treatment of ebv-hps. (ii) control of opportunistic infections particularly fungal and bacterial [71] infections in the setting of neutropenia. (iii) eradication of proliferating clonal and ebv containing t cells and nk cells. immunosup-virus associated haemophagocytic syndromes virus associated haemophagocytic syndromes 97 97 pressive medications inhibiting overactive t and nk cell responses, in conjunction with chemotherapeutic agents targeting dividing lymphocytes and mononuclear phagocytes are typically used to treat ebv-hps. early treatment is associated with markedly improved survival [47] . addition of agents that eliminate ebv infected b cells may decrease ebv viral loads and improve the efficacy of current therapeutic arsenal but are ineffective alone. rituximab [72] , a humanised anti-cd20 monoclonal antibody that targets mature b cells, was added to standard regimens to treat a few cases of ebv-hps [73] . hsct or bmt has become the treatment of choice for hlh as well as refractory cases of ebv-hps [74] . ebv-hps carries the worst prognosis among viral infections associated with hps. however, with the use of hlh-94 and hlh-04 protocols and, if necessary, bmt, the prognosis of ebv-hps has improved dramatically. in 78 patients with ebv-hps treated between 1992 and 2001, 75.6% were alive following a median follow-up of 43 months [75] . 85% of these patients were treated with etoposide-based regimens, 15% with bmt. 20% experienced a relapse. aside from ebv, the most common herpesviruses associated with hps are cmv and hhv8. cmv infection has been associated with hps (cmv-ahs) in different settings. it has been observed in healthy patients [76, 77] , premature infants [78] , patients with inflammatory bowel disease [79, 80] , rheumatological diseases [81, 82] , cancer [83] and in transplant recipients [84, 85] . in a prospective study of 171 patients undergoing hsct, 7 (4%) developed hps with cmv being the most common trigger in 3 cases [86] . the use of cmv hyperimmune globulin, foscarnet or ganciclovir has been associated with recovery in selected cases [77, [79] [80] [81] 85] . younger age may be associated with a worse prognosis, with fatal outcomes occurring in four of the five infants in the hlh japanese registry with cmv-ahs diagnosed at less than a year old [87] (1986-2002) . hhv-8 has been associated with hps, mostly in the setting of kaposi's sarcoma [88] , multicentric castleman's disease [89] or lymphoproliferative disorder [90] , in immunocompromised hosts (hiv [91] , transplant [92] ) and rarely in immunocompetent hosts [93, 94] . in a prospective cohort of 44 patients with castleman's disease and hiv, 4 (9%) had hps [89] . interestingly, in this series the cytokine levels of many known inflammatory markers were not elevated; however, il-8 and ifn were increased. in this study, all patients recovered after treatment with splenectomy, etoposide and rituximab [95] based regimens. ganciclovir and foscarnet have also been associated with recovery in some hhv8-hps cases. all other herpesviruses [96] [97] [98] [99] with the exception of hhv-7 have been associated with hps. hiv hps associated with hiv [100] infection is seen in several different settings. cases have been reported in acute or late hiv infection, in conjunction with immune reconstitution inflammatory syndromes (iris), in the setting of infections (both opportunistic and non opportunistic) [101] or malignancies. even though hemophagocytosis was observed in 20% of 56 autopsy cases of hiv positive patients [102] , clinically apparent hps is rare in this setting. this association, however, may be underreported and underdiagnosed since hiv and hps share many similar clinical and biological findings. to the best of our knowledge, 9 cases of acute hiv with hps have been reported in the literature [103] [104] [105] . there was a male predominance (m:f ¼ 8:1) with a median age at onset of 27. almost two thirds had a cd4 count < 200 cells/ml with a range of 63-500 cells/ml. treatment consisted mainly of ivig (n ¼ 3) and steroids (n ¼ 2); highly active antiretroviral therapy (haart) [104, 105] was only initiated during the acute phase in 2 cases. all cases reported in the literature survived. in a review of 39 hiv-hps cases [106] by bhatia and colleagues, 82% were male with a median age of 38 years (range . 80% had a cd4 less than 200 cells/ml with a mean of 132 cells/ml. a lower cd4 count was associated with a worse prognosis. eleven patients had no opportunistic infection or malignancy suggesting a possible pathogenetic role of hiv itself. most of the patients presented with fever, two thirds had hepatomegaly and more than half had splenomegaly; pancytopenia was very common. when information was available (n ¼ 15) regarding treatment, only three received haart and only one responded [107] to haart. two received chemotherapy inconsistent with the hlh protocol, six received ivig, five received corticosteroids and two underwent splenectomy. overall, recovery was noted in only 28% of patients with half of the deaths occurring within 1 month following the diagnosis of hps. four cases of hiv-ahs have been described in the setting of iris [108] [109] [110] . all patients were male and reported ages ranged from 33 to 45 years. symptoms occurred within 1 to 3 weeks following initiation of haart. iris hiv-hps has occurred in the setting of histoplasmosis [110] as well as hodgkin's lymphoma [109] . both patients with hodgkin's lymphoma were treated with chemotherapy [109] and one of them survived. the patient with histoplasmosis survived following treatment with ivig and amphotericin [110] . the fourth patient had a fatal outcome despite treatment with etoposide and corticosteroids [108] . the association of hps with influenza has been described in both immunocompromised [111] [112] [113] [114] and in immunocompetent patients [115, 116] . in a prospective pediatric study [117] , one fatal case of hps was observed among 32 children hospitalised with seasonal influenza. influenza associated hps has been seen with seasonal [111] [112] [113] [114] [115] [116] [117] , avian [118, 119] and swine (non-pandemic) influenza [120] . patients with severe h5n1 (avian) influenza infection have symptoms and laboratory findings similar to those seen in hps, mainly encephalitis [121] , organ dysfunction with haemophagocytosis [122] , bone marrow failure [122, 123] and cytokine storm [124, 125] . haemophagocytosis seen on autopsy and biopsy is the most common characteristic pathological finding [119, 125, 126] . the haemagglutinin found in h5n1 viruses may suppress perforin expression and reduce cytotoxicity of human cd8þ t cells including their ability to kill h5-bearing cells leading to marked lymphoproliferation and ifn-hyperproduction with macrophage overactivation [127] . with some viruses being resistant to amantadine [128, 129] and less commonly, even to certain neuraminidase inhibitors [130, 131] and since many similarities exist between hps and severe flu infection, some authors have speculated that the use of a modified hlh-94 protocol [132] with shorter course of eto-poside and dexamethasone should be considered in h5n1 infections associated with hps. one fatal case of swine flu associated hps in an immunocompetent patient has been reported in 1998 [120] . while it is possible that some fatal cases of the novel (swine-origin) h1n1 pandemic strain could also be due to hps, it is currently unclear how much hps contributes to the pathology of severe novel h1n1infection. approximately 30 cases [70, [133] [134] [135] [136] [137] [138] [139] of parvovirus b19 infection have been associated with hps. the most common underlying disease was hereditary spherocytosis. more than half of the patients were female and most of the cases were seen in adolescents and adults. the majority of patients with parvovirus-associated hps survived without any specific therapy suggesting that parvovirusassociated hps carries a better prognosis than other vahs. viral hepatitis fulminant viral hepatitis may mimic but can also be a cause of a true hps. hepatitis a virus is more commonly associated with hps than the other hepatitis virus including hepatitis b or hepatitis c. fifteen cases [140] [141] [142] of hav-hps have been described in the literature; all except 4 cases were reported from asia. the age range was 3-49 years with a median age of 28 and no gender predilection was noted. three patients had concurrent rheumatologic diseases (systemic juvenile idiopathic arthritis, still's disease), two patients also had hepatitis c and one patient was an alcoholic. four patients received no specific treatment but survived. most commonly, treatment consisted of corticosteroids ae ivig. eleven out of the 15 cases had a favourable outcome. there are 12 cases of enterovirus-associated hps that have been described in the english literature [143] : 5 cases occurred in infants less than 1 year of age, the oldest patient were 18 years old. underlying diseases were found in 4 cases (hodgkin's lymphoma, non-hodgkin's lymphoma, acute lymphoblastic leukemia and juvenile arthritis) and was associated with a higher fatality rate (75%). ten patients received ivig, six in combination with steroids. overall, seven patients survived. virus associated haemophagocytic syndromes virus associated haemophagocytic syndromes 99 99 other viruses found to be associated with hps include: adenovirus, bk virus, measles, mumps, rubella, parainfluenza virus, dengue, hantavirus, hemorrhagic fever and severe acute respiratory syndrome associated coronavirus [1] . haemophagocytic syndrome is a rare, often fatal disease frequently triggered by viral infections, most notably ebv. the treatment of the underlying infectious trigger alone tends to lead to suboptimal results. early recognition and prompt treatment have been shown to improve prognosis with severe cases often requiring chemotherapy with or without bmt. clinicians, therefore, need to be alert and suspect this entity in patients with high fever not responding to broad-spectrum 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patient with profound neutropenia secondary to virus-associated haemophagocytic syndrome use of rituximab in conjunction with immunosuppressive chemotherapy as a novel therapy for epstein barr virus-associated hemophagocytic lymphohistiocytosis a boy with fever, lymphadenopathy, hepatosplenomegaly, and lymphocytosis allogeneic hematopoietic stem cell transplantation for patients with hemophagocytic syndrome (hps) in japan longitudinal follow-up of patients with epstein-barr virusassociated hemophagocytic lymphohistiocytosis cytomegalovirus-associated hemophagocytic syndrome cytomegalovirus infectionassociated hemophagocytic syndrome cytomegalovirus infections associated hemophagocytic lymphohistiocytosis in a premature infant cytomegalovirus ileitis and hemophagocytic syndrome associated with use of anti-tumor necrosis factor-alpha antibody hemophagocytic syndrome caused by fulminant ulcerative colitis and cytomegalovirus infection: report of a case cytomegalovirus-associated hemophagocytic syndrome in a patient with adult onset still's disease systemic lupus erythematosus complicated by cytomegalovirus-induced hemophagocytic syndrome and colitis pediatric acute lymphoblastic leukemia complicated by secondary hemophagocytic lymphohistiocytosis cmv-associated, haemophagocytic syndrome following unrelated bone marrow transplantation successful treatment of cytomegalovirus-associated haemophagocytic syndrome following paediatric orthotopic liver transplantation hemophagocytic syndrome after hematopoietic stem cell transplantation: a prospective observational study occurrence of haemophagocytic lymphohistiocytosis at less than 1 year of age: analysis of 96 patients successful treatment with liposomal doxorubicin for widespread kaposi's sarcoma and human herpesvirus-8 related severe hemophagocytic syndrome in a patient with acquired immunodeficiency syndrome the successful treatment of haemophagocytic syndrome in patients with human immunodeficiency 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complicated by hemophagocytic lymphohistiocytosis and acute liver failure disseminated herpes virus (hsv-2) infection with rhabdomyolysis and hemophagocytic lymphohistiocytosis in a patient with bone marrow failure syndrome atypical varicella zoster infection associated with hemophagocytic lymphohistiocytosis human herpesvirus 6-associated hemophagocytic syndrome in a healthy adult haemophagocytic syndrome and hiv epstein-barr virus-associated hemophagocytic syndrome. a cause of fever of unknown origin in human immunodeficiency virus infection acquired immunodeficiency syndrome. clinicopathologic study of 56 autopsies hemophagocytic lymphohistiocytosis: an unusual initial presentation of acute hiv infection hemophagocytic syndrome in a patient with acute human immunodeficiency virus infection acute human immunodeficiency virus syndrome presenting with hemophagocytic lymphohistiocytosis candidiasisassociated hemophagocytic lymphohistiocytosis in a patient infected with human immunodeficiency virus successful recovery from human immunodeficiency virus (hiv)-associated haemophagocytic syndrome treated with highly active anti-retroviral therapy in a patient with hiv infection reactive hemophagocytosis associated with the initiation of highly active antiretroviral therapy (haart) in a patient with hemophagocytic syndrome after highly active antiretroviral therapy initiation: a life-threatening event related to immune restoration inflammatory syndrome? reconstitution inflammatory syndrome related to histoplasmosis, with a hemophagocytic syndrome in hiv infection influenza a and the virus associated haemophagocytic syndrome: cluster of three cases in children with acute leukaemia differential diagnosis of hemophagocytic syndrome: underlying disorders and selection of the most effective treatment an outbreak of influenza a in a neonatal intensive care unit virus associated hemophagocytic syndrome accompanied by acute respiratory failure caused by influenza a 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perforin expression in cd8þ t cells in response to hemagglutinin from avian influenza (h5n1) virus distribution of amantadine-resistant h5n1 avian influenza variants in asia detection of amantadine-resistant variants among avian influenza viruses isolated in north america and asia avian flu: isolation of drug-resistant h5n1 virus oseltamivir resistance during treatment of influenza a (h5n1) infection cytotoxic therapy for severe avian influenza a (h5n1) infection parvovirus b 19 associated hemophagocytic syndrome in a patient with hereditary sperocytosis spontaneous resolution of hemophagocytic syndrome and disseminated intravascular coagulation associated with parvovirus b19 infection in a previously healthy child parvovirus b19: a cause for aplastic crisis and hemophagocytic lymphohistiocytosis spontaneous resolution of hemophagocytic syndrome associated with acute parvovirus b19 infection and concomitant epstein-barr virus reactivation in an otherwise healthy adult acute liver failure and severe hemophagocytosis secondary to parvovirus b19 infection postrenal transplant hemophagocytic lymphohistiocytosis and thrombotic microangiopathy associated with parvovirus b19 infection tsutsugamushi disease with hemophagocytosis complicated by parvovirus b19 infection hepatitis a virus infection associated with hemophagocytic syndrome: report of two cases hemophagocytic syndrome associated with hepatitis a: case report and literature review hepatitis a-associated macrophage activation syndrome in children with systemic juvenile idiopathic arthritis: report of 2 cases enterovirus-associated hemophagocytic syndrome in children with malignancy: report of three cases and review of the literature key: cord-305746-svg3h2oi authors: mentis, a.‐f. a.; dardiotis, e.; grigoriadis, n.; petinaki, e.; hadjigeorgiou, g. m. title: viruses and endogenous retroviruses in multiple sclerosis: from correlation to causation date: 2017-05-23 journal: acta neurol scand doi: 10.1111/ane.12775 sha: doc_id: 305746 cord_uid: svg3h2oi multiple sclerosis is an immune‐mediated disease with an environmental component. according to a long‐standing but unproven hypothesis dating to initial descriptions of multiple sclerosis (ms) at the end of the 19th century, viruses are either directly or indirectly implicated in ms pathogenesis. whether viruses in ms are principally causal or simply contributory remains to be proven, but many viruses or viral elements—predominantly epstein‐barr virus, human endogenous retroviruses (hervs) and human herpesvirus 6 (hhv‐6) but also less common viruses such as saffold and measles viruses—are associated with ms. here, we present an up‐to‐date and comprehensive review of the main candidate viruses implicated in ms pathogenesis and summarize how these viruses might cause or lead to the hallmark demyelinating and inflammatory lesions of ms. we review data from epidemiological, animal and in vitro studies and in doing so offer a transdisciplinary approach to the topic. we argue that it is crucially important not to interpret “absence of evidence” as “evidence of absence” and that future studies need to focus on distinguishing correlative from causative associations. progress in the ms‐virus field is expected to arise from an increasing body of knowledge on the interplay between viruses and hervs in ms. such interactions suggest common herv‐mediated pathways downstream of viral infection that cause both neuroinflammation and neurodegeneration. we also comment on the limitations of existing studies and provide future research directions for the field. depth discussion of the viruses implicated in ms pathogenesis. we first consider viruses with the greatest evidence base, namely epstein-barr virus (ebv), human herpesvirus (hhv-6), vzv, human endogenous retroviruses (herv) and then go on to describe the potential roles for "minor" viruses in ms. we focus on the connection between viruses and ms pathophysiology rather than its clinical progression, and we highlight the limitations of existing studies and possible future research directions. the pubmed and google scholar databases were searched for articles published (or appeared "epub ahead of print") between 1 january 2006 to 31 december 2016 and the bibliographies examined. for initial screening, "multiple sclerosis," "infectious cause," "virus" or "viral model" were applied through the boolean operators "and" and "or". more specific terms were applied to different sections of the review based on their relevance. if and when an infectious agent had more than one name, all relevant search terms were applied. priority was given to original research articles and systematic reviews/metaanalyses over case reports or hypothesis/viewpoint articles and the most recent papers as applicable. some references prior to the above time period were included given their historical importance. studies referring to paediatric ms, infectious agents other than viruses and those not published in english were excluded. there is a lively ongoing debate on the role of ebv, the prevailing ms infectious risk factor and ms pathogenesis. [19] [20] [21] one hypothesis suggests that ms is caused by a genetically predisposed deficiency in eliminating previous ebv infection; ebv then persistently accumulates or even establishes itself in the brains of such patients. 7, 22 consistent with this theory, ebv might exercise a strong influence on the number of naïve and/or memory b cells and their differentiation status. 23 a competing hypothesis is that abnormal responses to ebv infection are secondary to and not a cause of ms. 24 at the epidemiological level, several systematic reviews clearly support an association between ms and ebv seropositivity. 3, 25, 26 practically, all ms patients are ebv seropositive, raising the question of whether ebv-seronegative ms patients even exist. 27 ebv seropositivity confers double the risk of ms than infectious mononucleosis (im) (or=4.56 vs or=2.17, respectively), 3 and im appears to have a stronger genetic component than ebv infection. 28 however, the reasons for this difference in risk between ebv seropositivity and im might be due to: (i) reporting bias for im; (ii) the molecular stochasticity of ebv-induced downstream events; (iii) the role of ebv latency; or, importantly, (iv) subclinical infection. high epstein-barr virus nuclear antigen (ebna) igg titres are associated with other ms risk factors such as non-hla gene loci and the hladrb1*15 allele (the most important genetic factor in ms). 7, 29 t cells restricted to the hladrb1*15 allele and linked to ms-related antigens seem to cross-react with the immunological response induced by the ebna-1 sequence. 30 however, the latest meta-analysis revealed an additive but not synergistic effect between the two risk factors, corroborating that hladrb1*15 carriage is not a confounding factor for ebv and ms. 31, 32 a highly synergistic increase (14-fold) in ms risk was reported for ebv detection or im combined with obesity, notably during adolescence. 7 however, there are conflicting results on the interaction between ebv and other well-established ms risk factors (reviewed also in 33 ). for instance, a prospective study found a positive association between smoking and ms development only in older patients and a negative one in patients less than around 30 years old, 34 whereas a later case-control study reported a negative, multiplicative interaction between im history and a prior history of smoking on ms risk. 35 with regard to vitamin d, some studies have failed to detect a statistically significant interaction, 32,36 while others have reported an interaction with either ebv antibodies or dna load. 37 mechanistically, observations that there is overlap between ebna-2a and vitamin d receptor (vdr) binding sites within ms-associated genomic regions and that ebna-3 binds to the vdr may provide further insights. 24, 38 in neuroimaging studies, mri (magnetic resonance imaging) markers of ms activity and grey matter atrophy were found to be associated with anti-ebv antibody levels. 39, 40 at the cellular level, cd8 + t cells specific for ebv lytic and latent antigens were more frequent in patients with active and inactive ms, respectively. 41 deep sequencing of t-cell receptorβ genes ("immunosequencing") showed intrathecally enriched ebv-reactive cd8 + t cells that were specific to patients with ms. 42 furthermore, in animal models using lymphocryptovirus (lcv), which is a close relative of ebv, lcv-infected b cells lost their ability to process the extrinsic pathogenic cd8 + t-cell epitope in myelin oligodendrocyte glycoprotein (mog). in doing so, they cross-presented this epitope to auto-aggressive cytotoxic t lymphocytes, a reaction that can initiate an autoimmune reaction and demyelination. 43 with regard to humoral immunity, the high antibody titres against ebna proteins in patients with ms might be due to intrathecal synthesis but it has yet to be clarified whether they result from highfrequency latent ebv-infected cells or, alternatively, have a concrete pathogenic role. 44 conversely, patients with im showed activation of mog-specific memory b cells. 45 furthermore, ebv genetic material has been identified in the csf and perivenular infiltrates of brain and spinal cord white matter, and, more recently, in the cortical grey matter and cervical lymph nodes of patients with ms. 44,46 ebv brain infection is likely to be limited to only a small number of b cells (approximately 5-3000 per 10 7 memory b cells). 23, 47 this could explain why histological studies for their detection are difficult, and it underscores the need for technologies such as massively parallel single-cell sequencing to detect these rare events in the future. 48 dual infection with ebv types 1 and 2 is more common in patients with ms compared to single infection. 49 mechanistically, ebv might act as an environmental trigger or by attacking the cns. 50 with respect to the former, an eae model with the murine ebv homologue gamma-herpesvirus 68 showed more pronounced ms-like clinicopathological features that were dependent on the latent life cycle of the virus. 51 there are a number of theories with regard to the latter mechanism of direct cns destruction by the virus: ms results from ebv infection of autoreactive b cells, which in turn produces pathogenic autoantibodies 44 ; (iv) the "mistaken self" hypothesis based on proteomic analyses shows a higher frequency of a peptide corresponding to an ebna-1 region sharing homology with the n-terminus of αb-crystallin in patients with ms. 52 overall, understanding these mechanisms paves the way for novel anti-ms strategies, notably ebv-specific adoptive immunotherapy. 22 it is also mechanistically intriguing how ebv plays a role in both cancer-a disorder of cellular proliferation-and ms-a disease characterized by neuronal cell death; however, recent reports of a genetic overlap between the ebv-related hodgkin lymphoma and ms could shed some light on this. 53, 54 in parallel, dogma that ebv cannot possibly be found in glial cells or neurons, the host immune response must remain the focus of studies, 53 or that ebv latency status underpins virus-mediated pathogenesis 24 should be re-examined in the light of recent observations that ebv can cause lytic infection in human primary neurons. 55 to summarize, in the context of discordance between the high rates of ebv infection vs low rates of ms worldwide, ebv is likely to be necessary but not sufficient to cause ms. 19 future studies on shared polygenic risk from genomewide association studies on ms cases with those with markers of increased ebv levels (eg ebna-1 56 ) are likely to shed further light on such host-pathogen interactions. a recent, inconclusive, non-systematic summary of evidence on the role of hhv-6 in ms 57 highlighted the need for a formal meta-analysis on this topic. furthermore, although hhv-6 has been detected mostly in acute demyelinating brain lesions in ms, detection rates are highly variable (hhv-6 dna in the csf ranging from 3% to 46% of patients). 58 additionally, other markers such as b-or t-cell reactivity, higher antibody responses or higher viral loads have not been consistently observed in ms patients' serum in different ethnic groups or prospective studies. 57, 59 some specific single nucleotide polymorphisms (snps; eg in cd46 and mhc2ta) are strongly associated with active replication of hhv-6 and, together, with worse clinical prognosis in ms. 60 at the edge of such gene-environment interactions lie the hervs (see below). one of their subtypes (herv-k18) was shown to be activated by hhv-6a, mainly in cell lines productively infected with the virus and followed by those with latently infected virus. these observations reinforce the notion that there is a common herv-mediated pathway downstream of viral infection in ms, 61 which might be therapeutically exploitable. 62 the marmoset (callithrix jacchus) hhv-6 model has been used to study viral neurotropism. 63 interestingly, in contrast to how the virus seems to gain entry to the human cns via olfactory pathways, 64 findings in marmosets revealed that only those with intravenous (and not intranasal) inoculation of hhv-6a (and not hhv-6b) developed neurological disease. 63 furthermore, in contrast to the global seroprevalence of >95% for hhv-6b, hhv-6a is more frequent in patients with ms than hhv-6b, which is certainly worthy of further investigation. 57 hhv-6a infection leads to apoptosis in the brain, induces autoimmunity in several ways 65 and activates antiviral genes in human astrocytes including some genes upregulated in ms. 66 varicella zoster virus (vzv)-induced encephalomyelitis is characterized by demyelination similar to that seen in ms, so vzv is suggested as an ms-triggering factor. 67 however, while some epidemiological studies reported no association between a history of varicella infection in childhood and ms risk, 68 others have observed an association, most notably for relapse-remitting (rr) and secondary progressive types. 69 a fourfold increase in ms risk in the year following herpes zoster infection has been observed in a region with a low ms prevalence. 70 regrettably, serological and molecular studies have not helped much in this area. vzv seropositivity was not significantly higher in patients with ms vs controls in two studies. 71, 72 moreover, while vzv dna was identified in the csf of patients with ms (particularly of rr type) in some studies, 73, 74 others failed to confirm these findings in the csf, blood or in acute ms lesions. 58, [75] [76] [77] more consistently, however, are the observations that the high levels of vzv dna in csf and pbmcs during relapse ultimately disappear during clinical remission. 78, 79 interestingly, the progressive ms type has been associated with vzv dna at levels between those found during the relapse and remission periods of the rr-ms type. 79, 80 the median fraction of intrathecal vzv-specific igg of total igg can differentiate patients with ms from those with vzv reactivation (35-fold higher in the latter). 81 this observation implies that low-level infection is present in at least some ms cases. it also helps address whether or not vzv detection in ms is due to reactivated, previously latent vzv infection; that is, "centripetal infection" from the neural ganglia towards the cns. 82 another theory suggests that vzv in ms is purely epiphenomenal due to leakage from destroyed sensory neurons; however, experimental evidence is lacking. 83 also, vzv has not been identified in "traditional" autoimmune diseases, implying a more specific connection with ms. 82 finally, vzv antigens induced and maintained activity of hervs in peripheral lymphocytes from patients with ms compared to controls; retroviruses, as explained below, are implicated as causal in ms. 84 although initially both were implicated in ms pathology since the 1980s, subsequent studies have continued to support a role for endogenous rather than exogenous retroviruses in ms. 85 hervs were integrated into the human genome relatively recently in evolutionary terms, that is some 30-40 million years ago, as a result of ancestral retroviral infections. in humans, they form up to 8% of the genome and constitute a notable category of long terminal repeat (ltr) retrotransposons. these transposable elements, also known as "jumping genes," change position within a genome and have repetitive sequences, explaining why it is more difficult to investigate their inheritance with classical genetics approaches. 86 despite these difficulties, the estimated 320 000 transcription factor binding sites (tfbss) regulated by hervs underscore their genomewide role. deciphering their pathophysiological roles will offer further insights into the molecular basis of disease beyond that offered by focusing exclusively on the exome. 87, 88 putative mechanisms of herv-related pathophysiology in ms are illustrated in figure there is also an established body of evidence that the envelope protein of the "ms-associated retrovirus" (msrv-env) in the herv-w family is causal for ms. 94 initially observed in leptomeningeal cells, the mrsv-env protein has been detected in ms plaques containing macrophages, microglia and perivascular cells in actively demyelinating lesions and in the astrocytes of inactive areas but not in control brains. 95 high mrsv-env dna copy number, transcript and antigen levels have recently been detected in the blood of over 70% of patients with ms 96 ; the increased dna copy number is indicative of herv-related reverse transcriptase activity. earlier studies suggested potential mrsv-env selectivity for the ms brain after observations of viral genetic material present at higher levels in the brain than in the blood of the same patients. 97 herv-w env expression is also increased on the surface of b cells and monocytes during the active phase of ms and parallels ms exacerbations. 98 this protein, a toll-like receptor 4 (tlr4) agonist, stimulates immune cells and enhances expression of markers of leucocyte adhesion to endothelial cells. the above raises interesting questions about the effect of mrsv-env on blood-brain barrier integrity. 99 in parallel, herv-w env impairs remyelination by inhibiting the differentiation of oligodendrocyte precursors to myelin-producing oligodendrocytes, potentially due to nitrosative stress. 100 the herv-w glycoprotein syncytin-1 also seems to be implicated in ms via a similar mechanism; it causes an endoplasmic reticulum stress sensor to induce inducible nitric oxygen synthase and, concomitantly, the release of oligodendrocyte cytotoxins by astrocytes (for further details, see 101 ). also, hervs can induce eae in mice, implying a role upstream of other mediators. 102 therefore, hervs seem to be implicated in both the neuroinflammatory and neurodegenerative components of the disease, rendering them promising therapeutic targets. the autoimmune mechanism may also lie in the fact that common viruses (including but not limited to hsv-1, hhv-6, ebv or influenza) can activate herv proteins 84, 103 (table 1) . as a "dual infection," ebv may be an exogenous and delayed cause for ms, with herv-w acting as a precipitant. 104 however, the mechanisms of transcriptional activation of hervs are generally obscure, as are the downstream events in human cells. a general framework might be that hervs and, more broadly, endogenous transposons act as a genomic defence response to external stimuli. 105 only a few studies have failed to find differences in the presence of herv nucleic acids or antibodies between ms cases and controls. 106, 107 in, 108 cells. this serological response may be associated with autoimmunity, although causality has yet to be established. (c) hervs are integral to the human genome but are epigenetically inactivated under normal conditions. herv expression may be induced by environmental triggers including hsv-1, hhv-6, vzv and ebv viruses to stimulate an immune response and autoimmunity. (d) the mrsv-env protein has been identified in ms plaques and is brain selective and immunopathogenic so may directly stimulate an autoimmune response. 1 t-cell responses to hiv. 111 there have been, to our knowledge, less than twenty hiv cases reported that describe demyelinating cns diseases including ms, with a disturbance in the cd8 + cytotoxic t-cell and cd4 + t regulatory cell ratio implicated as causal. 112, 113 this rarity of documented cases of hiv and ms is consistent with the largest relevant record linkage study, in which hiv patients-all presumed to have undergone highly active antiretroviral therapy (haart) therapy-were at a statistically significant reduced risk (relative risk=0.38)) for developing ms, with this relative risk including all recorded time intervals from first hiv record to the first ms record. 114 one explanation for this finding could be that hiv-induced immunodeficiency is protective against ms. alternatively, haart usually employs competitive or non-competitive reverse transcriptase (rt) inhibitors, and due to suspected similarity between the hiv rts and those of other viruses like hervs, these inhibitors might suppress expression of the latter. 115 the majority of epidemiological studies on cmv in ms are underpowered and inconclusive. 7,116 two synchronous but different metaanalyses suggested a protective role for cmv seropositivity in ms. 117, 118 at the molecular level, it seems that cmv is present in the cns including in some ms cases, but both exacerbating and protective roles are proposed. 116 for example, cmv-and brain-specific b cells are and histologically (ie the severity of the inflammatory cell infiltrate). 122 finally, cmv (betaherpesvirinae subfamily) and ebv (gammaherpesvirinae subfamily) might oppose each other with regard to the downstream immune cascade (the so-called "immune response competition"), which might explain their inverse epidemiological patterns in ms. 118 it has been also suggested that these herpesviridae viruses could both be required to elicit a "primate-specific autoimmune pathway". 116 the association between the measles virus and ms has been investigated for over 50 years, with ms postulated to be a host response to later measles infection. however, measles vaccination is not associated with ms, indicating that the measles virus is probably not connected with ms and supporting the evidence that measles vaccines are safe despite unjustified and well-publicized claims to the contrary. 123 however, it is worth mentioning that two cns complications of measles virus infection manifest with features of demyelination: acute disseminated encephalomyelitis, a differential diagnosis of paediatric ms and the very rare subacute sclerosing panencephalitis. 124 to our knowledge, recent research in this area has focused on the association between virus-specific csf-to-serum antibody indices (ais) and ms, not on virus detection using molecular techniques. the ais for measles, rubella and vzv, which form the "mrz reaction"high-specificity markers for "ruling-in" ms (reviewed in 125 )-are twofold higher than that for ebv. 126 in particular, the measles ai is higher in patients with ≥6 lesions on mri than those with fewer lesions in early ms. 127 another study showed that antimeasles virus antibody titres in the serum and csf of patients with ms increase according to the age and duration of the disease. 128 the phylogenetically close rinderpest virus has not been shown to be demyelinating or even neurotropic in its ruminant hosts. 124 interestingly, axonal damage precedes demyelination, prompting questions on the role of inflammation and astrocytes as intermediate players. 130 lymphocytic choriomeningitis virus (lcmv) can affect the human cns to cause paralysis and reduced consciousness. however, investigating its role in ms is more difficult due to low titres and short presence of lcmv in the csf. 131 in our opinion, this might indicate a "hit-and-run" mechanism. on the other hand, recent in silico predictions show high sequence and structural similarity between lcmv's nucleoprotein and specific myelin basic protein (mbp) residues. 132 murine models of chronic lcmv infection have given rise to two nobel prizes. 131 the virus is thought to activate microglia and astrocytes in the cns via a tlr2-mediated cascade. 133 moreover, lcmv blocks induction of type 1 interferon and consequential upregulation of hla class ii. this observation supports a potential virus-induced disturbance in the interferon-tumour necrosis factor balance, which is already known to trigger autoimmunity. 132 interestingly, lcmv infection limited to the periphery with concurrent cns measles virus infection can induce cns pathology via lcmv-specific cd8 + t-cell recruitment to the brain without the need for lcmv replication. the underlying reason why the brain, broadly considered "immuno-privileged", attracts these mis-recruited cells needs further exploration. 134 in rodents, certain coronavirus-family mouse hepatitis virus strains are neurotropic, disrupt the blood-brain barrier and cause immunemediated demyelinating-like lesions. 135 human coronaviruses (hcov) predominantly cause upper respiratory tract infections and are also neurotropic. recent epidemiological studies are lacking, while molecular analyses have shown the hcov-specific surface glycoprotein acts as a trigger for programmed cell death in a murine model of neurodegeneration. in addition, hcov-229e/mbp cross-reactive t cells have been isolated from patients with ms in single-cell analyses, implying a molecular mimicry mechanism (for a review, see 136 ) . in a mouse model of encephalomyelitis/demyelination induced by gliatropic murine coronavirus, the initial activation and accumulation of self-reactive cd4 + t cells were followed by a mechanism of host-mediated suppression that consequentially led to their decline, thus diminishing autoimmune phenotypes. 137 saffold virus (safv), a picornaviridae family member identified in 2007, was the first human virus in the cardiovirus genus to be described. 138 safv has a seroprevalence of over 90% in the adult population and is known to cause infection early in life. 139 safv is associated with both enteric and extra-intestinal diseases and, due to homology with tmev, is implicated in ms. 138 however, its ubiquity has created difficulties in deciphering any association between safv and ms. 17 safv was not detected in csf samples from patients with ms. 138 one hypothesis is that safv might cause low-grade persistent infection followed by inflammation rather than act as a "hit-and-run" trigger for autoimmunity. however, a recent study failed to find any safv in ms brains and only rare safv-specific oligoclonal bands in patients with ms and not different from controls. 17 there is, therefore, accumulated evidence that viruses may trigger or cause ms, with these organisms and the immune system interacting in several, potentially overlapping, ways. deciphering the epidemiological contribution of viruses to ms along with their pathogenic mechanisms may help in the development of effective targeted therapies to develop vaccines, treat the disease, prevent relapses and maintain remission. possible future research avenues include prospectively studying and monitoring carefully defined groups of patients, such as comparing patients with clinically isolated syndrome (cis) who went on to convert to ms with those that did not. although ebv has been studied in such cases, a broader causative role for viruses would be strengthened if any marker of viral presence (ie increased viral load and/or higher antiviral response) was observed in the first category. furthermore, the b-and t-cell receptor repertoires in ms samples need to be fully characterized, preferably in relation to viral detection and burden and perhaps using newer high-throughput technologies such as deep sequencing. this would be facilitated by the enrichment of immunosequencing databases with extensive experimental data on the repertoires induced by different human viruses. it would also be sensible to examine latentto-lytic switching of potentially existing viruses in ms biopsies. to complement previous efforts focusing on ebv-specific markers, 23 it would be interesting to analyse more recently proposed markers of cellular antiviral response with respect to the above switch. 142 finally, given that many viruses, not least ebv, express several proteins during different viral life cycle stages, the full spectrum of antibody responses to viruses over their infective course needs further exploration, perhaps using protein arrays methods for novel antigen discovery to overcome the limitations of current techniques. 143 animal models of multiple sclerosis evidence for viral etiology of multiple sclerosis environmental risk factors and multiple sclerosis: an umbrella review of systematic reviews and meta-analyses immunopathology of multiple sclerosis a basic overview of multiple sclerosis immunopathology variation in the human immune system is largely driven by non-heritable influences interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis genome, epigenome and rna sequences of monozygotic twins discordant for multiple sclerosis varicella zoster virus and relapsing remitting multiple sclerosis the search for the target antigens of multiple sclerosis, part 2: cd8+ t cells, b cells, and antibodies in the focus of reverse-translational research eae is not a useful model for demyelinating disease distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins infections and multiple sclerosis aetiology: neighbourhood watch igg antibodies against measles, rubella, and varicella zoster virus predict conversion to multiple sclerosis in clinically isolated syndrome virus-mediated autoimmunity in multiple sclerosis saffold cardiovirus and multiple sclerosis: no evidence for an association mycovirus-like dna virus sequences from cattle serum and human brain and serum samples from multiple sclerosis patients epstein-barr virus is a necessary causative agent in the pathogenesis of multiple sclerosis: yes epstein-barr virus is a necessary causative agent in the pathogenesis of multiple sclerosis: no eae: imperfect but useful models of multiple sclerosis epstein-barr virus and multiple sclerosis. from evidence to therapeutic strategies epstein-barr virus in the multiple sclerosis brain: a controversial issue epstein-barr virus and multiple sclerosis: association or causation? factors associated with onset, relapses or progression in multiple sclerosis: a systematic review risk factors associated with the onset of relapsing-remitting and primary progressive multiple sclerosis: a systematic review systematic review and meta-analysis of the sero-epidemiological association between epstein barr virus and multiple sclerosis evidence of genetic susceptibility to infectious mononucleosis: a twin study vitamin d, hla-drb1 and epstein-barr virus antibody levels in a prospective cohort of multiple sclerosis patients identifying patient-specific epstein-barr nuclear antigen-1 genetic variation and potential autoreactive targets relevant to multiple sclerosis pathogenesis a meta-analysis of interaction between epstein-barr virus and hla-drb1*1501 on risk of multiple sclerosis epstein-barr virus antibodies and vitamin d in prospective multiple sclerosis biobank samples association between human herpesvirus & human endogenous retrovirus and ms onset & progression the interaction between smoking and epstein-barr virus as multiple sclerosis risk factors may depend on age negative interaction between smoking and ebv in the risk of multiple sclerosis: the envims study anti-epstein-barr virus antibodies as serological markers of multiple sclerosis: a prospective study among united states military personnel ebv and vitamin d status in relapsing-remitting multiple sclerosis patients with a unique cytokine signature ebna2 binds to genomic intervals associated with multiple sclerosis and overlaps with vitamin d receptor occupancy antivirus immune activity in multiple sclerosis correlates with mri activity antibodies to epstein-barr virus and mri disease activity in multiple sclerosis increased cd8+ t cell response to epstein-barr virus lytic antigens in the active phase of multiple sclerosis high-throughput sequencing of tcr repertoires in multiple sclerosis reveals intrathecal enrichment of ebv-reactive cd8+ t cells lymphocryptovirus infection of nonhuman primate b cells converts destructive into productive processing of the pathogenic cd8 t cell epitope in myelin oligodendrocyte glycoprotein epstein-barr virus and multiple sclerosis: potential opportunities for immunotherapy infectious mononucleosis triggers generation of igg auto-antibodies against native myelin oligodendrocyte glycoprotein b-cell enrichment and epstein-barr virus infection in inflammatory cortical lesions in secondary progressive multiple sclerosis ebv infection and multiple sclerosis: lessons from a marmoset model somatic mutation in single human neurons tracks developmental and transcriptional history high frequency of co-infection by epstein-barr virus types 1 and 2 in patients with multiple sclerosis trigger, pathogen, or bystander: the complex nexus linking epstein-barr virus and multiple sclerosis latent virus infection upregulates cd40 expression facilitating enhanced autoimmunity in a model of multiple sclerosis high-density peptide microarray analysis of igg autoantibody reactivities in serum and cerebrospinal fluid of multiple sclerosis patients epstein-barr virus: the path from association to causality for a ubiquitous human pathogen meta-analysis of genome-wide association studies reveals genetic overlap between hodgkin lymphoma and multiple sclerosis gammaherpesvirus infection of human neuronal cells genetic loci for epstein-barr virus nuclear antigen-1 are associated with risk of multiple sclerosis evidence linking hhv-6 with multiple sclerosis: an update herpesviruses and human endogenous retroviral sequences in the cerebrospinal fluid of multiple sclerosis patients ebv & hhv6 reactivation is infrequent and not associated with ms clinical course herpesvirus active replication in multiple sclerosis: a genetic control? hhv-6a infection induces expression of herv-k18-encoded superantigen treatment against human endogenous retrovirus: a possible personalized medicine approach for multiple sclerosis novel marmoset (callithrix jacchus) model of human herpesvirus 6a and 6b infections: immunologic, virologic and radiologic characterization human herpesvirus-6 entry into the central nervous system through the olfactory pathway possible role of human herpesvirus 6 as a trigger of autoimmune disease transcriptome sequencing of neurologic diseases associated genes in hhv-6a infected human astrocyte varicella-zoster virus encephalomyelitis with a prominent demyelinating component a population-based casecontrol study on viral infections and vaccinations and subsequent multiple sclerosis risk association of a history of varicella virus infection with multiple sclerosis increased risk of multiple sclerosis following herpes zoster: a nationwide, population-based study the high prevalence of the varicella zoster virus in patients with relapsingremitting multiple sclerosis: a case-control study in the north of iran genetic and infectious profiles influence cerebrospinal fluid igg abnormality in japanese multiple sclerosis patients varicella-zoster virus at relapses of multiple sclerosis increased prevalence of varicella zoster virus dna in cerebrospinal fluid from patients with multiple sclerosis varicella zoster virus is not a disease-relevant antigen in multiple sclerosis low prevalence of human herpesvirus-6 and varicella zoster virus in blood of multiple sclerosis patients, irrespective of inflammatory status or disease progression detection of viral dna sequences in the cerebrospinal fluid of patients with multiple sclerosis varicella-zoster virus in cerebrospinal fluid at relapses of multiple sclerosis the participation of varicella zoster virus in relapses of multiple sclerosis varicella zoster virus in progressive forms of multiple sclerosis the fraction of varicella zoster virus-specific antibodies among all intrathecally-produced antibodies discriminates between patients with varicella zoster virus reactivation and multiple sclerosis on the viral hypothesis of multiple sclerosis: participation of varicella-zoster virus detection of varicella-zoster virus dna during medullary and brainstem relapses in multiple sclerosis activation of endogenous retrovirus reverse transcriptase in multiple sclerosis patient lymphocytes by inactivated hsv-1, hhv-6 and vzv endogenous retroviruses and multiple sclerosis-new pieces to the puzzle multiple sclerosis retrovirus-like envelope gene: role of the chromosome 20 insertion the multiple sclerosis-associated retrovirus and its herv-w endogenous family: a biological interface between virology, genetics, and immunology in human physiology and disease molecular functions of human endogenous retroviruses in health and disease a survey of endogenous retrovirus (erv) sequences in the vicinity of multiple sclerosis (ms)-associated single nucleotide polymorphisms (snps) the etiology of multiple sclerosis: genetic evidence for the involvement of the human endogenous retrovirus herv-fc1 human endogenous retrovirus herv-fc1 association with multiple sclerosis susceptibility: a meta-analysis endogenous retroviruses_auto-immune disease regulatory evolution of innate immunity through co-option of endogenous retroviruses multiple sclerosis between genetics and infections: human endogenous retroviruses in monocytes and macrophages human endogenous retrovirus w in brain lesions: rationale for targeted therapy in multiple sclerosis human endogenous retrovirus type w envelope expression in blood and brain cells provides new insights into multiple sclerosis disease quantitative analysis of human endogenous retrovirus-w env in neuroinflammatory diseases moller-larsen a. b cells and monocytes from patients with active multiple sclerosis exhibit increased surface expression of both herv-h env and herv-w env, accompanied by increased seroreactivity inflammatory response of endothelial cells to a human endogenous retrovirus associated with multiple sclerosis is mediated by tlr4 msrv envelope protein is a potent, endogenous and pathogenic agonist of human toll-like receptor 4: relevance of gnbac1 in multiple sclerosis treatment the human endogenous retrovirus envelope glycoprotein, syncytin-1, regulates neuroinflammation and its receptor expression in multiple sclerosis human endogenous retrovirus protein activates innate immunity and promotes experimental allergic encephalomyelitis in mice transcriptional derepression of the ervwe1 locus following influenza a virus infection expression and activation by epstein barr virus of human endogenous retroviruses-w in blood cells and astrocytes: inference for multiple sclerosis expression of long interspersed nuclear element 1 retroelements and induction of type i interferon in patients with systemic autoimmune disease comprehensive analysis of human endogenous retrovirus group herv-w locus transcription in multiple sclerosis brain lesions by highthroughput amplicon sequencing analysis of transcribed human endogenous retrovirus w env loci clarifies the origin of multiple sclerosis-associated retrovirus env sequences herv-k113 is not associated with multiple sclerosis in a large family-based study genomic analysis of ervwe2 locus in patients with multiple sclerosis: absence of genetic association but potential role of human endogenous retrovirus type w elements in molecular mimicry with myelin antigen hiv tat acts on endogenous retroviruses of the w family and this occurs via tolllike receptor 4: inference for neuroaids human endogenous retroviruses in neurologic disease multiple sclerosis and hiv-1 infection: case report of a hiv controller cns demyelinating disorder with mixed features of neuromyelitis optica and multiple sclerosis in hiv-1 infection. case report and literature review hiv and lower risk of multiple sclerosis: beginning to unravel a mystery using a record-linked database study does antiretroviral therapy for hiv reduce the risk of developing multiple sclerosis? cytomegalovirus: a culprit or protector in multiple sclerosis? cytomegalovirus and multiple sclerosis risk cytomegalovirus seropositivity is negatively associated with multiple sclerosis the correlation between the virus-and brain antigen-specific b cell response in the blood of patients with multiple sclerosis positivity of cytomegalovirus antibodies predicts a better clinical and radiological outcome in multiple sclerosis patients adaptive natural killer cell response to cytomegalovirus and disability progression in multiple sclerosis cmv infection attenuates the disease course in a murine model of multiple sclerosis multiple sclerosis incidence in the era of measles-mumps-rubella mass vaccinations involvement of morbilliviruses in the pathogenesis of demyelinating disease the mrz reaction as a highly specific marker of multiple sclerosis: re-evaluation and structured review of the literature intrathecal antibody production against epstein-barr and other neurotropic viruses in pediatric and adult onset multiple sclerosis measles igg antibody index correlates with t2 lesion load on mri in patients with early multiple sclerosis serum and csf measles antibody levels increase over time in patients with multiple sclerosis or clinically isolated syndrome pathogenesis and immunopathology of systemic and nervous canine distemper new aspects of the pathogenesis of canine distemper leukoencephalitis diseases of the central nervous system caused by lymphocytic choriomeningitis virus and other arenaviruses peptide motif analysis predicts lymphocytic choriomeningitis virus as trigger for multiple sclerosis lymphocytic choriomeningitis virus (lcmv) infection of cns glial cells results in tlr2-myd88/mal-dependent inflammatory responses cns recruitment of cd8+ t lymphocytes specific for a peripheral virus infection triggers neuropathogenesis during polymicrobial challenge brain invasion by mouse hepatitis virus depends on impairment of tight junctions and beta interferon production in brain microvascular endothelial cells neuroinvasive and neurotropic human respiratory coronaviruses: potential neurovirulent agents in humans self-reactive cd4(+) t cells activated during viral-induced demyelination do not prevent clinical recovery identification of cardioviruses related to theiler's murine encephalomyelitis virus in human infections saffold virus, a human theiler's-like cardiovirus, is ubiquitous and causes infection early in life human herpesvirus 6 infection as a trigger of multiple sclerosis consensus statement: virus taxonomy in the age of metagenomics epigenetics and genetics of viral latency the increased antibody response to epstein-barr virus in multiple sclerosis is restricted to selected virus proteins regulation of human endogenous retrovirus w protein expression by herpes simplex virus type 1: implications for multiple sclerosis two endogenous retroviral loci appear to contribute to multiple sclerosis the human endogenous retrovirus link between genes and environment in multiple sclerosis and in multifactorial diseases associating neuroinflammation human endogenous retrovirus-k18 env as a risk factor in multiple sclerosis viruses and endogenous retroviruses in multiple sclerosis: from correlation to causation a.-f.a.m. has been supported through an educational scholarship from the onassis public benefit foundation. the latter played no role in the design of the study, collection and/or interpretation of data, or writing of the review article. no financial conflict of interest exists. key: cord-290178-4i0z7ve8 authors: roncati, luca; lusenti, beatrice; nasillo, vincenzo; manenti, antonio title: fatal sars-cov-2 coinfection in course of ebv-associated lymphoproliferative disease date: 2020-05-24 journal: ann hematol doi: 10.1007/s00277-020-04098-z sha: doc_id: 290178 cord_uid: 4i0z7ve8 nan especially the elderly and those with pre-existing health issues or immunocompromised, covid-19 can progress from mild symptoms or signs, such as low-grade fever, h e a d a c h e , c o n j u n c t i v i t i s , r h i n o r r h e a , a n o s m i a , pharyngodynia, ageusia, cough, chills, myalgia, asthenia, skin rash, nausea vomiting, and diarrhea, to pneumonia, acute respiratory distress syndrome, renal insufficiency, disseminate intravascular coagulation, and multiple organ failure [1] . a marked lymphopenia has been observed in the most serious cases of the disease [2] . ebv belongs to the herpesviridae family and causes infectious mononucleosis as well as chronic active infections; besides, it can induce various pre-cancerous or cancerous lymphoproliferative disorders, such as mucocutaneous ulcer, hodgkin lymphoma, burkitt lymphoma, diffuse large b cell lymphoma, plasmablastic lymphoma, plasma cell myeloma, angioimmunoblastic t cell lymphoma, follicular t cell lymphoma, extranodal nk/t cell lymphoma, and aggressive nk cell leukemia, particularly in immunodeficient and/or post-transplanted patients [3] . in these subjects, the synergic action of ebv and sars-cov-2 is assumed to be burden by a very high fatality rate. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. fig. 1 an axial plan of the ct scan with contrast medium shows a 6.5 × 5.0 cm mass (a, yellow dashed circle) in the right axillary cable. the in situ hybridization for ebv (eber probe, × 10 objective) points out several positive blue-stained hodgkin cells inside the core needle biopsy, taken from the axillary mass (b, on the left), and in the bone marrow specimen (b, on the right). the chest x-ray performed at the patient's bed in isolation hospital room shows a diffuse bilateral interstitial pneumonia (c) clinical characteristics of 3,062 covid-19 patients: a meta-analysis lymphopenia predicts disease severity of covid-19: a descriptive and predictive study epstein-barr virus-associated lymphoproliferative disorders: review and update on 2016 who classification key: cord-307016-4hdsb5oq authors: allen, upton; green, michael title: prevention and treatment of infectious complications after solid organ transplantation in children date: 2010-04-30 journal: pediatric clinics of north america doi: 10.1016/j.pcl.2010.01.005 sha: doc_id: 307016 cord_uid: 4hdsb5oq effective prevention, diagnosis, and treatment of infectious diseases after transplantation are key factors contributing to the success of organ transplantation. most transplant patients experience different kinds of infections during the first year after transplantation. children are at particular risk of developing some types of infections by virtue of lack of immunity although they may be at risk for other types due the effect of immunosuppressive regimens necessary to prevent rejection. direct consequences of infections result in syndromes such as mononucleosis, pneumonia, gastroenteritis, hepatitis, among other entities. indirect consequences are mediated through cytokines, chemokines, and growth factors elaborated by the transplant recipient in response to microbial replication and invasion, which contribute to the net state of immunosuppression among other effects. this review summarizes the major infections that occur after pediatric organ transplantation, highlighting the current treatment and prevention strategies, based on the available data and/or consensus. upton allen, mbbs, msc, frcpc a,b,c, *, michael green, md, mph d,e organ transplantation is the most practical means of rehabilitating patients with a variety of forms of end organ dysfunction. this procedure is arguably the outstanding clinical biomedical accomplishment of the last 3 decades. potent immunosuppressive drugs have dramatically reduced the incidence of rejection of transplanted organs, but have also increased the susceptibility of patients to opportunistic infections. 1 thus, the success of organ transplantation is dependent in part on effective prevention, diagnosis, and treatment of infectious diseases after transplantation. to this end, emphasis is increasingly being placed on prevention. most transplant patients will have evidence of microbial invasion in the first year after transplant. the effects of this microbial invasion are diverse, resulting in direct and indirect consequences. the direct consequences result in a variety of clinical infectious disease syndromes such as mononucleosis, pneumonia, gastroenteritis, hepatitis, among other entities. the indirect consequences are mediated through cytokines, chemokines, and growth factors elaborated by the transplant recipient in response to microbial replication and invasion, which contribute to the net state of immunosuppression, the pathogenesis of acute and chronic allograft injury, and in some cases, the development of lymphoproliferative or malignant disorders. the risk of infection in the solid organ transplant patient is largely determined by the interaction of 3 factors: technical/anatomic factors that involve the transplant procedure itself, and the perioperative aspects of care such as the management of vascular access, drains, and the endotracheal tube; environmental exposures (box 1); and the patient's net state of immunosuppression (box 2). in the case of technical/anatomic mishaps, the best way to prevent infection is to correct the anatomic abnormality under coverage of appropriate antimicrobial therapy as antimicrobial treatment alone will not eliminate the risk of developing recurrent infections related to the uncorrected problem. as a consequence, the transplant recipient remains at high risk of subsequent infections with an increased risk of developing antimicrobial resistance until successful correction of the underlying abnormality. 1, 2 when one is considering therapy in the transplant patient, the concept of the therapeutic prescription package is useful. this package has 2 major components: an immunosuppressive component to prevent and treat rejection and an antimicrobial component to make it safe. thus, the nature of the antimicrobial program being administered must be closely linked to the nature and intensity of the immunosuppressive program required and the resulting net state of immunosuppression. 1, 2 there are 3 modes in which antimicrobial agents can be administered to the transplant recipient: a therapeutic mode, in which antimicrobial agents are administered in the treatment of established clinical infection; a prophylactic mode, in which antimicrobial agents are administered to an entire population before an event to prevent the occurrence of an infection important enough to justify this intervention: and a preemptive mode, in which antimicrobial agents are administered to a subpopulation noted to be at particular risk of clinically important infection based on clinical, epidemiologic, or laboratory markers. this review focus on preventive strategies (prophylactic and preemptive) and on the diagnosis and management of established infection. infection in the posttransplant period has a stereotyped temporal pattern, a timetable. although some clinical syndromes, such as pneumonia, can occur at any time point after transplant, the causes may be very different at different time points. fig. 1 delineates the timetable for the onset of infections after organ transplantation in the absence of effective preventative strategies. when preventative antimicrobial therapy fails to completely protect the patient, a common clinical effect is to extend the time period in which the infectious complication will likely appear. for example, in the case of cytomegalovirus (cmv) infection, in the absence of prophylaxis cmvinduced clinical disease is most common 1 to 3 months after transplantation. when prophylaxis is used, but fails, it is common for the disease to occur 4 to 8 months after transplantation (depending on the nature and duration of the prophylaxis and the immunosuppressive regimen). 2, 3 like all patients, the transplant recipient is at risk of acquiring infections in the health care and community settings. such infections are not necessarily transplant specific. fig. 1 is a graphical representation of the timing of infections during the posttransplant period. 2 in general, 3 time periods are recognized, each with differing forms of infection: [1] [2] [3] in the first month, there are 3 major causes of infection: (1) infection that was present in the recipient before transplant, with its effects now increased as a result of surgery, anesthesia, and immunosuppressive therapy; (2) infection conveyed with a contaminated allograft; and (3) the same bacterial and candidal infections of the wound, lungs, drainage catheters, and vascular access devices that are seen in nonimmunosuppressed patients undergoing comparable surgery. most (more than 95%) of the infections occurring in the first month after transplant fall into this last category; the main factor determining the incidence of such infections is the technical aspects of surgery as well as specific aspects of perioperative and postoperative care. this second time period is when the effect of immune suppression is most notable on the risk of infection. during this period, 2 major classes of infection predominate. the first of these is attributable to a group of viral pathogens that are associated with latent and/or chronic infections. examples include cmv, epstein-barr virus (ebv), human herpes virus 6 (hhv-6), and the hepatitis viruses (b and c); all of which may cause disease through acquisition of primary infection (typically from the donor) or secondary infection within the recipient under the pressure of immune suppression (secondary infection includes reactivation of latent pathogens and reinfection with a new strain). the second set of pathogens observed in this time period cause socalled opportunistic infections and include organisms such as listeria monocytogenes, aspergillus fumigatus, and pneumocystis jiroveci. development of infection with these opportunistic pathogens is attributable to the combination of sustained immunosuppression, which is often combined with the immunomodulating effects of viral infection creating a net state of immunosuppression great enough that these opportunistic infections can occur without an especially intensive environmental exposure. information describing infections occurring in children more than 6 months after transplant is limited because transplant recipients commonly return to their homes, which are often far from their transplant centers. accordingly, details regarding infectious complications occurring in this time period may be biased to include more significant infections resulting in hospitalization. despite this limitation, experience supports dividing individuals with infections during this last time period into 2 main categories: (1) most patients with a good result from transplantation (maintenance immunosuppression, good allograft function) are at greatest risk from typical community-acquired infections (such as influenza, parainfluenza, and respiratory syncytial virus); (2) a smaller group of patients with poorer outcomes from transplantation (excessive acute and chronic immunosuppression, poor allograft function, and, often, chronic viral infection). these patients remain at high risk for recurrent infections related to uncorrected mechanical problems as well as opportunistic infections attributable to organisms like pneumocystis jiroveci, listeria monocytogenes, cryptococcus neoformans, and nocardia asteroides. the time line of infections after transplantation outlines the wide spectrum of infections that occur after transplantation. among these infections, the major burden is represented by bacteria, candida species, cmv, ebv, adenovirus, varicella zoster virus, and community-acquired respiratory viruses. in addition, certain infections represent challenges for specific organ groups (eg, bk virus infection in renal transplant recipients and toxoplasma infection in heart/heart-lung transplant recipients). selected aspects of these infections are summarized later. as indicated earlier, bacterial infections are most commonly seen during the early posttransplant period. however, bacterial infections can occur at any time after transplantation. risk factors include the presence of indwelling catheter devices, including endotracheal tubes, foley catheters and central venous catheters. in this regard, hospital-acquired gram-negative organisms, coagulase-negative staphylococci and staphylococcus aureus are often encountered. the nature of these infections and the specific pathogens involved vary according to the organ transplanted, sites of infection, the microbiologic flora of the institution, and the pretransplant status of the patient. in general, the most common site of bacterial infection is at or near the site of transplantation. urinary tract infection, notably pyelonephritis, has been recognized as the most common infectious complication among renal transplant recipients. 4 infections after organ transplantation liver transplant recipients, the most frequent site of bacterial infection is within the intraabdominal space, often accompanied by bacteremia. 5, 6 intraabdominal and wound infections are also commonly seen in intestinal transplant recipients. bacteremia, which can be partly explained by disruption of the mucosal barrier associated with harvest injury or rejection, is commonly seen. 7, 8 infection of the lower respiratory tract (including pneumonia and lung abscess) is the most common site of infection reported in most, but not all, series of pediatric heart transplant recipients. 9-12 mediastinitis is another important infection after thoracic transplantation, particularly if reexploration of the chest is required. pathogens associated with mediastinitis include s aureus and gram-negative enteric bacilli. children undergoing lung transplantation because of cystic fibrosis experience a high rate of infectious complications as they often have preexisting colonization with resistant organisms, including pseudomonas species, burkholderia species and other bacterial pathogens. [13] [14] [15] [16] given the importance and difficulty in treating these often resistant organisms, transplant centers usually recommend a thorough microbiologic evaluation of heart-lung or lung transplant candidates before transplantation. the transplant patient is also at risk of developing infection as a result of community-acquired bacterial pathogens, the most important of which is streptococcus pneumoniae (pneumococcus). transplant recipients are known to be at increased risk of pneumococcal sepsis. 17 among these patients, heart recipients who have been transplanted at a young age seem to be at an increased risk compared with other pediatric organ recipients. 17 the frequency of fungal infections varies according to the type of organ transplanted. [18] [19] [20] for example, invasive fungal infections are uncommon after renal transplantation. for these patients, the most frequently encountered entity is candida urinary tract infection. similarly for liver, heart, and intestinal transplant recipients, the major fungal infections are also caused by candida species. for all of these patients, invasive aspergillosis and other mycoses occur uncommonly. the consequences of invasive aspergillosis and other noncandidal mycoses associated with invasive infections are frequently devastating. lung transplant recipients are unique in that they experience proportionately more infections with aspergillus species compared with other organ recipients. these infections are often seen in children undergoing transplantation as treatment of cystic fibrosis and reflect infection with aspergillus that was present in the recipient before transplantation. however, aspergillus is also frequently recovered from the lungs of transplant recipients with obliterative bronchiolitis (chronic rejection of the lung) regardless of the cause of their original lung disease leading to transplantation. 21 cmv cmv infection and disease remain important causes of mortality and morbidity among pediatric organ transplant recipients. 22 data on the precise burden in pediatric organ transplant recipients are limited, however, by wide differences in data collection and reporting. in addition, nonuniform approaches to the laboratory diagnosis and definition of cmv disease applied in retrospective studies affects the ability to interpret available data. in 5 centers in the united states, 10% to 20% of liver transplant patients experienced cmv disease within 2 years after transplantation. 23 a review of first-time pediatric lung transplant patients indicated that among at-risk subjects, the incidence of cmv viremia was 29% to 32%, whereas the incidence of cmv pneumonitis was 20% during the first year after transplantation. 24, 25 cmv disease is often associated with fever and hematologic abnormalities, including leucopenia, atypical lymphocytosis, and thrombocytopenia. visceral sites affected may include the gastrointestinal tract, lungs, and liver. central nervous system involvement, including chorioretinitis, is rare in organ transplant recipients. the diagnosis of cmv infection and disease in organ transplant recipients can be affected by the variable lack of sensitivity and/or specificity of different diagnostic tests. serology has no role in the diagnosis of active cmv disease after transplantation as it does not differentiate between prior infection and active disease. the interpretation of serologic results is further confused by the potential presence of passive antibody from blood products provided during or after the transplant procedure. in addition, the altered immune responses after transplantation might impair the patient's ability to mount predictable humoral responses. viral culture of blood for cmv has limited clinical usefulness for diagnosis of disease caused by poor sensitivity. there is no role for cmv urine culture in the diagnosis of disease caused by poor specificity. 26 a positive culture from bronchoalveolar lavage specimens may not correlate with disease. 27, 28 the presence of a positive measurement of cmv load in the peripheral blood (measured by either nucleic acid amplification techniques (nat) or pp65 antigenemia assay) in a patient with a compatible cmv clinical syndrome is strongly suggestive of cmv disease. however, the cmv load may be positive before the onset or in the absence of clinical disease and may be seen in the presence of disease from other causes. further, the cmv load in the peripheral blood may be negative in some patients with tissue invasive disease, especially cmv involving the gastrointestinal tract. given the variable usefulness of these tests, histopathologic examination of involved organs is essential to confirm the presence of cmv when the diagnosis of invasive cmv disease is being considered. intravenous ganciclovir (10 mg/kg/d, given twice daily) remains the preferred drug for the treatment of cmv disease in pediatric transplant recipients. reduction of immunosuppression is desirable unless concurrent evidence of rejection precludes this. ganciclovir therapy is sometimes accompanied by cmv hyperimmune globulin therapy in some centers. typically, a clinical response to treatments is expected in 5 to 7 days after treatment has been initiated. foscarnet and cidofovir may be considered in the setting of ganciclovir resistance. the optimal length of treatment should be determined by monitoring viral loads weekly. 22 treatment is typically continued until 2 consecutive negative samples are obtained. in cases of serious disease and in tissue invasive disease without viremia, longer treatment periods with clinical monitoring of the specific disease manifestation are recommended. data are emerging on the use of valganciclovir in the prevention and treatment of cmv infection/disease among adult transplant recipients. 29, 30 considerably less data are available for children. 31 a summary of the approach to prophylaxis is outlined in table 1 , including the roles of ganciclovir with or without immune globulin and suggestions on duration of their use, where indicated. although the most feared ebv-associated disease after transplantation is posttransplant lymphoproliferative disorder (ptld), patients may experience a broad range of clinical symptoms that do not meet the definitions of ptld. these might include the manifestations of infectious mononucleosis (fever, malaise, exudative pharyngitis, lymphadenopathy, hepatosplenomegaly, and atypical lymphocytosis), specific organ diseases such as hepatitis, pneumonitis, gastrointestinal symptoms, and hematological manifestations such as leucopenia, thrombocytopenia, hemolytic anemia, and haemophagocytosis. 32 ebv-associated leiomyosarcoma has also been described. 33 ebv disease is seen most frequently in patients experiencing primary ebv infections following transplantation. rates of ebv disease and ptld vary according to the organ transplanted with recipients of intestines and lungs being at the highest risk and those receiving liver, kidney, and heart at lower risk. as for cmv disease, serology is not useful for diagnosis in the posttransplant period. the presence of increased ebv viral load in the peripheral blood as determined by quantitative polymerase chain reaction (pcr) is widely accepted as an assay to predict or indicate the likely presence of ptld. however, these assays are limited in specificity and may remain persistently elevated in asymptomatic patients. the definitive diagnosis of ebv diseases, including ptld requires histopathologic examination of biopsy material. the use of ebv-specific assays (eg, ebv encoded rna [eber] staining) enhances the sensitivity and specificity of histologic examination in these patients. the approach to the treatment of ebv disease and ptld remains somewhat controversial. reduction of immune suppression is widely accepted as critical in the management of patients with these complications. the role of the antiviral agents acyclovir and ganciclovir are unproven, although many transplant clinicians use them in the treatment of ebv infection. 34, 35 treatment approaches are often modified from regimens used to treat cmv disease. currently, when antiviral agents are used to treat ebv, the agent of choice is ganciclovir, as in vitro it is 10 times more active against ebv compared with acyclovir. the controversy on the use of these agents for ebv/ptld arises because although these agents can suppress ebv lytic infection, infections after organ transplantation they seem to be of limited value in treatment nonlytic ebv proliferation, which is believed to be the dominant component of ebv-related ptld. increasing evidence (albeit anecdotal) supports the use of the anti-cd20 monoclonal rituximab in the treatment of ebv disease and ptld. however, the optimal timing and treatment strategy for this agent remain to be defined. additional alternative strategies such as the use of chemotherapy require collaborative input from oncologists familiar with the management of ebv-related disease in organ transplant recipients. the prevention of posttransplant ebv diseases, including ptld remains controversial. antiviral regimens have been modeled from the cmv scenario. to date, preemptive reduction in immunosuppression in the setting of increasing viral load may have the most supportive data and is increasingly being used (see table 1 ). adenovirus infection may be acquired by exogenous means or endogenously as a result of reactivation of latent infection. the clinical spectrum of infection and disease in pediatric transplant recipients is variable. 36 there are more than 51 serotypes that generally show some fidelity as this relates to the types of organs affected and the resultant syndromes. 37 among liver transplant patients, disease manifestations include self-limited fever, gastroenteritis, cystitis, hepatitis, and pneumonitis. these manifestations may occur in other transplant recipients, depending on the level of immunosuppression. adenovirus dna can be detected in the peripheral blood using qualitative or quantitative pcr techniques. in the appropriate clinical setting, the presence of adenovirus dna in the blood provides presumptive evidence of infection, with examination of tissue by histopathology providing more definitive evidence of infection. the management of adenovirus infection poses challenges because of limited effective treatment options. cidofovir is currently accepted as the drug of choice. however, this conclusion is primarily based on a retrospective review of historical experience and the agent is not approved for this indication by the us food and drug administration or similar agencies. nonetheless, ongoing experience continues to support a role for the treatment of adenoviral infections with this agent. before the advent of cidofovir, intravenous ribavirin was used with anecdotal reports of successes and failures. 38 although the major burden of bk virus infection is among adult renal transplant patients, the role of this virus in pediatric organ transplantation is becoming more clearly defined. most infections are as a result of reactivation in adults. primary infection may occur, notably among pediatric transplant recipients. the major clinical manifestation in the renal transplant recipient is tubulointerstitial nephritis. renal biopsy is required for definitive diagnosis. noninvasive testing modalities include screening of blood and urine for bk dna using pcr. 39 there is no firm consensus on the preferred approach to the management of bk nephropathy. early detection is a desired goal. to that end, quantitative pcr monitoring for bk dna is performed in some centers. this often provides opportunities to modulate immunosuppression. in situations where antiviral therapy is used, the agent most often used is cidofovir, for which there are reports of success. 40 however, at present no consensus exists supporting the therapeutic efficacy of this agent. varicella zoster virus (vzv) is a major threat to pediatric transplant patients and many individuals enter transplantation without immunity to this virus. 41 immunosuppressed individuals are at risk of severe outcomes from vzv infection. visceral involvement may accompany severe infection and clinicians should be reminded that disseminated disease can rarely occur in the absence of typical cutaneous vesicles. 42 pretransplant vaccination has been shown to provide sustained humoral immunity for at least 2 years after transplantation. 43 it is strongly recommended that transplant candidates be vaccinated before transplantation. given that this is a live vaccine, the minimum interval between vaccination and transplantation is recommended to be 4 to 6 weeks. although some centers have selectively considered the use of vzv vaccine in susceptible children after transplantation, this approach cannot be recommended at this time because of the lack of safety data, given the known risk of live vaccines in immunosuppressed individuals. families of transplant patients should be educated to be alert to potential exposures in settings such as schools and should report them promptly to health care providers to allow for postexposure prophylaxis. varicella-susceptible transplant recipients should receive varicella zoster immune globulin within 96 hours after a varicella exposure. 44 if this window has passed or if varicella zoster immune globulin is not available, there is the option for the use of postexposure chemoprophylaxis with acyclovir (80 mg/kg/d, given 4 times daily for 7 days; maximum dose 800 mg, 4 times daily) starting at day 7 to 10 after exposure. 44 in the absence of profound immunosuppression, no prophylaxis is usually necessary for exposed organ recipients who are immune to vzv as a result of prior infection or vaccination before transplantation. treatment of the transplant patient with vzv infection is usually initiated with intravenous acyclovir until there is evidence of clinical improvement (fever abates, no new lesions, lesions starting to crust, no visceral disease). outpatient treatment with oral acyclovir or valacyclovir has been used in children with mild infection, low levels of immunosuppression, and when there are no concerns regarding the adequacy of follow-up. famciclovir and valacyclovir are approved for use in adults. famciclovir is the prodrug of penciclovir, which has an extended half-life in infected cells. valayclovir is the prodrug of acyclovir and produces fourfold greater serum levels than those produced by acyclovir. pediatric formulations are current not available. most children who have undergone organ transplantation experience communityacquired viral infections and have no significant problems. however, it is well recognized that children who are significantly immunocompromised can have severe disease caused by these viruses, including respiratory syncytial virus infection, parainfluenza, and influenza viruses. 45, 46 for pediatric transplant recipients, the likelihood of more severe outcomes is greater during the early months after transplantation or during periods of peak immunosuppression. in 2009, the advent of a pandemic strain of influenza a (pandemic h1n1 2009) has been cause for concern. 47, 48 in general, the principles that govern the prevention and treatment of pandemic h1n1 in pediatric transplant patients are similar to those for seasonal influenza. transplant patients are among those who are known to be at increased risk of severe outcomes from pandemic h1n1. they are candidates for treatment with oseltamivir or zanamivir (where appropriate) if they have acute respiratory illness that is suspected or confirmed to be caused by h1n1. 49 like other immunocompromised patients, they are at an increased risk of having prolonged shedding of virus and the harboring of drug-resistant strains of influenza a, including pandemic h1n1. pediatric transplant patients are candidates for vaccination against this virus (as they are for seasonal influenza a) if they are greater than 6 months of age. most infections after organ transplantation experts currently delay vaccination until after the first months following organ transplantation. 49 pneumocystis pneumonia (pcp) is a recognized threat in the posttransplant period. 50, 51 the risk is greatest during the first 6 to 12 months after transplantation, with the time of onset being usually after the first month. trimethoprim-sulfamethoxazole remains the prophylactic agent of choice. 52 this agent is also preferred for initiation of therapy in individuals who develop pcp. although the optimal duration of pcp prophylaxis remains unclear, most experts provide pcp prophylaxis for a minimum of 6 to 12 months, with some recommending indefinite use, especially for solid organ transplant recipients requiring more prolonged periods of higher levels of immunosuppression. intravenous pentamidine is an alternative for treatment of pcp for patients who are intolerant of trimethoprim-sulfamethoxazole or whose disease has not responded to this agent after 5 to 7 days. 52 however, pentamidine is associated with a relatively high incidence of adverse events, including pancreatitis, renal dysfunction, hypoglycemia, and hyperglycemia. atovaquone may be used to treat milder forms of pcp among adults; however, pediatric data are limited. alternatives to trimethoprim-sulfamethoxazole for prophylaxis include oral atovaquone or dapsone. aerosolized pentamidine is recommended if children cannot tolerate these oral agents. another alternative is intravenous pentamidine, albeit at the risk of greater toxicity. 52 toxoplasma gondii infection is of greatest concern among heart transplant patients, but infection can occur in other categories of transplant recipients, including kidney and liver recipients. 53, 54 toxoplasma organisms can remain encysted within muscle tissue, such as cardiac muscle. thus, infection is acquired as a result of the reactivation of cysts that remain dormant in the donor hearts of toxoplasma seronegative children. clinical manifestations can occur as early as 2 weeks after transplantation. manifestations include pneumonia, fever syndrome, myocarditis, chorioretinitis, and central nervous system disease. current prophylaxis includes pyrimethamine/sulfadiazine for d1rã� patients. trimethoprim-sulfamethoxazole is typically used in r1 patients. however, some experts also recommend trimethoprim-sulfamethoxazole for d1rã� patients. the duration of prophylaxis is usually 6 months. infection with this parasitic worm is of relevance to individuals who previously acquired infection following a period of residence in endemic regions. 55, 56 donorassociated transmission of stronygloides has also occurred. asymptomatic immunocompromised persons, including transplant recipients are at risk of strongyloides hyperinfection, which results from dissemination of larvae via the systemic circulation, resulting in abdominal pain, diffuse pulmonary infiltrates, and septicemia or meningitis from enteric gram-negative bacilli. serologic screening is recommended for individuals from endemic regions ( table 2 ). ivermectin treatment is indicated for screenpositive individuals. tuberculosis (tb) is always a concern for immunocompromised hosts. 57-60 incidence rates are low in most transplant centers in the developed world, but outcomes of tb can be devastating in organ transplant recipients. before transplantation a careful history for tb exposure or infection, mantoux test screening, and a chest radiograph can assists in establishing the diagnosis of latent tuberculosis infection. the interferon-gamma release assays are currently being evaluated to define their role in settings where the tb skin test has poor utility. 61, 62 the use of antituberculous agents in transplant patients poses challenges because of the interaction between isoniazid and rifampin with immunosuppressive medications. however, this should not be seen as a contraindication to the use of antituberculous agents, which have to be used when warranted by the clinical situation. the pretransplant phase is arguably the most important phase of transplantation. a detailed history and physical examination are necessary to identify conditions that influence the risk or management of infections after transplantation. this assessment allows for the identification of preexisting conditions that require treatment or prophylaxis in the period before or after transplantation. table 2 summarizes screening tests that should be performed in the pretransplant period. immunizations represent an important strategy for preventing infections in the transplant patient. [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] wherever possible, vaccines should be administered in the pretransplant period to improve the chances of optimal immunologic take. a guideline on vaccinations for the transplant candidate/patient has recently been published. 73 in some situations, accelerated vaccination schedules may be used for selected vaccines. given differences in childhood vaccination schedules in different jurisdictions, clinicians should acquaint themselves with the appropriate schedules and the circumstances under which accelerated schedules could be used. when using vaccines after transplantation, one needs to be concerned about safety as well as efficacy. in general, all live virus vaccines should be avoided in the transplant recipient. the oral polio, yellow fever, and oral typhoid vaccines are live and are contraindicated in immunosuppressed patients. the live attenuated intranasal influenza vaccine is also contraindicated. measles, mumps, and rubella vaccines are somewhat contraindicated and their use should be limited to outbreak scenarios. the varicella vaccine is also somewhat contraindicated and is not approved for use in transplant patents. although limited published data support the potential use of this vaccine in transplant recipients, 72 most experts continue to advise against this practice. in the cases of the nonlive vaccines, the major concern is not safety, but efficacy. thus, in general, it is advisable to give nonlive vaccines at times when the level of immunosuppression would allow for immunogenicity. table 3 summarizes the vaccines that are indicated and contraindicated in transplant recipients. given the relative burden and importance of invasive pneumococcal disease in pediatric transplant recipients, the importance of pneumococcal vaccination should not be underestimated. 17, 63, 69 donor organ screening the organ donor is a frequent source of exposure to pathogens in the organ transplant recipient. accordingly, screening of the donor organ is a crucial aspect of the preventive strategies aimed at minimizing adverse outcomes from infections in the posttransplant period. despite a long-standing recognition of the importance of donor-derived infections, increased concern about this problem has emerged because of recent donor-related transmission of human immunodeficiency virus (hiv). this case, as well as concerns about the lack of sensitivity of serologic testing and the relatively long time period until seroconversion against hiv, hepatitis b virus (hbv), and hepatitis c virus (hcv), have led to interest in the use of nat-based testing for the pathogens hiv, hcv, and hbv. although arguments exist for and against the use of nat testing, a final international consensus addressing if and when to use these tests is only beginning to emerge. 74 decisions relating to the use of such tests must consider not only the reliability of this technology but also the feasibility of universal implementation of these testing procedures for all procurement organizations. recent cases of donor-associated transmission of lymphocytic choriomeningitis virus and west nile virus have also raised questions on whether the panel of routine tests performed on potential donors should be expanded to include these and other potential donorderived pathogens. to date, a consensus has not been reached on whether or not screening against these pathogens should be routinely included in donor testing panels. it is to be hoped that the implementation of working groups and committees focusing on the problem of donor-derived infections in north america and europe will lead to improved data to better inform subsequent recommendations regarding donor testing. current requirements for screening of nonliving donors are shown in table 4 . at present, no specific requirements have been implemented for screening allen & green of live donors. in general, testing strategies that are in place for deceased donors are applied to the use of these organs. the importance of documenting the presence of potential donor-transmissible pathogens is imperative not only to inform decision making regarding the use of potential donor organs but also because results of donor testing can inform specific preventative strategies even when donor-associated exposure to pathogens is unavoidable. various prophylaxis regimens are used in the posttransplant period. although there are common basic principles, the specific regimens vary across centers and by the type of organ transplanted. for most patients, the major targets of prophylaxis are infections after organ transplantation bacterial pathogens, herpes group viruses, and fungal pathogens, including pneumocystis. perioperative antibiotics are typically used for 48 to 72 hours to provide prophylaxis against surgical contamination. the burden of cmv infection in transplant patients is such that it represents the major focus of prevention in the posttransplant period, when intravenous ganciclovir is usually used with or without cmv hyperimmune globulin in selected patient groups. table 1 summarizes various pathogens and the regimens that are often used for prevention of infection in the posttransplant period. in the evaluation of the febrile transplant patient, clinicians should consider if the child's fever is related to common childhood infections or infections that are unique to the immunosuppressed transplant recipient. to this end, the timing of infections after transplantation (see fig. 1 ) provides guidance regarding the most likely pathogens. for example, as discussed earlier, the most likely causes of infection within the first month after transplantation are often bacterial or candidal and are largely similar to what is seen in nonimmunosuppressed patients who have undergone comparable surgery. the nature of the evaluation will depend on the clinical status of the patient and whether or not a source of infection has been identified. examination abnormal, focus of infection defined. admission to hospital may be indicated depending on the clinical status of the patient and the site of the infection. the diagnostic evaluation varies, but should include a minimum of a complete blood count and differential, blood, and urine cultures. additional investigations depend on the clinical assessment and the timing of presentation after transplantation. examination normal, no focus of infection defined. patients who are clinically unwell typically require admission for evaluation and treatment. the diagnostic evaluation should consider the likely differential diagnoses. consultation with infectious diseases is recommended. patients who are well may not necessarily require admission. however, this depends on several factors, including the adequacy of follow-up, the degree of immune suppression and the suspected diagnoses. the diagnostic evaluation should include a minimum of a complete blood count and differential, blood, and urine cultures. in all of these situations, clinicians need to be aware of the spectrum of viral infections that are associated with febrile syndromes without necessarily having a readily apparent organ focus of infection (eg, cmv virus). infection in organ-transplant recipients infection in the renal transplant patient the therapeutic prescription for the organ transplant recipient: the linkage of immunosuppression and antimicrobial strategies urinary tract infection in pediatric renal transplantation pediatric liver transplantation: patient evaluation and selection, infectious complications, and life-style after transplantation infectious complications in liver transplantation bacteremia after intestinal transplantation in children correlates temporally with rejection or gastrointestinal lymphoproliferative disease unique aspects of the infectious complications of intestinal transplantation infections in pediatric orthotopic heart transplant recipients pediatric heart transplantation at stanford: results of a 15-year experience heart transplantation during the first 12 years of life. loma linda university pediatric heart transplant group rejection and infection after pediatric cardiac transplantation pseudomonas cepacia empyema necessitatis after lung transplantation in two patients with cystic fibrosis burkholderia cepacia complex genomovars and pulmonary transplantation outcomes in patients with cystic fibrosis lung transplantation for cystic fibrosis, patients with burkholderia cepacia complex. survival linked to genomovar type update on the burkholderia cepacia complex invasive pneumococcal disease in pediatric organ transplant recipients: a high-risk population infections after organ transplantation risk factors for fungal infection in paediatric liver transplant recipients trends in invasive disease due to candida species following heart and lung transplantation australian candidemia study. candidemia following solid organ transplantation in the era of antifungal prophylaxis: the australian experience aspergillus colonization of the lung allograft is a risk factor for bronchiolitis obliterans syndrome on behalf of the transplantation society international cmv consensus group. international consensus guidelines on the management of cytomegalovirus in solid organ transplantation cmv-ivig for prevention of epstein barr virus disease and posttransplant lymphoproliferative disease in pediatric liver transplant recipients cytomegalovirus viremia associated with death or retransplantation in pediatric lung-transplant recipients the risk, prevention & outcome of cytomegalovirus after pediatric lung transplantation the prognostic significance of positive cmv cultures during surveillance of renal transplant recipients quantitative cultures of the cell fraction and supernatant of bronchoalveolar lavage fluid for the diagnosis of cytomegalovirus pneumonitis in lung transplant recipients the diagnosis of cmv pneumonitis in lung and heart/lung transplant patients by pcr compared with traditional laboratory criteria long-term outcomes of cmv disease treatment with valganciclovir versus iv ganciclovir in solid organ transplant recipients a multicenter study of valganciclovir prophylaxis up to day 120 in cmv-seropositive lung transplant recipients valganciclovir dosing according to body surface area and renal function in pediatric solid organ transplant recipients epstein-barr virus infections epstein-barr virus-associated multifocal leiomyosarcomas arising in a cardiac transplant recipient: autopsy case report and review of the literature management of epstein-barr virus-induced posttransplant lymphoproliferative disease in recipients of solid organ transplantation new developments in the diagnosis and management of posttransplantation lympholiferative disorders in solid organ transplant recipients adenoviral infections in pediatric transplant recipients: a hospital-based study adenovirus infections adenoviral infections and a prospective trial of cidofovir in pediatric hematopoietic stem cell transplantation sustained bk viruria as an early marker for the development of bkv-associated nephropathy: analysis of 4128 urine and serum samples quantitative viral load monitoring and cidofovir therapy for the management of bk virus-associated nephropathy in children and adults varicella zoster infection in solid organ transplant recipients: a hospital-based retrospective study varicella zoster virus sustainability of humoral responses to varicella vaccine in pediatric transplant recipients following a pre-transplantation immunization strategy varicella-zoster infections respiratory syncytial virus infections in pediatric liver transplant recipients parainfluenza and influenza virus infections in pediatric organ transplant recipients the first pandemic of the 21st century: a review of the 2009 pandemic variant influenza a (h1n1) virus outbreak of swine-origin influenza a (h1n1) virus infection -mexico guidance on novel influenza a/h1n1 in solid organ transplant recipients pneumocystis carinii pneumonia following heart transplantation unexpectedly high incidence of pneumocystis carinii infection after lung-heart transplantation. implications for lung defense and allograft survival pneumocystis jirovecii infections primary and reactivated toxoplasma infection in patients with cardiac transplants. clinical spectrum and problems in diagnosis in a defined population toxoplasmosis in pediatric recipients of heart transplants acute respiratory failure due to disseminated strongyloidiasis in a renal transplant recipient tacrolimus allows autoinfective development of the parasitic nematode strongyloides stercoralis mycobacterial infection after liver transplantation: a report of three cases and review of the literature mycobacterium tuberculosis after liver transplantation: management and guidelines for prevention mycobacterium tuberculosis infection in solid-organ transplant recipients: impact and implications for management american society of transplantation infectious diseases community of practice. mycobacterium tuberculosis interferon-gamma release assays improve the diagnosis of tuberculosis and nontuberculous mycobacterial disease in children in a country with a low incidence of tuberculosis performance of an interferon-gamma release assay for diagnosing latent tuberculosis infection in children seven-valent pneumococcal conjugate vaccine in pediatric solid organ transplant recipients: a prospective study of safety and immunogenicity a prospective, comparative study of the immune response to inactivated influenza vaccine in pediatric liver transplant recipients and their healthy siblings double-dose accelerated hepatitis b vaccine in patients with end-stage liver disease failure of hepatitis b immunization in liver transplant recipients: results of a prospective trial immunogenicity and safety of hepatitis a vaccine in liver and renal transplant recipients immunity to poliomyelitis, diphtheria and tetanus in pediatric patients before and after renal or liver transplantation safety and immunogenicity of the american academy of pediatrics-recommended sequential pneumococcal conjugate and polysaccharide vaccine schedule in pediatric solid organ transplant recipients report from the advisory committee on immunization practices (acip): decision not to recommend routine vaccination of all children aged 2-10 years with quadrivalent meningococcal conjugate vaccine (mcv4) pretransplant varicella vaccination is cost-effective in pediatric renal transplantation ast infectious diseases community of practice. guidelines for vaccination of solid organ transplant candidates and recipients safety and immunogenicity of varicella-zoster virus vaccine in pediatric liver and intestine transplant recipients special report. nucleic acid testing (nat) of organ donors: is the ''best'' test the right test? key: cord-000849-rrezynbs authors: kumar, rajesh; andrabi, raiees; tiwari, ashutosh; prakash, somi sankaran; wig, naveet; dutta, durgashree; sankhyan, anurag; khan, lubina; sinha, subrata; luthra, kalpana title: a novel strategy for efficient production of anti-v3 human scfvs against hiv-1 clade c date: 2012-11-15 journal: bmc biotechnol doi: 10.1186/1472-6750-12-87 sha: doc_id: 849 cord_uid: rrezynbs background: production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing hiv-1 infection, however only a few such antibodies have been generated till date. isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. this has been avoided by the process described in this paper, wherein, by combining the strategy of ebv transformation and recombinant dna technology, we constructed human single chain variable fragments (scfvs) against the third variable region (v3) of the clade c hiv-1 envelope. results: an antigen specific phage library of 7000 clones was constructed from the enriched v3positive antibody secreting ebv transformed cells. by ligation of the digested scfv dna into phagemid vector and bio panning against the hiv-1 consensus c and b v3 peptides followed by random selection of 40 clones, we identified 15 clones that showed v3 reactivity in phage elisa. dna fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. expression of the positive clones was tested by sds-page and western blot. all the 13 anti-v3 scfvs showed cross-reactivity against both the clade c and b v3 peptides and did not show any reactivity against other unrelated peptides in elisa. preliminary neutralization assays indicated varying degrees of neutralization of clade c and b viruses. ebv transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scfv generation, thus avoiding the problems of hybridoma technology. moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. after selection, the phage clones were propagated in a clonal manner. conclusions: this strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. this is the first study to generate anti-v3 scfvs against hiv-1 clade c. there is a rapid increase in the number of human immunodeficiency virus (hiv-1) infected individuals worldwide and so far we have met with little success in slowing down or preventing the progression of this pandemic disease. in order to use broadly neutralizing antibodies as effective reagents for passive immunotherapy to slow or to halt the disease progression in hiv-1 infected individuals and for immunogen design for vaccination to prevent the infection, the generation of large numbers of human hiv-1 specific monoclonal antibodies is desirable. although a few human broadly neutralizing antibodies (bnabs) to hiv-1 exist [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] , these antibodies have limited reactivity against non-clade b viruses, which are responsible for more than 85% of the infections worldwide [4] . few bnabs exist so far, that are effective against the clade c viruses, which include the 4e10, antibodies from the caprisa cohort and the recently isolated monoclonal antibodies pg9, pg16 and vrc01 [9] [10] [11] [12] . in order to evaluate their utility in combating hiv-1 infection, and to tackle the problems posed by the extensive diversity of hiv-1, it is essential to generate a large panel of human anti-hiv-1 antibodies of different specificities. further, it may be necessary to evaluate several antibodies to find rare but highly effective molecules. the methods used for the generation of human monoclonal antibodies include the hybridoma technology, recombinant technology by phage display and the recently employed techniques such as single b cell sorting followed by amplification of heavy and light chain genes [8, 13, 14] . generation of antibodies by the conventional hybridoma technology is not adequate enough to meet the challenge of assessing large numbers of human monoclonal antibodies from hiv-1 infected individuals at various stages of their clinical course. our approach to the problem has been to combine the antigen specific pre-selection of ebv transformed b cells with the construction of a phage library. phage display is a scalable method for antibody production against a wide variety of antigens [15] [16] [17] . investigators are using this technology for the production of antibodies with the desired isotype specificities and affinity for research, clinical and industrial applications. antibody gene variable regions are amplified and expressed on the surface of filamentous bacteriophage as a fusion protein [14] and a number of antibodies can be produced from a single library and can be expressed and produced in a prokaryotic system. the major drawback in this technology is the requirement for the construction of large sized phage libraries (10 9 -10 10 ) with an optimum diversity because the diversity of these libraries primarily determines the successful isolation of the desired antibodies. construction of such large libraries requires a number of ligation and electroporation reactions. screening of positive clones from these libraries generally require at least three to seven rounds of biopanning to select specifically binding clones [18, 19] . it is also difficult to maintain such large sized libraries. keeping all these factors in mind we have described here a simple and efficient method for the production of human monoclonal antibodies using a combinational approach of ebv transformation, antigen preselection and phage display technology. a similar approach was previously used by kempf et al. to isolate fabs to the gp120 of hiv-1 [20] . here, we adopted a modified strategy and isolated for the first time, human anti-v3 scfvs from the ebv transformed lymphocytes of a clade c hiv-1 infected patient. this method can be used to produce large panels of viral specific phage libraries in less time and in a cost effective manner. a limited number of monoclonal antibodies are generated against the hiv-1 clade c which is the most predominant subtype worldwide and in india [21] . the v3 region of hiv-1 gp120 is an important target to induce neutralizing antibodies against different strains of hiv-1. it exhibits a high degree of sequence variability in the most structurally conserved region of the hiv-1 envelope [22, 23] and it interacts with the co-receptors ccr5 and cxcr4 [7, 24, 25] . in this study, we constructed a scfv library from the ebv transformed b cells of an hiv-1 infected patient #254, reactive to the third variable (v3) region of gp120 of hiv-1 clade c. the plasma sample of this patient was previously tested and found to exhibit cross neutralizing activity against a standard panel of pseudoviruses of different clades (table 1 ) and also had cross reactive binding antibodies to clade c and b v3 peptides [26, 27] . the modified strategy used here employing ebv transformation; antigen preselection followed by phage library construction enabled the successful selection of human monoclonal recombinant scfvs specific to the v3 antigen of hiv-1. construction of human scfv phage library from these antigen preselected b cells led to an efficient and cost effective approach for the production of anti-v3 scfvs. the plasma antibodies of the hiv-1 infected patient #254 displayed neutralizing activity against 8 out of 10 hiv-1 viruses from subtype-a, b and c ( table 1 ). the plasma sample also showed a cross subtype-b and c binding reactivity in context of the anti-v3 loop directed antibodies ( figure 1 ). a total of 4.8 million pbmcs were isolated from the hiv-infected patient (# 254) and ebv transformed in 48 wells of a 96 well culture plate ( figure 2 ). after 2 weeks, the cultures were screened (first screening) for the presence of anti-v3 antibodies and 7/48 wells showed high v3 binding reactivity with od> 2. cells from these 7 v3 positive wells were expanded to a 24 well plate and after a second screening, cells from 5 out of 7 wells showing v3 binding with od > 0.6 were expanded in a six-well plate, pooled and finally transferred to a t-25 flask. in the 3rd (final) screening of the cells in the flask, the cells showed high reactivity to v3 (od > 1) ( table 2 ). these v3 positive (enriched) cells were processed for recombinant anti-v3 scfv generation. total rna from the v3 enriched antibody producing b cells of the patient #254 was isolated. a total of 200ng of rna was reverse transcribed to cdna. use of random hexamer and oligo dt in the cdna synthesis process was shown to be more effective than either of them alone or the 3 0 primers [15, 20] . it also enhanced the chances of amplification of the antibody genes even when the amount of rna is low [15] . a total of 54 combinations were used to amplify the heavy and light chains (24 for vh and 30 for vl). among the 24 combinations of heavy chains, 18 were successfully amplified. the heavy chains vh1, vh4, vh5 and vh6 were preferentially more expressed while vh2 and vh3 were very less expressed ( figure 3a ). all the 30 kappa light chain combinations were successfully amplified and expressed almost at the same level, except for vlk2, which were relatively less expressed ( figure 3b ). a heavy chain and light chain pool was made by mixing equal concentration of each combination (50ng) irrespective of their expression level, so that each combination of the heavy chain has an equal probability to combine with the light chain and vice versa and to avoid the predominant expression of one antibody subclass and finally to have new combinations of antibodies for a particular antigen that are naturally not elicited during the natural course of infection. a final pull through pcr was performed using the forward and reverse primers. these scfvs were then ligated into the pcantab-5e vector that contains the sfi1 and noti sites ( figure 3 ). the ligated product was transformed into tg1 using calcium chloride mediated transformation. the incubation time after the heat shock treatment during transformation was maintained for a maximum of 40 min to avoid repetition of similar types of clones in the library. we obtained 200-300 transformed colonies per plate. a total of 7000 colonies were obtained with 400ng of digested scfv dna that were ligated into 1 μg of phagemid vector. the ligated dna was transformed into e.coli tg1. to check the diversity of antibodies in the library, we randomly selected 10 clones from the unselected library. amplification of these 10 scfvs by pcr, followed by digestion with bstn1 and comparing their dna fingerprint patterns showed that 9/10 clones were distinct from each other. clone 3 and clone 6 showed identical dna fingerprinting patterns ( figure 4 ). we further confirmed the diversity of the antibody fragments by dna sequence analysis of 29 randomly selected scfv clones from the unselected library which showed high diversity of clones i.e. 73% (21/29) in the library. the heavy chain (vh) sequences showed a limited diversity, igvh4 (13/29) and igvh5 (15/29) were the most preferentially represented vh sequences that combined with all the representatives of ighd genes except for ighd1. diversity of the vl paratope was also limited with igkv1, igkv2 and igkv3 being most represented, except for one occurrence of igkv7 ( table 3) . as the size of the library was very small, one round of biopanning was done against the v3 c and v3 b peptides. forty clones were randomly selected and checked for figure 2 schematic overview of the strategy used for construction of antigen specific phage library. the peripheral blood mononuclear cells (pbmcs) were isolated from an antiretroviral drug naïve patient (#254) whose plasma exhibited neutralizing activity against a panel of viruses and also displayed cross clade reactive anti-v3 antibody binding potential. pbmcs were subjected to ebv transformation in 96 well culture plate, wells showing high level of anti-v3 antibodies were selected and expanded to 24 well stage and then to six well stage . the wells showing high titre of anti-v3 abs were pooled together and cultured in t25 flask. total rna was isolated from these antigen specific enriched b cells and cdna was synthesised. heavy and light chains were amplified and scfvs were constructed and cloned into a pcantab-5e phagemid vector. scfv phage library of 7000 clones was constructed. their binding, of which 15 showed positivity in phage elisa with both the peptides ( figure 5 ). four clones were randomly selected for elisa with purified phage. the binding specificity of these anti-v3 scfvs was checked with phage from the 24 well plate and peg precipitated purified phage. the 24 well elisa and pure phage elisa binding assays showed similar results. three clones exhibited almost similar binding in both the formats of elisa while one clone had lower binding reactivity in pure phage elisa although it was not completely lost (data not shown). dna fingerprinting analysis using bstn1 followed by sequencing revealed that 13/15 clones were distinct (table 4 ). all the distinct 13 anti -v3 scfvs that were finally selected, showed cross-reactivity against both the v3 peptides and did not show any reactivity against other unrelated peptides. one round of biopanning was found to be sufficient to get v3 positive scfv clones with diversity. the antigen binding clones showing positivity/binding in the phage elisa were further processed for soluble scfv expression by induction with 1mm iptg [28] . after induction, the periplasmic lysate, inclusion bodies, culture supernatant and whole cell extract were prepared and analysed for scfv expression on a 12% reducing sds-page. the scfv fusion protein was found to be expressed in the cell lysate, culture supernatant, periplasmic extract, with highest expression in the inclusion bodies. scfv expression was also observed in the uninduced culture ( figure 6 ). the scfvs were expressed in e. coli hb2151 cells by inducing for 6 to 8 h with 1 mm iptg at 24°c. the a total of 4.8 million pbmcs were isolated. 2 number of cells plated per well was 100000. 3 forty eight wells were subjected to ebv transformation. 4 all the 48 wells were successfully transformed (transformation efficiency 100%). 5 7/48(approx 14.6%) wells showed positive binding (od>2) with v3 peptide and expanded to 24 well stage. 6 antibody fragments were purified from the periplasmic extract and the purified product was analysed by sds-page. the scfv protein of 32kda was expressed in different elute fractions e1 to e5 ( figure 7c ) and protein samples were concentrated using ultrafiltration columns (ambion) over a 10kda cut off. expression of the scfv fusion protein (32 kd) in the periplasmic extract was confirmed by western blotting. the cell lysate of hb2151 was used as a negative control ( figure 7d ). the functional activity of the newly generated clones 3e6b and 3e7b was assessed by their binding to the v3 peptides of clade c and b in elisa. both the scfvs showed specific binding to the v3 peptides ( figure 8 ). in addition, these scfvs did not bind to other unrelated peptides like mper and id loop of hiv, peptide pool (unrelated viruses) and bsa. the purified scfvs were tested for the viral neutralization potential against a panel of pseudoviruses from clades a, b and c hiv-1 viruses (table 5) we have demonstrated in this study a strategy that successfully yielded recombinant human scfvs against the v3 region of hiv-1 using a pool of antigen selected ebv transformed b lymphocytes and phage display technology. the use of preselected b cells producing anti-v3 antibodies for phage scfv library construction proved to be an efficient strategy for the isolation of v3 specific clones. moreover, this approach has prevented any loss of unstable or slowly growing clones which may have useful and unique binding specificities (that often occurs during cloning and propagation). stringency during the initial stages helps in elimination of most of the false positive clones. beginning with the 96 well stage and up to the final screening of the cells in the t25 flask, we selected only those wells with high v3 binding reactivity for further expansion and finally for the phage library construction. by this approach, we obtained a phage library with an adequate number of v3 binding clones. amplification of the heavy chain gene showed that the vh1, vh4, vh5 and vh6 were preferentially more expressed than the vh2 and vh3 and corroborated with the previous reports [29] that anti-v3 antibodies more preferentially exhibit the above gene usages. it is well established that in hiv-1 infection, vh3 genes are less preferentially used [30, 31] . from the sequencing data, we observed that twenty nine randomly selected clones from the ebv transformed cells library exhibited the vh4 and vh5 gene usage suggesting that our library may be biased towards antigen specificity. in healthy individuals, vh3 genes are most frequently used and vh5 is used only by a low percentage of antibodies (http:imgt.cines.fr). the light chain did not show any preferential gene usage and all the light chains were successfully amplified. as heavy chain amplification was biased towards the expression of a set of heavy chain genes, to avoid predominant expression of one subset of genes in the phage library, we took an equal concentration of all the heavy chain genes which in turn increased the chances of the new combinations that are not expressed in natural infection. successful isolation of positive clones with higher affinity and specificity is possible when a library is more diverse and has less number of clones with incomplete scfvs sequences [15, 32] . if phage carrying incomplete scfvs are more in number, they overwhelm the library after multi-step panning thereby the presence of the full size insert will decrease, this being a common problem with phage libraries. interestingly, our library exhibited 90% diversity of clones and 100% clones had complete scfvs ( figure 7a,b) , this might be because of the optimum incubation time (30-40 min) maintained during the transformation process that reduced the chances of repetition of clones. in addition, the combination of the sfi1-noti site containing pcantab-5e vector that we used in the library construction may have reduced or eliminated the chances of getting incomplete scfv sequences because these restriction enzyme sites are rarely present in the scfv dna sequence [15, 33] . furthermore, none of our clones had internal sfi1-noti sites. stringency in the biopanning also reduces the number of false positive clones. initially, phage were allowed to bind to the plastic plate, next, to coated milk, followed by bsa and finally onto a pool of coated unrelated antigens in the plates. this stringency reduced the number of nonspecific phage binders and made the screening process more convenient and specific. kempf et al., using a similar strategy of ebv transformation, constructed a fab library of 10 7 clones from which they isolated three fabs after one round of biopanning. their scoring of positive clones however was less, probably because they selected cells positive for gp120 binding from the 96 well stage and also included wells that were low binders ( od> 0.1) [20] . we therefore selected only those wells with high v3 binding reactivity (od>0.6) at all stages for the phage library construction. this reduced the number of false positive clones and enriched the v3 specific cells. there are several reports on the generation of scfvs from different sources like pbmcs [34, 35] , bone marrow [36] , tonsils [37] , hybridomas [38, 39] , but none of them employed ebv transformed antigen specific cells to generate a scfv phage library. generation of phage library from ebv transformed lines after preselection with antigen is very useful for laboratories with limited resources because the antibody production by traditional hybridoma technology costs ranging between $8000 to $12000 and 80% of the costs are incurred in the post ebv immortalization steps like fusions and cloning. our strategy does not require the expensive process of electroporation because the cost of cuvettes and instrument mainly limits the construction of phage libraries with such a large size and diversity; also it eliminates the large number of biopanning rounds followed by cumbersome screening processes. such small libraries are easy to maintain and can be produced by routinely used calcium chloride mediated transformations. we have for the first time, generated anti-v3 human scfvs against clade c hiv-1. by antigen pre-selection, the phage library served as an adequate source of v3 positive clones. the combination of ebv transformation and selection of immortalized lines that exhibit the desired antigen binding characteristics with phage display technology provides a useful strategy for specific recombinant antibody generation. our study validated the generation of recombinant libraries as a powerful tool for the generation of diverse recombinant antibodies. the hiv-1 seropositive patient (# 254) enrolled for the construction of the anti-v3 scfv phage library was recruited after obtaining written informed consent from the department of medicine, aiims, new delhi. the study was approved by the institute ethics committee. whole blood was collected in edta vaccutainers. the plasma was separated from whole blood, aliquoted and stored at −70°c until tested. the peripheral blood mononuclear cells (pbmcs) were separated by phycoll hypaque centrifugation and processed immediately for ebv transformation. the plasma sample of the patient # 254 was previously tested and found to exhibit good neutralization potential against a diverse panel of viruses (table 1 ). it was also tested for the presence of binding anti-v3 antibodies and the data is shown in figure 1 . the neutralization efficiency of the plasma #254was tested against a standard panel of pseudoviruses of clades a, b and c obtained from the nih aids research and reference reagent program, by tzm-bl assay [40] . the standard panel of pseudoviruses has been categorized from tier 1 to tier 3, based on the decreasing order of susceptibility to neutralization by the known monoclonal antibodies [41] . the neutralization assay was carried out in 96-well tissue culture plates. briefly, 50 μl of the heat inactivated plasma/purified scfv, at different dilutions in duplicate, was added to 200 tcid 50 of the virus and incubated for 1 h. a cell control well containing culture media only and a virus control well containing both the culture media and the virus were tested in parallel. the rest of the procedure is the same as described for calculating tcid 50. the scfv (hepb), an scfv against the hepatitis b surface antigen [42] and 1418, a human antibody to parvovirus b19 protein, were used as negative controls. for all dilutions of test plasma, the percent neutralization was calculated based on the relative luminescence units (rlu) in the presence of plasma divided by the virus control. the cell control value was subtracted from the plasma rlu value as the background cutoff. the 50% neutralization titer (id50 titer) was determined for the plasma sample #254 against each virus by plotting percentage neutralization against the dilution of the plasma tested. a non-linear regression straight line was drawn by the method of least squares, and the reciprocal id50 titers were extrapolated. the experiments were performed in duplicate and repeated at least twice and the mean id50 titers were calculated. the anti-v3 antibody content in the hiv-1 seropositive plasma sample #254 and in the supernatants of the ebv transformed pbmcs in culture at different stages was determined using v3 peptide elisa. thirty five mer peptides of v3c (ctrpnnntrksirigpgqtfyatgdi igdirqahc) and v3b (ctrpnnntrksihigpgrafy ttgeiigdirqahc) were synthesised (sigma aldrich, usa), based on the consensus v3 sequences. the v3 peptides (1 μg/ml) were coated onto 96 well nunc-immuno plates (nunc: cat# 439454) using antigen coating buffer (150 mm na 2 co 3 , 350 mm nahco 3 , 30 mm nan 3 , ph 9.6) at 4°c overnight. plates were washed using phosphate buffered saline with 0.1% tween-20 (0.1% pbst) thrice using a plate washer. plates were then blocked with 100 μl of 15% fetal calf serum and incubated at 37°c for 1.5 h. following blocking and washing, heat inactivated plasma (100 μl, dilution range=300-100000) or 100 μl of supernatants from ebv transformed pbmc cultures was added to each well and incubated for 1hour at 37°c. after 3 washings with pbst (0.1%), the bound v3 specific antibodies were detected by addition of 100 μl of alkaline-phosphatase conjugated anti-human igg fc (1:2000 in pbst). immune complexes were revealed with ap-substrate in dae buffer and the colorimetric reaction was stopped by the addition of 6n naoh. the optical density was read at 405 nm. id 50 titers were calculated for the plasma sample against each of the peptide by plotting the absorbance at 405 nm against the dilutions of the plasma sample tested. a non-linear regression straight line was drawn by the method of least squares and the id 50 titers were extrapolated. b95 cell line comprises of ebv transformed human lymphoblastoid cells which secrete ebv in the supernatant. ebv transforms human b cells [43] . we obtained the b95 cell line from american type cell culture (cat. no. vr-1492). b95 cells were grown in t-75 tissue culture flasks, at 100,000 cells/ml at 37°c, 5% co 2, in complete rpmi media. after 10 days, the viral supernatant was centrifuged at 300 × g and filtered with 0.45 μm filters. peripheral blood mononuclear cells (pbmc) (100,000 cells/well) from the patient #254 were ebv transformed by mixing with 100 μl of the viral supernatant in a 96-well plate and cultured with a polyclonal b cell activator, cpg (2 μg/ml), which enhanced ebv infection and b cell transformation [44] and csa (0.5 μg/ml). the plate was incubated at 37°c in a 5% co 2 incubator overnight. next day, cells were fed with 100 μl of complete medium containing csa and cpg. cultures were fed twice per week and half of the culture supernatant was replaced with fresh complete media, 200 μl/well (no csa and cpg). after two weeks of culturing the b cells in the 96 well plate, we screened for the presence of v3 antibodies by v3 peptide binding elisa as described above. the positive b cell clones were further expanded to 24 well plates and screened in the 3 rd and 4 th week; v3 positive clones were transferred to a six well plate, pooled and further expanded to the t-25 flask ( table 2) . total rna from the v3 specific antibody producing b cells (from flask stage) was isolated by trizol reagent (sigma, usa) and then reverse transcribed to cdna, using the reverse aid mmulv reverse transcriptase (fermentas, usa). a total of 200 ng of rna was reverse transcribed in a reaction volume of 50 μl containing, 10ng of random hexamer, 20μm oligo-dt, 1.5 μl of rnase inhibitor (40u/μl), all dissolved in 1x rt buffer. the rna was heated to 65°c for 5 min and then immediately chilled on ice for at least five minutes. following the addition of the reaction mixture, the tube was incubated at 42°c for 60 min, at 70°c for 5 min and quickly chilled on ice and stored in −20°c. heavy chain variable region genes were amplified using a total of 24 combinations (6 forward primers and 4 reverse primers representing all human immunoglobulin subfamilies) and for light chain kappa, a total of 30 combinations were used (6 forward primers and 5 reverse primers) as described previously [15] . a total of 54 independent reactions were performed to generate the variable regions of heavy and light chains. the heavy chain 5 0 primers included a sfii site and the light chain 3 0 primer included a noti site. light chain 5 0 primer included part of the linker region (gly 4 ser) 3 and this was compatible with the heavy chain 3 0 primer. each variable heavy region was amplified using hot start taq dna polymerase (fermentas) in a pcr reaction of 50 μl containing 2.5 μl cdna, primers 1 μl (10 pmole each) both forward and reverse. pcr reaction was performed for 34 cycles (94°c for 3 min initial denaturation, 94°c for 1 min, annealing at 63°c for 1 min, extension at 72°c for 2 min) using eppendorf master cycler. light chain variable region were amplified with the similar protocol except the annealing temperature used was 57°c. each variable region gene was purified from the agarose gel using gel extraction kit (qiagen, germany). an equimolar mixture of pooled heavy and light chain dna was used in the second round assembly pcr. the assembly pcr reaction was cycled 20 times (94°c for 1 min, 94°c for 45 sec, 62°c for 50 sec, 72°c for 2 min) the assembly reaction was performed using pfu dna polymerase and without primers. full length scfvs were amplified using a pull through pcr reaction using taq dna polymerase and the following primers ptfw 5 0 cct ttc tat gcg gcc cag ccg gcc atg gcc 3 0 and ptrv 5 0 cag tca ttc tcg act tgc ggc cgc acg 3 0 (94°c for 1 min, annealing at 62°c for 1min, extension at 72°c for 1 min and final extension at 72°c for 5 min). the scfvs were agarose gel purified using gel extraction kit (qiagen, germany). the scfv dna fragments and pcantab-5e vector were digested with noti /sfii (new england biolabs, usa) respectively. the digested scfv dna fragments and pcantab-5e vector were gel purified using gel extraction kit (qiagen, germany). the scfv dna was ligated into vector at a 3:1 molar ratio using t4 dna ligase (new england biolabs, usa). the ligated dna was transformed into chemically competent cells of e.coli tg1 and placed on ice for 1h followed by heat shock treatment for 90 seconds and chilled on ice for 5 min. next, 800 μl of 2xyt media was added and incubated in a rotating shaker at 200 rpm for 40 min. the transformed cells were plated on to 2xyt medium agar plates containing ampicillin (50 mg/ml) and 2% glucose, and incubated overnight at 37°c. the following day, colonies were scraped into 1ml of 2xyt medium with 20% glycerol and stored at −70°c. sequence of the assembled scfv was confirmed by an automated abi prism sequencer using gene specific primers. the phage were rescued by infection with helper phage (m13-ko7), followed by precipitation with peg/nacl, resuspension in pbs and titration for the determination of phage concentration. the phage were then subjected to a single round of enrichment by bio-panning. the panning procedure was carried out in immuno 96 microwell plates. plates were coated with 100 μl of v3 peptide 1 μg/ml in 0.1 m nahco 3 (ph 8.6) overnight at 4°c. a phage library of 10 12 phage was incubated for 1h in milk coated wells to remove the non-specific binders. after one hour, unbound phage were transferred to the peptide coated wells for 30 min at rt. the unbound phage was eliminated by washing 10-15 times with pbs containing 0.1% tween 20. the bound phage was eluted with 0.2 m glycine ph 2.2 for 10 min at rt. the eluted phage were neutralized with 1m tris hcl ph 9.2 and immediately infected onto tg1 (od 0 .4 to .5) for 30 min at 37°c and for 30 min with shaking at 37°c. cells were spun down and plated on 2xty agar containing ampicillin (50 mg/ml) and 2% glucose. individual colonies were picked and grown in 96 well sterile culture plates (corning) and a glycerol stock was made and stored at −70°c. individual colonies were grown in 1ml 2xyt broth containing ampicillin and 2% glucose overnight with shaking at 37°c at 160 rpm. a small inoculum was transferred to 1ml 2xyt broth containing ampicillin (50 mg/ml) and 2% glucose at 37°c with shaking at 200 rpm till the od reached 0.4 to 0.5. helper phage were added and the plate was incubated at 37°c without shaking and then for 30 min with shaking at 180 rpm at 37°c. the cells were spun down at 1500 × g, the supernatant was discarded and pellet was washed with 2xyt broth. the pellet was then resuspended in 2xyt broth containing ampicillin (50 mg/ml) and kanamycin (100 mg/ml) (no glucose) with shaking at160 rpm at 30°c for 16-18 h. it was then centrifuged at 6000 × g and the supernatant was collected and stored at 4°c and an aliquot was tested in the phage elisa. the elisa plates were coated with 100 μl of v3 peptide (1 μg/ml) in 0.1 m nahco 3 (ph 8.6) and incubated overnight at 4°c. the plates were washed once with 1x pbs and blocked with 4% non-fat milk (titan biotech, india) for 2 h at 37°c. the plates were then washed three times with 1x pbs. phage supernatant (100 μl) was added to each well and incubated for 1h at rt. phage supernatant was discarded and the plates were washed four times with pbst(0.1%). 100 μl of anti m13 antibody (diluted 1:2000) was added (sigma) and incubated for 1h at rt. the plates were washed four times with pbst (0.1%). 100 μl of anti rabbit hrp (jackson) diluted 1:3000 were added and incubated at rt for 1h. the plates were then washed four times with pbst (0.1%). 100 μl of tmb substrate was added, and the reaction was stopped by adding 8n h 2 so 4. absorbance was measured at 450nm. individual bacterial colonies were grown in 5ml 2xyt medium containing ampicillin (50 mg/ml) and 2% glucose with shaking at 200 rpm at 37°c till the o.d reached 0.4 to 0.5. next, 1 μl of ko7 helper phage (10 18 ) was added and incubated at 37°c for 30 min without shaking, followed by 30 min shaking at 200 rpm. the cells were centrifuged at 2500 × g for 10 min, supernatant was discarded and the pellet was washed again with 2xyt broth. the pellet was resuspended in 50ml 2xyt broth containing ampicillin (50 mg/ml) and kanamycin (100 mg/ml) (no glucose) at 30°c with shaking at 160 rpm for 12-16 h. it was then centrifuged at 10000 × g for 20 min at 4°c. the supernatant was transferred to glass bottles and ¼ volume of peg/nacl was added and kept on ice for 4 to 6 h followed by centrifugation at 20000 × g for 20 min at 4°c. the supernatant was discarded and the pellet was dissolved in sterile and autoclaved 1x pbs and stored at 4°c. the transformation unit (tu) was calculated. twenty nine scfvs clones were randomly selected from the unselected library and sequenced by macrogen (south korea). the sequences were analysed using immunoglobulin blast [45] and v base software [46] . the diversity of the scfv repertoire was analysed by comparing the restriction digestion pattern of scfvs. ten clones were randomly selected from the primary phage library and the plasmid was isolated. the scfv sequences were amplified using primers ptfw 5 0 cct ttc tat gcg gcc cag ccg gcc atg gcc 3 0 and ptrv 5 0 cag tca ttc tcg act tgc ggc cgc acg 3 0 (94°c for 1 min, annealing at 62°c for 1min, extension at 72°c for 1 min and final extension at 72°c for 5 min). the amplified pcr products were digested with a frequent cutter restriction enzyme bstn1 (neb) and analysed on 2% agarose gel. clones showing positivity/binding in phage elisa were selected for soluble scfv expression. these were than transformed into hb2151 using calcium chloride mediated transformation for soluble scfv expression. the hb2151 cells carrying pcanta-5e plasmid were grown in 10 ml 2xty medium overnight at 37°c with shaking at 200 rpm. the next day, 1/100 th volume of the overnight culture was inoculated in 1 litre of 2xty medium and grown at 37°c with shaking at 240 rpm till the od reached 0.6 and then the culture was induced by 1 mm iptg for 6 to 8 h at 24°c [33] . cells were harvested and different fractions were prepared. scfvs were purified from the periplasmic fraction. briefly, the cells were harvested by centrifugation at 4000 × g for 15 min at 4°c. the supernatant was discarded and pellet was resuspended in 30 mm tris-cl. 20% sucrose, ph 8.0 at 80 ml/ gram wet weight. the cells were placed on ice for 20 min and 500 mm edta was added to a final concentration of 1 mm edta. the cells were spun down at 8000 × g for 15 min at 4°c. the pellet was resuspended in 5 mm mgso 4 and the cells were placed on ice for 10 min with slowly stirring, pelleted down at 8000 × g for 15 min at 4°c and the supernatant collected for purification. purification of e-tagged scfv was carried out using the recombinant phage antibody system (rpas) purification module (amersham biosciences) as per the manufacturer's instructions. the periplasmic extract was filtered through a 0.45 μm filter to remove any remaining cell debris. the anti-e tag column was regenerated by washing the column with 15ml of elution buffer (0.1 m glycine, ph 3.0) and was then equilibrated with 25 ml binding buffer (0.02 m phosphate buffer, 0.005% nan 3 , ph 7.0). the e-tagged scfv in the periplasmic extract was then allowed to bind to the column by passing the extract through the column. the unbound excess e. coli proteins were removed from the column by washing it with 25 ml binding buffer. the flow rate at each step was maintained at 5 ml/min through the column. finally, the bound scfv was eluted by 15ml elution buffer. several fractions of the eluted scfv (900 μl each) were collected in tubes containing 100 μl neutralization buffer (1 m tris, 0.05% nan3, ph 8.2). the amount of protein in each fraction was estimated using bca method and the fractions containing considerable amount of scfv were pooled together and concentrated. soluble elisa was performed, as described in the phage elisa. hundred microliter of soluble scfv periplasmic extract/purified scfv was added and incubated at room temperature for 1h. elisa plates were washed three times with 0.1% pbst and incubated with 1:1000 dilution of primary antibody in 2% mpbs. the plates were washed three times with 0.1% pbst and incubated with 1:2000 diluted anti rabbit hrp conjugated secondary antibody in 2% mpbs. the elisa plates were washed again as described above. 100 μl of tmp substrate was added and incubated at rt till the colour developed. reaction was stopped by adding 8nh 2 so 4. absorbance was read at 450 nm. sds-page was done as described [33] . proteins were separated on a 12% running gel and 5% stacking gel and visualized by coomassie brilliant blue (cbb) staining. for western blotting, gel was blotted onto nitrocellulose membrane using electroblotting, (100 v for 1h) and probed with primary antibody. anti-rabbit hrp was used as secondary antibody and colour was developed with dab as the substrate. broadly cross-reactive hiv-1-neutralizing human monoclonal fab selected for binding to gp120-cd4-ccr5 complexes the cross-clade neutralizing activity of a human monoclonal antibody is determined by the gpgr v3 motif of hiv type 1 efficient neutralization of primary isolates of hiv-1 by a recombinant human monoclonal antibody cross-clade neutralizing activity of human anti-v3 monoclonal antibodies derived from the cells of individuals infected with non-b clades of human immunodeficiency virus type 1 fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the cd4 binding site of human immunodeficiency virus type 1 gp120 an igg human monoclonal antibody that reacts with hiv-1/gp120, inhibits virus binding to cells, and neutralizes infection cd4-dependent, antibody-sensitive interactions between hiv-1 and its co-receptor ccr-5 broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals broad and potent neutralizing antibodies from an african donor reveal a new hiv-1 vaccine target rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1. science insensitivity of paediatric hiv-1 subtype c viruses to broadly neutralising monoclonal antibodies raised against subtype b potent and broad neutralization of hiv-1 subtype c by plasma antibodies targeting a quaternary epitope including residues in the v2 loop generation of human monoclonal antibodies to human immunodeficiency virus filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface a compact phage display human scfv library for selection of antibodies to a wide variety of antigens making antibodies by phage display technology monoclonal antibody fragment from combinatorial phage display library neutralizes alpha-latrotoxin activity and abolishes black widow spider venom lethality, in mice generating recombinant antibodies for research, diagnostics and therapy using phage display isolation of potent 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effective method for establishing human b lymphoblastic cell lines using epstein-barr virus an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the patient participant for providing blood sample. we profoundly thank dr susan zolla pazner from new york university for providing the antiparvovirus antibody 1418. we thank dbt (bt/pr 10511/med/29/66/2008) for the funds and for the junior research fellowship provided to rajesh kumar. we thank all india institute of medical sciences and national brain research centre for providing the facilities for conducting this study. the authors declare that they have no competing interests.authors' contributions kl and ss designed and conceptualized the study and finalized the manuscript. rk constructed the phage library, constructed the scfvs and wrote the manuscript. ra screened the plasma of patients for neutralization and performed the ebv transformation experiments. at provided valuable inputs in phage library construction. ssp, dd,as and lk helped in protein expression, purification and phage elisa. nw provided the patient samples. all authors have read and approved the final manuscript. key: cord-330698-9t24jo8s authors: wurdinger, thomas; gatson, natosha n.; balaj, leonora; kaur, balveen; breakefield, xandra o.; pegtel, d. michiel title: extracellular vesicles and their convergence with viral pathways date: 2012-07-25 journal: adv virol doi: 10.1155/2012/767694 sha: doc_id: 330698 cord_uid: 9t24jo8s extracellular vesicles (microvesicles), such as exosomes and shed microvesicles, contain a variety of molecules including proteins, lipids, and nucleic acids. microvesicles appear mostly to originate from multivesicular bodies or to bud from the plasma membrane. here, we review the convergence of microvesicle biogenesis and aspects of viral assembly and release pathways. herpesviruses and retroviruses, amongst others, recruit several elements from the microvesicle biogenesis pathways for functional virus release. in addition, noninfectious pleiotropic virus-like vesicles can be released, containing viral and cellular components. we highlight the heterogeneity of microvesicle function during viral infection, addressing microvesicles that can either block or enhance infection, or cause immune dysregulation through bystander action in the immune system. finally, endogenous retrovirus and retrotransposon elements deposited in our genomes millions of years ago can be released from cells within microvesicles, suggestive of a viral origin of the microvesicle system or perhaps of an evolutionary conserved system of virus-vesicle codependence. more research is needed to further elucidate the complex function of the various microvesicles produced during viral infection, possibly revealing new therapeutic intervention strategies. a wide variety of vesicles are actively released from living cells into the extracellular space with their contents reflecting the cellular composition and physiologic state (for review see [1] [2] [3] ). over the years, the different types of extracellular vesicles have been given a variety of names, including exosomes, shed microvesicles, ectosomes, microparticles, virosomes, viruslike particles, and oncosomes. the distinguishing features of each of the vesicle subtypes and the correct nomenclature are currently under intense study. here, we will refer to them under the general term, microvesicles. microvesicles carry rna [mrna, microrna (mirna), and noncoding sequences], cdna and genomic sequences, and a large component of proteins and lipids (see reviews above, as well as [4, 5] ). upon release these microvesicles can move within the extracellular space and are either taken up by neighboring cells or degraded. they can also enter adjoining bodily fluids, such as the systemic circulation and travel to distant sites. in fact, they have been found in abundance in blood (serum and plasma), urine, breast milk, sweat, saliva, ascites fluid, and cerebral spinal fluid (csf) [3] [4] [5] [6] [7] . at least two distinct release mechanisms for microvesicles have been described for two subtypes: (1) exosomes-derived from the multivesicular body (mvb) and (2) shed microvesicles-derived from the plasma membrane. interestingly, both mechanisms have considerable overlap with virus release and biogenesis (summarized in figure 1 and further discussed below). exosomes range from 30 to 100 nm in diameter and are generated by inward budding of the lumen of internal vesicular compartments derived from endosomes [8] . as vesicles accumulate within these endosome-derived compartments, they are referred to collectively as mvbs. these mvbs can either be targeted for degradation through the lysosomal pathway, or they can fuse with the plasma membrane releasing their interior vesicles into the extracellular space. the exact mechanism and kinetics of these fusion and release events are not fully elucidated and may vary among different cell types [9] . for example, depletion of hrs (an escrt-0 component) led to a decrease in exosome secretion in dendritic cells that were stimulated to release with ovalbumin and a calcium ionophore [10] . oligodendrocytes on the other hand seem to secrete exosomes by a mechanism that is escrt independent and ceramide dependent [11] . exosome release by hela cells has been found to involve rab27a/b [12] , and p53 is reported to play a role in exosome release in a nonsmall cell lung cancer cell line [13] . rab11 has also been shown to be involved in the release of exosomes from mvbs by acting in the tethering/docking of mvbs to the plasma membrane to promote homotypic fusion, in the presence of calcium [14] . in addition, tbc1d10a-c, a rab35 inhibitor, led to intracellular accumulation of endosomal vesicles and impaired exosome secretion [15] . shed microvesicles are released by outward budding directly from the plasma membrane and tend to be larger (>100 nm in diameter) and more heterogeneous in size [16, 17] . moreover, this release process is likely controlled by localized cytoskeleton dynamics, with small cytoplasmic membrane-covered protrusions detaching and being released into the extracellular space [18] by an activated gtpase, arf6 [19] . interestingly, recent observations indicate that virus-independent budding from the plasma membrane can be mediated by endosome to plasma membrane relocation of tsg101, a prominent member of the escrt-i complex, frequently noted as an exosome marker [20] . this type of budding is topologically identical to both the inward budding of the limiting membrane of mvbs and viral assembly at the plasma membrane, in that the outer surface of the plasma membrane is on the outer surface of the microvesicle. in fact, certain tumor cells shed retrovirallike vesicles, which can be abundant because of increased transcription of endogenous retroviral sequences [17, 21] , resulting from overall hypomethylation of the genome [22] . in general it seems that the clear distinctions between viruses and microvesicles based on composition and function are fading although they can be separated from vesicles released during the later stages of programmed cell death since these latter vesicles, referred to as apoptotic blebs [2] , are even larger in size [23] . the role of microvesicles in intercellular communication is currently receiving much attention. upon release from the donor cell, the microvesicles can either be taken up by neighboring cells or travel through bodily fluids for cargo delivery into recipient cells at distant sites. although many details are missing, cellular uptake of some microvesicles appears to depend, at least in part, on specific ligand-receptor recognition [24] , and can be mediated by direct fusion of the microvesicles with the plasma membrane or by endocytotic uptake of the microvesicles. for example, quah et al. [25] have shown that bystander naïve b cells are rapidly activated by acquiring the antigen from activated b cells through microvesicle-mediated membrane transfer. in a similar way cd41 is transferred from platelets to endothelial and tumor cells, resulting in increased proadhesive properties of the recipient cells [26, 27] . microvesicles also shuttle mrna between cells and influence the physiological state of the recipient cell, as well as the cellular response to external stress stimuli [28] . in addition, mirnas are transferred by exosomes [6, 29, 30] . for instance, mir-146a was shown to be transferred into recipient prostate cancer cells leading to the inhibition of their proliferation [31] , and recently mirnas which can modulate the immune response were detected in exosomes in breast milk [32] . furthermore, retrotransposon sequences are particularly enriched in tumor microvesicles, and tumor-derived human endogenous retroviral (herv) sequences can be transferred to normal human umbilical vein endothelial cells (huvecs) via microvesicles resulting in a prolonged increase in herv-k mrna levels [17] . this suggests that tumor cells transfer these mobile genetic elements via microvesicles to neighboring normal cells thereby modulating their genotype and phenotype. virology 3 microvesicular shedding of cellular membrane components and the release of internal endosomal-derived exosomes are important for cellular communication and modulation of immune responses [9, [54] [55] [56] [57] (table 1) . while release of microvesicles has been extensively investigated, recently the challenge has been to uncover the specific mechanisms that guide protein sorting and complexing into shed microvesicles and exosomes in various cell types. cells have been reported to secrete highly specified microvesicles after infectious exposure or under various cell activation conditions [5, 54, 56, 58] . through the packaging and transfer of functional proteins, mrna/mirna, and other cytosolic components, microvesicles have been found to be beneficial either to the host cell or to the infectious agent [37, 43] . virus-infected cells proved useful in early studies to elucidate the role of microvesicular shedding in intercellular communication [55, 56] . amongst the most extensively studied viruses with respect to microvesicles are herpes simplex virus (hsv), human immunodeficiency virus (hiv), and the tumorigenic herpes virus, epstein-barr virus (ebv). each virus possesses unique properties that afford protection from immune attack. here, we outline the important immune modulatory steps involved in virus-induced microvesicle sorting and release in these and other related viruses. preservation of the virus depends on microvesicle release of infected cells. microvesicles released by infected cells contain specific components of the cell and the virus, many of which facilitate the ability of virions to persist in a hostile antiviral immune environment [44, 55, 56, 58] . depending on the virus type, and, in some cases, the stage in the viral cycle, intercellular processes are well orchestrated to produce specific cellular and immune outcomes [56] : (1) evading the host immune system, (2) invasion, (3) replication, and (4) persistence (summarized in part in figure 2 and further discussed below). during primary viral infection, humoral and cell-mediated host immune responses such as production of neutralizing antibodies and cytotoxic t-cell attack on infected cells are employed to contribute to viral destruction. early evasion strategies adopted by viruses interfere with complete elimination of the virus, allowing it to persist. during hsv-1 infection the release of microvesicles, formerly known as l-particles containing viral tegument proteins and glycoproteins, can prime surrounding cells for productive infection and reduce immune rejection [48] [49] [50] . such virus-like vesicles lack both the viral capsid and dna and are thereby incapable of producing a replication-infective cycle in the cells on their own [49] [50] [51] . however, some of the viral tegument proteins contained within them are immediate early transcription factors that can produce rapid transcriptional activation of later arriving intact virions [48, 52] . another evasion strategy observed for hsv-1 is targeting of the mhcii molecule processing pathway by viral envelope glycoprotein b (gb) [37] . antigen-presenting cells (apcs) routinely sort the mhcii surface receptor hla-dr to mhcii compartments for processing. the primary role of this pathway is to present [37, [48] [49] [50] [51] [52] [53] peptide antigens to the immune system in order to elicit or suppress t-(helper) cell responses that stimulate b-cell production of antigen-specific antibodies [37] . hsv-1 gb couples with hla-dr, causing sorting through the exosome pathway as opposed to presentation on the cell surface. complexing of gb-dr effectively hijacks the cellular antigen presenting machinery, preventing further peptide loading and, in addition, increasing microvesicle production [37, 53] . this final step releases additional gb-dr complexes into the host immune microenvironment, promoting resistance of viruses to immune attack, and in some cases producing bystander t-cell tolergenicity or anergy [37, 53] . in the case of hiv, microvesicle packaging and spread of the virusencoded nef protein impairs proper endocytosis of the immature mhcii/invariant chain, antibody class switching, and lysosomal degradation of viral peptides allowing hiv virus to evade immune recognition [37, 38] . ebv, human cytomegalovirus (cmv) and hepatitis c virus (hcv) have also found means to evade immune responses by exploiting microvesicles, as discussed below. exosomes and shed microvesicles can both incorporate elements from the cell, as well as from the intruding virion [54] . upon circulation of these microvesicles, they encounter and enter susceptible cells and can sensitize them to viral infection thus increasing systemic spread of the virus to naïve cells. in the case of the human cmv, microvesicles released by infected cells present the c-type lectin family molecule expressed on dendritic cells-used in capture and internalization of pathogens-in complex with the cmv glycoprotein b. this complex can be subsequently distributed to other cells by microvesicles, thereby increasing the susceptibility of these cells to cmv [59] . a similar mechanism is found in the case of hcv. in hcv-positive patients, the cellular membrane protein cd81 associates with one of the hcv envelope glycoproteins, e2. extracellular release of the e2-cd81 complexes within microvesicles allows for increased virus-fusing ability and infectivity of previously naïve cells [60] . microvesicles bearing the e2-cd81 complex and containing hcv rna are of notable importance as they have been reported to be infectious even in the presence of neutralizing antibodies [60] . interestingly, hcv has been shown to release three phenotypically distinct types of microvesicles having variable infectivity from high to low [60] . however, differential release of these microvesicles during hcv pathogenesis remains to be elucidated. infection. conversely, microvesicular release can contribute to viral attack by the host immune system. for example, in early invasion steps of cmv, cmv antigens are transferred from infected epithelial cells (ecs) via ec-derived microvesicles to apcs [43] . these apcs are not detected as infected cells but are rendered more susceptible to infection with subsequent encounters with the virus [43] . while this is a primary infectious viral invasion and replication strategy, inadvertently transferred apcs bearing cmv antigens in transplanted organs serve as markers to the host immune systems to target nonself tissue. harboring of these susceptible apcs by the immune-compromised host and continued microvesicular shedding increases t-cell surveillance and influx into the grafted tissues, thereby exacerbating allograft rejection [43] . microvesicles can also promote the innate immune response to viruses, for example, as observed for hiv whereby transfer of a particular antiviral cytidine deaminase via exosomes inhibits hiv replication [61] . in addition, virus-like vesicles can be used as a vaccination strategy, and recently chimeric virus-like vesicles were engineered using a mixture of coronavirus and influenza proteins functioning as a potential severe acute respiratory syndrome (sars) virus vaccine [62] . viruses can use various microvesicle transport mechanisms as a survival strategy, while in other cases the host immune system can utilize microvesicles for cell signaling and host protection. microvesicles can directly activate or suppress cellular responses, induce or facilitate infection, and transfer material to improve or hinder host immune recognition [9] . these same strategies can be exploited in the development of virus-based therapies. oncolytic viruses armed with therapeutic genes are currently being evaluated for safety and efficacy for cancer therapy [63] [64] [65] . it would be of interest to determine whether microvesicles can alter the efficacy of oncolytic viruses, and other types of viral gene delivery vectors. recent work shows that microvesicles can be loaded with adenoassociated viral (aavs) vectors for more efficient gene delivery [66] , opening a new window into the microvesicle therapeutics field. several human pathogenic viruses are known for their ability to lie dormant in the host immune system, of which hsv and ebv are perhaps the best known examples. in the case of hsv this is due to the ability of the virus to enter a latent state in the nucleus of sensory neurons during which it expresses no viral antigens and does not disturb the physiology of the neurons. in latency a single transcript is generated which encodes a precursor for four distinct hsv, mirnas which act to suppress virus replication [67] . for human herpesvirus 4 (hhv4), better known as ebv, this is largely due to incomplete eradication of the virus after early primary infection. gamma herpesviruses, including ebv, have developed a variety of strategies to exploit host-cell regulatory pathways that lead to a permanent infection of their host. when these pathways are deregulated, what is usually an undamaging herpes infection can predispose to diseaseincluding encephalitis, autoimmunity, and cancer [68] . it was recently demonstrated that ebv exploits the endosomalexosomal pathway by balancing intracellular signaling in infected b cells [69] and controlling epigenetic changes in uninfected neighboring cells via microvesicles [30] . enveloped viruses of the herpes virus family, such as human cmv (hcmv/hhv5) and ebv, depend on the interaction with cellular endosomal membrane systems for replication [70] . interestingly, mature hhv-6 virions are released together with internal vesicles through mvbs by the cellular endosomal-exosomal pathway [71] . thus, many herpesviruses generally seem to exploit endosomal pathways and microvesicles for virus production, release, and immune evasion. however, the finding that viruses such as ebv modulate host-cellular pathways that are not directly involved in virus production needs further investigation. being the first human tumor virus identified, ebv is in many aspects an extraordinarily benign pathogen and is best known as the causative agent of "kissing disease" or infectious mononucleosis. it is estimated that over 90% of the world population is persistently infected with ebv. the ebv life cycle begins by exchange through saliva and ebv virions that seem to preferentially infect naïve resting b cells in secondary lymphoid organs, such as the tonsils. occasionally isolated epithelial cells also become infected and presumably sustain lytic replication [72] , which is required for viral shedding into the saliva for transmission to new hosts [73] . to reach its near universal prevalence without harming the host, ebv and related persistent herpesviruses have evolved complex strategies encouraging immune recognition in proliferative (potentially oncogenic) stages of its life cycle, while elegantly avoiding the immune recognition at other stages by "going into hiding" [74] . upon initial infection at the mantle zone of germinal centers (gcs), the newly infected naïve b cells undergo multiple differentiation stages and tight interactions with surrounding stroma and t cells [75] . interestingly, ebv facilitates these essential interactions for the maturation of b cells, for instance, by upregulation of crucial gc reaction-associated proteins, such as gp183 [76] . this integral part of the ebv life cycle (i.e., mimicking a gc-type reaction) requires tight growth regulation in a specific ebv latency gene expression program (latency iii) and promotes rapid growth and proliferation of these infected cells through nfκb activation. this strategy in expanding the infected pool of b cells without the need for lytic replication may be advantageous under normal conditions but raises the chances of turning-on malignant growth if the viral latency programs are not properly controlled. indeed, if these cells do not progress further into memory cells by shutting down this growth program, they can remain in the proliferative phase and give rise to ebvpositive lymphomas which can kill the host, thus, restricting further viral propagation and spread [77] . in addition, ebv infection at this stage may also predispose to autoimmunity as inappropriate survival signals may interfere with negative selection of self-reactive b cells. of note, immune-suppressed individuals are at increased risk for developing ebv-driven lymphomas, reflecting the importance of a lifelong potent anti-ebv t-cell response [78] . the ability of ebv to persist despite such vigorous t-cell responses indicates that ebv can escape from the adaptive immune system and may do so in part by exploiting the endosomal-exosomal pathway through the secretion of t-cell inhibitory exosomes [44] [45] [46] . when secreted by ebv-positive tumors, these exosomes carry immune-evasive proteins including the viral protein lmp1 [79] and high amounts of galectin 9 that cause massive apoptosis of ebv-specific cd4+ t-cells via specific interaction with t-cell immunoglobulin mucin-3 (tim-3), which can negatively regulate th1 t cell and macrophage activation. the inhibition of anti-ebv immune responses is believed to promote the progression of ebv-positive malignancies, such as hodgkin's disease (hd) [46] and nasopharyngeal carcinoma (npc) [80] . vallhov et al. [81] studied the interaction between exosomes secreted by ebv-driven lymphoblastoid cell lines (lcls) and peripheral blood b cells proliferating in vitro. lcls are 95% latent, but a small proportion of cells is in a lytic stage. exosome-cell interactions could be inhibited by specific antibodies against gp350 the major envelope protein of ebv or cd21 on b cells, indicating an interaction between cd21 on b cells and the gp350 on exosomes [81] . these specific exosome-cell interactions may be exploited for exosome-based anticancer therapies, for example, in delivering the cd154 protein to leukemic b blast cells rendering them immunogenic to t cells [82] . in addition to proteins, it is now clear that microvesicles from many cell types carry and transport functional rna molecules. ebv was the first virus discovered to encode its own small regulatory mirnas [83] . ebv encodes a staggering 44 viral mirna species, derived from two major gene clusters on the viral genome, which have an important role in ebv persistence [84] . next generation sequencing indicates that these ebv-encoded mirnas make up a large fraction (20-25%) of the total cellular mirna in ebv-infected cells, encompassing 300+ different mirna species [85] . similar results were found in the mirna profile of exosomes from ebv-driven lcl cells (pegtel et al., unpublished results) . this is consistent with the idea that viral mirnas manipulate gene regulation in host cellular pathways and also exploit the exosomal mirna communication pathways. indeed, the discovery of ebv-encoded regulatory mir-nas (ebv-mirnas) residing within the lumen of exosomes indicated a novel mechanism by which exosomes can exert inhibitory effects, namely, by translational repression of target genes in noninfected recipient cells via exosomal ebv mirnas [30] . earlier studies in mice had suggested that intact exosomes from ebv-infected cells had strong physiological effects in vivo, consistent with the idea that the luminal content of exosomes is biologically significant, apart from the proteins and lipids that make up their surface [86] . subsequent studies demonstrated that ebv-infected cancer ecs also secrete ebv-mirnas, presumably within exosomes [87] . due to the lack of an accurate in vivo model for human ebv infection it is difficult to investigate the mechanism controlling release of ebv-mirnas through exosomes and to determine whether this contributes to viral persistence in healthy infected individuals. however, ebv-encoded mir-nas are transported from infected b cells to noninfected (ebv-dna negative) t cells and monocytes, supporting the idea of horizontal mirna transfer in humans. thus, viral mirnas in exosomes may contribute to sustain persistent virus infection by delivery of such mirnas into noninfected responding t cells leading to their inactivation (anergy) [45] or destruction [44] . this is consistent with recent data suggesting that exosomes efficiently transport mirnas through the immunological synapse during interactions of t cells with apcs [47] , similar to what is known concerning antigen exchange [88] . studies are underway to establish whether ebv exploits these specialized intercellular contacts for efficient posttranscriptional control in neighboring responding immune cells as a possible mechanism for immune escape. hiv [56, [89] [90] [91] has been a discussion topic in the microvesicle field for many years. not only has it been hypothesized that hiv itself may have microvesicle features, but microvesicles also have been described to have immune modulatory functions on hiv-infected cells and to expand the infectivity of hiv. in 2003 gould et al. [92] postulated that hiv-an enveloped retrovirus-hijacks the microvesicle system to benefit its own assembly and subsequent exit. interestingly, 6 advances in virology inhibitors were identified that blocked the budding of both shed microvesicles and hiv particles [93] . in addition, peptides were identified that prevented interactions of hiv nef protein-a key protein in the hiv life cyclewith mortalin, a cellular heat shock protein, and resulted in inhibition of the release of hiv and nef-containing microvesicles [94] . careful analysis, however, has indicated that although hiv exploits certain proteins that also play a role in exosome formation via the mvb [95] , hiv assembly does not necessarily use the same logistics system as do exosomes. importantly, it has been established that hiv budding occurs mostly at the plasma membrane and not from within the mvb [96] [97] [98] [99] . interestingly, hiv recruits members of the mvb escrt complex for proper hiv budding from the plasma membrane [98] [99] [100] [101] [102] . while in cd4+ t cells hiv release appears to be independent of exosomes [103] , in monocyte-derived macrophages hiv can bud into endosomes [102, 104] . however, several studies highlight that hiv-1 budding also in macrophages occurs primary at the plasma membrane [105] [106] [107] . thus, the controversy about the site of productive virus assembly in macrophages mostly favors the plasma membrane. hiv release in dendritic cells may be triggered by signals similar to those for exosome release [102, 108, 109] , and secretion of hiv from endocytic compartments in dendritic cells can result in hiv release upon interaction with t cells [110, 111] . however, these endocytic compartments were also described to be connected with the extracellular space [112, 113] and suggested to be invaginated domains distinct from classical endocytic vesicles [114] . moreover, microvesicle release from t cells treated with ceramide inhibitors was not affected by such treatment [111] , as previously reported for hiv-1 [115] . however, both viruses and microvesicles produced from ceramide-deficient cells failed to be captured by mature dendritic cells [111] . therefore, more research is warranted on the specific sites of hiv assembly in particular cell types, and to what extent the endosomal compartments play a role in the hiv life cycle, as well as the possible convergence of hiv and shed microvesicle pathways. it seems likely that hiv has simply adapted to use certain host factors for different exit modalities, and that these may vary among different types of cells, as well as under different conditions. it will be of continuing interest to further study the retroviral family, including the endogenous retroviruses, in order to determine whether the microvesicle cargo systems are perhaps a remnant of previous retroviral infections that happened earlier in evolution-and elements of which are now used in an opportunistic setting by retroviruses, such as hiv [56, [89] [90] [91] 102] . this overlap in pathways and the consequence of using overlapping machinery for release can result in phenotypic similarities between microvesicles and retroviruses and potentially interfere with anti-hiv strategies. for instance, hiv released from t cells has similar glycome properties as t-cell microvesicles, arguing for a common origin and indicating phenotypic similarity [116] . more research in the convergence of microvesicle and hiv pathways may improve our understanding of these processes and propel the development of new antiviral drugs directed against hiv. the role of microvesicles during hiv infection has not yet been extensively studied, but they appear to be involved in both hiv infectivity enhancement and resistance depending on the cells of origin. microvesicles derived from hivinfected cells have been reported to contain hiv ccr5 coreceptors, allowing for enhanced hiv infection of other cells [34] . moreover, microvesicles from megakaryocytes and platelets contain cxcr4 and upon transference confer susceptibility to cells normally resistant to hiv infection [35, 117] . in addition, during hiv replication the hiv nef protein can alter the exosomal pathway by increasing the number of intracellular vesicles and mvbs [118] [119] [120] [121] . hiv nef-induced microvesicle release from infected and noninfected cells [39, 40] can induce apoptosis in cd4+ t cells [41] and convey resistance to hiv infection [61] . the transfer of nef or other viral components through microvesicles may represent an important mechanism for immune evasion by viruses. in addition, exosomes can contain apobec3g, a cytidine deaminase that is part of the cellular antiviral system against retroviruses, which upon transference to recipient cells via exosomes can inhibit hiv replication [61] . while cd45, cd86, and mhc class ii molecules have been found in microvesicles from hivinfected cells [42] , possibly serving to silence the immune response, microvesicles derived from cd8+ t cells can act to suppress hiv replication [33] . moreover, exosomes in association with hiv derived from dendritic cells significantly enhance hiv infection of cd4+ t cells [36] . in conclusion, microvesicles from hiv-infected cells as well as from noninfected cells play an important role in hiv replication and dissemination. therefore, interference with microvesicle-mediated signaling could possibly be harnessed to halt hiv infection. retrotransposon elements such as line, alu, and human endogenous retroviruses (hervs) make up about 45% of the human genome and have played an important role in genome evolution [122] . these viral-like elements infected germ cells in the human genome millions of years ago and then became a stable part of the inherited genetic material. although most line elements are inactive, a number of active ones remain and are able to "jump" to new locations in the genome, contributing to genomic instability [123] . these events can have important effects on our genome, for example, by inactivating genes, altering gene expression and facilitating random insertion of new cdna copies in the genome, as in integration of pseudogenes [124] . many tumor cells also release retroviral-like microvesicles that contain active retrotransposon sequences, such as herv-k [125] . recently, tumor-derived microvesicles have been shown to be enriched in retrotransposon elements such as line1, alu, and herv-k [17] . furthermore, herv-k was transferred through microvesicles to normal huvecs, which then showed an increase in herv-k levels 12 hours following exposure to tumor microvesicles. in addition, the mouse retroviral rna vl30 is packaged in retrovirus vectors by mouse packaging cell lines and transferred to human advances in virology 7 cells infected with those vectors [126] . the mouse vl30 has several stop codons in the regions encoding for genes such as gag, pol and env, thereby inhibiting its ability to encode functional proteins [126] . however, transfer of the vl30 mrna together with tissue factor (tf) to human melanoma cells served to induce their metastatic potential. this change in phenotype apparently occurs through formation of a complex with the protein-associated splicing factor (psf) protein which represses transcription of an insulin-like growth factor-1 (igf-1) inducible gene, with dissociation of this complex allowing transcription to proceed [126] . three of the 11 human genes affected by vl30 mrna were oncogenic, suggesting that the transfer of retroviral rna sequences can have catastrophic effects on recipient cells. song et al. [126] have identified human retrotransposon sequences that are >90% identical to the mouse vl-30 suggesting human vl-30 transferred through microvesicles could have similar effects on transcription [126] . long interspersed elements (lines)-most notably l1-comprise about 17% of the human genome. several studies indicate that a subset of l1 elements is still actively expanding in the number of sequences within the human genome by retrotransposition. this active subpopulation, termed transcriptionally active (ta), is approximately 2 million years old, and it has high levels of insertional polymorphism in the human population [127, 128] . some of these new insertions may be intolerable and lethal and therefore eliminated; others may result in phenotypically tolerable disease, such as in coffin-lowry syndrome and choroideremia [129] [130] [131] , while still others have been associated with the induction of cancer, for example, lung cancer [132] . the high level of polymorphism of l1 elements indicates that they continue to have profound effects on the human genome, and recent evidence suggests that microvesicles may be a potential route of delivery for these elements [17] . this microvesicle-mediated trojan horse-like [92] transferance of transposons could perhaps allow for a stealthy dissemination of retrotransposons, especially in a tumor setting, avoiding immune-recognition, and achieving "long distance" delivery. hervs also entered the human genome millions of years ago and comprise about 8% of the human genome. they consist of gag, pol, and env sequences, flanked by two long terminal repeats [133] . most of these sequences are now silent because of acquired mutations and deletions over the course of evolution, but herv-k113 can produce intact, albeit noninfectious, retroviral particles [134] . some of these sequences are still transcriptionally active and are associated with diseases, such as lymphoma and breast cancer [21, 135] . in cancer, hypomethylation of the genome seems to predominantly affect retrotransposon sequences (perhaps because they are highly abundant in the human genome), allowing increased transcription, especially in the case of the most recent entrants, which also happen to be the elements with the most intact coding potential [136] . indeed retroviral-like microvesicles have been found in cancer patients, notably those with lymphomas [21] , breast cancer [137] , and teratomas [138] . as expected, these patients also had high levels of reverse transcriptase, and viral gag and env proteins and rna in the tumor cells and retrovirus-like microvesicles released from them into the circulation [21] . tumor microvesicles from cultured tumor cells also have been shown to be enriched in retrotransposon rna, dna, and reverse transcriptase, suggesting that a subpopulation of these microvesicles may indeed be of retroviral origin [19] . in summary, this review deals with how extracellular vesicles-such as exosomes and shed microvesicles-share pathways with the assembly and release of retrotransposon elements and viruses. in figure 1 we summarize how herpesviruses such as ebv and hsv, originate from the nucleus and can merge with microvesicle pathways. several proteins used for exosome production are used by herpesviruses for functional release. also, the convergence of these pathways may explain the observations of virus-like particles, which can be exosomes or shed microvesicles containing viral proteins or nucleic acids. similar observations have been made for retroviruses and retrotransposon elements with circulating microvesicles containing retrotransposon rna found in some cancer patients. it remains to be investigated to what extent exosomes and shed microvesicles are remnants of previous retroviral colonization. in this review we note the observations of retroviral as well as retrotransposon elements in microvesicles, perhaps allowing further dissemination of such nucleic acid sequences. the use of microvesicle pathway elements by viruses such as hiv may be suggestive of an intricate coevolution of different endogenous and exogenous (retro)virus subtypes. viruses not only use microvesicle pathways for their own assembly and release but are also capable of exploiting the highly complex microvesicle communication system in an intercellular setting as simplified in figure 2 . during viral infection microvesicles can have various effects on different types of cells, either limiting viral infection or enhancing it. thus, a picture is emerging that viruses and microvesicles are codependent pleiotropic entities. more research is needed into the differential functions of different subtypes of microvesicles and their cross-talk in relation to the immune response and outcome of viral infection. shedding microvesicles: artefacts no more exosomes: extracellular organelles important in intercellular communication exosomes-vesicular carriers for intercellular communication brain tumor microvesicles: insights into intercellular communication in the nervous system the multifaceted exosome: biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities glioblastoma microvesicles transport rna and proteins that promote tumour growth and provide diagnostic biomarkers human saliva, plasma and breast milk exosomes contain rna: uptake by macrophages molecular characterization of dendritic cell-derived exosomes: selective accumulation of the heat shock protein hsc73 membrane vesicles as conveyors of immune responses exosome secretion of dendritic cells is regulated by hrs, an escrt-0 protein ceramide triggers budding of exosome vesicles into multivesicular endosomes rab27a and rab27b control different steps of the exosome secretion pathway the regulation of exosome secretion: a novel function of the p53 protein rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner regulation of exosome secretion by rab35 and its gtpase-activating proteins tbc1d10a-c intercellular transfer of the oncogenic receptor egfrviii by microvesicles derived from tumour cells tumour microvesicles contain retrotransposon elements and amplified oncogene sequences mechanisms for the formation of membranous nanostructures in cell-to-cell communication arf6-regulated shedding of tumor cell-derived plasma membrane microvesicles formation and release of arrestin domain-containing protein 1-mediated microvesicles (armms) at plasma membrane by recruitment of tsg101 protein human endogenous retrovirus k (hml-2) elements in the plasma of people with lymphoma and breast cancer hypomethylation of retrotransposable elements correlates with genomic instability in non-small cell lung cancer apoptotic bodies from endothelial cells enhance the number and initiate the differentiation of human endothelial progenitor cells in vitro platelet-derived microvesicles transfer tissue factor to monocytes but not to neutrophils bystander b cells rapidly acquire antigen receptors from activated b cells by membrane transfer modulation of monocyte-endothelial cell interactions by platelet microparticles platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment exosomes communicate protective messages during oxidative stress; possible role of exosomal shuttle rna exosome-mediated transfer of mrnas and micrornas is a novel mechanism of genetic exchange between cells functional delivery of viral mirnas via exosomes unraveling the mystery of cancer by secretory microrna: horizontal microrna transfer between living cells immune-related micrornas are abundant in breast milk exosomes noncytotoxic suppression of human immunodeficiency virus type 1 transcription by exosomes secreted from cd8 + t cells transfer of the chemokine receptor ccr5 between cells by membranederived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection plateletand megakaryocyte-derived microparticles transfer cxcr4 receptor to cxcr4-null cells and make them susceptible to infection by x4-hiv immature dendritic cell-derived exosomes can mediate hiv-1 trans infection the herpes simplex virus-1 encoded glycoprotein b diverts hla-dr into the exosome pathway hiv-1 evades virus-specific igg2 and iga responses by targeting systemic and intestinal b cells via long-range intercellular conduits massive secretion by t cells is caused by hiv nef in infected cells and by nef transfer to bystander cells genetic characterization of hiv type 1 nef-induced vesicle secretion hiv nef is secreted in exosomes and triggers apoptosis in bystander cd4 + t cells differential incorporation of cd45, cd80 (b7-1), cd86 (b7-2), and major histocompatibility complex class i and ii molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation cytomegalovirusinfected human endothelial cells can stimulate allogeneic cd4 + memory t cells by releasing antigenic exosomes blood diffusion and th1-suppressive effects of galectin-9-containing exosomes released by epstein-barr virus-infected nasopharyngeal carcinoma cells localization of the epstein-barr virus protein lmp 1 to exosomes galectin-1 mediated suppression of epstein-barr virus-specific t-cell immunity in classic hodgkin lymphoma unidirectional transfer of microrna-loaded exosomes from t cells to antigen-presenting cells the effect of herpes simplex virus type 1 l-particles on virus entry, replication, and the infectivity of naked herpesvirus dna comprehensive characterization of extracellular herpes simplex virus type 1 virions noninfectious l-particles supply functions which can facilitate infection by hsv-1 assembly of enveloped tegument structures (l particles) can occur independently of virion maturation in herpes simplex virus type 1-infected cells functional roles of the tegument proteins of herpes simplex virus type 1 herpes simplex virus type 1 targets the mhc class ii processing pathway for immune evasion exosomes and other microvesicles in infection biology: organelles with unanticipated phenotypes membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles microvesicles and viral infection b lymphocytes secrete antigen-presenting vesicles exosomal sorting of the cytoplasmic domain of bovine leukemia virus tm env protein pivotal advance: the promotion of soluble dc-sign release by inflammatory signals and its enhancement of cytomegalovirus-mediated cis-infection of myeloid dendritic cells association of hepatitis c virus envelope proteins with exosomes exosomes packaging apobec3g confer human immunodeficiency virus resistance to recipient cells chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix 1 efficiently form viruslike particles (vlps) that protect mice against challenge with sars-cov enhanced antitumor efficacy of vasculostatin (vstat120) expressing oncolytic hsv-1 antitumor efficacy of 34.5enve: a transcriptionally retargeted and vstat120-expressing oncolytic virus oncolytic viruses microvesicleassociated aav vector as a novel gene delivery system micrornas expressed by herpes simplex virus 1 during latent infection regulate viral mrnas viral mirnas exploiting the endosomal-exosomal pathway for intercellular cross-talk and immune evasion lmp1 association with cd63 in endosomes and secretion via exosomes limits constitutive nf-κb activation budding events in herpesvirus morphogenesis human herpesvirus-6 induces mvb formation, and virus egress occurs by an exosomal release pathway epstein-barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers the dynamics of ebv shedding implicate a central role for epithelial cells in amplifying viral output germinal center b cells latently infected with epstein-barr virus proliferate extensively but do not increase in number the intersection of epstein-barr virus with the germinal center ebi2 mediates b cell segregation between the outer and centre follicle persistence of the epstein-barr virus and the origins of associated lymphomas cellular responses to viral infection in humans: lessons from epstein-barr virus direct immunosuppressive effects of ebv-encoded latent membrane protein 1 functional inactivation of ebv-specific t-lymphocytes in nasopharyngeal carcinoma: implications for tumor immunotherapy exosomes containing glycoprotein 350 released by ebv-transformed b cells selectively target b cells through cd21 and block ebv infection in vitro ebv-gp350 confers b-cell tropism to tailored exosomes is a neo-antigen in normal and malignant b cells-a new option for the treatment of b-cll identification of virus-encoded micrornas epstein-barr virus micrornas are evolutionarily conserved and differentially expressed the viral and cellular microrna targetome in lymphoblastoid cell lines exosomes derived from il-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis human tumor virus utilizes exosomes for intercellular communication polarized secretion of lysosomes at the b cell synapse couples antigen extraction to processing and presentation exosomes and retroviruses: the chicken or the egg? hiv and mature dendritic cells: trojan exosomes riding the trojan horse? human and nonhuman primate lentiviral infection and autoimmunity the trojan exosome hypothesis identification of an inhibitory budding signal that blocks the release of hiv particles and exosome/microvesicle proteins secretion modification region-derived peptide disrupts hiv-1 nef 's interaction with mortalin and blocks virus and nef exosome release human immunodeficiency virus type 1 gag engages the bro1 domain of alix/aip1 through the nucleocapsid higher-order oligomerization targets plasma membrane proteins and hiv gag to exosomes exosomes and hiv gag bud from endosome-like domains of the t cell plasma membrane visualizing hiv-1 assembly dynamics of escrt protein recruitment during retroviral assembly live-cell visualization of dynamics of hiv budding site interactions with an escrt component the cell biology of hiv-1 virion genesis endosomes, exosomes and trojan viruses hiv-1 is budded from cd4 + t lymphocytes independently of exosomes evidence that hiv budding in primary macrophages occurs through the exosome release pathway plasma membrane is the site of productive hiv-1 particle assembly hiv-1 buds predominantly at the plasma membrane of primary human macrophages hiv-1 assembly in macrophages in macrophages, hiv-1 assembles into an intracellular plasma membrane domain containing the tetraspanins cd81, cd9, and cd53 lysosome-related organelles: a view from immunity and pigmentation dc-sign-mediated internalization of hiv is required for trans-enhancement of t cell infection capture and transfer of hiv-1 particles by mature dendritic cells converges with the exosome-dissemination pathway in vitro derived dendritic cells trans-infect cd4 t cells primarily with surface-bound hiv-1 virions the achilles heel of the trojan horse model of hiv-1 trans-infection hiv traffics through a specialized, surface-accessible intracellular compartment during trans-infection of t cells by mature dendritic cells the hiv lipidome: a raft with an unusual composition hiv-1 and microvesicles from t cells share a common glycome, arguing for a common origin enhanced activation of platelets with abnormal release of rantes in human immunodeficiency virus type 1 infection human immunodeficiency virus-1 nef expression induces intracellular accumulation of multivesicular bodies and major histocompatibility complex class ii complexes: potential role of phosphatidylinositol 3-kinase interactions between nef and aip1 proliferate multivesicular bodies and facilitate egress of hiv-1 nef-induced alteration of the early/recycling endosomal compartment correlates with enhancement of hiv-1 infectivity nef proteins encoded by human and simian immunodeficiency viruses induce the accumulation of endosomes and lysosomes in human t cells retrotransposons revisited: the restraint and rehabilitation of parasites great exaptations human line retrotransposons generate processed pseudogenes human endogenous retrovirus family herv-k(hml-2) rna transcripts are selectively packaged into retroviral particles produced by the human germ cell tumor line tera-1 and originate mainly from a provirus on chromosome 22q11.21 binding of mouse vl30 retrotransposon rna to psf protein induces genes repressed by psf: effects on steroidogenesis and oncogenesis l1 (line-1) retrotransposon evolution and amplification in recent human history identification and characterization of novel polymorphic line-1 insertions through comparison of two human genome sequence assemblies intronic l1 insertion and f268s, novel mutations in rps6ka3 (rsk2) causing coffin-lowry syndrome novel types of mutation in the choroideremia (chm) gene: a full-length l1 insertion and an intronic mutation advances in virology activating a cryptic exon retrotransposable elements and human disease natural mutagenesis of human genomes by endogenous retrotransposons endogenous retroviruses in the human genome sequence human endogenous retrovirus herv-k113 is capable of producing intact viral particles transcription of herv-e and herv-erelated sequences in malignant and non-malignant human haematopoietic cells cpg methylation directly regulates transcriptional activity of the human endogenous retrovirus family herv-k(hml retrovirus-like particles released from the human breast cancer cell line t47-d display type b-and c-related endogenous retroviral sequences induction of retrovirus particles in human testicular tumor (tera-1) cell cultures: an electron microscopic study the authors thank suzanne mcdavitt for help with the editorial process. t. wurdinger is financially supported by nwo-vidi, d. m. pegtel by nwo-veni, x. o. breakefield by nih/nci grants ca069246 and ca141150, and l. balaj by the huygens scholarship nl. key: cord-288945-c9ow1q5c authors: spengler, ulrich title: liver disease associated with non-hepatitis viruses date: 2019-11-01 journal: encyclopedia of gastroenterology doi: 10.1016/b978-0-12-801238-3.65782-3 sha: doc_id: 288945 cord_uid: c9ow1q5c hepatitis is commonly associated with certain viruses labeled as “hepatitis” viruses. however, many other viral infections can also affect the liver ranging from mild asymptomatic elevations of aminotransferases to fulminant hepatic failure. this article will provide a brief overview on a variety of different viral infections that may be associated with significant liver pathology at least under certain conditions, for example, immunosuppression. this overview discusses key virological features, clinical presentation of associated liver disease and provides some information on diagnosis and an outline of treatment options. thus, the overview can provide first orientation when infectious hepatitis is encountered in a patient that cannot be explained by the usual hepatitis viruses. the liver is a highly perfused organ and the first filter for blood coming from the intestines. thus, the liver is particular prone to become involved in blood-borne infections. apart from the so-called hepatitis viruses, many other viruses, primarily targeting other extrahepatic tissues, also lead to liver damage and hepatitis. damage can range from asymptomatic elevations of aminotransferases to fulminant hepatic failure depending on the virus and the host's immune response. when the immune system controls infection poorly, direct infection of hepatocytes and liver necrosis may occur. this situation applies to patients under severe immunosuppression or infections with particularly virulent agents such as the viruses that cause hemorrhagic fevers. alternatively, liver cells may become victims of collateral damage without direct infection when cytolytic cd8 þ t effector lymphocytes are expanded outside the liver and then recruited via liver-resident macrophages such as kupffer cells presenting viral antigens (polakos et al., 2006; schumann et al., 2000) . this type of liver damage is particularly associated with respiratory viruses such as influenza virus. however, since many viral infections expand cd8þ t lymphocytes, this by-stander mechanism may affect the liver more often (murali-krishna et al., 1998) . here, we provide a brief overview over viral infections primarily not designated as hepatitis viruses which may lead to liver disease and hepatic complications. this overview will largely focus on the hepatic aspects of viral infections. thus, readers interested in more detailed information should consult a comprehensive textbook in microbiology or clinical infectious diseases. approximately 8% of travelers to the developing world require medical care during or after travel, and fever is the underlying problem in 28% of them (wilson et al., 2007) . physicians evaluating returned travelers frequently suspect rare or exotic diagnoses. although exotic diseases, in particular viral hemorrhagic fevers are a severe threat in certain regions of the world, liver disease due to exotic infections such as ebola virus, rift valley fever or lassa fever have been reported only sporadically but do not represent a frequent health problem in returning travelers. viral hemorrhagic fevers share some epidemiological and clinical features and cause rather similar liver pathology. most viruses are transmitted via arthropod vectors. the viruses cause small vessel damage in multiple organs resulting in overt hemorrhage. the spectrum of diseases and their geographical distribution are listed in table 1 . much attention has been paid to abnormal liver function and altered hepatic pathology. nevertheless, clinically significant liver disease or death from liver failure are rare complications except in yellow fever. dengue fever is among the top three etiological agents, accounting for approximately 6% of febrile illnesses in the traveler (wilson et al., 2007) . of note, although malaria is the leading cause of systemic febrile illness worldwide, apart from sub-saharan africa and central america travelers returning from tropical or sub-tropical regions had dengue fever more frequently than malaria. chikungunya fever is an emerging novel virus infection recently expanding in asia and africa, which also causes fever, myalgia, arthralgia and skin rash in increasing numbers of patients. table 1 viral hemorrhagic fevers affecting the liver the dengue virus complex comprises four antigenically related but distinct flaviviruses termed dengue virus serotypes 1 through 4 (den-1 to . dengue viruses are transmitted by aedes aegypti mosquitos in epidemic and endemic outbreaks and cause acute infections. three to six days after a mosquito bite the virus spreads via the blood-stream, and among the various organs it can be isolated frequently from liver samples (rosen and khin, 1989) . dengue virus infection usually causes a flu-like illness with a rash-dengue fever (fig. 1 ). hepatomegaly and elevated serum aminotransferases, which are usually mild, are common in dengue virus infections (wahid et al., 2000) . clinically more severe diseases, for example, dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) can follow from secondary infection with dengue virus of different serotype. in 2009, the world health organization issued new guidelines and introduced a revised classification scheme consisting of the following categories: dengue without warning signs, dengue with warning signs, and severe dengue (world health organization, 2009 ). however, there are no reliable warning signs for severe dengue. in dhf there are widespread petechial hemorrhages together with multiple organ damage; in dss, which mostly affects children below the age of 15, there is extensive capillary leakage and severe fluid depletion leading to hypovolemic shock. if untreated, mortality approaches 50%. in fatal cases of dhf the liver is enlarged, pale from steatosis and shows multifocal hemorrhages. laboratory diagnosis of dengue virus infection is done by detection of viral components, for example, pcr in serum or indirectly by serology. the sensitivity of each approach depends on the duration and course of the patient's illness when the patient presents for evaluation. currently vaccines against dengue virus are being developed but have not become licensed, yet. there is no direct antiviral therapy available against the dengue viruses. thus, treatment is supportive, requiring meticulous fluid management and intensive care in dss. details for treatment are summarized in recent who guidelines (world health organization, 2012). chikungunya is an arthropod-borne toga virus initially endemic to west africa, which next spread to the indian ocean islands and southeast asia (charrel et al., 2007) . chikungunya fever was originally considered a disease of tropical and subtropical regions, until an outbreak in northern italy was recorded in 2007 (rezza et al., 2007) . since then chikungunya infections have become exported to other western countries, the americas and caribbean region via international travel. of note, dengue and zika viruses are transmitted by the same mosquito vectors as chikungunya. thus, the viruses can co-circulate in the same geographic region, and coinfections have been documented. transmission of chikungunya via blood products has been reported in france, where a nurse was infected by exposure to blood from an infected patient. chikungunya, might also be transmitted inadvertently by organ transplantation since viremia can exceed high levels prior to onset of symptoms. the infection begins abruptly with high fever, symmetric polyarthralgia and macular or maculopapular skin rash (taubitz et al., 2007) . pruritus and bullous skin lesions have also been described. previously chikungunya fever has been considered a self-limited disease. however, severe complications also comprising acute viral hepatitis and deaths have been reported in the recent outbreaks; particularly in elderly patients (> 65 years) and people with pre-existing chronic medical problems. some patients have persisting symptoms for a variable length of time after the acute illness. manifestations include arthritis/arthralgia, edematous polyarthritis of fingers and toes, morning pain and stiffness, and severe tendosynovitis. clinically chikungunya fever must be differentiated from dengue fever, which shares many symptoms and features. the diagnosis of chikungunya virus infection should be suspected in a patient with acute onset of fever and polyarthralgia who had possible exposure by travel to or residency in an epidemiological risk area. the diagnosis of chikungunya is established by detection of chikungunya viral rna via real-time reverse-transcription polymerase chain reaction (rt-pcr) or chikungunya virus serology (pan-american health organisation, 2017). chikungunya igm antibodies become detectable by elisa 5 days after the onset of symptoms. for certain regions simultaneous testing for dengue and zika virus infection is recommended. there exists no specific antiviral therapy for acute chikungunya virus infection. treatment consists of supportive care and includes rest, fluid replacement, and eventually the use of nonsteroidal antiinflammatory drugs (nsaids) or acetaminophen to relieve pain and fever. in patients suspected to have dengue virus coinfection nsaids should be avoided before the patient becomes afebrile for >2 days, in order to avoid bleeding complications associated with severe dengue virus infection. chikungunya infection cannot be prevented by vaccination. hanta viruses are examples of emerging viruses, which belong to the genus hantavirus within the bunyaviridae family. they are negative-sense single-stranded rna viruses, primarily harbored in rodents and shed in rodent urine, saliva and feces. the virus is inhaled by man as aerosols from dried rodent excreta, or in unusual circumstances, transmitted by bites (nichol et al., 2000) . hantaviruses exist in multiple serotypes worldwide, which differ in their virulence. some are considered apathogenic, while certain isolates can produce two distinct severe syndromes in humans: the hanta virus cardiopulmonary syndrome, mostly due to isolates in the americas; and the hemorrhagic fever with renal syndrome caused by isolates (seoul virus, dobrava virus, puumala virus, hantaan virus) in europe and asia (plyusnin et al., 2001) . in some instances, patients with hanta virus hemorrhagic fever suffered from severe acute hepatitis, whereas renal damage was rather mild (wong et al., 1987; chan et al., 1987; lledó et al., 2003) . hanta virus has also been incriminated as a potential cause of cryptogenic hepatitis in southwestern china. however, this hypothesis has not been confirmed by antibody studies in japan. hanta viruses, however, may still play some role, because hanta virus infection has been observed to trigger autoimmune liver disease (yotsuyanagi et al., 1998) . thus, this mechanism may contribute to community-acquired hepatitis (martin et al., 2008) . nevertheless, the precise role of hanta virus infections for human liver disease still awaits clarification. by the time symptoms are evident, patients uniformly have antiviral igm antibodies and most have igg antibodies. diagnostic assays to detect hanta virus antibodies include enzyme-linked immunosorbent assay (elisa), immunoblot test (sia), western blot, indirect immunofluorescence (ifa), complement fixation, and hemagglutinin inhibition as well as neutralization assays. hanta virus strains associated with hemorrhagic fever and hepatorenal syndrome are sensitive to ribavirin in vitro. a prospective, randomized, double-blind, placebo-controlled trial in the people's republic of china reported a sevenfold decrease in mortality among ribavirin-treated patients with serologically confirmed hanta virus disease (huggins et al., 1991) . however, ribavirin appeared to be less effective in hanta virus cardiopulmonary syndrome. thus, it has not yet been approved, but nevertheless may be tried as rescue treatment in emergency situations (sidwell and smee, 2003) . yellow fever is a member of the flaviviridae family and constitutes a single-stranded plus strand rna virus. it comprises a single conserved serotype and seven major genotypes reflecting distinct regions in west africa, central-east africa and south america (vasconcelos et al., 2004; barnett, 2007) . yellow fever virus is transmitted by a variety of different aedes vectors and causes endemic and epidemic outbreaks in africa and south america. yellow fever can be prevented by vaccination, and thus, has become rare in travelers. the spectrum of yellow fever virus infection ranges from subclinical infection to a life-threatening disease with fever, jaundice, renal failure and hemorrhage. usually yellow fever initially presents as an acute, flu-like illness of sudden onset with fever, myalgia and headache, which cannot be distinguished easily from other acute infections such as severe malaria, leptospirosis, fulminant viral hepatitis or dengue hemorrhagic fever. between 48 and 72 h after onset, aminotransferases start to rise in blood heralding the development of jaundice. the degree of liver abnormalities at this stage predicts the severity of liver disease during the course of the illness later on. next, a period of apparent remission lasting up to 48 h may follow the initial infection. patients with abortive infection recover at this stage. about 15% of patients will enter the third stage of intoxication characterized by the return of fever, prostration and organ dysfunction. patients suffer from nausea, vomiting, or epigastric pain, and develop jaundice and oliguria. bleeding can occur from the mouth, nose, eyes or stomach. serum aspartate transferase (ast) levels usually exceed those of alanine transferase (alt). the outcome of yellow fever infection is determined during the second week after onset, when many patients either rapidly recover, while between 20% and 50% of the patients, who have progressed to the stage of intoxication, will ultimately die from circulatory shock. convalescence may be associated with fatigue over several weeks, and occasionally jaundice and elevated aminotransferases may persist for months. the diagnosis of yellow fever is confirmed by demonstration of specific igm by elisa, by pcr or by isolating the virus from the blood. polymerase chain reaction (pcr) testing in blood and urine can detect the virus in early stages of the disease. in later stages, testing to identify antibodies is recommended. liver biopsies should be avoided due to a high risk of bleeding complications. there is currently no specific anti-viral drug to treat yellow fever but specific care to manage dehydration, liver and kidney failure as well as fever improves outcomes. ribavirin inhibits yellow fever virus in vitro. however, the extremely high concentrations of the drug needed cannot be achieved in vivo. recently there have been attempts to treat liver failure resulting from yellow fever infection by high urgency liver transplantation (song et al., 2019) . however, outcomes after liver transplantation were mixed and the few survivors had frequent postoperative bacterial and cytomegalovirus infections. a highly active attenuated live-vaccine is available, which induces seroconversion rates >95% and provides a high level of protection. due to potential risks associated with a live virus vaccine children below the age of 9 months, pregnant women and immunosuppressed individuals should not receive the vaccine, nor should subjects be vaccinated who are allergic to egg proteins. infrequently two serious vaccine-related complications may occur: a form of encephalitis termed yellow fever-associated neurotropic disease and a syndrome resembling natural infection designated as yellow fever vaccine-associated viscerotropic disease. adenoviruses have a worldwide distribution and cause febrile diseases. over 50 serotypes can be distinguished which are further subdivided into the six subgoups a through f. typical syndromes comprise conjunctivitis, upper respiratory tract infections such as pharyngitis and coryza, pneumonia and otitis media. in young children an acute diarrheal illness is caused by subgroup f type 40 and 41 adenoviruses. adenoviruses can persist in human tissue over prolonged periods (garnett et al., 2009) , and can cause a variety of clinical syndromes in immunocompromised individuals including severe hepatitis (kojaoghlanian et al., 2003) . in particular, transmission of latent adenovirus with the donated organ is a risk factor for adenoviral hepatitis in pediatric liver transplantation (michaels et al., 1992) . adenoviral hepatitis occurs in congenital and acquired immunodeficiency syndromes and resembles severe necrotizing hepatitis associated with herpes simplex virus infection. patients develop extensive areas of liver cell necrosis (fig. 2) , massively elevated aminotransferases, and a severe coagulopathy (south et al., 1982; janner et al., 1990; krilov et al., 1990) . ultimately, outcomes may be fatal. liver biopsy specimens may reveal typical intranuclear inclusion bodies in necrotic areas of the liver, but biopsies carry an extremely high bleeding risk. viral isolation and pcr techniques help to identify the causative viral strain. to date a proven therapy or vaccine to prevent adenoviral hepatitis does not exist, but ribavirin may be helpful in selected cases (wulffraat et al., 1995) . liver damage in fatal influenza has been considered immune-mediated, because high cytokine levels were detected (murali-krishna et al., 1998; peiris et al., 2004) . moreover, volunteers infected experimentally with intranasal influenza a/kawasaki/86 (h1n1) transiently developed elevated aminotransferases (polakos et al., 2006) . since the rise in liver enzymes occurred after pyrexia had settled, it was concluded that the host's immune responses rather than viral infection caused damage to the liver. immune mediated liver damage may also be the cause for elevated aminotransferases in other viral respiratory infections such as respiratory syncytial virus (peiris et al., 2004) . however, cardiovascular failure and hepatic ischemia must be considered in as alternative factors in patients with severe respiratory infections ( fig. 3) (eisenhut et al., 2004) . influenza viruses represent three genera in the orthomyxoviridae family. generally influenza a viruses is associated with more severe disease in humans than influenza viruses b and c. influenza a is further subdivided with respect to genetic variation in its hemagglutinin (h) and neuraminidase (n) genes. influenza viruses commonly cause a self-limited acute respiratory infection with fever, rhinorrhea, sore throat and occasionally gastrointestinal symptoms. therefore, aminotransferases are not monitored routinely. in the 2004 h1n5 influenza outbreak, however, about 60% of patients with pneumonia had deranged liver function tests with gastrointestinal symptoms such as vomiting, abdominal pain and diarrhea on initial presentation (yuen and wong, 2005) . although molecular evidence for viral liver disease was not found, autopsy revealed hepatic centro-lobular necrosis in some cases . in influenza virus infection the clinical presentation is dominated by fever and respiratory symptoms. the diagnosis be established by detecting viral antigen or antibodies, but nowadays the gold standard has become detection of viral rna in throat washings by pcr. influenza virus infection can be treated and prevented by neuraminidase inhibitors such as oseltamivir and zanamivir, which have been recommended especially for treatment in influenza h1n5 infection by the who in 2010 (schünemann et al., 2007) . however, their benefit is limited due to the appearance of resistant isolates in recent outbreaks. a preventive vaccine is adapted annually to the circulating strains. severe acute respiratory syndrome (sars) is caused by a novel coronavirus (sars-coronavirus, sars-cov), which caused outbreaks of severe infections of the lung and gastrointestinal tract in the far east and canada (ksiazek et al., 2003; drosten et al., 2003; lee et al., 2003; poutanen et al., 2003) . there was also other organ involvement. middle east respiratory syndrome coronavirus (mers-cov) first appeared in the arabian peninsula and meanwhile has occasionally been observed also in few travelers returning from risk areas. apart from viral pneumonia other internal organs may become affected including hepatitis (alsaad et al., 2018) , particularly when mers-cov hits patients with concomitant diseases, for example, diabetes mellitus. sars-cov and mers-cov have been detected in masked palm civets, dogs and cats as well as camel and thus represent zoonotic infections in man. approximately 25% of patients with sars had elevated liver enzymes at the onset of infection, and further 45% of patients with normal liver enzymes at initial presentation developed elevated aminotransferases later on, so that overall up to 70% of patients showed elevated liver enzymes during their illness (booth et al., 2003; choi et al., 2003; wong et al., 2003; chan et al., 2005) . jaundice was observed in <10% of cases. in most patients aminotransferases started to rise toward the end of the first week and peaked at the end of the second week. with resolution of sars aminotransferases normalized spontaneously in the majority of patients. severe liver damage (alt >5 times the upper limit of normal) was observed more frequently in male patients, and those with significant other comorbidities or elevated serum creatinine levels (chan et al., 2005) . diagnosis of sars-cov and mers-cov infection is suspected in persons with typical symptoms who had contact to risk areas. the diagnosis is confirmed in certified specialized microbiology labs by pcr from respiratory fluids. hygienic prevention measures have to be respected when handling samples and caring for patients with suspected coronavirus pneumonia. during sars outbreaks both ribavirin and kaletra (baby dose ritonavir/lopinavir) were tested as experimental therapy but showed limited success. herpesviruses form a large family of dna viruses, which comprises eight members that can cause disease in man (table 2 ). herpes simplex virus (hsv), varizella zoster virus (vzv), epstein-barr virus (ebv), cytomegalovirus (cmv) and human herpesvirus type 6 or 7 (hhv6, hhv7) can directly affect the liver and are infections in the human population usually acquired during childhood or adolescence. hhv8 can be transmitted sexually and presumeably also vertically from mother to child but has a more limited prevalence. in severely immunosuppressed patients hhv8 can cause kaposi sarcoma and body cavity lymphoma. herpesviruses persist life-long and can reactivate liver disease in immunosuppressed patients later on. primary herpes simplex infection produces characteristic oral (hsv-1) or genital (hsv-2) vesicular lesions. symptoms can be severe with fever and malaise but primary infections are frequently asymptomatic. fulminant hepatitis is a complication both of hsv-1 and hsv-2 infection (pinna et al., 2002) . organ transplantation and treatment for hematological malignancies are the most frequent underlying predispositions (johnson et al., 1992) . further individuals at risk include neonates, patients on steroids, hivinfected patients, and patients with cancer or myelodysplastic syndromes (pinna et al., 2002; johnson et al., 1992; kusne et al., 1991; zimmerli et al., 1988; frederick et al., 2002) . rarely fatal hsv-hepatitis has also been reported in immunocompetent adults (goodman et al., 1986) . varicella zoster virus (herpesvirus type 3) causes chicken pox; and shingles when latent infection is reactivated. after primary infection there is replication of varicella zoster virus in the epithelia of gut, respiratory tract, liver and endocrine glands. secondary viraemia then leads to infection of the skin and causes the typical rash. liver disease is rare and limited to patients with severe immunodeficiency. hsv-related hepatitis has a high (>80%) mortality and resembles septic endotoxic shock; jaundice is not always present (kusne et al., 1991) . patients suffer from fever, anorexia, nausea, vomiting, abdominal pain, leucopenia, and coagulopathy. typical oral or genital vesicular lesions may be present in only about 30% of patients (pinna et al., 2002) . some patients have disseminated further extrahepatic involvement, for example, lung, lymphnodes, spleen, and adrenal glands. in severe varicella zoster virus infection hepatic lesions are similar to herpes simplex hepatitis. varicella zoster virus has also been reported to trigger severe autoimmune type hepatitis (al-hoamoudi, 2009). the diagnosis of hsv-related hepatitis must be rapidly established. serologic assays are of little use. herpes simplex and varicella zoster viruses are detected preferentially by the polymerase chain reaction (finström et al., 2009) , or occasionally by viral isolation or immunofluorescence staining. prompt systemic treatment with acyclovir reduces hsv-associated morbidity and serious complications in hiv-infected patients. antiviral acyclovir prophylaxis has markedly reduced hsv re-activation after organ transplantation (seale et al., 1985; pettersson et al., 1985) . acyclovir resistance occurs in about 5% of immunocompromised patients and is negligible (<0.5%) in immunocompetent subjects (tyring et al., 2002) . valacyclovir is a prodrug of acyclovir, and famciclovir a prodrug of penciclovir, which have similar antiviral mechanisms as acyclovir. thus hsv isolates resistant to acyclovir are also resistant to these drugs (levin et al., 2004) . cidofovir and foscarnet are alternative choices to treat acyclovir-resistant hsv but are less well tolerated (safrin et al., 1991) . treatment and prophylaxis of varicella zoster hepatitis is similar to herpes simplex viruses because acyclovir also inhibits replication of varicella zoster virus. in immunocompetent hosts cytomegalovirus (cmv) infection may be rather asymptomatic but occasionally causes transient minimally symptomatic acute disease. congenital cytomegalovirus infection occurs in <2% of newborns and is encountered in (kylat et al., 2006) . when newborns or immunocompromised patients, for example, hiv infection, cancer, solid organ or bone marrow transplantation become cmv-infected, they may develop serious disease. cmv-related liver disease represents the commonest cause of viral hepatitis after organ transplantation. infection may result from re-activation of endogenous virus under immunosuppression, infection from the transplanted organ or blood transfusion of a cmv-positive donor. in liver transplantation, most cmv disease occurs at 1-4 months after transplantation. cmv infection is also a potential factor triggering acute and chronic rejection. vice versa, rejection therapy with corticosteroid boluses may induce endogenous cmv reactivation. although cmv re-activation is a frequent complication also in hiv-positive patients with advanced immunodeficiency (cd4 counts <200/ml), involvement of the liver seems to be rather rare (palmer et al., 1987) , but cmv occasionally causes bile-duct necrosis and a so-called hiv-cholangiopathy, a sclerosing cholangitis encountered in patients with terminal hiv-immunodeficiency (bonacini, 1992) . in about 10% of immunocompetent subjects primary cytomegalovirus infection produces an infectious mononucleosis-like syndrome, which is associated with elevated aminotransferases and a mild hepatitis (fig. 4) . liver histology may show focal hepatocyte and bile duct damage with lymphocytic infiltration into the sinusoids and occasionally epithelioid granulomas without necrosis, while cmv inclusion bodies or cmv immunostaining are only rarely seen (snover and horwitz, 1984) . fetal cmv infection has also been associated with obstructive biliary disease and neonatal hepatitis with giant cell transformation, cholestasis and viral inclusion bodies (finegold and carpenter, 1982) . in liver biopsies hepatocytes are swollen and may contain basophilic granules in the cytoplasm. a typical intranuclear amphophilic inclusion body can be present, resembling an "owl's eye" (fig. 3c ). both nuclear and cytoplasmic inclusions are full of virions (desmet, 1983) . however, in posttransplantation cmv hepatitis cytomegalovirus inclusion bodies are scanty. instead small foci of necrosis and inflammation (microabscesses) may be present (fig. 3b) . de novo appearance of cmv igm antibodies or a fourfold rise in igg antibodies herald cmv infection in immunocompetent individuals. however, serology is unreliable in immunocompromised patients and is replaced by quantitative molecular dna amplification assays (humar et al., 1999; caliendo et al., 2003) . meanwhile most transplant centers perform cmv surveillance by weekly quantitative determination of cmv dna. in most transplant units organ recipients at high risk for acquiring cmv disease receive immune prophylaxis with cmvhyperimmune antibodies and antiviral drugs (paya, 2001) . however, cmv infection and disease may still develop. cmv hepatitis should be treated promptly in patients with immunodeficiency. intravenous ganciclovir or oral valganciclovir over 3 weeks is the treatment of choice. drugs must be continued in reduced doses as chemoprophylaxis, if prolonged immunosuppression is anticipated (crumpacker, 1996) . fortunately, ganciclovir resistance is still rare (martin et al., 2008) , so that the more toxic alternatives cidofovir and foscarnet are rarely needed. epstein-barr virus (ebv) is shedded in oral secretions, and most primary ebv infections occur in adolescents. ebv accounts for 90% acute infectious mononucleosis syndromes. it persists life-long in a latent state, which results from a dynamic interplay between viral evasion strategies and the host's immune responses. while-unlike other herpesviruses-ebv reactivation-associated liver disease is not a prominent feature of persistent ebv infection, this herpesvirus is a potent cause for various malignancies such b-and t cell lymphomas, hodgkin lymphoma, and nasopharyngeal carcinoma. ebv has also been associated with an aggressive lymphoproliferative disease after liver transplantation. ebv intrauterine infection may lead to diverse congenital anomalies also comprising biliary atresia (goldberg et al., 1981) . however, only few pregnant women are susceptible, thus intrauterine ebv infection is rare. in infants and young children primary infection is frequently asymptomatic, while in adults it results in the infectious mononucleosis syndrome. patients develop malaise, headache, low-grade fever, before the more specific symptoms such as pharyngitis/ tonsillitis, swelling of cervical lymphnodes and moderate to high-grade fever occur. nausea, vomiting, and anorexia are frequent findings. a mild clinical hepatitis accompanies infectious mononucleosis in approximately 90% of patients (fig. 5) . splenomegaly is found in about half of patients, but hepatomegaly and jaundice are infrequent findings. patients show peripheral blood lymphocytosis with characteristic large abnormal lymphocytes in their blood smears. the vast majority of patients recover over 2-4 weeks, but fatigue may persist over several months after infection. ebv does not infect hepatocytes but lymphoid tissue. thus, liver damage is due to immune-mediated pathology and-when exceptionally done-biopsy specimens show diffuse lymphocytic infiltrates in the sinusoids but only occasionally focal apoptotic hepatocytes (fig. 3a ) (purtilo and sakamoto, 1981) . moreover, ebv-related immune activation can lead to several complications: patients with x-linked lymphoproliferative disease (xlp) caused by a mutation in the sh2d1a gene on the x chromosome are particularly vulnerable to the epstein-barr virus and may suffer fatal infections with extensive liver necrosis (seemayer et al., 1995) . in patients with severe immunodeficiency lymphomatoid granulomatosis is a further unusual complication of epstein-barr virus infection leading to granuloma formation in multiple organs including the liver, which may require interferon-alpha antiviral therapy (wilson et al., 1996) . in patients with hiv infection an ebv-associated lymphoproliferative disorder with hepatic infiltration of immunoblasts (beissner et al., 1987) , and a hemophagocytic syndrome have been reported (albrecht et al., 1997) . epstein-barr virus is also the major causative agent for the so-called posttransplant lymphoproliferative disease (ptld), which after organ transplantation may result in lymphocytic infiltration of the liver and other organs ranging from benign polyclonal b cell proliferation to malignant b cell lymphoma (hanto, 1995) . ptld occurs more commonly in children than in adults, depending on the degree of immunosuppression. it is primarily a complication in ebv-negative organ recipients, who develop primary ebv infection under immunosuppression owing to a graft from an ebv-positive donor. finally, ebv is a potent risk factor to develop lymphoma in later life even in patients without overt immunosuppression (fig. 6) . the clinical suspicion of epstein-barr virus infection is confirmed by detection of heterophilic or ebv-specific antibodies in infectious mononucleosis and quantitative polymerase chain reaction assays in patients with lymphoproliferative disorders (bruu et al., 2000; weinberger et al., 2004) . liver biopsy is not recommended for routine diagnostics. treatment of epstein-barr virus infection is primarily supportive. corticosteroid therapy can ameliorate symptoms. however, this option should be only considered in individuals with immune-mediated life-threatening complications, for example, imminent liver failure. because of theoretical concerns to suppress the immune system in an infection with a potentially oncogenic virus, corticosteroids are not recommended in general. acyclovir inhibits the ebv dna polymerase, and antiviral therapy with this drug has shortened virus shedding but failed to demonstrate a convincing clinical benefit even in severe acute epstein-barr virus infection (torre and tambini, 1999) . acyclovir antiviral therapy is not effective against latent ebv-infection. thus, reduction in immunosuppression, anticancer chemotherapy, and b-cell depleting antibodies are needed to treat ebv-related lymphoproliferative disorders. human herpesviruses types 6 (hhv6) and 7 (hhv7) hhv-6 exists in two variants, hhv6-a and hhv-6b, which infect t cells and various other cells types expressing the cd46 receptor (santoro et al., 1999) . although genetically clearly distinct from hhv-6, hhv-7 is another b-herpesvirus that shares many features with hhv-6. primary infection with either virus commonly occurs at young age and can lead to a febrile illness known as exanthema subitum or roseola infantum (leach, 2000) . pityriasis rosea reflects primary infection with hhv-7. hvv-6 also integrates into the host's genome and is transmitted via the germline. hhv6 and hhv-7 can reactivate each other (tanaka-taya et al., 2000) as well as cytomegalovirus leading to symptomatic cmv-disease in liver transplantation (humar et al., 2000) . the full spectrum of diseases caused by chronic hhv-6 and -7 infection is still unclear, but these viruses are putatively involved in a variety of different syndromes such as encephalitis, multiple sclerosis, pneumonitis, an infectious mononucleosis-like syndrome, postinfectious drug hypersensitivity as well as lymphoproliferative disorders and systemic disease in immunocompromised patients (stoeckle, 2000) . hhv-6 can cause severe and fatal hepatitis in neonates, children and adults (mendel et al., 1995; chevret et al., 2008; härmä et al., 2003) . hepatitis due to infection and reactivation of hhv-6 and hhv-7 can also complicate organ transplantation (dockrell and paya, 2001; härmä et al., 2006; ohashi et al., 2008) . in addition, hhv-6 has been associated with autoimmunity and giant-cell hepatitis and giant-cell transformation of bile duct cells (potenza et al., 2008) . hhv6-igm antibodies develop within a week after infection but are an unreliable marker, because false-positive test results in about 5% of healthy controls. beyond that serology does not distinguish between hhv-6a and hhv-6b variants and may cross-react with hhv7. the preferred method to diagnose hhv-6 and -7 infection is by quantitative dna amplification assays (deback et al., 2008) . detecting high viral loads in liver specimens or hhv-6 viremia is associated with approximately twofold increased mortality after liver transplantation (pischke et al., 2012) . in immunocompetent patients hhv-6 and -7 cause a benign self-limited infection, which does not require specific antiviral treatment. unlike hhv-6b both hhv-6a and hhv-7 are relatively resistant against ganciclovir, while foscarnet acts against all three viruses (yoshida et al., 1998; de clercq et al., 2001) . cidofovir may be a therapeutic alternative, but some resistant hhv-6 isolates have been identified (bonnafous et al., 2008) . human herpesvirus 8 is a g-herpesvirus, which has potential for malignant transformation. although primary hhv-8 infection can cause rash and fever in children and immunocompromised individuals, the onset of hhv-8-related diseases usually occurs several years after hhv-8 acquisition: kaposi sarcoma, body cavity lymphoma, and multicentric castleman's disease are the typical presentations of hhv-8 infection but bone marrow aplasia and multiple myeloma has also been described in association with hhv-8 infection (lee and henderson, 2001) . in autopsy studies kaposi sarcoma involved the liver in approximately 20% of patients with aids and was usually part of a widespread cutaneous and visceral disease. due to highly active antiretroviral combination therapy, kaposi sarcoma has become a rare complication of hiv infection. however, fulminant hepatic kaposi sarcoma may occur after organ transplantation (cahoon et al., 2018, fig. 7) . macroscopically there are dark-red tumors on the skin, the liver capsule and the parenchyma. under the microscope the typical lesion is a mesh of spindle-cell-like tumor cells and dilated thin-walled vessels (glasgow et al., 1985) . hepatosplenomegaly, fever, and weight loss are typical features of multicentric castleman's disease, a pre-malignant proliferation of b-lymphocytes (fig. 8a ). lymphocytes in multicentric castleman's disease and kaposi sarcoma seem to cooperate with each other, and thus the two hhv-8-related lesions occasionally occur within the same lymphnode (naresh et al., 2008) . ascites and pleural effusions are the hallmark of hhv-8-related body cavity lymphoma possibly giving rise to a false initial diagnosis of decompensated liver cirrhosis. however, abundant lymphoma cells in the aspirated fluids provide a pivotal diagnostic hint (fig. 8b ). hhv-8 can also cause solid organ lymphoma involving the liver (cesarman and knowles, 1999) . the diagnosis of hhv-8 associated malignancies is established from biopsies via their characteristic histopathological features. the virus itself is detected by various antibody assays or polymerase chain reaction (pcr) assays (chiereghin et al., 2017) . reconstitution of immune function is the primary goal for treatment of hhv-8 associated diseases. this can be achieved by highly active antiretroviral therapy in hiv infection and alternatively by antiproliferate m-tor (mammalian target of rapamycin) inhibitors for immune suppression in transplantation (barozzi et al., 2009) . immune stimulation with imiquimod and interferon-alpha (babel et al., 2008; van der ende et al., 2007) has been attempted. oncological therapy with liposomal anthracyclines or paclitaxel in kaposi sarcoma (di trolio et al., 2006; stebbing et al., 2003) , or rituximab in the case of castleman's disease and lymphoma are further potent treatment options (bower et al., 2007) . ganciclovir, cidofovir, foscarnet, adefovir and lobucavir but not acyclovir can block hhv-8 replication in vitro, and in a controlled crossover trial valganciclovir reduced oropharyngeal shedding of hhv-8 by 80% (casper et al., 2008) . this overview addresses the currently most relevant viral infections involving the liver. however, it provides only an outline and is in far not exhaustive. in rare instances, hepatitis occurred in the context of infections with enteroviruses (sun and smith, 1966) , measles (khatib et al., 1993) and rubella viruses (mclellan and gleiner, 1982) as well as parvovirus b19 (yoto et al., 1996; hayakawa et al., 2007) . the reader will find details of these rare infections in microbiology textbooks. beyond that, international travel and global warming are likely to introduce new exotic infections, which must be considered in the differential diagnosis of severe hepatitis. this problem is illustrated by the recent autochthonous crimean-congo hemorrhagic fever (cchf) virus infections in spain (negredo et al., 2017) , which did not occur in this country before. thus, hepatologists must be constantly prepared to face new challenges. epstein-barr-virus-associated hemophagocytic syndrome. a cause of fever of unknown origin in hiv infection severe autoimmune hepatitis triggered by varicella zoster infection histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection-clinicopathological and ultrastructural study development of kaposi's sarcoma under sirolimus-based 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asymptomatic hiv infection histology slides were kindly provided by prof. dr. hans-peter fischer, department of pathology, rheinische friedrich-wilhelms university of bonn, germany. key: cord-016255-kkko1xne authors: van der meer, j.t.m.; nouwen, j.l. title: 14 intravasale infecties en sepsis date: 2011 journal: microbiologie en infectieziekten doi: 10.1007/978-90-313-7944-6_14 sha: doc_id: 16255 cord_uid: kkko1xne infecties in het hart en de bloedbaan worden intravasale of endovasculaire infecties genoemd. de circulatie van bloed door het hart is essentieel voor de aanvoer van zuurstof en voedingstoffen naar weefsel en organen en voor de afvoer van afvalstoffen. in dit hoofdstuk worden infecties besproken die zich vooral afspelen in lymfoïde weefsels en meerdere orgaansystemen kunnen aantasten. cellen van het immuunsysteem spelen een belangrijke rol in de pathogenese van deze vaak chronische infecties. dit uit zich onder andere in gegeneraliseerde lymfadenopathie (lymfekliervergroting), een gemeenschappelijk klinisch kenmerk van deze ziekten. in dit hoofdstuk worden de pathofysiologische processen die aan deze lymfekliervergroting ten grondslag liggen kort samengevat en worden de belangrijkste infectieuze ziektebeelden met lymfadenopathie en hun verwekkers besproken. de lymfeklier verzamelt eiwitrijke vloeistof (lymfe) uit de extracellulaire ruimte van perifere weefsels. lymfe bevat antigenen, micro-organismen en fagocytaire cellen en vloeit continu via afferente lymfevaten naar de regionale lymfeklieren. vanuit de subcapsulaire ruimte van de lymfeklier bereikt de lymfe via weefselspletende sinussen -de hilus, waar zij de lymfeklier via de efferente lymfvaten verlaat. na passage van een aantal lymfeklierstations komt de lymfe uiteindelijk via de ductus thoracicus in de v. cava superior. tijdens de passage door de lymfeklier komt de lymfe in contact met een zeer groot aantal fagocyterende cellen die zich op een skelet van reticulinevezels hebben vastgezet. door fagocytose wordt de lymfe vrijwel volledig gefilterd van antigenen en micro-organismen. in de lymfeklier komen t-en b-lymfocyten in contact met antigene fragmenten die worden gepresenteerd door dendritische cellen. de lymfeklier fungeert dus behalve als mechanisch filter ook als kraamkamer van het specifieke immuunsysteem, waar door intensief contact tussen antigeenpresenterende cellen en lymfocyten een antigeenspecifieke cellulaire (t-cel) en humorale (b-cel) respons wordt gegenereerd. bij infecties kan zowel het niet-specifieke als het specifieke immunologische proces aanleiding geven tot lymfekliervergroting (lymfadenopathie). bij acute bacteriële infecties (zoals door staphylococcus aureus of streptococcus pyogenes) wordt de vergroting van de lymfeklier veroorzaakt door een toevloed van macrofagen en granulocyten, die in de lymfeklier doorgedrongen bacteriën afkapselen en fagocyteren. de vergroting is pijnlijk en gaat ook klinisch gepaard met andere ontstekingsverschijnselen (lymfadenitis), waarbij ook ontsteking van de afferente lymfvaten (lymfangitis) kan optreden. in de lymfeklier kan abcedering optreden. bij chronische virale infecties wordt daarentegen de lymfadenopathie veroorzaakt door een sterke toename van het aantal geactiveerde lymfocyten en immunoblasten. lymfeklieren bij chronische virale infecties zijn niet of nauwelijks pijnlijk. aspecifieke ontsteking en immuunactivatie kunnen ook naast elkaar voorkomen: in subacuut verlopende lymfadenopathieën, zoals veroorzaakt door bartonella henselae en chlamydia trachomatis, kunnen door histiocyten omringde microabcesjes optreden. lymfadenopathie door mycobacterium tuberculosis wordt gekenmerkt door het optreden van granulomen: haarden van epitheloïde cellen in palissadestand met veelkernige reuscellen en in het midden verkazende necrose. een overzicht van de belangrijkste ziektebeelden waarbij lymfadenopathie optreedt, met hun verwekkers en belangrijkste differentiële criteria, wordt gegeven in tabel 13.1. naast deze infectieuze oorzaken bestaan er tal van niet-infectieuze ziektebeelden die gepaard gaan met lymfadenopathie. hieronder vallen zowel systeemziekten (zoals bepaalde auto-immuunziekten en sarcoïdose) als maligne aandoeningen (primaire (non-)hodgkinlymfomen en secundaire lymfekliermetastasen). ten slotte kan lymfadenopathie optreden als reactie op bepaalde farmaca of corpora aliena (bijv. siliconen). mononucleosis infectiosa is een ziektebeeld met als belangrijkste klinische kenmerken moeheid, koorts, zwelling van (vooral de cervicale) lymfeklieren en een meer of minder uitgesproken faryngitis. de ziekte verloopt in de meeste gevallen subacuut en komt vooral voor bij adolescenten. in verreweg de meeste gevallen kan een recente infectie met epstein-barr-virus (ebv) worden aangetoond. ook kan het worden veroorzaakt door een recente cytomegalovirusinfectie (cmv-infectie) (5-10%), een toxoplasmose ( 1%), of een (primaire) hivinfectie. ebv en cmv behoren tot de humane herpesviridae (zie hoofdstuk 1). omdat lymfadenopathie tijdens de primaire infectie van ebv, cmv en hiv zo'n prominente rol kan spelen, zullen deze virussen in dit hoofdstuk uitvoeriger worden besproken. epstein-barr-virus (ebv, officiële taxonomische benaming: humaan herpesvirus 4, hhv-4) werd in 1961 ontdekt door epstein, achong en barr. zij zagen het virus met behulp van een elektronenmicroscoop in cellen die gekweekt waren uit een tumor die veel voorkwam bij kinderen in afrika (burkitt-lymfoom). vervolgens bleek dat de meeste volwassenen antistoffen hadden tegen ebv, passend bij een vroegere infectie. de toevallige observatie van de onderzoekers gertrud en werner henle dat zich antistoffen tegen ebv vormden bij een laboratoriummedewerker die mononucleosis infectiosa doormaakte, leidde tot de identificatie van ebv als een van de verwekkers van dit ziektebeeld. speeksel bevat wisselende hoeveelheden van dit virus, geproduceerd in de tonsilcrypten. primaire infectie via speekselcontact leidt tot productie van virus in de initieel geïnfecteerde epitheelcellen, waarop vervolgens de aanwezige b-cellen worden geïnfecteerd. infectie van deze b-cellen leidt tot een transformatie, wat in het laboratorium kan worden aangetoond met spontane uitgroei van lymfoblastoïde cellijnen. de infectie van b-cellen leidt tot een uitgesproken t-celrespons, waardoor in het bloedbeeld van patiënten een bont beeld van afwijkend gevormde (atypische) lymfocyten (geactiveerde t-lymfoblasten) wordt gezien. het kenmerkende symptomencomplex van mononucleosis infectiosa wordt niet rechtsreeks door virusreplicatie veroorzaakt, maar is het resultaat van de sterke t-celrespons (lymfadenopathie) en de daarmee gepaard gaande cytokine-en interleukineproductie, wat zich uit door extreme moeheid en koorts. deze t-celrespons is verantwoordelijk voor de onderdrukking van de door ebv geïnduceerde b-celproliferatie in vivo. ten gevolge van de virusspecifieke t-celrespons kan ebv na de primaire infectie alleen in latent geïnfecteerde b-geheugencellen voortbestaan. bij asymptomatische dragers is ongeveer 1 op de 100.000 b-geheugencellen geïnfecteerd. in deze cellen is weliswaar het ebvgenoom in de kern aanwezig, maar wordt slechts een beperkt aantal genen (10 van de ongeveer 100) tot expressie gebracht. hierdoor worden slechts weinig virale peptiden op het celoppervlak in de context van moleculen van hla-klasse i gepresenteerd, waardoor deze cellen niet door cytotoxische cd8+-t-cellen kunnen worden herkend. reactivatie van virusreplicatie treedt mogelijk op als gevolg van stimulatie van de b-geheugencel door herkenning van zijn specifieke antigeen. als gevolg van deze reactivatie kan bij 5-10% van de dragers uit speeksel virus worden geïsoleerd. het vermogen van ebv om b-celproliferatie te induceren kan leiden tot het ontstaan van b-celtumoren. aanvankelijk oligoklonale, in latere stadia vaak monoklonale b-cellymfomen kunnen worden gezien bij patiënten met een verstoorde t-celfunctie, zoals hiv-geïnfecteerde personen en ontvangers van orgaantransplantaten (post-transplant lymphoproliferative disorder, ptld). verder is aangetoond dat ebv-infectie een cofactor is in de pathogenese van burkitt-lymfoom en nasofarynxcarcinoom (frequent in china) en bij agressievere vormen van de ziekte van hodgkin. ebv komt wereldwijd voor. er is geen verschil in prevalentie tussen de geslachten of tussen etnische groepen. de leeftijd waarop infectie optreedt, wordt vooral bepaald door sociaal-economische factoren. in niet-westerse landen treedt infectie vaak al op de vroege kinderleeftijd op vanwege intensieve contacten; bij 5-jarigen ligt de seroprevalentie al boven de 50%, op volwassen leeftijd is uiteindelijk wereldwijd 90-95% geïnfecteerd. in zeldzame gevallen kan ebv-infectie aanleiding geven tot neurologische complicaties, zoals myelitis transversa, nervusfacialisparese, neuritis optica of cerebellitis. de soms heftig verlopende faryngitis kan naast slikstoornissen aanleiding geven tot pseudomeningisme door een verhoogde tonus van de cervicale musculatuur. respiratoire obstructie door zwellingen in hypofarynx en larynx komt incidenteel ook voor en kan ernstig zijn. karakteristiek is de rash die kan optreden tijdens ebvinfectie na empirische therapie met amoxicilline. een belangrijke hematologische complicatie is een hemolytische anemie op auto-immuunbasis. er zijn geen aanwijzingen voor betrokkenheid van ebv bij het chronisch vermoeidheidssyndroom. de diagnose ebv-infectie wordt gesteld op grond van serologisch onderzoek. waarschijnlijk ten gevolge van de massale polyklonale activatie van b-cellen kunnen tijdens de primaire ebv-infectie autoantistoffen en heterofiele antistoffen ontstaan (dat zijn meestal igm-antistoffen, gericht tegen erytrocytaire antigenen bij andere species). deze heterofiele antilichamen zijn dus niet gericht tegen ebv maar zijn een direct gevolg van polyklonale b-celstimulatie. de paul-bunnell-test, waarbij deze heterofiele antistoffen worden aangetoond in serum, is een specifieke surrogaattest voor ebv-infectie. deze test is positief bij 90% van de volwassenen met een primaire infectie, maar veel minder gevoelig bij jonge kinderen met een acute ebv-infectie. heterofiele antistoffen zijn vooral aantoonbaar in de eerste drie tot zes maanden na het begin van symptomen. voor screening, ook in de huisartsenpraktijk, zijn sneltests beschikbaar. specifieke ebv-serodiagnostiek kan ook worden verricht. hiermee kunnen igm-en igg-antistoffen worden aangetoond tegen het virale capsideantigeen (vca), een aantal vroege antigenen (early antigens, ea) en antigeen uit de latente cyclus van het virus (epstein-barr virus-associated nuclear antigen, ebna). de kinetiek van de verschillende antistofresponsen is weergegeven in figuur 13.2. aan de hand van het antistofpatroon kan een inschatting worden gemaakt van het moment van ebvinfectie. anti-vca-igg-antistoffen ontstaan tijdens de acute fase en blijven ook na herstel aantoonbaar. hoge titers anti-ea-antistoffen wijzen op een recente infectie. reconvalescentie gaat gepaard met het dalen van de anti-ea-titers en stijgende titers van anti-ebna-antistoffen. de kweek van ebv wordt niet gebruikt in de routinediagnostiek. de ontwikkeling van kwantitatieve moleculaire (pcr) technieken maakt het mogelijk om de hoeveelheid ebv in plasma te kwantificeren. bij patiënten die immuunsuppressieve therapie ontvangen vanwege een transplantatie wordt de hoeveelheid ebv gevolgd om ebv-gemedieerde posttransplantatie-lymfoproliferatieve ziekte vroegtijdig op te sporen en te voorkomen. de behandeling van een acute ebv-infectie is voornamelijk ondersteunend. aciclovir remt ebv-replicatie in vitro, maar heeft klinisch geen effect. dit past bij de beschreven vooral immunopathologische basis van het ziektebeeld. incidenteel kunnen corticosteroïden aangewezen zijn bij dreigende luchtwegobstructie of hemolytische anemie. een vaccin is nog niet voorhanden. bij ebv-positieve lymfoproliferatieve ziektebeelden speelt therapie gericht tegen b-cellen (anti-cd20 monoklonale antistoffen) een belangrijke rol. humaan cytomegalovirus (cmv, humaan herpesvirus 5) dankt zijn naam aan de reuzen-(megalo)cellen, die werden aangetroffen in organen van zuigelingen met een letaal verlopende congenitale infectie. humaan cytomegalovirus kent wereldwijd een groot aantal stammen met ongeveer 95% homologie op sequentieniveau; bij een groot aantal diersoorten (primaten, knaagdieren enz.) komen eigen cmv-soorten voor. besmetting met cmv treedt frequent prenataal of perinataal op (tijdens passage door het baringskanaal of door lactatie), of door contact met besmet speeksel of urine. monocyten kunnen tijdens de primaire infectie worden geïnfecteerd en produceren cmv na hun differentiatie tot macrofagen. op deze wijze dragen zij vermoedelijk bij tot de hematogene verspreiding van het virus. cmv infecteert onder andere epitheelcellen van nieren (proximale tubuli) en speekselkliercellen (ductusepitheel), endotheel en fibroblasten. het virus kan de placenta passeren en een intra-uteriene infectie veroorzaken (zie hoofdstuk 15). bij het onder controle krijgen van de primaire infectie spelen waarschijnlijk zowel specifieke cytotoxische t-lymfocyten als neutraliserende antistoffen een rol. de humorale en cellulaire respons leiden tot partiële immuniteit tegen andere cmv-stammen. in de latente fase kan cmv-dna worden aangetoond in monocyten en hemopoëtische stamcellen, maar niet in granulocyten, de cellen waarin het virus juist gedetecteerd wordt tijdens viremische episoden. actieve replicatie van cmv in de monocyten-/macrofagenreeks is waarschijnlijk beperkt tot gedefinieerde fasen in de differentiatie. de beperkte replicatie in de tussenliggende fasen draagt mogelijk bij tot de persistentie van de infectie. mogelijk geldt hetzelfde voor andere celtypen. bij immunocompetente dragers treedt episodisch een asymptomatische reactivatie op, waarbij het virus in urine en speeksel wordt uitgescheiden. lymfadenopathieën en hiv epidemiologie prenatale, intra-uteriene infecties kunnen ernstig verlopen (congenitale cmv, zie hoofdstuk 15), maar peri-en postnatale cmv-infecties verlopen vrijwel altijd asymptomatisch. overdracht vindt vooral plaats via speeksel, maar ook via moedermelk, urine, feces, bloed en sperma. tot 10% van de pasgeborenen wordt besmet met cmv tijdens de baring of via lactatie, maar intensief contact met leeftijdgenootjes die virus uitscheiden (crèches) zorgt voor een snelle toename van het aantal geïnfecteerden met de leeftijd. een tweede snelle stijging van de seroprevalentie wordt gezien bij jonge volwassenen. ook dan verlopen de meeste (90%) van de primo-infecties asymptomatisch. afhankelijk van de hygiënische omstandigheden varieert de seroprevalentie wereldwijd op volwassen leeftijd tussen 50 en 100%. ongeveer 10% van de primo-infecties op de volwassen leeftijd gaat gepaard met symptomen. deze treden op na een incubatietijd van gemiddeld zes weken, vergelijkbaar met ebv. ongeveer 5-10% van de klinische beelden van mononucleosis infectiosa wordt veroorzaakt door cmv. cmv-mononucleosis treedt doorgaans op wat latere leeftijd op dan ebv-mononucleosis. meestal blijven de symptomen beperkt tot twee à drie weken koorts. faryngitis en cervicale lymfadenopathie zijn minder gebruikelijk dan bij primaire ebv-infectie. het perifere bloedbeeld vertoont meestal een lymfocytose met atypische lymfocyten, en de leverfuncties kunnen gestoord zijn. volledig herstel treedt op na ongeveer zes weken. de paul-bunnell-reactie is kenmerkend negatief. zeldzame complicaties zijn hepatitis, pneumonie, aseptische meningitis en het syndroom van guillain-barré. ditzelfde ziektebeeld van koorts, leukopenie, atypische lymfocytose en splenomegalie kan optreden bij een cmv-seronegatieve ontvanger drie tot zes weken na bloedtransfusie met vers cmv-seropositief bloed. de kans op cmv-transmissie via bloedtransfusie (geschat op ongeveer 2,5% per unit getransfundeerd bloed) kan worden gereduceerd door gebruik van bloed van seronegatieve donoren, leukocytenarm bloed of bevroren bloed-of bloedproducten. ernstige ziektebeelden veroorzaakt door cmv treden vooral op bij intra-uteriene infectie (zie hoofdstuk 15) en patiënten met afweerstoornissen (zie hoofdstuk 17). bij orgaantransplantaties is cmv-ziekte een van de meest voorkomende complicaties. cmv-ziekte kan het gevolg zijn van reactivatie van het virus van de (seropositieve) ontvanger of infectie vanuit het donororgaan of door gedoneerd bloed. vanwege het beperkte aanbod van donororganen is het niet altijd mogelijk naast een goede hla-match ook de cmv-serostatus van donor en ontvanger op elkaar af te stemmen. cmv-infectie bij transplantatiepatiënten kan gepaard gaan met langdurige koorts, trombo-en/of leukopenie en gestoorde leverenzymen (hepatitis), spierpijn en ge-wrichtsklachten. daarnaast kunnen ernstige gastro-intestinale infecties optreden (oesofagitis, gastritis, colitis), die gepaard kunnen gaan met perforatie. cmvpneumonitis is vooral een levensbedreigende ziekte bij patiënten met een allogene beenmergtransplantatie. diverse vormen van cmv-ziekte kunnen optreden bij aidspatiënten, vooral bij een sterk gestoorde immuniteit (cd4+-cellen < 50/mm 3 ). naast pneumonitis, encefalitis en gastro-intestinale infecties is vooral retinitis een beruchte complicatie. de laatste manifestatie kan zich bilateraal voordoen en leidt bijna altijd tot blindheid, tenzij antivirale therapie wordt gegeven. incidenteel doet zich een ernstige cmv-colitis voor bij jonge volwassenen met een op het oog normale of hooguit licht gestoorde immuniteit (zoals bij zwangeren). diagnostiek de diagnose primaire cmv-infectie bij immuuncompetente gastheren wordt meestal gesteld aan de hand van het antistofpatroon. igm-antistoffen (in de vroege fase) en igg-antistoffen (levenslang) zijn aantoonbaar. een reactivatie van de cmv-infectie kan gepaard gaan met de hernieuwde vorming van igm-antistoffen maar dit is weinig betrouwbaar. een foutpositieve cmv-igm-test kan soms optreden bij een primaire ebv-infectie. het betreft hier antistoffen gegenereerd als gevolg van de tijdens de ebv-infectie optredende polyklonale b-celactivatie, of mogelijk antistoffen die gemeenschappelijke antigene determinanten van ebv en cmv herkennen. heterofiele antistoffen komen bij cmv-infectie echter niet voor. naast serologisch onderzoek bestaat de mogelijkheid om cmv als virus aan te tonen, door middel van kweek, door antigeendetectie of tegenwoordig vooral door cmv-dna-detectie. dit is vooral van groot belang bij de infecties van immuungecompromitteerde gastheren. cmv is in die gevallen als typisch systemische infectie aantoonbaar op vele plaatsen, zoals in de keel, in urine, in bronchiaal spoelsel en in leukocyten. ook bij congenitaal geïnfecteerde kinderen vindt soms nog jarenlang sterke cmv-uitscheiding in de urine plaats. positieve kweekresultaten wijzen niet noodzakelijkerwijs op een symptomatische cmv-infectie. asymptomatische uitscheiding van cmv komt regelmatig voor, in het bijzonder bij patiënten met een verminderde afweer. een goede indruk van de relevantie van een cmv-infectie is in die gevallen te verkrijgen door het meten van de hoeveelheid viraal dna in bloed of plasma. de drie middelen met klinisch bewezen werkzaamheid, ganciclovir, foscarnet en cidofovir, worden gekenmerkt door een hoge frequentie van toxische neveneffecten. ganciclovir is nauw verwant aan aciclovir, maar blijkt een veel effectievere remmer van de cmv-replicatie. dit is het meest toegepaste middel tegen cmv-infectie; het is ook oraal toepasbaar door middel van de prodrug valganciclovir. door het ter beschikking komen van snelle moleculaire tests voor de kwantitatieve bepaling van de hoeveelheid cmv in plasma wordt bij transplantatiepatiënten therapie vaak toegepast op geleide van de hoeveelheid cmv in het bloed. met deze strategie blijkt ernstige cmv-ziekte bij deze patiënten grotendeels te kunnen worden voorkomen (zie hoofdstuk 17). onderzoek naar preventie van cmv-ziekte door vaccinatie is gaande. immunisatie van ontvangers van niertransplantaten acht weken voor transplantatie met een verzwakte cmv-stam (towne) leidde niet tot een verminderde cmv-uitscheiding na transplantatie. de incidentie van cmv-ziekte was wel lager ten opzichte van de placebogroep en ook verliep de ziekte in die gevallen minder ernstig. naast onderzoek met dit type vaccin is ook onderzoek gaande met vaccins bestaande uit gezuiverd of recombinant gb, het voornaamste envelopglycoproteïne van cmv-virus. ook het voorkómen van schade door congenitale cmv-infectie is een belangrijk doel van vaccinatie. naar schatting 1% van de mononucleosis infectiosa-achtige ziektebeelden wordt veroorzaakt door toxoplasma gondii, een intracellulair protozoön. in hoofdstuk 18 zal nader op deze infectie worden ingegaan. casus 13.2 een 45-jarige vrouw wordt opgenomen in het ziekenhuis vanwege toenemende kortademigheid. tien jaar eerder werkte zij als reisleidster ruim acht maanden in tanzania, kort waarop zij een griepachtig beeld met lymfadenopathie ontwikkelde, dat toen werd geduid als mononucleosis infectiosa. twee jaar voor presentatie begon ze langzamerhand steeds meer last te krijgen van seborroïsch eczeem, genitale ulcera en eenmaal een ernstige gordelroos in het gelaat. de laatste maanden is ze 8 kg afgevallen. bij onderzoek blijkt er sprake van een verlaagde zuurstofspanning door een dubbelzijdige, interstitiële pneumonie. bij een longspoeling wordt pneumocystis in de alveolaire macrofagen herkend en wordt een hiv-antistoftest ingezet. deze blijkt positief. haar cd4-aantal blijkt 30/mm 3 . in 1981 werd in de verenigde staten een epidemie van longontstekingen met pneumocystis jirovecii beschreven, vaak gepaard gaand met een zeldzame vorm van huidkanker, het kaposi-sarcoom. al snel werd duidelijk dat een ernstige immuunstoornis aan deze verschijnselen ten grondslag lag en dat de slachtoffers allen in korte tijd overleden. aanvankelijk waren de slachtoffers jonge homoseksuele mannen, maar al snel werden vergelijkbare casus waargenomen bij heteroseksuele mensen uit haïti en bij ontvangers van bloedtransfusies. de aandoening werd aids genoemd (acquired immune deficiency syndrome) en gezien de epidemiologie vermoedde men al snel een infectieuze oorzaak. in 1983 isoleerden de latere nobelprijswinnaars (2008) barré-sinoussi en montagnier als eersten uit de lymfeklier van een franse aidspatiënt een nieuw retrovirus, dat uiteindelijk humaan immunodeficiëntievirus type 1 (hiv-1) werd genoemd. in 1986 werd een verwant virus, hiv-2, aangetoond bij aidspatiënten in west-afrika. inmiddels is duidelijk geworden dat beide virussen voorkomen bij primaten in afrika (als vormen van simian immunodeficiency virus, siv). men heeft aannemelijk kunnen maken dat de voorlopers van beide humane virussen in de jaren dertig (hiv-1) en veertig (hiv-2) van de vorige eeuw vanuit primaten bij de mens geïntroduceerd zijn op het afrikaanse continent. het begin van de epidemie in afrika is niet herkend, wel heeft men met terugwerkende kracht een geval van aids kunnen vaststellen bij een zeeman uit manchester in 1959, die vermoedelijk in afrika was geïnfecteerd. het virusdeeltje heeft een sferische vorm en is ongeveer 100 nm groot (figuur 13.3a). het virus heeft een envelop die bestaat uit de virale glycoproteïnen gp120 en gp41, en bestanddelen die afkomstig zijn van de gastheercelmembraan. deze envelop omgeeft de capside, met als voornaamste bestanddeel het virale p24-eiwit. binnen de capside bevinden zich twee identieke enkelstrengs rna-moleculen (het virale genoom) en een aantal moleculen van de enzymen reverse transcriptase, integrase en protease. het genoom is relatief klein (9 kb) en bevat negen genen, die in totaal coderen voor vijftien verschillende eiwitten. het genoom wordt aan weerszijden geflankeerd door een long terminal repeat (ltr), een niet-coderend deel van het genoom betrokken bij de regulatie van de virale expressie (figuur 13.3b). de genproducten zijn onder te verdelen in: 1 structurele proteïnen (o.a. de gag-genproducten (p24 en p17) en envelopglycoproteïnen (gp41 en gp120); 2 door het virus gecodeerde enzymen die nodig zijn voor de virale replicatiecyclus (reverse transcriptase, integrase en protease); 3 eiwitten die betrokken zijn bij de regulatie van de genexpressie (o.a. tat, rev en nef). hiv infecteert humane gastheercellen die op hun buitenmembraan een cd4+-receptormolecuul bezitten (figuur 13.4). deze cd4+-receptoren zijn uiteraard aanwezig op cd4+-positieve t-lymfocyten, maar in mindere mate ook op macrofagen en dendritische cellen. hiv gebruikt dit cd4+-molecuul als eerste receptor om de gastheercel te kunnen binden. door binding van het virale gp120 aan dit cd4+-molecuul ontstaat in de virusenvelop een conformatieverandering, waardoor het virus zich kan binden aan een tweede (co)receptor. deze tweede receptor komt uit de familie van de chemokinereceptoren, waarvan er twee belangrijk zijn voor hiv: ccr-5 en cxcr-4. de ccr-5-receptor bevindt zich vooral op macrofagen, dendritische cellen en cd4+-positieve t-lymfocyten, de cxcr-4-receptor komt vooral tot expressie op geactiveerde cd4+-positieve t-lymfocyten. op basis van hun coreceptorgebruik kan hiv worden onderverdeeld in varianten die vooral de ccr-5receptor gebruiken (r5-virussen, ook wel macrofagotrope virussen) en varianten die vooral de cxcr-4-receptor gebruiken (x4-virussen, ook wel lymfocytotrope virussen). er zijn ook varianten die beide receptoren kunnen gebruiken (duotrope virussen). het overgrote deel van de patiënten wordt geïnfecteerd met r5-trope virussen. gedurende de infectie kunnen virussen muteren met als gevolg dat bij ruim 50% van de geïnfecteerde individuen in de loop van de infectie virussen ontstaan die ook de cxcr-4-coreceptor kunnen gebruiken. na binding aan de coreceptor treedt een tweede conformatieverandering op en kan het envelopeiwit gp41 binden aan de gastheercelmembraan. binding van de virus-en celmembraan leidt tot fusie van beide membranen, waarop de nucleocapside het cytoplasma van de cel kan binnengaan. vervolgens worden met behulp van het enzym reverse transcriptase de twee enkelstrengs virale rna-kopieën omgezet in proviraal dubbelstrengs dna (figuur 13.4). dit dna migreert naar de kern en integreert in het gastheercel-dna met behulp van het reeds aanwezige virale integrase. het geïntegreerde dna noemen we een provirus. in feite is er nu sprake van een irreversibele ('ongeneeslijke') situatie: zo lang de geïnfecteerde gastheercel(populatie) overleeft, zal ook het geïntegreerde hiv-dna overleven. wanneer een provirus bevattende cd4+-cel geactiveerd wordt, wordt de transcriptiefactor nfkb geproduceerd. deze bindt aan de promotor van het provirus, waarop transcriptie wordt gestart. de virale eiwitten tat en rev die daarbij gemaakt worden, zorgen vervolgens voor een efficiënter verlopende transcriptie, waarop virale mrna's en de precursors van de structurele (glyco)proteïnen worden geproduceerd. de virale glycoproteïnen groeperen zich bij de celmembraan. het virale protease klieft het gag-precursoreiwit, waarop de eindproducten worden geproduceerd, onder meer de virale capside-eiwitten. samen met het genomisch rna assembleren deze eiwitten aan de binnenkant van de celmembraan tot nieuwe virionen, die zich door lokale uitstulping en afsnoering van de celmembraan (budding) buiten de cel begeven (zie figuur 13.4). men schat dat er bij een hiv-1-geïnfecteerde patiënt gemiddeld 10 8 -10 10 virusdeeltjes per dag worden geproduceerd, waarbij dagelijks 10 8 -10 9 cd4+-cellen worden geïnfecteerd. doordat het reverse transcriptase een slordig enzym is (1:10 4 nucleotiden wordt foutief gekopieerd), treden er mutaties op die leiden tot virusvarianten. men kan daardoor niet spreken van één virus maar van een viruspopulatie. afhankelijk van suppressieve factoren, zoals de gastheerimmuniteit tegen hiv of de aanwezigheid van medicatie, zullen vooral die virussen expanderen, die door hun mutatie(s) aan deze suppressieve druk kunnen ontsnappen (survival of the fittest). het kenmerk van een hiv-infectie is een geleidelijke afname van het aantal cd4+-positieve lymfocyten in het perifere bloed (figuur 13.5), waardoor uiteindelijk een functionele immuundeficiëntie ontstaat. aanvankelijk nam men aan dat de cd4+-daling een direct gevolg was van de celdood door virusreplicatie, dan wel door toegenomen geprogrammeerde celdood (apoptose), of door immuungemedieerde destructie. tegenwoordig neemt men aan dat de hiv-infectie een hyperactivatie van het gehele cd4+-compartiment veroorzaakt, waardoor de cd4+-cellen veel korter overleven en uiteindelijk onvoldoende kunnen worden aangevuld. aangezien de cd4+-lymfocyt een centrale rol speelt in de regulering van de immuunrespons, leidt het verlies van deze celpopulatie uiteindelijk tot deficiënties in die immuunrespons. vooral de cellulaire immuniteit is sterk afhankelijk van de aansturende rol van de cd4+-cel en is daarom als eerste gestoord. de cellulaire immuniteit controleert onder meer micro-organismen die in of op het lichaam latent aanwezig zijn, zoals schimmels, mycobacteria en herpesvirussen. wanneer de cellulaire immuniteit verminderd functioneert, kunnen deze micro-organismen tot ziekteverschijnselen leiden. men spreekt dan van opportunistische infecties (zie ook hoofdstuk 17). de humorale immuniteit ontwikkelt zich vooral in de lymfadenopathieën en hiv eerste levensjaren. de cd4+-cel speelt in die fase wel een belangrijke rol in deze ontwikkeling, maar als het humorale immuunrepertoire eenmaal gevormd is, wordt de rol van de cd4+-cel hierin minder belangrijk. een volwassen persoon met een laag cd4+-aantal kan gekapselde micro-organismen, zoals pneumokokken en stafylokokken, die vooral afhankelijk zijn van een humorale immuunrespons, daarom nog redelijk effectief bestrijden. jonge kinderen met hiv hebben daar duidelijk meer moeite mee door een nog onvoldoende gerijpt humoraal repertoire. vaccinatie, waarbij humorale immuniteit moet worden opgebouwd onder invloed van cd4+-cellen, is om diezelfde reden minder effectief bij hiv-geïnfecteerde patiënten met een laag cd4+-aantal. een hiv-infectie onderscheidt zich in het begin niet van andere virusinfecties waartegen het lichaam zowel een humorale als een cellulaire immuunrespons opbouwt. nadat het virus in de eerste weken vrij spel heeft gehad, waarbij grote hoeveelheden virus in het bloed kunnen worden aangetroffen (figuur 13.5), zorgen tegen hiv gerichte neutraliserende antistoffen en cytotoxische lymfocyten (ctl) voor controle over het virus, waarop de hoeveelheid celvrij virus in het plasma daalt. afhankelijk van de kracht van deze gastheerrespons en de mogelijkheid van het virus om hieraan te ontsnappen, ontstaat na een aantal weken een soort balans (set-point) waarbij hiv en het immuunsysteem elkaar in evenwicht houden. doordat het virus echter continu muteert, ont-staan virusvarianten die aan deze immuuncontrole ontsnappen, waarop een nieuwe immuunrespons tegen deze varianten de balans weer moet proberen te herstellen. doordat de daarvoor benodigde cd4+-cellen echter langzaam verdwijnen (en daardoor ook de ctlrespons tegen hiv vermindert), raakt de balans steeds meer verstoord en zal het virus deze strijd uiteindelijk winnen: de hoeveelheid virus in het plasma stijgt weer. in het algemeen geldt dat stijging van de hoeveelheid in het plasma (meer dan 100.000 viruskopieën per ml) gepaard gaat met een snellere cd4+-daling en dus een snellere ziekteprogressie. door de mutaties in de envelop kan het virus ook zijn tropisme veranderen: naast macrofagotrope virussen (r5) ontstaan duotrope (r5/x4) virussen, met als gevolg dat het virus in meer cellen kan repliceren. hierdoor stijgt de hoeveelheid virus snel en zal het aantal cd4+-cellen in korte tijd sterker dalen, wat gepaard gaat met snelle ziekteprogressie. het beloop van de hiv-infectie kent grote interindividuele verschillen: de duur van de asymptomatische eerste fase kan variëren van twee tot meer dan vijftien jaar. de genetische achtergrond, zoals de hla-typering van de geïnfecteerde persoon en ook andere genetische polymorfismen binnen het immuunsysteem, draagt daar belangrijk aan bij. zo zijn er individuen die een homozygote deletie hebben voor de ccr-5-receptor, waardoor infectie met r5-virussen niet kan plaatsvinden. bij personen met de heterozygote ccr-5-deletie kan een r5-virus zich minder gemakkelijk verspreiden en verloopt de ziekteprogressie trager. de kliniek rondom hiv-infectie is grofweg in drie fasen te verdelen: de acute hiv-infectie in de eerste drie maanden, daarna een relatief asymptomatisch verlopende fase, die twee tot meer dan vijftien jaar kan duren, en tot slot een symptomatische fase, die snel progressief kan verlopen en eindigt in aids. nadat iemand met hiv geïnfecteerd is geraakt, ontstaan in de acute fase klinische symptomen bij 80-90% van de personen, waarbij 50-60% medische hulp zoekt. de symptomen beginnen meestal twee tot vier weken na de infectie en lijken sterk op een griepbeeld of op mononucleosis infectiosa, dat eerder werd besproken bij de acute ebv-infectie. koorts treedt vaak op (> 90%), gepaard gaand met faryngitis (75%), lymfadenopathie (50-75%), spier-en gewrichtspijn (50-90%) en moeheid (80-90%). regelmatig worden bovenstaande verschijnselen begeleid door een uitgebreide maculopapulaire huiduitslag (30-70%), gewichtsverlies (50-70%), hoofdpijn (40%), darmklachten met diarree en/of braken (30-50%) en ulcera in mond, rectum en genitalia. bij bloedonderzoek ziet men vaak atypische lymfocytose (> 80%), leuko-en trombopenie (35-45%) en milde hepatitis (20%). meestal houden de klachten en afwijkingen niet langer dan twee weken aan. in deze fase van de acute hiv-infectie komt de immuunrespons tegen hiv op gang: er ontstaan antistoffen tegen alle hiv-eiwitten (seroconversie) en er ontstaat ook een sterke ctl-respons. initieel is er veel virus in het plasma aanwezig (hiv-rna viral load) en zullen de hiv-tests (die berusten op het aantonen van anti-hiv-antistoftests) nog negatief zijn; men spreekt van de window-fase. seroconversie treedt bij de meeste patiënten binnen acht tot tien weken op, zodat deze window-fase slechts enkele weken duurt. tijdens de acute hiv-infectie daalt het aantal cd4+-cellen in het bloed, wat zich meestal grotendeels weer herstelt. de daling kan soms zo diep zijn, dat ernstige opportunistische infecties kunnen optreden. de grootste daling van cd4+-cellen in de acute fase vindt echter plaats in het darmgeassocieerde lymfoïde weefsel (galt, gut associated lympoïd tissue). in dit compartiment vindt nauwelijks herstel van het aantal cd4+-cellen plaats. met het op gang komen van een krachtige immuunrespons daalt de hoeveelheid hiv-rna in het plasma en herstelt het aantal cd4+-cellen in het perifere bloed zich weer redelijk (figuur 13.5). na de acute fase volgt de al genoemde asymptomatische fase, waarbij virus en gastheer elkaar in evenwicht lijken te houden rond een soort set-point. algemene klachten zoals moeheid, nachtzweten en lymfadenopathie worden in wisselende mate gezien, evenals huidklachten zoals folliculitis en seborroïsch eczeem. bepaalde opportunistische infecties zoals herpes zoster en herpes simplex treden vaker op, evenals sinusitis en pneumokokkenpneumonieën, maar zo lang het aantal cd4+-lymfocyten hoger is dan 200/mm 3 zijn deze beelden mild en van voorbijgaande aard. zodra het cd4+-aantal duidelijk onder deze grens komt, worden de opportunistische infecties ernstiger. infecties door candida albicans van mondholte (stomatitis) en slokdarm (oesofagitis), longontstekingen met pneumocystis jirovecii (vroeger pneumocystis carinii genoemd) en diarree met giardia lamblia, cryptosporidium, isospora en microsporidia nemen in frequentie toe, naarmate het cd4+-gehalte lager wordt. bij cd4+-waarden onder de 100/mm 3 zorgen toxoplasmose (cerebraal abces), cryptococcus neoformans (meningitis, maar ook gedissemineerd), cmv (colitis en retinitis) en atypische mycobacteria (bacteriëmie, darmwand en beenmerg) voor ernstig verlopende en dodelijke infecties. los van deze cd4+-grenzen worden als belangrijke opportunistische problemen gewone tuberculose maar ook kwaadaardige aandoeningen als non-hodgkinlymfomen en kaposisarcomen gezien. wanneer een hiv-geïnfecteerde zich presenteert met een van deze opportunistische problemen, spreekt men per definitie van aids. onbehandeld is de gemiddelde overleving van een aidspatiënt meestal niet langer dan 18 maanden, afhankelijk van de ernst van de ziekte waarop de diagnose aids wordt gesteld. de belangrijkste aids-indicatordiagnosen zijn: -pneumocystis jirovecii-pneumonie (voorheen: pneumocystis jirovecii); -oesofageale candidiasis; -cerebrale toxoplasmose; -cryptosporidiose met > 1 maand diarree; -extrapulmonaire cryptokokkose; -cmv-ziekte van tractus digestivus, retinitis, pneumonitis, encefalitis; -hsv mucocutane ulcera > 1 maand, pneumonitis, oesofagitis; -pulmonale of gedissemineerde tuberculose; -gedissemineerde infectie met mycobacterium avium intracellulare of m. kansasii; hiv-wasting: > 10% gewichtsverlies en diarree, of langdurige febris e.c.i. en chronische malaise; -hiv-geassocieerde dementie; -kaposi-sarcoom; -non-hodgkin-of b-cellymfoom. sinds de eerste hiv-patiënten werden beschreven, is er al direct veel ontwikkeling geweest in het zoeken naar behandelingen. in eerste instantie bestond dit uit het steeds beter behandelen en voorkomen van opportunistische infecties. zo werd al snel duidelijk dat een van de beruchtste opportunistische infecties, de pneumocystis jirovecii-pneumonie (zie ook hoofdstuk 17) kon worden voorkomen als patiënten een profylaxe met co-trimoxa-zol ontvingen zodra hun aantal cd4+-cellen lager werd dan 200/mm 3 . vanaf 1987 werd het mogelijk de hiv-infectie zelf te behandelen met een middel dat reverse transcriptase remde: azidothymidine (azt). dit middel is een gemodificeerd nucleoside (thymidine), dat wanneer het in de dna-keten wordt ingebouwd verdere dna-verlenging (en dus uiteindelijk virusreplicatie) onmogelijk maakt. in eerste trials met dit middel was een duidelijk klinische verbetering aantoonbaar, maar het overlevingsvoordeel bleek van korte duur. nieuwe, vergelijkbare medicijnen, alle nrti's (nucleoside-analogue reverse transcriptase inhibitors) genoemd, kwamen op de markt, maar ook zij bleken in monotherapie geen aanwinst. duotherapie was effectiever, maar de echte doorbraak kwam pas met het gebruik van tripelcombinatietherapie. al snel werd het arsenaal medicijnen uitgebreid met een groep medicijnen die reverse transcriptase op een andere manier remmen (nnrti's, non-nucleoside reverse transcriptase inhibitors), maar ook met nieuwe nrti's en proteaseremmers. ook werd toen duidelijk waarom monoen duo-nrti-therapie niet werkten: deze middelen konden de virusreplicatie niet geheel remmen, omdat een of twee mutaties in het enzym reverse transcriptase al tot antivirale resistentie konden leiden. virussen met een tot twee mutaties waren al aanwezig in de viruspopulatie van onbehandelde mensen, met als gevolg dat deze mutanten werden uitgeselecteerd en konden repliceren. tripelcombinatietherapie (een proteaseremmer of een nnrti gecombineerd met twee nrti's) bleek wel tot volledige onderdrukking van de virale replicatie te leiden. hiervoor wordt het acroniem haart gebruikt (highly active anti-retroviral therapy). in enkele weken tijd kan de hoeveelheid hiv-rna in plasma tot onmeetbaar lage waarden worden teruggebracht, gevolgd door een snelle stijging van het aantal cd4+-cellen in het perifere bloed, deels als gevolg van reallocatie van deze cellen vanuit de lymfeklier, deels als gevolg van remming van virusgeïnduceerde celdood. met het stijgen van het cd4+-celaantal verdwijnen de ziekteverschijnselen snel en blijken de cd4+-waarden vervolgens geleidelijk te kunnen terugkeren tot relatief normale waarden, mits de virussuppressie voortdurend wordt gehandhaafd. belangrijk hierbij is dat haart dagelijks en goed geslikt wordt. met de eerstegeneratie-tripeltherapieën viel dit niet mee, aangezien zij bestonden uit grote aantallen pillen die meerdere keren per dag genomen moesten worden en bovendien frequent gepaard gingen met bijwerkingen als diarree, bloedarmoede en neuropathie. desalniettemin daalde in korte tijd de sterfte aan aids. ook bleek de virusoverdracht van moeder naar kind te kunnen worden geblokkeerd, door de hiv-positieve moeder tijdens het laatste deel van de zwangerschap een combinatiebehandeling te geven. na 2003 werden nog nieuwe klassen hiv-remmers geïntroduceerd: een remmer van de fusie van gp41 met de celmembraan (2003), een middel dat bindt aan ccr5-coreceptor (2008 ccr5-coreceptor ( ) en een integraseremmer (2008 , zodat het totale aantal geregistreerde antiretrovirale middelen in 2011 al ruim twintig telt. een bespreking van de aangrijpingspunten van antivirale therapie is te vinden in hoofdstuk 1 (paragraaf 1.7.2). meerdere combinaties van vrijwel steeds drie middelen kunnen op die manier worden samengesteld, waarbij ook resistente virussen goed kunnen worden geremd. behalve dit betere antivirale effect, maakten de nieuwere medicijnen combinaties mogelijk met een aanmerkelijk minder aantal pillen en ook met veel minder bijwerkingen. vooral deze laatste twee eigenschappen maakten dat patiënten daadwerkelijk hun medicatie goed konden blijven innemen. dankzij de haart lijkt de levensverwachting van hiv-geïnfecteerde personen inmiddels vergelijkbaar aan die van niet hiv-geïnfecteerden. langetermijnbijwerkingen, bijkomende ziekten (zoals chronische hepatitis b en c), maar ook ouderdomsverschijnselen spelen daarom een steeds grotere rol in de hiv-zorg anno 2011. met haart wordt gestart als het risico op opportunistische infecties begint te stijgen. aanvankelijk stelde men die grens onder een cd4+-aantal van 300/mm 3 , mede in overweging nemend dat de eerste combinaties ook veel bijwerkingen hadden. met de nieuwere combinaties is een vroegere start mogelijk en schuift de startgrens daarom op naar een hoger cd4+-getal (400/mm 3 anno 2010). haart is kostbaar en het bovenbeschreven succes kon aanvankelijk vooral worden bereikt in de rijkere landen. na 2000 is haart echter met vereende krachten ook geïntroduceerd in landen met de grootste patiëntenconcentraties. eind 2009 ontvingen zo ruim 5 miljoen patiënten over de hele wereld haart, weliswaar tegen een geschat totaal patiëntenaantal van 33 miljoen (2008). de bovenstaande beschrijving van het beloop en de behandeling van hiv-infectie betreft vooral hiv-1. wereldwijd is bij ongeveer 5% van de hiv-geïnfecteerde patiënten echter sprake van infectie met hiv-2. de prevalentie van dit virus is het hoogst in west-afrika, waar dit virus vermoedelijk rond 1940 door een meerkattensoort (zwarte mangabey) bij de mens werd geïntroduceerd. het ziektebeeld van hiv-2 verschilt niet van hiv-1, maar het beloop van een hiv-2-infectie is doorgaans veel milder en verloopt in een trager tempo. de hoeveelheid hiv-2-rna in plasma is veel lager dan bij hiv-1. dit draagt er waarschijnlijk toe bij, dat hiv-2 minder gemakkelijk kan worden overgedragen via seksueel contact of van moeder op kind. de diagnostiek van hiv-1 en hiv-2 verschilt enigszins: in de standaard hiv-serologie (elisa) zullen hiv-1-en hiv-2-infecties niet van elkaar onderscheiden kunnen worden. dit is pas mogelijk bij de zogeheten bevestigingstest (western-blot), waarbij specifieke antistoffen te-gen hiv-2-eiwitten apart worden gemeten. de pcr-test, die gebruikt wordt voor het meten van hiv-1-rna in plasma, detecteert hiv-2-rna niet. hierop dient men bedacht te zijn omdat een specifieke hiv-2-rna-test moet worden aangevraagd. ook therapeutisch zijn er verschillen tussen hiv-1 en hiv-2: non-nucleoside reverse transcriptase inhibitors (nnrti's) en fusieremmers zijn niet werkzaam tegen hiv-2 vanwege een andere structuur van het reverse transcriptase en gp41. het aantal behandelingsmogelijkheden tegen hiv-2 is daardoor beperkter. hiv kan worden overgedragen via seksueel contact, bloed-bloedcontact (zoals transfusie van bloedproducten en het gebruik van verontreinigde naalden) en door verticale transmissie (van moeder op kind). het risico op seksuele overdracht wordt vergroot wanneer er sprake is van slijmvliesbeschadigingen, zoals bij geslachtsziekten of bij agressieve c.q. traumatische seks (verkrachting), maar is ook afhankelijk van de seksuele techniek. zo is de transmissiekans groter bij anale seks ten opzichte van vaginale seks en ook zal de ontvangende partner eerder een infectie oplopen (tabel 13.2). de kans op overdracht wordt mede bepaald door de hoeveelheid aanwezig infectieus virus. bij een hoge virale load in het plasma, zoals vooral het geval is tijdens de acute hiv-infectie, is de transmissiekans veel groter dan bij een lage virale load. effectieve haart reduceert het transmissierisico tot bijna nihil. transmissie via bloed-bloedcontact verloopt efficiënter dan seksuele overdracht, maar is afhankelijk van het inoculum en ook weer van de hoeveelheid aanwezig virus. mondiaal gaat het hierbij vooral om hergebruik van injectienaalden bij intraveneus drugsgebruik, maar er zijn ook diverse voorbeelden van hergebruik van naalden en infuussystemen in medische setting (libië, china, roemenië), dan wel infusie van met hiv besmette bloedproducten. ook in nederland zijn vóór 1985 tientallen hemofiliepatiënten op deze manier met hiv geïnfecteerd, doordat er nog geen hiv-test beschikbaar was. het risico op verticale transmissie van hiv van moeder naar kind is 15-25% wanneer geen borstvoeding wordt geven, maar loopt op tot 40% als dat laatste wel gebeurt. de meeste kinderen raken geïnfecteerd in de laatste fase van de zwangerschap, vooral tijdens de partus. ook hier speelt de hoogte van de virale load weer een belangrijke rol. door alle zwangere vrouwen vroeg op hiv te testen en ten minste in het derde trimester van de zwangerschap met haart te behandelen (zie verder), is in de westerse wereld het risico op verticale transmissie tot minder dan 0,1% teruggedrongen. de incidentie van hiv-1 in de verschillende risicogroepen is sterk afhankelijk van de geografische lokalisatie en transmissieroute. in centraal-afrika, waar het virus eind jaren zeventig al wijdverbreid was, is heteroseksuele overdracht het frequentst en wordt het virus veel aangetroffen bij jonge mannen en vooral vrouwen. in sommige afrikaanse landen blijkt meer dan 30% van de zwangere vrouwen geïnfecteerd. ook infectie van pasgeborenen door verticale transmissie komt in afrika veel voor. in de verenigde staten, europa en australië komt een hiv-1-infectie vooral voor bij mannen die seks hebben met mannen (msm), ook al neemt het aantal heteroseksuele overdrachten geleidelijk toe. in oost-europa en veel aziatische landen, waaronder china en indonesië, is een snelle uitbreiding van de epidemie gaande, vooral als gevolg van intraveneus drugsgebruik. eind 2008 bedroeg het geschatte aantal in leven zijnde hiv-geïnfecteerden wereldwijd ongeveer 33 miljoen, waarvan het overgrote deel woonachtig was in afrika bezuiden de sahara. in nederland was eind 2010 het aantal in leven zijnde hiv-geïnfecteerden in de landelijke registratie 14.000, maar wordt het werkelijke aantal geschat op ruim 24.000. de belangrijkste transmissieroute bij de nederlandse patiënten is msm-contact (57,2 %), gevolgd door heteroseksueel contact (32%), (ex-)intraveneus drugsgebruik (3,2 %) en bloedproducten (1,5%). hoewel er direct na de ontdekking van hiv optimisme ontstond over het ontwikkelen van een beschermend vaccin, is hier tot op heden nog steeds geen zicht op. kandidaatvaccins bleken wel in staat tot het opwekken van hiv-specifieke immuniteit, maar dit bleek geenszins beschermend. toen uit een veelbelovende studie in 2008 bleek dat er wellicht meer hiv-infecties voorkwamen in de groep met het beoogde vaccin dan in de placebogroep, werden de verwachtingen nog verder getemperd, tabel 13.2 gemiddeld risico op hiv-1-transmissie bij eenmalig contact met een hiv-1-positieve, onbehandelde bron. al bleek er bij een intensieve gecombineerde vaccinatietrial in 2009 voor het eerst sprake van een zwak beschermend effect. goede voorlichting en vermijding van risicogedrag (veilige seks, schone naalden voor intraveneuze drugsgebruikers) blijven daarom vooralsnog de belangrijkste wapens in de strijd tegen de verspreiding van de hivepidemie. seksuele transmissie kan vooral voorkomen worden door goed en consequent gebruik van mannencondooms en ook mannenbesnijdenis blijkt de kans op transmissie te verlagen. geen van deze methoden biedt echter honderd procent bescherming. nieuwe methoden, zoals virucide vaginale crèmes en vrouwencondooms, bleken nog niet effectief. effectieve haart verlaagt ook het risico op seksuele overdracht, al maakt het daarmee condoomgebruik niet overbodig. transmissie via bloed-bloedcontact kan vermeden worden door goede screening van bloedproducten op hiv en het verstrekken van schone naalden aan drugsverslaafden. verticale transmissie kan worden voorkomen door het toedienen van antiretrovirale middelen aan de zwangere vrouw, waarbij in westerse landen ook de pasgeborene nog vier weken antiretrovirale therapie krijgt toegediend als een soort postexpositieprofylaxe (pep). in westerse landen worden daardoor weinig hiv-positieve kinderen meer geboren. in derdewereldlanden is een dergelijke benadering vaak niet mogelijk, maar lukt het dikwijls toch de transmissie terug te dringen door kort voor de bevalling een eenmalige dosis nnrti (nevirapine) toe te dienen. het aantal hiv-geïnfecteerde kinderen loopt hierdoor wel terug, maar de keerzijde van deze korte monotherapie zijn resistentieontwikkeling bij de moeders en de toch hiv-positief geboren kinderen. de resistentieprevalentie tegen nevirapine kan daarbij oplopen tot 60%. de genoemde postexpositieprofylaxe kan ook worden gegeven aan personen die door een incident (prikaccident, onbeschermd seksueel contact) aan hiv zijn geëxposeerd en mogelijk geïnfecteerd. hoewel hiervoor vooral dierexperimenteel bewijs bestaat, lijkt pep effectief als het zo snel mogelijk na het incident wordt toegediend. kernpunten -infecties met ebv, cmv, hiv-1 en t. gondii behoren tot de frequentste oorzaken van gegeneraliseerde lymfadenopathie. -een primaire infectie met ebv of cmv verloopt vaak asymptomatisch (kinderleeftijd) maar leidt op oudere leeftijd vaak tot een klinisch beeld met faryngitis, koorts, lymfadenopathie en atypische lymfocytose (mononucleosis infectiosa). -mononucleosis infectiosa kan ook gezien worden bij primaire infecties met toxoplasma gondii en hiv. -ebv en cmv zijn humane herpesvirussen die persisteren in latente vorm. vooral bij immuungecompromitteerde patiënten kunnen herpesvirussen ernstige systemische infecties of lymfoproliferatieve syndromen (ebv) veroorzaken. -een acute of doorgemaakte infectie met ebv of cmv is aan te tonen met serologisch onderzoek. moleculaire diagnostiek kan worden gebruikt om de hoeveelheid virusreplicatie te monitoren. -hiv is een retrovirus dat via seksueel of bloed-bloedcontact wordt overgedragen en een persisterende infectie veroorzaakt. ook perinatale transmissie komt voor. na de primo-infectie ontstaat een klinische latentieperiode die kan variëren van maanden tot jaren. -hiv-infectie leidt op den duur tot cd4+-t-celdepletie en een ernstige cellulaire immunodeficiëntie met als gevolg levensbedreigende opportunistische infecties en maligniteiten. -hiv-infectie kan worden aangetoond met serologisch onderzoek. de activiteit van de infectie kan worden vervolgd door seriële bepaling van het aantal cd4+-cellen en de hoeveelheid hiv in plasma. -hiv-infectie kan worden behandeld met een combinatie van antiretrovirale middelen (haart). haart leidt tot volledige virusonderdrukking met daarbij uiteindelijk goed herstel van de cellulaire immuniteit. -met behulp van levenslange antiretrovirale therapie kunnen patiënten een normaal leven leiden, met waarschijnlijk een bijna normale levensverwachting. hiv immunopathogenesis and strategies for intervention infectious diseases 3rd infectious mononucleosis principles and practice of infectious diseases clinical virology key: cord-031252-ji0ef0by authors: d'angelo, lawrence title: infectious disease problems in adolescents date: 2020-09-01 journal: j adolesc health care doi: 10.1016/s0197-0070(20)30007-3 sha: doc_id: 31252 cord_uid: ji0ef0by nan accounts for approximately 21% of the cases, and ebv infection, which accounts for 1-5% of the cases (12) . the rest of the cases are presumed to be viral, with the exception of a small percentage caused by neisseria gonorrhoeae (13) . it is difficult to determine the etiology of pharyngitis and to propose a course of therapy on the basis of clinical findings (14) . although illness is usually self-limited, its duration is influenced by the etiologic agent and the prescribed therapy. for example, the clinical course of a streptococcal infection is shortened significantly when appropriate antibiotics are prescribed early (15) . throat cultures are advisable for patients whose primary symptoms are throat pain, fever, and cervical lymphadenopathy or exudative pharyngitis. the optimal culture site is the tonsillar surface (16) . direct-swab rapid diagnostic tests for group a streptococcus, based on enzyme-linked immunosorbent assays (elisa technique), have been well evaluated for office use and allow the practitioner to initiate antibiotic therapy, when necessary, early in the cour.se of the illness (17) . therapy for pharyngitis is determined by the nature of the etiologic agent. the best example of a cause amenable to therapy is streptococcal infection, which ideally is treated with penicillin, either orally (250 mg of phenoxymethyl penicillin qid) or by a single intramuscular injection of benzathine penicillin (1.2 million units). gonococcal pharyngitis is best treated with 4.8 million units of intramuscular procaine penicillin g administered 30 minutes after a 1.0-g dose of oral probenecid. the most significant complication of pharyngitis is peritonsillar abscess (18) . primarily a disease of adolescents and young adults, peritonsillar abscess is characterized by dysarthria, trismus, and discomfort in swallowing. in addition to high doses of antibiotics (aqueous penicillin g, 2-4 million units q 4 hr), patients require incision and drainage, often followed by a tonsillectomy. otitis media is the second leading reason that patients under age 15 seek medical care, although it is far less common in older adolescents (19) . bacterial etiologic agents of otitis media include streptococcus pneumoniae, hemophilus influenzae, streptococcus pyogenes group a, branhamella catarrhalis, and staphylococcus aureus. a number of respiratory viruses have likewise been implicated as etiologic agents in this illness. as with many disorders, the median duration of symptoms depends upon the promptness of diagnosis and the method of treatment (20) . antibiotic therapy with amoxicillin (500 mg tid), trimethoprim-sulfamethoxazole (one double-strength tablet bid), or cefaclor (250 mg tid) is appropriate in the adolescent. with treatment, complications are rare. the sinuses are affected when the normal drainage of mucus and other secretions into the nasal passages is hindered. the usual pathogens include s. pneumoniae, h. influenzae, anaerobic organisms, mixed bacteria, s. aureus, s. pyogenes, b. catarrhalis, gram-negative bacteria, influenza, and parainfluenza. the incidence of these pathogens has varied from study to study. sinusitis can develop in the course of any upper respiratory infection. it is often associated with facial pain (periorbital or supraorbital) and purulent nasal discharge. headache and nasal obstruction, which alters the patient's sense of taste and smell, may also be present. only about half of patients have an elevated temperature. eyelid edema and tearing are indicative of ethmoid involvement. cough, particularly daytime cough, is an important sign (21) . the clinical diagnosis is based on the presence of sinus tenderness, purulent nasal discharge, and daytime cough of at least ten days' duration. although opacity of the sinus on transillumination is a helpful finding, transillumination is not a consistently reliable diagnostic technique (22) . radiography is the most specific test for sinusitis. mucosal thickening is usually visible, and the presence of airfluid levels in the sinus cavity confirms the diagnosis. once again, the duration of illness is determined by the speed of diagnosis and therapy. appropriate therapy includes antibiotics and local as well as systemic decongestants. with topical decongestants care must be taken to avoid the rebound phenomenon (rhinitis medicamentosa). amoxicillin (500 mg tid) is the most frequently prescribed antibiotic for initial oral therapy in outpatients. trimethoprimsulfamethoxazole and cefaclor serve as second-line agents. symptomatic relief of pain is best provided by acetaminophen. teenagers are at considerable risk of developing complications of sinusitis because their frontal sinuses are rather recently developed. complications include meningitis, brain abscess, orbital cellulitis osteomyelitis, and cavernous sinus thrombosis. for this reason, if a prompt response to outpatient oral therapy does not occur, hospitalization to enable in-travenous administration of antibiotics (i.e., chloramphenicol and oxacillin or cefuroxime) should be considered. the usual pathogens causing pneumonia include m. pneumoniae (which accounts for 50% of the cases), s. neul11oniae, and viruses such as influenza, parainfluenza, and adenovirus. pneumocusiis carinii, al-t~ough rare, must be considered in patients at high risk for human t-celllymphotropic virus (htlv)-iii infection. cough and fever are the most common clinical characteristics of pneumonia. the nature of the fever can sometimes suggest etiology. for example, fever associated with multiple chills suggests m. pneumoniae, whereas a single, shaking chill is more characteristic of s. pneumoniae. leukocytosis can also suggest etiology; it is less common with viruses and mycoplasmal infection than with bacterial pneumonia. the median duration of penumonia is 14 days, although adolescents may recover much sooner. teenagers can generally resume normal activities after being afebrile for 48 hours. diagnostic tests for pneumonia include a chest xray and sputum examination and culture. in adolescents, however, the routine sputum culture will not pr~vide a diagnosis for 70-80% of community-acquired pneumonias. similarly, a gram stain is often nondiagnostic and may be even less helpful than a routine culture (23) . even chest x-rays are less useful than clinical diagnosis based on an appropriate history and physical examination (24) . treatment of pneumonia is based on the most probable etiologic organism. most adolescents are candidates for an outpatient antibiotic regimen. erythromycin, 500 mg q 6 hr, is appropriate. patients with an underlying disease (i.e., sickle cell disease, malignancy) or concomitant bronchoconstriction, as well as those who fail to respond to an outpatient regimen, may require hospitalization. because complications from pneumonia are rare in adolescents, the presence of complications (i.e., empyema, lung abscess, or bacteremia) may be indicative of an unclerlying health problem. influenza is an acute, usually self-limiting, febrile illness that occurs in outbreaks of varying severity al-most every winter. adolescents play a major role in the spread of influenza and frequently account for the first cases (25) . for example, the first few cases of swine flu in new jersey in 1976 occurred in teenage army recruits at fort dix (26) . these cases gave rise to a national vaccination program. although two distinct strains of the influenza virus, a and b, can infect humans, they cannot be distinguished on the basis of clinical signs and symptoms. patients with either type of influenza usually have systemic symptoms, including fever, a chilly feeling or frank shaking chills, headache, myalgia, malaise, and anorexia. these symptoms progress rapidly to include dry cough, nasal discharge, and stuffiness. the median duration of illness is 10-14 days, depending on prior immunity and whether the patient receives the antiviral agent amantadine hydrochloride. diagnosis is based almost entirely on clinical findings as cultures are expensive and the results are not immediately available. preventing infection via strain-specific vaccine or prophylactic amantadine provides the most efficacious approach to influenza outbreaks (27) . adolescents with chronic cardiac or respiratory illness (including severe asthma), severe anemia, immunocompromise, or severe renal or metabolic illness should be given the vaccine. when prevention is impossible or unsuccessful, treatment with amantadine can shorten the duration of fever and systemic and respiratory symptoms by about 50% if the illness is caused by type a virus and the drug therapy is initiated within the first 48 hours of infection. although complications in adolescents are rare, influenza can cause croup, chronic obstructive pulmonary disease, bacterial pneumonia, myositis, and myocarditis. many of the 21 million deaths in the 1918-1919 outbreak were due to viral pneumonia in otherwise healthy young adults (28) . the most dramatic and dangerous complication in adolescents is reye's syndrome, which has been reported in the convalescent phase of the flu since the early 1970s (29) . there is apparently a strong epidemiologic association between salicylate intake and reye's syndrome (30) , with the majority of recently reported cases occurring in adolescents (8) . mycoplasma pneumoniae is responsible for 50% of cases of pneumonia in adolescents and young adults. this organism also causes many other clinical syndromes, including tracheobronchitis, myringitis, pharyngitis, gastroenteritis, hepatitis, meningitis, and myocarditis (31) . headache and multiple chills are the most important symptoms of pneumonia caused by mycoplasmal infection. the course of the illness is often protracted, and x-rays may not clear for two to six weeks (32) . diagnostic tests for pneumonia rarely reveal the etiology of the illness. x-rays may show a unilateral, lower-lobe, segmental bronchopneumonia. the peripheralleukocyte count is equivocal; it may be normal, depressed, or elevated. cold agglutinins are usually present, but in varying titer. complement fixation is the most specific diagnostic test, but it lacks sensitivity, particularly early in the illness. definitive diagnosis of mycoplasmal pneumonia is based on the combination of an antibody rise and a sputum culture showing m. pneumoniae. however, growth on agar may take two to three weeks, rendering results useless for initiating drug therapy. the treatment options for pneumonia caused by mycoplasmal infection are straightforward as the organism is sensitive to both tetracycline and erythromycin (500 mg of either medication qid). erythromycin is a particularly attractive therapeutic agent because it is equally effective against other common pneumonia pathogens. pleural effusions and spread of infection within the lung are the most common pulmonary complications of mycoplasmal pneumonia, but they are uncommon in the adolescent population. death from pneumonia in adolescents is extraordinarily rare. several points about anticipatory guidance must be emphasized. first, supportive therapy is the most important part of treatment for the adolescent with undifferentiated respiratory symptoms. most adolescents with respiratory infections will not require antibiotic therapy. second, adolescents with chronic health problems must be appropriately immunized. at this time, the most useful immunizing agent is for influenza. finally, because the salicylates are associated with an increased incidence of reye's syndrome (8, 29, 30) , adolescents with respiratory infections and/or fever should be treated with acetaminophen rather than aspirin. mononucleosis is an acute infection most commonly caused by the epstein-barr virus, a member of the herpesvirus group. the ebv was first isolated in tissue culture cell lines derived from burkitt's lyrn-phoma. the observation of ebv antibody seroconversion in a laboratory technician with infectious mononucleosis led to the etiologic connection between ebv and the mononucleosis syndrome (33) . the incidence and prevalence of infectious mononucleosis are greatest in closed groups of adolescents and young adults, i.e., at boarding schools, detention centers, colleges, and military reservations. the illness causes significant morbidity and even mortality in some individuals. the mononucleosis syndrome presents with a classic triad of sore throat, fever, and cervicallymphadenopathy, all of which can have severe clinical manifestations. other characteristic symptoms include malaise, headache, fatigue, and anorexia. splenomegaly occurs in approximately 50% of patients and is greatest during the second and third weeks of illness. illness usually lasts for two to three weeks, although fatigue and exercise-induced exhaustion can remain for several months. epstein-barr virus infections are a widespread phenomonon, particularly among people in lower socioeconomic groups and third-world countries (34) (35) (36) . in general, the incidence of mononucleosis is greatest in individuals who have not encountered the virus prior to adolescence, although ebv can cause illness in younger children (3) . individuals who enter a high-risk setting without previous exposure to the virus have a seroconversion rate of 12% per annum (37, 38) . by adulthood, 90-95% of all persons have antibodies to ebv (39) . initial viral replication takes place in the pharyngeal epithelial cells (40) , and the virus can be recovered in oropharyngeal secretions for prolonged periods (41) . exchange of infected saliva during intimate oral contact or from inanimate objects, such as eating and drinking utensils, is the usual mode of transmission (42, 43) . the ebv appears to be transmitted only minimally in standard living situations. like other members of the herpesvirus group, ebv can be maintained in a host in a latent form until it is activated or transformed, which often occurs under conditions of immunosuppression (44, 45) . this latency period accounts for the occurrence of illness or viral replication in renal transplant patients and may explain the recently described virologic findings in chronic mononucleosis syndrome (46, 47) . in adolescents and young adults, other infectious agents can cause illnesses that mimic ebv-induced mononucleosis. among these agents are cytomegalovirus, toxoplasma gondii, treponema pallidum (secondary stage syphilis), rubella virus, adenovirus, the hepatitis viruses, and s. pyogenes. noninfectious diseases that mimic ebv-induced mononucleosis include serum sickness, systemic lupus erythematosus, sarcoidosis, and lymphoma. mononucleosis is diagnosed by clinical manifestations and laboratory findings. diagnostic abnormalities in routine laboratory procedures include 1) relative or absolute lymphocytosis, 2) atypical lymphocytes (more than 10% of total lymphocytes), 3) relative or absolute neutropenia (48), 4) mild thrombocytopenia, 5) mild transaminitis, with or without overt hepatitis (49) , and 6) mild elevation of cryoglobulins. there are also specific tests for ebv antibody. heterophil antibodies, mostly of the igm class, develop transiently during the course of infectious mononucleosis and are seen in 90-95% of the cases associated with ebv. these antibodies, first described in 1932by paul and bunnell, are antibodies to antigens on cell surfaces of nonhuman species (50) . the monospot test, a single-dilution slide test that uses sensitized horse red blood cells, is almost as sensitive as the heterophil test (51) and is very useful in screening for these antibodies. although this test gives quick results and is generally specific for infectious mononucleosis, false-negative results can occur when heterophil antibody titers are low; false-positive results also occasionally occur. the clinical finding most perdictive of a positive heterophil test is the presence of axillary adenopathy (52) . the ebv-specific serologic tests may be diagnostic in the small percentage of patients who are heterophil negative (53) . these tests consist of measuring antibodies to the viral capsid antigen (vca), the viral early antigens (ea), and the viral nuclear antigens (ebna). the vca is the most common ebv-specific assay, and evidence of igm and igg antibodies is of value in making the serodiagnosis of primary ebv infection. the ea test measures two separate antigens and their respective antibodies-anti-d and anti-r-which are observed in 70-85% of patients in the acute phase of infectious mononucleosis (33) . the viral nuclear antigens appear late in the course of infectious mononucleosis, and positive ebna can document ebv infection when early serum samples are not available. supportive therapy with appropriate guidance is the best approach to treat infectious mononucleosis. sore throat and fever should be treated with antipyre tics, but because of the occasional presence of relative thrombocytopenia, aspirin should be avoided. steroids should be reserved for patients with severe, life-threatening complications, such as airway obstruction, neurologic complications, thrombocytopenia, hemolytic anemia, or myocarditis. antiviral agents have not yet been shown to have a therapeutic role, although intravenous acyclovir may eventually prove useful for serious infections (54, 55) . most complications of the mononucleosis syndrome resolve without significant sequelae. possible complications can best be grouped by organ system. hematologic complications, namely, autoimmu ne hemolytic anemia and severe thrombocytopenia, occur in up to 5% of patients (56,57). these disorders are usually self-limited but may respond to corticosteroids (58) . splenic rupture, a rare but serious complication, is most often associated with trauma (59), although it can occur spontaneously (60) . cardiorespiratory complications, including myocarditis, pericarditis, and pneumonia, have been reported in adolescents but occur more commonly in children (61, 62) . neurologic complications occur in less than 1% of cases of infectious mononucleosis (63) . when these complications do occur, they are frequently the primary manifestation of the illness. complications of the central nervous system include guillain-barre syndrome, bell's palsy, transverse myelitis, and meningoencephalitis myelitis. fortunately, most of these complications will remit without lasting neurologic deficit. death from mononucleosis is rare. it is most frequently associated with x-linked immunodeficiency (64) . viral-related malignancies such as african burkitt's syndrome (65, 66) and nasopharyngeal carcinoma (67) may also occur. the role of ebv as a cause of lymphomas and hodgkin's disease is under investigation (68, 69) . advising adolescents of ways to avoid contracting infectious mononucleosis is difficult. health practitioners can share certain information with patients and parents that will help them to cope with the disease in themselves and others. for example, supportive care is the hallmark of rehabilitative services. patients with infectious mononucleosis do not require isolation as the disease is poorly transmitted from person to person (70) . platelet abnormalities occur in a small percentage of patients, and therefore acetaminophen is preferred for symptomatic relief of fever and myalgia. finally, because of the risk of subclinical splenic enlargement, patients should avoid contact sports and strenuous activities for at least four weeks after signs and symptoms of infection remit. viral hepatitis is most frequently caused by one of three types of agent: hepatitis virus a, hepatitis virus b, and the so-called non-a, non-b agents, of which here appear to be at least two. individuals between the ages of 10 and 20 comprise 17.4% of hepatitis a cases, 9.3% of hepatitis bcases, and 11.1% of non-a, non-b hepatitis cases (70) . in 1983 alone, 1,360 cases of hepatitis a in teenagers were reported, 829 cases of hepatitis b, and 301 cases of non-a, non-b hepatitis (71) . all of these hepatitis forms have worldwide distribution. acquisition of the antibody to the hepatitis virus is closely linked to socioeconomic status and geographic location (72, 73) . as with mononucleo-, sis, individuals aquire antibodies for these viruses during childhood, usually via asymptomatic infecttion. ' personal contact with someone who has hepatitis a is the most important risk factor for acquisition of symptomatic illness (71) . for hepatitis b, the risk factors are personal contact with an infected individual, intravenous drug abuse, homosexual activity in males, recent dental work, and recent transfusion of blood or blood products. for non-a, non-b hepatitis, the risk factors are surgery, hospitalization, dental .work, and drug abuse. the classic mode of transmission of hepatitis a is by fecal-oral contact. attendance at day-care centers is currently the most important cause of the spread of this disease in both children and adults in north america, accounting for approximately 10% of cases (74) . young children acquire the infection (usually asymptomatically) and spread it to staff, siblings, and parents. hepatitis b and non-a, non-b hepatitis are usually spread through the blood or body secretions. hepatitis a is a more frequent cause of epidemics in adolescents than is hepatitis b (75,76). spread of hepatitis b among classroom contacts (77) and teenage wrestlers has been reported (78) . the type of hepatitis is difficult to distinguish by the clinical symptoms alone. all forms usually have a prodromal phase, characterized by fever, malaise, fatigue, headache, anorexia, nausea, vomiting, myalgias, and/or right upper quadrant tenderness. in general, the younger the patient, the less severe the manifestations of the illness (79) . nearly 80% of the patients with viral hepatitis develop jaundice (71) . in fact, because hepatitis is most specifically suggested by scleral and skin jaundice, many cases without jaundice may go undiagnosed or unreported. the clinical course of the illness varies but usually lasts for one to three weeks (80). although clinical manifestations alone will not reveal the etiology of the hepatitis virus, certain charac-,teristics are specific to each form of the illness. the incubation period for hepatitis a is 14-50 days, with a mean of 28 days. diarrhea is more frequent in type a than in any other type, occurring in approximately 20% of patients. upper respiratory symptoms, encephalitis, and aplastic anemia, although rare, occur more often with hepatitis a. hepatitis busually has an incubation period of 60-110 days (81) . more than the other types, it is associated with extrahepatic manifestations, including urticaria and arthritis, and, much less commonly, glomerulonephritis and vasculitis. in about 10% of patients with symptomatic hepatitis b, serologic tests continue to reveal markers of acute infection. most of these patients continue to have mildly elevated liver-associated enzymes, which are indicative of chronic persistent hepatitis (81) . this generally benign, nonprogressive disease is characterized by chronic portal inflammation, with little or no fibrosis. some patients develop cirrhosis. diagnosis of the non-a, non-b forms of hepatitis is essentially done by exclusion. the incubation period varies from two weeks to six months, with a mean of about eight weeks. half of the cases of non-a, non-b hepatitis are anicteric. although there are rare instances of chronicity, the course is usually benign (82) . hepatitis is usually diagnosed by clinical signs and symptoms-especially jaundice-and by laboratory evidence of elevated liver-associated enzymes. specific serologic tests for etiology of viral hepati-tis are now available, with certain limitations. the presence of serologic markers precedes the clinical illness itself. type a markers are antibodies directed at specific viral proteins. these antibodies are of igm and igg classes. the presence of the former suggests acute infection. the markers for hepatitis b are more complicated; they include the hepatitis b surface antigen (hbsag) and its antibody, anti-hbs, as well as core antigen antibody (anti-hbc), e antigen, and its antibody (anti-e). in the classic pattern both hbsag and anti-hbc are acutely positive, but this pattern may not occur in all cases. anti-hbc is almost always positive, but a single negative test for hbsag does not definitely exclude acute hepatitis b (83) . the persistence of antigen markers without concomitant antibody suggests chronic infection (84). specific serologic markers are available for non-a, non-b hepatitis. in the absence of specific treatment for acute viral hepatitis, the major emphasis is placed on support, symptomatic care, and prevention of transmission. there is no convincing evidence to justify the use of corticosteroids in acute hepatitis, regardless of its severity (85); prophylaxis for hepatitis a involves passive immunization with gamma globulin (86) for all close contacts of the infected patient. patients exposed to hepatitis b can be given a safe, effective vaccine (87) , which consists of highly purified and triple-inactivated hbsag obtained from the serum of chronic carriers (88). in some exposure situations, the hepatitis b vaccine is used in combination with a special high-titer immune globulin-hepatitis b immune globulin (hbig)-to allow for immediate as well as continuing protection (89) . the overall incidence of complications from viral hepatitis is low. complications are limited to those problems already discussed in the review of the specific clinical presentations. mortality rates vary from 0.2-2.0%. fatalities are less frequent with type a than with type bor non-a, non-b hepatitis. the overall mortality rate for viral hepatitis is about 1%. death can occur rapidly in patients with fulminant hepatitis (90) . to prevent hepatitis a: 1) limit the amount of raw shell fish eaten; 2) administer appropriate immune serum globulin (isg) when possible exposure has occurred; and 3) give prophylactic isg to patients traveling to third-world countries. to prevent hepatitis b: 1) promptly administer hbig and the hepatitis b vaccine to individuals with known exposure; 2) check the serologic status of adolescents with highrisk lifestyles (i.e., male homosexuals and intravenous drug users) and vaccinate those who are not protected; and 3) properly screen all blood products before use. at puberty, preadolescents undergo physiological changes that transform them into sexually mature individuals. the accompanying psychological and social changes frequently result in sexual activity. eighty percent of males and 70% of females have had intercourse during their teenage years (91) , and the average age of first sexual intercourse is 16. during these years of bodily change and emerging sexuality, symptoms attributed to the genital organs become a special concerrt of adolescents. some of the most common presenting complaints include urinary tract infections, vaginal discharge, urethral discharge, and genital lesions. in sexually active teen" agers, these complaints are especially important because they are frequently caused by sexually transmitted diseases (stds). an awareness of the most likely etiologies of each of these conditions will facilitate differential diagnoses arid medical management of urogenital disease. urinary tract infections (uti) are among the major reasons for physician visits by adolescent females. in women, the usual etiological organism is escherichia coli, but in adolescent and young adult females, staphylococcus saprophyticus is also common. chlamydia trachomaiis is a frequent cause of dysuria and acute urethal syndrome (92) . the risk factors for urinary tract infections include sexual intercourse, pregnancy, behavioral factors (i.e., diaphragm use (93) and voluntary urinary retention (94)), anatomical abnormalities, and underlying disease. the diagnosis of uti is. based on both clinical symptoms and laboratory criteria. the clinical symptoms of lower tract infection (cystitis and urethritis) include frequent urination, dysuria,â· burning pain during urination, suprapubic discomfort, and passage of cloudy and occasionally blood-tinged urine. when fever, flank or back pain, and rigors occur, upper tract involvement must be assumed to be present. because the symptoms of vaginitis often resemble those of uti (95) , it is important to determine quickly the site of infection. a bacteria count of over 100,000 organisms per milliliter of unspun urine is the old standard for making the diagnosis of a uti. new data suggest that this criterion is too restrictive and will not correctly identify women with overt infections (96) . if women are experiencing such symptoms, lower counts, 100-1,000 of organisms per milliliter of unspun urine, should be considered diagnostic of infection. treatment of utis depends upon the location of the infection within the urinary tract. lower-tract infections can usually be treated effectively with oral, single-dose therapy of trimethoprim-sulfarnethoxazole, two to four double-strength tablets. because single-dose therapy will not eradicate upper-tract infections (kidney), failure to respond to short-course therapy will establish a diagnosis of upper-tract in-.fection (97) . upper-tract infections require a 7-14-'day or longer course of therapy with trimethoprimsulfamethoxazole (one double-strength tablet bid for 14 days) or amoxicillin (500 mg po tid) for 14 days. if significant systemic signs and symptoms are present, the patient should receive intravenous antibiotics (gentamicin, 80 mg iv q 8 hr, or ampicillin, 1 g iv q 6 hr). prophylaxis is used to prevent recurrent symptomatic infection (98) . a lower-tract infection that has not been adequately treated can lead to pyelonephritis (99) . stones can result from recurrent infections. but sepsis is fortunately rare in adolescents. anticipatory guidance for utis includes the following: do not retain urine voluntarily, urinate after sexual intercourse, do not leave diaphragm in longer than necessary, drink plenty of fluids, and maintain good personal hygiene. vaginitis and cervicitis are also common in adolescent and young adult females. the rising incidence of vaginal discharge results from the physiological and behavioral changes that occur at puberty. hormonal changes and ovulation make the vagina and cervix more vulnerable to infection by a number of pathogenic organisms; sexual activity, which results from behavioral changes, makes the female even more susceptible to infection. a careful history of sexual activity and use of contraception must be obtained, and a complete pelvic exam should be performed. in adolescents the infections that cause vaginal discharge, in order of frequency, include the following: fungal vaginitis, usually caused by candida albicans; trichomoniasis; gardnerella vaginitis; gonococcal vaginitis, caused by neisseria gonorrhoeae; and nonspecific urethritis, caused by c. trachomatis. diagnostic features and therapeutic regimens for these infections are shown in table 1 . the risk factors for vaginitis and cervicitis include sexual activity and mutliple sexual partners, tight clothing, oral contraceptives, underlying illness (diabetes), poor hygiene, and pregnancy (100) . the chlamydia1and gonorrheal sexually transmitted infections can cause pregnancy loss (101, 102) , pelvic inflammatory disease (pid), and a variety of nonlocalized infections. the incidence of complications from vaginitis and cervicitis that are not sexually transmitted, however, is very small. vaginitis or cervicitis caused by either n. gonorrhoeae or c. irachomatis can ascend, causing endometritis, salpingitis, and peritonitis. any combination of these disease entities is commonly known as pelvic inflammatory disease (pid). the incidence of pid in adolescents is great; [20] [21] [22] [23] [24] year olds are the only age group with a higher rate than [15] [16] [17] [18] [19] year olds (103). adolescents face an increased risk of acquiring plo for several reasons. they tend to have a larger number of sexual partners (104) , and their immature anatomy is easily infected by a variety of sexually transmitted pathogens (105) . adolescents' infrequent use of barrier methods of contraception (106) contributes to the problem, as does poor follow-up of stds. diagnosis of pid is based on physical and laboratory findings. the major criteria for clinical diagnosis are a history of abdominal pain, lower abdominal tenderness (especially rebound), cervical motion tenderness, and adnexal tenderness. minor diagnostic criteria include fever, leukocytosis, elevated sedimentation rate, abnormal sonogram, culdocentesis revealing bacteria, and vaginal discharge. three or more major clinical features are necessary for diagnosis. if only three major criteria are identified, two or more minor criteria may be used as supplements (107) . adolescents with pid usually respond well to therapy. the decision to treat the individual as an inpatient or as an outpatient depends upon the severity of illness, a prior history of pid, coincidental pregnancy, the presence of an iud, inability to comply with the outpatient treatment, or a tubo-ovarian mass. some physicians feel that the consequences of inadequate treatment are so severe that all teenagers with pid should receive some of their antibiotic therapy as inpatients (103). inpatient and outpatient regimens for the treatment of pid are shown in table 2 . outpatients should be scheduled for follow-up visits on days 2, 7, and 14 to assess the progress of therapy. complica-tions of pid include tubo-ovarian abscess (108), recurrent infections (109), ectopic pregnancy (110) , and infertility (111) . most of the same infectious etiologies of vaginal discharge in adolescent females can cause urethral discharge and dysuria in adolescent males. as with the dolescent female, physicians must obtain a thorough history of sexual activity, preferences, and use of contraception in adolescent males. a complete genital exam must be performed, with particular attention to the testes and epididymis. urethritis is usually classified as gonococcal or nongonococcal urethritis (ngu). gonococcal urethritis classically presents with dysuria and a copious yellow-green discharge two to seven days after exposure. nongonococcal urethritis has a longer incubation period (one to three weeks) and, frequently, a more indolent onset with scantier discharge and less pain (112) . the symptoms of these two types of urethritis are similar regardless of microbiologic cause. the duration of symptomatic illness varies, depending upon when appropriate therapy has been administered. diagnostic features and therapeutic regimens for these infections are shown in table 3 . .complications of urethritis are rare in adolescents, although urethritis has been known to cause prostatitis (113) and epididymitis. also, in young males, chlamydial infection has been associated with reiter's syndrome in those genetically predisposed to this form of arthritis (114) . most genital lesions encountered in adolescents and young adults are due to stds. the most common lesions are vaginal warts and herpes simplex infection, usually with type ii virus. other causes include syphilis, chancroid, lymphogranuloma venereum, and granuloma inguinale. the clinical characteristics of genital ulceration varies, depending upon the etiologic organism, and diagnosis usually requires laboratory confirmation. diagnostic features and therapeutic regimens are outlined in table 4 . adolescent females account for a disproportionate number of the reported cases of toxic shock syndrome (tss), despite the fact that most teens do not use intravaginal catamenial products (115, 116) . those that do use these products, however, are at a higher risk for developing tss than women who are older and use the same feminine hygiene products, probably because of the immature anatomy of the adolescent genital tract. therefore, clinicians should provide anticipatory guidance aimed directly at preteen patients and their parents. because the adolescent's genital tract is not completely developed until ovulation occurs regularly, teenagers should be cautioned not to use intravaginal catamenial products until two years after the onset of menarche, except under special circumstances or for brief periods of time (117) . these products also should not be used for overnight protection. if intravaginal catamenial products are used, they should be changed at least every four hours regardless of the degree of menstrual flow, and patients who have experienced symptoms suggestive of tss should not use them for at least four years after the illness and only then after appropriate cultures have demonstrated that the s. aureus is no longer present. aids is relatively uncommon in adolescents. as of june 1985, a total of 59 cases had been reported for individuals between the ages of 10 and 19 (118) . risk factors include homosexual or bisexual practices, intravenous drug abuse, hemophilia, history of prior blood transfusion, and haitian heritage (119) . the clinical characteristics of aids are related to a deficiency in t-iymphocytes caused by infection with human t-celllymphotropic virus (htlv-lli). this deficiency in the immune system makes the host susceptible to a number of opportunistic infections, some of the most prominent being p. earl/ill pneumonia, disseminated infections with mycobacterium avium and iniracellularis, cryptococcal meningitis, c. albicans, cryptosporidium diarrhea, and disseminated herpes infections. malignancies such as kaposi's sarcoma and carcinoma of the rectum are seen in older persons but are rare in adolescents. the diagnosis of aids is based on appropriate laboratory findings, particularly the antibody to htlv-iii. there is no known treatment for the underlying immune defects in aids, and there have been no reports of spontaneous reversal of this condition. sexual preferences and orientation must be addressed honestly with teenage males because of the steady increase in the prevalence of aids in the adult male homosexual population. adolescent males need to be advised of the sexual practices that put them at high risk for this fatal disease, such as oralanal or genital-anal contact. males who have had homosexual experiences should be screened for hepatitis b antibody. when results are negative, patients should be offered the hepatitis bvaccine. using barrier contraception (condom) will prevent many sexually transmitted infec-tions in homosexual, as well as heterosexual, individuals. adolescents, like adults, need to be firmly warned about the dangers of sexually transmitted urogenital infections. in an attempt to limit the incidence and complications of stds, particular attention must be paid to a history of sexual activity and preferences, the menstrual cycle in the female, use of contraception in both males and females, and the coexistence of std syndromes. the importance of careful genital and pelvic examinations in the evaluation of adolescents cannot be overemphasized. finally, treatment of stds is incomplete without appropriate patient follow-up, therapy of partners, and counseling about prevention. the widespread use of vaccines to prevent measles and rubella has resulted in dramatic decreases in the incidence ofboth these illnesses in the united states (120) . the very success of the vaccination program has resulted, paradoxically, in a change in the epidemiology of these diseases. those who care for adolescents need to be aware of these illnesses, their manifestations, and approaches to their prevention. both measles and rubella were widely prevalent infections until the appearance of vaccines in 1963 and 1969, respectively. the vaccines abruptly reduced the incidence of these infections and changed their epidemiology. caused by rna viruses, measles and rubella are spread when droplet nuclei from infected patients are inhaled by susceptible hosts. once the virus is introduced into a susceptible population, the cases multiply quickly, as dramatically demonstrated in recent outbreaks of measles on a number of college campuses (121) . with minimal changes in production technique, the rubella vaccine has remained a highly efficacious, live attenuated vaccine (122) . the measles vaccine, on the other hand, has undergone a number of changes. the original measles vaccine, a killed vaccine, was available from 1963 until 1967. at that time, a live attenuated vaccine was introduced, which took the place of the killed vaccine. individuals who received the original killed vaccine appear to be still susceptible to measles infection and need to be revaccinated with the contemporary vaccine. also, the schedule for administering the vaccine has been altered with the realization that infants vaccinated before 15 months of age have a less reliable response to the vaccine. anyone given the vaccine as an infant needs revaccination to ensure protective antibody titer (123) . once exposed to the measles virus, a susceptible individual will develop illness in 10-14 days. the prodrome consists of fever, cough, coryza, and conjunctivitis. koplik spots-raised blue-gray specks on the buccal mucosa-appear 24 hr before the rash and are characteristic of measles. the rash is seen first on the neck and face; it proceeds downward to involve the whole body. the rash is maculopapular and erythematous. after three to five days, it fades slowly and eventually disappears. in individuals who have received killed measle vaccine, a syndrome characterized by a rash that begins on the extremities, high fever, and pneumonia can occur after exposure to measles (124) . this ill-ness, known as "atypical measles," is probably mediated by a hypersensitivity reaction to the virus in a partially immune host. in rare cases, atypical measles have been documented despite revaccination with live attenuated vaccine in persons previously given the killed vaccine (125). infection with the rubella virus produces a milder illness than that caused by the measles virus. the prodrome, consisting of fever and malaise, is similar to that of measles; mild coryza and conjunctivitis might also occur. the associated rash is less dramatic and, as with measles, it starts on the neck and/or face. the rash usually lasts three to five days. lymphadenopathy, a hallmark of rubella infection, occurs before the onset of the rash and frequently persists after the rash has subsided. the posterior auricular and suboccipital nodes are most likely to increase in size and are often the last to resolve. diagnosis of both of these rash illnesses rests primarily on recognition of the clinical symptoms. serologic confirmation of the presence of type-specific antibodies serves little function in terms of immediate diagnosis but is extremely important from a public health standpoint. both infections can be prevented by appropriate vaccination. vaccine efficacy ranges from 90-95% for both present vaccines (126) . prior to 1972, the recommendation of the advisory council on immunization practices of the ll.s. public health service was to vaccinate at 12 months. therefore, persons born before 1972 should be considered candidates for revaccination against measles. the measles and rubella vaccines can be administered singly, in combination with each other (mr), or with each other and mumps (mmr). systemic reactions, including fever and myalgia, are common but rarely severe. no specific treatment is indicated for clinical infections. although rubella remains a major public health problem because of the threat of congenital rubella syndrome in infants born to mothers who contract rubella in the first trimester of pregnancy, it rarely causes severe illness in adolescents. on the other hand, measles complications, including encephalitis, pneumonia, and subacute sclerosing panencephalitis, can be severe and even fatal in adolescents and young adults (127) . all adolescents born prior to 1972 should be considered candidates for revaccination with measles vaccine. those born before 1969 are candidates for rubella vaccine. health care providers should help ensure that students be excluded from schools unless they can prove that they are appropriately vaccinated or they have proof of prior infection. if measles or rubella is suspected in an adolescent, reassessment of vaccine status is necessary for all close contacts, particularly schoolmates. iii adolescents must be quarantined away from other potentially susceptible adolescents. although adolescents are a generally healthy group, infection can create significant morbidity and occasional mortality. as in all patients, early diagnosis and treatment can shorten the duration of infection 'and limit complications. physicians should provide teenage patients with guidance necessary to prevent transmission and recurrence. current estimates from national health interview survey: united states the national ambulatory medical care survey: united states primary epstein-barr virus infections in children clinical virologic and serologic evidence of epstein-barr virus 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studies of infectious mononucleosis with eb virus a prospective clinical study of the mononucleosis syndrome in a developing country prospective studies of a group of yale university freshmen. i. occurrence of infectious mononucleosis infectious mononucleosis at the united states military academy. a prospective study of a single class over four years differential reactivity of human serums with early antigens induced by epstein-barr virus epstein-barr virus replication in oropharyngeal epithelial cells andrews l-l. prolonged oropharyngeal excretion of epstein-barr virus after infectious mononucleosis the transmission of infectious mononucleosis release of infectious epstein-barr virus by transformed marmoset leukocytes oropharyngeal excretion of epstein-barr virus by patients with lymphoproliferative disorders and by recipients of renal homografts evidence for active epstein-barr virus infection in patients with persistent unexplained illnesses: elevated anti-early antigen antibodies persisting illness and fatigue in adults with evidence of epstein-barr virus infection granulocyte changes .in infectious mononucleosis the hepatitis of infectious mononucleosis: experience with 235 cases the presence of heterophil antibodies in infectious mononucleosis monospot: a differential slide test for infectious mononucleosis heterophil antibody in adults with sore throat epstein-barr virus specific diagnostic tests in infectious mononucleosis treatment of lifethreat ening epstein-barr virus infections with acyclovir threatment of infectious mononucleosis with intravenous acyclovir infectious mononucleosis plicated by hemolytic anemia due to anti-i severe thrombocytopenia in infectious mono hemolytic and other anemias in infectious mononucleosis ruptured spleen in infectious mononucleosis spontaneous rupture of spleen in infectious mononucleosis clinical, virologic, and serologic evidence of epstein-barr virus infection in association with childhood pneumonia mononucleosis and heart disease nervous system involvement in infectious mononucleosis: their heralding and/or major manifestation epstein-barr virus and the x-linked iyrnphoproliferative syndrome antibodies to epstein-barr virus in burkitt's lymphoma and control groups is burkitt's lymphoma related to perinatal infection by epstein-barr virus? antibodies to epstein-barr virus in nasopharyngeal carcinoma, other head and neck neoplasms, and control groups epstein-barr virus antibody in childhood hodgkin's disease central nervous system lymphoma related to epstein-barr virus failure to acquire epstein-barr virus infection after intimate exposure to the virus distribution of antibody to hepatitis a antigen in urban adult populations recent advances in the study of the epidemiology of hepatitis b a large outbreak of hepatitis a in a day care center hepatitis surveillance report 1985. centers for disease control an outbreak of type a hepatitis at the naval training center transmission of hepatitis b in a classroom setting an outbreak of hepatitis iun members of a high school sumo wrestling club infectious hepatitis: stud ies on the effect of gamma globulin on the incidence of inapparent infection the natural history of infectious hepatitis viral hepatitis. type b. studies on natural history and prevention re-examined non-a, non-b hepatitis: etiology and clinical course acute viral hepatitis type b hepatitis after transfusion with blood containing antibody to hepatitis b core antigen randornization of corticosteroid therapy in fulminant hepatitis prophylaxis and prevention of viral hepatitis hepatitis i3 vaccine: demonstration of efficacv in a controlled clinical trial in a high risk population in the~united states type b hepatitis after needle-stick exposure: prevention with hepatitis b immune globulin. a final report of the veteran's administration cooperative study recommendation of the immunization practices advisory committee: recommendations for protection against viral hepatitis fulminant hepatitis: mayo clinic experience with 34 cases teenage pregnancy: the problem that hasn't gone away causes of acute urethal syndrome in women association between diaphragm use and urinary tract infection behavioral factors and urinary tract infection dysuria in adolescent girls: urinary tract infection or vaginitis diagnosis of coliform infection in acutely dysuric women antibody-coated bacteria in the urine of infants and children with their first two urinary tract infections urinary prophylaxis with trimethoprim and trirnethoprim-sulfarnethoxasole. efficacy and influence on the natural history of recurrent bactiuria and cost control single-dose therapy for cystitis in women. a comparison of trimethoprimsulfamethoxazole, arnoxicillin, and cyclacillin symptomatic gonorrhea during pregnancy cervical chlamudia trachomatis and mycoplasmal. infections in pregnancy: epidemiology and outcomes acute salpingitis in the adolescent female incidence, prevalence, and trends of acute pelvic inflammatory disease and its consequences in industrialized countries gonorrhea in female adolescents. potential analogies to toxic shock syndrome barrier method contraceptives and pelvic inflammatory disease criteria for diagndsis and grading of salpingitis tube-ovarian abscess: a retrospective review laparoscopy in the diagnosis and management of patients with suspected salpingo-oohoritis ectopic pregnancy in the united states: 1970 through 1978 reproductive consequences of pelvic inflammatory disease nongonococcal urethritis: a clinical review prostatitis syndromes: pathophysiology and differential diagnosis chlamudia trachomatis infections in men with reiter's syndrome toxic shock syndrome surveillance in the united states epidemiology of toxic shock syndrome young teens and tampons: a crisis in waiting centers for disease control: unpublished data the acquired immunodeficiency syndrome: an update measles and rubella in adolescents and young adults some current issues relating to rubella vaccine need for measles revaccination in adolescents: correlation with birth date prior to 1972 atypical measles in adolescents and young adults failure of attenuated viral vaccine in prevention of atypical measles antibody response following measles-mumps-rubella vaccine under conditions of customary use measles outbreaks on university carnpuscs-s-indiana urogenital disease syndromes in adolescents. i. vaginitis, urethritis, and genital lesions urogenital disease syndromes in adolescents. 11. pelvic pain in the adolescent female std treatment guidelines key: cord-016932-bej10xbf authors: lum, lawrence g.; bollard, catherine m. title: specific adoptive t-cell therapy for viral and fungal infections date: 2018-06-19 journal: management of infections in the immunocompromised host doi: 10.1007/978-3-319-77674-3_20 sha: doc_id: 16932 cord_uid: bej10xbf despite advances in anti-infective agents, viral and fungal infections after hematopoietic stem cell transplantation (hsct) continue to cause life-threatening complications that limit the success of hsct. early adoptive t-cell immunotherapy studies showed that administration of allogeneic virus-specific cytotoxic t lymphocytes (vctl) can prevent and control viral infections and reconstitute antiviral immunity to cytomegalovirus (cmv) and epstein-barr virus (ebv). advances in immunobiology, in vitro culture technology, and current good manufacturing practice (cgmp) have provided opportunities for advancing adoptive cell therapy for viral infections: (1) t cells have been expanded targeting multiple pathogens; (2) vctl production no longer requires viral infection or viral vector transduction of antigen-presenting cells (apcs); (3) the source of lymphocytes is no longer restricted to donors who are immune to the pathogens; (4) naive t cells have been redirected with chimeric antigen receptor t cells (carts) or armed with bispecific antibody-armed t cells (bats) to mediate vctl activity; (5) these technologies could be combined to targeted multiple viral or fungal pathogens; and (6) pathogen-specific t-cell products manufactured from third parties and banked for “off-the-shelf” use post-hsct may soon become a reality. infections remain the leading cause of mortality and morbidity during the first 3 months after hematopoietic stem cell transplantation (hsct) [1] [2] [3] [4] . despite advances in prophylactic viral and fungal therapy to minimize the viral and fungal burden early after hsct, breakthrough viral and fungal infections remain life-threatening, and for some viral and fungal infections, there are no effective therapies [5] [6] [7] [8] [9] . vaccine strategies to induce immunity to cmv began in the 1970s but have been limited in their success [10] [11] [12] . the conditioning regimens for hsct that vary from non-myeloablative to myeloablative create an immunodeficiency that leaves the allogeneic hsct recipient susceptible to viral and fungal infections while immune reconstitution occurs during the first 6-9 months after hsct. immune reconstitution is further abrogated by intensive immunosuppression used to prevent and/or control gvhd. it is clearly established that the kinetics and rate of t-cell reconstitution are critical to controlling viral infections. factors that speed t-cell recovery will decrease the risk of viral infection during the first 3 months after hsct [2, 3, 13] . early studies showed that donor lymphocyte infusions (dli) given before t-cell reconstitution from the stem cell donor were effective for treating viral infections in hsct recipients but were associated with a high risk of gvhd [14] . since the early 1990s, investigators began to develop virus-specific cytotoxic t lymphocyte (vctl) for adoptive immunotherapy against specific targets early during immune reconstitution after hsct [15, 16] . advances in vctl therapy have benefited from (1) advances in understanding of immune responses to conserved t-cell epitopes for various pathogens [17] [18] [19] , (2) technological advances in ex vivo expansion of t cells and advances in the preparation of antigen-presenting cells [20] [21] [22] , and (3) assays that evaluate vctl activity and the mhc restriction of vctl [23, 24] . in this chapter, we review the following areas of how: (1) t cells have been expanded to target multiple pathogens; (2) vctl production no longer requires viral infection or viral vector transduction of antigen-presenting cells (apcs); (3) the source of lymphocytes is no longer restricted to donors who are immune to the pathogens; (4) naive t cells have been redirected with chimeric antigen receptor t cells (carts) to target pathogen-infected cells; (5) bispecific antibody (biab)-armed t cells (bats) can mediate vctl activity; and (6) pathogen-specific t-cell products can be manufactured by third parties and banked for "off-the-shelf" use post-hsct. we summarized the methodological approaches, clinical trials using vctl, promising preclinical studies, and early clinical trials of anti-pathogen ctls that have promise. these advances provide the rationale and impetus for future vctl adoptive immunotherapy. production of vctl as a guiding principle to decrease the risk of gvhd in allogeneic hsct recipients, strategies excluded alloreactive t cells by selecting virus-specific t cells. four major approaches were used: (1) stimulation with viral antigen(s) during ex vivo culture of donor t cells from peripheral blood mononuclear cells (pbmc), (2) direct selection of donor cells, (3) genetic modification of t cells to confer specific recognition of pathogen or pathogen-infected cells, or (4) arming of ex vivo expanded t cells with bispecific antibody to target the viral antigen ( fig. 20.1 ). numerous ex vivo culture approaches have been used to produce cytomegalovirus (cmv)-specific ctl or epstein-barr virus (ebv)-specific ctl [15, 16, [25] [26] [27] [28] [29] [30] . cmv viral-or peptide-specific stimulation in vitro expands single or multiple pathogenspecific vctl. the advantages of culture over cell selection are the generation and expansion of polyclonal vctl to clinically useful quantities of vctl from small amounts of blood [31] . however, the major disadvantages of this strategy is the daunting task of culturing and processing after stimulation to expand the vctl (up to more than 1 month) and the hlahistocompatibility requirement of finding a closely matched donor. during these longer-term cultures, the vctl may lose their capacity to self-renew and to persist in vivo, particularly after prolonged ex vivo culture [32] . it should be noted that clinical trials infusing ex vivo expanded vctl post-hsct showed prolonged persistence [33] and that ex vivo expansion using pathogen-specific stimuli decreased alloreactivity [19] . this may be due to selection of virus-specific clones and deselection of alloreactive clones. one study showed that residual alloreactivity seen in vctl is clinically insignificant [34] . the initial trials of vctl therapy required cmv lysates on apc, cmv-infected fibroblasts, or ebv-lymphoblastoid cells lines as a stimulant for expansion of donor-derived memory t cells [25, 27, 35] . the discovery of dominant and highly conserved antigens such as cmv-pp65 and adenovirus hexon and penton led to replacement of live viral stimulation with either 15-mer peptide pools spanning viral proteins or dna plasmidtransduced antigen-presenting cells [36, 37] . the newer approaches to rapidly expand and manipulate apcs enabled use of a less restricted population of donors and the targeting of an increased number of pathogens in a single culture [20, 38] . in a recent rapid vctl protocol, the addition of il-4 and il-7 leads to production of cd4+ t cells with a th 1 phenotype, whereas il-2 and il-15 tended to favor in vitro natural killer (nk) cell expansion [37] . the ideal population to adoptively transfer may be ex vivo expanded central memory t cells with a cd62l and cd45ra phenotype as these cells have a superior ability to persist in vivo after adoptive transfer [39, 40] . selection via cell capture sorting direct selection relies on cell sorting of immune donor pbmcs, usually after pulsing them with the antigen(s) of interest, to drive expansion of virus-specific t-cell clones [41] . this approach would not be viable for obtaining immune ctls from pathogen-naive donors. multimer selection is achieved by binding of hla-peptide complexes to t-cell receptors (tcrs) of known antigen specificity, followed by purification of bound cells, e.g., by magnetic column separation. alternatively, antiviral t cells expressing interferon-γ (ifn-γ) can be isolated using the gamma capture assay. direct selection methods have the advantage of rapid manufacturing time. unfortunately, these approaches require apheresis of donors in order to collect sufficient cells for sorting and processing for clinical appli-cations and pre-existing and detectable pathogenspecific t cells in the blood. multimer selection is major histocompatibility (mhc)-restricted and selects only cd8+ t cells of a limited specificity. this could possibly allow pathogen evasion and impair persistence of vctl in vivo [42] . earlier studies suggested that persistent binding of multimers to the tcr may impair t-cell function [43] . recent reversible streptamer technology for direct selection may overcome the problem of impaired function [44] . ifn-γ positive selection captures polyclonal antigen-specific cd4+ and cd8+ t cells and selects for a wider range of antigen-specific cells. combining direct selec-tion, culture expansion methods, and cytokine cocktails can optimize the selection of central memory t cells in vctl products and improve yields on targeted cellular phenotypes [37, 44] . tcr or car gene modifications t cells can be modified to redirect their specificity with retroviral and lentiviral vectors to introduce the transgenes for high-affinity tcrs or chimeric antigen receptors (cars) consisting of a single-chain variable fragments (scfvs). high-affinity tcr genes can be cloned and transduced into polyclonal t cells to generate a large population of (1) blood is obtained from donors (autologous, allogeneic, or umbilical cord blood) or is drawn or apheresis is performed to obtain a larger quantity of blood; (2) pbmcs are processed via: (a) cell selection panel using multimers with a pathogen-derived peptide associated with a type-i hla molecule or column selection after in vitro stimulation of t cells with antigens followed by binding of ifnɣ or cd154-expressing t cells with antibody-coated immu-nomagnetic beads; (b) cell expansion by stimulating the pbmc with apcs produced by antigenic peptide pools, viral transduction, or nucleofection; (c) genetic modification that involves the transfer of high-affinity pathogenspecific tcrs or cars to redirect the specificity of the t cells; and (d) polyclonal expansion of t cells for 8-14 days and arming with biabs directed at the pathogen of interest on one hand and the tcr on the other hand; (3) quality control and release testing; and (4) infusion into patients tcr pathogen-specific ctls [45] . a similar strategy was used to produce tumor-specific t cells after tcr gene transfer [18] . in contrast, cars have an extracellular region that consists of a scfv that binds to antigen, with an intracellular signaling complex composed of tcr zeta chain for first-generation cars, the tcr zeta chain and the cd28 for second-generation cars, and tcr zeta and cd28 or 41bb for third-generation cars [46] [47] [48] . the high-affinity tcr-transduced ctls have been used to target cmv-infected cells [49] , hpv-infected cells [50] , hepatitis b-infected cells [51] , hepatitis c-infected cells [52] , tuberculosis-infected cells [53] , sarsinfected cells [54] , chlamydia-infected cells [55] , and hiv-infected cells [56] . car t cells were used to target cd4 in hiv-infected cells [57] [58] [59] [60] and for recognition of β-glucans in fungi [61] . ex vivo ctl expansion is the most common method for producing clinical ctls for most clinical trials ( [25] . cmv has been the primary focus of the first virus targeted therapy trials and remains a primary focus in subsequent studies (table 20 .1). the first clinical report in which cd8 + cmvspecific ctls were isolated via tetramer selection [62] generated complete or partial clinical responses in nine patients, but there was limited data on long-term persistence of the infused cmv-specific ctls. ifn-γ column selection (gamma capture, miltenyi) to produce cmv-ctls was associated with partial and complete responses in 15 of 18 patients who were given one dose of cmv-ctls [63] . ifn-γ selection after stimulation with recombinant pp65 or an overlapping peptide pool of 15-mers covering the pp65 protein was used to produce cmv-ctl [64] . infusions of cmv-ctls administered prophylactically after stem cell transplantation successfully protected seven patients from the development of viral reactivation and disease. further, in vivo expansion of cmv-ctls was detected in 11 patients [64] . cmv-ctls from hsct donors using reversible streptamers with mhc-restricted pp65 peptides were used to successfully treat two patients with cmv reactivation after hct [44] . the strategy for using bispecific antibodies (biabs) to target cancer was nearly abandoned due to cytokine storm reactions. however, the last 10 years has seen a resurrection of interest particularly for targeting t cells to various cancer antigens. studies using retargeted t cells have been reported for her2 in breast and prostate cancer using anti-cd3 x anti-her2 biab atc [89, 90] ; egfr in colorectal, pancreatic, and lung cancer using anti-cd3 x anti-egfr biab atc [91] ; and cd20 in non-hodgkin's lymphoma using anti-cd3 x anti-cd20 biab atc [92] [93] [94] . since chemical or molecularly engineered constructs could be used to target the tcr on one hand and tumor-associated antigen (taa) on the other hand, we reasoned that cmv could be targeted by chemically heteroconjugating okt3 (anti-cd3, anti-tcr) with cytogam® (polyclonal donor-derived anti-cmv igg, designated cmvbi) to kill cmv-infected fibroblasts [95] . in this strategy shown in fig. 20 .1, anti-cd3 monoclonal antibody-activated t cells (atc) which expanded in low-dose il-2 were the t effector cells. atc alone do not kill cmvinfected targets. arming doses of cmvbi ranging from as low as 0.01 ng/10 6 atc to 50 ng/10 6 atc exhibited high levels of specific anti-cmv cytotoxicity in targets infected with cmv at multiplicities of cmv infection (moi) ranging from 0.01 to 1. the polyclonal nature of the cytogam may provide multiple antibody clones directed at multiple cmv epitopes on the cmv-infected targets leading to the increased potency at a low arming dose of cmvbi. cytotoxicity was evident at effector-to-target ratios (e:t) of 25:1, 13:1, 6:1, and 3:1 compared to unarmed atc alone. at an moi of 1.0, the mean % specific anti-cmv-specific cytotoxicities at e:t of 3, 6, and 13 were 79%, 81%, and 82%, respectively, whereas unarmed atc at the same e:ts killed <20%. unarmed atc, cytogam®, or cmvbi alone did not exhibit significant killing of uninfected or cmv-infected fibroblasts. furthermore, cultures of cmvbiarmed atc with cmv-infected targets induced cytokine and chemokine release from cmvbiarmed atc. this simple targeting strategy bypasses mhc-restricted cytotoxicity for treating viral disease in organ transplant and hsct recipients. it was shown that cmvbi atc do not react to alloantigens in vitro in a mixed lymphocyte culture, and they can be frozen and reinfused at different time points as an "off-the-shelf" drug. although promising, it is not clear from these data whether targeting cmv or other disease agents using this approach will be clinically effective. ebv-ctls have been used for prevention and treatment of post-hsct lymphoproliferative disease (ptld) as well as ebv + lymphoma. irradiated ebv-lymphoblastoid cells (ebv-lcl) were used to generate ebv-specific ctls in vitro for prophylaxis or treatment for ebv-ptld in 114 patients [27, 33] . remarkably, the first 26 patients received gene-marked ctls, and followup studies showed the gene-marked cells persisted up to 105 months after hsct (table 20 .1). hla-a2-specific pentamers and ifn-γ selection procedures were used to produce ebv-ctls. hla-a2 specific pentamers were used to produce ebv-ctls from the haploidentical mother of a patient with ebv-ptld who had received a cord blood transplantation [69] . a complete clinical response was obtained following two infusions of ebv-ctls. three of six patients with early ebv-induced ptld treated with ebv-ctls produced by ifn-γ selection achieved complete responses whereas three patients with advanced, multiorgan disease did not respond [70] . the latest strategy is to target ebv with multiviral ctl products (below) or third-party-derived ebv-ctls. most studies targeting adenovirus (adv) use multiviral ctls [21, 81, 83, 84] . a few exclusively target adv by selection technology. adv-ctls produced by ifn-γ selection was used for treatment of nine patients with drug-refractory adv infections [74] . there was in vivo ctl expansion in five of six patients and four patients cleared their disease. in all studies using cell selection, clinical benefit was observed in spite of very low doses of vctls infused (<5 × 10 4 cells/kg in most studies) [73, 74] . recent antiviral ctl therapy trials target multiple viruses (cmv, ebv, and adv as primary targets). cmv, ebv, and adv are the three leading causes of viral-associated mortality after allogeneic hsct. clinical-grade adv vector ad5f35pp65 contains the immunodominant cmv antigen pp65, providing a unique opportunity to transduce donor-derived dendritic cells or ebv-lcl to serve as apcs for the ctl cultures. triviral (cmv, ebv, and adv-specific) ctls were tested in a dose-escalation trial involving 26 patients [83] . there were no adverse effects at doses ranging from 5 × 10 6 to 1 × 10 8 cells/m 2 , and all patients were effectively protected against cmv, ebv, and adv. interestingly, although ebv-and cmv-specific ctls were detected by ifn-γ elispots, adv-ctls were not detectable except during infection. in a follow-up trial using ad5f35-transduced ebv-lcl to produce ebvand adv-ctls, 13 patients received prophylaxis or treatment for ebv and adv infections after hsct [21] . although the ctls provided protection in vivo, the adv-ctls could not be detected except in the setting of adv infection; these data suggest that levels of specific vctls below the limits of detection by ifn-γ elispots provide protection and infection induces clonal expansion. similarly, ad5f35pp65 transduced dendritic cells (dc) used to produce cmv-and adv-ctls were clinically effective in 12 patients after allogeneic hsct [84] . there were a few cases of cmv reactivation in the setting of lowdose prednisone. this approach was applied to 50 patients after allogeneic hsct with triviral (cmv, ebv, adv-specific) ctls using two methods: 10 were produced by pulsing donor dcs with the hla-a2 restricted cmv peptide nlvpmvatv and 40 were produced using ad5f35pp65-transduced donor dcs [87] . only 5 of 50 patients had cmv reactivation after ctl infusions and only 1 of 5 patients required antiviral drug therapy after steroid treatment for acute gvhd. advances in processing protocols have validated 5-mer peptide pools that include immunodominant viral antigens that replace viral transduction of apc thereby removing safety and regulatory barriers associated use of viral vectors [36] . the use of gas-permeable rapid-expansion (g-rex) bioreactors has simplified ctl culture [96] . these advances in technology led to the development of a rapid manufacturing protocol for expanding virus-specific t-cell products (vsts) that yield clinically relevant numbers of vsts in 10-12 days. further, vst products targeting multiple viral antigens have been shown to provide effective antiviral protection (against cnv, ebv and ad) in ten patients after hsct [37] . this rapid manufacturing protocol was subsequently adapted to produce five virus-specific ctls targeting ebv, cmv, adv, hhv6, and bk virus infections in a single t-cell product for patients following allogeneic hsct [88] . fourteen of 48 vst products manufactured from hsct donors recognized all 5 viral components while 35 (73%) recognized 3 or more by ifn-γ elispots. unexpectedly 22 of the donors were cmv seronegative and vsts produced predictably lacked cmv specificity. these vsts were used to treat 11 patients after hsct. the 3 patients treated prophylactically remained free of viral infections and 8 patients with 18 viral reactivations received vsts, with all experiencing partial or complete responses in their cmv, ebv, adv, or hhv6 infections. although there was intense interest in the use of ctl therapy for hiv, there was only limited success to date [97] . attempts to expand and reinfuse autologous hiv-specific ctls resulted in only transient improvements in viral load [75] . a larger number of clinical trials focused on genetically modified ctl to target hiv using transduction of a modified tcr or cars. these trials established safety, but exhibited limited antiviral efficacy [76, 77] . a major challenge for this approach is the outgrowth of escape mutants expressing alterations of the target epitope so the infected cell can no longer be targeted by the effector cells. a more successful approach has been inserting genes that would provide hiv resistance. this approach was clinically tested when antisense gene complementary to hiv env was transduced into t cells from 17 patients using lentiviral vectors [78] . the ctls persisted for 5 weeks, homed to gut-associated lymphoid tissue, and were well-tolerated with clinical toxicities. infusions of ctls in two of eight patients who underwent antiviral treatment interruption keep the viral load u ndetectable for 4 and 14 weeks. when ccr5-delta32 mutations were introduced to cd4enriched t cells through the use of a zinc-finger nuclease [79] , the ccr5-edited t cells were subsequently infused in 12 patients, and engineered t cells were detectable in the peripheral blood for up to 42 months post infusion. in six patients who underwent antiviral treatment interruption, the absolute number of gene modified cd4+ t cells decreased at a lower rate than non-modified t cells. recent studies showed that dual gene editing of cxcr4 and ccr5 via zinc-finger nucleases was successful in a t-cell line, and preclinical studies show that the t cells were highly resistant to hiv infection [98] . it is not clear whether this approach could prevent primary infection or have a clinical impact as an hiv cure strategy. there are a few studies that target other viruses with adoptive immunotherapy. the john cunningham virus (jcv) is a ubiquitous polyoma virus which can cause progressive multifocal leukoencephalopathy (pml), which occurs in immunocompromised individuals such as acquired immunodeficiency syndrome (aids), recipients of hsct or solid organ transplants, or primary immunodeficiency disorders. donorderived jcv-specific ctls were used in a 14-year-old patient with pml after prolonged steroid treatment for gvhd following hsct. cells were manufactured using 15-mer peptide pools that included jc antigens vp1 and lt and infused twice leading to clearance of jv-dna from the cerebrospinal fluid with improvements in neurologic status [80] . human papillomavirus (hpv) disease can be a late complication of hsct. peptide pools spanning the hpv e6 and e7 proteins were used to generate hpv-specific ctls from patients with oropharyngeal or cervical cancer that arise after hpv16 infection [99] . the ctls exhibited specific activity directed at hpv e6 and e7 and antitumor activity against the hpv16 cervical cancer cell line caski. adverse events after 381 infusions for 180 patients on 18 protocols by the groups at baylor college of medicine were reported [100] . side effects were limited to 24 mild adverse events observed within 6 h of infusion; nausea and vomiting were most common with 22 nonserious adverse events (fever, chills, nausea) that occurred within 24 h. no significant gvhd was attributed to ctl infusions. the only significant complications were rare reports of systemic inflammatory responses in patients with bulky ebv+ lymphomas following ebv-ctl therapy. seven cases of acute gvhd occurred in patients who had a greater degree of hla mismatch than controls after infusions of ebv-ctl. some of the cases of gvhd were attributable to reducing the corticosteroid dose prior to the cmv-ctl infusions [87] . for years, the selection or culture of anti-pathogen ctls was dependent on the presence of pathogen-specific memory t cells in the blood of donors, and, therefore, the approach could not help allograft recipients of pathogen-naive hematopoietic cell products after hsct. one strategy to address this problem is to provide "off-theshelf" pathogen-specific ctls derived from third-party donors. this strategy was first validated in a phase i trial involving 8 patients who received partially matched ebv-ctls for ptld that developed after solid organ transplantation [66, 67] and confirmed in a cohort of 33 patients in a phase ii trial [68] . the latter trial showed a response rate of 64% at 5 weeks and 52% at 6 months; the outcomes correlated with the degree of hla matching between the ctl donor and recipient. in the hsct patients, two patients with refractory ebv-ptld after cord blood transplantation (cbt) with third-party ebv-specific ctls [71] . a bank of 32 ctl lines with characterized activity against ebv, cmv, and adv were used to match for 50 patients with refractory viral infections. this strategy resulted in partial or complete antiviral responses in 74%, 78%, and 67% of those with cmv, adv, and ebv, respectively [85] . this is a marked improvement from standard therapy response rate of 13% in eight patients for whom a matched line could not be identified. despite partial hla matching at one to four loci, there were only two patients who developed grade i gvhd. clones that are responsible for gvhd have been selected against in the expansion culture and may exist at such low precursor frequencies after culture that they do not expand enough to cause clinically significant gvhd. the lower rate of response against ebv relative to cmv and adv may reflect selective expansion of t cells against immunodominant epitopes of the latter two viruses, thereby complicating the selection of an ideal third-party pathogen-specific line that fulfills the requirements of antiviral activity and mhc-restriction against multiple pathogens. the methods for producing third-party-virus-specific ctl include pentamer selection for ad, cmv, ebv [81] , and ifn-γ selection for adv-ctl [73] . a few studies reported transducing ctls with a virus-specific tcr [49, 101, 102] . a trial of transgenic ctls using a retroviral vector that expresses a cmv-specific tcr is ongoing in the united kingdom (morris e. et al. mrc# g0701703). alternatively, kumaresan et al. transduced t cells with the β-glucan receptor dectin [61] . since the carbohydrate β-glucan is found in the cell wall of most fungi [103] , investigators used its natural receptor, dectin-1, as a recognition receptor coupled to a cd28 (a key co-stimulatory molecule) and cd3-zeta transgene to initiate signaling and killing in t cells. the same group showed that the antifungal carts could mediate damage to hyphae in vitro and in vivo [61] . these novel approaches would allow creation of specific ctls from pathogennaive donors; however, they are subject to the regulatory challenges in gene transfer technology. furthermore, use of a single antifungal tcr allows for antigenic escape. a major advance in adoptive viral ctl therapy was development of virus-specific ctls from virus-naive donors. ctl could be produced from a 20% fraction from cord blood using donorderived dcs and ebv-lymphoblastoid cell line (lcl) as apc and ad5f35pp65 transduction as a source of cmv and adv antigens [20] . the resulting viral ctls exhibited specific anti-cmv, ebv, and adv ifn-γ elispots responses as well as specific 51 cr cytotoxicity with no alloreactivity. epitope mapping showed that the immunodominant epitopes recognized by cord blood-derived ctls were different from the immunodominant epitopes recognized by the cmv and ebv seropositive adult donors. the hla-a2-restricted epitope nlvpmvatv was notably absent in the cord blood-derived lines. ctls derived from cord blood were successfully infused in 12 cbt recipients in the ongoing act-cat trial (safety, toxicity and mtd of one intravenous iv injection of donor ctls specific for cmv and adenovirus, # nct00880789). recently, multiviral ctls were produced from cmv-naive adult donors using columnselected cd45ra+ naive t cells stimulated by donor dcs pulsed with cmv 15-mer peptide pools [38] . preclinical studies suggest that multiviral ctls will exhibit similar anti-cmv activity to dcs pulsed with cmv 15-mer peptide pools. ebv-ptld is a significant long-term risk in solid organ transplant recipients. rituximab can be effective, but treatment often requires reduction of immunosuppression which can lead to graft rejection. autologous ebv-ctls have been used in this setting [72] . several prophylactic infusions of autologous ebv-ctls reduced the ebv viral load without adverse reactions despite ongoing treatment with calcineurin inhibitors [65] . a heart transplant recipient who developed hodgkin's lymphoma-type ptld 8 years after transplant had remission after being treated with autologous ebv-ctls in combination with chemotherapy without alterations in his immunosuppression [72] . this observation supports the prior observations that calcineurin inhibitors block proliferation, but do not impair ctl activity. fungal infections are a major cause of morbidity and mortality in allogeneic hsct recipients, with gvhd being the major risk factor. candidal infections can range from mucocutaneous colonization of the skin and mouth to life-threatening systemic infections. aspergillus species are ubiquitous molds that cause invasive pulmonary infections as well as widespread infection including central nervous system dissemination in highly immunocompromised patients [104] . patients with inherited immunodeficiencies (e.g., chronic granulomatous disease), patients with prolonged neutropenia after repeated rounds of chemotherapy (e.g., for acute leukemia), and those receiving immunosuppression after lung transplant or allogeneic hsct are at the highest risk for mycoses [105] . the importance of t-cell immunity in defense against invasive aspergillosis and other filamentous fungi is not clear, since patients with these invasive fungal diseases usually have severe deficiencies in multiple components of the immune system. in patients with advanced aids, invasive aspergillosis is an uncommon complication and generally occurs when other forms of immune impairment (e.g., neutropenia and use of corticosteroids) are present. despite these unknowns, it may be clinically useful to target fungal infections with fungusspecific t cells after hsct. the adaptive immune response against invasive aspergillosis is believed to be orchestrated by cd4+ t cells. table 20 .2 summarizes preclinical studies that developed fungal-specific ctls against candida, aspergillus, and rhizopus (a member of the mucorales group) species. aspergillus-specific ctls were produced by stimulation of pbmc with antigens from aspergillus extracts, selection with ifn-γ secretion, and culture [106] . the ctls were predominantly cd4+, cd45r0+ memory cells that secrete ifn-ɣ in response to aspergillus and penicillium. the fungal-specific ctl enhanced hyphal damage by neutrophils and apcs. ifn-ɣ selection and stimulation with candida albicans, aspergillus fumigatus, and rhizopus oryzae extracts were used to produce multifungalspecific ctl lines, which were also nearly all cd4+ cd45ro+ hla-dr+ that exhibited activation markers of ifn-ɣ, cd154, and tnfα and enhanced oxidative activity of neutrophils when co-incubated with antigen and apcs [108] . several studies target the candida mp65 and aspergillus crf1 antigens. to produce multipathogen-specific t cells that secrete ifn-ɣ, proliferate, and kill cmv, ebv, adv, candida, and aspergillus, donor pbmcs were incubated with peptide libraries from cmv-pp65, ebv-lmp2, adv-hexon, candida mp65, and a 15-mer peptide from aspergillus crf1 [107] . however, it remains unclear what the significance of mp65 and crf1 is in antifungal immunity [117] [113] . expanded memory/effector th1 cells following stimulation with rhizopus extracts were used to generate memory/effector th1 cells for mucormycosis, and the product exhibited specificity to the original rhizopus oryzae extract as well as other mucorales species [118] . candida-specific t cells generated with cellular extracts of candida albicans released cytokines that caused hyphal damage and increased neutrophil activity against hyphae [111] . ctls produced by stimulation with inactivated conidia (spores) from aspergillus fumigatus resulted in clonal cd4+ ctls with anti-aspergillus activity by ifn-ɣ elispots [82] . these donor t-cell clones specific for aspergillus antigens were then infused in patients following haploidentical hsct. of 23 patients who developed invasive aspergillosis, 10 patients received anti-aspergillus ctls, while 13 patients did not. nine of 10 treated patients cleared their infections whereas only 7 of 13 untreated patients cleared their infections. aspergillus-specific ctls were detected in high frequencies in patients who received immunotherapy while they were barely detectable in untreated patients [82] . despite notable advances in antifungal ctls, a better understanding of the immunodominant t-cell targets that should be selected for various fungal species is needed, and standardized clinical-grade cgmp fungal antigen sources are needed to provide consistency between trials. although there have been major advances in producing pathogen-specific ctls, important questions remain regarding methods that affect potency and efficacy of the t-cell products. it is unclear whether manufacturing ctls to include more pathogens in a single culture will affect potency and specificity in the ctl cultures. although the proportions of virus-specific ctls for each virus decrease as the number of antigens increases, these effects have not seemed to impact clinical trials. ctls specific for 7 viruses (cmv, ebv, adv, bk, hhv6, rsv, and influenza) pro-duced using peptide pools for 15 antigens exhibited specific activity against all targeted viruses [37] . the question remains as to whether adding additional viral targets will skew specific cytotoxicity, alter potency for each target, induce alloreactive t cells, or compromise in vivo responses. a major challenge is achieving consistent and optimal culture conditions for generating the most effective ctl product. although multiple rounds of stimulation with antigen select and expand the specific antiviral clones, prolonged culture may lead to t-cell exhaustion. some groups have decreased production time using newer bioreactors [96] . identification of the "correct" subset of t cells for clinical use (however selected) will require well-designed randomized phase ii trials using a specific ctl product made by the same group or a common standard operating procedure (sop) in a homogeneous group of hsct patients. assays for measuring ifn-ɣ elispots and cytotoxicity need to be standardized and the timing of the studies needs to be the same. recently, a new population of healthy donors jolink [113] transgenic tcr-transduced cells tuberculosis murine cells feng [114] mhc-streptamer-enriched antigen-specific t cells listeria murine cells stemberger [115] (proof of principle) -targeting of ova-expressing parasites murine cells polley [116] transgenic trc-transduced cells chlamydia murine cells roan [55] "stem cell memory t cells" has been putatively identified -which possess characteristics ideal for use in adoptive immunotherapy. unfortunately, there are no randomized phase ii trials to date to support continued development and commercialization of clinically effective ctls. the presence of immunosuppression remains a barrier for optimal immunotherapy after allogeneic hsct and solid organ transplantation since most agents also suppress ctl functions. nearly all protocols require recipients to be receiving less than 0.5 mg/kg/day of prednisone and wait at least 30 days after anti-t-cell serotherapy to be eligible to receive ctl therapy. virtually all of the calcineurin inhibitors (cyclosporin a, tacrolimus, or sirolimus) at therapeutic doses impair ctl activity. ebv-specific ctls can be made resistant to tacrolimus by knockdown of fkbp12 via a retrovirally transduced specific sirna and exhibit anti-ebv lymphoma activity in the presence of tacrolimus [119] . similarly, ebv-specific ctls can be made resistant to both cyclosporine a and tacrolimus by mutating calcineurin [120] . the mutation does not alter the phenotype or antiviral activity of the ctls and mutated cells have a growth advantage in calcineurin inhibitors. although they have not been applied clinically, they have great potential for treating hsct and solid organ transplant recipients. there is one preclinical report of t cells used to target bacterial and parasitic infections [116] , but there are no clinical trials evaluating t-cell immunotherapy for bacterial and parasitic infections. despite numerous studies evaluating in vitro t-cell responses, there is no consensus on the role of t cells in defense against aspergillosis. infusions of anti-pathogen ctls in several hundred patients over the past several decades have been established as a safe and highly effective therapy following allogeneic hct. identifying preserved viral t-cell epitopes, probing the antigen limits in ctl monoculture, testing the clinical efficacy of immunosuppressive-resistant ctls, and improving conditions for rapid and specific expansion will further broaden the usefulness of this treatment strategy. as advances in protocols and methods for manufacture achieve acceptable clinical standards that can be supported commercially, ctl therapy may become an integral component of care offered to allogeneic hsct or immunodeficiency patients. late cytomegalovirus disease and mortality in recipients of allogeneic hematopoietic stem cell transplants: importance of viral load and t-cell immunity marked increased risk of epstein-barr virus-related complications with the addition of antithymocyte globulin to a nonmyeloablative conditioning prior to unrelated umbilical cord blood 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ebv can be expanded from naive t-cell populations in cord blood and will target a range of viral epitopes cytotoxic t lymphocyte therapy with donor t cells prevents and treats adenovirus and epstein-barr virus infections after haploidentical and matched unrelated stem cell transplantation large-scale expansion of dendritic cell-primed polyclonal human cytotoxic t-lymphocyte lines using lymphoblastoid cell lines for adoptive immunotherapy analysis of cd8 t cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides expansion of t cells targeting multiple antigens of cytomegalovirus, epstein-barr virus and adenovirus to provide broad antiviral specificity after stem cell transplantation reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of t-cell clones from the donor a phase i-ii trial to examine the toxicity of cmv-and ebv-specific cytotoxic t lymphocytes when used for prophylaxis against ebv and cmv disease in recipients of cd34-selected/t cell-depleted stem cell transplants infusion of cytotoxic t cells for the prevention and treatment of epstein-barr virus-induced lymphoma in allogeneic transplant recipients administration of neomycin-resistance-genemarked ebv-specific cytotoxic t lymphocytes to recipients of mismatched-related or phenotypically similar unrelated donor marrow grafts immunotherapy targeting ebv-expressing lymphoproliferative diseases autologous epstein-barr virus (ebv)-specific cytotoxic t cells for the treatment of persistent active ebv infection adoptive immunotherapy for posttransplantation viral infections acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred cd8+ t cells long-term outcome of ebv-specific t-cell infusions to prevent or treat ebv-related lymphoproliferative disease in transplant recipients allogeneic virus-specific t cells with hla alloreactivity do not produce gvhd in human subjects adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virusspecific t-cell lines generation of cmv-specific t lymphocytes using protein-spanning pools of pp65-derived overlapping pentadecapeptides for adoptive immunotherapy rapidly generated multivirus-specific cytotoxic t lymphocytes for the prophylaxis and treatment of viral infections naïve t-cell-derived ctl recognize atypical epitopes of cmvpp65 with higher avidity than cmvseropositive donor-derived ctl -a basis for treatment of post-transplant viral infection by adoptive transfer of t-cells from virus-naïve donors adoptive transfer of effector cd8+ t cells derived from central memory cells establishes persistent t cell memory in primates molecular signatures distinguish human central memory from effector memory cd8 t cell subsets the role of virus-specific adoptive t-cell therapy in hematopoietic transplantation evasion of cd8+ t cells is critical for 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genetically engineered cd4+ and cd8+ t cells expressing tcrs specific for a m. tuberculosis 38-kda antigen engineering t cells specific for a dominant severe acute respiratory syndrome coronavirus cd8 t cell epitope antigen-specific cd8+ t cells respond to chlamydia trachomatis in the genital mucosa reconstitution of anti-hiv effector functions of primary human cd8 t lymphocytes by transfer of hiv-specific alphabeta tcr genes t-cell engineering by a chimeric t-cell receptor with antibody-type specificity for the hiv-1 gp120 anti-hiv designer t cells progressively eradicate a latently infected cell line by sequentially inducing hiv reactivation then killing the newly gp120-positive cells characterization of t cell-expressed chimeric receptors with antibodytype specificity for the cd4 binding site of hiv-1 gp120 lentiviral vectors encoding human immunodeficiency virus type 1 (hiv-1)-specific t-cell receptor genes efficiently convert peripheral blood cd8 t lymphocytes into cytotoxic t lymphocytes with potent in vitro and in vivo hiv-1-specific inhibitory activity bioengineering t cells to target carbohydrate to treat opportunistic fungal infection adoptive transfer of cytomegalovirus-specific ctl to stem cell transplant patients after selection by hlapeptide tetramers adoptive transfer of pp65-specific t cells for the treatment of chemorefractory cytomegalovirus disease or reactivation after haploidentical and matched unrelated stem cell transplantation directly selected cytomegalovirus-reactive donor t cells confer rapid and safe systemic reconstitution of virus-specific immunity following stem cell transplantation reconstitution of ebv-specific t cell immunity in solid organ transplant recipients complete regression of posttransplant lymphoproliferative disease using partially hla-matched epstein barr virus-specific cytotoxic t cells treatment of epstein-barr-virus-positive post-transplantation lymphoproliferative disease with partly hlamatched allogeneic cytotoxic t cells allogeneic cytotoxic t-cell therapy for ebv-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial a novel haploidentical adoptive ctl therapy as a treatment for ebv-associated lymphoma after stem cell transplantation effective and long-term control of ebv ptld after transfer of peptide-selected t cells successful treatment of ebv-associated posttransplantation lymphoma after cord blood transplantation using third-party ebv-specific cytotoxic t lymphocytes successful treatment of a classic hodgkin lymphoma-type posttransplant lymphoproliferative disorder with tailored chemotherapy and epstein-barr virus-specific cytotoxic t lymphocytes in a pediatric heart transplant recipient thirdparty virus-specific t cells eradicate adenoviraemia but trigger bystander graft-versus-host disease safe adoptive transfer of virus-specific t-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation safety of autologous, ex vivo-expanded human immunodeficiency virus (hiv)-specific cytotoxic t-lymphocyte infusion in hiv-infected patients prolonged survival and tissue trafficking following adoptive transfer of cd4zeta gene-modified autologous cd4(+) and cd8(+) t cells in human immunodeficiency virus-infected subjects a phase ii randomized study of hiv-specific t-cell gene therapy in subjects with undetectable plasma viremia on combination antiretroviral therapy antiviral effects of autologous cd4 t cells genetically modified with a conditionally replicating lentiviral vector expressing long antisense to hiv gene editing of ccr5 in autologous cd4 t cells of persons infected with hiv polyomavirus jc-targeted t-cell therapy for progressive multiple leukoencephalopathy in a hematopoietic cell transplantation recipient rapid salvage treatment with virus-specific t cells for therapyresistant disease transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation monoculturederived t lymphocytes specific for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals prophylactic infusion of cytomegalovirusspecific cytotoxic t lymphocytes stimulated with ad5f35pp65 gene-modified dendritic cells after allogeneic hemopoietic stem cell transplantation multicenter study of banked third-party virusspecific t cells to treat severe viral infections after hematopoietic stem cell transplantation safety and clinical efficacy of rapidly-generated trivirus-directed t cells as treatment for adenovirus, ebv, and cmv infections after allogeneic hematopoietic stem cell transplant donor-derived cmv-specific t cells reduce the requirement for cmv-directed pharmacotherapy after allogeneic stem cell transplantation activity of broad-spectrum t cells as treatment for adv, ebv, cmv, bkv, and hhv6 infections after hsct targeted t-cell therapy in stage iv breast cancer: a phase i clinical trial phase i study of anti-cd3 x anti-her2 bispecific antibody in metastatic castrate resistance prostate cancer patients anti-cd3 x anti-egfr bispecific antibody redirects t cell cytolytic activity to egfr-positive cancers in vitro and in an animal model t cells armed with anti-cd3 x anti-cd20 bispecific antibody enhance killing of cd20+ malignant b-cells and bypass complement-mediated rituximab-resistance in vitro cd20-targeted t cells after stem cell transplantation for high risk and refractory non-hodgkin's lymphoma multiple infusions of cd20-targeted t cells and low-dose il-2 after sct for high-risk non-hodgkin's lymphoma: a pilot study targeting cytomegalovirus-infected cells using t cells armed with anti-cd3× anti-cmv bispecific antibody accelerated production of antigen-specific t cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (g-rex) t-cell therapies for hiv simultaneous zinc-finger nuclease editing of the hiv coreceptors ccr5 and cxcr4 protects cd4+ t cells from hiv-1 infection human papillomavirus type 16 e6/e7-specific cytotoxic t lymphocytes for adoptive immunotherapy of hpv-associated malignancies adverse events following infusion of t cells for adoptive immunotherapy: a 10-year experience human mhc class i-restricted high avidity cd4 t cells generated by co-transfer of tcr and cd8 mediate efficient tumor rejection in vivo cord blood t cells retain early differentiation phenotype suitable for immunotherapy after tcr gene transfer to confer ebv specificity beta-glucan recognition by the innate immune system immunity to fungal infections current challenges in the diagnosis and management of invasive fungal infections: report from the 15th international symposium on infections in the immunocompromised host generation of highly purified and functionally active human th1 cells against aspergillus fumigatus generation of a multipathogen-specific t-cell product for adoptive immunotherapy based on activation-dependent expression of cd154 clinicalscale generation of multi-specific anti-fungal t cells targeting candida, aspergillus and mucormycetes clinical-scale generation of human anti-aspergillus t cells for adoptive immunotherapy robust polyfunctional t-helper 1 responses to multiple fungal antigens from a cell population generated using an environmental strain of aspergillus fumigatus generation and characterization of anti-candida t cells as potential immunotherapy in patients with candida infection after allogeneic hematopoietic stem-cell transplant cross-protective th1 immunity against aspergillus fumigatus and candida albicans characterization of the t-cell-mediated immune response against the aspergillus fumigatus proteins crf1 and catalase 1 in healthy individuals cd4+ and cd8+ t cells mediate adoptive immunity to aerosol infection of mycobacterium bovis bacillus calmette-guerin lowest numbers of primary cd8(+) t cells can reconstitute protective immunity upon adoptive immunotherapy adoptive immunotherapy against experimental visceral leishmaniasis with cd8+ t cells requires the presence of cognate antigen biochemical and immunological characterization of mp65, a major mannoprotein antigen of the opportunistic human pathogen candida albicans characterization of the cellular immune responses to rhizopus oryzae with potential impact on immunotherapeutic strategies in hematopoietic stem cell transplantation generation of epstein-barr virus-specific cytotoxic t lymphocytes resistant to the immunosuppressive drug tacrolimus (fk506) generation of ebv-specific cytotoxic t cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease acknowledgments special thanks to the clinical coordinators for dedicating their efforts to serve the immunotherapy patients. we thank manley huang for his thoughtful reading of the chapter. the studies were supported in part by r01 ca140314 (lgl) and r01 ca182526 (lgl), translational grants #6092-09 (lgl) and #6066-06 (lgl) from the leukemia & lymphoma society, and uva cancer center support grant nci 5p30ca044579-24.lgl is a founder of transtarget, inc. cmb is supported in part by the nichd k12-hd-001399 award to mdk and cprit r01 rp100469 and nci p01 ca148600-02 awards to cmb. key: cord-023747-mvq6353a authors: ascherio, alberto; munger, kassandra l. title: epidemiology of multiple sclerosis: environmental factors date: 2009-12-25 journal: nan doi: 10.1016/b978-1-4160-6068-0.00004-8 sha: doc_id: 23747 cord_uid: mvq6353a this chapter discusses the environmental factors associated to epidemiology of multiple sclerosis. the epidemiologic evidence points to three environ­mental risk factors—infection with the epstein-barr virus (ebv), low levels of vitamin d, and cigarette smoking—whose association with multiple sclerosis (ms) seems to satisfy in varying degrees most of the criteria that support causality, including temporality, strength, consis­tency, biologic gradient, and plausibility. none of these associations, however, has been tested experimentally in humans and only one––vitamin d deficiency is presently amenable to experimental interventions. the evidence, albeit more sparse and inconsistent, linking other environmental factors to ms risk are summarized. epidemiologic clues to the hypothetical role of infection in ms are com­plex and often seem to point in opposite directions. the ecological studies, database/linkage analyses, and longitudinal studies of sunlight exposure and vitamin d are reviewed. biologic mechanisms for smoking and increased risk of ms could be neuro­toxic, immunomodulatory, vascular, or they could involve increased frequency and duration of respiratory infections. some other possible risk factors include––diet and hepatitis b vaccine. data. such has been the case, for example, with interventions to reduce lung cancer incidence by reducing exposure to tobacco smoke. as discussed in this chapter, epidemiologic evidence points to three environmental risk factors-infection with the epstein-barr virus (ebv), low levels of vitamin d, and cigarette smoking-whose association with multiple sclerosis (ms) seems to satisfy in varying degrees most of the criteria that support causality, including temporality (i.e., the cause must precede the effect), strength, consistency, biologic gradient, and plausibility. none of these associations, however, has been tested experimentally in humans, and only one (vitamin d deficiency) is presently amenable to experimental interventions. this chapter also summarizes the evidence, albeit more sparse and inconsistent, linking other environmental factors to ms risk. for many years, it appeared that the "who, where, and when" of ms epidemiology was well understood. however, some aspects of ms epidemiology may be changing, notably the observations of an attenuation of the latitude gradient 3, 4 and the increasing female-to-male ratio. 5 in this section, we discuss the "classic" view of ms epidemiology, some of which has been known for more than 50 years, and then some recent developments that may provide new clues to the etiology of ms. ms is the most common neurologic disease in young adults. incidence rates are low in childhood and adolescence (<6/100,000/year) high in the middle to late twenties and early thirties (11 to 18/100,000/year in high-risk populations), and gradually decline thereafter, with rates less than 9/100,000/year among those older than 45 years of age. 3, 6 women are approximately twice as likely as men to develop ms, 7, 8 and the lifetime risk among white women is about 1 in 200. 3, 9 ms exhibits a worldwide latitude gradient, with high prevalence and incidence in northern europe, 7 canada, 10,11 the northern united states, 3, 12, 13 and southern australia 14 and decreasing prevalence and incidence in regions closer to the equator. 15 exceptions to the latitude gradient exist and include a lower than expected prevalence in japan 16 and higher than expected prevalence and incidence in the mediterranean islands of sardinia and sicily. 7 kurtzke 17 summarized the early descriptive studies by depicting areas of high (≥30/100,000), medium (5 to 29/100,000), and low (<5/100,000) prevalence of ms; we have updated his figures with more recent prevalence estimates 7, 16, [18] [19] [20] [21] [22] [23] [24] (fig. 4-1) . a more comprehensive review of ms incidence and prevalence worldwide was published in 2005. 18 it is important to note that differences in estimated incidence across countries or time periods can result from differences in study design, case ascertainment, or diagnostic criteria, rather than from real changes in disease occurrence. differences in prevalence are even more difficult to interpret, because they may reflect increased survival or earlier diagnosis, both of which can occur even if the incidence is the same. 25 in spite of these limitations, the collective data do support a higher risk of ms at higher latitudes, both north and south of the equator. the existence of the latitude gradient alone is not enough to support an environmental component, because it could be explained by genetic differences. 18, 26 however, studies of ms incidence and prevalence among migrant populations also support a role for environmental factors. these studies have limitations, in that migrants may be different from nonmigrants in socioeconomic and health status, may not utilize local health care resources, and therefore they may be less likely to be diagnosed; in addition, enumeration of the immigrant population for disease statistics may be difficult or impossible. 25, 27 nevertheless, migrant studies on ms collectively support a decreased prevalence of ms among those who migrate from high-to low-risk areas, particularly if the migration occurs before 15 years of age. 27 moreover, one study found a decreased prevalence of ms in all age groups among immigrants from europe to australia, suggesting that the protective effect may extend into adulthood as well. 28 studies within the united states have also supported a decreased risk of ms among migrants from northern (>41° to 42° n), australia and new zealand europe figure 4-1 worldwide prevalence estimates of multiple sclerosis. blue, more than 90 cases per 100,000 population; purple, 60 to 89/100,000; green, 30 to 59/100,000; orange, 5 to 29/100,000; yellow, fewer than 5/100,000; white, insufficient data. an asterisk indicates that data for that region or country are older and should be interpreted cautiously. high-risk parts of the country to southern (<37° n), low-risk regions. 29, 30 the study of u.s. veterans 30 is particularly compelling because of its large sample size and rigorous design. in this study, kurtzke observed that individuals who were born in the northern united states but migrated to the southern part of the country before joining the military had a 50% reduced risk of ms compared with those who did not migrate ( fig. 4-2) . fewer studies have been conducted among migrants from low-to high-risk areas. in general, these studies have found that a low risk of ms is retained after migration, but that the offspring of migrants have a higher risk of ms, similar to that in the host country. 27, [31] [32] [33] [34] [35] in the u.s. veterans study, 30 individuals who were born in the southern part of the country and migrated to northern states before entering the military had a 20% increased risk of ms, and those migrating from the middle tier of states to northern regions had a 31% increased risk (see fig. 4 -2). more recently, in a study conducted in the french west indies (a low-risk area), an increased risk of ms was found among individuals who had moved to france (a high-risk area) and then returned to the west indies. the increase in risk was greatest among those who migrated to france before the age of 15 years. 36 the incidence of ms appears to have been relatively stable over the past 50 years in several high-risk areas, including denmark 6 and the northern united states, 37 but there is some evidence that ms may be increasing in japan 16 and in parts of southern europe, most notably in sardinia. 38 interestingly, the island of malta has continued to experience low, stable rates of ms 39 despite its proximity to sardinia and sicily and a high frequency of the ms-associated hla-drb1*1501 allele. 40 there is also evidence of an increased female-to-male ratio in ms incidence. in canada, the female-to-male ratio apparently has increased from approximately 2:1 among individuals born in the 1930s and 1940s to approximately 3:1 among those born in the 1970s. 5 this change is strongly correlated with, and could be at least in part explained by, a sharp increase in the female-to-male ratio in smoking behavior (unpublished data), because smoking is a strong risk factor for ms (see later discussion). an attenuation of the latitude gradient was observed independently in a population of u.s. nurses 3 and in u.s. military veterans. 4 among nurses born between 1920 and 1946 and among veterans of world war ii or the korean conflict, those living in the northern tier of states (>41° to 42° n) had a greater than threefold increased risk of ms compared to those in the southern tier (<37° n). among vietnam and gulf war veterans, however, this gradient was attenuated to less than twofold, and among nurses born between 1947 and 1964 it completely disappeared ( fig. 4-3) . because the methods used to determine rates of ms in the early and later cohorts were the same, and because the individuals in the cohorts had similar socioeconomic status 3 or access to health care, 4 this attenuation was unlikely to be due to artifact. a change of this magnitude over such a short period of time argues for an environmental, rather than a genetic, explanation of the latitude gradient; as discussed later, this environmental factor may involve changes in patterns of infection or sun exposure, or both. further, the attenuation was probably caused by an increase in ms incidence in the southern united states, because incidence rates in the northern states, based at least on data from the longitudinal study in olmsted county, minnesota, seem to have remained relatively stable. 37 an attenuation of the latitude gradient in europe has also been observed; however, no systematic studies have assessed this gradient within the same population over time, and the attenuation therefore may be due to improved study methodology and case ascertainment, particularly within the united kingdom. 18 the possibility of an infectious cause was considered early in ms history, and numerous viruses and bacteria were, at different times, implicated as likely etiologic agents. the results of early studies, based on microscopic examination of pathologic material and attempts to transmit the disease to animals, often were null or spuriously positive because of contamination and could not be replicated. later, numerous serologic studies were conducted, often demonstrating significantly elevated antibody titers against several viruses in ms patients compared with healthy controls, but these differences were probably an epiphenomenon of the immune activation rather than being of etiologic significance. 41 in part as a consequence of these investigations, many researchers became skeptical about the existence of an infectious agent causing ms, and this skepticism persists today. epidemiologic clues to the hypothetical role of infection in ms are complex and often seem to point in opposite directions. on the one hand, results of family studies, including investigations of half-siblings, adopted children, and spouses of individuals with ms, support a strong genetic component as the leading explanation of ms clustering within families and provide little evidence of person-to-person transmission. 42 on the other, there are well-documented, albeit controversial, 43 reports of epidemics of ms, most notably in the faroe islands, 44 that are most easily explained by the introduction and transmission of an infectious agent. to reconcile these findings, it has been postulated that ms is a rare complication of a common infection, with the disease occurring in genetically or otherwise predisposed individuals. in this scenario, the epidemics would be a consequence of the introduction of the ms-causing agent for the first time in remote, previously naïve populations. 45 two hypotheses as to the nature of this infection have been proposed: (1) the responsible microorganism is more common in areas of high ms prevalence (the "prevalence" hypothesis), and (2) the ms-causing agent is ubiquitous and more easily transmitted in areas of low ms prevalence, where infection occurs predominantly in infancy, when it would be less harmful and more likely to confer protective immunity. the latter proposal is called the "poliomyelitis" hypothesis, by analogy with the epidemiology of poliomyelitis before vaccination. 46 the poliomyelitis hypothesis is also consistent with the higher prevalence of ms in communities with better hygiene, 47 in individuals with higher education, 48, 49 and in those with late age at infection with common viruses, 50 as well as the general lack of increase in ms incidence among individuals migrating from low-to high-prevalence areas. 27 however, the poliomyelitis hypothesis cannot explain the reduced risk of ms among migrants from high-to low-risk areas and, in fact, would predict an increase in ms risk in this circumstance, whereas the prevalence hypothesis is consistent with the observations. failure to identify a specific microbe as the cause of ms, despite evidence that is consistent with some role for infection in at least modulating ms risk, has strengthened support for a third, more general, "hygiene" hypothesis, according to which exposure to multiple infections in childhood primes the immune responses later in life toward a less inflammatory and a less autoimmunogenic profile. 51 the hygiene hypothesis can explain all the features of ms epidemiology that are explained by the original formulation of the poliomyelitis hypothesis. in addition, the protective effect of migration from high-to low-ms areas, which is paradoxical under the poliomyelitis hypothesis, could be beneficial because of increased exposure of migrants to parasitic and other infections in the low-risk area. at the population level, prevalence of ms is positively correlated with high levels of hygiene, as measured, for example, by prevalence of intestinal parasites. 52 the improving hygienic conditions in southern europe in the last few decades could explain the increased prevalence of ms reported in multiple surveys (although whether there was a true increase in ms incidence remains unsettled). 7 it is also interesting that infection with intestinal helminths, which is highly prevalent in developing countries, had been reported to cause an immune deviation with attenuation of helper t-cell 1 cellular immune responses and remission of ms. 53 finally, the hygiene hypothesis provides a convincing explanation for the observations that infectious mononucleosis (im) is associated with an increased risk of ms (relative risk [rr] = 2.3; p < .00000001) 54 and that the epidemiology of im is strikingly similar to that of ms (table 4 -1). 55 because im is common in individuals who are first infected with ebv in adolescence or adulthood 56 but rare when ebv infection occurs in childhood, it is a strong marker of age at ebv infection, which is itself strongly correlated with socioeconomic development across populations and with socioeconomic status within populations. 57 an exception to this pattern is seen in asia, where ebv infection occurs uniformly early in life and im is thus rare. it is noteworthy that the incidence of ms remains relatively low in asian countries, including japan, despite the fast industrialization and reduction of infectious diseases, 58 although there is evidence that the incidence may be increasing in japan. 16 according to the hygiene hypothesis, the association between im and ms risk does not reflect a causal effect of ebv but rather the indirect manifestation of a common cause; that is, both ms and im are the result of high hygiene and a resulting low burden of infection during childhood. an important prediction of this hypothesis is that ms risk will be high among individuals reared in a highly hygienic environment, even if they do not happen to be infected with ebv later in life, whereas, if ebv has a causal role in ms, individuals who are not infected with ebv would have a low risk of ms. 59 the data on this point are unequivocal: individuals who are not infected with ebv, even though they have the same hygienic upbringing as those with im, have an extremely low risk of ms (odds ratio [or] from metaanalysis = 0.06; p < .00000001) ( table 4 -2). the contrast could not be sharper or more consistent: ms risk among individuals who are not infected with ebv is at least 10-fold lower than that of individuals who are ebv-positive, and 20-fold lower than that of individuals with a history of im. 59 because studies in pediatric ms 60,61 rule out a common genetic resistance to ms and ebv infection, 59 we can conclude either that ebv itself or some other factor closely related to ebv is a strong causal risk factor for ms or that ms itself strongly predisposes to ebv infection. temporality is the only truly necessary criteria for causality. the association between ebv infection and ms is strong and consistent across multiple studies in different populations, and there is to some extent a biologic gradient (higher risk associated with severity of infection, as indicated by history of im). until recently, all studies on ms and infection used a cross-sectional design and could not completely rule out the possibility that ebv infection was a consequence rather than a cause of ms. however, the results of four longitudinal serologic studies have now been published (table 4-3) . [62] [63] [64] [65] the most consistent finding across these studies is that, among individuals who will develop ms, there is an elevation of serum antibodies against the ebv nuclear antigen 1 (ebna1) that precedes the onset of ms symptoms by many years. the presence of anti-ebna1 antibodies is a marker of past infection with ebv, because titers typically rise only weeks after the acute infection. further, there is no evidence in clinical studies of acute primary ebv infection in individuals with ms. 66 taken together, these results indicate that ms is a consequence rather than a cause of ebv infection. until recently, ebv had not been found in ms lesions, 67, 68 and therefore the link between ebv and ms was postulated to be mediated by indirect mechanisms. the leading hypothesis was that the immune response to ebv infection in genetically susceptible individuals cross-reacts with myelin antigens (molecular mimicry). the discovery that ms patients have an increased frequency and broadened specificity of cd4-positive t cells recognizing ebna1 69 and the identification of two ebv peptides (one of which is from ebna1) as targets of the immune response in the cerebrospinal fluid of ms patients 70 provided support to the molecular mimicry theory. other proposed hypotheses included the activation of superantigens, 71 an increased expression of alpha b-crystallin, 72 and infection of autoreactive b lymphocytes. 73 however, in a recent, rigorous pathologic study, 74 large numbers of ebvinfected b cells were found in the brain of most of ms patients. these cells were more numerous in areas with active inflammatory infiltrates, where cytotoxic cd8-positive t cells displaying an activated phenotype were seen contiguous to the ebv-infected cells. alone, these pathologic findings provide only suggestive evidence for a causal role of ebv in ms, because the infiltration of ebv-infected b cells could be secondary to the inflammatory process that is the hallmark of ms, but their convergence with the epidemiologic evidence described earlier 59 is so striking that noncausal explanations become improbable. however, independent replication of these findings is needed before any conclusion can be drawn. the strong increase in ms risk after ebv infection and (if confirmed) the presence of ebv in ms lesions suggest that antiviral drugs or a vaccine against ebv could contribute to ms treatment and prevention. although antiviral drugs have been tried in the past for ms treatment with borderline results, 75-77 none of the treatment regimens used was sufficiently effective against latent ebv infection. several aspects of ms epidemiology cannot be explained by ebv infection, indicating that other factors must contribute. 59 genes are clearly important, and it is of interest that the association between anti-ebna1 titers and ms risk has been found in both hla-drb1*1501-positive and hla-drb1*1501-negative individuals. 78 variations in ebv strains could also play a role, although evidence in support of this hypothesis remains limited. 79, 80 many other infectious agents have been hypothesized to be related to ms, mostly because of pathologic studies or their role in animal models. recent candidates include chlamydia pneumoniae, 81-84 human herpesvirus 6, 85-87 retroviruses, 88, 89 and coronaviruses, 90 but there are no convincing epidemiologic studies linking these infections to ms risk. noninfectious factors may also be important, and prominent among them are vitamin d and cigarette smoking. one of the strongest correlates of latitude is the duration and intensity of sunlight, which in ecologic studies is inversely correlated with ms prevalence. [92] [93] [94] because exposure to sunlight is for most people the major source of vitamin d, 95 average levels of vitamin d also display a strong latitude gradient. ultraviolet b (uv-b) radiation (290 to 320 nm) converts cutaneous 7-dehydrocholesterol to previtamin d 3 . previtamin d 3 spontaneously isomerizes to vitamin d 3 , which is then hydroxylated to 25(oh)d 3 (25-hydroxyvitamin d 3 ), the main circulating form of the vitamin, and then to 1,25(oh) 2 d 3 (1,25-dihydroxyvitamin d 3 ), the biologically active hormone. 95 however, during the winter months at latitudes greater than 42° n (e.g., boston, ma), even prolonged sun exposure is insufficient to generate vitamin d, 96 and levels decline. 97, 98 use of supplements or high consumption of fatty fish (a good source of vitamin d) or vitamin d-fortified foods (mostly milk in the united states) may partially compensate for this decline, but few people consume large enough amounts of vitamin d, and seasonal deficiency is common. a link between vitamin d deficiency and ms was proposed more than 30 years ago as a possible explanation of the latitude gradient and of the lower prevalence of ms in fishing communities with high levels of fish intake 99 ; however, the immunomodulatory effects of vitamin d were not known, and the hypothesis did not generate much interest at the time. after the discovery that the vitamin d receptor is expressed in several cells in the immune system and is a potent immunomodulator, 100 a series of experiments revealed a protective role of 1,25(oh) 2 d 3 in several autoimmune conditions and in transplant rejection. 100 the effects in experimental autoimmune encephalomyelitis, an animal model of ms, were particularly striking: injection of 1,25(oh) 2 d 3 was found to completely prevent the clinical and pathologic signs of disease, 101,102 whereas vitamin d deficiency accelerated the disease onset. 102, 103 with vitamin d deficiency becoming a biologically plausible risk factor for ms, several epidemiologic studies were conducted to determine whether exposure to sunlight or vitamin d intake is associated with ms risk. the main results of these studies are shown in table 4 -4, and their strengths and limitations are discussed in the following paragraphs. as mentioned earlier, the results of ecologic studies support an inverse association between sunlight exposure and ms risk. however, because people living in the same area share many characteristics other than the level of sunlight, the consensus is that evidence from these studies is weak. in an exploratory investigation based on death certificates, working outdoors was associated with a significantly lower ms mortality in areas of high, but not low, sunlight. 104 in a separate study in the united kingdom, the skin cancer rate, a marker of sunlight exposure, was found to be about 50% lower than expected among individuals with ms (p = .03). 106 although the results of these investigations are consistent with a protective effect of uv light exposure, they could also represent "reverse causation" (i.e., individuals with ms could reduce their exposure to sunlight after disease onset). the results of case-control studies comparing history of sun exposure in childhood (presumed to be a critical period, mostly from the results of studies in migrants) between ms cases and controls have been conflicting. the results of one study were contrary to a protective effect of vitamin d, 106 and no association between sun exposure in childhood and ms risk was found in another. 107 in contrast, results consistent with a protective effect of sun exposure were reported in a study in tasmania in which information on time spent in the sun was complemented by measurement of skin actinic damage, a biomarker of uv light exposure, 108 as well as an investigation in norway 109 and a study of monozygotic twins in the united states. 110 in the norway study, an inverse association was also found between consumption of fish and ms risk. selection and recall biases are potential problems in case-control studies, but recall bias cannot explain the inverse association observed in tasmania with actinic damage, 108 and selection bias is unlikely in the twin study. the strongest evidence relating vitamin d levels to ms risk has been provided by two longitudinal studies, one based on assessment of dietary vitamin d intake, and one on serum levels of 25(oh)d. the relation between vitamin d intake and ms risk was studied in more than 200,000 women in the nurses' health study and nurses' health study ii cohorts. 111 dietary vitamin d intake was assessed from comprehensive and previously validated semiquantitative food frequency questionnaires administered every 4 years during the follow-up of the cohorts. 112, 113 total vitamin d intake at baseline was inversely associated with risk of ms: the age-adjusted pooled relative risk (rr) comparing the highest with the lowest quintile of consumption was 0.67 (95% confidence interval [ci], 0.40 to 1.12; p for trend = .03). intake of 400 iu/day of vitamin d from supplements only was associated with a 40% lower risk of ms. these rrs did not materially change after further adjustment for pack-years of smoking and latitude at birth. confounding by other micronutrients cannot be excluded, but adjustments for them in the analyses did not change the results. because dietary vitamin d is only one component contributing to total vitamin d status (the other being sun exposure), a determination of whether serum levels of vitamin d are associated with ms risk in healthy individuals would strengthen the evidence in favor of a causal role for vitamin d. the serum level of 25(oh)d is a marker of vitamin d status and bioavailability; therefore, if vitamin d is protective, high serum levels of 25(oh)d would be expected to predict a lower risk of ms in healthy individuals. this question was recently addressed in a collaborative, prospective case-control study using the department of defense serum repository (dodsr). 114 the study included 257 military personnel with confirmed ms and at least two serum samples collected before the onset of ms symptoms. risk of ms was 51% lower among white individuals with 25(oh)d levels of 100 nmol/l or higher, compared with those levels lower than 75 nmol/l, and the reduction in ms risk associated with 25(oh)d levels � 100 nmol/l compared with those levels < 100nmol/l was considerably stronger before the age of 20 years (16 to 19 years) than at ages 20 or older. an important question concerning vitamin d and ms is the age intervals during which vitamin d may be important. the results of migration studies suggest that more pronounced changes in ms risk are likely to occur among individuals who migrate in childhood. the age of 15 years, chosen as an arbitrary cutoff point in early studies, is usually quoted in the literature, but the reality is that data are insufficient to identify a meaningful threshold above which migration would not alter ms risk, 27 and in at least one study a reduction in risk was also observed among individuals who migrated as adults. 115 the results of the casecontrol study in tasmania suggest that exposure to sunlight is mostly protective in childhood. 108 further, vitamin d exposure in utero has been proposed as a possible explanation for the peak in ms incidence among individuals born in may (whose mothers were not pregnant during the summer, when uv light levels are higher) and the dip among those born in november, according to recent data from canada and sweden. 116 on the other hand, the results of the longitudinal studies support a protective effect of vitamin d also later in life. both the lower risk of ms among women taking vitamin d supplements 111 and the lower risk among men and women with higher levels of 25(oh)d 114 would be difficult to explain by a protective effect of vitamin d solely in utero or during childhood. therefore, it seems likely that, if vitamin d effectively protects against ms, levels during early adult life are also important. overall, the epidemiologic evidence of a causal association between vitamin d and ms is strong but not compelling, mainly because there are few studies based on prospective measurement of levels of exposure to sunlight, vitamin d intake, or serum 25(oh)d concentration. however, the public health implications of a possible causal association are enormous. if vitamin d reduces the risk of ms, supplementation in adolescents and young adults could be used effectively for prevention. based on studies among individuals with low sun exposure, supplements providing between 1000 and 4000 iu/day of vitamin d would increase serum 25(oh)d to the optimal levels. [117] [118] [119] [120] there is an urgent need to conduct further longitudinal studies, preferably in a large, randomized controlled clinical trial assessing whether vitamin d supplementation in the general population prevents ms. the trial would have to be very large, because ms is a rare disease, but the sample size could be reduced by oversampling individuals who are at high risk, such as those with first-degree relatives who have ms. alternative study designs might include national or multinational studies based on randomization of school districts or other suitable units. cigarette smoking was found to increase the risk of ms in some 121, 122 but not all 123, 124 case-control studies. a cross-sectional survey of the general population in hordaland county, norway, found an increased risk of ms in ever-smokers compared with never-smokers (rr = 1.8; 95% ci, 1.1 to 2.9). 125 four prospective studies on smoking and ms have been conducted. among 17,000 british women in the oxford family planning association study, those who smoked 15 or more cigarettes per day were compared with never-smokers and had an 80% increased risk of ms (rr = 1.8; 95% ci, 0.8 to 3.6). 126 a total of 46,000 women from across the united kingdom were enrolled in the royal college of general practitioners' oral contraception study, which found that women smoking 15 or more cigarettes per day had a 40% increased risk of ms (rr = 1.4; 95% ci, 0.9 to 2.2), compared with never-smokers. 127 the nurses' health study and nurses' health study ii cohorts included more than 200,000 u.s. women; those who smoked 25 or more pack-years had a 70% increased risk (rr = 1.7; 95% ci, 1.2 to 2.4; p < .01) compared with never-smokers. 128 in a prospective case-control study in the general practice research database, which included both men and women, ever-smokers had a 30% increased risk of ms, compared with never-smokers (rr = 1.3; 95% ci, 1.0 to 1.7). 129 the suggestion of an increased risk of ms among smokers was consistent across all four studies, and pooled estimates of the relative risk were highly statistically significant when never-smokers were compared with past and current smokers (fig. 4 -4a) or with moderate and heavy smokers (see fig. 4 -4b). additional support for a role of smoking includes a twofold increase in risk of pediatric ms among children exposed to parental smoking 130 and an increased risk of transition to secondary progressive ms among individuals with relapsing-remitting ms 129 ; however, the latter finding was not confirmed in a recent investigation. 131 biologic mechanisms for smoking and increased risk of ms could be neurotoxic, 132 immunomodulatory, 133, 134 or vascular (i.e., increased permeability of the blood-brain barrier), or they could involve increased frequency and duration of respiratory infections, 135 which may then contribute to increased ms risk. smoking also appears to increase the risk of other autoimmune diseases, including rheumatoid arthritis [136] [137] [138] [139] [140] and systemic lupus erythematosus, 141 arguing for a more general effect of cigarette smoking on autoimmunity. although several foods or nutrients were found to be related to be ms risk in ecologic or case-control studies, the results overall were inconsistent and unconvincing. in ecologic studies, positive correlations were found between ms and intake of animal fat [142] [143] [144] and saturated fat, 144 as well as consumption of meat, 145 milk, and butter, 143, 146 and inverse correlations were found with intake of fat from fish 143, 145 and nuts 143 (sources of polyunsaturated fat). an increased risk of ms with increasing animal or saturated fat intake and a protective effect of increasing polyunsaturated fat intake were also reported in a case-control study, 147 but otherwise the results of case-control studies have largely not supported an association between increased ms risk and milk or meat consumption, 121, [147] [148] [149] [150] or decreased risk and consumption of sources of polyunsaturated fat such as fish or nuts. 121, 147 however, in a recent study in norway, 109 fish consumption 3 or more times per week among individuals living at latitudes between 66° and 71° n was inversely related to ms risk. other results have included an inverse association of risk with intake of vitamin c and juice, 147 but no association with other antioxidant vitamins 147 or with fruits and vegetables 121, 123, 147, 151 has been reported. it is important to note that ecologic studies are prone to be confounding and in general provide only very weak evidence of the potential effects of diet on disease risk. retrospective case-control studies are also prone to bias due to both control selection and differential recall. the latter effect is particularly problematic, because even a modest difference in diet recall between cases and controls can cause a large bias in relative risk estimates. 152 this problem is compounded in ms by changes in diet that may occur in the early clinical or preclinical phases of the disease. therefore, although these studies have been important in drawing attention to several aspects of diet as potentially important risk factors for ms, their results, whether in favor or against an hypothetical association, should be interpreted extremely cautiously. understanding of the relation between diet and ms will require the conduct of large longitudinal investigations, with repeated assessment of diet using rigorous and validated methods and possibly measurements of biomarkers of nutrient intakes. so far, the only prospective studies of diet and ms were those conducted among women in the two nurses' health study cohorts. in this population, neither animal fat nor saturated fat was associated with ms risk, but there was a suggestion of an inverse association with intake of the n-3 polyunsaturated fat linolenic acid. 153 there were also no significant associations between intakes of dairy products, fish, meat, 153 vitamins c or e, carotenoids, or fruits and vegetables and ms risk. 154 however, participants in these studies were already 25 to 55 years of age at time of recruitment, and therefore they shed little light on the possible effect of diet earlier in life and ms risk. studies have also examined whether intake of polyunsaturated fats affects ms progression. n-3 polyunsaturated fat supplementation in doses ranging from 2.85 to 3.90 g/day administered for periods of 6 to 24 months did not have significant effects on disability levels in two randomized controlled trials that included a total of 339 patients with relapsing-remitting ms, 155, 156 although trends were in favor of the supplemented groups in both studies. results of three randomized controlled trials examining the effects of n-6 polyunsaturated fat supplementation (17 to 20 g/day for 24 to 30 months) on ms progression, including a total of 279 patients with relapsing-remitting ms, [157] [158] [159] and a meta-analysis of these studies 160 suggested that supplementation may reduce the severity and duration of relapses. in summary, there is no compelling evidence that dietary factors other than vitamin d play a causal role in ms, but neither can such a role be excluded, particularly for diet during adolescence or childhood, which may be important periods in the etiology of ms. estrogen has been hypothesized to protect against ms, because in high levels it appears to promote the non-inflammatory type 2 immune response, rather than the pro-inflammatory type 1 response predominately seen in ms, and because during pregnancy, when estrogen levels are high, women with ms experience fewer relapses than during the puerperium. 161 in prospective studies, 126, 127, 162, 163 neither oral contraceptive use, parity, nor age at first birth 162 was associated with ms risk. a decreased risk of ms during pregnancy followed by an increased risk during the first 6 months after delivery was shown in a study based on a general practice database in the united kingdom. 163 in the same study, recent use of oral contraceptives was also associated with a reduced risk. 163 collectively, these studies suggest that short-term exposure to estrogen may be protective against ms, but that this protection is transient. concerns that the hepatitis b vaccine may increase the risk of ms were raised after widespread administration of the vaccine in france, 164 but the results of most studies have not supported a causal association. studies in the united states conducted among subjects included in a health care database, 165 among nurses, 166 and among participants in three health maintenance organizations 167 found no association between hepatitis b vaccination and risk of ms. further, in studies of children and adolescents, no association was found between hepatitis b vaccination and ms risk 168 or risk of conversion to ms among children with a first demyelinating event. 170 however, a case-control study 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a review altered antibody pattern to epstein-barr virus but not to other herpesviruses in multiple sclerosis: a population based case-control study from western norway altered prevalence and reactivity of anti-epstein-barr virus antibodies in patients with multiple sclerosis a role of late epstein-barr virus infection in multiple sclerosis exposure to infant siblings during early life and risk of multiple sclerosis key: cord-323854-l8gfr19i authors: putz, austin m; schwab, clint r; sewell, alysta d; holtkamp, derald j; zimmerman, jeffery j; baker, kimberlee; serão, nick v l; dekkers, jack c m title: the effect of a porcine reproductive and respiratory syndrome outbreak on genetic parameters and reaction norms for reproductive performance in pigs date: 2018-12-27 journal: journal of animal science doi: 10.1093/jas/sky485 sha: doc_id: 323854 cord_uid: l8gfr19i the objective of this study was to estimate genetic parameters of antibody response and reproductive traits after exposure to porcine reproductive and respiratory syndrome virus. blood samples were taken approximately 60 d after the outbreak. antibody levels were quantified as the sample-to-positive ratio (s/p ratio) using a fluorescent microsphere assay. reproductive traits included total number born (tnb), number born alive (nba), number stillborn (nsb), number mummified (nbm), and number born dead (nbd). mortality traits were log transformed for genetic analyses. data were split into prior, during, and after the disease outbreak phases using visual appraisal of the estimates of farm-year-week effects for each reproductive trait. for nba, data from all phases were combined into a reaction norm analysis with regression on estimates of farm-year-week effects for nba. heritability for s/p ratio was estimated at 0.17 ± 0.05. heritability estimates for reproduction traits were all low and were lower during the outbreak for nba but greater for mortality traits. tnb was not greatly affected during the outbreak, as many sows that farrowed during the outbreak were mated prior to the outbreak. heritability for tnb decreased from 0.13 (prior) to 0.08 (after). genetic correlation estimates between prior to and during the outbreak were high for tnb (0.86 ± 0.23) and nba (0.98 ± 0.38) but lower for mortality traits: 0.65 ± 0.43, −0.42 ± 0.55, and 0.29 ± 1.39 for lnsb, lnbm, and lnbd, respectively. tnb prior to and after the outbreak had a lower genetic correlation (0.32 ± 0.33). in general, genetic correlation estimates of s/p ratio with reproductive performance during the outbreak were below 0.20 in absolute value, except for lnsb (−0.73 ± 0.29). based on the reaction norm model, estimates of genetic correlations between the intercept and slope terms ranged from 0.24 ± 0.50 to 0.54 ± 0.35 depending on the parameterization used, indicating that selection for the intercept may result in indirect selection for steeper slopes, and thus, less resilient animals. in general, estimates of genetic correlations between farm-year-week effect classes based on the reaction norm model resembled estimates of genetic correlations from the multivariate analysis. overall, compared to previous studies, antibody s/p ratios showed a lower heritability (0.17 ± 0.05) and low genetic correlations with reproductive performance during a porcine reproductive and respiratory syndrome outbreak, except for the lnsb. porcine reproductive and respiratory syndrome virus (prrsv) severely affects both the breeding and growing sectors of the swine industry. in the breeding sector, prrs causes abortions, stillborns, mummified piglets, pre-weaning mortality, and embryonic death . it was estimated that $302 million (~45%) out of the annual $663 million in costs associated with problems caused by prrs are due to losses in the breeding sector (holtkamp et al., 2013) . this was very different than estimates from a previous study by neumann et al. (2005) , where only ~12% was due to breeding herd losses. a relatively large amount of work has been done on the growing pig sector to reduce the economic impact of prrs (lunney et al., 2011) and less attention has been focused on reducing the effects of the disease in breeding herds . one reason is that, prior to the availability of high-density genotyping, genetic analyses required a pedigree, which is typically not available for sows at the commercial level due to pooling of semen. because nucleus and multiplier herds are managed to maximize biosecurity and minimize the risk of exposure to major pathogens such as prrs, studies on outbreaks in herds with pedigreed sows are rare. although pedigrees are not required with genomics, genotyping is still relatively expensive, and it still requires high-quality data, which is typically collected in nucleus herds. it is also much more expensive to set up experimental infection trials for reproductive traits in sows, as done in the prrs host genomics consortium nursery pig trials (lunney et al., 2011) . breeding for increased resistance to prrs is difficult in growing pigs and the problem becomes even more difficult for reproductive performance. antibody level in sows following an outbreak with a prrsv could be used as an indicator trait for selection. serão et al. (2014) demonstrated that antibody level measured as sample-to-positive (s/p) ratio from a commercial idexx elisa analysis of blood samples taken after a prrs infection was highly heritable (0.45) and had moderate-to-strong genetic correlations with many reproduction traits during the outbreak (~0.7 in absolute value for several traits). since antibody levels under a real challenge may be impractical for commercial breeding programs, serão et al. (2016) suggested that antibody following vaccination with a modified live virus (mlv) could be used as an indicator trait to select for reproductive performance under prrs (madapong et al., 2017) . to the best of our knowledge, serão et al. (2014) is the only study that has investigated genetic relationships between antibody level and reproductive performance under a prrsv challenge. therefore, it is necessary to validate these findings in a larger, independent prrsv outbreak study. to date, multivariate and reaction norm models are the two main methods that have been used for analysis of disease outbreak data. lewis et al. (2009) first split reproductive data from a commercial herd that experienced a prrs outbreak into healthy and prrs phases and found that splitting the data based on trait rolling averages was better than using diagnostic lab confirmation dates. estimated genetic correlations of reproductive performance between healthy and prrs phases ranged from −0.13 to 0.98, although many genetic correlations were moderate or low between phases (lewis et al., 2009) . lewis et al. (2009) only separated traits into two phases, while it is known that prrs is a persistent infection (wills et al., 2003; lunney et al., 2016) , suggesting that the post-outbreak phase may need to be considered as a separate phase, creating three phases (prior, during, and after the outbreak). in addition, estimates of genetic correlations between reproductive traits within phase may also give some insight into how disease changes the relationship among traits during the different phases of an outbreak. reaction norm models are a common way to model genotype-by-environment interactions (g × e) but they have only more recently been utilized for litter size in pigs. reaction norm models are an application of random regression (longitudinal) models that regress the response variable on a continuous environmental variable. these reaction norm models yield estimates of breeding values for an intercept term that is highly correlated to estimates of breeding values from routine genetic evaluations (knap and su, 2008) and estimated breeding values for a slope term that describes the additive genetic sensitivity to changes in the environment (when using an additive genetic relationship matrix). reaction norm models have been used to analyze disease outbreak data by regressing phenotypes on estimates of continuous farm-year-week effects or on an index of challenge load from multiple traits rashidi et al., 2014; herrero-medrano et al., 2015) . the objectives of the current study were to (i) estimate the genetic parameters of reproduction traits prior, during, and after a prrsv outbreak, and among traits within each phase, (ii) estimate heritability and genetic correlations of sample-to-positive (s/p) ratio from blood during the prrs outbreak with reproductive performance during the outbreak to validate findings from serão et al. (2014) , and (iii) evaluate a reaction norm model to model the effect of prrs on nba. blood sampling of sows was approved by the institutional animal care and use committee (iacuc, . reproductive data was retrieved from an existing database that included data collected as part of a routine breeding program and, therefore, did not require approval from an animal care and use committee. three breeding farms from the maschhoffs (carlyle, il), located in close proximity to each other in illinois, usa, broke with a prrsv strain in the spring of 2015. these farms included pedigreed purebred yorkshire (york) and landrace (lr) sows. farms 1 and 3 contained both breeds, while farm 2 contained only the lr breed. after suspect abortions, samples were sent for diagnostics and the 1-7-4 restriction fragment length polymorphism (rflp) pattern (strain) of prrs was confirmed by pcr analysis. this strain is a highly virulent strain. a nearby farm first broke with this prrsv strain. to protect some high indexing sows in farm 1, they were preemptively sent to a quarantine facility to be tested for prrs. after clearing these tests, these sows were then transferred to one of the other farms to farrow. farm 1 was confirmed positive on march 5 and was then depopulated to try to protect other nearby farms 2 and 3. subsequently, farms 2 and 3 broke with prrs and were then closed to new animals. farm 2 was confirmed positive on april 16 (42 d after farm 1) and farm 3 on april 9 (35 d after farm 1). after the initial confirmed outbreak with several positive samples, all sows on farms 2 and 3 were inoculated with live virus of a 1-7-4 rflp pattern (strain) that was isolated at each farm (same strain) approximately 3 wk later, on may 5 for farm 2 and on april 30 for farm 3. inoculation was intranasal at farm 2 and intramuscular at farm 3. all sows were injected with an mlv vaccine ~30 d after the inoculation. blood samples for antibody levels were taken from the anterior vena cava with vacutainer serum tubes from sows on farm 2 on june 18 and from farm 3 on june 16, 17, and 19. this was ~60 d after the initial outbreak. antibody levels taken at this time point should have plateaued for most animals . serum tubes were centrifuged at the farm and these serum samples were sent to kansas state university for analysis. antibody level against the prrsv n-protein was measured using a fluorescent microsphere immunoassay (luminex) and converted into a standardized sample-to-positive (s/p) ratio using positive and negative controls. this assay is conceptually similar to an indirect elisa. reproductive data obtained from routine data collection in these breeding herds included total number born (tnb), number born alive (nba), number stillborn (nsb), number mummified (nbm), and number born dead (nbd; the sum of nsb + nbm). raw weekly means for each farm are presented in figure 1 . for genetic analyses, mortality traits nsb, nbm, and nbd were all log transformed as ln(phenotype + 1) (lewis et al., 2009) and will be referred to as lnsb, lnbm, and lnbd, respectively. records on reproductive traits were separated into three phases (prior, during, and after the outbreak) based on estimates of farm-year-week (fyw) effects (extracted from the farrowing date) for each reproductive trait that were obtained from the following linear mixed model for each trait separately where par is the parity effect (i=1,…,8), farm is the farm effect (j=1,2,3), and breed is the breed effect (k=1,2), which were fitted as fixed effects, while fyw is the random fyw effect, assumed to follow ~n(0, iσ 2 fyw ) in which σ 2 fyw is the fyw variance and i is an identity matrix, and sow is a random sow effect, assumed to follow ~n(0, iσ 2 sow ) in which σ 2 sow is the sow variance (following rashidi et al., 2014) . to make all traits comparable, estimates of fyw effects for each trait were standardized by their respective overall sds (based on the variance estimated for fyw using lme4 in r; bates et al., 2015) and plotted over time ( figure 2 ). visual appraisal was used to split the data for each trait into three phases because it was known when the outbreak occurred (dates given above, similar to serão et al., 2014) . preliminary analysis showed only minor changes in variance component estimates (<0.1 for genetic correlations) when slightly different dates (by 1 or 2 wk) were used to split the data into phases because these different dates changed the data sets very little. the prrs outbreak phase for farm 1 was identified to be from march 12 to april 1 (20 d) and also from may 7 to may 27 (20 d), after sows were moved. the prrs outbreak phase for farms 2 and 3 was from may 7 to august 5 (90 d). based on these dates, data from each reproductive performance trait were separated into three traits (prior, during, and after), which were designated with subscripts p, d, and a, respectively, on the trait acronym (e.g., tnb p for tnb prior to the outbreak). transition periods were masked for this analysis by removing data just prior to and after the outbreak phase (following herrero-medrano et al., 2015) because these records represented a "grey area" for classification. for farm 1, data from 1 wk prior to the first outbreak and from the 3 wk between the two outbreaks were removed. for farm 2, data from 1 wk prior to and 2 wk after the outbreak were removed. for farm 3, data from 1 wk prior to the outbreak phase and 7 wk after the outbreak were removed. the latter were removed because rolling averages for tnb fluctuated continuously during these weeks, possibly from a rebound after the outbreak, which made it unclear how these data should be classified (see figure 2 ). variance components among traits were estimated both between phases (e.g., tnb p with tnb d ) and within phase (e.g., tnb d with nba d ) by basic bivariate animal models, using asreml4 (gilmour et al., 2015) . heritability estimates for a trait were averaged over the bivariate analyses. the model used for reproductive traits was as follows: where β included the fixed effects of parity (1 through 8), farms (1, 2, or 3), breed (york, lr), and farm-year-month (fym), and the 30-d rolling herd average of the trait analyzed as a fixed covariate (following lewis et al., 2009) . the vector u represents the random additive genetic effect of the sow [~n(0, aσ 2 sow )] where σ 2 sow is the sow variance and a is a matrix of additive genetic relationships among pigs, and the vector e represents the random residual term [~n(0, iσ 2 e )]. very few sows had repeated records for traits prior to and after the outbreak and in these cases, the second record in the dataset was removed such that a repeatability model was not needed. the final multivariate dataset included 2,014, 1,428, and 1,626 records for the prior, during, and after phases, respectively. the model used for s/p ratio (only recorded during the outbreak) also was a simple animal model, with parity, breed, date of sample collection (june 16 to 19), and the plate of the assay (96-well plates used) as fixed effects. collection date was confounded with farm (see above) and, therefore, farm was not fit in the model. random effects were the same as for the reproduction traits. the pedigree included at least three generations to calculate the numerator relationship matrix (a), for a total of 6,202 animals. animal models for variance components were analyzed with asreml 4 (gilmour et al., 2015) . reaction norms were used to analyze nba by regressing on estimates of farm-year-week (fyw) effects for nba (estimates ranging from −4.11 to 2.33) that were obtained using the animal models described above for each phase. for this analysis, the entire dataset was kept intact for each trait, without splitting it into phases. this dataset included 6,328 records from 3,378 sows. these sows recorded between one (1,397), two (1,209), three (575), and four (197) records (farrowings). the healthy phase started at approximately −1 on the fyw scale, which would include data from both prior to and after the prrs outbreak phase. the model used was as follows: where the fixed effects vector b included breed, farm, parity, status (prior, during, after), and the fixed covariate of fyw effect estimates for nba, with corresponding design matrix x. matrix q contains coefficients for the random additive genetic effects ( a), which included correlated random intercepts ( a i) and random slopes ( a s) on fyw effect estimates for each individual in the pedigree, connected through the pedigree relationship matrix. the variance-covariance structure of a was as follows: where g rn is the genetic (co)variance matrix, with σ a i ,a s , σ 2 a i , and σ 2 a s denoting the covariance and additive genetic variances for intercept and slope, respectively. three different scales were used for the random regression coefficients for the 170 unique fyw classes (φ matrix): (i) the raw scale of estimated fyw effects, (ii) legendre polynomial terms from the fyw effects (leg function in asreml), and (iii) orthogonal polynomial terms based on the pol function in asreml (gilmour et al., 2015) . no information is reported on model fit, as these were equivalent models. vector pe contains random permanent environmental effects for animals with records, with matrix z being diagonal with only an intercept term (1/0), as in a normal repeatability model. more complex models for the permanent environmental effects did not converge, possibly due to the low number of records per sow (meyer, 2005) . finally, vector e contains residuals which, after a preliminary analysis (fitting many different group sizes), were fitted using heterogeneous residual variances with five discrete classes based on the nba fyw effect estimates {−inf, −2, −1, 0, 1, inf}. thus, the residual variance was structured as follows: var estimates of genetic correlations of nba between fyw classes were obtained from the estimated genetic covariance matrix ĝ rn using φĝ rn φ , which results in a square, symmetric matrix with dimensions equal to the number of fyw effects that is used to calculate φ. estimated breeding values for each animal for each fyw level were calculated as φ (q×2)û (2×n), where û is a matrix of estimates of the random intercept and slope effects from the reaction norm model, where q is equal to the number of fyw levels (170), and n is the number of animals in the pedigree (n = 6,451). for the current data, this resulted in a 170 × 6,451 matrix of ebvs. table 1 shows means and sd for the five reproductive traits during the three phases (prior, during, and after). the average tnb was similar across phases, although slightly lower after the outbreak. all other traits were greatly affected by the prrs infection. the average nba dropped from 10.6 prior to the outbreak to 7.7 during the prrs phase. the average nsb rose from 0.5 to 1.2 per litter during the outbreak. the average nbm went from 0.3 prior to the outbreak to 2.6 per litter during the outbreak. finally, average nbd went from 0.8 to 3.8 during the prrs outbreak. all four traits (removing tnb) returned to their pre-challenge average after the outbreak. figure 1 displays the raw averages by fyw for each trait. the severity of the disease during the prrs outbreak in the spring of 2015 is evident. weekly means for tnb did not show large changes over time but did trend downward starting midway through the prrs phase. all other traits were affected much more severely by the prrs outbreak as expected. nba dropped below 7 pigs in all three farms. for farm 2, the mean for nbd was higher than the mean nba for 1 wk. farm 2 had a spike in nsb immediately after the outbreak and then returned to a normal level after approximately 5 wk. farm 3 was slightly less affected by the outbreak in terms of nba and nbd. an important note is that the majority of the in utero mortality during the prrs phase was due to mummies (68%) and not stillborns. prior to and after the outbreak, the percent of deaths due to mummies was 36% and 42%, respectively. standardized estimates of fyw effects followed the same trends as the raw means ( figure 2 ). the prss outbreak corresponded to spikes in mortality traits and drops in the estimates of fyw effects for tnb and nba. the most extreme standardized effects were from farm 2, where some estimates were as high as four. trends in estimates over time were similar for all traits, except for tnb. for farm 2, the nsb returned to baseline quicker than farm 3. heritability estimates were ≤0.13 for all reproductive traits for all phases ( of heritability for s/p ratio was also relatively low at 0.17 ± 0.05. heritability estimates for mortality traits (lnsb, lnbm, and lnbd) ranged from 0.01 to 0.06 prior to the outbreak, increased during the outbreak (0.06 to 0.13), likely because of the higher incidence of mortalities during the outbreak, and then reduced again after the outbreak. however, only the estimate of heritability of lnbm returned to its estimate prior to the outbreak, while estimates for both lnsb and lnbd remained slightly elevated after the outbreak (0.09 ± 0.04 and 0.06 ± 0.04, respectively). the estimate of heritability of tnb (0.13 ± 0.05) did not change during the outbreak but reduced to 0.08 ± 0.04 after the outbreak. estimates of genetic correlations of traits between the three phases are displayed in table 3 . estimates of the genetic correlation between prior to and during the outbreak for tnb and nba were >0.85, indicating similar genetic backgrounds. genetic correlations for tnb and nba were much lower between prior to and after the outbreak (0.32 ± 0.33 and 0.27 ± 0.42, respectively). again, this may be expected when sows are being bred during the outbreak. the genetic correlation between tnb during and after the outbreak was higher (0.72 ± 0.55) than for nba (0.21 ± 0.54). estimates of genetic correlations for mortality traits prior to and during the outbreak were inconsistent; they were positive for lnsb and lnbd but negative for lnbm (−0.42 ± 0.55). trends in estimates of genetic correlations for reproductive performance between prior to and after the outbreak were similar to those between during and after the outbreak. estimates of genetic correlations of s/p ratio with reproductive traits are presented in table 4 . prior to the outbreak, estimates of the genetic correlations ranged from 0.05 to 0.85, but with very large se for lnbm and lnbd. most estimates of the genetic correlation of s/p ratio with reproduction traits during the outbreak, which was of main interest, were close to zero, with the exception of lnsb, which had an estimate of −0.73 ± 0.29 with 1 tnb = total number born, nba = number born alive, lnsb = log number stillborn, lnbm = log number born mummified, lnbd = log number born dead, s/p = sample-to-positive ratio of the prrs antibody levels. 2 phases were split using a mixed linear model, fitting farm year week (fyw) as a random effect and extracting the predicted values. visual appraisal was used to split phases into prior, during, and after the prrs outbreak. 3 s/p ratio was only collected during the prrs outbreak. 1 tnb = total number born, nba = number born alive, lnsb = log number stillborn, lnbm = log number born mummified, lnbd = log number born dead. 2 phases were split using a mixed linear model, fitting farm year week (fyw) as a random effect and extracting the predicted values. visual appraisal was used to split phases into prior, during, and after the prrs outbreak. 3 completed with remlf90 from blupf90 programs in place of asreml due to convergence issues, no se available. s/p ratio. the negative genetic correlation estimates of s/p ratio with lnsb and lnbd during the outbreak were in the favorable direction (i.e., sows with higher antibody level are expected to have fewer stillborn pigs phenotypically/genetically). after the outbreak, estimates of genetic correlations of s/p ratio with reproductive traits were low (−0.20 to 0.05). negative genetic correlations may be as expected because producing more antibody during the infection may have diverted resources away from reproduction while the sow was cycling during the outbreak, leaving fewer embryos/fetuses to develop and be born after the outbreak. genetic correlations among reproductive traits within phase are displayed in table 5 . tnb and nba had high genetic correlations prior to and after the outbreak (>0.90) but the correlation dropped to 0.71 ± 0.16 during the outbreak, likely due to greater prenatal mortality during the outbreak. tnb was positively correlated, genetically, with all mortality traits during all three phases (0.23 to 0.56), as expected, but correlations were slightly stronger during and after the outbreak (0.56 ± 0.23 with lnbd during the outbreak). nba had close to zero genetic correlation estimates with mortality traits prior to and after the outbreak but slightly negative estimates during the outbreak (−0.14 to −0.22). estimates of genetic correlations among mortality traits within phase were all positive (0.23 to 0.98) for all phases. prior to and after the outbreak, lnsb and lnbd were genetically highly correlated, at 0.94 ± 0.07, likely because most mortalities at those times are due to stillborns rather than mummies. the estimate of the genetic correlation between lnsb and lnbd dropped to 0.73 ± 0.23 during the outbreak but the estimate of the genetic correlation between lnbm and lnbd increased from 0.68 ± 0.28 prior to the outbreak to 0.80 ± 0.15 during the outbreak, as a greater proportion of mortalities was due to mummies during the outbreak. the estimate of the genetic correlation between lnsb and lnbm was moderate prior to the outbreak (0.40 ± 0.47) and low during the outbreak (0.23 ± 0.48). all estimates of genetic correlations among mortality traits after the outbreak were >0.83. the three parameterizations of the reaction norm model only differed in estimates of genetic variances for the intercept and slope, and in estimates of the genetic covariance or correlation between intercept and slope (table 6 ). however, estimates of genetic variances and covariances for nba at given fyw levels were unaffected, as expected. estimates of the genetic variance of the intercept and slope ranged from 0.52 to 0.81 and from 0.07 to 1.21, respectively. all estimates of the genetic covariance between intercept and slope were positive (0.11 to 0.51). estimates of the genetic correlation between the intercept and slope were 0.54 ± 0.35, 0.24 ± 0.50, and 0.52 ± 0.36 for the raw, legendre, and the polynomial (pol) function of asreml, respectively. estimates of residual variance increased slightly from the first fyw level to the second (estimated from 12.21 to 12.73) and then reduced as the fyw effect increased (10.54, phases were split using a mixed linear model, fitting farm year week (fyw) as a random effect and extracting the predicted values. visual appraisal was used to split phases into prior, during, and after the prrs outbreak. 8.70, and 6.84). figure 3 shows estimates of genetic covariances (left) and correlations (right) from the reaction norm model for nba between fyw levels which, as indicated, were the same for all three parameterizations. estimates of genetic covariances (diagonals) showed the expected quadratic trend (given the first-order model) and were negative only between the most extreme fyw classes. estimates of genetic correlations for nba between fyw levels showed two fairly distinct blocks during the healthy and diseased phases (diseased in the top left, healthy in the bottom right). the transition from diseased to healthy started for nba fyw effects around −1 (with a very small overlap of the two phases, see figure 2 ). genetic correlations between the two blocks were moderate, except for the very extreme fyw levels, which was consistent with the multi-trait analysis of traits defined by phase. table 7 shows estimates of correlations of ebv from the multivariate analysis of nba by phase with ebv obtained from the reaction norm model using the raw fyw scale. correlations of ebv for the additive genetic intercept and slope terms with ebv for fyw levels equal to −4, −2, 0, and 2 are also shown, with the first two (−4 and −2) being during the outbreak and the other two (0 and 2) from the two healthy phases (representing a combination of prior to and after the prrsv outbreak). the ebv for the intercept terms from the reaction norm model had the highest correlation with ebv for nba p (0.82), while ebv for nba p and nba d were highly correlated with ebv from the reaction norm model at fyw equal to −2 and 0 (0.78 to 0.80). the ebv for the intercept was almost perfectly correlated with ebv for the reaction norm at fyw equal to 0 (as expected) and were also highly correlated with ebv for the reaction norm at fyw equal to −2. correlations between ebv at different fyw levels were very similar to the estimates of genetic correlations from the reaction norm model, showing decreasing correlations with increasing distance between fyw levels. estimates of heritability and genetic correlations for s/p ratio with reproductive performance during a prrs outbreak were mostly inconsistent with previous studies. serão et al. (2014) reported a heritability of 0.45 ± 0.13 for s/p ratio after a prrs outbreak in a multiplier herd in canada, which was validated in a more complex independent study (serão et al., 2016) . the estimate of heritability of s/p ratio from the current study was, however, substantially lower at 0.17 ± 0.05. estimates of genetic correlations of s/p ratio with reproductive traits also did not completely agree with previous results, except for the genetic correlation of s/p ratio with lnsb (serão et al., 2014) . although this is favorable, most of the prenatal mortality (68%) during the prrs outbreak in this study was due to mummified piglets, as mentioned above. this is important because although s/p ratio was more correlated with lnsb, it would not change overall mortality as much because more piglet mortality stems from mummified piglets. serão et al. (2014) found that s/p ratio tended to have moderate/strong genetic correlations with reproduction traits, ranging from −0.72 (nsb) to 0.73 (nba); the lowest estimate in absolute value was 0.27 (nbd). the only genetic correlation that was similar in the current study was for lnsb (−0.73). both tnb and nba were not strongly associated with s/p ratio in the current study. note, however, that these estimates come with large standard errors when dealing with small sample sizes and lowly heritable reproductive traits, therefore strong conclusions cannot be drawn until further studies are conducted. one notable difference between the current study and the studies of serão et al. (2014 serão et al. ( , 2016 is the use of different antibody assays for semi-quantification of antibody levels. the idexx prrs x3 elisa (at the same lab) was used in both serão et al. (2014) and serao et al. (2016) , while the luminex (luminex corp., austin, tx) microsphere assay was utilized in the present study. although the idexx is considered an industry/research gold standard for measuring prrs antibody (sattler et al., 2014) , the microsphere (or microbead) assay is rising in popularity because the luminex multiplex system allows for the detection of numerous analytes within a single biological sample, saving cost, time, and labor (lin et al., 2011) . the luminex assay was also used by the same lab in the study of hess et al. (2018) on nursery pigs following experimental prrsv infection, resulting in a moderate-to-high heritability estimate. the microbead assay is not a traditional elisa but is conceptually similar to an indirect elisa, as both measure antibodies against the nucleocapsid (n) protein (inside the complete prrs virus). lin et al. (2011) compared an earlier version of the standard single plex elisa (idexx herdchek prrsv 2xr kit) and the microsphere-based immunoassay and found the spearman rank correlation to be 0.72 for prrs antibody. the sensitivity and specificity between the assays were 91% and 93% for prrs in young pigs, respectively (κ coefficient of 0.67). commonly, young pigs are used for testing and validating assay results for several reasons (cost, ease of sampling, availability, etc.). in adult pigs, however, 64% (16/25) of samples were found to be positive by the luminex assay but negative by the idexx table 7 . correlations among ebv from the multivariate analysis of number born alive (nba) prior to, during, and after the outbreak (nba p , nba d , and nba a ) and with ebv obtained from the reaction norm (rn) model using the raw scale for farm-year-week (fyw) estimates, including ebv for intercept (rn int ) and slope (rn slope ), along with the ebv for nba for fyw estimates equal to −4, −2, 0, and 2 (rn −4 , rn −2 , rn 0 , and rn 2 ) herdchek prrs x3 assay (giménez-lirola et al., 2014) . giménez-lirola et al. (2014) used the newest idexx (herdchek prrs x3), the same test used in serão et al., (2014 serão et al., ( , 2016 . adults pigs (sows and boars) may have higher background reactivity than young pigs (giménez-lirola et al., 2014) , possibly due to a more mature immune system and antigens seen later in life and will need to be investigated further. although we do not have direct evidence that the differences in variance components estimated between the present study and serão et al., (2014 serão et al., ( , 2016 can be attributed to the differences between the idexx and luminex platform, it should be a major consideration in future research, along with the age of the animal being tested. there are several other possible reasons for the difference in estimates of genetic parameters for s/p ratio between the current and previous prrs outbreak studies. these could include other aspects of the assay such as in-house diagnostic target variations, time of year, the strain of the virus, sample processing, and other unknown environmental effects. in contrast to serão et al. (2014) , sows in the current study were inoculated 3 wk after the confirmed outbreak, followed by mlv vaccination. vaccination is not expected to impact antibody levels at 40 d after inoculation, as a secondary type of response in a relatively short time after infection is not expected due to the persistency of infection of the prrs virus discussed below) . however, it still could contribute to differences. typically, antibody response studies in pigs are conducted in designed experiments with one injection given simultaneously to all animals. in a natural disease challenge, this consistency is lost and sows in a large farm are consistently re-exposed to antigens, some possibly due to "rebound" animals (boddicker et al., 2012) . in serão et al. (2016) , antibody levels were measured on gilts following acclimation across many commercial farms, which represented a range of times following exposure, either through infection or mlv vaccination, again strengthening the idea that some of these other factors may not play a large role. multiple factors make determining the cause of differences between estimates of genetic parameters for s/p between studies hard to understand. these will continue to be an issue as what is best for measuring antibody response in research (i.e., this study) may not be optimal for production and clearing the virus from a commercial farm, such as inoculation and vaccination observed in the current study. this may provide some insight into the difficulty of conducting this type of research in field conditions and therefore alternatives will be needed (e.g., separate, carefully designed challenge studies in sows). perhaps other measures such as interferon-γ (ifn-γ) response after inoculation might be useful, along with antibody response. collecting antibody response at multiple time points may also be helpful to determine the approximate date of infection, but this would be expensive and not feasible on a commercial farm. ranges of heritability estimates for litter size and mortality traits were consistent with previous estimates (0.01 to 0.13; bidanel, 2011) . trends in heritabilities between phases generally followed results by lewis et al. (2009) . heritability for nba was lower during the outbreak, while estimates of heritability for mortality traits were higher during the outbreak, most likely due to the increased incidence of mortality under prrs challenge. heritability of tnb was not affected by the prrs phase like the other traits but was after the outbreak. biologically, this makes sense, as sows that farrowed during the outbreak were bred prior to the outbreak and all fetuses would be counted in the total born. however, sows bred during the outbreak farrowed later during or after the outbreak, which affects the total born observed due to possibly fewer oocytes being fertilized or fetuses being absorbed. there is no verification of this because pregnancies were not evaluated by ultrasound. low to moderate genetic correlations for reproduction traits between prior to and during the prrs phase likely indicate the influence of disease resistance qtl during the outbreak phase, making them different traits. serão et al. (2014) found similar trends as observed in the present study for estimates of heritability prior to and during the outbreak for nbm, nbd and nsb but their estimates of heritability for nba was higher during the outbreak. estimates of genetic correlations for reproduction traits between phases were fairly consistent with previous estimates (lewis et al., 2009; rashidi et al., 2014; herrero-medrano et al., 2015) , although these studies combined data from prior and after the outbreak into one trait. for instance, the genetic correlation from lewis et al., (2009) for nba was 0.56 between healthy and diseased phases, which would be a combination of the current estimates for nba between prior and during (0.98) and between during and after (0.21). rashidi et al. (2014) estimated genetic correlations for nba and nbd at 0.87 and 0.57 between healthy and diseased phases, respectively. herrero-medrano et al. (2015) estimated these same correlations at 0.75 and 0.74, respectively. differences between studies in estimates of genetic correlations for reproduction traits between diseased and healthy phases in prrs outbreak herds can also be due to other factors. the strain that caused the outbreak in the current study was a very severe strain of the prrs virus. more studies are needed to determine whether results from serão et al. (2014) also hold for other virus strains, such as those used in the prrs host genetics consortium and associated trials (hess et al., 2016; waide et al., 2018) . new viral strains develop and results from previous antibody studies may not apply. for instance, a new prrs strain that developed in china in the last decade shows very different clinical signs than normal strains (see figure 2 from tian et al., 2007) . diseases such as prrs can change and antibody measures as indicator traits need to continually be re-evaluated for effectiveness in a breeding program. to date, all studies have divided the reproduction data from prrs outbreak herds into only healthy and diseased phases. a finding from this research was that estimated genetic correlations may support keeping the time period after the prrs phase as a separate trait from prior to the prrs outbreak, although standard errors were large. prrs can be a persistent infection and, thus, it is possible that prrs still affects reproductive performance after the outbreak has cleared, perhaps subclinically. lunney et al. (2016) discussed the three stages of a prrs infection: acute, persistent, and extinct. the virus can persist in tonsils and lymph nodes and has been identified in animals as long as 175 to 251 d postinfection (wills et al., 2003; molina, 2008) , although most cleared within three to four months (wills et al., 2003) . in the present study, the after phase included ~4 mo of data. it is possible that the large farm sizes contributed to the persistent nature of the infection. it is also possible that the less than one genetic correlation between traits prior and after the outbreak was caused by genotype-by-environment interactions due to reasons such as seasonality, which will need to be investigated further in another study. the outbreak phase for the current study was during the late spring/ summer months. another reason why the after-period may need to be analyzed separately is that some sows that farrowed after the outbreak were bred during the outbreak, which could result in some residual effects. any sow bred during the prrs phase could suffer reduced tnb from reduced fertilization, embryos not surviving, or fetuses being absorbed. in contrast, most sows that farrowed during the outbreak phase were bred during the healthy phase prior to the outbreak and, thus, tnb should not be severely affected due to the piglets already being fully-formed, as observed in the present study, especially in farm 3. this was also reinforced by the low estimate of the genetic correlation for tnb between before and after the outbreak (0.32 ±0.03). thus, it may be better to consider leaving the after phase a separate trait or to remove this data for routine genetic evaluation. further research will be needed to determine how long this period extends. previous reproductive disease outbreak studies have separated reproduction data into two phases (healthy and diseased) but, to our knowledge, this is the first study to report estimates of genetic correlations within phase (e.g. nba p with lnsb p or tnb d with nba d ). the phase prior to infection represents typical variance components for litter size without major disease (su et al., 2007; putz et al., 2015) . not separating data from herds that experience disease outbreaks into three phases could affect estimates of genetic correlations between traits (e.g., tnb and nba or their genetic correlation with mortality traits). for example, the genetic correlation between tnb and lnbd was 0.28 ± 0.26 prior to infection and 0.56 ± 0.23 during the prrs outbreak. this should be as expected, as more total born during the outbreak would allow more pigs to be affected by disease and die prior to farrowing. one possible downside of the use of reaction norms for analysis of disease outbreak data is that they do not differentiate records obtained prior to and after the outbreak. separate stressors may cause dips in performance. this may be a disadvantage of the reaction norm models, especially for other situations such as outbreaks from different pathogens or different strains of the same virus (observed in herrero-medrano et al., 2015) . multivariate analyses may also not be able to disentangle causes if multiple pathogens are involved in the infection. in herrero-medrano et al. (2015) , both prrs and a coronavirus caused outbreaks that led to a high challenge load (described by mathur et al., 2014) of over 15 index units (the challenge load). the reaction norm model treats both of these outbreaks being very similar traits (in terms of regressor values, here the fyw effects), when in fact they most likely have different genetic backgrounds in terms of genetic resistance. for example, two fyw effects around −1 nba may be the result of separate environmental changes; one could be the result of porcine epidemic diarrhea and the other from heat stress for example. any fyw effect would be a combination of any management environments and challenges/stressors. regardless of this, given enough data, these models should still result in sows that are more resilient/robust to environmental challenges. the advantage of the reaction norm is that it would average over all of these effects in one parsimonious model without regard to specific causes. knap and su (2008) stressed the need to have large datasets for reaction norm models to be effective. the optimal breeding objective should include general resilience/robustness to any number of stressors, including different diseases, not to single diseases or stressors. therefore, for the reaction norm models to be effective, it would be advantageous to have a large number of environments classified from many different farms with as many different management practices as possible, such that the values used to regress on capture as many stressors and different environments as possible (knap, 2005) . when thinking about testing a sire, it would be best to have as many daughters in as many different farms/environments as possible. one issue is that there can be high leverage on the slope of the reaction norm slope for extreme fyw observations (pool et al., 2000) . it is known that different parameterizations of the regressor value (fyw estimates) lead to different variances and covariances for the intercept and slope terms. therefore, it can be dangerous to interpret these estimates, as was done by knap and su (2008) . another factor that can influence the (co)variances is using different contemporary group sizes (week, month, or season). knap and su (2008) utilized estimates for herd-year-season contemporary groups instead of herd-year-week contemporary groups used in the current analysis. the estimate of the genetic correlation between the intercepts and slopes from the three parameterizations used in the current study was different (as expected), although all were positive. this indicates that selection for improved nba of animals with the standard animal model (related to the reaction norm intercept) would result in animals with greater reaction to changes in the environment. again, as expected, estimates of genetic covariances and correlations between ebvs from each fyw from the reaction norm model were not affected by the parameterization of the model (figure 3) . the correlation between ebv for the intercept term from the reaction norm model and ebv from a typical animal model was high, which agrees with previous research (knap and su, 2008) . the current analysis expanded this by calculating the correlations of ebv from the multivariate phases (prior, during, and after) model with the reaction norm estimates of intercepts and slopes and ebv at discrete fyw levels. knap and su (2008) found ebv from the multivariate animal model and ebv for the intercept terms from the reaction norm to be correlated 0.78 to 0.85, similar to the current analysis, which found correlations between 0.75 and 0.82 (for prior, during, and after for nba). ebv from the reaction norm model at different levels of fyw was also correlated with ebv from the multivariate model. ebv from the reaction norm between −-2 and +2 were moderate to highly correlated with ebv from the multivariate analysis (between 0.64 and 0.80). the highest correlation between the multivariate ebv and the ebv at −4 from the reaction norm was for nba during the outbreak, as expected (0.38), however, this was at the very extreme of the outbreak phase. the ebv for the slope from the reaction norm was negatively correlated with the ebv at −4 (−0.55) and strongly positively correlated with ebv at 2 (0.91), which is as expected. animals with ebv for the slope that deviate from zero are considered sensitive to environmental changes. therefore, the optimum selection would be for animals with a high ebv for the intercept and an ebv for the slope close to zero, indicating high producing animals that produce uniformly (in ranking) across environmental gradients. the reaction norm can capture more than just health, which may contribute to the difference in genetic correlations observed between the multivariate and reaction norm models. guy et al. (2012) discussed resilience to not only health challenges but also other environmental challenges. in commercial data, challenges for pigs can encompass social, environmental, metabolic, immunological, and human interactions (martínez-miró et al., 2016) . seasonality encompasses effects of heat stress and disease and both affect fyw estimates. for instance, there is a positive seasonality effect during the summer months for pigs weaned/sow/ year (stalder, 2017) . sevillano et al. (2016) showed that seasonal infertility can be impacted by photoperiod and not just by ambient temperatures. so, as long as data are captured over long periods of time and plenty of heterogeneous environments, the reaction norm should also be thought of as general resilience, instead of only disease resilience. of course, the multivariate model could also pick up effects from other stressors. bishop and woolliams (2014) stated that the requirement to measure resistance phenotypes is a rate-limiting step in breeding for disease resistance. one problem for both the multivariate and reaction norm models is that it is difficult to get enough records in the diseased phase to obtain accurate ebv for disease resilience; most of the information for either model will come from correlated data, i.e., from the "healthy" phase in the multivariate case. for the reaction norm model, most data are from healthy weeks when an outbreak has not occurred for an extended period of time. for instance, in farm 1 of herrero-medrano et al. (2015) , only one outbreak occurred over a 6-yr span of the data and only five total outbreaks occurred in the three farms. the reaction norm is only observed on part of the fyw estimates for many animals, especially because many sows are culled early (especially in nucleus environments), although this is partially overcome by the use of the pedigree relationships. the use of random regression models for a reaction norm is different from many other situations in which random regression models are used for genetic analyses, such as milk yield in dairy cattle, growth or feed intake in pigs, and egg production in poultry. in those situations, animals have repeated records that span most of the lactation, growth period, or egg-laying cycle, leading to more accurate estimates of breeding values than obtained for the sparser reaction norm model (knap and su, 2008 ). in the current study, sows had between one and four records for the reaction norm model. meyer (2005) stated that using higher order polynomials when a substantial proportion of animals have fewer records than the order of polynomials fitted can lead to erratic and implausible estimates. one should be careful before applying complex models to this type of data. the total range in estimates of nba fyw effects was 6.43 on the original scale. a total of 47%, 70%, and 89% of sows had phenotypes in contemporary groups that ranged <0.5, 2.0, and 3.0, respectively, in nba fyw effects (i.e., the x-axis). this may contribute to the poor accuracy referred to by knap and su (2008) . there is some work needed prior to the swine industry adopting antibody response to prrs outbreaks or mlv vaccination. novel strains of prrsv are continuing to show up because of the high mutation rate of the prrs virus and predictive ability in terms of genetic correlations should be regularly checked. antibody tests continue to change over time and differences among labs exist, although the herdchek prrs x3 antibody test seems to be very repeatable within and across labs (kittawornrat et al., 2012) . this possible instability over time in other antibody assays such as the luminex (or future idexx assays) is risky for implementation into the swine breeding industry. one important validation needed is to send samples to multiple veterinary diagnostic labs and with multiple tests (e.g., idexx vs. luminex) to verify results for each test and each lab to make sure genetic analyses agree. perhaps even lab replicates will need to be performed to determine the repeatability. thus, at this point, it is unclear whether selection on antibody response (possibly to prrs vaccines) will be highly useful to the swine breeding industry. antibody level in sows to prrs following a prrs outbreak, measured as s/p ratio, was low to moderately heritable (0.17 ± 0.05) and had low genetic correlations with reproductive traits except for lnsb (−0.73 ± 0.29). standard errors for variance component estimates were large because of a relatively small dataset and lowly heritable traits, so no strong conclusions can be drawn. more research will be needed to understand why these results did not completely validate previous findings on s/p ratio heritability and genetic correlations with reproductive performance. it is possible that the differences in the antibody assay were the cause, but this is still unknown. the genetic correlation between reproductive performance prior to and during the prrs outbreak was high for both tnb and nba. the only negative genetic correlation between performance prior to and during the prrs outbreak was for lnbm. tnb had a genetic correlation of 0.32 between prior to and after the outbreak. it may be useful to consider reproductive performance several months after the outbreak as a separate trait from performance prior to the outbreak, as sows farrowing after the outbreak were bred during the outbreak. the reaction norm model for nba showed similar trends in genetic correlations as the multivariate model that considered reproductive performance prior, during, and after the outbreak as separate traits, although it considered data from prior and after the outbreak as having overlapping environments. overall, future work will need to address some of the differences from previous research observed in the current study. fitting linear mixed-effects models using lme4 biology and genetics of reproduction in genetics of the pig. rothschild, m. f. and a. ruvinsky, editors. the genetics of the pig genomics and disease resistance studies in livestock evidence for a major qtl associated with host response to porcine reproductive and respiratory syndrome virus challenge asreml user guide release 4.1 structural specification development and validation of a 4-plex antibody assay for simultaneous detection of igg antibodies against torque teno sus virus 1 (ttsuv1), ttsuv2, and porcine reproductive and respiratory syndrome virus types 1 and 2 selection of pigs for improved coping with health and 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with one of two porcine reproductive and respiratory syndrome virus isolates duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus we would also like to thank the maschhoffs for allowing us to take samples in their barns following the outbreaks and their farm staff for help with sample collection. several iowa state university undergraduate students also helped with data collection, including allysa koethe, jake baker, zach sauer, dylan lathrum, and kaylyn hodge. key: cord-304986-lk2ikxda authors: green, toni m.; santos, mark f.; barsky, sanford h.; rappa, germana; lorico, aurelio title: analogies between cancer-derived extracellular vesicles and enveloped viruses with an emphasis on human breast cancer date: 2016-08-27 journal: curr pathobiol rep doi: 10.1007/s40139-016-0116-4 sha: doc_id: 304986 cord_uid: lk2ikxda purpose of review: cancer cells utilize extracellular vesicles (evs) as a means of transferring oncogenic proteins and nucleic acids to other cells to enhance the growth and spread of the tumor. there is an unexpected amount of similarities between these small, membrane-bound particles and enveloped virions, including protein content, physical characteristics (i.e., size and morphology), and mechanisms of entry and exit into target cells. recent findings: this review describes the attributes shared by both cancer-derived evs, with an emphasis on breast cancer-derived evs, and enveloped viral particles and discusses the methods by which virions can utilize the ev pathway as a means of transferring viral material and oncogenes to host cells. additionally, the possible links between human papilloma virus and its influence on the mirna content of breast cancer-derived evs are examined. summary: the rapidly growing field of evs is allowing investigators from different disciplines to enter uncharted territory. the study of the emerging similarities between cancer-derived evs and enveloped virions may lead to novel important scientific discoveries. breast cancer, the leading cause of cancer-related deaths worldwide, is a very heterogeneous disease. while the classical distinction between the two main forms of breast cancer, ductal and lobular, is still valid, dna microarray analyses in the past decade have allowed the subclassification of breast cancer based on gene expression patterns in normal-like, basal-like, human epidermal growth factor receptor 2/neu-overexpressing, luminal a, and luminal b cells [1] . these subtypes have both prognostic and therapeutic relevance, although heterogeneity persists within each subtype. independent from its subclassification, metastatic spreading is mainly responsible for mortality of breast cancer patients, with overt metastases appearing in several cases 5-10 years after removal of the primary tumor. extracellular vesicles (evs), including ectosomes, also known as microvesicles (mvs), shed from the plasma membrane, and exosomes, derived from multivesicular bodies, are released from nearly all cells, but in increased quantities from cancer cells [2] , and have come to the limelight as biological entities involved in development and progression of breast cancer as well as most types of malignancies. moreover, their possible exploitation as breast cancer biomarkers and potential components or targets of novel therapeutic strategies is increasingly evident. enveloped viruses spread infection through the replication of viral nucleic acids, assembly of viral elements, budding of mature viral particles, and subsequent fusion to surrounding cells [3] . both viruses and cancer-derived evs contain proteins and nucleic acids that are transmitted to target cells in order to promote disease progression. common morphological characteristics, protein content, and entry and release pathways between viruses and evs produced from cancer cells, with a special emphasis on breast cancer, as well as the ability of viruses to utilize ev trafficking to spread pathogenesis are discussed in this review. a variety of techniques have been used to determine the morphology and size of evs, including electron microscopy, nanoparticle tracking analysis (nta), atomic force microscopy, and flow cytometry. both exosomes and mvs are spherical in shape [4] [5] [6] . although exosomes have traditionally been described as having a cup shape when viewed using transmission electron microscopy (tem), this observation is an artifact of sample preparation and does not accurately reflect exosome morphology [7] . cancer-derived exosomes are typically smaller than mvs, with the former often ranging from 30 to 100 nm in size [5, [8] [9] [10] , and the latter having a larger size distribution, frequently being described between 100 nm to over 1 lm in diameter [6, 10, 11]. recently, an even smaller subset of evs, 8-12 nm in diameter, called homogeneous nanovesicles from various cancerous cell lines and biological samples (human mda-mb-231 breast carcinoma cells, 4t1 mouse mammary carcinoma cells, plasma from colon cancer patients and from mice implanted with 4t1 mammary tumor cells) has been reported [12] . the size of evs also can also vary depending upon the originating cell line as well as type of cancer. for example, exosomes from oral squamous cell carcinoma were 50-200 nm as measured by scanning electron microscopy [13] , while using the same nta technique those derived from skbr3 breast cancer were 183 ± 34 nm and those from murine b16f0 metastatic melanoma were 162 ± 23 nm [7] . due to the size similarities between them, there are problems obtaining purified viral particles because they are often contaminated with evs and vice versa, so specific methodologies have to be employed when isolating virions or evs to ensure a pure preparation devoid of unwanted materials [14, 15] it is well known that viruses incorporate host proteins within the viral core as well as in the envelope [22] . however, there are also many proteins identified in evs, including those derived from breast cancer, that are observed in enveloped virions as well [23, 24, 25 •• , 26]. for example, proteins involved in endosomal trafficking pathways, such as syntaxin-12 and vamp3, have also been identified within pseudorabies virus [27], as well as in evs derived from ovarian, colon, and non-small cell lung cancer [28] [29] [30] . the epstein-barr virus (ebv) and human cytomegalovirus (hcmv) have been found in breast cancer patients, although to date, no viruses have been conclusively proven to induce breast cancer [31]. one interesting study examined the proteomics of skbr3 breast cancer-derived evs, using a 10,0009g centrifugation step to isolate larger plasma membrane-derived vesicles, 100,0009g centrifugation to isolate exosomes, followed by another 100,0009g centrifugation using a density gradient, or using size exclusion chromatography instead of centrifugations, prior to lc-ms/ms [25 •• ]. table 1 lists proteins that are common to both breast cancer-derived evs and enveloped ebv and hcmv viral particles. these virions and evs contained a variety of shared protein classes, including cytoskeletal (i.e., moesin, b-actin, and filamin a), those involved in endocytosis (i.e., clathrin, pik3c2a), autophagy (i.e., heat shock proteins, 14-3-3 proteins), and endocytic transport (i.e., annexins, rab1a, cd81). there are also proteins in common between skbr3 evs and ebv/hcmv that are involved with protein folding (i.e., t-complex 1 (tcp1), chaperonincontaining tcp1 (cct) proteins), cell adhesion/migration (i.e., galectin-1, a-enolase), and cell division [i.e., cell division cycle 42 (cdc42), ran binding protein 2 (ranbp2)], indicating evs and viral particles share more than transport and degradation routes, including proteins involved in significant cellular functions. additionally, ebv, which has been shown to support the growth of nasopharyngeal cancer (npc) [32], expectedly shares many proteins with evs derived from c666-1 (ebv-infected) npc evs (table 1) [33] [34] [35] . interestingly, there seem to be more cellular proteins associated with ebv particles that have also been identified in breast cancerderived evs compared to those that have been identified in npc evs (table 1) , further supporting a link between ebv and breast cancer. these studies suggest ebv and hcmv viral particles contain common cytoskeletal proteins, chaperones, and proteins involved in migration and cell division, as well as utilize similar intracellular trafficking routes as evs, with particular similarities to breast cancer-derived evs. in addition to morphology and protein content, enveloped viruses and evs also share cellular entry and release pathways. it is important to mention that evs and enveloped viruses often utilize multiple pathways for cellular entry [36-40], perhaps to increase the efficiency of uptake. figure 1 outlines cellular entry pathways shared by cancer-derived evs and enveloped viruses. endocytic vesicles interestingly, one common thread shared by evs and viruses is the recruitment of the endosomal sorting complexes required for transport (escrts) machinery to aid in cellular release [73] . however, within the two major types of evs-ectosomes and exosomes-the role of the escrt complexes differs. exosome biogenesis utilizes all four types of escrts, escrt-0, -i, -ii, and -iii. escrt-0 recognizes ubiquitinated proteins to be packaged into intraluminal vesicles (ilvs) within multivesicular bodies (mvbs) and recruits escrt-i [73] . escrt-i and escrt-ii also bind cargo and cluster at the endosomal membrane, prior to inward vesicular budding by escrt-iii [73] . one model suggests escrt-iii, along with vps4 atpase, constricts the membrane to then facilitate fission [74] . upon maturation of the exosomal cargoes, mvbs proceed toward the outside of the cell and release their contents through fusion with the plasma membrane. this process is facilitated by a host of proteins belonging to the rab gtpases family, notably rab11, rab35, and rab27a/b [75] . bobrie et al. [76] illustrated the importance of rab27a in the secretion of exosomes from ts/a (nonmetastatic) and 4t1 (metastatic) murine mammary carcinoma cells, as knockdown of this protein resulted in significantly decreased exosome numbers. in contrast, the biogenesis of ectosomes is not well established. generally, ectosomes are formed through the outward budding and fission of the plasma membrane. one particular study showed that the escrt-i subunit tsg101 may play a role in the direct release of ectosomes through plasma membrane budding [77] . upon recruitment to the plasma membrane, tsg101 bound to the arrestin 1 domain-containing protein 1 (arrdc1) via a tetrapeptide motif, psap, and, along with vps4 atpase activity, resulted in ectosome release [77] . the discovery of arrdc1's function in vesicle formation and release gives light to the similarity of viral recruitment of the escrt machinery to mediate their release. arrestin-related trafficking proteins such as arrdc1 and thioredoxin interacting protein that bind to escrt machinery are also found to also be recruited by viruses [78] . the gag structures in viruses employ specific tetrapeptide motifs, including p(t/s)ap, ypxl, and ppxy, to recruit escrt and escrt-associated proteins. similar to ectosome formation, gag ptap binds to tsg101, which along with vps4, are required for viral release [79] . additionally, ypxl and ppxy bind to alix and nedd4 proteins, respectively, which are important in viral release [80, 81] . the ability of viruses to mimic these interactions provides evidence of their efficiency in utilizing their host cells' endogenous vesicle-releasing machinery. there are conflicting reports about the involvement of actin in ev release. one study reported that ev release from cervical cancer cells was dependent upon actin, as treatment of cells with cytochalasin d markedly reduced the amount of secreted evs [82] . however, another group demonstrated a significant increase in ev release upon treatment of ht29 colorectal cancer cells with cytochalasin d [83] . another study supported this increase in ev release from ovarian cancer cells after addition of cytochalasin d [84] , suggesting non-actin-dependent mechanisms of ev release. this discrepancy could have to do with the type of cell from which the evs are released, with different cancer cell types utilizing separate release mechanisms. however, in mda-mb-231 cells, as well as other cancer cell lines, the inhibition of arf6, a regulator of erk/mlck actomyosin-based contraction, decreased ev release, further supporting an actin-based mechanism [85] and confounding the overall necessity for actin in the release of cancer evs. the role of actin in regard to virus release is more apparent, with actin depolymerization producing a decrease in enveloped vaccinia virus, measles virus, and respiratory syncytial virus (rsv) particle release [86] [87] [88] . ceramide is a sphingolipid that is located within lipid rafts and has a functional role in their organization and protein recruitment [89] . knockdown of neutral sphingomyelinase 2 (nsmase2), which produces ceramide, resulted in a decreased amount of evs released from 4t1 mammary carcinoma cells [90] . however, ev release from prostate pc-3 cells was not affected by inhibition of nsmase2 nor inhibition of ceramide synthase, although the knockdown of lipid raft proteins flotillin-1 and flotillin-2 altered the amounts of caveolin-1 and annexin a2 in exosomes [91] . therefore, the influence of lipid raft components on ev release varies from cell to cell. rsv [92] and mlv release [93] have also been reduced upon addition of mbcd, indicating viral release can also be dependent upon lipid rafts. due to the many similarities shared between evs and enveloped viruses, it is not surprising that viruses have been postulated to exploit the exosome transport pathway as a means to incorporate host proteins and avoid immune detection, which has been coined the ''trojan exosome hypothesis'' [94] . viruses also have the unique ability to transfer nucleic acids and proteins into host exosomes to promote infectivity. the utilization of the exosomal sorting and release pathway by viruses is especially evident in cancer cells, which secrete large amounts of evs. one such example involves the hiv-1 gag protein, which was localized to endosome-like regions and released into exosomes from infected jurkat and k562 chronic myelogenous leukemia cells [95] . viruses can take advantage of these evs and use them as a means to promote infectivity, as shown by viral transfer of sever fever with thrombocytopenia syndrome virus through evs to infect host hela cells [96] . in addition to proteins, viral nucleic acids can be transferred to host cells via evs, which was demonstrated through the transfer of functional ebv mirna to host monocyte-derived cells and resulting downregulation of mirna targets [97] . cancer-associated viruses not only use the exosome transport pathway to promote viral infection, but to also spread oncogenes and proteins that will promote cancer progression. npc-derived evs have the ability to transfer the ebv latent membrane protein 1 (lmp1) as well as viral mirnas to huvec cells and activate the akt and erk signaling pathways, which are known to promote cellular transformation and enhance tumor growth [35] . a separate study also demonstrated that lmp1 upregulated hypoxiainducible factor-1a (hif-1a) in evs derived from npc cells [98] . additionally, they observed ev-associated hif-1a was involved in the increase in levels of n-cadherin in hek293t cells, which is a marker of the epithelial to mesenchymal transition [98] . therefore, enveloped viral particles associated with traditional pathogenesis as well as cancer transmission both utilize evs within host cells to increase infectivity and enhance tumor growth, respectively. the mirna content, including those that target oncogenes as well as tumor suppressors, of evs has been shown to be altered by viral infection. for example, numerous ev mirnas with multiple targets were identified to have altered expression profiles in patients with chronic hepatitis b, including mir-520b, mir-149, and mir-150 [99] . while there is no direct correlation between ebv infection and ev mirna content, there are a number of mirnas that have been identified in viral-associated cancer (i.e., breast and prostate) evs that have also shown to be modulated by ebv infection. a variety of mirnas have been identified in metastatic and/or non-metastatic breast cancer evs, including oncogenic mir-21, -23b, -27b, -181a, -378, as well as mir-26a, -151-3p, 151-5p, and let-7i [100 • ]. these mirnas have been found to have differential expression upon ebv infection of both b cells and diffuse large b cell lymphoma, with upregulation of the former mirna group, and downregulation of the latter mirna group [101, 102] . additionally, ebv-infected host cells showed increased mir-146a and mir-34a levels, with the latter occurring through lmp1 expression and nf-jb activation [101] . mir-34a and mir-146a were also identified as two prostate cancer ev biomarkers, with significantly decreased expression of mir-34a observed in prostate cancer tissue compared to healthy controls [103] . taken together, these studies suggest that ebv, a virus not only causally associated with npc but also found in breast and prostate cancers, can regulate oncogenic or tumor-suppressive mirna levels secreted into evs, perhaps to enhance tumor growth in cancer patients. over the years numerous viruses including the mouse mammary tumor virus, hpv, the bovine papilloma virus, and ebv [31] have been associated with human breast cancer, although the association has not risen to causal significance. perhaps the reason for this is that some viruses may promote rather than initiate human breast cancer. the analogies mentioned in this review between mammalian evs and enveloped viruses may explain the promotional role of both in the pathogenesis of human breast cancer. for example, hpv dna sequences from the e6 gene region have been detected in human biopsy samples of invasive ductal carcinoma [104] . the transduction of e6/e7 into mcf-7 and bt-20 breast cancer cells increased cell invasion and metastasis [105] . a link between hpv and the content of cervical cancer evs has been observed by honegger et al. [106 • ]. this group showed ev-associated mirnas are regulated by the hpv oncogene e6/e7 within cervical cancer cells. for example, let-7d-5p, mir-378a-3p, and mir-92a-3p are significantly downregulated, and mir-21-5p, mir-100-5p, and mir-30c-5p are significantly upregulated in evs by inhibition of e6/e7 in hela and/or siha cells, which led to the conclusion that e6/e7 expression influences ev mirna content to enhance the levels of those with pro-tumorigenic properties [106 • ]. these mirnas that are influenced by e6/e7 are also expressed in evs from breast cancer cells, as mentioned above for mir-21 and mir-378 [100 • ]. evs from mcf-7 cells have been found to contain and transfer mir-100 to target cells [107 • ], and mir-92a, let-7d, and mir-30c have also been located in mcf-7 evs [108] . the combination of these studies raises the possibility that hpv within infected breast cancer cells can affect the mirna content of breast cancer-derived evs. future studies could determine if hpv-regulated ev mirnas or proteins have a direct promotional role in the progression of breast cancer. cancer-derived evs, including both exosomes and mvs, are membrane-bound particles that share multiple characteristics with enveloped viruses, including morphology, protein content, and entry and release pathways. the entry of evs and a variety of enveloped viruses into target cells is ph sensitive, dependent upon lipid rafts, and utilizes actin-dependent processes such as phagocytosis and macropinocytosis, among others. the release of both particle types includes similar mechanisms as those used for entry, including those involving actin as well as lipid rafts, but both viruses and evs utilize escrt machinery for cellular release as well. in order to disseminate infection, enveloped viruses exploit the endosomal trafficking pathway, incorporating host proteins during intracellular transport. viruses, including those associated with cancer, take advantage of the large quantities of evs released from normal and transformed cells, using them to incorporate viral oncogenes and nucleic acids into target cells, which can enhance the spread of infection and cancer development. in the case of human breast cancer, although a causal viral association has not been demonstrated, the presence of ebv and hpv dna in human breast cancer biopsies suggests a promotional role. many cancer-associated mirnas that are regulated by hpv are also present in breast cancer-derived evs, suggesting the possibility of viral-linked ev content and breast cancer promotion. furthermore, breast cancer-derived evs have a particularly large number of proteins, including those found in the endocytic pathway, which have also been identified in viral particles, supporting mutual cellular transport routes. the multitude of these similarities suggests evs, including those derived from breast cancer, and viruses may have evolved from a common origin, with both serving as tools for the pathogenesis of disease. the escrt pathway assembly and disassembly of the escrt-iii membrane scission complex biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression formation and release of arrestin domain-containing protein 1-mediated microvesicles (armms) at plasma membrane by recruitment of tsg101 protein multiple interactions between the escrt machinery and arrestin-related proteins: implications for ppxy-dependent budding tsg101 and the vacuolar protein sorting pathway are essential for hiv-1 budding structural and functional studies of alix interactions with ypx(n)l late domains of hiv-1 and eiav rescue of hiv-1 release by targeting widely divergent nedd4-type ubiquitin ligases and isolated catalytic hect domains to gag survivin is released from cancer cells via exosomes the protein interaction network of extracellular vesicles derived from human colorectal cancer cells lysophosphatidic acid stimulates fas ligand microvesicle release from ovarian cancer cells arf6-regulated shedding of tumor cell-derived plasma membrane microvesicles a36-dependent actin filament nucleation promotes release of vaccinia virus actin filaments disruption and stabilization affect measles virus maturation by different mechanisms cooperativity of actin and microtubule elements during replication of respiratory syncytial virus ceramide recruits and activates protein kinase c f (pkcf) within structured membrane microdomains neutral sphingomyelinase 2 (nsmase2)-dependent exosomal transfer of angiogenic micrornas regulate cancer cell metastasis regulation of exosome release by glycosphingolipids and flotillins cholesterol-rich lipid rafts are required for release of infectious human respiratory syncytial virus particles murine leukemia virus glycosylated gag (gpr80gag) facilitates interferonsensitive virus release through lipid rafts the trojan exosome hypothesis exosomes and hiv gag bud from endosome-like domains of the t cell plasma membrane extracellular vesicles mediate receptor-independent transmission of novel tick-borne bunyavirus functional delivery of viral mirnas via exosomes exosomal hif1a supports invasive potential of nasopharyngeal carcinoma-associated lmp1-positive exosomes identification of circulating microvesicles' microrna expression profiles and analysis of functional roles in chronic hepatitis b exosomemediated microrna signaling from breast cancer cells is altered by the anti-angiogenesis agent docosahexaenoic acid (dha) this article describes mirnas identified in a variety of breast cancer cell exosomes, and how their expression and resulting angiogenic regulation are altered by dha the epstein-barr virus (ebv)-induced tumor suppressor microrna mir-34a is growth promoting in ebv-infected b cells microrna profiling in epstein-barr virus-associated b-cell lymphoma mir-34a is an intracellular and exosomal predictive biomarker for response to docetaxel with clinical relevance to prostate cancer prediction identification of human papillomavirus dna gene sequences in human breast cancer e6/e7 of hpv type 16 promotes cell invasion and metastasis of human breast cancer cells this article describes how the hpv e6/e7 oncogene regulates intracellular and exosomal mirna content, specifically up-and downregulating specific mirnas which are known to effect tumor growth exosomes from drugresistant breast cancer cells transmit chemoresistance by a horizontal transfer of micrornas selective release of microrna species from normal and malignant mammary epithelial cells key: cord-340228-mvqoyror authors: al-herz, waleed; essa, sahar title: spectrum of viral infections among primary immunodeficient children: report from a national registry date: 2019-05-29 journal: front immunol doi: 10.3389/fimmu.2019.01231 sha: doc_id: 340228 cord_uid: mvqoyror objective: to present the frequency and spectrum of viral infections in primary immunodeficient children. methods: the data was obtained from the kuwait national primary immunodeficiency disorders (pids) registry during the period of 2004-2018. results: a total of 274 pid children were registered in knpidr during the study period with predominance of immunodeficiencies affecting cellular and humoral immunity, followed by combined immunodeficiencies with associated syndromic features and diseases of immune dysregulation. overall infectious complications affected 82.4% of the patients, and viral infections affected 31.7% of the registered patients. forty-five patients (16.4%) developed viral infections caused by at least 2 organisms, among those 20 patients were affected by three or more viral infections. there was a statistically significant association between viral infections and pid category. however, there was no statistically significant association between viral infections and gender or the patients' onset age. there was a total of 170 viral infections during the study period and the causes of these infections were predominated by cmv (22.2%), adenovirus (11.7%), ebv (11.1%), and enteroviruses (7.4%). cmv and parainfluenza infections were more common in the group of immunodeficiencies affecting cellular and humoral immunity while ebv and human papilloma virus (hpv) were more common in the immune dysregulation group and combined immunodeficiencies with associated syndromic features, respectively. the most common presentation was viremia (28.8%) followed by pneumonia (28.2%) and skin infections (17.6%). the most common causes of viremia were cmv followed by adenovirus and ebv, while the most common organisms causing pneumonia were cmv followed by rhinovirus and parainfluenza. there were 80 deaths among the registered patients, 10% were caused by viral infections. conclusions: viral infections are common in pids and result into a wide-range of clinical manifestations causing significant morbidity and mortality. primary immunodeficiency disorders (pids) are monogenic defects affecting the innate and/or adaptive immune systems (1) . patients are at increased risk of a wide range of manifestations including autoimmunity, immune dysregulation, and malignancies, but infectious complications are the commonest (2) (3) (4) . historically, pid patients used to die before recognition because of infections due to the lack of effective measures to either prevent or treat them. advances in public health and the discovery of antimicrobial agents made the diagnosis of pids possible (5) . although therapeutic interventions like intravenous immunoglobulins and hematopoietic stem cell transplants have helped to decrease morbidity and mortality, physicians caring for pid patients frequently struggle with treating infections which are usually recurrent or chronic, severe and are frequently caused by opportunistic organisms. among these microbes are viruses that in pid patients can be challenging with an increased risk of mortality and may predispose to malignancies (6) (7) (8) . pids may also lead to reduced clearance and prolonged shedding of certain viruses like rhinovirus and poliovirus (9, 10) . while most pids predispose to a wide spectrum of viral infections, certain diseases enhance vulnerability to specific viral infections (11) . whereas, the risk of viral infections in pids is well-established and was recently reviewed (12, 13) , we are not aware of any report that characterizes such infections in a large cohort of patients with different types of pids. in this report, we present the frequency and spectrum of viral infections in primary immunodeficient children from kuwait between january 2004 and december 2018. the data was obtained from the kuwait national primary immunodeficiency disorders registry (knpidr) which was approved by the research and ethics committee of the ministry of health in kuwait and the kuwait university health sciences center ethical committee in accordance with the declaration of helsinki. the patients were followed prospectively and classified according to the international union of immunological societies, primary immunodeficiency diseases committee report on inborn errors of immunity (2017) (1). secondary immunodeficiencies (drug induced, hiv induced, and immunodeficiency associated with metabolic disorders... etc.), were ruled out by obtaining a detailed history and by performing appropriate testing when these disorders were suspected. the clinical diagnosis was based on the standard of care depending on the patient's signs and symptoms supported by laboratory and/or radiologic findings. for example, patients who were diagnosed with pneumonia presented with respiratory signs and symptoms associated with radiologic findings. the diagnosis of herpes simplex virus (hsv) keratitis and stomatitis, warts caused by human papilloma virus (hpv), varicella-zoster virus (vzv) and molluscum contagiosum infections was based on clinical evaluation. in-house polymerase chain reaction (pcr) was used initially to test for cytomegalovirus (cmv), epstein-barr virus (ebv), herpesvirus 6 (hhv-6), adenovirus, respiratory and gastrointestinal viruses. this was replaced later by commercial kits as indicated below. for patients with gastrointestinal manifestations stool or colonic samples were collected. deep nasopharyngeal aspirate or bronchoalveolar lavage samples were collected from patients with respiratory manifestations using sterile nylon flocked swab or by bronchoscopy and placed in viral transport medium. serum and cerebrospinal fluid samples were collected for viral infections as required. all samples were labeled and transported the earliest to virology laboratory, faculty of medicine, kuwait university for day-to-day routine screening for viral infections and storage. viral nucleic acid from samples was extracted using an automated magna pure lc 2.0 system (roche diagnostic systems, branchburg, nj). hsv (2) keratitis (2) hpv (2) warts (2) cmv (8) viremia (4), pneumonia (6) adenovirus (3) viremia (2), pneumonia (1) enterovirus (3) hemophagocytosis (1), viremia (1), pneumonia (1) norovirus (1) immunodeficiency with centromeric instability and facial anomalies (2) sapovirus (2) enteritis (2) adenovirus (2) enteritis (1), viremia (1) predominantly antibody deficiencies (n = 6) btk deficiency (1) enterovirus (1) meningo-encephalitis µ heavy chain deficiency (1) enterovirus (1) (1) vzv (1) chickenpox lyst deficiency (1) ebv (1) hemophagocytosis cmv (2) colitis (1), viremia (2) ebv (2) stool samples were homogenized. prior to extraction, a 200 mg aliquot was suspended in 1 ml of nuclease-free water or 1 ml of stool transport and recovery buffer (roche diagnostics, meylan, france). then the suspension was immediately clarified by centrifugation at 11,000 g for 5 min. according to the manufacturer's instructions rt-pcrs was performed on 10 µl of nucleic acid by using lightcycler 480 rt-pcr thermocycler (roche, meylan, france). cmv, ebv, enterovirus, hadv, and hhv-6 detection were performed on serum, stool/colonic, respiratory or csf samples by lightmix r kits (tib molbiol, berlin, germany). amplifications were performed according to the manufacturer's instructions on lightcycler 480 rt-pcr thermocycler (roche, meylan, france). the presence of poliovirus rna in clinical samples was confirmed by one-step reverse transcription-pcr, followed by a direct sequencing of pcr products, as described previously (14) . data were processed using ibm spss, version 25 (ibm corporation, armonk, ny, usa 2017). pearson's chi-square test was used to assess the association between two categorical variables. the non-parametric mann-whitney u-test was applied to assess whether the patients' ages at onset of a symptom of pid have a significant effect on the risk of viral infection. the effect of age at onset was assessed both as quantitative and qualitative variables after dividing them into groups (0-5, 6-11, 12-24, 25-48, > 48 months). the p ≤ 0.05 was used as the cut-off level for statistical significance. a total of 274 pid children (142 males and 132 females) were registered in knpidr during the study period. the distribution of these patients according to pid categories is: immunodeficiencies affecting cellular and humoral immunity, 97 patients (35.4%); combined immunodeficiencies with associated syndromic features, 67 patients (24.5%); predominantly antibody deficiencies, 34 patients (12.4%); diseases of immune dysregulation, 47 patients (17.2%); congenital defects of phagocyte number or function, 17 patients (6.2%); autoinflammatory disorders, 1 patient (0.3%); and complement deficiencies, 11 patients (4%). no patients with defects in innate immunity were registered. seventy-one patients were treated with hematopoietic stem cell transplant (hsct) and 141 received intravenous immunoglobulins. it is important to mention that of the reported viral infections occurred prior to hsct in patients who received such treatment. overall infectious complications affected 226 patients (82.4%), and viral infections affected 87 patients (31.7% of the registered patients). forty-five patients (16.4%) developed viral infections caused by at least 2 organisms, mostly in the category of immunodeficiencies affecting cellular and humoral immunity (31 patients). among those, 20 patients were affected by three or more viral infections. there was a statistically significant association between viral infections and pid category after excluding patients who belong to congenital defects of phagocyte number or function, autoinflammatory disorders and complement deficiencies due to low numbers (p < 0.001) ( table 1) . however, there was no statistically significant association between viral infections and gender (p = 0.488), or the patients' onset age when assessed both as quantitative and qualitative variables (p-values 0.23 and 0.655, respectively). there was a total of 170 viral infections during the study period, 33% were detected at the time of pid diagnosis while 67% were documented after establishing the diagnosis. the causes of these infections were: cmv (22.2%); adenovirus (11.7%); ebv (11.1%); enteroviruses (7.4%), hsv and hpv (6.8% each); vzv and rhinovirus (6.2% each); molluscum contagiosum (5.5%) (figure 1) ; norovirus and parainfluenza virus (3% each); h1n1 virus (1.85%); rotavirus, rsv, sapovirus, hhv-6 and corona virus (1.2% each). a patient with severe combined immunodeficiency presented with myocarditis caused by poliovirus type 2. two patients (1 with severe combined immunodeficiency and 1 with mhc ii deficiency) had prolonged excretion of poliovirus type 1 in the stool. the details of the viral infections are presented in table 2 . figure 2 shows the number of patients affected by different viruses according to pid categories. the most prominent findings are that cmv and parainfluenza infections are more common in the group of immunodeficiencies affecting cellular and humoral immunity while ebv and hpv are more common in the immune dysregulation group and combined immunodeficiencies with associated syndromic features, respectively. the most common presentation was viremia (28.8%) followed by pneumonia (28.2%) and skin infections (17.6%) ( table 3) . the most common causes of viremia were cmv followed by adenovirus and ebv, while the most common organisms causing pneumonia were cmv followed by rhinovirus and parainfluenza ( table 3) . in the current study, we present the characteristics of viral infections in a large cohort of pid children who were followed prospectively over a period of 15 years. viral infections affected more than 1/3 of the registered patients, many of whom were affected by more than 1 virus. the patients were affected by a range of viral organisms but cmv, adenovirus and ebv were the culprits in almost half of the cases. the high frequency of cmv infections (>20%) in our cohort can be explained by the fact that most of the cases are affected by combined immunodeficiencies. patients with such defects are extremely susceptible to progressive infection with cmv (12) . our finding that cmv and parainfluenza infections are more common in the group of immunodeficiencies affecting cellular and humoral immunity has been documented previously (7, 15) . the observation that ebv is more common in the immune dysregulation group specifically triggering hlh is also welldocumented (8, 12) . we have found that patients with dock8 deficiency are particularly predisposed to mucocutaneous viral infections like molluscum contagiosum and hsv infections. this is probably since dock8 is an important regulator of the actin cytoskeleton that is critical for cell migration through collagendense tissue, hence playing an important antiviral immunity in the skin (16) . the presented cohort of patients are characterized by the high frequency of combined immunodeficiencies which are more severe with a higher predisposition to viral infections compared to other pid categories. another prominent feature of our cohort is that none of the registered patients suffer from adenovirus (12) ebv (10) enteroviruses (6) hhv-6 (2) pneumonia 28.2 cmv (18) rhinovirus (9) parainfluenza (5) adenovirus (4) h1n1 (3) coronavirus (2) rsv (2) ebv (2) enterovirus (1) skin infections 17.6 hpv (11) vzv (10) molluscum contagiosum (9) gastrointestinal infections 9.4 norovirus (5) enteroviruses (4) adenovirus (2) cmv (2) sapovirus (2) rotavirus ( increased susceptibility to specific viral infections. examples of such diseases are tlr3, trif, or unc93b1 deficiencies which predispose to hsv-1 encephalitis and epidermodysplasia verruciformis or cxcr4 deficiencies which predispose to hpv. it is important to stress that physicians should be aware of pids and consider them in patients with severe or recurrent viral infections. importantly, they should be aware that many pids result in poor antibody response, hence serologic testing should be avoided while testing a patient for infectious complications and antigenic detection method should be used instead. health care providers should also be aware of the recommendations for live viral vaccines in immunodeficient patients and their close contacts (17) . live vaccines, such as the chicken pox, measles, mumps, rubella (mmr), rotavirus, yellow fever, oral polio, and the influenza nasal spray should be avoided in certain types of pids. furthermore, any infants born into a family with a suspicious history of pid should avoid all live viral and bacterial vaccines until pids is ruled out. historically, oral polio vaccine (opv) was the only form used in the vaccination schedule in kuwait. since 2008 the first dose of opv given at the age of 2 months was replaced with the inactivated formulation. fortunately, only 1 patient from our cohort who was diagnosed with rag1 deficiency developed opv related complication (i.e., myocarditis). two more patients with cid had prolonged excretion of poliovirus type 1 in the stool. unfortunately, stool surveillance program of pid patients for vaccine derived polio virus is not available in the country. the present study has some limitations since we did not determine the true burden of viral infections in pids. this could be established by documenting the number of admissions to the intensive care unit and the type of care provided like mechanical ventilation and the use of inotropes during these admissions. other important variables that can be considered are the number of viral infection reactivations, the number of admissions to the hospital, the length of stay, and duration of using antiviral treatments. however, an important strength of the study is that the patients were followed prospectively by the same clinical immunologist. another important strength of the study is that most patients were diagnosed at the molecular level. this may help in determining the genotype-phenotype correlation. yet, collaborative efforts will be needed to collect a bigger number of patients. viral infections in pids should be treated aggressively with appropriate antiviral medications and definitive treatments like hsct when possible since failure to eradicate viral pathogens creates an inflammatory environment that promotes cell survival and proliferation and may predispose to malignancy (18) . innovative treatments like virus-specific t cells should be explored to improve clinical outcomes for this group of patients (19) . all datasets generated for this study are included in the manuscript and/or the supplementary files. the data was obtained from the kuwait national primary immunodeficiency disorders registry (knpidr) which was approved by the research and ethics committee of the ministry of health in kuwait and the kuwait university health sciences center ethical committee in accordance with the declaration of helsinki. wa-h: development of the research concept and goals, design of methodology, data collection and analysis, writing the initial manuscript draft, approval of the submitted manuscript, and agreement to be accountable for the content of the work. se: contributed to the research idea, writing of the manuscript and approval of the submitted manuscript and agreement to be accountable for the content of the work. knpidr was funded by kuwait foundation for the advancement of sciences. international union of immunological societies: 2017 primary immunodeficiency diseases committee report on inborn errors of immunity primary immunodeficiency disorders in iran: update and new insights from the third report of the national registry primary immunodeficiency disorders in kuwait: first report from kuwait national primary immunodeficiency registry primary immunodeficiency diseases in oman: 10-year experience in a tertiary care hospital history of primary immunodeficiency diseases survival and predictors of death among primary immunodeficient patients: a registry-based study patients with primary immunodeficiencies in pediatric intensive care unit: outcomes and mortality-related risk factors primary immunodeficiency diseases associated with increased susceptibility to viral infections and malignancies patients with primary immunodeficiencies are a reservoir of poliovirus and a risk to polio eradication virus shedding after human rhinovirus infection in children, adults and patients with hypogammaglobulinaemia human tlrs and il-1rs in host defense: natural insights from evolutionary, epidemiological, and clinical genetics recurrent and sustained viral infections in primary immunodeficiencies severe viral infections and primary immunodeficiencies echovirus type 9 is an important cause of viral encephalitis among infants and young children in kuwait respiratory virus infection in immunocompromised patients dock8 regulates lymphocyte shape integrity for skin antiviral immunity recommendations for live viral and bacterial vaccines in immunodeficient patients and their close contacts inflammation as a tumor promoter in cancer induction virus-specific t cells: current and future use in primary immunodeficiency disorders the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 al-herz and essa. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-023854-w8kx5n8k authors: schuster, v.; kreth, h. w. title: virusinfektionen date: 2019 journal: p&#x000e4;diatrie doi: 10.1007/978-3-662-57295-5_14 sha: doc_id: 23854 cord_uid: w8kx5n8k dieses kapitel behandelt klinik, komplikationen, diagnostik, prophylaxe und therapie der wesentlichen virusinfektionen im kindesund jugendlichenalter, u. a. virusinfektionen der herpes-gruppe (hsv1 und 2 [u. a. schwere neonatale infektionen, herpesenzephalitis]), vzv [u. a. neonatale vzv-infektionen, herpes-zoster], ebv, cmv, hhv6–7 [dreitagefieber], hhv8 [kaposi-sarkom]. unter den virushepatititiden spielen bei kindern die hepatitis a, b (chronische hepatitis b) sowie c (chronische hepatitis b) die größte rolle. weitere bedeutsame viren sind parvovirus-b19 (ursache der ringelröteln, einer aplastischen krise oder eines hydrops fetalis), humane papillomaviren (bedeutsam v. a. genitalwarzen, larynxpapillome und karzinome), rotaviren (bedeutsame gastroenteritiserreger), fsme, hiv sowie die „typischen kinderkrankheiten“ masern, mumps und röteln. häufige erreger des oberen und unteren respirationstrakts sind rhinoviren, das rs-virus, das humane metapneumovirus (hmpv), das humane bocavirus (hbov), das humane coronavirus (hcov) sowie influenzaund parainfluenzaviren. herpes-simplex-virus-infektionen j epidemiologie infektionen mit dem herpes-simplex-virus (hsv) treten ubiquitär auf. die ansteckung erfolgt bei kindern überwiegend durch virushaltige körperflüssigkeiten (speichel) und engen körperkontakt, seltener auch durch organtransplantation. die durchseuchung von hsv-1 schwankt zwischen 30% (länder mit höherem lebensstandard) und 90% (ärmere länder). die häufigkeit von hsv-2-infektionen korreliert mit der sexuellen aktivität der jeweils untersuchten bevölkerungsgruppe. die inkubationszeit beträgt 2-12 tage. j ätiopathogenese es existieren 2 herpes-simplex-viren, typ 1 (hsv-1) und typ 2 (hsv-2): 4 infektionen mit hsv-2 sind häufig mit erkrankungen im genitalbereich assoziiert. insbesondere sind infektionen des feten oder neugeborenen meist durch hsv-2 verursacht. 4 hsv-1-infektionen sind überwiegend im gesichtsbereich lokalisiert. hsv repliziert sich in mukosazellen (v. a. rachenraum, genitalschleimhaut) . anschließend dringt das virus in die nervenendigungen von peripheren sensorischen nerven ein und wandert in ihnen retrograd bis zu den spinalen hinterstrangganglien (bei hsv-1 meist ganglion des n. trigeminus, bei hsv-2 häufig sakralganglien; . abb. 14.1). an diesem ort liegt hsv in latenter form vor (keine produktion von infektiösem virus) und persistiert lebenslang im wirt. durch verschiedene faktoren (z. b. immunsuppression, stress) kann das virus jederzeit reaktiviert werden. nach einer solchen reaktivierung "wandert" hsv anterograd über die peripheren sensorischen nerven zur mukosaoberfläche des entsprechenden dermatoms und führt dort zur bläschenbildung mit aktiver virusreplikation (herpes labialis, rekurrierender herpes genitalis). entscheidend für die immunologische bewältigung einer hsv-infektion ist die zelluläre immunität. diaplazentar übertragene hsv-neutralisierende antikörper können bei exponierten neugeborenen eine hsv-infektion u. u. verhindern oder zumindest die schwere der erkrankung abmildern. dagegen können hsv-spezifische antikörper weder rekurrierende hsv-erkrankungen noch exogene hsv-infektionen verhindern. intrauterine herpes-simplex-virusinfektion in seltenen fällen kann es zu einer diaplazentaren, hämatogenen hsv-infektion des feten (überwiegend hsv-2) kommen. betroffene kinder sind meist hypotroph (85%), fast alle zeigen kurz nach geburt ein bläschenför-. abb. 14.1 pathogenese des herpes zoster und des rekurrierenden herpes simplex. oben: nach abklingen der primärinfektion mit vzv (varizellen) oder hsv (gingivostomatitis oder primärer herpes genitalis) wandern die viren retrograd entlang der sensorischen nerven zu den spinalganglien des rückenmarks, wo die viren lebenslang persistieren. unten: durch verschiedene faktoren (z. b. immunsuppression) kann vzv und hsv reaktiviert werden. beide viren wandern anschließend entlang der sensorischen nerven zu haut-bzw. schleimhaut (dermatom), wo es zur lokalen virusvermehrung und ausbildung von bläschen kommt: herpes zoster bei vzv, herpes labialis oder genitalis bei hsv die infektion beginnt mit unspezifischen symptomen (fieber, kopfschmerzen, krankheitsgefühl) . nach 1-7 tagen kommt es zu einer progressiven neurologischen symptomatik (fokale oder generalisierte krampfanfälle, verhaltensauffälligkeiten, vigilanzstörungen) bis hin zum koma. unbehandelt versterben 70% der patienten. verschiedene genetische faktoren prädisponieren für eine herpesenzephalitis (u. a. mutationen in den genen stat1, nemo, tlr3, traf3, . bildgebende verfahren (kraniales ct oder mr) und eeg zeigen im "typischen" fall fokale veränderungen uni-oder bilateral v. a. im bereich der temporallappen. im liquor findet sich meist eine pleozytose (überwiegend lymphozyten) und eine starke ei weißerhöhung. in bis zu 85% ist der liquor als folge der ausge dehnten nekrosen im zns hämorrhagisch. im frühstadium einer hsv-enzephalitis kann der liquor noch vollkommen unauffällig sein. die aciclovirtherapie hat die letalität auf ca. 29% gesenkt. eine vollständige ausheilung ohne residualfolgen findet sich in 38% der mit aciclovir behandelten enzephalitispatienten, bei kindern liegt der prozentsatz höher. bei unbehandelten patienten mit einer herpesenzephalitis dagegen kommt es nur in 2,5% der fälle zu einer restitutio ad integrum. chronisch rezidivierende verläufe, auch bei mit aciclovir behandelten patienten, kommen gelegentlich vor. ösophagus, der gastrointestinaltrakt, der respirationstrakt (pneumonie), das zns (enzephalitis) und andere organe (leber, nieren, milz, nebennieren) betroffen. j diagnose häufig kann die diagnose einer herpesinfektion der haut oder der schleimhäute aufgrund der typischen herpeseffloreszenzen klinisch gestellt werden. in zweifelsfällen wird hsv leicht aus bläscheninhalt, schleimhautabstrichen und bioptischem material isoliert. methode der wahl für die diagnose einer hsv-enzephalitis ist der hsv-genomnachweis im liquor mittels polymerasekettenreaktion. der serologische nachweis von spezifischen hsv-antikörpern im serum oder liquor spielt in der frühdiagnostik von hsv-infektionen nur eine sekundäre rolle. bei einer unklaren enzephalitis kann der nachweis von intrathekal produzierten hsv-antikörpern am tag 7-10 nach auftreten der symptome die ursache der erkrankung nachträglich beweisen. j therapie mittel der wahl bei hsv-infektionen im kindesalter ist das nukleosidanalogon aciclovir. zur behandlung von neonatalen hsv-infektionen sowie der herpesenzephalitis wird aciclovir in einer dosierung von 3-mal 15(-20) mg/kgkg/tag i.v. (frühgeborene nur 2-mal 10 mg/kgkg/ tag i.v.) für mindestens 14, besser 21 tage eingesetzt. bei zu kurzer aciclovirtherapie einer hsv-enzephalitis (<14 tage) kann es zu rezidiven kommen. neugeborene mit hsv-infektion und neurologischer beteiligung sollten im anschluss an die i.v.-aciclovirtherapie eine orale suppressionstherapie mit 3×300 mg/m 2 kö aciclovir über 6 monate erhalten. hierdurch wird das outcome hinsichtlich der neurologischen entwicklung verbessert. > bei klinischem verdacht auf herpesenzephalitis beginne man sofort mit einer ausreichend hoch dosierten intravenösen aciclovirtherapie, ohne die endgültige labordiagnostik abzuwarten. eine stomatitis aphthosa oder ein herpes labialis beim immunkompetenten kind wird im normalfall nur symptomatisch (z. b. mit bepanthenlösung oder -salbe) behandelt. bei allen komplizierten hsv-infektionen einschließlich dem herpes genitalis ist aciclovir derzeit das mittel der wahl (i.v., oral, topisch) . bei aciclovirresistenten hsv-stämmen (immunsupprimierte patienten) kann ein therapieversuch mit foscarnet unternommen werden. für die topische behandung einer hsv-keratokonjunktivitis stehen verschiedene wirksame medikamente zur verfügung wie acicloviraugensalbe und trifluridinaugentropfen. die therapie muss immer in enger zusammenarbeit mit einem diesbezüglich erfahrenen augenarzt erfolgen. bei patienten >18 jahren kann ein genitaler herpes (primärinfektion, rezidive) auch mit famciclovir oder valaciclovir behandelt werden. j prophylaxe bei schwangeren mit aktiver genitaler herpesinfektion (sowohl hsv-erstinfektion als auch -rezidiv) am geburtstermin sollte die geburt durch kaiserschnitt erfolgen, sofern der blasensprung nicht länger als 4-6 h zurückliegt. bei frauen mit rezidivierendem herpes genitalis in der spätschwangerschaft senkt eine orale aciclovir-oder valganciclovirtherapie die häufigkeit von hsv-2-rezidiven zum zeitpunkt der geburt. mütter mit florider hsv-1-infektion dürfen nur dann stillen, wenn die brust frei von frischen hsv-effloreszenzen ist und andere aktive läsionen abgedeckt sind. familienangehörige mit floridem herpes labialis müssen beim besuch eines neugeborenen immer einen mundschutz tragen und dürfen das kind nicht küssen. die labialen herpesläsionen müssen außerdem vorher mit aciclovirsalbe abgedeckt werden. eine langzeitchemoprophylaxe mit aciclovir kann bei immunsupprimierten und transplantierten patienten die häufigkeit (und schwere) von hsv-infektionen und -reaktivierungen signifikant senken. j epidemiologie varizella-zoster-virus (vzv) kommt ubiquitär vor und ist hochkontagiös. eine krankheitshäufung findet sich in den späten wintermonaten und im frühjahr. varizellen treten vorwiegend im kindesalter auf; bis zum 16. lebensjahr sind über 90% aller kinder infiziert. die ansteckung mit vzv erfolgt meist durch direkten kontakt von mensch zu mensch, seltener aerogen. die infektiosität bei varizellen beginnt 1-2 tage vor auftreten des exanthems und endet ca. 5 tage nach exanthemausbruch (immunkompetente kinder). der herpes zoster ist weniger kontagiös als varizellen. der kontakt mit einem zosterpatienten führt bei einer seronegativen person zu windpocken. die inkubationszeit bei varizellen beträgt meist 2 wochen (10-28 tage). j ätiopathogenese vzv gehört zur gruppe der herpesviren. eintrittspforte für vzv sind die schleimhäute der oberen atemwege. nach initialer virusvermehrung tritt nach 3-4 tagen eine erste virämie auf. hierbei wird vzv in t-zellen über den blutstrom im ganzen körper verteilt. in leber und milz findet anschließend eine massive virusvermehrung statt. am tag 6-7 post infectionem (p. i.) kommt es zur 2. virämie: hierbei wird vzv auch in die peripherie zur haut und zu den schleimhäuten transportiert. infizierte haut-und schleimhautzellen gehen bei der infektion zugrunde, es bilden sich die typischen bläschen mit virushaltigem inhalt. nach abklingen der varizellen wandert vzv retrograd entlang der peripheren sensorischen nerven zu den spinalganglien des rückenmarks (n. trigeminus, thorakale ganglien u. a.), wo das virus lebenslang persistiert (. abb. 14.1). in diesen spinalganglien liegt eine latente vzv-infektion vor, d. h. es wird kein komplettes virus produziert. vzv kann bei nachlassender zellulärer immunität sowie durch noch unbekannte mechanismen jederzeit reaktiviert werden: vzv wandert nun entlang der sensorischen peripheren nerven anterograd an die hautoberfläche, wo es im bereich der betroffenen dermatome zur virusvermehrung mit bläschenbildung (herpes zoster) kommt. . abb. 14.4 herpes labialis bei einem 8 jahre altem mädchen im gegensatz zu varizellen, bei denen es im rahmen der 2. virämie zu einem schubweisen auftreten von bläschen kommt (sternenhimmelbild mit verschiedenen stadien von effloreszenzen), befinden sich die bläschen beim herpes zoster im gleichen entwicklungsstadium: es liegt ein uniformes exanthem vor. für die immunologische kontrolle einer vzv-infektion ist das zelluläre immunsystem entscheidend. vzv-neutralisierende antikörper können die schwere des verlaufs von varizellen abmildern und u. u. auch eine vzv-infektion verhindern, insbesondere dann, wenn sie vor eintritt der primären virämie verabreicht werden. > windpocken treten nur einmal im leben auf. zweiterkrankungen sind sehr selten (ca. 1-2%). meist manifestieren sich windpocken als typisches bläschenförmiges exanthem (. abb. 14.5) mit nur leichtem fieber in den ersten 2-3 krankheitstagen. die effloreszenzen treten zunächst v. a. im gesicht, am behaarten kopf, und am stamm auf, weniger häufig kommt es zu einer zentrifugalen ausbreitung auf die extremitäten. die handinnenflächen sind meist ausgespart. frisch aufgetretene bläschen, die klare virushaltige flüssigkeit enthalten, trocken rasch ein und bilden häufig krusten. daneben treten immer wieder neue bläschen auf. diese hautveränderungen entwickeln sich schubweise mit einer dauer von bis zu 8 tagen und sind oft von einem starken juckreiz begleitet. durch kratzen kann es in betroffenen hautregionen zu exkoriationen und späterer narbenbildung kommen. bei der enzephalitis (häufigkeit ca. 1:10.000), die bereits früher im verlauf der varizellen auftritt und die im allgemeinen eine schlechtere prognose hat, kommt es zu schweren krampfanfällen und bewusstlosigkeit mit exitus letalis oder ausgeprägten defektheilungen. zerebrale insulte in form von akut auftretenden hemiplegien können erst nach monatelanger latenz nach einer vzv-infektion auftreten. andere seltene komplikationen sind peri-/parainfektiöse thrombozytopenische purpura (itp), purpura fulminans, myokarditis, arthritis, nephritis und reye-syndrom. bei zellulären immundefekten und immunsuppression (organtransplantation, hiv-infektion, immunsuppressive therapie, maligne grunderkrankungen) kommt es bei kindern häufig zu schweren progressiven varizellen mit viszeraler beteiligung wie pneumonie, meningoenzephalitis, hepatitis und pankreatitis. die letalität beträgt bis zu 20%. vari zellen im 1. und 2. schwangerschaftstrimenon (v. a. in der 13.-20. schwangerschaftswoche) führen in bis zu 2% zu einem konnatalen varizellensyndrom (cvs) mit hautnarben, gliedmaßenhypoplasien, dystrophie, katarakt sowie zns-schädigungen (. abb. 14.6). varizellen während der schwangerschaft (v. a. in der 16.-33. schwangerschaftswoche) können außerdem (in über 1%) zum auftreten eines herpes zoster im ersten lebensjahr führen. ein herpes zoster bei einer immunkompetenten schwangeren dagegen führt nur extrem selten zu einer konnatalen oder neonatalen vzv-infektion. j ätiopathogenese ebv gehört zur gruppe der herpesviren. eintrittspforte für ebv ist der rachenraum (waldeyer-rachenring, tonsillen), wo das virus zu einer sog. lytischen infektion des lymphoepithelialen gewebes (b-zellen, epithelzellen) mit anschließender produktion von infektiösem ebv führt (invasive phase). im weiteren verlauf (nach ca. 2 wochen) kommt es zur virämie oder mehreren virämischen phasen. hierbei werden ebv-infizierte b-zellen über den blutstrom in andere organe (leber, milz, knochenmark, lymphknoten, evtl. zns) transportiert, erst nach einer inkubationszeit von 10-50 tagen treten die klinische symptome auf. in den im blut zirkulierenden b-zellen kommt es zunächst noch zu einer lytischen ebv-infektion mit produktion von infektiösem virus, später liegt nur noch eine sog. latente infektion vor, d. h. es werden nur noch wenige virusantigene (kernantigene ebna1-6 und membranantigene lmp1 und 2) exprimiert. diese b-zellen werden hierdurch zu lymphoblastoiden zellen "transformiert" und erwerben die fähigkeit zur unbegrenzten teilung und vermehrung (immortalisation). beim immunkompetenten menschen werden nach einer ebv-infektion rasch aktivierte zytotoxische t-zellen vom cd8+-typ gebildet, die selektiv nur die ebv-infizierten b-zellen weitestgehendeliminieren. diese aktivierten t-zellen bilden einen großen anteil der typischen "pfeiffer-zellen" (syn. lymphatische reizformen, virozyten) und der teilweise extremen lymphozytose im blutbild von patienten mit akuter infektiöser mononukleose (. abb. 14.8). nach durchgemachter ebv-infektion persistiert ebv lebenslang in ruhenden b-zellen im knochenmark. ebv kann in diesen zellen jederzeit reaktiviert werden. bei eingeschränkter zellulärer immunität (z. b. nach medikamentöser immunsuppression, aids) können sich diese b-zellen -abhängig vom ausmaß der immunsuppression -expandieren und so zu schweren lymphoproliferativen krankheitsbildern und b-zelllymphomen führen. in 70-90% tritt initial eine ausgeprägte tonsillopharyngitis mit fibrinbelägen (. abb. 14.10) auf, die in der 2. krankheitswoche meist rasch abheilt. eine splenomegalie findet sich bei 50-60% der patienten in der 2. und 3. krankheitswoche. seltener (15-25%) ist eine hepatitis mit und ohne ikterus. in 5-10% treten meist flüchtige morbilliforme exantheme auf. lymphoproliferative krankheitsbilder kinder und jugendliche mit angeborenen zellulären immundefekten (x-chromosomale lymphoproliferative erkrankung, xlp u. a.), aber auch mit erwor bener immundefizienz (organtransplantation, immunsuppressiver therapie, hiv-infektion) zeigen eine eingeschränkte immunkompetenz gegenüber ebv. hierdurch kommt es zu einer verschiebung des virus-wirt-gleichgewichts zugunsten des virus. ebv-infizierte b-zellen können daher unkontrolliert auswachsen und zu polyoligoklonalen b-zell-lymphoproliferationen bis hin zu monoklonalen malignen lymphomen führen. die häufigkeit dieser komplikationen ist direkt abhängig von der schwere der immunsuppression. ebv-assoziierte maligne erkrankungen ebv findet sich zu 100% in tumorzellen des endemischen burkitt-lymphoms und des nasopharynxkarzinoms. darüber hinaus lässt sich das virus in geringerer häufigkeit auch in anderen malignomen (m. hodgkin, b-und t-zell-lymphome) nachweisen. die rolle von ebv in der tumor entstehung und/oder -progression ist noch weitgehend unbekannt. j diagnose die infektiöse mononukleose kann meist klinisch diagnostiziert werden. labor im blutausstrich lassen sich typischerweise zahlreiche aktivierte t-lymphozyten (pfeiffer-zellen) nachweisen (. abb. 14.8). in zweifelsfällen wird bei immunkompetenten patienten die diagnose serologisch gesichert (. abb. 14.11). der mononukleoseschnelltest zum nachweis von heterophilen antikörper ist im kindesalter sehr wenig sensitiv und spielt in der pädiatrie keine rolle. die bestimmung der viruslast im blut mittels polymerasekettenreaktion ist bei immunsupprimierten patienten mit ebv-assoziierten lymphoproliferativen krankheitsbildern sinnvoll. immunsupprimierte patienten bei angeborenen immundefekten (xlp etc.) kann eine frühzeitige stammzelltransplantation (nabelschnurblut, knochenmark) zu einer immunrekonstitution führen und so spätere komplikationen durch ebv verhindern. bei auftreten von ebv-lymphomen im rahmen einer immunsuppressiven therapie führt die rechtzeitige reduktion der medikamentendosis häufig noch zu einer rückbildung der tumoren. der einsatz von monoklonalen anti-b-zellantikörpern (anti-cd20-rituximab) führt bei ebv-assoziierten lymphoproliferativen krankheitsbildern in bis zu 60% zu einer kompletten remission. die präsymptomatische therapie mit ganciclovir oder valganciclovir kann bei kindern nach organtransplantationen (v. a. lebertransplantation) wahrscheinlich die häufigkeit von ebvassoziierten lymphoproliferativen komplikationen reduzieren. bei organtransplantierten patienten kann die infusion von ebv-spezifischen zytotoxischen t-zellen des organspenders ebv-positive lymphome zur rückbildung bringen oder die neuentstehung von lymphomen verhindern (sog. "adoptiver immuntransfer"). zytomegalievirusinfektionen j epidemiologie die durchseuchungsrate mit dem zytomegalievirus (cmv) in der bevölkerung ist v. a. abhängig vom alter und lebensstandard. in deutschland sind ungefähr 50% der erwachsenen gesamtbevölkerung seropositiv für cmv. cmv-infektionen als folge von organtransplantationen treten meist nach 4 wochen bis 4 monaten auf, als folge einer bluttransfusion bereits nach 3-12 wochen. cmv wird horizontal über infektiöse körperflüssigkeiten (speichel, urin, muttermilch), blut, blutprodukte oder transplantierte organe, sowie vertikal (konnatale infektion) übertragen. cmv-positive säuglinge und kleinkinder können über wochen infektiöses cmv ausscheiden. j ätiopathogenese cmv gehört zur gruppe der herpesviren. cmv repliziert sich in epithelialen zellen der speicheldrüsen und der nieren, bei schweren generalisierten infektionen auch in leber, genitaltrakt, lungen und anderen organen. die produktive cmv-infektion führt in diesen zellen zu typischen intranukleären einschlüssen ("eulenaugenzellen"; . abb. 14.12) und zu massiver vergrößerung der infizierten zellen ("zytomegalie"). während der virämischen phase(n) findet sich cmv überwiegend zellassoziiert in der fraktion der polymorphkernigen granulozyten. nach einer primärinfektion persistiert cmv lebenslang im blut in monozyten/makrophagen sowie in anderen infizierten organen (speicheldrüsen, nieren). das virus kann bei immunsuppression jederzeit reaktiviert werden. bei der immunologischen bewältigung einer cmv-infektion spielt die zelluläre immunität (u. a. cmv-spezifische cd8+-t-zellen) eine entscheidende rolle. neutralisierende cmv-spezifische antikörper können bei einer cmv-infek tion die schwere einer cmv-erkrankung abmildern. personen die meisten cmv-infektionen verlaufen bei immunkompetenten personen asymptomatisch bzw. subklinisch. in seltenen fällen (1:1.000) manifestiert sich eine cmv-infektion als mononukleoseähnliches krankheitsbild mit ähnlichen klinischen symptomen und blutbildveränderungen (lymphozytose, atypische lymphozyten). das risiko und die schwere einer cmv-erkrankung korreliert mit dem ausmaß der immunsuppression, der virusmenge im blut und anderen faktoren, die eine reaktivierung von cmv begünstigen (akute graft-versus-host-erkrankung, infektion mit anderen herpesviren, cmv-seronegativer transplantatempfänger eines cmvpositiven organs). . abb. 14.12 eulenaugenzellen in der niere eines verstorbenen kindes mit konnataler zytomegalie . abb. 14.13 cmv-chorioretinitis bei einem 14 jahre alten patienen unter immunsuppressiver therapie (mit freundl. genehmigung von prof. dr. handrick, frankfurt/oder) konnatale cmv-infektionen die zytomegalie ist die häufigste konnatale infektion (ca. 0,2-0,4% aller neugeborenen). eine cmv-primärinfektion in der schwangerschaft führt bei ca. 7-10% der infizierten kinder zu einer schweren generalisierten cmv-erkrankung. eine cmv-reaktivierung oder -zweitinfektion in der schwangerschaft führt dagegen nur sehr selten zu einer symptomatischen zytomegalie beim kind. das risiko für eine symptomatische konnatale zytomegalie scheint direkt mit der höhe von maternalen neutralisierenden cmv-spezifischen antikörpern sowie der cmv-virusmenge im blut zu korrelieren. etwa 90% aller neugeborenen mit konnataler cmv-infektion sind bei geburt klinisch symptomfrei. ein teil dieser kinder (7-15%) kann aber später eine bleibende hörstörung entwickeln. aus diesem grund sollten bei allen kindern mit konnataler cmv-infektion wiederholt hör-und sehprüfungen veranlasst werden. eine symptomatische konnatale zytomegalie (nach cmv-erstinfektion in der schwangerschaft) ist eine multisystemerkrankung mit hoher morbidität und letalität. erkrankte kinder zeigen eine ausgeprägte intrauterine wachstumsretardierung, ikterus, hepatosplenomegalie, thrombozytopenie mit petechien (77%), pneumonie sowie schwerste zns-schädigungen (bis zu 70%) mit mikrozephalus (53%), intrazerebralen verkalkungen, chorioretinitis, späterer taub-und blindheit und geistiger behinderung. die letalität liegt bei bis zu 30%. für die einschätzung von späteren neurologischen defiziten scheint ein schädel-ct oder mrt die derzeit sensitivste methode zu sein. der nachweis von cmv-dna sowie eine eiweißerhöhung im liquor sind wahrscheinlich mit einer schlechteren prognose hinsichtlich der neurologischen entwicklung assoziiert. die schlechteste prognose haben konnatale cmv-infektionen häufig dann, wenn sie im 1. trimenon auftreten. peri-und postnatale cmv-infektionen infektionen, die durch cmv im zervikal-und vaginalsekret bzw. durch infektiöse muttermilch übertragen werden, verlaufen bei reifgeborenen, immunkompetenten kindern meist asymptomatisch oder mild. bei sehr kleinen frühgeborenen kann eine perinatale cmv-infektion (v. a. via cmv-positive muttermilch) eine schwere interstitielle pneumonie, hepatosplenomegalie und ein sepsisähnliches krankheitsbild verursachen. die letalität liegt bei 24%. j diagnose sensitive und spezifische parameter für eine floride cmv-infektion sind der quantitative nachweis des cmv-antigens pp65 oder des cmv-genoms (pcr) im blut (oder anderen körperflüssigkeiten). beide methoden erlauben eine bestimmung der viruslast und somit auch das monitoring einer virostatischen therapie mit ganciclovir bzw. valganciclovir. der "klassische nachweis" von cmv besteht in der isolierung aus verschiedenen körperflüssigkeiten (urin, speichel etc.). herpesvirus-typ-6-infektionen zu den begleitsymptomen und komplikationen, die meist schon im frühstadium (tag 1-4) auftreten, gehören gastroenteritis (55-70%), lidödeme (bis 30%), nagayama-flecken (papeln auf dem weichen gaumen und der uvula 65%), husten (50%), zervikale lymphadenopathie (30-35%), vorgewölbte fontanelle (26-30%) sowie fieberkrämpfe (5-35%). die angaben über die häufigkeit eines exanthema subitum nach einer primärinfektion mit hhv-6 schwanken stark. primärinfektionen mit hhv-6 stellen insgesamt eine häufige ursache von hochfieberhaften infekten (mit und ohne exanthem) bei kleinkindern dar. fieberkrämpfe und andere klinische manifestationen während einer hhv-6-primärinfektion kommt es nicht selten zu einer invasion des virus in das zns. bei bis zu 40% von kindern mit florider hhv-6-infektion kann das virus im liquor nachgewiesen werden, wobei entzündliche liquorveränderungen (pleozytose, eiweißvermehrung) fehlen. fieberkrämpfe treten in bis zu 35% auf. zu den seltenen neurologischen komplikationen gehören die meningoenzephalitis und das guillain-barré-syndrom. in wenigen fällen kann eine hhv-6-infektion, v. a. bei älteren kindern, auch mit einer mononukleoseähnlichen symptomatik, einer fulminanten hepatitis, einem schwer verlaufenden hämophagozytosesyndrom sowie mit der verschlimmerung einer idiopathischen thrombozytopenie (itp) assoziiert sein. organtransplantation kommt es häufig (in bis zu 80%) zu einer reaktivierung von hhv-6, die möglicherweise zu einer vermehrten transplantatabstoßung führt. nach hhv-6-infektion bzw. -reaktivierung können folgende klinische komplikationen auftreten: interstitielle pneumonie, graft-versus-host-erkrankung (gvhd) mit und ohne exanthem, enzephalopathie und knochenmarksuppression (nach knochenmarktransplantation). inwieweit diese komplikationen tatsächlich ursächlich nur durch hhv-6, oder möglicherweise erst in verbindung mit zusätzlichen infektionen (hiv, cmv und andere herpesviren) hervorgerufen werden, ist derzeit nicht bekannt. j diagnose die diagnose eines exanthema subitum kann bei typischer ausprägung klinisch gestellt werden. labor am 3. und 4. fiebertag fällt im blutbild häufig eine leukopenie mit relativer lymphozytose (bis zu 90%) auf. eine vermutete hhv-6-primärinfektion wird durch den serologischen nachweis von hhv-6-spezifischen antikörpern (anti-hhv-6-igm und/oder anstieg von anti-hhv-6-igg) bestätigt. eine hhv-6-reaktivierung bei immunsupprimierten kindern kann bei akut ansteigenden anti-hhv-6-antikörpertitern (bei bekannten ausgangstitern) vermutet werden. hhv-6-dna kann mittels pcr nachgewiesen werden (speichel, blut, plasma, liquor, urin). j therapie, prophylaxe eine spezifische therapie existiert nicht. bei hohem fieber erfolgt eine adäquate symptomatische fiebersenkung. bei (immunsupprimierten) patienten mit schweren hhv-6-assoziierten komplikationen (pneumonie, enzephalitis) ist ein therapieversuch mit foscarnet und/oder ganciclovir, zu erwägen. herpesvirus-typ-7-infektionen j epidemiologie auch herpesvirus typ 7 (hhv-7) kommt ubiquitär vor. die durchseuchung in der bevölkerung liegt z. t. bei über 90%. in den ersten 6 lebensmonaten ist eine hhv-7-infektion sehr selten, am ende des 1. lebensjahr sind bis zu 30%, am ende des 6. lebensjahrs bis zu 86% der kinder seropositiv. die primärinfektion mit hhv-7 erfolgt i. a. deutlich später als die mit hhv-6. die übertragung erfolgt über infektiösen speichel, u. a. innerhalb der familie, und möglicherweise auch über infizierte muttermilch. j ätiopathogenese hhv-7 gehört zur gruppe der herpesviren. die pathogenese ist ähnlich wie bei der hhv-6-infektion. j klinik hhv-7 ist (neben hhv-6) der erreger des exanthema subitum (dreitagefieber, roseola infantum). im vergleich zu hhv-6 scheint hhv-7 insgesamt in einer höheren frequenz zu fieberkrämpfen zu führen. das mittlere alter bei symptomatischen hhv-7-infektionen liegt bei 26 monaten, bei hhv-6-infektionen bei ca. 9 monaten. gelegentlich führt eine hhv-7-infektion bei älteren kindern auch zu einem mononukleoseähnlichen krankheitsbild. in den meisten fällen verlaufen hhv-7-infektionen allerdings subklinisch oder gehen mit einem unspezifischen fieberhaften infekt einher. j diagnose, therapie die diagnose eines exanthema subitum erfolgt bei typischer symptomatik klinisch. nur in ausnahmefällen scheint eine weitere virologische abklärung gerechtfertigt. hhv-7 kann mittels polymerasekettenreaktion im speichel, im peripheren blut, in lymphatischem gewebe und teilweise auch in der muttermilch nachgewiesen werden. der nachweis von hhv-7-spezifischen serumantikörpern erfolgt mittels indirekter immunfluoreszenz oder elisa. zu berücksichtigen ist hierbei, dass antikörper gegen hhv-7 teilweise auch mit hhv-6 kreuzreagieren können. eine spezifische therapie oder eine impfung gegen hhv-7 gibt es nicht. bei schwerem klinischem verlauf erfolgt eine entsprechende symptomatische therapie. herpesvirus-typ-8-infektionen j epidemiologie das herpesvirus typ 8 (hhv-8) kommt ebenfalls ubiquitär vor. die seroprävalenz in der bevölkerung ist in afrikanischen ländern und in japan (bis zu 100%) deutlich höher als in europa und in den usa (20-30%). hhv-8 wird überwiegend (homo)sexuell übertragen. die ansteckung von kindern und jugendlichen erfolgt wahrscheinlich über infektiösen speichel. bei nierentransplantationen kann eine transmission von hhv-8 in bis zu 10% erfolgen. j ätiologie hhv-8 gehört zur gruppe der herpesviren. hhv-8 zeigt in vitro und teilweise auch in vivo einen tropismus zu cd19+-b-zellen, zu endothelialen zellen und ganglienzellen. gefährdet sind u. a. patienten mit gleichzeitig bestehender chronischer hepatitis c. das krankheitsbild ist gekennzeichnet durch ein schnell zunehmendes leberversagen. die letalität ist hoch. j diagnose die diagnose einer hav-infektion sollte immer erwogen werden, wenn bei mehreren familienangehörigen oder anderen kontaktpersonen ein ikterus und gastrointestinale begleitsymptome auftreten. labor die diagnose eine hav-infektion wird durch den nachweis von virusspezifischen antikörpern im serum (anti-hav-igm positiv und/oder anstieg von anti-hav-igg) gesichert (. abb. 14.14). mit ausbruch der erkrankung werden anti-hav-igm-antikörper gebildet, die für 3-12 monate im blut nachweisbar sein können. die anti-hav-igg-antikörper persistieren (wahrscheinlich) lebenslang und sind ausdruck von immunität. j therapie eine spezifische therapie gibt es nicht. bettruhe und spezielle diäten haben keinen einfluss auf den krankheitsverlauf und sind nicht indiziert. symptomatische maßnahmen richten sich nach den jeweiligen beschwerden. im seltenen fall einer fulminanten hepatitis ist eine lebertransplantation zu erwägen. j prophylaxe die hepatitis-a-impfung (2 injektionen im abstand von 6 monaten; für kinder zugelassen ab dem 2. lebensjahr) kann bei rechtzeitiger gabe eine symptomatische wildvirusinfektion wirksam verhindern. sie wird allen potenziell gefährdeten immungesunden personen (u. a. seronegatives personal in medizinischen einrichtungen und kindertagestätten, reisende in regionen mit hoher hav-prävalenz, patienten mit chronischer hepatitis c) empfohlen. bereits 2 wochen nach der 1. impfung sind über 95% der geimpften geschützt. diese impfung schützt z. t. auch dann noch, wenn sie in der frühen inkubationszeit gegeben wird (sog. inkubations-oder "riegelungsimpfung"). nach einer vorausgegangenen wildvirusexposition kann durch frühzeitige einmalige gabe von immunglobulinen der ausbruch einer hepatitis a verhindert oder der verlauf abgemildert werden (postexpositionsprophylaxe). werden die immunglobuline erst nach einem intervall von 10(-14) tagen gegeben, ist keine wirkung mehr zu erwarten. im durchschnitt gelten patienten in einem zeitraum von 2 wochen vor bis 2 wochen nach ausbruch der erkrankung als ansteckend. hospitalisierte kinder mit einer hepatitis a sollten, sofern dies aus medizinischer sicht vertretbar ist, nach diagnosestellung schnellstmöglich nach hause entlassen werden. ansonsten erfolgt eine isolierung (einzelzimmer, kohortierung) für max. 2 wochen. neugeborene hbsag-positiver mütter erhalten am besten noch im kreißsaal, ansonsten innerhalb der ersten 12 h post partum simultan 0,5 ml hepatitis-b-impfstoff i.m. sowie auf der kontralateralen seite 0,5 ml/kgkg hepatitis-b-immunglobulin i.m. nach 4 wochen und 6 monaten erfolgt eine weitere impfung. geimpfte neugeborene dürfen gestillt werden. mutter und kind müssen nach geburt nicht isoliert oder voneinander getrennt werden. bei nichtimmunen personen mit fehlenden oder erniedrigten anti-hbs-antikörpern wird nach kontakt mit virushaltigem blut oder sekret schnellstmöglich (spätestens innerhalb von 12 h) hepatitis-b-immunglobulin i.m. verabreicht (postexpositionsprophylaxe). gleichzeitig sollte möglichst auch aktiv geimpft werden. in den ersten 1-3 monaten einer akuten hepatitis c kann der antikörpernachweis noch negativ verlaufen. die hcv-serologie ist daher v. a. für die diagnose von bereits einige zeit zurückliegenden oder chronischen hcv-infektionen geeignet. das hcv-genom kann mittels pcr im blut oder in der leberbiopsie nachgewiesen werden. die bestimmung der viruslast im blut (und ggf. auch im leberbioptat) sowie eine genotypisierung der vorliegenden hcv-variante sind hinsichtlich der indikation für eine interferon-α-therapie sowie für das anschließende monitoring wichtig. j therapie für eine antivirale therapie der akuten hepatitis c liegen für kinder und jugendliche bisher keine daten vor. da von einer hohen chronifizierungsrate ausgegangen werden muss, sollte wie bisher bei erwachsenen vorgegangen werden. die kombinationstherapie mit normalem oder pegyliertem (= retardiertem) interferon-α (zugelassen bei kindern ab 3 jahren) plus ribavirin ist derzeit bei kindern noch die standardtherapie der chronischen hepatitis c. prädiktoren für einen therapieerfolg sind der hcv-genotyp (v. a. typ 2 und 3), die dauer der krankheit und möglicherweise die hcv-konzentration im serum. bei erwachsenen mit chronischer hepatitis c führen neue virustatika (z. b. epclusa in kombination mit sofosbuvir und velpatasvir) zu einer ausheilung von meist über 90%. es ist zu hoffen, dass diese therapieoptionenn in einigen jahren auch bei kindern zugelassen sind. diese gehen dabei zugrunde, wobei die erythropoese kurzzeitig unterbrochen wird. eintrittspforte für parvovirus b19 ist der obere respirationstrakt. nach 4-5 tagen kommt es zu einer virämie, begleitet von einer retikulozytopenie. etwa 1 woche nach der virämie tritt meist das typische exanthem auf, möglicherweise hervorgerufen durch antigen-antikörper-komplexe. die entstehung eines hydrops fetalis nach intrauteriner parvovirus-b19-infektion wird v. a. mit der infektion von fetalen erythroblasten und der daraus resultierenden anämie erklärt. in bestimmten fällen kann möglicherweise auch die infektion des fetalen myokards mit parvovirus b19 zu einer eingeschränkten herzfunktion mit der folge eines hydrops fetalis führen. j klinik ringelröteln diese typische "kinderkrankheit" (syn: erythema infectiosum, 5. krankheit, epidemisches megalerythem, großfleckfieber, kinderrotlauf) wird in 15-20% aller frisch infizierten personen beobachtet. nach einem 2-3 tage andauernden prodromalstadium mit unspezifischen symptomen wie fieber, kopfschmerzen und schüttelfrost (zeitgleich mit der virämie) und einem anschließenden beschwerdefreien intervall von ca. 1 woche tritt im bereich der wangen ein hochrotes, leicht erhabenes konfluierendes exanthem auf (. abb. 14.19a). gleichzeitig kann eine periorale blässe wie beim scharlach bestehen. an den folgenden tagen treten . abb. 14.19 ringelröteln. a typisches konfluierendes exanthem im bereich beider wangen ("slapped cheek") mit perioraler blässe, b girlandenförmige exantheme a b an den extremitäten und am rumpf makulopapulöse effloreszenzen auf, die konfluieren. durch zentrales abblassen entstehen die typischen girlanden-netzförmigen muster (. abb. 14.19b) . in den folgenden tagen und wochen können immer wieder neue pleomorphe exantheme auftreten, teilweise provoziert durch sonnenlicht oder hohe temperatur. das allgemeinbefinden der p atienten ist meist nur wenig beeinträchtigt. andere begleitsymptome (juckreiz, kopfschmerzen, fieber, gelenkbeschwerden, bauchschmerzen) sind bei kindern eher selten. bei adoleszenten und jungen erwachsenen kommen auch vaskulitische exantheme vor mit strenger begrenzung auf die hände und füße ("handschuh-socken-syndrom"). die parvovirus-b19-assoziierte polyarthritis, die bevorzugt knie-, fuß-und die proximalen interphalangealgelenke befällt und häufiger bei mädchen und jungen frauen auftritt, ist praktisch immer selbstlimitierend. hydrops fetalis eine parvovirus-b19-primärinfektion in der schwangerschaft führt in bis zu 35% auch zu einer infektion des feten. in den allermeisten fällen ist diese fetale infektion klinisch stumm, in bis zu 5% der fälle kommt es zum abort (meist innerhalb von 3-6 wochen nach der mütterlichen infektion). die entwicklung eines hydrops fetalis nach einer mütterlichen (und fetalen) parvovirus-b19-infektion ist insgesamt selten, sie liegt bei ca. 4%. trotzdem ist die parvovirus-b19-infektion wahrscheinlich die häufigste ursache des nicht immunologisch bedingten hydrops fetalis. die fetalen komplikationen sind am höchsten bei einer parvovirus-b19-infektion zwischen der 13. und 20. gestationswoche. j klinik eine rotavirusinfektion führt bei kleinen kindern praktisch immer zu fieber, erbrechen sowie enteritis mit häufigen wässrigen, nicht blutigen stühlen. die durchfälle können bis zu 5-7 tage andauern. in der mehrzahl der fälle kommt es hierbei zu einer unterschiedlich ausgeprägten, meist isotonen, selten einer hypertonen dehydratation. ein teil der kinder zeigt zusätzliche klinische symptome wie leichte vergrößerung der zervikalen lymphknoten, otitis und rhinitis. bei immunsupprimierten kindern kann eine rotavirusinfektion chronisch und teilweise sehr schwer verlaufen. in sehr seltenen fällen können rotavirusinfektionen auch zu einer enzephalitis und zu krampfanfällen mit und ohne fieber führen. j diagnose die praktikabelste und schnellste methode zum nachweis einer rotavirusinfektion ist der rotavirusantigennachweis im stuhl (eia). follikuläre konjunktivitis häufig, meist unilateral 1-7, 9-11, 15-17, 19, 20, 22, 37 haut exantheme (morbilli-, rubelli-und roseolaform) j ätiopathogenese rhinoviren sind rna-viren aus der familie der picornaviren (pico, klein). rhinoviren infizieren v. a. die schleimhäute der nase und des oberen respirationstrakts, seltener auch die unteren atemwege. hier vermehrt sich das virus im zytoplasma. anschließend werden die neu gebildeten rhinoviren in großer menge (bis zu 106 viruspartikel pro ml nasensekret), v. a. in den ersten 3 tagen der erkältung, sowie meist noch 2-3 wochen danach ausgeschieden. während einer rhinovirusinfektion kommt es meist nur zu minimalen epithelsschäden ohne zilienverlust sowie zu einer lokalen infiltration mit granulozyten und monozyten. j klinik in den meisten fällen führt eine rhinovirusinfektion zu einer "erkältung" der oberen luftwege ("common cold") mit einer nur geringen allgemeinen klinischen beeinträchtigung. hauptsymptome sind schnupfen, husten und halsschmerzen. die regionalen zervikalen lymphknoten sind in ca. 50% vergrößert. eine meist leichte otitis media tritt in ca. 20% der fälle auf. die klinischen beschwerden sind in den ersten 3 tagen am ausgeprägtesten, in den meisten fällen bilden sie sich innerhalb einer woche, seltener nach 2 wochen zurück. vor allem bei jüngeren kindern können rhinovirusinfektionen auch zu schweren verläufen mit bronchiolitis, bronchopneumonie und obstruktiver bronchitis führen. todesfälle sind beschrieben worden. in den meisten fällen entwickeln die betroffenen kinder einen zunehmenden keuchenden husten und dyspnoe bei häufig nur leicht erhöhten temperaturen. in schweren fällen finden sich eine tachypnoe mit nasenflügelatmen und thorakalen einziehungen, eine zyanose sowie eine tachykardie. das atemgeräusch kann (als indirektes zeichen der überblähung bei atemwegsobstruktion) abgeschwächt sein. die röntgenaufnahme der lunge zeigt als typischen befund bei einer bronchiolitis eine überblähung der lungen und ggf. einige infiltrate. die blutgasanalyse ergibt häufig eine leichte bis mäßige hypoxie, eine hyperkapnie ist ausdruck der zunehmenden ateminsuffizienz, die einer dringenden intensivmedizinischen betreuung bedarf. bei früh-und neugeborenen kann eine rsv-infektion mit plötzlich auftretenden teils massiven apnoen, irritabilität, trinkverweigerung und lethargie -ohne äußere zeichen eines atemwegsinfekts -einhergehen. rsv-reinfektionen verlaufen i. a. milder und manifestieren sich häufig als oberer atemwegsinfekt oder tracheobronchitis. bei kindern mit schweren grunderkrankungen (bronchopulmonale dysplasie, herzfehler, immundefizienz) kann jede rsv-infektion zu einem lebensbedrohlichen krankheitsbild führen. j diagnose die diagnose einer rsv-infektion basiert auf der klinischen symptomatik, auf dem zeitlichen hintergrund (winterhalbjahr? bekannte rsv-epidemie?), auf der altersverteilung (kind <2 jahre) und der positiven virusdiagnostik. für den klinischen alltag sind rsv-antigen-tests zum virusnachweis in nasopharyngealsekret von großer praktischer bedeutung. daneben ist der nachweis von rsv durch anzucht in der zellkultur oder mittels pcr möglich. die serologische diagnostik spielt in der praxis keine rolle. j therapie im vordergrund steht die intensivmedizinische betreuung (u. u. kontrollierte beatmung) der meist schwer kranken kinder. symptomatisch können β 2 -sympathomimetika, racemisches epinephrin oder adrenalin verabreicht werden. kortikosteroide, theophyllin und mukolytika sind unwirksam. ribavirintherapie als virostatikum steht ribavirin zur verfügung. aufgrund unterschiedlicher, z. t. enttäuschender studienergebnisse hinsichtlich der wirksamkeit, der umständlichen inhalativen applikation sowie potenzieller nebenwirkungen (teratogenität) ist die indikation für ribavirin seit 1996 stark eingeschränkt worden. in über 90% lässt sich in tumorgewebe hpv nachweisen. zu den kanzerogenen typen gehören v. a. hpv-16 und hpv-18. j diagnose die diagnose erfolgt in den allermeisten fällen klinisch anhand der typischen manifestation (warzen, papillome). in unklaren fällen sollte immer eine histologische abklärung erfolgen. der nachweis und die typisierung von papillomaviren im biopsat erfolgt -falls überhaupt erforderlich -mit molekularbiologischen techniken. bei genitaler krankheitslokalisation sind immer auch andere geschlechtskrankheiten auszuschließen. bei kleinen kindern sollte auch an einen sexuellen missbrauch gedacht werden. larynxpapillome verursachen bei kindern eine mehr oder weniger ausgeprägte dyspnoe, die diagnose erfolgt mittels laryngo-oder bronchoskopie. j therapie vor jeder therapieentscheidung ist die hohe spontanremissionrate von warzen im kindesalter zu bedenken! hautwarzen werden durch kontaktvereisung mit flüssigstickstoff oder durch eine keratolytische und antiproliferative lokalbehandlung mit salicylsäureund 5-fluorouracil-haltigen präparaten (z. b. verrumal) behandelt. die früher durchgeführte warzenentfernung mittels elektrokauter ist wegen der schmerzhaftigkeit und der möglichen narbenbildung obsolet. filiforme warzen können chirurgisch im hautniveau exzidiert werden. larynxpapillome werden, v. a. bei entsprechenden atembeschwerden, mittels laser-oder kryotherapie behandelt. unterstützend kann zur proliferationshemmung lokal oder systemisch interferon α appliziert werden. die behandlung von genitalen warzen kann sehr schwierig sein und sollte immer von einem erfahrenen gynäkologen durchgeführt werden. die lokalbehandlung erfolgt u. a. mit podophyllinlösung (5-25%), kryotherapie sowie mittels co 2 -laser. die prodromi mit fieber und katarrhalischen symptomen (8-12 tage p. i.) signalisieren den beginn der immunologischen abwehrreaktion. so ist auch das exanthem folge der explosiven auseinandersetzung zwischen virusspezifischen t-zellen und virusinfizierten epithel-und endothelzellen. spezifische antikörper scheinen bei der überwindung der akuten phase keine rolle zu spielen. > masern gehen immer mit einer immunschwäche einher, die wochen bis monate anhalten kann. hauttests vom verzögerten typ (tuberkulintest!) werden vorübergehend negativ. außerdem kommt es durch die immunschwäche zu bakteriellen zweiterkrankungen oder zur aktivierung chronischer krankheitsprozesse. die pathogenese der para-(post-)infektiösen masernenzephalitis ist bisher nicht geklärt. histopathologisch finden sich im gehirn perivaskuläre demyelinisierungen und perivaskuläre lymphozyteninfiltrate. virale antigene oder rna-sequenzen lassen sich aber nicht nachweisen. j klinik die erkrankung beginnt mit hohem fieber und uncharakteristischen katarrhalischen symptomen, wie schnupfen, halsschmerzen, heiserkeit und bellendem husten (prodromalstadium). die patienten sind aufgrund einer konjunktivitis und einer (milden) keratitis ausgesprochen lichtscheu. gleichzeitig oder 1-2 tage später treten feine, kalkspritzerartige stippchen, bevorzugt an der wangenschleimhaut gegenüber den molaren, auf (koplik-flecken; . abb. 14.25). außerdem entwickelt sich ein fleckiges, dunkelrotes enanthem am weichen gaumen. nach leichtem fieberabfall geht das prodromalstadium 3-4 tage später unter erneutem hohem fieberanstieg in das exanthemstadium über. das makulopapulöse exanthem beginnt hinter den ohren (. abb. 14.27) und im gesicht und breitet sich weiter zentrifugal über den ganzen körper bis zu den füßen aus (. abb. 14.28). nach dem 3. exanthemtag folgt bei unkomplizierten verläufen rasche entfieberung und abblassen des exanthems. meist besteht eine generalisierte lymphadenopathie, wobei auch die hilären, paratrachealen und mesenterialen lymphknoten betroffen sind. bei ca. 50% der infizierten treten pathologische eeg-veränderungen auf, die sich später in den allermeisten fällen zurückbilden. 4 mitigierte masern treten bei jungen säuglingen auf, die noch maternelle antikörper besitzen und auch bei kindern nach gabe von immunglobulinen. 4 bei patienten mit schweren t-zelldefekten kann das exanthem völlig fehlen ("weiße masern"). es entwickelt sich eine riesenzellpneumonie, die fast immer zum tode führt. j komplikationen am häufigsten sind bakterielle sekundärinfektionen; und zwar bronchopneumonien, otitis media und diarrhö. weitere komplikationen betreffen das zentrale nervensystem. im vordergrund steht die akute masernenzephalitis mit einer häufigkeit von 1:500-1:2000. die masernenzephalitis tritt bevorzugt am 3.-9. tag nach exanthembeginn auf. typisch sind bewusstseinsstörungen (somnolenz, koma), zerebrale krampfanfälle, neurologische herdsymptome (hemiplegien, hirnnervenparesen) und gelegentlich auch myelitische symptome. die masernenzephalitis hat auch heute noch eine letalität von 30% und eine defektheilungsrate von ca. 20%. eine weitere seltenere komplikation ist die subakute sklerosierende panenzephalitis (7 abschn. 14.23.1). masern sind immer eine ernste und gefährliche krankheit. todesfälle kommen besonders im säuglingsalter, bei älteren probanden und besonders bei immundefizienten patienten vor. die krankheit verläuft besonders schwer in entwicklungsländern bei unterernährten kindern. . abb. 14.25 koplik-flecken . abb. 14.26 masernexanthem bei einem 8-jährigen jungen (hinter den ohren beginnend) . abb. 14.27 generalisiertes masernexanthem bei einem älteren säugling j diagnose im rahmen einer epidemie wird die diagnose meistens klinisch gestellt. labor ein typischer laborbefund ist die leukopenie mit erniedrigung sowohl der granulozyten als auch der lymphozyten. bei einzelerkrankungen sollte die diagnose serologisch bestätigt werden. das masernspezifische igm ist meist nach den ersten 3 exanthemtagen mittels enzymimmunoassay (elisa) nachweisbar. bei trotz impfung an masern erkrankten ist nur ein 4-facher titeranstieg im igg-elisa oder im hämagglutinationshemmtest (hht) diagnosesichernd (bei geimpften findet sich oft keine igm-antwort). in fraglichen fällen, z. b. bei immunsupprimierten patienten oder bei verdacht auf riesenzellpneumonie ist zur diagnosestellung der virusdirektnachweis (pcr oder virusisolierung) erforderlich. die diagnose einer masernenzephalitis beruht allein auf dem zeitlichen zusammenhang der enzephalitis mit einer akuten maserninfektion (igm-nachweis!), da sich im liquor in der regel weder das virus noch eine intrathekale antikörpersynthese nachweisen lassen. j diagnose wegen der ähnlichkeit mit anderen viralen und nichtviralen exanthemen ist die klinische diagnose oft schwierig. charakteristische blutbildveränderungen (leukopenie mit relativer lymphozytose und auftreten von plasmazellen) können von diagnostischer bedeutung sein. ansonsten muss die infektion serologisch bestätigt werden. beweisend sind ein 4-facher titeranstieg im hämagglutinationshemmtest (aus 2 serumproben) oder der nachweis von rötelnspezifischem igm mittels enzymimmunassay (elisa). je nach empfindlichkeit der testmethode sind spezifische igm-antikörper mitunter lange (bis zu einem jahr) im serum nachweisbar. um z wischen einer primären infektion und der (seltenen) reinfektion bei schwangeren zu unterscheiden, stehen spezielle tests zur ver fügung. bei der akuten rötelnenzephalitis findet man im liquor eine leichte lymphozytäre pleozytose. das liquoreiweiß ist normal. virale rna und oligoklonale banden lassen sich in der regel nicht nachweisen. bei mumpsmeningitis zeigt der liquor eine mäßige lymphozytäre pleozytose (10-2.000 zellen/µl) bei normalem bis leicht erhöhtem eiweiß und normalem bis leicht erniedrigtem liquorzucker. im liquor treten 2-3 wochen später virusspezifische oligoklonale mumpsantikörper auf als ausdruck einer intrathekalen immunreaktion. j therapie, prophylaxe eine spezifische therapie existiert nicht. auch eine symptomatische behandlung ist selten erforderlich. bei schweren verläufen (mumpsenzephalitis, orchitis) sind u. u. kortikosteroide indiziert. alle kinder (und noch seronegative adoleszenten und erwachsene) sollten 2-mal gegen mumps geimpft werden (7 kap. 17). spezielle immunglobuline zur passiven immunisierung stehen nicht zur verfügung. gemeinschaftseinrichtungen dürfen 9 tage nach beginn der parotitis wieder besucht werden. seit 2012 ist mumps meldepflichtig. j diagnose die diagnose wird in der regel nach klinischen symptomen und dem eeg-befund gestellt. gestützt wird die diagnose durch den nachweis von neuronalen destruktions-und glialen aktivierungsmarkern im liquor (neuronenspezifische enolase, proteine 14-3-3, s100-β-protein). die genannten marker sind allerdings nicht spezifisch für cjk. ansonsten ist der liquor unauffällig. eine definitive diagnose kann nur durch die untersuchung von hirngewebe gestellt werden. j therapie und prophylaxe es gibt bisher keine wirksame therapie. iatrogene übertragungen durch chirurgische instrumente können durch adäquate dekontaminationsmaßnahmen vermieden werden (z. b. dampfautoklavieren bei 134°c für 1 h, behandlung mit 2,5-5%iger natriumhypochloritlösung oder 1-2 n natronlauge für 1 h. prionenverseuchte nahrungsmittel dürfen auf keinen fall in den verkehr gebracht werden. humane spongiforme enzephalopathien -außer familiärhereditären formen -sind meldepflichtig. j epidemiologie tollwut ist eine weltweit verbreitete zoonose. das tollwutvirus wird durch infektiösen speichel bei kratz-und bisswunden von infizierten tieren (füchse, hunde, fledermäuse, katzen u. a.) übertragen. die jährliche inzidenz von tollwut beim menschen wird weltweit auf 40.000-100.000 fälle geschätzt. deutschland gilt derzeit als "tollwutfrei". die inkubationszeit beträgt 5 tage bis mehrere jahre. sie ist abhängig von der lokalisation der bissstelle (cave: gesicht, augenregion!) und der inokulierten virusmenge. bei kindern ist die inkubationszeit kürzer. 14.29 frühsommermeningoenzephalitis j epidemiologie die frühsommermeningoenzephalitis (fsme, zeckenenzephalitis, zentraleuropäische enzephalitis) kommt europaweit, v. a. in russland, im balkan sowie in zentral-und nordeuropa vor. die endemiegebiete in deutschland liegen hauptsächlich in bayern, baden-württemberg und im saarland. fsme-erkrankungen treten v. a. in den monaten april bis november auf. das fsme-virus wird überwiegend durch zeckenstiche übertragen. wichtigster überträger ist ixodes ricinus, der "gemeine holzbock". > in endemiegebieten sind ca. 0,1-1% der zecken durchseucht. etwa 12-15% aller fsme-erkrankungen betreffen das kindesalter. die inkubationszeit beträgt ca. 10 tage (3-28 tage). nach einer fsme-infektion besteht lebenslange immunität. j ätiopathogenese das fsme-virus gehört zur familie der flaviviren. über die pathogenese ist relativ wenig bekannt. nach der infektion kommt es zu einer starken virusvermehrung im retikuloendothelialen system und in gefäßendothelien. während der virämie (3-14 tage nach zeckenstich) können bei dem patienten grippeähnliche symptome auftreten. nach zns-invasion des fsme-virus und anschließender lokaler virusreplikation kann es in einer 2. krankheitsphase zum auftreten neurologischer symptome kommen. j klinik in 70-90% verläuft eine fsme-infektion asymptomatisch. bei 10-30% der infizierten kommt es nach meist ca. 10 tagen zu einem grippeähnlichem krankheitsbild für ca. 3-7 tage ("sommergrippe"). bei ca. 10% dieser personen tritt nach einem kurzen beschwerdefreien intervall eine 2. krankheitsphase mit fieberanstieg und folgender neurologischer symptomatik auf: 4 meningitis (60%), 4 meningoenzephalitis (30%), 4 meningoenzephalomyelitis (10%). die restitutionsphase beginnt nach etwa 3 tagen mit kontinuierlicher klinischer besserung im verlauf von 1-3 wochen. eine defektheilung (kopfschmerzen, konzentrationsstörungen, motorische lähmungen, muskelschwäche) tritt v. a. bei älteren menschen in ca. 10% auf. die letalität beträgt ca. 1% (beim östlichen fsme-virussubtyp ca. 20%). kinder und jugendliche haben in der regel leichtere (meningitische) krankheitsverläufe. j diagnose bereits anhand der anamnese (zeckenstich, endemiegebiet, jahreszeit) und der klinischen symptomatik (biphasischer krankheitsverlauf) ergibt sich der verdacht auf eine fsme-erkrankung. die diagnose wird durch den serologischen nachweis von fsme-spezifischen serumantikörpern gesichert. die liquoruntersuchung in der 2. krankheitsphase ergibt typischerweise eine lymphozytäre pleozytose (<100-5.000 zellen/mm 3 ) sowie eine liquoreiweißer höhung. j therapie, prophylaxe eine antivirale therapie existiert nicht. die behandlung ist rein symptomatisch. zur aktiven immunisierung steht ein impfstoff auch für kinder zur verfügung. > zielgruppe für eine fsme-impfung sind alle personen, die sich in endemiegebieten vermehrt im freien aufhalten. meldepflichtig sind erkrankung und tod nach einer fsme-infektion. j ätiopathogenese das "human immunodeficiency virus" (hiv) gehört zur gruppe der retroviren. es existieren 2 haupttypen (hiv-1 und hiv-2). in europa und nordamerika kommen praktisch nur hiv-1-varianten vor, in teilen afrikas findet sich auch hiv-2 bei einem großen teil der bevölkerung. hiv benutzt zur infektion einer zelle das hüllprotein gp120, mit dem es sich an den cd4-rezeptor und korezeptor (ccr5 oder cxcr4) der zielzelle (t-helferzellen, makrophagen, monozyten, gliazellen u. a.) anheftet. anschließend fusioniert die hiv-hülle mit der zellmembran, 2 rna-stränge und mehrere virusenzyme werden in das zytoplasma freigesetzt. mittels des viralen enzyms reverse transkriptase (rt) wird die rna in doppelsträngige dna "übersetzt" (provirus). diese dna wird anschließend durch das viral kodierte enzym integrase in das wirtsgenom im zellkern eingebaut (viruslatenz). durch verschiedene aktivierende faktoren kommt es zu einer ausgeprägten virusreplikation mit freisetzung von neuen hiv-partikeln in die blutbahn. der verlust der t-zellfunktion führt zu einem zunehmenden zellulären und humoralen immundefekt mit der folge von schweren opportunistischen infektionen und dem auftreten von lymphomen. j klinik die klinische symptomatik bei horizontal hiv-infizierten jugendlichen (infektion durch geschlechtsverkehr oder durch kontaminierte blutprodukte) entspricht im wesentlichen dem bild bei erwachsenen. der natürliche verlauf bei vertikal hiv-infizierten kindern variiert sehr stark. ohne therapie erkranken bereits 25-30% dieser kinder sehr früh, meist schon innerhalb des j diagnostik virologische diagnostik als sicherer nachweis einer infektion gelten wiederholt positive hiv-kulturen, wiederholte nachweise des p24-antigens oder der hiv-rna (rt-pcr). die letztere me thode erlaubt die bestimmung der viruslast und hilft mit bei der indikationsstellung für eine antiretrovirale therapie und beim therapiemonitoring. erst der wiederholte nachweis von hiv-antikörpern (elisa, immunoblot) nach dem 18. lebensmonat beweist eine hiv-infektion. bereits vorher nachgewiesene hiv-antikörper können von der mutter diaplazentar übertragen worden sein. immunologische diagnostik im rahmen einer hiv-infektion kommt es im verlauf zu einem zunehmend schweren kombinierten (zellulären und humoralen) immundefekt. im rahmen der initialen statusdiagnostik und späterer verlaufskontrollen sollten bei allen hiv-infizierten oder -exponierten kindern u. a. folgende parameter im blut bestimmt werden: 4 quantitative bestimmung der cd4 + -zellen, 4 serumimmunglobuline (igg, iga und igm), 4 impfantikörper nach erfolgter grundimmunisierung (dtp, hib, hbv, polio-salk), 4 antikörper gegen verschiedene herpesviren (nach stattgehabter infektion)! diese untersuchungen sollten möglichst immer nur am selben immunologisch ausgewiesenen speziallabor durchgeführt werden. die viruslast eines patienten muss immer mit derselben bestimmungsmethode quantifiziert werden! aids-definierenden krankheiten (kategorie c) dagegen tritt bei einem großen teil der kinder die klinische verschlechterung erst im grundschuloder schulalter auf. prädiktive parameter für eine schlechte prognose sind u. a. eine hohe viruslast im blut und ein schneller bei fortschreitender infektion und störung im immunsystem treten klinische zeichen hinzu, die schon eher an einen immundefekt denken lassen (kategorie b) weitere atemwegsassoziierte viren das humane metapneumovirus (hmpv), das erst 2001 entdeckt wurde, ist bei kindern der zweithäufigste erreger der bronchiolitis. darüber hinaus verursacht hmpv auch bronchitiden und pneumonien. die hospitalisierungsrate ist wahrscheinlich ähnlich hoch wie bei rsv-und influenza-infektionen. das humane bocavirus (hbov) wurde im sommer infektionen mit humanen coronaviren (hcov) können v. a. bei kleinkindern zu erkrankungen der unteren und oberen luftwege führen. nicht selten finden sich koinfektionen mit anderen respirationstraktviren der nachweis aller drei erreger in infektiösen körperflüssigkeiten gelingt meist mittels polymerasekettenreaktion. die therapie ist in allen fällen symptomatisch helfen meist auch bei der hiv-assoziierten autoimmunthrombozytopenie.chemoprophylaxe die früher häufigste opportunistische infektion, die pneumocystis-jirovecii-pneumonie, lässt sich durch orale gabe von cotrimoxazol (trimethoprim 150 mg/m2 kof an 3 aneinander folgenden tagen pro woche) zu fast 100% vermeiden.allgemeine schutzimpfungen nur bei einer noch asymptomatischen hiv-infektion wird eine masern-oder mmr-impfung empfohlen. auch gegen varizellen kann geimpft werden, sofern die relative cd4-zellzahl ≥25% liegt. ansonsten sollten keine lebendimpfungen erfolgen. im übrigen sollten hiv-infizierte kinder entsprechend dem impfkalender mit inaktivierten impfstoffen/ toxoiden (polio-salk-impfstoff, hepatitis b, hib, dpt) und zusätzlich gegen pneumokokken, meningokokken und influenza geimpft werden. bei kindern, die regelmäßig mit immunglobulinen behandelt werden, sind aktivimpfungen dagegen nicht mehr sinnvoll. eine hiv-infektion ist namentlich nicht meldepflichtig. es besteht nur eine anonyme labormeldepflicht. j epidemiologie influenzainfektionen sind ubiquitär. alle 3-5 jahre treten influenzaepidemien durch antigendrift der zirkulierenden influenzasubtypen bei nachlassender immunität der exponierten bevölkerung auf. in größeren zeitabständen (10-20 jahre) treten durch rekombination zwischen humanen und animalen influenza-a-virusstämmen neue subtypen auf (antigenshift), die zu schweren pandemien führen können. in unseren breiten tritt die influenzagrippe üblicherweise in den monaten dezember bis april auf. die übertragung erfolgt überwiegend durch tröpfcheninfektion (niesen, husten), seltener durch kontaktinfektion. die kontagiosität ist kurz vor ausbruch der klinischen symptome am höchsten, sie dauert bis zu 1 woche an. die inkubationszeit beträgt in der regel 2-3 tage (1) (2) (3) (4) (5) (6) (7) .j ätiopathogenese influenzaviren gehören zur familie der orthomyxoviren. es existieren 3 typen (a, b, c). influenzaviren enthalten 8 rna-segmente, die von einer hülle von strukturproteinen umgeben sind. diese enthält spikes aus hämagglutinin (h) und neuraminidase (n). gegenwärtig zirkulieren die influenza-a-subtypen h3n2 und h1n1 sowie zwei verschiedene influenza-b-virusstämme. influenzaviren führen zu einer lytischen infektion des respiratorischen epithels. hierdurch kommt es zu einem verlust der zilienfunktion, zu einer verminderten schleimproduktion sowie zur nekrose der epithelschicht. es folgt eine entzündungsreaktion mit infiltration von lymphozyten, histiozyten und granulozyten. in den folgenden 2 wochen kommt es wieder zur regeneration und erholung der epithelzellschicht. die klinische symptomatik wie fieber, abgeschlagenheit, kopf-und gliederschmerzen ist immunologisch bedingt (u. a. durch freisetzung von zytokinen).j klinik bei älteren kindern und erwachsenen führt eine influenzainfektion typischerweise zur klassischen virusgrippe mit hohem fieber, kopf-und gliederschmerzen, abgeschlagenheit, trockenem husten, pharyngitis und konjunktivitis. nach einigen tagen tritt meist entfieberung ein, gefolgt von einer bis zu wochenlangen rekonvaleszenzphase. bei 10% der patienten finden sich klinische und radiologische zeichen einer pulmonalen beteiligung.bei kleinen kindern manifestiert sich eine influenzainfektion klinisch meist nicht unterscheidbar von infektionen durch andere respirationstraktviren (rsv, adenoviren, parainfluenzaviren u. a.), unter dem bild einer bronchiolitis, einer obstruktiven bronchitis, einer pneumonie, einer akuten subglottischen laryngotracheobronchitis (infektkrupp) oder einer unspezifischen atemweginfektion. darüber hinaus können bei kleinen kindern gastrointestinale symptome (durchfall, erbrechen) auftreten. häufig kommt es auch zu fieberkrämpfen. bei kindern mit chronischen grunderkrankungen (z. b. zystische fibrose, immunsuppression) können influenzainfektionen sehr schwer verlaufen. die meisten erkrankungen verlaufen insgesamt gutartig. bei immunsupprimierten kindern können erkrankungen mit parainfluenzaviren äußerst schwer und u. u. letal verlaufen (riesenzellpneumonie).j diagnose eine spezifische diagnostik ist meist nur bei schweren krankheitsverläufen erforderlich. parainfluenzaviren können im nasopharynxsekret nachgewiesen werden (immunfluoreszenztest, elisa, rt-pcr). der serologische nachweis von parainfluenzavirusspezifischen antikörpern spielt im klinischen alltag keine rolle.j therapie, prophylaxe eine spezifische antivirale therapie oder eine impfung existieren nicht. die behandlung der klinischen beschwerden ist symptomatisch. bei immunsupprimierten kindern mit schwerer parainfluenzavirusinfektion (pneumonie) kann ein therapieversuch mit ribavirin unternommen werden. bezüglich hygienemaßnahmen gelten die gleichen empfehlungen wie für rsv-infektionen. key: cord-342054-1u2fkwx3 authors: funaro, ada; gribaudo, giorgio; luganini, anna; ortolan, erika; lo buono, nicola; vicenzi, elisa; cassetta, luca; landolfo, santo; buick, richard; falciola, luca; murphy, marianne; garotta, gianni; malavasi, fabio title: generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune b cells date: 2008-11-12 journal: bmc biotechnol doi: 10.1186/1472-6750-8-85 sha: doc_id: 342054 cord_uid: 1u2fkwx3 background: human monoclonal antibodies (mabs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective. human cytomegalovirus (hcmv) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. the available therapeutic armamentarium (e.g. hcmv hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mab may be a decisive alternative in the prevention of primary and re-activated hcmv infections in these patients. results: the purpose of this study was to generate neutralizing mab against hcmv from the immunological repertoire of immune donors. to this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human b-lymphocytes are stimulated with tlr9-agonists and il-2; second, after both additives are removed, the cells are infected with ebv. using this strategy we obtained 29 clones secreting igg neutralizing the hcmv infectivity; four among these were further characterized. all of the mabs neutralize the infection in different combinations of hcmv strains and target cells, with a potency ~20 fold higher than that of the hcmv hyperimmune globulins, currently used in transplant recipients. recombinant human monoclonal igg1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated. conclusion: the technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mabs for immunotherapeutic uses. antibodies constitute the most rapidly growing class of human therapeutics and the second largest class of drugs after vaccines [1] . most of the growing number of antibodies entering the clinical trials are human [2] and are derived from phage-display technology [3] or from transgenic mice that express human immmunoglobulin genes [4] . however, the best mabs for clinical applications derive from natural human antibodies generated as a result of the in vivo immune response because they i) are products of the human and not animal repertoire, and ii) are of human origin, thus minimizing the risks of reactivity against self-antigens. lastly, iii) passive immunotherapy with human igg can confer immediate protection without the side effects linked to the use of chimeric or humanized mabs containing animal-derived amino acid sequences. furthermore, considerable evidence indicates that antibodies represent a new, although historically validated, approach to the development of therapies against bacterial and viral pathogens that causes disease in individuals with impaired immune response and/or for which there are few or no available drugs [5] [6] [7] [8] . ebv has long been used to immortalize human b-lymphocytes in vitro and to isolate human mab [9] [10] [11] , but the method was not routinely embraced due to its poor efficiency and because the resulting transformed cells are frequently unstable and fail to grow or secrete igg. agonists of toll receptor 9 (tlr9), e.g., cpg odn 2006 (synthetic oligodeoxynucleotides that contain immunostimulatory deoxycytidyl-deoxyguanosine motifs), have been reported to improve ebv infectivity and cloning efficiency [12] , however, there is no clear consensus as to the best conditions for their use and which subset of b-lymphocytes should be used. we applied the ebv immortalization procedure in the presence of different amounts of cpg with or without il-2 to 3 healthy donors selected on the basis of the high titer of cmv-specific igg in the blood and we obtained an immortalization efficiency not comparable to that recently reported [12] . therefore we re-evaluated all the steps crucial to cell immortalization, and established an optimized procedure of isolating human mabs from the repertoire of immune donors. the resulting method is highly reproducible, effective and rapid. further, it was validated by the successful isolation of clones of human b-lymphocytes that secrete high levels of igg that bind and neutralize the human cytomegalovirus (hcmv). hcmv is a ubiquitous opportunistic virus that infects 50-90% of adult human populations and persists for the life of the human host after primary infection. hcmv infection of an immunocompetent individual generally results in subclinical disease; by contrast, hcmv infection of immunocompromised individuals may cause lethal infections [13] . indeed, hcmv remains the single most impor-tant pathogen in hematopoietic stem cell transplantation [14] as well as, in organ transplantation [15] so that effective neutralizing mabs would make a major contribution to eliminate hcmv in these patients. the isolation of fully human mab from those generated during the natural human immune response is likely to identify the most effective therapeutic antibodies [6, 16] . many approaches have been used to generate human mab [17] , however, an efficient and reproducible process to isolate human mab with desired specificity from the natural repertoire is not available. agonists of tlr9 have been reported to improve ebv infectivity and cloning efficiency [12] . however, it is known that tlr9 agonists, such as cpg odn 2006, protect against a wide range of viral pathogens [18] [19] [20] . with this in mind, we reasoned that the presence of cpg during the ebv infection might exert negative effects and we decided to separate activation and ebv infection in two sequential phases. we developed a technology for raising human mabs for clinical applications based on two discrete steps (sequential, figure 1 ): first, purified human b-lymphocytes are treated with cpg2006 and il-2. next, after both additives are removed, the activated cells are infected with ebv in a distinct step. the new process was compared to a method using cpg2006 and il-2 in the presence of ebv [12] (combined) and a method using outline of the procedure for comparing ebv transformation methods figure 1 outline of the procedure for comparing ebv transformation methods. overview of the procedure for comparing the populations of ebv-transformed cells according to the sequential, combined, and basic methods. ebv alone (basic) (figure 1 ). different experimental settings were compared in pools of cells derived from 5 healthy donors, to minimize donor variability. the results clearly demonstrated that activation of cd22 + b-lymphocytes before ebv exposure leads to improved immortalization, as highlighted by the viability of cells following ebv transformation which is significantly higher than that obtained with both combined and basic conditions (figure 2a) . the short-term viability of b lymphocytes immortalized following the basic procedure is higher than those of b lymphocytes treated with the combined procedure. this is attributable to the polyclonal activation elicited by ebv itself which is not paralleled by an efficient immortalization. indeed, it is well documented that exposure of human b-lymphocytes to ebv results in cell death of a proportion of cells and that b-lymphocytes are activated but not immediately immortalized following exposure to ebv [21] [22] [23] . removal of the activating agents before immortalization significantly improves growth and viability of cd22 + blymphocytes allowing for a higher proportion of cells to be immortalized. indeed, when stimulation and ebv infection are performed separately rather than simultaneously, or with no stimulation at all, the cells exhibit increased proliferation potential and improved viability. in contrast, the combined presence of activating agents and ebv has adverse effects on both cell viability and ebv infectivity (figure 2a , b). the negative effects of the com-bined method on viability are surprising, given that cpg2006 induces robust polyclonal activation and proliferation of b-lymphocytes [24, 25] and that these effects are significantly enhanced by il-2 [26] . it has been reported that tlr-9 triggering resulted in higher transformation rates of b cells infected with ebv [12] but a comparison between ebv transformation in the presence of cpg2006 and ebv and after sequential exposure of b cells to cpg2006 and separately to ebv, was not investigated. the results obtained using different experimental setting on the same cell population clearly demonstrated that cpg2006 and il-2 used in combination with ebv exert negative effects on cell transformation. it is conceivable that the detrimental effects are related to the anti-proliferative activity of anti-viral cytokines induced by cpg2006 [27, 28] . indeed, the significant levels of tlr9 expressed by resting b-lymphocytes are further up-regulated by cpg2006 itself. increased tlr9 expression correlates with increased responsiveness to the ligand, indicating a positive feedback loop in which cpg2006 enhances its own effects [29] . kinetic studies demonstrated that optimal concentrations of cpg2006 combined with il-2 provide maximal cell stimulation after 2-5 days (data not shown). these observations are in line with cell-cycle analyses demonstrating that, in the same time frame, more than quantitative and qualitative comparison of different ebv transformation methods figure 2 quantitative and qualitative comparison of different ebv transformation methods. a) the b-lymphocyte subsets were prepared using the sequential, combined or basic methods, as outlined in fig. 1 . ten days after exposure to ebv, samples of each population were compared for the total cell number (black bars), and for the number of viable cells (white bars) measured microscopically by trypan blue dye exclusion. data are expressed as % of initial b cells exposed to ebv and represent the means ± s.d. of 5 cell counts for each condition. results are representative of two independent experiments. p < 0.001: sequential vs. combined; basic vs. combined (white bars); p < 0.05: sequential vs. basic (white bars) and sequential vs. combined; and basic vs. combined (black bars). b) ten days after infection with ebv, the cells prepared by the sequential, combined, or basic method were analyzed to identify viable lymphoblasts by propodium iodide (pi) exclusion (top panels) and cd23 expression (bottom panels) by flow cytometry. for each population of cells, the viable lymphoblasts (gated in regions r2, top panels) were defined as those with relatively high forward scatter (horizontal axes) and negative for pi fluorescence (vertical axes). r2 gated cells were then analyzed for cd23 expression. fluorescence was analyzed with a facscalibur flow cytometer and cellquest software (becton dickinson). background fluorescence was determined with a fitc-labeled, isotype-matched negative control mab. 10,000 events were measured for each condition. 95% of b cells enter the cell cycle independently from their memory or naïve phenotype [24, 30] . the sequential method does not modify the expression of cd21, which is the receptor for ebv [31] . the next step was to correlate the efficiency of ebv infection to the expression of cd23, which is directly upregulated by ebna2, one of the earliest markers expressed by ebv-infected b-lymphocytes [32] , it is expresses at high levels on ebv-transformed b cells [33] and its expression correlates with igg secretion [34] . the results demonstrated that cd23 expression (the number of positive cells and mean fluorescence intensity) is higher on cells exposed to the polyclonal activators and ebv separately than on those exposed simultaneously ( figure 2b ). the b-lymphocytes representing the whole repertoire of a selected donor immortalized with the sequential method show a significantly higher viability than those obtained with the combined method, and in consequence are easily clonable by limiting dilution, either in the presence or in the absence of polyclonal activators, such as cpg2006 and il-2. tlr9 expression is increased after ebv infection. thus bona fide, the addition of cpg2006 during the cloning phase enhances cell growth. however, we observed that cloning efficiency may be independent of cpg2006 and il-2 in some donors, suggesting that it is also dependent on the genetic background or physiological state of the donor. this issue warrants further investigation. the presence of cpg2006 and il-2 during the cloning phase may also have practical relevance for screening procedures. for instance, trace amounts of cpg2006 completely block hcmv infection in vitro, hampering screening by functional assays [20] . moreover, cpg2006 is a potent inducer of cytokines, such as il-12 and ifn-γ, potentially interfering with a number of bioassays [35] . the validity of the sequential method was evaluated by using it in a project to produce neutralizing human mabs against hcmv, a leading cause of morbidity and mortality in immunocompromised individuals and the most serious pathogenetic agent in transplant patients [15, 36, 37] . in the absence of other therapies such as vaccines [38] , these patients are treated with antivirals, which are often associated with considerable side effects and the emergence of drug-resistant strains. hcmv is also the leading viral cause of congenital infection associated to significant birth defects and neurological damage for which no effective therapies are available before birth [13, 39] . hyperimmune globulins specific for hcmv are beneficial in both conditions for the prevention and treatment of these infections [40, 41] . further, transfer of memory b cells from immune animals to severely immunodeficient recipients confers long-term protection from lethal murine cmv infection. this indicates that the humoral immune response is effective against cmv infection in the absence of a t cell-mediated response [42] . natural human mabs with stronger neutralizing activity than that of currently available immunoglobulin preparations may be a decisive alternative in the prevention of primary and re-activated hcmv infections in immunosuppressed patients, or during high-risk pregnancies [43] . using the sequential method, we obtained natural antibodies neutralizing hcmv infectivity by immortalizing b-lymphocytes from a healthy donor (cmv5) and from a patient recovering from acute hcmv infection (cmv7). high titers of anti-hcmv-specific igg coupled with the virus-neutralizing activity of the sera were the criteria for selecting the two donors from a larger panel ( table 1 ). the immortalized b-lymphocytes were cloned by limiting dilution. after 3 weeks, clusters of cells growing in wells were appreciable and the igg concentration in the clone supernatants was 10-40 μg/ml/10 6 cells, as determined by means of an immunoenzymatic method, measuring igg concentrations in the media normalized for cell densities. clones producing hcmv-specific igg were identified by different primary screening assays. in a first experiment a total of 1,664 clones were tested either by their binding to specific hcmv envelope glycoprotein domains (such as those from gb or gh, which have been shown to induce neutralizing antibodies) [44] , or by their ability to neutralize hcmv infectivity in vitro [45, 46] . the effect on hcmv infectivity was determined by an hcmv microneutralization assay based on the evaluation of the ability of clone supernatants to interfere on the infectivity of hcmv ad169 strain (a laboratory strain from atcc, cod. vr-538) in human embryo lung fibroblasts (helf). this strategy yielded 44 clones producing mabs specific for the gb, and 15 for gh. nine of these displayed a neutralizing activity of > 40%. those supernatants that were negative in the gb and gh elisas, or in the neutralization assay were also screened in an elisa that detects human igg that bind to hcmv virion proteins extract (beia-cmv), and 8 samples were further selected by this screening assay. in a second experiment, the immortalized blymphocytes were cloned by limiting dilution and after 3 weeks, 324 clones were screened directly for their neutralizing activity. of these, 20 produced igg with a neutralizing activity of > 40%, among these, 2 wells were found to bind gb, while the remaining 18 did not bind in either the gb or gh domains. four clones has been chosen as promising candidates for future clinical applications on the basis of their intrinsic characteristics: 10b7 and 8c10 showed a very strong binding to gb glycoprotein and 1f7 to gh glycoprotein; 26a1 has the strongest neutralizing activity. all clones were expanded in the absence of cpg2006, il-2 and a feeder layer, and further characterized. mabs10b7 and 8c10 proved to be specific for the hcmv envelope glycoprotein gb, mab 1f7 for the hcmv envelope glycoprotein gh and mab 26a1 recognized a viral antigen not yet characterized, as inferred from the reactivity on the hcmv virion proteins extract ( table 2 ). all of the antibodies neutralized the infectivity of the hcmv ad169 laboratory strain in human helf as well as that of the vr1814 clinical isolate in human umbilical vein endothelial cells (huvec), indicating that they are neither strain nor cell-specific. lastly, none of the antibodies bound the helf or huvec (data not shown). the specificity of the hcmv neutralizing activity was confirmed by the lack of significant neutralization of herpes simplex virus (hsv)-1 in vero cells (data not shown). clone stability was tested by maintaining the cells for 3 months in serum-free medium without cpg2006 or other additives, and the mabs recovered in the supernatant at a concentration of 20-50 μg/ml. all purified igg maintained their neutralizing activity; mab 26a1 demonstrated a potent neutralizing capacity, with a calculated inhibitory concentration 50 (ic 50 ) of 0.92 μg/ml against ad169 in helf (fig. 3a) , and of 1 μg/ml against vr1814 in huvec (fig. 3b) . the potency of 26a1 against both hcmv strains was ~20 fold higher than the reference anti-hcmv igg (ic 50 of 16 μg/ ml), currently used in transplant recipients. the inhibitory activity of mab 26a1 against the whole viral replicative cycle was confirmed by plaque reduction neutralization assay ( figure 3c ). the mechanism of virus neutralization is subject of ongoing studies. the broad and robust neutralizing activity of mab 26a1 encouraged us to generate recombinant igg suitable as prophylactic or therapeutic tools in clinical applications. to this aim, cells were submitted for 5' race pcr amplification, the pcr products of two amplification reactions were cloned using a eco ri restriction site in a sequencing vector and used for transforming top10 e. coli. a single vh and vl sequence was obtained from each cell culture, confirming that each original culture was clonal ( figure 4 ). the sequences encoding the vh and vl regions of mab 26a1 were cloned in expression vectors for the expression of mab 26a1 variable regions as recombinant igg1. the recombinant mab 26a1 confirmed the neutralizing activity of the natural mab 26a1 (figure 3 ). the sequential method is also effective when used with frozen cells. indeed, it was possible to select antibodies neutralizing hsv-1 and hsv-2 infectivity from frozen pbmcs from an hsv and hiv co-infected individual (not shown). neutralizing activity of natural (n) and recombinant (m) purified 26a1 igg in summary, this study provides proof of concept that good-quality, fully human mabs with desired specificity and biological activity can be generated with the sequential method. the strengths of this approach are: i) it allows the selection of human monoclonal igg to a variety of antigens, from a small sample of fresh or frozen peripheral blood, ii) it is rapid, iii) screening can be performed using a variety of assays, including functional assays, iv) the mabs of interest can be easily produced from the original clone as recombinant proteins suitable for clinical applications, and v) the generation of iggsecreting polyclonal populations can be considered as a library of antibody-secreting cells that can be used to select mabs with specificities not considered when cells were immortalized. the technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mabs for immunotherapeutic uses. it can be applied to the isolation of natural mabs from individuals affected by a variety of diseases, ranging from infectious diseases to cancer and autoimmune diseases. it allows the isolation of a large repertoire of human mabs with desired specificities to be selected from a small (~8 ml) volume of peripheral blood, and can be used with either fresh or frozen b-lymphocytes. moreover, direct cloning by limiting dilution and robust igg production allow the generation of panels of mabs with different specificities, thus allowing the development of mab cocktails for immunotherapy [47] . after informed consent was obtained, peripheral blood (8-10 ml) was collected from healthy blood donors or infectious disease patients. serum was screened for reactivity with hcmv glycoproteins and for hcmv neutralizing activity. peripheral blood mononuclear cells (pbmcs) were purified from heparinized peripheral blood by density gradient centrifugation on ficoll/hypaque (amersham pharmacia, milan, italy). cd22 + lymphocytes were purified by magnetic selection (miltenyi biotec, bologna, italy) from pbmcs pooled from 5 healthy donors, then cd22 + cells were divided into three aliquots that were exposed to different ebv-based methods for immortalization. for the basic process, cd22 + /igg + lymphocytes were isolated by depletion of igm + cells with a moflo ® high-performance cell sorter (dakocytomation, glostrup, denmark) and cultured (10 6 /ml) in 24 well plates in iscove's modified dulbecco's medium (imdm) supplemented with l-glutamine, 100 μg/ml streptomycin and 100 u/ml penicillin, non-essential amino acids and 10% fbs (complete medium) for 15 h, in the presence of ebv-containing supernatant (from b95-8 marmoset lymphoma cells, atcc, washington, dc, cat. no. crl-1612) 1:1 (v/v) at 37°c and 5% co 2 . the cells were then washed c. sera were tested in the hcmv microneutralization assay using the ad169 strain and helf [48] . numbers indicate dilution of serum required for 50% inhibition of hcmv infectivity. the number of viable lymphoblasts was measured microscopically by trypan blue dye exclusion. where indicated, the dna intercalating, fluorescent dye propidium iodide (pi, sigma aldritch) was used with a facscalibur flow cytometer and cellquest software (becton dickinson, franklin lakes, nj) to assess cell viability. viable cells were defined as those with a high forward and orthogonal scatter, characteristic of lymphoblasts, and excluding pi. min at 4°c with anti-human cd23-fitc (becton dickinson, milano, italy). after washing, fluorescence was analyzed using a facscalibur flow cytometer and cellquest software. cd23 staining was evaluated only on the viable lymphoblast population, gated using forward/orthagonal scatter and pi staining, as described above. background staining was determined with a fitc-conjugated isotypematched negative control mab. the number of cells analyzed was 10,000. the microneutralization assay was adapted from a previously reported technique [48] . the effect of b cell supernatants (or purified igg) on hcmv infectivity was measured by staining the hcmv immediate early antigens (iea, ie1+ie2) by indirect immunoperoxidase. iea-positive nuclei were counted under the microscope. b cell supernatants containing irrelevant igg antibodies were used as a negative control and a commercial preparation of human igg, purified from the serum of hcmv seropositive patients (cytotect; biotest, dreieich, germany), as a positive control, respectively. positivity was defined as ≥ 40% inhibition of iea + cells, compared to negative control wells. hcmv microneutralization assays were also performed with the endotheliotropic hcmv vr1814, a derivative of a clinical isolate recovered from a cervical swab of a pregnant woman [49] , and huvec, as described above. experiments were performed with cells at passage 2-6. plaque reduction assay was performed as reported [50] . selected experiments were done with igg purified on protein a columns (biorad, milano, italia) from serum-free supernatants (natural igg). antibodies were tested using the elisa assay for detection of igg binding to total proteins extract from hcmv virions (beia cmv igg quant kit; bouty, milano, italy) according to the manufacturer's instructions. the anti-hcmv recombinant gb igg elisa was purchased from biotest, ag (dreieich, germany) and used according to the manufacturer's instructions. the elisa makes use of the recombinant autologous interstrain fusion antigen cg3, corresponding to a combination of the gb antigenic domain 2 (ad2) from hcmv strains ad169 (swissprot acc. no. p06473) and towne (swissprot acc. no. p13201). the ad2 region contains a site (amino acids 70-81) conserved in different viral strains, that has been shown to be recognized by neutralizing antibodies [51, 52] . the gb-gst antigen was purchased from biodesign (saco, me, usa) and contains a gb immunodominant region fused to gst that reacts with hcmv-positive sera. it was used in elisa assays according to the manufacturer's instructions. the anti-hcmv recombinant gh-specific elisa was performed using the recombinant gh-gst antigen coated onto plastic. gh-gst corresponds to an in-frame fusion between the gh amino terminal region (amino acids 16-144) from the hcmv strain vr1814 and gst. the amino terminus of gh contains a linear antibody binding site between residues 34-43 that is recognized by neutralizing antibodies [44, 53] . the gh gene segment was pcr amplified from vr1814 dna with the following primers: vr1814 gh f: 5'-agtcatggatccctccttagtcac-ctca-3'; vr1814 gh r: 5'-actgatctcgaggcct-tcagctgctgc-3'. the 400 bp pcr product was then digested by xhoi and bamhi, the restricted fragment purified by agarose gel elecrophoresis and cloned into the pgex 4t3 expression vector (amersham) that had been restricted by xhoi and bamhi and gel purified. the recombinant gh-gst fusion protein was then expressed in e. coli bl21 and purified from the bacterial cell lysate on the basis of gst affinity. the variable regions of heavy chain (vh) and light chain (vl) of mab 26a1 were sequenced according the technology established by fusion antibodies ltd. briefly, frozen cells pellets (~5 × 10 4 cells) were used for extracting total rna with stat-60 rna extraction reagent. cdna was produced by reverse transcription with an oligo(d)t primer. pcr reactions were set up to amplify vh and vl regions with a mix of igg-and igk/λ specific primers, respectively. the pcr products of two amplification reactions were cloned using a eco ri restriction site in a sequencing vector (pcr2.1; invitrogen) and used for transforming top10 e. coli cells, following the manufacturer's instructions. a consensus sequence was determined from at least ten clones. the resulting dna sequences were aligned and translated into protein sequences enabling the generation of consensus dna and protein sequence for vh 26a1 and vl 26a1. the vh 26a1 and vl 26a1 protein sequences were compared and aligned with sequences present in the genomequest, geneseq, and ebi databases. the cdrs characterizing vh 26a1 and vl 26a1 protein sequences were predicted by the imgt database [54] . to generate a recombinant igg1, a consensus 26a1 vh sequence was amplified by pcr with the following primers: 5'-ttttttagatctcaccatgaacatactgtggag-catgctc-3'; 5'-ttttttagatcttgaggagacggtga ccagggttcc-3'. the primers contained a bglii restriction site (underlined) for ligation into the higg expression vector already containing human igg1 heavy chain constant domains. the in-house designed bicistronic mammalian retroviral expression vector pretmex (fusion antibodies ltd. belfast, ireland) was a dual promoter vector allowing expression of both antibody chains. the vh domain was cloned into the bamh1 site of multiple cloning site (mcs) 1 of the vector. a complete 26a1 recombinant vector was confirmed by dna sequencing. cho dg44 cells were adapted to serum-free suspension culture and seeded at 1 × 10 6 cells/ ml in 125 ml spinner flasks. 300 μg of plasmid dna was mixed with 900 μg of linear 25 kda pei and used to transfect the cell culture. the medium was harvested after 6 days. recombinant mab was purified with a protein g column on an akta prime chromatography unit following the manufacturer's standard programme (recombinant igg). values are expressed as means ± sd. data were analyzed for significance using a one-way analysis of variance (anova) with bonferroni post-test correction for multiple comparisons. data were considered significant at p < 0.05. therapeutic antibodies for human diseases at the draw of the twenty-first century monoclonal antibody successes in the clinic selecting and screening recombinant antibody libraries human antibodies from transgenic animals antibodies for the prevention and treatment of viral diseases passive antibody therapy for infectious diseases prophylactic and therapeutic efficacy of human monoclonal antibodies against h5n1 influenza human monoclonal antibodies by immortalization of memory b cells eb virus-induced b lymphocyte cell lines producing specific antibodies human monoclonals from antigen-specific selection of b lymphocytes and transformation by ebv synergy between anti-cd40 mab and epstein-barr virus in activation and transformation of human b lymphocytes an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus the human cytomegalovirus high risk of death due to bacterial and fungal infection among cytomegalovirus (cmv)-seronegative recipients of stem cell transplants from seropositive donors: evidence for indirect effects of primary cmv infection acceleration of allograft failure by cytomegalovirus human antibodies for immunotherapy development generated via a human b cell hybridoma technology potent antibody therapeutics by design local delivery of cpg oligodeoxynucleotides induces rapid changes in the genital mucosa and inhibits replication, but not entry, of herpes simplex virus type 2 immunotherapeutic uses of cpg oligodeoxynucleotides phosphorothioatemodified oligodeoxynucleotides inhibit human cytomegalovirus replication by blocking virus entry innate apoptosis of human b lymphoblasts transformed by epstein-barr virus: modulation by cellular immortalization and senescence epstein-barr virus provides a new paradigm: a requirement for the immediate inhibition of apoptosis steps involved in immortalization and tumorigenesis in human b-lymphoblastoid cell lines transformed by epstein-barr virus cpg motifs in bacterial dna trigger direct b-cell activation delineation of a cpg phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo activation of human b cells by phosphorothioate oligodeoxynucleotides interferon-alpha/beta receptor-mediated selective induction of a gene cluster by cpg oligodeoxynucleotide immune activation suppresses initiation of lytic epstein-barr virus infection the tolllike receptor repertoire of human b lymphocytes: inducible and selective expression of tlr9 and tlr10 in normal and transformed cells mechanism and function of a newly identified cpg dna motif in human primary b cells determination of the role for cd21 during epstein-barr virus infection of b-lymphoblastoid cells epstein-barr virus-induced b-cell transformation: quantitating events from virus binding to cell outgrowth b cell activation and the establishment of epstein-barr virus latency cell surface phenotyping and cytokine production of epstein-barr virus (ebv)-transformed lymphoblastoid cell lines (lcls) a minimal human immunostimulatory cpg motif that potently induces ifn-gamma and ifn-alpha production severe graft rejection, increased immunosuppression, and active cmv infection in renal transplant prophylaxis and treatment of cytomegalovirus disease in recipients of solid organ transplants: current approach and future challenges location, location, timing: analysis of cytomegalovirus epitopes for neutralizing antibodies preconceptional primary human cytomegalovirus infection and risk of congenital infections passive immunization during pregnancy for congenital cytomegalovirus infection intravenous immunoglobulin: appropriate indications and uses in hematopoietic stem cell transplantation protection from cmv infection in immunodeficient hosts by adoptive transfer of memory b cells the growth and potential of human antiviral monoclonal antibody therapeutics antibody-mediated neutralization of infectivity a model for neutralization of viruses based on antibody coating of the virion surface the antiviral activity of antibodies in vitro and in vivo recombinant human polyclonal antibodies: a new class of therapeutic antibodies against viral infections a rapid microneutralization assay for the measurement of neutralizing antibody reactive with human cytomegalovirus human cytomegalovirus immediate-early messenger rna in blood of pregnant women with primary infection and of congenitally infected newborns human cytomegalovirus stimulates cellular ikk2 activity and requires the enzyme for productive replication assembly of conformationdependent neutralizing domains on glycoprotein b of human cytomegalovirus an elisa using recombinant proteins for the detection of neutralizing antibodies against human cytomegalovirus the dominant linear neutralizing antibody-binding site of glycoprotein gp86 of human cytomegalovirus is strain specific imgt/v-quest, an integrated software program for immunoglobulin and t cell receptor v-j and v-d-j rearrangement analysis the authors would like to thank am gianni, m di nicola and a lazzarin for providing blood samples from cmv + patients, al horenstein for assistance with antibody purification, and g gerna for providing the hcmv vr1814 strain. gerrit hagens and guido poli are acknowledged for helpful discussion and intellectual contributions. this work was funded, in part, by grants from ribovax biotechnologies sa (geneva, switzerland), from miur (italian ministry for university and scientific research) (prin and 60% projects), from ricerca sanitaria finalizzata e ricerca scientifica applicata (regione piemonte), from compagnia sanpaolo (torino, italy) and from firms (international foundation for research in experimental medicine). a. funaro, m. murphy and g. garotta are inventors on a pct patent application (published as wo 2007/068758) describing the technology. a funaro, g. gribaudo and s. landolfo are inventors on unpublished patent applications describing specific hcmv-neutralizing antibodies that were isolated using the described technology. all patent applications are filed in the name of ribovax biotechnologies sa (petit-lancy, switzerland).data were analyzed for significance using a one-way analysis of variance (anova) with bonferroni post-test correction for multiple comparisons. data were considered significant at p < 0.05. af, ggr, mm and gga designed the study. al carried out the neutralization assays, nlb carried out the immunoassays, eo participated to the analysis and interpretation of data. ev, lc. selected antibodies neutralizing hsv-1 and hsv-2, sl and lf contributed to design the study. rb prepared the recombinant 26a1 igg, and af and fm wrote the paper. all authors read and approved the final manuscript. key: cord-327883-s9nbr5y8 authors: nan title: section virology date: 1990-03-31 journal: zentralblatt für bakteriologie doi: 10.1016/s0934-8840(11)80039-3 sha: doc_id: 327883 cord_uid: s9nbr5y8 nan s71 contains a human endogenous retroviral element related to simian sarcoma virus (ssv) and its associated virus ssa v. by sequence analysis and comparison with the corresponding ssvlssav sequences the genomic organization of s71 element was determined to be 5' gag-s-nrs-pol-ltr 3'. s-nrs represents a region of 1130 bp in s71 that consists of nonretrovira!'sequences. a probe containing the complete s71 element was used to screen two human cdna libraries under low-stingency conditions. 21 clones were isolated which had yielded strong hybridisation signals with the s71 probe. hybridisation of these clones with different fragments of the s71 genome revealed that 12 of these clones contain s-nrs related sequences. five of the isolated clones were sequenced and compared with s71. one of them contains a homologous to the u3-region of the s71 l tr. the outer four clones sequences with 65-82% identity to s71 s-nrs on nucleotide level. the deduced amino acid sequences of the four s-nrs clones are only 30-70% homologous to s71 s-nrs. none of the sequences represent a contiguous open reading frame. these results indicate that s-nrss probably do not have protein encoding function. hybridisation of human genomic dna shows that s-nrs belongs to a multicopy family of related sequences. human immunodeficiency viruses, human t lymphotropic viruses, and the human spumaretrovirus (hsrv) constitute the natural occurring human retroviruses. molecular cloning and sequencing of hsrv revealed four open reading frames (orfs) additional to gag, pol, and env. three additional orfs, bel 1-3, are located berween the env gene and the 3'ltr, one additional orf, sl, is loacted in the central region of the genome. functional analysis of the bel and s genes requires an infectious molecular clone of hsrv, which was not available so far. we have constructed such a clone (phsrv). the biological activity of the clone was confirmed by a variety of criteria: induction of characteristic cpe in susceptible cells infected with cell-free supernatant from cultures transfected with phsrv; indirect immunofluorescence; radioimmunoprecipitation of viral proteins and electron microscopy. furthermore, it is shown that phsrv is able to transactivate the hsrv-ltr. glycoprotein iv of bovine herpesvirus 1 (bhv-l) is one of the major immunogenic glycoproteins of the viral envelope and is involved in absorption and/or penetration of the virus. it is esential for the production of infectious virus and cannot be deleted from the viral genome. in contrast to the analogous proteins of herpes simplex virus (gd) and pseudorabies virus (gp50) the giv of bhv-1 shows a size heterogeneity among different strains. this indicates that in at least one part of the polypeptide backbone the aminoacid composition is variable. comparison of the nucleotide sequences of four bhv-1 strains revealed that the size heterogeneity in these strains is due to different repetitions of a 90 bp sequence, which is located upstream the sequence coding the membrane spanning domaine of giv. we are currently testing the possibility to introduce heterologous sequences into this part of giv to analyse whether the giv can contain antigenic epitopes of different viruses without affecting the functional activity of the giv. the molluscum contagiosum virus (mcv), a member of the family poxviridae, induces epidermal proliferation in man. the genome of mcv type 1 (mcv-l; 188 kbp) had been characterized by physical mapping using a defined gene library of the viral genome and by dna-dna hybridization. the physical maps of the viral genome were constructed for the restriction endonucleases bamhi, clai, ecori, and hindiii. detailed hybridization experiments revealed the presence of repetitive dna sequences located within the terminal regions of the viral genome, e. g. bamhi dna fragment b (18 kbp; 0 to 0.095 mu) and e (10.9 kbp; 0.944 to 1 mu). the fine mapping of these particular regions indicates that the repetitive dna sequences are located within the hindiii dna fragments jl (4.2 kbp; 0.962 to 0.985 mu), k (4.0 kbp; 0.014 to 0.036 mu), pi (2.7 kbp; 0 to 0.014 mu), and p2 (2.7 kbp; 0.985 to 1 mu). nucleotide sequence analysis was carried out. it was found that the hindiii fragments jl and k each contained an inverted repeat of 1682 bp and 1675 bp, respectively. the homology between the both repetitive dna elements was found to be 99%. the analysis of the coding capacity of the determined dna sequences revealed the presence of 9 and 5 open reading frames in the hindiii dna fragment k and the corresponding region in the hindiii dna fragment jl, respectively. positive in-patients (n = 307) and outpatients (n = 30) showed an increasing prevalence of antibodies to herpes viruses. the difference was particularly evident for cmv-igg (100% prevalence in hospitalized, 91.8% in out-patients and 63.5% in hiv negative patients). anti-hbc, with a low prevalence in controls (16.5%) was markedly associated with progression of hiv infection (69.5% prevalence in out-patients, 84.6% in in-patients). the antibody pattern to opportunistic viral infections may be regarded as a marker of pathogenicity in hiv infected patients. because of the strong crossreactivity of the two serotypes of herpes simplex virus (hsv) it is very difficult to distinguish between hsv-l and hsv-2-typespecific antibodies in patients' sera. although genital herpes can be caused by hsv-l in 14% of the cases, hsv-2 is the cause of most recurrent genital herpes (86%). the serological differentiation of past hsv-2 from past hsv-l infections is necessary for identifying pregnancies likely to be complicated by recurrent maternal hsv-2 infection. by improving the enzyme-linked immunosorbent assay (elisa) for hsv-2-antibodies and additional testing of sera by western blot, we were able to specifically identify hsv-l-and hsv-2-antibodies in serum samples. -for the elisa, hsv-2-antigen was immobilized on 96-well micro titer plates (nunc), and patients' sera were added. antibodies were detected by biotin/streptavidin peroxidase. -for the western blot the electrophoretically separated hsv-2-antigen was used. the antigen was blotted to a polyvinylidenedifluoride (pvdf) membrane (immobi-10n™, pharmacia) and incubated with the test sera. antibodies against typespecific glycoproteins of hsv type 2 (gg-92), forming a sharp band, were visualized by the application of biotinlstreptavidin peroxidase. our results showed that the elisa and the western blot correlated in 91.6%. serum samples with high antibody titers against hsv-l showed false positive reaction in the hsv-2-elisa in 8.4% of the cases. the optical density of these sera ranged ±0,2 around the cut-off value of the elisa. these samples could easily be identified by western blot. -serological studies showed, that 30% of the prostitutes (n = 20) have antibodies against hsv-l and 25% against hsv-2. in 40% both types of antibodies could be found. furthermore 70% of female patients in gynecological treatment (n = 20) had antibodies to hsv type 1 and in 10% of the cases against both serotypes. 20% showed no antibody reaction. roughly the same prevalence of hsv-l and hsv-2-typespecific antibodies was found in 20 patients from the dermatological department. a. m. eis-hubinger, k. kurowski, and k. e. schneweis inst. f. med. mikrobiologie u. immunologie, univ., d-5300 bonn 1 a nonneutralizing monoclonal antibody (mab), directed against glycoprotein gc of herpes simplex virus type 1 (hsv-l) and neutralizing mab to glycoprotein gb were evaluated for their ability to protect mice from genital hsv-infection. the nonneutralizing mab had only little effect on virus replication in the mucous membranes, but completely protected the mice from peripheral skin lesions, neurological illness and death. progression of the virus to the central nervous system was obviously inhibited by a modified course of the ganglionic infection: the number of ganglia presenting infectious virus and final latency were reduced, although early latency was not induced. in addition to the effects of the nonneutralizing mab, the neutralizing mab effectively shortened viral shedding from the vagina and converted proliferative ganglionic infection into latency. the different modes of action of the monoclonal antibodies are discussed in context to the possible defense mechanisms of the humoral immunity against virus proliferation in mucous membranes and in ganglia. the thymidine kinase (tk) gene of bovine herpesvirus 1 (bhv-l) can serve as an integration locus for foreign dna into the viral genome, because expression of a functional active tk is not essential for viral replication. the tk -gene of bhv -1 was located by conversion of tk--negative cells to the tk+ -phenotype and marker rescue experiments. it contains an open reading frame of 1068 bp. sequence comparison with other herpesviral tk-proteinsequences revealed, that only certain parts of the amino acid sequence as the nucleotide binding loop are highly conserved. hybridization of rna from infected cells with tk-dna showed, that tk-mrna has a size of 5.1 kb, whereas the size of tk-mrnas of other herpesviruses measures about 2 kb. 5'-and 3'-end of the tk transcription unit were characterized using nuclease 51-analysis. an open reading frame, when translated into protein, is homologous to the glycoprotein h (gh) of herpes simplex virus type 1 and lies downstream the open reading frame of the tk-gene. we conclude, that the expression of,bhv-l-gh correlates with the expression of thymidine kinase. preliminary results suggest, that transcription of both genes is initiated from one common promoter. in foot-and-mouth disease virus (fmdv) infected cells disappearance of the nuclear protein histone h3 and the simultaneous appearance of a new chromatin associated protein termed pi can be observed. we have sequenced the amino terminus of pi and clearly showed that protein pi derives from histone h3 by proteolytic cleavage. the 20 n-terminal amino acid residues are specifically cleaved off early during infection. in addition using an in vitro transcription/translation assay with different fmdv clones we showed that the histone h3 -pi transition is fmdv 3c protease dependent. this protease until now has only been found to be responsible for the processing of the viral polyprotein. the 3c protease (i) is the only fmdv protein required to induce this histone h3-pi transition, (ii) no other viral protein can perform this specific cleavage, and (iii) no viral precursor fusion protein is necessary for this specific cleavage, as it is reported for the processing of the poliovirus p1 precursorpolyprotein. the 3c mediated histone h3 cleavage is not restricted to chromatin derived from natural host cells. as the deleted part of the histone h3 corresponds to the domain presumed to be involved in the regulation of transcriptional active chromatin in eucaryotes, it is postulated that this specific cleavage of h3 is a mechanism which fmdv utilizes to switch off host cell rna synthesis, as is reported for picornaviruses. in combination with the reported mechanism of host cell translation shut off by cleavage of the cap-binding protein complex, this specific histone h3 cleavage could contribute to the almost complete breakdown of host cell functions during infection. the tk of hsv -1 contains three regions of homology to highly conserved sequences of other nucleotide binding enzymes. these are the residues 49 to 66 (nucleotide binding loop), residues 161 to 168 and residues 317 to 319. comparison of the viral and cellular tk protein sequences and 3-d structure analysis led to the hypothesis that the asp 161 might be involved in binding of acyclovir which is used as a therapeutic for hsv infection. site specific mutagenesis which replaced asp 161 by asn led to a polypeptide with no tk activity. the same was found when residues 184 to 306, which are not present in cellular tks, were deleted. both mutant genes were integrated into vaccinia virus to study the biochemical properties of the resulting polypeptides and to analyze the importance of the mutated sequences for the functional activity of the enzymes. . in contrast the human glioma cell line u138 and the adenovirus transformed kidney cell line 293 revealed replication of both organspecific variants as early as 48 hours p.t. nevertheless the amount of replicated dna of the kidney variant was clearly reduced in glioma cells and replicated dna was detected later than with the cns variant. from these data we conclude, thatjcv replication is regulated not only host type specific, but also in a cell type specific manner. young human volunteers were vaccinated with a killed hepatitis a vaccine produced in human embryo fibroblast cells. different amounts of vaccine were administered by the intramuscular route. 4 weeks after the first vaccination with a 0.3!-lg vaccine 36.6% of the volunteers seroconverted. 4 weeks after the second injection the seroconversion rate was 95.1 %. anti-hav was determined quantitatively and results showed anti-hav titers 20 to 100 times higher than those after gamma globulin administration. one year after the vaccination 19/19 volunteers still had anti-hav titers in their sera 20 to 50 times higher than those after gamma globulin administration. human papillomaviruses (hpv) associated with the epidermodysplasia verruciformis syndrome (ev) represent a group of closely related viruses, clearly distinct from other hpvs. the most interesting features of ev-viruses are: (i) high oncogenic potential of some specific virus types, (ii) extremely narrow host range. to get some insight into the molecular basis of processes controlling the viral expression we studied the sequence-specific dna-protein interactions within the genomic regulatory regions. using the nuclear extracts of hela cells and a combination of exonuclease iii-and dnase i-footprinting techniques the protein binding maps have been constructed for hpv8 and hpv19, the prototypes of viruses with high versus low oncogenic potential. the sequences in question showed a complex array of protein binding domains, covering almost the entire length of the regulatory regions. a cluster of prominent binding sites, which overlapped with the sequences of the motif 44 (m44), a highly conserved element in most of the ev-viruses, was investigated in more detail. in transient cat assays the m44 motif of hpv8 as dimer or trimer was found to act as a strong expression activator. the analysis of m44 sequences in band-shift tests revealed partially different sets of dna binding proteins, interacting with the elements from hpv 8 and 19. one of these proteins, at least in case of hpv8, was shown to be the regulatory factor api. a part of m44 sequences in the vicinity of the ap1 binding site display a homology to enhancer elements in other small dna viruses. a. gerritzen, j.-p. kleim, and a. friedrich inst. for med. microbiology, univ., d-5300 bonn semiquantitative detection of hepatitis b virus (hbv) dna in sera of infected individuals has become an important means of modern serological hepatitis diagnostics. it enables investigators to draw conclusions on infectivity, prognosis and therapeutical success. molecular hybridization using radioactively (i. e. 32phosphorus) labeled dna probes with subsequent autoradiography is characterized by both high sensitivity and specificity. alternatively digoxigenin-labeled probes which are detected by enzyme immunoassay have been employed. using this method 1-2 pg of hbv dna in aqueous solution have been detected with high specificity on membranes made of cellulose nitrate. testing sera of hepatitis patients 1 pg of hbv dna has been detected on nylon membranes of 1.2 11m pore size e 2 p: 0.1 pg). unfortunately specificity remained disappointing even after centrifugation, digestion by proteinase k and treatment of the sera with phenole/chloroform. objective. to assess the risk for laboratory personnel resulting from patients with unknown hiv infection, sera sent in to the institute for medical virology and immunology (imvi essen) for immunologic and/or virologic testing were anonymously screened for hiv antibodies. methods. between july and december, 1988, a total of 6252 sera from different departments of the university clinics of essen were selected for screening under code on the basis that no hiv antibody test was requested for diagnostic purposes at the same time, and sera from patients found to be anti-hiv positive during the previous 4 years were disregarded (n = 61). sera reactive in elisa (enzygnost-anti-hiv, behring) were further tested in a second (dupont) and third elisa (pasteur) and in a confirmatory test (western blot -wb-, dupont). results. in 11 of 6252 sera from patients not suspected for hiv infection, hiv antibodies were confirmed by wb (0.176%) (table) . an additional 4 sera showed less than 2 specific we bands and were considered indeterminate. sera from unknowingly infected patients were sent in most often from the internal (n = 5) and dermatology departments (n = 3), whereas sera with indeterminate hiv antibody results mostly originated from patients who received tumor treatment (n = 2). conclusions. the prevalence of 0.176% unknown hiv infections reflects a higher rate than generally assumed but possibly results from a selection in a university of patients with immunological disorders and underlying hiv infection. an overall prevalence of 1 % anti-hiv positive sera suggests the strict adherence to infection control measures in medical laboratories. bovine viral diarrhea virus (bvdv) is a member of the pestivirus group. in culture of bovine cells, a non cytopathic (ncp) and a cytopathic (cp) biotype can be distinguished. using radioimmunoprecipitation a monoclonal antibody (mab) detected a 125 kd nonstructural (ns) polypeptide in cells infected with ncpbvdv. in cells infected with cpbvdv an additional 80 kd protein was precipitated. a second set of mabs was directed against the 48 kd minor glycoprotein. the distribution of both proteins in cells infected with the two biotypes was analyzed by immunofluorescence analysis (ifa). none of the two proteins was detected on the surface of live, infected cells. the ns protein was homogenously distributed in the cytoplasm of cells infected with both biotypes. the second set of mabs displayed different staining patterns in cells infected with each of the biotypes. in cells infected with cpbvdv, a homogenous cytoplasmic fluorescence was visible. in cells infected with ncpbvdv, the stain was largely restricted to the perinuclear zone, giving a distinct staining with each of the mabs. the possible significance of these results for cytopathogenicity are discussed. in sera of patients with a variety of inflammatory rheumatic diseases autoantibodies to selfantigens (autoantigens) are found. one of the hypotheses on the molecular basis of autoimmune diseases which is discussed intensively since decades, assumes that structure-or sequence-related epitopes of virus and host proteins might be involved in initiation of autoimmune processes (molecular mimicry). -to search for crossreactive or sequenceidentical epitopes of cell and virus proteins, we started to map in detail antigenic regions and individual epitopes on autoantigens. a most detailed study has been performed for the ulsnrnp specific p68 protein which is the major target of autoantibodies in systemic rheumatic diseases such as mixed connective tissue disease and systemic lupus erythematosus. -by immunoblotting and elisa assays performed with recombinant p68 fusion proteins and peptides four antigenic regions and many patient specific epitopes could be mapped. in one of the antigenic regions an epitope 5 amino acids long could be identified which is also present on the matrix protein m1 of influenza b viruses. with affinity purified p68 specific autoantibodies the reaction with the m1 influenza b virus protein and vice versa could be experimentally verified. -these results demonstrate for the first time that autoantibodies from patients with rheumatic diseases recognize an epitope of identical sequence on a highly prevalent and pathogenic virus and a major auto antigenic target. whether the existence of this common epitope is by chance or causually related is currently investigated. detection of antibodies against cytomegalovirus (hcmv) induced "early" -antigens by immunoblotting contradictory informations about the diagnostic assessment of antibodies against the "early" -antigens of human cytomegalovirus (hcmv) have been numerously published. we investigated the correlation between the incidence of antibodies against hcmv "early"proteins and the state of infection. ninety-six hcmv-igg-positive sera (elisa) were tested for specific igg-antibodies against hcmv "early"-antigens by immunoblotting. three groups were examined: (a) 29 renal transplantation patients, (b) 33 aids-patients and (c) 34 randomly selected healthy individuals. each group yielded approximately the same percentage of positive immunoblots (59%,58%,59%). sera belonging to group (a) or (b) reacted stronger and recognized a greater number of polypeptides (6±4; 5±3) when compared to healthy persons (4±3). all immunoblot-positive sera reacted at least with the 66kd protein (major "immediate early"-protein). fourteen out of 19 hcmv-igm-positive sera (elisa) belonging to group (a) or (b) recognized "early"-antigens (74%). twenty-two out of 43 hcmv-igm-negative sera (51 %) reacted as well. we conclude that an acute hcmv infection does not cause the formation of antibodies against "early" -antigens in all individuals and furthermore these antibodies can persist during a subclinical/latent infection. berne virus (bev), isolated from a horse, is the prototype of the toroviridae, a proposed new family of positive-stranded rna viruses. bev virions consist of a peplomer-bearing membrane which envelops a tubular nucleocapsid of helical symmetry; this structure can be straight (resulting in a tubular particle) or bent into an open torus (conferring the shape of a kidney or biconcave disc to the virion). the nucleocapsid contains a single polyadenylated rna molecule of > 25 kb and the most abundant polypeptide, an 18.3kd basic phosphoprotein with nucleic acid binding properties. upon infection, a set of 5'-coterminal sub genomic mrnas is synthesized by leader-independent transcription; only the unique 3' sequences of each mrna are translated. -the combination of virion structure, nucleocapsid protein size and leader-independent transcription is unique in virology and justifies a family status for bev and related viruses. however, coronaviruses have a similar genome organization, a 5'-coterminal nested set of mrnas and sequence similarities in the second orf of the polymerase gene of bev. since the latter suggest common ancestry, toro-and corona viruses together may be considered as a third evolutionary cluster of positivestranded rna viruses, in addition to the alpha-and picornavirus superfamilies. w. ]ilg, h. mairhofer, c. markert, and h. wolf max v. pettenkofer-inst. for hygiene and med. microbiology, univ., d-8000 munich in order to characterize viral and nonviral structures responsible for the recognition of ebv-infected lymphocytes by the immune system, we studied the reactivity of ebv-specific cytotoxic t cells towards autologous ebv-positive lymphoblastoid cell lines (lcls). lcls were established from different ebv-positive donors by either spontaneous outgrowth of cells transformed by the donors own virus or by infection with a laboratory strain of ebv (b 95-8). these cell lines were used for the generation of ebv-specific cytotoxic t cells by weekly stimulation and addition of interleukin 2; cells were cloned by limited dilution. in chromium release assays these cd8 positive t cell lines and clones were able to discriminate between two autologous lymphoblastoid cell lines infected by either the "own" virus strain or the b 95-8 strain, respectively. further experiments using various concentrations of effector cells showed that structures recognized by different t cell clones were expressed only in a certain percentage of cells (20-40%) of each lcl, and that nonviral components differently expressed on different lcls also seem to playa role ctlllcl interactions. the large-scale production and purification of recombinant gp 160 leads to a native and biologically active protein, which bounds specifically to the cd 4 receptor. anti-hbs response of hbs vaccine recipients within an ongoing immunization course was studied in vitro. antigen specific antibody production and proliferative response as well as the expression of cd23 and cd25 and the secretion of scd23 were detected. it was the aim of the study to analyze the effect of various cell stimuli (mitogens, lymphokines and antibodies against cell surface antigens) on the antigen-specific immune response in vitro. pwm driven enhancement in hbs specific antibody production was shown to be time dependent. in contrast to iii and il6, incubation with il4 led to a significant increase in anti-hbs antibody synthesis. low doses of 114 led to a significant increase in hbs induced cd23 expression. moreover, 114 induced scd23 secretion is enhanced by hbs antigen. il2 receptor expression of hbs vaccine non-responder pbmc is reduced as compared to the responder group. cd25 receptor expression of responder pbmc is influenced by antigen as well as iii whereas no modulation can be seen with non-responder pbmc. the capacity of nonresponder cells to respond to hbs antigen is reduced whereas the capacity of these cells to respond to iii is markedly enhanced. in summary, our data show that the hbs specific immune response in vitro is influenced by lymphokines. 38.9% of sera from women (n = 560) and 32.2% of sera from men (n = 96) were found to contain antibodies directed against the l2 gene products of the hpv types mentioned above. 9.6% of female and 12.5% of male sera exhibited antibodies directed against the hpv-16 e4 and/or e7 gene products. on the other hand in an individual female serum antibodies directed against hpv-16 el and e2 could be found, and another one harboured antibodies directed against hpv-16 e6. all of the antibodies were of igg type. in order to specify whether antibodies against papillomaviruses are associated with sexual activity, we used sera from 100 female individuals of age 14 years old. we tested also the sera from the same individuals which were subsequently taken at time intervals of 3 and 10 years. it could be shown that l2 antibodies were present in the sera over the whole period of time. however, also an increase in the frequency of antibodies directed against hpv -16 ll with growing age of the women was observed. these data indicate that beside the distribution of human papillomaviruses by sexual intercourse other routes of papillomavirus infection may exist, for instance perinatal infection. further experiments revealed differences in salt stability and ph dependence between gd, gb, and gc with respect to their binding ability. thus, binding of gd could be disrupted at 500 mm nacl and had a ph optimum between ph6 and ph7, whereas binding of gc and gb showed a ph optimum near ph7 and was stable even at salt concentrations above 1000mm nacl. the results given above provide direct evidence for a functional role of gc, gb, and gd in binding of hsv-1 to the cell surface. to 4 h. as demonstrated by electron microscopy, the mechanism of this effect is due to irreversible binding of the metal ions to the viral membrane, presumably to mercaptan groups of the viral glycoproteins. adsorption of zinc inactivated virus is reduced to some extent only whereas a heavy impact has to be presumed on virus penetration. only in a small concentration range (100 to 200 i!m zn in culture medium) zns04 is inhibitory to the virus replication in infected cells without serious toxicity to the cells themselves. -therefore the mechanism of the in vivo efficacy of zinc preparations against hsv lesions of the mucosa is mainly based upon inactivation of free virus and only partly due to virustasis or inhibition of hsv adsorption. experiments done either with synthetic peptides or with cells transfected with the gene encoding the ebv latent membrane protein bnlfi suggested that this protein is a target for ebv specific cytotoxic t-cells (ctls). as such experiments do not closely resemble the natural situation we wanted to expand on this topic and established a system which should better reflect in vivo conditions. we constructed two recombinant vaccinia viruses, one carrying the complete bnlf l-ma reading frame, the other encodes a truncated form of bnlf i-ma containing the third exon only which was found on ebv infected burkitt lymphoma in addition to the downregulated full length protein. the continuous coding sequence of the complete gene which is necessary for expressing foreign proteins in recombinant vaccinia was cloned from genomic fragments and synthetic oligonucleotides. after infection of several cell types including freshly isolated pbls the expression of both forms of lmp was clearly demonstrated in immunoblots and by immunofluorescence. -ebv -specific cytotoxic t-iymphocytes (ctl) generated by repeated stimulation with autologous ebv infected lymphoblastoid cells in the presence of il-2 were reacted with autologous pha stimulated blasts infected with the two vaccinia viruses and the wild type virus as a control. ctls of two persons recognized the full length bnlfi protein but not the shortened protein whereas ctls of two other persons did not recognize any of the two bnlfi proteins. from these experiments we conclude that 1., the n-terminus of the bnlfi protein contains a determinant which is recognized by some ebv-specific ctls and 2., that there are additional proteins which are targets for other ebv-specific ctls. the expression of the epstein-barr virus regulation gene bmlfi is regulated by a promoter/enhancer complex located upstream from the short orf bslfi (transcribed as a spliced unit). the region 5'-proximal to the tata box contains consensus sequences for binding transcription factors (e.g. nf-l, ap-l). we analyzed the activity of this control region in different cell systems using appropriate reporter assays (chloramphenicol acetyltransferase and hepatitis b virus surface antigen as reporter genes). -a 127 bp aluilbsteii fragment including the tata box was shown to be sufficient to promote the transcription in raji cells induced to lytic ebv expression by different procedures. responsiveness to phorbolester was shown in ebv negative cells (3t3). the upstream region of the bmlfi promoter (1333 bp bsteiiissti fragment) was identified as regulatory segment in transfection assays showing both positive and negative effects depending on the presence and state of ebv. -in ebv negative cells (hela), the upstream element clearly responded to the ebv transactivator brlfl. this specific trans-activation of the distal control region by brlfi (expression vector pksvr kindly provided by a. sergeant, lyon) was down-regulated in latently infected cells (raji) while in ebv producer cells (p3hr-l) trans-activity by brlfi was detected. following insertion into a heterologous expression system the bmlf1 upstream control region enhanced transcription of the sv40 early promoter independent of its orientation. we have previously shown that mutants of prv that do not express the nonessential glycoprotein gill adsorb less readily to cells than does wildtype virus. we show here that the first interactions of prv with target cells occur via binding of the virus to a heparin-like component on the cell surface and that the viral protein mediating this binding is glycoprotein gill. this conclusion is based on the following findings: 1) heparin inhibits adsorption of wildtype prv effectively as measured by plaque formation as well as by attachment of radioactively labelled virus to cells. however, it affects adsorption of gill-mutants only slightly. 2) while wild-type prv binds well to matrix-bound heparin binding of a giiimutant is dramatically reduced. 3) pre-treatment of cells with heparinase reduces plaque formation and adsorption of wildtype pry by 90% but does not affect gill mutants. 4) of the pry membrane proteins glycoprotein glii binds most abundantly and specifically to heparin sepharose beads. our results indicate that binding of pry via glycoprotein glii to a heparin-like cellular component promotes efficient adsorption. in the absence of glii or of the heparin-like cellular receptor the virus adsorbs by an alternative less efficient mode. a. e. metzler and r. wyler inst. of virology, univ., ch-8057 ziirich, switzerland bovine herpesvirus 1 (bhv1) causes two well established entities, namely infectious bovine rhinotracheitis (ibr) and infectious pustular vulvovaginitis (ipv). encephalitis, a third entity, is less well understood. whereas ibr usually follows horizontal virus spread by aerosols, ipv is acquired by venereal contact. it remains controversial if the conditions were associated with distinct viruses. restriction endonuclease analysis of viral dna or evaluation of viral proteins following separation in sds-polyacrylamide gels may be used to distinguish between bhv1.1, bhv1.2 and bhv1.3. the proteins of european field isolates, recovered from distinct disease episodes within the time period 1960 to 1985, were compared with the proteins of established laboratory strains. of 74 field isolates 43 were classified as bhv1.1, 31 as bhv1.2 and none as bhv1.3. bhv1.2 was most regularly recovered from cattle with genital afflictions. however, some bhv1.2 strains originated from animals with ibr and others from aborted fetuses as well. the first recognition of bhv1.1 among the field isolates in 1972 coincided with sequential waves of severe ibr outbreaks throughout europe. results obtained with laboratory strains indicated that bhv1.1, obviously of limited pathogenic potential, must have existed at earlier times. together with published data the results suggest that recognition of a strain as bhv1.1 or bhv1.2 does not necessarily reflect a specific pathogenic potential nor a distinct organ tropism. nevertheless, the severe ibr incidences were clearly associated with a virulent bhv1. it has been demonstrated that the late gene products ll and l2 of certain papillomavirus types exhibit dna-binding activity. hepatitis b virus core proteins are also an example for the interaction of viral capsid proteins with nucleic acids. in this case dna binding is mediated by carboxy terminally located clusters of basic aa residues. similarly, the ll gene product of hpv-16 contains also groups of basic amino acids at its carboxy terminus. to examine whether this region is in fact the dna binding site of ll, we expressed different parts of this gene in e. coli as ~-gal fusion proteins. the vector system which we used allows the cleavage of the viral protein from ~-gal by collagenase digestion. out of the different expression products only the whole l1 protein and its carboxy terminal part bound dna. to specify the dna binding site, the coding sequence for the last 30 amino acids of l1 was fused to ~-gal. by this measure dna binding activity could be transferred to ~-gal, which did not formerly exhibit such property. collagenase digestion and use of l1 specific polyclonal antibodies ensured that dna binding was a genuine attribute hpv-16 l1 gene products. the latter enzyme differentiates between a 2,3-and a 2,6-linkages, cleaving preferentially sialic acid attached to galactose via an a 2,3-linkage. na-treated cells were resialylated using specific sialyl-transferases and cmp-sialic acid. only a transferase attaching sialic acid in an a 2,3-linkage to gal was able to restore receptors for bfdv. accordingly, cultured chicken embryo cells were resistant to bfdv infection following destruction of this receptor by na treatment. detection of cytomegalovirus (antigen) in body fluids has become increasingly important.-for that purpose, in our study blood and urine specimens from 60 immunosuppressed patients (52 renal transplant recipients, 2 heart and 2 liver transplant recipients, 4 patients with aids) were tested for hcmv. indicator cell cultures (foreskin fibroblasts) were inoculated with urine and leucocytes, respectively, and 2 resp. 7, 11, and 18 days after inoculation, subjected to an immunoperoxidase staining (ips) using a monoclonal antibody (dupont, f.r.g.) directed against hcmv early antigen. as controls, cell cultures inoculated with leucocytes were examined for hcmv-specific cytopathic effects (cpe) for 60 days. additionally, leucocytes from 30 patients were subjected to in-situ-hybridization with biotin-labeled hcmv ad 169-dna (eco ri j-fragment) probes. serum samples were tested for the presence of hcmv-specific antibodies of ig classes g, m., and a by elisa (behring, f.r.g.). -hcmv was detected in 6 blood and 8 urine samples from 11 patients. 2 blood samples were positive by both ips and cpe, 3 by ips only, and 1 by cpe only. hybridization assays were all negative. in virologically positive cases, titres of hcmvspecific antibodies amounted to (reciprocal titres): igg ;::: 640 (10 cases, with a significant rise of titre in 1 case), igm ;::: 40 (4 cases), and iga ;::: 320 (3 cases). -our results indicate that, for laboratory diagnosis of active hcmv infections, the detection of hcmv early antigen in urine is, with regard to practicability and rapidity, superior to the test for viremia and is to be considered a helpful extension of serological testing. moreover, with urine samples being easily obtainable, the detection of hcmv early antigen in urine is especially appropriate, too, for controlling the course of active hcmv infections. kai-olaf netzer, axel rethwilm, and volker ter meulen inst. f. virologie u. immunbiologie, univ., d-8700 wiirzburg the human spumaretrovirus (hsrv) is a distinct member of the foamy virus subfamily of retroviridae. its genome of about 11 kilo bases has been sequenced. it comprises the typical retroviral genes gag, pol, and env, and at least three more open reading frames possibly coding for regulatory proteins. thus far, not much is known about the gene products of hsrv. by means of radioimmunoprecipitation, we have identified and partly characterized the major immunogenic antigens of hsrv. radiolabeled viral proteins precipitated by hsrv-positive sera (but not by hsrv-negative control sera) were in the range of 32 to 170 kilodalton (kda) apparent molecular weight. labelling with 14c-glucosamine, or with 35s_ methionine in the presence of tunicamycin led to the identification of three viral glycoproteins of 170, 130 and 48 kda apparent molecular weight, respectively. these glycoproteins most likely represent env gene products. a phosphorylated protein of 60 kda may be related to the gag gene of hsrv. the results of this study show that radio-immunoprecipiation provides a powerful diagnostic and research tool to identify hsrv-positive sera. thomas nowak! and gerd wengler inst. f. virologie, univ., d-6300 gief~en, !present address: behringwerke ag, d-3550 marburg the primary structure of the nonstructural proteins ns1, ns2a, ns2b, ns3, ns4b and ns5 of the wnv has been determined. the nonstructural proteins were isolated from nuclear membrane fraction of wnv infected bhk cells. aminoterminal sequence data of these purified proteins were determined. together with the published amino acid sequence of the nonstructural coding genom region (castle et ai., 1986, virology 149, 10-26) we obtained the sequences of the nonstructural proteins ns1 (50 kd), ns2a (19 kd), ns2b (14 kd), ns3 (70 kd), ns4b (27 kd) and ns5 (97 kd). the gene order, the sizes of the virus coded proteins and the processing of the nonstructural proteins appears to be identical between the flaviviruses. it is well accepted that the functional impairment and killing of infected cells, hypersensitivity and autoimmunity contribute to the symptoms and pathology of viral diseases. we will discuss evidence for an additional mechanism can be defined as the uncontrolled activation of host effector functions not focused on viral antigens. "autotoxicity" is evident in the following situations: (i), certain paramyxoviruses and influenza viruses are capable of activating the generation of reactive oxygen species in phagocytes in the absence of antiviral antibody. this reaction is triggered by the binding of viral surface glycoproteins to the plasma membrane of the phagocytes. when injected into the blood stream, these viruses exert toxic effects independent of viral replication. -(ii), liver damage is observed in a model of murine influenza despite the apparent lack of viral replication in this organ. this effect may be mediated by cytokines. -(iii), in canine distemper, demyelination occurs despite the apparent lack of viral replication in oligodendrocytes, the cells which form myelin. degeneration of uninfected oligodendrocytes is also observed in dissociated brain cell cultures. as measured by luminol-dependent chemiluminescence, antiviral antibody stimulates the generation of reactive oxygen in microglial cells by linking fe receptors with viral antigen expressed on the surface of infected cells, e. g. astrocytes. in these cell cultures, oligodendrocytes can readily be destroyed by reactive oxygen species generated by xanthine/ xanthine oxidase. about 112,000 sera (january, 1984 to august, 1989) were analyzed retrospectively for hepatitis a or hepatitis b markers; 83,000 of these were evaluable. age distribution and seasonal distribution of acute hepatitis a virus infections in groups of patients with german or foreign names were determined. most cases of acute hepatitis a in the foreign population occurred between september and december, and most patients were below 15 years of age. in the german population no seasonal peaks could be found. however, there was one peak between 5 and 9, and another between 20 and 49 years of age. -we did not see seasonal peaks of acute infections with hepatitis b virus. no age was preferentially affected, but infections occurred earlier in foreign population than in german people. -in both groups cases of acute infections with both hepatitis a and hepatitis b viruses were only rarely found. more foreign people than german had markers for hepatitis a or hepatitis b (81 % vs.59%). the glycoprotein complex gil belongs to the essential membrane proteins of pseudorabies virus (prv). therefore mutational analyses of viral gil require growth of the respective mutant in a complementing, gil-expressing cell line. a cell line capable of supplying gil in trans was isolated after co-transfection of a genomic pry-dna fragment encompassing the complete viral gil-expression unit and the plasmid psv2neo conferring resistance against the antibiotic g418. the viral expression unit is usually silent but can be transactivated after superinfection by herpes simplex virus (hsv-l). transient expression of the pry "immediate early" protein is not sufficient to induce transactivation. by immunofluorescence and radioimmunoprecipitation using gil-specific monoclonal antibodies expression of authentic gil could be demonstrated. attempts to isolate a constitutively gil-expressing cell line failed, presumably due to the toxicity of the expressed glycoprotein. results regarding processing of gil without the context of a pry-infection will be presented. furthermore, availability of this cell line enables us to specifically mutate the gil-gene in the pry genome and characterize the resulting mutants biologically. cellular and humoral immune reactions are important for the pathogenesis of viral infections. in our animal model of mv encephalitis, lewis rats develop a subacute cns disease process (same) in the presence of only low levels of neutralizing antiviral antibodies. the contribution of the mv-specific cellular immunity to this disease is yet unknown. therefore, the in vitro reactivity of polyclonallymphocyte cultures to mv structural components was determined. additionally, the possibility to replace purified viral antigens by bacterially expressed fusion proteins was studied. t cells were primed by immunization with antigens in emulsified in freund's complete adjuvant. t cells prepared from regionallymphnodes were taken into culture 9-12 days later and lymphoproliferation was measured by the incorporation of tritiated thymidine in the presence of specific and irrelevant antigens with a panel of antigen-specific t cell lines. with all polyclonal t cell populations except those primed with recombinant haemagglutinin (pbd-h) a specific proliferation was obtained when either whole inactivated mv or the immunizing antigen was used for restimulation. while the nucleocapsid (n) or matrix (m)-specific cell lines recognize equally well pbd-n and virion purified n (nv), or pbd-m and mv respectively, such substitution for pbd-h and hv was not found. our observations indicate the induction of a differential t cell dependent immune response against the procaryotically expressed h compared to the h glycoprotein purified from virion. k. rittert, b. nellent, h. eiffertl, h. kratzin 2 , and r. thomssen 1 in the course of acute epstein-barr virus (ebv) infection igm antibodies always occur against two cellular antigens that were characterized as proteins with a molecular weight of 26 kd (p26) and 29 kd (p29), respectively. purified p29 was identified as a monomer of human triosephosphate isomerase (htpi). p26 is a so far unknown protein that possesses a high homology with triosephosphate isomerase of rabbits (rtpi). -the two autoantibodies are produced only as igm class antibodies, there is no switch to igg. presumably these antibodies are monoclonal in 40 percent of the patients. -in 25 percent of the cases with acute hepatitis a virus (hav) infection anti-htpiirtpi antibodies were found, too. acute ebv infection as well as acute ha v infection may be complicated by hemolysis of different extent. igm anti-htpiirtpi antibodies purified by affinity chromatography caused an increased 5tcr release from human erythrocytes (rh negatice, group 0). the contribution of these autoantibodies to hemolysis in acute viral infections is likely and will be further investigated. in first attempts we tried to express hbv pre-s(2) epitopes as aminoterminal fusions with hbc, all attempts resulted in no detectable expression due to instability or toxicity. we then inserted oligonucleotides coding for two overlapping pre-s(2) epitopes (dprvrglyfpa) o. immunol. 137: 2703 immunol. 137: , 1986 ) at the site where in duck hepatitis virus core antigens an insertion of 39 amino acid is found compared to the mammalian hepatitis viruses and predicted to be at the surface of particles. the insertion results in the expression of stable fusion proteins with pre-s2 antigenicity in western-blots. the chromatographic behaviour of hbdpre-s(2) chimaera on sepharose 4b is similar to that of authentic recombinant core particles, suggesting that they assemble to particles. a rabbit anti-pres(2) 120-145 antiserum (kindly provided by r. neurath) recognizes the hbdpre-s(2) particles in a non-denaturing elisa type assay, a monoclonal antibody to the second epitope does not, which shows that a part of the inserted sequence is accessible on the particles. since we can now express hbc particles carrying pre-s2 epitopes in e.eoli the immunologic properties (induction of t-cell and b-cell responses) of purified particles can be investigated. w. the characterization of an enzyme-linked immunosorbent assay, ®enzygnost-hsv (ag), for the identification and typing of hsv is discussed. -differentiation of hsv was evaluated against 26 laboratory hsv strains. it was shown that the typing indices were valid over a broad dilution range, which is essential for samples of both low and high antigen content. -in a preliminary clinical study (n = 93 specimens) direct testing revealed an identification sensitivity of 89.5%, a typing sensitivity of 90.9% and overall specificity of 93.9%. in a confirmatory test, a 100% agreement for both identification and typing was obtained. -on the basis of these results, it was concluded that ®enzygnost-hsv (ag) is a suitable alternative to the cell culture method, since it overcomes the failure of virus isolation due to possible inactivation resulting from improper transport and/or storage of the specimens. -in comparison with conventional hsv detection and typing test systems, ®enzygnost-hsv (ag) offers the following advantages: -ease of performance (within 4h), -less expensive, -subjective reading of test results using elisa photometers, -computer-controlled automation using microtitre plates is possible. sera of 48 persons in the age between 20-to 30-years were tested parallel with the biotin! avidin western blot (b/awb), with the biotin!avidin enzyme-linked immunosorbent assay (b/aelisa) and with the complement fixation technique (cft). antigens used in all tests of the study were preparations of the human adenovirus type 2. specific igg-antibodies directed against two immunoblotted group-specific major adenoviral polypeptides, the hexon epitope a-antigen and the penton base ~-antigen, were found in more cases (85%) than antibodies found with the b/aelisa (81 %) and with the cft (48%). the prevalences of igg-and iga-antibodies to human adenovirus were studied by b/aelisa in 579 serum samples obtained from 12 different age-groups of frankfurt/main, west germany. the lowest igg-antibody prevalence (52%) was measured in the six-to 12-month age-group, increasing to 95% in the six-to seven-years age-group. a lot of adenovirus positive sera with specific iga-antibodies were measured in the one-to two-(30% and two-to three-(16%) years age-groups. a second peak of iga-antibody prevalence (12%) appeared in the six-to seven-years and seven-to eight-years age-groups, where the highest prevalence of anti-adenovirus igg was seen. reverse transcriptase is necessary for viral replication, therefore it is an attractive candidate for antiviral therapy. detailed functional and structural analysis like e. g. cristallographic studies or neutron solution scattering are predisposition for the development of new inhibitors affecting reverse transcriptase activity. -for this purpose we have constructed a plasmid which allows high level expression of the active reverse transcriptase after introduction into e. coli. by partially adopting the e. coli codon usage and adding the original amino-and carboxy terminal sequence by synthetic oligonucleotides we were able to produce the authentic enzyme in e. coli in considerable amounts (up to 10% of the total e. coli protein) and in a very stable form, as shown by coomassie staining and by western blot analysis. mutants with additional amino acids fused at the aminoterminal sequence show a lower expression and an increased accessibility to the bacterial proteases. -the activity of the authentic and of the amino terminally altered enzyme was compared by standard methods and by an activity gel procedure. enzyme activity could be detected only at 66kd and 130 kd, wereas no activity could be detected at 51 kd. the recombinant produced enzyme can be used for purification and crystallographic studies. the genome of hog cholera virus (hcv) consists of an rna of 12 kb in length. the rna contains one large open reading frame(l) which is probably translated into a polyprotein and then processed proteolytically. -metabolic labeling and radioimmunoprecipitation with a polyspecific antiserum led to the identification of four different glycoproteins -gp55, gp48, gp44 and gp33 -in hcv infected cells (2) . inhibition of n-linked glycosylation through treatment of infected cells with tunicamycin resulted in distinct changes in migration behavior of these proteins in sds-page. -various cdna fragments located in the region coding for hcv encoded glycoproteins were expressed as fusion proteins in bacteria. the purified fusion proteins were used to prepare antibodies specific for the respective hcv glycoproteins. these serological reagents were used in radioimmunoprecipitation assays and western blots. the following conclusions can be drawn: 1.) gp48 and gp44 represent differently processed forms of a single protein. in patients with epidermodysplasia verruciformis (ev) hpv8 induces lesions with a higher risk of malignant conversion. there is evidence that the virus occurs also in the normal population. as hpv8 can not be propagated in tissue culture capsid antigens are not readily available for seroepidemiologic studies. we therefore expressed the major structural protein l1 of hpv8 and fragments thereof as ~-gal fusion proteins in bacteria. the expression vector pros encodes a fxa-cleavage site, which allows the separation of viral and bacterial moiety by proteolytic digest. purified viral antigens were used to test 360 sera by western blot analysis. 12% of the sera revealed antibodies against hpv8 l1 at a dilution of 1 : 20. in some cases the titers exceeded 1: 150. the antibody prevalence was similar in all age groups. western blots with l1 fragments showed that the humoral immunoresponse of different persons is not always directed against the same epitopes. the molecular basis for the lack of hbeag in viremic sera of some acute and chronic infected patients is not clear. in this study it was investigated whether this group of patients is infected with hbv variants which cannot synthesize hbeag. -viral dna isolated from sera of 5 hbeag negative chronic carriers was amplified (per) and sequenced directly or after cloning. in all 5 patient's sera hbv variants were found which had in common a stop codon in the precore region. -since the precore protein is the precursor for synthesis of the classical hbeag, one can conclude that the precore mutation of the hbv variants are responsible for the lack of hbeag in the serum of these patients. these results also implicate that the expression of hbeag is not essential for the viability of hbv. at least under immunosuppressive conditions the precore mutation seems not to drastically effect viral replication since a high hbv-dna titer was observed in the serum of one patient after liver transplantation. -whether the lack of hbeag expression is also responsible for the frequent severe and progredient course of infection and the lacking response in interferon therapy is currently investigated with a test specific for precore variant's infection and with animal models. inst. of virology, univ., d-6300 giessen a highly purified nucleoprotein (np) preparation from influenza virus infected cells yielded in addition to the commonly known 56 kd protein a 42 kd component which could not be detected in virus particles. among a series of np-specific monoclonal antibodies some reacted with both proteins and others were only bound by the 56 kd protein. among both types of np-specific monoclonal antibodies only a limited number were bound at the surface of murine cells infected with any type a virus. another category of antibodies bound to cells infected with a given subtype, but failed to react with the surface of cells infected with a different subtype. the results indicate that only restricted antigenic domains of the native np and perhaps np fragments are exposed at the surface of infected murine cells. additionally, the protective capacity of cell-associated np was determined by immunization of mice with the purified np preparation. in parallel, and in order to determine the immunogenic potency of newly synthesized np, mice were immunized with a vaccinia virus recombinant containing the gene for np prior to challenge with infectious virus. although immunized mice produced monospecific antibodies and a cytotoxic t cell response to the employed forms of np, they were not protected from influenza virus infection. the s segment rna of nephropathia epidemica virus (nev) strain hiillniis b1 was isolated by molecular cloning of the corresponding cdna. therna is 1785 nucleotides long with the 3' and 5' termini being complementary for 23 bases. the viral messengersense rna contains one major open reading frame (orf) with a coding capacity of 433 amino acids encoding a 49 kda polypeptide. compared to the hantaan s segment cdna sequencing there is a nucleotide homology of 60% and 61 % homology at the amino acid level. many of the amino acid differences are conservative exchanges. the c-termini of the nev and hantaan nucleocapsid proteins are nearly identical. the hydrophilicity profiles are very similar and most of the potential kinase dependent phosphorylation sites have been conserved. in contrast, the following differences are significant: the calculated isoelectric points of the nev and hantaan nucleocapsid proteins are 5.6 and 6.7, respectively. the most prominent antigenic determinants predicted by the hydrophilicity profiles are located close to the c-terminus of nev and close to the n-terminus of hantaan virus nucleocapsid polypeptides. -bmft project 0318973a. foot-and-mouth diesease virus-infected cells suffer from cytopathic effects. one causative agent is the virus protease 3c, as shown by transient expression of respective cdna in baby hamster kidney cells. in contrast to other edna-encoded virus proteins 3c is not detectable by indirect immunofluorescence. it is, however, detectable as de novo synthesized protein 16 hours after transfection by radioimmunoprecipitation. the enzyme is then indistinguishable in size from that found in virus-infected cells, indicating similar autocatalytical release from fused protein. transcription of protease 3c-encoding edna fragments is inhibited, as well as that of co-transfected fragments which do not encode protease 3c, as analysed by northern blot hybridizations. the shut off of transcription which is one of the cytopathic effects observed in infected cells correlates thus to the production of active protease 3c. the inhibitory molecular mechanisms may involve truncation of the nuclear protein histone h3 at its n-terminus as found by western blotting. this protein is found similarly truncated in virus-infected cells. virusprotein-specific antigenicity and immunogenicity of tissue culture rabies vaccines o. thraenhart, 1. marcus, and k. ramakrishnan inst. f. med. virologie u. immunologie, univ., d-4300 essen pre-and postexposure treatment with inactivated rabies vaccines prevent rabies virus infection without complications in contrast to vaccines of nervous tissue origin. a considerable diversity concerning virus strains (pm, flury lep, era), cell strains (wi-38, mrc-5, chick fibroblast, bhk) and concentration procedures (ultrafiltration, ultracentrifugation) exists between vaccines of different producers. the influence of the various criteria on the antigenicity and immunogenicity were tested in in vitro experiments using mono-and polyclonal antibodies against rv proteins with antigen-elisa, western blot and immuneelectronmicroscopy in unfractionated and fractionated vaccines after rate zonal ultracentrifugation. the immunogenicity was tested in vivo by protection induction (pi) in mice, the results were correlated with the virusspecific antigenicity (vps-ag) in tissue culture supernatants. vps-, immunglobulin-specific and functional antibody-(ab) and interferon-(ifn) induction was tested in human vaccinees. -rv glycoprotein (gp) and nucleocapsid (np) concentration in vaccines were correlated with pi in mice. the gp : np ratio was > 1 in tissue culture vaccines in contrast to brain vaccines. the harvest of gp and np in tc depends mainly on the cell strain. the virion-associated : soluble gp ratio is productionspecific. the concentration virion tc vaccines contain gp, np, m, l, which induce in vaccinees an early, high and long lasting vps-, igg specific ab-response; anti-gp and -np is induced as early as 3-7 days. ifn-induction is correlated with the applied dosis. no nonresponder was observed and all vaccinees had protecting levels of neutralizing ab from 7-14 days onwards and still after 1 year. the frequency and specificity of antibodies to p-gene encoded proteins of human hepatitis b virus was tested in sera of acute and chronically infected patients with and without hepatocellular carcinoma (hcc). for antibody detection an immunoprecipitation gel assay was performed with radioactively labeled polypeptides produced by in vitro translation of rna of different p-gene regions. thus, five antigenic regions were identified. all anti-p antibody positive sera reacted with carboxy terminal p-polypeptides, a subset with polypeptides of the aminoterminal and middle region, and none reacted with p-protein derived from the most sequence variable region. anti-p antibodies were detected at very high frequency in sera of acute (73 %) and chronically infected patients without hcc (87%), but less often in hcc patients (27%). anti-p antibodies appear early in infection and decline prior to hbsag/anti-hbs seroconversion. -these data indirectly demonstrate the expression of most hbv p-gene sequences and the high immunogenicity of p-proteins in vivo. moreover, they establish anti-p antibodies as a frequent serological marker of infection and identify the carboxy terminal region of the p-proteins (s) as immunodominant. the human polyoma virus jc produces brain tumors in hamsters. in these tumors jcv tantigen, the major product of the early genes is generally expressed. the protein plays a central role in the regulation of polyomavirus replication. since tissue culture as a major source for t-antigen is limited to human primary fetal glial cells we characterized jcv tantigen in the hamster cell line hjc derived from a jcv induced medulloblastoma. -southern blot analysis revealed the presence of jcv dna in hjc cells. the physical state was predominantly integrated. the transcription of early genes was demonstrated by presence of jcv specific mrna in northern blots. the nuclear localisation of t-antigen could be established by immunohistochemical staining and by immunoprecipitation with a crossreacting monoclonal sv40 antibody (pab 416) a molecular weight of 85 to 90 kd was determined. furthermore from western blot analysis it could be assumed that high molecular forms of jcv t-antigen are present in the hamster cell line. taken together these data demonstrate that the molecular weight and nuclear localization were as expected from sv40 transformed cell lines. therefore jcv t-antigen in hjc cells shall be used to characterize its dna binding capacity to the origin region of jc virus dna. current laboratory diagnosis of a coxsackie-b-virus (cbv) infection is mainly based on virus isolation, supported by the detection of rising or high virus-specific neutralizing antibody titers. since such high titres have also been found in apparently healthy peopleprobably originated from a subclinical infection and persisting for a year or more -cbv-igm detection may be a more reliable criteria for serological diagnosis. attempts to identify these antibodies in routine context using conventional techniques (nt, immunodiffusion) or solid phase immunoassays (eia, ria, reverse elisa, immunoblot) have failed, despite high sensitivity and specificity, due to high costs or technical disadvantages. in the present study we developed a modified western blot technique (modi-western blot), which enables a rapid and reliable identification of cbv-igm antibodies. in this assay the antigen was electrophoretically separated using an integrated electrophoresis system (phastsystemunit) and transferred to a solid matrix by diffusion blotting. subsequent immunodetection was performed using a biotin-avidin amplification and monoclonal antibodies. to appraise the efficiency of the method 51 human serum samples were analysed for virus-specific antibodies (igm, iga and igg subclasses) to coxsackie-b-viruses. -group specific igm responses could be found in 22 of 31 (70.9%) of igm seropositive cases. type-predominant igm antibodies (9/31) were detected in 3 cases against cbv1, in 5 cases against cbv2 and in 1 case against cbv4. moreover igg/lga assay in 8 patients seropositive for igg and igm revealed in all cases igg3 and iga antibodies, which supports the evidence of an acute cbvinfection. improvement of diagnosis of cytomegalovirus (hcmv) infections in immunosuppressed patients by detection of hcmv using a monoclonal antibody directed against hcmv-early d-6000 frankfurt primary or recurrent hcmv infections can cause severe clinical problems in immunocompromised patients. as far as active hcmv infections in these patients are concerned, the diagnostic significance of serological data can be restricted tiel: virology, in press 2 an unsatisfactory reproducibility and comparibility was observed when six available enzyme immune test kits for anti-hbc were evaluated by four german red cross bloodtransfusion centers. only 62 of 1838 blood donors were consistently positive, but 228 samples produced discrepant results, although five of the assays used the same inhibition procedure of labeled anti-hbc (caspari et ai., j. clin. microbiol., in press). we reexamined the clearly and discrepantly positive samples of this study (besides some negative controls) by an assay which measures the binding of igg to hbc antigen by peroxidase labeled anti-igg. practically all consistently positive samples were confirmed by the different test principle while practically all discrepant samples were negative. this shows that consistent results by different inhibition assays are indeed reliable while divergent results can be neglected. anti-hbc is used in some countries as surrogate test for hcv carriers. using an experimental test kit from ortho diagnostics, 2 of 64 confirmed anti-hbc positiive and 2 of 389 confirmed anti-hbc negative donors reacted repeatedly as anti-hcv positive. these data do not provide evidence that anti-hbc is useful for elimination of hcv carriers. in this study we compared directly the detection level, sensitivity, and specificity of the most sensitive radioactive and the most sensitive non-radioactive method for detecting hepatitis b virus (hbv) dna in patient serum by dot blot hybridization, based on our previous experience with 6 different assay systems. the former employed the 32p-labeled hbv rna probe included in the hepprobe kit (gibco-brl), detected autoradiographically. an advantage of this kit is the low level of radioactivity of the pre-labeled probe « 10 f,tc), thereby permitting its use even in those laboratories lacking a license for radioisotopes. the non-radioactive method involved the use of an hbv dna probe sulfonated with sodium bisulfite (chemiprobe kit, orgenics), followed by immundetection and an enzymatic color reaction. the detection level of the 32p probe was found to be 0.3 pg hbv dna, corresponding to 3 x 10 4 genomes in 50 f,tl serum, compared with only 2 pg with the sulfonated probe. subsequently, sera from 159 patients with various constellations of hbsag, and anti-hbe were tested with both methods. the concordance rate was 71 % (r = 0.42). compared with 32p results, sulfonation showed a sensitivity of 80% and a specificity of 67%. radiolabeling, therefore, still allows the most sensitive and reliable detection of hbv dna in serum. sulfonation could eventually provide a feasible alternative to radioactivity if the viral dna in serum could be sufficiently amplified in vitro, for example with the polymerase chain reaction. such studies are being planned in our institute. key: cord-022472-q2qtl26d authors: fishman, jay a.; ramos, emilio title: infection in renal transplant recipients date: 2009-05-15 journal: chronic kidney disease, dialysis, & transplantation doi: 10.1016/b978-1-4160-0158-4.50041-0 sha: doc_id: 22472 cord_uid: q2qtl26d nan successful management of infections in the immunocompromised renal transplant recipient is complicated by a variety of factors. 1 these include increased susceptibility to a broad spectrum of infectious pathogens and the difficulty in making a diagnosis of infection in the face of diminished signs and symptoms of infection, an array of noninfectious etiologies of fever (e.g., graft rejection, drug toxicity), and the possibility that multiple processes are present simultaneously. further, because immunocompromised patients tolerate invasive and established infection poorly with high morbidity and mortality, the urgency for an early and specific diagnosis to guide antimicrobial therapy is increased. given the primacy of t-lymphocyte dysfunction in transplantation, viral infections in particular are increased and contribute to graft dysfunction, systemic illness, graft rejection, and enhancing the risk for other opportunistic infections (e.g., pneumocystis and aspergillus species) and for virally-mediated cancers. the risk of infection in the renal transplant recipient is determined by the interaction of two factors: 1. the epidemiologic exposures of the patient, including those unrecognized by the patient or distant in time ( the prevention and treatment of infection is central to the optimal management of transplant recipients, given the adverse impact of infections on quality of life. consideration of the epidemiology of infection allows the clinician to establish a differential diagnosis for a given "infectious" presentation and to design the optimal preventive strategy for each patient. donor and recipient screening are critical components to the post-transplant health maintenance of the patient (table 37-3) . of these, consideration should be given to empiric therapy for purified protein derivative (ppd) positive patients, for strongyloides stercoralis in patients from endemic regions, and for patients known to have received organs from donors with acute bacterial and fungal infections. specific antiviral strategies stratified according to individual risk should be considered for all kidney recipients. exposures of importance can be divided into four overlapping categories: donor-or recipient-derived infections, and community-or nosocomial-acquired exposures. infections that are derived from the donor tissues and activated in the recipient are among the most important exposures in transplantation. some of these are latent while others are the result of bad timing-active infection transmitted at the time of transplantation. all of the known types of infections have been recognized in transplant recipients. the activation of these infections may reflect the intensity of immune suppression or result from the allogeneic response (graft rejection), which activates latent viral pathogens. three types of infection merit special attention. first, in donors who are bacteremic or fungemic at the time of donation, these infections-staphylococci, pneumococcus, candida species, salmonella, e. coli-tend to "stick" to anastamotic sites (vascular, urinary) and may produce leaks or mycotic aneurysms. second, viral infections, including cytomegalovirus (cmv) and epstein-barr virus (ebv), are associated with particular syndromes and morbidity in the immunocompromised population (discussed later in text). the greatest risk of such infections is in recipients who are seronegative (immunologically naïve) and receive infected grafts from seropositive donors (latent viral infection). third, late, latent infections, including tuberculosis, may activate many years after the initial exposure. disseminated mycobacterial infection is often difficult to treat once established due largely to interactions between the antimicrobial agents used to treat infection (e.g., rifampin, streptomycin, isoniazid) and the agents used in immune suppressive therapy. given the risk of transmission of infection from the organ donor to the recipient, certain infections should be considered relative contraindications to organ donation. given that renal transplantation is, in general, elective surgery, it is reasonable to avoid donation from individuals with unexplained fever, rash, or infectious syndromes. some of the common criteria for exclusion of organ donors are listed in table 37 -4. infections in this category are generally latent infections activated in the setting of immune suppression. it is necessary to obtain a careful history of travel and exposures to guide preventive strategies and empiric therapies. notable among these infections are tuberculosis, strongyloidiasis, viral infections (herpes simplex and varicella zoster or shingles), histoplasmosis, coccidioidomycosis, hepatitis b or c, and human immunodeficiency virus (hiv). vaccination status should be evaluated (tetanus, hepatitis b, childhood vaccines, influenza, pneumococcal vaccine). dietary habits should also be considered, including the use of well water (cryptosporidia), uncooked meats (salmonella, listeria), and unpasteurized dairy products (listeria). common exposures in the community are often related to contaminated food and water ingestion, exposure to infected children or coworkers, or exposures due to hobbies (gardening), travel, or work. respiratory virus infection due to influenza, respiratory syncytial virus, and adenoviruses and more atypical pathogens (herpes simplex virus, herpes zoster virus) carries the risk for viral pneumonia but increased risk for bacterial superinfection. community (social or transfusion-associated) exposure to cmv and ebv may produce severe primary infection in the nonimmune host. recent and remote exposures to endemic, geographically restricted systemic mycoses (blastomyces dermatitidis, coccidioides immitis, and histoplasma capsulatum) and mycobacterium tuberculosis can result in localized pulmonary, systemic, or metastatic infection. asymptomatic strongyloides stercoralis infection may activate more than 30 years after initial exposure due to the effects of immunosuppressive therapy. such reactivation can result in either a diarrheal illness and parasite migration with hyperinfestation syndrome (characterized by hemorrhagic enterocolitis, hemorrhagic pneumonia, or both) or disseminated infection with accompanying (usually) gramnegative bacteremia or meningitis. gastroenteritis due to salmonella species, campylobacter jejuni, and a variety of enteric viruses can result in persistent infection, more severe and prolonged diarrheal disease as well as an increased risk of bloodstream invasion and metastatic infection. nosocomial infections are of increasing importance because organisms with significant antimicrobial resistance predominate in many centers. these include vancomycin, linezolid and quinupristin/dalfopristin-resistant enterococci, methicillinresistant staphylococci, and fluconazole-resistant candida species. a single case of nosocomial aspergillus infection in a compromised host should be seen as an indication of the failure of infection control practices. antimicrobial abuse has resulted in increased rates of c. difficile colitis. outbreaks of infections due to legionella species have been associated with hospital plumbing and contaminated water supplies or ventilation systems. each nosocomial infection should be investigated to ascertain the source and prevent subsequent infections. nosocomial spread of p. jiroveci between immunocompromised patients has also been suggested by a variety of case series. respiratory viral infections may be acquired from medical staff and should be considered among the causes of fever and respiratory decompensation among hospitalized or institutionalized, immunocompromised individuals. the net state of immunosuppression is a measure of all of the factors contributing to the patient's risk for infection (table 37 -2). among these are: 1. the specific immunosuppressive therapy, including dose, duration, and sequence of agents. 2. technical problems from the transplant procedure, resulting in leaks (blood, lymph, urine) and fluid collections, devitalized tissue, poor wound healing, and surgical drainage catheters for prolonged periods. 3. prolonged airway intubation 4. prolonged use of broad-spectrum antibiotics 5. renal and/or hepatic dysfunction 6. prolonged use of vascular access or dialysis catheters presence of infection with one of the immunomodulating viruses, including cmv, ebv, hepatitis b (hbv) or c (hcv), or hiv. specific immunosuppressive agents are associated with increased risk for certain infections (table 37-5) . combinations of these agents may enhance this risk or cause toxicity (e.g., nephrotoxicity) and may further enhance risk. as immunosuppressive regimens have become more standardized, the specific infections that occur most often will vary in a predictable pattern depending on the time elapsed infection i in r renal t transplant r recipients 683 (figure 37-1 ). this is a reflection of the changing risk factors (surgery/hospitalization, immune suppression, acute and chronic rejection, emergence of latent infections, and exposures to novel community infections. 1 the pattern of infections will be changed with alterations in the immunosuppressive regimen (pulse dose steroids or intensification for graft rejection), intercurrent viral infection, neutropenia (drug toxicity), graft dysfunction, or significant epidemiologic exposures (travel or food). the time line reflects three overlapping periods of risk for infection: (1) the perioperative period to approximately 4 weeks after transplantation; (2) the period 1 to 6 months after transplantation (depending on the rapidity of taper of immune suppression and the type and dosing of antilymphocyte "induction" that may persist); and (3) the period beyond the first year after transplantation. these periods reflect the changing major risk factors associated with infection: (1) surgery and technical complications; (2) intensive immune suppression with viral activation; and (3) community-acquired exposures with the return of normal activities. the time line may be used in a variety of ways: (1) to establish a differential diagnosis for the transplant patient suspected of having infection; (2) as a clue to the presence of an excessive environmental hazard for the individual, either within the hospital or in the community; and (3) as a guide to the design of preventive antimicrobial strategies. infections occurring outside the usual period or of unusual severity suggest either excessive epidemiologic hazard or excessive immunosuppression. the prevention of infection must be linked to the risk for infection at various times after transplantation. routine preventive strategies from the massachusetts general hospital are outlined in table 37 -6. it should be noted that such strategies serve only to delay the onset of infection in the face of epidemiologic pressure. the use of antibiotic prophylaxis, vaccines, and behavioral modifications (e.g., routine hand washing or advice against digging in gardens without masks) may only result in a "shift to the transplantation 684 (1) a combination of atovaquone 1500 mg po with meals once daily plus levofloxacin (or equivalent fluoroquinolone without anti-anaerobic spectrum) 250 mg once daily; (2) pentamidine (300 mg iv or inhaled q 3-4 weeks); and (3) dapsone (100 mg po qd to biweekly) +/− pyrimethamine. each of these agents has toxicities that must be considered, including hemolysis in g6pd-deficient hosts with dapsone. none of these alternative programs offer the same broad protection of tmp-smx. continued right" of the infection time line, unless the intensity of immune suppression is reduced or immunity develops. during the first month after transplantation, three types of infection occur. the first type of infection is that present in the recipient prior to transplantation, was inadequately treated, and now has emerged in the setting of surgery, anesthesia, and immunosuppression. pre-transplantation pneumonia and vascular access infections are common examples of this type of infection. colonization of the recipient with resistant organisms is also common (e.g., mrsa). the first rule of successful transplant infectious disease is the eradication of all infection possible prior to transplantation. the second type of early infection was present in the donor before transplantation. this is often a nosocomial-acquired organism (resistant gram-negative bacilli and s. aureus or candida species) due to (1) systemic infection in the donor (e.g., line infection) or (2) contamination during the organ procurement process. the end result is a high risk of infection of vascular suture lines with resultant mycotic aneurysm. uncommonly, infections have been transmitted from donor to recipient, including tuberculosis or fungal (e.g., histoplasmosis) infection that may emerge earlier in the time line than would be predicted (i.e., in the first month). the third type and the most common source of infections in this period are related to the complex surgical procedure of transplantation. these include surgical wound infections, pneumonia (aspiration), bacteremia due to vascular access or surgical drainage catheters, urinary tract infections, or infections of fluid collections-leaks of vascular or urinary transplantation 686 table 3 37-6 renal transplantation antimicrobial protocols at the massachusetts general hospital, boston, massachusetts-cont'd prophylaxis is achieved with 50% of the therapeutic dose of ganciclovir or valganciclovir (corrected for renal function). in some patients, intravenous immune globulin (ivig or hyperimmune globulin) is used as an adjunctive therapy for prophylaxis. certain subgroups merit routine prophylaxis. these include: • solid organ transplant recipients who are naïve (seronegative) and receive an organ from a seropositive donor (d+/r−) • solid organ transplant recipients who are seropositive (r+) and receive antilymphocyte antibodies or other intensive immune suppression (e.g., for graft rejection) symptoms, fever/neutropenia mo (or valacyclovir 500 bid or acyclovir 400 tid) use of cmv-negative or leukocyte-filtered blood status unknown with als intravenous ganciclovir 5mg/kg iv for first dose and qd (corrected for renal function) until sero-status determined. neutropenia: the dose of antiviral and antibacterial therapies are not, in general, reduced for neutropenia. consider other options first! + als: antilymphocyte antibodies include any of the lytic, lymphocyte-depleting antisera *note: not fda approved at these doses prevention of mucocutaneous infection can be accomplished with oral clotrimazole (may increase cya levels) or nystatin 2 to 3 times per day at times of steroid therapy or in the face of antibacterial therapy. fluconazole, at a dose of 200-400 mg/day for 10-14 days is utilized in the treatment of prophylaxis failures. routine prophylaxis with fluconazole is used for pancreas transplants. anastamoses or of lymphoceles. these are nosocomial infections and, as such, are due to the same bacteria and candida infections observed in nonimmunosuppressed patients undergoing comparable surgery. however, given the immune suppression, the signs of infection may be subtle and the severity or duration may be greater. the technical skill of the surgeons and meticulous postoperative care (i.e., wound care, endotracheal tubes, vascular access devices, and drainage catheters) are the determinants of risk for these infections. also among the common infections is c. difficile colitis. limited perioperative antibiotic prophylaxis (i.e., from a single dose to 24 hours of an antibiotic such as cefazolin) is usually adequate with additional coverage only for known risk factors (e.g., prior colonization with mrsa). for pancreas transplantation, perioperative prophylaxis against yeasts with fluconazole is used in addition, bearing in mind the interactions between azole antifungal agents and calcineurin inhibitors and sirolimus (levels may be increased significantly). notable by their absence in the 1st month after transplantation are opportunistic infections, even though the daily doses of immunosuppressive drugs are at their highest during this time. the implications of this observation are important: the net state of immunosuppression is not great enough to support the occurrence of opportunistic infections unless an exposure has been excessive; this observation suggests that it is not the daily dose of immunosuppressive drugs that is of importance but rather the sustained administration of these drugs, the "area under the curve," in determining the net state of immunosuppression. thus, the occurrence of a single case of opportunistic infection in this period should trigger an epidemiologic investigation for an environmental hazard. infection in the transplant recipient 1 to 6 months after transplantation has one of three causes: 1. lingering infection from the peri-surgical period, including relapsed c. difficile colitis, inadequately treated pneumonia, or infection related to a technical problem (e.g., urine leak, lymphocele, hematoma). fluid collections require drainage. 2. viral infections, including cmv, hsv, shingles (vzv), human herpesvirus 6 or 7, ebv, relapsed hepatitis (hbv, hcv), and hiv. this group of viruses is unique: lifelong infection; tissue-associated (often transmitted with the allograft from seropositive donors); immunomodulating-systemically immune suppressive and, potentially, predisposing to graft rejection. it is also notable that the herpesviruses are prominent due to the attenuated ability of t cells to control these infections. among the other viral pathogens of this period must be included bk polyomavirus in association with allograft dysfunction and community-acquired respiratory viruses (adenovirus, influenza, parainfluenza, respiratory syncytial virus, metapneumovirus). the suppression of antibody production (e.g., using tacrolimus and mycophenylate mofetil or with lymphopenia) may predispose to other infections. 3. opportunistic infection due to p. jiroveci, listeria monocytogenes, t. gondii, nocardia species, aspergillus species, and other agents. in this period, the stage is also set for the emergence of a subgroup of patients, the "chronic ne'er-do-wells"-individuals who require higher than average immune suppression to maintain graft function or who have prolonged untreated viral infections and other opportunistic infections, predicting long-term susceptibility to many other infections (third phase, discussed later). such individuals may merit prolonged (lifelong) prophylaxis (antibacterial and/or antiviral) to prevent life-threatening infection. the specific opportunistic infections that occur, reflect the specific immunosuppressive regimen used and the presence or absence of immunomodulating viral infection. viral pathogens (and rejection) are responsible for the majority of febrile episodes that occur in this period. during this period, anti-cmv strategies and trimethoprim-sulfamethoxazole prophylaxis are effective in decreasing the risk of infection. trimethoprim-sulfamethoxazole prophylaxis eliminates p. jiroveci pneumonia (pcp) and reduces the incidence of urinary tract infection and urosepsis, l. monocytogenes meningitis, nocardia species infection, and toxoplasma gondii. transplant recipients who are more than 6 months past the procedure can be divided into three groups in terms of infection risk. the first group consists of the majority of transplant recipients (70%-80%) who had a technically good procedure with satisfactory allograft function, reduced and maintenance immunosuppression, and absence of chronic viral infection. these patients resemble the general community in terms of infection risk, with community-acquired respiratory viruses constituting their major risk. occasionally, such patients will develop primary cmv infection (socially acquired) or infections related to underlying diseases (e.g., skin infections in diabetes). the second group (~10% of patients) suffers chronic viral infection, which, in the absence of effective therapy, will lead inexorably to one of three results: • end organ damage (e.g., bk polyomavirus nephropathy, cryoglobulinemia, or cirrhosis from hcv-hbv being relatively well managed at present) • malignancy (post-transplantation lymphoproliferative disease [ptld] due to ebv, skin, or anogenital cancer due to papilloma viruses) • acquired immunodeficiency syndrome (hiv/aids) the third group of patients (~10% of all recipients) has less than satisfactory allograft function and requires excessive amounts of immunosuppressive therapy for recurrent graft rejection. this may be associated with chronic viral infection. this is the subgroup of transplant recipients, often termed the "chronic ne'er-do-wells," who are at highest risk for opportunistic infection with such pathogens as p. jiroveci, l. monocytogenes, n. asteroides, and cryptococcus neoformans. it is our practice to give these patients lifetime maintenance trimethoprim-sulfamethoxazole prophylaxis and to consider the use of fluconazole prophylaxis. also, this group is susceptible to organisms more often associated with immune dysfunction of aids (bartonella, rhodococcus, cryptosporidium, and microsporidium species) and invasive fungal pathogens (aspergillus, zygomycetes, and the dematiaceae, or pigmented, molds). minimal signs or symptoms merit careful evaluation in this group of "high-risk" individuals. guidelines for pre-transplant screening have been the subject of several recent publications including a consensus conference of the immunocompromised host society (ichs), the american society for transplantation (ast) clinical practice guidelines on the evaluation of renal transplant candidates, and the astp clinical practice guidelines on the evaluation of living renal transplant donors. [2] [3] [4] [5] [6] [7] [8] [9] the transplant donor the critical feature of screening for deceased donors is time limitation. a useful organ must be procured and implanted before some microbiologic assessments have been completed. thus, major infections must be excluded and appropriate cultures and stored samples obtained for future reference. as a result, bacteremia or fungemia may not be detected until after the transplant has occurred. such infections have not generally resulted in transmission of infection as long as the infection has been adequately treated, both in terms of use of antimicrobial agents to which the organism is susceptible and time. in recipients of tissues from 95 bacteremic donors, a mean of 3.8 days of effective therapy post-transplantation appeared adequate to prevent transmission; longer courses of therapy in the recipient are preferred, targeting known potential pathogens from the donor. 10 bacterial meningitis must also be treated with antibiotics that penetrate the csf before procurement. similarly, due to the limited time for testing, certain acute infections (cmv, ebv, hiv, hbv, or hcv) may be undetected in the period prior to antibody formation, and viral dna detection is preferred. as a result, the donor's clinical, social, and medical histories are essential to reducing the risk of such infections. however, in the presence of known infection, such infections must be treated prior to procurement, if possible. major exclusion criteria are outlined in table 37 -4. the differences in screening of the living donor and the cadaver donor are largely based on the different time frames during which this screening takes place. the living donor procedure should be considered elective-and, thus, evaluation completed and infections treated prior to such procedures. an interim history must be taken at the time of surgery to assess the presence of new infections since the initial donor evaluation. intercurrent infections (flu-like illness, headache, confusion, myalgia, cough) might be the harbinger of important infection (west nile virus, sars, rabies, trypanosoma cruzi). live donors undergo a battery of serologic tests (table 37-3) as well as ppd skin test and, if indicated, chest radiograph. the testing must be individualized based on unique risk factors (e.g., travel). of particular importance to the renal transplant recipient is the exclusion of urinary tract infection. whether focal infections in the donor outside the procured organs merit therapy remains unresolved. mycobacterium tuberculosis. this bacterium from the donor represented approximately 4% of reported post-transplant tb cases in a review of 511 patients by singh and colleagues. 11 active disease should be excluded in ppd positive donors, including chest radiograph, sputum cultures, and chest ct, if the chest radiograph is abnormal. urine afb cultures may be useful in the ppd-positive kidney donor. isoniazid prophylaxis of the recipient should be considered for untreated, ppdpositive donors. 12 factors mitigating towards prophylaxis include donor from endemic region, use of high-dose steroid regimen, or high-risk social environment. chagas' disease (t. cruzi). this parasitic disease has been transmitted by transplantation in endemic areas and recently in the united states. schistosomiasis and infection by strongyloides stercoralis are generally recipient-derived problems. epstein-barr virus. the risk for post-transplant lymphoproliferative disease (ptld) is greatest in the ebv seronegative recipient of an ebv seropositive allograft (i.e., d+/r−). this is most common in pediatric transplant recipients and in adults coinfected with cmv or on higher levels of immune suppression. monitoring should be considered for at-risk individuals using a quantitative, molecular assay (e.g., pcr) for ebv. 13, 14 ebv is also a cofactor for other lymphoid malignancies. varicella screening should be used to identify seronegative individuals (no history of chicken pox or shingles) for vaccination prior to transplantation. hsv screening is performed by most centers despite the use of antiviral prophylaxis during the post-transplant period. vzv serologic status is particularly important in children who may be exposed at school (for antiviral or varicella immune globulin prophylaxis) and in adults with atypical presentations of infection (pneumonia or gi disease). other herpesviruses may reactivate with hhv-6 and hhv-7 serving as cofactors for cmv and fungal infections and in endemic regions, kaposi's sarcoma-associated herpesvirus (hhv-8/kshv) causing malignancies. hepatitis b virus (hbv). hbsag and hbv core antibody (hbcab) are used for screening purposes with hbsab positivity indicating either vaccination or prior infection. hbcab-igm positivity suggests active hbv infection, whereas igg positivity suggests a more remote or persistent infection. the hbsag negative, hbcab-igg positive donor may have viral dna in the liver but may be appropriate as a donor for hbvinfected renal recipients. quantitative assays for hbv should be obtained to guide further therapy. the presence of hbsag negative, hbcab-igg positive assays may be a false-positive or reflect true, latent hbv infection. hepatitis c virus (hcv) infection will generally progress more rapidly with immune suppression and with cmv coinfection. hcv seropositive renal transplant candidates are more likely to develop cirrhosis and complications of liver failure. there is no good therapy for hcv infection; management is by quantitative molecular viral assays. hiv-infected donors have not been utilized. the progression of disease is rapid and outweighs the benefits of transplantation. donors may be excluded based on historic evidence of "high-risk" behavior for hiv infection. western blot testing and molecular assays (pcr) should be obtained prior to the use of tissues from any hivseropositive donor. human t-lymphotropic virus i (htlv-i) is endemic in the caribbean and parts of asia (japan) and can progress to htlv-i-associated myelopathy/tropical spastic paraparesis (ham/tsp) or to adult t cell leukemia/lymphoma (atl). htlv-ii is similar to htlv-i serologically but is less clearly associated with disease. use of organs from such donors is generally avoided. 15, 16 west nile virus (wnv) is a flavivirus associated with viral syndromes and meningoencephalitis and may be transmitted by blood transfusion and organ transplantation. 17, 18 routine screening of donors is not advocated other than in areas with endemic infection of the blood supply. donors with unexplained changes in mental status or recent viral illness with neurologic signs should be avoided. sars (severe acute respiratory syndrome) is a recently described coronavirus, thought to be associated with exposure to civets or other animals common to the diet of certain regions of china. tissue persistence is prolonged and infection of transplant recipients appears to be severe and often symptomatic. organ procurement should exclude patients with recent acute illnesses meeting sars criteria. the pre-transplant period is useful for a thorough travel, animal, and environmental and exposure history; updating immunizations; and counseling of the recipient regarding travel, food, and other infection risks. ongoing infection must be eradicated prior to transplantation. two forms of infection pose a special risk: 1. bloodstream infection: this is related to vascular access, including that for dialysis and pneumonia, which puts the patient at high risk for subsequent lung infection with nosocomial organisms. infected ascites or peritoneal dialysis fluid must also be cleared prior to surgery. urinary tract infection (uti) must be eliminated prior to transplantation with antibiotics with or without nephrectomy. similarly, skin disease that threatens the integrity of this primary defense against infection should be corrected before transplantation, even if doing so requires the initiation of immunosuppression prior to transplantation (e.g., the initiation of immune suppression to treat psoriasis or eczema). finally, the history of more than one episode of diverticulitis should initiate an evaluation to determine whether sigmoid colectomy should be carried out prior to transplantation. 2. tuberculosis: both the incidence of active disease and the occurrence of disseminated infection due to m. tuberculosis are far higher in the transplant recipient than in the general population. active tuberculous disease must be eradicated prior to transplantation. the major antituberculous drugs are potentially hepatotoxic, and significant drug interactions are common between the anti-tb agents and the agents of immune suppression. in patients with active infection, from endemic regions or with high risk exposures, tb therapy should be initiated in all ppd positive individuals prior to transplantation. some judgment may be used as to the optimal timing of treatment in individuals without evidence of active or pleuropulmonary disease. greater risk may include: • previously active tuberculosis or significant signs of old tuberculosis on chest radiograph • recent tuberculin reaction conversion • known exposure to active disease • protein-calorie malnutrition, cirrhosis, or other immune deficiency • living in a shelter or other group housing aids for those benefiting from haart, aids has been converted from a progressively fatal disease to a chronic infection controlled by complex regimens of antiviral agents. haart has been associated with reduced viral loads, improved cd4 lymphocyte counts, and reduced susceptibility to opportunistic infections. in the pre-haart era, organ transplantation was generally associated with a rapid progression of aids. as a result, hiv-infected individuals have been excluded at most transplantation centers. however, prolonged disease-free survival with haart has lead to a reconsideration of this policy. renal transplantation in hiv has been associated with good outcomes in individuals with controlled hiv infection and in the absence of hcv co-infection. 19 management requires some sophistication regarding both the immune suppressive agents and the various haart regimens. the spectrum of infection in the immunocompromised host is quite broad. given the toxicity of antimicrobial agents and the need for rapid interruption of infection, early, specific diagnosis is essential in this population. advances in diagnostic modalities (ct or mri scanning, molecular microbiologic techniques) may greatly assist in this process. however, the need for invasive diagnostic tools cannot be overemphasized. given the diminished immune responses of the host and the frequency of multiple simultaneous processes, invasive diagnosis is often the only method for optimal care. the initial therapy will, by necessity, be broad with a rapid narrowing of the antimicrobial spectrum as data become available. the first choice of therapy is to reduce the intensity of immune suppression. the risk of such an approach is that of graft rejection. the selection of the specific reduction may depend upon the organisms isolated. similarly, reversal of some immune deficits (neutropenia, hypogammaglobulinemia) may be possible with adjunctive therapies (colony stimulating factors or igg). co-infection with virus (cmv) is common and merits additional therapy. cmv is the single most important pathogen in transplant recipients, having a variety of direct and indirect effects. 1, 27 the direct effects include: • fever and neutropenia syndrome with features of infectious mononucleosis, including hepatitis, nephritis, leukopenia, and/or thrombocytopenia • pneumonia • gastrointestinal invasion with colitis, esophagitis, gastritis, ulcers, bleeding, or perforation • hepatitis, pancreatitis, chorioretinitis with the exception of chorioretinitis, the direct clinical manifestations of cmv infection usually occur 1 to 4 months after transplantation; chorioretinitis usually does not begin until later in the transplant course. although cmv is the most common cause of clinical infectious disease syndromes, its "indirect effects" are often more important. cmv infection produces a profound suppression of a variety of host defenses, predisposing to secondary invasion by such pathogens as p. jiroveci, candida and aspergillus species, and some bacterial infections. cmv also contributes to the risk for graft rejection, ptld, hhv6, and hhv7 infections. the mechanisms for this effect are complex, including altered t-cell subsets and mhc synthesis, and the elaboration of an array of pro-inflammatory cytokines, chemokines, and growth factors. transmission of cmv in the transplant recipient occurs in one of three patterns: primary infection, reactivation infection, and superinfection. 1 virus may be reactivated in the setting of an allograft from a seropositive donor transplanted into a seropositive recipient (d+r+). control of cmv infection is via mhc-restricted, virusspecific, cytotoxic t lymphocyte response (cd8+ cells) controlled by cd4+ lymphocytes. seroconversion is a marker for the development of host immunity. thus, the major effector for activation of virus is the nature of the immunosuppressive therapy being administered. the lytic antilymphocyte antibodies, both polyclonal and monoclonal, are direct activators of viral infection (mimicking the alloimmune response) and also provoke the elaboration of tnf and the other pro-inflammatory cytokines that enhance viral replication. cyclosporine, tacrolimus, sirolimus, and prednisone (other than pulse doses) have limited ability to reactivate latent cmv while azathioprine, mycophenolate, and cyclophosphamide are moderately potent in terms of promoting viral reactivation. these agents perpetuate infection once established. allograft rejection is a major stimulus for cmv activation and vice versa. thus, the cmv infection has been linked to a diminished outcome of renal and other allografts. as a result, reinke and colleagues 27 showed that 17 of 21 patients for whom biopsy revealed evidence of "late acute rejection" demonstrated a response to antiviral therapy. further, lowance and colleagues 28 demonstrated that the prevention of cmv infection also resulted in a lower incidence of graft rejection. clinical management of cmv, both prevention and treatment, is of great importance for the transplant recipient. it is based on a clear understanding of the causes of cmv activation and the variety of diagnostic techniques available. cmv cultures are generally too slow and insensitive for clinical utility. further, a positive cmv culture (or shell vial culture) derived from respiratory secretions or urine is of little diagnostic value-many patients secrete cmv in the absence of invasive disease. serologic tests are useful prior to transplantation to predict risk but are of little value after transplantation in defining clinical disease (this statement includes measurements of anti-cmv immunoglobulin m [igm] levels). should a patient seroconvert to cmv, this is evidence that the patient has been exposed to cmv and has developed some degree of immunity. however, seroconversion in transplantation is generally delayed and, thus, not useful for clinical diagnosis. the demonstration of cmv inclusions in tissues in the setting of a compatible clinical presentation is the "gold standard" for diagnosis. quantitation of the intensity of cmv infection has been linked to the risk for infection in transplant recipients. [29] [30] [31] [32] [33] two types of quantitative assays have been developed: the molecular assays and the antigen detection assays. the antigenemia assay is a semiquantitative fluorescent assay in which circulating neutrophils are stained for cmv early antigen (pp65), which is taken up nonspecifically as a measure of the total viral burden in the body. the molecular assays (direct dna pcr, hybrid capture, amplification assays) are highly specific and sensitive for the detection of viremia. most commonly used assays include plasma-based pcr testing and the whole-blood hybrid capture assay, noting that whole blood and plasma-based assays cannot be directly compared. the highest viral loads are often associated with tissue-invasive disease with the lowest in asymptomatic cmv infection. viral loads in the cmv syndrome are variable. either assay can be used in management. the advent of quantitative assays for the diagnosis and management of cmv infection has allowed noninvasive diagnosis in many patients with two important exceptions: 1. neurologic disease, including chorioretinitis 2. gastrointestinal disease, including invasive colitis and gastritis. in these syndromes, the cmv assays are often negative and invasive (biopsy) diagnosis may be needed. the central role of assays is illustrated by the approach to prevention and treatment of cmv (table 37-6). the schedule for screening is linked to the risk for infection. thus, in the high risk patient (d+/r− or r+ with antilymphocyte globulin) after the completion of prophylaxis, monthly screening is performed to assure the absence of infection for 3 to 6 months. in the patient being treated for cmv infection, the assays provide an end point (zero positivity) for therapy and the initiation of prophylaxis. prevention of cmv infection must be individualized for immunosuppressive regimens and the patient. two strategies are commonly used for cmv prevention: (1) universal prophylaxis and (2) preemptive therapy. universal prophylaxis involves giving antiviral therapy to all "at-risk" patients beginning at or immediately post-transplant for a defined time period. in preemptive therapy, quantitative assays are used to monitor patients at predefined intervals to detect early disease. positive assays result in therapy. preemptive therapy incurs extra costs for monitoring and coordination of outpatient care while reducing the cost of drugs and the inherent toxicities. prophylaxis has the possible advantage of preventing not only cmv infection during the period of greatest risk, but also diminishing infections due to hhv6, hhv7, and ebv. further, the indirect effects of cmv (i.e., graft rejection, opportunistic infection) may also be reduced by routine prophylaxis. in practice neither strategy is perfect. both breakthrough disease and ganciclovir resistance have been observed in both approaches. given the risk for invasive infection, patients at risk for primary infection (cmv d+/r−) are generally given prophylaxis for 3 to 6 months after transplantation. we utilize 6 months of prophylaxis in patients receiving lytic antilymphocyte antibodies. other groups are candidates for preemptive therapy if an appropriate monitoring system is in place and patient compliance is good. the standard of care for treating cmv disease is 2 to 3 weeks of intravenous ganciclovir (5 mg/kg twice daily, with dosage adjustments for renal dysfunction). in patients slow to respond to therapy and who are seronegative, the addition of 3 months of cmv hyperimmune globulin in seronegative individuals (150 mg/kg/dose iv) may be useful. relapse does occur, primarily in those not treated beyond the achievement of a negative quantitative assay. therefore, we treat intravenously until viremia has been cleared and following it with prophylaxis with 2 to 4 months of oral ganciclovir (1 g two or three times daily) or valganciclovir (based on creatinine clearance). this approach has resulted in rare symptomatic relapses and appears to prevent the emergence of antiviral resistance. a number of issues remain. first, the role of oral valganciclovir in treatment has not been well studied. this agent provides good bioavailability but is not approved for this indication. further, some relapses occur in gi disease because the assays used to follow disease are not reliable in this setting. thus, repeat endoscopy should be considered to assure the clearance of infection. the optimum dosing of valganciclovir for prophylaxis in renal transplant recipients is also unclear. many centers use 450 mg/day po (given reduced creatinine clearance) although the fda approved dosing 900 mg/day. it is worth measuring the creatinine clearance to ensure appropriate dosing. alternative therapies are available in intravenous form only. these include foscarnet and cidofovir. foscarnet has been used extensively for therapy of cmv in aids patients. it is active against most ganciclovir-resistant strains of cmv, although we prefer combination therapy (ganciclovir and foscarnet) for such individuals, given the toxicities of each agent and the antiviral synergy demonstrated. cidofovir has been used in renal transplant recipients, often with nephrotoxicity. both foscarnet and cidofovir may exhibit synergistic nephrotoxicity with calcineurin inhibitors. a newer class of agents (leflunamide) has been approved for immune suppression and treatment of rheumatologic diseases but also appears to have useful activity against cmv (and possibly bk polyomavirus). ebv is a ubiquitous herpesvirus (the majority of adults are infected) that has b-lymphocytes as a primary target for infection. in immunosuppressed transplant recipients, primary ebv infection (and relapses in the absence of antiviral immunity) causes a mononucleosis-type syndrome, generally presenting as a lymphocytosis (b-cells) with or without lymphadenopathy or pharyngitis. meningitis, hepatitis, and pancreatitis may also be observed. remitting-relapsing ebv infection is common in children and may reflect the interplay between evolving antiviral immunity and immune suppression. this syndrome should suggest relative over-immune suppression. ebv also plays a central role in the pathogenesis of posttransplant lymphoproliferative disorder or ptld. [34] [35] [36] [37] the most clearly defined risk factor for ptld is primary ebv infection that increases the risk for ptld by 10-to 76-fold. ptld may occur, however, in the absence of ebv infection or in seropositive patients. post-transplant non-hodgkin's lymphoma (nhl) is a common complication of solid organ transplantation. lymphomas comprise up to 15% of tumors among adult transplant recipients (51% in children) with mortality of 40% to 60%. many deaths are associated with allograft failure after withdrawal of immune suppression during treatment of malignancy. compared with the general population, ptld has increased extranodal involvement, poor response to conventional therapies, and poor outcomes. the spectrum of disease ranges from benign polyclonal, b-cell infectious mononucleosis-like disease to malignant, monoclonal lymphoma. 38 the majority is of b-cell origin, although t-cell, nk-cell and null cell tumors are described. it should be noted that ebv-negative ptld has been described and that t-cell ptld has been demonstrated in allografts, confused with graft rejection or other viral infection. ptld late (more than 1-2 years) after transplantation is more often ebv-negative in adults. the clinical presentations of ebv-associated ptld vary: 1. unexplained fever (fever of unknown origin) 2. a mononucleosis-type syndrome, with fever, malaise, with or without pharyngitis or tonsillitis (often diagnosed incidentally in tonsillectomy specimens); often no lymphadenopathy is observed. 3. gastrointestinal bleeding, obstruction, perforation 4. abdominal mass lesions 5. infiltrative disease of the allograft 6. hepatocellular or pancreatic dysfunction 7. central nervous system disease diagnosis serologic testing is not useful for the diagnosis of acute ebv infection or ptld in transplantation. thus, quantitative ebv viral load testing is required for the diagnosis and management of ptld. [39] [40] [41] [42] serial assays are more useful in an individual patient than specific viral load measurements. these assays are not standardized and cannot be directly compared between centers. there are some data to suggest that assays using unfractionated whole blood are preferable to plasma samples for ebv viral load surveillance. clinical management depends on the stage of disease. in the polyclonal form, particularly in children, reestablishment of immune function may suffice to cause ptld to regress. at this stage, it is possible that antiviral therapy might have some utility given the viremia and role of ebv as an immune suppressive agent. with the progression of disease to extra-nodal and monoclonal malignant forms, reduction in immune suppression may be useful, but alternate therapies are often required. in renal transplantation, the failure to regress with significant reductions in immune suppression may suggest the need to sacrifice the allograft for patient survival. combinations of anti-b-cell therapy (anti-cd20 rituximab), chemotherapy (chop), and/or adoptive immunotherapy with stimulated t cells have been utilized. [43] [44] [45] [46] polyomaviruses polyomaviruses have been identified in transplant recipients in association with nephropathy and ureteral obstruction (bk virus) and in association with demyelinating disease of the brain (jc virus) similar to that in aids. polyomaviruses are small nonenveloped viruses with covalently closed, circular, double-stranded dna genomes. adult levels of seroprevalence are 65% to 90%. bk virus appears to achieve latency in renal tubular epithelial cells. jc virus has also been isolated from renal tissues but appears to have preferred tropism for neural tissues. reactivation occurs with immune deficiency and suppression and tissue injury (e.g., ischemia-reperfusion). bk virus is associated with a range of clinical syndromes in immunocompromised hosts: viruria and viremia, ureteral ulceration and stenosis, and hemorrhagic cystitis. [47] [48] [49] [50] [51] [52] [53] [54] active infection of renal allografts has been associated with progressive loss of graft function (bk nephropathy) in some individuals. this may be referred to as polyomavirus-associated nephropathy or pvan. bk nephropathy is rarely recognized in recipients of nonrenal organs. the clinical presentation of disease is usually as sterile pyuria, reflecting shedding of infected tubular and ureteric epithelial cells. these cells contain sheets of virus and are detected by urine cytology as "decoy cells." in most cases, such cells are not detected and the patient presents with diminished renal allograft function or with ureteric stenosis and obstruction. in such patients, the etiologies of decreased renal function must be carefully evaluated (e.g., mechanical obstruction, drug toxicity, pyelonephritis, rejection, thrombosis, recurrent disease), and choices must be made between increasing immune suppression to treat suspected graft rejection and reducing immune suppression to allow the immune system to control infection. patients with bk nephropathy treated with increased immune suppression have a high incidence of graft loss. reduced immune suppression may stabilize renal allograft function but risks graft rejection. polyoma-associated nephropathy manifested by characteristic histologic features and renal dysfunction is found in about 1% to 8% of renal transplant patients. risk factors for nephropathy are poorly defined. nickeleit and colleagues 51, 52 found that cellular rejection occurred more commonly in patients with bk nephropathy than in controls. other studies have implicated high dose immunosuppression (particularly tacrolimus and mycophenolate mofetil), pulse dose steroids, severe ischemia-reperfusion injury, exposure to antilymphocyte antibody therapy, increased number of hla mismatches between donor and recipient, cadaver renal transplants, and presence and degree of viremia in the pathogenesis of disease. the role of specific immunosuppressive agents has not been confirmed. the use of urine cytology to detect the presence of infected decoy cells in the urine has approximately 100% sensitivity for bk virus infection but a low (29%) predictive value. 53, 54 it is, therefore, a useful screening tool but cannot establish a firm diagnosis. the use of molecular techniques to screen blood or urine has also been advocated but is more useful in management of established cases (viral clearance with therapy) than in specific diagnosis. [55] [56] [57] [58] [59] [60] hirsch and colleagues 53 showed that patients with bk nephropathy have a plasma viral load statistically significantly higher (>7700 bk virus copies per ml of plasma, p<.001, 50% positive predictive value, 100% negative predictive value) when compared to patients without such disease. 53 given the presence of viremia in renal allograft recipients, it is critical to reduce immune suppression when possible. however, the possible coexistence of rejection with bk infection makes renal biopsy essential for the management of such patients. renal biopsies will demonstrate cytopathic changes in renal epithelial cells without cellular infiltration with the gradual evolution of cellular infiltration consistent with the diagnosis of interstitial nephritis. fibrosis is often prominent occasionally with calcification. immunostaining for cross reacting sv40 virus demonstrates patchy staining of viral particles within tubular cells. there is no accepted treatment for pvan other than a marked reduction in the intensity of immune suppression. it is possible to monitor the response to such maneuvers using urine cytology (decoy cells) and viral load measures in blood and/or urine. the greatest incidence of bk nephropathy is at centers with the most intensive immune suppressive regimens. thus, it is unclear whether reduction of calcineurin inhibitors or antimetabolites should be considered first. given the toxicity of calcineurin inhibitors for tubular cells and the role of injury in the activation of bk virus, as well as the need for anti-bk t-cell activity, we have generally reduced these agents first. other centers have selected reduction of the antimetabolite first. regardless of the approach, renal function, drug levels, and viral loads must be monitored carefully. some centers advocate the use of cidofovir for bk nephropathy in low doses (0.25-1 mg/kg every 2 weeks). [61] [62] [63] [64] significant renal toxicity may be observed with this agent, especially in combination with the calcineurin inhibitors. retransplantation has been achieved in such patients with failed allografts, possibly as a reflection of immunity developing subsequent to reduction in immune suppression. 65 infection of the central nervous system by jc polyomavirus has been observed uncommonly in renal allograft recipients as progressive multifocal encephalopathy. this infection generally presents with focal neurologic deficits or seizures and may progress to death following extensive demyelination. pml may be confused with calcineurin neurotoxicity; both may respond to a reduction in drug levels. it is thought that these are distinct entities, but further studies are underway. in addition to the endemic mycoses, transplant recipients are at risk for opportunistic infection with a variety of fungal agents, the most important of which are candida species, aspergillus species, and c. neoformans. the most common fungal pathogen in these patients is candida, with c. albicans and c. tropicalis accounting for 90% of the infections and c. glabrata for most of the rest. mucocutaneous candidal infection (e.g., oral thrush, esophageal infection, cutaneous infection at intertriginous sites, candidal vaginitis) occurs particularly when candidal overgrowth is promoted by the presence of high levels of glucose and glycogen in tissues and fluids (e.g., with poorly controlled diabetes, high-dose steroid therapy) and by broad-spectrum antibacterial therapy). these infections are usually treatable through correction of the underlying meta-bolic abnormality and topical therapy with clotrimazole or nystatin. more difficult to manage is candidal infection occurring in association with the presence of foreign bodies that violate the mucocutaneous surfaces of the body (e.g., vascular access catheters, surgical drains, and bladder catheters). optimal management of these infections requires removal of the foreign body and systemic antifungal therapy with either fluconazole or amphotericin. a special problem in renal transplant recipients is candiduria, even if the patient is asymptomatic. particularly in individuals with poor bladder function, obstructing fungal balls can develop at the ureteropelvic junction, resulting in obstructive uropathy, ascending pyelonephritis, and the possibility of systemic dissemination. a single positive culture result for candida species from a blood specimen necessitates systemic antifungal therapy, because this finding carries a risk of visceral invasion of more than 50% in this population. fluconazole (400-600 mg/day, with adjustment for renal dysfunction), because of its better safety profile, is usually used as initial therapy, unless the patient is critically ill or a fluconazole-resistant species (e.g., c. glabrata or c. krusei) is present. in these instances, therapy is with caspofungin or amphotericin b, usually in a lipid preparation. flucytosine may be useful as an adjunctive therapy in resistant infections but must be guided by drug levels and attention to hematopoietic toxicity. invasive aspergillosis is a medical emergency in the transplant recipient, with the portal of entry being the lungs and sinuses in more than 90% of patients and the skin in most of those remaining. two species, a. fumigatus and a. flavum, account for most of these infections, although amphotericin-resistant isolates (a. terreus) are occasionally recognized. the pathologic hallmark of invasive aspergillosis is blood vessel invasion, which accounts for the three clinical characteristics of this infection: tissue infarction, hemorrhage, systemic dissemination with metastatic invasion. early in the course of transplantation, central nervous system involment with fungal infection is most often due to aspergillus species; more than 1 year after transplantation, other fungi (zygomycetes, dematiaceous fungi) are increasingly prominent. the drug of choice for this infection is probably voriconazole, noting the intense interactions between this agent and the calcineurin inhibitors and sirolimus. liposomal amphotericin is a reasonable alternative, and combination therapies are under study. of note, surgical debridement is often essential for the successful clearance of such invasive infections. central nervous system (cns) infection in the transplant recipient is an important differential for the clinician. the spectrum of causative organisms is broad and must be considered in terms of the timeline for infection in this population. many infections are metastatic to the cns, often from the lungs. thus, a "metastatic workup" is a component of evaluation of cns lesions, including those due to aspergillus, cryptococcus, nocardia, or strongyloides stercoralis. viral infections include cytomegalovirus (nodular angiitis), herpes simplex meningoencephalitis, jc virus (pml), and varicella zoster virus. common bacterial infections include listeria monocytogenes, mycobacteria, nocardia, and occasionally salmonella species. brain abscess and epidural abscess may be observed with methicillin-resistant staphylococcus, penicillin resistant pneumococcus and quinolone-resistant streptococci problematic. metastatic fungi include aspergillus and cryptococcus but also spread from sinuses (mucoraceae), skin (dematiaceae), and bloodstream (histoplasma and pseudoallescheria/scedosporium, fusarium species). parasites include toxoplasma gondii and strongyloides. given the spectrum of etiologies, precise diagnosis is essential. in particular, empiric therapy must "cover" listeria (ampicillin), cryptococcus (fluconazole or amphotericin), and herpes simplex virus (acyclovir) while awaiting data from lumbar puncture, blood cultures, and radiographic studies. included in the differential diagnosis are noninfectious etiologies, including calcineurin inhibitor toxicity and lymphoma, as well as metastatic cancer. biopsy is often needed for a firm diagnosis. cryptococcal infection is rarely seen in the transplant recipient until more than 6 months after transplantation. in the relatively intact transplant recipient, the most common presentation of cryptococcal infection is that of an asymptomatic pulmonary nodule, often with active organisms present. in the "chronic ne'er-do-well" patient, pneumonia and meningitis are common with skin involvement at sites of tissue injury (catheters) also being observed. cryptococcosis should be suspected in transplant recipients present with unexplained headaches (especially when accompanied by fevers), decreased state of consciousness, failure to thrive, or unexplained focal skin disease (which requires biopsy for culture and pathologic evaluation) more than 6 months after transplantation. diagnosis is often achieved by serum cryptococcal antigen detection, but all such patients should have lumbar puncture for cell counts and cryptococcal antigen studies. initial treatment is probably best with amphotericin and 5-flucytosine followed by high dose fluconazole until the cryptococcal antigen is cleared from blood and cerebrospinal fluid. scarring and hydrocephalus may be observed. the spectrum of potential pathogens of the lungs in transplantation is too broad for this discussion. however, some general concepts are worth mentioning. as for all infections in transplantation, invasive diagnostic techniques are often necessary in these hosts. the depressed inflammatory response of the immunocompromised transplant patient may greatly modify or delay the appearance of a pulmonary lesion on radiograph. focal or multifocal consolidation of acute onset will quite likely be caused by bacterial infection. similar multifocal lesions with subacute to chronic progression are more likely secondary to fungi, tuberculosis, or nocardial infections. large nodules are usually a sign of fungal or nocardial infection, particularly if they are subacute to chronic in onset. subacute disease with diffuse abnormalities, either of the peri-bronchovascular type or miliary micronodules, are usually caused by viruses (especially cmv) or pneumocystis jiroveci. 66, 67 additional clues can be found by examining pulmonary lesions for cavitation; cavitation suggests such necrotizing infections as those caused by fungi (aspergillus or mucoraceae), nocardia, staphylococcus, certain gram-negative bacilli, most commonly with klebsiella pneumoniae and pseudomonas aeruginosa. [68] [69] [70] ct of the chest is useful when the chest radiograph is negative or when the radiographic findings are subtle or nonspecific. ct is also essential to the definition of the extent of the disease process, the possibility of multiple simultaneous processes (superinfection), and to the selection of the optimal invasive technique to achieve microbiologic diagnosis. the risk of infection with pneumocystis is greatest in the first 6 months after transplantation and during periods of increased immune suppression. 1, 66, 67 the natural reservoir of infection remains unknown. aerosol transmission of infection has been demonstrated by a number of investigators in animal models, and clusters of infections have developed in clinical settings, including between hiv-infected persons and renal transplant recipients. activation of latent infection remains a significant factor in the incidence of disease in immunocompromised hosts. in the solid organ transplant recipient, chronic immune suppression that includes corticosteroids is most often associated with pneumocystosis. bolus corticosteroids, cyclosporine, or co-infection with cmv may also contribute to the risk for pneumocystis pneumonia. in patients not receiving trimethoprim-sulfamethoxazole (or alternative drugs) as prophylaxis, most transplant centers report an incidence of pneumocystis jiroveci pneumonia of approximately 10% in the first 6 months post-transplant. there is a continued risk of infection in three overlapping groups of transplant recipients: (1) those who require higher than normal levels of immune suppression for prolonged periods of time due to poor allograft function or chronic rejection; (2) those with chronic cytomegalovirus infection; and (3) those undergoing treatments that increase the level of immune deficiency, such as cancer chemotherapy or neutropenia due to drug toxicity. the expected mortality due to pneumocystis pneumonia is increased in patients on cyclosporine when compared to other immunocompromised hosts. the hallmark of infection due to p. jiroveci is the presence of marked hypoxemia, dyspnea, and cough with a paucity of physical or radiologic findings. in the transplant recipient, pneumocystis pneumonia is generally acute to subacute in development. atypical pneumocystis infection (radiographically or clinically) may be seen in patients who have coexisting pulmonary infections or who develop disease while receiving prophylaxis with second choice agents (e.g., pentamidine or atovaquone). patients outside the usual period of greatest risk for pcp may present with indolent disease confused with heart failure. in such patients, diagnosis often has to be made by invasive procedures. the role of sirolimus therapy in the clinical presentation is unknown. a number of patients have been identified with interstitial pneumonitis while receiving sirolimus; it is not known whether this syndrome is directly attributable to sirolimus or reflects concomitant infection. the characteristic hypoxemia of pneumocystis pneumonia produces a broad alveolar-arterial po 2 gradient. the level of serum lactic dehydrogenase (ldh) is elevated in most patients with pneumocystis pneumonia (>300 international units [iu]/ml). however, many other diffuse pulmonary processes also raise serum ldh levels. like many of the "atypical" pneumonias (pulmonary infection without sputum production), no diagnostic pattern exists for pneumocystis pneumonia on routine chest radiograph. the chest radiograph may be entirely normal or develop the classical pattern of perihilar and interstitial "ground glass" infiltrates. microabscesses, nodules, small effusions, lymphadenopathy, asymmetry, and linear bands are common. chest computerized tomography (ct-scans) will be more sensitive to the diffuse interstitial and nodular pattern than routine radiographs. the clinical and radiologic manifestations of p. jiroveci pneumonia are virtually identical to those of cmv. indeed, the clinical challenge is to determine whether both pathogens are present. significant extrapulmonary disease is uncommon in the transplant recipient. identification of p. jiroveci as a specific etiologic agent of pneumonia in an immunocompromised patient should lead to successful treatment. a distinction should be made between the diagnosis of pneumocystis infection in aids and in non-aids patients. the burden of organisms in infected aids patients is generally greater than that of other immunocompromised hosts and noninvasive diagnosis (sputum induction) more often achieved. in general, noninvasive testing should be attempted to make the initial diagnosis, but invasive techniques should be used when clinically feasible. the diagnosis of p. jiroveci infection has been improved by the use of induced sputum samples and of immunofluorescent monoclonal antibodies to detect the organism in clinical specimens. these antibodies bind both cysts and trophozoites. the cyst wall can be displayed by a variety of staining techniques; of these, the gomori's methenamine-silver nitrate method (which stains organisms brown or black) is most reliable, even though it is susceptible to artifacts. sporozoites and trophozoites are stained by polychrome stains, particularly the giemsa stain. early therapy, preferably with trimethoprim-sulfamethoxazole (tmp-smz) is preferred; few renal transplant patients will tolerate full-dose tmp-smz for prolonged periods of time. this reflects both the elevation of creatinine due to trimethoprim (competing for secretion in the kidney) and the toxicity of sulfa agents for the renal allograft. hydration and the gradual initiation of therapy may help. alternate therapies are less desirable but have been used with success, including: intravenous pentamidine, atovaquone, clindamycin with primaquine or pyrimethamine, and trimetrexate. although a reduction in the intensity of immune suppression is generally considered a part of anti-infective therapy in transplantation, the use of short courses of adjunctive steroids with a gradual taper is sometimes used in transplant recipients (as in aids patients) with severe respiratory distress associated with pcp. the importance of preventing pneumocystis infection cannot be overemphasized. low dose trimethoprim-sulfamethoxazole is well tolerated and should be used in the absence of concrete data demonstrating true allergy. alternative prophylactic strategies including dapsone, atovaquone, inhaled or intravenous pentamidine, are less effective than trimethoprim-sulfamethoxazole but useful in the patient with significant allergy to sulfa drugs. tmp-smx is the most effective agent for prevention of infection due to p. jiroveci. the advantages of tmp-smx include increased efficacy, lower cost, the availability of oral preparations, and possible protection against other organisms, including toxoplasma gondii, isospora belli, cyclospora cayetanensis, nocardia asteroides, and common urinary, respiratory, and gastrointestinal bacterial pathogens. it should be noted that alternative agents lack this spectrum of activity. due to concerns about the efficacy of vaccines following transplantation, patients should complete vaccinations at least 4 weeks beforehand to allow time for an optimal immune response and resolution of subclinical infection from live vaccines. vaccinations should include pneumococcal vaccine (if not vaccinated in last 3-5 years), documentation of tetanus and mmr (measles, mumps, rubella) and polio status, as well as vaccines for hepatitis b and varicella zoster (if no history of chickenpox or shingles) (see also . after transplant, influenza vaccination should be performed yearly or as per local guidelines. recommended schedules and doses for routine vaccinations can be obtained from the united states centers for disease control and prevention (cdc) at www.immunize.org or the cdc immunization information hotline, (800) 232-2522. infection in organ-transplant recipients pretransplant evaluation for infections in donors and recipients of solid organs prophylactic measures in the solid-organ recipient before transplantation organ donor screening for infectious diseases: review of practice and implications for transplantation cadaver donor screening for infectious agents in solid organ transplantation recipient screening prior to solid-organ transplantation american society of transplantation. the evaluation of renal transplant candidates: clinical practice guidelines ad hoc clinical practice guidelines subcommittee of the patient care and education committee of the american society of transplant physicians report of the crystal city meeting to maximize the use of organs recovered from the cadaver donor outcome of transplantation of organs procured from bacteremic donors mycobacterium tuberculosis infection in solid-organ transplant recipients: impact and implications for management isoniazid hepatotoxicity in renal transplant recipients diagnosis and management of posttransplant lymphoproliferative disorder in solid-organ transplant recipients serial measurement of epstein-barr viral load in peripheral blood in pediatric liver transplant recipients during treatment for posttransplant lymphoproliferative disease guidelines for counseling persons infected with human tlymphotropic virus type i (htlv-i) and type ii (htlv-ii) long-term results in human t-cell leukemia virus type 1-positive renal transplant recipients update: investigations of west nile virus infections in recipients of organ transplantation and blood transfusion update: investigations of west nile virus infections in recipients of organ transplantation and blood transfusion? michigan transplantation for patients infected with human immunodeficiency virus: no longer experimental, but not yet routine clinical presentation and outcome of tuberculosis in kidney, liver and heart transplant recipients in spain mycobacterial infections in renal allograft recipients mycobacterial infections in renal transplant recipients: report of five cases and review of the literature mycobacterial infections in renal transplant recipients: seven cases and a review of the literature mycobacterial infections after renal transplantation: report of fourteen cases and review of the literature typical and atypical mycobacterium mycobacterium tuberculosis infection in solid-organ transplant recipients: impact and implications for management late-acute renal allograft rejection and symptomless cytomegalovirus infection valacyclovir for the prevention of cytomegalovirus disease after renal transplantation. international valacyclovir cytomegalovirus prophylaxis transplantation study group comparison of quantitative cytomegalovirus (cmv) pcr in plasma and cmv antigenemia assay: clinical utility of the prototype amplicor cmv monitor test in transplant recipients cytomegalovirus (cmv) virus load kinetics to predict recurrent disease in solidorgan transplant patients with cmv disease multicenter comparison of the digene hybrid capture cmv dna assay (version 2.0), the pp65 antigenemia assay, and cell culture for detection of cytomegalovirus viremia prevention of cytomegalovirus disease in recipients of solid-organ transplants new strategies for prevention and therapy of cytomegalovirus infection and disease in solid-organ transplant recipients incidence of non-hodgkin lymphoma in kidney and heart transplant recipients epstein-barr virus-induced posttransplant lymphoproliferative disorders the diverse pathology of posttransplant lymphoproliferative disorders: importance of a standardized approach guidelines for the diagnosis and management of post transplant lymphoproliferative disorder in solid organ transplant recipients posttransplant lymphoproliferative disorders (ptld) pathology and genetics: tumours of haematopoietic and lymphoid tissues management of epstein-barr virus-induced posttransplant lymphoproliferative disease in recipients of solid organ transplantation quantitation of epstein-barr virus dna in the blood of adult liver transplant recipients epstein-barr viral load as a tool to diagnose and monitor post-transplant lymphoproliferative disease epstein-barr virus load monitoring: its role in the prevention and management of ptld immunotherapy for post-transplant lymphoproliferative disease anti-b cell and anti-cytokine therapy for the treatment of ptld: past, present and future treatment of epstein-barrvirus-positive post-transplantation lymphoproliferative disease with partly hla-matched allogeneic cytotoxic t cells interferon and cytotoxic chemotherapy for the treatment of post transplant lymphoproliferative disorder the persistence of papovavirus bk dna sequences in normal human renal tissue bk virus: discovery, epidemiology, and biology bk virus in solid organ transplant recipients: an emerging syndrome human polyoma virus-associated interstitial nephritis in the allograft kidney bk-virus nephropathy in renal transplants-tubular necrosis, mhc-class ii expression and rejection in a puzzling game polyomavirus infection of renal allograft recipients: from latent infection to manifest disease prospective study of polyomavirus type bk replication and nephropathy in renaltransplant recipients bk virus nephropathy? polyomavirus adding insult to injury morphological spectrum of polyoma virus disease in renal allografts: diagnostic accuracy of urine cytology prospective study of the human polyomaviruses bk and jc and cytomegalovirus in renal transplant recipients clinical course of polyoma virus nephropathy in 67 renal transplant patients bk virus infection in a kidney allograft diagnosed by needle biopsy polyomavirus reactivation in native kidneys of pancreas alone allograft recipients bk virus nephropathy diagnosis and treatment: experience at the university of maryland renal transplant program the use of vidarabine in the treatment of human polyomavirus associated acute haemorrhagic cystitis activities of various compounds against murine and primate polyomaviruses clinical pharmacokinetics of cidofovir in human immunodeficiency virus-infected patients quantitative viral load monitoring and cidofovir therapy for the management of bk virusassociated nephropathy in children and adults successful retransplantation following renal allograft loss to polyoma virus interstitial nephritis prevention of infection caused by pneumocystis carinii in transplant recipients prevention of infection due to pneumocystis carinii nocardiosis in transplant recipients recurrent nocardiosis in a renal transplant recipient central venous catheter-associated nocardia bacteremia: an unusual manifestation of nocardiosis key: cord-257299-z9u12yqb authors: mansi, n.; de maio, v.; della volpe, a.; ripa, g.; malafronte, l.; de filippis, c. title: ear, nose and throat manifestation of viral systemic infections in pediatric patients date: 2009-12-31 journal: international journal of pediatric otorhinolaryngology doi: 10.1016/s0165-5876(09)70006-0 sha: doc_id: 257299 cord_uid: z9u12yqb abstract objective/methods an exhaustive review of literature was performed to investigate available data and evidences regarding pediatric otolaryngologic manifestations of viral systemic infections. results/conclusions modern otolaryngologists should be familiar with viral systemic infections since many have head and neck manifestations. cooperation between otolaryngologist, paediatrician and virologist can be considered and excellent tool in diagnosis and treatment of these diseases in particular when complications occur. there are multiple systematic viral infections that can manifest themselves in orl related organs. their actions can work directly or indirectly causing an alteration in the human immune system and a consequent secondary bacterial invasion. notable advances in the diagnosis and treatment of viral infections have been mitigated by the appearance of new pathological processes, for example aids, which often has its initial manifestations in orl regions. table 1 is a list of illnesses affecting different anatomical sites and the viral etiologies that commonly strike each particular location. considering the vast nature of the subject, we subdivided our treatment into three parts corresponding to the same groups of interrelated viral illnesses: -viruses that can cause deafness. -viruses that can cause inflammation in the upper respiratory tract. -viruses particularly relevant to ent (infectious mononucleosis, papillomatosis, herpes infections). ascertaining specific viral causes of most infections is neither necessary nor cost-effective, and should be reserved only for specific cases. clinical and epidemiological acumen remain the basis for a presumptive diagnosis. when a specific diagnosis is necessary, diagnostic procedures based on biochemical and molecular biological processes provide sensitive, specific and rapid results [1] . in most viral infections, immunity to re-infection generally lasts a short period of time due to the host's limited immunological response or, rather for an antigenic change in the virus. viral pathologies that can cause deafness can be congenital, appear in either the pre-natal or postnatal period and can also be acquired upon contact with the pathogen [2] [3] [4] (table 2 ). in particular, the hearing damage caused by congenital infections can be part of a severe syndrome (such as "congenital rubella syndrome") but more frequently it is the first and only manifestation of intrauterine infection. common childhood viral infections, such as measles and mumps are probably an unrecognized cause of acute or progressive damage to hearing [5] . in prenatal deafness, a pathogen introduced during pregnancy can provoke an arrest or alteration of the normal development of the ear, even causing lesions on the already-formed hearing mechanism [6] . the most serious lesions manifest themselves in the first three months of pregnancy, especially between the seventh and tenth week, when the cochlea is developing; this would be considered a case of embriopathy. fetopathy refers instead to lesions that form between the fourth month of pregnancy and birth. since the hearing organ has already formed in these cases, patients do not generally suffer serious alterations although the inner ear is certainly sensitive. the viruses that most frequently cause prenatal deafness are rubella and citomegalovirus (cmv). rubella is caused by an rna virus of the togaviradae family of the rubivirus genus. congenital rubella is typically passed on to the fetus from a primary infection in the mother. the virus invades the upper airways of the mother causing viremia and spreading into different sites including the placenta. it has been hypothesized that in the first gestational phases, the rubella virus provokes a chronic intrauterine infection. fetal infection in the first trimester, particularly in the first 8-10 weeks, has an extremely high risk of malformations such as hypoacusia, cardiac and ocular defects (gregg triad); however, if the rubellum infection is contracted in the second or third trimester, it results in hypoacusia and pigmented retinas. thus, the more precocious the maternal rubellum, the greater the risk of fetal infections and the more serious the fetal malformations (100% in the first month, 80% in the first trimester, 70% in the second trimester and 30% in the third). this reduction is likely due to either a maturation of the placenta after the first trimester which limits the transfer of the virus, or the greater resistance of the differentiated cells [5] . the deriving hypoacusia is generally sensorineural and bilateral and at birth can already be progressive or it can manifest itself later. the hearing damage seems to be caused by a "teratogenic" effect of interference with the normal development of the organ at the cochlear level [6, 7] . unlike other congenital infections, rubella is easily prevented. between 12 and 15 months of age, a livevirus rubellum vaccine is administered along with a measles and mumps vaccination, giving the patient immunity to rubellum for about 15 years (mmr); a booster vaccination is administered before elementary or middle school. women of child-bearing age who are not immune to rubella must undergo vaccination and not get pregnant in the following three months. vaccination immediately after giving birth is advisable for mothers at risk of being infected. citomegalovirus (cmv) is a dna virus that belongs to the herpesviridae family. it can go into latency and then reactivate and has been isolated in various sites including saliva, urine, breast milk, sperm, brain fluid, and amniotic fluid. congential cmv infection is thought to be derived from transplacental infection from a primary or recurring maternal infection occurring in the first half of pregnancy. prenatal cmv infection is contracted by contact with infected cervical secretions, breast milk, or blood derivatives. it is believed that maternal antibodies have a protective function and that most of these newborns are either born asymptomatic or are not infected by the virus in the case of contact. many women who are infected by cmv during pregnancy are asymptomatic, but occasionally develop an illness similar to mononucleosis. it is still unclear if more serious lesions are a consequence of a precocious maternal infection or of a later one during the course of gestation. nearly 10% of children with congenital cmv infection are symptomatic at birth. manifestations include delayed intrauterine growth, premature birth, microcephalus, jaundice, petechia, hepatosplenomegalia, periventricular calcification, corioretinitis e pneumonia. the virus causes deafness by infecting the inner ear and altering the organ of corti. moreover, it can cause malformations of the labyrinth of ethmoid and at the same time also lesions on the auditory tract due to secondary toxicity. the hypoacusia that establishes itself is sensorineural, almost always bilateral, and profound, generally regarding acute tones. symptomatic newborns have a mortality rate of up to 30% and 70-90% of those who survive have neurological deficits such as hearing loss, mental retardations and visual disturbances [8] [9] [10] . a vaccine for cmv is still under research. exposure to the disease in non-immunized pregnant women must be controlled, despite the fact that cmv is ubiquitous everywhere. since it is frequent in children who attend preschools, pregnant women must observe all the common norms of good hygiene after contact with or being exposed to the urine or expectorate of such children [11, 12] . in post-natal deafness, numerous infective illnesses can be responsible for serious damage to the viii nerve and the cochlear apparatus. a large part of hearing defects arising in childhood can be traced back to the intrauterine period. the most frequent forms are: viral meningoencephalitis (arbovirus, herpesvirus, mixovirus, poxvirus, etc.), mumps, chickenpox and measles. meningoencephalitis can be primitive or constitute the secondary complication of a viral infection. the forms of primitive meningoencephalitis can be both epidemic (arbovirus, poliovirus, echovirus, coxsackievirus illnesses), and sporadic (herpes simplex, varicella zoster, parotite) [13] . secondary encephalitis, such as complications of a viral infection, probably have an immunological, pathgenic mechanism. secondary encephalitis to rubella, mumps, measles, smallpox, cow's pox and other less defined illnesses are all examples. currently, deafness caused by meningoencephalitis is not infrequent among the post-natal causes of deafness; in some cases it can be caused by a virus that, before birth, reach the cochlea via the external hearing conduct by way of the vascular system, causing a relatively symmetrical bilateral sensorineural hypoacusia which is either mild-serious or profound. in other cases, deafness is due to a meningoencephalital localization of liquor infection in the first four weeks of life as a complication of neaonatal sepsis (25%). deafness caused by meningoencephalitis occurs more often in males and is found in 2/10,000 neonates born at full term and in 2/1,000 low-weight neonates. no vaccines exist for the described viral forms. mumps (parotitis) is instead caused by a paramixovirus, spread through drops of infected saliva or through direct contact with material contaminated by infected saliva. the virus probably penetrates the body through the mouth. it can be found in saliva 1-6 days before the appearance of the swelling of the salivary glands and lasts throughout the duration of the illness (usually 5-9 days). an infection usually results in permanent immunity, even when there is unilateral swelling of the salivary glands. although the illness can occur at any age, most cases occur in children between 5 and 10 years of age; it does not usually occur in children under 2 years old. breast-fed children less than one year old are usually immune. the incubation period is 14-24 days. deafness can be a complication in 5/10,000 cases of mumps. in 80% of cases, deafness is sudden and unilateral in the context of an acute infection in association with aseptic meningitis and often accompanied by tinnitus, ataxia, and vomiting. hearing loss is profound and permanent for high frequencies and can go unrecognized. damage is confined to the cochlear duct and consists in the degeneration of the vascular strip of the corti organ, the degeneration of the upper membrane, usually more serious on the basal curve of the cochlea. active immunization is obtained through a single-dose, live-virus inoculation between 12 and 15 months of age with a mmr vaccine. a booster dose is administered before starting elementary or middle school. measles, caused by a paramixovirus, is extremely contagious and is spread primarily through either the nasal and oral excretions of an individual in the prodrome or precocious eruptive stage of the disease or through the nuclei of drops dispersed in the air. the contagious stage of the illness extends from 2 to 4 days before the appearance of the eruptions until 2-5 days after their appearance. the virus disappears from the nose and pharynx secretions as soon as the eruption on the skin has cleared up. the incubation period is 7-14 days. measles virus causes permenant, bilateral deafness in 1/1000 cases. deafness appears suddenly at the same time as the cutaneous rash. the viral infection of the inner ear spreads through the vascular strip and destroys the structures of the cochlea as well as most of the nervous and ganglionic and fibers [14] . measles infection can be avoided by administering a reduced, live-virus vaccine to children between the ages of 12 and 15 months (mmr). the vaccine confers long-term immunity and provokes an antibody response similar to that of natural measles. in some cases, the vaccine can provoke a light or asymptomatic infection that is not contagious. sensitive subjects at risk of contracting measles can be protected if the live-vaccine is administered within 2 days of exposure. in other cases, such as pregnant women, or children under one year of age, an immunoglobuline specific measles vaccine (mig) or a 0,25 ml/kg im dose of serum immunoglobulin may be administered. chickenpox is caused by the varicella-zoster virus (herpesvirus) and represents its acute, invasive phase, while the reactivation from its latent phase causes the herpes zoster illness. it is believed that chickenpox, which is extremely contagious, is transmitted through drops of saliva which are infected and even more infective during the brief prodromic period and the first phase of eruption. incubation period is 14-16 days and transmission is considered possible 10-21 days after exposure. the deafness it causes can lead to the destruction of the nervous and sensorial cells of the neuroepithelia through a process of neurolabyrinthitis which can result in severe bilateral, sensorineural hypoacusia [15] . chickenpox can be prevented by inoculating with a reduced, live-virus vaccine in all healthy children between 12 and 18 months of age, after which children who lack immunity to chickenpox may be vaccinated at any time. subjects over 13 years old who have not been immunized must receive two doses of the vaccine, with a period of 4-8 weeks between doses. the severity of the disease can be lessened by administering an immunoglobuline anti-zoster (zig) or anti-varicella-zoster (vzig) within 96 hours after exposure. its use, however, is restricted to subjects at risk, like those affected by leukemia, immunodeficiency syndromes or other serious pathologies, and pregnant women. neonates with mothers who were infected by chickenpox five days before giving birth or two days after are also candidates for such treatment [16, 17] . viral respiratory infection is almost always a benign pathology. its beginning is connected with the socialization of the child, and as such is most frequent during preschool. it noticeably interferes with the child's wellbeing and provokes significant medical-social costs.unfavorable environmental factors (atmospheric pollution, passive smoking, etc.), precocious socialization, and predisposing immunological factors with immunological immaturity could all be predisposing factors. viral respiratory infections are characterized by a series of acute episodes that can involve the entire respiratory system or a single sector (pharyngotonsillitis, otitis, rhinosinusitis, laryngitis, bronchitis, pneumonia) [18] [19] [20] [21] . the damage that a viral infection can inflict on the mucous membranes of the upper respiratory tract are influenced by the reduction of the mucous flux and phagocytes as well as the increase in the bacteria's adhesiveness to mucus cells [22, 23] . an upper-resipiratory viral infection is characterized by multiple processes. first, the virus replicates itself in the epithelium, spreading fragments of the disintegrated cells into respiratory secretions and demonstrating the presence of the virus, viral peptides, or viral nucleic acids [24, 25] . the host then responds to the infection by producing a range of cellular products. some, such as alpha-interferon, are specifically anti-viral. others, such as interleukin, are aspecific. specific antibodies are produced in sequence, for example igm followed by igg and iga; the comparison of a high level of igm without an increase in igg is an index of recent infection (the comparison of blood examinations confirms the diagnosis but is only clinically useful for epidemiologic purposes. acute rhino, pharengeal and tonsillar inflammation, caused by viral infections [26] are some of the most common deseases found in pediatric populations. less frequently, the pharangeal-tonsillar forms are accompanied by an involvement of the oral and or respiratory tract mucosa [27] [28] [29] . such infections, in anglo-saxon countries, the term, upper respiratory tract infection (urti) or "common cold" is used to describe an inflammation of the upper airways [30] . the episodes, which often reoccur, effect mostly preschool-age children and have socioeconomic repercussions, which are not related to the gravity of the pathology, but mostly to the increase in the requests for visits to the doctor, the costs incurred for treatment and the days of school and work lost by the children and the parents who must look after them. the etiology of the acute forms in the respiratory airways is, initially, of a viral nature in most patients, with later, secondary bacterial infections on the mucous lesions caused by the viral agents [31] . the transmission of the pathogens responsible for the urti frequently occurs in public locations by direct or indirect contact with the nasal secretions or plugge drops from infected subjects. seasonability, which could increase or reduce sensitivity to such infections, constitutional factors, and an incomplete maturation of the immune system of the child are hypothesized to be pathogenic mechanisms. multiple viruses are responsible for these infections as described in table 1 . the course of acute, unspecific viruses is quite variable but almost always concludes with a recovery within 2-5 days as long as other complications do not develop. the most common complications are infections such as otitis media (mostly in younger children), rhinosinusitis, satellite lymphoadenitis, spreading of the infection into the lower respiratory tract and obstructed respiration, and bronchial spasms in subjects with bronchial hyperactivity. occasionally respiratory viruses are responsible for clinical pictures described as the common cold [32] , caused by picornavirus (rhinovirus, echovirus and coxackievirus), and influenza syndrome [8, [33] [34] [35] , caused by influenza viruses that initially strike the epithelium of the respiratory mucous membranes which other viral strains attach themselves to, aggravating and complicating the original clinical portrait [36, 37] . both herpangina and laryngotracheitis have a unique clinical picture. herpangina is an extremely contagious illness caused by a coxackievirus characterized by a presence of a vesicular exanthema at the velopharyngeal mucous level and acute or croup laryngotracheitis [38] [39] [40] [41] when viral infections are associated. the infections are caused by diverse viruses but more frequently by type 1 and type 2 parainfluenza viruses [42] [43] [44] , which cause an inflammation of the tracheal and subglottic muscous membrane with a charateristic symptomology (inspiratory stridor and barking cough) occasionally relapsing with serious, obstructing respiratory complications [45] [46] [47] . the varicella-zoster virus (vzv) belongs to the herpesvirus group. it is a dna virus that gives rise to chickenpox as a primary infection and to herpes zoster (hz) as a localized relapse due to modifications of the pathogenic power of the virus and/or alterations of cellular immunity [38, 48, 49] . hz is an acute cutaneous-nervous illness that is locally circumscribed and provoked by a resurgence of the vzv acquired during infancy and latent in one or more of the more sensitive ganglia of the dorsal roots of the spinal marrow and/or cranial nerves for a prolonged period of time (often decades); during the latency period the virus does not replicate itself or give any sign or symptom of its presence [50] [51] [52] . in particular, at the auricular level, the illness takes on the name herpes zoster oticus but it is also described in the auricular zone as herpes zoster auris or ramsay hunt illness, in honor of the author who, in 1907, described its characteristics and its correlation with the geniculate ganglion [53, 54] . the illness is caused by a reactivation of the vzv in the geniculate ganglion of the facial nerve; through the sensitive nervous fiber, the virus reaches the skin and causes a characteristic centrifugal root vesicular eruption. we can clinically define four stages according to the involvement of the vii n.c. the diffusion of the virus from cell to cell must be impeded with the use of antiviral drugs (5 mg/kg acyclovir administered intravenously per day in three daily doses for 7-10 days followed by an oral administration for another 7 days); results of treatment with more recent antiviral drugs (such as famciclovir and valaciclovir) are promising, while a significant inflammatory reaction reactive in the nerve must be treated with cortisonebased anti-inflammatory medication (1 mg/kg/daily for ten days) and sometimes with surgical decompression [13] . analgesics and local antiseptics should be added to treament with cortisones and antiviral medication. laryngeal papillomatosis (lp) [55, 56] is caused by subtypes of the human papilloma virus (hpv) which is a member of the papova family of viruses [57] . seventy subtypes have been described, but only hpv 11, hpv 6, and more rarely hpv 16 are specifically associated with laryngeal papillomas [58] [59] [60] . typical of such a pathology is the multifocal nature of the lesion (>85% of cases), localized on the vocal cords in 60% of cases but also involving the upper-glottic plain (35%), the oropharynx, the bronchial tree and, rarely, the cervical esophagus. in the united states, the annual incidence rate of laryngeal papillomatosis is 4.3/100,000 in children and 1.8/100,000 in adults, with a prevelance of 5.7/100,000 [61, 62] . this data is substantially analogous to that revealed in a 1991 danish study [63] which registered an annual incidence in children of 3.6/100,000. histologically, papilloma cosists of a cartilaginous fiber scaffolding with the presence of connective vascular tissue its surface surrounded by squamous epithelium. keratinizing aspects are not observed on its surface. the course of this pathology is characterized by frequent relapses and aggravations that require frequent and repeated laryngoscopy and bronchoscopy to ablate the rapidly forming pappillose formations and to avoid obstruction of the airways [64] . even if the illness is usually resolved by a spontaneous recovery, some cases can move toward an unfavorable prognosis at any time for no recognizable reason and involve the trachea, the bronchi and the lungs [65] . it is believed that hpv 11 has a greater propensity for a distal, pulmonary diffusion and that, moreover, therapeutic action, such as tracheotomy or repeated endolaryngeal ablation can favor the distal insemination of papillomas. the tracheal, bronchial, and pulmonary involvement occurs, according to various authors, in 2-4% of cases. malignant degeneration of the laryngeal papillomatosis, is a rare, but serious event and leads to an unfavorable prognosis. most described cases involve adults who have other risk factors, such as the use of tobacco, long-term illnesses, and previous exposure to radiation due to radiation therapy for papillomatosis. there is a greater probability of malignant transformation with hpv 16 but also hpv 6 and 11 are capable of oncologically transforming the nature of a cell culture [66] . in surgical treatment of laryngeal papillomatisis, many techniques are used: asportation with tweezers via indirect laryngoscopy, electro-cauterization, cryosurgery, direct asportation in larynfissure, endoscopic asportation with microlaryngoscopy in suspension. currently, most surgeons prefer endoscopic laser surgery for its high precision and the haemostatic control which the technique permits [10] . more recent techniques include argon laser photo sensitization and hematoporphyrin derivatives, known as photodynamic therapy [67, 68] , and the technique with scaples and rf ("coblation") which seems to avoid the modest heat damage and the edema which results from the use of the laser [69, 70] ; the same advantage is shared by ablation with microdebrider which is also more rapid than excision by laser. that is why it is coming ever closer to replacing the co 2 laser as the ablative method of choice for use on children [71] . despite the radical nature of the treatment, relapse is the norm. this makes reiterated procedures necessary with the possibility, occasionally, of having to resort to a tracheotomy. such a procedure should be avoided for as long as is possible because of the recognized possibility of colonization on the part of the papillomas in the region of the tracheostomy and tracheobronchial tree. the risk of the tracheobronchial tree being colonized is also believed to be a consequence of the repeated intubations. the need to lengthen the amount of time between surgical asporation of the papillomas and the possibility of complete medical resolution have spurred many researchers to find adjuvant or resolving treatments. recent studies have supplied a way to identify adjuvant therapies to control papillomatosis and its relapses such as interferon-alpha [56] , acyclovir [72] , indolo-3-carbinolo [73] , retinic acid [74] , metotressato, cidofovir [75] [76] [77] [78] . even if the above therapies have sometimes significantly reduced relapses of papillomas, we believe the most effective to be intralesional injection of cidofovir associated with surgical treatment [76] . however, none of them are able to eradicate the hpv genome from the mucous cells of the respiratory tract [79] . the most promising therapies for pl are based on both therapeutic and prophylactic hpv vaccines that are currently in experiemental phases [56, 80, 81] . the epstein-barr virus (ebv) or human herpesvirus 4 is a ubiquitous gammaherpesvirus that infects more than 95% of the world's population. the most common manifestation of the primary infection of this organism is infective mononucleosis (im), a sometimes acute, but often asymptomatic clinical syndrome which more often strikes children, adolescents, and young adults [82] . it is a self-limiting lymphoproliferative illness connected to a first contact with the epstein-barr virus. the virus generally comes into contact with the mucous membranes of the oropharanx where it causes a localized primary infection from which it can circulate through the bloodstream.the period of incubation is not known, and to be the source of infection, is often misunderstood, even if it is known that it is mainly spread orally. in particular, the cells which host cells are mainly the b-lymphocytes and the cells of the human nasalpharanx are where the virus replicates itself. the b-lymphocytes transformed by ebv are the target of a multiform immune response. the immune response (production of antibodies) documents a primary ebv infection. the cellular immune response, consisting in part of the induction of an activated, postive t-lymphocyte cd8, is mostly responsible for atypical lymphocytes which is the consequence of a primary ebv infection. the virus can be found in the oropharangeal secretions of 15-25% of healthy adults who test positive for ebv. the reactivation of ebv is generally asymptomatic, the opposite of that of the herpes simplex and varicella-zoster virus. ebv is relatively labile. it has not been isolated from environmental sources and it is not very contagious. in the majority of cases, it is believed that the incubation period is 30-50 days. the virus can be spread by the transfusion of blood derivatives but is more frequently passed on by oropharangeal contact (kissing) between a non-infected subject and a healthy carrier that asymptomatically secretes the virus from the oropharynx. during early childhood, infection occurs more frequently in lower socioeconomic classes and in conditions of overcrowding [83] . ebv has also been associated with african burkitt lymphoma and some b-cell neoplasias in immunodepressed patients (especially transplant recipients, hiv or ataxia-teleangectasia patients) and to nasalpharangeal carcinoma [29, 84, 85] . these associations are based on seriologic evidence of an increased ebv activity and on proof of nuclear antigens (epstein-barr nuclear antigens, ebna) and of ebv dna found in tumor biopsies. it has been postulated that ebv places a role in some b-cell lymphomas, polyclonally stimulating and transforming the b-lymphocytes, making them more susceptible to a successive chromosome transfer to an evolution toward an oligoclonal or monoclonal lymphoproliferation. the classic symptomology of mi includes fatigue, fever, inflammation, lyphoproliferation, however, patients can also present all or only some of these symptoms. ebv infection in children is usually asymptomatic or with a light symptomology. usually patients present with an illness that has lasted several days to a week, followed by fever, inflammation, and adenopathy. fatigue is usually highest in the first 2-3 weeks. fever reaches its peak in the afternoon or early evening with a temperature of around 39.5â°c, but can even reach 40.5â°.when fatigue and fever are the dominant signs (the so-called typhoid form), the beginning and the resolution can take longer. inflammation can be serious, painful and sedating and can resemble streptococcica inflammation. lymphoadenopathy can involve almost any group of lymphnodes but is usually asymmetrical; anterior and posterior cervical adenopathy is often relevant. the enlargement of only one lymphnode or a group of lymphnodes can be the only manifestation; in these cases, studies of the heterophiles can forgo lymph nodal biopsy or help the interpretation of alarming histopathological aspects. splenomegaly, present in around 50% of cases, is at its maximum during the second and third weeks, manifesting itself through pain the upper left quadrant. slight hepatomegaly can also be present as well as a pain on the hepatic percussion. less frequent signs are malcularpapular eruptions, jaundice, periorbital edema, and palatal exanthema [40] . infective mononucleosis is usually self-limiting. the duration of the illness is variable, usually about 2 weeks, but generally 20% of patients can go back to school or work after a week, and 50% after two weeks. patients can usually begin their normal activites again after this period but sometimes the complete resolution of asthenia requires several weeks. only in 1-2% of patients does asthenia last months. the decline happens in less than 1% of all cases and is generally caused by complications of the primary ebv infection (encephalitis, rupture of the spleen, airway obstruction). generally, the diagnosis is clinical but it must always be confirmed by laboratory testing, and, in particular, identification of ebv. it should be mentioned that the tendency of a late positive score and the elevated possibility of false negatives. treatment of mi is generally supportive and consists of antipyretic and/or analgesic drugs. the use of antibiotics is controversial while therapy is underway with cortisones, which is considered by some to be routine, but considered by other aa to be exclusively reserved for the most serious cases or complications. mi is generally considered to be a benign and self-limiting illness. however, in some, rare cases, complications can arise putting the life of the young patient at risk. serious hepatic complications are those which progress toward cirrhotic forms, reye's syndrome or in extreme cases, toward duncan's syndrome, a syndrome characterized by massive hepatitis linked to the x chromosome and caused by a defect in the immune response to ebv. respiratory complications are, in general, obstructive and linked to adenotonsillar hypertrophy or to serious interstitial pneumonia. hematologic complications are particularly alarming and can lead to the bursting of the spleen as well as neurological complcations where encephalitis is the leading cause of death. in this regard, particular attention should be paid to guillain-barrã©'s syndrome which is an inflammatory, demyelinating form that can complicate infective mononucleosis and cause a progressive paralysis of the respiratory muscles or rather a more or less diffused involvement of the cranial nerves. none declared. evaluation of recurrent respiratory tract infection in children genetic evaluation guidelines for the etiologic diagnosis of congenital hearing loss temporal relationship between human parechovirus 1 infection and otitis media in young children immune-mediated inner ear desease and parvovirus b19 the patology of ribella deafness concurrent influenza a and group a beta-hemolytic streptococcal pharyngotonsillitis role of cytomegalovirus in sensorineural hearing loss of children: a case-control study tehran, iran laserchirurgia in otorinolaringoiatria syntomatic congenital cytomegalovirus infection: neonatal morbilitã  and mortalitã  the 4 ,4 -difluoro analog of 5 -noraristeromycin: a new structural prototype for possible antiviral 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papillomavirus in the oral cavity/oropharynx in a large population of children and adolescents hspe7 treatment of pediatric recurrent respiratory papillomatosis (rrp): interim results utilizing a laryngeal staging and severity scale (lsss). sentac (society for ear, nose, and throat advances in children) meeting recurrent respiratory papillomatosis laryngeal papillomas: the epidemiology in a danish subpopulation 1965-1984 recurrent respiratory papillomatosis: juvenile versus adult forms clinical and radiological features in three cases of pulmonary involvement from recurrent respiratory papillomatosis malignant transformation of recurrent respiratory papillomatosis associated with integrated human papillomavirus type 11 dna and mutation of p53 variable light-dose effect on photodynamic therapy for laryngeal papillomas treatment of recurrent respiratory papillomatosis with argon plasma coagulation radiofrequency coblation for the treatment of laryngotracheal papillomas soft-tissue 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organization clinical manifestations and quantitative analysis of virus load in taiwanese children with epstein-barr virus-associated infectious mononucleosis the merck manual of diagnosis and therapy nasopharyngeal carcinoma in tunisian children: retrospective epidemiological, clinical and biological study about 48 cases unilateral tonsillar lymphoepithelioma with ipsilateral parapharyngeal space involvement: a case report key: cord-289690-af6lsj1g authors: svobodova, tamara; mejstrikova, ester; salzer, ulrich; sukova, martina; hubacek, petr; matej, radoslav; vasakova, martina; hornofova, ludmila; dvorakova, marcela; fronkova, eva; votava, felix; freiberger, tomas; pohunek, petr; stary, jan; janda, ales title: diffuse parenchymal lung disease as first clinical manifestation of gata-2 deficiency in childhood date: 2015-02-10 journal: bmc pulm med doi: 10.1186/s12890-015-0006-2 sha: doc_id: 289690 cord_uid: af6lsj1g background: gata-2 transcription factor deficiency has recently been described in patients with a propensity towards myeloid malignancy associated with other highly variable phenotypic features: chronic leukocytopenias (dendritic cell-, monocyto-, granulocyto-, lymphocytopenia), increased susceptibility to infections, lymphatic vasculature abnormalities, and sensorineural deafness. patients often suffer from opportunistic respiratory infections; chronic pulmonary changes have been found in advanced disease. case presentation: we present a case of a 17-year-old previously healthy caucasian male who was admitted to the hospital with fever, malaise, headache, cough and dyspnea. a chest x-ray revealed bilateral interstitial infiltrates and pneumonia was diagnosed. despite prompt clinical improvement under antibiotic therapy, interstitial changes remained stable. a high resolution computer tomography showed severe diffuse parenchymal lung disease, while the patient’s pulmonary function tests were normal and he was asymptomatic. lung tissue biopsy revealed chronic reparative and resorptive reaction with organizing vasculitis. at the time of the initial presentation to the hospital, serological signs of acute infection with epstein-barr virus (ebv) were present; ebv viremia with atypical serological response persisted during two-year follow up. no other infectious agents were found. marked monocytopenia combined with b-cell lymphopenia led to a suspicion of gata-2 deficiency. diagnosis was confirmed by detection of the previously published heterozygous mutation in gata2 (c.1081 c > t, p.r361c). the patient’s brother and father were both carriers of the same genetic defect. the brother had no clinically relevant ailments despite leukocyte changes similar to the index patient. the father suffered from spondylarthritis, and apart from b-cell lymphopenia, no other changes within the leukocyte pool were seen. conclusion: we conclude that a diagnosis of gata-2 deficiency should be considered in all patients with diffuse parenchymal lung disease presenting together with leukocytopenia, namely monocyto-, dendritic celland b-lymphopenia, irrespective of severity of the clinical phenotype. genetic counseling and screening for gata2 mutations within the patient’s family should be provided as the phenotype is highly variable and carriers without apparent immunodeficiency are still in danger of developing myeloid malignancy. a prompt recognition of this rare condition helps to direct clinical treatment strategies and follow-up procedures. defects of transcription factor gata-2 have recently been identified in a few overlapping phenotypes associated with myeloid malignancies: dendritic cell, monocyte, b-and nk-cell deficiency; monomac syndrome (monocytopenia with mycobacterium avium complex infections); emberger syndrome (early onset primary lymphedema, multiple warts, sensorineural deafness, dysmorphism); and familial mds/aml with no additional known phenotype. these syndromes share autosomal-dominant inheritance with variable manifestation of immunodeficiency [1] [2] [3] [4] [5] [6] [7] [8] [9] . the respiratory tract is frequently affected by viral, fungal or mycobacterial infections. chronic lung tissue changes and pulmonary alveolar proteinosis (pap), as well as pulmonary arterial hypertension, have been described in adult patients [1, [7] [8] [9] . we present an adolescent male with gata-2 deficiency and early manifestation of diffuse parenchymal lung disease (dpld) as well as an atypical course of epstein-barr virus (ebv) infection. a 17-year-old caucasian male presented to the hospital with acute fever, malaise, headache, cough and dyspnea. a bilateral pneumonia with signs of systemic inflammation corresponding to bacterial infection (c-reactive protein 210 mg/l) was diagnosed and antibiotic treatment initiated. no causative microorganism was identified. despite rapid clinical improvement, chest x-ray showed persistent interstitial changes ( figure 1a) . a subsequent high-resolution computer tomography (hrct) revealed marked lung damage suggestive of bronchiectasis with peribronchitis, fibrotisation, subpleural cystic remodeling (honey-combing) and emphysema ( figure 1b) . interestingly, pulmonary function tests showed normal vital capacity, total lung capacity as well as diffusing capacity (table 1) . thus, we detected chronic lung figure 1 pulmonary changes in the index patient. a: diffuse bilateral linear and reticular opacities, compatible with interstitial pulmonary involvement (chest x-ray). b: diffuse subpleural fibrotic changeshoneycomb (black asterisk), areas of subpleural consolidations (blue asterisk) and bronchectasis (red asterisk) in the upper lobes (high-resolution computer tomography scan). c: chronic reparative and resorptive reaction: fibrosis and cystic rearrangement (green arrows) and cholesterol clefts (blue arrows) in the upper left lobe (hematoxylin and eosin tissue stain; original magnification 50x). d: thickened arterial wall, destruction of the elastic layer, thrombosis showing organizing vasculitis (red arrows) in the upper left lobe (elastin tissue stain; original magnification 250x). changes with no functional correlate during the first episode of pneumonia in a previously healthy boy. further investigations to unfold the cause for the diffuse parenchymal lung disease were initiated. a complete blood count showed leukocytopenia with marked monocytopenia (table 2) . immunological assays detected b-cell lymphopenia with predominance of memory b cells. despite the very low numbers of b cells, normal serum immunoglobulin levels of igm and iga and increased levels of igg were present (23.6 g/l). antibody response to routine vaccination was normal. no serum autoantibodies were found. functional testing of granulocytes (respiratory burst test: analysis of the ability of granulocytes to release reactive oxygen species after in vitro stimulation) and of t cells (evaluation of proliferative response of t cells to various in vitro stimuli) excluded chronic granulomatous disease and t-cell proliferation defects. serology corresponded with primary ebv infection (table 3) . however, the ebv viral load in peripheral blood was low. bronchoalveolar lavage (bal) showed ebv presence in the bronchial fluid. immunological analysis of the bal fluid showed lymphocytosis with predominance of cd8 pos with increased hla-dr expression (especially on cd3 pos 8 pos ); alveolar macrophages were present, cd1a pos cells were not detected. no pas (periodic acid-schiff) positive material was evident in the alveolar macrophages (table 4) . neither bacterial, fungal, mycobacterial (including nontuberculous mycobacteria) nor viral (cytomegalovirus, human herpes virus 6, varicella zoster virus, human herpes virus, respiratory syntitial virus, influenza, adenovirus, enterovirus, coronavirus, parainfluenza, human rhinovirus, human metapneumovirus, bokavirus and papillomavirus tested) infection was revealed via culture, serology or molecular genetic testing in peripheral blood and bronchoalveolar fluid. hence, the extensive microbiological analysis revealed only the presence of ebv in peripheral blood and lungs. histopathological investigation of the lung parenchyma was prompted. thoracoscopic lung biopsy from a severely affected region of the right upper lobe showed fibrosis, cystic rearrangement and cholesterol clefts with signs of organizing pneumonia and vasculitis (figure 1 c, d) . inflammatory infiltration was predominantly lymphoplasmocytic with presence of activated macrophages. no changes compatible with pulmonary alveolar proteinosis or other alveolar filling disorder were seen. despite an ebv presence (2300 copies/10.000 genomic equivalents, g.e.) in the lung tissue found with polymerase chain reaction, hybridization probes for ebv-encoded small rna (eber) were negative in the histology slides. thus, no clear relationship between ebv and the histopathological parenchymal changes could be stated. given the severe affliction of the lung parenchyma with fibrotic remodeling, ongoing inflammation with activated cd8 pos t cells in the bronchoalveolar fluid and lack of clear evidence for an infectious cause, a treatment with an oral steroid was initiated to suppress further tissue destruction. a prophylactic antibiotic (azithromycin) was added and the patient was closely monitored. immunoglobulin levels normalized and signs of systemic inflammation regressed. after 6 months a stable finding was documented via hrct and no clinical symptoms were present. the ebv viral load remained low in peripheral blood. minimal presence of the virus was seen in repeated bal. however, the serological signs of active ebv infection persisted and no ebna antibodies were detected at the follow up. no lymphoproliferation was present and the patient remained asymptomatic. the condition was classified as persistent ebv viremia accompanied by an atypical serological response. molecular genetic testing of sh2d1a was carried out. a normal result excluded x-linked lymphoproliferative disease, the most common inborn cause of abnormal immunological reaction to ebv infection. details on other possible genetic causes, not yet tested in our patient, are in the discussion. the patient has a history of occasional uncomplicated respiratory infections; at the age of 13 years he suffered from acute bronchitis, a chest x-ray was performed and retrospective analysis of the image showed some interstitial changes present already at that time. monocytopenia was documented as early as at the age of 10 years. the persistent profound monocytopenia and b-lymphocytopenia at follow up prompted gata2 sequencing. the diagnosis of gata-2 deficiency was confirmed by the finding of a known heterozygous pathogenic variation c.1081 c > t (p.r361c) [10] . myeloid malignancy was excluded by morphological, flow cytometric and cytogenetic analysis of the bone marrow aspirate. detailed immunophenotypic analysis of the bone marrow showed suppression of cd34 pos and cd117 pos precursors; impairment of b-cell lineage (only 1.4% b cells were present, out of those plasma cells constituted 42% and mature cd20 pos cd10 neg cells 37%, the precursors cd34 pos cd10 pos were scarce) and lower percentage of monocytes as well as their progenitors (cd14 high cd45 pos ssc med ). additional testing showed lack of myeloid and plasmacytoid dendritic cells. the immunosuppressive treatment was stopped and the patient was further treated with prophylactic antibiotics and antimycotics. vaccination against human papillomavirus (hpv) was performed as recommended [9] . he has been monitored closely, including regular checks of bone marrow aspirate for early detection of clonal myeloid proliferation. in case of myelodysplasia, transplantation of hematopoietic stem cells would be initiated. after two years of follow-up the patient did not develop any clinical symptoms. he was treated once for pseudomonas aeruginosa found in the bronchoalveolar fluid detected in the second bal analysis performed 6 months after the first one. otherwise, there were no clinical signs of increased susceptibility to infection. pulmonary function tests remained normal, no progression of the pulmonary parenchyma affliction have been detected so far (tables 1, 2 and 3) . the same heterozygous mutation in gata2 was found in the patient's 13-year-old brother and 45 year-old father, whereas his mother was healthy. the brother had been without any clinical symptoms so far, blood tests revealed leukocytopenia and marked monocytopenia. hrct scan showed normal parenchyma, no ebv activity was documented. the father suffered from bilateral ankylosing spondylitis (hla-b27 positive). apart from low b-cell numbers (2.4% cd19 pos cells of lymphocytes, with prevailing memory phenotype: 70% cd27 pos cells out of b cells; norm < 47%) no leukocyte count abnormalities were detected. the lymphocyte changes in the three family members carrying the gata2 mutation stimulated investigation of bone marrow output. newly emerging t and b cells can be assessed via t-cell recombination circle (trec) and kappadeleting element recombination circle (krec) analysis in the peripheral blood [11, 12] . as expected, both siblings had no detectable krec copies in the peripheral blood, indicating severe impairment of b cell development. trec analysis showed normal results. bone marrow examination of the younger brother also showed complete negativity of krec with normal trec copies. krec copies were absent in the peripheral blood of the father as well. the krec/trec copies were normal in the unaffected mother. interestingly, dna obtained from the newborn guthrie card of the younger brother was analyzed showing a normal amount of krec/trec copies. this indicates that the impairment in b lymphocyte development occurred postnatally. gata-2 deficiency is a protean disease with a broad spectrum of symptoms. most of the patients present with hematological abnormalities (cytopenias, early-onset myeloid malignancies) and an increased susceptibility to opportunistic infections [1] [2] [3] [4] [5] [6] [7] [8] [9] . in the recently published cohort of 57 patients treated at the national institute of health (nih, bethesda, usa) [9] 70% of the patients had severe viral infections, particularly infection with hpv (63%) presenting with recalcitrant warts, condylomata, and/or dysplasia. severe herpesvirus infections were present in 35% of patients: recurrent herpes stomatitis, esophagitis, genital infection, severe varicella in 11% of cases, and cytomegalovirus pneumonia or disseminated disease. interestingly, in 11% of patients persistent ebv viremia similar to our patient was documented; in 2 patients ebv-positive skin tumors occurred. infection with non-tuberculous mycobacteria was seen in 53%, severe bacterial infection was observed in 49% and severe invasive fungal infection in 16% of the patients. eighteen percent of patients showed no increased susceptibility to infection. additionally, vascular/lymphatic defects (venous thrombosis, lymphedema), sensorineural hearing loss, miscarriages and hypothyroidism were found [1] [2] [3] [4] [5] [6] [7] [8] [9] . pulmonary involvement in gata-2 deficiency is frequent, involving infections and pap [1, 3, 8, 9] , particularly in more advanced stages of the disease. in the nih cohort, 79% and 63% of the patients had diffusion and ventilatory defects, respectively. pap was found in 18% and pulmonary arterial hypertension in 9% of patients. structural abnormalities included nodules, reticular and ground glass opacities, subpleural blebbing, "crazy paving" , and paraseptal emphysema [9] . similar picture could be seen in our patient. the surprisingly normal pulmonary function test results in our patient could possibly be explained by localized affliction of the pulmonary tissue. the infiltrated and fibrotic tissue decreased the elasticity of the lung parenchyma, however, there was still enough normal tissue that kept the static volumes and transfer factor normal (table 1) . unfortunately, it is not possible to compare our findings with other pediatric patients as the data on pulmonary infliction in children are scarce. there were 24 children in the nih cohort. data on the pulmonary function tests were presented for only 6 of them (median age at testing 16 years, range 12-17 years; median time from disease manifestation 2.5 years, range 0-16 years). all those children suffered from myeloid malignancy, in four of them a chronic infection with herpesviruses or mycobacteria species was documented. mild to severe diffusion defects were found in all tested patients, in two children a bronchial obstruction was seen. no information on structural lung changes in the affected children was provided [9] . the median age at initial presentation in the nih cohort was 20 years but was highly variable (range 5 months -78 years). of note, four individuals (7%) had no apparent clinical manifestations as of the last follow-up (range 5-55 years). the proportion of patients without symptoms was 50% by age 20, 25% by age 30, and 16% by age 40 irrespective of the type of genetic change in gata2. the phenotype varied within families significantly [9] . this fact strongly argues for a substantial impact of epigenetic, infectious and environmental factors on disease manifestation. effects of germline or somatic mutations in other genes may play a role as well. this may explain the variability of symptoms in the three individuals carrying the same gata2 mutation within our index family. an intriguing issue is the etiology of the chronic diffuse parenchymal lung tissue changes in our patient in the absence of respiratory symptoms. the extensive investigations revealed only ebv presence in peripheral blood as well as in pulmonary tissue without specific tissue changes or clinically apparent ebv infection (e.g. mononucleosis-like symptoms, lymphoproliferation). as the patient presented with serological signs of acute ebv infection whereas the pulmonary changes were chronic, a decisive role of ebv in the pathogenesis of the pulmonary tissue changes in our patient was improbable. possibly repeated mild infections in an environment of impaired regulation of the endothelial nitric oxide synthetase expression [13] , defective phagocytosis and impaired gm-csf signaling in pulmonary macrophages [14, 15] played a role in the pathogenesis of the chronic pulmonary inflammatory changes. poor control of ebv replication resulting in persistent ebv viremia irrespective of lung involvement is a known phenomenon in gata-2 deficient patients [1, [7] [8] [9] . it has been shown that the inability to confine viral infections in patients with gata-2 deficiency correlates well with the extent of cytopenias, namely with the lack of dc-, nk-and cd4 pos t-cells. similarly, the defective antibody response at more advanced stages is associated with b cell lymphopenia [8, 9] . however, with regards to the possible oligogenic etiology of immunodeficiency in gata-2 deficiency, impact of other genes implicated in ebv control should be considered. we have excluded only the most common syndrome -xlinked lymphoproliferative disease type 1 caused by a defect in an adapter protein sap, involved in signalling of cell-cell interactions. other molecules implicated in ebv control encompass for example: the ubiquitously expressed xiap with both antiapoptotic function and multiple signalling pathway connections; the surface molecule cd27, important for intercellular communication; the nk cell activating receptor for antibody-dependent cell cytotoxicity (cd16), and minichromosome maintenance 4 (mcm4) crucial for nk-cell function; or il-2-inducible t-cell kinase (itk), coronin1a, serine-threonine kinase (stk)4 and magnesium transporter, magt1, indispensable for t-cell receptor signalling and tcell homeostasis [16] . the search for a gata-2 defect in our patient was prompted by the abnormalities in the leukocyte and lymphocyte counts. another serum marker useful in diagnostics as well as in monitoring of the disease progression (correlating with cytopenia) is the stem cell growth fmsrelated tyrosine kinase 3 ligand (flt3 ligand) [8] . we have shown that the newborn screening using krec/trec analysis [17] cannot be used to screen for gata-2 deficiency. the prognosis of individuals with gata2 mutations is difficult to establish due to high clinical variability, incomplete penetrance and lack of close phenotype-genotype correlation data [8, 9, 18] . antibiotics (e.g. azithromycin) and hpv vaccination are the recommended prophylactic measures [9] . use of steroids or other immunosuppressive therapy is not indicated and exclusion of immunodeficiency in dpld prior to use is warranted. a large proportion of patients will develop myeloid malignancy later in life [1, [7] [8] [9] . the only curative therapy is allogeneic hematopoietic stem cell transplantation. two patients with gata-2 deficiency with pulmonary involvement transplanted for advanced mds were reported to have profited significantly from this procedure [7] . diffuse parenchymal lung diseases are a heterogeneous group of disorders with an often insidious onset of symptoms [19] . the underlying immunodeficiency may not be apparent and an immunological and genetic work-up is required, in particular if abnormalities in peripheral leukocyte counts are revealed. as demonstrated in our patient, an aberrant immune response to common respiratory infections may result in diffuse lung disease with bronchial and bronchiolar damage, significant chronic changes of pulmonary parenchyma and fibrotic remodeling. the structural changes might be present prior to any severe infection. diffuse parenchymal lung disease may become the first manifestation of the gata-2 deficiency. early genetic diagnosis is critical to direct clinical management, prophylaxis, transplantation, and family screening. autosomal dominant and sporadic monocytopenia with susceptibility to mycobacteria, fungi, papillomaviruses, and myelodysplasia mutations in gata2 are associated with the autosomal dominant and sporadic monocytopenia and mycobacterial infection (monomac) syndrome the human syndrome of dendritic cell, monocyte, b and nk lymphoid deficiency mutations in gata2 cause primary lymphedema associated with a predisposition to acute myeloid leukemia (emberger syndrome) high frequency of gata2 mutations in patients with mild chronic neutropenia evolving to monomac syndrome, myelodysplasia, and acute myeloid leukemia gata2 haploinsufficiency caused by mutations in a conserved intronic element leads to monomac syndrome successful allogeneic hematopoietic stem cell transplantation for gata2 deficiency the evolution of cellular deficiency in gata2 mutation gata2 deficiency: a protean disorder of hematopoiesis, lymphatics, and immunity gata2 haploinsufficiency caused by mutations in a conserved intronic element leads to monomac syndrome factors affecting thymic function after allogeneic hematopoietic stem cell transplantation replication history of b lymphocytes reveals homeostatic proliferation and extensive antigen-induced b cell expansion molecular basis of cell-specific endothelial nitric-oxide synthase expression in airway epithelium a modular enhancer is differentially regulated by gata and nfat elements that direct different tissue-specific patterns of nucleosome positioning and inducible chromatin remodeling effect of transcription factor gata-2 on phagocytic activity of alveolar macrophages from pneumocystis carinii-infected hosts cellular immune controls over epstein-barr virus infection: new lessons from the clinic and the laboratory neonatal screening for severe primary immunodeficiency diseases using highthroughput triplex real-time pcr highly variable clinical manifestations in a large family with a novel gata2 mutation interstitial lung diseases in children written informed consent was obtained from the patient and the family for publication of this case report and any accompanying images. a copy of written consent is available for review by the editor of this journal. the authors declare that they have no competing interests.authors' contributions ts identified the patient and drafted the manuscript. em acquired and analysed the flow cytometry data and revised the manuscript. us and tf acquired and analysed the genetic data and revised the manuscript. ms analysed and interpreted the clinical and laboratory data and revised the manuscript. ph acquired and analysed the virology data and revised the manuscript. rm, mv and lh acquired and analysed data on pulmonary tissue pathology and revised the manuscript. md acquired and analysed the radiology data and revised the manuscript. ef and fv analysed the krec/trec data and revised the manuscript. pp acquired and analysed the pulmonary function tests and revised the manuscript. js provided supervision, analysed the clinical data and revised the manuscript. aj is the corresponding author, he initiated the study and wrote the manuscript. all authors read and approved the manuscript. key: cord-018017-c8myq6bi authors: iversen, patrick l. title: the threat from viruses date: 2018-09-30 journal: molecular basis of resilience doi: 10.1007/978-3-319-98164-2_3 sha: doc_id: 18017 cord_uid: c8myq6bi infectious disease represent the most significant threat to human health. significant geologic cataclysmic events have caused the extinction of countless species, but these “wrath of god” events predate the emergence of homo sapiens. pandemic infections have accompanied the rise of human civilization frequently re-occurring leaving a lasting imprint on human history punctuated by profound loss of life. emerging infections become endemic and are here to stay marking their presence with an annual death toll. each decade brings a new onslaught of emerging infectious agents. we are surprised again and again but are never prepared. the long-term consequences often remain unrecognized and are always inconvenient including cancer, cardiovascular disease and immune associated diseases that threaten our health. reliance on clusters of clinical symptoms in the face of diverse and non-descriptive viral infection symptoms is a foolhardy form of crisis management. viral success is based on rapid replication resulting in large numbers. single-stranded rna viruses with their high replication error rate represent a paradigm for resilience. contemplating recent career paths, i reviewed a broad range of scientific questions. most prominent was, why study an area of science that has no significant impact? in fact, why study anything that is not the most highly impactful area? this question demands a definition, what constitutes high impact? i decided to create a definition that impact and threat to life are related. further, threat to human life is the highest impact and it is likely that things threatening human life may also threaten all life. what presents the greatest threat to human life today? history should provide critical insights to answer this question. a comprehensive look would seek causes of mass extinctions over the past 3.5 billion years of life on earth. this perspective has been a trending subject in science with focus on five mass extinctions. there are inherent biases in focusing on mass extinctions such as these events fail to appreciate small living things such as single celled organisms or bacteria. another bias is in order to appreciate life in the past, the life form must occasionally produce a fossil when it dies. those concerns aside-geologic events have threatened the survival of living things (table 3 .1). the actual events causing these extinctions are frequently debated, for example, the asteroid impact 66 million years ago off the coast of mexico was accompanied by a massive tsunami that was responsible for mass extinctions in montana, the impact may have also triggered an enormous volcano in india resulting in ash and lava flow responsible for regional species extinctions, and the collective dust resulted in prolonged climate change which was probably responsible for even more species extinctions. the lesson remains the same, volcanic eruptions; climate change and asteroid impact threaten life in a highly significant way and are responsible for extensive selection pressure. unfortunately, these events are unpredictable and so enormous little can be done about them. a mass extinction may not represent a legitimate threat or selection pressure because large numbers of species are completely eradicated leaving few survivors to drive evolution. finally, outside of removing dinosaurs so that mammals could thrive, what impact did these events have on human life; humans were not yet on the earth when these events took place. refining the search to dramatic changes in human populations moves the period up to the last 100,000 years. mass migrations took place about every 23,000 years, which is in line with the precession of the earth leading to a possible linkage between earth's precession and human migration. this may have been due to regional climate change like ice ages in the northern hemisphere. however, human population changes on this timescale are not easily documented so estimates of climate and human population dynamics will be limited. time to refine the search. consider the exponential growth, outbreak, of tent caterpillars, malacosoma disstria, in montana reported by david quammen in his book spillover (quammen 2012) . the extensive caterpillar population consumed all of the leaves from the local elm and cottonwood trees in the summer of 1993. the caterpillar activity produced a crackle sound, "like a distant brushfire." the city attacked these insects with a broad arsenal of modern countermeasures but to no effect. however, a nuclear polyhedrosis virus (npv), uses the caterpillar host density like a critical mass in a nuclear weapon to explode destroying the caterpillars in an epic battle. the caterpillar population retreated to undetectable levels in a single season as a result of npv. human populations changed over the past 20,000 years with particular emphasis on the most recent 7000 years. a human population curve over this segment of time shows an exponential growth period interrupted in the middle ages by the plague. hypothesis: the plague and other pandemic infectious disease events appear to be the greatest threat to human life. infectious disease kills 15 million people each year, 26 percent of the total 57 million annual deaths in the global population (fauci and morens 2012) . a brief review of pandemic infections over the past 3000 years illuminates several events that reduced human populations. the plague of athens 430 bce killed 25 percent of the human population (table 3 .2). both bacterial and viral pandemic yersinia pestis is a bacterium causing plague. fleas can be infected with y pestis which transmit the bacterium to rodents, the primary hosts. changes in the environment may lead to the movement of rats into populated areas where humans become infected. homer points to such an infection in the iliad in his description of the trojan war in 1190 bce. plague has returned several times since the trojan war imposing enormous loss of human life (table 3 .2). the most recent plague epidemic killed over ten million people in india in the early 20th century. y pestis is still out there ready for favorable conditions to pounce on human populations but outcomes are likely to be less dramatic due to understanding of sanitation practices, quarantine, and availability of antibiotics. if infections are the greatest threat to human life, they should be critical drivers of evolution? clearly infections pose selection pressure on the human populations. origins of evolutionary thought did not include infection as darwin established key evolution concepts on the galapagos islands. these islands are isolated and an unlikely place for the spread of infections. the concepts speciation point to geographical separation of populations so infections would most likely be restricted to isolated populations. in many cases the survival selection pressure is not identified or ascribed to insufficient sources of food. unfortunately, common single-stranded rna viruses are so unstable that there are limited data for a viral fossil record. pandemic infections remain a threat to human survival in the presence of the information revolution, daily medical breakthroughs, and global travel. the human retrovirus hiv currently a global infection that infects up to 25% of the population in southern and eastern africa with a projected death toll of up to 100 million by 2025. measles killed 200 million people in the last 150 years and the development of an effective vaccine in 1963 reduced concerns for this infection but there were 777,000 deaths in the year 2000. vaccination programs are frequently disrupted due to complacence resulting from vaccine success, conflicts that shift healthcare focus, and social crisis such as the recent ebola outbreak in west africa. smallpox is also an ancient infection causing fever, skin lesions, and at times death. king ramses v of egypt is thought to have died from smallpox around 1200 bce. introduced into mexico in 1520, smallpox killed 3.5 million aztec indians or about half of the population in a period of 2 years and then proceeded to decimate the population of south america. variola is a highly infectious virus killing 300-500 million people during the 20th century inspiring the eradication campaign in 1967. variola was eradicated by december of 1979 (de cock 2001 , a rare triumph of public health. the who deserves acknowledgment for this unprecedented accomplishment and proof of concept that human suffering is not inevita-ble. however, variola is a dna virus with limited rate of mutation and a narrow host range so that animal reservoirs do not exist. the eradication of other viral infections will be more challenging. the world continues to confront a broad array of microbial threats. progress and preparedness make our engagement a likely success for those microbes that resurface and infections for which we have experience. medical and epidemiological uncertainties surround emerging infectious disease, those that challenge us with their novelty. pandemics dominate the infectious disease "fear factor" but each pandemic began as a much more frequent occurrence, an epidemic. most but not all epidemics come from emerging infectious agents, the most significant problem facing life on earth today. the concept of emerging is a human centric term as most of these infections are endemic in an animal host that serves as a viral reservoir. a 2005 report from the university of edinburgh identified 1407 human pathogens and 177 are emerging or re-emerging of which 75% are zoonotic, that is jump from an animal host to human. numerous emerging infections caused by viral agents have imposed high impact on human survival (table 3 .3). all the viral agents in table 3 .3 have genomes based on single-strands of rna except hbv which should focus scientific attention on rna. there are numerous questions that strike investigators as they ponder a collection of viral agents like those in table 3 .3. the viral polymerase errors in replicating single-stranded rna genomes are not corrected so the species are constantly changing. the apparent success of these viruses is that as they move from reservoir hosts to humans and as humans become immune to the initial infection, the population of diverse genomes offers multiple chances to adapt by finding a "fit" genome version which can propagate until the next transition requiring adaption. acquired immunodeficiency syndrome (aids) is caused by human immunodeficiency virus 1 (hiv-1), a retrovirus. these viruses have a single-stranded rna genome that is converted into dna, a paradigm shift in the flow of genetic information from dna to rna. the 36,000,000 human deaths caused by hiv-1 (table 3.3) is accompanied by a spectrum of clinical signs; eg. fever, diarrhea, peripheral neuropathy, pelvic inflammatory disease, cervical cancer, cytomegalovirus retinitis, kaposi's sarcoma, lymphoma, mycobacterium avium infection, recurrent pneumonia, and wasting syndrome. hiv-1 genome diversity includes base substitution, insertion, deletion, recombination, and gain or loss of glycosylation sites which all arises from the limited fidelity of the viral reverse transcriptase. these mutations are found in clusters or hypervariable regions indicating the fit virus is selected for from vast numbers of less-fit genome sequences. hiv-1 emphasizes key observations: (1) eid is a significant contemporary concern, (2) we are not prepared for the novel characteristics introduced by eid, (3) clinical signs can be diverse and often mimic symptoms of other diseases, and (4) the replication mechanisms are often error prone resulting in an array of fit viruses. dengue is a flavivirus with a positive sense single-stranded rna (+ssrna) genome carried by mosquitos to man. dengue has been a tropical disease for hundreds of years, typically a disease of young children but in 1953 an emerging severity was recognized in manila (table 3 .3), hemorrhagic fever (dhf) and dengue shock syndrome (dss). more than 2 billion people are at risk of dengue infection but only a small fraction of those infected will develop dhf or dhs. infected people develop antibodies that can lead to antibody-dependent enhancement (ade) in subsequent infections and more severe dhf or dss outcomes. it appears ade events occur in people that produce immunoglobins (iggs) with enhanced affinity to the activating fc receptor due to the igg1 subclass and lack of a fucose glycan modification of the igg (wang et al. 2017) aedes aegypti is the primary vector transmitting dengue but a new vector, aedes albopictus, now carries dengue to the southern united states. emergence of dengue in the southern united states is likely due to used tires imported from japan which provided a place for the asian tiger mosquito to live. outbreaks of dengue fever have become more numerous and more severe over the past three decades. viral "fitness" is constrained by the requirements imposed by the natural host so that it is a low probability event for a virus to move from the natural host to a human. while an insect frequently plays the role of vector carrying a virus from an animal reservoir to humans, several zoonotic viruses are transmitted by placing humans near rodents. several arenaviruses, minus sense single-stranded rna viruses, jump from their rodent natural hosts directly to humans through contact with rodent urine or saliva. notable arenaviruses are named for the hemorrhagic fever (hf) region of their zoonosis; bolivian hf is caused by machupo (macv), argentine hf is caused by junin (junv) and venezuelan hf is caused by guanarito (gotv). these south american outbreaks are caused by new world arenaviruses in contrast to lassa hf (lasv), an african or old world arenavirus. lasv is an endemic disease of west africa (table 3 .3) and can be confused with dhf or ebola infections. viruses that are transmitted by arthropod vectors are called arboviruses. in 1930, only yellow fever of six known arboviruses caused disease in humans. the discovery of arbovirus caused human disease expanded beginning in the late 1930s with western and eastern equine encephalomyelitis (weev, eeev) and st. louis encephalitis. both chickungunya and zika (table 3 .4) are arboviruses that have emerged to become global infections in the twenty-first century. zika not only became infamous for causing microcephaly in newborns of infected mothers and guillain-barre syndrome but is now sexually transmitted between humans. i worked on a therapeutic for the treatment of ebola infections from 2007 to 2013. the genome sequence recovered from each outbreak from 1976 to 2013 has been different. studies were conducted at usamriid in their bsl4 facility by expert ebola investigators. rhesus monkeys (macaca mulatta) were injected with 1000 plaque forming units (pfu) of zaire ebolavirus (zebov) kikwit into their thigh muscle. all of the untreated control monkeys died between days 8 and 10 following viral injection. we measured viral burden by quantitative polymerase chain reaction (qrt pcr) a measure of genome copies per milliliter (copies/ml) of plasma and plaque formation a measure of infectious viruses per ml (pfu/ml) plasma. we found an average of 1.7x10 8 genome copies per ml on day 8 post infection and 1.2x10 6 pfu/ml on day 8. nearly 100 genomes present for each successful virus in a nonhuvwas not observed, the dominant population of viral genome sequence did not change from one individual to another or from one time to another within the same individual. the existence of defective viral genomes may offer potential for rapid change but in a stable host, change was not rapid. when one considers the magnitude of differences in conditions as a virus jumps from a reservoir host to a human, rapid adaptability is a great advantage. a virus that kills all the hosts is not a successful virus. perhaps defective viral particles facilitate host immune responses giving them a chance to catch up to the rapidly proliferating virus. the ebola outbreak of 2014 illuminated the collateral damage that accompanies eid outbreaks. first, the first responders are local physicians and care givers are killed leaving a population lacking doctors and nurses. even physicians trained to exercise appropriate caution when interacting with patients such as dr. sheik humarr khan died of the ebola infection (bausch et al. 2014) . second, women are the main caregivers in their families and 75 percent of ebola deaths in the 2014 outbreak were women. this leaves families lacking traditional structure. third, the outbreak disrupts production, labor markets and trade resulting in scarcity and inflation of food prices. food security and nutrition are diminished which preferentially affects poor people as they spend 50 to 70% of their income on food. finally, public health measures are discontinued. before the 2014 outbreak, 97 percent of children in west africa were receiving routine vaccinations but that figure fell below 27 percent. the west africa loss of measles vaccinations was followed by measles out coronaviruses are unique in their large single-stranded rna genome (20,000 bases) and are often associated with mild disease, bronchitis and gastroenteritis. a 2003 outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in china rapidly spread to 8098 cases in 37 countries (table 3 .4). retrospective analysis of this outbreak linked zoonosis to a culinary trend in which people sought out exotic animals to eat. these nontraditional food animals appeared in street markets in urban areas bringing people close to the reservoir host triggering the outbreak. the civet cat was identified as the source of the first human cases but this is not the natural host. the natural host is the fruit bat, which infects a variety of small mammals including the civet cat. a point of intervention has been removal of the civet cat from markets but the natural bat host remains in the environment maintaining the virus for the next outbreak. a second coronavirus outbreak in 2014 of middle east respiratory syndrome coronavirus (mers cov) rapidly involved 23 countries including africa, western europe, and southeast asia. the number of cases is relatively low but the case fatality ratio is of great concern (table 3.4). the camel is reservoir host in this case but it may not be the natural host since camels often get sick as well. transition from animal-to-human transmission to efficient human-to-human transmission was observed quickly in the case of both sars-cov and mers-cov. this rapid adaptability of a highly infectious virus group should raise concern over endemic coronaviruses in companion animals since these viruses can clearly become pathogenic, can jump from animal to human and then become transmitted from human-to-human. an ongoing outbreak of yellow fever in africa (table 3 .4) is a striking reminder that eid can re-emerge into virtually naïve populations. some people recover from the acute symptoms but liver damage causes yellow skin, the reason for the name for the disease. the liver damage leads to bleeding and kidney damage and occasionally death. yellow fever is a flavivirus that originated in africa but was distributed to the world on barges and sailing ships to tropical ports. the virus arrived in the americas on slave ships. this virus interrupted the building of the panama canal which attracted the attention of walter reed, the military physician responsible for demonstrating transmission by mosquitos. an effective vaccine is used worldwide with immunity lasting over 10 years. the limitation of this vaccine like most is that populations need to be vaccinated regularly which in a world constantly changing due to political and economic drivers leads to vaccination gaps. cancer is one of the leading causes of human death but estimates are that 20 percent of all human cancers are directly caused by viruses (table 3 .5) (morales-sanchez and fuentes-panana 2014). the first tumor virus was identified by peyton rous in 1911. he took cell lysates from a chicken sarcoma which he passed through a filter known to hold back bacteria and then injected the filtered lysate into chickens. a tumor developed at the injection site. the virus is now known as rous sarcoma virus (rsv), a single-stranded rna virus. rous won a nobel prize in physiology or medicine for the discovery in 1966. rsv is a retrovirus that captured a tyrosine kinase, src gene, that triggers uncontrolled growth in infected host cells. the avian leukemia virus (alv) was discovered in denmark early in the twentieth century capable of causing disease in blood forming tissues of chickens. a strain of alv, avian myeloblastosis virus (amv) was described in 1952 that provided a convenient source of tissue for biochemical studies. during the 1930s a strain of inbred mice was found to develop leukemia between 6 and 18 months of (burkitt's lymphoma 2014) . after that he saw a second case at the jinja district hospital on the shores of lake victoria this became more than a curiosity. a review of hospital case notes confirmed the prevalence of these tumors of the jaw and they were accompanied by swellings in kidneys, ovaries, adrenal glands and liver. he assembled data from 38 patients and histological examination led to description of "lymphoma syndrome" or the "african lymphoma." burkitt received a grant in 1961 for ₤250 to visit 57 hospitals in eight african countries; uganda, kenya, tanzania, malawi, mozambique, zimbabwe, zambia, and republic of south africa to investigate african lymphoma. the tumor was found everywhere at the equator in areas where year-round temperature was above 60 °c but not in areas over 5000 feet in elevation. burkitt further determined the tumors only occurred where the annual rainfall was above 20 inches and not seen in the dry savannah of nigeria. burkitt contacted doctors in papua new guinea to discover these tumors were the most common childhood tumor in that country but only in wet coastal regions and not in the dry highland areas. burkitt's working hypothesis was that an insect transmitted virus infection was responsible for the lymphoma syndrome. he then observed adults with the lymphoma but only in individuals that moved into the "african lymphoma belt." these tumors were then observed in the united states and europe at a rate of 1-3 per million or about 100x less frequently than in africa. biopsy samples did produce viruses but none that came from insects so the working hypothesis was abandoned. a new hypothesis emerged between 1963 and 1966 in which p falciparum (a parasite causing malaria) was the causative agent for the lymphoma. it is believed that severe malaria does play a role in the development of burkitt's lymphoma. at the advice of peter clifford, burkitt administered methotrexate to treat patients which produced encouraging results. in 1961 burkitt gave a lecture at middlesex hospital, "the commonest children's cancer in tropical africa: a hitherto unrecognized syndrome." anthony epstein attended the lecture which changed his life and provides a punctuation mark in the history of science. epstein introduced himself after the lecture and the two sat down for tea. burkitt agreed to send tumor specimens to epstein and epstein received a grant from the us national institutes of health in 1963 for $45,000 which allowed him to employ two research assistants. yvonne barr was one of the research assistants that was able to propagate a virus after 26 tries with tumor specimens. the other research assistant was bert achong, an electron microscopist, who was first to identify the virus as a member of the herpes virus family. the virus bears the name epstein barr virus or ebv and is a causative agent of burkitt's lymphoma. an interesting historical note involves werner and gertrude henle at the children's hospital of philadelphia. they created antibodies to ebv infected cells and their survey of blood samples from american adults revealed over 90% to be ebv positive in 1966. they also showed that normal human lymphocytes could be made immortal by infecting them with ebv in 1967. we now know ebv silently infects children in the western world but in those infected a bit later in life become susceptible to infectious mononucleosis (the kissing disease) as adolescents. in 1972, a collaboration between george klein and the manolovs led to a discovery of a chromosome change that was specific to burkitt's lymphoma. this chromosome change was a translocation of 8:14 and rarely 8:2 or 8:22 (zech et al. 1976 ). the break points in chromosomes 2, 14, and 22 contained immunoglobin genes that become active in b lymphocytes as they produce antibodies during infection. a nobel prize in 1989 to michael bishop and harold varmus provided the significance to the break point in chromosome 8, bringing active immunoglobin genes near the oncogene c-myc. in 1970, a second cancer common in north and east africa as well as southern china was found to be ebv positive, carcinoma of the post-nasal space (nasal pharyngeal carcinoma or npc). the tumor is observed in epithelial cells not b-cells and is not geographically linked to endemic malaria regions. in this case, it appears that aerosol exposure to environmental carcinogens (possibly in preserved fish) may introduce mutations in nasal epithelial cells. the npc may be the result of silent ebv infection, carcinogen induced cellular mutations, and people carrying hla a*0207 and b*46 antigens. in 1975, several reports describing "fatal infectious mononucleosis" appeared. david purtilo described x-linked proliferative syndrome (xlp) based on an international registry of boys with fatal ebv infections (purtilo et al. 1977) . using genetic marker analysis from the international registry they narrowed xlp to a three million base pair segment on the x-chromosome. in 1998, a 384-base pair segment encoding the xlp protein of 128 amino acids was found to be lost or damaged in al xlp patients (nichols et al. 1998) . curiously, the xlp protein loss leads to a defect in nk and t lymphocytes which are necessary for recognition of ebv infected b lymphocytes. these individuals cannot make antibodies to ebv because their b cells require help from t lymphocytes. the development of potent immune suppressing drugs like cyclosporine a was linked to lymphoma after organ transplantation. two girls with acute lymphoblastic leukemia received bone marrow transplants from their hla-match brothers. the grafts were successful but they developed lymphoblastic leukemia from the transplant (male cells). these lymphomas do not present chromosomal translocation like burkitt lymphomas revealing a new path from ebv to lymphoma. the immune suppression upsets the ebv host homeostasis crippling t-cells that are required for keeping ebv under control. another situation where the virus can replicate without control is in hiv/aids patients and aids-lymphoma is yet another ebv induced casualty. ebv provides key insights into tumors associated with viral infections. first, the same virus is linked to multiple tumors in different populations including burkitt's lymphoma, hodgkin lymphoma, and nasopharyngeal carcinoma. second, tumorigenesis involves more than one mechanistic pathway but interest has focused on the latent membrane protein-1 (lmp1), ebv nuclear antigen 1 (ebna1), and bamh1-a reading frame-1 (barf1) genes. third, dissecting the association between infection with ebv and cancer has unfolded over a period of 30 years and led to a greater understanding of cancer, the immune system, and methods for immortalizing cells to study specific cell clonal populations. ebv has been a boon to scientific research while imposing a challenge to human survival. approximately 2 billion people have been infected with hbv and 360 million suffer from chronic infection. acute hbv infections are usually self-limiting associated with 5.2 million cases a year. chronic hbv infections lead to complications in 15-40 percent of cases resulting in 0.5 to 1.2 million deaths each year. hb v causes 60-80 percent of the world hcc cases which represents 5 percent of all cancers in the world. the hbx gene is considered key to oncogenesis. vaccination programs are effective in reducing mortality in infants and have reduced emerging prevalence of the disease. dietary exposure to aflatoxin enhances hbvrelated hcc (sun et al. 1999 ) as well as co-infection with hcv and excessive alcohol consumption. hepatitis b virus (hbv) was named due to differences in transmission: hepatitis a type virus follows fecal-oral transmission while b type virus is parenteral. the genome is circular dna that is not fully double-stranded with the end of the fulllength strand linked to dna polymerase. the genome is 3020-3320 nucleotides long (full length strand) and 1700-2800 nucleotides for the short strand. four genes are encoded including: (1) c the core protein (hbcag) synthesized by uorf aug to make pre-core protein, (2) p is the dna polymerase, (3) s is the surface antigen (hbsag) which has three aug start sites that divide the gene into pre-s1, pre-s2, and s, and (4) x is a gene that is not fully understood but may be a transcriptional transactivator. non-coding rna include hbv prealpha, hbv prebeta, and hbv rna encapsidation signal epsilon. there are 10 known genotypes which differ by 8 percent in sequence with distinct geographical distributions and are labeled a to j and at least 24 subtypes. type f is the most divergent form and found in central and south america, predominantly brazil. human hbv has a narrow host range infecting humans and higher primates, eg. chimpanzees. in the 1970s a woodchuck hepatitis virus (whv) was discovered which will not infect rodents. a ground squirrel form was identified (gshv) that is a distant relative of the marmot and woodchuck. indeed, dhbv infects ducks but not all species of duck, grey herons are infected with hhbv, the ross' goose with rghbv, the snow goose with sghbv, white storks with sthbv, and cranes with chbv. there are no hepadnaviruses in arthropods or insects. human t-cell lymphotropic virus (htlv-1) htlv-1 is a single-stranded rna retrovirus, defined by their use of reverse transcriptase, a polymerase, that makes a dna copy of the rna 7 kb viral genome. the dna viral genome is integrated into the host genome where it is referred to as a provirus and is replicated along with the host genome during cell division. only 1 percent of infected individuals will develop leukemia and this is observed 20 to 30 years after asymptomatic infection. the htlv-1 tax protein is likely to initiate cell transformation through interactions with transcription activators and cell cycle regulators. hepatitis c virus (hcv) hcv has infected approximately 3 percent of the world's population (~210 million) but screening of the blood supply has reduced prevalence. hcv is a flavivirus composed of a 9.4 kb single-stranded, positivesense rna. hcv is characterized by a single serotype but at least 6 major genotypes. genotype 1b is the most common genotype seen in the united states and taiwan. hcv becomes a chronic infection by evading host immune defenses through a combination of: (1) high replication rate (10 12 virions/day) and (2) lack of error proofreading by the viral polymerase leading to mutations in response to immune pressure. the genetic variability of hcv has limited efforts to design an effective vaccine. the polyomaviruses are small (3.4 kbp), double-stranded dna viruses. early studies with simian virus 40 (sv40) led to identification of the large tumor antigen (large t; lt). lt is also found in bk and jc viruses which are more suspect human tumor viruses and merkel cell polyomavirus (mcv) which is now well established as a human tumor virus (feng et al. 2008) . the n-terminus of lt contains an lxcxe motif that interacts with the retinoblastoma protein rb while the c-terminus contains an atpase/dna helicase domain that can inactivate p53. the ls-p53 complex activates insulin-like growth factor i (igf-i) which alone is capable of cell transformation. a recent report of morbidity and mortality reveals heart disease and cardiovascular events are the number one killer with neoplasia and infections following close behind. however, infections frequently cause neoplasia and cardiovascular disease leading to death. if we combine cardiovascular events and neoplasia caused by infection, then infectious disease is the most significant threat to human life and qualifies as the area of greatest impact. the picornavirus family are small (pico-), single-stranded, positive sense rna genome (7.4 kb) viruses that synthesize a single polyprotein that is cut into a small collection of functional proteins by virally encoded (2a and 3c) and cellular proteases. poliovirus is a picornavirus that has served as the prototype for the viral family. the enteroviruses are a group of picornaviruses that have been associated with cardiac disease. coxsackievirus b (cvb), coxackievirus a (cva), and echovirus infections lead to cardiac signs 3.2% of the time. about one-third of patients with acute cardiac disease (inflammation of the heart) are antibody positive for enteroviruses. while acute myocarditis is often self-limiting, chronic cardiac disease often leads to dilated cardiomyopathy (dcm) which is present with no heart inflammation. dcm is associated with heart failure which can be lethal (table 3 .6). these chronic infections lead to 50 percent of all cardiac transplants worldwide. the enteroviral 2a protease can also degrade dystrophin in the heart leading to cardiac necrosis, reduced ejection fraction, and then to dilated cardiomyopathy. transmission of these viruses is from contaminated food and water. the human herpesviruses are large double-stranded dna genome viruses. the human herpesvirus-5 (hhv5) or cytomegalovirus (cmv) has a 235 kbp genome encoding over two hundred genes. one problem in finding associations with cmv infections is that it is not an emerging disease, 50 to 99% of the population has been infected making comparisons to a control group challenging. most infections are observed in children and in newborns serious clinical findings can be observed. hence, most adults carry latent infections that are reactivated when individuals become immune suppressed following solid organ transplantation, malignant hematological disease, and aids. reactivation in immune suppressed individuals is associated with increased mortality. the association with cardiovascular disease has been demonstrated in two recent studies. in one, cmv infections were detected in 14.5 percent of coronary artery samples from bypass operations compared to 4 percent in of patients who needed cardiac surgery for reasons other than atherosclerosis hebar et al. 2015) . in the other, cmv reactivation (viremia) was detected in 16.5% of immunocompetent patients admitted for major heart surgery (roa et al. 2015) . the incidence of coronary artery disease is the major contributor to death from heart disease and if 14 to 16.5% of this disease is associated with cmv infections, this infection is a significant human health hazard. recognizing an emerging infectious disease involves well established strategies of surveillance; (1) identify unusual clusters of disease, (2) evaluate the spread of an outbreak, (3) estimate the magnitude of the problem, and (4) if possible identify the atheroscleotic plaque infectious agent. the strategy has proven valuable for known infectious and noninfectious diseases but has limited capacity to detect emerging infectious diseases. however, deviation from the traditional approach to surveillance is not likely to gain support. al smith, a veterinary virologist, approached me after a seminar i presented in 1997 in the college of veterinary medicine at oregon state university. i had just joined avi biopharma as the head of their research and development program. al was a veterinarian and professor and had devoted his career to the caliciviridae family of viruses dating back to his time in the naval research station in california. he had isolated the nonhuman vesivirus group of caliciviruses not only from suffering sea lions in the channel islands, but from reptiles in the san diego zoo and whales held in captivity. al and his capable technician, doug skilling successfully propagated the virus isolates in cell culture, an accomplishment not shared by other laboratories at the time. al developed nucleic acid probes and induced antibodies to these viral isolates. over decades of research he assembled an extensive collection of vesiviruses which were held in redundant −70° freezers. his singular vesivirus focus gave the appearance of a zealot but his ability to cultivate viral isolates, his extensive collection of isolates, and his one of a kind detection reagents made him a one of a kind virologist. al was well beyond retirement age and was an "old school" virologist ready at a moment's notice to take his bag into the real world and collect swabs from ailing animals. he maintained careful records of the condition of his patients, their location, and the setting of the animal in the community. every field trip led to work in the lab propagating virus from his swabs. al wanted to know if i would be interested in finding an antiviral agent for these viruses. my prime directive at avi biopharma was to explore the capabilities of our proprietary antisense technology and i had yet to investigate targeting a virus. the caliciviruses have positive sense single-stranded rna genomes which express three genes from a single polyprotein. we identified an active agent following an investigation with small collection of candidates (stein et al. 2001) . the vesivirus group of caliciviruses are considered animal only and are not believed to infect humans but al began exploring human samples with his collection of detection reagents. after several years making incremental progress, we found an association between human blood samples seropositive for vesivirus and markers of liver disease (smith et al. 2006) . we felt this evidence of an emerging infectious disease in humans and its potential to cause liver disease would be welcomed by the medical community. al and i made a trip to washington dc at our own expense to relate the findings in person. we meet with virologists at the national institutes of health but were met with judicious skepticism. harvey alter had been instrumental in the discovery of hepatitis c virus (hcv) and felt all viral liver disease is already accounted for by hepatitis viruses. hence, no interest in our findings. we met with the american red cross blood banking group in shady grove maryland and were met with concern. blood supplies are scrutinized by the nucleic acid test (nat) to eliminate viral contamination and any further elimination of blood samples would threaten an already limited inventory. our findings simply add complication to the blood supply finding an emerging infectious disease business. they asked for more compelling and more extensive data to add robustness to our claims. the only problem is that they were not interested in providing financial support for the recommended studies. the conundrum is all too common, you need more data to convince granting agencies to support the work but there is no support to add to the limited data. life on the cutting edge is frequently discouraging. our strategy took two paths: one to seek commercial support and the other to create proof of concept data for an antiviral solution. the most logical commercial solution was to meet with michael houghton at chiron. he was the driving scientific force in finding hcv and chiron might like adding to their reputation by finding another previously unrecognized hepatitis virus. indeed, dr. houghton invited us to bay area to discuss the project. he reviewed our data and the quality of al's detection reagents. chiron provided modest support and their in-house laboratory help to confirm our observations, a glimmer of hope. al created a company, calicitech, so that he could accept support and license his reagent patents from the university. testing an antiviral agent in humans would be expensive but in the absence of natural history of the infection would make human proof of concept impossible. however, al had been contacted by a cat rescue facility in atlanta, mommy cat, describing an outbreak of feline calicivirus (fecv) in their cattery. these cats had all been vaccinated with an approved fecv vaccine which failed to protect these cats. a veterinarian can decide to use alternative medicines if there are no alternatives and the owner oc the cats can sign the equivalent of informed consent. we provided our fecv specific antiviral to mommy cat along with a detailed protocol for treatment. the infected kittens we treated survived while those not treated died providing encouraging data. we then discovered a similar outbreak in the humane society facility in eugene oregon, our neighbors. again, we reached an agreement to treat infected kittens and were successful ). this was the first time we treated an infection based on symptoms in an "outbread" population of cats. with over 100 kitten patients and remarkable survival differences between treated and untreated, we had our proof of concept. there is no disease cluster to link to human vesivirus. norovirus is a calicivirus and is known to infect humans, in fact it is well known on cruise ships, day care centers, and extended care retirement facilities. our efforts to have human vesivirus recognized as an emerging infectious disease have failed. chiron was not impressed with our antiviral and no longer were interested in evaluating the detection reagents. if we are lucky, human vesivirus infections will remain mild clinical oddities. al smith has an interesting way of responding, "that virus is out there infecting humans. it won't go away." the existence of non-pathogenic viral infections led to the emergence of the study of the immune system, vaccination, gene therapy, and concern for future pandemics. we carry evidence of ancient retroviral infections in our genome from integration events that became vertically transmitted making up as much as 8 percent of the human genome (meyer et al. 2017) . these genomic fossils are called endogenous retroviral genomes (erv). most erv sequences accumulate sufficient mutations over evolutionary time that horizontal transmission is unlikely. these viral genome segments are trapped but can still be transcribed and encode some viral proteins. the significance of these integrated genomes is a current topic of investigation. adeno-associated virus (aav) is a single stranded dna virus that infects humans but are not known to cause disease. the lack of pathology has led to their use as viral vectors for human gene therapy. aav can infect dividing and quiescent cells and will persist extra chromosomally without integrating into the genome of the host cell. aav is a member of the parvoviridae family in the genus dependoparvovirus. is an arenavirus closely related to lassa virus and shares reservoir hosts. mv is naturally attenuated and nonpathogenic in humans. infection of humans with mv protects against lethal challenge with lv (wulff et al. 1977) . flaviviridae) named after g. barker, a surgeon, first identified in 1995. hgv infects one sixth of the world's population but does not cause human disease. a metaanalysis of 15 publications investigating gbv-c infections in hiv-positive individuals indicate coinfection with gbv-c slows the progression of hiv disease in individuals that have been seropositive for 2 years or more (zhang et al. 2006a) . two of the studies investigated gbv-c 5 years after documented hiv seroconversion estimate the hazard ratio of 0.36 (95% ci). is a retrovirus first identified in 1971 from a lymphoblastoid cell culture from a kenyan patient with nasopharyngeal carcinoma. hfv is homologous to primate foamy viruses and is most closely related to the chimpanzee foamy virus (sfvcpz). early studies raised alarm for association with autoimmune diseases but more extensive studies with more precise diagnostic reagents fail to find a disease associated with hfv. hfv is a rare human infection and concerns parallel sfv infections in humans. simian foamy virus (sfv; spumavirus-retroviridae) is a retrovirus infecting most primates born in captivity and people making contact with infected primates can become infected. human infections frequently occur in males probably requiring a bite from infected nonhuman primates but are harmless. the infected cells often fuse to form syncytia of giant foamy cells, which gives the virus its name. the error rate in sfv genomes is exceptionally low, 1.7 x 10 −8 substitutions per site per year, compared to hiv 10 −3 substitutions per site per year. since so-called crossspecies, infections have only been observed for a little over a decade the long term consequences are not known. these infections are watch and wait for everything from a zoonotic epidemic to identified disease clusters. perhaps this is exactly the sort of infection that will emerge as a significant human concern in the future. torque teno virus (ttv; alphatorquevirus) is a single-stranded, positive sense dna genome virus about 3.8 kb in size in the anelloviridae family. nearly 100 percent of even healthy individuals are infected in some countries. the virus was discovered in 1997 as the "transfusion transmitted virus (ttv)" in a japanese patient. it is often found in patients with liver disease but does not cause hepatitis on its own. closely related torque teno mini virus (ttmv) were isolated in 2007 and found to have smaller genomes of 2.8-2.9 kb. ttmv infections are also common but do not cause any described human disease. human adenovirus type 5 (rad5) is used to create an ebolavirus (ebov) vaccine encoding zaire ebolavirus glycoprotein failed to protect animals immune to ad5. a replication defective chimpanzee-derived adenovirus (chad3-ebo-z) provided protection against lethal ebov challenge in macaques but protection wane over several months. they boosted with a modified vaccinia ankara (mva-bn-filo) that led to durable protection (stanley et al. 2014 ). this vaccine progressed through phase i, single-blind, randomized human trials in mali between 2014 and 2015. a single dose of the chad3-evov-z is efficient as a prime vaccine strategy followed by mva-bn-filo as a boost was well tolerated in humans tapia et al. 2016) . is a 5229 base double-stranded dna virus infecting less than 5 percent of the human population. wu, named after washington university, is found as a co-infection in various respiratory infections but wu does not cause disease on its own. wu is closely related to ki virus that also is not known to cause clinical disease. however, related polyomaviruses that are clinically relevant include bk virus associated with nephropathy, jc virus associated with progressive multifocal leukoencephalopathy, sv40 virus associated with mesothelioma, and merkel cell polyomavirus associated with cancer. vaccinia virus (orthopoxvirus) is a large double stranded dna virus closely related to smallpox. edward jenner, the father of immunology, found the milkmaids exposed to cowpox (vaccinia) were immune to smallpox in 1798. this was the first vaccine (named after vaccinia) leading to the modern vaccine that has allowed for the eradication of smallpox. viral sequences are constantly mutating with no purpose other than seeking a survival/infectivity benefit. this means viruses with no current pathology represent a pre-mutation reservoir for the next catastrophic human pandemic. the popularity of rnaseq is likely to expand our catalog of nonpathogenic viral infections. however, the management of such information is in question. technology used to counter viral infections has resulted in over 90 approved drugs for the treatment of nine different human viral infections in just 50 years (de clercq and li 2016) . several different antiviral drug groupings have been reported, but the following arise from review of the mechanisms of action of antiviral drugs assembled in table 3 .7: (1) inhibition of viral attachment and entry, (2) inhibition of viral uncoating, (3) viral polymerase inhibitors, nucleotide analogues (ntrti) and non-nucleotide reverse transcriptase inhibitors (nnrti) and dna polymerase inhibitors, nucleic acid synthesis inhibitors and nucleotide pool size agents, (4) latency reversal agents, (5) integrase inhibitors, (6) protease inhibitors for both hiv and hcv, (7) neuraminidase inhibitors, (8) immune response modifiers, and (9) antisense inhibitors. administration of hyperimmune sera from immunized animals or human donors was the first effective treatment for infectious diseases. the practice has limitations but is still used to treat bacterial toxins and viral infections caused by cmv, rsv, hav, hbv, rabv, vzv and mev (keller and stiehm 2000) . the development of human or humanized monoclonal antibodies (humabs) has created a feasible way to rapidly generate novel antiviral therapeutics. humabs have advantages over serum therapy in that they are chemically defined reagents with minimal variability, greater activity per mass of protein, and they have no immunological consequences related to serum sickness. several mabs have been approved for treatment of infectious diseases including viral and bacterial pathogens (table 3 .8). the antiviral mab discovery field is exploding with activity particularly for hiv and hcv infections. a humanized mab targeting lymphocyte ccr5 receptors called pro140 has demonstrated potent and prolonged anti-hiv-1 activity and a large margin of safety (jacobson et al. 2010a) . administration of pro 140 by the subcutaneous route offers patients a way to self-administer the mab but importantly the mab is transported in the lymphatics providing enhanced access to binding to the cellular target (jacobson et al. 2010b) . the next generation of antiviral therapeutics are likely to be dominated by mabs. perhaps the only way to clear a viral infection involves a host immune response (table 3 .9). the innate immune response is particularly effective centered on a type 1-interferon pathway. unfortunately, many viruses carry mechanisms to evade host innate responses and innate immune effectors do not have the capacity for memory. the adaptive immune response can mitigate infection with antibodies, generally to surface antigens, which prevent the spread of the virus and t-lymphocytes, which can clear virus-infected cells. the first strategy will be to use an existing drug designed for another virus offlabel. this seems likely for antiviral drugs like cidofovir, foscarnet, and ganciclovir particularly for double-stranded dna viruses like ebv, hpv, and hhv6. ribaviran has been used for a number of single-stranded rna viruses including polio, junin, and lassa fever. secondary strategies will require investment of time and effort beginning with vaccine development and creation of monoclonal antibodies. platform technology rna-based therapeutics offer rational design for an expansive number of new antiviral strategies. this advantage is superimposed on the theoretical advantages of selectivity, specificity and affinity provided by watson-crick base pairing. rna-based therapeutics are expected to provide a substantially more narrow range of pharmacokinetic properties and toxicities thus are easier to compare to each other and ultimately combine into multi-agent cocktails. however, the mechanism of action may vary from rnase h or risc mediated degradation of the targeted rna or steric inhibition of rna function. the objectives of our antiviral program have been to exploit the broad understanding of rna-based therapeutics for antiviral activity with a common chemical type, the phosphorodiamidate morpholino oliogmer (pmo) and their enhanced derivatives. in this way the mechanism of action is common to all agents, which is steric blockade of rna function. studies reported by zamecnik and stevenson introduced the first approach to identification of an antisense antiviral agent stephenson and zamecnik 1978) . they used a 13-mer targeted to rous sarcoma virus. since the rous sarcoma virus pioneering efforts, antiviral rna based therapeutics have involved multiple oligomer chemistries with a variety of different mechanisms of action. chemical approaches to oligomers directed to hiv have been plentiful. a nonionic methylphosphonate oligonucleotide targeted to the splice acceptor site of hiv tat inhibited splicing of viral rna inhibiting syncytia formation and p24 synthesis at 3um concentration. poor aqueous solubility limited the utility of the methylphosphonate chemistry. phosphoramidate chemistry was investigated for inhibition of the splice-donor and splice-acceptor of hiv tat agrawal et al. 1988 ) and were more potent but these agents were cytotoxic and poorly water soluble. phosphorothioate chemistry targeting hiv-rev (matsukura et al. 1987 ) and hiv-tat were shown to be effective in inhibiting hiv replication, were not cytotoxic and were very soluble. further, the hiv-rev phosphorothioate oligodeoxynucleotide was stable in vivo with an acceptable pharmacokinetic profile (iversen et al. 1994) . a 25-mer phoshorothioate called gem91 targeting the initiation site of hiv-gag was evaluated in clinical trials (agrawal 1998 ) but the trials were discontinued. i focused on phosphorodiamidate morpholino oligomer chemistry which is both stable and net-neutral in charge at physiological ph. single stranded rna viruses with positive sense (ssrna(+)) these viruses are the most simple in terms of genome size, number of potential translated viral proteins, their genomes are all linear and they enter the cell ready for translation. the design of steric blocking rna-based therapeutics involves preventing translation, disrupting rna secondary structure and masking recognition sites for rna dependent rna polymerase (rdrp). the targeting of either the 5′-terminus and the first orf-aug are active. further, efficacy in animal challenge studies is observed with high fidelity when the most effective agent identified in vitro is employed. the family astroviridae with six different human astroviruses (huastv) responsible for 2-17% of all gastroenteritis. we found the 5′-terminus 1 to be the most effective site to target. this was also the optimal site for caliciviridae including vesivirus (vev; martin-alonso et al. 2005; stein et al. 2001), norovirus (nov; bok et al. 2008) , and feline calicivirus (fcv; smith et al. 2008) ; the flaviviridae including dengue (den; kinney et al. 2005; holden et al. 2006) , and west nile virus (wnv; deas et al. 2007; zhang et al. 2008) , arteriviridae including agriculturally important equine arterivirus (eav; van den born et al. 2005) , and porcine respiratory and reproductive virus (prrsv; zhang et al. 2006a, b) ; and the togaviridae exemplified by venezuelan equine encephalitis virus (veev; paessler et al. 2008 ). the family coronaviridae revealed a new active target site in the transcription regulatory sequence (5'-cgaac-3′) in both mouse hepatitis virus (mhv; neuman et al. 2004 ) and the severe acute respiratory syndrome virus (sars; neuman et al. 2005) . the picornaviridae active target site involved a highly conserved sequence in the internal ribosomal entry site (ires) in polio virus (pv; stone et al. 2008) , foot and mouth disease virus (fmdv; vagnozzi et al. 2007) , and the coxsackievirus (cvb3; yuan et al. 2006) . single stranded rna viruses negative sense (ssrna(−)) these viruses are generally more complex with respect to genome size, number of potential translated viral proteins, and multiple genome segments. the genome must be replicated prior to translation of viral proteins. the design of steric blocking rna-based therapeutics is similar to the positive sense rna genomes in that targets involve preventing translation and masking recognition sites for rna dependent rna polymerase (rdrp). we investigated 11 different targets in measles virus (mev), a member of the paramyxoviridae, finding the translation initiation start site of n the optimal target (sleeman et al. 2009 ). studies with the human respiratory syncytial virus (hrsv) found the translation site for l to be most active (lai et al. 2008) . the orthomyxoviridae studies investigated influenza a virus probing each of the 8 viral segments finding translation of pb1 active as well as the 5'terminal of vnp for h3n2 (ge et al. 2006 ) and h3n8 (lupfer et al. 2008 ) but a combination of targets was required in animal studies with a high pathogenic viral h7n7 isolate (gabriel et al. 2008 ). more extensive influenza a studies revealed a new target, the m-segment splice donor site. this target was evaluated in phase i clinical trials. the antisense platform technology has limitations in targeting viral sequences. inhibiting virally encoded proteins or blocking viral replication by interfering with the polymerase does not always work. the arenaviridae family proved difficult. we experienced some success with junin and lymphochoriomeningitis virus (lcmv) in cell culture observing 4 log 10 reductions in viral titer with a pmo targeting the highly conserved viral genome terminus. however, we failed to provide survival benefit to guinea pigs challenged with either junin or lassa virus. we then tried the mouse challenged with lcmv and failed again but we observed hemorrhagic disease in the mouse, a severe consequence of infection that mimics the worst aspects of arenaviral infection. we decided to try targeting host genes to mitigate disease in the mouse and found targeting il-17 provided a survival benefit (schnell et al. 2012) . the success in the face of failure puts targeting host genes at the center of attention for dealing with emerging infectious diseases. the antisense platform technology represents an excellent approach to rapid drug discovery for emerging infectious disease. rapid discovery demonstrated repeatedly but of course, the cost of advanced development is daunting. the application is best for immediate treatment of index cases, close contacts, and healthcare workers. we have an 800-pound gorilla in the room and everyone in the room considers it someone else's problem. the current course is to ignore the majestic creature until it starts tearing limbs from people and then the consensus is to kill the gorilla. agencies like the world health organization (who), the centers for disease control (cdc) and the national institutes of health (nih) can assemble highly skilled personnel and can confer with some of the greatest minds in the world. unfortunately, they are all aware of the potential problem that an emerging pandemic is likely to take us by surprise. significant speculation that a single-stranded rna virus will emerge killing tens of million people, costing hundreds of billions of dollars, and changing the course of human history. there is a high probability that the virus will be influenza a (h5n1 or h7n9) that will jump from a reservoir population of birds and establish human-to-human transmission. the pandemic will be a global event by the time an effective vaccine is available. neuraminidase (na) inhibitors as therapeutic and prophylactic agents in the setting of pandemic influenza a (flua) were called in to doubt in the past decade (michiels et al. 2013; jefferson et al. 2014) . indeed, 100% of isolates in the 2008/2009 a/h1n1 pandemic were found to be resistant to adamantanes, and resistance to oseltamivir (tamiflu; osl) was observed in virus recovered from individuals taking osl therapeutically or prophylactically (dharan et al. 2008; cdc mmwr 2009) , while effect on duration of shedding was not impacted. a recent outbreak of an influenza a (h7n9) virus caused 137 cases and 45 deaths in china revealed a novel na mutation r292k resulting in high level resistance to osl (wang et al. 2014) . therapeutic options for treatment of individuals with complicated influenza a are severely limited, perhaps no options. pandemic strains such as a/h1n1pdm09 carried significant morbidity and mortality, particularly in those who had not experienced h1-strain influenza in their lifetime. we are also witnessing more rapidly emerging highly pathogenic avian influenza strains that are resulting in human infection; some such as a/h5n1 and a/h7n9 appear to becoming more efficient in person-to-person transmission, and reports suggest osl resistance develops during treatment (de jong et al. 2005; lam et al. 2015) . we are not ready for an outbreak of avian flu or any other emerging single-stranded rna virus. why not? an estimate for the time and cost to develop a new drug is 10 years and $1 billion. the commercial use of a drug for an emerging infection is hard to estimate since by definition when you start development the infection has not emerged. most of us would be unlikely to use our retirement savings to invest in a drug development project with no reliable way to expect a return on our investment. it is a poor business model. when you consider there may be hundreds of emerging infectious diseases each times 10 years and $1 billion each the task is daunting. on which disease should we focus? consider the ebola drug avi-7537. the ebola therapeutic project began in 2004 following a laboratory accident at usamriid. we identified three compounds each with activity and when combined we observed unprecedented efficacy in a lethal challenge primate animal model (warfield et al. 2006 ). hundreds of experiments over the next 3 years optimized these agents (swenson et al. 2009 ). we were able to obtain research grants from the transformational medical technologies (tmt) division of the defense threat reduction agency (dtra) within the department of defense (dod) and we completed proof of efficacy studies (warren et al. 2010) . this led to submission of an investigational new drug application (ind) to the food and drug agency (fda) and phase i safety and tolerability studies were conducted i healthy human volunteers (heald et al. 2014) . after 11 years of continuous effort, we completed key aspects related to the fda approval process under the "animal rule" and we streamlined our treatment to a single agent, avi-7537 (warren et al. 2015) . however, shortly before the ebola outbreak in western africa, the us government "budget sequester" cancelled our project and the most advanced therapeutic on the planet was not deployed to treat those infected during the outbreak. as the outbreak continued, we found no viral resistance of avi-7537 unlike the monoclonal antibody therapy in use (khiabanian et al. 2014) . avi-7537 sits on a shelf, a political casualty and an unfavorable business model. the global virosphere may contain up to 10 31 virus/virus-like particles (suttle 2005) , the greatest reservoir of genetic diversity. the earth's atmosphere transports viruses all over the planet. viruses are found in soil at 1.5 x 10 8 to 6.4 x 10 8 particles per gram of dry soil (kimura et al. 2008) . the surface oceans carry approximately ten million viral particles in each milliliter of seawater, most of which are bacteriophages (phage). the small viral particles are easily carried into the upper environmental viral reservoirs atmosphere by up drafting winds. bacteria are deposited from the atmosphere at a rate of 0.3 to 8 x 10 7 per meter each day and viral deposition rates are 9-461 times greater (reche et al. 2018) . these phages influence bacterial lifecycles and play a role in natural energy and nutrient cycles fundamental to life on earth. the dynamics of phage-bacterial evolution drive changes in photosynthesis, phosphate, and nitrogen balance (breitbart 2012) . human accidental release of radioactive waste (discussed in chap. 2) and disposal of chemicals including potent antiviral and antibacterial compounds (discussed in chap. 7) may alter the eco-evolutionary dynamics producing unanticipated environmental consequences. the objective of this chapter has been to provide convincing evidence that infectious disease is the most significant threat to human health. the focus has been on viral infections because they rely on host ribosomes to produce their proteins, recent emerging infections have been from single-stranded rna genome viruses, and replication of rna viruses is error prone. pandemic infections have accompanied the rise of human civilization frequently re-occurring leaving a lasting imprint on human history punctuated by profound loss of life. emerging infections become endemic with an annual death toll. each decade brings a new onslaught of emerging infectious agents. we are surprised again and again but have never prepared for these inevitable catastrophies. the long-term consequences often remain unrecognized and are always inconvenient such as cancer, cardiovascular disease and immune associated diseases that threaten our health. reliance on clusters of clinical symptoms in the face of diverse and non-descriptive viral infection symptoms is a foolhardy form of crisis management. infectious disease will certainly continue to pose the most significant threat to human health in the age to cell phones, artificial intelligence, and global commerce. rapid replication of viral genomes combined with low fidelity polymerases provide the foundation for an unending source of new emerging infectious agents. these traits also make viral genomes sensitive to environmental contaminants in a way that may expand probabilities for zoonosis. infectious disease as part of our environment is not appreciated. the study of infectious disease is not a part of the curricula of students in environmental science/management. textbooks in in environmental studies do not include chapters in infectious disease. the integration of research at superfund sites focused on chemical contamination with infection and zoonosis would result in valuable insights into threat analysis. viruses with rna genomes lack sequence proofreading quality control during replication. the cumulative mutations in their genomes limits the genome size to under 30,000 bases. essentially, a larger genome would evolve out of existence, so called catastrophe evolution. the limited genome size makes these viruses exceptionally resilient to a changing environment. the virus must economize by combining functions. this means evolution and resilience are the same thing in the rna genome viruses. a unique insight is that in human evolution is restricted to the dna genome and resilience limited to rna, as it is in the rna genome viruses. antisense oligonucleotide-based therapy for hiv-1 infection from laboratory to clinical trials oligodeoxynucleotide phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus a tribute to sheik humarr khan and all the healthcare workers in west africa who have sacrificed in the fight against ebola virus disease: mae we hush inhibition of norovirus replication by morpholino oligomers targeting the 5'-end of the genome marine viruses: truth or dare cancer virus oseltamivir-resistant 2009 pandemic influenza a (h1n1) virus infection in two summer campers receiving prophylaxis-north carolina approved antiviral drugs of the past 50 years the eradicatio of smallpox: edward jenner and the first and only eradication of a human infectious disease oseltamivir resistance during treatment of influenza a (h5n1) infection in vitro resistance and in vivo efficacy of antisense oligomer against west nile virus outbreak of antiviral drug-resistant influenza a in long-term care facility the perpetual challenge of infectious disease clonal integration of a polyomavirus in human merkel cell carcinoma morpholino oligomers targeting the pb1 and np genes enhance survival of mice infected with highly pathogenic influenza a h7n7 virus inhibition of multiple subtypes of influenza a virus in cell cultures with morpholino oligomers safety and pharmacokinetic profiles of phosphorodiamidate morpholino oligomers with activity against ebola virus and marburg virus: results of two single ascending dose studies cytomegalovirus infection and atherosclerosis in candidate of coronary artery bypass graft inhibition of dengue virus translation and rna synthesis by a morpholino oligomer targeted to the terminal 3′ stem-loop structure pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev from human immunodeficiency virus type 1 in the adult male rat following single injections and continuous infusion phase 2a study of the ccr5 monoclonal antibody pro 140 administered intravenously to hiv-infected adults anti-hiv-1 activity of weekly or biweekly treatment with subcutaneous pro 140, a ccr5 monoclonal antibody oseltamivir for influenza in adults and children: systemic review of clinical study reports and summary of regulatory comments passive immunity in prevention and treatment of infectious diseases viral diversity and clonal evolution from unphased genomic data ecology of viruses in soils: past, present and future perspectives inhibition of dengue virus serotypes 1 to 4 in cell culture with morpholino oligomers inhibition of respiratory syncitial virus infections in cell cultures and in mice with morpholino oligomers dissemination, divergence and establishment of h7n9 influenza viruses in china health impact of globalization: towards global governance inhibition of influenza a h3n8 virus infections in mice by morpholino oligomers isolation and characterization of a new vesivirus from rabbits phosphorothioate analogs of oligodeoxyribonucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus endogenous retroviruses: with us and against us the value of neuraminidase inhibitors for the prevention and treatment of seasonal influenza: a systematic review of systematic reviews antisense morpholino oligomers directed against the 5'-end of the genome inhibit coronavirus proliferation and growth inhibition, escape and attenuation of sars coronavirus treated with antisense morpholino oligomers inactivating mutations in an sh2 domain-encoding gene in x-linked lymphoproliferative syndrome inhibition of alphavirus infection in cell culture and in mice with antisense morpholino oliogmers immunological disorders and malignancies in five young brothers spillover animal infections and the next human pandemic deposition rates of viruses and bacteria above the atmospheric boundary layer a prospective monitoring study of cytomegalovirus infection in nonimmunosuppressed critical heart surgery patients inhibition of acquired immunodeficiency syndrome virus by oligonucleotide methylphosphonates lymphocytic choriomeningitis virus infection in fvb mouse produces hemorrhagic disease ancient athenian plague proves to be typhoid inhibition of measles virus infection in cell cultures by peptide-conjugated morpholino oligomers vesivirus viremia and seroprevalence in humans virus specific antiviral therapy for controlling severe and fatal outbreaks of feline calicivirus infection chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge inhibition of vesivirus infetions in mammalian tissue culture with antisense morpholino oligomers inhibition of rous sarcoma viral rna translation by a specific oligodeoxynucleotide inhibition of multiple species of picornavirus using a morpholino oligomer targeting highly conserved ires sequence increased risk of hepatocellular carcinoma in male hepatiis b surface antigen carriers with chronic hepatitis who have detectable aflatoxin metabolite m1 viruses in the sea chemical modifications to phosphorodiamidate morpholino oligomer antisense molecules targeting vp24 modify their efficacy against ebola virus infection use of chad3-ebo-z ebola virus vaccine in malian adults with mva-bn-filo: a phase i, single-blind, randomized trial, a phase 1b, open label and double blind, doseescallation trial, and a nested, randomized, double-blind, placebo-controlled trial inhibition of foot-and-mouth disease virus in cell cultures with antisense morpholino oligomers antiviral activity of morpholino oligomers designed to block various aspects of equine arteritis virus amplification in cell culture pcr for detection of oseltamivir resistance mutation in influenza a(h7n9) virus igg antibodies to dengue enhanced for fcγriiia binding determine disease severity gene-specific countermeasures against ebola virus based on antisense phosphorodiamidate morpholino oligomers advanced antisense therapies for postexposure protection against lethal filovirus infections single component avi-7537 antisense compound provides greater protection than double component avi-6002 against lethal ebola virus infection in rhesus monkeys isolation of an arenavirus closely related to lassa virus from mastomys natalensis in south-east inhibition of coxsackievirus b3 in cell cultures and in mice by peptide-conjugated morpholino oligomers targeting the internal ribosomal entry site inhibition of rous sarcoma virus replication and transformation by a specific oligodeoxynucleotide characteristic chromosomal abnormalities in biopsies and lymphoid-cell lines from patients with burkitt and non-burkitt lymphomas effect of early and late gb virus c viremia on survival of hiv-infected individuals: a meta-analysis suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers west nile virus genome cyclization and rna replication require two pairs of long-distance rna interactions key: cord-356062-7q5n4t97 authors: nan title: cumulative pharmacological activity index volumes 1-30 date: 2005-12-31 journal: studies in natural products chemistry doi: 10.1016/s1572-5995(05)80101-2 sha: doc_id: 356062 cord_uid: 7q5n4t97 publisher summary this chapter lists the important subjects on pharmacological activity that are discussed in the publication studies in natural products chemistry, volumes 1–30, such as abdominal constriction test, acanthoic acid, acetaminophen, parkinson's disease, photodynamic activity, prostaglandins, and oleanolic acid. the terms are mentioned along with the page numbers in which they are discussed in the publication. use of psycotria colorata in 30:205 19(4--+ 3)-abeo-8 c~, 13(s)-epoxylabda-4(18), 14-diene 29:102 activity in ebv bioassay system 29:102 19-nor-abieta-4( 18),8,11,13-tetraen-7one 29:100 activity in ebv assay system 29:100 abieta-8,11,13-trien-7-one 29:99 activity in ebv assay system 29:99 18-nor11, 7a, 15triol 29:99 activity in ebv assay system 29:99 11, 15, activity in ebv assay system 29:99 abieta-8,11,13-triene-7o~,l 5,18-triol 7acetate 29:99 activity in ebv assay system 29:99 abietic acid 29:99 activity in tpa assay system 29:99 activity in crg (+)-5-epi-acetomycin 10:447 antitumor activity of 10:447 2(x-acetoxy-14-hydroxy-15-iso-valeroyloxy-9-oxo-costunolide 29:89 activity in nfkb assay system 29:89 activity in tnf(x assay system 29:89 9cx-acetoxy-4 ~, 15-epoxymiller-1 ( 10)zenolide 29:89 activity in nfkb assay system 29:89 3~-acetoxy-813-isobutyryloxy-reynosin 29:87 activity in mam-2 assay system 29:87 activity in dif assay system 29:87 15-acetoxy-9 ~-hydroxy-8 [3-methacryloyloxy-14-oxo-acanthospermolide 29:89 activity in nfkb assay system 29:89 15-acetoxy-9cx-methacryloyloxy-8 ~hydroxy-14-oxo-acantho-spermolide 29:89 activity in nfkb assay system 29:89 3-acetoxycostunolide 29:89 activity in nfkb assay system 29:89 15-acetoxy-eremantholide b 29:89 activity in nfkb assay system 29:89 activity in tnf~ assay system 29:89 9~-acetoxy-miller-1 ( 10) 30:191, 192, 203 development of 30:192, 203 use of 30:191 analgesic effects 26:401, 403, 404, 406, 416, 418; 30:205, 208 5:512,747;10:78, 117;11:134;12:398;14:145;20:245, 712 ;21:21,108,109,190,207,213,214, 230,237,262,266,267,275,284,601, 615 ;23:41,126,164,235,258,355,468, 375,472,473,531,534,800;24:573, 856,857;26:226,1002;28:140,688; 30:328 11:130;12:192; 17:283 ;21:188,202,223,239,364,437; 23:649;24:953-954 21:186,192,197, 203,206,213,214,218,221,226, 227,233-235,223,238,240,389, 391,399,404,405,435,599-662; 22:626;23:3,4,271,341,344,348, 468,473,798,801354,368 ;26:227, 228,553,1098;28:4;66,473,475,686; 30:327,738,629 against candida albicans 30:327,629 from hypericum roeperanum 30:629 of (e)-8(17), 12-1abdadiene-15,16-dial 23:798 of (e)-8b, 17-epoxylabd-12-ene-15,16-deol 23:798 of (e)-8 [3,17-epoxylabol-12-ene-15,1619:511,748;21:132, 133,149,252,269,664,665 ;23:316, 533,534,687,704 ;24:742,743 ;30:226, 396,397 13:647;17:137,376;18:775;21:130, 134,153,152,312,591,594,690,616, 656,657;22:93,119,307,345,361; 23:158,297,512,602,832;25:43; 26:340,341,343,391,398,400,401, 403,404,406,415,418,482,556,558, 741,759,1035 ;27:660,858 ;28:200, 257,293 ;671,844;30:206,207,224, 225,292,320,689 21:674,744; 26:229,790,813,887,789,790,791, 800,805,807,810,811,821,828,830 -microbial activity 5:752;10:443; 15:327-339;18:776-778;21:153, 235,257,401,571,573,591,596,597, 599,606;22:626;23:94,153,180,233, 237,249,262,268,267,346,353,522; 24:64,69,206,330,331-334,402,403, 404,406,417,418,443,470,485,859, 1091,1092,1189;26:64,330,401,402, 1189;27:807;28:62,224,257,293; 28:698 22:308,321,345 ;23:395,534,535,742, 772,773,845 ;25:59;30:173,224,225, 289,320,321,524,561,743,745; 26:250,255,509,556,672,763,768, 887,1005,1006;28:4,16,17,63,179, 257,293 1:275,316-320-408; 17:17 ;21:137,405,420,435,436,638, 704,733;22:676;23:74,96,126,132, 139,395,445,522,696,721; 24:272-275 ;24:288,845,847,849; 25:43,430,431,438,450-453;26:56, 482,706;27:803;28:211,517,519,559, 560,566;30:3,28,29,55,56,58,59,64, 65,224,314,395,589,690,777,840 :461,563;10:608, 609,619,620; 17:146 ;21:132,148, 150,393,435,600,615,662,690,718; 22:307,361 ;23:233,268,233,346,395, 473,512,531,703;25:114;26:169,222, 224,226,344,452,741,759;27:482; 28:257,293 ;30:224,324-326,393,394, 395,398,401-405,407,410-412 132,148,162, 223,236,238,263,265,269,284,297, 366,388,390,410,419,421,431,571, 578,591,598,601,603,620,694 ;23:63, 93,95,108,111,129,126,107-152,153, 158,179,267,271,285,395-453, 455-485,491,497,642-643 ;739;24:25, 799,845 ;27:178,377,431,421,443, 693,778,801,924 ;24:353-376; 27:185-225,332,659;28:109,212,228, 229,340;29:743 ;30:4,26,172,175, 200,224,303,310,395,404,483,484, 485,495,496,497,499,562,588,603, 637,831,840,843, acaricidal enzymatic activity 24:995-998 tx-cembra-2,7,11-triene-4,6-diol 29:100 activity in ebv assay system 29:100 activity in pkc assay system 29:100 activity in skin-1 assay system 29:100 activity in odc assay system 29:100 ~-cembra-2,7,11-triene-4,6-diol 29:100 activity in ebv assay system 29:100 activity in skin-1 assay system 29:100 activity in odc assay system 29:100 of (-)-deoxypodophyllotoxin 30:589 of (-)-hemone 30:590 of (-)-nymphone 30:590 of (-)-yatein 30:590 of (+)-corytuberine 30:589 of (+)-epiaschantin 30:590 of (+)-epimagnolin 30:590 of (+)-epiyangambin 30:590 of (+)-hernovine 30:589 of (+)-laurotetanine 30:589 of (+)-magnoflorine 30:589 of (+)-malekulatine 30:590 of (+)-n-formyldehydroovigerine 30:589 of (+)-n-formylnornantenine 30:589 of (+)-n-formylovigerine 30:589 of (+)-n-hydroxyhemangerine 30:589 of (+)-n-methylhemangerine 30:590 of (+)-ovigerine 30:590 of (+)-ovihernangerine 30:590 of (+)-vateamine-2' [3-n134,136,150, 174,257,259,260,262,263,265,267, 269,270,271,273,274,284,311,364, 410,634,643,656,661,690,704,744; 23:96,97,187,218,238,249,250,253, 255,272,274,277,290,292,299,308, 317,701,274 ;24:294,352,740,867; 26:212-213,1185 as anti-fungal agents 24:406 2 [3,5-epoxy-5,10-dihydroxy-6c~ange lo y-lo xy-9 [3-is obutylo xygermacran-8ot, 12-olide 29:89 activity in inos assay system 29:89 activity in nfkb assay system 29:89 7 a, 8 c~-ep oxy-6 or-hydro xyabieta-9(11), 13-dien-12-one 29:99 activity in ebv assay system 29:99 9,10or-epoxy-9,10-seco-abieta4-o-phenyl-2-propionyl-neu5ac-cx2me 27:123 4-t-butyl-dimethyl-silyl ether 27:127 5-azido-5-deamino-neu5ac-c~2me 27:119 5-n-benzyloxycarbonyl-neu-cx2me 27:119 5-n-propionyl-neu-c~2me 27:119 7-deoxy-neu5 ac-c~2me 27:123,127 8-amino-8-deoxy-neu5 ac 27:121,122 8-azido-8-deoxy-neu5ac 27:121,122 8-tosyl-neu5acme ester 27:121,122 9-amino-9-deoxy-neu5 ac 27:121,122 9-amino-9-deoxy-neu5ac-c~2me 27:119 9-azido-9-deoxy-neu5 ac 27:121,22 9-azido-9-deoxy-neu5ac-c~2me 27:119 9-o-acetyl-neu5 ac-( ,590 activity of (-)-deoxypodophyllotoxin in 30:589 activity of (-)-hemone in 30:590 activity of (-)-nymphone in 30:590 activity of (-)-yatein in 30:590 activity of (+)-corytuberine in 30:589 activity of (+)-epiaschantin in 30:590 activity of (+)-epimagnolin in 30:590 activity of (+)-epiyangambin in 30:590 activity of (+)-hernovine in 30:589 activity of (+)-laurotetanine in 30:589 activity of (+)-magnoflorine in 30:589 activity of (+)-malekulatine in 30:590 activity of (+)-n-activity of hydroxyhernangerine in 30:589 activity of (+)-n-formylnornantenine in 30:589 activity of (+)-n-formyldehydroovigerine in 30:589 activity of (+)-n-formylovigerine in 30:589 activity of (+)-ovigerine in 30:590 activity of (+)-ovihernangerine in 30:590 activity of (+)-vateamine-2'13-noxide in 30:590 activity of 7-formyldehydrohernangerine in 30:589 activity of 7-formyldehydronornantenine in 30:589 activity of 7-formyldehydroovigerine in 30:589 activity of 7-hydroxy-6-methoxy12-hydroxy -6,7-seco-abieta-8,11,13trien-6,7-dial 29:99 activity in ebv assay system 29:99 7-hydroxy-6-methoxy-1-methyliso22:510,512,513,516,518,519, 521,522,525-529,531,534 achyranthes aspera l 9,459,503,753,754,311; 17:313,441 ;20:108-113,273,613; 24:215,228-232,503-509,739-798; 26:229,790,813,887;30:560,562,567, 570,590 chemo-differentiation therapy 30:498 of acute monocytic leukemia chemopreventive activity 30:591,597, 766 in mouse mammary organ culture model 30:591 in vitro 30:597 in vitro bioassays for 30:591 of chlorella vulgaris 30:766 ofisoquinoline alkaloids 30:597 of lignans 30:597 chemopreventive agent 26:219 genkwanin as chemopreventive efficacy 27:386 of limonene carcinogenesis 27:386 chemoprophylaxis 26:674 chemosensitizers 30:595 antimalarial drugs as 30:595 chemotaxonomic markers 26:1128 chemotherapeutic agent 30:5,330,742 atovaquone 30:330 for colon cancer 30:5 for multi-drug resistant cancers 30:5 for ovarian cancer 5 for taxol-resistant breast cancer chemotherapeutic intervention 30:397 targets for chemotherapy drug 27:436 antitumor activity of 27:436 chest pain 30:203 use of ginseng root in 30:203 childhood disintegrative disorder (cdd) gram-negative bacteria 30:693 maytenus species against gram-positive bacteria 23:68;30:629, 693 actamycin against 23:68 antimicrobial activity of 30:629 inhibitor maytenus species against grandiflorolic acid 29:100 activity in ebv bioassay system grandiflorolic acid angelate 29:100 activity in ebv bioassay system grandisin 26:230 trypanosomicidal activity of grandmal seizures granulocyte phagocytosis 26:47,55 ofjenisseensosides c 26:47,55 ofjenisseensosides d green tea 27:417,420 biological activity of 757 effect of general analogues on pgf2 na-induced contraction griseofulvin 24:934 as anti-fungal agents grob-type fragmentation 24:728,731 growth 30:61 of tumor cells 30:61 growth inhibition 17:142 growth inhibitor activity 7:407 guaiazulene 5:363,369 as an anti-inflammatory agent guidimacrin 27:831 antitumor mechanism 584 neurodegeneration 30:224 reperfusion injuries 30:224 respiratory disorders 30:224 role of free radicals in 30:224 human growth hormone human hek 293 cell line 30:781 biological response of human hl-60 leukemia cells 30:496 differentiation of 30:496 inhibition of 30:496 proliferation of human leukemia cells 18:269 human leukocyte elastase 16:727 inhibitor human leukocyte elastase (hle) 30:830,831,843 homologous sequences of 30:831 plasminogen activator (u-pa) 30:830 human lung carcinoma 23:290;30:5 discodermolide against human lung carcinoma) 30:691 phenolic triterpenes against 30:691 human medulloblastoma 23:290 human melanoma 23:255 human mesangial cell proliferation 30:308 in vitro human monoblastic leukemia cells 30:498 effect of hydroxyurea (hu) 30:498 growth inhibition of 30:498 human monoblastic leukemia u937 cells 30:498 growth inhibition of human neuroblastoma nbla-n-5 cells tamoxifen activity against 12:390 human neuroblastoma sh-sy-5y cells 12:390 human neutrophils 12:390 human neutropil protein kinase c 12:389 human onchocerciasis 12:9 ivermectin for human papilloma virus 30:394 pathogenesis of 30:394 cause of hepatocellular carcinoma human papilloma virus (hpv) 23:97 human papilloma virus type 11 30:406 microbicidal compound against human papilloma virus type 40 30:406 microbicidal compound against 30:406 human platelets 12:390 study of protein kinase c in 12:390 by staurosporine 12:390 human rotavirus (hrv) 30:406,412 cause of dehydrating gastroenteritis 30:406 member of 30:406 reoviridae type of human serotonin transporter gene (slc6a4) 30:376 regulation of neuronal activity human spermatozoa motility 21:675 human synovial pla2 25:697 inhibitor anti-glycemic effects 24:902-904 anti-hyperglycemic effects of -o-methyl-20-hydroxyecdysone activity in 29:28 pinnatasterone activity in 29:28 polypodine b 2-o-cinnamate activity in muscarinic achr antagonists 21:56 muscarinic antagonist 22:19 muscarinic receptor 21:95 muscarinic receptor antagonists 22:734 muscarinic-l,2-receptor 21:95 muscle pain 24:898 aconitine for relieving activation effects of 24:906 medicinal uses of 24:906 protein kinase c activation effects 24:906 muscular p-388 (murine lymphocytic leukemia) 30:588 activity of (-)-deoxypodophyllotoxin against 30:588 activity of (-)-yatein against p-388 lymphocytic leukemia cell 25:764 p388 mouse leukemia 12:390 staurosperine activity against p-388 murine leukemia cells 21:411 pachyman 5:288,317 antitumor activity of 5:317 pachyrrhizus erosus 24:779,822-826 anti-feeding activity of 24:825 pachyrrhizus palmatilobis 24:822 biological activity of 24:823 paclitaxel 240 (+)-acetoxypinoresinol dimethyl ether as 26:241 arctigenin as 26:241 arctigenin methyl ether as 26:241 chamigrenal as 26:244 cinnamophilin as 26:240 dehydroschisandrol a as 26:245 denudatin as 458 fptase inhibition by 24:458 paw oederma 30:207 inhibition of 30:207 2-pectenotoxin 19:580 cytoxic activity of 19:580 pectinasic pectolinaringenin 30:725,748 antispasmodic activity of 30:748 pedunculariside 29:84 activity in cox assay system 29:84 activity in epp assay system pelargonidin (anthocyanidin) 29:585 effects on lela 29:585 effects on penates sp. 28:713 as ~-glucosidase inhibitor 28:713 penicillium turbatum 11:194 antibiotic a 26771b by 11:194 penstemide 7:441 activity against p-388 lymphocytic leukemia penstemon rosseus 24:838 anti-fungal activity pentacyclic triterpenes 30:207 protein kinases inhibition by 564 in lipid autoxidation 3,4,6-p enta-o-gallo yl-~ -d-g luc o s e (galloyl ester) 29:589 effects on 229 biological activity of 28:229 8-prenyleriodictyol 28:229 biological activity of 28:229 prenylflavones 28:225 anti-human immunodeficiency virus (hiv) activity of 28:226 prenylfiavonoid 30:207 antinocicieptive effect of antitumor promoting activities of 29:720 as cytomegalovirus protease inhibitors 29:720 gastro-intestinal problems 29:720 use in hodgkins disease 29:720 use in infections 29:720 use in lupus 29:720 use in osteomyelitis 29:720 use in parkinson's disease 29:720 use in psoriasis 29:720 use in respiratory problems 29:720 use in skin ulcerations 29:720 use in syphilis 29:720 6-prenylnaringenin prevention by chitosan 27:436 of myelotoxicity 27:436 primary biliary cirrhosis 25:463 primary granulosa cells 25:269 apoptosis in 25:269 primary solid-tumor growth 30:64 effect of triterpenoids on 30:64 pristimarin 30:692 cytotoxic activity of prl1 & prl2 proproteins 29:592 effects on cysteine protease 851 probes 27:332 activity of 27:346 procurcumenol 29:90 activity in tnfct assay system procyanidin b 1 (dimeric flavan-3-ol) 29:580 effects on ace procyanidin b2 (dimeric flavan-3-ol) 29:580 effects on ace procyanidin b-5 3,3'-di-o-gallate 29:580 effects on ace procyanidin c2 (trimeric flavan-3-ol) 29:580 effects on ace 29:580 procyanidin polymer (flavan-3-ol polymer) 29:580 effects on ace prodrug monotherapy 21:157 natural anthracyclines for 21:157 prodrugs 21:157 of natural anthracyclines pro-inflammatory cytokines 25:461 pro-inflammatory substances 30:206 in chronic painful inflammatory diseases 30:206 proliferation 30:368,378 effect of serotonergic neurotransmission on 30:368 serotonin action on 30:378 prolysine 25:388 prophylactic effects pro-nociceptive prostaglandins 30:192 in inflammation pro-nociceptive transmitter 30:194 release inhibition of 30:194 prooxidant activities 30:745 in thiobarbituric acid assays 30:745 of chemiluminescence 30:745 of tert-butyl hydroperoxide 30:745 prooxidant property 30:524 of carotenoids 30:524 prophylactic drugs prostaglandin e2-dependent flavonoids cytoprotection effect pgh2) 25:595 prostaglandin inhibitors 30:192 clinical use of 30:192 use in rheumatism 30:192 use in osteoarthritis 30:192 use in headache 30:192 use in dental surgery anticancer clavulones 16:366 as antiviral compound 24:541-544 as pain mediator 30:192 in mast cells 30:192 nociceptive effect of prostate cancer prevention 30:525 role of lycopene protease inhibitor proteins from plants 29:596 effects on metallo protease inhibitors 24:487,488 567 cereal bifunctionals as 29:567 chymotrypsin as 29:568 destructive potential of 29:568 in alzheimer's disease 29:567 in angiogenesis 29:567 in cancer inflammatory disease 29:567 in protozoal infection 29:567 in viral infection 29:567 kunitz as 29:567 mustard family of 29:567 of metallocarboxypeptidase 29:567 ofserine protease 29:567 pepsin as 29:568 phytocystatins as 29:567 potato type 29:567 proteins as 29:567 serpin as 29:567 trypsin as 29:568 proteases 24:1005;29:567 enzymatic activity 24:1005 in apoptosis 29:567 in blood clotting 29:567 in cell division 29:567 in digestion 29:567 in extracellular matrix digestion 29:567 in inflammatory responses 29:567 protein kinase a (pka) 192 in regulation of ionotropic receptors protein kinase activity 28:677 ofxestoquinolide protein kinase c (pkc) activator 30:70 protein kinase c inhibition 24:573; 25:46,488 by xestocylamine 24:573 protein kinase inhibition 30:207 by pentacyclic triterpenes protein phosphatase inhibitors 27:874 protein phosphatases 25:707 inhibitor of 25:707 protein synthesis 30:407 inhibitor of 30:407 protein transduction domain (ptd) proteinaceous receptor 25:371 proteinase activity 21:150 protein-coupled receptor 27:821 g-proteine kinase c activity 21:638 proteins protein-tyrosine kinase activity 27:842 offlavonoid aglycones 27:842 of glycosides 27:842 ofkoelreuteria henryi protein-tyrosine kinase inhibitory activities proteolytic activity 30:841 proteolytic systemin inactivation protium kleinii 30:206 anti-inflammatory effect of 30:206 antinociceptive effect of 30:207 protocatechuic acid (3,4-dihydroxybenzoic acid) 29:589 effects on proton pump inhibitors (ppis) 25:612 proton-translocating nadh:q oxidoreductase 28:435 high-affinity inhibitors protozoocidal activity 30:329,741,742 against leishmania 30:329 against plasmodium 30:329 against tryponosoma 30:329 genus baccharis 30:741 in vitro 30:329 of neo-clerodane diterpenoids 30:742 ofquinone derivative 30:329 protozoocidal compounds 30:740 use of proxyelocytic leukaemia (hl-60) prunella vulgaris 30:401 antiviral activity of psammaplin a (bisprasin) 28:693 effects on bacillus subtilis psammaplysilla purpurea 28:693 antimicrobial activity of 28:693 as tyrosine kinase inhibitor pseudobersana mossambicensis 20:478 bioactive steroids from pseudoguaiane 11 a,13-dihydrohelenalin acetate 29:90 activity in cro assay system 627 against cytomegalo virus 30:627 against human immunodeficiency virus-1 30:627 against influenza virus 30:627 as virucidal agents 30:627 retroviral activity of 7:421 pseudolaric acid b 13:653 pseudomonas aeruginosa 30:739 antibacterial activity against 30:739 antifungal activity against pseudorabies virus 30:404 in vitro replication of 30:404 role ofheparin 30:404 psii inhibitors 26:368,371 psk in mitosis 25:377 psk-receptor 25:378 psk-a activity 25:378 psoriasis 30:484 use of lc~,25(oh)2d3 30:484 psychiatric disorders 30:369 anorexia 30:369 bipolar disorder 30:369 bulimia 30:369 effect of circadian activity on 30:369 obsessive compulsive disorder 30:369 panic disorder 30:369 schizophrenia 30:369 seasonal effective disorder 30:369 unipolar depression 30:369 psychodelic drugs 26:820 cocaine as 26:820 morphine as 26:820 semisynthetic lsd as 26:820 psychovegetative disorders 22:643,684 psycotria colorata 30:205 to relieve abdominal pain puberulin a 26:243,244 as paf-induced inhibitor pulegone 29:83 activity in am assay system 29:83 pulmericin 16:299 antifungal activity of 16:299 antileukemic activity of 16:299 cytotoxic activity 16:299 pulmonary vascular injury 30:70 role of serine protease in 30:70 role of thrombin pumiliotoxin alkaloids 27:238,239 alio-ptx 323 b alkaloid 27:249,250 biological activity of punta toro virus 30:411 treatment of 30:411 purifying blood 26:50 herniaria hirsuta in 26:50 purine receptors putative receptor proteins 25:398 putative satiety factor 24:910 effect of cholecystokinin 24:910 pyranocoumarin 27:429 biological activity pyranoid carbasugars 29:466 as cellular messengers 29:467 as insulin release mediators 29:467 biological activities of 29:466 effects on glycosidase enzyme 29:467 effects on glycosyltransferase enzyme 29:467 galactosyltrans ferase inhibition by pyridoacridine alkaloids 28:639 cytotoxic effects on kb cells 524 glycosidase inhibitors of 10:524 pyrrolomycin antibiotics 25:794 pytressin-induced coronary spasm antileukaemic activity of 4'-quercetagetin (6-hydroxy quercetin hexahydroxyflavone) (flavonol) 29:573 effects on 4'-pentahydroxyflavone) 29:578;30:208,749 analgesic effect of quercetin 3-o-c~-l-arabinopyranoside 28:64 anti-oxidant activity quercetin 3-o-c~-l-rhamnopyranoside 28:64,65 anti-oxidant activity of quercetin 3-o-[3-d-galactopyranoside 28:64 anti-oxidant activity o-galloyl)-glucoside 29:580 effects on ace quercetin-3-o-ot-l-rhamnopyranoside 28:64 anti-oxidant activity quercitrin (quercetin-3-rhamnoside) 29:581 effects on ace quillaia 26:55 of adjuvant activity 26:55 quinidine 25:347 quinine 25:344 290 antitumor activity of 10:117 inhibitor of dna quinoline drugs 25:348 quinoline drug family quinoline-based antimalarials 25:344 quinoline-ring antimalarial drugs 25:327 quinolines 25:327 quinolizidine derivatives 27:284,285 testing against mycobacterium tuberculosis 541-544 as antiviral quinone methides 30:694 antiplasmodial activity of 30:694 quinones rabidaea platyphylla 22:513 for epilepsy 22:513 rabies virus 30:409 inhibitor of 30:409 rabies virus infection 30:409 in chicken-embryo-related cells 30:409 rabies virus replication 30:409 in nerve fibers rachitic bone healing potencies 484 of vitamin d2 30:484 of vitamin d3 30:484 of vitamin d4 30:484 of vitamin d5 30:484 of vitamin d6 30:484 of vitamin d7 radical scavenging activity 23:362 of coumarin 23:335 radioligands 30:814 from brain membrane synaptosomes ranitidine 25:612,617 ranp-triggered signal transduction raphe nuclei 30:368 role in cognitive functions ras oncogenes 22:621 rat brain synaptosomes 30:807 p-affinity rat mammary carcinogenesis model 30:592 cancer chemopreventive activity rat serum vitamin d-binding protein (dbp) 30:494 binding assay for rebaudioside a 29:101 activity in tpa bioassay system tpa bioassay system 8-receptor 22:296 ~-receptor 22:296 8-receptor affinity 30:814 binding properties of 30:814 ~t-receptor affinity 30:814 binding properties of 30:814 of glycopeptide 30:814 8-receptor agonists k-receptor agonists 30:797,806 pharmacological activity of -receptor agonists 30:797 pharmacological activity of 30:797 8-receptor antagonists 30:797 pharmacological activity of k-receptor antagonists 30:797 pharmacological activity of 30:797 ~t-receptor antagonists 30:797 pharmacological activity of 30:797 receptor binding 27:377 receptor binding assay 30:810 in vitro 30:810 mouse vas deferens (mvd) activity in 30:810 of guinea-pig ileum (gpi) 30:810 receptor binding properties 30:815 of tyr-d-ala-phe-[[3-d-glc(oac)4]tyr-pro-ser-nh2 30:815 of tyr-d-ala-phe-asp-val-val-gly-nh2 (deltorphin c) 30:815 of oac)4]-gly-nh2: 30:815 of tyr-d-ala-phe-gly-tyr-pro-ser-nh2 (deltorphin c) 30:815 of tyr-d-ala-phe-gly-tyr-pro-thr([3-d-glc)-gly-nh2 30:815 of tyr-d-ala-phe-gly-tyr-pro-thr c )-val-vai-gly-nh2 receptor tyrosine kinase (rtk) 25:512 receptors 27:363;30:421 and cell-cell adhesion 30:421 and cell-cell communication 30:421 antigenic determinants of 30:421 for bacteria 30:421 ofchiral proteins 27:363 with enzymatic activity 27:824 without enzymatic recombinant prourokinase (pro-uk) 30:839 as fibrinolytic enzymes recombinant tissue-type plasminogen activator 30:839 as fibrinolytic enzymes 30:839 red wine 27:430 biological activity of 27:430 redox enzymes redox inhibitors 30:225 of 5-1ipoxygenase repandusic acid (hydrolysable tannin) 29:573 effects on hiv-1 protease 29:573 repellent properties 28:395 of amblyomma variegatum 28:395 reperfusion injuries 30:224 role of free radicals rescorcinolic lipids 30:165-167 effects on enzymatic activity 30:165-167 interaction with proteins reserpine 25:538 as immunosuppressive drug resistance-modifiers 30:595 antimalarial drugs as resistant plasmodium species 30:595 in chloroquine sensitivity resorcinolic lipids 30:119,158,160,162-164 as antifungal fluids 30:160 as growth reulators 30:162-163 as hair restoration-lotion 30:160 used in gingival infections respiratory ailments 24:847 respiratory depression 30:799 due to analgesic alkaloid 30:799 respiratory disorder 30:224 role of free radicals in 30:224 respiratory infection 30:408 respiratory syncytial virus 30:395 respiratory toxins 24:1002,1063 respiratory tract infections 26:50 herniaria hirsuta in 26:50 response decay mechanism 25:491 response modifiers resveratrol 27:430 biological activity of 27:430 resveratrol 28:582 effects in llc-bearing mice 28:582 effects on tumor 597 treatment of 30:597 use of hernandia moerenhoutiana in 30:597 use of hernandia nymphaeifolia 596 antimalarial activity of 30:595 antiplasmodial activity of 30:596 as platelet aggregation inhibitor 30:594 effect on antiplasmodial activity 30:596 retinal 29:111;30:520,521 activity in am bioassay system 29:111 in visual signal transduction 30:520 retinoic acid 29:111 activity in am, ebv, skin-1, odc bioassay systems 29:111 retinoic acid 30:493,498,521 in cell differentiation 30:521 in development 30:521 in growth regulation 30:521 retinoid retinol 29:111 activity in am retinol acetate 29:111 activity in am bioassay system retinol palmitate 29:111 activity in am bioassay system 29:111 retroviral enzymes 30:397 inhibitor of 408 causative agent of immunodeficiency syndrome 30:394 inhibitor of rett's disorder (rtt) 30:372,373,384 by mutations in mecp2 gene 30:373 genetic basis of 30:373 reutericyclin 28:127 as antibiotic reverse transcriptase 30:226,395,397 role in hiv replication cycle 30:226 role in viral replication rhazinilams 29:362 as microtubule poisons rheedia gardneriana 30:207 antinocicieptive effect of 30:207 rheumatism 26:50 herniaria hirsuta in 26:50 rheumatism 30:192,691 use of non-steroidal antiinflammatory drugs (nsaids) 30:192 use of prostaglandin inhibitors 30:192 rheumatoid arteriosclerosis 30:70 rheumatoid arthritis 21:152;22:310; 26:170,486;30:70,204,225 in treatment of cph82 26:170 in rats 30:204 reagent-induced 30:204 treatment of 30:225 use of kalopanax pictus rhoifolin (5, 4'-dihydroxy -7-rhamnosyl-glucosyl-flavone) 29:573 effects on hiv-1 protease 29:573 rhombenone 24:456 fptase inhibition by ricinoleic acid 30:193 analgesic action of 30:193 rickettsial pathogens 28:394 cowdria ruminantium as rifamycin-type macrolides 24:545,546 antiviral microbial-derived compound 24 rift valley fever infection 30:411 treatment of rimantidine 27:108 for treatment of influenza in infections 814 activity of deltorphin b in 30:807 of glycopeptide 30:814 rna virus 30:394 arenavirus type of 30:394 bunyavirus type of 30:394 coronavirus 30:394 influenza virus 30:394 measles virus 30:394 parainfluenza virus 30:394 picornavirus type of 30:394 rabies virus 30:394 reovirus type of 30:394 rotavirus type of rna-dependent rna polymerase activity 30:407 virion-associated rna-directed dna-polymerases 29:127 inhibition by sf-9 cell line 29:33 activity in mam-3assay system 29:100 antileukemic activity 8:18 sarcoptes mites 28:410 cause of human scabies 28:410 symptoms sarcoptes scabiei 28:409 cause of mange mites 28:409 cause of scabies sargasm horneri 30:400,405 anti-cmv activity of 30:405 polysaccharide (ps) from 30:400 sarkomycin 8:150 as antitumor agent 8:150 satratoxins 30:743 antiviral activity of 204 scavenging effects 23:443 of tannic acid 23:443 sch 207278 24:451 fptase inhibition by 24:451 schisandra 26:183 display platelet activating factor antagonist activity 26:183 effect on cardiovascular system 26:183 effect on heart rate 26:183 for treatment of hepatitis schisandrol a 26:245 as paf-induced inhibitor schistosomiasis (bilharzia) 7:405 schistosomicidal activity 1:545 schizonticidal drug 26:837 schizophrenia 101 activity in crg bioassay system 29:101 from scoparia dulcis scropolioside a 29:84 activity in tpa assay system 29:84 scrovalentinoside 29:84 activity in tpa assay system scutellaria baicalensis 30:55,56,69, 254,289 in allergric inflammatory disease 431 sdb 21:695 inhibitory effect of 21:695 sdc 21:695 inhibitory effect of 21:695 sdz-249-665 30:201 antinociceptive activity of 30:201 anti-hyperalgesic activity of 30:201 seasonal affective disorder 30:369 effect of circadian activity on 30:369 seaweeds 30:404 anti-cmv activity of activity in ebv assay system 29:100 1,2-secoemetine derivatives 6:485 amoebicidal activity of 6:485 secoiridoids 26:330 antimicrobial activity of 26:330 second messenger 25:516 secondary metabolites 28:3,432,617 acaricidal activity of 28:432 biological activity of 28:617 secondary metastatic tumor growth 30:64 effect of triterpenoids on 599 arnica as 22:559 artocarpus heterophyllus as basilicum polystachyon for 22:514 sedative activity 21:673 sedative in convulsions 22:518 crocus sativus l. for 22:518 sedative in epilepsy 22:535 withania somnifera l. as 22:535 g-selective agonist 30:807 dermorphin 30:807 8-selective antagonist tyr-tic-phe-nh2 selective antitumor activity 15:355 selective cytotoxicity 13:648 8-selective opioid agonist 30:817 deltorphin b 30:817 g-selective opioid peptide 30:802 8-selective opioid peptide of ici 19944 30:811 ofk opioids 30:811 8,g-selectivity 30:819 in vivo 30:819 of glyco analogues semilicoisoflavone b 28:229 biological activity of 28:229 semiochemicals semisynthetic lsd 26:820 as psychodelic drug 10-dihydro xy-7-methoxy-naphtho sendai virus infection sensory neurons 30:194 hyperpolarisation of 30:194 8er264 inhibitors serine 27:850 activitiy of serine 30:836 c~2-macroglobulin inhibition by serine protease inhibitor proteins 29:617 arrowhead pis api-a & api-b as cp-thionin as 29:617 effects on barley malt cysteine endoproteinases 29:617 effects on chymotrypsin 29:617 effects on subtilisin 29:618 effects on subtilisin eleusine double-headed try-~x-amylase inhibitor i-2 as 29:617 from brassica nigra 29:618 from brassica napus 29:618 from cassia fistula 29:617 from eleusine coracana 29:617 from hordeum vulgare 29:617 from phaseolus angularis from sagittaria sagittifolia 29:617 from sinapis arvensis 29:618 from vigna unguiculata 29:617 hordeum lipid transfer proteins as 840 blood clotting factors as 29:570 cathepsin g as 29:570 chymase as 29:570 chymotrypsin as 29:570 granzymes as 29:570 in angiogenesis 29:570 in blood clotting 29:570 in cytosolic proteolysis 29:570 in digestion 29:570 in inflammation 29:570 in proprotein processing 29:570 in tissue remodelling 29:570 kallikrein as 29:570 plasmin as 29:570 prolyl endopeptidases as 29:570 role in blood coagulation 30:70 role in platelet activation 30:70 role in pulmonary vascular injury serine proteases (subtilases) 25:388 cysteine proteinase inhibitor 25:370 poly phenol oxidase 25:370 serine proteinase inhibitor serine proteinase inhibitor i 25:370 as defense proteins serine-threonine-specific receptor protein kinases serotonergic innervations 30:377 autoradiographic imaging 30:377 in cerebral cortex 30:377 in neonatal rats by immunohistochemical techniques 30:377 physiological role of 377 serotonergic neurons 30:368,376 in raphe nuclei 30:376 role in 30:regulation of neurogenesis 627 as hyperforin inhibitor 30:627 as neurotransmitter 30:367 effect on synaptogenesis 30:378 in apoptosis 30:367 in cell proliferation 30:367 in presynaptic neuron 30:381 morphogenic properties of 30:367 neurochemical connection of 30:368 receptor activity of -ht) receptors 30:376,378, 382,383 in brain 30:383 in limbic brain regions 30:383 pharmacological aspects of serotonin (5-ht) re-uptake inhibitors 30:368 mechanism of serotonin (5-ht) synthesis 30:370,376, 377 in autistic children serotonin (5-ht) 1ar receptor antagonists 30:378 in posmatal treatment -ht)sa receptor 30:375 in brain development 30:375 in phenotypic behavioral irregularities 30:375 in purkinje cells 30:375 mrna expression of 30:375 serotonin action 30:378 on apoptosis 30:378 on maturation 30:378 on neuronal functioning 30:378 on proliferation serotonin agonists 25:531 anticomplementary activity of 25:46 role of cyclic amp-dependent protein kinase 25:46 delayed-type allergy suppressant activity of 25 serotonin synthesis inhibitor 30:378 parachlorophenylalanine as serum tc 27:420 biological activity of 27:420 serum transaminase 27:415,417,420 biological activity of 27:415 serum transaminase activity 25:469 of curcumia longa l serum triglyceride 27:404 biological activity sesquiterpene lactone 7:426 sesquiterpene quinones 5:429 antimicrobial activity of 5:429 cytotoxic activity of sesquiterpenoids drimane-type synthesis 29:127 avian myeloblastosis virus (amv) inhibition by 127 by jauch 29:127 sex pheromone sexual potency 28:4 ofbroussoflavonol g 28:4 sgot 30:292 activities of 30:292 sgpt 30:292 activities of 21:654 shikimic acid derivatives 29:478 antitumoral activity of 29:479 shinpterocarpin 28:229 biological activity of 28:229 sialic acids 27:103 biological function of 27:103 model for sialyltransferase activity 16:81 side effect 30:194,799 of analgesic alkaloid 30:799 of opioids sigmoidin a 28:229 biological activity sigmoidin b 28:229 biological activity of 28:229 signal reception 30:377 in autistic children 30:377 signal transduction mechanisms 25:518 signal transduction pathways signal transductors/activators of transcription (stats) 25:519 silandrin 26:255 as antihepatotoxic agent 26:255 silchristin 26:255 as antihepatotoxic agent 26:255 sildenafil 30:155 used for erectile dysfunction silymonin 26:255 as antihepatotoxic agent 26:255 simocyclinon 29:319 antibiotic activity of 29:319 cytostatic effects of 29:319 sindbis virus 17:135 sinomenine 25:472,476 hepatoprotective effect glycoside) 29:590 effects on pep 29:590 skin cancer 5:747 skin diseases 30:732 skin inflammation 30:323 anthrones used for 30:323 skk moth 1:704-706 juvenile hormone from slaframine 27:252 biological activity slaframine alkaloids 27:250,255 as muscarinic agonist 27:255 sleeping sickness smenoquinone 5:434,425 antimicrobial activity of smooth muscle relaxation 24:875-925 as biological action 24:875 of kampo medicines snake bite 30:691 treatment of 30:691 use of crossopetalum gaumeri 30:691 sobrerol 29:83 activity in mam-2 assay system 29:83 activity in gst assay system 29:83 activity in ras assay system 29:83 social phobia sodium salt of caffeic acid tetramer 24:742,743 anti-hiv activity of 24:742 soilborne fungi 21:181 bioactive metabolites from soilborne phytopathogens 21:182 biological control of 21:182 solandelactones 24:455 fptase inhibition by solanidane-induced teratogenicity 23:573 solid tumors neovasularization 25:593 somatostain 25:265 as a~ adenosine agonists somatostatin receptor 21:72;22:26; 25:530 type of g-protein-linked receptor 25:530 sonodione 30:566 from hernandia sonora 30:566 sores 30:616 tricyclic acylphloroglucinols sorivudine 24:474,486 as anti-viral agent 24 sorocein f 28:230 biological activity of 28:230 sos chromotest 22:623 soyabean saponins 25:222,223 hypocholesterdemic effects soyasaponins 27:398 anti-obesity action of 27:398 biological activity of 27:399 soybean trypsin inhibitor (sbti) spasmolytic activity 23:358 ;28:257,293; 30:264,561,748 of hernandia moerenhoutiana 30:561 ofhernandia voyronii 30:561 of imperatorin 23:350,358 of plant polyphenols 28:257,293 of thymus satureioides 30:264 on guinea pig ileum 30:748 on smooth muscles specionin 10:425 antifeedant activity of 10:425 spermicidal activity 26:53 of gypsophila paniculata sphingolipid receptor ca 2+ channels 25:535 sphingolipid receptors 25:534 sphinxolide 10:153;17:17 antitumor activity of 17:17 spider mite 1:702 hatching inhibitor spiroalkyl analogs 28:357 biological activity spirulina platensis 30:399,404,408 anti-influenza activity of 30:408 biological activity of 30:404 calcium spirulan from 30:399 polysaccharide (ps) from spleen/adipose tissue weight 30:56,57, 66 1203 effect of fucoidan on 30:56,57 effect of oleic acid on 30:65 spontaneous apoptosis 26:926 spontaneous metastasis squarroside a 26:31,50 immuno-modulatory effect of 26:31 from vaccaria segetalis squash family serine protease inhibitors 29:614 bd-ti-ii as 29:614 cmti-i as 29:614 cmti-iii as 29:614 cmti-iv as 29:615 cpgti-i as 29:615 cpti-ii as factor xiia, kallikrein, trypsin 29:615 effects on lysyl endopeptidase 29:614 effects on trypsin 29:614 effects on trypsin 29:615 effects on trypsin 29:616 effects on xa, xiia, kallikrein, plasmin, trypsin 29:614 elti-i as 29:615 from bryonia dioica 29:614 from citrullus vulgaris 29:614 from cucumis melo 29:614 from cucumis melo 29:614 from cucumis sativus 29:614 from cucurbita maxima 29:614 from cucurbita maxima 29:614 from cucurbita pepo 29:615 from cucurbita pepo 29:615 from ecballium elaterium 29:615 from echinocystis lobata 29:615 from lagenaria leucantha 29:615 from luffa acutangula 29:615 from luffa cylindrica cucurbitaceae) 29:616 from tricosanthes 29:616 hmti-i as 29:614 lati as 29:615 lati-ii as 29:615 lldti-i as 29:615 lldti-ii as 29:615 mcei-i as stachenone 29:101 activity in ebv bioassay system 29:101 standishinal 29:100 activity in ebv assay system 29:100 standishinal diacetate 29:100 activity in ebv assay system staphyllococcus aureus 30:693 minimal inhibitory concentration (mic) of staphyllococcus epidermidis 30:693 minimal inhibitory concentration (mic) of staphyllococcus saprophyticus 30:693 minimal inhibitory concentration (mic) of staphyllococcus warnieri 30:693 minimal inhibitory concentration (mic) of staphylococcus aureus 30:628,739 antibacterial activity against 30:739 antibacterial properties of 30:628 antifungal activity against starfish fertilisation inhibitor 28:712 callyspongin b as 28:712 stavudin 24:474,486,488 as anti-viral agent 24:474 steganes 29:360,399 cytotoxicity of 29:360,399 tubulin assembly inhibition by 29:360 antitubulin activity of 29:399,400 microtubules assembly inhibition by sterculia urens roxb. 30:405 antiviral activity of 30:405 steroid hormones 27:823 steroid saponins 7:426 teratogenic metabolites of 7:21-24 toxic cardiac active steroids sterol biosynthesis 25:303 inhibitor of 25:303 6a-sterol sulfate 28:702 cytotoxicity against sterol sulfates haplosamate a 28:703 as hiv-1 integrase inhibitor 28:703 sterols 25:43 antitumor activity of 25:43 antiinflammatory activity of 25:43 stevia stevia extract 27:307,310,314 for diabetics stevia leaves 27:315 immunological activity stevia plant 27:315 allergenic activity steviol 27:304,305,307,312 acute toxicity tests of 27:307 effect on fertility 27:312 mutagenicity tests with tpa bioassaysystem 29:101 activity in skin-1 bioassay system 29:101 acute toxicity tests of 27:307 allergenicity problems of 27:315 chronic toxicity studies of 27:306,307,308 effect on fertility 27:309,310 for phenylketonuria 27:315 mutagenicity tests for stigmasterol 30:208,505,570,719 analgesic effect of stilbene derivatives 27:405,409;28:579 biological activity of 27:405,409 in llc-bearing mice 28:579 tumor growth inhibition by 28:579 stimulatory 24:845 biological activity 24:845 stimulant agent 23:642 ofteucrium sp stimulus-response mechanisms 25:515 streptococci 29:535 pathogenicity of 29:535 serological groups streptokinase/streptodomase 25:271 streptolygidin 28:130 antibiotic activity of 28:130 as bacterial rna polymerase 28:inhibitor 130 as terminal dna transferase striga asiatica 30:162 infection with ofbioactive metabolites 21:251 of scopadulan-type diterpenoids structure activity relationship studies (sar) 30:195,494,800 of anandamide 30:196 of aspirin 30:195 ofopioid analogues 30:800 of vitamin d analogues 30:493 structure dereplication 29:658 bioactive natural product database in 29:670 of melanin inhibition strychnine 25:530 as immunosuppressive drug 25:530 strychnopentamine 26:1062 anticancer activity strychnos bisindole 26:1062 anticancer activity strychnos monoindole 26:1061 anticancer/protozoal subacute toxicity 27:307 from animals 27:307 of stevioside 588 effects on furin 29:588 sudhl-4-1ymphoma 21:138 sugar cane cystatins 29:593 effects on cysteine proteases 29:593 suicide inhibition 27:348 of 8'-hydroxylase 27:348 suicide inhibitors 9 sulfatobastadin 13 28:695 as endothelin a receptor inhibitor sulfide-containing pyrroloiminoquinones 28:685 as antileukaemic agents sulfur-containing cyclic peptides 28:678 biological activities of 28:678 cytotoxicity of sulphated polysaccharide (ps) 30:398, 400,405 anti-cmv activity of 30:405 anti-hiv activity of 30:398 antiviral activity against hiv 30:400 effect on viral replication 30:400 from marine sources 30:400 inhibitory effect of 30:398 mechanisms of action sulphated polysaccharide (ps) extracts 30:399 inhibitory activity of sulphonylurea drugs 25:534 sunflower cystatin phytocystatin 29:593 effects on cathepsin h ficin 29:593 effects on papain 29:593 sunflower multicystatin 29:593 effects on papain supercritical fluid extraction 21:576 superoxide dismutase superoxide dismutase activity 21:669 suppressing activity 30:308 on cells proliferation zizyphus saponin i as 27:41 zizyphus saponin ii sweroside 25:471,476 serum alt activity of 25 symbiotic marine microorganisms 23:185 bioactive metabolites 23:185 sympathetic ganglia 30:782 blockade of 30:782 sympathetic nerve activity 30:787 sympathomimetic amines 12:411 synapic potential mediation 28:318 by non-nmda receptors 28:318 synaptic actions 30:381 of serotonin synaptic dopamine transporters 25:539 synaptic glycine transporters 25:539 synaptic serotonin transporters 25:539 synaptogenesis 30:368,377 effect of serotonergic neurotransmission on 30:368 role in rodents 30:377 role of serotonin in 30:377 syndrome type-la 26:1130 adipose tissue distribution as 26:1130 cerebellar dysfunction as 26:1130 liver insufficiency as 26:1130 peripheral neuropathy as 26:1130 psychomotor retardation as synergistic activity 21:673 synergistic effect 21:608,611; 30:498,596 ofhervelines b 30:596 ofhervelines c 30:596 of retinoids 30:498 of vitamin d derivatives 30:498 synergistic interaction 21:698 of acyclovir(acv) 21:698 ofganciclovir (gcv) 21:698 with ganciclovir synergistic purgative actions 30:306 of rheinanthrone 30:306 of aloe-emodin-anthrone 30:306 synthetic pharmacological agents 30:224 antioxidant activity of 30:224 synthetic podolactones 28:484 allelopathic activity synthetic psychotropic agents 24:1093 syringaresinol 24:741 anti-platelet aggregation activity of systemic wound response protein 25:402 systemin activity 25:373 assay for ]-val-val-gly-nh2 30:815 receptor binding properties of receptor binding properties tyr-pro-ser-nh2 (deltorphin c) 30:815 receptor binding properties of -d-glc)-gly-nh2 30:815 receptor binding properties of oac)4]-gly-nh2 30:815 receptor binding properties of -d-glc)-tyr-pro-ser-nh2 30:815 receptor binding properties of -d-g lc )-v a 1-v al-gly-nh2 30:815 receptor binding properties of tyrosinase inhibitory activity 21:587 tyrosine kinase tyrosine-specific kinase 19:178 inhibitor of tyr-tic-nh2 30:809 activity in functional bioassay tyr-tic-phe-nh2 30:806 as 5 selective antagonist 30:806 u2 66 myeloma cell 25:273 ucn-01-ucn-02 12:386 antitumor activity of 12:395 protein kinase inhibitor ulapualides a 19:609 antifungal activity unsaturated carbapyranoses 29:475 as enzyme inhibitors 29:475 as herbicidal 29:475 biological properties of 29:475 unsaturated ketonucleosides 4:253 tumor inhibition by u-plasminogen activator (u-pa) 30:844 urdamycin a 11:134 antifungal activity of 11:134 antitumor activity ureterolithiasis-drug-therapy 27:382 urinary tract infections 26:50 herniaria hirsuta in 26:50 urine retention 30:194 as opioid's side effect 30:194 uroterpenol 29:83 activity in mam-2 assay system 29:83 activity in ras assay system ursane triterpene 29:585 effect on chy 29:585 ursolic acid 28:18,40;29:588 effects on hiv-1 protease 29:575 effects on lela 29:588 inhibition of hiv-1 protease dimerization by ursolic acid methyl ester (ursene triterpene) 29:575 effects on hiv-1 protease 29:575 usambarensine 26:1062 anticancer activity of 26:1060 uterine tumor 30:27 effect of pironetin derivatives in 30:27 uusambarine 26:1062 anticancer activity uvaol (triterpene) 29:588 effects on urs-12-ene-3,28-diol) (ursene triterpene) 29:575 effects on vaccaria segetalis 26:50 imunomodulatory effect of 50 vaccination 30:408 for respiratory infection as neuraminidase inhibitors 29:487 as c~-amylase inhibitor 29:486 c~-glucosidase inhibitors of 7:46;10:518 yeast a-glucosidase inhibitor of 10:518 valinuoctins 24:459 fptase inhibition by valiolamine 13:189,195 yeast c~-glucosidase inhibitor valiolamines 29:484 as glucosidase inhibitor 29:484 biological potential of 29:484 in clinical trials 29:484 in treatment of diabetes 29:484 valproic acid 22:507 vancomycin 25:791 vancoresmycin 28:150 activity against gram-positive bacteria vanilloid receptor agonist 30:202 olvanil as 30:202 capsaicin as 30:202 structure of vanilloid receptor antagonist 30:201, 202 capsazepine as 30 varicella-zoster virus 30:394 cause of varroajacobsoni 28:387 toxicity of varroa mites 28:383 vascular physiological action 30:55,69 of natural products vasodilating activity 21:379 vasodilator 23:358 scoparone as vasodilator effect 25:595 of acetylcholine vasodilatory activity 30:275 ofsatujera obovata vasoprotective agent 22:443 vasoprotective effects 28:302 of green tea vateamine-2' [3-n-oxide 30:567, 590,595 activity in human cancer cell line 30:589 activity in murine cancer cell line 30:589 as platelet aggregation inhibitor 30:594 cytotoxic effect of 30:589 veinotonic activities veraguensin 26:230 trypanosomicidal activity verapamil 25:534,673 as caz+-channel blocker 25:487 vermisporin 28:125 antimicrobial activity vernonia amygdalina 28:400 tick toxicity of 28:400 verrucarins 30:743 arenavirus junin 30:743 herpes simplex virus type ii (hsv-2) verrucosin 26:228 ofantiviral activity 26:228 vertebrate toxicity 21:98 vesicant activity vesicular glutamate transporters 25:538 vesicular monoamine transporter type 2 (vmat2) inhibitor vesicular stomatitis virus (vsv) 30:743,752 replication of 30:743 vesititol 28:224 anti-microbial activity of 28:224 vestitol 28:243 anti-helicobacter pylori activity of 28:243 veterinary medicine 28:383 for ectoparasites control 28:383 vetiver grass 28:399 for controlling ticks viagra (sildenafil) 25:541 vicolides 29:89 activity in ctn bioassay system 458 anticancer activity of 14:805 viral diseases 30:732 viral dna 30:226 integration of 12:332;21:98,193, 263,270,312,398,430,585,589,591, 594,643,659,670,673,675,690;23:59, 216,808;26:217;28:481 ;30 :629 against bone resorption 21:720, 721 against h+,k+-atpase 21:719 against staphyllococcus aureus 30:629 mechanism action of 30:225 of (-)-(8,8a-di-epi-swainsonine 12:325 of (-)-swainsonine 12:327 o f (+) cyasterone activity in 29:32 2-deoxy-20-hyroxyecdysone activity in 29:32 14-deoxymuristerone a activity in 29:32 2 [3,3 [3-dihydro xy-513-chole st-7en-6-one activity in 29:32 3 [3,14a-dihydroxy-5 [3-cholest-7en-6-one activity in 29:32 ecdysone activity in 29:32 313-hydro xy-5 [3-cho le st-6-one activity in 29:32 20-hydroxyecdysone activity in 29:32 inokosterone activity in 29:32 kc cell bioassay as 29:30 makisterone a activity in 29:32 muristerone a activity in 29:32 podecdysone a activity in 29:32 ponasterones activity in 29:32 213,313,513,14 a-tetrahydro x ycholest-7-en-6-one activity in 29:32 2[3,3 ~ ,25-tridihydroxy-513cholest-6-one activity in 29:32 viticosterone e activity in 29:32 insect control 21:611 insect juvenile hormone analogues 14:391-397 from thujone 14:391-397 insect pheromones 11: [415] [416] [417] via diisopropylethanediol esters 11:415-417 insect repellant 5:757 insecticidal activity 18:229,704;21:252, 254,262,266,267,269,270,271,278, 285,379,595,611;23:666;667,670, 677,678;24:25,799,845,866;26:231, 232,426,471,479,485;28:477; 30:153-154 activity of (-)-deoxypodo phyllotoxin in 30:589 activity of (-)-hernone in 30:590 activity of (-)-nymphone in 30:590 activity of (-)-yatein in 30:590 activity of (+)-corytuberine in 30:589 activity of (+)-epiaschantin in 30:590 activity of (+)-epimagnolin in 30:590 activity of (+)-epiyangambin in 30:590 activity of (+)-hernovine in 30:589 activity of (+)-laurotetanine in 30:589 activity of (+)-magnoflorine in 30:589 activity of (+)-malekulatine in 30:590 activity of (+)-n-hydroxyhemangerine in 30:589 activity of (+)-n-formylnornantenine in 30:589 activity of (+)-n-formyldehydroovigerine in 30:589 activity of (+)-n-formylovigerine in 30:589 activity of (+)-ovigerine in 30:590 activity of (+)-ovihernangerine in 30:590 activity of (+)-vateamine-2' [3-noxide in 30:590 activity of 7-formyldehydrohernangerine in 30:589 activity of 7-formyldehydronornantenine in 30:589 activity of 7-formyldehydroovigerine in 30:589 activity of 7-hydroxy-6-methoxy1:467,468;2:278,279,286, 289;6:108;7:103,110,121-123; 17:235,244 ;29:87 ;30:193,206 activity in ebv assay system 29:87 activity in skin 1 assay system 29:87 activity in crg assay system 29:87 activity in cro assay system 29:87 activity in aa assay system 29:87 activity in brk assay system 29:87 activity in cps assay system 29:87 activity in htm assay system 29:87 activity in paf assay system 26:3,4,10,13,15, 18,27,30,42,54,57;30:407 anti-inflammatory activity of 26:27 antiviral activity of 30:407 saponins 1:305; 402; 7:155, 156, 190, [426] [427] [428] [429] [430] [431] [432] 434, 435; 18:649 ajugasterone c activity in 29:28 amarasterone a activity in 29:28 amarasterone b activity in 29:28 cyasterone activity in 29:28 ponasterone a 2p-glucoside activity in 29:28 pterosterone activity in 29:28 rubrosterone activity in 29:28 sengosterone activity in 29:28 stachysterone c activity in 29:28 capitasterone activity in 29:28 sarcophytol a 29:100 activity in ebv assay system 29:100 activity in colon-2 assay system 29:100 activity in liver-2 assay system 29:100 activity in lung-1 assay system 29:100 activity in mam-3 assay system 29:100 activity in pancr assay system 29:100 activity in skin-2 assay system 29:100 sarcophytol b 8:18;29:100 activity in skin-2 assay system 29:100 use of platycodon grandiflorum key: cord-001927-jt81i0uc authors: ali, abdelwahid saeed; al-shraim, mubarak; al-hakami, ahmed musa; jones, ian m title: epsteinbarr virus: clinical and epidemiological revisits and genetic basis of oncogenesis date: 2015-11-03 journal: open virol j doi: 10.2174/1874357901509010007 sha: doc_id: 1927 cord_uid: jt81i0uc epstein-barr virus (ebv) is classified as a member in the order herpesvirales, family herpesviridae, subfamily gammaherpesvirinae and the genus lymphocytovirus. the virus is an exclusively human pathogen and thus also termed as human herpesvirus 4 (hhv4). it was the first oncogenic virus recognized and has been incriminated in the causation of tumors of both lymphatic and epithelial nature. it was reported in some previous studies that 95% of the population worldwide are serologically positive to the virus. clinically, ebv primary infection is almost silent, persisting as a life-long asymptomatic latent infection in b cells although it may be responsible for a transient clinical syndrome called infectious mononucleosis. following reactivation of the virus from latency due to immunocompromised status, ebv was found to be associated with several tumors. ebv linked to oncogenesis as detected in lymphoid tumors such as burkitt's lymphoma (bl), hodgkin's disease (hd), post-transplant lymphoproliferative disorders (ptld) and t-cell lymphomas (e.g. peripheral t-cell lymphomas; ptcl and anaplastic large cell lymphomas; alcl). it is also linked to epithelial tumors such as nasopharyngeal carcinoma (npc), gastric carcinomas and oral hairy leukoplakia (ohl). in vitro, ebv many studies have demonstrated its ability to transform b cells into lymphoblastoid cell lines (lcls). despite these malignancies showing different clinical and epidemiological patterns when studied, genetic studies have suggested that these ebvassociated transformations were characterized generally by low level of virus gene expression with only the latent virus proteins (lvps) upregulated in both tumors and lcls. in this review, we summarize some clinical and epidemiological features of ebvassociated tumors. we also discuss how ebv latent genes may lead to oncogenesis in the different clinical malignancies epstein-barr virus (ebv) is classified as a γ-herpes virus [1] and contains a linear dna molecule about 172 kilo bases in length. it affects more than 90% of adult population worldwide [2] . first exposure to the virus, before or after adolescence, occurs via exchange of saliva or contact with the airborne virus [3] . if the infection does not become clinically silent, infectious mononucleosis is experienced by ebv-exposed persons. this infection was reported worldwide but predominantly in the developing countries and by around 18 months of age, the majority of children in developing countries are found exposed to the virus [4] . although ebv infection is lifelong, pathogenicity in the host is limitted unless the immune sysytem is compromised. reactivation of latent ebv results in recurrent infections of a clinical nature. the diseases include those of a lymphocytic nature, namely infectious mononucleosis, hodgkin's disease (hd), burkitt's lymphoma (bl), post-transplant lymphoproliferative disorders (ptld) and t-cell lymphomas and those of an epithelial nature such as oral hairy leukoplakia (ohl), nasopharyngeal carcinoma (npc) and undifferentiated gastric carcinoma [5] . the severity of ebv infection and level of virus titer vary with the carcinoma type with the highest titer for ebv being observed in undifferentiated carcinomas [6] . infectious mononucleosis (synonyms include pfeiffer's disease, filatov's disease, kissing disease, glandular fever or "mono" in north america) [7] is a primary self-limiting clinical syndrome that most frequently affects young adults, although other age groups can also be infected. the classic clinical symptoms of this condition include sore throat, fever, fatigue, malaise, vomiting and loss of appetite with common pathological lesions including lymphadenopathy, spleenomegaly, hepatomegaly and jaundice, all of which may be detected in older adults [8] . burkitt's lymphoma (or burkitt's tumor) is a tumor of the lymphatic system (specially b lymphocytes). the name was coined after denis parsons burkitt, who firstly described the disease in 1956 in equatorial africa [9] . currently, bl is divided into three main clinical forms: the endemic, the sporadic and the immunodeficiencyassociated bl and all forms related to ebv infectivity in african and non-african countries [10, 11] (fig. 1) will be described in detail later. hodgkin lymphoma (hl) is a cancer which originates from the germinal center of b cells with a unique histological picture that comprise inflammatory fields with a minority of neoplastic cells. it differs from the other b cell lymphomas with its characteristic clinical and pathological manifestations [12] . post-transplant lymphoproliferative disorder (ptld) is also a kind of b-cell immortalization attributed to immunosuppressive status following chemotherapy for organ transplantation surgery. such patients can suffer from infectious mononucleosis-like lesions or may develop polymorphic b-cells proliferation which later may become malignant due to additional mutational changes. this disease which is characterized by uncontrolled proliferation of b cells was known to have a strong association with ebv infectivity [13] . although, ebv was originally isolated from burkitt's lymphoma biopsies, a major b cell lymphoma, it is now considered to have a role in several other epithelioid malignancies [14] . oral hairy leukoplakia (ohl) is a mucosal tumor firstly recognized and described in 1984. it is a pathological lesion proved to be associated with ebv replication and mostly occurring in people with hiv infection [15] . it was also reported in hiv negative patients when firstly observed in 1996 in a patient suffering from systemic lupus erythromatosis [16] . nasopharyngeal carcinoma (npc) is the most common malignant tumor of the nasopharynx, worldwide, also known to be caused by ebv. this carcinoma varies from the others of the head and neck in incidence, causes, clinical pictures and management. it is more common in males than females and in the east asians and africans than other racial groups of people [17] . ebv infection was also known as the cause for a reasonable percentage of gastric carcinomas worldwide [18, 19] . the role of ebv in gastric carcinomas was confirmed by detection of the viral gene products like the ebv-encoded small rna (eber) in these tumors, in addition to the presence of clonal ebv [20, 21] . additionally, an in vitro ebv infection of resting b cells was known to lead to proliferation, immortalization and consequently to lymphoblastoid cell lines (lcl). these lcl were also shown to be latently infected with ebv, and hence provided a suitable laboratory model for investigation of ebv latency and virus-driven b cells carcinogenesis [22] . lcl have also served as ebv antigen presenting cells in several immunologic methods and tests [23, 24] including the development of human monoclonal antibodies [25, 26] . the efficiency of ebvmediated ctl proliferation increased with the use of mitogens like phytohemagglutinin and lipopolysaccharide [27] , pokeweed mitogen [28] and some immunosuppressive drugs like the cyclosporine a which prevents the t cell-mediated cytotoxicity of ebvinfected b cells [29 -31] . in a variety of studies, ebv was proved to perform its oncogenic capacity by an expression of what is known as latent genes, namely the latent membrane proteins (lmp1, lmp2a, and lmp2b) and the ebvdetermined nuclear antigens (ebna1 and ebna2). lmp1 was reported as the major oncogenic factor of npc development and were detected in 80%-90% of npc tumors [32] . in addition to its direct oncogenic potential, lmp1 was also known to play a role as an immunosuppressive agent against npc, which allows npc to develop quietly [33, 34] . the importance of lmp1 as an oncogenic factor was confirmed in several studies by demonstrating that tumor cells are much more sensitive to chemotherapeutic agents when lmp1 expression was inhibited [35] . previously, the exact contribution of lmp2 and ebna to cellular tumorgenesis was uncertain but later, due to more advanced research approaches, the role of these factors in the ebv-induced tumorgenesis is well-studied and recognized. early data suggested that lmp2 was required for tumor cell survival but the more recent data showed that lmp2 plays more diverse and critical functions in the process [36] . lmp2a was proved to downregulate the expression of the transcription factor of nf-κb-resulting in a decrease of lmp1 expression [37] . in addition, lmp2a is responsible for npc becoming more migratory and invasive [38] . ebna1 is the factor reported to bind the viral genome to the cellular genome, and in so doing, linking viral dna replication with the cells division [39] . ebna2 was known to serve as a powerful lmp1 transactivator [40] . many previous studies have led to the wellestablished fact that ebv is responsible for tumorgenesis in lymphoid and epithelial cells both during the natural course of infections as well as in the induced lymphoblastoid cell lines (lcl). in this review, we attempt to summarize the clinical outcomes and some epidemiological features associated with the different tumors induced by ebv in both lymphoid and epithelioid cells. we also demonstrate the exact genetic elements involved and the roles played by each separate genetic entity during the transformation process. the history of ebv infection goes back to 1958 when the english surgeon, denis burkitt, who was working in uganda, observed and reported a frequently occurring cancer affecting the children in his work area and equatorial africa in general [9] . this cancer was later officially named burkitt's lymphoma (bl) or burkitt's disease (bd) after the infection was recognized and scientifically well-established. a strong correlation between the distribution of bl and the climatic and geographical conditions in the infection areas was documented; consequently an idea of a vector borne virus responsible for the condition was suggested [41] . afterwards, using an electron microscopic examination of a biopsy from bl, epstein, achong and barr isolated and identified herpes virus-like particles and hence the virus name in 1964 [42] . as confirmatory evidence, it was shown by serology, in the late 1960s, that bl patients had high antibody titers to the antigens of ebv [43] . using serological assays, ebv was then also confirmed as the causative virus for infectious mononucleosis and it was also demonstrated as a common etiological agent for undifferentiated npc development and many other cancers [44, 45] . the transformation capability of ebv was later recognized and documented when the virus was shown to transform resting b cells efficiently in vitro and also cause tumors in other primates [46 -48] . subsequently, many research outputs have indicated that ebv is responsible for different human tumors of b cells origin such as hodgkin's disease (hd) and the lymphoproliferative disorders observed in immunosuppressed individuals. some t-cell lymphomas and epithelial malignancies were also linked to ebv as will be discussed later. multiple copies of the circular ebv genome were detected in the tumor cells of all these malignancies and ebv-latent gene expression was confirmed as a major marker in the development of these malignant conditions [49] . in conclusion, to confirm the role of the ebv in the neoplastic transformation of mammalian cells, many research studies pertaining to virology, clinical and the genetics of the virus were intensively carried out by researchers in many parts of the world. they are complied and summarized here for the purposes of this review. in total, herpes viruses have been studied intensively at the genetic levels at various points in time [50 -52] . ebv, in particular, received a special attention for genetic studies and analysis when recombination techniques became available for the virus in mammalian cells [53 -55] . the different genetic components of the ebv genome, and their functions were identified following generation of mutants through cloning techniques. when characterized some of these mutants were also found to have potentially useful medical properties such as those suggested as vaccine candidates [56] . the link between the various genes and biological behaviours such as the transforming property of the ebv was documented in many studies [57 -59] . the ebv genome consists of double-stranded, linear dna molecule coated by the capsid protein and surrounded by the tegument layer which is surrounded by an envelope composed of bilayer proteins sandwiched by lipids [60] . the size of the genome varies; it was first reported that ebv genome is about 172 kilo base pairs (kbp) in length [61] but later it was shown to be about 192 kilo base pairs and to encode about 85 genes [62] . among all herpes viruses, the ebv genome was the first to be sequenced completely for many strains of the virus [61, 63 -65] and recently mapped [66] . a series of repeated dna sequence accounts for 0.5 kb as terminal repeats at the end of the linear genome observed in the nucleus of cells during latency of the virus in infected cells. they are about 10-20 copies for each infected cell [67] . long and short sequence domains in the ebv genome were also reported as divided by internal repeat sequences (irs) and strain variation in the repeat copy numbers was also noted [68] . based on the sequence organization of the ebna 2, 3a, 3b, and 3c genes of ebv, two subtypes of the virus have been recognized [69] . the prototype strain of ebv, named as b95-8, was sequenced and the open reading frames (orfs) and the sites for the transcription processes are readily recognized and the virus has proved to have a coding potential of around 80 characterized and non-characterized proteins [61] . after analysis of the sequences of other ebv strains, more four open reading frames (bvlf1, bdlf3.5, bfr1a and bglf3.5) have been identified. the function of bvlf1 and bdlf3.5 are unknown so far and bfr1a was suggested to play a role in the dna packaging whereas bglf3.5 was recognized as a tegument protein [70] . generally, various aspects of the ebv genome have been intensively studied to correlate the basic genetic components and their expression with the biological behaviour of the virus, which include infectivity and pathogenesis, immune responses, latency and reactivation and the virus transformation potentia. these studies were especially possible after the advent of molecular approaches in virology. in order to replicate, ebv requires sequential steps of viral protein synthesis. the first expressed proteins are the immediate early proteins which serve as transactivators that enhance the synthesis of the early and late proteins required for dna replication and production of the progeny infectious virus. the two major immediate early protein transactivators are named zta and rta and they function in the initiation of the lytic ebv replicative cycle [36, 71] . the significance of these two transctivators for ebv replication has been well documented through mutants deficient in either zta or rta which showed only poor replication levels [72] . both lymphoid and epithelial cells can be infected by ebv through different attachment and entry mechanisms. to enter b lymphocytes, viral attachment protein (vap), also called gp350, binds to the cd21 receptor on the cell membrane; additionally, the viral gp42 also attaches to the mhc class ii molecules at the cell surface. both of these interactions allow the viral envelope to fuse to the cell membrane and consequently the virus gains access to b cells [73, 74] . for ebv entry into epithelial cells, viral protein bmrf-2 interacts with the β1 integrins in the cell membrane, and viral proteins gh and gl interact with the cellular αvβ6/8 integrins and these two processes enhance the fusion between the epithelial cell membrane and the viral envelope and ultimately result in the virus entering the cell [75]. following entry of ebv into the cell, an uncoating process takes place in the cytoplasm in which the virus sheds the protein coat and the viral dna will be transported to the cell nucleus. in both types of cells, ebv can undergo lytic replication cycle resulting in the production of infectious virions. the lytic replication of ebv in b cells is usually observed to occur following reactivation of latent virus whereas in epithelial cells it has been observed to happen directly in primary infection after viral entry to the cell [60] . the ebv circular latent genome turn to a linear form after reactivation to adopt the lytic replication cycle. three consecutive stages are required during ebv replication with three types of genes expressed; immediate-early (transactivators), early gene products include bnlf2 serving in replication, metabolism and blockade of antigen processing and the late gene products which express the structural viral proteins like the capsid and the bcrf1 protein, which helps the virus to escape the host immune response. generally, ebv lytic replicative cycle does not necessarily result into lysis of the cell as the virus is released from the infected cell by budding [60, 76] . thus while the ebv replicative cycle shows different features at the early phase of replication and entry processes, it adopts similar pathways to complete the cycle in either the lymphatic or epithelial cells. ebv is more likely transmitted from infected host to the susceptible host via saliva. following the primary infection, ebv can replicate extensively in epithelial cells of the nasopharynx and consequently cell lysis releases the virion, and the virus spreads to salivary glands and oropharyngeal lymphoid and epithelial tissues. b lymphocytes are then infected as they circulate in close to oropharyngeal epithelial cells. the virus then circulates throughout the body through the infected b cells. within the b cells, ebv may either lead to a series of lytic replication cycles or it may show differential expression of the latent genes which will allow the virus to become latent in the cells [77, 78] . as a result of this continuous replication of the virus, viremia will occur and consequently the lymphatic system such as the peripheral b lymphocytes, spleen, liver and lymph nodes will be infected so that ebv can remain latent in these sites as well [49] . when ebv establishes itself as a latent virus it does so as an episomal virus in which the viral genome is found as a self replicating extra chromosomal dna and is thus known as an episome and latency recognized as episomal latency [79] . depending on the differential expression of ebv-specific genes, three different types of latencies were recognized, latency i, ii and iii [49] . for several reasons relating to immunosuppression, ebv sporadically reactivates from latency [77, 78] . following reactivation, ebv shedding from saliva of infected host will occur intermittently and hence saliva constitutes the major source for transmission from infected to healthy individuals; however, transmission of the virus via blood transfusion, sexual contacts and tissue transplantation have been also reported [80, 81] . both in vivo and in vitro infection of ebv leads to transformation and immortalization of b cells [49] . the in vitro transformation of b cells can be diagnostically confirmed through the ebv-specific immunofluorescence assay [43] while the in vivo transformation resulting in several kinds of malignancies detailed below. subsequent to b cell immortalization by ebv infection, t suppressor lymphocytes were also observed to play a cytotoxic role and hence the viral counts are reduced with a dramatic reduction in the circulating b cells. as a herpes virus, ebv was known to establish latency after the primary infection in both b cells and epithelial cells where it persists as an episome in the nucleus without productions of virions. the linear dna molecule of the virus circularizes and only some ebv genes are expressed during latency stage. as mentioned earlier three different latency programs (i, ii and iii) were reported for ebv; during each program the virus expresses a set of a latency associated genes. the latency programs adopted by the virus in b cells are different from those in epithelial cells and for each type of latency, a distinct set of viral proteins and transcripts will be produced [82, 83] . within b cells, all three latency programs can take place, starting from latency iii to latency ii to latency i, whereas upon epithelial cells infection only the latency ii program is found. when ebv infects the a b cell, the latency iii program starts first with a considerable number of proteins expressed to cause cell transformation. less gene expression occurs in the move towards latency ii to differentiate the b cells into memory cells and finally lesser genes expressed to enter the stage of latency i [84]. the ebna-1 protein is expressed by the ebv during latency to allow the ebv genome to replicate during memory b cell division. the latency associated with ebv infection of b-lymphocytes is an essential mechanism for virus persistence. the replication of ebv in epithelial cells is necessary for the release of infectious virus into saliva. it was wellestablished that the ebv latency iii and ii infections is a characteristic features of b-cells whereas latency ii is that of the oral epithelial cells and nk-or t-cells all of which can result in different kinds of malignancies [85] . the ultimate and exact requirement of ebna2 and lmp1 in transformation of b cells in vitro studies was confirmed by using of an ebv recombinant deficient in some latent genes. a substantial role for ebna-lp, ebna3a and ebna3c in latency process was also suggested when these recombinants were used [86] . the expression of ebv-latency associated genes during the latency programs is summarized in table 1 . ebv latent infection in b cells can reactivate due to immunosuppression of the infected individual; it can also reactivate in vitro to switch to lytic viral replication and this has been accomplished by stimulating the b cell receptor through several mechanisms. it had been observed that patients with polymyositis or rheumatoid arthritis undergo ebv-associated lymphomas more frequently when treated with methotrexate (mtx) than when treated with other immunosuppressive agents. consequently, mtx was used for an in vitro reactivation and release of infectious ebv from latently infected b or epithelial cells [88] . in another study, the potential of monochloramine (nh2cl), produced during h. pylori infection to the gastric epithelia, to reactivate virus was also examined and the results suggested that nh2cl induces ebv lytic replication in gastric epithelium if latently infected with ebv [89] . several in vitro studies for ebv reactivation were done with cell lines derived from burkitt lymphoma as they are very permissive for the lytic cycle of the virus when treated with various immunosuppressive agents as compared to lcls [90 -92] . the issue of understanding ebv latency in infected cells and reactivation from latency as well as the mechanisms adopted for transformation of normal human b or epithelial cells into tumor cells are important for developing and establishing methods to prevent and pave the way for improved clinical management of these ebv-associated neoplasms. the various factors controlling ebv-associated oncogenesis can be categorized and summarized in the following: genetic: the oncogenic capacity and properties of ebv are recognized through its in vitro transforming effects. following infection of primary human b cells in vitro, ebv induces proliferation resulting into the development of lymphoblastoid cell lines (lcls). the genes of six ebv nuclear antigens namely ebna1, 2 3a, 3b, 3c and ebna-lp as well as three latent membrane proteins namely lmp1, 2a and 2b are expressed in these induced latently infected cells. likewise, these proteins are expressed at the early phases of natural infection [93, 94] . ebna2 is reported as the central transcription factor for both viral and cellular genes expression and is responsible for b cell proliferation and is expressed in lcls [95] . lmp1 expression, in turn, is regulated by ebna2 and serves as an active receptor for tumor necrosis factor, an essential apoptotic element [96] . lmp1was also reported to signal in the b cell similar to cd40-cd40 ligand interaction showing similar functional properties with activated cd40 [97] . however, in the lack of ebna2 in ebvproliferating b lymphocytes exists, cd40 activation and lmp1 expression leads to similar phenotypic characteristics, the continuous survival of the cell [98] . on the other hand, interestingly both the activated cd40 and lmp1 expression also prevented b cells apoptosis [99, 100] . additionally, experimentation with a transgenic mice revealed that lmp1 mimics cd40 signaling of b cells differentiation during natural infection [101] . generally, the role of different ebvencoded latent genes in tumor formation is recently complied [102] and summarized in table 2 . transactivator of viral latent genes and host genes; responsible for episome replication, segregation and persistence of viral genome; involved in p53 degradation and oncogenesis. transcriptional co-activator of ebna-2-dependent viral and cellular gene transcription; it is essential for ebv-mediated b-cell transformation. activates viral and cellular gene transcription for transformation. it is critical for ebv-mediated b-cell transformation. a co-activator of ebna-2, downregulate cmyc transcription and block ebna-2 activation effects; and induce cdkn2 and chemokines. it is essential for ebv-mediated b-cell transformation. a co-activator of ebna-2; dispensable for b-cell transformation; viral tumor suppressor; and up regulates cxcl10. ebna-3bknockout induces dlbcl-like tumors. ates with ebna-2 host cxcr4 and cxcl12 genes; overcomes ebv-infection-mediated dna damage response; promotes cell proliferation; induces g1 arrests; it is essential for ebv-mediated b-cell transformation. mimics the constitutively active form of cd40, activates nf-κb, jnk and p38 pathways; is critical for ebv-mediated b-cell transformation. responsible for constitutive activation of the erk/mapk pathway224; blocks antigen-dependent bcr signaling; induces bcell lymphoma in transgenic condition. it is important but not essential for in vitro primary b-lymphocyte growth transformation. augments colony formation and induces growth; confers cells resistance to pkr-dependent apoptosis; induces cytokines and modulates innate immune response; contributes to ebv oncogenesis. viral: strain variation was suggested to be the main factor for the increased incidence of ebv-associated malignancies in specific populations. some ebv strains indeed possess more distinct biological properties as compared to others. the difference between ebv types 1 and 2 is clearly observed in sequence variation and other ebv variants have been shown based on the viral genome sequence polymorphism of ebna 2 and 3 [103, 104] . changes among different ebv strains also reside in sequence changes of ebna1, bzlf1 and lmp1 [105 -107] . investigations into ebv strain variation using restriction fragment length polymorphisms (rflp) compared ebv strains predominately responsible for npc in southern parts of china [108] . similar ebv genetic polymorphism was also reported when npc samples from alaska and caucasian america were investigated; but no such polymorphism was observed for npc samples from the mediterranean and africa [103] . genetic analysis for ebv-associated malignancies indicated that the hiv co-infection is another potential factor regulating the incidence of the lymphomas as hiv infection is associated, in most cases, with multiple strains of ebv [109] . environmental: the endemic patterns of many ebvassociated tumors were initially apparent as ebv was originally isolated from samples of african burkitt's lymphoma (bl) [42] and this tumor was observed with high incidence in subequatorial africa [41] . endemic cases were also found associated with chronic malaria and ebv infectivity, whereas sporadic cases mostly occurs outside of africa and were associated with the ileocaecal region, with the jaw less often affected as in the endemic type. arboviral infection and plant extracted herbs used as therapeutic agents for some diseases have also been suggested as potent tumor-promoting cofactors for ebv induced oncogenesis [110] . the parotid gland tumors and the t-cell lymphomas, due to ebv have been found to develop with increased frequency among greenland eskimos and taiwanese and japanese respectively [111, 112] . as per the age and gender as factors, patients with t-cell lymphomas were found either middle-aged or elderly, and no gender preference for this disease has been observed [113] . study of the epidemiology of ebv-associated nasopharyngeal carcinoma (npc) indicated high incidence occurrence in the southern chinese and an elevated incidence in inuit populations and in mediterranean africa [114] . the prevalence rates of npc were particularly elevated in populations of southeast asia natives, the arctic region, north african arabs and some parts of the middle east. this was suggested to result from the consumption of preserved food during early ages of life which could predispose to a high risk of npc development in these populations. risk factors for npc development like cigarette smoking, exposure to formaldehyde and wood dust have been also recorded [114] . the co-factors that contribute to the development of hodgkin's lymphoma (hl) and gastric carcinomas are currently unknown but environmental and immune components due to immune impairment are likely factors. it is likely that environmental or genetic factors increased infection of distinct susceptible cell populations and activate cellular pathways that are highly synergistic with ebv genes expression which affect cell growth [115] . it has been documented in several reports in the medical literature that ebv is frequently linked with many b cells lymphomas, mostly observed in immunosuppressed individuals. although they look similar on pathological grounds, these ebv-linked bcells lymphomas were shown to heterogeneous on the molecular and clinical levels, as discussed in the following sections: burkitt's lymphoma: this lymphoma was the first malignancy known to be attributed to ebv. it was firstly observed in central africa and due to its strict correlation with malaria endemicity, it was suggested that an infectious organism might be involved. eventually and following ebv recognition, it was confirmed that ebv is the major cause of bl. the natural history of this association was recently reviewed by moormann et al. 2011 [116] . the precise contribution of ebv in the pathogenesis of this lymphoma has been extensively documented. despite the fact the ebv was detected in biopsies of some bl, some other studies suggested that ebv infectivity occurs before b cells proliferation to induce malignant conversion [117] . ebv is also suggested to have a powerful role in the sporadic form of bl as the episomal defective genome of the virus have been isolated from some sporadic cases of bl tumors in usa [54] . bl has been reported to occur with an incidence rate of about 5-10 cases per 100,000 children annually in equatorial and central africa where it became endemic [110] . higher incidences of this endemic form of bl were also for north africa, south america and the middle east regions [118] . less endemic situations were observed in african countries like egypt [119] and algeria [120] where the incidence of malaria is also at a low level. generally, sporadic bl occurred worldwide but at a lower frequency as compared to the endemic form [110] . although it can affects adults, bl most commonly affects children where it constitute the majority of b cell lymphomas during childhood, whereas in adults it accounts for less than 10% of all lymphomas [121] . the incidence is reported to peak at the ages of about 5-8 years old and is more commonly observed in males as compared to females [122, 123] . at the cellular level, chromosomal translocations within b cells are the consistent genetic feature of all bl tumors. these translocations occur in the region of the cmyc oncogene (mainly involves the 8q24 of the long arm in the chromosome number 8), and in the region of the immunoglobulin heavy chain gene (in the chromosome 14) or in the region of the immunoglobulin light chain genes (in chromosome 2 or 22). the activity of immunoglobulin genes together with the germinal centre of b cell proliferation stimulated through ebv infection were all suggested to be major attributing factors for selection, generation and proliferation of b cells which basically harbor a c-myc translocation [110] . at the virus level, the only protein factor being consistently expressed and detected during bl formation is the ebna1 protein [35, 124] . in another study, lmp1 together with the ebna2 were reported expressed in a small numbers of cells in some cases of the endemic form of bl [125] . in an in vitro culture of some ebvpositive bl tumors, other ebnas were also seen expressed beside the lmps [35] . clinical presentation of endemic bl mostly includes obvious tumors in the mandible and maxilla with the abdominal organs being involved less frequently. patients commonly showing swelling of the affected jaw bones and the lymph nodes in the neck, and jaws are rapidly enlarged without tenderness [126] . in the sporadic cases of this lymphoma, abdominal and pelvic organs (namely ileum, caecum and mesentry) are usually involved with the other abdominal and glandular tissues (e.g. tonsils and thyroid) seen affected in some cases but to a lesser extent [127] . patients of bl are commonly presented to the gastroenterology clinics with abdominal pain, ascites, abdominal distension and signs of intestinal obstruction. bl has also been observed to occur as a consequence of immunodeficiency, mostly in people with hiv/aids infections where it accounts for 30% to 40% of non-hodgkin lymphoma [128] , so much so that it used to be a clinical marker for aids. bl was also detected with conditions of congenital immunodeficiences and organ transplants patients who take immunosuppressive drugs [129] . for accurate diagnosis of bl, lesion biopsy is always performed and csf and bone marrow are usually examined. cytology of pleural fluid and ascitis was routinely employed to arrive to a definitive diagnosis. radiographic applications such as the magnetic resonance imaging (mri) and ct scanning are applied at diagnosis and then throughout the infection course. molecular biological techniques were mostly employed to distinguish between bl and the other large b cells lymphomas [130] . although endemic bl was known to be highly sensitive to chemotherapy, sporadic and immunodeficiency-associated bl are less sensitive [122] . different regimens of chemotherapy have been tried with different success rates as described by okebe et al. in 2006 [131] . intensive chemotherapy resulted in a very successful treatment for children with bl. in adults, good results were obtained when chemotherapeutic regimens were used in combination with rituximab (monoclonal antibody) [132] . non-intensive chemotherapy with immunosuppressive agents such as cyclophosphamide and methotrexate was proved useful to manage the endemic form of bl in developing countries in africa as reported by beogo et al. in 2011 [133] . in sporadic or immunodeficiency-related bl, lowintensity infused doxorubicin, cyclophosphamide with vincristine, prednisone and rituximab treatment was seen highly effective, particularly in adults [134] . bone marrow or stem cell transplants are proposed in recurrent cases of bl and surgery had also been proposed and used if intestinal obstruction is evident and if the abdominal tumors are small and absolutely resectable. the mainstay action for prevention of bl is the vaccine development for ebv and malaria, particularly where malaria is endemic [126] . hodgkin's disease (hd): the first report that suggested hd (also known as hodgkin's lymphoma, hl) is attributable to an infectious microorganism is made by macmahon in 1966 [135] . later, and due to detection of high levels of antibody specific to ebv antigens in the sera of patients with this lymphoma, the infectious agent incriminated as the cause of hd was claimed to be ebv [136] . furthermore, it was shown that high levels of antibodies to ebv antigens were detected several years before hd development [12] . as a matter of confirmation, several other investigations to confirm the association between hd development and ebv via in situ hybridization techniques were carried out and reported [57, 137, 138] . despite the fact that ebv was detected over the course of hd, cases of ebv-negative hd as a relapse of the previously ebv-positive hd lymphoma have been observed and reported [139] . explicit epidemiological and clinical differences between the ebv-associated and ebv-negative forms of hd tumors has been demonstrated and it has been claimed that hd due to ebv is characterized with multicellularity and commonly observed more in males than females [140] . another interesting epidemiological feature of hd is that, in usa, ebv-related hd was more commonly observed among hispanics and asians than whites or african americans populations [33] . similar findings were observed in studies in the uk where an intimate association between ebv-positive hd and south asian racial groups of people was noted especially in children [141] . as for age susceptibility, it was observed that ebv-positives cases of hd were more commonly seen in older patients (above 55 years) and younger children (below 10 years) whereas in young adults it is mostly ebv-negative [142] . this fact has led scientists to categorize hd into three disease forms based on the age group of patients: childhood hd which is ebv+ve (known as mc type), young adults hd which is ebv-ve (known as ns type) and older adults hd which is also ebv+ve (also known as mc type) [142] . the development of hd also correlated with acquired or congenital immunosupprression [39] and the overall incidence of hd was reported to be higher in hiv and/or aids patients with most of these lymphomas recognized as ebv-positive [143] . as for the genetic basis of this tumor, lmp1 proved to have an essential tumorgenesis of b cells and its high levels of expression was used to explain the obvious clinical and morphological variations between ebvpositive and ebv-negative forms of hd [140] . some observations had also showed an association between lmp1 and other genes expression suggesting that lmp1 upregulated these genes in an in vitro primary hrs cell cultures (e.g. il-10 and traf1). therefore, in contrast to ebv-negative hd forms, these genes are more likely to be expressed in ebv-positive hd [144 -146] . immunohistochemical assays and genetic studies using biopsies of hd also revealed that cultures of hrs cells of ebv-positive hd cases express lmp1 to high levels but not ebna2 [147 -149] . the most salient symptomatology of hd is the painless swelling in one or more lymph nodes especially those of the neck, underarm and the groin. some other patients have symptoms like fever, night sweats, fatigue, total body itching, weight loss, bone pain and increased susceptibility to infections as recently reviewed by movva [150] . detection of abnormal cells, referred to as reed-sternberg cells, by the pathologist following biopsy of an enlarged lymph node is the first line of hd diagnosis. radiographic imaging such as ct scanning, mri, or pet scanning may also be employed to determine the stage of hd. immunohistochemistry was also employed as the constitutively nuclear encoded nf-κb from the hrs cells can be detected [151] . the treatment of hodgkin lymphoma (hl) depends on the direct medical intervention using chemotherapy and radiotherapy; stem cells transplantations were also recommended in relapsed cases, although about 20% of patients were seen not responsive to that treatment [152] . brentuximab vedotin, which was approved in 2011, made great shifts in the treatment of hl and other novel therapeutics are expected to pave the way for future improved hd treatment [153] . a combination of brentuximab vedotin and rituximab was also implemented to help the immune system kill lymphoma cells with some side effects encountered [154] . radiation therapy to kill lymphoma cells had also been tried to shrink tumors and help control pain with better results obtained when combined with chemotherapy [155] . post-transplant lymphoproliferative disorders (ptlds): they are defined on pathological grounds as combinations of lesions progressing from atypical polyclonal b cells proliferation in immunodeficient individuals to more aggressive monomorphic lymphomas which continue as malignancy even after immunity reconstitution [156] . on clinical grounds, they are defined as lymphoproliferative disorders observed in the tissue transplanted patients as they are usually under immunosuppressive therapy and also in those who are congenitally immunodeficeint individuals such as those suffering from wiscott-aldrich syndrome and x-linked lymphoproliferative syndrome. they are also frequently observed in aids patients who may also experience similar tumors of b-cell origin [157] . although many of these b cell proliferation conditions derived from the patient himself, in some cases these proliferated b cells were reported originated from the donor [158] . the overwhelming majority of these ptlds were confirmed as associated with ebv infectivity and most of them characterized by the latency iii pattern of expressed genes, although latency i and latency ii forms of ebv were occasionally also observed [159] . in these ptlds, ebv-negative cases were also reported; however, they are more seen as t cell lymphomas than b cell tumors and characterized by cellular monomorphic picture, later presentation and more aggressive as compared to the ebv-positive tumors [160, 161] . the variations among the ptlds in their incidences and clinical manifestations were seen as dependent on the number of the immunosuppressive drug used and their doses, the period of the immunosuppressive status and the type of the tissues transplanted. common clinical presentations are frequently observed as diversified extranodal lesions such as those seen in the gastrointestinal tract or in the organ transplanted. the frequent occurrence of these ptlds in the organ transplanted was explained by the fact that continuous and chronic antigenic stimulation in the graft tissues stimulates the development and pathogenesis of these lesions. in another study involving scid mice, it was reported that t cells were also mandatory for ptld like tumors to develop and this suggests a crucial role of t cells in the growth of these b cells lymphoid tumors [162] . at the cellular level, chromosomal imbalances and genomic alterations in ebv positive ptlds were detected in some studies and were also proposed to play an important role in the development and progression of ebv induced-lymphoproliferations and their clinical significance [163, 164] . on the ebv side, lmp1 is the major proto-oncogene that is expressed in most ebvpositive ptlds and there is an intimate association of ebv lmp1 deletion mutants with the ptlds morphology, ptlds clonality and ebv strain subtype [165] . antiviral chemotherapy, cytokine therapy, cytotoxic chemotherapy, anti-b cells antibodies, cellular immunotherapy and local treatments have all been observed to successfully cure these conditions [166] . beside b cell lymphomas, ebv was also confirmed in several studies to be associated with multiple kinds of t-cell lymphoma. the ebv genome was detected in some t cell lymphomas in the sinuses and nasal cavity in patients from many parts of the world e.g. peru [167] , japan [168] , usa [169] , taiwan [170] , some european countries [171] and recently from china [172] . in other studies, ebv was detected to account for about 50% of t-cells lymphomas which indicates that the tumor establishment may precede ebv infection [173] . that was confirmed following the advent of more sensitive molecular biological techniques which investigated the role of the ebv in the development of these t cell lymphomas. the association of the ebv infectivity with the t-cells lymphomas was also reported to be sitespecific i.e. ebv was detected in 100% and 20% of the nasal and gastrointestinal lymphomas respectively and rarely in primary cutaneous t-cell lymphomas [174] . peculiar phenotypic and genotypic manifestations for the sinonasal t-cells lymphomas were observed; these include the expression of some cellular markers, absence of t cell antigens and receptor gene rearrangements. however, the latter was detected recently in some cases of skin t cells lymphomas [175] . it was also reported that most of the ebv-positive t cell lymphomas are extranodal and have a cytotoxic phenotypic features [176] . this suggests that such kinds of lymphomas may occur as a result of ebv infection of ctls during the killing action of ebv-infected cells. as a matter of etiology and clinical outcome, the nasal nk and t cell lymphomas are considered the most closely associated with ebv as well as the most aggressive subtype of non-hodgkin lymphoma (nhl) ever recorded. in a genetic study, the mrnas of ebna1, lmp1, lmp2 or bamhi a transcripts in the ebv-positive peripheral t cell lymphomas were detected suggesting their expression. this study also showed and indicated that ebv-positive t cell lymphomas were clonally expanded from a single ebv-infected cell with a gene expression pattern different from that observed in burkitt's lymphomas or ptlds but seen similar to that of the npc [177] . as mentioned earlier, despite the fact that ebvrelated post-transplant lymphomas (ptlpds) are predominantly b-cell lymphomas, a reasonable number of them are known to be t cells-associated. these t-cell ptlpds were observed to be inconsistently associated with ebv infectivity [178] . based on the site of their development, multiple clinical and epidemiological presentations were reported for ebv-associated t cell lymphomas: peripheral t cell lymphomas (ptcl): they are diversified types of t cells lymphoproliferative diseases characterized by different pathological and clinical manifestations. as compared to b cell lymphomas, ptcl are rare but more difficult to manage and treat. ptcl are the most frequently occurring among all types of t cell lymphomas. in north america, ptcl accounts for 10-15% of all non-hodgkin's lymphomas in and known to involve mature t cells or nk cells [179] . although most of ptcls were diagnosed and recognized confined to the lymph nodes, other tissues such as git, liver, skin and bone marrow were also seen involved; such kinds of ptcls are more aggressive and cannot resolve without combined chemotherapy when diagnosed. a particular subtype of ptcl known as the peripheral t-cell lymphomas non-otherwise specified (ptcl-nos) is the most commonly recognized subtype. ptcl-nos usually showing extranodal lesions in the liver, git, spleen and bone marrow [180] . the role of ebv in causation of these kinds of lymphomas has been documented [181, 182] . anaplastic large cell lymphomas (altcl): they are group of lymphomas characterized by proliferation of the large lymphocytes and peculiar growth characteristics and expression of the cytokine receptor cd30. they were firstly described in 1985 as different types of t cell lymphomas and known to account for 12-15% of all t-cell lymphomas in adults and around 10-30% of all lymphomas in children. depending on the site where they develop, altcl were categorized into three types: systemic lymphomas, which are usually present in lymph nodes, namely the anaplastic lymphoma kinase (alk) positive and alk negative lymphoma, based on the presence of the alk protein on the surface of the malignant cell and a third non-systemic type, appearing only on the skin, which is also called a primary cutaneous anaplastic large cell lymphoma. as compared to the skin type, the systemic lymphomas are mostly fast growing [183] . although the classification of the who for the lymphoid and hematopoietic tumors, 2008 edition, stated that alcl is consistently negative for ebv, some studies have reported that a correlation between ebv and alcl does exist [184, 185] . a case of alcl in immunocompetent 35-year-old male using an in situ hybridization was also proved positive for eber in the large neoplastic cells [186] . angioimmunoblastic lymphoma (ail): ebv had also been proposed as a possible causative agent involved in the pathogenesis of ails which is a progressive and often fatal lymphoproliferative disorder. it is a fast-growing t-cell lymphoma with a primary symptomatology including swelling of the lymph nodes as well as systemic symptoms like fever and rash. generally, this lymphoma is treated as for the other fast growing systemic lymphomas although it may only require mild therapy in certain conditions [187] . previous findings also suggested that in this type of lymphoma the t cells are very vulnerable to ebv persistent infection and that it is more likely to give a growth advantage to infected cells. it was concluded that the pathogenesis of these ail-tcls is mediated by three factors, ebv infectivity, cellular genetic defects and impairment in the immune system [188] . in addition to its role in the hematologic malignancies, ebv has also been known to be implicated in both epithelial and mesenchymal neoplasms. the mechanism whereby ebv enters epithelial cells, previously cited in this review, was debated for some time, and some studies have confirmed a role for cd21 as receptors for the virus in human epithelial cells [189, 190] . these receptors seen to play a similar role in the fresh tissues of the liver, skin, git, endothelium, renal tubules, striated muscles and parotid glands [191] . it was also reported that the secretory component-mediated iga transport, in which the mucosal polymeric immunoglobulin a (piga) binds to a secretory component, a transmembrane protein expressed by the epithelium; and is then endocytosed by the cell, was also proposed as a potential means for ebv infection of endothelial cells [192] . the mechanism of cell to cell contact between virus releasing cells and noninfected epithelial cells for ebv entering and infection was also reported [193] . nasopharyngeal carcinoma (npc): it is the first epithelial carcinoma known to be associated with ebv infectivity [194, 195] . this was confirmed by the elevated antibody titers against ebv in many cases of npcs [196] and later when ebv dna was identified in extracts of npc [197] . the ebv genome was also detected in npc tissues using southern blot technique suggesting that ebv infection precedes the clonally expanded malignant cells of npc [198] . it was well established that three factors are involved in the npc incidence, ebv infectivity, genetic predisposition and some environmental factors which may vary from one population group to another [199] . undifferentiated type of npc (unpc) was commonly observed in arctic, middle east (mainly north africa), china and south-east asia. it was also observed that people of chinese descent are the most common population showing higher incidences of npcs. beside these genetic and racial predisposition, an environmental factors such as food components (e.g. salted fish) are known to be important contributing factors in npc development [200] . other factors including history of respiratory infections, tobacco smoking and consumption of preserved foods were also considered environmental factors for npc development [201, 202] . as for gender as a co-factor, it was recognized and reported that npc incidences are 2 to 3 fold higher in males than females in the populations investigated [203] . based on histological bases, who classified npc into three types: type i (keratinizing squamous cell carcinoma); type ii (differentiated nonkeratinizing carcinoma) and type iii (undifferentiated npc) [204] . looking at the genetic basis of npc development, it was confirmed in some studies that individuals who inherit certain human leukocyte antigen (hla) gene alleles, that have reduced potential to present ebv antigen to immune cells, were proved to have an increased risk to develop npc, whereas individuals with hla alleles that present ebv efficiently may have a lower risk for npc [205, 206] . on the ebv side, lmp1 and lmp2 were proved to have a strong influence on the cells genetic expression and proliferation leading to highly invasive and malignant growth of npc. additionally, the establishment of ebv latency and transformation in epithelial cells is considered as a major contributing element in the development of npc [207] . in another former study, using selected human sera and immunoblotting, it was revealed that ebna2, ebna3 and ebna-lp were not expressed in npc tumor cells [208] . however, the ebna1 and ebers proteins were observed to be expressed in all ebv-positive npcs [208, 209] . expression of lmp2a gene in npc tumors was also detected using pcr while the lmp2a protein was not detected in npc tumors [210] . clinically, patients with npc may be presented with one or more of the following symptoms; nasal obstruction involving nasal discharge, congestion and bleeding. hearing changes which are usually attributed to the blockage of the eustachian tube may also be seen. palsies of the cranial nerve due to the extension of the npc tumor into the skull base causing headache and neck swelling were also observed in some cases as described by sham et al. (1990) [211] . some other malignancies with similar clinical features to npc in sites like tonsils, lungs, skin, thymus, salivary glands, stomach, breast and uterine cervix were also observed and referred to as undifferentiated type of npcs [212 -216] . gastric carcinoma (gc): diffuse gastric or intestinal adenocarcinomas were detected and also proved to be associated with ebv in about 10% of the total cases throughout the world [20, 217, 218] . ebv-negative gcs were also reported in the majority of cases [219] . ebv-positive gcs were more commonly observed to occur in males than females [20, 218] . no age preference for the occurrence of these kinds of carcinomas was observed, however, some studies' findings revealed more ebv-positive tumors in older people compared to children or young adults [38, 219, 220] . they were more likely to occur in the cardia or body of the stomach than the gastric antrum [6, 218, 221] and most of them were detected as undifferentiated tumors [6, 38, 218, 222] . ebv-positive gc is a non endemic disease detected in all parts of the world. however, high incidence rates of the disease were recognized in certain regions with the highest incidences recorded in germany and usa and the lowest in china [20, 223 -226] . in an epidemiological study in usa, ebv-positive gc was more commonly observed among hispanics than in whites or african americans. this indicates that the correlation of gc development with the ebv infectivity varies with the different ethnic groups of population [227] . advanced molecular studies for these type of carcinomas showed a restricted genetic expression which was confined to ebers, ebna1 and the bzlf1 with a reasonable expression of either lmp1 or the other ebnas [21, 228] . also, ebv-encoded small rnas were seen expressed in every gc cells, this indicates the importance of this molecule in development and persistence of the gc [229] . in another proteomic study, ebv-positive gc was found to have distinct protein expression profile and clinicopathological features as compared to ebv-negative carcinoma [230] . as for the clinical presentation of ebv-associated gcs, they appear as tumors in the non antrum part of the stomach accompanied by ulcers in the upper part of the stomach when endoscopy was used. it is unlike chronic gastritis and carcinoma attributed to helicobacter pylori (hp), which locates predominantly in the antrum of the stomach [229] . however, yani et al. in 1999 [231] showed that ebv-positive gcs are commonly observed adjacent to the mucosal atrophic border, where mild to moderately chronic atrophic gastritis (cag) is commonly observed. also ebv and hp were frequently detected in the stomach mucosa with moderate cag, the area where the inflammatory cells are in common and not the mucosa with obvious cag, where inflammatory cell infiltration is minimal. oral hairy leucoplakia (ohl): it is also known as hiv-associated hairy leukoplakia [232] and defined as white patches on either sides of the tongue with hairy appearance which cannot be scraped off. this carcinoma was firstly reported and intensively described by in 1984 greenspan et al. [233] . as a confirmation for the association of this malignancy with ebv, an electron microscopic examination revealed a herpes virus-like particle. later, the role of ebv in this tumor was confirmed using the dna hybridization and immunohistochemical techniques [234] . a cyclovir treatment of this tumor, an inhibitor of the herpesvirus dna polymerase, resulting in the regression of the ohl and hence ebv was implicated as a causative agent in its development [234] . this condition was confirmed to be due to ebv infectivity when the virus reactivated from latency because of immunosuppression [235] . ohl was frequently observed as common oral lesions with hiv/aids and oral candidiasis [236] . this tumor was reported as the most commonly hiv/aids associated condition and was known mainly to occur in adults rather than children and more in males than females [237] . firstly, ohl was believed is the disease of hivinfected and homosexual males but later was found to occur in people other than these two groups [237] . to determine the genetic behavior of ebv during ohl oncogenesis, a genomic study using northern blotting and sequencing to study the ebv gene expression dealing with clones from a cdna library constructed using rna isolated from ohl biopsies was made. this analysis revealed the expression of bdrf1, bcrf1, gp350/220, barf0, and bkrf4 reading frames whereas ebna1, ebna2 and ebna -3a rna were not seen expressed [238] . no exact symptomatology and epidemiological patterns connected to this tumor were reported. however, generally symptoms and signs linked to immunosuppression were the majors symptoms to be detected. lesions were described previously as white patches on the lateral surfaces of the tongue and may involve the dorsum of the tongue but they are rarely seen on the buccal mucosa, pharynx, esophagus and soft palate. these lesions textured as vertical corrugation (hairy), or as thick furrow or appears shaggy [239] . ebv immune evasion is the principal issue to be highlighted here as the well-known lifelong persistence of the virus in the infected cells which has been attributed to the virus' strong potential to escape from the host's immune responses. ebv was recognized as a powerful immunogenic virus upon infection. this was clearly demonstrated by the high levels of the immune responses elicited, especially during the primary infection. however, in order for the ebv to survive and successfully establish itself as a latent virus in the memory b cells of the adult, it has a farrago of mechanisms to escape the host immune responses. these include shut down of the most immune responsive proteins via the lytic proteins which interfere with antigen processing and with expression of the mhc molecules in the infected cells. additionally, viral homologues of human cytokines are produced by the infected cells to mimic host antigens. these strategies and mechanisms for ebv-mediated immune evasion were intensively reviewed by ressing et al. [240] . the limited subset of viral gene products during latency as compared to the expression of about 80 ebv gene products detected during the replicative phase is also considered a kind of immune evasion [240] . to specifically address this issue, a protein encoded by ebv, namely bnlf2a, had been identified and normally expressed in the early phases of the lytic replication cycle of the virus. this protein was known as an immune evasion protein. it was recognized to inhibit the peptideand atp-binding functions of the transporter associated with the antigen processing steps during the immune response. in addition, this protein expression resulted in decreased mhc class i expression and thus prevented the presentations of the viral antigens to cd8+ ctls [241] and ebna1, a major latent gene products is a poorly recognized protein by cd8+ t lymphocytes [240] . it was also recently shown that the bcrf1 genes encoded by the ebv, which are homologues of il-10, are immunoevasins in ebv's lytic cycle [242] . despite all these strategies adopted by ebv for immune escape, the development of the previously mentioned tumors has been attributed to some imbalances in the equilibrium between the inherent virus transforming properties and the host immune system [243] . overall, the evasion by ebv of the host's immune responses allows the virus to remain in the body as a latent infection for life whereas reactivation of the latent ebv comes as a consequence of immunosuppression and the reactivated virus may transform the infected cells. the present review provides comprehensive coverage of some epidemiological and clinical aspects of ebv-associated lymphoid and epithelial malignancies and relates the association of each of these tumors with ebv infectivity. the genetic basis of tumorigenicity, including both ebv and cellular gene expression underlying cancer formation was also reviewed for these malignancies. all of the tumors mentioned in this review were found to be ebv-positive; however, some of them may also have ebv-negative correlation e.g. hodgkin's disease and gastric carcinomas. different epidemiological patterns were reported for these tumors depending on the geographical distribution, ethnic group of people, host age and gender...etc. some of these tumors showed racial restrictions (e.g. npc was known to be having special affinity to asians as compared to other ethnic groups) while other co-factors like smoke, salted food intake and other kinds of food were also observed to be linked to some of these tumors as predisposing factors. generally, different clinical presentations were reported for different tumors and this was governed by the tissues and sites affected as well as the complications that develop. generally, the various genetic mechanisms adopted by ebv to induce tumorigenesis of infected cells are complex, but orchestrated processes. these include expression of some viral proteins and shut down of others. the expression of the latent genes of the virus, namely lmp1, lmp2a, lmp2b, ebna1 and ebna2, was important with lmp1 reported as the principal oncogenic factor of ebv while lmp2 as a mediator of tumor cell survival. ebna1 is the protein required to bind the ebv genome to host chromosomes and thus allow the transfer of the viral dna to the progeny cells during cellular multiplication. ebna2 is a transactivator of lmp1. these ebv-associated malignancies develop despite strong immune responses to the virus but the virus adopt several strategies for immune evasion including expression of latent genes during the latency stage. in conclusion, an improved understanding of the molecular mechanisms leading to the development of these malignancies, which has accumulated in recent years, may surely come up with better prognostic, treatment and management regimens for them in the future. molecular phylogeny and evolutionary timescale for the family of mammalian herpesviruses tyrosine 112 of latent membrane protein 2a 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diseases oral and maxillofacial medicine: the basis of diagnosis and treatment the significance of oral health in hiv disease epstein-barr virus gene expression in oral hairy leukoplakia oral manifestations in the era of haart epstein-barr virus evasion of cd8(+) and cd4(+) t cell immunity via concerted actions of multiple gene products stage-specific inhibition of mhc class i presentation by the epstein-barr virus bnlf2a protein during virus lytic cycle the ebv immunoevasins vil-10 and bnlf2a protect newly infected b cells from immune recognition and elimination the interplay between epstein-barr virus and the immune system: a rationale for adoptive cell therapy of ebv-related disorders declared none. the authors confirm that this article content has no conflict of interest. key: cord-281404-5a8au32c authors: gastaldello, stefano; chen, xinsong; callegari, simone; masucci, maria g. title: caspase-1 promotes epstein-barr virus replication by targeting the large tegument protein deneddylase to the nucleus of productively infected cells date: 2013-10-10 journal: plos pathog doi: 10.1371/journal.ppat.1003664 sha: doc_id: 281404 cord_uid: 5a8au32c the large tegument proteins of herpesviruses contain n-terminal cysteine proteases with potent ubiquitin and nedd8-specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. using as model bplf1, the homologue encoded by epstein-barr virus (ebv), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a bplf1-dependent decrease of nedd8-adducts and accumulation of free nedd8. expression of bplf1 promotes cullin degradation and the stabilization of cullin-ring ligases (crls) substrates in the nucleus, while cytoplasmic crls and their substrates are not affected. the inactivation of nuclear crls is reversed by the n-terminus of cand1, which inhibits the binding of bplf1 to cullins and prevents efficient viral dna replication. targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic n-terminus by caspase-1. inhibition of caspase-1 severely impairs viral dna synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by ebv reactivation in promoting virus replication. post-translational modification of proteins by covalent linkage of ubiquitin (ub) or ubiquitin-like proteins (ubls), such as sumo, nedd8, isg15, regulates diverse cellular processes, including cell cycle progression, dna repair, transcription, signal transduction and immune responses [1, 2] . cytosolic and nuclear proteins tagged with multiple lys48-linked ub moieties are targeted to the proteasome for degradation, whereas the attachment of single or multiple ub or ubls regulates a variety of non-proteolytic events, including protein-protein interactions and intracellular traffic [3] . conjugation of the modifiers is accomplished by an enzymatic cascade composed of activating enzymes (e1), conjugating enzymes (e2) and substrate-specific ligases (e3), and is reversed by substrate-specific cysteine or metallo-protease that control the turnover of the modification and play thereby a key role in determining the functional outcome. although each modifier is involved in unique cellular functions, important cross-talk has been highlighted by the demonstration that nedd8 and sumo regulate the activity of certain ubiquitin ligases. thus, the best characterized substrates of nedd8 conjugation are cullins that function as scaffolds for the assembly of cullin-ring ubiquitin ligases (crls) [4, 5] , while sumoylation is required for substrate recognition and subsequent ubiquitination by a family of sumotargeted ubiquitin ligases (stubls) [6] . furthermore, several deconjugases, including usp21 [7] , ataxin-3 [8] pfuch54 [9] , uch-l1 and uch-l3 [10] , exhibit dual specificity for ub and nedd8 conjugates, while senp8 is both a nedd8 and sumo deconjugase [11] . the significance of these multiple specificities in the context of biological processes remains, however, undefined. viruses rely on the host cell machinery for replication. given the key role of ub and ubl modifications in the regulation of cellular and immunological functions, modulation of these signaling pathways is essential for viral dna synthesis and for the survival of the virus during acute, chronic and latent infections [12] . two viral interference strategies have been documented in the infected cells. viral proteins were shown to regulate the expression or capture the activity of cellular components of the ub and ubl signaling networks and to redirect their function towards preferred cellular or viral substrates [13] . in addition, there are numerous examples of virus-encoded homologs of cellular ligases and deconjugases [14] . these viral enzymes are often multifunctional proteins that share little homology with their cellular counterparts and are therefore attractive targets for selective inhibition. adenovirus [15] , severe acute respiratory syndrome coronavirus [16] , and all members of the herpesvirus family [17, 18] , encode their own deconjugase. the homologs encoded in the n-terminus of the large tegument proteins of herpesviruses show very little sequence similarity but the residues implicated in the formation of the catalytic site are strictly conserved. all the tested homologs have potent ubiquitin-specific protease activity and their overexpression in transfected cells is associated with a dramatic decrease of polyubiquitinated substrates [17, 18] . expression of the active enzymes was confirmed during infection by human cmv (hcmv) [19, 20] , murine gamma-herpesvirus 68 (mhv-68) [12] , marek's disease virus (mdv) [21] , and pseudorabies virus (prv) [22] . although not essential for viral dna synthesis, disruption of the catalytic function correlated with severe impairment of viral replication in in vivo models of infection [12, 21, 22] . the epstein-barr virus (ebv) encoded homolog, bplf1, has very potent ubiquitin deconjugase activity in various experimental models. anchoring the enzymatic domain to the er membrane [23] or injection of the purified protein in semi-intact cells [24] promoted the dislocation of ubiquitinated erad substrates, resulting in their stabilization in the cytosol. overexpression of the n-terminus was associated with deubiquitination of the viral ribonucleotide reductase (rr) [25] and the cellular dna polymerase processivity factor pcna [26] , resulting in downregulation of the viral rr activity and attenuation of polg at dna damage sites. furthermore, expression of the catalytically active bplf1 was shown to correlate with deubiquitination of traf6 and inhibition of nf-kb signaling during productive ebv infection [27] . we have previously reported that, in addition to their deubiquitinating activity, bplf1 and the homologs encoded by hsv1, kshv and mhv-68 exhibit strong activity against nedd8 conjugates [28] . bplf1 hydrolyzes nedd8 conjugates in vitro and stabilizes several crl substrates in transfected cells, including the cellular dna-replication licensing factor cdt1. expression of bplf1 alone or in the context of the productive virus cycle induced the accumulation of cdt1 and arrest of the cells in s-phase, while re-expression of cdt1 was sufficient to revert the inhibition of virus replication induced by bplf1 knockdown. this phenotype is dependent on direct binding of bplf1 to crls via interaction of the conserved helix-2 of bplf1 with the c-terminal domain (ctd) of cullins, at a site that is also engaged by the crl regulator cand1 [29] . while the double specificity of this family of viral enzymes is experimentally documented, the importance of the ubiquitin and nedd8-specific deconjugase activities in infected cells is not easily understood since deubiquitination or inactivation of the specific ubiquitin ligase may have similar effects on individual substrates. thus, it remains unclear whether both the ubiquitin-and nedd8-specific deconjugase activities operate during virus replication and, if so, how the different functions are regulated or compartmentalized in the infected cells. here we report that induction of the productive virus cycle has no appreciable effects on the global levels of protein ubiquitination but is accompanied by a bplf1-dependent decrease of cullin neddylation and stabilization of nuclear crl substrates. targeting of catalytic nterminus of bplf1 to the nucleus is dependent on cleavage by caspase-1, and inhibition of the caspase halts virus replication and the release of infectious virus. the akata-bx1 cell line was used to study the contribution of the ub-and nedd8-specific deconjugase activities of the ebv large tegument protein bplf1 to the productive virus cycle. treatment of akata-bx1 with anti-igg antibodies promotes upregulation of the lytic cycle transactivator bzlf1, followed in temporal succession by the expression of immediate early, early and late viral gene products (supplementary, figure s1 ). conjugated and free ub and nedd8 were quantified in western blots probed with specific antibodies using cells lysates made in the presence of cysteine protease inhibitors ( figure 1 ). the levels of conjugated and free ub remained virtually unchanged over time ( figure 1a and 1b), whereas, consistent with the occurrence of deneddylation, the conjugated nedd8 progressively decreased in parallel with increase of free nedd8 ( figure 1c and 1d). in order to assess the involvement of bplf1, a previously characterized specific shrna [28] was transfected in akata-bx1 before induction of the productive cycle. as shown in figures 2a and 2b , the effect was abrogated in cells expressing the bplf1 specific shrna, confirming that the phenotype is dependent on bplf1 expression and supporting the conclusion that endogenous enzyme acts as a deneddylase during virus replication. transfection of the catalytically active bplf1 in hela cells promotes cullin deneddylation and their proteasomal degradation [29] . we asked therefore whether this phenotype is reproduced when the endogenous enzyme is expressed during virus replication. as illustrated by the representative western blot shown in figure 3a , induction of the productive virus cycle was accompanied by a gradual decrease of the cul1, cul3, cul4a and cul5 specific bands in akata-bx1. this was not due to a global impairment of protein synthesis since neither cul2, nor the crl subunit rbx1 were affected. furthermore, the decrease was rescued by treatment with mg132 confirming that cullins are degraded by the proteasome ( figure 3b ). this finding is consistent with a scenario where the inactivation of crls by bplf1mediated deneddylation of cullins, and their subsequent proteasomal degradation, are key requirements for efficient virus replication. however, the failure to degrade cul2 is surprising since the bplf1 binding site on cullins is highly conserved [29] . to explore the possible cause of this observation, the abundance of cul1, cul2, cul3, cul4a and cul5 was monitored over time in the nucleus and cytoplasm of the induced cells. the fractionation procedure was validated by probing of western blots with antibodies to parp, histone h1 and b-actin. in line with their known subcellular localization, parp and h1 were exclusively detected in the nuclear fractions, whereas b-actin was enriched in the cytoplasmic fraction ( figure 3c ). variable amounts of nuclear and cytoplasmic cullins were detected in untreated akata-bx1, with prevalent nuclear localization of cul1, cul3, cul4a and viruses rely on the host cell for replication and have evolved sophisticated strategies to manipulate and harness the cellular metabolic pathways and defense responses. a better knowledge of these viral strategies will provide new targets for antiviral therapies. the nterminus of the large tegument proteins of herpesviruses encodes an ubiquitin and nedd8-specific deconjugase, but the function of the enzyme during virus replication is largely unknown. here we report that, endogenously expressed bplf1, the homolog encoded by epstein-barr virus (ebv), promotes a dramatic decrease of nedd8conjugates and the accumulation of free nedd8 in cells entering the productive virus cycle. bplf1 exerts its deneddylase activity in the nucleus, which promotes the accumulation of cullin-ring ligase (crl) substrates that are required for efficient virus replication. targeting of the viral enzyme to the nucleus is dependent on processing of the catalytic n-terminus by caspase-1. inhibition of caspase-1 severely impairs viral dna synthesis and the release of infectious virus, pointing to an unexpected role of the cellular response to danger signals triggered by ebv reactivation in promoting virus replication. cul5, whereas cul2 was detected almost exclusively in the cytoplasmic fraction ( figure 3c ). induction of the productive virus cycle was accompanied by a progressive decrease of nuclear pool of cul1, cul3, cul4a and cul5, whereas the amount of proteins detected in the cytoplasm remained unchanged throughout the observation period ( figure 3c and 3d ). there was no detectable change in the expression of cytoplasmic cul2, further supporting the conclusion that only nuclear cullins are affected. we then monitored the abundance of known nuclear and cytoplasmic crl substrates. in agreement with the destabilization of the ligases, induction of the productive cycle was accompanied by the accumulation of several nuclear substrates of crl1 and crl4a, including p21, p27, cdt1 and cdc25a, whereas two cytosolic substrates of crl2, the rho gtp exchange factor vav [30] , and the hypoxia induced factor hif1a that is continuously degraded in normoxic conditions [31] , were not affected ( figure 4a and 4b) . ikba, a cytosolic substrate of crl1-btrcp, was progressively degraded, confirming that the ligase is inactivated only in the nucleus. the involvement of bplf1 in the degradation of nuclear cullins and stabilization of nuclear crl substrates was confirmed by shrna knockdown ( figure 4c ). thus, expression of the bplf1 specific shrna rescued the downregulation of cul1, cul3, cul4a and cul5 and promoted destabilization of their nuclear substrates p21, p27, cdt1 and cdc25a, whereas the levels of cul2, vav and hif1a (not shown) remained unchanged. interestingly, the levels of ikba were significantly increased in cells expressing the bplf1 specific shrna suggesting that the viral protein may indirectly regulate the activity of nfkb. we have previously shown that the capacity of bplf1 to promote the deneddylation and degradation of cullins is dependent on binding to cullins at a site overlapping with the binding site of the regulator cand1. the interaction is inhibited by overexpression of the n-terminus of cand1, which rescues cullin deneddylation and degradation [29] . in order to assess whether this regulatory interaction may operate during virus replication, the productive cycle was induced in akata-bx1 cells transiently transfected with plasmids expressing myc-tagged cand1 or the cand1 nterminus that compete for bplf1 binding to cullins, or, as controls, the cand1 c-terminus that binds to the opposite end of the cullin scaffold, and the empty vector ( figure 5a ). expression of comparable amounts of the transfected proteins was confirmed in western blots probed with a myc-specific antibody ( figure 5a , upper panels). in line with the above documented effects, low levels of cul1, cu4a and cul5 and high levels of the crl substrate cdt1 were detected upon induction of the productive cycle in cells transfected with the empty vector. the degradation of cullins and stabilization of cdt1 were reversed in cells expressing the full length or the n-terminus of cand1 that compete for binding of bplf1, whereas the c-terminus of cand1 had no effect ( figure 5a ). to assess the functional significance of this finding, the efficiency of viral dna replication was measured by q-pcrs in akata-bx1 transfected with cand1 or the deletion mutants using primers specific for unique sequences in the bzlf1 and ebna1 coding genes ( figure 5b ). induction of the productive cycle was associated with more than 10-fold increase of viral dna content in cell transfected with the empty vector or the cand1 cterminus while viral dna replication was strongly impaired in cells expressing the full-length cand1 or the cand1 nterminus. it is noteworthy that only the full-length cand1 that blocks both the n-terminal and c-terminal domains of cullins regulates the neddylation cycle in non-infected cells. thus, the capacity of the n-terminal domain to fully reverse the inactivation of crls in infected cells is consistent with a mechanism of action based on inhibition of the binding of bplf1 to cullins, and identifies cand1 as a potent and specific cellular inhibitor of ebv replication. the large tegument proteins of herpesviruses are huge proteins predominantly localized in the cytoplasm of the infected cells where they play a key role in the delivery of viral dna to the nuclear pore during primary infection and in the secondary envelopment and egress of mature virions [32] . however, the preferential effect on nuclear cullins and their substrates implies that the enzymatic activity of endogenous bplf1 is compartmentalized to the nucleus. to find a possible cause of this puzzling observation, the subcellular localization of bplf1 was investigated using a polyclonal rabbit serum raised against the catalytic nterminus (amino acids 1-325). to confirm recognition of the active enzyme, lysates of untreated and induced akata-bx1 were labeled with the ha-ub-vs and flag-nedd8-vs functional probes that covalently bind to the catalytic cysteine. western blots of anti-ha and anti-flag immunoprecipitates were then probed with antibodies to ha, flag and bplf1. as illustrated by the representative blots shown in figure 6a , the anti-ha and anti-flag antibodies detected two de-novo expressed enzymatic activities associated with polypeptides of .300 kd and approximately 38 kd in the lysates of induced cells. probing of parallel blots with affinity purified antibodies to bplf1 confirmed that the high molecular weight species corresponds to the full-length bplf1, while the 38 kd species is likely to represent an approximately 25 kd n-terminal catalytic domain cross-linked to the 11 kd probe. prediction algorithms were then used to screen the amino acid sequence of bplf1 for the presence of cleavage sites for known cytosolic endo-peptidases. several putative caspase-cleavage sites were identified in the first 2000 amino acids of bplf1 (supplementary, figure s2 ). in particular, a high-score caspase-1 cleavage site in position asp222 may generate a catalytically active fragment of the observed size. to test whether bplf1 is cleaved by capsase-1, the productive cycle was induced in akata-bx1 treated with the pan-caspase inhibitor zvad-fmk, and the specific caspase-1 inhibitors yvad-cho and small molecule vx-765. cell lysates collected after 48 h were labeled with ha-ub-vs (not shown) and flag-nedd8-vs. as shown in figure 6b , both the full length and the 38 kd species were readily detected by the anti-flag antibody, although the 38 kd species was significantly stronger, which may be due to more efficient cross-linking of the probe or to poorer transfer of the high molecular weight full length enzyme. the intensity of the 38 kd fragment was strongly decreased when the induction was carried out in the presence of caspase inhibitors while the intensity of the full length species was slightly but consistently increased, confirming that the catalytic nterminus of bplf1 is cleaved by caspase-1. having established the specificity of the antibody, we then turned to investigate the subcellular localization of bplf1. staining of akata-bx1 with the affinity purified rabbit serum revealed a diffuse cytoplasmic and nuclear fluorescence 48 h after induction ( figure 6c ), with essentially no background in cells stained with tritc-conjugated secondary antibody alone. the specificity of the staining was confirmed by its virtual abrogation in induced cells expressing a bplf1-specific shrna. the nuclear fluorescence was virtually abolished when the induction was performed in the presence of the pan-caspase inhibitor (not shown) or the caspase-1 inhibitors yvad-cho ( figure 6c , 6d) and vx-765 (not shown). thus, accumulation of the catalytic n-terminus of bplf1 in the nucleus is dependent on cleavage of the cytosolic protein by caspase-1. since the enzymatic activity of bplf1 is required for efficient ebv dna replication [27, 28] , we tested whether the latter is affected by inhibition of caspase-1. in line with the constitutive il-1 production of b-lymphoma cell lines [33, 34] , two bands of approximately 48 kd and 20 kd, corresponding to the pro-caspase and active caspase-1, were detected in western blots of untreated akata-bx1 probed with a caspase-1 specific antibody ( figure 7a ). the intensity of the 20 kd band increased upon induction of the productive cycle, which was prevented by addition of the caspase-1 inhibitors yvad-cho ( figure 7a ) and vx-765 (not shown). inhibition of caspase-1 did not affect the expression of the viral transactivator bzlf1, nor the subsequent expression of the early antigen borf2 and late antigen gp350/220 ( figure 7a ). nevertheless, the treatment abrogated the bplf1-dependent degradation of cul1 and cul4a and consequent stabilization of the crl substrates cdt1 and cdc25a ( figure 7b ). in line with the requirement of cdt1 stabilization for efficient viral dna replication [28] , the yield of viral dna was significantly impaired ( figure 7c ). similar levels of inhibition were achieved in cells treated with the pan-caspase inhibitor zvad-fmk and the caspase-1 specific inhibitors yvad-cho and vx-765. thus, caspase-1 appears to be the sole responsible for bplf1 processing. in the final set of experiments we asked whether the effect of caspase-1 is restricted to akata-bx1 cell line. to this end, the productive virus cycle was induced by culturing the prototype ebv producer cell line b95.8 in medium supplemented with 2% fcs, 20 ng/ml tpa in the presence or absence of increasing concentrations of vx-765. as shown in figure s4 , the marmoset cell line expresses a conserved caspase-1 species that is detected by the cross-reactive antibody used in our experiments, and the intensity of a polypeptide of approximately 20 kd, corresponding to the active caspase-1, increased upon induction of the productive cycle. inhibition of caspase-1 activity by addition of vx-765 resulted in a dose-dependent inhibition of viral dna synthesis, with maximal inhibition observed in the presence of 20 mm vx-765 ( figure 7d ). this was paralleled by a corresponding dosedependent inhibition of the release of infectious virus assessed by the capacity of spent supernatants to induce the expression of ebna2 in the ebv negative bjab cell line ( figure 7e ). our present study addresses an ongoing debate on the contribution of the deneddylase encoded in the large tegument protein of herpesviruses to virus replication, and provides a clear example of how, under physiologic conditions of expression, several lines of evidence support the notion that endogenously expressed bplf1 promotes ebv replication by acting as a deneddylase. the recombinant enzyme has potent ubiquitin deconjugase activity in vitro, and overexpression of the catalytic n-terminus induces a global decrease of ubiquitin conjugates in transfected cells [24, 28] . however, induction of the productive cycle was not accompanied by significant changes in the amount of ubiquitinated proteins or free ubiquitin in akata-bx1, whereas neddylated proteins decreased and free nedd8 increased in a bplf1-dependent manner (figures 1 and 2 ). while inconsistent with the potent deconjugase activity of bplf1 in experimental settings, our failure to detect appreciable change in the global levels of ubiquitination during productive infection does not formally exclude that the viral enzyme might selectively target few ubiquitinated substrates. yet, it should be stressed that evidence for the capacity of bplf1 to deubiquitinate specific substrates, such as the ebv ribonucleotide reductase [35] , pcna [26] and traf6 [27] , was in all cases obtained by overexpressing the catalytic n-terminus in transfected cells. in the experiments of saito et al. reconstitution of the viral enzyme in cells infected with a mutant ebv strain that lacks bplf1 was associated with deubiquitination of traf6 [27] . however, induction of the productive virus cycle was associated with strong traf6 deubiquitination also in the absence of bplf1, suggesting that other factors are primarily responsible for this effect and for the subsequent downregulation of nf-kb target genes. this interpretation is also supported by our findings that the nfkb inhibitor ikba was stabilized upon silencing of the endogenous bplf1 in induced akata-bx1 ( figure 4c ), which may be explained by failure to phosphorylate ikba due to a bplf1-independent inhibition of traf6 signaling associated with replicative cycle. similar to the effect of bplf1 in transfected cells [28, 29] , we found that endogenously expressed bplf1 is required for the selective degradation of cullins in productively infected cells and for the stabilization of several crl substrates that regulate the cell cycle and facilitate ebv dna replication (figures 3 and 4) . the reversion of this phenotype by overexpression of the crl regulator cand1 ( figure 5 ) strengthens the notion that bplf1 plays a key role in cullin deneddylation and degradation under physiological conditions of expression. we have previously reported that the conserved n-terminal domains of bplf1 and cand1 share a binding site on the c-terminus of cullins [29] . cand1 regulates the activity of crls by sequestering cullins that are deneddylated after substrate ubiquitination, which promotes the exchange of substrate-adaptor modules, broadening the substrate repertoire and allowing rapid adaptation to a variety of metabolic conditions [36] . cullins are the only known binding partners of cand1 and it is therefore reasonable to assume that the reduced ebv dna replication in cells overexpressing cand1, and in particular the truncated cand1 n-terminus that lacks the protein exchange function of the intact protein, is due to inhibition of the binding of bplf1 of cullins. collectively these findings support the notion that binding to the neddylated substrate determines the activity of this potentially promiscuous enzyme under physiological conditions of expression. this has two important implications. first, it underscores the possibility of experimental artifacts due to altered stoichiometry of the interacting partners in transfected cells. more importantly, it emphasizes the likelihood that interference with binding may have substantial downstream effects. in the case of ebv infection this could offer a new target for specific inhibition of virus replication. we have shown that endogenously expressed bplf1 acts on nuclear cullins and stabilizes nuclear crl substrates while cytosolic crls and their substrates are not affected. this nuclear compartmentalization is dependent on cleavage of the catalytic nterminus by caspase-1 ( figure 6 ). the processed fragment does not contain a putative nuclear localization signal but the size is sufficiently small for free diffusion through the nuclear pore that accommodates particles of up to 40 kd. processing of bplf1 is likely to be a key regulatory event in virus replication. it is noteworthy that a catalytically active n-terminal fragments of the bplf1 homolog ul36 has been detected in cells infected with hsv1 [17] , and preliminary results suggest that blockade of caspase-1 has a comparable inhibitory effect on hsv1 replication (gastaldello et al. unpublished) . although processing of the tegument protein was not detected in cells infected with hcmv [19, 20] , kshv [37] or mhv-68 [38] , and the full length enzymes encoded by these viruses are active dubs, we have previously shown that the catalytic n-terminus of kshv and mhv-68 share the cullin-binding capacity of bplf1 and inactivate crls in transfected cells [28, 29] . it remains to be seen whether the failure to detect processing of some tegument proteins is explained by different experimental procedures or whether it might reflect true differences in the interaction of these viruses with the infected cells. in the case of bplf1, it is tempting to speculate that, in addition to facilitating nuclear accumulation, cleavage of the catalytic nterminus may also activate the viral enzyme. this possibility is supported by the observation that the band corresponding to the processed bplf1 bound to the ub-vs and nedd8-vs functional probes was significantly stronger than the full length protein in lysates of induced cells ( figure 6a and 6b) . furthermore, cullins were not degraded in the cytosol, suggesting that the cytosolic enzyme is either inactive or cannot reach its targets. it is also possible that, while acting as a deneddylase in the nucleus, bplf1, or perhaps the unprocessed form of the enzyme, may act as an ubiquitin-specific deconjugase in the cytoplasm of the infected cells. experimental testing of this possibility remains an interesting focus for future work, pending the identification of substrates that are affected under physiologic conditions of expression. in this context it is noteworthy that cytosolic tegument proteins associated with the virion play important roles in the early and late phases of infection by contributing to the delivery of viral dna to the nuclear pore and to the secondary envelopment and egress of mature virions [32] . conceivably, a different set of cellular and viral substrates may be affected during these phases of the infection. an unforeseen outcome of our study is the demonstration that the infected cell may regulate the efficiency of virus replication via caspase-1 mediated processing of bplf1. caspase-1 is well known for its role as the converging target of danger signals such as physical stress, extracellular atp, bacterial and viral products, figure 6 . cleavage by caspase-1 releases the catalytic n-terminus of bplf1 and promotes its accumulation in the nucleus. a. a catalytically active n-terminal fragment of bplf1 is produced during ebv replication. lysates of control and induced akata-bx1 were labeled with the ha-ub-vs and flag-nedd8-vs functional probes and immunoprecipitated with ha-or flag-specific antibodies. western blots were probed with antibodies to ha or flag and with an affinity purified rabbit serum raised against the n-terminus of bplf1. cellular dubs labeled by the ha-ub-vs functional probe are indicated by asterisks. a non-specific band of approximately 65 kd detected by the anti-ha antibody in both untreated and induced akata-bx1 is indicated by an empty circle. one representative experiment out of four performed with different batches of affinity-purified antibodies is shown. b. inhibition of caspase-1 prevents the production of the catalytic n-terminal fragment. lysates of akata-bx1 induced in the presence of the indicated caspase inhibitors were labeled with the flag-nedd8-vs functional probe and flag immunoprecipitates were probed with a flag-specific antibody. one representative experiment out of three is shown. c. inhibition of caspase-1 prevents the accumulation of bplf1 in the nucleus. fluorescence micrographs of akata-bx1 induced in the presence of absence of caspase-1 inhibitors stained with the affinity purified antibody to bplf1. as controls induction was performed in akata-bx1 expressing a bplf1 specific shrna. scale bar 2 mm. representative cells are enlarged. d. quantification of nuclear and cytoplasmic fluorescence in 50 cells from 2 independent experiments. the images were analyzed using the imagej software. doi:10.1371/journal.ppat.1003664.g006 that are detected in the cytosol by sensing molecules and adaptors that promote the assembly of a multisubunit complex known as the inflammasome [39] . the inflammasome triggers the self-activation of caspase-1, which in turn mediates the maturation of proinflammatory cytokines like interleukin (il)-1b and il-18, and executes a rapid program of cell death known as pyroptosis [40] . additional cellular substrates of caspase-1 include the sterol regulatory element binding proteins (srebps) that are activated following changes in intracellular ions [41] . thus, caspase-1 plays pleyotropic roles in the activation of innate and adaptive immune responses as well as in processes that link changes of the intracellular environment with lipid metabolism, membrane biogenesis and cell survival. many viruses are known to inhibit the inflammasome or directly block the activity of caspase-1 in order to counteract antiviral responses [42] . our findings highlight a previously unrecognized role of the cellular response to danger signals triggered by ebv reactivation in promoting rather than inhibiting virus replication. this further illustrates the complexity and multilayer regulation of the interaction of ebv with its host where the capacity of the virus to adapt to and exploit physiologic cellular responses underlies the establishment of life-long persistent infections. . inhibition of caspase-1 prevents the inactivation of nuclear crls and hampers ebv dna replication. a. caspase-1 is constitutively active in akata-bx1 and is further activated during virus replication but is not required for expression of the lytic cycle genes. akata-bx1 cells were induced in the presence or absence of caspase-1 inhibitors and western blots were probed as indicated. the asterisks in the caspase-1 blot indicate residual igg heavy chains detected by the secondary antibody. one representative experiment out of three is shown. b. inhibition of caspase-1 prevents the degradation of cullins and stabilization of crl substrates. western blots of cell lysates produced as described in figure 7a were probed with the indicated antibodies. one representative experiment out of three is shown. plasmids encoding a bplf1 specific shrna [28] , and myctagged full-length human cand1, the cand1 c-terminus (cand1-c) and n-terminus (cand1-n) [29] were described previously. recombinant lentiviruses were produced in hek293t cells transfected with the recombinant plko.1 expressing a control scrambled or bplf1-specific shrna and the packaging plasmids, pspax2 and pmd2.g [43] (addgene, dr. didier trono, epfl lousanne, switzerland) by calcium phosphate precipitation and supernatant containing viral particles was collected after 48 h. virus titers were assessed by quicktiter lentivirus titer kit, a hiv p24 specific elisa (cell biolabs inc., san diego, ca). akata-bx1 cells that carries a recombinant ebv where the thymidine kinase gene was replaced by a cmv immediate early promoter driven gfp [44] the productive virus cycle was induced in akata-bx1 by incubating cell pellets for 1 h at 37uc with polyclonal rabbit antihuman igg (1:50, dako, denmark) and monitored by the increased of gfp fluorescence or by probing western blots with antibodies specific for the ebv transactivator bzlf1. for induction of the productive cycle in the b95.8 cell line the cells were placed in a six well plate at the density of 5610 4 cells/ml in 5 ml medium supplemented with 2% fcs and 20 ng/ml tpa and the spent supernatant was harvested after 2 weeks [45] . where indicated, the caspase-1 inhibitor vx-765 was added to the culture medium. quantitative-pcr was performed on dna extracted from the cell pellets and culture supernatant. total dna was purified with the dnazol reagent (invitrogen) and ebv dna content was assayed by qpcr with the kapa sybr fast qpcr kit (kk4604, kapa biosystems, cape town, south africa) and an abiprism 7000 sequence detection system using 100 ng of dna and the probes: ebna1: 59ggacgtggagaacagt-catc39/59cactcctgcccttcctcacc39, product 364 bp; bzlf1: 59cactaccaggtgccttttgt39/59gagactgg-gaacagc tgagg39, product 364 bp; gapdh: 59aaggtcg-gagtcaacggatt39/59ctcctggaagatggtgatgg39, product 224 bp. the cycling conditions were: initial step 50uc 2 min, denaturation 95uc 10 min, followed by 40 cycles of 95uc for 15 seconds, 60uc for 1 min, 95uc for 15 seconds, 60uc for 20 seconds and 95uc for 15 seconds. a final extension for 10 min at 72uc and melting curve between 65uc to 90uc, 1uc/second transition were incorporated. optical raw data were exported to microsoft excel for analysis. all qpcr reactions were performed in triplicate and ct values were averaged. the fold change in the target gene relative to the gapdh endogenous housekeeping control gene is determined by: fold change = 2 2d (dct) where dct = ct target -ct gapdh the subcellular localization of bplf1 was investigated by immunofluorescence in akata-bx1 cells 48 h after induction in the presence or absence of caspase inhibitors. eight 610 4 cells were deposited on glass slides by cytospin centrifugation, fixed with 4% paraformaldehyde and permeabilized with 0.5% v/v tritonx-100 in pbs for 20 min. the slides were then treated with blocking solution (0.5% bsa in pbs) for 2 h at rt and incubated with the anti-bplf1 antibody o/n at 4uc followed by incubation for 1 h with tritc conjugate anti-rabbit ig (1:200, r0270, dako) . the slides were mounted with vectashield mounting medium containing dapi (h-1200, vector laboratories) and images were captured with a zeiss lsm510 meta confocal microscope and analyzed with the imagej 1.42q software (wayne rasband, national institutes of health, usa). akata-bx1 cells were washed in cold pbs containing 0.2 m freshly added iodoacetamide and resuspended in hypotonic lysis buffer (10 mm hepes, ph 7.9, 1.5 mm mgcl 2 , 10 mm kcl, 0.1% np-40, 1 mm dtt, 20 mm iodoacetamide, 1 mm orthophenantroline, 10 mm nem and protease inhibitors cocktail. after incubation on ice for 30 min, the membranes were broken by passage through a 25-26 g needle and the nuclei were pelleted by centrifugation for 1 min at 10800 rpm. the supernatant was used as cytoplasmic fraction. the nuclei were washed three times with hypotonic buffer and lysed in buffer containing 50 mm tris-hcl, ph 7.5, 150 mm nacl, 1% np40, 0.5% doc and protease inhibitors cocktail. protein concentration was measured with a protein assay kit (bio-rad laboratories, ca). total cell lysates were prepared in lysis buffer (50 mm tris-hcl ph 7.4, 150 mm nacl, 1 mm dtt, 1 mm edta ph 8.0, 0.5% np40, 0.01% sds, 20 mm nem, 1 mm ortho-phenantroline, protease inhibitors cocktail) and protein concentration was measured with a protein assay kit (bio-rad laboratories, solna, sweden). twenty mg of cell lysate were denatured for 10 min at 100uc in loading buffer and fractionated in acrylamide bis-tris 4-12% gradient gel (invitrogen, carlsbad, ca). after transfer to pvdf membranes (millipore, bedford, ma), the filters were blocked in pbs containing 0.1% tween-20 and 5% non-fat milk or 3% bsa and incubated with the primary antibodies for either 1 h at room temperature or overnight at 4uc followed by incubation for 1 h with the appropriate horseradish peroxidaseconjugated secondary antibodies. the complexes were visualized by chemiluminescence (ecl, ge healthcare, uppsala, sweden). deconjugase activity was assayed by labeling with the ha-ub-vs and flag-nedd8-vs functional probes (boston biochem, boston, ma) as described [46] . ten 610 6 cells were lysed in 300 ml of buffer containing 50 mm tris-cl ph 7.4, 50 mm nacl, 1 mm dtt, 1 mm pmsf, 0.5% np40, 250 mm glucose, 5 mm mgcl 2 (lysis and labeling buffer, llb) followed by 20 strokes through a g30 needle. functional labeling was performed by addition of 2.5 mg of ha-ubiquitin-vs or flag-nedd8-vs followed incubation for 45 min at 37uc. the cross-linked proteins were immunoprecipitated with anti-ha-agarose or anti-flagagarose beads for 4 h at 4uc with rotation and eluted by competition with 25 mg/ml of the ha or flag peptides in llb. enzymatically active proteins were detected in western blots probed with anti-ha or anti-flag antibodies. putative caspase cleavage sites were searched in the bplf1 amino acid sequence using the grabcas software [47] . sites with the highest probability of cleavage were identified by setting the cut-off scores to .15. statistical analysis was performed using student's t-test. p-values ,0.05 were considered as significant. figure s1 kinetics of expression of immediate early, early and late antigens in induced akata-bx1. akata-bx1 cells were treated for 1 h with anti igg antibodies and western blots of cell collected at the indicated times were probed with antibodies to the immediate early antigen bzlf1, early antigen borf2 and late antigen gp350/220. western blots from one representative experiment are shown. figure s3 effect of caspase-1 inhibition on the nuclear localization of bplf1. representative localization profile of dapi and tritc fluorescence in induced akata-bx1 and cells treated with the caspase-1 inhibitor yvad-cho or bplf1 specific shrna. the bplf1 specific fluorescence was homogeneously distributed in the nucleus and cytoplasm of untreated cells but was excluded from the nucleus of caspase-1 inhibitor treated cells. background levels of bplf1 fluorescence were observed in cells expressing a bplf1 specific shrna. (tif) figure s4 induction of the productive cycle promotes the activation of caspase-1 in b95.8 cells. the productive cycle was induced in b95.8 cells by treatment with the indicated amounts of tpa or tpa and nabut in medium containing 2% fcs. induction of the ebv productive cycle was confirmed after one week by probing western blots of total cell lysates with antibodies specific for immediate early (bzlf1) early (borf2) and late (gp350/220) antigens. human caspase-1 specific antibodies detected a band of approximately 20 kd corresponding to the active caspase-1 in untreated cells and a stronger band was observed in the induced cells. the high levels of the active caspase-1 species detected in untreated cells is in line with the constitutive expression of the active enzyme in ebv transformed lcls and may be partly explained by spontaneous entry into the productive cycle. (tif) role of ubiquitin-and ubl-binding proteins in cell signaling ubiquitin-like proteins the ubiquitin-mediated proteolytic pathway: mode of action and clinical implications structural insights into nedd8 activation of cullin-ring ligases: conformational control of conjugation cullin-based ubiquitin ligase and its control by nedd8-conjugating system rnf4 is a poly-sumo-specific e3 ubiquitin ligase required for arsenic-induced pml degradation identification of a novel isopeptidase with dual specificity for ubiquitin-and nedd8-conjugated proteins nedd8: a new ataxin-3 interactor identification by functional proteomics of a deubiquitinating/deneddylating enzyme in plasmodium falciparum specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins desumoylating enzymes-senps a gammaherpesvirus ubiquitin-specific protease is involved in the establishment of murine gammaherpesvirus 68 infection targeting of host-cell ubiquitin pathways by viruses viral avoidance and exploitation of the ubiquitin system deubiquitinating function of adenovirus proteinase the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity a deubiquitinating enzyme encoded by hsv-1 belongs to a family of cysteine proteases that is conserved across the family herpesviridae epstein-barr virus encodes three bona fide ubiquitin-specific proteases cleavage specificity of the ul48 deubiquitinating protease activity of human cytomegalovirus and the growth of an active-site mutant virus in cultured cells highmolecular-weight protein (pul48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable a herpesvirus ubiquitin-specific protease is critical for efficient t cell lymphoma formation mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pul36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo enzymatic blockade of the ubiquitin-proteasome pathway a viral deubiquitylating enzyme restores dislocation of substrates from the endoplasmic reticulum (er) in semiintact cells positive reciprocal regulation of ubiquitin c-terminal hydrolase l1 and beta-catenin/ tcf signaling epstein-barr virus bplf1 deubiquitinates pcna and attenuates polymerase eta recruitment to dna damage sites epstein-barr virus deubiquitinase down-regulates traf6-mediated nf-kappab signaling during productive replication a deneddylase encoded by epstein-barr virus promotes viral dna replication by regulating the activity of cullin-ring ligases herpes virus deneddylases interrupt the cullin-ring ligase neddylation cycle by inhibiting the binding of cand1 suppressor of cytokine signaling-1 inhibits vav function through protein degradation hifalpha targeted for vhl-mediated destruction by proline hydroxylation: implications for o2 sensing the c terminus of the large tegument protein pul36 contains multiple capsid binding sites that function differently during assembly and cell entry of herpes simplex virus b-cellderived interleukin 1 (il-1)-like factor. i. relationship of production of il-1-like factor to accessory cell function of epstein-barr virus-transformed human blymphoblast lines b-cell-derived interleukin-1 (il-1)-like factor. ii. sources, effects, and biochemical properties the epstein-barr virus (ebv) deubiquitinating enzyme bplf1 reduces ebv ribonucleotide reductase activity cand1 promotes assembly of new scf complexes through dynamic exchange of f box proteins kaposi's sarcoma-associated herpesvirus encodes a viral deubiquitinase a functional ubiquitin-specific protease embedded in the large tegument protein (orf64) of murine gammaherpesvirus 68 is active during the course of infection inflammasomes and their roles in health and disease inflammasomes: caspase-1-activating platforms with critical roles in host defense caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival inflammasomes and viruses: cellular defence versus viral offence a lentiviral rnai library for human and mouse genes applied to an arrayed viral high-content screen inhibition of heavy chain and beta2-microglobulin synthesis as a mechanism of major histocompatibility complex class i downregulation during epstein-barr virus replication epstein-barr virus: transformation, cytopathic changes, and viral antigens in squirrel monkey and marmoset leukocytes a novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of usp14 grabcas: a bioinformatics tool for score-based prediction of caspase-and granzyme bcleavage sites in protein sequences we are grateful to drs kazuhiro iwai (department of biophysics and biochemistry, graduate school of medicine, osaka university, osaka, japan), ron t. hay (college of life sciences, university of dundee, dundee, uk) and jaap middeldorp (cca-vumc, amsterdam, the netherlands) for providing plasmids, antibodies and technical advice, and to teresa frisan for useful comments and critical reading of the manuscript. the contribution of the undergraduate students sandra zlodej, gianluca spaltro and jim baggen is gratefully acknowledged. key: cord-291063-de7v4e5s authors: moens, ugo title: silencing viral microrna as a novel antiviral therapy? date: 2009-05-28 journal: j biomed biotechnol doi: 10.1155/2009/419539 sha: doc_id: 291063 cord_uid: de7v4e5s viruses are intracellular parasites that ensure their existence by converting host cells into viral particle producing entities or into hiding places rendering the virus invisible to the host immune system. some viruses may also survive by transforming the infected cell into an immortal tumour cell. micrornas are small non-coding transcripts that function as posttranscriptional regulators of gene expression. viruses encode mirnas that regulate expression of both cellular and viral genes, and contribute to the pathogenic properties of viruses. hence, neutralizing the action of viral mirnas expression by complementary single-stranded oligonucleotides or so-called anti-mirnas may represent a strategy to combat viral infections and viral-induced pathogenesis. this review describes the mirnas encoded by human viruses, and discusses the possible therapeutic applications of anti-mirnas against viral diseases. viruses are common habitants of the human population, where they establish different forms of infection, including an acute, a chronic, or a persistent infection with production of low levels of virions. some viruses can exist in a true latent state in which infectious particles are only produced upon reactivation stimuli. viruses that reside harmlessly in their host can under certain conditions or in immunocompromised persons be responsible for malignant and nonmalignant diseases, which may even lead to the death of the host. a causal role for human polyomaviruses (hpyv), papillomaviruses (hpv), herpesviruses (hhv), hepatitis b virus (hbv), hepatitis c virus (hcv), and human t-cell lymphotropic virus type-i (htlv-i) and cancer is accepted (for recent reviews see [1] [2] [3] [4] [5] [6] [7] ). it is estimated that oncoviruses are associated with 15% of the human cancers [8] , while nonmalignant infections from human immunodeficiency virus (hiv), hbv and hcv alone cause more than 3 million deaths annually worldwide [9] . other viral infections (hiv no included) were responsible for the death of more than 6000 patients in japan in 2006, ∼7000 individuals in the usa in 2005, and 555 people in united kingdom in 2006 according the statistics of the world health organization [10] . thus the pathogenic properties of viruses necessitate the development of efficient antiviral therapies. viruses attempt to create a favorable cellular environment allowing viral replication or survival by establishing a lifelong latent infection through evading the immune system of their hosts. viruses can hide within a cell by restricting their activity to a minimum so as not to conceal their presence to the immune system and at the same time they will also try to avoid apoptosis. for these purposes, viruses have developed different strategies, one of which includes the posttranscriptional regulation of both cellular and viral gene expressions through modulating the host's rna-interference (rnai) machinery. viruses can suppress the rnai pathway by viral microrna (vmirna) targeting cellular or viral transcripts, or by viral proteins (e.g., human immunodeficiency virus tat protein, influenza virus ns1/ns2 protein, ebola vp35 protein, and vaccinia virus e3l protein) or viral rna (adenovirus va transcripts) that counteract the host's rnai machinery (for recent reviews see [11] [12] [13] [14] [15] [16] [17] ). this review summarizes the recent findings on virus-encoded mirnas and their described functions and briefly discusses the potential of antiviral mirna as a novel therapeutic strategy in combating virus infections. anti-mirna oligonucleotides (amos) are chemically modified synthetic oligonucleotides that are complementary to their target sequence and this will silence the action of the target. amos are modified with the dual purpose to stabilize them and to improve their affinity for their targets. one modification is the 2' sugar modification which implies a chemical modification of the 2'-o of the ribose residue (figure 1(a) ). the 2'-o -methyl amos have a methyl group linked to the 2'-o of the ribose residue, while the 2'-o -methoxyethyl amos contain a methoxygroup. this modification provides improved rnase resistance and binding affinity to rna compared to unmodified antioligonucleotides. however, 2'-o -methoxyethyl amos possess a higher affinity and specificity to rna than their 2'-o -methyl amos. other 2' sugar modifications that have been used include 2'-fluor and locked nucleic acid (lna). in lna-modified oligonucleotides, the 2'-ooxygen is bridged to the 4'-position via a methylene linker to form a rigid bicycle, locked into a c3'-end (rna) sugar conformation (figure 1(a) ). lnas give very strong duplex formation with their target sequences and they display excellent mismatch discrimination, hence avoiding off-target effects (for recent reviews see [28] and [29] ). lna injections against mir-122, a cellular mirna involved in lipid metabolism, resulted in efficient and long-lasting decrease in plasma cholesterol in african green monkeys without any evidence for toxicities [30] . a second type of modification is the phosphorothioate backbone which reduces the affinity to the target somewhat, but it confers significant stability to nuclease degradation (figure 1(b) ). a third generation of antisense oligonucleotides are phosphodiamidate morpholino oligomers (pmo) in which the ribose ring is replaced with a morpholine ring. adding an argininerich peptide (rxr) 4 further increased the stability and tissue retention of the pmo (reviewed in [31] ). an additional modification can be made to improve the cellular uptake of the amo. krützfeldt et al. linked a cholesterol moiety to their amos and referred to these anti-mirnas as antagomirs. antagomirs should be >19 nucleotides in length to provide highest efficiency in silencing target mirna [32] [33] [34] . the putative therapeutic potentials of antagomirs were recently demonstrated in treatment of lipid metabolic disease in animals [35] . an alternative class of amos is peptide nucleic acids (pna), which are synthetic oligonucleotides with n-(2-aminoethyl)-glycine replacing the deoxyribose or ribose backbone [36] . a study published in 2008 reported that pna can efficiently block the action of cellular mirnas [37] . finally, another approach in silencing mirna is the use of so-called microrna sponge, a synthetic mrna that contains multiple binding sites for a particular mirna and that is transcribed from a plasmid containing a strong promoter (reviewed in [28] ). in conclusion, different classes of amo have been shown to be efficient in silencing mirna and may be useful therapeutic tools (reviewed in [29, [32] [33] [34] 38] ). samples and they are referred to as kipyv wupyv [41, 42] . this year a novel human polyomavirus, merkel cell polyomavirus (mcpyv), was identified that is associated with merkel cell carcinoma [43] . the hpyv genome can be divided into three functional regions. the early region encodes the early proteins large t-antigen (lt-ag) and small t-antigen (st-ag), while the late region encodes the capsid proteins vp1-vp3 and the regulatory protein agnoprotein. both regions are separated by the noncoding control region that encompasses the origin of replication and the promoter/enhancer sequences for the early and late genes (reviewed in [44] ). the sv40 genome encodes a viral mirna (vmirna) of which both arms are complementary to the early viral mrnas and reduces expression of the early proteins (figure 2(a); table 1 ). cells infected with mutant sv40 lacking this mirna or with wildtype sv40 yielded comparable levels of infectious viruses, but the latter were less sensitive to lysis by cytotoxic t cells and produced less interferon-γ. thus sv40-encoded mirna allows the virus to evade the immune system [45] . the sv40 mirna is conserved in bkv and jcv and both mirnas generated from the precursor hairpin bind to the same target, that is, the early transcripts. the bkv and jcv mirnas serve the same role as sv40 mirna, that is, downregulation of early expression. jcv mirna, mir-j1, was readily detected in brain samples of pml patients, suggesting a biological role of this mirna [46] . the group of sullivan has also identified a mcpyv-encoded mirna, mir-m1, which does not share sequence identity with the known mirnas of the other polyomaviruses. mcpyv mir-m1 is located in the early region (figure 2 (a)) and can downregulate early gene expression. in accordance with sv40, this may allow the virus to evade the immune system. however, mcpyv is associated with merkel cell carcinoma and the viral genome is integrated in these tumours [43] . blocking mir-m1 by, for example, antagomirs will increase the expression of the viral oncoprotein lt-antigen and as such have little beneficial therapeutic effect [47] . expression of a corresponding or other viral-encoded mirna for the other hpyv wu and ki is lacking so far. human papillomaviruses (hpv) are nonenveloped viruses with a circular dsdna genome of approximately 8000 base-pairs. these viruses are associated with benign and malignant lesions of the skin and the genital tract. more than 100 different hpv genotypes have been identified and based on their association with benign warts or cancer, they are classified as low-risk and high-risk variants, respectively [1] . one study with hpv type 31 failed to clone vmirna from virus-infected cells [58] . however, this does not exclude that other strains may encode vmirna. moreover, the expression of vmirna may be regulated in a temporal and spatial manner, so that the experimental conditions for capturing vmirna may be tricky. in addition, the high mutation rate of hpv genome sequences may impede the prediction of the presence of putative vmirna. adenoviruses are naked dsdna viruses that can cause mild respiratory, gastrointestinal, urogenital, and ocular disease. more than 50 serotypes have been described in human. although there is no proof for a causative role in malignancies, adenoviruses can induce cancer in animal models and have been extensively studied to scrutinize viral mechanisms for cellular transformation [59] . adenovirus encodes small noncoding rnas, known as virus-associated rna or vai and vaii rna, which are generated by rna polymerase iii. this noncoding rna plays an important role for viral replication and neutralizes the antiviral action of interferon by blocking the dsrnainduced protein kinase (pkr), which phosphorylates and thereby inactivates the eukaryotic translational initiation factor 2 [60] . a recent study showed that a minor fraction of vai rna is processed by dicer into functional risc-associated ssrna which can act as mirna ( table 1) . blocking of vai-derived small rna by 2'-o -methyl amo complementary to vai decreased virion production [61] [62] [63] . in an attempt to identify potential targets for vai-derived mirna, computational analysis was used. putative targets include genes encoding apoptosis-related protein napor and pkr-activating protein pact. therefore vai rnaderived mirna may help adenovirus to escape the actions of the host defense mechanisms [64] . herpesviruses are enveloped dsdna viruses with a genome size ranging between ∼130 to ∼250 kilobase-pairs. they are divided into three subfamilies denoted α, β, and γ. approximately 130 different herpesviruses have been identified to date, including the human α-herpesviruses herpes simplex virus 1 (hsv-1 or hhv-1), herpes simplex virus 2 (hsv-2 or hhv-2), varicella-zoster virus (vzv or hhv-3), the β-herpesviruses cytomegalovirus (hcmv or hhv-5), hhv-6a, hhv-6b, and hhv-7, the γ-herpesviruses epstein-barr virus (ebv or hhv-4), and kaposi's sarcoma-associated virus (kshv or hhv-8). all human herpesviruses are able to establish latent infections with only a small subset of viral genes expressed (reviewed in [64] ). as will be discussed in the next section, among the viral transcripts that can be detected in latently infected cells are viral-encoded mirnas, which seem to be required to maintain a latent state of infecton, but may also contribute to the pathogenic properties of the virus. herpes simplex virus-1 (hsv-1 or human herpes virus 1; hhv1) infects the majority of the human population, but remains a latent cohabitant in most people. reactivation of the viruses usually results in cold sores, but it can also cause a spectrum of diseases from sightthreatening ocular infections in immunocompetent adults to more severe infections in newborns and immunosuppressed patients (reviewed in [89] ). an important gene in hsv latency is the lat gene. this gene encodes the latency-associated transcript (lat), which does not code for a protein. lat seems to promote cell survival of the infected cells [90] . gupta and coworkers proposed that the antiapoptotic activity of lat was achieved by an mirna-entrapped in the lat (mir-lat), which downregulates the expression of transforming growth factor-β (tgf-β) and smad3. the latter is a mediator of the signalling pathway induced by tgf-β, while tgfβ can prevent cell proliferation and induce cell death [67] . however, a later study revealed that the described mir-lat was not viral encoded, but in fact a cellular mirna expressed in sh-sy-5y cells [67] and the report describing mir-lat was retracted [91] . work by umbach et al. could also not confirm the existence of this mirna in hsv-1 infected sh-sy-5y cells [48] . later, it was shown that the hsv-1 lat exon encodes hsv-1 mir-lat-icp34.5, which can be detected in hsv-1 infected cells [89] . circa 120 base-pairs upstream in this region, a sequence with 77% homology to the hsv-2 mirna mir-i (see further) is present, but no mature mirna was detected in hsv-1 infected cells. however, the existence of this mirna during hsv-1 latency in vivo remains to be confirmed [68] . computational analysis predicted 24-mirna candidates in the hsv-1 genome, 8 of which were conserved in hsv-2, suggesting they may be functional mirnas [69] . the authors confirmed the expression of one mature mirna, designated mir-h1, in hsv-1 infected vero cells, where it was expressed late in productive replication. this mirna is encoded approximately 450 bp upstream of the transcription start site of the lat transcript, but a corresponding sequence is not conserved in the hsv-2. the function of mir-h1 6 journal of biomedicine and biotechnology downregulates the expression of cmv genes involved in its own replication process, for example, transactivators ie72 and ie86; ul120/121; ul114mhc class i-related chain b (micb), a cellular ligand for the activating receptor nkg2d; downregulation of ie-1 mir-ul148d-1 mir-us4-1 mir-us5-1 mir-us5-2 mir-us24 paramyxoviridae (measles virus) 1 predicted [65] remains to be established and no cellular target mrnas were identified [69] . umbach and coworkers detected in addition to mir-h1 five novel viral mirnas in trigeminal ganglia of mice latently infected by hsv-1, as well as in hsv-1-infected vero cells. these mirnas, that is, mir-h1 to mir-h6, are encompassed in the lat locus ( figure 2 (b)). by quantitative rt-pcr, the authors were able to roughly estimate the number of copies of each mirna during productive infection of vero cells. mir-h1 and mir-h6 were expressed at ∼1200 and 300 copies, respectively, while the other mir-hs were present at less than 40 copies per infected cell. in latently infected trigeminal ganglia, much higher levels were monitored with 63 000 copies of mir-h2, 8000 copies of mir-h3, 800 000 copies of mir-h4, 80 000 copies of mir-h5, and 40 000 copies of mir-h6. the large difference in numbers of mirna transcripts in latently infected cells compared to cells with productive hsv-1 infection indicates that mir-hs play an important role in establishing latent hsv-1 infection. indeed, mir-h2 expression diminished the protein levels of icp0, a viral transcriptional activator that promotes viral replication, while mir-h6 inhibits expression of icp4, which is required for expression of most hsv-1 genes during reproductive infection [48] . hsv-2 typically infects the genital region and establishes a lifelong latent infection. the prevalence of latent hsv-2 infection varies between 10-60%. reactivation can cause oro-facial and genital herpes, but hsv-2 infection can also cause encephalitis and neonatal herpes, and forms a risk factor for hiv acquisition [89] . the only detectable viral transcript during hsv-2 latency is the lat, but the molecular function of this transcript remains largely unknown [92] . hsv-2 lat exon encodes an mirna, referred to as mir-i, which is expressed during latent, as well as during acute infection. remarkably, several promoters regulate mir-i expression in different stages of the viral life cycle. this hsv-2 mirna efficiently diminishes the expression of the viral neurovirulence factor icp34.5, a multifunctional protein required for viral replication in neuronal cells in vivo, and with intrinsic neurovirulent properties [93] . thus, mir-i may affect the outcome of infection (latent versus productive) by modulating the protein levels of icp34.5. whether mir-i has other targets remains to be investigated, but an mir-i analogue is also expressed by hsv-1, indicating the importance of this mirna for these viruses [68] . tang and colleagues identified two new hsv-2 mirnas, mir-ii, which includes mir-ii-5p and mir-ii-3p, and mir-iii, both encoded by exon 2 of lat. the expression of mir-i, -ii, and -iii increased during infection of cells, but mir-iii displayed slower kinetics than the two other mirnas. similar to mir-i, mir-ii silences the expression of icp34.5,while mir-iii functionally resembles hsv-i mir-h2 in that it can downregulate the expression of icp0 [70] . the virus establishes a latent infection, but reactivation leads to herpes zoster, commonly referred to as shingles. acute vzv reactivation may lead to post-herpetic neuralgia [94] . no putative mirnas could be predicted in the vzv genome [65, 71] , but experimental studies to unambiguously proof the existence of vzv mirna are lacking. table 1 and figure 2(c) ). three viral mirnas, designated as ul23-5p, mir-ul23-3p, and mir-us24, were identified that are expressed during productive hcmv infection of permissive cells (human foreskin fibroblasts, astrocytoma u373mg cells, retinal pigment epithelial cells, and human microvascular endothelial cells). their putative cellular target genes include genes encoding transcription factors (e.g., hnf3 and tgif2), receptors (e.g., il-18 receptor 1 precursor; cd206), proteins implicated in t-cell activation (ahnak1), in signal transduction (e.g., rab2l), and in biosynthesis of leukotrienes that sustain inflammatory reactions (coactosin-like protein). whether these genes represent bona fide targets as well as the biological relevance of hcmv mirna-mediated silencing of these genes remains elusive [49] . hcmv usually establishes a lifelong persistent or latent state in healthy individuals by ensuring that infected cells avoid immune recognition. the hcmv-encoded mirna mir-ul112-1 seems to play a central role in helping the virus to hide from the host's immune system. this viral mirna targets mrna for mhc class i-related chain b (micb), and to a lesser extend mica. these proteins are cellular ligands for the activating receptor nkg2d, which is expressed on some natural killer (nk) cells, γ/δ t cells, and cd8+ t cells. during cellular stress (such as viral infection) micb is induced, thus activating nk-cells and t cells that can lead to the killing of infected cells. cells infected with mutant virus lacking this mirna were more susceptible to being killed in an nkg2d-dependent manner by nk cells [72] . the mir-ul112-1 also represses the expression of hcmv genes involved in its own replication process, in part by targeting mrna encoding immediate early proteins. one of them is the viral transactivator protein called immediate early 72 (ie72) that regulates the transcription of viral genes required for acute replication [50] . ie72 plays a pivotal role in controlling latency and reactivation. mir-ul-112-1 can thus restrict reactivation of the virus through negative regulation of ie72 expression [50, 95] . two separate studies showed that mir-ul112-1 also inhibited expression of the immediate early protein ie1. murphy et al. detected increased ie1 levels in cells infected with either a virus lacking mir-ul112-1 or with mutations in the seed sequence of the ie1 gene compared to cells infected with wild-type hcmv [73] . grey and coworkers demonstrated that addition of mir-ul112-1 rna prior to infection reduced ie1 protein levels and blocked viral replication [74] . ie1 is a crucial protein to ascertain lytic replication of hcmv, thus downregulation of ie1 may help the virus to establish latency. this strategy may be a common feature for herpesviruses because immediateearly genes may be putative targets for hsv-1 mir-lat, ebv mir-bart15 and mir-bhrf1-3, and kshv mir-k12-6-3p [73] . yet another target for mir-ul112-1 is the viral protein ul114, a homologue of the mammalian dna repair enzyme uracyl-dna glycosylase. ul114 is required for efficient viral dna replication. hence, mir-ul112-1mediated downregulation of ul114 may prevent viral dna replication and favor a latent infection state [65] . taken together, the actions of mir-ul112-1 seem to be associated with latent viral infection, a state which allows the virus to hide from the immune system. ablating expression of this viral-encoded mirnas by amos may therefore force the virus into a lytic cycle and provide the immune system the opportunity to get ride of the viral infection. -lymphocytes in more than 90% of the human population. ebv is associated with infectious mononucleosis and has been implicated in the pathogenesis of several malignancies including burkitt's and hodgkin's lymphomas, posttransplant and t-cell lymphomas, x-linked lymphoproliferative syndrome, nasopharyngeal, and gastric carcinomas [97] . latently ebv-infected cells are classified in stage i, ii, or iii, each of them characterized by distinct ebv gene expression [98] . among the latency stage-specific ebv transcripts are mirnas. more than 20 different ebv mirnas have been identified that are transcribed during latent infection (table 1 ; [25, 51, [75] [76] [77] [78] [79] ). the ebv mirnas are organized in two major clusters within the ebv genome (figure 2(d) ). one cluster resides in the bart (abbreviation for bamhi-a rightward transcripts) region. the bart region gives rise to multispliced transcripts and is highly expressed in ebv-positive cancers and in epithelial tissues, while there is low bart expression in b lymphocytes. the exact function of bart mrnas remains obscure [76] . the intronic region of bart also encodes the mirnas mir-bart1 to mir-bart20. the second region that encodes multiple mirnas is the untranslated region of the gene encoding bhrf1 (bamhi fragment h rightward open reading frame 1), a viral bcl-2 homologue that prevents apoptosis. bhrf1 encompasses the mirnas mir-bhrf-1-1 to mir-bhrf-1-3 [51, 75] . the expressions of ebv-encoded mirnas in clinical samples and computational analysis to predict putative targets were applied to unravel the biological functions of ebv mirnas. these approaches showed that the mir-barts are abundantly expressed in latently infected epithelial cells, nasopharyngeal carcinomas, ebv-associated gastric carcinoma cell lines and tissues, burkitt's lymphomas latency type i, ebv positive primary effusion lymphomas, and diffuse large b-cell lymphomas, but at a significantly lower level in b cells. this corresponds well with the expression pattern of bart multispliced transcripts (see above). higher levels of bhrf1-3 were measured in latency type iii burkitt's lymphomas and in diffuse large b-cell lymphomas [66, 79, [98] [99] [100] . another study demonstrated that induction of ebv replication in latency i-infected cells was associated with increased expression of mir-bhrf1-1, -2, and -3, but expression levels of mir-bart-1 and -2 did not change. on the other hand, induction of ebv replication in latency iii-infected cells did hardly change the expression levels of bhrf1-1, -2, and -3 [98] . these observations suggest that ebv mirnas may be implicated in the oncogenic properties of the virus, but also in regulating its replication. moreover, a precise knowledge of the latency state of ebv and the expression pattern of viral mirnas may improve the successful treatment of ebv infection with amos. the function of most ebv vmirna remains poorly understood, but some targets of ebv mirnas have been recently identified. several mi-barts prevent expression of viral latent infection membrane protein 1 (lmp1) protein (see table 1 ). lmp1 functions as a constitutively active tumour necrosis receptor [101] , and can activate several signalling pathways including nfκb, ap1, jak/stat, mek/erk, and pkc. lmp1 can also interact with p53 and affects cyclins, cyclin-dependent protein kinases (cdk), and the cdk inhibitors p16 and p27 (reviewed in [102] ). furthermore, lmp1 is expressed in all the ebv related malignancies and promotes cellular transformation. its oncogenic property makes lmp1 an attractive target for ebv therapy. interestingly, overexpression of lmp1 results in growthinhibitory and sensitization to apoptosis induced by stress or chemotherapeutic agents ( [76] and references therein). thus amo-mediated neutralization of mi-barts may lead to elevated lmp1 protein levels and render ebv-positive tumour cells more susceptible to chemotherapy. the viral mirna mir-bart2 can inhibit expression of viral dna polymerase balf5 and may thus interfere with viral replication and prevent lytic infection [51, 77] . silencing mir-bart2 could thus allow the virus the complete its life cycle and produce new infectious virus particles, which then could offer the immune system the opportunity to detect and eliminate ebv. using computational prediction programs such as miranda and rnahybrid (reviewed in [103] ) allowed choy and coworkers to envisage the cellular protein p53 up-regulator of apoptosis (puma) as a target for mir-bart5 [78] . the authors demonstrated that puma levels were decreased in cells expressing mir-bart5 compared to cells lacking mir-bart5. in accordance, when mir-bart5 was specifically inhibited with an anti-mir-bart5 oligonucleotide, puma protein levels decreased and apoptosis was triggered. thus, ebv may promote survival of infected epithelial cells by modulating the expression of an apoptotic protein through an mirna-mediated mechanism. this finding may have important implications in the development of anti-ebv agents such as amos directed against mir-bart5. fewer studies have been directed to determine the targets of mir-bhrf1s. mir-bhrf1-2 is involved in the cleavage of bhrf1 rna in the cytoplasm, but the biological relevance remains to be determined [98] . in another study, xia et al. observed that high levels of mir-bhrf1-3 were correlated with low levels cxcl-11, a potent interferon-inducible tcell attracting chemokine. mirna-mediated suppression of cxcl-11 may serve as an immunomodulating mechanism allowing the virus to survive in the host [79] . on the other hand, enhancing cxcl-11 expression in ebv-positive tumours by amos against mir-bhrf1-3 may increase susceptibility of the tumour cells to the immune system. in agreement with this, two recent studies reported antitumour activity for cxcl-11 in animal models [104, 105] . kaposi's sarcoma-associated virus, so named because it was detected in kaposi's sarcoma, belongs to the γ-herpesviruses and is also known as human herpesvirus-8 or hhv-8. hhv-8 is associated with kaposi's sarcoma, as well as with two rare forms of b-cell malignancy: primary effusion lymphoma (pel) and the plasma cell variant of multicentric castleman's disease. like other herpesviruses, kshv can establish a lifelong latent infection characterized by a limited viral gene expression [106] . a total of 17 kshv mirnas encoded by 12 distinct mirna genes have been reported and their sequences are highly conserved between different kshv genomes in pel cell lines and in clinical samples. however, some polymorphism was observed, particularly in mir-k12-5 and mir-k12-9 [80, 107, 108] . the entire kshv mirna cluster resides within an approximately 4 kilobase-pairs region between open reading frames orf k12 (kaposin) and orf 71 (figure 2(e) ). to elucidate the functions of the kshv mirnas, transcriptome analysis was performed from cells stably expressing the mir-k12 cluster. among the differentially expressed genes were genes encoding proteins implicated in proliferation, immune modulation, angiogenesis, and apoptosis. the gene encoding thrombospondin-1 was targeted by all ten kshv mirnas, but especially by mir-k12-1, mir-k12-3-3p, mir-k12-6-3p, and mir-k12-11. thrombospondin-1 possesses antiproliferative and antiangiogenic properties. other transcripts that were reduced corresponded to the genes for osteopontin, s100 calcium binding protein, plasticity related gene 1 product, and integral membrane protein 2a [80, 81] . the mrna for the bcl-2 interacting protein bclaf1 was identified as a target for mir-k12-5. additional inhibition of bclaf1 expression was obtained in the presence of mir-k9, -10a, and -10b. the exact biological relevance is not yet understood, but sirnamediated depletion of bclaf1 enhanced the frequency of spontaneous lytic reactivation of kshv. mirna-mediated reduction of bclaf1 expression would prevent permanent latency of the virus, a type of infection that represents a deadend pathway of viral spreading [108, 109] . a kshv mirna that has gained special interest is mir-k12-11 because its seed sequence, known to be critically important for mrna target recognition is 100% conserved with the cellular mir-155, suggesting that these mirnas may regulate common targets. the exact role of mir-155 remains unclear, but a number of b-cell lymphomas and solid organ tumors overexpress mir-155, while mir-155 transgenic mice develop b lymphomas [110, 111] . work by the groups of skalsky and cullen confirmed that mir-k12-11 indeed is an orthologue of mir-155, and that they target common transcripts [81, 82] . comparing the gene expression profiles in cells stably expressing either mir-155 or mir-k12-11 revealed that they regulate an analogous set of mrnas. the products of these transcripts include proteins involved in b-cell function (e.g., src-like adaptor or sla), innate immunity (e.g., iκb kinase and phosphoinositide-3-kinase), apoptosis (xiap associated factor-1; ldoc1), cell cycle regulation (e.g., fos), and gene expression (e.g., fos and bach1). bach-1 (btb and cnc homolog 1) is a bzip protein that can repress transcription through heterodimerization with the small maf proteins [112] , while c-fos can heterodimerize with the jun proteins to form the ap-1 complex. ap-1 is a multifunctional protein involved in cellular proliferation, transformation, and apoptosis [113] . for a complete list of mir-155/mir-k12-11 regulated genes, the reader is referred to the work of skalsky et al., and of gottwein et al. [81, 82] . treatment of latently-infected kshv with an antagomir against mir-k12-11 enhanced fos protein levels about 2.5-fold compared to untreated cells [81] . computational analyses further revealed seed sequence homology between the viral mirnas kshv mir-k12-6-5p, ebv-bart5, and hcmv ul70-5p with human mirnas mir-15a plus 16, mir-18a/b, and mir-340, respectively [82] . both mir-15a and mir-16 are believed to possess tumour suppressor activity and to induce apoptosis by silencing bcl2 expression, while mir-18 was demonstrated to be oncogenic [52, 114] . kshv-encoded mirnas seem to be crucial both in survival of the virus in its host, but also to play a causal role in viral-associated pathologies. amo-mediated silencing of kshv-encoded mirnas may thus be a strategy to counteract viral infection, but may also undesirably target cellular mirnas with identical seed sequences as the viral mirnas. poxviruses are dsdna viruses that replicate in the cytoplasm and have as such no access to the nuclear proteins involved in the biogenesis of mirna. nevertheless, mirna precursor sequences have been predicted in the genomes of the human poxviruses vaccinia virus and variola virus, but their existence has not been validated [25, 65] . whether the other human poxvirus, molluscum contagiosum virus, encodes mirna remains to be established. hepatitis b virus (hbv), an enveloped virus with a circular partial dsdna genome, persistently infects more than 300 million people worldwide. hbv can cause a spectrum of liver diseases ranging from mild liver dysfunctions to chronic hepatitis, cirrhosis, and hepatocellular carcinoma [53] . this makes efficient anti-hbv therapy highly vital. amo-based vmirna silencing is probably no option since no mirnas could be detected by computational analysis [65] , and expression of hbv mirnas has not been reported so far. one study identified a putative hbv-encoded mirna, but in vivo expression of this hbv mirna was not tested. computational screening for complementary sequences in the 3' untranslated regions of cellular mrna to this hbv mirna did not reveal putative target transcripts [54] . it therefore seems unlike that this is a bona fide viral mirna. hiv is the causative agent of acquired immune deficiency syndrome (aids), and it is estimated that >30 million people worldwide are infected with this virus. two species, hiv-1 and hiv-2, infect humans (for a recent review see [55] ). hiv utilizes reverse transcriptase to convert its ssrna genome into a dsdna provirus. during this process, the 5' and 3' ends of the viral rna genome are converted into long terminal repeats (ltrs). the ltrs play a pivotal regulatory role in establishing, maintaining, and overriding the latent state of the virus [115] . the central domain of the ltr is referred to the r region, which encompasses the (transactivation-response region) tar. tar binds the viral protein tat, a transactivator that plays an important role in the transcriptional activation of the provirus genome (reviewed in [116] ). the tar encodes proven and putative mirnas (figure 2(f) ). klase and coworkers described an mirna encoded by the hiv-1 tar element. this mirna causes hdac-1 to associate with the viral ltr, resulting in diminished viral gene expression. this suggests a role for hiv-1 mirna in maintaining viral latency [83] . in another report, ouellet and colleagues demonstrated the expression of two tar element-derived mirnas by northern blotting, primer extension, and rnase protection assay. the mirna derived from the left arm of the tar stem has been named mir-tar-5p, while the mirna originating from the right arm was designated mir-tar-3p. the latter appears to preferentially accumulate in hiv-positive cells [84] . the biological role of these mirnas remains to be elucidated, but they may contribute to modulating viral and/or cellular gene expression, with a potential impact on viral replication and/or host antiviral defense efficiency. the mir-tar-5p described by ouellet overlaps with the vmirna no. 1, while mir-tar-3p partially overlaps with vmirna no. 5 described by bennasser and coworkers [85] . they predicted by computer-directed analyses 5 pre-mirnas in the hiv-1 genome, which in principle could yield 10 mature mirnas. their expression has not been validated, nor has their biological role been addressed, but deduction of potential target transcripts resulted in the indentification of cellular genes encoding protein kinases, ion channels, proteins involved in protein synthesis and degradation, growth factors, and dna methylation [85] . tar dna of the long terminal repeat of hiv-1 encompasses an antisense rna (hivainr), which encodes hiv proteins, but that can also form a duplex with the 5' end of all sense hiv mrna, enabling the virus to control the expression of its gene [117] . hivainr can potentially code for several mirnas, referred to as haamirnas. putative targets for these mirnas are the mrnas for interleukin (il)-15, il-2 receptor γ chain, human fragile x mental retardation protein (fmrp), and il-1 receptor-associated kinase 1 (irak1). il-15 is important in the regulation of t-cell maturation, development and survival of natural killer cells, and survival of long-lived memory t cells, while the il-2 receptor γ chain is a common component of the receptors for il-2, -4, -7, -9, -15, and -21. aberant expression of this receptor leads to severe t-cell and nk-cell deficiencies. irak1 is a critical signalling mediator of innate immunity. downregulation the expression of il-15, il-2 receptor γ chain, and irak1 by hiv mirna would impair the immune system and favor survival of the virus in the host. fmrp is an rna-binding protein that is implicated in protein synthesis and mirna processing. thus hiv could use haamirna to deregulate the host mirna mechanism to dispose the virus by depleting fmrp ( [56] and references therein.) although the existence of these haamirnas has not been proven, it is tempting to speculate that, in accordance with other viruses, hiv encodes mirnas allowing hiv to survive in the host. a recent study examined the possibility of hiv-2 tar to encode mirnas. two putative mirnas, mir-tar2-5p and mir-tar-3p were identified, but their expression awaits validation [87] . besides tar, other regions of the hiv genome have been shown to contain mirna sequences. the nef gene of hiv-1 is located at the 3' end of the viral genome and is highly expressed during the early stages of virus replication. nef is a multifunctional accessory protein that is important for viral replication, but that also plays a key role in pathogenesis as nef can downmodulate cd4, cd28, and the class i major histocompatibility complex [86] . hiv-1 encodes a nef -derived mirna referred to as mir-n367 (figure 2(f) ). unlike classical mirna, this mirna does not affect gene expression at the post-transcription level, but rather at the transcription level by promoter interference. mir-n367 suppresses hiv-1 promoter activity via a negative responsive element in the 5'-long terminal region and via nef sequences in the 3' untranslated region [57, 118] . future studies are required to elucidate the precise mechanism by which mir-n3667 represses hiv-1 promoter activity. downregulation of nef expression may suppress hiv-1 replication and allow persistently low pathogenic or latent viral infection [57] . as the nef gene is conserved in hiv-2, hiv-2 may also apply a similar mechanism to maintain a low profile in the host. the identity and action of hiv mirnas remains to be scrutinized before amos-based therapy can be considered as anti-hiv drugs. however, computational alignment of the potential hiv-1 mirnas with specific human t-cell mrnas identified potential cellular targets including genes encoding cd4, cd28 and interleukin-2, il-3, and il-12 [119] . viral mirna-caused inhibition of the expression of these proteins seems advantageous for the virus, and therefore counteracting vmirna by amo may help the host to clear hiv infection. htlv-i persistently infects 10-20 million humans worldwide and is the etiological agent for adult t-cell leukaemia [5] . one study reported that t cells persistently infected with htlv-1 did not express viral micrornas [88] , but meticulous studies should be performed to rule out the existence of htlv-i mirna. none of these viral genomes seem to contain putative mirna sequences, except for measles virus and yellow fever virus, which each possesses a single putative mirna. however, the mirna in yellow fever virus could not be validated, while the existence of mirna in measles virus was not tested [25, 65] . intriguingly, the liver-specific mir-122 facilitates the replication of the oncovirus hcv, but the mechanism for this function of mir-122 in hcv replication is still unknown [120, 121] . numerous human diseases are caused by viral infections, but the intimate relation with the host makes the development of antiviral drugs difficult. vaccination has been proven to be very succesful to combat some viral infections, but mutations and diversity of virus strains has hampered the development of efficient vaccines against other viruses. new antiviral treatments are based on drugs that inhibit specific viral activities such as viral proteases or polymerases (for recent reviews see [122, 123] ). viral-encoded mirna that may be implicated in the viral life cycle and the pathogenic properties of the virus offers a novel attractive target for antiviral therapy. silencing the action of viral mirnas may enable the host cell or the immune system to gain control over the virus and even to eliminate the virus. the idea of targeting viral transcripts is not new, and rna interference has been demonstrated to efficiently mediate inhibition of replication of human pathogenic viruses such as hiv-1, hcv, dengue virus, severe acute respiratory syndrome (sars) coronavirus, poliovirus, human rhinovirus, influenza a virus, hepatitis d virus, hbv, hsv-1, hpv, jcv, ebv, and cmv in cell culture (reviewed in [12] ). besides recent studies have proven the potential of this rna interference as antiviral therapy in animal models [124, 125] , and even in clinical trials such as alnylam against respiratory syncytial virus and nuc b1000 against hbv (reviewed in [126, 127] ). however, anti-hiv rna interference studies revealed that escape virus variants could appear which could evade the inhibitory action of sirna [128, 129] . journal of biomedicine and biotechnology the use of amo to neutralize viral mirna adds a new twist to rna interference. amos are easy to produce and relatively cheap, and easy to administer locally (but not systemically). moreover, they possess low toxicity and are highly specific. most viral mirnas identified so far have little homology to each other and to known host cell mirnas (reviewed in [130] ). this reduces the risk of offtarget effects of anti-mirna oligonucleotides and increases the therapeutic potentials of mirna silencing. the mirna silencing action of both lna and antagomirs is sensitive to single nucleotide exchanges. for antagomirs, it was shown that this effect depends on the position of the mismatch. nucleotide substitutions at the very 5' end or in the centre did not prevent downregulation of mir-122 [33] . these data indicate that changes in the 3' end of the antagomir may abrogate its ability to destroy target mirna and should be taken into account when designing and testing antagomirs. another advantage of amos is that they probably can be used against all serotypes of a specific virus. although not meticulously investigated, mirna sequences between different viral strains seem to be conserved because of their importance for the viral life-cycle (see e.g., kshv-encoded mirnas; [80, 107, 108] ). however, polymorphism in viral mirnas has also been observed (see next paragraph). although amos may provide an attractive novel antiviral therapy, practical problems and other pitfalls may hamper the use of them. for example, antagomirs directed against mir-ul-112-1 could drive the virus towards acute replication and disrupted the inhibition of mcib expression, resulting in possible clearance of the virus by the immune system. however, there is a potential risk of severe pathological effects caused by acutely replicating hcmv, especially in immunocompromised patients [50] . another disadvantage of amos may be off-target effects. as mirna do not require full complimentarity to bind their target sequence, it can be imagined that an amo not only binds to its predicted mirna but also to other mirnas and even mrnas. in addition, ssrna oligonucleotides may interact with toll-like receptors 7 and 8 thereby stimulating the immune system. similar side effects have been reported for sirna (reviewed in [126] ). polymorphism in viral-encoded mirnas has been described in viral-infected cell lines and in clinical samples. for instance, mir-k12-5 of different kshv isolates contains mutations, which can affect maturation and biological activity of this mirna [25, 100, 107] . thus the mirna may not be expressed, in which case the amo will have no effect or the amo may not bind because of the mutations in its target mirna. another problem facing the use of amos in antiviral therapy is that the expression of a specific gene may be regulated by several viral mirnas. for example, translation of the transcript of thebachgene is prevented by three kshv viral mirnas: mir-k12-11, mirna-k12-1, and mir-k12-6. thus the effectiveness of an amo against, for example, mir-12-11 can be compromised. indeed, treatment of latently infected kshv virus with a specific antagomir against mir-k12-12 alone only modestly increased the amount of bach protein [81] . a cocktail of different amos directed against distinct viral mirna may help to overcome this problem. so far, such studies are lacking, but a recent study successfully applied sponge mirna to silence hbv transcripts. an expression vector encoding multiple mirnas targeting hbv hbsag mrna strongly reduced the expression of this protein [131] . another challenge is to improve in vivo delivery of the amos to viral infected cells and obtain long-lasting action of the antagomirs. aerosol delivery devices similar to the ones used for delivery of asthma therapeutics could be used for respiratory viruses [15] . other delivery strategies include intravenal or systemic injection, viral vectors, and lipid-and polymerbased vehicles [131] [132] [133] [134] . recently, sustained inhibition of hcv replication in cell-culture was obtained when celldegradable multilayered polyelectrolyte film (mpf) coated with sirna was delivered to infected cells. by this approach, a single regime of mpf-mediated sirna treatment was sufficient to inhibit hcv replication for 12 days. moreover, mpf-mediated delivery of sirna also protected uninfected cells from hcv infection. another advantage is the very low toxicity of mpf [135] . these promising observations in cell culture put mpf-based delivery of amos forward as an efficient antiviral tool. another limitation of anti-mirnas is the site of application. studies with antagomirs against mir-16 in mice revealed that when injected into tail veins, antagomirs were incapable of silencing mir-16, whereas local injection into the mouse cortex efficiently induced degradation of the target mirna [33] . another drawback of the use of amos is that the chemical modification can exert antiproliferative or other off-target effects such as been demonstrated for the phosphorothioate backbone, which can associate with cellular proteins [29] . antisense oligonucleotides such as lna and pmo have proven to efficiently inhibit rna and dna virus replication in cell culture and animal models, without toxicity for the cell or animal. however, these pmo were directed against viral protein-encoding mrna, and studies of pmo-mediated silencing of viral mirna have not been reported so far (see e.g., [31, [136] [137] [138] ). future viral mirna research is faced with important challenges before amos may enter the clinic. our comprehension on the functions of viral mirna and the interplay between viral infection and cellular mirna expression is just beginning to emerge. studies aimed at the identification of viral mirna and elucidation of their functions should be pursued. difficulties facing computational-based prediction are false positives, but also the shortcoming to detect genuine mirnas. moreover confirmation of expression of mirna by, for example, northern blot may fail to monitor mirna. expression levels of vmirna may be cell-specific, for example, ebv mir-bhfr1-2 had considerably lower expression levels in jijoye cells than in b95-8 cells [7] . dose-and time-dependent studies are required to determine the optimal therapeutic regime. such pharmacokinetics and pharmacodynamic studies are largely lacking [133] , but recent in studies in mice revealed 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tiwari, sneham; lapierre, jessica; ojha, chet raj; martins, kyle; parira, tiyash; dutta, rajib kumar; caobi, allen; garbinski, luis; ceyhan, yasemin; esteban‐lopez, maria; el‐hage, nazira title: signaling pathways and therapeutic perspectives related to environmental factors associated with multiple sclerosis date: 2018-09-11 journal: j neurosci res doi: 10.1002/jnr.24322 sha: doc_id: 345922 cord_uid: 3waid65i multiple sclerosis (ms) is an immune‐mediated demyelinating disorder of unknown etiology. both genetic‐susceptibility and environment exposures, including vitamin d deficiency, epstein‐barr viral and herpesvirus (hhv‐6) infections are strongly implicated in the activation of t cells and ms‐pathogenesis. despite precise knowledge of how these factors could be operating alone or in combination to facilitate and aggravate the disease progression, it is clear that prolonged induction of inflammatory molecules and recruitment of other immune cells by the activated t cells results in demyelination and axonal damage. it is imperative to understand the risk factors associated with ms progression and how these factors contribute to disease pathology. understanding of the underlying mechanisms of what factors triggers activation of t cells to attack myelin antigen are important to strategize therapeutics and therapies against ms. current review provides a detailed literature to understand the role of both pathogenic and non‐pathogenic factors on the impact of ms. almost all ages, although higher incidences are detected in younger adults as they are more susceptible to environmental factors. ms is more common in women, suggesting that hormones may also play a significant role in determining susceptibility (milo & kahana, 2010; nylander & hafler, 2012) . prevalence of ms is two to three times higher in women than in men ( figure 1 ) (ahlgren, oden, & lycke, 2012; compston & coles, 2002; orton et al., 2010; wallin et al., 2012) . during the initial stages of the disease, patients primarily experience a neurological complication referred to as clinically isolated syndrome (cis). this is the first demyelinating event, lasting for approximately 24 hr and presenting as monofocal or multifocal lesions in the cns (marcus & waubant, 2013) . possible symptoms of cis include optic neuritis, a brain stem/cerebellar syndrome, a spinal cord syndrome, or hemispheric dysfunction (miller, barkhof, montalban, thompson, & filippi, 2005) . later, the disease becomes more convoluted and characterized as progressing into four distinct variants. these subtypes are important for patient prognosis and treatment considerations. the most common type of ms is called relapsing remitting ms (rrms) and occurs in approximately 87% of ms cases (ransohoff, hafler, & lucchinetti, 2015) . rrms patients show neurological symptoms and have a short and temporary relapse phase where not only existing symptoms are aggravated, but new symptoms may also arise (weiner, 2008) . the inflammatory attacks during this period are unpredictable and are associated with visual and memory impairment, tingling and numbness and fatigue. this relapse stage is followed by a stage f i g u r e 1 multiple sclerosis (ms) epidemiology in the united states. prevalence of ms on the map is illustrated by different color. areas with low prevalence of ms are indicated in light pink, medium prevalence of ms (pink), moderate prevalence of ms (red), and high prevalence of ms (burgundy). inset shows the ratio between genders and that women are twice as likely than men to develop ms significance despite major research efforts in the past few decades, the extent to which environmental factors (including external pathogens) and genetic susceptibility contribute to the pathogenesis of multiple sclerosis (ms) is not clearly identified. this review examine the viral and bacterial as well as non-pathogentic environmental associations to ms progression and how these factors contribute to disease pathology. of remission (complete/incomplete) in which the patient may recover (fully or partially), resulting in asymptomatic disease for several months or years (ghasemi, razavi, & nikzad, 2017) . another variant is secondary progressive ms (spms) occurring in ~65% of patients with rrms, gradually transitioning into spms as neurological complications steadily advance over time. patients may have intermittent relapses, but no remission is observed (correale, gaitan, ysrraelit, & fiol, 2017; ghasemi et al., 2017) . a more uncommon variant, primary progressive ms (ppms), occurs in approximately 10%-15% of ms cases (ransohoff et al., 2015) . ppms is distinguished by a gradual progression from onset with deteriorating symptoms and no occurring relapses or remissions. these patients are primarily affected at the nerves of the spinal cord and present with fewer brain lesions compared to other subtypes. patients with ppms show ataxia with problems in walking, weakness, stiffness, and trouble with balance (ghasemi et al., 2017) . lastly, progressive-relapsing ms, another rare subtype (occurring in ~5% of ms cases) is marked by complications starting from the initial stages of the disease, and progresses with critical relapsing cases without any remissions, mostly without recovery resulting in exacerbating neurological injuries. symptoms in these patients include eye pain, double vision and depression, along with sexual, intestinal, and urinary system dysfunctions (ghasemi et al., 2017) . although ms is a disease common to adults, onset of the disease in children below 18 years of age is not uncommon, accounting for about 10% of total ms cases and is termed pediatric ms (pms) (ferreira, machado, dantas, moreira, & souza, 2008; jancic et al., 2016; schreiner, 2018) . pms is pathophysiologically distinct in terms of brain biopsy, cellular profiling in csf, and the immune molecules present when compared with adult ms patients (schreiner, 2018) . the psychological consequences of childhood ms include depression, fatigue, anxiety, panic disorder, bipolar disorders, and cognitive impairment (amato et al., 2010) . though the disability rate is lower than that of adult-onset ms, children diagnosed with pms pose a risk of developing neurological deficits during the active stage of life (renoux et al., 2007) . t-cells mature may express, on their cell surface, either a cd4 glycoprotein, found in t-helper (th) cells, or they may express a cd8 glycoprotein, present in cytotoxic t-cells (golubovskaya & wu, 2016) . exposure to specific cytokines, such as il-1, il-2, il-4, il-6, tnf, and inf-γ result in the differentiation of naïve cd4 + cells into a variety of subsets: th1, th2, th9, th17, th22, tfh, and treg. each subset possesses a unique cytokine profile, releasing cytokines specific to that subset, which may provide protective or anti-/pro-inflammatory properties (golubovskaya & wu, 2016) . th1 provides protection against intracellular pathogens, th2 defending against parasites, and th17 focuses on defending the host against extracellular pathogens. for example: exposure to il-1 results in the differentiation of a naïve cd4+ cell to a th17 subset which function as mediators of inflammation and produce il-17, protecting the host against fungi and bacteria (golubovskaya & wu, 2016; raphael, nalawade, eagar, & forsthuber, 2015) . th17 cells are known to mediate autoimmunity and play key roles in augmenting inflammation (raphael et al., 2015) . (saxena, martin-blondel, mars, & liblau, 2011) . proliferation and antigen-driven activation of these effector c8+ cells is mediated by th1 produced il-2, and may present as autoreactive t cells as a result of self-antigen presentation by apcs, potentially resulting in t-cell induced axonal damage (hwang, hong, & glimcher, 2005; rollings, sinclair, brady, cantrell, & ross, 2018; saxena et al., 2011) . expansion of cd8+ cells, detection of identical antigen-specificity in a population of t cell clones, and presence of predominantly il-17 producing cd8+ cells, all within active lesions in ms, support the concept of cd8+ cell mediated autoimmunity, in ms (saxena et al., 2011 ). is the common pathological factor between all ms subtypes. this response is facilitated by discriminatory activation of cd4 + helper and cd8 + killer t cells, accompanied by little to no activation of regulatory t cells (gandhi, laroni, & weiner, 2010) . as a result, inflammation is an alternate characteristic symptom of ms. t lymphocytes and activated macrophages infiltrate the brain and with the aid of activated microglia, induce immunological activation, leading to progressive myelin destruction (kasper & shoemaker, 2010) . disruption of the blood-brain barrier (bbb) also allows immune cells to enter the brain, where t lymphocytes attack the myelin sheath (compston & coles, 2002) . this triggers a subsequent inflammatory cascade, leading to the secretion of cytokines, including interferon (ifn)-γ, tumor necrosis factor (tnf)-α, and interleukin (il)-17, and the production of antibodies by b lymphocytes which can secrete their own cytokines, tnfα and transforming growth factor (tgf)-β (duddy et al., 2007) . these soluble factors cause further recruitment of immune cells, perpetuating inflammation and damage (nylander & hafler, 2012) . in addition, pro-inflammatory type 1 t helper (th1) and th17 cells display direct cytotoxic effects on oligodendrocyte precursor cells and obstruct remyelination (baxi et al., 2015; moore et al., 2015) . predisposition to genetic changes and its effect on ms susceptibility was derived from the characteristics of familial aggregation and the amount of genetic information they share (gejman, sanders, & duan, 2010; oksenberg, baranzini, sawcer, & hauser, 2008) . although ms is not a hereditary disease, predisposition to genetic variations increases the risk. studies have shown that the risk rate in monozygotic-twins that have 100% genetic familiarity is approximately 25% (ghasemi et al., 2017) . these risks are reduced on decreased genetic familiarity, with 5% in siblings, 2% in parents, (giovannoni & ebers, 2007) . single-nucleotide polymorphisms (snps) in il-7r markers identified in ms patients in sweden suggest that snps can lead to dysregulation in the activity of both t-and b-cells (lundmark et al., 2007) . by analyzing ms patients possessing snps in the serine-threonine kinase 11 gene (encoding liver kinase b1-lkb1), boullerne et. al. suggests it as a risk factor for ms (boullerne et al., 2015) . lkb1 has effects on t-cell activation, th1/th17 cytokine production, and modulate adenosine monophosphate kinase (ampk) activation. dysregulation of lkb1 function is implicated toward ms pathogenesis (germain et al., 2013) . it has been demonstrated using gsaw that multi high rates of discordancy in monozygotic twin studies led to the conclusion that genetic and epigenetic factors are not the only factors (giovannoni & ebers, 2007) , thus emphasizing the importance of environmental factors in developing ms. environmental factors such as smoking, vitamin deficiency, diet, and uv radiation exposure act as modifiable risk factors (sawcer et al., 2014) . the expression of the vitamin d receptor on most immune cells including apc is the risk of developing spms is 3-fold higher in smokers than in non-smokers who had relapsing-remitting clinical onset of the disease (healy et al., 2009; wingerchuk, 2012) . furthermore, tobacco affects the integrity of the bbb and triggers inflammatory responses that, lead to loss of membrane integrity (hawkes, 2007; hernan et al., 2005; riise, nortvedt, & ascherio, 2003) . marabita et. al. showed that smoking affects dna methylation in ms patients and gene expression of aryl-hydrocarbon receptor repressor (ahrr) is higher in smokers and contained hypomethylation at several cpg sites (marabita et al., 2017) . tobacco smoke induces the production of free radicals, such as nitric oxide (no) which exacerbates the clinical progression of ms by causing inflammatory lesions and axonal degeneration (hernan et al., 2005) . risk for ms development intensifies due to complex interplay of different pathogenic environmental factors including viral and bacterial infections (virtanen & jacobson, 2012) . infection with measles, herpesvirus (hhv-6) and epstein barr virus (ebv) at a young age increase the chances of developing ms (compston & coles, 2008; giovannoni et al., 2006) . virus-derived peptide sequences such as the epstein-barr nuclear antigen 1 (ebna1) (owens, gilden, antigens bind to autoreactive b cells that facilitate ebv replication, and augment ebv-specific t-and b-cell responses (ascherio et al., 2001; lunemann, kamradt, martin, & munz, 2007) . other viruses associated with ms include varicella zoster virus (vzv) and human endogenous retroviruses (hervs). a "multi-hit" model study illustrated how the gut microbiota creates a suitable proinflammatory condition for multiple insults (genetic disposition and environmental factors) to act together reinforcing ms disease (cekanaviciute et al., 2017) . akkermansia muciniphila and acinetobacter calcoaceticus induce proinflammatory responses, and are found in higher numbers within the gut whereas parabacteroides distasonis, which stimulates the anti-inflammatory response, is found in lower numbers in ms patients (mirza & mao-draayer, 2017) . it is also possible that individuals without early life exposure to infectious pathogens could mount abnormal responses during infectious exposure in their adult life (baranzini et al., 2010) . it is evident that multiple environmental factors play important roles toward increased risks of ms; however, the pathogenic contributions are still being unearthed and will be the focus of the remainder of this review. most pathogens associated with ms are also considered causative agents in the development of other neurological disorders. by defining the mechanisms behind ms-like symptoms linked to pathogenic infections, the similarities that may lead to onset of disease become more readily apparent. hhv-6 infection is associated with neurological disorders such as febrile seizure, epilepsy, and encephalitis (yao et al., 2009) , while ebv infection has been associated with cns vasculitis (kano et al., 2017) . these two pathogens share common mechanisms that can contribute to the onset of ms symptoms. both ebv and hhv-6 infection can trigger myelin-reactive antibodies specifically targeting myelin basic protein (mbp), an ubiquitous myelin membrane protein (deber & reynolds, 1991; tait & straus, 2008) . have not yet been characterized. in contrast, vzv utilizes an endosomal/autophagic recycling pathway similar to that used by myelin proteins and the iron-binding glycoprotein transferrin (winterstein, trotter, & kramer-albers, 2008) . it is through this pathway that vzv trafficks glycoprotein complexes from the endoplasmic reticulum (er) to the outer cell membrane to release new virus (buckingham, jarosinski, jackson, carpenter, & grose, 2016) . given that low serum transferrin levels have been linked to several chronic diseases, including ms (zeman et al., 2000) , trafficking of vzv alongside the transferrin receptor would aid in viral assembly, ultimately leading to increased immune activation. apart from demyelination, there is also evidence of axonal or neuronal injury in ms that have gained some importance in the ms field. the "inside-out" concept of induced demyelination suggests that pathogenic damage occurs at the unmyelinated axon, often in combination with enhanced oligodendrocyte apoptosis (tsunoda & fujinami, 2002) . several viral infections are accompanied by axonal damage like hiv (an et al., 1997) , hsv (martin, 1984) , and theiler's murine encephalomyelitis virus (tmev) (tsunoda & fujinami, 2002) , which has also been associated with ms. the pathogenesis of this axonal injury still requires much study. one concept suggests direct immune attack on the axon based upon the presence of antibodies to neurofilament and tubulin in some ms patients (silber & sharief, 1999) . separately, some viruses can be transported through the axons leading to axonal degeneration and dissemination of virus throughout the cns (kristensson, 1996) . in tmev infection, for example, axonal injury precedes demyelination which indicates in some cases demyelination may come secondary to axonal injury (das sarma, kenyon, hingley, & shindler, 2009 ). additionally, as has been discussed, there is also evidence on axonal injury in silent inactive plaques where few axons are degraded at a given point in time but will instead persist for a longer time in the cns (lassmann, 2003) . viral infections causing encephalitis (morfopoulou et al., 2016) , are also considered as a risk factor in developing ms (weiss, 1983) . although no definitive association between viral infections and ms have been reported so far, compelling evidence for this hypothesis exists, such as the presence of viral dna, rna, or proteins, and immune response elements in the body fluids or tissue of patients (donati & jacobson, 2002) . attempts to establish a definitive association between ms and viral infections has been restricted by many factors, including asynchrony between viral infection and demyelination, genetic, and immunological susceptibility of the individuals, and the involvement of polymicrobials as the causative agent of ms. this is further complicated by differences in the pathogenic mechanisms of different individuals with varying genetic backgrounds, resulting in different forms of the disorder (donati & jacobson, 2002) . even though different animal models of ms, such as the experimental autoimmune encephalitis (eae) model and tmev model aided in the understanding of possible underlying mechanisms including molecular mimicry and bystander activation, animal models for the most commonly associated viruses hhv-6 and ebv have yet to be developed (virtanen & jacobson, 2012) . studies on viral induced demyelination show cellular mechanisms of myelin destruction through persistence, latency, reactivation, and tissue damage by viruses. virus-induced demyelination disorders have different underlying mechanisms related to direct toxic effects or immune activation by virus, molecular mimicry, bystander activation, and epitope spreading (fujinami, von herrath, christen, & whitton, 2006) . examples of direct toxic effects from an active infection is jcv which directly causes demyelination after infecting oligodendrocytes, and canine distemper virus which infects astrocytes in white matter leading to demyelination (chalkias et al., 2014) . another mechanism underlying demyelination is molecular mimicry, where pathogenic proteins possess homologous amino acid sequences with self-proteins subsequently causing viral induced autoimmunity (fujinami & oldstone, 1985) . below, various viruses and their associations to the etiology or pathogenesis of ms will be discussed. measles is caused by the highly contagious paramyxovirus and is transmitted via respiratory route (laksono, de vries, mcquaid, duprex, & de swart, 2016; ludlow, mcquaid, milner, de swart, & duprex, 2015) . the clinical manifestation is relatively mild presenting as skin rashes, though, in rare cases, neurological complications can also occur. acute disseminated encephalitis (lundmark et al., 2007) , measles inclusion body encephalitis, and subacute sclerosing panencephalitis are all severe cases of measles which have been associated with cns disorders (laksono et al., 2016) . in ms patients, the most frequent anti-viral antibodies produced intra-thecally tend to be against measles, rubella and vzv, referred to as mrz reaction phenomenon. although this is not restricted to a direct association with these three viruses, it may be a response against different viruses such as hsv or ebv. evidence supporting the possible direct interaction or causal association of measles with ms is not well documented; moreover, unchanged prevalence of ms after vaccination against measles, shed further doubt on the possible association between the virus and disease (ahlgren, odén, torén, & andersen, 2009; donati & jacobson, 2002; jacobson, flerlage, & mcfarland, 1985; norrby, link, & olsson, 1974) . human herpesvirus 6 (hhv-6) is a dna virus belonging to β-herpes virus family and is widely sero-prevalent in adults. the two variants hhv-6a and hhv-6b have about 90% sequence homology, and are transmitted via saliva and close contact with infected parents during early years of life. hhv-6b is the causative agent of exanthema subitum (or roseola), a benign febrile illness with skin rashes while hhv-6a has not been definitively associated with any disease (reynaud, horvat, & horvat, 2013) . hhv-6 is able to enter cells through the cd46 receptor, with viral antigen able to be detected in oligodendrocytes and rarely in astrocytes. however, hhv-6 has been detected in the scar-like plaques on the myelin sheath of ms patients (reynaud et al., 2013) . accordingly, igm antibody against the p41/38 antigen and dna of hhv-6a was reported in serum from rrms patients. hhv-6a persists and may reactivate cns immune cells, leading to higher lymphoproliferative response against hhv6a which has been detected in ms patients. mechanistically, hhv-6 may be linked to pathogenesis of ms through a proline-rich integral membrane protein known as u24 (sullivan & coscoy, 2010) . the proline rich region of u24 possesses sequence identity with mbp at a key phosphorylation site which, if molecular mimicry were to occur, the mitogen-associated kinase pathway and the glycogen synthase kinase may be recruited and activated, resulting in lower phosphorylation of mbp. the potential of the proline-rich region of u24 acting as a molecular mimic may mediate cross-reactivity of t-cells and lead to targeting of mbp in ms patients. ebv causes infectious mononucleosis and remains latent in b cells (thorley-lawson, 2001) for approximately 30 years after infection (nielsen et al., 2007) . later onset of symptomatic ebv infection is extremely risky (ascherio & munger, 2007) . cd4 and cd8 t cells are the primary line of defense against ebv-infection. the association of ebv with ms is supported by numerous case reports where ebv infection was shown to cause axonal demyelination, and detectable ebv antibodies were reported in csf and meningeal lymphoid follicles (salvetti, giovannoni, & aloisi, 2009; thacker, mirzaei, & ascherio, 2006; tselis, 2012) . the major ebv antigen in ms cases is ebna1, with antibodies produced specifically in the csf compartment forming oligoclonal bands, a characteristic of viral infection in ms. epitope mapping techniques employed for testing the specificity of oligoclonal igg showed that ms-specific epitopes are derived from ebna1 and brrf2 (ebv lytic gene product) proteins, providing evidence of intrathecal production of ebna1-specific igg in ms (cepok et al., 2005) . viral capsid antigen (vca) and the early antigens (ea) are also produced against ebv. serum antibodies to vca show more variation and higher seroprevalence than antibodies to ebna-1. ebv transforms b cells controlled by host immunological control leading to ebv-associated malignancies. during the lytic stage, c-terminus of ebna1 binds to the episome (circular dna of ebv) and transmits virus into progenies. another link between ebv infection and ms corresponds to molecular mimicry for immunodominant myelin resultant motifs on t cells (libbey & fujinami, 2010) . peptides derived from ebv have sequences which are stimulatory for mbp derived cd4 t-cell clones. a cross-reactive t-cell clone detects the mbp peptide similar to the hla-dr2b and ebv bslf1 peptide similar to the hla-dr2a. t cells specific to ebna1 will provide immunity against ebv, but in ms patients, an ebna1specific response of t cells is distorted; yielding exacerbated t cell responses throughout infection. molecular mimicry amongst viral antigens and autoantigens support autoreactive t cells, such as the accumulation of ebna1-targeting t cells leading to autoimmunity associated with infection. ebv genes in turn excite autoimmune b cells which are also cross-reactive, serving as evidence that ebv has involvement in ms pathogenesis (compston & coles, 2008) ( figure 2 ). | 1837 vzv commonly causes encephalitis, meningitis, and myelitis. immune responses against vzv, measles, and rubella have been found in patients with cns vasculitis (graef, henze, & reiber, 1994) there is growing evidence that retroviruses play a role in ms. it is suggested that two cofactors of the hervs family, (a) ms-associated retrovirus (msrv) and (b) endogenous retroviral family w env(c7) member 1(ervwe1), whose envelope proteins (zappia e) show neuropathogenic features in vitro and in vivo, contribute to the inflammatory processes and ms pathogenesis (sospedra & martin, 2005) . herv-w env gene encodes syncytin-1 protein, which expresses in astrocytes, microglia and macrophages of ms patients. it promotes release of cytokines and reactive oxygen species in astrocytes leading to oligodendrocyte damage stimulating neuroinflammation. during the host response to infection, many bacterial components are released that can be recognized as targets for immune response. heteropolymer peptidoglycan (pg) released by bacterial cells serves as a ligand to activate inflammatory immune response (gupta, wang, vinson, & dziarski, 1999; weidemann et al., 1994) . pg has been shown to be present in immunocompetent apc in the brain tissue of ms patients suggesting its role in ms pathogenesis (schrijver et al., 2001) . the presence of pg in the brain tissue of ms patients has been shown in the absence of active infection and thus indicates that peripheral pg may translocate with the apc to the cns and serve to drive an inflammatory response in this location. chlamydia pneumoniae causes common respiratory infections, with up to 50% of the population showing seropositivity in late adulthood (grayston, wang, kuo, & campbell, 1989) . the first report of f i g u r e 2 activation of cross-reactive t cells by pathogen-derived peptides and self-epitopes via molecular mimicry or by stander activation. bystander activation involves a wide range of events that occur after viral infection such as production of cytokines and chemokines, expression of host otherwise inactive genes, and the unveiling of blocked self-antigens (donati & jacobson, 2002) . the process of epitope spreading implies the introduction of additional epitopes following an initial inflammatory response to one epitope, such as myelin basic protein (lehmann, forsthuber, miller, & sercarz, 1992) . the mechanism can be explained by self-sustained breakdown of myelin by continuous exposure of epitope during inflammatory reactions. the phenomenon of epitope spreading is well studied in the autoimmune encephalitis and tme models for demyelination induced by viruses. b-cells localized to the central nervous system present infectious agents on their surface and hence the infected epitope spreads to t cells and autoimmunity is promoted coincidental infection with c. pneumoniae in a patient with rpms was reported in 1998 (sriram, mitchell, & stratton, 1998) . upon broader examination it was found that over 97% of serum csf in patients with ms tested positive for the c. pneumoniae momp gene. additionally, c. pneumoniae was isolated from 64% of ms patients, showing coincidental infection (sriram et al., 1999) . c. pneumoniae has also been implicated in the pathogenesis of other neurodegenerative diseases such as alzheimer's disease and schizophrenia given that the pathogen can invade the blood stream, and when taken up by monocytes as carriers, can cross the bbb and access the cns (cahoon, 2009) . it must be recognized that the direct relation of c. pneumoniae with ms is controversial. likely, the bacteria acts as a co-factor and contributes toward persistent and prolonged infection leading to ms disease progression (fainardi, castellazzi, seraceni, granieri, & contini, 2008) . helicobacter pylori has shown the histological isolation from 86.4% of ms patients versus 50% of healthy control subjects. (gavalas et al., 2015) . gavalas et al. first reported a reverse correlation between h. pylori and ms with subsequent meta-analysis, concluding that patients with h. pylori infection had lower rates of ms than those without infection. this suggests that h. pylori infection may provide some protection against ms development or progression (jaruvongvanich, sanguankeo, jaruvongvanich, & upala, 2016) , mechanisms to which still remain unclear. counter to observed associations between bacterial infection and ms, staphylococcus aureus infection in a murine model of ms demonstrated a reduction in experimental autoimmune encephalomyelitis (fainardi et al., 2008) . this is counterintuitive to the model of bacterial inflammation driving ms development or progression as s. aureus produces systemic inflammation and stimulates the production of cytokines responsible for inflammatory response. this protective role was demonstrated by a reduction in demyelination and higher axon density in infected groups, as well as lower clinical scores, compared to uninfected groups. this protective role was mediated in part by an extracellular adhesion protein which was recovered in the blood of infected animals (kumar et al., 2015) . animal models are critical for diseases like ms, which have a complex etiology and lack a complete understanding of its mechanisms. due to its complexity, there is currently no single animal model that can reflect the whole spectrum of the disease. over the years, several animal models have been designed to study ms and reviewed effectively (procaccini, de rosa, pucino, formisano, & matarese, 2015) , however, the most frequently used model for etiological studies of ms is the eae model. in eae, an immune reaction to cns components is induced in susceptible animals with self-antigens that are derived from myelin protein. this was first described in monkeys stimulated with rabbit antigen (rivers, sprunt, & berry, 1933) , but later freund's adjuvant (freund & mcdermott, 1942) and pertussis toxin (munoz, bernard, & mackay, 1984) were added to induce a humoral immune reaction similar to as seen in rrms (kabat, wolf, & bezer, 1947) , and analogous to what is found in ms patients. although studies have been done on guinea pigs (freund, stern, & pisani, 1947) and monkeys (kabat et al., 1947) , the best models resulted from mice (olitsky & yager, 1949) and rats (lipton & freund, 1952) . the type of antigen introduced to the animal determined its particular presentation of eae. immunization of sjl/j mice with an epitope of proteolipid protein induces rrms (tuohy, lu, sobel, laursen, & lees, 1989) , while ms induced by myelin oligodendrocyte glycoprotein in c57bl6/j mice is chronic ms (tompkins et al., 2002) . apart from eae models, viral infection models which most effectively aid in the study of ebv related ms are tmev infection models (richards et al., 2011; tsunoda, iwasaki, terunuma, sako, & ohara, 1996) . utilizing tmev models also assist in studying new therapeutic methodologies targeted toward adhesion molecules and axonal degeneration (tsunoda & fujinami, 2010) . independent of introducing immunogens and viruses, certain chemical compounds have been utilized to cause an immune reaction leading to ms-like symptoms, like cuprizone and lysolecithin, which cause oligodendrocyte cell death and have been primarily used to study demyelination and remyelination in ms (jeffery & blakemore, 1995; matsushima & morell, 2001) . epigenetics, encompasses dna methylation, histone modifications, and small rna based mechanisms, may be a potential therapeutic avenue in ms treatment (huynh & casaccia, 2013) . due to observed epigenetic changes in ms, this therapy has emerged as a novel treatment alternative to ms. histone deacetylase inhibitors | 1839 like trichostatin a, vorinostat, and valproic acid have been used as therapeutic drugs, and have been shown to reduce cell infiltration in the cns and reduce overall inflammation through the suppression of dendritic cells and dendritic cell costimulatory molecules (camelo et al., 2005; ge et al., 2013; zhang, zhang, wu, & schluesener, 2012) . further, histone acetyl transferase inhibitors like curcumin were found to repress infiltration of inflammatory cells in the spinal cord (xie et al., 2009 ). more recently, 5-azacytidine, a dna methylation inhibitor, was shown to inhibit eae by induction of regulatory t cells in a mouse eae model (chan et al., 2014) . corticosteroids are commonly used drugs for treatment of acute ms relapses (karussis, 2013) . glucocorticoids strongly downregulate inflammation by inhibiting genes that affect the production of cytokines including il-1, il-2, il-3, this in turn, reduces the proliferative ability of t cells (abbruzzese et al., 1983; barkhof, hommes, scheltens, & valk, 1991; beck et al., 1992; miller et al., 1991; rose et al., 1970) , and induces the synthesis of lipocortin-1 to minimize eicosanoid production and reduce the permeability of bbb with steroids kesselring et al., 1989; miller et al., 1992) . studies have shown that both corticotrophin (acth) and intravenous methylprednisolone could be used in short-term treatment of the acute exacerbations of ms (berkovich et al., 2017 ). an alternative to corticosteroids are sex hormones, for example, testosterone supplements can be protective in males and combination of estradiol and progesterone is protective in case of females (golden & voskuhl, 2016) . the first line of treatment targeting t cells in both the clinic and mouse eae models was represented by type 1 interferons and glatiramer acetate (ga). ifn-β reduces relapses and slows progression of the disease (abreu, 1982) . ga is a synthetic co-polymer containing alanine, glutamine, lysine, and tyrosine (norohna, toscas, & jensen, 1990; rudick, carpenter, cookfair, tuohy, & ransohoff, 1993) . this compound shares immunologic similarities with the mbp molecule and can reduce inflammation by preventing lymphocyte sensitization (abreu, 1985) . ga is capable of downregulating antigen presentation through competitive inhibition for binding to mhc/hla, thereby inducing a th2 shift, in regulatory and cd8 suppressor gareactive cells (comi, filippi, wolinsky, & group, 2001) . b cell immunity plays an integral part in the development of ms because of its role in antibody presentation, cytokine production, meningeal inflammation, axonal degeneration, and grey matter f i g u r e 3 schematic diagram of environmental factors and host derived pathways in causation of the disease and therapeutic strategies associated with multiple sclerosis (ms) progression. although many correlations have been established between foreign pathogens and ms, there is no evidence for a direct association between any pathogen and the development or progression of ms. it is still unclear, whether bacterial or viral pathogens are capable of initiating ms in susceptible patients and exacerbate ms symptoms. as multiple species of bacteria and virus have been shown to associate with ms, it is possible that the mechanism of this association is a result of generalized inflammation caused by the immune response to these pathogens. the listed pathogens have been reported to cause one or more of the indicated pathways which culminate in the demyelination of neurons and progression of ms. the outlined therapeutic strategies include the existing treatment options as well as the focuses of current research demyelination (mclauchlan & robertson, 2017) . therefore, b celldepleting therapy as an alternative to t cell therapy is considered highly effective against relapsing ms, and disability worsening in ppms. the b cell-depleting therapy is divided into two categories; polyclonal and monoclonal antibody therapies. polyclonal antibody therapies inhibit cell-mediated immune reactions by causing general immunosuppression, albeit not commonly used in ms due to high toxicity (walker, hoehn, & kashiwagi, 1976) . on the other hand, numerous monoclonal antibodies including natalizumab, alemtuzumab and daclizumab have proven effective in early rrms (nguyen, gresle, marshall, butzkueven, & field, 2017) . in addition, three emerging monoclonal antibodies against cd20-positive b cells (rituximab, ocrelizumab, and ofatumumab) have shown overall promising effects in clinical phase ii and iii trials (abbruzzese et al., 1983; reff et al., 1994; stashenko, nadler, hardy, & schlossman, 1980) . other developing therapies against relapsing forms of the disease targets b cell cd19 antigen and b cell cytokine signaling molecules (schneider et al., 1999) . hematopoietic stem cell transplantation suppresses the immune system and mitigates the inflammatory responses generated by an insult (burt et al., 2009) . the entire immune system is reconstructed using the patient's own stem cells (burt et al., 2009) . in numerous clinical cases, the use of stem cell therapy was able to bring about an induction of remission and stabilization of ms (burt et al., 2009 ). in addition, stem cell therapy using embryonic and other types of adult stem cells, particularly mesenchymal stem cells, are currently being studied using the eae model (kassis et al., 2008; zappia et al., 2005) . electrical stimulation has emerged as a potential therapeutic technique for ms. a pilot study showed that intraluminal electrical pharyngeal stimulation can treat dysphagia caused by ms (restivo et al., 2013) . a randomized control trial showed that resistance training of light to moderate intensity over long periods of time increases muscle strength in ms patients, while concurrent use of electrical stimulation does not help to further improve the outcome (broekmans et al., 2011) . additionally, functional electrical stimulation has tremendous potential as a therapeutic strategy to aid with gait in ms patients (hausmann et al., 2015; sampson et al., 2016; van der linden, scott, hooper, cowan, & mercer, 2014) . although substantial resources have been devoted on finding a cure for ms, based on current knowledge about the etiology of the disease, further studies are required to determine the association between virus and bacteria in the pathology of ms development ( figure 3) . the most effective etiologic agent causing ms is still not known, though it is possible that multiple agents work in synchronization to hijack the immune system and aggravate the disease. as such, a deeper understanding of the underlying mechanisms is needed because a variety of factors associated with numerous diseases in completely different subsets of patients predisposed genetically for the disease have been shown. in addition, more studies on extent of interplay between ms and other non-pathogenic environmental factors like vitamin d levels, exposure to uvlight, consumption of tobacco, with respect to ms pathogenesis are required. this review was written as part of the curriculum for the course gms 6939, offered to the ph.d. graduate 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environmental factors associated with multiple sclerosis key: cord-291295-7og5umiq authors: xin, shuyu; du, shujuan; liu, lingzhi; xie, yan; zuo, lielian; yang, jing; hu, jingjin; yue, wenxing; zhang, jing; cao, pengfei; zhu, fanxiu; lu, jianhong title: epstein-barr virus nuclear antigen 1 recruits cyclophilin a to facilitate the replication of viral dna genome date: 2019-12-13 journal: front microbiol doi: 10.3389/fmicb.2019.02879 sha: doc_id: 291295 cord_uid: 7og5umiq epstein-barr virus (ebv) nuclear antigen 1 (ebna1)-mediated dna episomal genome replication and persistence are essential for the viral pathogenesis. cyclophilin a (cypa) is upregulated in ebv-associated nasopharyngeal carcinoma (npc) with unknown roles. in the present approach, cytosolic cypa was found to be bound with ebna1 into the nucleus. the amino acid 376-459 of the ebna1 domain was important for the binding. cypa depletion attenuated and ectopic cypa expression improved ebna1 expression in ebv-positive cells. the loss of viral copy number was also accelerated by cypa consumption in daughter cells during culture passages. mechanistically, cypa mediated the connection of ebna1 with orip (origin of ebv dna replication) and subsequent orip transcription, which is a key step for the initiation of ebv genome replication. moreover, cypa overexpression markedly antagonized the connection of ebna1 to ubiquitin-specific protease 7 (usp7), which is a strong host barrier with a role of inhibiting ebv genome replication. the ppiase activity of cypa was required for the promotion of orip transcription and antagonism with usp7. the results revealed a strategy that ebv recruited a host factor to counteract the host defense, thus facilitating its own latent genome replication. this study provides a new insight into ebv pathogenesis and potential virus-targeted therapeutics in ebv-associated npc, in which cypa is upregulated at all stages. epstein-barr virus (ebv) is a member of the gamma herpesviruses and was the first confirmed human tumor virus (ding et al., 2013; lu et al., 2017) . ebv ubiquitously infects more than 90% of the global population and is closely associated with the development of several malignancies, including burkitt's lymphoma and nasopharyngeal carcinoma (npc) (shair et al., 2008; yu et al., 2012; young, 2014; young and dawson, 2014; liu et al., 2017) . npc, which is primarily of epithelial origin, is a type of metastatic head-and-neck neoplasm that is highly prevalent in southern china and some other areas in east asia and africa (tang et al., 2016; tu et al., 2017; jiang et al., 2018) . ebv is able to establish life-long persistence in the human host (young and murray, 2003; . it contains a large genome approximately 172 kb in size and has two phases in its life cycle (the latent and lytic stages) (yu et al., 2011; hammerschmidt and sugden, 2013) . ebv mainly spreads through the saliva of human host, infects b cells through the oral mucosal epithelium, and then transforms b lymphocytes into resting memory b cells through a series of viral latent transcription programs, thus establishing a lifelong latent infection pattern (thorley-lawson et al., 2013) . the majority of ebv infections in vivo are latent (thorley-lawson, 2015) . during ebv latency, the ebv genome exists in the form of episome dna, few viral genes are expressed, and no virion is produced. ebv nuclear antigen 1 (ebna1) is the only viral protein that is expressed in all types of ebvassociated tumors (lu et al., 2010; tao et al., 2015) . determining how ebv is able to maintain its stable latent status in host cells is a topic of interest, because it may provide understanding about the pathogenesis of ebv and new targets to inhibit the persistence of ebv genome in the therapy of ebv-associated cancers. ebv replication is under the control of some host and viral factors that are not fully understood. ebna1 plays a key role in the replication and mitotic segregation of ebv dna episomes to daughter cells (yates and guan, 1991; frappier, 2012b) . ebna1-mediated s-phase episome replication depends on binding of ebna1 to the ebv origin of genome replication (orip) (reisman et al., 1985) . viruses are obligate intracellular parasites, and their replication cycles depend on some host cell factors. for example, some studies suggested that cellular origin recognition complex (orc) and minichromosome maintenance (mcm) complex are related to the ds element of orip, implicating them in the initiation of ebv dna replication (frappier, 2012a; capone et al., 2015) . these host factors may also be potential targets for antiviral therapy. cyclophilin a (cypa) is a protein with multiple functions as a typical member of the cellular peptidyl-prolyl cis-trans isomerase (ppiase) family (braaten et al., 1996; bahmed et al., 2012) . cypa was discovered initially as an intracellular receptor of the immunosuppressive drug cyclosporin a (csa) (braaten et al., 1996; bahmed et al., 2012) . studies have shown that cypa can use il-6 to induce cell signal conversion, activate tyrosine phosphorylation and nuclear transport of transcription factor 3, and can bind and activate nf-κb . cypa is involved in the life cycles of multiple viruses and plays a critical role in their successful infectivity and replication, including human immunodeficiency virus type 1 (hiv-1), hepatitis c virus (hcv), hepatitis b virus (hbv), vesicular stomatitis virus (vsv), vaccinia virus (vv), coronaviruses (covs), and feline coronavirus (bose et al., 2003; naoumov, 2014; jyothi et al., 2015; phillips et al., 2015) . the interaction between cypa and hiv protein promotes the replication and infection of hiv particles; cd147 is the main signal receptor of cypa, and the two interact to regulate the early steps of hiv replication (ciesek et al., 2009; tang et al., 2015) . conversely, cypa suppresses the replication of some viruses, such as rotavirus, infectious bursal disease virus and influenza virus (xu et al., 2010; liu et al., 2012) . however, the role and mechanism of cypa in the function of ebv remain unknown. our laboratory previously performed a proteomics study using npc tissues and found that cypa was upregulated from the early stages of npc (atypical hyperplasia and stage i) to the malignancy stages (yang et al., 2014) . since ebv infection also occurs during the early stage of npc (morrison et al., 2004) , we speculated that a potential relationship might exist between cypa and ebv, which initiated the present approach. recently, we have reported that the exosomal cypa level in npc is positively related that of exosomal latent membrane protein 1, which is another latent protein of ebv. the result suggests a relationship between cypa and ebv (liu et al., 2019) . ubiquitin-specific protease 7 (usp7) is a type of deubiquitinase that is also known as herpesvirus-associated ubiquitin specific protease (hausp) and has been documented as a host factor that inhibits ebv replication (holowaty et al., 2003a) . the usp7-ebna1 interaction was selectively disrupted by deletion of ebna1 residues 395-450 just upstream of the dna binding domain, and the resulting ebna1 mutant exhibited significantly increased dna replication activity (holowaty et al., 2003b) . usp7 also plays a role in the replication inhibition of kaposi's sarcoma-associated herpesvirus (kshv) by interacting with the latency-associated nuclear antigen 1 (lana), which is the homology of ebna1 (jager et al., 2012) . as it is known, kshv also persists in infected cells mainly in a latent state, and lana, is expressed in all latently kshv-infected cells (rainbow et al., 1997) . however, the mechanism by which ebv overcomes host suppression to maintain its own pathogenesis remains to be investigated. here, we demonstrate that ebna1 binds to and recruits cypa to the nucleus to support the function of ebna1 in the replication of the viral latent genome. cypa overexpression could antagonize the host barrier, usp7. the results revealed a strategy that ebv recruited one host factor to counteract another, thus favoring the viral dna replication and persistence in latent infection. the study provides novel insights into understanding ebv pathogenesis in epithelial cells. the p2089 plasmid (maxi-ebv), which contains the complete ebv genome of the b95-8 strain, was kindly provided by dr. w hammerschmidt (delecluse et al., 1998) . the human embryonic kidney hek293 cell line (hek293) was originally obtained from atcc and was used to establish latent infection of the whole ebv genome (p2089) by using hygromycin for the screening, resulting in the c2089 cell line that was previously described by our group (zuo et al., 2015) . subsequently, the cells were divided into groups of shrna (using prnat-u6.1-shrna-cypa treated cells), the nc shrna group (prnat-u6.1-shrna treated cells cypa nc), liposomal transfected cells are based on lipofectamine 3000 transfection reagent (invitrogen-thermo fisher; united states). the day after transfection, cells were treated with g418 was added (sigma-merck; germany) for cell selection. cells were subcultured every 2 days. the cells were grown in dulbecco's modified eagle's medium (dmem; sigma) supplemented with 10% fcs. the c666-1 cell line is an ebv-positive npc cell line (zuo l.l. et al., 2017) . all recombinant plasmids used for bimolecular fluorescence complementation (bimc) were constructed using standard cloning techniques according to the schematic diagram (supplementary figure s1 ) by using the pcaggs vector, which is a gift from dr. harty (lu et al., 2013) . cypa and cypb were amplified from 93 cells by rt-pcr. cypa or cypb gene was fused with the n-terminal fragment of the yellow fluorescent protein (yfp), constructing the plasmids of pcaggs-flag-cypa-ny (cypa-ny) and pcaggs-flag-cypb-ny (cypb-ny), respectively. the ebna1 protein was expressed as a fusion protein with the c-terminal fragment of (yfp), constructing the plasmid pcaggs-myc-ebna1-cy (ebna1-cy). the expression plasmids pcaggs-flag-cypa and pcaggs-flag-cypb were also constructed respectively. the myc tag sequence was added to the n-terminal of ebna1. myc-tagged ebna1 was generated by cloning into the sphi and nhei sites of the pcaggs vector to produce the pcaggs-myc-ebna1 plasmid. the truncations ebna1 (nt, 1-90) and ebna1 (607-641) were generated by pcr based on the plasmid pcaggs-myc-ebna1 using the primers listed in supplementary table s1 . the ebna1 deletion mutants ebna1 (nt, 90-376), ebna1 (376-459) and ebna1 (459-607) were generated by inverse pcr. for example, primer complementary to 243-67 bp (anti-sense) and 1129-1156 bp (sense) of ebna1 were used for amplification, and the resulting pcr. product was gel-purified and self-ligated, resulting in ebna1 ( aa 90-376). then a sphi-nhei fragment from ebna1 ( aa 90-376) was inserted into the sphi and nhei sites of the pcaggs-myc-ebna1 to produce the pcaggs-myc-ebna1 ( aa 90-376). the orip-sv40-luc expression vector was obtained by introducing the flanking sequence of orip amplified from the pc3-orip plasmid, which was a gift from prof. frappier (sivachandran et al., 2011) . the orip sequence was cloned into the saci and xhoi sites of the luciferase reporter vector, pgl3-enhancer. the rt-qpcr was performed as previously described (yu et al., 2012) . briefly, total rna was isolated from cells using the trizol reagent (invitrogen). first, trizol was added to lyse the cells, then 1/5 volume of chloroform was added to the lysate to separate the dna, protein and rna, followed by the isopropanol and 75% absolute ethanol to precipitate the rna. for rt, 1 µg of rna was reversely transcribed into cdna using the one-step gdna removal and cdna synthesis supermix kit (transgen biotech, beijing, china). real-time quantitative pcr (rt-qpcr) was then performed by using the kit of transstart r top green qpcr supermix (transgen biotech, beijing, china) according to the manufacturer's instructions. the following program was used for the rt reaction: 65 • c for 5 min, ice for 2 min, followed by 42 • c for 15 min and then 85 • c for 5 s. the cfx multicolor detection system (bio-rad) was employed for the detection. primers for rna detection by qrt-pcr were designed based on rna sequences, and bate-actin was used as an endogenous control. the following procedure was used for the rt-qpcr: 95 • c for 3 min, followed by 40 cycles of 95 • c for 12 s and 62 • c for 35 s. data obtained by the conventional method of calculating comparative method 2 (− ct) . repeated three times for each sample in parallel in each experiment, and the results were expressed as the mean of three independent experiments. the sequences of the qrt-pcr primers are provided in supplementary table s1. western blotting (wb) was performed using a standard protocol as described previously (zuo l.l. et al., 2017) . cells was lysed in ripa (50 mm tris, ph 7.4, 150 mm nacl, 1% np-40, 0.5% sodium deoxycholate, 0.1% sds, sodium orthovanadate, sodium fluoride, edta, leupeptin) followed by incubation on ice for 30 min, protein quantification according to the specification of bca kit (beyotime, shanghai, china). the cells lysates were centrifuged at 12000 × g for 20 min at 4 • c. supernatant fractions were used for detection. samples with equivalent amounts of denatured protein were separated using 10% sds polyacrylamide gels (epizyme, shanghai, china). after electrophoretic separation, the proteins were transferred to a polyvinylidene difluoride (pvdf) membrane (millipore, danvers, ma, united states). the membrane was blocked with 5% skimmed milk for 1 h at room temperature, followed by an overnight incubation with the primary antibody at 4 • c. after washing three times, the membrane was incubated with a secondary antibody for 1 h at 37 • c. finally, the proteins of interest were detected using the luminata crescendo hrp substrate (millipore) and viewed with the chemidoc xrs + molecular imager (bio-rad). the results were analyzed using the image lab software (bio-rad). the following antibodies were used for the immune detection: anti-ebna1 (santa cruz biotech, de, united states, sc-81579), anti-cypa (proteintech, chicago, il, united states, 10720-1-ap), anti-c-myc (sigma, st. louis, mo, united states, c3956), and anti-flag (sigma, h9658). gapdh (proteintech, chicago, il, united states, 10494-i-ap) and bate-actin (proteintech, chicago, il, united states, 66009-i-ig) were used as the loading controls. the secondary antibodies used for wb and if were as follows: hrp-conjugated anti-rabbit (cst, chicago, il, united states, #7074p2), hrp-conjugated antimouse (ge healthcare, united kingdom, na931v) and alexa fluor 488 donkey anti-rabbit igg (life technologies, united states, 1480770). c2089 cells were plated in a 96-well plate at a density of 1 × 10 3 cells per well. the next day, different csa concentrations as indicated were applied for treatment. at 24, 48 and 72 h posttreatment, the cell counting kit-8 (cck8) was used to test cell viability according to the manufacturer's instructions. the absorbance was detected at the 450 nm wavelength using a microplate reader. cyclosporin a (dalian meilun, china) is a type of immunosuppressive preparation that targets cypa (chatterji et al., 2009) . the drug was dissolved in dimethyl sulfoxide (dmso). ebv-positive cells were cultured in medium containing 40 µm of csa. after 48 h of treatment, the cellular proteins and total rna were extracted and subjected to detection. bimolecular fluorescence complementation assay was performed as described previously (liu et al., 2011; lu et al., 2013 lu et al., , 2014 . the schematic diagram of bimc assay is shown in supplementary figure s1 . in brief, human hek293t cells were grown on coverslips in six-well plates. the pcaggs-myc-ebna1-cy plasmid or the mutant constructs and the pcaggs-flag-cypb-ny plasmid were cotransfected into the cells using the lipofectamine 3000 transfection reagent (invitrogen) according to the manufacturer's instructions. approximately 0.25 µg of each plasmid was used for the transfection. transfection of a single plasmid for each used in bimc assay was performed as a negative control. after 24 h, the cells were gently washed for three times with pbs and fixed with cold 4% paraformaldehyde for 30 min at room temperature. then, the cells were washed and subsequently stained with hoechst 33342 (sigma) for 3 min at room temperature. finally, the cells were washed again, and the slides were observed for the specific yfp signal under a fluorescence microscope. a typical co-ip procedure was performed as described (liu et al., 2011; lu et al., 2013 lu et al., , 2014 . cell lysates were harvested directly or at 48 h post-transfection as indicated. the lysed sample was centrifuged at 13,000 rpm for 20 min at 4 • c. the supernatant lysates of about 1 mg were incubated with a primary antibody, such as an anti-flag monoclonal antibody (mab) (h9658, sigma, 1:100) or anti-mouse igg antibody (control) (sc-0025, santa cruz, 1:200), for 10 h at 4 • c. after centrifugation, the supernatant was transferred to a new tube containing 40 µl of protein g beads (transgen biotech, beijing, china) and incubated for 6 h at 4 • c. after extensive washes with cold lysis buffer, the immunoprecipitated proteins were eluted in sds sample loading buffer (aurigene biotech, changsha, china), separated by sds-page, transferred onto polyvinylidene difluoride membranes (millipore), and detected by wb (zheng et al., 2012) . three small interfering rnas (sirnas) targeting cypa (genbank accession number: nm 001166326) were designed and synthesized by guangzhou ribobio company. to evaluate the knock-down efficiency of the sirnas, 100 pmol of each sirna was transfected into hek293 cells. the cells were lysed with ripa as described by wb, and lysates were used for western blotting with anti-cypa and anti-bate-actin antibodies. the sirna with the best knock-down efficiency was chosen for the subsequent experiments. a scramble sirna-nc was used as the control. hek293t and vero cells grown on coverslips in six-well plates for 24 h were washed gently three times with pbs, fixed with 4% formaldehyde in pbs for 15 min, and then permeabilized with 0.5% triton x-100 in pbs for 5 min. subsequently, the cells were blocked with fresh 10% goat serum. the cells were incubated with an anti-cypa rabbit polyclonal antibody and anti-flag mouse mab overnight at 4 • c, respectively. after washing five times with pbs, the secondary antibody was added for 1 h of incubation at 37 • c. then, hoechst 33342 was applied for nuclear staining for 3 min at room temperature. the coverslips were finally mounted onto slides and observed under a florescent microscope (bx53, olympus, japan). the chip assay was performed to detect the binding of ebna1-orip according to previous reports (shen et al., 2016) . the immunoprecipitation kit (millipore) was used for the assay. hek293 cells were transfected with lipofectamine 3000 (invitrogen) according to the manufacturer's protocol. cells were collected after 48 h and lysed with lysis buffer (beyotime, shanghai, china) [20 mm tris (ph7.5), 150 mm nacl, 1% triton x-100, sodium pyrophosphate, β-glycerophosphate, edta, na 3 vo 4 , leupeptin]. the immunoprecipitated nucleoprotein complexes were eluted by incubation twice for 15 min at 25 • c with 200 µl of elution buffer (1% sds and 100 mm nahco 3 ), and the crosslinks were reversed by incubation at 65 • c for 4 h. the dna was extracted with phenol/chloroform and precipitated with ethanol. then orip dna was amplified by qrt-pcr using the primers listed in supplementary table s1 . the experimental procedure for the detection of orip transcription activation mediated by ebna1 was carried out according to previous description from other group . cells were seeded into 24-well plates 24 h prior to transfection. the following day, 1000 ng of the orip-sv40-luc reporter plasmid was transfected into the cells using lipofectamine 3000. the sv40-luc reporter plasmid was transfected as a control. cell lysates were collected at 24 h post-transfection and assayed for luciferase activity using a luciferase assay kit (promega, madison, wi, united states) on the panomics luminometer. the renilla luciferase activity was also measured using an enzyme assay kit (promega). the results were normalized to the renilla activity. three parallel repeats were performed for each sample in each experiment, and the results were expressed as the mean of three independent experiments. the detection of ebv copy number was performed as described previously (zuo et al., 2015) . dna was extracted from the c2089-shnc and c2089-shcypa cells using the general allgen frontiers in microbiology | www.frontiersin.org kit (cwbio, hunan, china) and quantified. the relative ebv copy number was determined by rt-qpcr using the ebv dna quantitative fluorescence diagnostic kit (sansure biotech, hunan, china) according to the manufacturer's instruction. in this product, bamhi-w fragment in ebv genome is designed as the specific primers and probes. the ebv copy number concentration (copies/cell) of the samples was calculated according to the level of internal reference. the experiment was repeated for three times. the statistical analyses were performed using graphpad prism 5 (graphpad software, ca, united states). differences between groups were determined using student's t-test or one-way analysis of variance (anova). the data are expressed as the means ± standard deviations (sds). single, double and triple asterisks indicate statistical significance ( * p < 0.05, * * p < 0.01 and * * * p < 0.001). by immunofluorescence (if) assay, cypa was detected to be mainly localized in the cytoplasm in vero and hek293t cells (supplementary figure s2) . to study the potential interaction between ebna1 and cypa, bimc assay was performed. the bimc assay is a useful tool for detection of protein-protein interactions in living cells, and the results can be visualized under a fluorescence microscope (hudry et al., 2012; yang et al., 2014) . a diagram of the basic principle of the bimc assay is shown in supplementary figure s1 . the ebna1 protein was expressed as a fusion protein with the c-terminal fragment of the yfp (pcaggs-myc-ebna1-cy), and cypa was fused with the n-terminal fragment of yfp (pcaggs-flag-cypa-ny). these two plasmids were co-transfected into hek293t cells, with the single pcaggs-flag-cypa-ny plasmid transfection as a negative control. the results showed that ebna1 and cypa interacted with each other in the nucleus (figure 1a , top). when the nuclear localization signal (nls) sequence of ebna1 was deleted, the proteins interacted in the cytoplasm ( figure 1a) . this translocation showed the specificity of the interaction mediated by ebna1 with an nls. cyclophilin b (cypb) is another member of the cyclophilin family, but we did not detect any binding signal for cypb-ebna1 in the bimc assay ( figure 1a) . there was no fluorescence signal in the cells transfected with only single plasmid which was a negative control (figure 1a , lower). ebna1 and cypa/b expression in the bimc assay was detected by wb ( figure 1b) . we further evaluated ebna1 and cypa intracellular localization in ebvpositive cells by if assays (figure 1c) . the result showed that cypa expressed in both cytoplasm and nucleus in ebv-positive c2089 cells, while mainly in the cytoplasm of the ebv-negative hek293 cells (figure 1c) . the co-immunoprecipitation (co-ip) assay was used to verify the interaction of ebna1 with cypa. these results showed that both endogenous and exogenous cypa interacted with ebna1 (figures 1d-f) . the plasmids pcaggs-myc-ebna1 and pcaggs-flag-cypa or pcaggs-flag-cypb were transfected in hek293 cells, myc-ebna1 was immuneprecipitated with anti-flag antibody. as shown in figure 1g , the result of co-ip assay further validated that ebna1 interacted with cypa but not cypb. to identify the contribution of the functional domains of ebna1 to the cypa-ebna1 interaction, we constructed five deletion mutants of myc-tagged ebna1 named ebna1 1-90, ebna1 90-376, ebna1 376-459, ebna1 459-607, and ebna1 607-641. the plasmid structures are illustrated in figure 2a . the bimc assay revealed that yfp was reconstructed in hek293t cells using wild-type ebna1 and four of the mutants (ebna1 1-90-cy, ebna1 90-376-cy, ebna1 459-607-cy and ebna1 607-641-cy) but not the ebna1 376-459-cy mutant (supplementary figure s3a) . each negative control with single plasmid transfection is shown in supplementary figure s3c . subsequently, we co-transfected pcaggs-myc-ebna1 or a pcaggs-myc-ebna1 mutant plasmid with pcaggs -flag-cypa into hek293 cells. a co-ip assay using a flag-tag antibody for the pulldown showed the same result (i.e., cypa did not interact with ebna1 376-459) ( figure 2b ). this domain, which is also the region containing the usp7binding domain in ebna1 (figure 2a) , was thus critical for the binding of ebna1 to cypa. co-expression of ebna1 376-459-cy (only this one domain of ebna1) and cypa-ny recovered their binding function based on the bimc assay results (supplementary figure s3b) . the nls (amino acids 379-386) is also included in the domain containing amino acids 376-459, and thus yfp was detected in the nucleus (supplementary figure s3b) . these results indicated that amino acids 376-459 of ebna1, which contain the usp7-binding domain, were essential for the interaction between ebna1 and cypa. we designed three sirnas to observe their efficiency on cypa protein expression in hek293 cells ( figure 3a) . the results showed that the knock-down of sicypa-1 was best, and this sirna was used in the subsequent experiments, whereas a scrambled control sirna (sinc) had no effect on cypa expression. as shown in figures 3b,c , ebna1 protein and mrna expression decreased in response to sicypa in ebvpositive npc c666-1 and c2089 cells. conversely, ectopic cypa overexpression in the ebv-positive cell lines resulted in an increase in the ebna1 expression levels detected by wb and qrt-pcr (figures 3d,e) . as ebv episomal genome is easy to be lost with culture passages in vitro (frappier, 2012b) , we investigated whether cypa depletion might expedite this process in consecutive passages. then we collected gdna from different generations figure 1 | detection of cypa-ebna1 binding by the bimc and co-ip assays. (a) detection of the ebna1-cypa interaction by the bimc assay. hek293t cells were transfected with the indicated plasmids, including cypa-ny, ebna1-cy, ebna1 nls-cy, and cypb-ny following by bimc analysis, and fluorescence was observed. transfection of a single cypa-ny plasmid did not lead to the production of fluorescence. scale bar, 50 µm. (b) the proteins expressed from the plasmids used in the bimc assay were detected by wb. (c) the detection of cypa and ebna1 in ebv-negative and ebv-positive hek293 cells by if assay. scale bar, 50 µm. (d) endogenous cypa interacts with ebna1 in hek293 cells. the plasmid pcaggs-myc-ebna1 was transfected into cells. an anti-myc antibody was used for the pull-down the cypa, and the wb assay was carried out for detection. (e) endogenous cypa interacts with ebna1 in c2089 cells. ebna1 was immune-precipitated with anti-cypa. ebna1 was detected by wb (f) exogenous cypa interacts with ebna1. hek293 cells were transiently transfected with flag-cypa alone or with myc-ebna1. flag-cypa was immune-precipitated with anti-myc antibody. igg was used as a negative control for the pull-down in the co-ip assay. flag-cypa was detected by wb. (g) co-ip assay for comparison of the interactions of cypa and cypb with ebna1. pcaggs-myc-ebna1 and pcaggs-flag-cypa or pcaggs-flag-cypb were transfected in hek293 cells. myc-ebna1 was immune-precipitated with anti-flag antibody. figure 2 | identification of the ebna1 domain required for binding to cypa. (a) diagram of the ebna1 deletion mutants. the start and end amino acid residues for each fragment are indicated according to a previous report (young and murray, 2003) . (b) validation of the interaction between each mutant ebna1 and cypa by the co-ip assay. pcaggs-myc-ebna1, pcaggs-myc-ebna1 mutants, and pcaggs-flag-cypa were transfected into hek293 cells. myc-ebna1 was immune-precipitated with anti-flag antibody. flag-cypa and myc-ebna1 were detected by wb. of cells and detected the copy number of each cell. as it shown in figure 3f , cypa depletion significantly facilitated loss of ebv copy numbers. ebna1-orip binding has been validated to be necessary for the replication initiation of ebv genome (shen et al., 2016) . here, we investigated the effect of cypa knockdown on the orip luciferase activity in cells transfected with an orip luciferase reporter in c2089 cells. ebv-positive c2089 and ebv-negative hek293 cells were stably transfected with a lentivirus expressing a short hairpin (sh) of cypa and a scramble control (shnc) (figure 4a) , and the results showed that cypa was successfully knocked down. ebna1 protein expression was restored in the c2089-shcypa cells transfected with the wild-type cypa expression plasmid ( figure 4b ). in these cells, an orip luciferase reporter, orip-sv40-luc was transiently transfected. after 24 h transfection, luciferase activities were detected. the ebna1 activated orip luciferase was decreased by 3.5-fold in the luciferase assay compared to that in the control cells (c2089-shnc) due to cypa depletion. in contrast, there was no effect on the transfection of an sv40-luc reporter plasmid with cypa depletion (p < 0.001, figure 4c ), ebna1 mrna was detected by qrt-pcr. when ebna1 was lacked, cypa knockdown did not affect luciferase activity of orip-sv40-luc ( figure 4c) . the results indicated that cypa enhanced the ebna1 activation of orip transcription. the chip assay was used to further validate that cypa played a role in ebna1-orip binding. pcaggs-myc-ebna1 and orip-sv40-luc were co-transfected into hek293-shnc and hek293-shcypa cells. as shown in figure 4d , cypa interference (shcypa) reduced the ebna1 binding to orip dna by approximately 50% compared to that of the control (shnc) in hek293 cells (p < 0.05). cypa is an intracellular receptor of csa, which in turn inhibits the ppiase activity of cypa through binding to the hydrophobic pocket of cypa (chatterji et al., 2009 ). based on above data about cypa regulation in ebv genome transcription initiation, we further investigated the possibility of csa against ebv replication. the working concentration of csa was considered as 40 µm according to its inhibition effectiveness and low cytotoxicity by the cell counting kit-8 assay ( figure 5a) . rescue experiments showed that ebna1 protein levels were restored compared to the csa treatment group (figure 5b ). ebna1 protein expression was evaluated by wb after treated or untreated with csa ( figure 5c, left) . rt-qpcr analysis showed that there was a significant reduction for the ebna1 following the decrease of cypa caused by csa (figure 5c, right) . subsequently, c2089 cells were transiently transfected with the cypa expression plasmid together with orip-sv40-luc. luciferase activities were increased at 24 h post-transfection compared with that of no cypa overexpression, but there was no change for sv40-luc (without orip), and ebna1-orip-luc activity increased in cypa overexpression ( figure 5d) . the protein level of cypa and the mrna level of ebna1 are detected ( figure 5d) . we further investigate the role of csa in ebna1-mediated orip transcription. after transfection of orip-sv40-luc, the cells were treated with csa. the result showed that csa decreased ebna-orip-luc activity compared to the untreated group, with a decrease in cypa and ebna1 ( figure 5e) . however, overexpression of cypa and treatment of csa in hek293 cells did not affect the activity of orip-sv40-luc ( figure 5f ). these data indicated that csa inhibited the ebna1-mediated orip activation. chip assay followed by quantitative pcr (chip-qpcr) was used to evaluate the the effect of cypa depletion on loss of ebv copy numbers during passages. ebv-positive c2089 was used for shrna stable transfection and selection. cell lines stably transfected with shrna-cypa or shrna-nc were established for the experiment. cell dispersal for each passage was performed at a ratio 1:2. the copy number of the c2089-shcypa cells was decreased compared with the c2089-shnc, with passage 10th of c2089-shcypa set to 1. * p < 0.05, * * p < 0.01, * * * p < 0.001. effect of cypa overexpression and csa treatment on ebna1 binding to orip in hek293 cells. myc antibody (for myc-ebna1) efficiently pulled down orip-dna from the above samples, and the results showed that csa treatment eliminates ebna1-orip binding, while cypa overexpression increases its binding ( figure 5g) . the results suggested that the ppiase activity of cypa was required for the role of cypa in ebv latent replication. restoration of ebna1 protein expression in c2089-shcypa cells transfected with wild-type cypa expression plasmids. cypa and ebna1 proteins were detected by wb. (c) effect of cypa knockdown on ebna1-orip-mediated transcription activity in the luciferase reporter assay. the ebna1 mrna was detected by rt-qpcr. (d) effect of cypa knockdown on binding of ebna1 to orip in the chip assay. cypa was depleted from hek293 with shrna. antibodies against myc and igg control were respectively used for the pulldown in chip assayshek293. the precipitation of orip dna was quantitated by rt-qpcr. cypa and ebna1 proteins were detected by wb. * p < 0.05, * * p < 0.01, * * * p < 0.001. ubiquitin-specific protease 7 has been implicated in strong inhibition of ebv replication through its tight connection with ebna1 (holowaty et al., 2003b) . in this study, as the above results demonstrated, the cypa binding domain was located within the domain containing amino acids 376-459, which spanned the usp7-binding domain in ebna1. because cypa played an opposite role in regulating ebna1 function compared with usp7, hek293-shnc and hek293-shcypa cells were transiently transfected with pcaggs-flag-cypa alone or with pcaggs-myc-ebna1, a co-ip assay was carried out. the results showed that usp7-ebna1 binding was strong in the hek293-shcypa and hek293-shnc cells. however, when cypa was overexpressed, the amount of usp7 bound with ebna1 was decreased remarkably (figure 6a) . in order to study the ppiase activity of cypa in this mechanism in ebv replication, a co-ip assay was performed to using the treatment of csa. the result verified that csa eliminated the antagonism of cypa on ebna1-usp7 interaction ( figure 6b) . to investigate the ability of cypa to influence the ebna1-orip connection through antagonizing usp7, in hek293 cells, orip-sv40-luc was transfected with pcaggs -myc-ebna1, pcaggs-flag-cypa or pcaggs-myc-ebna1 mutant, a chip assay was designed based on ectopic cypa expression. as shown in figure 6c , the interaction of orip and the ebna1 mutant with deletion of amino acids was significantly enhanced. in contrast, cypa overexpression increased ebna1-orip binding to a high level. the result demonstrated that the deletion of the binding site for both usp7 and cypa exhibited the enhancement of ebna1-orip binding ( figure 6c ). this was a priority effect of the release of usp7 inhibition but not cypa facilitation. only when cypa was overexpressed, could the effect of usp7 inhibition be reversed. the results further showed that cypa overexpression was essential to overcome the usp7 suppression in regulating ebna1 replication function. ebv latent infection is an important causative factor in the development of related cancers such as npc (dittmer et al., 2008; zheng et al., 2014 zheng et al., , 2018 , although the mechanism is largely unclear. viral replication and genome maintenance in host cells are important for the pathogenesis. cypa was found to be highly expressed in npc in the previous work from our laboratory (yang et al., 2014; liu et al., 2019) and played an unknown role related to ebv. herein, we reveal that cypa, especially when increasingly expressed, contributes to the replication function of ebna1. cypa first was recruited by ebna1 into the nucleus and then mediated ebna1-orip binding and replication activity. the mrna expression of ebna1 measured by rt-qpcr. ebv-positive c2089 cell lines were used for the test. (d) c2089 cells were transfected with orip-sv40-luc reporter plasmid and cypa expression plasmids. overexpressed cypa significantly increased ebna1-orip-dependent luciferase activity, but had no effect on sv40 promoter dependent luciferase activity (left). ebna1 mrna was measured by rt-qpcr (right) . (e) orip-sv40-luc reporter plasmid was transfected into c2089 cells, following the csa treatment. csa treatment greatly reduced ebna1-orip luciferase activity compared with untreatment. (f) csa and elevated cypa on ebna1-orip-mediated transcription activity in the luciferase reporter assay in hek293 cells. (g) chip-qpcr was used to determine ebna1-orip binding. csa treatment reduced ebna1-orip binding while elevated cypa increased binding. cypa and ebna1 proteins were analyzed by wb. * p < 0.05, * * p < 0.01, * * * p < 0.001. (c) effect of cypa overexpression and ebna1 376-459 mutation on the ebna1-orip binding detected by chip assay. * p < 0.05, * * * p < 0.001. figure 7 | schematic for the mechanism of cypa in supporting the replication function of ebna1. ebv genomic dna exists within the host genome in the form of extrachromosomal episomes. cytoplasmic cypa can be hijacked by ebna1 into the nucleus. overexpressed cypa can overcome the suppression of usp7 in binding to ebna1. nuclear cypa mediates ebna1-orip transcription, and thus contributing to the viral genome replication and maintenance. on the other hand, when cypa was upregulated, the usp7 suppression in ebna1-mediated replication could be reversed. this interaction is a type of quantity-driven winning for cypa, because its rival usp7 is too powerful. it was reported that the affinity of usp7-ebna1 was 10-fold higher than usp7-p53, implying the strong binding of usp7-ebna1 (saridakis et al., 2005) . the csa treatment demonstrated that the ppiase activity of cypa was required for this function. the schematic working model is shown as in figure 7 . cyclophilin a has been implicated in the life cycles of several viruses and plays a critical role in the successful infectivity and replication of these viruses, including some tumor viruses, such as hbv and hcv (bose et al., 2003; naoumov, 2014; jyothi et al., 2015; phillips et al., 2015) . it has not been reported for the relationship between cypa and kshv, another tumor virus in the same family of gamma herpesvirus as ebv. though cypa is involved in the regulation of several viruses, its function mode is different from that in other viruses depending on the different mode of viral infection and replication. for example, the interaction between cypa and hiv gag was proposed to facilitate disassembly of the viral rna containing core following virus entry and thus supports the efficient reverse transcription of the hiv-1 genome (luban et al., 1993; ott, 2002) cypa also enhances virus attachment to the host cell membrane through interactions with heparans (saphire et al., 2000) and after membrane fusion through interaction with cd147, thereby promoting viral infection. in the present study, for the first time, we showed that cypa was also utilized by ebv in the modulation of viral replication function in epithelial cells. thus, cypa is involved in the maintenance of the virus during its latency in host cells. ebna1 mediates dna episome replication from orip (frappier, 2012a) . our results revealed that cypa was recruited to influence ebna1-orip-mediated transcription. cypa depletion significantly facilitated loss of ebv copy numbers (figure 3f) , suggesting that impairment of ebna-orip binding further weakened successful replication and maintenance of the ebv genome. epstein-barr virus can naturally infected b cells and latently maintained in resting b cells (thorley-lawson et al., 2013) . as cypa is a multi-functional protein, which can be expressed in all kinds of cells, and ebna1 is the only expressed protein of ebv in all latency types. therefore, we think that both cypa and ebna1 may be expressed in resting b cells. how cypa plays a role in the function of ebv in b cells remains to be further investigated. the deubiquitinase usp7 is also known as a hausp and has been found to be associated with several herpesviruses, including hsv-1, ebv, and kshv (holowaty et al., 2003b; jager et al., 2012; hammerschmidt and sugden, 2013) . a previous study demonstrated that usp7 suppressed ebv replication (holowaty et al., 2003b) . here, we showed that ebv hijacked the host factor of elevated cypa to counteract usp7, thereby adding to the definition of hausp. additionally, our data showed that deletion of the binding domain in ebna1 for both usp7 and cypa resulted in significantly increased ebna1-orip binding activity (figure 6c) , mainly due to release of usp7 inhibition. the result was consistent with that of previous report (holowaty et al., 2003a) , demonstrating that the suppressive role of usp7 was strong enough to greatly overshadow the improved role of cypa. in summary, the study reveals that cypa is a novel critical host factor utilized by ebna1 in the viral dna replication in epithelial cells. elevated cypa levels remarkably antagonize usp7 in the interaction with ebna1. the results revealed a strategy that ebv recruited a host factor to counteract the host defense, thus facilitating its own latent genome replication and efficient persistence. our findings implied that ebv has evolved sophisticatedly. this study provides a new insight into ebv pathogenesis and potential virus-targeted therapeutics in ebvassociated npc, in which cypa is upregulated. the data used to support the findings of this study are available from the corresponding author upon request. we thank dr. wolfgang hammerschmidt (gsf-national research center for environment and health, germany) for kindly providing us the maxi-ebv system, which was used in the establishment of c2089 cell line. we thank dr. lori frappier (university of toronto, canada) for the kind gift, plasmid pc3-orip. we also thank dr. ronald n. harty (university of pennsylvania, united states) for providing us the pcaggs vector. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.02879/full#supplementary-material figure s1 | the schematic diagram of the bimc assay. figure s2 | cypa expression was detected by if assay in vero and hek293t cells. scale bar, 100 µm. after transfected the pcaggs-flag-cypa plasmid for 24 h in vero cells, the flag antibody was incubated overnight in 4 • c, followed by green fluorescent secondary antibody in 37 • c for 1 h, hoechst33342 stained nucleus. hek293t cells, cypa antibody incubated overnight, followed by red fluorescent secondary antibody, stained nucleus. extracellular cyclophilin-a stimulates erk1/2 phosphorylation in a cell-dependent manner but broadly 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epstein-barr virus genome in a plasmid based on a bacterial artificial chromosome epstein-barr virus microrna mir-bart5-3p inhibits p53 expression lactoferrin suppresses the epstein-barr virus-induced inflammatory response by interfering with pattern recognition of tlr2 and tlr9 inhibition of epstein-barr virus infection by lactoferrin the copy number of epstein-barr virus latent genome correlates with the oncogenicity by the activation level of lmp1 and nf-kappab an update: epstein-barr virus and immune evasion via microrna regulation cadherin 6 is activated by epstein-barr virus lmp1 to mediate emt and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma key: cord-000914-d0bk9gu5 authors: conant, katelyn l.; kaleeba, johnan a. r. title: dangerous liaisons: molecular basis for a syndemic relationship between kaposi’s sarcoma and p. falciparum malaria date: 2013-03-12 journal: front microbiol doi: 10.3389/fmicb.2013.00035 sha: doc_id: 914 cord_uid: d0bk9gu5 the most severe manifestations of malaria (caused by plasmodium falciparum) occur as a direct result of parasitemia following invasion of erythrocytes by post-liver blood-stage merozoites, and during subsequent cyto-adherence of infected erythrocytes to the vascular endothelium. however, the disproportionate epidemiologic clustering of severe malaria with aggressive forms of endemic diseases such as kaposi’s sarcoma (ks), a neoplasm that is etiologically linked to infection with ks-associated herpesvirus (kshv), underscores the significance of previously unexplored co-pathogenetic interactions that have the potential to modify the overall disease burden in co-infected individuals. based on recent studies of the mechanisms that p. falciparum and kshv have evolved to interact with their mutual human host, several new perspectives are emerging that highlight a surprising convergence of biological themes potentially underlying their associated co-morbidities. against this background, ongoing studies are rapidly constructing a fascinating new paradigm in which the major host receptors that control parasite invasion (basigin/cd147) and cyto-adherence (cd36) are, surprisingly, also important targets for exploitation by kshv. in this article, we consider the major pathobiological implications of the co-option of basigin/cd147 and cd36 signaling pathways by both p. falciparum and kshv, not only as essential host factors for parasite persistence but also as important mediators of the pro-angiogenic phenotype within the virus-infected endothelial microenvironment. consequently, the triangulation of interactions between p. falciparum, kshv, and their mutual human host articulates a syndemic relationship that points to a conceptual framework for prevalence of aggressive forms of ks in malaria-endemic areas, with implications for the possibility of dual-use therapies against these debilitating infections in resource-limited parts of the world. plasmodium falciparum (pf) malaria is one of the world's leading health challenges, and at least two million people, mainly children below the age of 5 years, die each year from clinical complications of the disease (snow et al., 2005) . the most severe manifestations of pf malaria occur as a direct result of parasitemia following invasion of erythrocytes by post-liver blood-stage merozoites, and during subsequent cyto-adherence of parasitized red blood cells (prbcs) to the vascular endothelium and other host cells and tissues. erythrocyte invasion is executed by a family of pf reticulocyte-binding-like homolog (pfrh) ligands displayed on the surface of merozoites. among at least five known members of this family, pfrh5 (rodriguez et al., 2008; lopaticki et al., 2011; tham et al., 2012) , which is indispensable for merozoite growth in cultures, is essential for invasion by all pf strains (baum et al., 2009; lopaticki et al., 2011) . the cognate erythrocyte receptor for pfrh5 was recently identified as cd147 [also known as basigin (bsg), extracellular matrix metalloproteinase inducer (emmprin), and leukocyte activation antigen, m6 (hereafter cd147); crosnier et al., 2011] . this discovery is notable for the fact that the cd147/pfrh5 pair is essential for invasion of all laboratory-adapted and field strains of pf, a crossstrain dependency that reveals opportunities for new anti-malarial therapies based on targeting this receptor/ligand interaction. following invasion, extensive replication within infected human red blood cells (rbcs) results in surface expression of the multidomain pf erythrocyte membrane protein-1 (pfemp-1) family of genes (baruch et al., 1995) , including pfemp-1 that serves as a platform for sequestration of prbc from the blood circulation by adhering to endothelial and other host cells and tissues (berendt, 1993) . the cyto-adhesive property of infected red blood cells to the microvasculature and sequestration within vital organ systems is an important survival strategy that allows the parasite to escape immune-mediated destruction (urban et al., 1999) . cyto-adherence is mediated by an orchestrated set of interactions between specific regions within the ectodomain of pfemp-1 [notable among them being the cysteine-rich interdomain region (cidr1α)], with a variety of host molecules on the surface of capillary endothelial cells. a well-characterized host receptor that mediates cyto-adherence of most pf isolates to the peripheral vasculature is human cd36 (ockenhouse et al., 1989) , although other cell adhesion molecules are also involved in execution of strain and tissue-specific cyto-adhesive events (ockenhouse et al., 1991; baruch et al., 1997 ; figure 1 ). recent studies have revealed that while erythrocyte invasion and cyto-adherence represent essential evolutionary strategies for parasite growth, survival, and persistence, they are also invariably associated with alteration of cellular physiology, which in turn may contribute directly to the defining clinical manifestations of pf infection (trossaert et al., 1991; fried and duffy, 1998) . however, less examined is the provocative hypothesis that malarial disease may not be solely attributable to complications associated with the various stages of the pf lifecycle alone; rather, the sequelae of illnesses associated with pf malaria is likely to be the collective manifestation of a multitude of complex interactions between pf, other co-pathogenic infections, and the human host. in this article, we highlight emerging evidence supporting the proposition that the signaling pathways anchored by basigin/cd147 and cd36, two of the known host receptors that control pf invasion and cyto-adherence, respectively, are also targets for functional subversion by kaposi's sarcoma (ks)-associated herpesvirus (kshv), an inherently persistent cancer-associated herpesvirus that is prevalent in malaria-endemic regions. we discuss a number of surprising nodes of pathogenetic figure 1 | basic interactions of p. falciparum with human erythrocytes during invasion (a) and with blood capillary endothelium during cyto-adherence (b). upon exit from the liver, invasion of red blood cells by the blood-stage merozoites leads to replication and subsequent surface expression of the multi-domain pf emp-1 molecule that mediates cyto-adherence via the binding activities of dbl1, cidr1, and dbl2 domains of pf emp-1 with various host adhesion receptors. shown here is a region of pf emp-1 encoded by fcr3s1.2-var1 (chen et al., 2000) . the cidr1 domain primarily binds to cd36 and to members of the immunoglobulin superfamily, including igm and cd31/pecam (platelet endothelial cell adhesion molecule), whereas the dbl2 domain binds mainly to cd31/pecam-1. interface between pf and kshv in this context, and evaluate the major implications of the apparent co-option, by both pf and kshv, of cd147 and cd36 signaling pathways as a means to promote pf persistence on one hand, and virus-induced regulation of the angiogenic phenotype, on the other. we then provide a synthesis of how the triangulation of interactions between pf, kshv, and their mutual human host represents the basis for a venerable syndemic relationship that may explain the co-incidence of aggressive forms of ks in malaria-endemic regions. the most important pathological manifestation of kshv infection is ks, a multifocal and highly angiogenic mesenchymal neoplasm characterized by profound inflammation and angioproliferative expansion of spindle cells believed to be of endothelial origin (bubman and cesarman, 2003) . ks occurs in at least four epidemiological forms, each with its own distinguishing clinical disposition determined by age, sex, geographical location, socio-economic status, previous exposure to parasitic infections, co-infection with the human immunodeficiency virus (hiv), and the extent of acquired or iatrogenic immunosuppression (bubman and cesarman, 2003; dourmishev et al., 2003; haverkos, 2008) : thus, iatrogenic ks is mostly associated with organ (especially renal) transplantation and is mostly seen as localized skin lesions among people from areas where kshv is endemic. epidemic hiv/aids-associated ks is more commonly seen among hivinfected individuals, while classical ks (cks) manifests among older men of mediterranean origin as red to purple skin plaques or nodules primarily on the lower extremities. endemic ks (eks), which is strikingly similar to cks in its clinical disposition, is highly prevalent in east and central africa, where it affects children and young adults as a cutaneous disease invading soft tissue and bone, or as a fulminant lymphadenopathy that can rapidly disseminate to visceral organs (hengge et al., 2002a,b) . eks is currently the most common cancer in adult east and central african men and follows only cervical and breast cancer in adult women (bassett et al., 1995; wabinga et al., 2000; casper, 2006 ). an important distinction is that whereas iatrogenic and aids-related ks are invariably associated with an immunosuppressed state, cks and eks are generally not (kestens et al., 1985) , implying that the development and/or propagation of the latter two types of ks (i.e., cks and eks) may be controlled by unique, geographically restricted cofactors unrelated to hiv or drug-induced immune suppression (pyakurel et al., 2007) . a number of non-competing hypotheses have been proposed to explain the contribution of socio-economic, behavioral, and environmental co-factors to the histogenesis of eks and cks, the two types of the lesion not strictly dependent on hiv or iatrogenic immunosuppression. for example, clinical studies have revealed that eks displays a notable predilection for the feet and legs of rural peasants and cultivators living in highland areas, thus inspiring the volcanic soil hypothesis first proposed by ziegler (1993) and subsequently supported by additional epidemiologic studies (montesu et al., 1995; montella et al., 1996 montella et al., , 1997 montella et al., , 2000 goedert et al., 2002) . this hypothesis proposes that walking barefoot allows soil-borne aluminosilicates, iron oxides, and other clay minerals to be taken up through sweat glands and abrasions by resident macrophages, dermal microvascular endothelial cells, and by the lymphatic system. the resulting chronic lymphatic irritation, inflammation, and immune suppression could in turn support primary infection through these portals, and/or reactivation of latent kshv within the epidermal aspects of skin. lin and colleagues also recently suggested that exposure to soil and water constituted an additional risk factor for ks development by promoting parasitic infections that could either reduce local immune reactivity or induce a condition of inflammation necessary for ks development as well (lin et al., 2008) . alternatively, the finding that some natural products of plants indigenous to the areas with eks could reactivate kshv (whitby et al., 2007) inspired the "oncoweed" hypothesis of ks development. subsequently, ruocco et al. (2011) noted that quinine, an anti-malarial drug used extensively as one of the mainstays for treatment of malaria in immunocompetent children, may trigger ks development via its immunosuppressive effects that could support virus reactivation. it is therefore noteworthy that although available evidence is not sufficient to explain the distinctive geographical distribution of ks, the four known types of the lesion have a multi-factorial etiology, with the unifying theme being that the potential co-factors in each case operate at the level of the molecular mechanisms that control kshv infection, dissemination, and the balance between virus replication and establishment of latency. this is a relevant link given the fact that a sustained state of low level lytic replication may be important for histogenesis and probably propagation of the ks lesion (grundhoff and ganem, 2004) . like burkitt's lymphoma, ks is one of the most prevalent childhood cancers in malaria-endemic areas. the incidence of ks displays a considerable degree of geographic variation that mirrors the prevalence of its causative agent, and may depend on etiologic mechanisms that are controlled by geographically restricted cofactors, including malaria endemicity (ziegler et al., 1984 (ziegler et al., , 2003 ascoli et al., 2001 ascoli et al., , 2006a coluzzi et al., 2003 coluzzi et al., , 2004b wakeham et al., 2011) . for example, ks incidence is particularly high in sub-saharan africa, a region with one of the highest rates of malaria deaths (snow et al., 2005) . epidemiological studies have also shown a disproportionately high incidence of ks among elderly men in greece, turkey, israel, and in italy where the greatest number of recorded cases are in the formerly malaria-endemic provinces of sardinia, sicily (vitale et al., 2001) , and in the po valley (ascoli et al., 2001) . such overlapping geographic clustering of pf malaria with eks or cks has inspired the co-pathogenesis hypothesis, which proposes that a previous or ongoing exposure to kshv in a setting of underlying parasite persistence (or vice versa) may result in molecular interactions between pf and kshv that could modify the overall disease burden exerted by both pathogens at a micro level. based on this attribution, we refer to the putative co-morbidity of ks and pf malaria (in settings for which the evidence for this linkage is strong), as representing an example of a classic syndemic relationship articulated by the display of co-incident clustering within defined geographic regions. in spite of evidence from case-control studies that show a disproportionately high ks prevalence in areas of currently or previously high malaria endemicity, a molecular link between ks pathogenesis and pf malaria has not been rigorously examined at a micro level, and the correlation is even more difficult to establish at a population level because of the indeterminate nature of the mechanisms by which malaria might influence ks pathogenesis outside the known clusters of endemic disease co-incidence. on one hand, the role of malaria as a co-factor for eks has been hypothesized based on the potential of the anopheles mosquito vector to contribute to person-to-person spread of kshv, and to establish an immunosuppressed state at the site of the mosquito bite, which would then create a local permissive environment for kshv infection (coluzzi et al., 2003; ascoli et al., 2006a,b,c) . however, we propose an alternative model based on the provocative hypothesis that both the malaria parasite and kshv exert bidirectional influences upon each other that operate at a much more complex level beyond the permissive benefits of immune suppression or the modifying effects of occupational, socio-economic, or environmental co-factors. as a conceptual basis for our hypothesis, consideration of some of the known molecular controls that regulate the kshv life cycle reveals a number of insights into the potential pathobiological link between ks and malaria. like epstein-barr virus (ebv) and related herpesviruses, kshv establishes both lytic and long-term latent infections, the balance between which determines the timing and intensity of pathologic outcomes in specific organ systems. interestingly, both kshv and ebv are highly prevalent in malariaendemic areas and also share many features in their life cycles, including the manner in which they regulate the molecular switch between latency and lytic replication. for example, expression of the major kshv replication and transcription activator (rta) is essential for virus replication and dissemination, and is dependent on activation of p38 and extracellular signal-related kinase 1/2 (erk1/2) mitogen-activated protein kinase (mapk; xie et al., 2008) . kshv rta is the genetic and functional homolog of ebv rta/brlf1 (bam hi fragment z rightward open-reading frame 1) which, in concert with zebra transcriptional activator/bamhi fragment z rightward open-reading frame 1 (zta/bzlf1), can initiate ebv lytic cycle (staudt and dittmer, 2007) . kshv rta positively regulates immediate and delayed early promoters as well as its own promoter by interacting with cellular transcription factors such as activator protein-1 (ap-1), octamer binding transcription factor 1 (oct-1), recombination signal-binding protein jkappa (rbp-jκ), and ccaat/enhancer binding protein alpha (c/ebpα; staudt and dittmer, 2007) ; in this way, rta is susceptible to a variety of signal transduction pathways that are likely to activate its promoter. in fact, kshv rta expression (and therefore lytic replication) can be induced in vitro using a variety of chemical compounds such as ionomycin (a calcium ionophore), sodium nbutyrate (nab, a histone deacetylase inhibitor) and phorbol esters such as 12-o-tetradecanoylphorbol-13-acetate (tpa), all of which can also be used to induce ebv lytic cycle (luka et al., 1979; renne et al., 1996; zhu et al., 1999; gao et al., 2001) . given the shared genetic and biological properties of ebv and kshv, it is not surprising that the two viruses can co-exist as latent episomes in certain peripheral effusion lymphoma-derived www.frontiersin.org cell lines (horenstein et al., 1997) . they also display similarities not only in the mechanism of induction of the lytic cycle but also in the distribution of endemic cancers associated with them in regions of high malaria endemicity, which perhaps reflects the contribution of malaria as a common co-factor in the pathogenesis of cancers associated with these viruses. for ebv, the epidemiological association between malaria and african endemic burkitt's lymphoma (ebl) is well established (moormann et al., 2011) , although the molecular mechanisms by which malaria modifies ebl and other pathobiological outcomes of ebv infection remain a matter of intense investigation (rochford et al., 2005) . interestingly, ebl is a b cell lymphoma propagated by a deregulation in the c-myc oncogene as a result of chromosomal translocation, and although ebv is a necessary etiological agent for ebl, it is clearly not sufficient in absence of other essential co-factors. it is also worth noting that whereas nearly all african children in endemic areas suffer from several malaria episodes as a result of chronic exposure to pf, only a fraction of them show signs of severe, life-threatening forms of the disease, implying that the biological processes underlying the progression of infection to disease are much more complex. interestingly, ebv is ubiquitous in the general human population, and in children living in malaria endemic areas, primary infection can occur within a few months of birth (piriou et al., 2012) , and may seroconvert within 3 years after primary infection, followed by a tightly orchestrated viral latency state in memory b cells that reflects the balance between viral replication and host immune control. emerging new evidence now suggests that the exit from latency to the viraemic state that supports ebl development may be impacted at a molecular level by the replicative blood-stage form of the parasite life cycle. thus, chene et al. (2007) recently demonstrated that interactions between the cidr1α domain of pfemp-1 on the surface of prbcs, with a cognate surface receptor(s) expressed on the surface of ebv-infected human memory b cells, stimulates b cell proliferation (as previously shown; donati et al., 2004 ) and reactivates ebv not only from the chronically infected akata cell line but also from latently infected b cells isolated from the peripheral blood and tonsils of healthy ebv carriers (chene et al., 2007) . the mechanism by which conjugation of pfemp-1-expressing prbc with ebv-infected b cells triggers virus replication in this model remains to be elucidated, but the consequences of this interaction can be readily appreciated as providing a possible explanation for the increased ebv viral load that constitutes risk for ebl among children living in areas of high malaria endemicity. remarkably, we have also discovered that cross-linking of cd36 on the surface of kshv-infected cells with mc179, a recombinant peptide derived from the cidr1α domain of pfemp-1 that normally interacts with cd36 to mediate cyto-adherence (ockenhouse et al., 1989 (ockenhouse et al., , 1991 baruch et al., 1997) , not only upregulated cd36 expression (figure 2a ) but also reactivated the virus from latency through transcriptional activation of kshv rta (figure 2b) , and that the molecular mechanisms that control this process overlap with those that putatively regulate pfemp-1dependent ebv reactivation from latently infected cells (chene et al., 2007) . remarkably, structural mimics of mc179, including the two helical heptad repeats (hr1 and hr2) derived from kshv entry glycoprotein b (gb) also upregulated cd36 figure 2 | (a) upregulation of cd36 by both the pf emp-1-cidr1α-derived mc179 peptide (lanes 5 and 6) and by kshv gb-derived heptad peptide ligands hr2 (lanes 3 and 4), and the less potent hr1 (lanes 1 and 2) are both blocked by a monoclonal anti-cd36 antibody. note that anti-cd36 did not block control upregulation of cd36 in response to treatment with sodium butyrate (nab; lanes 7 and 8). (b) activation of kshv rta by mc179 occurs via a cd36-dependent mechanism that can be blocked by anti-cd36 antibody fa6-152. methodology : briefly, melanoma-derived mel1700 cells were seeded in a six-well plate and either left untreated or pre-incubated with 10 μg/ml of anti-cd36 monoclonal antibody clone fa6-152 for 25 min. at 25 • c. after washing to remove excess antibody, cells were incubated with either 100 μg/ml of kshv gb-derived hr1, 200 μg/ml hr2 peptide, 100 μg/ml recombinant mc179, or 2 mm sodium butyrate (nab). forty-eight hours after treatment, total rna was isolated and used as template in semi-quantitative rt-pcr with primers to an internal fragment of human cd36, viral rta, or human glyceraldehyde 3-phosphate dehydrogenase (gapdh) loading control. (c) specific activation of the kshv lytic switch protein, rta, by the pf emp-1-cidr1αderived peptide mc179 from pf malayan camp strain, but not by peptides derived from cidr1α domains of a4tres (which binds icam-1) or the vietnam oak knoll strain (fvo). expression and further induced virus reactivation in a cd36dependent manner. indeed, like mc179, the effect of these helical peptides could be blocked by a monoclonal antibody to cd36 (figures 2a,b) , suggesting that they bind a region on the exoplasmic face of cd36 that overlaps with mc179. it is also insightful that the kshv lytic cycle was activated only by the pfemp-1-cidr1α-derived peptide from pf malayan camp strain, but not by peptides derived from the vietnam oak knoll (fvo) or a4tres strain [that preferentially binds intercellular adhesion molecule 1 (icam-1) and not cd36] (figure 2c) , demonstrating that reactivation of kshv by the parasite ligand displays some degree of strain specificity for pf that is associated with severe malaria in africa. since cd36 mediates parasite persistence by mediating cytoadherence and sequestration of parasitized erythrocytes away from immune surveillance, the ability of mc179 to stimulate kshv reactivation is significant, as it supports a model whereby in vivo cross-linking of cd36 on the surface of kshv-infected cells, either by its natural ligand(s) or upon conjugation with the cidr1α domain of pfemp-1 expressed on the surface of parasitized erythrocytes (as illustrated in figure 3) , represents a previously unrecognized mechanism by which kshv lytic replication could be induced in the context of a pf malaria co-infection. this model provides many new opportunities for experimental examination of the ability of parasite-derived antigens to reactivate kshv during cyto-adherence on the surface of infected blood endothelial cells and within tissues and organs: (i) biochemical analysis of structure/function relationships that control interactions of cidr1α (and its structural analogs) with cd36, should reveal potential targets for small-molecule inhibition of parasiteinduced sequestration or reactivation of kshv in co-infected individuals. (ii) elucidation of the signaling mechanisms that regulate cd36-dependent rta activation will require multi-pronged approaches that employ dominant negative versions of the srclike kinases such as yes, fyn, and lyn that control down-stream signaling events initiated by cd36 ligation (see figure 3 ). (iii) deletional mutagenesis, domain-swapping, functional complementation, and loss-of-function analysis of genetic mimics of the recently identified cd36 polymorphisms that lack the signaling motif (aitman et al., 2000; gelhaus et al., 2001; chilongola et al., 2009; fry et al., 2009) should elucidate the correlates of upstream signaling networks required for cd36-dependent activation of the kshv lytic cycle. cd36 is a class ii glycoprotein involved in multiple physiological functions including cell adhesion, fatty acid uptake, non-opsonic phagocytosis, and angiogenesis (mcgilvray et al., 2000; podrez et al., 2007; febbraio, 2007, 2009 ). the cd36 structure consists of a large extracellular loop and two short cytoplasmic tails at the n-and c-termini (silverstein and febbraio, 2009 ). the c-terminal tail is involved in signal transduction via association with src-like kinases, whereas the extracellular domain contains binding sites for thrombospondin-1 (tsp-1), a potent natural inhibitor of angiogenesis (bagavandoss and wilks, 1990; good et al., 1990; tolsma et al., 1993) , and a variety of other ligands including the cidr1α domain of pfemp-1 (ockenhouse et al., 1989) . although cd36 has never been directly associated with kshv pathogenesis, some of its pleiotropic functions are consistent with its potential contribution to the basic pathobiology of kshv. for example, cd36 is often expressed in association with signaling structures such as lipid rafts that also contain host receptors for kshv entry, including integrins and the kshv fusion receptor complex xct/cd98 (akula et al., 2002; kaleeba and berger, 2006; veettil et al., 2006) . since cd36-mediated signaling may culminate in activation of p38 and erk mapk pathways that overlap with one or more pathways necessary for kshv rta-dependent transcription of viral gene expression (yipp et al., 2003; cohen et al., 2006; xie et al., 2008) , our data suggests that figure 3 | hypothetical model of cd36-dependent rta activation. a motif displayed by pf emp-1 on the surface of parasitized erythrocytes interacts with its cognate epitope within the ectodomain of cd36 on the surface of microvascular endothelial cells. this interaction activates one or more of the src-like kinases, which in turn initiate a phosphorylation cascade that results in p38 and erk/mapk activation. this process culminates in activation of a cellular transcription factor, likely ap-1 (dimer of c-jun and c-fos), which translocates into the nucleus and stimulates kshv rta-dependent transcription of viral lytic cycle genes, starting with immediate early (ie), which then activate delayed early (de), followed by late structural genes involved in assembly of an infectious virus particle. cidr1α-dependent rta activation may occur via by signals transduced through cd36 (figure 3) . however, our model also raises many significant questions: (a) does cidr1α-induced kshv replication involve mechanisms that overlap with, or distinct from those that support cidr1α-induced ebv replication (chene et al., 2007) ? (b) does it require the traditional cd36-regulated recruitment of src-like kinases and subsequent phosphorylation of p38 and erk mapk? (c) does it directly result in downstream activation of a specific cellular transcription factor such as ap-1 that is known to activate kshv rta (or ebv zta) promoter(s) (e.g., see figure 3 )? (d) is it strictly dependent on interactions of cidr1α with a distinct epitope on cd36, or does it overlap with, and is therefore also inducible by, other cd36 ligands such as tsp-1? investigation of the role of tsp-1 in this context is relevant, given that tsp-1 is present in high concentrations in human saliva (crombie et al., 1998 (crombie et al., , 2001 shugars, 1999 ) that generally contains significant levels of kshv virions shed from the underlying oral zones of carriers (pauk et al., 2000; coluzzi et al., 2004a; chene et al., 2007; hadinoto et al., 2009) . critical new experiments that address these derivative questions represent an exciting area of research into the molecular basis for the emerging new paradigm of co-pathogenesis. it is also anticipated that isolation of polymorphisms in cd36 and other human genes that control host interactions with pf, ebv and kshv may open up additional opportunities for population-level studies aimed at explaining the www.frontiersin.org overlapping distribution of ks, burkitt's lymphoma, and malaria in areas where these diseases display coincident endemicity. angiogenesis, defined as the development of new blood vessels, is necessary for growth and proliferation of vascular tumors like ks, the extent of which is controlled by the balance between pro-angiogenic and angiostatic elements of the human hemostatic system. one of the regulatory components of this system is tsp-1, the angiostatic cd36 ligand known to inhibit endothelial cell proliferation, migration, and tube formation (iruela-arispe et al., 1991) . remarkably, kshv upregulates cd36 in melanomaderived cell lines but downregulates the protein in endothelial cells both at mrna and protein levels (figure 4) . the mechanism(s) by which kshv accomplishes these dichotomous, cell type-specific effects are not fully understood, but one study recently demonstrated that kshv-encoded micrornas can directly target tsp-1 mrna for degradation (samols et al., 2007) , ostensibly to promote an angiogenic growth state of infected cells via attenuation of the angiostatic effects resulting from interactions between tsp-1 and cd36. since the angiostatic signal is associated with viral lytic replication while the angiogenic phenotype is linked to the latency phase, virus regulation of cd36 expression and signaling in infected cells implies that the correlates of kshv latency are contextually linked to the angiogenic phenotype in diseaserelevant cells. they also provide a potential explanation for our recent findings that the kshv latency program is inefficient in melanoma cells from which the virus undergoes robust spontaneous replication, as opposed to endothelial cells in which the virus establishes a much tighter state of latency (fontana et al., unpublished findings) . in addition to direct targeting of tsp-1, kshv blunts cd36 signaling in endothelial cells by upregulating the endothelin (et-1) system. et-1 is a pro-angiogenic peptide secreted by the vascular endothelium and its deregulation is implicated in the pathogenesis of many malignancies (nelson et al., 2003) . et-1 polypeptides and their cognate receptors are expressed in ks lesions (nelson et al., 2003; basilico et al., 2004) , and et-1 receptor blockade limited ks cell invasion in an in vivo tumor growth model (rosano et al., 2003) . the interplay between the pro-angiogenic effects of et-1 and the pathophysiology of ks is also encountered in patients with complicated pf malaria in which plasma concentrations of big et-1, the precursor for bioactive et-1, are elevated (wenisch et al., 1996) as a direct result of cyto-adherence of prbcs to human endothelial cells, independent of the parasite strain and regardless of the origin of endothelial cells (basilico et al., 2004) . there is, therefore, a significant degree of molecular crosstalk between pf and kshv at the level of et-1 biology. considered in a broader context, it is conceivable that by suppressing the angiostatic effects of cd36 signaling either by reducing cd36 expression, downregulation of tsp-1, or via upregulation of et-1, kshv could establish long-term persistence by establishing a state of limited cd36-dependent viral reactivation, or reduce cd36-dependent cyto-adherence and consequently limit the frequency of illnesses associated with this aspect of the parasite life cycle. unfortunately, the latter outcome might be associated with an increase in the likelihood for parasite access to the extra-peripheral organs such as the brain, which may elevate the probability of cerebral malaria. although measurement of these parameters in vivo is not trivial, a number of guiding principles can emerge from in vitro scrutiny of these molecular interactions based on experimental approaches that might predict their occurrence in vivo. it was recently discovered that many people of african origin harbor a high frequency of mutations and single-nucleotide polymorphisms (snps) that cause a deficiency in the cd36 gene, yet semi-quantitative rt-pcr (a) and western blot (b) analysis of cd147 and cd36 expression in uninfected (−) versus infected (+) endothelial (lymphatic) or melanoma-derived mel1700 cells. kshv upregulates both cd36 and cd147 in melanoma-derived cells, whereas in lymphatic and telomerase-immortalized mixed dermal microvascular and brain endothelial cells, kshv upregulates cd147 (confirming a recent study; qin et al., 2010) but downregulates expression of cd36 (and its angiostatic ligand, tsp-1; data not shown); in vivo, these dichotomous effects are consistent with promotion of angiogenesis, invasion, and tumor metastasis in disease-relevant cell types. they still suffer from severe (particularly cerebral) malaria (aitman et al., 2000; gelhaus et al., 2001; chilongola et al., 2009; fry et al., 2009 ). the snps found in kenya and gambia introduce a premature stop codon that results in a truncated cd36 protein lacking the c-terminus and is therefore incompetent for signal transduction but can still bind its ligand(s), leading to the conclusion that mutations that cause cd36 deficiency may reduce cd36-mediated parasite sequestration in peripheral organs but they may not protect from severe cerebral malaria (aitman et al., 2000) that is associated with cyto-adherence to brain endothelium via interactions of pfemp-1 with icam-1 but not cd36 (ockenhouse et al., 1991) . this level of linkage in which mutations in cd36 -a molecule that is important for parasite persistencecause a deficiency that does not alter malaria pathogenesis, implies existence of selection pressures that may be induced or maintained in the population by an endemic infection (other than malaria) whose persistence is linked to this genetic output. the fact that the mutations and their phenotypes occur at high frequency in malaria-endemic areas with a high prevalence of viruses associated with endemic cancers underscores a pathobiological paradigm whereby an inherently persistent tumor virus such as kshv (or ebv) could provide the selective pressure for introduction or maintenance of such a mutation into the genetic registry of populations living in regions of high malaria endemicity. given that pfemp-1 interactions with cd36 result in induction of the viral lytic cycle, such a virus-induced genetic output would conceivably be designed to promote virus escape from immune surveillance by limiting virus reactivation that might result from interactions between pfemp-1 and cd36 on latently infected cells. the impact of such a genetic influence, can only be measured against the host's ability to restrict virus replication and dissemination, and it could be achieved by interrogating viral genomic variability or stability in a given population against a profile of polymorphisms within the genetic registries at the cd36 locus on a population basis. availability of patient samples with known patho-status and disease severity from regions in which the distribution of malaria overlaps with the incidence of virus-associated endemic cancers would facilitate such a retrospective analysis. cd147 is a widely expressed, type i integral membrane receptor that belongs to the immunoglobulin (ig) superfamily (biswas et al., 1995) . it is over-expressed in a variety of disseminated human solid cancers and is a major contributor to the malignant phenotype in a variety of human cancers (riethdorf et al., 2006) . signaling events transduced through cd147 are associated with survival, metastasis, and invasion of a variety of cancer cells, mainly because it stimulates enhanced stromal release of multiple matrix metalloproteinases (mmps) and vascular endothelial growth factor (vegf), which are among the key mediators of angiogenesis and metastatic transition (marieb et al., 2004; tang et al., 2005; bougatef et al., 2009; kanekura and chen, 2010) . cd147 promotes hyaluronan synthesis (marieb et al., 2004; slomiany et al., 2009a) , upregulates the wnt/β-catenin signaling pathway (sidhu et al., 2010) , and is also involved in epithelial-to-mesenchymal transition (emt; wu et al., 2011) . given the pleiotropic function of cd147, the significance of cd147/pfrh5 interactions in parasite invasion unlocks new avenues for investigating the nodes of pathogenetic intersection between pf malaria and other coinfecting agents such as kshv that have evolved mechanisms to subvert the cd147 signaling pathway to promote their existential success. several layers of this pathogenetic intersection are revealed by new data on the manner in which both pf and kshv have evolved to exploit cd147 and endothelial cell biology: a. whereas endothelial cells play a prominent role as a platform for cyto-adherence of prbcs, kshv displays profound tropism for this cell lineage that represents the basis for origination of the hyper-proliferating spindle cells characteristically found in ks lesions (bubman and cesarman, 2003; ganem, 2006) . b. recent studies demonstrated compelling evidence that de novo kshv infection of human endothelial cells as well as oral and fore-skin-derived fibroblasts results in upregulated expression of cd147 and that this effect is directly associated with various virological outcomes consistent with a pro-invasive, migratory, and pro-angiogenic phenotypes (qin et al., 2010; dai et al., 2012a,b) . we have also found that kshv upregulates cd147 not only in melanoma-derived cells but also in chronically infected lymphatic, microvascular, and brain endothelial cells (e.g., see figure 4 ). c. two recent reports showed that kshv promotes endothelialto-mesenchymal transition (endo-mt) through activation of notch-dependent signaling events that culminate in stimulation of an invasive phenotype analogous to that orchestrated by cd147 (cheng et al., 2011; gasperini et al., 2012) . kshv-induced endo-mt was dependent on the activity of membrane-type-1 mmp (mt1-mmp; cheng et al., 2011) , which is consistent with a role for cd147 in endo-mt since mt1-mmp is a cd147-stimulated endopeptidase involved in extracellular matrix remodeling. it is therefore conceivable that one of the mechanisms underlying kshv-induced endo-mt may involve viral induction of mmp activity , likely through upregulation of cd147. d. cd147 has been implicated in the entry processes of a number enveloped viruses including hiv-1 (pushkarsky et al., 2001) , measles virus (watanabe et al., 2010) , and severe acute respiratory syndrome coronavirus (sars-cov; chen et al., 2005) . based on our recent studies, we also have reason to believe that cd147 may regulate kshv entry as well, as an antihuman cd147 (neurothelin) antibody can potently block kshv glycoprotein-mediated fusion, consistent with existence of cd147 in the membrane of kshv-permissive cells as part of a molecular supercomplex (guo et al., 2000; yang et al., 2007; gallagher et al., 2009 ) that includes host molecules implicated in virus entry, such as integrins (akula et al., 2002) and the cystine transporter complex xct/cd98hc (xu and hemler, 2005; kaleeba and berger, 2006) . it is also noteworthy that, like cd36, cd147 also associates with the xct/cd98hc complex and confers resistance to some chemotherapeutic drugs (okuno et al., 2003; yang et al., 2007; zou et al., 2007) . interestingly, another independent study also demonstrated that the transport activity of xct/cd98hc, perhaps in association with cd147, is a critical correlate of resistance to cisplatin (huang et al., 2005; www.frontiersin.org singh et al., 2010; chen et al., 2011; riglar et al., 2011) . for kshv-infected individuals, one implication of these molecular associations could be that virus-induced upregulation of cd147 (as we and others have shown; qin et al., 2010; dai et al., 2012a,b) , could stabilize cd147-containing multi-partite complexes and in turn potentiate their drug efflux functions, which could blunt the efficacy of chemotherapeutic strategies that might be used in the treatment of ks and other virusassociated malignancies. in support of this view, qin et al. (2011) recently showed that the intrinsic resistance of kshvpositive peripheral effusion lymphomas to the cytotoxic effects of paclitaxel and doxorubicin depends on orchestrated interactions of cd147 with lymphatic vessel endothelial receptor 1 (lyve-1) and the homodimeric atp-binding cassette (abc)-g2/bcrp (breast cancer resistance protein) drug transporter that is highly expressed on the surface of primary effusion lymphoma (pel)-derived cell lines. e. available evidence suggests that "outside-in" signaling may be required for the malaria parasite invasion (singh et al., 2010; chen et al., 2011; riglar et al., 2011) , but it remains to be determined if the structural framework that supports erythrocyte invasion through cd147 is distinct from, or overlaps with, the epitope on the cd147 ectodomain that senses signals transduced to endothelial and other cells that express this molecule. if they are the same, pfrh5-mediated conjugation of merozoites with cd147 on normal or infected endothelial cell surfaces should result in so-called parasite-to-host "trans-signaling" events that may lead to untoward pathologic outcomes unrelated to parasite invasion itself. given that pfrh5 has neither a transmembrane domain nor a cytoplasmic tail, the probability of trans-signaling is likely to be high since a soluble form of pfrh5 [existing either as a monomer, as part of a bioavailable complex with another pathogen molecule such as pfripr (chen et al., 2011) , or as bound to a host "handler"] could initiate cd147-dependent signaling outcomes that are likely to modify pf malaria or kshv infection (marieb et al., 2004; xu et al., 2007; ruiz et al., 2008; slomiany et al., 2009b; sidhu et al., 2010; wu et al., 2011) . a first-line experimental testing of such a concept should seek to determine whether blood-borne merozoites or soluble forms of pfrh5 can indeed bind cd147 on kshv-infected cells and whether those interactions are directly associated with a cd147-dependent alteration in cellular behavior, including extracellular remodeling and induction of a pro-angiogenic phenotype that is one of the defining features of the infectious process of kshv (qin et al., 2010; cheng et al., 2011; dai et al., 2012a,b; gasperini et al., 2012) . in figure 5 , we highlight some of the important nodes of intersection between kshv and the malaria parasite in endothelial cells and skin-derived melanoma cells, along with their potential impact on the virus life cycle (i.e., reactivation), ks tumorigenesis, and malaria disease outcomes. in melanoma-derived cells (mel1700), kshv upregulates both cd36 (and its ligand, tsp-1) and cd147 (as shown in figure 4) . binding of the pf emp-1 cidr1α-derived peptide, mc179, to virus-upregulated cd36 prevents akt phosphorylation while inducing rta-dependent kshv reactivation via the mapk/p38 pathway. remarkably, structural mimics of mc179, such as the helical heptad repeat (hr) regions derived from kshv glycoprotein b (gb), can, like mc179, also induce virus reactivation in a cd36-dependent manner (as shown in figure 3 ). (b) in lymphatic (lec) and other endothelial cells such as mixed tolemeraseimmortalized dermal microvascular and brain endothelial cells (tdmb), kshv upregulates cd147 but unlike in melanoma cells, the virus downregulates both cd36 and its angiostatic ligand, tsp-1; in vivo, these effects are likely to promote angiogenesis, invasion, and tumor metastasis. (c) as a key receptor for the merozoite invasion antigen, pf rh5, kshv-induced upregulation of cd147 on the surface of infected endothelial cells increases the frequency of contacts between merozoite-bound or soluble pf rh5 and blood or dermal microvascular endothelial surfaces, resulting in induction of cd147-mediated signals that could alter the microenvironment and cause pathologic outcomes in a variety of physiological sites in the co-infected host. kaposi's sarcoma is one of the most frequent neoplasia diagnosed in malaria-endemic regions of africa, yet despite recent progress (casper, 2006; casper and wald, 2007) , the landscape of effective therapeutic strategies for ks remains limited. it is clear that ks presents in different epidemiologic and clinical forms dictated by a variety of modifying risk factors, but the lack of data on the relative contributions of these co-factors in any given epidemiological setting frustrates efforts aimed at developing new approaches for clinical management of the disease. surgical removal of isolated nodular ks does not eliminate latent kshv at secondary sites, while traditional chemotherapy is generally toxic and may have a high failure rate in hiv-infected patients in whom durable post-therapy immune reconstitution is improbable. for epidemic (hiv/aids-associated) ks, intervention with the highly active anti-retroviral therapy (haart) in hiv/hhv8 (human herpes virus 8) co-infected patients may have contributed to regression of ks lesions, but the initial success of haart-based therapy for ks has been eroded by several concerns. first, hiv is not necessary for endemic african or classical mediterranean ks. second, haart only limits the potentiating immunosuppressive effects of hiv but it does not remove the underlying etiology of ks. third, haart may reduce hiv viral load but it does not restore the entire t cell repertoire necessary for immunity against kshv. fourth, lack of access to haart, non-compliance with the treatment, failure to respond to treatment, and the development of drug-resistant strains of hiv confound the overall benefit of a strictly haartbased approach to ks management. fifth, the benefits of haart are not long-lasting and end up being more detrimental to many patients with advanced ks who may show no improvement while remaining in danger of developing post-therapy immune crisis (krown et al., 2004) . sixth, the probability of recrudescence of ks in patients treated with haart later in life is unpredictable. there is, therefore, an urgent need for a multi-pronged therapeutic approach aimed at developing strategies that are appropriate to the prevailing epidemiologic state of the disease, with the overall goal being improvement of the treatment outcome for ks patients in sub-saharan africa and other resource-limited parts of the world (mcallister et al., 2005) . the concept of "angio-therapy" designed to inhibit growth of angio-proliferative cancers like ks (tosetti et al., 2002a,b; ferrari et al., 2003; pfeffer et al., 2003) led to the surprising observation that the anti-malarial peptide artesunate has anti-angiogenic effects on ks-derived endothelial cell lines (dell'eva et al., 2004) . artesunate is already well tolerated as an anti-malarial drug, and because it has direct effects against transformed cells, the promise of its dual use for ks is attractive for co-infected individuals. if adopted as such, the clinical benefits of artesunate for treatment of ks would represent a classic illustration of one of the defining attributes of a syndemic relationship in which the overall clinical impact of two linked infections (i.e., pf and kshv in this case) can be blunted by targeting the molecular underpinnings that link the parasite with the disease-modifying influence of kshv. in addition, it has been proposed that quinine and its chloroquine and hydroxychloroquine derivatives -drugs that have been used widely to treat malaria -are immunosuppressive and may, as such, serve as co-factors for ks by stimulating kshv reactivation, which would not only promote virus dissemination but could also support ks histogenesis (ruocco et al., 2011) . however, the "oncodrug" hypothesis for ks is likely to be more relevant for individuals with severely altered immunity since, in immunocompetent hosts, the viremic state induced by quinine and its derivative drugs would concomitantly expose hematogenously disseminating virions to immune surveillance which could in turn limit virus spread. with respect to malaria control, approaches that interrupt the parasite life cycle are ideal, yet in spite of many multi-national efforts in this regard, successful elimination of the disease remains a major challenge, as more than half of the world's population still lives in areas where there is a risk of contracting the disease. there are many reasons for this sobering report card, chief among them being persistent endemicity as a result of drug and insecticide resistance, inadequate support for malaria control programs, poor environmental management, the complex biology of the disease, as well as the regional variability not only in the parasite but also in the nature of its impact on specific populations and age groups. another major challenge remains the lack of practical and affordable animal models that can faithfully reflect mechanisms of malaria pathogenesis and immunity in humans. such platforms would be valuable for evaluating the efficacy of drug and vaccine candidates, and for predicting the benefits of drug combinations that can maximize safety and efficacy while minimizing the development of drug resistance. given that the invasive asexual blood stage of the pf lifecycle is the form associated with symptomatic malaria, recent efforts toward a malaria vaccine have primarily focused on targeting this stage. in this respect, the discovery of the essential merozoite invasion receptor increases the number of potential targets for a second-generation malaria-specific vaccine based on cd147/pfrh5 interactions. however, the fact that deletion of other pfrh proteins also impairs invasion, albeit in a strainspecific manner, complicates vaccine design efforts, as it suggests that the pfrh5/cd147 interaction may be only one of many ligand-receptor recognition events that must occur during execution of the invasion process (cowman and crabb, 2006; tanne, 2011; tham et al., 2012 ). an approach that targets cd147 may not be feasible, as it could impact many important physiological processes controlled by this molecule. on the other hand, innovations oriented toward development of vaccine and therapeutic strategies based on the invariant aspects of pfrh5 and other "accessible" pf antigens may result in a more meaningful outcome associated with minimal impact on the human host. such strategies may include the combined use of nanovehicledeliverable peptide mimetics and single-chain antibodies that can be administered before or during active parasitemia, or therapeutic lentiviral vectors carrying immunogenic epitopes that can harness the host's immune capacity. these pathogen-centered approaches could be used alone or in conjunction with the rts,s vaccine that is based on the most prominent surface antigen of the pre-liver sporozoite stage and which has already shown some promise in phase iii trials (tanne, 2011) . in using pfrh5 as the target, however, the primary goal of any given approach [antibody-based (douglas et al., 2011) , or otherwise] would be to elicit the safest, most long-lasting and most efficacious outcome that limits the availability of pfrh5 to mediate invasion, but it must also be guided by the recognition that this antigen is predominantly located within the rhoptries and it is liberated and or revealed to the immune system only for a short period of time when the merozoite contacts the erythrocyte prior to invasion. infectious agents have long been implicated in the etiology of a variety of illnesses, and although recent studies have examined the role of microbial co-infections in many disease settings, it is not known whether "cooperative pathogenesis" is sufficient to provide the driving force behind strategies in which co-pathogenic agents co-evolve and forge a state of forbearance with each other and with their shared host to advance mutually exclusive existential goals. clearly, pf malaria has been linked to a variety of other infections that display co-pathogenic relationships with the parasite, notable among them being ebl (thorley-lawson and allday, 2008), but recent advances have revealed that many other important examples of these relationships do exist, and in most cases they provide a surprisingly informative conceptual window into how co-infections can modify each other's disease course. for instance, the recent elucidation of the molecular mechanisms of erythrocyte invasion and cyto-adherence has: (a) illustrated the extent to which pf can exert its impact on human physiology; (b) exposed how alterations in the expression and function of the host receptors that support these processes could not only dramatically change the dynamics of malaria but may also influence the pathogenesis of other coinfections such as kshv that exploit these pathways for existential benefit; and (c) generated new interest in the structural dispositions of these receptor/ligand pairs as potential multi-domain vaccine targets against specific stages of the pf life cycle. although malaria is not considered a typical opportunistic infection in the same way that kshv is, recent advances have revealed a surprising node of intersection between kshv and malaria pathogenesis at the level of the molecular controls that regulate the persistence of these two highly successful infectious agents. to the extent that such interactions can be measured at a micro level, the concept of co-pathogenesis establishes grounds for the expectation that kshv and pf can bidirectionally influence the clinical course of each other at many physiological levels leading to a variety of clinical outcomes (table 1; figure 5 ). for example, cd36-mediated cyto-adherence, by virtue of its ability to stimulate pathways that overlap with those required for kshv reactivation, may contribute to a transient increase in kshv viral load and dissemination, which in turn could increase the frequency of other defining correlates of kshv-associated disease that rely on a viremic state. on the other hand, the pathogenic mechanisms of a latent kshv infection, which include kshv-induced downregulation of cd36, could effectively alter the overall disease burden by limiting peripheral sequestration; this could in turn increase parasitic access to the central nervous system, leading to a higher probability for cerebral malaria. ultimately, the emerging picture supports a syndemic link which, however serendipitous, reveals a co-evolutionary paradigm centered at the putative dueling role of cd36 as a mediator of parasite sequestration on one hand and kshv replication on the other. in this regard, more extensive molecular and genetic analysis is required in order to determine the extent to which kshv (or ebv, for that matter) might provide the driving force for altering the genetic registry at the cd36 locus in regions of high malaria endemicity. it might also be necessary to analyze the cd36 locus in hematopoietic versus peripheral b cells that may serve as the vehicle for virus dissemination in vivo, in order to determine whether infection increases the propensity for a heritable lesion at this locus. in conclusion, recent advances have revealed several nodes of pathobiological intersection between malaria and a variety of clinically significant infections, and although substantial progress has been made, we are still in the "embryonic stage" of understanding how co-infections interact with each other in their mutual host. derivative new research emphasis that is inspired by these concepts should include the important goal of developing practical in vivo platforms (animal models) that could facilitate systematic, experimental integration of population studies with reductionist multi-component molecular data. although we are still a long way from developing such a platform for studying the malaria and kshv co-infection paradigm, attempts toward this goal are an essential step in elucidating the extent to which triangular interactions between pf, kshv, and their mutual human host might articulate a syndemic relationship that underlies the co-incidence of aggressive ks in parts of the world with endemic malaria. once the correlates of co-pathogenesis are isolated, innovative research efforts oriented toward development of effective "combined" therapies can be launched. malaria susceptibility and cd36 mutation integrin alpha3beta1 (cd 49c/29) is a cellular receptor for kaposi's sarcomaassociated herpesvirus (kshv/hhv-8) entry into the 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and volcanic soil in southern italy: a case-control study is volcanic soil a cofactor for classic kaposi's sarcoma? soil silicates and kaposi's sarcoma in sardinia the company malaria keeps: how co-infection with epstein-barr virus leads to endemic burkitt lymphoma the endothelin axis: emerging role in cancer molecular basis of sequestration in severe and uncomplicated plasmodium falciparum malaria: differential adhesion of infected erythrocytes to cd36 and icam-1 identification of a platelet membrane glycoprotein as a falciparum malaria sequestration receptor role of cystine transport in intracellular glutathione level and cisplatin resistance in human ovarian cancer cell lines mucosal shedding of human herpesvirus 8 in men antiangiogenic activity of chemopreventive drugs early age at time of primary epstein-barr virus infection results in poorly controlled viral infection in infants from western kenya: clues to the etiology of endemic burkitt lymphoma platelet cd36 links 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endothelin receptor blockade inhibits molecular effectors of kaposi's sarcoma cell invasion and tumor growth in vivo cd147 inhibits the nuclear factor of activated t-cells by impairing vav1 and rac1 downstream signaling kaposi sarcoma and quinine: a potentially overlooked triggering factor in millions of africans identification of cellular genes targeted by kshv-encoded micrornas endogenous mucosal antiviral factors of the oral cavity emmprin regulates the canonical wnt/beta-catenin signaling pathway, a potential role in accelerating lung tumorigenesis cd36-tsp-hrgp interactions in the regulation of angiogenesis cd36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior distinct external signals trigger sequential release of apical organelles during erythrocyte invasion by malaria parasites inhibition of functional hyaluronan-cd44 interactions in cd133-positive primary human ovarian carcinoma cells by small hyaluronan oligosaccharides hyaluronan, cd44, and emmprin regulate lactate efflux and membrane localization of monocarboxylate transporters in human breast carcinoma cells the global distribution of clinical episodes of plasmodium falciparum malaria the rta/orf50 transactivator proteins of the gamma-herpesviridae extracellular matrix metalloproteinase inducer stimulates tumor angiogenesis by elevating vascular endothelial cell growth factor and matrix metalloproteinases trial shows vaccine halves malaria episodes in children in africa erythrocyte and reticulocyte binding-like proteins of plasmodium falciparum the curious case of the tumour virus: 50 years of burkitt's lymphoma peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity the angiogenic switch in solid tumors: clinical implications angioprevention': angiogenesis is a common and key target for cancer chemopreventive agents cytoadherence of plasmodium falciparum and complications of malaria plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells rhoa-gtpase facilitates entry of kaposi's sarcoma-associated herpesvirus into adherent target cells in a src-dependent manner kaposi's sarcoma herpes virus and kaposi's sarcoma in the elderly populations of 3 mediterranean islands trends in cancer incidence in kyadondo county parasite infection is associated with kaposi's sarcoma associated herpesvirus (kshv) in ugandan women cd147/emmprin acts as a functional entry receptor for measles virus on epithelial cells big endothelin in patients with complicated plasmodium falciparum malaria reactivation of kaposi's sarcoma-associated herpesvirus by natural products from kaposi's sarcoma endemic regions hab18g/cd147 promotes epithelial-mesenchymal transition through tgf-beta signaling and is transcriptionally regulated by slug reactivation of kaposi's sarcoma-associated herpesvirus from latency requires mek/erk, jnk and p38 multiple mitogen-activated protein kinase pathways metabolic activation-related cd147-cd98 complex sirna targeted against hab18g/cd147 inhibits mmp-2 secretion, actin and fak expression in hepatocellular carcinoma cell line via erk1/2 pathway bridge linkage role played by cd98hc of anti-tumor drug resistance and cancer metastasis on cisplatin-resistant ovarian cancer cells src-family kinase signaling modulates the adhesion of plasmodium falciparum on human microvascular endothelium under flow identification of the immediate-early transcripts of kaposi's sarcoma-associated herpesvirus risk factors for kaposi's sarcoma: a casecontrol study of hiv-seronegative people in uganda endemic kaposi's sarcoma in africa and local volcanic soils kaposi's sarcoma: a comparison of classical, endemic, and epidemic forms inhibition of cd147 gene expression via rna interference reduces tumor cell invasion, tumorigenicity and increases chemosensitivity to paclitaxel in ho-8910pm cells the authors wish to express special thanks to dr. louis miller, dr. david narum, and dr. morris makobongo for expert advice and for providing recombinant mc179 proteins, and to anita marinelli and other members of the johnan a. r. kaleeba laboratory for technical assistance and constructive discussions. work in the authors' laboratory is supported by a grant from the u.s. department of defense (r073ns and r073rz to johnan a. r. kaleeba) through the intramural award program of the uniformed services university of the health sciences. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-015306-us58wwmp authors: nan title: abstracts for the ipna congress, 30 august 3 september 2013, shanghai, china date: 2013-06-21 journal: pediatr nephrol doi: 10.1007/s00467-013-2518-4 sha: doc_id: 15306 cord_uid: us58wwmp nan first 5 days after the diagnosis. dwi-mri was performed without application of contrast medium and without general anaesthesia. results: dwi-mri examination confirmed the inflammatory infiltration in kidney parenchyma in all our patients (100 %). on the other hand, static renal scintigraphy confirmed inflammation only in 15 children (60%). six months later, none of the two follow-up examinations showed any signs of inflammation or scarring in 17 children examined so far. conclusion: in conclusion, nuclear magnetic resonance (dwi-mri) imaging seems more beneficial and accurate in the diagnostics of acute pyelonephritis when compared with static renal scintigraphy. moreover, dwi-mri provides more accurate information on the extent of kidney damage. objective: to evaluate the most proper radiologic investigation algorithm in detecting high grade vesicoureteral reflux (vur) and renal cortical scarring after first febrile urinary tract infections in infants aged less than one year . methods: a total of 408 infants aged less than one year with a first febrile urinary tract infection who completed the diagnostic follow up of renal bladder ultrasound (rbus), voiding cystourethrography (vcug) and late 6 months technetium 99 dimercaptosuccinic acid renal scan (dmsa) were enrolled in the study.the most proper radiologic investigation algorithm that could highly detected both high grade vesicoureteral reflux and renal scar in infants after the first febrile urinary tract infection considering high benefit, low cost, low radiation exposure were assessed. results: abnormal renal bladder ultrasound (rbus) was identified in 101 (24.7 %) infants. vesicoureteral reflux (vcug) was identified in 92 (22.5%) with high grade reflux (grade 4 and 5) in 18 (4.4%) infants.abnormal renal parenchyma including renal scar was identified in 32 (7.8%) infants.the top down approach with late 6 months dmsa scan showed high benefit in detecting all abnormal renal parenchyma including renal scars, with high sensitivity ( 72%) in detecting high grade vesicoureteral reflux (vur) whereas reducing the unnecessary investigation for low grade vur (94.6%) and radiation exposure per patient (0.89 msv). conclusion: currently, there is no ideal diagnostic radiologic investigation after a first febrile urinary tract infection in infants and children.the study suggest performing the top down approach with late 6 months dmsa scan that could detect all renal scars and highly detection of high grade vur. abstract# p-sat010 association between postvoid urine bladder volume and urinary tract infection in infants and children: a retrospective cohort study orpheus monakil, ivy avilla department of pediatrics, da la salle university medical, philippines objective: to determine the association between post void urine bladder volume residual and urinary tract infection(uti) in children. methods: medical records of pediatric patients aged 0 to 18 years old with diagnosis of urinary tract infection were reviewed. demographic data, urinalysis and urine culture results and kidney and urinary bladder(kub) ultrasound findings were tabulated and analyzed using the 95% confidence interval. relative risks were computed with a level of significance of p value < 0.05. results: a total of 803 patients were included in the study. five hundred forty-four(67.8%) belongs to age group < 5 years old. majority were females with a 0.97:1 male to female ratio. escherichia coli is the most common organism isolated. patients with post void urine bladder residual are at risk for having growth in the urine culture. in sub group analysis for age and sex, statistically significant results were noted among patients belonging to the 3-5 years with the rr of 1.337, 95% ci (1.082, 1.651) p-value of 0.003 and males with rr 1.186 (95% ci 1.024, 1.374) p value 0.0168. conclusion: there is an association between the occurrence of urinary tract infection and the presence of post void bladder volume residual. males and those patients aged 3 to 5 years of age are more at risk for urinary infection. objective: to evaluate if a relationship exists between renal ultrasound and voiding cystourethrography (vcug) findings among children with uti and determine whether the renal ultrasound findings/results can serve as a guide if a procedure such as vcug is needed to be done in the patient. methods: medical records of infants and children diagnosed as having uti who underwent renal ultrasound and vcug were reviewed. demographic data, urine culture and results of imaging studies were tabulated and analyzed. kappa statistics was used to determine the agreement between renal ultrasound and vcug results. mcnemars test was utilized to determine the statistical significance of the agreement. level of significance was place at p value of < 0.05. results: a total of 146 patients were included in the study. thirtyeight(26%) had vesicoureteral reflux. there were more females with a male to female ratio of 1:1.3 while cases with reflux had a 1:1.5 male to female ratio. most common chief complaint was fever. e. coli is the most commonly isolated etiologic organism from the urine. twenty(52.7%) patients had primary vur while 18(47.3%) were secondary. fifty two of 146 children had abnormal sonogram; of duplex collecting system (dcs) diagnosed during postnatal usc on objective: the aim was to evaluate the relationship of laboratory investigations, therapeutic delay time (tdt), and therapeutic response time (trt) with acute renal damage and to verify these parameters in the presence of non refluxing and refluxing urinary tract infection (uti). methods: a prospective study was conducted in 67 children. all patients received voiding cystourethrography (vcug) and dimercaptosuccinic acid (dmsa) renal scintigraphy. statistical analyses were applied to assess all parameters with dmsa renal scintigraphy. results: abnormal dmsa renal scintigraphy was detected in 20/67 (29.9%) patients. there were no significant differences in peak temperature, tdt and treatment duration. however, white blood cells (wbc) count, percentage of serum polymorphonuclear cells (%pmn) and trt had significant differences at p-values 0.042, <0.001 and 0.001, respectively. the area under roc curve for wbc count, %pmn and trt was 0.653 (95%ci 0.509-0.798) at p-values 0.055, 0.799 (95%ci 0.681-0.888) at p-values <0.001 and 0.760 (95%ci 0.637-0.858) at p-value 0.001, respectively. overall, the optimal cutoff value for %pmn was 56.0 with sensitivity 84.2% (60. 4-96.4 ) and specificity 60. 9% (45.4-74.9 ). the optimal cut-off value for trt was 22 hours with sensitivity 80.0% (56.3-94.1) and specificity 63. 6% (47.8-77.6) . in 50 patients with no vesicoureteral reflux (vur), there was significant difference in trt at p-values 0.002. the area under roc curve for trt was 0.824 (95%ci 0.693-0.955) at p-value 0.004. the optimal cut-off value for trt was 25 hours with sensitivity 88.9 % (95%ci 51.7-98.2) and specificity 68.4% . in vur patients, there were no significant differences in tdt, %pmn and trt between normal and abnormal dmsa renal scintigraphy at p-value 0.750, 0.191 and 0.313, respectively. conclusion: %pmn ≥56% and trt ≥22 hours predict renal damage in the first episode of uti. however, in patients with no vur, trt ≥25 hours predict renal damage. dmsa renal scintigraphy in the first episode of uti should be considered in those patients. susceptibility data. knowledge of local antimicrobial susceptibility all children with the first episode of febrile urinary tract infection traditionally. nowadays it has been revealed that the presence of renal scar is more important than the presence of vur regarding renal and patient outcome. our aim was to assess the relationship between the severity of vur and the severity of dmsa scan changes which is performed in the first week of the first febrile uti. methods: children with the first febrile uti who were admitted in ali asghar children hospital were evaluated prospectively. all dmsa scans have been observed and graded by our nuclear medicine specialist without any knowledge of presence or absence of vur. renal damages in dmsa scan were classified to 8 grades as follows: grade 0: normal kidney, grade 1: decreased uptake in one pole with intact border, grade 2: decreased uptake in two poles with intact borders, grade 3: diffuse decreased uptake with intact border, grade 4: decreased uptake in one pole with scar, grade 5: decreased uptake in two poles with scar, grade 6: multiple scars, grade 7: diffuse decreased uptake with one pole scar, grade 8: diffuse decreased uptake with multiple scars. then dmsa findings in patients with no vur, with low grade vur and high grade or dilating vur were compared with each other. results and conclusion: one hundred and six patients with the first febrile uti were included in this study, thus 212 kidneys were evaluated for the presence or absence of vur and dmsa grading. the mean age of our patients was 4±2.9 years old. 12.3% of our patients were male. 44.8% of kidneys did not have any vur, low grade vur (grades 1,2,3) was seen in 34.4% of kidneys and high grade vur 9grades 4, 5) was found in 20.3% of kidneys. 76.2% of patients with low grade vur and/or without vur had dmsa scoring. abstract# p-sat040 fibronection in reflux nephropathy, is it a marker of grade of reflux? nahid rahimzadeh tehran university of medical science, associated professor, tehran, iran objective: vesicoureteral reflux (vur) is one of the most common urinary tract abnormalities in patients with urinary tract infection. nowadays noninvasive diagnostic methods are suggested to recognize vur and its severity. methods: we measured urinary and serum fibronectin in 51 children with vur. results: the mean serum fibronectin was 318.3±112.1 in children with low grade vur versus 356.1±189.9 in children with high grade vur (pv>0.05). the mean urinary fibronectin was also 31.5±12.9 in low grade vur and 25.9±14.2 in high grade vur (pv>0.05). thus we didn't find any association between the severity of vur and the amount of fibronectin in serum and urine of patients. we also didn't find any relationship between dmsa changes at the acute phase of uti and serum and urine fibronectin. conclusion: in contrast to some previous studies, we showed the serum and urinary fibronectin cannot preclude the severity and grade of vur and hence it is not suitable surrogate marker for imaging techniques for vur diagnosis. abstract# p-sat041 the use of serum procalcitonin level in the prediction of high grade vesicoureteral reflux in urinary tract infection nahid rahimzadeh tehran university of medical science, associated professor, tehran, iran objective: procalcitonin is a reliable and specific marker of bacterial infection such as urinary tract infection. some authors suggest measurement of serum procalcitonin as a predictor of vesicoureteral reflux. we investigated this association in children who admitted because of acute pyelonephritis. methods: forty eight children with the first febrile uti were included. twelve patients had low grade vur, nine patients had high grade vur ((≥ 3) and twenty seven patients didn't have any vur in their imaging assessment. results: there was a significant association between high grade vur and higher levels of procalcitonin (pv=0.04). the sensitivity of procalcitonin level ≥ 0.31 ng/ml was 90% and specificity was 32% for diagnosis of high grade vur. conclusion: we concluded that serum procalcitonin concentration is a sensitive and promising predictor of high grade vesicoureteral reflux. hydronephrosis, parenchymal scarring and to study the rate of resolution of vur on follow up. methods: this was a retrospective study conducted by reviewing case records of all infants and children with primary vur who had minimum follow up of 3 years, at our nephro-urology clinic over the last 10years.the imaging evaluation (renal ultrasound, voiding cystourethrogram, dmsa scan) was done based on the indian society of pediatric nephrology guidelines. severity of vur was classified as mild (grade i, ii), moderate (grade ii, iii) and severe (grade v) results: of the 218 children screened for primary vur, 107 with complete data were included for analysis. the mean age was 25.68 ±22.45 months with male to female ratio of 1.4:1. the age at presentation was significantly lower for moderate to severe vur as compared to mild vur (p=0.033). thirty children (28%) had abnormal antenatal scans. majorities (80%) of children were diagnosed to have vur during evaluation for urinary tract infection.among107 patients, six had solitary kidneys and thus the total number of systems evaluated was 208. of these, 164 systems had vur (24% had mild, 66% had moderate and 10% had severe vur). hydronephrosis was seen in 56% of patients with vur. of these, 45% were unilateral and 55% were bilateral.there was no significant difference in the presence of hydronephrosis and grade of vur. there was no significant difference in presence of scars between mild and mod-severe vur. at the end of 3 years of follow up, complete resolution of reflux was seen in 53%, 43% and 55% of children with mild, moderate and severe vur respectively. conclusion: severity of vur did not necessarily correlate with hydronephrosis and parenchymal scarring. over a 3 year follow up, complete resolution of reflux was seen in nearly 50% of children irrespective of the grade of reflux. abstract# p-sat044 growth in children with dilating vur -a follow up of the swedish reflux trial per brandstrom, sverker hansson pediatricuronephrologic center, university of gothenburg, gothenburg, sweden objective: the swedish reflux trial included 203 children, 1-2 years of age, with vur grade 3-4, diagnosed after uti in 194 and prenatal urinary tract dilatation in 9. dmsa was abnormal at start in 124 children (61%). the children were randomized to antibiotic prophylaxis, endoscopic injection with deflux™ or surveillance. there have been reports on growth retardation in children with vur and catch up after vur resolution. the mechanism behind these findings is unclear. the children of the swedish reflux trial constitute a high risk group with dilating vur, recurrent febrile uti and high prevalence of renal parenchymal defects. we have searched the growth pattern of these children for differences related to gender, treatment group, uti recurrence, vur grade at follow up or renal defects on dmsa. methods: height and weight z-scores, compared to standardized swedish growth charts, were registered during the 2 year follow up in 199 of the children in the trial and at outpatient visits thereafter in 129 patients to the age of 5.3 years (median, range 2.0-11.8). change in height z-score between first and last visit was used to measure growth over time. results: the first and last recorded height and weight z-scores were all within normal range. there was a larger gain of height in children with renal defects compared to those without (z-score difference 0.42 vs. 0.13, p=0.009). there were no differences in growth related to gender, treatment group, vur-grade at follow up or recurrent uti. conclusion: the children of the swedish reflux trial constitute a high risk group with dilating vur, recurrent febrile uti and high prevalence of renal parenchymal defects. they have normal weight, height and height gain at follow up for up to 10 years. there was no sign of growth inhibition in these children related to dilating vur. the larger height gain seen in those with renal defects seem to be due to their slightly shorter stature at study start, although within normal range, which could be related to more severe urinary tract and renal problems during their first 1-2 years of life. engin kose, caner alparslan, serdar saritas, cengizhan elmas, fatma mutlubas ozsan, onder yavascan, nejat aksu tepecik training and research hospital, pediatric nephrology, izmir, turkey objective: the management of vesicoureteral reflux (vur) is varied and remains controversial. conservative therapy is based on the understanding that vur can resolve spontaneously, mostly in young patients with low-grade reflux. in this study, we wanted to evaluate the spontaneousresolution rate of low-grade vur (grades i and ii) in children. methods: children with low-grade (i-ii) vur treated in our hospital from may 2010 to may 2012 were prospectively studied. patients with low-grade (i-ii) vur and those who showed normal dmsa findings were included into the study. initially, a dmsa scintigraphy was performed in all patients and those who experienced acute febrile urinary tract infection (uti) during the follow-up period. treatment success was defined as complete vur resolution. no patients were prescribed antibiotic prophylaxis. all parents were informed by being given an explanation of the clinical significance of personal hygiene methods used after urinating or defecating. our institutional review board approved to collect the data, retrospectively. statistical analysis was made by using ibm spss 20.0 software. results: the study sample comprised 21 infants (10 boys and 11 girls) all of whom showed low-grade reflux (grades i-ii). bilateral reflux was seen in 7 (33.3 %) of cases. median age at diagnosis was 8 months (range: 1-37 months). median follow-up time was 14 months (range: 9-28 months). the spontaneousresolution rate of reflux was 89.3 % (25 out of 28 renal units). the frequency of febrile uti was 0.74±0.7 episode/year (median: 0.8 episode/year). during the follow-up no patients with febrile uti experienced scar on dmsa scintigraphy. conclusion: infants with low-grade reflux show a low risk of febrile uti and a high spontaneous resolution rate without antibiotic prophylaxis. therefore, these children should be managed primarily by conservative therapy. ji-nan sheu 1,2 , hai-lun sun 1, 2 , shan-ming chen 1, 2 , yu-hua chao 1, 2 , min-sho ku 1, 2 , pen-fen liao 1 , ko-huang lue 1, 2 1 pediatrics, chung shan medical unversity hospital, taichung, taiwan 2 school of medicine, chung shan medical university taichung, taiwan objective: to assess the usefulness of procalcitonin (pct) as a marker for predicting dilating (grades iii-v) vesicoureteral reflux (vur) in young children with a first febrile urinary tract infection (uti). methods: children aged≤ 2 years old with a first febrile uti were prospectively evaluated. serum samples were tested for pct measurements upon admission to a tertiary hospital. all children underwent renal ultrasonography (us), 99m tc-dimercaptosuccinic acid renal scan, and voiding cystourethrography. the diagnostic characteristics of pct test for acute pyelonephritis (apn) and dilating vur were calculated. results: of 272 children analyzed (168 boys and 104 girls; median age, 5 months), 169 (62.1%) had apn. there was vur in 97 (35.7%), including 70 (25.7%) with dilating vur. the median pct value was significantly higher in children with vur than in those without (p< 0.001). using a pct cutoff value of ≥1.0ng/ml, the sensitivity and negative predictive value for predicting dilating vur were 94.3% and 95.4%, respectively, for pct, and 97.1% and 97.8%, respectively, for the combined pct and us studies, whereas the positive and negative likelihood ratios were 2.03 and 0.107, respectively, for pct, and 1.72 and 0.067, respectively, for the combined studies. by multivariate analysis, high pct values and abnormalities on us were independent predictors of dilating vur. conclusion: pct is useful for diagnosing apn and predicting dilating vur in young children with a first febrile uti. a voiding cystourethrography is indicated only in children with high pct values (≥1.0 ng/ml) and/or abnormalities found on a us. objective: to evaluate the accuracy of acute 99m tc-dimercaptosuccinic acid (dmsa) scan in predicting dilating vesicoureteral reflux (vur) among young children with febrile urinary tract infection (uti). methods: the medical records of children (age≤2 years), presenting with febrile uti between january 2000 and december 2011, were retrospectively reviewed. the sensitivity, specificity, positive and negative predictive value, positive and negative likelihood ratio of acute dmsa scan in predicting dilating vur in young children with febrile uti were calculated. results: a total of 523 children were included, of which 397(75.9%) had abnormal dmsa results and 178(34.0%) were identified as vur on micturatingcystourethrography (mcu). among all the patients, the number of dilating vur was 151(28.9%). the rate of abnormal results on dmsa of dilating vur group was significantly higher than the rates of non-vur and low-grade vur groups (p<0.01). in age<6 months group and age≥6 months group, the sensitivities of dmsa in predicting dilating vur were 96.15%, 100.0% respectively, while the negative predictive value were 97.26%, 100.0% and negative likelihood ratio were 0.0911,0.0000, respectively. conclusion: for children of age≤2 years with febrile uti, acute dmsa scan possesses certain values in excluding dilating vur. the possibility to detect dilating vur by mcu is rather low when the result of dmsa is negative. cakut: voiding disorders abstract# p-sat048 mono-symptomatic and non-mono symptomatic nocturnal enuresis: a clinical evaluation mitra naseri 1 , mehran hiradfar 2 1 dr sheikh children hospital/pediatric nephrology department, mashhad university of medical sciences, mashhad, iran 2 dr sheikh children hospital/pediatric surgery department, mashhad university of medical sciences, mashhad, iran objective: nocturnal enuresis is divided into mono-symptomatic nocturnal enuresis(mne) and non mono-symptomatic nocturnal enuresis(nmne).this study was conducted to review clinical and ultrasonography findings in enuretic children, and compare organic and functional pathologies of lower urinary tract (lut) in children with mono-mne with those who have nmne. methods: 111 neurologically normal children with chief complaint of enuresis enrolled in the study including 60 boys and 51 girls, aged 5-17 years old, 43 (38.8%) with mne and 68 (61.2%) with nmne. urine analysis, urine culture and kidney bladder ultra sonography was done for all .some patients underwent voiding cystoureterography (vcug), urodynamic study (uds), or both. results: patients were divided in to 3 groups: mne, nmne daytime incontinence and nmne +daytime incontinence. constipation, encopresis and urge incontinence were significantly more frequent in patients with nmne +daytime incontinence (p= 0.011, 0.003, 0.001 respectively) . bladder wall thickness was the most common us findings..one patient with mne and 9 with nmne+ daytime incontinence had vesico -ureteral reflux(vur) )(p=0.016).posterior urethral valve was reported in one patient with nmne. evidences of bladder dysfunction were noted in about half of the patients who underwent uds, with higher prevalence in cases with nmne + daytime urinary incontinence(p=0.297). bowel symptoms and vur were significantly more prevalent in cases with nmne +daytime incontinence. conclusion: we recommend doing vcug in enuretic children who have daytime incontinence.in addition our study revealed that symptoms suggestive of over active bladder are not good indicators for bladder dysfunction. abstract# p-sat049 correspondence between urinary calcium, ca 2+ concentration and osmolality in enuretic children agata korzeniecka-kozerska, tadeusz porowski department of paediatrics and nephrology, medical university of bialystok, bialystok, poland objective: among many factors predisposing to enuresis hypercalciuria may play an important role. hypercalciuria is be observed in patients both in patients with nocturnal polyuria and without. hence, we decided to assess urine concentration of calcium (mmol/l) and ca 2+ (mmol/l) in patients with monosymptomatic enuresis and answer the question if patients with enuresis present calcium balance disturbances methods: the study was conducted on 204 children (83 enuretic aged median 9.66 (4.16-16.98 ) yrs diagnosed with monosymptomatic enuresis after 6 months of unsuccessful non farmacological treatment and 121 healthy children aged median 9.99 (4.15-16.86 ) yrs. we collected 24-h urine samples from all children enrolled to the study. calcium concentration, ca 2+ , ph, osmolality of urine and additionally daily sodium excretion were estimated and compared between two groups. statistical analysis were performed using statistica ver. 10.0 (statsoft,tulsa, ok). the mann-whitney u test was used for comparisons between two independent parameters. correlations were made with spearman test. a p value of <0.05 was considered to be statistically significant. results: there were no differences in age, gender and parameters of physical development between both studied groups and between girls and boys among groups. urinary calcium concentration in enuretic children did not differ compared to reference group ( p=0.993). we found statistically significant differences in urinary ca 2+ concentration (p= 0.001) and osmolality (p=0.02) between both studied group. ca 2+ in urine correlated negatively with age and parameters of physical physical development only in enuretic patients. additionally, positive correlation was found between ca 2+ and calcium concentration (r = 0.7923; p<0.005) and between ca 2+ and osmolality (r=0.2795; p<0.05) in urine of enuretic children. positive correlation was also observed between ca 2+ and daily natrium excretion (r=0.318; p<0.05) in enuretic children. conclusion: disturbed calcium balance may play an important role in pathogenesis of monosymtomatic enuresis. it's a need to assess calcium and natrium excretion in enuretic children. abstract# p-sat050 usefulness of the application of questionnaires to detect attention deficit hyperactivity disorder (adhd) and other psychiatric disorders in children with functional voiding disorders sandra gautreaux pediatric nephrology, complejo asistencial universitario de leon, spain objective: although the relationship between functional voiding disorders (fvd) and the presence of a psychological problem remains controversial, the greater frequency of adhd among children with this condition is well known. the purpose of this study was to determine the diagnostic performance of the application of questionnaires to detect adhd and other psychiatric disorders in children with functional voiding disorders in general pediatrics consultations methods: the study was conducted on 32 children between 6 and 13 years of age (20 males) diagnosed with fvd (patient group) and 32 children of the same age (21 males) who had no urinary symptoms (control group). the parents of these children responded to the questions in two standardized questionnaires: the strengths and difficulties questionnaire (sdq) to screen for mental health disorders and the questionnaire for the detection of adhd of the dsm-iv psychiatric disorders of american academy of psychiatry manual. the variables obtained from the questionnaire responses were compared between the two groups of children using the student's t-test for unpaired samples when variables were quantitative and the chi-square test if the variables were qualitative. it was considered significant when p<0.05 results: no significant differences were found between the two groups in the sdq questionnaire in any of its sections (emotional symptoms, conduct problems, hyperactivity, peer problems and prosocial behavior) or global assessment test, in which only 2 patients of each group had an abnormal result. the questionnaire for the detection of adhd, presented an altered overall result of 17.18% and 14.06% patients in the control group (p=ns). there were also no differences between the two groups on the results of this test concerning the inattention or hyperactivity-impulsivity sections. conclusion: the results of the application of questionnaires to detect adhd and other psychiatric disorders in children with fvd are similar to those of the general population. the routine application of this type of questionnaires to all the patients in pediatrics consultations does not seem necessary. abstract# p-sat051 characterization of voiding dysfunction in chidren with attention deficit-hyperactivity disorder jun yonug kim, kun hee lee, jung won lee department of pediatrics, hallym university kangnam sacred heart hospital, seoul, korea objective: attention deficit hyperacitivity disorder (adhd) has been associated with impairment of frontal inhibitory function and catecholaminergic system. adhd is diagnosed in 3~5 % of children. children with adhd seem to suffer from various forms of urinary problems such as nocturnal enuresis, dysfunctional voiding and diurnal incontinence. however, no data exist to confirm in korean adhd children. we investigate the clinical findings of voiding dysfunction in adhd children. methods: between october 2009 and march 2011, a total of 63 children (33 children with adhd and the other 30 children with upper respiratory infection as control group) were enrolled in gangnam sacred heart hospital, hallymuniversity. adhd children were diagnosed under diagnostic and statistical manual of mental disorders (dsm)-iv criteria. a comprehensive survey of voiding and defecating were administered. results: the patient group included 28 boys and 5 girls, and the control group 15 boys and 15 girls. mean age were 9.09+/−2.8 year in adhd group and 8.58+/−3.1 in control group. children with adhd had statistically significant higher incidence of enuresis (p=0.017), urgency (p=0.017), urge incontinence (p=0.033) and constipation (p=0.007). there was no significant differences in straining, intermitteny, holding maneuvers (p>0.05). conclusion: children with adhd in korea have significantly higher rates of enuresis, urgency, urge incontinence and constipation than those without adhd. the psychological correlates of primary nocturnal enuresis weiran zhou, xiaomei liu, ying shen nephrology, beijing children's hospital, beijing, china objective: previous studies based on clinical samples have reported that enuresis in children is associated with behavioural problems and reduced self-esteem, anxiety, but the relationship remains controversial. this study investigated psychological correlates of enuresis in a group of children suffering primary nocturnal enuresis(pne). methods: this survey involved 98 parents and their children with pne aged 7-14 years. clinical datas of enuresis were collected through parents' reports and individual administrations to all children. parents completed the child behaviour checklist. children completed piers harris children'sself concept scale, the screen for child anxiety-related emotional disorders (scared)and depression self rating scale for children (dsrsc) . results: of the 98 children, 60 are boys and 38 are girls. 65 are monosymptomatic primary nocturnal enuresis and, 33 are nonmonosymptomatic. 72 children accord with the severest form of bedwetting(>=7 times/week). 64.3%(n=63)children have behaviour problems and, girls get higher scores in withdraw and internalizing problems than boys. boys are more aggressive. 42.9%(n=42) get positive results through scared. 9.2%(n=9) get positive results in dsrsc. children with the severest form of bedwetting are likely to have more complex form of psychological problems. they have greater social problems, get higher scores in behaviour problem (p=0.026) and lower scores in piers harris children's self concept scale (p=0.034). but children with or without the severest form of bedwetting have no significant difference in scared . mne have no significant difference with nmne in scores of all the three children's self-evaluated scales. conclusion: most children with pne had different psychological problems. the severer the symptoms are, the more complex the psychological correlates are. these preliminary findings support the view of enuresis closely related with psychological problems. psychological problems are affected by many factors except for enuresis, so further researches need to be conducted to determine whether there is a causal relationship between psychopathology and enuresis. the current situation of treatment of primary nocturnal enuresis in children xiaomei liu, lu chen, weiran zhou nephrology, beijing children's hospital, beijing, china objective: we would like to discuss the treatment of primary nocturnal enuresis in children. methods: children, diagnosed with primary nocturnal enuresis, paid outpatient visits to beijing children's hospital from january 2011 to may 2012, are enrolled in the study. the information of previous clinical experience, compliance and expected treatment goals are collected and analyzed. results: 623 children, with the mean age of 8.69±2.33 are enrolled. 449 children have visited doctors before, 295 have unsatisfied clinical experience, such as late intervention, less standardized treatment and over-treatment. 64.36% have poor compliance with doctor's suggestion of behaviour therapy and alarm therapy. 262 children have been suggested to take medicines such as chinese traditional medicine ddavp, but 17.8% of them refused the suggestion anxious of the side effects or stop the usage on their own. the primary expected treatment goal of parents is to improve the symptoms, accounting for 86.04%. excluding the underlying diseases, alleviating parents' burden, treating the symptoms of hyperactivity or inattention and avoiding adverse effects on intellectual and fertility are also included. for children, they'd like to improve the symptoms so they won't be published by parents or laughed by their fellows. most parents (79.94%) preferred medicines as the first choice. 12.40% parents and children are inconsistent with the behavior therapy (the rate of newly diagnosed and non newly diagnosed children were 8.04%, 14.03% respectly. χ 2 test p>0.05). 38 (6.10%) don't accept alarm therapy (the rate of newly diagnosed and non newly diagnosed children were 2.30%, 8.02% respectly. χ 2 test p>0.05). conclusion: children with primary enuresis need early intervention and standardized treatment. the effect of treatment largely depends on cooperation and joint participation of children and parents. this study found that the compliance of behaviour training and alarm therapy is lower than medicine. the goals of children and parents, living conditions and other factors should be fully considered in the treatment of enuresis to improve short-term remission rates and long-term cure rate of the disease. beware of the sleeping bladder in monosymptomatic nocturnal enuresis (mne) britt borg, konstantinos kamperis, birgitte mahler, soren rittig pediatrics, aarhus university hospital, skejbv, denmark objective: bladder reservoir function in children with nocturnal enuresis is assessed by maximal voided volumes (mvv) registered on frequency-volume charts. although a degree of association is evident, mvv does not always reflect the nocturnal bladder reservoir function in mne. we aimed to evaluate the nocturnal bladder reservoir function during the night in children with apparently normal mvv. methods: data from 557 children aged 5-15 treated for mne in a tertiary referral centre was analyzed. data from 135 children was excluded due to reduced mvv according to iccs standardization and 156 due to lack of home recordings. the remaining were divided into two groups, based on whether mvv was above (n=91) or below (n=175) the average nocturnal urine production during wet nights (nupw) . first morning voids were not included in mvv values. results: 34 % of the children with mne and a normal bladder capacity had an average nocturnal urine production during wet nights below their mvv, one third of these had nupw below 65 % of mvv expected for age. these children did not differ significantly in terms of demographic characteristics, frequency of wet nights or treatment time needed to achieve dryness. urine output during dry nights was not statistical significant between the groups (p=0.077, mean diff.=28.47 ml). the group with mvv>nupwet shared significantly higher mvv to mvv expected for age ratios (mean diff=0.16, p<0.05). urine output during wet nights was lower in the group of children with mvv>nupw (diff: 96.45 ml, p<0.05) . conclusion: children with mne and apparently normal bladder reservoir function during daytime may experience wet nights with urine volumes well below their mvv and mvv expected for age. the fact indicates bladder reservoir function abnormalities during sleep that is not assessed by day recordings. physicians treating children with mne should consider anticholinergic treatment. one year experience of a multidisciplinary investigation on nocturnal enuresis in a brazilian tertiary care facility objective: to characterize a cohort of children, 6-16 yrs old, with nocturnal enuresis, defined by 2010 iccs criteria on the basis of a multidisciplinary evaluation including renal, neurological, psychological and physical therapy approaches. chronic clinical conditions and genetic disorders constituted exclusion criteria. methods: after irb approval, families were invited to participate in the project through press releases, 130 children completed quality of life evaluation through clinically validated questionnaires, followed by a one -day multidisciplinary clinical evaluation. sleep, urinary and intestinal diaries were evaluated. urinary sonography, nocturnal polysomnography, urinary and blood analysis were scheduled. results: 96/130 participants (pts)were male (73,8%), mean age 8 ±1 yrs. twenty two children were excluded due to noncompliance /chronic clinical conditions. 118/130 pts were evaluated. 93/130 parents of children/adolescents, answered the cbcl questionnaire, 25/93 (27%) of which resulted in scores compatible with clinical psychological conditions. monosymptomatic enuresis (moe) was diagnosed in 89/118 children (75.4%). in the moe group, 67/89 pts were diagnosed with intestinal constipation; 7/89 pts with obstructive sleep apnea, 2/89 pts with hipercalciuria and 2/89 were characterized with adhd. area and velocity of center or pressure displacement (vm) were used for postural control evaluation and resulted in 3,67 ±2,95 cm 2 and 20,35±13,04 cm.s-1, respectively; 22/89 pts were also assessed for postural alignment exhibiting pelvic anteversion (10,27 ±3,71 grades) and head protusion (0,40±1,86 grades). these results corroborate with a smaller range of motion found for hip flexion (94,86±17,14 grades) and hip extension (2±3,89 grades) assessed using a goniometer. conclusion: multidisciplinary evaluation of enuretic children may be the key to optimize therapy on the basis of the underlying etiology of the process abstract# p-sat056 weight depending dosing of desmopressin (ddavp) in nocturnal enuresis pauline de bruyne 1 , ann de guchtenaere 1 , charlotte van herzeele 1 , ann raes 1 , jo dehoorne 1 piet hoebeke 2 , erik van laecke 2 , johan vandewalle 1 1 paediatric nephrology, ghent university hospital, ghent, belgium 2 department of urology, ghent university hospital, ghent, belgium objective: in children as well as in adults, a uniform starting dose of desmopressin is prescribed. this uniformity is based on the inability to detect a weight-dependent dose-concentration correlation as this correlation is probably blurred by the wide intra-and interindividual differences in plasma concentration for a fixed desmopressin dose. recently, a smaller variation in plasma concentration was shown for the oral lyophilisate formulation of desmopressin (compared to tablet formulation). therefore, this study assessed a possible correlation between weight-corrected dose and plasma concentration for both formulations. methods: 23 children with monosymptomatic nocturnal enuresis were recruited in a tertiary centre. two tests were performed on two separate days (at two weeks interval) in identical, standardized conditions: on day 1 desmopressin tablet 200μg and on day 2 desmopressin oral lyophilisate 120μg was administered. plasma concentrations were measured at one, two and six hours post dosing. the nonparametric spearman's rank correlation coefficient was used for assessing the correlation between weight corrected dose and plasma concentration. results: mean (sd) age and body weight of the patients were respectively 12.7 (2.9) years and 50.1 (15. 2) kg. a positive correlation between plasma concentration of ddavp was found for the oral lyophilisate formulation at 2 hours (p-value 0,021) and 6 hours (pvalue 0,005) post dosing. this is not the case for the tablet formulation. results are shown graphically in figure 1 and 2. figure 1 and 2: correlation of dose corrected by weight to plasma concentrations at 2 and 6 hours post dosing conclusion: to the best of our knowledge, this is the first pharmacokinetic study showing a significant dose (normalized for size) -concentration correlation for desmopressin. nevertheless, this correlation was only significant for the oral lyophilisate group. this result is clinically important as it is a strong indication for more predictable plasma concentrations for the oral lyophilisate formulation, and thus preventing elevated concentrations of desmopressin. objective: increased nocturnal urine production and/or bladder hyperactivity in primary nocturnal enuretic (ne) patients could possibly be associated with autonomic nervous system dysfunction. reports of studies on autonomic nervous system dysfunction in ne are limited. to investigate autonomic nervous system function in enuretic children by performing ambulatory blood pressure monitorisation (abpm) for 24 hours. methods: children with primary ne were enrolled in this study and they get 24 hour abpm. their results were compared with healthy children. urinalysis,urine electrolyte levels, urinary culture and urinary system ultrasound were carried out in all the children. they also requested to have a diary about daily fluid intake and volume of daily urine. results: the enuretic group consisted of 28 children (m/f:19/9) and the control group of 27 healthy children (m/f:14/13). the mean ages were 7.9±2.2 years and 8.77±2.65 years, respectively. the mean 24-hour bp and daytime dbp did not differ between the groups however, the mean systolic bp was significantly higher in enuretic children (p<0.05). the mean nighttime systolic bp, dbp and map were significantly higher as well in the patient group. daytime bp load did not found to be elevated in both groups.elevated night time systolic bp load was observed in 4 patients and was not observed in control group (14.2 % vs 0%) (p<0.01). nighttime diastolic bp load was elevated in 7 of 28 patients, compared with 1 of 27 control subjects (p<0.01). the nocturnal bp dip was significantly reduced in the patient group compared with control group for diastolic blood pressure ( 67.8 % vs 14.7 %, respectively; p=0.037). patients with elevated night time bp load was found to have higher frequency of urinary incontinence per week as well as per night while compared with enuretic children with normal night time bp load (r=0.72, p<0.01; r=0.69, p<0.01 respectively). conclusion: nocturnal bp loads were significantly higher in children with enuresis. these subtle abnormalities of circadian blood pressure regulation, loss of nocturnal dip and observation of decreased nocturnal pulse rate may reflect autonomic nervous system dysfunction and pathogenesis of en. objective: bladder dysfunction and especially oab plays a major role in nocturnal enuresis, not only in the non monosymptomatic (nmne) but as well in the monosymptomatic patients (mne) . if the enuresis is related to a mismatch in nocturnal diuresis and maximal functional bladder capacity, then the bladder volume should be a major parameter, but is sofar not taken as a parameter for subtyping into nmne in the iccs standardization. maximal voided volume in a diary is the golden standard, although relation with outcome is poorly studied. methods: aim of the study was to evaluate the optimal parameter for estimation of bladder volume as maximal voided volume in a diary, during forced diuresis, bladder volume during 3 uroflow + uroflow), correlating with cystomanometry (if indicated). studypopulation 398 patients age 5 to 18 years, >6/7days wet, only 48 cystomanometries. results: if we compare the data from the bladder volume against the reference frame from rittig (aarhus), then 60,4% of patients had a mvv in diary < 2,5% percentile demonstrating that a majority of patients had a small for age voided volume in their diary. correlation between mvvdiary, mvv forced diuresis, bladder capacity (uroflow+ residu) and cystomanometry show that there is a strong correlation between the 4, but especially for the last 3 parameters (r 2 0.48-0.62, p <0.01),but results with mvv diary are worse (r 2 0.34-0.48, p 0.04-0.018) . there is no sex or gender difference in this observation. correlation with response to therapy at 1 year shows a superior correlation with mvv forced diuresis and during uroflow than mvv diary (p0.04). since cystomanometry is only performed in refractory cases, this voided value had no correlation with clinical outcome. conclusion: bladder volume can be estimated in several ways, each with their advantages and pitfalls. our data demonstrate that the alternative non invasive methods during force diuresis and in center during 3 uroflow correlate best to each other and to the cystomanometricvalues. but as well to the one year outcome (p 0.04). abstract# p-sat060 evaluating nocturnal diuresis (polyuria) claire debusschere, delphine guenter, sophie wouters, johan vandewalle, ann raes, joke dehoorne pediatric, nephrology, gent, belgium objective: nocturnal enuresis is more than bedwetting, but a symptom of a disorder involving multiple pathogenetic factors in circadian rhythm of diuresis/solute excretion and bladder dysfunction. where the sum of diaper weight and morning voided volume, is the standard to evaluate nocturnal diuresis (polyuria), it does not give indices about pathophysiology. the aarhus concept : in center studies during standardised intake, offers the advantage of standardized conditions, and reliability of the values, but is restricted to specific research centres in limited patients for budgetary reasons). the ghent concept : a home based 24 hours concentration prophyle with 4 time daytime and 4 nighttime collections offers an alternative. this one day test was not validated against 14 days diuresis nighttime registration, and was criticized since waking up the patient overnight might increase diuresis and solute excretion, as is demonstrated in sleep deprivation aim of the study: to validate nocturnal diuresis and solution excretion to evaluate nocturnal polyuria against 14 days nighttime diary and 2 days daytime diary. methods: to the study the incidence of nocturnal polyuria, and to evaluate the value of the morning osmolality in the study of nocturnal polyuria. results and conclusion: 401 children (262m), mean age 8 y (5-18y) , 24 h diuresis 1023+/−445 ml, daytime 663+/−367 ml nighttime 365+/−203ml, no sex difference. correlation between volumes in diary and 24 h concentration prophyle during 24 h (p 0.036), night (r 2 0.336 p 0.013) and day (p 0.012). only 13% of patients have a nocturnal polyuria (&gt;130%ebv= absolute nocturnal polyuria), but up to 28% have a nocturnal diuresis higher than 100% ebv and &gt; than their mvv (relative polyuria). there is no correlation between early morning osmolality and nocturnal diuresis-rate (nocturnal polyuria). objectives, methods and results: we present a 16 months old albanian female with fever, urinary retention and constipation. this problems has occurred one week before hospitalization. no history of trauma. life history has no remarkable data. no history of uti or constipation before. physical examination: weight and stature on 50 centile. no respiratory problems. the big overfilled bladder was palpated. on the left sacral region, was a tumorous mass very painful and worm. perianal reflex, bulbocavernosus were weak. other reflexes were normal. there were a hard stools in the rectum. other systems examination was unremarkable. high es rate and pathological urine sed with infection indicators. other laboratory examination was unremarkable. ultrasound examination resulted with dilatation of the left kidney pyelon (9mm), distended and overfilled bladder lumen (>400ml) with thin bladder wall. rectum distended and full with fecal masses.lateroposterior there is a sonolucent cystic formation with (volume 7ml). that cystic formation was supected for ureterocellae first. the urinary catheter and rectal clismas were done. that formation was abscess and it was drained. and two weeks after was better. voiding cystourethrogram and computer tomography was done and resulted that are no anatomical defects in the urinary tract. because of the collapsed bowels there's not good visualization of the region around the sigma and rectum. irigografy resulted: dolihosigma and suspected for hirschprung. two months later the abscess relapsed and needed drainage again. conclusion: since the abscess has repeated the next step will be biopsy. hirschprung, tailgut cysts and other presacral masses should be included in the differential for patients with recurrent abscess in the perisacral space with clinical manifestation of urinary retention and constipation. abstract# p-sat062 treatment failure to enuresis alarms: challenges and factors influencing adherence to treatment indra ganesan 1 , jessica xj hooi 2 , jasmine jy goh 2 , yh ng 1 , sm chao 1 1 nephrology service, kk women's and children's hospital, singapore, singapore 2 department of pediatrics, kk women's and children's hospital, singapore, singapore objective: the study aims to identify factors influencing treatment failure to enuresis alarm and the factors influencing adherence to treatment. methods: all patients aged 6 to 16 years referred to the voiding clinic in kk women's and children's hospital, singapore with primary nocturnal enuresis who opted for enuresis alarm treatment, over 6 year period from 2007-2013, were prospectively studied. data, via direct interview and parental questionnaire was collected on demographics, age at presentation, presence of family history, frequency of nocturnal enuresis per week and per night, on whether child awakens after wetting, or associated constipation in the child and parental perception of why primary enuresis happens. results: seventy-nine of 111 (71%) children with primary nocturnal enuresis with complete data were included. mean age at presentation was 9.6±2.1 years and 45 (57%) were male. twenty-five children had a family history of nocturnal enuresis in a 1 st degree relative. eighteen children had constipation. fifty-one (64 %) children achieved the targeted 21 consecutive dry nights. in the remaining 28 children, the main reasons for treatment failure were non adherence to usage of enuresis alarms mainly due to an inability to wake up to the alarm in deep sleep (71%) and underlying neurobehavioural conditions (21%) (adhd, autism, depression and a chromosomal disorder). the only significant clinical factor predicting treatment failure was the presence of an underlying neurobehavioural condition (or 2.8, and developmental delay . factors such as male gender, family history in first degree relatives, frequency of enuresis, arousal when wet, and the presence of constipation, were not significant. conclusion: in our population, 64% of children achieved nocturnal urinary continence with enuresis alarms. treatment failure of enuresis alarms is higher in the presence of an underlying neurobehavioural condition and developmental delay. non adherence to treatment is mainly due to inability to arouse from deep sleep. long-term follow-up of children with nocturnal enuresis -do enuretics become nocturics? an-sofie goessaert 1 , bente schoenaers 2 , olivier opdenakker 2 , karel everaert 1 , johan vandewalle 3 1 urology, ghent university hospital, ghent, belgium 2 ghent university, ghent, belgium 3 pediatric nephrology, ghent university hospital, ghent, belgium objective: although an overlap between both nocturnal enuresis (ne) and nocturia is known, research on the occurrence of both conditions in one patient is lacking. this study aims to investigate the prevalence of nocturia and other urinary symptoms in patients who have suffered from ne. methods: a questionnaire was sent to 1265 patients treated more than 3 years ago in the university hospital of ghent for ne evaluating the history and current status of enuresis and validated questionnaires on urinary incontinence (iciq-ui) and overactive bladder symptoms (iciq-oab). medical files of all subjects were analysed on the history of enuresis. all subjects were asked to sign an informed consent. results: of the 516 (41%) subjects who completed the questionnaire, 183 reported nocturia (36%), with a sex ratio (m/f) of 101/81 in the nocturic group versus 230/103 in the non-nocturic group, mean age is 18 versus 17 years old, respectively. comparing the nocturic and nonnocturic group, retrospective analysis of the history of enuresis shows an older age at which the subjects were cured (10 vs 9.5, respectively) and a higher percentage of non-monosymptomatic ne (74% vs 63%, respectively) in the nocturic group. no differences in past treatment for ne were found between both groups. prospective analysis shows a significantly higher prevalence (p<0.001) of voiding frequency during daytime, urge and urinary incontinence in nocturics compared to nonnocturics. with an increase in number of nocturic episodes per night, the percentage of female subjects increases (p<0.001) and the percentage of subjects with non-monosymptomatic ne increases as well (p<0.011). conclusion: over one out of 3 former enuretic patients develops nocturia, often accompanied by other urinary symptoms and with significant bother. cure of nocturnal enuresis does not necessarily equals cure of the urological pathology and some of the nocturic patients, who remain to suffer from bothersome symptoms, might benefit from continuous treatment for the underlying urological condition, such as overactive bladder syndrome or nocturnal polyuria. objectives, methods and results: a 3 year old girl was admitted to our hospital due to unusual pattern of voiding. when she wanted to urinate, she was writhing in pain; she cried, became pale and sweated. since the newborn period parents have observed sudden episodes when the girl started to cry and curled up her legs. she stopped when she began to urinate. the girl's father occasionally had pains in the jaw between yawning. the girl's voiding was like parents described: when she was trying to begin urinating, she was squatting on the toilet seat, crying and became pale and sweated. when voiding occurred the girl seemed to relax. the voiding volume was small and the urinary stream was weak. all investigations results were normal. according to the clinical course we assumed that the girl had an unusual expression of paroxysmal extreme pain disorder (pepd). the diagnosis was confirmed by the detection of the heterozygous pathogenic mutation in the scn9a gene, a missense mutation in exon 5. the same mutation was also confirmed in her father. the girl was started on carbamazepine and the pain attacks almost disappeared. pepd is a rare autosomal dominant neuropathy linked to mutation in the scn9a gene which encodes voltage-gated sodium channels. abnormal pain sensitivity occurs due to changes in the properties of the channels. pepd's onset is in the neonatal period with the most characteristic clinical features being attacks of excruciating pain in the rectum, genitalia, face and limbs. in literature we did not find any citation of patients with attacks triggered by the voiding stimulus. carbamazepine has been effective in relieving symptoms but the response is often incomplete (1) (2) (3) , as in our patient. conclusion: the case described is interesting because of the rare clinical expression of pepd with pain triggered by voiding. the same mutation was recently found in patients with sfn (4) . the study of functional importance of this mutation showed that the result of the mutation is increased frequency of firing pain-signaling neurons (5) . rita pavione rodrigues pereira 1 , vera herminakalika koch 2 , simone nascimento fagundes 2 , aline rossi 1 , juliane de oliveira marques vieira 1 , clarice tanaka 1 1 physical therapy, university of são paulo, são paulo, brazil 2 communication disorders and occupational therapy, university of são paulo, são paulo, brazil objective: to assess the postural control in children and teenagers with enuresis compared to a control group methods: 22 enuretic (eg) patients (11 boys and 11 girls) with mean± sd of 10.52±2.77 years, ranging from 7 to 16 years old, and 19.39±3.50 of bmi were paired with assyntomatic kids (cg) of equal age, gender and bmi. three trials of 20 seconds were collected while standing on a forceplate at 60 hz of frequency with standard position of feet in four different sensorial conditions: (1) open eyes and stable surface; (2) closed eyes and stable surface; (3) open eyes and unstable surface, and; (4) closed eyes and unstable surface. the analized variables were area (a) and velocity (vm) of center of pressure displacement (cop). student t-tests were used to compare variables between groups for each sensorial condition. the level of significance was 95% for all the analysis. results: when compared to cg, the eg revealed larger area of cop displacement in the condition (1) (mean± sd of 2,46±2,11 cm 2 for cg and 4,16±2,76 cm 2 for eg; p=0,027), and in condition (4) (mean± sd of 4,29±2,45 cm 2 for cg and 7,33±6,46 cm 2 for eg; p=0,05). no difference in vm were identified. conclusion: when compared to assymptomatic control group, the enuretics children and teenagers presented larger area of cop displacement under normal sensorial input (condition 1) and when vestibular input alone was offered (condition 4). the hip and spine mobility is diminished in enuretic children and teenagers rita pavione rodrigues pereira 1 , vera herminakalika koch 2 , monica maria ribeiro goncalves 2 , thais de souza milhoratti 1 , daniela castro pacheco 1 , clarice tanaka 1 1 physical therapy, university of são paulo, são paulo, brazil 2 communication disorders and occupational therapy, university of são paulo, são paulo, brazil objective: to assess the hip and spine mobility in children and enuretic teenagers compared to a control group. methods: 22 enuretic (eg) patients (11 boys and 11 girls) with mean ± sd of 10.52±2.77 years, ranging from 7 to 16 years old, and 19.39±3.50 of bmi were paired with assyntomatic kids (cg) of equal age, gender and bmi. the range of motion of hip flexion and extension was measured using a goniometer. spine flexibility was measured using schober e stibor test and bank of wells. student t-tests were used to compare variables between groups. the level of significance was 95% for all the analysis. results: when compared to cg, the eg revealed diminished range of motion of hip extension bilaterally (2±3.89 for eg and 10±7.99 for cg at right side; p<0.01; ge 2.05±3.88 for eg and 10.4±7.22 for cg at left side; p<0.01). no difference was found for hip flexion range of motion, however compared to cg, eg showed lower values bilaterally (94.9±17.1 for eg and 100.32±8.52 for cg at right side; 98.3±11.7 for eg and 101±11.1 for cg at left side). the spine flexibility did not presente any difference for schober (14.88±3.18 for eg and 16.18±1.16 for cg; p =0.07), and stibor (50.21±6.2 for eg and 51.93±7.37 for cg; p=0.40) and bank of wells (25.58±7.67 for and 23.66±7.42 for cg; p=0.63). conclusion: when compared to assymptomatic control group, the enuretics children and teenagers presented diminished range of motion of hip extension suggesting transversal misalignment of the pelvis. more concern about enuresis children: an epidemiological study of primary nocturnal enuresis in elementary schools in shanghai yibing zheng 1 , yinv gong 1 , hong xu 1 , keli wang 1, 2 , zhonghui ni 1,2 , dandan he 1,3 1 nephrology and rheumatism, shanghai, china 2 xuhui health bureau, shanghai, china 3 minhang center for disease control and prevention, shanghai, china objective: (1) to assess the prevalence of primary nocturnal enuresis (pne) and its risk factors in children of elementary school age in shanghai. (2) to evaluate the impact of enuresis on these children and their parents, and to identify the methods and effectiveness of managing enuresis. methods: a randomly selected cross-sectional study was conducted in four elementary schools in two districts inshanghai. the parents of these children were asked to complete questionnaires anonymously which included items about the presence and frequency of enuresis, its risk factors and its perceived impact and management. the distress caused to the family by enuresis and the outcome of any management was evaluated using a 5-point visual analogue scale (vas). pns was defined as an involuntary voiding of urine during sleep, with a frequency of more than two times a week for three consecutive months, in the absence of congenital or acquired defects of the central nervous system. results: a total of 7600 questionnaires were distributed. the overall response rate to the questionnaire was 97.1%, girls 45.2% and boys 51.8%. the prevalence of pne declined with age from 6.3% at 7 years old to 0.5% at 11 years old. of all enuresis children, 10.1% had daytime urinary symptoms and 11.7% had a positive family history. 58.8% of parents were concerned and 17.6% were very concerned about enuresis. 41.2% of children were worried about and 5.9% were very worried about enuresis. only 35.3% of pne children sought for treatment and the common strategies (23.5%) were adjusting lifestyle such as restriction of water intake at night, 11.9% used alarm clock and 5.8% were take medication. 48.6% of parents felt that the current treatment was effective. conclusion: we conducted a relatively scientific epidemiological survey on the prevalence of pne in two districts in shanghai. we find enuresis still has great impact on children and their parents, but only a small part of them will seek for treatment. and only half of the parents feel that the current treatment is effective. therefore, it would be desirable to create a series of standardized management and follow-up processes for enuresis children. abstract# p-sat068 renal manifestations of tuberous sclerosis: a descriptive analysis sophy korula 1 , alka ekbote 2 , naresh kumar 1 , sumita danda 2 , indira agarwal 1 , swasti chaturvedi 1 1 paediatrics, christian medical college, vellore, india 2 clinical genetics, christian medical college, vellore, india objective: to describe the renal manifestations in children 0-18 years of age diagnosed with tuberous sclerosis complex (tsc) at a tertiary hospital in south india. methods: data of children with tsc who presented to christian medical college vellore hospital from january 2008 onwards, were analysed by a retrospective chart review. the cases were identified from outpatient records and underwent ultrasonography, urine analysis and serum creatinine to recognize renal involvement. results: twenty-five children with tsc were identified. two children did not imaging studies available and were excluded from the analysis. the age of included children ranged from 5 days to 15 years with a median of 8 years. seventy four percent (17/23) were males. ten of the 23 children had evidence of renal involvement (43.5%). of the ten children with renal involvement, six had angiomyolipoma (60%), five had renal cysts (50%) and one had suspected renal cell carcinoma. in two children both angiomyolipoma (aml) and cysts were noted. none were symptomatic. one child was found to have proteinuria and another had reduced creatinine clearance of 52.5 ml/min/m 2 with normal bp and urinalysis. the rest of children had no evidence of proteinuria and had normal creatinine clearance. conclusion: we conclude that all children with tsc should be screened for renal involvement and regular follow up should be arranged. abstract# p-sat069 sonographic growth charts for kidney length in normal korean children: a prospective observational study objective and methods: kidney length was measured by sonography in a prospective observational study of 343 normal children from 0 to 12 years of age. results and conclusion: there was a good correlation between kidney length and somatic values including age, weight, and height. the rapid growth of height during the first 2 years of life was intimately associated with a similar increase in kidney length. the values of weight and height showed good correlation with kidney length in children from 2 to 12 years of age. height should be considered the important factor to correlate with kidney length. for the children aged 2 years or older, the regression equation was obtained: kidney length (cm) = 2.677 + 0.046 x height (cm). for the younger than 2 years, the equation was: kidney length (cm) = 1.120 + 0.066 x height (cm). objective: the outcome of children with unilateral nonfunctioning mcdk. methods: 24 children (13 pts with right mcdk), afunction confirmed by renal scan, 12 boys, mean age of 6 years (range 1.5 month -21.5 years), mean observation period of 6.7 years with a range of 1.5 month -21.5 years. prenatally diagnosis was made in 83 %. abnormalities of the contralateral kidney were found in 4 /24 pts (13 %) -dystopia (n=2), hydronephrosis due to ureterovesical obstruction (n=1), vacter syndrome (n=1). in 2 pts mcdk had been removed (in period 1991-1997) . collected data include egfr (calculated by cystatin c), urine protein/creatinine ratio, microalbumin/creatinine ratio, a1microglobulin (a1m)/creatinine ratio, urine beta-2microglobulin (b2m), urine ngal, urine nag, blood pressure (bp), renal length. results: hypetrophy of the contralateral kidney (length >2 sds) was detected in 19/24 pts (79%) to 2 years of age. complete involution (in 33 %) or decrease of size (in 30%) of mcdk occured in the first 5 years. hyperfiltration (defined as egfr of 149 ml/min/1.73m2) was seen in 4 pts (17 %). none of 24 pts demonstrated decreased egfr, proteinuria (mean value of 24.8 mg/mmolcreatinine), microalbuminuria (mean value of 2.0 mg/mmolcreatinine).urine a1m (mean value of 5.8 mg/mmolcreatinine), urine b2m (mean value of 0.2 mg/l), urine nag (mean value of 7.3 ukat/mmolcreatinine), urine ngal (mean value of 10.4 ng/l) were in a normal range. all pts were normotensive -bp defined as <95th percentile for age and gender. the combination of hepatoblastoma with renal failure due to congenital dysplastic kidneys in children is extremely rare. we report two children in renal failure since birth due to congenital cystic dysplastic kidneys. hepatoblastoma was subsequently diagnosed in both of them. methods and results: case1 a male infant was born at 35 weeks gestation with an antenatal diagnosis of bilateral dysplastic kidneys and bladder outflow obstruction secondary to a prolapsing ureterocele, anhydramnios, and pulmonary hypoplasia. he underwent surgical repair of other congeniatal abnormality after birth. peritoneal dialysis was initiated at 3 months. treatment with erythropoietin (epo) was commenced at 6 weeks of age. case 2 a male infant with congenital cystic renal dysplasia, bilateral vesicoureteric reflux pulmonary hypoplasia and oligohydramnios was born at 36 weeks gestation. peritoneal dialysis was commenced at day 20 due to anuria from birth. epo was commenced at 6 months of age. both of them required high dose epo to maintain adequate haemoglobin. hepatoblastoma was diagnosed in both of them at 2-3 years, following an incidental finding in abdominal imaging done for different reasons. conclusion: hepatoblastoma has been reported in association with cystic renal dysplasia in 5 children in the literature to date. none of these children had evidence of impaired renal function prior to the diagnosis of hepatoblastoma. in our report both the child were treated with high dose erythropoietin prior to diagnosis of hepatoblastoma. erythropoietin (epo) is the primary regulator of erythropoiesis through specific interaction with its receptor (epo r). epor expression has been demonstrated in common paediatrictumour cells and such expression is reported to promote tumour cell survival through release of angiogenic growth factors. angiogenesis is the primary requirement for tumour growth. epo production has been observed in patients with hepatomas and in the hepg-2 cell line in hepatoblastoma. we report two cases of hepatoblastoma detected incidentally in children with established renal failure and propose a putative role of epo in the development and / or progression of hepatoblastoma in this population. objective: hypercalciuria is the most common metabolic cause of renal stone. long term immobilization is associated with hypercalciuria and bone loss. the effect of short term immobilization on hypercalciuria was the main objective of this study. methods: in a prospective study all orthopedic patients less than 40 years with pelvic fracture who were assigned for immobilization with traction were enrolled in this study. serum (calcium, phosphorous, alkaline phosphatase, sodium, potassium, uric acid, bun, creatinine)and fasting urine calcium, creatinine, sodium, potassium and uric acid were checked within 48hours of hospitalization and also in 1st,2nd and 3rd weeks of immobilization and then after 2to 3months of mobilization. student's t-tests used for statistical analysis. results: fifty five patients 45 male and 10 female with the mean age of 19.4 ± 12.7 years were studied. urine calcium /creatinine (u ca/cr)* ratio before immobilization u ca/cr0 was 0.13±0.06. one, two and 3 weeks following immobilization u ca/cr ratio was 0.17±0.11, 0.22±0.12 and 0.29±0.17respectively.multivariate tests revealed a significant rise in u ca/cr ratio during hospital stay when this value checked before immobilization and throughout immobilization as soon as end of 1st week and following in 2nd and 3rd week ( objective: importance of renal biopsy(rb) in diagnosis and treatment of kidney disease is the same for children as for adults. however there are some differences between institutions in practice of this invasive method. the aim of this study is to identify the current status and complications associated with the renal biopsy of childhood kidney disease. methods: a retrospective study was conducted based on medical records from all patients who had undergone native renal biopsies with 16-gauge needles from april 1997 to november 2012 in our center. we analyzed the number of experiments, sex, age, indication, the types of harvest (percutaneous or open) and anesthesia (general or local), histopathological findings, and complications. results: a total of 158 renal biopsies were performed in 141 patients (82 boys, 59 girls). the mean age at first procedure was 9.4 years. about 70 % of the indications for a biopsy showed moderate proteinuria and hematuria in the annual urinary screening program in japan, and approximately 20 percent of those were refractory childhood nephrotic syndrome. open renal biopsies were practiced in only 3 cases, and the rest of children underwent percutaneous ultrasound-guided kidney biopsies. the most procedures of patients aged 10 or younger were executed under general anesthesia due to the non-cooperation of them. about 30 % of the biopsy-proven kidney diseases showed immunoglobulin a nephropathy, and approximately 20 percent of those were henoch-schonlein purpura nephritis. for the post biopsy complications, 5 out of all performances administered hemostatic agents for moderate hematoma, but none of them required blood transfusion or surgical intervention. conclusion: we suggested that renal biopsy could be supportively performed under local anesthesia in the patients more than the upper grades of elementary school. in addition, 17 patients aged 3 or younger, including 12 months, could undergo the percutaneous ultrasound-guided renal biopsies without procedural and general anesthesia complications. therefore, our results revealed that this procedure was considered safe even in infants. abstract# p-sat084 fifteen years review of indications and results of renal biopsy in children from a single center in egypt there were 20 insufficient biopsies. in pathologically diagnosed 1226 specimens primary glomerulonephritis was the most common finding (n=826, 67.4%) followed by secondary glomerulonephritis (n=238, 19.4%) and end stage renal disease (n=50, 4.1%). the most common causes of primary glomerulonephritis were minimal change disease (n=267, 21.8%), diffuse proliferative glomerulonephritis (n=188, 15.3%), focal proliferative glomerulonephritis (n=164, 13.3%) and focal segmental glomerulosclerosis (n=129, 10.5%). lupus nephritis (n=209, 17%) was the most common cause of secondary glomerulonephritis followed by hemolytic uremic syndrome (n=16, 1.3%) and amyloidosis (n=10, 0.8%). only one mortality has been reported as a complication of renal biopsy (uncontrolled bleeding). conclusion: this study introduces the first biopsy based epidemiological information of pattern of renal diseases in egyptian children from a single tertiary pediatric center in which minimal change disease was the most common histopathological finding and steroid resistant nephrotic syndrome was the most frequent indication for biopsy. results: a total of 112 children were enrolled in the study. most of them (71%) had isolated microhematuria, and were proved mild lesion of glomeruli (60%) by renal biopsy. about 30% of them, however, might have progressive glomerulonephritis, such as iga nephropathy, focal segmental glomerulosclerosis, alport syndrome and so on. these asymptomatic children were found urine abnormalities due to either school urine screening study, health examination, or during diagnosis of non-renal diseases. in center a, iga nephropathy was the most case diagnosed, while in center b and c, minor glomerular abnormalities was the most case diagnosed. the difference among three centers was significant and this variation might be due to different indications of renal biopsy. conclusion: our findings confirm that urinalysis may help early detecting progressive glomerulonephritis in asymptomatic children. children with isolated microhematuria has relatively low risk of severe pathologic lesion of glomeruli, thus isolated microhematuria per se might not be suggested as indication of early renal biopsy. long-term follow-up with appropriate further examination is of great importance for these asymptomatic children. abstract# p-sat089 twenty-three-year review of disease patterns from renal biopsies: an experience from a pediatric renal center there were 59 episode of peritonitis with an incidence of 1 episode per 16.8 patient/months. the commonest organisms isolated were gram negative rods 12(20.3%), followed by staph aureus 11(18.6%). sixteen (27.1%) were culture negative and 3(5.1%) were fungal peritonitis. catheter removal related to peritonitis was performed in 20 patients in whom only one patient returned back to pd. exit site infection (21 episodes) and tunnel infection were reported in 20 and 10 patients respectively. catheter malfunction was the most common non infectious complication seen in 27 (61.3%) children. other noninfectious complications include haemorrahgic effluent 5 (11.3%) children while catheter leak, umbilical hernia and inflow pain where seen in 2 (4.5%) children each. at the end of the study 17 (18.7%) children remained pd active, 26 (28.6%) transferred to hd, 13(14.3%) transplanted, 9(9.9%) suspended from pd, 5(5.5%) lost follow up and 21(23.1%) children died. conclusion: in the setting of our country with limited resources and investment, capd is efficient and successful with complications comparable to most parts of the world. attempts to produce pd fluids locally and to train and educate health care workers will greatly improve the use of pd in developing countries. profile and response to therapy in patients with idiopathic membranous nephropathy from march 2010 to december 2012. renal biopsies were studied for light microscopy, immunofluorescence and electron microscopy. antibodies to the m-type phospholipase a2 receptor (pla2r), bovine serum albumin (bsa), and cationic bsa were measured by western blotting with patient sera (when available) and then detecting for both totaligg as well as specifically for the igg4 subclass. results: 80 renal biopsies were performed during the study period, out of which 11 patients had membranous nephropathy. five were secondary to sle and 6 patients (7.5 %) had idiopathic membranous nephropathy. mean age at presentation was 13.16+/-1.94 years with 50 % having hematuria and 66.6% hypertension at presentation. estimated gfr was 160.57+/-49.33. renal biopsy showed findings consistent with membranous nephropathy on light microscopy with with<5% of the interstitium showing tubular atrophy and interstitial fibrosis. immunofluorescence studies showed granular igg and c3 along the peripheral capillary walls. electron microscopy showed subepithelial deposits, with formation of basement membrane spikes between the deposits. few intramembranous deposits were also present. subendothelial and mesangial deposits were not present. there were no antibodies to bsa or cationic bsa detected in any of the five sera available. one of the five patients had circulating antibodies to pla2r. all patients received prednisolone, angiotensin converting enzyme inhibitors and oral calcium supplements. two patients continued to have nephrotic range proteinuria inspite of prednisolone and were started on cyclosporine. none of the patients developed any complications. conclusion: immunosuppressive therapy is beneficial in children with idiopathic membranous nephropathy. anti-pla2r antibodies can be seen as early as 13 years of age. abstract# p-sat108 hypercalcemic crisis and nephrogenic diabetes insipidus due to vitamin d intoxication methods: twenty-three children (46 kidneys) with first uti with p-fimbriatede.coli were enrolled in the study. children were aged between few months and 10 years. all children were treated with adequate antibiotic therapy. dmsa scintigraphy was performed in a few days after infection and control scintigraphy was performed 5 months to 36 months later. dmsa findings were categorised as normal, generally diminished uptake of activity, focally diminished uptake of activity and clearly pathologic with renal scars. results: the first dmsa scintigraphy immediately after infection was normal in 12 kidneys (26.1%), generally diminished activity was found in 21 kidneys (45.7%) and focally diminished activity in 6 kidneys (13% ). renal scars were present in 7 kidneys (15.2%) . on the control scintigraphy 20 kidneys (43.5%) had normal findings. generally diminished activity were found in 6 kidneys (13%) and focal diminished uptake in 8 kidneys (17.4%). significantly higher number of scars, in 12 kidneys (26.1%), despite of antibiotic treatment were found on the control scintigraphy (p< 0.01). conclusion: dmsa scintigraphy demonstrated that the infection with p-fimbriatede.coli can result in permanent renal damage, which clearly points to uti with p-fimbriatede.coli as a risk factor in renal damage. our results confirm obligatory scintigraphic follow-up of children with positive p-fimbriatede.coli infection. objective: efficacy of mannitol with furosemide was compared with that of albumin with furosemide in the treatment of diuretic resistant oedema in childhood nephrotic syndrome. methods: forty patients fulfilling the criteria for "resistant oedema" in nephrotic syndrome cases were enrolled in this descriptive cross sectional study. resistant oedema was diagnosed based on failure to achieve therapeutic response to diuretics or a weight loss of <1% body weight daily. all nephrotic syndrome patients with severe oedema, age 1 year to 15 years of both sexes were hospitalized and were managed with fluid restriction, no added salt and bed rest. beside these 2 mg/kg/day oral furosemide or combination of furosemide and spironolactone, were given for 3 days to achieve desired diuresis. those patients who did not get response were divided into two groups (group-a, group-b) in consecutive fashion. the group-a study population, was with intravenous mannitol 0.5-1 gm/kg/day in single daily dose over 1-2 hrs followed by intravenous furosemide 1 mg/kg/day for 5 days. the group-b study population was with intravenous albumin, 0.5 -1 gm/ kg /day in single daily dose over 1-2 hrs followed by intravenous furosemide 1 mg/kg/day in every alternate day, total 3 doses. conclusion: this boy presented with rhabdomyolysis, myohemoglobinuria and arf. (similar pr similar presentation can be rarely seen in human being, but often seen in dogs). blood exchange transfusion in time must be necessary for preventing "water fall effect" established, for it can eliminate inflammatory factor and toxic products such as bun, ccr, and creatinasein blood effectively. blood tranfusion in combination with azithromycin and clindamycin must be a good choice replace the "babesia-infected" rbc and eliminate pathogens in blood. beta-2-microglobulinuria in human immunodeficiency virus infected children objective: to determine whether hiv infection in children is associated with increased urinary excretion of β2-microglobulin, which is a marker of tubular defect. methods: a prospective observational study was done of hiv infected children attending outpatient immunology clinics to investigate urinary β2-microglobulin excretion by measuring urine β2-microglobulin to creatinine ratio. in addition serum sodium, potassium, urea, bicarbonate and creatinine levels were documented and urine dipstick test was done to quantify proteinuria. results: thirteen (13) children aged 2 months to 49 months (mean 20+/-4 months) were enrolled. all children had normal estimated glomerular filtration rate. eleven children were on different haart regimes (see table 1), one was receiving nevirapine prophylaxis and one was not on any treatment. the duration of haart use was 1-16 months (mean of 8+/-2 months). seven (7) children were on cotrimoxazole for pneumocystis jiroveci prophylaxis and 4 on anti-tuberculosis drugs, namely rifampicin, ethambutol, isoniazid and pyrazinamide. none received any nephrotoxic drugs. the hiv viral load was >1000 copies/ml in 8 children and <1000 copies/ml in 5 children. of 13 hiv-infected children, 4(31%) had no abnormal urine protein excretion and nine (69%) had elevated urinary levels of β2-microglobulin.of note only 2 (22%) of the 9 children with β2-microglobulinuria had associated proteinuria. ten children ( 76%) had metabolic acidosis with the mean serum bicarbonate (co 2 ) level of 16.9 mmol/l ( range 10-20 mmol/l). conclusion: proteinuria is believed to be the earliest finding for the diagnosis of hiv associated renal diseases. in our study β2microglobulinuria and metabolic acidosis were the prominent findings suggesting that hiv induces tubular dysfunction in children without clinical evidence of renal disease. objective: acute kidney injury (aki) is an important cause of mortality in sub saharan africa because access to renal replacement therapy is limited. there are few reports on haemodialysis in childhood aki in the sub region. we therefore performed a preliminary review of data on children who received intermittent haemodialysis for aki in our centre. method: a retrospective review of case records and haemodialysis registers of children in aki who received haemodialysis in our centre from january 2006 to december 2012. results: 62 children, including a child aged 3years, received haemodialysis for aki over the period but full details were available for 23 children and were further reviewed for this study. there were 13 males (56.5%). the children were aged 5-13 (8.4+/-2.4) years. the primary aetiology of aki was related to intravascular haemolysis (ivh) with massive haemoglobinuria (n=12), septicaemia (n=4), acute glomerulonephritis (n=2), malignancies (n=2), malaria (n=1), hiv (n=1) and haemolyticuraemic syndrome (n=1). the aetiology of ivh was secondary to glucose-6-phosphate dehydrogenase (g6pd) deficiency in 4 patients, autoimmune haemolyticanaemia in 1 and was unknown in the others. one of the patients with ivh secondary to g6pd deficiency also had malaria. the number of sessions of dialysis ranged from 1 to 5 sessions per patient with a modal value of 2 objectives, methods and results: a twelve-day old, formula-fed, full term neonate with unremarkable perinatal history was admitted for poor oral feeding and significant (12%) weight loss. physical examination was otherwise unremarkable. investigation showed normal anion gap metabolic acidosis, hyperkalemia (6.5mmol/l), hyperurecemia (14.5mmol/l) but normal creatinine (28umol/l). metabolic screen and other investigation was unremarkable. on further questioning parents has been feeding baby with goat milkbased formula since day 5. baby was switched back to normal cow milk-based formula with prompt resolution of metabolic disturbance and without recurrence of acidosis on follow-up. conclusions: goat milk is becoming increasing popular to parents because of the myths associated (organic, easier digested, hypoallergenic, better nutrient content etc). however there is good evidence suggesting against these misbelief and there are even reported morbidities associated with ingestion of goat milkbased formula. with increasing appealing but not necessarily accurate information available on the internet, what the public need is education. objective: there have been great inroads made in controlling the scourge of hiv in south africa. the national pmtct program has reduced the mtct rate to 3.5% and children infected prior to these interventions are now on effective treatment programs. these children are now surviving longer and so we are seeing increased numbers of hiv related renal pathology presenting to our clinics. before the widespread availability of arv's in south africa these children would have been denied access to our program but now we are able to offer them crrtand a transplant. the biggest challenge facing our program is one of the social disintergration of much of our indigent population. the vast majority of our patients come from social backgrounds that are incompatible with adhering to the rigours of a chronic renal program. this is compounded when the care givers are themselves ill or when the child has a second serious illness such as hiv to deal with. methods: we would like to present our experience with admitting 8 hiv positive children with ckd5 to our chronic program and will highlight the challenges that face these families and their health care providers. we will describe the social issues affecting each family and how they impact on the child's care. results: all 8 families had serious social issues on top of having to deal with their hiv disease. all 8 families had extreme difficulties in adhering to our program. 2/7 died as a result of non adherence and of the remaining 5 children only 1 is really coping well with the program. 3/7 children have no living parents and 1 is foster care. the rest are either with a single parent or with a family relative. conclusion: the huge difficulties facing these patients is forcing us to rethink the criteria that should be applied to admitting these patients to our program. restricting the program to children with these problem seems cruel and we feel that all options should be considered before denying these children access to life saving care. abstract# most of the patients presented during autumn and winter. the incidence of apsgn has decreased in the past 11 years in shenyang, but the proportion of the patients with acute kidney injury (aki) or nephroticrange proteinuria was increased. the proportion of children with macroscopic hematuria remained almost the same during these years. macroscopic hematuria resolved in 2-3 weeks and non-nephroticproteinuria resolved in 1-3 weeks. the treatment of apsgn patients with nephrotic-proteinuria was the same with the children with primary nephrotic syndrome and prednisone was withdrawn in 3-4 months. some aki patients have been followed up for 5 years and no patients with chronic renal failure. all patients with microscopic hematuria at onset and 30% patients with microscopic hematuria after 2 years. conclusion: the incidence of apsgn was decreased during recent years. the proportion of apsgn patients with aki or nephrotic-range proteinuria was increased. the short-term prognosis of apsgn was good and minority of the patients persisted with microscopic hematuria. abstract# p-sat122 analysis of clinical manifestations and prognosis in 43 children of acute poststreptococcal glomerulonephritis objective: crescentic glomerulonephritis (gn) is characterized by severe infiltration of massive inflammatory cells into glomeruli and crescent formation. although renin angiotensin system (ras) is the key player in renal injury, the impact of direct renin inhibitor on glomerular crescent formation is not elucidated yet. methods: to examine whether direct renin inhibitor ameliorate renal injury in crescentic gn, we investigated renal injury induced by antiglomerular basement membrane (gbm) antibodies in wistar kyoto rats treated with direct renin inhibitor, aliskiren. in addition, using cultured glomerular mesangial cell (mcs) and parietal epithelial cell (pecs), we examined whether recombinant renin could induce monocyte chemoattractant protein-1 (mcp-1) expression and cell proliferation, respectively. results: an anti-gbm nephritis model developed progressive proteinuria and glomerular crescent formation, accompanied by increased expression of mcp-1 and (pro)renin receptor. interestingly, (pro)renin receptor expressed strongly in the crescent formation area in diseased glomeruli. proteinuria was significantly reduced by the treatment of aliskiren. then, aliskiren markedly ameliorated renal injury (% glomerular crescent: 26.0 +/− 1.7 %) compared to vehicle treatment (59.6 +/− 3.6 %). excretion of urinary protein in a group of rats treated with aliskiren were significantly reduced compared to vehicle-treated rats. aliskiren treatment markedly decreased mcp-1 and (pro)renin receptor mrna levels in the diseased kidney. next, primary cultured mcs stimulated by recombinant renin showed significant increases of mcp-1 mrna expression. furthermore, primary cultured pecs showed an increase in recombinant renininduced cell proliferation. conclusion: these data suggest that therapeutic strategy of direct renin inhibitor may prove beneficial for crescentic gn by the suppression of the ras activation and the decrease of inflammation and cell proliferation in glomerular crescent via (pro)renin receptor. an update on management of acute glomerulonephritis in children objective: to analyze the clinical effects, safety, and significance of leflunomide combined with hormone in the treatment of children with refractory nephrotic syndrome. methods: collected 60 cases patients in our hospital in children with refractory nephroticsyndrome,and randomly divided into 30 cases of the control group and treatment group. control group was treated with mycophenolatemofetil (mmf)andhormone,the treatment group was treated with leflunomide (let) and hormone.measured and compared the changes before and after treatment of 24h urinary protein excretion,tc,alt,cr,bun clinical efficacy and complications. results: the two groups after 6 months of treatment, 24 h urinary protein, plasma albumin, total serum cholesterol, serum creatinine and blood urea nitrogen and other indicators were significantly better than before treatment and has a statistically significant (p <0.05); the overall response rate in the control group and treatment group were 83.33% and 86.67%. conclusion: the application of leflunomide combined with hormone treatment of children with refractory nephrotic syndrome can make the disease to be effectively alleviated, the clinical results were satisfactory, safe, and fewer complications, it is worthy of promotion. objective: post-streptococcal acute glomerulonephritis (psagn) is a familiar disease in children. misdiagnosis might occur when it is presented with atypical symptoms. here we report a 12-year-old girl presented with acute heart failure which was finally diagnosed comorbidity of psagn and graves' disease. methods: retrospectively review the history of the patient. the girl was admitted to our hospital with cough, shortness of breath and chest tightness, without any other obvious symptoms and history of illness. the examination of cardiac color ultrasound showed mitral insufficiency with severe mitral regurgitation and heart enlargement. other examinations also found she had hypertention, mild anomalies of urinalysis, hypocomplementemia, hypoalbuminemia, hyperthyroidism and elevated antistreptolysin o. first we diagnosed her illness as rheumatic disease. after two weeks treatment with rest, fluid and sodium restriction, controlling blood pression, diuresis and methimazole, the girl recovered very well. she had no any symptom with normal urinalysis and blood pressure, the result of reexamination of cardiac color ultrasound was normal, her hypocomplementemia and hyperthyroidism were improved significantly. finally she was diagnosed as co-morbidity of psagn and graves' disease. conclusion: psagn with atypical symptoms at presentation should be paid more attention, especially when it is also has co-morbidity. abstract# p-sat128 report of a child with idiopathic cryoglobulinemia idiopathic cryoglobulinemia is rare to see in child. here we report a child with typical manifestations of cryoglobulinemia. the subjected child was male, 6 years and 4 months, he had repeated rashes, itching, desquamation on his fingertips for more than two years, accompanied with recurrent miliary reddish rashes on the trunk and limbs for more than one year, transient arthralgia for 2 weeks, and edema and oliguria for 10 days before he was admitted to our hospital. he was treated as atopic dermatitis all the time. but the rashes were recurrent. erythema could be seen after desquamation. 2 weeks before admission, he had arthralgia on the right knee, 2 days later on left knee without swelling, both were retrieved spontaneously. x-ray of the knees revealed no abnormal. ten days before admission, edema and decreased urine output was notice without gross hematuria. physical examination in local hospital revealed blood pressure 140/108 mmhg, needlelike erythematous maculopapules in his hands and feet. urine test revealed protein 5g/l, rbc 164/ul, no casts. serum bun was 32.41mmol/l, cr 116umol/l, tco2 11mmol/l, c3 0.47g/l. he was sent to another hospital 2 days later. there, prednisone 25mg per day was given. as a result, the urine output increased gradually, but serum bun and cr remained high, so he was referred to our hospital. renal biopsy indicated membrane proliferation glomerular nephritis with abundant microthrombus. the diagnosis of cryoglobulinemia was suggested. after that serum cryoglobulin and serum protein electrophoresis were tested 18 days after prednisone was given, both were normal. etiological examinations, included hepatitis virus a, b, c, d, e, g were all negative; antibodies of hiv, syphilis, toxo, cmv, rvb, rsv, hsv, eb, adv, and rickett's organism were all negative, mp-igm 1:40. antinuclear antibodies of sle, anca and antiphospholipid antibodies were all negative. so idiopathic cyoglobulinemia was more favored. the treatment of prednisone 25mg per day was continued after admission. he restored gradually. prednisone was taped down gradually. now prednisone has been stopped for more than one month, he seems well during that period of time. methods: children with hsp over 3 years of age were enrolled in the study. they were evaluated for demographic, anthropometric, clinical and laboratory data including urinalysis, complete blood count, serum albumin, creatinine, iga levels. in addition, anti-tissue transglutaminase iga (elisa), anti-endomysium iga (ifat), antigliadin (gaf3x, deaminated) iga (ifat) and anti-gliadin (gaf3x, deaminated) igg (elisa) antibody levels were determined. seropositive patients were evaluated by endoscopic small bowel biopsy. the rate of cd seropositivity in hsp patients was compared to the rate in healthy turksih children by the test for the statistical significance of two percentages. results: celiac serology was evaluated in 42 children (25 male, mean age 11.2 +/-3.6 years) with hsp. there was no patient with growth failure or having symptoms associated with cd like abdominal pain, abdominal distention or diarrhea. in addition, none of the patients had iga deficiency, anemia or hypoalbuminemia. celiac serology was positive in 5 (12%) children. endoscopic evaluation was performed in 3 patients and one of them was diagnosed as cd. prevalence of cd in children with hsp was significantly higher compared to healthy turkish children (p<0.001). conclusions: celiac seropositivity was 12% in children with hsp and this rate is significantly higher than the rate in healthy children. although the number of children with hsp is small in this preliminary study, this result suggests that celiac screening may be considered in children with hsp. (14) groups. the glomerular and tubulointerstitial lesions were scored by katafuchi criteria and oxford classification of igan,respectively. results:1.the baseline data of different clincal or pathological groups were no significant differences (p more than 0.05),but 24h protienuria were significant increased in grade 3 and 4 groups than grade 2 group(p less than 0.05).2.according katafuchi critiria,the severity scores of glomerullar and tubulointerstitial lesions were positively correlated with either different clincal or pathological groups(p less than 0.01).3.according to oxford classification, only the severity scores of mesangial hypercellularity and segmental glomerulosclerosis/adhension were positively correlated with different clinical groups(p less than 0.01),but the severity of mesangial hypercellularity, endocapilarity hypercellularity, segmental glomerulosclerosis/adhension,tubular atrophy/interstitial fibrosis and cellular/fibrocellular crecents were all positively correlated with different pathological groups (p less than 0.01). [1990] [1991] [1992] [1993] [1994] [1995] [1996] [1997] [1998] [1999] [2000] [2001] [2002] [2003] [2004] [2005] [2006] [2007] [2008] [2009] were evaluated retrospectively. patients were analyzed comparing those hospitalized during the first and the second ten-year period.the long-term prognosis was evaluated in 60/64 patients (94%) with hsp nephritis followed-up for 1 to 11 years (mean 5.7±3.6 years) based on urine analyses, serum creatinine and blood pressure measurements. results: of 156 patients (mean age 6.9±3.5 years, range 1-17 years, m: f ratio 1.0) with palpable purpura (100%), other symptoms were more common during the first ten-year period including arthralgias (62% vs. 52%, p=0.282), gastrointestinal symptoms (63% vs. 44 %, p=0,018) and renal involvement (55% vs. 21%, p=0.001). among 64 patients (41%) with hsp nephritis, 42 had isolated hematuria and/or non-nephrotic range proteinuria (66%), 15 nephritic syndrome (23%) and 6 nephritic/nephrotic syndrome (10%). hematuria and/or nonnephritic proteinuria was less common (p=0.0005) in the second tenyear period while the incidence of nephritic/nephrotic syndrome was insignificant (p=0.807). recurrences of purpura had 11 patients within 2 years. 6 patients had renal biopsy including one with 30% of crescents and one another with iga nephropathy diagnosed seven years after initial presentation of hsp. long-term prognosis was favourable in most patients with abnormal urine analyses in 12 (20%), in one with hypertension but normal serum creatinine and in all with nephritic/nephrotic syndrome at disease onset. conclusion: clinical evaluation of patients with hsp nephritis treated at our institution showed a good long-term prognosis. urine and blood pressure abnormalities in followed-up patients were associated with nephritis at disease onset. -up (f-up) . egfr was estimated using schwartz formula; decline in renal function was defined as the slope of egfr over the f-up. results: 72% were males, with mean age at renal biopsy of 12.7 ± 3.7 y and a median f-up of 4.7 y (iq range 2.4-7.8 y); more than 80% presented with egfr > 90 ml/min/1.73m 2 . end-stage renal disesase (esrd) was reached by 4% of children, 50% loss of initial egfr by 5%; 7% reached the combined end point (esrd or 50% loss of initial egfr). at renal biopsy 57% presented with mesangial proliferation (m1), 24% with endocapillary proliferation (e1), 35% with segmental glomerulosclerosis (s1) and 9% with tubular atrophy/interstitial fibrosis (ta/if; t1/2). patients with segmental sclerosis and ta/if showed a significantly worse egfr slope over the f-up (s0 vs s1 p=0.04; t0 vs t1/2 p=0.003). at univariate linear regression, clinical data at renal biopsy (egfr, proteinuria and map) were not associated with renal function decline, while data at 6-12 and 12-24 months and ta-proteinuria and map significantly predicted egfr slope.a multivariate linear regression model (including proteinuria and map at 12-24 months together with the difference of egfr at renal biopsy and at 12-24 months as independent variables) performed well in predicting egfr slope (r 2 =0.39). this model was used to derive a formula able to estimate egfr slope with good performance (mean bias between estimated and really observed egfr slope of 0.05 ± 6.6 ml/min/1.73m 2 ). conclusion: the oxford classification of igan was well applicable to this pediatric population. a formula was developed that estimates renal function decline over the f-up based on proteinuria, map and egfr loss after 1-2 years of observation, which will need a validation on other cohorts. sixteen-year experience with pediatric iga nephropathy and validation of the oxford classification as a risk predictor (m1), segmental sclerosis (s1), endocapillary proliferation (e1), and tubulointerstitial fibrosis (t1 or t2) was 32 (35.2%), 15 (16.5%), 9 (9.9%), and 6 (6.6%), respectively. there was no significant decrease in gfr within the entire cohort. however, five patients showed decreased renal function, and of them, two reached stage iii or iv ckd. the five patients' changes in the k/doqi ckd stage were 2 to 4, 1 to 3, 1 to 2, 1 to 2, and 1 to 2. their pathological classifications were haas 4/m1 s1 e0 t0, haas 4/m1 s0 e1 t1 with global sclerosis, haas 1/m0 s1 e0 t0, haas 1/m0 s0 e0 t0, and haas 1/m0 s0 e0 t0 with global sclerosis. in the oxford classification, global sclerosis was the only factor correlated with decreased renal function, whereas the haas classification showed no significant correlation with renal function. conclusions: global sclerosis was correlated with decreased renal function. however, in our cohort, there was no significant decrease in renal function during the follow-up period (p = 0.216). thus, we could not clearly correlate the haas and oxford classifications with renal outcome. the value of the oxford classification as a predictor of renal outcome remains unclear in korean children with iga nephropathy. meng-jie jiang, xiao-yun jiang, ying mo, li-zhi chen, li-ping rong pediatric, the first affiliated hospital of sun yat-sen university, guangzhou, china objective: to analyze the clinicopathological characteristics of primary immunoglobulin a nephropathy (igan) manifested as macroscopic hematuria in children. the clinicopathological characteristics of 48 cases with primary igan manifested as macroscopic hematuria were analyzed retrospectively. according to the lasting time of macroscopic hematuria, 15 cases were assigned to group a (no more than seven days) and 33 cases were assigned to group b (more than seven days). results: the manifestations comprised: acute glomerulonephritis (40% in group a, 33.3% in group b), nephrotic syndrome (20% in group a, 15.1% in group b), hematuria and proteinuria (6.7% in group a, 12.1% in group b) and isolated hematuria (33.3% in group a, 39.4% in group b). 20.0% cases in group a and 21.2% cases in group b were accompanied with abnormal renal function. there was no significant difference between group a and b (p>0.05). the urinary lysozyme or β2-mg increased (6.7% in group a, 30.3% in group b). in group a, subclass ii was the most common histopathological type (53.3%), followed by iii (33.3%) and i (13.3%) while the predominant histopathological types in group b were subclass iii (45.5%), iv (27.3%), ii (21.2%) and i (6.1%). none in group a and 15.2% cases in group b showed crescent. besides, only one glomerulosclerosis was in group a while 8 (24.2%) cases of glomerulosclerosis were in group b. the difference between group a and group b was statistically significant (p<0.05).no differences were found in balloon stenosis between group a (6.7%) and group b (12.1%), as well as the renal tubular and interstitial damage (33.3% in group a, 39.4% in group b). 46.7% cases in group a and 60.1% cases in group b were found with simple iga deposition in mesangial area. there was no significant difference between group a and group b (p>0.05). the children with primary igan manifested as macroscopic hematuria lasting for more than seven days are easily appearing renal tubular damage, crescent formation and glomerulosclerosis. subclass iii and iv are the most common histopathological types. a case report of iga nephropathy accompanying crohn's disease akihiko shirasu 1 , akira ashida 2 , hideki matsumura 2 , hyogo nakakura 2 , tomoki aomatsu 2 , atsushi yoden 2 , motoshi hattori 3 , hiroshi tamai 2 , 1 pediatrics, hirakata city hospital, hirakata, japan 2 pediatrics, osaka medical college, takatsuki, japan 3 pediatric nephrology, tokyo wemen's medical university, tokyo, japan there have been several reports of iga nephropathy accompanying crohn's disease in which the clinical course of the two diseases was linked. we recently experienced a case of iga nephropathy with deterioration of renal function complicated by crohn's disease. a 15year-old boy diagnosed as having crohn's disease underwent total colectomy at the age of 7 years. the patient was referred to pediatric gastroenterology unit at the age of 13, and thereafter maintained a state of remission form crohn's disease while receiving 5-aminosalicylic acid (5-asa). two months prior to referral to our pediatric nephrology unit, when the patient was 15 years old, a school health examination had revealed microscopic hematuria and mild proteinuria. at that time, laboratory tests suggested deterioration of renal function. renal biopsy was performed and histological examination by light microscopy demonstrated focal segmental mesangial proliferation and moderate tubule atrophy with a diffuse interstitial inflammatory infiltrate consisting predominantly of lymphocytes. immunofluorescence assays revealed 2+ diffuse granular staining for iga and 1+ staining for c3 in the mesangium. therefore, the patient was diagnosed as having iga nephropathy and chronic interstitial nephritis. the 5-asa therapy was stopped considering the possibility that it had induced interstitial nephritis. the renal function of the patient has since remained quiescent for 7 months. patients with crohn's disease who present with abnormal urinalysis findings commonly have urological complications, such as urolithiasis and urinary tract infections. however, the possibility of renal parenchymal disease including iga nephropathy should also be considered, even if there is no apparent aggregative linkage of urinalysis findings with gastrointestinal symptoms of crohn's disease. the duration of heavy proteinuria determined long-term outcome of henoch-schönlein purpura nephritis in children results: among the 9 patients, 7 (77.78%) cases presented with hematuria and nephrotic syndrome (ns); 1 (11.11%) with hematuria and nephrotic range proteinuria (>50mg/kg/24h); 1 (11.11%) with hematuria and moderate proteinuria (25~50mg/kg/24h). the typical pathological features, such as the diffuse glomerular mesangial and endocapillary proliferation, mesangial interposition, and double contour formation, were shown in all specimens. moreover, the podocyte hypertrophy, shedding, and cytoplasmic absorption dropletswere also observed in most specimens under light microscopy. the percentage of small cellular crescents varied from 4.25-25%. 9 patients were followed up for 2 to 4 years, and all had recovered. conclusion: the predominant clinical manifestation of iskdc grade vi hspn in children was ns accompanying with hematuria. the well outcome might be associated with the prompt use of steroids and/or immunosuppressive drugs at the onset of iskdc grade vi hspn with very mild glomerulosclerosis and tubulointerstitial lesion. in our future study, all patients should be followed for five or ten years, and more specimens should be observed under electron microscope to investigate whether the podocyte lesions of iskdc grade vihspn should be taken into account in the clinical pathological report and a future new histologic classification of hspn. objective: the aim of the study was to assess efficacy of immunosuppressive treatment in children with iga nephropathy (igan) and henoch-schoenlein nephritis (hsn) based on clinical manifestation and histopathological oxford (o-c) and who (who-c) classifications. the study group consisted of 18 children with igan-9 and hsn -9, (mean age 10.44 ±3.94 years) , treated with azathioprine (aza) 2 mg/kg/day -12 months with prednison (pred) 2mg/kg/day (nephrotic syndrome-ns), 1mg/kg/day (nephritic syndrome -nps) 8 weeks, gradually diminished within 15 months. it recognize clinical remission at normal renal function and absence of proteinuria at the end of treatment. in all patients we estimated: proteinuria, gfrs (calculated using schwartz's formula) at the onset of study and after 15 months. they had performed renal biopsy on average 0.69 ± 0.95 years after the onset of illness and at the end of treatment. we were analyzed the histopathologic evaluation of renal biopsy specimens using the who-c (grade i-v) and oxford classification : (mmesangial hypercellularity, e-endocapillary hypercellularity, ssegmental sclerosis, t-tubular atrophy/interstitial fibrosis; absent =0, present=1). results: in patients with igan we observed: ns-1, nps-8; with hsn : ns-7, nps-2. after the treatment aza/pred we noted clinical remission in all children with igan and 89% with hsn. gfr was normal before and after treatment. at the onset of illness igan patients had: grade iii-6, grade iv-3 according to who-c; hsn had: grade ii -2, grade iii-6, grade iv -1; at the end of therapy we observed positive effect in who-c only in 2 (22%) igan patients and in 5 (56%) hsn , without progression properly 6 (67%) and 4 (44%). in o-c of igan patients we observed nobody with total regression of changes, only 3 children had m0 or e0 or s0 after treatment. in patients with hsn we noted total regression of changes in 4 (44%), in other 3-m0 or e0 or t0. conclusion: aza/pred therapy used in children with igan and hsn can make clinical remission in both groups, but histopathological regression-more frequent in hsn. oxford and who classifications are very useful for estimation of efficiency of treatment. 10-year follow-up of pediatric hennoch schönlein purpura with renal involvement elena tudorache 1 , christine azema 1 , stéphane decramer 2 , georges deschênes 3 , tim ulinski 1 1 pediatric nephrology, armand trousseau hospital, paris, france 2 pediatric nephrology, chu toulouse, toulouse, france 3 pediatric nephrology, robert debré hospital, paris, france objective: the aim of this study was to determine the long-term outcome in children with hsp nephritis who underwent a renal biopsy and to identify possible correlation between disease parameters and treatment. we included retrospectively all patients with renal biopsy proven iga nephropathy related to hsp of three pediatric nephrology centres over a 10-year period. results: 142 patients were included. nephrotic range proteinuria was present in 28% with grade ii, 60% with grade iii and 90% with grade iv lesions. renal function was impaired in 14%. significant proteinuria >500 mg/l was found in 9/48 patients 3 years post renal biopsy, in 8/25 patients at 5 years and in 3/14 patients at 10 years. there was no correlation between risk for proteinuria at 3, 5, or 10 years with initial histological lesions. there was a tendency to higher residual proteinuria in patients with nephrotic proteinuria at disease onset and also for patients who were not treated with steroid pulses at disease onset with 310 vs. 175 mg/l (p=0.06) at three months, 100 vs. 50 mg/l (p=0.93) at one year, 720 vs. 150 mg/l (p=0.1) at five years, and 520 vs. 355 mg/l (p=0.35) at ten years. there was a tendency to less proteinuria in the long term for those with early steroid pulses (<15 days vs. >16 days after proteinuria onset (p=0.16).in patients with acei/arb treatment within 15 days after renal disease onset, compared to those who were treated later (>one month), proteinuria was significantly lower at 6 (p=0.02) and 12 months (p=0.03). among 18 control kidney biopsies, 50% show fibrosis of different degree unrelated to initial histology. conclusion: there is a risk to underestimate long term disease severity in patients with low iskdc classes. there is need for prospective long term studies to explore the benefit of early acei/arb and/or steroid pulses. abstract# p-sat147 good outcome of biopsy-proven henoch-schonlein purpura nephritis in children in shanghai single center objective: severe hsp nephritis considered a risk factor for chronic kidneys disease development especially in patients with nephrotic syndrome (ns). methods: we present our experience with tacrolimus (tac) as a steroid sparing agent for a treatment of severe hsp nephritis in 17 years boy. our patient has been observing at the republic center of pediatric nephrology minsk for 5 years. he presented with a typical purpuric rash with recurrences after infections. one year later during acute pneumonia macrohematuria, proteinuria (1-1.5 g/24h) occured and ace inhibitors (iace) were prescribed. despite this, boy developed nephrotic range proteinuria (3.8-7.7 g/24h), biochemical protein 52g/l, albumin 27g/l, cholesterin 6,5mmol/l. renal biopsie showed mesangial proliferation with iga deposits. prednisolon (pred) 80mg/daily with heparin failed to decrease proteinuria and activity. cyclosporin a 200 mg/daily plus pred on alternate day were started and followed during 8 months with positive dynamic (proteinuria 1,7g/24h, decreased clinical and laboratory activity) after which pathogenic treatment was stopped. arterial hypertension (ag) was treated by iace. 6 months later proteinuria (3.5g/24h) and activity increased again. with the presence of ns resistance to steroids combined with hematuria and ag, medrol (64mg/daily) plus tac 1 mg twice daily were prescribed. duration of tac therapy was 5 months. tac was succesfully withdrawn with no rebound disease activity observed (during 9 months). at present the patient receiving iace only. our results indicate that tac may be a promissing steroid sparing agent for treatment of severe hsp nephritis. anti-proteinuric effect of cyclosporine a treatment for iga deposit diseases objective: to evaluate the therapeutic effect of cyclosporine a (csa) on nephrotic-range proteinuria in children with immunoglobulin a (iga) deposit diseases in mesangium such as iga nephropathy (igan) and henoch-schonlein purpura nephritis (hspn). methods: fifty four children (36 children with igan, 18 children with hspn) who were diagnosed with iga deposit diseases in mesangium at renal biopsy were analyzed retrospectively. all the patients developed nephrotic-range proteinuria (> 40 mg/m2/day) for more than 3 months. the starting dose of csa was 5 mg/kg per day that was given in two divided doses, and the drug level was maintained between 100 and 200 ng/ml. the degree of proteinuria was measured before and after csa treatment. steroids were tapered off and stopped gradually after initiation of csa. results: mean duration of csa treatment was 10.7 +/-5.6 months (range 1.4 -32.7 months). mean follow-up duration was 3.7 +/-3.1 yr (range 0.7 -15.2 yr) from the beginning of csa treatment. the mean ratios of protein to creatinine decreased from 3.7 +/-1.5 to 0.6 +/-0.4 after csa treatment for 12 months. thirty-two (59.2%) patients achieved complete remission. renal function was preserved in all patients 12 months after csa treatment. there were no severe complications in patients with csa treatment. conclusion: our findings indicate that csa is an effective agent to treat nephrotic-range proteinuria of iga deposition glomerular diseases such as igan and hspn and the duration of csa treatment should be at least more than 3 months for efficacy. objective: to observe the therapeutic effects of hemoperfusion combined with corticosteroid in treating children with henoch-schonlein purpura. methods: a total of 180 patients (101 inthe hemoperfusion group (hp) and79 inthe control group) were included and followed up for 12 months. both groups were treated with corticosteroids and other supportive therapy. patients in the hp group received 2 hours of hemoperfusion each day for 3 consecutive days. clinical features at the acute phase, relapses in a year and renal involvement at 1, 3, 6, 12 months were compared. the blood levels of iga and such cytokines as, tnf-alpha-il-1, il-6, ltb4 were measured by elisa technique before and 1, 2, 3 days after hemoperfusion. results: hemoperfusion significantly reduced the severity and duration of abdominal and joint pains. for patients with renal involvement at acute phase the hp group had quicker relief than the control (log rank p=0.000). for patients without renal involvement at acute phase the hp group had less occurrences of renal involvement compared with the control during follow-ups(log rank p=0.001). the rash was also alleviated after hemoperfusion though the difference was not significant. patients' levels of iga were elevated compared with the healthy control before treatment ( day0) patients'levels of cytokines also significantly increased compared with the healthy control on day0, they began to decrease 1 day after hemoperfusion and there were significant differences among those groups. less relapses were seen in the hp group compared with the control (p=0.011). conclusion: hemoperfusion combined with corticosteroid was more effective than corticosteroid alone in improving extrarenal symptoms, alleviating and preventing renal involvement. the effects may be achieved by iga and cytokine reduction after hemoperfusion. the treatment of henoch-schonlein purpura nephritis in children combined with mycophenolate mofetil and corticosteroid nephritic syndrome type, 13 cases(39.4%). about histological degrees, 32 cases were in grade 3 (mesangial proliferation, all accompanied by <50% crescent formation), 1 case was in grade 5 (moderate to severe mesangial proliferation, accompanied by 88% crescent formation). treated with new therapy, 30 cases were complete remission (91%), 2 cases were improved, and one died. the complete remission rate of this therapy was significantly higher than that of the past treatment. objective: steroid pulse therapy (spt) has been reported to be effective for improving urinary abnormalities and preventing renal deterioration in patients with iga nephropathy. however, some patients are refractory to spt or have relapse after spt, resulting in renal impairment. the efficacy of tonsillectomy combined with spt has been discussed. the aim of our study is to evaluate the efficacy of tonsillectomy combined with spt in childhood iga nephropathy. methods: 30 children, aged 3 to 14 years, who had been followed up for more than 3 years after the first biopsy between 2001 and 2009, were enrolled in this study. all patients received spt (methylprednisolone, 20~30 mg/kg intravenously 3 times per week for consecutive 3 weeks). 8 patients received tonsillectomy combined with spt (group a), and 22 patients received spt alone (group b). tonsillectomy was performed one month before or after spt. the therapy was followed by oral prednisolone and tapered off for 12 to 24 months, with warfarin, dipyridamole and ras inhibitor. all of 30 patients underwent repeat biopsy. clinical features and pathological findings were retrospectively analyzed. results: the disappearance rate of proteinuria was 87.5% for group a vs 100% for group b at 1 year, and that of hematuria was 100% vs 72.7% at 2 years. for group a, all the cases which of urinary abnormalities had improved by the treatment could maintain remission, but for group b, 27.3% of the cases had recurrence of proteinuria and 18.2% had recurrence or did not achieve disappearance of hematuria at the last observation. histlogically, the percentage of glomeruli that showed crescents was significantly reduced in both groups. but the cases which still showed crescent formation at the second biopsy were 0% for group a vs 22.7% for group b. conclusion: the differences of recurrence rate for urinary abnormalities after the treatment and disappearance rate of crescents at second biopsy may suggest that tonsillectomy can improve the long-term prognosis. objective: to observe the efficacy and security of tripterygium wilfordii polyglycosidium (twp) in treating children with severe henoch-schonlein purpura nephritis (hspn). methods: 25 children failed to intravenous pulse methylprednisolone for 6 days for their severe hspn entered our study and were given oral twp for 3-6 month. we evaluated the efficacy and security of twp through examinations of urinalysis, blood routine, function of liver and renal, myocardial enzyme series, level of sex hormone and electrocardiogram (ecg) before treatment, 1-2 month after giving twf and 4 weeks after stopping medication respectively. results: proteinuria disappeared in all patients. the remission time was 4 weeks in 22 children, 12 weeks in 2, 15 weeks in 1 respectively. during treatment, elevated glutamic-pyruvic transaminase and glutamic-oxaloacetic occurred in 2 patients and returned to normal after stopping medication. abnormal findings in examination of ecg were observed in 2 patients, one with left ventricular high voltage, another with knot parallel rhythm of the heart. after stopping medication, one patient still had t wave change in ecg. we didn't find any abnormalities of blood routine, renal function and level of sex hormone. conclusion: the twp has good efficacy and security on the treating children with severe hspn. the efficacy and side effects of glucocorticoids in treating children with henoch-schonlein purpura kai-yun liu, ling lu pediatrics, irst affiliated hospital of anhui medical university, anhui, china objective: to observe the efficacy and safety of glucocorticoid in the treatment of children with henoch-schonlein purpura. methods: 50 patients with hsp were divided into mild and severe group according to clinical manifestations. 30 children in mild group were given hydrocortisone sodium succinate (hcss) therapy. 20 children in severe group were given methylprednisolone ( mp ) therapy. a follow-up record system was set up for every patient. we observed the recovery time of clinical symptoms and side effects of glucocorticoid. results: symptoms of skin rashes, joint and gastrointestinal in all children were disappeared within 3 months of treatment. the urinalysis in 13 of 14 patients with renal involvement returned to normal in 4 weeks. the other patient failed to mp therapy was given oral tripterygium and got normal urinalysis in 8 weeks after disease onset. the body mass index (bmi) values in all children after glucocorticoid treatment were significantly higher than before treatment. after stopping medication, there was no significant difference in bmi value compared with before treatment and between two groups. during the treatment, 1 patient in each group had ecg abnormal, and their ecg returned to normal after stopping drug. we didn't find any abnormalities of blood routine, renal function and level of sex hormone. objective: to investigate the role of th17/treg imbalance in the pathogenesis of childhood henoch-schonlein purpura(hsp). and further explore the immunomodulatory affects of compound/lycyrrhizin(gl) on the th17/treg deviation in hsp. methods: 31 hsp patients were chose as gl-treated group, another 5 patients were used as conventional therapy group. besides (0.5-2)ml/kg/l was enjected in l-treated group, other treatments were same, any steroid or immunosuppressants treatment was prohibited. 15 age and sex-matched healthy children were used as healthy controls. blood samples of patients were obtained at the acute stage and after 5 days treatment. using intracellular staining, the frequency of peripheral cd3+cd8-il17+(th17) and cd4+cd25+foxp3+ treg cells was detected by flow cytometry all patients received methyl-prednisone pulses iv (1 g/1.73 m2 for 3 consecutives days) and subsequently oral prednisone with gradual withdrawal in six months, and mmf at dose of 10-20 mg/kg/12 h for 24 months. results: after six months of therapy all patients but one had persistent microscopic hematuria (me), only 3/11 patients had recurrent ma. patients with grade ii hspn showed significant reduction or normal proteinuria. only 3/11 patients with grade iii hspn had proteinuria still >1gr/24 h, 4/11 patients had proteinuria <1g/24 h, in 4/11 proteinuria was normal. mean period of follow-up was 3 years. after one year, only 1 patient had recurrent ma, 10 patients showed persistent me. two patients with persistent proteinuria >1 g/24 h showed a significant reduction (< 1g/24 h in 2 pts, normal in 1 pt). proteinuria did not reappear in all patients.the effect persisted after 2 years and after therapy withdrawal. no side effects was recorded. conclusion: our experience shows the efficacy and safety of mmf in hspn and has to be confirmed by larger controlled studies. the analysis of blood perfusion therapeutic effect on 45 cases of allergic purpura cheng guo-qiang xi 'an children's hospital urology department, xi'an, shanxi, china objective: to observe the auxiliary therapeutic effect on severe allergic purpura. methods: 45 children with severe allergic purpura were randomly divided into the control group (19 cases) and the treatment group (26 cases). the children of control group were treated with conventional therapy (anti-inflammatory, anti-allergic, anticoagulation and symptomatic treatment). the children of treatment group were treated with conventional therapy and blood perfusion. two groups of patients were both treated for 14 days, and were followed up for 9 months. the amelioration time and the regression time of clinical symptoms such as abdominal pain, bloody stool, rash, joint symptoms, visible hematuria and proteinuria, was compared in two groups before and after the treatment. the amelioration time of the clinical symptoms of children in the treatment group, such as acute abdominal pain, bloody stool, rash, joint symptoms, abdominal pain and bloody stool, visible hematuria, proteinuria, and other clinical symptoms, was shorter than the time in the control group (p < 0.05), and the late symptoms such as joint symptoms, abdominal pain and bloody stool iterations was also less than those of the control group (p < 0.05). methods: iga nephropathy model was established in male sprague-dawley rats. at week 10, rats in the model group were randomly assigned to either remain in the model group (n=5), or to receive treatment with rednisone (n=6), 1,25(oh) 2 d 3 group (n=6), or prednisone plus 1,25(oh)2d3 (n=6). at week 12, serum interleukin-17 level was detected by elisa, and the level of treg cells in the blood was assayed using a flow cytometry method. results: (1) the proteinurine and the number of blood cells in urine from rats in the model group was significantly higher than in rats in the control, prednisone, 1,25(oh)2d3, and prednisone plus 1,25(oh)2d3 treatment groups (p < 0.01). (2) serum interleukin-17 in the model group was significantly increased compared with control and treatment groups (p < 0.01), and the levels were decreased in turn in the prednisone treatment group, 1,25(oh)2d3 treatment group, and prednisone plus 1,25(oh)2d3 treatment group. in addition, the 1,25(oh)2d3 and prednisone plus 1,25(oh)2d3 treatment groups showed lower levels than the prednisone treatment group (p < 0.05). (3) the level of treg cells in the model group was significantly decreased compared with the control and treatment groups (p < 0.01), with the levels showing a slight increase in the prednisone treatment group, a larger increase in the 1,25(oh)2d3 treatment group, and the greatest increase in the prednisone plus 1,25(oh)2d3 treatment group when compared with the model group. furthermore, the 1,25(oh)2d3 group and prednisone plus 1,25(oh)2d3 treatment groups showed higher levels than the prednisone treatment group (p < 0.01). conclusion: a th17/treg disorder exists in rats with iga nephropathy, with the levels of interleukin-17 increased and the levels of treg cells decreased. vitamin d3 can regulate the th17/treg balance and reduce the level of protein and blood in the urine in rats with iga nephropathy. objective: to explore the therapeutic effect of prednisone (pred) combined with mycophenolate mofetil (mmf) and cyclosiporin a(csa) in children with severe purpura nephritis (hspn). methods: we collected 6 patients with severe hspn (from iskdc iiia to i) whose proteinuria symptom of nephrotic syndrome did not significantly relieve after 4 weeks which treated with oral pred and mmf (20-30 mg/(kg.d)); and (or) the gross hematuria did not disappear after 2 courses which treated with large doses of methylprednisolone. all patients were treated with oral csa 2-4mg/(kg.d) to induce remission therapy for 3-6 months, then gradually reduced csa to 1-3 mg/(kg.d). the follow-up was 8-30 months. results: combined therapy with immunosuppressant treated a month later, gross hematuria were disappeared in all 5 patients, 1 patient (17%) was partial remission (urinary protein and urine erythrocyte continued to decrease 25%-49% than before),5 patients (83%) were significantly remission (urinary protein and urine erythrocyte continued to decrease over 50% than before), 24-hour urinary protein was 30.50±19.35mg/kg, urine erythrocyte was 15.23±11.39×10 4 /ml.3 months later, 5 patients (83%) were significantly remission, 1 patient was complete remission (urinary protein was completely negative, urinary sediment red blood cells <8000/ml), 24-hour urinary protein was 18.67±10.10mg/kg, urine erythrocyte was 10.71±8.55×10 4 /ml. 6 months later, 5 patients (83%) were significantly remission, 1 patient was complete remission, 24-hour urinary protein was 7.66±6.31mg/kg, urine erythrocyte was 5.18±3.81×10 4 /ml. 4 patients were complete remission at last and the median duration of complete remission was 8.5±4.93 months. 2 patients had not been reached complete remission, which follow-up was 8-9 months. 3 patients were relapse during withdraw or reduction, other side effects included hairy (4 patients objective: increasing evidence suggest that the cosmc(1-beta-3galactosyltransferase-specific molecular chaperone ) might play a vital role in the pathogenesis of iga nephropathy (igan). however, the mechanism is not clear. methods: we investigated whether cosmc gene( c1galt1c1 )methylation is an important mechanism in igan in children. methods 33(m:f=25:8) primaryigan children and 39(m:f=28:11) healthy controls were enrolled randomly. the methylation status of the c1galt1c1 was detected using bisulfite-specific polymerase chain reaction (bsp)-based sequencing analysis. results: the male patients had obviousely high methylation percentage than the male controls(p=0.047). three significant hypermethylation sites were identified in the male patients: cpg site 20 (p = 0.034), site 21 (p = 0.039), andsite 26 (p = 0.047). in the female groups,the patients and the normal controls all had high level methylation without significance (p=0.947). conclusion: methylation of c1galt1c1 might have the vital significance in the susceptibility to the male igan patients. objective: the aim of this research is to investigate the value of serum and urinary tgf-β1and mmp-9 level, the noninvasive and easy repeatable biomarker, for assessing the severity and disease progression in igan children. methods: 54 children with biopsy-proven igan and 55 normal children(control group) were enrolled between july 2009 and january 2013.(fingure 1)the igan group was divided into 3 clinical groups according to their clinical features: isolated hematuria group (ih group, 20 patients), hematuria and proteinuria group (hp group, 16), and nephritic syndrome group (ns group, 18). patients were divided into two groups according to their lee's pathologic classification: grade i+ii (lee's i+ii, 39 patients), grade iii+ iv (lee's iii+ iv ,15) groups. igan group was classified according to the renal function into two groups(renal failure group vs normal group). igan group was also divided into four groups according to their immunophenotype: iga(7patients),iga+igg(9),iga+igm(10),iga+igg+igm(28) group. results: the level of tgf-β1 and mmp-9 inserum and urinary of the igan group was significantly higher than that in the control group (p<0.05). both the level of tgf-β1 and mmp-9 inserum and urinary of the three clinical groups in the igan children were not statistically different. the serum tgf-β1 and mmp-9 levels were significantly higher in lee' s iii+ iv group compared to that in lee's i+ii group(p<0.05). the difference was not statistically significant between urinary tgf-β1 and mmp-9 levels in the two lee's group.the serum tgf-β1 and mmp-9 levels of renal failure group were significantly higher than that of normal renal function group in igan children(p<0.05),and the urinary tgf-β1 and mmp-9 levels of the two group were not statistically significant different. the serum and urinary tgf-β1 and mmp-9 levels were not statistically different in all immune classification. conclusion: the serum tgf-β1 and mmp-9 plays an important role in the severity and progression of children with igan. tgf-β1 and mmp-9 may be two more noninvasive and easy repeatable biomarkers to be used in evaluating the progression of igan in children. abstract# p-sat163 glomerular ace2 expression is enhanced in pediatric iga nephropathy yusuke seki, maki urushihara, takahiro tayama, takashi nagai, ariunbold jamba, shuji kondo, shoji kagami department of pediatrics, the university of tokushima graduate school, tokushima, japan objective: angiotensin converting enzyme (ace)2 is a homolog of ace and is thought to be a potent counter-regulator against ace activity. while renin-angiotensin system (ras) plays a critical role in the progression of iga nephropathy (igan), the role of ace2 has not been investigated in pediatric patients with igan. this study was performed to examine the relationship between ace2 expression and the development of pediatric igan. methods: we performed immunohistochemical analysis of ace2 and ace in kidney tissues from 39 patients with pediatric igan and 14 patients with minor glomerular abnormalities (mga) using specific antibodies and examined the correlation with clinical parameters and pathological findings. in addition, we elucidated the effects of various cytokines on ace2 expression in cultured human mesangial cells (mcs) by quantitative real time pcr and western blot analyses. results: ace2 expression levels in glomeruli (r = 0.5732, p < 0.0001) and tubules (r = 0.4917, p = 0.0002) were positively correlated with the mesangial hypercellularity score, while ace expression levels in glomeruli (r = 0.1331, p = 0.3420) and tubules (r = 0.0743, p < 0.5959) are not. multiple regression analysis showed that the mesangial hypercellularity score correlated with ace2 expression level in glomeruli and urinary protein-creatinine ratio (r = 0.6295, p < 0.0001). in igan patients not treated with a ras blocker (ace inhibitors or angiotensin ii type 1 receptor blockers), ace2 expression levels in glomeruli were significantly increased compared to patients with mga (p < 0.0001). iga patients treated with a ras blocker did not show this increase in ace2 exrepssion. furthermore, ace2 mrna and protein expression was enhanced by interleukin-1 beta, a pro-inlammatory cytokine, and suppressed by transforming growth factor-beta 1 , a pro-fibotic factor in the progression of igan. conclusion: these data indicate that ace2 expression in glomeruli is associated with mesangial hypercellularity in the active phase of pediatric igan. abstract# p-sat164 increased serum interleukin-17 and peripheral th17 cells in children with henoch-schonlein purpura nephrology (hspn) xiaoshan shao nephrology, guiyang children's hospital, guizhou, china objective: interleukin (il)-17 and th17 cells have been involved in many autoimmune diseases. the aim of this study is to investigate the involvement ofil-17 and th17 cells in the pathogenesis of childhood henoch-schonlein purpura (hspn). methods: serum and supernatant levels ofcytokines and chemokines were analyzed by enzyme-linked immunosorbent assay (elisa). using intracellular staining, the frequency ofperipheral th17 and th1 cells was studied by flow cytometry. results: children with hspn had significantly higher serum levels of il-17, il-6and transforming growth factor-beta than healthy controls. the il-17 levels in culture supernatants of peripheral blood mononuclear cells with anti-cd3 and cd28 antibody stimulation were much higher in patients with hspn (212.2 +/− 71.4 vs. 34.7 +/− 12.6 pg/ml, p = 0.021). thepatients also had more th17 cells (1.47 +/− 0.23% vs. 0.61 +/− 0.10%, p = 0.012) but not th1 cells in peripheral blood. moreover, il-17 could promote human endothelial cells to produce chemoattractants il-8 and monocyte chemotactic protein-1. the increased frequency of peripheral th17 cells and serum il-17 levels are shown in childhood hspn that may in part contribute to vascular inflammation, suggesting cellular immunity is likely to be involved in the process of hspn. expression and significance of tgf-beta, smad7, fn, ub and smurf2 in the renal tissues of children with iga nephropathy chen li-zhi, jiang xiao-yun, ling yi-hong, mo ying, sun liang-zhong pediatrics, the first affiliated hospital of sun yat-sen university, guangzhou, china objective: in this study, we investigated the expression of tgf-beta 1, smad7, snon, fn, ub and smurf2 inthe renal tissues in children with igan, in order to explore the potential cross-talk between ubiquitinproteasome pathway and tgf-beta 1 signaling and even their role in the progressive renal fibrogenesis of igan. methods: sixty children with igan were divided into three groups according to their clinical features: isolated haematuria group (ihgroup, 20 patients), hematuria and proteinuria group (hp group, 21), and nephrotic syndrome group (ns group, 19) . patients were also divided into three groups according to pathologic grades: grade 2 (26 patients), grade 3 (20) and grade 4 (14) groups. six normal renal specimens (four was non-tumor kidney tissues from patients who had renal tumor and underwent nehprectomy, another two was healthmismatched donor renal tissues before renal transplantation) were used as the control group. the expression of ub, smurf2, tgf-1, smad7, snon, and fn in renal tissues were determined by immunohistochemistry (two-step powervisiontm) and semi-quantitatively analyzed by the software of image-pro plus 6.0. the degrees of glomerular and tubulointerstitial lesions were scored according to the katafuchi semi-quantitative criteria. results: the expression of tgf-beta 1, fn, ub and smurf2 inigan kidneys was significantly higher than that in the control group(pless than 0.01), while the increased expression of smad7 was only found in glomeruli but not in renal tubular interstitium(p less than 0.01). the highest expression of tgf-beta 1, fn, and ub were found in the ns and grade 4 groups(p less than 0.01), smurf2 in the ih and grade 2 groups(p less than 0.01), while the lowest expression of smad7 was found in the grade 4 group(p less than 0.01), closely associated with different clinical and pathological groups. (p less than 0.01) conclusion: these results demonstrate that upp and tgf-beta 1/smads signaling pathway were both activated in children with igan.both upp and tgf-beta 1/smads signaling pathway may play an important role in the progress of glomerular sclerosis, renal tubular injury and renal interstitial fibrosis in children with igan. abstract# p-sat166 objective: the proteasome (ps) plays a key role in the activation of transcriptional factors, cytokines and presentation of hla-i restricted peptides. in immune mature cells, particularly dendritic cells, the ps, under the action of interferon gamma and alpha, becomes an immunoproteasome (ips), by substituting 3 catalytic units beta1, beta2, beta5 with other low molecular weight proteins (lmp2 and lmp7) and an endopeptidase-like complex (mecl-1). this modification confers an optimal catalytic property for professional presentation of specific peptides to mhc class i. our group previously demonstrated that adult patients with iga-nephropathy (igan) have an increased expression of ips catalytic subunit, which correlated with the severity of renal disease.aim of this study was to investigate the switch from ps to ips in children with primary igan and with henoch-schoenlein purpura (hsp), a vasculitis sharing with igan several immune system abnormalities. methods: peripheral lymphomonocytes (pbmc) from 18 children with igan, 57 with hsp and 33 healthy control subjects (hc), isolated by centrifugation gradient, were tested with real time prc (taqman) to assess quantitatively the mrna levels of ps alpha subunit (constitutive), of the active subunit of ps (beta1, beta2, beta5) and of ips (lmp2, lmp7 and mecl-1). results: objective: recent studies suggest that dysregulated innate immunity plays an important role in the pathogenesis of iganephropathy (igan). interleukin-20 subfamily and its receptor, interleukin-22 receptor alpha-1 (il-22r1), were recently identified as immunomodulators in human diseases, acting as mediators of mucosal host defense. however, the potential role of il-22r1 in the pathogenesis of igan has not been explored. methods: in the current study, one hundred ninety four patients with igan and 287 normal controls were genotyped for coding polymorphisms of the il-22r1 gene and the association between the polymorphisms and igan was investigated. local expression of il-22r1 was examined in patients with igan and healthy controls using immunohistochemistry. results: our case-control analysis showed that genotypes of rs3795299 were associated with childhood igan. individuals with the cc genotype of rs3795299 had about three fold reduced risk of igan compared with those with the gg genotype in the codominant model (p=0.0028) and those with the genotypes containing g allele (gg or gc) in the recessive model (p=0.002).after bonferroni correction, the association between rs3795299 cc genotype and reduced risk of developing igan remained significant. furthermore, the renal expression of il-22r1 was significantly higher in healthy controls compared with subjects with igan. conclusion: our data suggest that the cc genotype of rs3795299 polymorphism in il-22r1 gene is associated with the reduced risk of igan, and this genetic association was supported by the higher renal expression of il-22r1 in healthy controls compared with patients with igan. abstract# p-sat168 toll-like receptor 9 gene polymorphisms contribute to development of proteinuria in childhood iga nephropathy henoch-schoenlein purpura (hsp) is the most common form of immune-mediated systemic vasculitis in children, which mainly affects skin, joints, gastrointestinal tract and kidney. the overall prognosis of hsp is favorable, but the long-term outcome is dependent on the degree of renal involvement. the incidence of renal involvement varies from 20 to 60% and there have been some reports showing that nephritis might be related to an older age at onset, persistent purpura (> 1 month), severe abdominal pain, and relapsing disease.recently, several studies have shown that galactose-deficient iga1 (gd-iga1) is recognized by anti-glycan antibodies, resulting in the formation of the circulating immune complexes and their mesangial deposition causing renal injury in hsp nephritis and serum galactose-deficient iga1 levels were highly inherited in children with hsp nephritis.regarding the treatment of hsp, one randomized double-blinded controlled study recently showed that patients with abdiminal pain or arthralgia may benefit from early treatment with prednisone, but the drug has not been proven to be capable of preventing the development of renal symptoms. however, it was effective in altering the course of renal involvementmild henoch-schoenlein purpura nephritis (hspn) generally does not require aggressive treatment due to a favorable course of the disease, but severe nephritis has a high risk of progression to end stage renal failure. although several intensive therapies, such as intravenous high-dose methylprednisolone pulse, immunosuppressive/cytotoxic drugs, fibrinolytic therapy, anticoagulants, antiplatelet agent and plasma exchange, have been used in children with severe hspn, treatment of hspn still remains controversial due to the rarity of randomized controlled studies in this field. expressions and significance of mtor, p70s6k1 and e-cadherin in glomeruli of children with iga nephropathy objective: to investigated the expressions of mtor, p70s6k1, and e-cadherin in glomeruli of children with igan to explore the role of mtor/p70s6k1 signaling pathway in the progressive renal glomeruli fibrosis of igan. methods: seventy-two children with igan were divided into three groups according to their clinical features: isolated hematuria group (ih group, 24 patients), hematuria and proteinuria(hp group, 26 patients), and nephritic syndrome group (ns group, 22 patients). patients were also also divided into three groups according to their pathologic grades: grade ii(33 patients), grade iii(25 patients) and grade iv(14 patients). six normal renal specimens were used as control group. the expressions of mtor, p70s6k1, and e-cadherin in glomeruli were determined by immunohistochemistry method and the relationship between these indexes and the clinical pathology indexes were analyzed. results: the highest expressions of mtor and p70s6k1 inglomeruli were found in the ns group, both of which in tubulointerstitium were higher in grade iii and iv group than that in grade ii group and control group while the lowest expressions of e-cadherin was found in the grade iv group. the glomerular lesion level degree was positively correlated with expressions of mtor and p70s6k1 inglomeruli. the quantitative urinary protein of 24 h was positively correlated with expressionsss of mtor in glomeruli. the concentration of serum iga was negatively correlated with the expressionss level of mtor in glomeruli. there was no significant correlation between the expressions of ang ii, mtor, p70s6k1, e-cadherin and α-sma in igan glomeruli and their courses from duration of illness as well as iga depositional strength. conclusion: the mtor/p70s6k1 signaling pathway was actived in the children with igan and it may play an important role in the progress of renal glomeruli fibrosis in children with igan. abstract# p-sat171 iga1 from hsp patients trigger apoptosis and inhibit cytoskeletal proteins in huvec objective: henoch-schonlein purpura (hsp) is the most common form of small-vessel vasculitis in children, and its etiology is believed to be associated with immune injury. a polymorphism in the gene encoding heat shock protein 70-2 (hsp70-2) is known to be associated with immune diseases. the purpose of this study was to investigate the correlation between the hsp70-2gene polymorphism (+ 1267 a/g) and hsp in children. the polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) method was used to detect the hsp70-2 polymorphism in 205 cases of children with hsp and 53 controls, and the association of this polymorphism with hsp and hsp nephritis (hspn) was analyzed. results: in patients with hsp, the a/a, a/g, and g/g genotypic frequencies at the +1267a/g position of hsp70-2 were 25.9%, 51.2%, and 22.9%, respectively. in the control group, the a/a, a/g, and g/g genotypic frequencies at the +1267a/g position of hsp70-2 were 30.2%, 60.4%, and 9.4%, respectively. thus, the g/g genotypic frequency in the hsp group was significantly higher than that in the healthy control group (χ2=4.764, p<0.05). the frequencies of the a and the g allele were 51.5% and 48.5%, respectively, in the hsp group and were 60.4% and 39.6%, respectively, in the control group. the frequencies of the a/a, a/g, and g/g genotypes in patients with hsp were 24.8%, 52.9%, and 22.3%, respectively. in hspn, the frequencies of the a/a, a/g, and g/g genotype were 27.4%, 48.8%, and 23.8%, respectively. conclusion: the +1267a/g polymorphism in the hsp70-2 gene is associated with hsp in children. the g/g homozygous genotype may be a genetic factor that predisposes individuals to hsp, but it is not significantly associated with the development of renal impairment. abstract# p-sat174 investigation of the correlation between tumor necrosis factoralpha gene polymorphism and henoch-schonlein purpura in children fei zhao 1,2 , songming huang 1, 2 , huaying bao 1 , guixia ding 1 , ying chen 1 , hongmei wu 1 , aihua zhang 1 1 nephrology, nanjing children's hospital, nanjing, china 2 institute of pediatrics, nanjing medical university, nanjing, china objective: to investigate the association between serum tumor necrosis factor-α (tnf-α) gene polymorphism and the occurrence and development of henoch-schoenlein purpura (hsp) in children. the polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) method was utilized to detect and compare the genotype and allele frequencies at the tnf-α -308 locus in 205 hsp child patients and 53 non-hsp child patients. furthermore, the relationship between the genotypic and allelic frequencies at the tnf-α -308 locus and the susceptibility to hsp and henoch-schonlein purpura nephritis (hspn) was analyzed. results: 1. the g/g, g/a, and a/a genotype frequencies at the tnf-α gene (−308g/a) locus in the hsp group and the control group were 59.5%, 32.2%, and 8.3% and 77.4%, 20.8%, and 1.9%, respectively; among them, the a/a genotype frequency in the hsp group was higher than that in the healthy control group, with a statistically significant difference (x 2 = 6.447, p < 0.05). the g and a allele frequencies in the hsp group and the control group were 75.6% and 24.4% and 87.7% and 12.3%, respectively. the a allele frequency in the hsp group was higher than that in the healthy control group, with a statistically significant difference (x 2 = 7.241, p < 0.05). 2. the g/g, g/a, and a/a genotype frequencies in the hsp non-renal impairment group and the nephritis group were 64.5%, 26.4%, and 9.1% and 52.4%, 40.5%, and 7.1%, respectively, but these differences were not statistically significant (x 2 = 4.474, p > 0.05). the g and a allele frequencies in the hsp non-renal impairment group and the nephritis group were 77.7% and 22.3% and 72.6% and 27.4%, respectively; the difference in the frequency of the a allele in the hsp non-renal-damage group and the nephritis group was not statistically significant (x 2 = 0.240, p > 0.05). conclusion: the tnf-α (−308g/a) gene polymorphism was associated with hsp in children. a/a homozygosity may be a genetic predisposing factor for hsp; however, this factor was not significantly correlated with the development of kidney damage. abstract# p-sat175 objective: the nephrotic syndrome (ns) is characterized by proteinuria, hypoalbuminemia and generalized edema. although the pathophysiological mechanisms of ns remain unknown, studies with animal models and patients have associated the ns with changes in immune response. the present study investigated the expression of molecules related to cell activation, such as the beta-2 integrin (cd18) and cd80 on peripheral blood leukocytes and renal production of reactive oxygen species in rats with ns induced by doxorubicin. meathods: male wistar rats, 250-300g, were divided into two groups: animals receiving intravenous injection of doxorubicin (7.5 mg/kg) (dox, n=32) and control animals that received saline (con, n=32). the animals were sacrificed at days 7, 14, 21 and 28 after injection, and 24 hour urine and blood samples were collected for biochemical and immunological analyzes. the phenotypic analysis of leukocytes was performed by flow cytometry. the expression of cd18 in monocytes, cd4+, cd8+ and nk cells and the expression of cd80 in monocytes were measured. in renal tissue samples, the oxidative activity was evaluated by tbars production and antioxidant activity of sod and catalase. results: the dox group animals showed significant increase in cellular cd18 expression and in the percentage of cytotoxic t lymphocytes, nk cells and monocytes that expressed cd18 as well as raised cd80 expression on peripheral blood monocytes when compared to the con group. the increased production of reactive oxygen species in renal tissue of dox group animals was positively correlated with the cd80 expression on monocytes and serum levels of creatinine. conclusion: these findings indicate a potential link between increased activation of peripheral monocytes and kidney damage in animals with ns. additional studies analyzing the effects of the blockade of integrins and co-stimulatory molecules may offer new therapeutic opportunities to treat human ns. abstract# p-sat182 cyclosporine a protects podocyte via upregulating the expression of cofilin-1 li xiaoyan, zhang xiaoyan, li xuejuan, wang xuejing, wang suxia, ding jie pediatric, peking university first hospital, beijing, china objective: podocyte foot process is dysregulated in nephrotic syndrome. the effacement of podocyte foot processes typically arises owing to perturbations in the actin cytoskeleton. calcineurin inhibitor cyclosporien a (csa) is currently used in the treatment of nephrotic syndrome. recent data suggest that the effects of csa on nephrotic syndrome are independent of its effects on the immune system. they identified that csa can stabilize the actin cytoskeleton through stabilizing synaptopodin in podocytes and thereby reduce proteinuria directly. other studies also showed that csa induced cofilin phosphorylation and promoted stress fiber generation in proximal tubular cells. however, whether the antiproteinuric role of csa is played by regulating cofilin-1 in podocyte has not been studied. methods: acute podocyte injury and nephrotic syndrome were induced by puromycin aminonucleoside (pan) injection in rats with or without csa. cultured podocytes were exposed to pan with or without csa treatment. cofilin-1, nephrin, synaptopodin expression were determined by western blot or immunofluorescence. the cofilin-1 specific effect was determined using cofilin-1 sirna. results: csa reduced proteinuria, restored expression of cofilin-1, nephrin, syanptopodin and repaired foot process effacement of pan induced nephropathy in vivo. in vitro studies showed that exposure of csa restored the expression of cofilin-1 and nephrin which decreased by pan. csa also repaired actin cytoskeleton impaired by pan. the protective effect of csa was disappeared partially when cultured podocytes were exposed to cofilin-1 sirna. objective: to find a probably mechanism of immune disorder in rsv nephropathy in rats which resembled the change of minimal change nephrotic syndrome. methods: 1. rsv mrnas (g, f, m2, ns1, ns2) and proteins (g and f) were detected with fluorogenic quantitative rt-pcr and indirect immunofluorescence assay (ifa) to find the evidence of viral persistence. 2. dc-sign mrna and protein were detected with rt-pcr and ifa to find the trend of its change. 3. the levels of il-12/il-10 and cd4/cd8 were measured by elisa and flowcytometry. results: the course could be divided into two stages: 1. first stage (4d-14d) the mrna of rsv f, g, m2, ns-1 and ns-2 in kidneys, lungs and spleens expressed and arrived at the highest on 14d, which accorded with the expression of rsv f and g proteins in ifa. the positively fluorescent luminance and mrna of dc-sign expressed stronger than that in the control, especially from 8d (ct kidney =18.50±3.12) to 14d (ct kidney =11.40±2.07). compared with the control, the secretion of il-12 in renal tissues was rising from 4d (1205.46±262.07pg/ml) to 14d (1054.68±154.18pg/ml). the expression of cd4 and cd8 in peripheral blood was lower than that in normal control. 2. second stage (30d-120d) rsv mrna and viral proteins in the tissues were persistently expressed; it showed that partial rsv survived through immune escape. after 30d, the fluorescent luminance of dc-sign was gradually reduced in the three tissues. the expression of dc-sign mrna in kidneys, lungs and spleens on 60d (ct kidney =14.10±8.32) and 120d (ct kidney =14.92±1.80) was higher, except that in spleens on 30d (ct= 21.90±1.43) was significantly lower than the control. the secretion of il-12 in tissues decreased after 30d (583.40±199.26pg/ml), but that of il-10 gradully increased after 30d (3832.38±88.20pg/ml). although the expression of cd4 and cd8 in peripheral blood was increased gradually after inoculation, the level was still lower than that in normal control. conclusion: there was rsv persistence in rats which likely attributed to the rsv-induced immune escape. rsv interacts with dc-sign to make dcs inhibit the immune ability of cytotoxic t cells, caused t cell immune dysfunction and finally induced the immune tolerance. abstract# p-sat185 evaluation of the migratory and regulatory profile of leukocytes from peripheral blood in pediatric patients with idiopathic nephrotic syndrome objective: dynamic regulation of the podocyte cytoskeleton plays an important role in maintaining the glomerular filtration barrier. severe glomerular disorders are associated with perturbed organization of the podocyte actin cytoskeleton. the therapy is based on immunosuppressive agents, in particular cyclosporine a, whose beneficial effect seems also related to the stabilization of the actin cytoskeleton in podocytes. previous studies of our group demonstrated, that the mtor inhibitor everolimus (ev) might similarly involve the recovery of the actin cytoskeleton in a puromycin (pan) experimental model of proteinuric disease. methods: in the present study, inhibitory effects of ev on mtor complex 1 (mtorc1) and −2 (mtorc2) were analyzed by western blotting in human differentiated podocytes. downstream of mtorc2 the activity of rhoa (gtpase pull-down assay) and myosin light chain (mlc) was studied. to identify further signaling pathways affected by ev, we performed affymetrix microarray expression analysis followed by verification using real-time rt-pcr. results: biochemical studies revealed a substantially decreased phosphorylation level of both mtor effector proteins mtorc1 (reduced p-akt) and mtorc2 (reduced p-p70s6k) by ev. objective: notch constitutes an evolutionarily conserved intercellular signaling pathway that determines cell fate in various organs. activation of notch1 and notch2 has been implicated in human glomerular diseases. notch1 was reported to play a critical role in development of glomerular diseases; however, a function of notch2 remains unclear. our aim of the study is to clarify notch2 pathway's contribution to developing proteinuria and glomerulosclerosis. methods: we injected either jagged1 antagonistic antibody (mab) or notch1 agonistic mab or notch2 agonistic mab intraperitoneally to mice with adriamycin (adr) nephropathy, a model of nephrotic syndrome and focal segmental glomerulosclerosis, and evaluated the levels of proteinuria and the ratios of sclerotic glomeruli. next, we treated cultured podocytes with adr in the presence or absence of notch2 agonistic mab, and we assessed the effect on cell survival and examined the pathways involved. then, we evaluated a correlation between notch2 activation and podocyte loss in human kidney specimen of nephrosis, minimal change disease (mcns) and focal segmental glomerulosclerosis (fsgs). results: administration of notch2 agonistic mab ameliorated nephrosis and glomerulosclerosis in mice with adr nephropathy, even when notch2 agonistic mab was administered therapeutically after the onset of nephrosis. in vitro, notch2 agonistic mab protected adr-damaged podocytes from apoptotic cell death. the specific knockdown of notch2 led to increased apoptosis in damaged podocytes. notch2 rescued damaged podocytes from apoptosis through akt pathway. human kidney specimens of nephrosis showed a positive linear correlation between the number of podocytes expressing activated notch2 and the number of residual podocytes. the glomeruli with mcns showed more activated notch2 and more podocytes, conclusions: whereas those with fsgs showed less activated notch2 and less podocytes. notch2 pathway has a pivotal role in preventing apoptosis of damaged podocytes. specific activation of notch2 may represent a novel clinical strategy for the amelioration of nephrosis and glomerulosclerosis. abstract# p-sat191 proliferation and apoptosis of tubular cells in initial and advanced stages of focal segmental glomerulosclerosis (fsgs) objective: to analyze processes of proliferation, apoptosis and ciliogenesis in proximal tubular cells during initial and advanced stages of fsgs associated with cystogenesis. methods: normal kidney tissues and tissues of fsgs kidneys were immunohistochemically analyzed using ki-67 proliferation marker and caspase-3 apoptotic marker. diameters of dilated and cystic proximal tubules were measured and correlated with proliferation index of tubular cells. data were analysed by the kruskal-wallis and dunn's post hoc test and expressed as mean+/−sd. significance was accepted at p<0.05. results: normal kidney tissue showed absence of proliferation in proximal tubules. initial stages of fsgs were characterized by intermingling areas of healthy non-proliferating proximal tubules, and pathologically changes, mildly dilated (15um diameter) proximal tubules showing proliferation index of 15.92%. advances stages of fsgs displayed different stages of cystogenesis: while mildly dilated tubules (diameter 12+/−1.4um) contained 36,18% of proliferating cells, their number increased to 45,23% in cysts with diameters from 21+/−1.8um to 54+/−1.1um, and then decreased to 13,76% in cysts with diameter 73+/−2.1um. while in the largest cysts proliferation index was lowest and the primary cilia short and distorted, in distal tubules and collecting ducts, primary cilia were extremely long and branching. apoptotic caspase-3 positive cells were observed within the tubular and interstitial cells. conclusion: deterioration of proliferation and apoptosis, and primary cilia formation characterizes cystogenesis in fsgs kidneys. we suggest that changes in primary cilia length might cause alterations in transfer of signaling pathways which control proliferation, differentiation and apoptosis of proximal tubules cells. advanced stages of fsgs are associated with cyst formation and increased proteinuria leading to nephrotic syndorme objective: to investigate effect of prednisone on the expression of ezrin and neph1 in rats with adriamycin-induced nephrosis. methods: adr model was induced by a tail intravenous injection of adr.the rats were divided into three groups, which were control group, model group and prednisone group. serum index and 24h urinary protein were measured at 4 and 8 weeks. observe pathologic changes of renal tissues at 8 weeks. the expression of ezrin and neph1 in glomerulus was evaluated by immunohistochemistry respectively. results: compared with control group, at 4 weeks, 24h urinary protein and total cholesterol in model group and prednisone group were significantly increased(p<0.05),albumin and total protein were significantly decreased(p<0.01),which indicated that the model was successfully established;compared with control group, at 8 weeks, the expression of ezrin and neph1 in the model group were significantly decreased(p<0.01).compared with the model group, the expression of ezrin and neph1 in prednisone group were significantly increased(p<0.01),24h urinary protein and total cholesterol were significantly decreased(p<0.01),albumin and total protein were significantly increased(p<0.05), renal pathology and ecm/ga were significantly increased(p<0.01). conclusion: the expression of ezrin and neph1 were reduced in rats with adriamycin-induced nephrosis which were negatively related to proteinuria and renal pathological damage. prednisone can reduce the proteinuria and relieve the renal pathological damage by improving the expression of ezrin and neph1. objective: the activation of the complement system plays an important role in various kidney diseases, such as antibody-mediated rejection or membranous glomerulopathy. the majority of the circulating complement components are produced in the liver. but in the last decade the local production of complement components by other cells is highly debated. the aim of our study was to proof the ability of human podocytes to produce and secrete complement components. methods: immortalized human podocytes were analyzed with western blot (wb), immunofluorescence (if) or/and pcr for their ability to produce components (c1/ c1q, c2, c3, c4, c5) inhibitors (factor h, mcp, cd 55, cd59) and activators (factor b, properdin) of the complement system. secretion of components was measured in the medium, and functionality of c3 was tested in a specific c3convertase assay. stimulation of the cells was done with interferon-γ, interleukin 6 and human albumin. results: pcr-studies revealed that human podocytes express on mrna level the components c2, c3, c4, c5, the inhibitors factor h, mcp, cd 55 and cd59, and the activators properdin and factor b. on protein levels c2, c3, factor b, properdin, factor h, cd 55 and cd59 were detected. in immunofluorescence all the components showed either intra-plasmatic, perinuclear or peri-membranous distribution. in addition, we could show that podocytes secrete the factors c2, c3, c4, c5, factor b and factor h into the medium. the secreted c3 was clearly functionally active and could further enhanced by stimulation with interferon-γ. objective: to construct the eukaryotic expression plasmid and its shrna plasmids of il-17 of mice, and to investigate their ability of expression or inhibitory effects on il-17. methods: the rna from spleen cells of bal b/c mouse was reversed to cdna, and the full-length cds fragment of il-17 was amplified. the target segment was cloned into the expression vector plvx-ires-zsgreen1, then, the recombinant plasmid plvx-il-17-ires-zsgreen1 was obtained. three pairs of shrna chains targeting il-17 gene and one pair control shrna chain were designed and synthesized, then annealed to form double strand ,and inserted into the expression vector plvx-shrna2. three shrna plasmids and one control plasmid were constructed ,which were called shrna1/shrna2/shrna3/shrnac, respectively. these plasmids were identified by restriction analysis and sequencing, and then they were transfected or co-transfected into 293t cells in mediation of liposome. the transcription levels of il-17 mrna were detected by fluorescent quantitative pcr, while expression levels of il-17 protein were detected by elisa. results: restriction analysis and sequencing proved that the recombinant plasmid were constructed correctly. the plasmid of plvx-il-17-ires-zsg reen1 could expression il-17; when cotransfected with expression plasmid, il-17 expression levels were significantly reduced in the group of shrna1/shrna2/shrna3 as compared with the shrnac group, and the group of shrna1 had the least expression. conclusion: il-17 eukaryotic expression plasmid and its specific shrna plasmids were constructed successfully, and plasmid shrna1 had the best inhibitory effects. abstract# p-sat195 reno-protective effect of all-trans retinoic acid on adriamycininduced nephropathy mice xiaoli wang, jingjing cui, qiu li nephrology and immunology department, children's hospital of chongqing medical university, chongqing, china objective: to investigate the reno-protective effect of all-trans retinoic acid on adriamycin-induced nephropathy mice. methods: balb/c mice were randomly divided into nephrosis(a single adriamycin 10.5mg/kg,n=20) and control(tail intravenous injection the same amount of saline,n=10). after two weeks nephrosis divided into atra-treated(intraperitoneal injection all-trans retinoic acid 20mg/kg, three times a week,n=10) and adr nephrosis. then, we chose five mice each group randomly after treatment 6 weeks and 10 weeks to collect 24-hours-urine, blood, kidney for detection. results: all-trans retinoic acid significantly decreased 24-h urinary protein excretion, serum tc and tg(p<0.05). fas/fasl shows by immunohistochemistry and fluorescence quantitative pcr is decreased sifnificantly(p<0.01). conclusion: all-trans retinoic acid has anti-apoptosis on adriamycin induced nephropathy mice to protect kidney damage and delay fibrosis process. abstract# p-sat196 molecular mechanism of ers induced by lipid accumulation in human mesangial cells (hmcs) objective: dehydroxymethylepoxyquinomicin (dhmeq) is a novel nf-κb inhibitor that potently inhibits the dna-binding activity of nf-κb, resulting in therapeutic effects for various pathological conditions. to elucidate the pathogenetic role of nf-κb in minimalchange nephrotic syndrome (mcns), we examined whether dhmeq could ameliorate the nephrosis in mice induced by puromycin aminonucleoside (pan), which is considered to be an animal model for mcns. methods: mice were injected with dhmeq or only vehicle 2 hours before pan injection on day 0. mice without pan injection served as controls. the dhmeq or vehicle was injected on 5 consecutive days. the daily urine was collected from day 0 to day 30, and the urinary concentrations of albumin and creatinine were measured. some mice were sacrificed 7 days after pan-injection, and their serum concentrations of cholesterol, total protein, albumin and il-6 were measured and the pathological changes of the kidneys were observed. results: pan injection without dhmeq in the mice induced albuminuria which gradually increased up to day 10 and gradually decreased thereafter. in contrast, pre-treatment with dhmeq was associated with no significant increase in the amount of albuminuria during the first 2 weeks. in the serological tests, pan-injection induced an increase in the cholesterol level, a decrease in the total protein, a decrease in the albumin and an increase in the il-6 in the serum compared to controls 7 days after injection, but dhmeq ameliorated these changes. an electron microscopic analysis indicated an effacement of the foot processes of the podocytes in the pan-injected mice, but this was rarely observed in the pan-injected mice pre-treated with dhmeq. further immunohistochemical analysis showed that dhmeq can inhibit the pan-induced translocation of nf-κb from cytoplasm into nucleus. conclusion: these results suggest that dhmeq can be a candidate therapeutic agent for mcns, because the activation of nf-κb of podocytes may be associated with pan-induced nephrosis. knocking-down stim1 gene expression inhibits some podocyte molecules jingjing yan, meigui wang, siguang lu the first people's hospital, lianyungang, jiangsu, china objective: to study the effect of knocking-down stim1 on nephrin,podocin,cd-2ap and α-actinin-4 in murine podocytes in vitro. methods: conditionally immortalized murine podocyte cells were cultured in rpmi1640 medium at 33°c permissive condition.then the cells were shifted to non-permissive condition at37°c and cultured for ten-fourteen days, and were transfected with stim1 small interfering rna(sirna) using transfection reagent lipofectamine 2000.transfection efficiency was measured by flow cytometer and inhibitory effect of stim1 sirna was determined by rt-pcr and western blot at fourty-eight, senventy-two hours after transfection respectively. results: (1)the transfection efficiency of fam-sirna was about 75.5%. (2)after transfection with specific sirna,the expression levels of stim1 mrna and protein were down-regulated by 80.7% and 49.8% respectively. (3) the expression levels of nephrin,podocin and α-actinin-4 mrna were decreased by 62%, 35%, 60% respectively, whereas cd-2ap showed no-change.both podocin and α-actinin-4 protein were decreased by 45%, 20% respectively. objective: one of the most common causes of nephrotic syndromes in children is the minimal-change-glomerulonephritis (mcgn). typically, mcgn results in foot processes effacement of podocytes. their plate-shaped flattening very likely results in dysfunction of the filtration barrier and may underlie the increase in urine albumin concentrations. notably, mcgn often disappears at the onset of puberty, thus in parallel with increasing peripheral hormone levels. this is particularly true in girls. methods: podocytes were studied by western blot analysis, immunofluorescence and by radioimmunoassay for their ability to secrete and bind estrogens. results: podocytes express estrogen receptors both, estrogen receptor alpha and beta, suggesting that increasing levels of estradiol during puberty potentially maintains and restores morphological integrity of podocytes. this protective function could be mediated by stabilization of the podocyte cytoskeleton by estradiol, as evidenced in vitro by the increase in phosphorylation of cofilin, very similar to the effects of estradiol in neurons. furthermore, we show that dissociated podocytes express aromatase, the final enzyme of estrogen synthesis, and that podocytes in fact synthesize estradiol, as evidenced by measuring estradiol content in the supernatant. conclusion: as it was shown that estrogen synthesis in cells other than those in gonads becomes stimulated during puberty, an autocrine mechanism of estrogen action could underlie decreasing frequency of mcgn at the onset of puberty. overexpression of pgc-1α inhibits aldosterone-induced podocyte phenotypic changes and detachment by blocking mitochondrial dysfunction objective: to explore the role of heparanase in the pathogenesis of rat respiratory syncytial virus (rsv) nephropathy. methods: 150~200g sprague-dawley(sd) rats (n=5 per group) were inoculated with 6×10 6 (plaque-forming units, pfu) rsv and sacrificed on days 4,8,14 and 28 postinoculation (rsv 4 ,rsv 8 ,rsv l4 and rsv 28 ). five normal sd rats inoculated with dulbecco's minimum essential medium were served as normal control. the expression level of heparanase protein and mrna in kidney of each group was determined by immunohistochemical staining and real-time quantitative rt-pcr respectively. the proteinurina was measured and the relationship between the expression level of heparanase and the 24hour urinary protein was studied. results: rats with rsv nephropathy exhibited higher proteinuria by comparison with normal rats. there was a significant difference between each group(rsv 14 >rsv 8 >rsv 28 >rsv 4 ). compared with normal control, rats with rsv nephropathy showed up-regulated expression of heparanase protein in glomeruli. the expression level of heparanase protein in rsv 8 and rsv 14 group was higher than that in rsv 4 and rsv 28 group. there was a linear positive correlation between the expression level of glomerular heparanase protein and the quantity of urinary protein(r =0.783,p<0.05).compared with normal control group, the expression level of heparanase mrna in kidney f r o m r s v 4 , rsv 8 , rsv 1 4 a n d r s v 2 8 g r o u p w a s elevated(rsv 14 >rsv 8 >rsv 4 >rsv 28 ).there was a linear positive correlation between the expression level of renal heparanase mrna and the 24-hour urinary protein(r=0.725,p<0.05). the increased heparanase in kidney may be important to the loss of glomerular negative charge in glomerular basement membrane and is involved in the pathogenesis of rsv nephropathy. podocyte morphology andautophagosomes were viewed using electron microscopy. podocyte numerical density was estimated by weibel-gomez method. expressions of autophagy markers and ersassociated proteins were analyzed by western blot. results: the expression level of ers-associated protein grp78 was up-regulated from day 4 to day 21 post-injection. the results also showed that autophagosomes were massively accumulated and the autophagy marker lc3 was up-regulated in the models on day 7 and 14. furthermore, rapamycin was given to phn rats to further explore the role of autophagy in the process of complement-dependent podocyte injury. results revealed that rapamycin, which enhanced autophagy in podocyte, could reduce proteinuria, lighten podocyte lesionsand prevent podocyte loss on day 21. conclusion: taken together, our results demonstrated that ers plays an important role in completment-dependent podocyte damage, and in another aspect, autophagy induced byers can alleviate injury as a protective mechanism. this provides an important basis for a thorough understanding of the role of autophagy in the process of podocyte damage and the pathogenesis of mn. abstract# p-sat209 the cytoprotective role of autophagy under oxidative stress in human podocyte objective: autophagy is a ubiquitous catabolic process involving selective degradation of cellular components. it shows cytoprotective effects in different cell types and helps to maintain cell homeostasis. some studies demonstrated that cell stress, generated by starvation or some chemical reagents, can initiate autophagy which responsively resists harmful stimuli at the early stage. however, little is known about autophagy in human podocytes under oxidative stress. since the apoptosis and autophagy pathways share some common molecules, we investigated the role of autophagy induced by puromycin aminonucleoside (pan) in human podocytes. methods: human conditional immortalized podocytes were treated with pan, the generation of reactive oxygen species (ros) was measured by immunofluorescence. then, autophagy was assayed by immunofluorecence staining for lc3 puncta and western blotting for lc3. in addition, the mammalian target of rapamycin (mtor) and its substrates which play critical roles in autophagy inhibition were investigated for elucidating the mechanism of pan induced autophagy. podocyte apoptosis was assessed by flow cytometry (yo-pro-1/pi and active caspase 3 assays). to study the effects of autophagy on podocyte apoptosis, 3-methyladenine (3-ma) and chloroquine were used to inhibit autophagy. results: autophagy was detected in pan-treated podocytes in a dose and time dependent manner and prior to apoptosis which was accompanied with increased ros generation. lc3 aggregates were observed in the cytoplasm and the expression of lc3 ii was significantly elevated. intriguingly, phospho-mtor and its substrates (phospho-p70 s6 kinase and phospho-4e-bp1) increased when the autophagy was activated. when autophagy was inhibited by 3ma and chloroquine, podocyte apoptosis increased significantly. conclusion: pan induced both apoptosis and autophagy in human podocytes. our results suggested that this in vitro model will be useful for the study of crosstalk between apoptosis and autophagy in podocytes. autophagy may be the adaptive cytoprotective mechanism for podocytes under oxidative stress. further studies directed at identifying the role of mtor are essential. abstract# p-sat210 impact of angptl3 knock-out on adriamycin-induced nephropathy mice objective: idiopathic focal segmental glomerulosclerosis (fsgs) is associated with recurrence after transplantation due to a circulating permeability factor or factors (n engl j med, 334:878-883, 1996) . we have shown the effects of fsgs plasma and its fractions on glomerular permeability in vitro and in vivo and have used state-of-the-art proteomics to identify cardiotrophin-like cytokine-1 (clcf1), a member of the il-6 family, as a candidate for the active substance. we hypothesize that a new model of fsgs can be based on the effects of clcf1 in mice. methods: rclcf1 (r&d systems) was injected intraperitoneally (ip), one dose, 10 μg/kg, or infused by minipump for 28 days, 40 μg /kg/day. a construct containing clcf-1 was administered by electroporation. all studies were done in c57bl6 mice. urinary albumin/creatinine, pjak1, pstat3, patk, and perk in peripheral blood cells (pbc) and in kidney homogenate were measured and glomerular histology was assessed. results: albuminuria was induced promptly by rclcf1 after either injection or electroporation. peak albuminuria occurred by 7 days of expression and was 3-5 fold increased vs. baseline. ip administration of rclcf-1 increased pjak2 and pstat3 of pbc within 15 min. and renal pjak1 and pstat3 remained upregulated for at least 72 hours after injection. kidney pjak2 and pstat3 as well as perk/12 and pakt were markedly increased after 28 days of infusion. mesangial matrix was increased at that time. conclusion: we conclude that clcf1 mimics many of the renal effects of the active fraction of plasma from patients with idiopathic fsgs and may play an important role in its etiology. its relationship to other candidates such as circulating urokinase receptor (supar) is not known. a murine model based on administration or overexpression of clcf1 may permit us to define mechanisms of injury and to test potential therapeutic agents prior to use in clinical trials. over-expression of myo1e in mouse podocytes enhance cellular endocytosis, migration, and adhesion objective: to investigate the effects of tacrolimus (fk506) on hepatocyte growth factor and transforming growth factor β1 in kidneys of fsgs rats. methods: establish unilateral nephrectomy combined adriamycin double tail vein injection of fsgs model in rats divided to model group and treatment group ,then treatment group rats were treated withtacrolimus 8 weeks. the protein expression and site of hgf and tgf-β1 in renal of each group were assayed by western blot and immunohistochemistry after animals were sacrificed. urine protein, serum albumin, blood lipoids and kidney function were tested automatic biochemical analysis system. results compared with the normal control group , model group and treatment group rats 24h urinary protein excretion , serum creatinine , blood urea nitrogen , cholesterol significantly increased serum albumin significantly reduced severe renal pathological changes and tgf-β1 protein expression was significantly increased , and the differences were statistically significant ( p <0.05 ) ; compared with the model group , in experiments 9 weekend , the treated rats 24h urinary protein excretion , relevant serum biochemical indicators of renal pathological changes varying degree of improvement in renal tissue hgf protein expression was increased and tgf-β1 protein expression was decreased , the above differences were statistically significant ( p < 0.05 ). objective: the cellular mechanisms of kidney injury caused by obstructive nephropathy are interstitial inflammation, fibrosis, and apoptosis. rodent models can be used to simulate obstructive nephropathy in the human kidney. we developed shear-thinning hydrogels for the local delivery of il-10 to abate the progression of inflammation and fibrosis that leads to ckd. methods: injectable dock-n-lock gels were developed as previously reported 1 (fig. 1) . il-10 was added to the gel or phosphate buffered saline solution at a concentration of 0.33 ug/ul. study design: eight cohorts were studied (7, 21, 35 days, n=4): healthy, sham operation, healthy injected with msa, healthy + gel, unilateral ureteral obstruction (uuo), uuo + il-10, uuo + gel, uuo + gel/il-10. 15 ul of il-10 solution, gel, or gel/il-10 was injected into the left kidney via retroperitoneal approach 3 days after the initial uuo or sham operation. histology: immunohistochemistry (ihc) was performed on paraffin sections to identify macrophages and apoptotic cells, and trichrome stain was used to evaluate fibrosis. cells and total area were quantified for ihc and total fibrotic area was quantified for trichrome. results: comparing the treatment groups to the untreated uuo, macrophage infiltration and apoptosis were significantly reduced at day 21 and 35. by day 35, adding the il-10 via gel injection reduced macrophage infiltration more than il-10 alone and il-10 alone did not reduce apoptosis. fibrosis was decreased by day 35 in all three treatment groups (fig. 2) . conclusion: injectable hydrogels were synthesized that permit facile local delivery of immunotherapy to both healthy and obstructed kidneys. renal inflammation and scarring was reduced in an animal model of ckd by using an injectable hydrogel for drug delivery. logistic regression reveled that apd was the main parameter significantly associated with surgery treated upjo cases (roc plot was 0.79). a possible threshold of 14mm apd may be used as a cut-off value of surgery treated upjo group with a sensitivity of 77% and a specificity of 69%. conclusion: apd dilatation was the strongest predictor of surgery treated upjo. pt and renal length also significantly discriminate the two groups and correlate with apd, only with lower predictive power. our findings expand the clinical knowledge in the field of prenatal consult by highlighting a threshold of apd, which predicts the need for surgery in prenatally detected hn cases. abstract# p-sat221 survival and renal outcome in fetuses with lower urinary obstruction (luto) with and without intra-uterine vesicoamniotic shunting.a ten years experience of a cohort. objective: ureteropelvic junction obstruction (upjo) is the most common cause of hydronephrosis in children. the significant role of the surgical relieving of high grade obstruction is indisputable. nevertheless, the effect of pyeloplasty on the function of the involved kidney remains controversial, especially in upjo with significantly reduced relative renal function (rrf) before surgery. to evaluate the effect of pyeloplasty on the relative function of kidneys in children with upjo and decreased rrf. methods: the records of children who underwent pyeloplasty for upjo during a 10 year period in schneider children's medical center of israel were reviewed. the study group included 40 children who underwent pyeloplasty for upjo and had an initial rrf <40%. children with bilateral upjo, solitary kidney or other genito urinary abnormalities were excluded. the control group included 38 children with non obstructive hydronephrosis and an initial rrf<40%. results: the average initial rrf in the study group was 26% (range 8-39%), and the final renal function was 32% (1-55%). in the control group, the initial rrf was 23% (2-40%), and the final function was 22% (0-44%). the final rrf of the study group was significantly higher comparing to the control group (p value<0.05). in the subgroup of patients with upjo and initial rrf<30% (24 patients) the average initial function was 20% (8-30%) and the final function was 27% (3-55%). in the control subgroup with initial rrf<30% (26 patients) the initial average function was 17% (2-30%) and the final function was 17% (0-44%). conclusion: pyeloplasty is associated with an improvement of renal function in children with upjo and an initial rrf <40%. this is also true for patients with rrf<30% before surgery. our results support the need for pyeloplasty in children with upjo and reduced rrf. objective: common cause of chronic kidney disease (ckd) in children. kidney damage occurs in utero, and kidney disease progresses postnatally. the objective of this project was to identify clinical biomarkers, in particular antenatal variables, which predicted long-term renal outcome in boys with puv. methods: this was a retrospective cohort analysis. primary outcome was the development of end stage renal disease (esrd) as defined as starting dialysis or preemptive transplantation. clinical variables studied included antenatal factors, postnatal renal function, and modifiable variables. continuous data for the two outcome groups were compared, receiver-operating characteristic (roc), kaplan-meier, and logistic regression analyses were conducted to assess the robustness of each candidate biomarker as a predictor of outcome. results: in this cohort 15 cases reached the primary outcome of end stage renal disease at a mean age of 7.0 ± 6.7 yrs. compared to those who didn't, those who progressed to esrd had younger age at diagnosis (0.6 ± 1.5 vs 3.3 ± 7.3 yrs, p<0.05), valve ablation ( . fsgs rate was found as 60% in biopsied subjects. nephrotic syndrome was defined by edema, massive proteinuria (>40 mg/m 2 per hour or a protein/creatinine ratio >2.0 mg/mg), hypoalbuminemia (<2.5 g/dl), and hyperlipidemia. remission was defined as a urinary protein excretion below 4 mg/m 2 per hour or a protein/creatinine ratio below 0.2 mg/mg for three consecutive days. steroid resistance was accepted as no achievement of remission in spite of treatment with prednisolone, 2 mg/kg per day for 4 weeks. if steroid resistance was seen, patients were also treated with cyclosporine a (csa) (3-5mg/kg per day for least 6 months) and, thereafter, if required, with cyclophosphamide(cp) (2.5-3.0 mg/kg per day for 10-12 weeks). renal failure was defined as a glomerular filtration rate (gfr) below 80 ml/min per 1.73 m 2 body surface area, and esrd was defined as a gfr below 10 ml/min per 1.73 m 2 or the necessity for any renal replacement therapy. bidirectional dna cycle sequencing analysis of entire coding exons and adjacent intronic segments of nphs2 gene was performed.nphs2 gene mutation analysis has been also performed in 100 healthy children. results: pathogenic nphs2 mutations were found in 127 patients (18%) from the totally 700 srns children group. mutation rate was 29% in familial group, and 16.5% in sporadic group. a total of 53 mutations were determined in the nphs2 gene, and 37 of which were characterized as a novel mutation presented at hgmd databank. in the mutation positive nphs2 group, most of the mutations were found out to harbor in exons 1, 4, and 5 while no mutation were found in exon 6 of the respective gene. patients with p118l, r138q, r138x, r168h, s211a, a212t, v218g, h228d, ivs7+5g>a, c.460-467inst, c.503delg mutations were progressed to end-stage renal disease (esrd). also, age at onset of proteinuria (years) was 4.2+/-0.5 in the mutation (-) (n=573) group; 3.6+/-3.08 in the mutation (+) (n=127) group. for the mutation negative patients, effects of other disease causing genes involving nphs1, wt1, trpc6, cd2ap, and actn4 for different molecular subtypes of srns may be considered. nphs1 mutation screening was performed for all the patients who had proteinuria up to two years of age. as a causative srns gene, we should consider nphs2 gene mutation screening in early diagnosis and the follow-up of the clinical course. conclusion: in relation to homozygous or compound heterozygous nphs2 mutated patients who have the lack of response to standard steroid therapy we suggest to perform nphs2 gene mutation analysis for every child (if consent can be obtained) soon after the first episode of ns. for the newly diagnosed patients, the crucial certain determination of the causative disease gene mutation will enable clinicians to avoid redundant immunosuppressive therapeutic trials. we then performed next generation sequencing in the 5 affected patients and in one of their unaffected relatives. exome enrichment was conducted using the agilent sureselect human all exon v4+ utrs capture kit. the multiplex libraries were then sequenced on an illumina hiseq2000 instrument with a 2x76 bp read length. results: we unexpectedly identified a novel lmx1b mutation segregating with the disease in the family. subsequently, we screened 74 additional unrelated families from our international cohort of autosomal dominant fsgs and found mutations of the same residue in 2 families. none of the probands had any sign of dysplasia of nails, patellae or elbows, iliac horns or glaucoma, or any ultrastructural changes suggestive of nail-patella-like renal lesions. lmx1b encodes a homeodomain-containing transcription factor that is essential during development. a lmx1b in silicohomology model suggests the mutated residue plays an important role in strengthening the interaction between the lmx1b homeodomain and dna. both mutations are expected to diminish such interactions. conclusions: our data demonstrate that isolated fsgs could be due to mutations in genes also involved in syndromic forms and highlights the need to include these genes in all next-generation sequencing diagnosis approaches in fsgs. .in 24/37 patients mutations were identified by ngs (detection rate 65%). in 8/24 patients sequence variants were found in two different genes (33%) suggesting oligogenic inheritance. in 6 of these 8 patients the causative mutation was located in a gene following an autosomal dominant inheritance pattern. these patients showed a more severe form of the disease compared to affected family members who showed only the causative mutation without additional modifier variants. one additional patient of this cohort with pierson syndrome had two single heterozygous mutations in nphs1 and lamb2, respectively (both genes following an autosomal recessive pattern of inheritance). a further patient showed two mutations in nphs1 and the non-neutral polymorphism p.arg229gln in nphs2. structural and/or functional analysis of all mutations identified in patients with mutations in two genes suggested impaired protein function of the particular gene products. conclusion: the influence of modifier genes or digenic inheritance seems to play an important role in the pathogenicity of srns. the application of ngs is therefore of special interest and highly efficient in the diagnostics of patients with srns. abstract# p-sat232 mutation analysis in japanese patients with congenital and infantile nephrotic syndrome objective: mutations in podocyte genes (nphs1, nphs2, wt1, and lamb2) are associated with congenital (<3 months) and infantile (3-12 months) nephrotic syndrome (ns). the purpose of this study is to investigate the frequency of causative mutations in these genes in ns manifesting in the first year of life in japan. methods: all exons and exon-intron boundaries were investigated in consecutive, unrelated 37 patients from regional pediatric kidney disease centers, by pcr-direct sequencing. results: we detected disease-causing mutations in 64.9% (24 of 37) patients (75% in congenital and 25% in infantile) (table) . objective: hypercoagulability along with thrombosis are prevalent complications of nephrotic syndrome. in cases of refractory nephrotic syndrome, intracardiac thrombus, although rare, is a serious complication because of its association with morbidity and high mortality herein, we report a case of a patient with a right atrial thrombus associated with nephrotic syndrome who responded well to corticosteroid therapy and attained quick remission. case presentation: a 12-year-old japanese boy was referred to our hospital for a right atrial thrombus associated with idiopathic nephrotic syndrome diagnosed 1 month ago. the patient had attained the partial remission within the 10 days of the corticosteroid therapy initiation. he was asymptomatic otherwise at the time of admission. the patient's vitals were stable and laboratory values were almost within the normal range. an echocardiogram showed a large isolated hyperechoic mass in the right atrium, which originated from the superior right atrial wall. the mass measured 31 × 26 mm in diameter and traversed through the tricuspid valve in a to-and-fro motion. the patient underwent emergency surgical thrombectomy on day 1 and anticoagulation therapy was started 7 days after the operation. the subsequent hospital stay was uneventful, and the patient was discharged on day 87. objective: nephrotic syndrome features proteinuria and severe sodium retention which is implicated in ascites and edema formation. previous works realized in nephrotic rats (pan) clearly highlighted that, the increase of sodium absorption in the ccd is associated with an increase activity of the na/k atpase; sodium absorption is independent of aldosterone, activation of epithelial sodium channel enac is not necessary and sodium absorption is inhibited by amiloride. our hypothesis is that, in nephrotic syndrome, another apical sodium channel sensitive to amiloride and independent of saar pathway is implicated in sodium absorption in the ccd. methods: transcriptome of ccds from control rats (ct) and pan treated clamped rats (adx pan) was analyzed using a high-resolution quantitative and comparative analysis of gene expression. subsequently, transcriptional (rt-qpcr) and proteic expression (western blot and immunohistochemistry) of channel were analyzed. sodium handling in vivo and in vitro microperfused collecting ducts in different conditions (ph 6 and 7.4, apical zinc 0.3mm and amiloride 0.1mm) was analyzed. results: metabolic study confirmed previous results: sodium absorption and ascites were similar in pan and adxpan rats suggesting that sodium absorption in nephrotic syndrome was independent of saar. transcriptome analyses highlighted an increased expression of accn1 mrna only in adx pan at day 6. rt-qpcr confirmed an increased expression of accn1 only in the ccd of adx pan (about eight time compared to adx ct). subsequently, protein shows an increase expression of accn1 (adx pan 0.72+/-0.12 vs adx ct 0.31+/-0.03, p 0.03) and immunohistochemistry on microdissecated ccd highlighted a strong apical expression of accn1 only in adx pan rats. schimke immuno-osseous dysplasia (siod) is a rare autosomalrecessive multisystem disorder, characterised by: disproportionate growth deficiency, defective cellular immunity, nephrotic syndrome and progressive renal disease.siod is caused by bi-allelic mutations of smarcal1 gene, which encodes the hepa-related protein (harp), a member of the snf2 family of atpases, acting as chromatin remodelers within multi-protein complexes. a 5-year-old patient was hospitalized in november 2011, after detection of proteinuria (1.5 g/day). at our first clinical examination: height-weightgrowth retardation, thinning hair, thin and hypopigmented skin, arched palate and normal neuro-psychological development.serological examination showed proteinuria (1.7 g/day), hyperchol. (col tot 242 mg/dl, ldl-col 165 mg/dl) and lymphocyte deficiency (17.6%).renal function and complement levels were normal. lymphocyte subpopulations showed a decrease of t lymphocytes and an increase of nk cells(cd16 + cd56 +).the patient had also a detour back-lumbar scoliosis and normal kidneys in size, but with reduced corticomedullarydifferentiation. we started therapy with ramipril 2.5 mg/day. the 24 hrs urine exams performed monthly showed persistent proteinuria (500-800 mg/day). at the end of april 2012, for the persistence of proteinuria, we performed renal biopsy, which showed focal and segmental glomerulosclerosis. at discharge, she started therapy with irbesartan 75 mg/day with significant reduction of proteinuria (140 mg/day). after 6 months of therapy, thepatient did not present proteinuria and her renal function was normal. the molecular genetic study of smarcal1 gene revealed that patient was compound heterozygous for two mutations: a novel missense mutation in exon 3, inherited by mother, and a nonsense paternally-derived mutation in exon 17, leading to a truncated smarcal1 protein. our data allow us to diagnose a schimke immuno-osseous dysplasia (siod). in most patients, life expectancy is limited to childhood or early adolescence, due to the onset of stroke, infections,hematopoietic bone marrow failure and renal failure. only patients with milder and late onset forms can survive until adulthood. gain of glycosylation in integrin-alpha-3 causes nephrotic syndrome and lung disease objective: congenital nephrotic syndrome and interstitial lung disease is a rare multiorgan disorder, characterized by disrupted basement membrane structures. itga3 gene mutations were recently identified as the genetic cause of this disorder, but the disease mechanism remains poorly understood. methods: we describe a patient who presented with neonatal respiratory distress, glomerulosclerosis, proteinuria, pulmonary hypoplasia and alveolar glycogenosis, who died 7 months after birth due to respiratory insufficiency. a genome-wide screening for deletions and duplications revealed a large homozygous region that included the itga3 gene. we sequenced the gene and then conductedin vitro characterization studies to investigate the effect of the variant on the protein function. results: a novel homozygous missense mutation was identified in the coding region of the itga3 gene, which introduces an n-glycosylation motif to the protein sequence. thereby, the mutant integrin alpha-3 protein becomes hyperglycosylated. functional studies demonstrated that the conformation of integrin alpha-3 is affected and the mutant alpha-3 precursor is targeted for degradation. consistent with these findings, alpha-3 integrin was not detected in the patient glomeruli. furthermore, integrin alpha-3 protein expression was absent in murine podocytes that lack endogenous integrin alpha-3 and transfected with mutant itga3. conclusion: our findings underscore the role of the integrin alpha-3beta-1 complex as the main regulator of podocyte basement membrane integrity. here, we show that hyperglycosylation of the integrin alpha-3 subunit, causing the complete lack of alpha-3-beta-1 expression on the basement membrane, is a new pathogenic mechanism underlying congenital nephrotic syndrome and interstitial lung disease. itga3 mutation screening was directly implemented in dna diagnostics, facilitating early diagnosis, recurrence risk estimation, and genetic counselling. the clinical study on pkhd1 gene-based testing for the diagnosis of arpkd objective: in order to study the implications of pkhd1 gene-based testing for arpkd diagnosis in clinical practice, we performed a prospective study to apply pkhd1-based genetic testing to the suspected arpkd patients. methods: 12 suspected arpkd patients from 10 unrelated families were detected on mutations of pkhd1 gene by pcr direct sequencing. these patients were evaluated by combining the testing results and their clinical materials. results: in the 6 detected children, 4 mutations (p137s, v836a, q1574h, l2658x) were novel and 7 mutations (t36m, r559w, r760c, n830s, a1262v, q3899r, q4048r) were previously described. 4 of the 11 variants were definite pathogenic mutations. two pathogenic mutations in both chromosomes were found in 2 out of 6 families (30%), which greatly aid to making definite diagnoses. the 4 families with perinatal presentation consisted of 2 fetuses and 2 couples. one definite pathogenic mutation (s3457c) was found in the wife of family no.9, however, it was still difficult in conforming the diagnosis owing to lack of the pathogenic mutation from the husband. for the other families, only some potential mutations were found.two in four children with congenital hepatic fibrosis were detected two pathogenic mutations in both chromosomes, and their liver functions were obviously lower than the other two children without pathogenic mutations. conclusion: pkhd1 gene-based testing is an effective means for the diagnosis of arpkd, at the same time it could improve the understanding of this disease by analysing the testing results and their clinical materials. objective: to present a case of schimke immuno-osseous dysplasia (siod) which is the first to be reported from egypt. methods and results: this article presents a case from egypt with mild form of siod presented at the age of 14.5 years with disproportionate short stature, srns (focal segmental glomerulosclerosis), laboratory evidence of cellular immune deficiency and radiologic characteristics of spondyloepiphyseal dysplasia and died at the age of 16.5 years with bone marrow failure and severe pneumonia. conclusion: we emphasize that siod is to be considered in children with growth retardation, srns and bone abnormalities and that inherited nephrotic syndrome may be presented in late childhood or even adolescence and siod suspected patients even before full picture development should be closely monitored for proteinuria, hypertension, cellular immunity and opportunistic infections especially with the need to start immune suppressive therapy for nephrotic syndrome. abstract# p-sat244 denys-drash syndrome presenting with polycystic kidneys objective: denys-drash syndrome (dds) is characterized by a congenital or infantile nephrotic syndrome due to diffuse mesangial sclerosis, male pseudohermaphroditism and a strong predisposition to develop wilm's tumors and gonadoblastoma's. the syndrome is caused by a dominant mutation in the wt1 gene. we report a case of dds associated with renal cysts, which has never been described so far. we report a case of dds in a 3 months old girl presenting with proteinuria and bilateral cortical cysts on renal ultrasound. autosomal recessive polycystic kidney disease was excluded by a normal liver biopsy. a kidney biopsy showed diffuse mesangial sclerosis. at the age of 7 months, she developed a unilateral wilm's tumor, which was treated by heminephrectomy and chemotherapy. the renal cysts were still present on ultrasound. histological examination of a cyst (located in the resected specimen, but separate from the wilm's tumor) showed one layer of flattened epithelial cells, without any evidence of malignancy. the combination of diffuse mesangial sclerosis and a wilm's tumor pointed to the diagnosis of dds. genetic analysis showed a de novo heterozygous missense mutation c.1186g>a (p.asp396asn) in the wt1 gene, previously described in patients with dds, confirming the diagnosis. because polycystic kidneys have never been reported in dds, we explored several genes responsible for these renal manifestations, such as hnf-1β, pax2, pkd1 and pkd2. remarkably, we identified a heterozygous missense variant c.12439a>g (p.lys4147glu) in the pkd1 gene. mutations in pkd1 lead to autosomal dominant polycystic kidney disease (adpkd). the pathogenicity of the newly identified missense variant in our case was evaluated by different mutation prediction software programs and was classified as being 'likely pathogenic'. the same variant was found in the patient's mother, having no renal cysts on ultrasound and in the grandfather, having bilaterally renal cysts. conclusion: this is the first case of dds in combination with polycystic kidneys. we hypothesize that pkd1 c.12439a>g variant might be an incompletely penetrant allele that can cause a severe phenotype of adpkd in association with a wt1 gene mutation. the phosphatase and tensine homolog (pten) gene is a tumor suppressor gene located on the long arm of chromosome 10 (10q22-23). the pten protein is broadly expressed in cells throughout the body, acting as both lipid and protein phosphatases, and regulates intracellular signaling via various pathways. reduced pten protein resulting from a pten gene mutation may enhance cell proliferation, resist from apoptosis. in addition, it has became clear that, in vascular endothelial cells, pten gene abnormalities lead to increased expressions of vascular growth factors, such as angiotensin ii (ang ii), that are essential to angiogenesis. recent reports revealed a germline mutation of the pten gene to cause several syndromes with generalized, multiple hamartomatous lesions of tridermic origin. thus, the new concept of pten hamartoma tumor syndrome (phts) has been proposed. to the best of our knowledge, we describe here for the first time a case of focal segmental glomerulosclerosis (fsgs) with phts. a 3-year-old girl was found to have proteinuria and hematuria on health examination and referred to us for persistent urinary abnormalities. igm nephropathy were diagnosed by renal biopsy, and she started taking an angiotensin-converting enzyme inhibitor. however, proteinuria was not reduced, and an oral angiotensin ii receptor blocker (arb) was administered. proteinuria persisted and she underwent renal biopsy again, yielding a diagnosis of fsgs. despite continued oral arb treatment, proteinuria is ongoing with a urine protein-to-creatinine ratio of approximately 0.5, and mild renal impairment. at age 13 years, 2 years ago, a neck mass prompted a detailed examination which revealed multiple thyroid nodules, ovarian cysts (mucinous cystadenoma), hemangiomas in the plantar and femoral regions, spinal cord lipoma, and renal nodular lesions, necessitating follow-up. subsequently, additional workup detected a pten gene heterozygous mutation (exon 8 codon 335 cga(arg) tga(stop)) and she was diagnosed with phts. methods: a 7-day-old girl, the first child of non-consanguineous chinese parents, was hospitalized due to edema, gross proteinuria, and progressive renal failure. she was delivered spontaneously at 36 weeks of gestation with a birth weight of 2700gand a body length of48cm. during the delivery, she was experienced mild asphyxia. placental weight was large, and the amniotic fluid was meconium-stained. pregnancy was unremarkable and had included routine prenatal ultrasound evaluation. the family history of proteinuria or renal failure was negative. on admission, the findings from physical examination showed the patient had bilateral microcoria and limb hypotonia. results of serological testing for torch infections were negative. since pierson syndrome was suspected, a kidney biopsy was performed when she was aged 10 days. meanwhile, all coding exons of lamb2 were analyzed by using pcr and direct sequencing. results: electron microscopy revealed some glomerular basement membranes were thick and thinning, with splitting of the lamina densa. in addition, the foot processes of podocytes were diffusely effaced, and the number of podocytes increased. two different novel nonsense mutations (trp16x and gln748x) of lamb2 were detected in the patient. these mutations were inherited from her parents respectively. conclusions: newborn with unexplained renal insufficiency or nephrotic syndrome should consider pierson syndrome. our report extends the genotypic spectrum of pierson syndrome. a familial wt1 mutation associated with incomplete denys-drash syndrome we report a familial wt1 missense mutation in exon 9 (1180c>t, r394w) in three members of one family. patient 1, a 2 years old boy, was born at ambiguous genitalia (46, xy karyotype), penoscrotal hypospadias and bilateral inguinal hernias. he was found proteinuria on the preoperative examination and renal biopsy showed diffuse mesangial sclerosis. patient 2, a 3 years old, is the older sister of patient 1, who has normal genitalia. she was found proteinuria and renal biopsy showed focal mesangial sclerosis. wilms's tumor was not found in both of them. a wt1 mutation was detected in both the two patients and their farther. the patients are considered as incomplete dds and they can inherit mutations from their father. so, this finding will provide new evidence for a better understanding of dds and further familial studies in patients of dds are need. ying chen 1 , songming huang 1, 2 , guixia ding 1, 2 , hongmei wu 1 , aihua zhang 1, 2 1 nephrology, nanjing children's hospital, nanjing, china 2 institute of pediatrics, nanjing medical university, nanjing, china objective: to investigate the correlations between peroxisome proliferator-activated receptor (ppar)-α, ppar-γ, and ppar-γ coactivator-1α (pgc-1α) gene polymorphisms and susceptibility to primary nephrotic syndrome (pns). methods: patient genotypes were determined using the polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) technique for the pro12ala and val290met polymorphisms of the ppar-γ gene, the gly482ser polymorphism of the pgc-1α gene, and the leu62val polymorphism of the ppar-α gene. the gene polymorphisms in 111 cases of pns in children and 111 normal controls (nc) were analyzed and compared to examine the differences in the clinical metabolism index, proteinuria, renal pathological types, and hormone treatment responses among pns children with different genotypes. results: the ppar-γ pro12ala and pgc-1α gly482ser mutations were not associated with the occurrence, blood pressure, total cholesterol, a decrease in glomerular filtration rate (gfr), urine protein excretion at the onset of disease, renal pathological type, or hormone treatment response in children with pns. although the homa-ir values for children with the a allele did not differ significantly from those of children with the pp genotype of the ppar-γ gene, their insulin levels were decreased and their isi values were significantly increased (p=0.012 and 0.006, respectively). the triglycerides (tg) levels of children with the aa genotype of the pgc-1α gene were significantly increased (p=0.026). in addition, the ppar-γ (val290met) and ppar-α (leu162val) gene polymorphisms did not show any mutations in the 111 cases of pns in children or the 111 nc children. conclusion: the pro12ala mutation of the ppar-γ gene may be correlated with a decrease in insulin secretion and an increase in insulin sensitivity and the pgc-1α (gly482ser) gene polymorphism may be a causative genetic factor for the triglyceride abnormalities in children with pns. the relationship between endothelin-1 gene polymorphisms and primary nephrotic syndrome in children objective: nphs1 mutations have been reported in cns. however, no hot mutation has been described in chinese family. in this study, nphs1 mutations were analyzed in a chinese family with two siblings died of cns. methods: genomic dna samples were extracted from peripheral blood of the proband, her parents, and 150 unrelated normal individuals. all 29 exons of nphs1 were detected by polymerase chain reaction(pcr) and direct dna sequencing. results: the proband, a 15-day-old girl, weighed 3500g at birth. the placenta was one time heavier than usual. she was hospitalized with edema of legs, heavy proteinuria(+++), and hematuresis(+++). two compound heterozygous mutations were identified in exon 15 (c.2020c>t, p.p674s), which was detected in her father, and in exon 16(c.2207t>c, p.v736a), which was also detected in her mother. these two new mutations were not found in the 150 chinese controls. conclusion: new compound heterozygous nphs1 mutations (c.2020c>t and c.2207t>c) were identified in a chinese family with two siblings of congenital nephrotic syndrome, which were suggested to be the causative mutations in this family. the compound heterozygous mutations (c.2020c>t and c.2207t>c) lie between ig6 and ig7 domain which has a free cysteine residues. these mutations might cause misfolding and defective intracellular transport, with consequent absence of the mutant nephrin on the plasma membrane. abstract# p-sat255 sporadic myh9-related disease: a case report and mutational analysis of myh9 gene .genetic analysis confirmed that these three cases had intron 9 (+kts) mutation.after confirmation of fs, cytogenetic analysis showed xy in girls though phenotypically there were females.abdominal ultrasound in them showed normal uterus with streak gonads and gonadectomy was done for both and histologically reported as dysgerminoma stage i. familial girl aged 12 entered esrd and received live related renal transplant from her mother. the sporadic girl aged 9 years also entered end stage renal disease rapidly and died. the third was a nephrotic boy aged 9 years with fs. he had bilateral hypoplastic testes. his chromosome was xy. renal biopsy showed mcd with igm deposits and partially responding to therapy. a six year old girl presented at the emergency department with a history of 3 episodes of vomiting in 24 hours. she had a history of polyuria and polydipsia with a current daily intake of 3 liters. physical examination revealed signs of mild dehydration. plasma electrolytes revealed hyponatremia (128 mmol/l), hypokaliemia (1.6 mmol/l), metabolic acidosis with normal anion gap (14 mmol/l), hypophosphatemia (0.86 mmol/l), hypouricemia (90 umol/l) and normal serum creatinine (29 umol/l). urinalysis confirmed the na, k, ph and acid uric losses: na (67 mmol/l, fractional excretion 2%), k (83 mmol/l, ttkg 30), tubular phosphate reabsorption of 18%, and fractional excretion of uric acid of 30%. cystine level in leucocytes was normal. she was discharged six days later with oral supplementation of potassium and phosphorus. laboratory studies 3 weeks later revealed surprising findings with persistent hypokaliemia (3 mmol/l), metabolic alcalosis (hco3 30 mmol/l), hypomagnesemia (0.65 mmol/l), and hypocalciuria (calcium/creatinine 0.06 mmol/mmol). we therefore sequenced the slc12a3 gene and we found that she is compound heterozygous for 2 known missense mutations (p.thr304met and p.gly439ser). conclusion: gs can have atypical presentations including severe polyuria; it should be suspected in case of severe hypokaliemia even in the absence of the other classical features. this case report emphasizes the need for repeated laboratory tests in unclear tubular disorders, especially outside of confusing intercurrent illness. a single basepair mutation causes cystinosis in the majority of western cape patients. conclusion: renaltube is being used by pediatric nephrologists over the world for the study of their patients with primary tubular disorders. renaltube will likely contribute to a better care of these children and to a better scientific understanding of primary tubular diseases. the creation and development of this sort of international collaborative efforts must be encouraged within the pediatric nephrology community. objective: to study molecular mechanism of hypercalcemia-induced nephrogenic diabetes insipidus (ndi) by identification of proteins in inner medullary collecting duct (imcd) responsible for this syndrome. methods: the effect on rat kidney medullary collecting duct of early onset ndi resulting from parathyroid hormone-induced hypercalcemia was studied using proteomics and phosphoproteomics in native inner medullary collecting duct (imcd) cells. imcd tryptic peptides and phosphopeptides were identified and quantified by mass spectrometry using a label-free methodology. the major findings were confirmed by selected reaction monitoring study, immunoblot, and immunohistochemistry. results: a total of 5,866 peptides corresponding to 1,107 proteins and 1,388 phosphopeptides of 580 proteins were identified, with significant changes in abundance of 69 proteins and 49 phosphopeptides in early onset ndi versus vehicle controls. gene ontology terms and pathway analysis revealed that hypercalcemiaaffected proteins and phosphoproteins are associated with integrin signaling, and actin cytoskeleton organization. immunoblot and selected reaction monitoring lc-ms/ms studies confirmed the hypercalcemia-regulated proteins (agrin, arcp1b, capg, erm, itgb1, lamb2, and lamc1) and phosphoproteins (myh9, add1, dync1li1, cgnl1, aqp2). hypercalcemia induced changes in abundance of vasopressin-regulated phosphorylation of aquaporin-2 (aqp2) at ser256 and ser261, but not total aqp2, and decreased abundances of slc14a2 urea transporters ut-a1, ut-a3, and phospho-ser486-ut-a1. the major findings were also identified in imcd of rats with vitamin d-induced hypercalcemia. filamentous actin aggregation was firstly demonstrated in imcd of rats with hypercalcemia-induced ndi from both pth and vitamin d. conclusions: early increasing in water excretion in response to hypercalcemia are dependent of changes in abundances of aqp2 phosphorylations, ut-a and its phosphorylations are consistent with prior literature pointing to key roles of the integrin signaling and the actin cytoskeleton in maintenance of collecting duct function. objective: to study molecular mechanism of hypokalemia-induced nephrogenic diabetes insipidus (ndi) by identification of proteins in inner medullary collecting duct (imcd) responsible for this syndrome. methods: the effect on rat kidney medullary collecting duct of early onset ndi resulting from hypokalemia was studied using proteomics in native inner medullary collecting duct (imcd) cells. imcd tryptic peptides were identified and quantified by mass spectrometry using a label-free methodology. the major findings were confirmed by selected reaction monitoring study, immunoblot and immunohistochemistry. results: a total of 2,477 peptides corresponding to 821 proteins were identified with significant changes in abundance of 189 proteins in early onset ndi versus vehicle controls.gene ontology terms and pathway analysis revealed that hypokalemia-affected proteins are associated with generation of precursor metabolite and energy and regulation of actin cytoskeleton. immunoblot and selected reaction objective: inherited distal rta (drta) is a rare condition and is almost always observed in children as a primary entity. mutations in genes encoding transporter or channel proteins operating along the renal tubule may result in a variety of functional defects. the identification of the molecular defects in drta may provide a basis for future design of targeted therapeutic interventions and, possibly, strategies for gene therapy of these complex disorders. for this reason, we evaluated mutations in slc4a1 and atp6v1b1 genes. methods genomic dna from 20 pediatric patients diagnosed with drta was extracted from peripheral blood samples. sequencing was performed for five exons of slc4a1 (14, 15, 17, 19, 20) and two exons of atp6v1b1 (1, 7). all the selected exons are considered hotspots for mutations associated to this tubular disorder. results silent mutations were identified in both studied genes. the mutation c.2688t>c was identified in exon 20 of slc4a1 in one patient and the second variation (c.27t>c) was found in exon 1 of atp6v1b1 in two patients. although previously described, these silent variations are not highly frequent in the overall population. also in slc4a1, exon 19, two nonsynonymous variations were identified in the same patient (p.pro854leu and p.val862ile). the p.val862ile variation was also found in three other patients. it has been shown that these variations are rare polymorphisms in the population, but they have already been associated to diego group erythrocyte antigen. in exon 1 of atp6v1b1 the variation p.met2? was presented in twelve patients and the mutation p.thr30ile was found in six patients. although they occur in the coding region of atp6v1b1, these variations have been described as highly frequent in the population. dent's disease is an x -linked renal proximal tubulopathy associated with mutations in clcn5 (dent's type 1) and ocrl1 (dent's type 2).ocrl1 mutations also cause the oculocerebrorenal syndrome of lowe. we report the case of a 5 year old boy with dent's disease associated with a novel hemizygous change, c.2t>c p.(met1) in exon 1 of ocrl. our patient presented at 3 years of age with incidental finding of non-nephrotic range proteinuria. he had elevated urinary beta -2 microglobulin, retinol binding protein and hypercalciuria. he was developmentally normal and is otherwise of good general health. slit lamp examination and vision were normal. he had normal renal function but had very poor renal accumulation of 99mtc-dmsa on renoscintigraphy. genetic testing identified a hemizygous change, c.2t>c p.(met1), in exon 1 of ocrl, which affects the translation initiation codon. his healthy 8 year old brother who was also found to have low -molecular weight proteinuria and hypercalciuria, is currently being investigated. to the best of our knowledge, the hemizygous change, c.2t>c p.(met1?) in exon 1 of the ocrl gene which was identified in our patient with dent's disease represents a novel variant which has not been previously reported in a dent's disease patient. abstract# p-sat274 enamel-renal syndrome associated to splenic and ovarian calcifications: a case report sameh mabrouk, noura zouari, houda ajmi, jalel chemli, menair tfifha, saida hassayoun, saoussen abroug , abdelaziz harbi pediatrics, university hospital of sahloul, sousse, tunisia enamel-renal syndrome (omim204690) is a rare condition characterized by amelogenosis imperfecta and nephrocalcinosis.we report a new pediatric case of this rare association with the particularity of associated splenic and ovarian calcifications. this report concerns a 13-year-old girl, born to consanguineous parents (first degree cousins). she has no family history of nephrocalcinosis or kidneydisease. her unique brother, aged 8 years, is in good health and has no dental or renal problems. she was first seen at the age of 12 years in dentistrydepartment because of dental abnormalities, the diagnosis of amelogenosis imperfecta was made and the patient was further investigated. a renal ultrasound showed a bilateral medullary nephrocalcinosis. ct confirmed bilateral nephrocalcinosis which was associated to ovarian and splenic calcifications. besides this young girl has a normal growth, her puberty began at the age of 12 years. laboratory findings including serum electrolytes, urea, creatinine, calcium, phosphate, parathormone and alkaline phosphatase were within normal ranges. on urine examination we found a hypocalciuria, with normal sodium and phosphate excretion. the patient is regularly seen in both dentistry department and pediatrics. the aim of this report is to highlight the important role of pediatricians as well as dentists in recognizing this rare and uncommon syndrome. objective: ipex syndrome, a hereditary (x-linked) immune dysregulation with autoimmune polyendocrinopathy and enteropathy, as the basic manifestations, presents a rare and severe disease. the objective of this case report is to highlight the pleomorphism of the syndrome. methods: the authors report the case of a male infant, with a family history of three male siblings affected by ipex syndrome. the patients' medical records were reviewed in order to describe the case of the youngest one. results: during the follow-up of the youngest of three siblings, who presented eczema and intestinal manifestation, without compromised pancreatic and thyroid function, different from other two siblings, it was noticed the pattern of dent's disease. we registered hypophosphatemia, hypercalciuria, glycosuria, low molecular weight proteinuria and ultrasound revealed second stage bilateral nephrocalcinosis. in this child there was no apparent glomerular involvement, as it was seen in the eldest sibling. conclusion: dent's disease is an x-linked renal proximal tubulopathy associated with mutations in the chloride channel gene clcn5 (xp11.22), which is next to foxp3 gene on the x chromosome (xp11.23-q13.3). it seems that in this sibling mutations occurred in exons of both of these genes. this case is to remind on pleomorphic potential of mutations that occur near the coding regions of the foxp3 gene. objective: multiple target organ defects (mtod) is a subgroup of pseudohypoaldosteronism (pha) type i and salt wasting is more severe in this form of pha i. it has a poorer outcome than the renal form, and therapy must be maintained throughout life. here, we reported an infant with mtod who had severe hyperkalemia resistant to classical treatment of pha. hyperkalemia was normalized only by peritoneal dialysis (pd). case: a 4 month-old boy was admitted to our hospital with persistent vomiting, metabolic acidosis, and severe hyperkalemia. there was consanguinity between parents and he had a healthy sister and brother. there was no history of medication. physical examination revealed the signs of volume depletion and acidosis. results: in laboratory examination, blood urea nitrogen was 71 mg/dl, creatinine 0.43 mg/dl, sodium 114meq/l, potassium 9.5meq/l, and chloride 97meq/l. blood ph was 7.33, base excess -12meq/l, and hco3 11meq/l. urinary analysis revealed low specific gravity (1003) with ph 8.0, and normal urinary sediment. urinary sodium was 68meq/l, potassium 1meq/l, and plasma renin activity 80.2ng/ml (2.7-37.0), aldosterone 2800pg/ml (50-900), cortisol 11.1 mg/dl (7-17.5), and acth 5.5pg/ml (10-469). renal ultrasonography did not show any abnormalities. sweet-test showed high sodium waste from sweet glands (144meq/l). the patient diagnosed as mtod. severe hyperkalemia persisted and was resistant to all treatment options for pha, including high-sodium and low-potassium diet, fluid resuscitation, potassiumbinding resins, indomethacin, fludrocortisone, and hydrochlorothiazide. nutritional status of the patient was also not good, and parents did not accept gastrostomy. hyperkalemia was corrected only after pd treatment, and his nutritional status improved dramatically. conclusion: in the case of type iv renal tubular acidosis resistant to therapy, mtod could be considered as a cause of acidosis. if the classical treatment options fail to correct hyperkalemia in these patients, pd may be a reasonable choice to normalize severe hyperkalemia. gülsün gülay yılmaz 1 erta yilmaz 1, 2 1 pediatric nephrology, md, antalya, turkey 2 akdeniz univercity, prof. dr, antalya, turkey tuberous sclerosis, which has an autosomal dominant hereditary disease with a high rate of spotaneous mutation, especially occupies the nervous system and many other organs like kidney, heart and lung. the case presented here was initially diagnosed as congenital heart and polycycystic kidney diasease, because of its characteristics which were quite different than expected for tuberous sclerosis. finally, it was evaluated at our pediatric department together with its dermatological and radiological aspects and defined as a tuberous sclerosis of delateddiagnosis. the case was discussed with its former and actual findings and presented as a warning for early diagnosis. abstract# p-sat285 the effect of bone marrow stem cells mobilization on expression of hif-1α and egf in ischemia/reperfusion-induced renal injury objective: to investigate the therapeutic effects of bone marrow stem cells which has been mobilized by g-csf and stem scf on expression of acute tubular necrosis hypoxia inducible factor-1a (hif-1α) and epidermal growth factor (egf), and to investigate the mechanism of scf and g-csf on the treatment of ischemia/reperfusion-induced renal injury. methods: 160 male sprague-dawley rats that are 8-10 weeks old were randomly allocated into 4 groups (n =40 ineach group): control group (group a), ischemic-reperfusion group (group b), scf + g-csf + ischemic-reperfusion group (group c) and scf + g-csf + control group (group d). detection index: hif-1α was measured by immunohistochemistry technique, the expression of egf and cd34 + cells in kidney was measured by strept avidin-biotin-peroxidase(sabc) and egf mrna by rt-pcr . results: (1) at 5 days postoperative, the cd34 + cells of group b and c was conspicuous higher than group a and d(p<0.05), group c was higher than group b(p<0.05). they gradually descended from 5 days with the time prolonged. (2)at 5 days postoperative, there was significant difference of the expression of hif-1α between group a and d (p<0.05). the expression of hif-1α of group b and c showed higher positive reaction at 5 days postoperative then decent to normal. at each time the expression of hif-1α of group c was significant higher than other groups (p<0.05). (3)the egf expression of group b and c showed higher positive reaction at 5 days postoperative and decent to normal with the time prolonged, group b peaks at 17 days and higher than group a, group c peaks at 10 days, and it is the highest (p<0.05 results: in contrast to model group, atn treated with adscs displayed: cells expressing green fluorescent protein were dectected in injured tubule in kidney . treatment group were significantly higher than model group, p<0.05; renal damage is lighter; kidney damages were relative gently and histopathologic lesion scores was relative lower; while ki-67 positivece cells in treatment group were more than those in model group (p<0.05); the expression of bax, bcl-2 and cell apoptotic index(ai) in treatment group was lower than those in model group , p<0.05, but bax/bcl-2 ratio was higher than those in model group. conclusion: allogenic adscs transplantation can accelerate proliferation of renal tubular epithelial cell and suppress apeptosis injury through up-regulating the ratio of bcl-2/ bax and down-regulating the expression of bax protein in acute kidney injury. objective: previously we showed that pretreatment with the antidepressant fluvoxamine (flu) improves postischemic survival and ameliorates functional and structural kidney damage after renal ischemia/reperfusion (ir). in heart ir injury flu is protective through activating the sigma-1 receptor (s1r) -nitric-oxide synthase (nos) system. here we studied the intrarenal vasoregulatory effect of flu and analyzed the renal s1r-nos cascade. methods: male wistar rats were were treated i.p. either with (1) vehiculum (veh); (2) flu (20 mg/bwkg; flu); (3) flu+ s1r antagonist ne-100 (1mg/bwkg; fn); (4) flu+ non-selective nos blocker l-name (10mg/bwkg); (5) flu + selective endothelial (e) nos blocker l-nio (20mg/bwkg); (6) flu and selective neuronal (n) nos blocker 7-ni (25mg/bwkg). 30 minutes later rats were either harvested or subjected to 50 minutes of ischemia followed by 24 hours of reperfusion. sham-operated animals served as controls (n=10/group). renal s1r, akt, enos and nnos protein levels were measured, the alteration of renal capillary diameters was determined in vivo using muliphoton microscopy. results: ir induced renal vasoconstriction, which was ameliorated by flu. this increase was neutralized by all nos blockers, mostly by 7-ni. after ir all measured protein levels increased. s1r expression was similar in all treatment groups. akt and enos levels were lower, while nnos levels were higher in the flu treated group compared to veh and fn. the acute vasodilatative effect of flu 30 minutes after treatment was suspended by l-name and 7-ni and even reversed by l-nio. s1r, akt and enos protein levels were elevated 30min after flu treatment, while nnos levels remained unchanged. discussion: pretreatment with the s1r agonist flu -used chronically without notable side-effects -improves postischemic renal perfusion through the activation of s1r -nos system in a time and nos isoform specific manner. based on this data one can hope to find a new therapeutic target in the treatment of renal ir damage through the modulation of the s1r. methods: 36 sd rats were randomly divided into model group, intervention group and normal group, 12 rats in each group. intraperitoneal injection with gentamicin for 7 days to establish renal tubular injury rat model. then the intervention group rats were transplanted with 4×10 6 /ml bm-mscs via tail vein. 7 days after transplanted with bm-mscs, took blood and kidney specimen of rats for testing. serum creatinine (scr), urea (bun), malondialdehyde (mda), superoxide dismutase (sod), glutathione peroxidase (gpx), heme oxygenase -1 (ho-1) were detected by spectrophotometry. he staining was used to evaluate the change of renal tissue pathology, then score the renal tubular injury according to the nangaku semiquantitative scoring method. tunel method was used to detect the apoptosis of epithelial cell rate in renal tubular, while immunohistochemistry was used to detect the proliferation of renal tubular epithelial cells (pcna labeling index). results: 1. scr and bun of intervention group rats were lower than those in the model group, the difference was statistically significant (p <0.01); 2. renal tubule pathological score of intervention group rats were lower than those in the model group, the difference was statistically significant (p< 0.01); 3. apoptosis ratio of renal tubular epithelial cell in intervention group rats were lower than the model group, the difference was statistically significant (p<0.01); 4. the pcna labeling index of intervention group rats were higher than those in the model group, the difference was significant (p<0.05); 5. the mda level was significantly reduced in intervention group rats than in the model group (p < 0.01); 6. the sod, gpx and ho-1 inintervention group rats were significantly increased than those in the model group (p < 0.01). conclusion: antioxidant factors such as sod, gpx and ho-1 were significant increased after bm-mscs transplanted into rats. it means that bm-mscs may have antioxidant effect to heal the renal tubular damage. the effect on regeneration and repair in renal tubular epithelial cell after injury deal with at2r antagonist pd123319 objective: to study the effect of proliferation of renal tubular epithelial cell after injury deal with at2r antagonist pd123319,and to explore the function and mechanism of at2r in the regeneration and repair after acute kidney injury. methods: to establish the human renal proximal tubule cells (hk-2 cells) hypoxia /reoxygenation model. hk-2 cells were divided into two groups. (1) (1) these results suggest that the renal proximal tubule epithelial cells after hypoxia/reoxygenation can be simulated tubular epithelial cell process damage; (2) the proliferation of renal tubular epithelial cell after injury was inhibited by pd123319.at2r may play an important role in the regeneration and repair in the kidney by means of promoting the proliferation of renal tubular epithelial cells. (3) arb have no significantly inhibit the proliferation of renal tubular epithelial cell after injury, suggesting that at1r may not have major role in regeneration and repair after acute kidney injury. the effect and mechanism on regeneration and repair in renal tubular epithelial cell after injury by the inhibitory proliferation effect of acei and arb objective: to study the effect of regeneration and repair of renal tubular epithelial cell after injury deal with fosinopril and valsartan, and to explore the function and mechanism of acei and arb induced acute kidney injury by suppressing proliferation. objective: ischemia-reperfusion injury (iri) is a major cause of acute kidney injury (aki) and both innate and adaptive immunity contribute to the pathogenesis. t cell immunoglobulin-3 (tim-3) has been reported as an important regulatory molecule and plays a pivotal role in several inflammatory diseases. however, it keeps unknown whether tim-3 is involved in renal iri. to investigate the expression of tim-3 on kidney mononuclear cells (kmncs) from mice with renal iri and explored its role in the pathogenesis of renal iri. methods: the left renal pedicle was clamped in c57bl6 mice for 45 min, followed by reperfusion. animals were sacrificed at baseline, day 1,3. flow cytometry was used to quantify tim-3 expression on kmncs ,cd4 + t cell,cd8 + t cell,foxp3 + tregs and cd19 + b cell. the levels of tnf-α, ifn-γ, il-4 and il-10 in kidney tissue were measured using elisa. results: at day 1, the increased expression of tim-3 on kmncs ,cd4 + t cell,cd8 + t cell,foxp3 + tregs in the injured kidney from mice with renal iri compared to those from uninjured kidney tissues and baseline (p<0. 05). percentage of tim-3 + cells in kmncs showed an inverse correlation with kidney injury score and kidney tnf-α level. similar negative correlations were found between kidney injury score and tim-3 levels on cd4 + t, cd8 + t cells. consistently, tim-3 expression on cd3 + t cells was further increased in mice at day 3. tim-3 expression on foxp3 + tregs negatively correlates with kindey tnf-α. conclusion: tim-3 might participate in the proceeding of renal iri by regulation on various cd4 + t cell subsets. tim-3 might be a potential new marker for assessing severity of renal iri. expression of galectin-9 and tim-3 in kidney of mice with renal ischemia reperfusion injury objective: t cell immunoglobulin-3 (tim-3) is a surface molecule expressed on various immune cells which plays a central role in immune regulation. identification of galectin-9 (gal-9) as a ligand for tim-3 has established that the tim-3/gal-9 pathway has been linked to several inflammatory diseases by regulating adaptive and innate immunity. to study the expression of gal-9 and tim-3 in kidney of mice with renal ischemia reperfusion injury (iri). methods: thirty c57bl6 male mice were randomized into renal iri groups with and without recombinant gal-9. the left renal pedicle was clamped in c57bl6 mice for 45 min, followed by reperfusion. animals were sacrificed at baseline, day 1,3,10, 21 after iri. gal-9 and tim-3 mrna levels in kidney tissues were determined using real-time rt-pcr. expression of gal-9 in kidney were detected by immunohistochemistry staining .the levels of tnf-α, ifn-γ,il-4 and il-10 in kidney tissue were measured using elisa. results: the expression of gal-9 and tim-3 mrna in the injured kidney tissues increased significantly compared with uninjured kidney tissues and baseline (p<0. 05). compared with the uninjured control and baseline control, the expression of tnf-α and ifn-γ increased significantly in the injured kidney. the expression of gal-9 and tim-3 mrna was positively correlated with renal il-4 and il-10 level (r=0.792, r=0.79 respectively; p<0. 05), but negatively correlated with kidney tnf-α and ifn-γ level (r=-0.69,r=-0.75 respectively,p<0.05). after recombinant gal-9 treatment for three days, the kidney injury ameliorated and inflammatory cytokines (tnf-α and ifn-γ) decreased. the expression of gal-9 and tim-3 in kidney tissues increase in mice with kidney iri. tim-3/gal-9 pathway are closely related to inflammatory process in renal iri. objective: we previously found that rgc-32(response gene to complement-32), a key factor in regulating cell cycle, plays an important role in dealing with epithelial-mesenchymal transition (emt). this study aimed to evaluate the effects of rgc-32 regulating cell cycle in renal tubular epithelial cells injury and repair. methods: (1) objective: autophagy is a lysosomal degradation pathway that is essential for cellular stress adaptation and normal homeostasis. increased level of autophagy has been reported in the post-ischemic kidneys by static analysis. this study aimed to understand the dynamics of epithelial autophagy in kidneys following acute ischemic injury and during renal repair. methods: taking the advantage of differential ph sensitivity of rfp (pka 4.5) and egfp (pka 5.9) fluorescence, we generated a new strain of cag-rfp-egfp-lc3 mice to distinguish early autophagic vacuoles from autolysosomes and to monitor autophagic process in the kidneys with ischemia-reperfusion injury (iri). results: renal epithelial cells responded to nutrient deprivation with easily detectable fluorescent puncta that represented autophagic vacuoles and corresponded to lc3-ii and atg5 protein levels. the majority of the egfp lost its fluorescence in the acidic environment of the autolysosomes where bright rfp signals remained. in normal kidneys, few egfp and rfp puncta were present in the nephron. iri led to dynamic changes in autophagic process in the proximal tubules with the number of egfp puncta reaching the peak at 1 day and returning to the control level at 3 days whereas rfp puncta persisted at a high level through 3 days post injury, indicating autophagy initiation at 1 day but autophagosome clearance at 3 days as kidneys were recovering. since rfp puncta persisted in cells with recent autophagy, we examined ki67 expression and found significantly lower proliferation in cells that contained rfp puncta, suggesting that autophagic cells were less likely to divide for tubular repair. furthermore, 87% proximal tubular cells with mtor activation indicated by p-s6 kinase expression contained no rfp puncta. inhibition of mtorc1 activity with rapamycin caused a 2-fold decrease in cell proliferation. conclusion: our results highlight the dynamic regulation of autophagy in post-ischemic kidneys and suggest a role of mtor in autophagy resolution during renal repair. abstract# p-sat297 novel mechanisms by which heparin can regulate the vascular activity of angiotensin ii (ang-ii) and fibroblast growth factor-2 (fgf-2) and affect the outcome of acute kidney injury (aki) objective: critically ill children treated with extracorporeal membrane oxygenation (ecmo) and/or cardiopulmonary bypass (cpb) frequently develop hypertension, endothelial dysfunction, and acute kidney injury (aki). these patients show high serum levels of ang-ii and fgf-2, and are treated with heparin to prevent clotting disorders. however, the mechanisms by which heparin may affect the vascular activity of ang-ii and fgf-2 are not clearly understood. we carried out this study to determine whether heparin can modulate the vascular activity of ang-ii and fgf-2 by affecting the rho-a, rac-1, src, and pkc signaling pathways. methods: normal fvb/n mice were injected with adenoviral vectors carrying a secreted form of human fgf-2 or lac z vectors, and treated with heparin (5,000 u/kg) or control buffer (n=5 per group). vascular contractility was studied in pressurized isolated resistance-sized mouse mesenteric arteries, in the presence and absence of ang-ii and fgf-2. the activation of the src, rho-a, rac-1, and pkc signaling pathways were assessed by pull-down assays and western blots, both in vivo and in vitro. permeability changes were explored in cultured human renal glomerular endothelial cells (hrgec) using fitc-dextran. results: heparin significantly enhanced the fgf-2-induced activation of rho-a and rac-1 in the kidney and isolated mouse mesenteric vessels. in addition, heparin antagonized the ang ii-induced contractility of isolated mouse mesenteric vessels through rho-a and pkc-dependent pathways. these changes were reverted by fgf-2, and abolished by the rho kinase inhibitor y27632. both heparin and fgf-2, acting in a synergistic manner, increased the permeability of hrec by activating the rho-a and src signaling pathways. conclusion: we conclude that heparin and fgf-2, acting in a synergistic manner, can modulate the activity of ang-ii in the kidney, isolated resistance vessels, and cultured hrgec. these findings clearly identify novel mechanisms by which heparin, acting alone or in combination with fgf-2, may affect the control of blood pressure, renal perfussion, and capillary permeability in critically ill children treated with ecmo and cpb. abstract# p-sat298 cisplatinum (cis) toxicity in immortalized human kidney (hk-2) proximal tubular epithelial cells is independent of dna strand breaks and has implications for biology and therapy of renal cell carcinoma (rcc) results: viability of huh-7 and hk-2 cells decreased similarly after ic50 doses of cis with morphology and mtt assays. many dna damage genes, including atm-related genes, were expressed less at mrna and protein levels in both cell types. however, p53 expression decreased in hk-2 cells. comet assays showed extensive dna strand breaks in huh-7 but not in hk-2 cells. atm promoter activity increased in only huh-7 cells. also, s and g2/m populations were depleted by facs in huh-7 but not hk-2 cells. conclusion: hk-2 cells transformed by hpv e6/7 oncogenes displayed independence from cis-induced dna breaks and atmmediated cell cycle arrest. such independence from genotoxicity will help explain mechanisms imparting resistance to chemo-or radiotherapy and invasiveness in rcc. abstract# p-sat299 renal neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 expression in children with acute kidney injury on henoch-schonlein purpura nephritis yue du, jinjie guo, ling hou, yubin wu, tingting sun paediatrics, post graduate trainee, china objective: to evaluate serum, urinary and renal neutrophil gelatinaseassociated lipocalin(ngal) and kidney injury molecule-1(kim-1) in children with aki on hspn by prifle or hspn with nephroticrange proteinuria. methods: we performed a prospective single-center evaluation of serum, urinary and renal ngal and kim-1 in a cohort of children. we recorded any relevant data including age, gender, weight, hemoglobin(hb), serum creatinine(scr), cystatine c(cysc), serum beta2-mg, albumine, urine beta2-mg, urine protein in all patients. each patient's estimated creatinine clearance was calculated using the original schwartz formula. blood sample and five ml of urine sample from each participating patient were collected for ngal and kim-1 using an enzyme-linked immunosorbent assay. analysis of proteins ngal and kim-1 using sabc immunohistochemical assay. results: twenty five patients were enrolled in the study. nine patients with aki-on-hspn(a-on-c) and six were done renal biopsy, sixteen patients with hspn with nephrotic-range proteinuria and ten were done renal biopsy. blood cystatinc, beta2-mg , scr, ngal and kim-1 were increased significantly in patients with a-on-c than those with hspn, and so do urine ngal, kim-1 and beta2-mg. there was no significant difference of proteinuria between the patient with a-on-c and the patients with hspn. immunohistochemical results showed that ngal and kim-1 were expressed in proximal tubule and their expression were significantly higher in a-on-c group than in hspn group. the correlation analysis showed that urine ngal and kim-1 were negative correlated with gfr and they were uncorrelated with proteinuria. conclusion: blood, urine and renal ngal and kim-1 were significantly increased in patients with a-on-c than those with hspn, and they were negative correlated with gfr. we may conclude that ngal and kim-1 may diagnose a-on-c patients more sensitive than scr. abstract# p-sat300 objective: neonatal period is an important stage in the development of renal function at the children. the frequency of renal damage in infants undergoing resuscitation is high, but non-specific clinical symptoms and low information existing survey methods impede their timely diagnosis. purpose -evaluation of diagnostic importance of determining the level of carbonic anhydrase ix (ca9) in the urine of newborns in critical states. methods: we evaluated human carbonic anhydrase ix in the urine of 40 newborns who had asphyxia at birth and receiving treatment in the intensive care unit -the main group. the control group consisted from healthy newborns (13 people). for the study used a sample of newborn urine collected at 1-2 days of age. carbonic anhydrase was determined using enzyme immunoassay kit. results: human carbonic anhydrase ix (ca9) -trans membrane protein, the main physiological function of which is to regulate the ph by the reversible hydration of carbon dioxide. the main pathogenic factor causing kidney damage of fetus is chronic fetal hypoxia. there is a single study hif-i alpha (hypoxia inducible factor-i alpha) in the blood, increased expression of which occurs during hypoxia. this is connected with damage of tubules and interstitial cells of the kidneys, their proliferation, synthesis of cytokines and extracellular matrix. in this case, activity of ca9 is reduced in the blood. normally, ca9 is missing in the urine, so its definition can be a marker of early renal dysfunction. the authentic increase of ca9 level in the main group was revealed in comparison with the control group ( and β2-mg, 59,723/ 32,616/ 1511/ 996/ 1084/ 600 μg/g cr. levels of each marker were very high in nb and 1m children, and subsequently decreased gradually. urinary α1-mg levels reduced the quickest, and became the same as at >=3 years old by 6 months after birth. conclusion: renal tubular function can be evaluated in children <3 years old using these normal values. the most stable and useful marker from early infancy seems to be urinary α1-mg. urinary biomarkers for gentamicin-induced acute kidney injury in the neonatal intensive care unit objective: gentamicin (gm) is an aminoglycoside frequently used in the neonatal intensive care unit (nicu). gm is nephrotoxic and may cause acute kidney injury (aki). serum creatinine (scr) appears to be an insensitive and unreliable marker for detecting aki. to determine whether urine biomarkers are useful for early detection of gm-induced aki in neonates in the nicu. methods: prospective, clinical, observational study. forty-six neonates (32m/14f) without pre-existent kidney disease were divided in a gm group (n=26) and a reference group (n=20). demographics, vital signs and clinical conditions were recorded. only neonates with a bladder catheter in place were included. urine samples were collected every two hours. biomarkers (gsta1-1, gstp1-1, kim-1, nag, ngal) were determined. residual blood samples were used to measure scr. results: the gm and reference group were comparable for gestational age, weight and mortality. neonates treated with gm are admitted longer than neonates in the reference group. treatment with gm resulted in higher scr compared to the reference group (58. 5 [44.8-58.5] vs. 34.0 [28.3-58.8]; p<0.05). higher levels of scr correspond with higher urinary excretion of all biomarkers, especially in neonates treated with gm. the average time until the highest peak was shorter for all biomarkers compared with scr (p<0.05). there was no difference in produced urine volume between the gm and the reference group. conclusion: higher scr levels correspond with higher urinary excretion of all biomarkers, especially in neonates treated with gm. gstp1-1 seems the most useful marker for early detection of aki in neonates. seong heon kim, sang wook mun, su young kim 1 1. pediatrics, pusan national university children's hospital, yangsan, korea objective: spontaneous tumor lysis syndrome (stls) before cancer treatment is rare and develop mostly in burkitt lymphoma and non-hodgkin lymphoma. here we report a case of stls secondary to tcell acute lymphoblastic leukemia(all) which presented with renal stone and subsequent aki. methods and results: a 6-year-old boy was admitted to our hospital for generalized tonic clonic seizure. one month ago, he visited other hospital for episodic right-sided flank pain and evaluations revealed microscopic hematuria, hyperuricemia and stone on right uretero-pelvic junction. his blood pressure was 160/90 mmhg and he did not have any dysmorphic features and he had developed normally. physical examination showed tender hepatomegaly (2 cm below costal margin) and no splenomegaly. his initial laboratory findings were as follows : bun 119.3 mg/dl, creatinine 4.77 mg/dl, uric acid 47.6 mg/dl, ldh 460 iu/l, wbc 11,900 /ul, hb 9.9 g/d l, platelet 242k /u l. there was no blast on his peripheral blood smear test. emergent hemodialysis was started because of aki, hypertension and seizure. his renal function, blood pressure, uric acid and electrolyte abnormalities gradually improved with appropriate therapy after 2 times of hemodialysis. but serum uric acid level increased again (from 6.1 mg/dl to 12.9 mg/dl), so allopurinol was added on. the cause of aki was unclear and we thought that aki presumed to be secondary to acute uric acid nephropathy caused by stls. after few days, bone marrow biopsy was done and demonstrated normocellular marrow without evidence of malignancy. after day 15 of hospitalization, abdominal pain with hepatomegaly was getting worse than before and uncontrolled high fever occurred. 20% of blasts were seen on his peripheral blood smear and subsequent second bone marrow biopsy demonstrated acute t cell lymphoblastic leukemia. then, he was referred to division of pediatric hemato-oncology on our hospital and induction chemotherapy was started. conclusion: stls with aki is very uncommon initial presentation of leukemia and stls presenting with renal stone is extremely rare. in a clinical situation of marked hyperuricemia with acute kidney injury, we need to consider occult malignancy and stls. the application of serum beta-2-microglobulin (beta-2-mg) and cystatin c(cysc)concentration to evaluation of renal function impairment in patients withneonatal jaundice yanan xin, cairong jiang, junfeng yang, hui xu, yuan zhang the fourth hospital of baotou, baotou, china objective: to explore the application of serum beta-2-microglobulin (beta-2-mg) and cystatin c(cysc)concentration to evaluation of renal function impairment in patients with neonatal jaundice. methods: 60 neonates with hyperbilirubinemia from jan. of 2012 to dec.of 2012 in pediatric department and 34 full term infants were chosen as research object, hyperbilirubinemia divided into mild group (bilirubin<256.5umol/l) and moderate severe (bilirubin> 256.5umol/l) group according to the level of bilirubin. the different of sex and age were not significant between neonates with hyperbilirubinemia and control group. serum beta-2-mg, cysc, cr and bun were measured in the two groups. results: serum beta-2-mg and cysc in 60 neonates of hyperbilirubinemia were significantly higher (p<0.01) than healthy neonates, meanwhile, serum cr and bun were significantly lower (p<0.01) than healthy neonates. after treatment, serum beta-2-mg and cysc of neonates with hyperbilirubinemia were significantly lower (p<0.01) than pretreatment. however, serum beta-2-mg were higher (p>0.05) abeta-d cysc were significantly higher (p<0.01) than normal group. but there was no significant difference between mild and moderate severe group in the level of serum beta-2-mg, cysc, cr and bun(p>0.05). the result implied that neonates with hyperbilirubinemia suffered for renal function to varying degrees. howerer, the damage can partially recovered after energetic treatment. clinical and pathological analysis of iga nephropathy with acute kidney injury minguang chen, xiaohua ye, qing yang nephrology, yuying children's hospital, wenzhou, china objective: to investigate the incidence, etiology, clinical pathological characteristics and prognosis in primary iga nephropathy(igan) children with acute kidney injury(aki). methods: retrospectively analysis the clinical and pathological manifestations and follow-up results of chlidren with primary igan and aki in our department from january,1996 to jun, 2012. results: there were 19 cases with aki in 196 chlidren with igan(9.7%), the peak serum creatinine were from 94.5umol/ to 282 umol/l. histological changes: with the formation of crescent in 10 cases, diffuse endocapillary proliferation in 5 example, 15 cases of renal tubular injury, 10 cases of red blood cell and protein cast, 1 cases with acute interstitial nephritis. multivariate logistic regression analysis showed: with massive proteinuria were independent risk factors of igan in children with aki (or = 27.370, 95% confidence interval was 3.151-237.740, p<0.01). the etiology of aki except with massive proteinuria, include: 1. iga nephropathy with severe glomerular damage, including crescentic glomerulonephritis and diffuse endocapillary proliferation; 2 complicated acute interstitial nephritis; 3 drugs causing decreased glomerular filtration rate; 4. renal tubular injury induced by gross hematuria. all of the patients were not on dialysis, hormone therapy in 13 cases (including 7 cases of methylprednisolone pulse therapy), 6 cases combined with cyclophosphamide treatment. except 1 cases no significant improvement, the renal functiones of all patients recovered or improved within 1-2 months after treatment. follow-up from 1 month to 7 years, 3 cases had renal function improved, but 2 were lost to follow-up and after 3 years enter to the chronic renal failure, 1 case with renal function loss after 32 months and repeated renal biopsy showed glomerular sclerosis of 31.6% during the follow-up period. conclusion: aki is not uncommon in children with igan, the causes are varied and massive proteinuria is independent risk factor among them. we should adopt different treatment strategy according to different causes and the short term prognosis is good. abstract# p-sat307 biopsy proven acute interstitial nephritis in children objective: biopsy proven acute interstitial nephritis (ain) is an uncommon cause of acute renal failure in children. the cause of ain most is commonly due to medications such as antibiotics or infections. we reviewed our experience of biopsy proven ain in children. the biopsy database of all native renal biopsies over a 15 year period was reviewed. all biopsies which listed acute interstitial nephritis were selected and clinical and laboratory data were extracted from clinical records. results: 16 cases of ain from 540 biopsies,(2.9%) was identified. median age 11.5 years, range 1-14 12/16 cases presented with acute renal failure without an obvious cause. 13/16 cases had non specific constitutional symptoms and oliguria was reported by 6 patients. antibiotics were the most common medications implicated (5/16 patients) three patients were on multiple medications with one having chronic epilepsy and the other crohn's disease. five patients required short term dialysis ranging from 2 to 7 days.13 patients were treated with corticosteroids and at last follow up, 4 had reduced gfr 45-74ml/min/1.73m 2 . conclusion: acute interstitial nephritis is uncommon in childhood but when it occurs, antibiotics are the single commonest cause. there is significant morbidity associated with the illness with not all patients making a full recovery. abstract# p-sat308 acute kidney injury as presentation of burkitt's lymphoma eva greta ter haar 1 , anne uyttebroeck 2 , marleen renard 2 , veerle labarque 2 , djalila mekahli 1 1 pediatric nephorlogy, university hospital leuven, leuven, belgium 2 pediatric hemato-oncology, university hospitals leuven, belgium objective: acute kidney injury (aki) has become increasingly prevalent. approximately 2-3% of children admitted to pediatric tertiary care centers present with this life-threatening condition. the most common causes are post-operative septic shock, organ or bone marrow transplantations and intrinsic renal disease. the latter comprises multiple disorders, but tumor invasion due to a lymphoproliferative malignancy is very exceptional. methods and results: we report two cases of aki caused by infiltration of burkitt's lymphoma. both four-year-old male patients presented with abdominal pain, nausea, vomiting, weight loss and overall weakness since a few weeks. clinical examination showed hepatosplenomegaly and impressive bilateral nephromegaly. both had malignant hypertension. blood analysis showed severe renal impairment in both patients. the first had creatinine of 2.9 mg/dl (egfr 21 ml/min/1.73m 2 ), urea of 97 mg/dl and uric acid of 8.9mg/dl. the second had creatinine of 5.97mg/dl (egfr 10 ml/min/1.73m 2 ), urea of 215mg/dl and uric acid of 12mg/dl. lactate dehydrogenase was extremely elevated in both cases (5860 u/l; 758 u/l respectively). urinary sediment was normal. ultrasound showed bilateral nephromegaly in both patients (patient 1:+12sd and +11sd; patient 2: +10sd and +9sd, for right and left kidney respectively). mri demonstrated a homogenous renal enlargement with features of an infiltrative lesion. bone marrow was inconclusive and diagnosis of burkitt's lymphoma was confirmed by a renal biopsy. after starting chemotherapy according to the inter-b-nhl ritux 2010 protocol, both children developed a tumor lysis syndrome and required hemodialysis. at last follow-up both children still had hypertension. nevertheless, renal size and function were normalized. conclusion: lymphomatous infiltration due to burkitt's lymphoma is a rare cause of aki. however, it should be considered in a patient presenting with unexplained renal failure and bilateral nephromegaly with normal urinary sediment. renal biopsy may be needed to confirm diagnosis. abstract# p-sat309 does malnutrition interact with acute kidney injury in children? objective: to investigate the interaction between malnutrition and acute kidney injury (aki) assessed by prifle criteria and to assess the effects of these factors on the outcomes of pediatric intensive care unit (picu) patients. methods: prospective cohort study conducted on children. outcome variables: mortality, need for dialysis, picu free days (the number of days alive from icu discharge to day 28) and ventilator-free days (the number of days alive and breathing without assistance from admission to day 28). exposure variables: malnutrition on admission (who growth standards) and any change in prifle criteria during the first icu 14 days. results: of 98 patients (median age =19 iqr =50 months, 35 girls) enrolled, 39 (40%) were malnourished and 34 (35%) developed aki. among the malnourished, 19/39 cases (49%) had aki, while this complication occurred in 15/59 (25%) of patients without malnutrition resulting in a risk ratio of 1.9 (95%ci 1.1 -3.3). the 28 days mortality rate was 10/98 cases (10%) and aki was associated with higher risk (8/34 versus 2/64 cases -rr=7.5 95%ci 1.7-33.5). concurrent malnutrition and aki was present in 19/98 cases (19%) producing a tendency (p=0.2) to a further increased risk of death (rr=9.4 95%ci 1.5-58.0). 9/98 (9%) patients needed dialysis and malnutrition increased the risk for this outcome (rr=5.3 95%ci 1.2-24.2). aki was associated with significantly higher icu free days (19±7 vs. 15±10) and ventilator-free days (22±9 vs. 13±12) but concomitant malnutrition did not reduce either of these outcomes. conclusion: malnutrition and aki are common in children admitted to the icu and the presence of malnutrition is associated with increased risk of developing aki. the need for dialysis was increased in malnourished patients and more powered studies are demanded to test whether the tendency to higher mortality observed in patients with concomitant aki and malnutrition is confirmed. urine erythropoietin level is associated with kidney and brain injury in critically ill neonates yanhong li, jie yan, xiaozhong li department of nephrology, children's hospital affiliated to soochow university, suzhou, china objective: erythropoietin (epo) is a glycoprotein hormone produced predominantly in the kidneys. the protective effect of exogenous epo in hypoxic-ischemic brain injury has been thoroughly examined in neonates. however, the metabolism of endogenous epo in neonates remains unclear. we aimed to evaluate the concentration of urinary epo (uepo) in critically ill neonates and to identify possible clinical and laboratory variables that may be associated with uepo levels. methods: the concentrations of epo, cystatin-c, microalbumin, and α 1 -microglobulin in the first available urine sample during the initial 72 hours of life were measured in 103 critically ill neonates. clinical and laboratory data were collected for each neonate. results: there was a positive correlation between uepo levels and urinary levels of cystatin-c (r =0.265, p =0.008), microalbumin (r =0.422, p <0.001), andα 1 -microglobulin (r =0.421, p <0.001). the concentration of uepo was elevated in neonates who developed acute kidney injury (aki) during the first week of life compared with those without aki (p =0.002) and was also elevated in neonates with brain injury, as demonstrated by ultrasound or magnetic resonance imaging, compared to neonates without brain injury (p =0.008). an increased log 10 uepo level was associated with the occurrence of aki (odds ratio of 2.70, p =0.007) and brain injury (odds ratio of 2.33, p =0.016). conclusion: an increased urinary epo level in the early postnatal period is significantly associated with kidney and brain injury in critically ill neonates. abstract# p-sat311 acute kidney injury following extracorporeal membrane oxygenation support and concomitant hemofiltration -the role of diuretics objective: fluid overload (fo) is common during extracorporeal membrane oxygenation (ecmo) and can be managed by continuous hemofiltration (hf) and/or diuretic therapy. although combination therapy of hf and diuretics in particular can be most effectively for fo removal during ecmo, it may also increase the risk for prerenal acute kidney injury (aki) post-ecmo. our objective was to describe the incidence of aki post-ecmo in patients treated with ecmo and hf who received concurrent diuretics. methods: in this cohort study all neonates (≤28 days after birth) treated with ecmo and concomitant hf between 2007 and 2011 were included. patients were divided into two groups based on diuretic regimen: group-1, patients who received no or one single diuretic dose during ecmo and group-2, patients who repeatedly received diuretics. both groups were compared using mann-whitney u test for nonparametric data and chisquare test for categorical data. aki was defined as the highest serum creatinine (scr)-based rifle class reached on post-ecmo day 1 up to post-ecmo day 4 (risk, injury, or failure being 150%, 200% or 300% of median scr reference values for age). results: 56 neonates received ecmo support with hf, 6 died immediately following decannulation and 6 others were lost to followup since they were transferred back to their referring hospitals within 3 days post-ecmo. of the 18 patients in group-1, 3 (17%) qualified as risk and 1 (6%) as injury. of the 26 patients in group-2, 3 (12%) qualified as risk, 4 (15%) as injury and 9 (35%) as failure (group-1 vs. group-2, p=0.039). patients in group-2 had an increased ecmo duration (p=0.001) and number of ventilator days (p=0.002). no differences were observed in baseline characteristics (e.g. age at the start of ecmo, underlying diagnosis, severity of illness scoring using the pim and prism risk adjustment systems), hf flow rate, and fluid balance between both study groups. conclusion: the incidence and severity of aki immediately post-ecmo was significantly higher in patients who repeatedly received diuretics in addition to hf during ecmo. valentina sitnikova, yuliya pashkova, tatiana zvyagina, elena kulakova, alexandra nastausheva voronezh state medical academy, voronezh, russia objective: it is actually to find the new markers of kidney damage and spread them in practice. cystatine c (cys) is one of these, but there are only few data about it in children with urinary tract infection (uti) according to the age. the aim of our study was to evaluate serum concentration of cys in children with uti of different age. we investigated 83 patients (47 girls and 36 boys) with uti from 1 month to 17 years among them 32 children were under 1 years old. methods: concentration of cys were measured by immunoenzime method (elisa) with test-system biovendor (check republic). results: the mean cys concentration in children with uti under 1 year was 1.43± 0.37 mg/l, in older children it was lower: 1.23 ± 0.36 mg/l, p= 0.025 (mann-whitney). in 16 girls before 1 year cys concentration was 1.47± 0.32 mg/l and in 16 boys of the same age it was 1.40±0.43 mg/l, p>0.05. in 31 girls over 1 year cys concentration in serum was 1.31± 0.41 mg/l, in 20 boys -1.3± 0.33 mg/l, p>0.05. the results of our study showed that among children with uti serum concentration of cys was higher in children of the first year of life and did not depend on sex. probably, higher level of cys in infants connects with low glomerular filtration rate in children of this age. microalbuminuria can prognosticate outcome in the critically ill children biplab maji, surupa basu, rajiv sinha paediatrics, post graduate trainee, kolkata, india objective: microalbuminuria [albumin creatinine ratio (acr) >30 mg/g] increases in acute inflammatory conditions as a result of glomerular endothelial dysfunction. the study evaluated its prognostic potential in the critically ill child. methods: an ongoing prospective observational study (oct 2012-mar 2012) of random urine acr (mg/g) estimated on day1 and day 3 of pediatric intensive care unit (picu) admission, of a tertiary care pediatric hospital. demographic, laboratory data, vasopressor use, mechanical ventilation, length of stay, outcome and pelod scores were recorded. results: of 113 children, 72 children (median age 4.5years, 68% male) recruited. the commonest cause of admission was pneumonia, infection associated hemophagocytic lymphohistiocytosis followed by meningitis. day1 acr (median 70.8 mg/g) significantly correlated with day 3 total leukocyte count (p=0.016). both day 1 and day 3 acr correlated with pelod scores [median 8] (p=0.002 & p= 0.003 respectively) and duration of mechanical ventilation (p=0.003 and p=0.001 respectively). both median day 1 acr, and day 3 acr were significantly different between survivors (n= 29) and non-survivors (n=11) (p=0.0001 and p<0.0001, respectively). roc curve analysis for mortality prediction revealed the highest area under curve (auc) of 0.963 for acr3, followed by 0.928 for acr1 and 0.867 for pelod scoring (p>0.05, non-significant for auc comparisions) (fig. 1.) . a cut-off value of day 3 acr of 102.4mg/g had a positive predictive value of 95.8% for negative outcome conclusion: microalbuminuria is an early inflammatory marker that correlates with organ dysfunction. significant levels reliably predict negative outcome early into intensive care admission, as accurately as pelod scores, which can help counsel patients, plan treatment and triage, and allocate resources judiciously. abstract# p-sat314 an unusual presentation of congenital nephrotic syndrome caused by wt1 mutation ann raes, sofie maebe, joke dehoorne, bert callewaert, johan vande walle pediatric nephrology, university hospital, gent, belgium objective, methods and results: a caucasian girl was admitted at the age of 28 days because of progressive lethargy, poor feeding, oliguria en peri-orbital edema. extreme hyponatremia (89 mmol/l), severe renal insufficiency (creat 1.9 mg/dl, p 10.7 mg/dl), low plasma albumin (1.5 g/dl), respiratory compensated metabolic acidosis (arterial ph 7.31 pco 2 27 mmhg, bicarbonate 13.5 mmol/l, be -10.9 mmol/l) and massive proteinuria (12 g/l). clinical examination was normal without anomalies of the external genitals or other dysmorphic features. despite supportive therapy (sodium, bicarbonate, albumin and furosemide) she progressed to respiratory failure and renal failure with need for mechanical ventilation and peritoneal dialysis. congenital infections and thrombosis was ruled out. histological examination showed diffuse mesangial sclerosis (dms). the combination of congenital nephritic syndrome and dms on biopsy was suggestive for mutations in the wilms' tumor 1 (wt1) gene. genetic analysis showed normal female karyotype and conformed a heterozygosity for the c.1223c>g or p.his441gln mutation within the zinc finger 2 domain. after a long and complicated icu episode, a stable situation with home choice peritoneal dialysis was achieved. due to the important risk for comparision of auc of pelod with acr 3, p= 0.766 for comparison with acr 1) for the prediction of mortality in the pediatric intensive care unit. development of wilms' tumor, bilateral nefrectomy was performed and kidney transplantation will be planned. we present an unusual case of congenital nephrotic syndrome with extreme hyponatremia at presentation and fast progression to esrd, due to mutation in wt1 gene. several genes have been implicated in congenital nephrotic syndrome. genetic testing is mandatory and can add important information such as risk of malignancy. renal histology can play an important role in the approach for appropriate mutational screening. mutations in the wt1 gene are associated with denish drash syndrome and frasier syndrome, but also with isolated and sporadic steroid resistant nephrotic syndrome. in the last category the majority of patients is female, the age of onset and time to esrd is variable, although the vast majority of cases are older at presentation and time to esrd is longer than in our case. objective, methods and results: a 8-year-old girl, affected by b thalassemia major, was admitted for proteinuria detected at the emergency unit. fever, hyporexia and diarrhea had been present in the last three days. on physical examination she presented with decreased skin turgor and hypertension. laboratory examinations showed: hyponatremia (124 meq/l), hypokalaemia (2.9 meq/l), hypophosphatemia (1.1 mg/dl), hyperazotemia (60 mg/dl), reduced creatinine clearance (crcl = 68 ml/min/1.73 m 2 ) and metabolic acidosis (hco 3 -12.8 mmol/l). urine analysis revealed: proteinuria, glycosuria, hypercalciuria, hyperuricuria, hyperphosphaturia and aminoaciduria. she had been under treatment for five years with deferasirox (dfx) 26 mg/kg/die. we stopped dfx and gave supplementation of potassium, phosphate, sodium bicarbonate and sodium chloride with electrolyte and renal function (crcl 130 ml/min/1.73 m 2 ) normalization in 6 days. after one month only minimal proteinuria persisted. conclusion: in so far a fanconi syndrome (fs) has been reported in 11 patients (6 children), while mild renal insufficiency (ri) has been documented only in 2 children. in our case dehydration seems to be responsible for the ri, while an interstitial nephritis cannot be excluded. close monitoring of renal function should be done in children on dfx. objective: to test the hypothesis that neutrophil gelatinase-associated lipocalin (ngal) and interleukin-18 (il-18) are early biomarkers for aki in critically ill patients and evaluate the predictive value of them in patients with established aki at inception of crrt. methods: children from picu and health examination center in guangzhou women's and children's medical center were divided into four groups: critically ill patients with aki recevied crrt group (group1 ), critically ill patients with non-aki recevied crrt group(group2) , critically ill patients with aki don't recevie crrt group (group 3),healthy control group (group4). 1.5 ml venous blood and urine specimens were collected and was kept under -70°c.from each patient in picu untill they were discharged, transferred or die . serum creatinine (scr) and urine ngal and urine il-18 were analyzed . results: compared with group2 and group4, the urine ngal and urine il-18 increases obviously in group1 and group3 (p<0.05). there is no significance of urine ngal and urine il-18 between group2 and group 4.(p> 0.05).the concentration of urine ngal increased more than10 times obviously 2 days before diagnosed of aki under the akin standard with auc 0.841(p <0.05) , and the concentration of urine il-18 increased more than 5 times 2 day before aki with auc 0.808(p <0.05). the auc was 0.943(p <0.01) when they were combined. the level of urine ngaland il-18 at initiation of rrt were higher in in non-survivors compared to survivors. conclusion: urine ngal and urine il-18 are useful indicators to predict and early diagnose aki. the level of urine ngal and il-18 at initiation of rrt.were negative correlation with the outcome of renal prognosis and survival rate . objective: the aim of this study was to evaluate the rate of early kidney injury in infants with congenital heart disease (chd). neutrophil gelatinase associated with lipocalin (ngal) which can be measured both in serum (sngal) and in urine (ungal), is at the moment the most promising marker directed to discover early kidney injury within the glomeruli and distal and proximal tubules. less known is cathepsine l as a marker of tubule necrosis. methods: the study group consisted of patients (22 boys and 12 girls) with congenital heart disease. all patients were under 3 years of age (mean 8.2 months). blood and urine samples were obtained during routine checkups. all patients had normal serum creatinine level at the day of collecting samples. the control group (n = 20) was age-and gender-matched. levels of sngal, ungal and cathepsine were compared in whole group. the study group was divided depending on the nature of the defect (cyanotic/acyanotic) and the treatment (surgical/conservative treatment). results: chlidren with congenital heart disease had significantly higher concentration of sngal, ungal and urine cathepsine than the control group. no significant difference was observed in the urine cathepsine and urine and serum ngal level between patients with acyanotic and cyanotic chd. there were also no significant differences in patients who underwent surgery and on conservative treatment in ngal level (both in urine and serum). cathepsine level was significantly higher in group after surgical treatment. conclusion: patients with chd are at higher risk of early kidney damage. this process is independent on the etiology and nature of the defect. ngal determined in the serum and urine of these patients may be used to detect kidney injury or monitoring disease progression. urine cathepsine is more efficient marker of early kidney injury in patients after cardiac surgery. abstract# p-sat318 tim-3 expression in kidney mononuclear cells and its relationship with foxp3+ tregs from mice with renal ischemia reperfusion injury yamei wang, yuhong tao, li ye department of pediatrics, west china second university hospital, chengdu, china objective: foxp3 + tregs participate in the repair of renal ischemia reperfusion injury. t cell immunoglobulin-3 (tim-3) plays a pivotal role in several inflammatory diseases by modulation of foxp3 + regulatory t (treg) cells. to detect the expression of tim-3 in kidney mononuclear cells (kmncs) from mice with renal iri and analyze the relationship with foxp3 + tregs, to explore the role of tim-3 in repair of renal iri. methods: the left renal pedicle was clamped in c57bl6 mice for 45 min, followed by reperfusion. animals were sacrificed at baseline, day 1,3,10, 21 after iri. tim-3 expression in kmncs were determined using real-time rt-pcr and flow cytometry. the percentage of foxp3 + treg in cd4 + t cells was quantified by flow cytometry. the levels of tnf-α、ifn-γ、il-4 and il-10 in kidney tissue were measured using elisa. the correlation among tim-3 expression, foxp3 + treg and cytokine level was analyzed. results: the expression of tim-3 expression in kmncs from injured kidney at acute stage(day 1) was significantly higher than those from uninjured kidney at acute stage and lower than those from injured kidney at repair stage (day 3,10, 21). the percentage of foxp3 + treg in cd4 + t cells was up-regulated with time. compared with the uninjured kidney, the expression of tnf-α and ifn-γ increased significantly in the injured kidney at acute stage and the expression of il-4 and il-10 increased significantly in the injured kidney at repair stage. the tim-3 expression in kmncs at iri repair stage was positively related with the level of foxp3 + treg, il-10 (r=0.81, r=0.79 respectively; p<0. 05), but negatively related with the level of tnf-α and ifn-γ. conclusion: the increased expression of tim-3 in kmncs may take part in the repair of renal iri and contribute to the development of foxp3 + treg. objective: (d+)hus is a critical health problem in argentina since it is the main cause of acute renal failure in children and the second cause of chronic renal failure. fecal contamination of food and drinking water by asymptomatic cattle is often the source although secondary infection through personto-person contact may also occur. occasionally in (d+) hus, the onset among affected siblings occurred within a short time of each other. to evaluate the risk and clinical severity of illness for childhood (d+)hus in siblings. methods: we retrospectively analyzed the clinical records of 133 children with d+hus that were admitted in our pediatric department between march 1997 and december 2012. results: (d+) hus occurred in 2 siblings in 4 of the 129 families studied (3%). 16 patients had an affected sibling with diarrhea and 4 progressed to hus (25%). family cases: mean age: 28 months, 5 were girls. the mean duration of interval between hus episodes was 4 days. second family members had prolonged oligoanuria (3 vs 12 days) and a most of them developed neurological complications. long-term renal complications were more frequent in this group, but differences were not statistically significant. conclusion: these findings emphasize the potential for extensive intra-familiar transmission of stec, especially between siblings. a second family member might also develop an even more severe hus episode, so siblings should be kept under close surveillance. objective: kawasaki disease (kd) is a common cause of systemic vasculitis in children. other than well known complications like coronary artery aneurysm, there have been few reports of this disease involvement in renal system. according to the study from wang jn et.al, 52% (26 out of 50) of the patients who suffered from kd showed renal inflammatory foci in dmsa renal spect which suggested a possibility of renal scar formation subsequent to kd. therefore, this study was performed to verify the renal inflammation following kd. methods: from march 2011 to october 2011, 15 patients who were diagnosed as kd at national health insurance service ilsan hospital were enrolled to the study. all of the patients underwent dmsa renal spect to evaluate renal involvement during their acute phase of kd. echocardiography was performed to assess cardiac involvement such as coronary artery aneurysm. complete blood cell counts, aspartate amino-transferase, alanin amino-transferase, albumin, c-reactive protein, and bun/creatinine were measured. also urine β2microglobulin was measured to assess renal tubular function. addition to fever of more than five days of duration, 4 of the following symptoms, rash, conjunctival injection, changes of lips or oral mucosa, erythema and swelling of hands and feet and cervical lymphadenopathy, was the diagnostic criteria for kd. results: among the 15 patients, 80% showed increased white blood cell s (wbc) and 47% showed elevated ast/alt level. serum albumin was below 4.0 g/dl in 93% of the patients. all of the patients presented normal renal function test. in urinalysis, hematuria and pyuria were observed in 13% and 33% respectively. echocardiography revealed coronary artery aneurysm in 33% of the patients. urine β2-microglobulin was elevated in 46%. regardless abnormal findings in urinalysis and elevated β2microglobulin, no significant findings were observed in dmsa renal spect. conclusion: according to the study, mild abnormality in the urinalysis and elevated β2-microglobulin were the only findings of renal involvement in kd. however there was no aggressive renal manifestation which could be detected in dmsa renal spect. objective: in this study, the question remains if ngal is a culprit or only bystander in aki due to sepsis in children. methods: twenty seven children, (m-17, f-10) admitted to intensive care diagnosed with sepsis, severe sepsis or septic shock were enrolled in this study. the concentration of ngal, protein c and s, antithrombin iii and basic parameters were measured in the plasma at diagnosis and after 10 days of treatment. results: mean creatinine concentration in septic children at admission was higher than in reference group (p <0.05). there was statistically significant difference in aptt, prothrombin time, prothrombin ratio, thrombocytes count, heart rate, and systolic blood pressure between study groups in first and after 10 days (p<0.005, p<0.001, p<0.001, p<0.01, p<0.05, p<0.05 respectively). the pc concentration in the septic patients was significantly lower than those of the references (p<0.0001), and during intensive 10 days treatment raised (p<0.01). also protein s concentration rose during the treatment (p<0.005), and was lower in septic children then reference group (p<0.0001). ngal concentration was significantly higher in study group on the admission day comparing with reference group (p<0.05). plasma ngal was correlated at admission and after 10 days of treatment with protein c (respectively: r=-0.48, p<0.00005; r=-0.53, p<0.05). multivariate regression revealed that ngal was significantly predicted by creatinine (β=0.39, p<0.01) and protein c (β=-0.38; p<0.01), yielding a model r2 =0.34 (p<0.001). conclusions: it is possible that kidney binding of pathogenic antibodies stimulates local expression of ngal, which plays a crucial role in the pathogenesis of aki. objective: the aim of the study was to assess the impact of perinatal risk factors (prfs) on serum ngal level in term neonates. methods: the study group consist of 62 term neonates with prfs, and 14 healthy neonatescontrol group (cg). serum ngal (elisa) was measured in samples of cord blood (cngal) and peripheral blood (pngal) taken within first 5 days of life, and stored in -80°c until elisa procedure was performed. prfs were divided into 3 groups: 1. pathology of pregnancy or mother: diabetes (d) n=23, infection (i) -elevated crp or wbc n=20, positive vaginal culture (vc) n=21, hypertension (ht) n=7, others n=5; 2. labor pathology: instrumental delivery (id) n=41, premature rupture of membranes, >6 hours (prom) n=26, fetal distress (fd) n=26; 3. neonatal pathology: intrauterine infection (ii) n=10, cakut n=5, perinatal asphyxia (pa) n=4. the results were shown as median; values of cngal, pngal were logtransformed before analysis. statistical analysis was performed with t-student, manova tests with help of statistica 10. statistical significance: p<0.05. results: in 62 term neonates, median cngal was 117.69ng/ml vs 64.37ng/ml in cg. mean log cngal was significantly higher (p<0.01) vs cg, pngal was ns. mean log cngal correlated positively with mean log pngal (r=0.36, p<0.01). univariate analyzes of log ngal in neonates with chosen prfs vs neonates without particular factor showed significantly higher (p<0.05) mean log cngal in id and fd group (median cngal: 126.47 vs 67.50 and 122.30 vs 77.01ng/ml respectively), whereas in ii group significantly higher were mean log cngal and mean log pngal (median cngal 209.69 vs 96.52ng/ml, pngal 457.35 vs 156.37ng/ml). multivariate analysis showed that the impact of id was significant in groups: d (f=9.84; p<0.002), i (f=4.56; p<0.04), prom (f=6.01; p<0.02) and fd (f=5.36; p<0.02). conclusion: 1. intrauterine infection may have a significant influence on ngal in cord and peripheral blood whereas fetal distress and instrumental delivery only in cord blood. 2. the instrumental termination of pregnancy has an import impact on ngal level in cord blood of neonates whose mothers had diabetes or infection, with prom or fetal distress. objective: acute kidney injury (aki) is a common problem and associated with significant morbidity and mortality in neonates. pediatric-modified rifle (prifle) classification system was developed to standardize the definition of aki in children. we aimed to evaluate the performance of prifle score diagnosis, severity and prognosis of aki in term and preterm neonates. methods: in this retrospective study, charts of 820 patients who were admitted to neonatal intensive care unit (nicu) over a 4 year period were reviewed for development of aki. a diagnosis of aki was determined for 254 patients (30.9 %) according to the prifle criteria results: of the 254patients included in this study, 101 of them were girls and 153-were boys. mean age was 4.12days (min-max: 0-49), mean birth week was 33.6 ±5.1 weeks (min-max: 24-42), mean birth weight was 2166 ± 1045 gr (min-max: 580-4600 gr), length of nicu stay was 37.3 ± 33.5(min-max: 2-165days). two hundred fifty four patients with aki were classified according to prifle criteria. ninetyeight patients (38.5%) achieved an 'r' level of aki severity, 86 patients (33.5%) an "i" level, 66 patients (26%) an "f" level, 4 patients (1,5%) an "l" level. the most common etiologies of aki -were prematurity, congenital heart disease, hypoxic ischemic injury, sepsis and usage of nephrotoxic agents. patients classified with a higher prifle score had a longer nicu stay and required a greater number of mechanical ventilation (p<0.05). renal replacement therapy (rrt) was needed for 30 patients (11.8%). mortality rate was 26% and all deceased patients were classified as prifle 'i','f' or 'l',p < 0.05) conclusion: using prifle criteria in neonates with aki is an efficient way to assess the severity and prognosis of the disease. objective, methods and results: i present a case of a 11 years albanian old boy, 25th centile of development. presented with abdominal pain, vomiting. four days after, was addmited in the surgery clinic for uregent apendicitectomy. wbc: 23.0, se: 64/, bun: 6.1, cre: 60, urine: normal. after the intervention medications that were used were: gentamycin, metronidazol, cephtriaxon, h2 blocators. two days after he was better. thereafter he had polyuria and frequent urinating. the fifth day he had oliguria to seventh day, when he couldn't urinate. nativ rtg of abdomen resulted normal. he was transferede to pediatric clinic-nephrology department as an akute kidney injory. he looked very sick, consious, stratified tongue, ecg: 50b/min, bp: 115/60 mmhg, breathing sounds were normal, periferal edemas and cold extremities. lab resultrs: cre: 624, bun: 16.6, k: 5.1, na: 131, egfr: 8.8 ml/min oer 1.73m2. ultrasound resulterd with urinary stasis: hydronephrosis, huge bladder. first was suspected for acute obstruction and aki couse of a medication. we changed a bladder cathether which we find out that was blocked and took out 2700 ml urine/24h. we dialysed him for three days and cre was stabilized to 266, when we stoped dialyse. bp was stabile, his weight droped from 32kg to 27.5 kg. he had proteinuria 2 g/24 h. schintigraphy was done (biopsy we couldn't do) and resulted: egfr was 12% less than adecuat for age, suspected for nephritis tubulointestialis. he had some toxoalergic exanthema spread to the body. repeted us resulted with ascaridosis in the gallbladder, which was treated with mebendazole and in the feces were found the parasites. imune ab were negativ, ige: positive, periferal blood smear had eosinophils, wastage of na and mg with urine. cre after four weeks was 58, bun:5, negative proteinuria. no changes of uveitis in the eyes. importance of this case is cause this is the first case in kosova that was dialysed and has recovered and now is free of dialyse (all other cases have traveled abroad until then). conclusion: we have to think for parasites as a cause of a tubulointesticial nephritis but also of a abdominal pain or fatigue. surgery team should have a close cooperation with pediatricians, in order to identifye in time complications of the surgery. urine output in first 48 hours after birth in relation to akin criteria saroj kumar patnaik, gaurav aggarwal, uday kumar, vempati venkateshwar, shamsher singh dalal pediatrics, command hospital air force, bangalore, india objective: applicability of akin urinary output (uo) criteria in neonates during first 48 hours of life remains contentious since nonpassage of urine during this time has traditionally been considered 'normal'. we hypothesize that 'physiological' oliguria in first 48 hours of life is mostly prerenal in origin related to poor fluid intake during transition and uo criteria should be applicable in the 1st 48 hours of life too. we aimed a) to investigate pattern of uo in babies admitted to nicu with underlying morbidity but regulated fluid intake versus roomed-in babies in postnatal ward on breastfeeds in first 48 hours of life; b) to relate uo till 48 hrs of life with development of aki methods: prospectively timing of first void and 6 hourly uo from birth till 48 hours age was compared between consecutive postnatal ward and nicu admissions. neonates with structural malformations and requiring ventilatory/vasopressor support were excluded. outcomes-primary : time of first void after birth secondary: uo in 1st 48 hrs in relation to subsequent aki results: amongst eligible 87 postnatal breastfed babies (mean bwt 2.86 kg ; sga 18; 27 lscs born) and 110 nicu admissions (median birthweight 2.01 kg,mean ga 34 wks (26-43 wks); 67 prematures (10 < 30 wks); 82 sga (6 elbw) recruited after informed consent, first voiding was significantly earlier for nicu babies (mean(95%ci) (hrs) postnatal 4.12 (3.29, 4 .93) vs nicu 2.31 (1.50,3.12) hrs). nonvaginal delivery(lscs 3.00 (1.89,4.11) vs vaginal 4.68 (3.60,5.75 ) and normal birthweight (aga 4.08(3.10, 5.06) vs sga 4.22(2.81, 5.63) ) had a trend for earlier voiding. 80% babies voided within 6 hrs of birth -more in nicu babies (90.4% nicu vs 76.7% postnatal (p<0.004) (fig) .asphyxia with mas in nicu and iugr and poor feeding in postnatal ward had delayed first void beyond 12 hours. 6 postnatal and 7 nicu neonates anuric in 1st 6hrs met stage i akin creatinine criteria. 2 asphyxiated babies anuric beyond 12 hours in nicu developed stage iii aki. conclusion: 'physiological' anuria in 1st 48 hrs of life is misnomer and is related to fluid intake. most babies are nonoliguric by 6 h; anuria >12 h needs investigation. akin urinary criteria should be applicable in neonates. odillha morales maglalang-reed nephrology, philippine children's medical center, quezon city, philippines objective: the precise mechanism of renal injury among dengue patients is not known. patients who have atn will usually require early dialysis. however, on admission to the hospital, it is difficult to distinguish dss patients with atn from patients with reversible prerenal causes that will respond to simple hydration. our understanding of the complex pathogenesis of tubular injury in dengue aki is very limited that until it is sufficiently increased, therapeutic strategies will continue to fail.therefore we sought to explore the limitations of serum creatinine in this setting. general objective is to determine the clinical and diagnostic factors which are predictive for the need for dialysis among dss patients at pcmc. specific objective is to determine if the following factors are predictive of the need for dialysis: decrease in estimated creatinine clearance by 75% or <35 ml/min/bsa with urine output of <0.3ml/kg/hr. x 24 hours or anuria of 12 hours, assess the usefulness of urinary sediment scoring (uss) in predicting the need for dialysis, methods: this retrospective study covered 60 newly admitted cases of dengue shock syndrome lll and lv at the philippine children's medical center between january 2010 to december 2011. results: data from 60 patients were available for analysis. comparison of the demographic characteristics between patients who required dialysis and those who did not showed no significant difference as proven by all p values >0.05. of the differentclinical and laboratory parameters, there was a significant difference in the hr, rr, o 2 saturation, bicarbonate and base excess as proven by all p values <0.05. the hr an rr were significantly higher among those who needed dialysis than those who did not o 2 saturation, bicarbonate and base excess were significantly lower among those who needed dialysis than those who did not. the estimated creatinine clearance was significantly lower among those who needed dialysis than those who did not. urine sediment score (uss) ≥3 was significantly higher among those who needed dialysis than those who did not. conclusion: our data indicate that a decrease in estimated creatinine clearance by 75% or <35 ml/min/bsa with urine output of <0.3ml/kg/hr. x 24 hours or anuria of 12 hours is not predictive of dialysis among dss-induced aki as well as other clinical and laboratory indices. but rather, relatively lower serum creatinine level among dssinduced aki who needed dialysis is associated with greater urine sediment scorecompared with those who did not. a uss ≥ 3 correlated with atn and was predictive of early dialysis.however, estimated crea < 50 is the only factor predictive of dialysis, since this is the only one that was positive after logistic regression. abstract# p-sat327 pre-operative fgf23 predicts acute kidney injury in pediatric cardiac surgery patients: a prospective study mark r hanudel, myke federman, barbara gales, georgina ramos, vicky campbell, kristen ethridge, mary scotti, brian reemtsen, isidro b salusky, katherine wesseling-perry pediatrics, ucla, los angeles, usa objective: fibroblast growth factor 23 (fgf23) is a phosphaturic hormone that predicts renal disease progression in ckd. however, in the setting of aki, there is a paucity of prospective data on fgf23, especially in the pediatric population. thus, we prospectively measured fgf23 levels in pediatric patients undergoing cardiopulmonary bypass (cpb) and assessed the ability of fgf23 to predict aki. methods: pediatric patients, age newborn to 21 years, without underlying ckd, undergoing cardiac surgery requiring cpb were eligible for the study. plasma fgf23 levels (2nd generation cterminal, immunotopics) were measured pre-operatively and at 2, 6, 12, 24, 48, and 96 hours post-reperfusion. serum creatinine was obtained at baseline and daily post-reperfusion. aki was defined by the akin criteria, estimated gfr was calculated using the schwartz formula, and cardiac surgery complexity was classified via rachs-1 score. results: of the 20 enrolled patients, 13 developed at least stage 1 aki. gender, weight sds, height sds, baseline egfr, cardiac surgery complexity, and cpb duration did not differ between the aki group and the non-aki group. patients who developed postoperative aki were younger than those who did not. preoperative fgf23 levels were inversely related to age (r = -0.67, p = 0.001). pre-operative fgf23 levels were significantly higher in patients who developed aki than in those who did not; this remained significant upon correcting for age. post-operatively, fgf23 levels increased in all patients. conclusion: pre-operative fgf23 levels predict the development of post-operative cpb-associated aki. fgf23 levels increase in non-ckd patients undergoing cpb, even in patients without significant changes in serum creatinine, suggesting that fgf23 may be a more sensitive marker of aki than serum creatinine. non-aki pateints p value objective: improved perinatal care increased not only the survival rate but also the frequency of acute kidney injury (aki) in newborns. we aimed to determine the frequency, etiology, clinical course and mortality of aki in a third level neonatal intensive care unit (nicu). methods: medical records of all patients admitted to a nicu in western turkey during 2007-2011 were evaluated and those having aki within 0-30 days of life were determined. birth weight, gestational age, mode of delivery, gender, maternal morbidity, hospitalization period, accompanying morbidities and mortality of all patients; and primary disease causing aki, highest serum creatinine and prognosis of patients with aki were recorded. results: there were 677 patients (m/f:392/285) and 94 (13,9%) had aki of which 80% developed during 0-7 days of life mostly due to birth asphyxia, hypovolemia, cardiac disease, sepsis and urinary system anomalies. aki incidence and total mortality rate were higher in patients with birth weight <1000 g and with gestational age <28 week. however, mortality in patients with aki was increased independent on birth weight (42,1% in <1000 g vs 32,1% in >1000 g; p=0,324) and gestational age (44,1% in <28 week vs 31,7% in >28 week; p=0,227). mortality tended to increase in the presence of aki independent on the underlying cause, but this was significant only for sepsis (33, 3% vs 5, 9%, p<0, 001; or 7, 9) and cardiac diseases (50,0 %vs 15,0%, p=0,001; or 5, 6) . hospitalization period was increased if aki was present (39,1 vs 20,5 days, p<0,001). serum creatinine in deceased patients with aki was higher than those who survived (2,0 vs 1,6 mg/dl, p<0,05). chronic kidney disease was developed in 6 (10%) patients of whom 3 had urinary tract anomalies and 3 had birth asphyxia. conclusion: aki incidence in nicu was 14%, and 80% of aki developed during the first week of life. aki is associated with low birth weight, prematurity, birth asphyxia, sepsis, hypovolemia, cardiac diseases and urinary tract anomalies. mortality in patients with aki is increased independent on the birth weight, gestational age and underlying etiology. objective: cisplatin (cddp) is one of the antineoplastic agents widely used for solid tumors in adults and children. an excessive dose due to medical error causes severe nephrotoxicity, ototoxicity and myelosuppression. previous reports described the effects of plasma exchange, sodium thiosulfate, n-acetylcysteine and other interventions. however, the strategy for over-dose patients, as regards the selection or intensity of therapies and the target of cisplatin removal, remains uncertain in children. a 12 year-old girl was admitted for headache and vomiting. she was found to have a right cerebellar tumor and underwent total extirpation of this medulloblastoma. next, we administered chemotherapy mainly using cddp. however, she developed renal insufficiency and hearing loss on the fourth day. we then realized that we had been administering cddp 90 mg/m 2 for 4 days, instead of the intended 1 day. we promptly discontinued the cddp and began plasma exchange and sodium thiosulfate administration. nonoligiric renal failure was confirmed by a urea nitrogen value of 63.6 mg/dl and creatinine level of 4.4 mg/dl. with 4 cycles of plasma exchange and 2 weeks of sodium thiosulfate, renal function improved and the cddp concentration decreased from 6.04 to 0.99 mg/ml. after 1 month, the concentration was 0.34 mg/ml. we switched from cddp to carboplatin, and chemotherapy was continued for 4 courses and then radiation therapy was added. ultimately, she had mild renal insufficiency and moderate hearing loss. we evaluated the relationship between renal function and the cddp concentration. severe renal failure and hyponatremia were observed at a cddp concentration of 1 mg/ml or more. creatinine clearance of 60 ml/min/1.73 m 2 was maintained at a cddp concentration below 0.5 mg/ml. conclusion: in children with a cddp over-dose, plasma exchange and sodium thiosulfate administration are effective. also, monitoring of cddp concentrations is recommended in patients with acute renal failure. in over-dose patients, early severe renal failure can be prevented when the cddp concentration is reduced to less than 0.5 mg/ml with these therapies. abstract# p-sat331 peritoneal dialysis in children with acute kidney injury: a developing country experience om p mishra 1 , aditya k gupta 1 , vishal pooniya 1 , rajniti prasad 1 , narendra k tiwary 1 , franz schaefer 2 1 pediatrics and medicine, institute of medical sciences, varanasi, india 2 division of pediatric nephrology, heidelberg university medical centre, heidelberg, germany objective: peritoneal dialysis (pd) is the preferred and convenient treatment modality for acute kidney injury (aki) in children and hemodynamically unstable patients. the present study analyzed the efficacy of pd in patients with aki and factors contributing to mortality. methods: the outcome of acute pd was studied in 57 children (39 males) with aki, aged 1 month to 12 years, at a tertiary care centre of a teaching hospital in india. results: there were 14 patients less than 1 year of age, 23 patients 1-5 years of age, and 20 patients more than 5 years of age. per the rifle criteria, 5 patients were classified at the risk stage; 13, at the injury stage; and 39, at the failure stage at the time of the decision to start pd. hemolytic uremic syndrome was the most common cause of aki (36.8%), followed by septicemia (24.6%) and acute tubular necrosis (19.3%). treatment with pd was highly effective in lowering retention markers (blood urea decreased by 40% and serum creatinine by 34% during the course of dialysis therapy, both trends significant at p < 0.001). overall mortality was 36.8%. deaths occurred 2-7 days after hospitalization. significantly higher proportion of non-survivors had fluid overload (66.7% vs 25%, p= 0.002) and septicemia (47.6% vs 11.1% , p< 0.001) than survivors at presentation. the risk of mortality by multivariate analysis was higher when patients were anuric [odds ratio (or) 8.2, 95% confidence interval (ci) 1.3-49, p<0.05), had septicemia (or 3.79, 95% ci 1.55-25.8, p<0.05), or severe infectious complications (or 8.2, 95% ci 1.5-42.9, p<001). conclusion: because of its simplicity and feasibility, acute pd is still an appropriate treatment choice for children with aki in resource-poor settings. septicemia and severity of aki are contributory factors to high mortality in pediatric acute kidney injury. outcome of acute kidney injury managed in a regional paediatric nephrology centre shivaram hegde, sabina pahari paediatric nephrology, university hospital of wales, cardiff, uk objective: this study reviewed the aetiology, treatment modalities and outcome of children with aki managed in our tertiary paediatric nephrology unit. method: retrospective analysis of referral practices, aetiology, and management of 38 children treated for aki over the last 5 years. children primarily treated in intensive care units were excluded. outcomes noted as complete recovery, residual renal injury, renal replacement therapy (rrt) dependency or death. they were followed up until their renal function normalised and any proteinuria or hypertension resolved. result: out of the total 38 children aged 5 months to 16 years, 34% were under 5 years. haemolytic uremic syndrome (hus) was the commonest aetiology in 18 cases (47%), 15 of them secondary to ecoli 0157 and 3 with atypical hus. obstruction was second most common (5) and renal function improved following relief of obstruction. supportive management sufficed in 23 (60%) cases and 15 (37.5%) received renal replacement therapy (rrt); peritoneal dialysis being the commonest mode. most children needing dialysis were oliguric (14). at discharge there were no deaths, 5 patients showed complete recovery of renal function, one was dialysis dependant and renal function was improving in the rest. at 3 months we found normal renal function in 26 (68%) children and chronic kidney disease (ckd) stage ii in 7 (18%) and ckd stage iii in 4. the dialysis dependent child underwent renal transplantation. based on the data from 12 patients currently under follow up (for 12-62 months, mean 41 months), 32 (84%) children have recovered completely and 5 have developed ckd; stage i in 2 and stage ii in 3. conclusion: prognosis following aki was excellent in our patients, probably because of lack of multiorgan dysfunction. hus was the commonest cause and urgent renal imaging needed when obstruction is suspected. oliguric patients are more likely to require dialysis and need early referral to the regional unit. all cases should have long-term follow up to ensure renal recovery and detect delayed complications. clinical course and outcome of acute kidney injury (aki) due to childhood haemolytic uremic syndrome (hus): a single centre experience shivaram hegde, sabina pahari paediatric nephrology, university hospital of wales, cardiff, uk objective: clinical data, along with the management and outcome of 15 children with aki due to hus, caused by shigatoxin-producing ecoli (stx-hus) are described. method: we analysed the data of children with stx-hus induced aki, managed in our unit over the last 5 years. outcomes noted as complete recovery, residual renal injury, dialysis dependency or death. all patients were followed up at least for a year and further monitoring continued until their renal function normalised and any proteinuria or hypertension resolved. methods and results: stx-hus was the commonest cause of aki in our unit, accounting for 15 of the total 38 cases (39.5 %) treated for aki during this period. 11 patients were less than 10 years of age. all children presented with blood in stool and 11 with oligoanuria. all showed microangiopathic haemolytic anaemia and thrombocytopenia. positive stool cultures (for ecoli 0157) were obtained in 11 and 4 had ecoli 0157 lipopolysaccharide serum antibodies. 10 patients required dialysis (peritoneal dialysis in 7, haemodialysis in 1 and both modes in 2) and one child needed plasma exchange. the remaining responded to supportive management. morbidities encountered included bowel perforation (1), hypertension (2), seizures (2) and diabetes mellitus (1) and three of these children needed intensive care management. at discharge there were no deaths, none with dialysis dependency or complete renal recovery but all showing improving renal function. at 3 months we found normal renal function in 12 (80%) and chronic kidney disease (ckd) stage ii in 2 and ckd stage iii in 1. a 13 year old girl with an unknown myopathy presented for the third time with clinical and laboratory features of rhabdomyolysis. on day two of admission, her renal function deteriorated with decreased urine output, increasing creatinine of 380 μmol/l and ck of 240,220 iu/l. she was commenced on haemodialysis and started on calcium supplements, as her calcium levels were 1.6 mmol/l. three week into her illness, she was symptomatic with high blood pressure of 170 mm of hg leading to a generalised seizure. her serum calcium was 4.01 mmol/l. the ct brain did not reveal any evidence of haemorrhage or infarction. there were no other trigger factors identified for the seizure and the hypertension, apart from the hypercalcaemia. the hypercalcaemia was managed with low calcium dialysate, calcitonin and sevalamer. despite the above treatments, she continued to be hypercalcemic and developed erythematous palms and soles and an injected conjunctiva. in view of the refractory hypercalcaemia, she received two doses of intravenous pamidronate (0.5 -1 mg/kg) when the creatinine was 317 mmol/l. following this, her calcium levels normalised to 2.59 mmol/l. her renal function improved and was discharged a week later. there was no nephrocalcinosis seen on follow up. bisphosphonates are used in children with caution, as there is little evidence on its safety and efficacy. they act by binding to the surface of calcium phosphate crystals and inhibiting osteoclast formation, aggregation and dissolution. it is nephrotoxic causing acute tubular necrosis and collapsing focal segmental glomerulosclerosis. pamidronate is usually used in children with chronic kidney disease but this was the first time it was used in a child recovering from acute kidney injury secondary to rhabdomyolysis. there were no complications with the use of pamidronate in our patient. chronic kidney disease during long-term follow-up in children treated with neonatal extracorporeal membrane oxygenation: do we need to worry? objective: acute kidney injury (aki) is a common complication in children receiving extracorporeal membrane oxygenation (ecmo) support. as aki may cause loss of a significant number of functioning nephrons, these children are at risk of developing chronic kidney disease (ckd) post-ecmo. therapeutic interventions might be needed to prevent further renal function deterioration or comorbidity of ckd in these patients. the objective of our study was to determine the prevalence of ckd during long-term follow-up (fup) of children treated with ecmo. methods: this was a cross-sectional study performed between 2010 and 2013. all children previously treated with neonatal ecmo who visited our fup clinic at the age of 1, 2, 5, 8, 12 and 18 years were screened for ckd. if more check-ups were available per patient, only the latter one was used for the study. ckd screening included height (ht) and blood pressure measurements (bp), and laboratory parameters including serum creatinine (scr) (schwartz formula [0.413*ht (cm)/scr (mg/dl)] or mdrd formula were used to estimate gfr) and urinary protein/creatinine (up/c) ratio. ckd was suspected in patients with hypertension (>95th percentile of reference values according to height and age), abnormal egfr (<90 ml/min/1.73m 2 ) or proteinuria (up/c ratio >30 mg/mmol creatinine). patients were excluded if scr was lacking. results: to date, 132 children visited the fup clinic. of these, 4 (3%) were excluded because of pre-existent kidney disease and 6 (5%) because of missing scr data. hence, 122 children (44% female) were screened for ckd. the number of patients per fup age category was 17 <5 years, 56 between 5-12 years, and 49 >12 years. bp was within normal ranges in all children. in 13 (11%) children either an abnormal up/c ratio or egfr was observed. up/c ratio was increased (median 36 [iqr 32-38 mg/mmol creatinine]) in 8 (7%) children, of which 1 was explained by low muscle mass. only 5 (4%) children had an abnormal egfr but all >60 ml/min/1.73m 2 . conclusion: the prevalence of ckd and its clinical implication in children previously treated with neonatal ecmo seems to be limited. future research will focus on identifying risk factors for ckd following ecmo support. abstract# p-sat336 10 years clinical retrospective analysis in children of acute poisoning inpatient man jiang, qiu li nephrology and immunology department, children's hospital of chongqing medical university, chongqing, china objective: acute poisoning is the common critical and emergency disease in children. since childhood is the special life stage with continuous growth, clinical features are different from the adult and changes were happened in resent years. the change rules, clinical features, treatments and the outcomes of acute pediatric poisonings inpatients were investigated in this article. methods: retrospective evaluated the 1005 cases of pediatric acute poisonings admitted to the children's hospital of chongqing medical university. cases were divided into 5 groups by ages, or divided into 2 groups based on different origins (urban or suburban), different causes and routes of poisonings in different groups were calculated. the clinical manifestations, treatments and prognosis in acute intoxication were also studied. results and conclusion: pediatric acute poisoning mainly happened in 1-4 years old children (50.02%), there was no statistical difference between genders. the common causes of poisonings were pharmacological poisoning (26.07%), food poisoning (19.50%), animal bites and stings (16.92%) and pesticide poisoning (14.73%). in the resent five years, pharmacological poisoning rose to be the top reason of poisonings, and was mainly composed by children≤3 years old (p < 0.001),and urban children were more than children from suburban(p < 0.001). neurological and psychiatric drugs were the most common (32.06%) in drug poisoning. rural children with animal bites and stings, pesticide poisonings and rodenticide poisonings were more than the urban children (p < 0.001, p < 0.001, p < 0.05). the main poisoning causes in 6-12 years old group and >12 years old group children were food poisoning (29.20%) and pesticide poisoning ( objective: little is known about cardiac surgery-associated acute kidney injury (cs-aki) in children in developing regions of the world. the study aimed to determine the prevalence of cs-aki, associated factors and its impact on mortality and utilization of hospital services. methods: hospital records of children aged 0-17 years that underwent cardiac surgery (other than device closure procedures and pacemaker insertion only) at an indian hospital between 2011 and 2012 were reviewed. cs-aki was defined as a rise in serum creatinine of ≥0.3 mg/dl in any 48 hours and or by urine output less than 0.5ml/kg/hr for an 8-hour period in the first 5 days after cardiac surgery. results: the study included 323 children with a median age of 1 year (0.04-17), of which 22 (6.8%) were neonates and 18.3% had single ventricle. about 60% of the children had rachs-1 1 or 2 interventions. cs-aki occurred in 39 children (12.1%), most often in the first 48 hours after cardiac surgery. on univariate analysis cs-aki was associated with sepsis and intra and post-operative hypotension. in-hospital mortality was 6-fold higher in children who developed cs-aki. cs-aki was associated with 2-3 days longer duration of mechanical ventilation, inotropic support and icu stay. conclusion: cs-aki occurs in children in developing countries but at a lower frequency mainly due to older children with less complex congenital heart disease undergoing cardiac surgery. cs-aki was associated with higher in-hospital mortality and increased utilization of hospital services. factors associated with cs-aki included intra and post-operative hypotension and sepsis. objective: data on long term effects of newborns after acute kidney injury is limited. the aim of the study was to evaluate long term effects of acute kidney injury (aki) in neonatal period. methods: inclusion criteria were as follows: oligo-anuria or plasma creatinine >1.5 mg/dl during first three postnatal days or >0.7 mg/dl after third postnatal day or ≥25% increase in plasma creatinine within 48 hours during hospitalization in neonatal period. three hundred sixty children who had aki during neonatal period (between january 2000 and december 2009) and survived to hospital discharge were invited; 106 of them accepted to participate. patients' characteristics during aki and during follow-up were recorded; a physical examination and laboratory studies, including acr (urine microalbumin/creatinine ratio) were performed. schwartz formula was used to estimate gfr; hyperfiltration was defined as a gfr >160 ml/min/1.73 m 2 . hypertension was defined as blood pressure ≥95 th percentile for age, gender and length. results: the mean age on evaluation was 6.78±2.9 years. 15.8-15.8-12 .2% of patients. all these children were dialyzed for more than 7 days on acute phase of the disease. at time of discharge from hospital proteinuria remained in 53 (85.5%), hypertension -in 47 (75.8%) patients. after 1 year, 5years and >10 years period proteinuria was detected accordingly to 26.3-7.9-33.3%, hypertension to 11.3-6.5-12.9% of patients. after >10 years from the onset of disease hypertension was detected more often in the group of children who were < 1 years old on acute phase of the disease (40% vs 11.1%, p=0.05). in the group of patients who hadn't proteinuria on the acute phase, after >10 years period proteinuria was observed to 25% of them. after 1 year period renal impairment of various degree was observed to 26.3%, after 5 years -to 39.5 %, after >10 years-to 33. . on admission his mean arterial pressure was 35 mmhg, his creatinine was 5.8, potassium 6.5 and sodium 121. usual medical management for aki was initiated but even by late d5 he continued to be anuric with a rising creatinine. his uric acid was grossly elevated at 14 mg/dl. dialysis was considered but as the parents were not in favour of it they were offered the option of rasburicase. rasburicase was given on late d5 and within 12 hrs he started to produce urine which peaked at 4 ml/kg/hr by d9. the creatinine also showed a concomitant fall and by discharge (d10) it was 2.1 mg/dl and normalized at 0.5 mg/dl by d30. uric acid was 0.5 mg/dl on d7 and 2.8 mg/dl on d9. case 2: 6 years old boy with multiple special needs was transferred from another hospital intubated, anuric and in multi organ failure (mof). creatinine was elevated at 3.7 mg/dl and uric acid was also found to be raised at 9.1 mg/dl. in view of mof along with disseminated intravascular coagulopathy (dic) dialysis was considered a risky process and parents were offered the option of rasburicase. rasburicase was given on d2 of admission and although the repeat uric acid was 0.3 mg/dl and he did produce 5 ml of urine within 6 hours no further improvement in renal parameters was noticed. dialysis (sled) was initiated on d3. although the creatinine improved with sled he continued to be oligo-anuric and died of mof by d5. conclusion: although rasburicase seemed to work in case1, it was not beneficial in case 2. the exact mechanism for rasburicase is still hypothetical and the few case reports have all documented positive results. although it seemed to have worked in case 1, to the best of our knowledge case 2 is the first documented case wherein it did not work. proper randomized control trial is needed before advocating rasburicase as a novel therapy for aki. results: age, sex, diagnosis, baseline and post-surgery hemoglobin, total leukocyte count, platelet count and biochemistry were recorded. baseline and post-operative, urea (mg/dl), creatinine (mg/dl), urine output (ml/kg/hr) and inotrope dose were also recorded daily. the duration of cpb was noted. post-operatively cardiac, renal, hepatic, neurological and respiratory dysfunctions were recorded. conclusion: fifteen (7.2%) children developed aki stage i, one child (0.5%) developed aki stage ii and four children developed aki stage iii (2%). all patients with aki had a longer stay in hospital. eight children required dialysis for aki; two required dialysis to maintain the fluid balance post operatively. none developed chronic renal impairment. using stepwise regression, younger age (<1 year), weight < 10kg, pump failure, sepsis and duration of cpb more than 60 minutes were significant risk factor for developing aki. results: most of the patients were male, with less than 28 days old and weighing less than 5kg.in the majority of cases (77%), arf was diagnosed in the first 6 days after surgery, demonstrating influence of the procedure itself. all of them were treated by peritoneal dialysis(pd) for a mean time of 255 hours; 58.3% of them died. there was no statistical significant difference between age, time on extracorporeal circulation and duration of dialysis, comparing the patients who survived and the deceased ones. conclusion: acute renal failure is a common complication in children with congenital heart disease and pd is a safe dialytic method. the mortality rate is high and influenced by aspects related to the child, underlying disease, type of surgery and many other associated aspects. abstract# p-sat344 acute kidney injury epidemiology and associated factors in a pediatric intensive care unit. conclusion: acute kidney injury (aki) is associated with significant morbidity and mortality in pediatric patients with critical illness. the main causes associated to aki were sepsis and shock. we found no association with cardiac surgery, probably explained by the low level of complexity that is performed in our center. almost half of our patients with aki were in failure and the mortality was high as previously reported. objective: fibroblast growth factor-23 (fgf-23), a phosphaturic hormone involved in calcium phosphate homeostasis, appears to predict renal disease progression in adults with non-diabetic ckd (fliser, jasn 2007) . this study aimed to determine renal survival according to fgf-23 serum levels in children with ckd stage ii-iv. methods: 232 children participating in the escape trial (age 11.5±4 yrs, gfr 45±18 ml/min/1.73m 2 ; underlying renal disease: hypo/dysplasia (71%), glomerulopathies (12%), hereditary or other (17%)) were analyzed. all patients received fixed dose ace inhibition and were followed prospectively by 2-monthly examinations for up to 5 years. the study endpoint was defined by egfr loss >50% from baseline, gfr <10ml or start of renal replacement therapy. fgf-23 levels were determined at baseline (c-terminal human fgf-23 elisa (immutopics, san clemente,ca,usa objective: the long-term outcome of patients born with unilateral renal agenesis (ura) and of those who underwent therapeutic unilateral nephrectomy (un) remains a topic of concern and debate. children with a solitary functioning kidney (sfk) have an increased risk of developing hypertension, albuminuria and chronic kidney disease in later life. the purpose of this study was to identify whether plasma symmetric dimethylarginine (psdma) is an useful biomarker reflecting the level of renal injury in children with solitary functioning kidney (sfk). methods: we measured circulating psdma in 51 patients with sfk and no other urinary defects. patients were subdivided for two groups: primary sfk (psfk)-unilateral renal agenesis (ura) and secondary sfk (ssfk) after unilateral nephrectomy. the control group (c) consisted of 21 healthy children, aged mean 9.92 ± 4.85 yrs. immunoenzymatic elisa commercial kits was used to measure psdma concentration. data analysis was performed using computer program statistica 9.0. results: the age and sex of studied children did not differ from healthy controls (p> 0.05). plasma sdma levels in sfk children were higher than in healthy participants (p< 0.05). there was no difference in psdma concentrations between psfk and ssfk patients (p> 0.05). sdma plasma levels correlated significantly with c cr (r= -0.32, p< 0.01) in all participants. roc analyses performed in order to define the diagnostic efficiency of serum creatinine and psdma in identifying children with c cr < 90ml/ min/ 1.73m 2 among sfk and healthy participants revealed no difference between all two aucs (p> 0.05). conclusion: in children with a solitary functioning kidney increased psdma levels were observed, however the sensitivity and specificity of this marker in detecting the decrease in c cr was not better than creatinine. objective: renal osteodystrophy encompasses a variety of skeletal disorder ranging from high turnover lesions of secondary hyperparathyroidism (shpt), to low turnover lesionsof diverse etiology that are usually associated with normal or reduced pth levels. so our aim was to assess by dexa which is a precise , rapid and noninvasive procedure the degree of osteopenia in patients with crf. methods: 53 children aged (11-3.84) years, 17 on conservative treatment and 36 on hemodialysis were included in the study bmd of lumbar spine and wrist were measured by (dexa) and compared with age and sex matched controls. results: shows that out of 53 patients, 25 (47%) are osteopenic 22 (88%) on hemodialysis and 3 (12%) on conservative treatment, of these 13 (24.4%) had severe osteopenia as regard bmd of the spine, while dexawrist shows 11 (21%) are osteopenic, 7 (64%) on regular hemodialysis and 4 (36%) on conservative iii, of these 3 (5.66%) had severe osteopenia ,correlation between z-score spine in the osteopenic group and different biochemical parameters shows non-significant correlation except -ve correlation with duration and age of the patients, while z-score wrist of the same group shows +ve correlation with bicarbonate. our result shows that group with ipth >= 200 pg/ml are more osteopenic than those with lower ipht levels, although difference did not reach level of statistical significance p > 0.05. conclusion: we can conclude that osteopenia is frequent in patients with crf more in the dialyzed group, with longer duration of the disease, older age and severe acidosis, irrespective of the severity of the disease. although, degree of osteopenia is not correlated with biochemical findings of (shpt) but still patients with (shpt) are more osteopenic and have lower cortical bone density. mineral and bone disorders were defined according to k/doqi guidelines. results: during the study period 95% of stage i-iv ckd patients and all esrd patients and renal allograft recipients had at least one type of mbd. high turnover bone disease with hyperphosphatemia, hyperparathyroidism and hypocalcemia was the most frequent type of mbd in stage i-iv ckd and esrd patient. adynamic bone disease was seen in only one esrd patient. hypophasphatemia was the most common disorder in renal allograft recipients. vitamin d deficiency/insufficiency was seen in 55% of stage i-iv ckd, 65% of esrd patients and all renal allograft recipients. all patients with stage i-iv ckd and esrd were treated with calsitriol and renal allograft recipients were treated with vitamin d3 supplementation. conclusion: mineral and bone disorders especially high turnerover type is common in children with ckd and esrd. although successful kidney transplantation corrects many of the metabolic abnormalities associated with the development of mbds, renal allograft recipients had increased risk of vitamin d deficiency/insufficiency. objective: calciphylaxis is a complication of chronic kidney disease that characterized by necrotic lesion in the skin and in histological examination reveals microcalcification of medium sized blood vessels. materials and methods: we report on a 21-month old girl with end stage renal disease due to diffuse mesangial sclerosis referred for tenckhoff catheter implantation. laboratory tests on admission were: bun = 150 mg/dl, cr= 5 mg/dl, uric acid = 10 mg/dl, calcium= 4 mg/dl, phosphate = 40 mg/dl, alkaline phosphatase= 1017, pth= 700 pm/l,vitd= 21 mg/ml. calciphylaxis process started when she received calcium gluconate iv the day before surgery and continued progressively when oral calcium carbonate and oral calcium gluconate were given. hourly peritoneal dialysis was started and all calcium containing medications were stopped. but the process continued progressively. surgical detriment of the necrotic tissue of right wrist was done. comfeel dressing had no effect and the circulation of tissue was poor. therefore the left wrist was kept as control. pamidronate 0.5 mg/kg/day prescribed for 6 days and then once a week for 5 weeks. after one week of starting pamidronate skin lesion began to heal, circulation improved and after six weeks all skin lesions completely recovered. as shown in figure one debridement cause skin scar on right wrist. conclusion: pamidronate is effective to stop calciphylaxis in children with advance renal insufficiency and severe calcium-phosphate imbalance. medical or surgical debridement are not suggested and lesions will recover without scar by pamidronate objective: fibroblast growth factor 23 (fgf23) is a key player in kidney-bone axis and regulation of calcium and phosphate homeostasis. most of the recent research has focused on fgf23 in chronic kidney disease and there is paucity of data in healthy children. hence we initiated a study on the various biomarkers of calcium-phosphate metabolism including fgf23 in healthy children and present here the interim report of our findings as of now. methods: a total 100 healthy school children and adolescents aged 6-16 years, were enrolled in this cross-sectional study, after a written consent from their parents. height, weight and pubertal staging were assessed. blood was collected and calcium, phosphate, pth and vitamin d were estimated by standard methods. plasma c-terminal fragment of fgf23 was quantitated using a commercial elisa kit (immutopics, usa). three day dietary recall was done to calculate mean daily calcium, phosphate and protein intake. results: of the 100 children enrolled 60% were males. the mean age was 12.3 yrs, mean height was144 cms and mean weight was 34kgs. the mean blood biochemistry values of the children tested so far for ca, p, pth and vitamin d shown in table 1. the parameters of the children were normal for their respective ages however fgf23 levels were found to be lower compared to reports in literature. conclusions: from the results available as of now all of the children had normal biochemistry for their ages. the fgf23 levels were found to be lower than reports in the literature. whether it can be attributed to the dietary differences from the children of the west needs further investigation. testing of the entire cohort will reveal if this lower fgf23 is a universal phenomenon among all children in our study. on the contrary, all dxa parameters showed a decreased in their mean zscore bmd values that reached statistically significance. finally, multivariate stepwise regression analyses showed that estimated glomerular filtration rate at the beginning of the study was the best predictor of the difference in bmd z-scores measured at lumbar spine. additionally, values of ipth at the beginning of the study and the change of ipth throughout the study predicted the 72.3% of the difference in z-score of sos measured at radius with an inverse relationship. conclusion: the use of two different techniques have shown bipolar changes of bone properties after renal transplantation. reversal of renal osteodystrophy and consequently correction of many of the underlying metabolic abnormalities, mainly normalization of ipth, strengths cortical bone as this is best illustrated by qus assessment. however, reduction in bmd, is predominantly considered as an adverse effect of steroid immunosuppression. age 3.3 yr) (group 1), 33 patients with ckd stage 5 on pd or hemo (mean age 9 yr, mean time on dialysis 2 yr) (group 2) and 29 patients with ckd stages 2-4 (mean age 7 yr, mean gfr 37 ml/min/1.73 m 2 ) (group 3). 25ohd levels were deficient in 20% and insufficient in 35% of the group 1 patients, 48% were deficient and 15% were insufficient in group 2, and 7 % were deficient and 17% were insufficient in group 3. results: intact pth (ipth) was above the target level by different ckd stages in 52 % of the group 1 patients, in 58% of the group 2 and in 52% of the group 3 and showed an inverse correlation with 25ohd levels (p<0.05, r=−0.25). there was inverse correlation with age in the complete cohort of patients (p=0.0005, r =−0.33) height standard deviation score (sds) was associated with 25ohd only in group 2 (p=0.025, r=0.45). there was direct correlation between 25ohd levels and gfr in group 1 and 2. conclusion: our data suggest that 25ohd deficiency is common in pediatric renal transplant and ckd children of any stage even in an area with year-long sunshine, especially in adolescents. hyperparathyroidism was also frequent in all groups. it would be advisable to monitor vitamin d status in these patients. 3) years, 28 with ckd 5 (2 on hd, 9 on pd and 17 tx) mean age 13 (5.4-17.5) and 113 healthy children, mean age 11.5 (5-17.9 objective: the aim of our study was to analyze twelve single nucleotide polymorphisms (snp) of gene lrp5 potentially associated with osteoporosis risk in children with routine steroidotherapy in the course of the idiopathic nephrotic syndrome. methods and results: glucocorticosteroids are important risk factors for drug induced osteoporosis. decrease in bone mineral density (bmd) and increased risk for pathological fractures are caused by direct division of steroids at the cellular level by inhibiting the replication of osteoblasts and stimulation of its apoptosis. also proven its effects on the inhibition of type i collagen synthesis. lrp5 is one of the wnt signaling pathway proteins coreceptors involved through the rank-rankl system in regulation of the osteoblasts function. lrp5 is included to the osteoporosis phenotype genes group and its selected single nucleotide polymorphisms can be responsible for bmd decrease in patients with glucocorticoid therapy. the study group was composed of 38 children with idiopathic nephrotic syndrome, 21 boys and 17 girls beetwen the age of five to twelve years old , 14 with osteoporosis and 24 with normal bone mass density. study also included the control group consists of 102 healthy individuals at the same age not treated with glucocorticoids. the analysis was carried out with polimerase chain reaction (pcr) and taqman molecular probe designed for 12 single nucleotide polymorphisms (snps) nearby investigated mutations in lrp5 gene that can be connected with osteoporosis fenotype. odds ratio value (or) was based on frequencies of single nucleotide polymorphisms in lrp5 gene and its haplotype analysis. the results showed significant differences in or value among the three groups. it was also found the differences in the lrp5 gene structure. based on gabriel algorytm single nucleotide polymorphisms pairs analysis in children with osteoporosis and nephrotic syndrome we proved the correlation between selected snps pairs presence and decretion of bone mineral density in studied group. objective: growth hormone (gh) and insulin-like growth factors are essential for normal growth and development. chronic renal failure (crf) results in major changes in the circulating growth hormone /insulin-like growth factor (igf) system. our aim isto study: to assess clinical and laboratory parameters of growth and osteodystrophy including igf1 and igfbp2 as part of the somatotropic hormone axis in egyptian children suffering from crf on conservative therapy. methods: 62 egyptian children (47 boys and 15 girls) with a mean age of 9.7y (0.47 to 21.12y) suffering from crf on conservative therapy and 21 controls were included in the study. ht, wt and tsf were measured ,pubertal staging was assessed and followed up for a period of 6 months. at the end of the follow up period serum for igf1 and igfbp2, renal function, electrolytes, ca, p ,and alkaline phosphatase and acid base balance were measured and an x-ray of the left hand and wrist was done to determine their bone age by tanner and whitehouse. results: our study shows that children suffering from crf in egyptian conservative therapy have growth retardation with a mean ht of -3.7 sds, a mean wt of -2.24 sds. tsf mean was -1.3 sds. on the average the patients had a delay of 2.95y (+/-2.0) in their bone age. their height was retarded more than their bone age with a height age/bone age of 0.8 (+/-0.18). alkaline phosphatase as a markers of renal osteodystrophy is significantly correlated to the height, height age , bone age and to the ph. the mean igf1sds (-0.6 +/-1.8) did not differ from that of controls while the mean igfbp2sds (2.4 +/-4.6) was significantly higher in patients with crf than in controls. height and weight were significantly correlated to igf1 but not igfbp2. there is a significant correlation between igfbp2 level and the glomerular filtration rate. conclusion: the imbalance between normal insulin-like growth factor-i (igf-i) and markedly increased igfbp2 plasma levels plays a pathogenic role for growth retardation in children with chronic renal failure. the lower the gfr the higher the igfbp2 level. the latters inhibitory action may provide hope for improving growth in cases of crf by reducing the level of igfbp2 or displacing igf1 from it. objective: anemia is one of the most common complications of chronic kidney disease (ckd) and renal transplantation. this study evaluated the prevelance and the etiology of anemia in children with stage i-iv ckd, end-stage renal disease (esrd) and renal allograft recipients. methods: between 2010 and 2012, we prospectively followed 21 pediatric stage i-iv ckd, 20 esrd patients and 27 renal allograft recipients. anemia was defined according to k/doqi guidelines as hb is less than the fifth percentile of the normal, adjusted for age and sex. results: during the study period52.3% of stage i-iv ckd, 100% of esrd patients and 48.1% of renal allograft recipients developed anemia. the mean hb levels were 11.30+/-1.72 g/dl, 9.62+/-1.94 g/dl and 11.95+/-1.88 g/dl in stage i-iv ckd, esrd patients and renal allograft recipients, respectively. the mean hb levels were significantly lower in esrd patients as compared to stage i-iv ckd patients and renal allograft recipients (p<0.0081, p<0.002). erythropoetine (epo) deficiency was the most frequent cause of anemia in stage i-iv ckd and esrd patients, followed by iron deficiency. in addition to epo and iron deficiency, bone marrow suppression and parvovirus infection were the causes of anemia in renal allograft recipients. erythropoietic stimulating agents were used in 27% of stage i-iv ckd, 100% of esrd patients and 23% of the renal allograft recipients. conclusion: although anemia is one the most common problems seen in children with ckd and esrd, the prevelance is also high in renal allograft recipients. these patients should be evaluated and treated accordingly to the underlying causes. abstract# p-sat375 determinants and prevalence of anaemia among preschool children in highly focussed states of india results: about more than 60 % child is anaemic among all eag states. the prevalence of severe anaemia is 6.7 % in rajasthan followed by uttar pradesh (3.6 %) and madhya pradesh (3.4 %). those children aged 12 to 17 months are 7 times significantly more likely to be severe anaemic as compare to the 36 to 59 months aged children. those mothers have severe anaemia, their children are also found to be severe anaemic (16 times more than not anaemic mothers). mothers who are highly educated and belong to richest quintile their children are less likely to be anaemic. the finding of the study shows that demographic and socio-economic indicators play significant role in determining the prevalence of anaemia in eag states which needs focused programme to reduce the prevalence of anaemia in preschool children. abstract# p-sat376 impaired renal growth hormone mediated jak/stat5 signaling in juvenile rats with chronic kidney disease objective: linear growth retardation is a major problem in children with chronic kidney disease (ckd) and is ascribed to gh insensitivity. treatment with exogenous gh has been accepted as standard therapy in children with ckd and short stature. however, concerns have been raised in the past on the potential fibrogenic effects of gh. there is no information regarding renal ghr signaling pathway in ckd. methods and results: to investigate this pathway, surgically 5/6 nephrectomized (ckd) and pair-fed control (c) juvenile (3 wk old) rats were sacrificed after 2 weeks of ckd. serum creatinine and albumin excretion were significantly elevated (associated with glomerulomegaly and early fibrosis) while body weight and length gain were reduced in ckd rats. serum igf-i levels were decreased in ckd, even though serum gh levels were unchanged. kidney ghr mrna and protein levels were reduced and phosphorylation of jak2 and stat5 was significantly impaired. supressor of cytokine signaling (socs3) mrna was increased in association of increase in renal il6 mrna. renal igf-1 mrna was unaltered in ckd. thus, in the remnant kidney of ckd growth retarded juvenile rats there is impaired gh mediated jak2/stat5 signaling. this defect may be due to a reduction in gh receptor expression and signaling together with an increase in socs3 expression. conclusion: we suggest that the insensitivity of the remnant kidney to gh may serve to protect against the potential adverse renal effects of exogenous gh in ckd patients. objective: concerns have been raised about possible adverse effects of growth hormone(gh) in short children with chronic renal diseases. six cases of short children with chronic renal diseases ckd were studied in our hospital, the microalbuminuria as a sensitive parameter of early glomerular damage was detected during the treatment of recombinant human growth hormone. the individual growth response to gh was also observed. methods: of the 6 cases, male were 4 , female 2, the onset age ranged from 4 to 14 years old, all were diagnosed as growth retarded children with various chronic renal diseases. the dosage of gh was 0.1 iu/kg/d. the follow-up period was from 3 months to 1years. microalbuminuria was measured by a commercially available elisa kit, as a sensitive parameter of early glomerular damage in children being treated with recombinant human growth hormone. serum insulin-like growth factor igf-i concentration and igfbp3 were also measured before and during the treatment with gh. results: there was no statistically difference on the level of microalbuminuria compared with that of pretreatment. linear growth were 1.0 cm/month with an increment in height sds by 1.5 at the end of the first year of gh treatment. growth retardation in children with ckd is associated with normal to slightly low concentrations of (igf)-i and igfbp3. objective: iron deficiency anaemia is common in chronic haemodialysis patients and is the most common factor of resistance to erythropoeitin treatment. kdoqi clinical practice guidelines and recommendations for anaemia in ckd suggest maintaining hb levels between 11-12 g/dl, tsats >20% and serum ferritin above 100 ng/ml. our unit's previous practice provided 1-2 mg/kg/week of elemental iron intravenously to our haemodialysis population and monitored blood parameters monthly. from july 2012, we changed our practice to withhold routine administration of iv iron, unless blood parameters (monitored fortnightly) suggested iron deficiency. we assessed the change in our practice in safely reducing the frequency of iv iron administration in our haemodialysis population. methods: we included all 9 children attending our haemodialysis unit (july 2012). we used %hypochromia of >4%, correlating with hb level of <11 g/dl as a marker for initiating iv iron sucrose (3mg/kg/week) until improvement of the above parameters. serum hb and %hypochromia were monitored at the start of change-over and fortnightly thereafter. all patients received routine erythropoietin at recommended doses of between 50-300 units/kg/week. results: at initiation of change-over, there were 9 patients with mean hb 11.3 g/dl, mean %hypochromia 1.2% and mean tsats 27.7%. from july 2012 -mar 2013, four patients required iv iron therapy (mean hb 9.2 g/dl, mean %hypochromia 6.2%, mean tsats 16.3%). three patients had 4 weeks and one patient had 7 weeks of iv iron sucrose 3 mg/kg/week to achieve normalisation of serum hb (mean 11.5 g/dl), %hypochromia (mean 2.73%) and tsats (mean 26%). at time of reporting (mar 2013), there were 7 patients with mean hb 11.22 g/dl, mean %hypochromia 2.07% and mean tsats 28.2%. two patients received transplants. conclusion: a novel change in our practice has resulted in a safe reduction of iv iron therapy in our population of children undergoing haemodialysis. from our findings, we suggest avoiding routine iron infusions in paediatrichaemodialysis, which is unlikely to have an adverse effect on the incidence of anaemia and will reduce the risk of inadvertent iron overload. abstract# p-sat380 reversible portal ascitis after bilateral nephrectomy in infant with polycystic kidney disease we report here on a newborn with moderate renal failure and severe hypertension due to autosomal recessive polycystic kidney disease (arpkd). antihypertensive therapy associating minoxidil, labetalol, amlodipine and lasilix were not able to control adequately blood pressure. due to massive kidney enlargement, inadequate respiratory function and inability to tolerate enteral nutrition, parenteral nutrition was initiated at 2 months to maintain adequate calories intake. at 4 months of life, the infant presented severe viral pneumonia complicated by cardio respiratory arrest . he required cardio respiratory resuscitation and hospitalization in icu for several days. unilateral right nephrectomy was decided after this severe complication in order to avoid similar accidents due to massive kidney volume and to improve respiratory and nutrition status. one week after unilateral nephrectomy, we noted rapid increase volume of the left kidney and important ascitis having the biological characteristics of portal hypertension. repeated aspiration of more than 100 ml of portal ascitis was performed every day. in parallel daily perfusion of albumin for more than 3 weeks and complete parenteral nutrition did not succeed in treating ascitis. faced to resistant ascitis and persisting difficulties in maintaining enteral nutrition and adequate respiratory ventilation , left nepherctomy was decided and performed. automated peritoneal dialysis was rapidly initiated in our anuric infant (weight after binephrectomy4kgs). portal ascitis disappeared in 48 hours with rapid normalization of respiratory ventilation and return to normal and well tolerated enteral nutrition. conclusion: to our knowledge, this is the first case reported of portal ascitis noted after palliative unilateral nephrectomy to facilitate pulmonary expansion and gastrointestinal function in arpkd. ascitis was resistant to daily albumin perfusion and repeated aspirations but was completely reversible after bilateral nephrectomy and initiation of peritoneal dialysis. early bilateral nephrectomy at 2 months of life was probably the ideal choice to avoid many complications in our infant due to abdominal and thoracic compression by massive kidney compression. ethical considerations, therapeutic possibilities, and parent consent should be discussed in such challenging and very difficult cases. abstract# p-sat381 serum hepcidin 25 levels and anemia in pediatric chronic kidney disease objective: hepcidin restricts the availability of iron from its stores for erythropoiesis resulting in a functional deficiency of iron and hyporesponsiveness to erythropoiesis stimulating agents. the objectives were to estimate the serum hepcidin 25 in children with chronic kidney disease stage ii to iv and correlate it with hemoglobin, iron status (serum ferritin and transferrin saturation), inflammation (c reactive protein) and estimated glomerular filtration rate (egfr). methods: it was a cross sectional study. children aged 2 to 18 years with ckd stage 2-4 on stable dose of iron and or erythropoetin for the last 4 weeks were included. children with anemia due to other causes, severe acute infection, chronic cardiac or respiratory conditions and those who received parenteral iron and or blood transfusion in last 4 weeks were excluded. hemoglobin, serum ferritin, transferrin saturation, c reactive protein were assessed. serum hepcidin 25 levels were estimated using a elisa based kit in the cohort as well as in 15 age and sex matched normal children to estimate the levels in normal population. multivariate regression analysis was used to assess the relationship between hepcidin and hemoglobin, iron status, inflammation and egfr. results: forty children were recruited into the study. the mean age of children was 9.1+ 4.5 years. around 30%, 28% and 42% of children had ckd stage ii, iii and iv respectively. the mean serum hepcidinlevels in children with ckd (55.87+14.27ng/ml) was significantly higher than controls (11.17+4.38ng/ml). conclusion: serum hepcidin levels showed an increasing trend with decrease in egfr, but this was not statistically significant. the mean haemoglobin was 10.76+1.59g/dl, the mean ferritin was 43.27+38.3ng/ml , the mean transferrin saturation was 21.68+11.5%. anemia and absolute iron deficiency was seen in 100% of our cohort and worsened with increasing stages of ckd. serumhepcidin levels did not correlate significantly with haemoglobin, ferritin or with c-reactive protein. objectives, methods and results: presenting a 16-year old armenian patient who developed a secondary amyloidosis on top of a familial mediterranean fever (fmf) and moved to germany 4 years ago. at the time of the move the patient had already a terminal renal insufficiency and received intermittent hemodialysis. despite the therapy with colchicine moderate attacks of the fmf occurred with an interval of 4 weeks and inflammatory parameters were permanently elevated. the dosage of colchicine was titrated up to 3 mg per day. as a consequence diarrhea occurred and the dosage had to be decreased. over the course the amyloidosis progressed and affected all internal organs including heart and bowel. the combination of inflammation, uremia and amyloidosis led to malnutrition (bmi 14 kg/m2). at the age of 16 years the most severe colitis with peritonitis and paralytic ileus occurred. the treatment with colchicine was paused in assumption of a toxic effect. after improvement of the diarrhea but still persisting abdominal pain the colchicine treatment was slowly started again. shortly afterwards a very severe attack of the fmf developed with an accompanied cardiovascular insufficiency. the treatment with a single high-dose of methylprednisolone and the inhibition of interleukin 1 with anakinra (1 mg/kg, given after dialysis) led only to a short remission. the high-dose methylprednisolone treatment was therefore repeated three times and anakinra was given on a daily basis. since then no new attack of the fmf occurred and the combination of lowdose colchicine and anakinra improved the general status of the patient the elevated inflammatory parameters including serum amyloid a have almost normalized. objectives: invasive aspergillosis is almost exclusively occurs in immunocompromised hosts. the central nervous system (cns) is one of the most frequent sites of invaziveaspergillozis after the lungs. we report a case of invasive aspergillosis in a boy with end stage renal failure. methods and results: a 15-year-old boy was admitted to the hospital for abdominal pain, fewer and cough. he suffered from chronic kidney failure (crf) known for a year and he was on peritoneal dialysis for seven months. he was initially treated with iv. ceftriaxone for lobar pneumonia, intraperitonealceftazidime and cefazolin for peritonitis. the peritoneal fluid was also cultured for fungus and mycobacteria and all cultures were negative. on the seventh day, the patient complained for lower extremity weakness, and bilateral hyperactive deep tendon reflexes and positive babinski reflex were found. cranial magnetic resonance imaging (mri) was normal, but the spinal cord was compressed between c7-t5 by a solid mass which was originated from right lung on spinal mri. computed tomography-guided tru-cut lung biopsy was performed for the solid mass. repeated peritoneal fluid culteres were also negative. two days later, when unconsiousness and convulsions were seen, intraparenchymal multiple hemorrhagic abscesses were found on his second mri. typical hyphae of aspergillusfumigatus was present in pathologic specimens and galactomannan test was positive. cerebral abscesses were evaluated as cns involvement of invasive aspergillosis. anti fungal therapy was began immediately as amphotericin b and caspofungin. the peritoneal catheter was removed and continuous veno-venous hemodiafiltration was performed. however his condition kept deteriorating and death ensued on the fourth day of anti fungal therapy. conclusion: invasive aspergillosis may rarely occur in immunocompetent patients and diagnosis may be missed or delayed due to lack of the particular clinical signs. we should be careful about diagnosis of fungal infections in patients with crf since early treatment before the invasion and dissemination of aspergillosis to adjacent tissues and/or organs offers a higher survival chance for the patient. questionnaire and to find a way to increase reliability for healthy children aged 8-12 years especially, according to iranian culture. objective: we lunch this study to compare the parent and children's behavior problem in pediatric patients suffering from ckd referring arak amir kabir hospital. methods: to perform this case control study, we recruit 116 children with ckd and compared them with other 116 non affected children age between 5 to 16 years old. the child behavior checklist cbcl4/18 for child behavior assessment and general assessment function gaf for the evaluation of their parent's behavior were completed by the parent's data was analyzed using qualitative variables and chi-square formula. results: among 116 patients with ckd, 10 case 8.6% showed behavioral problem while this figure was 3 case 2.6% in the control group, denoting a significant difference p-0.04. moreover 20 children 17/2% in the case group and 9 children 7.8% in the control group had internalizing problem p-0.02. 22 children 19% with ckd and 8 children 6.9% in the healthy group had externalizing problem which was also a significant difference p-0.0003. as a significant p-0.0001 the parent's average stress and behavior scores in case and control were 3.65 & 3.76, respectively. conclusion: the higher prevalence of behavioral problem in the children suffering from ckd and their parent's functional impairment highlights the important of early treatment and subsequently prevention of future behavioral problem in their sibling. abstract# p-sat391 development in 6-year-old children with and without ckd objective: ckd is a pathophysiologic process with multiple etiologies, resulting in the inexorable attrition of nephron number and function and frequently leading to esrd. a number of studies have suggested that children with ckd have problem in development than other children our goal was to compare development of children with ckd with normal children within the common age range for the disease. methods: in this descriptive-analytical study, we selected 30 children with 6-year-old children whom were diagnosed with ckd as our case group and studied their developmental status by ages and stages questionnaire (asq). the control group was selected from 6-year-old children who attended the clinic for reasons other than ckd. conclusion & application to practice: the eleven key areas of responsibility used to measure sc in a periodic evaluation demonstrated a strong correlation to the increasing extent of qpdo. additionally, as the nurses progressed to becoming expert a direct correlation to the qpdo was notable. the study became the foundation for staff training and developing a competency appraisal framework in renal nursing practice thereby promoting quality assurance procedures while attaining qpdo. objective: urinary incontinence is a child health problem that affect both the child and the family in psychological and social aspect and that leads to a decrease in quality of life of the child and his caregiver. patients with higher ckd stage had significantly lower qol score in all domains in the child-self reports, but not parent-proxy reports. whereas there was no difference between patents with stage i and stage ii. according to gender, boys had a tendency to present better qol than girls, but there was no significant difference between these two groups. age discrepancy was not a significant factor to decide qol in children with ckd. in addition, there was significant difference between parent-proxy reports and child-self reports and qol scores in the child-self reports was significantly higher than in the parent-proxy reports, especially in the domains of emotional, school functioning and psychosocial health score. conclusion: residual renal function in children with ckd is a important factor to decide qol. in addition, emotional and psychosocial support for their parents can be necessary to improve qol in children with ckd. abstract# p-sat398 results: the mean period of post-transplant follow-up was 4.7+/-2.1years(range: 0.8-9 years). in the first year of transplantation, 14 (15.2%) recipients had obesity obese patients had higher serum ggt levels than non-obese patients (21.2+/-6.2 u/l vs 14.9+/-3.8u/l, p=0.01). there was a positive correlation between bmi and ggt and tg levels in obese patients (r=0.61, and r=0.5, p<0.01 respectively). patients with dyslipidemia were found to have significantly higher mean ggt level (14.2+/-3.6 u/l vs. 19.2+/-6.2 u/l, p=0.016) and bmi (18.6+/-3.4 vs. 29.4+/-6.3 p=0.04). we could not find any correlation between bmi, and gender, post-transplant follow-up, duration of crf, primary disease, donor status and graft loss. in the first year of transplantation, gfr was significantly lower in obese patients than non-obese recipients (p<0.05) conclusion: our data has suggested that, even within its normal range, elevated serum ggt concentrations and hyperlipidemia are closely associated with obesity in transplanted patients and obesity has negative effects on graft functions. the relationship between the graft function and the serum and urine ngal in children with renal transplantation tehran university of medical science, associated professor, tehran, iran objective: the evaluation of graft function long term after transplantation is important. serum creatinine is the most common used marker for graft function in renal transplantation. the level of serum and urine ngal may predict graft function in renal transplant recipients. methods: we evaluated 21 pediatric renal transplant recipients by measurement of serum and urine ngal, serum creatinine concurrently. the gfr of the patients was also estimated by schwartz formula. results and conclusion: the time after transplantation was 3-12 years (mean: 6.8+/-2.47 years). the mean serum ngal was 140+/-94.2 ng/ml (15.4-324) . the mean urine ngal was 17.8+/-20 ng/ml (32-68). we didn't find any association between serum creatinine, gfr estimated by schwarz and serum ngal. we also didn'y find any association between urine creatinine, gfr estimated by schwarz and serum ngal. glomerular filtration rate measured by dtpa scan of fourteen patients were available. we also didn't any association between gfr measured by dtpa scan and serum and urine ngal. abstract# p-sat409 racial disparities in paediatric kidney transplantation in australia glomerulonephritis and late referral were more common in non-caucasian patients. among patients who did receive a transplant, caucasians had overall better hla matches to their graft and were less sensitised. conclusion: caucasian australian paediatric patients have greater access to kidney transplantation, particularly from living donors, and are likely to have the better graft survival. some racial disparities may be inevitable due to differences in kidney diseases, however further work is required to understand barriers to live donation and address disparities. abstract# p-sat410 cost-effectiveness of renal transplantation in paediatric and adult transplant recipients. [1.4-4.8] ), were associated with a lower inscription probability 6 months after reaching esrd. the national inscription rate was 69% with a significant center variability (median 69% [iqr 51-80]) that remained after adjustment on patient characteristics (p<0.0001). preemptive transplantation rate explained 19% of the inter-center variability (p=0.06). probability of transplantation 12 months after inscription decreased with time of waiting list inactivity (p<0.0001) and with the probability of finding a compatible transplant considering patient's hla and abo groups. underweight (bmi <-2sd) was associated with lower transplant probability (or 0.3 [0.1-0.9]). at 12 months, 81% of patients were transplanted with a significant center variability (median 80% [iqr 72-88] that remained after adjustment (p=0.02). conclusion: as expected patients' characteristics contribute to intercenter variability as well as centers' attitude towards the inscription of younger children, management of specific primary renal diseases and preemptive transplantation. despite national allocation rules we found a significant inter-center variability in the probability of being transplanted after inscription on the list, which was not explained by either the patient or center characteristics of our study. abstract# p-sat414 ten year outcomes of paediatric renal transplantation: the irish experience objective: focal segmental glomerulosclerosis is the most common cause of steroid-resistant nephrotic syndrome in children and adolescents. within 10 years of initial presentation, 50-70% of patients will progress to end stage kidney disease requiring kidney transplant. recurrence rate after transplantation is high and may be associated with early graft loss. methods: we analysed demographic and transplant data collected by the australian and new zealand dialysis and transplant registry (anzdata) on children and adolescents with a diagnosis of primary fsgs who were 20 years of age or younger at the time of transplant. kaplan-meier analysis was performed to compare graft survival according to recurrence of fsgs and donor characteristics. results: during the 20-year period from 1st january 1992 to 31st december 2011, 80 transplants were performed in 74 patients (age range 3-20 years, median 15 years). twenty seven patients developed recurrent fsgs, with one child developing recurrent disease in 2 grafts (overall recurrence rate 36%). the median time to recurrence was 15 days, with loss of graft function in 12/28 transplants (43%). median graft survival in patients with recurrence was significantly shorter than in patients with no recurrence (2.3 years vs >10 years, respectively; p<0.01). there was no significant difference in recurrence rate in recipients receiving deceased donor (dd) versus live donor (ld) transplants (p=0.24) and median graft survival was significantly better in recipients with live donors (11.7 yrs vs 1.6 yrs for dd, p<0.01) conclusion: the rate of recurrent fsgs and graft loss in this retrospective cohort study of children and adolescents transplanted within the australasian region, was similar to previously reported studies. however, in contrast to previous studies, there was no significant difference in rate of recurrence for live versus deceased donors. additionally, live donor recipients had longer median graft survival compared to deceased donor recipients. outcomes of calcineurin inhibitors conversion to mammalian target of rapamycin inhibitors in children with renal transplantation nuntawan piyaphanee, suroj supavekin, anirut pattaragarn pediatrics, siriaj hospital, bangkok, thailand objective: to study outcomes of pediatric renal transplant recipients who underwent cni elimination and conversion to mtor inhibitors. methods: we performed retrospective analysis of all pediatric renal transplant recipients who underwent cni elimination and conversion to mtor inhibitor at siriraj hospital, a tertiary care center in thailand. indications for conversion were defined. graft function prior and post conversion, history of acute rejection, and cni elimination failure were reviewed. results: nine of 32 pediatric renal transplant recipients underwent complete cni conversion to mtor inhibitor. triple-drug regimen including prednisolone, cni and mmf/mpa was basically prescribed to the recipients. eight with tacrolimus and 1 with cyclosporine were conversed to everolimus. mean ages at transplantation, conversion and last follow up were 12.1±2.4, 13.6±2.1 and 14.5±1.8 years, respectively. everolimus was initiated within 3 months in 4 patients. each patient due to cni induced hemolytic uremic syndrome, posttransplant diabetes mellitus, graft impairment with early onset nephrocalcinosis and graft impairment with renal artery stenosis. everolimus was initiated after 3 months in 5 patients due to 1 chronic cni nephrotoxicity, 2 chronic allograft nephropathy (can) and 2 unfavorable graft functions with history of delay graft function. median duration from transplantation to cni conversion was 4.5 months (range 0.8-88) and median time from conversion to the last follow-up was 8 months (range 2-26). mean creatinine clearance (crcl) prior to the cni elimination was 33.0±13.2, as compared to mean crcl of 49.0±16.3 ml/min/1.73m 2 at last follow up (p =0.069). three patients had acute rejection (ar) before the conversion, but only 1 patient had ar post cni elimination. there were no patients with graft loss or cni-elimination failure. conclusion: conversion from cni to everolimus was safe without increasing risk of acute rejection in pediatric kidney transplant patients who experienced early and late cni associated complications. objective: due to a severe shortage of suitable deceased-donor kidneys for children awaiting kidney transplants (kt), we have performed a series of abo-incompatible (abo-i) living kt since 1989. historically, abo-i kt was performed using several session of plasmapheresis (pp) to remove existing anti-a or anti-b antibodies, followed by splenectomy to prevent rebound of antibodies. because splenectomy had risks of surgical complications including bleeding and pancreatic pseudocyst, we introduced a new protocol, for abo-i kt, in january 2006. the new protocol without splenectomy utilizes the anti-cd20 monoclonal antibody (rituximab) and pp. this study retrospectivery examined the efficacy and safety of this protocol. methods: eight de novo abo-i kt (5 males and 3 females) were performed between january 2006 and december 2012. the mean age at transplantation was 15.3±4.0 years (range 9.9-20.5 years). the immunosuppressive protocol consisted of cyclospolin or tacrolimus, mycophenolate mofetil, and methylprednisolone. all patients received induction therapy with basiliximab. the preconditioning protocol included pp or double filtration plasmapheresis (dfpp) and a single dose of rituximab (average dosage 179 mg/m 2 ). all patients who underwent kt achieved a isoagglutinin titer less than 1:16 with 1-4 sessions of pp/dfpp treatment before transplant. results: the patients were followed for 5 to 82 months with a mean follow-up of 36 months. patient and graft survival rates were 100%. one non-adherent patient experienced antibody mediated rejection. figure1 ). in the rtr who had graft loss, there was no significant difference in graft survival time (6.0, 3.1 years, dsa negative vs dsa positive), grade 1 rejection (0.5, 0.7 episodes per patient), grade 2 rejection (0.5, 0.7 episodes per patient) or c4d staining (100%, 57% patients). one patient in each group had bk virus associated nephropathy. two patients had plasma cell infiltrates on biopsy and lost their grafts within 6 months of dsa detection. results: fifty-one kidney recipients (male 32 cases; female 19cases) were enrolled in this study. the median age was 15 years, the youngest kidney recipient was 3years old with a body weight of 10.0kg). there were 11 recipients less than equal 10 years, including one children with congenital renal dysplasia and one with congenital renal artery malformation. about 73.7% kidney recipients came from guangdongprovince. about 1 to 7 pediatric patients accepted renal transplatation each year from 2003 to 2011, but there were13 in2012, which showed a significant increase than in each previous sigle year.the policy that children had a kidney recipients priority may contribute to this increase. the kidney donors were cadaveric kidneys(72.5%) and living kidneys(27.4%), only one patient accepted auto-renal-transplatation. all kidney recipients were survival to present with the treatment of glucocorticoid and or immunosuppressants,such as csa, fk506, mmf,ect. only two patients accepted retransplatation due to losing function of the renal grafts. conclusion: renal transplantation in children increased significantly in 2012 in our hospital, and it seemed an effective treatment to children with esrd. recent adult studies have shown that low levels of immunosuppression (is) are associated with dndsa. however, limited data is available on significance of dndsa and its management in paediatric population. to assess relationship between dndsa with renal function, histological findings, immunosuppression levels and graft outcome in paediatric renal transplant recipients (rtr). in our centre, at the time of the study, dsa were tested if deterioration in graft function was found necessitating renal biopsy. methods: retrospective review of all rtrs in a single tertiary nephrology centre tested for dndsa. data collected included dndsa, histological findings, egfr as marker of renal function, baseline immunosuppression, subsequent treatment and outcome. results: 27/88 patients had dsa tested; 12/27 had dndsa detected. median time for measured of dndsa was 7.6 years post-transplant (range 1.07-13.02).histology: 8/12 biopsies in dndsa+ve subgroup had antibody mediated rejection (abmr) of which 6 were active abmr (3 were c4d positive) compared to 1/16 in dndsa-ve subgroup. the following treatments were used in the dndsa+ve group: increase in is+ pulsed methylprednisolone (mp) (5 patients), increase in existing is (2), rituximab (2), pulsed mp (1), sirolimus added+pulsed mp (1) . in one patient no change was made. at last follow up, in 9/12 patients with dndsa an improvement in egfr was observed. on re-testing for dndsas, 3/12 patients tested negative, 2/12 had lower and 3/12 increased levels. 3/12 had not yet had repeat dndsa testing. conclusion: despite previous reports of dndsa conferring poor prognosis, in our cohort with allograft dysfunction and positive dndsa an improvement in renal function was observed in 75%. however, larger prospective studies are required to further evaluate these findings. abstract# p-sat425 anuria since birth: does it impact outcome of kidney transplant in infants? shefali vyas, maria isabel roberti pediatric nephrology, saint barnabas medical center, livingston, usa objective: scarcity of data exists for allograft outcomes and urological problems in children with long term de functionalized bladders. however, even less is known about the outcome of infants anuric since birth and whose bladders underwent "forced rehabilitation" after a successful renal transplant. methods: in this retrospective study we compared urological events and allograft outcome in infants with esrd mainly due to urological problems. they were grouped according to the history of pre-txp urine out put: group a with urine output prior to txp and group b anuric since birth (all had visible small bladders by ultra sonogram). results: there were no significant differences regarding birth history or ethnicity . group a had 13 boys (87%) and group b had all girls; all but one child in group b received lrd txp. all received induction followed by triple therapy (tacrolimus, steroids and mmf). group a: 8/15 had v-u reflux and 13/15 were on pd prior to txp (mean time= 7 mos). all patients in group b were started on pd in the neonatal period (mean =9.8 days). pre txp native nephrectomies were done in 3 patients (2 in group a and 1 in group b). group a had 2 acute rejections and none in group b. conclusion: anuric young infants had higher rates of post txp utis and v-u reflux, a direct consequence of their inherent small bladders. however, the 3 year graft survival, patient survival and gfr remained excellent in anuric infants (group b )compared to young infants transplanted with higher rates of v-u reflux and dysfunctional bladders pre txp. abstract# p-sat426 kidney transplantation in a child with bladder dysfunction who underwent prior bladder augmentation: a case report 1 tepecik training and research hospital, pediatric nephrology, they were divided into 2 groups; 12pts with esrd on dialysis (d), 4/12 with history of kidney transplant, and 9 kidney transplant recipients with good graft function (t). all subjects were tested for anti-hla antibody 1 month prior to and 1 month and 6 months after administration of the combination vaccine of influenza a/ h1n1. results: among the t group, no pt tested positive for either anti-hla class i or class ii antibodies before or after influenza a/ h1n1 vaccine. in the d group, of the 4 pts with a history of graft failure who were sensitized before immunization; 2 showed no change in class i & ii, one patient had mild increase in class i after vaccination, and one patient had an increase in class i by 24%. [figure] conclusion: none of the t pts had clinical evidence of either cell mediated or humoral rejection after the influenza a/h1n1 vaccine. in the d group, no pt had any statistically significant increase in anti-hla antibody following vaccination. our study suggests that influenza a/h1n1 vaccination may be safe and tolerable in pediatric dialysis pts with or without a failed kidney allograft. the effect of dipping blood pressure status on structural and diastolic heart function in renal transplant recipients mitra basiratnia 1 , gholamhossein ajami 2 1 shiraz nephrology urology research center, shiraz university of medical sciences, shiraz, iran 2 department of pediatric cardiology, shiraz university of medical sciences, shiraz, iran objectives: non dipping has been linked to cardiovascular disease in adults, however the impact of non-dipping on cardiovascular status of the adolescents with renal transplantation has not been well established. the aim of this study was to evaluate the influence of non dipping status on left ventricular mass index and diastolic function in subjects with renal transplantation. methods: sixty six stable renal transplant patients (34 females, 32 males), aged 7 to 25 years (mean 17.4±4.3 years) were enrolled in this study. cardiac function assessed by tissue doppler echocardiography and blood pressure measurement performed using ambulatory method. dipping was defined as at least 10% bp decline during the night and was calculated as (mean daytime -mean night time/mean daytime)*% 100. left ventricular mass (lvm) was calculated by standard 2dimensional directed m-mode echocardiography according to the formula of devereux and was indexed to height in meters to the 2.7 power to allow the comparison between recipients of different sizes. left ventricular hypertrophy was defined as lvmi>38.6g/m 2.7 in patients younger than 18 years and 51g/m 2.7 in patients > 18 years. data analysis was performed by spss-15. a p<0.05 was considered statistically significant. results: non-dipping was identified in 48 (73%) patients. five recipients were systolic non-dippers, 1 diastolic non-dipper, and 42 both systolic and diastolic non-dippers. left ventricular hypertrophy (lvh) was found in 37.1% of the renal allograft recipients. lvh was present in 41% of the systolic nondippers and 27.8% of the systolic dippers (p=0.33) . forty five percent of the diastolic non dippers and 22.7% of the diastolic dippers had lvh (p=0.08) . there was no correlation between systolic and diastolic dipping status and lvh, respectively (p=0.33, p= 0.08). there were no significant differences in terms of diastolic function [measured by early diastolic inflow velocity (e), e/a ratio, and early diastolic mitral inflow velocity to earlydiastolic annular velocity (e/ea)] between dipper and non dippergroups(p= >0.05). conclusion: non dipping is common among renal transplant recipients, but is not always related to diastolic dysfunction and lvh. prospective longitudinal studies are required to determine the impact of dipping status on diastolic and structural heart function in renal transplant recipients. parsa yousefi chaijan, parvin soltani, farshid haghverdi, masood fazelimoslehabadee nephrology, arak university of medical sciences, arak, iran objective: nephrolithiasis in renal grafts is a relatively common phenomenon which can induce organ damage; hence early diagnosis and management of predisposing factors can preclude subsequent complications. the aim of this analytic cross-sectional study is to determine the contributory factors to nephrolithiasis after renal transplantation. method: 56 renal-transplanted patients (10-40 years old) were enrolled in the study, being divided into two groups of 28 transplanted patients suffering from nephrolithiasis (within first 5 years after surgery) and 28 transplanted patients free from renal stone with the same age and gender and similar gfr. data were collected and arranged in excellmicrosoft program and the statistic analysis was carried by spss v17. pvalue < 0.05 were considered statistically significant. result: the studied showed that male gender (p = 0.048), age group of 15-25 years (p=0.021), hyper cholesterolemia (p=0.007), hyper triglyceridemia (p=0.031), 3 rd year after surgery (p=0.023), hyperuricosuria (p=0.012) , hypocitraturia (p=0.001) and anemia (p=0.006) were significantly more common in patients with nephrolithiasis. conclusion: it is better to evaluate hyperuricosuria and hypocitraturia in kidney donors, moreover all patients had better undergo serial sonography for early screen and management of renal stone as well as treatment for hyperlipidemia and anemia. it also seems prudent to further assess the donors of transplanted patients suffering from renal stone and possbile relation between cni & nephrolithiasis ( regardinghyperuricusuria) objective: epstein-barr virus-associated smooth muscle tumor (ebv-smt) in immuno-compromised patients has recently been reported. but there were no evidence about the treatment of ebv-smt. we report a 6-year-old girl treated for multiple ebv-smt by using rituximab. methods: case report results: she was suffering from end stage renal disease due to congenital nephrotic syndrome induced by wt1 mutation. renal transplantation (tx) was performed at the age of 2 years, and immunosuppressive therapy was performed her. soon after performing tx, she was infected cytomegalovirus(cmv), bkv and ebv. she was reduced in amount of immunosuppresive therapy, and she recovered from viremia of cmv and bkv. her ebv titer did not become negative, but we did not perform any medication because of no symptom. four years after tx, she was pointed out 1cm of cholecystic polyp in a protocol abdomen ultrasonic examination. we soon performed cholecystectomy and extirpation of swelling mesenteric lymph nodes. the pathological finding of cholecystic polyp was ebv encoded small rna (eber) positive smooth muscle tumor but there was no finding about post-transplant lymphoproliferative disorder in mesenteric lymph nodes. positron-emission tomography (pet) showed accumulation of 1cm mass around 12th of thoracic vertebra and ct scan showed three small masses in the lung and one in the liver. we diagnosed her suffering multiple infection of ebv-smt. immunosuppression therapy was reduced, but there was no change about size of her tumor. rituximab was administered for keep away from repetition of tumor after proving infection of ebv only in b cells by using flow cytometry. after medication of rituximab, the ebv dna counts were normalized. ultrasonic examination detected three small masses in liver and one lymph node around main artery. but her mass around 12th of thoracic vertebra was disappeared and whole body ct scan detected only one small residual mass in the lung. we report a case of an ebv-smt presenting multiple infections, which was treated with rituximab. rituximab may be an effective treatment for multiple ebv-smt but careful observation is also needed for the reappearence of ebv-smt. the usefulness of monitoring of epstein-barr viral load after renal transplantation in pediatric recipients with ebv seronegative objective: the purpose of this study are to establish a protocol for monitoring epstein-barr virus (ebv) infection for identification of pediatric renal transplant recipients with a high risk of developing posttransplantlymphoproliferative disorder (ptld) and to predict the development of ptld. methods: peripheral blood mononuclear cells (pbmcs) and plasma ebv loads were measured by nested pcr (n-pcr) and real-time pcr (r-pcr) every 1-3 months after grafting in 17 pediatric recipients who were seronegative for ebv before grafting (4 with ebv-associated symptoms, including 2 with ptld (group a); 6 with asymptomatic persistent high ebv loads in pbmcs of >1,000 copies/ug dna for over 6 months (group b); and 7 with neither ebv-associated symptoms nor persistent high ebv loads in pbmcs (group c). ebv-ctls were also measured in 13 patients without ebv-associated symptoms. results: the ebv genome detected by n-pcr was present in plasma in 3 (75%), 1 (17%), and 0 (0%) in groups a, b and c (p<0.01 for a vs. b and a vs. c). ebv loads detected by r-pcr in pbmcs were significantly higher in groups a (p<0.05) and b (p<0.01) compared to group c. ebv genomes in plasma were detected by n-and r-pcr in only the 2 cases with ptld. one patient with lymphadenitis in group a and 1 patient in group b had ebv-dna in plasma based on n-pcr, but the viral loads using r-pcr were <250 copies/ml. the ctls' percentage was significantly lower in group b when ebv loads first rose above 100 copies/ugdna. conclusion: plasma ebv loads (over 250 copies/ml) estimated by r-pcr and ctls' monitoring may be useful to distinguish ptld from other ebv-associated diseases or asymptomatic viremia, and to avoid ptld as patients with asymptomatic persistent high ebv loads had higher ebv loads and lower percentages of ctls. abstract# p-sat437 risk factors for post-transplant lymphoproliferative disorder (ptld) in children with kidney transplantation (ktx) -a single center survey since the introduction of tacrolimus (tac) since pre-transplant evaluation showed complete obstruction of ivc/ iliac vein below diaphragm, she had been managed with peritoneal dialysis for 7 years until january 2012 when she received a living donor kidney allograft from her mother. the graft was transplanted in a left orthotopic position. venous drainage was to the left ascending lumber vein. in addition, a venous bypass was made using donor ovarian vein between graft vein and splenc vein after splenectomy. such double venous drainage was working very well after transplantation. there was no surgical complication and the serum creatinine (s-cr) during the hospital stay was 0.54mg/dl. at second month after the transplantation, she developed an increase of s-cr with edema and hypertention. however, biopsies of kidney allograft performed at 2 nd and 4 th month after the transplantation revealed no evidence of acute rejection. hypertention was difficult to control even with a maximum dose of calcium blocker, and a temporary increase of the s-cr up to 1.30 mg/dl was noted when angiotensin ii receptor blocker was administrated. by the ultrasonography, the maximum arterial blood flow of the kidney allograft was high enough (459cm/s) to suggest renal arterial stenosis. moreover, severe renal arterial stenosis was clearly shown for about 10mm length from the anastomosed site of aorta by the contrasting ct.for the treatment of artery stenosis of the kidney allograft, percutaneous transluminal renal angioplasty (ptra) was performed at 8 th month after the transplantation. the stenotic lesion was expanded using the special dilatation balloon from 1.7mm through 2.0mm in diameter. because intravascular ultrasonography showed no intimal thickening of the blood vessel, a vascular stent was not placed. after the ptra, s-cr was decreased to 0.5mg/dl, and the blood pressure became controllable by antihypertensive agents. moreover, the angiography performed 4 months after the ptra revealed no progression of arterial stenosis. the s-cr of this patient is now stable in 0.7 mg/dl. post-transplant encapsulating peritoneal sclerosis in children: a single center experience kei nishiyama tokyo women's mdical university, pediatric nephrology, tokyo, japan objective: a substantial proportion of encapsulating peritoneal sclerosis (eps) cases develop after renal transplantation (rt), an entity known as post-transplant eps. although risk factors for eps include prolonged pd, recurrent peritonitis, decreased ultrafiltration and prolonged administration of hypertonic glucose or icodextrin, the pathophysiology of post-transplant eps is largely unknown. however it has been postulated that the use of calcineurin inhibitors (cnis) after transplantation may promote eps, as these drugs are considered profibrotic. a recent scottish study showed that the contribution of post-transplant eps might be even as much as 50% of the total eps patients. by contrast pediatric cases are very rare. therefore we examined the incidence of post-transplant eps in pediatric renal transplant recipients. methods: in this study, we retrospectively investigated clinical records from 52 consecutive pediatric renal transplant recipients in our center between 2007 to 2012, who performed pd before transplantation. clinical parameters and pd-related risk factors of eps were collected at the time of rt. transplant-related variables were also collected. eps cases who met ispd diagnostic criteria including clinical feature and either radiologic and/or histopathological confirmation were examined. results: the median duration of pd was 2.0 yr (range 0.5 to 7.9). twelve patients (23.1%) had at least one episode of peritonitis. ten patients (19.2%) were administered hypertonic glucose and/or icodextrin. the median follow-up period after transplantation was 2.3 yr (range 0.3 to 5.8). all patients were administered cni, and 2 patients discontinued corticosteroid during the follow-up period. although 5 patients had ultrafiltration failure at the time of rt, there was no case who developed post-transplant eps. conclusion: the case of eps after rt was not seen in this study. possible explanation of this result might be associated with shorter durations of pd and small sample size. since post-transplant eps is rare but carries a high mortality, caution should be paid to developing post-transplant eps even in pediatric patients with a relatively long pre-transplant duration of pd. asli kantar, kaan gulleroglu, esra baskin, umut bayrakci, zafer ecevit, hande arslan, aydincan akdur, gokhan moray, mehmet haberal pediatric nephrology, baskent university, ankara, turkey objective: viral infections remain a significant cause of morbidity and mortality following renal transplantation. although cytomegalovirus is the most common opportunisticpathogenesis in transplant recipients, numerous other viruses may affect clinical outcome. viral infections are potentially severe complications of transplantation, as they not only induce specific diseases, but they also favor the development of allograft damage, opportunistic infections and acute rejection. we evaluated the major viral infections seen following kidney transplantation and allograft outcomes in our pediatric patients. methods: we evaluated retrospectively 94 pediatric renal transplant recipients for the occurrence of viral infections and compared outcomes among these patients. patients were divided in to two groups those who developed an infection and those who did not. inthese groups, we recorded induction therapy used at transplantation, immunosuppressive therapy given at discharge, acute rejection rate, patient and graft survival rates. results: the mean age of the patients was15.5±5.3 years. viral infection was found in 32 patients. cytomegalovirus infections were the leading causes; ebv and bk virus were following causes in our study. any significant correlation could not be shown between viral infections and immunosuppressive therapy. we did not observe any correlation between acute rejection and viral infections. lowest gfr at 6 th month was shown in patients with bk virus infection. a significant correlation was shown between viral infection and graftloss (r: 0.22 p: 0.028). conclusion: viral infections are common after kidney transplantation. patients should be monitored more carefully for provide against damage in transplanted kidney. objective: anemia is a frequent condition in kidney transplant recipients and it has a negative long term impact on graft and patient outcomes. recently it has been shown that treatment with angiotensinconverting enzyme inhibitors (acei), angiotensin ii receptor blockers (arb) and mtor inhibitors could be the leading causes of anemia in renal transplant recipients.to study the association of hemoglobin (hb) and ferric parameters with gfr, immunosuppressive drugs, acei, arb and clinical features of kidney recipients. method: hospital records of 94 (f/m:48/46) kidney recipients were reviewed retrospectively. the mean age of the study group was 15.5±5.3 years. the mean follow-up was 52.6±36.31 months. results: thirty six (38%) patients were found to be anemic. mean hb levels of anemic and non anemic patients were 10.2±1.15 mg/dl vs 13.87±3.41 mg/dl respectively. ferritin and iron levels as well as transferrin saturation index, rdw and mcv did not differ among the groups. anemia was not found to be correlated with immunosuppressive or antihypertensive drugs including acei and arb. donor status did not also have any influence on anemia. mean gfr of patients at posttransplant 6 months and 1 year follow-up was found to be significantly lover than patients without anemia (74.4±34.1 vs 99.1±28.9 ml/min/1.73m2 respectively). graft loss was also found to be significantly higher in anemic patients (16% vs 6%, p<0.018). conclusion: in this study we examined the prevalence of anemia and its risk factors in kidney transplanted patients. its incidence is quite high in our patients. we found that anemia in kidney recipients is neither related to iron status nor medications like acei, arb or immunosuppressive drugs. the major determinant of hb level, especially during the first year of transplantation is the graft function. the most important conclusion of this study is the considerable controversial impact of anemia on graft survival. there was no significant effect on blood pressure ; 4 patients 7±2 modigraf® administration days in the converted group. in the whole series, mean creatinine level was 0.7±0.5 mg/dl throughout the observational period, and no rejection episodes were detected. blood pressure was well controlled and no proteinuria was seen. the equivalent dose ratio between modigraf® and liquid tacrolimus was 1.28. conclusion: modigraf® appears to be a safe and sustainable way to administer tacrolimus in kidney transplanted infants. in our experience required modigraf® dose was 1.28 times the oral liquid tacrolimus one, and therapeutic levels were attained in one week. objective: prescribing of medications in paediatric practice is problematic as many drugs remain unlicensed. this is especially true of immunosuppressive medications. however, there is now a licensed product for tacrolimus called modigraf® with data available on bioavailability. we undertook a single centre prospective study of conversion to modigraf®, an oral liquid available in granule formulation (1mg and 0.2mg) that allows for dosing according to body weight. methods: all paediatric renal transplant recipients (rtr) under the care of a single centre were considered for conversion to modigraf® from their current tacrolimus regimen. inclusion criteria included all rtr under 18 years of age who were on tacrolimus suspension. exclusion criteria included recipients of multi-organ grafts, patients with lactose intolerance and patient choice to continue with their current suspension. patients were then seen by the multi-disciplinary team. after equivalent dose conversion, patients were monitored with blood tests one week after conversion with subsequent doses adjusted accordingly. patients continue to be monitored with blood levels over a 3 month period after conversion with renal allograft function, renal allograft loss and side-effect profiles recorded. results: forty-three (27% of 158) rtr were considered for conversion to modigraf®. the families of four patients requested to stay on their current tacrolimus suspension, three patients were deemed unsuitable due to lactose intolerance and three patients were excluded due to low doses (incompatibility with the granule dosing). thirty-three patients were then converted to modigraf® and closely monitored. after blood level monitoring one week after conversion, 12% (4) patients had their doses increased due to lower than anticipated 12-hour trough tacrolimus levels. there was stable renal allograft function without renal allograft loss after conversion. conclusion: conversion to modigraf® immunosuppression can be safely undertaken in paediatric rtr, although regular monitoring in the immediate period of conversion is required to provide accurate immunosuppression dosing. escort trial -effects of strict control of blood pressure in pediatric renal transplant recipients -baseline characteristics of patients from a randomized controlled trial tomas seeman, jiri dusek, nadezda simankova, karel vondrak, jakub zieg dpt. of pediatrics, university hospital motol, prague, czech republic objective: arterial hypertension is a known risk factor for impaired graft survival in patients after renal transplantation (rtx). strict control of blood pressure (bp <50 th percentile) delays progression of chronic kidney diseases in children (escape trial). it is not known whether strict bp control has renoprotective effect also in children after rtx. the aim of this randomized controlled trial was to investigate whether strict bp control can protect kidney graft in children after rtx. methods: all children from our pediatric renal transplantation center were screened for eligibility for the study (children 3-16 years at least 1 year after rtx, no acute rejection in the last 3 months, egfr>15 ml/min/1.73m2, 24hr mean bp >50 th percentile using ambulatory blood pressure monitoring abpm). altogether 23 children fulfilled the inclusion criteria. they were randomized to intensified bp control group (intens, target 24hr map <50 th percentile, n=12) or standard bp control group (stand, target 24hr map 50-95 th percentile, n=11). all antihypertensive drugs are allowed to reach the target bp. the study period is 3 years. the primary endpoint is the yearly change in egfr (schwartz formula, ml/min/1.73m2/year), the secondary endpoints are graft failure, change in proteinuria, left ventricular mass and safety of strict control of bp. results: the baseline characteristics of the patients are given in the allelic variants. tac dose (mg/kg/day) and tac exposure normalized for dose (tac co/d) (ng/ml/mg/kg/day) were analyzed with respect to cyp3a5 genotype and for interaction with azoles and corticosteroids, for a period of one year post-transplant. over time, tac co/d was significantly lower in recipients with a cyp3a5*1/*3 genotype compared to those being homozygous for the cyp3a5*3 allele (55.33 +/-21.30 versus 83.07 +/-9.9 ng/ml/mg/kg/day, p= 0.0068). the dose requirement was significantly higher in children with a cyp3a5*1/*3 genotype compared to those being cyp3*3/*3 (0.21 +/-0.02 versus 0.17 +/-0.01 mg/kg day, p=0.0079). the tac co/d was significantly higher for patients receiving azoles (n=17) than those not receiving azoles (n=40) (117.21 +/-1.06 and 62.01 +/-0.52 ng/ml/mg/kg/day, p= 0.002) and consequently the required tac dose was lower in patients receiving azoles overtime (0.08 +/-0.00 versus 0.22 +/-0.01 mg/kg/day, p < 0.001). in children receiving steroids without azoles the tac co/d was significantly different between the cyp3a5*1/*3and cyp3a5*3/*3 genotypes, respectively (52.86 +/-1.71 vs. 58.68 +/-0.85 ng/ml/mg/kg/day, p=0.0044) but the tac co/d was not different for those receiving steroid versus those not receiving steroid with cyp3a5*3/*3 genotype ( 84.91 +/-12.7 and 84.21 +/-1.41 ng/ml/mg/kg/day, p=0.16). conclusion: in conclusion, the tac dose is influenced by the cyp3a5 genotype and drugs such as azoles, while steroids may not impact tac dose. while therapeutic drug monitoring of tac remains necessary, integrated knowledge of patient genotype and comedication use provides the opportunity to refine tac dosing in children receiving kidney transplant. abstract# p-sat458 basiliximab induction therapy in pediatric renal transplantation, a double blind clinical trial hasan otukesh tehran university of medical science, associated professor, tehran, iran this is an open, single center, randomized study to compare induction therapy with basilixamb with no induction therapy in children with renal living transplantation. in this trial 20 pediatric renal transplant recipients enrolled randomly to one group with basiliximab as induction therapy and another group without basiliximab induction therapy. both group received prednisolone, cyclosporine and cellcept. we assessed graft function at 3 months after transplant and compared this item between these two groups. in the congress the data and results of this randomized trial will be presented. abstract# p-sat459 legalization of the organ donation and optimal utilization of young pediatric donor kidneys into pediatric recipients in china objective: china has started to establish a new national system for organ donation and transplantation since march 2010 by the ministry of health and the red cross society of china. the aim of this study was to describe our initial experience of pediatric renal transplantation using organ donations from pediatric patients no more than 6 years old. methods: the procedure of organ donation includes: 1. judgment of brain death, or circulatory death, or brain death followed by circulatory death by doctors; 2. organ donation informed consent form signed by family (children's parents); 3. approval by the hospital ethics committee; 4.organ donation to the red cross society and allocation by the china organ transplant response system (cotrs). the red cross society has been commissioned by the ministry of health to run this system. clinical data of 8 children who underwent renal transplantation using organ donations from pediatric patients no more than 6 years between september 2011 and march 2013 were retrospectively analyzed. results: the age at transplantation for these 8 patients was 4.5 years to 14 years and the weight was 14 kg to 35 kg. among these 8 cases, 7 donors were used aged from 33 days to 6 years and all diagnosed with circulatory death. 5 recipients received en bloc kidney transplantation and the other 3 recipients received one single kidney according to the size of both recipient and donor. the duration of follow-up after the transplantation was 1 month to 18 months. patient survival rate was 100% and graft survival rate was 7/9 (77.8%, one graft loss due to the hemorrhagic complication and the other one due to the thrombosis). at latest follow-up, the median serum creatinine level was 92umol/l and the median egfr was 80ml/(min·1.73m 2 ). conclusion: organ transplantation legislation is necessary to ensure the rights and obligations of donors, recipients and medical institutions. we believe young pediatric donors can be expanded further to increase the number of pediatric renal transplants. this pediatric to pediatric combination on the one hand efficiently lower the discarding ratio of the kidneys from small donor, and give more chances to the younger recipient on the other. to assess the normalization of serum 25 (oh)d level (>30 ng/ml) after standard treatment dosing among primary hypertension (ph) and chronic kidney disease (ckd) methods: we enrolled 144 patients aged 2-19 yrs we collected retrospective data on age, sex,race, cause of kidney disease, egfr, ht, wt, bmi, bp z-scores, lipid panel, 25(oh) d level, pth, calcium, phosphorus, magnesium, medications, type and dosing of vitamin d supplements and follow up serum vitamin d level three months post treatment. results: mean age (yrs) was (12.99±5.05). white(2) prevalence of vitamin d deficiency was (88%); 60% had level <20 ng/ml. mean pretreatment 25(oh) d (ng/ml) was (19.25±10.69) after completion of standard treatment almost 64 % patients had 25 (oh) d level <30 ng/ml; ph(28.02±9.22) and ckd(25.38±9.8). none were in toxic range. conclusion: the standard treatment dose of vitamin d doesn abstract# p-sat360 mineral metabolism in european children with end-stage renal disease marjolein bonthuis 1 the netherlands 2 pediatrics, nephrology and dialysis unit results: hypocalcaemia was found in 22% of hd, 18% of pd, and 48% of transplanted patients, with a mean time on transplantation of 4.3 years. hyperphosphataemia was found in 47% of hd, 37% of pd, and 5% of transplanted patients. pth was outside target in 56% of hd, 58% of pd and 14% of transplanted patients. in dialysis patients, calcium and pth were inversely associated with age; 53% of adolescents were hyperphosphataemic resulting in a significantly higher risk compared to 3-5 year olds (or:1.77, 95%ci:1.43-2.19). patients transplanted pre-emptively had a lower risk of hypocalcaemia compared fgf23 is modulated by calcium in children under chronic peritoneal dialysis azocar 1 , maria l. ceballos 1 , angelica m. rojo 1 luis calvo mackenna children's hospital human intact fgf-23 levels (pg/ml, immutopics) were determined through a 2-site elisa kit. klotho levels were determined by a solid phase sandwich elisa kit (pg/ml). descriptive statistics, univariate and multivariate analysis were performed methods: we measured serum calcium, phosphorus, intact parathyroid hormone, alkaline phosphatase (alp), 25-hydroxy vitamin d3 (25d3), 1, 25-hydroxy vitamin d3 (1,25d3), and fgf-23 from 233 children (male:female = 157:76, mean age 10.2 years) with ckd stage i-v predialysis in korea with 0.5-1 year intervals since 2011. results: hypocalcemia was observed in 23.8%, 32.4%, 21.8%, 22.4% and 31.8% of patients with cdk i to v (the rest is the same as above) 3%, 8.3%, 47.1%, 68.1% and 40.9%. 25d3 level was below fgf-23 (ru/ml) was 38.0, 44.9, 54.4, 67.3 and 119.6. serum p had positive correlation with alp (p < 0.0001), ipth (p = 0.0489) and fgf-23 levels (p < 0.0001) and was inversely correlated with urine phosphorus conclusion: the prevalence of hypocalcemia, hyperphosphatemia and hyperparathyroidism increased as ckd progressed. serum ipth and fgf-23 increased and 1,25d3 level decreased in proportion to the progression of ckd malaysia 6 national transplant resource centre, national transplant resource centre, kuala lumpur, malaysia objective: to analyse the cost-effectiveness of paediatric and adult living related renal transplantation (lrt) and deceased donor renal transplantation (drt) the time horizon was the lifetime of transplant recipient from transplant to death. data for survival analysis was obtained from national renal registry. statistical analysis was performed using stata se version 11.2. the costs was discounted at 3% p.a. results: we reviewed 206 medical records. there were 88 children (39 lrt, 49 drt) and 118 adults (63 lrt, 55 drt). the paediatric recipients mean age was 12.3 +/-3.4 (lrt) and 14.0 +/-2.4 years (drt), whereas the adult recipients mean age was 33.4 +/-10.3 (lrt) and 41.8 ±8.9 years (drt). the 5 and 10-year patient survival rates for both paediatric lrt and drt were 95% and 85% respectively. the 5 and 10-year patient survival rate for adult lrt was 92.5% and 90% respectively, whereas adult drt was 77.5% and 70% respectively. mean cost per paediatric transplant at first year was rm 81,000 (lrt) and 90,000 (crt), from second year onwards was at 20,000 per year (lrt) and 37,000 per year (drt) . the total lifetime cost was rm 650,000 (lrt) and 630,000 (drt) their serum creatinine, egfr, cholesterol, ldl, proteinuria, full blood counts and liver function were profiled to compare the pre-/post-conversion changes. common adverse effects, acute rejection, opportunistic infection and need of treatment for hypercholesterolaemia and proteinuria were investigated. results: we have 15 eligible patients with m: f = 7 : 8. the mean age at renal transplantation was 12 sirolimus was started at a mean age of 15.4 +/-5.0, at a mean time period of +/-2.5 years after renal transplantation, and used for a mean duration of we observed new onset hyperlipidaemia in 9 patients (60%) and needed statin treatment. we found significant proteinuria (spot pr/cr > 1.0 mg/mg) in 4 (27%) patients and needed acei treatment. there was no adverse effect on blood counts and liver function. there was no opportunistic infection observed after conversion to sirolimus in the study period. there was one acute cellular rejection (ia) one month after conversion to sirolimus and responded to pulse methylprednisolone. conclusion: converting from a cni based immunosuppressant protocol for paediatric renal transplantation patients to a sirolimus based one was effective and the benefit was shown up to 54 months post-transplantation one patient received a desensitisation regime with rituximab and plasmaexhange pre-transplant and iv immunoglobulin post-operatively. surgical complications occurred in 7 patients; 2 lymphocoeles, 3 ureteric leaks and 2 renal vein thromboses. early post-operative medical complications included hypertension (47), pulmonary oedema (13), seizures (9), reaction to basiliximab (3), sepsis (5), acute tubular necrosis (8), delayed graft function (5), acute rejection (2) and diabetic ketoacidosis (1). primary ebv infection occurred in 38 patients -1 developed ptld. there were 9 (12%) graft failures; renal vein thrombosis (2), acute humoral rejection (1), acute tubulointerstitial nephritis and tacrolimus toxicity (1), de-novo acute glomerulonephritis (1), chronic rejection (2) and non-compliance (2). graft survival rates were 95% at one year, 93% at 3 years, 89% at 5 years and 88% at 10 years. patient survival was 100% at 10 years. conclusion: our outcomes compare well with international figures a desensitisation program for paediatric renal transplant patients in nsw one child received atg in addition. results: complications/ patient outcomes:1: 2 yo boy, donation from mother. dsa antibodies mfi 658. no complications. no infections. good renal function, creatinine 45 at 2yrs. 2: 14 yo boy, previous transplant from biological father. donation from foster father. dsa antibodies mfi 2602. post operative complication of pseudomonas pneumonia/bronchiectasis, cmv, acute cellular rejection x2. good renal function creatinine 120 at 1 yr. 3: 17 yo boy, donation from father. dsa antibodies mfi 724. early e-coli urine infection. no other complications, creatinine 117 at 1 yr. 4: 14 yo girl, recipient of 3 liver transplants. maternal donor with positive b cell cxm and dsa mfi 6488. no complications. good renal function four of them had subsequently increasing needs in bicarbonate and/or sodium supplementation, and f was reintroduced in 3 of them. conclusion: f is effective in most cases of severe tubulopathy after rtx. however, side-effects can occur. further prospective studies are needed to validate this indication methods: eighty-one (f/m: 41/40)pediatrictransplant patients were included to the study.demographic characteristics and laboratory parameters were recorded.risk factors for hyperuricemia and the effects of plasma uric acid levels at 3 rd and 6t h months, 1 st and 3 th years on allograft outcomes were evaluated. results: mean age was 16.9±5.6 years.mean follow-up time after transplant was 3.5±0.47 years. hyperuricemia was detected in 17.6% of patients. a significant negative correlation was found between 6 th month uric acid leveland 3 th year of gfr value (r = -0.33, p = 0.04 and r = -0.33, p = 0.017). a significant positive correlation between 3 th and 6 th months uric acid levels and3 th year plasma creatinine level was demonstrated conclusion: uric acid levels may have predictive value in the long term assessment of renal function.posttransplanthyperuricemia can be used as a long term prognostic marker of poor renal outcome.patients with hyperuricemia should be monitored closely for renal functions abstract# p-sat447 tuberculosis in paediatric renal transplant patients -single centre experience methods: retrospective descriptive folder review of tb in paediatric renal transplantation at red cross children's hospital, university of cape town from 1994 -2012. results: 14 paediatric renal transplant with tb identified. male 11: female 3. ages 3.4 -18.8yrs(mean 12.0; median 13.2). all patients screened for tb prior to transplant polycystic kidney disease, 2 dysplasia, 2 chronic glomerular nephritis, 2 unknown cause, 1 systemic lupus erythematosis and 1 all patients were on steroids and 8 had recent intensification of immunosuppression. tb treatment included conventional drugs in all but 1 case. levels of calcineurin inhibitors were affected in 12/14 patients and required increased dosing(up to 3 times baseline) according to levels in all. rejection was seen in 7 patients(all biopsy proven). successful treatment of tb in all patients with retention of graft. graft and patient survival 100% post treatment. conclusion: tb is a significant problem in paediatric renal transplants in developing countries. our series shows that with careful investigation and diagnosis of tb as well as careful monitoring of immunosuppressant levels objective: to evaluate the reliability, validity and feasibility of the persian version of the pediatric quality of life inventory (pedsql tm 4.0tm 4.0) generic core scales in iranian healthy students ages 7-15 and chronically ill children ages 2-18. methods: we followed the translation methodology proposed by developer to validate persian version of pedsql tm 4.0tm 4.0 generic core scales for children. six hundred and sixty children and adolescents and their parents were enrolled. sample of 160 healthy students were chosen by random cluster method between 4 regions of isfahan education offices and 60 chronically ill children were recruited from st. alzahra hospital private clinics. the questionnaires were fulfilled by the participants. results: the persian version of pedsql tm 4.0tm 4.0 generic core scales discriminated between healthy and chronically ill children (healthy students mean score was 12.3 better than chronically ill children, p< 0.001). cronbachs alpha internal consistency values exceeded 0.7 for children self reports and proxy reports of children 5-7 years old and 13-18 years old. reliability of proxy reports for 2-4 years old was much lower than 0.7. although, proxy reports for chronically ill children 8-12 years old was more than 0.7, these reports for healthy children with same age group was slightly lower than 0.7. constructive, criterion face and content validity were acceptable. in addition, the persian version of pedsql tm 4.0tm 4.0 generic core scales was feasible and easy to complete. conclusion: results showed that persian version of pedsql tm 4.0tm 4.0 generic core scales is valid and acceptable for pediatric health researches. it is necessary to alternate scoring for 2-4 years old objective: bisphosphonates are widely used in the management of children with steroid induced osteoporosis (sio). with the increasing use of bisphosphonates, there have been reports of abnormal radiological findings in the growing skeleton. therefore, their use in pediatric patients remains controversial. the present study was conducted to evaluate the long term follow-up results of radiographic features especially metaphyseal sclerotic lines, associated with pamidronate therapy in pediatric patients with nephropathy. methods: twenty two children with nephropathy receiving oral calcium and pamidronate (mean duration: 7.9 months, dose: 125mg daily) were evaluated restrospectively. all patients had soi because of chronic glucocorticoid therapy for the treatment of nephropathy. biochemical tests, long bone radiography and bone mineral density (bmd) were performed before the treatment of pamidronate and followed up several years later. the physeal growth rates were estimated by measuring the distance that the sclerotic lines moved on the radiographs during the corresponding time intervals. results: the mean follow-up period was 9.5 years. in all patients, the well-defined sclerotic lines at the metaphyseal ends were observed and progressively moved from physeal plate to diaphysis on the radiographs of long bones. the mean moving rates of the sclerotic lines was 7.95 mm per year and in twelve patients, the lines disappeared. and the mean growth rate of height was 4.50 cm per year. conclusion: our long-term follow-up results suggest that the metaphyseal sclerotic lines associated with pamidronate treatment tend to disappear without affecting the skeletal growth. bisphosphonate treatment for soi in pediatric patients with nephropathy seems to be safe although further studies for larger number of patients are needed abstract# p-sat366 clinical effectiveness and safety of the high dose active vitamin d therapy on severe hyperparathyroidism in children with esrd eun gu kang, su-yon kim, joo hoon lee, young seo park department of pediatrics, asan medical center children's hospital, seoul, korea objective: a potent inhibiting effect of active vitamin d on parathyroid hormone is well known, but there is uncertainty on the dose to be used in hyperparathyroidism. the aim of this study is to assess the effectiveness and safety of high dose active vitamin d treatment on severe hyperparathyroidism in children with end stage renal disease (esrd). methods: fifty-four patients underwent dialysis for more than 1 year between may 2002 and feb 2013 in asan medical center. among them, patients who were administered high dose of active vitamin d (dose of alfacalcidol 1mcg/day to 3mcg/day) with severe hyperparathyroidism(intact parathyroid hormone (ipth) >800pg/ml) were selected. changes of ipth, plasma albumin-corrected calcium, phosphorus and 1,25 (oh)2 vitamin d were analysed. results: fourteen patients (10 boys and 4 girls) with median age of 12.5 years (6-19 years) were included. the mean duration of dialysis was 49±32 months and the median duration of the high dose alfacalcidol therapy was 5 months(3-22months). the ipth level significantly decreased in 10 patients (71%) from 1206.3±387.6 to 215.2±117 during the high dose alfacalcidol therapy (p<0.001). serum phosphorus (5.9±1.3mg/dl vs. 6.0±1.8mg/dl, p=0.88) and calciumphosphorus product (53.4±11.9mg2/dl2 vs. 59.8±18.4mg2/dl2, p=0.20) were not significantly changed and 1,25 (oh)2 vitamin d was low or normal after therapy. plasma albumin-corrected calcium significantly increased (9.4±0.64mg/dl vs. 10.2±0.75mg/dl, p=0.016). four patients (29%) had persistent severe hyperparathyroidism despite treatment with high dose of alfacalcidol, which was controlled after kidney transplantation (2 patients) or after parathyroidectomy (2 patients).conclusion: high dose active vitamin d therapy controlled severe hyperparathyroidism in the most children with esrd without significant adverse events. clinical features and treatment of ckd-mbd in children with ckd i-v predialysis objective: vitamin d is known to have multiple effects on the cardiovascular system, renal function, hyperparathyroidism and growth, its deficiency is common in adults and children with chronic renal disease (ckd), but data in kidney transplant children are scarce. the aim of the present study was to investigate the vitamin d status in 3 groups of children and adolescents with ckd and to establish the association between 25(oh) vitamin d (25ohd) levels, age, hyperparathyroidism, short stature, and renal function. methods: we recruited 105 children: 44 renal transplant patients with a functioning graft for at least 6 months (mean age 12 yr, mean graft abstract# p-sat386 the expression and significance of il-6 、ip-10 and il-17 in serum and synovial fluid with juvenile idiopathic arthritis. objective: to detect the disparity of three cytokines about interleukin-6(il-6),interferon-inducible protein 10(ip-10) and in peripheral blood(pb) and synovial fluid(sf) of patients with juvenile idiopathic arthritis (jia). method: serum concentrations of the three cytokines were measured in 27 patients with 13 systemic-onset jia(sjia), 14 polyarticular jia(pjia) and 28 healthy controls using enzyme-linked immunoabsorbent assays (elisa). 19 patients being no marked arthritis symptom or only temporary arthralgia enrolled in probable sjia group. sf from 18 patients with 7 sjia,11pjia were examined for cytokine levels. objective: the aim of the present study is to evaluate serum concentrations of ghrelin and leptin, and their associations with fat mass and insulin homeostasis in children undergoing chronic dialysis. methods: the study population consisted of 40 patients on maintenance dialysis (22 pd and 18 hd) aged between 5-19 years and 20 age-and sex-matched healthy children. serum levels of total ghrelin and leptin were measured in all patients and controls. fasting serum glucose and insulin levels were also measured in the patients; insulin resistance was estimated by the homeostasis model assessment of insulin resistance (homa-ir). nutritional status was assessed by measuring body mass index (bmi), triceps skinfold thickness (tsf) and multi-frequency bioimpedance analysis (bia). body fat mass was estimated by the bia method. results: the mean total ghrelin level was significantly higher in the patients than the controls (1671±1317 vs. 543±365 pg/ml, p<0.001). higher total ghrelin levels in dialysis patients were significantly associated with younger age (p=0.015), lower bmi-sds (p=0.050) and lower bia-based fat mass-z score (p=0.007). the mean leptin level was also higher in dialysis patients compared to the controls but the difference was not statistically significant (15.5±29.2 vs. 6.48±5.51 ng/ml). however, the ratio of leptin levels to fat mass was significantly higher in dialysis patients than the controls (1.16±1.31 vs. 0.50±0.12, p=0.041). serum levels of leptin in dialysis patients positively correlated with bmi-sds, tsf-z score and bia-based fat mass-z score (p<0.001 for all). serum leptin levels also had a positive correlation with serum insulin (p=0.001) and homa-ir (p<0.001), and an inverse correlation with total ghrelin level (p=0.005). conclusion: children on maintenance dialysis have high levels of total ghrelin that are closely related to decreased fat mass and poor nutritional status. in contrast to ghrelin, leptin is associated with increased fat mass and insulin resistance; however, these patients have inappropriately elevated leptin levels in relation to body fat mass that may be related to wasting. objective: this study investigated the influence of social support and other psychosocial factors upon mortality, adherence to medical care recommendations, and physical qol amongst hemodialysis patients. method: 272 hd patients were examined using the qol questionnaire to determine self-reported inclinations. logistics regression through weighted k was used to analyze data. results: 53.5% of patients reported health had interfered with their social activities demonstrating a strong associated with risk towards all-cause (sp=1.33) and cause-specific mortality including cardiac diseases (sp=1.28). these patients had a greater risk of withdrawing (sp=1.67) from treatment, non-adherence to phosphorus (sp=1.06) greater than 7.5 mg/dl and increased risk towards an albumin of less than 3.5 g/dl (sp=1.23). patients reporting dissatisfied with family support (12.0%) were at highest risk to non-adherence to intra-dialytic weight gain (sp=1.27), shortening the dialysis session (sp=1.21) and increased risk of potassium level greater than 6 meq/l (sp=1.14). however, patients reporting dissatisfied with staff support (14.1%) revealed a higher risk of decreased physical qol (sp=0.76). conclusion: this study demonstrated that physical qol was not only affected by medications and other laboratory work-ups but also with additional psychosocial support. the study led to the development of programs empowering patients and families to participate in their treatment plans. the program includes various counselling approaches directed to patient, families, and health team. objective: the aim of the study was to analyze health-related quality of live (hrqol) in children with chronic kidney disease (ckd) dependent on the ckd stage, treatment modality and selected social life elements in families of the patients. furthermore, potential differences between self-and parent/proxy reports and the factors influencing them were assessed. methods: 203 ckd children (on hemodialysis-hd, peritoneal dialysis-pd and conservative treatment-ct) and their 388 parentproxies were enrolled into a cross-sectional national study. a semistructured interview form was used to determine the demographic and social characteristics of the participants. we used the pediatric quality of life inventory (pedsql) 4.0 generic core scales to assess the hrqol in children. results: hrqol scores for all ckd groups were significantly lower in all domains compared with population norms, the lowest one being in the hd group. in ct children, hrqol did not depend on the ckd stage. children with ckd reported problems with education and emotional functioning. both parents assessed the hrqol of their children differently depending on their involvement in the care. there are differences between the hrqol scores of the children and their parents. conclusion: the hrqol in children with ckd is lower than in healthy children. this is already observed in the early stages of the disease. the disease itself influences the child's mental state. children on hd require special support on account of the lowest demonstrated overall hrqol. children's lower rating of the quality of life observed by their parents may render the patients unmotivated and adversely affect their adjustment to life in later years. it may also create conflicts between the parents and the children. objective: chronic medical illness is a significant risk factor for the development of psychiatric disorders. the aims of the study were: to investigate the level of anxiety in children with chronic kidney disease (ckd) and to identify factors associated with the presence of that emotional problem. methods: ckd children on hemodialysis (hd, n=22), peritoneal dialysis (pd, n=20,) and on conservative treatment (ct, n=95), and healthy subjects (n=100) were enrolled in the study. we used state-trait anxiety inventory (stai) for adolescents and stai-c for children. socio-demographic and physical factors were assessed. results: there was a significantly higher level of anxiety-state among hd children (8-12 years) compared with other groups of participants of the same age. the level of anxiety among adolescents (13-18 years), both anxiety-state and anxiety-trait, was significantly higher in the hd group compared with other groups, which did not differ among themselves. in the hd adolescents, there was a correlation between the anxiety-state and the duration of the disease as well as with the number of hospitalizations. pd adolescents in the mainstream education had higher levels of anxietystate and anxiety-trait compared with home schooled patients. conclusion: even though children and adolescents with ckd are at risk of developing a variety of emotional disorders, the level of anxiety among the researched group, with the exception of hd patients, was not significantly different than the level of anxiety among healthy subjects. adolescents on hd who present a high level of anxiety should undergo long-term psychological treatment. ipek akil objective: compare medication adherence and kidney graft loss rate before and after transition in a single-center cohort of pediatric patients. methods: records of the patients transplanted in our center between 1990 and 2011 were screened and patients who remained in the program by their 18th birthday were included in the analysis. adherence and graft function were assessed for the period of 2 years prior and 2 years after transition. undetectable and/or sub-therapeutic levels of calcineurin inhibitors and their level variability were used as measures of adherence. graft survival analysis was performed for the period of 4 years before and 4 years after transition. results: out of 197 screened patients, 71 were eligible and 25 of them were transitioned (tg -transitioned group). the remaining 46 patients were used as a comparison group (cg), 27 of them did not reach the age of transition by the time of the analysis. the median age at transplantation was 17.7 [15.1-19.1] years in the tg and 15.8 [13.5-17.6] years in the cg, median age at transition was 22. 5 [21.7-23 .3] years. overall, there was no significant difference in adherence within tg before and after transition (p=0.5 for low drug levels and p=0.6 for drug levels variability); however, patients with lower pre-transition adherence (tertile i) showed improvement of their adherence following transition when compared with those with high pre-transition adherence (tertile iii), p=0.02. there were 4 graft losses per 67.9 patient-years in tg vs 19 losses per 102.4 person-years in cg (p=0.02). the peak graft loss in all pediatric transplant recipients in our center (n=105 losses) occurred 3 years (sd 5.7 years) prior to the transition. conclusion: at our institution, transition was not associated with worsening adherence or graft loss. this may be in part do to the fact that the transition occurred at a later age than in other institutions. a report on 7-years experience in the use of mtor-inhibitor (sirolimus) in paediatric renal transplantation patients with calcineurin inhibitor (cni) toxicity the management of children with end-stage renal disease (esrd) deu to congenital urological abnormalities is more problematic and difficult than in patients with esrd due to other causes. kidney transplantation in neurogenic bladder patients with small capacity and defunctionionalized urinary bladders is a challenging issue in the field of pediatric transplantation. in these patients with severe bladder dysfunction, augmentation cystoplasty can protect the transplanted kidney by reducing intravesical pressure and creating an appropriate capacity. 8-year-old boy who presented urinary tract infection with fever at 2 year-old and while his investigation, stage 3 chronic kidney disease secondary to bilateral grade 5 reflux disease was found (blood urea: 81 mg/dl, creatinin: 1.5 mg/dl, creatinin clerance: 32 ml/min/1.73m 2 ). his mixiocystoureterographic and urodynamic study were showed bladder wall irregularity and trabeculation; decreased bladder capacity and compliance, respectively. at 8-month of followup he was putted into dialysis programme about 6 years. thereafter, normal bladder capacity was achieved after bladder augmentation implementation. he was achieved renal transplantation from his mother, after 6-month of augmentation operation. now, he still got uneventfull follow-up period about 24 months. consequently, bladder augmentation application with timely and rationally could be a chance to kidney transplantation in children with bladder dysfunction. the effect of anti-hla antibodies on renal graft functions esra baskin 1 , asli kantar 1 umut bayrakci 1 , kaan gulleroglu 1 , mahir kirnap 1, 2 , feza karakayali 1, 2 , aysegul haberal 1, 3 , gokhan moray 1, 2 , mehmet haberal 1, 2 1 pediatric nephrology, baskent university, ankara, turkey 2 general surgery, baskent university, ankara, turkey 3 immunology, baskent university, ankara, turkeyobjective: the identification of suitable donor kidneys for transplant candidates with high levels of circulating antibodies against human leukocyte antigen (hla) is a major challenge and results in adverse graft outcome. methods: seventy four kidney transplanted children without any shown hla antibody in the pre-transplant period were enrolled in the study. their anti hla antibody status was checked by luminex during post transplant period and its relation with the graft function and prognosis of the patients is studied. results: mean age of the patients was 13.5±5.2 years. mean follow-up time was 3.8±1.1 years. pre-transplant cytotoxicity tests and pra was negative in all patients. nine (12.1%) patients were found to have anti hla antibodies after kidney transplantation. mean time for the detection of antibodies was found as 11±4.8 months. patients with anti hla antibodies were similar with patients without antibodies in the terms of age, sex, hla mismatch, transfusions and immunosuppressive drugs as well as the presence of viral infections. mean serum creatinine level was found to be higher in patients with anti hla antibodies. the antibody mediated rejection rate was found to be 7.2% (5/65) in patients without anti hla antibodies while it was 55.1% (5/9) and remained dialysis dependent. all 6 were dialysis dependent by 6 months post diagnosis. time from anca gn diagnosis to kidney transplant (mean±sd) was 31±12 months (range 17 -48 months). all patients received induction therapy and maintenance immunosuppression with prednisone, mycophenolate mofetil, and tacrolimus. median duration of follow up post transplantation was 3.5 years (range 1.25 -6.9). egfr at last follow up was 71.9 ± 34.7 ml/min/1.73m 2 (range 5.7 -100.5). 1 patient lost her transplant to biopsy-proven, severe acute cellular rejection due to complete non-adherence to medications after 21 months of stable transplant function. no patient had recurrence of vasculitis. conclusion: short-term patient and allograft survival in paediatric patients with eskd secondary to anca gn is excellent despite aggressive disease, with no recurrence of vasculitis post transplant.abstract# p-sat429 en-bloc kidneys from infant donors less than 5 kg transplantation into pediatric recipients at school age objective: given the shortage of donor kidneys in china, the use of grafts from deceased infant donors (weight < 5kg) is a potential approach to expand the donor pool. in this study, we reviewed the results of the first cohort of en bloc kidney transplantation of infant donors to pediatric recipients at school age in our center.methods: from february 2012 to march 2013, 4 infant en bloc kidney transplants in pediatric recipients were performed in our center. en bloc kidneys from 4 infant donors (maastricht category iii) who died of severe congenital disease were recovered and donated to the red cross society of tianjin and allocated to our center by china organ transplant response system (corts). donor age ranged from 33 to 56 days with weight ranging from 2.5 to 5.0 kg. recipients included 2 females and 2 males with age ranging from 5 to 11 yr. the en bloc graft was implanted extraperitoneally in the right iliac fossa. the distal end of the donor aorta was anastomosed end-to-end to the internal iliac artery, while the donor vena cava was anastomosed to the external iliac vein. the donor ureters were implanted separately onto the bladder with double-j stents placement. after the operation, the recipients received basiliximab as an induction therapy. maintenance immunosuppression consisted of tacrolimus and myfortic. prophylactic anticoagulation with heparin was used for the first week after transplantation. results: patient survival was 100%. complications included delayed graft function in 1 patient (managed by pd for one week), urine leak in 1, and anticoagulation-related hemorrhage in 1. due to discontinued anticoagulation, one graft was lost early from vascular thrombosis. of the remaining 3 recipients, all had immediate and excellent long-term function with average creatinine of 1.13±0.31 mg/dl at 5 months follow-up (range 1-12 months). conclusion: this is the first report of en bloc kidney transplantation from infant donors into pediatric recipients in china. many improvements by our transplant teams had improved the survival of patients and grafts. based on our experience, albeit very limited, we concluded that favorable outcomes can be obtained from en bloc transplantation from infant donors. objective: to describe the rates and outcomes of renal transplantation in children with intellectual disability (id). methods: we performed a retrospective analysis of all children receiving a first kidney alone transplant in the united network for organ sharing (unos) dataset from january 1, 2008 to october 31, 2011. recipients with definite, probable, and without id were compared using chi-square and fisher's exact tests. kaplan meier curves were constructed for patient and graft survival. results: over the study period, 218 children with definite (90) or probable (128) intellectual disability underwent first renal transplant accounting for 17% of all first pediatric renal transplants (total n=1280). children with definite or probable id did not significantly differ from other recipients on the basis of gender or ethnicity but tended to be younger. children with definite id had higher rates of structural kidney disease and lower rates of glomerulonephritis. children with id were not significantly different than children without id with respect to rate of preemptive transplant, donor source, or number of episodes of acute rejection. graft and patient survival were similar between children with definite or probable id and without id. in cox regression, intellectual disability was not significantly associated with patient or graft survival. conclusion: in this first large-scale study, 17% of all first pediatric renal transplants are performed in children identified as having intellectual disability. early outcomes after transplant appear to be equivalent between children with and without id. further research is needed on long-term outcomes and quality of life effects of transplant in this population. mariana guerra duarte rosa de lima, ana cristina simoes e silva, nadine marcia de faria, eleonora moreira lima pediatrics, universidade federal de minas gerais, belo horizonte, brazilobjective: the aim of this study was to evaluate the clinical course of children and adolescents undergoing renal transplantation at federal university of minas gerais between 2000 and 2011 and to identify possible factors that could interfere with graft survival. methods: a retrospective observational cohort study through analysis of medical records of patients below 18 years submitted to renal transplant were performed. data were analyzed in spss, version 19.0. the results were expressed by descriptive variables and survival analysis was performed using the kaplan-meier method. comparisons between subgroups were made using the log-rank test. p level was set at below 5%. results: we analyzed 64 patients who underwent 66 transplants. the mean age was 10.5 ± 3.9 years. the main causes of chronic kidney disease were cakut (32.8%) and glomerular diseases (26.6%). most patients were submitted to dialysis before transplantation (81.9%) with a mean duration of 36.5 ± 25.5 months. deceased donor was used in 57.6% and the average cold ischemia time was 22.2 ± 6.3 hours. dialysis after renal transplantation was performed in 22.7% of cases. the median survival of patients 1, 5 and 10 years was 98.3%, 95.8% and 92.8%. there were four deaths in this population, two after graft loss. twelve patients lost their grafts (19.7%). the median survival of the graft was 9.2 years and 1, 5 and 10 years survival was 91.7%, 81.2% and 75.6%, respectively. no statistical difference was detected in graft survival between the deceased versus. living donor recipients, the different age groups, the occurrence of hypertension or not, the preemptive transplantation vs. preceded by dialysis (p=0.97) and those with more than three versus less than three hla mismatches. a significant reduction in graft survival was associated with cold ischemia time associated with 24h, need for dialysis after transplantation, early acute or late rejection and creatinine greater than 1mg per dl after the first year os transplantation. conclusion: in our center, graft survival was similar to that described in the literature. however, further studies are needed to address the variables that negatively impacted the results of transplantation. objective: the aim of this study is to assess whether there is an increase in anti-hla antibodies after the influenza a/h1n1 vaccine given to pediatric pts with esrd on dialysis (d) and with kidney transplant (t). methods: 21 pediatric pts were enrolled. mean age was 15.5 years old, 29% female, 71% male, 57% african american, 29% hispanic.objective: ptld is the most common malignancy and important complication of pediatric solid organ transplantation. the rates of pltd have been increasing through 1990s in ktx recipients. we conducted a survey of ptld in children who received ktx since the introduction of tac. methods: from 1975, ktx was performed in 463 recipients at our center. among them, we analyzed data from children who had undergone ktx since the introduction of tac and retrospectively studied incidence of ptld and risk factors, including immunosuppression protocol and pre-ktx ebv serology. results: we retrieved the data of 188 pediatric recipients since 1997 (110 boys, median age at ktx 8.2 years, living ktx 170, pre-emptive ktx 26, ebv-seronegative 82). among them, 11 cases (10 ebvrelated, 1 non-ebv) of ptld were diagnosed in 10 recipients (5.3%), and all cases were ebv-seronegative at ktx. the median duration from ktx to the onset of ptld was 7 months (range: 4-101 months). the most common presenting symptom of ptld was abdominal complaints in 9 cases, followed by fever in 3, superficial lymphadenopathy in 3 and nasal congestion in 1. immunosuppressants were reduced in all cases; the subsequent treatment consisted of antiviral agents + ivig (3/11), anti-cd20 monoclonal antibody (5/11), and anti-cd20 monoclonal antibody + chemotherapy (2/11). only one recipient died due to ptld. assessing the correlation between ptld and immunosuppressants in 82 ebv-seronegative recipients (median age at ktx 5.7 years, cya 39, tac 43, mzr 26, mmf 56, il-2 receptor antibody 56), a significantly higher incidence of ptld was associated with tac (9/43) than cya (1/39) [or 18.3, p=0 .003] in multiple logistic regression analysis. we compared the incidence of ptld, median age at ktx, and rate of ebv-seronegativity in 3 eras (a: 1997-2001, b: 2002-2006, c: 2007-2011) of the study period and found 4%/8.2 yrs/32% in a, 3%/7.1 yrs/43% in b, and 10%/9.0 yrs/55% in c. thus the incidence of ptld increased with the increase in ebv-seronegative recipients. conclusion: in our study, maintenance treatment with tac (compared with cya) is associated with a higher risk for developing ptld. attention should be paid to the increase in the ebv-seronegative recipients.abstract# p-sat438 acute graft dysfunction and encephalitis post renal transplant: the role of epstein-barr virus objective: epstein-barr virus (ebv) has been described as a rare cause of acute kidney injury and encephalitis in children. methods: we report a case of a 5 year old renal transplant recipient who developed acute graft dysfunction and a significant neurological insult with evidence of primary ebv infection. results: the patient had been diagnosed with posterior urethral valves prenatally and received a live related renal transplant aged 4 years. seven months post-transplant he presented fluid overloaded, oliguric, with significant uraemia and hyponatraemia. there was a history of lower urine output and sore throat 4 days before admission with evidence of anaemia with red blood cell fragments, thrombocytopenia, mild monocytosis, raised ldh, alt and splenomegaly on presentation. haemodialysis was commenced. primary ebv infection was subsequently detected by serology and pcr.renal biopsy showed a picture of acute glomerular thrombotic microangiopathy(figure above) and acute tubular injury secondary to haemoglobinuria.scanty eber+ lymphocytes containing ebvencoded small nuclear rna were also present. within 3 days of presentation patient deteriorated neurologically developing a reduced conscious level, dysarthria, decreased motor power peripherally and an inability to upward gaze. an mri brain demonstrated increased t2 signal and mild diffusion restriction in in the caudate nuclei, putamina and ventro-lateral thalami. this picture has been previously described in ebv encephalitis. there was a gradual neurological improvement and by day 7 of admission, and 6 doses of i.v. methylprednisolone, the patient had a full neurological recovery. the number of ebv copies has subsequently decreased on lower dose of immunosuppression. by week 4 after presentation he was no longer dialysis dependent with creatinine 3-fold higher than the previous baseline. conclusion: this is the first report of acute graft dysfunction and encephalitis due to primary ebv infection in renal transplant recipient.abstract# p-sat439 indoleamine 2,3-dioxygenase (ido) as a new immunological marker in kidney elisa loiacono 1 , barbara votta 2 , alessandro amore 1 , licia peruzzi 1 , maria paola puccinelli 2 , roberta camilla 1 , luca vergano 1 , giuliana guido 3 , maria elena donadi 1 , rosanna coppo 1 1 nephrology, cittàdella salute e dellascienza. regina margherita children's hospital, turin, italy 2 nephrology, cittàdella salute e dellascienza, turin, italy 3 nephrology, sapienza university of rome, rome, italyobjective: the enzyme indoleamine 2,3dioxygenase (ido), induced by interferon-gamma (ifn-gamma) and toll-like receptors (tlrs) ligands in dendritic cells, degrades the essential aminoacid tryptophan (trp) to kinurenine (kyn). its activity is estimated by the ratio of kyn to trp concentration (kyn/trp).t-cell activation and proliferation are affected by trp deprivation and accumulation of kyn. therefore, activating ido during immune responses counter balances mechanism of negative feedback loop of ifn-gamma and acts as a tool to downregulate overwhelming immune activation. ido activation has been reported to be increased in acute rejection and downregulated in vitro by the immunesuppressants adopted for organ transplantation. objective: to determine the prevalence of mycophenolic acid (myfortic) use as part of the immunosuppressant regime in our current paediatric renal transplant recipients in a single tertiary paediatric nephrology centre and examine the demography of these cases. method: case note and pharmacy record review of all paediatric renal transplant recipients' history, immunosuppressant medication and current renal allograft function in january 2013. indication for medication switch from mycophenolatemofetil to enteric-coated mycophenolic acid tablets (myfortic) was noted. data presented as median (range). results: 14 (10 male) out of a total of 88 patients with renal transplants are currently receiving myfortic in our centre. age and time from transplant was 12.3 (8. 1-17.4 ) years and 4.3 (0.68-10.6) years. the most common cause of end stage renal disease requiring transplantation in this subgroup was posterior urethral valves in 5/14 cases. two of these recipients had abo incompatible living related transplants. current egfr was 46.7 (20.9-82.3) ml/min/1.73m2. 13/14 patients receiving myfortic were also receiving prednisolone and tacrolimus as part of their immunosuppression regime. one patient was on tacrolimus and myforctic only (history of idiopathic intracranial hypertension). dosage of myfortic varied based on patient size from 180mg twice daily to 720mg twice daily. the indication for carrying out a medication switch from mycophenolatemofetil to myfortic was related to gastrointestinal symptoms in all cases; specifically diarrhoea in 9 cases, abdominal pain in 2 cases and both symptoms in 3 cases. these symptoms improved on myfortic with renal allograft function remaining stable, however in view of neutropenia in one patient her myfortic is currently suspended and under review. we have found that enteric-coated mycophenolic acid (myfortic) was a well-tolerated alternative in paediatric renal allograft recipients who developed gastrointestinal symptoms whilst receiving mycophenolatemofetil. its usage should be considered in such patients who are able to take tablet preparations. ferretti alfonso, ilaria luongo, bruno minale, gabriele malgieri, carmine pecoraro nephrology and urology, santobono children' hospital, naples, italyobjective: uti represent one of the main complications after kt and have an important role in graft funcion impairment. this retrospective report is aimed to evaluate the incidence of uti in a group of patients attending our hospital for post transplant usual medical controls. methods and results: we incuded in our study 75 children who underwent kt between 2000 and 2011: 42 m and 33 f, mean age 11.6 +/-5.3 . four children received a graft from a living related donor and 71 from a deceased donor. first morning sterile urine sample to the microbiology laboratory was obtained when attending the hospital for its usual medical control or immunesuppressive drug detection levels. results: in 41 patients (54.7%) esrd was secondary to cakut. thirty-six patients (48%) presented at least one episode of uti occurred in the first 6 months after kt, mainly in females (1.2:1) . uti developed in the first period were caused mainly by gram negative bacteria (91%). e. coli was the main agent (66.1%), the other uropathogens involved were: proteus (9%), enterobacter cloacae (9%), pseudomonas aeruginosa (6%), candida albicans (3.3%) and klebsiella (3.3%). later infections were caused mainly by candida, klebsiella, proteusansdenterobacterfaecalis. one patient at the 15th month post-transplantation manifested uti caused by corinebacterium urealyticum associated with concretions of baldder mucosae. eight patients (5m and 3 f) experienced the graft loss: 4 patients were affected by cakut and had febrile uti posttx. one patient left the second graft by a an acute deterioration of renal function during an uti by bkv. conclusion: uti in kidney transplanted children represent a main complaint as demonstrated by the high incidence (48%) in total population mainly in females. a significant role is played by primary uropathy and immunosuppression as demonstrated by the high frequency also in non uropathic patients. the role of uti in longterm outcome of kt remains controversial. factors associated with elevated pulse wave velocity in children after renal transplantation objective: even after successful transplantation children with underlying chronic kidney disease (ckd) still carry a high cardiovascular risk. this is also documented by cardiovascular death being the second leading cause of death after transplantation in this patient cohort.methods: in a cross-sectional approach 109 renal transplant recipients at three german transplant centres were enrolled. we measured pulse wave velocity (pwv), a strong predictor for cardiovascular events, as our primary end point. in all participants we assessed classical (e.g. blood pressure, cholesterol) and non-classical (e.g. crp, pth) cardiovascular risk parameters. results: patients were 13 ± 3 years of age and had received their transplant 5 ± 4 years ago. pwv-sds adjusted to height was 0.4 ± 1.5. in order to identify predictors of elevated pwv, we performed a univariate screen and introduced factors with a p-value below 0.2 into a stepwise forward linear regression analysis. we found that systolic blood pressure in the ambulatory blood pressure measurement and gfr independently predicted pwv-sds (table) . conclusion: our results show a large variability in aortic stiffness in children after renal transplantation. we found elevated pwv to be associated with classical as well as non-classical risk factors. the wide range of cardiovascular comorbidity seen in these patients highlights the need for individualized risk factor monitoring and management. objective: renal transplantation is the optimal treatment for end-stage renal disease in children,. however, it remains technically challenging in small recipients, especially with adult-sized graft. we report on two 4-year-old children, undergoing living donor kidney transplantation, and describe our management after failure to close the abdominal wall. method: both children weighed < 15kg and received their father's kidney. the recipient/donor weight ratios were 1/5 and 1/6. surgical procedure was an extra peritoneal approach to the iliac fossa, with vascular anastomosis on the aorta and vena cava. they recieved intensive fluid replacement during surgery to optimize hemodynamic status and graft perfusion. after initial successful reperfusion, all attempts to close the abdominal wall (even simple skin closure or superficial muscle layer closure) led to graft hypoperfusion, diagnosed on graft colour change and doppler ultrasound. closure with resorbablevicryl?mesh gave the same results and failure. finally, we had to close with a synthetic non resorbable plate (goretex?), without skin closure.results: the first patient presented acute tubular necrosis and required continuous hemofiltration from day 2 to 6. the second one had immediate diuresis and with a decrease of serum creatinine to 65μmol/l at day 2. in both cases, kidney was successfully replaced intra abdominally at day 6, with superficial closure of facia and skin in one child and with vicryl? mesh and skin in the second one. no effect on clinical graft perfusion or doppler parameters were observed after final closure. in patient 2, a limited necrotic area 3x3cm, on direct contact with the goretex?plate, was noticed during the second surgery procedure but it remained very superficial. finally, with a follow-up of respectively 1 year and 1 month, recipients and grafts are doing well without any other events and a serum creatinine of 40-50 μmol/l. conclusion: in conclusion, in small patients kidney size discrepancy and tissue oedema can lead to renal allograft compartment syndrome. in this situation, goretex? plate is an option for initial wound closure and graft salvaging. objective: amr in the transplant is one of the most complicated form of rejection, which do not respond to the standard therapy. survival of the transplant exposed to amr is still low. this is a clinical report of an amr-treatment in children in our centre. methods: 4 girls with amr after the deceased donor renal transplantation were observed of age10, 13, 15, 16 from 2009 to 2012 in our centre. all had a negative "cross-match" at the time of transplantation, the quantity of discordant antigens varied from 3 to 6. for 1 girl it was the 2 transplantation. 3 children had an immediate function of the graft,1-delayed. induction of the immunosuppressive therapy: polyclonal antibodies and methylprednisolone, maintenance: tacrolimus, prednisolone, mmf. 2 children had high level of preexisting antibodies (more than 30%) at the time of transplantation. there were morphological confirmation and decrease of the graft function for the moment of amr. the treatment of amr included: pulse therapy of methylprednisolone, polyclonal antibodies, rituximab, immunoglobulin iv. function of the graft was evaluated by the cr levels, gfr, the level of dsa.results: the cr of children was 483±256 umol/l for the moment of developing of amr. function of the graft has been restored to satisfied: cr fall to 121±28 umol/l, there were no proteinuria. it was noted a significant reduction of dsa i and ii class in 3 from 4 patients. conclusion: thus, treatment of an amr of the graft with methylprednisolone, rituximab and immunoglobulin intravenously let us achieve 100% 1-year graft and recipient survival after deceased donor kidney transplantation in children. since the risk of amrdevelopment is high in children with high levels of preexisting antibodies, addition of one-time rituximab and immunoglobulin intraveneously in therapy seems reasonable.abstract# p-sat454 tacrolimus granules use in kidney transplanted infants. dose and safety.ramon vilalta, enrique lara, alvaro madrid, marina munoz, sara chocron, gema ariceta paediatric nephrology, hospvalld'hebron, barcelona, spainobjective: tacrolimus in infants has been administered as an extemporaneously compounded oral liquid made by hospital pharmacy. modigraf® is a new tacrolimus formulation available as sachets containing granules (0.2 mg and 1 mg), that are made up with water, prepared by parents or caregivers. modigraf® is considered at least as safe and practical than the compounded oral tacrolimus liquid. furthermore, it is easier to be stored and ready to use, and promotes patient's autonomy, preventing potential medication errors. 9 kidney transplanted infants, 3 de-novo and 6 converted from oral liquid to modigraf® were evaluated to assess its safety and adequate dose. methods: after induction with basiliximab or atg, de novo's3 patients were treated with modigraf® associated with mmf and with tapered steroids. 6 infants with similar immunosuppression regimen were converted from oral tacrolimus to modigraf®. target tacrolimus through level was 8-10 ng/ml. mean patients' age at transplantation was 2.2 years old, and mean follow-up period with modigraf® was 10±8 months.results: in the de-novogroup the stable modigraf® dose needed to reach target levels was 0.28±0.12 mg/kg/day. in the converted group, the initial oral tacrolimus liquid dose was 0.20±0.12 mg/kg/day, whereas modigraf® stable required dose was higher: 0.30±0.10 mg/kg/day (p =0.0003). target levels of tacrolimus were reached after key: cord-004675-n8mlxe7p authors: nan title: 2019 cis annual meeting: immune deficiency & dysregulation north american conference date: 2019-02-26 journal: j clin immunol doi: 10.1007/s10875-019-00597-5 sha: doc_id: 4675 cord_uid: n8mlxe7p nan a 34 y.o. female was referred to our clinic with a history of multilineage cytopenias/evans syndrome, a history of idiopathic thrombocytopenic purpura, hemolytic anemia, chronic neutropenia, lymphopenia, and hypogammaglobulinemia treated with ivig. our patient was healthy until she was 8 years old; at that time, she developed joint pain, rash, and bruising. she was found to have evans syndrome with idiopathic thrombocytopenic purpura (itp), neutropenia, and lymphopenia. she was initially diagnosed with lupus and was given steroids. her bone marrow biopsy did not conclude myelokathesis. when she was 15 years old, she remained thrombopenic and was started on high dose of immunoglobulin replacement therapy. in 2012 (29 years old), she developed polyarthritis in her upper and lower extremities. in 2013 (30 years old), she had a severe nosebleed, for which she was admitted and treated with amicar twice; her platelets were found to be 2,000 k/ul. she received rituximab weekly for 4 weeks resulting in an increase of platelet count to 90-100k/ul. she recently (march 2017) had a splenectomy to remove her large spleen, and since then, her platelets have rebounded to 400-500k/ul. in 2015, she was placed on long-term immunoglobulin replacement therapy after being hospitalized for bilateral pneumonia for 5 nights requiring iv antibiotics for treatment. in 2017, she developed and was treated for another pneumonia. her family history is characterized by multiple members with autoimmune multilineage cytopenia as well as autoimmune diseases such as multiple sclerosis (mother), thyroiditis and enteropathy. on physical examination, she did not present with any warts and the remainder of her physical examination being unremarkable, except for her scar from her splenectomy and a cervical lymphadenopathy. immunologic evaluations showed igg 601 mg/dl, iga <5 mg/dl, and igm 208 mg/dl. cbc with differential and lymphocyte screen were as follows (cell/mm3): wbc 12.3 x103, hemoglobin 10.2 g/dl, platelets 503 x 103; 3 % neutrophils (anc: 300), 82% lymphocytes, 10% monocytes, 0% eosinophils; absolute total t-cell number was 8884 (750-2500 cells/mcl), cd4+ t-cells 6554 (480-1700cells/mcl), cd8+ t-cells 2185 (180-1000cells/mcl), natural killer cells 206 (135-525 cells/ mcl), and absolute number of b cells was 996 (75-375 cells/ mcl). she came to our clinic with her sister, who also had multilineage cytopenia and hypogammaglobulinemia, treated with monthly ivig; and her nephew whom had neutropenia. based on this family presentation all three underwent whole exome sequencing (wes). the patient, the patients sister and the patients nephew were all found to have a variant on cxcr4 (frameshift mutation on chromosome 2, p.val324fs; refnt: tca; altnt: t). as an important note, the patient had a bone marrow biopsy, which did not conclude myelokathesis. in summary, our patient with trilineage cytopenia and hypogammaglobulinemia, without any warts or myelokathexis, had whim syndrome (warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis), which was discovered by studying her wes. with the identification of her specific diagnosis, this allowed us to discuss the potential future indication of plerifaxor (antagonist of the alpha chemokine receptor cxcr4). and equally important, we discussed family planning and future pregnancies given that the mutation is autosomal-dominant. (4) submission id#555017 taha al-shaikhly, mbchb 1 , kathleen mohan, arnp 2 , matthew basiaga, do, msce 3 introduction: complement component-3 (c3) is shared by the classical, lectin and alternative complement activation pathways. c3, a major opsonin, facilitates phagocytosis of encapsulated microorganisms. inherited c3 deficiency is rare and is associated with increased risk of bacterial infections. subjects with connective tissue diseases (ctd) and c3 nephritic factors can have low and occasionally undetectable c3 levels, yet they are at an underappreciated infectious risk. we hypothesize that excessive c3 consumption in secondary complement deficiency disorders (scd) is associated with higher risk of bacterial infections similar to primary complement deficiency disorders (pcd). objectives: to compare the rate of bacterial infections between pcd and scd patients and evaluate the association between c3 level and bacterial infection risk. methods: we performed a retrospective cohort study. subjects with an undetectable complement activity (ch50) or any of the complement components measured at seattle childrens hospital from 2002-2018 were included in our study. we recorded the number of infections, observation periods, diagnosis (pcd, scd and its underlying etiology), lowest complement component levels, and the immunosuppressive agents used. the date of birth, and date of lowest c3 level were considered as start points to calculate the observation periods for pcd and scd subjects respectively. infections requiring hospitalization or parenteral antibiotics were categorized as serious bacterial infections (sbis). descriptive analyses were performed to determine medians and ranges for continuous variables. differences in rates of bacterial infection were assessed using the chi-square and kruskal-wallis tests when appropriate. among subjects with ctds, we treated every c3 measurement as a single observation (n=1,197) and studied the association between c3 concentration and the 30-day odds of having a sbi. multivariable logistic regression was performed to determine infection risk based on c3 level while controlling for contributing factors. results: we identified 14 subjects with pcd, and 52 subjects with scd. scd consisted of three subgroups (ctd-related (n=44), nephritic factor-related (n=2), and infection-related (n=6)). collectively, ctd subjects had a lower median rate of sbi compared to pcd subjects (p = 0.004). subjects with ctd and c3 level <40 have higher rate of bacterial infection (of any severity) (p = 0.002) and of sbi (p = 0.004) when compared to ctd subjects with c3 >=40 at the beginning of observation period ( figure 1 ). while controlling for immunosuppression level 1 pediatric resident, baystate medical center 2 faculty advisor, baystate medical center introduction: zap70 codes for a 619-amino acid enzyme, zap70, a member of the syk-protein tyrosine kinase family that plays an important role in t cell development and activation. zap70 is phosphorylated at tyrosine kinase residues upon t cell receptor (tcr) stimulation resulting in tcr-mediated signal transduction with src family kinases. zap70 deficiency results in a rare t+b+ nk+ severe combined immunodeficiency (scid). we report a novel compound heterozygous mutation in zap70 leading to presumed absent zap70 function in an infant with a normal trec newborn screen and scid. case description: the patient is a term, fully immunized female, born to non-consanguineous parents who was hospitalized for rsv bronchiolitis at 2 mo. at 4 mo she developed an erythematous, papular rash on her face and extremities, nonresponsive to topical antifungal therapy. at 6 mo she was re-hospitalized with rsv bronchiolitis and subsequently treated with multiple courses of antibiotics for presumed bacterial pneumonia followed by albuterol and oral steroids for possible reactive airways disease. during this course of treatment, her rash resolved. at 8 mo she presented with failure to thrive (wt <0.1% for age), multifocal pneumonia and respiratory failure requiring intubation. bronchial alveolar lavage confirmed pneumocystis jiroveci pneumonia prompting an immune evaluation. total immunoglobulins were normal for age, however antibody titers to tetanus, diphtheria and streptococcus pneumoniae were absent. lymphocyte enumeration revealed elevated cd4 t cells and markedly diminished cd8 t cells, normal b and nk cells. t cell proliferation to mitogens (pha, pwm) and antigens (candida, tetanus) was absent, however t cells proliferated normally to stimulation with pma and ionomycin. trec number was normal by newborn screening, but was 2 std deviations below the mean and would have resulted in a positive screen upon repeat. invitae 18 gene scid panel revealed two variants of unknown significance, c.109c>g (p.arg37gly) leading to substitution of arg with gly and c.1529_1532dupgcat (p.ile511metfs*65) resulting in a premature translational stop signal expected to disrupt the last 109 amino acids of zap70 protein. parental sequencing revealed these variants to be on opposite chromosomes. the patient was successfully treated for pjp pneumonia and has since successfully engrafted a 9/10 matched unrelated donor stem cell transplant. discussion: we report a novel compound heterozygous mutation in zap70 which we presume led to t+ b+ nk+ scid. our patients clinical presentation of failure to thrive, recurrent lower respiratory tract infections, dermatologic findings and pjp pneumonia are consistent with previously reported cases of zap70 scid. her paucity of cd8 t cells, abundance of cd4 t cells and absent proliferation to mitogens are also consistent with previously described cases of zap70. normal proliferation of t cells when bypassing the tcr by stimulating cells with ionomycin and pma confirms a defect in the tcr. we believe this is the second documented case of missed scid by newborn screen in ma since the implementation of trec screening in 2008. pediatric resident (pgy iii), goryeb children's hospital 2 attending physician, pediatric and adult asthma, allergy and immunology, llc introduction: acute otitis media (aom) is one of the most common reasons for antibiotic use in early childhood. we explored the challenges when aom fails traditional therapies and immunologic evaluation does not identify a commonly described immunodeficiency. case description: an eighteen-month-old male presented with 12 episodes of aom and recurrent purulent otorrhea requiring intravenous antibiotics. laboratory evaluation revealed a normal cbc, normal immunoglobulins (igg 588, iga 76, igm 63, ige 12) and igg subclasses. lymphocyte subset panel was normal. initial responses to dtap and prevnar boosters were normal, however, there was rapid decline to tetanus and pneumococcal antibody titers. a sub optimal response to haemophilus influenza type b vaccine was noted. although vaccinated twice for mmr, he never mounted mumps specific igg. mitogen response to pha was normal with decreased responses to cona and pokeweed and no detectable tetanus nor candida responses. further investigation revealed decreased non-class and class switched memory b-cells. the patient was recently vaccinated to pcv23 and at the present time has protective titers. discussion: it has been previously suggested that decreased memory b cells may contribute to decreased antibody responses to select vaccine antigens resulting in recurrent aom in children. our case supports the need to investigate beyond typical immunologic screening for immunodeficiencies. introduction: dna mismatch repair (mmr) system corrects replication errors in newly synthesized dna, and prevent recombination between dna sequences when they were not identical (1) . msh6 is a part of mmr genes, (2) (3) (4) . case: a ten-year-old girl presented with fever, brown spots on her skin, hair loss, recurrent pulmonary infections, arthritis on the left hand and right ankle. she has also been followed up with nf ( figure 1 ). there was a first-degree cousin marriage between her parents. physical examination revealed findings of pneumonia and nf. anti-nuclear antibody, anti-ndna, anti-dsdna, anti-histone, anti ro52 and anti-nucleosome antibodies were positive. in her immunologic assessment showed low igg and iga levels associated with high igm level ( table 1 ). the coexistence of nf, hyper igm syndrome, sle, were considered in the patient. intravenous ig (400 mg/kg, every 3 weeks) treatment was started due to hypogammaglobinemia. the frame shift mutation in exon 2 of the msh6 gene was detected in the boztug's laboratory. in the follow up period, she admitted at 11 years old with back pain. a mass in the left paravertebral area, related to the spinal canal and neural foramina, was detected at the l4-l5 levels in spinal mri. the lymphadenopathy around the liver and hilum and the left parietal bone lesions were developed within two months despite surgical excision of primary mass ( figure 2 ). as a result of pet examination; suvmax was found to be around 6.5 in the mass lesion in the paravertebral region and suvmax values did not exceed 2.5 in other lymphadenopathy and masses. atypical cellular infiltration suggesting neoplastic events, which were including small-medium size atypical pleomorphic mononuclear cells and t cells. since all these formations did not indicate definite cancer, chemotherapy was not started. interestingly, although chemotherapy was not given, progression stopped, and partial spontaneous regression was observed. discussion: the effect of msh6 mutations on patients may significantly vary with the inheritance pattern (2) . leukemias or lymphomas are not common in heterozygote mmr gene defects (5, 6) . however, homozygote mutations in mmr genes show a different pattern. wimmer and etzler proposed the new term constitutional mismatch repair-deficiency syndrome (cmmr-d) for patients who have a homozygous mutation in mmr (3) . cmmr-d characterized by development of childhood cancers, mainly hematological malignancies and/or brain tumors, as well as early-onset colorectal cancers, and neurofibromatosis type 1 (3) . bi-allelic germline mutations in any of the mmr genes in which msh6 is involved increases hematological malignancies by 15% (7, 8) . msh6 mutation has been associated with many cancers since its identification. leukemia, lymphoma, colorectal cancer, endometrial cancer, brain tumors are some of these cancer types (2) (3) (4) 9) . msh6 deficiency is an important disease that can affect different systems at the same time. there is a high risk of malignancy in the cases and therefore they must be closely monitored. this case has also shown that atypical lymphoproliferation may occur in msh6 homozygous mutant cases. (normal rage: 842-1943) background: advances in inborn errors of human immunity have supported the discovery of new syndromes that are marked by striking features of autoimmunity and immune dysregulation often associated with cytopenias, lymphoproliferation, and a predisposition to reticuloendothelial malignancies leading to evaluation with hematologists/oncologists. moreover, hematologists/oncologists have also seen an increasing use of effector cell-based therapies, checkpoint inhibitors, immunomodulatory and targeted therapies resulting in autoimmunity and hyperinflammatory complications. a working knowledge of clinical immunology could help practicing hematologists/oncologists in the identification and management of these conditions. objectives: to support the advancement of aspho members and the field by facilitating education regarding the best practices in diagnosis and management of immunological disorders. to create a platform for the development of collaborative clinical research in patients with hematological/oncological manifestations of immunological disorders or those requiring hematopoietic stem cell transplantation for a underlying immunological disorder. design/methods the aspho clinical immunology sig was initiated based on collaboration with the clinical immunology society (cis). aspho members who are pediatric hematology/ oncology clinicians, clinical researchers, and trainees are eligible to participate. we have established a steering committee with representatives from across the united states and canada with diverse clinical and research expertise. through regular teleconferences and annual in-person meetings, we have developed a platform to provide our members with a network of immunology resources to ensure a strong foundation of knowledge and tools to conduct clinical care and research pertaining to the diagnosis, evaluation, and treatment of patients with immunological disorders. results we currently support over 50 members within our online community. several educational initiatives have been successfully launched. we have submitted an invited review to pediatric blood and cancer which provides a case-based review of primary immune regulatory disorders. we hosted the first immunology for hematology oncology practice (i-hop) cased-based webinar series. this series features case-based discussions of patients with primary immunodeficiency disorders presented by fellow trainees and mentored by senior clinicians. we will also be hosting an aspho webinar focusing on the laboratory evaluation of primary immunodeficiencies and immune dysregulation syndromes. we have also begun the process of laying the groundwork for clinical research initiatives. conclusion: the aspho clinical immunology sig seeks to serve as a collaborative resource for pediatric hematology/oncology clinicians and researchers. through the development of educational and research initiatives, we envision improving the care of patients with immunological disorders that are often managed by pediatric hematologists/oncologists. moreover, we hope to broaden our understanding and application of clinical immunology within pediatric hematology/oncology. we hope that this successful initiative will serve as a blueprint for the development of future collaborations with other specialty societies and patient groups. autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced) is a rare autosomal recessive disease caused by aire gene mutations. clinical diagnosis is established by the presence of at least two components of the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and addisons disease. in europe, the classic presentation is widely recognized and nonendocrine autoimmune manifestations are rarely reported. a recent study of 35 american apeced patients demonstrated a more heterologous presentation, with many nonendocrine manifestations including urticarial eruption, hepatitis, gastritis, intestinal dysfunction, pneumonitis and sjogrens-like syndrome, all uncommon in european reports. within the american cohort, 80% of patients developed a mean of three non-triad manifestations before reaching the classic triad. finding of aire mutations and high-titer antiifn-autoantibodies is seen in both european and american cohorts. we present the case of two siblings, who demonstrate an apeced-like phenotype with both classical and atypical features. they share the same heterozygous c132+1_132+3delinsct aire mutation. the older, an eight-year-old boy, with history of prematurity, bronchopulmonary dysplasia and onychomadesis in infancy, came to medical attention at 16 months of age due to failure to thrive (ftt), in addition to fevers and urticarial rash lasting months after his mmr vaccine. the fevers resolved with anakinra, which was discontinued two years later due to pneumonia. from age 2-4 he developed an alps negative lymphadenopathy which self-resolved. lung issues include chronic cough, initially treated as asthma but with poor bronchodilator response, and frequent lung infections, including 1-2 pneumonias per year. at age five evaluation for ftt revealed growth hormone deficiency. two years later he was diagnosed with primary addisons disease. chronic abdominal discomfort, bloating, cyclical constipation/diarrhea, recurrent rashes, dystrophic nails, and sicca symptoms are also present. his sister, age five, shows ftt, but no growth hormone deficiency. at age one, she too developed a fever and rash syndrome lasting 3 months. severe gerd and constipation started in infancy and are ongoing. at age three she developed a transaminitis, initially diagnosed as ebv, but later thought to be autoimmune hepatitis. she has frequent viral respiratory infections, and pneumonia at age two. she has had a chronic cough, with poor bronchodilator response, for most of her life. evaluation of seizure at age three showed normal brain activity. brain mri revealed partial agenesis of the corpus callosum and microgyria. her brother has similar mri findings. both children have had developmental motor delay and poor tone. brain dysgenesis and neurodevelopmental delay has not previously been described in apeced. although there were both typical and atypical symptoms, the history in combination with genetic findings led to further investigation of an apeced-like syndrome. autoantibody testing confirmed high-titer antiifn-autoantibody typical of apeced in both children and hightiter bpifb1 autoantibodies found almost exclusively in apeced pneumonitis in the brother. whole exome sequencing and copy number variation analyses are underway to further evaluate the patients condition. this case demonstrates the importance of clinical presentation in the evaluation of genetic results and in the guidance of therapeutic management. (12) submission id#570047 rationale: infants with low t cell receptor excision circles (trec) born in queens, nassau, and suffolk counties are referred to our center for further evaluation. this study elucidates the demographic and laboratory characteristics of referred infants with transient or persistent idiopathic t cell lymphopenia (tcl) without clearly identified genetic or acquired etiology. methods: a retrospective analysis was performed from september 2010 (when trec screening started) through the end of december 2017. descriptive statistics were calculated for demographic and laboratory characteristics. t-test or mann-whitney tests were used to compare laboratory variables. pearson or spearman tests were used to determine correlation between initial trec levels and t cell counts. by definition, the cd3+, cd4+, and cd8+ populations of transient tcl patients normalize by age 1 year. results: eighteen infants with transient and 17 with persistent tcl were identified. males comprised 61.1% of the transient and 47.1% of the persistent tcl cohorts. whites comprised 11.1% of the transient and 35.3% of the persistent tcl cohorts. the mean initial trec levels did not differ between the transient and persistent cohorts (67.7 vs. 78.5 trecs/l of blood, p = 0.56). mean initial absolute counts of cd3+ (2149 vs. 1300 cells/l, p <0.0001), cd4+ (1462 vs. 922 cells/l, p <0.0001), and median initial absolute counts of cd8+ (524 vs. 309 cells/l, p = 0.0075), were higher for transient vs persistent cohorts. initial trec level did not correlate with initial cd3+, cd4+, or cd8+ absolute counts. the median age of resolution for the transient cohort was 121.5 days (range 23-244). the absolute cd3+, cd4+, or cd8+ counts rarely exceeded the reported median values for age, and remained closer or below the 5th percentile for age up to 1000 days of life. the majority of both transient and persistent tcl patients demonstrated unremarkable lymphocyte proliferation to mitogens. conclusion: our centers transient tcl cohort appears to be predominantly male and non-white, whereas the persistent tcl cohort is more evenly distributed by sex but still predominantly non-white. the transient cohort had lower initial trec levels, but higher initial t cell counts. both cohorts appear to have relatively intact in vitro function. introduction: primary immune deficiency disease (pidd) is typically considered a pediatric illness, although advances in treatment and diagnosis are changing this paradigm. currently, data on pidd in older patients are very limited. objectives: to characterize the prevalence of pidd among older individuals using a patient database maintained by the consortium of independent immunology clinics (ciic), comprised of 17 specialty immunology outpatient practices in the us. methods: patients with pidd were identified in the ciic database using icd-10 codes d80, d.80. 3, d80.4, d80.5, d80.6, d81.1, d81.2, d82.0, d82.3, and d83 .0. a total of 235 records from 11 geographically-diverse clinics were identified and characterized by age, gender, and pidd diagnosis. results: of the 235 pidd patients in the ciic registry, 73 (31%) were between 60-87 years of age (see figure) . within this age group, most patients were female (n=56, 77%). the most common diagnoses among patients >60 years of age included common variable immunodeficiency with predominant abnormalities of b-cell numbers and function (d83.0; n=41, 56%) and antibody deficiency with near normal immunoglobulins (d80. 6; n=14, 19%) . in comparison, the registry included 36 (15%) patients aged 0-19 years; this age group was predominantly male (n=23; 64%). the most common icd-10 codes within the younger cohort were relatively evenly distributed between hereditary hypogammaglobulinemia (d80.0), antibody deficiency with near normal immunoglobulins (d80. 6) , and common variable immunodeficiency with predominant abnormalities of b-cell numbers and function (d83.0). conclusions: our data suggest that pidd in patients over age 60 may be more prevalent than previously reported. additional research is needed to corroborate these findings, further characterize the nature of pidd in this population, and determine whether there are unique diagnostic and treatment considerations within this demographic. introduction/background: increased susceptibility to invasive infections with neisseria has been well documented in patients with deficiency of terminal complement proteins. the molecular attack complex is constructed with complement components c5 to c9. a deficiency in complement c6 has been described previously in both african american and south african populations. complement c6 deficiency is inherited in a co-dominant pattern, with multiple known mutations. we present a case of a 19-year-old, previously healthy male, who presented with invasive n. meningitides infection. he was found to have a novel mutation noted on genetic sequencing of the complement c6 gene. objective: we present the case of a 19-year-old, previously healthy male, who presented with invasive n. meningitides infection. on genetic sequencing, he was found to have three mutations of the complement c6 gene. two of which have been described previously, and a third novel mutation. methods: a 19-year-old male with no known history presented to us with a 3-hour history of emesis. he was found to be febrile, and quickly decompensated, developing septic shock. blood cultures were drawn, and within 12 hours grew n. meningitides. he was treated with broad spectrum antibiotics upon arrival, and subsequently narrowed to ceftriaxone. his hospital course was complicated by disseminated intravascular coagulation, as well as acute tubular necrosis, leading to endstage renal disease for which he is listed for kidney transplant. results: on immunodeficiency evaluation, he was noted to have an undetectable ch50 (<13, reference range 31-60). complement levels returned with c6 of 10.8 (reference range 28-69) and c1r of 41.5% (reference range 61-102%). complement c6 function screen returned at 0% (reference range 40.7-169%). all other complement levels were within normal limits. genetic sequencing showed the patient to be compound heterozygous for two of known four variants which have been reported to recur in african patients with complement c6 deficiency. this included c.821del and c.1879del, which are predicted to result in frameshift and premature protein termination. he was also found to be heterozygous for sequence c1202g>a, which results in amino acid substitution p.arg401lys. this variant is rare, with one large database reporting it in 6 of 276000 alleles, and not in a homozygous state. it has not been reported in a case of c6 complement deficiency previously. conclusions: we present the case of a previously healthy 19-year-old male with invasive meningococcal disease. he is compound heterozygous for two mutations that have been associated with total complement c6 deficiency; however, he was found to have subtotal c6 deficiency. furthermore, he has a third novel mutation of the complement c6 gene. further investigation is warranted on the significance of this finding and impact on relevance to possible kidney transplant. background: measuring the function of the classical pathway of complement activation is useful in several disease states, including complement deficiency, autoimmune conditions such as systemic lupus erythematosus and certain forms of nephritis. the original method for assessing classical pathway activity was the haemolytic ch50 method, but this assay can be time consuming and has reagent stability issues due to the use of sheep red blood cells. there can also be high lab-to-lab variability due to differences in the protocols used. here we report the assay characteristics of an automated, commercial, liposome-based assay to measure ch50 activity. we also compare the results obtained using the traditional haemolytic method with the automated, liposome-based method used on the spaplus turbidimetric analyser. methods: a linearity study was performed based on clsi guideline ep06-a. the linear range of the spaplus ch50 liposome assay was established by analysis of a series of sample dilutions and evaluation of results against pre-defined goals for recovery and %cv. precision was assessed based on clsi guideline ep05-a2 over 21 days. 4 samples with different ch50 activities (23.7-65.1 u/ml) were run in duplicate, with two runs per day using 3 reagent lots and 3 different analysers. interference analysis was performed by spiking haemoglobin, bilirubin, chyle, ascorbic acid or saline (as a control) into samples before measuring the ch50 activity. for the assay comparison study, sera from 125 routine patient samples were used. samples were collected from chulalongkorn hospital, faculty of medicine, chulalongkorn university, thailand. ch50 classical pathway activity was assessed using a haemolytic method and also using the liposome based ch50 assay for use on the spaplus turbidimetric analyser (the binding site ltd., birmingham, uk). c3 protein concentrations were also available for 116 of these samples. results: the liposome ch50 assay gives a linear response over the range 11.8-95.5 u/ml, covering the measuring range of the assay (12.0-95.0 u/ml) at the standard analyser dilution (neat). the within run, between run and between day %cvs were all 5.4%. the total %cv was 6.8% in all 4 samples. minimal interference was observed with the four common interferents tested. a significant correlation was observed between the two ch50 methods (p<0.0001, r=0.66, y=1.1xâ±0.1), with 90.4% agreement between the methods in determining whether patients were above or below the lower limit of the assay normal range. the 12 individuals in disagreement had normal ch50 results using the haemolytic method, and low ch50 values in the liposome assay. of these, c3 values were available for 10/12, and 5 had c3 concentrations below the lower limit of the assay normal range. conclusion: the liposome ch50 assay for use on the spaplus analyser has passed assay development guidelines based on those set out by the clsi for linearity, precision and interference, and there is a strong correlation between this automated assay and the haemolytic ch50 method used here. five additional patients with low c3 concentrations were defined as having a low ch50 using the spaplus liposome method compared to the haemolytic method. (18) submission id#580179 background/aims: rotavirus vaccine is a live viral vaccine that is part of the routine u.s. childhood immunization schedule. live viral vaccines administered to infants of mothers who received biologic medications during pregnancy can potentially cause vaccine-associated disease. infant death from disseminated mycobacterial infection after vaccination with bacille calmette-guerin (bcg) in infants whose mothers received infliximab during pregnancy has been reported. it is currently recommended that infants born to women who received biologic therapy during pregnancy not receive live viral vaccines, however there is a paucity of information regarding adverse events from live viral vaccines. we report two infants, born to mothers receiving infliximab during pregnancy, who tolerated the complete series of rotavirus vaccine. methods: two infants who received rotavirus vaccine and whose mothers received infliximab (monoclonal antibody against tumor necrosis factor alpha which blocks the inflammatory response) during pregnancy were identified and their charts were reviewed. each mothers chart was assessed for timing of the biologic doses during pregnancy and concurrent immunosuppressant therapy. results: the mother of the first infant had crohn's disease and received infliximab every 6 weeks throughout her pregnancy (final infusion at approximately 35 weeks estimated gestational age [ega] ). she did not take additional immunosuppressive drugs throughout her pregnancy. the infant was born at 39 weeks ega. the infant received rotavirus vaccine at 2, 4, and 6 months of age. the infant did not have coexisting medical conditions or recorded hospitalizations during the first year of life. there were no side effects from rotavirus vaccine documented during well child examinations. the childs growth was normal during the first year of life. the mother of the second infant also had crohn's disease and received infliximab infusions every six weeks during pregnancy until 27 weeks ega. additionally, she took mesalamine (anti-inflammatory) daily. the infant was born at 33 weeks ega. the baby had a brief and uncomplicated neonatal intensive care unit stay. she did not have medical conditions diagnosed at the time of birth, or in the first year of life. the child received rotavirus vaccination at 2, 4, and 6 months of chronological age, and the infant did not experience documented adverse reactions. the child presented to the emergency department twice in the first year of life: once for thrush at 10 months of age and once for viral gastroenteritis at 11 months of age. the childs growth curve was unremarkable. conclusions: we report two infants, whose mothers received infliximab during pregnancy, who safely tolerated the 3-dose series of rotavirus vaccination. neither infant in this case series suffered from minor or severe adverse events as a direct consequence of receiving rotavirus vaccine. this suggests that administration of rotavirus vaccine may be safe in infants whose mothers received biologic therapy. introduction: combined immunodeficiencies (cids) can arise from partial loss of function variants in recognized scid genes, which can lead to relative lymphopenia with poorly functioning and oligoclonal t cells. cids have been most commonly associated with variants of the rag genes, but other genes are also implicated. clinical symptoms may be less severe, and the onset generally is delayed, compared to typical scid presentations. case report: a 28-year-old female presented with a history of recurrent and progressively worsening infections involving multiple microorganisms and organs, starting in infancy and requiring frequent hospitalizations. bacterial or viral infections included rhinosinusitis, otitis media, herpetic stomatitis, dental abscesses, pneumonias, pulmonary mycobacterial abscesses, cmv hepatitis, urinary tract infections, dermal abscesses, and groin hidradenitis. fungal and yeast infections included cryptococcal meningitis, oral thrush, dermatophytosis of the face, osteomyelitis of a finger, and onychomycosis. laboratory tests in 2018 showed: mildly low t cell counts (791/ul) with a reversed ratio of cd4/cd8 t cells (0.22); almost absent b cells (2/ul) ; and low nk cell counts (19/ul). cd4+ t cells were mostly of the memory phenotype (87%). t cell development showed low counts of th17 cells. t-cell stimulation tests demonstrated poor proliferation responses (<30%) to concanavalin a, tetanus toxoid, and candida albicans, with near-normal responses to pokeweed (>13%) and pha (>84%). she had low ig levels (iga 72, igm 23, ige <2), except for igg (872mg /ml; due to replacement since early childhood). limited genetic evaluation at age 9 showed a heterozygous variant in the rag1 gene (g.36595918t>c, c.1064t>c, p.met355thr; nm_000448.2). discussion: loss of function variants in rag1 or rag2 genes are known to cause a t-b-nk+ type scid. more than 100 missense variants have been reported for rag1, with disease-associated variants predominantly in zinc binding regions. the rag1 missense variant in our patient also lies within the zinc binding region (amino acids 354-383). the variant is rare (mean allele frequency 0.0001521 in gnomad) and has been identified in at least one other individual with scid (t-, b cell-, nk+). although classified as a variant of unknown significance, occurrence in at least two individuals with deficiencies of t and b cells-within a functionally important rag1 domainsupports an interpretation that the variant may be pathogenic. most patients with cid with rag variants are either homozygous for a poorly functional allele or have one nonunfucitonal and a second, poorly functional allele. we detected only a single potentially pathogenic allele. our patient has decreased nk cells in addition to t and b cell defects. further genetic studies including whole exome sequencing, are planned to identify further variants in rag1 or other relevant genes. rationale: infants with low t cell receptor excision circles (trec) born in queens, nassau, and suffolk counties in new york were referred to northwell health for further evaluation after abnormal newborn screens. the demographic and immune parameters of infants with transient t cell lymphopenia (ttcl) without clearly identified genetic or acquired etiology are described. tcl is considered transient if the lymphopenia resolves by 12 months of age. similar data from the following infants with low lymphocytes (fill) program of the united states immunodeficiency network (usidnet) are presented. methods: a retrospective analysis of two separate patient cohorts with ttcl are described. cohorts include patients referred to a single center, northwell health, in ny from september 2010 to december 2017 and at usidnet using data tracked by fill from june 2011 to july 2018. results: out of 1,234 referrals at northwell, 18 infants with ttcl were identified. infants were predominantly male (61.1%) and non-caucasian (89.9%). out of 71 fill participants, 9 infants with ttcl were identified. infants were predominantly male (55.6%) and non-caucasian (55.6%). initial laboratory parameters for the northwell versus fill cohorts are summarized: a) median trec levels: 54.5 vs. 47.0 trec/l of blood; b) median absolute cd3+ count: 2135 vs. 1166 cells/l; c) median cd4+ count: 1460 vs. 777.0 cells/l; d) median absolute cd8+ count: 524.5 vs. 440.0 cells/l. initial naã¯ve cd4+ t cell information was available for 0 northwell and 5 fill infants (median 52%). mitogen proliferation studies were performed in 10 (55.6%) northwell and 6 (66.7%) fill infants with 90% of these northwell and 50% of these fill infants demonstrating normal proliferation. genetic testing, such as targeted genetic panels or chromosomal microarrays (cma), was performed in 5 northwell and 0 fill infants. no genetic or chromosomal aberrations were identified. whole exome sequencing (wes) was not performed in either cohort. 11 of 18 (61.1%) northwell and 7 of 9 (77.8%) fill infants did not receive the initial rotavirus vaccine. no fill infants were vaccinated but no adverse effects were reported in 5 of 18 (27.8%) northwell infants who received the first rotavirus dose. of these, 3 of 5 (60.0%) had normal mitogen proliferation while 1 (20.0%) had decreased proliferation to phytohemagglutinin. conclusions: identifying biomarkers for ttcl and developing evidencebased guidelines for the diagnosis and management of ttcl are important knowledge gaps. this descriptive study is limited by small sample size and the constraints of registry-based research. although there appear to be differences between these cohorts, our findings suggest that ttcl may disproportionately affect different segments of the population. ttcl infants with normal mitogen proliferation may be able to tolerate rotavirus vaccination. thus, routinely checking proliferation studies in all ttcl infants may help risk stratify these patients and minimize vaccinerelated adverse events. currently, there is insufficient evidence to recommend more extensive genetic testing such as genetic panels, cma, or wes. systematically collecting information about patient characteristics and outcomes, as well as encouraging increased participation in registries such as fill, may help address these shortcomings. background: systemic lupus erythematosus (sle) is a chronic, inflammatory disease that affects multiple organs. the measurement of anti-dsdna antibodies (abs) is a gold standard serological test used in the diagnosis and monitoring of sle, with higher serum levels associated with worse prognosis. however, not all anti-dsdna abs are pathogenic, and some patients have consistently high levels with low disease activity. one mechanism suggested for the pathogenicity of these antibodies is complement activation. here we describe an assay to measure the c1q binding activities of anti-dsdna abs in sle patients. materials & methods: the concentration of anti-dsdna abs was determined using the quantalite dsdna elisa kit (inova) as per the manufacturers instructions. in order to determine the c1q binding capacity of bound abs, samples were added to the pre-coated plate and incubated. bound anti-dsdna ab/c1q complexes were then detected using a biotinylated anti-c1q antibody (570 ng/ml) and streptavidin peroxidase (1 mg/ml). normal reference ranges were developed in serum samples from healthy controls, and upper limits of these normal ranges were used as cut-offs. the dsdna abs and c1q binding capacity of bound abs was then assessed in 49 sle patients, and compared to other markers and the sle disease activity index (sledai) score. results are displayed as absorbance at 450nm (au). results and conclusions: the 95th percentile ranges for anti-dsdna abs (0.068-0.137 au) and c1q binding activities (0.207-0.313 au) were developed from the measurements generated in 17 healthy serum samples. sle patients with an increased anti dsdna ab concentration (>0.137 au) were then separated into those with low (<0.313 au) and high (>0.313 au) c1q binding activities. patients whose dsdna abs had high c1q binding activity were found to have significantly higher sledai scores (mean 6.70 vs 3.19) . serum c1q concentration, serum dsdna abs (measured by another method) and serum c3 and c4 concentrations were not significantly different between the two groups. this assay suggests that dsdna abs from sle patients differ in their ability to bind complement, and that high complement binding activity of these antibodies may be linked to a more active form of disease. x-linked lymphoproliferative (xlp) is a primary immunodeficiency, caused by signaling lymphocyte activation molecule (slam)-associated protein (sap) deficiency. patients with xlp have severe immune dysregulation, usually triggered by ebv infection, leading to fulminant infectious mononucleosis, dysgammaglobulinemia and lymphoproliferation. without hematopoietic stem cell transplant (hsct) fatality is reportedly 100% by age 40. we report the natural history of xlp in a patient, and describe the lessons learned. our patient was healthy and developed normally until 6-years of age, when he developed progressive respiratory symptoms. lung biopsy revealed mature lymphoplasmacytic infiltrate in the alveolar septa, consistent with lymphoid interstitial pneumonia (lip). he received corticosteroids and cyclophosphamide with significant improvement. at age 12, he developed severe infectious mononucleosis (fever, hepatosplenomegaly, lymphadenopathy, lymphocytosis). he had a protracted clinical course, but eventually recovered and seroconverted to a typical convalescent pattern. he subsequently developed hypogammaglobulinemia, and was started on intravenous immunoglobulin (ivig). during the same year, his 10-year-old brother developed lip, and subsequently hemophagocytic lymphohistocytosis (hlh) and died within 4 months from overwhelming candidiasis. unfortunately, his youngest brother (age 7) then developed lip and died 2 months later from a massive gastrointestinal bleed. both siblings were treated with corticosteroids and cyclophosphamide; they did not have detectable ebv infection. at age 13 years, our patient experienced recurrent strokes and was found to have biopsy-proven cns vasculitis. he was treated with interferon-and recovered with residual left sided weakness, but was lost to follow-up. he continued on ivig, with no other immunomodulatory agents for several decades. he had progressive lung disease and recurrent seizures controlled with anti-epileptics. at age 43, he developed sudden vision change, headache and right-sided weakness, followed by a seizure. mri of the brain revealed small bilateral areas of acute infarction suggestive of a central embolic event, however, no primary thrombus was identified. he did not receive any immunosuppression but was anti-coagulated. eventually he was discharged home with resolution of weakness to his baseline. the patient was referred to our clinic after discharge and we re-evaluated him after 31 years. immune profiles at the time showed therapeutic igg troughs, low/undetectable igm/a/e, normal t/b/nk-cell counts, normal spontaneous, but decreased antibody-dependent nk cytotoxicity, 0% sap protein expression (on cd3+cd8+, cd3-cd56+ and cd3+ cd56+ cells), and deletion on the x chromosome encompassing the sh2d1a gene which encodes sap. his mother was a carrier of the same deletion. his functional status excluded the option of hsct. a year later, he had rapid deterioration with recurrent lung infections, liver failure, and thrombocytopenia. bone marrow biopsy revealed hodgkins lymphoma. he declined chemotherapy and died few days after diagnosis. our case represents a rare patient with xlp surviving to the fifth decade without hsct, particularly having experienced mononucleosis and non-ebv related cns vasculitis. our patient survived decades longer than his brothers (who most likely shared the same genetic defect) without evidence of somatic reversion (0% sap expression in cd3+cd8+) to explain his milder clinical phenotype. this case may help in understanding the natural history of xlp, and confirms that prognosis remains poor without hsct. haematology and oncology, chu de quã©bec ctla-4 is a major negative regulator of immune responses, and ctla-4 haploinsufficiency has been identified as a monogenic cause of primary immunodeficiency in patients presenting with a common variable immunodeficiency (cvid) phenotype with autoimmunity. here we present the case of pb, a 40-year-old man who had been followed by the immunology service of our center for 17 years. a diagnosis of cvid had first been made when the patient presented with atypical transverse myelitis, low immunoglobulin levels, and lymphopenia. over the years, his clinical picture was dominated by various forms of autoimmunity, namely inflammatory demyelinating disorder of the central nervous system, autoimmune haemolytic anemia, immune thrombocytopenia, cryptogenic organizing pneumonia, rheumatoidlike polyarthritis, chronic liver transaminitis with biopsy-proven moderate fibrosis, and lymphocytic colitis with malabsorption. immunoglobulin replacement therapy was started at diagnosis, and autoimmunity was sequentially treated with methotrexate, interferon beta 1-a, cyclophosphamide, mycophenolate mofetil, rituximab, and finally a combination of low-dose prednisone and sirolimus, with stabilization of his neurological condition, the most debilitating complication of his immune dysregulation syndrome. bone marrow transplant had been offered, but declined by the patient due to perceived good quality of life compared to transplant-associated risks. the patient was later referred to our hematology ward in july of 2018 for septic shock complicating febrile neutropenia, which was part of a twomonth, gradual-onset pancytopenia. the diagnosis of immune-mediated aplastic anemia soon became apparent, as demonstrated by a bone marrow biopsy performed in a peripheral center two days prior to admission. the underlying pneumonia and thereafter biopsy-induced staphylococcus aureus iliac osteomyelitis and soft-tissue abscess were treated with broad-spectrum antibiotics as well as multiple surgical interventions. the patient was started on eltrombopag, high-dose corticosteroids and cyclosporin a, the latter promptly switched to tacrolimus due to liver enzymes disturbances, all of which resulted in no significant hematologic response despite over seven weeks of treatment (with concurrent treatment of complicating infection, upper gastrointestinal bleeding, and intensive-care-unite myopathy). during that time, genetic confirmation of ctla-4 haploinsufficiency was received, and the patient was thereafter started on abatacept on day 48 of current hospitalization. administration of equine anti-thymocyte was initially foregone because of perceived infectious risk in the setting of poor iliac wound healing and superimposed adenovirus viremia; however, given the lack of response, it was given on days 52 through 54 of hospitalization. haematologic response began on day 67 of hospitalization with a steady rise in alllineage myelopoiesis up to a complete neutrophil response, platelet near-complete response as well as resolution of transfusion needs by day 101. while waiting for a well-matched bone marrow donor, isolated platelet decrease was observed and attributed to multiple factors, including low-grade thrombotic microangiopathy, inflammatory consumption and drug-related thrombocytopenia, but the patient remained well. to our knowledge, our patients presentation is one of the most severe manifestation of ctla-4 haploinsufficiency to have responded to targeted therapy with abatacept, as a bridge to hematopoietic stem cell transplantation, with resolution of both immune and infectious complications, showing that genetic diagnosis is helpful in optimizing the management of presumed cvid patients. hospital 12 de octubre health research institute (i+12), madrid, spain, dept. of immunology, university hospital 12 octubre. madrid. spain background: xlf/cernnunos deficiency is a rare primary immunodeficiency classified within the dna repair defects. these patients present severe growth retardation, microcephaly, lymphopenia and increased cellular sensitivity to ionizing radiation. here, we describe two unrelated cases with the same nonsense mutation in the nhej1 gene showing significant differences in clinical presentation and immunological profile but a similar dna repair defect. methods: missense nhej1 mutation was identified by targeted next-generation sequencing with an in-house designed panel of 192 genes. for foci experiments, primary skin fibroblasts were irradiated with ionizing irradiation (137cs) or treated with 20mm etoposide for 1 hour. after irradiation, the cells were seeded at a density of 1x104 cells/ml in t75 flasks in triplicate. to evaluate cell sensitivity to gamma-ir (1 and 3 gy),adherent cells were trypsinized and counted 11 days later. pbmcs from patient and healthy controls were irradiated with 10gy, fixed and stained for cd3, cd19 and phospho-histone h2ax. mean fluorescence intensities (mfi) of gamma-h2ax were evaluated on gated cd3+ lymphocytes. results:we report two patients harboring the same homozygous mutation in cernunnos/xlf/nhej1 gene. strikingly, their clinical phenotype ranges from severe combined immunodeficiency to isolated thrombocytopenia followed until escolar age (table 1) . they harbour the same c.169c>t mutation in nhej1 gene but different immunologic features (table 2) . p2 presented with mild t lymphopenia, hypersensitivity and nhej repair defect, typical for patients with xlf/nhej1 defects. on the other hand, p1 presented a more severe phenotype (t-b-) , however hypersensitivity and nhej repair defect was similar to p2.of note, p2 has survived into the first decade of live. both patients are alive and well after hsct. discussion: usually the repair defect in these disorders is assessed by immunofluorescence assays of irradiation-induced gamma-h2ax foci using skin fibroblasts. a high throughput, sensitive and reliable assay to quantify gamma-h2ax foci in pbmcs isolated from blood samples would be a valuable tool to diagnose these patients and perform hsct early. flow cytometry (fc) can be applied as a rapid diagnostic tool for dna repair disorders. patients with the same homozygous mutation (p.r178x) in nhej1 gene have been previously reported. two patients died at 1.5 and 4 years while another of the patients is already 8 years old and is alive (without hsct). however,none of these patients presented severe t lymphopenia as it has been observed in our first patient. conclusions: the assignment of a timely and accurate diagnosis is of paramount importance in the management of patients with defects in dna repair. in the era of nbs an abnormal trec assay should be followed by ngs approach as cernunnos deficiency may present early in life as scid,as other rs-scid defects. since genetic diagnosis takes time,functional radiosensitivity assays in peripheral blood may lead to the correct diagnosis and avoid exposure to alkylating agents during the conditioning regimen prior to genetic diagnosis. it would also be helpful in cancer patients to individualize and to guide the dosing of ionizing radiation (ir) and/or genotoxic agents to avoid accumulation of cells with genomic instability that could accelerate cancer development. figure 1 ). her skin lesions also significantly improved after starting the medication ( figure 2 ). her hospitalizations were complicated by fluid overload and hypertension. both fluid overload and hypertension resolved prior to discharge. she remains on 2mg prednisone daily, cetirizine, ranitidine, cromolyn and benadryl and hydroxyzine prn. to our knowledge, this is the youngest patient successfully treated with midostaurin and she is doing very well on therapy with no apparent side effects. she has had resolution of many of her systemic mastocytosis symptoms including skin lesions, axillary mass and improvement in her diarrhea and growth as well as objective improvements in her tryptase levels. case report: a two-year-old male presented to the hospital with a painful, non-pruritic facial and groin rash. the rash started one week prior to presentation. he had no associated fevers. his history was remarkable for failure to thrive (ftt) and chronic bilateral leg pain with antalgic gait. over the preceding months, he had been diagnosed with hand-foot-mouth disease and varicella. he had also had recurrent cervical lymphadenopathy (lad) for greater than one year requiring incision and drainage. gram stain and gomori methenamine-silver nitrate stain (gms) were negative and pathology showed only acute and chronic inflammation with areas of necrosis. his family history was negative for autoimmune disease or immunodeficiency. infectious exposure history was significant for an incarcerated father with unknown tuberculosis status and history of living in a shelter. on physical examination, the patient was well appearing with multiple erythematous papules, with superficial erosions and scabbing on the face (figure 1 ), lower abdomen, genital area, buttocks and proximal lower extremities. he had large, firm, non-tender submandibular lymph nodes. he also had small palpable axillary and inguinal lymph nodes bilaterally. his laboratory workup revealed normal white blood cell and platelet counts, but microcytic anemia, an erythrocyte sedimentation rate of 140 mm/hr, and c-reactive protein of 7.5 mg/dl. full body magnetic resonance imaging (mri) revealed bilateral cervical, supraclavicular, right hilar and inguinal lymphadenopathy and a patchy right upper lobe consolidation with at least one small area of cavitation ( figure 2 ) and an adjacent smaller area of ring enhancement. it also revealed three small nonspecific hypodense foci within the right lobe of the liver and borderline splenomegaly. given these findings, there was concern for granulomatous diseases. the patient underwent a liver biopsy ( figure 3 ) which showed non-specific evidence of necrotizing granulomatous disease. microbiological cultures and stains for bacteria, acid-fast bacilli and fungi were negative. his infectious work-up was negative for hsv, tuberculosis, hiv, syphilis, histoplasmosis, and toxoplasmosis. superficial bacterial cultures from the face and groin grew mixed gram positive and negative organisms, including methicillin-susceptible staphylococcus aureus (mssa). his immunologic workup revealed borderline elevated iga and igg with normal igm, normal t,b, nk-cell counts and pneumococcal and tetanus titers. a dihydrorhodamine (dhr) flow cytometric test was positive, consistent with a diagnosis of chronic granulomatous disease (cgd). genetic testing confirmed x-linked disease. he was treated with acyclovir and ceftriaxone with resolution of his rash. conclusion: we present a case of a two-year-old male with newly diagnosed x-linked cgd. though he had been seen by multiple healthcare providers for recurrent lymphadenopathy over the preceding year, he had no other history of recurrent viral or bacterial infections or significant family history that might implicate a primary immunodeficiency. at time of presentation, he had diffuse rash which could have caused his palpable lymphadenopathy on exam. a high index of suspicion for cgd in the setting of recurrent lad and ftt prompted sending the dhr, which led to the diagnosis. chronic granulomatous disease (cgd) is an inherited primary immunodeficiency (pid) which results in both inflammatory response dysregulation and an increase in susceptibility to certain bacterial and fungal infections. without curative treatment such as a bone marrow transplant, it remains a chronic disease with daily medication management, intermittent treatment and life-long surveillance. in general, chronic disease involves physical, psychological and social effects which can affect the patients quality of life. although some research has been done on how pid affects quality of life, there is little research in the united states about how cgd affects patients quality of life. to examine the effect of cgd on patients quality of life, as a part of a voluntary research protocol examining the natural history of immune deficiencies, we administered the who qol-bref instrument to adult cgd patients enrolled on a nih irb approved protocol and seen in the infectious disease clinic at the national institutes of health (nih) over a five-month period. the who qol-bref is comprised of 26 items, which measure the following broad domains: physical health, psychological health, social relationships and environment. each item is rated on 5point likert scale. it has been validated cross culturally and has been widely field tested. the survey was interview administered to 35 patients (23 males, 12 females) with genetically confirmed cgd. the age range was 18 -60 years old (mean age 37.6 years) with a distribution of 57 % x-linked cgd and 43% autosomal recessive cgd. results have been obtained and will be presented. rationale: common variable immunodeficiency (cvid) is the most common primary immunodeficiency with an estimated prevalence of 1:25,000. we aimed to analyze the clinical presentations and their associated comorbidities amongst cvid patients in usa. methods: data on 1,546 cvid patients reported in the united states immunodeficiency network (usidnet) from 1992 to 2018 were analyzed based on clinical, immunological and genetic factors. univariate analysis with spearman rank coefficients was done to analyze correlations between disease outcomes. observed survival was estimated using the kaplan-meier method. results: among the 1546 patients, 908 (58.7%) were female and 638 (41.3%) were male. median age at diagnosis was 29 years [mean (sd), 30.1 (20.2); range, 0-82; iqr, 12-47] with median age of onset of 14 years (mean (sd), 20. 3 (19.2) ; range, 0-81; iqr, . females showed a longer delay in diagnosis (9.5 vs. 6.6 years, p=0.006). higher body mass index (bmi) linearly correlated with the age of diagnosis (r= 0.46). in survival analysis, a 5-year delay in age at diagnosis increased the risk of death by 7.4% (hr: 1.07, 95% ci: 0.98-1.18, p=0.14). conclusions: our study suggests a longer delay in diagnosis in female subjects and a strong association with diagnosis of cvid in patients with higher bmi. females may have a longer period without symptoms leading to a diagnostic delay. gender-based and disparities-based inquiry into these trends may need additional study. the physical well-being of those with primary immunodeficiency (pi) and the physical maladies of those with pi are well-documented. since the 1950s, advances in identification and treatment of pi has for many led to lives where the physical infections of these groups of diseases are manageable. however, not as well understood are the emotional and mental health aspects of living with pi. as part of a larger survey project the idf 2017 national patient survey, this study aims to quantify any potential mental health issues or challenges faced by adults with pi. our hypothesis-those with pi, suffer from statistically higher rates of depression when compared to the u.s. general population. the 2017 idf national patient survey was a nationally distributed, unincentivized, mail-based survey of 4,500 persons in the idf patient database identified as being either adults with pi or the parent/caretaker of a child with pi. the questionnaire comprised approximately 44 main questions about pi as well as the validated sf-12v2, brief fatigue inventory and the patient health questionnaire-2 (phq-2) instruments. additional questions asked about current use of prescription medications for anxiety, depression, stress and pain. for the purpose of this study, only adult respondents with pi are included as the basis for analysis. the two-item patient health questionnaire (phq-2) meets the criteria for general screening of depression suggested by the u.s. preventive services task force. scored on a scale of 0-6, a score of three or higher is suggested as the cut-point for depressive screening. according to a 2014 ahrq study that utilized meps data, 2,139 of the 23,770 (9%) respondents scored three or greater. in our survey 211 of the 925 (23%) adults scored three or greater (2 <.05.) overall, those in our survey scored lower on the sf-12v2 mcs scale when compared to the u.s. population (44.3 v.50.0, p<.05) . further, adults with pi who scored three or higher on the phq-2 had an average mcs of 31.8. those who met the phq threshold in our survey were also more likely to report moderate to severe limitations in normal activities as a result of emotional problems than those that fell below the threshold (74% versus 13%, p <.05). not surprisingly, those that met the phq threshold reported much higher use of prescription medications for anxiety, depression, stress (69% versus 33% below threshold, p <.05) as well as a higher reported use of prescription pain medications (33% versus 17% below threshold, p <.05). though moderate to severe fatigue was reported by 68% of those below threshold, 99% of those with phq scores at threshold reported experiencing moderate to severe fatigue (p <.05). health care providers should consider including the phq-2 in the overall health assessments of their patients with pi. those scoring three or higher should be referred to the appropriate professional for further evaluation. (lek et al., 2016) . the w623l is a semi-conservative amino acid substitution, which may impact secondary protein structure. in-silico analyses supported a deleterious effect, located within the sh2 domain, which is a critical functional domain (chandesris et al., 2012; koskela et al., 2012) . it was thus determined that this variant is likely pathogenic. the patients prophylactic treatment was optimized with tmp-smx (800mg-160mg) twice daily for prevention of infections. she was also started on hibiclens (chlorhexidine) baths once per week. she was referred to pulmonology for optimization of pulmonary health in the setting of bronchiectasis and mild decline in dlco. she was advised to followup on a yearly basis to the primary immunodeficiency clinic to assess for recurrent infections and for changes in pulmonary health. finally, targeted testing and clinical evaluation of both of the patients parents was recommended to determine if w623l was inherited or arose de novo. the pathogenic role of the w623l missense change would be further supported if it had occurred de novo or if it segregates with the disease in the family. uploaded file(s) uploads pulmonary function testing results.pdf j clin immunol (2019) 39 (suppl 1):s1-s151 s20 introduction: lipopolysaccharide-responsive and beige-like anchor protein (lrba) deficiency is a rare autosomal recessive disease of the immune systems characterized by hypogammaglobulinemia and decreased ctla4 expression on t regulatory cell (t regs) due to defective intracellular trafficking of ctla4. previous in vitro study has shown a significant increase of ctla4 expression on lrba deficient t cells after overnight culture with chloroquine, an older anti-malarial agent. this effect is likely due to increasing lysosomal ph. however, there is no evidence of such effect in human subjects after administration of weight appropriate doses anti-malarial agents. we are presenting a set of siblings with lrba deficiency who had ctla4 expression measured before and four weeks after starting hydroxychloroquine. case reports: case 1 is a 14-year-old east-indian boy with autoimmune thyroiditis, type 1 diabetes mellitus (dm), short stature, autoimmune cytopenias, and lymphadenopathy. he was referred to immunology clinic at 9 years of age for suspicion of autoimmune lymphoproliferative disorder. primary immunodeficiency genetic panel was sent which revealed a homozygous mutation in lrba gene (c.6480_6481del). this novel variant resulted in a frameshift and created a premature stop codon 18 amino acids downstream from this location which may lead to absent or abnormal protein. lung ct scan showed interstitial lung disease. lung biopsy showed interstitial nodular and diffuse lymphoid proliferation. this diagnosis led to the testing of his sister (case 2) given her history of autoimmune illnesses and the family history of consanguinity. case 2 is a now 13-year-old girl with type 1 dm, autoimmune thyroiditis, lymphadenopathy, psoriatic arthritis, and seizures. her lung imaging showed pulmonary nodules without interstitial lung disease. both cases received hydroxychloroquine while waiting for insurance approval of abatacept. ctla4 expression on tregs was measured prior to and four weeks after starting hydroxychloroquine treatment. at baseline, 8.6% of case 1s cd4 cells were treg (foxp3+ve, cd25hi) and 51.4% of them expressed ctla-4 (in contrast to 94.1% tregs in the healthy control) with mean fluorescence intensity (mfi) of 335. this ratio and mfi did not change after 4 weeks of hydroxychloroquine treatment (6 mg/kg/day). soluble interleukin-2 receptor levels were measured: case 1 had a baseline level of 8510 pg/ml, which decreased to 2228 pg/ml after 4 weeks of hydroxychloroquine treatment. for case 2: 8.4% of her cd4+ t cells were found to be foxp3+cd25hi and 36.1% of these tregs expressed ctla-4. this ratio increased by 7% after one month of hydroxychloroquine. increase in mfi was also noted from 298 to 386. case 2 had a drop in soluble interleukin-2 receptor level from 1265 pg/ml to 950pg/ml after treatment. conclusion: in contrast to the previous in vitro assays, we did not find a significant increase in ctla4 expression on t regulatory cells in vivo after 4 weeks of 6mg/kg/day hydroxychloroquine. interestingly, soluble il-2 receptor levels improved dramatically with hydroxychloroquine. (36) submission id#592574 human nf-kappab2 defect results in defective intrinsic b-cell differentiation, function and class switching introduction/background: autosomal dominant heterozygous mutations in nfkb2 (encoding for the protein nf-kb2) have been identified in the etiology of a form of primary immunodeficiency disorder that presents with hypogammaglobulinemia, defects in b-cell maturation, endocrinopathy, and autoimmune manifestations. in humans, the effects of altered nf-kb2 and mechanisms of immune system impairment have not been fully delineated. objectives: to understand the mechanism of the antibody deficiency in patients with hypomorphic mutations in nfkb2 (c.2564dela; p.lys855serfs*7) by evaluating b-lymphocyte proliferation, differentiation, function, and gene expression. methods: immunophenotyping of primary b-cells from subjects with mutant nfkb2 was completed by flow cytometry. proliferation of b-cells was assessed by cfse stimulation of primary cd19+ b-cells from healthy and nfkb2 mutant subjects. differentiation of healthy and affected naã¯ve b-cells (cd27-cd38-) into plasmablasts (cd27+cd38+) following stimulation was assessed by flow cytometry. the supernatant from these cells were assayed for iga, igg and igm production by elisa. to study the defect in class-switch recombination, naã¯ve b-cells and ebvtransformed b-cells from affected and healthy individuals were stimulated and expression of the aicda gene was quantified by qpcr. in parallel experiments, ebv b-cells from wildtype and nfnb2 mutant individuals were stimulated and aid (activationinduced cytidine deaminase) protein levels were determined by western blot. results: patients with hypomorphic mutations in nfkb2 (c.2564dela) had low memory b-cell (cd19+ cd27+ igd-igm+) and class-switched memory b-cell (cd19+ cd27+ igd-igm-) numbers. in vitro, primary bcells from these patients demonstrated a 50% reduction in proliferation and cell division in response to cd40l and il-10 (p =0.01). compared to healthy naã¯ve b-cells, mutant naã¯ve b-cells had a significant reduction in plasmablast differentiation (p = 0.002) and secreted significantly lower levels of immunoglobulins in response to cd40l and il-21 stimulation. mutant naã¯ve b-cells and mutant ebv b-cells failed to increase aicda expression and aid protein levels in response to cd40l and il-21 stimulation. conclusions: our studies demonstrate that a hypomorphic nfkb2 mutation in humans affects intrinsic b-cell proliferation and differentiation. the mutation impairs transcription of the aicda gene that encodes aid, a key protein involved in b-cell class-switch recombination. the nfkb2 gene defect also impairs immunoglobulin production, as seen in common variable immunodeficiency-like cases. these studies provide unique translational insights into physiological activities of nf-kb2 in downstream immunologic outputs in humans, expanding those suggested by experimental observations in mice. background: few studies have evaluated the quality of life (qol) and patient reported outcomes of primary immunodeficiency disease (pidd) patients, and no studies have assessed medical provider perceptions of their pidd patients qol, neurocognition, physical well-being and psychosocial health. understanding provider beliefs regarding patient reported outcomes is essential to improving clinical management of pidds. here we report our pidd medical provider survey results. methods: providers were contacted via email with the assistance of the clinical immunology society. participants completed adult and/or pediatric-based likert scale survey questions via a secure online survey service. in addition to demographic information, survey questions assessed provider perceptions of patients overall qol and their impression of the impact of disease or its associated treatment on mental health, physical well-being, neurocognition, social relationships and school/work performance. clinicians were expected to make their assessments based on their pidd patient cohort as a whole rather than on specific diagnoses or patients. given the small sample size, a p-value < 0.1 was considered statistically significant; repeated measures anova and paired t-test analyses were used. results: study participants (n=58) were primarily from the united states (64%), born between 1965-1979 (44%) , and trained in allergy/ immunology (77%). 85% of survey takers practiced within an academic center, 52% were female and 95% cared for children with 42% of providers concurrently caring for adults. there was a statistically significant difference (p=0.07) in the perceived overall qol of pediatric versus adult pidd patients with 41% of providers feeling as though their pediatric patients had a good qol while only 25% believed their adult patients had a good qol. clinicians believed adult pidd individuals had more difficulties related to associated co-morbidities rather than their actual pidd compared to pediatric pidd patients (p=0.046). providers felt that the neurocognition and school performance of children were more often negatively affected by a pidd than the neurocognition and work performance of immunodeficient adults (p=0.1). clinicians believe children with pidd more frequently had difficulties related to their concentration than memory (p<0.01). 96% of those who care for pidd adults believe their patients work performance or daily mental functioning is at times negatively impacted. anxiety symptoms and social relationships were viewed as being more negatively impacted by a pidd diagnosis or treatment than anger or depressive symptoms in both children and adults (p<0.01). 38% of pediatric clinicians feel their pidd patients experience anxiety symptoms often or almost always. of physical health parameters, energy, rather than mobility or pain, was deemed to be more deleteriously influenced by an immunodeficiency in adult and pediatric patients (p<0.01). conclusions: our results show that medical providers perceive the overall qol of pediatric pidd patients to be superior to that of adults with pidd, but most clinicians feel a diagnosis or associated treatment regimen for pidd can negatively impact the physical well-being, psychosocial health, school/work performance and neurocognition of both children and adults. [cbm] complex is a critical signalling adaptor that regulates lymphocyte activation, proliferation, survival, and metabolism. primary immunodeficiencies affecting each component (termed cbmopathies) result in broad clinical manifestations ranging from severe combined immunodeficiency (scid) to lymphoproliferation. we present the laboratory and clinical findings of two canadian first nations patients found to be homozygous for the same novel card11 mutation (c.2509c>t; p.r837*). results: we have identified an 8-month-old boy who presented with a severe case of entero/rhinovirus bronchiolitis with interstitial lung disease and a 17-year-old boy with a history of severe pulmonary infections (including pjp), chronic sinusitis, candidiasis, invasive bacteremia, and severe ileo-colitis and oral ulceration requiring total colectomy. both patients possessed absent tregs, absent memory b cells, and hypogammaglobulinemia. however, only the 8-month-old had poor t cell proliferation to pha, cona, and cd3. both patients were found to be homozygous for the same novel variant of card11 (c.2509c>t; p.r837*). the mutation rendered card11 protein expression unstable and it was undetectable by immunoblot. to confirm card11 deficiency, we stimulated patient b cells with phorbol 12-myristate 13 acetate (pma) and ionomycin across a time-course and immunoblotted for various signalling proteins in both the nf-b (ikk/, ib, p65) and mapk (mek1/2, mkk4, jnk1/2, erk1/2) pathways as well as various cleavage substrates of the malt1 paracaspase (relb, cyld, bcl10, hoil1). nf-b and jnk activation were completely absent and malt paracapase activity was lost, but surprisingly, mkk4 (which acts upstream of jnk) was intact. furthermore, co-immunoprecipitation experiments revealed that card11 was required for optimal malt1 association with bcl10 in response to stimulation. conclusions: these two cases highlight the crucial role of card11 in regulating lymphocyte development, function, and humoral responses. in addition, we have identified the oldest known living individual with card11 deficiency and he presented uniquely with inflammatory gastrointestinal disease in addition to scid, further adding to the spectrum of phenotypes associated with card11-related primary immunodeficiencies. abstract: the usidnet registry began in 1992 with an niaid contract with the immune deficiency foundation, which continues today. it aims to provide a resource for clinical and lab research through enrollment of known immunodeficiency patients into a national registry, the usidnet. nih is a major national and international referral center for clinical trials on inborn errors of immunity, or primary immunodeficiency diseases. it is a mechanism for depositing nih data into usidnet. a registry of patient information may help us understand how many people have each disease. the information may improve how we diagnose and treat these conditions. the patient registry is designed to obtain longitudinal data on a large number of patients with primary immunodeficiency diseases who come to nih to participate in research. the data is collected from the nih electronic medical record system, cris and is deposited into a secure registry with restricted and monitored access. all medical information is anonymized for patient privacy. department of biochemistry, emory university, atlanta, ga oas1 is an intracellular sensor for dsrna that generates the second messenger 2'-5'-oligoadenylate to activate rnase-l as a means of antiviral defense. we describe four patients with a complex early-onset autoinflammatory and immunodeficiency disease caused by heterozygous de novo oas1 mutations. patients presented early in life with lung inflammation including pulmonary alveolar proteinosis and interstitial lung disease. they had febrile flares with dermatitis specifically with macular, pustule and bullous features often progressing to ulceration. infants had episodes of bloody diarrhea in 3 patients (assoc. with villous blunting and cryptitis in two patients and oesophagitis in one patient). immunoglobulin igm, igg, and iga levels were low while t cell, b cell, and nk cell numbers were generally in the normal range. exome sequencing identified de novo heterozygous oas1 missense mutations in all patients. one patient had a heterozygous de novo oas1 mutation p.ala76val, with mutant oas1 protein being expressed in ex vivo generated t cell blasts. in sorted primary patient monocytes and b cells, oas1 p.ala76val was associated with spontaneous rna degradation and apoptosis as determined by rna chip technology and flow cytometry, respectively, while t cells were not affected. monocytes displayed disturbed terminal differentiation and functioning as indicated by reduced gm-csf-r expression and signaling. b-cells display reduced class-switch-recombination. proliferation of allogeneic t-cells was reduced in response to sorted oas1 mutated monocytes and b-cells. activation of interferon response genes in pbmcs was detected. two further unrelated patients had a heterozygous de novo oas1 mutation p.cys109tyr, which appeared to compromise protein stability in transformed patient fibroblasts and when transfected. cells transfected with this mutant protein had reduced 2-5 oligoadenylate synthesis compared to wild type transfected cells. immortalized fibroblast lines demonstrated higher levels of inflammatory cytokines and spontaneous cleavage of rnas. a 4th patient with the clinical phenotype had a heterozygous de novo oas1 variant p.val121gly, but has yet to have formal validation of the variant. three patients underwent hematopoietic stem cell transplants in an effort to control their diarrhea and skin inflammation. one patient died with ongoing chronic graft versus host disease, while the two others (p.ala76val, cys109tyr) are alive and reasonably well with a followup of 0.5-7 years. the untransplanted patient died as a result of respiratory failure. in summary, patients with de novo heterozygous oas1 mutations have chronic ongoing inflammation of multiple organs. this is at least in part due to spontaneous rna cleavage, apoptosis and production of inflammatory cytokines and type i interferons. this defines a new category of autoinflammatory disorder. introduction: increased susceptibility to infections is the most common complication of chronic granulomatous disease (cgd). hemophagocytic lymphohistiocytosis (hlh) is a severe disorder resulting from hyperinflammation and hypercytokinemia that can lead to multi-organ system dysfunction (1) characterized by certain criteria: fever, splenomegaly, cytopenias, hypofibrinogenemia or hypertriglyceridemia, hyperferritinemia, increased soluble cd25/il-2ra, evidence of hemophagocytosis, or decreased/absent nk cell cytotoxicity (2) . secondary hlh occurs infrequently but often is preceded by smoldering infection in cgd (3, 4, 5) . we present a case of hlh in a 38-day old male, the youngest reported case with cgd. case: a 38-day old male with previously diagnosed x-linked cgd, due to known family history, presented with fevers. initial evaluation was unrevealing including chest x-ray, urinalysis, and blood and csf cultures. he was admitted and treated empirically with cefepime. ct demonstrated multiple multifocal nodules of the lungs and spleen. after lung nodule biopsy was performed, antimicrobial therapy was broadened to iv meropenem, voriconazole, and micafungin. despite this, he continued to have fever and developed new onset tachycardia, respiratory distress, and lactic acidosis. further decompensation with vasoactive refractory shock was treated with vasopressors and stress dose hydrocortisone. additional laboratory evaluation revealed rising liver enzymes (ast 1670u/l, alt 307u/l), cytopenias (hemoglobin 7 g/dl, anc 90/ul, platelets 96,000/ul), and coagulopathy (fibrinogen 93-135mg/dl). splenomegaly was present on abdominal ultrasound. a diagnosis of evolving hlh was considered and dexamethasone was administered. within 24 hours of clinical decompensation, the patient died of multiorgan failure. subsequent blood cultures returned with gram-negative rods (and ultimately burkholderia cepacia). autopsy confirmed hemophagocytosis within the bone marrow. no mutations were found in genes associated with primary hlh. discussion: patients with cgd are susceptible to infectious complications and auto-inflammation most commonly involving the lungs, gi, and gu systems (6, 7) . patients with cgd can be at increased risk of hyperinflammatory syndromes secondary to infections and chronic inflammation. as shown in the included case, hlh can present in infancy and can be deadly. early consideration and directed treatment of hlh is imperative, even in the setting of sepsis malignant proliferation of gamma-delta t cells include hepatosplenic t-cell lymphoma (hstl), primary cutaneous t-cell lymphoma and t-cell large granular lymphocytic leukemia (t-lgl). the former two have often been associated with splenomegaly and cytopenias. however, reactive proliferation of gamma-delta t cells in spleen mimicking malignancy has only been reported once and has a significant risk of misdiagnosis. a 30-year-old female presented with two years of unintentional weight loss, persistent leukopenia and thrombocytopenia, with leucocytes around 1-2 x 10^9/l and platelets around 100 x 10^9/ l. she also had associated macrocytic anemia (hemoglobin=10-11g/dl) with laboratory evidence of dat (direct anti-globin test) negative hemolysis. physical examination and computed tomography (ct) imaging showed splenomegaly. there was no hepatomegaly or lymphadenopathy. serum liver function test, auto-immune studies, hemolysis and hereditary diseases workup, viral and bacterial serologies were all normal or negative, except for mild hyperbilirubinemia and ldh elevation. bone marrow examination performed four months prior to the splenectomy revealed mildly hypocellular marrow (50%) with trilineage hematopoiesis. flow cytometric analysis and cytogenetics of the bone marrow aspirate and peripheral blood were normal except for small population of large granular lymphocyte and mild low absolute b cell counts in peripheral blood. a laparoscopic splenectomy was performed for diagnostic and therapeutic purposes due to patients worsening luq pain. there was no other treatment given prior to surgery. 24 hours postsplenectomy her leucocytes increased to 13.1 and platelets to 247. her three-month post-splenectomy wbc count and platelet count was 8.9 and 391, respectively. hemoglobin also improved to 14.9. pathology showed red pulp expansion by small lymphocytes (fig. 1 ) and subsequent ihc (immunohistochemistry) was positive for cd3 ( fig. 2) , cd2, cd7, tia-1 and negative for cd8, cd5 and cd56. cd4 was difficult to interpret. eber was negative. flow cytometry ( fig. 3) showed increased gamma-delta t-cell population (20%) with positive cd3, cd2 and cd 7 and negative cd 5, cd4 and cd 8. molecular studies by pcr didnt reveal any t-cell receptor gamma or beta gene rearrangement. cytogenetics was negative for isochromosome 7q or any other abnormalities. she was symptom free at 6 months from her splenectomy. the morphology and immuno-phenotype of these gamma-delta t cells show significant overlap with the malignant cells seen in hstl and t-lgl, such as loss or downregulation of cd5, cd4 and cd8. awareness of this reactive condition is necessary to prevent making a wrong diagnosis of a malignant disease with a potentially benign, spontaneously resolving disease. additional studies of similar cases is needed in order to establish more definitive criterion to separate benign from malignant processes and delineate the role of gamma-delta t cells. uploaded file(s) uploads fig 3. flow cytomtery.pptx background: sex steroids in the human thymic environment influence aire expression as well as interactions with its partners, i.e. genes coding for aire interactors. here we investigated the effects of sex steroids on these interactions during minipuberty the surge of sex hormones that occur along the first six months of life -and up to 18 months of life. we employed a network-based approach for investigating aire-interactors gene-gene relationships and how abundantly co-expressed thymic mirnas covariate with those genes. aire-interactors networks allowed the measuring of gender-related differences in gene-gene expression correlation disclosing relevant differences between minipuberty groups. methods: total rna was extracted from thymic surgical explants obtained from male (m) and female (f) infants -aged 0-6 months (groups mm and mf, for minipuberty) and 7-18 months (group nm and nf, for nonpuberty) and used in dna microarray assays. gene coexpression network (gcn) analyses were performed for aire and its interactors and for mirna-gene coexpression analysis. the set of genes coding for the aire-targeted proteins was previously identified in tecs by abramson et al. (cell 140:123-35, 2010) . aire-interactors networks were obtained for all groups (link strength cut-off for gene-gene > |0.80| and for mirnagene < -0.80). aire expression in mtecs was quantified by immunohistochemistry. these methodologies are described in moreira-filho et al. (sci rep 8:13169, 2018) . results: the mm x mf networks comparison showed that 16 abundantly expressed mirnas are interacting with the different aire interactor genes in both networks. it is interesting to note that network topology were more similar between nm and nf groups, although aire interacts with only one distinct mirna in each network (mir-150-5p in the nm group or mir-7977 in the nf group). conversely, in the non-puberty networks the sets of mirnas and their interacting genes are distinct for each network. immunohistochemistry analysis revealed a higher percentage of mtec aire positive cells in the minipuberty groups: i.e. there is a significant difference between mm x nm (p = 0.0006) and between mf x nf (p = 0.0060). conclusions minipuberty and genomic mechanisms shape thymic sexual dimorphism along the first 6 months of life. this process does not involve changes in aire expression between genders, but differences in the interactions of aire with its partners that persist throughout the non-puberty period, probably regulated by mirnas and also by genetic and epigenetic factors. introduction: neutrophils are presumed to defend against aspergillus species by releasing reactive oxygen species (ros) and neutrophil extracellular traps (nets) to degrade fungal hyphae. triazole antifungals synergistically enhance neutrophil mediated hyphal degradation. patients with cgd are particularly susceptible to aspergillus species likely due to their inability to create ros and nets, and in severe cases may not be amenable to antifungal therapy alone. objective: we present a case of severe disseminated aspergillosis in a patient with cgd in whom gt served as an important adjunct to antifungal therapy and bridge to transplant. results: a 6-year-old boy with known cgd, lost to follow up and nonadherent to prophylaxis, presented acutely with right-sided hemiparesis. neuroimaging revealed an embolic left middle cerebral artery infarction and cardiac magnetic resonance imaging showed extensive vegetations involving both right and left ventricles and atria, with an ejection fraction of 28%. the patient was admitted to intensive care, started on liposomal amphotericin b, meropenem and vancomycin, and underwent debulking of the intracardiac masses on post admission day (pad) 1. operative findings showed severe constrictive pericarditis with multiple abscesses and intracardiac vegetations. thorough debridement of the vegetations was undertaken, however some deep seated abscesses in the myocardium were not amenable. operative cultures were positive for aspergillus fumigatus. clinical status remained precarious, with ongoing requirement for inotropic and ventilator support. antimicrobial therapy was refined to voriconazole, with amphotericin b remaining on board until therapeutic levels of voriconazole were achieved. as effective neutrophils are integral in the immune response against aspergillus, the decision was made to start granulocyte transfusions to aid in clinical stabilization prior to hsct. interferon gamma infusions were not administered because of the risks of adverse effects and potentially increasing transplant rejection. gts were started on pad 6, at a dose of approximately 1x10^10 granulocytes, three times a week. the patient tolerated the infusions well, with no allergic or inflammatory response. neutrophil oxidative burst measured one hour post infusion showed 23.9% mean fluorescent intensity, compared to a baseline of 0% ( figure 1 ). clinical improvement was seen, with inotrope cessation on pad 12 and extubation to bipap on pad 41. human leukocyte antigen (hla) allosensitizaton was tested on pad 12, 6 days after the first gt, with no evidence of hla antibodies. a total of 28 gts were given over 3 months, prior to proceeding to a 10/10 hla matched related donor transplant (pad 69), with two transfusions given before neutrophil engraftment (anc 500) on day +14. the patient is now stable 13 months post transplant, with no evidence of graft rejection. he remains on chronic suppressive antifungal therapy, to continue until full lymphoid reconstitution. conclusion: gt may be a useful adjunct to antifungal therapy in patients with impaired neutrophil function with severe invasive aspergillosis, and potentially provide a life sustaining bridge to hsct. methods: subjects were enrolled in irb protocol 00051692 for rvt-802. rvt-802 was implanted into the quadriceps with immunosuppression. results: subject 1 was normal at 22q11.2 but had hypocalcemia, an asd, pda, and abnormal ears. the subject received a cord blood transplant mismatched at hla-b and hla-c alleles at age 3 months. subsequently mild graft-versus-host disease (gvhd) developed and was treated with antithymocyte globulin, steroids and cyclosporine. donor t cells developed in low numbers. twelve years later, the subject developed epstein barr virus lymphoma and suffered two relapses. while in remission, subject 1 received unmatched rvt-802. two weeks after rvt-802 implantation, the subject developed an adenovirus infection resulting in skin and gut gvhd, presumably from activation of the cord blood t cells. subject 1 was treated with corticosteroids, cyclosporine, cidofovir and infliximab. four years post rvt-802, subject 1 is healthy with 609 genetically recipient t cells/mm3 and 40% naã¯ve cd4 t cells. subject 2 was normal at 22q11.2 but had an asd, pda, hypoparathyroidism, and no t cells at birth. his genetic defect is unknown. subject 2 was treated with a ric myeloablative, allogenic, unrelated, 10/10 cord blood transplant, and a subsequent myeloablative, unrelated 9/10 cord blood transplant. hematopoietic chimerism was established without t cell development. rvt-802 expressed the one allele in the recipient that was not expressed by the second cord donor. the post-thymic transplant course included immune thrombocytopenia requiring rituximab and splenectomy and generalized adenopathy for 3 years but no gvhd. he failed weaning of immunoglobulin replacement. three years post rvt-802, he has 930 cd3, 750 cd4, and 105 cd8 t cells/mm3. he is active in school. subject 3 had absent trecs on newborn screening with 7 cd3+ t cells/ mm.3 a single mutation in foxn1 was identified; she has sparse scalp hair. subject 3 received a 9/10 matched unrelated umbilical cord transplant. the post-transplant course was complicated by significant morbidity, and no naã¯ve t cell development. rvt-802 expressed the one allele in the recipient that was not in the cord blood donor. the subject did not develop gvhd, is healthy and at 9 months has 98 naã¯ve cd4+ t cells. she had resolution of longstanding norovirus and sapovirus gastroenteritis. conclusion: rvt-802 can improve t cell immunity after poor or failed correction with allogeneic hematopoietic transplants. in subject 1, gvhd post rvt-802 was related to an acute viral infection; cord t cells attacked hla mismatches in the recipient. subjects 2 and 3 were given rvt-802 matched to recipient alleles that were not expressed in the hematopoietic donor. we hypothesize that thymocytes developing in rvt-802, if strongly reactive to the recipient-mismatched allele, are deleted by the bonemarrow-donor dendritic cells (that acquire recipient mhc from the recipient-allele-matched thymic epithelial cells) thereby preventing gvhd. rationale: ctla4 haploinsufficiency is an autosomal dominant immune dysregulation syndrome characterized by variable phenotypes. here we present a young woman diagnosed with evans syndrome and lymphoproliferation as a child, found to have a novel ctla4 variant as a young adult, and who developed hypogammaglobulinemia and a bacterial endocarditis while stabilized on ctla-4 replacement therapy. methods: sequencing of 207 genes, including ctla4, in primary immunodeficiency panel. results: our patient was diagnosed with evans syndrome at age 2 with manifestations of anemia and thrombocytopenia recalcitrant to treatment over many years with steroids, cyclosporine, and vincristine. bone marrow biopsy reportedly showed normal trilineage maturation and her symptoms responded for a short time to splenectomy at age 14. symptoms recurred at age 16 when she was also found to have pulmonary reticular opacities, prominent lymph nodes, and elevated b cells. repeat bone marrow and lymph node biopsies at that time were unrevealing. minor responses to treatment with ivig, rituximab, mycophenolate mofetil and gcsf were noted. at age 17, she developed varicella-related encephalitis shortly after vaccination. with a strong suspicion of an immune dysregulation syndrome, immune evaluation revealed normal immunoglobulins with good vaccine responses, elevated b cell numbers, normal t cell numbers, and normal mitogen proliferation. ctla4 sequencing revealed a mutation in exon 2 [c.420c>a, p.tyr140*] causing a premature translational stop signal, which was consistent with previously reported cases of ctla4 haploinsufficiency. she was started on rapamycin initially for her cytopenias but was then transitioned successfully to abatacept with almost complete resolution of her anemia, neutropenia, and pulmonary opacities. after 6 months of stable control, she developed a precipitous drop in her platelets and was eventually diagnosed with streptococcus viridans endocarditis of her native mitral valve. this responded to antimicrobial therapy, but eventually needed surgical intervention due to ongoing insufficiency. around this time, she was also found to be newly hypogammaglobulinemic, necessitating ongoing igg supplementation therapy. during successful replacement of her mitral valve with a biosynthetic prosthesis, it was noted that her aortic valve also had evidence of previous disease, implicating a prior endocarditis as part of her clinical syndrome as well. conclusions: in this patient, the presentation of recalcitrant cytopenias, lymphadenopathy, elevated b cells, vaccine-induced viral infections and lung findings precipitated concern for immune dysregulation syndromes and allowed for identification of a novel deleterious ctla4 mutation. in addition to previously reported clinical findings, our patient presents with the first reported case of repeated endocarditis in the setting of ctla4 insufficiency disease. given the finding in this patient of prior (unrecognized) disease, regularly screening patients with ctla4 insufficiency for evidence of cardiac affectation may be prudent. clinical research nurse, johns hopkins university background: the relationship between elevated serum alpha fetoprotein (afp) concentration and age, mortality, genotype and neurologic outcome in ataxia telangiectasia (a-t) patients has remained inconclusive over the past decades, leaving afp as a useful marker for disease diagnosis without further clinical significance. objective: to examine the relationship between afp levels and age, mortality, genotype and neurologic outcome using a data set larger than any prior study. methods: we retrospectively collected data on 280 a-t patients at johns hopkins medical center (0-34 years of age) with both classical (predicted protein null) and variant a-t. this included 459 serum afp measurements (179 serial levels in 50 a-t patients, max observations 9 per patient). mixed model compound symmetry covariance was used for statistical analysis to examine the effect of age at visit on afp levels. subgroup analysis by mutation type, mortality, feeding/swallowing scores as a surrogate for neurologic function, x-ray induced in vitro chromosomal breakage and serum transaminase levels were similarly analyzed. results: significant association between age and afp level was found such that for every 1 year increase in age, afp level increases 20 ng/ml (p<0.0001). subgroup analysis by mutation type found that the 12 patients with missense mutations showed a negative linear relationship be-tween log afp levels and age (r= -0.10, p=0.03). we found greater afp levels in patients who subsequently died, after controlling for age (least square mean afp level in log scale 0.67 greater in deceased patients versus living patients, p=0.002). we found a significant decline in feeding score by 0.18 units (score range 0-5) per 100 ng/ml afp increase (p=0.05) after adjusting for age. there was no significant relationship between afp levels and serum transaminase levels. conclusion: afp increases with age in a-t patients, though this may not apply to patients with missense mutations. there is a statistically significant increase in mortality and worsened swallowing scores with increasing afp levels, but this remains to be proven clinically significant. here we present a pediatric hae patient who had recurrent abdominal attacks in which constipation, secondary to the adhd medication dexmethylphenidate (focalin), appears to be a trigger. of importance, this is the first pediatric patient with hae to be described as having safely undergone a capsule endoscopy for direct visualization of the gastrointestinal tract. this was done to decrease the risks associated with the more invasive procedure of traditional endoscopy and colonoscopy. case presentation: the patient was an 8-year-old male with hereditary angioedema who presented with 1 day history of diffuse abdominal pain and nausea. in the ed, patient was in no acute distress. abdominal ultrasound showed severe circumferential thickening of the wall of multiple bowel loops and a large amount of simple ascites. x-ray revealed stool in the colon. he was admitted for pain control and hydration. in the next year, he visited the ed five more times for exacerbations of angioedema of his hand, penis, and bowel. each time, he presented he had underlying abdominal pain and constipation. he was seen by gastroenterology and had a workup that was negative for helicobacter pylori, parasites, and other gastrointestinal infections. to further evaluate his abdominal pain, capsule endoscopy was performed and well tolerated. during an admission in january 2016 he received a full inpatient bowel cleanout, after which, his angioedema finally improved. of note, he was diagnosed with adhd and started on dexmethylphenidate (focalin) just prior to this period of recurrent angioedema attacks, and he did not have attacks during the summer months when he was off the medication. discussion: abdominal pain is a common complaint in pediatric hospitals, and further workup consists of endoscopy and colonoscopy. this may be easily accomplished in the general population, however, in patients with hae, these procedures carry greater risk and may be avoided, leading to delayed diagnosis and treatment (2, 4) . a newer and less commonly used alternative for direct visualization of the gastrointestinal tract is capsule endoscopy. some benefits are that it does not require sedation, is less invasive, and is less likely to be irritating to the mucosa (3). additionally, since psychological stress may be a trigger for angioedema attacks, the decreased stress associated with a noninvasive procedure such as capsule endoscopy, makes it safer to use (1) . limitations of capsule endoscopy include dependence on battery life and its inability to biopsy or administer therapy if needed (3) . hereditary angioedema treatment consists primarily of avoiding triggers and managing acute episodes. in this first case of hae in a pediatric patient where capsule endoscopy was used, the procedure was well tolerated without any complications. recognizing constipation as a trigger and capsule endoscopy as a safe method of direct visualization of the gastrointestinal tract will help others to control and decrease the severity of their hae attacks as well. a 45 year old male with past medical history of common variable immune deficiency (cvid) and related autoimmune complications, including granulomatous-lymphocytic interstitial lung disease (glild), hepatosplenomegaly, leukopenia, and thrombocytopenia tolerated monthly subcutaneous immunoglobulin replacement as outpatient for several years with infrequent infectious complications. four months ago, he was found to have elevated liver enzymes on routine chemistry. a liver biopsy two months later showed pathology consistent with nodular regenerative hyperplasia (nrh) without overt cirrhosis. a hepatic venous pressure gradient (hvpg) of 21 mmhg was found, consistent with portal hypertension. his hepatitis viral markers were negative, he did not drink, and portal venogram was negative for thrombosis. in early october, the patient was admitted to the hospital with anasarca and tense ascites. he underwent a diagnostic and therapeutic large volume paracentesis and was also found to have spontaneous bacterial peritonitis (sbp) and bacteremia with group b streptococcus. the patients course was complicated by polymicrobial peritonitis, vre bacteremia, fungemia, variceal hemorrhage, hepatic encephalopathy, and hepatorenal syndrome. his hepatic complications from portal hypertension were out of proportion to his liver parenchymal disease. transjugular intrahepatic portosystemic shunt (tips) was considered to alleviate portal hypertension but was not feasible due to his degree of encephalopathy. immunosuppressants such as high dose steroids were given while in the hospital with plans to start rituximab to treat patients glild after he had recovered from the acute infections. unfortunately, after two months in the hospital, the patient succumbed to sepsis and progressive liver failure. this case emphasizes the importance of systematic screening and continued vigilance for hepatic complications in patients of cvid as studies have shown that nrh of the liver is present in more than 80% of cvid patients who undergo a liver biopsy (pmid: 23219764). a cross-sectional study of patients with primary hypogammaglobulinemia and hepatic dysfunction found that histological findings of nrh were present in 84% of cvid patients and was associated with portal hypertension in 75% of cases (pmid: 17998147). another study estimated the minimal prevalence of nrh in cvid patients as 12% (pmid: 18647320), stating that this was likely a gross underestimate as nrh may also be present in patients with normal liver function tests that are not routinely biopsied. therefore, liver enzyme levels may not anticipate the severity of liver involvement. there is currently no treatment for cvid-related liver disease. other causes of non-cirrhotic portal hypertension, including hepatic veno-occlusive disease and budd-chiari syndrome should be ruled out or treated in cvid patients presenting with hepatic disease. in the case of hepatic nrh in cvid patients, early detection could lead to earlier interventions (such as tips prior to hepatic encephalopathy), to mitigate complications. we describe the application of epigenetic quantification of t regulatory (treg) cells in addition to cd3+, cd4+, cd8+ t cells, b cells, nk cells, monocytes and neutrophils from as little as 50 î¼l of fresh, frozen or dried blood. the method yields identical results to flow cytometry from fresh blood samples of a healthy donor cohort, with the advantage of being more sensitive and precise with limited amount of blood and minimal sample preparation (sci transl med 2018). we have used this method 1) to immunophenotype patients with early onset immune regulatory disorders (pird) and primary immune deficiency (pid), and 2) to evaluate cell subsets reconstitution early after hematopoietic stem cell transplantation (hsct). patients with immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) and ipex-like pird were evaluated by analyzing the treg-specific demethylated region (tsdr) of the foxp3 locus in the total of cd3+ t-cells. despite the dysfunctional foxp3 mutated protein, ipex patients exhibited elevated treg/cd3+ cell ratios which seemed to correlate with disease severity. in contrast, most of the patients with ipex-like symptoms without foxp3 mutations exhibited decreased treg/cd3+ cell ratios -in line with the possible central pathogenic role of treg function and number in pird. using epigenetic quantification of cd3+/b-and nk cells, 23 out of 24 confirmed scid and xla cases were correctly identified within a cohort of 250 newborn dried blood spot (dbs) samples (96% sensitivity, 100% specificity). the method identified one delayed onset scid as well as a xla case that were missed by combined trec/krec testing. epigenetic immune cell quantification missed one scid case with maternal engraftment that was identified by combined trec/krec testing. abnormally elevated treg/cd3+ ratio was also detected in a dbs from a newborn who was subsequently confirmed to be affected with ipex syndrome. when applied to serial blood samples during engraftment and reconstitution post-hsct, the epigenetic method allowed identification of the different blood cell subsets, including treg cells, at earlier time points than flow cytometry according to current clinical practice. this opens the way to a better understanding of the correlation between early immune reconstitution events and graft vs. host disease or viral reactivation, earlier than with the current methods, in different types of hsct. these studies underscore the suitability of epigenetic immune cell quantification for accurately measuring multiple immune cell types from limited blood sample sources. we propose this method as uniquely suitable for novel molecular diagnostic applications in settings with limited fresh blood sample or limited cell number, at the point of care as well as for newborn screening. we evaluated a 5-year-old male with hyperpyrexia, hypertrichosis, conical hypodontia, and a history of illnesses concerning for nemodeficiency syndrome. starting at six months of age, he suffered recurrent episodes of acute otitis media (non-typeable hib and actinobacter iwolffli), pneumonia, and rsv bronchiolitis. whole exome sequencing demonstrated a de novo heterozygous c.1259g>a (p.r420q) mutation in the eda-receptor (edar) gene not present in the parental dna. his physical exam findings and mutation were consistent with hypohidrotic ectodermal dysplasia (hed), a rare genetic condition characterized by abnormal development of skin, teeth, hair, and sweat glands. hed is caused by defects in the ectodysplasin-a (eda)-nfkb signaling pathway but is not typically associated with immune deficiency. consistent with this, immunophenotyping showed normal sub-populations of t-, b-, and nk-cells. immunoglobulin and complement levels were quantitatively appropriate. he had normal mitogen-induced lymphocyte proliferation and normal antibody response to pneumococcal vaccination. nk-cell studies demonstrated robust cytotoxicity. however, nasal mucosa biopsy showed diffuse squamous metaplasia and the absence of ciliated epithelial cells. we hypothesize that recurrent infections in our patient arose from impaired mucociliary clearance due to a ciliary defect. this case raises the possible association between edar variants and ciliary dysfunction. it also underscores the importance of evaluating the immune status of hed patients with recurrent infections which could mimic nemo-deficiency and have broad implications about clinical management. the rapid pace of new gene discovery and phenotype expansion for primary immunodeficiency diseases (pidds) creates challenges for genetic testing and variant interpretation. whereas well-described clinical case reports in published literature have traditionally served as the source of phenotypic data used for variant interpretation, for pidds the causal variants are often private to the patients family and thus the sole source of phenotypic information for a novel genetic variant is frequently the history provided by the clinician on the test requisition form. taking into account such heterogeneous information during variant interpretation requires establishing objective criteria for its inclusion as part of the variant interpretation process. to this end, we adapted our laboratorys preexisting, evidence-based variant classification framework, called sherloc, by developing point-based criteria for the inclusion of clinical information such as a patients phenotype, familial segregation patterns, and whether the variant is inherited or de novo in the patient. as part of this process, we defined clinical criteria for 154 pidd genes. here, we illustrate the application of this method and the importance of integrating clinical information into variant interpretation. between april 2017 and october 2018, our commercial diagnostic laboratory performed 4057 immunological genetic tests, and information about the patients clinical history was provided in 2849 (70%) of these orders. restricting our analysis to just the 154 genes for which case report information is currently used in variant interpretation, these tests revealed 3868 variants, 370 (10%) of which were classified as pathogenic or likely pathogenic (p/lp). information from case report descriptions, segregation patterns, and de novo status were applied for 32%,15% and 4% of p/lp variants, respectively. in 37 (10%) cases, the clinical information provided by the clinician on the test requisition form was used as evidence in the classification of the patients variant as p/lp. ten variants were initially classified as being of uncertain significance and reclassified following receipt of further clinical information or testing of additional relatives. in addition, 35 suspicious variants of uncertain significance were identified in which one or two additional patient case reports would allow for reclassification from uncertain significance to p/lp. these data illustrate the importance of providing good quality clinical information to the genetic testing laboratory both at the time of sample submission and following the receipt of genetic test results. background: cartilage-hair hypoplasia (chh) is a skeletal dysplasia with combined immunodeficiency, variable clinical course and increased risk of malignancy, mostly non-hodgkin lymphoma and basal cell carcinoma. there is a paucity of long-term follow-up data, as well as knowledge on prognostic factors in chh. objective: we conducted a prospective cohort study in finnish patients with chh to describe clinical course and analyze risk factors for adverse outcomes. methods: we recruited 80 finnish patients with chh in 1985-1991 and performed clinical follow-up in 2011-2015. we obtained health information from finnish national medical databases (covering time period of 1969-2016), the finnish cancer registry and the cause-of-death registry of the statistics finland and analyzed all patients' health records. standardized mortality ratios (smrs) were calculated based on the population data. primary outcomes included immunodeficiencyrelated death (from infections, respiratory diseases or malignancies), the development of lymphoma and the development of skin cancer. results: the study cohort included 35 males and 45 females. median age at recruitment was 14.6 yrs (range 2 weeks -49.6 yrs) and median duration of follow-up for the surviving patients was 29.2 yrs (range 25.6 -31.0 yrs). half of the patients (46/80, 57%) had no symptoms of immunodeficiency, while 15 (19%) and 19 (24%) patients manifested symptoms of humoral or combined immunodeficiency respectively, including six cases of late-onset immunodeficiency. in a significant proportion of patients (17/79, 22%), clinical features of immunodeficiency progressed over time. of the 15 patients with non-skin cancer, eight had no preceding symptoms of immunodeficiency. altogether 20 patients had deceased (smr=7.0, 95% confidence interval (ci)=4. [3] [4] [5] [6] [7] [8] [9] [10] [11] including deaths due to pneumonia (n=4), malignancy (n=7, smr=10, 95%ci=4.1-21) and lung disease (n=4, smr=46, 95%ci=9. . malignancy was diagnosed in 21/80 (31%) patients, mostly lymphoma (n=9) and skin cancer (n=15). severe short stature at birth (compared to normal, smr/smr ratio=5. 4, , symptoms of combined immunodeficiency (compared to asymptomatic, smr=19 (95%ci=8.0-36) vs smr=4.8 (95%ci=2. 3-8.9 ), hirschsprung disease (odds ratio (or) 7.2, 95%ci=1.04-55), pneumonia in the first year of life or recurrently in adulthood (or=7. 6/19, , and autoimmunity (or=39, 95%ci=3.5-430) in adulthood associated with early mortality. in addition, recurrent pneumonia in childhood was associated with the development of lymphoma, while warts and actinic keratosis were associated with the development of skin cancer. birth length standard deviation score correlated significantly with the age at the diagnosis of first malignancy (p=0.0029), lymphoma (p=0.011) and skin cancer (p=0.014), demonstrating that patients with shorter birth length developed malignancies at an earlier age. conclusions: patients with chh have high mortality due to infections and malignancies, but also from lung disease. some subjects present with late-onset immunodeficiency or malignancy without preceding symptoms of immune defect, warranting careful follow-up and screening for cancer even in asymptomatic patients. we provide clinicians with the risk factors for adverse outcomes to assist in management decisions. autoimmune lymphoproliferative syndromes (alps and related disorders) are characterized by insufficient apoptosis due to defects in the fas apoptosis pathway. fadd deficiency (omim 602457) is an autosomal recessive disorder resulting from a mutation in fas-associated protein with death domain (fadd), the adaptor protein involved in fas signaling to caspases 8 and 10. we present a case of fadd deficiency identified by whole exome sequencing with a novel genetic mutation we describe two brothers with recurrent febrile episodes accompanied by seizures and respiratory compromise. the older sibling initially presented with status epilepticus following the measles mumps rubella vaccination later experiencing similar episodes until his demise at 18 months of age. the younger sibling, who is unvaccinated, presented at 14 months with fever, rash, vomiting, and diarrhea. he developed status epilepticus with respiratory depression that required intubation. he also had enlarged cervical lymph nodes that regressed with antibiotics and steroids. he recovered from that episode but subsequently had a series of similar illnesses with fevers, altered mental status and seizures. with the exception of elevated hhv6 igg, extensive infectious workup up in all instances was negative. previously described fadd deficiency patients demonstrate an alps like phenotype with increased circulating double negative t cells, lymphocyte apoptosis defects, elevated fas ligand and il10, encephalopathy, functional asplenism but no splenomegaly or lymphadenopathy. our patients clinical and laboratory findings were similar. he had normal igg and iga, decreased igm, and lack of isohemagglutinins. absolute cd3+ count is elevated, with elevated percent of cd3+ tcr+ cd4-cd8-. normal mitogen and antigen t lymphocyte stimulation, but with defect in pokeweed induced b cell proliferation. fas ligand and il10 level are increased (see table 1 ). no hepatosplenomegaly, but howell jolly bodies were detected in peripheral blood indicating functional hyposplenism. whole-exome sequencing revealed two different genetic alterations in the fadd gene: a maternally inherited nonsense mutation predicted to severely truncate the protein and a paternally inherited missense mutation in codon 105. although this paternal mutation has not been described as pathogenic, a different variant in same nucleotide of fadd has been associated with fadd deficiency (reference1). there are very few cases in the literature of fadd deficiency patients and the overall prognosis is poor compared to classical alps patients, as these patients are at significant risk of deadly sepsis from encapsulated organisms or death from neurologic complications. of the fadd deficiency patients described in the literature, several died prior to 5 years old. while pneumococcal prophylaxis may reduce the risk of sepsis, hematopoietic stem cell transplant has been reported for patients with fadd deficiency (reference2), and is being considered for our patient. rationale: hcuvp is a patient product-introduction program that provides cuvitruâ® (immune globulin subcutaneous [human], 20% solution [ig20gly]) free of charge for the first 4 infusions to eligible patients with primary immunodeficiency disease (pid). using patient data from this ongoing program, our analysis described the clinical characteristics and infusion parameters of pediatric and adolescent patients who were initiated on ig20gly through hcuvp. methods: hcuvp eligibility criteria were: patients aged 2 years old, with a primary icd-10-cm code verifying diagnosis of pid, and no current or prior use of ig20gly at program initiation. data from patients who received the first ig20gly infusion between january 1, 2017, and september 1, 2017 were included. data from patients receiving infusions after october 31, 2017 were censored. descriptive statistics were calculated for patients demographic and clinical characteristics and prescribed and actual infusion characteristics by age group (<18 years and 18 years). results: in total, 817 patients who completed all 4 infusions were included in the analysis, of whom 97 were aged <18 years. among those who previously received immunoglobulin (ig) therapy, a greater percentage of patients aged <18 years were treated with intravenous ig therapy (n=46; 73%) compared with adult patients (n=222; 62%) before initiating ig20gly. nine patients aged <18 years were treatment naã¯ve. the mean infusion volume per site was lower among patients aged <18 years (25 years: 17.9 ml; 611 years: 26.4 ml; and 1217 years: 34.6 ml) than among patients aged 18 years (1864 years: 38.5 ml and 65 years: 38.9 ml). however, the mean infusion rate per site was similar between patients aged <18 years ( xmen disease (x-linked immunodeficency with magnesium defect, epstein-barr virus infection and neoplasia) is a primary immune deficiency caused by mutations in magt1 and characterized by chronic infection with epstein-barr virus (ebv), ebv-driven lymphoma, cd4 t-cell lymphopenia, and dysgammaglobulinemia. magt1 gene codifies to magt1 protein, a mg2+-selective transporter, expressed in the human immune system, specifically in the spleen and the thymus. functional studies have established the key role of magt1 in t cells and natural killer (nk) cell activation. upon cd4+ t-cell receptor stimulation, magt1 mediates a transient mg2+ influx that is necessary for phospholipase c gamma 1 (plcy1) activation, which drives ca2+ rise and downstream signaling. this mg2+ influx also regulates cytotoxic functions of nk and cd8 t cells through nkgd2, reason why these patients have impaired cytolytic responses against ebv. eleven male xmen patients have been described. we present the case of a 1-year old hispanic infant with a pathogenic variant in magt1 gene that clinically manifested with early pneumocystis jirovecii and cytomegalovirus (cmv) interstitial pneumonia, and ebv chronic infection with good response to intravenous immunoglobulins supplementation without hematopoietic stem cell transplantation or gene therapy. laboratory study highlights low levels of nkg2d ligands. the objective of this case report is to broaden the spectrum of clinical presentation of xmen disease, that manifests initially as a combined immune deficiency (cid) and evolved with a favorable course of the disease with intravenous immunoglobulins supplementation therapy and chemoprophylaxis with trimethoprim-sulfamethoxazole. introduction: lysinuric protein intolerance (lpi) is a recessively inherited disorder of the cationic amino acids transporter subunit y+lat1 caused by variants in the slc7a7 gene. the disease is characterized by protein-rich food intolerance has a heterogeneous presentation. the clinical findings are a result of depletion of lysine, ornithine, and arginine. symptoms can include hyperammonemia, failure to thrive, protein aversion, neurologic disease, and lung disease. there is also evidence that inflammatory manifestations are mediated through upregulation of nfb, il1, and tnf that occur independent of intracellular arginine levels and can lead to lifethreatening episodes of hemophagocytic lymphohistiocytosis (hlh). case presentation: a 17-year-old male presented with history of anxiety, depression, eating disorder, delayed puberty and complex partial seizures. due to poor nutrition and failure to thrive, a gastrostomy tube was placed. following commencement of enteral feeds, he presented with altered mental status, bilateral mydriasis, hyperreflexia, and agitation which lead to a picu admission. ammonia peaked as high as 181 î¼mol/l and episodes ceased with cessation of enteral feedings. prior to enteral feeds, he had been self-restricting protein in his diet. biochemical testing was consistent with lpi and illumina next-generation sequencing revealed compound heterozygous variants in slc7a7 (p.s396lfs*122 and p.e465dfs*54). hyperammonemia resolved quickly with cessation of protein intake and high rate dextrose infusion without the need for ammonia scavenging agents. he was subsequently started on proteinrestricted enteral feeds. at diagnosis he did not have any respiratory symptoms, ct scan of chest showed patchy areas of groundglass opacification that was suggestive of early pulmonary alveolar proteinosis (pap). bronchoalveolar lavage demonstrated foamy, cloudy pink fluid and elevated bronchioalveolar macrophages on cell differential. his clinical course and slc7a7 genotype led to suspicion for smoldering hlh. the findings of elevated ferritin, hypertriglyceridemia, decreased fibrinogen, splenomegaly, elevated il-2 receptor, decreased nk cell function, along with hemophagocytosis on bone marrow biopsy confirmed the diagnosis. because of his pap and hlh, in addition to dietary modifications, a trial of il-1 beta inhibition (anakinra) at 3 mg/kg/day was initiated. follow up ct scan of chest 2 months after initiation of anakinra showed complete resolution of pulmonary groundglass opacifications and pap. bone marrow evaluation showed continued hemophagocytosis in spite of the normalization in ferritin, soluble il-2 receptor, nk function, and triglycerides levels. overall, he is significantly improved on daily anakinra and no longer meets criteria for hlh or pap. discussion: recent data has shown in y+lat1 models that thp-1 macrophages and a549 airway epithelial cells upregulate il1 and tnf regardless of intracellular arginine content. this suggests that inflammatory manifestations may continue independent of dietary modifications. we present a 17 year old patient with newly diagnosed lpi who was treated dietary modification and anti-il1 therapy resulting in resolution of hlh and pap. more research is needed to see if long-term il1 blockade that can consistently control both the immunologic and pulmonary manifestations of lpi and positively impact morbidity and mortality. learning objective: recognize that symptoms of bartonella endocarditis and associated complications can share features of certain immunocompromising conditions. case description: an 8-year-old caucasian boy with history of repaired pulmonary atresia and aortic root dilation was diagnosed with pancytopenia and splenomegaly during a brief hospitalization for atypical pneumonia. pancytopenia persisted, splenomegaly worsened, and five months after presentation, he developed hypertension and renal insufficiency. he was diagnosed with hypocomplementemic, diffuse sclerosing and crescentic glomerulonephritis and was started on mycophenolate mofetil with improvement in kidney function and stabilization of cytopenias. as part of a comprehensive immune work-up, alps (autoimmune lymphoproliferative syndrome) panel was sent and demonstrated elevated double-negative t (dnt) cells with 3 out of 4 positive immunologic criteria for alps. neither targeted sequencing for alps and alpslike disorders nor whole exome sequencing revealed pathogenic mutations. by age 10, the patient remained on mycophenolate, but developed failure to thrive, with weight dropping from 37th percentile to less than 3rd percentile. he was hospitalized again for low-grade fever, increased work of breathing, left shoulder pain and fatigue and was found to have right lower lobe pneumonia. pancytopenia worsened, and he was started on cefepime and azithromycin without improvement in symptoms. echocardiogram revealed vegetations in his pulmonary conduit and bilateral branch pulmonary arteries, but multiple blood cultures were negative. upon further history, the patient reported contact with kittens. bartonella henselae titers and polymerase chain reaction (pcr) from blood were sent and were both positive. he completed a 2-week course of gentamicin, 1-month course of ceftriaxone, and was transitioned to doxycycline and rifabutin. after initiating antimicrobial therapy, his weight and energy significantly improved, his blood bartonella pcr became negative, and his splenomegaly resolved. approximately one year later, the patient underwent pulmonary artery conduit replacement and bartonella pcr testing of the tissue specimen was positive. he has had sustained weight increase, resolution of hypocomplementemia and splenomegaly, decrease in dnt cell frequency from >2% to 0.9%, and improvement though not resolution of cytopenias. he currently remains on doxycycline and rifabutin and continues treatment with mycophenolate. discussion: alps is characterized by defective lymphocyte apoptosis and clinical features such as lymphadenopathy, splenomegaly, hepatomegaly, cytopenias, and glomerulonephritis. the hallmark laboratory finding is expansion of dnts. our patient met criteria for a probable alps diagnosis based on the presence of both required criteria (chronic splenomegaly and elevated dnt cells) and secondary additional criteria (typical immunologic findings noted on alps panel). pediatric cases of bartonella henselae endocarditis have been associated with splenomegaly, cytopenias, and glomerulonephritis which mimic many features of monogenic immune dysregulatory disorders. the diagnosis of bartonella endocarditis in our patient therefore raises the question of whether his immunosuppression predisposed him to infection or if his entire clinical presentation can be explained by bartonella endocarditis. physicians taking care of patients with immune dysregulatory disorders should consider bartonella endocarditis in the differential diagnosis of onset or exacerbations of immune dysregulation. rationale: while fever is considered a sign of infection, many individuals with primary immunodeficiency (pi) anecdotally report a lower than normal average body temperature. on immune deficiency foundation (idf) friends and idf pi connect research forum online, pi patients report a diminished fever response even when other signs of infection are present. there is limited knowledge about the average body temperature in persons with pi. however, the implications of missing an infection in those with pi is well established. methods: study investigators partnered with patient investigators to design a prospective cohort study to determine whether body temperature differed between persons living with and without pi. three hundred fifty adults with pi were recruited from idf and one adult household member without pi was also recruited. mckesson digital oral thermometers (model 01-413bgm) were provided and used to record temperatures in all participants three times a day for five consecutive days. descriptive statistics were calculated. median body temperatures were compared between the two cohorts at each time point using mann-whitney test. results: data from 254 households were used for analysis (72.6% participation rate). the pi population was largely female (85.8%) with a median age of 49 years and largely caucasian population (97.6%). the non-pi population was largely male (66.9%) with a median age of 53 years and largely caucasian population (92.9%). pi diagnoses included cvid (74.8%), hypogammaglobulinemia (12.6%), igg subclass deficiency (4.7%), selective iga deficiency (3.1%), specific antibody deficiency (3.1%), agammaglobulinemia (0.4%), chronic granulomatous disease (0.4%), combined immunodeficiency (0.4%), and complement deficiency (0.4%). a total of 123 individuals with pi (48.4%) reported a lower than normal non-sick body temperature, while 108 individuals with pi (42.5%) reported a normal (between 97â°f -99â°f) non-sick body temperature. a total of 172 individuals with pi (67.7%) reported absence of fever with infection, while 50 individuals (19.7%) reported a normal fever response with infection. the median body temperature was significantly higher for the pi patients in the morning, but not evening or bedtime, reading in 4 of the 5 days (monday: pi = 97.5â°f vs. non-pi = 97.2â°f, p = 0.0291; tuesday: pi = 97.4â°f vs. non-pi = 97.2â°f, p = 0.0020; wednesday: pi = 97.5â°f vs. non-pi = 97.2â°f, p = 0.0009; thursday: pi = 97.4â°f vs. non-pi = 97.2â°f, p = 0.0575; friday: pi = 97.4â°f vs. non-pi = 97.2â°f, p= 0.0008). conclusions: despite the limitations of this non-clinical study, individuals with pi are knowledgeable about their conditions and can offer unique insights and direction to researchers. this study demonstrates that collaboration with patient advocacy groups may facilitate patient-centered and patient-driven research with high participation among the target population. introduction: familial mediterranean fever (fmf) is a hereditary condition characterized by recurrent episodes of painful inflammation caused by mutations in the pyrin (mefv) gene. alterations in the mefv gene affect pyrin production leading to recurrent fevers and painful inflammation in the peritoneum, synovium, and pleura. amyloidosis may also develop as a complication. arabic, turkish, armenian, and sephardic jewish populations are most commonly affected. homozygosity for mefv mutations are associated with a more severe course. there is a paucity of information regarding pediatric fmf in the literature. case: we present a case of a 2-year-old male with minor speech delay diagnosed with compound heterozygous fmf. patient was initially referred due to recurrent fevers and infections. at 4 months of age, he was hospitalized with septic shock requiring intubation secondary to adenovirus. at 5 months of age, the patient began to have recurrent fevers every 3 to 4 weeks, leading to multiple blood draws and courses of antibiotics prior to referral. at 11, 12, and 22 months of age, he developed three separate episodes of febrile seizures. a total of 10-15 lifetime episodes of acute otitis media occurred prior to bilateral myringotomy tube placement. four episodes of streptococcus pyogenes pharyngitis confirmed by throat culture preceded tonsillectomy. no oral ulcers, joint pain, or abdominal pain were reported. no other infections such as pneumonia, sinusitis, uti, non-viral gastroenteritis, fungal infections, or skin infections were reported. both parents are ashkenazi jewish and a maternal history of early miscarriage was noted. family history was negative for immunodeficiency, malignancy, and autoimmunity. the patients vital signs and physical exam were unremarkable. serology indicated leukocytosis of 18.53 k/l with elevated monocytes of 1390 cells/l, elevated eosinophils of 1200 cells/l, and slightly elevated cd8 t cell count of 2653 cells/l. neutrophil, cd4 t cell, b cell, nk cell enumeration, and immunoglobulin panel were normal for age. tetanus, diphtheria, rubella, streptococcus pneumoniae, and haemophilus influenzae b titers were protective. genetic analysis identified that the patient was compound heterozygous for the e148q and v726 mutations in the mefv gene. family was instructed to keep a fever diary. colchicine 0.6mg once a day was given initially, then increased to 1.2mg once a day for inadequate response. loose stools were observed while patient was maintaining a lactose free diet so he was switched to colchicine 0.6mg bid with resolution of loose stools. apart from two occasions when his colchicine dose was missed, the patient remained afebrile at his follow up visits. conclusion: we present a pediatric case of compound heterozygous fmf (e148q and v726 mefv mutations) in an otherwise healthy 2-year-old male of ashkenazi jewish background, initially symptomatic at 5 months of age. individuals who are compound heterozygous for the e148q and a second mevf mutation are generally symptomatic, although severity cannot be predicted. additional pediatric research on symptomatic heterozygous and compound heterozygous fmf is recommended. natural killer (nk) cells are innate lymphocytes that play a key role in defense against virally-infected cells and in tumor surveillance. nk cells can be divided in two subsets. the majority of nk cells in peripheral blood expressed intermediate levels of cd56 and are referred to as cd56(dim). these nk cells are responsible for nk cell cytotoxicity. a minor population of nk cells express very high expression of cd56 and are referred to as cd56(bright). these nk cells are responsible for cytokine production and are precursors to cd56(dim) nk cells. a few immunodeficiencies have been described in which there are abnormal nk cell subsets, such as autosomal dominant gata2 deficiency where cd56(bright) nk cells are absent and irf8 where there is a paucity of cd56(dim) nk cells and relative expansion of cd56(bright) nk cells. here we present a patient with an absence in cd56(bright) nk cells secondary to cd27 deficiency. our patient is a 6-year-old african american female born to non-consanguineous parents. the patients past medical history is significant for chronic lung disease secondary to prematurity, recurrent acute otitis media, failure to thrive and congenital hypothyroidism. family history is significant for an older sister that presented at age 3 with ebv-associated hodgkin lymphoma whose treatment was complicated by chronic activated ebv infection and who ultimately underwent hematopoietic stem cell transplantation (hsct). our patient presented with pancytopenia, fever, lymphadenopathy and splenomegaly. she was found to have ebv viremia with greater than 550,000 copies in whole blood by pcr. she was treated with two doses of rituximab followed by etoposide and dexamethasone as a bridge to hsct. whole exome sequencing demonstrated a homozygous mutation in cd27. cd27 is a member of the tumor necrosis factor receptor family and influences the function of t cells, b cells and nk cells. in nk cells, cd27 is primarily expressed in cd56(bright) nk cells. cd27 deficiency is an autosomal recessive disorder associated with persistent symptomatic ebv viremia, including ebv-driven hemophagocytosis and lymphoma, hypogammaglobulonemia and specific antibody deficiency. our patients immune evaluation prior to initiation of chemotherapy and immunosuppression was notable for very elevated igg, iga and igm. despite hypergammaglobulonemia patient had only 3 out of 11 protective titers against streptococcus pneumoniae. the patient had pan-lymphopenia with appropriate percentages of lymphocyte subsets. assessment of her b cell subsets showed a slight increase in the percentage of transitional b cells/plasmablast and a nearly complete absence of cd27-expressing b cells. her nk cell phenotyping demonstrated a complete loss of cd56(bright) nk cells with reduced nk cell cytotoxicity, comparable to what has been previously reported in patients with gata2 deficiency. previous reports of patients with cd27 deficiency denote normal nk cell numbers with normal to moderately reduced nk cell cytotoxicity, however, cd27 deficiency causing a specific loss of the cd56(bright) nk cell subset has not been previously reported. cd27 deficiency should be consider in patients with ebv driven disease and abnormal nk cell studies. introduction/background: the transcription factor ikaros is encoded by the ikzf1 gene and plays a crucial role in lymphopoiesis. somatic, and more recently also germline mutations of ikzf1 are associated with a hematologic malignancies, most notably b-cell precursor acute lymphoblastic leukemia. germline mutation in ikzf1 was first reported as a monogenic cause of human disease characterized by marrow failure and immune deficiency in a single neonate in 2012. subsequently, mutations leading to haploinsufficiency were discovered to underlie a proportion of patients with cvid and low b cell numbers, and dominant-negative mutations have been observed to cause more severe combined immune deficiency phenotypes. at this time, there is very little known regarding allogeneic hematopoietic cell transplantation (hct) outcomes for patients with severe dominant-negative ikzf1 mutations. concerningly, ikaros deficiency has been observed to have a negative impact on graft versus host disease in mouse models. objective: to describe allogeneic stem cell transplant outcomes in patients with the dominant-negative ikaros mutation. methods: we collected transplant data from 4 patients who underwent allogeneic hct at transplant centers around the world. results: patients underwent allogeneic hct using a variety of conditioning regimens. patients received bone marrow (n=3) or cord blood (n=1) grafts from an hla-matched sibling donor (n=1) or single allele hlamismatched unrelated donor (n=3). neutrophil engraftment occurred between day +12 and +51 post-transplant. platelet engraftment occurred between day +8 and +167 except in one patient who did not have return of normal platelet counts due to underlying liver dysfunction. all patients were documented to have greater than 99% whole blood donor chimerism at a median of 28 days (range 12-51 days) following transplant and maintained >95% donor chimerism until last follow-up. only one patient developed grade ii acute gvhd. no patients developed chronic gvhd. one patient died approximately 1 year post transplant related to cryptosporidium cholangitis which existed prior to hct. at the most recent follow up of the 3 surviving patients (range: 0.99-7.2y), ivig had been discontinued, antimicrobial prophylaxis had been stopped, and patients had received routine vaccinations. they all had excellent performance status. conclusions: allogeneic hct may be a safe option to consider for patients with dominant-negative ikaros mutation as there does not appear to be an increased risk of death or gvhd. moreover, 3-out-of-4 of the transplanted patients are alive and well and show no features of the disease. however, because of the limited number of patients evaluated and the retrospective nature of this analysis, our data do not allow firm conclusions to be made, and further studies will be needed to evaluate outcomes in larger cohorts. introduction: when evaluating patients with t-cell lymphopenia, we often are concerned about defects in lymphocyte production and function, especially in the setting of frequent infections. here we outline a case demonstrating t-cell lymphopenia due to increased loss, which should be considered in the differential diagnosis. case report: we report a 13-year-old male who initially presented with recurrent, right-sided pneumonias requiring frequent hospital admissions including severe episodes necessitating intensive care unit admission. his work up for the pneumonias included a bronchoscopy revealing normal anatomy with minimal inflammation, and a chest ct with mild peribronchial wall thickening. as his pulmonary disease progressed, he developed a persistent, productive cough with expectorated mucous plugs that were plastic-like in appearance. while his pulmonary symptoms responded to steroids, his mucous plug production persisted. sputum cultures were intermittently positive, isolating cryptococcus neoformans and aspergillus niger. he underwent vats and wedge biopsy, concerning for recurrent aspiration. an immunologic evaluation initially demonstrated normal t-and b-cell counts, but serial evaluation of his lymphocyte population demonstrated low cd4+ cells (ranging 151-367 cells/cumm), and low normal cd8 cells (ranging 101-177 cells/cumm) with normal b-and nk-cell numbers. further t-cell evaluation revealed normal ratios of naive and memory p o p u l a t i o n s ( c d 4 c d 4 5 r a + 6 1 % , c d 4 c d 4 5 r o + 3 9 % , cd8cd45ra+ 74%, cd8cd45ro 33%), normal trec (7768 copies per 10^6 cd3 cells) and normal thymic emigrants (cd4cd31cd45ra+ : 158, normal 150-1500), indicative of sufficient thymopoiesis. mitogen and antigen stimulation assays demonstrated normal responses to phytohemagglutin, concanavalin a, and pokeweed mitogen, with a low lymphocyte response to candida. he had normal quantitative immunologlobulins, normal diphtheria, tetanus and streptococcus pneumonia titers. his dihydrorhodamine flow cytometry and fish for chromosome 21q11.2 deletion were negative. given normal function and thymic output, his immunologic profile was concerning for t-cell loss. our patient was registered with the undiagnosed disease network, and had a second review of his lung biopsy, concerning for plastic bronchitis. subsequent lymphatic imaging demonstrated abnormal lymphatics within the bilateral clavicular space, right greater than left, with questionable partial thoracic duct, explaining his unilateral symptoms. he was diagnosed with plastic bronchitis secondary to abnormal lymphatic drainage, with lymphatic fluid filling his airways and secondary t-cell loss. discussion: plastic bronchitis is a rare and potentially fatal disorder, seen commonly after the fontan procedure for congenital heart disease. this process has resulted in t-cell loss into the airway and subsequent t-cell lymphopenia. in patients with fontan-related protein losing enteropathy, multiple immune abnormalities have been described including reduced immunoglobulins, lymphopenia, and selective cd4 lymphocyte deficiency. similar findings have been reported in patients with lymphatic malformations. although the impact of t-cell loss on adaptive immunity is not entirely known, there is no indication of increased risk for atypical infections. given his normal mitogen assay, our patient did not start prophylactic antibiotics. he continues to have symptomatic episodes with lymphopenia, but has had no opportunistic infections, and remains stable with an aggressive pulmonary regimen. we conclude by reiterating the importance of considering t-cell loss in patients presenting with lymphopenia, particularly with evidence of normal thymopoeisis and t-cell function. introduction: granulomatous disease (gd) has been described with a variable incidence (8.0-22.0%) in patients with common variable immunodeficiency (cvid). an increase in malignancies has been reported in cvid patient cohorts, particularly for lymphoma, reported in 1.6-8.2% of the cvid patients depending on the cohorts. prior analysis of a cohort of 436 cvid patients included 59 patients with gd (gd+). in these, there was a suggestion of more cases of lymphoma (12.5%) when compared to cases without (gd-) (5.0%) although the difference was not statistically significant (p=.07). objectives: compare the frequency of lymphoma in gd+ and gd-patients in the cvid patient cohort from the usidnet registry. methods: we submitted a query to the usidnet registry requesting deidentified data for patients with the diagnosis of cvid, through august 2018. statistical analysis was performed on spss, with comparisons done with pearson chi-square or fisher's exact test, depending on the sample sizes, using an alpha level of .05. results: a cohort of 1395 cvid patients from the usidnet registry was analyzed. ninety-one patients (6.5%) were gd+. overall, 152 patients (10.9%) had a malignancy diagnosis, 47 of these (3.4%) with lymphoma. lymphoma was present in 6/91 gd+ patients (6.6%) versus 41/1304 gdpatients (3.1%) (p=.12). overall malignancy was present in 15/91 gd+ (16.5%) versus 137/1304 (10.5%) (p=.08). discussion: in the cohort of 1395 cvid patients from the usidnet registry, we found a frequency of lymphoma of 3.4%, which is in the range of previously described cohorts. the frequency of lymphoma was 6.6% in patients with gd, higher than the 3.1% frequency for gd-patients, but these differences were not statistically significant. our identified frequency of lymphoma in gd+ patients was lower than the one previously identified in the 436 cvid patient cohort, but with similar proportional differences between gd+ and gd-patients. despite no statistical significance, the frequency of lymphoma, as shown here and elsewhere, was higher in cvid patients gd+ than gd-in both studies, with no full understanding of this increased risk of lymphoma. expanding this analysis to larger groups of cvid patients may help to confirm, or deny a more robust association, which may have a meaningful impact in the outcomes of this particular population. introduction: patients with refractory pericarditis have been treated with intravenous immunoglobulin (ivig) or interleukin 1 receptor antagonist (anakinra) with limited and transient benefit. separate or combined therapy with subcutaneous immunoglobulin (scig) and interleukin (il) 1 inhibitor (rilonacept) for refractory pericarditis in a cohort of patients has not been previously described. case descriptions: 4 patients were referred for recurrent pericarditis refractory to traditional therapies at ages ranging from 16 to 54 years. they all had multiple serious sequelae of their pericarditis and abnormal immune parameters including hypogammaglobulinemia, poor responses to vaccines, poor mitogen induced lymphocyte proliferation, and/or b cell lymphopenia. the patients had varied past medical histories and associated conditions. patients were started on ig, with some initiated on ivig, though all were transitioned to hyaluronidase-facilitated scig (hyqvia). patients were then started on either anakinra or rilonacept with 3 patients continuing on rilonacept and 1 remaining on anakinra. all patients had complete or near complete resolution of their pericarditis on dual therapy for greater than 1 year. the markedly elevated il1 prior to therapy seen in all of the patients normalized post-therapy. some patients had elevated il6 prior to therapy that also improved post-therapy. 1 patient who has also been diagnosed with familial mediterranean fever (fmf) has stopped both therapies for greater than 1 year with no further episodes of her pericarditis. discussion: 4 patients with recurrent refractory pericarditis and signs of immunodeficiency and autoinflammatory disease on laboratory testing responded to dual therapy with hyqvia and rilonacept or anakinra resulting in resolution of pericarditis. inflammasome and immune abnormalities may be implicated or associated with recurrent pericarditis and may respond to targeted therapies. chief, laboratory of clinical immunology and microbiology, idgs, dir, niaid, nih, bethesda, md, usa hypomorphic recombination activating gene 1 (rag1) mutations result in residual t-and b-cell development in both humans and mice and have been found in patients presenting with delayed-onset combined immune deficiency with granulomas and/or autoimmunity (cid-g/ai). recent studies have shed light on how hypomorphic rag1 mutations alter the primary repertoire of t and b cells, but less is known about their effect on immune dysregulation in targeted organs. in order to investigate the role of these mutations in determining intestinal disease, we set out to evaluate gut immunity and microbiota interplay in rag1 mutant hypomorphic mice. we evaluated two mouse models carrying homozygous rag1 mutations (r972q and r972w), corresponding to human mutations (r975q and r975w, respectively) described in patients with cid-g/ai. both mutations fall in the coding flanksensitive region of the rag1 c-terminal domain. on the basis of aminoacid properties and in vitro studies, the r972q mutation has demonstrated a moderate effect on rag1 protein stability while the r972w mutation resulted highly disruptive. analysis of intestinal pathology in rag1 mutant mice (niaid animal protocol lcim 6e) revealed different degrees of spontaneous colitis, with the most severe inflammatory infiltrate observed in mice carrying the most disruptive mutation, r972w. colonic inflammation was characterized by crypt elongation, epithelial hyperplasia, and an abundant inflammatory infiltrate extending to the colonic lamina propria, with occasional crypt abscesses. a significant increase in activated cd44hicd62lcd4+ t cells expressing the gut homing receptor 47 was observed in mesenteric lymph nodes (mlns) of both mutant strains, and was especially prominent in r972w mutant mice. additionally, the proportion of mln cd4+ t regulatory (treg) cells was increased in both mouse models. finally, mln of mutant mice contained a high number of myeloid cells (cd11b+ ) along with a decreased number of b220+ b cells, and these abnormalities were also more prominent in r972w than in r972q mice. in summary, we have shown that rag1 mutant hypomorphic mice present with different degrees of inflammatory bowel disease, with the mouse model carrying the most disruptive mutation presenting with the most severe phenotype. we are currently performing studies to evaluate the impact of rag1 mutations on microbiome composition and diversity in these mouse models of cid-g/ai. background: hypogammaglobulinemia or low serum immunoglobulin g (igg) levels either inherited (primary) or acquired (secondary) is associa t e d w i t h i n c r e a s e d i n f e c t i o n r a t e s . p r i m a r y ( 1â°) hypogammaglobluinemia can be caused by many primary immune deficiencies (pid) including combined variable immune deficiency (cvid), while secondary (2â°) hypogammaglobluinemia can be caused by many acquired conditions such as lymphomas, leukemias, or chemotherapies and other immunosuppressive drugs. immunoglobulin replacement therapy (irt) has been the mainstay of treatment in patients with hypogammaglobulinemia by reducing infection through replenishing the quantitative igg. there are other applications of ig therapy such as in autoimmune diseases, where the mechanism of action is thought to be ig mediated immunomodulation. innate immune cells have shown to be involved in such mechanism, but whether irt modulates adaptive immune cells in patients with hypogammaglobulinemia is not well known. hypothesis: irt has an immunomodulatory effect on t-cell function and proliferation in patients with hypogammaglobulinemia. methods: blood from thirty patients with 1â°(n=12) or 2â°(n=18) hypogammaglobulinemia recruited from the immunodeficiency clinic at the ottawa hospital was drawn for peripheral blood mononuclear cell (pbmc) isolation, before starting irt and minimum 8 weeks after starting irt. data regarding igg level, number and type of infections after receiving irt was collected. pbmcs were analyzed using flow cytometry for quantitation of t-cell subset. cultured and anti-cd3/cd28 stimulated pbmc were also analyzed for extracellular and intracellular cytokine production, measured by e l i s a a n d f l o w c y t o m e t r y, r e s p e c t i v e l y. c o m b i n e d cytomegalovirus, epstein-barr virus and influenza virus (cef) peptides were used to study specific t-cell responses. anti-cd3/ cd28 stimulated pbmc were used for celltrace t-cell proliferation a s s a y s . d a t a w a s g r o u p e d b a s e d o n n a t u r e o f hypogammaglobulinemia i.e. 1â°or 2â°. results were compared between before and after irt using wilcoxon matched-pairs signed rank test. results: irt was not found to significantly alter proportion of treg, cd4+, or cd8+ t-cell populations or activation state as measured by cd45ra/r0 expression. however, irt was found to significantly increase expression of intracellular ifn-y in cd4+ and cd8+ t-cells post-cd3/cd28 stimulation in 2â°(p = 0.007), but not in 1â°h ypogammaglobulinemia patients. there was no change in extracellular il-10 and il-17 cytokine production in both groups. in contrast, cd8+ tcells in 1â°hypogammaglobulinemia patients showed significantly higher expression of intracellular ifn-y and tnf-a post-cef viral peptide stimulation (p = 0.027). cd3+ and cd8+ t-cell proliferation after cd3/cd28 stimulation was found to be decreased after irt for both groups (p = 0.025 & p = 0.049). conclusions: our results suggest that irt can alter cd4+ and cd8+ t-cell function with differential effect in patients with 1â°o r 2â°hypogammaglobulinemia in addition to replenishing serum igg level. more experiments assessing cytotoxicity of t-cells will be conducted to further study t-cell subset function as well as bcell function. these laboratory results will be analyzed for association with clinical outcomes. uploaded file(s) uploads background: severe congenital neutropenia (scn) is a rare immunodeficiency disorder characterised by the extremely low absolute neutrophils count (anc) less than 0.5x109/l. the clinical feature of scn is recurrent bacterial infections and the patients the risk of leukemia development. the incidence of scn is estimated to be 1 in 200 000 individuals. mutations in more than 20 genes have been described causing scn and it is either recessive, dominant or x-linked inheritance. case presentation: we described an 11 years old malaysian girl who presented with recurrent abscesses over the whole part of the body, recurrent oral candidiasis, growth failure and recurrent pneumonias since 4 months old. she also had history of a few episodes of acute tonsillitis, chronic suppurative otitis media and herpes zoster infections. throughout her age, she had persistent neutropenia less than 0.5x109/l but in few occasions, her anc elevated up to more than 1.0x109/l . she was treated as autoimmune neutropenia, respectively due to few positive results of autoimmunity workout such as antinuclear antibodies (ana) and double stranded dna (dsdna) but eventually later to be negative. later at the age 9 years old, whole exome sequencing was performed and confirmed by sanger s e q u e n c i n g , f o u n d a h e t e r o z y g o u s v a r i a n t i n e l a n e gene(c.640g>t; p.gly214ter), an autosomal dominant which was described to cause scn. both parents do not carry this mutation, hence, it is a de novo mutation. currently, she had few on and off recurrent infections. despite that, she is relatively well and on prophylaxis antibiotic. conclusion: to our knowledge, we report for the first time a malaysian girl with scn, with confirmed mutational analysis of the elane gene. the delayed diagnosis might be due to the insufficient awareness of the phenotypic presentation of this rare disease. moreover, the genetic analysis is not available in malaysia and need to be done outside of the country. this case demonstrates the importance of the genetic analysis which may help in improving the diagnosis and management of the patient. (69) submission id#600360 professor of paediatrics and immunology, university college london; great ormond street hospital nhs trust; orchard therapeutics, london, uk background: ada-scid is a rare genetic disorder which causes severe combined immunodeficiency. historically, ada-scid has been treated using enzyme replacement therapy (ert) followed by allogeneic hematopoietic stem cell (hsc) transplant (hsct) from a matched related donor (mrd) or, if none is identified, a non-mrd (matched/mismatched unrelated or mismatched related donor). we developed a self-inactivating lentiviral vector (lv), in which a codon optimized human ada cdna is driven by the short form of the elongation factor-1alpha (efs) promoter (efs-ada lv). the drug product (otl-101), composed of autologous hscs transduced ex vivo with the efs-ada lv, was evaluated in a prospective, historically-controlled phase i/ii clinical trial in ada-scid pediatric subjects. we report safety and efficacy at 24 months in 20 ada-scid subjects treated with lentiviral gene therapy (gt) compared to a historical cohort of 26 ada-scid patients treated with hsct. methods: twenty subjects (9 male, 11 female; 4 mo 4.3 yrs) were treated with gt. autologous cd34+ hscs were isolated from bone marrow and pre-stimulated with cytokines before transduction with efs-ada lv. busulfan was administered at a single dose (4 mg/kg) prior to infusion of otl-101. the control group included 26 patients (0.2 mo 9.8 yrs) treated with allogeneic hsct (mrds n=12, non-mrds n=14) at great ormond street hospital, uk (n=16) or duke university childrens hospital, usa (n=10) between 20002016. results: at 24 months, overall survival (os) and event-free survival (evfs), defined as survival in the absence of ert reinstitution or rescue allogeneic hsct) were statistically significantly higher in the gt group compared with the hsct group (table) . successful engraftment of genetically modified hsc was observed in all gt subjects at 6 months, which persisted over 24 months, based on vector gene marking in granulocytes (median 0.085 copies/cell [range 0.04-2.50] at 24 months) and peripheral blood mononuclear cells (median 0.843 copies/cell [range 0.13-1.86] at 24 months), and was associated with increased red blood cell ada enzyme activity and metabolic detoxification from deoxyadenosine nucleotides. over 24 months, none of the gt subjects required peg-ada ert reinstitution and 90% were able to stop receiving immunoglobin replacement therapy (igrt), whereas 38% hsct patients required rescue hsct or reinstitution of peg-ada ert, and 52% were able to stop receiving igrt (table) . nine subjects in the gt group experienced a serious adverse event (sae), most frequently infections and gastrointestinal events; only one was considered treatment-related. in the gt group, there were no events of autoimmunity during the study. due to the autologous nature of the product, there was no incidence of graft vs host disease (gvhd) in the gt group; whereas 5 patients in the hsct group experienced acute gvhd and 3 experienced chronic gvhd events, one of whom died. conclusions: treatment with lentiviral gt for ada-scid is well tolerated and has a favorable benefit-risk profile at 24 months based on sustained gene correction and restoration of immune function, as well as improved os and evfs compared with hsct (mrd or non-mrd) at 24 months. background: ada-scid is a rare genetic disorder that causes severe combined immunodeficiency, with minimal or absent b cell function. prior to, and often after, treatment with allogeneic hematopoietic stem cell (hsc) transplant (hsct) or autologous ex vivo hsc gene therapy (gt), patients are managed with enzyme replacement therapy (ert) and immunoglobulin (ig) replacement therapy (igrt). we evaluated a gt treatment with autologous hscs transduced ex vivo with a self-inactivating lentiviral vector (lv), in which a codon optimized human ada cdna is driven by an internal short form of the elongation factor-1alpha (efs) promoter ("efs-ada lv"). at 24 months follow-up, 20 pediatric ada-scid subjects treated with gt were compared to a historical cohort of 26 ada-scid patients treated with hsct. here, we report on b cell reconstitution in these cohorts. methods: twenty subjects (9 male, 11 female) aged 4 mo -4.3 yrs received gt. autologous cd34+ hscs were isolated from bone marrow and pre-stimulated with cytokines before transduction with efs-ada lv. genetically modified cells were administered after conditioning with single dose busulfan (4 mg/ kg). the control group included 26 patients aged 0.2 mo to 9.8 yrs treated with hsct at great ormond street hospital (uk) (n=16) or duke university children's hospital (us) (n=10) between 2000 -2016. the hsct patients received an allogeneic transplant from matched related donors (mrds) (n=12) or non-mrds (n=14). subjects continued to receive igrt post-gt until a clinical decision was made to stop, factoring in b cell reconstitution, general medical condition and seasonal infections. results: by month 12, in the gt group, 45% had stopped treatment with igrt compared to 38% in the hsct group overall. by months 18 and 24, higher proportions of gt-treated subjects had stopped igrt (70% and 90%, respectively) compared with mrd hsct patients (55% and 70%, respectively) and non-mrd hsct patients (42% at both timepoints) (table) . in the gt group, vector gene marking was detectable in peripheral blood mononuclear cells within 3 months and persisted at 24 months post-infusion (median 0.843 copies/cell [range 0.13-1.86]), suggesting successful gene modification. as evidence of b cell reconstitution, iga and igm levels in peripheral blood sera more than doubled by 18 months, from 18.5 mg/dl (range 8 to 95) to 48.0 mg/dl (range 20 to 110) and 32.5 mg/dl (range 16 to 107) to 69.0 mg/dl (range 20 to 180), respectively. additionally, antibody response following tetanus vaccination, was evaluated in 3 subjects. all 3 subjects mounted a protective response to the vaccine (median antibody response 3.2 iu/ml [range 0.1 to 3.5]), based on a normal threshold of 0.01 iu/ml (hammarlund clin infect dis 2016) and a laboratory reference range (0.10 to 2.9 iu/ml). conclusions: gt with autologous hscs transduced ex vivo with efs-ada lv resulted in b cell reconstitution, as evidenced by doubled iga and igm production at 18 months, cessation of igrt in 90% of patients by 24 months, and protective specific antibody responses to tetanus vaccine in patients that were evaluated. background: x-linked chronic granulomatous disease (xcgd) results from mutations in cybb encoding the gp91phox subunit of phagocyte nadph-oxidase. attempts to treat xcgd with gene therapy (gt) using transduced autologous hematopoietic stem cells (hsc) transduced ex vivo with a gammaretroviral vector have met with limited efficacy due to transient engraftment of gene corrected hscs, gene silencing, and vector insertion-mediated activation of oncogenes leading to myelodysplasia. we developed a novel self-inactivating (sin) lentiviral vector (g1xcgd lv) with a chimeric cathepsin g/cfes myeloid-specific promoter driving gp91phox expression from a codon optimized cdna. following transplant of g1xcgd lv ex vivo transduced autologous hscs into busulfanconditioned xcgd patients, there was long-term restoration of oxidase activity in peripheral blood polymorphonuclear neutrophils (pmn) at 12 months in 6 of 9 severely affected xcgd patients without evidence of genotoxicity. here we present data about the multiple assays used to assess quality and quantity of restoration of pmn oxidase activity. methods: similar trials of gt with g1xcgd lv were initiated in the uk (n=3, plus 1 compassionate use patient) and usa (n=5). all patients had histories of inflammatory disease and severe, persistent infections (some non-responsive to conventional therapy at time of gt). g-csf plus plerixafor-mobilized cd34+ hscs were transduced with ex vivo g1xcgdlv. subjects received myeloablative conditioning with singleagent busulfan, targeted to net area-under-the-curve of 70,000 ng/ml*hr. freshly prepared or cryopreserved quality-tested genetically-modified hsc, manufactured on-site, were administered intravenously. pmn oxidase activity post-gt was assessed by p-nitroblue tetrazolium (nbt) reduction, dihydrorhodamine (dhr) flow cytometry assay, and quantitative ferricytochrome c assay (ferric) measurement of superoxide generation. results: we report results for 7 patients (aged 2-27 years) with 1-2.5 years of follow-up; two additional patients were treated but died within three months of gt from complications deemed related to pre-existing diseaserelated co-morbidities (severe pulmonary disease and anti-platelet antibodies). within 1 month post-gt, oxidase (+) pmn were present in peripheral blood based on nbt testing and dhr flow cytometry. expression of the corrective transgene was confirmed by flow cytometry using antibody detection of gp91phox. quantitative biochemical measurements of oxidase activity were also confirmed in some samples using the ferric assay, demonstrating quantitative levels of superoxide production per corrected cell that were within the normal range. functional testing of oxidase burst activity using dhr fluorescent assays was applied serially to follow levels of corrected pmn where oxidase activity per corrected cell also were in the normal range. all patients had >15% pmn dhr+ within one month, which remained stable for most patients over the follow-up period ( figure) . follow-up demonstrated sustained stable persistence of 12-46% oxidase burst positive neutrophils in 6 of 7 surviving subjects at 12 months, with restoration to clinically beneficial levels (defined as 10% of pmn being dhr+) in these patients as of december 2018. conclusion: these results demonstrate corrected pmn function within 1 month in x-cgd patients treated with autologous gt. pmn oxidase activity was sustained at levels which restore biochemical function and provide clinically beneficial levels of immunity for 12 months in 6/7 patients. the formulation for igsc 20% was developed based on the knowledge acquired from the formulation of grifols currently licensed 10% immune globulin (human), gamunexâ®-c; however, the protein concentration was increased from 10% to 20% to facilitate efficient subcutaneous administration. gamunex-c has an extensive record of safety and tolerability when administered intravenously and subcutaneously for greater than 15 years in diverse patient populations. the igsc 20% manufacturing process employs the same purification steps as gamunex-c and was demonstrated to be robust and to provide an igg product with the required potency, purity, and quality. the formulation excipient characteristics and compatibility with the drug product have been well established. glycine has been an excipient of intramuscular immune globulin (human) for fifty years and intravenous immune globulin (igiv) for over twenty years. the igsc 20% formulation has low buffering capacity, and a low ph was selected to achieve a product with low aggregates, low fragments and viscosity suitable for subcutaneous administration. to improve visual clarity, the igsc 20% formulation contains a small amount of polysorbate 80 (ps80), which is widely used in biopharmaceutical products. subcutaneous administration of the igsc 20% formulation has been well tolerated in clinical studies. objectives: the goal was to provide the pid population with a new 20% immunoglobulin liquid product for subcutaneous administration (igsc 20%). methods: igsc 20% is manufactured using the current manufacturing process for gamunex-c, followed by an additional concentration step so that the product can be formulated at a higher protein concentration. igsc 20% and gamunex-c batches were produced at full industrial scale and then subjected to a series of analytical testing including assessment of purity, composition and neutralizing activity. results: the igsc 20% and gamunex-c manufacturing processes and formulations have preserved the igg integrity, molecular characteristics and potency. the manufacturing processes have eliminated lipids, alcohols, and acetate and coagulation factor impurities, including fxia, which were undetectable by either specific or global methods. the igsc 20% and gamunex-c batches were 100% gamma globulin by agarose membrane electrophoresis, and have a subclass distribution similar to normal plasma and acceptable specific antibody content. igsc 20% was shown to be primarily monomer plus dimer igg (99â±1%) with minimal aggregate or fragment, which confirms that appropriately gentle processing conditions were used during the concentration of 10% igg solutions to 20% igg. conclusions: igsc 20% is a highly concentrated igg solution with characteristics comparable to gamunex-c, but with twice the igg concentration in order to facilitate subcutaneous administration with reduced volumes and shorter infusion times. analytical testing demonstrates suitable potency, purity, and neutralizing activity for a number of specific antigens. funding: this study was funded and conducted by grifols, a manufacturer of 20% immunoglobulin for subcutaneous administration. disclosure: all authors are employees of grifols. frequent respiratory tract infections and seizures cause recurrent hospitalizations in these children and are typically considered a result of neurological impairment and poor airway clearance. evaluation of these patients for immunodeficiency is not a common clinical practice. here we report combined immune deficiency in 2 patients with mds and recurrent respiratory tract infections. case presentation case 1: a boy with mds was initially referred at age 2 months for an abnormal newborn screen with low t cell receptor excision circles (trec) for severe combined immunodeficiency (scid). initial evaluation revealed moderate cd3+ and cd4+ t cell lymphopenia (figure 1). initial immunoglobulins levels were normal. he was placed on antiseizure medications. he later developed recurrent and severe respiratory tract infections starting in infancy. at 12 months of age, he developed hypogammaglobulinemia ( figure 2 ). in addition, t cell counts progressively decreased and stayed around 600 cells/ul. immunoglobulin replacement therapy started at 18 months of age. hospitalizations due to respiratory tract infections significantly decreased. case 2: a 3-year-old boy with mds had recurrent bacterial and viral respiratory infections which required numerous hospitalizations including intensive care unit stays. newborn screening for scid was negative. he had been on anti-seizure medications. immunologic evaluation at 3 years of age revealed low total cd3+ cells and cd8+ t cells (cd3+: 1284cells/ul[normal range 1400-3700cells/ul], cd8+:278cells/ ul[normal range 490-1300cells/ul]), hypogammaglobinemia (igg: 252mg/dl[normal range 453-916mg/dl]), and non-protective igg levels to tetanus, varicella and pneumococcus serotypes. immunoglobulin replacement therapy started at 3 years of age which resulted in reduced frequency and severity of respiratory infections, and improved quality of life. discussions: t cell lymphopenia and hypogammaglobulinemia were seen in both our cases of miller-dieker syndrome. to our knowledge, immune deficiency has never been reported in mds. one of our cases suggests that low t cell counts may start as early as at birth and may be detected by newborn screening. hypogammaglobulinemia may be primary or secondary due to antiepileptics. both children had reduced frequency and severity of respiratory infections and improved quality of life after immunoglobulin replacement highlighting the importance of screening and early management of immunodeficiency. conclusion: miller-dieker syndrome is likely another syndromic primary immune deficiency disorder. a high index of suspicion with early screening and management of immunodeficiency may be beneficial for children with miller-dieker syndrome. uploaded file(s) uploads this prospective, multi-center, open-label study assessed the pharmacokinetic (pk), safety, and tolerability of immune globulin subcutaneous (human), 20% caprylate/chromatography purified (igsc 20%) in subjects with primary immunodeficiency (pi). the objectives were to determine a weekly subcutaneous (sc) dose of igsc 20% that is noninferior to the intravenous (iv) dose of immune globulin injection (human), 10% caprylate/chromatography purified (igiv-c 10%) and to determine the steady state trough igg levels after igsc 20% and igiv-c 10% infusions. there were 3 possible phases. if not on a qualifying igg regimen at enrollment, subjects (n=44) were required to enter the run-in phase, receiving igiv-c 10% to achieve steady-state before entering the iv phase to determine steady-state area-under-the-curve (auc) of iv infusions. subjects with a qualifying igiv-c 10% regimen (300-800 mg/kg) (n=9) directly entered the iv phase for steady-state iv pk assessments. upon completion of the iv pk assessments subjects entered the sc phase, receiving weekly doses of igsc 20% for up to24 weeks, with steady-state auc determined at the 13th dose. igsc 20% was not associated with any reports of serious local infusion site reactions (isrs). the majority of local isrs were mild-to-moderate. igsc 20% (at a dose conversion factor of 1.37) provided equivalent exposure to igiv-c 10% as assessed by steady-state auc0-7 days, with 33% higher mean igg trough values, lower fluctuations in igg concentrations and the flexibility of at home administration. igsc 20% was well tolerated with a safety profile comparable to igiv-c 10%. clinicaltrials.gov identifier: nct02604810 disclosure: kecia courtney, elsa mondou, and jiang lin are employees of grifols, a manufacturer of igsc 20%. grifols is the sponsor of this study. background: in 2014 two reports described the deficiency of adenosine deaminase 2 (dada2) as early-onset lacunar strokes, intermittent fevers, livedoid rash, and early onset polyarteritis nodosa (pan). since these first reports, the clinical spectrum has dramatically expanded to include antibody deficiency, liver disease, vasculopathy, pure red cell aplasia, cytopenias, and lymphoproliferative disease. methods: forty-two patients were enrolled in an irb approved study at the nih. sequencing of ada2, the gene encoding adenosine deaminase 2 (ada2), was performed in all patients. information was obtained by chart review of all clinical, serologic, and radiographic testing. results: all 42 patients had germline biallelic loss of function mutations in ada2, leading to absent or significantly decreased protein expression and function of ada2. the cohort comprises 20 females (48%) and 22 males (52%). there were 6 sibling pairs and 2 families with 3 affected individuals. twenty-seven patients had a history of at least one ischemic stroke and 6 experienced a hemorrhagic stroke. the average age at the time of first stroke is 5.6 years (range 4 months -24 years), and the average number of strokes is 3 (range 1-11). no new strokes have occurred in patients on anti-tnf therapy. skin manifestations occurred in 86% of patients and include livedo (74%), cutaneous vasculitis resembling pan (64%), and raynauds (19%). hepatomegaly (43%) and splenomegaly (55%) were also notable. portal hypertension was observed in 6 (14%) patients, with 1 patient requiring a spleno-renal shunt for a massive variceal bleed. abdominal mra revealed arteritis and aneurysm in 7/13 patients evaluated; 3 patients developed bowel necrosis. peripheral vasculopathy was seen in 3 patients, with one requiring amputation of gangrenous digits. the most common immune abnormality seen in this cohort is hypogammaglobulinemia (62%); 20 patients have low igg, 20 patients have low igm, and 14 patients have low iga. ten of these patients are on immunoglobulin replacement. specific antibody responses to vaccines were inadequate in 5/16 patients challenged. lymphocyte phenotyping revealed decreased class-switched memory b cells in 23/32 patients (72%) tested. however, there was no relationship between absolute number of class switched memory b cells and hypogammaglobulinemia or infection frequency. hematologic abnormalities include transfusion depended anemia (7%), neutropenia (7%), lymphopenia (5%), and thrombocytopenia (2%). seven patients developed pancytopenia, 1 presented with pure red cell aplasia, and 1 developed aplastic anemia. three patients have undergone bone marrow transplant, with two of those patients requiring a second transplant for graft failure. conclusions: the spectrum of dada2 has expanded from strokes, intermittent fever, and cutaneous manifestations to include portal and systemic hypertension, immune deficiency, cytopenias, vascular abnormalities, and bone marrow failure. while initiation of anti-tnf therapy improves inflammatory markers, and no new strokes have occurred while on therapy, cytopenias do not seem to improve. bone marrow transplantation should be considered in patients with findings of bone marrow failure, although transplant of our patients has been complicated by immune mediated neutropenia. disease manifestations are heterogenous, making a comprehensive evaluation critical to our understanding of this disease. given the increase in neonatal diagnosis of athymia, clinical care is provided by the referring medical centers prior to rvt-802 implantation and patients return to the referring centers earlier after rvt-802. this creates the need for clear, concise guidelines for the care of these patients. primary goals of pre-transplantation clinical care are (1) management of pre-existing medical needs such as feeding difficulties, airway obstruction, congenital cardiac defects and developmental disabilities; (2) management of symptoms related to oligoclonal recipient t cell expansion (autologous gvhd/atypical complete digeorge anomaly) and (3) prevention of infections. most deaths in the pre and early post-transplantation period are secondary to pre-existing infections. necessary surgical and medical procedures (ie cardiac surgery, hearing aids) should not be delayed. for the first 6 to 9 months after rvt802, patients have profoundly low naã¯ve t cell numbers and may require immunosuppression to prevent rejection of rvt-802 by oligoclonal recipient t cells. immunosuppression needs to be closely monitored and titrated for desired effect while minimizing side effects such as renal toxicity, electrolyte abnormalities and hypertension. t cell counts should be performed every 3 months and are used to guide weaning of immunosuppression. most patients with successful transplants develop greater than 100/mm3 naã¯ve t cells by 12 months post rvt-802. infection prevention, clinical stability and optimal nutrition are critical for lasting engraftment. clinical guidelines have been developed to address immunosuppression, management of autologous gvhd symptoms (gut, skin and liver), preservation of renal function, and developmental considerations. after the development of naã¯ve t cells, patients should continue to be monitored regularly by an immunologist. patients may develop autoimmune complications such as thyroid disease and transient cytopenias. while risk of complications related to viral infections is greatly decreased after development of naã¯ve t cells, patients with comorbidities (central venous access device dependence, tracheostomy, chronic lung disease) continue to require complex care from multidisciplinary teams. medical conditions associated with athymia but not alleviated by thymus transplantation, such as hypoparathyroidism or cardiac defects, may require lifelong medical care. lastly, patients must be evaluated for readiness for killed and live vaccines. transplant outcomes are influenced by the clinical condition at the time of rvt-802 implantation and optimization of immunosuppression, nutrition and clinical stability in the first 9 months following rvt-802. clinical care that maintains a well-nourished, clinically stable, infection free patient yields the best chance for successful t cell development. guidance documents supporting these goals ensure patients are best prepared to receive rvt-802 and develop long lasting thymic function. hemophagocytic lymphohistiocytosis (hlh) is a life-threatening disease of immune dysregulation characterized by unchecked inflammatory responses leading to end-organ dysfunction. primary hlh results from inherited mutations that impair capacity for immune regulation whereas secondary hlh arises from inappropriate response to an immune stimulus such as infection, malignancy or autoimmunity. we report a 9-monthold male who presented with symptoms of hlh as an initial manifestation of congenital disorder of glycosylation (cdg) due to mutations in the gene component of oligomeric golgi complex 4 (cog4) resulting in cog4-cdg (cdg-iij). a 9-month-old male with history of mild motor delay presented with 3 days of fever, vomiting, and diarrhea. initial evaluation identified highly elevated ferritin and triglycerides, transaminitis, coagulopathy, and hyperammonemia. he subsequently developed generalized seizures. liver and bone marrow biopsies demonstrated erythrophagocytosis consistent with hlh. immunologic evaluation was notable for mild hypogammaglobulinemia, neutropenia, thrombocytopenia, and anemia. serum cd25 levels and nk functional studies were later found to be normal. the patient was initially treated with ammonia-scavenger therapy and fresh frozen plasma (ffp) for coagulopathy with subsequent intravenous immunoglobulin and dexamethasone several days later. within 24 hours after starting ffp, the patients ferritin level declined sharply. hyperammonemia and transaminitis also resolved, and his fever curve improved. additional immunosuppression was considered, but not initiated due to the patients ongoing clinical improvement. over the next 3 months, the patient experienced two further acute episodes of fever, liver dysfunction, coagulopathy, and sepsis physiology. the second episode was successfully treated with ffp, though no clear infectious trigger was identified. the third episode occurred 4 days after routine vaccinations. the patient had prolonged hypotension requiring ionotropic support that resolved after receiving daily ffp, and hypoxia with pleural effusions that resolved after a single treatment with protein c concentrate. as the patient had met 5/8 clinical diagnostic criteria for hlh, but also had a history of hyperammonemia, he underwent concurrent biochemical and genetic evaluation for both primary hlh and inborn errors of metabolism. whole exome sequencing identified compound heterozygous mutations in cog4, part of an oligomeric protein complex involved in golgi apparatus structure and function. cog4 mutations have previously been reported in two patients with autosomal recessive cog4-cdg (cdg-iij), who were described to have similar clinical symptoms of hypotonia, seizures, coagulopathy, and liver dysfunction, as well as recurrent infections. subsequent immune phenotyping while the patient was healthy was notable for slightly low numbers of nk cells, but normal cd107a mobilization and perforin/granzyme b expression in vitro. our patient represents a novel presentation of cdg due to cog4 defect with associated immune dysfunction manifesting as recurrent episodes of inflammatory crisis with features of hlh. cdg and inborn errors of metabolism should be considered during diagnostic evaluation for patients with hlh symptoms, as cdg patients may develop acute episodes of severe inflammation, in the absence of cellular regulatory defects, for which ffp and protein c concentrate may have therapeutic value. of the 14 deaths with identifiable causes, 10 (71%) were related to infections. the rate of death per person-year was 0.044. the most common autoimmunity-related complication was sweets syndrome, seen in 29 patients (39%) with anti-ifn-g autoantibodies. sixteen of those patients (55%) had recurring sweets syndrome. additionally, 14 patients (19%) developed lymphatic obstruction, which continued to recur in 12 patients (86%). seven patients (9%) in this study did not have anti-ifn-g autoantibodies. the median [iqr] age of autoantibody-negative patients was 38 [27, 54] years and 3 patients (43%) were female. none of the autoantibody-negative patients developed new infections during follow-up. at the end of the follow-up period, none of the patients had active/progressive disease and 2 patients (29%) had died. conclusions: ninety-one percent of hiv uninfected thai patients with disseminated ntm infection with or without other opportunistic infections had detectable anti-ifn-g autoantibodies. about one third of patients with autoantibodies to ifn-g had recurrent infections during follow-up. after approximately 7 years of follow-up, 55% of patients with anti-ifn-g autoantibodies had inactive disease following multi-drug antibiotic therapy while 8% had active/progressive disease and 24% had died. patients with anti-ifn-g autoantibodies are at risk for recurrent infections and autoimmunity-related complications. therefore, longterm follow-up is recommended. life-long secondary antibiotic prophylaxis may be required to prevent recurrence of infection in the setting of persistent anti-ifn-g autoantibodies. the study of early t cell development in patients with severe t cell immunodeficiencies is challenging because of the rarity of these diseases, the difficulty to obtain hematopoietic stem cells (hscs), and limitations in the assays to assess in vitro differentiation of hscs to mature t cells. we recently developed a serum-free system that allows faithful analysis of sequential steps of t cell differentiation. in this system, artificial thymic organoids (atos) are generated, based on the 3d aggregation and culture of a delta-like canonical notch ligand 4 (dll4)-expressing stromal cell line (ms5-dll4) with cd34+ cells isolated from bone marrow (bm) samples of normal donors (nd). in this project, we set out to evaluate the possibility of using the ato system to study t cell differentiation in patients carrying t cell defects, in order to define the exact steps of t cell development affected by different genetic defects. using the ato system, we studied in vitro t cell differentiation from cd34+ cells obtained from patients carrying defects that are intrinsic to hematopoietic cells (rag1, rag2, ak2, il2rg) or that affect thymus development (digeorge syndrome, dgs). the ak2-deficient patient showed a markedly decreased viability in cd34+ cells and a very early defect in t cell development, already at the pro-t cell stage. this defect was very similar to that observed in a patient carrying a null il2rg mutation who was reported to show autologous reconstitution after unconditioned haploidentical hsc transplantation. in contrast, cd34+ cells from a patient carrying a missense il2rg mutation and with a leaky scid phenotype were capable of differentiating into mature t cells in vitro, although with 100-fold decreased efficiency as compared to normal donors (nd). interestingly, in the patient carrying the null il2rg mutation, we noticed very few cells that could reach full maturation, with an absolute number of cd3+ tcrab+ cells around 1000-times less than in nd. at variance with pro-t cells (that failed to express the gc protein), these mature t cells did express normal levels of gc, suggesting that they may have derived from residual cd34+ cells from the bm donor. in addition, cd34+ cells from the patients carrying rag1 and rag2 hypomorphic mutations were able to differentiate to cd4+cd8+ double positive cells, but not to cd3+tcrab+ cells. finally, the dgs patient showed a completely normal in vitro t cell differentiation, confirming that t cell deficiency reflected thymic abnormalities. in summary, our data show that the ato system could be extremely useful in determining whether the lack of t cells in patients with unknown gene defects reflect hematopoietic or thymic intrinsic problems, and may therefore provide critical evidence in deciding whether hsc or thymus transplantation is warranted, even without knowing the actual gene defect. introduction: ataxia-telangiectasia (at) is an autosomal recessive disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene, which aids in detection and repair of dna damage. at is characterized by progressive cerebellar ataxia, oculomotor apraxia, choreoathetosis, conjunctival telangiectasias, variable degrees of t-cell lymphopenia (tcl) and immune compromise. patients are at an increased risk for malignancy, particularly leukemia and lymphoma, and are unusually sensitive to ionizing radiation. with the advent of trecbased newborn screening (nbs) for scid, at patients are being recognized with asymptomatic tcl in early infancy. objectives: we present an older child with at and chronic granulomatous lesions and discuss how this may be avoided in individuals with at diagnosed following abnormal nbs. case report: a 12 y/o male was born at term following an uncomplicated twin pregnancy and delivery, prior to institution of scid nbs. he demonstrated mild gross motor and speech delay as an infant and was diagnosed with at at age 3. he had received all routine immunizations, including live vaccinations. he developed granulomatous skin lesions at age 1, initially small papules on his cheeks and ears, which subsequently formed large disfiguring plaques on sun-exposed areascheeks, arms and hands (fig 1) . following an extensive workup, his lesions were found to be secondary to a mutated vaccine-strain rubella (ra27/3) based on 739bp genotyping, previously described in other immunocompromised individuals [perelygina/sullivan et al. jaci 2016] . his lesions have been refractory to multiple treatments including nitazoxanide. he is currently on daily oral and topical steroids, tmp/smx and ivig. retrieval of his nbs for trec determination revealed that he would have screened positive [mallot/puck et al. j clin immunol 2013] . when first measured at age 3, cd3 t-cells were low, 443/ul, with cd4 227/ul and cd8 140/ul. b and nk cell numbers were normal. since april 2017, 4 cases of at were seen at ucsf in infants with non-scid tcl on nbs. these 3 males and 1 female were all born at term and discharged from well-infant nurseries. at was diagnosed at 2-7 months of age. their initial trecs ranged from 5-12/ul (normal with perkinelmer enlite kit >18), and all had low t-cells on initial flow cytometry (242-1612 cd3/ul, ref range>2500) with decreased cd4 (146-1178/ul) and cd8 (87-403/ul) t-cells; however naã¯ve t-cells were present, ruling out typical scid and raising concern for non-scid tcl. three infants also demonstrated low b-cells (<20-77/ul), while nk cells were normal in all. two are currently receiving ivig, one of whom is also on tmp/smx. all have avoided not only rotavirus but also mmr and varicella live vaccinations. conclusions: at is now often diagnosed in infants with low trecs on scid nbs, prior to neurologic manifestations. benefits of early diagnosis include avoidance of live vaccines, including mmr, which led to the debilitating granulomas in our older patient. additionally, patients receive prompt immunologic monitoring and treatment, avoidance of unnecessary radiation, specialty referrals and family genetic counseling. while there is no cure for at, ongoing research may bring neuroprotective treatments in the future. introduction: subcutaneous immune globulin 20%, ig20gly, was well tolerated in the phase 2/3 north american study in patients with primary immunodeficiency diseases (pidd). here we assess comorbidities, use of concomitant medications, infusion parameters, and tolerability in advanced age patients (60 y) treated with ig20gly in the north american study. methods: patients aged 2 years with pidd received weekly ig20gly infusions at volumes 60 ml/site and rates 60 ml/h/site for~1.3 years in the north american study (nct01218438). the medical history at baseline, medical conditions that were ongoing (defined as comorbid events), use of concomitant medications, adverse events (aes), tolerability, and infusion parameters were assessed by age: in advanced age patients (60 y; n=14), adult (16<60 y; n=39), and pediatric/adolescent patients (<16 y; n=21). results: the mean number of medical history events at baseline was higher in advanced age patients (28.7 events/patient; 402 events in 14 patients) versus adult (16.8 events/patient; 657 events in 39 patients), and pediatric/adolescent patients (6.5 events/patient; 137 events in 21 patients). of these, the medical conditions that were ongoing at baseline (comorbid events) were also higher in the advanced age patients (20.9 events/patient; 292 events in 14 patients) versus adult (12.4 events/ patient; 482 events in 39 patients), and pediatric/adolescent patients (3.4 events/patient; 71 events in 21 patients). in the advanced age patients, neurological comorbidities (51 events) were the most common, followed by those related to eyes, ears, nose, and throat (49 events), gastrointestinal (43 events), and musculoskeletal comorbidities (43 events). concomitant medications were given to treat a preexisting condition in all patients in the advanced age group (225 medications in 14 patients). despite the higher mean number of comorbid conditions, infusion parameters in the advanced age patients were comparable to those in the adult age group. median maximum infusion rates and infusion volumes/site were comparable in the advanced age patients (60 ml/h/site; 47.5 ml/site) and adults (60 ml/h/site; 44 ml/site); lower infusion rates and volumes/site were reported in the pediatric/adolescent patients ( . larger infusion volumes and faster infusion rates were not associated with increases in causally related local aes in the advanced age group, consistent with the trends seen in the pediatric/ adolescent and adult patients. conclusions: despite the higher mean number of comorbidities in advanced age patients with pidd, ig20gly was infused at relatively high rates and volumes and was well tolerated. introduction: hyqvia (ighy; immunoglobulin infusion 10% with recombinant human hyaluronidase [rhuph20]) is an immunoglobulin (ig) replacement therapy approved for patients with primary immunodeficiency diseases (pidd) that allows larger infusion volumes, up to 600 ml/site, and has improved ig bioavailability compared with conventional subcutaneous ig products. a post-authorization safety study is being conducted in the united states to acquire long-term safety data on ighy and to assess prescribed administration regimens in routine clinical practice. infusion characteristics and treatment-related adverse events from an interim analysis are reported here. methods: patients aged 16 years with pidd receiving ighy were included in this ongoing, prospective, non-interventional, open-label, uncontrolled, multicenter study. as a part of routine clinical practice, patients are treated with ighy according to standard medical care and their treatment regimen is at the discretion of the treating physician. adverse events (aes) are collected from enrollment to study completion/discontinuation using a subject diary and assessed at every study visit (every 3 months or standard practice). aes are assessed based on seriousness, severity, and causal relatedness to ighy. the presence of anti-rhuph20 antibody is evaluated on a voluntary basis. treatment preferences for various attributes of ig therapy were assessed annually using a treatment preference questionnaire. results: a total of 175 patients were enrolled at 26 us study sites (data cutoff date: august 21, 2017). infusions were self-administered at home (56%) or at the clinical site (44%) most commonly using 4-week infusion intervals (56.6%). the mean maximum ig infusion rate was 302.8 ml/h and the mean ig dose was 418 mg/kg bodyweight/4weeks. the mean number of infusion sites used for administration was 1.9 and mean infusion duration was 2.8 hours. most infusions (97.3%) were administered without a rate reduction, interruption, or discontinuation due to aes. there were no serious aes (saes) related to ighy. sixteen patients experienced a causally related non-serious local ae (9.1%; 0.43 events/patient-year, 0.07 events per infusion) and 25 patients experienced a causally related non-serious systemic ae (14.3%, 0.88 events/patient year, 0.14 events per infusion). seven of 113 patients who were tested for anti-rhuph20 antibody had 1 positive binding antibody test to rhuph20 (titer 1:160; maximum titer 1:10240 at enrollment, 1:5120 during the study); no neutralizing rhuph20 antibodies were detected. of the patients who responded to the treatment preference questionnaire at the end of year 1, the majority (38/52 [73.1%]) preferred to receive their ig therapy at home; 21.2% (11/52) preferred the doctors office; 3 patients preferred treatment at the hospital, had no preference, or indicated other. almost all patients (51/52 [98.1%]) indicated a preference to continue treatment with ighy. conclusion: this interim analysis of 175 patients with pidd treated with ighy in routine clinical practice supports previous observations that ighy is a well-tolerated and preferred therapy with no reports of treatment-related saes or neutralizing anti-rhuph20 antibodies. background: cartilage hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with variable immunodeficiency. pathogenic defects in rmrp, encoding the untranslated rna subunit of ribonucleoprotein endoribonuclease complex (rmrp), result in reduced mrna and rrna cleavage. rmrp c.70a>g is the most common variant, increased in finnish and amish populations. while cellular immunodeficiency is associated with increased morbidity and mortality, there is no established correlation between clinical and immunological phenotype. lymphocyte radiosensitivity has not been described. case: a full-term amish female infant had low trec copies on newborn scid screen. flow cytometry at 3 months-old demonstrated severe t and b cell lymphopenia (cd3+t-cells 413 cells/mcl, range: 2,300-6,500 cells/mcl; cd19+b-cells 214 cells/mcl, range: 600-3,000 cells/mcl) with normal nk quantitation (cd16/56+ 340 cells/mcl, range: 100-1,300 cells/mcl) and cd4+ memory t-cell expansion (33.2%) relative to the naã¯ve subset (67.0%). t-cell functional mitogen responses were normal. she was diagnosed with chh with homozygous rmrp c.70a>g mutation. lymphocyte subset (t, b and nk cells) radiosensitivity was evaluated by flow cytometric analysis of phosphorylated (p) atm, smc1 and gamma-h2ax after low-dose (2gy) irradiation. an increase in gamma-h2ax level was observed in a subset of non-irradiated t cells (17.66% v. 1.36% gamma-h2ax+) and nk cells (23.07% v. 1.04% gamma-h2ax+) in the patient, suggestive of a constitutive defect in dna repair. the relative distribution of t, b and nk cells expressing patm, psmc1 and gamma-h2ax at 1 hour postirradiation (ir) was not significantly different from the experimental healthy control (ehc) or pediatric reference range (prr). however, the kinetics of dephosphorylation at 24 hours post-ir was altered with residual gamma-h2ax expression in a subset of the patients t cells (delta 3.84%, mode ratio mean fluorescence intensity (mfi)=2.58; ehc: delta 0.10%, mode ratio mfi=1.39; prr: delta 2.16%, mode ratio mfi=2.42). a similar finding was observed in a subset of patient b-cells for gamma-h2ax (delta 11.35%, mode ratio mfi=1.48; ehc: delta 0.82%, mode ratio mfi=0.86; prr: delta 1.95%, mode ratio mfi=1.19). the frequency of the patient's lymphocytes with residual gamma-h2ax persistence at 24h post-ir was prominent, with 8.29% t-cells demonstrating persistence of gamma-h2ax (compared to 0.82% in the ehc, and 2.60% in the prr), and 18.02% b-cells gamma-h2ax+ (compared to 1.80% in the ehc, and 2.96% in the prr). there has been lack of follow-up, but verbal report suggests no significant immunological or infectious concerns at 1 year of age. discussion: lymphocyte radiosensitivity is a novel finding in chh with t and b cell lymphopenia. the ability of rmrp to associate with telomerase reverse transcriptase (tert) and function as an rna-dependent rna polymerase, yielding distinct silencing rna sequences, may underlie radiosensitivity in rmrp mutants. systematic characterization of lymphocyte radiosensitivity and immunological phenotype could provide useful information on whether this could serve as a biomarker for the magnitude or complexity of immunodeficiency. assessment of radiosensitivity has implications in conditioning regimen selection for patients requiring allogeneic hematopoietic cell transplantation. we recommend lymphocyte radiosensitivity assessment in chh infants identified by nbs scid and chh patients with significant immunodeficiency and/or malignancy. novel primary immunodeficiency with lymphoproliferative disease due to biallelic defects in nckap1l background: three children from 2 non-consanguineous families and different ethnic backgrounds developed lymphoproliferative disease by 2 years of age. they also had recurrent infections, including pneumonia and bronchiectasis, otitis media, and skin pustules. immune phenotyping revealed low cd4+ t cell percentages, an accumulation of memory-like cd8+ t cells, impaired t cell proliferation, and low total nk cell numbers. methods: the affected individuals, unaffected parents, and other unaffected family members underwent exome sequencing. results: all 3 affected cases had rare and bioinformatically damaging biallelic variants, with appropriate familial segregation, in nckap1l, which encodes hem1. hem1 is an essential component of the wave2 regulatory complex (wrc). immunoblotting confirmed destabilization of the wrc in all patients. immunofluorescence microscopy demonstrated defective f-actin and wave2 localization to immune synapses in nk cells. significant abnormalities were identified in patient lymphocyte and neutrophil migration and morphology, consistent with altered wrc-mediated cytoskeletal dynamics. all patients exhibited impaired inside-out integrin activation. knockdown of hem1 produced deficient proliferative responses and mtorc2-mediated akt activation in control t cells. conclusions: the immunologic and clinical phenotype in the affected individuals recapitulates the phenotype observed in hem1-deficient mice. biallelic defects in nckap1l therefore result in a novel human primary immunodeficiency disease characterized by lymphoproliferation and susceptibility to infections. background: concurrent existence/significance of immunodeficiency with new onset lymphoproliferative disease remains understudied. just two studies to date have evaluated the prevalence of hypogammaglobulinemia in chronic lymphocytic leukemia (cll) and neither studied prevalence and impact of ige deficiency on outcomes in cll [1, 2] . therefore, the objective of this study was to examine the prevalence of hypogammaglobulinemia, examining all isotypes, in newly diagnosed cll patients and to test the hypothesis that patients with hypogammaglobulinemia have a distinct clinical profile and outcome. methods: using the banked sera of 150 newly diagnosed, treatmentnaã¯ve, cll adult patients from the lymphoma molecular epidemiology resource (l-mer), ig (igg, iga, igm and ige) levels were measured. the l-mer was initiated as an observational cohort study of prospectively enrolled newly diagnosed lymphoma patients evaluated at the mayo clinic (rochester, mn) and the university of iowa (iowa city, ia) [3] . igg/a/m levels were measured using immunoturbidimetric assay whereas the ige level was determined using electrochemiluminescence immunoassay. the associations between ig deficiencies and clinical factors were evaluated with wilcoxon rank sum and chi-squared (fishers exact, where appropriate) tests. cox regression models were used to assess the effects of clinical variables on overall survival (os). time was calculated from biopsy to death due to any cause; patients still alive were censored at last contact. all tests were two-sided and assessed for significance at the 5% level using sas v9.4 (sas institute, cary, nc). results: the mean age (sd) of the selected cll cohort was 63.8 (11.0) years with a male predominance (69.3%). 96.2% of the patients were white. with a median follow-up of five years, there were 50 deaths. hypogammaglobulinemia in newly diagnosed, treatmentnaã¯ve cll was common in our cohort with 88 (58.7%) patients having a measurable isotype deficiency. the most common ig deficiency was igm (44.0%, 95% ci 35.9-52.3%), followed by igg (34.7%, 95% ci 27.1-42.9%), ige (16.7%, 95% ci 11.1-23.6%) and iga (12.0%, 95% ci 7.3-18.3%). multiple deficiencies in the same patient were common ( figure 1 ). iga and ige deficiency were associated with higher rai stages (grading system for cll) at presentation (p<0.01 and 0.04 respectively) as well as with higher white blood cell counts at presentation (p=0.02 and 0.01 respectively). a higher proportion of iga deficient patients needed second treatment during follow-up (61% compared to 36%, p=0.04). when comparing predictors of overall survival, higher rai stage [3-4 vs 0, hazard ratio (hr) 2.43, 95% ci 1.08-5.46, p=0.03] and age (hr 1.08, 95% ci 1.05-1.12, p<0.01) correlated with worse overall survival. individual immunoglobulin deficiencies did not correlate with overall survival. conclusions: a significant proportion of treatment-naã¯ve patients with cll have underlying ig deficiencies-both in isolation and a combination of different isotypes. a deficiency of iga or ige was associated with severe disease at presentation. the underlying relationship between these two immunologic disorders deserves further study. background: patients with primary immunodeficiency (pid) have an increased risk of developing autoimmune diseases, including rheumatoid arthritis (ra). management of these patients is challenging as immunomodulators can further increase their risk for infections. additionally, patients with ra that undergo therapy with drug modifying antirheumatic drugs (dmards) may develop a secondary immunodeficiency. there are few studies reviewing the characteristics of patients with a pid who later develop ra, and no studies have been reported comparing these patients to those who develop an immunodeficiency after starting dmard therapy for ra. methods: 65 patients were identified as having inflammatory arthritis and a concomitant immunodeficiency (id) at our institution between 1/1/2000-10/03/2017 using icd-9 and 10 codes. manual chart review was performed to confirm and identify the timing of diagnosis of these disorders. patients were excluded if either there was no definitive diagnosis of id or ra (clinically diagnosed by a practicing allergist/immunologist and meeting acr 2010 criteria for ra with a score of 6 or higher, respectively), or rituximab was administered prior to diagnosis of id . clinical symptoms, treatment, and laboratory data were extracted. fishers exact test was used to compare the categorical variables between the groups; ttest was used to compare the continuous variables. results: 10 patients met the inclusion criteria. 5 patients were diagnosed with an id and developed ra later in life (group 1), and 5 patients were diagnosed with ra and subsequently developed a clinically significant id (group 2). the mean ages of diagnosis of id and ra in group 1 patients were 32.0 years (sd â± 26.9) and 42.6 years (sd â± 19.0), respectively. in group 2, the mean age of diagnosis of ra was 37.8 (sd â± 14.2), compared to 54.8 years (sd â± 12.7) for the diagnosis of id. most patients in both groups were female (60% in group 1 and 80% in group 2). all patients in both groups had a humoral id, including common variable immunodeficiency (cvid) (40% of group 1 patients), specific antibody deficiency (sad) (20% of group 1 and 60% of group 2 patients), and hypogammaglobulinemia (20% of group 1 and 40% of group 2 patients). all patients in group 2 were seropositive for rheumatoid factor (rf) or anti-cyclic citrullinated peptide (anti-ccp), whereas only 20% of patients in group 1 were positive for rf or anti-ccp (table 1 ). most patients in both groups were treated with immunoglobulin replacement therapy. treatment of ra in both groups was similar, but combination dmard therapy was not used in group 1 patients in contrast to group 2 patients. conclusions: our study indicates that even though clinical characteristics and management are similar in patients with coexisting id and ra, rf and anti-ccp are usually negative in patients who develop ra after id, possibly due to impaired antibody production in immunodeficient patients. assistant professor of allergy and immunology, arkansas children's hospital, university of arkansas medical sciences introduction/background: complement deficiencies are relatively rare, comprising less than 1% of primary immunodeficiencies. they are associated with increased risk for infections with encapsulated organisms and autoimmunity. of all complement deficiencies, the rarest are defects in the alternative complement pathway. properdin deficiency is the most commonly described alternative pathway deficiency, with factor b and factor d deficiency more rarely described. fewer than 5 patients with factor d deficiency have been reported with all reported cases being children of consanguineous parents who succumbed to meningococcal sepsis. objectives: to describe a case of factor d deficiency associated with recurrent respiratory infections with streptococcus pneumoniae pneumonia with associated lung abscess and empyema. methods: retrospective chart review was conducted. laboratory investigations included lymphocyte immunophenotyping by flow cytometry, lymphocyte proliferation to mitogen, quantitative serum immunoglobulins, vaccine titers, complement assays and functional evaluation, and genetic evaluation by next generation sequencing. results: a 2 year old marshallese male was transferred from an outside hospital to our facility for further evaluation of worsening pneumonia and was found to have right-sided pleural effusion and pulmonary abscess in the right lower lobe. the abscess was drained and was found to be positive for streptococcus pneumoniae via polymerase chain reaction. he improved after chest tube placement and treatment with intravenous antibiotics. his medical history was significant for recurrent acute otitis media and prior hospitalization out-of-state for pneumonia with empyema secondary to streptococcus pneumoniae, which required chest tube placement and admission to the pediatric intensive care unit at 18 months of age. immunologic work up revealed age-appropriate lymphocyte subpopulations, lymphocyte proliferative responses to mitogens, quantitative immunoglobulin levels, pneumococcal/tetanus/diphtheria titers, and ch50 complement assay. ah50 complement assay was decreased to 44 units/ml. complement testing was repeated -with normal ch50 and ah50 of 0 units/ml. further evaluation revealed normal levels of factors b, h, i and properdin. factor d level was 0.12 mcg/ml, and factor d function was decreased to 2 units/ml, indicating a diagnosis of factor d deficiency. sequencing of the cfd gene revealed a previously undescribed homozygous deletion (c.721_723del and p.lys241del). the parents were not agreeable to personally undergoing genetic evaluation to determine if this was a de novo mutation. the patient was managed with pneumococcal and meningococcal immunizations, prophylactic amoxicillin and intravenous gamma globulin (ivig) without any further infections. unfortunately, after two ivig infusions, he was lost to follow up. conclusion: factor d deficiency is an extremely rare alternative complement pathway deficiency, described in less than 5 patients. all infections described thus far have been secondary to neisseria meningitidis. this case represents not only a novel mutation in the cfd gene leading to factor d deficiency, but also the first description of a patient with factor d deficiency developing invasive infection secondary to streptococcus pneumoniae. background: viral infections are a significant cause of morbidity and mortality in patients with primary immunodeficiency disorders and following hematopoietic stem cell transplantation. adoptive immunotherapy using virus specific t-cells (vsts) has been shown to prevent and treat viral infections in immunocompromised hosts. human parainfluenza virus-3 (hpiv3) is a common cause of severe respiratory illness in immunocompromised patients and has no approved antiviral therapies and has not previously been used as a target for t cell therapeutics. introduction: we previously reported that fatigue is increased in common variable immunodeficiency (cvid). however, in previous studies, fatigue was not defined using validated tools. our aim from this study is to identify the prevalence of patient-reported fatigue, using validated questionnaires, and determine the factors predisposing to fatigue in cvid methods: data from cvid who responded to the idf 2017 patient national survey a were analyzed. fatigue was measured using the brief fatigue inventory (bfi) questionnaire, which includes seven items to identify fatigue, and measure fatigue severity. a total of 555 patients with cvid and responses to bfi were enrolled. demographics, co-morbidities, immunoglobulin replacement therapy (iggrt) route and dose, co-morbidities, infections, depression, quality of life (qol) (using the sf-12v2) and disability were compared between fatigued and non-fatigued. logistic regression was used to identify the significant variables. ebv reactivation without ptld, treated with rituximab. alive and well. j clin immunol (2019) 39 (suppl 1):s1-s151 s58 granulomas are the most significant day-to-day problem for cvid patient management. currently, there are limited options for their treatment and the optimal therapy is unknown. in case reports and small series, infliximab has been reported effective while others found it useless. we here describe a 26yo white male referred for monthly ivig in august 2016. at age 1, he developed large areas of erythematous polymorphic plaques in his cheeks, arms and legs. a skin biopsy showed tuberculoid granulomas negative for bacteria, baar and fungi, with infiltrating cd4+ lymphocytes. a prolonged course of steroids did not improve his skin. he also had multiple pneumonias and bronchiectasis, and oral candidiasis. he received all vaccines, including bcg with no complications. with low immunoglobulins and a poor response to pneumococcal polysaccharides and tetanus toxoid he was diagnosed as cvid and placed on ivig at 7yo with excellent infectious control since then. at age 8, his skin lesions persisted and deepened to the bone on his left leg. broad spectrum antibiotics for 3 months were unsuccessful. at 16yo to 18yo, skin grafts were performed on his arms, legs and both cheeks. two ulcers persisted on his left leg until august 2018 that increased in size, deepened and became erythematous and extremely painful (fig. 1) . in september, two new ulcers appeared on his right cheek and right gluteus, respectively. one week later a third ulcer was found on his left calf. on september 28th, infliximab 5mg/kg (300mg) was administered. on the second infliximab dose, october 12th, the pain was completely gone and all ulcers were shrinking, and those ones in the cheek, gluteus and calf almost completely resolved. by the third dose, on november 23rd the ulcers in his right leg were almost closed (fig. 2) . infliximab 300mg treatment continues every 8 weeks. lab test remained unchanged from 2016 till 2018, when his wounds got worsened. (table 1 ) granulomatous disease in cvid is a challenge. both b and t cell directed therapies are encouraged. we add a new case of an infliximab responsive patient to others already reported. over 20 genes have been reported to cause monogenic cvid. a 4 year old girl presented with recurrent pneumonias and a diagnosis of cvid. the parents sought a second opinion. born at 33 weeks gestational age, she was "always smaller and sicker than her friends," and in the prior 8 months she had 3 episodes of pneumonia with fever to 104f requiring emergency department treatment. two of these were associated with rsv and metapneumovirus, respectively. laboratory evaluation confirmed low levels of igg (326 mg/dl) iga (7) and igm (6) congenital tuberculosis (ctb) is a rare disease most often associated with maternal genitourinary (gu) tuberculosis (tb) or disseminated tb. due to infertility caused by gu tb, ctb is rarely reported even in endemic countries. infants can acquire tb hematogenously via the placenta or umbilical vein or by fetal aspiration of infected amniotic fluid. presenting symptoms include respiratory distress, fever, hepatosplenomegaly, poor feeding, lethargy, and low birth weight. we report a premature female infant conceived via in vitro fertilization (ivf), who was born to indian immigrant parents at 29 weeks of gestation due to preterm premature rupture of membranes. maternal history was significant for pulmonary tb at 9 years of age. she denied abdominal or gu symptoms. infants nicu course was complicated by opacifications in the right lung and leukocytosis with neutrophil predominance, identified during evaluation of frequent apnea and bradycardia episodes at 1 month of age. clinical improvement was noted after treatment with vancomycin, amikacin and piperacillin-tazobactam; however, leukocytosis of unknown etiology persisted. at 1.5 months of age she was discharged to inpatient rehabilitation. at 3 months of age, she was readmitted for fever and respiratory distress. during this admission, an immune evaluation was undertaken due to persistence of symptoms along with unresolved leukocytosis with a peak of 58,000 cells/l with neutrophilia to 42,850 cells/l, and chest ct evidence of progressive multifocal lung disease worse in the right upper lobe despite empiric treatment with broadspectrum antibiotics. infectious work-up was negative, including acid-fast bacilli testing from bronchoalveolar lavage. due to the pronounced and persistent leukocytosis and neutrophilia, a primary immune defect was suspected. immune evaluation included: normal immunoglobulins (ig) g, a, and e, elevated igm, vaccine-specific antibody titers protective to diphtheria and 9 of 13 streptococcus pneumonia strains, mildly elevated t and b cells, a normal flow cytometry for dihydrorhodamine, myeloperoxidase stain and glucose-6-phosphate dehydrogenase level, as well as a peripheral smear with no giant azurophilic granules. her primary immunodeficiency genetic panel was unrevealing. she underwent lung biopsy via video-assisted thoracoscopic surgery, which showed noncaseating granulomas and eventual growth of multi-drug-resistant mycobacterium tuberculosis (mtb). upon treatment with an appropriately adjusted anti-tuberculosis regimen, she showed rapid clinical and laboratory improvement. endometrial samples obtained from mother showed gu tb, confirming the diagnosis of ctb. the slow-growing nature of mtb that resulted in delayed diagnosis, along with the presence of non-caseating granulomas and persistent neutrophilia, prompted an immune work up that was completely normal. this case demonstrates the importance of considering ctb in the differential diagnosis of an infant presenting with severe lung infection, persistent neutrophilia, suboptimal response to broad-spectrum antibiotics and relevant epidemiologic risk factors. furthermore, in the setting of appropriate parental exposures and infertility prompting the use of ivf, maintaining a high level of suspicion of ctb can aid in earlier diagnosis of affected neonates. 15-year-old caucasian male who initially presented with recurrent otitis media, persistent hsm, lad, and hypogammaglobinemia (igg <170 mg/dl) at 2 years of age. he was diagnosed with common variable immunodeficiency (cvid) and chronic arthritis when he was 6 and 9 years of age, respectively. subsequently, he developed hepatitis and recurrent pneumonia with mycobacterium avium complex (mac). his arthritis partially responded to anti-tumor necrosis factor (tnf) agents and tofacitinib, but did not respond to anti-interleukin-6 treatment. a combination of anti-tnf inhibitor, tofacitinib, and low dose prednisone was required to control his arthritis. hypogammaglobulinemia (igg <110 mg/dl), recurrent otitis media, pneumonia, crohn's disease, celiac disease, lad and failure to thrive at 2 years of age with more recent development of hsm. he required only immunoglobulin replacement therapy. case#3 is a 9-year-old caucasian male, the half-brother of case#2, who initially presented with recurrent pleural effusion and bilateral pulmonary infiltrates, hsm, lad, abdominal distension and ascites at 7 years of age. a transbronchial lung biopsy revealed chronic eosinophilic pneumonitis. liver biopsy showed increased eosinophils in the sinusoids with diffuse enlargement of hepatocytes, but without hepatitis. colon biopsy revealed minimal colonic eo-sinophilia. his pulmonary infiltrates and pleural effusion responded to prednisone, and he has not required additional treatment for past 1.5 years. conclusions: the clinical manifestations of the same genetic variant may be variable and unpredictable even in the same family. stat3 gof syndrome should be considered in children with multisystem autoimmune diseases, lad, hsm and low switched memory b cells regardless of presence of hypogammaglobulinemia or history of recurrent infections. background: patients with primary immune deficiencies characterized by severe t lymphopenia and/or poor t cell function and patients posthematopoietic cell transplantation are at high risk of severe viral infections. antiviral medications are expensive, not always effective and associated with significant toxicity and/or long-term side effects. as such, there has been increasing interest in the use of donor-derived or thirdparty virus-specific t cells (vsts), and several studies have demonstrated efficacy of vsts generated using various manufacture strategies. however, in depth immunologic and metabolic characterization of vsts has not been reported, limiting correlative investigations into efficacy. methods: ebv-vsts were generated from apheresis t cells collected from healthy donors using three methods: (1) stimulation and expansion with hla-matched ebv-lymphoblastoid cell lines (lcls) purchased from astarte biologics or sigma-aldrich over a period of 4 weeks, (2) stimulation with ebv peptivator from miltenyi followed by expansion over 9-12 days with different cytokines, and (3) stimulation with ebv peptivator followed by isolation of activated cells using the ifn-gamma capture system from miltenyi. immunophenotyping by flow cytometry was performed using the miltneyi macsquant analyzer. the nanostring ncounter system was used to measure gene expression for metabolic pathway analysis, and the agilent seahorse xf cell mito stress test system was used to measure mitochondrial respiration. results: ebv-vsts generated using lcls or peptivator plus il-15 both resulted in a high percentage of cd8 t cells skewed to the effector memory and terminal effector memory phenotype with high expression of the exhaustion markers pd-1, tim-3, and lag-3. conversely, ebv-vsts generated using peptivator plus il-4 and il-7 and the ifn-gamma capture system resulted in a mixed cd4 and cd8 t cell population with a high number of central memory t cells and lower percentage of cells positive for pd-1, tim-3, and lag-3. stimulation with peptivator followed by expansion with il-2 resulted in an intermediate immunophenotype. nanostring results demonstrated upregulation of the glycolytic pathway in ebv-vsts stimulated with peptivator followed by expansion with il-2 or il-15 compared to ebv-vsts generated using the other manufacture approaches. the seahorse mito stress test demonstrated that the peptivator plus il-2 ebv-vsts had a significantly lower spare respiratory capacity than other ebv-vsts and a low extracellular acidification rate despite upregulation of the glycolytic pathway. the peptivator plus il-4 and il-7 ebv-vsts had the highest basal oxygen consumption rate, atp-linked respiration, and extracellular acidification rate. conclusions: manufacture of ebv-vsts using the various approaches currently employed clinically results in t cell pools with different immunophenotypes and different metabolic profiles. ebv-vsts stimulated with peptivator followed by expansion in il-4 and il-7 and ebv-vsts isolated using the ifn-gamma capture system have immunophenotypes and metabolic phenotypes suggestive of potential greater in vivo persistence, whereas ebv-vsts expanded in il-2 and il-15 have characteristics correlated with increased effector function. however, these vsts are more likely to be short-lived and to have impaired metabolic fitness. these phenotypes will enable better correlation with clinical results and suggest combinatorial approaches depending on clinical indication. introduction: majority of patients with primary immunodeficiencies (pid) require life-long replacement therapy with immunoglobulins (ig) to prevent severe infections and irreversible complications. in addition to safety and efficacy, tolerability and convenience of administration of ig products are essential factors for patients. a new 16.5% ig preparation octanorm (octapharma, lachen; tradename cutaquigâ® in north america) has been developed for subcutaneous administration (scig) derived from the established manufacturing process of octapharmas intravenous ig (ivig) brand octagamâ®. objectives: primary outcome was assessment of the efficacy of octanorm in preventing serious bacterial infections. main secondary endpoints included (among others) evaluation of tolerability and safety of octanorm, the number and rate of other infections, number of days missed at work, and use of antibiotics. methods: a prospective, open-label, non-controlled, single-arm phase 3 study involving 25 adult patients with pid was conducted at 5 russian centers. patients treated with at least 4 infusions of ivig prior to enrollment and with igg trough levels 5.0 g/l underwent an 8-week wash-in/wash-out period followed by a 24week efficacy period. during the study, patients received weekly administrations of octanorm at the same monthly dose as during previous ivig treatment (monthly ivig dose divided by 4 for weekly dose). in total, each patient received 32 scig infusions. results: twenty-four patients completed the study. one patient terminated early (after infusion 7, during wash-in/wash-out phase; personal reasons). mean age was 35.24 years (range 18-64 years). fifteen patients (60%) were female and 10 patients (40%) male. no serious bacterial infections were recorded. during the efficacy period a total of 26 non-serious infections was observed in 14 patients. seventeen infections in 11 patients were of mild and 9 infections in 5 patients of moderate intensity. the infection rate per person-year was 2.37. in total 25 patients received 775 infusions of study drug. the average dose of cutaquigâ® was 0.11 g/kg/week. during the entire study, 59 systemic adverse events were reported (including 34 infections). three of these systemic adverse events were rated as related to study drug, all were non-serious. there was no serious or significant adverse event nor was there an adverse event leading to withdrawal. infusion site reactions were reported for 15% of infusions. serum igg trough levels were nearly constant during the efficacy period. median igg trough levels were 8.15 g/l at screening, 9.52 g/l at the end of wash-in/wash-out period and 10.71 g/l at the termination visit. one patient had a trough level 5g/l at 2 visits during the efficacy period and the dosing was subsequently adjusted for this patient. during the primary treatment period 10 patients (41.7%) used antibiotics in 19 treatment episodes (total of 229 treatment days; range 5-76 days) and 3 patients had 4 absences from work or school due to infections (total of 14 days of absence). conclusion: this study demonstrated that the new subcutaneous human normal immunoglobulin 16.5% is well tolerated, safe and effective in adult patients with pid. background: children with chronic granulomatous disease (cgd) are at high risk for fungal infections (especially with aspergillus species) and these infections usually have contiguous site involvement. most patients have pulmonary presentation. infective endocarditis and fungal osteomyelitis of skull are distinctly unusual. we report one such case. case: a 6-year-old boy, born out of a non-consanguineous marriage, presented with soft tissue swellings of skull for 2 months. his past history was significant with an episode of pneumonia at 1 year and recurrent soft tissue swellings all over the body since 1â½ years of age. on examination he was wasted, had signs of micronutrient deficiency, rickets, pallor, cervical lymphadenopathy and two abscesses, 12x4 cm on right temporo-parietal region and 4x3 cm over left frontal region. he was also found to have hyperdynamic precordium with an ejection systolic murmur. investigations revealed hemoglobin 85g/l; platelet count 7.34x109/l; total leukocyte count 13x109/l(n60/l23/m13/e1); elevated c-reactive protein( 244 mg/l) and a raised erythrocyte sedimentation rate(104 mm 1sthr). chest x ray revealed cardiomegaly (cardiothoracic ratio 67%) and 2d echocardiography showed vegetation of 6x3 mm over the anterior mitral leaflet suggestive of infective endocarditis. blood and urine cultures were sterile. culture from pus over the temporo-parietal abscess showed growth of aspergillus fumigatus. human immunodeficiency virus serology was non-reactive. immunoglobulin profile revealed elevated igg 21.20g/l (5.40-16.10g/l) and iga 5.66 g/l(0.5-2.4g/l); igm was 1.63 g/l(0.50-1.8g/l). in view of strong suspicion of cgd, nitroblue tetrazolium dye reduction test (nbt) was carried out-it revealed no reduction and dihydrorhodamine (dhr) assay showed a low stimulation index (4.34). flow cytometry for gp 47 phox and gp 67 phox was normal and dhr of mother did not reveal x linked carrier state. contrast enhanced computerized tomography (cect) of head showed osteomyelitis of the calvarial bones. contrast enhanced magnetic resonance imaging (cemri) brain showed heterogeneously enchancing soft tissue lesion in the scalp at right fronto-parietal region and left frontal region with underlying bony destruction suggestive of osteomyelitis. he was given intravenous antimicrobials (ceftriaxone, gentamycin, cloxacillin, voriconazole). after 6 weeks of therapy, he showed resolution of findings on mri brain and a repeat 2d echocardiography showed significant decrease in size of mitral leaflet vegetation. conclusion: this case highlights a rare presentation of cgd with infective endocarditis and skull osteomyelitis due to aspergillus fumigatus. to the best of our knowledge, this has not been reported previously. background: genetic defect in il12r1 affect cellular immunity, underlie mendelian susceptibility to mycobacterial disease (msmd) and inflammatory bowel disease (ibd) through different pathways. we present for the first time a patient with il-12r1 deficiency from a consanguine family with two different phenotypes. initially diagnosed as crohn's disease prior to the msmd diagnosis. method and material:patient was referred to the clinical immunology and allergy clinic at the at alzahra university hospital for immunological and genetic evaluation . blood samples from patient, his family and healthy donor controls were collected upon informed consent. in this study, we investigated effect of il12r1 mutation in il-12/ifnaxis by evaluation of patients whole blood cell response to il-12 and ifn-, il-12r1 expression in pbmcs and t cell blasts. also wholeexome sequencing has been performed. result and discussion: a 26 years old male from consanguine family , with history of right sub-axillary bcg lymphadenitis, recurrent mouth ulcers , chronic diarrhea in childhood and appendectomy at age of 5 was investigated. based on his clinical presentation abdominal pain, significant weight loss, chronic and bloody diarrhea , endoscopic and pathological findings treatment for crohn's disease (cd) was started at the age of seven . unfortunately, protracted patient's symptoms ends up to resection of his colon and colostomy two years later. he was presented with multi focal osteomyelitis at the age of 13 . although no bacteria was detected in pcr and tissue culture of the bone biopsy and the patient was not responded to antibacterials , he had a dramatic response to empirical anti mycobacterial treatment and his severe bone pain and lesions were healed. even though the bone manifestations were completely controlled, he continuously was under treatment for his gastrointestinal symptoms. genetic analysis was confirmed segregation of homozygous mutation in 3splice site of exon 15 in il-12r1. expression of gene was completely abolished in pbmcs of patient and the surface expression of il12rb1 was not detectable in t cell derived pbmcs of the patient compared to healthy control. furthermore, did not response to il12 stimulation since we could not detect increase of inf-after stimulation with il12 and bcg. our patient received bcg vaccination at birth and had bcg lymphadenitis as an infant, cd and mycobacterial multifocal osteomyelitis as a child. furthermore there are some evidences which indicate the role of atypical mycobacterial infections as a trigger for cd. conclusion: we reported for the first time contemporary msmd and ibd in 26 years old patient, who had impaired il-12 signaling and abolished il12 r1 expression in pbmcs and t cell blast. however, mycobacterial osteomyelitis is a typical phenotype of msmd patients with deficiency in ifn-r1 or stat, there were no mycobacterial osteomyelitis reported in il-12r1 deficient patients. background: advanced genetic studies help explain the occurrence of many undiagnosed, rare conditions. recently, nbas variants were identified as a causative basis of recurrent liver failure in infants (infantile liver failure syndrome 2, ilfs2). the nbas (neuroblastoma amplified sequence) gene encodes a protein involved in golgi to er retrograde transport. nbas functions seem to be broad and loss of function variants in nbas have been associated with multisystem manifestations. case report: a 5y 9m old chilean male presented to the er with a three day history of vomiting, diarrhea and one day of fever (38.3â°f). on examination he was pale, lethargic, and tachycardic. a chemistry profile revealed markedly elevated liver enzymes, increased bilirubin, and coagulopathy, consistent with the acute hepatic failure (alt 6291, ast >4000, total bilirubin 3.49 (2.82 db), ggt 52, and inr of 2.1). he was hospitalized, given vitamin k, and kept on intravenous fluids, ursodiol, and antipyretics. his liver function improved significantly within 5 days of admission (alt was down to 980, ast 45, total bilirubin 1.62). work-up of possible etiologies including autoimmunity and infectious hepatitis was negative. liver sonogram was normal, but liver biopsy was consistent with acute hepatitis with some necrosis. urine organic acid and plasma amino acid screens were not consistent with any inherited metabolic disorders. his parents recalled two previous episodes of liver failure at ages 3 and 4 years. both were preceded with a mild febrile illness and non-specific symptoms including fever, coughing, vomiting, diarrhea, lethargy, and decreased po intake. these subsequently were followed by jaundice and marked elevation of liver enzymes. flu a and adenovirus were identified as causes of febrile illnesses of the two previous episodes. for this admission, adenovirus was found in the respiratory secretions and a mild ebv viremia was also detected. genetic evaluation in chile was reportedly normal. after a literature review we obtained sequencing of nbas which revealed two variants: c.2827g>t,p.glu943* and nbas c.2951t>g, p.iie984ser. both variants have been previously reported in patients with an infantile onset, recurrent liver failure syndrome. his other clinical features include developmental and speech delays, failure to thrive, and facial dysmorphism. he also has a history of recurrent ear infections and has had 3 sets of tympanostomy tubes. further testing was limited due to the lack of insurance coverage. conclusion: nbas deficiency is a newly described syndrome of recurrent acute liver failure that occurs early in life. once individuals have survived to adulthood they do not seem to develop liver failure with illness. typically, liver crisis is triggered by a common childhood febrile illness. the mechanism of disease is thought to be thermal instability of hepatocytes which improves over time in most cases. however, although spontaneous recovery can occur following the crises, each episode can be fatal or result in permanent liver damage required liver transplantation. increased awareness of this disease will lead to the early establishment of the diagnosis. appropriate and timely management of fever at the onset of illness can significantly improve outcome in this potentially fatal disease. associate prof., infectious diseases and tropical medicine research center, isfahan university of medical sciences, isfahan, iran background: pre-eclampsia, a pregnancy-specific complication, has been shown to be associated with cytomegalovirus (cmv) infection. cmv specific t-cell response plays the major role in cmv infection or disease .we explored whether a change in cmv-specific cell-mediated immunity (cmi) is related to the development of preeclampsia. method: cmv-specific cmi was assessed using cmv-quantiferon (qf-cmv) assay in serum from 35 women with pre-eclampsia as well as 35 normal pregnancy controls retrospectively. participants were matched for gestational age individually. proportion of reactive results, mean value of interferon-level produced in mitogen and antigen tubes were compared between the cases and controls via chi-square, wilcoxon rank-sum tests, respectively. odds ratio (or) and confidence interval (ci) were calculated as well. result: no significant differences observed between demographic characteristics of the case and control groups. the qf-cmv assay turned reactive (qf-cmv [+]) in 22 of 35 of patients (63%) vs. 32 of 35 controls (91.4%) (p = 0.004). women with pre-eclampsia had lower mean ifn-levels in antigen tube (1.57 â± 1.79) compared with normal pregnancy controls (2.40 â± 2.21) (p = 0.028). there was no statistically significant differences in this value of mitogen tube between cases (3.53 â± 1.67) and controls (3.53 â± 1.67) (p = 0.209). women with suppressed cmv-cmi were 6.3 times more likely to manifest pre-eclampsia (or= 6.30, 95% ci: 1.60-24.7). this result even strengthened after adjustment for age, gestational age and gravidity (or = 12.86, 95% ci: 2.68-61.6). conclusion: our finding support an association between suppressed cmv specific cmi and pre-eclampsia. introduction: the triad of susceptibility to infections, auto-inflammation, and cancer in a patients personal and family history are always suggestive of an underlying primary immunodeficiency; however, in some cases the diagnosis might be delayed for years. furthermore, the results of immunological and inflammatory evaluation can also be affected by ongoing immunomodulatory therapy initiated by different specialists upon clinical diagnosis. objective: to describe a unique presentation of auto-inflammatory disease with combined immunodeficiency in an adult patient. case presentation: we report here the case of a 64 year old male, who had a long history of infections including recurrent sino-pulmonary bacterial infections starting during childhood, osteomyelitis at 7 years of age, recurrent tonsillitis requiring tonsillectomy at 21 years of age, recurrent cellulitis, an episode of prostatitis with septicaemia, as well as recurrent varicella zoster and warts. the patient was also diagnosed with sclerosing mesentheritis, and reynauds phenomenon, recurrent oral ulcers, arthritis, uveitis, autoimmune thyroiditis, lung fibrosis and suffered repeated episodes of abdominal pain. furthermore, there is a family history of early childhood death, multiple soft tissue cancers, crohns disease, and autoimmune thyroiditis. upon physical examination, the patient had multiple telangiectasia, baseline erythroderma, and flushing. immunological evaluation showed lymphopenia with significant reduction in both circulating b and t cells, however, assessment of humoral immunity revealed low igg and decreased igm with normal iga levels. at the time of the evaluation he had been on low dose daily prednisone (7.5mg), colchicine, and methotrexate as immuno-modifying therapy. genetic evaluation revealed a heterozygous mutation in nod2 as well as compound heterozygous mutations in the mefv gene. discussion: mutations in nod2 have been described in association with blau syndrome a multisystem auto-inflammatory syndrome which may explain many of the features experienced by our patient. to our surprise next generation sequencing revealed a second aberration in the mefv gene which causes familiar mediterranean fever, another multisystem auto-inflammatory disease, which might lead to the phenotype observed in the patient. conclusion: this is the first report of genetic lesions in two different genes leading to a severe course of auto inflammation. monogenic autoinflammatory syndromes (mais) are a diverse group of disorders characterized by primary over-activation of the innate immune system. induction of the inflammasome complex by innate immune sensors and increased production of il-1b are implicated in the pathogenesis of mais. macrophage activation syndrome (mas) is a life-threatening illness defined by acute hyper-inflammation and unopposed cytokine release. it is considered an acquired condition secondary to infection, rheumatoid disease or malignancy. the early therapeutic use of il-1b inhibition has profoundly improved the prognosis mas. it has recently been shown that increased free il-18 levels in the blood are causatively linked to the development of mas. significant overlap in clinical presentation and laboratory markers between patients with mais and mas led us to explore the role of free il-18 and therapeutic use of il-1b inhibition in a patient with cdc42 mutation. here, we report the case of an 18 months-old female who presented with hydrops fetalis in utero, and later developed failure-to-thrive, splenomegaly, anemia, thrombocytopenia, arthralgias, rashes, frequent febrile episodes and mild facial dysmorphism along with massive increase in crp, esr and ferritin. whole exome sequencing (wes) identified a heterogenous likely pathogenic de novo variant in cell division control protein 42 homolog (cdc42) c.563g>a (p.c188y). cdc42 encodes a small rho family gtpase that regulates multiple signaling pathways controlling cell polarity, migration, endocytosis and cell cycle progression. single allele mutations in the cdc42 gene were recently reported to cause takenouchi-kosaki syndrome manifesting with growth retardation, developmental delay, facial dysmorphism, and thrombocytopenia however systemic autoinflammation has not been described. cdc42 closely interacts with the wiskott-aldrich syndrome protein but little is known about the mechanism underlying immune abnormalities associated with cdc42 mutations. our patient had an inflammamosopathy-like syndrome. because of significant clinical overlap to mas, we measured il-6, il-18, free il-18 and il-18 binding protein, all of which were significantly increased. this increase in free il-18 heightened her risk of developing mas. her il1b level was normal, but an increase in il-1b is hardly ever detectable in the serum despite playing a critical role in this type of inflammation. indeed, chronic il-1b excess in the tissues promotes systemic inflammation and is associated with chronically elevated crp and esr. with this rationale we started the il-1 receptor antagonist anakinra. within 48 hours from starting anakinra, the parents observed an increase in appetite, resolution of arthralgias and improved mobility. over the course of the following weeks, fever, anemia, thrombocytopenia and rash disappeared, the spleen massively decreased in size and the patient started to meet developmental milestones. crp, esr eventually normalized while ferritin and free il-18 are still trending down. conclusions: significant increase in free il-18 and extremely encouraging clinical response to therapy with anakinra in a patient with novel cdc42 mutation suggests a link between mas and defects in cdc42. elucidating the mechanism of inflammasome activation and the drivers of il-18 increase in mas and mais more broadly may shed light on novel therapeutic targets like the use of human recombinant il-18 binding protein. j clin immunol (2019) 39 (suppl 1):s1-s151 s69 maintenance; smarcal1 is enriched in cells that maintain telomeres via the alternative lengthening of telomeres pathway and smarcal1decifient cells demonstrate telomere instability with replication fork collapse and increased telomere-associated dna damage. [1, 2] telomere analysis of 4 siod patients, including one patient who received a hematopoietic stem cell transplant (hsct) 20 years prior, as well as 5 heterozygous family members revealed significantly shorter telomeres in siod patients compared to heterozygous family members and compared to agematched, healthy controls. methods: peripheral blood mononuclear cells were isolated using a ficoll-hypaque density gradient, cryopreserved, then sent to repeat diagnostics in north vancouver, bc. telomere length measurements were performed at a single-cell level using flow-fluorescence in situ hybridization as previously described. [3] telomere length was measured in total lymphocytes, naive and memory enriched t cells, b cells, and nk cells and compared to reference samples from age-matched, healthy individuals. results: compared to age-matched healthy controls, three siod individuals had mean telomere lengths (mtls) less than the 1st percentile for age across all lymphocyte subsets (total lymphocytes, b cells, nk cells, naã¯ve and memory t cells). in comparison, three unaffected family members had normal mtls (10th percentile< x <90th percentile) across all subsets, and two unaffected family members had low mtls (1st< x <10th percentile) in some subsets. the siod individual who received a matched-sibling hsct 20 years prior, had normal mtl in nk cells (10th < x <90th percentile) but low mtls (1st< x <10th percentile) for all other subsets. conclusions: these data show that siod patients have significantly impaired telomere lengths across multiple lymphocyte lineages and support a limiting role for smarcal1 deficiency in telomere maintenance. in comparison, unaffected family members, heterozygous for smarcal1 mutations, have mean telomere lengths that are normal or slightly low for age. this suggests that abnormally short telomeres are seen in individuals with homozygous but not heterozygous smarcal1 mutations. for the individual who received a hsct, we do not have pre and post-hsct telomere data, but these results support obtaining pre and post-hsct telomere length analysis in future cases. abnormally short telomeres have been linked to widespread perturbation of gene expression. [4] we hypothesize that smarcal1 deficiency, by the effect of stalled forks and shortened telomeres, leads to perturbation in the transcriptome of affected tissues. shortened telomeres may explain the reduced hematopoietic bone marrow production in siod, as bone marrow failure is a cardinal feature of dyskeratosis congenita, a disorder of impaired telomere maintenance. future studies to investigate the role of telomere maintenance in siod include measurement of telomerase activity in polyclonally activated t cells and transcriptome analysis using rna-seq background: yellow fever is a potentially fatal disease for which only supportive treatment is available. vaccination is the primary strategy for prevention of this disease and the vaccine is extremely effective, but there are a few specific populations where it is contraindicated. regarding iga deficiency (the most frequent primary immunodeficiency), current recommendations in the literature are controversial. there are no specific studies in this disease, so case series addressing the safety or possible adverse events after vaccination are essential for decisionmaking during epidemic scenarios, as experienced in brazil in the last years. in this context, this study aimed to describe adverse events after the use of the yellow fever vaccine in iga deficient patients. method: a retrospective cross-sectional study was conducted including iga deficient patients followed at a specialized pediatric outpatient clinic between 2017 and 2018. all patients had at least one year of follow-up. immunoglobulin levels, antibody response to vaccines and lymphocyte subset count were evaluated to exclude other immunodeficiencies or the presence of abnormalities that could contraindicate vaccination. demographic data, the presence of infections and comorbidities, use of immunosuppressive medication and adverse events after vaccine administration of the vaccine were described. results: thirty-eight patients with iga deficiency were included in the study and 18 received the vaccine. vaccinated patients had a mean age at the time of the study of 13.7 years (sd â± 3.5y). six out of the 18 presented comorbidities: thyroiditis (n=3), type 1 diabetes mellitus (n=1), celiac disease (n=1) and juvenile rheumatoid arthritis (n=1). all patients were atopic and only one had recurrent infections in the last year despite the use of antibiotic prophylaxis. all 18 patients had normal igg and igm levels for their age, positive vaccine responses for measles, rubella and mumps, and age-appropriate lymphocyte subset count. after 6 months of observation, no immediate or late adverse events were reported. among the 20 non-vaccinated patients, only one had a formal contraindication (systemic erythematosus lupus using immunosuppressive therapy). five out of the 20 non-vaccinated patients reported being afraid of receiving the vaccine, 7 still intended to receive it and for other 7 patients data regarding vaccination was unavailable. conclusion: despite the small number of patients, the absence of adverse events in this case series suggests that immunization with yellow fever vaccine may be safe in iga deficient patients, excluded other contraindications. more studies are essential to confirm the safety and help the decision-making process regarding the vaccine administration for iga deficient patients, especially in this yellow fever outbreak scenario. introduction/backround: immunodeficiency, centromeric instability, and facial anomalies syndrome (icf) is a rare group of autosomal recessive disorders involving the triad of hypogammaglobulinemia, centromeric instability, and facial anomalies. the majority of patients have hypo-or agammaglobulinemia, but t cell defects have also been reported. we present the case of a child with icf-2 who presented with nk deficiency and ultimately developed an ebv-driven malignancy and was successfully treated with bone marrow transplant. methods: whole exome sequencing and nk cell function via 51-cr cytotoxicity assay and phenotyping via flow cytometry were performed at baylor college of medicine and texas childrens hospital. centromeric banding studies were performed at university of pittsburgh medical center. results: the female patient presented at 3 months of age with cmv pneumonitis and persistent cmv viremia requiring treatment followed by prophylaxis with valgancyclovir. she initially had hypogammaglobulinemia and low t, b, and nk cells; she had normal trecs, lymphocyte mitogen proliferation responses and zap 70, mhci and mhcii expression. the hypogammaglobulinemia and t-and b-cell lymphopenia resolved within 9 months after initial presentation as she clinically improved from her cmv infection. she was found to have nk cell deficiency on three separate commercially tested samples. whole exome sequencing revealed a homozygous variant in zbtb24 indicative of icf-2 syndrome that was confirmed with sanger sequencing (c.1492_1493del, p.q498vfs). repeat nk cell studies confirmed impaired function, and phenotyping showed an increase in cd56-bright and a decrease in cd16-positive cells, suggesting either impaired transition from immature to mature nk cells or impaired survival of mature cells. her karyotype and centromeric banding studies were normal, as were centromeric instability studies. she later developed a memory b-cell defect and presented at 34 months of age with persistent fever, respiratory distress, loss of vaccine titers, hypogammaglobulinemia and low b and t cells. she was found to have ebv viremia and an eber-positive diffuse large b-cell lymphoma in her right lung. due to tenuous clinical status, she received rituximab for treatment of ebv prior to definitive lymphoma diagnosis. she was treated with chemotherapy per protocol anhl1131, group b (pre-phase with cop, courses 1 and 2 with copadm, and courses 3 and 4 with cym) and her course was complicated by seizures attributed to methotrexate toxicity. she ultimately underwent reduced intensity conditioning with hydroxyurea, alemtuzumab, fludarabine, mephalan, and thiotepa followed by a cd-34 selected, hla-matched, unrelated donor peripheral blood stem cell transplant. her early post-transplant course was complicated by adeno-, ebv, and cmv viremia, all successfully treated with antivirals and a donor lymphocyte infusion. she is now greater than 8 months posttransplant, off immunosuppression with 100% donor engraftment, no evidence of organ toxicity or gvhd, and with excellent immune reconstitution. conclusions: this is the first reported case of impaired nk cell function and phenotype and ebv-driven malignancy in a patient with icf-2. this case expands the phenotype of icf-2 and suggests that early bone marrow transplant should be considered in these children. it also demonstrates a novel requirement for zbtb24 in human nk cell maturation and function. rationale: common variable immunodeficiency (cvid) is a disorder that affects the production of immunoglobulins and is associated with development of autoimmunity. multiple mutations have been described that are associated with cvid, but plcg2 mutations have only been described in patients with phospholipase c gamma 2 (plc2) associated antibody deficiency and immune dysregulation (plaid) and autoinflammatory plc2 associated antibody deficiency and immune dysregulation (aplaid). we present a case of a 44 y/o male cvid patient with recurrent upper respiratory tract infections, steroid-dependent autoimmune thrombocytopenia, low b cell count, hepatosplenomegaly, and restrictive lung disease. he was found with a variant of unknown significance at the plcg2 gene. in contrast to plaid our patient does not exhibit cold urticaria. method: case presentation of a cvid patient followed in our clinics. patients chart and previous laboratories were reviewed. sequence analysis and deletion/duplication cvid panel testing was performed using invitaeâ© discussion: genetic testing has revolutionized the diagnosis of immune deficiencies, but variants of unknown significance are being increasingly reported. in this case, a variant of uncertain significance was identified which replaces threonine for alanine at codon 829 of the plcg2 protein. this codon is located at the sh3 domain, which is part of a region that provides auto-inhibitory enzymatic functions. plaid mutations have been identified in sh2 domain, but it has been known that both sh2 and sh3 domains facilitate plcg association with other proteins. studies with deletion of plcg2 gene have shown functional abnormalities in b cells, natural killer cells and mast cells. to our knowledge, there has not been any previous report of a cvid patient with a variant mutation at the sh3 domain of the plcg2 gene without being diagnosed as plaid or aplaid. our patient has immunodeficiency, recurrent upper respiratory tract infections, steroid-dependent recurrent autoimmune thrombocytopenia, rheumatoid arthritis, hepatosplenomegaly, early-osteoporosis and restrictive lung disease. he does not have cold urticaria as seen in plaid, but exhibits autoimmunity not observed in aplaid. conclusion: conclusion: plcg2 is an important protein in the pathway of b cell development. a novel mutation in the sh3 domain of the plcg2 gene may be associated with the cvid phenotype of low b cells and autoimmunity. this could lead to a gain-of-function mutation as seen in plaid but without early-onset cold urticaria. functional studies are required to confirm the significance of this mutation. primary (or familial) hemophagocytic lymphohistiocytosis (hlh) is a rare, life-threatening hyper-inflammatory disease affecting mainly young children. it is caused by mutations in genes involved in the granule-dependent cytotoxic pathway, and is characterized by extreme inflammation and massive tissue infiltration by activated t cells and macrophages. to this day, hematopoietic stem cell transplantation is the only available curative treatment with a transplantrelated mortality of 30%. thus, the development of new, more efficient anti-inflammatory treatments would be a significant advancement in the treatment of hlh. here, we hypothesize that combination therapies targeting both jak-dependent and independent cytokines will be more effective than either one alone to reduce the lifethreatening symptoms induced by this pathology. using a perforin-deficient mouse model of hlh, we first compared the effect of targeting individual cytokines with blocking antibodies on the progression of the disease. we show that blocking ifng and il-18, but not il-6, significantly reduces the severity of hlh. targeting the jak-stat signalling pathway with ruxolitinib, a specific inhibitor of jak1 and jak2, downstream of ifng and il-6, but not il-18, is similarly beneficial. more importantly, combination therapies using ruxolitinib and blocking antibodies to either ifng or il-18 show synergistic effects, further mitigating the progression of the disease. these results suggest that jak-dependent and independent cytokines drive the pathogenicity of hlh in perforin-deficient mice. it further supports that ruxolitinib, although effective in reducing the symptoms of hlh, should be used in combination with anti-ifng and/or anti-il-18 antibodies to prevent hlh progression. this is particular relevant since the former were recently approved for the treatment of hlh while the latter (il-18 binding proteins) are in clinical trials for il-18-dependent macrophage activation syndromes. despite the increased risk of opportunistic lung infection in patients with severe t cell dysfunction (e.g. cd40l deficiency) and/or severe cd4 t cell lymphopenia, we are not aware of any reports of disseminated pneumocystis jiroveci infection in non-human immunodeficiency virus (hiv) patients with primary immunodeficiency (pid). we report the first case, to our knowledge, of disseminated pjp in a patient with cvid like/ctla4 haploinsufficiency. he had been diagnosed with common variable immunodeficiency (cvid) in 2009, approximately eight years prior to being referred to us, and was on intravenous immunoglobulin (ivig). he was also diagnosed with multilineage evans syndrome in 2015. his medical history was also significant for potential granulomatous lymphocytic interstitial lung disease (glild) (lung biopsy in the remote past with interstitial disease), significant splenomegaly (29.4 cm), severe portal hypertension, nodular liver disease (likely nodular regenerative hyperplasia) complicated by anasarca, history of chronic diarrhea (potential enteropathy), lymphadenopathy s/p biopsy with nodular lymphoid hyperplasia, and a history of multiple pneumonias. in 2017, he had developed disseminated pjp with lung, liver, and bone involvement. the t2 vertebra pjp invasion was confirmed with a bone biopsy; gomori methenamine silver staining and pcr were performed and concluded pjp. he was treated with trimethoprim sulfamethoxazole (tmp-smx) and steroids, then was continued on tmp-smx prophylaxis. due to his liver damage and his chronic neutropenia, tmp-smx was replaced by atovaquone as a secondary prophylaxis for pjp. his laboratory studies were significant for an absolute neutrophil count of 1.54 k/ul, absolute lymphocyte count of 0.61 k/ul, hemoglobin of 12.7 g/dl, platelets of 78 k/ul, total bilirubin of 2 . t-cell receptor beta chain repertoire analysis showed an oligoclonal distribution. severe combined immunodeficiency panel through ambry genetic testing was negative as was genetic testing for cd40l deficiency. given his complex clinical history, whole exome sequencing was obtained and detected an autosomal dominant heterozygous missense mutation (c.436g>a) implicated in ctla-4 haploinsufficiency and previously reported by schwab et al. our patient is currently undergoing therapy with abatacept (ctla-4 fusion protein), which has been reported to improve glild, splenomegaly and enteropathy in patients with ctla-4 haploinsufficiency. he is improving on this regimen. he has met with the stem cell transplant team, but at this point of time, due to his abnormal lung function, his liver damage and his significant splenomegaly, he is not a good candidate. defects in the nf-b signaling pathway are implicated in the pathogenesis of several primary immune deficiencies in humans. the clinical features of these conditions vary significantly, reflecting the complexity of the pathway, and its broad role in innate and adaptive immune responses, and the development and differentiation of lymphoid organs. here we report the first case of a human pid caused by a homozygous mutation in nfkbid in a 30 year-old male. he was the second child of consanguineous parents, and was diagnosed with possible cvid at the age of 16, after recurrent episodes of pneumococcal pneumonia. however the clinical features have evolved over time; he developed severe ebv infection at age 18, causing hepatitis and pancreatitis. at the age of 20, he presented with an anca-negative systemic vasculitis, manifesting as pulmonary haemorrhage, and acute necrotizing pauci-immune glomerulonephritis. pulsed methylprednisolone and cyclophosphamide induced an initial remission, however, relapse a year later led to end-stage renal failure. he is now dialysis-dependent, and due to the underlying pid, and chronic cmv viraemia, is not a candidate for renal transplantation. genomic dna was subjected to whole-exome sequencing. variants were filtered using a model of autosomal-recessive inheritance and functional analysis of primary cells was performed. we identified a novel, homozygous, single-base deletion resulting in a frame-shift, and premature stop in nfkbid. nfkbid encodes ibns, a non-classical inhibitor of nf-b signaling. at diagnosis the patient had reduced levels of igg2, iga and igm, elevated ige, with absent humoral immune responses to pneumococcal polysaccharide vaccine, and an intact response to tetanus. lymphocyte numbers were initially within normal reference ranges, albeit with an increased proportion of cd4+:cd8+ t cells. however, over time there has been a significant reduction in b cells and cd8+ t cells. cd4+ t cells demonstrated a skewing towards a central memory phenotype (cd45ro+/ccr7+), and cd4 t cell proliferative responses to pha were comparable to a healthy control. functional analysis of primary cells from the proband revealed a complete absence of bns protein expression, dysregulated nf-b signaling, and elevated pro-inflammatory cytokine production. the patient is currently receiving a trial of targeted therapy to modulate the aberrant immune responses. this novel pid highlights the importance of regulation of nf-b signalling, in orchestrating an appropriate immune response, maintenance of self-tolerance, and protection against viral pathogens. primary immunodeficiency diseases (pid) are a heterogeneous group of conditions with variable clinical features that are frequently associated with significant diagnostic delay. accurate diagnosis has significant therapeutic benefit and may lead to personalized therapies. we established the immunology flagship of melbourne genomics health alliance in australia to determine the clinical utility of genomic sequencing for diagnosis and management of individuals with suspected and confirmed cases of pid. 198 adults and children with suspected or confirmed pid (n=153), autoinflammatory disease (n=33) and hereditary angioedema (hae, n=11) were recruited to the melbourne genomics immunology flagship. whole-exome sequencing (wes) was performed, with targeted gene analysis. variant curation and reporting was performed according to the american council of medical genetics guidelines. overall, wes was diagnostic in 15% (30/198), confirming a preexisting diagnosis in 7% (14/198), and offering a new or more specific diagnosis in 8% (16/198). variants of uncertain significance were identified in a further 28 patients (14%) in genes known to be associated with their clinical diagnosis, that warrant further functional validation. in the hae group, diagnosis was confirmed in only 5 patients (45%), suggesting that wes may not be the appropriate technique for genetic diagnosis in this condition. a higher diagnostic rate was observed for autoinflammatory disorders (20%; 8/40) compared to pid (12%; 18/146). of those who received a diagnosis, immediate changes to patient management and treatment occurred for 17/29 patients (59%), including hsct for 3 and specific targeted therapy for 11 (38%) individuals. we have demonstrated the utility of wes for accurate diagnosis of complex immune diseases, with the potential to change diagnoses, guide therapeutic intervention and provide opportunities for genetic counseling. further longitudinal analysis will determine clinical outcomes and health economic implications of genomic sequencing for diagnosis and management of immunological conditions in australia. at birth he had neonatal asphyxia and cerebral palsy. at 4 years old he had presented involuntary movements, left paresis, bilateral horizontal nystagmus. at 8 years of age, he had a right nasal obstruction. it was resected by otorhinolist and informed by biopsy: inflammatory polyp and chronic sinusitis. he has had 3 pneumonias, sinusitis and diarrhea. at the age of 13 years, the ataxia telangiectasia was confirmed by sequencing with pcr (62 exons, 91711 bp) of the atm gene: transition g> a, nucleotide position 2250, codon 750, affecting splicing. alpha fetoprotein 572-606.90 u/ml. brain mri, say cerebellar atrophy. he had igg 685 mg / dl -734 mg / dl, iga 0.00 mg / dl, <1 mg / dl, igm 268 mg / dl -315 mg / dl, ige 0.10 -<1 iu / ml. subclasses of igg: igg3: 0.05 g / dl, igg4: 0.04 gr/dl, low. igg anti hepatitis b 6,22. no seroconversion. hiv negative tcd3 + lymphocytes: 32,40%, = 553 cells / mm3, ltcd4 +: 23,78% = 413,21 cel / mm3, ltcd8 +: 7,69% = 133,5 cells / mm3, cd4 / cd8: 3.09. for all of the above, common variable immunodeficiency was diagnosed. he receives human immunoglobulin. at 16, i arrived at this hospital due to fever, respiratory distress and lymphadenopathy in the neck. ct showed ganglionic conglomerate on right side neck. lymph node biopsy: strong tumors with cd20 and bcl2, weak and moderate diffuse pax-5; negativity with cd68, cd3 and cd10, and a cell proliferation index with ki67 of 50%, diagnosis: diffuse large b cell lymphoma. treated with rituximab and chemotherapy. lymphoma completely remitted. conclusion: the association ataxia telangiectasia and lymphoma is frequent. by contrast, cvid and ataxia telangiectasia are extraordinarily rare. introduction: chronic granulomatous disease (cgd) is a primary immunodeficiency wherein affected patients are susceptible recurrent infections caused by specific bacteria and fungi as a result of defective nadph activity. additionally, inflammatory complications involving the bowel and lungs can cause significant morbidity. currently the only proven permanent cure to cgd remains hematopoietic stem cell transplant. case: a 25-year-old patient was diagnosed in infancy with x-linked cgd. at age 5yrs he received a nonmyeloablative peripheral blood stem cell transplant from his 10/10 non-carrier sister as previously reported (nejm 344:881, 2001) . conditioning was cyclophosphamide (60 mg/kg) on d-6 and d-7; daily fludarabine (25mg/m2) on d-5 through d-1; antithymocyte globulin at 40mg/kg on d-5 through d-2. posttransplant immunosuppression consisted of cyclosporine on d-4 through d+100. he received 7.8x106 cd34+ peripheral blood stem cells which were t-cell depleted with 1x105 add back of cd3+ cells on day 0. after 10 days of neutropenia (anc <500) there were signs of engraftment. per protocol, he received donor peripheral-blood lymphocytes containing 2.0x106 cd3+ cells/kg on d+ 30 after transplantation. since donor t cells constituted less than 60 percent of his circulating cd3+ t cells and he had no graft versus-host disease, he received 1.0â¬107 cd3+ cells/kg on d+60. after the discontinuation of cyclosporine, he received a total of three donor-lymphocyte infusions (1.0â¬107 cd3+ cells/kg) at 90-day intervals achieving 100% t cell and myeloid engraftment at 26 months post-transplant with no acute nor chronic gvhd. at last follow-up 6 years post-transplant (2004) he had 100% and 98% lymphoid and myeloid peripheral chimerisms, respectively. the patient and family declined further periodic followup. then, in october 2018 he presented with malaise, cough and fevers. he eventually was found to have a large consolidation and a bal grew burkholderia cepacia. his dhr showed 12% activity and peripheral blood myeloid and lymphoid chimerisms were 12% and 60%, respectively. discussion: this late graft failure following peripheral blood transplant occurred following a conditioning regimen which is not the current standard for transplant in cgd. in the case series in which this patients transplant is reported (nejm 2001), another patients myeloid chimerism fell to 15% by 3 years post-transplant, remaining stable at that level of chimerism without any serious infections over regular periodic follow up to the present time. current regimens typically include busulfan to enhance engraftment and prevent graft failure. this case reinforces the need for prolonged monitoring of primary immune deficiency patients after transplantation. introduction: with the introduction of severe combined immunodeficiency (scid) newborn screen (nbs) in the state of kansas in 2017, a case of complete digeorge syndrome (dgs) was discovered in an infant born to a diabetic mother with atypical features. this is the first dgs case diagnosed after adding the scid nbs, which emphasizes the need to establish scid nbs in all 50 states. case presentation: the female infant was born via spontaneous vaginal delivery at 39 1/7 weeks to a 31 year old g1 now p1 mother. maternal history was significant for chronic hypertension, obesity, insulin dependent type 2 diabetes, anxiety, depression, and scoliosis. the infant was noted to have a left sided abdominal wall defect and hernia, imaging identifying left renal agenesis, and was initially suspicious for vater syndrome. fortunately, the infant's scid nbs revealed low t cell receptor excision circles (trecs). her initial white blood cell count was 14.1 with an absolute lymphocyte count of 2.679 k/ul. ebv pcr, cmv pcr, and hiv studies were negative. chest imaging discovered absent thymus, abnormal vertebrae with only 10 ribs on the right and 12 ribs on the left, and abnormally formed thoracic vertebrae (t7). echocardiogram detected an atrial septal defect measuring 0.32 cm, possible pfo versus secundum asd. endocrinology was consulted for management of labile calcium and phosphorus levels. fish was negative for 22q11.2 deletion. microarray r evealed a variant of unknown signif icance arr[grch37]2p11.2(86285942_86506132)x3. sequence analysis of combined and severe immune deficiency genes showed a variant of uncertain significance c.544c>a (p.leu182met). management and outcome: additional evaluation included: cd3 67ul (1700-3600ul), cd4 51ul (1700-2800ul), cd8 19ul (800-1200ul), cd45ra 14 cells/ul (1100-5200cells/ul), normal cd 19, and cd 16/ 56, normal immunoglobulin g level, and normal dihydrorhodamine assay. skeletal survey, ct abdomen and chest, and hla typing were performed in preparation for thymic transplant. discussion: patients with complete dgs, a form of scid found in less than 1 percent of patients with 22qds, have absent thymus and a t cell count <3 standard deviations below normal for age (typically <50 naã¯ve cd3+ t cells/mm3). in a large series of patients with complete dgs, only 52 percent had an identifiable 22q11.2 deletion [1] . infants of a diabetic mother have various genetic and syndromic associations including diabetic embryopathy. [2] despite the importance of immunological aspects in pregnancy, few studies have reported on the cellular immune modifications of diabetic embryopathy. diabetes during pregnancy may affect the development of the thymus and thus maturation of the immune system in the offspring. [3] the recent addition of a trec assay to newborn screening can identify such a subset of infants with atypical presentations. scid nbs uses an assay for trecs, a biomarker of t cell development. [4] [5] [6] this initial presentation now places the immunologist in the role of "first responder" with regard to diagnosis and management of these patients, who may present with atypical features. newer genetic and molecular techniques now allow for earlier identification of immune defects in such disorders with life-long clinical concerns. [7] references: introduction/background: goods syndrome is a rare cause of combined b-and t-cell immunodeficiency occurring in association with a thymoma. affected patents are susceptible to bacterial, fungal, viral, and opportunistic infections. an association with autoimmunity has also been reported. current knowledge of goods syndrome is primarily limited to case reports and small series. objectives: to examine the spectrum of clinical and laboratory features of a major cohort of goods syndrome patients in the us. methods: we conducted a retrospective analysis of patients with goods syndrome in the usidnet registry and the mount sinai hospital (msh) cohort. r e s u l t s : we i d e n t i f i e d 2 0 p a t i e n t s w i t h t h y m o m a a n d hypogammaglobulinemia (usidnet, n=11; msh, n=9; median age: 60 years; female: 45%), representing data from 151 patient years. the median age at diagnosis of thymoma and hypogammaglobulinemia were 52 years (range 31-85), and 50.5 years (range 28-86), respectively. two patients were deceased (at age 65 and 70 years, cause unspecified). all patients had low igg (median 313mg/dl, range 47-699). iga and igm were reduced in 90% and 45% of patients, respectively. low cd19+ b cells (median 0.5/mm^3, range 0-28) were reported in all available records. the absence of cd19+ b cells was observed up to 21 years postthymectomy. a wide range of additional laboratory abnormalities were identified: low cd4+ t cells (n=5), low cd8+ t cells (n=2), low cd4/ cd8 ratio (n=6), low nk cells (n=6), and absent peripheral eosinophils (n=8). the most common sites of infections were lower respiratory (70%), upper respiratory (55%), and gastrointestinal (35%). in addition, sepsis (15%), meningoencephalitis (5%), osteomyelitis (5%), and urinary tract infection (5%) were also observed. identifiable infectious agents included: bacteria (35%), virus (35%), fungus (25%), parasites (10%), and protozoa (5%), with opportunistic infections recorded in 25% of patients. opportunistic infections were significantly associated with absolute cd4 lymphopenia (p=0.048, fishers exact test). enterovirus was identified as a previously unreported cause of meningoencephalitis in this population. autoimmune manifestations were reported in 45% of patients, with a higher prevalence of inflammatory colitis (20%) than previously reported. hashimoto thyroiditis, fibromyositis, and bronchiolitis obliterans organizing pneumonia (n=1 each) were identified as previously unreported autoimmune/inflammatory conditions in this population. a case of alopecia areata was also observed. additionally, bronchiectasis was recorded in 20% of patients. all patients were initiated on immunoglobulin replacement, with antibiotics prophylaxis in 20%, and immunosuppressive medications employed in 10% of patients post diagnosis of immunodeficiency. conclusion: goods syndrome is a combined immunodeficiency, with a wide range of autoimmunity in a subset of patients. we expanded upon the spectrum of associated infectious and inflammatory complications through a major us cohort. persistent immune dysregulation was observed up to 2 decades post-thymectomy. introduction: primary immunodeficiencies (pids) constitute a large group of rare disorders that affect the immune systems function. some pid patients develop autoimmunity in addition to having increased susceptibility to infections due to their impaired immunity [917] . (1) case presentation/ management: a 43 year old caucasian female with history of bipolar disorder, factor v leiden deficiency, anti thrombin 3 deficiency, pulmonary embolism, endometriosis, and seasonal allergies was evaluated for chronic granulomatous disease (cgd) in 2007. the main symptoms were inflammatory breast lesions necessitating 4 surgeries on the right breast, and back, facial, genital, ocular, mouth, and scalp sores. biopsy with cultures of the wounds was positive for corynebacterium, coagulase-negative staphylococcus, enterococcus, bacteroides species, and provatella. neutrophil oxidative burst was ordered by the infectious disease specialist and showed normal and abnormal neutrophil populations, a finding consistent with cgd carrier. patient was started on interferon gamma-1b after failing multiple courses of antibiotics. her symptoms were well controlled on interferon gamma-1b 100mcg/0.5ml sq every other day, trimethoprim 100mg tab (2tabs in am and 1 tab in pm), cefixime 400mg once daily, and topical mupirocin as needed except for her recurrent genital ulcers. cgd can be rarely associated with oral ulcers however there is a limited literature describing associated genital ulcers. according to the international study group diagnostic criteria published in 1990 (2), the patient was diagnosed by a rheumatologist as having behcets disease (bd). there are no pathognomonic laboratory tests in bd; as a result, the diagnosis is made clinically. patient failed a trial of colchicine and was later started on cyclosporine, which resulted in decrease of her mouth and genital ulcers. discussion: bd is a rare disease mostly seen along the silk road. the prevalence has been reported as 0.12 (usa) to 370 (in a single village, northern turkey) for 100 000 inhabitants. (3) cgd is a primary immunodeficiency caused by defects in any of the five subunits of the nadph oxidase complex responsible for the respiratory burst in phagocytic leukocytes. patients with cgd are at increased risk of life-threatening infections with catalase-positive bacteria and fungi, and inflammatory complications such as cgd colitis. (4) reports of cgd female carriers with discoid lupus erythematosus, photosensitivity rashes, and other autoimmune phenomena have been published [48, 49] (4) . to the best of our knowledge, this is the first case to report bd in an affected cgd carrier. the treatment of inflammatory disease in patients with cgd poses a difficult balance between therapeutic immunosuppression and the increased risk of severe infection. (5) . high dose intravenous immunoglobulin, and targeted therapies such as ctla4-ig for t cell mediated pathologies, rituximab for b-cell mediated pathologies, and anti-tnf for ibd, may be preferable over the broad immunosuppressive activity of glucocorticoids. in addition, emerging evidence suggests that hematopoietic stem cell transplantation has indication for cases that have been difficult to control using immunosuppression. (1) given all that, our case emphasizes the need to maintain suspicion for autoimmune disorders / immune dysregulation in patients with pid. introduction: cd40-ligand deficiency is an x-linked combined immunodeficiency, characterized by susceptibility to infection, often with associated neutropenia, malignancy, and autoimmunity. central nervous system (cns) manifestations are less commonly reported than respiratory or gastrointestinal complications, but are most often attributed to infection. herein we describe a challenging case of gradual onset episodic memory loss, confusion, and unilateral hemiplegia in a young male with cd40ligand deficiency. case presentation: the patient is a 13-year-old male with cd40-ligand deficiency on immunoglobulin replacement therapy presenting with recurrent, episodic altered mental status (ams) and gradual neurocognitive decline. initial neurologic symptoms began at age 11 years, and included fever, nausea, and eyelid fluttering. initial comprehensive infectious workup at this time, including blood and urine cultures, lyme antibody, serum pcr for hsv, cmv, ebv, respiratory viral pcr including atypical viruses, csf studies including culture, lyme eia, pcrs for enterov i r u s , v z v, e b v, c m v, h s v 1 / 2 w e r e u n r e v e a l i n g . electroencephalogram (eeg) and mri displayed generalized slowing and global atrophy, respectively. definitive diagnosis was not made. the patient continued to decline with worsening developmental delay and memory loss. one year later, at age 12 years, he had a recurrent episode of ams with repeat negative infectious workup including blood and urine cultures, respiratory virus pcr including atypical viruses, csf culture including acid fast bacillus and fungi, cryptococcal antigen, viral encephalitis panel by pcr, and serum pcr for ebv and hhv-6. eeg at this time showed left hemispheric epileptogenic potential, consistent with seizure activity. his presentation, at age 13 years, was notable for right-sided hemiplegia with facial numbness, dysarthria, nausea, and fever. he was found to have anello virus on pcr of csf, abnormal left temporal region on eeg, and global atrophy with stable, diffuse generalized volume loss on mri. he was diagnosed with occult anello virus-induced encephalitis with hemiplegic migraine and discharged on valproate. discussion: here we present the first reported case of anello virus detected by pcr in a cd40-ligand deficient male with neurocognitive manifestations, attributed primarily to hemiplegic migraine. given the anello virus prevalence and relatively avirulent character, it is presumed to be unlikely culprit for encephalitis; however, the significance of this finding is as yet unknown. this case highlights diagnostic challenges in immunodeficiency: infection may go undetected by standard diagnostic techniques; however, the significance of infections identified with advanced techniques may not yet be understood. background: henoch-shã¶nlein purpura (hsp) is an iga-mediated small vessel vasculitis that presents with a tetrad of abdominal pain, arthritis, glomerulonephritis, and purpura. hsp is typically a selflimiting disease of childhood following a viral illness. there is no universal treatment for patients with chronic or recurrent hsp. we report a chronic refractory case of hsp that was successfully treated with a tumor necrosis factor inhibitor (tnfi), etanercept. etanercept functions as recombinant protein that consists of a tnf-alpha receptor ligand-binding region that links to the fc portion of human igg. it is currently approved for use in 5 diseases: juvenile rheumatoid arthritis, rheumatoid arthritis, ankylosing spondylitis, plaque psoriasis, psoriatic arthritis. tnfi are categorized into two broad categories, recombinant receptors (etanercept) and neutralizing antibodies (ex. infliximab and adalimumab). there have been prior case reports of hsp associated with tnfi agents during the treatment of other autoimmune conditions in the adult population. to our knowledge, there have been 3 prior etanercept related hsp reports, one report associated with adalimumab, and one with infliximab. however, there has been no prior report of etanercept use successfully treating chronic refractory hsp. case presentation: a 16-year-old native american male with 3 year history of chronic hsp, hla-b27 positive, and enthesitis related arthritis who was initially treated with steroids, sulfasalazine and methotrexate for symptoms of joint pain and purpura. his iga level was 545 mg/dl prior to therapy. despite treatment for one month of steroids, eight months of sulfasalazine exclusively and eight months of methotrexate and sulfasalazine, he continued to have persistent purpura on bilateral extremities without improvement. he was subsequently initiated on etanercept 50mg weekly and methotrexate was discontinued. approximately one month later, his rash significantly improved. his rash and joint pain recurs when he misses a dose of etanercept. punch biopsies were taken 3 months after initiation of etanercept. the biopsies of a lesion from his left arm showed early leukocytoclastic vasculitis and from his left leg showed weak granular deposition of iga, igm and c3 within vessel walls. there is controversy whether this is a true iga vasculitis. however, we believe that his clinical presentation and the deposition of iga and c3 within blood vessel walls seen on biopsy correlates with chronic henoch-shã¶nlein purpura. conclusion: there is no standard treatment of chronic hsp, but there are reports of benefit with nsaid and corticosteroids. per our literature review, there are no prior reports of etanercept use in the treatment of chronic hsp. tnf inhibitor, etanercept should be considered as a treatment for chronic refractory hsp in the pediatric population as it has showed rapid resolution of purpura in this case report. further studies of etanercept in the treatment of chronic hsp should be conducted given the controversial literature of anti-tnf ab induced hsp during the treatment of other autoimmune diseases. although clinical manifestations of iron overload appear to be quite uncommon in patients who are heterozygous carriers of hfa mutation, we present cases that appear to suggest an increased risk non allergic rhino-sinusitis. case report: we present a 66 year old gentleman with perennial colored rhinorrhea, with facial pressure and tenderness, constant post nasal drip, dry cough and bilateral congestion that had been going on for the past several years. he also had a frequent urge to clear his throat and had frequent episodes of sore throat despite having no history of gerd or lpr. he reported to have multiple sinus infections every year that would progress to pneumonia and eventually require long courses of oral antibiotics. all started in his 40s intensified in the recent past. he had 3 other siblings; one died in his 40s due to liver complications of hh and had a carrier sister and brother with a hx of sino nasal problems exactly similar to the patients. his exam was remarkable for bilateral narrowed nasal passages and moderate edema of the mucosa. his rhinolaryngoscopy showed significant edema and purulent drainage, most notably from bilateral middle meati. his skin test was negative. his cbc showed a wbc count of 6.7/ml with 2% eosinophils and his immunoglobulin panel showed an iga of 236 mg/dl, igg of 1190 mg/dl and ige of 31 mg/dl. patient was placed on alkalol sinus rinses and azelastine nasal spray, which he reported to work pretty well. he left for costa rica and is expected to return back with his siblings to a&i clinic in the coming months. discussion: hh is one of the most common inherited disorders in people of northern european descent with an incidence of 1:200 and carrier rate of 1:10.. most affected hh patients are homozygous for the mutation designated c282y at the hfe gene located at the 6th chromosome. unlike hereditary hemochromatosis, clinical manifestations of iron overload appear to be quite uncommon in patients who are heterozygous carriers. hh patients are at risk for a number of infections with bacteria whose virulence is increased in the presence of excess tissue iron. hh is also a risk factor for acute fulminant frs . here the mechanism is postulated to be due to quantitative or qualitative neutrophil defects as this condition is mostly seen in patients with dm, aplastic anemia, and can happen in patients undergoing antineoplastic chemotherapy. no known increased susceptibility for infections through either mechanism is postulated for patients with the heterozygous carrier state. here we present 3 hh carrier patients who present with recurrent rhinosinusitis with no allergen sensitizations and normal ige levels. since most fungal immunity is at the tissue level and is cytokine driven, it can be speculated that increased tissue levels of iron might interfere with mechanisms of innate immunity. chief, human immunological diseases section, laboratory of clinical immunology and microbiology, niaid, nih, bethesda, md background: dedicator of cytokinesis 8 (dock8) mutations are associated with a combined immunodeficiency disorder marked by atopic features, infectious susceptibility with a striking preponderance of cutaneous viral disease, and a risk for the development of malignancy including lymphoma. almost all cases can be diagnosed by documentation of the loss of dock8 protein expression. methods: we describe a 22-year-old male with a diagnosis of pre-b cell acute lymphoblastic leukemia (all) followed by epstein-barr virus (ebv) associated diffuse large b cell lymphoma (dlbcl). compound heterozygous mutations in dock8 were documented following the completion of whole exome sequencing (wes). the pathogenicity of the variants was assessed. flow cytometric quantification of intracellular dock8 protein was completed. dock8 protein function was assessed by evaluating the morphology of patient lymphocytes when migrating in a 3d collagen matrix. results: a concern for a primary immunodeficiency was raised due to a history of recurrent otitis media which began at 12 months of age. by 4 years of age, numerous warts were noted on his fingers; however, they were transient for a duration of only 2 years. no atopic features were appreciated. at 15 years of age, a diagnosis of pre-b cell all was made. during all therapy, infectious complications were severe including an intestinal perforation, osteomyelitis, and sepsis. at 22 years of age, still in an ongoing remission from his all, an incidental finding of a lung nodule led to a diagnosis of ebv-associated dlbcl. during therapy, however, infectious complications were again severe including a soft tissue infection and sepsis. wes was performed and compound heterozygous mutations in dock8 (c.1128_1132del and c.4474-1g>c) were documented. flow cytometric quantification of intracellular dock8 protein was normal when compared to a normal control. nevertheless, additional functional assessment of dock8 protein was completed. when migrating through a 3d collagen matrix, 45% of the patient lymphocytes studied demonstrated abnormal elongation (stretch ratio > 8 defined by length/width) compared with 10% of lymphocytes from a normal control. he is being evaluated for hematopoietic stem cell transplant. conclusion: autosomal recessive mutations in dock8 are a rare cause of a combined immunodeficiency marked by atopic features, infectious susceptibility with a striking preponderance of cutaneous viral disease, and a risk for the development of malignancy including lymphoma. here, pre-b cell all followed by the development of a subsequent malignant neoplasm (ebv-associated dlbcl) led to the discovery of dock8 deficiency. hence, as our case underscores, for rare instances of high clinical suspicion despite normal dock8 protein expression, additional functional testing is crucial to make a definitive diagnosis and plan treatment. understanding the spectrum of dock8 mutants and their phenotypes will improve our understanding of dock8 deficiency. background: autosomal dominant hyperimmunoglobulin e syndrome (ad-hies) is a rare primary immunodeficiency caused by heterozygous loss-of-function mutations in the signal transducer and activator of transcription 3 (stat3) gene. ad-hies classically characterized by recurrent cold staphylococcal abscesses, pneumonia, eczema, and an elevation of ige level. other additional clinical manifestations of hies have been recognized including skeletal dysplasia (scoliosis, pathologic fractures, delayed dental deciduation), pneumatoceles, coronary-artery aneurysms, brain lesions, and chiari malformations. objective: to describe a unique case of abdominal abscesses in a patient with ad-hies. method: a 22-year-old female with known ad-hies (c.1144 c>t (p.arg382trp)) and a complicated history of early pneumococcal pneumonia and meningococcemia resulting in bilateral amputation below the knees along with loss of several digits, presented for evaluation of skin infection. she had a history of recurrent staphylococcal skin abscesses and presented with inability to use her prostheses due to pain from inflammation around her amputation sites. she underwent imaging and was found to have bilateral extremity abscesses with an associated osteomyelitis of her l tibia (which was found to be mrsa after incision and drainage). while receiving intravenous antibiotics for her osteomyelitis, she developed intractable abdominal pain. imaging showed a thick-walled, multi-septated, paranephric abscess as well as several smaller abscesses scattered throughout her abdomen. she underwent multiple drain placements and drainage of retroperitoneal fluid collections via interventional radiology (ir). purulent fluid from the abdominal abscess drainage grew mrsa. the patient continued to have re-accumulation of abscesses despite multiple drainages. repeat imaging noted increased paranephric abscesses which were not communicating with drains. given lack of response to several ir-placed abdominal drains and to 6 weeks of intravenous antibiotics, she had an open surgical washout with minimal improvement. hospital course was further complicated by development of a left lower lung lobe consolidation and sub-segmental pulmonary embolism necessitating treatment with heparin. finally, after several weeks of escalating antimicrobial therapy and with additional drain placements, the retroperitoneal abscesses started to recede. repeat abdominal imaging several months later while asymptomatic revealed slow but continuing resolution of the abscesses. conclusion: the present case raises awareness of an unusual location for infection in a patient with ad-hies. although the majority of complications of ad-hies are sinopulmonary and skin infections, recalcitrant intra-abdominal abscesses should be considered in the differential of infections in hies. introduction/background: the recent epidemiologic studies have revealed that primary immunodeficiencies (pids) are more common than previously thought. however, there are very few data on epidemiology of pids in korea. objectives: we attempted to estimate the pid epidemiology and disease burden in korea and provide the background information for pid registry for future. methods: to review the previously reported scientific studies, pubmed, koreanmed, google scholar were searched. any studies on pids reported in scientific journal (korean or international) from january 2001 to november 2018 were searched. both korean and english reports were searched. diagnosis for pid was categorized from group i to group xi according to 2017 iuis phenotypic classification. study period was divided into two periods: period 1 from 2001 to 2005 and period 2 from 2006 to 2018, because there was a multicenter study to estimate pid epidemiology from 2001 to 2005. in addition, the number of pid patients and the cost for care were estimated among patients who requested reimbursement to health insurance review and assessment service (hira) korea for one year in 2017. results: a total of 334 pid patients were identified in 75 reports. one hundred and ninety-nine patients (20 reports) and 135 patients (55 reports) were found in period 1 and period 2, respectively. the pids were reported in 11 patients for immunodeficiencies affecting cellular and humoral immunity, 23 patients for combined immunodeficiency with associated or syndromic features, 143 patients for predominantly antibody deficiencies, 33 patients for diseases of immune dysregulation, 113 patients for congenital defects of phagocyte, 1 patient for defects in intrinsic and innate immunity, 4 patients for auto-inflammatory disorders, 6 patients for complement deficiencies, and none for phenocopies of pid. from hira reimbursement data, the number of pid patients were 42 for combined immunodeficiency, 486 for predominantly antibody deficiency, 47 for common variable immunodeficiency, 135 for functional defect of neutrophils, 238 for immunodeficiency associated with other major defects, 272 for other immunodeficiencies. a total of 1,220 pid patients were treated for 14,316 days and $3,351,678 was reimbursed in 2017. conclusions: we performed a systematic review on published studies for pid in medical journals and national open data system of hira to estimate the pid disease burden for the first time in korea. to obtain more information on true pid epidemiology and disease burden in korea, a national multicenter study for pid registry is required in the future. micro-thrombocytopenia is one of the most serious challenges for wiskott-aldrich syndrome (was) and x-linked thrombocytopenia (xlt) patients. thrombocytopenia leads to severe, potentially life-threatening, bleeding episodes, which require frequent transfusions and account for 23% of deaths in patients experiencing was mutations. the gold standard treatment for was patients is hematopoietic stem cell transplantation (hsct) from an hla-identical donor but more recently a number of gene therapy (gt) trials in europe and usa showed promising results. in particular, it has been shown that was patients receiving lentiviral mediated gt, consisting of autologous cd34+ cells transduced with lentiviral vector encoding the human was gene under the control of the endogenous promoter, in combination with a reduced intensity conditioning regimen, have a significant increase in platelet (plt) counts. even though plt counts do not reach normal levels, treated patients decreased the severity and frequency of bleedings. here, in a cohort of 4 xlt and 16 was patients, fifteen treated with gt, the plt phenotype and function were analyzed by electron microscopy, flow cytometry and proteomic profile. the aim of the project is to assess the presence of plt defects in was untreated patients and the impact of gt treatment on the correction of plt behavior. we demonstrate that plts of untreated was patients have reduced size and abnormal ultrastructure along with hyperactivated phenotype at steady state, showing increased expression of cd62p, activated iib3 integrin and cd40l; conversely, activation response to agonist and aggregation capacity are both decreased. analyzing plt samples isolated from treated patients, we found that gt restores plt size and ultrastructure very early after treatment and reduces the hyperactivated phenotype proportionally to was protein (wasp) expression and follow-up length. plts isolated from gt treated patients showed a normal activation response to agonists and restored aggregation capacity in 5 out of 7 analysed patients. by proteomics, various protein pathways were found downregulated in untreated plt samples, mainly involving cytoskeletal-rearrangement proteins, integrins, signal transduction molecules, vesicles-transport proteins; additionally, decreased metabolic capacity were observed. these results are in line with the functional defects observed in plts in terms of activation and aggregation. conversely, the expression of protein-pathways found downregulated in untreated patients is comparable to healthy controls in gt-treated plt samples, reflecting the amelioration of plt phenotype and function. overall, our study highlights the coexistence of multiple defects in the activation and aggregation responses occurring in was patient plts in absence of wasp. gt was able to normalize the plt proteomic profile followed by consequent restoration of plt ultrastructure and phenotype, suggesting gt is responsible for the observed reduction of bleeding episodes in treated patients. introduction: pik3cd is an autosomal dominant genetic disorder of the immune system that results in persistent activation of pi3k. signaling through pi3k is essential for immune cell regulation of metabolism, migration, proliferation and differentiation, leading patient to present with lymphadenopathy, immunodeficiency and senescent t cells. the mutated protein causes t cells to over activate and mature too quickly leading to their death, this over activation also blocks the maturation of b cells. case presentation: a 51-year-old female with a childhood history of failure to thrive, asthma, chronic rhinitis and common variable immunodeficiency on intravenous immunoglobulin replacement, was seen in immunology clinic to establish care. she reported frequent episodes of pneumonia and bronchitis in her childhood. her family history was significant for family members with leukopenia, but no diagnosed immunodeficiency. patient had 1 son who did not report symptoms concerning for immunodeficiency. physical exam was within normal limits with no lymphadenopathy. laboratory examinations exhibited normal iga (185 mg/dl), igg (800 mg/dl), and igm (100 mg/dl). while flow cytometry showed normal absolute cd3 687 (570-2400 cells/ul), cd4 (540 cells/ul), nk cells (151 cells/ul), cd19 (179 cells/ul), cd45ra (160 cells/ul), cd45ro (311 cells/ul), cd2 (757 cells/ul), and hla-dr (173 cells/ul), nonswitched memory cells (9 cell/ul) and class-switched memory cells: (15 cells/ul). (4-62 cells/ul). vaccine response was not pursued as patient had been on ivig. genetic testing was pursued, and revealed a mutation in pik3cd gene, specifically a mutation in the c.2320g>a; p.val774met variant (rs370932461). this mutation though seen in databases, is not currently reported in medical literature as associated with this condition. based on these, ct chest was ordered to screen for bronchiectasis, adenopathy and lymphoma. ct showed no cardiopulmonary disease or adenopathy, but did show an incidental adrenal mass which is now being worked up. while the pattern of inheritance of this mutation is autosomal dominant, her son is asymptomatic and testing of her son has not been pursued, though it was advised for her cousins given history of leukopenia. patient has continued on igg replacement therapy. conclusion: recent publication by the clinical immunology society suggests consideration for next generation sequencing when it can affect future family planning or it has treatment and prognostic implications. this case highlights all aspects of the importance of genetic testing as part of the diagnosis of cvid, since it can affect progeny, it offers the possibility of treatment with immune modulating agents and has implications on screening, since patients are at increased risk for malignancies. background: abnormal v(d) j recombination activity in patients with mutations in the recombination-activating genes 1 and 2 (rag1/2) results in markedly reduced usage of distal vand j genes at the t cell receptor alpha (tra) locus. mucosa-associated invariant t (mait) cells express a semi-invariant t cell receptor containing the distal trav1-2 gene. mait cells can be identified by flow cytometry using a mab directed against valpha 7.2, which recognizes the product of the trav1-2 gene. by performing high throughput sequencing (hts) of tra rearrangements and flow cytometry, we have confirmed lack of t cells using distal valpha genes in patients with known rag mutations. we now report that flow cytometry with mab against valpha 7.2 successfully identified rag deficiency in two patients with an atypical presentation. methods: tra rearrangements were analyzed by hts using gdna from sorted t cell subsets from rag-mutated patients and healthy donors. distal valpha usage was measured in whole blood by flow cytometric analysis with an anti-valpha 7.2 antibody. rag mutations were detected by sanger sequencing. patients were enrolled in niaid protocol 18-i-0041. results: hts of tra rearrangements revealed lack of distal trav and traj gene usage in patients with rag1/2 mutations. the presence of circulating mait cells in controls and patients with known rag1/2 mutations and various clinical phenotypes was analyzed by flow cytometry using mab against valpha 7.2. we found a virtual lack of valpha 7.2 expression in rag mutated patients (<0.5%) compared to controls (2-8%) . we used the valpha 7.2 assay to test two patients with unknown immunodeficiency manifesting as skin granulomas and autoimmune cytopenia, and found nearly absent expression (0.14% and 0.08%). targeted sequencing of rag1/2 revealed that both patients were compound heterozygous for rag1 mutations: p.r112h/p.c328y and p.r410w/p.r507q, respectively. conclusions: patients with mutations in rag1/2 demonstrate a skewing of their tcralpha repertoire. the reduction in recombinase activity in these patients does not allow for rearrangements of the most distal valpha segments. rapid identification of patients lacking valpha 7.2+ t cells by flow cytometry may prompt sanger sequencing and identification of rag1/2 mutations in a matter of days. this assay represents a simple but powerful tool to reduce the cost and time associated with other analysis methods. acknowledgements: supported by dir/niaid/nih. director, centro de inmunologã­a clã­nica dra.bezrodnik y equipo introduction: the fate of effector t cells is strongly dependent on the expression of bcl-6 or blimp-1, which are inhibited reciprocally through a complex signaling pathway. several studies have shown that bcl-6 is a key transcription factor for differentiation towards the follicular helper t cells (tfh) lineage able to collaborate with b lymphocytes (bl). on the contrary, the transcription factor blimp-1 is highly expressed in t lymphocytes th1, th2 and treg, thus regulating the differentiation towards tfh. materials and methods: whole fresh blood and peripheral mononuclear cells from a patient with homozygous mutation in stat5b were analysed by flow cytometry. analysis of ctfh (cd4+cd45ra-cxcr5+), ctfh1 (cxcr3+), ctfh17 (ccr6+), ctfh2 (cxcr3-ccr6-), naã¯ve bl (lb igm+igd+cd27-), memory (mbl) (lb igm+ igd-cd27+), switched (mbl-sw) (igd-igm-) and plasmablast (pbc) (cd27+cd38++) cells was performed. immunoglobulins were measured in serum. results: the patient with stat5b deficiency showed increased values of ctfh (38%) (healthy donors p10-p90: 7,9-17,8 %) that presented an activated phenotype (icos+ and pd-1+) with a skewed to a th17 profile (ccr6+), consistent with her hipergammaglobulinemia and the marked and sustained increase in the switched mbl and pbc subpopulations in peripheral blood over the years. discusion: this immunological phenotype described in the patient with stat5b deficiency could explain in part the pathophysiology of the autoimmune disorders. this patient (as well as the other two patients with mutations in stat5b previously described by our group), have had chronic hypergammaglobulinemia, autoantibodies and consequently autoimmune processes (psoriasis, hypothyroidism, eczema, alopecia and celiac disease, among others). we believe that the link between this clinical symptomatology and the molecular defect relies in the fact that the absence of stat5b promotes a greater expression of bcl-6, which generates a bias towards the production of ctfh cells, that give rise to a greater activation of lb, generation of lbm and plasma cells (dysregulation in the cg), events that manifest as hypergammaglobulinemia and autoimmunity. in summary, we provide promising evidence of the mechanisms that lead to autoimmunity in this type of patients that could also be a consequence of the defect in the regulation of gc, highlighting the crucial role of stat5b in the humoral immune response and maintenance of the tolerance of the immune system. background/introduction: the term primary immunodeficiencies (pid) encompasses a phenotypically and genetically diverse group of conditions. genetic testing for these conditions can guide treatment, reduce morbidity and mortality, allow for genetic counseling, and identification of additional at-risk family members. however, this testing can be complicated by a number of factors, including pseudogenes, high homology, methodology limitations, and the heterogeneous nature of pids. methods: mayo clinic laboratories launched their first set of nine pid next generation sequencing (ngs) tests approximately one year ago. these tests include one single gene assay for gata2 deficiency and eight targeted next generation sequencing panels for: atypical hemolytic uremic syndrome (ahus), autoinflammatory disorders, b-cell disorders, monogenic irritable bowel disease (ibd), phagocytic defects, severe combined immunodeficiencies (scid), and severe or cyclic neutropenia. herein we summarize our first year of experience with these ngs tests, with a focus on the eight targeted panel tests. results: from march 2018 through november 2018 we performed testing for 341 cases. our highest volume of tests was for the ahus panel (127/341 cases, 41%). a variant was reported in 76/341 cases (22.29%). these variants included variants of uncertain significance, likely pathogenic variants and pathogenic variants. the indication with the highest percentage of cases where a variant was reported was scid (9/13 cases, 69.23%). the number of cases that were considered solved, where the genotype likely explains the patients phenotype, varied widely by indication. twenty cases were found to have a pathogenic or likely pathogenic variant or variants; however 2/20 cases were heterozygotes for an autosomal recessive condition and were not considered solved cases. the panel with the highest percentage of solved cases is our scid panel (4/13 cases, 30.77%). conversely, we have yet to solve an autoinflammatory, irritable bowel disease, or telomere defects case; however 20% of cases in each of those three panels have had a variant of uncertain significance reported. we hypothesize that one of the reasons for the low detection rate for these three panels is inappropriate test orders. we are also actively looking for ways to update all 8 panels to increase detection rates and clinical utility, for example expanding the gene list of our ibd panel, including large deletion/duplication detection, and including ncf1, a difficult gene to capture by ngs, on the phagocytic panel. finally, we present the molecular findings from a number of interesting cases that were solved using our targeted ngs panels. conclusions: the launch of our pid ngs tests in march of 2018 has allowed us to aid patients by confirming diagnoses and providing molecular diagnoses that will enable more accurate genetic counseling and risk assessment. we have also uncovered areas for improvement, both on the clinical side: provider education is important to enable better identification of patients who can benefit from molecular genetic testing for pids, and on the laboratory side: introduction of more expanded panels and additional methodologies. the progressive decrease of red blood cells, platelets or neutrophils via a self-directed immune process is jointly termed as autoimmune cytopenias. while autoimmune cytopenias, including autoimmune hemolytic anemia (aiha), immune thrombocytopenic purpura (itp), and autoimmune neutropenia (an), are a common presentation of autoimmunity in the general population, they are particularly frequent and can appear as the first sign in patients with primary immunodeficiencies (pids). possible causes of cytopenia in pids comprise mainly immune dysregulation, bone marrow failure (bmf) and myelodysplasia. our goal is to investigate possible immune mediated mechanisms underlying chronic cytopenia in children in order to achieve an early diagnosis and consequently offer timely and appropriate therapy. we selected 24 patients affected by chronic cytopenia, evaluated with immunophenotyping by flow-cytometry; data were subjected to multivariate analysis by principal component analysis (pca). next generation sequencing (ngs) analysis of genes frequently implicated in pids was performed. among the patients, 5 were affected by bone marrow failure, of which 2 were diagnosed with fanconi anemia and severe congenital neutropenia; 12 were affected by immune-mediated cytopenia and 7 by idiopathic cytopenia. the immunephenotyping showed a typical pattern of cd8 t cell subpopulations expression in patients compared with healthy donors with an increase of naã¯ve t cells and a reduction of central memory (cm) and effector memory (em) t cells levels. we observed a decrease in total b cells, b switched and b memory cells and an increase in cd21low cells. pca showed an overlap between groups, however it revealed a peculiar trend of some single patient, suggesting the pathway involved in immune defect. preliminary results from ngs studies revealed genetic variations in genes previously associated with pids in 10 out of 11 patients investigated. in particular we identify one patient with a mutation in fas, one with a mutation in aire and one with a mutation in ikaros. concerning the remaining patients further studies are ongoing to validate the pathogenicity of the genetic variations. pca is a very effective tool to analyze several parameters at the same time, highlighting patients whose phenotype shows the main peculiarities. the presence of specific lymphocyte subpopulation patterns can be important indicators of immune-mediated cytopenias and helpful signs of specific pids that should promptly be investigated with genetic analysis. the rapid of discovery of novel, monogenic primary immunodeficiencies has been made possible by the broad availability of clinical whole exome sequencing (wes). however, clinical wes has major shortcomings that should be understood by practicing immunologists. focusing on the 2017 iuis list of~330 monogenic primary immunodeficiency genes, we show here limitations in coverage that could significantly impact clinical interpretation. on the agilent whole exome capture kit, the most common wes platform, there are a number of genes with exons that are poorly covered. specifically, there are at least 94 genes with less than 100% exonic coverage, 26 with less than 99% coverage and 5 with less than 90% coverage (e.g. ikbkb, ncf1, taci, unc93b1 and tbx1). beyond this challenging technical issue, there are more subtle issues as well. these include the presence of pseudogenes in at least 17 of our genes (e.g. ak2, c1qbp, cd46, cftr, cr2, msn, ncf1, ncstn, ikbkg, nhp2, pms2, pten, rnaseh2c, rps, sbds and was), which can make accurate sequencing very challenging. finally, there are many known causative intronic (e.g. btk, ctla-4, wasp) and copy number variant mutations (e.g. rag1 and xiap) as well as large deletions (e.g. dock8) that we cannot expect to be optimally covered using wes. this list of genes requires consideration even with a negative exome and may require additional approaches including whole genome sequencing, sanger sequencing, cnv arrays and/or long-read ngs sequencing. wes is a powerful genomic diagnostic tool, but to avoid missing key diagnostic insights using these alternative approaches may be critical when certain genes are in the differential diagnosis. going forward, as pid phenotypes continue to broaden, these issues remain fundamentally important even if these genes are not obviously implicated in a given clinical phenotype. more physicians are utilizing targeted genetic panels to reach a definitive diagnosis for their patients with immunodeficiency. however, this increase in testing also has led to the discovery of many more variants of uncertain significance (vus) in the genes tested. these findings can often leave the patient and the physician with more questions than answers. we present a patient with recurrent infections found to have multiple variants of uncertain significance in several genes associated with primary immunodeficiency. a 13-year-old female who was diagnosed with crohns disease at age 9 after intestinal perforation and jejunal resection experienced two discrete episodes of epstein barr virus (ebv) meningoencephalitis and septic shock. the first episode was diagnosed when patient had fever and altered mental status and occurred prior to her crohns disease diagnosis and the second episode was complicated with altered mental status, disseminated intravascular coagulation (dic) and hypotension requiring picu admission. aside from these two major infections, the family denied any other infections requiring antibiotics in the last 5 years and reported a remote history of repeated streptococcal pharyngitis that have not recurred. immunology was consulted at the time of the second episode of meningoencephalitis and work up was mainly unremarkable with normal immunoglobulins, adequate vaccine response to hib, tetanus, diphtheria, rubella, measles and pneumococcus (18 out of 22 protective titers). she had normal t cell numbers with slightly decreased natural killer numbers for age. neutrophil studies showed normal dihydrorhodamine (dhr) analysis, glucose-6-phosphate dehydrogenase levels and myeloperoxidase (mpo) stain. commercial testing of her toll like receptors (1) (2) (3) (4) (5) (6) (7) (8) showed normal function. invitae primary immunodeficiency panel demonstrated a heterozygous variant in nod2 (c2.104c>t; p.arg702trp) as well as heterozygous variants of uncertain significance in il7r (c.662g>t; p.ser221ile) and tlr3 (c.889c>g; p.leu297val). the patients nod2 variant is known to be associated with an increased risk for crohns disease. even with our patients presentation with recurrent severe viral infections and ibd, it is not immediately clear how these genetic results explain the pathology. innate immune defects probably contribute to her presentation and it is currently unclear if and how the combination of multiple genetic variants has left her immunologically vulnerable. we use this case to demonstrate that even when genetic testing does not elucidate a clearcut diagnosis of primary immunodeficiency, it can still provide helpful insight into a patients underlying immune phenotype. introduction: xiap deficiency is a rare primary immune deficiency characterized by hemophagocytic lymphohistiocytosis, recurrent fever and inflammatory syndromes, inflammatory bowel disease, hypogammaglobulinemia, recurrent infections, and other manifestations. loss of xiap results in abnormal tnf receptor signaling and nlrp3 inflammasome actvity which leads to dysregulated production of il-1beta and il-18. we hypothesized that suppressing the nlrp3 inflammasome with either targeted deletion or pharmacologic inhibition would suppress abnormal production and secretion of inflammatory il-1beta and il-18. methods: bone marrow derived macrophages (bmdms) from control, xiap-deficient, and xiap and nlrp3 double knock-out mice were derived with 1 week of culture in l929-cell conditioned media. bmdms were stimulated with a variety of tlr agonists or tnf-alpha, with or without a variety of inhibitors including the nlrp3 inhibitor mcc950, the cathepsin b inhibitor ca-074, and quercetin, which is a natural flavonoid (antioxidant) found in many fruits and vegetables, and available as a nutritional supplement. il-1beta, il-18, and tnf-alpha were measured in supernatants by elisa, and cell death was evaluated by flow cytometry using pi exclusion. results: as expected, bmdms from xiap deficient mice had markedly increased tlr-agonist-or tnf-alpha-induced il-1beta production compared to normal bmdms. genetic deletion of nlrp3 and the pretreatment of cells with the nlrp3 inhibitor mcc950 greatly reduced abnormal il-1beta production; residual production of il-1beta could be inhibited by caspase-8 inhibition. pre-treatment of cells with the cathepsin b inhibitor ca-074 also decreased cytokine production but was toxic at higher concentrations. quercetin reliably abrogated il-1beta, and also il-18. quercetin was found to inhibit priming of the nlrp3 inflammasome (decreased upregulation of pro-il1beta and nlrp3) and also decreased tnf-alpha secretion following tlr agonist stimulation. conclusion: quercetin suppresses the nlrp3 inflammasome and may be a promising therapeutic option for patients with xiap deficiency. it prevents il-1beta and il-18 secretion. it is a particularly appealing option given that it is a naturally occurring antioxidant, has a great safety profile, and is readily available as a nutritional supplement. human studies are needed. recently, single cell rna sequencing (scrnaseq) analysis in mice has disclosed an unexpected complexity of thymic stromal cells, and medullary thymic epithelial cells (mtecs) in particular. however, the developmental origin, hierarchy, and function of these subpopulations remain ill-defined. moreover, although cortical tecs (ctecs) are thought to represent a more homogeneous population, their characterization has been largely restricted to the adult thymus. we have previously shown that impaired lymphostromal cross-talk in the thymus of patients with combined immunodeficiency (and of corresponding mouse models) is associated with abnormalities of thymic architecture and tec maturation. here, we sought to compare tec distribution and gene expression in wild-type (wt) and in mice carrying rag1 hypomorphic mutations observed in patients with combined immune deficiency and immune dysregulation. methods: multi-color flow cytometry and scrnaseq were used to analyze composition and distribution of ctec and mtec subpopulations in wt and rag1 mutant mice at various weeks of age (niaid animal protocol: lcim-6e). results: we observed that rag1 mutant mice have an excess of ctecs, and that their mtec compartment is predominantly represented by cells with high levels of mhc class ii (mhc-ii) expression, recapitulating the phenotype of neonatal wt thymi. while mhc-iihi mtecs are thought to represent a minor fraction of mtecs in adult wt mice and include mature aire+ cells, a relative abundance of mhc-iihi mtecs is observed also at neonatal age, where they are thought to represent immature mtecs. to define more precisely tec maturation, we performed scrnaseq on sorted cd45-epcam+ cells, and identified 8 and 10 distinct clusters of tecs in wt and rag1 mutant mice, respectively. a large proportion of cells in rag1 mutant mice could be ascribed to the ctec compartment, confirming our previous flow cytometry and histopathology results. furthermore, scrnaseq analysis also disclosed a different distribution of mtec subsets in wt and rag1 mutant mice. to address the hypothesis that this difference in ctec and mtec abundance and subset distribution may reflect different maturation stages in tec development in wt and rag1 mutant mice, we will perform lineage tracing and transplantation experiments, and we will also extend tec scrnaseq analysis to wt and mutant mice of embryonic and neonatal age. in parallel, to evaluate the contribution of thymocyte maturation in shaping the stromal populations, scrnaseq will be performed on thymocytes. conclusions: we have further refined the complexity of tecs, and shown that impaired development of t cells in combined immune deficiency (as exemplified by rag1 mutant mice) has profound effects on the composition and maturation of tecs and may thus contribute to abnormalities of immune tolerance that are often associated with these conditions. the advent of next-generation sequencing (ngs), with the development of whole-exome sequencing (wes) in particular, has allowed the identification of unknown genetic lesions for many diseases and the implementation of specific therapeutic strategies. primary immunodeficiencies (pids) are a group of rare diseases which have benefited from ngs, with the discovery and molecular characterization of previously genetically undefined diseases and the identification of novel molecules involved in the regulation of the immune system. pids are often associated with autoimmune disease due to the dysregulation of the immune system as a whole. the clinical phenotypes are heterogeneous and often overlapping. while a monogenic cause of disease has been identified in a most subsets of patients, the recent application of whole-genome sequencing has found that a polygenic cause is likely. our aim is to investigate the genetic background of patients with immunedysregulations and autoimmunity and to evaluate the possible pathogenicity of the identified gene variants through extensive functional studies. we select 19 patients with sign of immunedysregulation and autoimmunity, extended immunophenotyping and next-generation sequencing (ngs) analysis of 50 genes frequently implicated in pids was performed. in six of them we identify a single gene as responsible of the clinical feature. in particular, we identify two patients with gain of function mutation in stat3, one patient with a mutation in ctla4, one patient with an activating pik3cd mutation, one with a rag1 mutation and one with a fas mutation. in most of them variants in multiple genes have been detected. interestingly, we find that some genes are recurrently mutated in more then one patient such as was, dock8, casp10, casp8, nfatc2 and fcgr3a. further studies are ongoing to validate the effect of the variations identified. our results strongly suggest that the old hypothesis, based on a single gene mutation as a cause of illness, should be revised in favor of the concept that "is the sum that causes the effect" and that a different point of view on pids now seems inevitable. physician, omni allergy, immunology, and asthma introduction/background: immunoglobulin replacement therapy (igrt) may be optimized to reduce the severity and incidence of infections and potentially delay or abrogate the development of pulmonary complications of primary immune deficiencies. pulmonary complications including bronchiectasis are common in common variable immune deficiency (cvid) and contribute significantly to morbidity and mortality in these patients. it remains unclear whether continued obstructive bronchial changes are a result of repeated respiratory infections, associated inflammation and immune dysregulation, or simply lung-damage that is irreversible by the time therapy is initiated. it has also been suggested that under-treatment in addition to the diagnostic delay may contribute to the development of bronchiectasis in patients with pid. lower serum igg levels with any given dose of immunoglobulin replacement therapy have been demonstrated in patients with bronchiectasis compared to those pid patients without this complication. in addition, earlier studies have shown that greater doses of ig (600 mg/kg/ month) may reduce the frequency and duration of infections and help prevent or slow progression of chronic lung disease. objective: to evaluate the prevalence of bronchiectasis in a cohort of patients with a diagnosis of cvid and identify associated ig dosing patterns and clinical outcomes. methods: data were analyzed from the ideal (immunoglobulin, diagnosis, evaluation, and key learnings) patient registry. this is a prospective, longitudinal registry study of patients receiving ig replacement therapy in the home or ambulatory infusion suite with one national home infusion provider. nursing and pharmacy standard of care forms were collected, and dose, infection rate, and prevalence of bronchiectasis were evaluated in patients with a diagnosis of cvid (icd-10 codes: d83.9, d83.1) results: there were 310 patients in the registry with cvid, 14 (4.5%) of which bronchiectasis was also observed. seventy-nine percent (n=246) of the study population was female, and 50% (n=7) of the cases of bronchiectasis were observed in females. the mean age of the patients with concurrent bronchiectasis was 65â±15.8 at start of care compared to 57â±15.8 in those without this observed bronchial obstruction. most bronchiectasis patients (n=11) received igrt subcutaneously every week with a mean dose of 123.8â±22.8 mg/kg/wk. the mean dose of ig in the 3 remaining patients receiving ig intravenously was 506.8â±82.0 mg/kg/month. the average annual rate of infection in ivig and scig patients with bronchiectasis was 1.6â±1.0 and 2.2â±1.3, respectively, however many were serious bacterial infections. at time of analysis, 7 of the bronchiectasis patients remained active in the registry and 7 had withdrawn. reasons for withdrawal included stopping igrt due to the following: patient decision (n=3), physician decision (n=1) insurance change (n=1), and patient expired (n=2). conclusions: there were 14 documented cases of bronchiectasis in our cohort of cvid registry patients, and dosing patterns aligned with standard doses despite the presence of bronchial obstruction. further studies are necessary to assess evolution of lung damage with respect to ig dosing in patients with cvid. background: activated phosphoinositide 3-kinase syndrome type 1 (apds1) is a combined immunodeficiency resulting from gain-offunction (gof) mutations in pik3cd, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (pi3k). this form of pid is characterized by recurrent respiratory tract infections, susceptibility to herpes virus infections, impaired antibody responses, lymphoproliferation and autoimmunity. previous studies showed that patients with apds1 have b cell defects that contribute to the clinical phenotype. furthermore, these patients display t cell abnormalities, including increased numbers of memory t cells and t follicular helper cells (tfh), reduction of naã¯ve t cells and impaired t regulatory cell (treg) function. whether these t cell abnormalities are also associated with perturbations of t cell repertoire in unknown. objective: we aimed to investigate the effects of increased pi3k signaling on the t-cell repertoire of patients with apds. methods: high throughput sequencing was used to study composition and diversity of t-cell receptor (tra) and t-cell receptor (trb) repertoire in sorted treg, tfh, conventional cd4+ (tconv), and cd8+ t cells from 4 patients with pik3cd gof mutations and healthy controls. results: treg cells of patients with apds1 show restriction of tra and trb repertoire diversity, and increased clonality. no repertoire restriction was detected in tfh, tconv, and cd8+ t cells from the same patients. however, the trb repertoire of treg and cd8+ cells was enriched for the presence of hydrophobic amino acids in position 6 and 7 of the cdr3, a biomarker of self-reactivity. conclusion: these data demonstrate that the t-cell repertoire of patients with apds1 is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition. furthermore, our result support the notion that the pi3k pathway is a key regulator of treg cell development and homeostasis in humans. j clin immunol (2019) 39 (suppl 1):s1-s151 s87 (4), iii. predominantly antibody deficiencies (2), i. immunodeficiencies affecting cellular and humoral immunity (1), vii. auto-inflammatory disorders (2), ix. phenocopies of pid (1) . two non related cases of ataxia-telangiectasia and one case of schimke syndrome (smarcal1 compound heterozygous mutation) were diagnosed in the last year. we observed a wide range of age (we evaluate adult and pediatric population) with a male:female ratio close to 1: immunodeficiency, immune dysregulation, and systemic autoimmunity. clinical diagnosis of these disorders is complicated by overlapping phenotypes. in april 2017, a 207-gene next generation sequencing (ngs) panel inclusive of copy number variation analysis was launched by a commercial laboratory to facilitate clinical diagnosis of primary immunodeficiency (pid), monogenic autoimmunity and autoinflammatory disorders. we assessed the outcomes of genetic testing utilizing this panel on a cohort of pediatric patients with immunohematologic phenotypes evaluated at our tertiary care center during an 18-month period (5/1/17-10/31/18). eligible subjects were evaluated by at least two of three providers from a multidisciplinary pediatric hematology-immunology team, including a hematology physician, immunology physician and a geneticist or genetic counselor. twenty-three patients met inclusion criteria; 20 (87%) were caucasian, 12 (52%) were male with an average age of 11.7 years. the two most common phenotypic diagnoses included cytopenias, single-or multilineage (leukopenia, neutropenia, anemia, thrombocytopenia) primarily attributed to autoimmune causes or hypogammaglobulinemia. five (22%) were given a definitive genetic diagnosis as a result of panel testing, though in two of these cases, the causative mutations were listed as variants of uncertain significance (vus). diagnoses included common variable immunodeficiency due to a pathogenic variant in nfkb2, stat3 multiorgan autoimmunity due to gain-of-function mutation, and familial cold autoinflammatory syndrome due to a pathogenic mutation in nlrp12. biallelic dnmt3b vus were found in a patient whose phenotype and further laboratory studies (including karyotype) were consistent with immunodeficiency-centromeric instability, facial anomalies syndrome. further, a stat3 vus was identified in a patient with multiorgan autoimmunity and his father with hypothyroidism; studies from an outside research laboratory were consistent with gain-of-function with this variant (private communication). an additional three patients had vus identified that were suspected to be related to their phenotype, prompting eligibility for research studies. four (17%) patients had increased risk alleles in nod2, conferring an increased risk of crohns disease. three (13%) patients had pathogenic or likely pathogenic carrier findings warranting genetic counseling. in addition, 47 vus (an average of 2 per patient) thought to be unrelated to phenotype were identified, necessitating further investigation and counseling. the use of an ngs panel in a cohort of pediatric patients with immunohematologic disorders led to a definitive diagnosis in 22% of previously undiagnosed patients and prompted further research investigation in several more. genetic testing also led to the identification of clinically significant carrier findings, risk alleles and 47 vus unrelated to phenotype, necessitating genetic counseling. our experience illustrates the value of genetic testing for diagnosis of immunohematologic disorders, and the importance of multidisciplinary care, including genetic counseling, for the proper evaluation and management of these patients. background: allogeneic hematopoietic cell transplantation (allohct) is curative for primary immune deficiencies (pid). however, many patients lack a fully-matched unaffected sibling, or may have an unknown underlying genetic defect, rendering it undesirable to use related donors. many pid patients have significant comorbidities at the time they are referred to allohct, precluding the use of myeloablative conditioning. the use of alternative donors with reduced-intensity conditioning (ric) has historically led to increased rates of graft failure, graft-versus-host disease (gvhd), and transplant-related mortality (trm). posttransplantation cyclophosphamide (ptcy) as gvhd prophylaxis immunomodulates the graft through the preferential sparing of regulatory t cells and hematopoietic stem cells from its cytotoxic effects, thus allowing for robust donor engraftment that overcomes the hla barrier while effectively preventing severe acute and chronic gvhd. we report the outcomes of two institutions using a ric allohct regimen with alternative donors and ptcy in patients with pid. design: we transplanted 35 pid patients (table 1) using alternative donors and ric, either serotherapy-free (n=21) or alemtuzumab-based (n=14). all patients received ptcy for gvhd prophylaxis on days +3 and +4, either alone (n=3), or combined with mycophenolate mofetil and either sirolimus (n=21) or tacrolimus (n=11). donors included haploidentical family members (n=16), matched unrelated (n=15), and mismatched unrelated (n=4). stem cell source was t cell-replete bone marrow (n=33) or peripheral blood stem cells (n=2). results: the median follow-up is 17 months (range 0.5-8 years). at 17 months, overall survival is 91%, and event-free survival (defined as alive without graft failure) is 83%. the median days of neutrophil and platelet engraftment are 17 (range 14-42) and 28 (range 15-110), respectively. there were 10 patients who developed acute gvhd, grade 1 (n=5) or grade 2 (n=5), and there were no cases of grade 3 or 4 agvhd. seven of eight patients treated with systemic corticosteroids responded, and one was corticosteroid-dependent, then responded to second-line therapy. one patient developed skin-only chronic gvhd, which responded to corticosteroids and puva light therapy. five patients developed graft failure, either primary (n=1) or secondary (n=4), and four were successfully re-transplanted and remain engrafted. one patient with secondary graft failure had autologous recovery and has not required a second allohct given some durable infection control gained during initial engraftment. there were three deaths prior to day 180 due to infection, and one death at 1.5 years secondary to presumed overdose. in ongoing follow-up of engrafted survivors (n=30), evidence of phenotype reversal has been demonstrated in all patients, with complete or ongoing resolution of some or all of their underlying disease manifestations, including infection, transfusion-dependence, autoimmunity, malignancy, and/or immune dysregulation. discussion: we have observed high rates of engraftment, low rates and severity of acute and chronic gvhd, and low trm in 35 patients with pid transplanted using alternative donors, ric, and ptcy-based gvhd prophylaxis. ric allohct with ptcy shows promise for curing pid, and its use minimizes toxicity and widely expands the donor pool, thus allowing us to offer this curative therapy to many more patients with pid. chronic granulomatous disease (cgd) is a primary immune disorder that involves mutations in the nicotinamide adenine dinucleotides (nadph) oxidase complex (deffert, cachat, & krause, 2014) . two-third of cgd cases are caused by loss-of-function mutations in the cybb gene that encodes the gp91pox subunit of the nadph. the increased in patients' life expectancy thanks to progress in diagnosis and management has underlined the burden of inflammatory manifestations occurring independently of infectious agents (dunogue et al., 2017; marciano et al., 2018) . cgd patients develop inflammatory granulomatous disorders, notably colitis, as a consequence of a dysregulated inflammasome activation. the treatment of inflammatory manifestations remains challenging, as it can be associated with an increased risk of infections. thus, understanding the pathophysiological mechanism of auto-inflammation in cgd could help improve the therapeutic arsenal for the management of these manifestations. to reveal the precise pathophysiological mechanism of auto-inflammation in cgd, we have developed a cellular model that reproduces the cgd phenotype in phagocytic cell. through crispr-cas9 gene-editing we generated a thp-1 c e l l l i n e h a r b o r i n g t h e p r e v i o u s l y d e s c r i b e d mu t a t i o n c.90_92delccginsggt (p.tyr30ter) in the cybb gene responsible for gp91phox knock-out by early termination of translation. this cell line recapitulates the phenotype of cgd phagocytes: (i) decreased h2o2 production (ii) and enhanced inflammatory responses after pma stimulation as evidenced by increased il-1, il-6 and tnfa secretion levels (kuijpers & lutter, 2012) . these features were rescued by complementation through lentiviral transduction of a wild type cybb gene. this new model will help us to investigate the auto-inflammation reported in cgd patients and also to propose new therapeutic targets of inflammatory manifestations in this disorder. interleukin-1 (il-1) driven responses. children with irak-4 deficiency are predisposed to recurrent and invasive infections secondary to streptococcus pneumoniae, staphylococcus aureus and other pyogenic bacteria with high mortality rates in early childhood. the frequency and severity of infections is thought to decrease with age due to the acquisition of humoral immunity and immunologic memory, however due to the rarity of the disease, the natural history of this condition beyond early childhood is not well described. objectives: we present three unrelated irak-4 deficient patients with persistent chronic rhinosinusitis with nasal polyposis that developed in childhood. cases: patient 1 is a 15 y/o male with compound heterozygous mutations in irak4 (p.g75afs*14/c.717-1g>t) with a history of recurrent s. pneumoniae osteomyelitis (left hip at age 9 and left knee at age 10) and c. septicum sepsis at age 9 following acute bowel perforation. additionally, he experienced recurrent aom during infancy and recurrent uti since age 9. despite prophylactic antibiotics and ivig, he has had recurrent polymicrobial (mrsa, s. pneumoniae, h. influenzae, p. aeruginosa, a. fumigatus) rhinosinusitis with nasal polyposis since age 4 refractory to medical management requiring surgical intervention and prolonged courses of iv antibiotics. patient 2 is an 11 y/o female with homozygous deletions (exons 10-12) in irak4 with a history of ruptured appendicitis complicated by pseudomonas abscess and bacteremia at age 2, culturenegative sepsis with septic arthritis and osteomyelitis of the right leg at age 3, and septic shock secondary to mssa bacteremia complicated by rhabdomyolysis and dic at age 5. she has a history of chronic rhinosinusitis, and despite ivig and prophylactic antibiotics, she developed polymicrobial (h. influenzae, b. fragilis) rhinosinusitis with associated nasal polyposis pending surgical management. patient 3 is a 10 y/o female with homozygous mutations in irak4 (q293x/q293x on exon 8) with a history of s. pneumoniae meningitis at 3 months, m. catarrhalis epiglottitis and neck cellulitis at 4 months, rsv bronchiolitis at 6 months, enterococcus bacteremia at 8 months, s. pneumoniae sepsis at age 2 and streptococcus lymphadenitis at age 9. despite ivig and prophylactic antibiotics, she developed recurrent polymicrobial (h. influenzae, b. fragilis, mssa, v. cholera, p. aeruginosa, a. fumigatus) rhinosinusitis refractory to medical management requiring surgical intervention and iv antibiotics. conclusions: in our centers experience, irak-4 deficient patients continue to suffer from infectious complications, most prominently recurrent polymicrobial sinus infections beyond early childhood. the consistent presence of sinonasal polyps in these children is unusual, as it is not typically found in uncomplicated pediatric chronic rhinosinusitis. these infections have occurred despite antimicrobial prophylaxis and ivig, highlighting the role of irak-4 in sinopulmonary epithelium. additionally, the infectious organisms identified in our patient cohort are not commonly associated with irak-4 deficiency. further study of chronic rhinosinusitis and nasal polyposis in a larger cohort of irak-4 deficient patients and other innate immunodeficiencies may help identify pathways for targeted treatment of these patients. introduction: chronic granulomatous disease (cgd) is an inherited phagocytic defect associated with inability to clear catalase positive organisms. infections in patients with cgd are severe and recalcitrant. commonest infections are pulmonary followed by soft tissue infections and suppurative lymphadenitis. osteomyelitis is an uncommon infection in patients with cgd. it poses several diagnostic and therapeutic challenge. we herein report our experience of osteomyelitis in cgd over the last 10 years. material and methods: review of records was carried out to describe the profile of osteomyelitis in cohort of patients with cgd at pediatric immunodeficiency clinic, advanced pediatrics centre, postgraduate institute of medical education and research, chandigarh, india. the diagnosis of cgd was based on nitroblue tetrazolium dye reduction test (nbt) and dihydrorhodamine reduction (dhr) assay. results: of the 63 patients with cgd, 8 (12.7%) had osteomyelitis (6 males and 2 females; age range 1-10 years). most patients had their first episode of serious infection in early childhood (mean age: 1.5 years). stimulation index (si) of dhr assay ranged from 1 to 4.58. mutational analysis was done in 5/8 patients (3 x-linked; 2 autosomal recessive). site of involvement was variable ribs-4; vertebrae-2; radius-1; skull-2; tibia-1. aspergillus fumigatus was the most common isolate (62%; 5/8); others had aspergillus flavus, aspergillus terreus and serratia marcescens each. all 4 patients with rib osteomyelitis had concurrent pneumonia, and fungus was isolated in all of them (aspergillus fumigatus-2, aspergillus flavus-1, zygomyces spp.-1). antifungals (intravenous amphotericin b) were given for a duration of 4-6 weeks and were followed by oral voriconazole in therapeutic doses for 3 to 6 months in majority of them. debridement and resection of ribs was required in one patient, while other patients were managed conservatively. out of 8 patients, 2 (25%) succumbed to pneumonia and respiratory failure. conclusion: osteomyelitis in the context of cgd is usually caused by aspergillus spp. involvement of ribs and vertebra usually occurs with the contiguous spread of infection from the lungs. therapy often requires prolonged duration of anti-microbials, and may require surgical debridement in addition to it. a 29-year-old woman with history of hypogammaglobulinemia and acute liver failure a 29-year-old woman with a 7-month history of nausea, vomiting, and abdominal pain was admitted to an outside hospital with new onset of jaundice and anasarca. liver biopsy was thought most consistent with alcoholic steatohepatitis, and she was discharged with counseling on alcohol cessation and medical management of liver disease. she presented to our facility for a second opinion. over the following days, she developed further rise in direct hyperbilirubinemia up to 19.2 mg/dl, new coagulopathy with an inr 2.06 and hypoalbuminemia to 1.7 mg/ dl in the absence of ongoing alcohol consumption. liver sonography revealed course echotexture and patent vessels. pcrs directed against multiple hepatotropic viruses were negative and copper studies were normal. due to a history of moderate alcohol consumption, she was started on high-dose corticosteroids due to a presumptive diagnosis of alcoholic hepatitis. additional history raised concern for a possible primary immunodeficiency, including idiopathic thrombocytopenic purpura at 11 years of age, multiple episodes of sinusitis treated with antibiotics and sinus surgery, one episode of suspected bacterial pneumonia, and one hospitalization for influenza a during which she developed neutropenia. in her 20s, she developed refractory genital warts, prompting infectious diseases evaluation. initial immune evaluation had revealed low immunoglobulins (iga <7 mg/dl, igg 198 mg/dl, igm 13 mg/dl) with very low responses to tetanus and diphtheria, despite a recent booster dose, and b and t cell lymphopenia (cd19+ 89 cells/î¼l, cd3+ 567 cells/î¼l, cd4+ 345v, cd 8+ 244 cells/î¼l, cd16/56+ 236 cells/î¼l); antigen and mitogen proliferation were not assessed. intravenous immunoglobulin replacement was initiated but discontinued by the patient due to infusion-related adverse effects, and she was lost to follow up until she presented with liver failure. both parents were deceased from cardiovascular disease in their 40s and she had no siblings. she had limited knowledge of family history but no known immune diseases. due to suspicion for genetic etiology of immune disorder and liver disease, we performed next-generation sequencing of a panel of over 200 genes implicated in primary immune deficiencies. patient was heterozygous for a nucleotide substation (c.1752+1g>a) within a splice site at the exon 16/intron 16 boundary of the nfkb1 gene. during the hospitalization, immunoglobulin replacement and trimethoprim-sulfamethoxazole prophylaxis were initiated. an attempt was made to refer the patient for additional immunological evaluation and transplantation evaluation but unfortunately, she developed worsening liver failure and multiple complications, including extended-spectrum beta-lactamase (esbl)-producing e. coli bacteremia, hypotension requiring vasopressors and extensive bowel ischemia, and died in the hospital. in summary, this case highlights both the risk of diagnostic delay in adult patients presenting with a primary immune deficiency and potential for genetic testing to clarify the diagnosis. while the particular genetic change has not been described, other splice site and predicted loss-offunction mutations have been reported as pathogenic in this gene, which have been implicated in autosomal dominant common variable immunodeficiency. this case further expands on the genetic causes and spectrum of disease associated with changes in the nfkb1 gene. introduction: malnutrition and micronutrient deficiency are underrecognized causes of acquired immunodeficiency in adults, and may occur even in patients with high body mass index (bmi). methods: a 46-year-old woman with a medical history significant for one remote urinary tract infection presented to the emergency department after sudden onset of severe right flank pain. the pain was accompanied by urinary frequency and not relieved by ibuprofen; she denied fevers or chills. she was diagnosed with pyelonephritis and discharged on ciprofloxacin, which was later changed to trimethoprim-sulfamethoxazole after her culture grew resistant e. coli. her pain continued despite treatment, prompting her to return to the hospital three days later. upon presentation, she was afebrile with blood pressure of 128/88 mmhg and heart rate of 86 bpm. her body mass index was 32.4 kg/m^2. her physical exam was otherwise notable for right costovertebral angle tenderness. laboratory studies revealed a leukocyte count of 14,300/ul with 83% neutrophils; alkaline phosphatase of 146 units/l and albumin of 2.7 g/dl, but otherwise normal liver function tests; normal lactic acid; and urinalysis with 3,000 wbc/hpf, 40 rbc/hpf, moderate bacteria, and the presence of wbc clumps. ct scan of the abdomen and pelvis demonstrated an obstructing 13 mm right renal stone with hydronephrosis and a right renal abscess contiguous with a right-sided hepatic abscess measuring 7.8 x 6.0 x 7.5 cm. she was treated with ceftriaxone and metronidazole, and underwent imaging-guided drainage of the abscesses. abscess cultures again grew resistant e. coli. she was discharged from the hospital with drains in place and a plan to continue trimethoprim-sulfamethoxazole until definitive management of her nephrolithiasis with ureteroscopy and lithotripsy. discussion: there remained the question of how an ostensibly immunocompetent patient had developed such severe intraabdominal infection with little systemic inflammatory response (e.g. no fever and only mild leukocytosis). a hiv antibody screen was negative. on further interview, she described a 200lb intentional weight loss over the preceding 2 years, accomplished by dietary restriction to less than 600 calories per day. nutritional assays revealed prealbumin, vitamin c, and vitamin b6 levels below the threshold of detection. she had low-normal b12 and b1. out of concern for an acquired immunodeficiency resulting from malnutrition with micronutrient deficiency, balanced nutrition was discussed with the patient who agreed to liberalize her diet. background: the past decade has brought dozens of new mendelian disorders of immunity. yet, the genetic contribution(s) to diverse disorders of the immune system remain largely unelucidated. the majority of research participants referred to the national institute of allergy and infectious diseases (niaid) for what may be a mendelian disorder evade molecular diagnosis. making progress in this area requires a coordinated, systematic, and transparent approach to clinical genomics research which leverages the unique environment at the national institutes of health clinical center (nih cc). methods/design: this study is designed to systematically apply exome sequencing and related technologies with clinical grade interpretation and reporting to niaid research participants at the nih cc under a single protocol in order to facilitate research and clinical genetics care across niaid. we are recruiting approximately 1000 participants per year from approximately 35 intramural clinical investigators. we generate genomic data, collect standardized phenotyping and report clinical interpretation in the medical record, all while providing linked genetic counseling. results: to date, we consented 1287 participants, we sent out 1058 samples for exome sequencing and 183 samples underwent copy number variant analysis. we have completed analysis for 359 families (502 individuals) and finalized and resulted 177 cases. here we present a case series illustrating some of our findings. case 1: a 10year-old female was referred to niaid for neonatal onset multisystem inflammatory disease (nomid). developmental delay and mild intellectual disability were appreciated on clinical evaluation. exome sequencing detected a mosaic novel likely pathogenic variant in nlrp3. chromosomal microarray analysis (cma) showed 㣠5 mb interstitial deletion of chromosome 12 previously associated with developmental delay and intellectual disability. case 2: a 10year-old ukrainian male was referred to niaid for the clinical diagnosis of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced). exome sequencing and cma did not detect pathogenic variants in aire, but did find a de novo variant in fam111b. defects in fam111b are associated with poikiloderma with tendon contractures, myopathy, and pulmonary fibrosis (poiktmp). the clinical features of the patient were consistent with poikmp. case 3: a 63-year-old man had a history of brain, liver and kidney nocardiosis, disseminated mac infection, prostate cancer and lymphoma. family history was significant for prostate cancer. exome sequencing showed a heterozygous pathogenic variant in brca2, associated with susceptibility to breast-ovarian, male breast, pancreatic and prostate cancer. conclusion: this case series illustrates that multiple diagnoses, unexpected diagnoses, secondary genomic findings, and data sharing helped identify variants in candidate genes. process standardization supports data integrity and efficiency while accommodating the need for investigator flexibility and providing tailored patient care. rationale: activated pi3 kinase delta syndrome (apds) is a primary immunodeficiency caused by dominant mutations that increase activity of phosphoinositide-3-kinase (pi3k). the catalytic subunit p110 is mainly expressed in cells of the hematopoietic system, primarily lymphocytes and myeloid cells, and mutations affect both b-and t-cells. we sought to further evaluate the role of the t-cell receptor (tcr) repertoire in immune dysregulation and the pathogenesis of autoimmunity and lymphoproliferation in patients with apds. methods: we evaluated the tcr repertoire in the peripheral blood in 3 patients with pik3cd mutations and compared these to the peripheral tcr repertoire in 26 patients with common variable immunodeficiency (cvid) and 50 healthy controls to investigate the role of the tcr in disease. the tcr repertoire in affected tissue of 2 patients with pik3cd mutations was also evaluated (tissue included lymph nodes for both patients, in addition to gastrointestinal tract and lung tissue in one patient). a fixed number of tcrs were subsampled (35,000 for blood and 5,000 for tissue) and diversity was calculated using the gini and shannon indexes. results: using the shannon and gini diversity indexes, the tcr repertoire in patients with pik3cd mutations had less diversity/ increased clonality as compared to healthy controls and those with cvid ( figure 1 ). for the two apds patients with biopsy tissue available for analysis, the diversity of the tcrs in tissue was increased as compared to the peripheral blood tcr repertoire ( figure 2 ). conclusions: pi3k plays an important role in the development and function of both b-and t-cells. patients with apds were found to have decreased tcr repertoire diversity in the circulating t-cell compartment compared to healthy controls and other cvid patients. the increased tcr diversity in the affected tissues compared to peripheral blood implicates the pi3k/akt signaling pathway with t-cell trafficking and tissue immune homeostasis, and suggests this pathway may play a role in the development of inflammatory and lymphoproliferative complications in these patients. gain-of-function mutations in pi3kd result in a human primary immunodeficiency, named apds (activated pi3k-delta syndrome), characterized by lymphopenia, lymphoproliferation, respiratory infections and inefficient responses to vaccination. however, what promotes these immune disturbances at the cellular and molecular level remains unknown. we have recently published a mouse model that recapitulates major features of this disease and used this model and patient samples to probe how hyperactive pi3kd fosters aberrant humoral immunity. we found that mutant pi3kd alters the intrinsic function of t and b cells, leading to icos-independent increases in t follicular helper (tfh) and germinal center (gc) b cells, disorganized gcs, and poor class-switched antigen-specific responses to immunization. these phenotypes were associated with increased phosphorylation of akt and s6 in t and b cells, and lower threshold of activation, with altered regulation of foxo1 and bcl2 family members. moreover, b cells showed enhanced responsiveness and proliferation to both antigens and innate stimuli, accompanied by reduced cell death. strikingly, aberrant responses were accompanied by increased reactivity to gut bacteria, and a broad increase in autoantibodies that were dependent on commensal microbial stimulation, as demonstrated by striking reduction of self-reactivity upon antibiotic treatment in mutant mice. we now have further examined b cell function in these mice and demonstrate that altered foxo1 plays a major role in disruption of both b and t cell function. we further provide evidence for altered activation of metabolic pathways in b cells, compared to wt cells, that may contribute to the dysregulated b cell reactivity. our findings suggest that proper pi3kd regulation is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome. this research was supported in part by the intramural research program of the nih, nhgri and niaid. autoimmune cytopenias are seen in a significant proportion of patients with immunodeficiencies affecting antibody production. previous b-cell maturation studies using fluorescence-activated cell sorting (facs) have associated various phenotypes of primary immunodeficiency diseases affecting antibody production with differing levels of b-cell differentiation. in this study we analyzed the peripheral b-cell compartment of 84 patients with a hypogammaglobulinemia and >1% b-cells with and without a history of autoimmune cytopenias. b-cells were isolated from peripheral blood using monoclonal anti-cd19 and these cells were gated to identify the proportion of memory b cell (cd19+cd27+ ), igm+ memory b (cd27+igm+), marginal zone b-cells (igm+ igd+), isotype-switched memory b-cells (cd27+igm-igd-) and transitional cells (igmhicd38hi). pid patients with a history of aic had decreased proportions of total cd27+ b-cell (11.6% vs 25.6%; p=0.0003) and igm memory b cells (8.3% vs 18.4%; p = 0.0018). conversely, the proportion of marginal zone b-cells was increased in this group (82.0% vs 66.5%; p = 0.0043). consistent with previous reporting, the proportion of isotype-switched memory b-cells was significantly lower in the aic group (0.75% vs 2.8%; p = 0.0003). statistically significant inter-group difference was not seen within the transitional b-cell subset. our data suggest that maturation arrest of marginal zone (cd27+igm+ igd+) b-cells may be implicated in the development of autoimmune cytopenias in humoral immunodeficiency. (159) submission id#601984 taissa de matos. kasahara 1 , sudhir gupta, md 2 1 phd student, state university of rio de janeiro and university of californis irvine 2 professor, university of california at irvine, irvine, ca, usa introduction/background: common variable immunodeficiency (cvid) is the most frequent form of primary hypogammaglobulinemia with decreased serum igg and iga levels and variable levels of igm in adults. in addition to decreased serum immunoglobulins, 25-30% of cvid patients present autoimmune manifestations. the mechanisms that lead to a breakdown of selftolerance in cvid are not completely understood. however some differences in b and t cells subsets and autoreactive b and t cells can be detected. elevated expression of surface igd and downregulation of igm receptor are hallmarks of anergic naã¯ve b cells that contain autoreactive receptors in human peripheral blood. moreover, memory b cells that have class switched to igd and present an igd+igm-phenotype are also highly reactive to self-antigens in healthy individuals. the role of these autoreactive naã¯ve and memory b cells in the immunopathogenesis of cvid has not been evaluated. here we investigated the frequency of cd27-and cd27+ b cells expressing igd and igm in peripheral blood of cvid patients. methods: peripheral blood mononuclear cells (pbmc) from cvid patients (n=29) and health subjects (n=32) were separated by ficollhypaque and incubated with anti-human cd19-percp, cd27-fitc, igd-bv510 and igm-apc to identify different subsets of b cells by flow cytometry. cd19+cd27-igd+igm-and cd19+cd27-igd+ igm+ b cells were sorted, loaded with cfse and cultured with cpg and ant-cd40 for 5 days to evaluate the proliferation. results: among the compartment of cd27-b cells, cvid patients showed an increased frequency of igd+igm+ cells and a lower frequency of igd-igm-cells as compared to control group. no differences were observed in the frequency of igd+igm-cells in cd27-b cells between cvid patients and controls. in contrast, in the compartment of cd27+ b cells, cvid patients showed an increased frequency of igd+igm-, igd+ igm+ and igd-igm+ cells and a lower frequency of igd-igm-cells when compared to health subjects. when the patients were divided in two groups based on autoimmune manifestations, the group with autoimmune disease showed an increased frequency of igd+igm+ and igd-igm+ cells in cd27-b cells when compared to the control groups. both patient groups showed an increased frequency of igd+igm-, igd+igm+ and igd-igm+ cells and a lower frequency of igd-igm-cells when compared to health subjects. regarding the proliferation, naã¯ve b cells from cvid patients showed a reduced proliferative capacity in response to in vitro stimulation as compared with naã¯ve b cells from health subjects. conclusion: our results suggest that the increase of cd27+igd+igm-b cells can be related to the susceptibility of autoimmunity in cvid patients. introduction: immunoglobulin g4-related disease (igg4-rd) is a group of immune-mediated conditions where tissues are affected with dense lymphoplasmacytic infiltrations with a predominance of igg4-positive plasma cells and storiform fibrosis, usually in the setting of elevated serum concentrations of igg4. common presentations include autoimmune pancreatitis, sclerosing cholangitis, retroperitoneal fibrosis, salivary gland disease, and orbital disease, among others. symptoms of asthma or allergy are present in approximately 40 percent of patients and they typically exhibit a good initial therapeutic response to glucocorticoids. case presentation: a 61-year-old female with a history of gastroparesis, cutaneous lupus erythematosus and suspected autoimmune pancreatitis was referred to allergy/immunology clinic for evaluation of elevated igg4. she reported a 15-year history of recurrent abdominal pain attributed to recurrent pancreatitis based on previous mild lipase elevations. prior endoscopic ultrasound (eus) of the pancreas revealed edema. there was concern for gallstone pancreatitis but ercp followed by cholecystectomy, biliary and pancreatic sphincterotomy had no change in her symptoms. in 2016, she was noted to have a positive ana and high serum igg4, per patient (values from osh records could not be obtained). symptoms improved with a course of steroids, hence suspicion for autoimmune pancreatitis. in 2018 she developed a rash on her arms and face. biopsies of the affected areas revealed cutaneous lupus erythematosus on the arms and a basal cell carcinoma on the face, which was excised. ana was only 1:80 at that time. at the visit, she complained of severe allergic rhinitis, joint pains, as well as a malar rash, which responded to intermittent courses of prednisone by prior providers. laboratories obtained at initial visit were significant for thrombocytopenia (135 thou/cu mm), positive lupus anticoagulant (56 sec) and elevated igg4 (95 mg/dl; normal range 4-86 mg/dl). c3, c4, c1q, ana, anti-double stranded dna, anti-smith antibodies, antiphospholipid panel, upep and spep were all unremarkable. ct chest and abdomen were also normal. given the patient's history of cutaneous lupus erythematosus, plaquenil was started as a steroid sparing agent. eus of the pancreas with possible biopsy was ordered in an attempt to obtain a histopathologic diagnosis of igg4-rd. conclusion: this case exhibits the association between elevated igg4, pancreatitis of unknown origin, allergic rhinitis, and cutaneous lupus erythematosus, highlighting the value of identifying a pathologic connection between seemingly unrelated disorders in patients with elevated igg4, as they may be manifestations of igg4-rd. in order to make the diagnosis, histopathologic findings showcasing lymphoplasmacytic tissue infiltration consisting mainly of igg4-positive plasma cells and small lymphocytes is essential. the majority of patients respond to glucocorticoids, and while the duration of response is variable, most patients flare during or after glucocorticoids are tapered, as noted in this patient. rituximab has been shown to be effective in some patients and will be considered in this patient if symptoms persist. (161) submission id#602042 rationale: pnp deficiency is an autosomal recessive disorder due to defective purine metabolism leading to severe combined immunodeficiency (scid) and neurological deterioration. newborn screening utilizing t-cell receptor excision circle (trec) assay can detect affected patients before complications arise. herein, we describe an infant initially identified by newborn screening with pnp deficiency and congenital cmv, a previously unreported presentation. methods: cmv quantitative pcr (qpcr) was performed by nebraska medicine, pnp enzyme activity by duke and genetic sequencing by invitae. results: a small for gestational age (sga) male infant was reported to have an abnormal trec assay on day of life (dol) 7. he was hospitalized for further evaluation. initial studies revealed profound lymphopenia, normal lymphocyte proliferation to mitogens and no evidence of maternal engraftment. additionally on dol 10, he had cmv viremia and viruria; thus with sga, failed unilateral hearing screen and head ultrasound with bilateral parenchymal calcifications, congenital cmv was suspected. pnp enzyme activity was abnormal. cmv treatment was initiated with ganciclovir on dol 10. foscarnet was added on dol 13. cmv qpcr levels decreased below the limit of detection by dol 30. genetic testing found a pathogenic homozygous mutation in pnp (c.286-18g>a). the infant has a 10/10 hla-matched, unaffected, cmv positive sibling and will proceed to hematopoietic stem cell transplantation. conclusions: to our knowledge, this is the first reported case of pnp deficiency identified through newborn screening. this novel case of congenital cmv and pnp deficiency highlights the importance of cmv screening and need for treatment strategies for congenital cmv in scid. despite a dramatic increase in the use of next generation sequencing over the last decade, the majority of the more than 50 million identified human genomic variants do not have well-established clinical implications. progress is being made on this complex challenge through multiple approaches, including data sharing. to maximize our understanding of genomic data, platforms that enable effective and responsible data-sharing are essential. this means that genotypic and phenotypic data must be findable, accessible, interoperable, and reusable under conditions that are ethical and transparent. to highlight innovations in data-sharing and their potential to advance discovery, we present three data-sharing mechanisms. for each platform, we will present a case highlighting its key functionality and discuss opportunities and challenges that may arise as each platform is scaled up. (1.) genomic research integration system (gris) is a collaborationengendering web application that facilitates the identification of genetic variants associated with rare immunological disorders. users can access integrated and standardized phenotypic and genomic data that is analyzable within the platform. gris enables systematic and automated capturing, and links patient data from disconnected systems and paperbased records. standardized annotations allow for the comparison of data from different clinical studies. the main goal of this tool is discoverability of other affected individuals enrolled in separate protocols within the niaid intramural research program. this internal database was used to find a second family with a rare variant in a candidate gene. (2.) the genomic ascertainment cohort (tgac) is a resource that aims to improve our understanding of the phenotypic consequence of genetic variation by providing access to aggregate, de-identified genomic data from large nih intramural and related cohorts. participants have provided informed consent to be re-contacted for additional phenotyping in the future. the main goal of this tool is to enable further study of the clinical consequence of variants in a large, unbiased cohort of patients ascertained for many indications. this database was used to investigate findings in participants with previously published pathogenic variants in genes associated with primary immune deficiency based on medical record review. (3.) clingen is dedicated to building an authoritative central resource that defines the clinical relevance of genes and variants for precision medicine and research. through the sharing of genetic and health data, clingen seeks to answer whether a given gene is associated with a disease (clinical validity)?; whether a given variant is causative (pathogenicity)?; and whether the information is actionable (clinical utility)? this resource is meant to convene disease-and gene-specific expert groups to curate the medical literature on mendelian disease to better define gene-disease and variant-disease relationships using many lines of evidence. this resource was used to clarify clinical validity of disease-gene assertions. together these efforts help create a clinical research ecosystem that maximizes the value of clinical research data and ultimately improves patient care. this research was supported by the intramural research program of the nih, niaid. introduction: according to the population reference bureau, the number of elderly americans, defined as age 65 and older, is projected to more than double from 46 million to 98 million by 2060, rising from 15% to 24% of the total population. the impact of immunodeficiency in this important segment of the population remains understudied. methods: the usidnet registry was queried to obtain demographic, clinical data of elderly patients defined as age 65 and older. descriptive analyses were performed on the data. results: 373 participants (7.2%) were eligible out of 5176 total registry participants. the median age of the cohort was 70 years and predominantly female (74.7%) and white (78.0%) with a median bmi of 26.6 â± 6.6.the majority (81.8%) of subjects were living. humoral deficiencies comprised the majority of diagnoses (94.6%), with common variable immune deficiency being the most frequent (76.9%). of the remaining non-humoral diagnoses, immune dysregulation (1.3%) and immunodeficiency with myelodysplasia (1.1%) were the most frequent. the majority (79.1%) of subjects reported having received immunoglobulin replacement therapy (igrt) at some point, with 51.7% reporting via iv route. of the 1275 infections that occurred in this cohort, sinopulmonary infections were the most commonly reported, specifically sinusitis (18.5%), pneumonia (13.8%), upper respiratory infection (6.7%), and otitis media (5.5%). in this cohort, 107 autoimmune, 49 cardiovascular, and 11 granulomatous complications were reported . the number of patients with malignancy was 89, with some patients diagnosed with multiple malignant disorders. of the reported malignancies, the majority (69.9%) were solid tumors. conclusions: compared to the age-matched non-immunodeficiency united states population, this cohort had more females 74.7% (usidnet) versus 56.0% (us population) and fewer whites 78.0% (usidnet) vs 86.0% (us population. humoral immunodeficiencies, specifically cvid, were most common diagnoses, similar to other age groups of immunodeficiency patients. majority of these patients have received igrt, with approximately half via iv route. this cohort reported living with a variety of non-infectious complications, including autoimmunity and malignancies. more research which specifically focuses on elderly patients with immunodeficiency is needed. clinical microbiologist and infectious disease physician, university of calgary x-linked agammaglobulinemia (xla) is a primary immunodeficiency caused by mutations in the bruton tyrosine kinase gene which leads to b cell maturation failure and defective antibody production. this puts patients at risk of recurrent sinopulmonary infections, gastrointestinal infections, and recurrent skin infections including infections caused by helicobacter sp. helicobacter sp are gram negative bacilli commonly found in the gastrointestinal tract of various animals. helicobacter sp. have been linked with gastritis most notably helicobacter pylori causing gastric ulcers in humans. helicobacter sp. has been found in rare cases to cause disseminated infections including pyodermic gangrenosum and cellulitis notably in patients with agammaglobulinemia. infections caused by helicobacter bilis are challenging to diagnosis due to difficulties with culturing the pathogen as well as poor guidelines for antimicrobial management. case report: the patient was diagnosed with x-linked agammaglobulinemia at the age of 16 months with a history of recurrent sinusitis and was started on ivig q3weeks. despite regular ivig, he developed bronchiectasis. at 11 years of age in 2013, he developed a chronic rash around his left knee resembling erythema nodosum. by 2014, he had developed a left knee effusion associated with left sided calf pain. his knee pain was found to improve during courses of ciprofloxacin to treat recurrent lung infections. given case report data of h. pylori causing erythema nodosum in patients with agammaglobulinemia, he was treated empirically for an h. pylori infections with no improvement. in 2015 he was found to have progressive cellulitis with pyomyositis of the left leg. a skin biopsy of a calf nodule was found to be culture negative but 16s pcr was positive for h. bilis. he was started on treatment with ertapenem and levofloxacin with subsequent resolution of his rash. his left ankle pain progressed and by late 2015 and was found to have possible osteomyelitis of the left ankle on mri. in 2016 he was found to be bacteremic with h bilis. due to progressive symptoms with significant impact on function and rising inflammatory markers despite 12 months of antimicrobial treatment, doxycycline and flagyl were added leading to clinical improvement and normalization of his inflammatory markers. he was continued on oral doxycycline and flagyl for 12 months for a chronic osteomyelitis. discussion: h. bilis is a slow growing pathogen which is challenging to culture in the laboratory often requiring special agar plates and prolonged incubation. in patients with agammaglobulinemia and associated chronic skin infections or erythema nodosuma, h bilis should be suspected as a possible pathogen. due to challenges with culturing, 16s pcr or amplification of the 16s ribosomal subunit should be considered to try to identify the pathogen. there are poorly delineated clinical antimicrobial breakpoints to help guide therapy with minimal evidence. case reports suggest prolonged therapy with aminoglycosides and penicillin. other studies have successfully treated patients with a carbapenem, azithromycin and levofloxacin. in the absence of sensitivity data, prolonged treatment (12months) should be considered with a combination of antimicrobials. patients should be followed closely as recurrent infections are not uncommon. chief, human immunological diseases section, laboratory of clinical immunology and microbiology, niaid, nih, bethesda, md introduction: dock8 deficiency is a combined immunodeficiency characterized by eczema, recurrent sinopulmonary infections, viral skin infections, malignancy and early mortality. in recent years, liver disease and vasculopathy have been increasingly recognized as a complication of dock8 deficiency. we clinically characterized our cohort of dock8 deficient patients, with a specific focus on these newly identified areas of disease involvement. methods: chart reviews were performed on patients seen at nih with genetic and clinical diagnosis of dock8 deficiency. patients were all enrolled on irb approved niaid protocols. results: we identified 52 patients from 40 families with dock8 deficiency in our nih cohort, ranging in age from 6-44 years. of the 40 families, 17 had homozygous mutations. of the 52 patients, food allergy was diagnosed in 31 (60%), eczema in 49 (94%), and asthma in 30 (58%). chronic or recurrent viral skin infections were seen in 49/52 (94%). chronic ebv viremia by pcr positivity was seen in 18/46 patients (39%); only 2 patients were known to be ebv immune without viremia. cmv viremia was infrequent. sinopulmonary infections were common, with bronchiectasis occurring in 23 /50 (46%) with available imaging. liver disease was diagnosed in 14 (27%), with 7 having biliary tract abnormalities on imaging and stool positive for cryptosporidia; most patients with cryptosporidia were without diarrhea. the incidence of cryptosporidia is likely under-represented due to more recent availability of sensitive assays for cryptosporidia detection. other liver abnormalities included fatty liver, metastatic disease from malignancy and medication related hepatitis. vasculopathy, predominantly of the aorta and cerebral arteries, was diagnosed in 7, with patients in the last 5 years being prospectively imaged. autoimmunity was rare (5%) including autoimmune cytopenias and hypothyroidism. 36 of 50 with follow-up are alive (70%) with age range 6-44 years. of the 36 living patients, 28 (78%) have had a hsct. causes of deaths include malignancy (6), infection (1) , and hsct complications (7) . long-term follow-up of patients with hsct (up to 6 years) has revealed resolution of the infection susceptibility and eczema, no new cancers, and stabilization of vasculopathy. conclusions: in addition to the well described manifestations of dock8 deficiency including eczema, allergy, recurrent sinopulmonary infections, skin viral infections and malignancy, our cohort revealed a relatively high incidence of liver disease, frequently associated with stool positivity for cryptosporidia, as well as vasculopathy. both of these clinical manifestations should be considered during preparation for hsct as they may affect management through transplant. autoimmunity has likely been over-estimated in prior descriptions of dock8 deficiency. long-term follow-up after hsct is needed to determine the prognosis from the vasculopathy, liver disease, and malignancy risk. (166) submission id#604115 yasuhiro yamazaki 1 , stefano volpi 2 , luigi d. notarangelo 1 introduction/background: extl3 (exostosin like glycosyltransferase 3) is an exostosin family member which initiates heparan sulfate (hs) chain biosynthesis and elongation. we have reported homozygous extl3 hypomorphic mutation (r339w) as a cause of immunoosseous-dysplasia syndrome. fourteen patients who have extl3 homozygous mutation were reported so far. eight of them manifested t cell lymphopenia, and 5 presented with severe combined immunodeficiency (scid) or omenn syndrome. using patient-derived induced pluripotent stem cells (ipscs) as a model, we have previously reported that extl3 mutations affect differentiation to thymic epithelial progenitor cells as well as expansion of hematopoietic progenitor cells. consistent with the latter, previous studies have suggested that mutations in other genes involved in hs biosynthesis affect hematopoietic stem cell (hsc) differentiation. however, the exact mechanisms by which extl3 mutations affect hematopoiesis are not known. objectives: we tried to clarify gene expression difference in hscs derived from wild-type, extl3 hypomorphic and extl3 knock-out (ko) human ipscs. methods: the control bj ipsc line was engineered by crispr/cas9 gene targeting. extl3 ko ipscs were obtained which carried compound heterozygous extl3 mutations (c.1003_1004inst; c.1005_1006insgatattt). hsc differentiation was induced using the stemdiff hematopoietic kit (stemcell technologies). bulk rna from each ips cells and each differentiated cd34+cd43+cd45+ was analyzed by rna sequencing. results: as compared to control ipscs, patient-derived cells showed slightly lower capacity to generate cd34+cd43+cd45+ cells. on the other hand, extl3 ko cells showed no differentiation into cd34+ cd43+cd45+ cells. gene set enrichment analysis showed enriched expression of genes involved in hematopoietic progenitor cell differentiation, regulation of hemopoiesis, and positive regulation of hemopoiesis in both control and patient-derived cd34+cd43+cd45+ cells compared to parental ipscs. moreover, these gene sets were more abundantly enriched in control than in patient-derived cd34+cd43+cd45+ cells. the gene set of response to type i interferon was significantly enriched in control versus patient-derived cd34+cd43+cd45+ cells. conclusions: these results confirm that extl3 plays an important role for hsc homeostasis in human cells. because type 1 interferons play a role in hsc proliferation, the decreased type i interferon signature may account for the reduced number of hscs that we have previously reported upon in vitro differentiation of extl3-mutated versus control-derived ipscs. this study was supported by the division of intramural research, niaid, nih, under protocol 16-i-n139. a case of autoinflammatory syndrome with osteoporosis and specific antibody deficiency autoinflammatory syndromes are inherited disorders with an exaggerated inflammatory response with no specific trigger. the clinical phenotypes of variants of autoinflammatory syndromes may overlap. we report a case of a 13 year old male with prior diagnosis of specific antibody deficiency, periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (pfapa) syndrome, arthralgia and moderate atopic dermatitis. he was diagnosed at 3 years of age with specific antibody deficiency based on persistently low pneumococcal titers against repeat immunizations. due to recurrent infections, he was placed on immunoglobulin replacement therapy (igrt) at 8 years of age. igrt was discontinued at 13 years of age due to full resolution in infections and patient demonstrated robust response to immunizations. patient had lifelong history of recurrent fevers (every 5 weeks) associated with pharyngitis and aphthous ulcers consistent with diagnosis of pfapa. as he became older these episodes became less frequent. last episode of fever was over a year ago. the father had similar symptoms of recurrent fevers and oral ulcers as a child but currently remains asymptomatic. paternal grandfather died of kidney disease. patient has been generally in good health until recent year with intermittent abdominal pain, arthralgia and several long bone fractures with no history of prior trauma. a bone density scan revealed osteopenia and osteoporosis with a z score of -2.2 of lumbar spine, -4.0 of left femoral neck, -3.1 of left hip. given history of familial autoinflammatory disease, and antibody deficiency genetic testing was obtained which identified a pathogenic heterozygous variant of taci and mefv c.2082g>a (p.met694lle). taci mutation has been linked to antibody deficiency syndromes. genetic study for family members is pending. the mefv gene is associated with autosomal recessive familial mediterranean fever (fmf) and has been reported in autosomal dominant fmf as well. fmf is characterized by recurrent episodes of fever associated with serositis, arthralgia, and arthritis. patients with fmf have elevation in acute phase reactants during attacks with most returning to normal levels during the episode-free periods. multiple studies have shown that patient with fmf have lower bone mineral density and zscores than the general population. inflammation in fmf is thought to be mediated by several different cytokines (il-1, il-2, il-6, il-7, il-8, il-11, il-15 and tnf-). these same cytokines play a role in osteoclast activity and bone resorption. it has been suggested chronic inflammation during acute attacks and subclinical inflammation during the disease-free period lead to bone loss and osteoporosis. regular use of colchicine, the main treatment for fmf, may slow down osteoporosis. beside careful monitoring of clinical and laboratory phenotype, genetic evaluation is an important step in distinguishing between overlapping entities and can prevent complication and promote targeted intervention. a 5 year old previously healthy boy was referred for periodic fever/ pfapa and mosquito bite hypersensitivity. eight weeks earlier he developed fever to 104f, mouth sores and exudative tonsillitis; a rapid strep screen was negative. one week later he developed moderate cervical lymphadenopathy and had a positive ebv early antigen antibody.. one month later he had several severe local reactions to mosquito bites. each manifested 6-8 cm of erythema and induration with a 1+ cm bullae which left an ulcer after rupture and healed with a hypopigmented scar. the bites were accompanied by fever to 104f for 4 days. one febrile episode was treated with low dose prednisolone for presumed pfapa, and the fever resolved within hours. his past history was positive for nasal allergy and mild asthma. his parents are not related: mom is of european-indonesian and dad european-african (creole ancestry. testing prior to this visit showed normal igg, iga and igm, elevated ige (12,000 u/l) and normal cbc. lymphocyte subsets revealed cd3+ 23% (1538/mcl), cd4+ 17% (1109/mcl), cd8+ 6% (363/ mcl), cd19+ 9% (587/mcl), nk cells 67% (4435/mcl). on examination he appeared well with height at 86th%ile and weight at 58th%ile. there was no lymphadenopathy, hepatosplenomegaly or inflammed skin lesions; there was a 1cm round scar on the right plantar surface at the site of a prior mosquito bite. laboratory studies confirmed nk lymphocytosis 64% (5459/mcl) and elevated ige (29,600 u/l). lymphoproliferation to mitogens, cd3/cd28, cmvand hsv were normal, but absent to tetanus and candida antigens. ebv antibodies reflected past infection (vca-igg+, vca-igm-, ebna+); quantitative ebv pcr was >5,000,000 copies/ml whole blood. nk cytotoxicity and cd107a expression were decreased. bone marrow nk analysis suggested conality. the patient was diagnosed with "hypersensitivity to mosquito bites with ebv-associated t-/ nk lymphoproliferation." this disorder represents a subset of chronic active ebv (caebv) that is rarely seen outside of east asia. the lack of organomegaly or lymphadenopathy with hyper-ige and nk lymphocytosis and decreased nk function support the likelihood that nk cells are the target of ebv infection in this patient. this diagnosis may be a precursor to hemophagocytosis, liver necrosis or lymphoma/leukemia, and the only curative treatment is bone marrow transplantation. the patient's sister is a 10/10 hla match. she is seropositive for past ebv infection, and she has no history of extreme reactions to mosquito bites. genetic mutations that cause familial hemophagocytic lymphohistiocytosis have not been reported in caebv, and to the best of our knowledge familial cases of this disorder have not been identified. the response to bmt in this patient is pending. introduction/background: a number of case reports have described symptomatic hypogammaglobulinemia following administration of anti-epileptic drugs (aeds), specifically lamotrigine, carbamazepine, and levetiracetam. the mechanism by which symptomatic hypogammaglobulinemia develops is unclear. we evaluated the prevalence and the clinical significance of hypogammaglobulinemia associated with use of these aeds. objectives: our aim was to characterize the prevalence of aed-induced hypogammaglobulinemia, identify specific aeds associated with hypogammaglobulinemia, and characterize the timeline to development of hypogammaglobulinemia after initiation of therapy. methods: a retrospective, multicenter, electronic medical record review spanning 18 years identified patients with hypogammaglobulinemia who were on aed therapy (lamotrigine, carbamazepine, or levetiracetam). patients were excluded if they had a pre-existing primary immunodeficiency (pid), malignancy, protein-losing enteropathy, or significant proteinuria. patients on chronic immunosuppressive therapy, those without laboratory criteria for hypogammaglobulinemia, or those on one of the aeds for less than one month were also excluded. results: of the 316 cases reviewed, 5 patients met our inclusion criteria. the median age was 35; 80% were adults, 80% were female, and 80% were white. lamotrigine was implicated in 3/5 of the cases, carbamazepine in 2/5, and levetiracetam in 1/5. tetanus and pneumococcal titers were available for 4/5 patients. of those patients, 3/4 had protective titers to both per report with responses to >70% of the serotypes. only one patient reported severe, recurrent infections while the remaining four had little to no symptoms. interestingly, the patient with severe infections did have protective titers. of the five laboratory proven hypogammaglobulinemia patients, one died of an infection, two have continued on the medication due to refractory seizures responsive only to these medications, and two are currently being tapered off of their aed. conclusion: while it appears that aed-induced hypogammaglobulinemia is quite rare, it should be considered in a patient without other secondary causes of hypogammaglobulinemia on aed therapy. many antiepileptics downregulate nfkb signaling suggestive that patients who develop symptomatic hypogammaglobulinemia may have hypomorphic mutations in the nfkb signaling pathway. (170) submission id#604503 autoimmune lymphoproliferative syndrome (alps) results from defective apoptosis of lymphocytes mediated through the fas/fas ligand (fasl) pathway. the hallmark lab finding is an expansion of t cells that express the alpha/beta t cell receptor, but lack both cd4 and cd8 (double negative t cells) in the setting of normal or elevated lymphocyte counts. patients present with chronic, nonmalignant, noninfectious lymphadenopathy or splenomegaly. for definitive diagnosis, patients need to have (1) a pathogenic mutation in fas, fas ligand or caspase 10 or (2) a defective fas-induced lymphocyte apoptosis. we describe a probable case of alps with heterozygous mutation in fas c.287a>g(p.his96arg), a variant that has not been previously reported (his lymphocyte apoptosis assay is pending). unique to this case is the patients castleman disease-like features on pathology. a 15 year-old male referred from hematology clinic presented with an 8 year history of chronic lymphadenopathy, splenomegaly, anemia, and no underlying diagnosis. malignancy had previously been excluded by bone marrow aspirate and biopsy 8 years prior. however, he had a right sided lymph node that had increased in size for the past 4 months. he was otherwise asymptomatic. a lymph node biopsy 7 years prior was reportedly normal. his exam demonstrated significant bilateral lymphadenopathy, greater on right, with an approximately 8 x 6 cm mobile right neck mass. he had splenomegaly palpated 7 cm down and across to midline. he was therefore admitted for excisional lymph node biopsy to evaluate for possible malignancy and labs were sent to evaluate for alps. labs were supportive of alps. he had elevated t cell receptor alpha beta double negative t cells (tcr a/b dntcs) in blood (10.5%). b12 level was elevated (>1000 pg/ml). plasma soluble fasl level was elevated (5517 pg/ml). interleukin-10 (il-10) and il-18 levels were elevated (88 and 909 pg/ml respectively). he had multilineage cytopenias: anemia with hgb of 9.5 g/dl and neutropenia (absolute neutrophil count of 1380 k/ul). he had hypergammaglobulinemia with an igg level of 2010 mg/dl. broad infectious work-up was negative, including hiv, quantiferon, cocci, bartonella, toxoplasma, coxiella burnetii, ebv pcr and, cmv igm. lymph node biopsy showed no evidence of malignancy. immunostains and flow cytometry showed the presence of expanded tcr a/b dntcs in the lymph node, consistent with alps. interestingly, lymph node histology showed morphologic features typical of plasma cell variant castleman disease. numerous castlemanlike follicles showed typical regressive changes with onion-skinning morphology. paracortical hyperplasia with sheets of plasma cells was noted. there was negative staining for hhv8 (a well-known cause of plasma cell variant castleman disease). the diagnosis of idiopathic multicentric hhv8-negative castleman disease was excluded by definition in the setting of alps, per evidence-based consensus criteria published in 2017. in addition, our patient did not show any symptoms typically associated with it, such as fever, night sweats, weight loss, weakness or fatigue. should his fas-induced lymphocyte apoptosis be defective (in 2 separate assays), this would confirm his alps-fas diagnosis and we would start the patient on sirolimus. head of immunology unit, children' s hospital ricardo gutierrez introduction: slc46a1 gene encodes the proto-couple folate transporter (pcft), which supports intestinal folate uptake, and participates in folate transport into the central nervous system. slc46a1 mutations cause pcft defects, resulting in low folate levels in serum and cerebrospinal fluid. hereditary folate malabsorption (hfm) is a rare, autosomal recessive disorder with pcft deficiency resulting in cerebral folate deficiency. most of the patients present megaloblastic anaemia, moderate pancytopenia in the first few months of life, failure to thrive, diarrhoea and/or later onset neurological symptoms including seizures and developmental delay. i m m u n o d e f i c i e n c y i n h f m c a n m a n i f e s t i t s e l f w i t h hypogammaglobulinemia with normal t-cell function. b-cell precursor compartment seems to be particularly vulnerable to folate deficiency in some hfm patients. this immunodeficiency can be restored with specific treatment with folic acid. aim: to describe a female patient with a homozygous pathological variation in the slc46a1 gene. results: a 17 months old girl, born of non-consanguineous parents. she started at 3 months old with diarrhoea due to rotavirus, low weight and bicytopenia with normal bone marrow aspiration. she presented low levels of folic acid 1.5ng/ml (nv 3.1-20.5 ng/ml) at first thought due to secondary to malnutrition. treatment with folic acid supplementation was administrated, improving platelets counts. at 5 months old she presented steatorrhea with severe perianal panniculitis which required surgical treatment. no germs were rescued after a skin biopsy. moreover, she suffered from a respiratory infection due to picornavirus with two episodes of pneumothorax which required intensive care. at that moment ivig treatment was administered due to hypogammaglobulinemia and clinical severity. chronic diarrhoea worsened with bloody depositions. three rectal ulcers were found in the gut biopsy. bowel inflammatory disease was suspected and mesalazine administration was started with weight improvement. furthermore, at 10 months old she presented 3 status epilepticus, with pathological eeg and normal mri; one of them related to a cmv infection, successfully treated. in the immunological evaluation igg and iga were low with normal igm and igd. the protein-antibody response was not evaluated. she presented normal lymphocyte and t cells extended populations, t cells proliferation assay, dhr, treg cells, complement, cd107a expression, alpha-fetoprotein, without autoantibodies a molecular panel testing was done by ngs and a homozygous variant in slc46a1 gene was found, causing impaired intestinal folate absorption. conclusion: hfm should be considered in the diagnosis of patients with cytopenias and hypogammaglobulinemia in order to provide specific treatment. hfm has wide clinical manifestations, not only with megaloblastic anaemia and neurological impairment but also with gastrointestinal and skin manifestations. with folate treatment, clinical and immunological defects can be normalized. introduction: multifocal epithelial hyperplasia (meh), or hecks disease, is a rare, benign infection of the mucosa caused by human papilloma virus (hpv). clinically, meh manifests as numerous painless, soft, sessile papules or plaques, and typically occurs in the labial, lingual, and buccal mucosa. meh lesions are usually associated with hpv types 13 and 32, and seen more commonly in patients of caribbean or central/south american descent. prior studies in adults have shown that tumor necrosis factor alpha (tnf) promotes hpv, and may influence duration of hpv infection. case: we present a five-year-old full term male of haitian descent referred for assessment of multiple flesh colored, papular lesions on the buccal and labial mucosa that had persisted and quantitatively increased over one year, although some lesions regressed. he had no pain or difficulty eating. medical history significant for one seizure; negative for infection. no family history of infection, immunodeficiency, consanguinity, or miscarriage. head and neck examination failed to reveal cervical lymphadenopathy, masses, or hypertrophy in the salivary glands. intraoral examination revealed multiple papular nodules, mostly flat although some were corrugated. the greatest concentration was noted on the lower left labial surface extending to the mucosal vermillion interface, not involving the vermillion or commissure region. lesions extended into the mandibular vestibule and the left buccal mucosa. no other lesions were noted on extremities, genitalia, or any other visualized mucosal surface. based on history and exam, he was diagnosed with meh. white blood cell count, neutrophils, lymphocytes, cd4 and cd8 t cell, b cell, nk cell enumeration, and immunoglobulin panel were normal for age. tetanus and streptococcus pneumoniae titers were protective. cytomegalovirus igg and igm were negative. epstein-barr virus igg was positive, igm and early antigen ab negative. serology was significant for elevated tnf (84 pg/ml; reference range <22pg/ml) while interferon gamma and interleukins 1, 2, 4, 5, 6, 8, 10, 12, 13 , and 17 were normal, as was il-2 receptor cd25. one month after the initial visit, lesions were stable and unchanged. nine-valent hpv vaccination was considered, but not administered. conclusions: meh is a rare but benign disease caused by hpv. awareness of the disease and its course is important to prevent unnecessary expanded immunodeficiency work-up and possible procedures to eliminate lesions. although mucosal immunity can be site specific, especially with hpv, our understanding of t-cell cytokine and chemokine responses to hpv in cervical and laryngeal lesions may be instructive. the mechanism which allows hpv persistence in meh is not characterized, but it likely is due to increased viral persistence and an inability for the host immune response to successfully induce viral latency and successful containment. elevated tnf levels, with normal levels of il-2, il-6, il-8, il-10, may correlate with decreased clearance of hpv and prolonged duration of meh. it remains unclear if viral persistence is the cause of, or the sequela of, increased tnf. longitudinal monitoring of cytokine (tnf, il-2, il-6, il-8, il-10) and chemokine (ccl17, ccl18, ccl19, ccl20, ccl21, and ccl22) serum concentrations may be useful biomarkers for disease resolution. introduction: autosomal dominant hyper ige (jobs) syndrome is a rare primary immunodeficiency characterized by eczema and sinopulmonary infections as well as musculoskeletal and vascular complications. as in all chronic illnesses, patient education is an ongoing need. in the rare disease population, patient education is especially important as patients must be able to explain their unique healthcare concerns in a variety of medical settings. we focused on ad-hies, due to our relatively large cohort of patients, the frequent lack of classic signs of illness often impairing diagnosis of severe infection, and the diverse nonimmunologic clinical features of this disease. objectives: we aimed to increase understanding of the clinical manifestations of ad-hies to promote earlier recognition of symptoms and to increase self-efficacy for symptom management in the adult hies population. methods: adult patients were asked to participate in a patient education project. demographic information was collected from participants. they also completed a 12-item multiple choice test about symptom recognition in ad-hies and promis self-efficacy for managing symptoms, an 8item validated survey. then, patient education handouts that focused on pulmonary symptoms, eczema, bone health, and cardiovascular complications were reviewed with the participant. six weeks later, participants were asked to repeat the 12-item test and the self-efficacy survey. the demographic information, test, and self-efficacy were collected anonymously. results: 33 participants provided demographic information, completed the test and the self-efficacy survey. of the 33 participants, 15 were male and 17 were female. participants ranged in age from 18 to 66 years. 22/33 (67%) reported looking for information about ad-hies using search engines and most patients (91%) report that they have been given information about ad-hies from a doctor. 19/33 (58%) participants identified pulmonary symptoms as the symptom that concerns them most and 10/33 (30%) participants identified more than one symptom of concern. 25 participants returned the second test and second survey. the mean test score increased from 9.08 to 10.28 with 23/25 participants achieving a score of 9/12 or higher. the self-efficacy scores were unchanged with a mean score of 50.08 before reviewing the patient education handouts and 50.13 after. conclusions: participant feedback to this project was generally positive. ad-hies patients are seeking information and an educational intervention can improve their understanding of disease. self-efficacy results were mixed and unchanged overall, but suggest that ad-hies patients manage symptoms as well as other patients with chronic illnesses. patient education should continue at each encounter. this project can be expanded to include more topics, pediatric patients, and other rare disease populations. funded by the nci contract no. introduction: bcl11b plays an important role in the development and maintenance of the immune system and the central nervous system. expression of bcl11b represses nk and myeloid factors while inducing t cell lineage genes in thymocytes at the dn2 stage. conditional loss of bcl11b expression in murine thymocytes leads to t cell deficiency while complete knockout of bcl11b was fatal within a few days of birth. recently, specific heterozygous bcl11b mutations have been reported in 11 individuals with global development delay. however, only 2 of these cases, both carrying heterozygous missense variants, had low trec values with 4 other cases having frequent infections. little is known regarding the impact of bcl11b on human nk and t cell function. methods: we identified a novel heterozygous truncating mutation in bcl11b in an infant who was first detected by trec newborn screening. she subsequently developed severe autoimmune hemolytic anemia at the age of 3 months. we used standard immunoblotting and flow cytometry methods to assess protein expression and the impact of this bcl11b mutant on t cell and nk cell development and function. results: the patient has a novel single base-pair deletion in the bcl11b gene, which is predicted to produce a truncated protein with the loss of 3 of 6 zinc finger domains in bcl11b. immunoblotting of t cell blast lysates revealed a reduced bcl11b expression in the patient consistent with the heterozygous defect in bcl11b but also generated a novel band with a smaller molecular weight that we postulate represents the truncated protein product. while mitogen responses to cona and pha were normal, both cd4+ and cd8+ t cell counts were decreased, especially cd4+ naã¯ve and cd4+cd31+ naã¯ve t cells, suggesting reduced thymic output. the function of th1 cells was skewed with reduced il-2 production but increased ifn levels after pma and ionomycin stimulation. moreover, t regulatory cell counts were below normal range. nk cell counts were normal but these were mostly cd56bright nk cells. of the few cd56dim nk cells that presented, approximately half did not express cd16, the fc receptor for adcc. perforin was only present in cd16 expressing nk cells. as such, anti-cd16 stimulation understandably led to low but not defective nk cell degranulation. function after stimulation with k562 cells was normal when controlled for nk cell counts. conclusion: we report a novel bcl11b truncating mutation with a leaky scid phenotype that manifested with t-cell lymphopenia and autoimmunity. lowered thymic-derived naã¯ve t and regulatory t cells, skewed th1 cytokine response, and incomplete nk cell development suggests that bcl11b is important for the development and differentiation of multiple lymphocyte lineages. introduction: chronic diarrhea is one of the most common gastrointestinal complaints in patients with common variable immune deficiency (cvid) and can lead to life-threatening complications such as malabsorption and malnutrition. chronic diarrhea in cvid could be caused by infections, an inflammatory bowel disease-like picture, as well as malignancy. giardia lamblia is one of the most common parasites causing diarrhea in cvid (up to 40%), and can be refractory in these patients, leading to villous atrophy, weight loss, and failure to thrive. case report: a 41-year-old female with a history of cvid presents with chronic diarrhea and significant weight loss. her cvid was diagnosed by hypogammaglobulinemia (low levels of igg, igm, and iga), inadequate responses to protein and polysaccharide-based vaccines, decreased memory b cells (cd19+cd27+ 0.5%), and recurrent sinopulmonary infections. she was started on immune globulin replacement therapy and had significant improvement in her rate of infections. four years before her presentation to our center, she developed chronic, severe diarrhea. work up revealed giardia lamblia infection on endoscopy and colonoscopy. biopsy showed intraepithelial lymphocytes, villous blunting, and atrophic gastritis with rare plasma cells concerning for non-infectious enteropathy related to her cvid, in addition to the high burden of giardia organisms. she was initially treated with metronidazole for several weeks. however, her diarrhea did not improve, and she developed significant peripheral neuropathy leading to lower extremity weakness and limited mobility. her diarrhea persisted and was associated with approximately a 20-pound weight loss. repeat endoscopy and colonoscopy two years later showed persistent high burden giardiasis of the small intestine, as well as reactive lymphocytic infiltrates and atrophic gastritis. she was treated with nitazoxanide but continued to have diarrhea, and her stool continued to show trophozoites. given the significant inflammation and the lack of response to multiple antiparasitic agents, she was referred to our center for further evaluation. she was started on oral budesonide (9 mg daily) and oral immune globulin (20 grams weekly for 12 weeks). with this regimen, she had significant improvement in her diarrhea with a 10-pound weight gain. repeat colonoscopy showed considerable improvement in inflammation and resolution of her giardia infection, though her stool antigen continues to be positive. conclusions: persistent diarrhea in our patient is most likely due to a combination of cvid enteropathy and giardiasis. a prolonged course of metronidazole and later nitazoxanide did not control her diarrhea and led to significant side effects. switching to an immunomodulatory approach significantly decreased the inflammation in her bowel and may even have helped to reduce the burden of giardia in the gut. targeting both underlying bowel inflammation as well as active infection in cvid patients with chronic diarrhea might be needed to control symptoms. introduction: sphingosine-1-phosphate (s1p) is a lipid chemoattractant that is critical for lymphocyte egress from lymphoid organs. following a s1p concentration gradient maintained by s1p lyase ubiquitously expressed in tissues, lymphocytes within lymphoid organs are drawn to efferent lymph and blood unless their s1p receptor is internalized or downregulated. owing to diminished degradation of not only s1p, but also other sphingoid bases, deleterious mutations in sgpl1 (encoding s1p lyase) perturb sphingolipid catabolism in numerous tissues. correspondingly, human s1p lyase deficiency results in multiorgan dysfunction including kidney, skin, endocrine gland, and neurologic impairment alongside expected lymphopenia. although severe t cell lymphopenia (<300 cells/microliter) rivaling that of severe combined immunodeficiency (scid) can be seen in patients with s1p lyase deficiency, no such patients have been identified by newborn screening of t cell receptor excision circle (trec) counts, which are a surrogate measure of effective t cell production. herein, we describe an infant boy with an undetectable trec count at birth who was found to have two novel, biallelic sgpl1 mutations resulting in s1p lyase deficiency. case description: a 1-day-old boy with a preceding history of fetal hydrops is born at a gestational age of 36 weeks and presents with renal failure, anasarca, and respiratory failure. trec analysis of a dried blood spot obtained at 24 hours of life reveals zero copies/microliter. subsequent peripheral blood studies show profound lymphopenia, with diminished cd3+ t (129/microliter; 96 cd4+, 27 cd8+), cd19+ b (130/microliter), and cd16/56+ natural killer (124/microliter) cell counts. recent thymic emigrants are reduced (11.3% of cd4+ t cells are cd45ra+cd31+), as is the ratio of naã¯ve-to-memory cd4+ t cells (63% cd45ra+, 37% cd45ro+). expedited whole genome sequencing identifies two novel variants in sgpl1 a paternally inherited splice site variant (c.1566+2t>c) predicted to impact a canonical splice donor site, and a maternally inherited missense change (c.854g>a; p.cys285tyr) located in a well-established functional domain of s1p. in addition to nephrotic syndrome and lymphopenia, the patient displays evidence of adrenal insufficiency and has increased plasma levels of sphingoid bases and ceramides. before further analyses could be pursued, the infant dies at 40 days of age due to ongoing complications of renal failure and eventual cardiorespiratory failure. summary: we report the first case of s1p lyase deficiency identified by newborn trec screening for scid. as sgpl1 is not included in most commercially-available, scid-tailored gene panels, s1p lyase deficiency would be missed by conventional genetic testing. therefore, analysis for variants in sgpl1 should be considered in neonates with low-to-undetectable trec counts, nephrotic syndrome, and other suggestive sequelae. w a r t s , hypogammaglobulinemia, recurrent infections, and myelokathexis) is a rare autosomal dominant primary immunodeficiency. it is caused by a defect in the gene encoding the chemokine receptor cxcr4. this receptor, along with the associated ligand cxcl12, regulates leukocyte migration. we present the case of a 40-year-old female, who presented after she self-identified the signature signs of whim syndrome in herself and multiple family members. objectives: we present the case of a 40-year-old female who presented with a history of recurrent warts, leukopenia of unknown cause, and recurrent infections as a child. as a child, she experienced multiple ear and sinus infections, along recurrent warts on her upper and lower extremities that have persisted to this day. furthermore, during a routine examination when she was 14-years-old, she had a complete blood count drawn significant for leukopenia. no further workup was undertaken at that time. when continued leukopenia was noted at the age of 30, referral to a hematologist and a bone marrow biopsy was completed. bone marrow was significant for myelokathexis with borderline hypercellular marrow for patient age (80% cellularity), and normal cell line quantity. a trial of neupoegen was undertaken, without significant improvement. her family history is significant for father and brother with both leukopenia and recurrent warts. results: genetic analysis showed a heterozygous pathogenic variant in the cxcr4 gene, c.1012_1015dup (p.ser339phe fs*6). recent complete blood count was significant for a total wbc count of 1.0 k/ul, with a differential consisting of 30% neutrophils and 57% lymphocytes. lymphocyte subsets were significant for quantitatively low cd3+, cd8+ and cd19+ subsets, with normal numbers of cd4+ and nk cells. immunoglobulin levels revealed an igg of 835 mg/dl, iga of 145 mg/ dl, and igm of 54 mg/dl; igg anti-diphtheria and tetanus titers were protective, however, none of the 23 s. pneumoniae serotype titers were > 1.3 ug/ml. mitogen (pha, cona and pwm) and antigen (candida and tetanus) stimulation of lymphocytes were normal for all stimuli. conclusions: we present the case of a 40-year-old female with a history of recurrent infections, warts, and myelokathexis. on genetic analysis, she is noted to have a pathogenic mutation of the cxcr4 gene. the substitution of a phenylalanine for a serine decreases one of the seven serine phosphorylation sites in the carboxy tail of the molecule that occurs upon binding to its ligand, cxcl12 (sdf1). additionally, the variation generates a premature stop condon terminating the remainder of the carboxy terminal amino acids including ser346-7, known to have a role in carboxy terminial beta-arrestin binding. failure to generate adequate beta-arrestin binding sites leads to prolonged cxcr4 cxcl12 interaction resulting in myelokathexis. background: lacking protective antibodies, patients with primary antibody deficiencies (pad) suffer from frequent respiratory infections leading to chronic pulmonary damage. macrolides prophylaxis has been proven effective to successfully manage chronic lung diseases as cystic fibrosis, bronchiectasis, copd. we conducted a trial to evaluate the efficacy and safety of orally low-dose azithromycin prophylaxis when added to the usual care in pad patients. methods: a 3-year, phase ii, prospective, multicenter, randomized, double-blind, placebo-controlled trial on pad patients (age 18-74 years) with chronic infection-related pulmonary disease. patients received azithromycin 250 mg or placebo once daily three-times a week for 24 months. the primary outcome was the decrease of annual episodes of respiratory exacerbations. secondary endpoints included: time to the first exacerbation, number of hospitalizations, additional doses of antibiotics, health related quality of life measures, and safety. results: forty-four patients received azithromycin and 45 patients received placebo. the mean number of exacerbations was 3â·6 per patientyear (95%ci 2â·5-4â·7) in the azithromycin arm, and 5â·2 (95%ci 4â·1-6â·4) in the placebo arm (p=0â·02). in the azithromycin group the hr for having an acute exacerbation was 0â·5 (95%ci 0,3-0â·9, p=0,03) and the hr for hospitalization was 0.5 (95%ci 0,2-1â·1) (p=0â·04). the rate of additional antibiotic treatment per patient-year was 2â·3 (95%ci 2â·1-3â·4) in the intervention and 3â·6 (95%ci 2â·9-4â·3) in placebo groups (p=0â·004). improvement in hrqofl was observed in intervention group. azithromycins safety prole was comparable with placebo. conclusion: in pad with respiratory exacerbation, azithromycin prophylaxis led to reduction of exacerbation episodes, of additional courses of antibiotics, and of risk of hospitalization. given the deleterious effects of respiratory diseases adding azithromycin to pad treatment should be considered as a valuable option. background: the autosomal-dominant hyper-ige syndrome (hies), is a primary immunodeficiency caused by mutations in signal transducer and activator of transcription 3 (stat3) that leads to defective th17 immunity. adverse reactions following 23-valent pneumococcal polysaccharide vaccine (ppsv23) have been reported in 75% of stat3-hies patients, including severe local reactions that appear to be specific to this vaccine. case report: we present the case of a six-year-old girl, second child of nonconsanguineous healthy parents, that developed an extensive inflammatory skin reaction at the vaccination site following a single dose of ppsv23. the vaccine was prescribed due to history of recurrent respiratory tract infections and an incomplete vaccine calendar with no previously administered pneumococcal vaccines. the reaction began after 2 hours with local erythema and edema at vaccination site, expanding in 48 hours to a phlyctenular lesion with no well-defined borders. within the first 3 weeks, it progressively evolved to a deep necrotic lesion that required surgical debridement. the subsequent skin defect required surgical repair with a split-thickness skin graft from her right thigh as the donor site. the complete wound healing process took about 5 months, leaving a large scar ( figure) . the patient had a longstanding history of recurrent infections with multiple hospitalizations including severe neonatal pneumonia that required respiratory support, a colon perforation with secondary peritonitis and septic shock that required a hemicolectomy at 8 months of age, recurrent oral candidiasis, recurrent pneumonias of different lobes, recurrent acute otitis media, a cervical phlegmon, three episodes of dental abscess and multiple kidney abscesses due to gram-negative bacteria treated with intravenous antibiotics and surgical drainage. family history is notable for an older sibling that died due to sudden infant death syndrome. the patients mother has large and wide nose suggestive of stat3-hies phenotype, but no history of infections. immunological work up showed mild eosinophilia (850 cells/ mm3), elevated ige (1850 mg/dl), normal igg, iga, igm and lymphocyte subsets (cd3, cd4, cd8, cd16, cd56). peripheral th17 cells were markedly decreased (0.6% vs. 3.7% of normal control). specific pneumococcal antibodies evaluated 1 month after psv23 revealed 5/10 serotypes in protective levels. high resolution thorax ct showed multilobar bronchiectasis. echocardiogram and total spine x-rays were normal. stat3-hies was suspected with a national institutes of health score of 40. a novel heterozygous missense variant in stat3 affecting the src homology 2 (sh2) domain (p.lys591glu) was found by next-generation panel sequencing. a variant in the same position (p.lys591met) has been previously reported in a hies patient (clinvar). currently, she is on monthly ivig and prophylactic antibiotics (cotrimoxazole, azithromycin and fluconazole). conclusions: the case presented raises awareness on the risk of severe local adverse reactions to ppsv23 in stat3-hies patients. the etiology of such reactions is unclear and warrants further study. the benefits and risks of immunizing stat3-hies patients with ppsv23 should be weighed carefully by medical providers. abstract (max 500 words) introduction: dock8 deficiency is a rare primary immunodeficiency characterized by susceptibility to viral infections, atopic eczema, defective t-cell activation and th17 differentiation, impaired eosinophil homeostasis and dysregulation of ige. to date, there are no reported cases from malaysia. objective: we aimed to describe the clinical, immunological profile and mutational analysis of three siblings of consanguineous parents, presented with hyper-ige and lymphopenia between the years 1998 and 2012, which were solved by mutational analysis of the second and third siblings. methods: clinical data and investigation results were collated from the medical record. scoring of the symptoms and physical examination findings using nih score was performed. t, b, nk lymphocyte subsets and serum igg, iga, igm, total ige quantification, lymphocyte proliferation test and pneumococcal specific antibody response were performed. mutational analyses were performed in freiburg, germany. result: three siblings presented at different time points over a 20-year span with raised ige levels, recurrent infections, eczema, hypereosinophilia and bronchiectasis. the nih scores for hyper-ige syndrome (hies) ranged from 39 54. we also documented two serious infections in the siblings, which were disseminated cryptococcus neoformans and salmonella sp. immunological results showed t-cell lymphopenia, defective t-cell proliferation, decreased igm, raised ige, hyper-eosinophilia and defective pneumococcal antibody responses present but not in all 3 siblings. we identified a large deletion in dock8 starting from exon 30-48 in 2 of the siblings from mutational analysis performed. we will proceed with next generation sequencing and dock8 protein assay in malaysia to further characterize the defect. conclusion: our on-going study is the first description of dock8 in a family from malaysia. the diagnosis of dock8 should be suspected in cases with raised ige levels, recurrent infections and lymphopenia, despite no warts infection in the history. this study emphasized the importance of international research collaboration and networking in solving complicated cases. the index patient presented at the age of 4 years with increased susceptibility to lower airway and gastrointestinal infections (hospital admissions 5x/year until puberty). she suffered from mumps and varicella disease despite immunization, as well as from recurrent local, partially destructive hsv infections. she was diagnosed with common variable immunodeficiency (cvid) at age 13 and started on immunoglobulin replacement therapy. following a hypoglycemic seizure at age 20, the patient was diagnosed with isolated acth insufficiency with secondary adrenal insufficiency requiring hormone substitution. during and following her first pregnancy at age 25, she suffered from recurrent bronchopneumonias including pneumocystis jirovecii infection, resulting in bronchiectases documented on chest ct at age 30. currently, chronic lung disease is severely limiting her quality of life (table 1) . her daughter was noticed to be hypogammaglobulinemic soon after birth and failed to develop antibody responses to inactivated vaccines. she was started on immunoglobulin replacement therapy. she has not suffered from severe lower airway infections, but developed alopecia totalis at age 10 and nail dystrophy. w h o l e e x o m e s e q u e n c i n g r e v e a l e d a h e t e r o z y g o u s c.2553_2554insacccgag (p.lys855profster33, nm_001077494) mutation in exon 22 of nfkb2 in both mother and daughter. this monoallelic loss-of function frameshift mutation was not found in gnomad, gvs washington or clinvar databases. as previously published, a monoallelic mutation in this c-terminal domain leads to impaired phosphorylation and subsequent reduced nuclear translocation of the nfkb2/p52 active form. pediatricians and internal specialists need to be aware of the combination of hypogammaglobulinemia, acth deficiency, immune dysregulation and ectodermal dysplasia which is unusual for cvid -possibly indicating nfkb2 deficiency. this clinical syndrome may overlap with symptoms and signs found in both apeced/ aire (ar) and eda-id/nfkbia (ad) deficiencies. besides ig and hormone replacement therapy, curative treatment with hematopoietic stem cell transplantation is a therapeutic option for patients with nfkb2 deficiency, although the experience is limited. table 1 introduction: the modes of immunoglobulin (ig) administration for primary immunodeficiency diseases (pidd) differ in pharmacokinetics, infusion parameters, and tolerability. during 3 consecutive clinical studies, a cohort of 30 patients with pidd experienced all 3 modes of administration with the same ig 10% product in sequence from intravenous (iv) to subcutaneous (sc), then to hyaluronidase-facilitated sc (ighy), providing a unique opportunity to assess each administration modality within the same patient cohort treated and observed at the same sites. here we report the rates of infections stratified by igg trough levels, and the rates of adverse events (aes) with the 3 modes of ig administration (ivig, scig, ighy) within this patient cohort. design and methods: this analysis included patients with pidd aged 4 years who participated in 3 clinical studies: in study 1 (nct00546871) patients received ivig 10% every 34 weeks followed by weekly scig 10%; in study 2 (nct00814320), patients were treated with ighy every 34 weeks; in study 3 (nct01175313; extension of study 2), patients continued with the same ighy dose. to assess a potential association between the administration route at comparable igg trough levels and the infection rate, igg trough levels were categorized as 500 <700mg/ dl, 700<900mg/dl, 900<1100mg/dl, 1100<1300 mg/dl, 1300 <1500 mg/dl and 1500 mg/dl. periods where patients had trough levels within these strata were assessed, and the infection frequency was calculated. the time periods for this analysis were 3 months for ivig and 12 months each for ighy and scig 10% (2.25 years) treatments. in order to account for differences in the frequency of administration, rates of systemic and local aes were assessed as aes/patient-year for each mode of therapy. results: for igg trough levels of <1500 mg/dl, the associated annual infection rates were lower or similar for ighy than scig ( the treatment involves the control of infections and immune dysregulation with chemotherapeutic regimens followed by definitive treatment with hematopoietic stem cell transplant (hsct). aim: to describe a female patient with a pathogenic variation in stx11 with normal cd107a expression. results: she was a 2 years old female, the 5th daughter of nonconsanguineous parents, without relevant personal or family records. she was admitted due to a prolonged febrile syndrome, lymphoproliferation, pancytopenia and hepatitis, with hhv6 rescued in bone marrow and blood. gancyclovir treatment started with good response. she was admitted one month later with similar clinical symptoms with relapsed hhv6 infection. furthermore, hemophagocytosis was found in the bone marrow and evaluation of nk cell cytotoxicity demonstrated slightly reduced cytotoxic activity. functional studies for primary fhl were performed: perforin expression and cd107a surface expression were normal. she fulfilled criteria of fhl, and treatment with gancyclovir and steroids was administered. despite this treatment, she persisted with activated macrophagic parameters, and started with hlh2014 treatment protocol. she improved the clinical symptoms and laboratory parameters, but persisted with hhv6 low viremia. three months later, when immunosupression was decreased, she was readmitted with similar clinical manifestations and added neurological symptoms (facial paralysis, abnormal movements and sleep tendency). cerebral spinal fluid was pathological with hhv6 positive rescue. immunosupresive treatment was adjusted, but hhv6 copies in blood increased markedly. foscarnet treatment was administered and immunosupression was suspended for 2 days in order to control viral infection. unfortunately the patient died 6 days later. although specific functional tests were normal, sequencing of stx11 gene by ngs revealed a homozygous variation in c.581_584deltgcc, which is a previously reported mutation responsible for fhl. conclusion: despite the fact that cd107a was normal, the strong clinical and laboratory results must keep the fhl diagnosis in mind and intensive treatment should be early administered; in order to give the patient the opportunity to achieve the curative treatment. objectives: to report and characterize the clinical course of a patient with apeced and specific antibody deficiency. methods: retrospective chart review was performed. the patient was enrolled in niaid irb-approved protocol 11-i-0187. results: the patient is a 13 year-old-girl with apeced caused by homozygous aire c.967_979del13, who manifested cmc, hypoparathyroidism, adrenal insufficiency, sjogrens-like syndrome, autoimmune hepatitis, intestinal dysfunction and autoimmune pneumonitis. she suffered from recurrent sinusitis and severe pneumonias requiring hospitalization and administration of intravenous antibiotics several times per year. at age 9, she presented to our institution with fever and cough, a computed tomography (ct) of the chest revealed bilateral pulmonary infiltrates and bronchiectasis. bronchoscopy showed mucopurulent secretions in the bilateral lower lobes with culture of the bronchoalveolar lavage fluid growing streptococcus pneumoniae. further evaluation for an underlying disorder such as primary ciliary dyskinesia and cystic fibrosis including exome sequencing and sweat chloride testing was unrevealing. quantitative immunoglobulins were normal. despite prior vaccination, specific antibody testing showed negative rubeola igg and protective levels (> 1.3 mcg/ml) to only 3 of 23 pneumococcal serotypes. lymphocyte enumeration showed normal b cell subsets. as approximately 15% of apeced patients may experience asplenia, splenic ultrasound was performed confirming the presence of a 7 cm spleen and peripheral blood smear did not reveal howell-jolly bodies. serotyping of the s. pneumoniae isolate confirmed serotype 33f, which is part of the 23-valent vaccine. follow up vaccine challenge with the 23 valent pneumococcal polysaccharide vaccine showed an inadequate response. hence, she was started on monthly immunoglobulin replacement and over the following 4 years she has experienced a single methicillin sensitive staphylococcus aureus pneumonia. she has missed very few school days and other parameters including linear growth have improved, she is now along the fifth percentile for height and along the tenth percentile for weight. although she continues to experience intermittent cough she remains active participating in sports without limitation. conclusions: we report the evaluation, treatment and outcome of a patient with apeced complicated by autoimmune pneumonitis and specific antibody deficiency. as infectious susceptibility of apeced classically pertains to the signature infectious disease, cmc, patients with invasive or recurrent infections should be evaluated for underlying immune deficiency. investigation should include assessment for asplenia, quantitative immunoglobulins and specific antibodies with response to antigens. in patients with predominate respiratory symptoms, autoimmune pneumonitis should be evaluated given the near 40% prevalence of pneumonitis observed in american apeced patients. acknowledgements: supported by dir/niaid/nih introduction: autoinflammatory diseases are genetically heterogeneous disorders of innate immunity characterized by recurrent fever, rash, and/ or serositis, which generally are considered distinct from autoimmune diseases. we report a case of a patient with lupus-like disease and a mutation of nucleotide-binding oligomerization domain-containing protein 2 (nod2 r702w, yao syndrome) suggestive of an overlap between autoinflammatory and autoimmunity processes. case presentation: a 72-year-old man was evaluated for recurrent pleural effusions, morning stiffness, erythematous rashes, and fever up to 103â°c. history was notable for hashimotos thyroiditis and multiple admissions for presumed pneumonia with recurrent bilateral lung infiltrates and pleural effusions. transbronchial biopsy showed nonspecific pneumonitis and organizing pneumonia. antinuclear and anti-dsdna antibodies were positive. he received prednisone for presumed lupus pneumonitis leading to improvement. prednisone was tapered and hydroxychloroquine was started, but his fevers, pleuritic pain and pleural effusion reoccurred. genetic testing revealed a nod2 sequent variant (r702w) associated with autoinflammatory disease. hydroxychloroquine was stopped and colchicine was added to his regimen, allowing prednisone to be tapered without recurrence of symptoms. further immunological testing revealed increased signaling through the type i interferon receptor (interferon signature). conclusion: although this patient had several clinical (serositis, arthralgia) and immunological (antinuclear and anti-dsdna antibodies, interferon signature) manifestations of lupus, his clinical presentation also was consistent with yao syndrome. in retrospect, he had been having recurrent inflammatory symptoms for many years. recent studies in both mice and humans suggest that inflammasome activation and il-1 production are involved in the pathogenesis of lupus. this case provides further support for the idea that lupus and hashimotos thyroiditis, prototypical autoimmune diseases, may have overlapping autoinflammatory features. background: the implementation of severe combined immunodeficiency (scid) newborn screening by trec assay has played a pivotal role in identifying these patients early in life. the screen has also led to the identification of infants with other immunologic abnormalities, of which the clinical implications have been unclear and there are limited data on their outcomes. objective: to review immunologic and genetic outcomes of infants referred to an immunology service of a tertiary care center with abnormal newborn scid screens. methods: we retrospectively reviewed charts of infants with positive scid screen from july 2014 to november 2018. we excluded patients who had positive screen at <36 weeks corrected gestational age. we classified outcomes into 3 groups including scid, non-scid t-cell lymphopenia (nscid-tcl) and normal t-cell count. idiopathic t-cell lymphopenia was defined as nscid-tcl (cd3+ < 2,500 cells/mcl) with negative chromosome microarray and negative whole exome sequencing/or genetic panel (either genedxâ® scid panel or invitaeâ® primary immunodeficiency panel). results: of 119 infants, 78% were male, 56% were caucasian, and 37% were african-american. fifty-four % and 46% of infants were identified by illinois and missouri screens, respectively. the mean age at initial evaluation was 22 days (4-122 days). 69% of infants had a normal tcell count (n=80) or normal repeat newborn screen (n=2), 25% had nscid-tcl, including mild (cd3+ 1,500-2,500 cells/mcl, n=20) and moderate (cd3+ 300-1,500 cells/mcl, n=10) tcl, and 6% had scid (n=5), leaky scid (n=1) or complete digeorge (n=1). genetic etiologies of nscid-tcl included 22q11 deletion (n=4), trisomy 21 (n=1), and mutations of tbx1 (n=2), foxn1 (n=1), and cd3e (n=1). three of these infants had novel variants at the time of diagnosis. secondary causes of tcl were identified in 1 infant (thoracic infantile fibrosarcoma). one infant had idiopathic tcl. eighteen infants with nscid-tcl were followed clinically without complete genetic testing performed. for scid, mutations were found in jak3 (n=2), ada (n=1), il2rg (n=1), and rag1 (n=1). the patient with leaky scid had negative whole exome sequencing. all patients with scid and leaky scid underwent hematopoietic stem cell transplantation at a median age of 5 weeks (3 weeks -4 months), with successful engraftment in all but 1 patient. of 19 idiopathic and nscid-tcl cases followed clinically, 12 had at least one follow-up visit at median age 5 months (2.6 months 2.2 years) and the majority had improved or stable lymphocyte count without serious infections requiring intravenous antibiotics, though 1 had a hospitalization for rsv infection. the mysm1 patient died after cord blood transplant from unclear etiology. our study had limitations. half of infants with nscid-tcl did not have a complete genetic workup, and only a fifth of patients with nscid-tcl were inpatients, potentially explaining the relatively low number of infants with secondary lymphopenia. conclusions: in our cohort, one-fourth of infants with abnormal scid screen had nscid-tcl. although the majority of nscid-tcl did well, approximately one-third of them had underlying genetic abnormalities associated with their t-cell lymphopenia. (189) submission id#606320 introduction: accumulation of intracellular adenosine and deoxyadenosine nucleotides (daxp) due to adenosine deaminase deficiency results in profound lymphopenia and severe combined immunodeficiency. left untreated this form of scid is uniformly fatal. while allogeneic hematopoietic cell transplant (hct) and autologous gene corrected stem cell therapy (gt) are potential cures for ada-scid , initiating enzyme replacement therapy (ert) immediately upon diagnosis regardless of definitive treatment is standard of care. hct and gt are not therapeutic options for all ada-scid patients and ert offers immediate therapeutic intervention for these patients leading to partial immune reconstitution, and durable survival in most patients treated. adagen (pegademase), approved by the fda in 1990 in the usa, is a pegylated bovine ada (nada) with the enzyme harvested from bovine intestines. this unsustainable production process led to the development of a recombinant enzyme source based on the bovine protein sequence and an improved pegylated linker by using succinimidyl carbamate (revcovitm-(elapegademase-lvlr). methods: a phase ii/iii clinical trial was performed at 5 us sites under institutional irb approval. eligible ada-scid subjects were stable on adagen and without complicating underlying conditions. demographics, medical history, lymphocyte counts, immunoglobulin levels, trough plasma ada activity and rbc daxp measurements were collected. patients were treated with adagen as a single, weekly im dose adjusted to achieve a trough plasma ada activity of > 15 mmol/hr/l and rbc daxp < 0.02 mmol/l (protocol target levels). once patients had achieved this level (3-9 weeks), a seven-day pk on adagen was done and the patients were transitioned to revcovi based on the formula for enzyme equivalent activity of 1mg revcovi = 150 units adagen. after 5 weeks on revcovi, trough ada and daxp were assessed and a seven-day pharmacokinetic study was conducted at week 9. patients were assessed periodically for clinical and laboratory values and evaluation of the study endpoints was done at week 21. subjects subsequently continued on revcovi and were assessed periodically. results: six patients, ages 16-37 entered the trial with initial adagen dosing at 7.7-42.9u/kg/wk (see table 1 ). adagen dosing was adjusted to target endpoints of ada trough activity (>15mmol/hr/l) and rbc daxp (<0.02 mml/l). patients transitioned to weekly revcovi using the aforementioned conversion formula at doses of 0.17-0.285 mg/kg/wk. the spectrum of clinical manifestations range from infections to autoimmunity and inflammation among patients with hypomorphic recombination gene 1 and 2 (rag1/2) pathogenic variants. auto-antibodies targeting cytokines ifn-alpha, ifn-omega and il-12 were reported in a large proportion of these patients and their occurrence often coincides with viral infections. we report the time of emergence and relative frequency of anti-cytokine antibodies in children and adults, and their persistence among patients with hypomorphic rag deficiency. antibodies were measured from plasma samples of patients by enzyme linked immunoassay (elisa). our rag cohort includes 28 patients with rag1 (n=17, 61%) and rag2 deficiency (n=11, 39%). antibodies targeting ifn-alpha (75%) were most common followed by il-12 and ifn-omega (40% each). two asymptomatic patients who were detected by newborn screening for scid and received hematopoietic stem cell transplantation had no detectable anti-cytokine antibodies. in the cohort of young children (ages 11 mo-7 years, n=9), all patients had detectable antibodies to ifn-alpha, prior history of severe viral infection and subsequently developed autoimmune cytopenias. other anti-cytokine antibodies were less common (ifn-omega 44%, il-12 33%). similarly, children between 10-18 yo age (n=9) also had high fraction of anti-ifn-alpha antibodies (89%) with prior history of infections (66%) and continued to have other anticytokine antibodies less commonly (ifn-omega 37%, il-12 62%). in the adult cohort (n=8, ages 25-39 years) the frequency of anti-ifnalpha anti-cytokine antibodies were lower (62%,) and il-12 and ifnomega (50% each) continued to persist. three adult patients had anticytokine (ifn-alpha, ifn-omega and il-12) antibodies tested at multiple timepoints and elevated titers persisted up to 4 years. our data demonstrates that anti-cytokine antibodies, especially those targeting ifn are frequent and emerge early in life in association with viral infections in patients with rag deficiency. a lower fraction of adult patients have detectable anti-cytokine antibodies, and maintain these over several years. anti-ifn-alpha may serve as a useful biomarker for identifying partial rag deficiency among young and adult patients with history of viral infections and autoimmune cytopenias. the role of these antibodies to cytokines is yet to be determined but a specific signature of these antibodies may help to identify an underlying immunodeficiency and initiate early definitive treatment with bone marrow transplantation. anti-cytokine antibodies appear to be a novel tool in evaluation of autoimmune diseases including rag deficiency. introduction: norovirus is one of the most common pathogens causing gastroenteritis in immunocompromised patients, often leading to chronic infection, causing villous atrophy, malabsorption, weight loss, organ failure, need for parenteral nutrition, and death. norovirus treatment in immunocompromised patients is challenging. oral immunoglobulin (poig) has been used to treat norovirus gastroenteritis with variable success. our aim in this study was to determine the outcomes of treating norovirus gastroenteritis in immunocompromised patients methods: electronic medical records were reviewed for patients with norovirus infection confirmed by rt-pcr since january 2012. our initial cohort was focused on patients with primary immunodeficiency (pid), lung, and liver transplant. data on demographics, immunological phenotype, treatment with poig, the number of bowel movements (bm), and virus clearance were collected. descriptive statistical methods were used to describe treatment outcomes. further analysis of patients immunophenotype, immunosuppression medications, and co-morbid illnesses is underway. results: twenty-six immunocompromised patients (27 norovirus infection episodes, as one patient had reinfection) were analyzed twelve females, age range 7 months-50 years. twelve patients had pid diagnosis (3 common variable immunodeficiency, 2 severe combined immunodeficiency, 1 x-linked agammaglobulinemia, 1 wiskott-aldrich syndrome, 1 digeorge syndrome, 1 hyper-igm, 1 stat3 gain-of-function, 1 nemo and 1 lymphopenia in a patient with trisomy 21), 13 patients were status-post liver transplant, and two patients were status-post lung transplant. 13 of26 patients were on ig replacement therapy at the time of the norovirus infection. the average number of bm/day in all patients was 8. 4 (range 2-20) . eight patients received poig (250-500 mg/kg) weekly for a duration from 1-12 weeks. three of those received additional nitazoxanide and 2 received ribavirin. 2/8 patients in the poig group were receiving total parenteral nutrition (tpn), and 4/19 on no treatment group received tpn. the average number of bm/day in poig before treatment was 9.5 (range 4-16), and 8.6 (range 2-20) in those who did not receive any treatment. 5 of 8 (62%) on poig vs. 11 of 19 (57%) in the no treatment group cleared the virus. the average number of weeks to return to baseline bm was 2.6 (range 1-7) in the poig group vs. 1.5 (range 3 days-5 weeks) in the no treatment group. 2 of 8 on poig continued to have chronic diarrhea that is still ongoing. conclusion: despite anecdotal reports suggesting successful use of poig in immunocompromised patients, our data did not show a significant decrease in stool output in patients treated with poig, compared to no treatment. however, poig led to a higher rate of virus clearance. a study with larger sample size might be warranted to identify the patients who benefit from poig in the context of norovirus infection and ensure the appropriate use of ig products, especially given the concerns for the national shortage of ig products. chief medical officer, novimmune sa primary hemophagocytic lymphohistiocytosis (phlh) is a life-threatening, immune regulatory disorder characterized by immune hyperactivation that is driven by high production of interferon (ifn)-. patients with hlh typically develop fever, splenomegaly, cytopenias and coagulopathy. until recently, there have been no fda approved treatments for hlh, and standard dexamethasone/etoposide-based treatment has not evolved significantly in 20+ years. emapalumab-lzsg (ni-0501) is a fully human, anti-ifn-monoclonal antibody that neutralizes ifn-and which was recently approved (november 2018) by the fda for the treatment of adult and pediatric (newborn and older) patients with phlh with refractory, recurrent, or progressive disease or intolerance with conventional hlh therapy. results of the pivotal trial supporting this approval are presented herein. methods: this open-label pivotal study (nct01818492) includes patients 18 years with a diagnosis of phlh and active disease. data presented were from 34 patients, of whom 27 had failed conventional hlh therapy prior to study entry. the initial emapalumab-lzsg dose was 1 mg/kg given intravenously every 3-4 days. subsequent doses could be increased up to 10 mg/kg based on the evolution of response parameters. dexamethasone was administered concomitantly at 5 to 10 mg/m 2 /day and could be tapered during the study. treatment duration was 8 weeks, with possible shortening to a minimum of 4 weeks, or extension up to the time of allogeneic hematopoietic stem cell transplantation (hsct). the primary efficacy endpoint was the overall response rate (orr) at end of treatment, assessed by pre-defined objective parameters, including normalization or at least 50% improvement from baseline of fever, splenomegaly, cytopenias, hyperferritinemia, fibrinogen, d-dimer, central nervous system (cns) abnormalities, and with no sustained worsening of scd25 serum levels. the primary analysis used an exact binomial test to evaluate the null hypothesis that orr be 40% at a one-sided 0.025 significance level. patients were eligible to enter an extension phase for follow-up after completing the main study (nct02069899). the data cut-off applied is july 20 2017. results: patient characteristics are summarized in table 1 and efficacy is summarized in table 2 . disease at study entry was consistent with the broad spectrum of phlh abnormalities. over 30% of patients had signs and/or symptoms of cns disease. orr was significantly higher than the pre-specified null hypothesis of 40%, meeting the primary endpoint. the response rate based on investigators clinical judgement was 70.6%. emapalumab-lzsg infusions were in general well tolerated, with mild to moderate infusion-related reactions reported in 27% of patients. the observed safety events (pre-hsct conditioning) mostly included hlh manifestations, infections or toxicities due to other administered drugs. infections caused by pathogens potentially favored by ifn-neutralization occurred in 1 patient during emapalumab-lzsg treatment (disseminated histoplasmosis), and resolved with appropriate treatment. no off-target effects were observed. conclusions: treatment with emapalumab-lzsg was able to control hlh activity with a favorable safety and tolerability profile in a very fragile population. the majority of patients proceeded to hsct with favorable outcomes. our results indicate that emapalumab-lzsg should be considered as a new therapeutic option in phlh thanks to its targeted mode of action. results: a total of 360 genes were differentially expressed between t cells of 22qds patients (n=13) and healthy controls (n=6) (log 2 fold change range (-2.0747, 15.6724)).when these 360 genes were tested for pathway enrichment, the top 5 pathways in t lymphocytes based on their p value included communication between innate and adaptive immune cells, cross talk between dendritic cells and natural killer cells, allograft rejection signaling, dendritic cell maturation, and b cell receptor signaling. the top 10 biological processes with differential expression included 36 immune response, 31 inflammatory response, 33 apoptotic process, 12 interferon gamma mediated signaling pathway, 14 nucleosome assembly, 16 defense response to virus, 8 lipopolysaccharide mediated signaling pathway, 7 positive regulation of nf-kappa b import into nucleus, 10 type i interferon signaling pathway, and 10 neutrophil chemotaxis genes. we compared gene expression between 22qds participants with low t cell counts (n=7) and 22qds participants with normal t cell counts (n=6) and found 94 genes that were differentially expressed (q<0.05) (log2 fold change range (-4.5445, 5.1297) patient began experiencing recurrent high fevers and developed splenomegaly. elevated transaminases and concern for lymphoproliferative disease prompted a splenectomy and liver biopsy. both the spleen and liver biopsy were positive for ebv but were negative for malignancy. bone marrow biopsy was unrevealing. genetic testing identified a pathogenic variant in xiap/ birc4 (1141c>t), and the patient was treated with high dose oral steroids resulting in an improvement in symptoms. subsequently, therapy with anakinra was started and steroids were tapered. during the steroid taper, he noticed a change in the vision of his left eye consistent with naion, as well as worsening of his colitis. there was loss of the inferior visual field and fundoscopic exam was significant for left optic disc swelling. oct noted superior retinal nerve fiber layer thinning. oral steroids were restarted with improvement in optic disc swelling, but without improvement or change in vision. as of his most recent exam, the patient has persistent bilateral inferior visual field defects with segmental optic nerve atrophy typical of naion. he has continued therapy with anakinra, and subsequently tapered off of prednisone; though he remains on a physiologic dose of hydrocortisone. conclusions: this case demonstrates an unreported ocular manifestation in a patient with xiap deficiency, which clinically appeared sensitive to immunomodulation. our patient is an unusual candidate for naion due to his young age, the average age of onset being the mid to late 60s, and lack of vascular risk factors. we hypothesize that his hyper-inflammatory condition contributed to irreversible vascular damage in the optic nerve head, resulting in naion. therefore, it may be useful to consider the involvement of systemic inflammatory and immune dysregulatory conditions when treating patients with atypical naion. additionally, naion should be considered in patients with xiap deficiency and sudden unilateral vision loss. the importance of de novo mutations in causing severe sporadic immune disease is well described, yet significance of such a variation in less severe and later onset of immune disease is poorly investigated. whole exome sequencing has been a powerful tool to resolve and explain the genetic basis of novel syndromes in immune related diseases. however, proving causation can be complicated due to low number of the affected individuals. we performed whole exome sequencing in a cohort of patients with noncongenital immune defects, along with detailed cellular biochemical phenotyping. we report and describe a novel non-congenital combined immune deficiency arising from a de novo gain-offunction mutation in ikbkb(c.607g>a). this gene encodes ikk2, and activates canonical nfkb signalling. cellular and biochemical studies of the proband revealed that ikk2v203i results in enhanced nf-kb signaling, as well as t and b cell functional defects. ikk2v203 is a highly-conserved residue, and to prove causation, we generated a crispr/cas9 mouse model that carry the precise orthologous missense mutation. we show that mice and humans carrying this missense mutation exhibits remarkably similar cellular and biochemical phenotypes. dysregulation in patients. total rna isolated from cryopreserved peripheral blood mononuclear cells was reverse transcribed to generate cdna. we selected four known gata2 transcriptional targets, gata1, gata2, tal1 and zfpm1 (encoding fog1) and used droplet digital pcr to quantify transcript levels normalized to the low-expressing gene tbp1. we used samples from 9 individuals with wild-type gata2 (wt), 5 known gata2 mutation patients (mut) and two individuals suspected of gata2 deficiency but without identified mutation or allelic imbalance (unk1, unk2). transcript analysis revealed significantly decreased transcript levels of gata1, gata2 and tal1 in mut pbmcs compared to wt. most wt samples had higher zfpm1 transcripts than gata2 mutated patients however it did not reach statistical significance. strikingly, we were able to use this analysis for two individuals suspected of gata2 deficiency. in the first case (unk1) a 51 yr old female with primary lymphedema, hypogammaglobulinemia, recurrent infections and possible family history of leukemia was referred for gata2 testing. no mutation was identified however it was noted that she was homozygous across the gene preventing allelic evaluation. the second patient (unk2), a 24 yr old female, had erethemya nodosa on legs, mycobacteria kansasii and cytopenias. in each of the targets analyzed, transcript levels from unk1 were lower than the wt samples and in a similar range as the gata2 mutation samples while unk2 had a profile consistent with the wt samples. we propose the use of gata2 targets as surrogate markers in cases where a mutation is not identified and allelic expression analysis is uninformative. are often under-reported and under-recognized. we sought to further understand and evaluate the prevalence, type, and association with serum immunoglobulin e (ige) for cvid patients with atopic manifestations. methods: we performed a retrospective analysis of cvid patients with atopic manifestations in the partners healthcare cvid cohort. we evaluated baseline patient characteristics, atopic diagnoses, and serum ige levels. results: in the partners cvid cohort, the average age was 52 years old (â±17) and 64% female. 92/175 (52.6%) of patients had a diagnosis of asthma, with the majority of these diagnosed by an allergist (65%) or pulmonologist (16%). eczema/atopic dermatitis was diagnosed in 47/175 patients (26%), by either an allergist (53%) or a dermatologist (8%). allergic rhinitis was diagnosed in 50/175 (28.5%) with positive skin prick testing in 52% of these patients. food allergy was diagnosed in 5 patients (2.9%). the median cohort serum ige was 7.5 iu/ml. the median serum ige was higher in patients with 2 or more atopic complications compared to those with one or less atopic condition (9 vs. 5 iu/ml), which was statistically significant (p=0.01). conclusions: we report higher rates of atopy than previously described in other cvid cohorts. consistent with previous reports, we find a low median cohort serum ige level in cvid patients compared to the general population. however, we identify a subset of patients with a predisposition towards atopy and higher ige levels within the broader characterization of cvid, and these patients may have a more specific molecular diagnosis that leads to elevated ige and atopic conditions. whole exome sequencing is underway to further evaluate this hypothesis. whim (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome is a primary immunodeficiency with autosomal dominant inheritance. in most patients, the genetic cause of the disease is a gain-offunction variant in c-x-c chemokine receptor type 4 (cxcr4) that results in arrest of neutrophil migration from the bone marrow. most patients develop hypogammaglobulinemia and early waning of antibody response with vaccination. however, the exact origin of aberrant humoral immunity in whim syndrome patients is yet to be clarified. here we describe a 4-year-old iraqi female with a heterozygous cxcr4 p.ser338ter variant, which is presented with haemophilus influenzae meningitis, history of tetralogy of fallot, early onset intermittent neutropenia, lymphopenia, recurrent bacterial and viral infections. immunologic evaluation revealed hypogammaglobulinemia, elevated igm level and a lack of protective vaccine titers after tetanus and prevnar vaccinations. a bone marrow biopsy was consistent with myelokathexis. immune phenotyping, functional studies and apoptosis assays were performed on peripheral blood cells by flow cytometry in our whim patient and controls. although we found that all lymphocyte compartments were reduced, naã¯ve cd4 t helper cells and switched memory b cells were predominantly affected. spontaneous apoptosis was most pronounced in b rather than t cell compartments in whim patients. in addition, naã¯ve b cells easily activated and died upon activation in vitro. cxcl12, a ligand of cxcr4, induced elevated t helper cell migration and increased actin polymerization in p.ser338ter mutant cells. we conclude that intrinsic b cell abnormalities, such as increased rate of apoptosis and altered activation, might be responsible for defective antibody response in whim patients. although most individuals effectively control herpesvirus infections, some suffer from unusually severe and/or recurrent infections requiring anti-viral prophylaxis. a subset of these patients possesses defects in nk cells, innate lymphocytes which recognize and lyse herpesvirus-infected cells; however, the exact genetic etiologies are rarely diagnosed. plcg2 encodes a signaling protein in nk cell and b cell receptor-mediated signaling. dominant-negative or gain-of-function mutations in plcg2 cause cold urticaria, antibody deficiency, or autoinflammation. however, loss-of-function mutations and plcg2 haploinsufficiency have never been reported in human disease. we examined 2 families with autosomal dominant nk cell immunodeficiency with mass cytometry and whole-exome sequencing to identify the cause of disease. we identified two novel heterozygous loss-of-function mutations inplcg2 that impaired nk cell function, including calcium flux, granule movement, and target killing. although expression of mutant plcg2 protein in vitro was normal, phosphorylation of both mutants was diminished. in contrast to plaid and aplaid, b cell function remained intact. plcg2+/-mice, as well as targeted crispr knock-in mice, also displayed impaired nk cell function with preserved b cell function, phenocopying human plcg2 haploinsufficiency. we report the first known cases of plcg2 haploinsufficiency, a clinically and mechanistically distinct syndrome from previously reported mutations. therefore, these families represent a novel disease, highlighting a role for plcg2 haploinsufficiency in herpesvirus-susceptible patients and expanding the spectrum of plcg2-related disease. we pursued genetic diagnosis, which identified bi-allelic frameshift mutations in the rag1 gene which had not been previously described: c.967delg (p.v323sfsx22) and c.1048_1075del128insaaaagagtg (p.v350kfsx47). taken together, his presentation suggested significant immune dysfunction had evolved since transplant leading to extensive pulmonary nontuberculous mycobacterial infection and possible bronchiolitis obliterans. he therefore will undergo a subsequent unconditioned cd34+ stem cell boost from his sister, the original donor, once he completes mycobacterium abscessus treatment. this case highlights the potential long-term immune dysfunction which may evolve after unconditioned allogeneic stem cell transplant for scid, in which full engraftment in all myeloid and lymphoid compartments is not expected. it also highlights the importance of guideline-driven follow-up of these patients to monitor for said dysfunction, to prevent serious infection and long-term sequelae. somatic hypermutation (shm) in the b cell receptor (bcr) heavy (igh) and light chain genes promotes affinity maturation and also mutation away from self-reactivity, therefore serves as an important peripheral tolerance checkpoint. as an example, unmutated bcr ighv4-34 genes give rise to antibodies that bind to i/i antigen on red blood cells (rbc) and may elicit cold agglutinin disease (cad), a variant of autoimmune hemolytic anemia (aiha). in case of healthy individuals, frequent shms in the i/i binding site of bcr ighv4-34 genes decrease rbc reactivity and cad. patients with primary immunodeficiencies (pid) paradoxically develop autoimmune diseases, including autoimmune cytopenias, especially aiha. it is unclear if impaired shm of bcr, in particular mutation away from i/i binding, is relevant in the development of rbc reactivity and consequently aiha in a pid background. our studies focus on pid patients with hypomorphic recombination activating gene (rag1 and 2), combined immunodeficiency phenotype and history of autoimmunity, in particular aiha (rag cid/ai). we detected increased frequency of unmutated ighv4-34 bcr in memory b cell repertoires of rag-cid/ai patients as well as elevated titer of unmutated ighv4-34 antibodies in the patients' plasma. lower level of shm likely reflect abnormal germinal center (gc) reaction. as rag1 and 2 heterotetramer primarily shapes the pre-immune t and b cell repertoire, we studied the interaction of follicular helper t cells (tfh) and naive b cells via in vitro co-culture experiment. interestingly, tfh cells from rag cid/ai patients exhibited highly activated phenotype with increased expression of cd40l and il-21 compared to healthy controls and were able to initiate exaggerated response (class switching and shm) of healthy donor naive b cells. on the contrary, in vitro activated naive b cells from rag cid/ai patients showed impaired proliferation, class switching and decreased level of shm with diminished induction of genes involved t cell co-stimulation (cd40, il-21r) and shm (aicda, repair enzymes) compared to healthy donor naive b cells indicating intrinsic defect in patient b cells. furthermore, b cells from rag cid/ai patients also showed increased apoptosis and accumulation of gamma-h2ax foci at steady state indicating reduced cellular fitness. these findings suggest that the development of aiha is a multifactorial process in partial rag deficiency. our studies highlight that impaired germinal center reaction is an important tolerance checkpoint with the inability of patient's b cells to respond to hyperactive tfh cells and introduce proper level of shm. hence, we propose that b cell fitness is compromised which impairs proper gc interaction, shm, including mutation away from self and sustains rbc reactivity in hypomorphic rag deficiency. introduction/background: the forkhead box n1 (foxn1) transcription factor is an essential regulator of t cell development, affecting the differentiation and expansion of thymic epithelial cells (tecs). autosomal recessive mutations in foxn1 cause a t-b+nk+ lymphocyte phenotype due to a thymic aplasia in conjunction with alopecia universalis and nail plate dystrophy resulting from keratinocyte dysregulation. this is a classic nude/scid (omim # 600838) phenotype. we report on the identification of two independent patients, identified through newborn screening with absent trecs and with a t-nk+b+ scid phenotype who presented with a t cell lymphopenia who had compound heterozygous mutations in foxn1. notably, these individuals had normal hair and nail beds. objectives: to determine whether distinct compound heterozygous mutations in foxn1 cause a novel t-nk+b+ phenotype in the absence of a classic nude presentation. neutralizing autoantibodies (autoabs) against cytokines increase the susceptibility for selected infections (e.g. anti-ifn-autoabs for nontuberculous mycobacteria and non-typhoid salmonella, anti-il-17-autoabs for mucocutaneous candidiasis and anti-gm-csf-auotabs for infections by cryptococcus, nocardiae and aspergillus spp). however, the role of anti-il-6-autoabs is less clear. il-6 is a key mediator of the acute-phase response and released early in bacterial infections. patients with impaired signaling or affected production of il-6 are at increased risk for severe bacterial infections. only three patients with high-titer and neutralizing anti-il-6-autoabs who suffered from severe infections caused by s. aureus, s. intermedius and e. coli have been described so far. to investigate the prevalence of anti-il-6-autoabs in patients with bacterial infections, we investigated a cohort of 350 patients and identified three further patients, all previously healthy, with neutralizing auotabs against il-6 who hardly developed an acute-phase response. the first patient suffered from life-threatening pneumonia caused by s. pneumonia, the second patient developed a submandibular abscess and septic arthritis caused by s. pyogenes and the third patient suffered from life-threatening pneumonia caused by s. aureus. we also discovered neutralizing anti-il-6-autoabs in two adults among a cohort of patients with autoimmune diseases (n = 564), in one adolescent among a cohort of obese individuals (n = 455) as well as in three mothers of neonates with impaired il-6 signaling. so far none of the later individuals developed a severe bacterial infection. this suggests that naturally occurring and neutralizing anti-il-6-autoabs are a risk factor for severe bacterial infections yet with incomplete penetrance. (215) submission id#606931 persistent transaminitis in copa syndrome 1 researcher, immunodeficiencies research unit, national institute of pediatrics, mexico city 2 social service intern, immunodeficiencies research unit, national institute of pediatrics 3 pediatrics resident, pediatrics hospital, 21st century national medical center, mexican institute of social security 4 researcher, data science department, mexican autonomous institute of technology 5 researcher, department of research methodology, national institute of pediatrics background: inborn errors of immunity constitute a heterogeneous group of over 400 individually rare congenital diseases that involve genes coding for proteins of the immune system, and which result in increased susceptibility to infection, inflammation, autoimmunity, allergy and cancer. the complexity of the diagnostic task, and the intrinsic biases and limitations of the human mind, can be aided by computational tools. among the available machine learning approaches, decision tree algorithms select the best node to split based on entropy and information gain; random forests build hundreds or thousands of decision trees randomly (bootstrapping), to improve accuracy and reduce overfitting. aim: to implement a machine learning-assisted clinical decision support system for the diagnosis of inborn errors of immunity (iei). methods: with a local database of patients with suspected iei, we built a decision tree using c4.5 dtc, and a random forest on python 3 (jupyter notebook, scikit, mathplotlib, pandas, numpy). the database was obtained by conducting an electronic search on medsys of patients with the term immunodeficiency in their electronic medical records, and then hand-picking cases in which an iei had been confirmed or ruled out. it consisted of 234 patients, of which 201 had been diagnosed with iei. we first split the dataset randomly into training (70%) and testing (30%) sets. the decision tree was tasked with classifying correctly pid or not. after running the algorithm in the training set, we evaluated in the testing set. the random forest classified all cases by majority vote into nine groups (0 to 8), according to the iuis pid group. next, we repeated the process on a larger scale with a dataset of 2,400 patients from usidnet. accuracy was assessed by out-of-bag (oob) error estimates. results: accuracy was greater than 95% for the local dataset (pid/ not, 9 groups), and for the usidnet dataset (9 groups). we provide a list of decision nodes and a diagnostic route with those questions that achieved a greater information gain and less entropy. this might help clinicians direct their interrogation and diagnostic approach of suspected iei patients. discussion: we built two classification models. decision trees lend themselves more easily to learning and deriving rules of thumb from their sequences. random forests are more robust and better suited for categoric (as opposed to binary) classification. we next want to develop a chatbot that will ask relevant questions in optimal sequence, and extract undiagnosed patients with suspected iei, based on statistical red flags. 13 researcher, immunodeficiencies research unit, national institute of pediatrics, mexico city dna repair defects are inborn errors of immunity that result in increased apoptosis and oncogenesis. dna ligase 4-deficient patients suffer from a wide range of clinical manifestations since early in life, including: microcephaly, dysmorphic facial features, growth failure, developmental delay, mental retardation; hip dysplasia, and other skeletal malformations; as well as a severe combined immunodeficiency, radiosensitivity and progressive bone marrow failure; or, they may present later in life with hematological neoplasias that respond catastrophically to chemo-and radiotherapy; or, they could be asymptomatic. we describe the clinical, laboratory and genetic features of five mexican patients with lig4 deficiency, together with a review of 36 other patients available in pubmed medline. four out of five of our patients are dead from lymphoma or bone marrow failure, with severe infection and massive bleeding; the fifth patient is asymptomatic despite a persistent cd4+ lymphopenia. most patients reported in the literature are microcephalic females with growth failure, sinopulmonary infections, hypogammaglobulinemia, very low b-cells, and radiosensitivity; while bone marrow failure and malignancy may develop at a later age. dysmorphic facial features, congenital hip dysplasia, chronic liver disease, gradual pancytopenia, lymphoma or leukemia, thrombocytopenia and gastrointestinal bleeding have been reported as well. most mutations are compound heterozygous, and all of them are hypomorphic, with two common truncating mutations accounting for the majority of patients. stem-cell transplantation after reduced intensity conditioning regimes may be curative. 1 department of laboratory medicine, clinical centre 2 immunology, allergy and rheumatology division, department of pediatrics, baylor college of medicine, texas children's hospital, houston,texas, usa 3 laboratory of clinical immunology and microbiology, fungal pathogenesis section, national institute of allergy and infectious diseases, 4 department of intramural research, national institute of allergy and infectious diseases (niaid), national institute of health, bethesda maryland, usa card9 deficiency is an autosomal recessive primary immunodeficiency known to underlay increased fungal infection susceptibility mostly presenting as invasive cns candida infections (in infancy or adulthood) and dermatophyte infections. more recently, a rare card9 variant (c.1434+1 g>c, leading to exon 11 skipping, card9del11) showed a significant protective association towards inflammatory bowel disease (ibd) when present in heterozygosity. at the nih we studied an 8-year-old male patient (p1) born to a non-consanguineous marriage who presented as an infant with recurrent/severe thrush, candida esophagitis, and an episode of tinea pedis; p1 also has mild hypogammaglobinemia (igg 500mg/dl at age 8y). p1s gdna was tested by whole exome sequencing and showed a card9 c.1434+1 g>c mutation in homozygous state. segregation analysis and sanger confirmation determined that both parents and p1s elder brother carried the same variant in heterozygosity, while his asymptomatic younger brother (p2) was also homozygous. as previously described, this variant caused card9 exon 11 deletion as determined in p1 and p2s pbmcs by cdna sequencing and by a lower molecular weight card9 protein by immunoblot evaluation. p1 and p2s pbmcs, as well as the heterozygous parents cells, showed a defective cytokine generation (tnf-, il-1, il-6 and gm-csf) in response to heat killed candida (hkc), but not to lps. while patients pbmcs failed to induce phospho-erk and phospho-p-38 upon hkc-stimulation but presented an intact response to pma+ionomycin; the parents cells responded normally to both stimuli. moreover, t-cell activation and proliferation was affected in response to hkc but not to pha in both patients, whereas the parents exhibited normal results under the same conditions. when hek293 cells were transiently transfected with wt or card9del11 vectors together with a trim62 plasmid (e3-ubiquitin ligase, naturally associated to card9), we confirmed that card9del11 failed to bind trim62 by immunoprecipitation. furthermore, malt1, bcl10 and trim62 were only co-precipitated by wt card9, but no by card9del11, strongly suggesting trim62 is an integral part of the card9/bcl10/malt1 -cbm-complex. in summary, herein we demonstrate that the card9del11 allele fails to bind trim62, and in turn is unable to conform a complete/functional cbm complex. our data also show that card9del11 acts in a dominant negative fashion in terms of cytokine generation (previously reported), but one wt allele seems sufficient to generate normal levels of hkcinduced p-erk and p-p-38, as well as t-cell proliferation. while decreased cytokine generation associated with card9del11 in heterozygosity has been described to be sufficient to protect towards ibd, other defective pathways are affected in homozygosity and likely necessary to confer increased susceptibility to fungal infections. altogether these results suggest that card9del11 acts through a gene dosage mechanism that can dissect pathways that associate ibd protection and fungal infection susceptibility. further work is warranted to explore card9del11 role, if any, in b-cell and t-cell biology. professor, endocrinology, university of michigan medical school background: acquired generalized lipodystrophy (agl) syndromes are a heterogeneous group of diseases characterized by selective dysfunction and loss of adipose tissue after birth. this causes ectopic lipid deposition and deficiency of the adipokine leptin, which promotes metabolic dysfunction through impaired glucose handling resulting in insulin-resistant diabetes mellitus, dyslipidemia and steatohepatitis. while the metabolic effects of altered adipokine secretion are known, the molecular mechanism is less clear. many agl cases are suspected to have an autoimmune etiology. effector and regulatory t cells, dendritic cells and macrophages reside in normal adipose tissue. t cells within adipose tissue highly express pd-1 and regulatory t cells express ctla4, which limits immune activation in the adipose tissue under normal circumstances. thus, inhibition of these immune checkpoints may hypothetically cause immune activation, leading to adipocyte dysfunction and autoimmune destruction. we have encountered two cases that raise clinical concern for this process. patient cases: patient 1 is a 16-year-old female who presented with failure to thrive at 6 months. she was diagnosed with insulin-resistant type 1 diabetes and hypertriglyceridemia at ages 2 and 4 years with progressive subcutaneous fat loss and low leptin levels culminating in a diagnosis of agl. her childhood clinical course was complicated by hypertrophic cardiomyopathy, hepatomegaly, autoimmune hemolytic anemia with massive splenomegaly and severe chronic diarrhea secondary to autoimmune enteropathy. she presented at 14 years with acute liver failure, thrombotic microangiopathy, nephrotic syndrome and progressive kidney insufficiency. evaluation for her multi-faceted autoimmune presentation identified a familial heterozygous pathogenic variant in the ctla4 gene (c.4_5insgttgg,p.ala2glyfster14). despite aggressive immune therapies, including ctla4-ig (abatacept), her kidney disease and enteropathy have progressed. patient 2 is a 55-year-old male diagnosed with localized malignant melanoma of the right neck in july 2014. he underwent excisional biopsy and regional lymph node dissection with negative margins. he relapsed in november 2017 and underwent a modified radical neck dissection with 1 lymph node positive for disease and received external beam radiation from january-february 2017. additionally, he was started on anti-pd-1 therapy with the humanized antibody drug pembrolizumab in april 2017 but discontinued the drug in february 2018 in the setting of toxicities including hypothyroidism. subsequently, he developed up to 7.5% weight loss with progressive loss of subcutaneous fat first in his face, then generalized to the rest of his body. in the ensuing months, imaging with pet-ct demonstrated loss of subcutaneous fat concurrent with elevations in alt and triglyceride levels plus a low leptin level consistent with agl. conclusion: these cases raise concern that inhibition of the immune checkpoints ctla4 and pd-1 may facilitate the development of agl. we hypothesize that these defects significantly increase t cell autoimmune activity in the adipose tissue and/or alter t cell metabolism resulting in agl. disorders of immune dysregulation should be considered in the etiology of agl. similarly, patients with either genetic or pharmacologic inhibition of immune checkpoints should be monitored for the development of agl with careful physical exam and periodic monitoring of glucose and triglyceride levels. background: rosai-dorfman disease (rdd; also known as sinus histiocytosis with massive lymphadenopathy) is a rare non-langerhans cell histiocytosis. it is characterized by proliferation and accumulation of activated histiocytes in affected tissues. classically, rdd presents with bilateral, non-tender, and often markedly enlarged cervical lymphadenopathy. case presentation: a 2-year-old female presented with a 6-week history of asymptomatic, persistent and bilaterally enlarged cervical lymph nodes. she was otherwise healthy with no significant past medical history. operative excision biopsy of the largest lymph node confirmed the diagnosis of rdd. three months following diagnosis, routine bloodwork revealed that she had developed lymphopenia (lymphocyte count 1.4 x 109/l). between 1-year and 2-and-a-half-years post-diagnosis, the patient was hospitalized and treated with intravenous antibiotics for 2 presumed episodes of osteomyelitis and 2 presumed episodes of lymphadenitis. given the recurrent presumed infections and persistent lymphopenia, the patient was referred to immunology for evaluation. she received a full immunologic work-up. lymphocyte immunophenotyping revealed low cd4 (288 cells/mm3) and low cd8 (228 cells/mm3) counts. the rest of her immunologic work-up was within normal limits. approximately 3-and-a-half-years post-diagnosis, the decision was made to initiate treatment for rdd. she was started on a 6-week tapering course of prednisone therapy. within 2-weeks of starting corticosteroid therapy, the lymphadenopathy had diminished, and by 6-weeks, the lymphopenia completely resolved. at her most recent clinic visit, she had been free of serious infections for more than 3-years, and her lymphocyte counts had remained stable and within normal limits for over one year. discussion: in the literature, immune system dysfunction has been reported in rdd, with both auto-antibodies and cellular immunodeficiency implicated. in this patient, the persistent lymphopenia and recurrent episodes of presumed infections appeared consistent with an immunodeficiency. given the known association of rdd with immunologic dysfunction, this was certainly a reasonable assumption; however, when these issues resolved following corticosteroid therapy, we questioned whether her clinical presentation could instead represent a manifestation of her underlying rdd. this case highlights the diagnostic challenge of differentiating between an infection and an rdd exacerbation. the episodes of presumed infections were considered probable but not confirmed with microbiologic or histopathologic specimens. the mechanism underlying lymphopenia in rdd is not clear but may involve decreased production, increased destruction, or sequestration of lymphocytes. to our knowledge, this has not been specifically studied in rdd in the past, however lymphopenia has been linked to lymphocyte maldistribution in other diseases. for example, studies have shown that experimentally altering either the surface of the lymphocyte or the environment through which the lymphocyte travels through can cause sequestration of lymphocytes in various lymphoid organs including lymph nodes. conclusion: we describe the case patient with rdd that developed persistent lymphopenia, and multiple episodes of presumed infections resulting in hospitalization and intravenous antibiotic therapy. her lymphopenia resolved and she had sustained remission of rdd following treatment with corticosteroids. we hypothesize that lymphocyte sequestration in enlarged lymph nodes may have resulted in lymphopenia. this, combined with recurrent rdd exacerbations that clinically resemble infections created a presentation that mimicked an immunodeficiency. background: there is an expanding spectrum of immunodeficiency phenotypes linked to dna repair defects, and some patients may not be diagnosed until adulthood. the most well recognized genetic defect linked to dna repair is in the gene, ataxia telangiectasia mutated (atm), which causes ataxia telangiectasia, characterized by combined immunodeficiency, neurodegeneration, radiation sensitivity, and ocular telangiectasias. however, there are several other dna repair defects associated with immunodeficiency, including some syndromic and severe combined immunodeficiency (scid) disorders. objective: we present the case of an adult patient with prolonged history of recurrent infections, facial abnormalities, and autoimmunity who was found to have radiosensitivity suggestive of a dna repair defect. methods: retrospective chart review, immunodeficiency evaluation, flow-based radiosensitivity assay, gene sequencing. results: a 68-year-old female was referred to our clinic due to a complex history of recurrent infections and immune dysregulation. the patient had a lifelong history of sinopulmonary infections and panhypogammaglobulinemia with low vaccine responses, leading to a diagnosis of common variable immunodeficiency (cvid), necessitating treatment with immunoglobulin replacement. clinical features were also notable for congenital dysmorphia (strabismus, thin and angular face, high arched palate, nasal septal defect, small mouth, missing dentition, clinodactyly, severe equinovarus, and scoliosis). she was subsequently diagnosed with autoimmune features of vasculitides requiring trial of cyclophosphamide, azathioprine, rituximab and belimumab, which was later discontinued due to neutropenia and worsening sinopulmonary and skin infections despite immunoglobulin replacement. in the course of our evaluation she was revealed to have severe b cell lymphopenia (1%), cd4 naã¯ve t cell lymphopenia, persistent iga and igm deficiency one-year post rituximab therapy, and elevated alpha fetoprotein (afp). radiosensitivity assay revealed decreased atm phosphorylation and elevated levels of h2ax 24-hours after low-dose (2gy) radiation in her lymphocyte subsets (t, b and nk cells) . due to the evidence of radiosensitivity and elevated afp levels, there was concern for an atm or other genetic defects in a dna repair pathway. therefore, a targeted (primary immunodeficiency genes) panel was pursued for genetic testing (207 genes, invitae, san francisco). the evaluation did not identify a variant in the atm gene but rather a variant of uncertain significance was identified in the chd7 gene, in exon 38, c.8440g>a (p.gly2814arg), which may be mosaic. this variant has not been reported in population databases. chd7 is typically associated with charge syndrome, and while this patient has some dysmorphic features, she is not typical for charge syndrome. currently, studies on copy number variation (cnv) and deep intronic variants in atm are pending. conclusion: dna repair defects may occur in adult patients with a primary diagnosis of cvid. our patient exhibits some phenotypic features of both a chd7 variant, and atm leading to possible abnormal dna damage responses (ddr). the exact cause of the immune deficiency in our case remains presently unsolved. this case highlights the relevance of both functional studies and genetic evaluation of complex cases of immune dysregulation, for improving our understanding of the phenotypic variability in these immunological disorders. background: womens health issues in patients with immunodeficiency are largely underrepresented in the literature. there are no studies assessing for fertility issues in patients with antibody deficiencies, and there are few sizable studies examining pregnancy and outcomes on progeny in the same cohort. the two largest studies of pregnancy in antibody deficiency, an idf survey and a study of the czech population, provide conflicting data about the safety of pregnancy for these patients. immunoglobulin replacement has been shown to be safe and beneficial in pregnancy for patients with cvid, however, dosing strategies are unguided. we sought to further understand these and other issues associated with fertility and pregnancy in a large cohort of patients with antibody deficiencies. methods: we performed a retrospective chart review of over 100 patients with icd9 and/or icd10 codes of cvid or another antibody deficiency from january 2005 to december 2018. inclusion criteria also comprised of having reached at least 16 years of age, the beginning of child bearing years. data collected included disease characteristics, comorbidities, laboratory values, and outcomes. this was followed by a phone survey to elucidate data regarding fertility, pregnancy, delivery complications, and outcomes of children. this study was irb approved. results: the current age of women included ranged from 16 to 88 years of age, currently being in childbearing years to being post-menopausal. forty percent of the women had been pregnant, delivering an average of 2 babies per woman who had been pregnant. fertility issues were not a prominent factor for women who never became pregnant. a majority of women who had babies (64%) did not receive a diagnosis of antibody deficiency until after their child bearing years. recurrent upper respiratory tract infections, bacterial sinusitis, and urinary tract infections during pregnancy were common even in those not yet diagnosed with antibody deficiency. immunoglobulin levels and dosing of intravenous and/or subcutaneous replacement were recorded for a subset of patients with recent pregnancies. the data re-enforced that increases in dosing are needed in the third trimester. cord blood igg levels were also recorded for baby and were the same or higher than the mothers most recent igg prior to delivery. it was rare for children of our patients to be diagnosed with antibody deficiency or a related condition, although cvid, hypogammaglobulinemia, combined immunodeficiency, lymphoma, rheumatoid arthritis, and other diagnoses were found. conclusion: this is the largest report of outcomes before, during, and after pregnancy for patients with antibody deficiencies in the united states. this report highlights the importance of closely monitoring women during pregnancy for recurrent infections regardless of whether a diagnosis of antibody deficiency is present. it also highlights that close monitoring of igg levels during pregnancy is necessary for women with antibody deficiencies. backgrounds: autoinflammatory diseases (aids) are a group of disorders with an inborn error of innate immunity, characterized by recurrent episodes of fever and inflammatory attacks. the spectrum of aids is expanding, but no data on clinical presentation and symptom variability exist for the iranian population for timely precise diagnosis. this study aims at establishing the first autoinflammatory registry of an iranian population focusing on the clinical and laboratory features that may help clinicians toward a better understanding and diagnosis of these disorders. methods: clinical and laboratory characteristics of patients who clinically and or genetically diagnosed with aids collected. we used the updated version of classification criteria from the eurofever registry for the clinical diagnosis. results: in our retrospective study, clinical and laboratory characteristics of the participants collected. mean age of disease onset, disease course manifestation, the mean duration of episodes, atypical symptoms, laboratory and imaging studies as well as complications, and response to treatment also reviewed. data resulted in 26 patients of whom 16 were male. their age ranged from 2 to 68 years. 5 out of 26 were genetically diagnosed. familial mediterranean fever (fmf) was the most common clinically and genetically approved diagnosis. there were also patients suspected of nlrp12 and nod2 mutations. age at disease onset differed variably and ranged from the neonatal period to adulthood. fever was present in all the participants and the duration of episodes was 1-10 days. the frequency of attacks was between 3 to more than 12 per year. some of the common clinical manifestations were as follows: myalgia or fatigue (77%), arthralgia and arthritis (70%), abdominal pain (65%), aphthous stomatitis (38%), chest pain (34%), chronic gastrointestinal symptoms (38%), skin lesion ranging from urticarial rash and severe nodular acne to pyoderma gangrenosum (50%), exudative and or erythematous pharyngitis (46%), consanguineous parents (42%), symptoms of a type of allergy (84%), lymphadenopathy (27%), splenomegaly (27%), increased acute phase reactant (54%), elevated liver function test (19%) . 10 out of 26 of the individuals reported positive family history and in one of the cases, a patient carrying the homozygous mutation in the mefv gene has shown no clinical manifestation. conclusion: this study highlights the most common manifestations of aids in the population of iranian origin and can be used as evidencebased clinical criteria for their diagnosis. background: the term benign ethnic neutropenia (ben) is used to describe patients of african/arabic descent with absolute neutrophil counts (ancs) less than 1500 cells/ul in the absence of other causes. historically, race has been used to support the diagnosis of ben, but self-reported race is notoriously imprecise. the duffy null phenotype (fya -/fyb-) is a known molecular cause of ben and may be a more reliable marker of ben than self-reported race. in addition, although the anc is known to be lower in patients with ben, the lower limit of ancs is poorly described. it is important to differentiate patients with ben from primary immunodeficiency diseases (pidd) and to recognize their expected anc values. methods: eligible subjects included patients less than 21 years seen at the university of michigan between january 2010-july 2018. duffy null (fya -/fyb-) patients were identified using electronic medical record search engine (emerse) software and search terms duffy and fyab. 105 potential subjects were identified; 67 patients met inclusion criteria including duffy null status and the absence of other conditions or medications, potentially impacting ancs. 251 unique healthy anc values were recorded from the 67 duffy null patients. age and sex matched controls were identified using emerse software with search terms tonsillectomy, department of anesthesiology and absolute neutrophil count. subjects with conditions or medications that might impact the anc or of african/arabic descent were excluded from the control group. asian and caucasian patients included as controls were presumed to be duffy null given that <1% of these populations are expected to be duffy null. 363 control subjects were identified; 134 met inclusion and exclusion criteria. statistical analysis was performed using two-sided two-sample t-test, anova and onesample t-test. results: the median age of the duffy null cases was 4.78 years (iqr: 1.68-11.48) with 61.2% (n=41) male and all of african or arabic descent. mean anc for duffy null patients was 1190 cells/ul (n=251, sd= 650) while mean anc for controls was 4300 cells/ul (n=134; sd=1600) with a mean difference between controls and duffy null cases of 3100 cells/ul (95% ci: 2950-3380; p=0.0001). the anc levels between duffy null individuals and controls were evaluated by five age categories (p=0.0001 for all age categories). however, there was no difference in anc levels between duffy null cases at different age categories (anova, p=0.14196). 54 (21.5%) duffy null cbcs had anc levels in the nonneutropenic range (>1500 cells/ul), 99 (39.4%) cbcs had mild neutropenia (1001-1500 cells/ul), 70 (27.9%) cbcs had moderate neutropenia (500-999 cells/ul), and 28 (11.2%) cbcs had severe neutropenia (<500 cells/ul). conclusions: although neutropenia can be associated with pidds and is often a sign of a compromised immune system, duffy null patients have a wide range of values that are often much lower than previously appreciated. the degree of neutropenia related to duffy null phenotype appears to persists throughout childhood and young adulthood. in the context of patients of african/arabic descent presenting with asymptomatic neutropenia, duffy null status should be assessed, and ben should be considered in the differential. complications, hypogammaglobulinemia and a unique characteristic of decreased susceptibility to enveloped viral infections. objective: to investigate the role of impaired host n-linked glycosylation on viral susceptibility to ebola virus. methods: to mimic the condition observed on cdg-iib patients, we tested in vitro three proprietary iminosugars (emergentbiosolutionsâ©), uv4b, uv001, and uv00128, which act as competitive inhibitors of -glucosidase i and ii. their ability to inhibit the trimming of n-glycans was compared to known n-glycans modifiers as castanospermine, tunicamycin, as well as the bacterial enzyme peptide-nglycosidase f (pngase-f). ebola virus envelope protein gp1 was chosen as a prototype glycoprotein, as it is heavily glycosylated with 15 nglycosylation sites. hek 293t cells were seeded at 1x10^5 cells/well in 12 well plate. after 18h, cells were transfected with pflag-ebolavirus gp1 by coupling with effecteneâ®. after 24h, cells were treated with the inhibitors and harvested 24h after treatment. trimming of n-glycans was evaluated via molecular weight assessment by western-blot. results: all three inhibitors had comparable effectiveness in inhibiting trimming of nglycans from ebola gp1 glycoprotein compared to castanospermine. a greater molecular weight shift was seen with tunicamycin and pngase f as expected. conclusions: chemical inhibition of the n-linked glycosylation pathway was successfully achieved using three new mogs inhibitors. this approach merits further investigation on potential applications on antiviral therapies. investigator, laboratory of human genetics of infectious diseases, necker branch, inserm u1163, necker enfants malades hospital, paris, france 5 head, immunodeficiencies research unit, national institute of pediatrics stat1 gof mutations are associated with infections, autoimmunity and inflammatory manifestations; the rosacea is one of the manifestations described in this disease, however, the etiology rosacea is not clearly established. the characteristics of rosacea are not described in stat gof in the different clinical series. we describe the different characteristics of rosacea in a family with 8 affected members with stat1 gof. a family with eight members with stat1 gof mutation were diagnosed through a first affected member affected with tuberculosis and onychomycosis. seven members more had a clinical history of mycobacterial, viral and fungus infections and autoimmunity disease, in all the seven, was documented the same mutation stat1gof. in six of these adults patients, we documented rosacea, it started after adolescence, it was localized in the face and/or eyes, was progressive and not ameliorated with medical treatment and caused nose deformity. rosacea has been described previously as a unique manifestation, and the etiology is not clear, an autoimmune hypothesis has been proposed. the fact that is present in patients with stat1 gof could suggest that have effectively an autoimmune component. physicians face the patients with rosacea must look for other manifestation presents in stat1 gof mutations. genetic studies in rosacea patients could evidence an new gene defect. introduction: homozygous mutations causing loss of function of the transcription factor forkhead-box n1 (foxn1) underlie autosomal recessive severe combined immunodeficiency with congenital alopecia and nail dystrophy (nude scid). affected humans, like the scid mouse, have small or absent thymus, absent or severely diminished t cells, alopecia, and nail dystrophy. infants with nude scid have had neonatal lymphopenia and severe, life-threatening infections. studies of heterozygous carriers of foxn1 mutations are limited, some having been reported with no phenotype or mild disease manifestations, such as nail dystrophy without lymphopenia or recurrent infections. objective: we describe six infants, including two brothers, with t-cell lymphopenia (tcl) following abnormal california newborn screens (nbs) for scid. each had a single heterozygous variant in foxn1. case reports: six infants (3 female, 3 male) were referred for evaluation after abnormal california nbs for scid (table 1) , with t-cell receptor excision circle (trec) counts from undetectable to 12 (normal >18). all infants were well at the time of initial evaluation. five infants with absolute cd3 t cell counts >400 cells/ul and cd4 t cell counts >250 cells/ul began evaluation as outpatients on home isolation. patient 5, with undetectable trecs, cd3 t cell count 78, and cd4 t cell count 65 was urgently admitted for inpatient evaluation and management and immediately started on antimicrobial prophylaxis. patient 5 further evaluation was significant for lymphocyte proliferation to mitogens that was initially normal but waned with time, prompting treatment with a paternal haploidentical hematopoietic cell transplant at 6 months of age. patients 3 and 5 developed neutropenia within 6 weeks of birth treated with granulocyte colony stimulating factor (gcsf). patient 3 remains well on gcsf but has had persistent growth failure under continued evaluation. patients 1, 2, 4 and 6 remain stable off antimicrobial prophylaxis, but with persistent moderate tcl. as part of an immune evaluation, patients 1 and 3-6 had gene panel testing revealing heterozygous variants in foxn1. only the variant of patient 1 (presumed shared by patient 2, his brother) was predicted to be pathogenic; patient 1 had dystrophic nails and sparse hair most evident after 2 years of age, features shared by his mother and his brother, patient 2. the other patients lack the clinical features of the previously described phenotype of nude scid. their heterozygous foxn1 variants are of unknown significance; the functional role of these variants in the patients clinical phenotype is unknown. conclusion: six infants with abnormal nbs for scid had lymphopenia and heterozygous variants in foxn1. for these infants, variation exists in level of tcl and presence of hair and nail findings. heterozygous variants of unknown significance in foxn1 have been uncovered in others, including infants with abnormal nbs for scid, highlighting the need for functional studies to address the possible role of each heterozygous foxn1 variant in congenital lymphopenia and neutropenia. more work is needed before attributing tcl to a novel foxn1 variant of unknown significance in the absence of family history, abnormal hair or nails, or functional evidence. remains poorly understood. we characterized the intestinal microbiome and metabolome in patients with cgd to determine if intestinal microbiome and metabolomic signatures could distinguish subpopulations of patients with cgd while using the metabolome to add a functional dimension to observed microbiome signatures. methods: clinical metadata and fecal samples were collected crosssectionally from healthy volunteers (hv; n=16) and patients with cgd (n=77). metabolomic profiling and 16s rrna (v4) sequencing was performed on fecal samples (total samples: 108; reads/sample: 15,254 to 191,415; median: 60,816) . results: samples from patients with cgd had distinct intestinal microbiome signatures and metabolomic profiles depending on genotype, presence of cgd-ibd and specific interventions (e.g. treatment with an elemental diet). notably, samples from patients with active cgd-ibd (compared to samples from patients without a history of cgd-ibd) had significantly different alpha-and betadiversities, and were enriched for enterococcus spp. signal transducer and activator of transcription 1 gain of function (stat1-gof) is a primary immunodeysregulatory disease in which a subset of patients have features of autoimmunity and autoinflammation. enteropathy with growth failure and nutrient wasting is a more common feature of immunodysregulation. ruxolitinib is a janus kinase-stat inhibitor that has been shown effective for the treatment of immunodysregulatory features in stat1-gof. our patient is a 13 year old male with stat1-gof (c.983a>g p.h328r) with severe total parenteral dependent enteropathy that led to growth failure (weight 28.5kg). treatment with ruxolitinib led to resolution of diarrhea, return of normal diet, and catch up growth. a dose of 12.5mg twice daily was initially started but was decreased to 12.5mg every morning and 10 mg every evening due to elevated transaminases and thrombocytopenia. over the following year the patient thrived gaining 7.5kg with normal every other day stools. despite weight gain, he remained stable on the same dose of ruxolitinib. as he outgrew his dose, he developed an increased frequency of upper respiratory infections (parainfluenza, coronavirus, rhinovirus). one year after initiation of ruxolitinib, he again developed profuse watery diarrhea that was norovirus positive (weight 36kg, bsa 0.9). he was placed on bowel rest and ruxolitinib was dose escalated with a goal of 15mg/m2/day. when he reached 15mg twice daily, enteropathy completely resolved but liver function tests began to rise. he gained weight and began thriving after 2 weeks of therapy. six months later, enteropathy is controlled, and transaminases have remained elevated (alt 88 iu/l, ast 73 iu/ml) but stable. the appropriate dose and pharmacokinetics for ruxolitinib for the treatment of immunodysregulatory symptoms in pediatric patients has not been thoroughly studied. the dose used was extrapolated from data on the use of ruxolitinib in pediatric myelofibrosis. a dose of 15mg/m2/day appears to provide the most benefit with tolerable adverse effects. this dose should be maintained in order to prevent recurrence of disease related manifestations. abstract clathrin-mediated endocytosis (cme) is the major endocytic pathway by which eukaryotic cells internalize cell-surface cargo proteins and extracellular molecules, thereby allowing for a broad range of biological processes, including cell signaling, nutrient and growth factor uptake, and cell fate and differentiation1. the fbar domain only proteins 1 and 2 (fcho1/fcho2) are involved in the initiation of clathrin coat pit formation. whether fcho1 and fcho2 are functionally redundant or have distinct functions is unclear. we report here the first cases of a severe immunodeficiency due to a genetic defect affecting cme. by using whole exome sequencing and genomic analysis of a targeted pid gene panel, we have identified biallelic loss-of-function fcho1 mutations in five patients from unrelated families of italian (p1), turkish (p2, p3, and p5) and algerian (p4) origin with severe t cell lymphopenia manifesting as recurrent and severe infections of bacterial, mycobacterial, viral and fungal origin. p3 developed ebv-associated diffuse large b cell lymphoma. three patients (p3-p5) died in childhood, whereas p1 and p2 are alive with full donor chimerism at 13 and 1.5 years after allogeneic hematopoietic stem cell transplantation, respectively and have cleared pre-transplant infections. patients p2, p3, and p4 carried homozygous frameshift mutations predicted to cause premature termination. western-blotting analysis of ha-or flag-tagged fcho1 constructs showed expression of truncated products in p2 and p3, whereas no protein was detected in p4, presumably due to mrna decay. p1 and p5 carried homozygous splice-site mutations at the invariant -1 and +1 positions, respectively, leading to skipping of exon 6 in p1's fcho1 cdna. qpcr analysis demonstrated differential expression of the fcho1 and fcho2 genes, with the former being predominantly expressed in lymphoid cells, whereas fcho2 was more abundantly expressed in fibroblasts and k562 cells. analysis of t cell activation in p2 (the only patient for whom pre-transplant pbmc were available) revealed reduced t cell proliferation. while tcr internalization in response to cd3 cross-linking was normal (consistent with recent evidence that tcr internalization occurs through a clathrin-independent pathway), chase experiments demonstrated that transferrin internalization was abolished in activated t cells from p2. we had previously reported that a missense mutation in tfrc, encoding transferrin receptor 1, impairs transferrin internalization and intracellular iron delivery, causing a combined immunodeficiency with defective t cell proliferation. our data identify the first form of severe immunodeficiency due to defects of clathrin-mediated endocytosis, and provide additional evidence in support of the critical role played by iron cellular metabolism in t cell function and homeostasis. natural history of anti-interferon-gamma autoantibody-associated immunodeficiency syndrome in thailand submission id#601826 centralized sequencing initiative at niaid: year 1 therefore, we set out to investigate the pneumococcal-specific responses of igg, igg2, iga and igm to prevnar13â® in igg subclass deficient (iggscd) patients in this study. pneumococcal responses were measured using the vacczyme pneumococcal capsular polysaccharide igg, igg2, iga and igm elisas (the binding site group, birmingham, uk) in control (n=10, median age 57 years, range 27-64) and iggscd patients (n=10, median age 55 years, range 25-65) recruited from the immunodeficiency unit at the karolinska university hospital iga and igm antibodies in response to pcv13 vaccination was observed 4 weeks post vaccination in iggscd patients (median, 2.5th and 97.5th percentile these median concentrations were lower than those observed in control patients (median, 2.5th and 97 pcv13 igg2 71 mg/l, 14-90 however, percentage changes between pre to post vaccination concentrations of igg, igg2 and iga in response to pcv13 in iggscd patients were not significantly different to the control patients u/ml vs 17.1 u/ml, respectively) iga 26 u/ml and pcv13 igm 39 u/ml) responders and non-responders of pcv13 igg iga and igm in response to pcv13 in iggscd patients were generally lower compared to the control population. these results support the fact that in addition to igg and igg2, measurement of iga and igm could also provide useful information for the clinician gain-of-function ikbkb mutation causes human combined immune deficiency submission id#606903 neutralizing anti-il-6-autoantibodies are a risk factor for pyogenic bacterial infections national institutes of health, national institutes of allergy and infectious diseases service of immunology and rheumatology, garrahan national pediatric hospital copa mutations impair er-golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis copa syndrome: a novel autosomal dominant immune dysregulatory disease analysis of pulmonary features and treatment approaches in the copa syndrome expanding the phenotype of copa syndrome: a kindred with typical and atypical features the forest and the trees: machine learning to classify cases of suspected inborn errors of immunity using decision tree and random forest algorithms submission id#607035 card9î�11 gene dosage: from mono-allelic protection to ibd, to bi-allelic increased fungal infection susceptibility yamanaka d 3 , walkiewicz m 4 , lionakis m 3 and rosenzweig s 1 stim1 mutation associated with a syndrome of immunodeficiency and autoimmunity a novel hypomorphic mutation in stim1 results in a late-onset immunodeficiency clinical, histological and genetic characterisation of patients with tubular aggregate myopathy caused by mutations in stim1 gain-of-function mutation in stim1 (p.r304w) is associated with stormorken syndrome gain-of-function mutations in stim1 and orai1 causing tubular aggregate myopathy and stormorken syndrome stormorken syndrome caused by a p.r304w stim1 mutation: the first italian patient and a review of the literature by studying ecs-pre and ecs-post patients we were able to describe the bona-fide effect of gcs on the immune system in general, and t lymphocytes in particular. decreased lymphocyte/thymic output, as well as increased apoptotic tcell death underlies lymphopenia in ecs/chronic gcs-exposed patients. under such conditions, il-21 was significantly decreased in plasma and our in-vitro studies showed that il-21 replenishment was able to increase bcl2 (anti-apoptotic molecule) and bcl6 expression, and efficiently counteract the apoptotic effects of gcs. recombinant il-21 has been explored as a co-adjuvant treatment for multiple human cancers and may offer a treatment option for lymphopenia and its genetic counselor, co-director of personalized medicine, division of hematology/oncology/bmt and the institute for genomic medicine, nationwide childrens hospital 2 genetic counselor, division of hematology/oncology/bmt, nationwide children's hospital acknowledgments. genetic sequencing was kindly provided by drs. raif geha and janet chou at the division of immunology, allergy, rheumatology and dermatology, boston children's hospital, harvard medical school. the following grants are acknowledged: 1. rui 1.1001/cippt/812036 (usm) 2. bmbf 01 eo003 (freiburg) the authors would like to thank the director general of health of malaysia for permission to publish this scientific presentation. while severe viral infections may also be an initial presentation of primary immunodeficiency, an immune evaluation is not always obtained in this scenario. patients with xla have an increased susceptibility to severe enterovirus infections, manifesting as chronic meningoencephalitis, which can be fatal. the following case describes a patient with newly diagnosed xla presenting as suspected coxsackievirus and confirmed hhv-6 meningitis, pseudomonas meningitis and bacteremia. this may be the first reported new diagnosis of xla presenting with both severe bacterial and viral coinfection. case description: a 2 year old, partially vaccinated, hispanic male with a history of febrile seizures presented to the emergency room with fever, oliguria, watery diarrhea, lethargy, meningismus, ecthyma gangrenosum and lower abdominal pain. eight days prior to presentation, he was seen by his pediatrician for facial rash and low grade temperature, and was diagnosed with hand-foot-and mouth disease. he worsened on empiric antibiotics. he had no history of sinopulmonary infections. he did not attend daycare. his vaccines were delayed due to parental choice, and he had not received live vaccines (rotavirus, mmr or vzv). full sepsis evaluation was performed. csf demonstrated pleocytosis, and he was started on empiric antibiotics and transferred to picu. due to worsening abdominal pain, ct of the abdomen was performed, which was consistent with ruptured appendicitis and septic emboli at the lung bases. csf pcr panel was positive for hhv-6 and he was started on gancyclovir. csf and blood cultures subsequently grew pseudomonas aeruginosa. immune evaluation was performed. serum immunoglobulins were undetectable. in addition to iv antibiotics, he received 500 mg/kg ivig and lymphocyte subsets revealed profound b cell lymphopenia (0.23 %, 5 cells/ul). btk protein analysis revealed hemizygous btk pathogenic variant confirming the diagnosis of x-linked agammaglobulinemia. the hospital course was further complicated by brain abscesses and pyoventriculitis. he was treated with 3 additional doses of 500 mg/kg ivig and iv antibiotics. repeat mri of the brain nearly 4 weeks after admission demonstrated significant improvement. there was significant clinical recovery. he was discharged home at baseline neurological status. his igg level upon discharge home was 605 mg/dl with the plan to increase dose to 600 mg/kg per month with close monitoring. conclusion: both severe opportunistic bacterial infections and severe viral infections as the initial presentation of xla have been well reported in the literature. this case describes the first reported severe pseudomonas aeruginosa and hhv-6 co-infection in a newly diagnosed xla patient. this case further highlights the necessity for an increased index of suspicion of primary immunodeficiency in a patient who presents with a severe first infection, despite lack of recurrent infections. we present two patients with dock8 deficiency due to compound heterozygous variants including a copy number loss at chromosome band 9p24.3 spanning approximately .107 mb with partial deletion of the dock8 gene and a novel c.2603c>t (p.ser868leu) missense variant [chr9:379933 (grch37) nm_203447] in dock8. functional data is presented to support the pathogenicity of the missense change, along with a review of the literature on dock8 variants. the proband is a 14-year-old female with elevated serum ige, severe atopic dermatitis, mild persistent asthma, food allergies, and seasonal allergic rhinitis. she is currently healthy following haploidentical bone marrow transplant in june 2018. she has a 17-year-old brother with dock8 deficiency with the same compound heterozygous variants. the brother had later onset of symptoms and a milder presentation of intermittent asthma and seasonal allergic rhinitis. each of the parents is heterozygous for one of the two variants. we evaluated the pathogenicity of the c.2603c>t missense variant with western blots of dock8 protein expression, intracellular flow cytometry, and dock8 stretch assays. flow cytometry showed decreased dock8 protein expression and stretch assays revealed t cells that were stretched in collagen gels. notably, dock8 is a large gene containing 47 exons spanning 190 kb and it is relatively common to be a carrier of a rare missense change. in fact, gnomad has approximately 1500 individuals with rare (<0.002 frequency) missense alleles in dock8. therefore, it is important to demonstrate the potential pathogenicity of any given rare missense change, since few pathogenic missense variants in dock8 have been reported. of the 168 published dock8 variants listed in the human gene mutation database (hgmd) only 13 are missense. the majority are gross deletions, 97 of which were reported in hgmd. the remaining reported dock8 variants include 19 nonsense, 15 splicing, 13 small deletions (all frameshifting), 3 small insertions (all frameshifting), 2 small indels, and 5 gross insertions/duplications. this case demonstrates the relatively infrequent but important contribution of missense changes to pathogenic dock8 alleles. functional validation of missense alleles is critical in the complex evaluation of dock8 deficiency. background: hsct is the only known curative option currently for cd40l deficiency, an x-linked disorder. in cd40l deficiency and other x-linked immune deficiencies, there is an ongoing debate regarding the use of a carrier female sibling or mother as hsct donor. skewed lyonization despite complete donor chimerism has raised concerns for incomplete disease control post-hsct. no data exist regarding the efficacy of related female carrier as hsct donor for cd40l deficiency. we herein report outcomes of three patients with cd40l deficiency who underwent hsct using a related female carrier donor. method: retrospective review of patients who received hsct from carrier female related donor at three separate institutions. results: three patients with cd40l deficiency underwent hsct between 2016-2018. patient 1 had recurrent episodes of pneumocystis jiroveci pneumonia (pjp) despite being on bactrim and immunoglobulin replacement. patient 2 presented with pjp and severe neutropenia. patient 3 presented with acute respiratory failure from severe respiratory viral infections, cmvand had severe neutropenia requiring g-csf treatment. age at the time of hsct ranged from 0.5-15 yrs. all three underwent reduced toxicity hsct with busulfan and fludarabine-based preparatory regimens. two of them received matched sibling bone marrow hsct and one received tcr and cd19 depleted mobilized maternal pbsc haploidentical hsct. donor cd40l expression varied from 37% -67% on activated cd4 cells. immunoglobulin profile and lymphocyte subset were done in two of donors, they were within normal range for age, and none had significant infection history. no history of intermittent neutropenia or oral ulcers noted in donor and the absolute neutrophil count of the donor varied between 2500 6520 /l. donor age ranged from 3.2 yrs 48 years. cd34 dose ranged from 6.1 x 106 -23.1 x 106 cells/kg and cd3 dose ranged from 1 x 105 22.1 x 107 cd3+ cells/kg. gvhd prophylaxis consisted of csa/mmf (n=2) and tcr-a/b depletion and no csa (n=1). neutrophil engraftment ranged from 11-18 days and platelet engraftment ranged from 13 28 days. none of the patients developed acute or chronic gvhd. all three patients maintain full donor myeloid chimerism at the latest testing (9 months 18 months); t cell chimerism was 100% in one and mixed in two patients (91% at nine months, 80% at 12 months). all three patients had excellent t cell immune reconstitution; two patients came off immunoglobulin replacement 5 -11 months post hsct, whereas the 3rd patient is ivig dependent, though iga level was 25 mg/dl at nine months post-transplantation. latest evaluation, 9 18 months post-hsct, revealed 27% -63% cd40l expressing activated cd4 t cells, which correlated with donor cd40l expression and t-cell chimerism. conclusion: our data suggest that hsct utilizing x-linked carrier appears to be safe and results in durable engraftment with excellent humoral and cellular immune reconstitution in patients with cd40l deficiency. longer follow-up and data from a larger cohort is needed to make a definitive determination of safety and efficacy of utilizing female carrier as hsct donors in this disease. chief, immunology service, department of laboratory medicine, nih clinical center, bethesda, md, usa background: ikaros belongs to a hematopoietic-specific zinc-finger (zf) family of transcription factors. after dimerizing and dna binding to pericentric-heterochromatin (pc-hc) regions, ikaros is described as a central regulator of lymphocyte differentiation. somatic mutations/ deletions affecting ikaros n-terminal zf have been identified in b-acute lymphoblastic leukemia (all) patients, and germline n-terminal mutations were reported in cvid patients with progressive lack of b cells, hypogammaglobinemia, autoimmune diseases and b-all. methods: we performed targeted sequencing panel for known inborn errors of immunity disease-causing genes in a previously healthy male pediatric patient with burkitt lymphoma, followed by benign lymphoproliferation, thrombocytopenia and neutropenia. b-cells and immunoglobulin levels were normal. ikaros dna-binding, nuclear localization and protein binding were evaluated by emsa, fluorescence microscopy and immunoprecipitation. protein modeling was also performed. results: a novel heterozygous germline mutation in ikaros c-terminal zf6 dimerization domain (p.r502l) was detected in this patient. this mutant showed normal pc-hc localization but dna-binding was markedly reduced in terms of ikaros dimerization and multimerization. moreover, reduced wt-mutant binding was also detected. mutant/wt cotransfection experiments suggest a haploinsufficient defect. geometry based docking of wildtype ikaros predicted that r502 is within the homodimer interface and may abolish cation-pi interactions and destabilize the ikaros-zf6 dimerization domain. conclusion: a novel germline ikaros c-terminal mutation affecting homodimerization/multimerization and resulting in reduced dna binding to its dna consensus site was detected in a patient with burkitt lymphoma, benign lymphoproliferation and cytopenias. further studies are warranted to formally establish the casual connection between this genotype and phenotype.(210) submission id#606894 patricia pichilingue-reto, md 1 , prithvi raj, phd 2 , igor dozmorov, phd 3 , quan-zhen li, md, phd 4 , edward wakeland, phd 5 , nancy kelly, md 6 , maria teresa de la morena, md 7 , nicolai s. van oers, phd 8 methods: mice were generated by crispr/cas technology to genocopy the foxn1 compound heterozygous mutations identified in one of the human patients. thymopoiesis and hair follicle extrusion was analyzed in the various heterozygous and homozygous mutant mice. gene expression analyses of the hypoplastic and normal-sized thymii and the developing skin were performed. in addition, a structure-function analysis was performed with luciferase reporter assays using 9 distinct and previously unreported foxn1 mutations uncovered in patients who presented with low trecs. results: mice harboring compound heterozygous mutations in foxn1 that match the human patient phenocopy the t-b+nk+ scid phenotype with normal hair and nails. a functional characterization of the diverse foxn1 mutations suggests that the severity of the block in thymopoiesis depends on whether the mutations affect the dna binding or transactivation domains of foxn1. a 5-amino acid segment at the end of the dna binding domain appears to be essential for tec development. however, this segment is not required for normal keratinocyte functions in the skin and nail plate. gene expression comparisons are revealing key targets of foxn1 that suggest a dichotomy in its function in the thymus versus the skin. conclusions: novel compound heterozygous mutations in foxn1 are causal to a t-nk+b+ phenotype with normal hair shaft extrusion and nail plate extension. this differs from the classic nude/scid (omim # 600838) reported for individuals with autosomal recessive mutations in foxn1. assistant professor of medicine and pediatrics, department of allergy and immunology, uva introduction: copa syndrome is a recently described monogenic immunodysregulatory syndrome. the cop protein, encoded for by the copa gene, is expressed in all cell types and is involved in trafficking from the golgi complex to the endoplasmic reticulum (1) . the most common clinical features of copa syndrome are interstitial lung disease, pulmonary cysts or follicular bronchiolitis, pulmonary hemorrhage, arthritis, glomerular disease, and autoantibody development (2, 3) . atypical features of copa syndrome identified thus far include: extrapulmonary cysts in the liver and kidney, renal and neuroendocrine malignancies, autoimmune neurological disorders such as neuromyelitis optica, and infections, such as meningitis (4) . clinical case: we present a case of a 2 year-old male with copa syndrome (de novo heterozygous mutation in exon 9, c.715g>c; p.ala239pro) manifesting as lymphocytic interstitial pneumonitis, peripheral blood b-cell lymphocytosis, mediastinal lymphadenopathy and persistent transaminitis (alt and ast 100-400 u/l, nl ast<35 u/l, alt <55u/l) with normal bilirubin, alkaline phosphatase and pt/inr. the transaminitis was noted prior to diagnosis of copa syndrome, and has persisted despite seven months of therapy with pulse dose steroids, two cycles of rituximab and maintenance therapy with hydroxychloroquine and prednisone. he has had a normal ck and aldolase excluding muscle injury as a source of his transaminitis. a congenital cholestasis panel was normal. markers of autoimmune liver disease including ana, anti-liver kidney microsomal antibody and anti-smooth muscle were negative. serum ceruloplasmin and alpha-1-antitrypsin level were normal and celiac serologies, were negative. liver ultrasound was normal. a liver biopsy did not demonstrate inflammatory changes, hepatocyte necrosis, mononuclear cell infiltrates or fibrosis. nonspecific biopsy findings included occasional intraparenchymal neutrophils. it is unclear if these scattered neutrophils and the transaminitis are due to an early as yet unidentified autoimmune process, perhaps in response to hepatocellular stress exacerbated by the copa mutation. discussion: liver involvement has not been reported in copa syndrome. we describe a child with copa syndrome who has had chronic transaminitis with no clear alternative cause. if the phenotypic spectrum of copa syndrome involves the liver, it may limit immunomodulatory options for the treatment of this disease. background: in humans, biallelic stat1 lost-of-function (lof) mutations lead to a very low or complete absence of the wild-type (wt) protein. whereas, heterozygous mutations can lead to partial loss of function. these patients are susceptible to mycobacteria and herpes virus infections. on other hand, heterozygous gain-of-function (gof) mutations in the stat1 gene result in a hyperphosphorylated state where patients develop recurrent or persistent chronic mucocutaneous candidiasis (cmc), other cutaneous mycosis, bacterial infections, disseminated dimorphic fungal infections, viral infections and autoimmune disease. methods: in this study, we evaluated 4 novel stat1 mutations, three gof and one lof. in vitro, pbmcs from these patients were stimulated with ifn-and ifn-for 30, 60, and 120 minutes and levels of phospho-stat1 were measured by flow cytometry. the stat1 phosphorylation and activity (firefly and renilla luciferase activities) were evaluated in u3a-stat1 deficient cells transfected with a reporter plasmid (for luciferase), wt or mutant-stat1 plasmids. results: we observed higher levels of stat1 phosphorylation after two hours of stimulation from three gof mutations compared to wt. however, a lof mutation showed absent stat1 activation at baseline and in response to ifn-and ifn-. luciferase reporter assay confirmed gain of function and loss of function stat1 activity observed by flow cytometry. conclusions: using flow cytometry followed by a luciferase assay, we confirmed four novel stat1 mutations. measuring phosphorylation of stat1 by flow cytometry is sufficient to determine whether the stat1 mutation is disease causing. this assay can be translated to a clinically accessible test for stat1 related disease. background: variants in recombination-activating genes (rag) are common genetic causes of autosomal recessive forms combined immunodeficiencies (cid) ranging from severe combined immunodeficiency (scid), omenn syndrome (os), atypical scid (as) and cid with granulomas and/or autoimmunity (cid-g/ai). the clinical and immunological presentation is broad, ranging from severe infections secondary to near absence of t and b lymphocytes and hypogammaglobulinemia to the occurrence of autoimmunity with late manifestations with partly preserved immune subsets and near normal immunoglobulin levels and broad spectrum of autoantibodies. objective: we aim to estimate the incidence, clinical presentation, genetic variability and treatment outcome with geographic distribution of patients with the rag defects in populations inhabiting south, west and east slavic countries. due to shared ancestry, we also investigated our cohort for founder variants in rag1 and rag2 genes. methods: demographic, clinical and laboratory data were collected from rag deficient patients of slavic origin via chart review, retrospectively. results. based on the clinical and immunologic phenotype, our cohort of 80 patients from 66 families represented a wide spectrum of rag deficiencies, including scid (n=19), os (n=36), as (n=21) and cid-g/ai (n=4). sixty-six (82.5%) patients carried rag1 and 14 patients (17.5%) carried rag2 biallelic variants. we estimate that the minimal annual incidence of rag deficiency in slavic countries varies between 1 in 180,000 300,000 live birth and it may vary secondary to health care disparities in these regions. in our cohort, 70% of the patients carried rag1 p.k86vfs*33 (c.256_257delaa), either in homozygous (n=17, 26%) or compound heterozygous (n=29, 44%) form. the majority (77%) of patients with homozygous rag1 p.k86vfs*33 originated from vistula watershed area in central and eastern poland, and compound heterozygote cases distributed among all slavic countries except bulgaria. clinical and immunological presentation of homozygous rag1 p.k86vfs*33 cases was highly diverse suggestive of strong influence of other genetic and/or epigenetic factors in shaping the final phenotype. survival of rag deficient patients without hematopoietic stem cell transplant (hsct) (n=3, 8.8%) is poor and dramatically improved in the last decade with access to hsct and tailored conditioning regimens. conclusion: we propose that rag1 p.k86vfs*33 is a founder variant originating from the vistula watershed region in poland, which may explain a high proportion of homozygous cases from central and eastern poland and the presence of the variant in all slavs. our studies in cases with rag1 founder variants confirm that clinical and immunological phenotype only partially depend on the underlying genetic defect. hsct is becoming available for rag deficient patients in eastern europe with improving outcome. clinical immunologist, centre hospitalier universitaire de montrã©al (chum) background: acute gvhd following solid organ transplantation is a rare complication. intestinal and liver transplantation have the greatest risk of gvhd among solid organs due to high number of donor lymphocytes in these organs. prevalence of acute gvhd after liver transplantation is estimated to be around 0,1-2% and has a poor prognosis (1) . chronic neurological gvhd is a rare form of gvhd with three subtypes described: cerebral vasculitis, demyelinating disease and immune mediated encephalitis. acute neurological gvhd has no clear definition and is still considered a controversial entity. case presentation: a 63 year-old male underwent cadaveric liver transplantation for alcoholic cirrhosis and hepatocellular carcinoma. the donor was a 70 year-old man who died from anoxic brain injury. the receiver was induced with basiliximab and then put on prednisone, azathioprine and tacrolimus. he was readmitted 10 weeks later for myalgia, headache, fever and neutropenia. clinical state initially improved with empiric antibiotics. he then developed a skin eruption, colitis and dic. the latter was thought to be tacrolimus-induced. he was switched to cyclosporine. skin and rectosigmoid biopsies were compatible with acute gvhd. he received basiliximab and ivig and developed a refractory convulsive state. csf analysis showed elevated proteins and slight pleocytosis. cerebral mri showed non-specific white matter lesions and conventional angiography was normal. chimerism on peripheral blood was 0% but was 45% donor on csf. with the presence of chimerism on csf, evidence of cutaneous and digestive gvhd and no infectious cause, neurological gvhd was considered the most likely diagnosis. brain biopsy showed non specific change including neuropil spongiosis, microglial activation and reactive gliosis; but no signs of vasculitis or demyelinating disease. he was treated with atg, highdose systemic corticosteroids, cyclosporine, ivig and intrathecal methotrexate and corticosteroids. csf pleocytosis, proteins and chimerism improved with treatment (45% to 2% donor). no improvement was noted regarding his neurological state and he developed pancytopenia. he was then transfer to palliative care and died shortly after (4 month and a half after liver transplant). discussion: to our knowledge, there is only one prior case published of neurological gvhd following liver transplantation (2) . both patients were old, had hepatocellular carcinoma and had at least one hla match. age >50 year, hepatocellular carcinoma and shared hla antigen are known risk factors for gvhd following liver transplantation (1). our patient had only one hla match with the donor. this case is intriguing as there was a great discrepancy between blood and csf chimerism. acute neurological gvhd following transplantation is a real complication. it must be taken into consideration in patients with neurological involvement after transplant, even solid organ transplantations. introduction: hyper-igm syndrome are rare. although no data are available on the frequency of activation-induced cytidine deaminase (aid) deficiency, this disorder is estimated to affect less than 1:1,000,000 individuals. by the year 2012, 110 cases worldwide (1) with such mutation have been described. we describe a patient with hyper igm by mutation in the aicda gene. case report: mvv, 5-year-old boy, born to consanguineous parents, was referred with recurrent pneumonia, which started shortly after discontinuation of breastfeeding at 6 months old. repetitive otitis evolved with bilateral tympanic and partial hearing loss. he was submitted to adenoidectomy without improvement. immunological evaluation showed normal numbers of b and t cells with cd3+ (1290/mm3, 65%), cd4+ (547/mm3, 28%), and cd8+ (259/mm3, 13%). immunoglobulin concentrations were: igg = 138mg/dl (p97). treatment with intravenous immunoglobulin and prophylactic antibiotic was initiated and he had no infections during the follow up except for one episode of sinusitis. at 10 years of age, molecular evaluation was performed and a mutation in homozygosity in the aicda gene (omim * 605257) at position chr12: 8.757.821 was found, confirming the clinical suspicion. conclusion: the role of aid in the immunoglobulin class-switch recombination (csr) and somatic hypermutation (shm) have not been fully elucidated. summarizing within the shm and csr processes, aicda mutation can induce dna lesions in directed sequences in the s and v regions required for dna cleavage. recurrent infections and consanguinity raised the suspicion of inborn errors of immunity in this patient. the literature described late diagnosis as in the second or even the third decade of life. it was suggested that high levels of igm antibodies may provide effective defense, at least, against some infectious agents. it is important to emphasize that the impossibility to obtain genetic diagnosis did not prevent to introduce therapy. * aicda: activation induced cytidine deaminase gene patients with chronic granulomatous disease (cgd) are at risk for recurring infections and non-infectious inflammation, reduced quality of life and life expectancy. conventional treatment with life-long anti-bacterial and antifungal prophylaxis prolongs lifespan but does not eliminate the lifelong risk of infection and inflammation. allogenic stem cell transplantation is currently the only curative option for this disease. although sct with reduced intensity conditioning has improved treatment-related mortality and efficacy, it remains a matter of debate whether all patients with cgd benefit from sct, whether pre-existing infections and non-infectious inflammation are risk factors and at what age sct should be performed. we compared patients with cgd on conventional treatment with those after stem cell transplantation for their prognosis and evaluated potential risk factors for stem cell transplantation outcome followed up in six european centers. frequency of infections, inflammatory complications, hospitalizations, operations and immunomodulative/immunosuppressive therapy, height and weight were compared in patients on conventional treatment /before stem cell transplantation versus patients after sct. correlation between transplantation outcome and patient characteristics or medical history was tested. 105 patients were recruited, 55 on ct, 50 after stem cell transplantation. before/without transplantation 98% of patients suffered from at least one infection, 84,8% from inflammatory complications. patients on conventional treatment developed infection/inflammation/ hospitalization/surgery at a median of 2,28 (range [0,29-21,82] , iqr 2,79) per year, versus 9 (range , iqr 8,5) in the first year after stem cell transplantation but 0 (range [0-15], iqr 0,53) after the first year post stem cell transplantation. there was a significant decrease of all complications after stem cell transplantation (p < .05). growth improved significantly after stem cell transplantation (z-score weight -1,692 versus -0,846 (p.017), z-score height -1,906 versus -1,064 (p.029)). nevertheless, complications post stem cell transplantation are frequent: 88% of patients had at least one infection, 8% had severe acute gvhd, 12% chronic gvhd, 16% had graft rejection, 12% died. preexisting active mold infection increased the risk for complications after stem cell transplantation. in summary infections and non-infectious inflammation are common in patients with cgd on conventional treatment, their growth is significantly impaired. stem cell transplantation, if successful, significantly reduces the risk for infections and non-infectious inflammation. however, treatment related mortality of stem cell transplantation in patients with cgd remains considerable. introduction: development of a diverse t cell repertoire is essential for full immune recovery following definitive treatment for severe combined immunodeficiency (scid), whether by allogeneic hematopoietic cell transplantation (hct); autologous gene therapy (gt); or, in the case of adenosine deaminase deficiency, enzyme replacement therapy (ert). however, the time course and depth of diversity of t cell receptor rearrangements have been difficult to measure directly, necessitating estimates from total and naã¯ve t cell counts and from spectratyping, in which t cell receptor (tcr) beta chain diversity is estimated by the length distributions of cdna amplicons between a series of tcr beta chain variable (v-beta) segments that have productively recombined with the tcr beta-chain constant region. analysis of the actual sequences of rearranged tcrs could indicate more precisely the status of the t cell compartment of these patients, and might reveal oligoclonal expansion of dysregulated t cells, t cell insufficiency, or t cell exhaustion. objectives: we wished to ascertain whether deep sequencing of individual tcr v-beta rearrangements in peripheral blood could be performed sequentially following diagnosis and treatment of scid to differentiate satisfactory immune reconstitution from incomplete or skewed repertoire development that might require further cellular therapies. methods: equal amounts of total rna were obtained from peripheral blood of controls and scid patients pre-hct and at 100 d, 6 and 12 mo, and yearly post-treatment(s). cdna was used as template to semi-quantitatively amplify rearrangements at the tcr-beta locus (trb). raw sequences were filtered to remove pcr errors, and resulting fastq files were converted into fasta format (seqtk software, github, inc), filtered for productive rearrangement, and analyzed for v, d, and j gene composition and length (imgt highv-quest software). the vdj statistics file (past program) was used to calculate a shannon entropy (h) index to measure repertoire diversity, taking into account both abundance and richness of the overall repertoire; and a gini-simpson index of unevenness, measuring inequality in the relative representation of species in a given sample. graphical representations of repertoire diversity were generated by hierarchical tree maps of the trb repertoires (irepertoire software): each dot represents a unique sequence and the dot size corresponds to frequency of that sequence in the total sample. results: tcr v-beta sequence analysis of 3 scid patients (image) showed (top) baseline poor diversity due to pre-treatment ada deficiency followed by improvement to normal complexity (shannon h >7.0) after receiving peg-ada and autologous lentivirus gene therapy at age 3 m; (middle) increasing diversity in xscid after maternal t-depleted unconditioned hct, although b cells did not recover; and (bottom) failure of initial unconditioned maternal t-depleted hct in another xscid patient at 12 m, followed by autologous lentivirus gene therapy with subsequent improvement (shannon h increasing from 3.8 to 6) 12 months later. conclusions: tcr v-beta diversity sequence analysis provided a detailed assessment of repertoire diversity in response to cellular therapies for scid. this method could become a useful predictive tool to measure successful t cell immune reconstitution, both as early as 100 d and in the years following treatment. background: the stim1 (stromal interaction molecule 1) protein, encoded by the stim1 gene, is involved in calcium regulation in the endoplasmic and sarcoplasmic reticulum. pathogenic variants in this gene are associated with three different disorders. homozygous loss-of-function (lof) pathogenic variants in stim1 have been reported to cause autoimmune cytopenias, lymphoproliferation, enamel defects, anhydrosis, and iris hypoplasia. the first described cases had frequent mortality in early childhood due to recurrent life-threatening infections and development of kaposi sarcoma (1), while recently discovered cases have had more prolonged survival, though still with recurrent serious infections (2) . heterozygous gain-of-function (gof) pathogenic variants in stim1 have been associated with both tubular aggregate myopathy (tam) and stormorken syndrome. tam is a clinically heterogeneous progressive muscle disorder with a variable age of onset. muscle biopsy characteristically demonstrates tubular aggregates, with type ii muscle fiber atrophy (3) . stormorken syndrome has a phenotype that includes miosis, thrombocytopenia, intellectual disability, mild hypocalcemia, muscle fatigue, asplenia, and ichthyosis (4) . the thrombocytopenia has not been reported to be immune-mediated; rather it is due to abnormal platelet calcium regulation (5). we report a patient with stim1 pathogenic variant presenting with tam and immune-mediated thrombocytopenia, along with lymphoproliferative features, arthritis, and a mild immune deficiency. case: the patient is a 16-year-old with a history of congenital thrombocytopenia (platelets ranging 60,000-100,000) who presented with acute arthritis of bilateral hand joints after exposure to cold temperatures, which resolved with naproxen. he had back pain without muscle weakness, and preceding sore throat and general fatigue. labs were significant for leukocytosis and elevations in his inflammatory markers and creatine kinase. mri of his lower extremities was negative for inflammatory myositis, but did demonstrate bilateral hip and knee effusions, and significant inguinal lymphadenopathy and hyperintense linear signal changes in the mid-and distal femurs with patchy red marrow signal. abdominal ultrasound could not identify a definite spleen. bone marrow biopsy was negative for malignancy but significant for toxic granulation of neutrophils, evident of inflammation. alpha-beta double negative t cells were not elevated. interferon-gamma was mildly elevated. flow cytometry demonstrated normal t, b, and nk cell absolute counts. circulating antibodies against platelets (both igg and iga) were detected. on lymphocyte antigen and mitogen proliferation testing, he did not exhibit any proliferation when stimulated with tetanus toxoid even though he had been fully vaccinated against tetanus. muscle biopsy demonstrated large vacuoles consistent with tam on both light and electron microscopies. invitaes primary immunodeficiency panel identified a pathogenic variant in stim1 (c.910c>t; p.arg304trp), consistent with a diagnosis of autosomal dominant stim1-related conditions, including stormorken syndrome (6) . conclusion: this patient expands the phenotypic spectrum of stim1 related disease. based on previous evidence, gof pathogenic variants in stim1 are associated with tam and stormorken syndrome, while lof pathogenic variants in stim1 are associated with immune deficiency. however, our patient with a stim1 gof pathogenic variant has features of lymphoproliferation and immune dysregulation in addition to tam. stim1 gof pathogenic variants should be considered in the differential of patients with immune thrombocytopenia and lymphoproliferation. references: introduction / background: card11 is critical for protein binding upstream of nf-kb (nuclear factor kappa b) and mtorc1 (mammalian target of rapamycin complex 1) the signaling pathway involved in t-cell activation and inflammatory response. prior testing of card11 mutations demonstrated variable t-cell dysfunction. in vitro studies have demonstrated reduced interferon gamma cytokine production, interference of t-cell receptor (tcr) signaling, and th2 phenotype skew in t-cells with card11 defects. while homozygous mutation causes severe combined immunodeficiency deficiency, heterozygous card11 defect is associated with atopy by way of inappropriate th2 skewing. heterozygote atopy is characterized by eosinophilia, elevated ige, and severe dermatitis. despite multiple studies demonstrating in vivo consequences of card11 on t-cell function, little is known of the clinical significance. moreover, few studies have demonstrated the impact of card11 mutations on b-cell maturation and development, despite the recognized tcr and interleukin 2 signaling deficits. objectives: this case demonstrates a card11 defect that evolved from atopy to combined immunodeficiency requiring intravenous immunoglobulin therapy. it highlights the poorly understood effect of card11 mutation on t-cell function, and the downstream impact on b-cell quality. methods: 53-year-old male, with past medical history of t-cell lymphoma and no evidence of disease status post autologous stem cell transplant, was found to have card11 e57d missense mutation by genetic testing. consistent with previous literature regarding heterozygous card11 defects, the patient suffered from frequent asthma exacerbations, aeroallergen sensitivity, and eczema. lab work was consistently positive for elevated ige and eosinophilia. family history was positive for a son born with congenital molluscum, and multiple other children with recurrent infections. one child was also identified with card11 mutation. the patient had flow cytometry demonstrating 4% of circulating cells with atypical immunophenotyped cd3+ t-cells, and positive gene rearrangement studies. his qualitative immunoglobulin levels were significant for consistently low igm, but normal quantity igg. in the patients adulthood, he had recurrent bronchitis and pneumonia requiring hospitalization and intravenous antibiotics. given his recurrent infections, the patient underwent immunodeficiency evaluation. despite previous infection with herpes zoster, the patient did not have protective titers. additionally, the patient had received the pneumococcal conjugate vaccine once, and the pneumococcal polysaccharide vaccine four times. the most recent vaccination was one year prior to evaluation. despite repeated vaccinations, titers were unprotective. consequently, the patient was diagnosed with combined immunodeficiency, and initiated on intravenous immunoglobulin therapy. results: in summary, card11 defect is a cause of atopy, observed to become less severe with age. studies of card11 heterozygote mutations have demonstrated in vitro deficiencies in t-cell activation, likely secondary to skewed or decreased inflammatory cytokine production and tcr activation. our patient demonstrates that the variable t-cell dysfunction seen in vitro can have significant clinical implications evidenced by his inadequate vaccine response, and recurrent infections. his combined immunodeficiency poses a connection between card11 defects and, not only t-cell, but also b-cell function. conclusions: further studies are needed to determine deficits in t-cell and b-cell function in the setting of card11 defect, as this case suggests the clinical implications span further than atopy. genetic variants in the scaffold gene card11 cause disorders of the immune system. the clinical course and treatment depends on whether the card11 variant causes gain-or loss-of-function. however, lymphocyte immunophenotyping and proliferation assays in cells expressing card11 variants don't easily distinguish between gain-and loss-of-function. to address this challenge in variant interpretation, we used multiplexed genome editing in a lymphoma b cell line (tmd8) to generate cell populations expressing all possible singlenucleotide variants in the n-terminal 140 amino acids of card11. to assess function in each variant, we tracked its relative abundance over multiple conditions using dna sequencing. since card11 is required for survival of tmd8 lymphoma b-cells, cells expressing clinically identified gain-of-function variants grew faster relative to cells expressing other variants, even in the presence of upstream pathway inhibitors. upon evaluation of the relative abundance of each variant in genomic dna and mrna, we found that clinically identified loss-of-function variants were depleted in mrna, which could be attributed to alterations in splicing or to nonsensemediated decay. to address the impact of splicing, we modeled a newly-identified splice donor mutation (c.358+1g>a) found in two patients from one family diagnosed with combined immune deficiency, autoimmunity and atopy that was also observed in our screen. we show that the variant causes deletion of exon four and that card11 missing exon four exerts a dominant-negative effect leading to decreased nf-kb signaling and cell growth. these experiments demonstrate the utility of multiplexed functional assays for determining variant effect in clinically-relevant genes, which will improve diagnosis and treatment in patients. mutations in the rag1 and rag2 genes in humans cause a wide spectrum of phenotypes, ranging from severe combined immunodeficiency (scid) with lack of t and b cells to omenn syndrome (os), atypical scid (as) and combined immunodeficiency with granulomas and/or autoimmunity (cid-g/ai). here, we sought to investigate the molecular basis for phenotypic diversity presented in patients with various rag1 mutations. methods: we have recently described a novel flow-cytometrybased assay in which mouse rag1-/-pro-b cells containing an inverted gfp cassette flanked by recombination signal sequences (rss) are transduced with a retroviral vector expressing either wild-type or mutant human rag1 (hrag1). the green fluorescent protein expression directly relates to the activity of rag proteins, representing a quick and powerful tool to correlate between defective activity of hrag1 mutant and severity of the clinical phenotype. the genetic variants of hrag1 analyzed in this study were affecting the various domains of the protein: ring, zinc finger ring type domain (amino acids 168-283); nbr (amino acids 387-461); hbr (amino acids 531-763) and the core domain (amino acids 385-1011). using this sensitive assay, we tested the recombination activity of 27 human rag1 variants that have been reported in patients. results: we have demonstrated correlation between the recombination activity of the mutants and the in vivo clinical phenotype of patients. in particular, similarly low levels of recombination activity were observed in patients with scid and os, whereas patients with as and especially those with cid-g/ai carried mutations that retained significant residual levels of activity. conclusions: these data provide a framework to better understand the phenotypic heterogeneity of rag deficiency. here we report a case of a child with b. cepacia lymphadenitis, ultimately diagnosed with takayasu arteritis. takayasu arteritis is a large vessel vasculitis which may have a nonspecific clinical presentation in childhood possibly leading to difficulty in diagnosis. case: a 16-month-old female presented with two weeks of fever, respiratory distress, and lymphadenopathy, and was treated with ivig for presumed atypical kawasaki disease. imaging studies performed due to worsening respiratory distress revealed retropharyngeal abscess with bilateral cervical lymphadenopathy, culture-positive for prevotella oralis and melaninogenica, with improvement following incision and drainage and antibiotic therapy. recurrence of fever and respiratory distress prompted ct imaging of her neck significant for worsening lymphadenopathy. cultures from lymph node biopsy grew b. cepacia. following treatment, she was readmitted with respiratory distress requiring chronic steroid treatment and found to have candida albicans on bronchoalveloar lavage and necrotizing granulomatous inflammation on lung biopsy. an immunologic evaluation was notable for two normal dhr assays. cgd genetic panel was negative for pathogenic variants in cybb (p91), ncf1 (p47), cyba (p22), ncf2 (p67). testing was also notably negative for hiv pcr, bartonella pcr, cryptococcal antigen, histoplasma antigen, bal afb stain and mycobacterial cultures, cmv pcr, ebv pcr, anca, serial blood cultures, and sweat test. lymphocyte subsets were normal for age. mitogen stimulation test, myeloperoxidase antibody igg, serine protease3 igg, c4 level, lad panel, and cytokine panel were normal. autoimmune lymphoproliferative disorders (alps) panel was negative. whole exome sequencing demonstrated heterozygous mutations in cfi and jak3, not considered to be clinically relevant given the patients clinical picture and laboratory evaluation. the patient was then lost to follow-up for over a year. at the age of 3 years, the patient presented with fever and back pain. imaging revealed severe large vessel vasculitis involving the aorta and subclavian, vertebral, mesenteric, and renal arteries. she also had evidence of cardio-embolic strokes on brain mri. she had had no significant interval infections, and her immunologic evaluation remained unrevealing. in the context of her new vasculitis, evaluation for deficiency of ada2 (dada2) was negative. she was ultimately diagnosed with takayasu arteritis and has begun therapy with systemic corticosteroids, aspirin, and etanercept. conclusions: we describe a case of b. cepacia infection in a child without identified immunodeficiency, ultimately diagnosed with a large vessel vasculitis. the presence of b. cepacia infection warrants a thorough investigation. burkholderia has been previously associated with giant cell arteritis, another type of large vessel vasculitis, though causation has not been established. to our knowledge b. cepacia infection has not been associated with takayasu arteritis. christopher santaralas, valentine jadoul, jacqueline squire, john cannon, jessica trotter, susan aja, neil goldenberg, david graham, jennifer leiding background: chronic granulomatous disease (cgd) is a primary phagocytic immunodeficiency secondary to mutations in any of the components of nadph oxidase. in addition to infection susceptibility, patients with cgd can develop auto-inflammatory disease that is difficult to manage. metabolomics is the systematic study of small molecule biomarkers of the clinical phenotype of disease. we sought to investigate plasma metabolic profiles in cgd as we hypothesized that unique signatures may differentiate patients with cgd. methods: plasma collected from 15 subjects with cgd (9 x-linked, 4 p47phox-deficient, 2 p22phox-deficient) and 2 x-linked cgd carriers was analyzed using a targeted multiplex assay by liquid chromatography mass spectrometry (lc-ms) and simultaneously a profiling assay by lcms. sufficient signal was present for 34 metabolites. x-linked cgd and p47phox-deficient groups were sufficiently sized for multivariate and univariate analyses in metaboanalyst. twelve patients had a single time point of plasma metabolomics analysis and three had multiple time points, including one in whom both pre-and post-hematopoetic cell transplantation time points were assessed. post-hoc comparisons were also performed for those with, versus without, clinical comorbidities of autoinflammation. results: plasma from patients with x-linked and p47phox deficient cgd had a differential metabolomic signature at baseline. many metabolites as measured by ion intensity were present at high levels, particularly homocysteine, kyneurine, tryptophan, citric acid, carnitine, methionine, and adenosine. increased values of metabolites reduced to that of normal (compared to post hct). homocysteine levels were elevated among patients with (mean 1.5x105), versus without (mean 6.8x104), clinical comorbidities of auto-inflammation (i.e., colitis, lupus). baseline samples showed elevated kynurenine among all cgd patients, relative to historical normal controls (unmatched, separate analysis). patients with colitis had elevated citric acid levels that were higher among patients with (mean 2.1x106), versus without (mean 4.5x105), colitis irrespective of genotype. conclusions: preliminary data with a small patient subset suggest that patients with cgd have metabolomic signature distinguishable by phenotype. citric acid cycle metabolites are elevated in crohns disease and ulcerative colitis. based on our data, citric acid may too act as a biomarker for inflammatory bowel disease in cgd. analyzing a larger number of samples, across time points, will likely describe a metabolomics profile for cgd and identify biomarkers for auto-inflammation in cgd. no significant medical history in mother; paternal history is unknown and unavailable.no significant medical history in mother or father. rationale: ataxia telangiectasia is a disorder with variable phenotypes characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity, and cancer predisposition which may correspond to the degree of atm protein expression and/or radiosensitivity. we used in vitro cytometric assessment of atm, smc1 and h2ax phosphorylation to assess dna damage in response to radiation and found that two siblings with the same copy number gain in atm have variable clinical neurologic and immunologic phenotypes. methods: chart review and radiosensitivity assays using cytometric assessment of patm, psmc1, and h2ax expression after irradiation with 2gy. results: patient a is a 6 month old male identified after having low trecs on newborn screening, then found to have lymphopenia and elevated igm. he has diffuse cafã© au lait macules and no neurologic symptoms. his 9 year old sister, patient b, was being followed by neurology for several years for ataxia. she has selective iga deficiency, normal lymphocyte counts, lymphocyte proliferative responses, gammaglobulins, and vaccine specific antibodies. both patients have a 4 copy number gains in atm (exons 48-61). mother and father both have 3 copy number gains in atm and are healthy without neurologic symptoms or recurrent infections. both patient a and b have normal atm protein expression. phosphorylated atm, smc1, and h2ax was assessed in lymphocyte subsets (t, b, and nk cells) after low-dose irradiation to induce dna double-stranded breaks (dsbs). these parameters were assessed at 1 hour post-irradiation when they are expected to be maximal and at 24 hour post-irradiation, when under conditions of normal and effective dna repair, the phosphorylation state returns to baseline. patient a had abnormal patm and psmc1 but normal h2ax expression 1 hour and 24 hours after irradiation of t, b, and nk cells. patient b had normal patm, psmc1, and h2ax expression in t cells but abnormal patm and psmc1 expression in b and nk cells 1 hour after irradiation. patient b, however, had abnormal atm phosphorylation at 24 hours after irradiation of t, b, and nk cells.conclusions: our results indicate that a unique copy number gain in atm within a family can correspond to different clinical and immunologic phenotypes as well as variable degree of radiosensitivity. the persistence of h2ax at 24 hours post-irradiation and impaired phosphorylation of atm and smc1 at 1 hour post-irradiation demonstrates defects in dna dsb repair, and this is variably altered in different lymphocyte subsets. correlation between atm phosphorylation in lymphocytes with outcomes may be an area for future studies and particularly important in counseling patients regarding outcomes. antibodies have been implicated in both protection and pathology of dengue virus infections. however, much of this data is gathered from serum/plasma responses that is a cumulative of historical and ongoing infection. to precisely understand the role of antibodies with respect to the ongoing dengue virus infection, we employed the cutting edge approach of generating of human monoclonal antibodies from individual plasmablasts from peripheral blood of dengue patients that allows us to probe for answers at a single cell level. this method involves ex vivo single cell sorting of plasmablasts from peripheral blood of well-characterized dengue infected patient followed by single cell molecular cloning of immunoglobulin heavy-and light-variable regions into expression vectors containing the defined constant region followed by transient cotransfection of hek 293a cells with the heavy and light chain expression vectors made from genes arising from the same cell. thus far, using this powerful technology, for the first time in india, we have made 140 number of human monoclonals, of which 80 are specific to dengue and 14 neutralize dengue virus at various concentrations. all the neutralizing antibodies are dengue-envelope specific and bind the highly conserved fusion loop of the dengue virus envelope. together, with the ongoing comprehensive analysis of the b cell repertoire and somatic hypermutations, these studies provide a detailed understanding of the dengue-specific plasmablast cell response at a single cell level and create a platform for testing these antibodies for basic research, diagnostic, prophylatic and as well as therapeutic applications. surviving. six of the 13 (46.2%) surviving patients remain dependent on ig replacement despite robust donor chimerism of 99-100% and no active gvhd. all but two received rituximab pre-hsct. of the patients who are independent of ig replacement, only one (14.2%) received rituximab post-hsct, whereas 5/6 of the ig dependent patients received rituximab post-hsct. t cell immune profiling revealed that the absolute numbers of lymphocyte subsets, cd4+ naã¯ve t cells, and cd4+ recent thymic emigrants were not statistically different between ig independent and dependent patients ( figure 1 ). however, there was a marked decrease in the number of total b cells, the percentage of memory b cells (cd27+ b cells), and classswitched memory b cells (cd27+ igd-igm-cells) in ig dependent patients ( figure 1 ). t follicular helper (tfh) cell populations (cd4+cd45ra-cxcr5+pd1+) were evaluated in four patients and the frequency was similar to healthy controls (4.5+/-1.2 vs. 3.9+/-1.4%). the ability of the patients naã¯ve b cells to class-switch was assessed following exposure to il-21, anti-cd40 antibody, and anti-human igm, and revealed normal b cell class-switching and differentiation to plasmablasts ( figure 1) . additionally, t cell ability to provide b cell help was assessed by coincubating naã¯ve b cells with activated cd4+ t cells. this revealed comparable b cell class switching to that of healthy controls. conclusion: the high incidence of poor long-term functional b cell reconstitution following allogeneic hsct for xlp-1 could be related to the use of rituximab in the post-hsct setting rather than pre-hsct. normal tfh numbers and function, and ability of b-cells to class-switch in-vitro suggest that persistent hypogammaglobulinemia is these patients is unlikely from a b or t-cell intrinsic defect. the possibility of rituximab induced acquired lymph nodal stromal defect in these patients is being explored. further studies are needed to understand the biology of persistent hypogammaglobulinemia in xlp-1. additionally, due to the high incidence of persistent hypogammaglobulinemia, exposure of rituximab should be limited post-hsct. background: tandem mass spectrometry (ms/ms) has emerged as a primary platform for many clinical and newborn screening laboratories. the application of ms/ms mainly focuses on the quantification of accumulated small metabolites in plasma resulting from various metabolic defects. however, many disorders do not yield such metabolic markers and would benefit from the direct quantification of intracellular target proteins. unfortunately, the extremely low (e.g., pmol/l range) protein concentrations in blood cells limit their detection via ms/ms. in recent years, peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-srm) has emerged as a promising technique for the quantification of low abundance proteins in complex matrices, including dried blood spots (dbs). our lab has demonstrated that immuno-srm methods are able to reliably distinguish affected patients from the normal controls for wilson disease (wd), wiskott-aldrich syndrome (was), severe combined immunodeficiency (scid), and x-linked agammaglobulinemia (xla) (j. proteome res., 2017 and front. immunol., in press). these results demonstrate the utilization of immuno-srm as a sensitive platform for multiplexed quantification of signature peptides in the low pmol/l range. methods: several candidate peptides for each protein were selected based on uniqueness using in silico blast tools and lc-ms/ms response. monoclonal antibodies (mabs) were then generated for peptide enrichment from dbs. blood from normal controls, wd, xla, scid, and was patients was spotted onto filter paper, dried, and stored at -20â°c until use. proteins were extracted from dbs, digested with trypsin, and enriched using mabs bound to magnetic beads. the enriched peptides were then eluted and analyzed using srm mode with a waters xevo tq-xs. results/conclusions: to date, immuno-srm methods have been generated for wd, was, scid, xla, and cystinosis. preliminary data shows immuno-srm methods are able to reliably quantify target proteins using signature peptides and accurately distinguish affected patients from normal controls. analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (was and btk: p = 0.0001, scid: p = 0.05). intra and inter-assay precision ranged from 11 -22% and 11 -43%, respectively, and the multiplexed assay showed a broad linear range (1.39 2000 fmol peptide) . in a blinded sample set of 42 pidd patients and 40 normal controls, immuno-srm-predicted diagnoses showed excellent agreement with clinical or genetic diagnoses. every molecularly-confirmed case of was and btk was also diagnosed by immuno-srm analysis. in addition, 62 randomly selected samples provided by the nbs laboratory of washington state were tested and peptide concentrations were found to be within normal ranges. efforts are underway to validate and incorporate peptide biomarkers for adenosine deaminase deficiency, dock8 deficiency, and ataxia telangiectasia, as well as general markers for nk cells and platelets into a single multiplexed assay. in addition, scid, was and xla samples continue to be run while we focus on reducing assay costs, time, and necessary sample input. our data herein provides proof of concept for the immuno-srm workflow to be extended to various other genetic diseases as potential multiplexed newborn screening methods.(250) submission id#617782the background: the long-term effects of glucocorticoids (gcs) on the immune system have been extensively studied in patients with different underlying conditions (e.g, malignancies or autoimmune conditions), as well as in healthy volunteers receiving short-term courses of these drugs. although these approaches provided highly relevant data, neither of them answered the unbiased/bona-fide effect of long-term gcs use on the immune system. endogenous cushing syndrome (ecs) may be caused by pituitary or ectopic acth-producing adenomas, or by tumors or hyperplasia of the adrenal cortex. patients with ecs present with different gcsdependent manifestations, including those affecting the immune system as neutrophilia and lymphopenia. when tumors are removed, most of the effects of gcs tend to progressively regress. methods: paired samples from 15 patients with ecs due to acth-producing adenomas (age range 7-16y, 8 females) were studied before (ecs-pre) and 6-12 months after tumor removal (ecs-post). extended lymphocyte phenotypes and apoptosis in different cell subsets were evaluated by flow cytometry. cytokine production (elisa) and responses, as well as their effects on cell proliferation and viability, were evaluated using cell trace violet and annexin-v staining. results: among multiple immunophenotypic changes, ecs-pre patients showed significantly reduced naã¯ve t cells and recent thymic emigrants (rte) as well as increased apoptosis in t cells when compared to themselves (ecs-post) or age matched healthy controls. moreover, significantly increased exhausted cd8 t cells were observed in ecs-pre patients. interestingly, ecs-post patients showed full cellularity recovery of t cells and rte with increased proliferation and reduced apoptosis, in addition to correction of most of the other changes evidenced. significantly lower il-21 plasma levels were also detected in ecs-pre when compared to ecspost patients. to determine the role of il-21 in an ecs-resembling condition, healthy control pbmcs were treated with gcs in-vitro and the effect of il-21 and other cytokines was tested. a significant reduction in apoptosis was observed in the il-21-treated cells that almost completely countered the pro-apoptotic effects of gcs; il-21 was also significantly more efficient than il-2, il-7, ifn-alpha and ifn-gamma in rescuing cells from apoptosis. il-21-specific upregulation of bcl2 and bcl6 expression was evidenced in these cells.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-024651-578c9ut5 authors: nan title: 2020 cis annual meeting: immune deficiency & dysregulation north american conference date: 2020-05-11 journal: j clin immunol doi: 10.1007/s10875-020-00764-z sha: doc_id: 24651 cord_uid: 578c9ut5 nan abstract/case report text oral lichen planus (olp) is a t-cell mediated chronic inflammatory tissue reaction in which presentation can range from asymptomatic plaques to painful, erosive, bullous, or ulcerative lesions. here, we present a 15 year-old female with a novel ctla-4 variant, multiple autoimmune conditions, and unusual tongue lesions. our patient was healthy until 9 years of age when she developed hashimoto's thyroiditis. at 11, she developed psoriasis. at 13, she was diagnosed with alopecia totalis and epstein-barr virus (ebv) with resultant and persistent anemia, thrombocytopenia, lymphopenia and neutropenia. she had chronic abdominal pain and diarrhea since age 13. esophagogastroduodenoscopy revealed lymphocytic esophagitis and active duodenal inflammation with increased intraepithelial lymphocytes. colonoscopy revealed mildly active chronic colitis with eosinophils. whole exome sequencing revealed a heterozygous c.239dela (p.q80rfs*2) pathogenic mutation in exon 2 of ctla-4. family history is remarkable: father (splenomegaly and psoriasis) and brother (autoimmune hemolytic anemia) have ctla4 haploinsufficiency with the same mutation. abatacept was initiated with re-growth of hair, improvement in cytopenias, improvement in psoriasis, and some reduction of gastrointestinal symptoms. since her abdominal pain persisted repeat endoscopies after six months of abatacept revealed persistent active lymphocytic esophagitis with some improvement in inflammatory injury in her duodenum and colon. physical exam revealed glossitis with a gel-like coating and ulceration on her tongue, xerosis along her face and scalp without other abnormalities ( figure) . she denied recent dental procedures, appliances, or tongue biting. her wbc ranged from 3-4 x10^9 cells/l and hemoglobin 9.4-12.7 g/dl. absolute lymphocyte count ranged from 1.0-1.7 x10^9 cells/l. immunologic evaluation revealed low iga and pan-low lymphocyte subsets (table) . ebv pcr ranged from 430-1,700 copies/ml. tongue scraping revealed candida dubliniensis and she responded to 5 days of fluconazole. two months later, she developed painful white patches along her tongue and subsequent 4 kilogram weight loss recalcitrant to viscous lidocaine, antacids, and 14 days of fluconazole. incisional tongue biopsy revealed ulceration with underlying granulation tissue with lymphocyte and plasma cell infiltration consistent with olp ( figure) . periodic acid-schiff diastase stain and grocott stain were negative. aerobic culture was normal. no fungus was isolated within 14 days. epstein-barr encoding region in situ hybridization was negative. two weeks of topical dexamethasone lead to temporary improvement. her tongue lesions waxed and waned over the following months. due to persistent psoriasis, methotrexate was initiated without worsening in her tongue lesion. to our knowledge, this is the first case of olp reported in a patient with ctla-4 haploinsufficiency. ctla-4 haploinsufficiency may present with variable clinical phenotypes including increased risk of ebv viremia and malignancies. therefore, after ebv and malignancy are ruled out, olp may be a prudent diagnosis to consider in a ctla4 insufficient patient with unusual oral lesions. mutations of bcl6b appear to be associated with lymphoproliferation and autoimmunity as well as susceptibility to herpes virus infections. additional research focusing on characterization of dna binding sites of bcl6b as well as the downstream expression of associated target genes is needed. these data combined with longitudinal analysis of additional patients with confirmed bcl6b mutations, will help clarify determinants of bcl6b pathogenesis and highlight potential therapeutic strategies. consanguineous marriages in tribal cultures, such as that in the united arab emirates significantly increase the prevalence of autosomal recessive disorders. premarital genetic screening and counseling, thus, are expected to reduce the frequency of these diseases. in this pilot study, diagnostic exome sequencing was used in the premarital screening program to identify recessive pathologic variants preventable by premarital counseling. a total of 487 pathologic or likely pathologic variants were identified in 176 studied emiratis (88 couples), averaging 2.8 variants per person. four percent of the persons had negative diagnostic exome sequencing; the remaining had one to eight variants per person. of the 351 distinct variants, 162 (46%) were novel. twenty (23%) couples had pathologic or likely pathologic variants of inborn errors of immunity (iei). two couples (10%) had iei pathologic or likely pathologic heterozygous variants in both partners imposing risk for autosomal recessive disease in the offspring. other eighteen couples (90%) had pathologic/ likely pathologic heterozygous variants present in only one person of the couple. total of sixteen (4.5%) iei variant identified and eight (50%) were novel. fourteen known phenotypic iei diseases were recognized (table. 1 ). these preliminary results support a need for nationwide premarital genetic screening, and primary immunodeficiency registry to identify common and novel pathogenic variants with high heritability rate. these results will aid adopting a preand post-connectional reproductive carrier counseling to reduce autosomal recessive diseases. also, it will assist the diagnosis of these complex diseases in our community. table 1 pathologic or likely pathologic variants of primary immunodeficiency (pid). abstract/case report text background: chronic granulomatous disease (cgd) is a primary immunodeficiency disorder caused by defects in the phagocytic nadph oxidase complex, leading to increased susceptibility to infection and inflammatory or autoimmune disease. up to 50% of patients have gastrointestinal (gi) involvement and meet diagnostic criteria for inflammatory bowel disease (cgd-ibd). objectives: we analyzed cgd patients from the united states immunodeficiency network (usidnet) registry to determine whether ibd may change the presentation, treatment, and outcomes of cgd patients, as compared to those without ibd. methods: a retrospective evaluation of cgd cases from the usidnet registry was completed. cgd-ibd was defined as the presence of any major physician-reported inflammatory, non-infectious gi tract disease manifestation, including crohn disease, ulcerative colitis, ibd endoscopy findings, gi fistulas, gi strictures, gi obstruction, and proctitis. demographic information, genotypes, symptoms and conditions, infections, antimicrobial therapies, immunomodulator use, and allogeneic hematopoietic stem cell transplantation (hsct) data were analyzed. results: 194 patients with a diagnosis of cgd were identified. 96 met criteria for ibd; 98 were categorized in the non-ibd group. crohn disease and colitis were the most common gi disease manifestations in the cgd-ibd group (n=79), followed by gi fistulas (n=22). cgd-ibd patients had an increased average frequency of infections (10.6 events/patient) compared to the cgdnon-ibd group (5.1 events/patient). in both groups, lower respiratory tract infections were the most common infection type and aspergillus was the most common organism. enteric organism infections were more common in ibd patients. temporal data regarding the timing of infections were not available. immunomodulators, including biologics and interferon-gamma, were used at a significantly higher rate in ibd patients compared to non-abstract/case report text down syndrome (ds) is characterized by the occurrence of three copies of human chromosome 21 (hsa21). these patients often develop chronic mucocutaneous candidiasis (cmc) and autoimmune thyroiditis, mimicking patients with heterozygous gain-of-function (gof) stat1 mutations, which enhance cellular responses to the three types of interferon (ifn). hsa21 contains a cluster of four interferon receptor (ifn-r) genes: ifnar1, ifnar2, ifngr2 and il10rb. a gene dosage effect at these four loci may contribute to the infectious and autoimmune manifestations observed in individuals with ds. we report high levels of ifn-αr1, ifn-αr2 and ifn-γr2 expression on the surface of monocytes and ebv-transformed-b (ebv-b) cells from ds patients. levels of ifn-ɣr1, encoded by a gene on chromosome 6, were similar in the immune cells of ds patients and healthy controls. total and phosphorylated stat1 (stat1 and pstat1) levels were constitutively high in unstimulated and ifn-α-and ifn-γ-stimulated monocytes from ds patients, although less so than those in patients with gof stat1 mutations. following stimulation with ifn-α or -ɣ, but not with il-6 or il-21, pstat1 and ifn-ɣ activation factor (gaf) dna binding activities were significantly higher in the ebv-b cells of ds patients than in controls, this response resembling the dysregulated responses observed in patients with stat1 gof mutations. plasma type i ifns concentrations were high in about 12% of the ds patients tested. a genome-wide transcriptomic analysis involving principle component analysis and a comparison of interferon modules was performed on circulating monocytes. it showed that ifn-stimulated genes (isgs) were expressed more strongly in ds than in controls. ds monocytes have intermediate levels of ifn-α-and ifn-γ-induced isgs relative to monocytes from healthy controls and from patients with gof stat1 mutations. by contrast to patients with gof stat1 mutations, circulating th17 counts were normal and the proportion of terminally differentiated cd8+ t cells was high in ds patients. the constitutive upregulation of type i and type ii ifn-r, at least in monocytes of ds patients, may therefore contribute to the autoimmune diseases observed in these individuals. scid screens (positive screen defined as trec values less than 250 units/ul per statewide criteria). results forty nine neonates were identified with low trec values. 46 (94%) of these infants had repeat trec screening, 14 (29%) of which were found to have a positive second trec screen. lymphocyte subsets were evaluated in 23 of these infants and 5 of which were noted to have lymphopenia (defined as absolute lymphocyte count less than 2500). 2 infants were noted to have low cd4 levels (defined as < 300 cells/ul) and 6 were noted to have low cd8 levels (defined as less than 500 cells/ul). of note, 50% of the infants with cd4 and cd8 lymphopenia had normal repeat trec levels. 14 of the infants were noted to have low b cell levels (defined as < 610 cells/ul). 12 infants had quantitative immunoglobulin levels and of these two were noted to have igg levels less than 100. of note one infant was diagnosed with partial digeorge syndrome via microarray. in our study population, no infants were diagnosed with scid. discussion our study shows that testing for trec levels on newborn screen may be beneficial in identifying not only scid, but also other immunologic conditions. infants in our study had evidence for both cell mediated and humoral immunodeficiency which necessitated further workup and follow up from allergy and immunology specialists. it may be beneficial to develop further programs to track infants identified with abnormal trec levels on newborn screens to determine if they develop signs of immunodeficiency syndromes later in life. abstract/case report text the patient was transferred to our adult clinical immunology transition clinic for low igg and iga, elevated igm and b cell lymphopenia, treated with subcutaneous gamma globulin. his medical history was relevant for recurrent respiratory infections since two years-old, failure to thrive and developmental delay. he also developed chronic auto-immune hemolytic anemia (aiha) at ten years old, accompanied by prominent lymphoid hyperplasia. our initial evaluation at the age of twenty years old showed massive polyadenopathy and splenomegaly. work-up confirmed flair-up of aiha. at that point, the diagnosis of apds was raised. he also had mild intellectual impairment and dysmorphic features such as a mild degree of ocular depression, deep-set eyes, vaguely triangular face, small chin, but had normal stature. a customized panel for usual genes involved in classic hyperigm syndromes, apds, noonan and kabuki syndromes came back negative. at 23 years old, an urgent coloscopy was performed because of acute abdominal pain and showed diffuse ileal lymphoid hyperplasia. biopsies confirmed reactional lymphoid hyperplasia without infection nor malignancy. a second 232 genes ngs panel associated with pid identified a heterozygote mutation in tap1; expression of hla class 1 was normal on flow cytometry. the patient was then started on sirolimus for an "apds-like syndrome" despite the lack of genetic confirmation. six months after introduction of mtor inhibitor, his abdominal pain had completely disappeared. tep scan showed complete resolution of axillar, retroperitoneal and inguinal lymph nodes and significant regression of splenomegaly. a third large non-biased 6700+ genes ngs panel revealed a 30 pb de novo deletion that included the splice site of pik3r1 exon 11 typically involved in apds2: c.1392_1425+4del p.(asp464glufs*5) , which was missed by the first two panels. indeed, oligonucleotide-selective sequencing technology used for the previous panels was associated to mapping errors of short reads and difficult detection of large deletions. interestingly, the patient also presented some but not all dysmorphic features of short syndrome which is related to pik3r1 haploinsufficiency. in this new era of genetic testing, this case is a reminder that we need to be aware of the pitfalls of genetic tests and that clinical judgment is still our best diagnostic tool. abstract/case report text intro: patients with chronic granulomatous disease (cgd) are theorized to have a lower risk of malignancy related to their lack of free radical formation. there are relatively few reports of malignancy described in patients with cgd. we report three cases of malignancies in the large cohort cgd population at the national institutes of health followed between 1972-2018 to add to the seven cases described in the literature. case 1: a 48-year-old man with x-linked chronic granulomatous disease with a history of severe inflammatory bowel disease, who presented with progressive left-sided chest pain in 2017, decreased appetite and weight loss. transthoracic lung biopsy showed atypical cells and a pet/ct showed abnormally dense mesentery and widespread hypermetabolic abnormalities. a mesenteric biopsy showed metastatic pancreatic adenocarcinoma. palliative care was initiated. patient expired four months after diagnosis. case 2: a 24-year-old man with x-linked chronic granulomatous disease and severe inflammatory bowel disease requiring total proctocolectomy and who had been remotely treated with infliximab, presented in 2015 with right upper quadrant pain. abdominal ultrasound and mri of the liver showed multiple liver lesions. biopsy of these lesions revealed hepatocellular carcinoma. patient underwent two courses of radiolabeled itrium spherules. however, his disease progressed and he expired approximately five months after diagnosis. c a s e 3 : a n 1 8 -y e a r-o l d m a n w i t h x -l i n k e d c h r o n i c granulomatous disease and inflammatory bowel disease who presented in 2007 with fevers, abdominal pain and pancytopenia. during the course of his hospitalization, he developed sepsis which led to his demise. on autopsy, an incidental finding of papillary thyroid carcinoma was made. discussion: these three patients all had poorly controlled inflammatory bowel disease. additionally, patients with cgd are typically exposed to higher doses of radiation, leading one to expect higher rates of radiation induced malignancies. however, there are still relatively few case reports of cancer in the cgd population. tissue biopsy is necessary for diagnosis. due to end organ damage secondary to the underlying disease in the first two cases, treatment options were limited. managing infections during chemotherapy can be complex due to drug interactions with chemotherapeutic agents. abstract/case report text myeloperoxidase (mpo) deficiency is the most common inherited defect of phagocytes that impairs microbial killing since the toxicity of the respiratory burst is dampened without myeloperoxidase release from the azurophilic granules. a significant portion of these patients remain asymptomatic, however there is a clinically variable phenotype that can present if they do become symptomatic. fungal infections with candida strains appear to be the most frequently reported. we present an adulthood case of recurrent invasive candidal disease due autosomal recessive myeloperoxidase deficiency from a pathogenic missense variant in the mpo gene (c.1705c>t (p.arg569trp)). a 23-year-old caucasian male was in his normal state of health without any major illnesses until 18 years of age when he was diagnosed with candida osteomyelitis of the heel, followed by cryptococcal meningitis the following year which ultimately required a ventriculoperitoneal shunt. in 2019, he had a prolonged hospitalization after presenting with lethargy, headache and vomiting that culminated in seizure activity and prompted an emergency room visit. imaging at the time showed ventriculomegaly, and fluid from the shunt revealed yeast, but no bacteria. he was started on broad spectrum antifungal therapy and admitted for further management. cerebral spinal fluid and blood cultures confirmed invasive candida albicans meningitis. during this hospitalization, he also developed sepsis secondary to serratia marcescens. because of the pathogens that were being isolated, our service was consulted. of note, our patient does not have diabetes mellitus. a neutrophil oxidative burst assay showed an absent respiratory burst compared to control. a primary immunodeficiency panel to identify genetic variants was also sent to invitae. variants in cyba, cybb, ncf2, and ncf4 were not identified, making chronic granulomatous disease less likely. peroxidase staining was negative on neutrophils and normal on eosinophils, suggesting a diagnosis of mpo deficiency. this led to mpo gene sequencing for deletion and duplication analysis. a homozygous pathogenic variant consistent with a molecular diagnosis of a mpo related condition was identified. immunoblotting of patient-derived immune cells demonstrated an absence of mature enzyme. although not typically indicated, given the severity of his presentation, our patient remains on fluconazole for long term prophylaxis. his younger brother also had a history of invasive disease with candidaosteomyelitis and meningitis. a neutrophils oxidative burst assay showed similar results in his brother and similar results with peroxidase staining, also suggesting a diagnosis of mpo deficiency. confirmatory genetic testing has not been performed yet. their father, who reported severe skin infections with candida, had peroxidase stains performed on neutrophils and eosinophils which were both normal. we have presented a patient without a significant history of diabetes mellitus who developed invasive disease from candida and serratia and was ultimately diagnosed with myeloperoxidase deficiency. abstract/case report text introduction: juvenile xanthogranuloma (jxg) is an often benign, histiocytic proliferative disorder of the mononuclear phagocytic system. patients typically present with localized cutaneous lesions. systemic disease, especially central nervous system involvement, rarely occurs but has significant morbidity and mortality risk. no standard evaluation nor therapy regimen exists for systemic jxg and little is known about the genomic alterations underlying its pathology. case report: a full-term male infant presented at 4 months of age with post-prandial abdominal pain, fevers, altered mental status and weight loss. abdominal ultrasound and ct identified renal masses. a chest ct was obtained showing a paraspinal mass with possible neural foramina extension. mri brain and total spine was consistent with diffuse leptomeningeal disease involving the left frontal convexity, brainstem, cerebellum, and multiple cranial nerves. abnormal enhancement was also present along the entire surface of the spinal cord extending into the cauda equina with additional enlargement of the cervical/upper thoracic cord with intramedullary enhancing masses and a right paraspinal mass. renal biopsy yielded a pathologic diagnosis of disseminated jxg. integrative clinical sequencing of the mass identified a somatic driving alk rearrangement (kif5b-alk in-frame fusion). tumor and matched germline dna sequencing did not detect any alterations in the ras/mapk pathway. bone marrow biopsy was negative for disease with cerebrospinal fluid analysis showing numerous monocytes and macrophages consistent with jxg. the patient was started on therapy consisting of systemic dexamethasone, intrathecal methotrexate/hydrocortisone and systemic intravenous cytarabine. his first cycle was complicated by pseudomonas aeruginosa bacteremia and gangrenous cellulitis of the perianal region, treated with systemic/topical antibiotics and topical gm-csf. following completion of the initial cycle of therapy, the patient was noted to have declining neurologic status, including seizure-like activity. repeat mr imaging revealed worsening cns disease with new subdural fluid collection and progression of leptomeningeal enhancement and intramedullary cervical lesion. in light of disease progression, the decision was made to continue dexamethasone treatment, but add adjunct intrathecal cytarabine, and transition to targeted alk inhibition via daily oral ceritinib, given its predicted cns penetrance followed by ceritinib in combination with systemic intravenous clofarabine. significant clinical and radiographic improvement was noted with the new targeted treatment regimen. ceritinib therapy was tolerated well overall after a 25% dosing reduction made for initial grade 2 gastrointestinal toxicity and grade 4 hypertriglyceridemia (non-life threatening but level > 1000 mg/dl). following continued treatment with daily ceritinib and completion of 12 cycles of clofarabine therapy, our patient experienced complete disease remission. he continues to do well on daily ceritinib monotherapy with plan to complete an additional year of therapy. conclusion: our report highlights the potential benefit of real-time integrative clinical sequencing in the management of systemic histiocytic lesions, specifically non-langerhans cell conditions. it has the potential to identify novel somatic genetic alterations, other than the typical lchassociated braf mutations of the mapk pathway, that may be therapeutically targetable. treatment with 2nd generation alk-inhibition in our pediatric disseminated jxg patient was a novel, biologicallyrationale management approach with minimal toxicity and potentially contributed to his complete remission. abstract/case report text background: c3 glomerulonephropathy (c3gn) is a progressive kidney disease with the predominant pathological feature of c3 deposits around the glomerular capillaries. c3gn patients suffer from dysregulated activation of the alternative pathway as the result of autoantibodies or congenital genetic defects that stabilize cleavage of c3. despite therapy involving immunosuppression and complement-pathway inhibition, the prognosis for c3gn is poor. we report a patient with autoantibody-mediated, refractory c3gn who demonstrated no improvement on rituximab but achieved sustained remission on bortezomib. follow up studies after one year demonstrated clearance of the culprit autoantibody, normalization of c3 levels, and improved pathologic appearance of the kidneys. this case supports the idea that c3gn is frequently driven by pathogenic autoantibodies that may not clear with rituximab alone. plasma cell directed therapy has the potential to clear these autoantibodies and halt the progression of disease. case presentation: we report the case of a hispanic male with chronic renal dysfunction initially diagnosed with membranoproliferative glomerulonephritis on renal biopsy at 12 years of age. despite cellcept and prednisone, over the next 4 years, he had worsening proteinuria and an increase in protein-to-creatinine ratio. renal biopsy suggested c3gn, and lab studies revealed a factor-h autoantibody and c3 level below the assay limit of detection. after initiating eculizumab, the proteinuria temporarily improved; however, the proteinuria eventually worsened, and he was referred to immunology. we hypothesized that the factor h-binding autoantibody was the cause of dysregulated c3 cleavage and disease progression, and blocking the terminal complement pathway with eculizumab would not halt upstream c3-mediated kidney injury. at the age of 20, rituximab and plasmapheresis were administered to clear the factor-h autoantibody. three months after rituximab administration, the factor-h autoantibody level decreased to the normal range, but he continued to have significant proteinuria with low serum albumin and undetectable c3 level. we concluded that the relevant autoantibody was not solely produced by differentiating memory b cells, so we decided to target the plasma cell compartment. bortezomib was started at the age of 21, and eculizumab was continued given his initial response to treatment. after adding bortezomib, factor h autoantibody levels dropped below prior levels and serum c3 level normalized. renal biopsy at the age of 22 showed evidence of imp r o v i n g c 3 d e p o s i t i o n a n d l e s s p r o m i n e n t g l o m e r u l a r hypercellularity, with stable mesangial hypercellularity, interstitial fibrosis, tubular atrophy, and sclerotic glomeruli. although his proteinuria did not worsen, it remained persistent, suggesting that earlier introduction of bortezomib could have prevented disease advancement. there has been no further progression of kidney failure. conclusions: the majority of c3gn patients harbor autoantibodies to components of the alternative pathway of complement. this case provides evidence that at least some of these autoantibodies are indeed the cause of complement dysregulation, and thus are prime targets for therapy. b cell targeting therapies may be inadequate to decrease autoantibody levels for some patients. early initiation of bortezomib, or other plasmacell directed therapy, may effectively induce complement normalization and disease remission in these cases. inhaled corticosteroid and a long-acting bronchodilator. he has no family history of immunodeficiencies or congenital disorders. computed tomography(ct) chest showed bronchiectasis. his complement studies and isohemagglutinin titers were also normal. his serum immunoglobulin(ig) and lymphocytes on presentation are shown in table 1 . he had a poor response to polysaccharide pneumococcal vaccination. he was diagnosed with combined igg2/igg4 subclass/iga deficiency and was started on immunoglobulin replacement therapy and prophylactic rotating antibiotic therapy. thereafter, his clinical course markedly improved with a reduction in the frequency of rti's as well as the number of bronchiectasis exacerbations. there was high suspicion for an underlying genetic disorder based on his constellation of neurodevelopment disorders and immunodeficiency. cytogenetic evaluation with array comparative genomic hybridization(cgh) analysis showed duplication of xq25 and xq28 consistent with mds. discussion: mds is caused by duplications involving the mecp2 gene locus of the x chromosome at xq28. it has a 100% penetration rate in males whereas females act as carriers and are usually unaffected. rarely, cases of de novo mutations causing mds have been reported. chromosome microarray analysis is currently the best initial clinical test when mecp2 duplication syndrome is suspected. management needs a multidisciplinary approach involving geneticists, neurologists, ophthalmologists, physical medicine and rehabilitation specialists, psychologists, gastroenterologists, and allergy and immunology specialists. prophylactic treatment with ivig and antibiotics has been the standard of care for immunodeficiency in these patients. prognosis is guarded and most male patients die in the mid to late 20's because of severe rti's secondary to immunodeficiency. conclusions: this case confirms the association of mds with combined iga and igg subclass deficiencies. clinicians should consider pursuing genetic evaluation for mds in patients with neurodevelopmental disorders and immunodeficiency because the diagnosis of the syndrome can change the overall approach to management and expectations in prognosis. abstract/case report text introduction: c3 nephritic factor is an autoantibody that binds to the alternative pathway c3 convertase (c3bbb). this results in unchecked overactivation of the alternative complement pathway, which can lead to renal disease, partial lipodystrophy, retina disease, and frequent infections. in this case, we present a patient with partial lipodystrophy and low c3, subsequently found to have c3 nephritic factor. case description: a 5 year old female presented with a 19 month history of low c3 levels. she was diagnosed 21 months ago with poststreptococcal glomerulonephritis (psgn) after presenting with hematuria and elevated aso titers. she had c3 levels drawn 1-2 months after time of diagnosis and c3 level was low at 27 (normal range 81-157), which was consistent with psgn. it was rechecked 3 months after time of diagnosis and was still low. she was referred to rheumatology at this time and was found to have a positive ana titer 1:160. tests for lupus and anti-phospholipid syndrome were negative. c3 normalized to the low-normal range at 6 months after time of diagnosis to 81. her pediatrician checked to make sure it remained normal around 17 months after initial diagnosis and c3 was low again at 22. c4 was normal at 26. she was referred to immunology for further evaluation. during this time she was asymptomatic with no fevers, infections, hematuria, rashes, joint pain, or joint swelling. she has no history of hospitalizations other than the first for psgn. mother denied family history of autoimmune disorders. physical exam: physical exam was notable for abnormal subcutaneous facial fat with normal fat distribution in the rest of her body. the rest of the exam was unremarkable with normal cardiac, pulmonary, abdominal, and skin exam. testing: c3 level was rechecked and low at 26. c3 nephritic factor was elevated at 1.07 (normal range 0.00-0.26). alternate pathway complement (ah50) was confirmed twice and was undetectable, < 10 (normal level greater or equal to 46). total hemolytic complement (ch50) was low at 22 (normal level 42-95). other complement levels were checked and c1q, c2, c4, c5, c6, c7, c8, and c9 complement were within normal range. discussion: the overactivation of the alternative complement pathway by c3 nephritic factor can result in various clinical manifestations, such as c3 glomerulopathy and acquired partial lipodystrophy in predominantly the face and the upper torso. the exact mechanism of how c3 nephritic factor is related to facial and upper body lipodystrophy is not known. one proposed mechanism is that adipocytes in the face and upper body produce more factor d, which is a complement protein utilized by c3 nephritic factor. overactivation of the alternative complement pathway on the adipocyte then leads to formation of the membrane attack complex, resulting in adipocyte lysis. eye disease, such as retinitis pigmentosa and macular degeneration can develop. c3 nephritic factor can also lead to more frequent infections and renal disease. patients need to be closely monitored. if patients develop c3 glomerulopathy, they may need to be considered for immunomodulatory therapy, such as steroids and other immunosuppressants. 4 project manager/ucsf benioff children's hospital 5 senior clinical research associate/ucsf benioff children's hospital 6 associate professor/department of clinical pharmacy, ucsf 7 staff research assistant iv/ucsf benioff children's hospital 8 senior supervisor/ucsf benioff children's hospital 9 laboratory specialist/ucsf benioff children's hospital 10 research specialist/ucsf benioff children's hospital 11 assistant professor/ucsf benioff children's hospital 12 clinical professor/ucsf benioff children's hospital 13 assistant professor/ucsd rady children's hospital 14 staff pediatrician/tuba city indian health service 15 staff pediatrician/phoenix children's hospital 16 associate professor/seattle children's hospital 17 chief, genetic immunotherapy section/niaid, nih 18 professor/university of minnesota 19 professor/ucsf benioff children's hospital abstract/case report text background: artemis-deficient scid (art-scid) represents 3% of all scid, but occurs in 1/2000 births in navajo and apache native americans. artemis protein, encoded by dclre1c, is essential for repairing dna double-stranded breaks, including those generated during v(d)j recombination of antigen receptor genes as t and b cells develop. artemisdeficiency causes not only t-b-nk+ scid, but also increased sensitivity to alkylating drugs and radiation. art-scid is the most difficult scid to treat with allogeneic hematopoietic cell transplantation (hct) due to high rates of rejection and gvhd, incomplete immune reconstitution, and toxicity following intensive conditioning regimens. as an alternative, we developed a self-inactivating lentiviral vector containing the human artemis promoter and dclre1c cdna (aproart). we are evaluating its toxicity and efficacy in a phase i/ii gene transfer trial in art-scid patients. methods: newly diagnosed infants with art-scid and older patients with insufficient immunity despite prior allogeneic hct were eligible if organ function was acceptable. infants needed to have no matched sibling donor and be at least 2 months old at conditioning. cd34+ cells were isolated from bone marrow or cytokine-mobilized peripheral blood, cultured with cytokines, transduced x2 with aproart, and cryopreserved. patients received 2 daily doses of busulfan, targeted for a cumulative exposure (cauc) of 20mg*hr/l, with infusion of thawed cells on the following day. results: we treated 5 newly diagnosed infants (art001-3&007-8) with median age 2.6m (range 2.3-3.7) and 3 previously-treated patients (art004-6) (5.5y, 12.7y and 20.9y), with a median follow-up of 9.6m (range 1.6-17.2). the mean (sd) bu cauc was 19.4±1.0 mg*hr/l. patients received a median of 6.5x106 aproart-transduced cd34+ cells/kg (range 3.9-12.4). the average vector copy number (vcn) and transduction efficiency in the marrow grafts exceeded those in the pbsc grafts: 2.1±1.0 copies/cell vs 0.83±0.1 (p=0.02) and 75±9% vs 59±4.6% (p=0.03), respectively. there were no serious busulfan side effects. all patients had transduced peripheral blood leukocytes by 4w and 7 of 8 developed gene marking in t, b, nk and myeloid cells by 8w (fig. 1) . gene-corrected cd3, cd4, cd4/ 45ra/ccr7, cd8 and cd19 cells appeared in 7 of 8 patients (fig. 2) , with art005 having t, nk and myeloid marking without b cells at 6m post infusion. normalization of lymphocyte proliferation to pha occurred in the 3 evaluable (>12w) infants (fig. 3) , all 3 now outpatients off isolation. two infants and 1 previously treated child developed autoimmune hemolytic anemia (aiha), with 2 requiring immunosuppressive therapy. infections included rhinovirus at presentation in art001 that resolved with t cell reconstitution. after discharge art001 acquired and recovered from norovirus and art002 acquired and recovered from cmv and rotavirus. analyses of insertion sites and t cell receptor diversity are pending. conclusion: infusion of aproart-transduced autologous cd34 cells into art-scid patients pretreated with very low exposure busulfan resulted in multilineage engraftment of transduced cells with evidence for t and b cell immune development. aiha, the only complication to date, occurred early and appears to resolve following restoration of t cell immunity. these encouraging results suggest potential effectiveness of ex vivo gene therapy for art-scid. (26) submission id#798344 mailan nguyen, md 1 , susan canny, md, phd 2 , andrea ramirez, md 1 , ivan chinn, md 3 abstract/case report text background: systemic lupus erythematosus is a heterogeneous disorder of the immune system. systematic genetic evaluation of patients with childhood-onset sle (csle) has begun to identify phenotypic clusters of csle patients with classic sle-causing genetic variants, as well as revealed unexpected genetic mimics of lupus. we report 3 patients diagnosed with csle with similar typical and atypical lupus features, who were subsequently found to carry pathogenic nras variants that are the cause of ras-associated autoimmune leukoproliferative disorder (rald). cases: all 3 patients (2 females, 1 male) presented at < 3 years of age (average age 21.7 months, range 17-27 months) with antinuclear antibodies, anti-double-stranded dna antibodies, autoimm u n e h e m o l y t i c a n e m i a , s e v e r e t h r o m b o c y t o p e n i a , antiphospholipid antibodies, hypocomplementemia and nephritis. additionally, the patients all displayed fevers, organomegaly, lymphadenopathy and hypergammaglobulinemia. two out of 3 patients had a malar rash, leukopenia, lymphopenia, anti-smith antibodies, serositis or arthritis. no patient had oral or nasal ulcers or photosensitivity. despite the fevers, lymphoproliferation and systemic autoimmunity, the patients did not display overwhelming immune dysregulation (peak ferritin 128-623 ng/ml). interestingly, the patients were found to have monocytosis (19-45%), as has previously been reported in rald. double negative t cells were within normal range in the 2 patients in which this was tested. all 3 patients required aggressive immune modulation for control of their disease manifestations. two of the 3 developed severe infections, specifically pneumococcal sepsis, during therapy. current follow-up covers an average of 9.2 years (range 0.6 to 17 years). the patients responded to corticosteroids and were given sequential trials of various steroid-sparing therapies. in general, they appeared to benefit from both b cell depletion and t cell-directed modalities (cyclosporine, rapamycin), which are not first line therapy in csle. unfortunately, 1 patient developed a fatal pulmonary infection while on treatment; her underlying disease was felt to be quiescent. due to the early-onset of disease, each patient was selected for genetic evaluation (2 by exome sequencing, 1 by gene panel). this lead to the discovery of pathogenic nras variants (c.38g>a, p.g13d) in all patients, assumed to be somatic, although this was confirmed in only 1 case. conclusion: ras-associated autoimmune leukoproliferative disorder can present indistinguishable from csle with positive autoantibodies, immune cytopenias, arthritis, nephritis and hypocomplementemia. clinicians should consider evaluating for rald in csle patients who present at an early age ( < 3 years) with predominant features of lymphoproliferation and hematologic abnormalities, particularly monocytosis. t cell-directed therapy with cyclosporine or rapamycin should be considered for rald. (1) human ctla4 loss-offunction causes dysregulation of foxp3+ regulatory t (treg) cells, hyperactivation of effector t cells, and lymphocytic infiltration of target organs. patients also exhibit progressive loss of circulating b cells, associated with an increase of predominantly autoreactive cd21(lo) b cells and accumulation of b cells in non-lymphoid organs. inherited human ctla4 loss-of-function demonstrates a critical quantitative role for ctla4 in governing t and b lymphocyte homeostasis. (2) this case highlights the importance of next generation sequencing (ngs) in diagnosing and managing complex presentations with multi-system involvement. case presentation: patient was diagnosed with diffuse large b cell lymphoma at age 22 and treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop) in 9/2012. he underwent autologous stem cell transplant with preparative carmustine, etoposide, cytarabine, and melphalan (beam) in 10/2012. he subsequently developed recurrent giant condyloma acuminata following transplantation requiring 9 surgical resections, refractory immune thrombocytopenic purpura (itp) requiring aggressive systemic steroids and high dose ivig at least yearly, experiencing hypogammaglobulinemia, recurrent sinopulmonary infections, disseminated herpes zoster, and kaposi sarcoma. in 6/2014, he underwent ct/pet, revealing extensive hypermetabolic lymphadenopathy and splenomegaly. bone marrow biopsies were negative for lymphoma, although showed a slightly hypocellular marrow (40-60% cellularity), 2% blasts, and eosinophilia without peripheral eosinophilia. repeated evaluations for hiv, syphilis, histoplasma, cmv, hhv6, hhv8, bartonella, coxciella, brucella, htlv, toxoplasma were negative. htlv1 and 2 antibodies had been negative prior to transplantation. tonsillectomy 12/2015 due to progressive enlargement showed reactive follicular hyperplasia with focal acute tonsillitis without granulomas or viral inclusions. repeated lymphocyte enumeration and proliferation studies were normal. a repeat pet scan 3/2016 revealed persistent diffuse lymphadenopathy involving the neck, chest, abdomen, and pelvis. left lung biopsy revealed non-caseating granulomas without lymphoma. stains for ebv were negative. repeat pet scan 11/2016 indicated disease progression prompting a left axillary excisional lymph node biopsy, revealing ebv lymphadenitis with large, reactive follicles with interspersed inflammation and loosely formed granulomas and cd20 positive b cells within the follicles. ebv blood pcr was negative. afb and fungal stains were negative on all biopsies. in 1/2017, his igg was 615 (762-1488), iga 33 (70-390), and igm 58 (38-328) with only 1 out of 23 protective serotypes to pneumococcus post-vaccination at 1.3 or greater. in 3/2018, igg was 380 (762-1488). custom ngs panel showed a heterozygous missense variant in ctla4 c.534c>g (p.s178r) located in the transmembrane domain. this variant of uncertain significance is suspicious and strongly suggests the diagnosis of ctla4 -related autoimmune lymphoproliferative syndrome . patient was referred to the national institute of health, where he received a bone marrow transplant. conclusion: primary immunodeficiency diseases comprise a group of highly heterogeneous immune system diseases and around 300 forms of pid have been described. ngs has recently become an increasingly used approach for gene identification and molecular diagnosis of human diseases guiding treatment to patients who may otherwise have poor outcomes. (3) (28) submission id#799299 erik newman, md 1 , cullen dutmer, md 2 1 allergy and immunology fellow/university of colorado and children's hospital colorado 2 assistant professor of pediatrics/section of allergy & immunology, children's hospital colorado, university of colorado school of medicine, aurora, co, usa abstract/case report text introduction: inherited defects of the complement system are rare disorders that can result in unique susceptibility to infections with select bacteria. patients with a deficiency of a complement protein early in the complement pathway (affecting c1qrs, c2, c3, factor h, or factor i) have increased susceptibility to infection with encapsulated bacteria, most notably streptococcus pneumoniae and neisseria species. in contrast, patients with a deficiency of a complement protein at the terminal end of the complement pathway (affecting c5, c6, c7, c8 alpha/beta/gamma, or c9) almost universally present with severe, recurrent, or disseminated neisseria species infections. most genes encoding complement proteins are found on autosomes, in which specific complement deficiencies result from biallelic mutations. although particular complement deficiencies occur at higher frequencies in certain populations, the prevalence of specific complement deficiencies is unknown in many parts of the world, especially in underdeveloped regions, including sub-saharan africa. herein, we describe a young congolese boy with an atypical presentation of c8 alpha deficiency. case description: a 17-month-old congolese boy with consanguineous parents (first cousins) presented with recurrent infections. prior to an evaluation of his immune system, he was hospitalized five times. his infections included episodes of acute otitis media, bacterial pneumonia, and viral pneumonitis. a bronchoscopy revealed diffusely edematous airways and growth of candida albicans, moraxella catarrhalis, and streptococcus pneumoniae in bronchoalveolar lavage cultures. concurrently, his respiratory pcr panel was positive for adenovirus. his initial immune evaluation included assessments of his serum immunoglobulin levels, vaccine titers (tetanus, diphtheria, haemophilus influenzae, and streptococcus pneumoniae), neutrophil oxidative burst, lymphocyte subsets, and ch50, in which only his ch50 was abnormal (16 u/ml). his ch50 remained low on repeat assessment (16 u/ml), at which time an ah50 was pursued and also returned with a low result (1% of normal). a complement system genetic panel identified a homozygous intronic variant in c8a (c.856-12g>a). functional confirmation of the variant revealed that the patient had a significantly decreased c8 level (18 mcg/ml) and absent c8 function. discussion: we present a case of a young congolese boy with c8 alpha deficiency and a clinical presentation atypical for defects in terminal complement proteins. our patient presented primarily with recurrent respiratory infections, including streptococcus pneumoniae pneumonia, but without a preceding history of meningococcal disease. while it is well established that patients presenting with terminal complement pathway defects have an increased susceptibility to meningococcal disease, it is less clear if they have increased susceptibility to pneumococcal infections. occurring between exons 5 and 6, the homozygous intronic variant in c8a identified in our patient is predicted to result in abnormal splicing. while the allele frequency of this mutation is relatively high in the african population (0.017), functional confirmation of the variant demonstrated a decreased c8 level and absent c8 function that support the pathogenesis of the mutation. the discrepancy between the allele frequency and reported disease cases could be explained in part by varying clinical manifestations seen in c8 deficiency or underrecognized disease in sub-saharan africa. abstract/case report text rationale: scid is a syndrome characterized by profound t, b, and (in some cases) nk cell defects that is universally fatal unless immune reconstitution is achieved. a total of 177 scid infants have been given allogeneic bone marrow transplantation at duke university medical center without pre-transplantation chemotherapy or post-transplantation graft-versus-host disease (gvhd); 90% received t cell-depleted haploidentical parental marrow and 47(26%) are known to be deceased. post-transplantation follow-up ranged from 22 months to 37 years. the aim of this cross-sectional study is to characterize the clinical status of a large cohort of survivors treated at a single medical center. methods: clinical status was assessed by detailed questionnaires delivered by mail or electronically. adult (≥ 18 years old) and pediatric questionnaires were based on patients' age. patients were also contacted by telephone and evaluated at clinic visits. molecular type of scid, demographics, type, date and age at transplant were obtained from a clinical database. results: fifty questionnaires were completed to date from survivors ranging in age from 5 to 37 years. twenty-nine/50 were adults ≥18 years at the time of the questionnaire. genetic defects were known for all 50 patients-xlinked scid was the cause in about half ( figure 1 ). twenty-three of 50 patients were on immunoglobulin replacement. thirty of 50 reported having received immunizations, and about half of those received live vaccines. fifteen of 50 reported they were taking no regular medications; 11 reported taking prophylactic antibiotics. >we found substantial scholastic achievement, with 17/29 adult patients reporting college attendance. two had post graduate education including doctorate level degrees. occupations included physician, nurse, factory worker, musician, teacher, and engineer. one patient had 3 children. twenty-seven /29 adult patients shared their height and weight and 78% (21/27) had a healthy bmi (bmi 18.5-24.9), while 15% (4/27) were overweight, and 7% (2/27) were underweight (< 18.5) . in pediatric patients, the average age and sex-adjusted bmi was at the 47th percentile and only 3 had a bmi that was < 5th percentile. thirty-four/50 patients reported seeing an immunologist regularly. in the adult group, 38% reported no longer seeing an immunologist. the health conditions reported were similar to those common in the general population, and included rashes, warts and mouth ulcers. most reported these were transient, self-resolving issues. thirteen of 50 (26%) reported having adhd, higher than nih reported rates which estimate adhd in 8.1% of adults and 11% of children). ten of 50 (20%) reported having anxiety, similar to the nih reported prevalence of 19.1% in the general population. 34/50 (~68%) reported having no active concerns about their health. conclusions: overall, our findings are consistent with those in the last update done by railey et al, j. peds. 155: [834] [835] [836] [837] [838] [839] [840] 2009 in this population. patients are doing well with most problems similar to those common in the general population. most have a healthy bmi. adhd had a higher prevalence than in the general population. more than 1/3 of scid patients are not seeing an immunologist regularly, and a majority do not have any active concerns. . genetic causes of scid in the 50 patients whose questionnaire data are presented. x-linked scid was the most common, followed by ada and il-7r deficient scid clinical fellow/national institute of allergy and infectious diseases (niaid/nih) 2 research nurse/nih-nhgri 3 professor of pediatrics and allergy and immunology/ann & robert h. lurie children's hospital of chicago 4 chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih abstract/case report text rationale: myopathy has been occasionally documented in patients with primary immunodeficiency (pid). however, data on frequency and patient characteristics associated with myopathy are lacking. we performed a descriptive analysis of patients with primary immunodeficiency (pid) in the usidnet having myopathy as a feature of their primary disease. methods: the usidnet registry was queried for the spectrum of myopathic disorders in pid patients that had been entered into the registry as of november 26, 2019. results: a total of 63 pid patients with myopathy were identified, 36 of which (57.1%) were female. median age at onset of symptoms related to pid was 4 years (range 0.1-72 years, iqr 0.5-25 years). median age of diagnosis of pid was 8.3 years (range 0.4-77 years, iqr 3-32.5 years). age at onset of myopathic disorders was not known. twenty-eight (44.4%) patients had a diagnosis of common variable immunodeficiency (cvid), 6 (9.5%) had agammaglobulinemia, 3 patients each (4.8%) were diagnosed with severe combined immunodeficiency, hypogammaglobulinemia, or 'hlh and pigmentary disorders', while 2 patients (3.2%)were reported in each category of combined immunodeficiency (cid), autoimmune lymphoproliferative syndrome (alps), and autoinflammatory disease. thirty-five patients (55.6%) had a causative gene variant identified attributable to pid. the most common variant identified was btk (6 patients) followed by aire, lyst, cybb (3 patients each) and pi3kcd (2 patients). eighteen individual patients had other variants identified ( figure 1 ). 15 patients had cellulitis or skin/ subcutaneous tissue infection, 7 patients had a 'skin or subcutaneous tissue abscess', 2 had pyoderma gangrenosum, and 1 patient with eczema herpeticum. within this cohort of 63 patients, the most common myopathy listed was myositis (18) followed by 'muscle weakness' (11), dermatomyositis (8) , myalgia/s (6) , myalgia/myositis (4), myopathy (4), polymyositis (2) , steroid-induced myopathy (2) . no patient had an infectious myositis or muscle abscess listed. eighteen patients (28.6%) had a myopathic disorder at the time of diagnosis of their pid. thirtyone patients (49.2%) received prednisone, 11 (17.5%) received hydrocortisone and 2 (3.2%) received dexamethasone. two patients had a diagnosis of adrenal insufficiency. nine patients (14.3%) underwent hematopoietic stem cell transplantation. nine patients (14.3%) died; median age of death 16 years (range 0.4-40.6 years, iqr 3.6-21.1 years). ). one patient with chronic granulomatous disease had myopathy due to duchenne muscular dystrophy, which was listed as a cause of death. no other myopathic disorders were listed as a cause of death for the other patients. conclusion: myopathy and inflammatory myopathic disorders occur at relatively high frequency in pid, and may be present even at the onset of clinical symptoms. the underlying etiology can be speculated to be multifactorial. further subgroup analysis is warranted to elucidate possible variant-specific or treatment-associated characteristics of myopathy in pid. laboratory studies at 4 years revealed normal igg/iga/igm but markedly elevated serum ige (42,550 ku/l), anemia (hb 8.7 g/dl), thrombocytopenia (78 x109/l) and lymphopenia (520 cells/l), with low t and b cell counts, very low proportion of naïve t cells, skewed repertoire of cd8+ t cells, undetectable trec levels, and impaired t cell proliferation to mitogens and antigens. there was an elevated percentage of circulating plasmablasts (7.4%) and of dysreactive cd21low cd38low b cells (53.8%). at the age of 5, hsct with reduced intensity conditioning was performed from her phenotypically hla-matched father, with improvement of t and b cell count and function. whole exome sequencing (wes) identified a homozygous missense variant in the mannosidase alpha class 2b member 2 (man2b2) gene (p.asp38asn), that segregates with disease in the pedigree. the man2b2 asp38 residue is evolutionary conserved. the p.asp38asn allele has a minor allele frequency of 0.0002687 in gnomad, with no homozygotes. the cadd score for this variant is 28.300, significantly higher than the mutation significance cutoff score (3.313). man2b2 is involved in the lysosomal degradation of glycoproteins and demannosylation of free n-glycans. in particular, man2b2 cleaves man2glcnac1 to generate man1glcnac1. serum n-glycan profiling revealed elevated man5/man6 and man5/ man9 in the patient. n-linked and free glycan profiling by mass spectrometry (ms) showed accumulation of man2glcnac2, man2glcnac1 and man3glcnac1glycans in patient fibroblasts as compared to control cells, consistent with defective lysosomal glycoprotein degradation. lentiviral transduction of wild-type man2b2 into patient fibroblasts led to normalization of the n-linked glycan profile, with reduction of man2glcnac2 from 8.4 to 0.9 times control levels, and of man2glcnac1 from 26.9 to 1.5 control levels, indicating rescue of the impaired deglycosylation ( figure 1a ). western-blotting demonstrated defective n-glycosylation of lamp2 and icam1 proteins in patient fibroblasts ( figure 1b) , which were corrected upon lentiviral transduction of wild-type man2b2 ( figure 1c ). overall, our results indicate that loss of man2b2 enzymatic activity leads to dysregulation of deglycosylation and abnormal mannosylation of glycans. in conclusion, we have demonstrated that man2b2 deficiency accounts for a novel autosomal recessive cdg with prominent features of immune deficiency and immune dysregulation. abstract/case report text heme oxygenase-1 (hmox1) is a rate-limiting enzyme that catalyzes the degradation of heme to carbon monoxide, ferrous iron, and biliverdin, which becomes bilirubin. these byproducts are implicated in inflammation, cell homeostasis, and antioxidant defense(1). hmox1-deficiency is an extremely rare autosomal recessive disorder with a complex presentation of a wide spectrum of symptoms, including hemolytic anemia and hyperinflammation, requiring genetic testing for confirmed diagnosis (2) . we report the fifth known case of hmox1-deficiency (3) (4) (5) , a boy who presented at 4 years of age with aspects of the characteristic phenotype, but also had early onset asplenia, interstitial lung disease, and previously undocumented immune deficiency. patient's presentation was notable for hyperinflammatory exacerbations triggered by viral and bacterial infections as well as vaccinations. episodic flares occurred every few months lasting weeks to months with fevers of 102-104f, hypoxia, leukocytosis above 40,000/mm3, hemolytic anemia with negative coombs, thrombocytosis exceeding 1 million/ mm3, transaminitis, hemoglobinuria, hyperferritinemia to 4,000ng/ml, and elevated ldh to 28,000iu/l. immune evaluation revealed normal immunoglobulin levels and adequate vaccine titers to both protein and carbohydrate antigens. although class switched populations were normal, b-cell phenotyping showed absent immature and transitional b-cells, low mature memory, and reduced cd27+ memory b-cells at 6% (normal >8%). mitogen stimulation with phytohemagglutinin and anti-cd3 were decreased (24.7% of control and 21.5% of control, respectively). t-cell phenotyping demonstrated cd4 population heavily skewed to immaturity with 65% of cells with naïve phenotype cd45ra+cd27+ccr7+. there were few effector-memory t cells and the cd8 population was skewed towards immaturity with >65% of the cells naïve. liver biopsy was performed secondary to hepatomegaly yielding mild to moderate sinusoidal fibrosis. bone marrow biopsy revealed a normocellular marrow with 3% blasts, increased megakaryocytes, and extensive hemophagocytosis. natural killer cell function was very low, while soluble il-2ra level was normal. further workup for hemoglobinopathies, metabolic defects, congenital disorders of glycosylation, lysosomal storage disorders, wilson's disease, autoimmune hepatitis, inherited and autoimmune hypercoagulability disorders, connective tissue disorders, myositis, and myopathies were all unremarkable. imaging demonstrated asplenia and howell-jolly bodies were present. hemophagocytic lymphohistiocytosis (hlh) genetic testing showed no variants. he was suspected to have systemic juvenile idiopathic arthritis (sojia) with episodes of macrophage activation syndrome. the frequency of his autoinflammatory flares increased such that he was corticosteroid dependent by age 9, having failed methotrexate, azathioprine, and anakinra. he was started on tocilizumab with laboratory improvements, but his lung disease progressed and became oxygen dependent. lung biopsy confirmed nonspecific interstitial pneumonitis (nsip) with cholesterol granulomas also seen in sojia. ultimately, chronic lung disease led to his death at age 10. whole exome sequencing yielded a paternal frame shift hmox1 and maternal splice donor hmox1 resulting in absence of protein. bone marrow transplantation (bmt) in hmox1 deficient mice have rectified phagocytotic defects and thereby their autoinflammatory phenotype, but no human reports for bmt treatment of hmox1-deficiency has been described. here we describe a phenotype expansion for hmox1deficiency to include not only asplenia and hepatomegaly, but also interstitial lung disease with cholesterol granulomas and inflammatory flares. abstract/case report text introduction: mutations in the gene encoding signal transducer and activator of transcription 3 (stat3) cause autosomal dominant hyperimmunoglobulin e syndrome (ad-hies) characterized by recurrent skin and sinopulmonary infections, atopic dermatitis, and elevated serum immunoglobulin e (ige) levels. treatment is largely aimed at controlling symptoms and preventing infections with no standard of care. there is a paucity of literature describing the utilization of biologic therapies in the ad-hies patient population. we present 3 patients from one family with ad-hies successfully treated with monoclonal antibody therapies targeted at il-5, il-4 and il-13. case descriptions: patient 1: 13-year-old female with stat3 lof c.1003c>t (p.arg335trp) with a history of atopic dermatitis and asthma requiring 2-5 steroid courses per year with frequent school absences. she developed a severe pruritic rash covering her upper body 18 months ago that failed to respond to antihistamines and topical antibiotics prescribed by her primary care provider. given her poorly controlled asthma and our concern for a follicular type morphologic variant of atopic dermatitis, dupilumab was initiated. her scoring atopic dermatitis (scorad) prior to initiation of biologic therapy was 53.8 and improved to 4.5 following 12 doses (24 weeks) of dupilumab with clear dramatic improvement in her skin and quality of life ( figure 1 ). she also reports decreased asthma severity with no steroid courses, reduced albuterol usage, and significant decline in school absences since initiation of dupilumab. patient 2: 17-year-old female with stat3 lof c.1003c>t (p.arg335trp) who is the sister of patient 1. she has a history of severe asthma requiring frequent emergency department visits, hospitalizations, and 4-5 steroid courses per year despite therapy with high-dose fluticasone-salmeterol. spirometry prior to april 2017 demonstrated an obstructive pattern with an fev1 ranging from 51-64%. she was initiated on mepolizumab in april 2017. subsequent spirometry demonstrates an fev1 average of 115% with a range of 96-126%. she had one hospitalization in early 2018 but otherwise no hospitalizations for asthma since initiation of biologic therapy. patient 3: 15-year-old female with stat3 lof c.1003c>t (p.arg335trp) who is the paternal first cousin of patients 1&2. she has a history of severe atopic dermatitis with associated pruritus and picking behaviors, poorly controlled despite daily triamcinolone application. she previously failed ultraviolet therapy and crisaborole. she also has a history of severe asthma requiring 2-5 steroid courses per year despite high-dose fluticasone-salmeterol. dupilumab was started in may 2019. scorad prior to initiation monoclonal antibody therapy was 80.3 and declined to 21.2 following 13 weeks (7 doses) of dupilumab therapy with marked improvement in skin appearance and pruritus ( figure 2 ). discussion: we present three cases of ad-hies caused by stat3 loss-of-function mutations treated successfully with monoclonal antibody therapies targeted at il-5 or il-4 and il-13. to the best of our knowledge, there is no published data describing the use of these biologic agents in the treatment of ad-hies. future studies are needed to clarify the role of these cytokines in the pathogenesis of ad-hies and to elucidate clinical indications for biologic therapy in this patient population. informed consent was obtained from all individual participants included in the study. abstract/case report text ctla-4 is a potent inhibitor of t cell proliferation that competes with costimulatory receptor cd28 for its ligands cd80 and cd86 expressed on antigen presenting cells. heterozygous loss-offunction mutations in ctla-4 have been identified in patients with lymphocytic infiltration of multiple nonlymphoid organs (lo et al). the patient is a 2-year-old jordanian male born at term to nonconsanguineous parents, hospitalized at 1 mo for lll pneumonia, and at 2 mo and at 4 mo he was evaluated in the ed and diagnosed with non rsv-bronchiolitis with lll infiltrate thought to be secondary to atelectasis. at 2 yo he developed lll pneumonia and respiratory failure requiring picu admission. he was treated with ceftriaxone. after discharge, he had 6 weeks of intermittent fever, progressive fatigue, productive cough, and ftt. he received courses of cefdinir, clindamycin, and tmp-smx without improvement. chest ct revealed left lung consolidation, lll bronchiectasis, lul tree in bud opacities, and hilar lymphadenopathy. bronchoscopy with bal revealed no bacterial growth and no acid-fast bacilli. 16srrna ngs was positive for h. influenzae. lung biopsy demonstrated acute and chronic bronchiolitis with bronchiolitis obliterans and intraluminal polyps, with lymphocytic infiltration involving the bronchi and bronchioles. the lung parenchyma showed airspace filling with foamy macrophages and chronic interstitial inflammation. acid fast and fungal stains were negative. he was treated with systemic steroids for bronchiolitis obliterans with noted improvement. the severity of lung disease at such an early age prompted an immune evaluation. sweat test, anca, anti-pr3 and hiv were negative. total immunoglobulins were normal for age, and titers to s. pneumoniae, diphtheria and tetanus were protective. lymphocyte enumeration revealed elevated t and nk cell numbers for age. lymphocyte proliferation to pha, pwm, candida and tetanus were normal. dihydrorhodamine assay was normal. b cell phenotyping was normal. there was normal expression of cd69, hla-dr and cd25 on activated t cells; of note the patient was on systemic steroids when tested. invitae 207 gene pidd panel revealed a variant in ctla4: c.326g>a (p.gly109glu) that has been shown to be pathogenic in one patient (schawb et al). flow cytometry showed normal frequency of t follicular helper cells and t regulatory cells compared with controls, however ctla-4 expression by t regulatory cells was lower than control. due to the severe and progressive nature of the patient's lung disease, therapy with 3x weekly azithromycin and abatacept 50mg sq weekly was initiated. we report a case of ctla-4 haploinsufficiency presenting with recurrent pneumonia and bronchiolitis obliterans in a 2-year-old child. based on patient registry data, our patient appears to be the youngest child diagnosed with ctla-4 haploinsufficiency reported in the literature to date (schawb et al). notably, our patient l a c k s o t h e r f e a t u r e s c o m m o n l y d e s c r i b e d i n c t l a -4 haploinsufficiency, including autoimmune cytopenias, gastrointestinal disease, lymphoproliferation, and hypogammaglobulinemia. this case illustrates the importance of consideration of this diagnosis in young children with severe lung disease without other evidence of immune dysregulation. our hope is that prompt recognition and early treatment administration will prevent disease progression and further decrease in pulmonary function. splenectomy, he had an episode of pneumococcal meningitis at age 35, and sepsis of unknown origin at age 55. over the past several years he has developed chronic tinea corporis, onychomycosis, and otitis externa infections despite numerous antimicrobial regimens. at age 47, the patient developed urinary retention, walking, and balance difficulties. he was found to have diffuse white matter changes on mri, elevated wbc, and positive oligoclonal bands. initially, he was diagnosed as progressive ms treated with steroids with partial improvement. csf microbiology studies including afb stains, bacterial, fungal, mycobacterial cultures, cryptococcal antigen, vdrl, t. pallidum particle agglutination (tppa), as well as, pcr for cmv, ebv, vzv, enterovirus, hsv 1-2, jc virus and t. pallidum were all negative. peripheral blood studies included mycobacterial blood culture, pcr for cmv, ebv, hhv-6, in addition to serology for cryptococcal antigen, and coccidioides species, all of which were negative. additional neurological complications include granulomatous uveitis and oscillopsia, which he developed around age 47. immune evaluation performed at age 51 revealed low igg2 and igm, and the patient was started on 30 grams of monthly ivig. cbc with differential was notable for normal monocyte count and thrombocytopenia, mild neutropenia (table1). immunophenotyping revealed absent b cells and nk cells, while the cd8 t cells were elevated. cd4 t cells were normal (table 1) . at age 55, whole-exome sequencing identified a heterozygous missense mutation in gata2 c.1186c>t, p.(arg396trp). after the diagnosis of gata2 haploinsufficiency, he was found to have myelodysplastic syndrome with multilineage dysplasia (mds-mld) on bone marrow biopsy. he is currently awaiting bone marrow transplant discussion: we present a 55-year-old male with cytopenias, splenomegaly, leukoencephalomyelopathy, granulomatous uveitis, and recurrent fungal infections found to have a pathogenic heterozygous missense mutation in gata 2. leukoencephalomyelopathy in gata 2 haploinsufficiency has been associated with jc virus and ebv infection. our patient did not have any evidence of a chronic csf infection. to our knowledge, myelopathies have not been reported with gata2 c.1186c>t, p.(arg396trp). this case highlights the variable nature of presentation in gata 2 haploinsufficiency, and the need for clinical awareness of this entity in order to facilitate early diagnosis and appropriate therapy. immunoglobulins* white blood cells 4.3 x10*9 /l (n: 4-11) magnetic resonance imaging (mri) of the brain and spine showed numerous enhancing parenchymal nodules (figure one). brain or spinal biopsy was requested, but not recommended by our neurosurgery service. lumbar puncture evaluation was performed. cerebral spinal fluid showed no bacterial or fungal elements. both quantiferon gold for tuberculosis and three consecutive sputum cultures for acid fast bacilli were negative. he continued his sirolimus and the intravenous immunoglobulin replacement was increased to two grams/kg. the patient was cleared from respiratory isolation and discharged after two weeks in our facility with mild improvement in his neurologic status. within five days he was sent to a nationally renowned hospital. at this facility, extensive evaluation for his neurologic deficits were performed including culture and pcr for bacteria, virus, mycobacteria from csf bone marrow, lymph node, blood, and induced sputum. these were noncontributory. he was given high dose corticosteroids for two days. one of our facility's sputum cultures was reported with acid fast bacilli. but sputum mycobacterium tuberculosis pcr was negative. repeat mri scans of the brain and spine showed improvement (figure 2), so no brain biopsy was performed. he was sent back to our facility with the recommendation to start a targeted pi3kinase inhibitor on compassionate grounds as he was not eligible for the clinical trial because his weight was less than 45 kg. but pretreatment abdominal ct revealed multiple low-density lesions scattered throughout the liver (figure 3) not previously seen on prior noncontrast ct four months prior. discussion: although this gain in function mutation of the pi3kδ signaling pathway disorder has been well characterized, this is a rare report of a patient with pasli immunodeficiency with central nervous system and later liver lesions pet imaging showed subcarinal, mediastinal, retroperitoneal lymphadenopathy, splenic enlargement to 23cm and bilateral lung nodules ( figure 1 ). excisional biopsies of left axillary and left lower lobe of lung were performed and showed low-grade b-cell lymphoma (mucosal) and underlying lymphoproliferative disease. invitae alps and cvid panels (37 genes) revealed a heterozygous variant of unknown significance in exon 3 of fas (c.323a>g(p.asp108gly) unlike most fas mutations causing alps, this mutation is in the extracellular region rather than the death domain2. the c.323a>g variant has been reported in a single patient with alps phenotype, affecting fas protein function by inhibiting binding to fas ligand (fas-l), reducing fas-l induced apoptosis2. this fas c.323a>g mutation was found in alps affected brother and was absent in the unaffected father. the patient's mother passed away prior to testing. since diagnosis, the patient's malt lymphoma has been treated with rituximab weekly for the first month and then monthly. he continues to receive monthly ivig for hypogammaglobulinemia. after two years, ct demonstrates a significant decrease in pulmonary nodules and splenomegaly ( figure 2 ). the patient's forced vital capacity (fvc) improved from 3.78l (67% predicted) to 4.39l (79% predicted). conclusion: we report the first case of malt lymphoma seen in a patient with alps. it is unknown whether the unique fas c.323a>g mutation in the non-death domain contributes to malt lymphoma progression. we propose malt lymphoma is a malignant transformation of chronic inflammation that has the potential to occur in patients with alps. in the future, improved knowledge of mechanistic pathways of inflammation in lymphoma development and progression is important in the optimal management of alps. abstract/case report text background wiskott-aldrich syndrome (was) is a rare x-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, infections, autoimmunity and increased risk of hematological malignancies. gene therapy (gt) using autologous cd34+ cells is an emerging alternative treatment with possible advantages over standard allogeneic hematopoietic stem cell transplant. we report the outcomes of a phase i/ii clinical trial in which 5 was patients underwent gt using a self-inactivating lentiviral (sin-lv) vector expressing the human was cdna under the control of a 1.6kb fragment of the human was promoter. subjects and methods: five patients with severe was (clinical score 3-5) were enrolled (table 1) . cd34+ cells were transduced ex-vivo and reinfused after conditioning with busulfan and fludarabine. two subjects (p4, p5) had autoimmunity pre-gt, manifested as skin vasculitis and autoimmune cytopenias. results: all subjects were alive at median follow-up of 5.1 (range 2.8-6.3) years. multi-lineage vector gene marking was sustained over time. all had clinical improvement of eczema, infections and bleeding diathesis. was protein (wasp) expression was increased over baseline but remained below normal levels. proliferation of t cells in response to anti-cd3 improved post-gt. humoral immune deficiency improved, with normalization of igm, and independence from ig replacement and vaccine responses in those tested. platelet levels increased to >50 x 103 cells/ul in only the two subjects with a vcn ≥2 in transduced stem cells. podosome formation in monocyte-derived dendritic cells was near absent pre-gt and improved in all subjects post-gt, but only reached healthy control levels in the 2 subjects with highest vcn. in contrast to other trials using this sin-lv, two patients (p4 and p5) had flares of autoimmunity post-gt, offering the opportunity to study the poorly understood mechanistic features of immune dysregulation in this disease. selfreactive vh4-34-expressing b cells and cd21lo b cells remained elevated in most patients. however, despite wasp expression in foxp3+ tregs, those with autoimmunity had poor numerical recovery of t cells and tregs at the time of clinical symptoms ( fig 1a) . in addition, il-10 producing regulatory b cells (bregs) were highly deficient pre-gt, recovered in subjects who did not experience autoimmunity, but failed to recover in p4 and p5 ( fig 1b) . moreover, transitional b cells, which are enriched in bregs and are potent inducers of treg populations, also recovered poorly in those two subjects ( fig 1c) . there have been neither severe gt-related adverse events nor abnormal clonal expansion in transgene-marked cells to date. conclusion in summary, our data confirm and extend the safety and efficacy of gt in correcting disease manifestations associated with was, with the longest overall follow-up reported so far in studies using sin-lv. in addition, our findings suggest that higher vcn is needed in order to correct myeloid compartments such as platelets and monocytes. finally, we report the novel finding of the restoration of bregs and suggest that recovery of this compartment, along with tregs, is protective against development of autoimmunity post-gt. overall, these data suggest a mechanism for breakdown of immune tolerance in was with important therapeutic implications and prognostic value. this is an 8-year-old hispanic female who initially presented with failure to thrive, recurrent fevers and intermittent cough with episodes of perioral cyanosis. symptoms started at age 6 months and were attributed to recurrent viral and bacterial infections. at 14 months old, she was hospitalized with fever and hypoxemia (o2 saturations 70%). cxr showed prominent interstitial lung markings and she was diagnosed with pneumonia. ct scan confirmed cxr findings and ruled out anatomical anomaly. she was lost to follow up for 2 years, and re-presented with worsening respiratory status. a repeat ct scan demonstrated worsening interstitial thickening. immune workup, including quantitative immunoglobulins, ch50, lymphocyte subsets and vaccine response titers (pneumococcal and tetanus), was unremarkable, except for elevated igg levels. genetic testing for surfactant dysfunction mutations was negative. thoracoscopic lung biopsy revealed interstitial fibrosis, pas-positive granular alveolar proteinosis, type ii cell hyperplasia, and lymphoid follicles. at age 6, she was admitted for a pericardial effusion. rheumatology was consulted for evaluation frequent fevers and persistently elevated inflammatory markers, with concern that the pericarditis was autoinflammatory. she had an elevated ana (>1:2560 homogeneous pattern), il-6 (378.88 pg/ml), and igg (2020 mg/dl) at that time. she had an atypical anca pattern with positive myeloperoxidase antibodies. anti dsdna, smith and scl70 were negative. she was treated with steroids and hydroxychloroquine with some improvement in her oxygen requirement. one year later she had an additional episode of pericarditis, treated with colchicine. a few months later, she was admitted with newonset gross hematuria and elevated serum creatinine (to 2 mg/ dl). kidney biopsy showed anca vasculitis with glomerulonephritis (75% crescents, no scarring or fibrosis). she provisionally received a diagnosis of microscopic polyangiitis, with lung and kidney involvement. she did not have peripheral vasculopathy. she was started on cyclophosphamide, rituximab, and iv steroid pulses. cyclophosphamide was discontinued due to recurrent episodes of posterior reversible encephalopathy syndrome (pres) after infusion. her igg level decreased as she developed nephrotic range proteinuria. a primary immunodeficiency genetic panel was sent to evaluate for monogenic immune dysregulation syndromes and revealed a tmem173 gene mutation (c.463g>a) which has previously been reported in 4 other subjects with savi (stingassociated vasculopathy of infancy syndrome). sting is a cytosolic dna sensor that leads to type i interferon production upon stimulation. this gain-of-function mutation was confirmed by measuring interferon signature gene expression at the nih (fig 1) , and her diagnosis was revised accordingly. the patient was started on a jak-inhibitor (tofacitinib) to block interferon signaling. unfortunately, the patient is now deceased, due to overwhelming infection and multi-organ system failure. conclusion: genetic testing can be crucial in aiding the diagnosis of complex patients with immune dysregulation and can provide an opportunity for targeted therapy, which should be employed as soon as able to stop disease progression. abstract/case report text background: activated phosphoinositide 3-kinase δ syndrome (apds-1) was first described in 2013 as a monogenetic immune dysregulation syndrome with a variable phenotype. increased sinopulmonary and herpesvirus infections are well described, but fungal infections such as candidiasis have been rare. to date, disseminated histoplasmosis has not been described. history: a 38 yo caucasian male who was previously diagnosed with common variable immunodeficiency (cvid) in late childhood due to recurrent sinopulmonary infections presented with recurrent fever, pancytopenia, severe splenomegaly, and lymphadenopathy. urine histoplasmosis antigen and beta-d-glucan were elevated. a bone marrow biopsy demonstrated granulomatous inflammation. transbronchial biopsy of a subcarinal lymph node was consistent with granulomatous disease. this led to a diagnosis of disseminated histoplasmosis. he was treated with amphotericin b and then 11 months of itraconazole, with improvement of his symptoms. he was admitted to the hospital about 7 years later when he presented with fatigue, fever, chills, dark urine, and scleral icterus. he was found to have an acute worsening of chronic anemia with a hemoglobin of 4.7 g/dl. due to elevated ldh, presence of schistocytes on peripheral smear, and undetectable haptoglobin, he was diagnosed with autoimmune hemolytic anemia, despite a negative direct coombs. a bone marrow biopsy specimen was hypercellular with marked erythroid predominance, with normal flow cytometry and no blasts identified. infectious workup was negative. ct chest during the workup revealed new right hilar and mediastinal lymphadenopathy, in addition to calcified right hilar and subcarinal lymph nodes, bronchiectasis, and stable hepatosplenomegaly. transbronchial biopsy of lymph nodes showed benign lymph nodes with calcified necrotizing granulomata and presence of non-viable fungal species, presumably "old" histoplasmosis. family history: family history was significant for mom dying at 29 years-old from undefined cns infection. immune labs: · panlymphocytopenia: absolute lymphocyte count of 380/ul, cd3+ t cells 283/ul, cd4+ 174/ul, cd8+ 99/ul, cd19+ 65/ul, cd16+cd56+ 25/ul. cd4+/cd8+ ratio 1.8 · decreased class-switched memory b cells and plasmablasts · elevated t central memory cells and activated (hla-dr+) cd4+ and cd8+ t cells · hemoglobin 10.3 g/dl, platelets 77,000/ul, anc ranging from 360/ul to 1610/ul · iga 107 mg/dl, igm 273 mg/dl; reportedly had low igg prior to initiating ivig in childhood · ebv pcr and cmv pcr negative genetics: · pik3cd (c.3061g>a), consistent with diagnosis of autosomal dominant apds-1. discussion: gain-of-function variants leading to increased pi3kδ activity have been shown to cause both b and t cell dysfunction, leading to impaired immunologic responses to bacterial and viral infections. recurrent sinopulmonary infections and herpesvirus infections are commonly seen and while mucocutaneous candidiasis has been reported in cohorts of patients with pik3cd, other fungal infections are not common. severe disseminated histoplasmosis infections have been described in primary immunodeficiencies characterized by signaling defects in the il-12/ifn-γ pathway, stat3 deficiency, cd40l deficiency, gata2 deficiency and in stat1 gain-of-function mutations. to our knowledge, disseminated histoplasmosis has not been previously reported in patients with pik3cd immunodeficiency. abstract/case report text the reported case represents the first case of nbas disease detected by newborn screening program for primary immunodeficiency, based on krec assay. the patient came to our attention due to the complete absence of krecs and normal trecs on dbs (dried blood spot) while hospitalized for low weight at birth (1,520 g), intolerance for enteral feeding, hepatosplenomegaly, slightly elevated liver transaminase, head and face eczematous dermatitis. during the 1st month, he also presented klebsiella pneumoniae urinary tract infection and methicillin-resistant staphylococcus aureus sepsis. peculiar phenotypic features including triangular face, proptosis, flat philtrum, mild retrognatia, hirsutism, loose and slightly wrinkled skin, and apparent reduction of subcutaneous fat were noticed at birth. complete blood count showed lymphocytopenia, marked hypereosinophilia. serum immunoglobulin g (igg) were markedly decreased, iga and igm were undetectable. extended immune-phenotyping showed complete absence of cd19+ cells, low count of cd8+ lymphocytes, and reduced natural killer (nk) levels. at 9month of age a colonoscopy was carried out for persistent diarrhea and reduced tolerance to enteral feeding. the histological examination of mucosal intestinal biopsies showed signs compatible with autoimmune enteropathy. for this reason immunosuppressive therapy with rapamycin was started without consistent clinical amelioration. many cvc-sepsis occurred in the last months, associated with persistent gastrointestinal symptoms and severe growth restriction. despite the absence of experience data in literature for nbas syndrome, we retain that hsct represents the only resolutive therapy for him. abstract/case report text case: a 31-year-old female with asthma and allergies presented to immunology clinic with a history of chronic fatigue and sinusitis. fatigue occurred daily every 2-3 weeks and was described as not feeling rested even after 12 hours of sleep. chronic sinusitis required 4-5 prolonged antibiotic courses per year. nasal cultures grew methicillin-sensitive and -resistant staphylococcus aureus and haemophilus influenzae type b (hib). three separate sinus surgeries over the prior few years reduced her sinus symptoms. other infectious history was significant for recurrent urinary tract infections with e. coli and klebsiella, recurrent otitis media as a child, and a diagnosis of transient hypogammaglobulinemia of infancy that resolved at 4 years of age. review of systems revealed axillary lymphadenopathy for 2-3 days twice per year not related to infection. she had longstanding eczema that responded to topical tacrolimus, multiple environmental allergies, and recently diagnosed asthma that improved with inhaled budesonide/formoterol. as a teenager she received allergy immunotherapy for a few years but stopped due to frequent adverse reactions. family history revealed that father died from cancer. physical exam was unremarkable. laboratory evaluation demonstrated normal igg 874 mg/dl, igm 133 mg/dl, iga 166 mg/dl, elevated ige 457 mg/dl, normal t and nk cell enumeration, mildly low total b cells (180 cells/mcl, 5.6%), and normal b cell subsets (cd19+igm+cd27-67%, cd19+igm+cd27+ 17%, cd19+igm-cd27+ 13%). tetanus antibody titer was protective, but hib antibody titer was undetectable at < 0.15 mcg/ml with marginal response after vaccination (0.53 mcg/ml). pneumococcal serotype specific igg levels (mayo) were mostly undetectable with 3 of 23 serotypes protective at baseline and only 4 protective post vaccination with pneumovax 23. cd3/ cd28 blastogenesis was poor. due to poor antibody response and continued sinus infections she was started on igg replacement. her fatigue and sinus symptoms improved moderately but she continued to require antibiotics and sinus ct scans continued to demonstrate significant disease. a focused exome sequencing panel was pursued and a novel heterozygous card11 variant was found (c.215g>t, p.r72l). discussion: the card11/bcl10/malt1 (cbm) complex is a critical signaling adapter that facilitates several downstream immune responses predominately through nf-kb. mutations in several different domains of card11 result in a clinical entity collectively referred to as card11-associated atopy with dominant interference of nf-kb signaling (cadins). cadins is associated with a broad range of clinical manifestations but most have marked atopy with infections, poor t cell proliferation, and varying levels of poor antibody response. both our variant (p.r72l) and a previously reported pathogenic variant in the same amino acid (p.r72g) involve a change from a charged arginine to a non-polar amino acid in the critical bcl10/ card11 binding interface. iκbα degradation was not present in b cells from our patient ( figure 1 ) confirming the functional defect in nf-kb signaling. thus, we present a novel variant that fits cadins both clinically and genetically. clinicians should be aware of cadins when patients present with recurrent infections in the setting of significant allergic disease. i b degradation assay. stimulation: 200 μl of whole blood was stimulated in a 5 ml facs tube with 50 ng/ml phorbol 12-myristate 13-acetate (pma; sigma, cat# p1585) at 37°c for 5, 10, or 20 min, at which point 4 ml of pre-warmed 1x lyse/fix buffer (bd, cat# 558049) was added. cells were fixed for 10 min at 37°c, centrifuged and washed twice with facs buffer (pbs supplemented with 2% fbs and 1 mm edta). staining and permeabilization: fc receptors were blocked for 5 min at rt (human trustain fcx; biolegend), followed by a 20 min stain on ice with anti-cd4 af647 (clone rpa-t4; biolegend), and anti-cd19 bv421 (clone hib19; biolegend). cells were washed with facs buffer and permeabilized for 30 min on ice with 1 ml phosflow perm buffer ii (bd biosciences, cat# 558052) that had been precooled to -20°c. after permeabilization, two ml facs buffer was added and the samples were centrifuged. after three additional washes, the cells were stained with anti-iкbα pe (clone 25/ikba/mad-3; bd biosciences) for 30 min at rt. samples were washed three times and data were collected on a cytek dxp10 flow cytometer. data were analyzed with flowjo software. abstract/case report text introduction: wiskott-aldrich syndrome (was) is a rare, but well-defined x-linked disorder. loss-of-function mutations in the was gene result in classic was and x-linked thrombocytopenia (xlt), while gainof-function mutations lead to x-linked neutropenia (xln). classic was phenotypic features include recurrent infections, microthrombocytopenia and eczema along with increased susceptibility to autoimmune disorders and malignancy. most males with classic was are diagnosed in early childhood and early death can result from its various clinical manifestations. case: we present a 32-year-old male who was referred to immunology for hypogammaglobulinemia. as an infant he had moderate eczema, and at the age of two was diagnosed with immune thrombocytopenia (itp) with baseline platelets of 50-60 x 10^9/l. infectious history was notable for one episode of pneumosepsis and recurrent otitis media, influenza, and herpes labialis infections. around the age of 22, he was diagnosed with common variable immunodeficiency (cvid) based on the finding of low immunoglobulins. he developed diffuse large b cell lymphoma at age 26, and was treated with cyclophosphamide, doxorubicin, vincristine, prednisone and rituximab (chop-r). at age 32 he developed abdominal pain with bloody stools. investigations confirmed an endoscopic and pathologic diagnosis of ulcerative colitis. due to the severity of his disease, he has required maintenance therapy with vedolizumab. he was again noted to have hypogammaglobulinemia at which point he was referred to immunology at our centre. there was no significant family history of immunodeficiency, malignancy or autoimmunity. blood work was notable for normal white blood cell and lymphocyte counts, platelets of 60 x 10^9 g/l, low igg at 4.82 g/l (7.0-16.0 g/l) with normal iga, igm and ige. lymphocyte subsets including t, b and nk cells were within the normal range. genetic testing was performed and he was found to have a known pathogenic mutation in the was gene (c.1453g>a, p.asp485asn) which has been previously reported in association with was and xlt. he has since been placed on immunoglobulin replacement and has been referred for consideration of hematopoietic stem cell transplantation. discussion: was is a rare syndrome that can have a similar phenotype to other immunodeficiency disorders including cvid, omenn syndrome and ipex (immune dysregulation, polyendocrinopathy, x-linked). individuals with cvid present with hypogammaglobulinemia and recurrent infections, and these individuals also have an increased susceptibility to autoimmune disorders, gastrointestinal disease and malignancies, especially lymphoma. although eczema is a common disorder, its presence in addition to features of early onset thrombocytopenia, immunodeficiency, autoimmunity and/or malignancy in male patients should heighten the suspicion for was. it is important to make the diagnosis of was as hematopoietic cell transplantation and gene therapy are potentially curative treatment options. abstract/case report text secondary immune deficiencies (sid) are caused by varied mechanisms and are common in patients with hematological malignancies such as chronic lymphocytic leukemia (cll) and multiple myeloma (mm). in this setting, both the disease and its treatment (such as b cell ablation therapy) contribute to the development of secondary antibody deficiency. infections remain a major cause of morbidity and mortality in cll and mm patients. this underscores the need for early recognition and stratification of risks in order to guide appropriate treatment, including immunoglobulin replacement therapy (igrt). new guidelines for the use of human normal immune globulins in sid patients were implemented by the european medicines agency (ema) in 2019. despite these new guidelines, significant variations remain across european countries in the assessment and approaches aiming to achieve reduction in infection burden, including different strategies for initiation, dosing and discontinuation of igrt. the same is true for north america where igrt is widely used off-label to prevent infections in patients with sid due to hematological disease or other reasons. in order to address this variability, a task force comprising both immunologists and hemato-oncologists drafted 20 statements aiming to test for consensus. statements were related to six major areas: definition of infections, measuring igg levels, initiating igrt, igrt dosing, scig usage and discontinuing igrt. this was followed by an international delphi consensus exercise in three rounds which aimed to develop recommendations on how to diagnose, treat and follow-up patients with antibody deficiency associated with hematological malignancies. the first delphi round consisted in testing the 20 statements with a panel of sid specialists and subsequently their comments were used by the task force to refine the statements. in the second delphi round, the refined statements were presented via phone interviews to the same panel to assess their level of agreement with each statement (ranging from 1 "i totally disagree" to 6 "i totally agree"). consensus was considered to be reached per statement if 70% of the experts agreed with each statement overall. the cut-off for overall agreement was 4 "i somewhat agree". if the expert chose level 4 or less the reasons underpinning his/her choice were discussed. consensus was achieved for all statements on level 4 ("i somewhat agree"). only 5 statements did not achieve consensus on level 5 "i mostly agree". in delphi round 3, panelists who had not "mostly agreed" with these five statements were given the opportunity to reconsider their assessment based on the feedback from other panelists, which was shared with them. the panelists then chose to maintain or refine their assessment. analysis of the full results on the six key areas identified by the task force will be presented at the conference to offer recommendations and help guide the management of sid in patients with hematological malignancies. abstract/case report text introduction: mast cells (mcs) are hematopoietic-derived immune cells, whose precursors migrate within tissues reaching maturation and differentiation. masitinib, a selective tyrosine kinase inhibitor, is efficient in controlling the survival, differentiation, and degranulation of mcs. aim: to optimize mast cell-differentiation from human bone marrow (bm) hematopoietic stem cells, and to find best cell culture conditions for proliferation, differentiation, and maintenance of mcs, which is important when studying particularly mcs' response to cytotoxic compounds. material-methods: to produce mcs in vitro, the first method (m1) we used was a modified semi-solid culture method (1). briefly; human bm mononuclear cells (mncs) were obtained with ficoll gradient from bm sample of a patient with idiopathic thrombocytopenic purpura. colonyforming unit (cfu)-mast was developed from mncs in methylcellulose medium supplemented with scf (200ng/ml) + il-6 (50ng/ml), and il-3 (1ng/ml; only first week). 5-6 weeks later mast cell colonies were transferred into suspension cultures, in which mcs matured and multiplied up to 7-8 weeks and were used in experiments till 10th week of culture. on the other hand, in our second method (m2); mncs were separated by ficoll, seeded in 6 well-plates with imdm containing fbs 2%, pen/ strep, and a little amount of methylcellulose, and incubated at 37 o c, 5%co 2 . cultures were then supplemented with imdm (fbs 1%) + scf (100ng/ml) + il-6 (50ng/ml) on day 4; and imdm (fbs 2%) + scf (100ng/ml) + il-6 (50ng/ml) + il-3 (1ng/ml) on day 9. beginning on day 18 till the end, imdm (fbs 2%) + scf (100ng/ml) + il-6 (50ng/ml) were added to cultures. for both methods, morphological assessment of colonies/cells were evaluated under an inverted microscope (figure 1 and 2). verification of mcs was performed by immunoflorescence staining for anti-tryptase andchymase antibodies, and by toluidine blue staining. macrophages were verified by anti-cd-68 immunoflorescence staining. mcs were exposed to masitinib or dmso for the evaluation of dose-related effects of masitinib, and cytotoxicity was evaluated by mtt assay. results: in m2, culture conditions were easier to handle compared to m1. in m2, high amounts of mcs in immature and pre-mature forms were appeared as early as 15-18 days, and peak levels of proliferation rate was around 2-4 weeks of culture, which was about 3 weeks earlier than m1. culture could be maintained till 10 weeks in both methods. although mcs are non-adherent cells, in liquid method adherent bm cells such as fibroblasts, endothelial cells and mesenchymal stem cells have adhered to the plate and grown up, providing an attachment site for mcs and serving as a natural bm nest, mimicking in-vivo environment, for mcs to grow and proliferate ( figure 2 ). attachment of mcs has provided medium exchange available without changing culture dishes. when mcs were exposed to masitinib (0.5, 1, and 2 μm/μl), approximate survival rates were 75%, 72%, 69%, respectively. discussion: in our liquid medium method, the adherent bm cells not only provided a natural nest supporting mc development and differentiation, they also served as an attachment site for mcs. as the cells slightly adhered, when trypsinized shortly, they easily detached and used for experiments. and we also report for the first time that adding a little amount of methylcellulose to the liquid medium provides ease of aggregation of cfus, and easy development of mcs. we suggest that our liquid culture may be superior to semi-solid method, that it is faster and easier to handle. in 2 studies subjects crossed over to subcutaneous (sc) igiv-c 10%, and in the third study crossover was to immune globulin sc (human), 20% caprylate/chromatography purified (igsc 20%). a total of 95 pi patients from these 3 studies were included in the poppk analysis and 1841 serum igg concentrations were included in the final pk analysis. the pk of igg following iv and sc administration was adequately described by a two-compartment model with first-order elimination from the central compartment. administration of igiv was modeled as an infusion directly into the central compartment. absorption of exogenous igg from the depot site of sc infusions into the central compartment was modeled as a first-order process with an absorption rate constant (ka). the full model was constructed by incorporation (forward selection process) of covariates of interest into the model. after completion of the covariate model development, the final model showed that igg pk was not influenced by (a) the igsc formulation used in the different studies (10% vs. 20%), (b) gender, and (c) age (pediatric vs. adult). body weight was identified as a significant covariate having an effect on clearance and volume of distribution. based on the final pk results, serum clearance of igg for the reference population was estimated to be 0.150 l/day. the volume of distribution of the central and peripheral compartments accounted for 3.06 l and 1.93 l, respectively. the intercompartmental clearance was 0.474 l/day, and the absorption constant from the depot (ka) was 0.246 day-1. the absolute bioavailability of igg after sc administration was calculated as 70.5%. the developed method was used to evaluate alternative dosing intervals following sc administration. the equivalent of a weekly igsc maintenance dose administered 1, 2, 3, 5, or 7 times per w e e k , o r b i w e e k l y p r o d u c e d o v e r l a p p i n g s t e a d y -s t a t e concentration-time profiles and similar area under the concentration versus time curve (auc), maximum concentration (cmax), and minimum concentration (cmin) values. the results of the evaluation and simulations for igg exposure following a switch from igiv-c 10% dosing (every 3-or 4-weeks) to sc dosing further suggest that a range of dose-adjustment factors (daf), from 1:1 to 1:1.37 would be sufficient to provide clinically effective trough igg concentrations throughout the course of treatment at various treatment frequencies. current us product labeling for igsc 20% specifies a daf of 1:1.37 for transitioning immune globulin dosing from iv to sc, and specifies igsc 20% dosing frequencies of weekly or more frequently (2-7 times per week). in this poppk analysis all sc dosing regimens evaluated theoretically would provide viable alternative administration options for maintaining adequate immunoprotection in pi patients with dosing flexibility over a range of regimens. however, in 50% or more, no causative gene can be found going down to undefined inflammatory syndromes (uis). anti-il1 drugs (ail1d) revolutionized some il-1 mediated diseases, such as traps, caps, hyper-igd/mkd and fmf. nevertheless, treatment response among disorders are not the same as well as no specific study was designed for uis. papa et al, 2019, recently suggested the use of anakinra for the treatment of uis, especially those refractory/intolerant to colchicine with severe or very symptomatic phenotype. this paper aims to retrospectively report for the first time the experience with canakinumab in monogenic and multifactorial disorders in a single, private center in brazil. patient and methods: patients's records that received canakinumab from january 2016 to december 2019 at clinica croce, ima-brazil, were revised. demographic and clinical data were extracted and descriptively described. all statistical analysis are presented as: average (minimal; maximum; standard deviation). results: a total of 23 patients with autoinflammatory diseases were enrolled and 65% (n=15) are female. of them, 65% (n=15) patients had a monogenic disease: 26% (n=6) caps, 21% (n=5 fmf), 4% (n=1) mkd, 4% (n=1) homozygous nlcr4 and 4% (n=1) pami syndrome. multifactorial disorders were 39% (n=9) patients : 8% (n=2) recurrent idiopatic pericarditis, 8% (n=2) schnitizler syndrome and 21% (n=5) uis. the average age of the first symptoms was 12,51 years (0;69;19,78) and the average age of diagnosis was 24,43 years (0;72;21,76) while the aveage of diagnosis delay was 11,59 years (1;59;15,16). all patients had used, prior to anti-il1, corticosteroids with 100% prevalence of cushing syndrome and 69% (n=16) tried at least one steroid sparing agent without clinical success due to: intolerance or non-effective disease control or side effects. in the fmf group (n=5) 100% tried colchicine prior to canakinumab and this drug was not effective to 60% because of amyloidosis status and in 40% colchicine-induced hepatitis was observed. canakinumab was effective for disease control in 75% (n=23) considering: control of clinical manifestations, amyloidosis reversion and normalization of acute reactants markers. the only side effect observed during the follow up were acute flu-like symptoms and psicomotor agitation (33,34% , n = 8). the average time of follow up is of 12,51 months (1;37;12,46). canakinumab could be discontinued in just one patient with uis. conclusions: this is the first report of canakinumab use for autoinflammatory disorders in brazil. canakinumab is an effective and safe drug for monogenic and multifactorial disorders control. no serious adverse effect could be observed in the 3 years maximum follow up of this drug. neither, no specific infectious disease more prevalent in south america, such as yellow fever, dengue, zika or chikungunya was observed. abstract/case report text a 21-year-old caucasian male with autosomal recessive hyper igm syndrome type 2 (higm2) due to aicda mutation, diagnosed at age 1, presented with a newly developed mediastinal mass. he receives routine ivig, pulmonary function tests (pft's) and chest x-rays. at age 16, patient was noted to have cervical and inguinal lymphadenopathy. ct scan indicated left mediastinal, hilar and pleural lymphadenopathy with soft tissue infiltration around the descending thoracic aorta and esophagus. biopsy indicated no evidence of a lymphoma or infection. 5 years after initial workup, routine pft's showed a declining diffusion capacity by 50%. patient complained of intermittent chest pain but displayed no clinical symptoms of cough, dyspnea, dysphagia or reflux. ct scan which revealed an extensive illdefined soft tissue mass extending from the thoracic outlet to the level of the esophageal hiatus that encased vascular structures resulting in narrowing and occlusion of left upper lobe pulmonary artery and left lower lobe pulmonary arteries respectively. imaging demonstrated homogenous ventilation to bilateral lungs and decreased perfusion in the left lung compared to the right lung. infectious workup was negative for atypical infections. biopsy revealed miced cellular infiltrate with no predominenant cell type or evidence of malignancy, consistent with previous lymph node biopsy 5 years prior. cd20 and cd3 stains revealed aggregates and scattered b-cells and t-cells, respectively. removal of mass was proposed but due to the ambiguous borders and location, surgical excision was not possible. patient was given 4 doses of rituximab (75g), 3 doses 10mg/kg pulse steroids 18 hours apart, and daily sirolimus (level was adjusted based on sirolimus level). follow-up ct scan indicated significant interval improvement with 50-60% reduction of the soft tissue mass. blood flow in the left lower lobe pulmonary artery has still not returned. this may be due to collaterals and may be a separate problem from compression due to the mass. cytotoxic t-lymphocyte antigen 4 (ctla-4) is an inhibitory immune regulator critical for governing t and b cell homeostasis. heterozygous ctla4 mutations can cause a syndrome of immune dysregulation with a variable clinical phenotype including hypogammaglobulinemia , autoimmune cytopenia and endocrinopathies, lymphoproliferation, predisposition to malignancy, tissue specific lymphocytic infiltration of brain, lung and gi tract as well as colitis. methods: we retrospectively reviewed medical records of all patients with ctla4 haploinsufficiency evaluated at the nih between 2014-2019. a pathologic variant in ctla4 was confirmed in all patients. we analyzed frequency of campylobacter species detected in the stool samples by pcr based biofilm rapid array as well as reflex bacterial stool and blood cultures when available. results: forty-six patients aged 8-75 years were evaluated at the nih between 2014 and 2019. six of 46 patients (13%) had at least one episode of campylobacter species associated acute or worsening diarrhea, with one patient also having campylobacter bacteremia. all patients with positive campylobacter species in stool samples had clinical histories and/or endoscopic biopsy findings consistent with enteropathy or colitis predating the incidence of campylobacter infection. two of the six patients (33%) had recurrent or chronic campylobacter infection, while four of the six patients (67%) had multiple gastrointestinal pathogens detected by stool pathogen screening at various times. conclusions: campylobacter species infection of the gastrointestinal tract seem to occur at an increased incidence in our ctla4 haploinsufficient cohort. to the best of our knowledge, this is the initial report for the association between ctla4 haploinsufficiency and campylobacter species infection of the gastrointestinal tract. although ctla-4 is a critical immune checkpoint involved in mucosal immune homeostasis and gut microbiota-immune system cross talk, the underlying mechanism predisposing to campylobacter infection in ctla-4 deficient patients remains to be explored. our study suggests screening of stool for campylobacter species in patients with ctla4 haploinsufficiency associated enteropathy. (62) submission id#806771 abstract/case report text ikaros transcription factor and ikaros family members are critical for development of lymphocytes and other blood cell lineages. full length ikaros (isoform 1) contains six c2h2 zinc fingers (zf), four nterminal dna binding zf and two c-terminal dimerization zf. somatic ikaros mutations and deletions have been associated with increased predisposition to b-acute lymphoblastic leukemia (all) as well with poor disease prognosis. recently, germline ikaros mutations affecting the n-terminal dna binding domain and acting in a haploinsufficiency or dominant negative manner were reported to be associated with common variable immunodeficiency (cvid) and combined immunodeficiency (cid), respectively. herein we describe a novel set of germline heterozygous ikaros allelic variants affecting the c-terminal dimerization domains in four unrelated families. clinical manifestations include hematopoietic cytopenias presenting as evans syndrome, and hematologic malignancies including t-cell all and burkitt lymphoma; other manifestations observed were b-cell lymphopenia and hypogammaglobinemia, but recurrent or severe infections were not prevalent or characteristic. we demonstrate that mutants affecting dimerization abolish ikaros homodimerization as well as heterodimerization with ikaros family members aiolos and helios. these variants also affect dna binding at dimerization sites and pericentromeric targeting. opposed to previous allelic variants reported, dimerization changes alter post-translational sumoylation and gene transcription regulation. our data show that mutations affecting ikaros dimerization are mainly associated with cytopenias and/or malignancies, have a different mechanism of action than previously reported variants, present with incomplete clinical penetrance, and contribute to the growing spectrum of genotype-phenotype ikaros associated diseases. introduction: there has been much discussion regarding the return of secondary findings in genetic sequencing research. opinions differ on whether researchers should return secondary findings to participants at all and if so, what the best method is to do so. we have opted to systematically identify and return pertinent secondary findings to participants in our cohort of patients with immune-mediated diseases that undergo exome sequencing. additionally, exome sequencing may determine multiple or other genetic diagnoses in addition to the primary diagnosis, which we call "incidental findings." here, we discuss the secondary and incidental findings discovered in our cohort thus far. methods: individuals in our protocol underwent consent for exome sequencing, including a discussion of the possibility of secondary findings. exome sequencing data was analyzed, and variant pathogenicity was scored using the acmg criteria (richards et al); variants determined to be likely pathogenic, pathogenic, or otherwise clinically important were confirmed via clia-certified sanger sequencing. confirmed variants were returned to participants. we then queried internal databases for cases involving secondary and incidental findings. results: as of november 2019, exome sequencing, interpretation and reporting had been completed for 629 participants. we detected a total of 18 secondary findings in 17 (2.7%) participants, including variants in apob, brca1(2), brca2 (5), dsp, fbn1, kcnh2, ldlr, mybpc3 (2), ryr1, pkp2 (2), and vhl. additionally, we detected possible dual/multiple genetic diagnoses in 18 (2.9%) participants, some of which explained an unusual clinical presentation or symptom. these included individuals with variants in multiple immune-related genes, including one individual with variants in gata2 and tnfrsf1a, and those with variants in genes related to multiple organ systems, including an individual with variants in ifngr1 and sco2. discussion: exome sequencing in this cohort detects not only important secondary findings, but also discovers a significant portion of individuals with multiple genetic diagnoses. notably, exome sequencing may provide further context or explanation for unusual phenotypic presentation and help determine specific symptom etiology even when a primary genetic etiology is already known. additionally, these secondary and incidental finds may be important to consider when delineating risks and symptoms of novel or recently-discovered conditions. abstract/case report text background: immune dysregulation and lymphoproliferative disorders including alps like disease, hlh ebv driven lymphoproliferative disease leading to rare lymphomas require a multidisciplinary approach utilizing expertise in immunology and hematology/oncology to care for these patients as we learn the molecular etiology of their underlying disorders. at texas children's hospital, the immunology lymphoproliferative evaluation and diagnostic (ilead) clinic was created to provide a comprehensive clinical and research approach to caring for patients with these rare disorders. in an effort to streamline care and access, we recently on-boarded an advanced practice provider (app) . methods: a chart review was conducted 6 months before and after onboarding the app for ilead patient visits. we reviewed the following patient care and access parameters to determine increase in efficient and effective patient care as well as improved access to the clinic. these parameters included: referral process, time of referral placement to appointment, number of patient visits, wait time in clinic, lab interpretation and reporting time for disseminating results to families, and collaboration process with other specialties. results: within 2 months of the app starting our average wait from placing the referral to first appointment fell by an average of 45%. in addition, we created an algorithm to prioritize patients with immediate need to be seen. by streamlining the referral process and patient priority, we developed a "pre-clinic" conference process by which all patients are reviewed and preliminary plans are made prior to the patient's arrival. this has translated into our ability to increase the number of patients seen in clinic from 4 to 6 and decreased the wait time in clinic by approximately 30 minutes. since the app started, no patient has been in clinic for more than 60 minutes. this has also led to an increase in rvu generation. in terms of efficiency in patient care, all labs are now ordered while in the room with the patient by the app and physician providers. in turn, all labs are resulted directly to the app who reviews labs, collaborates with physicians for care and reports to families in a timely fashion within 1-2 weeks of labs being resulted compared to greater than 1 month previously. to improve collaborator communication and post visit plans, a post-visit clinic summary was created. this has been effective in reducing the time to other specialty referrals, follow up visits and effective care for ongoing clinical needs. conclusions: the addition of an app in our ilead multidisciplinary clinic which provides specialized care for patients with immune dysregulation and lymphoproliferative disorders effectively increases work productivity of providers and enhances patient care by increasing access to care, decreasing wait time in clinic and time of reporting of results and future plans. the app with knowledge and expertise in immunology and immune dysregulation is a cost effective way to enhance provider and patient support. with the overwhelmingly positive results, future plans include expanding our multidisciplinary clinic to other services that care for patients with suspected immune deficiency. abstract/case report text introduction: pediatric lymphoproliferative disorders represent a clinically and genetically heterogeneous group of conditions. misdiagnosis and delayed diagnosis can contribute to substantial morbidity and mortality. identification of molecular etiologies and underlying disease mechanisms may facilitate timely interventions and guide targeted or curative therapies. methods: the study was performed through retrospective chart reviews in accordance with all local ethics and irb committees. the study was designed to investigate a cohort of pediatric patients who met criteria for non-malignant lymphoproliferative disorders from texas children's hospital and collaborating centers for underlying genetic etiologies. results: a total of 51 affected individuals from 47 families met criteria. distribution between male and females was nearly equivalent: males (n = 26) and females (n = 25). approximately half of the cohort was hispanic (n = 25). overall kaplan meier survival was 67% (n = 39). whole exome sequencing was performed in all subjects and available family members. likely disease-causing genetic defects were identified in 29 of 47 families (62%). within these 29 families, 20 (69%) carried variants in genes in international union of immunological societies established primary immunodeficiency diseases. potential novel genetic causes of immune deficiency or immune dysregulation were also discovered. mechanistically, all of the implicated genes had roles in modulating lymphocyte activity; initial activation, cytoskeletal organization, or apoptosis of lymphocytes; or regulation of inflammation. all subjects less than one year of age had an identified gene in one of the three mechanistic categories with the dominant mechanistic genetic category being defective control of lymphocyte signaling (57%). in addition, 72% of patients between 1 and 8 years of age were found to have a potential genetic diagnosis underlying the lpd, with a more equal distribution of mechanistic categories compared to patients greater than 15 years of age where only 33% have a genetic cause. other important disease manifestations identified were ebv-associated disease in 21 subjects (41%) and 15 subjects (24%) met hlh-2004 criteria. conclusion: primary immunodeficiency diseases and other genetic abnormalities of the immune system underlie a significant percentage of pediatric lymphoproliferative disorder cases. greater than 72% of patients less than 8 years of age have a genetic etiology underlying the lymphoproliferative disorder. many of these gene defects can be treated with targeted therapies or hematopoietic stem cell transplantation. genetic testing therefore plays an essential role in the diagnosis and management of children with these conditions. abstract/case report text a 21-year-old gentleman with a history of immune dysregulation polyendocrinopathy enteropathy x-linked (ipex) syndrome with known pathogenic variant in foxp3 presented to our emergency department with two witnessed episodes of tonic-clonic seizures earlier that day. he has had a longstanding history of recurrent infections and autoimmune conditions since birth, and was being treated with monthly ivig infusions and sirolimus while awaiting bone marrow transplantation. his symptoms on admission included foaming at the mouth, generalized shaking, bladder incontinence, and tongue biting that lasted about five minutes. family reported recent sores inside his mouth and lips, but denied any recent fevers, neck pain, headaches, chest pain, abdominal pain, nausea, vomiting, and sick contacts. he lives on a farm with livestock and reportedly had recent tick exposure. his last ivig infusion was two weeks prior to admission, at which time he also received inactivated flu vaccine. in the ed, a third seizure was witnessed by multiple medical providers. he subsequently received lorazepam and lacosamide with interval improvement. he underwent diagnostic lumbar puncture, as well as extensive evaluation for infections. he was started on empiric antibacterial and antiviral meningitis coverage. analysis of the csf showed a lymphocytic pleocytosis; bacterial cultures and hsv 1/2 pcr were negative, as was a 14-pathogen meningitis/ encephalitis panel performed by pcr. eeg was negative for seizure-like activity, and brain mri showed mild atrophy without sclerosis in the left hippocampus. subsequently, anti-infectious therapy was stopped, and patient was discharged with outpatient followup scheduled for suspected non-infectious aseptic meningitis that was potentially triggered by flu vaccination versus ivig. on day four post-discharge, however, pcr for ehrlichia chaffeensis in the serum returned positive, and he was started on oral doxycycline. ehrlichiosis is a rare tick-borne illness that may cause various nonspecific symptoms including fever, headaches, myalgias, and generalized malaise. most prevalent in the mid-atlantic regions of the united states, tick-borne ehrlichia spreads through the mononuclear phagocytic system and can infiltrate many organs including the kidney, liver, lungs, and heart. csf penetration can cause sometimes fatal meningoencephalitis. aseptic meningitis due to ehrlichiosis has been described in recent literature. cases in hiv patients and transplant patients on chronic immunosuppressive therapy have been severe, resulting in organ dysfunction in many instances and death in a few. however, this case marks the first documented ehrlichia infection in a patient with primary immunodeficiency. this patient's presentation of aseptic meningitis and clear exposure history fits the clinical picture. his relatively benign course could be due to preserved t effector function not seen in persons with hiv or transplant patients with significant immunosuppression. patients with ipex usually present with autoimmunity and allergies, but are also prone to significant infections. it is important to perform a comprehensive workup, including testing for atypical infections, in patients with immune dysregulation syndromes who present with symptoms of unclear etiology. special attention should be paid to patients who live in areas with known endemic exposure risks. empiric antibiotic therapy may need to be considered early to prevent delays in treatment. abstract/case report text introduction autosomal recessive hypomorphic mutations in pgm3 have been described to result most commonly in either hyper-ige or severe combined immunodeficiency (scid) clinical phenotypes in humans, with one report of an individual with combined immunodeficiency without atopy. herein, we describe a series of individuals newly diagnosed with pgm3 deficiency functionally confirmed using lectin-based flow cytometric analysis of peripheral blood mononuclear cells, that broadens the associated clinical phenotypes to confirm cid without atopy and childhood evans syndrome. in addition, we present 3 new disease-causing pgm3 variants, and functionally confirm the pathogenicity of a fourth (p.i350t). classical hies phenotype cases 1.1 and 1.2 identify 2 sisters of spanish descent with a classical hyper-ige phenotype. the younger sibling demonstrated severe atopic dermatitis, mild-moderate asthma, multiple food allergies, one episode of itp, and adhd. the older sibling demonstrated atopic dermatitis, skin infections, and c. albicans otomastoiditis. the siblings were found to have the damaging compound heterozygous variants p.t492i and p.q506x in pgm3. case 2 is a 15 year-old guatemalan boy with prominent atopy including asthma, allergic rhinitis, food allergy, elevated ige, atopic dermatitis, as well as oral hsv who was found to be homozygous for the damaging pgm3 variant p.i350t. cid phenotype with a paucity of atopy case 3 is a 10-year-old turkish girl who is the daughter of a consanguineous union. she presented with infantile nephrotic syndrome at 6 months of age, and subsequently developed leukopenia, neutropenia, and low igg. complications include bronchiectasis, sinusitis, pseudomonas urinary tract infection, and inflammatory skin lesions without atopy. she was found to be homozygous for the damaging pgm3 variant p.r69h evans syndrome case 4 is a 5-year-old girl from guatemala. she developed multilineage autoimmune cytopenias including immune thrombocytopenic purpura (itp), autoimmune hemolytic anemia (aiha) and autoimmune neutropenia (ain) at the age of 2 years, refractory to multiple treatments and finally responding to mycophenylate mofetil. she has a history of mild eczema but is without other atopy and suffered from multiple invasive bacterial infections. an additional patient, case 5, was diagnosed with coombs positive aiha and itp at age 3 years refractory to multiple treatments and finally responsive to cyclosporine. cytopenias recurred 1 year later, resulting in hypoxic brain injury. he died of infectious complications at the age of 7 years. both patients were found to be homozygous for the damaging pgm3 variant p.i350t. discussion this is the first report of pgm3 deficient individuals presenting with evans syndrome as a primary presentation without additional pathology. while disease-associated mutations appear to cluster around the 4 key conserved domains of the protein, no clear genotype-phenotype correlation is readily observed. in addition to autoimmune cytopenias, pgm3 deficient individuals have also been reported with splenomegaly, lymphoma, and ebv viremia. thus, in particular for children with lymphoproliferative disease, pgm3 deficiency should also be considered in the differential diagnosis. abstract/case report text introduction: meningitis is a life-threatening manifestation of cryptococcus neoformans (c. neoformans). it occurs in increased frequency in those with impaired cell-mediated immunity, especially those with hiv/aids. infection with c. neoformans has been seen in previously healthy individuals diagnosed with idiopathic cd4 lymphopenia (icl). icl is defined by an absolute cd4+ count of less than 300 cells/m3 on multiple occasions, usually 2 to 3 months apart, without other apparent cause such as hiv infection, immunodeficiency, or immunosuppressive medications. case description: our patient is a previously healthy 50-yearold female with cryptococcus meningitis and fungemia. her course was complicated by elevated intracranial pressure requiring extraventricular drain. she was treated with amphotericin and flucytosine for 1 month. notably, the patient was also found to have moraxella catarrhalis (m. catarrhalis) bacteremia without identifiable source. she denied history of environmental risk factors, was not up to date on cancer screening, and recently returned from a trip to italy. initial evaluation revealed lymphopenia (422 cells/ul), low cd3+ (283 cells/ul), cd4+ cells (42 cells/ul), and cd16/56+ (31 cells/ul), but normal cd8+ (234 cells/ul) and cd19+ cells (121 cells/ul). hiv, ana, leukemia/ lymphoma flow cytometry panel was negative. she also had a normal lymphocyte proliferative responses to pha (66.7%), normal cd45ra:ro, and protective tetanus titers (2.31 iu/ml), but only 1/23 protective pneumococcal serotypes. initial immunoglobulins demonstrated slightly low igg (616 mg/dl). laboratory studies 2 months after presentation demonstrated improved lymphopenia (800 cells/ul) continued low cd4+ cells (86 cells/ul), but normalized igg levels (645 mg/dl). followup labs also demonstrated decreased cd19+ b cells (44 cells/ ul) and insufficient response to polysaccharide vaccine (9/23 pneumococcal serotypes). three months after discharge, she is continued on daily fluconazole without recurrence of infections although she still has diplopia and headache. discussion: in a review of cryptococcosis in 53 patients with icl, 7 of them had cryptococcal infection in both the cns and blood. of these 7 patients, 1 was cured, 2 improved, 3 relapsed and then improved, and 1 died. three of these patients were treated with amphotericin and flucytosine. five of these patients had underlying disease and 3 had notable infections with vzv, tb, or hpv, however other infections such as m. catarrhalis were not mentioned. m. catarrhalis bacteremia has been described in children with underlying immune dysfunction and respiratory infection as well as secondary to pneumonia with m. catarrhalis. in 24 cases of m. catarrhalis bacteremia in adults, most had underlying malignancy and/or neutropenia, predisposing respiratory factors, or source for infection. conclusion: this report of c. neoformans meningitis and m. catarrhalis bacteremia in the setting of icl is unusual in that to our knowledge, m. catarrhalis bacteremia has not been reported in icl. cases like this also raise the question as to whether some laboratory abnormalities are secondary to infection, treatment, or underlying disease. it is important to report these cases with icl in order to group disease phenotypes, as continued monitoring and data collection of these cases may lead to discovery of new disease processes. abstract/case report text cytokines play critical roles in regulating the development, survival, differentiation and effector function of immune cells. cytokines exert their function by binding specific receptors on the surface of immune cells and typically activating intracellular jak/stat signaling pathways, resulting in induction of specific transcription factors and regulated expression of target genes. in order to differentiate into an appropriate effector fate, lymphocytes need to integrate multiple signals often provided concomitantly by numerous cytokines that activate shared transcription factors. how these signals are balanced and regulated to yield the optimal class of immune response remains to be completely determined. inborn errors of immunity, or primary immunodeficiencies (pids), result from germline mutations in defined genes, leading to loss-of expression, loss-of function, or gain-of function of the encoded protein. pids are characterised by defects in immune cell development, or their differentiation into effector cells during immune responses, thereby rendering patients not only highly susceptible to infectious diseases, but also autoimmunity, autoinflammation, allergy and cancer. pids are thus an unprecedented model to link defined monogenic defects to immune dysregulation in clinical settings. indeed, pids have unequivocally revealed non-redundant roles of single genes, molecules, signaling pathways and lymphocyte subsets in host defense and immune regulation, and formed the basis of better therapies for immunopathologies. our indepth analysis of inborn errors of immunity of cytokine signalling pathways have identified fundamental requirements for generating long-lived humoral immune responses in humans. here, i will present data relating to our recent studies of how inactivating mutations in il21r, il6r, znf341, stat1, stat3, and stat5, disrupt or dysregulate the generation and function of human memory b cells and tfh cells, thereby precipitating humoral immunity, as well as allergic disease and autoimmunity. abstract/case report text introduction: flow cytometry is a powerful diagnostic tool for detecting hematologic malignancies in a variety of patient specimens including body fluids and lymph node aspirates. cytopathologists are frequently confronted with lymphocyterich effusions, and the definite decision of whether the lymphocytosis is of a purely reactive nature or a presentation of an indolent lymphoma may be an extremely difficult based on microscopy alone. moreover, small proportions of malignant cells that may be missed out by routine morphology can be detected by flow cytometry. objective: the purpose of this study was to evaluate the usefulness of multiparametric flow cytometry immunophenotyping (fci) to confirm the presence of leukemia or lymphoma cells in body fluids and fna specimens. methods: body fluids and fna specimens simultaneously obtained for fci, cytologic analysis and real time pcr from 30 patients were submitted to our flow cytometry laboratory from january 2017 to september 2019. the samples studied were 16 body fluids ( 11 pleural fluids and 5 ascitic fluids) and 14 fna samples (13 enlarged lymph nodes and 1 lung mass).four color fci method was performed and the following fluorescent monoclonal antibodies were used: cd45, cd19, cd5, cd20, cd22, cd23, cd79b, fmc7, kappa and lambda light chains, cd200, cd123, cd10, cd11c, cd2, cd1a, cd3, cd5, cd7, cd4, cd8, tdt, cd52, cd25, cd30, cd40, cd56, cd95, bcl2, cd34. fci analysis was performed on a beckman coulter cytomics fc500 flow cytometer using software cxp to analyze data. the cases were diagnosed as leukemia or lymphoma as per tuberculosis (1 case). ascitic fluid (n=5) samples showed positivity for angioimmunoblastic t-cell lymphoma (4 cases) and dlbcl (1 case). fna of lymph nodes (n=13) were positive fort-lymphoblastic lymphoma (2cases), angioimmunoblastic tcell lymphoma (2 cases), dlbcl (3 cases), hodgkin lymphoma (1case), nodular lymphocyte predominant hodgkin lymphoma (1 case), peripheral t-cell lymphoma (nos) (1 case), splenic b-cell marginal zone lymphoma(1 case), tuberculosis (2 cases). one fna of lung mass were tumor of neural cell origin. both immunophenotype and cytomorphology positive for malignancy were in 19/30(63.33%) cases.cytomorphology was negative/ suspicious in 11/30(36.67%) cases, of which both cytomorphology a n d i m m u n o p h e n o t y p e n e g a t i v e w e r e 3 ( 1 0 % ) a n d cytomorphology negative but immunophenotype positive cases were 8(26.67%). mtb dna was detected in pleural fluid in 1 case and fna sample in 2 cases. conclusion: multiparametric flow cytometry by using comprehensive panel of monoclonal antibodies is a useful diagnostic test to evaluate body fluids or fna as it can demonstrate small malignant populations that may be missed out by routine cytomorphology. clinical laboratory geneticist/department of genetics, university of groningen, groningen, the netherlands abstract/case report text the phenotypes of primary antibody deficient (pad) patients range from milder (e.g. specific antibody deficiency) to severe (e.g. x-linked agammaglobulinemia) deficiency of the immune system. pad patients form a clinically, immunologically as well as genetically heterogeneous group. often, the genetic background has not been elucidated; it probably is not monogenetic in a large subgroup of patients. pad patients suffer most frequently from recurrent bacterial infections of the respiratory or gastrointestinal tract due to immune deficiency, but may also have varying degrees of autoimmune and lymphoproliferative comorbidities due to immune dysregulation. unfortunately, a standardized description of pad phenotypes is lacking rendering robust definitions of pad-subtype diagnoses, including cvid, difficult. this impairs the formation of homogeneous cohorts that can form the starting point for future clinical and genetic research. the pad subgroup of the human phenotype ontology (hpo) immune mediated disorders consortium supported by ern rita and esid is addressing the gaps in standardized phenotypic description of pads. using the hpo dataset, literature mining, and esid, iuis and omim classifications, we aimed to reevaluate and complete the pad-related hpo terms to allow efficient data exchange and matching of phenotypically similar pad patients. as a principle, it was decided to avoid the ongoing variance in pad-subtype definitions and to build the pad-related hpo tree based as much as possible on unambiguously interpretable items. 'hypogammaglobulinemia' was deleted as hpo term, and replaced by separate hpo terms such as 'decreased total igg in blood', subdivided in 'transient' vs. 'chronic', and '(near) absent' vs. 'partially decreased' (the same for igg1, igg2, igg3, igg4, iga and igm). 'decreased specific antibody level in blood' was specified further into 'decreased natural antibody level to blood group antigens in blood', subdivided in '(near) complete' vs. 'partial' absence (the same for protein, polysaccharide and protein-conjugated polysaccharide vaccination). relevant hpo terms related to infection and to specific organ manifestations like bronchiectasis, autoimmunity and lymphoproliferation were re-evaluated and completed, and will be linked to pad diseases in the hpo online system by the pad subgroup experts. once finalized, existing pad cohorts will be classified according to the new hpo pad-related terms, and studied by clustering technologies (example of two patients shown in figure 1 ; white = absent, color = present). acceptance and widespread use of this pad-related hpo tree for standardized phenotyping will be essential to empower future multicenter clinical research and related genetic discoveries as well as support clinicians in diagnosing pad through the linkage of hpo terms to pad disease entities. senior investigator oral immunity & infection section/nih/nidcr abstract/case report text leukocyte adhesion deficiency type 1 (lad1) is an autosomal recessive disorder characterized by the inability of granulocytes to emigrate from the bloodstream to sites of inflammation. lad1 is caused by mutations in the itgb2 gene (21q22.3), encoding the beta-2-integrin, cd18, which is essential for firm adhesion of leukocytes to the endothelium. in lad1 survival is compromised, morbidity from inflammatory lesions is high, and treatment is poor. the moderate form of lad1 is often managed with antibiotics for prophylaxis and during acute infections. after infancy severe gingivitis and chronic periodontitis are universal. periodontal findings affect primary and permanent teeth, causing intense oral mucosal (gingival) inflammation and destruction of tooth supporting bone, which are hallmarks of the disease periodontitis. blocking the il-23/il 17 cytokines, which are up regulated in lad1 gingiva, can reduce bacterial load and resolve inflammatory gingivitis. ustekinumab binds to the shared p40 subunit of human il-12 and il-23, cytokines that modulate lymphocyte function, including t helper (th) 1 cells and th17 subsets, thereby blocking them. objective: explore the effect of ustekinumab on lad1 inflammatory disease. method: prospective study using ustekinumab for oral inflammation. patients receive five doses over 1 year, 45-or 90 mg depending of weight. results: (two patients have enrolled, p1 is >1 year post treatment, p2 is still on study) patient characteristics · patient 1: age at diagnosis, 9yrs.(itgb2 mutation c.2070delt (null)); cd18(%pmn control):32.6%; cd11a(%pmn);6.6%. at the initiation of the protocol (17yrs old) wcc:7.41k/ul; crp: 6.10 · patient 2: age at diagnosis, 4yrs.(itgb2 mutation c.850a>g,p.g284s c.809c>t,p.a270v) cd18(%pmn control): 13%; cd11a(%pmn): 3.9%. at the initiation of the protocol (17yrs old) wcc:7.41k/ul; crp: 6.10 response: patient 1: oral ulcers before treatment: episodes every two months. during ustekinumab therapy: · oral ulcers: 1 episode in a year · reduction in bleeding on probing: 57.5% · gingival index reduction: 95% patient 2: oral ulcers before treatment : monthly. during ustekinumab therapy: · oral ulcers: none in first 4 months · reduction in bleeding on probing : 68.3% · gingival index reduction: 37.5% safety: no significant adverse events were documented during the therapy p1 had a previous skin lesion that flared leading to iv antibiotics. p2 had a previous sebaceous cyst drain spontaneously. discussion: two patients showed improvement in chronic periodontitis and a substantial decrease in oral ulcers while on ustekinumab. no clear safety signals were seen. durability of these findings is still unknown. ustekinumab in lad1deficiency appears to be safe and potentially effective. post doctoral fellow/servicio de inmunología, inst. multidisciplinario de investigación en patologías pediátricas (imipp), hospital de niños ricardo gutiérrez. 2 chief resident/servicio de inmunología-hospital de niños "dr. r.gutierrez" 3 immunologist/centro de inmunología clínica "dra. liliana bezrodnik y equipo"-servicio de inmunología htal. de niños "dr. r.gutierrez" 4 immunologist/centro de inmunología clínica "dra. liliana bezrodnik y equipo" 5 immunologist/servicio de inmunología-hospital de niños "dr. r.gutierrez" abstract/case report text introduction: primary immunodeficiencies with dysregulation associate defects in the immune homeostasis leading to inappropriate immune response (lack or excess) that causes autoimmunity, allergy and/or inflammation. impairment of different subsets of t and b compartments may be associated with these pids. aim: 1) describe t and b memory compartment of 18 pid patients (pts) with dysregulation: 1 cd25 deficiency, 3 stat1 gof, 1 stat5b deficiency, 4 ctla4 variant, 2 pi3kcd variant and 6 cvid-like (with no molecular defect) and compare them with a group of healthy donors (hd). 2) associate ctfh profile with b cell compartment impairment. results: 1) pts showed a significant decrease of naïve cd4+ t cells (cd45ra+cd27+) (52,2% vs 17,6%) (p < 0.0001) with expanded central memory t cells (cd45ra-cd27+) (32.8% vs 60.8%) (p < 0.0001); cd4+ t cells had higher levels of activation markers (cd4+hla-dr+) (6,1%vs 27,4%) (p < 0,0001). pts showed a significant increase of circulating follicular t cells (ctfh) (cd45ra-cxcr5+) compared with hd (mean 30,5% vs 11,6%) (p < 0,0001) with pd-1 overexpression (p < 0.0001). stat1 gof, ctla4, pi3kcd and cvid-like pts showed a skew towards ctfh1 (cxcr3+). regulatory t cells (cd4+cd25++ foxp3+) were absent in cd25 and stat5b deficiency and decreased in the other pts. within cd8+ cells, although effector memory (cd45ra-cd27-) (p < 0.002), temra (cd45ra+ cd27-) (p < 0.009) and hla-dr+ cd8+ (p < 0.002) subsets showed a significant increase compared with hd, the behaviour was variable between different mutations. regarding b cell compartment, pts with stat1gof, pi3kcd and cvid-like showed a severe impairment of switched-memory b cells (sw-mbl) (cd27+igd-igm-); the stat5b deficient patient had increased frequencies of this subset, while ctla4 pts had a variable b defect. 2) lower sw-mbl values were significantly associated with lower values of ctfh17 cells (p < 0.007) (r=0.6975). cd21low b cells were exclusively high in cvid-like pts, and transitional b cells were increase in pi3kcd and almost all cvid-like pts. discussion: in summary, patients with dysregulatory syndromes associate a defect of t and b homeostasis (survival, activation and differentiation). specific mutations can differentially affect the quantity and/or the quality of ctfh. there is a strict association between the differentiations of tfh with th17 profile with the generation of sw-mbl. these alterations may play a role in the pathophysiology of primary immunodeficiencies with b lymphocyte functional impairment. immune monitoring of lymphocyte subsets of patient with dysregulation may approach to the diagnosis of specific monogenic mutations. objective: the purpose of this study is to increase awareness and improve diagnosis of primary immune deficiency (pid) in the heterogenous group of patients with autoimmune cytopenia (aic) by identifying clinical characteristics and laboratory biomarkers that distinguish those with underlying pid, disease activity and guide mechanism-based targeted therapy. methods: patients with aic (autoimmune hemolytic anemia (aiha), immune thrombocytopenia (itp), or autoimmune neutropenia (ain)) were referred to our immune dysregulation team and prospectively enrolled during 2016-2019. detailed immune phenotyping (igg, iga, igm, lymphocyte subsets, vaccine titers, lymphocyte proliferation to mitogens/ antigens), serum lipopolysaccharide (selps) and autoantibodies were measured and/or collected by chart review and genetic testing for pid was pursued. results: from 2016 to 2019, 93 patients were enrolled; two subjects were removed due to parental request or lack of aic diagnosis. of the 91 remaining patients, 43 (46%) were classified as "aic-pid" based on genetic testing and/or immune phenotyping; 41 (45%) were classified as aic-only, and 7 (9%) were asymptomatic family controls. the patients were predominantly children (ages 1-82 years, average age 17.3 years); 47% (44/93) were male. among patients who have had genetic testing to date (n=66)(72%), pathogenic genetic mutations were confirmed in 23/66 (35%) of patients. mutations include fas/fasl (n=8, including 3 family members without aic), ctla4 (n=4), 22q11 (n=4), and one patient each with nfkb1, was, pole-1, pi3k, casp10, card11, and cgd; the remainder of aic-pid patients were classified as combined immune deficiency or common variable immune deficiency based on immune phenotyping. lymphocyte subsets (cd4+t, cd8+t, cd19+b, cd56+ nk) and immune globulins (igg, iga, igm) tended to be lower in aic-pid patients vs aic-only (p < 0.05). evans syndrome was more common in aic-pid patients (13/43, 30%) compared to aic-only (4/41, 10%). lps was elevated in the serum of aic patients compared to healthy controls (mean 719 vs 87 pg/ml, p < 0.001). excluding partial digeorge syndrome patients (average lps 222pg/ml), selps levels were significantly higher in genetically-defined untreated pid patients (average 1463 pg/ml) vs. other pid (average 444 pg/ml)(p=0.02) or patients with aic alone (average 667 pg/ml)(p=0.03). studies are ongoing on specific subsets that are linked to immune dysregulation (switched memory b cells, t-regulatory cells, double negative t cells, t follicular helper cells) and the use of soluble il-2 as a biomarker of disease activity. conclusions: a high fraction of aic patient were identified with underlying pid in our study. basic immune evaluation with immunoglobulin levels and lymphocyte subsets expedited diagnosis of pid. genetic evaluation distinguished a group of patients with aic-pid and highly elevated lps level, reflecting high bacterial load, which may distinguish them from the rest the aic cohort. the source of bacterial lps can be multifactorial and is yet to be determined. our studies continue focusing on biomarkers that can be applied to the heterogenous group of patients with aic. this will allow early detection and timely initiation of targeted therapies. investigator/dermatology branch, niams, nih 6 head, dermatology consult service/dermatology branch, niams, nih 7 chief, fungal pathogenesis section/laboratory of clinical immunology and microbiology, nih abstract/case report text introduction/background: autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (apeced) is a monogenic autoimmune disease resulting from biallelic mutations in the aire gene. although typically characterized by the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism and adrenal insufficiency, we recently reported that the clinical spectrum of the syndrome is far broader and that incorporation of an adjunct triad of apeced rash, intestinal dysfunction, and enamel hypoplasia in the classic triad could lead to earlier diagnosis (ferre et al., jci insight, 2016). among the adjunct triad manifestations, apeced rash occurs in 66% of american apeced patients by age 3, most often developing in the first year of life. objectives: to report and describe the clinical features of apeced rash as the first manifestation in a 10-month old patient with apeced. methods: following enrollment in a niaid irb-approved protocol (11-i-0187) the patient was evaluated with history and physical examination, aire sequencing, measurement of interferon-autoantibodies, and skin biopsy with immunohistochemical analyses. results: a 10-month-old girl with a family history of genetically confirmed apeced in her 13-year old sister developed discrete circular, maculopapular erythematous lesions on her torso that spread to the face, arms, and legs while sparing the palms and soles. the rash was partially blanching, non-painful and non-pruritic and was preceded by low-grade fever (38□c) without other accompanying symptoms. she had not received medications or vaccinations prior to the rash onset. the lesions increased in size with associated central clearing and resolved 2.5 months after onset. the rash recurred with similar appearance 10 times over 14 months with each recurrence lasting between 10 days and 2.5 months. as with the first rash episode, recurrences were often preceded by fever (38-39□c) without accompanying symptoms or inciting factors. neither topical nor oral antihistamines improved the rash. aire sequencing identified the same compound heterozygous mutations (c.967_979del13 and c.769c>t) that the sister has. high titers of interferon-□ autoantibodies were measured in serum. skin biopsy revealed superficial perivascular chronic inflammation and intraepidermal lymphocytes composed predominantly of mixed cd4 and cd8 t lymphocytes with few perivascular b lymphocytes. no eosinophils or vasculitis was observed. myeloperoxidase immunostaining revealed extensive karyorrhexis. laboratory studies revealed normal white count and esr, negative anti-ige receptor antibody, and positive anti-ige antibody. at 22 months, she developed oral candidiasis as second manifestation of apeced, thus reaching a diagnostic dyad when applying our proposed expanded diagnostic criteria. she has not developed hypoparathyroidism or adrenal insufficiency; thus, she has not yet reached a classic diagnostic dyad. systematic screening for these endocrinopathies will be needed to avoid lifethreatening complications of acute endocrine failure. conclusions: we report the clinical and histologic features of apeced rash manifesting as the first disease component of apeced in a 10month old girl. apeced should be considered in the differential diagnosis of recurrent erythematous maculopapular urticaria-like eruptions characterized by mixed lymphocytic and neutrophilic infiltration unresponsive to antihistamines. our case illustrates the clinical utility of incorporating the expanded diagnostic criteria of apeced rash, enamel hypoplasia and intestinal dysfunction into the classic diagnostic triad, which can lead to earlier apeced diagnosis. who presented with prolonged severe neutropenia despite g-csf and seven hospitalizations for febrile neutropenia in the span of ten months. prior to his neutropenia, patient was on monthly ivig, with igg trough 700-900 in the past year. he was evaluated for bmt in 2015 but declined. patient was first found to be neutropenic in aug 2017 when he was admitted with pseudomonas thigh abscess, hsv stomatitis and rhinovirus infection. he was treated with broad-spectrum antibiotics with improvement in neutropenia. the following month, he was hospitalized again with neutropenic fever, left axilla pseudomonas abscess and rhinovirus infection. he underwent bone marrow biopsy revealing left shifted myeloids with decreased maturing forms and t cell predominant lymphoid aggregates, suggestive of autoimmune neutropenia vs. hyper igm syndrome associated with neutropenia. anti-neutrophils antibodies were negative. he was then admitted the following month (10/2017) with febrile neutropenia with cxr concerning for viral pneumonitis vs. atypical pneumonia. he was started on g-csf therapy with significant initial response in anc. however, this response was short-lived as he was again admitted in 12/2017 with febrile neutropenia and upper respiratory rhinovirus infection. he was continued on daily g-csf. due to persistently normal anc for approximately three weeks, he was weaned off g-csf in 1/2018. in 3/2018 and 4/2018, he had two more hospitalizations for febrile neutropenia. g-csf was restarted with dose uptitrated to 8mcg/kg during his hospitalization in april. he was found to be thrombocytopenic with splenomegaly on abdominal ultrasound. anti-platelet antibodies and repeat anti neutrophil antibodies were not detected. he was discharged with close follow up with immunology and hematology. due to his age, he was transitioned to penn allergy/immunology in 4/2018. there was close communication between chop allergy/immunology, chop hematology and penn allergy/immunology during this transition period. patient was admitted to hup in 5/2018 with febrile neutropenia (despite higher dose of g-csf), rhinovirus infection, pseudomonas sinusitis and ct chest findings suggestive of possible fungal pneumonia. due to persistent neutropenia refractory to g-csf treatment, hematology was consulted and repeat bone marrow biopsy showed hypercellular bone marrow with markedly left shifted granulocytic hyperplasia, compatible with g-csf therapy. flow cytometry showed no evidence of plasma cell neoplasm. dose of ivig was adjusted and increased based on his weight. per hematology, he also received an additional high dose ivig 1g/kg x 2 days for presumed immune mediated neutropenia with immediate increase in anc. despite anc of 0 for 8days,anadditional1g/kgofivigimprovedhisancto>1000within12hours of his first dose. thrombocytopenia also improved to normal range. since then, patient has been on monthly 500-600mg/kg ivig with no recurrence in neutropenia. this patient's prolonged persistent neutropenia with immediate response to high dose ivig is suggestive of autoimmune neutropenia, which should be taken into consideration in hyper igm patients with persistent neutropenia. abstract/case report text background: children with digeorge anomaly (dga) represent a heterogenous group, often classified as either partial dga (pdga) or complete dga (cdga) based upon the degree of thymic hypoplasia. this paucity of t-cell parameters and function has serious implications for infection risk, autoimmunity, and malignancy. however, there are limited studies stratifying children with dga by these subgroups, especially in regard to immune function and subsequent infection risk. study design: single-center, retrospective cohort analysis evaluating the relationship between pdga and cdga to infection risk with particular focus on infection-related hospitalization, pathogenic organism identification, and antimicrobial resistance profiles. the source population includes all pediatric patients < 18 years of age diagnosed with either pdga or cdga while receiving care at duke university from january 1, 2014 to june 30, 2019. the final analysis sample included 145 patients. methods: to evaluate the differences in immune function between dga subgroups, we will report the proportion of low ( < 10th percentile for age) t cell immune biomarkers for both subgroups and compare populations using a chi-squared test. to compare per year incidence of infectionrelated hospitalization for dga subgroups, a poisson model with number of hospitalizations per patient as the outcome, an offset equal to the time at risk for hospitalization, and either pdga or cdga diagnosis as the exposure will be used. models will be bivariate. we will report an incidence rate ratio (irr) and 95% confidence interval (95% ci). to evaluate the impact of cellular and humoral immune function on infection-related hospitalization, we will use poisson models where the outcome is the number of hospitalizations per patient, an offset equal to the time at risk for hospitalization, and low immune biomarker as the exposure. all models will be bivariate. we will report an irr and 95% ci. infection type and resistance profiles will be completely descriptive. results: as expected, children with cdga had a significantly higher probability of a low ( < 10th percentile for age) values for total t cells (cd3+), helper t cells (cd3+cd4+), cytotoxic t cells (cd3+cd8+), and naïve helper t-cells (cd4+cd45ra+cd62l+) as well as a significantly lower probability of low pan memory t-cells (cd3+cd45ro+) compared to children with pdga. no differences were detected in the percentage of low natural killer (nk) cells (cd15+cd56+) or b cells (cd19+) between subgroups. cdga patients had a significantly higher incidence of hospitalization per year (1.52 (0.61, 3.80)) compared to pdga patients (0.20 (0.12, 0.32)). the irr is 7.62 (2.71, 21.3). across both subgroups, the incidence of hospitalization was higher in dga patients who had low helper and naïve t-cells. there is ongoing analysis into hospital-related infection and resistance profiles. notable frequencies include bacteremia ( > 5%), invasive viral disease ( > 1%), and opportunistic infections ( > 1%). conclusions: children who had cdga were 662% more likely to have an infection requiring hospitalization than children who had pdga, emphasizing the need for thymus transplant for cdga. further analysis of infection type and patient outcomes is critical to enhancing management of this unique patient population. abstract/case report text antibody cross-reactivity among flavivirus has been documented. in recent times zika virus has been emerging in pockets of the mosquito-infested regions, while southwestern saudi arabia is known for arthropod-borne viral diseases and we do not know the incidence or even presence of zika virus in this region. it is restricted to predict the igm and igg antibody detection ranges owing to limited data and colossal cross-reactivity among the zika and other flaviviruses. we tested sera from 217 pregnant women irrespective of their clinical presentation for zika and dengue igm, igg respectively. the zika positive samples were further confirmed by plaque reduction neutralization tests (prnt). from our results, 3.6% (8) cases were positive for zika igm against 1.8 % (4) positivity to igg. when these samples were assessed for dengue igm and igg, we observed 1.3% (3) seropositivity for igm and igg respectively. there was no single sample positive for both igm and igg of zika or dengue. however, we observed one sample positive for both zika and dengue igm. upon mapping the overlapping serotiters, there was no significant correlation observed between the dengue igm and igg. whereas zika igg positive sample showed high serotiter for dengue igg indicating the contribution of cross-reactivity for observed zika positivity. screening for the incidence of zika, therefore, becomes particularly hard in a population that has the presence of pre-exposure of dengue and this cross-reactivity makes it hard to determine the zika incubation and antibody prevalence confounded with other flaviviruses. abstract/case report text introduction humoral pid diagnostic protocol includes the analysis of the immune response to different protein and polysaccharide antigens (ags) (1) . although the analysis of the immune response against the polysaccharides vaccine from pneumococca has been the standard method, the use of s. typhim vi vaccine has appeared as a good alternative (2) . in this report we show the results obtained with the use of s.typhim vi in 27 adults patients attending the pid outpatient clinic. material and methods patients with humoral-suspected pids were challenged with typhoid polysaccharide vaccine (typhim vi®; sanofi-pasteur). serum was obtained on basal and after 4 weeks of vaccination. specific igg levels against s.typhim were measured using "vacczyme tm human anti-salmonella typhi vi igg enzyme immunoassay kit" (binding-site). results a total of 90 adult patients attended the pid clinics during nov 2018-nov 2019. from those, 27 patients were fully evaluated using a humoral-suspected pid algorithm that includes the s.typhi vaccination. in total 13 male and 14 female patients completed the protocol and were analyzed. twenty patients were considered as responders (ratio pre/ post >3x) whereas 7 patients were non-responders. discussion the main advantage of assessing polysaccharide immune response using s.tyhpim is the usual lack of specific igg at the moment of the initial evaluation. in this serie, just one patient has a high basal level (#2-vaccinated in the past). thirteen patients had basal levels below the detection limit of the test (7,4u/ml) and 13 patients between 7,4 and 23,35u/ml, that has been described as a cut otf level for nonimmunised individuals (personal experience,3,4). regarding the polysaccharide immune response as a tool to distinguish pid vs non-pid patients, the results showed a good correlation between those non-responders with more clinical relevant pid diagnostics. seven non-responders patients were subsequently diagnosed with a primary (* on table i ) and/or secondary id (** on table i ). despite this, there were 16 patients that we could have classified as strong responders (ratio >3x, absolute specific igg postvaccination level >100u/ml) and 4 patients considered as weak responders (ratio post/pre >3x, absolute specific igg postvaccination level < 100u/ml). strong responders were considered non-pid after including other clinical investigations and laboratory tests (cell subpopulation study, pcp response) whereas weak responders group consisted in some "minor" forms of pid, like isolated igm or ig subclasses deficits. more patients are needed to confirm this functional classification of pid patients regarding their s.typhi immune response. abstract/case report text activated pi3kδ syndrome (apds) is a primary immunodeficiency characterized by recurrent respiratory infections, as well as increased risk of chronic viremia with herpes family viruses, benign lymphadenopathy and b cell lymphoma. it is caused by heterogeneous germline gain-of-function mutations which ultimately lead to the hyperactivation of the phosphoinositide-3-kinase δ (pik3δ). pik3δ exists as a heterodimer composed of a catalytic and a regulatory subunit. it interacts with b cell receptors, t cell receptors, costimulatory and cytokine receptors, and is a key player in a signaling pathway involved in cell growth, proliferation and survival. apds1 is caused by mutations in the pik3cd gene, affecting its protein product p110 δ (catalytic subunit). apds2 is caused by mutations in the pik3r1 affecting p85a (regulatory subunit). short syndrome is a rare multisystem disorder characterized by short stature, hypertextensible joints, ocular depression, reiger anomaly and tooth eruption delay. the primary causes of short syndrome are heterozygous loss-of-function mutations in the pik3r1 gene. the combination of apds2 and short syndrome is very rare, with only few cases described in the literature. in this report we present a teenager with a pathogenic variant in the pik3r1 gene, and phenotypic characteristics of both apds2 and short syndrome. our patient is a 17-year-old female with a history of growth delay and short stature, delay tooth eruption, recurrent sinopulmonary infections and hypogammaglobulinemia. evaluation performed at a prior institution for recurrent infections revealed low igg levels. she did not initiate therapy at that time and was lost to follow up for several years. at the time of our initial evaluation she reported continued recurrent episodes of upper respiratory infections and sinus infections requiring antibiotic treatment that often did not clear the infections. her physical exam was relevant for short stature (1%ile, z=-2.33), low weight for age ( < 1%ile, z=-2.69) and hyperextensibility. her facial features were significant for prominent forehead and triangular face. given concern for immune deficiency, a complete immune evaluation was obtained. her workup revealed low igg levels, with igm and iga within normal limits. she did not have protective titers to s. pneumoniae, h. influenza or diphtheria and tetanus. after administration of vaccine boosters, she was able to generate a response to all vaccines except for tetanus. she had remarkably low absolute b cells (34 cells/ul) and percentage (1 %), and low cd4:cd8 ratio (0.84). she was started on amoxicillin prophylaxis and monthly ivig replacement therapy. invitae immunodeficiency panel genetic testing was sent and revealed a pathogenic loss of function variant in an intronic splice site in the gene pik3r1 (c.1425+1g>c). after initiating treatment with ivig, her sinus infections significantly improved and she has not had any further episodes. igg levels have remained within normal limits with monthly ivig therapy. this pathogenic variant had been previously associated with apds2; however, it had not been associated with short syndrome. the mechanisms that link both conditions is yet to be identified. this case report emphasizes the importance of screening for comorbidities associated with short syndrome in apds2 patients, and vice versa. finding the genetic diagnosis for patients with suspicion of primary immunodeficiency (pid) is becoming increasingly important in the management of primary immunodeficiency and estimating the risk for family members. we constantly increase the diagnostic yield for pids by improving the sequencing technology, updating the panels with new genes discovered related to pid, and finding diagnoses from difficult to sequence regions and regions with high homology. here we report our experiences with nearly 1700 patients suspected with pid. moreover, we provide a case example, how we increase the diagnostic yield by developing unique techniques for specific genes which cannot be reliably analyzed by ngs alone. diagnostic yield including all immunology related panels was 14.9% (253/1698). the majority of the tested individuals were males (1343/1698, 79.1%) and the most common age of testing was between 10 to 20 years (468/1698, 27.6%). the highest diagnostic yield 20.6% (75/364) is in children from ages 0 to 5 years, whereas in patients over 60 years of age the diagnosis was found for only 6.5% (4/62) of the patients. in two patient cases, our cnv detection algorithm indicated a homozygous deletion in the index patient samples potentially covering the whole ncf1 gene. additional bioinformatic analysis targeting specifically two coding positions that differ between the ncf1 gene and the two pseudogenes showed that all reads in those positions originated from the pseudogenes. homozygous deletion in the ncf1 gene was further confirmed by sanger sequencing two regions in ncf1 with primers that specifically bind to either ncf1 or the pseudogenes. while clean ncf1 sequences from both regions were obtained for a control sample, no ncf1-specific amplification product was obtained for the index patient samples. pseudogenes were amplified and sequenced successfully in both index patient samples and positive control samples. loss-of-function of ncf1 is a well-established mechanism leading to cgd and by overcoming the difficulties regarding ncf1 deletion detection by ngs, we can improve diagnostic rate in individuals affected with cgd. gata2 deficiency can lead to a broad spectrum of clinical and hematological phenotypes; in some cases, nk cell deficiency is the primary manifestation, resulting in a greatly increased susceptibility to viral infections and malignancy. gata2-deficient patients, particularly those who suffer from severe viral infections, have reduced frequencies of peripheral blood nk cells and loss of function in the existing nk cells. specific loss of the less mature (cd56^bright) nk cell subset is a hallmark of the immune phenotype in gata2 deficiency, suggesting that generation or survival of nk cell precursors is impaired. given the remarkable spectrum of clinical phenotypes in gata2-deficient patients and the poorly understood biology underlying their nk cell defect, we sought to characterize circulating nk cells on a single-cell level. we performed single-cell (sc)rnaseq of lineage-depleted innate lymphocytes from a patient with gata2 deficiency. as expected from flow cytometric phenotyping of peripheral blood cells from this and other patients, scrnaseq revealed decreased representation of canonical cd56^bright cells. within the cd56^dim population, we identified two nk cell populations that were seemingly unique to the gata2 deficient patient relative to a healthy donor. pathway analysis defined the first of these populations (population 1) by the expression of genes associated with cellular response to stress, extracellular stimulus and inflammation, as well as programmed cell death and regulation of proliferation and apoptosis. the second population (population 2) was defined by genes associated with nk cell chemotaxis, cytokine responses and interferon signaling. to extend our findings, we performed scrnaseq of 6 additional healthy donors and analyzed an additional gata2-deficient individual who was clinically asymptomatic (yang et al. 2019). of note, we detected population 2 in seemingly healthy cmvnegative individuals, suggesting it was not uniquely a result of gata2 deficiency but associated with an inflammatory response and not related to adaptive nk cells generated in response to cmv infection. population 1, on the other hand, only appeared in our symptomatic gata2-deficient patient. we additionally performed bulk gene expression analyses from an unrelated gata2-deficient patient that confirmed the altered expression of genes associated with both novel cell populations. current efforts are focused on better defining the functional response of nk cells in these patients and confirming the identification of our novel populations by mass cytometry (cytof). together, our data define the heterogeneity and complexity of nk cells in gata2 deficient and healthy individuals. perforations have been reported with tocilizumab, a monoclonal antibody of the interleukin 6 (il-6) receptor, suggesting that il-6 signaling plays a role in intestinal wall integrity. as il-6 signals through stat3, we sought to investigate the potential association between lof stat3 and intestinal perforations, as well as the incidence and outcome in our patient cohort. methods: we performed a retrospective chart review of patients with lof stat3 (n=158) followed at our institution, looking for those with non-malignancy associated spontaneous gastrointestinal perforations. the demographic information, stat3 mutation, comorbidities at the time of perforation, clinical presentation, management, and clinical outcomes were compiled results: ten lof stat3 patients were identified as having documented intestinal perforations, an approximate rate of 6%. one perforation was the initial presentation of diffuse large b cell lymphoma (dlbcl) of the duodenum and liver, and was excluded from the rest of the analysis. the other nine perforations occurred between 4 to 60 years old (mean:26), and 55% were female. stat3 mutations were localized to the dna binding domain (n=4) and the sh2 domain (n=5). two of the perforations occurred while inpatient for lung infection. another occurred while recovering from pneumonia at home. two perforations were associated with the initial diagnosis of diverticulitis (at age 26 and 60). one perforation occurred in the terminal ileum, one in the cecum, one in the transverse colon, and six in the sigmoid. five patients underwent primary closure of the bowel. four patients required a temporary ostomy, with subsequent successful ostomy reversal. only one patient has since died of pulmonary hemorrhage, the other patients are alive with a mean of 6 years post perforation follow-up, with no recurrence of perforation. one patient with prior sigmoid resection required ileal resection 6 post perforation due to as massive intestinal bleed. conclusion: spontaneous gastrointestinal perforations occurred in our lof stat3 cohort at a rate of approximately 6%. one case was associated with malignant infiltration of the gastrointestinal tract and two cases were associated with diverticulitis both known risk factors for perforation. although the pathogenesis of the perforations in lof stat3 remains unclear, the connective tissue phenotype likely contributes as well as the association with diminished il-6 signaling, as has been demonstrated with the perforations and tocilizumab. abstract/case report text background granulomatous and lymphocytic interstitial lung disease (glild) is a life-threatening complication that occurs in patients with common variable immunodeficiency (cvid) and monogenic cvid-like disorders, but the optimal treatment is unknown. objective to determine if the use rituximab and azathioprine (rtx-aza) or rituximab and mycophenolate mofetil (rtx-mmf) would improve the radiographic abnormalities as determined by high-resolution computed tomography (hrct) of the chest and/or pulmonary function tests (pfts) in patients with cvid and glild. methods this is a retrospective study of patients seen from july 2006 to december 2018 with cvid and glild who completed immunosuppressive therapy (rtx (375mg/m2) for 4 weeks, repeated at 6-month intervals for 3 or 4 total courses, and aza (0.5-2.0 mg/kg/day) or mmf (250mg-1000mg-bid) for 18 months). complete pfts and hrct scans were performed prior to therapy, at the conclusion of therapy, and periodically thereafter. hrct scans were blinded, randomized, and scored independently (in pairs) by two radiologists. all patients underwent whole exome sequencing (wes). number (percentage) and median (interquartile range) were reported for categorical and continuous variables, respectively. differences between pre-and post-treatment and between relapse and post-relapse hrct scores and pft parameters were analyzed with wilcoxon signed ranks test. kaplan-meier survival curves were also done. unadjusted one-sided p-values < 0.05 were considered statistically significant. results the glild cohort (n=39) had a 2:1 female predominance, and age at glild diagnosis was 36 (25-43) years (table 1) . autoimmunity was present in the majority of patients, with thrombocytopenia (28 (72%)) the most common manifestation. enteropathy (3 (8%)), inflammatory bowel disease (1 (3%)), and nodular regenerative hyperplasia of the liver (6 (15%)) were also present. splenomegaly (31 (79%)) was present in the majority, but polyarthritis (0 (0%)) was notably absent. twenty (51%) patients had been previously treated with systemic steroids. hrct scores substantially improved between pre-and posttreatment for rtx-mmf (p=0.0005) and rtx-aza (p < 0.0001, figure 1 ). fev1 (p=0.019), fvc (p=0.0011), and tlc (p=0.0071) also improved, but dlco (p=0.060) was unchanged (figures 2 and 3 ). excluding two (5%) patients who died 2.3 and 2 years after therapy of respiratory failure (1 (3%)) and septicemia (1 (3%)) respectively, 9/37 (24%) patients relapsed 3.2 (2.8-3.5) years following therapy with an estimated 60% relapse rate after 5 years ( figure 4 ). as of december 2018, 6 of 9 patients that relapsed showed improvement in hrct scores (p=0.031), and the remaining 3 patients are still undergoing retreatment ( figure 5 ). four (10%) pneumonias occurred during immunosuppressive therapy, all with severe restrictive lung disease . eight (21%) patients had a damaging mutation in a gene known to predispose (tnfrsf13b, n=3 (8%)) or cause a cvid-like primary immunodeficiency (ctla4: 2 (5%); kmtd2: 2 (5%); birc4: 1 (3%)). immunosuppressive treatment improved the hrct scores regardless of the absence (p < 0.0001) or presence of a damaging mutation (p=0.0039) ( figure 6 ). conclusion combination chemotherapy appeared to be effective in improving the radiographic abnormalities and pulmonary function of patients with cvid and glild. a majority of patients had sustained remissions, regardless of the presence or absence of a monogenic disorder. iqr=interquartile range, cvid=common variable immunodeficiency, glild=granulomatous and lymphocytic interstitial lung disease, vats=video-assisted thoracoscopic surgery, tbx=transbronchial biopsy, ms=mediastinoscopy excluding b cell malignancy, pft=pulmonary function test, h/o=history of, dz=disease, nrh=nodular regenerative hyperplasia, ibd=inflammatory bowel disease table 1 . baseline patient characteristics since its first description the number of cases has increased progressively [1] . although described as a predominant antibody deficiency [1] , various complex phenotypes have been associated with mutations in this gene [2] . case description. female patient with no remarkable history until 2 years old, when she suffers from a persistent fever associated with purulent abscesses in venipuncture areas and hyperleukocytosis with neutrophilia, so she was treated for about 8 months in 3 hospitals in the city of barranquilla before she was referred to our institution. the patient's clinical picture consisted of persistent fever unresponsive to broad-spectrum antibiotic treatments, skin abscesses, left subphrenic abscess and toes osteomyelitis. the microbiological studies documented a bacteremia by acinetobacter baumannii and isolation in bone marrow of candida parapsilosis. during her care stay in barranquilla, she was approached as a chronic granulomatous disease versus job's syndrome, she received two doses of immunoglobulin with partial control of symptoms. due to the recurrence o f fever, abs ces ses and hyperleukocytosis, they decided to refer to our institution for further studies. upon admission to our institution, the patient presented nutritional compromise, with spontaneous resolution of fever but persistence of high acute phase reactants, with significant improvement of leukocytosis. all the cutaneous lesions she presented were debrided at the site of remission. immunoglobulin levels, lymphocyte populations and dyhidrorhodamine test were normal. she remained with no weight gain, constipation, abdominal distension and hepatic involvement with elevated liver enzymes and prolonged coagulation times. new bone compromise was documented. inflammatory bowel disease, neoplastic or chronic infectious disease involvement was ruled out. during the stay in our institution no microbiological isolation was documented. skin, colon and bone tissue biopsies were performed and extra-institutionally performed liver biopsy were examined, showing as a single common finding leukocytoclastic vasculitis in all tissues. given the heterogeneous nature of the condition, the diagnostic possibility of an immune dysregulation disorder was considered and a therapeutic trial with nsaids and prednisolone at 1mg/kg/day was started, as well as genetic studies by exome sequencing. the exome results documented a novel mutation in the pik3cd gene [c.1895t> a (p.phe632tyr)] as probably pathogenic. the patient has presented a clinical improvement and a significant decrease in inflammation markers. at the moment, we are waiting for the performance of functional tests to define the definitive therapy for this patient. conclusions. this case description highlights the diagnostic difficulties that face in developing countries, where the nonavailability of functional testing has implications on the diagnosis opportunity and establishment of optimal therapeutic for patients with complex diseases such as primary immune regulatory disorders abstract/case report text background: autoinflammatory syndromes, a wide family of diseases, defined as attacks of inflammation that are unprovoked (or triggered by a minor event) and are primarily related to dysregulation of the innate immune system. periodic/recurrent fever syndromes were the former name of these diseases. however, only in two conditions: cyclic neutropenia (cn) and periodic fever, aphthous stomatitis, pharyngitis, and adenitis (pfapa) are febrile episodes truly periodic. for pfapa, although diagnostic criteria differ and there is no consensus research definition, patients are usually not difficult to recognize based on clinical course and presentation1. high index of suspicion and understanding the parental experiences and descriptions of febrile episodes is imperative in facilitating early recognition and timely diagnosis. aims: to standardize and summarized the clinical presentation of pfapa, based on parental descriptions and providers observation of febrile episodes. methods: utilizing a query for the icd-10 diagnosis code m04.8( + ) we identified a cohort of children diagnosed and managed for periodic fever, excluding those with monogenetic mutation (e.g. blau, majeed) and those with chronic illness. we reviewed the charts for documented parental report and provider observation of febrile episodes. standardized signs and symptoms were recorded for each patient [ table 1 ]. results: a cohort of 75 children, 35 boys (47%) and 40 girls (53%) with documented, cyclic episodes of fever >101, was identified. the average age at diagnosis was 3.77 +/-2.34 years. classic symptoms were reported or observed in 85% of patients (64). more than half (68%, 51 patients) had documentations of other symptoms, usually reported by parents to occur sporadically during some fever episodes. decreased oral intake and general "ill appearance" was reported by parents in 91% of patients. when reporting the time intervals, parents usually reported similar length for each episode, typically between 3-5 days, and regular interludes, typically between 3-4 weeks. the findings are summarized in table 2. discussion: the findings presented here are in concordance with previously published data describing pfapa as a syndrome affecting young, generally healthy children with identical episodes of fever lasting for a few days, recur with regularity. our data support the approach that parental observation is fundamental in identifying the unique pattern of illnesses. engaging the parents with directed interview is crucial to establish this clinical diagnosis in a timely fashion, prevent misdiagnoses of future febrile episodes as presumptive infections, and avert unnecessary antibiotics courses. this cohort adds the observation that up to 15% of patients who display this identifiable pattern of illnesses, do not present with aphthous stomatitis, pharyngitis, or adenitis. these classic symptoms although common, are not the rule. the cyclic chronicity of febrile episodes associated with general ill appearance (but not lethargy), and decreased po, in an otherwise healthy child is the clinical gold-standard of this condition. abstract/case report text heterozygous mutations in nfkb1 are frequently identified among immunodeficient patients with highly variable clinical symptoms. in a world-wide collaborative effort, we characterized the clinical and cellular phenotype and the management of 251 of these patients harboring 106 distinct nfkb1 variants. nfkb1 encodes the transcription factor precursor p105 which is processed to p50 (canonical pathway). known pathogenic variants cause p50 haploinsufficiency (due to protein decay) or p105-skipping (with expression of p50-like forms). most variants however are single amino acid changes with yet unknown effects. all sequence changes were assessed in silico for their probability of pathogenicity including 32 variants which were additionally tested in vitro. these analyses include the sub-cellular protein localization (microscopy), protein expression, stability and processing (western blotting), transcription factor activity (reporter assay) and dna-binding ( figure 1 ); the associated unadjusted odds ratio (or) was not significant, suggesting igrt had restored cases to a similar baseline infection risk as the controls. in a multivariate conditional logistic regression model adjusting for the significantly higher occurrence of risk factors in cases compared with controls in the preindex period, cases were associated with a 31% lower adjusted odds of major/severe infections in the post-index period versus controls (or=0.69; p=0.04). conclusions: patients who required treatment with igrt (privigen®/ hizentra®) had previously experienced more major/severe bacterial infections than those not needing treatment with igrt. however, igrt was associated with a reduction in the risk-adjusted odds of major/severe bacterial infections compared with non-igrt treated sid patients with haematological malignancies. abstract/case report text introduction: real-world data are lacking as far as identifying patients with secondary immunodeficiency (sid)/hypogammaglobulinemia who may benefit most from interventions to protect them from potentially fatal infections. this study aimed to identify risk factors for major/severe infections in patients with sid with underlying haematological malignancies. methods: a retrospective database analysis was conducted using the iqvia real-world data adjudicated claims -us database (study period: january 2010-september 2018). inclusion criteria were adults newly diagnosed with sid (first diagnosis termed the index date), with ≥12months continuous health plan enrolment pre-index (baseline period) and a minimum of 3 months' data post-index (mean: 615 days), with chronic lymphocytic leukaemia, multiple myeloma and/or non-hodgkin's lymphoma and without claims for any ig therapy in the 12-month baseline period. patient characteristics in the 12-month baseline period were assessed. over the post-index period, antibiotic/antiviral use and frequency of infections were assessed. the frequency of major/severe infections was determined using diagnosis codes for bacterial, viral, fungal, parasitic, other or unspecified causal pathogen infections. major/severe infections were defined as those requiring inpatient hospitalisation with an infection diagnosis code and/or use of intravenous (iv) antibiotics or iv antivirals in an outpatient setting. a multivariate cox proportional hazards (ph) model evaluated baseline patient characteristics associated with risk of major/severe infections post-index. results: a total of 4,066 patients met the inclusion criteria. the mean age of patients was 57 years and 56.0% were male. in the 12-month baseline period: 75.6% of patients received cancer treatments and 87.0% of patients received antibiotics (27.1% iv antibiotics). a total of 79.5% of patients experienced any infection, 65.6% experienced ≥2 infections and 30.4% experienced major/ severe infections. the mean number of infections over the baseline 12 months was 9.5 for any infection (at the unique diagnosis code level) and 0.7 for major/severe infections (unique hospitalisations with any infection diagnosis code and/or unique days with an outpatient iv antibiotic or iv antiviral). in the post-sid diagnosis period, 46.4% of patients had major/severe infections; of the major/ severe infections, 34.0% were identified as bacterial, 11.6% were viral, 6.3% were fungal, while 48.2% did not have a causal pathogen specified. a total of 21.3% of patients experienced one severe/ major infection and 10.5% experienced ≥4 severe/major infections ( figure 1 ). the mean annualised number of major/severe infections post-index was 1.5. receiver operating characteristic (roc) curve analysis to optimise sensitivity versus false positives in identifying those at risk of major/severe infections post-index identified a cutoff point of three bacterial infections in the baseline pre-index period as a potential optimal trigger to consider treatment to avoid major/severe infections post-index ( figure 2 ). the multivariate cox ph analysis suggested that hospitalisations, infections (≥3), or antibiotic use in the 12-months pre-index (prior to sid diagnosis) were predictive of major/severe infections post-index (post-sid diagnosis) (all p < 0.0001). conclusion: infections are common in patients with haematological malignancies and sid. key baseline predictors for major/severe infections in patients with an sid diagnosis were a history of infections, hospitalisations or antibiotic use. unfortunately, ms/ms detection is limited by the extremely low (e.g., pmol/l) protein concentrations in blood cells. peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-srm) is a robust method for quantification of low abundance proteins in complex matrices, including dried blood spots (dbs). in a study of 37 patients, immuno-srm reliably identified wiskott-aldrich syndrome (was) and x-linked agammaglobulinemia (xla) patients using direct quantification of proteins responsible for disease (front. immunol., 2018). we further expanded our approach for x-linked chronic granulomatous disease (x-cgd), ada and dock8 deficiency. marker proteins representing platelets, nk cells, and t-cells have also been analyzed to provide additional information about disease processes. these results demonstrate the utilization of immuno-srm as a sensitive platform for multiplexed signature peptide quantification and its potential for pidd newborn screening and clinical diagnosis from dbs. methods: candidate peptides were selected based on ms/ms sensitivity and uniqueness in the proteome. anti-peptide monoclonal antibodies (mabs) were then generated for peptide enrichment from dbs. blood from normal controls, xla, was, xl-cgd, dock8 and ada deficiency patients was collected after consent on filter paper, dried, and stored at -20°c. proteins were extracted from dbs, digested with trypsin, and enriched using mabs bound to magnetic beads. the enriched peptides were then eluted and analyzed with a waters xevo tq-xs. results: a multiplexed immuno-srm panel has been generated for screening eight signature peptides representing five pidd-specific and three cell-type specific proteins from dbs. limits of detection and quantification were femtomoles of peptide, the assay showed a broad linear range, and intra-assay and inter-assay coefficients of variation were < 20%. in samples from 13 xla, 8 was, 3 xl-cgd, 1 dock8 and 1 ada deficiency patients, signature peptides are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. also included in the multiplex panel are cell specific markers for platelets (cd42), t-cells (cd3ɛ), and nk cells (cd56). diagnostic cutoffs for each peptide concentration have been established. in was patients, cd42 levels were significantly reduced consistent with characteristic thrombocytopenia. immuno-srm also has the ability demonstrate the effects of pidd treatment. a was patient analyzed before and after bone marrow transplant showed normalized was protein and cd42 after treatment. two ada deficiency patients showed normal levels of ada enzyme after rbc transfusion. finally, a high-throughput (ht) immuno-srm method screens pidd-specific peptides in a 2.5-minute runtime meeting high volume nbs workflow requirements. this ht method returned identical results to the standard immuno-srm pidd panel. conclusions: the data herein demonstrate the feasibility of using immuno-srm as a broad clinical diagnostic for identifying and studying pidd patients from easily collected and shipped dbs. significantly, ht immuno-srm workflows represent a promising potential option for nbs of pidds and other congenital disorders. 7 chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih abstract/case report text we have previously used the artificial thymic organoid (ato) system, based on the 3d aggregation and culture of a delta-like canonical notch ligand 4-expressing stromal cell line (ms5-dll4) with cd34+ cells, to study t cell differentiation from cd34+ cells obtained from patients carrying defects that are intrinsic to hematopoietic cells (rag1-2, ak2, il2rg) or that affect thymus development (digeorge syndrome). we now report results of in vitro t cell differentiation of cd34+ cells obtained from patients with either haploinsufficiency or dominant negative (dn) mutations of the ikzf1 gene. ikzf1 is an essential transcription factor expressed throughout hematopoiesis and involved in both lymphocyte and myeloid differentiation. heterozygous germline mutations in ikzf1 give rise to distinct clinical phenotypes, depending on the nature of the mutation. in particular patients with ikzf1 haploinsufficiency present with common variable immunodeficiency (cvid) associated with b cell immune deficiency, b-all susceptibility, and autoimmune manifestations. no clinical t cell defects are evident among these patients, except for elevated naive and central memory cd3+cd8+ t cells. in contrast, patients carrying dn ikzf1 mutations present with combined immunodeficiency (cid) characterized by the presence of an increased proportion of naïve t cells, associated with defective generation of memory t cells, impaired t cell activation, signaling and proliferation, reduced t-helper (th) polarization, and susceptibility to pneumocystis pneumonia. different mouse models of ikzf1 mutations have been developed, however their phenotype does not fully match what reported in patients, and in some models indicates a more severe defect in t cell development. to address these controversies and to gain novel insights into the effects of distinct ikzf1 mutations on human t cell development, we used the ato system to analyze progression of t cell development from cd34+ cells obtained from one patient with ikzf1 haploinsufficiency and one patient with dn ikzf1 mutation. both patients showed a similar early block in t-cell differentiation a t p re -t c el l s ta g e. ho w ev e r, th e p a ti e nt wi t h i kz f1 haploinsufficiency showed a more pronounced leakiness, with a residual production of cd3+tcrab+ cells, which could account for the milder t-cell phenotype presented in this type of patients. interestingly, the dn patient presented an increased accumulation of cd4-cd8b-cd8aa+ cells. these results show an unexpected role for ikzf1 in humans in early stages of t-cell differentiation and indicate ikzf1 as a necessary factor for the induction of cd8b expression in t cells. abstract/case report text background: pediatric acute liver failure without an identifiable cause (indeterminate palf/ipalf) is associated with increased rates of liver transplant and mortality. aplastic anemia (aa) may develop weeks after the diagnosis. the immunologic mechanisms that contribute to disease pathogenesis have not been clearly elucidated. we report detailed immunophenotyping of a patient with ipalf/aa. case: a previously-healthy 7-year-old male was admitted for acute hepatitis presenting with jaundice and hepatosplenomegaly. evaluation for infectious, toxic, metabolic, autoimmune, and rheumatologic disorders was negative. he was pan-lymphopenic (cd45+alc 445 cells/μl) with an inverted cd4:cd8 ratio of 0.2 on admission. liver biopsy showed severe portal, interface, and lobular inflammation characterized by activated sinusoidal macrophages and perforinexpressing cd8+t-cells. compared to a healthy control, the percentage and number of peripheral blood cd8+t-cells expressing perforin (20%v.94%, 85 v.153 cells/μl) and granzyme-a/b (15%v.90%, 66 v.147 cells/μl) was also increased, while percentages of perforin+ (96%) and granzyme-a/b+(98%) nk cells were normal. bone marrow (bm) showed 75% cellularity with rare hemophagocytosis. serum cytokine analysis demonstrated il-18 4052 pg/ml, il-18-binding-protein 23003 pg/ml, cxcl9 4436 pg/ml, and sil-2rα 7979 u/ml, consistent with smoldering hemophagocytic lymphohistiocytosis (hlh), but he did not meet hlh diagnostic criteria. genetic sequencing did not identify pathogenic variants in 207 genes associated with primary immunodeficiencies. he was diagnosed with ipalf and treated with three doses of anakinra and two weeks of ruxolitinib, followed by prednisone 1-2 mg/kg/day and intravenous immunoglobulin 1g/kg/month. immunophenotyping performed after two months of therapy showed persistent inversion of the cd4:cd8 ratio with small expansions of cd3+cd4-cd8-cells and tcr··+t-cells. in the cd4+t-cell subset, there was a substantial paucity of naïve cells, with effector memory t cells (tem) being more abundant than central memory t cells (tcm). in the cd8+t-cell subset, the majority of cd45ro+cells were tem with no detectable tcm, and 49% of all cd3+t-cells were cd8+temra. in both cd4+ and cd8+t-cell subsets, activated (hla-dr+) and senescent (cd57+) subpopulations were increased, and the majority of cells expressed the exhaustion marker pd-1. the hepatic inflammatory infiltrate similarly reflected repetitive antigenic stimulation, with expansion of cd103+cd8+t-cells. quantitative immunoglobulins and total memory b-cells and plasmablasts (cd19+cd27+) were normal for age. however, there were no circulating iga-memory b-cells and a reduced number of iggswitched memory b-cells (table 1) . given the severity of his phenotype and bm hypocellularity (5%), allogeneic hct was performed using a matched-related-donor (10/ 10) with conditioning of flu+cy+alemtuzumab. at d+30, he shows improved liver function but persistent pancytopenia, with transfusion-dependence for platelets. discussion: to our knowledge, this is the first description of detailed immunophenotyping in blood from a patient with ipalf/ aa. other studies have identified distinguishing hepatic infiltrates and cytokine/chemokine profiles that suggest excessive activation of cytotoxic t-lymphocytes and macrophages contribute to disease pathogenesis (alonso et al, 2017). our preliminary data supports this hypothesis and expands the spectrum of immune dysregulation in the t and b cell compartments, proposing a primary immune etiology. immune dysregulation may be concordant with hyperinflammation and cytokine storm, the latter offering potential therapeutic targets. early diagnosis and treatment of immune dysregulation may prevent development of aa. background: x-linked agammaglobulinemia (xla) is one of the first inborn errors of immunity identified, with thousands of patients described to date. infections originally dominated the clinical phenotype, but early diagnosis and immunoglobulin replacement allowed for long term survival as well as recognition of late-onset complications. nodular regenerative hyperplasia (nrh) of the liver is a silent cause of non-cirrhotic portal hypertension. nrh underlying pathophysiology remains blurry and the disease has no specific treatment. nrh has been increasingly reported in primary immunodeficiency but data in xla are very limited. objectives: to assess and characterize nrh in patients with xla. methods: we retrospectively reviewed the medical records of all xla patients referred to the nih between 1994 and 2019. hepatology evaluation and liver biopsies were performed when clinically indicated. patients were stratified into nrh+ or nrhgroups, according to their nrh biopsy status (patients with no liver biopsies were classified as unknown). laboratory values are presented as medians. fisher's exact test and mann-whitney test were used to compare categorical and continuous variables, respectively. results: twenty-one xla patient records were reviewed, with a median age at start of follow-up (f/u) of 17y and a median duration of f/u of 10 years. eight patients underwent at least one liver biopsy of whom 6 (29% of nih xla cohort) were nrh+. the median age at nrh diagnosis was 20y (17-31). among patients who had liver biopsies, alanine aminotransferase (alt) levels were mildly elevated in all, while alkaline phosphatase (alp) levels were only increased in nrh+ patients (p=0.04). both nrh+ and nrhgroups had similar aspartate aminotransferase (ast) levels at baseline but higher values were observed at the end of f/u in the nrh+ group (85 vs. 32 u/l, p=0.04). persistently low platelet count ( < 100k/μl for more than 6 months), mildly to highly elevated hepatic venous pressure gradient (hvpg) and either hepatomegaly and/ or splenomegaly were present in all nrh+ patients. in opposition, neither persistently low platelet counts, nor hepato-or splenomegaly were present in the two nrh-patients evaluated. hvpg was normal in the only nrh-patient tested. all-cause mortality was higher among nrh+ patients (5/6, 83%) than in the rest of the cohort (1/15, 7% among nrh-and unknown patients, p=0.002). conclusions: based on our retrospective analysis, nrh appears as an underreported, frequent and severe late-onset complication in xla, which is highly associated with increased mortality. persistent thrombocytopenia, elevated alp, elevated hvpg, hepato-and/or splenomegaly were common in liver biopsyproven xla/nrh+ patients and distinguish them from xla/ nrh-patients. based on nrh prevalence, severity, lack of specific treatment and poor outcome in xla, immune-reconstitution (rather than igg replacement and infectious prophylaxis) should be considered early in this population in order to prevent fatal long term complications. abstract/case report text introduction: barth syndrome (bths) is an x-linked recessive disorder caused by a mutation in the tafazzin (taz) gene resulting in an inborn error of cardiolipin phospholipid metabolism (an important mitochondrial inner membrane lipid). it is commonly characterized by intermittent neutropenia and cardiac and skeletal myopathies. we present a case of bths with associated lymphopenia and hypogammaglobulinemia, which has not been previously described in the literature. case report: a two-month old male, born full term with normal newborn screening, was first admitted for rsv bronchiolitis. at this time, patient underwent an echocardiogram given his older brother with hydrops had died hours after birth and on autopsy was found to have dilated cardiomyopathy (dcm). patient was similarly noted to have dcm and thus had whole exome sequencing done that showed a hemizygous mutation in the taz gene (c.639g>a). this novel variant resulted in early termination of the protein (p.trp213ter) with concern for loss of function. in regard to patient's first year of life, he had frequent uri symptoms, 6 episodes of acute otitis media requiring tympanostomy tubes, but no documented pneumonias or other serious bacterial infections. patient also had gross developmental delay, particularly motor, and feeding difficulties with persistent failure to thrive requiring g tube placement. his absolute neutrophil count ranged from 900-6800 cells/mm3 in the first year. at age 13 months, patient was found to be in acute decompensated heart failure with concern for myocarditis (ck 15,313 u/l, troponin i 3.006 ng/ml) as well as acute hypoxic respiratory failure with respiratory cultures growing pseudomonas. he was incidentally found to have an igg level of 188 mg/dl (normal for age 345-1213) and treated empirically with ivig. when seen by immunology, further workup showed persistent b cell lymphopenia (absolute cd19 of 167-377/mm3). he also had a low initial nk cell count (78-84/mm3, later normal) with normal cd8 and cd4 t cell counts. tetanus and hib titers could not be assessed as he had recently received ivig. his igg trended up to 931 mg/dl a few days after initial ivig and then subsequently dropped to 632 mg/ dl, with a level of 241 mg/dl two weeks following initial dose. workup for gastrointestinal or renal losses of immunoglobulin were negative. he also shortly after developed enterobacter bacteremia. his igg levels at this time continue to remain around 270 mg/dl. he subsequently required a heart transplant at age 14 months for his dcm. after transplant, he continued to improve from a cardiac standpoint, but his lymphopenia persisted and each time he was weaned off ivig, his hypogammaglobulinemia persisted at 275-372 mg/dl thus requiring additional ivig replacement over the course of the next 8 months. the remainder of immunoglobulins were normal initially, but the igm slowly dropped over time to 31-33.8 mg/dl. patient was started on weekly subcutaneous immunoglobulin replacement at 22 months, doing well clinically at age 24-month follow up. conclusion: here we present a patient with bths, with a novel variant, who had b-cell lymphopenia as part of his presentation with persistent hypogammaglobulinemia requiring ivig replacement. year fellow/ucla 2 associate professor/division of allergy, immunology, and rheumatology, university of california los angeles 3 chief of pediatric allergy and immunology/harbor-ucla 4 chief, laboratory of clinical immunology and microbiology/national institute of allergy and infectious diseases, niaid/national institutes of health, nih 5 biologist/laboratory of clinical immunology and microbiology, division of intramural research, national institute of allergy and infectious diseases, national institutes of health 6 project scientist/ucla abstract/case report text a 28-year-old female presented for combined immunodeficiency. at 2 years of age she was diagnosed with rag1 hypomorphism and started on ivig. as a child, she was hospitalized for pneumonias and cryptococcal meningitis. she suffered sinusitis, hepatitis, tooth abscess, cmv and herpes stomatitis. later, she experienced recurrent cutaneous abscesses, utis, vaginal yeast infections, and hidradenitis. she twice hospitalized recently for pneumonias and diagnosed with mycobacterium abscessus on bronchoscopy. she suffers onychomycosis, osteomyelitis and oral and esophageal candidiasis with odynophagia. on exam, she had white plaques on tongue and buccal mucosa. she had hyperpigmented plaques on forehead and cheeks and thickened nails. immune evaluation was significant for lymphopenia with alc 770 and thrombocytopenia with platelets 102k. b cells were nearly absent (2 absolute count) and nk cells were low at 19 absolute count. ige was absent, igm 23 mg/dl, iga 72 mg/dl and igg 872mg/dl (on replacement). her total cd3+ count was 791, cd4+ t cells were low at 17%, but cd8+ cells normal at 78%. the cd4+ t cells were mostly memory phenotype, which probably reflects lymphopenia-induced proliferation of a small number of clones. her cd8+ t cells also had an elevated amount of memory cells for age, but still had presence of naive cd8+ t cells. as expected with perpetual lymphopeniainduced proliferation, there was evidence of terminal memory (temra) in the cd8+ lineage. proliferative responses of t cells were modest. cd4+ t cells did respond to pokeweed, but less to pha and cona. there were no antigen specific responses. trecs were normal. esr was mildly elevated at 31. of note, her liver enzymes were elevated with alkaline phosphatase 556 and ast 116, presumably secondary to prolonged fluconazole use. w es r e v e a l e d a k n o w n p a t h o g e n i c v ar i a n t i n s tat 1 (nm_007315.3: c.537c>g (p.n179k)) as well as a heterozygous variant in rag1 p.m355t. the stat1 mutation is de novo and was previously published as a gain of function mutation. however, when we performed validation studies to evaluate cd4+ cells with stimulation to ifna, the patient had decreased pstat1 as compared with control. va7.2 analysis was performed to evaluate rag1 defect and showed 12% of t cells with va7.2 expression confirming that the rag1 defect is not clinically significant. she developed severe thrombocytopenia refractory to platelet transfusions and ivig. she was started on ruxolitinib which improved platelet counts. however, she presented with shortness of breath, persistent tachycardia and was found to have cmv carditis and hepatitis significant for echocardiogram with ef 23%. cmv pcr is improving with last check 815 iu/ml after 1 month of therapy with ganciclovir. we now are looking for evidence of socs1 to explain the decreased stat1 phosphorylation. genetic testing is critical when evaluating a patient with immunodeficiency. our patient demonstrates that genetic mutations cannot be taken at face value and should be evaluated and validated fully to optimize patient care. fellow/university at buffalo / oishei children's hospital abstract/case report text opportunistic infections (oi) are commonly seen in patients undergoing hematopoietic cell transplantation (hct). different strategies for antimicrobial prophylaxis are often employed in the transplant setting to reduce the likelihood of encountering infection. the predisposing risks for infections include the expected neutropenia and lymphopenia following conditioning, prolonged defects in cell-mediated and humoral immunity during the engraftment period, and iatrogenic immunosuppression by medications for graft versus host disease (gvhd). we report the case of a 4-year-old male with acute lymphoblastic leukemia, which relapsed to chronic myelogenous leukemic blast crisis, and failed a subsequent allogeneic hct with central nervous system relapse. he was subjected to a second allogeneic hct. his immediate post-second transplant course was complicated with skin and gut gvhd, and infection and/or reactivation of coronavirus, respiratory adenovirus, epstein-barr virus, and human herpesvirus 6. while the herpesviral infections were controlled with antivirals and rituximab, adenovirus c1 infection proceeded to involve the gastrointestinal tract, and proved persistent over several months despite use of cidofovir. the patient's gvhd and transplant-associated thrombotic microangiopathy necessitated use of further immunosuppressants, including the complement protein c5-binding eculizumab (an inhibitor of formation of the terminal c5b-9 complex), ruxolitinib (a janus kinase [jak] 1/2 inhibitor) and low-dose interleukin-2. h i s c l i n i c a l c o u r s e w a s f u r t h e r c o m p l i c a t e d b y stenotrophomonas maltophilia gut colonization and subsequent bacteremia, as well as multiple gram-positive bacteremia courses. at around day +190, there was a life-threatening pericarditis with pericardial effusion and respiratory distress, associated with pneumocystis and stenotrophomonas being isolated from bronchoalveolar lavage. this occurred despite the patient being on pentamidine prophylaxis. the patient eventually recovered on trimethoprim/sulfamethoxazole therapy. we discuss the various risk factors potentially contributing to each oi in this illustrative case. in particular, complement and jak inhibitor therapy are fairly new drugs approved for other indications, whose off-label use in transplant patients is increasing. both have recently been associated with certain oi in the literature, as they are in this patient. abstract/case report text background: autoimmune lymphoproliferative syndrome (alps) is characterized by chronic nonmalignant lymphadenopathy, splenomegaly, hepatomegaly, cytopenias, and other autoimmune manifestations. typically, the biomarker profile of patients with alps includes elevated tcr αβ+ dnt cells, serum igg, serum b12, serum il10 and soluble fas ligand (sfasl). hdl cholesterol can also be significantly low. alps is caused by lymphocyte accumulation due to defects in the fas-mediated apoptosis signaling pathway. these defects cause resistance to physiological apoptosis in lymphocyte populations that results in chronic lymphoproliferation. the molecular defect underlying most alps etiologies is attributed to heterozygous germline or somatic (limited to dnt cell subpopulation) pathogenic single nucleotide variants (snv) in fas. we describe copy number variants (cnvs) at the fas locus underlying alps in 3 unrelated families. methods: through the centralized sequencing initiative at at the national institute of allergy and infectious diseases (niaid), patients undergo genomic workup to identify molecular defects contributing to clinical phenotypes of immune system disorders. all patients receive exome sequencing and a subset of patients also receive array-cgh analysis. patients and results: we performed exome sequencing on 132 patients with a clinical diagnosis of alps. for 30 patients with no molecular defect through exome, we performed cnv analysis. in this cohort, we identified three patients with a copy number variant involving the fas locus. all patients presented with splenomegaly and lymphadenopathy in childhood with ages of onset ranging from 8 months to 9 years old. all patients experienced anemia, autoimmune neutropenia, and thrombocytopenia. they had biomarker evidence showing elevated serum b12 levels, sfasl levels, and elevated αβ+dnt cell populations. they were found to have very low hdl cholesterol in early childhood ranging from 5-8mg/dl (38-55 mg/dl). all patients had negative family histories for lymphoproliferative disorders and immunodeficiency. these patients had clinical presentations and biomarker profiles similar to alps patients with germline and somatic fas variants. patient 1: we detected a~1.03 mb copy number loss encompassing all of fas. parental studies were not performed. patient 2: we detected a~1.004 mb copy number loss encompassing all of fas. parental studies showed this to be maternally inherited. in addition, prior karyotype testing of the bone marrow showed the same deletion. patient 3: we detected a~0.044 mb copy number loss encompassing exons 7-9 of fas. parental studies were not performed. these results are consistent with the pathogenic nature of copy number variant losses involving fas. the mechanism of disease in these patients is consistent with haploinsufficiency. in family 2, the mother harboring the fas deletion is unaffected. this is consistent with prior observation of reduced penetrance within a family in alps. conclusion: these three cases harbored causative deletions in fas in the presence of biomarkers indicative of alps and negative results for germline and somatic genetic variant testing. these patients demonstrate that copy number variant analysis should be pursued if there is robust clinical and biomarker evidence of alps as it can lead to a molecular diagnosis and appropriate treatment when exome or next generation panel based fas sequencing is inconclusive. abstract/case report text rationale: the thymus is essential for the development of tcells. patients with thymoma have decreased aire expression and have an abnormal thymic microenvironment where the negative selection of t-cells is compromised, resulting in a broad spectrum of autoimmune-mediated diseases. besides myasthenia gravis, which is found in 15 to 20% of patients with thymoma, other autoimmune diseases have been reported including erythroblastopenia, systemic lupus erythematosus, inflammatory myopathies, thyroid disorders and good's syndrome. recent studies have described additional autoimmune conditions such as pneumonitis in thymoma patients. we identified a patient who developed chronic cough post-thymectomy and was found to have lymphocytic pneumonitis with associated autoantibodies against lung antigen kcnrg and lung immunopathology consistent with apeced pneumonitis, which implies a common pathogenic mechanism between these conditions. methods: we describe a patient with thymoma who developed autoimmune pneumonitis associated with kcnrg autoantibodies and a characteristic pattern of immunopathology recently described in patients with monogenic disorder caused by primary aire deficiency (apeced) and secondary aire deficiencies (thymoma, rag deficiency). results: patient is a 32-year-old male with no significant past medical history who was in good state of health until age 20 when he was diagnosed with and received treatment for guttate psoriasis (resolved with uv therapy) and alopecia areata. at age 30, he developed severe abdominal pain and weight loss. he had an abdominal ct performed that showed chronic pancreatitis and thymoma. one month later, the patient underwent thymectomy and subsequently, underwent ercp and pancreas biopsy, revealing atrophic pancreatitis with negative staining for lgg and lgg4. at that time, he was started on pancreatic enzymes with improvement of abdominal symptoms. following thymectomy, he developed persistent dry cough and recurrent symptoms of sinusitis which did not respond to several courses of oral antibiotics to treat his positive culture for pseudomonas. he had a negative work up for vocal cord dysfunction and cystic fibrosis, and negative autoantibodies against ifn-gamma, il-17a, and gm-csf. for work up of chronic cough, the patient underwent ct imaging of the chest which revealed diffuse peri-bronchial thickening, mucus plugging, and tree-in-bud nodularity through most of his lungs. he underwent bronchoscopy with bal which revealed n o r m a l b r o n c h i a l m u c o s a a n d a i r w a y n e u t r o p h i l i a . endobronchial biopsies showed basement membrane thickening and dramatic lymphocyte infiltration in intraepithelial and submucosal areas. his bal cultures revealed mycobacterium intracellulare/chimaera. patient was also tested for autoantibodies against lung-specific bactericidal/permeabilityincreasing fold-containing b1 (bpifb1) and the potassium channel regulator kcnrg that have been associated with the development of pneumonitis in patient with apeced, thymoma and rag deficiency, and was found to have kcnrg-targeted autoantibodies. conclusions: thymoma is a disease associated with secondary aire deficiency. this case illustrates common clinical, radiographic, histological, and autoantibody features in thymomaassociated and apeced-associated pneumonitis, indicating that disorders with primary and secondary aire deficiencies may have common pathogenetic mechanisms. bpifb1 and kcnrg should be included in the autoantibody profile testing of patients with thymoma and lung disease. immune suppression and antimycobacterial antibiotic treatment are planned. abstract/case report text introduction/background: activated phosphoinositide 3-kinase δ (pi3kδ) syndrome (apds) is a primary immunodeficiency caused by a gain-of-function mutation in the pik3cd gene that encodes the p110δ catalytic subunit of pi3kδ. it is characterized by recurrent respiratory tract infections, lymphoproliferation, nodular mucosal lymphoid hyperplasia, enteropathy, ebv and/or cmv infection, reduced t cell function and high levels of igm. there is not evidence of this disease in peruvian patients. methods: a case series of two pediatric patients with apds. results: the first patient is a girl of non-consanguineous parents. family history shows four maternal uncles died at pediatric ages with unknown diagnosis. at the age of 2, she presented lymphadenopathy and fever being treated as cat scratch disease without improvement of symptoms. 8 months later, she was hospitalized due to anemia, mild hepatosplenomegaly, ascites and chronic diarrhea and diagnosed with gastrointestinal tuberculosis (tb). a hepatic biopsy only showed reactive hepatitis. however, the patient did not improve her symptoms despite anti tb treatment. 2 years later, she was hospitalized for lymphadenopathy, pancytopenia, chronic diarrhea, ascites and severe hepatosplenomegaly. cmv igg was positive and lymph node biopsy revealed paracortical and follicular lymphoid hyperplasia due to ebv infection without neoplastic proliferation. low cd4+ t and cd19+ b cells and high igg levels were found (table 1) . at this time, it was suggested the diagnosis of apds which was confirmed by next generation sequencing (ngs) identifying a heterozygous mutation in the pik3cd gene (c.3061g>a, p.glu1021lys). she was treated with sirolimus and ivig for 2 years. the symptoms persisted despite treatment and died at the age of 6. the second patient is a 6-year-old girl also of non-consanguineous parents. family history includes eczema (father) and colorectal cancer (mother). she has had recurrent respiratory infections, chronic diarrhea and poor weight gain since 4 months old receiving symptomatic treatment only. at the age of 3, she was hospitalized for persistent pneumonia ( p s e u d o m o n a s p o s i t i v e ) , l y m p h a d e n o p a t h y a n d m i l d hepatosplenomegaly. a ct scan showed bilateral bronchiectasis and the sweat chloride test was negative. based on this, a diagnosis of cystic fibrosis was made and treatment was started. however, a genetic study only showed heterozygous mutations in the cftr gene (g551d and g542x). 1 year later, she presented a neck-located skin abscess. at the age of 5, she was hospitalized for complicated pneumonia, diarrhea, lymphadenopathy, ascites and severe hepatosplenomegaly. multiple polyps in the duodenum and colon with lymphoid hyperplasia were detected, ebv igm and igg were positive and a lymph node biopsy showed paracortical hyperplasia without neoplastic proliferation. cd8+ t cells and igm levels were increased (table 1) . a diagnosis of apds was suspected and ivig was started. ngs showed the same mutation as the first patient (c.3061g>a, p.glu1021lys). conclusion: apds should be considered in patients with recurrent respiratory tract infections, lymphoproliferation, enteropathy and abnormal immunologic function without another explanation. ngs is a useful tool to identify these cases in low-income countries. acknowledgments: we thank drs. raif geha and janet chou, division of immunology, boston children's hospital, harvard medical school for the genetic diagnosis. background: granulomatous-lymphocytic interstitial lung disease (glild) is an increasingly recognized pulmonary complication associated with common variable immunodeficiency (cvid) but the natural history and long term prognosis remains poorly defined. imaging findings with computed tomography (ct) are heterogeneous and visual features do not consistently predict a patient's progression to fibrotic lung disease. computer-aided lung informatics for pathology evaluation and rating (caliper) provides an objective analysis of lung parenchymal texture and quantifies the extent of normal lung, along with abnormal features such as honeycombing, reticular/consolidative and groundglass opacity. this may be useful in cvid patients to monitor changes in character or extent of disease and may facilitate early intervention before the disease becomes more aggressive or advanced. case description: our patient is a 46-year-old non-smoking female with cvid who has been followed for her cvid and associated interstitial lung disease. for more than twenty years, she has had varying abnormalities found on chest ct and these appear consistent with glild. specifically, she has had variable regions of mixed consolidation, ill-defined nodularity and septal thickening. the changing morphology and distribution made assessment of overall severity and extent of fibrosis versus parenchymal infiltration inconsistent. for clinical decision support we used caliper to analyze the current ct (2019) and compared caliper results for previous ct data. caliper provided a comprehensive analysis of the extent and characteristics of parenchymal features, and objectively determined normal and abnormal regions, some of which were not visually apparent. the caliper color overlay was able to highlight subtle regions of ground-glass opacity in areas that visually were regarded as uninvolved lung and quantify the extent of the reticular densities/ consolidation over time. caliper does not differentiate reticulation from consolidation, does not detect nodularity or septal thickening, and ct imaging cannot distinguish inflammation from fibrosis. however, caliper has the power to quantitatively assess overall disease extent and demonstrate subtle abnormalities that would otherwise have been dismissed as normal, given relative sparing compared to other regions. caliper may also provide evidence for disease progression or therapeutic response that is not otherwise radiographically apparent. conclusion: caliper assessment may be a useful tool as an adjunct for a patient with glild to help quantify the extent and character of lung parenchymal involvement. this information may serve as an important guide for clinicians in the assessment of successful management and early intervention to prevent irreversible fibrosis. patients had a trial of fingolimod without any beneficial changes in immune status. both patients receive pneumocystis jirovecii pneumonia prophylaxis with sulfamethoxazole-trimethoprim. conclusions: these results indicate that s1pl deficiency due to sgpl1 mutations is a syndromic primary immunodeficiency leading to profound lymphopenia and hypogammaglobulinemia. our data emphasize the importance of sphingolipid metabolism for an efficient immune response and the need for more studies to delineate the exact mechanisms on how this happens in humans. after 1d at 4°c there was a median -9% (range -44% to +3%; p=0.017) change in activity. after 2d -11% (range -48% to +8; p=0.006), 3d -13% (range -55% to +4%; p < 0.001), 4d -16% (-60% to +6%; p < 0.001) and after 7d -21% (-68% to +2% p < 0.001). a 4°c stability of 3d was determined from the median percentage reduction; total allowable error adjusted stability data indicated a 4°c stability of 5d. samples stored at -20°c following repeat freeze thawing saw a freeze/thaw cycle dependent decrease in ch50 activity. after 1 freeze/thaw cycle there was a median -9% (range -22% to +1%; p=0.018) change, 2 cycles -11% (range -26% to +2%; p=0.003), 3 cycles -13% (range -38% to +2%; p < 0.001), 4 cycles -23% (range -69% to -6%; p < 0.001) and after 5 cycles -25% (range -68% to -7%; p < 0.001). allowable error adjusted stability data indicated a maximum of 3 freeze/thaw cycles. conclusion: sample storage and handling can have a significant impact on functional complement assessments. room temperature storage should be avoided unless samples will be analysed on the day of collection, 4°c storage is tolerable providing that assessment is within 3d; freezing samples at -20°c with limited freeze/thaw analysis would be optimal. however, further investigations into longer-term storage at -20°c and -80°c would be beneficial. conclusions: gi disease is common in cvid affecting 63% of patients in our cohort. gi+ cvid patients have a higher frequency of autoimmune manifestations than those without gi complaints. the odds of itp, hypothyroidism, and evans syndrome all showed significantly increased odds in the gi+ group. the results of our study may have implications for both gastroenterologist and immunologist. recurrent infections especially those of the sinopulmonary tract are often the trigger for cvid evaluation. autoimmune and gi symptoms however may be the initial presentations of cvid and overlooked until other more recognizable manifestations evolve. the combination of gi issues and autoimmunity especially thrombocytopenia, evans syndrome, and hypothyroidism should include cvid in the gi differential. for the immunologist, a cbc is standard in the work-up of cvid and may reveal autoimmune cytopenia. evaluation for autoimmune disease and in particular hypothyroidism is not. given our findings an initial immune work up specifically for thyroid disease may be indicated. is the transcriptional factor for many cytokines such as il-17, responsible for t cell and neutrophil defense again fungal infection. stat3 mutation leads to defect of neutrophil proliferation and chemotaxis to inflammatory site as well as production of antimicrobial peptides by respiratory epithelial cells. the poor tissue repair in the cavitary lesions and bacterial superinfection in patient's lung created a culture dish for fungal growth and dissemination. traveling to the endemic area and patient's noncompliance to antifungal prophylactic treatment further increased the risk of histoplasmosis infection. pediatric immunologist/john hunter children's hospital abstract/case report text we present the case of a 20 month old boy, the first child to his nonconsanguineous parents of european descent. he first presented at 9 months of age with a cellulitis of his right fourth finger culture positive for staphylococcus aureus which responded to a prolonged course of flucloxacillin. at 12 months he presented with norovirus positive gastroenteritis leading to a brief admission and slow resolution. the first of two severe episodes of oral stomatitis and respiratory distress occurred at 13 months of age. hsv1 was isolated from the oral lesions and blood culture during that admission was positive for kingella kingae. no cardiac or bone involvement was identified. a more severe episode of oral stomatitis occurred two months later (age 16 months) swab positive for an enterovirus (not typed). due to airway compromise and rapid deterioration he was admitted to the pediatric intensive care unit. again, kingella kingae was cultured from blood cultures with no obvious focal systemic source. the only notable clinical finding was rapid deterioration and, in retrospect, the absence of any significant recorded fever ( < 38oc). crp elevation was observed (max. 188 mg/l) and neutropenia was found with each of the more severe infectious presentations but recovered in the interval. baseline immunological investigations were normal (lymphocyte subsets, naïve t cell populations, lymphocyte proliferation, serum immunoglobulins and vaccine responses). serial measurement of circulating neutrophils did not identify a cyclical pattern and they were morphologically normal. a panel of genes relevant to primary immunodeficiency (invitae©) revealed a homozygous mutation in irak4 ((c.877c>t (p.gln293*)) which leads to a premature stop codon. this is a known pathogenic mutation leading to disease and is most prevalent in the european population (allele frequency (gnomad) = 0.0005). prophylaxis with sulfamethoxazole / trimethoprim and amoxicillin was commenced along with monthly ivig. he has been well since diagnosis with no further severe infectious presentations. functional testing is underway to assess in vitro host viral defence in our patient and potential novel mechanisms relevant to this rare innate immunodeficiency. case studies will be presented on the five cases of fmp that were diagnosed and treated in 2019. potential exposures were identified in four out of five cases: gardening exposure in one case and vaping exposure in three cases. all five were male, age range 14-45. four were gp91 deficient, and one was p47-phox deficient. historically, the vast majority of cases of fmp could be traced to a significant gardening exposure such as lawn mowing or spreading mulch. this was the first year that we saw patients with no identifiable gardening exposure in the setting of significant vaping exposure. with vaping at epidemic levels, especially among teenagers and young adults, it is important to consider that a vaping history is potentially a risk factor for fmp and counseling regarding the potential risks of vaping should be included in infection risk modification for all patients with cgd. abstract/case report text background: granulocyte-macrophage colony-stimulating factor (gm-csf) plays a critical role in macrophage and dendritic cell maturation and host defense against fungus. autoantibodies to gm-csf are associated with susceptibility to cryptococcus and nocardia infections as well as pulmonary alveolar proteinosis (pap) in otherwise healthy individuals. we report a case of a 23-year-old previously healthy female who presented with cryptococcal meningitis and was found to have autoantibodies against gm-csf. case presentation: 6 weeks prior to admission, our previously healthy 23-yearold taiwanese female developed a headache associated with tinnitus and visual changes. the headache worsened over the next few weeks and she developed photophobia, phonophobia, and severe nausea/vomiting. at presentation, her exam was notable for papilledema, bilateral cn vi palsy and right foot & left hand paresthesia. mri brain showed ring-enhancing lesions in the anterior frontal lobe, caudate head, and the inferior globus pallidus. she underwent a diagnostic and therapeutic lp. opening pressure was elevated at 60 and csf studies were notable for low glucose, elevated protein, pleiocytosis (94% lymphocytes) and positive cryptococcal antigen. csf culture grew cryptococcus gattii. ct chest revealed a right upper lobe and a left lower lobe nodule. workup: cbc with diff was unremarkable. hiv was negative. lymphocyte subsets were unremarkable with only mildly decreased nk cells, normal immunoglobulin panel including ige, protective titers to tetanus, diphtheria, and ppsv23. targeted genetic sequencing did not identify any known mutations in primary immunodeficiency. notably, anti-gmcsf autoantibodies were detected by elisa and were able to neutralize gm-csf phosphorylation of stat5 detected by flow cytometry. autoantibodies to ifn-γ were not detected. management: patient was initiated on a 6-week course of liposomal amphotericin b and flucytosine. her csf cultures were cleared of cryptococcus after 20 days of treatment, but her hospital course was complicated by persistently symptomatic intracranial hypertension, worsening pleiocytosis, and elevated cytokine levels in the csf, all of which were consistent with post-infectious inflammatory syndrome (piirs). she received therapeutic lps 2-7x/week until subsequent ventriculoperitoneal shunt placement. concurrently, methylprednisolone was administered for 7 days with a gradual prednisone taper. these interventions led to improvements in her symptoms, including diplopia, and reduction in opening pressures and inflammatory markers in the csf. lifelong fluconazole prophylaxis was recommended. from a pulmonary standpoint, she remained asymptomatic without signs of pap and has had normal pulmonary function tests (normal dlco) and stable chest imaging. conclusion: in otherwise healthy hiv-negative patients presenting with extrapulmonary cryptococcus or nocardia infections, autoantibodies to gm-csf should be suspected and testing for functional autoantibodies to gm-csf (and ifn-γ) should be sent, as genetic testing will not pick up this disease entity. genetic testing should be considered to rule out gata2 deficiency and x-linked cd40l deficiency. idiopathic cd4 lymphopenia can be ruled out with lymphocyte enumeration. immediate treatment of cryptococcosis is not necessarily different from patients without gm-csf autoantibodies. long-term prophylaxis (fluconazole if presenting with cryptococcus; trimethoprim-sulfamethoxazole if with nocardia) is likely warranted in addition to monitoring for the development of pap. recognizing piirs in patients with cryptococcal meningitis and management with corticosteroids are critical steps. abstract/case report text background: non-infectious complications cause most morbidity and mortality in common variable immunodeficiency (cvid). cvid with complications (cvidc) is defined by elevated t helper 1 (th1) responses attributed to increased circulating microbial products resulting from mucosal iga deficiency. however, complications do not uniformly occur in those with iga deficiency. objective: we tested whether cvidc occurs preferentially in those with hyper-responsiveness to microbial stimuli, manifested by elevated nf-κb-driven cytokines and resultant th1 responses in cvid patients with increased circulating microbial products. methods: we applied unbiased high-throughput seromics and mass cytometry, cellular and molecular biology approaches, and clinical record review in a 78 subject cvid cohort. results: cvidc was defined by increased nf-κb-driven cytokines that promote th1 immunity in blood in association with elevated soluble cd14, a marker of circulating microbial products, and elevated tnf production by peripheral blood mononuclear cells stimulated with lipopolysaccharide. this cytokine upsurge was associated with mutation of full-length nfkb1 p105 gene product (1375delt) but not mutations that also involved the nfkb1 p50 product involved in transactivation. cytokine elevation corresponded with increased cd14+cd16-monocytes expressing higher cd86 and hla-dr and more central and effector memory cd4+ t cells, t cell chemoattractants, and t cellpredominant tissue pathology. those with granulomatous or neutrophilpredominant, rather than t cell, pathology had the highest tnf. tnf antagonism improved neutrophilic gastritis in cvid with nfkb1 1375delt after t cell targeted therapy failed. conclusion: nf-κb dysfunction underlies th1 immunopathology and tnf-associated innate inflammation in cvidc. both forms of nf-κb immune dysregulation may divergently shape cvid immunopathology. staff clinician/laboratory of clinical immunology and microbiology, immunopathogenesis section, national institute of allergy and immunology, national institutes of health, abstract/case report text introduction: patients with autoantibodies to ifn-γ develop severe and progressive infections with intracellular pathogens, despite aggressive antimicrobial treatment. we describe the use of daratumumab (anti-cd38, targeting plasma cells) in a patient with autoantibodies to ifn-γ and progressive disseminated mycobacterium avium infection. she had progressive disease despite treatment with multi-drug antimycobacterials rituximab, and bortezomib. methods: clinical symptoms, total cd19/cd20, anti-ifn-γ autoantibody titers, and specific imaging were obtained before and after treatment with daratumumab. anti-ifn-γ autoantibody titers were determined by serial 10-fold dilutions of plasma and measuring anti-ifn-γ autoantibody levels by a particle-based technique as previously described. results: a 31-year-old filipino woman had progressive disseminated m. avium with extensive bone and soft tissue involvement (calvarium, ribs, bilateral arm soft tissue, paraspinal muscles, bilateral glutei, left inferior pubic ramus, bilateral iliac bones, sacrum, and bilateral humeri) and a tracheo-esophageal fistula. she received bedaquiline, azithromycin, ethambutol, tedizolid, moxifloxacin, clofazimine and meropenem as well as rituximab 1g once monthly for 5 months. despite these she had progression of clinical and radiographic disease. bortezomib 1.3 mg/m2 twice weekly for 8 weeks was added, but discontinued for ast and alt elevations. rituximab was continued to maintain cd20 numbers undetectable but clinical and radiographic disease progressed. while on rituximab, total igg level and anti-ifn-γ autoantibody levels decreased from 1521mg/dl to 1069mg/dl and 3058 to 2504, respectively. while on bortezomib, total igg levels remained stable (1031mg/dl to 1051mg/dl) and anti-ifn-γ autoantibody levels fell slightly (2504 to 1275). after starting daratumumab, there was clinical and radiographic improvement, with reduced pain and disappearance of multiple soft tissue lesions. igg levels decreased from 1100mg/dl to 434mg/dl and anti-ifn-γ autoantibody levels decreased from 1275 to 157. adverse effects of daratumumab were urticaria, pruritus and shortness of breath after the first infusion and aseptic meningitis after the 5th infusion. conclusions: daratumumab resulted in clinical and radiographic improvement of disseminated m. avium in a patient with rituximab and bortezimib-refractory autoantibodies to ifn-γ. daratumumab is another potentially effective therapeutic agent for anti-ifn-γ autoantibodies. abstract/case report text next-generation sequencing (ngs) is now routinely used as a clinical diagnostic tool. however, regions of high sequence homology continue to be a major challenge for short-read technologies. regions within ikbkg, ncf1, sbds, c4a, c4b, coro1a, fcgr3a, fcgr3b, pms2, slfn11, slfn13, stat5b, unc93b1, and ups18 are not available by standard ngs. we discuss strategies for analysis of these special regions. we have developed a strategy for supplementing our disease targeted panels which are performed using capture chemistry and a standard reference file. the supplemental method uses gene specific long range amplicon and a special gene specific reference file for alignment. the genes of interest are separated from their homologous counterparts using specific long range amplification primers. multiple amplicons may be pooled together and prepared for sequencing on an illumina miseq instrument using truseq nano dna library prep. bioinformatic analysis proceeds with a custom reference file in which non-specific regions of homology have been removed. this allows reads to be uniquely mapped despite significant homology; a requirement for variant calling. we prepared specific amplicon for several homologous gene targets including the ikbkg gene and the ikbkg pseudogene (ikbkgp1). both amplicons were sequenced in separate reactions and were compared with the standard capture method. variants which are not called in the standard-capture method due to poor mapping scores (non-uniquely mapped reads) are called in the amplicon method. in the capture method, the variants are visualized in the bam as a mixture of gene and pseudogene, while gene and pseudogene variants are clearly separated and identified in the amplicon method. due to high variability in alignment, many homologous regions do not provide reliable copy number variant (cnv) results and must be removed from cnv analysis. however in some situations, we are able to creatively leverage cnv analysis to identify alleles that mis-align to the pseudogene. the pathogenic ncf1 gt deletion in exon 2 appears to resemble a copy number deletion event when present as reads from one allele mis-align to the ncf1b and ncf1c pseudogenes. complement genes, c4a and c4b, share alignment due to their high homology with each other. cnv analysis in normal samples represents four alleles rather than two alleles. cnv events may have a weak signal with no indication of which gene is affected. variant frequencies from the capture and supplemental pcr analysis can be used in tandem with cnv analysis to detect events and may indicate which gene is affected. we plan to include these strategies in our new inborn errors of immunodeficiency gene panel (ieigp) which will enable us to provide a more comprehensive analysis than is currently available. the im diagnosed were inflammatory bowel disease-like (n = 5, with perianal fistula in 2/5), mouth ulcers (n = 1), discoid lupus (n = 1), autoimmune dermatitis (n = 1) and eczema (n = 1), chronic lung disease (n = 2) and granulomas (pulmonary n = 2; ocular n = 1; bladder n = 1; oropharynx n = 1). three patients presented more than one site of inflammatory disease. all patients were treated with systemic or topical immunosuppressive or immunomodulatory therapy, most of them corticosteroids. five patients underwent hematopoietic stem cell transplantation (hsct), median age at hsct was 13 years (4 -17), and two died 1 month after hsct. conclusions: although infections are more frequent and have a major impact on patient morbidity and mortality, im are increasingly prevalent in patients with cgd. awareness regarding this possible comorbidity is of major importance, since earlier diagnosis and adequate treatment may be crucial for patients survival and quality of life. there is a gap in clinical knowledge regarding associations between specific pid and different rheumatological diseases. in this study, we are reporting the incidence of various rheumatological conditions reported in a large pid population using the usidnet (united states immunodeficiency network) registry. methods: we used the retrospective usidnet registry to conduct the analysis. we included all primary immunodeficiency patients with physician diagnosed rheumatological diseases. results: the total number of pid patients in our query was 5058. 278 (5.49%) patients had a diagnosis of rheumatological disease. this cohort included 172 (61.8%) female and 106 (38.2%) male patients. rheumatologic complications were highest in the interferonopathies (66.6%), complement deficiencies (14.2%) and autoimmune lymphoproliferative syndrome (alps) (13.7%). additionally, disease patterns were noted to be different in each pid. dermatomyositis was found to be the most common rheumatologic condition in patients with x-linked agammaglobulinemia (xla) with a rate of 1.65%, which was remarkably higher than the reported prevalence in the united states (0.005%). alps patients had a higher (6.85%) numbers of sjogren syndrome diagnoses as compared to the general population (0.01-0.09%). systemic lupus erythematosus was increased in patients with mucocutaneous candidiasis (7.41%) as compared to the general population (0.001%) and other pids. rheumatoid arthritis (ra) was reported in patients with specific antibody deficiency (3.66%), common variable immunodeficiency (cvid) (2.93%) and alps (2.74%). wiskott-aldrich syndrome patients had the highest numbers of cases diagnosed with vasculitis (6.50%). 0.29% of patients with severe combined immunodeficiency (scid) had reported rheumatologic disease. juvenile rheumatoid arthritis (jia) and systemic sclerosis were reported in 0.19% of patients with digeorge syndrome. conclusions: this study reports that higher numbers of rheumatologic diseases are diagnosed in pids compared to the general population. the incidence of different rheumatological disease was variable based on the pid diagnosis. early diagnosis of these diseases is crucial, given the high risk of irreversible complications. limitations of our study include possible selection bias as majority of cases were enrolled from tertiary care centers. abstract/case report text background disorders of immune dysregulation are associated with autoimmune features. this feature could potentially have an impact on the outcome post hematopoietic stem cell transplantation (hsct). hsct, although curative, can be challenging with the underlying immune dysregulation resulting in significant morbidity and mortality. we present the journey through hsct for these children and the factors affecting the outcome. we analysed the data on children up to the age of 18 years diagnosed to have a disorder of immune dysregulation through gene mutation analysis and who underwent hsct at our centre from 2015 to 2019. results 1. xiap mutation a 10-year-old boy underwent a haploidentical hsct from his father using fludarabine, treosulfan, and 2 gray radiotherapy with post-transplant cyclophosphamide. after initial complete chimerism and cytomegalovirus reactivation responsive to valganciclovir, he developed progressive diarrhoea almost 15 months post-hsct. a rectal biopsy confirmed cmv reactivation and features of inflammation. he has since been treated for the same and is on follow up for inflammatory bowel disease. his chimerism had dropped to 67% and has remained stable. the second child is a 1-year-old girl who underwent tcr alpha/beta depleted haplo sct and is 12 months post-hsct, with no features of gvhd or infections, and is doing well with complete chimerism. 2. il10r deficiency three boys aged eight months, one year, and two years of age, diagnosed to have il10r deficiency underwent hsct. all three children needed nasogastric tube feeding, parenteral nutrition, and vigilant monitoring for electrolyte disturbances. in the first two children, we had performed tcr alpha/beta depleted pbsc transplants from their haplo matched fathers. the 1-year-old engrafted by d+17 and is doing well two years post hsct with complete chimerism, no gvhd, and infections. his autoimmunity, including recurrent skin scarring, has resolved entirely. the 8-month-old, however, had primary graft failure and succumbed to his illness. the 2-year-old boy underwent matched unrelated donor hsct and engrafted by d+14 with completed chimerism documented on three occasions. he, however, had secondary graft failure around d+60, and he succumbed to the illness. 3. lrba deficiency an 18-month-old girl with lrba deficiency had presented at four months of age with excessive sweating, hepatosplenomegaly, and recurrent chest infections. she was started on monthly intravenous immunoglobulin replacement and abatacept. she received myeloablative conditioning with thiotepa, treosulfan and fludarabine and underwent a matched sibling donor hsct. she engrafted by d+17 and has been well ten months post hsct with complete chimerism, no gvhd, and infections. conclusion disorders of immune dysregulation are a heterogeneous group with a varied spectrum of immune dysfunction. myeloablative conditioning is essential, and there is a high risk of cytokine release syndrome and the need for supportive care. the autoimmune features need to be followed for progression in organs other than the hematopoietic system and may require interventions. as long-term data evolves, more precise definitions for patient and donor selection will enable improving outcomes. we performed a retrospective observational analysis of case records of children up to 18 years of age, diagnosed to have variants of scid, and underwent hsct at our centre from 2002 to 2019. results 1. zap 70 deficiency a 6-month-old girl presented with oral thrush and submandibular cellulitis from one week of life with failure to thrive. she underwent a tcr alpha/beta depleted haploidentical hsct. conditioning included treosulfan/thiotepa/fludarabine/anti-thymocyte globulin. she engrafted by d+15; now three years post-hsct with complete donor chimerism without gvhd or infections. 2. orai-1 mutation a 15-month-old girl presented with failure to thrive, generalized hypotonia, oral thrush, and recurrent respiratory infections. she underwent haplo-sct with post-transplant cyclophosphamide with pbsc from her haplo-matched father. conditioning included fludarabine/treosulfan. she had cytokine release syndrome grade 4, which responded to tocilizumab. she had hypertension throughout the peri-engraftment period and had an episode of pres with seizures. her symptoms abated with neutrophil engraftment by d+ 17. the post-transplant period was complicated by grade 2 skin gvhd and cytomegalovirus reactivation. she has remained disease-free with complete chimerism three years post-hsct. her hypotonia is steadily improving with physiotherapy. 3. cernunnos-xlf deficiency a 27-year-old male presented with recurrent infections from 14 years of age, aplastic anemia diagnosed at 20 years of age, subsequent transformation to acute myeloid leukemia at 27 years of age. he had developed multiple fusarium abscesses during the neutropenic period post-chemotherapy for aml. he was referred for a matched sibling sister hsct when in remission. conditioning included fludarabine/treosulfan. he engrafted by d+15 with complete chimerism. he developed progressively worsening skin, gut, and liver toxicity secondary to chemotherapy and succumbed to the illness two months post-hsct. 4. ikzf mutation an 18-month-old girl presented with failure to thrive, massive splenomegaly, persistent pneumonia, anemia, and thrombocytopenia. she underwent a matched sibling donor pbsc transplant after myeloablative conditioning with thiotepa/treosulfan/fludarabine. she engrafted by d+20, following which all her symptoms abated. she had secondary graft failure two months post-hsct and succumbed to her illness. 5. mhc class ii deficiency (bare lymphocyte syndrome) three children, aged 18 months, two years, and four years underwent matched sibling donor hsct. myeloablative conditioning with thiotepa/treosulfan/fludarabine resulted in engraftment. the first child died of invasive intestinal aspergillosis 30 days post-hsct. the other two children are well 11 months post-hsct with complete chimerism without gvhd or infections. the two-year-old girl received one cycle of pre-transplant immunosuppression with fludarabine/dexamethasone to prevent graft rejection pre-hsct as she was referred for a second transplant. conclusion children with scid have traditionally been transplanted using reduced intensity (ric) conditioning with immunomodulation. scid variants require myeloablative conditioning with a vigilant follow up for the detection of graft rejection. radiation sensitive scid associated with dna breakage repair defects require ric and close monitoring for gvhd. advances in hsct, including supportive care and haplo-sct, have provided a ray of hope for these hitherto rare conditions. j clin immunol abstract/case report text immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) (omim #304790) is a monogenic autoimmune disorder that occurs due to loss of function variation in foxp3 causing dysfunctional t regulatory cells. although immunosuppression is a mainstay of treatment for autoimmunity, ipex treatment is frequently limited by insufficient response to therapy or side effects of immune suppression. we present a 28 year old male with ipex whose prior immunosuppressive treatment was complicated by inefficacy and medication side effects, requiring a new approach to treat his colitis and erosive dermatitis. he initially presented with infantile diabetes and subsequently developed dermatitis, squamous cell carcinomas, alopecia totalis, and colitis. his clinical diagnosis of ipex was confirmed by foxp3 sequencing, demonstrating known pathogenic variant c.1150g>a (p.ala384thr). this variant has been described in ipex affected individuals in multiple publications (ref 1). his variant affects at the frkhead domain of foxp3 and has been associated with others with severe psoriasiform dermatisis and alopecia universalis (ref 2). his prior immunosuppressive therapies included at different times combinations of corticosteroids, tacrolimus, sirolimus, azathioprine, infliximab, adalimumab, rituximab, dupilumab, and oral mesalamine. the relative efficacy of these agents based on experiences in a cohort of ipex patients was reviewed in 2018 (ref 3), with the exception of duplimab, which was not listed in that review. for our patient, management of his widespread autoimmunity has been limited by toxicity or lack of efficacy of medications. notably, his dermatitis had no improvement with duplimab, consistent his low total ige and lack of allergic manifestations. at age 27, after initiation of treatment with sirolimus, he had spontaneous colonic perforation requiring descending colectomy. after stabilization of his colonic perforation, his multi-disciplinary team of allergy-immunology, gastroenterology, and dermatology initiated tofacitinib. tofacitinib is small molecule inhibitor of janus kinase (jak) signaling pathways that mediate cytokine driven autoimmune activation. it is fda approved to treat rheumatoid arthritis, psoriatic arthritis and ulcerative colitis. the decision to use this jak inhibitor was due to its fda approved use for ulcerative colitis, to target our patient's colitis and his other autoimmune manifestations, specifically his dermatitis. its off label for primary immune dyregulatory disorders including candle, stat1-gain of function and stat3-gain of function disorders has been published (ref 4), but thus far its use to treat autoimmunity due to ipex has not been published. he experienced leukopenia while on 15 mg of tofacitinib, which resolved after lowering his dose. currently, he has had improvement in his colitis and dermatitis, and partial improvement in alopecia. he has been on tofacitinib 10mg daily for 11 months, with only prednisone 10mg daily as additional immune suppression. as the number and types of selective immune modulators increases, there is continued need to share the experiences of treating physicians of which therapies have been successfully able to decrease disease manifestations with tolerable side effect profiles. we present a 28 year old male with ipex syndrome with severe dermatitis and colitis complicated by colonic perforation despite standard immunosuppressive therapy, who is safely and effectively being treated with tofacitinib. is not frequently associated with autoimmunity, likely due to impaired il-6 and il-17 pathways. however, in our relatively large cohort of lof stat3 patients, we have noted an increased incidence of systemic lupus erythematosus (sle) diagnoses and sle-like symptoms. herein, we characterized the clinical and laboratory features of the patients in our cohort with sle and sle-like disease, with the aim to better understand the pathogenesis by evaluating ifn stimulated genes and neutrophil net formation. methods a retrospective chart review was performed of patients with lof stat3 to identify those with sle and sle-like presentations, and included clinical features, laboratories including inflammatory markers, auto-antibodies, and complement levels. rt-pcr was performed for interferon stimulated genes (isgs) from neutrophils and pbmcs of lof stat3 patients with and without sle, and healthy controls. neutrophil net formation was assessed for lof stat3 patients with and without sle, and healthy controls. results out of a cohort of 158 patients, five patients (ages 12-39) were identified who carried the diagnosis of sle, and 4 with slelike disease (ages 15-34). for those with sle, age of presentation was 8-21 years, 4 of 5 were female. clinical features included nephritis (4), alopecia (2), autoimmune cytopenias (2), arthritis (3), discoid rash (2), and raynaud (1). all had positive auto-antibodies, and 4 of 5 had low c3 and/or c4. for those with sle-like disease, age of presentation was 12-24, and 2 of 4 were female. clinical features included alopecia (1), autoimmune cytopenias (1), raynaud(1), and nephritis (2). all had positive autoantibodies, and 1 of 4 had low complements. lof stat3 patients with and without clinical features of sle had increased expression of isgs from both pbmcs and neutrophils. increased spontaneous net formation was observed for lof stat3 patients both with and without sle symptoms. discussion although autoimmunity is not a common finding in lof stat3, we have identified sle or sle-like disease in about 6% of our cohort, with a high incidence of kidney disease, including one patient who required kidney transplant. the interferon signature and net formation were unexpectedly high in both the patients with and without the sle features. ongoing studies include whole exome sequencing for possible second mutations or modifiers, the role of ige in the kidney disease, and further autoantibody detection. the increased ifn signature raises the question about jak-stat modulation for therapy. 8 chief, genetic immunotherapy section/niaid, nih 9 abstract/case report text chronic granulomatous disease (cgd), a rare immunodeficiency with decreased reactive oxygen species (ros) production, increased susceptibility to infection, and increased mortality is caused by mutations in any one of 5 distinct phagocyte oxidase (phox) components of the nadph oxidase, nox2. in the past, identification of the specific protein defect was primarily determined by immunoblotting using specific antibodies to the phox proteins. recently, however, we have shown using fluorescenceactivated cell sorting (facs) analysis of neutrophils in whole blood permeabilized and stained with specific anti-p47phox antibody that p47phox protein expression was absent in p47phox cgd patients and significantly reduced in p47phox cgd carriers [kuhns et al. 2019. blood adv. 3(2):136-147]. these findings demonstrated that determination of phox protein expression by facs analysis provide an alternative to immunoblotting and can aid in the identification of p47phox cgd patients and carriers. we now have extended these studies to patients and carriers with p67phox cgd. facs analysis of p67phox expression in permeabilized neutrophils demonstrated that p67phox expression was absent in four patients with different mutations in ncf2 [two patients homozygous for c. e12(+1) g>a, one patient homozygous for c.287_289 del aag, p.glu96 del; and one patient compound heterozygous for the mutations, e3(+1) g>a and c.55_63 del aagaaggac]. moreover, the expression of p67phox in nine p67phox cgd carriers was significantly reduced >50% compared to expression in neutrophils from healthy volunteers. another cytosolic phox protein, p40phox, has been shown to associate with p67phox in a 1:1 molar ratio [tsunawaki et al. 1994 . biochem biophys res comm. 199(3): 1378-1387]. the expression of p40phox was reduced in both carriers and patients with mutations in ncf2. despite reduced expression of p67phox and p40phox, neutrophils isolated from carriers of p67phox cgd exhibited normal dihydrorhodamine (dhr) oxidation after stimulation with phorbol ester and fell within the normal range for ros production (measured by luminol-enhanced chemiluminescence) after stimulation with either fmlf, opsonized zymosan, or phorbol ester with one notable exception. included in this cohort of p67phox carriers was a p47phox cgd patient (homozygous for a gt deletion at the start of exon 2 in ncf1) who also carried a heterozygous damaging mutation in ncf2 [c.1256 a>t; p. asn419ile]. normal ros production in the presence of reduced p67phox and p40phox expression suggest that these proteins are not rate-limiting components for maximum nox2 activity in neutrophils. finally, determination of the expression of specific phox components by facs analysis of permeabilized neutrophils from whole blood provides a rapid and alternative approach to immunoblotting to determine the specific protein defect in cgd, and, importantly, one that could be easily established in most clinical labs. funded by nci contract no. 75n910d00024. the original clinical observation that defined patients with hyper-ige syndrome (hies) was the presentation of cold abscesses ("job's syndrome"), which indicated a deficient inflammatory response. mutations in the stat3 gene have now been identified in most classic autosomal dominant hies patients, but we do not fully understand how these mutations cause the clinical presentation. since the discovery of stat3 mutations, research on hies focused largely on the adaptive arm of the immune system and suggested that the innate immune defects could be secondary. for example, the discovery that there is a th17 cell and il-17 cytokine deficiency in hies provided a possible explanation to the neutrophil chemotaxis defects in hies, as il-17 is one of the chemokines critical for neutrophil recruitment in vivo. the goal of this study was to investigate myeloid cells from hies patients. first we used c5a, fmlp, il-8, cxcl1, and cxcl2 to study neutrophil chemotaxis in vitro. responses to c5a, fmlp, and il-8 were equally robust in hies compared to healthy controls, demonstrating that neutrophils from patients are capable of efficient directed migration in vitro. neutrophils from all hies patients responded to cxcl1 and cxcl2 significantly below that of the healthy controls. cxcl1 and cxcl2 are cxcr2-specific chemokines. these results indicated a neutrophil intrinsic cxcr2-specific defect. we also found that patient-derived cells express comparable levels of cxcr2 on the cell surface, suggesting a cxcr2 chemokine receptor signaling defect. after identifying a neutrophil defect in hies, we wanted to get a broader view of myeloid cells in hies in addition to identifying the cxcr2specific defect. stat3 is a transcriptional regulator, therefore we performed transcriptional profiling of hies and healthy control-derived neutrophils and monocytes. as it was shown before, the expression of stat3 was not different between patients and controls, since hies is usually caused by the decrease in stat3 activity not by decrease in expression. we found, however, an increase in stat1 and stat2 expression as well as significant changes in the expression of genes regulated by interferons. increased expressions of stat1/2 in both neutrophils and monocytes likely provide and explanation for the increase in interferon regulated genes. multiple genes were identified as potential regulators of cxcr2 signaling. the balance between the stat3 and stat1 signaling has long known to be a regulator of immune cell activation, especially in t cells, but less studied in myeloid cells. stat3 and stat1/2 signaling pathways crossregulate each other in healthy cells. we propose that in hies the decreased stat3 signaling leads to not only changes in expression of effector (e.g. inflammatory) genes, but also decreases expression of genes in the regulatory (negative) feed-back loop, which are required for decreasing stat1/2 activity. therefore, the immune cell defects caused by decreased stat3 activity are compounded by the increase in stat1/2 activity. increase in stat1/2 signaling can cause pathologies in the absence of stat3 defects, as well as further decrease stat3 signaling, thus contributing to hies. interfering with stat1/2 signaling in hies may represent a therapeutic opportunity. abstract/case report text rationale: t-cell receptor excision circles (trecs) testing on newborn screening (nbs) has been vital for identifying patients with severe combined immunodeficiency (scid). we aimed to determine whether one or more abnormal trecs result on a nbs might predict higher mortality rates despite the absence of an identifiable underlying etiology. methods: newborns with a positive trecs nbs result without the diagnosis of scid or 22q11.2 deletion syndrome born from october 2011 to december 2014 were included (n=467). newborns were divided into three groups: group 1 infants had a subsequent normal repeat screen (n=375); group 2 infants did not undergo repeat screening as the majority expired before a repeat screen could be conducted (n=36); group 3 infants had a normal initial screen but subsequent abnormal screen (n=56). cases were matched 3:1 to controls on gestational age, birth weight, nicu status, race, birth quarter, and birth year. nbs records were linked to birth and death certificate records. demographic characteristics were compared and mortality rates were calculated between the groups. results: the mortality rate of group 1 was 2.4%, group 2 was 91.7% and group 3 was 46.4%. when compared with matched controls, there was no difference in the mortality rate of group 1 when compared to the control group. there was a significant difference in the mortality rate between cases and controls in both group 2 (p < 0.001, 95% ci 0.711, 0.950) and group 3 (p < 0.001, 95% ci 0.256, 0.551). the apgar scores in group 1 infants were comparable to their matched controls. infants in group 2 (p = 0.01) and group 3 (p = 0.003) had significantly lower apgar scores than the controls. the majority of the infants in all three groups were less than 37 weeks gestation, however, group 2 had a higher percentage of infants born very premature (less than 32 weeks). there was no significant difference in maternal age, maternal education, prenatal care status, cigarette use, or maternal steroid use between the cases and controls in all three groups. conclusions: infants with an initial abnormal screen who had a subsequent normal repeat screen did not have an increased rate of mortality compared to their matched controls (group 1). however, group 2 infants (with unresolved repeat screen) and group 3 infants (with a first abnormal value on a repeat screen) did have increased mortality rates when compared to their controls. overall, an abnormal trecs level on nbs without a confirmed negative repeat screen, was associated with higher mortality in our study population. further studies will be needed to determine if the trecs assay can serve as a predictor for mortality in newborns with an abnormal screen. abstract/case report text introduction: primary immunodeficiency refers to a heterogeneous group of diseases characterized by altered function or composition of the immune system, and are grouped into adaptive or innate system defect. immunoglobulin g subclass immunodeficiencies (iggscs) are classified as a b-cell-related adaptive system disorder and are therefore associated with recurrent sinopulmonary infections with encapsulated bacteria, presenting with pneumonia, recurrent bronchitis, rhinosinusitis, and herpes zoster. its primary mechanisms are still unclear, although the cause for this deficiency might be related to gene deletions, transcription errors, or be an effect of allotype. igg4 immunodeficiency reaffirms its association with the patient's clinical condition and is often associated with igg2 deficiency. objective: to evaluate the prevalence of igg4 immunodeficiency in ferraroni's clinic, classify it by gender, age, igg4 dosage and other subclasses, correlate it with igg2 immunodeficiency and the clinical presentations presented by the patients under analysis. method records of 24 patients with igg4 immunodeficiency whose clinical pictures were followed throughout 15 years were evaluated, patients aged from 4 to 85 years. all tests were done at the same laboratory and all patients have consented to be part of this study, which has been approved by the ethics committee. results twenty-four patients with igg4 deficiency, 79,16% (n=19) were women and 20,83% (n=5) were male, with average of 47 and 28 years, respectively. the average of igg4 was 7.76 mg/dl, and that of igg2 was 299 mg/dl. of the patients evaluated, 62.5% had upper airway infections (sinusitis, rhinitis, otitis and tonsillitis), 25% herpes simplex, 33.3% asthma. less prevalent cases were reported as 4.1% of patients had bronchiectasis, 12.5% candidiasis and 4.1% herpes zoster. 41.67% presented the association of igg4 and igg2 deficiency. discussion: the role of specific igg4 deficiency in the infectious setting is still unknown, but it usually occurs in association with other isotypic deficiencies and sinopulmonary infections. furthermore, the igg4 subclass is relevant on the study of environmental antigens -suggesting its involvement with allergic disordersand has been described in association with other diseases, such as chronic mucocutaneous candidiasis, ataxia-telangiectasia and allergic colitis. igg2 deficiency is related to increased susceptibility to bacterial infections. studies show a correlation between igg2 and igg4 immunodeficiency that generally imply clinical features characterized by recurrent infections by encapsulated bacteria. the data obtained through the analysis of patients' charts corroborated this information, since it was evident that most of the patients had really similar clinical conditions. conclusion: igg4 deficiency has a direct correlation with higher prevalence of upper airway infections, such as rhinitis, sinusitis and pneumonia, and with an increased incidence of allergic disorders, here presented by our cohort. additionally, research suggests that hies may cause impaired cd8+ t cell function. we hypothesized that a low percentage of both th17 and th1 cells would be predictive of hies and would differentiate hies from atopic disorders. to evaluate this hypothesis, we examined the percentage of th17, th1, and ifng+cd8+ t cells, laboratory parameters, and genetic diagnoses from a large cohort of patients to determine which parameters distinguish patients with stat3 loss-of-function variants. methods: we conducted a retrospective, multi-institutional chart review of over 200 patients who received a th17 assay at the medical college of wisconsin clinical immunology research laboratory. the th17 assay is performed by activating pbmcs with pma/ionomycin/brefeldin a and staining for cd4, cd8, ifng and il-17a. the following parameters were included in the chart review: the percentage of th17, th1, and cd8+ifng+ cells, immunoglobulin levels, atopy scores, infectious history, and genetic diagnoses. results: using logistic regression, we demonstrated that the percentage of th17, cd8+ifng+, and th1 cells were positively correlated with age, and percentage of cd8+ifng+ cells was higher in females than males. we found that the percentage of th17 and th1 cells were decreased in both atopic disease and hies, with hies having the lowest values. interestingly, one subject with a stat3 gain-of-function (gof) variant had an elevated percentage of th17 and th1. in addition, we determined that ige levels were inversely correlated with the percentage of th17, cd8+ifng+, and th1 cells, while iga and igm were positively correlated with the percentage of th17 cells. several different monogenic defects characterized by increased fungal infections exhibited a low percentage of th17 including tatton-brown-rahman syndrome and cornelia de lang syndrome. conclusions: we confirmed that the percentage of th17 cells is low in both hies and atopy in a large cohort of subjects, and that the percentage of th1 cells may be helpful in distinguishing hies from atopic disease. however, since the percentage of th17, cd8+ifng+, and th1 cells correlate with age, caution should be used when testing young children. the inverse correlation between ige levels with th1 and th17 responses suggests that similar pathway(s) may drive both hies and atopy. additionally, the decreased th1 responses in stat3 lof and increased th1 responses in stat3 gof hies raise questions about the role of stat3 in regulating ifng levels. we also identified patients with different genetic disorders with fungal infections in which the th17 percentages were low, suggesting that the th17 test may be useful in evaluating individuals with unusual fungal infections. abstract/case report text background:primary and secondary autoimmuune neutropenia (pan/san) are well described entities. several autoimmune neutropenias do not fit the criteria of either pan or san showing peculiar characteristics mainly for older age at onset and/or for duration of the disease; moreover they are not associated , at least at the beginning, with autoimmune markers/diseases aim of the study: to describe a cohort of subjects affected with autoimmune neutropenia, defined as "atypical" (aan), registered in the italian neutropenia registry (inr) and to compare these data with those from subjects diagnosed with pan still in the inr. patient and methods: subjects with neutropenia and positivity of indirect antibodies against neutrophils (registered in the inr from 2013 to 2019) lasting for more than 3 years, or diagnosed after 5 years of age ( up to 18 y), without any associated autoimmune, signs/markers were considered eligible for the present study. results: data from 248 patients were collected: 79/128 subjects (32%) were defined as aan and 169/248 (68%) as pan. among 79 aan affected patients 61%, were "long lasting" aan, while 39% were defined as "late onset" aan .the degree of neutropenia in aan group was mild in 20 %, moderate 44 % and severe in 36 % of the subjects . leukopenia at onset was a common hall mark seen in 46% of aan patients (median values 3700/mm³ ; range 2750-5140/mm³ ) especially in the "late onset" aan if compared with pan and " long lasting" one ( p=0.003). as for clinical features, almost half of the aan cohort suffered from recurrent or "significative infections", while severe episodes (namely sepsis, meningitis , osteomyelitis , pneumonia, deep abscess or flemmon) were shown in 28% being more frequent, but non significantly higher than those reported in the pan group (6% )( p=ns) interestingly, recurrent apthae were significantly more seen in the "late onset" aan group if compared with the "long lasting" aan (p=0.006). during follow up, markers and/or symptoms of autoimmunity appeared in 31% of the aan cohort, being another element of peculiarity in respect to pan (p < 0.0001). as for immunological pattern in aan, immunoglobulin values were lower than the references for age in 12%,while were above them in 17% of the cohort . lymphocytes subsets evaluation showed decreased value of cd3+cd19+ cells in 40% of cases, followed by depletion of cd3-cd16+cd56+ subtype in 33%, cd3+cd4+ in 24 % of cases and cd3+ cd8+ in 17% . preliminary study on b memory and t-reg cells values, showed a quantitative deficiency respectively of in 59% and 27% of the studied subjects. mutation analysis performed by ngs in 20% of the subjects identified pathogenic variants of : taci (2), tinf 2 (1) and lrba (1) .comparison between pan and aan is detailed in table 1 . conclusions atypical neutropenia in childhood is a disorder which show many difference with pan; indeed appears an epiphenomenon of a complex immunological disturbances rather than a disease itself. occasionally mutations of genes of immunodeficiency/disimmunity can be demonstrated abstract/case report text background: medications treating ra typically include systemic corticosteroids used to treat inflammation flares, and disease modifying therapies (dmards). traditional dmards include methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine. recently, biologic/immuneresponse modifiers have come to the forefront for overall therapeutic benefit, however, an unfortunate side-effect may be the risk of increased immunosuppression. this study seeks to determine the occurrence rates of immune deficiencies among patients initiating ra therapies. methods: using the pharmetrics plus commercial claims database from 2012-16, ra patients (icd-9 714 and -10 codes: m05, m06) over the age of 18 were indexed on their first use of a new biologic therapy. all patients were required to have enrollment six-month pre and one-year post index. cohorts of patients were grouped by medication: methotrexate, adalimumab, etanercept, and rituximab. ra patients receiving adalimumab, etanercept, and rituximab were allowed concomitant use of methotrexate, but could not use any other biologic medications in the post period. a minimal adherence of 40% was required of all biologic treated ra patients. an additional cohort of ra patients untreated with biologic therapies was indexed on their first ra diagnosis within the time window and used as a control. ra patients with comorbid conditions who would also require biologic treatment were excluded including crohn's disease and ulcerative colitis. between group comparisons were made with the no treatment group as the referent. to account for differences in age, gender, and elixhauser comorbidity conditions patients in each cohort were matched 1:1 to the rituximab group. results: 52,013 ra patients met inclusion criteria: 6,608 in the methotrexate group, 2,826 receiving etanercept, 2,808 receiving adalimumab, and 467 receiving rituximab. a total of 12,709 in the treated groups and 39,304 in the no biologic treatment group. demographic information including age and gender were significantly different but numerically similar between the groups, with rituximab group having the highest proportion of female patients but limited dispersion with the lowest proportion being in the etanercept group. healthcare utilization metrics highlighted a significantly higher average number of office visits (21.18, sd: 13.48 vs no treatment 15.61, sd: 13.29, p < 0.01) and a higher proportion of rituximab patients being hospitalized (14.35% vs no treatment 10.37%, p < 0.01). the diagnosis of immune deficiency was highest among the rituximab group with 7.92% followed by methotrexate 2.91%, adalimumab 2.88%, etanercept 2.80%, and no treatment 2.80%. after matching, similar rates were seen for healthcare utilization to the pre-match results. the post-match odds of being diagnosed with immune deficiency were significantly greater for the rituximab group (or 3.76, ci: 1.61-8.85) than the no treatment group. conclusions: the purpose of dmards is to modulate the immune system and decrease autoimmunity in ra. however, this treatment may lead to significant immunosuppression. this study suggests that treatment with certain biologic/immune-response modifier therapies may be associated with higher rates of healthcare utilization. in particular, the increased post-treatment diagnostic coding of immune deficiency demonstrates the heightened awareness among healthcare providers of the chronic immunosuppressive potential of rituximab. evaluation of potential secondary immunodeficiency pre-and post-dmard use should be incorporated into routine practice. abstract/case report text introduction: autoimmune lymphoproliferative syndrome (alps) is a rare inherited disorder of lymphocyte homeostasis due to a fasmediated apoptosis and characterized by non-infectious and nonmalignant lymphoproliferation, autoimmunity, and secondary malignancies (national institute of health criteria). in spite of recent progress, one third of alps patients still remain gene orphan and they have been previously categorized as alps-u. in some cases, patients fitting alps diagnostic criteria have been shown to carry mutation on genes involved in other immune-dysregulation syndromes. aims: the aim of this study is to compare the clinical and immunological features, and the outcome of a cohort of alps patients with mutations on the typical causing genes (fas, fasl, fadd and casp 10)-here defined as alps-g -vs the ones without a molecular diagnosis or carrying mutations on other genes (both defined as alps-u). patients and methods the demographic, clinical, biochemical, genetic informations and details about treatment are derived from the alps italian network. search of mutations was performed with sanger pcr and/or next generation sequencing techniques (extended to immunodeficiency genes panel). results: 68 alps patients were registered in our data base; the genetic analysis was performed in 42 subjects (62%): 14/42 pts (33%) were alps-g and the remaining 28 (66%) alps-u. six-teen out of 28 (57%) alps-u patients resulted to carry mutations on other genes (lrba, stat3+cecr, ctla4, baffr, taci, nmlrc4, ikbkg, gaucher), and the remaining 12 (43%) were negative. the alps-u subjects showed a more complex phenotype compared to the alps-g group, which was characterized by multi-organ involvement (p=0.003) and positivity of autoimmune markers (p=0.002). (table 1 ). cytopenia affecting one or more haematopoietic lineages was present in both groups (69% and 82%) with no significant difference, apart from lymphocytopenia that was more frequent in alps-u group (p=0.03) ( table 1) . as for lymphocyte subets and immunoglobulin dosage no differences were shown within the two groups. vitamin b12 and il-10 were more frequently raised in alps-g group (p=0.01, p=0.001) (table 1). four out of 42 (9%) patients did not require any treatment. first-line treatment (steroid or intravenous immunoglobulins) controlled the disease only in 4/38 (10%) cases. the response rate to second line therapy -micofenolate mofetile (mmf) or rapamycin-was 100% and 40% in alps-g and alsp-u group, respectively. moreover, target therapies or drug combinations were more commonly applied in alps-u subjects (p=0.02) ( table 1) . conclusions: our study showed that alps-u subjects, despite the alps phenotype, represent distinct clinical entities and that genes associated with other immune-dysregulation syndromes are frequently represented in this group (16/ 28, 57%). the identification of such disorders is crucial for the management of second-line treatment and/or the administration of target therapies abstract/case report text background: we have shown previously that allergic reactivity to ovalbumin (ova) could be regulated in mice following perturbation of immune networks using combinations of an immune ig along with antiidiotypic ig. we have explored features of this regulation including: its persistence after cessation of administration of combined igs; the ability of heterologous igs to produce immunoregulation; a role for treg induction in regulation; and the ability to attenuate responses in mice presensitized to an allergic stimulus. methods: balb/c mice were sensitized to ova. mice also received 5 weekly injections of immune ig or anti-idiotype ig (at separate sites) from either homologous (mouse) or heterologous (human) sources. in the latter case pooled ivig (given im, hence hereafter imig) was used as a source of anti-idiotype ig, and human anti-tet as immune ig. injections of the ig were given from the time of ova sensitization (to attenuate development of immunity), or after pre-sensitization of mice (to attenuate existing allergic responses). all mice were assayed for development of ova-specific serum ige and igg, as well as the production of ova-induced il-2, il-4, il-13, il-31 and il-33 in splenocytes cultured for 72hrs. in studies examining possible mechanism(s) responsible for inhibition of immunity mice received, in addition to the ig treatments described, infusion of depleting anti-cd4, and/or anti-cd8 antibodies, or a mab to tnfsfr25, known to expand tregs implicated in regulation of allo immunity. results: combinations of both heterologous and homologous immune igs and anti-idiotype igs attenuated ova allergic responses in both naïve and pre-sensitized mice. this attenuation persisted in mice greater than 14 weeks after cessation of treatment with the igs used. finally, depletion of either cd4 or cd8 cells ameliorated the suppressive effect seen, while the combination of anti-cd4 and anti-cd8 essentially abolished suppression. suppression was further enhanced by anti-tnfsfr25 mab. conclusions: we conclude that the combine ig treatment protocols used produced a long-lasting suppression of allergic immunity, even in pre-sensitized animals. the effects seem to depend upon induction and expansion of tregs and represents a novel approach to treatment of allergic disease in humans and other animals. abstract/case report text background wiskott-aldrich syndrome protein (wasp) is found in the cytoplasm of hematopoietic cells but can transit to t lymphocyte nuclei at distinct developmental timepoints. wasp deficiency is a rare, x-linked combined immunodeficiency disease. affected patients display qualitative but not quantitative t cell defects. we report two immune deficient subjects with nearly identical exon 11 frameshift mutations in was, the gene encoding wasp. one subject lacked circulating t cells, the other possessed several distinct cd8 t cell populations each expressing quantitatively different amounts of wasp. objective to determine how similar was mutations can cause scid in one person and generate b and t cells with heterogenous wasp expression in another. methods to identify somatic was mutations, we deeply sequenced was exons, introns, promoters and 5' untranslated regions at 10,000 read depth in genomic dna from various b and t cell populations of each subject and their unaffected relatives. we confirmed genomic variants were transcribed and translated by sequencing was transcripts and analyzing wasp in primary cell lysates, both fractionated and not. to model our subjects' diseases we transfected primary cells and cell lines with mutant was transcripts and then measured viability and nuclear localization via confocal microscopy. results deep sequencing of genomic dna revealed all of subject one's cells carried the same germline exon 11 frameshift was mutation. the mutation was incorporated into subject one's was transcripts and translated into a truncated form of wasp, which was relegated primarily to the cell nucleus. subject two possessed three distinct cd8 t cell subsets that each carried either the germline exon 11 frameshift was mutation or a variety of somatic mutations that circumvented frameshift wasp expression. evasion strategies included exon 11 skipping, adoption of a cryptic exon 11 splice site and reversion to wild type amino acid sequence. subject two incorporated somatic mutations into was transcripts which encoded either stable near full-length proteins or unstable non full-length ones. subject one's sister and subject two's mother, who both carried the germline exon 11 frameshift mutation, produced only wild type transcripts and proteins. conclusion we report two patients with was mutations encoding truncated wasp. if expressed, truncated wasp localized to the cell nucleus, and this was associated with t cell developmental arrest and severe combined immune deficiency. if, through a variety of epigenetic and somatic strategies, t cells could avoid expression of truncated wasp, they would survive but display phenotypical abnormalities and functional defects. patients with pidds show a higher susceptibility to hematopoietic malignancies, in particular to non-hodgkin lymphomas (nhl) that, generally, account for approximately 6-7% of paediatric cancers and their incidence increases with age. recently new gene defects responsible for pidds with lymphoproliferation as a key clinical sign have been identified. our goal is to investigate possible immune-mediated mechanisms underlying malignant lymphoproliferation in children who did not show other typical symptoms of pidds. we retrospectively selected and reviewed the clinical history of nine patients with nhl (6 burkitt lymphoma, 2 large b cell lymphoma and 1 lymphoblastic tcell lymphoma). immunophenotyping and exome analysis of known pidds genes were performed after lymphoma remission. six out of nine patients showed a mild hypogammaglobulinemia at time of presentation, not noticed before. moreover, one patient had history of recurrent respiratory infections, one of hematologic autoimmunity and two of nine were ebv-positive at diagnosis. preliminary results show an aberrant b cell phenotype in four patients; exome analysis reveals a novel heterozygous genetic variation in ikzf1 gene in one patient with burkitt lymphoma and autoimmune cytopenia was identified. concerning the remaining patients, further studies are ongoing. a detailed review of clinical history of paediatric patients affected from nhl as well as an impaired immunophenotyping can be important indicators of immune-mediated disorder underlying lymphoproliferation and helpful signs of possible pidds that should promptly be investigated by genetic analysis. this will allow an appropriate diagnosis and disease management. abstract/case report text introduction: caspase activation and recruitment domain 11 (card11) encodes a scaffold protein that links antigen receptor activation to intracellular signaling. dominant heterozygous loss of function (lof) mutations in card11 cause a syndrome of severe atopic dermatitis, elevated ige, and allergic disease. atopic dermatitis can be difficult to control leading to substantial morbidity. dupilumab is a humanized monoclonal antibody that blocks il-4 and il-13 signaling approved for treatment of refractory atopic dermatitis. we present a case of a 10-year-old female with card11 deficiency successfully treated with dupilumab. case: a 10-year-old puerto rican female with history of recurrent sinopulmonary infections with 5 episodes of pneumonia, moderate persistent asthma, food allergies, recurrent skin boils, and severe atopic dermatitis was referred for further management and evaluation for autosomal dominant hyper-ige syndrome (ad-hies). her atopic dermatitis was refractory to conventional therapy with topical corticosteroids, twicedaily emollient use, and bleach baths; it was also refractory to immunosuppression with mycophenolate mofetil and cyclosporine. on exam the patient exhibited coarse facial features and a high palate. she had eczematous lesions on the face, trunk, and extremities (scorad 84). laboratory evaluation showed: eosinophilia (1400 cells/ul), elevated ige (>2000 ku/l), low igm (20mg/dl), and elevated iga (568 mg/dl). lymphocyte subsets and mitogen response were normal but antigeninduced proliferation was abnormal. autosomal dominant hyper ige score was 42 indicating a high likelihood of ad-hies. no mutations in stat3 were identified and th17 cell expression was elevated. dedicator of cytokinesis 8 (dock8) deficiency was also considered but dock8 protein expression was normal. further genetic testing revealed an 18 base pair deletion in card11 (c.518_535del) predicted to be pathogenic. the combination of the patient's phenotype and large deletion was consistent with card11 deficiency. despite continued immunosuppression with cyclosporine and aggressive skin care, the patient's atopic dermatitis was still severe and poorly controlled. off label (patient < 12 years) treatment with subcutaneous dupilumab 300mg every 2 weeks was initiated. at last follow-up, 4 months after dupilumab start, the patient had substantial improvement in dermatitis with clear skin on the face, trunk, and extremities (scorad 40). cyclosporine was discontinued and topical medications were applied less frequently. discussion: hypomorphic heterozygous dominant negative loss of function mutations in card11 have recently been associated with severe atopic dermatitis and allergic disease. treatment of atopic dermatitis in card11 deficiency remains challenging, but dupilumab appears to be an effective alternative to refractory disease. longer follow-up and a larger cohort of card11-lof patients treated with dupilumab are necessary to understand the long-term efficacy and safety for use of dupilumab in these patients. 6 abstract/case report text purpose: the micromilieu within premalignant respiratory papillomas supports persistent hpv6/11 infection and disease recurrence in recurrent respiratory papillomatosis (rrp). these patients show polarized (th2-/treg) adaptive immunity in papillomas and blood, enriched immature langerhans cell (ilc) numbers, and overexpressed cox2/pge2 in the upper airway. to better understand the adaptive and innate dysregulation in rrp, we studied blood-derived monocytes, ilcs, and tissue-derived ilcs from rrp patients and controls. experimental design: monocyte subpopulations were isolated, differentiated into ilcs, activated, and then assessed by flow cytometry. monocytes were induced to differentiate into ilcs with/ without added pge2, and then activated by il-36γ, pge2, pge2+il36γ, or lps. ilc cd83 expression was identified by flow cytometry. monocyte-derived ilcs, papilloma, foreskin, and abdomen skin ilcs, were also analyzed by qpcr for select chemokine/cytokine mrna expression after isolation, 24 hrs later in culture, and again after poly(i:c) or tnfα stimulation. results: the three monocyte sub-populations differed between patients and controls, and patients' monocytes generated fewer ilcs. classical monocytes generated most, but not all ilcs. pge2 levels were higher in rrp plasma, and added pge2 reduced control, but not patients' monocyte-ilc differentiation. pge2 had no effect on ilc maturation identified by cd83 expression. papilloma-derived ilcs expressed low ccl-1, and high ccl-20 mrna and were unresponsive to poly(i:c) or tnfα. tissuespecific cytokine/chemokine responses between ilcs from papillomas, foreskin and abdominal skin differed. only papilloma ilcs expressed il-36γ after isolation, and they up-regulated ccl1 mrna 24 hrs later without further stimulation. conclusions: monocyte/ilc innate immunity is impaired in rrp, in part due to increased pge2 exposure. the immunosuppressive papilloma micromilieu likely alters ilc responses that skew, hpv6/11-specific th2/treg adaptive immunity in rrp. abstract/case report text introduction: familial mediterranean fever is a hereditary auto inflammatory disorder that typically manifests with recurrent fevers, abdominal pain and in some patients there is an associated with amyloidosis leading to eventual renal failure. while there are several common mutations in the mefv gene that when homozygous give these classic symptoms, patients with atypical mutations or heterozygous mutations often have a different clinical course. we present identical twin siblings with compound heterozygous mefv mutations but differing clinical phenotypes. case description: the index patient is a 3 year old girl, conceived via ivf, who began having fevers at age 2.5. her fevers occurred every 4 weeks for 4 months before she was referred to immunology for evaluation. her parents describe her as happy and otherwise not ill appearing during these episodes. genetic testing for familial mediterranean fever revealed compound heterozygous e148q and p369s mutations in the mefv genes. initiation of colchicine therapy in the affected sibling has resulted in a complete resolution of her symptoms. a trial off colchicine resulted in return of cyclic fevers. her identical twin sister was also tested, and carries the same mutation, but is still asymptomatic. this created great concern amongst their parents who had genetic testing prior to undergoing ivf that revealed no parental mutations in mefv. in consultation with genetics the mother was tested again through the same laboratory that had performed testing on the children. this revealed an identical mutation in mom who is also asymptomatic. conclusions: although classic homozygous mefv mutations have resulted in well described fever syndromes, there is considerably less data on heterozygous and compound heterozygous mefv mutations. in these two identical siblings only one patient has a classic manifestation of familial mediterranean fever. while it is possible that the other twin will develop similar symptoms later on in life, it is also possible that another factor is necessary to trigger symptoms in this unusual genetic presentation of fmf. in addition this case highlights the importance of understanding the testing method used by the laboratory performing the genetic testing. while the mother was initially reported as negative the laboratory that performed her testing only tested for the most common mefv mutations. more complete testing, that included the entire gene sequence, revealed that she did contain an mefv mutation in e148q, which although more rare is thought to be pathologic when combined when combined with a second mutation. abstract/case report text there are several lines of evidence that link the pi3k/akt/mtor signaling pathway to primary immunodeficiencies. hyperactivation of the pi3k/akt/mtor/s6k signaling pathway in immune cells can be the consequence of dominant gain-of-function mutations in the genes encoding for pi3kδ that cause the activated pi3kδ syndrome (apds). patients with these mutations may develop immunodeficiency and immune dysregulation as well as neurodevelopmental delay and growth retardation. in addition, mutations of genes within the pi3k-akt-mtor pathway were also known to cause megalencephaly and segmental cortical dysplasia. mutations in akt3, a member of the akt family of proteins and a downstream effector of pi3k-mediated signaling, was shown to be associated with autosomal dominant megalencephalyassociated syndromes. here we describe a 4 year old girl, born to consanguineous healthy parents, who presented with megalencephaly, developmental delay, hypotonia, cervical lymphadenopathy and hepatosplenomegaly. the patient had recurrent hospital and icu admissions for idiopathic thrombocytopenia (treated with ivig), recurrent laryngitis, recurrent peritonsillar abscess, preorbital cellulitis, conjunctivitis with purulent discharge, otitis media, pneumonia with pleural effusion (required drainage), metapneumovirus pneumonia with respiratory failure, recurrent skin cellulitis, and abscesses that grew mrsa (required drainage). in addition, the patient is known to have asthma and allergic rhinitis. mri of the brain showed megalencephaly, ventriculomegally, thin and dysplastic corpus callosum, a normal cerebellum, and myelination appropriate for age. immunoglobulin levels, lymphocyte subsets and the oxidative burst test were all within normal limits. cmvand ebv were not detected. bacterial cultures grew mrsa (skin), strept. pneumoniae, h. influenzae, and e. coli (urine). extensive metabolic workup was done, which was inconclusive (metabolic/mitochondrial diseases). whole exome sequencing identified an akt3 variant c.958g>a; p.(asp320asn) in exon 10. the variant was identified in the patient but not in the parents and it was confirmed by sanger sequencing. further molecular testing concluded that the variant is caused by a de novo mutation during early development. although pathogenic variants in akt3 gene were shown to be associated with megaloencephaly-associated syndromes, no associations with immune deficiency have be reported. functional studies will be pursued to confirm the link between the clinical phenotype and the identified variant in the akt3 gene. complex is a critical signalling adaptor that regulates lymphocyte activation, proliferation, survival, and metabolism. primary immunodeficiencies affecting each component (termed 'cbm-opathies') result in broad clinical manifestations ranging from combined immunodeficiency (cid) to atopic disease or lymphoproliferation. we present the laboratory and clinical findings of two canadian first nations patients found to be homozygous for the same novel card11 mutation (c.2509c>t; p.r837*) causing complete card11 deficiency. results: we recently identified an 8-month-old boy who presented with a severe case of entero/rhinovirus bronchiolitis with interstitial lung disease and a 17-year-old boy with a history of severe pulmonary infections with bronchiectasis (including pjp), chronic sinusitis, candidiasis, invasive bacteremia, and severe ileo-colitis and oral ulceration requiring total colectomy. testing of both patients demonstrated absent tregs, elevated naïve b cells with absent memory b cells, and panhypogammaglobulinemia. next generation sequencing revealed that both patients were homozygous for the same novel variant of card11 (c.2509c>t; p.r837*), which rendered card11 protein undetectable by immunoblot. card11 deficiency was confirmed by stimulating patient b cells with phorbol 12-myristate 13 acetate (pma) and ionomycin and immunoblotting for signalling proteins in both the nf-κb (ikkα/β, iκbα, p65) and mapk (mek1/2, mkk4, jnk1/2, erk1/2) pathways as well as cleavage substrates of the malt1 paracaspase (relb, cyld, bcl10, hoil1). nf-κb and jnk activation were completely absent and m a lt p a r a c a p a s e a c t i v i t y w a s l o s t . f u r t h e r m o r e , c oimmunoprecipitation experiments revealed that card11 was required for optimal malt1 association with bcl10 in response to stimulation. to define the impact of card11 deficiency on the b cell transcriptome, rna-seq experiments were performed. this revealed an inability to upregulate critical genes involved in immunity and tolerance (e.g. cd40lg, ctla4, il2, il10), decreased enrichment in cytokine pathways (e.g. ifn-α, il-6, tgf-β), and decreased enrichment in malt1-dependent genes. furthermore, rna-seq confirmed the developmental block observed in patient b cells and suggested that b cells were halted at the centroblast to centrocyte transition. both patients ultimately underwent hematopoietic stem cell transplantation (hsct), which restored lymphocyte signalling and activation as measured by nf-κb, jnk, and malt1 paracaspase substrate cleavage. conclusions: we have presented the most comprehensive clinical and molecular characterization of human card11 deficiency to date. these two cases highlight the crucial role of card11 in regulating b cell development, function, and humoral responses, as confirmed by signalling and transcriptomic analyses. furthermore, hsct is potentially helpful for these patients as assays performed on post-transplant cells demonstrated restored signalling and activation. abstract/case report text introduction: primary immune deficiencies (pid) can have a significant impact on the quality of life of patients and their families. as more patients with pid are surviving to adulthood, the need to monitor them closely and ensure they are transitioned appropriately is even more crucial. we compared the perspectives of pediatric and adult immunologists toward the transition of patients with pid at our institution. methods: pediatric allergy/immunology providers at lurie children's hospital and adult allergy/immunology physicians at northwestern university both in chicago, il completed respective surveys anonymously (www.surveymonkey.com). questions were derived from the validated 'attitude' and 'quartt' instruments for transition. respondents were asked to rate their level of agreement on a 5-point likert scale, ranging from strongly disagree to strongly agree. results: overall, 9 pediatric and 11 adult providers participated (response rate 71.4%). of total respondents, 55% thought the transition process should be initiated at age 18-20. about 36% of the adult immunologists selected 21 and older, whereas pediatric providers would begin earlier; 33.3% of pediatric providers note they would initiate transition at age 15-17. both pediatric and adult immunologists agreed that patients should be transferred when the provider felt they were ready (80%) and when they were in stable condition (70%). both adult and pediatric immunologists selected transfer of complete medical file as a preferred communication method for transition. other strategies preferred by adult providers were a referral letter with brief summary of medical history (90.9%) and staff meeting with pediatric and adult immunologists (63.6%), whereas pediatric providers would prefer a joint outpatient dedicated transition clinic (77.8%). pediatric and adult immunologists, patient, parent, and transition liaison were considered the most important active participants in the transition process. the most prominent barriers to a formal transition were unavailability of a transition coordinator or nurse specialists (100%) or of all disciplines of the interdisciplinary team (95%), and limited time (95%). on the other hand, limited demand (too few patients) was strongly rejected as a barrier. all participants agreed during transition patients should be educated about medications and their side effects, their condition and related potential future complications, and symptoms that require seeking health care. over 95% of participants also agreed that education about how to set up ivig and further insurance needs were important. all participants agreed that the transition process should include assistance on how to promote the patients' independence and self-management skills, medication management/adherence, and understanding of immunoglobulin replacement and side effects. other important transition components included knowing how frequently lab draws are required for monitoring (95%) and having a written individualized transition plan (70%). pediatric providers also thought having an email or telephone help line would be beneficial. conclusion: this study adds to the growing body of literature examining attitudes of immunologists toward transition, and it highlights important transition components and barriers. further work is ongoing to determine the transition needs identified by patients and parents and define markers for successful transfer in order to build a transition policy at our institution specific to immunodeficiency patients. abstract/case report text introduction: bronchiectasis (bq) is an abnormal and irreversible dilatation of bronchi secondary to repeated cycles of airway infection and inflammation. predominantly antibody deficiency is the main group of primary immunodeficiencies (pid) in adults and had been reported up 10% of subjects with non-cystic fibrosis bronchiectasis (ncfb). hypergammaglobulinemia (igg level higher than 1,600 mg/dl) had been observed in 9.2 % of ncfb cases (retrospective data). diagnostic delay and inappropriate management of patients with predominantly antibody deficiency can lead to irreversible lung damage or even death from serious infections. the effect of hypergammaglobulinemia on ncfb is unknown. here we present the frequency of immunoglobulin abnormalities (pad and hyperigg) in adults with ncfb in cali, colombia. methods: we present preliminary data of a descriptive prospective study that will include 260 patients with ncfb. women and men >14 and < 65 years old will be included. all volunteers will be evaluated by a clinical immunologist, complete blood count and serum igg, iga, igm and ige levels will be determined. according with clinical suspicious, igg subclasses, anti-pneumococcal igg response and b cell subpopulations will be performed. the project will be executed in 24 months. written informed consent has been obtained for all subjects included. this project count with irb approvals at universidad del valle and hospital universitario del valle. results: a total of 103 ncfb cases have been included in the study. the mean age was 46.7 years (14-65 years) with a female:male ratio 64:39. moderate-severe dyspnea was observed in 17/103 cases (medical research council -mrcdyspnea scale 4 to 5). recurrent pneumonia was found in 33/103 cases (32%). the main etiologies of bronchiectasis were: post-infection 30/103 (29%); idiopathic 13/103 (12%); autoimmunity 12/103 (12%); primary immunodeficiency 11/103 (10%) asthma 11/103 (10%); copd 4/103 (3.8%); primary ciliary diskinesia 3/103 (3%); reflux 3/103 (3%) and others. primary immunodeficiencies, 10.6% of ncfb cases, were classified as: predominantly antibody deficiency (10cases) including cvid 5 cases, igm deficiency 2 cases, igg subclasses deficiency 1 case, selective iga deficiency 1 case and hypogamaglobulinemia 1 case. combined immunodeficiency (dock8 deficiency) 1 case. interestingly igg hypergammaglobulinemia was observed in 29/103 cases (28.1%) suggesting humoral immune response deregulation. conclusion to the best of our knowledge this is the first prospective study evaluating the etiology of non-cystic fibrosis bronchiectasis (ncfb) in colombia. hypergammaglobulinemia and predominantly antibody deficiencies affect 39% of adults with ncfb in colombia. our study reinforced the necessity to evaluate humoral immune response in patients with bronchiectasis. conflict of interest: authors disclosure any potential financial conflict of interest related to this abstract. acknowledgements: this investigator-initiated research was supported with a grant from baxalta us inc, a member of the takeda group of companies bt16-35583/iir-col-bxlt-001923. abstract/case report text background: early-onset inflammatory bowel disease (eoibd) is defined as ibd diagnosis in children less than 10 years of age. the occurrence of autoimmune disease in children (where it is relatively rare, compared to adults) may be caused by a highrisk predisposition gene (monogenic disorders). mayo clinic children's center has a unique care model, where a patient who is referred for eoibd meets with a team of physicians, including gastroenterology, immunology, genetics, and nutrition. we describe our experience of our eoibd clinic from an immunologic perspective. methods: we conducted a retrospective cohort study through emr chart review of pediatric patients who were referred to our eoibd program (2011 -2019). first diagnosis of ibd under the age of 10 was the inclusion criteria. we assessed the presentation, clinical correlates, and immunologic evaluation. approval was obtained from mayo's institutional review board. data abstraction and analysis was done using the software jmp. results: 51 pediatric patients met the inclusion criteria, with 31(61%) males and 20(39%) females. the median age of ibd diagnosis was 5 years (3-7 years range). median values and the distribution of variables used in the nutritional and immune evaluation were assessed (fig 1) . nutritional assessment was remarkable for low to low normal hemoglobin and ferritin levels. vitamin d and albumin levels were overall within the normal range. growth parameters indicated that the median bmi percentile was 57 (28-77). with immune and genetic screening, one patient was found to have x linked chronic granulomatous disease (cgd). immune evaluation of other patients was overall within normal limits. fecal calprotectin served a reliable non-invasive biomarker for inflammation with the median being 220.5 (62.7-499.3). 41 of these patients underwent gi pathogen panel testing of which 17 (41%) tested negative, four (10%) tested positive for c. diff, and two (5%) others to shiga toxin-producing e. coli. it was also noted during the chart review that most patients had poor disease control despite undergoing treatment with various anti-inflammatory and immunosuppressive drugs. the patient diagnosed with cgd underwent bone marrow transplantation. a higher proportion of patients referred to our program in recent years underwent a more comprehensive multispecialty evaluation. conclusion: awareness of monogenic causes of inflammatory disorders in children has increased in recent years. it is also important to rule out intestinal infections that can act as ibd mimic. identifying monogenic disorders and other ibd mimics helps with targeted therapy and symptom improvement in these patients who have a difficult-to-treat disease. the group of children with eoibd, regardless of whether there is an inborn error of immunity, suffers from very high morbidity and a high burden of disease. comprehensive immune-nutrition assessment of eoibd patients paves way for further in-depth immunogenic assessments and allows for global management. abstract/case report text background: chronic granulomatous disease (cgd) is a primary immunodeficiency (pid) affecting the nadph oxidase system in phagocytes resulting in increased susceptibility to catalase-positive organisms. holland presented the first report of dual impact of cgd and hiv in a patient with disseminated nocardiosis. because their cgd patient admitted to history of iv drug use, he was frequently screened for hiv. case: a 19-year-old african american male with known cgd tested positive for hiv1 by western blot in the ed in 2009 when he presented with complaints of intermittent fever and cervical lymphadenopathy. his cgd was diagnosed by nbt blood testing at 4 years of age. he had frequent skin infections and fever prior to diagnosis. clinically, he did so well that his cgd diagnosis was questioned by his immunologists. however, cgd was confirmed by 2 additional abnormal nbt tests and, ultimately, dhr flow cytometry testing. during his second infectious disease consultation for hiv, at age 19, he disclosed that he was bisexual. previously, the patient was screened for hiv1 and hiv2 antibodies in 2003 due to anal fissure. he was screened again in 2007 for marked cervical and supraclavicular lymphadenopathy. his cd4+ t cell absolute count was noted to be low (293/mm3) in 2006 at age 17. until 2009, his prior hiv screenings were negative. during his cgd treatment course as an adult, he was known to be variably adherent with administration of interferon gamma due to adverse effects, particularly pain at the site of injection and malaise. at the time of his positive hiv western blot in 2009, his cd4+ t cell count was 237/ mm3. after starting hiv antiretroviral treatment, his viral load became undetectable. at age 23, he had burkholderia cepacia pyelonephritis resulting in left nephrectomy. sepsis from b. cepacia was fatal (positive blood cultures without known primary source) in 2019 at age 29. his recent viral load was still undetectable and cd4+ count was 240/mm3. summary: our case reveals the complexities of treating a patient with both primary and acquired immune deficiencies. it illustrates the importance of taking a thorough social and sexual history starting in adolescence, including those patients with pid. patients with pid should be followed closely by a primary care physician, in addition to an allergist-immunologist and infectious diseases specialist, to ensure age appropriate medical and developmental screening. the recognition of pids is improving due to better screening, awareness, and treatment. currently, the rate of hiv infection is highest among young homosexual african american males. it remains important to understand the epidemiology of primary and acquired immunodeficiencies to best identify those at highest risk. (155) submission id#812008 phosphatase and tensin homolog (pten) hamartoma tumor syndrome identified by newborn tcell receptor excision circle screening for severe combined immunodeficiency δ) subunits that are critical for cellular signaling. heterozygous gain-offunction (gof) mutations in pik3cd (encoding p110δ) result in activated pi3k δ syndrome 1 (apds1), while heterozygous loss-of-function (lof) mutations in pik3r1 (encoding p85α) result in activated pi3k δ syndrome 2 (apds2). given its role as a negative regulator of the pi3k signaling pathway, heterozygous lof mutations in pten (encoding phosphatase and tensin homolog, pten) result in a clinical phenotype that approximates that of apds1/apds2 and is therefore referred to as activated pi3k δ syndrome-like (apds-l). however, sequelae of heterozygous pten lof mutations extend beyond the immune system and include a group of disorders collectively known as pten hamartoma tumor syndrome (phts). although severe t cell lymphopenia at birth would be unexpected in apds1, apds2, or apds-l, below normal t cell receptor excision circle (trec) counts have been reported in apds1, but only in individuals outside of the neonatal period. herein, we describe an infant girl with a low trec count at birth who was found to have phts. case description: a 1-day-old girl, born at a gestational age of 39 weeks, was found to have a low trec count of 22/microliter (normal => 40). a second trec count obtained at 2 weeks of age resulted as 16/microliter. arguing against a diagnosis of severe combined immunodeficiency (scid), flow cytometric analyses performed at 3 weeks of age revealed only a modestly diminished cd3+ t cell count (1599/microliter; 1240 cd4+ and 277 cd8+) with a normal percentage of naïve and memory cd4+ t cells (78% and 22%, respectively). by 7 months of age, her cd3+ t cell count dropped to 828/microliter, which was accompanied by a significantly decreased percentage of naïve cd4+ t cells (56%). sequencing and deletion/duplication analysis was pursued via a commercially available 207-gene panel aimed at genetically defined primary immunodeficiency (pid), in which no clearly pathogenic mutations were identified. over the following months, the patient was noted to have macrocephaly, tall stature (99th percentile), axial hypotonia, and gross motor delays. sequencing and deletion/duplication analysis was then pursued via a commercially available 29-gene panel aimed at genetically defined macrocephaly and overgrowth syndromes, in which a hemizygous pathogenic mutation in pten (c.512a>g, p.gln171arg) was identified. subsequent flow cytometric analyses demonstrated findings characteristic of apds-l, including expanded transitional and cd21lo b cells, decreased isotype switched memory b cells, increased effector memory t cells, a lowered threshold for intracellular calcium mobilization upon b cell receptor engagement, and increased basal akt (protein kinase b) and s6 (ribosomal protein s6) signaling. discussion: we report the first case of phts identified by newborn trec screening for scid. as pten is not included in most commercially available, scid-or pid-tailored gene panels, phts would be missed by conventional genetic testing. therefore, analysis for variants in pten should be considered in neonates with low trec counts, macrocephaly, developmental delay, and other suggestive sequelae. abstract/case report text primary (or familial) hemophagocytic lymphohistiocytosis (hlh) is a rare, life-threatening hyper-inflammatory syndrome affecting mainly young children. it is caused by mutations in genes involved in the granule-dependent cytotoxic pathway, inducing extreme inflammation and massive tissue infiltration by activated t cells and macrophages. standard chemotherapy-based treatment regimens are toxic and induce remission in only 80% of patients. to this day, hsct is the only available curative treatment, but the inability to efficiently control the inflammation in many patients prior to transplantation often leads to graft failure, with transplant-related mortality around 25%. thus, the development of new, more potent and less toxic anti-inflammatory regimens would be a major advancement in the treatment of hlh. here, we hypothesize that combination therapies targeting several jak-dependent cytokines will be more effective than monotherapy to reduce the life-threatening symptoms induced by this pathology. using a perforin-deficient (pko) mouse model, we first tested the effects of blocking antibodies against ifnγ, the dominant cytokine secreted during hlh, in combination with antibodies targeting other highly elevated cytokines, such as il-6 and il-18, on the manifestations of the disease. we found that anti-il-6r and anti-il-18 antibodies, when used in combination with anti-ifnγ antibodies, did not significantly improve the symptoms of hlh compare to anti-ifnγ antibodies alone. further, we found that targeting the jak-stat signaling pathway with ruxolitinib, a specific inhibitor of jak1 and jak2, molecules downstream of ifnγ and il-6, but not il-18 signaling, was as beneficial as anti-ifnγ monotherapy. next, we tested the efficacy of ruxolitinib in combination with anti-il-18 antibody, as this later cytokine is not jak-dependent and was shown to drive macrophage activation syndrome in other contexts. unfortunately, this combination did not result in better symptom resolution than the use of ruxolitinib only. in contrast, combination therapy using ruxolitinib and anti-ifnγ antibodies showed a striking synergistic effect on the resolution of most disease manifestations, to such an extent that our pko mice presented a clinical phenotype indistinguishable than that of a c57bl6 control mice. our findings demonstrate that jak-dependent cytokines are the main cytokines driving the progression of hlh in pko mice. collectively, our results suggest that anti-ifnγ antibodies and ruxolitinib, although effective independently, should be used in combination to more efficiently suppress hlh progression. these results are particularly relevant since the emapalumab, an anti-ifnγ monoclonal antibody was recently approved by the fda for the treatment of hlh while ruxolitinib will soon be in clinical trials for this indication. this project was supported by funds from the fondation de cancérologie charles bruneau and the canadian institutes of health research (mop-130469). abstract/case report text introduction: transcription factor 3 (tcf3), also known as transcription factor e2-alpha (e2a), is a helix-loop-helix transcription factor which plays a critical role in lymphopoiesis. tcf3 is required for b and t lymphocyte development. defects in tcf3 have been associated with agammaglobulinemia 8, autosomal dominant, characterized by low levels of immunoglobulin and early onset recurrent bacterial infections. deletion or diminished activity of tcf3 may also play a role in lymphoid malignancies. runs of homozygosity (roh) are contiguous stretches of homozygous genotypes at consecutive polymorphic dna marker positions. roh are important reservoirs of homozygous deleterious variation. the homozygosity heterogeneous hmm (h3m2) algorithm was specifically developed for analyzing whole exome sequencing (wes) data. the branch point sequence (bps) is an essential splicing signal located 15-55 bases upstream of splice acceptor sites. while bps variants are rare, they may result in aberrant pre-mrna splicing and genetic disorders. these variants may be overlooked by standard wes analysis methods because they are intronic and the mammalian bps is a degenerate motif. objective: describe the method used to identify a bps variant in consanguineous brothers with immunodeficiency, including early onset recurrent infections and b-all, hypogammaglobulinemia, t and nk lymphocytosis, low b cells, and low naïve t cells. methods: wes was performed for all family members. data were analyzed using standard read mapping, variant calling and annotation methods. roh were analyzed using the h3m2 algorithm (magi et al. 2014). roh from the siblings was intersected (bedtools) and the output was submitted to the genomic oligoarray and snp array evaluation tool (v3.0). variants were confirmed by sanger sequencing. results: no candidate disease variants were detected in the coding regions, 5' or 3' splice sites, or utrs for both brothers; however, analysis of intersected roh revealed a homozygous tcf3 intronic variant within a putative bps (tcf3 c.1451-18a>t). sanger sequencing of the mutant cdna revealed activation of a cryptic splice site. heritability for routine childhood vaccines has been shown to range from 38-89%. the genetic component of vaccine response suggests we should be able to predict vaccine response in infants with biomarkers. methods: multi-center study of 93 infants born vaginally at full term and followed through 12 months of life. cord blood was collected at birth & peripheral blood was collected at 6 and 12 months of life. six-month collection was 2 weeks post administration of routine vaccinations, while 12-month collection was immediately prior to receiving the 12-month booster vaccines. b cell subsets were analyzed with flow cytometry. vaccine titers and cytokines were measured via multi-plex elisa. study was irb approved. results: our data confirmed the immaturity of the newborn humoral immune system with a lower overall b-cell abundance, a predominance of naïve b cells, an inability to class-switch and produce igg or iga, and a th2 bias. maturation was observed over the first year with increasing overall b-cell abundance, frequency of memory b-cells producing iga, igg, and igm, and frequency of plasmablasts. scd14 levels also increased throughout the first year due to microbial translocation reflecting establishment of the microbiome. conversely, cord blood contained high levels of baff, april, scd40l, il-4, and il-21, with levels decreasing thereafter. all infants displayed evidence of humoral immune system activation after getting 6-month vaccines. total plasmablast levels peaked 2 weeks after receipt of 6month immunizations. a decrease in total plasmablasts was evident between 6 and 12 months, although levels remained above those at birth, corresponding with the need for 12-month booster vaccinations to maintain long-lasting immunity. il-21 and ifn-gamma had a significant positive correlation with memory b cells and plasmablasts at subsequent time points suggesting that these cytokines play a role in b cell differentiation and vaccine response. baff and april cytokines were elevated at birth, consistent with germinal center formation and underwent a compensatory decrease thereafter. april & scd163 levels in cord blood significantly correlated with higher tetanus titers at 12 months suggesting that vaccine response may be predicted by cytokine biomarkers at birth. response to vaccines was also dynamic with il-2 levels being significantly correlated with tetanus titers at 2 weeks after receipt of 6 month immunizations. conversely, scd40l levels did not correspond to b cell development consistent with a known b cell hyporesponsiveness to cd40l in infants. conclusion: humoral immune development is both predictable and dynamic. biomarkers in the cord blood, produced by the infant, are predictors of b cell development and vaccine response in infancy. background: adult-onset immunodeficiency with anti-ifnɣ autoantibodies is a newly described immunodeficiency syndrome characterized by disseminated nontuberculous mycobacterial and other opportunistic infections in previously healthy middle-aged individuals typically from southeast asia. it is caused by the presence of autoantibodies directed against the cytokine ifnɣ, which is required for intracellular pathogen killing by macrophages as well as phosphorylation of the transcription factor stat1, which is involved in cell survival gene expression. successful treatment of the immunodeficiency has been described in prior case reports with immunomodulatory therapies, including rituximab. however, there are no standard recommendations for dosing or timing of these agents, or recommendations for longterm monitoring of disease activity. case presentation: a 49-year-old laotian woman with a history of type 2 diabetes and possible prior hepatitis c infection presented to the immunology clinic for evaluation of immunodeficiency. in the two years prior to presentation, the patient was diagnosed with mycobacterium avium infection involving the parotid gland and lymph nodes of the neck, mycobacterium avium complex bacteremia, histoplasma capsulatum involving the lymph nodes of the neck, and leukocytoclastic vasculitis of the lower extremities. as a child and young adult, she had no severe or recurrent illnesses and did not suffer from any chronic disease. preliminary immunologic testing demonstrated normal t, b, and nk cell subsets, elevated immunoglobulin g, a, and m levels, and protective titers to tetanus, diphtheria, and 23/23 pneumococcal serotypes. measurement of anti-ifnɣ autoantibodies was positive, which led to the diagnosis of adult-onset immunodeficiency with anti-ifnɣ autoantibodies. the patient was treated with four doses of monthly rituximab with resolution of the anti-ifnɣ autoantibodies, restoration of normal stat1 phosphorylation, and depletion of cd19-positive b cells. after a 4-year period of being lost to follow-up, during which she continued to receive rituximab every 6 months at the direction of a local provider, the patient re-presented to the immunology clinic to re-establish care. at that time, the patient had no evidence of anti-ifnɣ autoantibodies based on titers and normal stat1 phosphorylation. cd19-positive b cells remained depleted. the patient also confirmed subjective clinical improvement and denied any interim infectious complications. conclusion: this case provides an example of successful treatment of a patient with adult-onset immunodeficiency with anti-ifnɣ autoantibodies with rituximab. it also highlights the utility of ifnɣ functional testing with stat1 phosphorylation, which may be used to monitor disease activity and to make decisions about ongoing immunomodulatory treatment. it is necessary to monitor additional immunological markers to refine the diagnosis of a secondary post-lt immunodeficiency to take preventive measures before infections occur. we sequentially measured t lymphocytes and antibodymediated immunity in a 67-year-old male receiving a lung transplant for idiopathic pulmonary fibrosis to determine which immune indicators could improve the identification of a secondary immunodeficiency. methods: t and b cell numbers, igm, igg, iga, ige and igg subclasses and 13 specific antibodies to s. pneumonia capsular polysaccharides were assessed over a 5 months-long post lt period. the clinical progress, infections and pulmonary function were monitored prior to transplantation and at regular intervals thereafter. results: the patient had progressive idiopathic pulmonary fibrosis starting with an episode of pulmonary hypersensitivity 4 years earlier. he developed increasing respiratory failure progressing to complete 02 dependency requiring a lt in june 2019. he was treated with prednisone, 20mg/ day continuously for 3 months, then decreased to 15 mg/day. other immunosupressants included mycophenolic acid and tacrolimus and on/ off antibiotics that eventually led to severe tendinitis at 4-5 months post lt. at 4½-month post lt he developed an early onset bronchiolitis obliterans syndrome (bos) that was controlled by increasing the prednisone dose. sequential immunologic evaluation showed his igg dropping from 1,400 mg/dl to 800 after two weeks and then remaining stable at that level for the rest of the observation period. igm and iga had minor variations and ige remained very low. igg2 fell from 700-800mg/ml pre-lt to 180-200 in 2 weeks and remained stable at that level thereafter. antibodies against s. pneumoniae polysaccharides started high, between 5-15 ·g/ml for all 13 serotypes and fell rapidly in the first 2 weeks post lt, then continued a steady decline with > 50% serotypes falling < 1.3·g/ml at 5 months. twelve of 13 pneumococcal serotype antibodies increased above 1.3·g/ ml after igg replacement at 5 months. cd4 t lymphocytes decreased from ±1,000 cells/ul to 500-600 at 1 month, remaining at that number after that. cd4/th17 cells increased from 4-32 cells/ul to 178 at 5 months when prednisone was tapered down and then decreased to 12 after increasing the prednisone dose again. this decrease coincided with a reduction in bos manifestations. conclusions: immune monitoring revealed an independent decrease of immunoglobulins with a stronger decrease in igg2 and specific pneumococcal antibodies. the role of th17 cell increase in developing bos needs further investigation. stat3 is a frequent target of cancer therapies due to its role in certain malignancies for cell proliferation and metastasis. with lof stat3, decreased incidence of some cancers may be expected, however increased rates of lymphoma are described. we sought to describe the incidence and spectrum of malignancy in our relatively large lof stat3 cohort. methods: we performed a retrospective analysis of 158 lof stat3 patients evaluated at the nih clinical center to determine the type of malignancies diagnosed, treatments received, and outcomes following therapy. results: a total of 9 patients with 10 malignancies were identified (cancer incidence 6%). six patients (4%) were diagnosed with non-hodgkin lymphoma (nhl); 5 with diffuse large b-cell lymphoma (dlbcl) and 1 with burkitt lymphoma (bl) with age at diagnosis ranging from 4 years to 66 years with median age of 31 years. pathology staining for ebv was available in four patients; all of whom were negative by eber. all 5 dlbcl patients received da-epoch-r for 3-6 cycles, and all achieved complete remission. five of 6 patients with lymphoma are alive and disease-free. one patient died of heart failure 16 years post chemotherapy without disease relapse. two patients were diagnosed with papillary thyroid carcinoma at ages 26 and 27, one of whom was subsequently diagnosed with nhl. two other patients were diagnosed with basal cell carcinoma of the skin at ages 42 and 48; both of whom had prior voriconazole exposure. conclusion: malignancy, most commonly nhl, occurs in patients with lof stat3 mutations. nhl should be considered in patients with progressive lymphadenopathy, and thyroid carcinoma should be considered in patients with thyroid nodules. patients treated with voriconazole are at an increased risk of skin cancer and require careful skin monitoring. as survival increases, it will be important to monitor the incidence of malignancies diagnosed, as it is possible that decreased stat3 signaling may prove to be protective of some cancers, such as colon and breast carcinoma, in which increased stat3 signaling is implicated in pathogenesis. abstract/case report text introduction: recurrent pneumonia is defined as 2 or more episodes of pneumonia in one year or more than 3 pneumonias throughout life (with radiological resolution between episodes). in retrospective studies, up to 30% of adult subjects with recurrent pneumonia coursed with primary immunodeficiencies (pid). prospective studies evaluating the etiology of recurrent pneumonia are scarce. diagnostic delay and inappropriate management of patients with pid (predominantly antibody deficiency for example) could lead to irreversible lung damage or even death from serious infections. here we present the frequency of primary immunodeficiencies in adults with recurrent pneumonia in cali, colombia. methods: we present preliminary data of a descriptive prospective study that will include 100 patients with recurrent pneumonia. women and men >14 and < 65 years old will be included. all volunteers will be evaluated by a clinical immunologist, complete blood count and serum igg, iga, igm and ige levels will be determined. according with clinical suspicious, igg subclasses, anti-pneumococcal igg response and b cell subpopulations will be performed. the project will be executed in 24 months. written informed consent has been obtained for all subjects included. this project count with irb approvals at universidad del valle and hospital universitario del valle. results: a total of 52 recurrent pneumonia cases have been included in the study. the mean age was 38.8 years (14-65 years) with a female:male ratio 28:24. moderate-severe dyspnea was observed in 4/52 cases (medical research council -mrcdyspnea scale 4 to 5). non cystic fibrosis bronchiectasis was found in 33/52 cases (63%). the main etiologies of recurrent pneumonia were: primary immunodeficiency 17/52 (32%); asthma 4/52 (7.6%); autoimmunity 3/52 (5.7%); primary ciliary diskinesia 3/52 (5.7%) and others. hypergammaglobulinemia represented 14/52 (27%) of cases. primary immunodeficiencies, 32% of recurrent pneumonia cases, were classified as: predominantly antibody deficiency (15cases) including cvid 7 cases, igm deficiency 3 cases, selective iga deficiency 2 cases, igg subclasses deficiency 1 case, hypogamaglobulinemia 1 case and agammaglobulinemia 1 case. combined immunodeficiency: dock8 deficiency 1 case and ataxia telangiectasia 1 case. conclusion: to the best of our knowledge this is the first prospective study evaluating the etiology of recurrent pneumonia in colombia. predominantly antibody deficiencies and igg hypergammaglobulinemia affect 60% of adults with recurrent pneumonia in colombia. this study allows us diagnosed more than 10 new cases of adult onset pid. immunological evaluation is critical in the assessment of patients with recurrent pneumonia. conflict of interest: authors disclosure any potential financial conflict of interest related to this abstract. acknowledgements: this investigator-initiated research was supported with a grant from baxalta us inc, a member of the takeda group of companies bt16-35583/iir-col-bxlt-001923. abstract/case report text introduction: lysinuric protein intolerance (lpi) is an autosomal recessive metabolic disorder due to pathogenic mutations in slc7a7. it is distinguished by decreased plasma concentrations and increased urinary excretion of lysine, arginine and ornithine and can present with multiorgan involvement and a spectrum of immune deficiency. we present a five-year-old female with lpi, early-onset juvenile systemic lupus erythematous (sle), hemophagocytic lymphohistiocytosis (hlh), and granulomatous skin lesions that were positive for vaccine-strain rubella. methods: retrospective chart review was conducted. laboratory investigations included lymphocyte immunophenotyping by flow cytometry, lymphocyte proliferation to mitogen, quantitative serum immunoglobulins, vaccine titers, autoantibodies, metabolic studies, and genetic evaluation by next generation and whole exome sequencing. results: a five-years-old female of mixed native american and african american race presented at 3 years of age with severe failure to thrive, history of recurrent fevers, joint swelling, recurrent skin lesions, severe anemia and neutropenia, and hypergammaglobulinemia. upon further evaluation, she demonstrated hyperferritinemia and ana, rnp, smith, and ss-a autoantibodies and was diagnosed with early-onset juvenile sle. laboratory immune evaluation revealed age-appropriate lymphocyte subpopulations and lymphocyte proliferative responses to mitogens and antigens, markedly elevated igg, iga, igm with no associated monoclonality, and protective tetanus and pneumococcal titers. given her severe clinical manifestations at an early age, concern for immunodeficiency prompted further genetic evaluation with next generation dclre1c sequencing, which was negative, and whole exome sequencing, which revealed two heterozygous mutations in slc7a7, consistent with lpi. laboratory metabolic evaluation was also consistent with a diagnosis of lpi. she continued to experience recurrent cutaneous lesions on her upper and lower extremities. biopsy findings were consistent with a granulomatous lesion and subsequently identified by the cdc to have vaccine-strain rubella infection. due to recurrent pneumonia and concern for pulmonary alveolar proteinosis (pap), pulmonology was consulted and eventually confirmed pap, and she has required home oxygen supplementation. given her history of recurrent infections and vaccine-strain rubella infection, supplemental ivig was initiated. her sle has been fairly refractory to medical management, including systemic corticosteroids, mycophenolate, rituximab, and cyclosporine. she recently developed hlh at 5 years of age and is currently maintained on canakinumab, mycophenolate, and systemic corticosteroids, yet continues to have sle and pap that has been difficult to control. conclusion: the range of clinical and immunologic findings in lysinuric protein intolerance has varied widely in the literature. our patient presented with early-onset juvenile sle and did not develop hlh and pap until 2 years after initial presentation. despite relatively normal cellular immunity by laboratory evaluation, our patient was identified to have chronic infection with vaccine-strain rubella virus, indicating severe t cell dysfunction, and poses challenges for future immunomodulatory treatment. introduction: x-linked immunodeficiency with magnesium defect, ebv infection, and neoplasia (xmen) disease is caused by lossof-function (lof) mutations in the magnesium transporter 1 (magt1) gene. it is a rare x-linked combined immunodeficiency and selective congenital disorder of glycosylation. clinical manifestations include chronic ebv viremia, recurrent bacterial and viral infections, lymphadenopathy, splenomegaly, autoimmunity, liver and central nervous system (cns) abnormalities. magt1 deficiency was first noted to result in chronic ebv infection and an increased susceptibility to ebv+ lymphomas. we recently recognized merkel cell carcinoma at a very young age in two xmen patients, leading to our review of the malignancies in this cohort. methods: we reviewed the records of 25 male patients (22 seen at the nih) with confirmed hemizygous lof mutations in magt1 for diagnosis of malignancy, therapy, and outcome. results: we identified malignancy in 11 patients of 25 with magt1 deficiency (44%). four patients had hodgkin's lymphoma (hl) (ages 15-29 years), three had non-hodgkins lymphoma (nhl) (ages 7-57 years), one had kaposi sarcoma (5years) , one patient developed eber-negative liposarcoma (age 27 years) after receiving chemo and radiotherapy for severe lymphoproliferative disease (lpd) at age 13 years and two had merkel cell carcinoma at exceedingly young ages (14 and 22 years). all patients had chronic ebv viremia. they all received treatment according to established protocols. currently, all except for three patients are alive and in remission, including one post-hsct. overall malignancy survival of 73%. it is important to note that three patients who did not have malignancy had ebv lpd so severe that it warranted treatment with a malignancy protocol, with one mistaken as having lymphoma. conclusion: xmen immune deficiency, an x-linked glycosylation disorder, is a multisystem disease associated with increased susceptibility to malignancies. initially, ebv driven lymphoproliferation and lymphoma was described with xmen; however, with increasing diagnoses, more malignancies are being recognized. all the recognized malignancies are associated, at least in part, with dnaviruses, including ebv, hhv-8, and merkel cell virus. understanding the clinical phenotype and pathogenesis of this disease will improve monitoring and early diagnosis of malignancies for patients with magt1 deficiency. abstract/case report text background: primary immune deficiencies (pid) constitute a heterogeneous group of over 400 individually rare congenital diseases that involve genes coding for proteins of the immune system, and which result in increased susceptibility to infection, inflammation, autoimmunity, allergy and cancer. the complexity of the diagnostic task, and the intrinsic biases and limitations of the human mind, can be aided by computational tools. among the available machine learning approaches, decision tree algorithms select the best node to split based on entropy and information gain; random forests build dozens or thousands of decision trees randomly to improve accuracy and reduce overfitting. aim: to implement a machine learning-assisted clinical decision support system for the diagnosis of pid. methods: with a local database of patients with suspected iei, we built a decision tree using c4.5 dtc, and a random forest on python 2.7 (jupyter notebook, scikit, mathplotlib, pandas, numpy). the database was obtained by conducting an electronic search on medsys of patients with the term "immunodeficiency" in their electronic medical records, and then hand-picking cases in which a pid had been confirmed or ruled out. it consisted of 234 patients, of which 185 had been diagnosed with iei. we first split the dataset randomly into training (60%) and testing (40%) sets. the decision tree was tasked with classifying correctly pid or not. after running the algorithm in the training set, we evaluated in the testing set through cross-validation. results: accuracy was greater than 80% for the dataset (pid/not). 0.819 for the dtc with 15 levels. the attribute with the lowest gini coefficient was low iga (0.044). accuracy for the random forest classifier was 0.808 with 25 trees. feature importance was highest for lung infection (0.054), high igg (0.043), low iga (0.041), skin infection (0.046), no isolate (0.057), and allergy (0.043); it was lowest for consanguinity, high igm, central nervous system infection, parasites and no infections. during the random generation of trees, accuracy reached up to 87%. discussion: we built two classification models. decision trees lend themselves more easily to learning and deriving rules of thumb from their sequences. random forests are more robust and better suited for categoric (as opposed to binary) classification. we next want to develop a chatbot, currently under construction, that will ask relevant questions in optimal sequence, and extract undiagnosed patients with suspected iei, based on statistical "red flags". we also have preliminary results of this process applied to a usidnet database with over 3,000 patients, and are also working on multinomial logistic regression and naïve bayesian classifiers for this and other databases. abstract/case report text objectives: acute viral respiratory infections (avri) are associated with significant healthcare resource use and cost. the use of intravenous immunoglobulin (ivig) may be an effective treatment for immunosuppressed patients and reduce overall healthcare resource utilization. the goal of this study was to assess hospital resource utilization associated with ivig use among patients hospitalized for avri. methods: using data from the 2011-17 premier hospital database, we identified patients hospitalized with a diagnosis of avri [respiratory syncytial virus (rsv), parainfluenza virus, rhinovirus, or metapneumovirus], and who had an immune deficiency (chemotherapy treatment, transplant, primary immunodeficiency disorder (pidd), specific antibody deficiency, other immunodeficiency, or disorders of the immune or lymphatic systems). patients receiving ivig within the first 48 hours were compared to patients who did not receive ivig at all. due to the nature of the need to better understand the treatment effect associated with ivig, we used an inverse probability weight-based regression model. since there were substantially more controls than cases, we randomly drew 5,000 controls. a logistic regression model was developed to adjust for factors associated with the probability of ivig use within 48 hours of admission. this propensity score was then used to weigh subsequent models to assess length of stay (total and icu) using negative binomial models and logistic regression for inpatient death. results: a sample of 1,927 immunocompromised inpatients were identified, 65 receiving ivig within the first 48 hours of admission and 1,862 who did not receive ivig. the ivig group was older (mean age 54 vs 35, p < 0.001), had more antiviral use (40% vs 22%, p < 0.001), and had less cancer (40% vs 75%, p < 0.001). after adjustment for immunity type (transplant, cancer), rsv, pidd, age, prednisone, antiviral use, ribavirin use, urban hospital setting, teaching status, intubation and lung disease, patients with ivig use had 3.24 less days of hospitalization (p=0.027) and 1.83 less days in the icu (p=0.003) than non ivig users. conclusions: this data analysis suggests that hospital length of stay and icu length of stay were significantly shorter for immunocompromised patients hospitalized for acute viral respiratory infections who were administered ivig within the first 48 hours of admission, as compared to patients who did not receive ivig. it is possible that ivig use may have an impact on hospital resource utilization and costs. future prospective studies would help further assess the role of ivig in patients hospitalized with acute viral respiratory infections. associate professor/yale university abstract/case report text background: cd40 ligand deficiency is an x-linked combined immunodeficiency associated with opportunistic infections and increased risk of malignancies. expansion of memory cd8+ t-cells with senescent features is known to be associated with chronic immune stimulation including aging, chronic infection and malignancy. cd8+ t-cell characteristics of cd40l deficient (cd40ld) patients in relation to their clinical history have not been described. objective: we studied correlation between cd8+ t-cell senescence with clinical histories of cd40ld patients. methods: we analyzed the frequency and phenotypic characteristics of peripheral cd8+ t-cell subsets in four cd40ld patients (5, 28, 33 and 34 years old (yo)) and healthy controls (hcs). t cell excision circle (trec) counts and telomere lengths of the patients and hcs were measured using quantitative pcr. in-depth analysis of cd8+ t-cells of the 5 yo patient and hcs was done using high-dimensional cytometer time of flight analysis (cytof). results: three patients (5, 28 and 34 yo) with histories of recurrent infections and poor compliance with immunoglobulin therapy (ivig) showed an increased frequency of effector memory cd8+ t-cells with the senescent phenotype compared to age matched hcs. whereas 33 yo patient with excellent ivig compliance starting at infancy did not show any senescence phenotypes of the cd8+ t-cells. the telomere length and trec count of each patient correlated with the degree of cd8+ t-cell senescence and their current ages, respectively. in-depth analysis showed similar expression patterns of molecules related to senescence and cytotoxicity in cd8+ t-cells including cd57, t-bet, eomes, granzyme b and perforin in the 5 yo patient and mid-elderly hcs. conclusion: our findings suggest that prompt diagnosis and compliance with ivig starting at the infancy may prevent early onset cd8+ t-cell senescence in cd40l deficiency. abstract/case report text introduction: immunoglobulin g4-related disease (igg4-rd) is an immune-mediated fibroinflammatory condition that affects multiple organs. when igg4-rd is found in the ocular adnexa, the term "igg4related ophthalmic disease (igg4-rod)" is used. objective: our case describes a patient with igg4-rod without systemic involvement. case: mr. x is a 46-year-old male with a pmh of cml (on imatinib) and allergic rhinitis who presented to clinic with orbital swelling for twenty years. his swelling had always been responsive to steroids, but would return once steroids were tapered. patient was diagnosed with biopsy proven cml in 2011 and is currently taking imatinib. because his peri-orbital edema persisted, a right lacrimal gland biopsy was done which showed "marked lymphocytic infiltrate of soft tissue with lymphoid follicles, many plasma cells, and eosinophils. no atypical histiocytes." flow cytometry was negative for malignancy. results: crp 4.12 mg/l. esr 14 mm/hr. igg4 elevated at 655 mg/dl. ct chest from 2013 and ct chest, abdomen, pelvis from 2019 were without fibrotic changes. assessment: when diagnosing igg4-rd, we categorize diagnosis into three levels (possible, probable, or definite) by three criteria (clinical manifestation, elevated serum igg4, and histopathology). this is detailed as follows: clinical exam showing organ specific swelling or masses, elevated serum igg4 (>135 mg/dl), and histopathology with either lymphocyte and plasmacyte infiltration and fibrosis or infiltration of igg4+ plasma cells (ratio of igg4+/igg+ cells ≧40 % and ≧10 igg4+ plasma cells per high power field). not all these components are required for diagnosis, but meeting histopathologic criteria makes diagnosis more probable. our patient's disease was localized to his eye, and patients with igg4-rod have unique diagnostic criteria. these criteria are similar to the criteria for igg4-rd, but emphasize enlargement of the ocular adnexa, less frequent fibrosis, and ≧50 igg4+ plasma cells per high power field. our patient's histopathology revealed a lymphoplasmacytic infiltrate, but lacked storiform fibrosis or obliterative phlebitis. his serum igg4 level was 655 mg/dl, and his biopsy was positive for an igg4+/igg+ ratio of 50% and more than 100 igg4+ plasma cells per high power field. based on this, he meets criteria for igg4-rod. conclusion: igg4-rod is a rare condition that is usually associated with systemic organ involvement. our case is unique, as no systemic disease has been detected. we also suspect our patient has been living with igg4-rod for several years, as his orbital swelling began in high school. it is important to note that he has been on imatinib, a tyrosine kinase inhibitor, for treatment of his cml. imatinib inhibits c-abl and platelet-derived growth factor receptor, tyrosine kinases involved in profibrotic pathways. patient's lack of fibrosis could also be due to his longstanding use of this drug. it is also possible that he has a rare form of igg4-rod without systemic involvement. a limited number of such cases have been reported, but no consensus has been made on why disease course was localized. our patient was started on rituximab, and his serum igg4 decreased to 295 mg/dl after the first cycle. we hope his disease achieves remission. informed consent: informed consent was obtained from all individual participants included in the study. abstract/case report text platelet abnormalities with eosinophilia and immune-mediated inflammatory disease (plteid) is a recently discovered combined immunodeficiency with inflammatory and allergic manifestations with few cases reported. we describe a female patient with compound heterozygous mutation in arpc1b gene with suggestive clinical findings of plteid. a 2-year-old girl presented with chronic diarrhea since neonatal period, with bloody stools and failure to thrive. she also presented atopic dermatitis, recurrent cutaneous and mucosal ulcers, recurrent respiratory infections (4 episodes of otitis media, 2 pneumonias) and many episodes of mucocutaneous candidiasis. family history revealed a sibling deceased in the second month of life, who presented a similar clinical picture and a paternal uncle and second degree cousin that died in the first year of life. there is no history of consanguinity. laboratory evaluation revealed peripheral eosinophilia (1000/mm3), normal platelet numbers with low platelet volume (8,3 fl -reference value 9,4-12,4 fl), normal igm levels with elevated igg (1114 mg/dl-rv 453-916 mg/dl), iga (517 mg/dl -rv 20-100 mg/dl) and ige (1141 iu/ml -rv a) associated with plteid. t h e i n f a n t r e c e i v e s a n t i m i c r o b i a l p r o p h y l a x i s w i t h sulfamethoxazole-trimethoprim and fluconazole, intravenous immunoglobulin replacement and was referred to hematopoietic stem cell transplantation (hsct). this case was the first one described in brazil and highlights the importance of seeking for a genetic diagnosis in patients with complex clinical phenotypes. precise diagnosis can impact on treatment approach. live vaccines are generally contraindicated in patients with combined immunodeficiency (cid). however, in less severe cid, such as partial dgs, those vaccines can be considered depending on the immunologic status of the patient. there are recommendations regarding to measles, mumps, rubella (mmr) and varicella vaccines, but yellow fever vaccine (yfv) is generally contraindicated in this population. considering the severity of the yellow fever disease and the absence of specific treatment, the use of this vaccine is an important topic for debate in cases of patients from endemic areas. objective: this study aimed to describe the use of yfv and other live attenuated vaccines in patients with dgs, associating it with their immunological profiles and the presence of adverse effects. methods: retrospective study of medical records of patients with dgs confirmed by mlpa or fish, followed in a pediatric reference center for primary immunodeficiencies between 2009 and 2019. collected data included: demographic characteristics, medical history, history of immunization with live vaccines, postvaccination adverse reactions and immunological profile, including immunoglobulins levels, serologic vaccination responses, lymphocyte immunophenotyping, lymphocyte proliferation responses to mitogens and prophylactic treatments (antibiotic or immunoglobulins). results: thirty-five patients with confirmed dgs and median age of 12 years (2-21y) were included (22m:13f). thirty-three children (94%) received mmr vaccine: nine presented t lymphopenia. two of the 9 patients had cd4 < 300, one of them with normal mitogenic proliferation response and the other was not tested. three of the 33 patients had low immunoglobulins levels (2/33 low igg, 2/33 low igm and 1/33 low iga), and one of them received intravenous immunoglobulin (ivig). twentynine of 33 had normal serologic vaccination responses. adverse effect was only reported by one patient, who had one episode of fever after the administration of all vaccines. yellow fever vaccine was administrated to 14 children (40%): 2 had t cell l y m p h o p e n i a ( b u t c d 4 > 5 0 0 ) , a n d a n o t h e r p a t i e n t h a d hypogammaglobulinemia and received ivig and prophylactic antibiotics. twelve of 14 showed adequate serologic responses to mmr and hepatitis b. only 1 patient reported mild reaction (tremors) two days after the yfv administration. the same patient had normal t cells, immunoglobulins and vaccine responses. twenty patients (57%) received bacillus calmette-guerin vaccine (bcg), 15 (42%) received oral polio, 9 (25%) rotavirus and 8 (22%) received varicella vaccine. no severe adverse events were documented in any patient that received live vaccines, and no patient developed measles, mumps, rubella or yellow fever diseases as a consequence of administration of the vaccine. conclusions: in this cohort of pediatric patients with dgs, yfvand other live vaccines were well tolerated, and no severe adverse events were reported, suggesting that widespread contraindication of yfv may endanger unvaccinated patients with less severe phenotype living in endemic areas. immunological evaluation and individualized decisions are always recommended, and further studies are needed to assess the safety of the yfv in this pediatric population. abstract/case report text introduction: lad-i is a rare inherited disorder of leukocyte (primarily neutrophil) adhesion to endothelial cell surfaces, migration, and chemotaxis resulting from itgb2 gene mutations encoding for the β2-integrin component, cd18. severe lad-i (i.e., cd18 expression on < 2% of neutrophils) is characterized by recurrent serious infections, impaired wound healing, and childhood mortality. although allogeneic hematopoietic stem cell transplant (allohsct) is potentially curative, its utilization and efficacy are limited by hla-matched donor availability and risk of graftversus-host disease (gvhd). rp-l201-0318 (clinical trials.gov # nct03812263) is a phase 1/2 open-label clinical trial evaluating the safety and efficacy of autologous cd34+ cells transduced with a lentiviral vector (lv) carrying the itgb2 gene encoding for cd18 (chim-cd18-wpre) in severe lad-i. methods: pediatric patients ≥ 3 months old with severe lad-i (demonstrated by cd18 expression on < 2% neutrophils and at least one prior significant bacterial or fungal infection) are eligible. peripheral blood (pb) hematopoietic stem cells are collected via apheresis after mobilization with granulocyte-colony stimulating factor (g-csf) and plerixafor. cd34+ hspcs are selected, transduced with chim-cd18-wpre lv, and cryopreserved. myeloablative conditioning with busulfan (therapeutic drug monitoring (tdm) dosing with adjustments to enable target area under the curve (auc)) is administered over 4 days, followed by infusion of the thawed investigational drug product (rp-l201). patients are followed for safety assessments including replication competent lentivirus (rcl) and insertion site analysis (isa), and for efficacysurvival to age 2 (24 months) and at least 1-year post-infusion without allohsct, increase in neutrophil cd18 expression, pb vector copy number (vcn), decrease in infections and/or hospitalizations, and resolution of skin or periodontal abnormalities. results: an initial lad-i patient (age 9 years) with recurrent severe infections and documented itgb2 mutations has been treated as of november 2019. baseline cd18, cd11a, and cd11b expression were < 1%. mobilization and apheresis procedures were performed successfully and busulfan conditioning was administered at the target auc. investigational product was comprised of 4.2x10e6 cd34+ cells/kg with vcn of 3.8 copies/cell (liquid culture), and was infused without complications. no serious treatment-emergent adverse events were reported. neutrophil engraftment (3 consecutive days of anc ≥ 500) was observed 18 days post-infusion. pb pmn cd18 expression 3 months posttreatment was 44.9% with comparable cd11a and cd11b expression levels; pb cd15 (myeloid) vcn at 2.5 months was 1.5. safety and efficacy data 6 months post-treatment will be available at the time of presentation, in addition to preliminary data regarding a potential additional patient. conclusion: preliminary evidence demonstrates that rp-l201 enables itgb2 genetic correction with robust cd18/cd11 neutrophil expression in this frequently fatal primary immunodeficiency. abstract/case report text introduction: the complement system plays an integral role in the innate immune system and links innate and adaptive immunity. complement deficiencies, hereditary or acquired, are rare. acquired deficiencies are more prevalent, occurring in nephrotic syndrome, reduced hepatic synthesis or transiently in sepsis/viremia. they are also seen in the presence of autoantibodies known to cause depletion of complement factors, such as c3 nephritic factor (c3nef). c3 deficiency is associated with infection susceptibility, particularly to encapsulated bacteria, and immune complex disease. case description: a 47 year old male was evaluated for recurrent infection. in childhood, he had recurrent sinusitis, otitis media requiring tympanostomy tube placement and persistent pharyngitis despite tonsillectomy. as a teenager, he developed glomerulonephritis, progressing to end stage renal disease and requiring transplant at age 22. the kidney allograft failed 4 years later, with biopsy demonstrating recurrent glomerulonephritis. the patient was transitioned to peritoneal dialysis and later hemodialysis, due to recurrent pd-related infections. his adult course was complicated by recurrent methicillin sensitive staphylococcal aureus (mssa) catheter and soft tissue infections (cellulitis and abscess), sinusitis, sepsis (streptococcal, mssa and tularemia), multifocal pneumonia and a left below knee amputation for osteomyelitis that required revision surgery. patient reported other autoimmune phenomena including a presumptive diagnosis of vasculitis and possible lupus-like syndrome. the constellation of recurrent infections and autoimmune features was most concerning for an early complement deficiency. prior work up was notable for low c3, ch50 and ah50 with normal c4, factor h and factor i. extensive laboratory work up revealed normal c1q, c4 level and function, serum immunoglobulins, vaccine titers, factor b and factor d levels. atypical hus (ahus) panel revealed a heterozygous silent variant in exon 17 of cfh and a heterozygous polymorphism within an intron in mcp/cd46, seen with increased prevalence in the patient population with ahus. wes was notable for a variant of uncertain significance in the vcl gene only. c3 level and function were markedly decreased, alongside low ch50 and ah50. both sc5b-9 level and c3 nephritic factor were elevated. a diagnosis of acquired c3 deficiency due to c3nef was made and patient was started on bactrim prophylaxis. he has remained free of serious infection since starting antibiotic prophylaxis. discussion: c3nef stabilizes the alternative pathway c3 convertase, c3bbb, increasing its half-life and blocking dissociation. this leads to unregulated consumption of c3 with subsequent deficiency. c3nef has been associated with c3 glomerulopathy, infection and partial lipodystrophy. however, there is marked heterogeneity in clinical phenotypes with reported asymptomatic individuals. our patient's glomerulonephritis likely represents c3 glomerulopathy. case reports and series of successful treatment of c3 glomerulopathy with rituximab and eculizumab have not commented on immune outcomes beyond the kidney. other potential therapeutic strategies include plasma cell depletion with either bortezomib or daratumumab. further study is needed to evaluate these therapies influence on both reversal of c3 depletion and overall impact on immune function in the setting of c3nef. abstract/case report text introduction: patients with heterozygous signal transducer and activator of transcription 1 (stat1) gain of function (gof) pathogenic variants exhibit an array of phenotypes including susceptibility to viral, bacterial, fungal and mycobacterial infections, autoimmunity, and cancer predisposition. progressive disseminated histoplasmosis (pdh) is well-described to affect infants. however, no reports have evaluated underlying monogenic immune dysregulation in previously healthy infants presenting with pdh. we report an infant who presented with pdh and associated hemophagocytic lymphohistiocytosis (hlh) leading to the diagnosis of a heterozygous stat1 gof mutation. case report: a previously healthy 9-month old male presented with persistent fever, pancytopenia, transaminitis, elevated ferritin, hepatosplenomegaly and coagulopathy. his clinical and laboratory evaluations were concerning for hlh syndrome. he had no prior history of immune hyperactivation or atypical infections. secondary causes of hlh were investigated, and patient was diagnosed with pdh based on marked histoplasma antigenemia. targeted genetic testing did not reveal a genetic etiology of familial hlh. he was successfully treated with a pulse and taper of dexamethasone as well as liposomal amphotericin b with transition to itraconazole. immunologic evaluation at the time of initial presentation demonstrated increased mean channel fluorescence for both perforin and granzyme noted in his nk cells. his nk function was decreased; however, he had a normal cd107a degranulation assay. his b-cell panel demonstrated low non-switched memory b-cells, low switched memory b-cells and low total memory b-cells. given his extreme immune activation with histoplasmosis, abnormal immunologic testing, and persistent lymphopenia despite resolution of his infection, a primary immunodeficiency next generation sequencing panel was sent. the results demonstrated a pathogenic variant in stat1 (c.800c>t; p.ala267val). this single nucleotide variant has been previously shown to be pathogenic (clinvar). abstract/case report text introduction: cytotoxic t lymphocyte antigen-4 (ctla-4) is known to have an important role as a negative regulator of immune responses, participating in the control of regulatory t cells and effector t cells. in mice its absence is associated with fatal autoimmunity and several ctla-4 mutations, leading to low or absent ctla-4 expression, have been shown in humans to be associated with a phenotype that includes hypogammaglobulinemia (with recurrent respiratory infections) and several manifestations of autoimmunity (enteropathy, granulomatous lymphocytic interstitial lung disease, organ infiltration, splenomegaly, autoimmune cytopenias, lymphadenopathy, amongst others), in an autosomal dominant mode of transmission. one of the published mutations, c.c257t, that results in an alanine to valine substitution (p.a86v), with a highly conserved alanine at that position, had a cadd score of 24 and was associated with the phenotype above, and was shown to be associated with a low expression of ctla-4 on regulatory t cells and with low ctla-4 function (reduction of ctla-4-mediated transendocytosis). methods: after irb approval, we searched for ctla-4 mutations present in the biome biobank· biorepository, containing whole exome sequencing data on 30845 patients, with data obtained using illumina· v4 hiseq 2500 sequencing platform. sifting through all the ctla4 mutations in the data, we identified four patients with the c.c257t mutation described above. extensive chart review of the four patients was performed. results: four patients were found with the ctla-4 c.c257t mutation. none of them had any of the described phenotypical characteristics of ctla-4 deficiency. patient 1 is a 68-year-old male with history of coronary artery disease, atrial fibrillation, stroke, hypertension, brain aneurysm, chronic kidney disease, gout and depression. patient 2 is a 29-year-old female with history of morbid obesity. patient 3 is a 51-year-old female with history of hypertension, obesity, pre-diabetes, dyslipidemia and iron deficiency anemia. patient 4 is a 70-year-old female with history of peripheral artery disease, hypertension, dyslipidemia, chronic kidney disease and lung cancer. conclusion: prior literature has attempted to characterize the clinical penetrance of ctla-4 mutations, suggesting it to be around 67%, with that number applying to 45 different mutations in 133 ctla-4 mutation carriers. we screened a large biorepository of more than 30 thousand patients for ctla-4 patients and identified four patients that carry one of the best described ctla-4 mutations, previously validated from a functional standpoint and associated with a severe phenotype. none of the four patients demonstrated any of the previously described phenotypical characteristics, and all four have ages above the median age of onset of 11 years. with the increasing use and broad population application of genetic studies, it is crucial to define the value of identifying presumed pathogenic variants in the absence of the adequate phenotype, with all the prognostic, therapeutic and ethical considerations it may imply. prior case reports of pil patients with b-cell malignancies have discussed treatment regimens with chemotherapy, radiation, and/or surgery, but neither the use nor the outcomes of allogeneic hematopoietic stem cell transplantation (hsct) in the management of recurring b-cell malignancies have been readily reported. case description: a 21-year-old man with pil and an accompanying history of lymphopenia, hypoproteinemia, hypoalbuminemia, and hypogammaglobulinemia was diagnosed with diffuse large b-cell lymphoma (dlbcl) of the liver following a preceding history of burkitt lymphoma of the ileum at 6 years of age and dlbcl of the liver at 16 years of age, in which each malignancy was genetically distinct. in addition, the patient had a history of benign nodular adenomatoid hyperplasia of the thyroid at 17 years of age that required a hemi-thyroidectomy. treatment considerations for the patient included chimeric antigen receptor t-cell therapy, autologous hsct, and allogeneic hsct, in which allogeneic hsct was ultimately pursued. prior to hsct, the patient was lymphopenic (670 cells/microliter) with significant t-cell lymphopenia (290 cells/microliter) and an increased proportion of memory t-cells (67% of his cd4+ t cells were cd45ro+), as well as hypogammaglobulinemic (igg 296 mg/dl; iga 47 mg/dl; igm 62 mg/dl). immediately following treatment of his dlbcl with rituximab, ifosfamide, carboplatin, and etoposide, the patient underwent a matched-related sibling donor hsct with a preparative regimen of busulfan, thiotepa, and fludarabine. now 18 months status-post hsct, the patient has maintained full-donor chimerism and has no evidence of graft-versus-host disease or malignancy. as expected, hsct has not corrected abnormalities in certain parameters associated with his pil, as he continues to display significant hypoproteinemia, hypoalbuminemia, and hypogammaglobulinemia, but he has an improved lymphocyte count (1,530 cells/microliter). discussion: there is no definitive or curative treatment for pil; furthermore, the genetic etiology of pil remains unknown. supportive regimens to help mitigate or offset manifestations of pil exist, such as adherence to a low-fat diet with medium-chain triglyceride supplementation, but there are no therapies available to prevent or reduce the risk of developing bcell malignancies in this patient population. although previous case reports have detailed successful treatment of b-cell malignancies in pil patients with chemotherapy, radiation, and/or surgery, there are no published consensus guidelines regarding management of b-cell malignancies in the setting of pil, especially if recurrent in nature. for non-pil patients with chemotherapy-refractory disease, or recurrent disease following autologous hsct, allogeneic hsct is a potentially curative option. herein, we describe a pil patient with a history of multiple b-cell malignancies who underwent a successful allogeneic hsct, indicating that allogeneic hsct may be an effective treatment option for similarly affected patients. abstract/case report text introduction diarrhea in young infants is common and generally self-limited. in persistent cases, the differential diagnosis is broad and includes infections, food protein-induced allergic proctocolitis, congenital diarrheas and enteropathies. in addition to monogenic inflammatory bowel diseases, many cellular, humoral, and combined immunodeficiencies should be considered, including but not limited to cvid, ipex and ipex-like phenotypes, lad, dyskeratosis congenita, intestinal lymphangiectasia, omenn syndrome, cartilage hair hypoplasia, cgd, il-10 axis defects, aid deficiency and wiskott-aldrich syndrome. case presentation a full term infant born after an uncomplicated pregnancy to nonconsanguineous honduran parents presented with non-bloody, non-bilious vomiting and dehydration at 21 days of life. the infant later developed frequent loose stools, some of which were bloody, and failure to thrive. his family and prior medical history, including newborn screen, were normal. an extensive workup was initiated which showed: -persistent and severe anemia with a hemoglobin nadir of 3.5 mg/dl -hypoalbuminemia requiring multiple infusions -elevated alpha-1-antitrypsin and calprotectin level in stool -profound hypogammaglobulinemia with normal iga, igm and ige for age -normal gross and histologic findings on esophagogastroduodenoscopies and colonoscopies besides a gastric ulcer thought not be the cause of his anemia -normal abdominal imaging including ultrasound, ct angiography and mri -no source of bleeding on meckel scan or exploratory laparotomy -normal dhr assay, g6pd level and positive myeloperoxidase stain -immunophenotyping showing t cell lymphocytosis affecting cd4+ more than cd8+ compartment, with normal lymphocyte proliferation to mitogens -normal sweat chloride level genetic testing was initiated with a targeted immunodeficiency panel which showed variants of unknown significance in adar, dock8, lyst, ptprc and tbx1 genes, none of which adequately explained his presentation. whole exome sequencing showed that he was a compound heterozygote in the dgat1 gene. a pathogenic variant c.751+2t>c (ivs8 + 2t>c) was inherited from the father and a likely pathogenic variant c.1073g>c (p.r358p) was inherited from the mother. patient was diagnosed with dgat1 deficiency, an inborn error of lipid metabolism resulting in protein-losing enteropathy (ple). under gastroenterology's guidance, a low fat diet was initiated, resulting in rapid improvement in stool consistency, weight gain, albumin level and stool alpha-1-antitrypsin level. he remains on subcutaneous immunoglobulin replacement therapy for ongoing hypogammaglobulinemia. conclusion: protein-losing enteropathies commonly present with intractable diarrhea and significant laboratory derangements due to malabsorption including hypogammaglobulinemia. as a result of these findings and since many of the etiologies are immunologic in origin, immunologists are an integral part of the evaluation of such cases. in cases where immune system interrogation reveal normal results, genetic testing is crucial in guiding the diagnosis. in our case, whole exome sequencing not only provided the diagnosis but also characterized a variant that was previously of unknown significance as likely pathogenic. dietary management provided rapid improvement in growth and nutritional status. ongoing monitoring will reveal if this management also assists in igg level maintenance and hematologic abnormalities or if even more stringent control of dietary fat will be required abstract/case report text background: foxp3 gene mutations are associated with immune dysregulation polyendocrinopathy x-linked (ipex) syndrome, a rare xlinked monogenic disease of immune dysregulation and autoimmunity. the classic presentation consists of severe enteropathy, dermatitis, and endocrinopathies (commonly early onset insulin dependent diabetes mellitus). clinical presentation and severity can be variable even in family members with the identical variant. we present a patient with ipex symptomatology and a hemizygous variant in the polyadenylation (polya) signal of foxp3 that is classified as a variant of uncertain significance (vus). this specific variant was reported in a single case study in which the patient improved after hematopoietic stem cell transplantation (hsct). case presentation: a 2 month-old ex 34-week gestation boy was admitted with lethargy, hypovolemia, electrolyte disturbances, and acute kidney injury. he developed persistent diarrhea and vomiting after receiving rotavirus vaccine. his family history is significant for early deaths of three maternal unclesone stillborn, one death at 6 months and another at 2 years from unknown gastrointestinal problems. he demonstrated peripheral eosinophilia (to 4.26 k/ul), elevated ige, and anemia requiring multiple transfusions. he developed severe enteropathy with hypoproteinemia requiring total parenteral nutrition, continual albumin infusions and maintenance of npo. he had generalized edema, respiratory distress requiring high flow nasal cannula, and repeatedly spiked fevers with negative infectious evaluation. acute kidney injury improved but renal ultrasound showed persistent nephrocalcinosis. endoscopy yielded biopsies demonstrating duodenitis with severe villous atrophy, scanty isolated intraepithelial eosinophils and neutrophils, a few crypts with mucin, reactive epithelial changes and increased lamina propria eosinophils. colon biopsies showed mucosa with focally increased lamina propria eosinophils with scanty neutrophils and surface epithelium without cryptitis. esophagitis with reactive epithelial changes, spongiosis, and many intraepithelial eosinophils was also present. the patient's lymphocytes showed unremarkable proliferation to pha and pwm. cd4+ cd25+ t cells demonstrated intracellular foxp3 expression by flow cytometry. a commercially-available immunodeficiency targeted panel revealed that he was hemizygous for a vus in foxp3 (exon 12, c.*878a>g non coding). this variant is also referred to as an aauaaa>aauaag or aataaa>aataag change in the polya site. he was also heterozygous for vus at these additional loci: cd79a c.341c>t abstract/case report text introduction: implantation of allogeneic cultured thymus, partially depleted ex vivo of t cells, can result in naïve t cell development in patients with complete digeorge syndrome (dgs). in a few patients, early and transient skin rash, often characterized as "atypical dgs" or late autoimmune manifestations have been reported following implantation. here we describe a patient with complete dgs who developed immune reconstitution inflammatory syndrome (iris) or atypical dgs following thymus implantation. case description: a female patient was diagnosed at birth with complete dgs due to absent t cell receptor excision circles (trec), hypoplastic thymus, profound hypocalcemia with hypoparathyroidism and cardiac defects. the patient also had microretrognathia, oral motor dysfunction, sialorrhea, recurrent aspirations and reflux requiring a gastro-jejunum feeding tube, low-set ears with right ear microotia, semicircular canals atresia, alopecia and mal-rotated kidneys. prior to thymus implantation, the patient was thriving, had no skin rash, no eosinophilia and no t cells. detailed genetic analyses, did not reveal a cause for her syndrome. at 9 months of age pulmonary aspergillosis was diagnosed presumptively. at 10 months of age the patient received an allogeneic t-cell depleted thymus implant from a male donor, without prior conditioning or post-implantation immune suppressive medications. the procedure was uneventful and the patient returned home after 7 days. results: four months after implantation, a pruritic maculopapular rash appeared on the head and trunk that spread to the extremities including the palms and soles. there was no lymphadenopathy or splenomegaly. an infectious etiology could not be found. eosinophilia and an increase in liver enzymes were noted. there was an increase of cd4+ and cd8+ t cells with predominantly memory phenotype, which had been undetectable 1 month earlier. analysis of t cell diversity showed a restricted repertoire with expansion of two v-beta families. there was no evidence of donor cells to suggest graft versus host disease. skin biopsy showed minimal superficial perivascular inflammatory infiltrate composed mainly of cd163+ histiocytes and rare cd3+ t cells. the patient was treated with prednisone and cyclosporine. a liver biopsy was performed 3 weeks after initiation of treatment that showed moderate and diffuse peri-portal ductular reaction but no duct associated lymphocytic infiltrate or significant duct epithelial injury or ductopenia. the skin rash rapidly resolved with desquamation, while the liver enzyme abnormalities persisted for two more months. cyclosporine and prednisone were weaned over 2 months. t cell numbers, their response to stimulation and diversity have since normalized, as well as trec and naïve t cell production. the patient is producing appropriate antibodies to protein and polysaccharide vaccines. sixteen months after implantation the patient developed grave's disease with markedly elevated free-t4, undetectable tsh and elevated antibodies to the thyroid receptor, which rapidly normalized with ongoing methimazole treatment. the patient is currently 30 months after the implantation and is free of infections, thriving and developing appropriately. conclusions: this patient developed atypical dgs or iris, often associated with autologous and allogeneic hematopoietic stem cell transplants, organ transplants or effective treatment of hiv, after successful thymus implantation for complete dgs. abstract/case report text congenital disorders of glycosylation are a rare group of genetic disorders due to defects in protein glycosylation. phosphoglucomutase 3 (pgm3) is an enzyme necessary for the synthesis of uridine diphosphate n-acetylglucosamine, an important precursor for protein glycosylation. patients with autosomal recessive pgm3 deficiency have a multisystemic disorder characterized by a neurologic impairment and clinical features classically observed in autosomal dominant hyper-ige syndrome due to stat3 mutations; including recurrent pneumonias, skin abscesses, elevated levels of ige, and abnormalities in connective tissues and bones. we hypothesized that gp130, a highly glycosylated protein and coreceptor of the cytokine il-6, would be weakly expressed on pgm3deficient cells, due to impaired glycosylation. we studied 6 pgm3-deficient patients from 3 kindreds and showed that il-6-driven stat3 phosphorylation was impaired in their pbmcs and ebv-transformed b cells. accordingly, the induction of socs3 target gene was significantly decreased. in contrast, the patients had normal stat3 phosphorylation and socs3 induction downstream of il-10, a cytokine whose signaling is independent of gp130. flow cytometry and immunoblotting showed significantly lower gp130 expression in peripheral t-cells and ebv-transformed b cells from pgm3-deficient patients compared to healthy donors. we did also show that in vitro inhibition of n-glycosylation, using tunicamycin in ebv-transformed b cell line from healthy donor, alters gp130-mediated signaling. collectively, our findings demonstrate that defective glycosylation in pgm3-deficient patients results in reduced expression of gp130 and consequently, impaired gp130 dependent stat3 phosphorylation and defective il-6 signaling. this may account for the overlapping clinical features shared by pgm3 and stat3 deficient patients. abstract/case report text introduction: there are no known effective therapeutic modalities for patients hospitalized with moderate to severe acute viral respiratory infections, and treatment is primarily supportive. intravenous immunoglobulin (ivig) has been reported in limited cases to be used in this setting, especially in immunocompromised patients. the primary objective of this retrospective study is to compare clinical and economic outcomes among immunocompromised patients hospitalized with viral respiratory infections who received ivig to those who did not receive ivig at a large academic center hospital. methods: we performed a double-center, retrospective cohort study of all immunocompromised patients who were hospitalized for acute documented respiratory viral infections between 2011 and 2016. we divided patients into two groups: those who received ivig therapy for respiratory infections, and those who did not receive ivig therapy. data on age, gender, immune status, viral type, immunosuppression type, respiratory support, microbiological data, length of hospital stay (los), icu los, as well as death and readmission rates were extracted from medical records. in order to adjust for severity bias typically present in observational data such as these, we employed inverse probability weighting (ipw) using all collected baseline covariates. outcomes (death, length of stay in hospital and icu, readmission) were examined using a series of logistic and poisson regression models adjusting for baseline covariates and employing ipw. results: a total of 282 individual hospital admissions were analyzed; 99 patients received ivig and 183 did not receive ivig. there were no significant differences between the two groups in terms of mean age, gender . average age was 40.3, 50% were female, 74.5% were transplant patients of which 26.6% had lung transplant, 26.6% had liver transplant, 23.1% had bone marrow transplants (bmt), 8.5% had kidney transplant, 7.8% had heart transplant and 4.3% had both solid organ and bmt. 32.3% of patients had a hematologic malignancy, and 2.5% had a primary immunodeficiency. the most common isolated respiratory virus w a s r h i n o v i r u s ( 5 1 . 4 % ) , f o l l o w e d b y r s v ( 2 5 . 9 % ) , parainfluenza (11.4%) and metapneumovirus (10.6%). overall, the use of ivig as associated with a significantly shorter icu length-of-stay, with an (or=-2.46, p=0.001), and a higher hospital readmission rate. in the sub-analysis of patients who received ivig within the first 48 hours of hospitalization (n=39), ivig use was associated with a significantly shorter icu los (or=-6.01, p=0.0), significantly shorter overall hospital los (or=-4.341, p=0.008), and no significant change in readmission rate. conclusions: to our knowledge, this is the first retrospective cohort analysis evaluating the effect of ivig in immunocompromised patients hospitalized with respiratory viral infections. the results suggest that immunocompromised patients receiving ivig may have a shorter hospital and icu los, especially if ivig is provided within the first 48 hours of admission. this may result in reduced healthcare costs. this study is limited by its retrospective nature, and the potential bias that patients treated with ivig are sicker to start with. future prospective studies are suggested to further evaluate these findings. (1, 2) . the most common precipitant in children is medication, followed by infection (2, 3) . although a clear association between mycoplasma pneumoniae and sjs has been established, there is a scarcity of literature exploring the role of this infection in recurrent sjs in children (2) (3) (4) . case presentation: a 15-year-old female with prior history of sjs was admitted for mucosal and skin lesions in the setting of community acquired pneumonia. her past medical history included sjs with eye involvement, secondary to mycoplasma pneumoniae (ig m positive), occurring five years prior to this admission. she also had frequent episodes of acute otitis media and sinusitis in early childhood. family history was negative for immunodeficiency. her clinical presentation included respiratory symptoms and fever for 8 days treated with ceftriaxone, followed by cefdinir and levofloxacin. her fever improved the day prior to admission, but she developed conjunctival injection, ocular pain, and ulcerative lesions in her mouth and nares. on physical examination, she had low grade fever with mucosal lesions including conjunctival erythema with serous discharge, painful blisters and denudated skin in lips, perioral area, nares, tongue and oropharynx. initial testing included negative blood hsv pcr, blood culture, rapid antigen testing for group a streptococcus and influenza a/b, and elevated crp in 4.4mg/dl and esr 55mm/hr. right lower lobe p n e u m o n i a w a s c o n f i r m e d w i t h a c h e s t r a d i o g r a p h . nasopharyngeal pcr and serum igm were positive for mycoplasma pneumoniae. she had a mildly elevated anticardiolipin igm (17 mpl), a mildly decreased c3 (75 mg/dl) and a negative ana. she was diagnosed with recurrent sjs secondary to mycoplasma pneumonia infection. she completed treatment with levofloxacin for mycoplasma pneumonia, and received cyclosporine and high-dose methylprednisolone. she had bilateral amniotic membrane transplantation to prevent corneal ulceration . she was discharged after clinical improvement, and recurrent oral lesions were noted at followup. immunological work up as an outpatient revealed normal serum immunoglobulins, normal lymphocyte subsets and low pneumococcal titers with adequate response post-vaccination. sjs secondary to mycoplasma pneumonia infection has predominance of mucosal involvement over rash, which was observed in our patient (1, 2) . some case series reported a recurrence of sjs up to 18% within a 7-year follow up. almost half of patients with recurrent sjs developed multiple sequelae (2, 3) . early diagnosis of sjs, especially in those with prior history of sjs, helps to provide appropriate supportive care, monitoring of complications and treatment of possible superinfections (5, 6) . conclusions: there is limited information in the literature regarding the role of mycoplasma pneumoniae associated recurrent sjs in children. it is possible that these episodes are triggered by and/or immune predisposition. there is ongoing discussion regarding whether these clinical presentation should be labeled sjs secondary to mycoplasma pneumonia infection or, depending of the skin involvement, m. pneumonia-associated mucositis (mpam) and m. pneumonia-induced rash and mucositis (mirm) (5,6,7). mycoplasma should be treated appropriately in patients with recurrent sjs. abstract/case report text background: growing access to genetic testing has facilitated the genetic evaluation of primary immunodeficiencies but has also greatly increased the number of variants of uncertain significance (vus) encountered in clinical practice. interpreting the significance of vus requires multiple lines of evidence. w e d e s c r i b e a n e u t r o p e n i c i n d e x p a t i e n t w i t h hypogammaglobulinemia, unusual hpv susceptibility, and dual heterozygous pathogenic loss-of-function nfkb1 and heterozygous missense cxcr4 vus. family analysis showed the nfkb1 variant was inherited from his mother, while the novel cxcr4 variant was present in his father and sister. all four patients presented with recurrent infections, warts, and hypogammaglobulinemia. (figure 1 ) the nf-κb1 gene encodes p50/p105 transcription factor of the canonical nf-κb pathway, the most common autosomal dominant monogenic cause of common variable immunodeficiency (cvid). cxcr4 is a g-protein-coupled chemokine receptor with cxcl12 as cognate ligand. autosomal dominant pathogenic gain-offunction cxcr4 variants lead to impaired receptor downregulation and retention of neutrophils and other leukocytes in the bone marrow defining whim (warts, hypogammaglobinemia, infections, and myelokathexis) syndrome. all cxcr4 pathogenic variants truncate the carboxyl-tail of the cxcr4 receptor, a region responsible for receptor internalization, with the exception of one missense non-truncating variant p.e343k. case series: the proband (p1) is a 19-year-old male with history of recurrent bacterial respiratory tract infections, warts, moderate neutropenia, thrombocytopenia and hypogammaglobulinemia requiring immunoglobulin replacement therapy (igrt). bone marrow biopsy didn't show myelokathexis. next-generation panel sequencing identified a novel heterozygous missense cxcr4 (c.1022c>a, p.s341y) vus. the serine residue is highly conserved up to zebrafish. this variant was present in heterozygous form in two cases in gnomad database (277,984 alleles). additional whole-exome sequencing revealed a heterozygous pathogenic nfkb1 variant (c.980dup, pa328sfs*12) located in the nterminal rel homology domain, consistent with nfkb1 loss-offunction. both, the patient's sister (p2) and their father (p3), carry the heterozygous cxcr4 vus but not the pathogenic nfkb1 variant, and have history of warts, hypogammaglobinemia, and recurrent infections. the hpv susceptibility is particularly striking in p3 manifesting by genital warts and hpv-positive oropharyngeal cancer. bone marrow evaluation didn't identify myelokathexis in p2 (p3 is pending). the mother of the index case (p4) has cvid requiring igrt and immunomodulation. she shares the nfkb1 variant with p1 but is negative for the cxcr4 vus. extensive t and b cell phenotyping revealed low class-switched memory b cell count (0-7 counts/ul) in all subjects, and loss of transitional and mature naïve b cells in p1 and p4 with nfkb1 variant. proband b cells showed the highest tendency for apoptosis (35-55%) within the family. we describe members of a family with similar presentation (infections, hypogammaglobinemia, warts), however variable combination of nfkb1 and cxcr4 variants, where either genetic defect or their combination could explain the clinical phenotype. biochemical consequence of our novel cxcr4 variant is pending. as the proband showed the most severe immune phenotype and neutropenia, we hypothesize that cxcr4 has a synergistic effect on nkfb1 loss-of-function. the contribution of cxcr4 vus of the clinical phenotype of the two other family members is yet to be determined. background: the yield of diagnosis by exome sequencing for some primary immunodeficiencies (pid) has been less than the typical diagnostic rate for clinical exome analysis (~30-35%). the relatively low diagnostic rates for certain subtypes of the pids may be attributed to variable expressivity and/or an incomplete understanding of the genetic basis, among others. additionally the extent of multiple diagnoses and phenotypyic expansion in pid is not well explored. cohorts with highresolution clinical and genetic data are instrumental for exploring these questions. we evaluated the use of human phenotype ontology (hpo)annotated datasets to systematically address the prevalence of these issues using a cohort of 1000 individuals with pid who participated in research exome sequencing at the niaid. results: we generated a phenotype dataset of 1000 individuals with pids by annotating the clinical features of these subjects obtained from electronic health records (ehr) with hpo terms. exome sequencing of these 1000 individuals identified 313 probands with a pathogenic or likely pathogenic (p/lp) variant in a gene associated with the respective clinical presentation. we identified 118 probands where the same gene harbored a p/lp variant in at least three unrelated individuals. we used the clinical and genetic data of 118 individuals in the following areas: 1) we identified p/lp variants in aire, pik3cd, nlrp3, fas, ctla4, gata2, cybb, stat1 and tnfrsf13b in at least ten patients that explained their clinical presentations. this dataset allowed us to characterize variable expressivity of diseases associated with these genes by capturing the variability in the observed hpo terms among probands with p/lp variants in the same gene. dimensional reduction of clinical features of probands allowed us to cluster patients sharing similar phenotypic profiles. we found clinical presentation of individuals with monoallelic p/ lp variants in aire were relatively less variable and clustered more compactly compared to that of individuals with gata2 variants. 2) the extent of multiple diagnoses in pid is not well explored. the benchmark cohort we developed allowed us to identify candidates for multiple diagnosis or phenotype expansion by comparing the phenotype profile of each patient expressed in hpo terms to the hpo terms typically observed for a given pid. for example, we identified gain-of-function pathogenic variant in pik3cd in a patient that explained the clinical features of the pid observed in this patient. however, the patient also displayed developmental delay, congenital hemiplegia, cerebral palsy and absent speech. these features are not known to be associated with pik3cd variants, making this individual a candidate for >1 genetic diagnoses. conclusions: we developed a benchmark dataset where clinical features of patients were described using hpo terms. this dataset allowed us to quantify variable expressivity for certain pid subtypes and to systematically identify potential candidates for multiple diagnosis or phenotypic expansion. abstract/case report text warts, hypogammaglobulinemia, recurrent infections and myelokathexis syndrome is a rare combined immunodeficiency due to autosomal dominant gain-of-function mutations of cxcr4 chemokine receptor. the late diagnosis of whim syndrome in two ukrainian adolescents highlights the diagnostic challenges in this disease. patient 1, 12 year-old girl, had recurrent pneumonia since the first year of age; overall she had 7 episodes of pneumonia. she has suffered from chronic bronchitis for last several years. she had recurrent otitis media and chronic pyelonephritis. neutropenia was revealed when she was 3 year old. during episodes of bacterial infections she occasionally had normal value of neutrophils. the girl does not receive any treatment. patient 2, 14 year-old boy, had three episodes of pneumonia when he was 2, 7 and 14 year old. others symptoms include recurrent herpetic infection, warts on the hands. since 2 years of age he has haven persistent low neutrophil counts. the child was followed by hematologist and since 5 years of age he has received g-csf (5 mg/kg) twice a month. both children have leukopenia 750 -1200 cells/mm3, neutropenia -100 -300 cells/mm3, lymphopenia 560-830 cells/mm3, low number of bcells -30-50 cells/mm3. hypogammaglobulinemia was not prominent in both children, they have slightly decreased level of igg (7,1 g/l), normal level of igm (1,16 -1,44 g/l), patient 1 has low level of iga 0,24 g/l. patient 1 does not have protective level of antibodies to diphtheria and tetanus anatoxin, and anti-hbs antibodies were absent despite complete immunization. bone marrow aspirate revealed hypercellular marrow with granulocytic hyperplasia which was characterized by hypersegmented nuclei and cytoplasmic vacuolization of neutrophils. on molecular analysis of cxcr4, heterozygous mutation c.1000c>t (p.arg334*), known as r334x mutation, was detected in both patients, confirming the diagnosis of whim syndrome. replacement therapy with intravenous immunoglobulin was started in both children together with antibacterial prophylaxis and g-scf. vaccination with 4-valent vaccine against hpv infection was recommended for both patients. whim syndrome is very rare immunodeficiency but may be underdiagnosed. the awareness about rare forms primary immunodeficiency is very important in clinical practice for early diagnosis and treatment. methods: clinical providers recruited from nicer institutions electively completed web-based survey questions related to provider characteristics as well as initial diagnostic evaluation of itp, aiha, ain and es via securequestionpro® software. likert scales ranging from 1 ("rarely" < 20%), 2 ("sometimes" 21 to 40%), 3 ("half the time" 41% to 60%), 4 ("frequently" 61 to 80%), and 5 ("almost always" 81 to 100%) were used to ascertain frequency of evaluation for each diagnostic study. statistical analysis and plotting was done using rv3.6.1. plots were created using packages ggplot2, v3.2.0 and ggiraphextra v0.2.9. mean likert scale scores were calculated for each study for each suspected disease and plotted on radar charts. results: the survey was completed by 93 providers, including hematology/oncology (48.6%), rheumatology (16.4%), allergy/ immunology (17.1%) and other sub-specialties (17.9%). a slight majority of physicians (51%) were fellows or within 5 years of graduation; physician extenders and clinical pharmacists were also respondents. the majority (62.4%) of respondents indicated that ≤ 50 new immune-mediated cytopenia patients were seen at their institution annually. the vast majority of respondents (90.4%) reported evaluating ≤ 25 new es patients per year at their institution with 60% evaluating ≤ 10 cases annually. collated data from all respondents showed that in all disease states, the primary evaluation was focused on peripheral destruction mechanisms; the majority of patients are only "sometimes" or "rarely" evaluated for bone marrow failure syndromes, connective tissue disease, immunodeficiency and non-malignant lymphoproliferative disorders, but when done were more likely in es ( figure 1 ). evaluations were biased by sub-specialty with higher degrees of connective tissue focus by rheumatology and immunodeficiencies by allergy/immunology (table 1) . genetic sequencing was "frequently" or "almost always" sent in 4.5% of itp, 7.0% of aiha, 6.2% of ain and 32.2% of es p a t i e n t s . p e r s o n a l o r f a m i l y h i s t o r y o f a u t o i m m u n e / hyperinflammatory disease, malignancy or cytopenias most strongly influenced the decision to send genetic testing. lack of insurance coverage/negative financial impact on the patient and concerns about the inability to resolve variants of uncertain significance were the biggest barriers for obtaining genetic testing. conclusions: current practices in the evaluation of immunecytopenias are heterogeneous by sub-specialty and globally limited in scope with few patients being evaluated for underlying etiologies. in particular, despite a known high frequency of pathogenic variants in es, less than a third of patients are undergoing sequencing, highlighting a need to reduce barriers to genetic testing. development of a consensus guideline with multi-disciplinary engagement to harmonize an optimal evaluation for patients with immune-mediated cytopenias is needed. interferon regulatory factor-2 (irf2) binding protein-2 (irf2bp2) was originally identified as a transcriptional co-repressor of irf2(1). mutated irf2bp2 was identified in a 3-member family with recurrent sinopulmonary infections, progressive hypogammaglobulinemia, and poor response to protein vaccines (2) . we have now identified 10 additional families (18 subjects) with irf2bp2 mutations. clinical histories show an expanded phenotype with 15/21 having chronic gastrointestinal disease; 7 with gastrointestinal manifestations as the initial clinical complaint. five had granulomata in liver(x2), spleen, lung(x2) and gastrointestinal tract. five out of six tested had poor pneumococcal vaccine responses and four patients reported viral infections including varicella zoster(x2), influenza a and sapovirus. irf2bp2 is a 589 amino acid protein containing a highly conserved cterminal protein-protein interaction ring domain (rd). constraint metrics from gnomad indicate mild tolerance to missense changes and intolerance to loss-of-function alleles. we identified 3 categories of mutations: rd mutation or deletion (n=9 patients), null alleles (n=3) and non-rd missense changes (n=9). functional studies assessing the ability to affect nfatdriven luciferase expression were performed. rd mutations (4/5) had more profound loss-of-repression than wild-type, while missense changes had lesser, but still measurable effects. further, mutation categories and functional studies correlated with clinical phenotypes. of 9 patients with rd mutations, 8/9 had infections as presenting symptoms, 6/6 tested had hypogammaglobulinemia and 7/9 were diagnosed with cvid. one patient with a missense rd mutation had only an infectious phenotype (pulmonary mycobacterium avium) with slight decrease in immunoglobulins; in functional studies this mutation had the least effect of the rd mutations. haploinsufficient patients reported respiratory infections (3/3), recurrent urinary tract infections (2/3), gastrointestinal disease (2/3) and hypogammaglobulinemia (3/3). in contrast, 8/9 patients with non-rd missense changes presented with gastrointestinal complaints while only 2 patients had infections (recurrent bronchitis, shingles). gi disease prevalence is consistent with high levels of irf2bp2 expression in the colonic crypt cells (human protein atlas). to confirm this, immunohistochemical staining of colon biopsies from two patients was performed, identifying epithelial and glandular cells of the colon. irf2bp2 is involved in multiple processes, including the negative regulation of nfat signaling(3), tcr signaling(4), inflammatory macrophages (5) , and pd-l1 transcription (6) . interaction with the glucocorticoid receptor affecting anti-inflammatory and metabolic transcription (7) has also been reported. these observations highlight the irf2bp2 response to type-i interferons (irf2) and tcr stimulation (nfat), regulation of inflammatory macrophages and co-regulation of glucocorticoid receptor mediated signaling. the expanding role of irf2bp2 in multiple biologic systems correlates with the broad clinical presentation we observed in our patients. further studies utilizing irf2bp2 mutation knock-in mice will help characterize the gastrointestinal, lung and immune pathology seen in our cohort. abstract/case report text common variable immunodeficiency (cvid) is a disorder of antibody deficiency arising from over 20 genetic lesions. the clinical presentation of patients with cvid varies from recurrent, severe infections to autoimmunity. the immune dysregulation in cvid is especially difficult to treat and the lifespans of patients suffering from autoimmunity are much shorter than those without such complications. unfortunately, we have no way to identify which patients fall into which categories, or even know how many sub-categories of cvid there are. therefore, the field requires a method to classify patients into categories to precisely recognize and aggressively treat the more severe phenotypes. we address this goal by integrating analyses of patient exomes with analyses of cellular signaling. by analyzing stimulation assays with phospho-protein mass cytometry and high-dimensional data analytics, we aimed to elucidate signaling and phenotyping deficiencies in patients with cvid. importantly, our panel identifies all circulating immune cell subsets in whole blood. in eosinophils, we found amplified responses of pp38, pstat3, and cleaved caspase-3 in response to tlr1/2 stimulation. we found additional amplified responses of pstat3 and pstat5 in cd16lo monocytes. this finding suggests a previously unidentified role for eosinophils and cd16lo monocytes to contribute to the pathophysiology of cvid. we found abormal numbers of memory b cell counts, total switched b cell counts, and igm+, cd38+ b cell (plasmablasts) counts between cvid patients and healthy controls. cd21 expression on b cells was significantly reduced in cvid patients as well. these b cell results mirror findings from prior, seminal studies on cvid. notably, we have found higher pd-1 expression in the effector cd8 t cells of patients. integrating phenotype data, genetic analysis, and mass cytometry data will provide a deeper understanding of each patient's phenotype and how the are clustered. we also expect that a better understanding of alterations in the exomes and functions of the circulating immune cells of cvid patients will lead to new therapeutic approaches. abstract/case report text objectives: primary atopic disorders are monogenic disorders leading to profoundly dysregulated allergic responses. studying patients with these disorders has been instrumental in expanding our understanding of the pathogenesis of allergic inflammation with therapeutic implications for common polygenic versions of allergic disease. clinical findings: we have identified a now 8-year old boy who presented with severe eczema, extremely high blood eosinophil counts (5.8x109 cells/l, normal range: 0-0.85x109 cells/l) after birth and very high serum ige levels (2645υg/l, normal range: 0-500ug/l) since birth. known allergic disorders and parasitic infections are ruled out. given the extreme phenotype, whole exome sequencing was performed on the trio of patient and parents, and the patient was found to have a homozygous mutation in the evolutionarily conserved fibronectin iii domain of the osmr gene (c.1307t>a, p.v436d) (figure 1 ). osmr encodes oncostatin m receptor-beta, a component of both the osm type ii receptor and the il31 receptor, and is important for keratinocyte cell proliferation, differentiation, apoptosis and inflammation. mutations in osmr have been reported in association with familial primary localized cutaneous amyloidosis, however this condition was ruled out in this patient through skin biopsy which showed no amyloid deposits. methods and results: we modelled the c.1307t>a osmr mutation in hek293 cells and observed a loss of expression of the osmr receptor on the cell surface (with normal intracellular protein levels). this observation was mirrored in primary fibroblasts obtained from the patient. signal transduction through phosphorylation of stat1 and stat5 and gene expression (il6 and ccl2 measured via qpcr) was absent after stimulation with osm in patient fibroblasts. these signaling defects were rescued using a lenti-viral transduction approach to introduce the wild-type (wt) osmr gene. whole transcriptome analysis using rna sequencing confirmed that osm mediated jak-stat signalling pathways were deficient in the patient fibroblasts and were rescued after lenti-viral transduction of wt osmr. rna sequencing analysis also suggested significantly enhanced expression of genes in the nf-κb signalling pathway (e.g.: il18 and cxcl1) and decreased expression of genes in the tgf-β signalling pathway (e.g.: smad6 and smad7) in patient fibroblasts at baseline. this was also rescued upon lentiviral transduction. conclusion and future directions: our findings shed light into the disease mechanism of a novel primary atopic disorder, caused by a homozygous missense mutation in osmr. abstract/case report text 32-year-old caucasian female presented to immunology clinic with hypereosinophilia, eosinophilic esophagitis, peptic ulcer disease, severe gi bleeds, and chronic hepatitis. healthy throughout childhood, with minimal infectious history. in adolescence developed chronic severe myalgias and nsaid overuse, to which the peptic ulcer disease and bleeding were attributed. parents healthy and non-consanguineous. son with severe bleeding episodes and small stature. on exam she weighed 82lb, bmi 16. sclerae anicteric. tongue deeply furrowed. cervical nodes palpable. heart and lung exam normal. no hepatosplenomegaly. no clubbing of the digits or edema. skin was clear. wbc 11,600/ul, eosinophils 4730/ul, hemoglobin 10g/dl, normal platelet count. however, platelet aggregation testing abnormal. bone marrow normocellular, and flow cytometric and molecular analysis did not show hematolymphoid malignancy, primary hypereosinophilic syndrome, or systemic mastocytosis. lymph node biopsies did not show lymphoma or aberrant t cell populations. noted to have chronically elevated creatine phosphokinase, ranging from 706-3164u/l over two years at our institution. deltoid muscle biopsy showed non-specific myelopathic changes. an adult dystrophy immunostaining panel was normal. ultrastructure examination showed no abnormal storage material. a genetic panel for metabolic myopathies failed to reveal a cause. total igg, iga and igm normal. ige elevated at 934ku/l, and igg subclasses showed igg4 elevated at 354mg/dl. flow cytometry showed normal t, b and natural killer cell numbers. normal proportions of naïve, mature and activated t cells. vaccine response assessment was normal. evaluation for autoimmune/rheumatologic diseases was negative. liver biopsy demonstrated findings consistent with primary or secondary sclerosing cholangitis (without increased igg4 staining). given her inflammatory phenotype, additional genetic analysis was sent, assessing for primary immunologic disorders. this identified heterozygous variants of uncertain significance in ctla4 (c.553t>a; ps185t), zap70 (c.981c>g; p.d327e), and stim1 (c.752t>c; p.l251s). analysis of the ctla4 variant in vitro revealed that it was expressed normally. foxp3 expressing regulatory t cells were present in normal proportions in vivo and appeared phenotypically normal. this variant was found in her unaffected father. the zap70 variant is present in population databases (rs201605654, exac 0.07%), and was felt unlikely to be clinically relevant. the stim1 l251s variant, although not shown previously in human patients, has been previously shown in vitro to be a gain of function mutation [1] [2] [3] . furthermore, familial analysis revealed that this was a de novo mutation arising in the patient, and present in her son. humans with other gain of function mutations in stim1 and the orai1 channel it activates have overlapping syndromes including storkmorken syndrome, tubular aggregate myopathy and york platelet syndrome, characterized by chronic myopathy and platelet aggregation defects [4] . the stim1 l251s mutation is predicted to cause constitutive stim1 activation and calcium influx and likely provides an explanation for the patient's chronic myopathy and abnormal platelet aggregation. neither eosinophilic disease, nor cholangitis, have been described previously in stim1 gain of function-related diseases. it is unclear whether these issues are related to this novel stim1 mutation, or to other genetic or environmental influences. treatment of diseases caused by overactive crac channels is challenging as no pharmacologic inhibitors are yet clinically available. nomid/cinca syndrome is one of the periodic syndromes associated with cryopyridines. it is a defect in the innate immune system causing excessive activation of the inflammasome, with consequent il-1 secretion and neutrophil recruitment. clinically, damage occurs to organs such as the skin (neutrophilic urticaria), central nervous system (meningitis and deafness) and joint (arthritis). levy et al. (2015) evaluated a large series of 136 patients and median onset age was 0.8 years, while the median age at diagnosis was 15 years, although the symptoms initiate in the first days of life. treatment includes corticosteroids, which act by nonspecifically blocking all inflammatory cytokines, or by blocking il-1 specifically. if early diagnosis and treatment of the disease is not made, natural evolution leads to motor and adaptive disability and death in 20% of cases already in adolescence due to infection, neurological complications or secondary amyloidosis. we report a 10-month-old male child from nonconsanguineous parents who presented shortly after birth, multiple scaling and erythematous lesions throughout the body, evolving with following symptoms: abdominal abscess, hepatitis, meningitis and pioarthritis. laboratory tests showed elevation of inflammatory tests (esr, crp, amyloid protein a) and leukocytosis. the diagnosis was suspected at the nursery where the patient remained hospitalized for 51 days. a personalized multigene panel was requested. it was identified the variant p.gly309val, heterozygous for nlrp3 gene, not described in the literature, confirming the diagnosis of cinca/nomid syndrome. after discharge, it was introduced prednisolone (1,5mg/kg/day) and antiinterleukin-1 (il-1). after the second dose, skin lesions and joint edema regressed, weight gain, and neuropsychomotor development improved. this case reports a very early diagnosis of nomid/cinca syndrome. it warns neonatologists and pediatricians about the need of precocious recognition of the syndrome, probably improving the prognosis of the patient. professor/university center health abc abstract/case report text background: leprosy affects more than 208,000 people worldwide. brazil represents the 3rd. country in the world in leprosy frequency and maranhão state is an hyperendemic region. the city of imperatriz (ma) stands out as a reference center in the care of these patients. according to few reports, lectin pathway of complement system may play a role in susceptibility to leprosy. mannose binding lectin (mbl) and ficolins (fcns) recognize patterns of sugars and acetylated residues (pamp), respectively, in a wide variety of pathogens, including m. leprae. high levels of ficolins and mbl may act unfavorably promoting the spread of m. leprae. the present study evaluated the role of ficolin 3 and mbl in m.leprae patients and contacts. methods: a cross-sectional case-control analytical study was carried out, evaluating clinical and epidemiological data and serum levels of mbl and fcn3 (elisa) from july 2018 to april 2019. the study was approved by ethics committee and informed consent forms were signed before sample collection. data analysis was performed using the spss 22.0 for windows statistics program. results: we evaluated 169 serum samples (90 patients and 79 healthy family contacts), 54.4% were female, 32% under 15 years old, 78% african-brazilian, 65% of the families had more than 3 contacts at home. clinical data showed multibacillary forms in 73.3%; dimorphic (51%) and virchowian clinical forms (22.2%), up to 03 affected nerves in 26 (64.4%) and more than 5 lesions in 38 (42.2%). it was observed that 32 (35.6%) had a reaction, being type 1 (75%) more predominant. disability grade 2 was found in 16 patients (17.8%). in children under 15 years, 62.8% were multibacillary, 48.5% dimorphic and 20% undetermined; 11 (31.4%) also had reactions, 90% type 1 reaction and degree of disability 2 in 11.4% of children with the disease. the evaluation of serum fcn3 and mbl levels for the patients (n = 90) and contacts (n = 79) were 363.36ng/ml and 365.81ng/ml, (p = 0.76), and 3035.91ng/ml (p) and 2744.66ng/ml (c) (p = 0.29), respectively. there were lower values of fcn3 in patients with type 1 reaction (sudden and intense inflammatory processes) versus no reaction (337.23 ng/ml vs 372.86 ng/ml) (p = 0.03) and in patients with disability grade 2 (severe sequelae) versus disability grade 1 (319.74 ng/ml vs 343.25 ng/ml) (p = 0.003). higher fcn3 values was observed in patients with no disability (383.82 ng/ml) (p = 002). mbl concentrations were higher for patients above 15 years in comparison with patients below that age (3482.69 ng/ml vs 2626.04 ng/ml)(p = 0.02)) and correlated with the occurrence of a multibacillary clinical form. conclusions: mbl and fcn3 levels were not different in the patients and contacts of m. leprae, nevertheless the presence of severe forms with sequelae (reaction type 1 and disability grade 2) were associated with lower levels of fcn3. in addition, it is possible that lower mbl levels could influence the higher frequency of multibacillary disease below 15 years old. abstract/case report text introduction: hyper ige syndrome (hies) is a primary immunodeficiency characterized by elevated ige levels. symptoms can range from severe eczema, recurrent skin infections or pneumonias, and typical dysmorphic facies. there have been wide non-immunologic presentations in patients with hies, including retained primary teeth, scoliosis, craniosynostosis, arterial aneurysms and joint hyperextensibility. an association between hies and autoimmune hemolytic anemia (aiha) has further been described in the literature. however, there have been no reported cases of hies in association with iron deficiency anemia and concurrent pica. we present a unique case of a patient with a history of eczema, recurrent skin infections and pica found to have hies and iron deficiency anemia. case presentation: a 5-year-old boy with a history of allergic rhinitis presented to the allergy & immunology clinic for evaluation of chronic eczema and recurrent skin infections. the patient had a history of multiple hospitalizations requiring intravenous antibiotics for cellulitis and superinfected eczema since he was an infant. symptoms were refractory to the use of multiple skin barrier ointments and oral antihistamines. his mother further noted that for the past two months prior to initial evaluation, he developed a fixation with eating crayons, baby powder and chewing on drywall. physical exam was notable for a dysmorphic face, broad based nose, pale nasal mucosa with ample clear discharge, high-arched palate and lower incisor supernumerary teeth. his skin was characterized by generalized dryness, lichenification and scaly desquamation with boils on extensor surfaces of knees and elbows. initial screening for hies via t-helper 17 functional assay was consistent with decreased expression of il-17. genetic testing revealed stat3 s614g missense pathogenic variant consistent with hies. cbc was also notable for decreased hemoglobin at 9.9 g/l and mcv of 69 fl. patient was diagnosed with concurrent hies and pica in the setting of iron deficiency anemia. iron supplementation was started and patient's pica improved. discussion and conclusion: our patient with hies had a peculiar initial presentation with the classic signs and symptoms of hies and pica. the diagnosis of hies can often be delayed due to the wide range of clinical presentations. to our knowledge, the association of hies with iron deficiency anemia and pica has been underreported in literature. screening for anemia should be considered when evaluating patients with hies in order to rule out comorbid iron deficiency anemia which can be easily treated with iron supplementation. abstract/case report text introduction: common variable immunodeficiency is a primary immunodeficiency with variable and diverse phenotypic presentations. the two main phenotypes include a group which primarily exhibits recurrent infections and a group with or without infections and primarily inflammatory and autoimmune complications. the latter, may lead to a delay in diagnosis and is associated with poorer outcomes and higher morbidity and mortality. (1) another group of patients present with t-cell defects, lung disease, autoimmunity, and infections and may be diagnosed as having cvid but instead can have mutations in lrba or pi3 kinase. this subset of patients has been referred to as "cvid-like" in the literature. (2) case presentation: patient is an 11 year old female who initially presented to an outside facility due to 2 days of fatigue, fever, and abdominal pain. upon presentation, she was found to have massive splenomegaly, hepatomegaly, and an abnormal chest x-ray showing mediastinal lymphadenopathy and pleural effusion. laboratory results demonstrated pancytopenia, hypogammaglobulinemia, and low b cells, t cells, and nk cells via flow cytometry. she was transferred to our institution for further work up. she did not have any prior history of recurrent infections, asthma/lung disease, or autoimmune conditions. initial ct of the chest was consistent with granulomatous lymphocytic interstitial lung disease. patient was diagnosed with common variable immunodeficiency with granulomatous lymphocytic interstitial lung disease and was treated initially with high dose ivig, corticosteroid taper, rituximab, and imuran. she had interval worsening of pft and lung disease as shown by ct scan. genetic panel for cvid and related conditions revealed 2 variants of unknown significance. one heterozygous mutation in blnk gene (c.616g>a) and one heterozygous mutation in lrba gene (c.3914g>a). she was started on infliximab with plans to repeat ct scan in 6 months. discussion: mutations in both blnk and lrba have been associated with primary immunodeficiency. mutations in blnk, which is located on chromosome 10, have been associated with autosomal recessive agammaglobulinemia. homozygous or compound heterozygous mutations in lrba on chromosome 4, can lead to lrba deficiency which encompasses a wide range of clinical presentations including hypogammaglobulinemia, autoimmune disease, inflammatory bowel disease, antibody deficiency, organomegaly, and recurrent infections. (3) without genetic testing, the clinical presentation can be difficult to distinguish from common variable immunodeficiency. the patient presented has clinical features that can be seen with mutations in both blnk and lrba, however she is heterozygous for both mutations. further analysis, including measurement of lrba protein expression, is needed to further define her underlying immunodeficiency so appropriate treatment can be administered. abstract/case report text a 5 month-old, previously healthy, unvaccinated male presented with one week of diarrhea and cough and was admitted for dehydration and hypoxemia. his mother and sister both had a history of incontinenti pigmenti (ip). on physical exam, he was alert, afebrile, with tachypnea and subcostal retractions. enterovirus/rhinovirus and parainfluenza 3 were detected, but he became progressively hypoxemic and eventually required intubation and high-frequency oscillatory ventilation. chest x-ray showed multifocal bilateral airspace opacities. empiric treatment for pjp with trimethoprim/ sulfamethoxazole and glucocorticoids was started. tracheal aspirate pcr confirmed p. jiroveci. hiv rna pcr was negative. ivig was started due to suspicion for primary immunodeficiency. although his respiratory status gradually improved, he subsequently developed multiple skin lesions. skin biopsy grew mycobacterium szulgai. m. szulgai osteomyelitis of the right fibula and the left nasal bone was also detected, indicating hematogenous spread of the infection. he was started on four-drug anti-mycobacterial therapy and interferon-gamma (actimmune) at doses ranging from 50 μg/m^2 three times weekly to 100 μg/m^2 qod. immune work-up revealed t-cell lymphopenia [cd3+/cd4+ 202/μl (1400-5,100/μl) and cd3+/cd8+ 222/μl (600-2,200/μl)] with an abnormally increased proportion of memory cd4 t-cells compared to naïve cells for age. b-cell numbers were normal, and nk cells were decreased [cd56+cd16+/cd3-17/μl (100-1000/μl)]. nk cell lytic function by k562 lysis was normal, whereas cd107a degranulation was decreased. the serum igm level was normal [93 mg/dl (31-103 mg/dl) whereas iga [124 mg/dl (8-83 mg/dl)] and igg [1020 mg/dl (165-781 mg/dl)] were elevated. mononuclear cell cytokine response to ligands for tlr2-tlr1, tlr2-tlr6, tlr3, tlr4, and tlr7-tlr8 was normal. dna sequencing revealed a novel nonsense mutation in exon 5 of the ikbkg (p.gln201ter (q201x) (cag>tag): c.601 c>t, confirming the diagnosis of nemo deficiency, which was suspected based on the infectious disease presentation and the maternal history of ip. the diagnosis was further supported by signs of ectodermal dysplasia of teeth that appeared starting at 10 months of age. he underwent hsct using bone marrow from a 10/10 matched unrelated donor after conditioning with atg, busulfan, fludarabine and rituximab. actimmune therapy was continued until 10 days prior to transplant. for gvhd prophylaxis, he received tacrolimus and low-dose methotrexate. he achieved full donor chimerism post-transplant and has had no significant gvhd. interesting features of this case include the prominence of ip in mother and sister, which is usually due to female heterozygosity for an ikbkg null allele. such null alleles when inherited by the male fetus are embryonic lethal. our patient's nonsense mutation would be expected to result in severely impaired ikbkg protein expression and function. however, the fact that he had was born at term and initially was healthy coupled with his preservation of normal tlr function suggests that his ikbkg allele is likely to be a hypomorphic mutation. studies are in progress using ebv-transformed b-cell lines from the patient to evaluate ikbkg expression and function. also of interest, our patient was able to tolerate relatively high doses of interferon-gamma therapy without inflammatory side effects or an adverse impact on engraftment or gvhd. abstract/case report text background: primary atopic disorders are caused by genetic mutations that skew the immune system towards severe allergic disease. germline gain-of-function (gof) mutations in jak1 are a newly described monogenic cause of severe atopy, with affected patients demonstrating profound eosinophilia and allergic inflammation. our initial report of this novel condition identified a dramatic clinical response to the combined jak1/2 inhibitor ruxolitinib. we aimed to determine the long-term clinical response to ruxolitinib in patients carrying a germline jak1 gof mutation, and to characterize the effect of enhanced jak1 signaling on t lymphocyte effector functions and hematopoiesis. methods: clinical outcomes were evaluated in two pediatric patients carrying the c.1901 c>a (p.a634d) gof mutation in jak1 after 3.5 years of ruxolitinib treatment. t cell phenotyping was performed using extracellular surface marker and intracellular cytokine staining by flow cytometry, and by gene expression signature profiling of rna sequencing data. to evaluate the effect of enhanced jak1 activity on myelopoeisis, we reprogrammed jak1 gof patient-derived peripheral blood mononuclear cells into induced pluripotent stem cells (ipsc) and performed directed myeloid differentiation. rna sequencing was performed on rna collected during ipsc myeloid differentiation and from whole blood of affected patients before and after ruxolitinib treatment. results: long-term use of ruxolitinib was associated with improved growth, reduced eosinophilia, and control of allergic inflammation without significant infectious complications, however, anemia represented a dose-limiting adverse effect. t cell immunophenotypic analysis revealed severe t helper (th) cell skewing towards a th2 phenotype preruxolitinib treatment, in keeping with the allergic clinical manifestations. analysis of myeloid differentiation revealed an increased myeloid to erythroid ratio in colonies derived from jak1 gof ipscs compared to controls. rna sequencing analysis of jak1 gof human whole blood and ipscs compared to controls revealed upregulation of cytokine and cytokine receptor genes implicated in allergic inflammation and early eosinophil precursor commitment, including csf-1 and the interleukin-33 receptor. reactome pathway analysis of genes upregulated in both jak1 gof ipsc and whole blood compared to controls showed enrichment of several pathways including interferon alpha/beta, interleukin-4/-13 and interleukin-33 signaling. conclusions: this work demonstrates a critical role for jak1 in atopic immune dysregulation, specifically driving a th2 phenotype and eosinophilia. combined jak1/2 inhibition can reverse much of the allergic inflammation, with dramatic clinical effects. this has important implications for our understanding of the pathogenesis and potential therapeutic targets for early life allergic immune dysregulation. had severe combined immunodeficiency (scid) and/or severe disease in association with their combined immunodeficiency (cid) necessitating haematopoietic stem cell transplantation (hsct). we present clinical and laboratory features of 4 new zealand patients from the same family with a novel heterozygous missense variant in rac2 [c.62t>g, p.ile21ser (i21s)]. the index patient (p1 -age 8 y, m) has a history of infectious gastroenteritis, staphylococcal aureus conjunctivitis, recurrent otitis media and recurrent herpes simplex virus (hsv)-1 cutaneous infections. his 2 siblings (p2age 9 y, m; p3 -age 6 y, f) and his mother (p4age 42 y, f) all have a history of recurrent viral (hsv-1) and bacterial (staphylococcal aureus, streptococcal pyogenes) cutaneous infections and/or recurrent sinopulmonary infections that respond to empiric antimicrobial therapy. their neutrophils all had enhanced superoxide production in response to stimulation by fmlp and pma as compared to healthy controls'. these findings suggest that rac2 i21s is an activating mutation causing notable abnormalities in neutrophil morphology and nadph oxidase activation similar to other recently reported mutations. this novel mutation expands the phenotypic spectrum of rac2 activating mutations. clinical management of affected patients needs to be tailored to their phenotype and disease severity. background and aims: heterozygous mutations in cytotoxic tlymphocyte antigen-4 (ctla4) are associated with recurrent infections, lymphoproliferation, autoimmunity and lymphocytic infiltration of target organs. disease penetrance can be highly variable even among related family members carrying the same ctla4 mutation. our evaluation of a subset of the ctla4 patient cohort followed at the national institutes of health (nih) revealed that 50% of ctla4 mutation carriers have gastrointestinal (gi) manifestations which include diarrhea and diffuse lymphocytic enteropathy. our aim was to determine whether the intestinal microbiome, metagenome and metabolome could distinguish patients with ctla4 haploinsufficiency (ctla4-h) based on disease severity, and the presence or absence of gi manifestations. methods: clinical metadata and fecal samples were collected from healthy individuals (n=16) and patients with ctla4-h (n=32). patients with ctla4-h were classified as having minimal (n=7, only endocrine and/or dermatological manifestations) or systemic disease (n=25, hematological and multi-organ involvement). they were further classified based on whether they had a history of enteropathy (n=18) or active gi disease ( < 2 bowel movements per day and/or blood or mucus in stool) at time of sampling (n=8). metabolomic profiling (using a panel of 150 metabolites) and 16s rrna gene sequencing (v4 region) was performed on fecal samples (total samples: 62; number of reads/sample: 18,341 to 226,027; median: 74,857). a subset of 20 samples were subjected to shotgun metagenomic sequencing based on findings from the 16s rrna gene sequencing analysis. results: all patients with ctla4-h and a history of enteropathy or active gi disease also had systemic disease. fecal samples from patients with a history of enteropathy had a distinct microbial community structure (fig. 1 ) which was significantly less diverse (fig. 2 ) compared to healthy individuals and patients with minimal vs. systemic ctla4-h. patients with a history of enteropathy had significantly higher relative abundance of 5 bacterial taxa including shigella-escherichia (fig. 3) . shotgun metagenomic sequencing confirmed that samples from patients with a history of enteropathy were dominated by subsets of 12 identified escherichia coli strains, all of which share 30 genes coding for specific types of virulence factors such as curli fibers (facilitate uptake into host cells), flagellar proteins (increase motility) and enterobactins (increase bacterial iron transport). meanwhile, samples from patients without active gi disease at the time of collection were enriched for several taxa including bacteroides nordii and akkermansia muciniphila compared to patients with ctla4-h and active gi disease (fig. 4) . metabolomic analyses showed that asparagine, 3-hydroxybutyrate, cytosine and cystine were enriched in samples with abundant e. coli, whereas samples without e. coli were enriched in metabolites involved in pyrimidine (holm p=0.0005), purine (holm p=0.004), and alanine/aspartate/glutamate metabolism (holm p=0.01) (fig. 5) . conclusions: fecal samples from ctla4-h patients with a history of enteropathy were heavily colonized with e. coli strains that are associated with a specific metabolomic profile and that share virulence factor genes that may facilitate host invasion. these data suggest that the microbiome and metabolome can distinguish patients with ctla4-h and gi disease, and support the potential use of antibiotics or even antimetabolites to treat ctla4-hrelated enteropathy. the dna polymerase delta (pol δ) complex is essential for leading and lagging dna strand synthesis. its catalytic subunit (pold1), carries both polymerase and exonuclease activities and plays a crucial role in dna replication and repair. heterozygous pold1 mutations have been associated with inherited colorectal cancer and mandibular hypoplasia, deafness, progeroid features and lipodystrophy (mdpl) syndrome. more recently a biallelic loss of function mutation in pold1 (p.r1060c) that impairs the stability of the pol δ complex, has been reported in 3 related subjects with recurrent infections, deafness and combined immunodeficiency (cid) with t-cell lymphopenia, cd8+ t cell oligoclonality but preserved b cell proliferation. we report here a second family in which a novel biallelic missense mutation in pold1 gene was associated with cid. the proband is a 9-year-old boy born to consanguineous pakistani parents. since infancy he suffered from failure to thrive and recurrent infections, including 5 episodes of pneumonias, multiple otitis media, sinusitis, recurrent cellulitis at the g tube site, bk viruria and shingles. live and dead vaccines were well tolerated. at 2 years of age sensorineural hearing loss together with profound leukopenia (anc 260 cell/μl, alc 680 cells/μl) and hypogammaglobulinemia (420 mg/ dl) were identified. intermittent ivig replacement and antimicrobial prophylaxis were initiated. immunophenotyping at 9 years of age showed severe t cell lymphopenia (122 cd3+ cells/μl, 68 cd4+ cells/μl, 39 cd8+ cells/μl, figure 5 . volcano plot of metabolites present in fecal samples enriched with e. coli vs. samples without e. coli. c e l l s / μ l , 1 3 c d 4 + c d 2 5 h i f o x p 3 + c e l l s / μ l ) , a n d hypogammaglobulinemia (igm 23 mg/dl, igg 276 mg/dl, iga < 10). physical exam was remarkable for multiple acquired nevi in the groin area, teeth abnormalities and global developmental delay. whole exome sequencing analysis revealed a homozygous pold1 missense variant (nm_001256849 c.3175c>g, p.q1059e) absent in public databases (cadd score of 24). parents were heterozygous. tcr-vβ family expression was normal in both cd4+ and cd8+ t cells, but the proportion of t cells expressing vα 7.2 (encoded by the distal trav1-2 gene) was markedly reduced (less than 1%), consistent with impaired vdj recombination at the tra locus and/or with defective thymocyte survival. constitutive expression of γh2ax was observed in t and nk cells after 1 h and 24h of culture in unirradiated conditions. at 1 h post-irradiation (2 gy), reduced levels of p-atm were detected in t and nk cells, and lack of atm, smc1 and h2ax phosphorylation was observed in a subset of b cells, suggesting inability of these cells to mount an effective dna repair response. bone marrow examination showed normal trilineage hematopoiesis but decreased proportion of cd10-cd20+ mature b cells and increased proportion of pre-b cells. conclusion: we report the second mutation associated with autosomal recessive pold1 deficiency. our findings broaden the understanding of the mechanisms underlying the immune defect in this disease to include b cell maturation arrest in the bone marrow and a dna repair defect that may support the generation of a restricted tcr repertoire in the thymus and increased malignancy risk. abstract/case report text following allogeneic hematopoietic cell transplantation (hct) for scid, the development of a diverse t cell repertoire is essential for optimal immune recovery. high-throughput sequencing (hts) of the trb repertoire is the best tool for the evaluation of clonotype dynamics during immune reconstitution as compared to cdr3 spectratyping and staining of vβ families. we investigated whether longitudinal hts analysis of trb would accurately assess development of tcr repertoire diversity over time and reflect the quality of t cell reconstitution following hct for scid. we wanted to study the effect of conditioning regimen, scid genotype, donor type on tcr diversity post hct. we hypothesized that repertoire diversity may represent an early biomarker to predict long-term immune reconstitution vs. need for a second intervention. we assessed if the trb repertoire post-hct carried a molecular signature of selfreactivity. methods: the composition and diversity of trb repertoire of 27 scid infants, pre-hct and at 100 d, 6 and 12 mo and yearly posttreatment(s) was studied by hts. median time of follow-up was 48 mo. subjects were part of a prospective study of scid by the primary immunodeficiency treatment consortium. equal amounts of total rna extracted from peripheral blood was used as template to semi-quantitatively amplify trb rearrangements. the vdj statistics file (past program) was used to calculate a shannon entropy (h) index of repertoire diversity and simpson (1-d) index of repertoire clonality. results: trb sequence analysis of 27 scid patients showed poor diversity at baseline, followed by improvement to normal complexity (h index >8.0) after hct. similar kinetics of development of trb diversity were seen in patients with il2rg, jak3, and il7r defects (n=16) as in those with rag and artemis defects (n=14). in the latter group, however, hct with no conditioning or immune suppression only was associated with persistently lower diversity than hct with conditioning (p < 0.01), a difference not found in the il2rg/jak3/il7r group (fig.2) . hct from a matched donor (6/13 conditioned) correlated with higher diversity than hct from a mismatched donor (5/15 conditioned) (p=0.01). having > 500 cd4+ t cells/ul at 6 mo post-hct correlated with higher trb diversity at 24 and 36 mo post-hct (p < 0.01). the trb repertoire 100 d post-hct was enriched for the presence of central cysteines at the apex of the cdr3 (p < 0.001), a biomarker of self-reactivity ( fig.1 ). an h-index of 4.7 or lower at 100 d after hct predicted need for second intervention (hct or gt) (fig.3) . conclusions: analysis of trb diversity allows for detailed assessment of development of a diverse t cell repertoire following cellular therapies for scid and confirms the need for patienttailored treatment strategies based on scid genotype. t-cell repertoire 100 d post-hct is characterized by a molecular signature that may contribute to the increased rate of autoimmunity early post-transplant. furthermore analysis of trb diversity at 100 d post-hct may identify patients at risk for failure of sustained immune reconstitution, thus prompting a second intervention without delay. abstract/case report text background atopic dermatitis is a chronic, multifactorial, relapsing inflammatory skin condition which is one of the main known health problem worldwide. atopic dermatitis lesions are frequently colonized by staphylococcus aureus and staphylococcus epidermidis. their susceptibility to form biofilms, ability to form adhesive skin colonies which lead to extremely resistant to antibiotics and immune responses. formation of skin biofilm resulted in complex bacterial communities that have unique effects on human keratinocytes, mouse fibroblasts and host immunity. aims: the aims of this study to confirm the specificity of s. aureus or its secreted factors in induction of pro-inflammatory cytokines il-33, tslp and toxicity on human keratinocytes and mouse fibroblast. the second aim to study the inhibitory effect of co-culture of s. epidermidis with s. aureus in term of production of pro-inflammatory cytokines and toxicity. method and materials: human epidermal keratinocytes and mouse embryonic fibroblasts cell lines from 3t3 were used as a control strain to examine production of inflammatory response (il-33 and tslp) and cell death induced by s. aureus in the presence and absence of s. epidermidis. tslp and il-33 were detected by elisa and the apoptosis of s. aureus and s. epidermidis on these cells was evaluated by flow cytometry. result: recent findings propose the important role of skin biofilms in the pathogenesis of atopic dermatitis. s. aureus have been found to induce secretion of pro-inflammatory cytokines and cause apoptosis of human keratinocytes and mouse fibroblasts. presence of s. epidermidis as skin biofilm found to protects the human keratinocytes and mouse fibroblasts from induction of proinflammatory cytokines and cytotoxicity. conclusions and future work: s. aureus are essential in production of inflammatory response and cell death of mouse fibroblasts and human keratinocytes. future work will be carried out to identify the soluble factors that responsible in induction of pro-inflammatory cytokines. in addition, more studies are needed to be able to understand the mechanism by how s. epidermidis reduce the induction and cytotoxicity caused by s. aureus. j clin immunol in adult patients, in whom arbitrarily defined diagnostic criteria for antibody deficiency syndromes are not fulfilled, is subject to interpretation and decision differences reported by immunologists world-wide. in this study, we explored whether training in one particular program would decrease the variability in diagnostic and treatment approaches seen in the responses to two nationwide questionnaires in the uk and the usa. methods: a 10-minute online survey originally administered to a cross-sectional sample of 203 us allergists/immunologists (usa/i) in january, 2019, was also answered by 43 a/i subspecialists who had trained in the last 25 years at the louisiana state university health science center allergy immunology training program in new orleans (laa/i). respondents were asked questions on patient assessment, antibiotic use, initial igrt, and immune response assessment in decisionmaking to prescribe igrt. usa/i participants were recruited from the dynata physician professional panel. laa/i participants were recruited by the louisiana primary immunodeficiency network (lapin). results: overall, laa/i had consensus responses to the various practice questions close to 90% of the time, but outliers were always present, as was also observed in the usa/i. there was a higher frequency in the reported care of patients as described in the questionnaire by laa/i. over 98% of laa/i assessed vaccine responses prior to commencing igg replacement vs only 90% of usa/i p < 0.5. all la a/i used the pneumococcal vaccine for assessment purposes while few used tetanus and hemophilus influenza, and none used meningitis or salmonella vaccines. these vaccines were still used by some of usa/i. a high level of concordance was observed among all respondents in that only few regarded pneumococcal antibody testing as the definitive test to commence igrt. high resolution chest ct scan was used more often by laa/i before starting igrt. assessment of effectiveness of igrt was decided after only 3 months by more usa/i, vs laa/i, who tended to wait 6 months to decide to continue or discontinue igrt. conclusions: all a/i responders saw a significant number of patients who do not conform to strict diagnostic criteria for antibody deficiency syndromes. there is diversity in the approach of usa allergists/ immunologists in determining the indication for igrt for non-classical antibody deficient patients. laa/i responses made it obvious that post graduate influences always play a role in shaping the way a/i practice evolves after graduation. drawing on clinician experiences through questionnaires offers a valid contribution to developing consent approaches to improve patients' clinical conditions. diagnostic criteria and treatment guidelines would benefit from practice-based realistic recommendations based on a/i experience. abstract/case report text background: autoimmune lymphoproliferative syndrome (alps) is a rare genetic disorder secondary to a defective fas-mediated apoptotic pathway of mature lymphocytes. it is characterized by chronic nonmalignant lymphoproliferation in the form of lymphadenopathy and/or splenomegaly, autoimmune manifestations such as cytopenias, increased risk of lymphoma, and expansion of tcrαβ+ cd4-/cd8-(dnt)t-cells. germline or somatic pathogenic variants in fas, fasl, and casp10 are well described genetic defects associated with alps. the definitive diagnosis for alps, based on the revised 2009 nih diagnostic criteria, include both required criteria (chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, or both and elevated tcrαβ+ dnt t-cells) and one of the primary accessory criteria (defective lymphocyte apoptosis or mutation in the genes mentioned above). patients who do not meet the current diagnostic criteria are considered for alps-related disorders. case presentation: we report a 2-year-old male who presented with recurrent infections, splenomegaly and chronic lymphadenopathy since 8 month of age. due to its chronicity he was evaluated by multiple specialists for malignant and infectious causes. hematological workup including bone marrow biopsy was unremarkable except for an elevated ldh level. infectious workup identified a past cmv infection. clinical course is pertinent for chronic splenomegaly which was identified incidentally at 1.5 years of age during an evaluation for intussusception. family history is pertinent for a father with recurrent infections, paternal grandmother with thrombocytopenia of unknown cause requiring platelet transfusions, and paternal cousin with neutropenia. there is no family history of lymphomas. history of chronic lymphoproliferation and recurrent infections prompted an evaluation for lymphoproliferative disorder. full immune workup was notable for elevated plasma il-10 and il-18, normal immunoglobulin levels, lymphocytes subsets, vitamin b12 level, soluble fasl, and relative frequency (%) but borderline increased absolute count of tcrαβ+ (dn) t-cells. in addition, he was noted to have presence of anti-platelet antibodies, poor lymphocyte proliferation to antigens, and low pneumococcal antibody titers. genetic testing with a 207 pid gene panel identified a likely pathogenic heterozygous variant in prf1 c.487del (p.his163thrfs*96), a heterozygous variants of uncertain significance in casp10 c.683c>t (p.pro228leu) and stim1 c.304a>g (p.thr102ala). the casp10 variant is present in 63 alleles in gnomad (282k total allele count) and reported deleterious by sift. discussion: unlike the typical alps presentation, characterized by dominantly lymphoproliferation and autoimmunity, our patient's clinical phenotype is striking for recurrent infections, abnormal t-cell function, and poor antibody response. our patient does not the meet diagnostic criteria for alps due to normal relative frequency of dn t-cells. however, presence of elevated of il-10, il-18, platelet autoantibodies raise concern for alps-related disorder. in addition, family history of recurrent infections and cytopenias raises concern for familial autoimmunity and alpslike phenotype. although casp10 is associated with autosomal dominant and autosomal recessive alps, the role of this vus is yet to be determined. conclusion: we continue to investigate the pathogenicity of our novel casp10 vus. further studies include pedigree analysis, fas apoptosis assay and apoptosis pathway testing to assess for the etiology of this alps-related disorder. (word count 487, max 500) abstract/case report text rationale: ocrelizumab is a recombinant anti-cd20 monoclonal antibody, which binds to a different, but overlapping cd20 epitope than rituximab. there have been increasing reports evaluating hypogammaglobulinemia and morbidity and mortality in patients receiving rituximab, but there is a paucity of data on hypogammaglobulinemia in patients treated with ocrelizumab. methods: we performed a retrospective review of patients who received ocrelizumab in our healthcare system. we evaluated the demographics, indication for ocrelizumab, frequency of immunologic evaluation, and h y p o g a m m a g l o b u l i n e m i a p r e -a n d p o s t -o c r e l i z u m a b . hypogammaglobulinemia was stratified as mild (igg < 600mg/ dl or less than lab reference range), moderate (igg < 400mg/ dl) or severe (igg < 200mg/dl). results: we identified 185 patients who received ocrelizumab for multiple sclerosis (average number of ocrelizumab cycles = 4; range 1-9 cycles). there were 120 (65%) female patients, with a mean age of 49 years old (range 23-74; standard deviation ±13 t-cells. tnfα, ifnγ, and il-10 were not statistically different between cgd patients and healthy controls. tnfα, ifny, il-10, and il-17a expression in patients with cgd who had active colitis or history of colitis were increased as compared to cgd patients without a history of colitis but did not reach statistical significance. in two patients, il-17a expression that was elevated pre-hct normalized post-hct. discussion: the mechanism for increased susceptibility to inflammatory disorders in patients with cgd has not been well elucidated. our results agree with previous studies demonstrating increased il-6 and il-17a production from cd4+ t-cells in patients with cgd indicating a proinflammatory state in these patients at baseline. also, there appears to be an increase in tnfα, ifny, il-10, and il-17a expression from cd4+ t-cells that correlates with presence of inflammatory disease vs. those without inflammatory disease indicating that these cytokine perturbations may be able to serve as biomarkers of disease activity. a larger sample size with prospective collection will be analyzed in the future. abstract/case report text background: glucose-6-phosphatase catalytic subunit 3 (g6pc3) deficiency, is characterized by severe congenital neutropenia, recurrent bacterial infections, mild intermittent thrombocytopenia and a high incidence of congenital cardiac and uro-genital defects. we report the case of a 28-yo male with chronic neutropenia and thrombocytopenia, who was found to have homozygous pathogenic variants in g6pc3 (c.210del, p.phe71serfs*46). unique to this case is the patient's long history of misdiagnosis of evans syndrome (chronic autoimmune neutropenia with thrombocytopenia). case presentation: a 28-yo male with reported diagnosis of chronic autoimmune neutropenia and thrombocytopenia since age 7 was referred to our a b clinic with concern for an underlying immune dysregulation syndrome. he had a history of oral ulcers, gingivitis, recurrent bacterial infections (otitis media, pneumonia, skin abscess) concomitant with severe neutropenia ( < 500 cells/ul), for which he had received treatment with systemic steroids and g-csf since he was 9 years old. he also had history of asthma and short stature, thought to be secondary to his chronic systemic steroid use. we present the first chilean patient with stat1 gof immunedysregulation . moreover, to our knowledge this is the first stat1 gof patient presenting with lymphomatoid granulomatosis. this is a severe pulmonary disease in which primary immunodeficiencies including stat1 gof should be considered in the differential. in this case rituximab successfully resolved pulmonary nodules and respiratory symptoms. there was persistence of mild hepatosplenomegaly but otherwise clinical stability and monitored expectantly until the age of 12. at that time he presented with fever, left knee and ankle arthritis. he underwent arthrocentesis of the left knee and left ankle, both aspirates were sterile, with notable leukocytosis with heavy neutrophilic predominance. an extensive rheumatologic and infectious workup was non-diagnostic. both sil-2r and il-6 were elevated, 5,026 units/ml ( < 1105) and 44pg/ml ( < 6), respectively. he was treated with systemic corticosteroids, ultimately arthritis resolved after 2 months. at age 13 he presented for the first time with periorbital pain and conjunctival injection of the left eye that persisted after minor trauma. he was found to have nongranulomatous uveitis, which responded ultimately to systemic corticosteroid. he then presented at age 14 with fever and right knee and great toe arthritis. again he underwent arthrocentesis which revealed aseptic arthritis, and at that time was started on anakinra (anti il-1β) and prednisone. there was clinical improvement over several weeks followed by return of right knee arthritis, coupled with onset of symptomatic uveitis of the left eye. despite systemic corticosteroids and anakinra and il-1 blockade, the patient was again admitted shortly thereafter to the hospital with arthritis, fevers, rash and abdominal pain. there was concern for evolving hlh and the patient was ultimately transferred to cincinnati children's for further evaluation and treatment. pertinent inflammatory biomarkers at that time included sil-2r of 3,394 units/ml and il-18 level of 9,624 pg/ml. in addition to anakinra and systemic corticosteroids, the patient was started on tadekinig alfa (recombinant human il-18 binding protein) as part of a prospective study. hlh flare ultimately resolved without use of antineoplastic agents, and the patient was discharged home. soluble il-2r levels since normalized, and il-18 levels decreased to less than 1000 pg/ml. the patient has been doing well on anakinra and tadekinig alfa, though continues to experience mild to moderate right knee effusion. this case suggests il-18 inhibition may be an effective therapeutic approach for patients with xiap deficiency. in the absence of neurological involvement and infectious trigger, ruxolitinib was initiated at a dose of 50 mg/m 2 /day, in combination with dexamethasone (10 mg/m 2 /day). this treatment led to rapid normalization of the neutropenia (48 hours), complete resolution of the splenomegaly (10 days) and disappearance of hlh biological markers (triglycerides levels in 1 week, activated hla-dr+ cd8 t cells in 2 weeks, fibrinogen levels in 1 month), without the need for etoposide or serotherapy. dexamethasone was weaned every two-weeks and stopped after 8 weeks. ruxolitinib was well-tolerated with no side effects. while in complete remission of her hlh, the patient then received alemtuzumab (0.5 mg/kg total dose) and a fludarabine-based myeloablative conditioning regimen. ruxolitinib was weaned over one week, and a 9/10 unrelated transplant was performed with success. the immediate posttransplant period was complicated by a veno-occlusive disease that responded rapidly to defibrotide and a corticosteroidresistant skin and ocular graft-vs-host disease (gvhd) despite a prophylaxis with ciclosporine and mycophenolate mofetil. gvhd was controlled by the reintroduction of ruxolitinib. at 3 months post-hsct, her chimerism is 100% donor. to our knowledge, this case is the first description of a patient with primary hlh successfully treated in first intent by a combination of dexamethasone and ruxolitinib prior to hsct. our observation suggests that this targeted and less-toxic treatment regimen, that does not include etoposide nor high-dose alemtuzumab, is effective, well-tolerated and could be used in first intent to treat primary hlh. abstract/case report text presentation a 15-year-old girl, presented in ambulatory consultation, with a 3-year history of recurrent fever, influenza-like symptoms (sore throat, malaise), associated with self-limited painful genital ulcers (just within the period of fever). the first episode was characterized for an fournier's infection, requiring in-hospital treatment, multiple surgical procedures, antibiotics and hyperbaric oxygen therapy. after that catastrophic debut, she was diagnosed approximately 6 episodes per year pharyngitis (with fever, malaise and sore throat) treated with corticoids, antibiotics and topic medication. the last year, noticed that every episode of fever (total of 4) were associated with one or several genital lesions. the patient had no relevant medical history, she didn't receive long-term medication, she received all immunizations, she was sexually inactive, and hadn't apply any topic medication or product on the vulva, there was no trauma history, psychological medical history or sexual abuse. episodic gynecologic examination showed her labia minor several lesions, fibrinous, soft ulcerations on their inner aspect, these lesions had a symmetrical appearance, known as kissing lesions; no vulvar swelling, vaginal discharge or lymphangitis were noticed. there were no other skin or mucous membrane lesions (figure 1 ). investigations viral (hiv, hbv, hcv, ebv, cmv) and treponemal (tpha-vdrl) serologies were negatives. erythrocyte sedimentation rate (esr) and pcr analysis within ulcers episodes were positive. specific antibodies (cardiolipin, anti ro/ss-a, anti la/ss-b, anti ccp, anti ena, anti gliadin, anti tpo anti tpo) serologies were negative. otherwise, important elevation immunoglobulin d was observed (9,3 mg/dl, twice the normal value): mild elevations of immunoglobulin m and immunoglobulin a were observed. serum subtypes of immunoglobulin g and immunoglobulin e were normal. leukocytosis with monocytes elevation and an increase of lymphocytes b were present. (table 1 ). discussion lipschütz ulcers are uncommon and an often unknown entity for physicians, but it is important to recognize and include it in the differential diagnosis of vulvar ulcerations. this condition is characterised by self-limited painful ulcerations of the vulva or lower vagina in adolescent or young women, non-sexually transmitted, and usually preceded by influenza or mononucleosis-like symptoms. hyperimmunoglobulin d syndrome (hids) is characterized for unremitting fever lasting four to seven days and the presence of palpable tender lymphadenopathy, splenomegaly, arthralgia/arthritis, abdominal pain, and mucocutaneous manifestations. laboratory findings suggestive of hids include elevated age-specific serum immunoglobulin d (igd) and/or immunoglobulin a (iga) levels, elevation of acute phase reactants, and urinary excretion of mevalonic acid during, but not between, attacks. the diagnosis is established if an elevated age-specific level of igd is detected. iga levels are typically measured at the same time but are not required for diagnosis. elevated serum igd is not specific for hids and can occur in patients with certain neoplastic, infectious, heritable, and idiopathic disorders. in the present case report, the patient was treated with colchicine, with favorable evolution and free from new events. levels of ig d, platelets and monocytes remain high. we describe a young female patient presenting recurrent lipschütz ulcers, fever and elevation of serum immunoglobulin d, suggesting that hids could be associated with genitalia ulcers. (2), and transmission in vivo in extremely low birth weight infants was considered (3) . based on these considerations, the risk/benefit was considered favorable for restarting pasteurized donor breastmilk feeds. given his small size and young age, we had significant concerns about using ganciclovir prophylaxis and opted to hold this and monitor weekly cmv pcr. the child has been titrated up on these feeds, is gaining weight appropriately, and has had weekly cmv pcrs which are negative x5 since restarting donor breastmilk. t cells, last checked at 7 weeks of age (37 weeks gestational age) remain essentially absent. given the lower risk of nec in premature infants with breastmilk-based enteral feeds, a broader, multi-institutional study is warranted to best examine the safety of pasteurized donor breastmilk in infants with scid and complete digeorge syndrome. transfected with a reporter plasmid (for luciferase), wt or mutant-stat1 plasmids. nk cell cytotoxicity was measured by cr 51 release assay. we used multiparametric immune profiling to dissect the effect of stat1-gof mutations on nk cell developmental phenotype. results: similar to our previous studies, we observed higher levels of stat1 phosphorylation after two hours of stimulation from the dbd mutation compared to the ccd mutations. the stat1 activity assay confirmed gain of function observed by flow cytometry, but this activity was higher in k388q mutant and d292e mutant (ccd-closer to dbd) than v266i mutant. all patients demonstrated low nk cell lytic unit compared to healthy donors. interestingly, we observed a correlation between low lytic unit and lower numbers of cd56 dim perforin + cd16 + nk cells; much lower in patient with k388q mutation. stat1-gof patients showed a significant decrease in total nk cell numbers and impaired nk cell maturation was characterized by low expression of cd57, and higher levels of immature nk cell markers (cd117, nkg2a, cd158b). conclusions: these data suggest that impairment of nk cell function is affected by the location of the stat1 mutation and continues to be the case in novel mutations identified. the identification the genotype/ phenotype correlation in the spectrum of the nk cell defect in stat1 gain-of-function mutants may help to better understand the molecular basis for stat1 activation and/or function to predict clinical manifestations of disease and ultimately treatment regimens. mutation specific analysis after an amniocentesis showed that one twin was a carrier (l152m) and the other was a compound heterozygote (r156c and l152m). after birth, twin a had a mildly low erythrocyte ada level (12.8 nmol/h/mg; normal range 63+41) with normal metabolites and a normal immunophenotype, similar to both parents. twin b showed a normal absolute lymphocyte count and mitogen proliferation, normal t lymphocyte subsets, mildly low b and nk cells with 85% naïve t cells and a normal trec assay. erythrocyte ada levels were absent in peripheral blood, with mildly elevated metabolites [daxp=0.041 μmol/ml rbc (normal < 0.002) and %axp=0.9 (normal < 0.2)]. weekly recombinant ada enzyme replacement therapy (ert) was started at 1 week of life with subsequent normalization of the metabolites by week 14. absolute lymphocyte, t cell subsets were normal at birth but continued to rise slightly above normal range after starting ert. b and nk cell counts were mildly low at birth but normalized by week 3. genetic testing confirmed the prenatal genotypes in the twin girls. the patient is now 11 months old and doing well with no history of infections. her twin was not an hla match and family is currently awaiting gene therapy approval. discussion: ada deficient patients show substantial clinical and metabolic heterogeneity that tends to correlate with the genotype but phenotypic discordance occurs even within the same genotype. we describe an infant with prenatally diagnosed compound heterozygous mutations in the ada gene (grade-i: r156c and grade-ii: l152m). ada alleles are graded from 0-iv with increasing ada expression and decreasing severity respectively. there are reports of children with grade i/iii allele combinations with delayed, late and partial phenotypes. two siblings have been reported with l152m allele (grade iii) in combination with a different grade i allele (r235q), presenting with combined immunodeficiency at 1 and 13 months. the specific allele combination from our patient has not been previously reported, however, we expected that the grade-i allele likely would be more deleterious than the grade-iii allele. in our case, predicting a future phenotype remains a challenge, creating a dilemma regarding management strategies. however, with only mild metabolite elevations in our patient after birth, we may speculate whether the prenatal diagnosis with early ert precluded the development of a full immunophenotype and it remains to be seen whether nonimmune sequeli may be prevented. conclusion: children with compound heterozygous mutations in the ada gene can pose diagnostic and therapeutic challenges, especially due to the associated metabolic and clinical phenotypic variability. early recognition and treatment may potentially alter long-term morbidity and mortality. (ipex)-like phenotype. immunodeficiency is often combined with impairment of the humoral and cellular compartments. hematopoietic cell transplant (hct) can resolve disease-related manifestations in stat1-gof, but overall survival is poor and there is a high rate of secondary graft loss in transplanted patients. jakinibs are a class of medications that block cytokine-induced jak/stat activation. ruxolitinib preferentially inhibits jak1 and jak2 and has been used as precision-directed therapy for treatment of stat1-gof related manifestations with success in stabilizing and in some cases reversing organ-specific manifestations. the utility and safety of jakinibs for long term treatment of stat1-gof and in the prevention of disease-related manifestations is not known. as such, hct is often pursued for patients once disease-related manifestations are controlled with jakinibs. we present a patient with stat1-gof mutation with gradual secondary graft loss following hct 10 years ago, that has had continued disease progression despite chronic ruxolitinib treatment. case presentation this is a 14 years-old male diagnosed with a de novo heterozygous stat1 mutation (c.983a>g/a) at age 9, 5 years following hct for ipex-like disease. he has been treated with ruxolitinib for the last three years. this patient initially presented at 5 months of age with wasting enteropathy, failure to thrive, early-onset type 1 diabetes and hypothyroidism. he had frequent upper respiratory infections during childhood including mycobacterium fortuitum mediastinal lymphadenitis. at 4 years he underwent 10/10 matched, unrelated bone marrow transplant following reduced-intensity conditioning. mixed donor chimerism was present in the first 100 days following hct, and he continued to have a slow progressive decline of donor chimerism with full graft loss (0% whole blood donor chimerism) by age 13. at age 11, enteropathy returned leading to cachexia and tpn dependence. concurrently, he had recurrent upper respiratory tract infections, lymphopenia, and hypogammaglobulinemia. imaging showed bronchiectasis and lung function was consistent with obstructive lung disease (fev1:1.88 l fvc:1.96 l dlco:13.9 ml/min/mmhg). initiation of ruxolitinib at age 11 resolved his enteropathy with discontinuation of tpn and >25-pound weight gain. enteropathy has not returned. pulmonary clearance measures have also been employed. dlco initially improved (dlco: 19.11 ml/min/mmhg) but obstructive lung pattern continued (fev1: 1.91 l fvc: 2.08 l). after initial improvement, dlco began to decline. over the last 2 years and despite treatment with ruxolitinib, lung function has deteriorated with worsened fev1 (1.64 l), fvc (2.03l), and dlco (17.91 ml/min/mmhg). with this progressive decline, the family is now pursuing second hct. discussion jakinibs apply precision-directed therapy for immune dysregulatory features of stat1-gof. their use leads to substantial disease control and clinical improvement but does not prevent disease progression. jakinibs should be used as a bridge to definitive therapy with hct in patients with stat1-gof mutation. a recent large registry study showed a higher risk of infections (hr 1.42, 95% c.i 1.33-1.52) in children with thymectomy as compared to surgery controls, in addition to demonstrating differences in the risk of cancers, autoimmunity and atopy. limited small studies have described some risk factors for altered immune consequences; however, specific predictors of infections among children with congenital heart disease (chd) undergoing thymectomies have not been systematically assessed. among children with chd and thymectomy, we sought to characterize children with and without reported infections within 4 years postthymectomy and identify predictors of bacterial and viral infections. methods: using a retrospective chart review (institutional irb approved) from 7/2/2013 and 3/31/2017, we identified children with chd that underwent thymectomy and excluded any known conditions associated with immunodeficiency and those with less than 6-month follow-up post-thymectomy. first absolute lymphocyte count (alc) after thymectomy was stratified using a cutoff at 50% of the lower limit of age-adjusted normal values (alc value < 50% vs alc value >50% of the lower limit of age-adjusted normal levels). we sought to assess predictors of reported bacterial (positive blood, cerebrospinal fluid, respiratory cultures and chest-x-ray confirmed pneumonia) and viral infections (positive viral pcr tests) within 4 years postthymectomy. results: we identified 128 children with chd who had thymectomies, of which, 65% (84/128) were male. the median age at thymectomy was 6 months (interquartile range 2 months-2.2 years); 51% (66/128) underwent a complete thymectomy; and 3% (4/128) developed a chylothorax within 1 week post-thymectomy. a substantial proportion of children had an alc below 50% of the lower limit of age-adjusted normal levels after thymectomy (2% [3/127] pre-thymectomy vs 65% [82/127] postthymectomy). among children with chd post-thymectomy, 51% (65/ 128) and 45% (57/128) reported bacterial and viral infections within 4 years, respectively. children with post-thymectomy alc values below 50% of the lower limit of age-adjusted normal levels had higher odds of reported bacterial (or 3.44, 95% c.i 1.37-8.64, p=0.008) and viral (or 5.86, 95% c.i 2.02-16.96, p=0.001) infections post-thymectomy as compared to those with an alc greater than 50% of the lower limit of ageadjusted normal levels (multivariate logistic regression). there was no association with the type of thymectomy (partial vs complete), age at thymectomy, weight at thymectomy, sex or prematurity. conclusions: among children with congenital heart disease with no known immunodeficiency undergoing thymectomy, alc below 50% of age-adjusted normal levels post-thymectomy may be associated with higher odds of bacterial and viral infections. a retrospective study design with a small sample size poses several limitations; however, this study suggests that post-thymectomy absolute lymphocyte values may be a potentially useful marker to identify higher risk patients in this population. radiological assessments esp. in the ct chest is commonly performed, but has associated radiation exposure and pulmonary function testing, at times, maybe insensitive to small changes in lung pathophysiology. many pids may have overlapping features with short telomere syndromes (sts) a, which are accelerated aging syndromes affecting hematopoietic, pulmonary, hepatobiliary and/or immunological systems, unified by a high cell turnover in these organs. clinical assessment of ageappropriate telomere length (tl) is performed using flow cytometry & fluorescence in-situ hybridization (flowfish). methods: we retrospectively analyzed telomere lengths in lymphocytes and granulocytes using the flow cytometry and fish method .flowfish testing was done at reference laboratories in johns hopkins university (jhu, usa).approval was obtained from mayo's institutional review board. data abstraction and analysis was done using the software jmp. results: 24 patients were included in our analysis with 13 females (54%) and 11 males (45%).the median lymphocyte count of our cohort was 0.98 (0.51-1.78).the telomere length was strongly associated with the presence of lung disease (p=0.02*) and the presence of interstitial lung disease closely paralleled the changes in telomere length (delta-as compared to age adjusted normal percentiles lengths). shorter lymphocytic telomere length was associated with more severe reduction on total lung capacity (tlc; p=0.006*). conclusion: shorter lymphocytic telomere length served as a reliable biomarker for interstitial lung disease in pid patients. this may open up newer avenues for assessment of aging pathways in pid and may offer the option of using senolytic therapies in pids. mutations in the il-2 receptor common gamma chain gene (il2rg) result in x-linked severe combined immunodeficiency (scid). the common gamma chain is shared by il-2, il-4, il-7, il-9, il-15 and il-21 receptors. x-linked scid typically presents with low or absent t and nk cells and normal or elevated numbers of b cells. we report a case of x-linked scid with elevated b and nk cell numbers (t-b+nk+). the male patient had an abnormal newborn screen for scid in north carolina. lymphocyte enumeration performed at 12 days of life showed 8 cd3+ cells/mm3, 370 b cells/mm3, and 997 nk cells/mm3. he had no naïve t cells. repeat lymphocyte enumeration two weeks later showed that the cd3+ count had increased to 389/mm3. only 1.5% (6 cells/mm3) were cd45ra+ naïve t cells. he continued to have elevated b cell and nk cell numbers. chimerism studies revealed the presence of 6% female cells in mitogen-stimulated pbmc by fluorescence in situ hybridization, indicating the presence of transplacentally transferred maternal cells. lymphocyte proliferation responses to pha and cona mitogen stimulation were very low (less than 10% of normal). immunoglobulin levels were igg 921mg/dl, igm 18mg/dl, and undetectable iga and ige. genetic studies revealed a missense mutation in il2rg, c.467c>t, resulting in an amino acid substitution (p.ala156val) in the extracellular domain. family testing showed that the patient's mother was a carrier for this variant. the father and the two healthy older brothers did not have this variant. of note, the family history was significant for lateral maternal male early deaths. at 7 weeks of age, the patient received an unfractionated bone marrow transplant from his hla-identical brother without conditioning or gvhd prophylaxis. at the time of this report's submission, he is 4 weeks post-transplantation and has had successful engraftment (whole blood-cd3+ fraction was composed of >95% donor cells). he also now has normal t cell proliferation in response to mitogens and normal levels of all immunoglobulins. genetic defects that cause primary immunodeficiency can have variable phenotypic presentations. the patient's phenotype was atypical in that he had elevated nk cell numbers. to further evaluate these cells, we checked for stat4 phosphorylation following il-12 stimulation of abstract/case report text background: stat3 gain-of-function (gof) mutations cause a multisystem disease of early onset autoimmunity and lymphoproliferation, severe post-natal growth restriction, and recurrent and/or invasive infections. treatment of the autoimmune and auto-inflammatory features of stat3 gof patients relies heavily on immunosuppression and is often challenging. the full scope of phenotypes, treatments and outcomes may be broader when analyzing a substantially larger cohort than those already reported. methods: we gathered and analyzed data on 144 patients from 56 centers world-wide with confirmed gof mutations in stat3. retrospective chart reviews were performed in accordance with all local ethics and irb committees to determine clinical manifestations, immunophenotype, treatment regimens, success of treatment methods, and overall survival. funcitonal transcriptional activity was assessed by luciferase reporter assay on each individual mutation. results: fifty-nine individual mutations were identified and all conferred gof by a validated luciferase assay. there were 5 mutations in the nterminal domain, 17 in the coiled-coil domain, 28 in the dna binding domain, 6 in the sh2 domain, and 3 in the transactivation domain with the overwhelming majority being missense mutations. median age at presentation was approximately 5 years; 56% of subjects are male and 44% are female. immunodysregulatory features presented in all patients. autoimmune cytopenias were the most common occurring in 72% of subjects (n=105), followed by lymphoproliferation in 69% (n=100) with increased frequencies of double negative (cd4-cd8-)t cells being found in 71% of of patients tested, enteropathy in 53% (n=76), endocrinopathy in 35% (n=50), interstitial lung disease in 45% (n=65), dermatitis in 41% (n=42), and inflammatory brain disease in 6.25% (n=9). growth failure was present in 54% (n=77) with half of those patients having concurrent enteropathy. infections were reported in 60% of the cohort to include recurrent and/or invasive viral, bacterial, opportunistic, fungal, and mycobacterial infections. prominent abnormalities of immunophenotyping included t cell (54%) and b cell (36%) lymphopenia with reduced t cell proliferation in response to mitogens or antigens in 30% of those evaluated patients. fifty-nine percent of the patients hypogammaglobulinemia while 40% exhibited poor specific antibody responses to recall antigens. overall survival was 86% at data collection.treatment of stat3 gof patients often included multiple agents: ivig , chronic and pulse steroids, mtor inhibitors, calcineurin inhibitors, rituximab, mycophenolate mofetil, alemtuzumab, tocilizumab, and jakinibs. those started on jak inhibition showed improvement in clinical symptoms and, to date, there are 20 stat3 gof patients on targeted jak inhibition. thus far, 18 patients have undergone bone marrow transplant with a 61% survival rate. discussion: stat3 gof mutations were first reported in 2014 to cause a heterogeneous syndrome of autoimmunity and lymphoproliferation with immunodeficiency and infection susceptibility. earlier treatment with targeted therapy such as jak inhibitors has led to reduced disease morbidity. we report the largest cohort of stat3 gof patients collected through a multi-national collaboration of the longitudinal data and natural history of stat3 gof disease. understanding the heterogeneity of presentation and key features that will lead to proper diagnosis and early treatment in an effort to prevent long term disease associated sequelae. we present the case of 5 month old male with a novel heterozygous mutation in tcf3 and two previously unreported phenotypes: 1) absent circulating cd19+ b cells yet preserved immunoglobulin synthesis and vaccine responses and 2) significant thrombocytopenia that improved with immunosuppression. there is also a striking family history of two half-sisters who died during early infancy with similar clinical and lab findings and the same genetic change. the infant boy was born at term with respiratory failure and generalized rash. at birth he had thrombocytopenia (24k/ul) and lymphopenia (465/ ul). initial absolute cd3+ t cell count was low (442/ul), yet he had normal thymic output and proliferative responses to mitogens ruling out scid. cd19+ b cells were < 5/ul and bone marrow biopsy revealed decreased hematagones, yet he had a normal igm level (23.1 mg/dl) elevated iga (56 mg/dl), and elevated ige (27 iu/ml). igg levels were initially obscured by maternal igg and ivig; in turn he made positive titers to diphtheria and tetanus vaccination. the infant has maintained his own igg production. rapid genome sequencing revealed a heterozygous predicted deleterious vous in tcf3, in the second transactivation domain (c.1138 c>t, p.pro380ser). the same change in tcf3 was identified in the deceased half-sisters as well as the father: all 3 infants had different mothers, suggesting autosomal dominant inheritance. the sisters had similarly severe thrombocytopenia and absent circulating b cells; their causes of death were not completely understood. the 33 year-old father has normal platelet levels, very low cd19+ b cells (35/ul), elevated igg (1,580 mg/dl) and ige (1,030 iu/ml), and normal levels of iga and igm. the father also has an elevated number of cd3+ t cells (3,455 /ul) with an increased percentage of t cells expressing hla-dr (46%). in the months after birth, the infant boy continued to require frequent platelet transfusions. despite the persistent t lymphopenia, there was evidence of increased t cell activation with elevated levels of soluble il-2r (2510 pg/ml) and increased percentage of cells expressing hla-dr (30%) cd95 (98%), cd25 (86%), cd71 (55%), and cd69 (14%). a 10-day trial of prednisone was associated with an increase in his platelet count to >100k/ul. he was switched to rapamycin as a steroid-sparing agent, and his platelet count has remained > 200k/ul for several weeks without transfusions. interestingly, his b cell counts also improved after the steroid trial (50/ul) and his absolute lymphocyte count is normalizing on rapamycin. a potential mechanism could be rapamycin decreasing t cellmediated destruction of platelets or b cells. reassessments of t cell activation markers and b cell phenotyping while on rapamycin will be done in the future. in contrast to multiple published cases of tcf3 mutations associated with complete agammaglobulinemia and absent b cells, we present a case of an infant with absent b cells yet preserved humoral function as well as severe thrombocytopenia responsive to rapamycin. in collaboration with colleagues at nih, studies are underway to understand whether/how the unique change in tcf3 is related to either phenotype described above. abstract/case report text background: childhood-onset, chronic, multi-system inflammatory diseases are increasingly being characterized as monogenic inborn errors of immunity. arpc1b deficiency is a recently described, rare combined immunodeficiency characterized by recurrent/severe infections, a variety of autoimmune manifestations and platelet defects. we describe a case of arpc1b deficiency identified in an adult patient with recurrent ulcers/ bechet-like disease, non-malignant lymphoproliferation and intermittent microthrombocytopenia. patient case: at 1 year of age, our female patient was diagnosed with behcet disease based on a history of bloody stools at 3 months, oral ulcers at 9 months and vulvar lesions at 1 year. she underwent rheumatology evaluations for inflammatory arthritis, episcleritis, eczema, vasculitic ulcerating nodules of the trunk, perineum and extremities, and verrucae forming flat plaques similar to epidermodysplasia verruciformis without a unifying diagnosis. other infections include otitis media, sinusitis, pseudomonas ecthyma gangrenosum, cervical lymphadenitis, and pneumonia. at 28 years old, the patient was referred to our immuno-hematology comprehensive program clinic with a concern for malignancy versus a primary immune regulatory disorder (pird). she had a 6-month history of drenching night sweats, urticarial plaques, edema in her extremities and diffuse cervical, axillary and inguinal lymphadenopathy. past complete blood counts showed intermittent mild microthrombocytopenia. lymph node biopsies were negative for a neoplastic process but identified plasmacytosis, including focally increased iga-kappa+ plasma cells. expert review of the lymph node biopsy, and further evaluation excluded multicentric castleman disease. consideration was also given to autoimmunune lymphoproliferative syndrome (alps)-like disorders; however, her alps flow cytometry panel was nondiagnostic. her basic immune evaluation showed severe t cell lymphopenia (cd3+ 229 cells/ cm, cd4+ 222 cells/cm, cd8+ 52 cells/cm) with adequate b and nk cells, normal lymphocyte proliferation to pha and pwm, and dysgammaglobulinemia with igg 1579 g/dl, iga 638 g/dl, igm 50 g/ dl and ige 592 g/dl. due to concern of an underlying pird, a primary immunodeficiency panel was sent for 207 gene analysis with negative results. however, trio clinical exome sequencing identified biallelic variants in the gene arpc1b. one allele has a truncating, nonsense pathogenic variant in exon 8 denoted as c.898g>t, p.glu300ter. the other allele has a likely pathogenic variant in intron 4 denoted as c.393-2a>g, resulting in disruption of the canonical splice acceptor for exon 5. this is predicted to cause exon skipping, with an in-frame deletion of amino acids coded by exon 5. conclusion: this case highlights the value for evaluation for pirds in patients presenting with behcet-like disease, particularly in the context of other autoimmune manifestations and/or microthrombocytopenia. it also underscores that patients with arpc1b deficiency may present with chronic non-malignant lymphoproliferation. moreover, this patient emphasizes the value of exhaustive genetic testing for complex immunologic phenotypes. abstract/case report text lipoyltransferase 1 gene defect is associated with severe mitochondrial dysfunction disrupting lipoic acid biogenesis. clinical manifestations associated with early seizures, hypotonia, cardiomyopathy and pulmonary hypertension and encephalopathy. early neonatal death due to sepsis and cardiovascular collapse is commonly seen. the patient is a 36 week preemie male with congenital heart disease who developed severe intractable lactic acidosis on day of life 1 with increased excretion on organic acids of 2-methyl-2,3dihydroxybutyric acid. a mitochondrial disorder , echs1 or hibch deficiency was suspected. at 3 mo of age the patient was admitted for apneic spells and respiratory compromise. he was found to have elevated crp associated with rhinovirus infection and gram-negative bacteremia. due to the history of failure to thrive and sepsis, immunology was consulted. immunologic work up indicated normal b, t and nk cells with normal dhr, but showed agammaglobulinemia. the patient was started on ivig and whole exome sequencing was done. molecular analysis showed compound heterozygote mutations in the lipt1 gene: c.293g>a (p.arg98gln) and c.635t>g (pval212gly). subsequent biochemical analysis also showed biochemical abnormalities consistent with lipt1 defect. lipoyltransferase 1 is an enzyme involved in activation of a number of enzymes requiring lipoic acid. it is involved in lipoic acid synthesis. lipoic acid is required for the activity of pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched-chain alpha-ketoacid dehydrogenase. the literature indicates that most patients with lipt1 defect have a severe, often fatal course. the patient is now almost 3 years old and has stable clinical course without any major infections. he certainly has significant hypotonia and developmental delay. in conclusion, we are presenting the first case of lipt1 gene mutations associated with agammaglobulinemia who responded well to ig supplementation therapy. our immunologic findings in this case highlights the importance of immunodeficiency work up in challenging cases. as we see more cases lipt1 gene mutations, we will better understand the clinical spectrum. abstract/case report text a now 8-year-old male was initially evaluated for concerns regarding food allergy, eczema, food protein-induced enterocolitis syndrome, and failure to thrive. he had reactions of varying severity to multiple foods. these usually involved immediate urticaria or prolonged vomiting, diarrhea, and abdominal pain. ige and skin prick testing was performed to suspected foods and was positive to milk, egg, pork, wheat, peanut, pecan, coconut and corn. these foods had historically caused reproducible immediate symptoms. testing was negative to other suspected foods. he developed an oral aversion and extremely restricted diet. symptoms of abdominal pain, hematochezia, rashes, arthralgias, headaches, fatigue, dyspnea, and palpitations increased. urticaria and severe abdominal pain with vomiting and diarrhea continued intermittently without identifiable triggers on a restricted diet. laboratory markers demonstrated elevated inflammatory markers, anemia, iron deficiency, vitamin b12 deficiency, and vitamin c deficiency (scurvy). gastroenterology work up did not identify any pathology. gastrointestinal symptoms did not respond to treatment with multiple gerd medications or oral steroids. baseline tryptase was elevated. low histamine diet was initiated and repeat tryptase remained elevated. fractionated tryptase revealed normal mature (beta) tryptase with elevated total tryptase, negative genetics for c-kit mutation, normal urine prostaglandins. family members had tryptase levels drawn. one parent and sibling had elevated tryptase levels, while the other parent's tryptase was normal. hereditary alpha tryptasemia syndrome is defined by elevated blood tryptase levels and symptoms involving multiple organ symptoms. patients with elevated tryptase levels without symptoms are defined as having hereditary alpha tryptase trait. there is significant variability regarding which patients are symptomatic. organ symptoms that may be involved include skin, gastrointestinal, neurologic, connective tissue, cardiac, neuropsychiatric. severe allergic reactions such as anaphylaxis can occur. increased blood levels of the protein tryptase are caused by extra copies of the alpha tryptase gene (tpsab1). treatment is usually directed at specific symptoms, antihistamines, and mast cell stabilizers. research continues into additional treatment options. this patient was started on cromolyn and long-acting antihistamine. his gastrointestinal symptoms and rash/urticaria improved, and he began tolerating a small, but increased, variety of foods. the majority of his constitutional symptoms of fatigue, arthralgias, weakness resolved as he began gaining weight, and hemoglobin, vitamin c and b12 normalized. his sibling was evaluated and noted to have food allergy, asthma, abdominal pain, gerd, and eczema. she was also started on cromolyn and antihistamines which improved her gastrointestinal symptoms. parent with elevated tryptase was recommended to be evaluated further with allergist. this is an example of a patient with elevated tryptase and multiple organ system involvement. some of his signs and symptoms responded to mast cell stabilizing and antihistamine medications. patients with history of recurrent episodes of allergic reactions to foods and multiple constitutional symptoms would benefit from baseline tryptase levels. family members should also be tested if the patient has elevated tryptase. multiple studies have been published looking at the rates of scid in the united states. the estimated rate of scid prior to screening was 1 per 100 000 live births. post screening implementation, on average rates of scid were found to be closer to 1 in 60 000 live births. results : development of a t cell receptor excision circles (trec) scid screen in alberta involved the screening of 4000 anonymous term neonates using quantitative pcr for trecs. the cycle threshold for the control gene, rnasep, was set at 30.5 as 95% of our population had a cycle threshold < 30.5 (90% ci [30.4,30.6]). from those bloods spots with adequate dna, a final trec cut off of 40 was chosen, as it would give an accuracy of 99.6%, and fairly low false positive rate of 0.4% (95% ci [0.002, 0.006]). since starting a population based screen for scid in june of 2019, we have identified 4 cases of scid and 13 cases of low trec not caused by scid. to date we have detected one case of reticular dysgenesis, 2 cases of ada scid and one case of x-linked scid. other causes of lymphopenia in the neonatal period detected with abnormal trecs include one syndrome associated with variably affected cellular immunity (charge) and 12 cases of secondary lymphopenia including four cases of prematurity, three cases of diaphragmatic hernia or gastroschisis, four patients with underlying cardiac disease, and one patient with severe hydrops. discussion : canada has multiple unique populations with increased risk of scid. the estimated rate of scid in canada prior to implementation of a population based screen was 1.4 per 100 000 live births. the rate within canada's first nations, métis and inuit populations is 4.4 per 100 000 live births. prior to scid screening, alberta had 13 cases of scid identified between 2005-2014 with an estimated rate of 1 per 60 000 live births. to date, our screen in alberta has identified 4 cases of scid with a rate of 1 per 7000 live births which is significantly higher than previously estimated. given that early diagnosis and definitive management through bone marrow transplant or gene therapy has been shown to reduce mortality this screen will help reduce morbidity and mortality in this vulnerable population. abstract/case report text introduction: human herpesvirus 6 (hhv-6) has the ability to integrate its genome into host telomeres. if this integration occurs in gametes, then the virus can be genetically transmitted and offspring will carry a copy of chromosomally integrated hhv-6 (cihhv-6) in each somatic cell. this can lead to false attribution of infectious and non-infectious presentations of hhv-6, and make the diagnosis of active hhv-6 infection difficult. we present the case of a patient with meningoencephalitis attributed to hhv-6 and persistently elevated blood levels of hhv-6 by pcr concerning for primary immunodeficiency who was discovered to have cihhv-6. case description: a 7-year-old female who carried a past diagnosis of hhv-6 meningoencephalitis was seen in immunology clinic for follow up of persistently elevated levels of hhv-6 dna in her blood by pcr. she was born at 35 weeks and as an infant had failure to thrive (ftt), anemia and a varicella like rash after varicella immunization. at the age of 3, she received flumist vaccine and developed a fever the following day. over the next few days, she developed lethargy, altered mental status, headache, photophobia, seizure, papular rash and oral ulcers. she was admitted to the hospital and csf studies were consistent with viral meningoencephalitis (270 wbc, 30l, 63m, 100 rbc) although hsv, cmv, ebv and enterovirus were negative. she was treated for presumed hsv encephalitis with 21 days of iv acyclovir and 5 days of high dose steroids. one week after discharge, she again developed papular rash on feet, headache, oral ulcers and lethargy. she was admitted and csf studies this time showed only 3 wbc but positive hhv-6. blood and skin swab were also positive for hhv-6. immunology was consulted while admitted and work up for primary immunodeficiency was initiated. her work up was normal including responses to vaccine titers, complement studies, moderate in t cells, monocytes, neutrophils, lung and muscle, and low in skin, liver, heart and kidney tissues. analysis of each individual gene expression level by nanostring in comparison with healthy controls demonstrated significantly higher levels of the following irgs: ddx60, epsti1, gbp1, ifi6, isg15, ly6e, oas1, oas2, oas3, rsad2, rtp4 and socs1. whole blood rna-seq was performed in 3 patients and pathway analysis with the differentially upregulated genes demonstrated an enrichment of intraluminal vesicle formation and negative regulation of apoptotic signaling pathways. stimulation of pbmcs with the tlr ligands poly i:c, odn, and lps induced a 200fold increase in ifi27 and a 30-fold increase in ifna1 and ifnb1 transcription compared to baseline. one patient had constitutive upregulation of stat1 and stat6 in monocytes and of stat1 and stat3 in t cells. conclusion: we describe a novel immunedysregulatory disease caused by de novo truncating variants in samd9l that presents similar to candle with neutrophilic pannicultis and points to an important role of samd9l on regulation of adaptive and innate immune responses. acknowledgements: this work was supported by the nih irp of niaid abstract/case report text introduction: ataxia telangiectasia (at) is caused by a defect in the atm gene which is responsible for repair of damaged dna. it is a rare, devastating neurodegenerative disease that results in ataxia and telangiectasias, particularly of the sclera and skin. those with at are at increased risk for immunodeficiency and cancer. the immunodeficiency is variable and may result in deficiency in humoral and cellular immunity in some patients. here we present a patient with ataxia telangiectasia and hypogammaglobulinemia on immunoglobulin therapy who developed recurrent urinary tract infections (uti) and sepsis. case presentation: the patient is a 23-year-old female with ataxia telangiectasia and hypogammaglobulinemia who presented with three episodes of uti, one of which resulted in prolonged hospitalization due to sepsis and acute kidney injury (aki). she presented with 4 days of flank and back pain and was hospitalized for 3 days for e coli uti. she improved on iv antibiotics and was discharged home to complete treatment with oral ciprofloxacin. due to persistent emesis, she was readmitted 2 weeks later with urosepsis and aki with a creatinine of 2.06 mg/dl, over 5 times her baseline creatinine. after additional antibiotics and iv fluids, she improved clinically, renal function normalized and she was discharged home. renal ultrasound was unremarkable with no anatomical abnormalities. she was relatively healthy prior to this with only one episode of bacterial pneumonia in 2006. she receives weekly subcutaneous immunoglobulin therapy dosed at 120 mg/kg with normal igg levels (1023, 931, 1078 mg/ dl). at baseline, she had high igm (550 mg/dl) and low iga ( < 4 mg/ dl) levels, as well as decreased t cells but normal nk and b cells (489 cells/ul cd3+, 329 cells/ul cd4+, 107 cells/ul cd8+, 211 cells/ul cd16+cd56+, and 357 cells/ul cd19+). she has hyperglycemia (on metformin), hypertriglyceridemia (on atorvastatin) and hypertension (on losartan). she is thin and wheelchair bound with bilateral telangiectasias to the sclera, neck, and chest. she has occasional eye bleeding and epistaxis, presumably from her telangiectasias. she has good hygiene and good adherence to medications. she voids voluntarily, has no indwelling urinary catheter and is not sexually active. discussion: patients with ataxia telangiectasia may have frequent viral and bacterial infections, most frequently upper and lower respiratory tract infections, as well as wart and skin infections. based on our review, this is the first reported case of ataxia telangiectasia with hypogammaglobulinemia on immunoglobulin therapy with recurrent utis complicated by urosepsis and aki. despite adequate igg levels on immunoglobulin therapy, our patient continued with recurrent utis. it is uncertain whether her non-ambulatory status, hyperglycemia or related immunodeficiency are the causes for her increased susceptibility to utis. the literature reports patients with at and bladder wall telangiectasias can result in significant hematuria, and perhaps this may be a source of entry for bacteria and consequent development of uti. this suggests that patients with ataxia telangiectasia and recurrent utis may benefit from renal ultrasound and possible cystoscopy to better visualize telangiectasias. we recommend consideration of workup for recurrent utis in patients with ataxia telangiectasia. abstract/case report text objectives: patients with partial rag deficiency frequently present with humoral autoimmunity suggesting breach in tolerance mechanisms and subsequent expansion of autoreactive b cell clones. here we aim to trace polyreactive b cells and their descendants at b cell developmental stages through our in-house bioinformatic pipeline, immchaintracer (ict). methods: the b cell receptor (bcr) was expressed as monoclonal antibodies from single sorted mature naive b cells (n=30-50 per donor) from patients with hypomorphic rag deficiency and healthy donors. xthe recombinant monoclonal antibodies were screened for polyreactivity (dsdna, insulin, lps and ifnα) by elisa. in parallel, igh repertoires were deep sequenced from sorted mature naïve, activated naïve and memory b-cell compartments. our in-house assembled bioinformatic pipeline called immchaintracer (ict) was applied to track down the descendants of cloned autoreactive igh sequences in repertoires of subsets above. results: igh sequences (n=125 including 45 polyreactive, 80 nonpolyreactive clones) from mature naive b cell from six patients with partial rag deficiency and 3 healthy donors (n=91 including 7 polyreactive, 84 non-polyreactive) were analyzed with our novel inhouse bioinformatic approach to track lineage fate in repertoire at specific developmental stages. interestingly, 28.8% of the patients' sequences and their descendants were identified in their mature naive, activated naive or memory b cell repertoires, while none of the analyzed healthy donor clones were found at later subsets. furthermore, genealogical analyses of related clones revealed lineage expansion and progressive positive antigen selection of the autoreactive clones in the patients. conclusions our findings demonstrate that peripheral tolerance checkpoint is broken in hypomorphic rag patients. our novel method enables tracing the fate of autoreactive naive b cells in the effector repertoires. we have shown that impaired b cell tolerance allows the expansion and combination of sirolimus and rituximab therapy controlled his autoantibody production which was an important goal for his autoimmune condition. we present a new treatment approach for anti-nmda receptor encephalitis. rituximab was tried on these cases before but the combination of sirolimus and rituximab therapy was never given before. we now recommended that on refractory cases of anti-nmda receptor encephalitis, combination of rituximab and sirolimus therapy can be tried. (247) submission id#812639 mosaic variants in immune function genes identified through exome sequencing stable with no interim infections and is doing well on thyroid hormone replacement. the current plan is for the patient to undergo a stem cell transplant for the arpc1b deficiency as he is at high risk for recurrent infections and severe disease. although this gene mutation is rare, review of the current literature describes patients with this condition that have undergone stem cell transplant and have done well. at this time, this seems to be the best option for management, and it may potentially be curative. abstract/case report text introduction: common variable immunodeficiency (cvid) is a disorder characterized by impaired immunoglobulin production and frequent or recurrent infections, but also associated with an increased risk for developing malignancies such as lymphomas. although intravenous and subcutaneous immunoglobin g replacement has been successful in reducing the number of bacterial infections and prolonging survival, it fails to address other complications that arise from this disorder. we report a case of a patient with cvid who developed mycosis fungoides (mf). mf is a rare form of cutaneous t-cell lymphoma, occurring in about 1 in 100,000 to 350,000 individuals, lack of treatment could potentially be fatal. case presentation: a 44-year-old caucasian woman with a history of ulcerative colitis, allergic rhino-conjunctivitis and tonsillectomy was referred to the immunology clinic for evaluation of low serum immunoglobulins. there was no family history of infections or immune deficiencies, but paternal grandfather had colon cancer and maternal grandmother had lung cancer. the patient reported frequent episodes of bronchitis, and sinus infections. immunizations were up to date for her age. medications included azathioprine, cetirizine, fluticasone nasal spray, hyoscyamine, montelukast, lactobacillus, omeprazole, and olopatadine ophthalmic solution. no history of frequent use of systemic steroids. initial serum immunoglobulins revealed normal ige and igm but low iga (72 mg/dl, normal range 81-463 mg/dl) and low igg (522 mg/dl, normal range 694-1618 mg/dl). cbc with differential, lymphocyte subsets, c3 and c4 levels were normal. while she had adequate protective titers against haemophilus influenza type b, diphtheria and tetanus, titers against pneumococcus were < 50% protective and she failed to mount an adequate response to pneumococcal polysaccharide vaccine. given her diagnosis of cvid, she started scig (500 mg/kg every 2 weeks). a year later, she developed a bilateral nonpruritic rash in the abdomen and upper trunk. initial skin biopsy suggested a drug reaction. a subsequent biopsy revealed a superficial perivascular lymphoid infiltrate with focal epidermotropism and positive t cell receptor gamma gene rearrangement, consistent with mf. testing for cell t receptor beta gene rearrangement was negative. while mf treatment consisted of triamcinolone 0.1% ointment, treatment for ulcerative colitis transitioned from azathioprine to vedolizumab. conclusions: cutaneous t cell lymphomas, although uncommon, can be seen in cvid. there are several reasons for the increased risk of lymphoma in cvid. the role of chronic infections and the development of lymphoma as of yet, is not clear. skin reactions to scig products in the areas of infusion are relatively common and resolve promptly. high index of suspicion is crucial in obtaining tissue sample to confirm or rule out malignancy therefore avoiding delaying proper treatment. abstract/case report text patients with lipopolysaccharide responsive beige-like anchor protein (lrba) deficiency present with a plethora of immune related defects including a defective humoral response characterized by low numbers of switched memory b cells and plasma cells, as well as an impaired production of antibodies, leading to recurrent infections. however, the molecular mechanisms behind the defective b cell response remain unknown. to gain better insights into the possible roles of lrba in b cell physiology, we screened for lrba-interacting proteins using computational predictions. twenty-seven proteins involved in vesicle trafficking and autophagy were identified as potential lrba-interacting partners. to validate those potential lrba interactions, we performed coimmunoprecipitations and proximity ligation assays (pla), finding that endogenous lrba interacts with the phosphoinositide 3kinase regulatory subunit 4 (pik3r4) in b cells. pik3r4 (aka vps15) is the regulatory subunit of vps34, the catalytic subunit of the pi3k-iii complex, which acts as a positive regulator of autophagy by producing phosphatidyl inositol-3 phosphate (pi(3)p). autophagy is a catabolic mechanism essential for cell survival and plasma cell differentiation. in fact, we observed that reduced lrba impaired the production of pi(3)p upon autophagy induction. in addition, we observed in both lrba-deficient hela and b cells reduced mobility, abnormal accumulation and increased size of autophaghosomes, accompanied by an atypical lysosomal positioning. these abnormalities are due to a blockade of the autophagosome-lysosome fusion, as detected by reduced lc3-ii lipidation upon autophagy induction in the presence of lysosome inhibitors. interestingly, lrba-deficient hela and b cells exhibited enhanced activity of mammalian target of rapamycin complex 1 (mtorc1) signaling, a key suppressor of autophagy whose activation possibly contributes to defective autophagy. taken together, b lymphocytes lacking lrba can form autophagosomes but they fail to fuse with lysosomes. thus, we propose a role of lrba at late stages of autophagy through the binding to pik3r4. abstract/case report text apds caused by gain-of-function mutations (gof) in the genes (pik3cd and pik3r1), encoding for the p110δ and p85 subunits of phosphoinositide 3-kinase δ (pi3kδ), results in hyperactivation of the pi3k/akt/mtor/s6k pathway and lead to immune dysregulation, lymphoproliferation and immunodeficiency. apds manifests with respiratory tract infections, bronchiectasis, susceptibility to herpes group viruses, autoimmunity, cytopenia, lymphoproliferation and lymphoma. gastrointestinal system manifestations include enteropathy, colitis, and liver disease. eosinophilic esophagitis (eoe) or eosinophilic gastrointestinal disease (egd) have been under diagnosed in reported apds cohorts. objectives: to review the incidence, demographics and relevant clinical data for eosinophilic gastrointestinal disease in a single center apds cohort. methods: review of clinical and laboratory findings from 70 apds patients followed at the nih clinical center, from 2005 to 2019. results: 12 patients were either historically diagnosed or actively studied at our center for egd. incidence of all egd is 17 % in our cohort and all patients had mutations in pik3cd, none in pik3r1. most patients also had multiple gi manifestations. conclusion: immunopathology and genetic predisposition leading to eoe is complex. eosinophilic gi disease including eoe appears to represent significant gi pathology in apds. this implies that activation of pi3k pathway may be directly involved in the etiology of eoe. abstract/case report text introduction: dominant negative mutations in stat3 (lof stat3; job's syndrome) cause a primary immune deficiency characterized by eczema, recurrent skin and lung infections, and connective tissues and skeletal abnormalities. over the last several years, vascular abnormalities causing tortuous and aneurysmal middle-sized arteries have increasingly been recognized. our institution has been imaging prospectively the coronary and cerebral arteries since 1999 -2019 for brain imaging and from 2004-2019 for heart imaging. the purpose of this review is to provide an update on the extent of clinical manifestations noted in hies (continued) pain improved but rapidly worsened with tapering of steroids. the pet/ ct at that time, showed extensive hypermetabolic areas in lymph nodes, femurs, left acetabulum, left pubic ramus, right ischium, sacrum, both iliac bones, eight rib, t4 and t8, both clavicles, both humeral, manubrium, and extension into musculature. initial biopsies were culture negative, 16s was positive for mac. she was referred to the national institutes of health where she was diagnosed with autoantibodies to interferon-γ . prior to referral, she initially was treated with azithromycin, ethambutol (need to discontinue due adverse effects), amikacin, rifampicin, linezolid, with not no evidence of clinical response. she underwent debridement of epidural anterior abscess to t8. surgery involved t8-9 laminectomy, and curettage on several bones. she presented unable to walk secondary to pain and neuropathies. initial laboratory: crp:90mg/l; wc 13.51; hgb: 8.2g/dl; ana :4.6(strongly positive) cd20: 373 ul her first course of rituximab consisted of 7 doses of 1gm at d0, 14, 42 and monthly thereafter for a total of 7 doses with clinical and radiographic improvement. she was maintained on optimal antibiotics therapymeropenem, rifampin, azithromycin, moxifloxacin, and clofazimine. two years after she completed rituximab, she presented to her home hospital with increased left hip pain and biopsy grew mac. retreatment with rituximab failed to show clinical improvement. her medical regimen was augmented with tedizolid and bedaquiline, however no iv antibiotics were added. rituximab was reinitiated, after the progression of symptoms despite treatment with rituximab; bortezomib, was trialed using the schedule based on the multiple myeloma literature. she completed 5 full cycles (two at the nih, three at home). she subsequently has had clinical improvement and is working again and has not had progressive neurologic decline. the titers of antibodies to interferon-· did not follow the clinical improvement. however, we plan to keep her cd20+ cells zero and continue the bortezomib given her clinical and radiologic improvement. abstract/case report text for patients with primary immunodeficiencies (pid), finding a genetically defined diagnosis can be critical for prognosis, treatment, and counseling. however, for many patients, determining a genetic etiology remains elusive despite routine gene panel and exome sequencing because of an inability to resolve variants of uncertain significance (vus). crispr-based genome editing could be used to address this need by introducing patient-derived vus into primary human immune cells for further study; however, existing techniques are limited by poor efficiency. we recently developed novel non-viral techniques for large gene editing in primary human t cells and hematopoietic stem cells (hscs). we achieve up to 8fold greater efficiency than existing tools using crispr cas9 ribonucleoprotein nanoparticles that are non-covalently linked to homology directed repair (hdr) template dna. whereas mutation analysis has previously been limited to expensive and time-consuming animal models and transformed cell lines, we now have the ability to rapidly recreate any mutation in the native gene locus in otherwise healthy primary human cells. we demonstrate this ability by using our technique to knock-in well characterized loss-of-function mutations in jak3 and il2rg and gain-of-function mutations in jak3 that are known to cause severe combined immunodeficiency and tumor growth, respectively. we show that these recreated mutations have the expected effects on t cell proliferation and intracellular signal transduction in the setting of il-2 stimulation. we then use our technique to investigate a prototype case of an adult patient with the unusual combination of common variable immunodeficiency, inflammatory arthritis and uveitis, and neutrophilic urticaria. genetic testing in this patient had previously revealed heterozygous coding vus's in four genes previously associated with a pid disease, including jak3, but none of the specific variants have been previously reported. it is thus not clear which mutation (if not more than one) causes this patient's dysregulated cell activation, which limits targeted treatment options with kinase inhibitors or future gene or cell therapy. by knocking our patient's jak3 variant into primary human t cells and comparing these cells to those carrying wild-type, known loss-of-function, and known gain-of-function mutations, we are able to rapidly characterize the functional impact of our patient's variant and isolate its effect on his complex phenotype. this in vitro genetic engineering approach thus allows patient-specific vus to be modeled directly in primary human immune cells with a rapid turnaround time that is relevant for clinical applications, including molecular diagnosis and screening of pharmacologic or gene therapies. further, similar strategies could be leveraged as a potential basis for future gene correction therapy. abstract/case report text definition : "at variants" comprise a heterogeneous group characterized by the later onset of clinical symptoms, a slower progression, a prolonged lifespan compared to most patients with at and decreased levels of chromosomal instability and cellular radiosensitivity. in these patients, telangiectasia and / or immunodeficiency may be absent, while neurological features are present.(1). material and methods :a 4 years old girl, born out of a non-consanguineous parents with clinical picture consisting in progressive alteration of the march (15 months), associated to exotropia, no weight gain or height, alteration of balance while she is sitting, she walks by herself. she has 1 acute bronchiolitis. results and discussion: brain magnetic resonance without alterations. abdominal ultrasonography and cpk normal. ophthalmologist assessment found exotropia, he did not find telangiectasia. motor and sensitive neuro-conduction were reported normal. alpha fetoprotein 102 ng / ml increased. karyotype xx, non-structural alterations. normal auditory-visual evoked potentials. whole exome sequencing (wes): identified the small homozygous pathogenically deletion ¬c.5496 + 2_5496 + 5del taag; p.? have a spelling effect in the splicing. it is not present in the population database or not as a known variant in function mutation of smad4 has been shown to increase smad phosphorylation in the nucleus in fibroblasts. further research is needed to examine the role of this mutation in t and b lymphocytes, given the interesting immunological phenotype of this patient. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. outbreak of mycoplasma pneumoniae-associated stevens-johnson syndrome characterization of children. with recurrent episodes of stevens johnson syndrome recurrent stevens-johnson syndrome secondary to mycoplasma pneumoniae infection recurrence and outcomes of stevens-johnson syndrome and toxic epidermal necrolysis in children syndrome after mycoplasma pneumoniae infection a review of causes of steven-johnson syndrome and toxic epidermal necrolysis in children submission id#812369 expanded phenotype and genotype-phenotype correlations bsc(hons) msc bmbs mrcp 6 dominant activating rac2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects an autosomal dominant scid form due to a gain of function mutation in the rac2 gene national institute of allergy and infectious diseases (niaid) professor biochemistry/molecular pharmacolog/nyu langone health canada 5 head of the ircm bioinformatics core facility/montreal clinical research institute (ircm) associate investigator/national human genome research institute (nhgri) division of intramural research (dir), national institute of allergy and infectious diseases (niaid) translational and functional genomics branch/chief and senior investigator of the translational and functionanational human genome research institute (nhgri) division of intramural research (dir), national institute of allergy and infectious diseases (niaid) formula versus donor breast milk for feeding preterm or low birth weight infants cytomegalovirus (cmv) inacɵvaɵon in breast milk: reassessment of pasteurizaɵon and freeze-thawing prevenɵon of cytomegalovirus transmission via breast milk in extremely low birth weight infants submission id#812554 the costa rican registry for primary immunodeficiencies department chief/department of pediatric immunology and rheumatology, national children´s hospital "dr. carlos sáenz herrera national children´s hospital "dr. carlos sáenz herrera" cid 13% (n=38), cid with syndromic features 54% (n=161, at 132), antibody deficiencies 13% (n=38, xla 15) ada deficiency, 1 cd40l-, 2 flh-4 (syntaxin 11 deficiency), 1 ipex and 8 osteopetrosis. nineteen patients developed malignancies, mainly non-hodgkin lymphomas among at patients conclusions: during the study period, 298 pid were registered, mainly at and osteopetrosis cases. scid represented 12% and xla 11% of patients allergy and immunology/ department of pediatric immuunology and texas children's hospital director immunogenetics program/department of immunology, allergy and rheumatology at baylor college of medicine 8 associate professor, director food allergy program/department of pediatric immuunology shearer center for human immunobiology 10 associate professor/department of pediatric immuunology, allergy, and retrovirology granuloma faciale: successful treatment of nine cases with a combination of cryotherapy and intralesional corticosteroid injection intralesional steroid injection: a novel method to treat the symptoms of idiopathic granulomatous mastitis long-term effectiveness of intralesional triamcinolone acetonide therapy in orofacial granulomatosis: an observational cohort study sarcoidosis in the periocular scar as the first finding of systemic sarcoidosis: clinical-radiological characteristics a case of scar sarcoidosis of the eyelid submission id#812563 null mutations: a gene dosage effect shubham goel, phd 1 , hye sun kuehn clinical centre, nih, 2 staff scientist/immunology service certified molecular geneticist/national institute of allergy and infectious diseases, national institutes of health genetic counselor/national institute of allergy and infectious diseases, national institutes of health division of intramural research; chief, immunopathogenesis section/laboratory of consultant/university hospital freiburg center for pediatrics and adolescent medicine cci -center for chronic immunodeficiency 16 medical director professor/division of blood and marrow transplantation and cellular therapies new south wales, australia. 23 professor of medicine and pediatrics/icahn school of medicine at mount sinai 24 senior investigator/laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health 25 functional nk assay, t/b/nk panel, tlr assay phd 11 staff scientist/translational autoinflammatory disease section, lcim, niaid, nih, bethesda, md 2 research fellow/translational autoinflammatory disease section post-baccalaureate fellow/translational autoinflammatory disease section pediatric rheumatologist/department of pediatric infectious diseases and immunology shupyk national medical academy for postgraduate education alder hey children's nhs foundation trust hospital, liverpool, uk 13 pediatric rheumatologist/department of pediatrics dra. liliana bezrodnik y equipo dra. liliana bezrodnik y equipo"-servicio de inmunología htal. de niños hospital de niños ricardo gutiérrez 19 pathology department/children's hospital of philadelphia, philadelphia, pa 20 post-doctoral fellow/translational autoinflammatory disease section results: we identified 6 patients with 4 de novo frameshift variants in samd9l (c.2626dela, p.i876lfs*15 reduced toxicity allogeneic hct with a busulfan, fludarabine regimen: a promising approach for non-cgd primary immune deficiencies requiring myeloablation? phd 2 , blachy davila saldana college of medicine 2 assistant professor/division of bone marrow transplant, immunedysregulation and immuno-hematology program, aflac cancer and blood disorders center 6 instructor of pediatrics/division of bone marrow transplantation and immune deficiency, cincinnati children's division of bone marrow transplantation and immune deficiency, cincinnati children's national institutes of health 2 certified molecular geneticist/national institute of allergy and infectious diseases genetic counselor/national institute of allergy and infectious diseases, national institutes of health operations manager and genetic counselor/national institute of allergy and infectious diseases collaborative bioinformatics resource/national institute of allergy and infectious diseases genome in a bottle analysis team/genome in a bottle consortium division of intramural research (dir), national institute of allergy and infectious diseases (niaid) medical genomics and metabolic genetics branch/national human genome research institute, national institutes of health table 1 clinical and demographic characteristics, severity measures, and previous treatments for the pid cohort geographic region, n (%) north central 66,736 (17.5) 406 (15.6) 3,022 (19.7) northeast 69,027 (18.1) 302 (11.6) 2,179 (14.2) south 9) 601 (23.1) 4,497 (29.3) high-potency oral antibiotics 130,153 (34.1) 967 (37.1) 4,720 (30.8) systemic high-dose corticosteroids copd, chronic obstructive pulmonary disease cd4 / cd8: 2.73 , lymphocytes b 161 , natural killer 314 cell/mm3. conclusions: although the atm activity corresponding to this new splicing mutation is unknown, it is presumed that it has some residual function, since splicing mutations is associated with better neurological prognosis have been reported (pidd) . at the immunodeficiencies research unit we discuss and pursue the molecular and genetic diagnoses for patients with suspected pid from all around the country. starting this year, we are processing and analyzing our own patients' wes results at the unit. evaluating the diagnostic yield of wes, as a measure of effectiveness or quality control, may result in process optimization and perhaps allow for better patient selection and resource allocation. objective: to describe and characterize our patients with suspected pidd whose dna samples were sent out for whole-exome sequencing; to analyze and compare our wes diagnostic yield after the first 3 batches of patients; to identify patient attributes that may predict a positive diagnostic wes result. methods: genomic dna was obtained from whole-blood samples of patients with suspected pidd from hospitals in mexico city, monterrey and puebla. wes was performed using a ng sequencer (illumina hiseq) in new jersey (admera health, llc), with 90% coverage and a 50x depth of the idt xgen library, human genome version 38 (december 2013). two fastq files for each patient sample were transferred back to our unit, where the bioinformatic workflow was completed. we used galaxy in the cloud for quality control, mapping & alignment, and detection of variants; variant effect predictor to process, map, annotate and filter variants; and igv (broad institute) and genome browser (ucsc) for visualization. we defined diagnostic yield as the proportion of patients with a genetic diagnosis after analysis of their wes results. we performed multivariate logistic regression, tree partitioning algorithm and linear abstract/case report text a 15-year-old male with x-linked chronic granulomatous disease (cgd) complicated by severe perianal disease and proctocolitis presented with two weeks of open draining lesions on the thighs, bilateral inguinal regions, and gluteal cleft. the wounds became excruciating and prevented normal ambulation. the patient was admitted for iv antimicrobial therapy, local wound care and systemic steroids. wound cultures, throughout his course, yielded growth of klebsiella pneumonia, candida parapsilosis, malassezia globose, escherichia coli, enterococcus faecalis and staphylococcus epidermidis allowing for directed antibiotic and antifungal therapies. despite improvement, the wounds persisted after several weeks of treatment. faced with recalcitrant cutaneous lesions despite aggressive systemic and topical therapies, we looked to alternative options. noting that other granulomatous diseases show response with intralesional corticosteroid therapy, we considered this for our patient (1, 2) . for example, patients with idiopathic granulomatous cheilitis had a complete response after three monthly injections of intralesional corticosteroids (3) . sarcoidosis patients also improve with intra-granuloma corticosteroid injection (4, 5) . our patient received 20 mg triamcinolone acetonide injections two separate occasions, administered in multiple open lesions at eight-week intervals. the cutaneous lesion improvement was gradual and complete resolution of the first open wound was noted fifty-two days from initial steroid injection. to our knowledge, intralesional glucocorticoid therapy has not previously been used to treat cutaneous disease in cgd patients. we are reporting the first cgd patient with successful lesion resolution following steroid injection as part of therapy. as such, we believe this case is significant and suggests that direct lesion injection with glucocorticoids can add to treatment options for cgd patients with recalcitrant cutaneous disease. serum igg, alone or in combination with iga and/or igm, were reduced in all patients. t-follicular helper cells were reduced only in patients carrying biallelic mutations (a.ii.1, a.ii.2 and a.ii.3) but not in any patients with monoallelic tcf3 null mutation. t cell enumeration and function by means of proliferation was normal in all mutation+ individuals. no mutated (truncated) protein expression was detected from patients with either biallelic or monoallelic tcf3 null mutations. however, wildtype tcf3 protein was detectable in about half amount in heterozygous patients. cdna data showed either 0/100 or 50/50 wt/mutated transcripts ratios in homozygous or heterozygous individuals, respectively, suggesting mutated proteins instability; and all together, protein haploinsufficiency for the heterozygous cases. ex-vivo, cd40l and il21-induced plasmablast differentiation was found to be reduced in 4/5 patients tested (1 biallelic patient a.ii.2 and 3 monoallelic patients a.i.2, b.i.1 and c.i.1). moreover, decreased igg, iga and igm production in vitro correlated with reduced plasmablast cell differentiation.in conclusion, all individuals carrying either mono-or biallelic null mutations have immunological penetrance of the b cell defect. however, while clinical penetrance was complete in patients with biallelic mutation, it was partial for those with monoallelic tcf3 null mutation suggesting a gene dosage effect for clinical penetrance. in addition, our study emphasizes that tcf3 is relevant to the plasmablast differentiation process as well as for ig production. further studies are being conducted to evaluate the individual roles of e12 and e47 on the immune and clinical features. background: heterozygous gain-of-function (gof) mutations in the stat1 gene result in a hyperphosphorylated state where patients develop recurrent or persistent chronic mucocutaneous candidiasis (cmc), other cutaneous mycosis, bacterial infections, disseminated dimorphic fungal infections and autoimmune disease. furthermore, the nk defect we characterized illustrated an immature cd56 dim nk cell subset with decreased expression of cd16, perforin, cd57, and impaired cytotoxic capacity associated with increased susceptibility to viral infections observed in these patients. methods: in this study, we evaluated 2 patients with novel stat1 mutations (d292e mutation, located in coiled-coil domain and k388q mutation, located in dna-binding domain). a third patient with the previously reported v266i mutation (ccd) was also recruited for this study. in vitro, pbmcs from these patients were stimulated with ifn-α for 30, 60, and 120 minutes and levels of phospho-stat1 on cd56 dim nk cell subset were measured by flow cytometry. the stat1 activity (firefly and renilla luciferase activities) was evaluated in u3a-stat1 deficient cells abstract/case report text background: t cell lymphopenia associated with genetic syndromes can be identified with low t cell receptor excision circle (trecs) up on the newborn screen for severe combined immune deficiency (scid). jacobsen syndrome (js), 11q terminal deletion, is a rare genetic disorder seen in 1/100,000 births characterized by facial dysmorphisms, platelet abnormalities, neurologic complications, immune system abnormalities including t and b cell defects. we report an infant with js found to have low trecs on nbs and review the immune phenotypes of our cohort of 7 patients with js. methods: a retrospective chart review of all patients with js seen by the allergy immunology service at a large tertiary referral center from 12/1/ 14-12/1/19 was performed in accordance with irb standards. result: the index patient had two newborn screens 24 hours after birth and two weeks later in accordance with texas state law. the first nbs resulted normal trecs while the second nbs had low trecs. a third nbs was done per protocol and again showed low trecs. subsequently, lymphocyte subsets at 2 months of age showed severe t-cell lymphopenia: cd3 575 cells/ dl (61%), cd4 351 cells/dl (21%), cd8 208 cells/dl, and low recent thymic emigrants (cd4+cd45ra+ccr7+cd31+) 42 cells/dl (12%) with normal lymphocyte mitogen proliferation. a chromosomal microarray (cma) revealed a 11q deletion known to cause js.over the five year study period we evaluated seven patients with js referred to our center. the majority of patients (85%) presented to clinic with history of recurrent infections including recurrent pneumonia, sinusitis, otitis media, skin abscesses and warts. t-cell lymphopenia was found in 3 of 7 (43%), 2/7 (29%) had abnormal lymphocyte proliferation (mitogens and antigens) and 2 met criteria for pjp prophylaxis. in addition, 4/ 7 (57%) had antibody deficiency requiring igg replacement therapy. of the 7 cases reviewed, only 2 patients were born during the period of time that texas was performing the nbs. conclusion: jacobsen syndrome can present with a spectrum of immune defects most notably t cell lymphopenia and antibody deficiency. these patients can present at birth with low trecs. this cohort analysis highlights the importance of considering chromosomal genetic syndromes with features of primary immunodeficiency in evaluating patients with low trecs. further evaluation of larger cohorts gathered from neurology or genetics clinics at multiple centers would be helpful for future study in identifying those who need close immunology care. abstract/case report text introduction: recognized as sentinels of the immune system, mast cells (mcs) and dendritic cells (dcs) are both derived from hematopoietic/ progenitor stem cells in bone marrow (bm). the crosstalk and direct contact between these cells have been well documented and play an important role in modulating immune response. we showed that presence/absence of il-3 directs cell fate; whether progenitor cells will be differentiated into mcs or dcs. we also report an easy method in which in vitro generation of dcs is possible without external induction of gm-csf or il-4. material-methods: to produce mcs and dcs in vitro, briefly bm samples were collected from patients with idiopathic thrombocytopenic purpura., bm mononuclear cells (mncs) were seperated by ficoll gradient, and seeded in plates with imdm medium containing fbs 2%, pen/ strep, and a little amount of methocult, and incubated in 37oc, 5% co2 (day 0). the treatments on the following days were as follows: day 4, imdm (fbs 1%) + scf (100ng/ml) + il-6 (50ng/ml); day 9, imdm (fbs 2%) + scf (100ng/ml) + il-6 (50ng/ml) + il-3 (1ng/ml). on day 18, 3 groups were formed: group i: imdm (fbs 2%) + scf (100ng/ml) + il-6 (50ng/ml) + il-3 (30ng/ml), group ii: imdm (fbs 2%) + scf (100ng/ml) + il-6 (50ng/ml), group iii: dmem + fbs 10%. the cultures were evaluated every 2 days under an inverted microscope. verification of mcs was performed by toluidin blue and tryptaseimmunoflorescence staining. macrophages were verified by cd-68 immunoflorescence staining. dendritic cells of different stages of abstract/case report text introduction a reduced toxicity busulfan, fludarabine regimen with alemtuzumab or anti-thymocyte globulin (gungor et al.) was efficacious in patients undergoing allogeneic hct for cgd. we report our experience with a similar approach for patients with non-cgd primary immune deficiencies needing a reduced toxicity myeloablative approach. methods we retrospectively reviewed records of consecutive patients who underwent allogeneic hct for primary immune deficiencies with a preparative regimen containing busulfan, fludarabine and alemtuzumab or anti-thymocyte globulin(atg), at three transplant centers between 2015-2018. busulfan was given either every 6 hours over 4 days with target auc of 875 to 1025 μmol/min (based on q6 hr. dosing) or twice daily over 4 days with target auc of 1800 to 2000 μmol/min (based on q12 hr. dosing) or once daily over 4 days with a target auc of 3600-4400 μmol/min (based on q24 hr. dosing). fludarabine 150 mg/m2 to 180 mg/ m2 was given divided over 4-6 days. serotherapy included alemtuzumab 0.3 -1.0 mg/kg or atg 7.5 mg/kg given divided over 3 days. gvhd prophylaxis consisted of cyclosporine and mycophenolate mofetil. results forty patients (was=12, hlh=10, cd40l deficiency=7, ipex/veoibd=4, scn=2, ifngr1 def./cid/x-scid/msn1/ lad=1) received busulfan, fludarabine and alemtuzumab or atg for allogeneic hct (first hct in 31 patients and second hct in the 9 patients with hlh). median age was 2.0 years (range, 0.25 years -19.8 years). patients received a graft from an hla-matched related (n=11), unrelated (n=27), or single allele mismatched related or unrelated donor (n=2). all except one patient engrafted at a median of 13 days (range,11-34 days). one patient developed veno-occlusive disease and two patients developed diffuse alveolar hemorrhage. notably, it was the second transplant for all 3 patients. eight patients (20%) developed grade 2-3 acute gvhd and 2 patients (5%) developed chronic gvhd. one patient developed primary graft failure and two patients secondary graft failure. nineteen patients (48%) maintained full donor (>95%) chimerism following allogeneic hct. twenty patients (50%) developed mixed chimerism, predominantly in the t-cell lineage, but t-cell donor chimerism progressively increased post-hct. at 1-year post-transplant, 15 of 20 patients (75%) with mixed chimerism had donor myeloid chimerism >90% and t-cell chimerism >75%. two patients underwent a second transplant for graft failure. there were 6 deaths in the cohort. overall survival was 85%(34 of 40) and event free survival was 80%(32 of 40) at 1 year. conclusion our experience suggests that a reduced toxicity busulfan, fludarabine regimen with alemtuzumab or atg as serotherapy offers a promising approach with low toxicity, durable myeloid engraftment, low incidence of grade 2-4 gvhd and excellent survival and can be considered for a variety of primary immune deficiencies where myeloablative hct is desired. abstract/case report text this is an 18-year-old patient who has the diagnoses of anti-nmda receptor encephalitis with associated catatonia. the patient has history of a pineal gland germinoma diagnosed in august 2018 after 8 months of double vision. however, after several months, the patient developed difficulty in sleeping, anxiety and nightmares. the patient presented to emergency department in february 2019 with personality changes. he was diagnosed with nmda encephalitis based on the clinical findings as well as presence of elevated serum and csf anti-nmda antibodies. the patient was initially treated with high dose systemic steroids with poor response. due to the worsening of his clinical condition, he was started on plasmapheresis, but had poor response to this therapy as well. these treatments are known standard treatment options for this condition. during his hospital stay, different therapies were discussed. cyclophosphamide was one of the treatment options, but because of the side effect profile and severe toxicity, we recommended different treatment modality. we started the patient on rituximab as well as sirolimus therapy to suppress both t and b-cell responses. after receiving two doses of rituximab in addition to daily sirolimus, the patient showed improvement of his symptoms. and declining nmda receptor antibody titers. this treatment plan was chosen for autoantibody mediated encephalomyelitis due to the fact that rituximab has inhibitory effect on naive b cells, but not on the proliferation of memory b cells and sirolimus has profoundly inhibiting role on memory b cells as well as a t-cell responses. a mosaic gene variant is one which is present in some, but not all, cells within an individual. they are increasingly associated with a number of diseases, including a number of primary immunodeficiency disease (pid). here we systematically analyzed mosaic variants in known or putative immunodeficiency genes from exome sequencing data from 998 individuals. lofreq was used to identify mosaic variants with a variant allele fraction (vaf) of 0.05-0.30 (0.50 is the vaf for a non-mosaic, heterozygous variant). we removed variants from extreme read-depth (> 2 standard deviations), unmappable, repeat-rich, and duplicated genomic regions. the average number of detected variants per mb was 3.2 and the total number of genomic locations with variants was 65,446. mosaic variants were underrepresented in exonic regions, suggesting that coding variants may be deleterious. more mosaic variants were detected in saliva exomes compared to peripheral blood exomes (p-value < 1 x 10-5), suggesting tissue-specific mosaic variants in buccal epithelial cells and white blood cells of saliva. to understand the clinical relevance of these findings, the variants were further filtered to include only nonsynonymous variants found in fewer than 1% of samples, leaving a total of 6,808 variant locations. of the remaining variant locations, 40 had a pathogenic assertion in the human gene mutation database, and 140 were in international union of immunological societies pid genes. further variant interpretations and clinical correlations are underway. these data suggest that mosaic variants in pid genes are common, vary by location of collection, and may have clinical diagnostic relevance. abstract/case report text introduction/background: the arpc1b (actin related protein 2/3 complex subunit 1b) gene is a protein coding gene prominently expressed in blood cells and is necessary for the assembly and maintenance of the human actin-related protein 2/3 complex (arp2/3). actin polymerization plays a central role in many immune functions including proliferation and differentiation of immune cells, migration, intercellular and intracellular signaling and activation of both innate and adaptive immune responses. defects in the actin cytoskeleton affect hematopoietic cells in the bone marrow and the immune response giving rise to a distinct primary immune deficiency, which is phenotypically similar to wiskott-aldrich syndrome (was). arpc1b deficiency clinically presents as a severe multisystem disease which includes platelet abnormalities, recurrent infections, failure to thrive, inflammatory changes in the intestine, eczema, cutaneous vasculitis, eosinophilia, and elevated inflammatory markers. arpc1b deficiency is rare and has only recently been described in the literature. here we present a clinical case of a patient found to have a pathogenic arpc1b mutation via whole exome sequencing. case report: here we describe a 15 month old somalian boy who presented to the immunology team at 9 months of age with hematemesis, hematochezia, melena, failure to thrive, atopic dermatitis, hypothyroidism, autoimmune thrombocytopenia and recurrent infections concerning for a primary immune deficiency. family history was notable for parental consanguinity and an older sibling with a similar presentation of hemorrhagic gastroenteritis who died in kenya around 3 months of age due to complications of his symptoms. the initial primary immunodeficiency evaluation revealed normal inflammatory makers, normal igg and protective vaccine (prevnar and tetanus) response. iga and ige were elevated at 364 mg/dl and 99.6 ku/l respectively. flow cytometry was remarkable for t cell lymphopenia (cd3 1541, cd4 1163, and cd8 363) with reduced naïve cd4 and cd8 t cells. b and nk cell count were normal. alps panel and was protein expression was unremarkable. whole exome sequencing was performed and revealed homozygotic mutation of arpc1b c.392t>c, ivs4+2t>c which was predicted to be a pathological variant. subsequently, dhr flow cytometry with fmlp showed significant increase of dhp fluorescent (mfi 9.7 when compared to control mfi 3.7) consistent with findings from other arpc1b deficiency patients. at the time of the most recent clinic visit, the patient has remained abstract/case report text introduction: activation of the nod-like receptor family cardcontaining 4 protein (nlrc4) leads to the formation of an inflammasome. the inflammasome, a large cytosolic multiprotein complex of innate immunity, promotes proteolytic cleavage, maturation, and secretion of pro-inflammatory cytokines, including il-1 and il-18. a gain-of-function mutation (gof) in the gene encoding nlrc4 or nlrc4-inflammasomopathy is characterized by hyperinflammation with persistent elevated il-18, infantile enterocolitis, and early-onset macrophage activation syndrome (mas). objectives: to describe phenotypic variation among three siblings with a novel nlrc4 gof variant and to expand our current understanding of the clinical manifestations of the disease methods: clinical and laboratory features were studied in three siblings of a hispanic family with a novel nlrc4 gof variant. results: a novel variant, c.1475g>a (p.arg492gln) on the nlrc4 gene, was identified in a 3-year-old male with recurrent febrile episodes since one year of age. his laboratory findings showed highly elevated esr, crp, il-18, and fecal calprotectin. his endoscopic finding was unremarkable. the recurrent fever partially responded to canakinumab. a 10-year-old sister with ileocolonic crohn's disease for two years was found with the same nlrc4 variant and highly elevated il-18. crohn's disease was well controlled after adding infliximab infusion to methotrexate therapy. a15-year-old sister, who has been asymptomatic and healthy, was tested with the same positive nlrc4 variant and highly elevated il-18. the nlrc4 variant is inherited from their father, who currently has a diagnosis of psoriasis vulgaris. the il-18 levels of the three siblings show in the figure 1 patients with flh-4 develop the classic hlh phenotype early in life, with periods of remission. the pathogenesis is associated with a defect of perforin-dependent cytotoxicity. t-ctl and nk cells fail to remove abnormal cells, consequently, an uncontrolled proliferation and activation of cd8+ t cells and macrophages develops and generates an inflammatory cytokine storm and a solid organs infiltration. mortality of fhl-4 is very high without treatment; allogeneic hsct is the only curative therapy. we report the outcome of a child with fhl-4, four years after his treatment with an allogeneic hsct using an hla haploidentical donor. case report: a boy born in december 2007, previously healthy, the only child of parents without consanguinity. he has a normal family and perinatal history. when he is 7 years old, he begins suffering from with fever, arthralgia, hepatosplenomegaly and pancytopenia. he meets the diagnostic criteria for hemophagocytic lymphohistiocytosis (hlh) and received treatment with iv methylprednisolone and entered in remission of its hlh. six months later, he restarts his clinical picture of hlh and j clin immunol receives treatment with the hlh-2004 protocol, which leads to remission. ayear later he presents a new episode of hlh that responds to ivig, cyclosporin a and dexamethasone. the possibility of primary hlh is then suggested. when he is 8 years old, the molecular diagnosis is made with the identification of a mutation in the stx11 gene, the case was classified as fhl-4 due to syntaxin 11 deficiency. in october 2015, a hsct was performed. at the age of 12, his clinical status is evaluated and laboratory studies show that his immunodeficiency due to syntaxin 11 deficiency was cured. his 42-year-old mother, hla haploidentical was used as a donor and a protocol developed for the treatment of osteopetrosis was applied. the hsct protocol did not use a tcell depleted graft. hstc conditioning was done with melphalan days -11 and -3, fludarabine days -7 to -2, anti-thymocyte globulin days -7 and -6, and cyclophosphamide day -2. (figure) . clinical and demographic characteristics, including the use of a novel claims-based weighted algorithm (risk vital sign; rvs) and those initiating ivig and scig, were described. stratified analysis based on pid diagnosis codes was performed. probability of receiving available ig treatments based on baseline characteristics was evaluated by logistic regression and propensity score methods. results: selected clinical and demographic characteristics, severity measures, and previous treatments between the overall pid population (n=382,131), pid patients initiating scig (n=2,604), and pid patients initiating ivig (n=15,327) are presented in the table. patient characteristics and previous treatments tended to be stable, although hypertension, obesity, and corticosteroid use increased during the study period. new ig users tended to be older and female, with increased depression, dyslipidemia, and hypertension than all pid patients. new scig users had more diagnoses of respiratory (e.g., asthma, copd) and inflammatory (e.g., arthritis, fibromyalgia, inflammatory bowel disease) comorbidities and less cancer than all pid patients. new scig users compared with new ivig users had increased asthma and copd, fibromyalgia, and inflammatory bowel disease and decreased cancer and peripheral vascular disease. previous corticosteroid use was higher in ig users than all pid patients. among scig users, prior pid treatments of iv antibiotics, and oral high potency antibiotics were similar to all pid patients. ivig users had higher iv antibiotic and antifungal use. rvs was initially developed to identify patients likely to have undiagnosed pid. this analysis applied rvs to patients diagnosed with pid to assess severity. rvs based on 1-year history in the overall pid cohort was predominantly low, with only 18.0% of patients scoring in the medium and high ranges. rvs was increased in incident ig users, with (37.4% medium/high for scig; 46.4% medium/high for ivig). in other markers of severity, scig users had more sinusitis and ivig had more pneumonia than all pid. ig users had fewer abscesses, cellulitis, and otitis media than the full pid cohort. conclusions: this exploratory analysis showed a trend toward increased hypertension, inflammatory and respiratory comorbidity, higher rvs, and previous corticosteroid treatment in patients initiating on ig compared with all pid patients. results could be confounded based on pid diagnosis codes used and warrants further research. author disclosures: mp, cas, and zh are employees and stockholders of the takeda group of companies. jo is a consultant to takeda. jbl is an employee of rti health solutions, an organization funded by takeda to conduct this research. mer was an employee of rti health solutions at the time this research was conducted.presenting author: colin anderson-smits submission topic: immunoglobulin replacement therapy patients in a larger cohort group and to determine if there are patterns of disease not previously reported. methods: we performed a retrospective chart review of patients with lof stat3 (n=81) followed at the national institutes of health. we specifically looked at tortuosity, aneurysms, and dilation of both coronary and cerebral arteries. epidemiologic information, stat3 mutation, co-morbidities, and laboratory information were reviewed along with imaging studies, specifically brain mri, brain mra, heart mri, and coronary ct. results: most recently, patients with hies are found to have vascular abnormalities including tortuosity, dilatation, narrowing, and aneurysms of middle sized, cerebral, and coronary arteries. in an effort to determine the extent of vascular involvement in addition to miscellaneous organ involvement, we are reviewing a cohort of 81 patients with hies who were evaluated at the nih. of these 81 patients, 53 are women and 28 are men. of these 81 patients, two have passed away due to vascular events leading to their deaths. there are four patients under the age of 20, 43 patients between the ages of 21 and 40, 33 patients between the ages of 41 and 60, and three patients above the age of 60 (age range 15-68, mean age 35.6).of the 81 patients, five of these patients were found to have abnormal brain mri/mra at an approximate rate of 6.2%. two of these patients were found to have at least one cranial aneurysm, two of these patients were found to have a level of narrowing or stenosis, and one patient was found to have dilatation.in terms of coronary abnormalities, 36 of the 81 (44.4%) patients were noted to have at least one coronary abnormality including dilatation, aneurysm, or tortuosity on heart mri or coronary ct. eight patients (9.8%) were found to have dilatation of which four patients were female and 4 patients were male. of the 81 patients, ten patients (12.3%) were found to have at least one aneurysm. there were 30 patients (37%) that were found to have at least mild tortuosity. conclusion: vascular abnormalities in our lof stat3 patients occurred at an exceedingly high rate-cerebral and coronary artery, 6.2% and 44.4% respectively. due to this, patient's with lof stat3 should be considered for screening with brain and heart imaging. currently, there are no guidelines which outline the appropriate timeline for screening in these patients however following these patients over time will allow us to determine the most appropriate interval for imaging follow up. abstract/case report text 63 year old woman with personal history of severe and recurrent upper and lower respiratory infections, chronic pulmonary disease with bilateral bronchiectasis and several micro nodules, chronic diarrhea without diagnosis (colonoscopy with mild colitis, without cmv. no bacterias no parasits were found in stools), mild osteopenia, focal lesion in right hepatic lobe, atopic dermatitis and anemia. she was followed up in other center and in 1996 she was diagnosed with common variable immunodeficiency (cvid) and started treatment with intravenous immunoglobulin (ivig), but with low adherence to it. she did not have referred history of lymphoproliferation nor significant viral infections. she had a daughter with spherocytosis who required esplenectomy and also had bronchiectasis and cvid diagnosis, she deceased at 28 years old due to pulmonary infection. one 30 years old son has anemia. her other daughter and son are healthy. objective: to describe unique management of recurrent viral respiratory infections in a 22qds patient. case: 5-year-old male with a history of 22qds complicated by truncus arteriosus, vsd, hypoparathyroidism, asthma, and autism was followed for a history of "frequent pneumonias." he had daily rhinitis and a history of frequent otitis media which resolved after tympanostomy tube placement at age 3y. however, over the next two years, he had 11 admissions with various respiratory viral infections resulting in respiratory distress and prolonged oxygen need. viruses detected during these separate admissions varied and included parainfluenza 3, metapneumovirus, rsv, b and coronavirus nl63, coronavirus hku1, and rhino/enterovirus (5 times total). he was previously on a prophylactic course of antibiotics which made no difference in his symptoms. laboratory evaluation showed protective adaptive immunity with normal immunoglobulin numbers for age (igg 589 mg/dl), along with normal b and t cell numbers. he had protective pneumococcal titers and mounted a normal mitogen response. while his nk cell numbers were normal, his nk t cells were low. his tlr functioning also appeared normal. in order to decrease his overall illness burden and to keep him out of the hospital, ivig infusions (~500mg/kg) were initiated monthly. shortly after initiation of treatment, his nasal purulence and drainage resolved and his family noted that he became more active and playful. treatment was continued for 18 months, during which time he had only one episode of influenza infection needing inpatient management. he has been off of ivig for 12 months without recurrence of his viral infections. conclusion: in this patient, severe recurrent viral respiratory infections despite apparently normal adaptive and cellular immunity presents a unique management dilemma. this was not an issue of recurrent bacterial infections as prophylaxis did not make a difference in the frequency of his infections. his nk t cells were low, which could have contributed to his frequent viral infections as nkt cells are known to play a role in viral immunity. the successful use of ivig treatment in his case points to a different use for ivig, namely for the anti-respiratory virus antibodies which are presumably contained within the formulation.given this finding, it may be prudent to consider ivig in management of 22qds patients, even with normal immune evaluation, in order to decrease risks of complications associated with severe and recurrent viral respiratory infections. abstract/case report text introduction: smad 4 is a critical downstream signaling molecule for transforming growth factor-β (tgf-β) and bone morphogenic protein (nmp1). initially, smad4, also known as dpc4 deleted in pancreatic cancer locus 4, was described as a tumor suppressor gene, and somatic deletion of the smad4 is seen in 90% of pancreatic carcinomas. subsequently, a germline point mutation in the smad4 gene (p. i500v) was reported to cause myhre syndrome (mim#139210). myhre syndrome is an autosomal dominant disease characterized by cognitive impairment, hearing loss, and musculoskeletal anomalies. the immunological phenotype of these patients has not been previously described, despite the critical role of tgf-β in regulating t cell response and the prevention of excessive inflammation. case report: a 9-year-old boy with myhre syndrome was referred to immunology clinic for evaluation of recurrent ear infections. he developed acute otitis media infections as an infant and had tympanostomy tubes placed at one year of life. he also had a recurrent sinus infections. at two years of age, he was diagnosed with autism and sensory neuronal hearing loss. brain mri showed a mildly hypoplastic pituitary gland, and a thickened corpus callosum with decreased myelination. given these findings, whole-genome sequencing was performed, which revealed a heterozygous de novo mutation in smad4 (p.i800v / c.1498 a>g) consistent with the diagnosis of myhre syndrome. through age 5, he was in the 5th percentile for height until he was started on growth hormone, which he responded to robustly. he is now he is in the 50th percentile for height. he had adenoidectomy due to sleep-disordered breathing at the age of 7 years. he is maintained on montelukast and inhaled corticosteroids for treatment of rhinitis and mild persistent asthma. he is on atenolol for the treatment of primary hypertension. on physical exam, he has facial dysmorphisms, thickened skin, and contraction of the fingers consistent with myhre syndrome. immunologic elevation showed significant hypogammaglobulinemia (igg 311 mg/dl), low iga (11 mg/dl) and normal igm 50 mg/dl. igg subclasses showed low igg1 and igg2 at 227 mg/dl and 58 mg/dl respectively. although he was fully vaccinated, his tetanus antibody was low at 0.14 iu/ml. however, this improved after repeat vaccination to 4.38 iu/ml. total t and b lymphocyte counts were normal; however, his memory cd4 and cd8 t cells were low for age at 2.26% and 0.5%, respectively. additionally, his switched memory b cell count was low at 1.9%. conclusion: smad4 gain of function (myhre syndrome) can lead to impaired memory t and b cell formation with significant hypogammaglobulinemia and low iga. although the patient was able to respond to protein vaccination (tetanus), it is not clear if he will be able to maintain a long-term response. in a previous study, a similar gain of key: cord-006466-e1phpqes authors: nan title: 2018 cis annual meeting: immune deficiency & dysregulation north american conference date: 2018-04-23 journal: j clin immunol doi: 10.1007/s10875-018-0485-z sha: doc_id: 6466 cord_uid: e1phpqes nan mutations in the genes encoding proteasome subunits (psmb8, psmb4, psma3 and psmb9) have been identified as the cause of candle syndrome. these mutations lead to malfunction of the proteasome, which results in buildup of cellular waste products. it is hypothesized that dysregulation in the interferon (ifn) signaling pathway in response to this waste is the driving mechanism of the inflammatory response, and may serve as a therapeutic target in these patients. objectives: to describe a case of suspected candle syndrome successfully treated with tofacitinib. methods: retrospective chart review was conducted with respect to diagnosis, treatment and response. results: a 16-month old caucasian male was admitted to the hospital for evaluation of profound anemia. his medical history was significant for extreme prematurity (born at 22 weeks from premature labor), intraventricular hemorrhage grade iv that resulted in hydrocephalus needing ventriculoperitoneal shunting, and developmental delay. he was noted to have a hemoglobin of 6.2 g/dl during a neurosurgical evaluation for routine shunt revision. he developed hemodynamic decompensation and required hospital admission for packed red-blood cell transfusion. review of systems was remarkable for intermittent pruritic macular rash, daily temperature fluctuations (fever to hypothermia), joint pain/swelling/ stiffness of multiple sites, poor weight gain, irritability, irregular breathing, abdominal distention, and regression of gross motor milestones (no longer rolling over, sitting without support, or pulling up to stand). workup excluded infections and lymphoproliferative malignancies, and he met clinical criteria for systemic juvenile idiopathic arthritis. his initial laboratory studies showed systemic inflammation (wbc 14x 10^3/ul, hgb 6.0 g/dl, platelets 558 x10^3/uul, crp 14.2 mg/dl, sedimentation rate 70 mm/h, ferritin 2370 ng/ml). imaging studies revealed serositis with right-sided pleural and pericardial effusions. he also had myositis supported by imaging and elevation of muscle enzymes (ast 52 u/l, aldolase 30.6 u/l). the patient was started on pulse iv methylprednisolone 30mg/kg daily x3 days, followed by oral prednisolone 2mg/kg/day and anakinra 2mg/kg/day with partial improvement and he was discharged home. he was readmitted 3.5 weeks later due to concerns for macrophage activation syndrome (ferritin 12,362 ng/ml) in the setting of a gastrointestinal infection and anakinra was increased to 4.5mg/kg/day. however, he continued to have persistently elevated inflammatory markers and so the dose was increased again to 7mg/kg/day. three months after initial presentation, he had an upper respiratory and ear infection and became ill with generalized rash, increased work of breathing, and poor perfusion. anakinra was considered a treatment failure at that time. he required several doses of pulse steroids and initiation of tocilizumab 12mg/kg iv every 4weeks with improvement on systemic symptoms. methotrexate 15mg/m² weekly was added soon after for persistent arthritis and inability to wean systemic steroids. he continued to have abnormal inflammatory indices, including ferritin (1,586 ng/ml) and il-18 levels (35,588 pg/ml, normal 89-540). proband only whole exome sequencing revealed a single heterozygous mutation in the psmb4 gene (c.-9g>a), a published pathologic variant. based on this finding, the patient was started on tofacitinib 2.5mg orally twice a day with a dramatic response. laboratory markers of inflammation normalized, and he was able to walk within the first month of treatment. further genetic testing to detect an additional proteasome subunit variant, as well as functional testing on a research basis to demonstrate an interferon signature are being pursued. conclusions: this case highlights the value of early genetic studies in patients with autoinflammation so that initiation of targeted therapy is not delayed in efforts to achieve control of symptoms and evade future complications. this case also illustrates the challenges in diagnosing monogenic autoinflammatory disorders in young patients that present with recurrent fevers, generalized rash, arthritis, and systemic inflammation that mimic systemic juvenile idiopathic arthritis. our experience contributes to the understanding of janus kinase inhibition in type i interferonopathies. of his recurrent infections and etiology of myasthenia gravis. results of the ct chest are notable for a thymoma. thymectomy with biopsy reveals benign pathology with a mixture of type a and b cells. he continues to have persistent fatigue, generalized weakness, diplopia, diarrhea and recurrent respiratory infections after thymectomy. immunoglobulin and lymphocyte subset panels reveal hypogammaglobulinemia with absent b cells. endoscopy reveals villous atrophy and blunting without evidence of celiac disease, inflammatory bowel disease or infection suggesting autoimmune enteropathy. the constellation of clinical and laboratory features are consistent with good syndrome with evans syndrome, seronegative myasthenia gravis and autoimmune enteropathy. the patient is started on immunoglobulin replacement therapy and pyridostigmine with resolution of recurrent infections and improvement of fatigue, generalized weakness and diplopia. three years later his fatigue and evans syndrome recur with new onset loss of appetite and a thirty pound weight loss. repeat immunologic labs were notable for elevated cd3, borderline low cd4 and highly elevated cd8 cells with low absolute number and fraction naïve cd4 and cd8 cells suggesting worsening combined immunodeficiency with peripheral t cell expansion. a bone marrow biopsy reveals large granular lymphocytic (lgl) leukemia and he is started on methotrexate. serum antibodies targeting ifn, and il-12 are negative four years after removal of thymoma. conclusions: this case is consistent with a classic presentation of good syndrome represented by thymoma, t and b cell-mediated immunodeficiency, increased susceptibility to infections and autoimmune manifestations of evans syndrome, myasthenia gravis and autoimmune enteropathy. in this case the combination of evans syndrome, autoimmune enteropathy and lgl leukemia as malignancy further worsen prognosis and is typically not seen together in good syndrome. this case depicts well the crossroad of infection, autoimmunity and malignancy in late onset immunodeficiencies. introduction/background: dedicator of cytokinesis 8 (dock8) deficiency is a known cause of autosomal recessive hyper-ige syndrome with a combined immunodeficiency. most of the mutations in dock8 are lossof-function homozygous or compound heterozygous point mutations or deletions. dock8 deficiency has been associated with low lymphocyte counts with impaired antibody responses, as well as eosinophilia, recurrent bacterial and cutaneous viral infections, malignancies, and severe atopy. we report the case of a 47 year old man with history of hyper-ige syndrome, severe atopy, eosinophilia, and antibody deficiency, phenotypically atypical for dock8, who was noted to have two variants of unknown significance in the dock8 gene. objectives: we report the case of a 47 year old man with history of hyper-ige syndrome, severe atopy, eosinophilia, and antibody deficiency, phenotypically atypical for dock8, who was noted to have two variants of unknown significance in the dock8 gene. methods: a 47 year-old man presented to us for evaluation of known hyper-ige syndrome. he had a long history of elevated ige, peripheral eosinophilia, severe atopic dermatitis, food allergies, asthma, severe eczema since early childhood which failed to respond to methotrexate, mycophenolate, cyclosporine, and omalizmuab, but ultimately responded to intravenous immunoglobulin (ivig). his infectious history included mrsa skin infections and one episode of pneumonia, and he reported a history of fungal skin infections but the history was unclear. initial immune workup revealed eosinophilia of 2898, ige level 3540 (as high as 20,000), igg level 603, igm level 106, and iga level 124. he had no random antibodies to streptococcus pneumonia 13 serotypes, but he had protective antibodies to diphtheria and tetanus. lymphocyte subsets showed cd3 1874, cd4 1597, cd8 277, cd19 85. he had normal mitogen stimulation to pha but decreased mitogen stimulation to candida. dna testing for a stat3 mutation was negative. results: we found two missense variants of uncertain significance in the dock8 gene (1.p.v194i, nm_203447.3:c.580g>a and 2.p.l1330v,nm_203447.3:c.3988c>g ). the first variant had previously been reported in the clinvar database as a variant of uncertain significance, and the second variant had not been previously reported in the literature to our knowledge. our assay could not determine if the two dock8 variants were on the same allele or on different alleles. dock8 protein expression testing is currently pending. conclusions: our patient presented with history of elevated ige, eosinophilia, atopy, severe eczema, and cutaneous mrsa and fungal infections. he was noted to have variants of uncertain significance in the dock8 gene. homozygous or compound heterozygous pathogenic variants in dock8 are associated with an autosomal recessive hyper-ige syndrome and combined immunodeficiency with clinical features of recurrent bacterial infections, cutaneous viral infections, severe atopic disease, as well as susceptibility to malignancy. our patient does not have all the typical features of dock8 deficiency and he seems to have a less severe phenotype. notably, he does not have the cutaneous viral infections or malignancy often seen in dock8 mutation hyper-ige cases. our case demonstrates new missense mutations, which have not previously been described in the literature, possibly causing a milder phenotype of dock8 deficiency. a case of igm deficiency and adult-onset still's disease negative. previous biopsy of her cervical and thoracic lymphadenopathy was unremarkable for malignancy. during her hospitalization, serum immunoglobulins were performed, which demonstrated normal levels of igg and iga, with igm level of <10 mg/dl (reference range 40-230 mg/dl), consistent with selective igm deficiency. liver function tests revealed an elevated aspartate aminotransferase (ast) of 177 u/l and an alanine aminotransferase (alt) of 177 u/l with a total bilirubin of 1.7 mg/dl and an alkaline phosphatase of 146 u/l. her ferritin was elevated at 349 g/l. the patient fulfilled yamaguchi criteria for aosd with three major criteria of evanescent rash, intermittent fevers in a quotidian pattern, bilateral arthralgias in the hips, knees, and ankles. she also met two minor criteria of liver abnormalities and lymphadenopathy. conclusions: selective igm deficiency is an uncommon immunodeficiency disorder associated with increased risk for autoimmune disorders. the recognition of co-morbid autoimmune illnesses in an immunodeficient patient is often complicated by a paucity of examples in the literature and potential confounding of laboratory serology analysis. we report the first case of a patient with selective igm deficiency and aosd. introduction/background: anaphylaxis to protamine is an uncommon but life-threatening complication of cardiac surgery and insulin therapy. here we present a case of recurrent protamine hypersensitivity during vascular surgery. objectives 1. recognize clinical signs of protamine hypersensitivity 2. recognize recurrent hypersensitivity to protamine as a serious complication of anesthesia methods: a 63 year old man with a history of diabetes, previously on nph insulin, hypertension, hyperlipidemia, chronic smoking, and peripheral artery disease with multiple vascular interventions was admitted to undergo a right lower extremity saphenous vein graft bypass. three years earlier during a similar intervention, the patient had developed intraoperative hypotension after protamine sulfate administration. protamine was subsequently held for additional surgeries, however the patient was able to tolerate protamine with slower infusion one year later. for the current vascular surgery, the patient was pretreated the day of surgery with diphenhydramine and dexamethasone, and a test dose of protamine was infused prior to full dosing. the patient initially appeared to tolerate the full protamine dose, but quickly developed facial erythema and angioedema. due to concern for laryngeal edema he remained intubated and was transferred to the surgical intensive care unit, where he received additional diphenhydramine and dexamethasone. his symptoms resolved and he was successfully extubated the next morning. results: anaphylaxis to protamine is an uncommon but lifethreatening complication of cardiac surgery and insulin therapy. protamine sulfate is a polypeptide used widely to neutralize heparin anticoagulation during cardiac and vascular surgeries, and in nph insulin. severe anaphylactic or anaphylactoid reactions caused by injection of protamine sulfate are well documented in literature, and the product contains a black box warning for such. the pathophysiologic mechanisms underlying these reactions are not clear, but ige-mediated hypersensitivity appears to play a role in many reactions, and prior sensitization or cross-sensitization (eg, to fish) have been suggested. type b adverse drug reactions are idiosyncratic drug reactions and are often unpredictable, as in our patient who previously tolerated protamine but subsequently developed an adverse reaction. hypersensitivity reactions during anesthesia should be thoroughly studied to identify the responsible drug and minimize exposure in recurrent surgeries. conclusions: this case illustrates the potential for severe reactions even with newer protamine formulations, and highlights the unpredictable nature of type b adverse drug reactions. it is important for clinicians to exhibit awareness of the potential adverse effects of protamine sulfate in such situations. introduction/background: x-linked lymphoproliferative syndrome type 2 (xlp-2) is a rare primary immune deficiency caused by loss of function in the x-linked inhibitor of apoptosis protein (xiap). common reported manifestations include recurrent hemophagocytic lymphohistiocytosis, splenomegaly, crohns-like inflammatory bowel disease, and transient hypogammaglobulinemia without reductions in major t cell or b cell repertoires, with the exception of inkt cells and mait cells. however, with only~100 known cases worldwide, we are likely only beginning to understand the phenotypic spectrum of this disease. objectives: to describe additional manifestations of xlp-2 that expand our current understanding of its phenotype. methods: a 26 year-old male with adult-onset, treatment refractory ulcerative colitis was evaluated in the immunology clinic for a history of recurrent sinopulmonary infections, skin abscesses, and recurrent ebv and vzv infections. extensive laboratory testing was performed in the course of his evaluation, including lymphocyte immunophenotyping, lymphocyte proliferation and cytotoxicity studies, quantification of total immunoglobulin levels and specific antibody function, hiv testing, and genetic testing. results: laboratory testing was significant for persistent cd4 lymphocytopenia ranging from 380-459 cells/mcl (rr: 5031736 cells/mcl). total b cell count was normal but b cell subsets showed an elevation in the percentage of naïve b cells (range: 85. .7%), low non-switched memory b cells (range: 4.7-6.0%, rr: 7.0-23.8%), and low to low-normal switched memory b cells (range: 7.2%-8.3%, rr: 8.3-27.8%), a pattern that has been seen in some autoimmune diseases. genetic testing with a commercial immune deficiency panel (invitae corp) showed a pathogenic mutation in xiap [exon 2, c.664c>t (p.arg222*)]. this mutation has previously been reported to cause a premature stop codon and reduced xiap function. the patient was referred for hematopoietic stem cell transplant and is currently awaiting transplant with a matched unrelated donor. conclusions: xlp-2 is typically reported as having normal t cell, b cell, and nk cell counts, but the presence of persistent cd4 lymphocytopenia in this patient illustrates that this is not always the case. our patient also had abnormalities in his b cell repertoire that have not been previously reported in xlp-2. additionally, xlp-2 has been associated with crohns disease and celiac-like bowel diseases, while our case indicates that the phenotype may also include ulcerative colitis. (9) submission id#420740 a case report: enteroviral encephalitis as a consequence of partial humoral immunodeficiency in a chronic lymphocytic leukaemia patient treated with rituximab hadeil morsi, st3 immunology st3, oxford university hospitals introduction/background: enteroviral (ev) infections are prevalent and usually self limited or cause mild gastrointestinal manifestations. however , in the context of primary antibody deficiency , rare cases has been reported to develop meningoencephalitis and been linked to poor outcome with fatality or chronic course. ev meningoencephalitis is even far rare reported in the era of rising secondary humoral immunodeficiency as a consequence of b cell depleting therapy e.g. rituximab and lymphoproliferative malignancies. limited treatments for ev encephalitis are available to date, apart from intravenous immunoglobulin replacement which has variable efficiency. objectives: studying such rare cases of ev meningoencephalitis as a consequence of antibody deficiencies would help to develop guidelines for intravenous immunogobulin replacement for treating these infections to improve outcome as well as predicting patients at higher risk who should be considered for prophylactic immunoglobulin therapy. methods: herein, we report a rare case of proven enteroviral meningoencephalitis following rituximab based therapy for b-cell chronic lymphocytic leukaemia and an uneventful six months period of follow up. he was found to have persistent absent b cells six months after completing six cycles of fludarabine, cyclophosphamide and rituximab therapy. interestingly, he had partial pneumococcal igg serotypes deficiency, whilst his total igg, igm and iga were all within normal limits throughout the course of the disease. the patient was treated empirically with intravenous immunoglobulin when his subtle confusion progressed to overt behavioural changes. initially his level of consciousness continued to deteriorate and he was not communicating. results: fortunately enough, the patient did have a remarkable improvement of gcs within couple of days and a slower recovery of higher mental functions e.g. memory and calculations in the next couple of months. conclusions: early suspicion and detection of entervorial meningoencephalitis in patients at risk of secondary antibody deficiency is crucial for timely ivig replacement and better outcome. patients with haematological malignancies and those on b cell depleting immunotherapy should be screened for pneumococal igg serotypes as part of secondary immunodeficiency workup. further studies on enteroviral neurological meningo/encephalitis are required to optimise ivig therapy and prognostication. furthermore, such studies provide an important asset to reveal the underlying mechanisms for humoral/b-cell mediated protective response against ev compared to other t-cell mediated viral immunity, whilst highlighting the mechanisms of immunodeficiency in cll and immunotherapy. (10) submission id#418733 a comparison of immune reconstitution following human placenta-derived stem cells (hpdsc) with umbilical cord blood transplantation (ucbt) vs. ucbt alone in pediatric recipients with malignant and non-malignant diseases introduction/background: ucbt is a safe and effective treatment in children (geyer/cairo et. al bjh, 2011) . however, due to a limited concentration of hematopoietic progenitor cells (cd34+) in ucb, ucbt has been associated with delayed hematopoietic reconstitution and a higher incidence of engraftment failure. hpdscs contain a rich population of hpcs, are low in hla class i/ii expression and t-cells, and have regenerative, anti-inflammatory, and immunosuppressive properties (cairo et al bmt, 2015) . objectives: to determine whether ucbt + hpdsc (vs. ucbt alone) is associated with enhanced hematopoietic and immune cell reconstitution in children with malignant and non-malignant diseases. methods: immune cell reconstitution at days +100, 180, 270 and 365 was assessed in children who received ucbt with hpdscs at nymc (nct01586455, ind#14949). minimum tnc was 5 x 10^7/kg (4/6 hla match) or 3.5 x 10^7/kg (5-6/6 hla match). immune cell subset counts at these time points were compared to those from a historical population of pediatric recipients of ucbt alone (geyer/cairo et. al bjh, 2011) . results: twenty four patients 18 years were enrolled. mean age was 6 (range, 0. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] years. malignant diseases =14, non-malignant diseases =10. fourteen patients received myeloablative conditioning (mac) and ten patients received reduced toxicity conditioning (rtc). there were no severe adverse events associated with hpdsc infusion. two patients with non-malignant disease receiving rtc using alemtuzumab experienced primary graft failure. probability of neutrophil engraftment was 91.6 %, median day 22 . of evaluable patients at day 100, the probability of platelet engraftment in neutrophil engrafted patients was 100%, median day 43.5 (20-98) . at days 30, 60, 100 and 180, mean percent donor chimerism in whole blood was 94, 98, 95, and 99%, respectively. average percent of whole blood hpdsc chimerism was 1% at day 30 and <1% at beyond day 60. one patient with malignant disease relapsed. 12 month overall survival was 83.3%. there was no significant difference in cd3, cd4, cd8, cd19 and cd56 immune cell reconstitution following ucbt + hpdsc vs. ucbt alone (image 1). conclusions: these results suggest that ucbt ± hpdsc results in similar immune cell reconstitution. a larger cohort with extended follow-up would be required to confirm these preliminary findings. supported by a grant from celgene cellular therapeutics. a decade of disseminated abscesses due to mycoplasma faucium in a patient with activated pi3k syndrome 2 (apds2) introduction/background: pik3r1 monoallelic mutations are known to be responsible for apds-like 2 syndrome, a rare form of primary immunodeficiency presenting as combined immunodeficiency or hyper-igm like phenotype. this study reports a patient carrying heterozygous pik3r1 mutation with early onset and long-term disseminated abscesses due to mycoplasma faucium in both peritoneal abscess and skin, with generalized involvement in neck and both upper extremities. objectives: we describe clinical management of retroperitoneal and skin abscesses before molecular diagnosis was available in a patient with a primary immunodeficiency. identification by rdna 16s in so-called sterile abscesses may confirm the clinical suspect of an oportunistic infection. furthermore, this study offers insight on the pik3r1 suspicion even in the absence of higm-like phenotype. methods: a 16-year-old girl with 2-year history of recurrent peritoneal effusion, which had been drained repeatedly was admitted in our institution for a 10-year history of multiple supurative cutaneous and lymph node-abscesses (fig1&2a-c). she had prior diagnosis of agammaglobulinemia under standard subcutaneous immunoglobulin replacement therapy and subcutaneous interferon-gamma treatment. on physical examination at the age of 16 years, she was stunted (weight and height below the 3rd percentile), with facial, arm skin abscesses and right fistulized axillary lymphadenopaties, 5-6cm hepatomegaly and giant splenomegaly results: she had her first immunological work up at the age of 6 years during one isolated episode of knee arthritis and first episode of skin abscesses. serum immunoglobulins revealed panhypogammaglobulinemia (igg<7.3 mg/kg, iga <5.8 mg/kg, igm <4.3 mg/kg) with low b cell count. on her back, there was a 10x15cm, elastic, neither painful nor tender mass. after proper assessment by ct scan and mri she had her retroperitoneal abscess drained percutaneously, and healed with sclerotherapy (percutaneous alcohol and polidocanol instilation) by the interventional radiologist ( fig 2b) . analysis of drained pus as well as pus of skin abscesses was made by 16s rdna pcr, having coincidence of 99.9% with mycoplasma faucium . combination antibiotic therapy (doxycycline and ciprofloxacin) was started with favourable response. unfortunately skin abscesses then relapsed. t-cell phenotype only showed t-cell lymphopenia with senescent (tem & temra expansion) phenotype. whole exome sequencing revealed a heterozygous mutation, previously reported (c.1425+1g>t) conclusions: in summary, this report emphasizes the suspicion of a combined immunodeficiency in the presence of multiple abscesses by mycoplasma, the usefulness of rdna 16s in order to achieve proper objectives: we describe a 15-year-old male patient with novel heterozygous mutation of ep300 gene; his first manifestations were initially characterized by infections, cytopenia and hypogammaglobulinemia suggesting a common variable immunodeficiency (cvid), but later on, persisting lymphopenia was suggestive of a combined immunodeficiency. methods: the patient was born to unrelated healthy italian parents at 34 weeks gestation with adequate weight for gestational age. shortly after birth, he underwent several surgical procedures due to interventricular defect, aortic coarctation, double outlet right ventricle, open botallis duct, and gastroesophageal reflux. at the age of four, he came to our attention due to stomatitis. clinical examination revealed dysmorphisms (microcephaly, wide forehead, sparse eyebrows, high nasal root, low-hanging columella, thick lips, micrognathia), splenomegaly (spleen diameter 11.6 cm at abdominal ultrasound), and severe developmental delay. in the course of the infectious episode, blood tests showed leukopenia associated with neutropenia (white blood cells 2.210/mm3; neutrophils 20/ mm3) and thrombocytopenia (platelets 1.000/mm3). analysis of bone marrow aspirate revealed normal differentiation of both myeloid and erythroid lineages. treatment with high doses immunoglobulin resulted in increase of platelet counts (up to 44000/mm3 after 1 month), while neutrophil counts spontaneously returned to normal when the infection resolved. however, thrombocytopenia relapsed (2000/mm3) after 2 months and intravenous high-doses of corticosteroids did not achieve normal platelets count. despite oral corticosteroid treatment started at the age of six, two episodes of autoimmune hemolytic anemia occurred. during the following six years of follow-up, the patient experienced recurrent infections (stomatitis, upper respiratory tract infections, and skin abscesses), but none of the episodes has required hospitalization. but, at the age of ten, he was admitted to the hospital because of severe cultures negative diarrhea. despite immunoglobulin replacement therapy was started at the age of fourteen, he was admitted twice due to bilateral pneumonia requiring continuous positive airway pressure and, a few months later, acute respiratory failure with evidence of mycoplasma pneumoniae and rhinovirus infections. immunological evaluation under chronic corticosteroid treatment at different time points showed persisting lymphopenia, with lymphocyte counts ranging from 350/mmc3 to 2100/mm3 , thrombocytopenia (platelets ranging from 0/mm3 to 308000/mm3), undetectable anti-diphteria and anti-tetanus toxoid antibodies, and splenomegaly. interestingly, analysis of isohemoagglutinins, revealed low titers of anti-a (1:8 ) at 4 years of age, but normal immunoglobulins (igg 685 mg/dl, iga 73 mg/dl, and igm 83 mg/dl). at the age of seven, reduced mitogen proliferation, hypogammaglobulinemia (igg 274 mg/dl; iga 17 mg/dl, igm 185 mg/dl), increased cd3+tcr+cd4cd8 t-cell counts (2.7/3.6%) and impaired fas mediated apoptosis as measured in two separate assays (table 1 ) . at the age of fourteen, evaluation of b-cell subsets showed increase of cd21locd38lo cells and reduction of switched memory b-cells. analysis of t-cell compartment unveiled a decreased proportion of cd31+ccr7+cd45ra+ recent thymic emigrants (rte) cells and ccr7+cd45ra+ naive cells, with prevalence of effector memory t-cells (ccr7-cd45ra-) ( table 1) . interferon signature gene expression showed borderline levels of ifi27 (data not shown). because of the decrease of igg and the infectious episodes ivig treatment was started at age of fourteen. a molecular investigation performed by whole exome sequencing (wes) revealed a novel heterozygous missense mutation (nm_001429. 3:c.4763t>c , p.met1588thr) in the exon 29 of the gene ep300 encoding the histone acetyltransferase (hat) protein p300. results: few immunological reports are available in rsts patients1-4. in keeping with previous data1,3,4, our patient presented with progressive b-and t-cell lymphopenia, hypogammaglobulinemia with poor antibody response but also reduced naïve t cells, evans syndrome, splenomegaly, and defective lymphocyte apoptosis with increased dnt. at the age of seven, the patient presented the features of cvid. flow cytometry revealed expansion of cd19hicd21locd38lo b cells, that is frequently associated with splenomegaly in cvid patients7, and reduced switched memory b-cell, previously reported in a rsts patient with crebbp mutation4 (fig. s1 ). lougaris et al. reported expansion of cd21locd38lo b-cells in nf-kb1 haploinsufficiency 8, and this suggests in our opinion that alterations in the nf-kb pathway due to ep300 mutations may affect b-cell differentiation. compared to healthy controls and cvid patients with predominant infectious complications, upregulation of interferon responsive genes in cvid subgroup with noninfectious complications (i.e hematologic autoimmunity, lymphoproliferation) and lymphopenia with reduced total b cells and switched-memory b cells has been demonstrated9. borderline levels of ifi27 expression under corticosteroid treatment represent a novel finding in rsts. interferon signature may identify and better characterize subgroup of rsts patients with autoimmune cytopenias and lymphopenia. at the age of fourteen, analysis of lymphocyte subsets revealed decreased total cd3+, cd4+, cd8+, and of both naïve cd4+ and cd8+ cells. elevated and persisting igm levels were also observed (fig. s2 ). according to our data, increased igm levels may be related to high proportion of terminal differentiated igm+ cells. these data (infections requiring hospitalization, immune dysregulation, lymphopenia, reduced naïve t cells, and reduced proliferation to mitogen) together with clinical history (fisher evans syndrome and lymphoproliferation) and exclusion of known syndromic immunodeficiencies, suggested a diagnosis of combined immunodeficiency10. conclusions: our case underlines the value of wes in patients with difficult phenotype-genotype correlation. no rsts typical traits were present and, prior to wes, several syndromes and immunodeficiencies were excluded. our report expands the phenotypic spectrum of ep300 mutations, thus in syndromic patients with clinical and immunological overlap between cvid and cid ruling out ep300 mutation should be advisable. furthermore, immunological work-up should be taken into consideration in rsts patients, in order to early identify immunological abnormalities that may lead to severe immune-hematological complications. introduction/background: interferon gamma receptor (ifngr1)-related disorders are rare variants of mendelian susceptibility to mycobacterial diseases. although hematopoietic stem cell transplantation (hsct) is curative, it is complicated by high rates of delayed or failed engraftment thought to be due to high concentrations of interferon (ifn)-gamma. umbilical cord blood transplantation additionally increases risk of graft failure. objectives: describe a pediatric patient with non-functional ifngr1 who successfully underwent umbilical cord blood transplantation. methods: direct clinical care of described patient with additional electronic medical record chart review. results: the patient is a 19-month-old boy of yemeni descent who initially presented with significant hepatosplenomegaly and extensive lymphadenopathy, including a large mediastinal mass. he then developed salmonella enteritidis sepsis requiring numerous antimicrobials, vasopressor support, intubation and continuous renal replacement therapy. his evaluation showed a hyperinflammatory state with elevations in ferritin, ifn-gamma, scd25, il-10, il-13, il-17, il-6 and il-8 levels. maximal ferritin and ifn-gamma levels reached 12065.4 ng/ml and 463 pg/ml (normal <5 pg/ml), respectively. flow cytometry revealed normal expression of ifngr1 and il12r but absent ifn-gammastimulated stat1 phosphorylation, suggesting defective ifngr1 signaling. genetic testing showed a previously unreported homozygous mutation in ifngr1 (c.373+2t>c) which affects a donor splice site in intron 3 and is predicted to cause absent protein function. dexamethasone and a single dose of alemtuzumab (0.2 mg/kg) were given to decrease inflammation. he then underwent allogeneic hsct using a 5/6 human leukocyte antigen matched umbilical cord unit following a reduced-toxicity conditioning regimen of alemtuzumab (0.6 mg/kg), fludarabine (180 mg/m2) and busulfan (auc 55 mg/l*h). plasma ifn-gamma was undetectable prior to starting conditioning and on the day of transplant. neutrophil engraftment occurred on day +14 with day +30 posttransplant chimerism analysis of peripheral blood myeloid cells showing the presence of donor cells only. conclusions: these early results suggest that umbilical cord blood transplantation may be feasible in patients with ifngr1-related disorders provided adequate control of inflammation is gained prior to transplant. introduction/background: introduction: btk is a cytoplasmic tyrosine kinase that activates phospholipase c2 (plc2) via phosphorylation, which ultimately leads to the activation of nfk, which is essential for b cell development and survival. mutations in btk lead to x-linked agammaglobulinemia (xla). in addition to the pleckstrin homology and tyrosine kinase domains, btk contains two src homology domains, sh2 and sh3, which are essential for btk function. we describe a novel btk mutation (c.1197a>t) resulting in v335a substitution in the sh2 domain that results in aberrant xla function with nearly normal btk protein expression. objectives: case report/results: the male proband presented with recurrent otitis media, persistent fevers and neutropenia beginning in the first year of life with an igg level of 46 mg/dl and a lack of b cells (0 cell/mm3), as demonstrated by flow cytometry. btk protein expression in monocytes, also determined by flow cytometry, was equivalent to controls. family history is significant for a maternal uncle with history of recurrent sinus infections and pneumonias with low iga and igm, low to normal ige, and an absent vaccine response. flow cytometry also showed an absence of b cells and essentially normal btk protein expression in his monocytes compared to controls. targeted high throughput sequencing of both proband and the uncle revealed a previously unreported missense mutation in exon 12, leading to the substitution of an aspartic acid residue for a valine (v335d.) the mutation is in the highly conserved sh2 domain of btk (conservation phylop conservation score 2.8.) the probands mother and his sister were shown to be carriers of the same mutation and had normal serum immunoglobulin levels and normal numbers of b cells. methods: we hypothesized that if the btk v335d mutant protein was non-functional, the female carriers of the mutation would only express wild type (wt) btk in their b cells while their monocytes would express both wt and mutant btk. to test this hypothesis cd19+ b cells and cd14+ monocytes were purified from pbmc by fluorescence activated cell sorting (facs) from the sister, cdnas were generated from the respective populations of cells, and the btk cdna was sequenced using high-throughput sequencing. results: at a read-depth greater than 16,000, cd19+ b cells demonstrated btk expression only from the wt allele (~99% wt btk) whereas both the wild type and mutant allele of btk were expressed at approximately equal levels in monocytes. conclusions: discussion: these results define a novel mutation in btk that nominally affects protein expression, but alters function. the v335a substitution is found in the d structural element of the sh2 domain that is part of a hydrophobic phosphotyrosine binding pocket. a mutation in the adjacent residue, y334s, has been shown to alter protein conformation and decrease binding affinity to plc2 by roughly 4-fold resulting in xla. our study, using a carrier harboring the c.1197 a>t mutant btk, demonstrated that in contrast to mononuclear cells, b cells only expressed the wt allele. this is consistent with the loss of function of v335a btk protein, thereby causing xla in both the proband and affected uncle. introduction/background: pyoderma gangrenosum (pg) is often associated with systemic autoimmune diseases but it has rarely been reported with common variable immune deficiency (cvid). while genetic analysis has been increasing in both disease domains, there has been little investigation into the genetic components associated with the cooccurrence of these entities. heterogeneous nfkb1 mutations have recently been identified in familial cases of cvid, though rarely have they been associated with pg. objectives: this case describes a novel nfkb1 mutation that may link both cvid and pg, and bolsters the recent identification of heterogeneous nfkb1 mutations in cvid. methods: a 24-year-old woman with a history of frequent skin infections in childhood presented with persistent, infected wounds following cholecystectomy. upon admission, she was started on broad spectrum antibiotics but continued to have fevers and leukocytosis. labs were also notable for elevated crp (340; normal (n):0.1-3) and esr (120; , with low c3 (60; n:81-145), c4 (<10; n:16-39) , and ch50 (<10; n:>74). wound cultures grew multi-drug resistant coagulase negative staphylococcus. despite broad spectrum antibiotics, the wounds failed to heal. dermatology was consulted and punch biopsy revealed a dense neutrophilic infiltrate and no identifiable pathogens, which supports the diagnosis of pg. the patient was started on high dose steroids (1mg/kg/ day) and had a rapid response with decreased skin inflammation and lesion expansion. unfortunately, the patient developed posterior reversible encephalopathy syndrome (pres) on steroids and therefore pg treatment was changed to infliximab as recommended by dermatology. further laboratory testing found that the patient also had low igg (499; n: 700-1600) and iga (34; , and a diagnosis of cvid was made given her clinical history of recurrent skin infections. genetic testing was pursued to evaluate additional pg therapy options and this revealed a heterozygous mutation in nfkb1 (c.a2415g; p.q805q), located 2 base pairs upstream of the splice donor site for exon twenty-one. while this mutation has not been previously identified as a pathogenic variant, similar mutations in this gene have been linked to autosomal dominant cvid. the patients father also carried a similar mutation but without any evident clinical phenotype. results: nfkb1 plays a crucial role in both immune and inflammatory responses. this case highlights a novel mutation in nfkb1 that has not been previously described as a disease-causing change. other mutations resulting in nfkb1 haploinsufficiency have been associated with cvid and rarely with concurrent pg, as in this case. based on the location of the mutation, it is expected that the variant causes obliteration of the normal splice site and therefore results in defective mrna that encodes p50/ p105. interestingly, studies on p50 knockout mice show decreased levels of igg, iga, and ige but not igm and our patient similarly had low levels of igg and iga but normal igm. conclusions: further studies are needed to determine if there are other links to this novel nfkb1 mutation in patients with cvid and pg. (17) submission id#412594 a rapid flow cytometric analysis of dna repair proteins reveals a radiosensitive phenotype in bcl11b deficiency associated with severe combined immunodeficiency (scid). introduction/background: we present the second report in the literature of a patient with immunodeficiency, dysmorphic features, growth retardation, and a homozygous variant in the dna ligase i (lig1) gene with associated absence of full-length lig1 protein. results: this is a now 2 year old girl who was the fourth child of parents who are first cousins. she has one healthy older brother, a second older brother who died within 12 hours of birth of meconium aspiration, and an older sister who died at six months of age of an upper respiratory illness / pneumonia after a history of congenital anemia, poor weight gain, cardiomegaly and hepatomegaly. she was born at 37 weeks and spent the first five weeks of life in a neighboring hospital neonatal intensive care unit (nicu) for hepatomegaly, mild cardiomegaly (previously identified on fetal ultrasound) and congenital anemia (requiring transfusions). her exam was and remains notable for weight, height, and head circumference below 3rd percentile, prominent forehead, hypotelorism with epicanthal folds, downslanting palpebral fissures, and low set, posteriorly rotated and prominent ears. after discharge to home from the nicu, her course was subsequently complicated by poor weight gain, chronic diarrhea beginning after her first rotavirus vaccine, and multiple deep vein thromboses. she then became critically ill at 6 months of age with respiratory failure, and was transferred to our institution for respiratory oscillator support. absolute lymphocyte count on admission to our institution was 0.3 k/μl (total wbc 4.0 k/μl, anc 2.7 k/μl) with agammaglobulinemia. she was diagnosed with and treated for pneumocystis jirovecii pneumonia, gradually weaned from oscillator to room air, and was discharged home five weeks later on 0.4 mg/kg every other week igg replacement. she has had no serious infections requiring hospitalization in the 18 months since. alc has remained persistently below 1.0 k/μl with a corresponding uniform deficiency of t, b, and nk cells and no detectable trec positive t-cells. whole exome sequencing identified homozygosity for a c.914g>a coding region variant in the lig1 gene not previously reported in the literature. a fibroblast cell line was successfully established and western blot shows an absence of full-length lig1 protein. further molecular characterization is in progress. conclusions: this second reported case provides further evidence for dna ligase i deficiency as a distinct clinical entity comprising immunodeficiency, dysmorphic features, and growth retardation. introduction/background: chronic mucocutaneous candidiasis (cmc) is associated with a heterogeneous group of primary immunodeficiencies. autosomal dominant stat1 gain-of-function (gof) mutations have been identified in up to 50% of patients with cmc. these mutations lead to impaired il17a/f t cell immunity although the underlying mechanism is unclear. there seems to be no genotype-phenotype correlation. recently, jak inhibitor therapy has been reported to improve cmc and autoimmunity in patients with stat1 gof mutation. objectives: we describe an infant with cmc associated with a novel stat1 gof mutation. results: a 7-month-old girl was referred to our immunodeficiency clinic with chronic diaper rash since 2 weeks of life, failure-to-thrive, and history of labial abscess complicated by rectolabial fistula. she was subsequently diagnosed with food protein-induced enterocolitis syndrome triggered by cows milk-based formula. laboratory evaluation revealed normal cbc with differential, lymphocyte subsets, mitogen response, immunoglobulin levels, antigen response to candida, and neutrophil oxidative burst assay. whole exome sequencing identified a de novo heterozygous variant in stat1 (c. 1627t >c, p.cys543arg) . further evaluation of this mutation revealed increased gas (gamma activation sequence) reporter activity in response to ifng stimulation suggesting that this is a gain-of-function mutation. the patient later developed significantly elevated liver enzymes while on fluconazole treatment, candida parapsilosis sepsis, granulomatous lesions in the liver, splenic lesions, intermittent thrombocytopenia and n o r m o c y t i c a n e m i a . s e p s i s a n d l i v e r l e s i o n s r e s o l v e d on amphotericin treatment but other findings, including tpn dependency persisted. we are planning to initiate a jak inhibitor therapy, ruxolitinib. conclusions: this case is a possible genotypic and phenotypic expansion of cmc due to stat1 gof. professor, the university of british columbia introduction/background: b cell cll/lymphoma 11b (bcl11b) is a zinc finger protein transcription factor with a multitude of regulatory functions in the integumentary, central nervous, cardiac, and immune systems. it is critical for t cell lineage commitment, development, differentiation, survival, and function. in addition, it also specifies the identity and function of innate-like lymphocytes, including t cells, innate lymphoid cells (ilcs), and invariant natural killer t cells (inkt). however, little is known about its function in the human immune system, especially in the context of immune disorders. objectives: to understand the immunopathogenesis of a novel p.c826y bcl11b variant. methods: research study protocols were approved by our institutional research ethics board. two members of the family were enrolled (the index patient and her father). written informed consent for genetic testing and participation was provided by the parents for the child. genetic, bioinformatic, proteomic, and biochemical analyses were performed. results: we have identified the second described case of immune disease caused by a de novo heterozygous damaging variant of bcl11b (p.c826y). this young girl presented with intellectual disability, microcephaly, severe atopy, eczema, alopecia totalis, and brittle nails. extensive clinical immunophenotyping of patient blood showed initially unremarkable b and t cell populations. however, the patient possessed abnormal rare innate-like lymphocyte populations (inkt, dn t cells). using mass cytometry (cytof), a technique capable of concurrently analyzing 40 parameters in a single cell, we were able to examine various innatelike lymphocyte populations, including t cells, ilc1-3, and nk cells. we found that the patient possessed severely compromised numbers of t cells, thus potentially implicating the p.c826y variant in t cell development and function. conclusions: the identification of decreased t cells in a patient with a p.c826y variant of bcl11b suggests that bcl11b is important for human t cell development and provides novel insights into the roles of both bcl11b and t cells in regulating atopy and autoimmunity. introduction/background: adenosine deaminase 2 deficiency caused by mutations in ada2 gene is a newly recognized disorder. it is associated with a spectrum of vascular and inflammatory phenotypes, ranging from early onset recurrent stroke to systemic vasculopathy or vasculitis. objectives: we describe a 13 year old female patient with features of early onset immune thrombocytopenia (itp), autoimmune hemolytic anemia (aiha), chronic splenomegaly and variable abdominal lymphadenopathy. she was diagnosed with evans-syndrome and treated with rituximab at 19 and 27 month of age. from 3 years of age she developed recurrent infections, hypogammaglobulinaemia with specific antibody deficiency, progressively decreasing class-switched memory b cells, and increased cd3+cd4-cd8-//t cells (4%). differential diagnosis included common variable immunodeficiency (cvid) or autoimmune lymphoproliferative syndrome and therefore a broad search for causative genetic defect was initiated. the parents are first cousins of middle-eastern origin suggesting an autosomal recessive inheritance. patient was stable on long-term mycophenolate mofetil (mmf) and immunomodulatory dose (1g/kg/ month) ivig treatment. methods: genomic dna of the patient was sequenced with next generation sequencing technology. a panel of 250 genes linked to primary immunodeficiency was analyzed. the identified variant was confirmed by sanger sequencing. results: genetic testing revealed a homozygous pathogenic mutation in the ada2 gene with one base pair duplication in exon 2 (c.144dup. p.arg49alafs*13) that creates a frame shift starting at codon arg49. the new reading frame ends at a stop codon 13 positions downstream, likely resulting in a truncated protein. plasma ada2 activity of the patient was markedly reduced (0.2 mu/ml, normal 4.8-27.2) and confirmed the diagnosis of ada2 deficiency. the parents of the patient are heterozygous carriers of the same mutation. unlike most previously reported cases, this patient had an extended phenotype with no neurological evidence of vascular pathology, however brain mri revealed two silent lacunar infarct or vasculitis related changes. we speculate whether the long-term mmf or ivig therapy might be protective against vasculitis. conclusions: ada2 deficiency may present with a wide spectrum of clinical phenotypes beyond classical vasculopathy. the diagnosis should be considered in patients with hematological autoimmune disease, splenomegaly and/or cvid like presentation. better understanding of pathophysiology of ada2 deficiency may help diagnosis and targeted treatment. professor, university of california, los angeles ca introduction/background: adenosine deaminase (ada) deficiency as a cause of severe combined immunodeficiency (scid) is distinct from other forms of scid in several ways. historically, survival and clinical outcome of infants with ada scid have been inferior compared to infants with other scid genotypes. there are multiple treatment modalities available for ada-scid, including enzyme replacement therapy (ert), allogeneic hematopoietic cell transplant (hct) and experimental autologous transplant of gene corrected cells (in recent years preceded by low dose busulfan), designated as gene therapy (gt). in addition, there is a growing body of evidence for effects of ada deficiency on non-immunologic organ systems that may contribute to the historically poorer outcomes of these infants. therefore, it is important to evaluate the cohort of patients with ada scid separately from other scid cases. objectives: to capture incidence and treatment trends and to compare outcomes following available treatments for this rare inborn error of metabolism and other forms of scid, the primary immune deficiency treatment consortium (pidtc), a network of 45 north american immunology and transplant centers, has collected standardized data for analysis. methods: 118 ada scid patients, first treated between 1977 through 2016, were enrolled from 26 centers (range 1-25 subjects/site). ada accounted for 13% of the 926 total pidtc scid patients treated during that time. patients were entered into either a retrospective protocol (pidtc 6902, n=96) or a prospective protocol starting in 2010 (pidtc 6901, n=22) . ada-scid patients who received an hct as first therapy entered either of two strata, as with other scid patients in pidtc studies, based on whether their initial presentation met definitions for typical (n=46) or leaky scid (n=6); in contrast, patients initially treated with either ert or gt were entered into a separate stratum (n=66). results: sixty-four patients (54% of all ada-scid enrollees) had ert as first therapy, but only 6 in this cohort received ert as sole therapy; 58 went on to have subsequent hct (n=30) or gt (n=28). there were various combinations of treatment cycles among these ada-scid patients. most received hct {+/-subsequent treatments} (44%), ert followed by hct {+/-subsequent treatments} (25%), or ert followed by gt {+/-subsequent treatment} (24%); several patients received multiple successive treatment modalities, representing either failure of initial treatment or planned progression from ert to cellular therapy. two-year survival has improved over time from 69% in (1977-2000 to 92% 2001-2016 (p=0.007) (figure 1 ). the survival for all other non-ada scid patients registered by pidtc over these two eras were: 78% (1977-2000) and 81% (2001-2016) . hct (either as sole therapy or after ert) accounted for >95% of cellular therapies between 1986 and 2007; in contrast, since 2007, gt was used as commonly as hct (n=27 vs. n=29, respectively). there was a trend toward better two-year survival for patients receiving gt as first cellular therapy since 2001 (100%, n=28, all after initial ert) compared to those receiving hct over the same time period (87%, n=40, either as first therapy or after ert), although this did not achieve statistical significance (p=0.073). conclusions: this study reveals the improved prognosis for patients with ada scid in recent years and the emergence of gt as a new treatment modality. further analyses are investigating the impacts of prior infection and treatment modality, including effects of conditioning, on outcomes (survival, event free survival, clinical outcomes and completeness of immune reconstitution) for ada-scid in successive eras. this study may identify optimal treatment approaches for future ada scid patients. sponsored by the pidtc, a member of the rare diseases clinical research network (rdcrn) and funded by u54 ai 082973 (niaid and ordr, ncats, nih). dbk has potential financial conflict of interest as a member of the scientific advisory board for orchard therapeutics and an inventor on intellectual property which ucla has licensed to orchard therapeutics related to gene therapy for ada scid. jp discloses that her spouse is employed at invitae, a dna sequencing company. intern, imam abdulrahman bin faisal university introduction/background: patients with diabetes mellitus are immunologically vulnerable population to develop different types of microbial infections. immunization has an important role in infection prophylaxis. in fact, vaccines containing thymus-dependent antigens protect patients with diabetes as they produce massive and complex immune response and feature immunologic memory. the recommended vaccinations for patients with diabetes mellitus are influenza vaccination yearly and pneumococcal vaccination. in observational studies, influenza vaccine has been shown to be similarly effective in adults <65 years of age with diabetes as in older patients with or without diabetes [1] . among immunocompetent elderly, vaccine efficacy of the 13-valent pneumococcal conjugate-vaccine (pcv13) was modified by dm with higher vaccine efficacy among subjects with dm [2] . the hepatitis b vaccination should be given to unvaccinated adults with diabetes mellitus who are ages 19 to 59 years. for older patients administration only after assessment of benefits and risks of acquiring hepatitis b virus (hbv). in fact, one review suggests that dm is associated with the progression of severe liver outcomes in adults with hbv [2] . on the other hand, tetanus and diphtheria vaccinations should be updated. in addition to, vaccinations such tick-borne encephalitis, meningococcal infections and other infections that put in risk diabetic patients travelling abroad. accordingly, theres a variability of vaccines that can offer a preventive method to reduce morbidity, mortality, and medical expense. in our multicenter study among eastern province saudi arabia evaluated the degree of adherence of the physicians to the immunization recommendations for adult patients with diabetes mellitus type 1 and type 2 to increased awareness of the immunization importance in diabetic patients. objectives: to increased awareness of the immunization importance in diabetic patients. in fact, patients with diabetes mellitus are immunologically vulnerable population to develop different types of microbial infections. immunization has an important role in infection prophylaxis. methods: this is a cross sectional study involving 500 adult patients with type 1 and type 2 diabetes mellitus using a questionnaire. patients will be recruited from outpatient clinics including the primary care clinics and inpatient words of king fahd hospital-al khobar and other centers in the eastern province saudi arabia. after an informed consent, baseline data will be collected. patients will be then asked if they received the recommended vaccines and who was the provider. their knowledge regarding the needed immunization will be also tested. they will then be asked about frequency of upper respiratory tract infections and pneumonia they had over the last 2years. results: we are expecting to find low adherence to the recommended immunizations by the physician. we may also find that patients who received the vaccines has low incidence of related infections. conclusions: increased awareness of the immunization importance in diabetic patients and adherence to immunization as part of standard care of adult patients with diabetes mellitus that offer a preventive method to reduce hospitalizations, mortality, and medical expense. 1-10, 11-20, and 21-30 bases. repeatability and reproducibility of the assays were 0.994 and 0.998, respectively. 99.5% of the target regions were covered with over 15x sequencing depth. we showed that the assays had 0.748 sensitivity to detect single-exon deldups and 1.000 sensitivity to detect copy number aberrations covering two or more exons. using acmg guidelines for variant classification a diagnosis was established in 20% patients that were sent for comprehensive panel analysis. conclusions: conclusions: our results demonstrate the analytic validity of the developed tests and show that the technology is well-suited for clinical diagnostics of inherited eye disorders. it also demonstrated a cost-effective diagnostic tool to simultaneously diagnose various types of mutations from snvs to copy number variations. introduction/background: loss of function (lof) and null mutations in orai1 and stim1 cause a rare autosomal recessive immunodeficiency by abolishing calcium release-activated calcium (crac) channel function and store-operated ca2+ entry. the clinical presentation is characterized by scid-like disease, dental enamel defects, muscular hypotonia and anhidrotic ectodermal dysplasia. objectives: here we present the outcome of calcium assessments performed on lymphocytes from an adult patient with unusual infections and a purported novel single pathogenic variant in orai1. methods: the ca2+ response in lymphocytes following activation by varying concentrations of non-cross-linked anti-cd3 was assessed by flow cytometry. results: the patient was a 47 year old female with a history of seasonal and medication allergies and recurrent sinus infections, who had recently developed an acute infection of her right first metatarsal joint. cultures from the joint space grew neisseria gonorrhea and atypical mycobacteria at two different time points. hiv screening was negative. follow-up testing with the mantoux test and quantiferon gold suggested that she also had latent tuberculosis infection, for which she was started on rifampicin therapy. immunologic evaluation revealed normal complete blood count and differential, normal t, b and nk cell counts, normal immunoglobulin g, a, and m levels, as well as normal responses to polysaccharide vaccines and normal t cell proliferative responses to mitogen and antigen stimulation. however, given the identification of atypical organisms from joint fluid cultures as well as latent tuberculosis (despite the lack of significant risk factors), genetic testing was recommended by her local physicians to rule out an underlying primary immunodeficiency. an invitae primary immunodeficiency panel identified a single novel pathogenic variant in orai1. she was then referred to our institution for further evaluation. so far, individuals identified as heterozygous for lof or null mutations in either orai1 or stim1 have lacked any phenotype associated with crac channelopathy. however, there have been reports of abnormalities in calcium response in parents of a number of these patients, who are heterozygous for the disease-causing mutation. to examine the functional effect of the observed mutation in our patient, her freshly isolated pbmcs were loaded with indo-1 and the ca2+ response of her lymphocytes were assessed by flow cytometry. this showed a dose-dependent decrease in the patients t cell response to non-cross-linked anti-cd3 in comparison to the normal control, i.e. there was a clear decrease in the patients ca2+ response at 1 microgram/ml in comparison to the normal control, but the decrease was rectified upon stimulation with 5 microgram/ml or more of anti-cd3. conclusions: these findings provide functional support for the identification of a new pathogenic mutation in orai1. nevertheless, it is not yet clear if the mutation has any mechanistic role in the patients recent clinical presentation. introduction/background: patient 1: 10 years old boy, born to nonconsanguineous parents, with hypothesis of autoimmune encephalitis (vasculitis), which was not characterized. csf has already been routed to barcelona 2 times and to vienna 1 time. refractory epilepsy remains (uses 5 drugs yet). he had continuous fever during the entire hospitalization. he had adhd (took ritalin for 2 years) and central auditory processing deficit. one year ago he began to have fever for 3 days, improving later but evolving with bilateral otitis, predominantly on the left ear, accompanied by sinusitis. soon after that he began presenting epilepsy that worsened severely. he was interned and took acyclovir ev, associated with hydantoinate (had stevens-johnson by the drug, suspending and improving quickly). in march had uti + amo, several infections requiring meropenem + vanco, among others. received flebogamma 500 mg/ day/5 days, twice; he also received weekly rituximab for a few months. in use of carbamazepine, clobazan, phenobarbital, levotiracetam and vigabatrin 3 times a day. personal antecedents: allergic rhinitis, bronchial asthma, recurrent otitis media, iga deficiency. patient 2: male, 9 years old, born to non-consanguineous parents, with history of repeated infections in the upper respiratory tract from one year of age with prolonged dry cough and sore throat in all episodes, treated with dexamethasone without improvement, followed by antibiotics with resolution of the condition. at age five, in february 2012, he had a new episode of sore throat and cough that lasted three months, improving spontaneously thereafter. in june 2012, new episode of sore throat with elevated fever and whitish plaques in the tonsils, being prescribed benzetacil, without improvement in three days. he returned to the same emergency room, the antibiotic was replaced by zinnat (axetil-cefuroxime), but within the hospital he began to convulsionate, entering into an epileptic crisis, being hospitalized for 33 days in this service and being transferred to another hospital specialized in pediatrics, intensely investigated and treated with partial improvement of the condition. he remained in coma, not walking and talking for some months, recovering slowly with physical therapy and speech therapy. of relevant exams have: reduced iga, but before it was normal (probably induced by anticonvulsants); full-body magnetic resonance imaging demonstrates generalized lymphadenomegaly and hepatosplenomegaly; pet-ct showing signs of hypoperfusion in temporal (right), occipital and cerebellum regions (suggestive of hypoperfusion -vasculitis?) my first impression was of possible mevalonate kinase (mvk) or autoimmune lymphoproliferative syndrome (alps) deficiency. with these tests described above, i believe that the first diagnostic suspicion is that the epilepsy was triggered by hemophagocytosis in the central nervous system and consequent extremely severe epilepsy, triggered by ebv infection. objectives: to compare the clinical and genetic similarities and differences of both patients. methods: both patients wer submitted to whole exome sequencing looking for the genetic alterations associated to the disease of these patients. results: wes of patient 1 showed an allelic variant in the gene of rai1 (c.4625g.a; p.arg1542gln) possibly pathogenic and that could be related to the clinical features of the patient. wes of patient 2 showed an allelic variant in the gene of cd27 (c.281g>a; p.arg94his) heterozygous and of uncertain significance. another allelic variant was found in the gene of btk (c.707g>a; p.arg236gln) classified as of uncertain significance and hemizygous (as btk is in chromosome x). the expression of both proteins evaluated by flow cytometry is normal, decreasing but not abolishing the possibility of pathogenicity. conclusions: the similarity of the clinical presentations is striking, but the genetic alterations are totally different, leading to the presentation of this abstract. (inf-) have been associated with adult onset immunodeficiency in patients of asian origin. pathogens that cause infections in these patients include mycobacterium avium-intracellularae (mai), non-typhoidal salmonella, cytomegalovirus, penicillium marneffei, and varicella zoster virus. methods: chart review of one patient results: we present a thirty-two-year-old filipino female, with sjogrens syndrome, penicillin and vancomycin allergy, and shellfish allergy suffering from recurrent mai spinal osteomyelitis. after three months of conservative management of back pain, mri showed an abscess at l4/l5 and l5/ s1 vertebrae, which was diagnosed as acid fast bacilli on biopsy. she was treated for mycobacterium tuberculosis with rifampin, isoniazid, pyrizinamide, and ethambutal with subsequent change in antibiotic therapy after six weeks once cultures grew mai. mri showed spread of abscess to l3-s2 vertebrae. three months into treatment, she was found to have a new abscess at a different spinal site, and antibiotics were again changed. two months later, she had recurrence of disease with multiple large iliopsoas abscesses, cutaneous fistulas, insufficiency fractures of the sacrum bilaterally, and osteonecrosis of the l4 vertebra, requiring extensive surgical debridement. medications were adjusted and she was referred to immunology eight months after initial presentation to infectious disease. laboratories were notable for elevated igg (2580 mg/dl) and iga (474 mg/dl) and decreased cd4 (425 cell/ul) and cd16/56 (49 cell/ul) cells. serum electrophoresis showed low albumin, elevated gamma fraction, and polyclonal gammopathy. specific antibody titers, lymphocyte proliferation assay, ch50, and immunofixation were within normal limits. cytokine panel was significant for elevated il-2 receptor cd25 (3860 pg/ml), il-6(15 pg/ml), and il-13 (12 pg/ml), and normal tnf, inf, il-1, il-2, il-4, il-5, il-8, il-10, il-12, and il-17. further serologic testing was positive for autoantibodies to inf-. patient continues treatment with iv antibiotics and is awaiting enrollment in a rituximab trial. conclusions: autoantibodies to inf-should be considered in patients of asian origin presenting with adult onset immunodeficiency, particularly those with severe or recurrent infection with mai. a high level of suspicion is required to make this diagnosis: failure to consider this disease entity leads to delay in diagnosis with potentially significant consequences for the patient. allergy/immunology, university of south florida at johns hopkins all childrens hospital introduction/background: autoimmune and inflammatory conditions are common in cvid. these have been associated with increased morbidity and mortality. objectives: we sought to further understand and evaluate the prevalence of autoimmune and rheumatologic manifestations in patients with common variable immunodeficiency (cvid). methods: we performed a retrospective analysis of cvid patients with rheumatologic/autoimmune complications in the partners healthcare cvid cohort. we evaluated baseline patient characteristics as well as autoimmune and rheumatologic complications in this cohort of patients. results: in the partners cvid cohort, 120/210 (57%) had autoimmune or rheumatologic disease. autoimmune cytopenias were reported in 37/210 (18%) patients, including coombs positive autoimmune hemolytic anemia (n=18), idiopathic thrombocytopenic purpura (n=30), and autoimmune neutropenia (n=10). autoimmune thyroid disease was reported in 43/212 (20%) patients, including hypothyroidism (n=25) and hashimotos thyroiditis (n=18). inflammatory arthritis was present in 35/210 (17%), most commonly seronegative rheumatoid arthritis (ra) (n=18), followed by inflammatory arthritis (n=8), seropositive ra (+rf or +ccp antibody) (n =6), psoriatic arthritis (n=2), and juvenile idiopathic arthritis (n=1). systemic autoantibody disease was diagnosed in 26 patients (12%), with diagnoses including vasculitis (n=7), systemic lupus erythematosus (n= 6), polymyalgia rheumatica (n=4), antiphospholipid syndrome (n=3), mixed connective tissue disease (n=2), crest/ scleroderma (n=2), myositis (n=2), sjogrens syndrome (n=2), and discoid lupus erythematosus (n=1). inflammatory neuropathy was diagnosed in 21 patients, with small fiber polyneuropathy (n=10), uveitis (n=3), myasthenia gravis (n=2), bells palsy (n=2), and multiple sclerosis (n=1). autoimmune skin conditions were diagnosed in 24 patients with diagnoses including psoriasis (n=13), alopecia (n=8), and vitiligo (n=3). while the mean igm was higher in the patients with autoimmune/rheumatologic manifestations than in other cvid patients (83 vs 57mg/dl), this difference did not reach statistical significance (p=0.15). conclusions: autoimmune and rheumatologic complications are present in over half of patients with cvid. increased vigilance for autoimmune and rheumatologic complications is important as survival outcome are worse in cvid patients with non-infectious complications as previously described. further evaluation of these patients to understand the mechanism of immune dysregulation is essential, as this may promote targeted therapies and improve clinical outcomes. introduction/background: immunoglobulin concentrates have been successfully used for decades to treat patients with primary or secondary immunodeficiency disorders. this treatment has substantially decreased the frequency of life-threatening infections in these patients. octanorm is a newly developed maltose-formulated subcutaneous immune globulin (human) 16.5% liquid for the treatment of patients with primary immune deficiency (pid) and secondary immune deficiency (sid). objectives: biochemical and physico-chemical properties were investigated. methods: molecular size distribution of monomers, dimers, polymers and fragments were determined (according to european pharmacopeia (ep) monograph 8.0) by size exclusion chromatography (sec). igg and igg subclass concentrations were quantified by respective nephelometric methods. functionality of the igg was demonstrated by measurement of fc function, opsonophagocytosis and fc gamma receptor binding assays. dynamic light scattering measurement and size exclusion chromatography were used to characterize the integrity of the igg molecule. measurement of potential procoagulant activity was done by natem and tga (fxia-like activity). the capacity of the octanorm manufacturing process to robustly inactivate/remove pathogens was investigated in spiking experiments with prions and viruses. results: octanorm contains more than 96 % of human igg and is characterized by an especially low content of polymers and aggregates, low viscosity, low isoagglutinin titres, low iga and igm contents with a broad spectrum of antibodies against infectious agents. it has a distribution of immunoglobulin g subclasses closely proportional to that in native human plasma. in the final product, potential procoagulant activity is not detectable. functionality and physico-chemical properties of the igg molecules were demonstrated by state-of-the-art methods. virus safety of octanorm is obtained via a combination of three validated orthogonal methods as part of the manufacturing process: cold-ethanol fractionation, solvent/ detergent (s/d) and ph 4 treatment. a substantial depletion of prions during the manufacturing process was demonstrated. conclusions: octanorm is a state-of-the-art subcutaneous immunoglobulin. based on the excellent stability the intended shelf life of octanorm is 24 months stored at +2°c to +8°c protected from light. within its total shelf life the product can be stored at room temperature up to +25°c for up to six months. efficacy and very good tolerability of this new subcutaneous normal immune globulin 16.5% were shown in a clinical phase iii study performed in 18 centers in north america and europe. professor, sapienza university of rome introduction/background: primary antibody deficiencies (pad) are characterized by defective ig production resulting in high susceptibility to bacterial infections, especially caused by s. pneumoniae and h. influenzae. there is a limited evidence on the rate of microbial airway epithelial colonization and on the role of bacterial carriage on the development of recurrent respiratory tracts infections in such populations. objectives: the aim of this study was to investigate the prevalence of s. pneumoniae and haemophilus influenzae colonization in pad adults in italy and its clinical and immunological correlates. methods: nasopharyngeal and oropharyngeal swabs were obtained from 93 cvid and 16 patients with idiopathic primary hypogammaglobulinemia (iph) over 18 years of age and under ig replacement treatment during the period october 2016-april 2017. presence of s. pneumoniae and h. influenzae was investigated using conventional cultural methods and rt pcr. s. pneumoniae isolates were serotyped by the quellung reaction; capsular type of h. influenzae isolates was determined by pcr. the pattern of associations between the two species and potential risk factors were investigated. respiratory infections rate was recorded over 12 months of follow up. results: among cvid prevalence of carriage assessed by traditional culture was 11% and 27% for s. pneumoniae and h. influenzae, respectively. rt pcr allowed to identify a higher rate of carriage of s. pneumoniae and h. influenzae compared to standard culture. cvid and iph had not different rate of pneumococcal colonization, whereas cvid had higher rate of h. influenzae carriage identified by a culture methods and rt pcr. no synergistic association between s. pneumoniae and h. influenzae colonization was observed. among cvid, s pneumonia and h. influenzae carriage were associated to low iga and igm levels. cvid under antibiotic prophylaxis did not have an increased prevalence of carriage. no association was found between carrier status detected by culture and having chronic lung disease, bronchiectasis or the rate of infections during the follow up. rt pcr identified merely the association between igm levels and h. influenzae carriage. antibiotic resistance from isolated stains was also assessed. conclusions: this is the first study assessing the prevalence of s. pneumonia and h. influenzae carriage in cvid. objectives: to discuss clinical challenges in a subject with congenital hair hypoplasia detected on newborn screening with preserved t-cell mitogen responses but declining naive t-cell counts. parents are jehovah's witnesses, which adds complexity to clinical decisionmaking. methods: immune evaluation included complete blood count, lymphocyte subsets, flow cytometry to define t-cell subsets, immune globulins, repeat trec assay from peripheral blood, human immunodeficiency virus (hiv) and cytomegalovirus (cmv) dna pcr, screen for maternal engraftment, chromosome microarray analysis, lymphocyte proliferation to mitogens, t-cell proliferation to interleukins, t-cell receptor v-beta diversity analysis by spectratyping, and thymic ultrasound. results: repeat trec assay from peripheral blood on day 17 confirmed undetectable trecs. cd4+ thymic emigrants were low at 147 (reference 733-3181 cells/ul). naïve cd4+ and cd8+ t cell compartments declined as did naïve cd4+ t cells from 48 to 17 cells/ul and naïve cd8+ t cells from 33 to 5 cells/ul. lymphocyte proliferation to mitogens was preserved except for the cd19+ response to pokeweed mitogen. interleukin proliferation of cd45+ lymphocytes was slightly decreased after stimulation with anti-cd28 (14%, normal >38%). however, t-cell proliferation with other interleukins (il-2, anti-cd3+il-2, anticd28 as %cd3) was preserved. the t cell receptor repertoire had intermediate diversity. antibody replacement therapy was prescribed for declining igg with no history of severe infections except for rhinovirus prior to discharge from the nicu at 8 weeks, during which she required continuous positive airway pressure (cpap) therapy. the viral load for cmv and hiv dna were undetectable. breastfeeding was discontinued early because the mothers cmv serology revealed positive cmv igg. the child has had intermittent anemia, which is common in patients with chh. [2] tests for autoimmune hemolytic anemia were not performed because the patient required ivig from an early age. the parents of the child are jehovahs witnesses, complicating requests for blood transfusions. conclusions: this case highlights challenges in clinical decision making for a newborn with chh identified on nbs. t-cell function is preserved, but declining naïve t cell counts and absent trecs lead us to consider hematopoietic stem cell transplant (hsct). indication and timing of elective hsct is unclear and may depend on the natural progression of disease, including infections and anemia division of immunology and allergy, the hospital for sick children introduction/background: cartilage-hair hypoplasia (chh), caused by mutations in the ribonuclease mitochondrial rna-processing (rmrp) gene, is associated with diverse immune abnormalities including combined immune deficiency (cid). most patients with chh are managed with supportive measurements, while few have received allogeneic hematopoietic stem cell transplantations (hsct). the progression of the immune abnormalities and the impact of hsct in patients with chh and cid have not been well characterized. objectives: to characterize the progression of the immune abnormalities and the impact of hsct in patients with chh and cid methods: the clinical and laboratory findings of 2 siblings diagnosed in infancy with chh and cid due to the common 70 a>g mutation in rmrp, including the effects of hsct performed in 1 of them, were compared. results: both patients suffered from recurrent respiratory infections at early age with reduced t cells numbers and responses. patient #1 immune function continued to deteriorate leading to hsct from an hla-matched sibling at 4.5 years of age. the patient suffered acute and chronic graft versus host disease of the skin with residual mild joint contractures and scleroderma-like skin changes. seven years after hsct patient #1 has normal immune function. immune evaluations of patient #2 in the first years of life indicated mild improvement. the patient did not have a suitable related hsct donor and the family elected to continue with supportive care. at 7 years of age, patient #2 is clinically well and thriving with persistent t cell abnormalities. conclusions: close monitoring of immune function in early life for patients with chh and cid as well as the availability of suitable donors assists in determining management, including hsct introduction/background: leukocyte adhesion deficiency (lad) represents a group of distinct inherited disorders, which inhibit the normal extravasation of neutrophils and their recruitment to sites of infection or inflammation. objectives presentation of case: the patient is a girl of 8 years old with no history of primary inmunological disease (pid) in family members, including a healthy brother (currently 12 years old). she received all the immunization schedule until 1 year old (according to peruvian schedule). she had several admission to the hospital since newborn; the 1st hospital admission was at 15 days of life with diagnosis of: sepsis, pneumony, onphalitis (leukocytes count: 70 000), she had 3 more hospital admission and the leukocytes count were always above 25000. in summary, she presented other infections besides the ones admitted at hospital like: 2 episodes of sepsis, 1 episode of pneumony, 5 episodes of cellulitis (left eye, left elbow, right thigh and vaginal (2), 27 episodes of otitis, 7 episodes of tonsillitis, 6 episodes of diarrhea, 5 episodes of rhinoadenoiditis, 5 episodes of sinusitis, 3 episodes of gingivitis and 1 episode of whooping cough. she received different antibiotics for treatment, even broad-spectrum as vancomycin, meropenem and cefepime. the diagnosis of leukocyte adhesion deficiency (lad) was made at 1 year 9 months by clinical features like delaying in separation of umbilical cord, recurrent infections and persistent leukocytosis>25000. flow citometry was taken at 3 years old, resulting cd11b/cd18: 0,21%. results: discussionf: or the severe phenotype, in which leukocytes express <1% of normal levels of cd18, death occurs at an early age because of severe infection unless patients receive bone marrow transplant. however, it does not happened with her so we suspected in a possible reversion in lad1 so we took a second flow citometry at 5 years old and the results were: total leukocyte: 27870; linfocyte: 6777 cd11b+/cd18+: 0,04%; granulocyte:17438 cd11b+/cd18+: 1,41% and the conclusion was c3 receptor (cd11b/cd18) absent in linfocyte, monocyte and granulocyte. molecular study was taken at 7 years old, resulting a homozygous substitution c.562c>t identified in exon 5, causing a nonsense mutation: p.arg188, confirming lad1 diagnosis. she is, currently, receiving profilactic antibiotic and antimicotic which has reduced considerably recurrent infections. it seems that our patient may have a mixed phenotype due to a clinical expression, which may not require hematopoietic cell transplantation (hct) despite features of the severe type. conclusions conclusion: we conclude that the clinical evolution of this patient is unsual because she has severe lad1, she has not transplanted yet, profilaxis treatment has improved to decrease frecuency of infections, she exceeded life expectancy and last flow citometry confirmed that lad1 was not reverted. , ph.d. 5 , luigi d. notarangelo, md 6 , troy r. torgerson, m.d. ph.d. 7 , ph.d. 8 , hans d. ochs, m.d. 9 , m.d., ph.d. 10 introduction/background: patients with x-linked hyper-igm syndrome (x-higm) due to cd40 ligand (cd40l) deficiency often present with low blood neutrophil counts. however, even when not neutropenic and despite immunoglobulin (ig) replacement therapy, cd40l-deficient patients are susceptible to life-threatening infections by opportunistic pathogens, suggesting impaired function of phagocytes, and requiring novel therapeutic approaches. objectives: to analyze whether peripheral neutrophils from cd40l-deficient patients display functional defects and to explore the in vitro effects of recombinant human interferon (rhifn)-on such cells. methods: we investigated the microbicidal activity, respiratory burst and transcriptome profile of neutrophils from cd40l-deficient patients. in addition, we evaluated whether the lack of cd40l in mice also affects neutrophil responses. results: neutrophils from cd40l-deficient patients exhibited defective respiratory burst and microbicidal activity which were significantly improved in vitro by rhifn-. similar to humans with cd40l deficiency, cd40l-deficient mice were found to have defective neutrophil responses. moreover, neutrophils from cd40l-deficient patients showed reduced cd16 protein expression and a dysregulated transcriptome profile suggestive of impaired differentiation. conclusions: our data suggest a non-redundant role of cd40l-cd40 interaction in neutrophil development and function that could be improved in vitro by rhifn-, indicating a potential novel therapeutic application for this cytokine. methods: in this study, through the use of 23 healthy livers, paired peritumoural tissues (pt) and intratumoural tissues (it) from 236 hcc patients. results: increased expression of cd96 on nk cells was observed in intratumoral but not peritumoral regions, along with increased expression of its ligand cd155 and a poor prognosis. human cd96+ nk cells exhibited functional exhaustion, showing decreased ifn-and tnf-productions, impaired cytolysis in response to in vitro stimulation, and high gene expression of il-10 and tgf-1 with low expression of t-bet, il-15, perforin and granzyme b by global transcriptomic analysis of sorted cd96+ and cd96-nk cells. blocking tgf-1 specifically inhibited cd96 expression and reversed the dysfunction of nk cells. in addition, we compared other two receptors, cd226 and tigit, which share common ligand cd155 with cd96, and found cd96 plays a more important role in nk exhaustion. conclusions: these findings indicate that human cd96+ nk cells have features of functional exhaustion, suggesting that cd96-cd155 blockade has the potential to restore immunity against liver tumors by reversing nk cell exhaustion. introduction/background: mutations in ncf1 (encoding protein p47phox of the nadph oxidase) result in an autosomal recessive form of chronic granulomatous disease (cgd), a rare genetic disease with impaired phagocyte production of reactive oxygen species and recurrent infections. diagnosis of p47phox cgd is based on abnormal dihydrorhodamine assay and absence of p47phox protein by immunoblotting; however, these assays fail to diagnose carriers of ncf1 mutations. instead, carrier status is inferred after the birth of a child with p47phox cgd. furthermore, identification of the specific genetic defect in patients with p47phox cgd is complicated by two highly conserved (>98%) pseudogenes. the ncf1 gene has a gtgt at the start of exon 2, while the pseudogenes (ncf1b and ncf1c) delete one gt (gt). in p47phox cgd, the most common mutation in ncf1 is gt causing c.75_76delgt; p.tyr26fsx26. sequence homology between the wild type gene and pseudogenes precludes using standard sanger sequencing to identify specific mutations in ncf1. objectives: to identify phenotypic and genotypic differences that facilitate the diagnosis of patients and carriers with p47phox cgd. methods: expression of p47phox in neutrophils is determined by fixing and permeabilizing whole blood with intraprep, and then incubating with either anti-p47phox antibody or its corresponding isotype. alexafluor 488-conjugated secondary antibody is used to detect the target antigen. expression of p47phox is based on the mean fluorescence intensity of cells within the neutrophil population gated using forward and side light scatter. differential expression of p47phox by flow cytometry is validated using quantitative immunoblotting. to screen for the gt mutation, a droplet digital polymerase chain reaction (ddpcr) with two distinct probes recognizing either the wild-type gtgt sequence or the gt sequence was used to quantitate the ratio of gtgt vs. gt copies. a second ddpcr reaction established copy number by comparing one probe for an invariant region of ncf1/ncf1b/ncf1c and a second probe for the single-copy telomerase reverse transcriptase gene, tert. the results of these two assays were combined to determine the total number of gtgtcontaining and gt-containing ncf1 copies. results: analysis of p47phox expression in permeabilized neutrophils determined that neutrophils from p47phox cgd patients had negligible p47phox expression; neutrophils from p47phox cgd carriers exhibited~60% of p47phox expression compared to healthy volunteers independent of the mutation in ncf1. of all p47phox cgd patients tested by ddpcr, 83.2% (109/ 131) exhibited 0 copies of gtgt, 6.9% (9/131) exhibited 1 copy of gtgt (compound heterozygotes with 1 non-gt mutation), and 9.9% (13/131) exhibited 2 copies of gtgt (two non-gt mutations). moreover, ddpcr can identify the carriers among kindreds within the gt p47phox cgd families. unexpectedly, among normal subjects tested, only 78.8% exhibited the expected 2 copies of gtgt per 6 total ncf1/ncf1b/ncf1c copies, designated 2/6; a significant number exhibited more than two copies of gtgt (14.6% with 3/6, 1.9% with 4/6); others exhibited ncf1/ncf1b/ncf1c copy number variation (2.8% with 2/7 and 1.9% with 2/5). conclusions: flow cytometric analysis of neutrophil intracellular p47phox staining provides a quick method to identify patients and carriers of p47phox cgd. droplet digital pcr can be used to identify patients and carriers of p47phox gt, the most common mutation in p47phox cgd copy number variation is observed at the ncf1 locus among normal subjects tested. introduction/background: defects in the cd3 subunits of the tcr/cd3 complex account for a small percentage of the scid presentations. cd3 deficiencies are characterized by profound and t-cells lymphocytopenia, and normal numbers of b-and natural killer (nk) cells in the peripheral blood (t-b+nk+ scid). thymocyte development from double negative stage to double positive stage results arrested. affected individuals typically present with severe and opportunistic infections in the early infancy. objectives: to evaluate possible underlying immunodeficiency disorder in the setting of chronic ebv infection. methods: here we report our experience of a patient with an atypical presentation of cd3 deficiency. results: a 7-year-old girl previously healthy, first generation americanborn of gambia immigrants was referred to our clinic for further evaluation of chronic ebv infection. one year before presentation, she developed bilateral parotid enlargement, cervical, axillar, and hilar lymphadenopathy, bronchiectasis, and pulmonary nodules. relevant previous studies included: ebv viremia (pcr quant 63,000 copies), ebv igg and ea positive whereas igm and ebna were negative. peripheral blood phenotyping was not suggestive for malignant process and cervical lymph node biopsy was consistent with a reactive process without evidence of a clonal lymphoproliferative disorder and bal studies positive for ebv only. hiv and tb both negative. she was otherwise thriving with no history of recurrent infections and family history was negative for consanguinity or immunodeficiency. our evaluation revealed: normal blood counts, ebv pcr quant 206,677 copies, normal immunoglobulin levels and vaccine titers. she had a normal lymphocyte subsets, but skewed cd45 ra/ro consistent with low thymic output (very low cd4 naïve t cells (5.8%), low cd31+ naïve t cells (36.7%), and elevated temra (42.6%)), poor mitogen, and antigen responses. all these findings were suggestive of a scid like phenotype; therefore, scid next generation sequencing panel was pursued, and showed a novel homozygous splice site mutation (c.55 +1 g>t) in the cd3 gene. conclusions: a homozygous mutation in the cd3 gene might not necessarily imply profound t-cell lymphopenia. though this patient did not present with classic clinical course, and immune findings; her inability to clear ebv with persistent significant viremia does support a t-cell immune deficiency. she remains at risk for ebv-associated lymphoproliferative disorder, infections, and autoimmunity. bmt will be a curative treatment option. however, it is difficult to predict evolution of clinical phenotype given the atypical presentation. this case illustrates the importance of contextual interpretation of clinical findings, laboratory data, and genetic analysis for treatment approach. introduction/background: pol is multi-subunit polymerase that includes both pole1 with catalytic activity and additional pole2, 3 and 4. this holoenzyme plays a key role in proofreading damaged dna and is required for proper dna replication in proliferating cells, such as lymphocytes. germline mutations are linked to rare cause of primary immunodeficiencies whereas somatic mutations are described in colon cancer. primary immunodeficiencies are reported pole-1 deficiency in members of a large consanguineous french kindred and a palestinian female with fils (facial dysmorphism, immunodeficiency, livedo, and short stature). all reported cases with pole1 deficiency have homozygous intronic splice site variant (c.4444+3a>g) that result in a deletion of exon 34 which lead to subsequent frame shift (from p.s1483v onwards) and a premature stop codon at position 1561; this transcript results in a degraded product. the proportion of the pole1 transcript in t lymphoblasts is significantly lower (10%) in patients then carriers or healthy individuals. objectives: hereby we describe the clinical progression and treatment challeneges of the palestinian female with pole-1 deficiency secondary to homozygous g.g4444+3 a>g substitution. results: initially patient presented with viral and recurrent ear infections and cmv viremia. with age, patient had less episodes of infections even with intermittent pause in immunoglobulin replacement therapy (igrt), however had multiple admissions for fever of unknown origin with negative cultures but increased ferritin level (3300 ng/ml) and low platelet count (46,000 count/ml). autoimmune and inflammatory complications were not reported among the french kindred. her skin also worsened with poor wound healing and scarring throughout that hinders igrt via subcutaneous route. furthermore, igrt with intravenous administration has resulted in symptoms of aseptic meningitis likely related to the underlying inflammatory state. in addition, patient shows decline in immune dysfunction. initially, she had normal immunoglobulin g (igg) however by 5 years of age, she developed hypogammaglobulinemia with low igm unlike in the french family with patients. also, pneumococcal, diptheria, tetanus titers were non-protective off of immunoglobulin replacement therapy (igrt). beyond low switched memory b cells, patient also developed low b cell by 5 yo age. t cell dysfunction continued to decline from decreased lymphocyte proliferation to antigens at 2 yo age to fully absent lymphocyte proliferation to antigens and mitogens. naïve cd4 and cd8 compartments continue to be preserved. currently management challenges include treatment strategies for thrombocytopenia, inflammation and progressive skin disease that complicates the proper selection of route for optimal igrt. conclusions: beyond progression of immunological decline, our patient developed inflammatory phenotype with age. the progression of her immunological decline may be related to further decrease in the proportion of the wild type pole1 transcript and yet to be examined. overall, clinical follow up is essential in patients with pole-1 deficiency as phenotype can change with age and may pose new challenges. longitudinal follow up studies are needed to uncover the potential role of germline pole1 and pole2 pathogenic variants in cancer susceptibility. introduction/background: x-linked hyper-igm syndrome (xhigm) is one type of primary immunodeficiency diseases, resulting from defects in the cd40 ligand/cd40 signaling pathways. objectives: here, we retrospectively reviewed clinical, laboratory and genetic characteristic of xhigm in chinese population, thus further improving diagnosis and treatment for xhigm. methods: we collected and analyzed 47 chinese patients, who were diagnosed and followed up in hospitals affiliated to shanghai jiao tong university school of medicine from 1999 to 2016. targeted gene capture combined with next-generation sequencing technology and sanger sequencing were used to find out related gene mutation. results: the median onset age of these patients was 7 months (range: 20 days66 months). thirty-six percent of them had positive family histories, with a shorter diagnosis lag. the most common symptoms were recurrent sinopulmonary infections (40 patients, 85%), neutropenia (22 patients, 47%), protracted diarrhea (21 patients, 45%), and oral ulcer (19 patients, 40%). ten patients had bcgitis. six patients received hematopoietic stem cell transplantations and four of them had immune reconstructions and clinical remissions. twenty-seven unique mutations in cd40l gene were identified in these 47 patients, with 18 novel mutations. conclusions: to our knowledge, this report provides the largest cohort of patients with xhigm in china. mutation analysis is an important tool for xhigm diagnosis. infants with scid are asymptomatic at birth and unless prompt diagnosis of the disease is made they may horrifically be vaccinated. simultaneous appearance of two live vaccine associated infections in one person is rarely reported. objectives: in this study we present two infants with scid, who received bcg and oral polio vaccines early in life before the diagnosis of immune deficiency was made. both patients developed localized and disseminated infections originating from the bcg vaccine (bcgitis and bcgiosis, respectively) and in addition were diagnosed with chronic fecal secretion of vaccine-derived polio virus (vdpv); alarmingly, in both cases, the vdpv underwent reverse mutation of attenuated sites to the neurovirulent genotype. the rarity of concomitant infection from two live vaccines in one recipient, together with the multiple complexities originating from these infections in immunodeficient infants, led us to report these cases and to inquire the pathogenesis that underlies this unique condition. methods: immunological evaluation:: cell surface markers of peripheral blood mononuclear cells (pbmcs) were measured by immunofluorescent staining and flow cytometry serum concentrations of immunoglobulins were measured using nephelometry. quantitative analysis of the tcr v repertoire was performed by means of flow cytometry. quantification of t cell receptor excision circles (trecs) was determined by real-time quantitative (rq)-pcr. genetic analysis: genetic diagnosis of scid was made for patient 1 by direct sanger sequencing of candidate genes and retrieval of mutation in rag2 , and for patient 2 by wes (whole exome sequencing), followed by validation of the dna cross-link repair 1c (dclre1c) mutation using sanger sequencing. poliovirus detection and characterization: stool samples were collected monthly and transported to the national poliovirus laboratory located at israel central virology laboratory (icvl) for polio detection and characterization. results: in both patients, immunological workup revealed undetectable serum iga and igm levels with normal igg levels (table 1) , lymphocyte immune-phenotyping using flow cytometry revealed complete absence of t and b cells with presence of nk cells (table 2) . trecs, a dna marker of naive t cells and thymic output, were absent in both patients. the diagnosis of scid was made. we initiated prophylactic antibiotic (trimethoprim-sulfamethoxazole) and anti-fungal (fluconazole) treatment, as well as monthly intravenous immunoglobulin (ivig) infusions. genetic workup: the t-b-nk+ scid phenotype in patient 1 led us to search for a mutation in the rag complex genes. indeed, a sanger sequencing of the rag2 gene, revealed a g104t homozygous mutation which predicts an amino acid substitution from glycine to valine in position 35 (fig.1 ). for patient 2, who had a similar t-b-nk+ scid phenotype, we identified a homozygous mutation in the dclre1c gene (del.4bp, c.817-cttt) (fig. 2) , using wes. the genetic evaluation confirmed the diagnosis of scid due to rag2 deficiency in patient 1 and artemis deficiency in patient 2. clinical course: during hospitalization patient 1 developed disseminated bcg related disease with skeletal lesions involving the phalanx, tibia and maxillary bones, as well as involvement of the spleen, liver and pancreas. anti-tubercular therapy with isoniazid, rifampicin, ethambutol and ciprofloxacin was initiated. due to lack of response, empirical trial of g-csf (granulocyte colony-stimulating factor), in order to enhance macrophage activity . the patient showed good response to this combination therapy. patient 2 developed a palpable rigid mass on her left shoulder with surrounded redness at the site of the bcg vaccine at the age of 5 months. the clinical diagnosis of bcgitis was established and triple antitubercular therapy with isoniazid, rifampicin and ethambutol was initiated with good response. throughout their hospitalization, both infants suffered from intermittent diarrhea. pcr for enterovirus was performed and detected the presence of type 3 and type 2 vaccine-derived poliovirus (vdpv) in patient 1 and 2, respectively. during the follow-up, stool samples collection revealed accumulation of several polio virus mutations and some of the neuro-virulence attenuation sites were reverted to the neuro-virulent genotype .fortunately, both patients did not show any signs of flaccid paralysis. precautionary measures of isolation were taken to prevent spread of the vdpv. eventually, both patients underwent allogeneic bone marrow transplantation (bmt): patient 1 had bmt without pre-conditioning, from a matched sibling donor. due to engraftment failure, a second bmt was repeated, this time successfully. on follow up examination her t cell repertoire showed a normal tcr v polyclonality and trec was detected, indicating the emergence of new t cells. due to ongoing low immunoglobulin levels this patient is still on regular ivig-infusions and prophylactic antibiotic treatment, as well as isoniazid. currently, she is well, her bcgitis is not active and her stool specimens are negative for polio. patient 2 underwent an urgent haplo-identical bmt with alpha-beta t cell depletion without pre-conditioning, due to her unstable medical condition. flow cytometry analysis six months post bmt revealed lymphopenia of 7.7% (1334/mm³) with low mature t cells (cd3 32%) and absent b cells. trecs were barely detected. microsatellite analysis as a marker for engraftment revealed a stable donor chimerism of 10%. the patient is still on immunosuppressive therapy doing well, her bcgitis is not active and her stool specimens are negative for polio. conclusions: these cases highlight the importance of early recognition of scid by neonatal screening or thorough family anamnesis, and the need to further defer the timing of administration of live vaccines. introduction/background: measurement of b cells, b cell subsets and specific antibodies produced in response to vaccination are key tests used to investigate immune system function. specific antibody production may indicate b cell functionality. objectives: in this study the correlation between the different b cell subsets and antibody responses to pneumovax® in an immunocompromised population was investigated. methods: b cell subsets were assessed by flow cytometry and pneumococcal responses measured using the vacczyme pneumococcal capsular polysaccharide (pcp) igg, iga and igm elisas (the binding site group, birmingham, uk) in 39 primary immunodeficiency patients (pid) vaccinated with pneumovax®. lower limits of normal were defined as follows: b cells: 6.6%; naive b cells: 65.6%; non-switched memory b cells: 7.4%; switched memory b cells: 7.2%; pcp igg: 50 mg/l; pcp iga: 125 u/ml; and pcp igm: 140 u/ml. results: the correlation coefficients between percentage of b cells/b cell subsets and igg, iga or igm pneumovax® responses ranged from -0.61 to 0.39. the percentage of the cohort achieving a normal b cell and normal igg, iga or igm response to pneumovax® was 45%, 29% and 40% respectively. b-cell responses were measureable in the remaining patients but they did not produce normal concentrations of pcp igg, iga or igm. further stratification of patients who achieved normal percentage of switched or un-switched b cells but who failed to achieve normal igg, iga or igm responses to pneumovax® were 32%, 55% and 50%, respectively, for un-switched and 22%, 47% and 33%, respectively, for switched b cells. conclusions: the combined measurement of b cells and response to vaccination are required to provide a detailed insight into these disorders. introduction/background: this is a 2-year-old boy of african american heritage with multiple congenital malformations who was diagnosed with very early-onset inflammatory bowel disease, which proved to be resistant to treatment. subsequent testing revealed a heterozygous mutation in exon 1 of ctla4. this variant has not been previously reported in the literature in individuals with ctla4-related disease. heterozygous mutations in ctla4 cause a disease of immune dysregulation. clinical presentation is variable and may be characterized by enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung disease, lymphocytic organ infiltration in non-lymphoid organs, autoimmune cytopenias, and recurrent infections. early onset colitis has been reported with ctla4-related disease and is unique to our patient's initial clinical presentation. objectives: this is a 2-year-old boy with cloacal exstrophy of the urinary bladder, omphalocele, imperforate anus, polydactyly, and sacral agenesis who was diagnosed with very early-onset inflammatory bowel disease at six months of age. he underwent cloacal exstrophy closure, omphalocele repair, and colostomy placement in the first week of life. at six months of age, he presented with dark tarry stools. upper endoscopy and colonoscopy revealed polyps, ileitis, and colitis. he was p-anca positive and started on sulfasalazine. unfortunately, he continued to have symptoms suggestive of active colitis, prompting a change to prednisone and azathioprine. despite therapy, his colitis persisted leading to chronic bloody diarrhea and growth failure. initial immune evaluation consisted of a normal complete blood count, serum immunoglobulin, lymphocyte subsets, and neutrophil oxidase burst assay. foxp3 analysis by flow cytometry showed a moderately elevated percentage of foxp3+cd25+ cells in the cd4+ t cell population, but the regulatory t cell immunophenotype was normal. because suspicion was high for a monogenic immunologic disease to explain his symptoms, genetic sequencing was performed. a candidate gene panel was sequenced by next generation sequencing, and a heterozygous mutation in exon 1 of ctla4 (c.23g>a; p.arg8gln) was found. this variant has not been previously reported but is predicted to be pathogenic in exac and polyphen databases. results: the patients diagnosis of ctla-4 haploinsufficiency associated with very early-onset inflammatory bowel disease has provided opportunity for targeted treatment of his specific molecular defect. given his poor response to treatment thus far, the patient will be started on abatacept. abatacept is fda approved for the treatment of rheumatoid arthritis but has been used successfully for the treatment of disease-related manifestations of ctla-4 haploinsufficiency. abatacept is a ctla-4 fusion protein formed by the igg1 fc region linked with the extracellular domain of ctla-4; it replaces the defective protein in ctla-4 haploinsufficiency. in addition, given other manifestations of ctla-4 haploinsufficiency including lymphoproliferative disease in non-lymphoid organs, particularly the brain and lung, we have initiated further evaluation of these organs to evaluate for disease-specific manifestations. conclusions: the protein cytotoxic t lymphocyte antigen-4 (ctla-4) is an essential negative regulator of t cells. heterozygous mutations in ctla4 cause a disease of immune dysregulation. clinical presentation is variable and may be characterized by enteropathy, hypogammaglobulinemia, granulomatous lymphocytic interstitial lung disease, lymphocytic organ infiltration in non-lymphoid organs, autoimmune cytopenias, and recurrent infections. inflammatory bowel disease may be associated with certain variants in ctla4, but the literature remains limited, both by number of papers published as well as by ethnic subsets studied. clinicians who are presented with children who have early-onset colitis, and particularly inflammatory bowel disease that is difficult to treat, should consider possible genetic abnormalities, such as ctla-4 haploinsufficiency, as these can impact therapeutic decision-making and outcomes. introduction/background: dock8 deficiency is an autosomal recessive combined immunodeficiency syndrome associated with recurrent infections, eczema and other atopic diseases. the infections are usually viral, bacterial and fungal resulting in predominantly cutaneous and sinopulmonary manifestations. homozygous or compound heterozygous deletions or mutations in the dock8 gene (9p24) lead to abnormal cytoskeletal organization and impaired function of dendritic cells and lymphocytes. an aniline derivative belonging to the group of synthetic sulfones, dapsone has been employed in the treatment of chronic skin diseases characterized by an accumulation of neutrophils and eosinophils. methods: chart review of one patient results: we present a four-year-old male with severe eczema, persistent asthma, allergic rhinitis, as well as peanut and egg allergy suffering from recurrent skin abscesses and prurigo nodularis. abscesses began at age 18 months and required prolonged courses of antibiotics, eight in total prior to presentation. other infectious history included otitis media and lymphadenitis. there was no history of pneumonia or other severe infections. skin abscesses responded to oral antibiotics, but recurred shortly after completing extended courses of treatment. laboratory results including quantitative immunoglobulins, specific antibody titers, myeloperoxidase staining, neutrophil oxidative burst and complement were within normal limits. laboratories were notable for elevated ige (10080 iu/ml) and eosinophilia (1200 eosinophils/microl). lymphocyte immunophenotype was significant for mild elevations in cd3 and cd4. dock8 genetic sequencing by genedx revealed a heterozygous missense mutation in exon 17 (c.1979 c>a, amino acid change p.ala660asp). abscess cultures grew methicillin sensitive staphylococcus aureus (mssa) and enterococcus faecalis. mssa was sensitive to ampicillin/sulbactam, cefazolin, gentamycin, moxifloxacin, oxacillin, rifampin, tetracycline, and vancomycin. isolate was resistant to ciprofloxacin, clindamycin, erythromycin, levofloxacin, penicillin, and trimethoprim/ sulfamethoxazole. the patient was initially treated with emollients, mupirocin washes, topical steroids, anti-histamines, bleach baths, and cephalexin three times a day. he improved clinically but was unable to tolerate cephalexin for more than ten days secondary to abdominal pain. cephalexin, with addition of probiotic, was attempted several months later and again had to be discontinued because of abdominal pain and vomiting. due to limited antibiotic options, dapsone was started after ruling out glucose-6-phosphate dehydrogenase deficiency. dapsone was initiated at a dose of 1.5mg/kg, and the patient was monitored weekly for hemolytic anemia. after two weeks of treatment, anemia was noted and the dose was decreased to 1mg/kg. he has continued on dapsone 1mg/kg once a day, with significant improvement in abscess number and severity. he has not required other systemic antimicrobials since starting dapsone. conclusions: dapsone may be considered as a treatment option for children with heterozygous dock8 mutation and recurrent abscesses, particularly those requiring prophylaxis, long term treatment and lacking antibiotic options. introduction/background: autosomal dominant hyper-ige syndrome (ad-hies) is a rare complicated primary immunodeficiency disease (pid). signal transducer and activator of transcription 3 (stat3) gene mutation is found to cause ad-hies. tlr7/9 signaling plays multiple roles in b cell proliferation, activation, class-switch recombination, and cytokine and antibody production. however, little is known about b cell response to tlr7/9 agonist in patients with ad-hies. objectives: here, we aim to study the response of b cells from ad-hies patients to the tlr7/9 agonist. methods: pbmcs were isolated from peripheral blood of 5 ad-hies patients and 10 age matched healthy controls. pbmcs were stimulated with tlr7 and tlr9 agonist (r848 and cpg odn 2006, respectively), then b cells were analyzed for proliferation, the expression of certain surface markers (cd40, cd80 and cd86), intracellular immunoglobulin levels (igm and igg) and intracellular cytokine levels (il-6 and il-10) by flow cytometry. results: in response to tlr7/9 agonist, proliferative capabilities of b cells were reduced in ad-hies patients compared with those in age-matched healthy controls. besides, defective costimulatory molecule cd80 expression was observed in b cells from ad-hies patients. furthermore, significantly lower igm and igg levels, and il-10 production was detected in b cells from ad-hies patients. however, there was no significant difference in b cell apoptosis between ad-hies patients and healthy controls. conclusions: these data demonstrated that stat3 gene mutations in ad-hies patients contributed to impaired b cell tlr7/9 signaling, and further affected b cell proliferation, activation, cytokine secretion and antibody production. introduction/background: antibody function is most commonly measured by a rise in antibody titers in response to antigen introduced by vaccination or natural infection. specific antibody deficiency is defined as normal serum levels of immunoglobulins with reduced or absent antibody response to antigens, often after administration of the pneumococcal vaccine polyvalent (pneumovax® 23). a paucity of information exists about measurement of antigen-antibody binding, or avidity, as a measure of antibody function, including persons with recurrent sinopulmonary infections who have normal response to immunization with pneumococcal vaccine polyvalent. objectives: the aims of this study are to identify and evaluate children with recurrent sinopulmonary infections who had appropriate rise in pneumococcal antibody titers following immunization with pneumococcal vaccine polyvalent but low response by avidity, and to assess response with igg replacement therapy in these patients. methods: a retrospective chart review involved eight children with recurrent sinopulmonary infections with discordant pneumococcal antibody and avidity results following vaccination with pneumococcal vaccine polyvalent. these eight children subsequently received igg replacement therapy. results: the mean age of subjects was 9.75 (range 2 -15) years. the mean number of serotypes with a normal antibody response (>1.3 ug/ml) among 8 children following immunization with pneumococcal vaccine polyvalent was 18.1 (range 12-22) of 23 serotypes while the mean number of serotypes with a normal avidity response (1.0) was 4.4 (range 2-7) of 23 serotypes. igg replacement was administered subcutaneously in 8 children. the mean igg level was 720 mg/dl. local reactions were all mild and observed in 4/8 (50%) children. no serious adverse events were reported. all 8 children experienced a marked reduction in respiratory illnesses while on igg replacement therapy. conclusions: discordance between pneumococcal antibody titers and pneumococcal avidity titers was identified in eight children with recurrent respiratory illnesses. in children with recurrent sinopulmonary infections despite normal antibody response to pneumococcal vaccine polyvalent, measurement of pneumococcal avidity may identify patients with poor pneumococcal antibody function. igg replacement in these children was well tolerated and associated with a decrease number of respiratory infections. introduction/background: exome sequencing (es) is a powerful genomic tool that can be used to identify novel molecular causes of disorders with multiple etiologies. immunologic disorders are clinically and genetically heterogeneous, and therefore present unique diagnostic challenges both in the clinic and in the laboratory. objectives: the objective of this study was to assess the utility of es for determining the genetic etiology of immunological disorders and describe diagnostic yield and outcomes of exome sequencing (es) for patients with immunologic disorders and immunologic phenotypes. methods: a retrospective review was performed of 315 individuals referred for clinical es for primary abnormalities of the immune system and individuals with additional phenotypes where multiple immunologic features were reported as part of the clinical picture, as determined during internal curation. analysis of clinical es data was performed by boardcertified clinical geneticists and all variants reported were confirmed by a secondary methodology. positive es outcomes required a pathogenic or likely pathogenic variant in a gene with autosomal dominant or x-linked inheritance, or compound heterozygous or homozygous pathogenic or likely pathogenic variants in a gene with recessive inheritance. results: the most common clinical indications for es in this cohort were hypogammaglobulinemia (16%), neutropenia (14%), immune dysregulation (10%), lymphopenia (9%), and combined immunodeficiency (8%). the gender distribution was 59% male (n=185) and 41% female (n=130); 76% of cases were pediatric (<18 years, n=240) and 24% were adult (>=18 years, n=75). positive results were reported in 83 cases (26%), comparable to the overall diagnostic yield of es at our laboratory (retterer et al., 2016) . this included 20/90 (22%) of cases submitted as proband-only, 47/178 (26%) submitted as a trio, and 16/47 (34%) submitted as duo, quad or alternative family structure. diagnostic results spanned 70 different genes and recurrently reported genes with identified pathogenic variants included flg (n=10), rag1 (n=5), sbds (n=4), lrba (n=3), stat1 (n=3), and pik3cd (n=3). variants possibly associated with the phenotype, but not considered diagnostic, were reported in 58% of cases (n=183), while 13% of cases (n=41) had reportable findings in a candidate gene. conclusions: these results support that exome sequencing for individuals with immunologic-based phenotypes has similar diagnostic utility as the overall rate for clinical es. immunologic es cases with trio family structures have a higher diagnostic yield than proband-only cases, as inheritance information improves confidence in classification of variants as pathogenic or likely pathogenic. genetic heterogeneity, as demonstrated by the large number of distinct genes represented in this cohort of diagnostic es cases and rapid candidate gene discovery make es a valuable tool for genetic diagnosis in patients with immunological disorders. introduction/background: vaccination response to the 23-valent polysaccharide vaccine (ppsv23) is often used in the diagnosis of common variable immunodeficiency (cvid). unfortunately, ppsv23 titers are often difficult to interpret and many cvid patients are started on igg replacement therapy (igrt) before adequate evaluation. unlike ppsv23 titers, the enzyme-linked immunosorbent spot assay (elispot) is independent of igrt and can provide an ex vivo functional measurement of specific antibody production on the b cell level. objectives: develop and test an elispot assay to better determine vaccination response to ppsv23 compared to ppsv23 titers in cvid patients on igrt, healthy controls, and igrt patients without immunodeficiency. methods: an elispot assay was successfully optimized and used to evaluate the ppsv23-specific b cell response in 8 healthy adult controls. elispots were performed on day 1, and day 7 (when plasmablasts are best evaluated). ppsv23 titers were measured on day 1, day 7, and day 30. for igrt patients, flow cytometry for b cell subpopulations will be performed to further validate the assay. results: normal controls demonstrated a significant increase in ppsv23 antibody spot forming units (sfu) between day 1 and 7 after ppsv23 vaccination. ppsv23 titers showed generally robust initial titers at day 1, and no significant change at day 7, with day 30 results pending. conclusions: here we optimized an elispot assay that functionally measures the specific antibody response to ppsv23 in normal controls with ppsv23 titer results pending. we are actively recruiting patients on igrt (both with cvid and without immunodeficiency) for comparison. we hope to validate our assay as a useful alternative to ppsv23 titers that may be particularly useful when patients are on igrt. previous studies have demonstrated bal may be a sensitive diagnostic method for treatment failures of clinically diagnosed pneumonias, even if performed under treatment with empiric antibiotics, and can lead to a culture-directed change in antimicrobial therapy in the majority of cases. however, it has also been reported that at least in one piddchronic granulomatous disease (cgd)the diagnostic yield of bal was inferior to that of other diagnostic methods (marciano et al., 2015) . further information on the diagnostic yield of bal and other invasive procedures to obtain a specific organism diagnosis in pidd patients with suspected pulmonary infection is needed. objectives: to characterize the yield of diagnostic procedures used in pidd patients with pneumonia or other suspected pulmonary infections at the ucsf benioff childrens hospital. methods: we screened our database of pidd patients (encompassing patients seen from september 1, 1998 to september 1, 2017 cared for by the pediatric immunology service at the university of california, san francisco to identify patients with history of at least 1 bal or other invasive diagnostic procedure (fna or open lung biopsy) for etiologic diagnosis of suspected pulmonary infection. if multiple bals were performed during a single episode of illness, only the first was used for this analysis. results: we identified 22 pidd patients with history of at least one bal or other invasive diagnostic procedure, for a total of 55 events. most procedures (n=53) were performed at our institution, with 2 documented in outside hospital records. patient diagnoses included cgd (11), nemo deficiency (4), cvid (3), stat3-deficient ad-hies (2), dock8 deficiency (1) , and mhc class ii deficiency (1) . of 49 bals, 24/49 (49%) grew a predominant organism, but only 18/49 (37%) were positive for an organism believed to be causative by providers and/or for which the overall result of the bronchoscopy affected antimicrobial treatment. bal yield was highest in patients with a clinical and/or radiologic diagnosis of pneumonia (13/27, 48%). yield was poor (3/16, 19%) in minimally or chronically symptomatic patients referred for bal for interval changes on chest ct (i.e. suspected fungal infection). our nemo deficiency cohort had the highest rate of positive organism isolation by bal (6/11, 66%) for diagnosis of pulmonary infection. in our cgd cohort, 11/ 26 (42.3%) bals grew a causative organism. lung biopsy yielded positive organism isolation in 2/3 cases (2/2 in cgd, burkholderia cepacia and nocardia cyriacigeorgica; 0/1 in cvid) and fna in 1/2 cases of cgd (aspergillus fumigatus). fna or biopsy was done concurrently or after bal in 5 cases; in 4/5 (80%), fna or biopsy, but not bal, was positive for a causative organism. conclusions: at our institution, bal overall had a 37% rate of causative organism isolation in pidd patients, but had up to a 50% rate of organism isolation in those with clinical and/or features of pneumonia. our rate of causative organism isolation was slightly higher than in previous reports. however, in specific instances, biopsy was still required to make a definitive diagnosis. bal may have limitations in certain populations of pidd patients, such as in cgd, but it may be a reasonable starting point in the diagnosis of pneumonia or worsening pulmonary disease in pidd. prospective research is needed to evaluate whether fna or lung biopsy, though more invasive, could result in overall shorter time to institution of appropriate directed therapy and shorter hospitalizations for specific pidd patients. introduction/background: prior to the introduction of newborn screening, cases of severe combined immunodeficiency often presented with severe or disseminated infections. herein, we report an infant from india who presented for evaluation and treatment of a periorbital mass, presumed to be malignant. however, he was found to have disseminated bacille calmette guerin (bcg), as well as multiple other infections, and was eventually diagnosed with x-linked scid. results: a 4-month old boy was born to unrelated parents in india. he initially presented with growing periorbital mass and fever for two weeks. pet scan showed hypermetabolic areas in the bone marrow, spleen, mesenteric lymph nodes, and left shoulder, along with the periorbital mass. biopsy of the mass revealed numerous b lymphocytes without malignant transformation. there was no evidence for malignancy on bone marrow biopsy, though numerous granulomas and a significant decrease in t lymphocytes were seen. peripheral blood flow cytometry showed a complete lack of t cells. the bone marrow biopsy was reexamined, and innumerous acid-fast bacilli were found. cultures grew mycobacterium bovis, and he was diagnosed with disseminated bcg disease. genotyping revealed a novel splice site variant in il2rg, consistent with x-linked scid. further evaluation revealed multiple other infections. these included extended-spectrum beta lactamase-produce e. coli bacteremia, human metapneumovirus, cytomegalovirus, and pneumocystic jiroveci. he was also tested for vaccine-associated poliovirus because he had received the oral polio vaccine, but this was negative. four-drug therapy was started for the bcg, and the periorbital lesion completely resolved within several weeks. additionally, he was treated with intravenous ribavirin for the human metapneumovirus, and sulfamethoxazole-trimethoprim for the p. jiroveci. although the cmv was initially treated with ganciclovir, the virus eventually developed resistance and required treatment with foscarnet. due to his many infections, he underwent haploidentical stem cell transplant plus donor lymphocyte infusion from mom. this transplant failed to engraft, but a matched unrelated donor was eventually found, and our patient received a second bone marrow transplant. he currently is showing signs of engraftment, and is continuing to be treated for his multiple infections. conclusions: disseminated mycobacterial disease due to the bacille calmette-guérin (bcg) vaccination has been noted in cases of xlinked scid previously, often consisting of lymphatic, skin, and pulmonary manifestations. however, there is a paucity of published cases presenting with multiple other co-infections. nor are there reports of disseminated bcg presenting as a localized mass. this case highlights the unique considerations when evaluating a patient with immunodeficiency from another country, where vaccination practices and epidemiology differ. specifically, unusual presentations of infections or masses may warrant investigation for severe immunodeficiency. prototypic t-b+nk+ immune phenotype is caused by mutations in the iil7ra gene. the il7 signaling has an important role during t-cell development in the thymus, contributing to cell proliferation and survival. in addition, in mouse models, the rearrangement of the t cell receptor genes, specifically the gamma locus (trg), has been shown to be regulated by il7 signaling. similar to other scid phenotypes, patients with il7ra deficiency are predisposed to acquire opportunistic infections early in life and to display poor outcome and death, unless their immune system is restored by mean of hematopoietic stem cell transplantation (hsct). while most patients with il7ra mutations have full il7ra deficiency resulted in a severe t cell depletion, some have a partial deficiency with residual cells or leaky phenotype, or even present symptoms later in life. objectives: here we report two non-related infants detected by the israeli national newborn screening program for scid. despite having similar il7ra missense mutation (f40l) they displayed distinct clinical and immunological course, resulted in a completely different treatment approaches; observation in one patient with an unusual recovery, and hsct in the other patient methods: patient lymphocytes were examined for subset counts, thymic output (via excision circles), t cell receptor repertoire diversity (tcrb) and il7ra expression and function. the pathogenic il7ra mutation was found by whole exome sequencing (wes). high throughput immunesequencing was performed to characterize the trg repertoire. results: we established patients' diagnosis by validating the pathogenic il7ra mutation, and showing profoundly impaired t cell immune work up and abnormal il7ra expression and function, determined by stat5 phosphorylation assay. all these measurements improved over time for the patient with less severe clinical presentation, while remained low in the patient with the severe phenotype. characterization of their trg immune repertoire using high throughput immune-sequencing revealed restriction of t cell receptor repertoires of both patients upon their initial diagnosis, compared to healthy controls. however, skewed usage of variable (v) gene segments and abnormalities of the cdr3 length distribution were more prominent in the patient with the severe phenotype. conclusions: these studies illustrate the gap that exists in our understanding of other non-genetic parameters that may influence disease course and severity in patients harboring a similar genetic defect. furthermore, the results reinforce the role of il-7 signaling not only in cell proliferation but also in trg rearrangements introduction/background: dock8 immunodeficiency syndrome is primary immunodeficiency disease caused by loos of function mutations in the dock8 gene, which was known to play a critical role in the survival, proliferation and function of several types of immune system, especially lymphocyte. dock8 immunodeficiency syndrome is the most common cause of autosomal recessive hyper-immunoglobulin e syndromes (hies) and mainly expressed as recurrent infections and severe allergic disease affecting the skin. in addition, autoimmune features including systemic lupus erythematosus, hemolytic anemia or idiopathic thrombocytopenic purpura may be presented in dock8 immunodeficiency syndrome. objectives: we report a case of 16-month-old boy diagnosed with dock8 immunodeficiency syndrome, which was initially expressed as sle without recurrent skin infections. methods: a child with atopic dermatitis was admitted to another hospital because of fever lasting more than 3 days accompanied by swelling of the hands and foot. he developed whole body edema, perioral purpura and oliguria. complete blood count was normal and blood urea nitrogen , creatinine and albumin levels were normal on his laboratory findings. however, c-reactive protein level was high at 9.85 mg/dl, coagulation parameters were abnormal (prothrombin time 14.8 sec; activated partial thromboplastin time 67.0 sec, d-dimer 5.82 ug/ml). so he was transferred to our hospital for further examination and treatment on the 5th day of fever. additionally, ulceration of the tonsil and maculopapular rashes on the abdomen and both legs were observed in physical examination. we suspected a meningococcal infection and administered antibiotics. however, no bacterial isolates were identified in the blood and csf culture test. fever persisted despite the administration of antibiotics. we checked immunoglobulin level, complement level and autoantibodies based on fever of unknown origin. immunoglobulin g, m and a were normal, complement fractions c3, c4, and ch50 were low at 80.0 mg/ dl, 6.7 mg/dl and 13.3 u/ml, respectively. antinuclear antibodies were positive at 1:128 with homogenous fluorescence. anti-ds dna antibody was positive at 84.4. the tests for anti-ssa, anti-ssb, anti-ribonucleoprotein, anti-scleroderma 70, and anti jo1 antibodies were all negative. he developed leukopenia and thrombocytopenia over time. results: he was treated with steroid satisfied diagnostic criteria for systemic lupus erythematosus (sle) and fever subsided. he was confirmed lupus nephritis by renal biopsy later. because of onset of sle at the young age, we performed diagnostic whole exome sequencing and multiplex ligation-dependent probe amplification assays. conclusions: he was confirmed dock8 gene deletion (a deletion on one allele and point mutation on the other allele). he is preparing for hematopoietic stem cell transplantation due to autoimmunity and nonreversible parenchymal organ damage form infections although he has not yet experienced a life-threatening infection. introduction/background: during immunological investigation, it is important to distinguish those individuals who may hav e hypogammaglobulinemia (hypo) without fulfilling the criteria for the severe antibody deficiency common variable immunodeficiency (cvid) e.g. unspecified hypogammaglobulinemia from those with cvid. objectives: since low igg3 concentrations may support a diagnosis of cvid, we sought to investigate whether the measurement of additional igg subclass antibodies (iggsc) may provide further discrimination between patients with cvid and those with hypo. methods: iggsc concentrations were measured in serum samples from cvid patients (n=15, 1:1.3 m:f, median age 41.5 years, range 20-78) and hypo patients (n=19-21, 1:1.3 m:f, median age 41.5 years, range 20-78). results: cvid patients had lower median iggsc concentrations for all iggsc: igg1 290mg/dl (range 46-478) vs 365mg/dl (range 174-601); igg2 -88mg/dl (range 8-190) vs 116mg/dl (range 13-546); igg3 -15mg/dl (range 7-58) vs 30mg/dl (range 15-62); igg4 6mg/dl (range 0.2-25) vs 11mg/dl (range 0.31-108). this was significantly lower for igg1 and igg4 (p=0.04 and 0.01 respectively). a higher percentage of cvid patients had iggsc below the lower limit of the normal range compared to hypo patients: igg1 (80 vs 57.9%); igg2 (100 vs 90.5%); igg3 (60 vs 28.6%); igg4 (26.7 vs 10.5%). 100% of cvid patients had low concentrations of 2 or more iggsc vs only 68.5% of hypo patients (p=0.02). 5.2% and 26.3% of hypo patients had low levels of 0 or 1 iggsc, respectively. conclusions: igg subclass measurements may have some utility in distinguishing cvid patients from hypogammaglobulinemia patients. introduction/background: the thymus is often removed during cardiac surgery for repair of congenital heart disease, but the extent of tissue removed varies between procedures and surgeons. previous studies have shown decreased t cell counts after thymectomy but there is limited data on the effect of thymectomy on t cell receptor excision circle (trec) levels and infection risk. objectives: to determine the effect of partial and complete thymectomy during cardiac repair surgery on trec levels and infection risk. methods: a retrospective study of electronic medical records was performed on children who received cardiac surgery before age one at new york presbyterian/morgan stanley childrens hospital between 7/1/2013 and 3/31/2017. patients with heart transplant or primary immunodeficiency were excluded. data was recorded on trec levels (abnormal trec < 200 copies/μl on new york state newborn screen), number of positive cultures, viral pcr panels, and infiltrates on chest x-ray. patients were followed for a minimum of six months after cardiac surgery. study was irb approved. results: cardiac surgery was performed on 256 patients and data was available for 133 patients. of patients included, 65 had a partial and 68 had a complete thymectomy. trec levels after surgery were recorded for 79 patients. only 4% of patients had an abnormal trec level on newborn screen. there was no difference between partial and complete thymectomy on risk of abnormal trec (p = 0.58) and mean total number of infections at 6 months (p =0.42). conclusions: thymectomy rarely causes low trec levels in children undergoing cardiac surgery. complete thymectomy does not significantly increase infection rates in these children compared to partial thymectomy. these findings are possibly due to presence of ectopic thymic tissue or thymic regeneration and are reassuring for children undergoing complex cardiac surgeries. however, long-term follow-up of these children will be necessary to determine residual function of the thymus and clinical response. introduction/background: new sequencing techniques have revolutionized the identification of the molecular basis of primary immunodeficiency disorders (pid), not only by establishing a gene-based diagnosis, but also by facilitating defect-specific treatment strategies, improving quality of life and survival, and allowing factual genetic counseling. because these techniques are generally not available for physicians and their patients residing in developing countries, collaboration with overseas laboratories has been explored as a possible, albeit cumbersome, strategy. objectives: we sought to determine whether blood collected by guthrie cards could be shipped across continents by regular airmail to a cliaapproved laboratory for confirmatory testing. methods: blood was collected and blotted onto the filter paper of guthrie cards by completely filling three circles. we enrolled 20 male patients with presumptive x-linked agammaglobulinemia (xla) cared for at the vietnam national children's hospital, their mothers and several sisters for carrier analysis. dbs were stored at room temperature until ready to be shipped together, using an appropriately sized envelope, to a cliacertified laboratory in the us for sanger sequencing. the protocol for sanger sequencing was modified to account for the reduced quantity of gdna extracted from dbs. results: high-quality gdna could be extracted from every specimen. btk mutations were identified in 17 of 20 patients studied, confirming the diagnosis of xla in 85% of the study cohort. type and location of the mutations were similar to those reported in previous reviews. the mean age when xla was suspected clinically was 4.6 years, similar to that reported by western countries. two of 15 mothers, each with an affected boy, had a normal btk sequence, suggesting gonadal mosaicism. conclusions: dbs collected on guthrie cards can be shipped inexpensively by airmail across continents, providing sufficient high-quality gdna for sanger sequencing overseas. using this method of collecting gdna we were able to confirm the diagnosis of xla in 17 of 20 vietnamese patients with the clinical diagnosis of agammaglobulinemia. introduction/background: znf341 is a positive regulator of stat3 expression. it has recently been described that nonsense mutations in znf341 account for the stat3-like phenotype in four autosomalrecessive kindred. patients presented with reduced stat3 expression and diminished th17 cell numbers, in absence of stat3 mutations. objectives: here, we decribed a turkish case having nonsense mutation in znf341 developed dual malignancy in 2 years. results: a 26-year-old female patient presented with severe eczema, recurrent cold skin abscesses, herpetic skin lesions, sinopulmonary infection, otitis media and hearing loss. the parents are cousins. two younger sisters of the index case had eczema and recurrent skin infections since their infancy period. physical examination of the patient revealed severe eczema, high palate, micrognathia, maxillary hypoplasia and hearing loss. laboratory findings showed reversed cd4/cd8 ratio, high serum ige level (13.916 u / l) and low igg2 level (186 mg / dl). the patient was diagnosed as hyper ige syndrome and ivig therapy (400 mg / kg, every 3 weeks) was initiated because of igg subgroup deficiency and recurrent sinopulmonary infections. at the age of 32, a polypoid mass filling the left nasal cavity was detected in her examination. paranasal sinus ct revealed a mass obliterating the left nasal cavity, left ethmoid sinus and frontal sinus. immunohistochemical stains showed a small round cell malignant tumor in the nasal cavity. she was treated with chemoradiotherapy successfully. a homozygous nonsense mutation has been detected at exon 8 in the znf341 gene (c.1156c> t) (kindly provided by grimbacher's lab) very recently. she developed papillary thyroid carcinoma two years after completing the cancer therapy. conclusions: the relationship between znf341 defect and cancer development is unknown. the development of a second malignancy in this patient for a short time of completing the therapy might imply us a tendency for malignancy in znf 341 patients. additionally, the likelihood of increased radiosensitivity in these patients should be taken into consideration. introduction/background: prometic 10% igiv contains purified igg, 95% as monomer; with a distribution of igg subclasses proportional to that in native human plasma. we report the interim results from a phase 3 trial in the usa of prometic 10% igiv in adults and children with pidd. objectives: this was a phase 3, single-arm, open-label, multicenter trial to evaluate the safety, tolerability, and efficacy of prometic 10% igiv in adults and children with pidd. methods: adults and pediatric subjects with pidd on a stable dose of igg replacement therapy (200-900 mg/kg) for at least 3 months with serum igg trough levels > 500 mg/dl were included. subjects received prometic 10% igiv every 3 to 4 weeks for approximately 1 year at the same dose and schedule as their previous igg replacement therapy. results: an interim analysis was conducted when data were available on 50 adult subjects who received at least one dose of prometic 10% igiv (total of 461 infusions), with 15 subjects receiving at least 6 months of treatment (exposure = 27.72 subject years). at this time, pediatric exposure was only 4.29 subject years. there were no serious bacterial infections (sbis) reported, and rate/yr of infections other than sbis was 2.49 which was comparable to rate while on commercial product (2.80) all subjects achieved an igg trough level > 500 mg/dl. there were no deaths, and no subject had a study drug-related serious adverse event or an adverse event that resulted in permanent discontinuation of study drug. a total of 145 adverse reactions (ar) (0.315/infusion) occurred in 32 subjects (64.0%), with 92 infusions (20.0%) associated with an ar. most infusions (97.0%) were completed without a rate reduction. most ars were mild or moderate in severity, with 6 severe ars (0.013/infusion) occurring in 3 subjects (6.0%). the most frequent ars were headache (20.0% subjects or 0.056/infusion) and fatigue (14.0% subjects or 0.017/infusion). conclusions: in adults treated with prometic 10% igiv, there were no sbis and infusions were well tolerated. director, sean n. parker center for allergy and asthma research at stanford naddisy foundation, professor of medicine and pediatrics, stanford university introduction/background: desensitization to food allergies is being studied in clinical trials using oral immunotherapy (oit). there are limited data regarding the immune changes associated with successful oit. epigenetics involves heritable changes in gene function without modification of the underlying dna sequence. this is mediated by methylation, histone modification, or changes in microrna. objectives: to study methylation changes in the loci of four key genes of immune cells involved in allergy, interleukin 4 (il-4), interferon gamma (ifn-g), forkhead box protein 3 (foxp3), and interleukin 10 (il-10), comparing baseline to post-oit. methods: we completed a phase 2, randomized, placebo-controlled, multi-food oit trial using omalizumab, an anti-ige biologic, to facilitate desensitization for 48 multi-food allergic individuals. double-blind, placebo-controlled food challenges (dbpcfcs) to multiple foods were conducted at entry and after 36 weeks of treatment, the primary endpoint. omalizumab (n=36) or placebo (n=12) was administered for 16 weeks, with oit for 2-5 foods starting 8 weeks after the beginning of omalizumab or placebo. after 36 weeks (28 weeks of oit), participants underwent dbpcfcs to their offending foods. treatment failures (n=11) were offered open-label omalizumab. pyrosequencing of bisulfite treated genomic dna purified from pbmcs from each participant at baseline and post-oit was undertaken to investigate changes in methylation. results: forty-four participants achieved successful desensitization, defined as passing dbpcfcs to 2 or more foods following oit. we found that the -48 cpg site in the il-4 promoter region is hypermethylated over time during successful multi-food oit (fdr-adjusted p < 0.001 by wilcoxon signed-rank test). the median % of methylation at baseline was 81.6 (interquartile range 2.1%) and was 84.5 post oit (interquartile range 2.05%). there were no statistically significant (with a significance level of fdr adjusted p value of 0.001) changes in the il-10, foxp3, or ifn-g loci in the cpg sites we studied. conclusions: these preliminary results suggest that one immune mechanism involved in successful desensitization may involve suppression of th2 function by hypermethylation of il-4 in immune cells in the peripheral blood. introduction/background: atopic dermatitis (ad) is a common chronic inflammatory skin disorder afflicting from infancy to adults with itching, scratching, and lichenification. objectives: we investigated the effects of esculetin from fraxinus rhynchophylla on atopic skin inflammation. methods: for induction of atopic skin inflammation, we exposed the ears of female balb/c mice with house dust mite (dermatophagoides farinae extract, dfe) and 2,4-dinitrochlorobenzene (dncb) during 4 weeks. results: oral administration of esculetin reduced dfe/dncbinduced atopic skin inflammation symptoms based on ears swelling and scratch numbers. the immunoglobulin (ig) e, igg2a, and histamine levels in serum were decreased and inflammatory cell infiltration in skin tissue was reduced by the esculetin. it suppressed th1, th2 and th17 responses by inhibiting the production of inflammatory cytokines such as tumor necrosis factor (tnf)-, interferon (ifn)-, interleukin (il)-4, il-13, il-31 and il-17 in the ear tissue. further, we investigated the effects of escueltin on activated keratinocytes, one of the most representative cells for studying pathogenesis of acute and chronic atopic skin inflammation. as results, esculetin suppressed gene expression of th1, th2 and th17 cytokines and activation of nuclear factor-b and signal transducer and activator of transcription 1 in tnf-/ifn-stimulated keratinocytes. conclusions: taken together, the results imply that esculetin attenuated atopic skin inflammation, suggesting that esculetin might be a potential therapeutic candidate for the treatment of ad. introduction/background: autoimmunity is often seen in common variable immune deficiency (cvid) with immune thrombocytopenic purpura (itp) being the most frequent manifestation at a prevalence of 8-14% in cvid patients. in such patients, itp is often recognized and treated long before cvid, the implications of which are unknown. primary itp is a clinicopathologic diagnosis which includes an evaluation of other conditions that may mimic it including cvid. currently, it is unknown how frequently cvid is evaluated during the diagnostic workup of itp and what percentage of those patient actually have cvid. objectives: the two main objectives of this study were to determine the number of itp patients that had an igg level checked during their clinical course and if the globulin fraction can be used as a marker for hypogammaglobulinemia in itp patients at the time of diagnosis. methods: a retrospective chart review was undertaken at a large academic medical center of patients with a new diagnosis of itp between january 2009 and january 2017. igg levels were collected and globulin fractions were calculated as the difference between serum total protein and albumin within 30 days of the initial itp diagnosis. results: six hundred and twenty-three patients were found to have a new diagnosis of itp in the given timeframe. of these, only 61 (9.79%) had igg levels checked at any point during their clinical course. twelve of the 61 (19.7%) had hypogammaglobulinemia with only 3 of the 12 (25%) having a formal immunologic follow-up evaluation. two were diagnosed with a primary immunodeficiency (1 cvid and 1 ctla4 deficiency). globulin fractions were calculated on 52 patients at the time of itp diagnosis. mean calculated globulin fraction in hypogammaglobulinemic patients was 2.64 (range 2.0 3.7), versus 3.92 (range 2.4 7.5) in patients without hypogammaglobulinemia (p=0.0004). conclusions: the diagnosis of cvid is often delayed from the onset of symptoms which can include autoimmune conditions such as itp. our data indicate that clinicians do not routinely check igg levels at the time of itp diagnosis which should be considered standard of care based on the current guidelines. our data suggest that although calculated globulin fractions were significantly lower in hypogammaglobulinemic patients, the variability was substantial and hypogammaglobulinemic patients would be missed using this as an indicator of low immunoglobulins. future directives include a prospective study using igg levels checked at the time of itp diagnosis, with formal evaluation for cvid in any hypogammaglobulinemic patients to evaluate the true prevalence of cvid among itp patients. introduction/background: autoimmune lymphoproliferative syndrome (alps) is a disorder characterized by immune dysregulation due to the rupture of lymphocyte homeostasis, which occurs as a result of mutations in the apoptotic pathway mediated by fas. this disease is sometimes misdiagnosed due to its variable phenotypic expression and the overlapping of symptoms with many other hematological and immunological disorders. (1) a patient diagnosed with evans-fisher syndrome (sef) was referred to the laboratory for immunity studies. this is a patient who from infancy had multiple admissions for severe sepsis, with severe anemia and severe thrombocytopenia. in the evaluation of the history of the disease by the work team, a series of clinical data was observed that suggested the possibility of the patient presenting an alps, so it was decided to incorporate as part of the study, the quantification of the cells t cd3 + cd4-cd8-(double negative t cells or dnt) that express tcr +. the differentiation pathways of dnt tcr + and the role of fas in this process are not clear, some authors hypothesize that they may be represented by direct descendants of chronically activated positive simple t cells, with deregulated co-receptors, in a state of differentiation in which they were destined to perish by fas-mediated apoptosis. (1) (2) (3) (4) the population of tcr + dnt cells required for diagnosis must be derived from the particular study of each laboratory in their populations, but several working groups agree that the pathological limit values are 1,5% in total lymphocytes or 2,5% in cd3 + lymphocytes. (4) evidence shows that dnt alps cells are not simply accumulated in senescent withdrawal; they and their precursors remain active and proliferate under the influence of activation signals. (5) although numerous genetic deficiencies lead to lymphoproliferation of t cells, only that caused by a defective fas pathway is dominated by negative double t cells. (2) in clinical immunology, the distinction between cd45ra + and cd45ro + cells is particularly useful for determining the state of the "naive" cell compartment in relation to its thymic origin. primary immunodeficiency disorders are characterized by a decrease / absence of thymus performance, and often involve a decrease in cd45ra + t lymphocytes. scientific evidence suggests the important role of the "naive" subpopulation in the origin and maintenance of self-reactive effectors in the periphery. (5) regulatory t cells are able to effectively control autoreactive t cells, especially when negative thymic selection is defective. its differentiation and function is controlled by foxp3. the decrease or absence of regulatory t cells leads to autoimmune diseases, specifically those mediated by cd4 + t cells, and to lymphoproliferation characterized by multiorgan inflammation and other autoimmune disorders. (4, 5) objectives general: establish the diagnosis of a pediatric patient with suspected primary immunodeficiency due to dysregulation, an autoimmune lymphoproliferative syndrome (alps). specific: evaluate the tcr + dnt population, relevant in the diagnosis of alps. quantify cell populations that exhibit markers of activation, differentiation and regulation of the relevant t cells in the study of this disease. methods: monoclonal antibodies (mab) cd4fitc / cd8pe / cd3pc5 triple labeled (beckman coulter), tcrapc, cd4pe, cd45rapercp, cd45rofitc, cd8fitc, cd45rope, cd4apc, cd25f, cd56pe, cd19percp, cd3pe, cd5pe, cd45percp, cd4fitc, cd38fitc were used , hla dr and the cd4fitc / cd25pe / foxp3apc regulatory t cell kit, all from miltenyi biotec. the trial included the use of a healthy control. both samples were processed in unison to ensure reproducibility. the samples were acquired in a beckman coulter gallios cytometer, with the use of the kaluza program, version 1.2. the analysis strategy included the formation of overlapping "gate" windows for the quantification of dnt tcr + and regulatory t cells. results: the patient studied showed 11.60% of tcr + negative double t cells, in relation to 1.45% of the healthy control. the presence of this population of t cells in patients diagnosed with evans syndrome, even in the absence of lymphoproliferation, is consistent with alps. (5) (graph 1) the presence of increased tcr + dnt and lymphoproliferationexpressed as lymphadenopathies and splenomegaly of noninfectious or malignant cause, of at least 6 months of evolution; plus the typical immunohistological findings found in the patient's lymph node biopsy (paracortical hyperplasia), the presence of autoimmune cytopenias (hemolytic anemia and thrombocytopenia) and hypergammaglubulinemia, led to the "probable diagnosis" of alps in the patient studied. the accumulation of dnt in the lymph nodes and other peripheral organs is accompanied by qualitative changes in the composition of the t cell repertoire, so that the immunophenotype performed included markers of activation, differentiation and regulation. the evaluation of the activation on t lymphocytes and nk cells showed that there was a slight decrease in the expression of the receptor for il-2, cd 25. the cd19 + cd25 + population was expressed in greater percentalthough discretely-in the patient than in the healthy control, which was directly related to the presence of hypergammaglobulinemia, elevated iga and the presence of autoantibodies. it was also highlighted by the high expression of the cd19 + cd5 + autoreactive population in the patient studied (11.73% vs 0.39%). in order to know the impact on cell differentiation, the subpopulations of effector and memory cells for cd4 + and cd8 + t lymphocytes were evaluated by combining the cd45ra and cd45ro isoforms. cd45ra is expressed in naive t lymphocytes. particularly, the cd4 + cd45ra + population has an essential function as a suppression inducer, and it is diminished in the patient studied, in relation to the control. (29.11% vs. 62.75%, respectively) the same behavior was shown by the cd4 + cd45ro + memory and effector cells, 53.38% vs73.37%. in the cd4 + population itself, it was possible to confirm the presence of a clone that expressed both receptors (cd45ra and cd45ro), probably a temra population (terminally differentiated effector memory cells). when comparing the results, it was unexpectedly found that there was a slight decrease between patient and control (2.70% vs 4.0%). as the characteristic phenotype of this cell population could not be corroborated, conclusive assessments could not be made. the combination of cd45 isoforms was also used to study cd8 + t lymphocytes, but similar behavior between patient and healthy control was observed. (the lab results are shown in table 1 ) the analysis of the regulation of the immune response showed a decrease in the cd4 + cd25 + population and the expression of the foxp3 transcription factor in the patient with respect to the control. (11.16% vs. 6.30%, 0.00% vs. 1.05%, respectively) (graph 2) conclusions 1. the markedly high quantification of cd4-cd8-tcr + t lymphocytes allowed to define the "probable diagnosis" of autoimmune lymphoproliferative syndrome in the patient under study. 2. the increased activation of cd19 + cells and the presence of the cd19 + cd5 + self-reactive population, largely responsible for autoimmunity and lymphoproliferation in the alps, could be confirmed. 3. the decrease in the cd4 + cd45ra + t-cell suppressor-inducing population evidenced corroborated its involvement in this disorder by primary immunodeficiency, in relation to the thymus dysfunction that originates and maintains self-reactive effectors in the periphery. 4. the decrease in the expression of the foxp3 transcription factor observed, points towards a low regulation of the response that leads to autoimmunity and to lymphoproliferation, specifically mediated by cd4 + t cells. allergy and immunology arnp, nicklaus children's hospital 6 allergy and immunology division director, nicklaus children's hospital introduction/background: specific antibody deficiency syndrome is characterized by a weak antibody response to bacterial polysaccharide antigens when no other immune system abnormalities can be found. low titers to pneumococcal vaccine have become one of the most frequently recognized immune abnormalities in pediatric patients with recurrent sinopulmonary infections. nonetheless, insufficient data and lack of consistent testing of the response to pneumococcal polysaccharides continues to affect the optimal diagnosis and management of this specific antibody deficiency. objectives: to characterize the pre-and post-immunization igg antibody trend for each specific serotype included in the pneumococcal 13-valent conjugate vaccine (pcv13), as well as others that are routinely tested, in a cohort of pediatric patients with recurrent sinopulmonary infections. secondarily, to understand differences in the immune response to the vaccine booster between age groups. methods: this retrospective review identified 182 patients with recurrent sinopulmonary infections. in this cohort, 131 required an immune workup, and 99 were found to have low pneumococcal titers needing a pcv13 vaccine booster. baseline pneumococcal serotype-specific antibody titers at initial visit and 6 weeks after the vaccine booster were obtained. patients were categorized by age: 2 years, 3-5, 6-10, and 11-18. an adequate response to the pneumococcal conjugate vaccine was deemed to be a 4-fold increase over baseline and/or a post-immunization titer of 1.3 μg/ml or greater. results: overall, pcv13 booster provided a significant improvement in the number of protective titers, increasing from 3.6 (95% ci: 3.2-3.9) serotypes at baseline to 11.1 (95% ci: 10.7-11.5) serotypes at 6 weeks (p < 0.001). this increase correlated with improved clinical outcomes81% showed no signs of recurrent infection after the first booster and 94% after a second dose. all those who did not improve clinically suffered from co-morbidities (genetic abnormalities and rheumatologic diseases). post-immunization antibody concentrations were significantly higher than at baseline for all serotypes (p < 0.05) and only 8, 9n, and 12f did not exhibit a greater than 4-fold increase (p > 0.05) 6 weeks following the booster. across age groups, only 1, 7f, and 9v showed pre-immunization differences in titers. there were no differences between ages in post-immunization titer levels for all serotypes. similarly, all age groups had a comparable number of baseline titers (p = 0.63) and at follow-up (p = 0.10). conclusions: in pediatric patients with recurrent sinopulmonary infections, an additional pneumococcal booster proved to be effective in the protection of these children from further infections. the pcv13 booster substantially increased titer levels and concentrations, and significantly improved clinical outcomes, independent of age. this investigation has provided us with a better understanding of the response after booster vaccination, and its role in the protection of patients from recurrent sinopulmonary infections. further studies are needed to elucidate whether a fifth dose of pcv13 should be optional as part of the vaccination schedule of patients with recurrent sinopulmonary infections. introduction/background: many primary immunodeficiencies (pids) share overlapping presentations, complicating clinical diagnosis. due to the ability to include many genes in one assay and the rapid turnaround time that these panels allow, expanded next-generation sequencing (ngs) panels are valuable in facilitating the diagnosis of patients with pids. objectives: we aimed to determine the clinical utility of an expanded ngs panel for the genetic diagnosis of patients with suspected pids. methods: we performed a retrospective analysis of the clinical utility of a 207-gene pid ngs panel used in a clinical diagnostic laboratory. from april to october 2017, 260 panels were ordered for patients with suspected or known pids. results: seventy-four pathogenic or likely pathogenic (p/lp) variants were identified in 63 (24.2%) patients. eight (10.8%) of the p/lp variants were copy number variations (6 deletions, 2 duplications). fifty-two patients (20%) had 1 p/lp variant, and 11 patients (4.2%) had 2 p/lp variants. of positive patients, 31.7% (n = 20) were heterozygous carriers for autosomal recessive conditions where a second variant was not identified. twenty-two patients had p/lp heterozygous variants in genes with autosomal recessive and autosomal dominant inheritance patterns, in which the positive findings may or may not explain the patient's phenotype. for example, 13 variants in this category were heterozygous variants in tnfrsf13b (taci). genetic diagnoses were established or likely in 30% of patients with p/lp variants (7.3% of all patients). five patients were heterozygous for a single p/lp variant and a variant of unknown significance in the same autosomal recessive gene. four patients in whom a genetic diagnosis was determined were also heterozygous carriers for a second, unrelated condition. one patient was found to have two distinct genetic diagnoses. variants of uncertain significance (vus) were identified in most (94.2%) patients. the average turnaround time from test requisition to return of results was 18 days. in total, 63% percent of genetic diagnoses were for conditions that are treatable with hematopoietic cell transplantation. conclusions: these results illustrate the utility of broad ngs panels for the diagnosis of patients with pids. introduction/background: severe combined immunodeficiency (scid) is a life-threatening immune deficiency manifest by extreme susceptibility to infection. early diagnosis and definitive treatment with either hematopoietic cell transplant (hct) or, in select cases, gene therapy (gt) has been shown to significantly improve survival. newborn screening for scid has allowed for the opportunity to promptly identify these patients before significant infections occur. at ucsf, newly diagnosed infants with scid are admitted to the hospital for management and remain in isolation for definitive treatment and until adequate immune reconstitution occurs. previous work by our group demonstrated up to 60% of these parents experience psychosocial trauma manifested by depression and post-traumatic stress disorder (ptsd). the psychosocial challenges that contribute to depression and ptsd in these parents have not been qualitatively described. objectives: to understand the range of experiences and feelings of parents/caregivers of infants with scid diagnosed by newborn screening throughout their prolonged hospitalization and isolation in order to better support patients, parents, and their families. methods: voluntary participation was elicited from parents of children with scid who were status post hct/gt for one year or longer. semi-structured, in-person interviews lasting approximately 45-60 minutes were conducted with 11 parents; interviews were recorded and transcribed. parents were asked to discuss their experiences from first notification of an abnormal screening result through discharge after hct or gt. emerging themes were identified from the transcribed interviews. results: we interviewed 6 mothers and 5 fathers of 7 infants with scid. six infants received hct, while one underwent gt for ada-scid. all children were alive and well at the times when interviews were conducted. overall, once admitted, parents reported feeling well supported by the medical team and support staff. however parents identified a number of stressful events. uniformly reported key stressors included: receiving the first phone call regarding their childs abnormal newborn screening results, preparing for hct, coping with prolonged isolation, and transitioning from hospital isolation to care with ongoing isolation at home. other challenges described reflected the additional stressors of caring for a newborn, including coping with postpartum depression. overall, we identified three major themes encompassing the challenges faced by parents of hospitalized scid patients: (i) loss of normalcy and control over multiple aspects of life; (ii) prolonged waiting periods (especially the wait between diagnosis and hct and between hct and evidence of t cell engraftment); and (iii) perceived lack of guidance on realistic expectations during the hospital stay . parents sought and relied on peer support from other scid parents to learn about coping with the nuances of daily life as a parent of an infant with scid. conclusions: we identified multiple psychosocial stressors and challenges uniquely faced by parents and caregivers of infants diagnosed with scid by nbs. parents described barriers to caring for their own physical and mental health, which can be especially harmful to parents experiencing postpartum depression. recognizing these challenges allowed for the identification of opportunities to improve both healthcare delivery to their children and institutional support for future families affected by scid (table i) . emphasis should be placed on providing parents with scid-specific resources as early as the time of diagnosis, connecting parents with scid support networks, and facilitating access to psychosocial and mental health services for caregivers introduction/background: chronic granulomatous disease (cgd) is caused by a genetic defect that impairs phagocyte function. this disease results in recurrent infections and granuloma formation. rarely do patients develop cutaneous symptoms, unless associated with autoimmune disorders such as systemic erythematous lupus. previously described cutaneous findings include granulomas, abscesses, photosensitivity, malar rash, discoid lupus, vasculitis, and rarely vesicular rashes. here we describe two term infants diagnosed with x-linked cgd who present, in addition to frequent infection, with a unique papulopustular skin rash initially diagnosed as non-classic appearing eczema refractory to usual eczema treatment and antibiotics. objectives: to characterize cutaneous findings in x-linked cgd and emphasize the importance of considering further work in patients who present with similar rashes in conjunction with concerning features for primary immunodeficiency. methods: each infant was diagnosed with cgd based on abnormal dhr testing and/or genetic evaluation. after obtaining consent from both families, we have documented photographs of the development of rash in two newly diagnosed infants with cgd. one infant underwent cutaneous biopsy with resulting pathologic evaluation. results: our first patient presented at 8 months of age with episodic fever with chronic leukocytosis, iron deficiency anemia, thrombocytosis, elevated inflammatory markers, proctocolitis with elevated calprotectin, and rash. egd with flexible sigmoidoscopy showed no signs of inflammatory bowel disease, the left colon had macroscopically raised erythematous lesions but was microscopically normal with no signs of active colitis. he was initially diagnosed with fpies and eczema in the setting of diagnosis of various infections (otitis, upper respiratory illness). the skin rash was described as non-pruritic, generalized pink-purple papulopustular lesions with a pink base, most prominent on upper and lower extremities, mostly sparing the trunk. skin biopsy histopathology revealed an essentially unremarkable appearing epidermis and within the dermis, there was a superficial perivascular lymphohistiocytic infiltrate. eosinophils and plasma cells were not abundant. the histologic changes were thought to be non-specific, but could be seen in a drug reaction or urticaria. a viral exanthem could also demonstrate these changes. giemsa stain and a stain for mast cell tryptase revealed normal numbers of mast cells in the biopsy specimen. he was not on any oral medications at the time and did not respond to treatment with topical moisturizers, oral antihistamines, topical steroids, or any standard for eczema care. he had a maternal uncle who passed away in infancy from infection. heightened clinical suspicion for primary immunodeficiency led to obtaining a neutrophil respiratory burst assay which was consistent with cgd and genetic testing was positive for x-linked cgd. pathogenic mutation nucleic acid change was c. 469c>t; hemizygous; amino acid alteration was p.157arg>stp within the cybb gene locus. he had no other features of auto-immune disease including negative ana obtained later in his clinical course and his colitis diagnosed as cgd-associated colitis. his rash did not respond to systemic treatments for his colitis, oral antibiotics, and during hsct. our second patient presented at 14 months of age with persistent fevers, inguinal lymphadenopathy, leukocytosis, elevated inflammatory markers, non-bloody diarrhea and rash. there was a family history of autoimmune gi disease. he presented to the hospital with fever of unknown origin with concern for infection, atypical kawasakis, and drug rash. his rash was described as a blotchy, pink, papular rash most prominent on the upper arms but also present on lower arms, legs, and chest, faint on face. there is some induration and thickness to the rash in some areas (confluent on the arms with more erythema and induration) and more faint pink papules (scattered on legs) elsewhere. a biopsy of his inguinal lymph node showed granulomatous lymphadenitis with neutrophilic abscess formation, and culture was positive for serratia marcescens. neutrophil respiratory burst assay was consistent with cgd and genetic testing was positive for x-linked cgd, mutation with the cybb gene. pathogenic nucleic acid change was c. 141+5g>a; hemizygous; amino acid alteration was p. deletion of exon 2. the rash remained unchanged with treatment for infection, initiation of antifungal and bacterial prophylaxis, along with topical steroid therapy. conclusions: in patients who present with frequent infections and who have unusual cutaneous findings that do not fit with common infant rashes, consideration of work up for cgd should be pursued. specifically, generalized papulopustular rash with a negative skin biopsy can be misdiagnosed as atopic dermatitis, and in the right clinical context cgd should be considered. introduction/background: chronic lung disease in common variable immunodeficiency (cvid) is heterogeneous, and it is a leading cause of morbidity and mortality in this population. currently, the diagnosis and monitoring of chronic lung disease in cvid rely on radiographic findings, biopsy and/or pulmonary function tests. fractional exhaled nitric oxide (feno) is a noninvasive biomarker of airway inflammation, and it has been widely utilized to aid the in-office diagnosis, characterization, and management of inflammatory airway disease, such as asthma. however, exhaled nitric oxide in cvid patients with chronic pulmonary complications has not been examined. objectives: we aimed to determine fractional exhaled nitric oxide levels in cvid patients. methods: we measured exhaled nitric oxide in cvid patients with or without chronic lung disease. results: feno measurements were obtained in 13 cvid patients (mean age: 53, range 34-68). five patients had no known lung disease; 8 patients had chronic lung disease (bronchiectasis, n=3; lung granulomas, n=4; hepatopulmonary syndrome, n=1). four patients were on inhaled steroid (bronchiectasis, n=3; lung granulomas, n=1), 2 patients were on systemic steroid (lung granulomas, n=1; hepatopulmonary syndrome, n=1), and 1 patient was on oral budesonide (no known lung disease). feno was elevated (> 25 parts per billion [ppb]) in 9 of 13 patients, 6 of whom had known chronic lung disease. patients with granulomatous lung disease had higher feno (mean 50.8 ppb, range 41-59 ppb) compared to patients without known lung disease (mean 31 ppb, range 23-38 ppb, p=0.005), patients with bronchiectasis (mean 18.3 ppb, range 8-25, p=0.005), and the patient with hepatopulmonary syndrome (18 ppb). feno levels remained elevated in granulomatous lung disease in patients with inhaled or systemic steroid use. conclusions: this is the first report of feno measurements in cvid patients. feno is elevated in a subgroup of cvid patients, and it may differ according to the underlying lung pathology. further investigation is warranted to determine the utility of feno in the diagnosis and management of chronic lung disease in cvid. professor of pediatrics, university of british columbia introduction/background: whole exome sequencing (wes) has revolutionized the discovery, diagnosis, and treatment of primary immune deficiency diseases (pids), a group of disorders with high genetic and phenotypic heterogeneity. current bioinformatic analysis approaches for wes rely heavily on population databases to exclude variants present in the populations represented by these databases. this approach may confound the ability to detect true disease causing variants, and conversely, flag variants that are absent from population databases only by virtue of originating from minority populations. ultimately, the pathogenicity of novel variants can only be mechanistically established through rigorous biochemical and functional validation. objectives: to evaluate the pathogenicity of a novel homozygous variant in caspase recruitment domain family member 11 (card11) (c.1798g>t, p.c600y), in a patient with features of combined immunodeficiency (cid). methods: research study protocols were approved by our institutional review board. six members of one family (the affected child, healthy siblings and their parents) were enrolled. written informed consent for genetic testing and participation was provided by the parents for their children. genetic, bioinformatic, biochemical and immunological investigations were performed. results: targeted sanger sequencing confirmed the presence of the homozygous card11 variant c.1798g>t, p.c600y in the index patient (and subsequently in one apparently healthy sister). each parent was found to be a heterozygous carrier of the variant. nfkb activation in vitro was found to be normal for both the index patient and sister, and was indistinguishable from unaffected family members. conclusions: although multiple in silico tools predicted the c.1798g>t, p.c600y card11 variant to be pathogenic, the patients b and t cells did not have aberrant nfkb activation in vitro, as would be predicted given the central role of card11 in nfkb activation. this practical experience highlights how imperative it is to functionally characterize novel variants found by wes, even when variants are predicted to be damaging. chief, clinical immunology -faculdade de medicina do abc introduction/background: gata2 is a zinc finger transcription factor essential for embryonic and definitive hematopoiesis. heterozygous mutations leading to gata2 deficiency were first described in 2011. the age of clinical presentation ranges from early childhood to late adulthood, with most of them in adolescence to early adulthood. patients present clinical findings as monocytopenia, nontuberculous mycobacterial infections, myelodisplasia, viral (mainly hpv and ebv), fungal and bacterial infections. patiens may arise with many phenopytes, showing how complex is the effect of this transcription factor. objectives: we report a patient with gata2 mutation. methods: report: a 34 year old female patient was referred due to a mycobacterial non-tuberculosis pneumonia. she was a healthy child until puberty. at the age of 15 she presented genital herpes and recalcitrant vulvo-vaginal warts (hpv). at 18, she complained of lower back pain with no diagnosis for months, medication was unnefective, and she developed ischemic stroke. hemiparesia was mild and temporary. endocarditis with no agent identified was also diagnosed. therapy with acetilsalicilic acid was mantained until the age of 28. five years later, vaginal hpv spread through vulve and anal region evolving to neoplasia. she was submitted to surgery, radio and chemotherapy until october 2016. one year later, profound cytopenias led to hospitalization. during that time, she had cough and fatigue and pulmonary tuberculosis was diagnosed. despite therapy with rifampin, isoniazid and pyrazinamid, there was no improvement. bronchoalveolar lavage was positive for mycobacterium avium. main immunological evaluation showed: monocytes 162 (6%), t cells /mm3; cd4+: 28 (1,5%), cd8+: 74 (4%); b cells 2/mm3 (0,1%); nk: 9/mm3 (0,5%); normal immunoglobulin levels; incomplete response to pneumococcus serotypes; igm and igg positive for cmv; negative serologies for hiv, ebv and htlv1/2. molecular analysis identified an heterozygous mutation c.1061c>tp.(thr354met) in gene gata2. results: we report a patient with gata2 mutation. the same gata2 mutation was previously described in national institute of health nih-usa (n=5) and in australia (n=3). conclusions: it took almost 20 years for diagnosis suspicion. gynecologists should be warned about this diagnosis in order to improve patients prognosis. early diagnosis is crucial with adequate prophylaxis, prompt treatment of infection, surveillance of malignancy, and, moreover, family screening and genetic counseling. this is the first report of this mutation in latin america. introduction/background: severe combined immunodeficiency (scid) due to adenosine deaminase (ada) deficiency was the first human monogenic disease to be approached with gene therapy, and ongoing research advances over 25 years led to the approval by the european medicines agency of a stem cell gene therapy product for its treatment using a gammaretroviral vector (gv), strimvelis®. despite the high success rate using gvs for ada gene transfer without vector-related complications, the development of leukoproliferative complications from the use of gvs in gene therapy of other disorders led us to develop lentiviral vectors (lvs) to deliver the corrective ada sequence/cdna (corrigan-curay et al, mol ther 2012; modlich et al, mol ther 2009.). lvs, typically derived from hiv-1 and devoid of all viral genes, can be produced in a self-inactivating (sin) configuration, in which the viral long-terminal repeat enhancers are absent, eliminating the major identified cause of insertional oncogenesis due to gvs. we developed a lv (efs-ada) that carries a normal human ada cdna (codon-optimized) and demonstrated in pre-clinical studies its efficacy to transfer and express the ada protein, with evidence of significantly decreased potential for insertional mutagenesis compared to gammaretroviral vectors using the immortalization (ivim) assay and in murine bone marrow transplant models (carbonaro et al, mol ther, 2014) . this new lv was evaluated for safety and efficacy in parallel clinical trials of gene therapy for ada scid performed at sites in the u.s (university of california, los angeles and the national institutes of health {nih} clinical center) and u.k. (university college london/great ormond street hospital) enrolling subjects between 2012 and 2016. we report here results from the 20 patients treated in the u.s. between 2013-2016. objectives: this is a prospective, non-randomized phase i/ii clinical study to assess the safety and efficacy of efs-ada lentiviral vector stem cell gene therapy in ada-scid subjects older than 1 month. methods: 20 subjects with ada-scid were enrolled, screened to document eligibility and underwent bone marrow harvest (15-20 cc/kg). bone marrow was processed to isolate cd34+ cells, which were pre-stimulated by overnight culture in serum-free medium containing c-kit ligand, flt-3 ligand, and tpo followed by culture with the efs-ada lentiviral vector overnight. subjects received a single dose of busulfan (4 mg/kg) iv for reduced intensity conditioning. cells were removed from culture, washed, formulated and administered by iv infusion at least 24 hours after busulfan administration. enzyme replacement therapy (ert) with pegylated bovine ada (peg-ada) was continued for one month post-transplant and then stopped. subjects were followed over 24 months to assess safety and efficacy end-points. results: with 17-54 months follow-up, overall survival is 100%. eventfree survival has also been 100%, as all subjects are alive, remain off peg-ada ert, and none have required a second transplant. successful engraftment of gene-corrected cells was observed in all subjects at 6 months, and persisted over the 24 months of observation, based on vector gene marking in granulocytes and peripheral blood mononuclear cells (pbmcs), changes from baseline in rbc ada enzyme activity, and levels of metabolic detoxification of deoxyadenosine nucleotides. immune reconstitution was observed in all subjects and was sustained over the two years of observation, based on improvement of peripheral blood absolute lymphocyte counts and lymphocyte subsets (t, b and nk cells, and naïve cd4+ t cells). eighteen of 20 patients have been able to stop receiving immunoglobulin replacement therapy. subjects who had routine infections all recovered with standard of care treatment, and there were no severe or opportunistic infections. conclusions: conclusions: gene therapy using the efs-ada lv has a favorable safety profile and was efficacious in this trial. a current followon trial at ucla is using a cryopreserved formulation of the cell product and pharmacokinetic-adjusted busulfan dosing, sponsored by the california institute for regenerative medicine (clin2-09339) and orchard therapeutics. acknowledgements: this study was supported by research grants from the national institutes of health (u01 ai100801; 2p01 hl073104-01; nhlbi gtrp rsas #1101 and 1129) and the california institute for regenerative medicine (cl1-00505; fa1-00613); support from the ucla david geffen school of medicine human gene and cell therapy program and the ucla eli & edythe broad center of regenerative medicine and stem cell research; and funding from the nhgri intramural program. dbk has potential financial conflict of interest as a member of the scientific advisory board for orchard therapeutics and as an inventor on intellectual property which ucla has licensed to orchard therapeutics related to gene therapy for ada scid. introduction/background: x-linked severe combined immune deficiency (scid-xl) is a rare monogenic primary immunodeficiency disorder (pid) where male infants are born without an adaptive and innate immune system. it is a life-threatening disease due to patients inability to fight viral and bacterial infections. scid-xl is driven by any of the two hundred known pathogenic mutations in the interleukin 2 gamma receptor (il2rg) gene, which function is required for proper development of t, b and nk cells. the most effective treatment for scid-xl, if performed in the first few months of life, is allogeneic hematopoietic stem cell transplantation (allo-hsct). this treatment is limited by absence of match donors, incomplete immune reconstitution, graft versus host disease and the need for long-term immunosuppression. an alternative curative treatment for scid-xl would be genome editing-based gene therapy by ex vivo genome correction of the patients long-term hematopoietic stem cells (lt-hscs) prior to autologous stem cell transplantation (auto-sct). objectives: here we report a proof-of-concept genome editing-based approach for correcting scid-xl disease. methods: using a crispr/cas9-raav6 platform, we deliver a fulllength codon optimized il2rg complementary dna (cdna) at the endogenous start site in cd34+ hematopoietic stem and progenitor cells (hspcs). results: using an optimized genome editing protocol we achieved >80% genome editing as early as 48h and a median of 45% genome targeting while retaining >80% viability and promoting greater than 70% ex vivo expansion of cd34+ hspcs from healthy donors. we demonstrate that our approach retains proper il2rg signaling function in t-cells derived from healthy male donors and rescues the lymphopoietic defect from a patients derived mobilized cd34+ hspcs both in vitro and in vivo. we further show robust in vivo primary human engraftment potential and multi-lineage hematopoietic reconstitution of il2rg gene targeted hspcs: a median of 26% (bone marrow), 47% (spleen) and 37% (liver) of il2rg genome targeted engrafted cells was achieved at four months following primary transplantation into nsg mice and a median of 9.5% -20% was detected at 8 months from secondary transplants of il2rg targeted hspcs, thus achieving clinical levels of editing of lt-hscs. lastly, our observation of (1) an intact hematopoiesis derived from il2rg targeted cd34+ hspcs combined with (2) a normal karyotype analysis and (3) our deep analysis of potential off-target activity that showed only 2 off-target sites of no known functional significant with < 0.3% frequency of off-target activity presents strong evidence for the safety of our genome editing approach. conclusions: in sum, this pre-clinical study provides specificity, toxicity and efficacy data supportive of continued development of genome editing to treat scid-xl. objectives: to determine the risk of gvhd in msd hsct for scid patients compared to matched related donor (mrd). methods: retrospective cohort study comparing msd with mrd and the outcome of gvhd in all scid patients who underwent hsct between 1993 and 2013. all statistical analysis was done using ibm spss statistic software. results: 145 scid patients underwent 152 hsct, 82 (54%) received gvhd prophylaxis. gvhd occurred in 48 (31.5%); 20/48 (42%) had gvhd prophylaxis compared to 28/48 (58%) that did not, p value = 0.022. acute gvhd occurred at a higher rate in msd 22/120 (18.3%) compared to mrd 2/32 (6.2%) p value = 0.095. we analyzed outcome also according to period of hsct. first periods was 1993 to 2003; 48 hsct, msd: 43, mrd: 5; all had gvhd prophylaxis and there was no difference in gvhd. the second period was 2004 to 2013:104 hsct, 77 had msd and 27 had mrd, gvhd prophylaxis was used in 22.1% in msd and 63% in mrd, p value = 0.000. gvhd was significantly higher in the msd (40.2%) compared to mrd (18.5%) odds ratio of 2.9 (95ci 1.01 to 8.66) p value = 0.041. conclusions: gvhd prophylaxis in msd transplant may have a role to be considered in scid patients. introduction/background: xiap deficiency (also known as x-linked lymphoproliferative disease type 2 xlp2; mim: 300635) is an x-linked primary immunodeficiency associated with mutations in the gene encoding the x-linked inhibitor of apoptosis (xiap; mim: 300079). the pathophysiology is characterized by immune dysregulation, usually triggered by epstein-barr virus (ebv) infection. primary ebv infection is followed by hemophagocytic lymphohistiocytosis (hlh) with high grade persistent fever, splenomegaly, hematologic cytopenias and hepatitis. most patients die during this acute phase, and those who survive usually evolve with hypogammaglobulinemia, recurrent infections, cytopenias, inflammatory bowel disease (ibd) and low counts of inkt cells. dysbiosis of intestinal microbiota is believed to fuel ibd and possibly contribute to the initiation and/or perpetuation of the disease. experimental studies have provided solid evidence to support a role for the indigenous gut microbiota in the pathogenesis of autoimmune diseases, thereby raising the possibility that an altered gut microbiota is an environmental risk factor for xiap disease. objectives: in this study, we sought to investigate the identity and abundance of the bacteria in gut microbial communities in a 28 years old male patient with xiap deficiency. case presentation: at 15 years of age, the patient presented with positive serology of active ebv infection, hlh, severe hepatitis, encephalitis and myocarditis. after recovery, the patient evolved well with few manifestations for several years. approximately 3 years ago, the patient showed slow progression of hypogammaglobulinemia predisposing him to infections of the upper and lower respiratory system that required intravenous immunoglobulin replacement. immunological evaluation revealed reduction (but not absence) of inkt cells. at that time, the patient presented with intermittent diarrhea and abdominal pain that became more frequent and severe. after evaluation as ibd, the patients had been treated but without much improvement of diarrhea or resolution of his pain. after approximately 18 months, the patient presented noted pain and fistulas lesions at the scrotal and gluteal regions. the exact causes of ibd in xiap deficiency are not known, but the abnormal activation of the mucosal immune system due to exaggerated response to the commensal bacteria associated to the dysregulation of nod1 and nod 2 signaling might play an important role in the development and maintenance of the inflammatory status. methods: the gut bacterial composition was assessed by targeted metagenomic from the patients stool sample, collected before initiation of ibd antibiotics therapy. the 16s rrna amplified and its sequences were analyzed using a bioinformatic pipeline based on mothur software. we determined the bacterial community composition using 100,000 filtered reads using illumina miseq platform. results: according to the ezbiocloud database, the obtained dataset included 548 operational taxonomic units at 3% dissimilarity, distributed among the following groups: bacteroidetes (67.3%) with 37.5% of b. dorei, 18,1% b. vulgates, and 15.3% b. fragilis, firmicutes (24.8%), proteobacteria (7.7%), bacteria_uc (0.06%), actinobacteria (0.04%). conclusions: increased abundance of bacteroides species including b. dorei and b. vulgatus have been implicated in inflammation in several gut diseases such as ulcerative colitis, irritable bowel disease, and celiac disease. although this experience is limited to a single patient, the results of the present study suggest an association between altered gut microbiota and the pathogenesis of ibd in xiap disease and may be of relevance to the future development of novel therapeutic strategies for xiap deficiency. (77) submission id#427718 hematopoietic stem cell transplantation in patients with primary immune regulatory disorders: a primary immune deficiency treatment consortium (pidtc) and inborn errors working party (iewp) study introduction/background: primary immune regulatory disorder (pird) is a newly recognized group of immune-mediated diseases with prominent features of autoimmunity, autoinflammation, and non-malignant lymphoproliferation in addition to immunodeficiency. the clinical manifestations of pirds are frequently difficult to manage and hematopoietic cell transplantation (hct) can be considered as a treatment option, often in those with the most severe disease. we sought to aggregate data from patients who have undergone hct for genetically defined pird or features of immune dysregulation. objectives: we sought to aggregate data from patients with pird who have undergone hct in order to determine the quantity of patients, clinical manifestations, indication and hct, and overall outcome. methods: a questionnaire based survey was sent to all primary immunodeficiency treatment consortium sites and 3 hct referral centers in europe to determine the quantity and characteristics of patients with pirds who have undergone hct. the survey captured clinical manifestations, timing and indication of hct, strategy of hct, and outcomes from 1982-2017. results: 224 patients from 34 centers (31 in north america and 3 in europe) were included with either known genetic defects or considered to have immune dysregulation regardless of gene defect. known genetic defects were identified in 170 subjects, while 54 had symptoms of immune dysregulation but lacked a genetic diagnosis. the mean age of onset of disease was 2 years (range 0-20 years). clinical manifestations included gastrointestinal disorders (69%), failure to thrive (67%), dermatitis (51%), hematologic cytopenias (49%), and lymphoproliferative disease (39%). recurrent infections (66%), immunodeficiency (63%), autoimmunity (48%), and autoinflammation (38%) were also common. organ specific autoinflammation occurred most commonly in the lung (39%) and brain (17%). the median age of hct was 9 years (0-64 years). graft sources included matched unrelated donors (47%), matched related donors (20%), mismatched unrelated donors (16%) and haploidentical donors (3%). reduced or minimal intensity conditioning was used in 44% of transplants. five-year overall survival was 61% and the majority of survivors had resolution of symptoms that led to transplantation. among those patients that died, infection was the most common cause. conclusions: based on our survey data, pird patients commonly develop clinical features of autoimmunity, autoinflammation, and susceptibility to infection at a young age. hct can be successful and lead to disease resolution. however, further studies to define the appropriate patient, timing of hct, donor selection and pre-hct conditioning regimen are necessary to improve outcomes of patients with pird. introduction/background: pathogenic variants in tnfrsf13b (taci) are relatively common (found in about 1% of the population) but have been seen in about 7-10 % of cvid patients, interestingly in both homozygous and heterozygous states. recent articles suggest that the heterozygous state increases the risk of developing autoimmunity due to an effect on autoreactive b cell selection and activation. objectives 1) illustrate the importance of genetic testing in patients with difficulty to treat inflammatory disease 2) present a case report of inflammatory bowel disease associated with a pathogenic mutation in the tnfrsf13b gene results: 16 yo wf was diagnosed with crohns disease due to the chronic abdominal pain, vomiting, and bloody stools since 13 years of age. intestinal biopsy revealed inflammatory changes in the entire intestine with active, submucosal lymphoid hyperplasia, neutrophilic cryptitis with focal areas of crypathic damage, and submucosal epithelioid granulomas more predominantly seen in the colon and rectum. she was started on immunosuppressant medications but developed anaphylactic reaction to both infliximab and adalimumab. she was then treated with azathioprine, mesalamine, methotrexate, and oral budesonide. despite these medications, she continued to have frequent relapses, 4-5 episodes a year and required periodic systemic corticosteroid bursts. other biologics, vedolizumab and ustekinumab, were also tried without success, and she subsequently underwent a colectomy. her postoperative course was complicated by ards, poor abdominal wound healing, and sepsis. due to her complicated clinical course, immune work up was performed which revealed a normal cbc and lymphocyte subpopulations, but hypogammaglobulinemia with low isohemagglutinin titers and specific antibody levels. comprehensive genetic testing ruled out chronic granulomatous disease and other known primary immunodeficiencies but revealed a rare missense mutation in tnfrsf13b (taci). this variant, c310t (p.cys104arg) (rs34557412, exac 0.5%) is likely pathogenic. this heterozygous variant has been seen in both cvid cases and unaffected relatives but significantly more common among cvid patients. moreover, the studies on b cells of these relatives showed impaired function. increased number of autoreactive b cells were also found in the bone marrow of heterozygous individuals and these cells could give a risk of developing autoimmunity. conclusions: in difficult-to-treat autoimmune diseases, identifying the underlying immune defect may aid in the treatment decision. in this case, b cell targeted treatment such as anti-cd20 monoclonal antibody could be beneficial. introduction/background: defects in immunoproteasome caused by biallelic or digenic loss-of-function mutations in proteasome catalytic subunits cause an autoinflammatory disease identified as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (candle) associated to an increased interferon type i gene signature. the proteasome maturation protein (pomp) is a chaperone for both standard and immuno-proteasome assembly and is critical for the incorporation of catalytic subunits. here, we characterize and describe pomp-related autoinflammatory immunodeficiency disease (praid) in two unrelated patients and identify the underlying genetic mechanism of disease. objectives: determine the genetic cause and mechanism of disease in two patients with pomp variants . methods: whole-exome sequencing (wes) was performed to identify a genetic cause of our patients dysregulatory syndrome. proteasome assembly and catalytic function was assessed by sds-page and native gel respectively, using patient derived cell lines. expression of interferon type i-induced genes was measured by rt-qpcr. pomp protein was identified by western blot. results: we identified two unrelated individuals with a unique syndrome characterized by neonatal onset autoinflammation, neutrophilic dermatosis, autoimmunity, and combined immune deficiency with severe systemic viral and bacterial infections. immunologic evaluation for both individuals revealed elevated immunoglobulins, low cd8+ t cell numbers and extremely low b cell counts with persistently high titers of autoantibodies and increased expression of interferon type i-induced genes. in both individuals, truncating heterozygous de novo frameshift variants in pomp were identified by wes and confirmed by sanger sequencing. most mrna transcripts with premature termination codons should undergo nonsense-mediated decay (nmd), however in both of our patients, cdna sequencing revealed these transcripts escaped nmd. the expression wildtype and truncated versions of pomp protein was further confirmed by western blot. transfection of mutant constructs into an otherwise healthy cell line recapitulated an increased interferon signature suggesting a dominant negative mechanism. conclusions: we define praid in two unrelated individuals characterized by neonatal onset immune dysregulation and combined immunodeficiency caused by truncating variants in pomp in which transcripts that escape nmd result in a truncated protein that leads to a dominant negative (i.e antimorphic) allele. to our knowledge, praid is the first inherent defect of immunity mechanistically characterized by nmd escape. introduction/background: parvovirus viremia may occur in pediatric heart transplant patients who underwent thymectomy and developed secondary t cell lymphopenia. high dose intravenous immunoglobulin (hdivig) has been used to treat parvovirus infection in these cases. objectives: we aim to review different routes of immunoglobulin treatment in pediatric cardiac transplant patients with parvovirus viremia and compare the patients immunological phenotype. methods: data from three pediatric heart transplant patients with parvovirus viremia in a tertiary care center was reviewed including t cell counts, parvovirus viral load, route, dosage, and frequency of immunoglobulin treatment. results: all three patients received hdivig. patient 1 and 2 tolerated the treatment and viremia improved. patient 3 developed recurrent aseptic meningitis from hdivig treatment and his viral load remained >1 million copies/ml. compared to the other two cases, patient 3 had a much lower t cell count that likely contributed to the persistence of viremia. to improve his quality of life and reduce healthcare costs, a facilitated subcutaneous immunoglobulin (scig) treatment option was explored. scig treatment was well tolerated and led to a dramatic decrease in parvovirus viral loads in patient 3. conclusions: most pediatric cardiac transplant patients with persistent chronic parvovirus viremia respond well to hdivig, scig may serve as an alternative treatment option in refractory cases, especially in those with severe t cell lymphopenia. severe combined immunodeficiency (scid), by detecting tcell receptor excision circles (trecs) from dried blood spots (dbs) on routine newborn screening (nbs). this has lead to improved estimates of the incidence and prevalence of scid, decreased diagnostic delay, and improved patient outcomes. preliminary studies outside the us have demonstrated that nbs can be adapted to include screening for b-cell deficient infants before symptom onset by quantifying kappa-deleting recombination excision circles (krecs) on dbs. objectives: we report results of the initial characterization of a high throughput triplex trec/krec/rnasep assay run in 9,994 samples in new york state (nys). methods: dbs from 9994 anonymous, de-identified infants were included in the current study. dna from patients with confirmed primary immunodeficiencies (n=32), including, but not limited to, x-linked agammaglobulinemia (n=11) and scid(n=13) were obtained from the centers for disease control and prevention, and clinical immunologists working with the nbs programs in massachusetts, minnesota, wisconsin, and nys, were used as positive disease controls. all dbs were extracted and processed according to current nys nbs protocols. a trec/krec/rnasep triplex assay was designed and optimized to minimize reagent use, and maximize target amplification. cycle threshold (ct) was determined, and error detection cutoffs were identified to optimize sensitivity. results: pcr efficiency, assay quantification, intra-assay reproducibility, and error detection rates all met nys nbs standards. error detection rate for the triplex trec/krec/rnase p assay is 6%, comparable to the current error detection rate of 5% for the current duplex trec/rnase p assay. samples falling into the error detection range are repeated (analysis in process) to determine a receiver operating characteristic curve. conclusions: we show that the high-throughput trec/krec/rnase p triplex assay is feasible in a large, racially and ethnically diverse population in nys. compared to the current duplex assay, this assay has favorable performance characteristics and provides additional immunologic characterization. due to assay optimization, we were able to add the krec test at no additional cost. work is underway to further characterize other assay parameters such as sensitivity and specificity, in preparation for adoption of the triplex assay as part of routine nys nbs. highly accurate wiskott-aldrich syndrome diagnosis via rapid flowbased was protein staining samuel chiang 1 , sue vergamini 2 , ammar husami 3 , marianne ifversen 4 , kejian zhang 5 , jack bleesing, md, phd 6 , yenan bryceson 7 , rebecca marsh, md 8 introduction/background: wiskott aldrich syndrome (was) is a rare xlinked hemizygous disease commonly associated with symptoms of immune deficiency. diagnosis is based on clinical parameters including thrombocytopenia and reoccurring infections, but currently does not include any disease specific marker. as the only permanent treatment for was is hematopoietic stem cell transplantation, it is imperative that a swift and accurate diagnosis be made. absent or lowered was protein (wasp) levels have been reported in sporadic was cases. however, no systemic evaluation exists to date on the accuracy of wasp quantification for was diagnosis. objectives: to determine the accuracy of wasp staining in predicting was genetic abnormalities. methods: we retrospectively evaluated results from a rapid whole blood flow cytometry based assay on a cohort of suspected was patients and compared relative wasp staining levels to was genotype. roc curves as well as accuracy calculations were generated. results: a total of 59 patients with normal and 49 patients with a genetic abnormality in was were collected. missense mutations were most common but insertions, deletions, and gross mutations were also found ( fig a) . comparing was sequencing results to whole blood wasp expression levels provided an 82.6% sensitivity and 100% specificity for a combined accuracy of 95.3% when juxtaposed against genetic sequencing. when 3 variants of unknown clinical significance (vucs) were removed, the sensitivity improved to 89.1% (fig b) . conclusions: staining for wasp is a quick, simple, and accurate assay for the prediction of genetic was defects. introduction/background: cytotoxic t lymphocyte antigen 4 (ctla4) is an inhibitory co-receptor essential for regulatory t cell (treg) function and a central regulator of t cell proliferation and expansion. ctla4 haploinsufficiency is a recently described autosomal dominant disease, in which heterozygous ctla4 mutations result in severe immune dysregulation with variable age of onset and a wide array of clinical manifestations. herein we describe atypical findings in a patient with a novel pathogenic variant in ctla4. objectives 1) to understand whether the novel ctla4 variant identified is pathogenic and 2) to describe eosinophilic gastrointestinal inflammation and exocrine pancreatic insufficiency as possible manifestations of ctla4 haploinsufficiency. methods: next generation sequencing (blueprint genetics ©) was used to identify the ctla4 variant. polyphen and sift (blueprint genetics ©) were used for in silico analysis for the prediction of the effect of this genetic variant on protein structure/function. flow cytometry was used to evaluate ctla4 expression of regulatory t cells. results: a 10-year-old boy with type 1 diabetes mellitus and autoimmune thyroiditis presented with abdominal pain, diarrhea, and weight loss. initial studies revealed markedly elevated peripheral blood eosinophils (>4000 cells/μl) and exocrine pancreatic insufficiency (<50 μg elastase per gram of stool). prominent eosinophilic inflammation was appreciated in biopsies of the stomach, duodenum, jejunum, and terminal ileum. no parasitic infection or inciting drug/food trigger was identified. additional blood studies revealed normal total quantification of t cells but with increased memory t cells (45%, cd3+cd4+cd45ro+) and decreased treg (1%, cd3+ cd4+cd25hifoxp3hi). b cell quantification and serum immunoglobulin (ig) levels were unremarkable, save a modestly elevated ige level (74 iu/ml). comprehensive next-generation sequencing of 232 genes associated with primary immune deficiency revealed a novel heterozygous missense mutation (c.457g>a, p.asp153asn) affecting the last nucleotide in the ligand binding domain (exon 2) of ctla4. subsequent analyses revealed decreased ctla4 expression in the patients t cells compared to healthy controls as well as evolving hypogammaglobulinemia. treatment consisted of methylprednisolone and parenteral nutrition followed by sirolimus and abatacept to which the patient responded favorably. conclusions: we report a 10-year-old boy with a history of type 1 diabetes mellitus and autoimmune thyroiditis presenting with hypereosinophilia, eosinophilic gastroenteritis, and exocrine pancreatic ins uff iciency as unique m anifestat ions o f ctla4 haploinsufficiency. although not previously reported in individuals with ctla4 haploinsufficiency, peripheral blood eosinophilia and eosinophilic inflammation of the gastrointestinal tract have been observed in patients receiving ipilimumab (ctla4 blocking antibody) suggesting a potential mechanism for the aforementioned findings. severe exocrine pancreatic insufficiency is a rare but observed manifestation in individuals with type 1 diabetes mellitus. whether severe exocrine pancreatic insufficiency would be expected t o o c c u r m o r e f r e q u e n t l y i n i n d i v i d u a l s w i t h c t l a 4 haploinsufficiency and type 1 diabetes mellitus is unclear; however, our reported case and surveillance of others with ctla4 haploinsufficiency could elucidate incidence and prevalence of this manifestation. introduction/background: mild asymptomatic hypogammaglobulinemia during pregnancy is a well-described phenomenon due to hemodilution. in patients with known humoral primary immunodeficiency such as common variable immunodeficiency, women require an upwards titration of their immunoglobulin replacement dose. isolated symptomatic hypogammaglobulinemia during pregnancy in a patient is not well described in the literature. objectives 1. understand the physiology of igg during pregnancy. 2. define a rare entity with a likely genetic predisposition that manifests as hypogammaglobulinemia isolated in pregnancy. 3. management of this entity with defining goals of treatment. results: 33 year old gravida 5 para 4 female presented with progressive hypogammaglobulinemia restricted to pregnancy starting with 3rd pregnancy, recurrent otitis media requiring 3 sets of myringotomy tubes, recurrent sinusitis, and streptococcal pharyngitis. her son has common variable immunodeficiency (cvid) requiring immunoglobulin replacement (igrt). prior to conception, her igg was 849 mg/dl. during 13th week of pregnancy, her igg level was 627 mg/dl. during 21 weeks of pregnancy, she complained of fatigue, and developed an episode of sinusitis that required 2 different antibiotic treatments. at that time, her igg was 567 mg/dl. she was started on subcutaneous igrt maintain igg troughs of >650 mg/dl. she remained infection-free during her pregnancy and igrt was stopped a few months after delivery with her serum igg level returning to pre-pregnancy levels. patient was initially evaluated during her 3rd pregnancy with recurrent streptococcal pharyngitis. at that time, her igg was 682 mg/dl. diphtheria, haemophilus influenza, mumps, measles and rubella titers were protective, tetanus titer was non protective with 1/14 antipneumococcal titers protective. she was vaccinated with pneumovax and tdap with development of protective titers and remained infectionfree. igrt was not given and post-delivery, her igg levels improved to 792 mg/dl. she was seen one and half years later, for prenatal counseling for her 4th pregnancy. her immune evaluation included an igg 915. she demonstrated protection to tetanus, diphtheria and streptococcal pneumoniae. at 16 weeks of gestation, she developed recurrent upper respiratory infections requiring antibiotics. her igg was 697 mg/dl. at 28 weeks of gestation, her igg was 537 mg/dl. igrt was recommended, but patient refused at that time. after delivery, igg improved to 849 mg/dl. conclusions: decreased immunoglobulin levels during pregnancy are welldescribed phenomenon which can be attributed to hemodilution of pregnancy. igg transport to fetus generally begins in the second trimester and reaches its pinnacle in the third trimester. patients with known cvid require dose adjustments (higher) during pregnancy as they can be more symptomatic during this time. here we describe a patient who has an almost normal immune evaluation except for mildly low iga during absence of pregnancy, but during pregnancy, develops recurrent infections with significantly low igg level. her family history of a son with cvid, new daughter with low igg levels like to be transient hypogammaglobulinemia of infancy (thi), another daughter with thi suggests a genetic b-cell defect that manifests as cvid with mildly low iga and hypogammaglobinemia during metabolic stress such as pregnancy. there is only one similar report of a single pregnancy of transient symptomatic hypogammaglobulinemia during pregnancy. such patients should be adequately worked up and treated during pregnancy with igrt to decrease maternal and fetal mortality. introduction/background: card11 encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to nf-b, jnk, and mtorc1. germline mutations in card11 are known to give rise to distinct primary immune disorders in humans, including scid (null mutations), b cell expansion with nf-b and t cell anergy (benta; gain-of-function mutations), and severe atopic disease (loss-of-function, dominant interfering mutations). objectives: here we report our experience with an expanded cohort of patients harboring novel heterozygous card11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with card11 mutations. methods: cell transfections and primary t cell assays were utilized to evaluate signaling and function of card11 variants. results: we demonstrate that in addition to severe atopy, heterozygous missense mutations in card11 associated with dominant negative activity can present with immunologic phenotypes similar to those observed in stat3lof, dock8 deficiency, common variable immune deficiency (cvid), congenital neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome. evaluation of rare or novel card11 variants found in affected patients showed that dominant negative activity was largely confined to the card or coiled-coil domains, but did not always manifest in atopic disease. conclusions: these results illuminate a broader phenotypic spectrum associated with card11 mutations in humans, and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity. introduction/background: increasing number of states have been screening for severe combined immune deficiency (scid) as part of the expanded newborn screening program for nearly a decade. in the era of newborn screening, patients with scid present often asymptomatically and are prepared for early hematopoietic stem cell transplantation (hsct). with advances in genetic testing, mutations in over 20 genes have been associated with development of the scid or leaky-scid phenotype. objectives: to present 2 unique cases of hypomorphic x-linked scid where no known pathogenic mutations were identified on initial genetic testing, the il2r gamma-chain as protein was expressed, but subsequent testing months later revealed pathogenic il2rg mutation affecting either translation or protein function. to expedite earlier identification of pathogenic il2rg mutation, we propose screening with x-inactivation studies in maternal t lymphocytes and assessing gamma-chain function by evaluating il21r signaling in select male infants with abnormal newborn screen for scid, specifically those with scid phenotype but no identified pathogenic gene mutation on initial genetic testing, and the presence of gamma-chain expression. male patient a presented during the first week of life after his newborn screen was found to be abnormal with undetectable t cell receptor excision circle (trec) count. a phenotype of t-b+nk-scid was established and the patient was sent for bone marrow transplant evaluation. genetic testing revealed a novel hemizygous missense mutation in il2rg (p.glu59gln), which was a variant of unknown significance. the patient had common gamma-chain expression by flow cytometry on b and nk cells. he underwent haploidentical hsct with his father as donor. unfortunately, his transplant was complicated by prolonged neutropenia, slow t cell reconstitution, and eventual graft failure. to review his next treatment options, it was necessary to prove that his il2rg mutation was pathogenic. male patient b presented with concern for an underlying immune deficiency after being hospitalized for peumocystis jirovecii pneumonia. his newborn screening for scid was inadequate at birth and follow up was delayed. retrospective analysis of the newborn screening card at birth confirmed absence of trecs. his phenotype was also t-b+nk-, consistent with x-linked scid. he also proceeded to hsct with a haploidentical parent donor. despite hhv-6 viremia pre-hsct which persisted posttransplant, he has had appropriate t cell engraftment. comprehensive genetic testing on whole exome level did not reveal any known mutations contributing to his phenotype. patient did have expression of gamma-chain by flow cytometry on t, b and nk cells. however, further testing revealed an il2rg 3 utr deletion of aa that, based on similar findings from a prior study can possibly lead to mrna abnormalities.1 to expedite the association of scid phenotype with x-linked disease, implying gamma-chain pathology, we obtained x-inactivation studies in maternal t-cells that showed severe skewing in both cases. furthermore, il21r signaling was impaired on b-cells in each case. the combination of these two assays proved that both patients carry a pathogenic il2rg mutation. conclusions: in the era of newborn screening for scid, we are discovering that phenotypic variability of scid patients can be very broad and caused by hypomorphic mutations in the common chain gene. exonbased genetic testing cannot exclude all variants and novel variants of unknown significance have to be evaluated by additional assays, including functional studies for causal effect. it is important to expedite early proof of association with gamma-chain pathology, especially in the era of gene therapy. we propose that in male infants with abnormal scid newborn screening and no known or previously described pathogenic mutation on genetic screen, evaluation continues for hypomorphic il2rg mutations. the probability of this process can be increased by a simple screening test for x-inactivation of maternal t lymphocytes. allergy / immunology, allergy / immunology associates inc. / case western reserve university introduction/background: igg4-related disease (igg4-rd) is an immunologic disorder with multiple clinical presentations previously thought unrelated. it is characterized by the frequent presence of tumor-like swelling of the affected organs and several histopathological findings including tissue lymphoplasmacytic infiltrates with predominantly igg4-positive plasma cells and lymphocytes, storiform fibrosis and obliterative phlebitis. humoral immunodeficiency is a term that encompasses several disease entities associated with impaired antibody production. it is suspected in patients who present with recurrent, frequently severe, sinopulmonary infections with encapsulated bacteria, which leads to evaluation of quantitative immunoglobulin levels and vaccine responsiveness. despite a few reports of primary immunodeficiency in patients with serum igg4 elevation, no adult case has been reported of igg4-rd in a patient with concomitant humoral immunodeficiency. objectives: to present a unique case with the presence of concomitant igg4-related disease and humoral immunodeficiency. methods: comprehensive chart review of our patient and all performed exams. literature review for igg4-related disease, igg4 elevation in humoral immunodeficiency and concomitance of igg4-related disease with humoral immunodeficiency. results: our patient is an 84-year-old caucasian male with relevant past medical history of chronic bronchitis who was referred to our practice after several episodes of pneumonia in the previous years, with six courses of antibiotics just in the year prior for recurrent sinopulmonary infections. blood tests revealed hypergammaglobulinemia and low level of vaccine responsiveness. chest ct showed multiple bilateral pulmonary nodules and hilar and mediastinal lymphadenopathy. sinus ct showed left maxillary sinus opacification. pulmonary function testing was normal. he later presented with left eye edema, proptosis, diplopia, and painless submandibular salivary gland enlargement. laboratory investigation showed an igg of 1730 mg/dl, igg4 of 771 mg/dl. the patient denied any history of pancreatitis or abdominal pain and abdominal ultrasound was normal. biopsy of a salivary gland was normal. mri of the left orbit was obtained, showing lacrimal gland enlargement. based on the patients recurrent infections, lack of response to tetanus immunization, and limited, non-sustained response to pneumococcal immunization, the patient was started on ivig therapy. the patient was also diagnosed with possible igg4-rd based on his salivary gland enlargement and orbital disease in association with hypereosinophilia and increased plasmablast levels. oral prednisone 40mg daily was started for four weeks, later followed by slow steroid taper (reducing 10mg every two weeks) with considerable improvement in left eye swelling and proptosis. a few months after discontinuation of the steroids, the orbital disease returned to its previous severity. left lacrimal gland biopsy confirmed igg4-related disease, with many areas showing greater than one hundred igg4-positive plasma cells per high-power field. after another course of steroids, oral prednisone was weaned to a maintenance dose of 10mg daily and the patient became asymptomatic from his ophthalmologic complaints with normalization of his ophthalmologic exam. his last checked igg was 1105 mg/dl. igg4 was still elevated at 324.8 mg/dl , but given controlled symptoms the patient was spaced to monthly ivig infusions and continued on that daily steroid dosage. conclusions: our patient, initially diagnosed with a humoral immunodeficiency, was later also diagnosed with biopsy-proven igg4-related disease, which is a novel association of this two diseases in an adult patient. previous rare reports of association between elevated serum levels of igg4 and patients with concomitant humoral immunodeficiency were in the presence of isolated igg4 elevation and not in the presence of igg4related disease. this novel association creates a therapeutic dilemma since the patient in question is hypergammaglobulinemic, yet needs ivig, which can lead to side effects such as thrombosis due to a hyperviscous state. the description of additional concomitant cases of both diseases and further understanding of their pathophysiology will be crucial to create awareness and obtain earlier diagnosis, to refine therapeutic options and design adequate treatment protocols. igm and iga anti-pneumococcal capsular polysaccharides as prognostic tool for common variable immunodeficiency: a longitudinal study. professor, sapienza university of rome introduction/background: the clinical spectrum of cvid ranges from a poorly symptomatic form to severe phenotypes characterized by high susceptibility to infections, autoimmunity, granulomatous inflammation, lymphoproliferative disorders, and malignancies. due to high prognosis heterogeneity, prognostic factors are required. objectives: with the aim to identify additional prognostic factors, we evaluated the anti-polysaccharide iga and igm responses by elisa assay in 75 cvid in a longitudinal study over a 6-year period. methods: patients were immunized at baseline with the 23-valent pneumococcal polysaccharide vaccine (pneumovax®). twenty healthy donors (hd) were also included. results: as expected, cvid patient had lower igm/iga response than hd. for cvid, four immunological phenotypes were identified by postvaccination igm and iga levels: igm and iga responders (11%), igmhigh responders (4%), igm-low responders (20%) and non-responders (61%). to simplify, we analysed igm-high group with igm and iga responders and igm-low with non-responders. during the follow up, concomitant cvid-related conditions, immunoglobulin serum levels, respiratory infections and outcome were recorded by medical files. cvid igm-low/non-responders developed more frequently respiratory, gastro enteric and autoimmune manifestation and malignancies in comparison to igm-high/igm and iga responders (respectively, pneumonia: 62% vs 25% ; chronic diarrhea: 33% vs 18%; autoimmunity 38% vs 9%). autoimmune cytopenias were not found in the igm-high/igm and iga responders group. eleven (15%) patients died during the study time. survival analysis according to the igm/iga responder status showed that the 6-years estimated survival for igm-high/igm and iga responders vs igm-low/non-responders group was respectively: 100% vs 100%, 100% vs 95%, 100% vs 92%, 100% vs 89%, 100% vs 85%, 100% vs 85%, 100% vs 82%. interesting, in our series only two deaths were due to infective complications: five were consequent to malignancies, one to autoimmune cytopenias and three to not-cvid related conditions. conclusions: in conclusion, even if patients could not raise the protective humoral level, in cvid the anti-polysaccharide iga and igm responses could represent a prognostic factor, individuating groups of patients with less immunological impairment, lower risk of comorbidities and better survival. introduction/background: gaucher disease (gd) is a rare autosomal recessive disorder characterized by a defective function of the catabolic enzyme -glucocerebrosidase (gba) leading to a progressive accumulation of its substrate-glucocerebroside (gc) -in various organs in particular in mononuclear phagocite system. hepatosplenomegaly and cytopenia represent the most common features of the disease. moreover, gd patients also show hyperinflammatory features -secondary to machrophages engorgement and actviation-hypergammaglobulinemia, and a immune-dysregulation involving b , t and nk cells. since clinical phenotpye can be subdolous, symtoms can overlap with alps, however, few data are available on specific immunity pattern in these patients. objectives: to evaluate immune-phenotype and other alps parameters in a cohort of patients with gd methods: we evaluated lymphocytes subsets, immunophenotypic and serological features of alps (dnts, tcr alfa/beta b220, b-memory cells, tregs/hla-dr ratio, il-10, il-18), and test of apoptosis in a cohort of patients with gd followed-up at igg. results: 35 patients (28 in treatment, 5 not) were studied. dnts and tcr alfa/beta b220+ resulted to be >1.5% of t-lymphocytes and > 60% in 6/32 (19%) and in 7/32 (22%), respectively. b-memory cells and t-regs/hla-dr ratio were <15% and <1 in 11/32 patients (34%). 3/32 evaluable (9%) had all these parameters concomitantly alterated. 4/19 (21%) evaluable patients were resistant to apoposis. il-18 was pathological in 26/29 (89%) patients. all patients had normal levels of il-10 and sfas. conclusions: this study shows that some patients with gd may present an immune-dysregulation pattern that can overlap with alps features. therefore, the differential diagnosis of gd should be taken into consideration by clinicians during diagnostic work-up of patients with an alpslike phenotype. introduction/background: allogeneic hematopoietic stem cell transplantation (hsct) using unrelated and haploidentical donors is complicated by increased rates of graft-versus-host disease (gvhd) and slow immune reconstitution. selective depletion of alpha/beta t lymphocytes and b cells is a recently developed method of graft manipulation that retains mature natural killer (nk) and gamma/delta t lymphocytes, both of which may exert a graft-versus-leukemia effect and protection against life-threatening infections. objectives: to describe the rate and quality of immune reconstitution, incidence of transplant-related complications, including viral reactivation and gvhd, and overall outcomes following tcr-alpha/beta-and cd19depleted hsct for hematologic malignancy in pediatric patients. methods: forty patients of median age 11.2 years (1.7-21.7) underwent hsct for acute myeloid leukemia (n=25), acute lymphoblastic leukemia (n=12), and myelodysplastic syndrome (n=3). grafts were from unrelated (n=29) and haploidentical (n=11) donors. tcr-alpha/beta and cd19 depletion was performed with the miltenyi clinicmacs plus system. median cd34+ cell dose was 10.1 x 106/kg (4.7-15), and median cd3+ cell dose was 2.3 x 107/kg (0.21-43.3). conditioning was with myeloablative busulfan or total body irradiation, cyclophosphamide, and thiotepa. twenty of 29 unrelated donor hscts and 11/11 haploidentical hscts also included antithymocyte globulin x 3. no patient received post-transplantation gvhd prophylaxis. all but 5 patients received rituximab on day +1 per protocol for recipient positive epstein barr virus (ebv) serology. results: all patients engrafted. median time to neutrophil engraftment was 13 (8-30) days, and median time to platelet engraftment was 16 (12-40) days. one patient experienced graft rejection on day +18, and twelve patients relapsed at a median of 173 (47-625) days. overall survival was 28/40 (70%) at a median of 24.2 (5.8-34) months follow-up. two (5%) patients developed grade iii or higher acute gvhd, and 2 (5%) patients developed extensive chronic gvhd. cumulative incidence of cytomegalovirus (cmv) and adenovirus reactivation were 11/40 (27.5%) and 5/40 (12.5%), respectively. nine (22.5%) patients developed bk hemorrhagic cystitis +/-viremia. ebv reactivation was not observed. median total, myeloid, t cell, and b cell donor chimerism were all 100% (ranges 50-100%, 93-100%, 41-100%, and 99-100%) at 1 year post-hsct. immune reconstitution of all cells lines was rapid ( table 1) . eighteen of 26 (69%) patients had detectable t cell receptor excision circles (trecs) by 4 months with a median trec count of 1226 (0-5713) per 10^6 cd3 t cells, and recovery of the naïve t-cell compartment was observed by 8 months in 19/26 (73%) of patients. t cell function as measured by response to pha was normal by 8 months in 15/23 (65%) patients and continued to increase steadily with time. despite rituximab on day +1 for 35/40 (87.5%) patients, there was rapid b cell reconstitution. nineteen of 23 (82.6%) patients had present switched memory b cells at 8 months, and 22/28 (78.5%) surviving patients are off immunoglobulin replacement at a median of 4 (4-18) months. conclusions: selective tcr-alpha/beta and cd19 depletion of haploidentical and unrelated grafts results in high engraftment and rapid immune reconstitution with low incidence of gvhd in children with hematologic malignancy. cd19 depletion and routine post-hsct rituximab on day +1 are effective at preventing ebv reactivation. introduction/background: hyper-igd syndrome (hids; 260920) is an autosomal recessive disorder characterized by recurrent episodes of fever associated with lymphadenopathy, arthralgia, gastrointestinal disturbance, and skin rash. the diagnostic hallmark of hids is a constitutively elevated level of serum immunoglobulin d (igd), although patients have been reported with normal igd levels. the disease is associated with mutations in the gene of mevalonate kinase. objectives: male patient, 30 years old, born to non-consanguineous parents, with a history of severe diarrhea since childhood, followed by respiratory infections and pneumonias. moreover he presented several episodes of severe abdominal pain, with intestinal obstruction since seven months old, needing surgical intervention due to acute abdomen. this picture repeated several times until 10 years of age, being submitted to new surgeries due to intestinal suboclusion. at 16 and 17 years he had gastroenteritis and then pancreatitis. 2 years ago new severe acute gastroenterocolitis with suboclusion, submitted to laparotomy and resection of a little part of the gut. he has frequent diarrheas, triggered by coffee and tea. usually evacuates 5 times a day. he was evaluated by several pediatric immunologists in childhood, who distrusted alpha heavy chain disease. he had an intense reaction to the bcg vaccine, and then did not make any more vaccines (only opv (sabin) in the campaigns). he was tonsillectomized at 2 years of age. he had measles, chickenpox, and mumps (at 5 years old). cellulitis at 7 years, repeating several times since then. he presented improvement of pneumonias but still has sinusitis and otitis (approximately 8 times a year introduction/background: pitthopkins syndrome is a rare neurological disorder caused by mutations the tcf4 gene on chromosome 18q21. clinical features include severe intellectual disability, constipation, microcephaly, and seizures. features distinguishing it from other neurodevelopmental syndromes such as rett syndrome and angelman syndrome include breathing abnormalities (either apneic episodes or hyperventilation) and atypical facial features. typical facial dysmorphism includes bitemporal narrowing, deep-set eyes, an m shaped upper lip, and widely spaced teeth. although a very rare diagnosis (slightly over 200 reported cases), it is not known to be associated with underlying humoral or cellular immunodeficiency. there is only one report of igm abnormalities described in a patient with pitt-hopkins syndrome. objectives: to present a case of pitthopkins syndrome with humoral immunodeficiency. methods: this is a case presentation of a patient with pitt-hopkins syndrome requiring immunoglobulin replacement therapy. results: 15 year old female with genetically diagnosed pitt-hopkins syndrome who presented to our office for immunological evaluation in the setting of recurrent sinopulmonary infections. she was placed on chronic antibiotics by the department of otholaryngology for approximately two years prior to presentation. she was found to be hypogammaglobulinemic (igg: 398 mg/dl, iga: 21 mg/dl, igm: 40 mg/dl), had non-protective titers to streptococcus igg antibody (6/23 titers protective greater than 1.3 mcg/ml) despite booster vaccination with pneumovax, and had borderline tetanus (0.18 iu/ml) and diphtheria titers (0.11 iu/ml). given this laboratory evaluation and her recurrent illness, immunoglobulin replacement therapy (igrt) was started. whole exome sequencing was completed to assess for any other genetic cause of her immunodeficiency. the only abnormality was her previous known pathologic variant p.q670hfsx40 c.2010_2011delga (gln670his) in exon 19 in the tcf4 gene. she continued to have infections despite therapeutic igrt. chronic antibiotic treatment was initially tapered, however needed to be reintroduced as ivig alone was not stopping her infections, despite a igg level over 1000 mg/dl. conclusions: humoral immunologic deficits are not known to be associated with pitt hopkins syndrome. there has been one case report of a patient with poliomyelitis-like syndrome following an asthma attack in a patient with pitt hopkins syndrome, which was treated with igrt and resulted in a nearly complete recovery. however, igrt was not used for reasons of underlying immunodeficiency. to our knowledge this is the first patient with pitt hopkins syndrome with persistent hypogammaglobulinemia and frequent infections requiring immunoglobulin replacement therapy. it remains unclear why the patient continued to have infections despite igg levels > 1000 mg/dl, yet with the combination of igrt and prophylactic antibiotics the patient remains healthy. introduction/background: gata2 deficiency is a rare disease that typically presents in late childhood or early adulthood with heterogeneous phenotypes including emberger syndrome. emberger syndrome is characterized by lymphedema and predisposition for myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). over 75% of patients with gata2 mutations have immune deficiencies. definitive diagnosis of gata2 deficiency is made by gene sequencing, and treatment includes infection control and potentially hematopoietic stem cell transplant (hsct). objectives: the goal of this report is to contribute data to the small documented cohort of patients with gata2 deficiency to aid in diagnosis and management of this rare, heterogeneous disorder. methods: we present a case series of three siblings with identical gata2 mutations with variable phenotypes. a usidnet query resulted 51 patients with gata2 mutations. results: three siblings (12 year-old female (a), 15 year-old male twin (b), and 15 year-old female twin (c)) and their mother had congenital deafness. clinical symptoms include (a) h1n1 influenza requiring mechanical ventilation, warts, and hypogammaglobulinemia, (b) streptococcus pyogenes neck abscess, warts, acne, and mds, and (c) lymphedema and acne. all three patients had absolute monocyte count (amc) of 0 and lymphopenia without documented lymphoid cell dysfunction. a mutation on exon 5, c.1062delg, confirmed the diagnosis. all three patients were started on mycobacterial prophylaxis with azithromycin and recommended hpv vaccination. the usidnet query average age of symptom onset was 22 years, and average age at diagnosis was 31.5 years. 35.3% (18/51) of patients had a family history of gata2 deficiency, 49% (25/ 51) of patients had warts, 7.8% (4/51) had lymphedema and only 1 patient had sensorineural deafness. 38% (19/50) of patients had amc of 0. functional data was limited. conclusions: life threatening infections as well as hematologic malignancies have been reported in patients with gata2 deficiency, which can be successfully treated with hsct. to our knowledge, the gata2 mutation detected in this family has not been previously reported. the clinical presentation in these three patients was heterogeneous despite identical genotypes, and diagnosis occurred years after initial symptoms. variable phenotypes were found in the usidnet gata2 deficiency cohort as well. a high index of suspicion for the disorder and early recognition of clinical manifestations and laboratory abnormalities may aid in timely diagnosis of gata2 deficiency, with potential for improved outcomes. consulting medical advisor, immune deficiency foundation introduction/background: as individuals with antibody deficiencies age they are susceptible to developing shingles. patients with antibody deficiencies are advised not to receive live viral vaccines such as zostavax, the shingles vaccine. objectives: our survey aimed to determine the frequency of shingles and use of zostavax in common variable immunodeficiency (cvid) and hypogammaglobulinemia patients. methods: 11,533 email invitations delivered to members of the immune deficiency foundation database requesting participation in an online survey about zoster, influenza and varicella experiences. data from 881 individuals age 19 years old or older with cvid (n=760; mean age 54 years old; 83% female; 92% white non-hispanic) or hypogammaglobulinemia (n=121; mean age 54 years old; 83% female; 93% white non-hispanic) were analyzed. results: close to one fifth (18%, n=105) of adults age 50 or older with cvid or hypogammaglobulinemia (n=568) had received shingles vaccination. the majority of those who were vaccinated reported receiving zostavax once (92%, n=97), while 5% (n=5) received a booster vaccination as well. mild side effects (e.g., skin rash and muscle pain) were only reported by 18% (n=18) of vaccine recipients after receiving their first vaccination. no side effects were reported after receiving the booster vaccination and no hospitalizations were reported as a result of receiving zostavax. when comparing shingles diagnosis and shingles vaccination, of the 568 adults age 50 years old and older, 35% (n=196) had been diagnosed with shingles, and of those diagnosed 82% (n=160) did not receive zostavax. of adults age 19 years old or older, 32% (n=279) reported a shingles diagnosis. similarly, the cdc reports almost 30% people in the united states will develop shingles during their lifetime. more than half (56%, n=157) of those ever diagnosed with shingles reported experiencing shingles once, 18% (n=51) had shingles twice and 24% (n=68) had shingles three or more times. respondents with more than three shingles episodes were more likely to report their rash lasted more than two months and their blisters became infected. conclusions: almost 20% of adults with cvid or hypogammaglobulinemia reported receiving zostavax despite recommendations against vaccinations for immunodeficient individuals. however, side effects in those pi patients who received the shingles vaccine appears minimal. though it is possible these individuals were vaccinated prior to diagnosis of their pi; additional patient and physician education on live vaccines and immunodeficiency may be needed as well. the approval of the new non-live virus component varicella zoster vaccine may be of benefit to patients with pi. introduction/background: inclusion body myositis (ibm) is a rare disorder characterized as an inflammatory myopathy with endomysial inflammation and numerous red-rimmed vacuoles seen on biopsy. five cases of ibm have been described in the literature in patients with common variable immunodeficiency (cvid). objectives: to make immunologists aware of ibm as a complication of cvid, that may be incorrectly diagnosed as myositis or autoimmune neuropathy. methods: case description results: we report a 62-year-old man with common variable immunodeficiency on gammaglobulin replacement who presented complaining of progressively worsening lower extremity pain, weakness, and fatigue. he states that over the last couple of years, it has become difficult climbing and descending steps, arising from a seated position, and has begun experiencing frequent falls. his creatinine kinase level was found to be elevated at 293u/l and he continued to be lymphopenic with total lymphocyte counts ranging from 300 to 800k/ul (cd4+ t-cells 34%, cd8+ t-cells 18%, cd19+ bcells 2%). his esr ranged from 25 to 60mm/h. anti-glutamic acid decarboxy antibody (gad) was initially elevated at 1.3u/ml and rose to 2.3u/ml within 6 months suggestive of a neuropathy. based on an electromyography (emg) and a muscle biopsy, he was diagnosed with polymyositis. he was treated with high dose steroids with no improvement. his intravenous gammaglobulin dose was then increased from 45mg every 3 weeks to 45mg per day for 3 straight days every 2 weeks. 4 months later, his creatinine kinase level dropped into the normal range (<200u/l), however, he continued to complain of worsening weakness. physical exam showed decreased muscle bulk in forearms and quadriceps bilaterally, lack of a quadriceps tendon reflex, strength 4/5 in flexor digitorum profundus and 4/5 in hip flexors, and a broad-based gait. histology and electron microscopy of a repeat muscle biopsy identified rimmed muscle vacuoles as typically noted in inclusion body myositis. conclusions: inclusion body myositis is a potential rare complication of common variable immunodeficiency. it can mimic polymyositis and inflammatory demyelinating disorders. high dose steroids and ivig are of no clinical benefit in ibm, despite decreasing serum creatinine kinase levels, which may raise a false impression of a clinical benefit. objectives: in this abstract we present the case of the development of generalized cmv infection in a child with scid. girl n. at the age of 3 months entered the children's infectious clinical hospital with complaints of cough, high febrile temperature for 5 days, refusal to eat. from the anamnesis of life the girl from the 1st pregnancy, 1 birth, was born full term in 40 weeks gestation, birth weight 4640g. for 3 months of life, a bad increase in body weight was noted and at the time of admission, the weight in 3 months was 5400 g. according to the parents, the child had atopic dermatitis. from the anamnesis of the disease on 08.01.2017, the temperature rose to 38.2°c, there was a cough and a mucous discharge from the nose. then the child refused to eat, the body temperature rose to 39.2°c. at this time, the girl's mom was borne by the ari. january 14, 2017 patient was hospitalized in the hospital with a diagnosis: acute respiratory viral infection, acute rhinitis, pharyngitis, acute bronchitis, toxicosis of 1-2 degrees. acute pneumonia? atopic dermatitis, infant form. on january 15, 2017, due to the worsening of the condition associated with the increase in oxygen (o2) -dependence, the child was transferred to the department of anesthesiology and resuscitation. methods: in the general analysis of blood upon admission, leukocytes are 14.2x10 9 / l, hemoglobin is 105 g / l, platelets are 172 x 10 9 / l, esr is 3 mm / hour, stabs are 4% (abs. -0.58 x 10 9 / l), segmented -60% (abs-8.64 x 10 9 / l), lymphocytes -24% (abs 3.46 x 10 9 / l), monocytes -10% (abs -1.44 x 10 9 / l). in a biochemical study, the total protein is 51 g / l, total bilirubin is 4.7 mol / l, urea is 7.4 mmol / l, creatinine is 60 mol / l, lactate dehydrogenase is 2249 u / l, alt is 131 u / l, asat -257 e / l, crp -5.8 mg / l. radiography of the lung from 14/01/2017 -data in favor of interstitial pneumonia. the study of the acid-base ph state is 7.367, pc02 is 34.3 mmhg, po2 is 40.1 mmhg, lactate is 1.4 mmol / l. a blood test was performed using the elisa and pcr method for markers of hsv, cmv, enterovirus and toxoplasmosis. ultrasound of the abdominal cavity revealed moderate hepatomegaly, signs of thickening of bile, splenomegaly. moderate diffuse changes in the renal parenchyma (toxic-inflammatory?). the minimum amount of free fluid in the abdominal cavity. ultrasound of the brain revealed signs of subependimal microcyst on the right. according to the immunogram, a sharp decrease in cd3 + 26% (58-85%) was detected, activated t-lymphocytes (cd3 + hla-dr +) were 19.9% (3-15%), t helper / inducers (cd4 + cd8 -26.6% (30-56%) and t suppressors / cytotoxic (cd8 + cd4-) 0.5% (18-45%), a high ratio of tx / tc (cd4 + cd8 +) was detected 53.2% (0.6-2.3), cytotoxic non-t cells (cd3-cd8 +) -1,2, an increase in the number of b-lymphocytes (cd19 +) -58.9% (7-20%), natural killers (cd16 + cd56 +) -6.6% (5-25%), natural t-killers (cd3 + cd16 + cd56 +) -0.3 (0-5%), leukocyte gates (cd45 + cd14-) -99% (95 -100 %). the absolute content of t-lymphocytes was 0.15 x 10 9 / l, b -lymphocytes -0.35 x 10 9 / l. the number of thymic migrants (cd45 + cd45ra + cd31 +) was not detected (0%). according to the results of the immunogram the diagnosis is made: severe combined immunodeficiency (t-b + nk +). 01/17/2017 ct scan of the chest was diagnosed ct signs of a polysergic two-sided inflammatory process in the lungs (figure 3 ). when blood was sown for sterility on january 19, 2017, staphylococcus epidermidis was isolated in an amount of 10 3, sensitive to linezolid, gentamicin resistant to amoxicillin, amoxicillin / clavulonic acid, and ciprofloxacin. on january 19, 2017, cmv dna was detected in an amount of 7.6 × 10 6 copies / ml. results: since the arrival clarithromycin was administered at a dose of 15 mg / kg per day in 2 divided doses from 14/01/2017 to 15/01/2017. from 16/01/2017 to 17/01/2017. change of antibacterial therapy for azithromycin intravenous at a dose of 10 mg / kg per day once a day.17.01.2017 -01/18/2017 the state of the child is very severe with negative clinical and laboratory dynamics despite the ongoing therapy. antibacterial therapy was changed to meropenem in a dose of 60 mg / kg intravenously every 8 hours, linezolid at a dose of 30 mg / kg per day and oseltamivir at a dose of 6 mg / kg per day from 17/01/2017 to 20/01/2017. 19.01.2017-20.01.2017 substitution therapy with an octagam in a dose of 0.4 g / kg was intravenously dripped. the patient's condition without significant dynamics. based on the results of pcr on cmv, ganciclovir was administered at a dose of 10 mg / kg intravenously drip 2 times a day. 01/23/2017 due to a decrease in platelet count, the platelet mass is transfused and there was a rash all over the body at night, which is associated with the development of the "graft versus host" reaction (gvhr). despite the ongoing therapy, a fatal outcome occurred. the main diagnosis: primary immunodeficiency (severe combined immunodeficiency, t0 b + nk +). complications: sepsis. septic shock. spon: ards, renal failure, dis, thrombocytopenia, anemia 3. two-sided lower-lobe pneumonia. generalized cmv infection. gvhd, acute dermal form. concomitant: atopic dermatitis, infant form. conclusions: the peculiarity of the described clinical case was that the patient's first symptoms of scid developed in the first months of life and were manifested by a bad weight gain, atopic dermatitis and the development of a life-threatening generalized cytomegalovirus infection with the development of bilateral low-grade pneumonia, respiratory insufficiency and acute cutaneous gvhd form, after transfusion of unirradiated platelet mass. an expanded immunological study confirmed the diagnosis of scid. methods: this study included 5 patients (2 boys and 3 girls) aged 2 months to 5 years. the reasons for entering the hospital were manifestations of severe hepatitis (in 2 children), acute respiratory infection (2 children) and 1 patient with symptoms of infectious mononucleosis. all patients were examined according to clinical protocols and given the severity and atypicality of the course of any infectious diseases, patients underwent immunological examination of the blood and they were consulted by an immunologist. all children were diagnosed with congenital immunodeficiency. results: in 3 cases, the trigger for the realization of the immunodeficiency state was infection (e. meningoseptica + kl. pneumonia + b. pertussis; cmv; veb); in 2 patients, giant cell hepatitis occurred. in 2 patients, despite the ongoing therapy, the disease had an unfavorable (lethal) outcome (1 patient with hepatitis and 1 patient with generalized cmv infection). conclusions: thus, it should be noted that timely diagnosis of a congenital defect of the immune system and thus timely therapy will avoid adverse outcomes. interferon gamma (actimmune®) effects on severe burkholderia cepacia pneumonia in variant x-linked chronic granulomatous disease 4 professor of pediatrics, university of utah 5 associate professor of clinical pediatrics, keck school of medicine at the university of southern california 6 professor of medicine, university of utah introduction/background: interferon gamma (ifnγ; actimmune®) has been proven to significantly decrease the overall number of infections in patients with chronic granulomatous disease (cgd) when given prophylactically (nejm 324:408, 1991) . therapy with ifnγ has also been employed to treat severe overwhelming infections in some instances, such as severe aspergillosis with success (jid 613:908, 1991) . we report here, two patients with very severe burkholderia cepacia (b. cepacia) infection, one of whom was placed on a respirator for approximately two weeks and another who was on extracorporeal membrane oxygenation for an extended period of time. both were treated with ifnγ (actimmune®) in addition to appropriate antimicrobial therapy in an attempt to affect these lifethreatening infections. objectives: the objective of this presentation is to describe two very severe variant x-linked cgd patients with b. cepacia pneumonia who were treated with ifnγ. in addition, we measured both super oxide production as well as nitric oxide production in the stimulated or unstimulated phagocytes from these patients in the presence or absence of interferon gamma. methods: case histories of both patients were reviewed in respect to the severity of their infection, the time spent on a respiratory or extracorporeal membrane oxygenation, the antimicrobial therapy administered, and the clinical results following the administration of interferon gamma as adjunctive immunomodulatory treatment. a standardized neutrophil oxidative burst assay was employed using cytochrome c reduction to measure super oxide production. in addition, nitric oxide was measured in the phagoctyes of the patients after stimulation with phorbol myristate acetate (pma) in the presence or absence of ifnγ using daf-2 fluorescence dye to detect the production of intracellular nitric oxide. results: a 2-year-old male developed a left lobar pneumonia and was admitted to primary children's medical center's intensive care unit and treated with iv cefotaxime, clindamycin, later, vancomycin and azithromycin were added. ct scan revealed a left-sided pneumonia and moderate parapneumonic effusion. subsequently, the patient decompensated, was intubated, and placed on respirator therapy. broncheoalveolar lavage and blood grew b. cepacia. neutrophil dihydrorhodamine fluorescence (dhr) demonstrated an intermediate broad peak of fluorescence with a small peak of unactivated cells, while the mother's dhr showed a broad intermediate peak suggesting the carrier state of variant x-linked cgd. both the patient, a 2.5 month old younger brother, and the carrier mother were found to have a g to a splice site mutation in exon 3 of the gp91-phox gene at position c.252 confirming the diagnosis of x-linked variant cgd. after approximately 2 weeks on the respirator, ifnγ (actimmune®) therapy was instituted with significant improvement of the patient's lung function. he was taken off the respirator approximately 3 days after the ifnγ therapy was instituted. following addition of ifnγ to his pma-stimulated neutrophil, there was a 22% increase in superoxide production and a 20 fold increase in nitric oxide in his monocytes. the second patient was a 9-year-old male who presented with fever and cough and was diagnosed with right-sided middle and lower lobe pneumonia with cavitations. bronchoalveolar lavage grew b. cepacia and nocardia. following increasing ventilatory and circulatory collapse he was placed on extracorporeal membrane oxygenation and treated with 4-5 antimicrobial agents. after 9 days of such therapy, ifnγ therapy was initiated and he was weaned from ecmo after 3 days and has remained essentially healthy since then when he is on ifnγ prophylaxis. dhr revealed a broad intermediate peak in the patient and normal and intermediate peaks in the mother suggesting variant x-linked cgd in the patient and the carrier state of x-linked variant cgd in the mother. targeted sequencing revealed a g to a splice site mutation in exom 3 of the cybb gene at position c.266 confirming the diagnosis of x-linked variant cgd. following addition of ifnγ to this patient's pma stimulated phagocytes, there was a 150% increase in superoxide production and a 200% increase in monocyte nitric oxide production. conclusions: two male variant x-linked cgd patients with splice site mutations in the cybb gene and severe life-threatening b. cepacia pneumonia, one on a respirator and one on ecmo were administered ifnγ (actimmune®) and each responded dramatically within 2-3 days recovering their respiratory capacity and coming off of assisted ventilation and ecmo, and have continued to do well when on ifnγ (actimmune®) therapy. introduction/background: hyqvia is a recombinant human hyaluronidase (rhuph20)-facilitated subcutaneous immunoglobulin (ighy) 10% replacement therapy for patients with primary immunodeficiency diseases (pidd). objectives: to acquire long-term safety data on ighy, and assess prescribed treatment regimens and administration in routine clinical practice, a global postauthorization safety study (pass) is being conducted. methods: this is an ongoing prospective, non-interventional, open-label, uncontrolled, multicenter study initiated in the united states in november 2015 to assess local and systemic effects of ighy within a routine clinical setting. patients aged 16 years with pidd who have been prescribed and/ or have started ighy are eligible for enrollment. patients are followed according to standard clinical practice and their treatment regimen is at the discretion of the treating physician. the presence of anti-rhuph20 antibody titers is evaluated on a voluntary basis. results: as of august 2017, 175 patients had been enrolled at 26 us study sites. there were no serious aes which were deemed treatment related. sixteen patients experienced a causally related non-serious local ae (9.1%; 0.43 events/patient-year, 0.07 events per infusion) and 25 patients experienced a causally related non-serious systemic ae (14.3%, 0.88 events/patient year, 0.14 events per infusion). of the 113 patients with immunogenicity data, 7 had 1 positive binding antibody test to rhuph20 (titers 1:160); no neutralizing rhuph20 antibodies were detected. conclusions: this interim analysis of prospectively-collected data of ighy use in routine clinical practice indicates that ighy is well tolerated with no treatment-related saes and has not been associated with neutralizing anti-rhuph20 antibodies in patients with pidd. introduction/background: idiopathic thrombocytopenic purpura (itp) and/or hemolytic anemia accompanied by splenomegaly occurs in up to 25% of patients with common variable immunodeficiency (cvid). treatments include steroids, other immune suppressants and rituximab. however, in some that do not respond, splenectomy may be performed. while splenectomy is known to be associated with an increased risk of infections or thromboembolic events, studies in other conditions (hemolytic disorders, hereditary spherocytosis, etc), also suggest an increased risk of pulmonary arterial hypertension (pah) after this procedure. objectives: while splenectomy is known to be associated with an increased risk of infections or thromboembolic events, studies in other conditions (hemolytic disorders, hereditary spherocytosis, etc), also suggest an increased risk of pulmonary arterial hypertension (pah) after this procedure. methods: we report three cases of pah following splenectomy for cytopenias in patients with cvid. results: the first is a 40 yo female, with long standing cvid complicated by interstitial lung disease, nodular regenerative liver disease and itp post splenectomy. the second is a 48 yo man with severe bronchiectasis, cirrhosis/nodular regenerative liver disease and itp post splenectomy. the third is a 54 yo woman with cvid (taci compound) complicated by cirrhosis/nodular regenerative liver disease, lung nodules and evans syndrome. all developed severe pah requiring chronic medications. pah in these patients is best classified as multifactorial, group v. conclusions: whether due to thrombus formation, continued cytopenias, and/or vascular changes, we suggest that pah may be a long-term complication of splenectomy in complex cvid. connective tissue, skeletal and vascular abnormalities. two isoforms exist, stat3, a 770 amino acid protein, and stat3, a 722 amino acid protein produced by alternative splicing of exon 23 resulting in a frame shift and truncated protein at the c-terminus. objectives: we follow 4 patients from 2 families with stat3 mutations leading to altered c-terminal proteins. the patients have high ige but milder features of ad-hies. methods: clinical data were collected. stat3 sequencing, stat3 functional assays as well as lymphocyte phenotyping were performed. results: patient 1 is a 58 year old man diagnosed with hies as a child due to eczema, recurrent boils and high ige (5000s iu/ml). he has tortuous and dilated coronary arteries, however denies lung infections, cmc, retained teeth, scoliosis, minimal trauma fractures, or hyperextensible joints. as an adult he developed avascular necrosis of both hips. whole exome sequencing revealed a novel splice mutation, c.2144+1g>t at the end of exon 22 causing skipping of the 43 nucleotide exon as well as utilization of the stat3 alternative splice acceptor in exon 23, resulting in a 93 nucleotide deletion. the mutant stat3 protein product has a 31 amino acid, in-frame deletion encompassing both y705 and s727 phosphorylation sites. no stat3 is made from the mutant allele. lymphocyte phenotyping was unremarkable, however total stat3 protein levels were decreased in ebv transformed b cell lines and there was decreased y705 phosphorylation after stimulation. patient 2 (family 2) was healthy until diagnosed with severe, refractory coccidiodies pneumonia complicated by pneumothorax with prolonged bronchopleural fistulae at age 16 years. this led to an immune evaluation in which he was found to have elevated ige (878 iu/ml, ref 0.0-91.0 iu/ ml). he had one perianal abscess, primary teeth requiring extraction and mild scoliosis, but denies cmc, bacterial pneumonias, eczema, or minimal trauma fractures. stat3 sequencing revealed a single base insertion in the transactivation domain, c.2185_2186insc causing a frameshift in the stat3 isoform, p.r729pfsx11, occurring immediately after the s727 phoshporylation site. the deletion occurs within the alternatively spliced region of exon 23, providing an intact, wild-type stat3. stimulation with il-6 or il-21 showed reduced pstat3 at y705, and elevated pstat1which is often seen in other ad-hies patients. lymphocyte phenotyping was unremarkable and th17 cell analysis showed low-normal levels of th17 cells while ebv transformed b cells showed reduced total stat3 levels. his mother and infant sister also share this mutation -the 8 month old infant had normal ige, and intermittent rashes; his mother has high ige (1193 iu/ml), recurrent sinopulmonary infections (complicated by tobacco use) but without bronchiectasis or pneumatocele, and denies cmc with the exception of pregnancy related vaginal candidiasis. conclusions: loss of function and gain of function mutations in stat3 lead to distinct syndromes, but it appears that stat3 mutations affecting the isoform expression, such as those reported here, can also lead to immune dysregulation with incomplete features of ad-hies. these stat3 mutations will allow us to better understand the relative roles of the isoforms stat3 and stat3 in somatic and immune cell signaling. physicians and is associated with immunological defects aswell. mutationsin the kmt2d and kdm6a genes are the most common genetic changes that lead to kabuki syndrome but for many cases the genetic basis remains unknown. objectives: recognize the varied presentation for a unique immunodeficiency syndrome methods: this is a case series. results: this is a case series describing three patients with ks and their clinical presentations which predominantly involve immunodeficiency and autoimmunity. our first patient is a 31-year-old male with autoimmune hemolytic anemia (aiha) at a young age, recurrent respiratory infections and hypogammaglobinemia which led to a diagnosis of cvid at the age of six. in addition, he has dysmorphic facial features, intellectual disabilities, and short stature but it was not until his late twenties where he was found to have a missense mutation in kmt2d (p.arg5048cys) which has been described in patients with ks. the second patient is a 34-year-old female with hypogammaglobinemia, evan's syndrome, short stature, and severe complications which include granulomatous-lymphocytic interstitial lung disease, pulmonary hypertension, and chronic kidney disease. she was also found to have a missense mutation in kmt2d (p.arg5048cys) in her early thirties and passed away from complications of her disease. the third patient is a 27-year-old female with a history of low iga/igg and poor vaccine titers, aiha, neutropenia, pulmonary nodules, and developmental delay who was diagnosed with cvid in her mid-twenties. for her immunodeficiency and autoimmunity, she was treated with immunoglobulin replacement, rituximab and cyclosporine and was found to have a missense mutation in kmt2d (p.cys1471trp). this mutation was a de novo mutation in this patient and has also been reported in another patient with ks. conclusions: these cases highlight that the presentation of ks is varied and frequently includes immunodeficiency and autoimmunity in addition to the characteristic short stature and developmental delay. a diagnosis of ks remains challenging due the diversity of symptoms and disease severity, the need for genetic testing, and due to overlapping clinical presentations with other developmental conditions. thus, often times, like in these cases, the diagnosis of ks is delayed. chief medical officer, rocket pharma introduction/background: lad-i is a rare disorder of leukocyte adhesion, resulting from itgb2 gene mutations encoding for the beta-2 integrin component cd18. cd18 deficiencies prevent integrin dimerization and endothelial leukocyte adhesion, essential for extravasation and antimicrobial activity. severe lad-i (<2% of normal neutrophil [pmn] cd18 levels) is characterized by recurrent serious infections and early mortality unless treated by allogeneic hematopoietic stem cell transplant (hsct). mortality for severe lad-i was reported as 75% by age 2 in an initial 1988 multicenter retrospective study. moderate lad-i (2-30% of pmn cd18 levels) is more indolent; although most patients (pts) survive childhood with recurrent skin and mucosal surface infections; mortality by age 40 can exceed 50%. lad-i is characterized by umbilical cord complications (delayed separation and omphalitis), poor wound healing and leukocytosis. objectives: reports regarding lad-i have been published in recent decades but no recent comprehensive prognostic assessments are available. we sought an updated understanding of severe lad-i with emphasis on prognosis in the absence of hsct, hsct outcomes and association of cd18 expression with clinical features. methods: we created a database of all published lad-i cases via pubmed searches and review of available references. results: three hundred twenty-three lad-i cases were reported between 1975-2017 in 107 publications (68 case-reports; largest series n=36). the nations reporting the most cases were iran (n=65), usa (n=50), and india (n=45); the highest number of publications were from us centers (25). 113 pts were considered to have severe lad-i, 63 moderate and 147 were not classified. pmn cd18 expression levels was reported for 265 cases and was <2% in 135 patients (51%) and >=2% in 130 pts. four pts with cd18 >2% were considered to have severe lad-i (cd18% range 2.4 17.3). gender was noted for 282 pts; 148 (52%) were male. age at presentation was reported for 146 cases. for 63 pts with cd18<2%, median presentation was age 1 m (range 0.03-18m); for 62 pts with cd18 >=2%, median presentation was age 6m (range 0.03-192m). infection details and cd18% were available for 154 (48%) cases. the most frequent infections in pts with cd18 <2% were respiratory tract (39%), sepsis (29%) and otitis media (27%) and for pts with cd18 >=2% they were periodontal (52%), otitis media (365%) and sepsis (25%). perianal skin infections and necrotic skin ulcers were noted in >10%. umbilical complications were more frequent in severe lad-i (92 of 110 pts with cd18<2% [84%] and 47 of 81 with cd18 >=2% [58%; p = 0.0002]). for severe lad-i pts with 2 years of follow-up (or death prior to 2y), there was correlation between absence of umbilical complications and survival to 24 m (p < 0.001). wbcs were reported in 143 cases (median 45 x 109/l; range 10 150 x 109/l). there were limited correlations between cd18 expression and wbc (r < 0.1) and between cd18 and cd11 expression (r < 0.5). mutation analyses were reported in 139 cases with >20 gene locations noted and mutations on exons 5, 6 and 7 accounting for 44% of specified cases. in 18 cases, cd18 expression was >30%; in 8 of 12 cases where cd11 expression was noted, at least one cd11 moiety was reported as <2%. we sought to understand whether prognosis for severe lad-i in the absence of hsct is similar to the initially-reported 25% survival to age 2. there were 66 severe lad-i cases (per investigator assessment or cd18 <2%) for whom survival to 2 years was reported, 40 of whom died prior to age 2 (61% mortality). mortality was similar for the subset of 43 cases reported since 2000 (56%, 24 deaths). early mortality was substantially lower in patients with cd18 >=2% and the majority of pts with cd18 >4% survived to adulthood. outcomes for 101 pts who received hsct were consistent with recent series; phenotypic correction was reported in 83% of pts with hla-matched sibling donors. mortality was 19% overall (11% for hlamatched sibling recipients). for 22 pts receiving haploidentical hsct there was 32% mortality and 55% received 1 subsequent hsct. conclusions: severe lad-i remains a life-threatening condition with limited 2-year survival in the absence of allogeneic hsct. umbilical complications and granulocytosis are frequent early manifestations; respiratory tract, ear, sepsis, oral and skin infections are common. hsct is potentially curative; transplant-mortality and other complications are frequent, especially in haploidentical recipients. diverse itgb2 mutations result in lad-i, and genetic evaluation may be valuable for diagnosis and prognosis. rapid identification of pts with potential lad-i (unusual or severe infections in infancy, granulocytosis and umbilical complications) is essential to enable referral to centers with disease expertise. objectives: to determine the phosophorylation of stat3 in ad-hies patients with known stat3 mutations. methods: peripheral blood mononuclear cells (pbmcs) were collected from ad-hies patients under irb-approved institutional research protocols. pbmcs were then stimulated with il-6 or il-21 for 15, 30 and 60 minutes. cells were surface stained for cd3, cd4, cd8, cd56 and cd19. they were then fixed, permeabilized and stained with anti-y705-stat3 to evaluate for phosphorylation of stat3 at position y705. cells were then washed and data acquired using flow cytometry. results: pbmcs from one ad-hies patient with a stat3 sh2 domain mutation (p.y657c) demonstrated normal y705 phosphorylation after il-6 stimulation. pbmcs from another ad-hies patient with a dbd mutation (p.h437y) exhibited highly reduced stat3 phosphorylation after il-6 stimulation and absent phosphorylation after il-21 stimulation. conclusions: these findings highlight that, in the context of ad-hies, the domain location of the stat3 mutation does not predict stat3 phosphorylation potential following stimulation and challenges the current paradigm. (106) submission id#426423 cheng sun 1 1 associate professor, institute of immunology introduction/background: as the predominant lymphocyte subset in the liver, natural killer (nk) cells have been shown to be highly correlated with the outcomes of patients with hepatocellular carcinoma (hcc). previously, we reported that nk cells were decreased and functional deficiency in hcc. however, the mechanism underline remains unknown. objectives: in this study, through the use of 23 healthy livers, paired peritumoural tissues (pt) and intratumoural tissues (it) from 236 hcc patients, methods: we have evaluated the expression of cd160 and its co-ligand receptor btla on hepatic cd8+ t cells and nk cells. results: decreased expression of cd160 on nk cells was observed in intratumoural but not pt regions, along with nk cell dysfunction, poor prognosis and tumour metastasis. human cd160+ nk cells exhibited functional activated, high capacity of ifn-secretion and nk mediated immunity by global transcriptomic analysis of sorted cd160+ and cd160-hepatic nk cells. blocking tgf-1 specifically reversed the ifn-production of cd160+ nk cells. in addition, this decreased cd160 expression is predominantly on cd56bright nk cells. conclusions: these findings indicate that cd160 expression reduction contributes to nk cell exhaustion and tumour immune escape, suggesting that cd160 has the therapeutic potential for fighting liver cancer. introduction/background: the low number of circulating lymphocytes in the blood is a marker for cellular immunodeficiency in young children. ethnicity also affects the lymphocyte count and ethnicity-specific lymphocyte norms have been used in many countries. this study analyzed the lymphocyte counts in a large cohort of infants and young children from the arabian peninsula. objectives 1-to define the normal lymphocyte counts in arab children 2-to define the possible cutoff lymphocyte count that define lymphopenia. methods: this is a cross-sectional analysis of the lymphocyte counts in 11,237 arab children. the age groups were: 1 day, 1-6 months, 6-12 months, 1-2 years, 2-3 years, 3-4 years, 4-5 years, and 5-6 years, 47% females. we analyzed the first blood count performed during their visit to the abu dhabi seha ambulatory healthcare services between april 2008 and october 2013. the median, 10th percentile and 90th percentile counts were calculated. the 10th percentile lymphocyte count was used to define lymphopenia. the kolmogorov-smirnov test, a non-parametric test, was used to compare lymphocyte counts between groups. statistical significance was defined by a two-tailed p<0.05. results: the median counts were higher during infancy. the variability (disparity) of the counts (reference intervals) progressively decreased from birth to 6 years of life. the 10th percentile lymphocyte counts were relatively constant from birth to 2 years (2.5-3.0 x109/l) and from 2 to 6 years (1.4-1.8 x109/l), table 1 . the lymphocyte counts were similar in boys and girls. the lymphocyte counts were compared to those from five other studies. conclusions: arab children have lower lymphocyte counts (10th percentile) than children in the united states, brazil and south africa, but their counts are similar to children in china and uganda. our study results support the development and use of ethnicity-specific lymphocyte count standards. the implication of our results is that using these lower cutoff values for lymphopenia will prevent a large number of arab children from having unnecessarily investigated for immunodeficiency. introduction/background: chronic granulomatous disease (cgd) is a rare phagocytic defect caused by mutations in the nadph oxidase 2 system leading to reduced or absent reactive oxygen species production. in addition to specific infectious susceptibility, patients with cgd are predisposed to hyperinflammation in response to infectious agents, autoimmunity, colitis, and other forms of autoinflammation. cgd patients with mutations in p47phox are at increased risk of diabetes and cardiovascular disease. hyperinflammatory and auto-inflammation responses are often difficult to predict and manage. intracellular adhesion molecule-1 (icam-1) and e-selectin are endothelial adhesion markers that facilitate the adhesion and transendothelial migration of leukocytes and elevations in these markers have been associated with cardiovascular disease, glomerular injury, and thrombotic events. expression of adhesion molecules is induced by pro-inflammatory cytokines and are associated with a hyperinflammatory state. objectives: to determine if e-selectin and icam-1 are elevated in patients with cgd and to determine of endothelial adhesion markers could serve as a biomarker of inflammatory disease in cgd patients. methods: thirty-eight pediatric and adult subjects with cgd (14 xlinked (xl-cgd), 21 p47phox deficient and 1 p22phox deficient, and 2 x-linked cgd carriers were enrolled. e-selectin (pg/ml) and icam-1 (ng/ml) were measured from the plasma of patients via sandwich elisa. results: all 38 cgd patients had histories of severe infection, active infection, chronic colitis, or other autoimmune disease at time of evaluation. nine p47phox deficient cgd patients had history of diabetes and/or early onset cardiovascular disease. one subject with x-linked cgd had undergone hematopoietic stem cell transplantation (hsct). plasma levels of e-selectin were significantly elevated above healthy controls (median 19,648 pg/ml) in subjects with xl-cgd (median 45,990 pg/ ml, p<0.001), p47phox deficient cgd (median 23,707 pg/ml, p=0.0017) and p22phox deficient cgd (80,108 pg/ml). plasma levels of icam-1 were also elevated above healthy controls in subjects with xl-cgd (median 553.2ng/ml), p47phox deficient cgd (median 241.7ng/ml), and p22phox deficient cgd (383.1ng/ml), although none were statistically significant. plasma quantities of e-selectin and icam-1 increased further in those p47phox deficient patients with diabetes and/or cardiovascular disease (median e-selectin 37,143 pg/ml, icam-1 356.7 ng/ml) but neither reached statistical significance. e-selectin and icam-1 quantities in female carriers of xl-cgd and in 1 xl-cgd cured by hsct were similar to values found in healthy controls. conclusions: immune dysregulatory features and hyperinflammation in cgd can be difficult to predict and manage. the endothelial adhesion markers e-selectin and icam-1 are elevated in patients with xl-cgd and p47phox deficient cgd that worsens with presence of early onset cardiovascular diseases and resolves post-hsct. elevations in e-selectin and icam-1 in the serum of cgd patients may serve as surrogate markers of inflammation and suggest a chronic endotheliopathy in cgd patients. introduction/background: the phenotypic presentation of ctla4 haploinsufficiency was only recently described. the management of these patients in the medical literature is limited to anecdotal case reports. we aimed to detail our experience with short and long term immunomodulatory therapy in treating autoimmune cytopenias in the background of ctla4 impairment. objectives: we aimed to assess the efficacy of mtor inhibitors in treating autoimmune cytopenias in patients with ctla4 haploinsufficiency. methods: we retrospectively identified 7 patients with proven ctla4 mutations and documented refractory autoimmune cytopenias while receiving care at nih clinical center (from july 2011 to august 2017). the complete (cr) and partial (pr) clinical response was assessed after six weeks of treatment and defined as hgb >10 g/dl, platelets >100k/ul and hgb >8g/ dl, platelets >50k/ul, respectively without transfusion requirement. results: all analyzed patients failed or exhibited disease recurrence on at least one prior medical therapy, including: corticosteroids, rituximab, romiplostim and eltrombopag. the initial response rate to evrolimus and/or sirolimus was 71% (3 cr and 2pr). one of the partial responders had recurrence of idiopathic thrombocytopenic purpura at four months while on rapalogs. overall, we used mtor inhibitors in 22 patients for a total of 37 patient years to treatm multiple modalities. the top three most common recorded adverse events were: clostridium difficile colitis (n=9, in 4 patients), lipid abnormalities (n =7, 3 patients required treatment) and bacterial pneumonia (n=6, in 4 patients). conclusions: our limited retrospective data suggests that mtor inhibitors might be efficacious in the treatment of autoimmune cytopenias in ctla4 haploinsufficent patients. further prospective studies are required to assess safety and efficacy of mtor inhibitors in this patient population. association with frequent upper respiratory tract infections and pharyngitis. from 2 years of age she continued to have recurrent febrile episodes in the absence of infection, with fever of 39-40 degrees celsius on a monthly basis, normally lasting 1-2 days. she also developed episodes of urticaria, temporally unrelated to her febrile episodes. the rash was noted to be induced by exposure to the cold, and would generally last 1-3 days with some clinical response to antihistamines and naproxen. her clinical picture then progressed and she developed joint pain and swelling, particularly affecting her hands and feet. over the following years the patient continued to have recurrent episodes of urticaria as well as progressive joint involvement and limitation. there was some initial improvement with naproxen and prednisone. her symptoms were refractory to subsequent treatment with methotrexate, leflunomide, infliximab and tocilizumab and she remained corticodependent. immunological work up including lymphocyte phenotype, immunoglobulins, vaccine responses to both protein and live vaccines and ch50 were normal. there was no evidence of raised inflammatory markers with esr 3-11mm/h, crp <0.2mg/l and ferritin 39ug/l. autoimmune workup including ana, ena, anti-dna, anca and c3 and c4 was normal. the initial differential diagnoses included systemic juvenile arthritis, periodic fever syndrome or cryopyrin associated periodic syndromes (caps). the patient therefore had a periodic fever gene panel that identified a heterozygous mutation in exon 3 of nlrp12, c.466c>t (p.arg156trp). whilst mutations in nlrp12 are known to be associated with familial cold autoinflammatory syndrome, this was reported as a variant of unknown significance. we therefore proceeded with functional in vitro testing to demonstrate pathogenicity. the patients monocytes showed increased secretion of il1b upon stimulation with lps as compared to healthy donors. when tested in a luciferase reporter assay, the mutated nlrp12 partially lost the capacity to inhibit the nfkb pathway. overall these in vitro studies show that this nlrp12 mutation results in defective regulation of the inflammatory response. the patient was commenced on anti-il1 therapy with canakinumab, with no clinical improvement so was therefore discontinued. we report a case of familial cold autoinflammatory syndrome due to a mutation in exon 3 of nlrp12, that to this point has remained refractory to multiple treatment modalities. functional testing was able to demonstrate mutation causality and defective regulation of the inflammatory response. objectives: authors aim to evaluate the spectrum of clinical phenotypes associated with nemo hypomorphic mutations within the usidnet registry. methods: investigators obtained demographic, laboratory, and clinical data on patients with a defect in nemo within the usidnet registry. results: there were 19 male patients within the usidnet registry with a diagnosis of eda-id attributed to nemo hypomorphic mutation. of these, 9 were associated with a known variant in nemo (e391x, f312l, m407v), and 4 were associated with previously unreported variants (d113v, e287del, c.1-16g>c). for 6 patients, a mutation was not specified. most reported having an affected family member (n=14, 74%). median age of symptom onset and diagnosis were 1 year (iqr 0.5-2y) and 2 years (iqr 1-10y), respectively. median age at most recent visit was 11 years (iqr 9-16y). infections, additional clinical features, treatments, and outcomes are summarized in table 1 . skin manifestations (n=13, 68%) and pulmonary complaints (n=10, 53%) were common, with eczema (n=11, 58%) and asthma being most prevalent (n=5, 26%). gastrointestinal conditions (n=9, 47%) were also frequently reported, and included non-specific diarrheal illness, enteropathy, colitis, enteritis, and inflammatory bowel disease. neurologic features (n=8, 42%), including seizures, hearing defect, peripheral neuropathy, and encephalopathy, were unexpectedly common, suggesting a previously unrecognized disease association. conclusions: we observed that allergic diseases, including asthma and eczema, were common in patients with nemo mutation. notably, varied neurologic features were more prevalent than previously reported. this study highlights the potential of cross-institutional registry analysis to deepen our understanding of extremely rare genetic diseases. coordinated effort across institutions is required to better characterize the spectrum of clinical phenotypes associated with hypomorphic mutations in nemo. mycobacterial lysate (ml) and purified protein (ppd) in the diagnosis of patients with mendelian susceptibility to mycobacterial disease (msmd) elimination of this infection depends mainly on the success of the interaction between macrophages and infected t lymphocytes. patients with mendelian susceptibility to mycobacterial disease (msmd) present severe and recurrent infections due to impaired signaling of the ifn/il-12 axis. objectives: our aim was to evaluated the ifn/il-12 axis of patients with clinical history suggestive of msmd using mycobacterial lysate (ml) and purified protein (ppd) by elisa assay. methods: samples of patients (n=43) with a clinical history suggestive of msmd arrived in our laboratory. for the diagnosis, 3 ml of blood diluted (1:2) in rpmi 1640 culture medium supplemented were used. the samples were distributed in two different plates, one used in the dosage of il12 (24 h) and the other in the ifn dosage (48 h). thus, they were stimulated with ml (2 ug/ml for the 24 h plate and 10 ug/ml for the 48 h plate) and with ppd (10 ug/ml for the 24 h plate and 2 ug/ml for plate of 48 h). at the same time, in half of the wells stimulated with lm and ppd, the cytokine ifn (1000 iu/ml) or il12p40 (2 ng/ml) were added in the plate. next, the plates were incubated at 37°c and 5% co² and the supernatant were collected and quantified by the elisa assay. the results were evaluated by the statistical mann whitney u test. results: six of the 43 patients presented alterations in the evaluation of the ifn/il-12 axis. the age of the diagnosis of male patients ranged from 2 to 16 years. the only two female patients diagnosed were 36 and 40 years old. the clinical history was heterogeneous: 4 had lymph node hyperplasia, 2 pneumonia, 1 colitis, 1 herpes zozter, another had a urinary tract infection and 1 bcgitis. the pathogens isolated were the m. tuberculosis, m. abscessus, m. gordonae, m. genavense and m. konsossi species found in 5 different patients. statistical analysis of the ifn-/il-12 axis evaluation by elisa was performed.the results of the dosage of ifn were significant in samples with lm and lm plus il-12 (***). similar results were observed in samples treated with ppd and ppd plus il-12 (**) when compared with healthy controls. in the il-12 dosage, statistical difference was observed in the samples with lm (***) and with lm plus ifn (*). in samples stimulated with ppd, the results did not show statistical differences (ns.) but ppd + ifn (*) were significantly different. conclusions: six patients were diagnosed by the evaluation the il-12/ ifn axis. the use of micobacterial lysate (ml) showed reliable results to the diagnosis of patients with il12/ifn pathway defects. the genetics diagnosis will be performed. introduction/background: invasive infections due to mycobacterial species are a feared complication in patients with t-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. cd4+ th1 t-cell immunity is known to be critical to antimycobacterial defense; accordingly, adoptive t-cell immunotherapy with mycobacteria-specific t-cells (mst) may be a beneficial therapy for combating these infections. objectives: to determine if ex vivo expansion of t-cells targeting common mycobacterial antigens is feasible from healthy donors, and whether the same antigens are recognized by patients with primary immunodeficiency (pid) and invasive mycobacterial infections. methods: peripheral blood mononuclear cells (pbmc) from healthy donors were pulsed with overlapping 15-mer peptide libraries encompassing five mycobacterial antigens (ag85b, ppe68, esat-6, cpf10, adk) and expanded for 10 days with cytokines il-4 and il-7. expanded msts were tested for specificity against the targeted antigens via ifn-g elispot, multiplex cytokine analysis, and flow cytometry. pbmcs from pid patients with invasive mycobacterial infections were similarly tested for presence of t-cells recognizing the tested mycobacterial antigens. a minimum of 20 spots per 1x105 cells above negative control was considered specific on elispot. results: ten healthy donors and eight patients with pid were tested. specificity against 1-5 mycobacterial antigens (median 3) was confirmed in all ten healthy donors, with a mean 6.6-fold cellular expansion during the 10-day culture. msts were predominantly cd4+ t-cells (mean/sd: 55 +/-6%), with both central memory (mean/sd 17.7 +/ -6.4%) and effector memory (mean/sd 77.4 +/-7.6%) populations. there was no clear difference in antigen specificity between bcg immunized (n=4) and bcg naïve (n=6) healthy donors. six of 8 pid patients had no detectable immunity to tested mycobacterial antigens. one patient with combined immunodeficiency has a low detectable specificity to ag85b (mean 47 spot forming colonies[sfc]), and a patient with nfkb1 haploinsufficiency mounted a response against ag85b (mean 388 sfc) and ppe68 (mean 40 sfc). conclusions: mycobacteria-specific t-cells can be rapidly expanded from healthy donors utilizing a protocol that could easily be translated to a good manufacturing practices facility. the majority of tested pid patients lacked immunity to the targeted antigens. adoptive immunotherapy with msts derived from third-party healthy donors may be a beneficial adjunctive therapy for pid patients with invasive mycobacterial infections. is an innate immune deficiency, primarily affecting the phagocytic compartment, and presenting with a diverse phenotypic spectrum ranging from severe childhood infections to monogenic inflammatory bowel disease. dihydrorhodamine (dhr) flow cytometry is the standard diagnostic test for cgd, and correlates with nadph oxidase activity. while there may be partial genotype correlation with the dhr flow pattern, in several patients, there is no correlation. objectives: in such patients, assessment by flow cytometric evaluation of nadph oxidase-specific (nox) proteins provides a convenient and rapid means of genetic triage (table) . methods: we performed dhr flow cytometry and nox flow cytometry on granulocytes and monocytes of cgd patients. results: phenotypic and laboratory patient data shown in table. *p9 had decreased p22phox (% and mfi) monocytes, but not in granulocytes. all other siblings (p6, p7, and p8), and mother (p10) had relatively higher p22phox (%) monocytes, but still lower than the healthy control. p7 had normal %p22phox monocytes, comparable to control. however, p6-p8, and p10 had normal p22phox (mfi) in monocytes and granulocytes. p6-9, and p10 have normal %gp91phox+ granulocytes, while p6and p9 have modestly decreased %gp91phox in monocytes. p6-9, and p10 have all moderately decreased gp91phox protein (mfi) in granulocytes and monocytes compared to healthy control, with a single population for protein expression. p6, p9 and p10 have bimodal populations for gp91phox in monocytes but not in granulocytes, with a larger positive population, and a much smaller negative population. the data from p6-9 suggest that the amount of gp91phox does not necessarily correlate with neutrophil oxidative burst, as measured by dhr. also, not all cybb variants affect p22phox protein expression, though both proteins are membrane-bound. the cyba vus in p8 does not appear to have affected p22phox protein expression in either monocytes or granulocytes. conclusions: the atypical clinical presentation of some cgd patients can make genotype-phenotype correlation with dhr flow data challenging. genetic testing, while necessary, can take several weeks. however, nadph-oxidase specific-protein flow assessment offers a rapid alternative to identification of the underlying genetic defect, and can be utilized as a reflex test to an abnormal dhr flow. further, it can provide insight into correlation between oxidative burst relative to protein expression in granulocytes and monocytes. head, division of immunology and allergy, the hospital for sick children introduction/background: adenosine deaminase (ada) is a ubiquitous enzyme important for purine metabolism. few studies have indicated that ada deficiency, in addition to causing profound lymphopenia and susceptibility to infections, is associated with neutrophils abnormalities. objectives: determine whether ada deficiency directly affects neutrophils and what are the mechanisms involved. methods: peripheral blood (pb) and bone marrow (bm) from 2.5 weeks old ada-deficient (ada-/-) mice that closely recapitulate the phenotype observed in ada-deficient patients, as well as ada+/-littermates were used to study neutrophils development and function. some experiments were supplemented with 6-week old ada-/-mice, maintained until 4 weeks of age with ada enzyme replacement, and littermates. results: the number of neutrophils in pb of ada-/-mice at 2.5 and 6 weeks of age was similar to ada+/-mice. the function of pb neutrophils from ada-/-mice, determined by oxidative burst, was also normal. the percentage of lin-/c-kit+/sca1+ hematopoietic progenitor cells in bm demonstrated significant reduction in ada-/-mice compared to littermates (0.93±0.23% and 1.21±0.33%, respectively, p=0.013). moreover, expansion of bm isolated from ada-/-mice in methylcellulose resulted in significantly less cd11b+/ly-6g+ neutrophils compared to healthy controls (13.6±8.4% compared to 25.3±10.3%, respectively, p=0.003). proliferation of bm cells, determined by brdu incorporation into cells dna, was higher in ada-/-mice than in littermates, possibly contributing to the normal neutrophil numbers in pb of ada-/-mice. conclusions: ada deficiency directly affects neutrophil development. further studies will help understand the significance of these effects and potential therapies for ada-deficient patients. objectives: we present here the clinico-pathologic features of a 10-yearold female with a heterozygous fancd2 gene deletion/mutation with evidence of cellular and humoral immune dysregulation. methods: we evaluated the patient using standard immunology anatomic, cellular, and biochemical functional assays. results: the patient has multiple dysmorphias including total anomalous venous return (repaired), mesomelia, absent ear canal, radial ray dysplasia, and short stature. her medical history is significant for an episode of pneumococcal sepsis despite adequate vaccination. whole exome sequencing demonstrated deletion of exons 2-17 and a pathologic mutation (c.2444g>a, p.arg815gln). repeated blood samples and immunophenotyping demonstrated severe lymphopenia. there were markedly low cd4+ t-cell counts with a low cd4:cd8 ratio (0.42). changes in the composition of the b-cell population included: significantly diminished absolute total b-cells, elevated immature cells, low levels of transitional cells, and undetectable advanced b-cell populations. there was no immunogenic response to pcv-13 or varicella/tetanus/ diphtheria vaccination. the nk-cell count was unaffected and demonstrated normal spontaneous and stimulated cytotoxic response. bone marrow analysis demonstrated hypocellularity without dysplasia. conclusions: we report here a pediatric patient with a novel fancd2 deletion/mutation presenting with severe lymphopenia in two cell compartments (b and t cells) and susceptibility to invasive bacterial infection. the findings are suggestive of combined immune deficiency. the cellular immune profile suggests that fancd2 may be involved in the transition of immature b and t cells to mature cells, a process that requires substantial dna recombination. additional genetic and biochemical evaluation is needed to further characterize this rare clinical finding. introduction/background: familial hemophagocytic lymphohistiocytosis type 2 (hlh) is a rare fatal condition due to a mutation in the pfr1 gene on chromosome 10q21-22 inherited in an autosomal recessive pattern which results in overactivation of the immune system. symptoms usually manifest before 1 year of age. a 17-year-old previously healthy male presented with acute onset of bilateral lower extremity pain and weakness, with subsequent inability to ambulate over a 10-day period. neurological exam demonstrated decreased lower extremity power, sensation, absent patellar and achilles reflexes, and a wide-based gait. objectives: not applicable methods: not applicable results: laboratory data was significant for neutropenia and thrombocytopenia, elevated levels of ferritin and serum cd25, and a positive ebv pcr. patient was initially treated with ivig for possible ebv-driven guillain-barre syndrome with some improvement in neurologic symptoms as well as thrombocytopenia. a mass of retroperitoneal lymph nodes were noted on spinal mri. testing was negative for alps and bone marrow biopsy was negative for leukemia/lymphoma. further work-up revealed absent perforin expression in cytotoxic cells and normal sap protein expression on staining, with poor nk function. genetic testing revealed a pathogenic mutation in the pfr1 gene with 1 additional variant of unknown significance. the patient was treated with rituximab for persistent ebvand returned with worsening lower extremity weakness. on mri, focal enhancements were found in the brain as well as worsening mass compression of the lumbosacral nerve roots. nerve root biopsy showed histiocytes with hemophagocytosis and a dense lymphohistiocytic infiltrate which stained positive for cd3, cd2, and granzyme b, with some loss of cd5 and no perforin. bone marrow biopsy was negative for hemophagocytes. conclusions: the patient was diagnosed with worsening familial hlh with cns involvement. hlh-directed chemotherapy (dexamethasone, cyclosporine, etoposide, intra-ommaya methotrexate/hydrocortisone) was started and hsct was performed. familial hlh is a rare and often lethal disorder that generally presents at a very young age. the acute onset and severity of presentation as occurred in this previously healthy adolescent is uncommon. familial hlh should be considered even in older patients with unexplained overactivation of the immune system. is the most profound form of primary immune deficiency, and is usually fatal in the first year of life without treatment. newborn screening for scid using quantitative analysis of t-cell receptor excision circles (trecs), has become the accepted method to facilitate early diagnosis and treatment in most of the united states, as scid babies typically do not make trecs. ikbkb deficiency is a rare form of autosomal recessive scid found in the northern cree first nations people of canada, where t cells develop normally but are non-functional. trec analysis is expected to be normal in ikbkb scid, and does not identify these cases. objectives: the objective of our study was to determine the feasibility of targeted genetic newborn testing for ikbkb deficiency. methods: we implemented a pilot project of prospective targeted genetic testing for the previously described homozygous ikbkb mutation (c.1292dupg) in newborns from 2 small northern manitoba communities. between 2013 and 2017, dna was extracted from dried blood spots of 724 newborns, and targeted sanger sequencing of the mutationharbouring ikbkb exon 13 was performed. results: all 724 infants born in the 2 selected communities underwent testing. fifty-five infants (7.6%, or 1/13.5) were found to be heterozygous carriers. one affected infant was identified, and underwent hematopoietic stem cell transplant before onset of infections. our findings are consistent with the predicted homozygosity for this mutation (1/13.5 x 1/13.5 x 1/4 = 1/729 births). conclusions: we demonstrated that targeted newborn testing for ikbkb deficiency was feasible, and provided the first prospective estimate of the ikbkb mutation carrier frequency in select manitoba northern cree first nations populations. we suggest that if we are to capture all babies with scid in manitoba, future newborn screening should be universal and include both trecs and direct mutation testing for population-specific mutations, including the first nations ikbkb mutation. high throughput analysis for trecs and targeted mutations will be introduced for universal newborn screening in manitoba. introduction/background: immunoglobulin class-switch recombination (csr) and somatic hypermutations (shm) are prerequisites of antibody and immunoglobulin receptor maturation and diversity within the adaptive immune system. the mismatch repair (mmr) machinery, consisting of homologues of mutsa, mutla, and mutsb (msh2/msh6, mlh1/pms2, and msh2/msh3, respectively) and other enzymes, is involved in csr, e.g. as backup of nonhomologous end-joining repair of activation-induced cytidine deaminaseinduced dna mismatches, and furthermore, in addition to errorprone polymerases, in the repair of shm-induced dna breaks. in line, a varying degree of antibody deficiency, from iga or selective igg subclass deficiency, to common variable immunodeficiency and hyper-igm syndrome have been shown in small numbers of patients with constitutional mmr deficiency (cmmrd) in addition to the known severe cancer predisposition due to genomic instability of patients with biallelic loss-of function mutations in one the mmr components. objectives: to elucidate the clinical relevance of primary immunodeficiency (pid) in cmmrd, we collected history and laboratory data of a novel cohort of 14 consecutive patients from 14 families with homozygous mutations in pms2 (n=7), msh6 (n=5), and mlh1 (n=2) reported to the consortium care for cmmrd (c4cmmrd) between 2014 and 2017, most of whom manifested with typical malignancies during childhood. methods: retrospective chart review according to a specific questionnaire and extended routine immunological analyses were performed with irb approval from the medical university of graz, austria. results: none of the presented patients fulfilled any classical or extended clinical warning signs of pid (infections, immune dysregulation, inflammation). furthermore, analyzing multiple specific laboratory parameters of the humoral and cellular immune system, we could not detect a uniform pattern of abnormalities. importantly, our data do not confirm previous suggestive evidence of iga or igg subclass deficiency, a specific antibody formation, or a b memory cell maturation defect. results of next generation sequencingbased detection of impaired class switch recombination and somatic hypermutations are pending. the t cell subsets and receptor repertoires were unaffected. together, neither clinical nor laboratory parameters were suggestive of pid in the present series of novel cmmrd patients. conclusions: we conclude that patients with cmmrd do not generally show a clinically relevant pid that could facilitate early diagnosis. on the contrary, these data support the prospect of potentially successful immune therapy of malignancies in the context of cmmrd. introduction/background: ada-2 deficiency is immune dysregulation diseases caused by an autosomal recessive mutation on cecr1 gene characterized by polyarteritis nodosa, childhood-onset, early-onset recurrent ischemic stroke and fever. objectives: report a new mutation on the cecr1 gene resulting in ada-2 deficient children. methods: female, 6-year-old, presented history of multiple ischemic strokes at one-year-old associated with recurrent fever and livedo racemosa. she has no siblings and parents are not consanguineous. results: the laboratory evaluation shows red cell=4.91x106/ml, hemoglobin=12.4g/dl, leucocytes=6.1x103/ml, neutrophils=4.3x103/ ml, lymphocytes=1.2x103/ml and plateletes=217 x103/ml. ige<25ui/ml, igg=467mg/dl, igm=13.8mg/dl and iga=21.3mg/dl. subsets lymphocytes shows cd3+=81.2%, cd4/cd8=1.41, cd19+ =10% and cd16/56=8.8%. due the clinical history, we performed cecr1 gene sequence homozygous substitution located at position -2 of acceptor splice site intron 6, c.847-2 g>a. this is a high conservative region with no alteration along phylogenetic studies and predicted to be pathogenic. to confirm the functional alteration, the ada-2 activity was tested in dried plasma spot showing 0.0 mu/g protein confirm the gene loss of function and the mutation pathogenicity. conclusions: the authors presented a novel mutation of cecr1 gene, the first one described in splice site causing gene loss of function and confirmed by extremely reduced ada2 activity. introduction/background: severe combined immunodeficiency (scid) is the most severe form of primary immunodeficiency characterized by severe, life threatening infections during early infancy. scid is a medical emergency associated with significant mortality if hematopoietic stem cell transplantations is not instituted early in the course of the disease. scid is a genetically heterogeneous disease caused by mutations in more than 30 different genes. different genes are implicated in different ethnic populations and geographical locales depending on the rates of consanguinity and endogamy in these populations. however, following the institution of newborn screening of scid in almost all state of the us and the widespread use of next generation sequencing in primary immunodeficiency diseases ar-scid due to mutations in rag1 and rag2 genes are found to be more prevalent than reported earlier. objectives: we performed a retrospective analysis of scid cases diagnosed at our centre and referred to us from other centres to determine the clinical, immunological and genetic basis of the disease in these cases. genetic variants both recurrent and novel were analysed in detail. methods: fifty six (56) of the 70 suspected patients met the esid diagnostic criteria. the clinical features, immunological defects and the gene sequencing results of these patients were analysed. gene sequencing was performed at the our centre and other collaborative centres at dept of pediatrics and adolescent medicine, queen mary hospital, hong kong, national defense medical college, saitama japan, kazusa dna research centre, chiba, japan and duke medical university centre, usa. mutations were detected in 36 of the 56 patients. mutations were classified as recurrent or novel after checking different databases such as exome aggregation consortium (exac), human gene mutation database (hgmd) and other relevant scid databases. the effect of novel, previously unreported mutations was determined using in-silico prediction tools such as sift and polyphen2. functional studies were also performed in few cases to determine the effect of novel mutations. results: mutations were detected in 36 patients. mutations were more common in genes causing autosomal recessive form of scid than the x-linked variant. mutations were detected in il2rg gene in 8 patients followed by mutation in the rag1 gene in 7 patients, dclre1c in 6 patients and rag 2 in 5 patients. mutations were also detected in ada gene (4 patients, 6 mutations), il7r (3 patients), stim1, pnp and nhej1 (1 patient each). nine novel mutations were detected. three in il2rg gene, 2 in rag1, 2 in ada and one each in the nhej1 and il7r gene. conclusions: autosomal recessive form of scid was more common in our cohort compared to x-linked form of the disease. mutations in rag1 and rag2 genes were the commonest (12 patients) followed by mutation in il2rg gene (8 patients). nine novel mutations in 5 different pid genes were detected in our cohort of scid patients introduction/background: rasgrp1 is a guanine-nucleotide exchange factor which phosphorylates ras-gdp to the activated form ras-gtp in response to t-cell receptor stimulation, resulting in ras activation. mutations in the gene coding for rasgrp1 have been recently described in four patients with profound t-cell deficiency, resulting in recurrent bacterial and viral infections, autoimmunity and malignancy. here we describe a two-year-old male presenting with recurrent sino-pulmonary infections, found to have two variants in rasgrp1, one not previously described. objectives 1. to describe a case of combined immunodeficiency with two pathogenic compound heterozygous rasgrp1 mutations. 2. to compare the clinical phenotype of rasgrp1 deficiency of our patient with that of previously described cases. 3. to argue for early hematopoietic stem cell transplantation in view of the increased susceptibility to epstein barr virus (ebv) induced lymphoma in patients with rasgrp1 deficiency. methods: a two-year-old male was referred to seattle children's immunology clinic for recurrent otitis media and two episodes of pneumonia. the diagnosis of combined immunodeficiency was considered based on a profound t-cell deficiency during immune evaluation and he was started on azithromycin and tmp/smx prophylaxis. at age 3 ½ years he was hospitalized with a bladder outlet obstruction and found to have two abdominal masses. biopsies were obtained and he was diagnosed with an ebv driven b cell lymphoproliferative disorder. in addition, his csf and bone marrow were considered positive based on pcr and staining, respectively. treatment with cyclophosphamide, prednisone, rituximab, and intrathecal methotrexate was initiated. due to poor csf ebv clearance, intrathecal therapy was escalated to rituximab. results: initial laboratory evaluation showed elevated igg (2290 mg/dl), normal number of cd19 b-lymphocytes, and adequate response to tetanus, prevnar-13 and varicella vaccine. b-cell phenotyping showed elevated immature/transitional b-cells. a profound t-cell defect was identified with cd4 t-cell lymphopenia (361/mm3), elevated cd8 t-cells (2074/mm3), inverted cd4/cd8 ratio (0.3) and absent proliferation in response to mitogens (pha, anti-cd3) and antigen (tetanus). forty-three percent of peripheral blood t-cells were / positive. t-cell phenotyping revealed decreased cd4 and cd8 naïve t-cells with elevated proportion of cd8+ t-effector memory t-cells. cervical lymph node and retroperitoneal mass biopsies showed atypical lymphoproliferation without malignant transformation. exome sequencing revealed two variants in rasgrp1. the first variant (c.1428+1g>a) is located at a splice site predicting an unstable transcript targeted for degradation. the second variant (c.1780c>t) is a novel mutation resulting in a stop codon. conclusions: recurrent sino-pulmonary infections are often a presentation of antibody deficiency. in this case, further investigation showed a profound t-cell defect, resembling that reported in 2 patients with homozygous nonsense mutations in the catalytic domain of rasgrp1 and 2 patients with homozygous insertion mutations leading to a premature stop codon at the bzip domain. our patient had biallelic mutations in rasgrp1 downstream of the catalytic domain, both leading to unstable transcripts. he developed an ebv induced atypical lymphoproliferative disorder, a complication reported in one rasgrp1 deficient patient whose disease progressed to b cell lymphoma and unsuccessful hsct. another patient developed ebv induced lymphoproliferative disorder after hsct for ebv-positive hodgkin lymphoma. two additional patients presented with recurrent infections, developed b-cell lymphoma and one was successfully transplanted. we are preparing the patient for hsct after chemotherapy for his lymphoproliferative disease to correct the underlying immune defect given that these patients are at high risk of developing lymphoma following ebv infection. introduction/background: immunodeficiency-centromeric instabilityfacial anomaly is a group of rare genetic disorders typically involving agammaglobulinemia. type four is caused by variants in the hells gene. the five patients previously reported with icf4 have fit the phenotype of agammaglobulinemia. here we report a patient with novel phenotype including neutropenia and neuroblastoma. objectives: describe a unique presentation of immunodeficiencycentromeric-instability-facial anomaly syndrome 4 to further expand our understanding of this disease. methods: retrospective chart review results: six-month old male was transferred to our tertiary care facility for ongoing chronic respiratory infection, chronic diarrhea, and failure to thrive. his past medical history was significant for 31-week prematurity due to rupture of membranes requiring a two month nicu stay for bronchopulmonary disease. upon discharge, he was bottle feeding and on room air. he had recurrent congestion for three months with two courses of antibiotics and one and a half weeks of diarrhea leading up to admission for difficulty breathing. he was found to have multiple infections including rhinovirus and parainfluenza virus on nasal wash, pjp pneumonia, norovirus, and pseudomonal cellulitis of his nose causing significant destruction. although previous laboratory studies revealed a normal absolute neutrophil count (anc), his anc quickly dropped to 170 cells/ul. his igg, iga, and igm were undetectable. while his total b cell count (451 cells/ul) was normal, he lacked any switched memory b cells. he had near normal total t cell count (cd3 2579 cells/ul) and cd4 count (2373 cells/ul) and a markedly decreased cd8 count (181 cells/ul) and poor proliferative response to low concentrations of phytohaemagglutinin and pokeweed mitogen. a 0.9cm x 1.6cm x 1.9cm paraspinal mass was found on chest ct, which was subsequently characterized as mibg-avid with a curie score 1 neuroblastoma. metastatic evaluation including bone marrow aspirate and biopsy was negative for malignancy. however, marked granulocytic hypoplasia and maturation arrest were present suggesting severe congenital neutropenia or, less likely, immune-mediated. whole exome sequencing detected homozygous variant of unknown significance in the hells gene (p.m223t). he was treated with intravenous immunoglobulin and g-csf with clinical and laboratory improvement. his neuroblastoma was initially observed, then subsequently removed due to a >50% increase in size. pathology confirmed mycn non-amplified favorable histology. he remains in remission 4 months after resection. he is currently awaiting bone marrow transplant for his immunodeficiency. conclusions: the significance of this case report is the novel presentation of icf4. neutropenia and malignancies have been reported in immunodeficiency-centromeric-instability-facial anomaly syndrome 2 (icf2) but not icf4. this case report thus expands upon the clinical picture of icf4 patients to include neutropenia and malignancies, and further describes the immunodeficiency. associate professor, university of south florida -johns hopkins all childrens hospital introduction/background: identification of newborns with severe combined immunodeficiency using state wide newborn screening (nbs) began in florida in 2012. abnormal results require extensive confirmatory diagnostic testing and prophylactic antimicrobial medications are needed to effectively evaluate and treat the infant. several barriers have been identified within government-sponsored health insurance programs that impede delivery of these evaluations and medications, often resulting in delays and/or inpatient hospitalization in order to provide timely and appropriate care. objectives: determine the cost differential between the initial evaluation and treatment of a scid patient detected by newborn screening in the inpatient versus outpatient setting. methods: the cost utilization of inpatient versus outpatient management of newly identified scid patients from nbs were analysed to include the cost of confirmatory testing and initiation of prophylaxis within the inpatient versus outpatient setting. laboratory tests included assessment of t cell immunity with quantitative and functional assessment, immunoglobulin measurement, genetic testing for scid variants, evaluation for maternal engraftment, and hla typing. medications included ig supplementation, pentamidine, fluconazole, and acyclovir. we compared the actual cost of inpatient stay and inpatient evaluation versus the approximate cost that would have been accrued if the patient were not admitted to the hospital. results: from 2012-2016, 4 infants with government-sponsored health insurance had abnormal nbs and were confirmed to have scid after evaluation at our institution. all 4 infants were admitted into the hospital for initial evaluation and initiation of appropriate medications for an average of 7 days. total average cost of medication administration for 7 days was $1,623, total cost of laboratory testing was $12,297, and average inpatient stay averaged $22,158 per patient. conversely, the cost that would have been accrued in the outpatient setting for medication would have been $1,097 for 7 days. laboratory testing costs would be no different as an outpatient. in total, the cost for inpatient evaluation was $36,708 versus $13,389 as an outpatient. conclusions: standard laboratory assessments and medications are necessary for infants identified with scid by population based nbs. despite government sponsoring of the florida nbs program, unnecessary barriers exist by government sponsored insurers that lead to a delay appropriate care. inpatient admission alleviates these barriers, but significantly increases cost. we advocate that standard ambulatory scid outpatient evaluation and initial treatment be authorized in children identified with scid through nbs without delay. patients with cd3g mutations reveal a role for human cd3g in treg diversity and suppressive function. introduction/background: integrity of the tcr/cd3 complex is crucial for positive and negative selection of t cells in the thymus, and for effector and regulatory functions of peripheral t lymphocytes. genetic defects that reduce, but do not abrogate tcr signaling, are associated with a variable degree of immune deficiency and immune dysregulation. in particular, while cd3d, cd3e, and cd3z gene defects in humans present mainly with severe immune deficiency, cd3g mutations lead to milder phenotypes, mainly characterized by autoimmunity. however, the role of cd3, encoded by cd3g, in establishing and maintaining immune tolerance has not been elucidated. objectives: we aimed to investigate abnormalities of treg cell repertoire and function in patients with genetic defects in cd3g with evidence of clinical autoimmunity. methods: high throughput sequencing (hts) was used to study composition and diversity of the t cell receptor (trb) repertoire in treg, conventional cd4+ (tconv), and cd8+ cells from 6 patients with cd3g mutations and in healthy controls. treg function was assessed by studying their ability to suppress proliferation of tconv cells. results: treg cells of patients with cd3g defects had reduced diversity, increased clonality, and reduced suppressive function. the trb repertoire of tconv cells from patients with cd3g deficiency was enriched for hydrophobic amino acids at position 6 and 7 of the cdr3, a biomarker of self-reactivity. overlap between treg and tconv cell repertoires was observed in cd3g mutated patients. conclusions: the treg and tconv cell repertoire of patients with cd3g mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition. introduction/background: a common concern with b cell-depleting therapies is their potential effect on humoral immunity. although there have been reports of prolonged hypogammaglobulinemia in adult patients receiving rituximab, little is know about this phenomenon in children. objectives: we sought to assess humoral immunity in children receiving rituximab and determine risk factors leading to low immunoglobulin levels and infections. methods: we conducted a retrospective study on all pediatric patients ( 18 years) who received rituximab for the first time between january 2014 to december 2016 in a single tertiary pediatric hospital. charts were reviewed and data was collected prior to rituximab treatment and at 6, 12, and > 12 months after treatment. patients who received rituximab after hematopoietic cell transplantation (hct) or for a malignancy and those with an underlying primary immune deficiency (pid) at the time of treatment were excluded. results: in total, 106 patients received rituximab during the study period. of those, 38 were excluded (hct: n=31, lymphoma: n=6, pid: n=1). sixtyeight patients were eligible. indications for rituximab treatment were renal disease (n=23), neurologic disease (n=18), hematologic disease (n=12), rheumatologic disease (n=10), ebv control (n=3), other (n=1). one patient who died from autoimmune encephalitis 8 days after rituximab was excluded from the follow-up study. at any time after rituximab treatment, low igg was present in 24/62 (38.7%), low iga in 6/61 (9.8%), and low igm in 36/61 (59.0%) of patients. over a year after their last rituximab dose, 12/29 (41.4%) of patients still had low b cell counts for age, and 10/10 (100%) had low memory b cell counts (cd27+ among cd19+ cells: mean value = 2.9% +/-1.9% sd). hospitalisation for infection was required in 13/67 (19.4%) patients in the year following rituximab treatment, which was associated with having either low igg (33.3% vs 15.2%, p=0.03) or low iga (50.0% vs 18.2%, p=0.04), but not with low igm levels (19.4% vs 24.0%, p=0.33). also, receiving a treatment with more than one rituximab cycle was a risk factor for low igg (63.0% vs 20.0%, p=0.0003). conclusions: hypogammaglobulinemia following rituximab treatment was frequent, and the presence of low igg and iga were associated with a higher risk of serious infection in this context. introduction/background: subcutaneous immunoglobulin (scig) replacement therapy for patients with primary immunodeficiency (pid) is usually administered once a week. however, a variety of dosing regimens can be used to provide flexibility for patients. objectives: we used pharmacokinetic (pk) analysis to evaluate the pk characteristics of weekly and biweekly (once every 2 weeks) scig administration in patients with pid. methods: this pk substudy was part of a prospective, open-label, phase 4 study (nct02711228) in patients with pid treated with igpro20 (hizentra®, csl behring, bern, switzerland). a noncompartmental analysis of serum igg concentrations was used to calculate pk parameters and compare pk outcomes on weekly and biweekly dosing. results: of the 17 patients included in the pk substudy, 15 provided samples for both weekly and biweekly regimens. the dose-adjusted area under the concentration-time curve was comparable for both treatment regimens: 0.24 and 0.25 (h*g/l)/mg for the weekly and biweekly regimens, respectively. the igg clearance was also similar, being 4.41 for the weekly and 4.14 ml/h for the biweekly regimen. median peak igg concentrations occurred later with the biweekly regimen (3.02 days) compared to 2.00 days for the weekly regimen. igg trough levels were close for both treatment regimens, with arithmetic means slightly lower for biweekly than for weekly regimens, at 10.13 vs. 10.21 g/l respectively. the minimum igg concentrations within a dosing interval were also comparable, with arithmetic means of 9.60 and 9.83 g/l for the weekly and biweekly treatment regimens, respectively. conclusions: biweekly and weekly hizentra® administration at the same total monthly igg doses resulted in similar igg exposures. pharmacokinetics, efficacy, tolerability and safety of a new subcutaneous human immunoglobulin 16.5% in primary immune deficiency introduction/background: patients with primary immune deficiencies (pid) require life-long replacement therapy with immunoglobulins (ig) to prevent severe infections and irreversible complications. in addition to safety and efficacy, tolerability and convenience of administration of ig products are essential factors in patient acceptance. a new 16.5% ig preparation (octapharma, lachen) was developed for subcutaneous administration (scig) derived from the established manufacturing process of octapharmas intravenous ig (ivig) brand octagam®. objectives: primary outcome was to assess efficacy of a new 16.5% subcutaneous human immunoglobulin preparation in preventing serious bacterial infections. secondary endpoints included evaluating tolerability and safety, determining the pk profile, the number and rate of other infections and changes in quality of life measurements. methods: a prospective, open-label, single-arm phase 3 study involving 61 patients was conducted at 18 centers in north america and europe. pid patients who were stable on ivig treatment for at least 6 months and with igg trough levels 5.0 g/l underwent a 12-week wash-in/wash-out period consisting of weekly scig doses 1.5 times the previous ivig dose (based on published conversion rates for marketed scig products), followed by a 52-week efficacy period (64 scig infusions in total). 22 of 61 patients enrolled had complete pharmacokinetic assessments at different time points: before the switch from ivig to scig (pkiv), after the wash-in/wash-out phase (pksc1) and at week 16 of the efficacy period (pksc2). results: 61 patients (age: 2-73 years; mean age 32.2 years; 54.1% female) receiving a total of 3,497 scig infusions (0.135 g/kg/week in young children (2 years and <5 years of age) and 0.185 g/kg/week in adults; average overall: 0.175 g/kg) were included in the full analysis sets. no serious bacterial infections were recorded. among the 188 other infections observed during the efficacy period only one infection was graded as severe (bronchiolitis due to rsv virus), which led to hospitalization (2 days). all other infections were mild (72.3%) or moderate (27.1%) in intensity. infection rate per person-year was 3.43. of the 233 reported adverse events, only 14 were assessed as being related to the study drug; all of these events were non-serious. five non-study drug related serious adverse events were reported in 4 patients (6.6%). serum igg trough levels were nearly constant during the study with a minimum trough level of 6.1 g/l and mean trough plasma concentrations of 11.4 ± 3.3 g/l and 11.6 ± 3.4 g/l for pksc1 and pksc2. median igg trough levels after scig treatment were 1.9 to 2.6 g/l higher compared to ivig treatment prior to enrollment. a dosing conversion factor (dcf) of 1.37 was determined by auc (area under the curve) measurements, allowing dose adjustment to achieve bioequivalence between ivig and scig dosing. improved quality of life measurements, utilizing sf-36v2, were observed in both physical and mental health parameters when compared from the first to last scig infusion. conclusions: this study demonstrated that the new subcutaneous human normal immunoglobulin 16.5% is well tolerated, safe and effective in patients with pid. introduction/background: atopic dermatitis (ad) is a chronic, relapsing, inflammatory skin disorder with associated pruritus that affects 11 percent of children in the united states. severe atopic dermatitis refractory to conventional therapy can be concerning for an underlying immunodeficiency, especially in infants. increased incidence of hypogammaglobulinemia has been associated severe ad. a handful of cases describe a correlation with transient hypogammaglobulinemia of infancy (thi), but a thorough immunological evaluation is often missing to further understand this relationship between ad and characterization of thi. objectives: define various phenotypes of thi with their clinical presentation and laboratory findings. compare thi vs. thi associated with severe a.d. methods: a case series of six patients was conducted at a single academic center from 2/1/2014 to 12/1/2017 for patients with severe atopic dermatitis with low igg levels. all available immunological laboratory data were retrospectively collected during this time period. descriptive statistical analysis was utilized for data comparison. results: of the six patients, four had no infectious history. of the remaining two, one had recurrent skin abscesses associated with his poorly controlled atopic dermatitis requiring oral antibiotics and one patient had two episodes of staphylococcus aureus superinfection of the eczema. at the time of presentation, the mean age was 7 months with mean igg of 164 mg/dl and mean ige of 2,273 ku/l. iga and igm were within normal age cut offs. all patients had normal protein and polysaccharide specific antibody titers after completion of vaccination series. mean cd19+ count was 2,200/ul with normal cd3+, cd4+, cd8+ and cd1656+ cell counts. three patients were tested for lymphocyte mitogen proliferation and complement function which were normal. two patients, who were tested, had normal phagocyte work up. mean age of igg improvement to igg> 217 mg/dl, was 10 months. patients had total protein and albumin levels with mean 5.1 ku/l and 3.5 ku/l, respectively which eventually normalized. four patients improved with skin care and dietary modification to hypoallergenic formula. one patient improved with extensively hydrolyzed formula and three patients improved with amino acid formula. one patient improved with aggressive skin care. one patient, who was noncompliant with dietary recommendations and aggressive skin care, did not improve. conclusions: one prospective study described an increased incidence of hypogammaglobulinemia in patients with atopic dermatitis compared to controls regardless of the severity. no further prospective studies have characterized hypogammaglobulinemia in this population. according to the primary immunodeficiency practice parameters, children with thi often present with frequent viral and bacterial respiratory illnesses, low igg levels and normal vaccine responses. in one study, the period of hypogammaglobulinemia spontaneously corrects to normal by mean age of 27 months with all patients reaching normal levels by 59 months. these patients may have low igm or iga and decreased t or b cells, which eventually normalize. management is often with antibiotic prophylaxis and if refractory to prophylaxis or unable to tolerate, igg administration (igrt) based on severity of symptoms is recommended. we describe a less severe variant of thi associated with severe atopic dermatitis and characterized by very transiently decreased igg levels with earlier resolution than typical thi, normal igm and iga levels, normal specific antibody levels and normal t, b and nk cells. these patients typically do not manifest recurrent viral or bacterial respiratory illnesses. they also do not require antibiotic prophylaxis or igrt. severe atopic dermatitis maybe a positive prognostic indicator for patients with thi and is associated with an earlier self-resolution of hypogammaglobulinemia, lack of typical infections, normal iga, igm and normal t and b cell numbers. a possible mechanism for thi associated with severe ad may be transdermal loss of protein which was supported with mildly low total protein and albumin levels rather than immaturity of the immune system in patients with true thi. introduction/background: in recent years it was found that heterozygous mutation in pik3cd gene produces an autosomal dominant primary immunodeficiency characterized by onset of recurrent sinopulmonary and other infections in early childhood with defects in both b-and t-cell populations and a special susceptibility to uncontrolled viral infections. many patients develop chronic lymphoproliferation and there is also an increased susceptibility to b-cell lymphomas. here we present a patient assumed as a common variable immunodeficiency with heterozygous mutation in pik3cd gene. objectives: to describe a case of a patient assumed as a common variable immunodeficiency with heterozygous mutation in pik3cd gene. results: 60 year old woman with personal history of severe and recurrent upper and lower respiratory infections, chronic pulmonary disease with bilateral bronchiectasis, chronic diarrhea without diagnosis, mild osteopenia, focal lesion in right hepatic lobe, atopic dermatitis and anemia. she was following up in other center and in 1996 she was diagnosis with common variable immunodeficiency (cvid) and started treatment with intravenous immunoglobulin (ivig), but she referred low adherence to it. she did not referred history of lymphoproliferation nor significant viral infections. she has a daughter with spherocytosis who required esplenectomy and also had bronchiectasis and cvid, she deceased at 28 years old because pulmonary infection. other daughter and 2 sons referred healthy. in our first immunologic studies we found severe hypogammaglobulinemia (igg 428 mg%, no dosable iga and igm) with absent of b cells in peripheral blood. we started with high doses of ivig (800 mg/k/month) and antibiotic prophilaxis with improvement of the functional respiratory test and without new infections. we are planning colonoscopy to study her chronic diarrhea. thinking that her clinical picture could be other than cvid we order a genetic study. a nextera exome capture and next generation sequence with illumina hiseq was made and an heterozygous mutation in pik3cd gene (chr1:9.775.746, p.pro97ala) was found. family and functional studies are still pending. conclusions: due that clinical presentations of primary immunodeficiencies are becoming more complex, its diagnosis is a challenge for immunologist now a days. studies with next generation sequence is a very useful tool in indefinite cases, especially when more than one member in the family are involved. chief, laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health introduction/background: heterozygous gain-of-function mutations in pik3cd as well as heterozygous pik3r1 mutations that affect interaction of p85 with p110 lead to constitutive hyperactivation of the pi3k pathway and cause activated pi3k delta syndrome type 1 or type 2 (apds1, apds2), respectively. we describe a female with apds2 with short stature, diffuse lymphadenopathy, recurrent upper respiratory tract infections, elevated igm, persistent ebvand cmv viremia and disseminated toxoplasmosis who gave birth to a genetically affected daughter with severe congenital toxoplasmosis. objectives: to characterize the molecular and cellular defects underlying severe toxoplasmosis in this family methods: investigation of the molecular basis of the disease was performed through whole exome sequencing, and results were validated by sanger sequencing. functionality of the pi3k pathway was assessed by analyzing akt and s6 phosphorylation in freshly isolate b cells with and without stimulation with anti-igm. an excisional lymph node biopsy from the affected mother was stained with anti-pd1 antibody to detect t follicular helper cells, and with igm and igg specific antibodies to analyze the proportion of isotype-specific b and plasma cells results: whole exome sequencing of maternal dna with targeted analysis of 362 pid genes identified a heterozygous mutation at an essential donor splice site of pik3r1 (nm_181523.2:c.1425+1g> a). sanger sequencing confirmed the presence of this mutation in both the mother and her child. functional studies on b cells freshly isolated from both patients confirmed an increase in baseline akt and s6 phosphorylation, suggesting constitutive activation of the pi3k-mtor signaling pathway. a lymph node biopsied from the mother contained numerous pd-1+ tfh cells and igm+ plasma cells conclusions: toxoplasma gondii is an obligate intracellular parasite that is usually only symptomatic in immunocompromised hosts. severe toxoplasmosis has been reported in the following primary immunodeficiencies: cd40 ligand deficiency, tap deficiency, cvid, nfkb2 deficiency, and immunodeficiency due to anti-ifn-autoantibodies. importantly, toxoplasma infection was previously reported in a 9month-old infant with apds1, and ocular involvement has been described in a 36-year-old patient with apds2. this, however, is the first report of systemic and severe congenital toxoplamosis in a mother and child with apds. to evade innate host defenses, t. gondii induces the activation of the pi3k/akt signaling pathway, reducing intracellular reactive oxygen species and creating an intracellular environment that is hospitable to parasite survival and proliferation. therefore, we postulate that the pik3r1 mutation may lead to a hospitable cellular environment for toxoplasmosis replication. this work was partially supported by the division of intramural research, niaid, nih (protocol # 05-i-0213). additional authors of this work are: ottavia delmonte and kerry dobbs, from the laboratory of clinical immunology and microbiology, niaid, nih. introduction/background: aplaid (autoinflammation and plc-gamma-2-associated antibody deficiency and immune dysregulation) is a term that was proposed for the newly discovered autoinflammatory condition resulting from a pathogenic missense variant, ser707tyr, in the cterminal sh2 (csh2) domain of plc-gamma-2 in order to distinguish it from plaid, a distinct clinical entity that results from intragenic deletions of portions of the csh2 domain of the same protein. plc-gamma-2 is a phosphodiesterase that is predominantly expressed in hematopoietic cell lines and acts on pip2 to produce ip3 and dag in the pkc and ras/raf/ erk pathways. the only formally published case of aplaid described a father-daughter pair with an autoinflammatory clinical syndrome affecting the skin, mucosa, eyes, pulmonary and gastrointestinal systems. objectives: to describe the aplaid phenotype resulting from a novel genetic variant of the phosphodiesterase plc-gamma-2 in two unrelated families methods: clinical review and case presentation results: we report a 33-year-old female with a long-standing history of recurrent pneumonia, cellulitis and cystitis with obstructive lung disease characterized as bronchiolitis and dynamic airway collapse, with negative alpha-1-antitrypsin testing. she had a history of childhood onset granuloma annulare and pressure-induced urticaria, as well as episcleritis. immune testing revealed low igm (37 mg/dl), elevated baff levels and a low percentage of cd27+ memory b-cells, prompting sequencing of plcg2, which identified a c.3422t>a, p.met1141lys variant in the calcium binding c2 domain. her 15-month-old daughter, who has a history of bullous skin lesions, failure to thrive, febrile episodes and recurrent respiratory infections was likewise found to have this plcg2 variant. like her mother, the daughter also has low igm and elevated baff levels. similar symptoms of recurrent sinopulmonary infections and hypogammaglobulinemia, have been described in an unrelated family that shares this same plcg2 variant in a doctoral thesis by rozmus. this work also describes functional analysis, in which this particular variant of plc-gamma-2 was demonstrated to have dysregulated plcgamma-2 activity leading to aberrant intracellular calcium signaling and increased apoptosis of immature b-cell subsets. conclusions: we describe our experience in evaluation and treatment of this family with a previously undiagnosed disorder. together, these new cases add to the expanding body of knowledge regarding plc-gamma-2 and its importance for the development of autoinflammatory and primary immunodeficiency conditions. (133) submission id#427357 present a case.non-celiac gluten sensitivity is an emerging entity with symptoms similar to celiac disease, but without positivity in specific diagnostic tests. it is considered more common than celiac disease patients with sigad have a greater risk of concomitant autoimmune disorders than health individuals. sigad was previously to be associated with celiac disease, but not usually in non -celiac gluten sensitive. objectives: describir a case ataxia non celiac gluten sensitivity, methods: clinic case description. results: he patient has negative serology test for gluten, but clinically respond as celiac disease. conclusions: all patient has negative serology test for gluten, but clinically respond as celiac disease, could has non-celiac disease, this case described here ,it suggest than this entity should to thought before than the celiac disease, since not-celiac disease is it more common. associate professor, federal university of rio de janeiro introduction/background: primary cutaneous actinomycosis is a rare condition caused by gram-positive filamentous bacteria and generally occur after traumatic inoculation. objectives: to report an unusual etiology of skin lesions in a patient under anti-tnf-alpha therapy. methods: we report the case of a 30-years-old female receiving conventional doses of adalimumab for ankylosing spondylitis who presented, two weeks before referral for dermatological assessment, with an erythematous nodule with purulent discharge on right pretibial region and pruritic erythematous plaques with scaling and peripheral pustules in the trunk and nose tip. results: a fungal etiology was suspected and scraping specimens from several cutaneous lesions were submitted to direct microscopic examination and culture, which were negative for fungi. cutaneous biopsy of the pretibial lesion was sent for histopathology and microbial cultures. adalimumab was interrupted and empirical therapy with oral terbinafine (250 mg/day) was started with close clinical follow-up. a folliculitis reaction pattern without granulomas was observed during histopathological examination and gram-positive cocci were isolated from biopsy sample and further identified as saccharopolyspora sp. by molecular typing. sulfamethoxazole+trimethoprim was added and discontinued after one week due to a cutaneous rash. terbinafine (250 mg/day, p.o) was used for 12 months with complete clearing of all skin lesions and very good tolerability. spondyloarthritis signs and symptoms were unremarkable and no anti-inflammatory or immunomodulating treatment was necessary. conclusions: increased risk of skin actinomycotic infections in patients under anti-tnf therapy is not consistently reported in the literature. nevertheless, they should be included as a differential diagnosis of atypical skin lesions in individuals under anti-tnf therapy. where we aim to determine the prevalence, incidence, characteristics, treatment and outcomes of primary immunodeficiency disease (pid) patients in qatar. pids are rare heterogeneous disorders of the immune system that result in an increased susceptibility to infection, immune dysregulation and occasionally, to cancer. objectives: determine the range of pids with important epidemiological data in qatar after analyzing the database and creating a registry. methods: this is a retrospective study of pid patients followed at hamad medical corporation from 1989 to 2017 using medical records. all patients who were diagnosed with a pid irrespective of age were included. patients were classified according to the international union of immunological societies expert committee on pid. the data is captured under 5 sectionsa) patient demographics including age, gender, ethnicity b) clinical presentation, c) immunodeficiency profile including age at diagnosis, type of immunodeficiency, family history d) treatment modality and e) lab/genetic data. results: we registered 150 patients (60 females and 90 males) over a span of four years. mean age at onset, diagnosis and diagnostic delay were 2.6, 4.6 and 2 years respectively. majority of the patients were arabs 77% followed by people from the asian subcontinent 12%. antibody deficiency was seen in 32%, immune dysregulation 28%, well defined immunodeficiency (at, hige, digeorge,wiskott aldrich) 25% and t/b cell cid 12%. rare diagnoses (ipex, msmd) were recorded whereas no cases of toll like receptor and complement deficiency were seen. consanguinity rate was (n = 97, % = 65) and first degree cousin marriage (n = 38, % = 40). family history was positive in 50% (n= 75) of the patients. maximum diagnostic delay was seen in scid (33% >3 months) and agammaglobulinemia (66% >3 months).during the patients life, infection was the most common presenting complaint (47%) followed by sinopulmonary disease (16%) and gi-tract manifestations (7%). the most common infections were pneumonia (46 %), otitis media and conjunctivitis (42 % each) followed by failure to thrive (30%) and sepsis (21%).microbial isolates particularly seen as causative agents of infections were p.aeruginosa and salmonella (14%) each, mrsa and e.coli (9%) each. a genetic defect was confirmed in 57% of ataxic telangiectasia and 65% of scid patients. active infections were treated and prophylactic antibiotics were prescribed in 76 cases (51%). prophylactic antibiotics were prescribed to 34% of patients with immune dysregulation and to 25% of well-defined syndromes. out of 18 patients who had hsct, 72% had successful transplants. ivig was given to 42% of the total pid patients with humoral immunodeficiency patients receiving the most (45%). one patient had gene therapy and two required interferon gamma treatment for msmd. mortality rate at 1st year of life was 4% whereas total mortality rate was 12%, excluding cases that passed away before pid was diagnosed. conclusions: the estimated prevalence of pid in qatar is found to be 6 per 100000. over the years, physicians have become increasingly aware of pid and survival rate has improved. initiation of newborn screening for scid and agammaglobulinemia will lead to earlier diagnosis and initiation of therapy with better outcomes. introduction/background: agammaglobulinemia is typically associated with a near absence of b cells secondary to a developmental block in bone marrow, and, usually but not always, manifests in early life. most of such patients are males with x-linked agammaglobulinemia (btk deficiency). females with agammaglobulinemia, of either autosomal recessive or dominant inheritance, are rare. objectives: we present and discuss the differential diagnosis for the conflicting clinical and immunological phenotypes of two adult females who present with infections, cytopenias, and agammaglobulinemia with low to normal b cell count. methods: retrospective chart review of clinical and laboratory data results case 1: a 22-year-old female who, at age 17 years, had evans syndrome (autoimmune thrombocytopenia and hemolytic anemia) that resolved after steroid and high-dose gammaglobulin treatment. at age 21 years, immunologic workups revealed a complete absence of serum immunoglobulins (igg, iga, igm) and low b cells 43 (3%) (normal range 100-500). however, three years prior, patient had detectable igm (218 mg/ dl). b cell subset analysis showed an expansion of cd19hi21lo b cells (30%, normal 0.2-8.6%), with a marked decrease in switched memory b cells (0.2%, normal 7-61%). additional immunophenotyping revealed a reduced frequency of naïve cd4+ (3.6%, normal >50%), cd8+(42%, normal >50%) t cells, and normal lymphocyte proliferative responses to mitogens and anti-cd3, anti-cd3/anti-cd28, and anti-cd3/il-2. case 2: a 30-year-old female with recurrent upper respiratory tract infection, undetectable igg, iga, igm and ige, and no response to vaccinations (tetanus and pneumococcal). thrombocytopenia (38x103 count/microliter) was noted once during her thirdtrimester pregnancy without evidence of pre-eclampsia. postpartum cd4+ t cell count was low (442 cell/microliter, normal 500-1400 cell//microliter). immunophenotyping revealed normal b cell count with reduced frequency of naïve cd4+ (17.6%, normal >50 %) and cd8+ (18%, normal >50 %) t cells. conclusions: we report two adult females who present in early adulthood with recurrent infections and cytopenias. both had agammaglobulinemia and decreased naïve t cells suggestive of late-onset combined immunodeficiency (locid). the presence of peripheral b cells makes autosomal recessive defects in b cell receptor signaling (lamda5, iga, igb, igm, blnk) less likely. differential diagnosis of locid with cytopenias includes ctla-4 haploinsufficiency, gain-of-function pik3cd mutations, ikaros defects and autosomal recessive rag deficiency. both patients remain at risk for developing autoimmune complications. a molecular diagnosis will facilitate targeted therapy for the underlying defect. professor, director of the laboratory of childhood immunology, ku leuven introduction/background: complement factor properdin (cfp) is a soluble glycoprotein which has a unique known role as a positive regulator of the alternative complement pathway by binding and stabilizing the inherently labile c3/c5 convertase enzymes. mutations in the cfp gene lead to aberrant protein expression or to expression of a dysfunctional protein which results in high susceptibility to pyogenic infections especially neisseria meningitidis. properdin-deficient individuals are at greater risk of fulminant meningococcal disease, with mortality rates as high as 75%. objectives: case report: a 5 year old belgium boy from nonconsanguineous parents presented with achronic purulent cough and recurrent infections of upper and lower airways. he suffered from a severe pneumonia at the age of 5. ct thorax showed bronchiectasis of the right middle lobe and left lower lobe. in addition, he had recurrent acute otitis media since the age of 1 year old. immunological work-up showed an absent ap50 activity, with normal ch50, c3 and c4. results: genetic and functional analysis: sanger sequencing of cfp gene identified a hemizygous c.961t>g, p.y321g (cadd score 8.081, msc_cadd 0.102) mutation in the patient and his mother. properdin elisa (hycult biotech) showed absent and 50% of the normal healthy control value of serum properdin concentrations in patient and the healthy mother, respectively. conclusions: conclusion: we diagnosed properdin deficiency in a 5y old boy presenting recurrent lower and upper respiratory tract infections. ap50 testing, added to ch50, should therefore be part of initial workup for patients with recurrent severe respiratory tract infections. indeed early diagnosis allows for appropriate prolonged antibiotic prophylaxis and immunization to reduce the risk of fatal meningococcal disease, to reduce or prevent organ damage and to allow for genetic counselling in the family. 2-4, 5-7, 8-12 and 13-18) and child depression inventory (cdi) were implemented to both children and parents in addition to sociodemographic data form; beck depression inventory (bdi), beck anxiety inventory and zarit caregiver burden scale were implemented only to parents. results: the depression inventory parent and child form values of the patient group with primary immunodeficiency were significantly higher than the control group (p=0,004 and p=0,032). according to beck depression and anxiety inventories, it was seen that the depression and anxiety inventory scores of the parents of the patient group were higher than the control group (p=0,009 and p=0,022 respectively). it was determined that the cdi parent form scores of the patients with hospitalization history were statistically higher than the patients without hospitalization history (5,4±0,5 and 4,8±0,6; p=0,009). bdi scores were significantly higher in the group which received ivig (p=0,024). while the quality of life of the patients compared to their parents was perceived as worse than the healthy children in all dimensions (p=0,003 p<0,0001 and p<0,0001), the quality of life of the children was worse only in psychosocial and total quality of life fields (p<0,0001 p=0,002 and p=0,158). it was seen that quality of life of children physical health scores of only the patients with hospitalization history were statistically significantly lower (15,5±1,9 and 14,3±2,3; p=0,04) . while no statistically significant difference was found in terms of quality of life of the child scores between the groups which received and did not receive ivig replacement treatment, psychosocial and total qualify of life scores of parents were statistically significantly lower in the group which received ivig replacement treatment (p=0,017 p=0,033). zarit care giver burden scale scores were similar in patient and control groups. although both groups were on the limits of mild to moderate caregiving burden, it was seen that the scores of the group which received ivig were significantly higher than the group which did not receive ivig (p=0,026). conclusions: as a conclusion, we think that it will be appropriate to inform and monitor the entire family in relation to psychosocial difficulties and care giving burden they may experience in time as well as the medical aspects of the disease in order to develop a holistic approach to children with primary immunodeficiency. introduction/background: chronic lung disease is the most common complications of cvid, affecting 30-60% of patients. it includes bronchiectasis affecting 50% of patients and granulomatous lymphocytic interstitial lung disease (glild) in 10 to 55%. both are associated with an increased morbidity and mortality. pulmonary functional studies and ct scans have been proposed as screening procedures for lung involvement in cvid. the definitive diagnosis of glild is established histologically, but it is too invasive in patients with radiological abnormalities and no symptoms. objectives: we aim to describe the pulmonary complications of our cohort of patients with cvid comparing them with subjects affected with other types of hypogammaglobulinemia. methods: we reviewed all clinical records of the patients with a diagnosis of hypogammaglobulinemia of any cause until december 2016. we looked at all pulmonary function tests (pft), 6-minute walk tests and ct scans performed. we classified patients according to the ct scan pulmonary disease pattern. we compared the demographic data and pulmonary characteristics of each group and intended to describe similarities and differences between them. results: we collected 34 patients and 70 ct scans from 26 patients. patients were grouped for further analysis as follows: no ct performed: 8; normal ct: 9; bronchiectasis: 9; glild: 7 and bronchiectasis + glild: 1. eight patients (24%) had no ct including six with cvid, one with x linked lymphoprolipherative disease (xlp) and one had a syndromic deletion in chromosome 11. the median age of symptom onset was 23.5yo, with a median delay to diagnosis of 1 year. nine subjects (26%) had ct scans with no chronic disease, six had cvid, 2 rituximab induced hypogammaglobulinemia (rih) and 1 mgus with hypogammaglobulinemia (mguswh). the median age of symptom onset was 58 years old with a median delay to diagnosis of 2 years. nine patients (26%) presented persistent bronchiectasis, 7 with cvid, 1 with an igg3 deficiency (sigg3d) and 1 with xlp. median age at symptoms onset was 14yo and median delay to diagnosis 13 years. seven patients (21%) had glild; all afected with cvid. median symptoms onset was 30 years old, and median delay to diagnosis was 10 years. one patient (3%) was included in the bronchiectasis and glild group. she was referred with a diagnosis of rih, in the context of a pulmonary malt lymphoma. however, her ct did not show a typical ct lung lymphoma pattern, lymphocyte monoclonality was never shown and she had a reduced pre-treatment low gamma globulin concentration and a history of respiratory infections since adolescence, we believed her diagnosis is cvid. pft with an obstructive pattern identified patients with bronchiectasis (p<0.01) and patients with glild had a significantly lower basal and post 6 minute walk o2 saturation (p<0.01, n=22) conclusions: using a systematic approach, we identified that roughly 50% of patients with hypogammaglobulinemia have chronic pulmonary ct abnormalities. half of them had bronchiectasis, that were associated to hypogammaglobulinemia of any cause, a longer disease course and a reduction in fvc and fev1 with a shorter distance reached in the 6 minute walk test. the other half had glild, spirometries in this group were useless, but o2 saturation was significantly lower, basal and after the 6 minute walk test. we found a high frequency of pulmonary disease in our cohort and a disease progression study is now in place. hies) is a primary immunodeficiency characterized by eczema, sinopulmonary infections, and musculoskeletal and vascular abnormalities. care of patients with this disease is largely supportive with the use of prophylactic antibiotics and topical eczema therapies. the use of replacement immunoglobulin is increasing. hematopoietic stem cell transplant (hsct) is being considered more frequently, but many questions remain regarding which patients should undergo transplant. as pulmonary complications are a leading cause of morbidity and mortality in this disease, and potentially improved with replacement immunoglobulin and hsct, we sought to examine more closely the patients with more frequent pulmonary hospitalizations and structural lung disease. objectives: to determine the rates of pulmonary complications in our large cohort of ad-hies patients, and examine the relationship between immunologic markers and pulmonary disease. methods: we retrospectively reviewed the records of 110 ad-hies patients seen more than one time at nih between 2010 and 2016. there were 36 pediatric patients under the age of 18 years. we reviewed the number of and cause for hospitalizations, and reviewed radiology reports for structural lung disease including bronchiectasis and pneumatoceles. we correlated these findings with specific antibody responses and lymphocyte phenotyping, such as the number of memory b lymphocytes. results: the patients range in age from 2 years to 66 years, with a median age of 23 years. 106 patients had chest cts, with 42 percent having bronchiectasis, and 41 percent having cavitary lesions. 31 percent of patients had both. these abnormalities were more prevalent in patients with low memory b cells. 31 percent of patients receive replacement immunoglobulin. in patients not receiving replacement ig, 42 percent had appropriate response to pneumococcal vaccine. during this time period, there were 190 hospitalizations, the majority of which were associated with pulmonary infections. the rate of overall hospitalization was higher in the group with low memory b cells (p=0.006), but there was no difference associated with age. conclusions: immunologic abnormalities may assist in determining the long-term prognosis of patients with ad-hies, and can be considered in management plans such as the use of immune globulin and consideration for hsct. introduction/background: purine nucleoside phosphorylase (pnp) deficiency is an autosomal recessive disorder affecting the purine salvage pathway causing a (severe) combined immunodeficiency disorder, autoimmunity, and neurological symptoms. patients typically present in the first few years of life with frequent infections and a failure to thrive. decreased plasma levels of uric acid are suggestive, while neutropenia is a rare complication. prognosis is generally poor with few patients making it to adulthood without treatment. treatment options are limited to general supportive care and hematopoietic cell transplantation. methods: we present the cases of three patients, one sister and two brothers, from a consanguineous french canadian family. they presented in early adulthood with nearly identical histories of repeated pneumonias and chronic rhinosinusitis. one patient had necessitated filgrastim to treat a parainfectious neutropenia. they did not report any neurological abnormalities or symptoms of autoimmunity. results: a primary immune deficiency was suspected. initial evaluation showed normal immunoglobulin levels, a lack of response to vaccination, positive ebv ebna iggs, and auto-antibodies. the patients were severely lymphopenic with reduced numbers of t, b and nk cells ( l: 300, cd3+81%, cd4+71%, cd8+5%, cd19+12%, cd3-cd56+7%). in particular, they had a severe depletion of naive t-helper recent thymic emigrant cells (cd4+cd45ra+cd31+2%). additional studies revealed increased urinary inosine, guanosine, deoxyinosine, and deoxyguanosine, severely reduced erythrocyte pnp activity (3% residual activity), and a normal uric acid level. a homozygous, missense mutation, c.769 c>g (p.his257asp) was found and described as pathogenic in silico. the diagnosis of pnp deficiency was made; tmp-smx prophylaxis and ivigs were started. conclusions: theses cases are atypical for a number of reasons. the first is the relatively benign immunological course, the lack of neurological symptoms, and the advanced age at the time of the diagnosis, possibly due to residual enzymatic activity. in addition, pnp deficiency has been classically described as causing a marked reduction of t cells with a relative sparing of b cells. however, b cell lymphopenia have been reported, particularly with latter presentations. while this specific mutation had been identified once previously in the literature, as a compound heterozygote, these are the first confirmed cases of homozygotes. the pathogenicity of the mutation was not only suggested in silico, it was confirmed biochemically. in addition, in vitro functional studies have demonstrated the importance of his257 for the binding of pnp to its substrate, particularly in the formation of the early transition state. indeed, pnp (p.his257asp) has been shown to have a greatly reduced affinity for its substrates and catalytic activity. further study is needed to identify the optimal management for these patients. introduction/background: whole exome sequencing has become an integral part of diagnosis and treatment of rare immunodeficiency diseases. one of these diseases is tetratricopeptide repeat domain 7a(ttc7a) mutations; a rare disorder associated with multiple intestinal atresias and severe combined immunodeficiency. histologic assessment of organ biopsies of patients with ttc7a deficiency suggest it may play a role in multiple organs as it is expressed in the cytoplasma of intestinal, thymus and pancreatic cells. many patients with ttc7a deficiency have moderate to severe combined immunodeficiency. nine patients described in the literature with ttc7a mutations have been treated with hematopoietic stem cell transplant, mostly at a young age. objectives: this case report describes a rare presentation of an adolescent with ttc7a deficiency, her clinical presentation, immune evaluation and treatment with eventual referral to bone marrow transplant at the age of 16. methods: retrospective chart review under irb approval was performed. whole exome sequencing performed at baylor texas childrens hospital and mutations were confirmed by sanger sequencing. extensive immune and nk cell phenotyping were performed by flow cytometry and nk cell function was tested using standard cr51 release cytotoxicity assays. results: a 13 year old female was referred to immunology for severe refractory warts, history of severe diarrhea with epithelial dysplasia but no atresia, failure to thrive requiring parenteral nutrition until 6 years old and multiple infections with staphylococcus aureus and candida. at 16 years of age, she developed rapidly worsening pulmonary function that improved with monthly pulse methylprednisone. her immune phenotype demonstrated a combined immunodeficiency including severely low cd4, cd8, b and nk cells. she had no igd-cd27+ memory b cells and undetectable igg, iga and igm, isohemagglutinins and vaccine titers to diphtheria and tetanus. her mitogen proliferation response was low and she had abnormal tcr v-beta repertoire. she was referred to the texas childrens hospital center for human immunobiology for the nk cell evaluation and reasearch clinic (near) the patient was found to have impaired nk cell lytic function and terminal maturation. this was demonstrated by the decreased frequency of cells that matured to the cd56dim subset and was accompanied by decreased expression of the lytic effector molecule perforin and the fc receptor cd16. this phenotype was conserved when we isolated cd34+ hematopoietic precursors and performed nk cell differentiation in vitro. whole exome sequencing demonstrated a compound heterozygous mutation in the ttc7a gene, including a c.1001+3_1001+6aagt mutation, which has been previously described as a pathologic founder mutation in the french canadian population. the second mutation is a previously unreported c.211g>a (p.e71k), a variant of unknown significance. mutations were confirmed by sanger sequencing, and parents are carriers of the mutations. she was referred for hematopoietic stem cell transplantation with a 10/10 unrelated donor option. conclusions: ttc7a deficiency is a genetic mutation associated with high morbidity and mortality leading to gut atresia and dysfunction in combination with severe immunodysfunction. we have described a unique case of ttc7a deficiency with a novel mutation which is associated with intestinal epithelial dysplasia with no atresias and a late presentation and evolution of combined immune deficiency. nk cell assessments show significantly impaired terminal maturation suggesting a critical role for ttc7a in human nk cell development. introduction/background: ras-associated autoimmune leukoproliferative disorder (rald) is a rare condition with significant overlap of clinical and laboratory findings with malignant disorders, notably juvenile myelomonocytic leukemia (jmml) and chronic myelomonocytic leukemia (cmml), but with much better prognosis without specific therapy. we report a case of rald in a 4month-old male infant originally suspected to have jmml. results a 4-month-old male infant, born to an unrelated ethiopian father and turkish mother, presented with splenomegaly and leukocytosis (26,000/l), monocytosis (7,800/l), and thrombocytopenia (14,000/l); hemoglobin was 10 gm/dl and fetal hemoglobin concentration 1.9%. family history indicated early death of three paternal uncles, two at age 6-7 months and one at age 10 years. jmml was suspected and bone m a r r o w a sp i r a t i o n w a s p e r fo r m e d ; h ow e v e r, r e s u l t s o f immunophenotypic testing with flow cytometry analysis and cytogenetic testing with fish did not support this diagnosis. patient had a normal b12 level. serum igg (1884.8 mg/dl [nl 694.0 -1618.0 mg/dl]) and igm (139.0 mg/dl [nl 35.0 -102.0 mg/dl] levels were elevated and iga was normal. lymphocyte subset analysis showed 37% cd3 [nl 51-74%], 20% cd4 [nl 34-53%], 51% cd19 [nl 17-37%] positive cells with normal percentages of cd8 and cd16/cd56 cells; there were no cd4-/ cd8-t cells detected. given the findings of leukocytosis, monocytosis, splenomegaly, hypergammaglobulinemia, and a dearth of cd4-/cd8-t cells, the patient appeared to meet criteria for rald. mutation testing with next generation sequencing showed an nras missense mutation [c.37g>t; pgly13cys] in 46% of t cells and 48% of myeloid cells. the presence of the nras mutation, in the absence of other typical jmml findings or rasopathy syndrome features, supported the diagnosis of rald. there was no therapeutic intervention and, at one year of age, the patient was reported to remain clinically well; he relocated to ethiopia with his parents. conclusions rald is a nonmalignant clinical syndrome originally classified as a subtype of autoimmune lymphoproliferative syndrome (alps) but subsequently distinguished to be a separate entity due to lack of double negative t-cells, a mutation in fas/fasl/caspase-10, or consistently elevated serum b12 levels. though also present in 25% of jmml patients, a somatic mutation in ras signaling protein (kras or nras), which controls b-cell tolerance and production of autoantibodies, is present in all reported cases of rald. while all previously reported cases of rald had somatic kras or nras mutations, our patient's nras mutation was present in more than 40% of t cells and myeloid cells, suggesting a heterozygous germline mutation; this has not previously been reported in rald and needs to be further confirmed (145) the primary objective of this study was to evaluate hct outcomes in patients with was who underwent hct since 2005 in north america. we hypothesized that survival after hct from alternative donors such as cord blood has improved compared to published results, but that overall survival would be superior in patients who undergoing hct at a young age. methods: patients were enrolled on pidtc protocol 6904, a multicenter retrospective natural history study of patients treated for was in north america since 1990. clinical features, disease status, hct type and post-hct outcomes were analyzed in 129 patients who underwent hct at 29 pidtc centers between 2005-2015. descriptive statistics such as median and range for continuous variables and counts and percentages for categorical variables were used to summarize characteristics of the study population. in addition, kaplan-meier curves were used for estimating survival probabilities results: diagnosis of was was confirmed by an expert review panel for eligibility. mutation in the was gene was available for 118 patients, including nonsense (n=27, 23%), frameshift (n=32, 27%), missense (n=31, 26%), splicing (n=20, 17%), gross deletion (n=5, 4%), in-frame deletion (n=2, 2%), pr complex (indel) (n=1, 1%). donor types included matched sibling (n=22, 17%), unrelated adult volunteer bone marrow or peripheral blood stem cells (n=66, 51%), umbilical cord blood (n=39, 30%) and other related donors (1 each, phenotypically matched related, haploidentical) . median age at time of hct was 13.6 months (range, 2.1 260.5 months). the vast majority of patients received busulfan containing conditioning regimens (87%), with some receiving other myeloablative (2%) or reduced intensity regimens (11%). with a median follow-up of 4.6 years, overall survival was excellent with 1-year and 5-year survival probabilities of 92% (95% ci, 86-96%) and 91% (95% ci, 84-95%), respectively. survival was similar at 1 year for recipients of hct from matched sibling (95%, 95% ci, 72-99%), matched unrelated donor (92%, 95% ci, 82-97%) or umbilical cord blood (90%,95% ci, 75-96% see figure) . importantly we confirmed that survival at 1 year was better in patients who were <5 years old (n=118) compared to those who were 5 years old (n=11) at the time of hct (95% versus 62%, respectively p=0.032). this difference persisted when only unrelated donor recipients were analyzed (97% vs 57%, p=0.036). overall the percentage of patients in our study who underwent hct at a young age was high (91%) compared to the literature (84% in moratto et al, 2011, blood) . the rate of second hct was only 4% (n=5). cumulative incidence of acute grade 2-4 at 100 days, acute grade 3-4 at 100 days and chronic graft-versushost disease at 1 year were 26% (95% ci 18-34%), 15% (95% ci 9-22%) and 14% (95% ci 8-21), respectively. conclusions: outcome of hct for was since 2005 shows excellent overall survival for all donor types, including umbilical cord blood with very low rates of second hct. importantly, hct at a younger age (<5 years old) continued to be associated with superior survival supporting the provision of hct earlier in the course of the disease. further analysis of the complete cohort is planned to determine whether age at hct has decreased in the modern era compared to pre-2005 and to analyze factors associated with platelet and immune reconstitution, donor chimerism and autoimmunity. introduction/background: comel-netherton syndrome is a rare disease hallmarked by congenital ichthyosis, atopy, trichorrhexis invaginata (bamboo hair) and, within the past 10 years, has been defined as a primary immunodeficiency. specific, rare genetic polymorphisms (c.230t>a, pl66h) in the fc receptor iiia (fcgr3a) on natural killer (nk) cells have been shown to decrease nk cell function. patients with these defects present with susceptibility to severe and recurrent viral infections, however, not all fcgr3a mutations result in this clinical phenotype. here we report on a child with a mixed genotype, presenting with congenital ichthyosis, atopy and recurrent bacterial and viral infections. methods: immunophenotyping of lymphocyte subpopulations were evaluated by flow cytometry. genomic dna was sequenced using next generation sequencing. all detected variants were then sequenced using sanger sequencing. cd16 dual epitope assay and evaluation of nk cell maturation was also performed. intravenous immunoglobulin replacement therapy was initiated. results: our patient presented at 9 months of age for evaluation of food allergy. she has congenital ichthyotic erythroderma with pruritic atopic dermatitis-like skin eruptions and a history of hypernatremic dehydration immediately following birth, thin and easily broken hair and a history of failure to gain weight with appropriate catch-up growth following formula fortification. she has had multiple episodes of acute otitis media and recurrent upper respiratory tract infections associated with wheezing. t-, b-and nk cell quantitation by flow cytometry was normal, including naïve and memory b cells. immunoglobulins and vaccine antibody levels to haemophilus influenza type b, tetanus and streptococcus pneumoniae were normal. lymphocyte proliferation to mitogens was normal. natural nk cell cytotoxicity was initially decreased, however, subsequent cytotoxicity testing performed at 13 months of age was normal. whole exome sequencing revealed a homozygous mutation in spink5 (c.795-11a>g) and a homozygous missense mutation in the first immunoglobulin domain of fcgr3a (c.305t>a), both variants of unknown significance and under additional investigation. this homozygous mutation in spink5 was not detected in the patients healthy, unaffected sibling. conclusions: this is a case of an infant with a clinical phenotype consistent with comel-netherton syndrome, however genotyping has revealed homozygous mutations in spink5 and fcgr3a, suggestive of a possible mixed genotype. a recent large study investigating the use of whole exome sequencing to identify variants implicated in primary immunodeficiency found that in 11% of families, more than 1 gene contributed to the immunodeficiency phenotype. it should therefore be kept in mind that variability in an individuals clinical phenotype may be attributable to the presence of a mixed genotype. introduction/background: cartilage-hair hypoplasia (chh) is a rare autosomal recessive disease caused by mutations in the rmrp gene and can manifest with scid. allogeneic hct is a curative therapeutic option for scid associated with chh. bordon et al. reported an overall survival of 62% (10/16 patients) following hct with a predominantly myeloablative conditioning regimen with busulfan and cyclophosphamide. data on outcomes of hct using a ric regimen are limited. objectives: we herein report our experience with allogeneic ric hct for scid associated with chh. methods: we reviewed records of all patients who underwent allogeneic hct for scid associated with chh at our institution, with a ric regimen containing alemtuzumab, fludarabine and melphalan. results: five patients (3 male, 2 female) underwent allogeneic ric hct for chh at median age of 8 months (range, 4 months 4 years). all patients had biallelic mutations in the rmrp gene, and met pidtc criteria for scid, prior to hct. two patients were diagnosed by newborn screening for scid. one patient received serotherapy only (rituximab 375 mg/m2 daily x 3 days, anti-thymocyte globulin 1.5 mg/kg daily x 4 days) conditioning for initial hct but developed graft failure and subsequently received a ric regimen for second hct. all patients received a ric regimen consisting of alemtuzumab 1 mg/kg over 5 days(n=4) or 3mg/kg over 4 days (n=1) and fludarabine 5 mg/kg (weight <10 kg, n=4) or 150 mg/m2 over 5 days (n=1), and single dose of melphalan 4.7 mg/ kg(weight <10 kg, n=4) or 70 mg/2(n=1, dose reduced by 50% for preexisting sclerosing cholangitis with grade 3 liver fibrosis). patients received matched (n=3) or 1-2 allele mismatched (n=2) bone marrow grafts and all but 2 grafts were from unrelated donors. all patients received cyclosporine and steroids for gvh prophylaxis. all patients engrafted with full donor chimerism. three patients developed mixed chimerism, but continue to maintain donor t cell chimerism > 90%. two patients developed vod of the liver (one patient developed mild vod whereas the patient with pre-existing sclerosing cholangitis developed severe vod). one patient developed grade 2 skin gvhd and one patient developed limited chronic skin gvhd. none of the patients developed liver or gi-gvhd. all patients remain alive at a median follow up of 4 years (range 11 months-11 years) with good t-cell and b-cell immune reconstitution. conclusions: our experience suggests that allogeneic ric hct with alemtuzumab, fludarabine and melphalan for scid associated with chh is curative, offers durable t-cell engraftment, low gvhd along with excellent survival and might be preferable over a myeloablative conditioning regimen, to further limit toxicity in young infants, especially in the era of newborn screening. refractory thrombocytopenia in a patient with wiskott-aldrich syndrome despite hematopoietic stem cell transplantation: eltrombopag as a therapeutic option. mauricio chaparro-alzogaray 1 , marcela estupiñan-peñaloza 1 , gisela barros-garcía 2 , oscar correa-jimenez 3 1 pediatric hematologist/oncologist, hsct unit, fundación homi hospital de la misericordia 2 pediatric hematologist/oncologist, fundación homi hospital de la misericordia 3 pediatrics resident, universidad nacional de colombia introduction/background: wiskott-aldrich syndrome (was) is an xlinked disorder characterized by: immunodeficiency, eczema and hemorrhage due to thrombocytopenia [1] . hematopoietic stem cell transplantation is the treatment of choice, with an overall survival of approximately 90% regardless of the source of the stem cells [2] . eltrombopag has been used as a pre-transplant stabilization therapy [3] . in our knowledge, there are no reports of its use for the management of post-transplant autoimmune cytopenias in was. objectives: to present a clinical case in which the complexity of diagnosis and management of wiskott-aldrich syndrome is highlighted, as well as the usefulness of eltrombopag in post-transplant persistent thrombocytopenia. methods: clinical case presentation and review of literature. results: clinical case: 9-month-old infant with a history of thrombocytopenia identified at the third day of life (positive serology and viral load for cmv), bacteremia due to serratia marcescens at 4 months, intermittent diarrhea from 4 months, multiple platelet transfusions, ivig cycles, and ambulatory management with corticosteroids; who consulted the er for a 5-day of bloody diarrheic stools, generalized petechiae and fever. he was irritable with generalized petechiae in the lower extremities, diaper area dermatitis with signs of superinfection. admission cbc: wbc: 20180/mm3, neutrophils 330/mm3, lymphocytes 17250/mm3, monocytes 2360/mm3, hb 11.2 g/dl, ht 34.1% mcv 80.2fl, mch 26.3 g/dl, mcmh 32.8%, platelets 15000/mm3 mpv 8 fl. bone marrow aspiration was performed that ruled out proliferative syndrome. immunological profile with normal immunoglobulins and flow cytometry showed t lymphocytosis with cd4/ cd8 inversed ratio, direct coombs was positive. he progressed to refractory thrombocytopenia, with intracranial bleeding and transfusion platelet requirement every 12h. molecular diagnosis of wiskott-aldrich syndrome was made and it was decided to carry out an allogeneic transplant of unrelated umbilical cord. he showed adequate response, 100% chimerism; however, he persisted with important cytopenias, without response to management with ivig and corticosteroids, so that on day +68 he restarted eltrombopag that he received prior to transplantation. from day +97 he received rituximab for 6 weeks. he continued with eltrombopag 10 months post-transplant. currently without cytopenia and without complications, he completed the post-transplant year without additional complications. conclusions: was represents a great diagnostic challenge in pediatric clinical practice. despite the therapeutic option of transplantation of hematopoietic stem cells, patients may persist with different complications, within these; autoimmune cytopenias will require additional therapies. eltrombopag, in addition to being used as a pretransplant transient measure, is useful for the management of post-transplant persistent thrombocytopenia in these cases. references: 1. immunol allergy clin north am. 2010; 30 (2) methods: retrospective study of medical records in two centers of immunology results: in our center we have 1205 p with pid, with made retrospective study of medical records, up today we have registered 1155p (96%). according to this record, these diseases are distributed in the following way: predominantly antibody disorders: 890p (77%), predominantly t cell deficiencies:88p (7,6%), phagocytic disorders:41p (3.5%), complement deficiencies:26p(2.2), other well pid:53p(4.5), autoimmune and immune dysregulation syndromes:8p(0.6%), unclassified immunodeficien cies:57p(4.9%). predominantly antibody deficiencies are the most common pid, which comprise more than half of our all p. these group is represented by: specific iga deficiency (sad):332p, specific igg deficiency:164p, transient hipogammaglobulinemia:206p, common variable immunodeficiency (cvid):96p, agammaglobulinemia linked x:33p, agammaglobulinemia unknown causes:8p, secondary hipogamma globulinemia:23p, selective igm deficiency:1p, subclasses deficiency:10p, cd40l deficiency:7p, hyper igm unknown causes:32p, other hyogammaglobulinemia:3p. among them, selective iga is the most common pid.in our cohort of unclassified immunodeficiency, we have 25p with auto inflammatory syndrome, such as family mediterranean fever, hyper igd syndrome and candle like-syndrome and 6 p with nk deficiency. in all our pid 244p, are under replacement gammaglobulin (gg)treatment, 171p use intravenous gg and 71p use subcutaneous gg conclusions: the lasid registry model represents a powerful tool to improve health policies, showing that are under diagnosed and should receive more attention. more data are needed to define the exact prevalence of pid to avoid underestimation of these diseases due to under reporting. as different reports in different countries, in our centers predominantly antibody deficiencies are the most prevalent. although the number of patient diagnosed with pid, is growing. many physicians still know little about these disorders. introduction/background: a number of anticonvulsant medications have been shown to cause hypogammaglobulinemia. lamotrigine is a phenyltriazine anticonvulsant medication that is approved to treat seizure disorders and bipolar disorder. objectives: we describe a patient who developed hypogammaglobulinemia secondary to lamotrigine use. methods: we performed a chart review and case-based literature review. results: a-27-year old female presented to immunology clinic for evaluation of panhypogammglobulinemia. she was initially evaluated by general internal medicine for lightheadedness and fatigue and was found to have serum igg of 382 mg/dl (767-1590 mg/dl), igm 26 mg/dl (37-286 mg/dl) and iga 33 mg/dl (61-356 mg/dl). the patient reported a history of recurrent sinus infections occurring around twice per year. she reported resolution with oral antibiotics. she denied any history of pneumonia and was never hospitalized for treatment of infection. she did report increased number of recurrent upper respiratory infections; around 4-5 per year for the last several months. she denied any family history of primary immune deficiency. she was never prescribed corticosteroids or immuno-modulators. her past medical history was significant for bipolar disorder type 2 for which she was prescribed lamotrigine 100 mg oral twice daily seven months prior to her initial visit. the medication was prescribed prior to the onset of recurrent sinus infections. she had protective post-vaccination titers against tetanus, diphtheria, and acellular pertussis and non-protective pre-vaccination pneumococcal titers. post vaccination pneumococcal titers were not obtained due to the cost of the pneumonia vaccine. the patient was started on prophylactic azithromycin three times weekly. lamotrigine was discontinued and the patient switched to lurasidone due to concern for anticonvulsant-induced panhypogammaglobulinemia. her serum immunoglobulin levels increased after 2.5 months off of lamotrigine (igg 416 mg/dl, igm 46 mg/dl, iga 34 mg/dl), and she is currently asymptomatic without further infections. conclusions: lamotrigine is likely responsible for the reversible hypogammaglobulinemia in this patient. serial immunoglobulin levels should be checked in all patients who experience recurrent sinopulmonary infections while on lamotrigine. in two separate reports, lamotrigine induced hypogammaglobulinemia began within 7 months and 13 months of starting therapy respectively. in another study, hypogammoglubinemia was reported in 28% of 74 patients taking lamotrigine. further studies are needed to accurately describe onset and frequency of hypogammgeobulinemia in these patients. currently, it is unclear whether post vaccine titers are protective in patients with lamotrigine induced hypogammaglobulinemia. introduction/background: the association of vaccine strain rubella virus with cutaneous and sometimes visceral granulomatous disease has been reported previously in 19 patients with various primary immunodeficiency disorders (pids). the majority (14/19) of these pid patients with rubella positive granulomas had dna repair disorders, namely ataxia telangiectasia (at) (n=9) or nijmegen breakage syndrome (nbs) (n=3) or rag1 (n=1) and rag2 (n=1) deficiency. objectives: to support this line of inquiry, we provide additional descriptive data on the previously reported nbs patients as well as additional previously unreported patients with rubella virus induced cutaneous granulomas and dna repair disorders as well as additional previously unreported pid patients with rubella virus induced cutaneous granulomas. methods: we provide in-depth descriptive data on the previously reported nbs patients as well as 5 additional previously unreported patients with rubella virus induced cutaneous granulomas and dna repair disorders including at (n=4) and dna ligase 4 deficiency (n=1). we also provide in-depth descriptive data on 4 additional previously unreported pid patients with rubella virus induced cutaneous granulomas, including cartilage-hair hypoplasia (n=1), mhc class ii deficiency (n=1), whim syndrome (n=1), and coronin-1a deficiency (n=1). results: the median age of the patients is 10.5 years (range 3-33). the majority are females (83%). cutaneous granulomas have been documented in all cases while visceral granulomas (spleen and liver) were observed in 3 cases. t cell and b cell lymphopenia as well as hypogammaglobulinemia or impaired antibody formation were present in most patients. all patients had received rubella virus vaccine. the median age at presentation of cutaneous granulomas was 84 months (range 14-377). the median duration of time elapsed from vaccination to the development of cutaneous granulomas was 19 months (range 12-152). the diagnosis of rubella was made by pcr in 83% of patients and by immunohistochemistry in the remainder. one patient was confirmed to have vaccine strain rubella virus. hematopoietic cell transplantation was reported in three patients. rubella associated complications did not contribute to death among those patients who died (16%). conclusions: of the now 28 cases, 19 (67%) share the diagnosis of a dna repair disorder and confirm that chronic rubella virus infection is associated with cutaneous granuloma formation. analysis of patients with dna repair disorders and other pids with this complication will help clarify determinants of rubella pathogenesis, identify specific immune defects resulting in chronic infection and may lead to defect-specific therapies. introduction/background: introduction/background: hizentra® is a 20% liquid igg product approved for subcutaneous administration in adults and children greater than two years of age who have primary immunodeficiency disease (pidd). limited information on use of hizentra® is available for children who have received hematopoietic stem cell transplantation (hsct). objectives: objectives: the aims of this study are to determine the safety and efficacy of hizentra® in pediatric patients post hsct, and to characterize reasons for switch from intravenous igg (ivig) to subcutaneous (scig) delivery following hsct. methods: methods: a retrospective chart review involved 13 pidd infants and children (mean age 6.3 [range 3.1 to 14.5] months) status post hsct who received hizentra®. ten patients received hizentra® by pump administration, and 3 patients by manual push. results: results: hizentra® administered weekly to 13 children included an average of 144 infusions (range 14-416). the mean dose was 722 mg/kg/4 weeks. the mean igg level was 889 mg/dl while on hizentra® in 11 patients compared to a mean trough igg level of 563 mg/dl in 6 patients during immunoglobulin administration prior to hizentra® of most children. four patients naïve to igg therapy were started on hizentra®. average infusion time was 4.7 (range 3-15) minutes for manual entry and 71 (range 60-90) minutes for pump entry, and the average number of infusion sites was 1.9 (range 1 to 2). local reactions were mild and observed in 3/13 (23.1%) children. ten patients had no local reactions. no serious adverse events were reported. the rate of serious bacterial infections (sbi) was 0.12 per patient-year while receiving hizentra®, similar to reported efficacy studies. the reasons for switch from ivig to scig in 13 patients (some patients had multiple reasons) were improved igg serum levels and physician desire for steady state serum igg levels (n=6), loss/lack of venous access (n=2), patient/caregiver preference (n=7), home site of care preference (n=4), and physician preference for scig (n=5). conclusions: conclusions: hizentra® is a safe and effective option in children who have received hsct. reasons for switch from ivig to scig included improved serum igg levels, desire for steady state serum igg levels, and patient/caregiver preference. consulting medical advisor, immune deficiency foundation introduction/background: it is currently unknown how many persons with primary immunodeficiency (pi) receive the seasonal influenza vaccination, and what proportion becomes ill. additionally, the effect of immunoglobulin (ig) replacement therapy on the frequency of influenza diagnosis and severity of symptoms is not known. objectives: the current study sought to measure the prevalence of seasonal influenza vaccination and diagnosis among persons with pi, specifically those with antibody deficiencies: x-linked agammaglobulinemia (xla), common variable immunodeficiency (cvid) and hypogammaglobulinemia. methods: 11,533 email invitations were delivered to members of the immune deficiency foundation database requesting participation in an online survey regarding their zoster, influenza and varicella vaccination experiences. data from 1009 persons with xla (total n=41; children age<18; n=19; adults age>17, n=22; 100% male; 81% white non-hispanic), cvid (total n=824; children age<18, n=57; adults age>17, n=767; 79% female; 92% white non-hispanic), and hypogammaglobulinemia (total n=144; children age<18, n=23; adults age>17, n=121; 77% female; 92% white non-hispanic) were analyzed for the 2016-2017 influenza season. results: overall, 74% (n=749) of the sample received a seasonal influenza vaccination during the 2016-2017 influenza season. persons with xla were less likely to receive an influenza vaccination (54%, n=22), than persons with cvid (76%, n=628) and hypogammaglobulinemia (69%, n=99). a fifth of respondents (21% overall, 34% of children and 19% of adults) who attend school and/or work stayed home at some time to avoid seasonal influenza. though the majority (81%, n=821) were not diagnosed with influenza, when stratified by age, children were twice as likely to be diagnosed than adults. of the 99 children sampled, 30% (n=30) were diagnosed with seasonal flu compared to 15% (n=139) of the 910 adults that were sampled. the role of replacement ig therapy in protection against flu can best be examined in the 41 patients with xla since these patients cannot make specific antibodies to vaccine and only 54% (n=22) received the vaccine. although 59% (n=24) of the individuals with xla were exposed to flu, only 10% (n=4) were diagnosed with influenza; two of whom were not receiving ig therapy at time of diagnosis. concerning influenza severity, individuals receiving ig therapy at the time of flu diagnosis tended to have modestly milder symptoms than those not receiving ig treatment (e.g., less likely to report sore throat (78% versus 92%, p<.05)), but the sample size is too low to draw firm conclusions. conclusions: a high proportion of antibody deficient persons received a seasonal influenza vaccination for the 2016-2017 influenza season. in addition to vaccination, many individuals attempted to avoid influenza infection by remaining home from school and/or work. there was a suggestion that ig replacement therapy may partially protect xla patients from symptomatic flu, although the role of cell mediated immunity in protection against flu is not clear. a prospective study could determine if ig replacement therapy partially protects against clinical flu symptoms in xla and cvid patients using two groups those that receive the current flu vaccine vs. those that do not receive the flu vaccine. introduction/background: fanconi anemia (fa) is an autosomal recesive or x-linked genetic disorder, characterized by cytopenia or bone marrow failure, this will lead to a severe anemia, neutropenia and thrombocytopenia, requiring frequent interventions with diversified therapies, including hematopoietic stem cell transplant (hsct). a complete immune reconstitution is requiere for a success transplant. one of the main upcoming events asociated after the hsct, is the secundary immunodeficiency, which is asociated with a significant morbility and mortality in the patients. to rich a successful alogenenic-hsct, is impending a complete reconstitution of the t cells immunity, for this it is crucial the presence of factors like; thymic activity of the hsct recipient, biological features of the allograft (eg, degree of histocompatibility, number and type of infused donor t cells) and preparative regimens. objectives: describe the kinetics of the immune reconstitution in fa patients after alogenic-hsct, as well as the infections associated during this process and other comorbilities. methods: we decribed the lympocyte population (t, b and nk) in pb measured by flow cytometry in fa patients on days +90, +120, +150, +180, +210 and +360 post alogenic hsct from a match related donor. the conditioning was base on fludarabine 150mg/m2, cyclophosphamide 10mg/kg and antithymocyte globulin rabbit 20mg/kg. infectious-disease survillance for virus was determined by the quantification by dna pcr-rt for cmv, ebv, adenovirus, as well as the presence of galactomannan and candida sp antibodies, or isolation of fungus or bacterial culturing in the cases of infectious disease of known or uknown aetiology. results: the lymphocyte population mesurement was performed in a total of five fa patients who undergo to an allogenic hsct, all of them received stem cells from a match related donor and the source was bone marrow. successful engraftment was observed in all 5 patients, there were no deaths reported. after the hsct was performed, the kinetics of recovering for the distinct lymphocytes subsets was the the following: nk cells (cd16+cd56+) were the first to recover, followed by cd+8 t cells, b lymphocyte and finally cd+4 t cells (figure 1 ) all of them rich normal values and remain stable. three out of five patients presented infectious disease: two of them were cmv positive, one patient has a concurrent detection of adenovirus, and in the other was detected aspergillus, in both of them it was presented at day +90. the third one developed acute gvhd which progressed to a chronic gvhd, at day +153 was diagnosed on him listeria monocytogenes meningitis, at that momento he has just cd+16 +cd56 and cd8+ reconstituded. there is no new infectous disease detected in any of the patients after they reconstituted cd4+ t cells. conclusions: complete immune reconstitution is the decisive for the presence of several morbilities and mortalities, mainly because of oportunistic infecctions and gvhd. we show that the kinetics of recovery of the different populations of lymphocytes follows those patterns also described for patients with other hematological malignancies: early recovery of nk cells, followed by effector cytotoxic t cells and b cells, and finally, cd4+ t-helper cells. the utility of post-transplant monitoring of pb-lymphocyte subsets for improved follow-up of patients undergoing bmt and prevent opportunistic infections. introduction/background: early detection of primary immunodeficiency diseases (pids) before serious infection is of utmost importance and a question of patient survival. one of the main organs involved in pids is skin with mucocutaneous manifestations being one of the common complaints in pids. the presenting skin symptoms may serve as an essential element in early diagnosis of pids. objectives: this study aims to determine characteristics, frequency, nature and incidence of skin manifestation in pid patients seen in qatar. methods: this retrospective study was conducted at hamad medical corporation the only tertiary hospital in qatar from january 2009 to july 2017.the subjects included were pid patients < 14 years old, who had dermatological complaints. information collected included dermatological diagnosis, gender, age at onset of signs and symptoms, age at definite diagnosis, family history of related disease, any lab results such as pathology or viral studies obtained from the clinical medical record. patients were diagnosed and classified according to clinical and laboratory criteria by the international union of immunological societies primary immunodeficiency committee. results: a total of 109 patients were studied and skin/mucocutaneous manifestations were found in 65 patients (59%) with male to female ratio of 1.5:1. age at onset of skin manifestations ranged from 0 to 12 years. skin manifestations were divided in two categories according to presentation before pid diagnosis (primary manifestations n=32, 47%) and during the disease period (secondary manifestations n=35, 53%). the type of pids predominantly having primary manifestation were scid (n=5, 15%) cvid and cgd (n=4, 12%, each), xla, at and griscelli syndrome (n=3, 9%, each).various manifestations which were atypical in their presentation and persistently found in patients who did not respond to any effective treatment (n=13) led to the basis of clinical immunological study and diagnosis including rare diagnosis such as ipex. whereas secondary manifestations were primarily reported in scid (n=9, 25%) cgd, at and digeorge syndrome (n=5, 14%, each). the nature of skin manifestations varied in both groups, primary manifestations notably had 41% cutaneous infections comprising of thrush (30%) stomatitis (17%) viral rashes (26%) bacterial rashes (13%) impetigo (8%) and fungal rash (4%). eczema/atopic dermatitis were 31% and other minor miscellaneous skin alteration cases found were apthous mouth ulcer (5) erythroderma (4) gvhd like rash (1) alopecia (1) psoriasis (1) and scleroderma (1) . secondary manifestations were identified as 53% cutaneous infections 20% eczema and infantile seborrheic dermatitis (1) pruritus (1) gvhd and alopecia (1) each. the causative microorganisms were confirmed in 44% of the cases via lab cultures whereas common infections such as chicken pox and herpes were validated through clinical symptoms. none of the cases were biopsied. overall, highest skin infection was seen in ataxia telangiectasia (11). other pids with prominent cutaneous manifestations included cgd (9), digeorge syndrome (5), hyper ige syndrome (4) and scid (rag 1 or 2, 4 cases). more than two types of skin manifestations were found in 47% patients over the due course of their illness. conclusions: an awareness of various cutaneous and skin disorders associated with pid which are persistent and unresponsive to treatments among dermatologist and family physicians is crucial to raise suspicion for early detection, timely management and prevention of complications. introduction/background: ataxic telangiectasia (at) is a rare neurodegenerative autosomal recessive disease associated with immunodeficiency, poor coordination and disability. ataxia telangiectasia has a diverse clinical heterogeneity which may often lead to an incorrect diagnosis or late detection of the disorder resulting in exposing the patient to unnecessary radiation from various sources. objectives: we present a female child from the asian subcontinent that was seen for the first time with ulcerative skin lesions, failure to thrive and marked lymphopenia. methods: a search of the pubmed database was carried out, using different combination of the terms "ataxia", "telangiectasia", typical , atypical and "presentation" results: four-year-old pakistani girl, product of first degree nuptial presented with generalized vesicular rashes mainly on abdomen and scalp which turned into ulcers with residual hypo pigmented lesions and diarrhea. she had short stature, failure to thrive (weight and height < 5th percentile) and no previous infection or family history of primary immunodeficiency diseases (pid). mild delay in milestones was observed especially in speech. immunizations were up-to-date including bcg. on examination she had mild ocular telangiectasia but no cutaneous involvement. investigations revealed lymphopenia (1.6) and eosinophilia. workup for diarrhea including duodenoscopy revealing subtotal villous atrophy however the biomarkers for celiac disease were negative. immune system workup showed high igg levels, normal iga, ige and igm level. igg subclasses: low igg 2, and 3, antibody titers to h.influenza and pneumococcus vaccine were relatively low. lymphocyte subsets showed low cd4,cd19 and high nk cells (40%), low naïve t cells (2%) and inverted cd4/cd8 ratio.t cell function with post pha was 18.7% but had normal response to cd3. alfa feto protein was requested due to continued low cd4 count and the level was found to be high: 260.9 iu/ml. next-generation sequencing (ngs) showed homozygous mutation for trp1750fs in chromosome 11 of atm gene confirmed by whole exome sequencing. immediate treatment with ivig was started leading to mark improvement of skin lesions, diarrhea and weight gain. conclusions: the index of suspicion of at should be highlighted when deciphering lymphocyte subset. currently there is no neonatal screening for pid diseases and next generation sequences in qatar. once implemented it may prove beneficial in discovering cases from early infancy and reaching upon a definite diagnosis. introduction/background: chronic granulomatous disease (cgd) is a genetic disorder of the nadph oxidase complex in phagocytes which results in impaired production of microbicidal reactive oxygen species that can lead to recurrent life-threatening bacterial and fungal infections. the majority of affected patients in the united states have a x-linked defect in the cybb that encodes gp91phox protein followed by an autosomal recessive defect in the ncf1 gene that encodes p47phox . x-linked female carriers show 2 populations of neutrophils and following lyonization, and severe skewing of x-chromosome inactivation can get cgd type infections. a recent study has shown that the lower dihydrorhodamine (dhr) percent in female carriers predicts a higher infection risk but carrier state on its own predicts autoimmunity results: a 39-year-old female presented for an evaluation of recurrent fevers. she had a history of several lobar pneumonias in childhood. at age 32, she developed erythematous bumps on her abdomen that were initially non-bothersome but later became painful. she then developed recurrent fevers, chills, and rapid weight gain. biopsy of the skin lesions showed subcutaneous panniculitis-like t cell lymphoma. she was treated with chemotherapy and found to be in complete remission. she had no previous family history of recurrent infections. at the completion of her chemotherapy, her 1-year-old son became septic in the hospital and was then diagnosed with cgd from mutated cybb gene. post chemotherapy, the patient had a prolonged course with an atypical lung infection that required bronchoscopy and video thorascopic surgery, however no bacteria was ever identified. she did improve after a long course of ivantifungals. evaluation for immunodeficiency was done after she had a recurrence of low grade fevers, cough, and fatigue. dhr flow cytometry with phorbol myristate acetate (pma) was done which showed 3.6% neutrophil oxidative burst activity after stimulation, consistent with a highly skewed lyonization pattern in x-linked cgd. conclusions: this case demonstrates an interesting lesson in a woman with recurrent infections since childhood whose clinical picture was confounded by her history of malignancy. the abnormal lung infections prior to malignancy were alarming and gave cause to do additional immunology testing. however, it begs the questions, if she did not have a son, would she have been diagnosed with severe x-lined carrier disease. introduction/background: measurement of the specific antibody response following vaccine challenge provides clinicians with a better understanding of the adaptive immune response in individuals undergoing immunological evaluation. objectives: we hypothesised that after classification of primary immunodeficiency (pid) patients based on vaccine response (vr), further division using igg subclass (iggsc) 1-3 measurements may identify additional patients with abnormal b cell function and patients with different frequencies of infection at presentation. methods: vrs and serum iggsc concentrations were quantified using the vacczyme anti-pneumococcal polysaccharide (pcp) igg elisa and human iggsc liquid reagent kits (the binding site group limited, uk) in 23 pid patients (1:1.3 m:f, median age 41.5 years, range 20-78). all patients were immunised with pneumovax®23 (sanofi pasteur msd). the lower limits of published normal iggsc ranges were used as cut-off values (igg1 3.2 g/l, igg2 1.2g/l and igg3 0.2g/l). pcp concentrations equal to or less than 70 mg/l post-vaccination was considered an abnormal response. results: agreement between vr and iggsc measurements was 48% (p=1.00). the frequency of respiratory tract infections at presentation among pid patients with a normal pcp igg vr was 45% and 55% among patients with an abnormal vr. subsequently, four separate groups could be identified by including iggsc measurements. the frequencies of infections at presentation were for the vr+/iggsc+ group (n=4) 36% vs. 48% for the vr+/iggsc-group (n=10); and 33% for the vr-/iggsc+ group (n=2) vs. 63% for vr-/iggsc-individuals (n=7). conclusions: these results confirm that vr and iggsc measurements are independent serum biomarkers of humoral immunity. taken together the vr and iggsc results provide more detailed information about the immune status that may influence diagnosis, treatment and monitoring decisions. introduction/background: hizentra® is a 20% liquid igg licensed for subcutaneous administration in adults and children greater than two years of age who have primary immunodeficiency disease (pidd). subcutaneous immunoglobulin (scig) use in autoimmune conditions is reported. stiff person syndrome (sps), a rare neurologic disorder characterized by fluctuating muscle spasms and rigidity, is mediated by autoantibodies to glutamic acid decarboxylase (gad). symptoms of sps have been shown to improve after administration of intravenous immunoglobulin (ivig) however, there is a paucity of information regarding use of scig in sps. objectives: the aim of this study is to describe the use of hizentra® in patients with sps, including indications for scig, clinical characteristics of patients, and clinical and laboratory response to scig. methods: a multicenter retrospective chart review examined 2 patients with stiff person syndrome treated with hizentra®. results: sps was diagnosed in 2 patients at 12 and 25 years. both patients were started on ivig, steroids, and rituximab prior to initiation of weekly hizentra® (ages 15 and 27 years). the average dose of hizentra was 125mg/ kg weekly. the average igg level while receiving ivig was 1,164.5 mg/dl, similar to the average igg level while on hizentra was 1,250 mg/dl. the number of administration sites ranged from 2-4, and duration of infusions ranged from 60-120 minutes. serum anti-gad antibody levels prior to hizentra® were 845 u/ml and 134 u/ml respectively. anti-gad antibody level during treatment with hizentra (available for one patient) was >5,000 u/ml, and 132,240 u/ml following discontinuation of hizentra®. one of the reasons for switching to hizentra® was lack of response while on ivig. the average number of hizentra infusions were 138. patients reported improvement in spasticity related to sps while on hizentra®, and one patient had improvement in seizures. one patient discontinued hizentra® in favor of intravenous immunoglobulin (ivig) due to physician preference. the most common side effects were local reactions including pain, pruritus, and redness. no serious adverse events were reported. conclusions: hizentra® was associated with improved symptoms in sps in both patients including decreased spasticity, and improved seizure frequency in one patient. serum anti-gad levels did not decrease following administration of hizentra. hizentra® was well tolerated in patients with sps, with most side effects reported as mild. hizentra® may be considered as an alternative to ivig treatment in patients with sps. introduction/background: combined immunodeficiency (cid) has been associated with a spectrum of secondary gastrointestinal manifestations, including infectious and non-infectious causes. abatacept, a soluble ctla4-igg1 fusion protein targeting t cell activation, has demonstrated benefit in the treatment of autoinflammatory manifestations in patients with cid due to underlying heterozygous germline mutations in ctla4 and lrba. objectives: herein, we report two patients with cid characterized by low immunoglobulin levels, low class-switched memory b cell counts, and low peripheral naïve cd4+ t cell counts. both patients suffered from severe and persistent diarrhea complicated by protein-wasting and malnutrition, ultimately diagnosed as biopsy-consistent autoimmune enteropathy. the clinical history of patient 1 was also notable for granulomatous lung disease and autoimmune cytopenias. methods: a t-regulatory cell disorder was considered in both cases, however, ctla4 and lrba were found to be unaffected by whole exome sequencing (patient 1) and/or flow cytometry (patient 2). due to progressive worsening of the autoimmune enteropathy despite management with chronic steroids (both patients), combination rituximab/mycophenolate mofetil (patient 1), and total parenteral nutrition (tpn) complicated by recurrent infections (patient 2), abatacept was trialed at 125 mg sc weekly. results: in both patients, the start of abatacept therapy produced a dramatic improvement as measured by decreased stool frequency, improved weight gain, and decreased protein-wasting. patient 1 has been maintained on this monotherapy for over one year, with improvement in the gastrointestinal as well as pulmonary autoinflammatory complications. the only documented adverse event has been hepatitis b reactivation, managed with tenofovir and abatacept continuation. patient 2 has required azathioprine/abatacept combination therapy for clinical stabilization and is without a significant adverse event to date. conclusions: we conclude that abatacept may be a relatively safe and effective therapeutic in the management of severe autoimmune enteropathy in the background of cid, even when used outside of the classical clinical context of ctla4 or lrba haploinsufficiency. objectives: as increasing the outbreak of allergic diseases, study for treatments comes to the force. in this research, we aimed to assess the effects of sg-sp1, a derivative of gallic acid, on mast cell-mediated allergic inflammation using various animal and in vitro models. methods: ovalbumin-induced systemic anaphylaxis and immunoglobulin e (ige)-induced passive cutaneous anaphylaxis are standard animal models for immediate-type hypersensitivity. oral administration of sg-sp1 hindered the allergic symptoms in both animal models. these inhibitions were deeply related to the reductions of histamine and interleukin-4. results: sg-sp1 reduced degranulation of mast cells and expression of inflammatory cytokines in a dose dependent manner. sg-sp1 showed better anti-allergic effects compared to gallic acid and dexamethasone. down regulations of intracellular calcium level and nuclear factor-b activation by sg-sp1 were causative of the reduction of allergic mediators. to anticipate the exact target of sg-sp1, phosphorylation of proteins involved in mast cell signalling was assessed. sg-sp1 suppressed the activations from lyn and was aggregated with high affinity ige receptor (fcri). conclusions: from these results, we assured that sg-sp1 directly interact with fcri. all together, we propose that sg-sp1 might be a therapeutic candidate for allergic disorders. (162) submission id#425361 systematic assessment of pain in patients with primary immune deficiency using validated pain questionnaires: a prospective study. introduction/background: the number of primary immunodeficiency (pid) patients is rising dramatically because of good medical care as well as increased awareness. around 1 in 1200 live births are affected. more than 300 disorders have been discovered so far and this number is expected to rise in the coming years. as with any other chronic illness, pid patients are also prone to acute and chronic pains. chronic pain is a big challenge and lack of understanding of the etiology and underlying mechanisms limit our ability to diagnose or treat it effectively. objectives: to systematically assess chronic pain in patients with pid using validated questionnaires and to try to understand the underlying mechanisms of neuropathic versus non neuropathic pain. methods: short-form mcgill pain questionnaire (sf-mpq) is a recognized way to ascertain different pain characteristics as well as severity. a validated arabic version of the sf-mpq was used to prospectively assess chronic pain in patients with pid. furthermore, a validated arabic version of the neuropathic pain questionnaire-short form (npq-sf) was also used to assess neuropathic pain and to differentiate it from nonneuropathic pain. a total of 27 patients with pid were included. results: males: females were 33.3%: 66.6% respectively. mean age was 29.73 years. commonest diagnosis was combined immune deficiency in 37% of the patients followed by common variable immune deficiency which was 25.9 %. chronic pain was found in 64% of the patients that participated in the study. 50% of the patients who complained of pain found it to be tiring and exhausting. 35% each had aching or heavy or sharp pain. 28% had cramping pain, 25% had tender pain and 21% characterized their pain as throbbing or burning. 18% felt shooting pain, 14% had splitting pain and 7% had gnawing pain. 28% found it to be frightening and 17% described it as being sickening in itself. the commonest pain complaint was abdominal pain in 14% of the patients followed by headache 11 % and chest pain 11%. pain attributed to neuropathy was present in about 17.8% of the study population. most patients described that they had been experiencing pain for at least 2-3 years. conclusions: this is the first international study to understand the prevalence, duration and severity of chronic pain among pid patients. a significant number of patients reported ongoing pain. this is the first time any kind of pain has been studied systematically in the patients with primary immune deficiency. treating pain should have a major impact on improving the patients quality of life. introduction/background: primary immunodeficiency (pidd) patients, particularly those with severe t cell defects, are at increased risk of infections. bone marrow transplant also contributes to significant t cell lymphocytopenia, which can be associated with similar risks. several prophylactic measures, which vary per institution, are placed on these patients in order to prevent infections. we report on a likely outbreak of rapid growing nontuberculous mycobacteria (ntm) at our institution, with a suspected association to tap water objectives: to recognize nontuberculous mycobacteria as a threat to patients with immunodeficiency disorders methods: case series of consecutive patients admitted to one institution with similar infections results: in a period of 16 months, 4/2016 to 8/2017, 5 patients admitted to our institution were found to have rapid growing ntm species on central line culture or bronchoalveolar fluid. these cultures were obtained due to fever and symptoms of systemic infection. per institutional practices no peripheral blood samples were obtained at the time of initial culture. all had significant t cell dysfunction: wiskott-aldrich syndrome (1), an undefined combined immunodeficiency (1) , and three patients post bone marrow transplantation (bmt), one 6 months (173 days) post an unrelated cord blood transplant for farber syndrome, one 120 days out of a matched related transplant for epstein barr virus (ebv)-associated lymphoproliferative disorder, and one patient with high risk neuroblastoma 106 days out of his second tandem autologous transplant. both allogeneic transplant recipients had received serotherapy. all species were rapid growing, identified as mycobacterium mucogenicum (n=3), immunogenum (n=1), or abscessus-chelonae complex (n=1). the patients had not shared rooms, caregivers, invasive procedures, or medications from the same batch. three (60%) cases met cdc criteria for hospital acquired infections (hai). all patients were in general ward rooms, and were on standard precautions given diagnosis had not been established (n=2) or they were considered outside the typical window of strict bmt precautions (n=3). ntm species were subsequently isolated from hospital tap water. this has resulted in a significant increase in infection precautions, including the use of sterile water only for cares, as well as bottled water for drinking (with no use of ice machines), for all patients being evaluated for pidd or with significant lymphocytopenia. conclusions: ntm are a threat to patients with pidd. tap water is a potential source of mycobacterial infections in pidd patients. minimizing exposure risk to water sources containing ntm is very important in this population. patients with concern for pidd or significant t cell lymphocytopenia should take steps to avoid ntm exposures, including the use of sterile water for cares, and bottled water for drinking. introduction/background: purine nucleoside phosphorylase (pnp) deficiency is a rare autosomal recessive condition leading to severe combined immunodeficiency and neurologic impairment, typically presenting in early childhood. the condition is progressive and typically fatal in the first or second decade of life without hematopoietic stem cell transplantation (hsct). objectives: to illustrate the clinical and laboratory presentation of pnp deficiency with a novel mutation in the pnp gene via a case study. results: case: a four year old female of hispanic descent, with a past medical history of spastic diplegia, initially presented with chronic nasal congestion, recurrent sinusitis, and cough. laboratory studies were significant for an alc 500 cells/mcl (2000-8000 cells/mcl). she was lost to follow-up for two years, before returning to medical attention for pneumonia. evaluation of lymphocyte subsets at age 6 years revealed cd3+ 145 cells/mcl (1200-2600 cells/mcl), cd4+ 119 cells/mcl (650-1500 cells/ mcl), cd8+ 28 cells/mcl (370-1100 cells/mcl), and cd19+ 44 cells/mcl (270-860 cells/mcl). t cell mitogen stimulation to pha measured by flow cytometry was 30% of control. pnp activity was nearly absent and urine guanosine and inosine were significantly elevated. gene sequencing revealed a homozygous c.655a>g mutation in the pnp gene. prophylactic antimicrobials were started. despite strong recommendation for hsct, the patient was again lost to follow up until age 10 years, at which time she was found to have progressive lymphopenia, bronchiectasis, and developmental delay. t cell mitogen stimulation to pha measured by thymidine uptake assay was 9% of control and pnp functional activity was undetectable. the patient underwent a 9/10 matched unrelated hsct six years after her initial presentation. one year post-transplant, the patient continues on immunoglobulin replacement therapy. her course was complicated by cutaneous gvhd, treated successfully with topical corticosteroids. conclusions: this case study illustrates the progressive nature of pnp deficiency in the first decade of life. our patient is notable in that she survived without significant medical intervention to the age of 10 years. her presentation at age 4 years was not unlike those previously reported in the literature, with muscle spasticity, ataxia, and recurring bacterial infections. to the authors knowledge, this case reports a novel mutation in the pnp gene. introduction/background: severe congenital neutropenia (scn) is a primary immunodeficiency disease characterized by early onset recurrent infections, persistent severe neutropenia and congenital genetic defect. severe idiopathic neutropenia (sin) is a rare disease defined by persistent severe neutropenia, in the absence of an identifiable etiology. objectives: here, we aim to find out clinical, laboratory, genetic characteristic and remission status in children with scn and sin in chinese population. methods: in this study, we enrolled 39 chinese children who experienced severe neutropenia longer than 6 months without any virus infection or auto-immune antibodies from june 2008 to july 2017 in hospitals affiliated to shanghai jiao tong university school of medicine. their clinical, laboratory and molecular characteristics were analyzed and the patients were followed up to observe their remission status. targeted gene capture combined with next-generation sequencing technology was used to find out related gene mutation. results: patients in this study had a mean age of 29.21±27.94 months. molecular analysis revealed that 7 patients had associated mutations of scn, including elane and g6pc3. among 26 patients with continuous follow-up, one died for unknown reason. ten patients have recovered from sin (r-sin) with mean neutropenia duration of 19.40±10.91 months. scn patients had more frequent infection (5.86±1.57 times per year) than sin (3.95±1.05 times per year, p=0.008) and r-sin patients (p=0.005, 3.89 ±1.27 times per year). scn patients had significantly higher count of anc and monocytes than sin (p=0.015) and r-sin patients (p=0.029). however, there was no difference in anc and monocytes counts between sin and r-sin patients. bone marrow examinations demonstrated a myeloid maturation arrest at the myelocyte-metamyelocyte stage in scn patients, while most of sin and r-sin patients were normal. conclusions: our study indicated that, patients with mild infection, lower anc, monocytes count and normal bone marrow are likely to be sin. whereas others with relatively more severe infection, higher anc, monocytes count and maturation arrest in bone marrow are inclined to be scn. introduction/background: patients with qualitative and/or quantitative defects of humoral immunity often require immunoglobulin (igg) replacement therapy (igrt). usual starting doses range between 400-600 mg/kg and the dose is adjusted as needed depending on the patient. there is a paucity of information about whether and how extreme bmi (obese or underweight) and route of administration may affect a patients rate of infections. objectives: the objective of the study is to determine whether rate of infection is associated with bmi, dose or route of administration in patients receiving igrt. methods: this is a retrospective chart review from december2000-october 2017. we included patients between the age of 1 month and 100 years old who were evaluated initially and had at least one follow up evaluation. data reviewed included the route of administration, dose, infection history, igg serum levels, height and weight to calculate bmi, gender, age and diagnosis requiring immunoglobulin replacement therapy. participants were excluded if the type of immunodeficiency is unknown or if the participant had incomplete data for the requested data fields. the number of infections between visits was modeled using poisson regression as a function of dose, route of administration and the bmi with the log of the follow up interval as an exposure offset. results: eighty-five patients were eligible, and preliminary results for 50 patients are presented here. the mean infection rate per 100 weeks of follow up was 1.9 in obese patients, 3.0 in overweight patients and 1.5 in underweight/normal weight patients adjusted for route of administration; however these rates do not differ significantly from one another. the mean infection rate per 100 weeks of follow up differed by administration route; 1.4 infections for ivig versus 3.1 infections for scig when adjusting for bmi (p<0.0131). the mean infection rate per 100 weeks of follow up was not associated with dosage. conclusions: overweight patients may experience more infections than obese or underweight patients, regardless of administration route. ivig patients may have a lower rate of infections compared to scig patients regardless of bmi. work is ongoing to complete analyses for the remainder of the eligible patient population. (167) submission id#421931 introduction/background: primary immunodeficiencies (pids) are a rare group of genetic disorders associated with a tendency to infectious diseases and an increased incidence of cancer. there is no data regarding the prevalence of malignancies in patients with pid in turkey. objectives: in this study, we aimed to evaluate patients who diagnosed as pids and developed malignancies, and calculate the estimated frequency of malignancies associated with pids in turkey. methods: forty five patients who were diagnosed with malignancy in the follow-up period of pid at the four tertiary immunology clinics between 1992 and 2017 years were included in the study. the data were obtained retrospectively from the hospital records and the database of esid online patient registry. results: the prevalence of malignancies in patients with pid was found as 1.05% (45/4285). the male to female ratio of the patients was 29/16, the median age was 13.5 years (minimum: 1.5, maximum: 57) and the median age at which patients get diagnosed with malignancy was 9 years (minimum: 1.5, maximum: 51) . there was no cancer history in their family members. the most common type of pid which associated with malignancy was ataxia telangiectasia (n=15, 33.3%). non-hodgkin lymphoma was the most common malignancy (n = 25, 55.5%) in our group (table 1) . ebv quantitative pcr was positive in 6 lymphoma cases (17.6%). the median number of x-rays and ct scans in patients with at and bloom syndrome before malignancy developed were 5 (minimum: 1, maximum: 24) and 1 (minimum: 0, maximum: 3), respectively. two cases had dual malignancies (papillary thyroid cancer and anal adenocancer). twenty cases were treated with chemotherapy, 7 cases with hematopoietic stem cell transplantation, 5 cases with radiotherapy, and 5 cases with surgical treatment, treatment information of 17 patients was not reached. remission was detected in 16 cases, while resistance to therapy in 2 cases and recurrences in two patients were observed. four patients are still on chemotherapy. twenty cases died. conclusions: the tendency of malignancy in patients with pids is due to the deficiency in the immune response that lead to failed surveillance against oncogenic viruses, premalignant/malignant cells, or both. lymphoid malignancies are the most common malignancies associated with pids. pids-associated malignancy incidence has increased in recent years because of that improved survival of the patients. this study is the largest cohort investigating the association of malignancy in patients with pid in turkey. additionally, we first reported tendency to malignancy in a patient with znf 341 deficiency. introduction/background: next-generation sequencing (ngs) has become an integral tool in the evaluation of primary immunodeficiency disorders (pid). we describe a patient with a previously described pathogenic foxp3 variant who met clinical and laboratory criteria for cvid. objectives: describe a patient with a previously described pathogenic foxp3 variant who met clinical and laboratory criteria for cvid. results: case description: an 8-year-old male born premature at 32 weeks presented with a history of recurrent infections. family history was negative for immunodeficiency. the patient developed recurrent acute otitis media beginning at 1 year of age, three episodes of pneumonia beginning at 3 years of age, and recurrent sinus infections requiring treatment on average four times a year beginning at 5 years of age. initial immunologic evaluation at age 8 was notable for: igg 305 mg/dl (reference 673-1734 mg/dl), igm 15 mg/dl (reference 47-311 mg/dl), iga 173 mg/dl (reference 41-368 mg/dl), ige 166 iu/ml (reference 0-90 iu/ml). lymphocyte subpopulations were normal. specific responses to vaccines showed: protective antibody titers to diphtheria, but not to tetanus or pneumococcal antigens. he did not respond to booster vaccination and was started on ivig with significantly reduced frequency of infections. at age 10, while on ivig, he developed oral ulcers (biopsy consistent with ulcerative eosinophilic granuloma), abdominal pain, and recurrent arthralgias involving ankles, elbows, hips and the sacroiliac joint. magnetic resonance imaging (mri) was consistent with sacroilitis. subsequent imaging was consistent with chronic relapsing osteomyelitis (crmo). gastrointestinal biopsies showed severe active chronic pangastritis with antralized oxyntic gastric mucosa with enterochromaffin cell hyperplasia; suggestive of autoimmune gastritis. plasma cells were present throughout the gastrointestinal tract. ngs (bcm-ngs, baylor miraca genetics laboratory, houston tx) identified a hemizygous pathogenic missense variant, c.1190g>a (p.r397q) in the x-linked foxp3 gene, that has been reported previously in patients with immunodysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome. flow cytometry studies showed a decreased percentage of treg cells of total cd4 expressing cells, 2.6% (reference 4.2-9.9%), but normal foxp3 expression within these cells, 68% of treg cells with foxp3 expression (reference 55-81%). interestingly, treg cell subset phenotyping obtained at the same time showed a normal percentage of natural tregs, 3.2% cd4 t cells (reference 0.7-4.0%), as well as normal percentage of naive tregs, 5.6% cd4 t cells (reference 0.6-9.0%). up to this point, he had not had any signs of diabetes, thyroid disease or frank enteropathy involving the small or large intestine. conclusions: we report a pathogenic foxp3 variant, occurring in a patient with a cvid-like phenotype, autoimmune gastritis, and an association with crmo. this case demonstrates the increasing utility of ngs, which can profoundly impact prognostic and therapeutic considerations. (169) submission id#413429 the natural history of patients with profound combined immunodeficiency (pcid): interims analysis of an international prospective multicenter study. immunodeficiency in this patient group. so far, 48 patients were transplanted after enrolment, overall 19 patients died (9 in the hsct group, 10 in the non-hsct group). analysis of the hsct decisions revealed the divergent decisions in patients with similar disease burden, favoring an ongoing prospective matched pair analysis of patients with similar disease severity with or without transplantation. so far, neither the genetic diagnosis nor simple measurements of t-cell immunity emerged as good predictors of disease evolution. conclusions: the p-cid study for the first time defines and characterizes a group of patients with non-scid t-cell deficiencies from a therapeutic perspective. since genetic and simple t-cell parameters provide limited guidance, prospective data from this study will be an important resource for guiding the difficult hsct decisions in p-cid patients. (170) submission id#419587 the plasma contact system and its role in common variable immunodeficiency: an explorative study. introduction/background: a growing body of evidence suggests that the contact system is involved in the activation of various vascular and immunological pathways and acts as an interface to help regulate allergic reactions, coagulation, complement, innate immunity and inflammation. as demonstrated in mice experiments, contact activation and high molecular weight kininogen (hk)-derived peptides increased homing of t and b lymphocytes into lymph nodes, which suggests an important area of research for understanding the contact systems role, specifically fxii, in immune-mediated inflammation and immune dysregulation. this novel mechanism prompted further inquiry into its role of various human disease states characterized by inflammation. plasma hk cleavage has been proposed as a useful and minimally invasive biomarker in various inflammatory disease states. this pathway has not been explored in cvid, in which inflammatory complications are found in one-third of patients with an unidentified genetic cause. characterizing the contact system biomarkers in cvid patients could elucidate a role in pathogenesis. objectives: assess the presence of contact activation at baseline in sera from cvid patients with and without inflammatory complications compared to healthy controls. methods: cvid patients were recruited in the outpatient setting and the measurement of cleaved plasma hk (chk) levels was determined by western blot analysis, under reducing conditions, with quantitation of total and chk bands using an odyssey imaging system (licor). a one-way anova test for differences among the 3 studied groups will be applied. c1 inhibitor levels, c3 and c4 levels and high-sensitivity crp were also measured as comparable biomarkers for inflammation. results: to date, 9 cvid patients were studied, 7 with and 2 without inflammatory complications. repeated determinations of cleaved hk% (chk%) revealed an average of 1.20% (range: 0.46-2.66%) in cvid patients with inflammatory complications and those without complications averaged 1.07% (range: 0.79-1.35%). healthy controls had an average chk of 1.15% (n=10, range: 0.60-2.10%). conclusions: cleaved kininogen detected in the sera of cvid patients was found at similar levels compared to healthy controls (chk<5%). findings suggest that systemic activation of the contact system might be absent in cvid, however, future considerations include developing detection methods for local tissue activation. (171) submission id#428729 the underlying primary immunodeficiencies and lung diseases, and low cd3 and cd4 counts are associated with recurrent pneumonia in hiv negative lymphopenia patients. 1 1 clinical fellow, yale university school of medicine introduction/background: lymphopenia can be considered as primary or acquired immunodeficiency. lymphopenia is associated with a considerable increase in susceptibility to infections and the treatment focus for lymphopenia is mainly consists of prophylaxis and treatment of opportunistic infections. aids is the most well known cause of acquired lymphopenia and the cd4 count serves as an effective surrogate marker for disease progression and guideline for prophylaxis for hiv positive lymphopenia patients (hplp). hiv negative lymphopenia patients (hnlp) have been following the same prophylaxis guideline for hplp patients. however, it is unclear whether same prophylaxis guideline will be appropriate for both groups since the underlying immune mechanisms are different between these two groups. objectives: we aimed to define the optimal treatment and prophylaxis guideline for hnlp. in this study, we compared the clinical phenotypes and absolute counts of lymphocyte subsets between hnlp with recurrent pneumonia (pna), recurrent upper respiratory infection (uri) and no pulmonary infection. methods: electronic medical records of hnlp (n=29) seen at an academic immunology clinic between the year of 2012 and 2017 were reviewed retrospectively. lymphopenia was defined as absolute cd3 count less than 1000/μl. the age, absolute counts of cd3, cd4, cd8, cd19, nkt and nk cell counts, history of antibiotic or antiviral prophylaxis use, autoimmune disease, lung disease, immunosuppressive therapy use, hypogammaglobinemia and ivig therapy use were compared between patients with recurrent pna (n=8), recurrent uri (n=13) and no pulmonary infection (n=8). results: this study showed that patients with recurrent pna had significantly lower absolute cd3, cd4, nk cell counts and age compared to the patients with recurrent uri ( table 1) . none of the clinical phenotype was significantly different between these two groups. when we compared the patients with recurrent pna vs no infection, all lymphocyte subset counts and age were similar between these two groups except the frequency of underlying lung disease which was significantly higher in the recurrent pna group (table 1) . lastly, we grouped the recurrent pna and uri groups together as the pulmonary infection group. when we compared the pulmonary infection group with the no infection group, lymphocyte subset counts were not significantly different, however, infection group showed significantly higher incidence of pid and the trend of lower rate of is therapy use than the no infection group (table 1) . conclusions: the initial study has several limitations. this was a retrospective study from a single clinic and patient population was limited to 29 patients. despite these limitations, we believe that this study provides valuable messages regarding prophylaxis guideline for pulmonary infection in nhlp; the underlying pids including icl and cvid, lung diseases particularly bronchiectasis and the absolute cd3 and cd4 counts less than 500/μl and 300/ μl, respectively, are associated with recurrent pna and the patients with these risk factors likely will benefit from antibiotic prophylaxis. in addition, patients with acquired lymphopenia due to chronic use of is therapies without underlying pid or lung disease less likely develop pulmonary infection despite of low cd3 and cd4 counts. further investigations are crucial to elucidate the clinical significance of our initial observation by increasing the patient population and analysis of detailed immunologic and genetic profile of these patients which will likely reveal immunological markers and genes that are involved in the pathogenesis of both primary and hiv negative acquired lymphopenia. director, neuro-oncology program, division of hematology/oncology, ucsf benioff children's hospital oakland, ca introduction/background: gata2 is a hematopoietic transcription factor, required for the development and maintenance of a healthy stem cell pool. heterozygous mutation of gata2 has been associated with different but overlapping syndromes affecting both myeloid and lymphoid cell lines including aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia, pulmonary alveolar proteinosis and lymphedema. it is also associated with immunodeficiency and susceptibility to mycobacterial, fungal, and viral infections. hematopoietic stem cell transplantation (hsct) is the only available cure which should ideally occur before patients develop neoplasia, severe infections or lung disease. objectives -to describe the clinical presentation of a genetically confirmed case of gata2 mutation -to describe the characteristic hematological and immunological profile of patients with gata2 mutation -to emphasize the importance of high index of suspicion and early diagnosis in improving outcome with hsct methods: case: a 17-year-old female presented with prolonged fever, fatigue, nonspecific rash, and unremarkable clinical exam. laboratory evaluation was significant for mild pancytopenia with profound monocytopenia. bone marrow analysis was remarkable for hypocellularity without evidence of dysplasia or malignancy. peripheral blood flow cytometry showed decreased t-and b-cells and absent nk cells. results: the constellation of pancytopenia, marked monocytopenia and absent nk cells, were suggestive of gata2 deficiency. sanger gene sequencing of gata2 revealed a heterozygous nonsense mutation (p.arg337*). conclusions: mutation of gata2 is the underlying defect in overlapping clinical syndromes and is associated with immunodeficiency and malignant predisposition. incidence of organ dysfunction, infections and neoplasia increases with age. confirming the diagnosis during the phase of marrow hypocellularity/monocytopenia and pursuing hsct prior to malignant transformation may improve patient outcome. introduction/background: reduction in child mortality has kept pace with improved immunization and nutritional status. however, there are children with severe infections who have no identifiable reason. the search for children with pid was initiated in a tertiary care referral hospital from a region with no documentation of the prevalence of this disorder, but with a high rate of consanguinity. financial constraints, poor availability of laboratory facilities or therapeutic options locally, limited awareness among pediatricians and vast distances between premier centres were major obstacles. objectives to identify children with primary immune deficiency and study the spectrum of pid in north kerala to prevent infectious and other complications in these children to provide curative therapy whenever feasible methods 1. acquisition of knowledge and skills to diagnose and manage pids 2. establishment of an immune deficiency clinic 3. liaison with centres across the country offering diagnostic tests and stem cell transplant 4. formation of project team and submission of a project to the central government 5. improvement of diagnostic facilities at home institution results: 180 children with recurrent, severe or persistent infections or with two or more esid warning signs were screened for pid. severe combined immune deficiency was diagnosed in 3 children, wiskott -aldrich syndrome in 5 children, chronic granulomatous disease in 6 children and x linked agammaglobulinemia in 11 children and leucocyte adhesion deficiency in 3 children. prophylaxis with ivig was initiated in 22 children and stem cell transplant was done for 8 children. conclusions: in a country with resource constraints, limited awareness among health care providers and vast distances, it is possible to make a difference to the lives of families of children with pid by networking across centers with expertise in immunological and molecular genetic diagnostic methods and life -saving therapeutic modalities like stem cell transplantation. introduction/background: complete thymic aplasia is a rare cause of scid, and requires thymic transplantation for curative treatment. because thymic transplantation is not widely available, there can be significant delay between diagnosis and curative therapy placing the infant at risk of invasive life threatening infections. objectives: describe modalities of therapy for adenoviremia in a patient with scid due to thymic aplasia. methods: chart review was performed. treatment including hlapartially matched third party cytotoxic t cell therapy and matched related hematopoietic cell transplant were assessed for treatment of life threatening adenoviremia in a scid patient with thymic aplasia. we identified an infant with a mutation in tbox transcription factor 1 (tbx1) (c.1176_1195dup20) by way of state newborn screening for scid. she had severe t cell lymphopenia and abnormal t cell function. at 7 months age, she developed adenoviremia with associated fulminant hepatitis, and an initial viral load of 5 million copies/ml. despite prolonged therapy with cidofovir, viral load increased to as high as 264 million copies/ml. treatment with two infusions of partially hlamatched third-party cytotoxic t cells specific to adenovirus (5/10 and 4/10 hla matched, with hla class ii-mediated antiviral restrictions) led to partial clinical improvement without viral clearance. due to continued severe adenovirus-related hepatic dysfunction, unmanipulated bone marrow from an hla-identical sibling was infused without conditioning (10 million cd34+ cells/kg and 5.2x107 cd3+ cells/kg), at 9 months of age. after an initial surge in adenoviral loads attributed to massive viral lysis, the degree of viremia progressively declined and was <1,000 copies/ml within 7 weeks of marrow infusion. antiviral tcell activity against adenovirus was detected at low level in peripheral blood via ifn elispot at 3 weeks post-marrow infusion. she subsequently developed acute cutaneous and hepatic gvhd responsive to tacrolimus and steroids without recrudescence of viral illness. conclusions: delay in curative therapy in scid substantially increases risk of invasive life threatening infections. one strategy of allogeneic hct can be to eradicate severe infection in scid by providing the necessary t cell directed therapy against infectious agents. antigen-specific partial immune reconstitution can be achieved with hct in patients with thymic aplasia but concern regarding the development of full immunologic t cell diversity in athymic patients remains. senior technician, sanquin bloodsupply amsterdam introduction/background: chronic granulomatous disease (cgd), an inherited disorder of granulocyte function caused by a failure of intracellular superoxide production, normally presents as severe recurrent bacterial and fungal infections in the first years of life. the majority of affected individuals are diagnosed before the age of 2 years, although patients may remain undiagnosed until adulthood despite the early onset of the symptoms objectives: investigation of fungal infection in adult cgd patients methods: nbt and dhr 123 for detection of cgd and molecular analysis for detection of type of mutation in cgd and fungal characterization. results: we report here the detection of causative fungal infections in 2 adult patients with cgd. in the first patients we found paecilomyces formosus infection in an adult patient (18 years old female) with undiagnosed cgd who was referred to the shahid beheshti university hospital (tehran, iran) complaining of cough, dyspnea and fever for 5 weeks prior to admission. microscopic analysis revealed branching solitary phialide with ellipsoidal conidia with long chain arrangement and many chlamydospores which mostly resemble botryotrichum species but during subcultures on potato dextrose agar (pda) and sabouraud dextrose agar (sda), phialides typical of paecilomyces species appeared. typically, paecilomyces spp. rarely cause infections in humans and if these fungi are detected in blood urine or cerebrospinal fluid cultures they are considered as contaminants. the second patient was a 26-year-old man was referred to the hospital with weight loss, fever, hepatosplenomegaly and coughing. he had previously been diagnosed with lymphoadenopathy in the neck at age 8 and prescribed antituberculosis treatment. bal and serum galactomannan tests were negative. low, subnormal levels of ros were produced following stimulation of purified neutrophils with the phorbol ester pma. genomic dna was extracted and gene scan was used to determine the ratio between the number of exon 2 sequences of neutrophil cytosolic factor 1 (ncf1) gene, which encodes p47phox, and the number of -ncf1 exon 2 sequences. in addition, the fungal culture was disrupted with glass beads and dna was extracted. the dna sequence results were blasted using the ncbi genebank database, which showed 99% similarity to an aspergillus terreus isolate in the gene bank fungal library with accession no 1168. conclusions: thus, despite its current relative rarity in older patients, the presence of fungal infection is changing our understanding of the diagnosis, management and outcome of cgd. greater appreciation of the potential of fungal infection in older cgd patients is important in regions, such as iran, where tuberculosis is endemic and that sarcoidosis and cgd are considered as differential diagnosis. the demonstration of the successful patient-orientated treatment after using sequencing to confirm cgd and to identify the presence of the specific infectious agent emphasises the importance of adopting this approach across the region. introduction/background: primary immunodeficiencies (pi) represent a heterogeneous group of over 350 genetically distinct disorders which interrupt normal host-defense mechanisms and predispose to significant morbidity and mortality. presently, we are only able to screen for severe t-cell deficiencies at birth; however, the most common forms of pi often go undetected for years leading to adverse patient outcomes and excessive healthcare spending. given the relatively high incidence of pi in the general population, informatic measures could be useful for determining individual risk for pi and facilitating earlier, correct diagnosis and appropriate treatment across the spectrum of pi. objectives: the purpose of this study was to test the jeffrey modell foundations spirit (software for primary immunodeficiency recognition intervention and tracking) analyzer on the texas childrens health plan. major aims were to identify individuals with medium-high risk for pi, assess the clinical characteristics of at risk patients and determine if risk identification led to diagnosis of pi over a 12 month period. methods: after removing all known pi diagnosed patients from the database, 185,892 individual texas childrens health plan enrollees were screened for risk of pi with the spirit analyzer using relevant, weighted icd9 and icd10 codes. patient characteristics are shown in table 1 . following identification of medium-high risk (mhr) individuals, letters were sent to their primary care physicians to alert them of patient risk. a second analysis of the mhr individuals was performed 12 months later. detailed chart reviews were conducted on 769 mhr individuals to further assess clinical features of this group. the study was approved by the baylor college of medicine institutional review board (study h-38501). results: of the original cohort, 2188 (1.2%) were identified as mhr for pi. from that group, 1068 (0.6%) were accessible for analysis and 769 (0.4%) had electronic health records for review. in the 12 months following the first analysis, 43 (0.02%) were diagnosed with a pi. (figure 1 ) another 61 patients had concerning diagnoses coded warranting further investigation. (figure 2 ) concerning diagnoses included: cellulitis(18), abscess (14), recurrent otitis media(11), recurrent sinusitis (5), osteomyelitis (2), and mastoiditis (1) among others. in total 104 patients had a pi diagnosis or a history concerning for pi (0.06% of main cohort; 13.5% of mhr cohort). conclusions: the spirit analyzer is effective at identifying persons at risk for pi and facilitates diagnosis. potential mhr yield by the analyzer is over 10%. these patients are treatable and will benefit from targeted intervention once identified. the analyzer can also highlight concerning conditions worthy of additional assessment. future work should focus on longitudinal healthcare outcomes for patients diagnosed with pi via spirt and physician perspectives on the utility of the tool. introduction/background: genetic variants in card11 contribute to several diseases caused by dysregulation of the adaptive immune system. dominant-negative, loss-of-function and gain-of-function variants in card11 variants lead to primary immunodeficiency disease. however, primary immunodeficiency disease caused by card11 gain-of-function variants often progresses to b-cell malignancy. the clinical course and treatment options depend on the type of card11 mutation. unfortunately lymphocyte immunophenotyping and traditional proliferation assays can't distinguish the variant effect or predict the likelihood of malignancy. objectives: to use multiplexed functional assays for determining variant effect to distinguish between dominant-negative, loss-and gain-offunction effects in card11. our ultimate goal is to generate a variant function map that can be used to guide diagnoses and treatment of immune dysregulation caused by mutations in card11. methods: we co-delivered crispr/cas9 ribonucleoprotein complexes with libraries of single-stranded oligonucleotide repair templates thus generating lymphoma cell populations containing all possible singlenucleotide variants (~2400 different protein coding changes) in the nterminal 140 amino acids of card11. following culture with and without bcr pathway inhibitors, we used next generation sequencing to quantify variant abundance before and after culture, both with and without bcr pathway inhibitors. results: due a requirement for card11 in these lymphoma cells, those with dominant-negative and loss-of-function card11 variants grow more slowly, whereas those with gain-of-function variants grow faster in the presence b cell receptor pathway inhibitors. by tracking the relative abundance of each variant in the population by next generation sequencing over multiple conditions, we determined the functional effect of each. we assessed the functional effects of thousands of card11 variants in parallel. this enabled us to confirm several previously reported gain-offunction and dominant-negative variants in card11, as well as identify several additional novel variants. finally, we evaluated previously undescribed dominant-negative, loss-of-function and gain-of-function variants during differentiation of primary human b cells and during nf-b signaling. conclusions: the results of our experiments demonstrate the utility of multiplexed functional assays for determining variant effect in proteins where distinguishing between dominant-negative, loss-and gain-offunction effects are required to guide diagnoses and treatment. introduction/background: primary immune-deficiencies (pids) are a heterogeneous group of nearly 300 monogenic inborn errors of immunity. in recent years, whole exome sequencing (wes) became a valuable diagnostic approach for the identification of molecular defects in patients with clinical manifestations suggestive of pid. this approach provides a definitive diagnosis and may help in genetic counseling, prenatal diagnosis and pre-symptomatic identification of patients with a potentially lethal disease. the diagnostic yield using wes was found to be~25% in rare mendelian disorders and~40% in pids. we present here a markedly higher yield, of 65%, in pids with a high percentage of consanguinity (65% in this study). objectives: using the whole exome sequencing (wes) approach in 55 israeli pid patients with high percentage of consanguinity to identify the genetic causes underlying their diseases for better diagnosis and clinical management. methods: wes was performed on genomic dna obtained from wbc of 55 immune deficient patients with potential genetic causes. the sequencing data was analyzed bioinformatically. each of the discovered mutations was validated and the familial segregation was confirmed, using sanger sequencing. results: the 55 probands (32 males and 23 females) ranged in age from 2 weeks to 26 years with a mean of 4.4 years. of them, 20 patients are jewish (36%), 34 palestinians (62%) and 1 (2%) is from a greek ethnicity (cyprus). based on their clinical and immunologic phenotype at the time of initial evaluation, patients were assigned to one of seven pid groups: (i) humoral immunodeficiency 5 patients (9%); (ii) combined immunodeficiency (cid) 16 (29%); (iii) cid with syndromic features 10 (18%); (iv) scid 9 (16%); (v) congenital defects of phagocytes 6 (11%); (vi) immune dysregulation 7 (13%) and (vii) phenocopy of pid 2 (4%). we identified 67 mutated alleles, all in coding regions, that are highly likely to be causative in 36 of the 55 patients, achieving a 65% molecular diagnostic yield. among the 36 patients, the mode of inheritance in 28 patients (78%) is autosomal recessive and in 3 is compound heterozygote (8%). four patients (11%) harbor a non-inherited mutation on one allele, either de novo or somatic. the inheritance of the mutation in one patient (3%) is x-linked. the high rate of bi-allelic inheritance (93% of the alleles) is mainly due to the high frequency (65%) of consanguinity among the studied cohort. twenty eight mutated genes were identified in this study. of them, 6 were found to be novel in causing an inherited disease in man. interestingly, some genetic defects in known genes were found in patients with atypical phenotypes. in 23 patients (42% of the total number of patients and 64% of the wes diagnosed) the discovery of the genetic cause led to a change of therapy, towards a more targeted and personalized one. the revised treatments included bone marrow transplantation, conditioning protocols, reduced intensity of immune suppression, and prevention of unnecessary treatments due to their possible deleterious outcome. conclusions: except of being a useful tool for diagnosing and deciphering novel or atypical forms of pids, our wes study demonstrates an immediate and powerful impact on patient therapy in pids. early intervention with bone marrow transplant or gene therapy is critical and best when the infant in uninfected. newborn screening for scid and t-cell lymphopenia has been implemented in 45 states. the screening test is performed from dried blood spots collected at birth and involves pcr quantification of circular dna byproducts of t-cell receptor gene rearrangement, t-cell receptor excision circles (trec). trecs are generated during t-cell maturation in the thymus and are indicative of naïve t-cell output. assays and protocols to measure trecs vary by state and there is no standardized guideline at this time. washington state is unique in that it is one of few states where all newborns undergo two or more independent newborn screens, the first by which igrt treatment helps in these patients is unreported. here we present the first case of an mbl deficient patient with other complicating conditions (low igg and multiple cf polymorphisms, ciliary dyskinesia) who did not show improvement on igrt, thus refuting current literature. results: a 28-year-old caucasian male patient presented due to recalcitrant warts of the hands and feet. he had childhood otitis media status post tympanoplasty, mild molluscum contagiosum, and eczema. also, history of migraines with a negative brain mri. a trial of weekly pegylated interferon alpha 80mcg for his warts was discontinued due to significant neutropenia and depression. physical exam was notable for bulky skin colored warts on the lateral and dorsal fingers and dorsal hands including periungual skin. clusters of verrucae were noted on the feet. initial laboratory testing was notable for mild hypogammaglobulinemia with protective specific antibodies. lymphocyte subset analysis revealed a predominantly t cell lymphopenia with decreased naïve and memory cd4 and cd8 subsets, normal absolute numbers of b cells but with low memory subsets, and normal numbers and function of nk cells. whole exome sequencing ultimately revealed homozygous r169q mutations in the cecr1, a mutation previously described as causing ada2 deficiency. ada2 activity was about 40%. conclusions: ada2 deficiency is a relatively newly defined genetic defect, with a clinical phenotype that continues to evolve with newly diagnosed cases. our patient had not had evidence of vasculitis or stroke, but had recalcitrant warts with lymphopenia as his primary presentation. approach to therapy for those without vasculitis or significant cytopenias remains unknown. introduction/background: whole exome sequencing has added greatly to our library of primary immune deficiencies. however, interpretation of findings is not always straightforward. clinicians need to understand the limitations of this diagnostic tool and be familiar with the next steps in order to achieve a diagnosis. objectives: a 7 year old male presents for evaluation of bronchiectasis of bilateral lower lobes and poor growth. past medical history was significant for gerd with poor weight gain during infancy and childhood, oral thrush, recurrent respiratory infections and bilateral partial hearing loss. the bal showed abundant neutrophils and was positive for strep pneumoniae and haemophilus influenza. methods: serum immunoglobulins were normal with an elevated iga (369) and mildly elevated ige (361). tetanus igg was protective (0.4 iu/ml) but post-vaccine responses to the pneumococcal polysaccharide vaccine were poor with protective titers achieved to 3/14 serotypes. lymphocyte phenotyping was remarkable for a complete absence of b and nk cells. cd4:cd8 ratio was inverted (0.25) and the majority of the cd45+ t cells were memory phenotype. mitogen proliferation was essentially normal. due to the absence of b cells and despite normal total serum immunoglobulins, the patient was started on sc ig weekly and antibiotic prophylaxis with trimethoprim-sulfamethoxazole and fluconazole. clinically he demonstrated good weight gain and a reduction in cough and respiratory symptoms. results: whole exome sequencing was performed. a single base mutation in ada1 (c.320 t>c) was identified in one allele. this mutation (p.l107p) is known to be deleterious and when homozygous is associated with typical scid. however, this mutation was not present on the other allele. secondary analysis looking for large deletions and duplications failed to identify any abnormality in sequence in the normal allele. but the expression of this allele was markedly diminished causing us to suspect that its function might be impaired. functional testing was performed and demonstrated that there was no ada activity in the patients red blood cells, thus confirming a diagnosis of combined immune deficiency due to ada deficiency. conclusions: null mutations in ada result in an absence of ada function with profound cd3 cell lymphopenia and dysfunction. clinically affected infants have typical scid. hypomorphic mutations may lead to partial function of ada with more variable immune defects. interestingly, most patients with ada deficiency have neurologic complications, frequently hearing loss. we believe that most likely this patient has had some degree of spontaneous reversion of the mutation in the normal allele leading to a less severe phenotype. as reported previously in a case involving a mutation in il2rg chain, normal function was not restored and the patient remains with a combined immune deficiency. this case highlights an important limitation of whole exome sequencing and the need for confirming the impact of a genetic defect with a functional assay. introduction/background: the autosomal dominant hyper-ige-syndrome (ad-hies) is a rare primary immunodeficiency and multisystem disorder resulting from heterozygous loss-of-function mutations in the stat3 gene. ad-hies is characterized by skeletal dysplasia, recurrent pulmonary and skin infections (e.g. staphylococcal abscesses, eczematoid dermatitis) due to an increased susceptibility to bacteria and fungi. since many patients do rather well on anti-infective prophylaxis and supportive care, and early case reports suggested no benefit of allogeneic hematopoetic stem cell transplantation (hsct), ad-hies patients are rarely referred for hsct. the literature still contains only a handful of patients who underwent hsct. all these patients had experienced severe disease related complications before hsct and consequently the benefit of hsct was reported to be variable. objectives: currently, the general consensus is to only consider patients with severe pulmonary disease for hsct. it could however be postulated that transplanting patients earlier in their disease course and correcting their immunodeficiency before permanent organ damage due to infectious complications has occurred may extend life-expectancy and improve the quality of life of ad-hies patients. methods: we report on a 15 year old female ad-hies-patient who presented with bronchiectases following recurrent pneumonias, one pneumatocele, and normal pulmonary function tests. her past medical history also included recurrent skin infections, serious haemoptysis, pathological fractures and atopic eczema. considering that lung infections are the major life-limiting complication in ad-hies and are potentially positively influenced by hsct, our patient had enquired about a transplant. after extensive discussion of the pros and cons and obtaining full informed consent from her and her family, she underwent an elective hsct. she received bone marrow from an hla-matched sibling donor after a reduced-intensity conditioning consisting of alemtuzumab (2x0,2mg/kg), treosulfan (3x14g/ m2), fludarabine (5x30mg/m2) and thiotepa (2x5mg/kg), and graftversus-host disease (gvhd) prophylaxis with cyclosporine a (csa) and mycophenolate mofetil (mmf). results: the peri-transplant course was complicated by acute lower gastrointestinal tract bleeding and renal failure of unknown origin. continued kidney function impairment led to early tapering and discontinuation of csa on day +132 after hsct in the absence of any acute gvhd. neutrophils and platelets engrafted on days +15 and +26 respectively. she is currently on day +150, free of gvhd or infection, exhibiting full donor chimerism and recovered kidney function. in view of the preexisting pulmonary damage she currently remains on antibiotic prophylaxis and inhalation therapy. conclusions: this ad-hies patient who underwent hsct with few pre-hsct disease complications and relatively little permanent organ damage may add to our understanding of whether early hsct will lead to improvement of quality of life and possibly increased life-expectancy in ad-hies patients. it remains to be elucidated whether her rather uncommon peri-hsct complications are connected to her underlying disease. future research should be directed at identifying ad-hies patients at high risk of severe pulmonary complications early, so these could be referred for timely hsct. objectives: this is a case study of a patient who had refractory ibd symptoms and recurrent infections who was found to have xiap deficiency and mefv variant mutations. methods: a 37 year old male presented at age 16 with recalcitrant ibd unresponsive to multiple medications including steroids, mesalamine, azathioprine, infliximab, adalimumab, methotrexate, and vedolibumab. in addition, he developed severe infections from a combination of immune dysregulation and immunosuppressant medications. currently he is on ustekinumab but still has severe abdominal symptoms. patient does not have a history of hemophagocytic lymphohistiocytosis (hlh). family history was significant for ibd in both his mother and sister. he has had persistent lymphopenia which ranged between 210 to 838 cells/ mm3. t/b/nk panel showed decreased cd3 t cells (596 cells/mm3) with normal cd4, cd8, and cd4/cd8 ratios. b and nk cells were normal in quantity. t cell antigen/mitogen assays showed normal response to all mitogens (pha, pwm, con a) and most antigens (tetanus, candida, hsv, vzv, adv) but low response to cmv antigen. igg was elevated at 2,750, but iga, igm, and ige were normal. his ebv dna pcr is negative. results: exome sequencing revealed a novel xiap hemizygous variant at position c.693g>a (p.=?) of unknown significance and a mefv heterozygous variant. functional testing performed at medical college of wisconsin showed no expression of xiap on lymphocytes and a defect in nod2 pathway. given the xiap deficiency, bone marrow transplant was discussed as an option for refractory ibd and prevention of hlh. rituximab was also offered to decrease the possibility of hlh. currently the patient is in the decision making process of both treatment options. conclusions: genetic evaluation with clinical exome in early onset refractory ibd, family history of ibd, and recurrent infections demonstrated a novel mutation in the xiap gene with possible contribution of mefv variant as well. the absence of xiap protein expression and abnormal functional assay of the nod2 pathway confirm the pathogenicity of the mutation. identification of this genetic variant will help guide future therapeutic options and prognosis for this patient. introduction/background: x-linked agammaglobulinemia (xla) is a primary immunodeficiency disease caused by mutations of bruton tyrosine kinase (btk), which is essential in b cell maturation. xla is typically associated with bacterial infections of upper and lower respiratory systems, enteritis and increased risk for malignancies. objectives: to present the evolution and treatment of a complicated flexispira (helicobacter bilis) infection with delayed diagnosis in an xla patient. methods: clinical and laboratory features of a patient with xla evaluated at the national institutes of health. results: a 29-year-old male patient with xla presented for initial evaluation of indurated lower extremity and torso lesions. he was diagnosed with xla at age 2 years secondary to bacterial sepsis pneumonia and empyema. after starting ivig at age 2, he was well without significant infections except for recurrent otitis media. at 12 years of age, he developed right leg edema below the knee, which progressed to patchy skin thickening and discoloration. a tissue biopsy at 15 years of age revealed marked fibrous thickening of the subcutaneous septum with diffuse infiltrate of eosinophils with negative cultures for bacterial, fungal, and mycobacterial infections and thought to be consistent with eosinophilic fasciitis. mri demonstrated infiltration in the superficial and deep muscle compartments with fibrosis. symptoms persisted despite empirical treatment with iv antibiotics and steroids. at 16 years of age his left leg became involved with similar findings, while under treatment including multiple immunosuppressants (dapsone, methotrexate, tacrolimus, hydroxychloroquine, iv cyclophosphamide, remicade, cytoxan, and enbrel) targeting eosinophilic fasciitis. at age 28, he developed ulcerations over the left shin and ankle and was diagnosed with chronic multifocal osteomyelitis based on mri findings. physical exam was notable for bilateral leg swelling below the knees with woody appearance and induration, hyperpigmentation, and tenderness. indurated and nodular lesions without discoloration were noted above the waist line, on right forearm and above right nipple. skin biopsies of right lower extremity and right forearm were positive for numerous spirochetal-like organisms with warthin-starry stain. treatment was initiated with meropenem and gentamicin. gentamicin was discontinued due to vestibular ototoxicity six weeks later and doxycycline was added. initial response was followed by worsening of symptoms and intolerance to treatment, which led to the addition of tigecycline and azithromycin. with continued progression, seven months into initial evaluation, chloramphenicol and nitazoxanide were added to the regimen. due to the persistence of flexispira organisms on skin biopsy warthin-starry stains, fresh frozen plasma (ffp) was introduced four months later (11 months after initial evaluation) as an adjunctive treatment. cultures performed at the cdc were negative; however, pcr and sequencing resulted in identification of helicobacter bilis. 6 units of ffp was infused weekly for over three years, then reduced to every two weeks, with goal igm levels > 40mg/dl. subsequently, labs including cytopenias, inflammatory markers, and immunoglobulins improved as well as a negative warthin-starry stain. symptoms have improved with almost complete resolution of findings with only residual small areas of discoloration over both lower extremities. conclusions: xla immune deficiency is associated with flexispira (helicobacter bilis) infections, with typical appearance of discoloration and induration, which may evolve to osteomyelitis due to delayed treatment. although typically observed over the lower extremities, immunosuppressive treatment may lead to further expansion above the waist line. approach to therapy with weekly to bimonthly ffp infusions in addition to antibacterial treatment has proven to be beneficial in controlling the infection. higher igm levels resulting from the ffp may also provide antibacterial effects. introduction/background: x-linked severe combined immunodeficiency (scid) is a well described primary immunodeficiency associated with mutations in the common gamma chain. patients with x-linked scid classically present with profoundly low or absent t cells and nk cells with a variable number of b cells. the lymphocytes that are present typically have a proliferation index <10% control when stimulated with mitogens and antigens. patients must undergo corrective therapy with bone marrow transplant (bmt) or gene therapy to avoid the life-threatening infections that are associated with the nearly absent adaptive immune system. objectives: a 2-week-old boy presented to childrens national immunology clinic for initial evaluation of critical result on newborn screen. methods: targeted partial exome sequencing was performed on a 7-dayold patient who was picked up via trec assay on the maryland newborn screen. flow cytometry was completed at childrens national and proliferation studies completed at cincinnati childrens hospital diagnostic immunology lab. results: flow cytometry revealed markedly decreased lymphocytes with nearly absent cd8+ t cells and low cd4+ t cells with a r e l a t i v e i n c r e a s e i n c d 4 + c d 4 5 r o t c e l l s ( r a t i o cd45ra:cd45ro: 18%:12%). the b and nk cells were within the reference range for age. mitogen proliferation studies showed a mild decrease to pha and normal responses to pokeweed and cona (35587 cpm, 40924 cpm, and 82651 cpm, respectively). partial exome sequencing revealed a hemizygous nonsense substitution in il2rg (c.982c>t, p.r328) . maternal engraftment accounted for 3% of the t cells. the patient was started on prophylaxis with ivig, bactrim, and fluconazole with the plan to proceed with bone marrow transplant. as patient approached bmt maternal engraftment became absent in whole blood and repeat proliferation studies revealed normalization of the response to pha (stim index) with continued normal responses to cona and pokeweed. the patients flow cytometry values and ratios remained unchanged. patient completed a reduced intensity preparative regimen of busulfan, fludarabine and alemtuzumab prior to receiving his 8/8 matched unrelated donor bone marrow transplant. conclusions: it remains to be determined why initial proliferation studies showed >10% function with improvement over time in a patient with a well-described genetic mutation causing scid (187) director, division of intramural research, nih/niaid introduction/background: the advent of next-generation sequencing (ngs) has led to a proliferation of newly discovered genetic diseases and expanded phenotypes of known immunodeficiencies. availability of specific gene panels or whole exome sequencing (wes) with targeted analysis based on broad phenotypes, coupled with clinicians increasing awareness has led to higher utilization of ngs. published reports from high throughput sequencing labs indicate exome analysis identifies causative mutations in only 20-30% of probands. results: we have seen several patients who underwent high quality ngs in whom causative mutations were not identified. two separate families with multigenerational histories of leukemia, aplastic anemia, myelodysplastic syndrome, and cytopenias suggestive of gata2 deficiency had myeloid gene panel screens at commercial labs without causative mutations identified. targeted gata2 sequencing in the first family identified a novel change in gata2, c.1017g>t, p.l339l. cdna analysis demonstrated this synonymous variant resulted in aberrant splicing leading to a frameshift and premature termination. the wes bioinformatics pipeline failed to recognize the splice mutation. in the second family, pcr amplification spanning the 2 terminal exons revealed a shortened pcr product with a 426 base deletion fully encompassing the penultimate exon and leading to a 42 amino acid in-frame deletion. the deletion spanned all capture probes for the exon resulting in only the wild-type allele being captured and sequenced. additionally, capture kits targeting only coding regions of genes fail to capture deep intronic mutations such as those seen in gata2 (hsu, 2013) or in the 5 untranslated region of ikbkg, encoding nemo, (mooster, 2014; hsu, submitted) . lastly, even with good capture and sequencing, the presence of pseudogenes may confound downstream sequence alignment as seen in ncf1, encoding p47phox preventing recognition of disease causing mutations. conclusions: with ngs becoming more widely available as a clinical diagnostic tool, it is important to remember that wes results, unlike many laboratory tests, are not binary. inadequate bioinformatics pipelines, deletions, intronic or untranslated mutations, and pseudogenes can all mask the presence of causative mutations. targeted panel captures or analysis will miss novel genes. astute clinicians need to recognize the limitations of the current technology and pursue alternate assays when suspicions warrant. a cell based assay for the detection of autoantibodies to il-17 in human serum. matt phillips, phd 1 , vijaya knight, md, phd 2 1 senior scientist, national jewish health 2 director of immunology and complement adx labs, national jewish health introduction/background: patients with chronic candida infections are typically deficient in some aspect of il-17 signaling. one mechanism, which has recently come to light, is through the production of il-17 autoantibodies, particularly in patients who already suffer from specific autoimmune diseases. objectives: our objectives are to develop a diagnostic assay to accurately and easily detect il-17 autoantibodies in patient serum. methods: we developed a cell-based reporter assay using hek blue il-17 cells (invivogen) to detect the ability of patient serum to block il-17 receptor signaling. once stimulated with il-17, the cells secrete alkaline phosphatase (ap) into the surrounding media which is detected by invivogen hek blue media and an absorbance reader. addition of serum containing blocking antibodies inhibits secretion of ap. results: we were able to demonstrate, using a single patients serum with known il-17 autoantibodies, the inhibition of il-17 signaling in hek blue il-17 reporter cells. we further characterized the sensitivity of our assay with a commercially available anti-il-17 monoclonal and found it to be sensitive to between 1.4 x 10-6 m and 6.7 x 10-7 m. conclusions: loss of il-17 signaling can lead to problematic immune deficiencies including difficulty in clearing extracellular pathogens such as candida. some people who have an immune deficiency in the il-17 pathway may have developed autoantibodies to il-17 and thus have difficulty generating an appropriate immune response. we have developed a relatively low maintenance, cost effective, and simple test for detecting il-17 autoantibodies in human serum. alternations in repertoire of t and b cell subsets in patients with partial recombination activating gene (rag) deficiency with autoimmunity and history of viral infections introduction/background: patients with partial deficiency of recombination-activating genes 1 or 2 (rag1/2) can present with a wide spectrum of primary immunodeficiencies including combined immunodeficiency with granuloma and/or autoimmunity (cid-g/ai). prior case reports have highlighted alterations in b and t cell compartments; however comprehensive characterization of t and b cell receptor repertoires of lymphocyte subsets regarding diversity and autoreactivity has not been reported. objectives: defects in v(d)j recombination due to rag deficiency results in a skewed t and b cell repertoire that may be further modified by viral infections and promote inflammatory or autoimmune phenotype. methods: peripheral t and b cell compartments were sorted from two patients with combined immunodeficiency secondary to hypomorphic rag1 and rag2 mutations. b cells were stimulated with cd40l, cpg and il-21 to transition to antibody secreting cells (ascs), mimicking viral infection. repertoire analysis and single cell cloning of bcr heavy and light chain variable regions from sorted b cell populations has been performed. repertoire of t cell subsets (treg and follicular helper) were also examined results: we noted skewing towards proximal j usage in all b cell compartments (mature naïve, marginal zone, cd21-/low and memory) of two rag deficient patients compared to healthy controls. b cell clones with v4-34 v genes with low rate of somatic hypermutation expanded during b cell development. after in vitro stimulation mature naïve b cells from rag deficient patient were capable of transitioning to antibody secreting cells and enriched for polyreactivity to dsdna, insulin, lps and ifn cytokine (25 to 36%) compared to healthy control (5 to 14%). in connection to altered b cell compartments, restricted repertoire of regulatory t cells and an expanded and skewed follicular helper t subset were detected. conclusions: our data indicate that patients with partial rag deficiency have skewed t and b cell subsets that can further be altered towards antibody secretion and polyreactivity after stimulation such as viral infections. chief of the division of allergy and immunology, division of allergy immunology, the childrens hospital of philadelphia, philadelphia, pa usa introduction/background: the clinical features of 22q11.2 deletion syndrome include virtually every organ of the body. t cell lymphopenia, as a consequence of thymic hypoplasia, is the most commonly described immunologic feature and is most prominent in childhood. later in life, t cell exhaustion may be seen and secondary deficiencies of antibody function have been described in patients with 22q11.2 deletion syndrome. objectives: the role of deletion breakpoints in determining 22q11.2 deletion syndrome immunophenotype is unknown. in this study, we examined the effect of 22q11.2 deletions with and without tbx1 on lymphocyte counts. methods: lymphocyte counts were compared between 52 total 22q11.2 patients with tbx1-containing deletion (a-b, a-c, a-d deletions), and a total of 8 patients with tbx1-noncontaining deletion (b-d, c-d, d-e, d-f deletions). lymphocyte counts of patients with 22q11.2 deletions were compared to a set of 6 patients with a 22q11.2 duplication including the tbx1 locus. lymphocyte subset counts for each group were analyzed by t-test. results: cd3 counts were significantly lower in the tbx1-deleted cohort compared to the other two cohorts (mean 2169 cells/mm3 in tbx1 deleted cohort, 3709 cd3 cells/mm3 in the non-tbx1 deleted cohort, and 3657 in the duplication cohort, p<0.01 for all). similarly, cd4 counts were lower in the tbx1-deleted patients compared to the other two cohorts. there were no significant differences in cd8, cd19, and nk cell counts between the three cohorts. conclusions: these represent the first data to examine t cell counts in 22q11.2 deletion syndrome patients with different breakpoints. our data highlights an important role for tbx1 or other genes in the a-b region in regulating t cell production. paracoccidioidomycosis associated with a heterozygous stat4 mutation and impaired ifn-immunity introduction/background: mutations in genes affecting ifn-immunity have contributed to understand the essential role of this cytokine in the protection against intracellular bacteria and fungi. however, inborn errors in stat4, which controls il-12 responses, have not yet been reported. objectives: to determine the underlying genetic defect in a family with a history of paracoccidioidomycosis (pcm) disease. methods: genetic analysis was performed by whole-exome sequencing and sanger sequencing. stat4 phosphorylation and translocation from the cytosol to the nucleus, as well as ifnrelease by patient lymphocytes were assessed. the effect on stat4 function was evaluated by site-directed mutagenesis using a lymphoblastoid b cell line (b-lcl) and u3a cells. microbicidal activity of patient monocytes/macrophages was also analyzed. results: a heterozygous missense mutation, c.1952 a>t (p.e651v) in stat4 was identified in the index patient and her father. patients and fathers lymphocytes showed reduced stat4 phosphorylation and nuclear translocation as well as impaired ifn-production. in accordance, b-lcl and u3a cells carrying the stat4 mutant displayed reduced stat4 phosphorylation. patient's and father's pbmcs and macrophages (alone or in the presence of t cells) displayed impaired fungicidal activity compared with those from healthy controls that improved in the presence of recombinant human (rh) ifn-, but not rhil-12. conclusions: our data suggest autosomal dominant stat4 deficiency as a novel inborn error of il-12-dependent ifn-immunity associated with susceptibility to pcm disease. profound b cell lymphopenia in gof-stat1 that improves post ruxolitinib associate professor, university of south florida -johns hopkins all childrens hospital introduction/background: subjects with gain of function signal transducer and activator of transcription (gof-stat1) mutations have a variable clinical phenotype including combined immunodeficiency (cid). ruxolitinib, a janus kinase 1/2 inhibitor has been successful at treating immune dysregulation in subjects with gof-stat1. two subjects with profound b cell and/or t cell lymphopenia as a major manifestation are described, one of which was successfully treated with ruxolitinib. objectives: to discuss gof-stat1 mutations, their effect on the immune system, and the potential benefit of ruxolitinib in these subjects. methods: retrospective chart review was performed. results: subject 1 (c.494a>g) is a 21 year old male with a history of recurrent shingles, chronic mucocutaneous candidiasis (cmc), pneumocysitis jiroveccii pneumonia, varicella zoster meningitis, severe enteropathy, cerebral aneurysm and lymphoproliferation, and autoimmune hypothyroidism. he has profound lymphopenia predominantly affecting t and b cells (cd3+ 456 cells/ul, cd4+ 104 cells/ul, cd8+ 296 cells/ul, cd56+ 42 cells/ul, cd19+ 0 cells/ul). subject 2 (c. 1053g>t) is a 12 year old male with a history of severe cmc, recurrent pneumonia, enteropathy, and autoimmune thyroiditis. he had severe b cell lymphopenia (cd3+ 2168 cells/ul, cd4+ 1461 cells/ul, cd8+ 589 cells/ul, cd56+ 47 cells/ul, cd19+ 24 cells/ul). treatment with ruxolitinib 12.5mg bid led to clinical improvement of enteropathy and increased b cell counts in subject 2 (cd19+ 124 cells/ul). ruxolitinib has not yet been initiated in subject 1. both subjects were treated with anti-microbial prophylaxis and immunoglobulin supplementation. conclusions: combined immunodeficiency with variable degrees of b and t cell lymphopenia and hypogammaglobulinemia can be profound in subjects with gof-stat1 mutation. despite proper anti-microbial prophylaxis, this immunodeficiency can lead to severe infections. in addition to treating the autoimmune and immune dysregulatory features, treatment with ruxolitinib can improve the cid present and potentially reduce infectious susceptibility. igg4-related disease (igg4-rd), its common mimickers and response to anti-il5-(reslizumab) treatment rachel eisenberg, md 1 , arye rubinstein, md-phd 2 1 fellow in allergy and immunology, montefiore medical center 2 chief division of allergy and immunology, albert einstein college of medicine and montefiore hospital, bronx, ny, usa introduction/background: we describe a complicated case of igg4 related disease (igg4-rd) both in its presentation and novel treatment objectives: to review the common mimickers of igg4-related diseases which often lead to delayed diagnosis and treatment. to discuss novel therapeutic treatments for igg4-related disease results: a 70-year-old woman with a history of thyroid disease, sicca symptoms, lipodystrophy, relapsing parotid enlargement, asthma and erdheim chester syndrome initially presented with recurrent bacterial and fungal sinusitis despite multiple sinus surgeries. immunologic workup was notable for lymphopenia of 300/ml, cd4 count of 132 (677-1401 cells/ul normal range) and elevated igg4 of 511 (4.0-86.0 normal range). imaging was notable for nasal septal perforations and hypoplastic maxillary sinuses. there was high suspicion for igg4 disease however the patient was lost to follow up during which time she developed cachexia, eosinophilic pleural effusions (80% eosinophils), lung mass and a parotid mass with predominant t cell infiltrate misdiagnosed as follicular lymphoma. features consistent with igg4-rd included >50% ratio of igg4/igg and a predominant t cell infiltrate a biopsied lung mass showed igg4 plasma cell >50/hpf also consistent with igg4-rd. bone marrow biopsy was within normal limits. the patient was treated with rituximab, an effective treatment for igg4-rd. on treatment her igg4 levels normalized, however she developed recurrent large eosinophilic lung effusions requiring repeat drainage. fractional exhaled no (feno) was elevated to 86ppb. she was started on reslizumab at a dose of 5mg/ kg resulting in marked improvement in her respiratory status along with normalization of peripheral eosinophilia and reduction of feno to the normal level of 12ppb. conclusions: igg4-rd is a fibro-inflammatory condition which can affect any organ system and is diagnosed via tissue histology showing igg4 positive plasma cells and a typical morphologic pattern. this case outlines the common mimickers of igg4-rd often leading to a delayed diagnosis. before the final diagnosis of ig4-rd was made by us, the patient carried multiple diagnoses including: thyroid disease, recurrent parotid enlargement and seronegative sjogrens. these diagnoses in hindsight may have been mikuliczs syndrome, kuttners tumor and/or riedels thyroiditis which are common manifestations of igg4-rd. chronic sinusitis, atopic diseases, peripheral eosinophilia and destructive osseous lesions as noted in our patient are seen in up to 40% of patients with igg4-rd. destructive bony lesions and eosinophilia can mimic granulomatous polyangiitis, which was ruled out in our patient. her cachectic appearance and diagnosis of lipodystrophy can be explained by destruction of osseous tissue in the craniofacial skeleton which was later confirmed on imaging. lymphoid inflammatory infiltrates are commonly seen in igg4-rd and are can be misdiagnosed as a follicular lymphoma as in our case. a novelty in this case is the successful treatment with reslizumab targeted at the eosinophilic component of the disease. on reslizumab our patients asthma was for the first time controlled, pleuritis improved, fractional exhaled no (feno) normalized and her cachexia is improving. treatment with reslizumab should be considered in patients with igg4-rd who manifest with eosinophilic respiratory disease. introduction/background: ikbkb deficiency (c.1292dupg in exon 13) is a rare autosomal recessive form of severe combined immune deficiency (scid) originally described in canadian infants of northern cree descent. ikbkb scid is characterized by normal lymphocyte development, but impaired t-cell activation, along with innate immune defects. objectives: to report the clinical presentation, immunologic phenotype, and outcomes for patients with confirmed or suspected ikbkb scid due to this founder mutation. methods: we retrospectively reviewed hospital records dating back to 1973 of patients with confirmed homozygous ikbkb mutations, as well as patients suspected to be affected due to their clinical presentation and family relations to molecularly confirmed cases. results: fifteen patients were included. they presented early in life (average age 2 months) with invasive and disseminated viral, bacterial, mycobacterial, and fungal infections. patients had concurrent and multiple infectious organisms, with a notable predilection for candida, gram negative organisms, and mycobacteria. four patients in our cohort received bcg vaccination at birth, resulting in fatal disseminated m. bovis infection in all, and 2 additional patients succumbed to atypical mycobacterial infection following hematopoietic stem cell transplant (hsct). one newborn was identified in 2016 through new initiatives for targeted newborn screening for the mutation. immunologic features at presentation included normal to elevated lymphocyte counts with normal to elevated cd3, cd4, and cd8 t cells. when tested, response to pha varied from absent (1/10), to low (4/10), to normal (5/10). most patients had hypogammaglobulinemia, most often of the igg isotype (10/14). six had assessment of trec levels, and all had values above thresholds for screening programs. eight patients died before they could undergo hsct, and 6 received transplants in the setting of ongoing severe, lifethreatening infections. only 1 patient underwent hsct prior to the onset of infection. in our cohort, there are only 2 long-term survivors. conclusions: ikbkb deficiency is a severe form of scid with early onset of invasive and disseminated multi-organism infection. the immunologic phenotype is characterized by normal to elevated lymphocyte numbers which do not meet pidtc criteria for scid, variable (and sometimes normal) mitogen response, normal trec levels, and low igg levels. the disease is universally fatal without hsct, however, conclusions regarding efficacy and long-term outcomes of hsct are uncertain given the small sample size. immune -dysregulation mimicking systemic lupus erythematosus in a patient with lysinuric protein intolerance introduction/background: lysinuric protein intolerance (lpi) is an inherited aminoaciduria caused by defective amino acid transport in epithelial cells of the intestine and kidney due to bi-allelic, pathogenic variants in slc7a7. the clinical phenotype of lpi includes failure to thrive and multi-system disease including hematologic, neurologic, pulmonary and renal manifestations. individual presentations are extremely variable, often leading to misdiagnosis or delayed diagnosis. here we describe a patient that presented with suspected immunodeficiency in the setting of early-onset systemic lupus erythematosus (sle), including renal involvement, who was subsequently diagnosed with lpi post-mortem. objectives: describe a clinical a patient with lysinuric protein intolerance that presented as early-onset systemic lupus erythematosus (sle), including renal involvement and primary immunodeficiency. methods: after informed consent was obtained, dna samples were obtained from the proband and his parents. trio whole exome sequencing was performed to identify a cause of early onset autoinflammation resembling systemic lupus erythematosus. results: the male proband had a history of failure to thrive starting at 12 months of age, recurrent bacterial otitis media, and one episode of severe bacterial pneumonia requiring hospitalization. he presented at 30 months of age with multifocal pneumonia, anemia (hgb 8.2 mg/dl) and mild thrombocytopenia. initial laboratory studies revealed low albumin (2.8 mg/dl), elevated ldh (718), and mild hepatomegaly. renal and liver function testing was initially normal. immunologic evaluation for suspected primary immune deficiency showed normal immunoglobulin titers, low c3 (60) and low c4 (6.3). lymphocyte phenotyping revealed low b cell counts (0.6% of total lymphocytes) with t cells and nk cells within the normal range. despite antibiotic therapy, the patient worsened, developing fevers, a generalized erythematous rash, edema and nephrotic syndrome with oliguria. renal biopsy uncovered glomeruli with accentuated, global thickening and diffuse, peripheral capillary loops, as well as focal spiculated defects, there was endothelial swelling and other signs of acute damage including epithelial flattening, adluminal irregularity and extensive intraluminal proteinaceous detritus. endocapillary proliferative lesions, extracapillary crecents or tubular atrophy was not observed. immunofluorescence studies were positive for c3, igg and c1q granular deposits, mainly at the mesangium, interpreted as lupus nephropathy. endothelial swelling and massive, subepithelial electron-dense deposits with spike formation from the basement membrane were noted on electron microscopy, mimicking a stage ii membranous pattern of injury. autoantibodies included ana (1:320), anti-dsdna, smith, ssa and rnp were positive in agreement with the diagnosis of sle. immunosuppressive therapy with high dose iv corticosteroids and cyclophosphamide was initiated. despite this, the patient developed pancytopenia, elevated ferritin levels, increased triglycerides, and low fibrinogen. bone marrow biopsy displayed erythrocyte phagocytosis by macrophages, confirming a diagnosis of hemophagocytic lymphohistiocytosis (hlh). the patient subsequently died despite aggressive immunosuppression with high dose methylprednisolone and high dose iv immunoglobulin and dialysis. samples were collected from the deceased patient and his parents for research whole exome sequencing. trio analysis identified compound heterozygous missense variants in slc7a7. ammonia levels were not evaluated during the patients hospitalization. conclusions: lysinuric protein intolerance is a severe metabolic disorder that can present with protean systemic features including primary i m m u n o d e f i c i e n c y. i m p a i r e d l y m p h o c y t e f u n c t i o n , hypocomplementemia, immune-mediated glomerulonephritis, autoantibodies, and hlh are known complications of lpi. exactly how ineffective amino acid transport triggers these systemic inflammatory features is not yet understood. lpi should be considered in the differential diagnosis of early-onset sle, particularly in the absence of response to immunosuppressive therapy. director, national institute of immunohaematology introduction/background: chronic granulomatous disease (cgd) is a primary immunodeciency disorder with recurrent pyogenic infections and granulomatous inflammation resulting from loss of phagocyte superoxide production. mutations in any one of the five structural genes of the nicotinamide adenine dinucleotide phosphate (nadph) oxidase complex viz. cybb and cyba encoding for membrane bound gp91phox and p22phox; and ncf1, ncf2, and ncf4 encoding for cytosolic components p47phox, p67phox, and p40phox respectively, have been found to cause cgd. the relative incidence of these gene defects varies significantly depending on the ethnic background of the population. identification of molecular defect is important for patient management as well as for prenatal diagnosis in the affected families. the present study was aimed at studying the pattern of underlying genetic defects in a cohort of indian patients affected with cgd. objectives 1.to identify the underlying genetic defect in patients with chronic granulomatous disease in india 2. clinical,immunological and molecular characterisation 3. to utilise this information for genetic counselling and prenatal diagnosis of the affected families methods: eighty-seven (n=87) patients with abnormal nbt and dhr were included in this study. in case of male patients, mothers were first screened for carrier status to rule out x-linked cgd (xl-cgd). those patients where mother is not showing mosaic pattern were suspected for autosomal recessive cgd (ar-cgd) and were screened for the ratio of ncf1 gene to pseudo ncf1 gene by genescan analysis. additionally, evaluation of nadph oxidase components expression by flow cytometry also helped us to determine the underlined genetic defect and it is validated by dna sequencing of respective genes. results: eighty-seven patients were molecularly characterized to identify disease causing mutation which includes: 12 novel mutations in cybb, 1 in cyba, 2 in ncf2 gene in our cohort. 29.8% (n=26) of the patients belonged to xl-cgd. 58.6% (n=51) of the patients are suspected to have ncf1 gene defect among which; homozygous delgt mutation was identified in 39 patients. 6.8% and 4.5% patients showed abnormal p22phox and p67phox expression suggesting defect in cyba gene and ncf2 gene respectively. spectrum of mutations involve: 41% of delgt mutations, 15% nonsense, 14% missense, 10% deletion, 2% insertion, 14% other than homozygous delgt mutations. male to female ratio is 1.87:1. consanguinity is noted in 30% of the patients. conclusions: despite the male predominance ar-cgd is more common (70%) as compare to xl-cgd (30%) in this cohort of indian patients, which is distinct from the western data. 17% are the novel mutations suggesting, a wide heterogeneity in the nature of mutations in indian cgd patients. flow cytometric evaluation of nadph oxidase component is used as a secondary screening test to identify cgd sub-group. molecular characterisation of cgd genes was not only used in the confirmation of diagnosis but also in genetic counselling and pre-natal diagnosis in affected families. novel nlrc4 gain-of-function mutation presenting with neonatal enterocolitis and autoinflammation, with positive clinical response to rapamycin and anakinra. senior investigator, viral immunology section, national institute of neurological disorders and stroke 8 senior investigator, translational neuroradiology section, national institute of neurological disorders and stroke 9 staff clinician, neuroimmunology clinic, national institute of neurological disorders and stroke 1 0 staff clinician, laboratory of clinical immunology and microbiolology, niaid, nih introduction/background: cytotoxic t-lymphocyte antigen-4 (ctla-4) is an essential negative regulator of the immune response and its function is critical for immune homeostasis. uni-allelic mutation in the ctla4 gene leading to reduced function or expression of ctla-4, termed ctla-4 haploinsufficiency, can lead to systemic immune dysregulation with wide spread clinical disease, but with variable clinical penetrance. the neurological manifestations of ctla-4 haploinsufficiency are not known. objectives: to perform detailed phenotyping of the neurological manifestations of ctla-4 haploinsufficiency. methods: a retrospective review and prospective collection of clinical, imaging, cerebral spinal fluid, and pathological specimens was performed in a cohort of genetically confirmed patients (n=50) with ctla4 mutations who are followed at the national institutes of health. neurological symptoms and exams were collected on patient visits and from historical records. the data collected included 289 brain mris and 53 spinal cord mris that were visually inspected for evidence of inflammation. cerebral spinal fluid values were obtained from 14 patients including flow cytometry in 10 patients. pathological tissue from brain biopsies of inflammatory lesions was examined from 10 patients. results: central nervous system (cns) inflammation was found in 14/50 (28%) of the cohort. common clinical manifestations from the 14 patients with cns inflammation were headaches 13/14 and seizure 8/14. focal deficits were rare. mri findings included contrast enhancing neuroinflammatory lesions in the brain 14/14, brainstem/cerebellum, and spinal cord 8/12. figure a -c show representative inflammatory lesions. lesions were multifocal in 13/14 patients and 12/14 had recurrent inflammatory lesions on longitudinal follow up. lesions were, at times, extremely large, 5/14 with a lesion > 25 cm^3. leptomeningeal enhancement (lme) was seen in 10/13 patients and clearly preceded intraparenchymal lesion development in 3 patients. figure d shows a site of lme (green chevron) that develops into an intraparenchymal lesion (yellow chevron), mris are separated by 11 days. spinal fluid analysis showed a lymphocytic pleocytosis (mean 32 cells/mm^3) with the presence of oligoclonal bands in 6 patients. pathological features included a mixed cellular infiltrate, predominantly lymphocytes or plasma cells, with little evidence of demyelination or necrosis (figure e). conclusions: the neurological manifestations of ctla-4 haploinsufficiency include recurrent and, at times, severe neuroinflammation. however, even large lesions and lesions in eloquent anatomical locations had little to no focal clinical defects resulting in a striking clinical-radiological dissociation. future studies into the mechanisms of cns-related disease may reveal important information related to peripheral and central immune system functioning. conditioning with anti-cd45 immunotoxin in a mouse model of hypomorphic rag1 deficiency allows complete reconstitution of the immune system with lack of toxicity enrica calzoni, md 1 , cristina corsino, technician 2 , marita bosticardo, phd 3 , yasuhiro yamazaki, md phd 1 , hsin-hui yu, md phd 4 , lisa ott de bruin, md 5 , john manis, md 6 , rahul palchaudhuri, phd 7 , david scadden, md 8 , luigi d. notarangelo, md 9 treated mice and 80% in cd45-sap/200 treated mice. at sacrifice, in the bm we observed strong selective advantage for donor b cells at all developmental stages, but in particular in the most mature subsets, in both cd45-sap and cd45-sap/200 treated mice, rescuing the block in development at pre-b cell stage found in untreated f971l mice. donor engraftment in bm hsc reached levels around 70% and 80% in cd45-sap and cd45-sap/200 treated mice, respectively. donor chimerism in t and b cells in the spleen was also higher than 90% in both cd45-sap and cd45-sap/200 groups. in the thymus, full donor chimerism was achieved in both cd45-sap and cd45-sap/200 treated mice, starting at the dn4 stage and persisting at dp, sp4 and sp8. importantly, in both treatment groups, t cell development was corrected both in terms of subset distribution and absolute numbers to levels comparable to those of wt mice. finally, the thymic epithelial cell compartment was also fully reconstituted, with a normal number, distribution and maturation of both cortical and medullary thymic epithelial cells. conclusions: in conclusion, we show here that conditioning with cd45-sap immunotoxin, alone or in combination with 200rads tbi, is safe and leads to full reconstitution of the immune system in rag1 hypomorphic mice, suggesting that this conditioning regimen should be considered for testing in clinical setting. introduction/background: foxp3 is a key transcription factor for the maintenance of immune tolerance. foxp3 mutations result in dysfunction of foxp3+ regulatory t cells (tregs) causing immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome, a severe early onset autoimmune disease, which can be fatal if not promptly diagnosed and treated. our recent international study analyzing the longterm outcome in 96 i.e. patients of the two currently available treatments, pharmacological immune suppression and allogeneic hematopoietic stem cell (hsc) transplantation, showed poor long-term disease-free survival or overall survival limitations, respectively (barzaghi f. et al, jaci, 2017) . ipex syndrome is a good candidate for gene therapy as it has been demonstrated that reconstitution of wild-type treg cells can control the disease. however, foxp3 expression is highly regulated, and its safe and physiological expression in treg and teffector (teff) cells is challenging. lentiviral-mediated (lv) foxp3 gene transfer successfully converts ipex patients-derived cd4+ t cells into treg-like cells (cd4lv-foxp3 t cells) with stable suppressive capacity (passerini l. et al, sci transl med, 2013) . these ex vivo converted tregs are ideal as a short term cell-based therapy for ipex patients, but this approach does not reestablish regulated foxp3 expression in teff cells, that also likely contribute to the ipex pathology. thus, we are further characterizing cd4lv-foxp3 t cells and, at the same time, developing gene editing strategies for ipex, whereby autologous t cells or hscs are genetically modified or corrected, respectively, and reinfused into the patients. objectives: to provide more effective treatments for ipex patients, we are i) optimizing lv-foxp3 gene transfer in t cells to be suitable for clinical use, and ii) establishing a novel foxp3 gene editing in hscs and testing both approaches in preclinical models. methods: lv-foxp3 gene transfer can be obtained in cd4+ t cells activated polyclonally or in an antigen-specific manner. the vector construct is bidirectional, foxp3 expression is under the ef1a-promoter and the truncated form of ngfr, used as marker gene, is under cmv promoter. foxp3 gene editing is performed using a combination of crispr/cas9, a chemically modified sgrna targeting foxp3, and an aav6 packaged homologous donor dna template and the efficacy and safety of the resulting construct is tested in different cell types in vitro and in humanized mice. results: we demonstrate that cd4lv-foxp3 t cells can successfully be generated specific to different antigens. this result opens to new potential clinical benefit of cd4lv-foxp3 t cells with more safe and specific regulatory effect than polyclonal cd4lv-foxp3 t cells. we are currently adapting the protocol to optimal in vitro production for clinical use and assessing dose, survival and efficacy of the cd4lv-foxp3 t cells using different in vivo models. due to the wide distribution of identified mutations throughout the foxp3 gene, we have designed a gene editing strategy that uses homology directed repair to insert the coding sequence of the foxp3 gene at the start codon of the endogenous mutated foxp3 gene. this strategy permits regulated expression of the inserted wild-type, functional foxp3 protein in patient cells independent of the location of the downstream mutation. using this site-specific gene knock-in, we find that the system effectively targets expression of foxp3 in different cell types, namely tregs, teff cells and primary human cord blood-or bone marrow-derived hscs. gene editing of normal donor and ipex tregs and teff cells allowed us to test for regulated gene expression and for establishment of normal treg suppressor function and t cell proliferation upon activation. additionally, preliminary results demonstrate that gene edited hscs can be transplanted into nsg mice for long-term reconstitution. conclusions: our results show the feasibility of different gene therapy approaches for ipex syndrome. in addition, they suggest that cd4lv-foxp3 t cells, either polyclonal or antigen-specific, could be applied not only in ipex but also in immune mediated diseases of different origins. the results from the foxp3 gene editing support the use of crispr/ cas9 to treat ipex syndrome patients with autologous edited hscs. this gene editing approach may also be applied to treat other pediatric monogenic blood and immune disorders. human pi3kgamma deficiency with humoral defects and lymphocytic infiltration of barrier tissues andrew takeda, bs 1 , william comrie, phd 2 , yu zhang, phd 3 , paul tyler, bs 4 , koneti rao, md 5 , carrie l. lucas, phd 6 4 graduate student, yale university 5 clinician, niaid, nih 6 assistant professor of immunobiology, yale university introduction/background: the phosphatidylinositol 3-kinase (pi3k) signaling pathways play a key role in transducing signals from a diverse array of stimuli by producing the pip3 second messenger. class ib pi3k is primarily activated by g protein-coupled receptors (gpcrs), and this class is comprised of the p110gamma catalytic subunit in complex with the p84 or p101 regulatory subunit. in contrast to the class ia pi3k subunits, inherited mutations in the genes encoding the class ib subunits have not been described. objectives: given the leukocyte-restricted expression pattern of p110gamma, we hypothesized that mutations affecting this kinase may be found in cohorts of patients with rare immunodeficiency disorders. our objective was to identify such mutations and determine molecular, biochemical, and cellular derangements in patients with mutated p110gamma. methods: we used whole-exome sequencing of families to identify inherited gene mutations and determined the mechanistic basis of disease using biochemical assays to assess effects on protein function and cellbased assays to define functional defects with disease relevance. results: we identified a patient (here called a.1) harboring compound heterozygous mutations in pik3cg, the gene encoding p110gamma, who presented in early life with autoimmune cytopenias and eczema and, at the age of 9 years, developed cryptogenic organizing pneumonia and prominent t cell infiltration of the lungs. she also has a history of skin infections, lymphadenopathy/splenomegaly, eosinophilia, defective antibody production, and more recently, lymphocytic colitis. she inherited a frameshift pik3cg mutation from her mother and a missense mutation resulting in an r1021p amino acid substitution from her father. expression of p110gamma protein was lost, and stability of its p101 binding partner was reduced. despite defective t cell signaling responses to chemokines (i.e., gpcr stimulation), chemotaxis of patient t cell blasts in vitro was normal. intriguingly, the frequency of peripheral blood treg cells was low in patient a.1, and her cd4 t cells more frequently expressed the tissue-homing cxcr3 chemokine receptor. consistently, serum levels of cxcr3 ligands were elevated in patient a.1. moreover, we found augmented inflammatory cytokine production from m1-polarized macrophages differentiated from patient a.1 monocytes or from thp1 cells treated with p110gamma inhibitor or stably expressing pik3cg shrna. conclusions: we report the first human with loss of pi3kgamma activity and present her clinical presentation with notable t cell infiltration of barrier tissues. based on our analyses, we propose that loss of p110gamma activity in humans causes t cell-intrinsic effects of reduced tregs and increased tissue-homing propensity and the t cell-extrinsic effect of augmented inflammatory responses in macrophages. together, these consequences of p110gamma deficiency drive aberrant accumulation of t cells in lung and gut. introduction/background: pulmonary disease is a frequent complication across many primary immunodeficiencies (pidds), however its impact on the quality of life (qol) in pidds is not well characterized. objectives: to ascertain the types of infectious and non-infectious pulmonary complications occurring in pidds and to determine how these complications affect qol. methods: we analyzed the pulmonary complications, disability descriptions, and clinical status of 3610 subjects with pidds in the usidnet registry using descriptive statistics. karnofsky or lansky performance indices (n=1267) and promis29 qol data (n=120) were also analyzed. the t-test/mann-whitney test and chi square test were utilized to compare continuous and categorical variables, respectively. results: infectious pulmonary disease was reported in a majority of subjects (52.2%), most commonly pneumonia (42.3%) and bronchitis (17.9%). non-infectious pulmonary disease was reported in 30.8% of all subjects, most commonly asthma/reactive airway disease (21.7%), bronchiectasis (7.0%) and interstitial lung disease (4.2%). pulmonary insufficiency was listed as a cause of disability in 4.3% of all subjects with pidds, with highest rates of this disability in subjects with immune dysregulation (15.6%). lower karnofsky/lansky performance scores were observed in subjects with pneumonia, lung abscess, bronchiectasis, interstitial lung disease, and emphysema/copd as compared to without these disorders (p<0.001). promis29 qol metrics were largely similar among subjects with and without pulmonary disease, although physical function scores were significantly worse in those with copd/emphysema (mean= 37.5 +/-5.1) as compared to without (mean= 43.6 +/-8.4, p =0.007). promis29 physical function scores were also worse in subjects with non-infectious pulmonary disease (mean =40.3 +/-6.81) compared to those with infectious pulmonary disease only (mean =45.2 +/-8.53, p=0.046). a significantly greater percentage of patients with a history of copd/emphysema (24.0% vs. 8.95%) or interstitial lung disease (16.7% vs. 8.9%) were deceased as compared to those without a history of these disorders (p<0.0001). conclusions: both infectious and non-infectious pulmonary disorders cause significant morbidity in pidds and are associated with higher mortality in this population. infectious and non-infectious pulmonary complications were often associated with worse karnofsky/lansky scores while there was limited impact on promis29 qol measures. latin-american consensus on the management of patients with severe combined immunodeficiency, part 1: supportive measures during the time from diagnosis to definitive treatment. juan carlos bustamante ogando 1 , armando partida-gaytán 2 , francisco espinosa rosales 3 , lasid "consensus on scid" study group 4 1 pediatric allergy and clinical immunology specialist, clinical immunologist and researcher at primary immunodeficiency research unit, national institute of pediatrics 2 pediatric allergy and clinical immunology specialist, researcher at primary immunodeficiency research unit, national institute of pediatrics 3 pediatric allergy and clinical immunology specialist, president, fundación mexicana para niñas y niños con inmunodeficiencias primarias (fumeni) result in a majority of patients with late diagnosis, more comorbidities and reduced access to curative treatments. the interventions during such period are vital to keeping optimum health status to improve the probability of success of curative therapies. many interventions are not supported by clinical trials, are based mainly on clinical experience, and there are no clinical guidelines to standardize such treatments. objectives: to generate a consensus on the supportive care of patients with scid, from the diagnosis until a curative treatment is given, under a latin-american perspective taking into account particular challenges for our region. methods: in a first step, we gathered available information about scid diagnostic and therapeutic guidelines from two sources: a) literature search and b) personal communications with pid experts from europe and usa. next, we developed an expert consensus through a modified delphi technique (electronic and anonymous). we used google® forms® to gather the information and microsoft office excel® for the analysis of agreement through kappa coefficient and rounds concordance through repeated measures analysis of variance (anova). results: we gathered an expert panel of 34 subjects from 6 latin-american countries (argentina, brazil, chile, costa rica, mexico, and peru) including the primary centers caring for scid patients. we generated a document with 123 agreed diagnostic and therapeutic interventions grouped in 8 topic-domains (i.e. protective and isolation methods to decrease the risk of infections, antimicrobial prophylaxis, immunoglobulin treatment, immunizations, nutritional aspects, antimicrobial treatment, blood derivatives use, routine laboratory workup, imaging and other studies, conventional multidisciplinary approach). we also included 38 nonagreed interventions, but where relevant arguments are shared, to allow for particular clinical scenario decisions. conclusions: this is the first document of its type, and it intends to standardize clinical care of latin-american patients with scid, reduce disease burden and ultimately improve health outcomes. we see this effort as a starting point for the continuous improvement of our professional care to such patients and is intended to help as a tool not only for immunologists but for primary care physicians and other specialists involved in scid patient's care. this work will hopefully be published during 2018 as a lasid collaborative work, and it will help as a guide for clinicians caring for scid patients not only in latin america but in other world regions. also in the future, this consensus may be improved by collaboration from immunologists worldwide. no significant difference in hospitalizations one year before vs. year after treatment for prophylactic antibiotics (p=0.85) or igrt (p=0.07). baseline igg was higher in prophylactic antibiotics vs. igrt (770.6 vs. 914.4 mg/dl, p=0.03) . sex, severity of sad, igg subclasses deficiency, and lymphocyte counts were not significantly different between treatment groups. conclusions: prophylactic antibiotics are not inferior to igrt in preventing infections in some sad patients. while, clearly some patients with sad will need igrt, our date indicate that larger prospective studies are needed to identify patients who will benefit most from igrt vs prophylactic antibiotics alone. richard and barbara schiffrin presidents distinguished professor of microbiology and director, institute for immunology, university of pennsylvania introduction/background: t cell thymic development is dependent on signals received via the pre-tcr complex and we here report the first case of pre-tcr alpha (ptcra) autosomal recessive t cell immunodeficiency in an infant with a positive scid newborn screen (nbs). objectives: we sought to uncover the mechanistic links between ptcra mutations and immune dysfunction. methods: the patient was tracked clinically, with serial clinical immunophenotyping and t cell function testing. in addition, we performed deep immunophenotyping with mass cytometry and single cell rna sequencing to delineate the molecular circuitry underlying her immune phenotype. results: the patient presented with t cell lymphopenia and impaired response to mitogen stimulation. hsct was considered, but she did not meet clinical criteria and remained healthy, so she was watched closely on prophylaxis while awaiting genetic testing. response to serial mitogen stimulation remained between~20-100%, response to serial cd3/cd28 activation was normal and tcrv spectratyping was normal. whole exome sequencing revealed two mutations in ptcra. no prior human cases of ptcra deficiency have been published, but a mouse model bears a striking resemblance to this case (fehling et al, nature, 1995) , with elevated t cells and decreased t cells. her mitogen stimulation responses became persistently normal around 2 years of age with stable t cell lymphopenia, elevated t cells and normal switched memory b cells. anti-fungal prophylaxis was halted, and she remained on atovaquone alone with persistent t lymphopenia. she was able to mount an antibody response to rabies vaccine at 2.5 years and was weaned off scig replacement and is planned to initiate vaccination. deep immunoprofiling with mass cytometry (cytof) demonstrated a unique immunophenotype. single cell rna sequencing confirmed normal cd4 and cd8 tcr clonotypic diversity but increased clonotype diversity in t cells and increased and transcript levels. in addition, cd4 naïve, cd4 memory and cd8 naive t cells demonstrated both increased numbers of expressed genes and transcriptomic diversity, with altered cytoskeletal and tcr proximal signaling pathways across t cell subsets versus control. this may reflect a peripheral role for ptcra, a durable imprint of thymic signaling events mediated by ptcra, evidence of homeostatic proliferation or a combination of the above. conclusions: scid nbs led to identification of homozygous variants in ptcra causing a novel t cell immunodeficiency characterized by t cell lymphopenia, altered proximal tcr and cytoskeletal signaling and increased number of altered t cells. we will continue to pursue the mechanism of these mutations by developing ipsc and studying their t cell differentiation capacity in vitro, as well as further defining her immunometabolic phenotype. rag1 hypomorphic mouse mutants show partial preservation of thymocyte development but peculiar abnormalities of thymic epithelial cell phenotype introduction/background: the recombination-activating gene (rag) 1 and rag2 proteins are essential for v(d)j recombination. in the absence of these proteins, the development of b and t cells is blocked at early progenitor stages, resulting in severe combined immunodeficiency (scid). hypomorphic mutations in rag1, allowing residual activity, result in delayed-onset combined immunodeficiency with residual development of t and b lymphocytes, associated with autoimmunity and/or granulomas (cid-g/ai). objectives: to study in details the effect of rag1 hypomorphic mutations at the early stages of t cell development, we have generated 3 mouse models carrying mutations described in patients with cid-g/ai (r972q, r972w, f971l) (niaid animal protocol: lcim 6e). methods: we performed an extensive evaluation of the thymic phenotype in the 3 mouse models. results: the number of total thymocytes was found to be drastically reduced in all three models. however, two of these mouse models (r972q and f971l) retained a significant level of rag1 activity, and resulted in the development of mature t cells in the thymus, while the mouse model carrying the r972w mutation had minimal rag1 activity and presented a phenotype more similar to that of complete rag1 knockout mice. in r972w mice, almost all thymocytes were blocked at the double negative (dn)3 stage and there were virtually no mature t cells, as found in rag1 ko mice. on the other hand, r972q and f971l mice presented double positive (dp) and single positive (sp)4 and sp8 cells. the cross talk between t cells and thymic epithelial cells (tec) in the thymus is fundamental for the development and maturation of both types of cells. in rag1-/-mice, and consequently in the absence of mature t cells, tecs cannot complete their maturation, and the mtec subset is virtually absent. these results were also observed in the r972w mouse model. instead, in r972q and f971l mice, the residual rag1 gene activity allows development of a reduced number of mature t cells. although the number of tecs was markedly reduced in r972q and f971l mice, ctecs and mtecs were both present, but with an excess of ctecs. furthermore, mtecs were predominantly mhc-iihigh (mtechi), and only a minority of mtecs were mhc-iilow (mteclo) cells, the opposite of what found in adult wt mice. finally, mtechi cells from rag1 mutant mice were found to express aire to levels and frequencies comparable to those of wild-type (wt) mice. conclusions: our results show that tec in mouse models carrying rag1 hypomorphic mutations are affected both in terms of absolute numbers and in terms of subset distribution and maturation state. to further investigate the functional consequences of impaired cross-talk between thymocytes and tecs in rag1 mutant mice, we have performed rnaseq in sp4 and tecs sorted from r972q, f971l and wt mice. analysis of the gene expression profile in tec may thus provide novel insights in the mechanisms that govern normal and pathologic thymic t cell development. vedolizumab for autoimmune enteropathy in primary immunodeficiency: a case series of outcomes introduction/background: gastrointestinal complications are common in patients with primary immunodeficiency. infections are the leading cause, but autoimmune enteropathies including inflammatory bowel disease (ibd)-like colitis, sprue-like enteropathy, and nodular lymphoid hyperplasia (nlh) have been recognized in a subset of these patients. to date, there is no established treatment for these noninfectious disorders. vedolizumab is a humanized monoclonal antibody that binds to the alpha-4 beta-7 integrin, inhibiting the migration of memory tlymphocytes across the endothelium into inflamed gastrointestinal parenchymal tissue. it is fda approved as first-line therapy for inflammatory bowel disease. the safety and efficacy of treating autoimmune enteropathy with vedolizumab in patients with concurrent primary immunodeficiency (pid) has not previously been reviewed. objectives: to review the outcomes of a series of patients with hypogammaglobulinemia and autoimmune enteropathy following vedolizumab therapy methods: 7 patients (3 male, 4 female) at mount sinai with enteric biopsies demonstrating inflammatory enteropathy with t cell infiltrates have been treated with vedolizumab. results: five of the seven patients completed induction therapy. one patient was recently started on therapy. therapy was aborted in one patient who developed acute hepatitis during induction. another developed severe cytomegalovirus enteropathy, prompting discontinuation. two patients discontinued therapy due to response failure. at present, two patients remain on therapy at 12 months with symptomatic improvement. conclusions: vedolizumab was effective in 2 cases, but had no benefit or deleterious side effects in 4 subjects. its effectiveness in another patient is presently under investigation. introduction/background: ataxia telangiectasia (at) is an immunodeficiency most often associated with t cell abnormalities and abnormalities in serum immunoglobulin levels, primarily iga. there is a subset of patients with a hyper-igm phenotype, some with cutaneous granulomas, which may reflect a distinct clinical phenotype. a 5 yearold female presented for evaluation of concern for immunodeficiency because of frequent illnesses, presumed to be viral. she was found to have an ataxic gait, some speech delay, mild ocular and ear pinna telangiectasia, and an ulcerative rash on the left upper and right lower extremity. initial blood work showed elevated -fetoprotein levels (50 ng/ml), elevated serum igm (719 mg/dl), low igg (<75 mg/dl), and iga (0.9 mg/dl). objectives: to determine if the atm mutations in this patient are associated with perturbations in the frequencies, distributions and functions of b and t cell subsets which account for the observed phenotype. methods: next generation sequencing was used to identify the mutations in the atm gene. b and t cells were purified from the patients peripheral blood by positive selection. intracellular staining for foxp3 and t-bet was performed. b cells were activated in the presence of polyclonal f(ab)2 rabbit anti-human igm, multimeric, soluble, recombinant-human cd40l, gardiquimod (tlr7 agonist), or cpg (tlr9 agonist). the treg suppression assay was carried out by co-culturing cd4+cd25hicd127lo tregs and cd4+cd25cd127+ responder t cells at a 1:1 ratio in the presence of beads loaded with anti-cd2, anti-cd3, and anti-cd28 for 4.5 days. results: next generation sequencing revealed two pathogenic mutations in the atm gene, a novel mutation creating a premature stop codon [c.237dela,(p.lys79asnfs370)], and a nonsense mutation [c.3372c>g, (p.tyr124ter)]. proliferative responses of pbmc to mitogens (pha, cona, pwm) were reduced to roughly half of the control responses; the response to tetanus was normal whereas the response to c. albicans was absent. serum cytokine analyses demonstrated elevations in levels of tnf (14.5 pg/ml) and il-10 (7 pg/ml); levels of ifn, il-2, il-5, il-6, and il-12 were below the limits of detection. b cell abnormalities included markedly increased percentages of cd38locd21lo cells (40%) expressing t-bet and fas. activation of these cd21/low b cells through the b cell receptor, tlr7 and tlr9, and cd40 was decreased in response to all of the stimuli as evidenced by a lower percentage of b cells expressing the activation markers cd69 and cd86 relative to healthy control samples. the frequency of unswitched cd27+igd+ memory b cells was also increased (54%). among the naive b cells, the proportion of cd19+ cd27cd21cd10+igmhi transitional b cells that newly emigrated from the bone marrow (bm) was found to be diminished to 1.1% of the naive b cell compartment. in the t cell compartment, there was a decreased frequency of total cd3+ cells but normal absolute numbers of cd3+cd4+ t cells. there was also a decreased proportion of naive cd3+cd4+cd45rocd62l+ t cells and a striking increase in the cd3+cd4+cd45ro+ memory t cells (90%). this appeared to be largely attributed to the increased proportion of cd3+cd4+cd45ro+cd62l effector memory t cells (63%). the circulating t follicular receptor (ctfh) cell frequency in the patient was 5-fold higher (19%) than the average for healthy donors but icos expression levels were normal. treg frequency was decreased but suppressive capacity was not impaired. conclusions: the mutations in atm described here add to the growing understanding of the heterogeneity in degree and complex nature of the immunodeficiency seen in patients with at. these mutations resulted in perturbations in frequencies and distributions of normal and atypical b and t cell subsets, which can explain some immunologic aspects of the clinical phenotype in this patient. the immunophenotype seen here may also differentiate at patients with granulomas from those without cutaneous lesions. supported in part by grifols, the joanne siegel memorial fund, the dreizessen fund (to ewg), grants from niams t32 ar007107-41 (km) and niaid r01 ai071087 (em). introduction/background: a 10-month-old male presented with pancytopenia, b cell deficiency and developmental delay. he was born at 36 weeks with weight of 2.3kg. he was severely anemic with a hb of 7.4, and transfused on day 2 of life. he received hep b and bcg vaccines without complications. a month later he had a hb of 3.6 with a febrile illness. a bone marrow aspiration performed at 45 days, showed dyserythropoiesis without hemophagocytosis, and normal numbers of precursors. t and b cells were decreased. further evaluation with repeat bone marrow showed decrease in all 3 cell lineages. exome sequencing of the family showed homozygous variant in mysm1 (c.899_902delp. (lys300arg/s*11) omim: *612176) in the patient. both parents and hla-matched sister, were heterozygous for the same variant in mysm1. treatment consistent of replacement immunoglobulin, packed rbcs, and g-csf. there was no history of recurrent viral or severe bacterial infections except for 2-3 episodes of urinary tract infections, which were treated with antibiotics. physical exam revealed low set ears, sunken and wide set eyes, depressed nasal bridge, mild micrognathia, frontal bossing, and 1 cm x 1 cm cafeau-lait spot noted behind left knee. he was pancytopenic with a wbc count ranging 600/mcl to 6300/mcl, and anc ranging from 10/mcl to 4500/mcl (on intermittent g-csf). hb = 7.7 gm/dl requiring transfusions every 3-4 weeks, and platelet count was 91,000/mcl. b cell deficiency was confirmed with total b cell count of 36 cells/mcl. b-cell maturation was essentially normal. t cell counts were normal with ageappropriate distribution of naïve and memory t cells, and t cell function. there was normal t cell receptor repertoire diversity. objectives: to assess defect in dna repair using a flow cytometry-based assay in a patient with mysm1 deficiency. methods: patients with mysm1 deficiency are reported to have increased genomic instability. deb testing, and telomere length analysis revealed normal results. defects in the dna repair pathway were assessed using a flow cytometry-based assay measuring phosphorylation of atm (patm), smc1 (psmc1) and h2ax (gh2ax) without irradiation, or 1h or 24h after low-dose (2gy) radiation using a cs137 source. the analysis was performed in t, b and nk cells. results: the patient had higher patm and psmc1 in t cells compared to the experimental controls (hc) at 1h post-irradiation. also, the amount of gh2ax in nk cells was significantly higher than hc at 1h post-irradiation. interestingly, the patients b cells showed approximately 12% of b cells with constitutive gh2ax even without irradiation, and this subset increased slightly to 16% at 1h after irradiation. the mfi (amount) of gh2ax also increased at this time-point. at 24h post-irradiation, there was normal dephosphorylation in healthy control lymphocyte subsets. however, the patients t cells did not de-phosphorylate completely and showed higher residual patm, and psmc1 in both t and b cells. also, both t and b cells, at 24h, demonstrated a small subset of t cells (1%) with constitutive gh2ax without irradiation, which increased to 4% after irradiation. there was also an increase in gh2ax mfi in the irradiated sample. in b cells, 6% showed constitutive gh2ax without irradiation at 24h, and this increased to 37% after irradiation, with a corresponding increase in mfi. conclusions: in summary, this rapid flow analysis revealed defects in the dna repair pathway, including higher patm, psmc1 and h2ax phosphorylation in t, b and nk cells at 1h post-irradiation. at 24h, only t cells showed a residual subset with patm expression. but, psmc1, a downstream target of atm, revealed higher levels in t, b and nk cells at 24h post-irradiation. this assay, which allows lineage-specific analysis, permitted dissection of dna repair defects, in individual lymphocyte subsets revealing heterogeneity within the cell subset to radiation susceptibility. the practical benefit of this rapid multi-parameter flow assay is selection of appropriate conditioning regimen for hematopoietic transplantation, as was the case with this patient. this has significant practical implications for treatment of patients with radiosensitive immunodeficiencies. ctla-4 haploinsufficiency-associated inflammation can occur independently of t-cell hyperproliferation introduction/background: cd28 and ctla4 provide opposing proliferative signals to t cells. we identified an 18-year-old female subject (s1) with heterozygous deletions of cd28 and ctla4 and multi-organ inflammatory disease characterized by a lack of t cell infiltrates in affected organs. inflammatory disease was remarkably responsive to s1 ctla4-ig therapy. objectives: our goal was to characterize the immunologic consequences of combined deletion of cd28 and ctla4, specifically assessing t cell proliferation and treg function in comparison to patients with alps5associated ctla4 haploinsufficiency. we further sought to explain how this s1s inflammatory diseases could occur without a pathologic t cell infiltrate and why they were amenable to ctla4-ig therapy. methods: we performed phenotypic analyses of subject t cells and innate lymphoid cells (ilcs). we functionally characterized subject t cells. we created serum cytokine profiles. we stained and analyzed tissue biopsies. results: cd28 and ctla4 expression on s1 t cells were half that of control t cells. s1 t cells were hypoproliferative. s1 tregs were scarce and lacked suppressive function similarly to alps5 tregs. s1 tregs could suppress autologous t responder cells, likely due to their poor proliferative capacity. s1 colonic biopsies featured significantly fewer infiltrating intraepithelial lymphoid cells than biopsies from an alps5 patients. unlike alps5 patients whose colonic gland infiltrates were overwhelmingly t cells, s1 intraepithelial lymphoid cells were neither t cells nor b cells, suggesting the presence of ilcs. indeed, a greatly expanded population of type 3 innate lymphoid cells (ilc3) and prototypical ilc3 cytokines were identified in s1 peripheral blood. ilc3 frequency and cytokine levels decreased in response to treatment with ctla4-ig, corresponding with marked improvement in enterocolitis, hepatitis and pericarditis. conclusions: we report a novel genetic syndrome of combined cd28/ctla4 deletion and describe the immunolopathologic correlates of this disease. dual ctla4-and cd28-haploinsufficiency results in a phenotype of multi-organ inflammatory disease characterized by ilc3 expansion in the setting of t-cell hypoproliferation and quantitative and qualitative treg defects. our patients clinical response to ctla4-ig parallels published mouse studies and suggests the existence of additional stimulatory b7 receptor(s) preferentially expressed on ilc3s over conventional t-cell populations. diagnosis of radiosensitivity and dna repair defect in dna ligase iv deficiency with a rapid flow cytometry assay. introduction/background: dna ligase 4 deficiency (lig4-scid) is one of several monogenic defects affecting dna repair, and causing lymphopenia (t-b-nk+) and a radiosensitive scid (rs-scid) phenotype. the assignment of a timely diagnosis is vital in the management of patients with rs-scid. laboratory assessment of radiosensitivity is laborious, and utilizes fibroblasts (non-hematopoietic) or lymphoblastoid cell lines, and can take several weeks to months for results. objectives: we demonstrate for the first time, the application of a flow cytometric-based kinetic analysis of phosphorylated h2ax (h2ax) in lymphocyte subsets, especially nk cells, for the diagnostic assessment of lig4-scid. methods: simultaneous measurement of multiple dna repair markers phosphorylated (p) atm, smc1 and h2ax (h2ax) was performed by flow cytometry to assess dna repair defects in a 3-year-old korean female. the patient was evaluated for recurrent fevers, chronic respiratory tract infections, chronic diarrhea, and rash. genetic testing revealed compound heterozygous variants (nm_001098268, c.1341g>t, p.trp447cys and nm_001098268, c.1103a>t, p.asp368val) in lig4. functional assessment (phosphorylation) was measured in t and nk cells (b cells were absent), before irradiation (background control), or after low-dose (2gy) irradiation (1 and 24 hours). results: we observed maximal h2ax generation at 1 hour post-irradiation, with progressive dephosphorylation at 24 hours post-irradiation in healthy controls. the patient showed normal frequencies (%) of t cells and nk cells positive for h2ax (95.62% and 99.40% respectively); (controls (n=2) t cells = 99.29% and 99%; nk cells = 99.6% and 99.11%), but increased intracellular levels (mean fluorescence intensity, mfi) of h2ax (t cells = 50.67 and nk cells = 52.41) compared to controls (t cells = 24.86 and 19.58; nk cells = 41.33 and 35.03) at 1 hour post-irradiation. however, more importantly, at 24 hours post irradiation there was a lack of dephosphorylation in a substantial proportion of lymphocytes (64% of t cells and 99% of nk cells) compared to healthy controls (t cells= 1.75% and 1.51%; nk cells = 9.27% and 17.83%). further, while there was dephosphorylation of h2ax at 24h in patient lymphocytes as compared to 1h, the amount, as measured by mfi, remained elevated at 24h (t cells = 8.97, nk cells =7.57) compared to controls (t cells =1.71 and 2.25; nk cells = 3.18 and 2.75). the data from patm and psmc1 were uninformative for the evaluation of lig4-scid. conclusions: flow-based kinetic analysis of h2ax is a useful marker for the diagnosis of lig4-scid, and can be performed with a small amount (5cc) of blood, and provides a result in 3-4 days, facilitating rapid assessment of radiosensitivity in this condition. human plcg2 haploinsufficiency results in nk cell immunodeficiency and herpesvirus susceptibility objectives: we aimed to investigate the cause of disease in three patients from two kindreds with recurrent or severe herpesvirus infections and nk cell dysfunction. methods: we used exome sequencing and mass cytometry (cytof), as well as traditional immunologic techniques, to investigate the genetic causes, immune cell subpopulations/signaling, and nk cell function of these patients. we additionally used mouse models, crispr cell lines and in vitro assays to assess the role of plcg2 haploinsufficiency in disease. results: kindred a consisted of two patients presenting with hsv1 susceptibility and autoimmunity. kindred b consisted of one patient with severe cmv myocarditis and adenoviral hepatitis. both kindreds were evaluated for nk cell function and showed reductions in target killing in spite of normal cytotoxic granule degranulation against the same target. microscopy analysis suggested that granule mobility was reduced in at least one kindred. cytof revealed reductions in plcg2 phosphorylation after receptor crosslinking in the nk cells of both kindreds. kindred a also presented with a reduction in naïve b cells without perturbations in immunoglobulin output, b cell memory formation or class switching. trio whole exome sequencing was performed and revealed rare heterozygous plcg2 mutations in both kindreds. functional analysis, as well as mouse and crispr models, support a functional haploinsufficiency as a cause for nkd in these patients. conclusions: heterozygous loss-of-function point mutations in plcg2 have not been previously investigated as a cause of nk cell deficiency or recurrent herpesvirus infection. thus, these patients represent a novel immunodeficiency involving plcg2 haploinsufficiency, nk cell dysfunction, and herpesvirus susceptibility. hypomorphic rag1 mutations alter the pre-immune repertoire at early stages of lymphoid development introduction/background: human rag deficiency is associated with a spectrum of clinical phenotypes. while the most severe forms of rag deficiency manifest with severe combined immune deficiency or omenn syndrome since the first weeks of life, more recently patients have been identified who present to medical attention at a much older age predominantly with symptoms of autoimmunity and/or inflammation. many of the mutations associated with this atypical syndrome are found in the c-terminal domain (ctd) of the rag1 gene and allow residual development of t and b cells. these patients have an abnormal peripheral t and b cell repertoire, but how this is affected by abnormalities in the composition of the pre-immune repertoire vs. antigen-mediated selection and homeostatic proliferation in the periphery is unknown. objectives: in order to investigate whether mouse models with hypomorphic mutations in the rag1 ctd recapitulate the phenotype observed in patients with cid-g/ai, and to study how these mutations affect repertoire composition, cell selection and survival during t and b cell development, we generated three mouse models carrying homozygous rag1 mutations (f971l, r972q, and r972w), corresponding to human mutations (f974l, r975q, r975w) previously reported in patients with late-onset combined immune deficiency with granuloma and/ or autoimmunity (cid-g/ai). methods: mice were generated using crispr/cas9 mediated gene editing. t and b cell development, including apoptosis was studied by flow cytometry. immunoglobulins in naïve mice, baff levels and specific antibody responses were measured by elisa. serum igm autoantibodies were measured using a microarray (utsw). analysis of t cell receptor (trb) repertoire in several sorted t cell populations and immunoglobulin heavy chain (igh) repertoire in pre-b cells was performed by adaptive biotechnologies. in order to be able to detect both dj and vdj rearrangements, pro-b cells and spleen b cells were sequenced using high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (htgts-rep-seq). analysis of vk-jk rearrangements in pre-b cells was performed by pcr amplification. results: immunological characterization showed partial development of t and b lymphocytes, with persistence of naïve cells, preserved serum immunoglobulin, but impaired antibody responses and presence of autoantibodies, thereby recapitulating the phenotype seen in patients with cid-g/ai. by using high throughput sequencing, we identified marked skewing of igh v and trb v gene usage in early progenitors, with a bias for productive rearrangements after selection occurred, and increased apoptosis of b cell progenitors. this suggested that more alleles remained in germline configuration. moreover, in the rearranged igh loci, the distal v gene segments were preferentially rearranged already at the earliest stages of b cell development, a finding that has not been previously reported. in addition, rearrangement at the igh locus was impaired, and polyreactive igm antibodies were detected. conclusions: in conclusion, this study demonstrates that hypomorphic rag1 mutations reported in cid-g/ai cause abnormalities of the primary b and t cell repertoire. these changes may affect survival and selection of t and b cells, and thereby contribute to the immune dysregulation often seen in patients with cid-g/ai. senior clinician, nih/niaid/lcim introduction/background: autosomal dominant hyper ige syndrome (ad-hies) is a primary immunodeficiency due to loss of function stat3 mutations. disease manifestations include recurrent skin and pulmonary infections, eczema, mucocutaneous candidiasis, as well as tion and in vitro studies demonstrated that the enhanced signaling could be controlled by ruxolitinib, an approved jak1/2 inhibitor. informed by these experimental data, the patients were treated with ruxolitinib with remarkable improvement in a variety of clinical end-points, including hematological profiles and growth parameters. conclusions: this characterization of a human jak1 gain-of-function mutation expands our current understanding of the role of jak1 in eosinophil biology, hematopoiesis and immune function. chronic granulomatous disease, ornithine transcarbamylase deficiency and x-inactivation is a primary immune deficiency characterized by defects in the nadph oxidase enzyme complex resulting in a susceptibility to a narrow spectrum of bacteria and fungi. mutations in cybb encoding gp91phox and located at xp21.1 are associated with the most common form of cgd. deletions and rearrangements in this region are associated with other genetic diseases such as mcleod syndrome (xk), retinitis pigmentosa (rpgr), duchenes muscular dystrophy (dmd), ornithine transcarbamylase deficiency (otc), and x-linked mental retardation (tspan7). patient phenotype depends on the extent and position of the deletion, creating a "contiguous x-chromosome gene deletion syndrome. objectives: we report a four-year-old female, who presented with symptoms of x-linked cgd and otc deficiency with a large, contiguous multi-gene deletion on the x-chromosome. methods: we report a four-year-old female, who presented with symptoms of x-linked cgd and otc deficiency with a large, contiguous multi-gene deletion on the x-chromosome. the patient is the second born of a set of non-identical triplets from a clomiphene assisted pregnancy at 35 weeks gestation. (weight at birth: 5lbs 2oz). after a 7 day stay in the nicu, and cpap for one day she was discharged home. by the second month, she began having problems gaining weight and had persistent vomiting. at 3 months, she was admitted with a pneumonia diagnosed as methicillin resistant staphylococcus aureus by lung biopsy. during that hospitalization, the mother noted an enlarging lesion on the infants left hand which was biopsy proven serratia marscecens osteomyelitis and was treated with intravenous cefepime for 6 weeks. at this point a dihydrorhodamine assay (dhr) showed only 15.1% positive cells (nl 80-100%) and she was diagnosed as a carrier of x-linked cgd. bactrim was initiated for antibacterial prophylaxis but it was discontinued due to recurrent diarrhea; she was unable to tolerate antifungal prophylaxis as well due to liver function abnormalities. she continued having frequent vomiting episodes, irritability, failure to thrive and development delay. ulcerations in esophagus were confirmed by egd and colonoscopy, probably related to persistent emesis. diarrhea was unresolved. at 30 months of life she was admitted with acute encephalitis, a serum ammonia level of 218 and elevated urine orotic acid. results: given the demonstrated carrier status for cybb and apparent otc deficiency, comparative genomic hybridization was performed, revealing a deletion from xp21.1 to xp11.4 this 3.9mb loss includes 18 genes that are known to cause disease. since the diagnosis, the patient has been on reduced protein diet, resolving her diarrhea, she has subsequently grown and is on 5th percentile for weight and height. her dhr is now 28% positive. at 3 years she was diagnosed with cone rod dystrophy. currently she is on bactrim for cgd prophylaxis and l-citrulline for the otc deficiency. she continues to gain weight, and has shown great improvement in her development delay and no new cgd-related infections have recurred. conclusions: this case reminds us that x-linked carriers with large deletions may be symptomatic and genetic analysis to determine other affected genes can be important for medical management. introduction/background: wiskott-aldrich syndrome (was) is a rare and severe x-linked disorder with variable clinical phenotypes correlating with the type of mutations in the was gene. the long-term prognosis of this syndrome is generally poor, with hematopoietic stem cell transplantation (hsct) remaining the only curative choice. the syndrome is poorly characterized in china. objectives: we retrospectively reviewed patients with was referred to our hospital from 2004 to 2016, and summarize their clinical manifestations and genetic features. methods: sixty-four children suspected to be was from 62 unrelated families were enrolled in this study. the clinical data of children were reviewed in the present study. distribution of lymphocyte subsets from peripheral blood and was protein (wasp) expression in peripheral blood mononuclear cells was examined by ow cytometry (fcm). wasp mutations were identified by direct sequencing of pcramplified genomic dna results: among 725 patients with primary immunodeficiency diseases (pid), 40 (5.52%) were finally diagnosed as was with gene identified. the mean time of diagnosis was 6.25 months (range, 0.4-9.63). the common onset clinical manifestation was diarrhea, and most patients had recurrent upper respiratory tract infection, otitis media, pneumonia, and skin abscess. one patient had nephrotic syndrome and no patient with malignancy. all patients had classical was phenotype with was clinical scores 3-5. total 36 mutations in wasp were identified, including 14 novel mutations. six patients received hsct, five survived, with one died because of gvhd. compared with the other 24 patients without wasp mutations, was patients had lower numbers of cd4+ t cells and b cells, and higher eos and ige level. there was a negative association between the number of b cells and the was clinical scores. conclusions: in china, diagnosis of was has improved over the last decade, although a much higher number of cases had been expected. establishing more diagnostic centers dedicated to the care of pid will facilitate early, correct diagnosis and better care of was in china. regulatory cells (tregs) are precisely quantified by measuring demethylation of the treg-specific-region of foxp3. more recently, we have identified highly cell type-specific dna regions of demethylation for further cell populations. objectives: this novel technology allows the implementation of differential immune phenotyping into the newborn screening procedure. here, we aimed at epigenetically quantify multiple immune cell types in different biological samples including dried blood spots and samples from patients with pid with immundysregulation, where currently no approach is available. methods: using cell type-specific demethylation sites, we developed epigenetic qpcrs for quantification of t-, treg, b-, nk-, monocyte and granulocyte cell population. epigenetic qpcr is applicable for relative and absolute quantification in whole blood and dried blood spots using isolated, bisulfite converted dna. results: we demonstrated >95% concordance with flow cytometric analyses of the same fresh blood samples from healthy subjects. we have validated the method in 30 children with symptoms of immundysregulation resulting from monogenic defects, including for example foxp3, cd25, stat1, ctla4, and leading to treg/teffector cell imbalance where treg deficiency has been difficult to assess by flow cytometry. furthermore, we tested 250 dried blood spot (guthrie card) samples from healthy newborns and 30 patients with diverse pids and correctly identified 29/30 pid patients, indicating that this method holds promise for newborn screening. conclusions: the method we established for immune cell quantification based on epigenetic cell type-specific markers, is feasible and reliable in biological samples either fresh, frozen or archived, including dried blood spot. the analysis of further immune cell types introduces an innovative opportunity to diagnose a variety of pids and immunodysregulatory disorders as early as newborn screening. pancytopenia and immunodeficiency with mds in an infant due to samd9l mutation we present our data on behalf of the pcid study consortium of the inborn errors working party worth profound combined immunodeficiencies (p-cid) are inherited diseases with impaired t-cell function leading to infections, immune dysregulation or malignancies. genetic, immunologic and clinical heterogeneity make patient specific decisions on indication and timing of hematopoietic stem cell transplantation (hsct) difficult. objectives: since 2011 the pcid study recruits non-transplanted p-cid patients aged 1-16 years to prospectively compare natural histories of age and severity-matched patients with or without subsequent transplantation and to determine whether immunological and/or clinical parameters may be predictive for outcome methods: our prospective/retrospective international observational multicenter study recruits pediatric p-cid patients to identify biomarkers and clinical parameters that are predictive of outcome. results: so far >130 growth hormone deficiency comprised the majority of neuroendocrine cases (65%, 11/16). other notable neurologic diagnoses included headache (17%, 211/1227), seizure (5%, 62/1227), cerebrovascular accident (2%, 25/1227), and neurologic tumors (0.4%, 5/1227). in addition to somatic neurologic conditions, many cvid patients (37.7%, 462/1227) had reported diagnoses of depression, anxiety, and post-traumatic stress disorder. conclusions: our findings suggest that neurologic diagnoses are more common in cvid patients than previously recognized. patients with neurologic autoimmune disease appear to have a more severe phenotype with earlier age at symptom onset. many cvid patients had depression, anxiety clinical outcomes of human herpesvirus 6 reactivation after hematopoietic stem cell transplantation prognostic factors and outcome of epsteinbarr virus dnaemia in high-risk recipients of allogeneic stem cell transplantation treated with preemptive rituximab cytomegalovirus in hematopoietic stem cell transplant recipients daratumumab controls life-threatening post-hsct autoimmune haemolytic anaemia objectives: we present a 17 month old pancytopenic male who was diagnosed with myelodysplastic syndrome (mds) with monosomy 7 due to samd9l mutation. methods: whole exome sequencing was performed on a male, who presented at 8 months of age with findings concerning for a bone marrow failure (bmf) syndrome despite a normal bmf genetic panel. results: the patient presented at 8 months of age, with severe pancytopenia, fevers, e. coli bacteremia, pancolitis, and echtyma gangrenosum. bone marrow showed severe aplasia with occasional macrophages.he was treated with antibiotics, as well as steroids, etoposide and cyclosporine for presumed hemophagocytic lymphohistiocytosis (hlh). a bone marrow failure and hlh genetic panels were normal conclusions: this is one of the first reported cases of samd9l mutations causing mds since its initial discovery earlier this year. samd9l mutation should be considered in patients who present with pancytopenia and monosomy 7 mds. as new defects continue to be identified, further evaluation outside of typical bmf panels may be relevant primary immune deficiency disease in patients over age 60: an analysis from a proprietary immunology patient registry roger ucla school of medicine 2 director objectives: to characterize the prevalence of pidd among older individuals using a patient database maintained by the consortium of independent immunology clinics (ciic), comprised of 17 specialty immunology outpatient practices in the us. methods: patients with pidd were identified in the ciic database using icd-10 codes d80 conclusions: our data suggest that pidd in patients over age 60 may be more prevalent than previously reported introduction/background: the patient is a 6-month-old boy, born at 35+4 weeks gestation to non-consanguineous parents of italian origin. he was admitted to the intensive care unit at 12 days of age with profuse bloody diarrhoea, weight loss, severe metabolic acidosis and acute renal failure. he had a rapid respiratory deterioration necessitating intubation, ventilation and inotropic support. the patient developed features of macrophage activation syndrome with: (i) prolonged fever > 38.5°c, (ii) hepatosplenomegaly, (iii) bicytopenia (anaemia and thrombocytopaenia), (iv) hypertriglyceridemia, (v) high ferritin (14,7000) and (vi) haemophagocytosis on bone marrow smear. he also presented with three interesting features (i) a macular erythematous rash that slowly resolved and was replaced by reticulo-livedoid rash, (ii) a marked hypereosinophilia and (iii) no significant elevation of hladr/cd8+ t cells on lymphocyte immunophenotyping (7-15%). the patient underwent rectal biopsy which confirmed the presence of eosinophils, but without significant inflammation or architectural changes. stool microscopy showed presence of partially necrotic intestinal epithelial cells. immune work up demonstrated global t lymphopaenia without balanced subpopulation and eliminated a familial hemophagocytic lymphohistiocytosis (normal perforin and cd107a expression on cd8+ t-cell and nk cells). circulating foxp3+ cd25+cd127lowcd4+ t cells were within normal range. a dihydrorhodamine reduction assay was normal. 9 chief, laboratory of clinical immunology and microbiology, national institute of allergy and infectious diseases, national institutes of health introduction/background: hematopoietic stem cell transplantation (hsct) has been used for the treatment of hematologic malignancies and primary immunodeficiencies (pid), for several decades with increasing efficacy. however, toxicity related to conditioning regimens based on the use of chemotherapy and/or irradiation to ensure engraftment of donor cells, remains a significant problem. recently, an alternative, potentially low-toxicity, approach has been proposed, which makes use of an immunotoxin targeting cd45-expressing cells, which include hsc and more mature leukocytes. this approach is particularly attractive for leaky forms of severe combined immune deficiency (scid), with residual production of dysfunctional t and/or b cells, such as atypical forms of rag deficiency. objectives: we have developed a mouse model carrying a hypomorphic mutation in the rag1 gene (p.f971l) resulting in a combined immunodeficiency with signs of autoimmunity, recapitulating the phenotype seen in patients. methods: using this model, we have tested the efficacy of conditioning with an anti-cd45 immunotoxin (cd45-sap) alone or in combination with low irradiation (200rads; cd45-sap/200), and compared these regimens to a myeloablative dose of irradiation (800rads) [niaid protocol lcim 6e]. following conditioning, the mice were transplanted with wild-type (wt) bone marrow (bm) lineage-negative cells and followed over time to evaluate immune reconstitution. results: conditioning with cd45-sap alone or with cd45-sap/200 led to a consistent engraftment of donor t and b cells in the peripheral blood (pb) of f971l that increased overtime, reaching 90% donor chimerism at 16 weeks. myeloid (cd11b+) and nk cells in pb of f971l mice also showed high level of donor engraftment that remained stable at around 70% in cd45-sap around 24 hours of life and the second after one week. there is no data on the utility of one versus two screens for scid screening. here we present our data evaluating whether patients with scid or t-cell lymphopenia were identified on the first or second trec screen. objectives 1. determine the benefit of a second trec screen in identifying scid and t-cell lymphopenia at birth. 2. examine outcomes of trec screening in washington state since implementation. methods: results of scid newborn screening performed in washington state between january 2014 and june 2017 were reviewed retrospectively with the staff of the washington department of health laboratory where the trec assay is performed. trec thresholds (copies/μl) were defined as follows: absent (20), low (21-60), borderline (61-80) and normal (>80). all trec assays were run with a beta-actin control to assure sample adequacy. a screen is considered abnormal if there is one low/ absent trec or two borderline trec. newborns with abnormal trec screening have follow-up diagnostic testing consisting of lymphocyte flow cytometry to evaluate numbers of naïve and mature t cells, b cells, and nk cells, performed at seattle childrens hospital. results: a total of 68 positive trec screens were found in washington state between january 2014 and june 2017. five patients who did not have diagnostic flow cytometry testing were excluded from the analysis (one protocol deviation, one lost to follow-up and three who died before testing could be performed). the first screen was abnormal in forty three patients, while the second screen was abnormal in 16 patients. five patients had an abnormal third or fourth trec drawn for other newborn screen follow-up. three patients with scid were identified, all with abnormal values on first screen. fortyfive patients with t-cell lymphopenia were identified; 30 from the first screen and 11 from the second screen. there was one patient with mhc ii deficiency was missed by both first and second screens because she did not have t-cell lymphopenia. the false positive rate with the first screen was 21% versus 28% with subsequent screens. the false positive rate dropped to 3% with two abnormal trec. the positive predictive value of scid or t-cell lymphopenia with the first abnormal trec was 82% versus 96% with two abnormal trec. average age of collection among infants with a positive screen was 30.5 hours for the 1st nbs and 11.5 days for the 2nd nbs. live viral vaccines were postponed in three patients who had an abnormal secondary screen (one with idiopathic t-cell lymphopenia, one with ectrodactyly-ectodermal dysplasia-clefting (eec) syndrome and one with 22q11.2 deletion). one of these was started on pjp prophylaxis. conclusions: the practice of obtaining a second nbs from all newborns in washington state has led to increased identification of patients with tcell lymphopenia but did not result in identification of additional patients with scid. the false positive rate of the first and subsequent newborn screens was similar and decreased in patients with two abnormal trec. interventions including delaying live viral vaccines and pjp prophylaxis were instituted in patients who had a normal initial trec but abnormal secondary screen and documented t cell lymphopenia. two trec screens in all newborns can result in identification of additional patients with t-cell lymphopenia who may require intervention and additional follow-up. it is not yet clear whether the cost of a second mandatory scid newborn screen is balanced by the additional sensitivity gained by this approach. introduction/background: mannose-binding lectin (mbl) is a multimeric lectin that recognizes a wide array of pathogens independently of specific antibody, initiates the lectin pathway of the complement system, and acts as a proinflammatory mediator. mbl deficiency is reported to increase the frequency of infections in patients with impaired immune systems or cystic fibrosis (cf) patients. mbl replacement is experimental and unavailable. immunoglobulin replacement therapy (igrt) is controversial in the treatment of mbl deficiency; however, there are no reports of its efficacy or role in this condition. we describe a cf carrier patient with mbl deficiency, ciliary dyskinesia and mildly low igg who did not respond to igrt. objectives 1. understand when mbl deficiency can be symptomatic. 2. define the relationship between mbl deficiency and cf. 3. describe the treatment options of mbl deficiency. 4. define the efficacy of igrt and mbl deficiency. methods: a single case report. results: 11 year old girl with mbl deficiency, cf carrier state with polymorphisms (2752-26 a>g cf variant with 7t/7t and m470v polymorphism) and ciliary dyskinesia (diagnosed by ciliary biopsy) who initially presented at eight years of age with recurrent sinusitis, recurrent otitis media status post tympanostomy tube placement, reactive airway disease and tracheomalacia. she had nine episodes of recurrent sinusitis with six negative sinus cultures and one positive for pseudomonas aeruginosa. she required a total of nine courses of antibiotics. labs showed several mbl levels <50 ng/ml on three occasions, normal ch50, ah50, igg 577-590 mg/dl (low normal for age), normal iga, igm, t cells, b cells, nk cells, and robust specific antibody titers. despite adequate pulmonary hygiene including nebulized levalbuterol, budesonide, dornase alfa, ipratropium, hypertonic saline and compression vest twice daily, she continued to have recurrent bronchitis and cough. in addition, even with sinus rinses and intranasal corticosteroid, she continued to have 6-8 episodes of sinusitis yearly. for this reason, she underwent bilateral total ethmoidectomies and maxillary antrostomies with modest reduction in frequency of sinus infections and symptoms. however after two years, her bacterial sinusitis recurred. four episodes were positive for methicillin staphylococcus aureus or pseudomonas aeruginosa. this did not improve significantly with a trial of intranasal mupirocin. due to increased frequency of bacterial sinusitis refractory to traditional therapy, she was started on subcutaneous igrt dosed approximately 350 mg/kg/month dosing maintaining igg troughs around 700 mg/dl. after a six month trial, she did not have improvement in the frequency of sinusitis, bronchitis and otitis media. she required 3-4 courses of antibiotics for bacterial upper respiratory tract infections and igrt was stopped. prophylactic antibiotics and repeat sinus surgery were instituted. conclusions: majority of the patients with low/deficient mbl levels do not manifest significant symptomology due to the redundancy of the innate immunity. the increased susceptibility to infections is thought to be due to additional factors that compromise other components of the immune system. specifically in cf patients, mbl deficiency is associated with earlier colonization with pseudomonas, more rapid decline in lung function and earlier death secondary to end-stage lung disease. there are no reports of combined mbl and cf carrier symptomatic patients similar to this patient. there are no validated age-corrected values for mbl levels in pediatric patients. the clinical relevance of these levels to infection frequency, severity, or treatment of mbl deficiency remains to be proven. it has been proposed that <100 ng/ml is considered deficient in children. mbl therapy is still experimental and not commercially available. management when provided for severe or frequent infections includes prompt treatment with antibiotics, prophylactic antibiotics, appropriate vaccinations, and a trial of igrt. there are no reported cases describing the efficacy of igrt in mbl deficiency, and the mechanism subsequent genetic analysis identified the presence of a de-novo heterozygous mutation in the nucleotide binding domain of nlrc4 (c.1021g>c, p.val341leu). a mutation involving this amino-acid position has already been described but with a different substitution pattern in a boy and his father who presented with mas (p.val341ala) (1) . in order to prove the causality of this mutation, our team generated thp-1 cell lines expressing the two different mutations through gene-editing with the crispr system. in this system the mutation p.val341leu, as well as the p.val341ala mutation, were responsible for spontaneous activation of caspase1 , as evidenced by flica assay. the patient was treated with iv methylprednisone 2mg/kg/day. he continued to have progression of his inflammatory state, and was therefore commenced on anakinra. following confirmation of mutation, he was started on rapamycin, reasoning that (i) through autophagy induction, rapamycin could potentiate the action of anakinra (2, 3) and (ii) through mtor inhibition counteract the effect of il-18 on t-cells (4). with a combinatory therapy of anakinra up to 15mg/kg/day and ramapycin (with trough levels of 10-15 ng/l), the patient showed a marked clinical improvement, allowing weaning of steroids and establishment of enteral feeds. ferritin levels reduced to 800-1000 ng/ml. we observed a significant decrease in il-18 plasmatic level following treatment initiation (pre-vs post-treatment levels of 82844 pg/ml and 10055 pg/ml, respectively). we report a novel nlrc4 gain-of-function mutation, presenting with neonatal enterocolitis and autoinflammation with improvement under combinatory therapy of anakinra and rapamycin. to our knowledge this is the first case to report the use of rapamycin in this disease, with what appears to be encouraging results. further studies are required to elucidate the potential role of rapamycin in the management other inflammasome disorders. introduction/background: allogeneic hematopoietic stem cell transplantation (hct) is currently standard treatment for patients with severe combined immunodeficiency (scid), with 2-year overall survival >90% for typical scid (heimall blood 2017). previous studies revealed that poor clinical outcomes correlated with poor long-term t cell reconstitution, including low cd4 t cell counts and low naïve cd45+ t cell counts (pai nejm 2014) . we hypothesized that t cells developing in a poorly reconstituted immunologic environment would show features of chronically activated t cells with increased expression of inhibitory co-receptors. we further hypothesized that the intensity of the conditioning regimen would correlate with the expression of inhibitory co-receptors. objectives: to characterize the t cell phenotype of scid patients at >2 years post-hct and to investigate the impact of conditioning regimen on the quality of t cell reconstitution and the expression of inhibitory coreceptors methods: we analyzed 34 scid patients 3-28 yrs (median 14 yrs) after hct. we excluded from the analysis patients with chronic graft-versushost disease (gvhd), chronic dna viral infections or patients who had received donor lymphocyte infusion or boost in the 6 months prior to study. scid genotypes (n) included il2rg/jak3 (20), rag1/rag2/ dclre1c (5), ada (2), il7r (1) and other/unidentified (4). nine patients had received a reduced intensity (ric) or myeloablative (mac) conditioning regimen, while 25 had received either no conditioning or immunosuppression only (none/is). poor t cell reconstitution was defined as cd4 t cell counts below 500 cells/mm3 (21 patients). t cell phenotype, including expression of inhibitory co-receptors, was assessed by flow cytometry. results: compared to patients with cd4 counts above 500 cells/mm3, patients with low cd4 counts had low naïve cd45ra+ccr7+ t cells (p=0.0004) and high cd45ra-ccr7-t effector memory (tem) cells (p=0.02), low numbers of naïve thymic cd45ra+ cd31+ t cells, low numbers of trecs and a less diverse t cell repertoire (p<0.0001). additionally, they had an increased frequency of cd8 t cells expressing pd1 (8% vs 3%), ctla4 (5% vs 1.5%), cd160 (25% vs 5%, p=0.01), and 2b4 (63% vs 29%, p=0.0001) inhibitory co-receptors. increased inhibitory receptor expression was associated with a differentiation profile skewed toward a tem phenotype, reduced t cell diversity, increased markers of t cell activation, and the development of a highly exhausted cd39high pd-1high t cell population. more importantly, a fraction of ccr7+ cd45ra+ cd8 t cells expressed pd1 (6% vs 1%, p=0.03) and 2b4 (27% vs 4%, p=0.01) in patients with low cd4 counts, suggesting that some naïve t cells of poorly reconstituted patients were chronically activated. inhibitory receptor expression did not increase with hla disparities between the donor and recipient, a history of gvhd after transplant or the infection status of the patient prior to transplant. however, inhibitory co-receptor expression was correlated with conditioning regimen, with increased frequency of 2b4+ cd8 t cells in unconditioned patients (54% vs 28% in none/is and ric/mac patients respectively, p=0.01). conversely, ric/mac conditioning was associated with higher naïve cd8 t cell numbers (p=0.01), higher naïve thymic cd45ra+ cd31+ t cell numbers (p=0.001), a more diverse t cell repertoire (p=0.001) and low expression of inhibitory co-receptors. ric/mac conditioning was also associated with improved naïve t cell generation and limited expression of inhibitory receptors in il2rg/jak3 patients (23% vs 50% for 2b4, 1% vs 11% for cd160 in ric/mac versus none/is il2rg/ jak3 patients respectively), a genotype permissive to t cell engraftment. conclusions: collectively, our results suggest that the expression of inhibitory co-receptors may be a biomarker of poor t cell reconstitution in transplanted scid patients. further, we propose that lack of conditioning limits t cell reconstitution, which correlates with increased expression of inhibitory receptors on circulating cd8 t cells. antibodies targeting inhibitory receptors are now available in clinical trials to treat cancer and viral infections. it will be necessary to evaluate the relationship between inhibitory receptor expression, t cell function and clinical outcome, to see if a selected group of scid patients could benefit from these immunotherapies. wallace chair, chief of allergy immunology, children's hospital of philadelphia introduction/background: prophylactic antibiotics (abx) and immunoglobulin replacement (igrt) are commonly used to treat specific antibody deficiency (sad), but the optimal therapy is not established. objectives: to compared outcomes (number of infections and hospitalizations) in sad treated with igrt vs. prophylactic antibiotics. methods: two-center, retrospective chart review of sad patients from jan 2012-may 2017. we excluded patients with hypogammaglobinemia and/or other immunodeficiency diagnosis. characteristics and treatment were reported, rates of infections/hospitalizations among treatment groups were compared using linear regression model. results: 78 sad patients included. mean age was 18 years, 54% were females. 22 (28.8%) received prophylactic antibiotics, 45 (57.6%) received igrt, 11 (14.1%) did not receive any specific treatment. number of infections decreased from 8.2 (year before treatment) to 2.0 (year after treatment) in prophylactic antibiotics group (p=0.008), and from 7.2 to 2.3 in igrt group (p<0.001). various musculoskeletal and vascular abnormalities. little is known of gynecologic-obstetric complications, but with improved therapies women are living longer making reproductive health more pertinent. objectives: to learn more about obstetric and gynecological health in women with stat3 loss of function. methods: we prospectively interviewed and retrospectively reviewed medical records of adult women with ad-hies evaluated at the nih between 2000-2017. results: of 61 patients aged 18-66 years (mean 35 years), 47 women were interviewed, and chart reviews were performed on 14. age of menarche in our cohort was consistent with the national average (13.05 vs. 12.5 years). five of 30 patients (16.7%) reported having worsening lung symptoms with menstruation, and 14 of 34 participants (41%) reported worsening eczema during menstruation. with regard to routine health maintenance, 21 of 30 women reported having regular cervical cytology testing; 7 (23%) reported an abnormal result. of these 7, 5 had hpv that responded to treatment or was hpv only not requiring treatment; 2 had reactive changes due to yeast and 1 ascus that was subsequently normal. mastitis and breast abscesses occurred in 14 women. eleven women reported vulvar cysts/abscesses requiring drainage. nine women had progestin-releasing iuds placed, in some to suppress menses-associated vulvar eczema/abscess flares; no infectious complications were reported from progestin iud use. over 30% of women chose not to conceive given underlying disease. of 16 women with pregnancies, 15 women had 26 live births, 6 of 16 (38%) had miscarriages, and 3 of 16 (19%) experienced recurrent pregnancy loss. three women whose pulmonary symptoms worsened during pregnancy were diagnosed with progression of parenchymal lung disease post-partum. one woman experienced worsening of skin manifestations. other reported postpartum complications included one wound infection (after caesarean) and one hemorrhage leading to hysterectomy. conclusions: as women with ad-hies are living longer, significant infectious and disease-related exacerbations related to both menstruation and pregnancy were observed in this patient population. it is important to focus on maintenance of their gynecologic and obstetric health and carefully monitor for these morbidities. finally, while these women may choose to attempt pregnancy, the risk of recurrent pregnancy loss and worsening disease warrants discussion. rna sequencing identifies aichi virus 1 as the cause of chronic infection with lymphoproliferation in a patient with x-linked agammaglobulinemia objectives: we here present an xla patient with a complicated course in whom we detected aichi virus 1 (aiv1). methods: case report: the patient was diagnosed with xla at age 3 years, based on agammaglobulinemia with absent b cells and a known pathogenic mutation in btk (c82c>t, p.r28c). he had been suffering from recurrent respiratory and gastrointestinal infections since the age of 2 months. he was started on immunoglobulin (ig) substitution, which resulted in complete control of infections with igg trough levels at 8 g/l. however, at 6 years of age he developed unexplained fever, refractory temporal epilepsy, hepatitis, progressive nephromegaly with chronic renal failure, splenomegaly, episodic diarrhea and growth failure. ultrasound identified multiple focal lesions in the liver, spleen and kidney. a liver biopsy showed severe chronic hepatitis with initial perisinusoidal fibrosis. serial kidney biopsies showed variable oligoclonal cytotoxic t cell infiltrates, suggestive of chronic viral infection. standard diagnostics failed to reveal a pathogen in blood or stool samples and on biopsies. results: we finally resorted to rna sequencing on a kidney biopsy sample. this technique identified aiv1, a kobuvirus of the family picornaviridae, with a high read number. subsequently pcr confirmed the presence of aiv1 in both liver and spleen. results for cerebrospinal fluid, blood and feces are pending. conclusions: aiv1 is a picornavirus responsible for self-limiting gastroenteritis in humans. confirmatory pcrs on blood, csf and stool samples are ongoing. however, given the unequivocal result of the rna sequencing and confirmatory pcrs in other affected organs, we believe that the complications in this xla patient can be explained by aiv1 chronic infection. these findings confirm the potential of next generation sequencing techniques to identify infectious agents in patients with primary immunodeficiency. characterization and successful treatment of a novel autosomal dominant immune dysregulatory syndrome caused by a jak1 gain-offunction mutation. introduction/background: janus kinase 1 (jak1) plays an essential, nonredundant role in the jak/stat signaling cascade, a key pathway in the control of hematopoiesis and immune function. significant progress has been made in elucidating the role of jak1, but gaps in our knowledge still persist. to date, somatic gain-of-function mutations in jak1 have been linked to t-cell acute lymphoblastic leukemia. objectives: to understand and treat human jak1 gain-of-function mutations. methods: research study protocols were approved by our institutional review board. four members of the family (the affected children and their parents) were enrolled. written informed consent for genetic testing and participation was provided by the parents for their children. genetic, bioinformatic, biochemical and immunological investigations were performed. results: we describe the first known patients carrying a germ-line gainof-function mutation in jak1. the clinical phenotype includes severe atopic dermatitis, markedly elevated peripheral blood eosinophil counts with eosinophilic infiltration of the liver and gastrointestinal tract, hepatosplenomegaly, autoimmunity, and failure to thrive. functional analysis established the gain of function phenotype caused by the mutaintroduction/background: herpesviridae infection after hsct for hematologic malignancies, specifically cytomegalovirus (cmv), epstein-barr virus (ebv) and human herpes virus-6 (hhv-6), have been associated with various outcomes including post transplant lymphoproliferative disorder (ptld), graft-versus-host disease (gvhd) and mortality (1) (2) (3) . patients with primary immunodeficiency may have different incidence and outcomes with respect to herpesviridae given their differences in age at transplant, conditioning choices and underlying disease susceptibility to this viral family. objectives: the objective of this study was to describe the incidence and outcomes of cmv, ebv and hhv-6 post hsct for the primary immunodeficiency population. methods: a single center retrospective chart review of primary immunodeficiency registry patients (research ethics board protocol no. 1000005598) who received hsct from january 2000 december 2016 was undertaken. patients who received gene therapy were excluded. antiviral prophylaxis was given according to institutional protocol. demographic and clinical data were collated and analyzed with microsoft excel . the primary outcome was incidence and time to dnaemia for cmv, ebv and hhv-6. results: sixty one patients who underwent hsct for primary immunodeficiency from january 2000-december 2016 were reviewed. diagnoses are noted in table 1 . the average age of transplant was 18.0 months (range 1.9-93.8). 51 transplant recipients received busulfan and cyclophosphamide conditioning (2 with anti-thymocyte globulin (atg), 2 with alemtuzumab added), 3 transplants received busulfan, fludarabine and either atg or alemtuzumab, and 1 received atg alone. six transplants were unconditioned. 70.5% (43/61) of patients developed gvhd requiring systemic immune suppression. the overall incidence of cmv, ebv and hhv-6 post hsct for primary immunodeficiency was 9.8% (6/61), 59.0% (36/61) and 24.6% (15/61) respectively. in those with severe or profound combined immune deficiency the incidence of cmv was 7.0% (3/43), ebv 53.5% (23/43), and hhv-6 18.6% (8/43). in the 6 patients with chronic granulomatous disease, cmv incidence was 17% (1/6), and ebv and hhv-6 were both 50% (3/6). in recipients with pre-transplant negative pcr for ebv, cmv and hhv-6 (r-), time to dnaemia post transplant is seen in figure 1 . ptld was seen in 2 patients with rag1 and il2r, both of whom were d+r-for ebv status with ebv dnaemia, and one of whom died attributed to ptld. two year mortality for all patients was 23% (14/61), with mortality 24.4% (11/45) for those with either cmv, ebv or hhv-6 dnaemia vs 18.8% (3/16) in those without (p=0.64). disseminated cmv prior to transplant was attributed to cause of death for one case. conclusions: cmv incidence was rare, likely from screening for cmv negative hsct donors. cmv, ebv and hhv-6 dnaemia was not associated with differences in mortality in this cohort. ebv incidence was common, and ptld incidence was similar to previously published outcomes for hsct for other disease (2) . the advent of cytotoxic t cell therapy for ebv may help abrogate this risk. professor, senior physician, ulm university medical center, pediatrics, germany introduction/background: new-onset aiha occurs in 2-6% of pediatric patients post-hsct. incomplete immune recovery may predispose to immune dysregulation following hsct including autoimmune cytopenias. although prednisolone or other immunosuppressive drugs control most episodes, some patients respond incompletely to first or second line therapies including rituximab. objectives: we describe an innovative therapy for post-bmt aiha refractory to proteasome inhibition. three patients responded to anti-cd38 antibody (daratumumab) therapy after failing treatment with bortezomib. methods: we retrospectively evaluated data from three patients treated with daratumumab for post-transplant aiha. patients 2 and 3 were treated according to the positive response reported for patient 1. results: aiha occurred between 4-9 months following hsct. daratumumab was curative in 2 patients, the third one had only transient response and relapsed 5 months after this treatment had been initiated. following daratumumab patients no longer required any prbc transfusions. conclusions: in potentially life-threatening aiha in the context of hsct daratumumab may be an effective rescue therapy in combination with rituximab. early post-natal thymus development is strictly dependent on the level of foxn1 expression in tec these infants present severe t cell lymphopenia early in life, but in most cases their immune system gradually normalizes. however, the role of foxn1 haploinsufficiency in causing this phenotype is unclear. objectives: to analyze t cell development and tec phenotype in nu/+ mice of various age, in order to investigate whether foxn1 haploinsufficiency in mice results in impaired thymic development early in life, followed by progressive normalization, thereby recapitulating the human phenotype. methods: we analyzed 3 groups of nu/+ and +/+ littermates, divided by age: 1 day, 4-7 days, 3 weeks. the number of etp and the distribution of cortical and medullary tecs (ctecs, mtecs) were analyzed by flow cytometry. maturation of mtecs was further assessed by staining for mhc-ii and aire. real-time pcr was used to analyze the expression of ccl25, cxcl12, dll4, and scf, four key foxn1 target genes. the study was performed in accordance to niaid animal protocol lcim 6e. results: at 1 day and 4-7 days of life, nu/+ mice showed a dramatic reduction of etps, both in terms of frequency and absolute numbers, as compared to +/+ mice. however, by 3 weeks of age, the frequency and count of etps were comparable in nu/+ and +/+ mice. a slight but significant reduction in the frequency and absolute count of mtecs was observed in all three groups of nu/+ mice. additionally, the ratio between mtec expressing high levels of mhcii (mtechi) and those expressing low levels of mhcii (mteclo) was always higher in +/+ mice as compared to nu/+ mice. moreover, mtechi cells in nu/+ mice expressed lower levels of aire, the gene crucial for thymic negative selection of autoreactive t cells. finally, as compared to wild-type littermates, nu/+ mice showed reduced thymic expression of ccl25, cxcl12, dll4, and scf at day 1. reduced expression of ccl25 persisted at day 5, but normalized at 3 weeks. conclusions: these data indicate that foxn1 haploinsufficiency in mice leads to impaired thymic colonization by etps and abnormalities of tec differentiation and maturation early in life, followed by progressive normalization, thereby recapitulating what observed in newborns with heterozygous foxn1 mutations. these observations have important implications for the management of these infants, who should be monitored closely without rushing to definitive treatment for scid. introduction/background: foxn1 is the master regulator gene for the development and maturation of the thymic epithelial cells (tecs). by inducing the expression of chemokine receptors such as ccl25 and cxcl12, foxn1 also allows the migration of early thymic progenitor (etp) cells from the bone marrow. lack of foxn1 leads to the nude (nu)/severe combined immunodeficiency (scid) phenotype in humans and mice. recently, a number of newborns have been identified with low t cell receptor excision circles (trecs) at birth, associated with heterozygous foxn1 mutations. these infants present severe t cell lymphopenia early in life, but in most cases their immune system gradually normalizes. however, the role of foxn1 haploinsufficiency in causing this phenotype is unclear. objectives: to analyze t cell development and tec phenotype in nu/+ mice of various age, in order to investigate whether foxn1 haploinsufficiency in mice results in impaired thymic development early in life, followed by progressive normalization, thereby recapitulating the human phenotype. methods: we analyzed 3 groups of nu/+ and +/+ littermates, divided by age: 1 day, 4-7 days, 3 weeks. the number of etp and the distribution of cortical and medullary tecs (ctecs, mtecs) were analyzed by flow cytometry. maturation of mtecs was further assessed by staining for mhc-ii and aire. real-time pcr was used to analyze the expression of ccl25, cxcl12, dll4, and scf, four key foxn1 target genes. the study was performed in accordance to niaid animal protocol lcim 6e. results: at 1 day and 4-7 days of life, nu/+ mice showed a dramatic reduction of etps, both in terms of frequency and absolute numbers, as compared to +/+ mice. however, by 3 weeks of age, the frequency and count of etps were comparable in nu/+ and +/+ mice. a slight but significant reduction in the frequency and absolute count of mtecs was observed in all three groups of nu/+ mice. additionally, the ratio between mtec expressing high levels of mhcii (mtechi) and those expressing low levels of mhcii (mteclo) was always higher in +/+ mice as compared to nu/+ mice. moreover, mtechi cells in nu/+ mice expressed lower levels of aire, the gene crucial for thymic negative selection of autoreactive t cells. finally, as compared to wild-type littermates, nu/+ mice showed reduced thymic expression of ccl25, cxcl12, dll4, and scf at day 1. reduced expression of ccl25 persisted at day 5, but normalized at 3 weeks. conclusions: these data indicate that foxn1 haploinsufficiency in mice leads to impaired thymic colonization by etps and abnormalities of tec differentiation and maturation early in life, followed by progressive normalization, thereby recapitulating what observed in newborns with heterozygous foxn1 mutations. these observations have important implications for the management of these infants, who should be monitored closely without rushing to definitive treatment for scid. introduction/background: immunoglobulin g4rd is an immunemediated disease most commonly seen in middle-aged and older men. clinical features include autoimmune pancreatitis, salivary gland disease, orbital disease and retroperitoneal fibrosis. pathologic features include a lymphoplasmacytic infiltrate enriched in igg4-positive plasma cells and fibrosis in a storiform pattern. laboratory evaluation usually reveals an elevated serum igg4 concentration, and glucocorticoids are often used as treatment in early stages of disease. however, disease may recur off of steroids, and prolonged illness or steroid non-responsive disease is usually treated with rituximab. objectives: the objective of this case presentation is to discuss a presentation of a igg4rd is a young adult male. results: 16 year old male adopted from thailand, initially presented to hospital with five days of intermittent abdominal pain, night sweats and worsening fatigue. ct abdomen and pelvis revealed bronchiectasis in lung bases, hepatic masses and soft tissue infiltration in the porta hepatis extending into the liver, pulmonary nodules (read as possible metastases), bilateral renal masses, retroperitoneal nodes and ileocolic intussusception secondary to possible lymphoma. one year prior to presentation he developed new onset bilateral cervical lymphadenopathy. biopsy revealed a heterogenous lymphoid population and lymphoma was ruled out. he had a repeat lymph node biopsies during the admission, which was all negative for malignant cells, and was compatible with reactive lymphoid tissue with plasmacytosis. immunophenotype showed 59% lymphocytes with normal cd4/cd8 ratio, and 8% cd4-,cd8-, tcr ++ t cells. immunohistochemical stain of a lymph node fine needle biopsy showed a mixture of cd20+ b and cd3+ t cells, abundant cd138+, igg+ plasma cells(pcs) , with some igg+ pcs(ct of the neck and chest was completed for possible staging and revealed cervical lymphadenopathy, nasal polyposis, a soft tissue mass vs. enlarged lateral rectus muscle (left orbit), and mediastinal lymphadenopathy. a diagnosis of autoimmune lymphoproliferative syndrome (alps) was presumed by the hematologic/oncologic team given his elevated igg (6,682 mg/dl) and elevated vitamin b12 (>2000) with multiorgan involvement. alps panel revealed a mutation in faslg (allele 1, c.-2c>t) which is a variant of uncertain clinical significance. alps criteria/scorewas + for 1/4 criteria (cd3+cd25+/hla dr ratio < 1.0). he had been referred to the nih for further management of alps before initial presentation to our office. immunologic work up revealed immunoglobulins of igg: 6,600 mg/dl, iga: 83 mg/dl, igm: 71 mg/dl; 13/23 protective streptococcal titers (> 1.3 mcg/ml); hib: 0.46 mg/l; negative quantiferon gold, and hiv-serology. at that point igg4rd vs. castleman disease was suspected instead of alps. further work up showed a normal il-6 level, and human herpes virus 6 was also negative. igg subsets showed an elevated igg1 (2670 mg/dl), igg2 (1450 mg/dl), igg3 (355 mg/dl) and normal igg4 (20.9 mg/dl). excisional lymph node biopsy was recommended to rule out castleman syndrome or igg4rd. a left salivary gland was removed and showed mainly igg4 positive pcs with no multicentric lymphocytic infiltrates. dilution of the patients serum to determine if there was a prozone affect that minimized the igg4 level showed an elevated igg4 level of 2700.9 mg/dl. this is in the 1st percentile of all cases of igg4rd in terms of serum igg4 concentration. he was diagnosed with igg4rd, a form previously called mikulicz disease, which is comprised of lacrimal and parotid gland enlargement. rituximab was given initially because of the severity of his disease. after two cycles he showed decreased lymphadenopathy, notable weight gain and marked decrease in fatigue. conclusions: igg4rd is a rare and complex immunologically based disease process rarely seen in children or adolescents. patients with igg4rd often undiagnosed at initial evaluation. normal serum igg4 levels are seen in 40% of patients with igg4rd and thus a normal igg4 level should not be used as a biomarker to make the diagnosis of igg4rd or in treating this disease. furthermore, the possibility of having a prozone affect in measuring igg4 needs to be considered and evaluated. meticulous correlation of clinical, pathologic, and imaging findings is required to make the diagnosis. modelling human immune deficiency from novel missense mutations with orthologous heterozygous mutations engineered in mice by crispr/cas9 introduction/background: next generation sequencing has resulted in substantial progress in identification of mendelian immune deficiency syndromes. in some cases, however, putative causal mutations occur in single kindreds, or even individual patients. under these circumstances, functional analysis of patient derived cells combined with in vitro analysis of genetically manipulated cell lines can provide additional evidence in support of genetic causation, but this might not be conclusive. objectives: understanding how genetic defects result in complex syndromes of immune deficiency and immune dysregulation can be impossible to achieve in vitro. one method for overcoming these obstacles is to generate accurate mouse models of human immune deficiency methods: mouse models of human immune deficiency are a valuable tool in which the murine genome is engineered to introduce a mutation orthologous to that discovered in the patient. we have applied this strategy to elucidate causation and mechanism of immunological defect in several mutations affecting the nf-kb pathway. results: so far, defects in both canonical and non-canonical pathways of nf-kb activation have been shown to cause immune deficiency, often associated with immune dysregulation. we describe a known defects and novel putative defect identified in the canonical nf-kb pathway conclusions: crispr-cas9 mouse models can be used to elucidate mechanism of disease and provide compelling evidence that mutations are causative. introduction/background: primary immune deficiencies (pid) with or without immundysregulation are rare diseases resulting from monogenetic aberrations leading to either infections or autoimmune manifestations or both. early diagnosis and treatment are crucial for reducing morbidity and mortality. beside genetic diagnosis not commonly yet performed, current standard methods for early diagnosis are dependent on either fresh samples or limited to certain cell types. to overcome those limitations, especially in newborn screening, a novel technology of methylation-based qpcr can be applied. among the known epigenetic modifications, dna demethylation is the most stable and genomic loci with highly cell type specific demethylation sites can be identified. differential methylation can be measured from blood samples of limited availability, or with suboptimal storage. we previously showed that thymic-derived t population, and determine whether there are unique diagnostic and treatment considerations within this demographic. whitney goulstone 1 , elizabeth tough 2 1 executive director, canadian immunodeficiencies patient organization 2 lpn, alberta health services introduction/background: primary immunodeficiency diseases (pids) represent a significant collection of immune system disorders that increase susceptibility to infection, which in some cases are serious or life-threatening. patients with pid often require immunoglobulin g (igg, commonly referred to as ig) replacement therapy to prevent infections and associated comorbidities. pid treatment, in addition to symptoms and associated social and emotional impacts, has a significant impact on patients quality of life (qol). information available on the real-world diagnosis, management, and outcomes of the canadian pid patient population is limited. objectives: to better understand diagnosis and treatment of canadian patients with pid, we surveyed canadian patients with pid associated with the canadian immunodeficiencies patient organization (cipo) the primary goal of the survey was to gain insight into the canadian pid patient population with regards to demographics, diagnosis, treatment, including regimes, qol, and communication and support . methods: the authors conducted a cross-sectional survey to measure health-related qol in a cohort of patients with pid. eligible participants were identified through the canadian immunodeficiencies patient organization (cipo). the questionnaire consisted of 61 questions that covered patient-reported outcomes including diagnosis, qol, treatment regimes, and communication. results: surveys were returned by 149 patients with pid. participants conveyed significant impact on qol including personal, occupational, financial, emotional, and social impacts as a result of pid symptoms, risks, and treatment logistics, limitations, and side effects. the most common diagnoses were related to b-lymphocyte disorders (60.4%). common treatments include intravenous immunoglobulin (ivig; in hospital) and subcutaneous immunoglobulin (scig; at home), in addition to antibiotics and antifungals as required. respondents reported feeling average before treatment on a scale of 0-10 (mean 5.46 ± 2.23) with increased health after treatment (mean 6.66 ± 2.32). respondents felt current treatment was convenient (mean 8.22 ± 1.94) and were comfortable with self-infusions (mean 6.96 ± 3.46). conclusions: patients with pid are not uncommon in the canadian community, and in these patients pid is associated with a significant impairment in qol. experiences range with regards to a particular treatments advantages and disadvantages, cost, travel, and convenience. respondents hope to achieve improved qol through the following solutions: better treatment, improved infusions, gene modification, more research and clinical trials, a cure, and education and outreach. improved financial, medical, and social supports were also requested. introduction/background: severe combined immunodeficiency (scid), the most severe form of t cell immunodeficiency, is detectable through quantification of t cell receptor excision circles (trecs) in dried blood spots (dbs) obtained at birth. for many professional, humanitarian and financial reasons, newborn screening (nbs) for scid is warranted. implementation of this screening test is highly important where high frequency of consanguinity is known to exist. objectives: since october 2015, israel has conducted national scid nbs. this important, life-saving screening test is available at no cost for every newborn in israel. methods: herein, we describe two years results of the israeli scid newborn screening (nbs) program. validation includes cbc, lymphocyte subsets, trec (in a different method), t cell receptor (tcr) repertoire and response to mitogenic stimulation. whole exome sequence (wes) for genetic detection, and next generation sequencing (ngs) to demonstrate tcr clonality are used as well, in some unsolved cases. results: of 396,159 births screened, 719 (0.18%) had abnormal trec in their first screen (448 terms, 271 pre-terms), 69 (0.016%) had a repeated abnormal trec also in their second screen and were referred for a validation process. fourteen scid patients were diagnosed, so far, through the nbs program in its first years, revealing an incidence of 1:28,000 births in the israeli population. consanguine marriages and muslim ethnic origin were found to be a risk factor in affected newborns, and a founder effect was detected for both il7r and dclre1c deficiency scid. other diagnoses were made as follows: 8 cases were found to have t cell lymphopenia; 16 cases were diagnosed with syndromes; 4 cases were found to have secondary t cell lymphopenia; 10 cases were pre-term infants and 18 cases were considered as false positive with normal evaluation. lymphocyte subset analysis and trec quantification in the peripheral blood appear to be sufficient for confirmation of typical and leaky scid and ruling out false positive results. detection of secondary targets (infants with non-scid lymphopenia) did not significantly affect the management or outcomes of these infants in our cohort. we could also report several perspectives regarding t cell development in non immunodeficient newborns that emerged from the accumulated data. conclusions: already in a short term, trec nbs in israel has achieved early diagnosis of scid and other conditions with t-cell lymphopenia, facilitating management and optimizing outcomes. this program has also enabled gaining insights on t cell development in health babies. (hct) . surprisingly, 76% of these infections were acquired during the interval between confirmation of the scid diagnosis and hct (heimall, blood 2017). to investigate further, in late 2017 we surveyed pre-hct management practices for scid patients at pidtc centers. 51 physicians representing 43 north american centers responded, including 18 immunologists, 25 transplant specialists and 7 who identified as both. 33% of centers lacked a standard procedure for the management of infants with a positive nbs result. to confirm the scid diagnosis, basic t, b and nk cell flow cytometry was performed by most. testing for naïve t cells was generally included, but testing of lymphocyte mitogen proliferation was inconsistent. specialists were notified of a patients positive nbs test at median age 7.5 days (2-30 days) , and management of patients as scid was started at median age 9 days (0-90 days). when unconditioned hct was anticipated, 72% of respondents began planning as soon as possible after diagnosis, while 20% awaited genetic testing results before hct. respondants consistently implemented pre-hct prophylaxis with trimethoprim/sulfamethoxazole (84%), fluconazole (80%) and immunoglobulin infusions (96%), although timing of initiation varied. palivizumab was used by 26%. there was little consensus regarding viral monitoring. although most physicians screened for cmv by blood pcr (85%), only about half routinely screened for ebvor adenovirus. while 44% of physicians started prophylaxis against double-stranded dna viruses in all patients, 46% did so only in selected situations, such as active genital hsv at the time of delivery, or when the mother was cmv-seropositive. although all centers used only acyclovir as antiviral prophylaxis, doses and timing varied widely: from 25-90 mg/kg/day, divided into 2 or 3 doses, continued in most centers until immune reconstitution. finally, 84% of physicians recommended that cmvseropositive mothers stop breast-feeding. there was no consensus on where patients should reside prior to hct. hospital or home were favored equally, although need for a reliable family was indicated by 88% as a criterion for home-based management. for hospitalized patients, 62% of centers required patients to be in a reverseisolation/positive-pressure room and over half required staff to wear gown, gloves, and mask. with regard to visitors, 75% required parents/ relatives to perform hand hygiene. approximately 75% did not require a gown, gloves, or mask for visitors. finally, there was no consensus on allowing siblings or friends to visit, but the majority permitted grandparents or other adult relatives. this survey revealed wide variability in the diagnostic pathway, viral surveillance, and isolation practices for scid patients, although pretransplant prophylaxis with immunoglobulin, fluconazole, and trimethoprim/sulfamethoxazole were utilized consistently. we conclude there is considerable opportunity to develop diagnostic and pre-hct management pathways for scid. prospective tracking of management practices could reveal which are important for avoiding pre-hct infections. evidence-based practice guidance is needed to maximize the potential to bring each scid patient identified via nbs to hct infection-free. ikaros/ikzf1 is an essential transcription factor expressed throughout hematopoiesis. in humans, somatic mutations in ikzf1 are linked to b-cell acute lymphoblastic leukemia (all), and germline heterozygous haploinsufficient mutations cause common variable immunodeficiency-like disorder with incomplete penetrance. herein, we report seven unrelated patients with an earlyonset novel combined immunodeficiency associated with de-novo, fully-penetrant, germline heterozygous dominant negative mutations affecting amino acid n159 in ikzf1 dna binding domain. patients presented with different infections, but pneumocystis jirovecii pneumonia was common to all. one patient developed a t-cell all. additional findings included decreased b-cells, neutrophils, eosinophils and myeloid dendritic cells as well as t-cell and monocyte dysfunction. t-cells exhibited a profound naïve/recent thymic emigrant/ t-helper 0 phenotype and were unable to evolve into effector memory cells; monocytes failed to respond to different stimuli or facilitate t-cell activation. this new defect expands the spectrum of human ikzf1-associated immunodeficiency diseases from haploinsufficient to dominant negative. the beta subunit of the il-2 receptor (il2rb; cd122) is essential for il-2 and il-15 mediated signal transduction in a variety of hematopoietic cell types including t and nk cells. here we report the clinical and immunologic phenotypes of two siblings born to consanguineous parents harboring a variant in il2rb, resulting in autoimmunity, with lymphoproliferation of cd8+ t and cd56hi nk cells, and decreased regulatory t cell frequency. the proband presented with inflammatory enteropathy, failure to thrive, and disseminated cmv infection at 2 months of age, and later developed atopy, lymphocytic interstitial pneumonitis, and red blood cell autoantibodies. his younger sister presented with severe autoimmune hemolytic anemia and cmv viremia at 2 months of age, and later developed lymphocytic interstitial pneumonitis. whole exome sequencing and chromosomal microarray studies revealed a homozygous deletion within the highly conserved wsxws motif of the extracellular domain of il2rb (c.665_673delcctggagcc, p.pro222_ser224del). the deletion results in reduced il2rb expression (cell surface and intracellular) in t and nk cells. the functional consequences of the defect include complete impairment of stat5 phosphorylation through the il-2 receptor but only partial impairment through the il-15 receptor, as well as a compensatory increase in serum il-2 and il-15 levels (>100 pg/ml). cd8+ t cell proliferation responses to in vitro t cell receptor (tcr) stimulation were reduced compared to an age-matched, healthy control, but partially rescued by supraphysiologic levels of il-2 and il-15. despite reduced cd8+ t cell proliferation responses, the proband displayed a t cell population skewed toward cd8+ t cells, with an oligoclonal expansion of effector memory cells. arguing against effects of pervasive cmv infection alone, the sister displayed similar phenotypic and functional abnormalities at birth, prior to her cmv infection. our data suggest that the identified hypomorphic mutation in il2rb results in a survival advantage for those cd8+ t and cd56hi nk cells most sensitive to the exuberant serum il-2 and il-15 levels produced in response to the defect. these surviving cd8+ t and cd56hi nk cells have great lymphoproliferative potential, leading to multisystem autoimmunity and inflammatory complications. therefore, we describe il2rb deficiency as a novel primary immunodeficiency disease with prominent immune dysregulation and selective cd8+ t and cd56hi nk cell lymphoproliferation. key: cord-000718-7whai7nr authors: nan title: esp abstracts 2012 date: 2012-08-22 journal: virchows arch doi: 10.1007/s00428-012-1284-1 sha: doc_id: 718 cord_uid: 7whai7nr nan ofp-01-002 intraoperative evaluation of breast cancer sentinel lymph node metastasis with an automated molecular detection method as an alternative to standard pathological evaluation f. beca * , e. rios, p. pontes, i. amendoeira * centro hospitalar de são joão, anatomic pathology, porto, portugal objective: one-step nucleic acid amplification assay (osna) from sysmex® is an automated system to detect sentinel lymph node (sln) metastasis in breast cancer patients. this assay is based on the detection of cytokeratin 19 mrna amplification by reverse-transcription loop-mediated isothermal amplification. traditionally intraoperative evaluation of sln is accomplished by cytology and/or frozen section, accounting for heavy workload to pathology department. the objective of this study is to compare the osna assay with our standard processing for intraoperative sln evaluation. method: 82 sln from 45 patients with ck19+ and invasive primary carcinomas where simultaneously evaluated by cytology and/or frozen section and by osna assay. results where compared with gold-standard definitive h&e evaluation (serially sectioned at 150 μm intervals). results: the sensitivity and specificity of osna assay was 92.3 % and 98.2 %, respectively with a positive predictive value (ppv) and a negative predictive value (npv) of 96 %. for standard intraoperative evaluation, specificity was 100 % and sensitivity was 73.0 %. conclusion: osna assay showed similar specificity and higher sensitivity than standard pathologic examination. these findings suggest it can be used as an accurate tool for intraoperative evaluation of slns metastasis and possibly contribute to reducing the need for re-intervention for axillary lymph node dissection. objective: antracycline-based chemotherapy represents a standard of care in breast cancer patients especially who overexpress her-2. topoisomerase 2a has been considered a molecular target for antracyclines and its co-amplification with her-2 genes has been proposed. in our study we investigated if topoisomerase 2a overexpression can be used as a molecular marker on predicting response to antracycline-based chemotherapy. method: breast cancers tissues from 40 patients underwent to antracycline-based neoadiuvantchemotherapy and 267 treated with adiuvant therapy were collected into 4 tissues microarrays, and classified according to pathological stage, er, pr, her-2 and topo2a status. the expression of topoisomerase 2a has been correlated to response to chemotherapy. results: our results show that topo2a expression is significantly higher in neoplasms with larger size, nodal metastasis, scant er end pr receptors, and amplification of her-2 gene (p < 0,00, t-test). cancers in witch high level of topo2a has been found exhibit a better clinical response to treatment with anthracycline (p <0,0000, t-test). therefore topo2a expression could be considered both as predictive and prognostic marker. conclusion: the employment of topo2a as a predictive marker in clinical practice may be useful for the medical therapy of non-endocrine-responsive patients candidate to undergo treatment based on anthracycline, that is no free from adverse effects as cardiomyopathy and leukemia. ofp-01-004 stem cells in triple negative breast cancer and associated in situ lesions m. comanescu * , m. dobre, g. bussolati * institutul victor babes, dept. of pathology, bucharest, romania objective: triple negative breast cancer (tnbc) (negative for expression of estrogen and progesterone receptors (er, pr) and her2/neu protein) represent a subtype of breast cancer associated with poor prognosis and highly aggresive behaviour. genetic characterization of stem cell (cd 44, ....) in this type of carcinoma might provide important data concerning origin and evolution. data of the literature focused specifically on tnbc but, despite the interest shown to stem cells recently, there are no data concerning the genetic characterization of stem cells in the context of cell biology of tnbc as compared with associated dcis. method: we investigated, through immunohistochemistry, the expression and distribution of several stem cell/related antigens, exploring the association of tnbc with dcis and comparing the presence of stem cells in the invasive and in the in situ component. results: the multiplicity of parameters to be detected for characterising stem cells and of the diagnostic materials that are available would prevent a straightforward approach and a sequential combination will have to be planned. conclusion: optimization of detection, identification and characterisation of tumourigenic breast cancer stem cells might permit further identification of targeted treatment. over-and undergrading of breast cancer on core biopsies in comparison to surgical specimens t. decker * , c. focke, d. gläser * d. bonhoeffer medical center, dept. of pathology, neubrandenburg, germany objective: to evaluate the relevance of histological grading (hg) based on core biopsies (cb) for clinical decision making in breast cancer (bc) we evaluated the concordance with hg from surgical specimen (ssp) and the reasons for under-or overgrading. method: cb and related ssp of 398 bcs were prospectively graded according to the nottingham grading system (ssp: 65 g1, 162 g2 and 171 g3). cb/ssp agreement rates and positive predictive values (ppv) of cb based hg were calculated. rates of over-and underestimation of glandular differentiation (gd), nuclear pleomorphism (np) and mitotic activity (ma) were calculated. results: cb/ssp agreement rates came out with 95 % for g1, 73 % for g2, and 56 % for g3. the ppvs of cb based hg were 56 % for g1, 61 % for g2 and 100 % for g3. the overgrading and undergrading rates on cb were 1 % and 37 %, respectively. main causes for undergrading in cb were underestimated ma (40 %) and a combination of underestimated np and ma (32 %). conclusion: whereas overgrading on cb is an exception, undergrading derives largely from underestimation of proliferation. agreement of hg between cb and ssp ranges from almost perfect for g1, moderate for g2 to slight for g3 tumours. ofp-01-006 comparative histopathology of mammary alterations in women and dog v. deckwirth * , p. kronqvist, m. lintunen, a. sukura * university of helsinki, dept. of veterinary pathology, finland objective: companion animals such as dogs share the same environment with people. mammary tumours are the most prevalent spontaneous neoplasia in intact bitches. more animal models are needed for human mammary tumour research. our objective was to describe equivalent non-neoplastic and neoplastic mammary gland alterations in woman and dog. method: formalin-fixed, paraffin-embedded archived human and canine mammary gland tissue samples. epidemiological data were collected and analyzed from human (n= 1935) and canine (n=161) patients for the years 2003-2005. histological comparison was performed with he-stained tissue sections and immunohistochemistry. applied antibodies included ck5/6, erα, pr, ki67, her2, p63, sma, ecadherin and calponin. results: several comparable benign and malignant mammary gland alterations were identified, consisting of lobular, tubular and ductal as well as mesenchymal of origin. most of these have not been described previously. the epidemiological results show also shared characteristics. conclusion: although there are canine specific lesions there also exist equivalent entities. our results indicate the female canine as a suitable model for translational research. ofp-01-007 st. gallen intrinsic subtyping of breast cancer: influence of proliferation assessment methods c. m. focke * , d. gläser, k. finsterbusch, t. decker * dietrich bonhoeffer klinikum, abt. pathologie, neubrandenburg, germany objective: the st. gallen conference 2011 strongly recommends intrinsic subtyping of breast cancer (bc) for therapeutic decisions. we analyzed the influence of different proliferation assessment methods on the bc subtype distribution. method: intrinsic subtyping was performed according to the st. gallen criteria on 225 bcs. proliferation was assessed by mitotic activity index (mai) (area: 1,59 mm², cut-off ≥10) and by several variants of ki67-labeling-index counting any nuclear staining of (1) 100 tumor cells within the hot spot (ki67-100), (2) 1020 tumor cells in 3 hpf in the tumor periphery, including the hot spot (ki67-1020periphery), and (3) 1020 tumor cells in 3 hpf, including hot spot, cold spot and an intermediate area (ki67-1020spectrum) using the recommended cut-off of <14 %. results: of 225 carcinomas, 10 % were triple-negative, 2 % her2+, 6 % luminal b her2+, and 9 % special type. the rates of bcs after different proliferation assessment methods were: mai 49 % vs. 24 %; ki67-1020spectrum: 34 % vs. 39 %, ki67-1020 periphery: 24 % vs. 49 % and ki67-100: 10 % vs. 63 %. conclusion: subtyping of luminal a and luminal b her2neg bcs according to st. gallen 2011 is highly influenced by the method of proliferation assessment used. its biological relevance seems to be not unequivocal. ofp-01-008 the utility of cytological smears allied to the osna method for intraoperative analysis of sentinel lymph-node metastasis in breast cancer patients s. foreid * , m. martins, h. pereira, m. oliveira * loures, portugal objective: one-step-nucleic-acid-amplification (osna) is a recently introduced pcr method to evaluate intraoperative sentinel lymph-node metastasis in breast cancer. it assesses the whole lymph-node in a 20-minutes protocol. many studies have reported it to be more sensitive than frozen section and/or cytology. yet, it is more expensive and takes longer to perform. method: between 2010 and 2012 at chlc, we have performed the intraoperative analysis of sentinel lymph-node using both osna and cytological smears in a group of patients. results: 35 lymph-node evaluations were done. 16 cases were osna positive (5 micrometastasis and 11 macrometastasis). of those, 11 were also positive for tumor cells on cytological smears. 19 cases were negative by both methods and 5 cases were detected by osna but not on cytological smears. importantly, all macrometastasis diagnosed by cytology were confirmed by osna. of notice, in this series, 69 % of the positive cases detected by osna were also detected by cytological smears. conclusion: like previously reported, osna showed a greater sensibility than cytology to diagnose sentinel node metastasis. however, the majority of metastasis can be detected by cytological smears alone. our results suggest that a less expensive and faster cytological analysis should precede osna, and if positive, the latter method could be disregarded. tuesday, 11 september 2012, 17.00-19. objective: tumor markers may be concentrated intraductally in early breast cancers. method: to test this hypothesis, her2-ecd and ca15-3 of the needle washout fluid from 29 benign lesions, 26 dcis, and 95 invasive carcinomas of the breast were measured using chemiluminescence. the measuring limit for her2-ecd (0.5 ng/ml) and that for ca 15-3 (4 u/ml) were used as the cutoff values, respectively. results: the proportion of patients with any biomarker elevation was 6.9 % in cases of benign lesions, 46.2 % in dcis, and 15.8 % in invasive carcinomas. thus, biomarker elevation was most frequent in dcis, followed by invasive carcinomas (p<0.01). her2-ecd values over 6 ng/ml or ca 15-3 values over 25 u/ml were s een exclusively in dcis or invasive ductal carcinomas with an extensive intraductal component, supporting the hypothesis. conclusion: our approach may be useful to identify a subset of breast cancer patients who are suitable for intraductal molecular targeted therapy. ofp-02-002 biological significance of proliferation and her2 overexpression in luminal/oestrogen receptor-positive breast cancer d. jerjees * , a. green, a. benhasouna, a. alshareeda, r. abduljabbar, f. barros, c. nolan, i. ellis, e. rakha * university of nottingham, dept. of oncology, united kingdom objective: in this study, we have compared the biological significance of proliferation; assessed using ki67 labelling index (ki67-li), with respect to her2 expression in oestrogen receptor positive (er+) breast cancer to assess the impact on growth fraction and biological characteristics of luminal breast cancer. method: 1562 well-characterised er + breast cancers were assessed for expression of a large panel of biomarkers (no=30). results: 53 % of the cases showed high ki67-li (>13 %) and 12 % showed her2 over-expression and both were positively associated with younger age, higher tumour grade, lymph node stage and shorter outcome. both markers were associated with up-regulation of er-coactivators (cd71, carm1, pi3kca&tk1), p-cadherin, p53 and with lower levels of er expression and down-regulation of er-related genes (pgr, ar&gata3) and brca1. neither of the two markers was associated with expression of basal-associated cytokeratins. high ki67-li was associated with down-regulation of luminal enriched markers including luminal cytokeratins, muc1, gcdfp-15, fhit, and transcription and differentiation errelated genes (foxa1 and tff3). in contrast, her2 expression was associated with up-regulation of luminal cytokeratins, gcdfp-15, fhit and e-cadherin. conclusion: unlike high ki67-li, her2 overexpression is not associated with the down-regulation of luminal enriched genes. increased growth fraction in er + tumours may be driven by different mechanisms in her2+&her2-disease. ofp-02-003 loss of drosha expression is associated with poor survival in breast cancer patients s. khoshnaw * , t. abdel-fatah, c. nolan, z. hodi, e. rakha, d. macmillan, i. ellis, a. green * university of nottingham, breast cancer pathology, united kingdom objective: drosha is a protein that plays a key role in the biogenesis of micrornas which are well known to be deranged in human breast cancer. the purpose of this study was to investigate the hypothesis that drosha is significant in the development and progression of breast cancer and has clinical relevance as a potential predictive and prognostic target. method: we have examined drosha protein expression, using microarray and immunohistochemistry, in a well-characterised series of unselected invasive breast cancer patients (n=750) with long-term follow-up and documented expression characteristics for a large range of biomarkers of known relevance in breast cancer. furthermore, a smaller cohort of selected breast cancer cases (n=24) was also investigated for differential drosha protein expression in distinct stages of tumour progression, including in situ (dcis), invasive and metastatic components. results: drosha cytoplasmic and nuclear expression was lost with cancer progression, and was associated with loss of brca1 expression. multivariate statistical analyses showed that drosha cytoplasmic expression was an independent predictor of breast cancer specific survival, tumour recurrence and metastases. conclusion: drosha cytoplasmic expression in breast cancer is an independent predictor of patient outcome, tumour recurrence and metastasis. these observations imply that alterations in mechanisms of mirna regulation can influence tumour behaviour. objective: determination of proliferation in breast carcinomas is highly prognostic and can have treatment implications. however, there is no consensus on issues such as counting area or cut-off values. our aim was to examine and compare ki-67 counts in various tumor areas, the novel mitosis marker phh3, and standard mitotic count. method: we examined a nested case-control series (n= 190) as part of the population-based norwegian breast cancer screening program. mitotic count (mitosis per mm2) was assessed on h&e-sections. the percentage of ki-67 positive nuclei among 500 tumor cells was recorded for both hot-spots (hs; highest proliferation) and cold-spots (cs; lowest proliferation). phh3 counts (per mm2) were evaluated by immunohistochemistry. results: the upper quartiles for ki-67-hs, ki-67-cs, phh3 and mitotic count consistently showed the strongest associations with known unfavorable tumor features (high histologic grade, er negativity, her2 positivity, ck5/6+, p-cadherin+). univariate survival analysis showed comparable prognostic strength of ki-67-hs, phh3 count and mitotic count. in multivariate analysis, mitotic count was the strongest prognostic factor among these. conclusion: by upper quartile, ki-67-hs, phh3 and mitotic count all showed strong associations with various tumor features. in multivariate survival analysis, mitotic count was the strongest proliferation marker. objective: ki67 li is a good marker of breast cancer prognosis. we aimed at comparison of performance of 5 ki67 antibodies. method: 283 breast cancer cases were retrieved. immunohistochemical staining for 5 ki67 clones (30-9, b56, mib1, polyclonal, sp6) performed in tma-s in automated immunostainer. evaluation performed on digital slides utilizing linear 0-10 frequency score. objective: in breast cancer, classification systems are used to assess pathological response to neoadjuvant chemotherapy. biomarkers are still needed to better predict the efficiency of primary systemic therapy. dysregulation of connexin (cx) channels (gap junctions) is involved in carcinogenesis and tumor progression. method: we have correlated cx expression and the pathological response in neoadjuvant treated breast cancers. fifteen of the 21 human cx isotypes were pre-screened. cx26, cx32, cx43 and cx46 were found widespread and tested in tissue microarrays of 96 breast cancers prior to and after neoadjuvant chemotherapy. pathological response was characterized according to the ewgbsp, cps-eg, miller-payne, sataloff, and nsabp classification systems. results: only the cps-eg classification had prognostic relevance by score 1-2 cases presenting better overall survival (p=0.015) than those scoring 3-5. cx43 levels prechemo correlated with hormone receptor status pre-and post-chemo and had inverse correlation with her2 levels pre-chemo. reduced cx26 expression (<5 %) post-chemo correlated with better overall survival (p=0.011). moderate or higher cx46 expression (>20 %) pre-and post-chemo was also associated with better survival in the ewgbsp tr2b (ppre=0.006), sataloff tb (ppre=0.005; ppost=0, 029) and miller-payne g3 (ppre=0.002; ppost=0.012) subgroups. conclusion: classifications combined with testing for cx46 and cx26 expression can improve prediction of neoadjuvant chemotherapy in breast cancer. ofp-02-008 atypical vascular lesions after radiation therapy for breast cancer: a clinicopathologic study and outcomes of 24 cases h. gobbi * , c. fraga-guedes, m. mastropasqua, m. kneubil, e. botteri, g. viale * ufmg, dept. of anatomic pathology, belo horizonte, brazil objective: we assessed clinicopathologic features and outcomes of 24 cases of atypical vascular lesions (avl) in patients submitted to radiation therapy for breast carcinoma. method: data from all patients were retrieved from the files of the european institute of oncology (eio), milan. histopathological review of all cases was carried out. results: all patients were women (median age: 58 year, range: 36-81 year). median lesion size was 0.5 cm (range: 0.3 to 1.1 cm). median latency interval from radiation to avl diagnosis was 49 months (range: 17 to 124 months). 62.5 % of cases presented as an erythematous unique papule confined to superficial/middermis with proliferation of thinwalled, vascular channels, lined by a single layer of bland endothelial cells. no prominent nucleoli, mitoses or blood lakes were seen. patches of chronic inflammatory infiltrate accompanied all avl. one patient (4.2 %) developed local recurrence, and two patients (8.3 %) developed angiosarcoma in the previous biopsy site, 19 and 89 months later. margin involvement was associated to unfavorable outcome. conclusion: our data suggest that post-radiation avl and angiosarcoma may represent a spectrum of the same disease process. the development of local recurrence and subsequent angiosarcoma indicate that avl must be completely excised, with free surgical margins, and patients must be accompanied by clinical exams and imaging. objective: gastric dysplasia is classified as gastric/foveolar and intestinal/adenomatous according to morphological (architectural and cytologic) features. the immunophenotypic classification of dysplasia, based on the expression of mucins and cd10, recognizes three major types: gastric (muc5ac and/or muc6); intestinal (muc2 and/or cd10); hybrid (gastric and intestinal markers). cdx2 is a transcription factor responsible for early intestinal differentiation. aldh1 is recognized as a stem cell marker in several organs. method: nineteen cases of dysplasia were classified as gastric/foveolar (n=8) or intestinal/adenomatous (n=11) and graded as low-grade (n=9) and high-grade (n=10). immunophenotypic classification showed gastric (n=5; 26 %), intestinal (n=6; 32 %) and hybrid (n=8; 42 %) phenotypes. results: cases classified by morphology as gastric/foveolar-type displayed gastric (50 %) or intestinal (50 %) immunophenotype, whereas intestinal/adenomatous-type cases displayed gastric (9 %), intestinal (55 %) or hybrid (36 %) immunophenotype (p=0.023). high-grade dysplasia was identified in cases with gastric (80 %), intestinal (17 %) and hybrid (63 %) immunophenotype (p=0.085). cdx2 expression was significantly lower (p=0.044) in gastric (60 %) than in intestinal and hybrid immunophenotypes (100 %). aldh1 expression was displayed only in gastric (80 %) and hybrid (50 %) immunophenotypes (p=0.023). conclusion: gastric dysplasia is heterogeneous and morphology is insufficient to elucidate cell differentiation. high-grade dysplasia, decreased cdx2 expression and high aldh1 expression are associated with gastric immunophenotype. ofp-03-002 tumor budding in pancreatic cancer: are we missing important prognostic information? d. born * , i. zlobec, m. montani, b. gloor, a. lugli, e. karamitopoulou-diamantis * universität bern, inst. für pathologie, switzerland objective: tumor budding is defined as single tumor cells or small cell clusters at the invasive front of gastrointestinal (including colorectal, gastric and ampullary) carcinomas and is linked to adverse prognosis. to date it has not been reported in pancreatic ductal adenocarcinomas (pdacs). we assessed the presence and prognostic importance of tumor budding in pdac. method: whole-tissue sections of 120 pdacs with full clinico-pathological and follow-up information were stained with pancytokeratin. tumor budding was assessed in 10 high-power fields (hpfs) by two pathologists and considered high-grade (hg) when an average of >10 buds was counted. measurements were correlated to the patient and tumor characteristics. results: inter-observer agreement was strong (icc=0,72). hg-budding was found in 70.3 % of cases and was linked to advanced pt-stage (p=0.0463), lymphatic invasion (p=0.0192) and decreased disease-free and overall survival (p<0,0001 and p=0,0005). there was no association with pn, pm, r-stage or vascular invasion. in multivariate analysis the prognostic effect of hg-budding was independent of lymphatic invasion, pn and r-stage [p<0,0001, hr (95 % ci)=3,65(2,1-6,4)]. conclusion: tumor budding occurs frequently in pancreatic cancer, is an indicator of worse outcome and adds independent prognostic information. routine use of tumor budding would help to better stratify patients into prognostic subgroups. ofp-03-003 epstein-barr virus and p53 in the development of gastric cancer v. genitsch * , m. cathomas, l. terracciano, l. tornillo, a. lugli, a. marx, g. sauter, f. carneiro, f. hofstädter, n. willi, g. cathomas * kantonsspital baselland, abt. pathologie, liestal, switzerland objective: epstein-barr virus (ebv) is associated with a subset of gastric cancers (gc) . in this study, the prevalence of ebv and its presence in the development of gc were assessed. method: ebv was evaluated in gc by tissue microarrays using in situ hybridization for eber (n=610). non-tumorous, dysplasia, intramucosal and invasive carcinomas and metastasis of ebv positive gc were analyzed (n=22). p53 expressen was evaluated in ebv positive and a subset of 84 ebv negative gc. results: 4.9 % of gastric cancers were positive for eber. all invasive cancers showed diffuse eber positivity and only 2 lymph nodes revealed a focal loss of eber. in 13 gc with foci of atypia/low grade dysplasia, no or only focal eber positivity was observed. p53 was overexpressed in 24 of 84 ebv negative but not in ebv positive gc (28.6 % vs. 0 %; p=0.007). conclusion: ebv is diffusely expressed in invasive gc and only occasionally lost in metastasis. in low grade dysplasia, however, ebv is often focal or missing, indicating that ebv infection may be associated with progression from low grade dysplasia to invasive cancer and this progression is p53 independent. ofp-03-004 p2x7r attenuates colitis associated carcinogenesis p. hofman * , t. juhel, m. ilie, o. bordonne, o. boyer, x. hebuterne, s. adriouch, v. vouret-craviari * chu de nice, lpce, france objective: it is now well established that patients with inflammatory bowel disease presented a higher risk to develop a colon cancer. our previous works characterized the link that exists between bacterial infection and inflammation and highlighted the role of the microenvironment on the onset of chronic inflammation. here, we wished to understand the role of inflammasome, a complex of proteins that is activated by microbial motifs and the purinergic receptor p2x7 and involved in the maturation of il-1β. method: we treated p2x7r knockout mice to induce acute colitis (3 % dss) or colitis associated colon cancer (aom and dss). results: we showed that p2x7r ko mice are more resistant than wild type mice to a dss-induced colitis. further, we demonstrated that p2x7r ko mice are highly susceptible to inflammation driven colon tumorigenesis. in addition, we showed that tumors from p2x7r ko contained more neutrophils (cd11b+/ly6g + cells) and less macrophage (cd11b+/ly6g-) than tumors obtained from wt mice. conclusion: we will present results on the characterization of the pro tumoral tumor associated neutrophils and the molecular mechanisms that are linked to the presence of such immune cells. ofp-03-005 iel counts and distribution in normal duodenum, non-gse ielosis and gse: do we need a cut-off? b. a. karabork * , s. yuksel, b. savas, a. ensari * ankara university of medicine, dept. of pathology, turkey objective: intraepithelial lymphocytosis (ielosis) is a characteristic feature of gluten-sensitive enteropathy (gse), though, the cut-off is still debatable. we aimed to evaluate the number and distribution of iels in normal duodenum, and in non-gse ielosis and gse. method: the study group comprised of a spectrum of normal duodenal biopsies (n=31), non-gse ielosis (n= 30), type 1 (n=22) and type 3 (n=21) serologically diagnosed gse cases. the number of iels/100 enterocytes and distribution on h&e and cd3-immunostained sections, were assessed for each group. chi square test was used for statistics. results: iels increased significantly through the spectrum (15, 55 in normal, 25, 37, 00 in type 1, 67, 10 in type 3) on h&e and cd3, respectively (p<0,001). iel counts ≥20 on h&e and ≥25 on cd3 had a sensitivity of 95,35 % and 100 %, and a spesificity of 40,98 % and 32,79 %, respectively, for gse. iel distribution was diffuse in type 1 (95,5 %), type 3 (100 %) but focal in non-gse (80 %) (p<0,001). conclusion: the current cut-off for iel counts seems to be sensitive for ielosis, though, not specific for gse and needs to be accompanied by the diffuse distribution of iels. ofp-03-006 geographic analysis of the protein expression of metastasis suppressor gene rkip and its clinical relevance in colorectal cancer v. koelzer * , e. karamitopoulou, h. dawson, a. lugli, i. zlobec * university of bern, inst. of pathology, switzerland objective: loss of raf-1 kinase inhibitor protein (rkip) expression has been implicated in disease progression in several tumor types. the aim of this study was to evaluate rkip expression in colorectal cancer stratified by histological "zones" and to determine the zone responsible for its clinical relevance. method: immunohistochemical expression of rkip was assessed in 100 colorectal cancers using whole tissue sections. four different areas were evaluated using 10 highpower-fields (hpfs) each: normal mucosa, tumor center, invasion front and tumor buds. the average expression for each zone was assessed for its clinical relevance. results: expression of rkip was diffuse in normal mucosa and progressively lost towards the tumor center and front (26 %, 15 % and 8 % rkip-positivity, respectively; p<0.0001). only 3 % of tumor buds were rkip-positive. rkip loss in the tumor center only corresponded to more frequent lymph node positivity (p=0.0766), distant metastases (p=0.0243), lymphatic invasion (p=0.0533) and more advanced tnm stage (p=0.0278). rkip loss was highly prognostic (hr (95 % ci): 0.45 (0.2-0.9); p=0.0288) independently of tnm and therapy. conclusion: the clinical relevance of rkip is restricted to the tumor center where it acts as an independent prognostic factor. its absence in tumor buds provides further evidence to support their hypothesized metastatic potential. ofp-03-007 outcomes of neoadjuvant chemoradiation for rectal carcinoma g. ozgun * , f. oz atalay, n. ugras, o. yerci * uludag university, dept. of pathology, bursa, turkey neoadjuvant radiation and chemoradiation is currently the treatment of choise for patients with locally or advanced carcinoma of the rectum. patients treated with neoadjuvant chemoradiatheraphy with good clinical response and tumor regression present a controversial management dilemma. to assess the effects of chemoradiation on tumor regression and disease free survival, consecutive series of 27 patients receiving neoadjuvant chemotheraphy or chemoradiation and a control series of 32 patients without pretreatment were studied. the overall survivals and disease free survivals were compared in terms of variables such as tumor regression grade, stromal response in tumor bed, state of lymph node metastasis, and the localization of the tumor in between patients treated with chemoradiation and the control group. neoadjuvant treatment is one of the treatment modalities in rectal carcinomas. we want to present our experience between 2005 and 2011 in our institution. key words: rectal carcinoma, neoadjuvant treatment, pathologic complete response ofp-03-008 identification of tissue microvascular invasion biomarkers in hepatocellular carcinomas by maldi imaging mass spectrometry n. poté * , t. alexandrov, j. le faouder, s. laouirem, j. belghiti, j. m. camadro, v. paradis, p. bedossa * paris, france objective: microvascular invasion (mvi), a major predictive factor of tumoral recurrence and mortality in patients with hepatocellular carcinoma (hcc), is only detectable on pathological examination. so far, there is no reliable tool to identify mvi prior to surgical procedures. maldi imaging mass spectrometry (ims) represents a new analytical tool to provide the relative abundance and distribution of the whole proteins expressed in a tissue section. the aim of this study was to compare, using maldi ims, the tissue proteome of hcc without and with mvi in order to identify surrogate biomarkers of mvi. method: a total of 56 hcc samples obtained from surgical specimens, for which frozen samples were available, were retrospectively collected. two groups of tumors were defined (26 hcc without mvi; 30 hcc with mvi) and were analysed by maldi ims. a statistical comparative analysis of acquired mass spectra was then performed in order to identify protein peaks differentially expressed between the two groups. results: 30 protein peaks were differentially expressed between the two groups, all overexpressed in hcc with mvi. protein characterization of some of these peaks is in progress. conclusion: these results highlight the potential of maldi ims to uncover new biomarkers in liver carcinogenesis. the identification of mvi biomarkers would be helpful in the therapeutic strategy of patients with hcc. ofp-03-009 a histopathological scoring system can predict the recurrence of hepatocellular carcinoma in liver transplant patients treated with transarterial chemoembolization f. rosini * , f. vasuri, d. malvi, p. baldin, w. f. grigioni, a. d´errico-grigioni * university of bologna, dept. of anatomic pathology, italy objective: aim of this study is to define the histopathological features predictive of hepatocellular carcinoma (hcc) recurrence in patients treated with orthotopic liver transplantation (olt) after tumoral transarterial chemoembolization (tace), in order to establish a predictive "score" applicable by pathologists for recipient risk stratification. method: we retrospectively enrolled 110 patients (276 total neoplastic or necrotic nodules) who received tace for hcc downstaging before olt. the following data were collected: number of neoplastic/necrotic nodules, microscopic thrombosis, the residual neoplastic tissue (rnt), and dysplastic nodules. sizes, percentage of necrosis, edmondson's grade, clear cell features were also collected for each nodule. results: median follow-up was 1230 days: 14 (13 %) hcc recurrence cases were recorded. statistically rnt (best cutoff 3 cm3, p=0.032), the total nodule number (best cut-off ≥ 3, p=0.001), and the neoplastic thrombosis (p=0.011) were the only variables predictive of hcc recurrence. scoring 1+ each parameter, recipients with 0 (n=43) had 0 % recurrence rate; recipients with 1+/2+ (n=49) had 12 %; recipients with 3+ (n=18) had 44 % recurrence rate (p<0.001). we were able to stratify olt recipients with tace-treated hcc in 3 risk groups based only on histopathological analysis. ofp-03-010 discovery of a new histopronostic factor in rectal adenocarcinoma treated by radiochemotherapy followed by surgical resection a. sannier * , j. lefèvre, f. bretagnol, d. cazals-hatem, y. panis, p. bedossa, n. guedj * paris, france objective: neoadjuvant radiochemotherapy (rct) followed by surgical resection became the treatment for locally advanced rectal cancer. pathological diagnosis is important for the prediction of prognosis and adjuvant treatment. study aim was to identify histopronostic factors in a consecutive series of patients treated by rct and surgery. method: 113 patients were included, follow-up, tumor morphologic pattern and modified rectal cancer regression grade (m-rcrg) were assessed. univariate and multivariate analysis were used to assess predictors of disease-free survival (dfs). results: 5-years dfs was 58 %. in univariate analysis, ypt, budding, calcifications, circumferential margin, node involvement, invaded margin, vascular emboles and perineural involvement were pronostic factors (p<0.05). in multivariate analysis, presence of calcification in tumor bed (p=0.027) and small circumferential margin (p=0.037) were the only two independent factor of worse dfs. mrcrg was not correlated to dfs. among the 50 mrcrg1 tumor, dfs was significantly better in patient with ypt0 than in other ypt stages (p=0.003). conclusion: presence of calcification in tumor bed is a new major histopronostic factor described for the first time in rectal cancer. our results raised the question of whether ypt stage or histological tumor regression is more important in the prediction of patient prognosis. tuesday, 11 september 2012, 17.00-19. objective: the most widely used system for histological grading of colorectal cancer (crc) is based on the degree of gland formation, despite significant interobserver variability and low prognostic value. herein we analyzed the prognostic significance of a grading system based on the presence of poorly differentiated clusters in stage i crc. method: poorly differentiated cancer clusters were assessed by two independent pathologists in stage i crc characterized or not by disease progression. tumors with <5, 5 to 9, and >10 clusters were classified as g1, g2, and g3, respectively. the prognostic value on disease-free survival and the association with other clinicopathologic characteristics of the conventional and novel grading systems were analyzed. results: k statistics for inter-observer variability in the assessment of histological grade based on poorly differentiated custers was 0.728 (good). high histological grade assessed with the novel system, but not with the traditional one, represented a negative significant prognostic factor for disease-free survival and it was significantly associated with venous invasion, lymphatic invasion, budding, invasive growth and nodal micrometastases. conclusion: we suggest that a tumor grading system based on the number of poorly differentiated clusters has a stronger power to stratify stage i crc patients by prognostic outcome than conventional grading. ofp-04-003 cytokeratin 7: a marker for braf mutated colorectal carcinomas? s. gurzu * , z. szentirmay, i. jung * university of targu mures, dept. of pathology, romania objective: cytokeratin20+/cytokeratin7− (ck) is used as the characteristic immunophenotype of colorectal carcinomas (crc) . some new studies suggested that aberrant pattern ck20/ck7 can be identified in colorectal cancer with microsatellite instability (msi). our aim was to establish which factors may determine changing in this immunophenotype. method: in 70 crc, randomly selected, we performed immunohistochemical stains with ck20 and ck7 and analyzed the microsatellite status and braf mutations with real time pcr. results: from the 70 crc, 15 were msi and 55 mss (microsatellite stable) cases. 90 % of mss cases diffusely expressed ck20 without ck7 expression. from the 15 msi cases, 6 presented braf mutations. in msi cases with braf mutations, ck20 was focally expressed or negative and ck7 was diffusely expressed. conclusion: cytokeratin7 positivity may be used to select braf mutated msi colorectal carcinomas. both ck7 and ck20 should be used for differential diagnosis of colorectal cancer. ofp-04-004 intratumoral budding in preoperative biopsies predicts local and distant metastasis in colorectal cancer patients a. lugli * , m. hädrich, d. inderbitzin, m. borner, i. zlobec * medizin. universität bern, inst. für pathologie, switzerland objective: in 2011, the term "intratumoral budding, itb" was used to describe the presence of tumor buds within the main tumor body and was correlated to worse clinical outcome in colorectal cancer patients. here, we further elucidate the potential clinical role of itb in pre-operative biopsies using pan-cytokeratin stained tissues and a quantitative scoring system. method: 139 pre-operative biopsies from patients with colorectal cancer underwent immunohistochemistry for pancytokeratin (ae1/ae3). itb were counted in the area of densest budding (40×) and classified as high-grade when >10 buds/hpf were observed based on receiver operating characteristic (roc) curve analysis. results: high-grade itb occurred in 26.6 % of cases and was associated with right-sided tumor location (p= 0.0356), more advanced pt (p =0.0198) and pn (p< 0.0001) classifications, distant metastasis (p = 0.0164), higher tumor grade (p=0.0037) and lymphatic invasion (p=0.0445). the specificity and positive predictive value for lymph node metastasis was 86.7 % and 75.6 %, respectively. disease-free survival was significantly worse in patients with high-grade itb (5-year survival=25 %) in comparison to patients with low-grade itb (5-year survival= 55 %) (p=0.0157). conclusion: the assessment of itb in pre-operative biopsies is predictive of local and distant metastasis in corresponding resections and should be considered in daily management of colorectal cancer patients. ofp-04-005 alpha-methylacyl-coa (amacr) in gastric cancer -correlation with clinicopathological data and disease free survival a. mroz * , m. kiedrowski, z. lewandowski * cmkp, gastroenterology and hepatology, warsaw, poland objective: diagnostic and prognostic significance of alphamethylacyl-coa (amacr) has been established in many human cancers. its correlation with clinical and pathological data in gastric cancer has not been fully elucidated and its impact on surveillance has not been studied thus far. method: we analyzed consecutive gastric cancer cases in terms of amacr immunohistochemical expression and clinical/pathological characteristics and followed patients' postoperative history. results: amacr was expressed in 94/164 gastric cancers (57.3 %). we did not find correlation between amacr expression and gender, age, location, histological type, ptn staging, vascular and nerve sheaths invasion. overall disease-free survival tended to be worse in amacr positive patients (p=0.062), and in adenocarcinoma subgroup it was significantly shorter (p=0.021). conclusion: amacr expression might represent promising adverse prognostic factor in gastric cancer, particularly in adenocarcinoma histological type. ofp-04-006 development of a rabbit monoclonal antibody for determining hent1 status and predicting response to gemcitabine in pancreatic ductal adenocarcinoma m. raponi * , j. isaacson, j. ranger-moore, j. clements, b. richardson, s. ormanns, k. winter, a. allen, r. andersson, v. heinemann, a. p. dicker * clovis oncology, san francisco, ca, usa objective: human equilibrative nucleoside transporter 1 (hent1) is the primary membrane channel through which gemcitabine (gem) enters pancreatic tumor cells, and patients whose tumors have low expression of hent1 may derive little benefit from gem therapy. method: an analytically validated rabbit monoclonal antibody immunohistochemistry (ihc) in vitro diagnostic (ivd) for assessing the degree of hent1 expression in pancreatic adenocarcinoma using an automated ihc platform has been developed. results: using primary tumor samples (n=201) from a randomized controlled clinical adjuvant trial (rtog 97-04), we developed and verified a hent1 scoring methodology and cut-off for identifying patients least likely to benefit from gem (hent1-low). the distribution of hent1 expression was similar between these samples and an independent set of 130 pancreatic adenocarcinoma specimens from the aio-pk0104 study of which 77 were confirmed metastatic biopsies. overall, using this cut-off, approximately two thirds of pancreatic tumors displayed low-hent1 expression across the different data sets. conclusion: a robust hent1 ihc ivd has been developed using a rabbit monoclonal antibody and identifies two thirds of primary and metastatic pdac cases as having low expression of hent1. ofp-04-007 hypoxia activates pancreatic stellate cells: development of an organotypic culture model of thick slices of normal human pancreas v. rebours * , m. albuquerque, p. ruszniewski, p. lévy, a. sauvanet, v. paradis, p. bedossa, a. couvelard * beaujon hospital, pancreatology unit, clichy, france objective: pancreatic stellate cells (psc) promote oncogenesis by modulating cell proliferation. aim-to evaluate the early activation of psc in case of hypoxia in normal human pancreas. method: an organotypic culture model of thick sections of human pancreas has been developed and validated. slices of pancreas (300 μm) were prepared from surgical specimens and cultured in hyperoxia conditions. half of the samples underwent an initial phase of culture in normoxia (21 % o2) to reproduce hypoxia. the total duration of culture was 72 h. cell viability, hypoxia, apoptosis and activation were monitored. results: 30 sections per specimen were cultured. analysis was performed at baseline, 24 h, 48 h and 72 h. morphological analysis showed gradual appearance of ductal/acinar dedifferentiation. at 72 h, foci of necrosis were identified. hypoxia was confirmed by the expression of hif1 and ca9 at 48 h (10 % and 50 % of labeled cells). apoptosis was limited, acinar cells expressed caspase 3 at 48 and 72 h. analysis of proliferation using ki67 index showed significant activation of psc at 48 h (×5/baseline) and at 72 h (×6/ baseline). activation of psc was confirmed by smooth muscle actin immunochemistry. conclusion: organotypic culture of normal human pancreas is possible with optimized cell viability at 72 h. hypoxiainduced activation of psc occurs very early. objective: ipn are still poorly characterized precursor lesions of bile duct adenocarcinoma. we present a multicenter study with focus on morphology, subtyping and prognosis and provide a detailed molecular analysis in the stepwise progression from low-grade to high-grade lesions and invasive carcinoma. method: fourty-five patients with biliary ipn were included into the cohort, twenty-two patients with conventional adenocarcinoma of the bile duct served as control cohort. subtyping was performed based on histomorphology and expression profile of mucins and cdx2 expression. furthermore, low grade lesions (n=14), high-grade lesions (n=38) and invasive components (n=13) of ipn as well as conventional bile duct adenocarcinomas (n=22) were investigated individually for alterations of common oncogenic pathways using immunohistochemistry, real-time polymerase chainreaction and direct sequencing. results: ipn are mainly detected at preinvasive stage (71 %), pancreato-biliary and intestinal type are the most common subtypes (36 and 29 %, respectively). detailed molecular analysis showed that p53 overexpression, k-ras mutation, loss of heterozygosity of p16 locus and nuclear ßcatenin expression are early events and loss of smad4 and overexpression of egfr and her2 are later events in the tumor progression of ipn. conclusion: stepwise alterations of common oncogenic pathways are required for progression of biliary ipn from non-invasive precursors to invasive carcinoma. ofp-04-009 influence of braf mutations and rac1b/rac1 mrna ratio or protein expression on outcome in patients with metastatic colorectal cancer (mcrc) treated with firstline chemotherapy m. cuatrecasas * , v. alonso-espinaco, p. jares, c. horndler, a. castells, j. j. lozano, j. maurel * hospital clinic, barcelona, spain objective: metastatic colorectal cancer (mcrc) patients with braf mutation v600e present poor overall survival (os). rac1b, a rac1 spliced variant, is overexpressed in crc and impairs apoptosis by activation of nuclear-factor-kb. we evaluated if rac1b was an independent prognostic factor in mcrc. method: we examined braf (v600e) mutational status, rac1b by immunohistochemistry, rac1b/rac1 expression ratio by mrna rt-pcr, and mismatch repair (mrr) deficiency by microsatellite instability (msi) analysis, in 186 mcrc patients treated in firstline therapy with folfox or capox from three spanish institutions. we assessed whether these biomarkers were independently predictive of progression free survival (pfs) or os. results: 7 % crc had braf mutations, 5 % msi-h, 20 % high rac1b/rac1 ratio, and 25 % rac1b protein overexpression. five of 11 crc (46 %) with braf mutations had high rac1b/rac1 ratio/protein vs 25/144 (18 %) without braf mutation (p=0.036). all braf mutated or rac1b/rac1 high were mss. low rac1b/rac1 ratio and braf wt had higher response rate (68 % vs 43 %; p= 0.035). multivariate regression analysis identified ecog ps as a significant variable for pfs and ldh levels, ps and high rac1b/rac1 ratio for os. conclusion: high rac1b/rac1 expression ratio or immunoexpression constitutes a marker of poor os and a potential marker of acquired chemo-resistance in mcrc treated with oxaliplatin-based therapy. objective: annually more than 120.000 new cases of papillary thyroid cancer, that is 1 % of malignant tumors, are registered all over the world. method: there are clinical and morphologiсal methods: immunohistochemistry (mmp-2, mmp-9, timp-1, timp-2), light, transmission, electron, probe and scanning microscopy with the trace element analysis. it was studied 1541 cases of thyroid cancer since. results: at the papillary thyroid cancer progression matrix metalloprotease and specific tissue inhibitors of metalloprotease (timp-2) increased in the cytoplasm of tumor cells, but the concentration of timp-1 decreased. tumors with low degree of ultrastructural differentiation were presented by cells with electron-dense dark cytoplasm and cells with poor cytoplasm organelles and differently formed nucleus with numerous deep invaginations. high level of some matrix metalloproteaseexpression (ммр-9) and low level of timp expression (тimp-1) were observed in current cases more (45 % and 58 % respectively), than at more differential papillary thyroid cancer (23 % and 37 %). at performance of atomic force microscopy it was shown that connection between cells had become weak at the decreasing of malignancy degree. it was revealed the significant increasing of oxygen, magnium, potassium, calcium in tumor nodes. conclusion: conducted comlex morpho-chemistry analysis display diagnostic patterns of papillary thyroid cancer. objective: cytological features of btn-ph, sometimes interpreted as papillary carcinoma (pc) on fine-needle aspiration biopsies (fnab), have not been studied extensively. method: available material from 48 btn-ph was reviewed retrospectively and compared with 15 pcs. results: fnab diagnoses were: non-diagnostic (n=1), benign (n=18), aus/flus (n=10), suspicious for follicular neoplasm (n=4), suspicious for malignancy (s-pc) (n=13), and malignant (p-pc) (n=2). the extent of papillary hyperplasia on histology was higher in s-pc/p-pc diagnostic categories (50 %; p=0.019; range: 5-90 %). on cytology, papillary structures, present in 53 % of bpn-ph cases, were more frequent and numerous in the s-pc/p-pc group (89 %; p= 0.0093). focal nuclear atypia was seen in all s-pc/p-pc cases, including rare grooves (87.5 %), enlargement/crowding (75 %), chromatin clearing/rare pseudoinclusions (25 %), but were less than in pcs (p<0.0001). nuclei were smaller in btn-ph than in pcs (mean diameter/range (μm): 8.2/6-12 vs 14.2/7-26; p=0.0001). all btn-ph tested (n=9) were immunonegative for hbme-1, galectin-3, and keratin-19. conclusion: btn-ph diagnosed as s-pc/p-pc often show papillary structures and focal nuclear features of pc but they are significantly less than in classical pcs, stressing the need to apply strict criteria for pc diagnosis. ofp-05-003 genetic alterations in glucagon cell adenomatosis t. henopp * , m. anlauf, s. biskup, g. klöppel, b. sipos * medizin. universität tübingen, inst. für pathologie, germany objective: glucagon cell adenomatosis (gca) was recently recognized by us as a multifocal neoplastic disease of the endocrine pancreas unrelated to men1. multiple micro-and a few macrotumors are found on the background of a hyperplasia of glucagon cells. the disease may cause unspecific abdominal symptoms and only rarely a glucagonoma syndrome. recently a mutation in the glucagon receptor (gcgr) gene was described in one gca patient. method: the extracted dna of five patients with gca was sequenced and the gcgr gene analyzed for mutations. results: sequencing of the gcgr gene revealed germline mutations in three out of five patients. one patient shows two different heterozygous point mutations in the hyperplastic alpha cells as well as in the non-tumorous tissue leading to two premature stop codons. one patient harbors a homozygous stop mutation. the third patient shows two homozygous missense mutations of the gcgr gene that most likely also led to a dysfunction of the gcgr. in the two other patients no germ line mutations of the gcgr gene were detected. these variants were not identified in healthy subjects. conclusion: the finding of germ line and somatic "loss of function" mutations of the gcgr gene in three of five patients with gca suggests that a change in the signalling function of the gcgr may cause glucagon cell adenomatosis via glucagon cell hyperplasia. ofp-05-004 timp-1 expression and hypoxia in papillary thyroid carcinoma: relationship to brafv600e mutation and clinical behavior m. ilie * , s. lassalle, p. brest, c. bonnetaud, a. bozec, n. guevara, j. haudebourg, i. birtwisle-peyrottes, j. santini, p. hofman * chu de nice, lpce, france objective: brafv600e causes up-regulation of timp-1, promoting cell invasion in papillary thyroid carcinoma (ptc). hif-1α is regulated by hypoxia but also by brafv600emediated signaling pathway in ptc. we assessed the impact on clinical behavior in ptc of timp-1, hif-1α and the hypoxia-inducible caix and caxii. method: the protein expression was assessed by western blot in two cell lines, tpc-1/brafwt and bcpap/ brafv600e. tma-immunohistochemistry analysis was used to study protein expression in 114 ptc samples. braf status was analyzed by pyrosequencing. data were correlated with clinicopathological variables of patients. results: higher expression of all proteins was detected in bcpap exposed to hypoxia. timp-1 expression displayed 87 % sensitivity and 83 % specificity for identifying braf mutation (p<0.001), and was associated with pt-stage (p= 0.001), pn-stage (p= 0.02), and multifocality (p=0.03). hif-1α expression was correlated to pt-stage (p=0.05). caix expression was related to pn-stage (p=0.02), and both caix (p=0.004) and caxii (p=0.05) were highly associated with vascular invasion. conclusion: timp-1 protein expression is reliable surrogate of braf mutated status in ptc. timp-1 and hypoxiaregulated proteins have potential for future use as predictors of the malignant change in ptc, and warrant further investigation as new therapeutic targets for the treatment of highly aggressive form of ptc. objective: mtc are usually aggressive tumours which account for 3-5 % of all thyroid carcinomas. most of mtc are sporadic, the remaining tumours are due to hereditary forms with germline activating mutations of the ret proto-oncogene. the aim of this study was to assess the microrna profiling expression in both sporadic and hereditary forms of mtc. method: a total of 40 frozen mtc from patients with welldocumented clinico-pathological parameters were used for microarray analyses (14 hmtc and 26 smtc) using human microrna mircoarray kit version 2 (agilent technologies). after identification of a set of mirna of interest, validation was done using qpcr. results: when comparing hmtc harbouring germinal ret mutation with smtc, seven mirnawere significantly deregulated (mir-96, −10a, −376c, −15a, −7-2*, −106b, −132). the most highly discriminative mirna in tumoral samples (>3 fold change) identify mir-375, −129-3p, −136, −376c and −451. moreover, 5 mirna could segregate mtc with good prognosis and better clinical outcome (mir-137, −144, −224, −144 and −224) . conclusion: a specific microrna signature in hmtc can be identified. moreover, detection of micrornas in tissue samples might be of interest for improved clinical management and treatment of patients with mtc. ofp-05-006 focus on the follicular variant of papillary thyroid carcinoma: combination of the immunohistochemical markers ck19, hbme1 and tpo is associated with histopathological diagnosis, better than molecular markers objective: the follicular variant (fvptc) is the major variant of papillary thyroid carcinoma (ptc), ranging from 9 % to 22 % of all ptc. it remains however a problematic entity. some consider that the encapsulated fvptc (enfvptc) have an excellent prognosis like follicular adenomas (fa); others showed that enfvptcs had potential for hematogenous spread like follicular carcinoma. its place in the spectrum the multivariate cox regression the flt3-itd mutation (p= 0.001) was an independent poor prognostic factor, whereas the goodness-of-fit of the minkowski fractal dimension an independent favorable prognosticator (p=0.025). the karyotype had no influence on survival. conclusion: flt3-itd and chromatin fractal characteristics were more important as prognostic factors for overall survival than karyotype and gene methylation status. financial support: fapesp and cnpq. objective: the prevalence of cyclin d1 (cyd1) positive bcells with mantle cell lymphoma (mcl) phenotype in the mantle zones of reactive lymph nodes ("in situ" mcl/mclis). and related minimal mcl infiltrates in reactive lymphoid tissues of healthy individuals and in mcl patients are unknown. method: all 1.292 reactive lymph nodes from unselected consecutive surgical specimens of 131 patients without a history of lymphoma obtained over a 3 months´period were stained for cyd1. additionally, all morphologically reactive lymph nodes and extranodal lymphoid infiltrates of mcl patients from 2000 to 2011 were studied. samples predating the lymphoma diagnosis for at least 2 months were available from 37/423 (8.7 %) patients. results: a mclis was not identified. however, in four mcl patients, an early manifestation of mcl with mantle zone growth pattern was detected retrospectively, antedating the lymphoma diagnosis for 3-86 months. in six mcl patients, only small groups of cyd1 positive cells in morphologically reactive extranodal infiltrates were detected more than 2 months before the diagnosis of mcl (range 3-59 months). conclusion: mcl "in situ" is an extremely rare phenomenon in morphologically reactive lymph nodes. in mcl patients, however, immunohistochemically detectable infiltrates of mcl cells antedating the lymphoma diagnosis were found in a significant proportion of cases (10/37=27 %). ofp-07-003 e2f-1's relationship with tumor growth in hodgkin's lymphoma is regulated by p53 status e. georgiadi * , g. dimtsas, t. p. vassilakopoulos, v. g. gorgoulis, i. a. doussis-anagnostopoulou * larisa, greece objective: e2f-1 is a member of the e2f family group of transcriptional factors that play a major role in cell cycle progression and arrest, as well as in apoptosis. in different tumors e2f1 can demonstrate opposing roles, acting either as an oncogene or as a tumor suppressor gene. method: we have previously established e2f-1's immunohistochemical expression in hodgkin lymphoma (hl) and its correlation with p53 expression, although no relationship with either proliferation or apoptosis was demonstrated. a further investigation in a larger series of 100 cases of primary hl with the addition of new apoptotic techniques and the use of p21 expression as an indicator of p53 functional status was undertaken. results: following stratification of our cases based on p53 functionality, e2f-1 was inversely correlated with the proliferation marker topoiia. independently of p53, high levels of e2f-1 expression showed a linear trend with apoptosis and an inverse trend with proliferation (borderline p value in both cases). conclusion: both e2f-1 and p53 activity is upregulated in hl, however it seems that p53 status is a major regulator of the relationship between e2f-1 expression and tumor growth. alternatively, e2f-1 may use p53-independent pathways to induce apoptosis. objective: ptld represent a spectrum of usually ebv-driven lymphoplasmacytic proliferations in the settings of immunodeficiency associated with allograft. ebv infects cells in latent or lytic forms. although viral oncogenes are expressed in latency programs, lytic ebv replication is required to develop lymphomas in mouse models. the role of intratumoral ebv replication in ptld has never been addressed before. method: a series of 35 ptld was reviewed and ebv latency genes were explored in all cases and included eber1-2, lmp1 and ebna2. moreover, two early lytic genes involved in ebv replication, bzlf1/zebra and ead11 were also analyzed. all clinical and pathological data were collected. results: the median age was 50 years (27-77) with a male predominance (26 m: 9 f). ebv infection was observed in 28 cases (80 %). ebv replication was observed in 40 % of the cases. these were more likely to be polymorphic ptld with latency iii in the context of stem cell transplantation. moreover these cases had atypical clinical presentations such as brain involvement or disseminated disease with aggressive behavior (os: 11 months vs. 48 months). conclusion: ebv replication occurs in tumor cells of ptld-patients associated with poor outcome and may be share common morphological and phenotypical features. in routine praxis, their differential diagnosis might represent a challenge requiring a specific work-up of immunohistochemical (ihc) and fluorescent in situ hybridization (fish) analyses. method: paraffin sections from 17 cd20+/cd5+ blastic b-nhl were examined for cyclin-d1, cd10, bcl-6 and mum-1 ihc expression together with fish method using probes for ccnd1 rearrangement and/or igh/ccnd1 translocation. the final diagnosis was established according to the criteria of who classification. results: 11/17 cases were diagnosed as bmcl showing cyclin-d1 positivity and ccnd1 rearrangement, other analysed ihc markers were negative. five of these cases were polyploid. 6/17 cases were diagnosed as cd5+ dlbcl in spite of coexpression of cyclin-d1 in 2 of them, as they showed various cd10, bcl-6 and mum-1 expressions and missed ccnd1 gene rearrangement. conclusion: cyclin-d1 positivity may appear in other than mcl lymphomas due to changes at transcriptional or posttranscriptional level. in spite of overlaping phenotypes, fish analysis of ccnd1 represents an effective tool to increase the diagnostic accuracy to distinguish cd5+ dlbcl from bmcl. supported by vega grant nr. 1/ 0378/12 and projects ceprii (imts: 26220120036) and mdcc (imts: 26220220113) co-financed by eu sources. ofp-07-008 still's disease associated lymphadenopathy: report of two cases resembling malignant lymphoma with review of possible morphological findings k. kamarádová * , m. nova, m. kalinova, v. campr * prague, czech republic objective: still's disease (or systemic onset juvenile idiopathic arthritis, sojia) is a systemic inflammatory disorder characterized by fever, rash, and arthritis. it has a peak of incidence in early childhood with rare occurence in adulthood (adult-onset still's disease, aods). several other symptoms as hepatosplenomegaly, leukocytosis and lymphadenopathy may be associated possibly mimicking malignant lymphoma. method: we present two cases of still's disease associated lymphadenopathy in a child and in an adult simulating morphologically peripheral t-cell lymphoma and classical hodgkin lymphoma respectively. results: histological, immunohistochemical and molecular examination revealed non-neoplastic lymphadenopathy with atypical paracortical t-cell hyperplasia with immunoblastic reaction in the former and burnt-out histiocytic pattern in the latter, both falling into a broad spectrum of reactive lymph node changes associated with still's disease. the lymph node lesions in still's disease can be histologically divided into four major categories: 1) simple atypical paracortical hyperplasia, 2) burnt-out histiocytic pattern, 3) exuberant immunoblastic reaction in paracortical hyperplasia and 4) follicular hyperplasia. the second and the third pattern may closely resemble malignant lymphoma. extended and careful clinical examination with relevant laboratory findings and possible complex histochemical and molecular analysis are necesarry to rule out the suspition of lymphoma. objective: intravascular large b-cell lymphoma (ivlbcl) is a rare form of diffuse large b-cell lymphoma characterized by exclusive or preferential intravascular growth. ivlbcl often goes clinically unrecognized. we report three clinically unsuspected cases of classic variant ivlbcl. method: cases diagnosed as ivlbcl were collected from the pathology files of germans trias i pujol hospital. the diagnosis was reviewed in agreement with who 2008 criteria and immunophenotypic and clinical data were gathered. results: a 71-year-old male and an 87 year-old-male showed neurological symptoms leading to progressive deterioration and death. a 79-year-old female presented with fever of unknown origin and died 2 days after admission. at autopsy there were no gross findings suggestive of a neoplastic process in any instance. histologic study showed intravascular large b cells with vesicular nuclei involving predominantly the central nervous system in the first two cases and the adrenal glands, liver, and spleen in the third case. conclusion: the three cases of aggressive, advanced-stage lymphoma herein described illustrate how the clinical diagnosis of ivlbcl remains elusive due to its low frequency and variegated clinical presentation and how the pathologic diagnosis of ivlbcl continues to be highly dependent on autopsy examination. objective: the world health organisation (who) currently recognises four categories of ptld, which are lymphoid/ plasmacytic proliferations occurring due to immunosuppression after organ transplantation. the aim of this study was to examine the clinicopathologic characteristics of ptld occurring after renal transplantation in the republic of ireland. method: the national renal transplant registry from 1991 to 2000 (inclusive) was cross referenced with the national cancer registry to identify renal transplant recipients who had developed ptld up to the present day. review of pathology and chart review was conducted for each case. results: of 2,441 patients (2,667 allografts), 34 cases of ptld were identified, with pathology review possible in 33 cases. there were 26 (79 %) monomorphic ptld (23 diffuse large b cell {dlbcl}, 1 anaplastic large cell, 1 burkitts-like, 1 myeloma], 3 hodgkin-type ptld, 1 (3 %) polymorphic ptld and 1 (3 %) early lesion. in addition, there was 1 mantle cell lymphoma and 1 lymphomatoid granulomatosis. the annual incidence of ptld was estimated as 0.2 % (95 % ci 0.14-0.28). median time to diagnosis was 8.7 years (range 2 to 19.2 years). conclusion: similar to other studies, monomorphic ptld is the most common type, with dlbcl representing the majority of cases. of interest, early onset ptld was very uncommon in this study. monday, 10 september 2012, 17.00-19 .00, forum hall ofp-08 oral free paper session head and neck pathology ofp-08-001 human papilloma virus associated oropharyngeal carcinoma is strongly correlated with cancer of unknown primary s. ihrler * , g. assmann, p. zengel, m. mollenhauer * medizin. universität münchen, institut für pathologie, germany objective: human papillomavirus (hpv) associated oropharyngeal carcinoma has been identified as a distinct entity within squamous cell carcinoma of the head and neck and is preponderantly restricted to palatinal tonsils and base of tongue. these two primary locations have for long -and hpv-associated carcinomas have recently -been associated with the clinical situation of cancer of unknown primary (cup). method: to investigate the relationship between hpv and cup in detail, we studied 26 consecutive patients who initially presented as cup and were finally diagnosed with carcinomas of these two locations. results: twenty-one carcinomas (81 %) proved to be positive for high-risk hpv (p16 and polymerase chain reaction). they were frequently very small (smallest: 0.3 cm; 6 cases: ≤ 0.6 cm; on average 0.9 cm) and located in a deep, submucosal position. conclusion: this demonstrates an overrepresentation of hpv-associated carcinoma in patients who were initially diagnosed with cup, supporting a strong causal relationship between hpv-association and cup. the frequent manifestation as cup presumably is caused by the unusual predisposition for small size and submucosal location, combined with frequent and early lymphatic metastization. in order not to miss these small, clinically occult carcinomas, consequent interdisciplinary cooperation and meticulous histological work-up is mandatory. ofp-08-002 methylthioadenosine phosphorylase inactivation depends on gene deletion in laryngeal squamous cell carcinoma a. nadal * , l. conde, i. vilaseca, l. alos, m. bernal-sprekelsen, a. cardesa * hospital clínic barcelona, dept. of pathology, spain objective: methylthioadenosine phosphorylase is an essential enzyme for the methionine and adenosine salvage pathway in normal cells, frequently inactivated in many different human cancers. the mtap status could be important for tumor cell sensitivity to adjuvant chemotherapy. to date there are no reports on mtap status in laryngeal carcinoma. method: a series of 31 laryngeal squamous cell carcinomas was investigated for mtap mrna expression through reverse transcription and quantitative pcr (qpcr) as well as for mtap gene deletion and/or promoter hypermethylation through qpcr and methylation-specific pcr respectively. results: low mtap mrna expression was found in 32 % of cases, associated with mtap gene deletion in 70 % (p< 0.001) but not to mtap promoter hypermethylation, indicating that in this tumors gene deletion is the main mechanism of mtap inactivation. neither low mrna expression nor gene deletion was associated with any of the clinicopathologic parameters investigated. conclusion: given the significance of mtap status for cell sensitivity to different chemotherapeutic regimes, our results suggest that determination of mtap inactivation could be useful for adjuvant therapy selection in laryngeal squamous cell carcinomas. ofp-08-003 ercc1, p16 and ki-67 immunohistochemical expression as predictive and prognostic markers in head and neck squamous cell carcinoma treated with platin-based induction chemotherapy h. roussel * , m. housset, h. tournat, p. ravel, s. hans, c. badoual * hegp, dept. de anatomie-pathologie, paris, france objective: ecc1 enzyme has been associated with resistance to platinum-based chemotherapy. the purpose of this study was to evaluate the role of ercc1 expression with p16 and ki-67 as predictive and prognostic markers in the response to platin-based induction chemotherapy in patients with head and neck carcinoma (hnscc). method: 208 patients treated from 2000 to 2006 by an induction chemotherapy regimen for hnscc were included retrospectively. we assessed response to treatment, progression-free survival (pfs) and overall survival (os). results: 68 % and 81.5 % of hnscc showed low expression of 8 f1 and fl297 ercc1. no correlation was found between the two clones (p=0.1). in the 129 patients treated with cisplatin-5fu chemotherapy, a low expression of 8 f1 ercc1 was associated with a better response (p=0.027). over expression of p16, (34.5 % of cancers of the oropharynx) was correlated with a better os (p=0.0007) and a better pfs (p=0.01). conclusion: these results suggest that ercc1 expression might be a useful predictive marker of hnscc in patients treated by cisplatin-based chemotherapy. the 8 f1 ercc1 clone appears to be the best for immunohistochemistry. our study confirms the prognostic value of the over expression of p16 in carcinoma of the oropharynx. objective: tumor-stroma ratio or proportion of tumor (pt) has been presented as a prognostic factor in colorectal and breast adenocarcinomas, but there is no information about squamous cell carcinomas (scc) and laryngeal carcinomas in particular. method: eighty-five laryngeal carcinoma cases were included in this series. five digital images of the tumor sections were obtained (h&ex20). percentage of epithelial tumor component was determined by software allowing the pathologist's selection of tiny areas as carcinomatous and stromal region for statistical analysis as a prognostic marker. results: median follow up was 48 months (range 3-194). the mean pt was (48,63+18,18) . there was no difference for pt when tumor grade and stage were considered. although statistically insignificant, the mean pt was the lowest (37,46+12,49) for subglottic carcinomas and cases with perinodal invasion (44,72+20,23), and highest in pn0 cases (50,05+17,34) but there was no statistical significance. the over all and disease free survival analysis did not reveal significance for pt. only pathological stage was an independent factor for over all survival (p=0,08). conclusion: although there might be an association with adverse prognostic factors and low tp, the findings in this series does not support pt as a prognostic marker in laryngeal carcinomas. ofp-08-005 tumoral microvasculature in ameloblastoma subtypes c. h. siar * , k. h. ng * university of malaya, dept of. oral pathology, kuala lumpur, malaysia objective: angiogenesis is essential for tumoral growth and progression. the ameloblastoma is a benign but locallyinvasive odontogenic neoplasm with distinct behavioural characteristics of its subsets. whether angiogenesis contributes to a more aggressive course in these variants remains unclear. the aim here was to determine and compare the tumoral microvasculature in different ameloblastoma subtypes and to speculate on their significance. method: immunohistochemical staining for 4 vascular markers (cd31, cd34, cd105 and vegf) was performed on archival tissues of 40 cases solid/multicystic (sma), 20 unicystic (ua), 3 desmoplastic (da) and 14 recurrent ameloblastoma (ra). mean microvessel density (mvd) at the tumour advancing front and centres were obtained. results: vegf was heterogeneously expressed in the tumoral epithelium in all ameloblastoma subtypes. protein localization was membranous/cytoplasmic. mean mvd was slightly higher in sma compared to ua but the difference was not significant (p>0.05). mean mvds were not significantly different between primary and recurrent ameloblastoma; and between tumoral centre and advancing front of each subtype (p>0.05). conclusion: although ameloblastoma subtypes are distinctive in their clinicopathologic presentations and behaviour, their tumoral microvasculature is not different. this suggests that angiogenesis is not a major factor influencing the progression of these ameloblastoma subsets. ofp-08-006 an immunohistochemical study of e-cadherin and snail expression in laryngeal squamous cell carcinoma a. tanoglidi * , m. tsopanomichalou, c. barbatis, c. kostopoulos, o. pantzartzi, v. gorgoulis * hellenic red cross hospital, dept. of pathology, athens, greece objective: snail 1 and 2 (slug) are zinc-finger transcription factors, repressing the e-cadherin in epithelial tumors. published data in head and neck squamous cell carcinoma show either inverse relationship or unrelated co-expression, but e-cadherin reduction/loss and snail overexpression seem to confer an aggressive phenotype. method: thirty cases of laryngeal squamous cell carcinomas (lsccs) (gradei: 11, gradeii: 8, gradeiii: 11) and one large cell neuroendocrine carcinoma (nec) were studied using double staining immunohistochemical method for e-cadherin (mab-dab) and snail + slug (pab-ap). the colocalization of both molecules was assessed and the expression was compared to the adjacent normal or dysplastic epithelium and the histological grade. results: normal squamous and dysplastic epithelium show strong nuclear snail expression and complete e-cadherin membranous positivity. reduction/loss of snail with or without e-cadherin membranous expression is observed in 14/30 (47 %) lsccs with a tendency for inverse relationship with e-cadherin at the invasive front. 16/30 (53 %) lsccs strongly express nuclear snail, without absolute relationship to the reduction/loss of e-cadherin, with a tendency for snail negativity in grade ii (75 %). the nec is nuclear snail negative but cytoplasmic positive. conclusion: snail and e-cadherin may be retained, reduced or lost in lsccs, without a definite inverse relationship. these immunophenotypic combinations need further investigation. ofp-08-007 non-sebaceous lymphadenoma of salivary glands: proposed development from intraparotid lymph nodes and risk of misdiagnosis c. weiler * , a. agaimy, p. zengel, s. ihrler * munich, germany objective: non-sebaceous lymphadenoma (nsla) is a recently described, rare benign salivary tumour composed of lymphoid and epithelial components, definitionally lacking sebaceous differentiation. method: nine cases of nsla were immunohistochemically stained for ck5/6, ck7, ck14, ck18, p63, and ki67. results: all tumours (6 males, 3 females, mean age 50 years) were located in the parotid gland and showed intimate intermingling of lymphoid tissue with islands of epithelium with a wide spectrum of histological differentiation. the immunohistochemical profiles mirrored the epithelial differentiation; tumours with basaloid or lymphoepithelial differentiation strongly expressed ck5/6, ck14, p63, while tumours with ductal differentiation showed strong positivity for ck18/ck7 and ck5/6/ck14/p63 in luminal and basal cell layers, respectively. a hilus structure with salivary inclusions or d2-40 (podoplanin) positive marginal sinus were identifiable in 4 and 9 of the cases, respectively, confirming origin within intra-/periparotid lymph nodes. six cases were initially misdiagnosed as other benign (n=4) or malignant (n=2) tumours. conclusion: our study provides strong evidence that nsla belongs to the group of salivary tumours that pathogenetically develop from embryonic salivary inclusions in intra-/periparotid lymph nodes. knowledge of the wide histological spectrum of this rare tumour is important in order to avoid misdiagnosis. ofp-08-009 crtc1/maml2 fusion transcript in central mucoepidermoid carcinoma of mandible: diagnostic/ histogenetic implications d. bell * * md anderson cancer center, dept. of pathology, houston, tx, usa objective: mec typically arises from major/minor salivary glands. intraosseous salivary carcinomas are extremely rare (2-3 % of all mecs reported). the t(11; 19) and its crtc1/ maml1 fusion transcript have been identified in mec at different sites and are associated with development of a subset of these tumors. we report 9 examples of central mec of the mandible, including a case with a history of primary retromolar mec. method: rt-pcr and dna sequencing analyses used to study microdissected components of 9 central mec and 1 noncentral mec. of the central mec tumors, 5 arose from ectopic salivary rests; others appeared to be of glandular odontogenic origins. results: we identified crtc1/maml1 in 5 central mec arising from ectopic salivary rests. this fusion transcript was not detected in non-central mec or in another 4 central mec arising from a glandular odontogenic etiology. conclusion: central mec can manifest the fusion transcript in a subset of central mec originating from ectopic salivary rests, and may have diagnostic/histogenetic roles in the future analysis of this entity, given the absence of the fusion transcript in mec with glandular odontogenic precursors. since the initial clinical and radiological diagnosis in three central low-grade mecs was a benign odontogenic cyst, our findings support a future role for the fusion analysis in initial diagnostic efforts. ofp-08-010 salivary duct carcinoma: morphological and immunohistochemical study of 15 cases m. vazmitel * , a. dubrovskij, r. smoljakova, s. rjabceva * minsk, belarus objective: an evaluation of morphological and imunohistochemical features of salivary duct carcinoma (sdc). method: 15 cases sdc were retrieved from tumor archives collected at belarusian cancer centre from 2002 to 2011 years. immunohistochemistry, fish. results: a man to women ratio was 10:5; median age 61,5 years (average . tumors arose in the parotid (n=13), submandibular (n=2) glands. sdc developed from pleomorphic adenoma (n=3) or de novo (n=12), there were foci of ductal carcinoma in situ (n=4), sialodochodysplasia (n=4) and pagetoid spread (n=1) additionally to usual features of sdc. as well, tumors demonstrated acinic cell carcinoma-like (n = 2) and micropapillary growth patterns (n=1), oncocytic (n=4), clear cells (n=3), apocrine (n=1) and mucinous (n=1) changes. immunohistochemically, tumors were characterized by ar-expression (n= 9), her2/neu overexpression (n=7), of them score 2+ in 2 cases. ck8 and s-100 positivity, stat5 negativity was detected in one case with acinic cell carcinoma-like pattern. foci of sdc in situ were highlighted by ck14 and calponin. amplification of c-erb2-gene was found in 1 case, etv6/ntrk3 gene was not identified in acinic cell carcinoma-like sdc. conclusion: salivary duct carcinoma, like breast cancer, is characterized by wide variability of morphological patterns and immunohistochemical features reflecting possible molecular heterogeneity, which need further investigation. sunday, 9 september 2012, 17.00-19. objective: quantification of protein expression based on immunohistochemistry (ihc) is an important step for translational research and clinical routine. however, routinely used eyeballing scoring systems are time-consuming and subject to significant intra-and interobserver variability. aim of our study was to explore, whether an image analysis software proves sufficient as an alternative tool to assess protein expression. method: 630 prostate cancer specimens were stained with one nucleus specific marker (i.e., erg), one cytoplasmic specific marker (i.e., slc45a3), and one marker expressed in both compartments (i.e., tmprss2). a pathologist visually quantified all stainings, applying a four-step scoring system. for digital quantification, an image analysis software (tissue studio v.2.1) obtained a continuous spectrum of staining intensity. results: for each of the three antibodies we found a strong correlation of the eyeballing protein expression score and the score of the image analysis software (correlation coefficient of 0.94, 0.92, and 0.90 for erg, slc45a3, and tmprss2, respectively, p<0.01). conclusion: our data suggest that tissue studio is a powerful tool for the quantification of protein expression in ihc stainings. further, since the digital analysis is precise, it might help to overcome intra-and interobserver variability and increase objectivity of ihc based protein assessment. ofp-09-002 is quality of histological and cytological slides a palatable issue?: the taste project m. comanescu * , g. bussolati, a. sapino, g. butur, f. schmitt, f. feoli, t. tot, e. ovcin * institutul victor babes, dept. of pathology, bucharest, romania objective: the taste (technological platform and repository of high quality images and slides) project aims at developing a novel tele-pathology system by means of modern icts (information and communication technology) and is addressed to technicians and doctors. method: histological and cytological preparations are the basis for pathological diagnosis performed in daily practice throughout europe in a number of several millions per year and correctness and reproducibility of such diagnosis are heavily dependent on the technical quality. yet, quality is variable in different places and countries, related to school level, technicians' dedication, standard of apparatuses and reagents. variation in technical quality of the preparations prevents their open circulation at european level and precludes optimal diagnosis. results: the taste project tackles these problems by building-up an ict environment taste system where professionals from different countries will submit, via web, "virtual slides" of their own preparations to a panel of internationally recognized experts who will give comments and suggestions on the quality of the preparation. conclusion: the present approach (unprecedented at world level) will fuel a web-based community, aimed to a levelling and improvement of histopathological and cytopathological preparations, thus leading to an innovative training and more reproducible diagnosis, a basic requisite for disease treatment. of the whole atlases. virtual microscope positions can be stored in the memory and users can navigate quickly back and forward similarly to web pages. all these features are available through any web browser without need to install any additional software. results: in addition to the current atlases (dermatopathology, fetopathology, pathology of the newborn and bone marrow), an interdisciplinary atlas of pathology for pregraduate students of medicine is under development. it will contain full size annotated virtual slides. the atlases are free of charge, but registration is required. in order to ease the access, the atlases are connected to 15 national academic identity federations. the members of institutions connected to identity federations can use theirs home credentials without revealing them to the atlases. conclusion: the atlases can serve not only to the students as a source of information, but they can be used by teachers as a source of teaching material. proving experiment of international virtual slide telepathology, japan-vietnam and japan-china trial i. mori * , s. aida, y. osamura * international university of health, dept. of pathology, tokyo, japan objective: the communications between asian countries are growing. in the field of medical information and technologies, the necessity of communication is also required. in this work, as communication of pathology field, we tried to prove that international remote pathology diagnosis is possible using virtual microscopy (vs) telepathology. method: we put vs scanner and server to 1) our hospital, tokyo, japan, 2) cho-rai hospital, ho-chi-minh city, vietnam, and 3) chinese rehabilitation research center, beijing, china. pathologists go and look the vs through leased optical line circuit and make diagnosis. discussion was made through teleconference system. results: the vs image maintained adequate quality for pathology diagnosis even looked cross-border. the diagnoses matched well between hospitals. the response of vs viewer was also good this time. when we use too much band width to the teleconference system or we sent too many data in the background, the response deteriorate, and was not good enough for diagnosis. conclusion: it was confirmed that the international vs telepathology diagnosis is now reached to a stage of actual use at least when connected by leased optical line circuit. challenges and solutions in the setup of a findings database for a large scale tissue collection r. reihs * , h. müller, s. sauer, k. zatloukal * medizin. universität graz, inst. für pathologie, austria objective: many medical centres have acquired through the last years huge data collections of great relevance for biomedical research. in order to utilize this knowledge it is necessary to analyse this records in a structured way. in our use case the starting point were approximately 1.4 million pathological findings of a non-selected patient group. method: we developed software tools for the classification of medical records using a phonetic a multi-level spell correction module and an ontology term extraction and decision tree text classification system. with the help of a visual editor a comprehensive decision tree (4174 nodes) was set-up by a team of bioinformaticians and medical experts. results: with our tool set we achieved in the icd-10 classification a f-score of 89,7 % (precession 83,2 % and recall 97,5 %) for the interpretation of the classification results we developed visualization tools and applied the whole software suite at several test cases, e.g. the analysis of a colon data-set with 216.000 findings over 28 years. conclusion: we developed an automatic classification and visualization system and applied the results in a series of research projects. the software suite is currently used in several research projects and can be easily adjusted to specific phrases used in different institutions and languages. an image repository for decision support o. eichhorn * * aperio, vista, usa objective: pathology is a visual field, and pathologists make decisions based on visual information. an online repository of easily searched, well-categorized pathology images can be of immense use to pathologists, scientists, educators, and students. method: individuals and institutions all over the world have accumulated collections with thousands of slides representing a huge variety of pathologies. some collections are digitized and available online, while others remain libraries of glass slides, with a variety of formats for case information and other metadata. working with collectors to publish their slides and aggregating them together can yield an amazing decision support resource. results: long-term archival storage is a key issue for organizations adopting epathology. a cloud-based repository can provide an inexpensive and scalable solution. epathology is increasingly used for remote consultations. cases managed by online consultation networks can be de-identified and contributed to a common repository, increasing the value of the decision support tool while providing a long-term archive. conclusion: this talk will present concepts for an image repository for decision support, and discuss technical and business considerations for making it a reality, including ways to curate image metadata, organizational principles and search tools, relationships with users and contributors, access methods, and security and privacy considerations. ofp-09-007 perfect diagnostic accuracy using pathomorphological diagnosis spectra constructed with the help of whole slide imaging k. furuya * , t. maeda, k. kito * ehime prefectural central hospital, dept. of pathology, matsuyama, japan objective: pathomorphological diagnosis (pd) is important for estimation of clinical trials and new medical equipment such as elastography. however, interpretation of pathomorphological findings is often subjective, and pathology publications show only a limited number of photographs. because pathomorphological changes occur in succession, we attempted to construct pd spectra to improve pd accuracy. method: pd spectra are defined as the series of pathomorphological images arranged in the order of disease progression or cell cycle procession. the images from the whole slide imaging (wsi) server were arranged in order in power point files at a fixed magnification, and 4 pd spectra were constructed: chronic hepatitis staging, nuclear grading of hepatocellular carcinoma, nuclear grading of breast cancer, and ki-67 labelling index (li). the numbering of all images in the pd spectra corresponded with that of the wsi images so that the whole slides could be viewed alongside. next, these tools were tried out by 6 people. results: these tools brought on perfect accuracy with regard to staging and grading, and very high precision of ki-67 li for breast cancer. conclusion: pd spectra are valuable not only for pd but also as learning tools and have a worldwide applicability for measuring pd online. ofp-09-008 color correction of immunohistochemistry stained tissue section images by histogram transfer according to control tissues s. sarioglu * , s. seyrek, m. sakar * dokuz eylul university, faculty of medicine, izmir, turkey objective: for semiquantitative and quantitative analysis, previous normalization attempts of images of tissue sections are not satisfactory. in this study we evaluated, if color correction can be achieved by histogram transfer depending upon control tissue image (cti) differences. method: images from colon and placenta sections stained by anti-cd34 were used as cti and/or sample tissue images (sti). thirty-four but one (standard stained slide with images scti and ssti), was stained for different durations and dilutions than the standard procedures. digital images taken by a ccd camera connected to a light microscope without normalization was stored at a computer. software was prepared in order to find the histogram difference between two ctis and transfer the difference to the sti for achieving a corrected sti (corsti). ssti (one image) and sti and corsti (34 images each) were semiquantitatively scored by two observers in blind fashion and the sti and corsti scores were compared with ssti score. results: the wkappa was 0,59 for two observers. the sti semiquantitative score was same as the ssti in 23,5 % of the sti but this was 76,35 % for corsti. conclusion: it seems histogram transfer depending upon ctis may be a valuable tool for color correction of tissue section images. sunday, 9 september 2012, 14.15 -16.15 objective: there is a crucial need for identification of markers to choose the best treatment schedule and monitor the effectiveness of treatment in metastatic melanoma (mm) . the detection of circulating tumor cells (ctc) correlates with prognosis in different cancer subtypes. ctc can be detected by direct and indirect methods such as cytopathological approach, rt-pcr or immunomagnetic separation using the cellsearch (cs) method. method: from june 2011 to december 2011, blood was drawn from 27 patients with mm and 10 controls patients and processed into 72 h by the cs method. the primary end-point was the overall survival (os) of mm patients. results: enumeration of at least 1 cmc was effective in 61.5 % of patients with mm (n=16) and in 20 % of controls patients (n=2). nine patients (35 %) had ≥ 2cmc. none of the controls patients had more than one cmc. mean os time was 5.6 months among patients with mm and <2 cmc (n=8) and mean os time was 1.7 months among patients with mm and cmc ≥2 (n=26) (p=0.002). conclusion: our results demonstrate that detection of cmc correlates with os in melanoma. thus, cmc monitoring should be developed to ensure the best treatment follow-up for patients with mm. ofp-10-002 significance of circulating tumor cells (ctcs) detection using the cellsearch system in patients with locally advanced head and neck squamous cell carcinoma (hnscc) a. bozec * , m. ilie, e. long, o. dassonville, g. poissonnet, j. santini, e. chamorey, f. peyrade, k. benezery, a. sudaka, e. selva, p. hofman * centre antoine lacassagne, dept. of surgery, nice, france objective: the significance of ctcs in patients with hnscc is debated currently. we evaluated the potential detection of ctcs using the cellsearch (cs) assay tm (veridex, nj, usa) in hnscc patients and to identify the clinical factors predictive of the presence of ctcs in this population. method: forty-nine patients with locally advanced hnscc were included. the presence and number of ctcs were determined using the cs system in all patients prior to the initiation of therapy, in 21 patients 3 months after treatment, and in 10 healthy individuals. results: the cs system was able to detect the presence of ctcs in 8 of 49 patients (16 %) before therapy and in 4 of 21 disease-free patients (19 %) after therapy. no ctc was found in the control group. when considering the presence of ctcs before or after therapy, the presence of ctcs was statistically associated with patients' age (p=0.04; t-test) and n-stage (p=0.02; fisher's exact test). conclusion: ctcs are identified in a relatively low proportion of patients with locally advanced hnscc and correlated with initial lymph node involvement. ctc detection could be a future useful prognostic tool to adapt the treatment intensity in hnscc patients. the diagnostic utility in identifications of an aneuploid chromosomal (ch.) pattern by using urovysion™: a fluorescence in situ hybridization (fish) commercial test, to improve the efficacy of cytology in peritoneal effusions l. baron * , m. postiglione, c. trombetta, f. quarto * p.o.s. leonardo, s.o.c. di anatomia patologica, castellammare di stabia, italy objective: the challenge in diagnosis of effusion is in differentiation of reactive and neoplastic mesothelial or metastatic cells and identification of primary neoplasia. the difficulties in this differential diagnosis derived from the low sensitivity (~50 %) and specificity of effusion cytology. by identification of tumor associated aneuploidy fish could enhance the cytodiagnostic yield in effusions. method: we used urovysion™ (abbott), a fish commercial kit, designed for identifications of ch. 3, 7, 17 polisomy and 9p21 loss in urothelial tumors. it has been applied to detect chromosomal aneuploidies as a marker of malignancy in 64 effusions with or without known source primary tumors. also was evaluated reproducibility of molecular alterations with primary tumors and any their specific aneuploidies. results: fish positivity was observed in 44 (69 %) of cases, whereas 31 were positive at cytology (48 %). overall results of cytology and fish together rised to 75 %. conclusion: urovysion™ test could be an useful tool to distinguish malignant cells in inconclusive effusions cytology but it doesn't seem to be used to provide definitive indications about primary neoplasia. ofp-10-004 loss of imprinting of isulin-like growth factor 2 (igf2) in colon carcinomas leads to the cell cycle genes activation -hints to intense proliferation d. belharazem * , j. kunanz, a.-k. henne, p. kienle, p. ströbel * inst. of pathology, university of medicine, mannheim, germany objective: the insulin-like growth factor 2 (igf2) gene is regulated by imprinting in normal tissues. the loss of imprinting (loi) results in the bi-allelic expression of igf2. its increased activity has been associated with many cancers including colorectal cancer (crc). to investigate the significance of igf2 in crc, normal tissue and tumor biopsies from 400 patients were analyzed and correlated with clinical outcome and the kras and pik3 status. method: the igf2 820 g/a gene polymorphism and imprinting status were examined by restriction fragment length polymorphism of dna derived from normal tissues and tumors then of rna from heterozygous cases. igf2 protein analysis as well as an rna microarray were performed. results: 41 % of the analysed cases were heterozygous. loi was detectable in both normal tissues and tumors in 60 % of cases. tumors with loi had significantly higher igf2 protein levels. gene expression analysis revealed significance of cell cycle progression and mitosis genes in loi. conclusion: igf2 loi is a common and early change in patients with crc and in normal tissues, provides possibly a preneoplastic change. however increased igf2 protein levels as well as a different cell cycle specific gene expression profile were detected in tumors with loi. future studies must be performed to find out, whether such subsets of tumors differ in their response to chemotherapy or alternative therapies. objective: novel high-throughput technologies has revealed numerous genomic abnormalities in b-cell non-hodgkin lymphomas (nhl). this study aimed to detect point mutations involving ezh2 and cd79b in a population of b-cell nhl by applying a multiplex mutation assay to minimal residual material from needle aspirates, and to determine the evolution of their mutational status overtime using all sorts of available samples from these patients. method: dna was extracted from residual cytological material stored on fta® cards as well as from archived cytological and histological specimens. the presence of point mutations was investigated by a specifically developed assay utilizing massarray spectrometry and confirmed by direct sequencing. results: all 121 samples from 80 b-cell nhl cases were successfully analyzed. mutations in ezh2 (y641) and cd79b (y196) were detected in 13.2 % and 8 % of the samples, respectively. mutational status varied in one third of the positive cases, with either gain or loss of mutations over the course of time. conclusion: minimal material from lymph node fine needle biopsy is a reliable source for high-throughput multiplex analysis for the detection of point mutation involving ezh2 and cd79b. whenever possible, the most recent sample should be tested to verify the current status of the disease. ofp-10-006 microrna expression profile identifies a distinct molecular signature between myc translocation-positive and negative burkitt lymphoma cases g. de falco * , a. onnis, f. morettini, f. fuligni, c. bellan, e. rogena, p. p. piccaluga, l. leoncini * university of siena, dept. human pathology and oncology, italy objective: the molecular hallmark of burkitt lymphoma (bl) is a dysregulation of myc oncogene, due to one of the three translocations leading to ig-myc fusion. however, about 10 % of bl cases lack an identifiable myc rearrangement, although no significant difference of myc expression among cases has been observed. additional mechanisms alternative to translocations were explored by microrna profiling. method: ten myc translocation-positive and ten myc translocation-negative formalin-fixed and paraffin-embedded bl specimens were used for this study. for microrna and gene expression profile, the samples were hybridized on the mircury™ lna array and the illumina dasl whole genome assay, respectively. results: our results identified a panel of four micrornas which are differentially expressed between the two groups. importantly, these micrornas control relevant biological processes, such as the angiogenesis, apoptosis and cell proliferation, according to gene ontology categories. furthermore, the impact of microrna dysregulation on the gene expression pattern identified genes which more likely are regulated by the selected microrna. conclusion: using this approach, we showed a clear-cut microrna and gene signature between myc translocationpositive and negative bls. the identification of specific altered micrornas may represent an alternative molecular mechanism leading to myc over-expression in the absence of genetic alteration in cancer. ofp-10-007 droplet digital pcr: a highly sensitive assay for b-raf, k-ras and egfr mutation detection j. frampton * , c. thorne, v. spivey, j. goodall, c. lowe * horizon discovery, dept. of diagnostics, cambridge, united kingdom objective: droplet digital™ pcr (ddpcr™) enables the absolute quantitation of nucleic acids in a sample. horizon discovery's patented gene editing technology enables it to manufacture reference material that is genetically defined and validated for the allelic frequency of the mutation. these reference standards were used to test the sensitivity of bio-rad's qx100™ droplet digital™ pcr platform. method: a panel of defined genomic dna allelic standards and formalin fixed paraffin embedded (ffpe) cell line samples were used in this study. this panel covered egfr (g719s, t790m, l858r and l861q), k-ras (codon 12) and b-raf (v600e and v600k) mutations containing allelic frequencies between 0.05 % and 50 %. droplet digital™ pcr was performed using mutation specific taqman custom snp assays. results: there was a strong correlation between the predicted and actual allelic frequencies even down to 0.05 %. the allelic frequency called by the ddpcr™ platform was highly reproducible between experiments particularly in the 1-50 % dilution range. conclusion: this study has demonstrated that the droplet digital™ pcr qx100™ platform can detect allelic frequencies down to at least 0.05 % using genomic dna purified from cell lines and down to at least 3.5 % using genomic dna extracted from ffpe cell lines. ofp-10-008 chromothripsis and focal copy number alterations determine poor outcome in malignant melanoma t. gaiser * , d. hirsch, r. kemmerling, j. camps, t. ried * medizin. universität mannheim, inst. für pathologie, germany objective: genetic changes during tumorigenesis are usually acquired sequentially. however, a recent study showed that in 2-3 % of all cancers a single catastrophic event, termed chromothripsis, can lead to massive genomic rearrangements confined to one or a few chromosomes. method: in order to explore whether the degree of genomic instability and chromothripsis would influence prognosis in cancer, we applied high-resolution array comparative genomic hybridization to 20 malignant melanomas (mm) that were, despite comparable conventional clinical and pathological parameters, defined by a profoundly different clinical course. results: we observed a striking association between both number and structure of chromosomal aberrations and outcome. mm associated with good prognosis showed only few chromosomal imbalances (average 1.6 alterations per case), predominantly presented as whole chromosome or chromosome arm gains and losses. mm with poor prognosis harbored significantly more chromosomal aberrations (13.9 per case; p=0.008). these aberrations were mostly focal events, which culminated in two cases in a pattern consistent with chromothripsis. conclusion: here, we describe for the first time the phenomenon of chromothripsis in primary mm and reveal a link between focal copy number alterations and chromothripsis with poor outcome in mm patients (p=0.0002), providing a genetic approach to predict outcome in histopathologically indistinguishable mm. ofp-10-009 targeting endometrial stromal sarcoma: histone deacetylase and pi3k/akt/mtor signaling p. quan * , e. lederer, i. halbwedl, h. denk, k. zatloukal, j. haybaeck * institute of pathology, medical university of graz, austria objective: endometrial stromal sarcoma (ess) is a gynecological malignancy with few therapeutic options. up-regulated histone deacetylase (hdac) 2 was observed in ess patients. the hdac inhibitor saha reduced ess cell growth by inhibiting mtor. the pi3k/akt/mtor signaling cascade, vital to cancer growth is a critical target in cancer therapy. this study aimed at investigating if hdacs interact with the pi3k signaling in ess pathogenesis and thus might serve as targets for ess therapy. method: tissue microarrays were used to determine hdac expression in ess specimens. western blots revealed protein expression levels of hdacs and pi3k related molecules in two independent ess and one control cell lines (ess-1, mes-sa and hesc). results: elevated hdac1 and 2 levels were found in ess tissues and cell lines. increased cell growth and akt/mtor cascade hyperactivation were detected in ess cells. saha reduced growth of all cells, and induced cell death in ess cells. saha reduced phosphorylated 4ebp1 in all cells, but only inhibited activation of akt and p70s6k in ess-1 cells. conclusion: hdacs are linked to pi3k signaling during ess pathogenesis. saha reduced cell growth via inhibiting pi3k/akt/mtor components suggesting that a combination of saha with pi3k inhibitors might be effective in treating ess. ofp-10-010 usefulness of linking biobanking field and animal model: high successful rate of human primary non-small cell lung carcinoma (nsclc) xenografts in a model system separated by distance and time m. ilie * , l. blot, v. hofman, e. long, m. nunes, c. butori, e. selva, a. merino-trigo, n. vénissac, j. mouroux, p. vrignaud, p. hofman * chu de nice, lpce, france objective: with the ongoing need to improve therapy for nsclc, there has been an increasing interest in the development of reliable preclinical models to test novel therapeutics. the aim of this study was to evaluate the rate of establishment of patient-derived nsclc xenografts in the context of a long-distance research network. method: fresh surgically resected nsclc specimens were addressed from the human biobank hospital (nice) to the animal facilities (sanofi, vitry-sur-seine). shipment was performed in aqix medium at room temperature. within 24 h post surgery, tumour fragments (~63 mm3) were subcutaneously implanted in female scid mice. the growing tumours were passaged in new mice (≤10 passages). the xenografts were histologically checked to eliminate human or murine lymphoma. results: overall, 98 nsclc samples were implanted leading to 32 (33 %) nsclc xenografts. the rate of tumour growth was higher in non-adenocarcinoma specimens (23/45; 51 %): 20/38 (53 %) for squamous cell carcinoma, 2/4 (50 %) for large cell carcinoma, and 1/3 (33 %) for pleomorphic carcinoma, when compared to adenocarcinoma samples (8/51; 16 %). conclusion: we report a high successful rate of xenotransplantation established from patient-derived nsclc tissues. our biobanking model system, regardless to extended time and distance, provides a stable and reliable animal model in human lung cancer research. monday, 10 september 2012, 14.15 -16.15 objective: we tried to interpret "the job" of mf in different stages of tumor growth by monitoring the dynamics of their distribution. method: on a rat model of experimental tumorigenesis using bp6 fibrosarcoma cells injected intraperitoneally, we performed a semiquantitative analysis of the recruitment of mf in the tumor compartment (microenvironment) as well as in peripheral blood. results: we observed a slow increase of mf count in the initial phase of tumor growth followed by a slow decrease in the late phase within the tumor compartment. simultaneously peripheral blood showed constant increase throughout the experiment resulting in typical leukocytosis accompanying developed tumors. conclusion: increased mf in the initial phase could reflect their enrollment to "prepare a suitable microenvironment for tumor seeding". their decrease began after the tumor size was macroscopic, thus it was time for them to carry out a "different job". in the mean time their amount in peripheral blood started becoming significant, this is also the time when tumor -host interactions reach a systemic level. in futher experiments we will attempt to verify our assumption: when the tumor flourish in the host organism, it uses informed, transformed and recruited macrophages for growth and building construction of the tumor, and for communication with distant organs on a regulatory level. ofp-11-002 wwtr1 and cyr61 are early markers of barrett's esophagus malignant progression m. mesquita * , j. cardoso, a. pereira, s. braga, m. bettencourt-dias, p. chaves, j. pereira-leal * ipolfg, dept. of pathology, lisboa, portugal objective: barrett's esophagus (be) is the major risk factor for esophageal adenocarcinoma (ea). be has a low risk of progression to ea, being imperative to identify markers to stratify the risk. our aim was to look for potential early biomarkers of be malignant progression. method: three available microarray datasets were analyzed with r statistical computing software complemented with bioconductor. biomarker prioritization included: 1) expression barcode 2.0; 2) filtering using differential expression analysis. candidate genes were validated by qrt-pcr on rna from paraffin embedded samples (rna-pes) of be patients with high-grade dysplasia/adc diagnosed during surveillance and of their index endoscopy dysplasia-free samples. as controls, we used samples from be patients who have not progressed. results: under conservative criteria, we identified 19 upregulated genes that distinguish ea-progressed from nonprogressed be samples. a second filter, followed by qrt-pcr validation, trimmed the candidates to 2 markers (wwtr1 and cyr61). qrt-pcr on time-series rna-pes of ea-progressed and ea-free be samples showed these genes are up-regulated years before the development of ea as compared to patients who did not developed ea. conclusion: wwtr1 and cyr61 were identified as early risk markers for be neoplastic progression and may have a potential role in be risk stratification. combined microrna in situ hybridization and immunohistochemical detection of protein markers b. nielsen * , t. møller, k. holmstrøm * bioneer a/s, dept. of molecular histology, hørsholm, denmark objective: micrornas are small noncoding rnas that constitute a novel group of biomarkers with exciting functions in cell differentiation, proliferation and apoptosis by mediating degradation or destabilization of target mrnas. mir-21 is highly prevalent in malignancies. previous reports on mir-21 in situ hybridization (ish) in colon and breast cancer identified expression in the stromal cell population that was related to recurrence and cell proliferation (nielsen et al., clin exp metastasis 2011, 28:27; rask et al., apmis 2011, 119:663) . here, we present a histological assay platform that allows parallel localization of micro-rnas and protein markers. method: routinely processed paraffin embedded breast cancer samples were analyzed. microrna ish probes were detected with fluorescent tyramine reagents and primary antibodies with compatible immunofluorescence. the reagents were introduced on a semi-automated platform, histoflex, which has a precise temperature control unit and utilizes continuous liquid flow. results: by combination of mir-21 ish and pdcd4 immunofluorescence we found differential expression in many but not all mir-21 positive cells. using the histoflex, the double fluorescence assay was reduced from 7 to 2 h without loss of sensitivity and specificity. conclusion: a 3-fold faster assay platform was developed and our in vivo findings support the assumption that pdcd4 is a target of mir-21. the tnf-superfamily members april and baff and their receptors (baffr, bcma, taci) are differentially expressed in tumors and normal tissues and exert specific effects related to cell fate and differentiation v. pelekanou * , v.-i. alexaki, g. notas, m. kampa, e. stathopoulos, a. tsapis, e. castanas * university of crete, pathology, school of medicine, heraklion, greece objective: classically, tumor immune-related microenvironment was synonym of inflammatory cell infiltrate. recently, the detection of synthesis and secretion of immune mediators by tumor cells highlighted tnf-superfamily (tnfsf) members as key counterparts of cell fate-related signals. here, we assayed the synthesis-expression-function of a subset of tnfsf ligands and receptors (baff, april, baffr, bcma, taci) in normal epithelial tissues and tumors and resident adult mesenchymal stem cells. method: tissues (breast, cns, kidney, skin, adipose tissue) have been assayed by means of immunohistochemistry and real-time pcr. cell models were used for signaling, transcriptomics and functional assays. results: all tumors expressed this tnf-sf subset. baffr is absent, while its specific ligand baff is ubiquitously expressed; contrairewise, april, bcma and taci are expressed in tissue-specific manner. april relays on tumor evolution/grade and is involved in tumor proliferation/differentiation/apoptosis signaling through nfκb-related, or a novel pathway, implicating jnk/foxo3a/gadd45, triggering transcriptional events. conclusion: autocrine/paracrine effects of april and its receptors bcma and taci in normal/tumoral parenchyme and the recruitment/attraction of immune-related cells could orchestrate tissue response, regulating cell fate, differentiation, inflammation, tissue remodeling and cancer, with potential tailored-therapy application. objective: mutation analysis of kit and pdgfra genes is currently used in gastrointestinal stromal tumors (gist) as an important step in diagnostic protocol. method: dna samples obtained from parafin-embedded biopsy material of 278 gist patients diagnosed in 2004-2011 were screened for mutations in exons 9, 11, 13 and 17 of kit and 12, 14 and 18 of pdgfra. results were tested for associations with clinical parameters of the tumor. results: causal mutations (according to in silico analysis with polyphen-2 predictor) were identified in 83.8 % of patients, most frequently in kit exon 11 (62.95 %). the kit exon 9, pdgrfa exons 18 and 12, kit exon 13, pdgfra exon 14 and kit exon 17 mutations appeared with frequencies 8.3 %, 7.6 %, 2.5 %, 1.4 %, 1.1 % and 0.0 % resp. genotype-phenotype correlation analysis revealed statistically significant association between intestinal localization of tumors and presence of kit exon 9 p.503-504_dup2 mutation and gastric localization of tumors and presence of pdgfra exon 18 p. d842v mutation. conclusion: centralized and standardized gist diagnosis offers clinically and prognostically relevant informations and is necessary for the indication of appropriate targeted therapy of gist patients. supported by projects cepr ii (imts 26220120036) and mbcc (imts 26220220113) at cu jfm co-financed by eu and by grant novartis slovakia. objective: an association between cystic fibrosis transmembrane regulator (cftr) gene mutations and infertility may occur. this study investigated the frequency of mutations in the cftr gene, of a group of consecutive patients candidate for assisted reproductive techniques with the aim of identify subjects carriers of the most severe ones. method: we screened 11208 healthy subjects (5943 females and 5265 males) for 56 cftr gene mutations and ivs8-polit polymorphism utilizing the cftr inno-lipa amplification kit including both general and italian regional strips. results: cftr mutations were detected in 6.2 % of the patients, a percentage similar to that reported in the general population. the most common mutation was δf508/n observed in 0.9 % of patients. no difference in the gender distribution was evidenced. in the large group of patients analyzed 87.7 % were wt, 11.8 % carrier of one mutation, and interestingly 0.5 % compound heterozygous. conclusion: our data support the relevance of an accurate determination of mutations in the cftr gene in order to inform the couple of their carrier risk and the possibility on having affected child. moreover our findings highlight the potential of genetic screening as a tool to identify possible compound heterozygous subjects without cf-like symptoms. objective: a patient's response to cancer treatment can depend on their biomarker status. therefore it is important that laboratories perform their biomarker tests well. in 2009 an international external quality assessment scheme for kras mutation analysis on ffpe slides was set-up. method: this scheme was run for 3 years by the coordination center, a medical and technical expert and scheme organizers under the umbrella of esp. participants received 10 samples. genotype results were analyzed and labs with good performance were listed on the esp website. written reports of 2011 were evaluated in detail. results: in 2011, 79 % of participants identified all samples correctly. results seemed to improve over the years from 9.49/10 to 9.62/10. genotype results from laboratories that participated for 3 years appeared to improve overtime, although this couldn't be statistically proven. in the reports some important elements weren't always present. for example, the patient's name was only present in 80 %, correct hgvs nomenclature in 90 % and interpretation of results in less than 40 %. conclusion: kras-eqa probably facilitates improvement of laboratory testing. the quality of reports of kras tests needs to be improved. a guideline for reporting molecular pathological results would be a good instrument for both participants and assessors. objective: microsatellite instability (msi) due to mismatch repair (mmr) deficiency is reported in a fraction of colorectal cancers (crcs) (5-10 %) complicating ibds (ibd-crcs). our recent findings argued for the existence of yet unknown mechanisms underlying mmr-deficiency in ibd-crcs, different from those involved in sporadic and hereditary msi crcs. here we hypothesized that over-expression of mir-155 and mir-21, two inflammation-related mirnas that target core mmr proteins, could constitute a mechanism underlying mmr deficiency in ibd-crcs. method: we compared the expression of both mirs in ibd-crcs and non-ibd hereditary or sporadic crcs using nonneoplastic ibd and healthy mucosa samples as controls. results: overall, mir-155 and mir-21 were significantly over-expressed in both non-neoplastic and neoplastic mucosa of ibd patients as compared to healthy controls. mir-155 alone was preferentially expressed in msi vs. mss ibd-crcs. in contrast, mir-155 was poorly deregulated in non-neoplastic and neoplastic tissues of non-ibd msi or mss crcs. conclusion: mir-155, alone or synergistically with mir-21, may favor the emergence of msi ibd-ccrs. since its deregulation was not limited to neoplastic tissues but extends to non-neoplastic normal mucosa as well, the hypothesis of a mir-155 field defect promoting mmr deficiency and yet msi-driven transformation can be proposed. ofp-11-009 pi3k, akt and pten expression in enucleated brazilian uveal melanoma cases using tissue microarray g. freeman * , d. begnami, a. damascena, j. neves, s. nonogaki, f. soares * são paulo, brazil objective: in the world literature, little has been published on the molecular pathways in primary uveal melanoma or subsequent development of metastases. the pi3k pathway has been implicated in regulation of apoptosis, cell cycle regulation, transcription and translation. method: we used immunohistochemistry with tma to investigate expression of pi3k, akt and phos-akt, and pt in over 200 formalin fixed paraffin embedded blocks of uveal melanoma from the a.c. camargo hospital, são paulo, brazil from 1988 to 2005 . fish was performed for pten copy numbers. results: in this study, neither immunohistochemistry nor fish results showed a statistically significant difference between the primary tumors and the tumors which metastasized. statistical analysis of all tumors together (tma) revealed only one molecular marker (pten), which gave results close to statistical significance. conclusion: this is the first large study of brazilian patients for pi3k, pten akt expression by immunohistochemistry and pten using fish. expression values for molecular markers chosen did not reach statistical significance, although pten values were close. use of fish to distinguish metastatic from non metastatic cases also came close to statistical significance. these results suggest that the expression of pten in uveal melanoma may be a good topic to investigate further. objective: chronic antibody-mediated (camr) and chronic t-cell-mediated rejection (ctcmr) represent predominant reason for late graft dysfunction. both categories share similar morphological features. we ask the question whether there are differences between camr and ctcmr on the molecular level. method: graft biopsies (>3 m) were performed and evaluated according to the banff classification. biopsy specimens with camr (n=13), ctcmr (n=9) and from protocol biopsies with normal histology (n=10) were stabilized in the rna-later. using the taqman low density array, the intrarenal expressions of 378 genes relating to immune response (b-cell activation, t-cell activation, chemokines, growth factors, immune regulators and apoptosis) were analyzed. results: both categories of chronic rejection were associated with up-regulation of many genes in comparison with the control group: chemokines (ccl4, ccl5, cxcl9, cxcl10, cxcl11), growth factor tgfb1, mhc class ii (hla-dma, hla-dmb, hla-dr, ubd), and in t-cell dependent mechanisms (cd3, cd86, lag3), including cytotoxic t-cell associated transcripts (gbp1, gzmk). in hierarchical clustering, camr and ctcmr gene expression profiles were similar. conclusion: chronic rejection very probably involves prolonged cooperation of innate immunity and allospecific immune response. our study showed that ctcmr and camr do not differ on the molecular basis. ofp-12-002 podocyte loss and glomerulosclerosis in inducible mouse model of podocin mutation-related nephrotic syndrome i. simic * , m. tabatabaeifar, g. mollet, c. antignac, s. weber, f. schaefer * universitätskinderklinik, nephrologisches labor, heidelberg, germany objective: mutations in nphs2 gene, encoding podocyte specific protein podocin, cause hereditary nephrotic syndrome. knock-in mice carrying the r140q mutation, murine analogue of the most common human mutation r138q, show developmental arrest of podocytes and renal failure at neonatal age. the aim of this study was to quantify renal histopathological changes in mice with postnatally induced r140q hemizygosity. method: c57bl/6 mice with nphs2flox/r140q, cre + genotype were injected with tamoxifen for 5 days to induce hemizygosity for r140q-mutant podocin. tissue samples were collected at defined intervals after induction. renal morphology was evaluated by quantitative histology, immunohistochemistry and electron microscopy. results: animals developed proteinuria within 1 week that progressed into renal failure and advanced uremia at week 12-16. the number of podocytes per glomerulus started decreasing at week 2 (42±19 vs. 98±31, p= 0.05), whereas glomerular sclerosis index increased from week 4 (1.5±0.15 vs. 0.25±0.08, p<0.00001). interstitial changes included fibrosis (up to 20 % of section area in end stage renal disease), tubular atrophy and dilatation. conclusion: our results implicate that the expression of mutated podocin in induced r140q-podocin mice leads to podocyte loss that precedes interstitial damage and glomerulosclerosis. quantification of histological changes will enable better evaluation of the efficacy of different pharmacological approaches directed to improvement of podocyte viability and attenuation of glomerulosclerosis. ofp-12-003 heavy chain deposition disease in kidney biopsies a. vizjak * , j. mraz, j. lindic, d. ferluga * university of ljubljana, faculty of medicine, slovenia objective: heavy chain deposition disease (hcdd) is a rare and not yet fully explored monoclonal immunoglobulin-related disorder. method: we studied the histopathology in 4 kidney biopsy cases of hcdd, representing a 0.07 % prevalence among 5481 native kidney biopsies. results: hcdd was diagnosed in kidney biopsies of 4 women (mean age 73.0 years). light microscopy showed diffuse nodular glomerulosclerosis (4/4), associated with mesangial proliferation (3/4) and capillary aneurysms (4/ 4). immunofluorescence showed abundant mesangial deposits and ribbon-like deposits along glomerular, capsular, tubular and vascular basement membranes, positive for heavy chain igg3 (3/4) and igg1 (1/4), with deleted gamma ch1 domain (4/4). complement c3 and c1q stained positive in all cases. by electron microscopy, punctate and powdery electron-dense deposits were found on the same locations. conclusion: immunofluorescence examination of kidney biopsies, including testing for immunoglobulin heavy and light chains, is crucial for diagnosis of hcdd. our study confirmed that hcdd is peculiar among monoclonal immunoglobulin deposition diseases not only because of its rarity but also because of uniform histomorphologic pattern of nodular glomerulosclerosis with pronounced capillary aneurysms and significant proliferation due to complement activation. deletion of the heavy chain ch1 domain and its significance in the pathogenesis has to be emphasized. ofp-12-004 two cases of anca associated vasculitis with geographical necroses in the kidney tissue a. bartonova * , e. honsova, r. rysava * ikem, dept. of pathology, praha, czech republic objective: anca associated vasculitides (aav) are rare systemic autoimmune diseases affecting small to mediumsized blood vessels. in kidney biopsy samples, aav usually demonstrate pauci-immune necrotizing crescentic glomerulonephritis (gn). method: among 198 cases of aav evaluated at ikem during 10 years, 2 had a very unusual pattern of renal involvement. results: the first patient had been treated for recurrent otitis media with unilateral hearing loss. he also suffered from artralgias and hematuria. an ultrasound study revealed a tumor-like mass in his kidney. during exploratory surgery, the lesion appeared to be infiltrative and a nephrectomy was performed. the second patient presented a granulomatous inflammation in a scar. a month later she developed multiple kidney and spleen "abscesses" with negative hemoculture, and underwent a nephrectomy with splenectomy. in both cases, the final pathological diagnosis was necrotizing granulomatous vasculitis of small and medium-sized vessels, with large geographical necroses of kidney tissue simultaneously with necrotizing crescentic gn. tests for anca antibodies were positive. no microbial pathogens including mycobacteria were detected. objective: polyomavirus nephropathy (pvn) is a common complication after renal transplantation. virus control is achieved by a reduction of immunosuppression allowing an effective t cell-mediated antiviral immune response. the morphology of resolving pvn has not been investigated. method: 101 protocol biopsies of 34 patients with pv viremia treated by reduction of immunosuppression only were included and scored according to banff criteria. the extent of interstitial inflammation was estimated as % of cortex. the number of tubular cross sections with sv40+ cells per mm of biopsy length was counted. findings were grouped as pre-, increasing, decreasing, and post-viremia. results: during the phase of decreasing viremia, we found a significant increase in the tubulitis score, the extent of tubules with intraepithelial lymphocytes, and interstitial inflammation (p<0.001). these, to a lower extent, persisted after virus clearance. the number of sv40+ tubules correlated with the virus load in the serum, but sv40 immunohistochemistry was frequently negative (33/55 cases), especially if viremia was below 106 geq/ml. conclusion: resolving pvn is characterized by a self-limiting acute interstitial nephritis. our findings are important because the diagnosis of interstitial rejection depends on the same morphological criteria. therefore, acute interstitial rejection cannot be diagnosed with certainty during pv viremia. ofp-12-008 neural cell adhesion molecule and fibroblast growth factor receptor positive interstitial cells increase in interstitial fibrosis in different renal diseases j. markovic-lipkovski * , c. müller, s. cirovic, s. tatic, d. mitrovic, g. müller * university of belgrade, faculty of medicine, serbia objective: ncam + cells with dendritic morphology are rarely present in normal renal interstitium. the aim of this study was to evaluate presence of ncam + cells in kidney biopsies of different renal diseases with and without interstitial fibrosis (if). further immunophenotyping of ncam + renal interstitial cells was performed. method: 97 kidney biopsies, after routine diagnosis, were stained applying antibodies against ncam clone123c3.d5 or cloneeric1. for double immunofluorescence, antibodies against ncam cloneep2567y and fgfr1, alpha5beta1 integrin, alphasma, cadherin9, cadherin11 were used. by rpcr, different ncam isoforms (120, 140, 180) were detected using specific primers in 11 renal tissues with and without if. results: ncam + interstitial cells were identified in 48 cases: 69 % lupus nephritis, 64 % focal segmental glomerulosclerosis, 64 % iga nephropathy, 55 % membranoproliferative glomerulonephritis, 50 % membranous glomerulonephritis. ncam + cells coexpressed fgfr1 and alpha5beta1 integrin, and were increased in if. ncam + cells did not coexpress alphasma, cadherin9 and cadherin11, although they were closely colocalised. all normal renal tissues tested for rpcr showed presence of all ncam isoforms, however ncam180 lacked in some tissues with if. conclusion: ncam + renal interstitial cells coexpress fgfr1 and alpha5beta1 integrin and are increased in renal diseases with if, mostly in diffuse proliferative lupus nephritis. in contrast to normal kidneys, in renal tissue with if ncam180 is present in a lesser extent. reproducibility for c4d immunohistochemistry in renal allografts: results from the banff trial m. mengel * , s. chan, j. climenhaga, p. randhawa, h. regele, y. kushner, r. colvin * university of alberta, laboratory of medicine, edmonton, canada objective: detection of c4d is crucial for diagnosing antibody mediated rejection, yet formal reproducibility studies are limited. method: we conducted an international multi-center trial to assess the reproducibility for c4d immunohistochemistry on paraffin-sections. a tissue microarray (tma) was constructed comprising 44 kidney allograft specimens representing negative, focal, and diffuse c4d positive cases. participants stained the tma slides, evaluated their stains and entered their scores online. stained slides were returned for centralized panel scoring. weighted kappa statistics were used to determine reproducibility. results: inter-institutional reproducibility, i.e. the product from variability between observers and staining methods was low (mean kappa 0.17). inter-observer reproducibility was fair (kappa 0.39), while inter-laboratory reproducibility was moderate (kappa 0.49). inter-observer reproducibility could be significantly improved by omitting the banff c4d grading schema and only considering +/− calls (kappa 0.60). scoring only c4d+/− inter-laboratory reproducibility improved considerably (kappa 0.78). higher dilutions of the primary antibody were associated with worse reproducibility. fixation <1 h or fixation in ethanol had significant negative impact on inter-laboratory reproducibility. conclusion: c4d results reported from paraffin section are highly variable between institutions. simplification of the grading schema would improve reproducibility between observers. technical reproducibility between laboratories is acceptable but could further be improved by standardizing protocols. objective: heparanase is a predominant mammalian enzyme that cleaves heparan sulfate, the key polysaccharide found in the basement membranes and at cell surface. heparanase is overexpressed in the diabetic kidney; however, its role and mode of action in diabetic kidney disease remains largely unclear. method: applying heparanase-null mice we found that deletion of the heparanase gene protects diabetic mice from diabetic nephropathy (dn). recombinant heparanase enzyme and a specific heparanase inhibitor (sst0001) were used to explore its mode of action in several in vivo and in vitro models. results: there is essential involvement of heparanase in the pathogenesis of dn. deleting the heparanase gene protects diabetic mice from dn and administration of specific heparanase inhibitor decreases the extent of albuminuria. in vitro, heparanase enhances macrophage activation by diabetic milieu components, and thus increases the kidneydamaging properties of macrophages. conclusion: our results validate the role of heparanase in dn and reveal the mechanism of heparanase action emphasizing its function in coupling chronic inflammation, macrophage activation and diabetic kidney injury. these findings will help in developing effective strategies to disrupt the heparanasedriven sequence of events in diabetic kidney disease, and in designing novel therapeutic interventions in dn. sunday, 9 september 2012, 17.00-19 .00, terrace 1 ofp-13 oral free paper session neuropathology ofp-13-001 microvascular angiogenesis occurs in a subset of only the arteriovenous types of vascular malformations and is more abundant in men than in women l. b. meijer-jorna * , c. m. van der horst, c. m. van der loos, a. c. van der wal * medical center alkmaar, symbiant pathology center, netherlands objective: episodic volume expansion may complicate the very slow growth pattern of congenital vascular malformations. we investigated the role of microvascular angiogenesis in this process of sudden growth. method: 100 resection specimens of symptomatic vascular malformations were screened for presence and extent of sheets or clusters immature microvessels, interpreted as microvascular angiogenesis. microvessel density (mvd), mast cell density (mcd) and ki67 labelling index of endothelial cells (ec) were assessed immunohistochemically and quantified. extent of angiogenesis was correlated with the type of vascular malformation and clinical characteristics. results: of 107 cases, 71 were arteriovenous malformations (avm), 21 were venous (vm), and 8 were lymphatic (lm). microvascular angiogenesis was observed in 30 % of all vascular malformations, of which 94 % appeared to be avm. mvd, mcd and ki67 labelling indexes of ec were significantly higher in immature vessel areas. moreover, in affected patients these angiogenic responses were far more extensive (in terms of area involvement) in men than in women (p<0.05). conclusion: microvascular angiogenesis appears a specific feature of the arteriovenous type of vascular malformations, and is much more extensive in men than in women. we suggest that these microvascular responses may contribute to onset of symptoms due to a mass forming effect. ofp-13-002 the impact of ventricular assist device prior to transplantation on acute cellular rejection and antibody-mediated rejection in cardiac allografts with due consideration of seasonal behaviour: a prospective study k. wassilew * , e. potapov, c. knosalla, t. krabatsch, m. hummel, r. hetzer * deutsches herzzentrum berlin, germany objective: this study evaluated the impact of bridge-to-transplant ventricular assist device support on development of acute cellular (acr) and antibody-mediated rejection (amr) with due consideration of seasonal behaviour in cardiac allografts. method: we studied 263 consecutive right ventricular endomyocardial biopsies (emb) between 01/2011 and 03/2012 prospectively. paraffin-embedded sections were evaluated for acute cellular rejection, endothelial cell swelling and capillary deposition of c4d, c3d and iga/m/g. the effects of vad (n= 101) on acr and amr, classified according to the ishlt, were studied for seasonal effects and compared to results of emb harvested from patients without vad support (n=162). results: our results did not reveal significant differences between the two groups in any given parameter. a positive correlation was found for endothelial cell swelling and capillary c4d deposition; the data failed to show a correlation between c4d and c3d deposition or c3d deposition and endothelial swelling. complement and immunoglobulin depositions seemed to be more pronounced but without statistical significance in autumn and winter. conclusion: our results demonstrate only statistically insignificantly more pronounced capillary complement deposition in autumn and winter. the use of vad did not predict development of amr or acr. the c3d staining does not add to the pathological diagnosis of amr. relevant changes of the molecular profile in the recurrence of glioblastomas with respect to the correspondent primary tumors m. idoate * , j. echeveste, r. diez valle, m. montanana, j. sola, t. labiano * university of navarra, dept. of pathology, pamplona, spain objective: in the literature, there is not enough information about changes of relevant molecular parameters in the recurrent glioblastoma. method: the study included a total of 11 grade iv astrocytomas (oms) and their correspondent recurrences in a series of patients treated by 5-ala guided surgery. all patients received similar treatment. a comparative histologic and molecular study which included loh of pten region, egfr amplification sish, methylation of mgmt by msp-pcr and sequencing of egfr variant iii mutation on representative tumor samples was obtained. results: the recurrences appeared adjacent to surgical cavity in seven cases. the mean time between the diagnosis of primary and the recurrence was 500 days (215-1670). the primary glioblastomas showed loh10q23 in 82 % of cases, hypermethylation of mgmt in 50 %, egfr amplification in 45 % and egfrviii in 37 %. in five cases (45 %) the molecular profile of the recurrences was different to the primaries and most of them (80 %) in the group of vaccines treated glioblastomas. the molecular profile change included one to several of the studied parameters. in one case all of the molecular parameters had changed. conclusion: the molecular profile change of the recurrences of glioblastomas in respect to the primaries is a frequent event that could be due to the selection of tumor cells due to both heterogeneity and treatment effect. objective: there is not literature reference on the prognostic significance of the proliferative activity in the border of tumor. a large series of patients with glioblastomas operated by fluorescence guided surgery with uniform treatment and enough follow-up have been studied. method: a total of 207 samples of different fluorescence quality from 67 glioblastomas, 44 primaries and 23 recurrences, were studied. for each tumor, the maximum value of ki-67 was determined by a semiquantitative counting by two observers and by an autoanalyzer. these values were compared with relevant oncologic parameters. results: the results of ki67 according both counting methods were concordant. the ki67max values of red (center), pink (intermediate) and blue (border) fluorescence samples obtained by quantitative analysis were 28 % 11 % and 6 %. in the cox regression analysis for overall survival, the ki67 value was an independent prognostic factor, stronger than other relevant clinical parameters studied (p=0.002). in the kaplan-meier for a ki67 cut-off of 5 %, the median overall survival was 25.1 months against 13.0 months (p=0.023). for the patients with recurrent glioblastomas the overall survival according to ki67 values was also significant (p=0.017). conclusion: the evaluation of the proliferative activity by ki-67 in the border of tumor defined by 5-ala fluorescence is a relevant independent prognostic marker of primary and recurrent glioblastomas. objective: according to the levels of estrogen receptor (er) expression, primary breast carcinoma (bc) cases are divided into two groups: er-positive and er-negative (godhirsch et al. 2011) . in metastatic bc, and especially in brain metastases (bm) of bc, the role of er expression is not clearly defined. aim: investigate er expression levels in brain metastases of bc and evaluate its prognostic significance. method: surgically obtained tumor specimens from sixty bc female patients (median age 52, ranges 29-73) with bm bc were stained with antibodies to er (dako). ihc expression was evaluated according to the semi-quantitative method. survival was estimated by means of kaplan-meyer curves. statistical analysis was performed using staitis-tica 8.0 software. results: er expression was detected in 52 % of patients (31/60), and 48 % (29/60) were er-negative. median time period between primary diagnosis and bm development was significantly (p<0.05) longer in er-positive group compared to er-negative group (48 and 23 months, respectively). recurrent bm developed in both groups with a similar frequency (32 % and 34 %, respectively). general survival was significantly (p=0,026) higher in er-positive patients compared to that of er-negative group. conclusion: er expression is considered to be an important prognostic factor for development of bmbc. nevertheless, further studies involving more patients with bmbc are required. ofp-13-006 dopamine and alpha synuclein interplay in neurodegeneration: a rat animal model g. stoica * , g. lungu, n. bjorklund * texas a&m university, veterinary pathobiology, college station, usa objective: a spontaneous autosomal recessive rat model for neurodegeneration was developed in our laboratory. these rats demonstrate progressive increases in alpha synuclein (α-syn) in the brain mesencephalon followed by loss of dopaminergic terminals in the basal ganglia and motor impairments. method: histology, immunohistochemistry, transmission electron microscopy were used for morphological analyses. for α-syn assessment real-time pcr and western blot were used. dopamine measurements were performed by ion mobility mass spectral (imms). results: the severity of pathology is directly related to the overexpression of α-syn and parallel decrease in dopamine (da) level in the striatum (st) of affected rats. the neurodegeneration in this model is characterized by the presence of perikarya and neurites lewis bodies (lb) and diffuse marked accumulation of perikaryal α-syn in the substantia nigra (sn), brain stem, and striatum along with neuronal loss. light and ultrastructural analyses revealed that the process of neuronal degeneration is a "dying back" type. the disease process is accompanied by gliosis and release of inflammatory cytokines. conclusion: decrease dopamine and overexpression of α-syn in the brain mesencephalon may provide a naturally occurring animal model for parkinson's disease and other synucleinopathies that reproduces significant pathological, neurochemical, and behavioral features of the human disease. sunday, 9 september 2012, 14.15 -16.15 objective: the mitotic index in thin primary melanomas replaced clark level in the 2009 staging system of the ajcc. however, the recommended quantification of proliferation by hot spots on he stains is criticized. an alternative may be the immunohistochemical proliferation marker phosphohistone h3 (phh3) visualizing all mitotic cells. and, when combined with the melanocytic marker mart1, phh3 insures quantification of proliferating melanocytes, only. method: 153 primary melanomas with a median follow-up of 12 years for patients with event-free melanoma were included. phh3/mart1 stains were performed by an indirect sequential immunoenzymatic technique. the number of phh3/mart1 positive cells was counted in a fixed 1-mm2 frame in the dermal area with the highest concentration of positive cells (hot spots). results: in multivariate analysis, phh3 in hot spots was a strong independent prognostic marker for recurrent disease (hr=3.7, 95 % ci, 1.4 to 9.6; p=.008) and melanoma-specific death (hr=3.4, 95 % ci, 1.3 to 9.0; p=.013), when corrected for primary tumor thickness and ulceration. conclusion: cellular proliferation is an independent prognostic marker in primary cutaneous melanoma. however, accurate quantification is crucial for correct clinical staging. for this purpose, proliferation indices of the novel phh3/mart1 double stains seem very promising. objective: some of the more common benign vocal fold lesions are polyps, nodules and polypoid corditis (reinke's edema). the majority of vocal fold pathology develops in the mucosal layer of the vocal fold, precisely in reinkes space. basic science and clinical research over past decades has led to advances in our understanding of benign laryngeal lesions. the aim of our study was compared contact telescopy findings with histological analyzes in a patients with reinke's edema. method: the our study included 80 consecutive patients with clinical diagnosis of reinke's edema. videoassisted contact telescopy was performed in all cases under general anesthesia and mucosa was stained by methylene blue. tissue biopsies were taken from areas exhibiting visible pathological changes and sent to routine histopathology and immunohistochemical analyses. we analyzed expression of pancytokeratin antibodies, vimentin and cd 34. results: in the all case of reinke's edema, contact telescopic scans showed a change of direction or a disappearance of regular distribution and morphology of blood vessels (irregular shapes, positions, and patterns, apparently running in random directions) in vocal fold mucosa. marked pathological changes were noted by contact telescopic scans, confirmed with histopathology analyses. conclusion: contact telescopy is a useful additional diagnostic tool regarding reinke's edema. ofp-14-005 volunteering in malawi: a snapshot of surgical pathology in sub-saharan africa s. berezowska * , t. tomoka, s. kamiza, d. a. milner jr., r. langer * universität bern, institute of pathology, switzerland objective: the breadth of material found in surgical pathology services in african countries significantly differs from the common spectrum of "the west". we report our experience in the pathology departments of blantyre and lilongwe, malawi. method: during a six-week period 405 cases were processed (378 histology, 27 cytology). results: the vast majority of cases showed significant pathological findings (n=369; 91.1 %): 175 (47.4 %) were non-tumoral conditions and 39 (10.6 %) benign tumors or tumor like lesions. the large group of malignancies (n=140; 37.9 %) comprised 11 pediatric tumors (e.g. rhabdomyosarcoma, small blue round cell tumors), and 129 adult tumors. amongst women (n=76), squamous cell carcinomas (scc) of the cervix uteri predominated (n=25; 32.9 %), followed by breast carcinomas (n=12; 15.8 %), and esophageal scc (n=9; 11.8 %). males (n=53) most often showed scc of the esophagus (n=9; 17.0 %), and scc of the urinary bladder (n=7; 13.2 %). lymphomas (n=7) and kaposi's sarcomas (n=6) were less frequent. conclusion: providing pathology service in a low resource country may be handicapped by lack of personal, inadequate material resources, or insufficient infrastructure. rotating volunteers offer a bridge for capacity building of both personnel and the local medical service; in addition, the volunteer's horizons are broaden professionally and personally. objective: antinuclear antibodies (ana) are routinely tested by indirect immunofluorescence (if) and immunoblot. in this paper we aim to retrospectively analyze results of both methods. method: on two cohort groups: a (a total of 5518 samples), and b (a total of 2615 samples) we performed if on commercially obtained hep-2 cell, and immunoblot on (strips euroline). results: if revealed at least 14 different morphological patterns of ana staining, which we grouped into 5 easily distinguished categories: speckled nuclear fluorescence, homogenous nucleoplasmic fluorescence, multiple nuclear dots, nucleoli staining, and scattered nucleoplasmic dots. we compared these groups according to frequency, type of antibody and correlation with immunoblot resuts. we summed the characteristics of each if category, and noticed that both cohorts revealed 7.4 % and 6 %, respectively of sera, which did not show positive if at an acceptable dilution (only by 1:10) or were fully negative in if test, but were clearly positive in immunoblot test. this was especially alarming with anti-scl-70 antibodies, which could be barely recognized in if test, but were clearly present in 53 of 407 positive stained strips (cohort a) and/ or 14 out of 156 positive stained strips (cohort b). ofp-14-007 macrophages and their subtypes in tumorigenesis and growth o. el-hassoun * , l. maruscakova, z. valaskova, m. kopani, j. jakubovsky, i. hulin * alphamedical laboratories, dept. of pathological anatomy, martin, slovakia objective: it has been established that macrophages (mf) play a basic role in the tumor microenvironment. still, the definition of the function of mf with relation to tumors is still vague, mainly when mf are observed acting occasionally as pro tumor and elsewhere as anti tumor. method: in this paper we attempt to sum up the accumulating observations of mf function with respect to the microenvironment and the tumor entity. results: through the different stages of tumorigenesis and growth, macrophages are engaged in various signaling cascades that tame them to perform a certain function in a given microenvironment. this makes mf a multi-program cell with unique adaptivity. we assume that without the macrophages tumors cannot progress. conclusion: the available mf subtype analysis is still inadequate and doesn't bear into account the dynamics of tumorigenesis and growth. our presented mf schemes provide an insight on the tumor microenvironment with respect to stage. a complex view on the signaling cascades affecting mf is needed to formulate a novel classification of mf based on their programming. this consequently can be the basis of the development of "anti-mf therapy" or on a larger scale "anti-tumor microenvironment therapy". ofp-14-009 lodox x-ray is an invaluable asset in autopsy procedures l. liebenberg * * university of cape town, dept. of forensic medicine, south africa objective: the field of autopsy performance has faced numerous challenges internationally. minimal background information on the deceased, various religious and personal objections regarding cutting a deceased body as well as health hazards face the pathologist regularly. lodox x-ray is a huge help for the pathologist. method: lodox x-ray (low dose x-ray) using the statscan has proven invaluable in busy forensic pathology laboratories in cape town, south africa. the digitally captured radiology images render a wealth of clear, recognizable, easily documented and user friendly images for use in diagnosis and court evidence. results: the use of the statscan has revolutionized the process of forensic autopsy procedures in cape town, south africa. the radiology images obtained in an unusually user friendly and time efficient way have improved efficacy, efficiency, accuracy and pro-active occupational health safety in our forensic laboratories. conclusion: lodox x-ray is a proven invaluable special investigation in forensic, and also academic, autopsy examination procedures. sunday, 9 september 2012, 17.00-19. objective: castration resistant prostate cancer (cr-pca) is the most aggressive form of prostate cancer (pca) posing a significant therapeutic challenge. our aim was to perform whole exome-sequencing on 5 cr-pca/normal paired formalin fixed paraffin embedded (ffpe) samples using the solid4 next generation sequencing platform (ngs). we identified two promising genes-ywhaz and ptk2, that could serve as novel potential therapeutic drug targets in pca. method: genomic dna was sequenced from 5 cr-pca/ normal paired ffpe samples. a set of amplified/deleted genes were validated using fluorescence in-situ hybridization (fish) assays using a pca progression cohort consisting of 138 cases for localized pca, 105 patients with primary pca and corresponding ln metastasis, and 39 samples for crpca. results: exome-sequencing identified regions of deletions/ amplifications, including nkx3.1, pten, cmyc and ar genes, known to play a role in pca. we identified several amplified genes as druggable targets such as ptk2 and ywhaz. for ywhaz we identified 3.5 % amplification in localized pca, 24.2 % amplification in ln metastasis and 45.5 % amplification in crpca. for ptk2 we 4 % amplification for localized pca and 34 % for both ln metastasis and cr-pca. conclusion: this is the first study to use exome-sequencing approaches on ffpe cr-pca to understand the biology of disease and its plausible treatment options. a study of d-np63 (p40) expression in tumours of stratified epithelium d. nonaka * * the christie hospital, dept. of histopathology, manchester, united kingdom objective: p63 can be expressed by a minority of adenocarcinomas. δnp63 (p40), isoform of p63, has recently been reported as more specific in lung squamous cell carcinoma (scc) than p63, and it appears to be a more reliable marker for scc. there is no comprehensive study on p40 expression in tumours of different histotypes. method: 460 tumours of various hisotypes were studied, including tumours of stratified epithelium such as scc (84 cases) of the skin, head/neck, lung, cervix, and oesophagus, urothelial carcinomas (18), cutaneous basal cell carcinomas (5), thymomas (20) and thymic carcinomas (5), adenocarcinomas (210) from multiple organs, germ cell tumours (25), brain tumours (17), lymphomas (14), and sarcomas (12). p40 immunostains were performed on tissue microarrays and staining extent was evaluated as focal (1-50 %) and diffuse (50-100 %). results: all but one tumours of stratified epithelium diffusely expressed p40. the negative case represents spindle cell scc. other tumours except for basal/squamous component of two teratomas, one breast metaplastic carcinoma, and squamous component of 5 adenosquamous carcinomas were all negative for p40. conclusion: p40 is specific in tumours of stratified epithelium and its sensitivity appears comparable to p63. p40 can serve as a marker for tumours of stratified epithelium. objective: although formaldehyde is the standard fixative used in routine histopathology, it is not considered an "ideal" fixative. formaldehyde presents carcinogenic properties, a slow fixation and produces cross-links with proteins and nucleic acids. formaldehyde-free fixatives are commercially available. method: from 2002 to 2012 we used glyo-fixx (thermo scientific, us), a glyoxal-based fixative, as formaldehyde substitute, analysing 54314 surgical pathology specimens and 205 autopsies. all samples were processed and stained routinely. moreover we performed immunohistochemistry by an automated immunostainer and special stains with standard techniques, where needed. in tumoral cases we fixed 1 cm3 sample in neutral buffered formalin and compared the results with gyo-fixx fixation. results: glyo-fixx fixation needed less hours than formaldehyde and not harden the tissues. on gross examination of the adipose tissue, lymph nodes were easily found, due to a more marked whitish appearance. morphologically we have not found differences between formalin and glyo-fixx, except for the eosinophils degranulation. special stains resulted similar to those with formaldehyde fixations. almost all antibodies not required pretreatment, but needed adjusts in the standardized protocol for formaldehyde. conclusion: our experience demonstrated glyo-fixx is a good non-toxic alternative to formaldehyde in routine pathology, capable to preserve morphology and protein integrity of the tissues. liposarcoma with solitary fibrous tumor-like dedifferentiated areas: clues on differential diagnosis m. aizpurua * * huvh/ics, dept. of pathology, barcelona, spain objective: well-differentiated liposarcoma (wdls) can undergo dedifferentiation to nonlipogenic sarcomas. solitary fibrous tumour (sft) characteristics have not been highlighted as a pattern of dedifferentiated liposarcoma (dls). however, this does occur and could be the cause of diagnostic pitfalls in atypical locations or partial tumoral resections. the aim of this comparative study is to demonstrate that some dls's may show morphologic features resembling sft's, and furthermore to describe the clue features supporting differential diagnosis. method: study comprised 11 wdls-dls and 12 sft (4 malignant). histological features were systematically reviewed, and cd34/cd99/s100/bcl2 immunostains and mdm2 fish-analysis were evaluated in all cases. results: there are many overlapping characteristics between dls and sft (table 1 ). significant differences between both tumor types were observed in lipoblast-like cells (lpc); mature adipocitic cells within tumor (mac); stromal bands surrounding adipose tissue with atypical cells (sac); patternless (pa); keloidal-collagen (kcm) and haemangiopericytoid-pattern (hp). diagnostic accuracy of different features is shown in table 2. conclusion: the dedifferentiated component in some liposarcomas may resemble sft. in the dls/sft differential diagnosis, immunohistochemistry may be confusing. presence of wdls areas is the main diagnostic clue and mdm2 fish amplification is successful in distinguishing dls from sft. osteoblastoma and diagnostics pitfalls w. ouahioune * * ehs douera, dept. of pathology, algiers, algeria objective: osteoblastoma is a rare benign osteoblastic tumor with a potential for local bone destruction and aggressiveness. the most common site of osteoblastoma is the vertebral column particularly the posterior elements and the sacrum. method: in this study we present nine cases of osteoblastoma and the principal differential diagnosis. results: on a period of 10 years, nine cases of osteoblastoma had been diagnosed including seven males and two females. the age of those patients ranged from 5 to 29 year old. the tumor was localized in the spine in five of the nine cases and the other ones in the long bones. the radiological diagnosis of osteoblastoma was made in just two cases. the diagnosis was made by histology in eight cases. all our patients had been treated with curettage. on the nine patients, just one of them had developed two successive recurrences. conclusion: osteoblastoma is a rare benign tumor which is rarely diagnosed by radiology alone. the pathologist should always suspect an osteoblastoma in front of a vertebral localization of any tumor. objective: congenital hepatic fibrosis (chf) is a developmental disorder of the portobiliary system. clinical findings usually include enlarged liver, well-preserved hepatocellular function and portal hypertension. chf is frequently associated with hepatorenal fibrocystic disease. method: a 29-year-old woman was referred to our hospital for further evaluation of hepatomegaly, and portal hypertension was found. laboratory tests were all negative. mri identified hepatosplenomegaly with hypertrophic left medial segment, high uptake nodular areas and homogeneous boundary liver. needle biopsy was performed. results: the biopsy demonstrated slight portal fibrosis and fibrous septa with proliferation of numerous biliary ducts, some of them containing inspissated bile. it was also noticeable the few number of portal vein branches. there was no inflammation or hepatocyte necrosis. therefore, our diagnosis was congenital hepatic fibrosis. we correlated these results with radiology. magnetic resonance cholangiopancreatography showed peripherally dilated biliary intrahepatic branches contrasting with preserved caliber of main intrahepatic branches. these findings were also diagnostic for chf. conclusion: the low prevalence of chf makes it hard to think about this entity in patients with portal hypertension and normal laboratory findings. the histological differential diagnosis must be done mainly with cirrhosis, but also with idiopathic portal hypertension and, in small biopsies, with biliary hamartoma. results: a total of 97 cases of digestive tract lymphoma were reported (64 males, 33 females) with a sex-ratio of 2.0. the patient age ranged between 6 and 82 years with a median age of 48 years. stomach location was the most frequent with 80 % of cases followed by intestine (15 %) and colon (5 %). among the 97 digestive tract lymphomas, 48 % were of marginal zone type (malt) and 47 % were large b-cell lymphoma. according to the ann arbor staging system, 85 % of patients were diagnosed at stage i and ii. conclusion: lymphomas of the digestive tract are still frequent in the centre of tunisia. the malt type lymphoma is main reported histological type. the association with the helicobacter pylori should be considered. collecting duct carcinoma in the west of ireland: a rare experience a. shalaby * , c. e. connolly * university hospital galway, dept. of histopathology, ireland objective: collecting duct carcinoma is a rare renal neoplasm arising from the epithelium of bellini's ducts in the distal part of the nephron. method: we describe our experience of this entity in university hospital galway with a series of four cases diagnosed between 2003 and 2011. results: three patients aged between 35 and 62 years presented with either lymph node or bone metastases without evident abdominal mass. in each case the biopsy from the metastatic lesion showed adenocarcinoma with an immunohistochemical profile suggestive of renal origin and a renal mass was subsequently confirmed by radiology. the fourth patient was an 81 year old male who presented with haematuria. abdominal ct scan revealed a 5.6 cm mass in the right kidney extending into the renal vein. biopsy of the renal mass in all cases confirmed adenocarcinoma with a tubular pattern with positive cytokeratin and vimentin staining and cd10 negative staining. one patient was treated with nephrectomy with post-neoadjuvant chemotherapy and died 25 months after surgery. the other three patients died few months after their diagnosis. conclusion: collecting duct carcinoma is a rare renal neoplasm that can be difficult to diagnose on core biopsies, however, it can be identified based on radiological findings, gross, microscopic, histochemical and immunohistochemical features. objective: wilms tumors (wt) is the most frequent renal tumor of children. genetic alterations have been suggested as associated factors but the exact pathogenesis of wt is not fully characterized. tissue factor (tf) is a glycoprotein which happens to be a key receptor for factor vii/viia and a primary initiator of coagulation. tf has also been associated with angiogenesis. recent evidence pointed out an important role for tf in cancer progression and metastasis. method: in the present investigation the differential expression of tf in wt was assessed by real-time pcr of rna retrieved from paraffin sections using microdissection. results: different histological components of wt were analysed. the results revealed that tf was upregulated in blastema and epithelial components as compared to nonneoplastic tissue (14.38 and 16.02-fold respectively, p< 0.001). stroma and non-neoplastic tissue presented low levels of tf expression. tf expression in wt metastatic lesions was also significantly upregulated as compared to non-metastatic wt. microvessel density was positively correlated with tf expression (r=0.721). conclusion: as described in other tumors, tf seems to play a significant role in the behavior of wt. further investigations are warranted to better understand the pathways by which tf exerts its effects on tumor progression and its potential as a target for therapy. objective: biliary atresia (ba) is the most common neonatal cholestatic disorder and the prime indication for liver transplantation (ltx) in children. histopathological markers in liver biopsies emerge promise as indicator of early ltx in patients with ba. method: ductular proliferation, ductal plate malformations and type i, iii, iv and v collagen deposition were evaluated on kasai portoenterostomy (kpe) and liver transplantation (ltx) biopsies from 36 children with ba. formalin fixed and paraffin embedded liver biopsies were stained with hematoxylin-eosin, picrosirius-polarization and immunofluorescence methods. there were analyzed liver histoarchitecture, biliary ductus and collagen deposition in hepatic compartments. pathologic findings were graded according to a 5-point semi-quantitative severity-based scoring system. impact of these markers was tested on ltx time (<2 year and >2 year). results: median age of kpe was 12 weeks (range 6-20) and of ltx was 27 months (range 6-120). in kpe liver biopsies, ductular proliferation, ductal plate malformations and collagen deposition were increased but these parameters presented no association with clinical evolution for early ltx. furthermore, collagen v prominent deposition was found along of hepatic sinusoids and type i, iii and iv were more frequent in portal compartment. conclusion: these results suggest that histopathological parameters evaluation presented may not determine early ltx in biliary atresia. the role of hofbauer cells on villous vasculature in early fetal losses e. özer * , y. arman karakaya * dokuz eylul university, dept. of pathology, izmir, turkey objective: the aim of this retrospective study is investigate the role of hofbauer cells in early fetal losses. method: the slides obtained from archieval blocks of missed abortion (ma, n=15) and blighted ovum (bo, n= 15) cases and unwanted pregnancies materials (control group, n=15) were stained by immunuhistochemical methods using cd68 and cd31 antibody to label hofbauer cells and endotelial cells, respectively. results: the mean number of vilous hofbauer cells was found to be significantly higher in both bo and ma in contrast to the control group (p=0.005 and p<<0.001, respectively). however it was not significantly different between bo and ma (p=0.04). chalkey method revealed no statisticaly significant difference in the control group in comparison with ma and bo in (p=0.29, p=0.09, respectively). higher microvessel scorring were found in ma in contrast to bo and the control group (p=0.003 and p= 0.003, respectively). however, there was no difference between the control group and bo (p=0.54). conclusion: we think that hofbauer cells may be of biological importance in early fetal losses and play a role on defective vasculature formation in ma. placental vegf and its receptors expression patterns in preeclampsia k. pavlov * , e. dubova, a. shchegolev, g. sukhikh * v.i. kulakov scientific center, dept. of pathology, moscow, russia objective: placental angiogenesis anomalies play an important role in some complications of pregnancy development, including preeclampsia (pe). vegf and its receptorsone of the key placental angiogenic factor. aim: to evaluate patterns of vegf and its receptors (vegfrs 1, 2 and 3) expression in placentas from pe complicated pregnancies. method: we performed complex morphological and immunohistochemical study of 9 term placentas from mild preeclampsia (mpe) cases (1st group), 6 term placentas from severe preeclampsia (spe) cases (2nd group) and 10 term placentas from uncomplicated pregnancies (control group). results: we revealed significantly increased syncitial vegf expression levels in preeclamptic placentas terminal villi and these changes were much prominent in spe. we also detected insignificantly increased endothelial vegf expression levels in preeclamptic placentas terminal villi. vegfr-1 syncitial expression levels in spe terminal villi were significantly higher in compare to the mpe and control groups. vegfr-2 endothelial and syncitial expression levels were significantly lower in both pe groups terminal villi and these changes were much prominent in mpe. patterns of vegfr-3 expression in preeclamptic and control groups were multidirectional. conclusion: revealed patterns of vegf and its receptors expression point on altered placental angiogenesis in pe and reflect different degree of such alteration in mild and severe pe. objective: chorionic villi vascularity disturbances, caused by abnormal expression of angiogenic factors could correlate with maternal and fetal complications in diabetic pregnancies. method: aim: to evaluate the patterns of vegf receptors (vegfr1, vegfr2 and vegfr3) expression in placentas from gestational diabetes (gd) and type 1 diabetes (d1) pregnancies and correlation between them and some clinical parameters in newborn (placental weight (pw), newborn weight (nw), placental/newborn weight index (pnwi),1st day of life blood glucose test results (1bgtr)). 29 term placentas from d1 (n=16) and gd (n=20) and 12 term placentas from normal pregnancy (control group) were studied. results: in both d1 and gd group we revealed significantly higher level of vegfr1 (p=0.0001), vegfr2 (p=0.001) and vegfr3 (p=0.01) endothelial expression in terminal villi. difference in vegfr1 expression among d1 and gd groups was also significant (p=0.0009) as difference in its expression between d1 and control group (p = 0.002). among clinical parameters we only revealed significant increase in 1bgtr in diabetic groups (p = 0.03) with marked difference between d1 and control group (p = 0.002). we revealed multidirectional correlation between vegf and vegfr2 expression and nw in d1 and gd groups. conclusion: revealed changes reflect placental angiogenesis disturbances influence on intrauterine fetal development. microvillus inclusion disease (mvid) is a disorder of defective intracellular trafficking and disrupted epithelial cell polarity c. thoeni * * division of cell biology, medical university innsbruck, austria objective: mvid is a congenital enteropathy characterized by loss of microvilli and formation of microvillus inclusions (mi) in enterocytes. mvid is caused by mutations in the myo5b gene, coding for the myosin vb (m5b) motor protein involved in intracellular transport and maintenance of epithelial cell polarity. we examined the effects of loss of m5b in enterocytes of mvid patient and in a caco-2 cell model. method: the expression and localization of cell membrane transporters (cd36, na/k atpase) and various cell organelle markers : endosomal rab gtpases [rab5, 7, 8, 9, 11] ; early (eea1) and late endosome (lamp2), and golgi (giantin)) were analyzed using multilabel immunoflourescence and confocal microscopy of a duodenal biopsy from mvid patient and in caco-2 cells following m5b si rna knock-down (hum mut 31:1-8,2010). results: depletion of m5b in both enterocytes and caco-2 cells resulted in disruption of epithelial cell polarity with loss of apical microvilli, formation of mi; mislocalization of transporters as well as aggregation of epitopes for different rab gtp ases, early and late endosomes. conclusion: m5b plays a critical role in polarized organization of enterocytes in mvid -a disorder characterized by defective intracellular trafficking and altered endosomal sorting. caco-2 cells provide an excellent model to study the pathogenesis of mvid. ofp-16-007 beta-catenin expression and mutational analysis of ctnnb1 gene in pediatic adrenocortical tumors (act) r. alaggio * , p. dall'igna, a. martines, e. lalli, r. boldrini, v. d´onofrio, g. esposito * università di padova, dipt. di pathology, italy objective: act have an unpredictable clinical behaviour, and no liable histological or molecular parameters are available to predict outcome. the activation of wnt/β-catenin pathway, involved in tumor growth and progression in adult act has been explored only in brasilian pediatric act. the aim of this study is the investigation of the possible prognostic role of β-catenin accumulation and/or ctnnb1 (β-catenin gene) mutations also in italian pediatric act. method: β-catenin immunostaining and mutational analysis of ctnnb1 gene at exons 3 and 5, when possible, were carried on a series of 19 act (7 malignant and 12 benign, according to wieneke classification), from patients enrolled in the italian pediatric rare tumor (trep) study. results: immunostaining for β-catenin showed membrane/ cytoplasmic staining in 9 cases, 5 benign and 4 malignant, and nuclear staining in 1 malignant. no mutations of ctnnb1 gene were found in the 8 tumors analyzed. conclusion: ctnnb1 gene mutations do not appear to be involved in pathogenesis of pediatric act. the accumulation of protein might be related to different mechanisms. its presence in the majority of malignant act suggests a possible role in tumor progression. ofp-16-008 neuroblastoma presenting like a wilms' tumor with thrombus in inferior vena cava: a case series h. sartelet * , g. gaetan, a. ouimet, c. lapierre, p. teira * chu sainte justine, dept. of pathology, montreal, canada objective: neuroblastomas and wilms' tumors are frequent pediatric solid tumors. the first is frequently detected in the adrenal gland and the second develops in the kidneys. the extension through the vena cava and the lung metastases are frequent in wilms' tumors and are rarely in neuroblastoma. we present the cases of three children with abdominal tumors with thrombus in the inferior vena cava and pulmonary metastases were discovered yet demonstrated a stage 4 neuroblastoma. method: the three male patients were between 23 and 48 months old. they presented an abdominal mass, near the superior pole of the kidney. thrombus of the vena cava was evoked on imaging studies in all cases and pulmonary metastases were always found. catecholamine metabolites were present in the first case and negative in the two others. two out of three patients had a radical nephrectomy. results: the pathological analysis always found a neuroblastoma poorly differentiated or undifferentiated without mycn amplification and confirmed the tumoral thrombus in the second case. the evolution of the two first patients was unfavorable and the third is alive. conclusion: invasion of the inferior vena cava and pulmonary metastases in children with neuroblastoma is uncommon and can modify the surgical management. ofp-16-009 epcam -a marker for tufting enteropathy (te) and a useful tool in the differential diagnosis of congenital enteropathies c. thoeni * * division of cell biology, medical university innsbruck, austria objective: congenital enteropathies (ce) are characterized by villous atrophy and disruption of apical microvilli in duodenal enterocytes. te is characterized by focal eptithelial tufts and mutations in the epcam gene, coding for epithelial cell adhesion molecule, important for cell-cell contacts. microvillus inclusion disease (mvid), shows loss of apical microvilli and formation of intracytoplasmatic microvillus inclusions. mvid is caused by mutations in the myo5b gene, important for intracellular transport and organization of epithelial cell polarity. in this study, we identified a new epcam mutation in a patient with te and used anti-epcam antibody as a marker for the diagnosis of te and distinction from mvid. method: we used immunohistochemistry (ihc) with monoclonal epcam antibody (mouse monoclonal ab for epcam, ncl esa/leica microsystems) on routinely processed duodenal biopsies from patients with te, mvid and age matched controls. immunostaining for e-cadherin served as reference. results: epcam expression was completely absent in the biopsy from te patient homozygous for a novel epcam mutation (c227 c>g, ser 76x). e-cadherin showed normal expression and distribution pattern in the enterocytes. in mvid, expression and distribution pattern was comparable to controls for both epcam and e-cadherin. conclusion: loss of epcam is specific and a sensitive marker for confirmation of te. epcam antibody is useful in the differential diagnosis of ce. objective: in pulmonary adenocarcinoma egfr mutation analysis has become an important part of the diagnostic work-up. frequently the tissue of bronchoscopies is of limited diagnostic value. the aim of the present study was to evaluate the impact of rose on brochoscopy specimens with regard to egfr mutation. method: rose was used for adequacy of the specimen and for separating adenocarcinomas and carcinomas nos from other neoplasms in the lung. if it was not possible to retrieve material for histology, cytology and cellblock were made. if adenocarcinoma or carcinoma nos was suspected in rose, further two biopsies or two cell blocks were obtained. egfr mutation analysis was performed by pcr (light cycler 480; egfr rgq pcr kit; qiagen). results: malignancy was diagnosed in 93 of 354 cases by histology or on cytology with and without cell blocks. adenocarcinomas (n=30) and carcinomas nos (n=9), scc (n =26), sclc (n = 13), and rare tumors (n=15) were diagnosed, respectively. among the group of adenocarcinomas and carcinomas nos (n = 39), egfr mutation analysis was performed in 29 (74,3 %) cases and showed mutations in 3 (10,3 %) and "wild type" in 26 (89,7 %) tumors. no material was left in 10 (25,6 %) cases. conclusion: rose supports bronchoscopic procedures to retrieve adequate specimens for tumor diagnosis and subsequent egfr mutation analysis. objective: patients with fibroblast growth factor receptor 1 (fgfr1) amplified squamous cell lung cancers (l-scc) are treated in phase i clinical trials using small molecule inhibitors (smi). scc of the lung share common molecular alterations with squamous cell head and neck cancers (hn-scc). aim of our study is to assess if hn-sccs also harbor fgfr1 amplifications. furthermore, we aim to inhibit cell proliferation of fgfr1 amplified hn-scc cell lines using a smi. method: the cohort consists of 227 patients suffering from hn-scc, 97 of these suffering from metastatic disease. primary tumors and metastatic tumors were assessed for fgfr1 copy number status using fluorescence in-situ hybridization (fish). we tested cell lines for fgfr1 amplification status and inhibited these with smis. objective: the pulp of pequi has high levels of antioxidants properties. method: eighteen male balb/c mice divided: 14 animals received by gavage 0,5 μl/mg/day of pequi oil (control + cbcoil=4) during 75 days. after 15 days, 10 of these mices received two doses of 1,5 g/kg intraperitoneal of urethane (urethane + cbc oil=10). the other 4 animals were only submitted to two doses of 1,5 g/kg intraperitoneal of urethane (urethane group=4). after 75 days, groups were sacrificed. antioxidant activity was evaluated in the lung tissues by tbars (thiobarbituric acid-reactive substances), cat (catalase) and sod (superoxido dismutase) test. dna damage was estimated by comet test. results: the lung parenchyma from urethane groups without oil and with oil showed neoplasic formations induced by the chemical carcinogenesis in contrast with control + cbc oil group. the results of tbars test showed a significant decrease of lipid peroxidation in the urethane + cbc oil, than urethane group. the cat and sod test didn't show a significant difference. comet assay showed a significant decrease of dna damage in urethane + cbc oil when compared with urethane group. conclusion: the antioxidant components in the pequi oil diminish the oxidative stress status and dna damage in chemical carcinogenesis, suggesting that this type of strategies may have a greater impact in lung cancer treatment. financial support: fapesp. ofp-17-004 an algorithm for gene mutation analysis in lung cancer m. comanescu * , c. iosif, m. dobre, l. buburuzan, g. bussolati, f. vasilescu, c. ardeleanu * institutul victor babes, dept. of pathology, bucharest, romania objective: somatic alterations of k-ras, egfr and alk which are increasingly requested in order to predict response to personalized therapies in lung cancer are mutually exclusive and are represented in over 50 % of lung adenocarcinomas. the approach to be followed for planning analyses is not standardized in the literature. method: we have studied by molecular methods (pcr-rflp for k-ras, direct sequencing for egfr and fish for alk) 185 formalin fixed, paraffin embedded cases of lung adenocarcinomas of different subtypes. results: based on rational, biological and economic considerations we started with k-ras analysis by pcr-rflp, which showed mutations in 38 cases (37 with mutation on codon 12 and one case with mutation on codon 13), the 147 residual cases were all analyzed for egfr mutations. fish for alk translocation followed in egfr-wild cases. conclusion: the analysis for k-ras mutations allows to select the significant percentage (approximately 25 % of adenocarcinomas) when egfr mutation analysis by direct sequencing can be postponed. k-ras and egfr wild cases will then undergo fish analysis for alk translocation. acknowledgements study conducted with the support of the following project: project persother -smis-csnr: 549/12.024; with the support of sectoral operational programme "increase of economic competi-tiveness" priority axis 2: research, technological development and innovation for competitiveness. ofp-17-005 gene mutation analysis in adenosquamous carcinomas of the lung m. comanescu * , g. bussolati, c. ardeleanu, l. daniele, g. gaina, c. luca, a. sapino * institutul victor babes, dept. of pathology, bucharest, romania objective: adenosquamous carcinomas of the lung constitute a rare and aggressive variant of lung cancers. the pressing interest in evaluating the mutational status in lung carcinomas for predicting responsiveness to targeted therapies is presently focused on adenocarcinomas (of different sub-types), which makes preliminary histological typing a crucial step in order to select cases to be genetically analyzed. hence the interest in deciding if adenosquamous carcinomas should be included among adenocarcinomas or, viceversa, if they should be interpreted as a variant of squamous cancers and excluded from the process of gene analysis. method: we have thus collected, from our two institutions a large number (45 cases) of cancers showing the histological definition of adenosquamous carcinomas according to the who criteria and performed gene analysis for k-ras (codons 12, 13) and egfr (codons 18, 19 and 21) mutations. the detection of rearrangements of the alk gene by fish was also performed. the results indicate that k-ras and, specifically, egfr mutations are detectable in a fraction of these tumors. in conclusion, adenosquamous carcinomas should not be denied the chance of genetic analysis eventually leading to a targeted therapy. acknowledgement project persother -smis-csnr: 549/12.024 romania. ofp-17-006 pathology of the lung progenitor cells and their niches in idiopathic interstitial pneumonias and lung sarcoidosis s. demoura * , e. kogan, v. tuong, o. kichigina * moscow, russia objective: the aim of the study was to investigate the morphological and the molecular-biological changes of the lung progenitor cells and their niches in idiopathic interstitial pneumonias (ipf) and lung sarcoidosis (ls). method: we performed an immunohistochemical study on open lung biopsies from 250 patients (usual ipf -87, desqvamative interstitial pneumonia-37, nonspecific ipf -42, cryptogenic ipf -20, ls -32, control -34 patients). immunochemistry was done on step paraffin sections with monoclonal and polyclonal antibodies: apo-cas, pcna, 18, 19, pdgf, fgf, igf ii, cd 34, mmp 1, 2, 7, 9, timp 4, cd 68, sma, ema. results: deep injury of clara cells together with pneumocytes ii and their nishes were found in usual ipf, sk with prominent ipf and desqvamative interstitial pneumonia. myofibroblast proliferation, neoangiogenesis, adenomatosis and fibrosis with high production of tnfα, tgf-β, pdgf, fgf, igf ii, cd 34, mmp 1, 2, 7, 9, timp 4 accompanied proliferation of these epithelial cells. while in other variants of ipf pathological changes were localized in the interstitium, vessels and basal bronchiolar epithelial cells. conclusion: localization of injury and inflammation in progenitor cells niches results in pathologic reparation, sclerosis and precancer lesions. ofp-17-007 a small immunohistochemical panel allows for accurate diagnosis of primary and metastatic lung cancer in biopsy specimens d. felizardo * , r. henrique, a. l. cunha * instituto de oncologia porto, dept. of pathology, coimbra, portugal objective: precise subclassification of lung cancer, mostly performed in biopsy or fine needle-aspiration specimens, is required for appropriate therapy. moreover, distinction between primary and metastatic carcinoma is critical. thus, the role of immunohistochemistry (ihc) has been emphasized, although an optimal ihc diagnostic algorithm has not been firmly established. herein, we evaluated the performance of the ihc panel used at our institution in subclassification of lung cancer and identification of metastatic carcinoma. method: cases of non-neuroendocrine lung carcinoma, diagnosed from march 2011 to april 2012 were selected. ihc was performed for ck7, ck20, ttf-1 and p63. resection specimens were compared with the respective biopsy. results: of 184 cases analysed, 73 (39.7 %) were diagnosed as lung adenocarcinoma, 59 (32.1 %) as epidermoid carcinoma, 22 (12 %) as other forms of nsclc, and 17 (9.2 %) as metastasis. importantly, 26 % of primary lung adenocarcinomas were initially suspected to be metastasis. in 26 cases submitted to surgical resection, 22 (84.6 %) were correctly diagnosed in the biopsy, revealing a substantial agreement (κ-value=0,757). conclusion: our ihc panel allows for reliable subclassification of lung carcinomas in most cases and is decisive for appropriate diagnosis in patients suspected of lung metastasis, which is critical issue in a cancer institute. ofp-17-008 rationale for treatment of metastatic squamous cell carcinoma of the lung using fgfr1 inhibitors f. göke * , a. franzen, a. schroeck, v. scheble, r. kirsten, r. menon, d. goltz, d. boehm, w. vogel, s. perner * universitätsklinik bonn, inst. für pathologie, germany objective: we previously identified amplified fibroblast growth factor 1 (fgfr1) as a therapeutic target for small molecule inhibitor (smi) therapy in squamous cell lung cancer (l-scc), resulting in currently running clinical trials treating patients with stage iii disease. as most patients present with metastatic stage of disease, we attempt to demonstrate fgfr1 amplification in lymph node metastases of amplified primary tumors. our study aims to give a rational to include these patients in a targeted smi therapy. method: we assessed 75 formalin-fixed paraffin-embedded (ffpe) primary l-scc samples. 46 samples were primary tumours with corresponding ffpe lymph node metastasis. the biotin-labelled fgfr1 target probe (8p11.23 to 8p11.22) was used to determine the fgfr1 amplification status performing fluorescence in situ hybridization (fish). results: of 39 assessable metastatic l-scc, 7 samples displayed fgfr1 amplification (18 %). all of these primary tumors also harbored fgfr1 amplification in their lymph node metastasis. non-amplified tumors never displayed fgfr1 amplification in corresponding metastases. conclusion: we found fgfr1 amplification not only in primary l-scc, but also in corresponding lymph node metastasis, suggesting that this genetic aberration is a clonal event in tumor genesis. our study provides data indicating new therapeutic possibilities for patients suffering not only primary, but also metastatic fgfr1 amplified scc lung cancer disease. kras mutation was a g to t transversion in 80 % of the smoker nsclc population and a g to a transition in 98 % of the non smoker nsclc population. mutations were noted at codon 12 (90 %), codon 13 (8 %) and codon 61 (2 %). a kras mutation was a negative prognostic factor with a hazard ratio for death of 1.38 (95 % confidence interval, 1.16-1.63). a mucinous histological subtype was observed in more than 75 % of kras mutated tumours. conclusion: kras oncogene substitution must be accurately determined in primary lung adenocarcinoma for correlation with tumour behaviour and clinico-pathological parameters. objective: there is an urgent need for diagnosis of nsclc at its early-stages and for improving the survival rate of patients. micrornas, small non-coding rnas, are frequently deregulated in nsclc. this study aimed to explore plasma micrornas for diagnostic value, and evaluated the correlation between expression profiles of plasma micro-rnas and disease-free survival (dfs) in nsclc patients. method: we selected eighteen most frequently expressed micrornas in nsclc. total plasma rna including micrornas was isolated and reverse-transcribed into cdnas. the level of micrornas was determined by quantitative real-time rt-pcr in 42 resectable nsclc patients and 10 matched cancer-free individuals. the correlation between the expression of micrornas in plasma and patient dfs were examined by log-rank and cox analysis. results: expression levels of mir-320,−296,−145,−199a, −191,−223,−24,−152,−126, and let-7f in the plasma of nsclc including stage-i patients were significantly higher compared with controls (p<0.0001). the combination of these micrornas yielded 87 % sensitivity and 90 % specificity (auc=0.934) in discriminating nsclc patients from controls. the levels of mir-155, −152, −20a, −223, −126 and mir-199a were significantly associated with dfs (p<0.05). conclusion: our results suggest that high expression of 6plasma mirnas signature would provide potential noninvasive blood-based biomarker for the prognosis of nsclc. monday, 10 september 2012, 14.15 -16. objective: intratumoral disorganized neo-vasculature induces oxygen fluctuations which contribute to tumour growth and metastatic potential. although the activation by hypoxia of the carbonic anhydrases caix and caxii is well known, responses of these proteins under reoxygenation remain to be elucidated. method: in this study we evaluated the effects of hypoxiareoxygenation on caix and caxii expression and cell proliferation in a549 and h1975 lung adenocarcinoma cell lines. we further investigated by immunohistochemistry on tissue microarray the value of the combined expression of these proteins to predict outcome in 552 nsclc patients. results: caix expression was maintained at high level after reoxygenation in contrast of the rapid caxii downregulation, whereas the cell proliferation rate was significantly increased. survival analyses showed that high caix/ low caxii was associated with high cumulative incidence of relapse and with poor overall survival of nsclc patients (p<0.05). conclusion: our results provide insight into understanding dynamic responses of caix and caxii expression under tumour cells reoxygenation and demonstrate a critical role for reoxygenation on caix and caxii levels that may select for aggressive lung cancer phenotype. these findings suggest that caix and caxii play selective roles in tumour progression and emphasize their significant prognostic and potential therapeutic value. ofp-18-002 alk-gene rearrangement: a comparative analysis on circulating tumour cells (ctcs) and tumour tissue from lung adenocarcinoma patients m. ilie * , e. long, c. butori, v. hofman, c. coelle, v. mauro, k. zahaf, c.-h. marquette, j. mouroux, p. paterlini-bréchot, p. hofman * chu de nice, lpce, france objective: until now the alk status in ctcs isolated from lung cancer patients has not been characterised. we assessed the alk status in ctcs detected in lung cancer patients and correlated the results to the alk status defined in the corresponding tumour tissue. method: 87 lung adenocarcinoma patients showing ctcs isolated using the isolation by size of epithelial tumour cell method were screened for their alk status both in tumour samples and in ctcs. alk break-apart fluorescence in situ hybridisation (fish) and immunochemistry using an anti-alk antibody were done on ctcs and compared with results obtained on corresponding tissue specimens. results: 5 patients showed alk-gene rearrangement and strong alk protein expression in ctcs and corresponding tumour samples. negative results were found for 82 patients in ctcs and corresponding tumour samples. conclusion: we demonstrate that the alk status can be determined in ctcs from lung cancer patients both by immunocytochemistry and fish analysis. a strong correlation was found for the alk status obtained from corresponding tissue specimens. these results favour non-invasive, alk-gene status pre-screening on a routine basis on ctcs isolated form lung cancer patients and open new avenues for real-time monitoring for adapted targeted therapy. ofp-18-003 detection of egfr mutations and eml4-alk rearrangements in lung adenocarcinomas using archived cytological slides e. imyanitov * , n. mitiushkina, a. iyevleva, a. poltoratskiy, a. ivantsov, a. togo, i. polyakov, s. orlov, d. matsko, v. novik * n.n. petrov institute of oncology, st. petersburg, russia objective: while the molecular analysis of egfr and alk in archival lung cancer tissues is relatively well established, the acceptability of genetic investigation of cytological material in clinical routine remains a subject of debate. method: islets of malignant cells were visually located on the archived cytological slides, lysed in situ by the drop of sds-containing buffer, and subjected to the standard dna and rna extraction. examination of paraffin-embedded tissue blocks from the same patients was done in parallel. results: 75 cytological/histological lung adenocarcionoma sample pairs underwent the analysis for egfr mutation. 2 cytological and 1 morphological samples failed to produce dna. concordance for the wild-type and mutation status was observed in 53/72 and 14/72 informative pairs, respectively; 1 pair was non-interpretable; 3 and 1 pairs had mutation only in the cytological or histological material, respectively. rna extraction followed by rt-pcr analysis for the eml4-alk translocation was done for the 51 pair; failures were observed for 3 cytological and 8 histological samples. 34/40 informative pairs were concordant for the norm, 3 contained identical translocations, and 2 were noninterpretable. 1 pair demonstrated alk rearrangement in the tissue block but not in the histological slide. conclusion: archived cytological slides appear to be well suitable both for egfr and alk analysis. objective: non-small cell lung cancer (nsclc) represents a heterogeneous group of cancers consisting mainly of squamous cell carcinoma (scc) and adenocarcinoma (ad). increased sirtuin (sirt-1) expression leads to deacetylation of p53 that could be important in the pathogenesis of lung cancer. differences of the molecular mechanisms in nsclc subtypes may follow subtly different pathways to tumorigenesis. the aim of our study was compare sirt-1 expression in lung adenocarcinoma, squamous cell carcinoma and control group. method: 25 patients with stage 2 lung cancer were enrolled in the study. 14 patients had adenocarcinoma, but 11 patients had squamous cell carcinoma. lung tissue for control group were selected from 15 autopsy cases. immunohistochemical and western blot methods were used to evaluate sirt-1 expression in lung tissue. results: obtained results showed that patients with lung cancer had increased sirt-1 expression compared to control group (146±111 vs. 21±16 cells/mm2, p<0.0001). in addition, patients with squamous cell carcinoma had increased sirt-1 positive cells compared to patients with adenocarcinoma (203±143 vs. 95±49 cells/mm2, p=0.03). conclusion: lung cancer is characterized by an increased sirt-1 expression which ir more prominent in squamous cell carcinoma. objective: transplantation is the only treatment for several end-stage lung diseases but limited by chronic allograft dysfunction particularly obliterative bronchiolitis (ob) and its correlate bronchiolitis obliterans syndrome. the development of preclinical models is crucial to better identify immunological/non-immunological mechanisms leading to ob. [group b and c] post-transplant, animals were sacrificed. in animals from group c, cyclosporine was administered at sub-optimal dose. lung rejection was graded according to the working formulation of ishlt and the presence of circulating donor-specific (dsa) antibodies determined by flow cytometry. results: in group a, acute rejection (ar) or ob occurred in 33 % and 17 % of animals, respectively. ar occurred in 33-44 % of animals in group b and c respectively. ob was detected in 33 % and in 44 % of group b and c respectively. high levels of dsa igg were observed in cases with ar. conclusion: a novel model of pulmonary ob was developed in the rat. to obtain a reproducible onset of ob, shortterm and sub therapeutic cyclosporin administration appears indispensable, at least in our species combination. ofp-18-007 epithelial dysplasia and lung cancer in end-stage idiopathic pulmonary fibrosis: padova experience n. nannini * , f. lunardi, e. balestro, e. rossi, m. loy, m. saetta, f. rea, f. calabrese * university of padova, italy objective: idiopathic pulmonary fibrosis (ipf) is associated with increased risk of lung cancer. the prevalence of high grade dysplasia/lung cancer was studied only in a small number of ipf patients. the aim of our study was to investigate the prevalence of precancerous/cancerous changes and their relationship with both metaplastic changes and clinical data. method: native lungs from 66 ipf patients were studied. the degree of honeycomb changes and squamous, cuboidal and bronchial cell metaplasia were graded (score: 0-3). the presence or absence of precancerous/cancerous changes were also evaluated. results: three patients showed neoplastic transformation (4 %) and nine high grade dysplasia (14 %) ("cancer" group). the "cancer" group had similar smoking history, sex, age and duration of disease than the "no cancer" group. all lungs showed metaplasia, the score of squamous (p= 0.0001), cuboidal (p=0.018) and bronchial cell (p=0.018) metaplasia was significantly higher in the "cancer" than in the "no cancer", while the honeycomb score was similar in the two groups. conclusion: advanced ipf patients have a high prevalence of high grade dysplasia/lung cancer, complex epithelial metaplasia, particularly squamous type, is more frequent in "cancer" group, independently from all clinical parameters, including smoking history. ofp-18-008 primary pulmonary adenocarcinoma with enteric differentiation, morphologically indistinguishable from metastatic colorectal adenocarcinoma: case report with a history of metastatic colon adenocarcinoma s. percinel * , p. celepli, h. nalbant, y. yuyucu karabulut * ankara, turkey objective: according to a computerized medline search in the english literature, the present case is thought to be the first primary pulmonary adenocarcinoma with enteric differentiation, completely resembling metastatic colorectal adenocarcinoma morphologically, with a history of metastatic colon adenocarcinoma. results: a 62 year old man with a history of colon adenocarcinoma which was detected in 2007 underwent radiologic evaluation. a chest computed tomography scan revealed a 7 mm solitary nodule in the left lower lobe which enlarged in 1 year. the patient underwent left lower lobe wedge resection. macroscopically, an irregular whitish nodule of 1.5 cm in greatest diameter was detected. microscopically, the nodule was entirely composed of glandular and papillary structures, some of which had a cribriform pattern, lined by tumor cells that were cuboidal to tall columnar with nuclear pseudostratification, eosinophilic cytoplasm, brush-border, luminal necrosis and nuclear debris. tumor cells were diffusely positive for cytokeratin (ck)7 and negative for thyroid transcription factor-1, surfactant protein-a, cdx-2 and muc2. only a very few tumor cells stained for ck20. conclusion: enteric morphology with consistent expression of ck7 and a scattered positivity for ck20 helped in the distinction from metastatic colon adenocarcinoma and favored the diagnosis of primary pulmonary adenocarcinoma with enteric differentiation. expression of cxcr4 and cxcl12 in pulmonary carcinoids s. seiwerth * , l. brcic, a. sepac, a. zanko * zagreb school of medicine, pathology, croatia objective: cxcr4 and its chemochine ligand cxcl12 seem to play an important role in the process of tumor metastasis and, possibly, homing of metastatic tumor cells. the chemochine seems to be responsible for creating microenviromental predispositions for survival of metastatic cells, in a similar way it is done for developing immuno-competent cells. in contrast, expression of cxcl 12 in primary tumor cells, is thought to be associated with lower metastatic potential. cxcr4 is expressed in a wide range of tumors, and is thought to be crucial for the metastatic process and tissue-specific spread firstly of breast and prostate cancer. the role of cxcr4 signaling has been poorly evaluated in carcinoids in general and not at all in pulmonary carcinoids. method: immunohistochemically we investigated the expression of cxcr4 and cxcl12 in pulmonary carcinoids. results: together 60 tumors (47 typical and 13 atypical) where investigated. in 6 there was a metastatic process (in 4 typical and 2 atypical). ligand cxcl12 expression was negative in all of the metastatic tumors and in two without known metastasis in contrast to only two non-metastatic tumors showing negative reaction. cxcr4 positivity was found in both metastatic and non-metastatic carcinoids. the other investigated parameters where present in both metastatic and non-metastatic tumors. objective: to evaluate col v and decorin expression in pulmonary tissue and to characterize biochemical profile of colv from lung fibroblasts culture from ssc patients. method: we evaluated col v and decorin expression and tridimensional reconstruction (3d) of 6 patients with ssc without pulmonary hypertension that underwent surgical lung biopsy and as control was obtained lung fragments from 6 normal individuals who died from trauma. col v amount in lung sections was evaluated with immunofluorescence. to biochemical characterization of col v from lung fibroblasts culture was used quantitative immunoblot. results: it was found that the structure of col v fibers was distorted and strongly thickened in lung tissue from ssc patients compared with thin fibers pattern in the healthy controls. decorin was distributed around col v fibrils in the bronchovascular interstitium and vascular walls. histomorphometric analysis of ssc lung demonstrated increased expression of both col v and decorin when compared to the control (p < 0.01). the semiquantitative imunoblot detected an increased high molecular weight colv fraction in patients when compared to the control. conclusion: the over expression and unusual organization of colv fibers with biochemical changes associated to increased decorin indicates that matrix signalization pathway is involved in colv fibrillogenesis process in ssc pulmonary fibrosis. objective: probe-based confocal laser endomicroscopy (pcle) is a new method used during bronchoscopy by means of special miniprobe alveoflex and based on the visualization of intraalveolar structures which possess autofluorescence. aim: to compare the visual signs of a healthy and pathologically changed lung tissue received at pcle in patients with infiltrative and local lung nodules with the diagnosis, delivered by light microscopy. method: an autopsy and surgical material was fixed in 10 % neutral formalin solution and was analyzed by studying with a new method for visualization of structures. histological specimens were studied at that spaces, where the pcle was applied. we compared our results by using qualitative method. results: normal lung tissue structures include alveolar septum with the high light emission and light-negative spacesthe alveolar spaces. in case of pneumonia alveolar septum were saved, but the light density of alveolar spaces was higher in compare with the normal tissue. in case of alveolar proteinosis we observed unique globules, which had the highest light emission. we found out several authentic signs, which are representative for each kind of pathological feature. moreover, we revealed some other characteristics, which help us to distinguish some types of lung cancer. conclusion: pcle can be used as an additional method of noninvasive diagnostics in vivo. increased copy nember of alk gene is not associated with increased immunoreactivity of alk protein in lung adenocarcinomas t. balharek * , a. farkasova, z. kviatkovska, k. scheerova, p. tilandyova, z. hutka, l. plank * comenius university, dept. of pathology, martin, slovakia objective: anaplastic lymphoma kinase (alk) gene rearrangements represent an important predictive marker and promissing therapeutic target in small subset of non-small cell lung carcinomas (nsclc). immunohistochemical (ihc) screening of alk abnormalities in nsclc was reported to have variable reliability. we analyzed association between alk immunoreactivity and increased number of alk gene copies often seen in nsclc. method: we examined 10 clinically selected egfr negative lung adenocarcinomas. alk protein expression was detected by ihc using antibody clone alk1 (dako). alk gene status was assessed by fluorescent in situ hybridization using lsi alk dual color rearrangement probe (abbott) and spec alk/eml4 tricheck probe (zytovision). results: increased alk gene copy number was identified in 7 cases, 3 of them showed cytoplasmic alk positivity. in 2 of these cases we detected rearrangement of alk locus, once represented by alk-eml4 fusion combined with more complex cytogenetic abnormalities. remaining third alk + case was negative for alk rearrangement. conclusion: ihc seems to be useful method for initial screening of alk rearrangements in nsclc. there is no clear association between alk protein expression and number of alk gene copies. prognostic relevance of alk copy gains or amplification in lung adenocarcinomas remains to be determined, together with role of alk-inhibitors in those cases. expressions of egfr, ercc-1, ß tubulin iii, rrm-1 in advanced non-small cell lung carcinoma n. bassullu * , e. namal, i. turkmen, r. yasar, p. y. korkmaz, z. akcali, g. demir, g. b. dogusoy * istanbul bilim university, dept. of pathology, turkey objective: egfr, ercc1, rrm1 and βtubuliniii predicts sensitivity to therapeutic agents and provide prognostic information in nonsmall cell lung cancer (nsclc) which is the most frequent worldwide cause of cancer death. method: we investigated the expressions of egfr, ercc1, βtubuliniii, rrm1 immunohistochemically in 42 advanced nsclc cases and their correlations with other pathologic features. results: the age distribution was 30-82 with an average of 63,02. male/female ratio was 34/8. ercc1 protein was detected in nuclei of carcinoma cells in 38 patients (90,5 %) (h score >1). βtubuliniii expression was detected in cytoplasm of cancer cells in 41 patients (97,6 %) (score ≥ 50). ercc1 and βtubuliniii expression were not associated with pathological factors. rrm1 was negative (score<9) in 29 (69 %) cases and negativity was significantly correlated with male gender (p<0,032). egfr was negative (score<200) in 40 (95,2 %) cases and negativity was nearly correlated with absent necrosis (p<0,065). no significant correlations were found between egfr, ercc1, βtubuli-niii, rrm1 and the pathological parameters. conclusion: in our study, as a first step, egfr, ercc1, βtubuliniii, rrm1 showed no significant correlation with pathologic features. we look forward to obtain further results in the next study consisting of correlations with follow ups. proliferative markers in idiopathic pulmonary fibrosis: clinical, radiological and functional significance e. parra * , m. cornati, v. capelozzi * fmusp, dept. of pathology, são paulo, brazil objective: natural course of idiopathic pulmonary fibrosis (ipf) could be predicted by proliferative markers of the fibrotic process, such as myofibroblasts and interleukins (il)-13 and il14. our primary aim was to determine whether these proliferative markers influence the course of ipf course measured by a radiological/functional score. method: twenty-eight patients with biopsy-proven ipf disease, who underwent pulmonary evaluation by high-resolution computed tomography (hrct) fibrosis score and pulmonary function tests were studied. five normal lung tissues (nlt) were included biomarkers in lung tissues were detected by immunohistochemistry and quantified by histomorphometry for myofibroblasts alpha-smooth muscle actin (α-sma), anti-interleukin (il)-4 and il-13. results: myofibrobalst amount, il-4 and il-13 expression were higher in ipf than in nlt (p<0.01). myofibroblast expression of α-sma was positively correlated to il-14 and il-13 expression. lung tissue from patients with high hrct fibrosis scores expressed significantly greater α-sma+, il-4 and il-13 when compared with patients with low hrct fibrosis scores (p<0.05). negative correlations were found between myofibroblasts α-sma + and vc and dlco. conclusion: proliferative markers, detected by immunohistochemistry, in lung tissue allowed recognizing a dichotomous distribution of hrct fibrosis course and influenced pulmonary function tests, suggesting that they may be promising markers of prognosis in these patients. objective: epithelioid mesothelioma is the most common histologic type of the diffuse malignant pleural mesothelioma, also having the best prognosis. although a great variety of histological patterns within epithelioid type has been described, a clear impact of histological subtyping on clinical outcome is unknown (kadota et al., 2011.) . here we compared median survival of six histological subtypes of epithelioid mesothelioma. we examined hematoxylin and eosin-stained slides of 74 patients diagnosed with epithelioid mesothelioma. according to previously described predominant histological features we grouped them into six subtypes: trabecular, solid, microglandular, tubulopapillary, micropapillary and pleomorphic. results: the median survival of all 74 patients with epithelioid mesothelioma was 14.5 months. the best median survival was in trabecular and micropapillary subtypes (each 18 months, n=20 and n=5, respectively), followed by tubulopapillary (17 months, n=18), microglandular (16 months, n=7) and solid (11 months, n=19) subtypes. the worst median survival was in pleomorphic subtype (5 months, n=5). conclusion: epithelioid type of diffuse malignant pleural mesothelioma shows a great diversity of histological patterns that likely have an impact on the clinical outcome and patient's survival. further investigations of genetic variations among different subtypes may provide valuable information for better understanding of pathogenesis of these tumors. objective: genetic instability resulting in both aneuploidy and polyploidy are discussed to be involved in prostate cancer (pca) development and progression. aim of this study was to comprehensively characterize the ploidy status and proliferation levels in pca with regard to disease progression. method: using fish, we assessed 112 localized pca, 75 pca with 125 corresponding lymph node metastases, and 42 hormone-refractory distant metastases for losses and gains of all 24 chromosomes. the proliferation rate was assessed using phh3 and ki67 immunohistochemistry. results: we observed significant increases in aneuploidy with advancing tumor stage (p<0.05). chromosomes x, 21, y, 14, 16, and 8 were most frequently numerically altered. increased levels of proliferation were significantly associated with the extent of aneuploidy and tumor stage (p<0.01). combining aneusomy of chromosomes 4, 6, 20, and x with phh3 immunoreactivity resulted in a prediction model for lymph node metastases with a sensitivity of 73.3 % and a specificity of 72.6 %. conclusion: we present evidence that genomic instability leading to aneuploidy is an important factor in pca progression. furthermore, we demonstrate that increased ki-67 and phh3 expression are potential indicators of metastatic disease. lastly, we suggest a new approach to preoperatively determine lymph node metastasized disease in pca. ofp-20-002 erg protein expression and genomic rearrangement status in primary and metastatic prostate cancer: a comparative study of two monoclonal antibodies m. braun * , d. goltz, z. shaikhibrahim, w. vogel, d. boehm, a. dobi, n. wernert, g. kristiansen, s. perner * universitätsklinik bonn, inst. für pathologie, germany objective: overexpression of the erg protein is highly prevalent in prostate cancer (pca) and most commonly results from gene fusions involving the erg gene. recently, an n-terminal epitope targeted mouse and a cterminal epitope targeted rabbit monoclonal anti-erg antibody have been introduced for the detection of the erg protein. here, we are the first to compare the mouse erg-mab to the rabbit erg-mab for their concordance on the same pca cohort. furthermore, we assessed if the erg protein expression is conserved in lymph node and distant metastases. method: we evaluated tissue microarrays of 278 specimens containing 265 localized pca, 29 lymph node, 30 distant metastases, and 13 normal prostatic tissues. we correlated the erg protein expression with the erg rearrangement status using an erg break-apart fluorescence in-situ hybridization (fish) assay and ihc of both erg antibodies. results: erg protein expression and erg rearrangement status were highly concordant regardless of whether the mouse or rabbit erg-mab was used (97.8 % versus 98.6 %, respectively). conclusion: this is the first study to comprehensively compare the two erg-mabs. by demonstrating a broad applicability of ihc to study erg protein expression using either antibody, this study adds an important step towards a facilitated routine clinical application. ofp-20-003 improved method of detecting the erg gene rearrangement in prostate cancer using combined dual-color chromogenic and silver in-situ hybridization m. braun * , j. stomper, d. boehm, w. vogel, n. wernert, g. kristiansen, s. perner * universitätsklinik bonn, inst. für pathologie, germany objective: the recently detected erg rearrangement revealed as a recurrent and prevalent prostate cancer (pca) specific event. to detect this alteration, fish is the method of choice. however, fish harbors disadvantages for widespread adoption in clinical practice. subsequently, the chromogenic in-situ hybridization (cish) and the enzymatic metallography silver in-situ hybridization (sish) emerged as promising bright-field alternatives. we aimed to develop a combined cish and sish (cs-ish) gene break-apart assay on the example of the erg gene. method: we assessed and compared 178 pca and 10 benign specimens for their erg rearrangement status applying a dual-colour fish and cs-ish erg break-apart assay on consecutive sections. results: we observed a highly significant concordance (97,7 %) between fish-based and cs-ish-based results (pearson's correlation coefficient 0.955, p<0.001). conclusion: we demonstrate that the erg rearrangement status can reliably be assesed by cs-ish. further, we confirm that the cs-ish technique combines the accuracy and precision of fish with the ease of bright field microscopy. we developed a tool which allows a broad spectrum of applicants to study the biological role and clinical utilization of erg rearrangements in pca. moreoever, our study is the first proof-of-principle for bright-field cs-ish gene fusion or break-apart assays. ofp-20-004 rearrangement of the ets genes etv-1, etv-4, etv-5 and elk-1 is a clonal event during prostate cancer progression m. braun * , z. shaikhibrahim, w. vogel, d. boehm, r. menon, n. wernert, g. kristiansen, s. perner * universitätsklinik bonn, inst. für pathologie, germany objective: ets gene rearrangements are frequently found in prostate cancer (pca). recently, we observed that erg rearrangement in primary pca transfers into lymph node metastases, suggesting it to be a clonal expansion event during pca progression. here, we investigated whether this also applies to the less frequent ets genes, etv-1, etv-4, etv-5 and elk-4. method: using break-apart fish assays, we evaluated the status of above mentioned ets gene rearrangements on a cohort comprising of primary pca, corresponding lymph node and distant metastases. results: etv-1, etv-4, etv-5 and elk-4 rearrangements were found in 10 %, 6 %, 1 % and 2 % of the primary pca, respectively, and in 8 %, 6 %, 6 %, 1 % of the corresponding lymph node metastases, respectively. rearrangements of etv-1 and etv-5 were not found in any of the distant metastases cases, whereas etv-4 and elk-4 rearrangements were found in 4 % and 4 % of the distant metastases, respectively. conclusion: our results suggest that rearrangement of the less frequent ets genes is a clonal event during prostate cancer progression. our findings provide insights into potential clonal expansion events during pca progression and may have significant implications in understanding the molecular basis of the metastatic cascade of pca. ofp-20-005 comparative analysis of two prostate biopsy systems: a study of 120 cases f. e. costa * , r. dias, j. r. vizcaíno * centro hospitalar do porto, braga, portugal objective: it's expectable that the higher the number of prostate biopsies the greater the probability of prostate cancer detection, presumably improving the diagnostic accuracy and treatment decision. method: under this assumption, since may/2010, santo antónio's hospital implemented a prostate mapping system, increasing the number of biopsies performed from 8-12 to 16-18 and processing each core independently. we intend to establish a comparison between the traditional prostate biopsy system (ts) and the prostate mapping system (ms). for this study, all biopsy slides and corresponding surgical specimens from 60 patients subjected to ms and 60 patients subjected to ts were reviewed. gleason score, percentage of tumor present in prostatic tissue (expressed as small ≤ 5 %, medium 6-19 % and large ≥20 %) and presence of tumor in prostate apex and base were analyzed. results: comparing ms with ts, the correlation coefficient was 53,5 % and 31,9 % for gleason score and 46,9 % and 37,4 % for the percentage of tumor present in prostatic tissue, respectively. mapped biopsies were able to detect the tumor in the apex and base in 72 % and 80 % of cases. conclusion: these results suggest that prostate ms improves the diagnostic accuracy of prostate cancer and has a good ability to predict the presence of tumor in prostate apex and base. ofp-20-006 frequency and prognostic significance of tmprss2-erg gene fusion in lymph node positive prostate cancers a. fleischmann * , i. zlobec, t. visakorpi, g. thalmann * universität bern, inst. für pathologie, switzerland objective: tmprss2-erg incidence and prognostic significance in lymph node positive prostate cancer are virtually unknown. method: a tissue-microarray was constructed from 119 hormone-naïve nodal positive, surgically treated prostate cancers containing samples from all gleason patterns (gp) present in every primary tumor (pt) and corresponding lymph node metastases (met). tmprss2-erg status was determined by fluorescence in-situ hybridization and correlated with various histomorphological tumor features (gleason score, stage, cancer volume, nodal tumor burden) and biochemical recurrence-free, cancer-specific and overall survival. results: tmprss2-erg fusion was present in 43.5 % (homogeneous 25.2 %, heterogeneous 18.3 %) of the pt and in 29.9 % (homogeneous 28.7 %, heterogeneous 1.2 %) of the met. percentage of tmprss2-erg in gp3/4/5 of pt and met were: 38 %/37 %/24 % and 23 %/30 %/39 %. concordance in tmprss2-erg status between pt and met was poor (kappa 0.39) showing 20.9 % and 8.1 % of cancers with gene fusion solely in the pt and met, respectively. tmprss2-erg fusion was not correlated with histopathological tumor features and predicted late biochemical recurrence independently (p=0.041) when present in pt. conclusion: tmprss2-erg fusion in pt is more frequent and its distribution more heterogeneous compared to met. the gene fusion in primary tumors independently predicts late biochemical recurrence. ofp-20-007 wnt and shh pathways activation in penile carcinoma a. silva * , t. almeida, f. soares, m. buim * hospital ac camargo, investigative pathology, são paulo, brazil objective: penile carcinoma (pc) is rare in developed countries, accounting for less than 1 % of all neoplasms in men, and biological characteristics of this tumor are poorly known. wnt and shh pathways are important for cell proliferation, differentiation and survival, and therefore, play a role in carcinogenesis of various organs. the goal of this study is to investigate the expression profile of wnt and shh pathway proteins in pc, characterizing the expression of target proteins from wnt (wnt-1, wnt-2, wnt-2 gsk3β, β-catenin, d1 cyclin, mmp7, c-myc, cd44) and shh (shh, smo, gli, egfr) pathways. method: for that, 18 samples of pc were obtained from the files of anatomic pathology department from a.c. camargo hospital (brazil) and submitted to immunohistochemistry. results: we observed that wnt-1 and wnt-2 were expressed in 15 and 16 cases, respectively, out of the 18 evaluated samples. strong shh expression was detected in 16/18 cases, whereas weak and negative expression was seen only in one case each. smo and gli-1 proteins were expressed in almost all cases (17/18), and also d1 cyclin, b-catenin and egfr were frequently expressed. conclusion: these preliminary results suggest that wnt and shh pathways may be active and participating in the progression of penile cancer. ofp-20-008 expression of the multidrug resistance protein 4 correlates with longer psa relapse free survival and androgen receptor and forkhead box a protein expression in prostate cancer m. montani * , g. kristiansen * medizin. universität bern, inst. für pathologie, switzerland objective: multidrug resistance protein 4 (mrp4) a transmembranary transport protein has shown to be expressed in prostate cancer cell lines and cancer cell specimens and turned out to be among the highest upregulated genes in an arraybased transcription analysis. its expression is regulated in an androgen dependent manner, possibly modulated by forkhead box a (foxa), an androgen receptor (ar) co-activator. therefore, we investigated its expression in a large cohort of neoplastic and non-neoplastic prostate tissues (n = 441) and evaluated its prediction of psa relapse free survival (rfs). method: tma (n=441) stained for mrp4, foxa, ar, er, psa, ki-67: radical prostatectomies (rpe) (n=332), castrate resistant prostate cancer (crpc) (n=26), metastases (n=39) and non-neoplastic (n=187). psa-rfs of 258 patients; mean 70 months. results: mrp4 expression decreases with tumor progression into castrate resistant disease, correlates with psa, ar and foxa expression and inversely correlates with gleason score. moreover, a strong mrp4 expression is significantly associated with a longer psa rfs in rpe patients. normal tissues from the transitional zone show a weak mrp4 expression compared to the peripheral and central zones. conclusion: mrp4 expression seems to predict psa relapse free survival in prostate cancer patients. since its expression is androgen dependent it decreases with tumor progression into crpc. ofp-20-009 synchronous angiomyolipoma and renal tumors in patients without tuberous sclerosis: clinico-pathological study of 18 cases f. j. queipo * , r. a. carías, án. f. panizo, m. l. gómez-dorronsoro, f. j. pardo * clínica universidad de navarra, pathology, pamplona, spain objective: simultaneous existence of aml and renal neoplasia is frequent in tuberous sclerosis (ts) patients, but uncommon in non-ts cases. method: a total of 18 cases of coexistent renal neoplasia and aml in non-ts patients were identified. clinico-pathological features were studied. results: 18 patients: 10 m/8 f (mean age 62,77 year; range: 35-83) . mean aml size: 0,82 cm (range: 0,2-3,4). the main size of the renal neoplasms was 5,09 cm (range 1,2-15). aml morphology: 6 leiomyomatous, 4 classic-triphasic, 4 lipomatous, and 4 epithelioid. three cases had multifocal aml. 15 cases had ipsilateral tumors associated with aml: 6 ccrcc (1 sarcomatoid), 4 chrcc, 1 ro, 1 tfe3 rcc, 1 hybrid (ro-chrcc) renal-cell tumor, 1 urothelial carcinoma, and 1 case had 2 tumors: mtsc-rcc and concomitant prcc. 2 cases had contralateral tumours associated with aml: 1 chrcc and 1 ro. one patient had bilateral tumors associated with aml: an ipsilateral ro and a contralateral ccrcc. the median follow-up was 36,85 mths (range 0,4-199,8) : all patients were alive without disease. conclusion: the coexistence of renal tumors with aml is a rare event, usually incidental. if aml is found incidentally together with other renal tumors, it is important to exclude ts retrospectively. ofp-20-010 primary renal synovial sarcoma: a clinicopathologic, immunohistochemical and molecular genetic study of 16 cases j. schoolmeester * , j. cheville, a. folpe * mayo clinic, dept. of anatomic pathology, rochester, mn, usa objective: primary renal synovial sarcoma (r-ss) is a rare malignant neoplasm. approximately 45 cases have been described, chiefly as case reports or as part of relatively limited small studies. we studied the clinicopathological, immunohistochemical (ihc) and molecular genetic features of 16 well-characterized r-ss. method: all available slides for 16 institutional and consultation cases of r-ss were reviewed. an ihc panel (tle1, bcl2, cytokeratins, s100, cd34, ini1) was performed. rt-pcr for ss18-ssx1/2 or fish for ss18 rearrangement was performed. follow-up was obtained. results: the patients (9 m, 7 f) ranged from 17 to 78 years (mean 46). all tumors were of monophasic fibrous type. ihc results: all cases were strongly positive for tle1 and bcl2; focally positive or negative for cytokeratins; negative for s100 and cd34; and showed retained ini1 expression. molecular genetic results were: ss18-ssx1 (5 cases), ss18-ssx2 (10 cases), syt rearrangement (1 case). follow-up (5 cases): 3 dead of disease, 2 alive without disease. conclusion: our data show a striking overrepresentation of the ss18-ssx2 fusion subtype among r-ss, in contrast to other ss, in which the ss18-ssx1 fusion subtype accounts for two-thirds of cases. the reasons for this difference are unclear. the prognosis for r-ss appears similar to that of ss of more common locations. objective: new data related to the diagnosis and therapy of lung cancer have also affected the position of the pathologist in this area. principal changes in its classification have appeared in relation to adenocarcinoma and partly also in the large cell type of lung cancer. method: presented data represent a five-year retrospective study of a series of 546 cases of lung cancers analyzed in our institute. besides routine he staining the following immunohistochemical reactions were used: antibody against ttf-1, surfactant a, chromogranin, synaptophysin, ck7, ae1/ae3, p63, 34βe12, cd56 and napsin a. results: using the who, 2004 classification of lung cancer, an unusual phenomenon of the return of squamous cell cancer (scc) prevalence over adenocarcinoma has been documented, which in the earlier years has equated the scc. the difference in obtaining the tissue samples, and changes in the geographical origin of certain patients of eastern slovakia may partly explain these differences. though the new travis classification (j thorac oncol, 2011) was not used, there prevailed mixed forms also in our series. conclusion: it is important to respect new information regarding the classification of lung cancer, especially in the category of adenocarcinoma. the constructive collaboration of pathologists with clinicians is recommended. objective: extranodal marginal zone lymphoma malt lymphoma and carcinoid are neoplasms occurring most frequently in the gastrointestinal tract and respiratory system. although each of them occurs relatively frequently and separately, the simultaneous appearance of these two neoplasms is exceptionally rare. method: we report an exceptional synchronous association of malt lymphoma and typical carcinoid in the lung. results: a 52-year-old male with sjögren's syndrome presented with cough and chest pain with no improvement after antibiotherapy. bronchial fibroscopy was normal. computed tomography showed a diffuse interstitial pneumopathy with a persistent left lower lobe nodule suspected of malignancy. a surgical resection of the nodule was achieved. histopathologically, it measuring 1.2 cm in diameter and was composed of association of malt lymphoma and typical carcinoid tumor which were focally admixed. immunohistochemical stains were strongly reactive to endocrine marker in carcinomatous component. tumor cells were diffusely cd20 positive in lymphomatous component. assessment of extent of lymphoma revealed a gastric location. the patient received chemoptherapy. he's still alive since 8 months. conclusion: the best of our knowledge, this is the first report of such a collision lung tumor at the same anatomical site. the aim of this study is to describe the pathogenesis and clincicopathological feartures of such exceptional association. objective: primary mucoepidermoid carcinoma is rare comprise less than 1 % of all lung tumors. it is characterized by the presence of squamoid cells, mucin-secreting cells and intermediate type. on the basis of morphological and cytological features, it is divided into low and highgrade types. method: we report a retrospective study of 15 cases diagnosed during a 15-year period. diagnosis was made by histological examination of specimen obtained from lobectomy in 13 cases and pneumonectomy in 2 cases. no patient had history of salivary mucoepidermoid carcinoma. results: there were 13 men and 2 women ranging in age from 10 to 76 years. computed tomography showed a well circumscribed tumor arising in bronchus in all cases. a bronchial fibroscopy showed a main, lobar or segmental mass. bronchial biopsy revealed a non-small-cell carcinoma in only one case. histologically, tumors were classified into 7 low grade mucoepidermoid carcinoma and 8 high grade mucoepidermoid carcinoma. patients with low grade carcinoma remain alive after surgery alone. patients with high grade carcinoma received chemotherapy, four of them developed distance metastasis. conclusion: mucoepidermoid carcinoma is a rare primary malignancy of the tracheobronchial tree which is difficult to diagnose by limited biopsy. the prognosis is variable and depends upon the histological grade. objective: primary pulmonary malt lymphoma is considered to originate from malt of the bronchus secondary to autoimmune or inflammatory processes. although, it comprises more than two-thirds of all primary non-hodgkin's lymphoma of the lungs, it is a rare entity and accounts for less than 1 % of all lymphomas. method: we report a retrospective study of 6 cases of malt pulmonary lymphomas diagnosed during an 8-year period. diagnosis was made on surgical pulmonary biopsy in 2 cases, bronchial biopsy in 3 other cases and on lobectomy in one case. results: there were 4 women and 2 men ranging in age from 48 to 74 years. two patients had a history of sjoren's syndrome and one had a history of previous mammary carcinoma. computed tomography revealed bilateral nodules in 3 patients, lung mass in 2 patients and an area of consolidation in one patient. fiberoptic bronchoscopy showed bronchial stenosis in only 3 cases. morphologically, the neoplasms had features typical of malt lymphoma. one patient was treated with surgery alone and 5 received chemotherapy. five patients remained alive while one patient presented with recurrence. conclusion: pulmonary malt lymphomas are characterized by an important dissociation between clinical expression and radiological pattern. therefore, histological documentation is mandatory to ensure diagnosis. objective: malignant mesothelioma (mm) is an aggressive tumour with a poor prognosis. carbonic anhydrases and their inhibitors offer an opportunity for developing novel anticancer drugs, as well as diagnostic and prognostic tools. carbonic anhydrase ix (caix) is a membranous metalloenzyme involved in cell adhesion and ph homeostasis. it is a direct target of hypoxia-inducible factor and serves as a marker of hypoxia. this study was designed to assess systematically caix expression in mm of pleura and peritoneum, and their benign counterparts. method: 47 mms of pleura (41 epithelioid, 1 biphasic, 2 sarcomatoid) and peritoneum (3 epithelioid), and 14 normal or reactive pleural samples were analyzed. caix expression was determined using immunohistochemistry. membranous immunoreactivity was evaluated semiquantitatively. specimens were divided into five subgroups according to the staining pattern and intensity. results: 95,7 % (45/47) of mms expressed caix. all epithelioid mesotheliomas showed at least a weak focal (8,5 %, 4/47), but predominantly a strong diffuse (70,2 %, 33/47) staining with caix antibody, without any perinecrotic pattern. sarcomatoid mesotheliomas were negative. normal mesothelial cells were diffusely positive. conclusion: these data suggest that mechanisms of caix overexpression in mm are different than due to hypoxia and appear promising in prospective use of specific therapeutic caix targeting in advanced mesothelioma. objective: eml4-alk crizotinib therapy needs validation at lower cost and rapid answer in pathology routine. method: histological/who 2004 and ck7, ttf1, ck5.6, cd56/chromogranin and vimentin panel classifications with alk (clone 5a4, novocastra laboratories ltd, newcastle, united kingdom) were applied to paraffin sections of 35 bronchial-pulmonary carcinomas: 20 adenocarcinomas, 6 epidermoid carcinomas, 4 pleomorphic carcinomas (mixed type adenocarcinomas with large/giant/fusiform cells), 4 neuroendocrine carcinomas (nec) (1 combined large cell nec with adenocarcinoma and 2 with epidermoid carcinomas; 1 sclc chromogranin positive combined with adenocarcinoma) and 1 adenosquamous carcinoma. results: the applied antibodies specified bronchial pulmonary carcinomas subtypes clearly. in 3 over 60 years old nonsmoking females mixed type adenocarcinomas alk expression was over 50 %: acinar, solid, micropapillary and microacinar patterns; one glandular mucinous pattern (mucinous ba pattern) and one ba pattern, all expressing ttf-1. conclusion: in this study, 3/20 adenocarcinomas of older women had alk protein expression, only one with a mucinous pattern. as protein positivity cases comprise a lower number, fish described by s. lantuejoul seems to be the most appropriate method. it is now necessary to decide whether kras and egfr mutations have to be determined together and/or select ttf-1 positive adenocarcinomas (from terminal respiratory unit) raised by this approach. objective: the 5 year survival of bronchial-pulmonary carcinomas remains poor, between 6 % and 14 %/men and 7 % to 18 %/women. treatment orientation is influenced by clinical staging and morphological classification in biopsies of 70 % of the cases. method: this study comprised 41 surgical specimens where we compared immunohistochemistry in-between adenocarcinomas (18), epidermoid carcinomas (12) and the heterogeneous groups of large cell neuroendocrine carcinoma (3), small cell lung cancer (1), large cell carcinoma (2), adenosquamous carcinoma (2) and pleomorphic carcinomas (3) with max 18 f-fluordesoxiglucose (fdg), a clinical parameter based in pet to preview diagnosis and prognosis. results: we found significant differences (p=0.028) between ttf-1 positive and negative adenocarcinomas where the 18 f-fdg capture was lower in ttf-1 positive cases, indicating lower metabolic activity. ttf-1 negative adenocarcinomas have similar and higher metabolic activity as epidermoid carcinomas. the other histological types have fdg capture similar in between the two defined groups. conclusion: immunohistochemical and 18 f-fdg analysis correlate with clinical differences between adenocarcinomas and epidermoid carcinomas, where ttf-1 negative adenocarcinomas are biologically similar to epidermoid carcinomas, requiring a different medical approach as well as molecular pathology particular interpretation. these results strain the classification of bronchial ttf-1 negative adenocarcinomas because they are different from the terminal respiratory unit ttf-1 positive adenocarcinomas. objective: according to researchers pneumonias are characterized by increase number of alveolar macrophages population after increased migration of monocytes from bone marrow and blood into the alveolar space. method: we examined 60 observations of pneumonia with bacteriological, histological and blood monocytes examination in all cases. bacteriological study was performed at the institute of antimicrobial chemotherapy. histological examination was performed in the smolensk regional institute of pathology. results: in the foci of pneumonias we discovered mainly mixed gram positive and gram negative microorganisms. ten observations were characterized by an increase in the absolute number of blood monocytes. fifty observations did not reveal excess levels of blood monocytes. in all cases with an increase in the number of blood monocytes, gram negative bacteria were isolated. in cases with normal number of blood monocytes some of the microorganisms were gram positive. conclusion: gram negative bacteria result in activation of the monocytes and their entry from the bone marrow into the blood more than gram positive bacteria. objective: bronchial carcinoids and schwannomas are uncommon, slowly growing, low-grade neoplasms. carcinoid tumors are thought to arise from neuroendocrine/kulchitsky's cells of bronchial epithelium. schwannomas are thought to originate from schwann cells. clinically they may be asymptomatic or present with non resolving recurrent pneumonia, hemoptysis, respiratory distress. conclusion: we present two cases of respiratory distress in two teenage patients. pulmonary hamartomas mimicking metastatic carcinomas h. erdem * , l. yilmaz aydin, a. k. uzunlat, m. oktay, a. n. annakaya, u. yilderim, f. basar * duzce university, dept. of pathology, turkey pulmonary hamartomas are usually found incidentally and they mimick metastic tumours. we report a case of a 47 year-old man who admitted to chest disease service for cough. he had laryngectomy operation formerly and diagnosed as squamous cell carcinoma. chest-x-ray and computed tomography revealed pulmonary nodules. it was removed but there was not metastatic carcinomas. this case was presented because it can be difficult to make diagnosis when the patient has malignancy. objective: experimental models of pulmonary fibrosis (pf) has been proposed and its later phase tend to the resolution in different degrees depending on the drug/ strain/inflammatory-pattern. thus the maintenance of these mechanisms may participate in the pf-progression. our objective was to determine the immune-fibroticpattern in models of pulmonary fibrosis in the late stage (21d). method: distinct animal models were used, including balb/c, c57bl/6 and il17ra-ko-c57bl/6 mice by bleomycin and paraquat. we analyzed the amounts of total peribronchiolar-interstitial collagen (tpc-tip) by picrosirius and col3-col5 by immunofluorescence through the morphometric evaluation. these data were validated by rt-pcr. results: the tpc did not differ between the treated groups. tic was higher in the c57bl/6 strain, independent of the absence of il-17ra. the col5-immunoexpression was higher in control and blm-treated il17ra-ko-c57bl/6 than in wild-mice and lower in blm-balb/c mice. the col3immunoexpression was higher in blm-balb/c. likewise, the col5 gene expression was higher in the il17ra-ko mice and lower in the blm-balb/c. conclusion: the perpetuation of fibrosis in pf-susceptiblemice can be modulated by il-17-dependent col5-hiperexpression and by col3-subexpression. this suggests that col5 is an important component responsible for the development of pf. the prognostic role of filamin a protein expression in patients with non-small-cell lung cancer m. gachechiladze * , j. skarda * palacky university, pathology, olomouc, czech republic objective: an actin-binding protein filamin a (flna) serves as a scaffold in various signaling pathways. recently, it has been reported that flna interacts with brca1 protein and is required for efficient regulation of early stages of dna repair processes. as dna repair proteins are important prognostic markers for non-small-cell lung cancer (nsclc) patients, we aimed to investigate the role of flna protein expression in nsclc, as well as its correlation with brca1 protein expression. method: we performed a preliminary study of fnla and brca1 protein expression in 50 nsclc patients. formalin-fixed paraffin-embedded tissue sections were stained by immunohistochemistry using antibodies against flna c-terminus (ep2405y, lsbio), brca1 n-termunus (ms110) and against phosphorilated forms of brca1 at ser1524 and ser1423 (abcam). staining intensity was estimated semi-quantitatively and correlated with all available clinico-pathological factors. results: flna expression was significantly higher in cancer, compared to normal lung tissue. positive correlation has been revealed between flna and brca1 phospho-ser1423 expression. also, 5 year overall survival rate was higher in patients with strong flna expression. conclusion: according to our preliminary study results the prognostic role of flna protein expression deserves to be a subject for further studies in patients with nsclc. ps-01-014 pulmonary nodular lymphoid hyperplasia ("pulmonary pseudolymphoma") -the significance of increased numbers of igg4 positive plasma cells d. guinee * , a. gerbino, s. murakami, m. koss * virginia mason medical center, pathology, seattle, usa objective: pseudolymphomas of the skin, breast, and lacrimal glands show an increase in igg4 positive plasma cells. we hypothesized that a similar increase in igg4 positive plasma cells occurs in pulmonary nodular lymphoid hyperplasia (pnlh). method: immunohistochemical stains for igg4 and igg were performed in 2 cases of pnlh, 7 cases of balt lymphoma, 8 cases of intraparenchymal lymph nodes (ipl) and 1 case of follicular bronchiolitis (fb). the mean number of igg4-and igg-positive plasma cells and the igg4/igg ratio was obtained from a manual count of three separate high power fields (hpfs) of areas of highest cellularity. results: the average number of igg4 positive plasma cells and the igg4/igg ratio was increased in pnlh (case 1: 225/hpf, ratio: 0.39, case 2: 280/hpf, ratio: 0.59). in comparison, average igg4 positive plasma cells per hpf were much lower in balt lymphoma (range=0-2.3/hpf, ratio=0-0.03), ipl (range igg4/ hpf=0-102, ratio=0-0.23) and fb (1/hpf, ratio=0.03). conclusion: the increase in igg4 positive plasma cells and igg4/igg ratio in pnlh aids in diagnosis and supports our current understanding of pnlh as a distinct form of reactive lymphoid proliferation. the relationship between pnlh and igg4-related sclerosing disease requires further study. ps-01-015 calretinin, ck5/6, ttf-1, cea, ber-ep4, and cd15: a useful combination of immunohistochemical markers for differentiating pleural mesothelioma from pulmonary adenocarcinoma n. gursan * , m. calik, e. demirci, s. sipal, b. gundogdu, c. gundogdu * ataturk university, medical faculty, erzurum, turkey objective: the distinction between pleural epithelial mesothelioma and peripheral lung adenocarcinoma involving the pleura is still an important diagnostic problem for surgical pathologists. the aim of our study was to identify the most specific and sensitive markers for the positive identification of mesothelioma. method: paraffin-embedded blocks from surgical material of 19 pleural epithelial mesotheliomas and 22 pulmonary adenocarcinomas were retrieved from the files in our department. the primary antibodies used in each case were the following: antibody anticalretinin, ema; cea, berep4, ttf-1 and cd15. results: of the mesotheliomas, 100 % stained for calretinin, 63.2 % for ck5/6, 78.9 % stained for ema and ae1/ae3. of the lung adenocarcinomas, 9.1 % cases showed reactivity for calretinin, 27.3 % for ck5/6, 77.3 % for cd 15, all for ttf-1, 50 % for cea, 59.1 % for ber-ep4, 90.1 % for ema, and all for ae1/ae3. conclusion: calretinin were the highly specific positive mesothelial markers, whereas ck 5/6 showed high sensitivity but low specificity. among negative markers, we advocate the use of ttf-1, cea and cd15 which were the most specific in differentiating mesotheliomas from adenocarcinomas. cyclooxygenase-2 expression in nsclc i. kern * , e. sodja, m. rot * university clinic golnik, slovenia objective: overexpression of cox-2 correlates with aggressive disease of nsclc. aim of our study was to determine the cox-2 expression levels by two methods. method: analysis was done on 24 consecutive surgical specimens of nsclc fixed in paxgene tissue system. relative quantification of cox-2 mrna expression was performed by quantitative rt-pcr using intron-spanning primer-probe set. cox-2 protein expression was assessed by immunohistochemistry (ihc). scoring was performed using an intensity-extent system, both parameters on the scale 0-3 and multiplied to give ihc index. results: there were 12 cases of adenocarcinoma, 11 cases of squamous cell carcinoma and one typical carcinoid. cox-2 mrna expression was detectable in all specimens. the median cox-2 mrna expression value, normalized against he internal reference gene gadph, was 0,53 (range 0,08-12,49). we observed cytoplasmic and membranous immunohistochemical reaction patterns. average ihc index was 3,8. there was positive correlation between mrna and protein cox-2 expression (r2=0,31). adenocarcinoma cases had average relative mrna expression value 2,45 and ihc index 5,08, while squamous cell carcinoma had average relative mrna expression value 0,67 and ihc index 2,73. conclusion: cox-2 is expressed in nsclc with various ihc reaction patterns. adenocarcinoma and squamous cell carcinoma have different cox-2 expression levels. objective: the akt/mammalian target of rapamycin (mtor) pathway is up-regulated in many human cancers, and agents targeting the mtor pathway are in various stages of clinical development and application. method: expression of pakt and mtor was studied by immunohistochemical analysis of 574 surgically resected nonsmall cell lung cancer (nsclc) specimens on a tissue microarray (tma). results: the results were correlated with clinicopathological features. expression of mtor showed a strong correlation with the expression of pakt (p<0.001) and was significantly associated with female gender, tumor size ≤3 cm, adenocarcinoma (adc), non-smoker status, and lower pathological stage. expression of pakt was correlated with older age (≥ 65), adc, non-smoker status, and lower t stage. univariate survival analysis revealed that the mtor and pakt positive group had a significantly longer cancer-specific survival than the mtor and pakt negative group (p=0.038 and p=0.024, respectively). coexpression of pakt and mtor correlated with better prognosis than either single or double negative pakt and mtor groups (p=0.016). however, multivariate analysis proved that mtor and pakt expression are not independent prognostic factors for cancer-specific survival. conclusion: expression of pakt and mtor expression is more significantly associated with adc than squamous cell carcinoma (scc) and expression of these proteins is associated with better prognosis. ps-01-018 pak 6 immunohistochemistry marker in non-small cell lung cancer (nsclc) a. kucukosmanoglu * , o. d. i̇lara colakkadıoglu, k. bakir * university of gaziantep, pathology, turkey objective: pak 6 is a member of p21-activated kinase family. many human tumor express and activate this family. because of their role in cell transformation, they become therapeutic targets. they have important roles in cell survival, cell proliferation and cell migration. they affect cell growth and tumor invasion. method: 175 cases with nsclc reported in gaziantep university pathology department between 2000 and 2010, reviewed retrospectively. immunohistochemically nuclear and cytoplasmic pak 6 stainings were considered positive and scored according to degree of staining. results: our cases included 110 squamous cell carcinoma, 49 adenocarcinoma, 9 adeno squamous carcinoma and 7 large cell carcinoma. there was no significantly associated between tumor types and pak6 staining. 38 of 175 patients showed recurrence and of 23 cases showed nuclear staining but there were no statistically significance (p:0,267). 31 cases died from the disease and in 21 of these cases nuclear stainig was very intresting, inspite of statistical results. conclusion: in this study pak 6 staining patern and score were compared with tumor type, size, recurrence, mortality and lymph node involvement in nsclc. and we found no statistically significance. 150 of 175 case with nsclc stained with pak 6. these results suggest that pak 6 was expressed in lung like prostat, plasenta and breast. pleural fibroelastosis is a similar spectrum of histopathology in chronic fibrosing lung disorders l. marcal * , e. r. parra, l. antonangelo, v. capelozzi, f. vargas, e. c. nascimento, v. teodoro, k. c. silva * lia junqueira marçal, são paulo, brazil objective: parenchymal fibroelastosis in chronic fibrosing lung disorders has been much investigated, but little attention has been directed at the visceral pleura (vp) participation in these situations. our aim was to verify whether elastic deposition accompanies collagen deposition in the repairing process of chronic lung injury. in this work we studied the distribution of these fibrous components of vp in bullous disease type i and ii, smoking-related interstitial fibrosis and usual interstitial pneumonia (uip). method: we employed histochemical methods on conventional histological slides. we measured, by image analysis, the content of fibres of the collagenous and elastic systems of the visceral pleura in histological slides sampled from surgical lung biopsies and bullectomy, using the picrosirius-polarization method and weigert's resorcin-fuchsin stain, respectively. results: four groups were studied: i, 7 patients with spontaneous pneumothorax due to type i bullous disease; ii, 12 patients with spontaneous pneumothorax due to type ii bullous disease; iii, 5 smoking-related interstitial fibrosis; and iv, 5 patients with idiopathic pulmonary fibrosis. the first two groups were used as controls. patient diagnosed with idiopathic pulmonary fibrosis (2009), in which experimental transplantation was performed of type ii pneumocytes, ingress to the hospital for further study groundmass of the right lung. in the postmortem study a 2340 gr. mass was identified, the mass was formed by sarcomatous cells habit, undifferentiated and pleomorphic, with the presence of multiple implants and adhesions in rib cage, diaphragm, pericardial fat. ihc profile was performed: vimentin (+), ema focally (+), wt1, ck8, ttf1, ckae1/ae3, s100, cd34, cd31 and cd45 (−) and low proliferation index, the diagnosis undifferentiated malignant tumor. with inconclusive ihc profile, so blocks were sent to clinic barcelona hospital, for study. the estimated prevalence of idiopathic pulmonary fibrosis is 20 cases per 100,000 population in men, with a mortality of 50 % at 5 years of diagnosis. cancer rate of in transplanted patients is within the range between 4 % and 18 %. could the appearance of this tumor be related either to transplanted pneumocytes or it is inmunosupression the main risk factor? current treatments for idiopathic pulmonary fibrosis are not effective, which requires the study and development of new therapeutic options, using fibroblast proliferation inhibitors, as well as prospective studies, in the long term, the evolution of these patients and the development of secondary entities to it. objective: mediastinal hemangiomas are rare tumors accounting for 0,5 % of all mediastinal tumors. these tumors are challenging because of the lack of clinical and radiologic specific signs. their diagnosis is mainly based on microscopic study. method: we report a 10-year-experience of a single institution. we describe 5 cases of mediastinal hemangioma. results: our study contained 2 men and 3 women with a mean age of 60.4 years. symptoms consisted mainly in chest pain. neurologic signs were observed in 2 patients. surgical treatment was performed in all patients dealing with a total resection in 4 patients. the most used surgical approach was a posterolateral thoracotomy. video-assisted thoracic excision was tried in one patient and was converted into a median sternotomy. microscopic examination concluded to a mediastinal hemangioma in all cases. all the patients presented no complications after a follow up periods varying from 9 months to 2 years. conclusion: mediastinal hemangiomas are rare tumors whose diagnosis is based on microscopic findings. their surgical management may be challenging because of their connection to the adjacent structures. video-assisted thoracic excision is being more frequently used by experienced surgeons. these tumors are benign with a good behavior. objective: actinomycosis is an infectious disease caused by actinomyce israeli in 85 % of the cases. it is mainly observed in alcoholic patients and affects mainly cervicofacial, abdominal and thoracic regions. pulomnary actinomycosis accounts for 15 % of the localizations and is caused by the inhalation of septic particles causing granulomatous lesions, the extension to the adjacent organs and a cutaneous fistulae. it represents a real pitfall and mimics malignant lesions, tuberculosis or other infectious diseases. method: we report a retrospective study about 6 cases of pulmonary actinomycosis diagnosed over a 17-year-period. results: we describe the cases of 6 men aged between 35 and 46 years who presented with respiratory signs. radiologic investigations showed in all cases parenchymal masses with irregular margins mimicking malignant processes. positivie diagnosis was based on microscopic examination. the treatment was based on surgical excision associated to a medical treatment without complications after a follow up period ranging from 3 months to 18 months. conclusion: pulmonary actinomycosis shares usually the same clinical and radiologic features as malignant lesions. positive diagnosis is based on microscopic findings. it inducesusually surroundng organs and may affect, in some cases, the vital prognosis. over expression of hyaluronan syntehase-2 activity has impact in the remodeling process and survival evolution in patients with idiopathic pulmonary fibrosis e. parra * , v. capelozzi * fmusp, dept. of pathology, são paulo, brazil objective: the idiopathic pulmonary fibrosis (ipf) is a terminal illness characterized by unremitting extracellular matrix (ecm) deposition in the lung. in this regard, the myofibroblasts and the ecm components such as collagen and hyaluronan (ha) have an important role in the fibrosis. we analyzed the expression of has1 (ha synthase 1), has2, has3 and hyaluronic acid receptor (cd44) by epithelial and myofibroblasts cells in patients with ipf and we correlated with a survival. method: has-1, has2, has3 and cd44 epithelial and myofibroblast expression were evaluated in 27 surgical lung biopsies from patients with ipf in minimal and severe fibrosis by the point-counting technique. impact of these markers was tested on pulmonary functional tests and follow-up until death from ipf. results: has2 and cd44 expression were significantly increased and directly associated with severe fibrosis. myofibroblast has2 activity was indirectly associated to dlo/va (r= −0.584; p=0.05). kaplan maier curves determined a higher risk of death for patient with high ha2 (>6.83 %) expression than in low expression (log rank p=0.05, figure) . conclusion: the increased has-2 activity in epithelial and myofibroblast cells have impact in the remodeling process and the survival evolution, suggesting that strategies aimed at preventing the effect of this ecm component may have a greater impact in patient's outcome. financial support: fapesp, cnpq. ps-01-024 viral antigens are more frequently observed in acute interstitial pneumonia than in other types of idiopathic interstitial pneumonias e. parra * , g. c. dos santos, f. weisshaupt stegun, c. dos santos cirqueira, m. i. seixas duarte, v. l. capelozzi * fmusp, dept. of pathology, são paulo, brazil objective: the etiology of idiopathic interstitial pneumonias (iips) remains unclear. many researches assumed that viruses can represent an important factor of aggression to the lung provoking fibrosis. in this regards, we researched the presence of virus infections in patients with iips. method: biopsy samples from 13 patients with idiopathic interstitial pneumonia (ipf); 8 patients with nonspecific interstitial pneumonia (nsip); 13 patients with acute interstitial pneumonia (aip) and 4 patients with idiopathic cetrilobular fibrosis (iclf) were used to investigate by microarray and immunohistochemistry the presence of measles virus (mv), hepatitis-c virus (hcv), adenovirus (adv), respiratory syncytial virus (rsv), epstein-barr virus (ebv), herpes i and ii viruses (hvi and hvii). results: we detected the epithelial alveolar infection by mv and cmv in 30.8 % and 15.4 % in aip, respectively. endothelial cmv infection was observed in 25 % of iclf group. when we compared with the age of these patients, patients with ≤ of 43 years old had more infection by mv and cmv than the group with ≥ 72 years old. the other viruses were not detected in the different groups. conclusion: the viral infections observed in aip and iclf groups reinforce the possible viral participation in these pulmonary diseases. financial support: fapesp. abnormal up regulation of cyclooxygenase-2 is observed in idiopathic pulmonary fibrosis e. parra * , f. lin, w. teodoro, a. p. velosa, v. l. capelozzi * fmusp, dept. of pathology, são paulo, brazil objective: several factors modulate fibroblast proliferation and collagen deposition in pulmonary fibrosis diseases, including cyclooxygenase (cox). the main of this study was observed the relationship between cox1 and cox2 in surgical lung biopsies from patients with idiopathic pulmonary fibrosis (ipf). method: twenty four patients (64±8.9 year) were characterized with ipf. immunohistochemistry and histomorphometry were used to evaluate the amount of cox1 and cox2 expression in lung specimens. the expression of these markers was tested with their pulmonary function tests. results: significantly a higher amount of cox-2 was observed in ipf patients principally in normal and vascular areas (p=0.05) when compared with control group (figure) , contrasting with similar amounts of cox-1 in both groups. an important negative correlation was observed between total lung cox-2 expression and dco/va (r=0.694, p=0.01) in ipf patients. conclusion: higher vascular expressions of cox2 probably mediated the inflammatory reaction in patients with ipf and have an important impact with pulmonary function tests, suggesting that a participation in the pathway of ipf. financial support: fapesp. error barr shows the correlations between cox-2 expression in normal and vascular areas of ipf compared with control group ps-01-026 multiplex ligation probe-dependent amplification (mlpa) as an ancillary method for the diagnosis of malignant pleural effusion e. parra * , d. rosolen, l. kulikowski, r. dutra, v. l. capelozzi, f. vargas, m. acencio, l. antonangelo * fmusp, dept. of pathology, são paulo, brazil objective: a definitive diagnosis provided by the finding of malignant cells in pleural fluid (pf) can be established in around 50 % of patients with pleural malignancy. however, underdiagnosis risk in cytological suspicious cases is high, which makes the cytological diagnosis quite limited. this is an important clinical problem, especially if we consider that some patients, in bad clinical conditions, can not be submitted to a guided thoracoscopic biopsy. method: using multiplex ligation probe-dependent amplification (p315-mrc-holland) we have studied sequence variations of egfr gene and amplifications/deletions of chromosomal regions frequently associated to tumors (atg4b, pahs, pros, nsd1, and cdgif genes). results: forty-three malignant pf samples from patients with different cancers were evaluated, even in those cases with scarce pellet cells. four benign pleural effusions were used as control. gene sequence changes were observed in 13 (30.2 %) cases, while others copy number abnormalities were found in 19 (44.2 %). conclusion: the findings suggest that mlpa could be considered an alternative tool to detect molecular genetic changes in malignant pleural effusions, since this technique is relatively low expensive and not time consuming. our next challenge is to find the best combination of probes capable to recognize malignant cells of any origin in fresh samples of pf. multiple primary pulmonary myopericytoma: case presentation n. petre * , f. pop, d. leonte, s. bedereag * medcenter bucharest, dept. of pathology, romania objective: myopericytoma is described as a perivascular myoid tumor which usually develops as a solitary mass in the subcutaneous tissues of the extremities, in adults. method: the chest radiography of a 52-year-old woman showed multiple, bilateral lung nodules. after the surgical excision of one of them, the histopathologic examination revealed a well circumscribed nodule, composed of fusiform or oval cells, with eosinophilic cytoplasm and ovoid nuclei, without atypia, in a concentric pattern, intimately associated with thin-walled vessels, within a myxoid stroma. results: the spindle cells were diffusely positive for vimentin, smooth muscle actin (sma) and occasionally for desmin. cd31 and cd34 were positive only in endothelial cells. ki 67 was positive in less than 4 % of neoplastic cells. conclusion: the histological and immuno-histochemical findings led to the diagnosis of multiple primary pulmonary myopericytoma. ps-01-028 five cases of lung pneumocytoma: clinico-pathological, immunohistochemical and ultrastructural study i. rodriguez * , a. panizo, b. larrinaga, i. amat * c. hospitalario de navarra, dept. of pathology, pamplona, spain objective: lung pneumocytoma (lp), also called sclerosing hemangioma, is an uncommon tumor of uncertain histogenesis. method: we analyzed the clinical, morphological, immunohistochemical and ultrastructural features of 5 lp. results: all 5 patients were women, with median age of 48 year (range: 29-73). mean size was 26 mm (range 15-45 mm). all tumors were located within the right lung (upperlobe: 1; middle-lobe: 2; lower-lobe: 2). all cases showed solid, papillary, sclerotic, and hemorrhagic patterns. the tumors were composed of 2 cells types: pale polygonal and surface cells. ema, ttf1, and pr were observed in both type cells. the surface cells showed also positivity for ck7, napsin-a, and surfactant-a, whereas the polygonal cells for vimentin. surface cells had short microvilli and lamellar bodies in their cytoplasm. polygonal cells contained abundant microfilaments and rough endoplasmic reticulum. all patients were alive and well without recurrence at the last follow up. conclusion: lp is likely to be an epithelial tumor with a differentiation toward type ii pneumocytes, which exhibits various architectural patterns. the ihc profile provides useful clues for the diagnosis of this lung neoplasm when typical features are absent. all our cases had an excellent prognosis with no evidence of recurrence following surgery. rare tracheobroncheal lesions diagnosed by small bronchoscopic biopsies a. sasmaz * , s. yuksel, h. nalbant, p. celepli, e. kadan, s. percinel, s. dizbay sak * ankara, turkey objective: six different, rarely seen tracheobronchial lesions, taken by bronchoscopy as small fragments were discussed with an emphasis on morphological findings and differential diagnosis. results: six patients were men and two patients were women with an age range of 50 to 70 years. most of the lesions were symptomatic and discovered as small masses by bronchoscopy. the tracheobronchial lesions varied from polypoid, nodular, well-demarcated, fragile masses to nodular thickenings. microscopically, a polyp composed of a fibrovascular stroma with scattered mononuclear inflammatory cells was compatible with fibroepithelial polyp. mature adipocyte proliferation in two cases, s-100 and cd68 positive polygonal or ovoid cells with abundant eosinophilic, granular cytoplasm having small, hyperchromatic nuclei, smooth muscle actin and desmin positive spindle cells in interlacing fascicles within a myxoid and collagenous stroma with mononuclear inflammatory cells, cd34 and cd31 positive thin-walled vascular channels, filled with red blood cells and s-100 positive spindle cells having palisading of wavy nuclei in two cases, located in the submucosa were main findings of lipoma, granular cell tumor, inflammatory myofibroblastic tumor, cavernous hemangioma and schwannoma, respectively. conclusion: tracheobronchial biopsies, even with small fragments of lesions can allow to render rare specific histopathological diagnoses, some of which need to be confirmed by immunohistochemistry. objective: small cell lung carcinomas (sclc) are aggressive neoplasms that give very early distant metastases. matrix metalloproteinases (mmps) are a family of endopeptidases which degrade extracellular matrix and modulate cell adhesion, playing a crucial role in cancer cell invasion and metastasis. the aim of the present study is to investigate the immunohistochemical expression of mmp-2, mmp-9 (type iv collagenases) and mmp-1 (interstitial collagenase) in patients with sclc. method: formalin-fixed, paraffin-embedded lung biopsy specimens from 40 patients with sclc (m:f=29:11, median age=66.7) were immunostained for mmp-1, mmp-2 and mmp-9 (anti-mmp-1 polyclonal antibody, anti-mmp-2 72 kda collagenase iv antibody, anti-mmp-9 92 kda collagenase iv antibody, spring bioscience). results: immunohistochemical expression of mmps was detected in the cytoplasm of neoplastic cells. immunoreactivity was designated positive when >10 % of neoplastic cells were stained. overall, mmps were expressed in the majority of sclc. specifically, mmp-1 was positive in 90 % (36/40), mmp-2 in 87.5 % (35/40) and mmp-9 in 82.5 % (33/40) of sclc. non-neoplastic lung parenchyma did not stain for mmps. conclusion: the majority of sclc express immunohistochemically mmp-2, mmp-9 and mmp-1. given the role of mmps in cancer progression and metastasis, their expression in sclc may contribute to their aggressive course. objective: number of pulmonary adenocarcinoma (adc) patients incresease. treatment of non-small cell pulmonary carcinoma depends of its histological type in era of target molecular therapy particulary of adc: diagnosis of adc is established on small-sized pulmonary biopsies obtained on bronchoscopy and by transthoracic needle pulmonary biopsy. the aim of this study was to evaluate optimal panel of monoclonal antibodies in diagnosis of pulmonary adc on small-sized biopsy pulmonary samples. method: analysis of 50 small-sized biopsy pulmonary samples. diagnosis of adc was established on hematoxillyn-eosin stained (h&e) samples and confirmed immunohistochemicaly by thyreoid transcription factor-1 (ttf-1), napsin-a, surfactant b and cytokeratin7 (ck7). descriptive statistical method (%) was used. results: ttf-1 specificity was 86 %(43/50), napsin-a-82 %(41/50), surfactantb -56 %(28/50) and ck7-90 %(45/50) in adcs. two monoclonal antibodies were positive in 24 % (12/50) adcs, one of them was necessary ttf-1 or napsin-a. three monoclonal antibodies were positive in 40 %(20/50) and 4 in 36 %(18/50) adcs, respectively. there is not statistical significans in number of monoclonal antibodies for diagnosis of adc. conclusion: no one monoclonalantibody is specific for one histological type of carcinoma and its origin. ttf-1, napsin-a, surfactantb and ck7 belong in optimal panel for diagnosis of pulmonary adenocarcinoma. ps-01-032 expression of cd44, e-cadherin and bcl-2 in lung cancer i. strumfa * , a. abolins, j. gekis, k. pavlovs, a. vanags, j. grusina-ujumaza, g. volanska, j. gardovskis * riga stradins university, dept. of pathology, latvia objective: lung cancer represents major problem in oncology as the incidence and mortality is high (parkin et al., 2005) . molecular studies could reveal additional targets for intervention or prognostic evaluation. however, the published data are controversial (renouf et al.,2009; leung et al.,2010; maraz et al.,2011; ko et al.,2011) . method: consecutive lung cancer cases (278) were retrieved by retrospective archive search. the diagnostics has been performed systematically in accordance with who classification (travis et al.,2000) . the expression of cd44, e-cadherin and bcl-2 was analysed by immunohistochemistry and evaluated semiquantitatively. results: the intensity of bcl-2 expression was highest in small cell cancer, reaching the mean value 2.25. no or very low bcl-2 expression was found in adenocarcinoma, carcinoid and squamous cell cancer. the membranous expression of e-cadherin showed significantly higher mean intensity in adenocarcinoma (1.93) and carcinoid (1.90) than in small cell cancer (0.14) and squamous cell cancer (0.85). cytoplasmic e-cadherin expression was observed in small cell cancer. cd44 expression was widespread and frequent in non-small cell lung cancer. conclusion: the main histological types of lung cancer show different immunophenotype. bcl-2 expression is found in small cell cancer. the expression of e-cadherin is characteristic in adenocarcinoma and carcinoid. cd44 is expressed in non-small cell lung cancer. localized nodular pulmonary and thoracic cage amyloidosis i. strumfa * , a. abolins, j. gekis, k. pavlovs, a. vanags, b. strumfs, g. volanska, j. gardovskis * riga stradins university, dept. of pathology, latvia objective: localised amyloidosis represents a rare differential diagnosis of neoplasms. however, in some cases amyloid deposits are manifestation of tumour itself. to increase awareness about these uncommon but important differential diagnostic issues, we show here three well-characterised cases of thoracic cage or pulmonary localised nodular amyloidosis. method: the cases were identified by systematic retrospective archive search, 2000-2011. congo red stain, polarisation microscopy and immunohistochemical analysis was applied. results: there were 2 males and 1 female among the patients diagnosed with thoracic cage or pulmonary amyloidosis. the patients were 67-84 years old and had no history of previous malignancy. clinically, either lung cancer or sternal osteochondroma was suspected. radiologically, 1-2 nodules measuring 1.5-9.0 cm were found in lungs (2 cases, one of these being bifocal) or sternum (1). surgical treatment was applied in all cases. amyloid deposits were identified by congo red stain and polarisation microscopy. al type of amyloid was confirmed. in case of bone lesion, the plasma cells were monoclonal justifying the diagnosis of multiple myeloma. conclusion: localised amyloidosis can involve lung or sternum as tumour-like mass. the lung lesions can be single or multiple. association with haematologic neoplasm is likely if bone is affected. clonality analysis of plasma cells surrounding amyloid deposits is mandatory. pleural localization of castleman disease: a rare entity e. tastekin * , f. oz puyan, e. isler, u. usta * trakya university, dept. of pathology, edirne, turkey objective: castleman disease (cd) comprises a heterogeneous group of disease with various clinical presentations and prognosis. by definition; cd is a lymphoproliferative disorder of the mediastinal lymph nodes and extranodal tissues with follicular and interfollicular abnormalities. pleural localization is an unusual presentation of cd. method: a 27-year-old man applied to the hospital because of cough. chest x-ray and ct images showed an irregular, 35×56×67 mm sized intrapleural-extrapulmoner mass in the paramediastinal region. surgical specimen was consisting of a 7×5×3.5 cm sized, irregular-round shaped, gray colored calcified mass. microscopically, a lymphoid proliferation with distorted follicles and increased dendritic cells (with cd21) was detected. lymphocyte depleted germinal centers resembling "onion skin" appearance with penetrated sclerosing hyalinized blood vessels were also seen. interfollicular region was composed of proliferating blood vessels lined by plump endothelial cells and few lymphoplasmositer infiltration. there was heterogeneous positivity with cd3 and cd20. expanded mantle zones showed diffuse bcl2 positivity. ki67 proliferation index was low. the mass was diagnosed as "castleman disease, hyaline vascular type". typical appearance of the follicular and interfollicular abnormalities allowed definitive differentiation from mesothelioma and solitary fibrous tumor of the pleura. conclusion: pleural cd is a rare lesion but should be considered especially on young patients in the differential diagnosis of pleural masses. objective: to evaluate foxp3 expression in bronchus-associated lymphoid tissue (balt) and correlate with the inflammatory process and collagen content in the lung tissue in an experimental model of scleroderma (ssc) after type v collagen (col v)-induced nasal tolerance. method: female new zealand rabbits (n=12) were immunized with 1 mg/ml of col v in freund's adjuvant (im). after 150 days, six immunized animals were nasally tolerated with col v (25 νg/day), during 60 days (im-tol). animals (n = 6) only tolerated served as control (ct). foxp3 expression in balt and inflammatory cells in pulmonary interstitium were evaluated by point counting method. types i, iii and v collagen gene expression were evaluated by real-time pcr. results: im-tol when compared to im presented decreased lymphocytes, macrophages and monocytes and types i (p=0,002) and v (p=0,009) collagen mrna expression in pulmonary tissue. t lymphocytes foxp3 were expressed in 100 % of im-tol and 33,3 % of ct (p= 0,03). additionally, balt was higher expressed in im-tol in relation to ct. conclusion: col v-induced nasal tolerance in ssc model induces foxp3 regulatory t cells in balt which can trigger an immune regulatory mechanism resulting in decreased inflammation and collagen expression. it suggests that col v tolerance could be a promising therapeutic for human scleroderma treatment. igg4-related lung disease t. tichy * , h. hornychova, j. jankova, j. skarda, b. krajsova * university hospital olomouc, inst. of pathology, czech republic objective: igg4 -related lung disease is one of the manifestations of igg4-related systemic disease. obliterative vasculitis is considered an organ specific feature of igg4-related disease in the lung. we report a case of a 78-year-old man with multifocal subpleural consolidations in both lungs. histologic examination revealed igg4-related lung disease with obliterative lymphoplasmacytic vasculitis. method: histological sections from paraffin blocks were used for hematoxylin-eosin and special stains (elastica-masson trichrome, pas, geimsa, grocott), for immunohistochemistry (antibodies against kappa and lambda light chains, cd20, cd3, igg, igm, iga, igg4, lmp1) as well as for eber in situ hybridisation and pcr. results: the lung tissue was irregulary fibrotic with dense lymfoplasmacytic infiltration concentrated in the walls of pulmonary arteries. some arteries were obliterated by intimal fibrosis. infiltrating lymphocytes were small and without atypias. the majority of lymphocytes were cd3 positive, nearly all plasmocytes showed igg positivity. igg4/igg ratio was 60 %. eber in situ hybridisation was negative and monoclonal reaarangement of t cell receptor or immunoglobulin heavy chain genes were not detected. conclusion: igg4-related lung disease is rare. obliterative vasculitis and high percentage of igg4 positive plasmocytes are main histological features of this entity. objective: primary lung carcinoma with heterotopic osteocartilaginous formation is exceedingly rare. to date only 38 cases have been reported in the literature and the pathogenesis is not fully investigated. we report a case of pulmonary adenocarcinoma with osteocartilaginous formation with immunohistochemical staining of bone morphogenetic proteins (bmps). method: a 59-year-old man was admitted to our hospital for a lung tumor in his left upper lobe. an open lung biopsy followed a lobectomy was done. the resected tissue was fixed with 10 % formalin and embedded in paraffin. sections, cut from paraffin blocks, were subjected to hematoxylin and eosin (he) staining and masson-goldner staining, in addition to immunohistochemical staining. results: the tumor was poorly differentiated adenocarcinoma with heterotopic ossification. some of the tumor cells were positive for bmp-2, 6, and 7. some of mesenchymal cells in tumor interstitium were positive for osteoblast (ob)-cadherin or bmp receptor-ia. conclusion: we report a case of primary lung adenocarcinoma with heterotopic osteocartilaginous formation and reviewed previously published reports. to our best knowledge, this was the first case that not only bmp was expressed in tumor cells but also ob-cadherin or bmp receptor in some mesenchymal cells of tumor interstitium. expression of transforming growth factor ß1 and e-cadherin in lung adenocarcinoma j. yoo * , s. y. park * st. vincent's hospital, pathology, suwon, republic of korea objective: there is evidence supporting the concept of tumor progression from pulmonary adenocarcinoma in situ (formerly bronchioloalveolar carcinoma, bac) to adenocarcinoma with varying degrees of invasion. the aim of this study was to investigate the role of tgfß1in tumor invasiveness in lung adenocarcinoma, and to determine the potential relationships between its expression and immunophenotypes of cell adhesion molecules. method: tumor samples (n=40) from adenocarcinoma in situ (n=13), minimally invasive adenocarcinoma (formerly bac with ≤5 mm invasion, n=2), and lepidic predominant invasive adenocarcinoma (formerly mixed adenocarcinoma showing non-mucinous bac features with >5 mm invasion, n=25) were examined for the expression of tgfß1, e-cadherin, n-cadherin, and h-cadherin proteins using immunohistochemistry. results: twenty-five tumors (63 %) were positive for tgfß1. the frequency of immunoreactivity in patients with adenocarcinoma in situ, minimally invasive adenocarcinoma, and lepidic predominant invasive adenocarcinoma was 23 % (3/13), 50 % (1/2), and 84 % (21/25), respectively (p=0.001). loss of e-cadherin expression was more frequently observed in invasive adenocarcinomas than in adenocarcinomas in situ (p=0.034). tgfß1 expression showed a statistically significant correlation with h-cadherin expression (p=0.040), but not with e-cadherin expression (p=0.752). conclusion: these results suggest that tgfß1 and e-cadherin may play an important role in invasive progression of lung adenocarcinoma through regulating epithelial-to-mesenchymal transition. ps-01-040 lin28a expression as prognostic indicator in lung adenocarcinomas v. zolota * , v. tzelepi, g. psiouri, i. lilis, n. panagopoulos, c. sirinian, c. scopa * medical school of patras, dept. of pathology, greece objective: lin28a is implicated in stem cell pluripotency by blocking let-7mirnas and oct-4 is a transcription factor highly expressed in embryonic stem cells. the present study investigates the prognostic significance of both markers in lung adenocarcinomas. method: we evaluated, by immunohistochemistry, lin28a and oct-4a expression in formalin-fixed, paraffin-embedded tissues from 92 lung adenocarcinomas. immunoreactivity was scored as 1+, <1 % positive tumor cells (ptc); 2+, 1-5 % ptc; 3+, 5-10 % ptc; and 4+, >10 % ptc and was compared with clinicopathologic features and overall survival. results: regarding lin28a expression 23 % of tumors were recorded as 1+, 51 % as 2+, 14 % as 3+ and 12 % as 4+. the stratification for oct-4 expression was 19 % (1+), 50 % (2+), 23 % (3+) and 8 % (4+). lin28a and oct-4 expression was not significantly associated with age, gender and tumor grade or stage. lin28a 4+ expression predicted a shorter overall survival (p<0.05) on univariate analysis. oct-4 was not associated with patients' prognosis. cox multivariate analysis showed that age, tnm and lin28a 4+ expression were independent prognostic factors of survival. conclusion: lin28α expression appears to be an independent predictor of poor outcome in lung adenocarcinomas. further studies are warranted in order to investigate its role in stratifying patients at increased risk for poor outcome. comparison of the mutation status of egfr and kras on pulmonary adenocarcinoma and corresponding brain metastasis objective: egfr and kras mutation statuses have been known associated with the sensitivity of tyrosine kinase inhibitor treatment in pulmonary adenocarcinoma. however, the mutation analyses were usually performed on the primary tumors only, due to the availability of the tumor tissue. method: to compare the kras and egfr mutation statuses between pulmonary adenocarcinomas and corresponding metastases, we performed direct sequencing, followed by scorpion arms method on wild-type cases for egfr analysis, and allele-specific real-time pcr for kras analysis, on the paired samples of pulmonary adenocarcinoma. results: thirty-one (63.3 %) pulmonary adenocarcinomas and 30 (61.2 %) brain metastases out of the 49 paired specimens have egfr mutations, and 30 (61.2 %) paired specimens were concordant for the egfr mutation status between the primary and metastasis. in addition, 17 (51.5 %) pulmonary adenocarcinomas and 17 (51.5 %) brain metastases out of the 33 paired specimens have kras mutations, and only 16 (48.5 %) paired specimens were concordant between the primary and the metastasis. conclusion: the status of egfr mutation is relatively consistent between primary and metastasis comparing to that of kras mutation in pulmonary adenocarcinomas. however, discordance for the mutation statuses does happen. accordingly, repeat analysis is recommended if tissue from metastatsis or recurrence is available. objective: we tried to measure and analyze characteristics of neuroendocrine tumors in lung by image analysis and help to diagnose them. method: it was analysed that sixteen cases of typical carcinoid tumors, five cases of atypical carcinoid tumors, fifteen of small cell carcinomas, fifty one cases of large cell neuroendocrine carcinomas. we analyzed the nuclear area, perimeter, major axis and minor axis using i-solution image analyzer software package. results: the mean nuclear area was 488.00 μm2 in the typical carcinoid tumors, 499.30 μm2 in the atypical carcinoid tumors, 481.48 μm2 in the small cell carcinomas, and 684.05 μm2 in the large cell neuroendocrine carcinomas. after the statistical results, every method was effective to distinguish large cell neuroendocrine carcinoma from other tumors and the circumferences of nucleus was the most effective to distinguish among them. conclusion: pulmonary neuroendocrine tumors were the nuclear morphologic differences of each tumors. therefore, diagnosis that considers morphologic differences of pulmonary neuroendocrine tumors contributes to increase reproducibility and accuracy. sunday, 9 september 2012, 09.30 -10.30 an abundant growth of lactobacilli may result in lysis of vaginal epithelial cells, named as cytolytic vaginosis. this cytolytic process may cause the symptoms as seen in candidiasis. we observed pap smears to evaluate if cytolysis has a relationship with infertility. method: in the pathology department of mardin maternity hospital, 2011-2012 period, we examined pap smear cases suffering from the similar syptoms as candidiasis. of the 4672 smears, 82 were diagnosed as "cytolytic vaginosis". no growth was observed in cultures. results: the number of the cases suffering from the infertility was 261 (%5.58) among 4672 cases. in the cytolytic vaginosis group this ratio was %32.9 (n=27). the ratio of the infertil cases of cytolytic vaginosis group over general population was significantly higher (p<0.05). conclusion: our results are in favour of supporting the hypothesis of the relation between cytolytic vaginosis and infertility. lactobacilli are thought to have inhibitive role in fertility by changing the vaginal ph and adhering to the epithelial cells so inhibiting the sperm penetration. penicillium and aspergillus spp. on pap smears from the surprising origin i. i. akgun * , b. a. borsa * mardin maternity hospital, dept. of pathology, turkey objective: fungal organisms are commonly seen on smears. some unusual species as aspergillus and penicillium are so rare and generally seen via contamination. in this study, we investigated the origin of the many extraordinary fungal organisms seen on pap smears in series in only a few months. method: in pathology department of mardin maternity hospital, we observed both smear and vaginal discharge materials, came from the same hospital but different clinicians, for 3 months. results: 149 smears came from clinician a and 335 smears from clinician b. curious fungal organisms with huge branching hyphas and macrochonidias were seen in 15 smears among 149 smears (2 aspergillus and 4 penicillium species were recognized morphologically). no growth was observed in cultures. interestingly, among all 335 smears came from clinician b, there was no abnormal funguses. the smear samples with unusual fungal components all came from the same gynecologist. this made us strongly consider the probability of contamination. conclusion: penicillium and aspergillus spp. are extremely rare in vaginal smears. in our study, these funguses are thought to be airborne passed from the thick layer of the mold spreading on the ceiling of the office. objective: nearly all cervical cancers are related to human papillomavirus (hpv) infection. hybrid capture 2 -hc2 (qiagen, hilden, germany) is the method to determine the presence of high risk hpv (hr hpv) in cytology samples with the best clinical sensitivity. method: in our study we compared the results of the hpv test in 742 thinprep® cervical samples using first hc2 and secondly cervista (hologic, madison). samples were divided in two groups for the statistical analysis. the first group were hsil cases confirmed by biopsy (n =65) and the second group cytology samples reported as negative, asc-us or lsil (without biopsy or biopsy, not hsil) (n=677). results: overall, concordance between the two techniques was 92 % (683/742). there were 23 cases hc2+ cervista−, and 36 cases hc2 −cervista+. the average hc2 viral load in discordant cases was 2.83. in our series the hc2 sensitivity for ≥ cin2 was 100 % (65/65) and the specificity was 85.4 % (578/677). cervista results were 98.5 % (64/65) and 83.3 % (564/677), respectively. in the hsil cases the coincidence was 98.5 % (64/65). conclusion: cervista results showed good clinical sensitivity and high concordance with hc2, with a slightly lower specificity. in conclusion, according to our data the determination of hpv with cervista is comparable with hc2. mammary analogue secretory carcinoma of salivary glands: 2 cases including cytology, histology, ihc, em, rt-pcr and fish p. farrajota * , e. tani, c. carvalho, j. wejde, g. elmberger * centro hospitalar do porto, dipt. do anatomia patologica, portugal objective: mammary analogue secretory carcinoma (masc) of the salivary glands was described in 2010 by skálová et al. in a series of 16 cases. we present 2 new molecularly confirmed cases with focus on detailed cytomorphological features and correlation with special studies. method: a 75 year old man and a 68 year old female presented with history of slow growing parotid and submucosal buccal tumors, respectively. two fine needle aspiration biopsies (fnab) were performed in each case. the surgical resection specimens were routinely processed and special studies applied on paraffin tissue section. the fnab smears were retrospectively studied. results: all smears showed similar features of abundant proteinaceous background, moderate cellularity including macrophages and irregular and variable sized groups of cells with slightly enlarged round hypercromatic nuclei and abundant bluish vacuolated cytoplasm frequently containing small reddish granulations. the first fnab reports confirmed salivary origin and the second ones alert for possible neoplastic nature, advising surgical resection. electron microscopy performed in the parotid case caracterized the granules as mucigen. subsquent pathologic evaluation and molecular confirmation was carried out. conclusion: masc is a low grade malignant tumor capable of simulating a benign salivary gland process so it is important to know its cytological characteristics. adenosquamous lung carcinoma: the importance of cyto-histologic correlation d. felizardo * , a. l. cunha, r. henrique, c. lobo * instituto de oncologia porto, dept. of pathology, coimbra, portugal objective: adenosquamous lung carcinomas (asc) are uncommon and aggressive tumors. identification of a squamous component within a nsclc is critical as it excludes anti-vegf therapy. likewise, recognition of an adenocarcinoma component is also relevant because it prompts the search for egfr mutations. since 70 % of lung cancers are diagnosed in small biopsies/cytological specimens, cytohistological correlation is crucial for correct diagnosis. thus, we aimed at determining the relevance of combining cytologic and histologic findings in initial diagnostic workup of asc. method: we searched for cases of asc diagnosed at our institution from march 2011 to april 2012. cytologic and histopathologic findings were evaluated and their accuracy for diagnosing asc was assessed. results: of 184 cases of lung cancer in biopsy, 11 (6 %) corresponded to probable asc and in 9 cases cytologic examination was also performed. in 7 cases, cytology was not conclusive, but in 2 cases it demonstrated cytomorphological features of probable asc. we emphasize one case in which only the squamous component was valued by histology, but cytology provided clues that prompted immunohistochemical analysis which led to the diagnosis of probable asc. conclusion: cyto-histological correlation augments the diagnostic accuracy in asc of the lung, emphasizing the complementarity of both procedures. the impact of the bethesda system for reporting thyroid cytopathology (tbsrtc): a retrospective study of 828 aspirates with emphasis on the prior "indeterminate" objective: the material of this study is consisted of selected fna biopsies performed in the last 10 mounths period and followed by the endocrinology department. method: the bethesda system of reporting thyroid cytopathology was used. slides were stained with papanicolaou preparations. results: totally 380 cases were examined, 288 of which were female and 92 were male. age of the patients varied between 5 and 88. 121 cases were diagnosed as nondiagnostic or nonsatisfactory. 211 cases diagnosed as consistent with a benign follicular nodule. only 4 cases were diagnosed as a consistent with lymphocytic thyroiditis in corelation with the clinical features. only 1 case diagnosed as a granulomatous thyroiditis. diagnosis as "atypia of undetermined significance or follıcular lesion of undetermined significance "was made only in 2 cases whereas "follıcular neoplasm or suspicious for a follıcular neoplasm" was diagnosed in 1 case. "suspicious for malignancy" diagnosed in 15 cases. 2 cases were diagnosed as malignant. conclusion: although diagnostic terminology and morphologic criteria for cytologic diagnosis of thyroid lesions are well established in the bethesda system, difficulty in categorizing the cases and underestimation or overdiagnosis are quıite frequent issues. to solve this problem, retrospective studies which include surgical and cytopathologic reports, to examine slides by at least two different pathologists in different times will be helpfull. metastatic pulmonary adenoid cystic carcinoma: report of a case diagnosed by fine-needle aspiration cytology p. karabagli * , s. ozbek * selcuklu medical faculty, dept. of pathology, konya, turkey objective: a rare case of adenoid cystic carcinoma of ceruminal gland with pulmonary metastasis presented with characteristic cytological and histological findings. method: the biopsy specimen of the patient, a 45 year-old man with painfull, growing mass in his external auditory canal was interpreted as an adenoid cystic carcinoma. he was treated surgically and received post-operative radiotherapy. after 8 years, a distant metastasis relapse was observed. computed tomography (ct) of the chest depicted multiple spiculated masses suggestive of metastases throughout both lung fields, the diameter of the largest measuring 2 cm. ct guided percutaneous transthoracic fine needle aspiration biopsy was performed from the largest lesion. results: cytological examination of the aspirates revealed large spherical hyaline globules representing basement membrane material surrounded by neoplastic cells. the cells were cohesive, closely packed, and had uniform round to oval hyperchromatic nuclei with scanty cytoplasm. these features were suggestive of an adenoid cystic carcinoma. conclusion: patients with adenoid cystic carcinoma could be frequently encountered with disease recurrence confined to the lung. fine-needle aspiration cytology provided a conclusive diagnosis of adenoid cystic carcinoma. objective: often some of a fine needle aspiration (fna) sample is left behind in the hub of the needle when the specimen is ejected on the slides ( figure 1a) . method: we have designed a device that collects this material. it consists of a plastic adaptor that has a core of polyvinyl alcohol foam protruding from its lumen at one end ( figure 1b) . the other end of the adaptor then fits on to the syringe (figure 1 c) . as the fna is being performed the sample material flows up the lumen of the needle's shaft and emerges into the hub of the needle where it is absorbed into the tip of the foam core ( figure 1d ). smears are made by ejected the specimen from the needle onto a slide by air pressure from an attached syringe. the air passes through the foam forcing some of the sample in the foam back down the needle but some remains behind. the device is then removed and placed in a formalin specimen pot. once fixed the core is pulled from the adaptor ( figure 1e ), processed and sectioned as for routine histology specimens ( objective: the presence of dcs has been studied histologically in ptc, but their presence and potential diagnostic value in thyroid fnas has not been evaluated. method: we assessed the presence of dcs in cytological samples of histologically confirmed ptcs (n=31) and benign thyroid nodules (btn) (n = 29) using cd1a. corresponding ptcs (n=11) and btn (n=10) from surgical excisions were stained with both cd1a and langerin. results: cd1a + dcs were identified in 30/31 ptcs on cytology (97 %). they were either isolated in the background or more typically closely associated with tumor cell clusters. the 3 ptc cases with the least dcs corresponded to the follicular variant on histology. in contrast, most btn (69 %) lacked cd1a + dcs. when dcs were present, they were primarily isolated in the background although 5/29 cases (17 %) contained rare dcs among tumor cells. tumor-infiltrating dcs and background dcs were both higher in ptcs than in btn, but only the former was statistically significant (p<0.0001 and p=0.1173 respectively). similar findings were found on histology where all ptcs contained cd1a + and langerin + dcs while only 2/10 btn (20 %) contained rare dcs. conclusion: dcs are present in fnas of ptc, typically among tumor cell clusters, while they were absent or rare in fnas of btn. thus dcs may be useful as an additional diagnostic marker for ptc. significance of p 16 immunostaining in postmenopausal women with atypical squamous cells a. repse fokter * , k. gornik kramberger, s. hutter čelik, s. sramek zatler * celje general hospital, dept. of pathology and cytology, slovenia objective: the evaluation of postmenopausal pap smears can often be challenging. degeneration associated with atrophic vaginitis, hyperchromatic crowded groups, parabasal cells with organophilic cytoplasm or variations in nuclear size may be falsely interpreted as squamous atypia or even more severe lesion. method: the study included 25 postmenopausal papanicolaou patients (26 smears) with the initial cytological diagnosis of asc-us or asc-h. the smears were decolorized and immunostaining for p16(ink4a) was applied. in 21 patients (22 smears) tissue biopsy and histological examination was performed and four asc-us cases had cytological follow up only. results: among cases with histological examination positive p16 reaction was found in 17 patients (18 smears). the histological diagnoses were: cin1 (3), cin 1-2 (2), cin2 (1 case), cin3 (5 patients/6 smears), ais (1 case) and invasive squamous carcinoma (2 patients). two p16 positive patients with three smears and initial negative histology had cin3 after 2 years. among p16 negative patients there were two with cin3 and two with normal histology. four asc-us patients without histological examination had normal cytological follow up. conclusion: p16 is a useful marker in detecting of clinically significant cervical lesions in postmenopausal women. endoscopic-ultrasound guided fine needle aspiration cytology of intraductal papillary-mucinous neoplasms of the pancreas: report of two cases and review of the literature e. tejerina gonzález * , a. lópez garcía, c. gonzález lois, f. fernández garcía, a. herreros de tejada, e. sanz * hu puerta de hierro-majadahonda, dept. de anatomía patológica, spain objective: intraductal papillary-mucinous neoplasms (ipmn) of the pancreas are mucin-producing tumors recently recognized as a distinct entity among other mucinous pancreatic tumors. endoscopic-ultrasound guided fine needle aspiration (eus-fna) is a sensitive technique which can be very useful in the accurate diagnosis of these neoplasms. method: we present a case of a 30-year-old female with recurrent episodes of acute pancreatitis and a 49-year-old male with abdominal pain. tc showed a 26 mm-cystic tumor at the uncinated process of the pancreatic head and cystic dilatation of the main pancreatic duct, respectively. in both cases eus-fna was performed. results: smears showed medium-to-large, cohesive groups and complex papillary clusters set in a clean, mucoid background, composed of cuboidal or columnar epithelial cells with abundant clear or mucinous citoplasms. a honeycomb arrangement was occasionally seen. although nuclei were predominantly medium-size and uniform, some groups showed some nuclear enlargement and pleomorphism with visible nucleoli. a diagnosis of "mucinous neoplasm with papillary pattern" was rendered in both cases, followed by partial pancreatectomy. the histologic examination was consistent with ipmn. conclusion: the cytologic features of intraductal mucinous papillary neoplasms of the pancreas, evaluated by eus-fna, seems to be quite characteristic of these tumors and allow to suggest a correct preoperative diagnosis. objective: the human igh locus at chromosome 14q32 is frequently involved in different translocations of non hodgkin lymphoma (nhl), and the detection of any breakage involving the igh locus should identify a b-cell nhl. the split signal igh fish-cish dna probe (ifcd) is a mixture of two fluorochrome-labeled dnas that binds the telomeric and the centromeric segments, on the igh breakpoint respectively. we tested the capability of the ifcd to detect igh translocations and diagnose b-cell nhl on cytological samples. method: fifty cytological specimens from lymphoproliferative processes were tested using the ifcd and the results compared to light chain assessment by flow cytometry (fc), igh status by pcr, and to clinical-histological data. results: ifcd analysis detected 29 positive, 15 negative and 6 inadequate cases; there were 29 true positive (66 %), 9 true negative (20 %), 6 false negative (14 %) and 0 false positive cases (0 %). comparing the sensitivity of the ifcd with fc and pcr, the highest sensitivity was obtained by fc followed by ifcd and pcr. conclusion: the ifcd is effective in detecting any translocation involving the igh locus; it can be employed on different samples from different b-cell nhl whereas it is not useful to classify the specific entities. sunday, 9 september 2012, 09.30 -10. objective: morphometric analysis of the glands and inflammatory cells proportions in colon mucosa may be crucial for distinction of subtle changes. the most important step is a recognition and proper segmentation of the separate glands, even branched, atrophic or distorted. method: new sequential algorithm has been proposed, based on mathematical morphology transformations, such as extended regional maxima, hit-or-miss, opening and closing operations. for lumen opened glands snake algorithm with closing has been applied to surrounding elements with sequential elimination of the closed contours under decreasing area criterion. proposed method has been used to evaluate 47 cases of diversion colitis to compare specimens sampled from functional part of colon and defunctioned distal one. results: the appropriate results were obtained for specimens with different section planes with efficiency 89 %. in comparison of the b to a was observed decreased participation of epithelium in the glands area measured in deep region (an average 57 % instead 68 %). in addition, the area of the gland was slightly reduced from 51 % to 46 % of the mucosa. conclusion: the proposed method can be useful to automatic morphometry analysis of the gland shape in colon mucosa. the recognition of the opened glands improves previously designed algorithms. objective: quantitative analysis of the immunohistochemical histological samples plays an important role in the diagnosis and prognosis of many cancers. the commercial and non-commercial software's to computerized image analysis of microscopic images based in several approaches. method: the advance segmentation scheme usually consists of preprocessing, nuclei extraction and classifier blocks. when the preprocessing step includes filtering and conversion to the most discriminative representation and the classifier can be realized by various tools, especially support vector machine as a state-of-art, nuclei extraction is the most crucial part of any algorithms. we compare single thresholding methods such as otsu, kurita, minimum error, entropy thresholding, with the sequential thresholding approaches combined with area criteria, local threshold value or extended regional maxima. all of them are accompanied by watershed method and filtration. results: for ki-67 stained meningioma specimens in the fields of view without overlapping nuclei the most advance methods are adequate. when increasing the cell number, only sequential thresholding with area criteria and extended regional maxima give acceptable results. the mean relative error is 2.6 ± 2.2 and 5.4 ± 6.4 % respectively. conclusion: the results confirm the efficiency of using sequential methods for segmentation of cell nuclei. for best results, use sequential thresholding with area criteria. ps-03-003 nhs improvements: enhanced it project for histopathology f. mayall * * musgrove park hospital, dept. of cellular pathology, taunton, united kingdom objective: many histopathology laboratories are using antiquated software that hobbles their performance. we developed open source filemaker pro based histopathology reporting software with innovations based on lean principles to enhance work flow. these include: -colour coded visual workflow control -"one-click" extrawork requests with order tracking -user defined template reporting -reporting of complex cases using benchmark profomas -exportable data-sets using open database connectivity -easy local customisation and enhancement by the user. results: the software was used by 15 staff to report more than 7000 histopathology specimens at two histopathology laboratories. many of these cases were complex cancer resection cases requiring key data capture. the histopathology staff using the software were surveyed on their experience of using the software. the key steps in lean it development: -start with a small idea -develop software with multiple plan, do, study, act (pdsa) cycles -ask users for more ideas for enhancements -recruit others into the project by allowing them to use the software and experience its benefits -standardise new reporting process by achieving agreement between users -design a project that requires minimal financial investment this open source software is available for download from www.freedp.org. sunday, 9 september 2012, 09.30 -10. objective: myxopapillary ependymomas (mpe) often occur at the filum terminale but occasionally occur outside the central nervous system (cns). common site for these extraneural tumors is sacrococcygeal area. method: a cocygeal mpe with radiologic, gross and microscopic features will be presented. a 39 year old female admitted neurosurgery department with painful coccygeal mass. magnetic resonance imaging revealed a coccygeal, well-demarcated, non-enhancing mass of 4×5 cm in diameter. the mass did not have any connection with the spinal cord. it was completely excised and grossly the cut surface of the encapsulated mass was soft. microscopically the tumor was comprimised of myxoid stroma with pseudopapillary structures around hyalinized vessels. the tumoral cells showed intracytoplasmic dot-like positivity with ema. there were no mitosis, and the ki67 labeling index was low. conclusion: extraspinal mpes are rare and the most common location is sacrococygeal area. these masses are often preoperatively misdiagnosed as pilonidal sinus. unlike the cns mpes, extraspinal mpes have a potential to metastasize even if they do not show any anaplastic morphological feature. long term follow-up is recommended as metastases can occur up to 20 years after initial presentation. objective: cerebellar liponeurocytoma (cl) is a rare neoplasm with neuronal, astrocytic and lipomatous differentiation arising in adults. the entity is considered as a grade ii neoplasm in the last who classification of central nervous system tumours. we show two typical cases of this entity stressing the spectrum of histological changes and its differential diagnosis. method: patient 1 is a 72-year-old woman with a history of headaches and instability. ct scans and mri showed a 5 cm in diameter relatively ill-defined, poorly enhanced, round tumour mass in the left hemisphere. patient 2 is a 56-yearold woman with a history of headaches and instability. mri showed a 6.5 cm in diameter well-defined triangular lesion in the right hemisphere. results: complete surgical resection was achieved in both cases. both tumours showed highly cellular neoplasms composed by round to polygonal cells with scant cytoplasm and round nuclei with fine chromatin pattern. mitoses were not seen. well differentiated lipomatous cells grouped in irregular nests were intermingled with the predominant neuronal component. conclusion: cerebellar liponeurocytomas are located in the cerebellar hemispheres and pursue a non aggressive clinical course, with local recurrences detected mainly after incomplete resections. the typical adipous tissue is the result of a lipidization process and not a true metaplasia. objective: giant cell tumor of bone is an uncommon neoplasm that accounts for about 5 % of all bone tumors. it usually involves long bones and is very rarely encountered in the skull where it mostly involves the sphenoid and temporal bones. it is a benign neoplasm but can be locally aggressive. method: we present a case of a 32 year old woman with history of multiple sclerosis whose routine ct scan revealed a mass with corrosive features and calcification situated at the petrous portion of her left temporal bone. a biopsy was sent to our laboratory. results: histopathologic examination revealed a neoplasm composed of sheets of oval mononuclear cells, with uniformly distributed nuclear chromatin, evenly intermixed with numerous giant cells that contain a variable number of nuclei with features similar to those of mononuclear cells. cytoplasm of either cell type was eosinophilic and cell borders were indistinct. mitoses were scarce and regular. foamy histiocytes, hemosiderin and microscopic nodules of cartilage formation were also noted. conclusion: although giant cell tumor is a benign neoplasm its' localization in this case posses a threat that should be managed surgically and by adjuvant radiotherapy if complete excision is unobtainable. leptin and leptin receptor expression in pituitary adenomas g. ayranci * , t. avsar, a. seker, t. kilic, s. bozkurt * marmara university, dept. of pathology, istanbul, turkey objective: leptin is a regulatory hormone which is mainly synthesized by adipocytes and regulates body fat mass. leptin also has regulatory function on anterior pituitary. in this study, we have investigated the expression levels of leptin (ob) and its receptor (ob-r) in various types of pituitary adenomas and normal anterior pituitaries. method: 50 pituitary adenoma cases between 2006 and 2011 were selected. immunohistochemistry for leptin (ob) was performed for each 10 cases of null cell, gh, acth, prolactin and fsh/lh secreting adenomas. western blot was performed for leptin receptor (ob-r) in 9 cases consisting of all five subtypes. results: immunohistochemical staining showed greater immunoreactivity for leptin in normal pituitaries compared to adenomas. except for acth-secreting pituitary adenomas, all other four subtypes showed widespread and intense staining for leptin (p<0.0001). null and fsh/lh-secreting subtypes were mainly showed cytoplasmic and nuclear staining. western blot analysis showed leptin receptor expression in all types of adenomas except null cell. objective: primary malignant brain tumors often present local invasive growth. the study aimed to estimate the expression of kai1 protein, mmp-2, cd44v6 and their correlation in gliomas of different grade of malignancy. method: expression of mmp-2, cd44v6 and kai1 was evaluated on 154 formalin fixed paraffin-embedded tissue blocks divided into: pilocytic astrocytoma (n=15), fibrillary astrocytoma (n=17), anaplastic astrocytomas (n= 27), anaplastic oligodendrogliomas (n=27), glioblastoma multiforme (n=54) and normal brain tissue (n=14) using immunohistrochemistry. results: mmp-2 and cd44v6 was observed frequently in gliomas with high grade of malignancy. kai1 immunoreactivity was mainly observed in specimens with low degree of malignancy. mmp-2 and cd44v6 expression was significantly increased when the degree of malignancy of the gliomas increased (p<0.05). whilst kai1 expression increased when the degree of gliomas malignancy decreased (p = 0.03). positive correlations between mmp-2 and cd44v6 (p=0.01) and inverse between kai1 and mmp-2 expression (p=0.02) were found in gliomas. these results indicate that association between mmp-2 and cd44v6 expression may increase dissemination of tumor cells. whereas, high expression of kai1 protein might suppress the function of mmp-2 in gliomas. pediatric glioblastoma: a case report a. barin * , s. ekici, c. altunkaya, s. yilmaz, g. s. yalcin, g. hatipoglu, m. caydere, h. ustun * ankara education hospital, dept. of pathology, turkey objective: glioblastoma is the most common malignant tumor of central nervous system in adults which characterized by malignant pleomorphic astrocytic cells with marked nuclear atypia, mitotic activity, necrosis and/or microvascular proliferation. glioblastoma may manifest at any age but usually affects adults especially older than 50 years. in this case, we present a pediatric glioblastoma case because it has seen rarely under 20 years and it is so important owing to its prognosis. method: an 11-year-old boy has had headache for 20 days. according to radiological imagings 23×20 mm in diameter infiltrating mass has seen in temporal lobe contains 15× 7 mm in diameter cystic component and contrast enhancement was noted. clinically grade 3 astrocytoma has thought primarily and he has been operated. results: in microscopic examination nuclear atypia, mitotic activity and microvascular proliferation has seen without necrosis. objective: gliosarcomas are biphasic neoplasms composed of a glioblastoma admixed to a sarcomatous component with different lines of differentiation. histogenesis of these tumors is still discussed. our objective is to specify pathological characteristics of this neoplasm its related to or not to glioblastoma. method: 3 cases of gliosarcomas diagnosed in our department of pathology. clinical, radiological, therapeutic and follow-up data were reviewed. histological features and immunohistochemical results were also established. results: 3 female patients with a median age of 46 years (ranging 30-69 years). intracranial hyperpressure and paralysis are main symptoms. the brain imaging showed frontal, fronto-parietal and intra ventricular expansive process, respectively. microscopically, a biphasic pattern, with anaplastic astrocytic cells alternated with malignant mesenchymal areas, demonstrating a fibrosarcoma pattern, in two cases and rhabdoïd pattern in one case. immunohistochemical stain confirmed the diagnosis of gliosarcoma in each case. patients were treated by surgical excision; one patient was lost on follow-up. one patient died by postoperative complications. conclusion: clinical, radiological and follow-up features of gliosarcomas share great similarities with glioblastomas. histopathological, histochemical and immunohistochemical studies are helpful in accurate diagnosis. cytogenetic and molecular data support a monoclonal origin for these tumors. objective: case of biphasic synovial sarcoma of nerve in a 59 year-old female located between flexor muscles and radial diaphysis and adherent to median nerve and ulnar artery is presented. method: tissue was fixed in formalin, embedded in paraffin and stained with haematoxylin and eosin. periodic acid-schiff stain without diastase pre-digestion was obtained. ventana antibodies were employed: ttf-1, er, ema, s100, ck 14, ck 7 and mnf 116. ss18-ssx fusion gene transcript was detected with conventional rt-pcr and qrt-pcr. results: macroscopically nodule had smooth circumscribed borders, grey-yellowish colour and measured 3,7×3×2,5 cm. histologically it was delimited by a dense sclero-hyaline capsule and constituted of numerous glands lined by one layer of cuboidal-columnar cells showing eosinophilic cytoplasm and round to ovoid nuclei with a single small nucleolus. they contained dense eosinophilic strongly pas and ema positive material. glands were immersed within tightly packed spindle cells. mitoses were scanty. necrosis was absent. ema strongly stained most of the glandular as well as scanty spindle cell elements. chromosomal reciprocal translocation t (x; 18) with positive signal for ss18-ssx1 transcript was seen. conclusion: synovial sarcoma of nerve is a rare condition that has to be distinguished from histologically similar lesions. rabdoid meningioma: presentation of a rare case i. dimitriadis * , a. heva, i. matzarakis, a. sekouli, p. sakellariou, l. sakkas * hospital of thessaloniki, dept. of pathology, greece objective: in 1998, keppes and later perry suggested the term rhabdoid meningioma which was adopted into the who classification (grade iii) in 2000. we present a rare case of a rabdoid meningioma and study the clinicopathological characteristics of the neoplasm. results: α 68-year-old woman was submitted in surgical excision of an endocranial tumour (d:3 cm) in our hospital. the histological examination revealed entirely rabdoid morphology. the neoplastic cells expressed the following immunohistochemical phenotype: s100+, vimentin+, ema+, gfap−, pr+, hmb45−, a1/ae3+(focally), ck8/18+(focally), sma−, ki-67 (<1)%. conclusion: rabdoid phenotype represents an indicator of malignant transformation regardless of the tumor's histogenesis. expression of ki-67 antigen correlates with recurrence even in meningiomas with minimal atypical features, which should prompt closer surveillance after excision. rabdoid meningiomas behave aggressively and have bad prognosis contrary to the classic meningioma. prognosis is influenced by it's complete excision and the expansion within the intracranial cavity. it has to be distinguished from glioma, melanoma and metastatic carcinoma. the patient after 1 year is in generally good health, and is followed up by oncologists. atypical extraventricular neurocytoma (case report) s. ersöz * , u. yazar, a. reis, k. kuzeyli * karadeniz technical university, pathology, trabzon, turkey objective: central neurocytomas are rare intraventricular tumors with neuronal differentiation, typically located in the ventricles while tumors located outside the ventricles designated as ''extraventricular neurocytomas'' (evn). neurocytomas are classified as atypical if they exhibit a mib-1 labeling index >2 % or atypical features, like focal necrosis, vascular proliferation, and increased mitotic activity. we here report a case of atypical evn of the left frontal lobe. method: a 60-year-old woman patient admitted to hospital for headache. ct scan showed a large partially calcified cystic mass. mr imaging showed circumscribed, solid mural nodule within the cystic mass. the patient underwent a left frontal craniotomy. specimens were fixed in 10 % formalin and stained with hematoxylin and eosin. for immunohistochemistry, sections were incubated with monoclonal antibodies to synaptophysin, neurofilament protein, map-2, nse and gfap. the proliferative index was assessed with mib-1. results: histopathological examination revealed a neuronal neoplasm composed of uniform cells in solid sheets. small necrosis areas and mitotic figures were evident. immunohistochemical examination revealed positive results for synaptophysin, neurofilament protein, map-2, and nse. gfap staining was negative. mib-1 index was calculated as 5 %. conclusion: in general, central and extraventricular neurocytomas have a good prognosis. atypical extraventricular neurocytomas are quite rare like this case with malignant histopathological properties. meningioma and schwannoma associated with neurofibromatosis type 2: a case report of rare genetic disorder o. e. gürer * , r. tuncer * akdeniz university, dept. of pathology, antalya, turkey objective: neurofibromatosis is a group of genetic disorders referred as phakomatoses. there are two main forms called nf1 and nf2 which have different incidence, molecular characteristics, and clinical exhibitio. both have higher risk for the development of certain rare malignant tumors that occur in the brain, nerves or spinal cord. method: here, we present a rare case in which multiple meningiomas and vestibular schwannomas diagnosed in the same operation. results: a 23 year old woman presented with hearing loss clinically. she underwent surgical treatment for tumoral lesions in two different localization in the same session. one of them resected from pontoserebellar location was diagnosed as a schwannoma and the other one located on dura was diagnosed as a meningioma respectively. conclusion: nf2 is far less common than nf1 and strict diagnostic criteria have been described each of them. since difficulty to detect genetic abnormalities and no single criteria is pathognomonic, clinical characteristics much more valuable for differentiation. knowledge of associated clinical features will help to correct diagnosis and to predict much more devastating clinical course for nf2. diagnostic confusion: central neurocytoma n. gursan * , e. demirci, m. calik, f. daloglu, c. gundogdu * ataturk university, medical faculty, erzurum, turkey objective: central neurocytoma is a rare intraventricular brain tumor that affects young adults and presents with increased intracranial pressure secondary to obstructive hydrocephalus. they are usually located in the supratentorial periventricular region. because of some clinical and radiological findings cns neurocytomas were confused with other intraventricular lesions. method: a 37-year-old male was admitted with chronic headache with 6 months duration on july 21, 2011, when there were no neurological deficits or physical abnormalities. computed tomographic (ct) scans showed 51×40 mm a mixed density mass with amorphous calcification in the right lateral ventricle, which was irregularly enhanced by contrast medium. light microscopy showed sheets of monotonously neoplastic cells with uniform round-to-oval nuclei and inconspicuous nucleoli. the cytoplasm was clear or eosinophilic with indistinct border. capillary networks were well developed and divided the tumor cells into groups. no nuclear pleomorphism or mitosis was seen. we present a case of intraventricular neurocytomas confirmed by immunohistochemical studies. conclusion: this rare tumor causing diagnostic confusion, discussed by the literature. a case of oligodendroglioma with neuronal differentiation t. hirose * , s. nobusawa, y. nakazato, a. sasaki * tokushima pref. center hospital, dept. of diagnostic pathology, japan objective: we report a case of oligodendroglioma showing marked neuronal differentiation. method: a 46-year-old female visited an emergency room because of an attack of convulsion. imaging analyses disclosed a calcified, 3.5×3 cm tumor in the right frontal lobe. results: the resected tumor was composed of a mixture of oligodendroglioma-like (olg) and gangliocytoma-like (gc) areas. in the former, fried-egg appearance and more cellular nodules were recognized. in the latter, there were numerous small neuron-like cells with basophilic cytoplasm. olg areas showed immunoreactivity for olig2, gfap and mutant idh1, while gc areas were positive for synaptophysin and neun. ki-67 labeling index was about 10 % in cellular nodules of olg areas. at least one allelic loss of 1p/19q was detected in 53.4/61.2 % cells in olg areas and 47.2/40.6 % in gc areas with fish analyses. furthermore, an identical mutation of idh1 (g395a, r132h) was demonstrated in both olg and gc areas. conclusion: genetic abnormalities including 1p/19q loss and idh1 mutation indicate that this case is an oligodendroglioma associated with prominent ganglion cell differentiation. oligodendrogliomas with neuronal differentiation like our case may suggest a close relationship between oligodendroglial progenitor cells and neuronal cells. gemistocytic astrocytoma with granulomatous inflammation o. ipci * , e. atik dogan, t. ozgur, h. gokce, n. yilmaz, a. yilmaz * mustafa kemal university, dept. of pathology, hatay, turkey objective: the coexistence of granulomatous inflammation and astrocytoma is extremely rare. hemostatic agents are used to control intraoperative bleeding in many surgical subspecialties, including neurosurgery. there are occasional reports of granulomatous reaction to biomaterials. method: we aimed to present a 35 year old female patient, who was operated because of the tumor at left frontoparietal region. tumor was histopathologically reported as oligoastocytoma. however, no granulomatous inflammation was detected beside the tumor. after 6 months, patient was reoperated because of tumor recurrence, abscess and inflammatory necrotic tissue beside the tumor. histopathologically, gemistocytic astrocytoma with granulomatous inflammation and abscess formation was reported. no micro-organism had grown in any kind of cultures. at our neurosurgery department surgicel is used after craniotomy as hemostatic agent. it's raw material is cellulose and absorbed in about 3 months. results: in the literature, germinoma accompanied by granulomatous inflammation have been reported. however, astrocytic tumors don't express such an association. the patient doesn't use any drugs forming granuloma reported in the literature. conclusion: we encountered that surgicel causing granulomatous inflammation in very few cases in the literature. the findings might be secondary to surgicel that used in the first operation. objective: astrocytic tumors show various molecular and genetic alterations in its development and progression. glioblastoma multiforme (gm) develops as either de novo or by progression from diffuse astrocytomas (da) or anaplastic astrocytomas (aa) by genetic alterations. method: loss of heterozygosity (loh) and microsatellite instability (msi) was investigated in 40 cases of astrocytic tumors (das [n=12], aas [n=15] and gms [n=13]) with 14 microsatellite markers and 5 microsatellite markers harboring p53 and pten, respectively. the patients' age ranged from 27 to 77 years (mean, 50 years). results: loh was statistically significant in aas and gms, compared to that of das (p=0.023). loh for 17p13 and loh-h group for 10q23 were statistically significant in aas and gms (p=0.045 and p=0.001). msi rate in das, aas and gms was detected in 16.7 %, 13.3 %, 23.1 %, but it showed no significant correlation with prognosis factors. msi and msi-h rates were correlated with younger (〈50 years) group (p= 0.040, p=0.011, respectively). conclusion: alterations on pten and p53 may contribute to the development and progression of astrocytic tumors. especially, loh-h for 10q23 and 17p13 is considered as clinical application of discriminating the aas and gms from das. msi might be involved in the tumorigenesis of relatively young patients. objective: charcot-marie-tooth disease (cmt) iii or dejerine-sottas syndrome is a severe hereditary demyelinating motor and sensory neuropathy presenting in infancy or early childhood with delayed motor milestone and extremely slow nerve conduction velocities. method: among 530 unrelated korean cmt patients, mutational screen for well known cmt genes reveals three patients with pmp22 de novo mutation showing dss. the histopathological findings of distal sural nerves are analyzed in all patients twice; cases 1 and 2 with 10 years interval and case 3 with 17 years. results: on semi-thin transverse sections, all the remaining axons (thinly myelinated or demyelinated) are surrounded by classic or basal lamina onion bulbs (obs). the first biopsy of case 1 (2 years-old) shows obs (4,452/mm2), which consists of 2,295 (51.55 %) mfs with obs and 2,157 (48.45 %) demyelinated axons with obs. the second biopsy shows obs (2,275/mm2), consisting of 425 (18.68 %) mfs with obs, 489 (21.49 %) demyelinated axons with obs, and 957 (42.07 %) obs with no discernible axons. cases 2 and 3 show similar histopathologic findings with main differences in numbers of obs. conclusion: although the differences noted in two biopsies are probably due to age of the patients, correlations with clinical symptoms will be helpul for management of the patients. objective: two mutations of the seipin gene (n88s and s90l) have been reported and known to cause neurodegenerative disorders. we performed whole exome sequencing in two charcot-marie-tooth families and identified three patients with two causative heterozygous mutations (n88s and s90w) and s90w is a novel mutation. method: the histopathological findings of distal sural nerves are analyzed in two patients (cases 1 and 2) with s90w novel mutation (fc305) and a patient (case 3) with n88s mutation (fc51). results: transverse semi-thin sections reveal increased numbers of myelinated fibers (mfs) (11,898/mm2; 11,429/ mm2; 9,430/mm2) with increased small mfs forming regenerative axon clusters and loss of large mfs. histogram shows unimodal distribution pattern. cases 1 and 2 show the similar ranges and average of diameter of mfs (1.461 3.24 μm, 5.13 μm). case 3 shows more numbers of small mfs (0.82~10.50 μm, 3.73 μm). ultra-structural examination reveals mfs with myelin abnormalities, pseudo-onion bulb formation including single axon or axon clusters, and thick mfs. conclusion: the findings are consistent with axonal neuropathy with features of demyelination. additional cases will be necessary to know the significance of differences between these cases. immunohistochemical subtyping of primary and secondary glioblastomas k.-s. lee * * seoul national university hospital, republic of korea objective: glioblastomas (gbls) may develop de novo (primary gbl; p-gbl) or through progression from lowgrade or anaplastic astrocytomas (secondary gbl; s-gbl). for subtyping of gbls, we tried immunohistochemical anaysis. method: we collected 150 cases of gbls in snubh and snuh. the mean age at the time of the primary surgery was 58.8 years (range 19-85). immunohistochemical studies were performed for egfr, p53 and idh-1. results: according to clinical history, gbls consisted of 146 primary and four secondary gbls. however, egfr(+)/ p53(−) immunohistochemical features of p-gbl consisted 41.3 % and egfr(−)/p53(+) immunohistochemical features of s-gbl consisted 28.6 % among 150 cases. egfr(+)/p53 (+) was noted in 20.6 % and egfr(−)/p53(−) in 9.3 %. immunohistochemical expression of idh-1 was noted in 14 cases (9.7 %) out of 144 cases. expression of idh-1 and egfr(−)/p53(+) showed positive correlations with young age. characteristic features of s-gbl were noted in 3.5 % of clinically p-gbl. conclusion: we analyzed immunohistochemical subtypes of korean glioblastomas. combination of egfr and p53 was unsatisfactory but combination of egfr, p53 and idh-1 can be a good tool for immunohistochemical subtyping of glioblastomas. (also known as erbb-2) is a 185-kd transmembrane glycoprotein with tyrosine kinase activity. her2 is expressed in some human malignancies and can be a potential target for therapeutic intervention with selective inhibitors. there are only a few studies on the relationship between meningioma and her2 expression, and the results are different as well. the aim of this study was to determine this relationship. method: seventy-two paraffin blocks of meningioma were selected randomly and immunohistochemical staining was then performed for each specimen. results: thirty-one of the 72 meningiomas were her2-positive. her2 expression was observed in 11 (55 %) of the 20 grade ii/iii, and 20 (38.5 %) of the 52 grade i meningiomas. conclusion: consequently, her2 expression was detected in 43 % of meningiomas. no significant difference was seen between grade i and ii/iii meningiomas, primary and recurrent tumors, and males and females from the point of view of her2 expression. objective: in our previous papers we studied accumulation of iron and proteoglycans in the brain. the aim of this analysis is to build on our previous results alongside other authors to investigate the reason for the presence of excessively accumulated iron observed in parkinson disease (pd). method: postmortem samples taken from the globus pallidus were prepared for light microscopy for iron and proteoglycans detection. patients had neither iron metabolism disorders nor had clinical signs of neurodegeneration. the samples were analysed using electron paramagnetic resonance (epr) to investigate the bounds between iron and proteoglycans. results: epr measurements confirm the presence of complex bounds between fe(iii) and proteoglycans. conclusion: previous data showed a possible dependance between accumulated iron, proteoglycans, ferrireductive alpha-synuclein and lewy bodies. we propose a mechanism of toxic iron accumulation in the brain in pd. this cyclic process of amplification is presented by onward steps. it is completed by our own observations into one cycle. the initiator of this vicious circle is probably an impirement of the equilibrium in the presence of useful amount of iron. understanding these relations could bring new insights to the neurodegenerative disease. role of pdgfr overexpression in schwannoma, and its diagnostic and therapeutic implications t. neuman * , y. fellig * hadassah medical center, dept. of pathology, jerusalem, israel objective: platelet-derived-growth-factor-receptors (pdgfr) are transmembrane tyrosine kinase receptors that function as relay points for signaling pathways. they play a key role in numerous processes that affect cell proliferation, tumor genesis, cancer invasion, metastasis, and modulation of apoptosis. recently, pdgfr has been demonstrated to be overexpressed in schwannoma in vivo, and in a small series of acoustic neuromas. however, its phenotypic expression in different variants of schwannoma remains largely unclear. method: immunohistochemical staining was used to detect pdgfr expression in archived formalin-fixed, paraffin-embedded schwannoma tissue samples (n=24 objective: pancreatic ductal carcinoma (pdc) has one of the worst prognoses. up to 90 % of pancreatic cancers are diagnosed at the locally advanced or metastatic stage. however, brain metastases from pancreatic carcinoma are extremely rare since less than 20 cases have been reported in the literature since 1978. method: we report a case of a brain metastasis from pancreatic adenocarcinoma occurred in a 66 years old women. results: 62-year-old woman developed severe neurological symptoms and progressively decreased consciousness. ct revealed cystic tumor in cerebral peduncle and medulla oblongata. stereotactic biopsy revealed metastasis of carcinoma. the patient died from of the twelfth day after stb because of recurrent hemorrhage. autopsy revealed well-differentiated papillary adenocarcinoma in the head of pancreas and with multiple metastases in liver and two metastases in medulla oblongata and cerebral peduncle. ihc staining for cytokeratins 7, 18 and 19, mucin 1 and 5 ac types was strongly positive. ki67 labeling index was 14 %. conclusion: we reported an autopsy case of brain metastases from pancreatic cancer, in which the patient initially developed symptoms of a neurologic disorder without exhibiting any symptoms of pancreatic disease. the use of bone morphogenetic proteins (bmp2) with nunostrucnure grey implant in reparation of bone tissue defect t. pavlova * , l. pavlova, a. nesterov, i. goncharov, d. kolesnicov * belgorod state university, dept. of pathology, russia objective: the matters of materials development of scull bones defect plastic are obviously necessary this time. the experience in use of nanocostructive titans, composites, bone morphogenetic proteins was accumulated. method: implant, made of nanoconstructed titan grey, covered by 1 layer (gelatin, dextran), 2 layers (1-gelatin, dextran, 2-hydroxyapatite, collagen, dextran) and by 2 layers with use of bone morphogenetic proteins bmp-2. the experiment was conducted on 240 rat-males. in order to study the regeneration processes it were used light, fluorescence, probe and scanning microscopy with the trace element analysis. results: the most active changes were registered in the experimental group with use of composite from titan grey with 2 layers of covering and bmp-2. the covering compound increase the rate of regeneration processes due to creation calcium and phosphorus ion depot and perform support function for again-formed tissue. increase in the concentration of calcium, phosphorus, sodium, magnesium in trabeculae of bones in comparison with matrix bone at term of 12 weeks testify about active regenerative processes; the mature smooth surface with structured relief of bone trabeculae and diploic veins were detected. conclusion: results can be used in neurosurgery, traumatology-orthopedics, dentistry, plastic and cosmetic surgery. alpha-synuclein (as) pathology of the peripheral autonomic nervous system (pans) in parkinson disease (pd) and other lewy body diseases (lbd) t. ribalta * , e. tolosa, j. navarro otano, e. gelpi * hospital clinic of barcelona, dept. of anatomic pathology, spain objective: pd and other lbd have been associated with as aggregates in the central nervous system (cns). however, autonomic dysfunctions may appear at any time in the course of the disease. our objective was to investigate the distribution of as in the pans in synucleinopathies. method: we examined pans structures obtained at post mortem from 28 subjects (19 female, age range 69-93, mean 81) at our brain bank with a diagnosis of pd (10); lewy body dementia (5); alzheimer's disease (9); mixed dementia or other (4). a complete neuropathological study of pans was performed, including dorsal spinal ganglia, vagus nerve, paraspinal sympathetic ganglia, mesentery, adrenals, digestive and genitourinary systems, heart, and skin. routine and immune stains for as and tyrosine-hydroxylase were applied. cases were semiquantitatively assessed. results: 71 % of cases showed as pathology in the cns. of them, as aggregates were additionally present in the pans in 80 %. a gradient in involvement was observed, being the paraspinal chain (particularly the stellate ganglion) the most constantly involved structure, followed by digestive system, adrenals, and gu system. conclusion: these findings indicate that as aggregates are extensively found in pans in synucleinopathies. the highest expression is found in the paraspinal ganglion chain and decreases in other pans regions. our results provide valuable information about potential development of new diagnostic and therapeutic strategies. objective: chordoid meningioma is an uncommon histopathological variant of meningioma. method: we report one case of chordoid meningioma occurring in adult patient. paraffin embedded tissue was stained with hematoxylin-eosin. we used immunohistochemical markers such as cea, ema, vimentin and tle3. results: a 81-year-old woman was hospitalized with clinic of an intracranial hemorrhage. axial computed tomography (ct) of the brain revealed a 3.1*5.2*4.9 cm lesion with perilesional edema in the left temporoparietal region. foci of intratumoral hemorrhage were also seen. compression of the left lateral ventricle and midline shift to right side was seen. the patient underwent a left temporoparietal craniotomy for resection of the tumor and died 3 days after surgery. histologically, sections revealed sheets, trabeculae and lobules of tumour cells scattered in a pale basophilic myxoid matrix. some of these cells exhibited characteristic cytoplasmic vacuolization. a typical focal meningiomatous pattern was also observed. the tumor cells were diffusely positive for epithelial membrane antigen, vimentin and a strong nuclear immunoreactivity for tle3. these cells showed negative immunostaining for cea. conclusion: chordoid meningioma should be distinguished from chordoma, chondrosarcoma, metastatic mucinous carcinoma and other variants of meningioma. morphology of non-specified encephalopathy in cases of polymerase chain reaction proved presence of human herpesvirus-6 s. roga * , i. strumfa, s. kuleznova, s. chapenko, s. rasa, m. murovska * riga stradins university, teaching department, latvia objective: the morphology of non-specified encephalopathy is a complex medical problem. human herpesvirus-6 (hhv-6) infection can be discussed as a predisposing factor. the aim of the present study was to investigate the presence of beta hhv-6 in non-specified adult encephalopathy cases. method: the blood, meninges and brain tissue were obtained in adult (aged 42-74 years) autopsies including 21 cases with encephalopathy and 23 cases in the control group. tissues were submitted for routine histology including haematoxylin-eosin stain. the presence of hhv-6 genome (dna) was analysed by nested polymerase chain reaction (npcr), hhv-6 variants by restriction endonuclease analysis. results: the gross and microscopic structure did not reveal any specific changes. in the encephalopathy group, hhv-6 dna sequence was found in meningeal tissues (16/21 cases; p=0.0036), in brain tissues (15/21 cases; p = 0.0007), and both in brain and meningeal tissues (10/21 cases; p=0.0174). in the control group, the viral dna was identified in meningeal tissues (7/23 cases), in brain tissues (4/23 cases), both in brain and meningeal tissues (3/23 cases). hhv-6b variant was detected in all cases. conclusion: on the basis of the present study it can be concluded that hhv-6 is a pathogenic factor that can predispose to encephalopathy. pathological variants of brain metastases of breast carcinoma and their prognostic and predictive role in different age groups d. objective: it is extremely rare that the intramedullary spinal cord metastases (iscm) are the first manifestation of cancer. method: in database of neurosurgery biopsies from 2002 to 2011 two cases of iscm were found without preoperative signs of primary neoplasm. surgically removed tissue was stained with hematoxylin-eosin and immunohistochemically using following markers: ae1/ ae3, ck7, ck20, mammaglobin, ttf-1, gfap, s-100 and vimentin. results: one patient was male aged 51 and another female aged 44. they presented with neck pain and rapidly progressive upper and lower limb weakness. imaging analysis showed contrast-enhancing mass at the level c4-c6 in male and c2-c4 in female, reported as an ependymoma. grossly, tumor tissue was of soft consistency and grey white color with yellowish foci of necrosis. histopathology revealed moderately differentiated adenocarcinoma with distinct border to surrounded glial tissue. in both cases, tumor cells were immunopositive for ae1/ae3, ck7 and ttf-1 indicating primary lung origin. postoperatively primary cancer was found in upper lobe of the left lung in both cases, without evidence of lymphadenopathy and other distant metastatic lesions in female. conclusion: diagnosis of iscm in both cases without preoperative signs of neoplasm in other organs was surprising. immunohistochemical analysis was essential for determination of cancer origin. objective: tetraspanin cd151 is a positive effector of cancer invasion and metastasis in many tumor types. method: we investigated the protein expression of cd151 in 211 cases of who grade i to iv gliomas. additionally, we performed o6-methylguanin-dna methyltransferase (mgmt) methylation analysis using real-time methylation-specific pcr in 36 glioblastomas, and the prognostic significance of these biomarkers in glioblastomas was evaluated. results: cd151 was overexpressed in a significant proportion (55.6 %) of glioblastomas, while it was not deetected in most of grade i to iii glial tumors except for rare overexpression in anaplastic astrocytoma (2/10, 20 %) and oligoastrocytomas (3/23, 13 %). cd151 overexpression was closely associated with mgmt methylation (p= 0.014), and it was a prognostic factor for predicting worse overall survival (os; p=0.002) and progressionfree survival (pfs; p=0.043). combination of cd151 overexpression and mgmt methylation better stratified the patients' os (p=0.001) and pfs (p=0.009). in multivariate analysis, cd151 overexpression was an independent prognostic factor for predicting os over mgmt methylation (p=0.012). conclusion: cd151 seems to have a critical role for high grade progression in astroglial tumors. cd151 is a good tissue marker for predicting worse prognosis in glioblastomas. results: a gross total resection of the tumor was achieved. histological examination revealed a paucicellular tumor with lobulated architecture and abundant myxoid stroma, containing stellate or spindle cells lacking mitotic activity. alcian blue and mucicarmin histochemical stains were diffusely and strongly positive. immunohistochemical analysis showed diffuse reactivity for vimentin and scattered cells positive for cd34. stains for ema, gfap, s100 protein, cytokeratin and smoothmuscle actin were negative. conclusion: primary intracranial myxoma should be distinguished from other myxoid intracranial tumors such as myxomatous meningioma, epithelioid hemangioendothelioma or sarcoma through appropriate pathological and inmunohistochemical analysis. a metastasic cardiac myxoma should also be ruled out through cardiac evaluation including an echocardiography. the distinction between this entity and the reported neurothekeoma of the meninges needs to be reevaluated. holocord pylocitic astrocytoma associated to syringomyelia j. trillo-tinoco * , t. garibay-huarte, e. gómez-apo, m. a. rodríguez-florido, l. chávez-macias, j. e. olvera-rabiela * hospital general de mexico, dept. of surgical pathology, mexico city, mexico objective: spinal intramedullary tumors sometimes extend both superiorly and inferiorly along almost the entire cord, and those diagnosed as pilocytic astrocytoma are rare. the syringomyelia is an ependimary or periependimary cavitation of the spinal cord and is considered a suffering of degenerative and irreversible type, 58 % of the cases is associated with intramedullars tumors. method: a three-year-old girl with history of headache, progressive weakness, 3 months before, she was hospitalized for pneumonia, had impairment of neurologic functions and died of cardiac arrest. the autopsy was performed. results: the neuropathologic study revealed a intramedullary holocord tumor with secondary syringobulbia, cervical and lumbar syringomyelia. histopathological examination of all specimen resulted in diagnosis of a pilocytic astrocytoma. although no signs of atypia were present, an elevated proliferative activity of endothelial vessels was noted. conclusion: gross total resection of holocord and longitudinally extensive intramedullary spinal cord tumors can be achieved with preservation of long-term neurological function and also solved the syrinx. objective: epithelial cellular adhesion molecule (ep-cam) has been studied in many tissues and neoplasm, including thyroid and thyroid tumors. this is a preliminary study to assess the immunohistochemical expression of ep-cam in thyroid tumors using ber-ep4. we examined the expression of ep-cam using the monoclonal antibody ber-ep4 in 36 cases of thyroid tumors, including 4 adenomas, 11 papillary carcinomas, 5 follicular carcinomas, 8 medullary carcinomas, 2 poorly differentiated carcinomas, and 6 anaplastic carcinomas. we assessed the positivity as a predominantly membranous staining of the cells, and was scored according to the estimated percentage tumor cells in the total tissue section (negative: 0-10 %; positive: ≥10 %). results: ber-ep4 expression was detected in normal thyroid tissue (perilessional), in all the adenomas, follicular carcinomas, and medullary carcinomas. papillary carcinomas showed in 36 % lacking areas of expression, coinciding histopathologically one case with poorly differentiated component. all of the poorly differentiated and the anaplastic carcinomas were negative. conclusion: the expression of ep-cam, using ber-ep4, was related to normal thyroid tissue and well differentiated neoplasms. our study suggest that lost of expression is associated to dedifferentiation. this results match up with the literature. in addition, clinical data and follow up are required to correlate focal areas of lost of expression of ber-ep4 with dedifferentiated areas of the tumor. the pathological effects of estradiol valerate on testis tissue: size and weight in male rat f. bidhendi * , r. ahmadi * imamkhomeini hospital, dept. of radiology, tehran, iran objective: studies show that estrogens can influence reproductive system differentiation. the main aim of this study was to determine pathological effects of estradiol valerate on testis histology and morphology in male rats. method: adult male albino wistar rats were divided to control and estradiol valerate (200 μg/kg/day) receiving groups. estradiol valerate was applied subcutaneously. after 4 weeks, testes were excised and studied morphologically and histologically. data were statistically analyzed and compared between groups using anova. results: our findings revealed that estradiol valerate injection resulted in reducing of testes weight and size (p<0.05). semeniferous tubules were apparently deformed in estradiol valerate receiving animals and cellular density was also reduced. number of spermatocytes, spermatids and sperms was decreased in estradiol valerate receving rats compared with control animals (p<0.001). conclusion: estradiol valerate has considerable pathological effects on testes morphology and histology in male rats. key words: estradiol, testes. are tumor-dimension, galectin-3 and cyclind3 useful to characterize oncocytic adenomas and carcinomas?: mutations in about 45 % of cases. the first clinical manifestation of this tumour is often represented by lymphatic metastases. method: twelve cases of ptc and respective lymph node metastases were retrieved from our archive from 2010 to present. formalin fixed paraffin embedded tissue sections were stained with routine hematoxilin-eosin and representative tissue areas for both tumour and metastases were microdissected and dna was extracted. after pcr amplification the mutational status of braf and ras was determined by dna sequencing. results: seven cases of twelve (58 %) showed a braf-v600e mutation. interestingly, only in three these cases (43 %) there was concordance in the mutational status between primary tumour and metastases. moreover, all metastase of wild type carcinomas were also wyld type. conclusion: even with an overall good prognosis, ptc is characterized by high incidence of lymph node metastases. as braf mutational status correlates well with prognosis and tumour progression, a correct molecular assessment is of paramount importance. our data may indicate that, because the possibility of discrepancy in the mutational status between primary tumour and metastases, molecular analysis should be performed on the primary tumour. objective: diabetes may cause chronic and non-healing diabetic foot ulcers (dfu) decreasing the welfare of patients. some neuropeptides, substance p and neurotensin (nt), may act as inflammatory modulators and improve wound healing. natural biopolymers, chitosan derivatives, are receiving great attention as powerful wound dressing materials for wound healing applications due to their favorable properties. the work aim was to use 5-methyl pyrrolidinone chitosan (mpc) as a platform for the delivery of nt. method: diabetes was induced by intraperitoneal injection of 200 mg/kg streptozotocin. mice were anesthetized and two 6 mm excision wounds were created dorsally. mpc alone, nt alone, mpc loaded with nt or pbs were placed daily on wounds. histological analysis of skin, at days 0, 3 and 10, was done through h&e and masson´s trichrome stainnings. results: in diabetic mice, the healing process was slower, showing an engulfment of acute inflammatory cells that triggered macrophage activity when compared with controls. at day 3, mpc treatment leaded to a faster healing with retraction of the wound site, nt induced a slower noncontracting healing and combined application delayed inflammatory repair with persistent neutrophils. conclusion: at day 10, all treatments induced a total healing however, mpc + nt reduced neutrophils infiltration compared with mpc alone. acth producing nasal paraganglioma: case report j. cassis * , a. galzerano, m. chorão * hospital egas moniz, dept. of anatomy pathology, lisboa, portugal objective: female patient with chronic sinusitis and hypertension presents with lipothymia, dizziness, polyuria and polydipsia. laboratory studies showed hyperglicemia, leucocytosis, hypokalemia, hypercortisoluria and elevated serum acth levels. head mri revealed tumor in the right nasal cavity with implantation at the cribiform plate of the ethmoid bone. method: microscopically the tumor had lobular architecture with big, round cells and with eosinophilic granular cytoplasm. these cells were nse+, synaptophysin+, cd56+, cd57+ and acth+. surrounding the tumor lobules were several spindle cells s-100+ (sustentacular cells). results: the lobular growth pattern, the presence of sustentacular cells (s-100 positive) , and the acth producing granules favours an acth producing nasal paraganglioma diagnosis. conclusion: head and neck paragangliomas account for 0,12 % of the tumors in this region, and are mainly located in the carotid bodies. paragangliomas arising in the nasal cavity are rare, only 23 cases reported so far. they affect the middle or upper turbinates, and also the etmoid, maxillary and sphenoidal sinuses. we report the third case in literature of a acth producing paraganglioma in the nasal cavity. sporadic aggressive silent somatotroph pituitary adenoma in the young: report of two cases l. chinezu * , a. vasiljevic, s. achard peyregne, g. raverot, e. jouanneau, j. trouillas * emergency county hospital, pathology, tg. mures, romania objective: silent pituitary adenomas are infrequent tumors in adults and exceptional in young. we describe two cases of 20-year-old girls with pituitary adenoma without genetic history, revealed by visual field defects, with panhypopituitarism in one patient. in both cases, mri showed giant pituitary adenoma, with invasion of the cavernous and sphenoidal sinuses. hormone assays revealed normal gh, prl and igf1 plasma values. surgery was incomplete in both cases. results: both tumors presented a diffuse pattern. the cells exhibited large nuclei with prominent nucleoli. by immunocytochemistry, focal and low percentages of gh-and prl-immunoreactive cells were observed respectively in both tumors (gh=1-30 % and prl=1-5 %). cga was positive. cytokeratin and others antibodies against pituitary hormones were negative. both tumors had high proliferative indexes: ki-67>3 % (4-10 %) and elevated mitotic number (1-13 mitoses). detection of p53 was also positive (0.5-5.7 %). the cases were diagnosed as atypical adenomas, according to the who classification. conclusion: the pathological diagnosis of these aggressive gh-prl tumors has to be taken into account by the clinician to choose the optimal therapeutic strategies. despite of the important side effects of the radiotherapy, this aggressive treatment might be proposed earlier, even for the young patients, to avoid the tumoral progression. intrauterine growth retardation with high fat diet in rats markedly disturbs islet morphology characterized by peri-islet inflammation, fibrosis and haemosiderosis j. dahlstrom * , v. delghingaro-augusto, l. madad, a. chandra, e. bean, c. simeonovic, c. j. nolan * act pathology & anums, dept. of anatomical pathology, garran, australia objective: pre-and post-natal factors such as intrauterine growth retardation (iugr) and high fat (hf) diet contribute to type 2 diabetes (t2d). our aim was to determine if iugr and hf diets interact in t2d pathogenesis. method: a surgical model of iugr (bilateral uterine artery ligation) in sprague-dawley rats with sham (sh) controls was used pups were fed either hf or chow (ch) diets from weaning. serial measures of body weight and glucose tolerance were made. at 25 weeks, rat pancreases were harvested for histological assessment. results: iugr vs sh pups weights were 17 % lower. hf diet caused excess weight gain, dyslipidaemia, hyperinsulinaemia and mild glucose intolerance not further aggravated by iugr. markedly abnormal islet morphology was evident in 0/6 sh-ch, 5/8 sh-hf, 4/8 iugr-ch and 9/9 iugr-hf rats (chi-sq, p=0.008). abnormal islets were characterised by larger size, irregular shape, peri-islet inflammation with cd68 positive cells and marked haemosiderosis. overall beta-cell mass was not altered by iugr, with a trend for it to be mildly increased in both hf-fed groups. conclusion: hf and iugr independently and together contribute to islet injury. the marked islet haemosiderosis associated with iugr and hf diets warrants further investigation as iron is toxic to β-cells. objective: adreno-hepatic fusion (ahf) is defined as adhesion of the liver and right adrenal gland with or without a fibrous capsule dividing both organs. we report a surgical case of ahf in which a virilising malignant adrenocortical tumour protruded into the liver mimicking a hepatic mass. method: a 36-years-old woman presented with mid-right abdominal pain and marked hirsutism. computed tomography (ct) revealed an apparently intrahepatic solid tumour suggestive of a giant hepatic adenoma. the right adrenal gland was not visible on ct and the right kidney showed marked downwards displacement. a radical right adrenohepatectomy was performed, with a satisfactory outcome. results: grossly, a giant, partially encapsulated mass was seen to protrude from the right adrenal gland into the liver. microscopically, the tumour was constituted by atypical clear cells with nuclear polymorphism, necrosis and moderate mitotic activity. tumour cells expressed vimentin and melan-a protein. conclusion: to our knowledge, this is the first description of a malignant adrenal tumour in the setting of ahf. as illustrated by this case, awareness of ahf as an entity and attention to its distinctive gross and histologic features are essential to avoid confusion between adrenal and hepatic lesions, especially when imaging studies have provided misleading findings. objective: cushing's syndrome (cs) is a rare disease, resulting in the majority of cases from acth hypersecretion. 70 % is of pituitary origin, 20 % of adrenal origin, and only 10 % from ectopic acth production. method: we reported a rare cause of ectopic acth dependent cs, caused by a pheochromocytoma. results: a 54-year-old male was hospitalized for severe high blood pressure, depression and weight loss. biological investigation revealed serious hypokaliema, severe acth dependent hypercortisolism with elevated urinary free cortisol secretion, loss of diurnal variation, and excess plasma acth level. ct-scan revealed a nodular lesion in the right adrenal gland with hyperplasia in the left one. the nodular lesion was assumed to be a pheochromocytoma based on the elevated serum and urinary catecholamine and metabolites and local uptake in right adrenal gland in 131 i-mibg scan. right adrenalectomy was performed. macroscopic examination revealed a 4 cm, well-circumscribed, tan-brown tumor, associated with diffuse adrenocortical hyperplasia. histological examination confirmed the diagnosis of pheochromocytoma, without signs of aggressiveness. the tumor cells were immunopositive for chromogranin a, synaptophysin and acth. after surgery, catecholamine secretion returned quickly to normal level. biological and clinical cs regression was noted. conclusion: despite the rare association of cs with pheochromocytoma, preoperative diagnosis is required to an appropriate therapy. a rare case: extralobar pulmonary sequestration mimicking neuroblastoma g. emiroglu * , n. özsan, s. tiryaki, a. çelik, y. ertan * ege university, dept. of pathology, izmir, turkey objective: extralobar intraabdominal pulmonary sequestrations are rare congenital malformations which are characterized by disorganized and nonfunctioning pulmonary parenchyma. these lesions have no communication with the bronchial tree and pulmonary arteries. they receive their blood supply from the systemic circulation. method: we describe a 5 days old male infant admitted to the hospital with a left subdiaphragmatic, 50×40×35 mm, hiperechogenic and, solid mass that was identified during ultrasonography on the 26th week of gestation. results: abdominal computerized tomography demonstrated a 48× 37× 33 mm, solid, vascularized, encapsulated, mostly cystic suprarenal mass with no calcification reported to be highly suspicious for neuroblastoma. the mass was completely excised. gross pathologic examination revealed a well circumscribed spongy lesion that mimicked a lung tissue. on microscopic examination, a disorganized lung tissue that was composed of alveoli, alveolar ductus and bronchioles was seen. normal adrenal tissue was not observed. later, we learnt that the lesion's arterial blood supply was from the abdominal aort. based on these findings, our diagnosis was intraabdominal extralobar pulmonary sequestration. conclusion: intraabdominal extralobar pulmonary sequestrations should be kept in mind in cases of the adrenal masses as the surgical resection is the adequate treatment method for these lesions. axillary lymph node metastasis of papillary thyroid carcinoma showing anaplastic transformation with cutaneous metastasis s. erkilic * , a. ozkur, u. elboga, f. celenk, m. kanlikama * gaziantep university, medical faculty, turkey objective: papillary thyroid carcinoma (ptc) rarely metastasizes to axillary lymph nodes. although anaplastic transformation (at) may occur in the cervical lymph node metastasis from ptc, it is rarely observed in the metastatic axillary lymph nodes. furthermore, cutaneous metastasis from anaplastic thyroid carcinoma (atc) is extremely rare. method: a 65-year-old male patient who had operated for multinodular goiter 8 years ago presented with neck swelling on the right side. residual thyroid tissue was detected and the lesion was removed surgically in combination with a right neck dissection. histopathologic examination was consistent with ptc and metastatic lymph nodes. the patient received radioactive iodine therapy. afterwards, the patient presented with a right axillary mass 14 years after the first operation and underwent surgical excision. results: histopathologic examination showed ptc with anaplastic transformation. histopathologically confirmed multiple skin metastases from atc emerged in thoracic and abdominal regions 3 months after the last operation. patient died 3 months after the diagnosis of anaplastic carcinoma. conclusion: although most ptcs show an indolent course and have a favorable prognosis, distant metastasis and anaplastic transformation of the metastatic lymph nodes may occur even more than many years after the primary treatment. local "bystander"effect of gene therapy on human tumor cells of medullary thyroid carcinoma in vivo l. feketeova * , m. poturnajová, l. kucerová, p. babál * comenius university, inst. of pathology, bratislava, slovakia objective: gene therapy acts on change of prodrug into cytotoxic drug inside tumor cells transfected by foreign enzyme. it uses also "bystander" effect on surrounding tumor cells without enzyme. we used yeast cytosine deaminase (ycd) in combination with 5-fluorocytosine (5fc) converted into 5-fluorouracil (5fu) and its metabolites. medullary thyroid carcinoma (mtc) is a malignant tumor often with therapy resistant distant metastases. patients with sporadic form have metastases at the time of diagnosis, mostly in bones, lungs and liver. the aim of this study was to evaluate the efficiency of ycd/5fc in mtc treatment using xenotransplants derived from model tt cell line in nude mice. method: tumors were immunohistochemically stained with polyclonal antibody anti-egfp (enhanced green fluorescence protein) and monoclonal antibodies anti-pcna and anti-ki67. positivity was semiquantitatively evaluated. results: diffuse positivity for pcna was seen in untreated and treated tumors, respectively. positivity for ki-67 was diffuse in untreated and only sporadic in treated tumors. conclusion: diffuse pcna positivity in treated tumors suggests that the tumor cells stopped in s phase. scattered positivity of ki-67 after treatment document suppressed proliferation mediated by ycd/5fc gene therapy, which implies therapeutic application in patients with metastatic mtc. (itms 26240220052, vega 2/008/11, vega 2/0146/10). ps-05-015 e-cadherin/ß-catenin immunoexpression in thyroid carcinomas k. ivanova * , e. onal, j. ananiev, t. vlaikova, m. gulubova * trakia university, general and clinical pathology, stara zagora, bulgaria objective: the aberrant activation of wnt signaling pathway may be a common denominator for the development of thyroid tumorigenesis. it was announced that the loss of ecadherin rather than β-catenin mutation represents a crucial event in determining the degree of differentiation of thyroid carcinomas. the aim of the study was to evaluate the expression of e-cadherin and β-catenin in the thyroid cancer tissue and to correlate these data with some histological and clinical parameters of the tumors. method: we investigated 57 patients, having thyroid tumorspapillary, follicular, anaplastic and oncocytic carcinomas immunohistochemically with antibodies against e-cadherin and β-catenin. survival analyses were done. results: e-cadherin expression was focally retained in the tumor cell membranes and in the tumor cell cytoplasm of the papillary, follicular and oncocytic thyroid cancers, weather in anaplastic cancers it was almost lost (p= 0.0042, and p= 0.019, respectively, fisher's exact test). the expression of β-catenin in tumor cytoplasm and membrane in papilary cancers was higher as compared to that in the other tumors (p=0.111, and p=0.0104, respectively). conclusion: not surprisingly, the presence of aberrant expression of e-cadherin and/or β-catenin in thyroid cancer has been associated with better patients' prognosis and more well differentiated tumor histology. correlation between estrogen receptor and progesterone receptor with some prognostic factors in papillary thyroid carcinoma m. jalali nadoushan * , r. amirtouri, a. davati * shahed university, dept. of pathology, tehran, iran objective: the more prevalence of papillary thyroid cancer in women shows the probability of the role of sex hormones in the cancer. the aim of this study was determination of relation between sex hormones receptors and some prognostic factors. method: we studied 92 patients with pathology report of papillary thyroid carcinoma after thyroidectomy between 2006 and 2009. the specimens were stained immunohistochemically for er and pr. the other informations such as sex, age, tumor size and lymph nodes involvement obtained from the patients documents. results: the average age of patients was 39.32+16,93. 46.7 % t of samples were er positive while this was 6.5 % for pr. the percentage of lymph nodes involvement was 23.9 %. the size of tumors was 3.60+2.21 cm. there was a direct relationship between female sex and positivity of er (p≤0.014). but there was no significant relationship between er and pr with age, tumor size and lymph nodes involvement. conclusion: it seems to be that er is more prevalent in females but for showing of its role in prognosis, further studies are recommended. objective: type 2 diabetes mellitus (t2dm) treatment aims to control metabolic effects, preserve pancreatic function and reduce complications, such as nephropathy. this study intends to evaluate the effects of sitagliptin, a dipeptidyl-peptidase-4 inhibitor, in pancreatic and renal lesions in zucker diabetic fatty (zdf (fa/fa)) rats, an animal model of t2dm. method: male obese diabetic zdf (fa/fa) rats, 20-weeksold, were treated with vehicle or sitagliptin (10 mg/kgbw/ day) for 6 weeks, and compared with lean control zdf rats (n=8 each). biochemical parameters as well as pancreatic and renal lipid peroxidation and histopathology profile were assessed. specimens we stained with haematoxylin-eosin and periodic acid of schiff, and examined by light microscopy. lesions were evaluated by a semiquantitative rating. endocrine/exocrine pancreas and renal glomerular, tubulointerstitial and vascular lesions were assessed and scored (0 3/0-2). results are mean ± sem; anova and duncan's post-hoc analysis (p≤0.05 was considered as significant). results: sitagliptin improved metabolic parameters, reduced lipid peroxidation (p<0.001) in both organs and significantly prevented major diabetic pancreatic and renal lesions in obese diabetic zdf rats. conclusion: sitagliptin seems to comply with the three main objectives of t2dm therapeutic management. underlying molecular mechanisms deserve further elucidation, but could be related with metabolic improvement and reduction of oxidative stress. objective: thyroid tumors of uncertain malignant potential (tt-ump) include follicular and well-differentiated tumors of ump (ft-ump/wdt-ump), as it refers to the presence of questionable capsular/vascular invasion or incompletely developed papillary thyroid carcinoma (ptc)-type nuclear changes. however, a diagnosis of tt-ump is difficult in most cases. we aimed to investigate whether immunohistochemistry (hbme-1, cytokeratin-19, galectin-3, cd56 and p63) provides additional information concerning such lesions. method: we performed an immunohistochemical analysis on 14 wdts-ump and 4 fts-ump. results: in the wdt-ump group, hbme-1 was positive in 6/14 (42.9 %) cases. cd56, a marker whose expression is reduced in thyroid carcinoma, showed a "malignant" profile (no expression) in 9/14 (64.3 %) cases. 6/14 (42.9 %) cases were positive for both antibodies. one case showed the coexpression of hbme-1, cd56, galectin-3 and cytokeratin-19. only one ft-ump case was positive for hbme-1. the follow-up data revealed no distant metastases or persistent disease. conclusion: tt-ump demonstrated very heterogeneous immunohistochemical profiles. wdts-ump revealed a certain tendency toward a ptc profile, suggesting a possible pathogenetic link between these two entities. however, immunohistochemistry is to be regarded more as a supporting factor, while morphological criteria should always prime in the diagnostic decision. results: there was a positive correlation between tpcfv and hmwck, ck 19, hbme1, galectin 3, fibronectin (p< 0.05), but there was no correlation with tpcfv and ret/ ptc (p>0.05). hbme-1 and ck 19 stained strong and diffuse positive in tpcfvs but weak and focal in fas. our study suggests that morphologic features combined with immunohistochemical panel of hmwck, ck19, hbme-1, galectin-3 and fibronectin can help to distinguish benign and malign thyroid neoplasms and tpcfv from follicular adenomas. ret/ptc expression has been nonspecific but its detection can be a useful tool combined with immunohistochemistry for diagnosing tpcfv. morphological features of the system mother-placentafetus during pregnancy on diabetes mellitus t. pavlova * , v. petrukhin, e. malutina, a. nesterov, l. pavlova, i. goncharov, d. kolesnicov * belgorod state university, dept. of pathology, russia objective: combination of diabetes mellitus and pregnancy has a special concern. method: it were studied 50 pregnants with diabetes mellitus type 1 and 29 pregnant with gestational diabetes mellitus (gdm). the methods were used: light, transmission, electron, probe and scanning microscopy with microelement analysis. results: the pregnancy at diabetes mellitus type 1 and gdm proceeds with complications: treat of pregnancy termination (64,0 % and 50,0 %), hydramnion (27,0 and 62,5 %), preterm birth (36,0 and 12,5 %). it was displayed decreasing of oxygen in mother's erythrocytes. at the diabetes mellitus in uterus of women in birth there are violations of spiral arteries as well as circulatory disorders (stasis, sludge, thrombosis and diapedetic bleedings), that is significantly expressed at gestosis. tendency to cell form changing is observed in erythrocytes at diabetes mellitus, especially at diabetes mellitus type 1. the frequency of placental insufficiency in pregnant with diabetes mellitus type 1 were 75 %, at gdm-50 %. the gestosis accession were resulted to birth's increasing of children with prenatal injury of central nervous system at diabetes mellitus type 1 in mother and diabetic fetopathy at gdm. the clinical and morphological parallels of the system mother-placenta-fetus were presiced ad diabetes mellitus type 1 and gdm. sclerosing mucoepidermoid carcinoma of the thyroid in a 13 year-old female a. polónia * , l. santos, c. eloy, r. celestino, p. soares, m. jácome, c. lobo * ipo-porto, pathology, rio tinto, portugal objective: sclerosing mucoepidermoid carcinomas (smecs) are low-grade malignant tumours with both squamous and mucinous differentiation representing less than 1 % of thyroid malignancies. smecs are usually associated to hashimoto thyroiditis and classically disclose eosinophilia. results: a 13 year-old female with hashimoto thyroiditis presented a 2.5 cm nodule in the left lobe of the thyroid. the total thyroidectomy specimen disclosed a firm, well circumscribed, non-encapsulated tumour. microscopically, the tumour was composed by anastomosing clusters of squamoid cells in a sclerotic background without eosinophils. intra-cytoplasmatic pas/diastase positive material was focally found. the tumour cells expressed ae1.ae3, ttf-1 and p63 whereas no expression was observed for thyroglobulin, calcitonin and cd5. the ki-67 labeling index was 5 %. in the molecular analysis no mutations were detected in braf or (k-, n-, h-) ras genes nor ret/ptc or pax8/ppargamma rearrangements. the remaining thyroid showed hashimoto-type thyroiditis. a cervical lymph node metastasis was identified. the patient was treated with radioactive iodine and is alive without signs of disease after 10 months of follow-up. conclusion: smecs may not disclose the classical eosinophilia and represent rare low-grade tumours that can give rise to metastases. the most frequent molecular alterations found in thyroid tumours were not detected in this case. ps-05-024 alk1 and bmp-9 overexpression as a cause of ossifying papillary thyroid carcinoma objective: ossification is an often encountered finding in papillary thyroid carcinoma (ptc). we hypothesized that osteogenic signaling may be related to osteogenesis of ptc. bone morphogenic protein (bmp)-9 is the most osteogenic subtypes among bmps. and as a cellular receptor, activin receptor-like receptor (alk)1 has been emphasized in bmp-9 induced osteogenic signaling. we investigated the expression of alk1 and bmp-9 and their correlation with ossification in ptc. method: alk1 and bmp-9 expression were investigated by immunohistochemistry in tumors and adjacent normal follicles of 78 ptcs with bone formation and 64 ptcs without bone formation. alk1 and bmp-9 expression were further verified by quantitative real time polymerase chain reaction in each group of 15 cases of ptcs with or without bone formation. results: alk1 and bmp-9 immunoreactivity were increased in ptc with bone formation when compared to those without bone formation (p<0.001 and p=0.001). both mean values of alk1 and bmp-9 mrna expression were elevated in ptcs with bone formation compared with those without bone formation (p=0.037 and p<0.001). conclusion: alk1 and bmp-9 overexpression may be underlying the molecular alteration that accounts for osteogenesis in ptc. objective: thyroid is an extremely rare site of metastases. renal clear cell carcinoma (rccc) is one of the most frequent primary malignancies causing thyroid metastases as a single nodule (77 %) or multiple nodules (23 %). we report the case of a 62-years-old female patient affected by multinodular goiter, who was diagnosed with rccc, treated with nephrectomy. method: due to compressive symptoms, 6 years after nephrectomy, the patient underwent thyroidectomy and the thyroid was sent for histology. results: histology showed multinodular goiter. in the largest hyperplastic adenomatous nodule (left lobe, diameter mm 53), multiple solide yellow-orange areas were detected grossly. these areas were microscopically composed of sheets of large cells with clear cytoplasm and small hyperchromic nuclei, with a focally infiltrative growth pattern. at immunohistochemistry clear cells were negative for ttf1, hbme1, galectin-3 and positive for cd10. conclusion: this is a rare case of multiple rccc metastases within an adenomatous nodule in goiter. clinical diagnosis of rccc metastases to thyroid is extremely difficult, even more if metastases grow in multinodular goiter. it should be always suspected in patients with a clinical history of rccc. objective: follicular carcinoma, oncocytic variant, is a rare type of thyroid carcinoma. we examined a case of oncocytic carcinoma misdiagnosed as medullary carcinoma on fineneedle aspiration (fna) because it was associated with amyloid material. method: a 73-year-old female rheumatoid arthritis (ra) patient had a 4.0 cm mass in the right lobe of her thyroid, which showed no enhancement effect on ct scan and was diagnosed as a "cyst". fna was performed, and she subsequently died of unrelated causes. an autopsy was performed. results: cytology: large and small round-shaped tumor cells were present with round nuclei with granular chromatin. double-or triple-nucleated cells and amyloid material were observed. histology: oncocytic round cells proliferated diffusely in the fibrous capsule. extra-capsular invasion and vascular infiltration by the tumor cells were recognized. electron microscopy: the cytoplasm of tumor cells was full of mitochondria. therefore, we diagnosed this as follicular carcinoma, oncocytic variant. amyloid deposition was also observed in several other organs. conclusion: amyloid deposition was caused by amyloidosis secondary to ra. narrowing of the tumor feeder artery due to amyloidosis may have prevented early enhancement on the ct image. objective: whipple's disease is an infectious disease caused by tropheryma whipplei, an ubiquitous gram-positive actinobacteria. the incidence of the disease is less than 1 per 1 milion. we report five new consecutive cases, four males and one female, diagnosed in our clinic from august 2002 to january 2012. method: diagnosis was reached with the help of gastroduodenal endoscopy and histopathological examination of the duodenal biopsies, by lymph node biopsy and by electron microscopy. results: the main symptoms were arthralgia, weight loss and diarrhea. the endoscopic aspect of the small bowel mucosa varied from congestion, granularity of the mucosa to whitish plaques. all patients showed pas positive, diastase resistant, ziehl-neelsen negative macrophages in the lamina propria of the duodenal mucosa and, in one patient, suspected for lymphoma, in an abdominal lymph node. the diagnosis was confirmed by electron microscopy in all cases. classic whipple's disease was the diagnosis in all five cases, but one patient showed involvement of the endocardium and two patients showed lymphadenopathies. conclusion: clinical evolution was favorable under long-term antibiotics (cephtriaxone/trimethoprim-sulfamethoxazole) and follow-up biopsies in three patients showed a normal endoscopical mucosa and a reduced but persistent number of pas positive macrophages in the duodenal mucosa. results: eleven cases of nen of gastrointestinal tract were identified. they were 6 men and 5 women with a mean age of 53.09 years. the most common localization was the appendix (7 cases). the 4 other cases were localized in the small intestine, the ileo caecal valve, the fundus and the bulb. the mean tumor's size was 13.8 mm. mitoses were absent in the majority of cases (72,7 %). all the tumors were well differentiated classified as grade 1 in 9 cases and grade 2 in 2 cases. the tumor was aggressive in 2 cases. the small intestine's net was multifocal infiltrating the subserosa with lymph node metastasis classified pt3 n1. the ileo caecal valve's net was also aggressive with lymph node and hepatic metastasis classified pt4n1m1. all patients underwent surgical treatment. conclusion: gastrointestinal nens are complex tumors whose incidence is rising and whose treatment requires precise classification and risk stratification. multiple duodenal stromal tumors associated with neurofibromatosis-1 g. benkhedda * , y. lamouti * chu frantz fanon, dept. of pathology, blida, algeria objective: gastrointestinal stromal tumors (gist), most commonly occur sporadically, but there seens to be some increased tendency for these tumors to develop in patients with neurofi-bromatosis1(nf1). there is no histological difference between the nf1 assocaciated cases and the sporadic cases. however, tumors associated with nf1 frequently show multiplicity. method: a case of multiple duodenal gastrointestinal tumor arising in a 45 year old mal with nf1 is reported. the abdominal exploration revealed multiple solid nodules in the duodenum, and the pancretecticoduodenectomy was performed. results: macroscopy: the resected segment of the duodenal showed seven suberosal solid masses. the largest mass measured 3,5 cm×2 cm×3 cm, and is coupled with the bors lower pancreas but remains limited by a capsule. the cut surface was smooth and white in appearance. microscopy: the tumors were composed of interlacing fascicles of the uniform spindle cells with elongated cytoplasm. the tumor cells lacked pleomorphism, and mitotic figures were absent. immunohistochechemistry: the tumors cells were diffusely positive for cd117, cd34, and negative for desmin, aml and ps100. conclusion: gist are rarely noted in association with neurofibromatosis-1. duodenal gist are most frequently diagnosed in the workup of symptoms not specific to these masses. duodenal resection is rarely indicated except in the case of duodenal gist and early-stage adenocarcinoma or if the tumor appeared involve the pancreatic parenchyma on preoperative imagings. periampullary adenomyoma: a true trap diagnosis g. benkhedda * , y. lamouti * chu frantz fanon, dept. of pathology, blida, algeria objective: adenomyoma is a term generally applied to nodular lesions showing proliferation of both epithelial and smooth muscle components. it is usually presented as biliary obstruction. most cases are misdiagnosed as adenoma or carcinoma by preoperative endoscopic or radiologic. therefore, it is frequently treated with extensive surgery. method: we report a case of a 28 year-old man with an adenomyoma located in the ampulla of vater diagnosed by endoscopic piecemeal resection. results: on histologic examination, the lesion consisted of hyperplastic glandular lobules, mainly located in the muscle layers of the vaterian system. the lobular formations consist of small glands surrounded by myofifroblastic, fibroblastic proliferation, sparse capillaries and inflammatory cells. ihc: ki67: rare cells with a positive nuclear staining were presenting in the epithelial and mesenchymal components aml: the myofibroblastic of most spindle cells was confirmed by a strong cytoplasmic expression. conclusion: real incidence of adenomyoma of the vaterian system is difficult to appreciate as different names (adenomyoma, adenomyomatosis, myoepithelial hamartome) are used to designate the same histological lesion. adenomyoma was diagnosed only in adult patients -mean age: 63 y). the histogenesis is still a subject of controversy. the most widely accepted hypothesis is that the lesion may represent a form of incomplete heterotopic pancreas. adenomyoma is considered as benign and slow growing, but its potential neoplastic nature cannot be excluded. however, the histogenesis of these tumors is subject to further study. carcinosarcoma of the digestive organs has been reported to exhibit aggressive behavior. carcinosarcomas in digestive organs have been reportedly associated with a poor prognosis.12,13 however, some cases that have been treated with a curative operation showed long-term survival. analysis of her2 expression level in gastric carcinomas m. bialas * , a. sinczak-kuta, a. lazar, k. urbanczyk, k. galazka, j. szpor, s. demczuk, d. adamek * jagiellonian university, dept. of pathomorphology, krakow, poland objective: the expression of her2an oncoprotein belonging to tyrosine kinase family belongs to recently evaluated prognostic and predictive factors in gastric cancer. overexpression of her2 is observed in about 8-27 % gastric cancers. according to many authors, overexpression and/or amplification of her2 correlates with poor prognosis. the aim of our study was to analyze her2 expression in gastric cancer in material routinely examined in pathomorphology department, jumc. method: we have analyzed 50 cases of gastric cancers. the material came from 19 females and 31 males, age: 21-86. immunohistochemical reactions were performed automatically on benchmark bmk classic (ventana) using path-way her-2/neu (4b5) antibody. scoring system for her2 expression was ranged from 0 to 3+ where: 0 and 1+ were regarded as a negative, 2+ as equivocal and 3+ as positive. results: most cases of gastric cancers were her2-negative (45/50): including 0 score (38 patients) and 1+ (7 patients). three cases were equivocal and two cases showed 3+ expression level. the lower level of her2 expression in the analyzed material in comparison with literature could be related to a relatively small group of cases but one cannot exclude that there exist some other factors that stand behind this and surely the investigations should be continued. ps-06-012 mapkap kinase 2 overexpression influences prognosis in gastrointestinal stroma tumors, associates with copy number variations on chromosome 1, and expression of p38 map kinase and etv1 p. birner * * medizinische universität wien, institut für klinische pathologie, austria objective: etv1 has been proposed to be activated by kitmutations in gastrointestinal stromal tumors (gists). aim of the study was to evaluate the role of etv1 and associated proteins in gist. method: expressions of etv1, mapkap kinase 2 (map-kapk2), phosphorylated p38 map kinase (pp38), phosphorylated msk1, phosphorylated rsk1, cop1 and kit were determined immunohistochemically in 139 gists. sequence analysis of kit, pdgfra and mapkapk2 and fish of etv1 and chromosome 1 was performed. results: prominent etv1 expression was seen in 50 % of gists, but no correlation with clinical outcome was found. correlation of etv1 expression and kit mutation was seen in 60 % of cases. mapkapk2 overexpression (n =62/ 44.6 %) correlated with pp38 expression (p=0.021) and alterations of chromosome 1 (p=0.024). in one cases with high makapk2 expression, a mapkapk2 gene mutation was found. all relapsing gists with very low/low risk showed high mapkapk2 and kit expression. map-kapk2 overexpression was an independent prognostic factor for disease free survival (p=0.006). conclusion: etv1 is not universally overexpressed in gist and seems to be induced also by other pathways than kit-mutation. mapkapk2-overexpression is associated with shorter survival in gist. patients with low risk gists overexpressing mapkapk2 might profit from adjuvant tyrosine kinase inhibitor therapy. gastrointestinal stromal tumors: comparison of two risk stratification systems in a multicenter study of 1963 turkish cases g. bulbul dogusoy * * gayrettepe florence nightingale, dept. of pathology, istanbul, turkey objective: a nationwide database was performed for gastrointestinal stromal tumors (gists) in a large series of primary gists surgically treated at centers all around turkey. the aim of this multicenter study was to analyze and compare the performance of the national institute of health (nih) and armed forces institute of pathology (afip) risk criteria to determine the ideal risk stratification system. method: statistical analysis of a nationwide database is consisted of age, gender, location, risk groups, histopathologic features and the results of cd117, cd34, desmin, sm actin, s-100 protein, and ki67 immunohistochemistry. results: in 1965 cases registered in database, male to female ratio was 1.20 and mean age was 57.64 years. most common location was stomach (47.5 %) followed by small intestine, omentum-peritoneum, large intestine, and esophagus (30.3 %, 12.1 %, 9.1 %, 1.0 % respectively). comparison of the two risk-stratification systems demonstrated that proposed modified afip seems to be better when compared with nih system. many histopathologic and immunohistochemical findings showed significant correlation with risk groups of afip, even with 'not sufficient data' group (p<0.005). the results of this multicenter study demonstrates that although follow up results are not provided, afip risk criteria seems to be more useful in prognostication for gists among the two systems. objective: her 2/neu overexpression or amplification is an important biomarker for identifying patients with intestinal-type gastric cancer who respond to therapy with trastuzumab. moreover, intestinal type gastric cancer shares many phenotypic and molecular genetic changes with colorectal cancer. in particular a progression from chronic gastritis to intestinal metaplasia, dysplasia, and finally malignant transformation is probably the sequence of gastric carcinogenesis. somatic mutation of k-ras gene is common in colorectal cancer, being found in more than one-third of cases, but it seems to have, mostly in intestinal-type gastric cancer, a low incidence (7-20 %). the purpose of this study is to assess her2 gene amplification and k-ras mutational status in a series of intestinal-type gastric carcinoma patients. method: twenty paraffin embedded gastric cancer specimens were tested for her2 amplification by chromogenic in situ hybridization (cish) and k-ras mutational status (codon 12 e 13) by pcr-rflp. results: six (30 %) cases were her2 amplified. only one case (5 %) was found to have k-ras mutation (codon 12), but it was her2 not amplified. conclusion: the frequency of k-ras mutation and her2 amplification are in agreement with other studies on this topic. in our study, the two seem mutually exclusive events. objective: gastric carcinoma is related with cancer genetic susceptibility that can be investigated as single nucleotide polymorphisms (snps) and as cytokine genes are known to predispose to malignant disease, several polymorphisms of interleukin-6 (il-6) gene have been reported to in some may be associated with tumour progression including inhibition of malignant epithelial cells apoptosis and stimulation of angiogenesis. method: the aim of this study was to clarify the association between il-6 polymorphisms and the risk of gastric cancer and chronic gastritis development or maintenance. pcr-ssp genotyping for il-6 -174 c>g polymorphism was performed in 100 biopsies of gastric carcinoma and in 100 biopsies of chronic gastritis. results: there was association between il-6 -174 c allele (p = 0,0466) and -174cc, low producer, genotype (p = 0,0466) and gastric carcinoma, whereas il-6 g allele (p= 0,0278) and il-6gg (p<0,0001), high producer, genotype was associated with gastritis. conclusion: we conclude that il-6 -174, low producer genotypes, may have an important role in gastric carcinogenesis and the polymorphism study of this molecule could be a good marker for gastric carcinoma susceptibility when high grade dysplasia is seen in biopsies. objective: in helicobacter pylori gastritis, constant antigenic stimulation triggers a sustained b-cell proliferation. errors made during this continuous dna replication are corrected by the dna mismatch repair mechanism. failure of this mechanism has been described in hnpcc and results in a replication error phenotype. inherent to their instability during replication, microsatellites are the best markers of this replication error phenotype. we aimed to evaluate the role of defects in the dna mismatch repair mechanism and microsatellite instability (msi) in gastric mucosa-associated lymphoid tissue (malt) lymphoma. method: we examined 10 microsatellite loci (bat25, bat26, d5s346, d17s250, d2s123, tgfb, bat40, d18s58, d17s787 and d18s69) for instability in 28 patients with malt lymphomas. in addition, these tumors were also immunostained for mlh1, msh2 and msh6, as well as screened for the presence of t(11;198)(q21;q21) by real time polymerase chain reaction (rt-pcr). results: we found msi in 5/28 (18 %) lymphomas, with one tumor displaying high levels of instability. msi occurred in both t(11;18)(q21;q21)-positive and -negative tumors. conclusion: our data suggest that a mmr-defect may be involved in the development of gastric malt lymphomas, and mutations in the msh6 mmr gene or hypermethylation of the msh6 promoter might be associated with msidriven gastric lymphomas. mitosis-specific marker phospho-histone h3 in the diagnosis of gastrointestinal stromal tumors a. dolzhikov * , a. tverskoi, k. hizhnyakov * regional pathology hospital, dept. of oncomorphology, belgorod, russia objective: the assessment of proliferative activity is one of the major parameters in the proper grading of gastrointestinal stromal tumors (gists). in the low level of mitotic activity it is difficult to calculate dividing cells correctly. phospho-histone h3 (phh3) is a recently introduced immunomarker for mitotic cells. method: immunohistochemical study of 46 cases of gists of different malignant potential, statistical analysis. results: the count of phh3-immunoreactive cells demonstrates the strong correlation (gamma 0,756; p<0,05) with malignant potential of gists detected by standard parameters. it clearly separates true mitotic cells from apoptotic and piknotic nuclei. in all cases phh3 count was slightly higher than the mitotic index in h&e stained slides. we have found that pph3 demonstrated the two types of staining: nucleosomal dot-like type and mitotic homogenous type. the first type, probably, reflects the fraction of cells just before the prophase, which cannot be identified in h&e slides. conclusion: immunostaining of pph3 is a useful additional marker in detection of proliferative activity in gists, helping to identify true dividing cells correctly. ps-06-019 her2 assessment in gastric carcinoma using ihc and fish p. drev * , b. gazic, j. contreras * institute of oncology, dept. of pathology, ljubljana, slovenia objective: her2 in gastric cancer (gc) should be assessed following guidelines -recommended testing algorithm employs ihc as a screening tool and is followed by ish to clarify equivocal cases. reported incidences of her2+ gc vary substantially and discordant results are frequent. in a series of gc her2 was assessed by ihc and fish to determine incidence of her2+ gc, concordance of the methods and reevaluate the recommended algorithm. method: her2 was assessed by both ihc (pathway 4b5™) and fish (pathvysion™) in 164 gc samples. reactions were evaluated according to guidelines. her2 was considered positive in case of strong protein expression (3+) and/or gene amplification (ratio ≥2.0). frequencies of ihc and fish scores, incidence of her2+ gc and concordance (chi-square) of applied methods were analysed. results: ihc score distribution: neg (0) objective: gastric glomus tumors are rare neoplasms originated from modified smooth muscle cells of the glomus bodies. such lesions present a diagnostic challenge in biopsy material. herein, we report a case of gastric glomus tumor in a 35-year-old woman. method: a 35-year-old woman presented with refractory epigastralgy. a gastrointestinal stromal tumor of the gastric antrum was suspected. the diagnosis was made after ultrasound-guided endoscopic biopsy, followed by an endoscopic submucosal resection. results: the biopsy showed tight convolutes of capillarysized vessels surrounded by collars of small, rounded cells set in a hyalinized, myxoid stroma. immunohistochemically, these cells were positive for smooth muscle actin and type iv collagen (pericellular pattern), synaptophysin (focal positivity) and negative for cd34, cd117, desmin, s100 and cytokeratins. the diagnosis of gastric glomus tumor was rendered. the resection specimen revealed a submucosal well-circumscribed 2×1,5×1,5 cm nodule with identical histological features. the patient was disease-free 12 months postoperatively. conclusion: visceral glomus tumors are rare neoplasms. when they arise in the gastrointestinal tract, the stomach (antrum) is the most frequent location. the differential diagnosis includes net, epithelioid gist and hemangiopericytomas. they usually behave in a benign fashion, although malignant cases have been reported. surgical excision is the standard treatment. immunohistochemical evaluation of replication protein-a1 in gastric cancer: clinicopathological associations h. gakiopoulou * , e. fourtziala, g. levidou, a. stofas, n. alexakis, p. korkolopoulou, e. patsouris * university of athens, 1st dept. of pathology, greece objective: replication protein a1 (rpa1) is required for stabilization of single-stranded dna at early and later stages of dna replication being thus critical for eukaryotic dna replication. in this study, we investigated for the first time the immunohistochemical expression of rpa1 protein in a series of 73 gastric carcinomas in relation with clinicopathological parameters (age, sex, lauren's histologic classification, histologic grade, lymphovascular invasion, tumor size, depth of invasion (t), lymph node metastasis (n) and stage). method: a standard immunohistochemical method and a semi-quantitative evaluation for the detection of rpa1 labeling index (li) were applied. results: nuclear rpa1 immunoreactivity was seen in all carcinomas with a mean value of 26.5 %. statistical significant correlations emerged between: 1. rpa1 li and low tcategory (p=0.009) 2. rpa1 li and absence of lymph node metastasis (p=0,014). rpa1 li was higher in cases without lymphovascular invasion; however this association did not reach statistical significance. conclusion: the widespread expression of rpa1 in gastric carcinomas suggests that this protein is implicated in gastric cancer growth. the observed significant associations between rpa1 li and low t as well as n0 tumors could imply that rpa1 might offer a growth advantage in the early stages of gastric cancer progression. pigmented histiocytic "pseudotumoral" reaction due to endoscopic tattooing of the duodenum with india ink r. hadhri * , c. objective: india ink has been used for endoscopic tattooing to facilitate localization of a luminal abnormality at the time of surgery or repeat endoscopic examination. recognition of this phenomenon is important to prevent misinterpretation of this _nding as other cause of black tissue deposits. some of them can be easily excluded by microscopic examination; the others, however, necessitate special stains or, as illustrated by our observation, an adequate clinical information! method: a 76-year-old man was referred for enteroscopy for microcytic anemia. the procedure revealed a 2 cm flat umbilicated lesion of the duodenum. endoscopic tattooing was performed to guide surgical excision. results: histologic examination of the surgical specimen revealed a tubulovillous adenoma with low-grade dysplasia. the submucosa showed numerous aggregates of large cells containing a heavy black pigmentation. special histologic stains were inconsistent with melanin or iron. this submucosal cellular infiltration was linked to the preoperative use of india ink when the notion of tattooing was "kindly" provided by the surgeon! conclusion: despite a striking and misleading appearance, the microscopic finding of such phenomenon does not represent a pathologic state. nevertheless, communication between enterologists, surgeons and pathologists are mandatory to assure prompt recognition and avoid unnecessary investigations. objective: gastritis cystica polyposa is a unique lesion found on occasion at the stoma of a gastrojejunal anastomosis. however, gcp is rarely found in an unoperated stomach. method: a 51-year-old woman with abdominal discomfort and vomiting. physical examination, routine hematological examination and biochemical tests were within normal limits, exept mild anemia. results: upper gi endoscopic examination revealed a pedanculated polyp, in the greater curvature. endoscopic ultrasonography revealed a polypoid heteroechoic mass with cystic area and 20×22 mm diameter in posterior wall of gastric body. polypectomy was performed without any complications. histological examination of the protruding lesion revealed some misplaced cystic glands were entrapped in dense disorganized bundles of smooth muscle of muscularis mucosa surrounded by a rim of lamina propria. these findings were consistent with gastritis cystic polyposa. objective: human papillomavirus (hpv) infection is a known risk factor for the development of squamous cell carcinoma (scc) of the cervix, the oropharynx and the anogenital region. the aim of our investigation was to assess the prevalence of hpv dna in patients with esophagus cancer in the western population. method: formalin-fixed paraffin-embedded blocks from 37 consecutive patients who underwent esophageal endoscopic mucosal resection or esophagectomy for scc were tested for the presence of hpv dna by polymerase chain reaction using consensus primers gp5+/gp6+. viral genotyping was determined by type-specific primers. results: among the total of 37 cases, no hpv dna was detected in 33 cases (89.2 %). 4 cases were tested hpv positive (10.8 %) , of which 1 was hpv 16+, 1 was hpv 18+ and 2 were non-high-risk hpv type. the non-high-risk hpvs were detected in patients with previously treated scc of the mouth (n=1) and the oropharynx (n=1). patients with high-risk hpvpositive tumors had no history of oropharyngeal cancer. conclusion: our study revealed the presence of oncogenic hpv genotypes in a subset of esophageal cancer. the low rates of viral infection detected suggest that hpv unlikely represents a significant etiologic factor in esophageal carcinogenesis. further studies are needed to confirm these data in larger populations. objective: gastric cancer is the second leading cause of cancer mortality in the world. amplification of her-2/neu oncogene has become an important biomarker for identifying patients who will respond to her-2 targeting therapy. the rate of her2 positivity in gastric cancer is variable, ranging from 6 % to 35 %. objective: gastrointestinal stromal tumors (gists) are primary mesenchymal tumors that arise in the gi tract. only 3-5 % of gists occur in the duodenum. here, we report a rare case of duodenal gist with extramural growth that mimicked a pancreatic tumor. method: a 57 years old male patient was admitted to our hospital for duodenal ulcer and gastrointestinal bleeding which was diagnosed in an another clinic by endoscopy. abdominal mr of the patient showed a 5×5,5 cm necrotic mass in the uncinate process of the pancreas which was excised by whipple operation. results: macroscopically, the resected specimen consisted of solid mass that was connected to the patient's duodenal wall but not to the parenchyma of the pancreas. microscopic examination of the tumor showed spindle shaped and epithelioid cells with mild nuclear pleiomorphism. immunohistochemistry revealed that the cells are strongly positive with cd117, with focal expression of cd34 and sma. conclusion: gists are low-grade malignant mesenchymal tumors of the gi tract and are believed to originate from the neoplastic transformation of the cajal cells, which are located between the longitudinal and circular layers of the muscularis propria. they most frequently arise from the second part of the duodenum where they push or infiltrate into the pancreas. in our case report, the patient's tumor exhibited extramural growth and mimicked a pancreatic tumor. mantle cell lymphoma (mcl) is relatively a rare subgroup of non-hodgkin lymphoma. we have experienced an uncommon case of mcl. a 65-year-old man was admitted to emergency service for upper gastrointestinal bleeding as melena. gastric corpus wall thickness found increased and splenomegaly determined at whole abdominal ultrasonography. endoscopic examination revealed subsequently, 1 cm diameter polypoid lesion at corpus anterior wall and 2 cm dimensioned elevated and vascularised lesion at corpus posterior wall. endoscopic biopsy had reported as active gastritis and mucosal reactive hyperplastic changes with ulcerative background. after than total gastrectomy and splenectomy specimens examined diffuse infiltration of monotonous medium-sized, atypical lymphoid cells with hyperchromatic nuclei. these cells were positive for cd20, cd5, cd43, bcl-2 and cd79a, but negative for cd10, cd23 and bcl-6. atypical lymphoid cells were present even in splenic hilus and lesser curvature lymph nodes. we reported this case as primary gastric mantle cell lyphoma according to morphologic and immunohistochemical staining features. we report a 74-year-old man who had been searched for bicytopenia (anemia and thrombocytopenia). bone marrow examination revealed extensive bone marrow metastasis of signet ring cell carcinoma. pas, d-pas and mucin histochemical markers performed to bone marrow sample which were all positive. immunohistochemical markers were positive with muc-1, muc-5 ac, muc-2 (focal staining), ck7, ck20, ema, cea and negative with vimentin, cd10, chromogranin. according to morphologic and immunohistochemical results we thought primarily gastrointestinal system malignancy, particularly stomach carcinoma. upper gastrointestinal endoscopy performed subsequently, hemorrhagic ulcerative lesion had seen and taken biopsies. endoscopic biopsy results showed metastatic signet ring cells origin from stomach. we report this case because bone marrow metastasis can be found commonly in some malignant tumors but diagnosing a nonhematologic malignancy from bone marrow is an unusual event. the pathological effects of salvia officinalis extract on serum level of alkaline phosphatase in male rats e. abdollahi * , r. ahmadi * tehran university, iran objective: studies show that there is association between salvia officinalis extract administration and liver or heart functions. the main aim of this study was to determine the effects of salvia officinalis extract on serum level of alkaline phosphatase in male rats. method: male wistar rats were randomly divided into control, normal saline receiving and salvia officinalis extract (100, 150 or 200 mg/kg/day) receiving animals of 5 in each group. after a period of 6 weeks, blood samples were collected using cardiac puncture method. following serum collection, serum alkaline phosphatase levels were measured by spectrophotometry method. data were statistically analyzed and compared between groups using anova. results: the results indicated that serum alkaline phosphatase levels were significantly increased in salvia officinalis extract (100, 150 or 200 mg/kg/day) receiving animals compared with control rats. conclusion: our findings show that salvia officinalis extract is enhancer of serum alkaline phosphatase according to which, impairing effect of the extract on certain tissues is conceivable. the pathological effects of waterpipe smoking on serum levels of cea, alkaline phosphatase or creatine kinase in male and female rats r. ahmadi * , m. mafi * islamic azad university, dept. of physiology, hamedan, iran objective: various studies show that smoking can influence serum levels of tumor markers such as carcino embryonic antigen (cea) and kinase or phosphatase enzymes. the main aim of this study was to determine the pathological effects of waterpipe smoking on serum levels of cea, alkaline phosphatase (alp) or creatine kinase (ck) in male and female rats. method: male and female wistar rats were randomly divided into control and waterpipe smoking groups of 5 in each. after a period of 10 weeks, blood samples were collected using cardiac puncture method. following serum collection, serum cea, alkaline phosphatase or creatine kinase levels were measured. intestinal tissue was also examined histologically. data were statistically analyzed and compared between groups using anova. results: the results indicated that serum cea, alp or ck levels were significantly increased in male and female waterpipe smoking animals (p<0.01, p<0.01 or p<0.001, respectively). there was not gender effect on serum cea, alp or ck levels. there were also histological changes in intestinal tissue including increased tissue plasma cells infiltration and inflammation. conclusion: our findings show that waterpipe smoking is an enhancer factor of serum cea, alkaline phosphatase or creatine kinase levels, according to which, damaging effects of waterpipe smoking on various tissues, particularly intestine should be considered seriously. the effects of acute or chronic immobilization stress on serum level of creatine kinase and alkaline phosphatase in male rats m. alinavaz * , r. ahmadi, m. mafi * islamic azad university, nutrition, tehran, iran objective: studies show that immobilization stress has a variety of effects on serum levels of liver enzymes. the main aim of this study was to determine the effects of immobilization stress on serum level of creatine kinase and alkaline phosphatase in male rats. method: 50 male wistar rats weighing 200±30 g were randomly divided into control, acutely or chronically immobilized animals of 5 in each group. animals were immobilized for 2 h/day or 8 h/day for a period of 3 weeks or 1 week in chronically or acutely immobilized groups, respectively. blood samples were collected using cardiac puncture method. following serum collection, creatine kinase or alkaline phosphatase level was measured by spectrophotometery method. data were statistically analyzed and compared between groups using anova. results: the results indicated that serum creatine kinase level was significantly increased in rats enduring acute or chronic immobilization compared with control animals (p< 0.001), however, there was not significant difference between serum alkaline phosphatase levels in immobilized animals compared with control rats. conclusion: immobilization stress may leave pathological effects in liver or other organs leading to enhanced serum creatine kinase level. immunoexpression of lactoferrin in bone metastases and corresponding primary carcinomas v. barresi * , a. ieni, g. giuffrè, g. branca, g. tuccari * university of messina, dept. of human pathology, italy objective: with reference to primary bone tumors, we previously found lactoferrin (lf) immunoreactivity in chondroblastomas, chondromyxoid fibromas, giant cell tumours and osteoid osteomas, while no immunoexpression for this protein was detected in chondrosarcomas and osteosarcomas. herein we aimed to analyze lf distribution in bone metastases from cancers of different sites. method: lf immunohistochemical expression was investigated in 25 formalin-fixed and paraffin-embedded specimens of human bone metastatic lesions as well as in the primitive corresponding carcinomas. the primitive sites of carcinomas were: breast (8), prostate (4), kidney (4), lung (3), colon-rectum (2), uterus (4). a lf intensity-distribution (id) score was calculated for each case by multiplying the values of the area staining positivity and the intensity staining. results: lf immunostaining, with variable id scores, was encountered in 11/25 (44 %) metastatic bone lesions. immunoreactivity for lf was found in primary carcinomas with a percentage of neoplastic cells ranging between 50 tand 75 %, although this positivity decreased in breast carcinomas (37.5 %) and was totally absent in lung cancers. the immunohistochemical concordant evidence of lf in bone metastases and corresponding primary carcinomas strongly supports the hypothesis of an autoctone production of this protein by the neoplastic elements themselves in order to get a greater availability of iron for their increased turnover. results: it was observed a negative correlation between il1/il6 and il17/foxp3 concentration in the sf as measured by fc, before the treatment. in the sm, il6 and mmp9 were strongly expressed in macrophages, fibroblasts, endothelial cells and the extracellular matrix of control cases, but reduced significantly in the treated cases (p< 0, 05). il 17 and foxp3 positive cells remained costantly in a reduced number in the ms. conclusion: as the proinflammatory cytokines decreased after the therapy, we can conclude that these are more efficiently influenced than some immune cells participating in the autoimmune process. histological evaluation of the spleen after acute bleeding followed by blood replacement with two different physiologic solutions m. cabral * , a. l. ortiz, c. venâncio, j. objective: chondromyxoid fibroma is a rare benign lesion accounting for less than 0.5 % of all primary neoplasms of bone. a study was conducted to assemble a national study group for rare entities and share the experience of different centers. method: the data was collected from the pathology archives of 13 referral hospitals in turkey. results: among the 61 cases enrolled in the study, 57.4 % (n=35) of them were male. median age was 35±18 yearsold (range: 6-67). the most frequent localizations were tibia (28 %, n=17), femur (n=10, 16.4 %) and pelvic bones (n=9, 14.8 %), followed by feet bones (11.5 %), fibula (8.2 %), humerus (4.9 %), hand bones (4.9 %), cranium (3.3 %), costa (3.3 %), radius (1.6 %), scapula (1.6 %), and vertebrae (1.6 %). although the cases with appendicular skeleton involvement was younger than the others (median age: 29.5±17.03 vs 53±11.5 respectively) no significant correlation was found between age, gender and localization. conclusion: cases displayed a wide age-range with a slight male predominance. the most frequent localizations were tibia, femur and pelvic bones. rare localizations such as temporal frontal bones, vertebrae and scapula were also observed. the study, which may serve as a preliminary work for future studies was presented to share our experience on this rare entity. histological changes and granulocytes redistribution in adjuvant arthritis l. feketeova * , p. jancová, p. objective: surgical treatment options of malignant tumors of the knee includes reconstruction with incorporation megaprosthesis. inguinal lymphadenopathy due to the lymphatic uptake of metal debris has been described, and may be clinically confused with tumor metastasis. method: we report the case of a 39-year-old woman with inguinal lymphadenopathy caused by metallic debris from a knee mega prosthesis for malignant fibrous histiocytoma. results: the histopathological changes seen in lymph node were metal debris containing sinusoidal macrophages in a background with numerous epithelioid granulomata in the remaining lymph node. conclusion: the identification of regional lympadenopathy in patients with past history of malignancy usually indicates metastatic disease. post-prosthesis lymph node histiocytosis resembling metastatic disease is described, and that is why we need resect and examine lymph nodes with the use of polarized light microscopy to identify birefringent particles of prosthetic debris for an accurate histologic diagnosis. objective: chordoma is a slow-growing malignant bone tumor that exhibits notochordal differentiation. nearly 90 % of cases occur in the sacrococcygeal region and in the base of the skull. the remaining cases develop in the mobile spine, predominantly in the cervical and lumbar vertebrae. we report a rare case of paravertebral mediastinal chordoma without bone destruction. method: case report. results: a 47-year-old japanese woman was admitted to hospital after a tumor was incidentally detected on a plain chest x-ray image. the tumor was located in the paravertebral region of the mediastinum and did not show any destruction of the thoracic vertebra radiologically. the tumor was clinically diagnosed as a benign neurogenic tumor and the tumor was easily removed surgically. microscopically, the tumor mainly consisted of tumor cells with extensively vacuolated cytoplasm, arranged in cord-and nest-like fashion in a myxoid matrix background. immunohistochemically, the tumor cells showed diffuse positivity for pancytokeratin (ae1/ae3), vimentin. the tumor cell nuclei were positive for brachyury, which is a key transcription factor of notochordal development. conclusion: these results confirmed the tumor to be an extraosseous chordoma in the paravertebral mediastinal region, which is rather an extremely rare location for usual chordoma. ps-07-020 testicular papillary mesothelioma: a case with borderline features s. mavropoulou * , z. tatsiou, p. nasos * general hospital, laboratory of pathology, xanthi, greece objective: well-differentiated papillary mesothelioma occurs rarely in the paratesticular region, with only a few published case reports. method: we describe a case of a 37-year-old man who initially presented with discomfort in the left testis and underwent resection of a hydrocele in the left testis. a hydrocelectomy was performed, during which a pedunculated mass, 2,5 cm in greatest dimension, was found attached to the testis. results: microscopically, the mass was composed of multiple branching papillary structures with fibrovascular cores covered by a single layer of low cuboidal to cuboidal cells with predominantly bland nuclear and cytologic features and rare microscopical necrosis. immunohistochemical staining for calretinin and cytokeratines 5/6 was positive and proliferative marker ki67 was <2 %. accordingly the diagnosis of a well-differentiated papillary mesothelioma was made. the patient has not received additional therapy and is disease free 15 months after diagnosis. conclusion: in conclusion, we report a rare case of a welldifferentiated papillary mesothelioma of the tunica vaginalis of the testis. the combination of benign and semimalignant characteristics can make the diagnosis of such a lesion problematic and pathologic distinction from malignant mesothelioma is crucial, although it may be difficult because of the variability of associated histologic features. objective: lipoma of the bone is a rare benign adipocytic tumor that arises intraosseous and rarely on the cortex or on the surface of the bone (parosteal lipoma). parosteal lipoma affects the long bones diaphysis of adults over 40 years old. method: we present the case of a 23 years old female with clinical diagnosis of femoral exostosis. conventional radiographs shows the presence of a 5 cm length area of lucency on the femoral metaphyseal surface with a periostal reaction at the base of the lesion. results: gross examination reveals a 5 cm osseous tumor, whitish on the surface and yellow on the section. on microscopic examination there are mature adipocytes with small foci of bone scattered throughout the adipocytes and hyaline cartilage at the perifery of the lesion. the gross and microscopic examinations correlated with conventional radiographs led us to diagnosis of parosteal lipoma. conclusion: this case is interesting being a rare bone tumor, the young age of the patient and the location on the surface of the bone. application of scanning acoustic microscope to evaluate lymph node lesions k. miura * * hamamatsu university, school of medicine, japan objective: a scanning acoustic microscope (sam) is a device that uses ultrasound (frequency, >20 khz) to image an object or tissue. because it is known that the harder the tissue, the more the speed of ultrasound, sam can provide data on the elasticity of cells and tissues. method: we compared lymph node lesions between acoustic and light microscopic images to evaluate the usefulness of sam. results: sam system discriminated lymph node components and demonstrated distinct acoustic images of the lymph nodes such as cancer metastasis, lymphomas, granulomatous diseases, and deposition diseases such as amyloidosis. areas with desmoplastic reactions associated with cancer invasion or post-inflammatory fibrosis showed the greater speed of sound than normal lymph nodes. these results corresponded well to those obtained using the conventional microscope. conclusion: sam provides the following benefits: (1) images are acquired in only few minutes without requirement for staining, (2) imaging pattern is similar to that of light microscope, and (3) speed of sound from each lesion is digital and statistical analysis is possible among diseases. although resolution of sam is little lower than that of light microscope, the sam can be an ancillary tool for histological diagnosis and clinical research. objective: parkinson's disease (pd) is considered one of the major neurological disorders of the population, and there is increasing data provides enough evidences confirming the involvement of free radicals and other reactive oxygen species (ros) in a number of physiological and pathological processes. the aim of the present study was to evaluate, the effect of therapy in patients with parkinson disease on biomarkers of oxidative stress, such as products of lipid peroxidation by two different methods: electron paramagnetic resonance and visible spectrophotometry. method: the study was performed in blood samples of patients with pd -with therapy, patients with pd -without therapy and healthy volunteers as controls. the products of lipid peroxidation were measured as malondialdehid (mda), spectrophotometrically by thiobarbituric acid (tba) method. the levels of lipid radicals were determinated ex vivo at room temperature on an x-band emx-micro spectrometer, bruker, germany. results: by the present study we reported higher levels of oxidative stress in pd patients without therapy compared to those with therapy. these results were comfirmed by the epr method. conclusion: the increase of oxidative stress, in pd patients' might be an additional reason for many secondary complications. objective: primary aneurysmal bone cyst (abc) is a histologically complex and mainly cystic lesion that accounts for 2 % of all primary bone tumors. information regarding its clinical presentation and management in hands and feet remains sparse. method: the medical records abc in hands or feet in hospital la paz pathology department from 1966 to 2011 were retrospectively reviewed and compared with existing data. we also propose pathological criteria for differential diagnosis between abc and giant cell reparative granuloma (gcrg). results: ten abc in hands or feet were identified, out of 78 (12.8 %), five tumors were in metacarpals, 4 in metatarsals and 1 in phalanx. radiographs showed expansile lytic lesions in metadiaphyseal region, sometimes with aggressive appearance. histologically, abc showed a mixture of blood-filled spaces with connective tissue septae containing osteoclast giant cells and foci of osteoid. scattered areas of so called "blue bone" were present in 5 cases (50 %). there were two solid variants. five patients underwent resection and 5 curettage, three of which relapsed (30 %). conclusion: abc should be considered in the radiologic differential diagnosis of hands and feet tumours because these lesions can even mimic malignancies. although clinicopathologically some authors consider gcrg is related to the abc solid variant, we believe they are different entities. carb-3 is the superior anti-cd15 monoclonal antibody in optimized protocol settings r. røge * , s. nielsen, m. vyberg * inst. of pathology, aalborg, denmark objective: immunohistochemical detection of cd15 is important in diagnosis of hodgkin lymphoma and may be relevant in classification of renal tumours. in four tests with 71-121 participating laboratories conducted by the nor-diqc external quality assessment scheme only 50-76 % of cd15 stains were sufficient, mainly because of too diluted primary antibody concentration, insufficient hier and less successful antibody clone. the purpose of this study was to evaluate three anti-cd15 antibodies based on vendor and inhouse optimized protocols. method: multitissue blocks with various malignant lymphomas, renal tumours and normal tissues (n=218) were stained with three concentrated (conc) antibodies (carb-3, mma and by87) according to predetermined in-house optimized protocols on two staining platforms. ready-to-use (rtu) solutions of carb-3 and mma were also examined. extension and intensity of stains was scored using the h-score method. results: carb-3conc with an in-house optimized protocol gave the highest h-scores in classical hodgkin lymphoma, renal tumours and normal kidneys. carb-3rtu and mmaconc gave slightly lower scores, while mmartu and by87conc gave the lowest scores and a large proportion of false negative reactions. all in-house optimized protocols gave better staining results than vendor protocols. conclusion: the importance of antibody selection and protocol optimization in immunohistochemical laboratories is emphasized. objective: studies show that there is association between stress and pathophysiological changes in reproductive system. the main aim of this study was to determine the pathological effects of immobilization stress on testes tissue and serum testosterone level in male rats. method: wistar rats were randomly divided into control, acutely or chronically immobilized groups of 10 in each. animals were immobilized for 2 h/day or 8 h/day for a period of 3 weeks or 1 week in chronically or acutely immobilized groups, respectively. after 6 weeks, blood samples were collected using cardiac puncture method. following serum collection, testosterone level was measured by radioimmunoassay method. the effect of immobilization stress on testes histology was also examined. data were statistically analyzed and compared between groups using anova. results: serum level of testosterone was decreased in acutely or chronically immobilized rats compared with control animals (p<0.001). in histological study, semeniferous tubules were significantly deformed and cellular concentration was reduced in immobilized rats compared with control animals (p<0.05). the number of spermatocytes, spermatids or sperms was also decreased in immobilized rats (p<0.001) and this pathologic change was more prominent in acutely than chronically immobilized rats. conclusion: this report underlines the importance of studying the archive material in order to thoroughly comprehend a single museum object. this handling of matters will help to turn anatomical collections into a unique teaching tool for modern medical practice and a noteworthy documentation of scientific, artistic and historical value. objective: extramedullary plasmocytoma is an uncommon plasma cell tumor localized preferentially in the upper aerodigestive tract, with no evidence of underlying multiple myeloma. it accounts for less than 1 % of head and neck tumours. method: we report a case of a 57-year-old male patient with reccurent left-sided epistaxis and nasal obstruction. ct-scan showed a left maxillary mass eroding the lateral wall of the nasal cavity. the tumor was surgically removed. results: histologic examination showed a diffuse infiltrate of neoplastic cells in the submucosa, arranged in a scant vascularized stroma. the neoplastic cells were large to medium-sized with amphophilic cytoplasm, irregular nuclei and proeminent, eosinophilic nucleoli. mitotic figures were frequent. imunohistochemical stains were performed in order to make a differential diagnosis: the tumor cells were positive for cd38 and cd138 and they expressed cytoplasmic immunoglobulin with kappa light chain restriction. most of the tumor cells were also cd56 and ema positive. conclusion: even though extramedullary plasmacytoma of the sinonasal tract is rare, it should be included in the differential diagnosis. the cooperation among the otorhinolaryngolists, pathologists and hematologists are required to manage the patients effectively in order to provide optimal treatment. objective: sinonasal malign melanoma is a rare antity, mostly arising from nasal cavity and accounting for <5 % of all sinonasal tract neoplasms. method: we report two patients complaining of nasal obstruction and epistaxis. endoscopy revealed partially haemorrhagic mass obliterating nasal passage. in one patient diagnosis was done as a plasmacytoma by pathologist in an another hospital. multiple biopsies were taken. microscopically, the tumour was consisted of medium sized cells, which had high nuclear to cytoplasmic ratio with pleomorphic nuclei containing eosinophilic nucleoli and intranuclear cytoplasmic inclusions. the cytoplasms contained variable amount of melanin pigment. mitosesincluding atypical forms-were easily detectable. there were mild inflammatory infiltrate against the tumour and rare tumour necrosis. one was showed pseudopapillary growth pattern and lymphovascular invasion. results: immunhistochemically, tumour cells showed immunreactivity for s-100, vimentin, hmb-45 and melan-a(mart-1). tumour cells were negative for cd-20, cd-3, cd-138, cd-56, sma, pan-ck and desmin. submandibulary lymph node metastasis was observed in one patient. this patient has undergone chemotherapy of temozolomide and 23 cures of regional radiotherapy. for the other patient, there were no metastasis detected by pet-ct. conclusion: sinonasal malign melanoma is a rare malignacy, but this entity could be kept in mind to avoid missdiagnosis with other tumors sharing similar morphology. objective: significant changes in the voice occur after 50 years. vocal quality is dependent on the vocal fold (vf) tissue biomechanics that derive from the extracellular matrix composition and organization. we studied muscularlamina propria interface of human vf in the aging. method: two authors evaluated density of vessels and the thickness of the deep and muscle layer by he and ihc (collageniv) with semiquantitative score. these data were validated by point-counting morphometric method. results: with the aging of the vocal folds we identified increased of vessels density in the muscle and deep layers, increasing the matrix density and thickness of the deep layer, and "dissection" of muscle fibers by dense connective tissue. the progressive structural changes in musclelamina propria interface play a crucial role in the remodeling and vocal quality. the increased density of vessels and matrix in aging may contribute to the preservation of vocal function by the physiological repairing. ps-09-012 "lymphoepithelioma-like" thymic carcinoma in parotid gland m. bugdayci * , g. atay, b. sozeri, g. guler tezel * hacettepe university, pathology, ankara, turkey objective: we report a 27-year-old woman presented with a slow growing painless mass beneath the right ear. a fineneedle aspiration revealed whartin tumour. the patient underwent parotidectomy and histopathological examination showed thymic carcinoma with areas of thymoma. method: histopathological examination was done according to conventional protocols. immunohistochemical stainings were performed using lsab methods. panck, cd3, cd20, ck5/6, ck7, vimentin, ki-67, cd5, bcl-2, cd1a, s100, sma, tdt antibodies were used. results: histopathologically, a well circumscribed, nodular, hybride neoplasm finely seperated from parotid gland had two components, uniform spindle cells in basaloid form lobulated by fibrous septae and large epitheloid cells with vesicular nuclei and meganucleoli, suggesting thymoma and thymic carcinoma, respectively. prominent lymphocytic reaction with germinal centers, high mitotic activity, small necrotic foci and microabcesses were also seen. immunohistochemisty revealed panck, vimentin, cd5 and cd1a positivity. the diagnosis of "lymphoepithelioma-like" thymic carcinoma was made. conclusion: thymic carcinoma is a rare tumor most commonly located in the anterior mediastinum. thymic carcinoma which originates from ectopic rests of thymic tissue caused by defective migration of the embryonic thymus, is extremely rare. in the presented case a thymic carcinoma was found in parotid gland which is an unusual site. objective: recent in vitro and in vivo studies have shown that several malignant tumors express an alternatively spliced variant of the receptor of growth hormone -releasing hormone (sv1), which operates by a ligand-dependent and independent manner. method: nine (9) adenocarcinomas, 11 pleomorphic adenomas and 11 warthin tumors were studied by immunohistochemistry for sv1 expression and visualized by diaminobenzidine staining. results: sv1 expression was cytoplasmic and was detected in 6/8 malignant (75 %) and 11/11 (100 %) warthin tumors. however, only 1/11 (9 %) pleomorphic adenomas expressed sv1 (p<0.05, x2-test). immunoreactivity ranged from mild to intense in all positive specimens, with the exception of warthin tumors at which only intense immunoreactivity was recorded. conclusion: our study, for the first time reports the presence of anti-sv1 immunoreactivity in tumors of the salivary glands. furthermore, the high association of sv1 expression with the malignant as opposed to the benign neoplasms implies a role of sv1 in the progression of the disease. a surprising finding of our study was the high positivity for sv1 exhibited by the warthin tumors that implies biological similarities between these histopathological entities of the salivary glands. these results imply that the use of antagonistic analogs of ghrh merit further investigation. objective: sialadenoma papilliferum (sp) is a rare, benign neoplasm of salivary gland origin which manifests as an exophytic papillary excrescence of the mucosa. indeed, sp is both an exophytic proliferation of papillary stratified squamous epithelium above the mucosal surface and an endophytic salivary ductal proliferation beneath the mucosa. it arises predominantly in minor salivary glands and usually affects patients in the age range of 32-87 years, with reports in young patients being exceedingly rare. method: we report a case of a previously healthy 20 yearold man diagnosed with a nodular mass in the upper lip buccal mucosa. the tumour was excised and submitted for microscopic examination. results: histologic examination revealed a biphasic proliferation of papillary stratified squamous and salivary ductal epithelia, both underneath the mucosal surface. conclusion: in this unique case, as the classical sp, the tumour has a biphasic proliferation of squamous and ductal epithelia. however, unlike the classical sp, both epithelia grow under the mucosal surface. as a result, it didn't manifest as an exophytic proliferation, but as a nodule. we excluded squamous papilloma, inverted ductal papilloma, intraductal papilloma and mucoepidermoid carcinoma, the principal entities in the differential diagnosis of sp, and concluded it to be a sp with inverted pattern. interest of histopronostic classification in three grades in the therapeutic management of primary epithelial parotid carcinoma v. results: microscopic examination showed a biphasic tumor with a prominent myxoid stroma and tumor cells with clear cytoplasm involving pleural tissue. immunohistochemistry showed tumor cells positive for keratin, s100 protein, smooth-muscle actin, p63 protein, vimentin. histological and immunohistochemical features confirmed the diagnosis of epithelial-myoepithelial carcinoma. the patient underwent a right parotidectomy 3 years earlier for removal of 3.5 cm mass diagnosed as emc. conclusion: epithelial-myoepithelial carcinoma of salivary gland is a rare low-grade malignant neoplasm with a potential for local recurrence and metastases. rare metastases for lungs, kidney and brain were reported. we described an additional case of pleural metastasis. objective: salivary defense system recognized as an important protective factor of the child oral environment. research data reveal that bacterial infection, dental caries and periodontal diseases have a higher incidence in cases of salivary glands dysfunction. the aim of present study was to find out the morphological peculiarities of the fetal parotid gland in cases of restrictions of its intrauterine growth (iugr) at late gestation. method: parotid gland of twenty human fetuses with iugr from the late spontaneous abortions material were compared with fifteen fetal glands in cases of induced abortions due to psychological reasons (control group). tissue samples were stained with hematoxylin and eosin. stereological examination was done to find out volume fractions of parotid gland structural components. results: results have shown the reduction of the area of acini, large collected ducts, striated and intercalated ducts in iugr group. the volume fractions of vessels were also lower than on controls. the foci of immature secretory lobes in cases of growth restriction occupied wider zones within loose, poorly cellular, fibrous stroma. conclusion: our study demonstrates the delay of the parotid gland structural maturation in pregnancies complicated with iugr. impaired growth and secretory gland's dysfunctions may cause pathological changes in oral ecosystem of a child. objective: dermal fillers are injectable products commonly used in aesthetic medicine. this type of treatment, which often replaces traditional surgical procedures provides satisfactory cosmetic results. it is known, however, that there is a risk of undesired effects at the site of injection of the product or even at a distance. literature reports numerous cases of orofacial injuries caused by dermal fillers. the aim of this study is to evaluate two of the most widely used facial dermal fillers for aesthetic purposes in an effort to identify adverse reactions they may produce. method: two of the most commonly used materials for aesthetic purposes by dermatologists and plastic surgeons, polymethylmetacrylate and hialuronic acid were injected in rat tongues: 10 % polymethylmethacrylate (n = 16) and 20 mg/ml hyaluronic acid (n =18), compared to an inert solution for control (n=16). after 7, 60 and 90 days, local clinical and histological alterations were analyzed. results: the following factors were verified: intensity of the inflammatory response (h&e), amount of newly formed blood vessels (ihc) and macrophages and collagen fibers density (picrosirius). results showed that both filling materials triggered local inflammatory response to a greater or lesser degree. objective: the aim of this study was to compare clodronate and zoledronic acid regarding their influence on the repair of surgical wounds in maxillae (soft tissue wound and tooth extraction) and their relation to osteonecrosis. method: thirty-four wistar rats were allocated into three groups according to the treat-ment received: (i) 12 animals treated with zoledronic acid, (ii) 12 animals treated with clodronate and (iii) 10 animals that were given saline solution. all animals were subjected to tooth extractions and surgically induced soft tissue injury. histological analysis of the wound sites was per-formed by means of hematoxylineosin (h&e) staining and immunohistochemical staining for receptor activator of nuclear factor-kb ligand (rankl), osteoprotegerin (opg), von willebrand factor, and caspase-3. results: the zoledronic acid group showed higher incidence of non-vital bone than did the clodronate group at the tooth extraction site. at the soft tissue wound site, there were no significant differences in non-vital bone between the test groups. rankl, opg, von willebrand factor, and caspase-3 did not show significant differences between the groups for both sites of surgical procedures. conclusion: both of the bisphosphonates zoledronic acid and clodronate are capable of inducing maxillary osteonecrosis. immunohistochemical analysis suggests that the involvement of soft tissues as the initiator of osteonecrosis development is less probable than has been pointed out. objective: the aim of this study was to evaluate the expression of cd1a, cd4, cd8, cd20, caspase-3, tryptase and basement membrane thickness in oral lesions of lichen planus (lp) and lupus erythematosus (le). method: oral lesions of lp (n=21) and le (n=23) were biopsied. after confirmation of diagnosis, the specimens were submitted to pas and immunohistochemistry. results: cd1a expression was significantly greater in epithelium and connective tissue in lp. epithelial cd4 and cd20 did not differ between the diseases, but were greater in connective tissue in lp. cd8 expression was greater in both epithelium and connective tissue in lp. caspase-3 did not differ between the groups, whereas tryptase was greater in le epithelium. basement membrane thickness did not differ between lp and le. conclusion: cd1a, cd4, cd8, cd20, caspase-3 and tryptase expression and the basement membrane thickness are not definitive criteria for differential diagnosis of oral lichen planus and lupus erythematosus. objective: odynophagia may be caused by inflammatory/ infectious conditions and tumors arising within the oropharynx. we present an unusual case of a follicular dendritic cell sarcoma (fdcs) of the tonsil manifesting with odynophagia. method: an 80 year-old man presented with odynophagia refractory to medical therapy; a 0.9 cm left tonsillar mass was noted on inspection. bilateral tonsillectomy was performed. a panel of antibodies was applied on paraffin tissue section. results: grossly, the left tonsil featured a 1.2 cm well circumscribed grey lesion. histologic examination showed a proliferation of spindle to ovoid cells arranged in fascicles with swirling qualities admixed with scattered lymphocytes. frequent mitoses and apoptotic bodies were seen but no "tumoral" necrosis was recognized. tumor nests were separated by thin stromal septi containing a prominent glioblastoma-like microvascular proliferation with focal formation of glomeruloid tufts. the tumor cells were immunoreactive for cd21, cd23 and vimentin. objective: salivary gland tumors reveal a broad morphology and immunohistochemistry may be helpful. method: we examined 12 mucoepidermoid (mec), 8 adenoid cystic (adc), 3 asinic cell (acc) and 4 salivary duct carcinomas (sdc); 2 myoepitheliomas (me), 5 basal cell (bca), 31 pleomorphic adenomas (pa) and 18 warthin tumors (wt) for sma, calponin, s100, cd10, gfap, p63, cea, gcdfp15, glut1, 34be12, ck14, ck19, cd117 and galectin3, using tissue microarray. results: tumors expressing myoepithelial markers were adc (sma, calponin), bca (sma, calponin, p63), pa (sma, calponin, s100, cd10, gfap, p63), me (s100, cd10, gfap), mec (p63) and wt (p63); pa being the only tumor expressing all of the markers in the panel. gfap, s100, ck14, p63, ck5/6 and galec-tin3 expressions were higher in benign and ck19 was higher in malignant tumors (p < 0.05). some of our findings helping in differential diagnosis are as follows: in differentiation of pa from adc: gfap, cd10, gcdfp15 positivity; higher expression of s100; lower expression of ck14 and cd117 favors pa. in differentiation of pa from bca: gfap and cd10 positivity favors pa. in differentiation of mec from sdc: cd10, ck14, p63 and ck5/6 positivity favors mec. conclusion: we conclude that salivary gland tumors may be well characterized by using distinct markers in each differential diagnosis. objective: lobulary capillary hemangioma (lch) is a benign, rapidly growing vascular lesion of the skin and mucous membrans. it may rarely present as a mass of considerable size and thus entirely fill the nasal cavity. it occasionally appears in the nasal region as a pedunculated or broad base mass. trauma and hormonal influences are the most common presumed etiologic factors. lch usually involves the gingiva, lips, tongue, and buccal mucosa. the most common symptoms are epistaxis and nasal obstruction. method: in this report; a case of 14 years old boy operated surgically of lcd of the nasal septum, which occurred post delivery was described. results: on examination, a red pedunculated bloody swelling 3,5*2 cm was noted arising from the posterior part of the nasal septum to nasopharinx. histopathologically lch has characteristics consistent with lobular prolifeation of capillaries in a fibromyxoid stroma. conclusion: we emphasise that rarely seen lch must be kept in mind in the differantial diagnosis of a rapidly growing mass, who is cured surgically. follow up 2 months later showed no recurrance. although the head and neck is not an uncommon region, the nasal cavity is extremely rare sites for lch in children. objective: claudins are integral transmembrane proteins of tight junctions, critical for maintenance of cell adhesion and polarity. altered claudin expression has been detected in carcinomas and correlates with tumour progression. actinic cheilits is a pathologic condition that affects the lower lip, caused by chronic exposure to solar radiation; it corresponds to the early phase of squamous cell carcinoma. we investigated claudin patterns in phases of actinic cheilitis, as alterations in adhesion molecules are considered important during the progression of this process. method: immunohistochemistry against claudins −1, −3, −5, −7 and −11 was performed in 100 cases of actinic cheilitis/squamous cell carcinoma; results were analysed qualitatively. results: actinic cheilitis (low grade intraepithelial squamous cell carcinoma), and the invasive front of superficially invasive squamous cell carcinoma and in situ squamous cell carcinoma were negative for claudins −1, −3 and −7. claudin −5 was present on all epithelial layers in most cases evaluated. claudin −11 was present in all epithelial layers in actinic cheilitis, but negative in cases of superficially invasive and deeply invasive squamous cell carcinoma. conclusion: altered expression of claudins is present in ac from its incipient phase throughout the progression of the disease to invasive squamous cell carcinoma. objective: metastases to the jaw bones are uncommon and are most likely to arise from primary lung, breast, prostate or kidney tumours. jaw bone metastases from a primary oesophageal carcinoma are especially rare, with only seven reports published in the literature. method: here, we describe the case of a 69 year-old male patient where 7 years elapsed between the diagnosis and successful treatment of a poorly differentiated, stage pt2n0 primary oesophageal adenocarcinoma and re-presentation with jaw pain due to a metastatic mandibular deposit. results: the morphological appearance of the metastasis and immunohistochemical positivity with ck20, ck7 and cdx2 strongly supported an adenocarcinoma of upper gastrointestinal tract origin. conclusion: this case is of particular interest as there is an unusually long time between the detection of the primary oesophageal adenocarcinoma and diagnosis of metastatic disease. the longest period of time we have found for this reported in the literature is 9 months, although it is known that some oral metastases may appear more than 10 years following the primary tumour diagnosis. melanotic oncocytic metaplasia of the nasopharynx -a report of three cases and review of the literature j. s. lee * , j. y. na * chonnam national university, hwasun hospital, pathology, jeollanam-do, republic of korea objective: melanotic oncocytic metaplasia of the nasopharynx is a rare condition which is characterized by the presence of usually a small, brown to black colored pigmented lesion around the eustachian tube opening. although it is a benign lesion, it may be clinically misdiagnosed as malignant melanoma. microscopically, melanotic oncocytic metaplasia is a combination of oncocytic metaplasia of the epithelium of the gland and melanin pigmentation in its cytoplasm. method: in our present study, we report three cases of melanotic oncocytic metaplasia of the nasopharynx. results: all the three cases occurred in men and were presented as multiple black pigmented lesions around the torus tubarius. microscopically, mucous glands with diffuse oncocytic metaplasia and numerous black pigments were observed. no cellular atypia was observed. immunohistochemically, the scattering of s-100 proteinpositive, and hmb-45-negative dendritic melanocytes was evident. conclusion: this is the first report of cases of oncocytic metaplasia of the nasopharynx in korea. objective: angiokeratoma is a rare cutaneous lesion. mucosal involvement is occasionally found as part of a more generalized cutaneous disease. isolated angiokeratoma in the oral mucosa is extremely rare with only a few cases reported thus far. method: we report the case of a 61-year-old female presented with a painless lesion on the left bucal mucosa, of 4 months duration. on clinical examination, a solitary purple lesion of approximately 4 mm in diameter was found. results: the lesion was excised and the histological examination revealed a tumour involving lamina propria, composed of large dilated vascular spaces, lined by normal appearing endothelium and filled with blood or with fibrin thrombi. the overlying epithelium showed variable degree of acanthosis and hyperkeratosis. accordingly the diagnosis of angiokeratoma was made. on clinico-laboratory examination no angiokeratomas were found anywhere on the skin as well as no other malformation or metabolic disorder. the patient received no further treatment and 2 years later remains disease free. conclusion: in conclusion we report a case of a solitary angiokeratoma of the oral mucosa. although rare, it should be included in the differential diagnosis when evaluating any lesion in this location and further investigations should be performed to rule out a metabolic or systemic disease. results: histopathologically in macroscopy, the cystic lesion was determined as a small part of tissue in white -grey color, in sizes of 0,6×0,5×0,3 cm. in microscopic evaluation, the epithelial tumor cells were formed mostly tubular, partially in solid form and broadly making pseudopapillary pattern in stromal tissue. in the luminal structures formed by tumor cells, there has been mucin as dark basophilic stained with pas/alcian blue (ph: 2, 5). the tumor cells are strong and have common positivity with ck7, and have negativities with ck20 (b-sa method). in the focal area that the tumor cells got the solid pattern, synaptophysin and chromogranin to evaluate the neuroendocrine differentiation wasn't determined. conclusion: the lesion in the medial part of the tympanic membrane of the patient was reported as colesteatom and the lesion in the middle ear was reported as middle ear adenoma. histopathologic evaluation for helicobacter pylori as a possible etiopathogenic factor in chronic tonsillitis e. özgün * , d. altinel, a. albayrak, a. tan, s. sayhan, n. bozlak * nevehir devlet hastanesi, dept. of pathology, turkey objective: helicobacter pylori is the major gastric pathogen which has an important role in the etiopathogenesis of chronic gastritis. we investigated the presence of helicobacter pylori as an extragastric reservoir in the tonsillectomy specimens to display if it is an etiologic factor in the development of chronic tonsillitis. method: in the current study, 100 cases with chronic tonsilitis were examined in bilateral tonsillectomy specimens. objective: salivary duct carcinoma (sdc) is a rare, aggressive malignancy with poor prognosis. its histomorphology is distinctly reminiscent of the ductal carcinoma of the breast. method: a 48-year-old man was admitted with a mass of the right preauricular area. the mass had been enlarging steadily for the past 7 months. computed tomography (ct) of the neck revealed 4×3×3 cm contrast enhancing solid mass with irregular borders at the right parotid region. thorax ct, abdominal and thyroid ultrasonography were unremarkable. as the aspiration cytology was malignant right parotidectomy and right cervical lymphadenectomy was performed. results: histopathological examination showed a mixture of ducts, nests and cords of cells often embedded in a desmoplastic stroma with comedonecrosis of some ductal structures. the tumor cells were polygonal with vesicular nuclei, prominent nucleoli and eosinophilic, oncocytic cytoplasm. the tumor margins were lobulated and irregular with perineural invasion. mucin stains were negative. we noted high proliferative index and cerbb2 overexpression. the tumor was classified as a salivary duct carcinoma, oncocytic variant of parotid gland. the resection margins were negative and the lymph nodes were reactive. conclusion: as high proliferative index and cerbb2 overexpression are predictive factors, close clinical follow up is recommended for the risk of local recurrences and metastasis. immunohistochemical expression of bcl-2 and ki-67 in oral lichen planus and leukoplakia with different degrees of dysplasia f. pigatti * , l. a. de assis taveira, c. t. soares * university of são paulo, oral pathology, brazil objective: the oral lichen planus (olp) is a chronic inflammatory disease of unknown cause, and its malignant potential is a very controversial issue. therefore, the aim was to evaluate the immunohistochemical expression of apoptosis-related proteins and cell proliferation in olp and epithelial dysplasia in order to investigate changes related to carcinogenesis and emphasize the importance of long-term follow-up of patients with olp. method: for this purpose, we selected 14 samples of olp, 14 samples of leukoplakia with epithelial dysplasia, and 09 samples of normal oral mucosa as controls. the evaluation of the expression of bcl-2 and ki-67 was conducted in accordance with the immunoperoxidase technique. results: there was also a high expression of blc-2 protein in inflammatory cells in olp lesions and leukoplakia with epithelial dysplasia. the expression of ki-67 marker was higher in all analyzed tissue levels in the lesions of olp and leukoplakia with epithelial dysplasia when compared with the control group. objective: pleomorphic adenoma (pa) is the most common benign salivary gland neoplasm of the major and minor salivary glands. pleomorphic adenoma was shown sometimes to undergo malignant transformation in its natural course. carcinoma ex pleomorphic adenoma (cpa) is a rare salivary gland malignancy that may develop from either a long-standing primary or a recurrent pa. the genetic mechanisms involved in the progression of adenoma to a carcinoma is still unclear. to identify the predictors of disease, more knowledge about their genetic profiles is necessary. this study aimed to characterize alteration in the dna copy number of pa and cpa. objective: leukoplakia is an oral premalignant white lesion. although its etiology may vary, smoking has been implicated as a possible risk factor. the p27 protein has been shown to inhibit kinases, and it is known that its expression is decreased during carcinogenesis. the reduced expression of p27 has been correlated with poor prognosis in carcinoma. in this study a role for the smoking habit in the expression of this protein was investigated. method: forty cases clinically diagnosed as oral leukoplakias and that presented a mild to intense degree of epithelial dysplasia and could not be diagnosed as any other disease were selected. twenty cases were from current smokers (more than 20 cigarettes/day for at least 1 year) and neversmokers. thirty-six cases of leukoplakia without dysplasia were used as controls. histological sections of each lesion were subjected to the estreptoavidin biotin immunohistochemical method for detection of p27. results: a semi quantitative analysis was performed and the results showed that the expression of p27 was independent of the smoking status of the patient (p=0, 5237), using kruskal-wallis and mann-whitney tests. although not statistically significant, due to the small number of cases, the results indicate that the counting of p27 in leukoplakia correlates inversely with degree of epithelial dysplasia. low-level laser therapy may influence on the akt/mtor signaling pathway in oral cancer cells f. sperandio * , f. giudice, l. correa, d. pinto jr., s. de sousa * university of sao paulo, dept. of oral pathology, brazil objective: distinct cells respond differently to low-level laser therapy (lllt), while the exact molecular mechanisms involved in cell proliferation or growth inhibition, after light stimulation remain poorly understood. although lllt has shown promising results in accelerating wound healing and preventing or treating oral mucositis, there is no evidencebased consensus of what this energy could cause in cancer cells. this should be highly pondered when an oral cancer patient is treated with lllt due to oral mucositis, for example. method: two tongue squamous cell carcinoma cell lines (scc9 and scc25) were utilized to find the effect of low-level laser irradiation on the akt/mtor signaling pathway. laser irradiation (660 and 780 nm) consisted on 40 mw of power and energy densities of 2, 3 and 6 j/cm2. after a single irradiation the most significant energy densities found with mts assay were employed to analyze akt/mtor signaling pathway related proteins through immunofluorescence and western blotting. results: beyond modifying the growing rates of cancer cells, low-level laser irradiation was able to induce different variations in the studied pathway, however a direct correlation among the proteins was not found. conclusion: lllt may act on akt/mtor/cyclin d1 signaling pathway, which has a widely recognized role in head and neck cancer progression. mucins as predictors of recurrent pleomorphic adenoma of salivary glands: an immunohistochemistry analysis of over 60 cases t. teshima * , r. ianez, c. coutinho-camillo, s. lourenço * são paulo, brazil objective: pleomorphic adenoma (pa) is the most common tumor of salivary glands and, despite its benign behavior, the recurrence after primary surgery is significant. in attempt to find a marker capable to predict the recurrence of this lesion, this study aims to analyze the expression of mucins muc 1, 2, 4, 5 ac and 6 in 62 cases of pa, considering that mucins have been related to tumour growth of some organs. method: this study was performed in 62 cases in pa, which 5 of them presented recurrence after the initial surgery. all the primary tumors were histologically processed and submitted to the immunohistochemistry reaction. the antibodies for muc 1, 2, 4, 5 ac and 6 were used and then analyzed with conventional optical microscope. results: muc1 was the only mucin significantly expressed in most of all cases (88 %), being present within ductal lumen and cytoplasm. the other mucins showed a focal positivity in few cases, where muc 2, 5 ac and 6 were cytoplasmic, while muc4 was expressed in ductal lumen and blood vessels. objective: drug resistance remains a major problem in the treatment of nsclc cancer patients for both, conventional chemotherapeutic and novel biological agents. intrinsic or acquired resistance is caused by a variety of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. recent data showed that drug resistance mechanisms might also be regulated by micrornas (mirnas). method: we tested 65 lung cancer samples. the total rna was isolated from individual specimens and then rt-pcr and pre-amplification were performed. we detected levels of mir-21, mir-23a, mir-23b, mir-126, mir-205, mir-335*, mir-3163, mir-491-3p, mir-548p, mir-548x, mir-576-5p, mir-590p, mir-655, mir-656 and mir-944 using taq-man® microrna assays by lightcycler® 480 real-time pcr system. we assigned obtained results using statistical methods. results: our results suggest that mir-590-5p and mir-655 are involved in apoptosis. the higher levels of mir-590-5p correlate with bcl-2 positivity and pro-apoptotic protein bax seems to be regulated by mir-655. the level of lrp protein responsible for drug resistance in lung cancer patients seems to be regulated by mir-255. conclusion: within our cohort of nsclc patients we did not find any correlations between the expression profiles of the abovementioned mirnas and survival. the modified methacarn fixation as an excellent preservation of histology, protein immunoreactivity and rna integrity in paraffin embedded tissue specimens p. babal * , r. milcheva, p. janega, p. celec * comenius university, dept. of pathology, bratislava, slovakia objective: fixation techniques preserving morphological fidelity, immunoreactivity and integrity of nucleic acids may have a high impact on both basic and applied research and diagnostic pathology. we investigated the effect of formalin, absolute ethanol and methanol; ethanol supplemented with acetic acid and modified methacarn fixative on the tissue morphology and immunoreactivity of different types of tissues and the preservation of rna fragments of different lengths. results: the modified methacarn fixation provided a histomorphological quality comparable to the formalin-fixed tissue specimens. the immunoreactivity was superior in the buffered than in the untreated formalin and the preservation of protein antigenicity in normal and pathologically changed tissues tested with several antibodies was excellent with the use of alcohols with acetic acid. the acidic ethanol and the modified methacarn fixative showed the best preservation of the integrity of rna in satisfactory quantity and quality of fragments up to 577 bp, which was reliable for relative evaluation of gene expression. objective: cflip prevents the apoptosis by caspase 8 inhibition, its overexpression correlates with the progression of different tumors. thymomas and thymic carcinomas are thymic epithelial tumors, in which the regulation of apoptosis is still unknown. we investigated the role of cflip in regulating the viability of thymus carcinoma cell line 1889c using an rna interference. the cell line hacat was used as a control. method: 1889c and hacat cell lines were transfected with an established "short hairpin" pires/puro cflip-shrna expression vector. cflip suppression and its effect on pro and antiapoptotic molecules were analyzed using q-pcr and western blot. apoptosis was quantified using the flow cytometry. results: the 1889c-shcflip, but not the shcflip-hacat, showed sensitivity to trail (tumor necrosis factor-related apoptosis-inducing ligand), accompanied by an overexpression of both the pro-apoptotic protein noxa (p<10-4) and the anti-apoptotic proteins birc2, birc3 and xiap compared to the non-transfected cells (p<10-4). conclusion: cflip shrna seems to induce apoptosis in the thymus carcinoma cell line. simultaneously antiapoptotic proteins (aips) birc2, birc3 and xiap were up regulated in order to protect the cell from apoptosis. could these aips provide the way to escape the cell death? aips selective inhibitions could represent a promising therapeutic approach for malignant thymic carcinomas. objective: nucleolin is a multifunctional dna-, rna-and protein-binding protein, involved in fundamental aspects of transcription, cell proliferation and growth. it is located in the nuclei/nucleolus, cytoplasm and on the cell surface. the present study aimed at optimizing the histological identification of nucleolin. method: mamary invasive ductal carcinoma 3 μm paraffin sections were pretreatead with pronase at room temperature (5-10 min) to epitope exposure. biotinilated peptide against nucleolin was applied (1/1.5 mm dilutions), incubated at room temperature for 30 min/overnight at 4°c; after phosphate-buffered saline, peptide binding was identified by peroxidase-conjugated streptavidin and 3,3-diaminobenzidine tetrahydrochloride applied according to manufacturer's instructions before haematoxylin counterstaining. results: pretreatment for 5 min at 1.5 mm of biotinilated peptide showed only nuclear expression, as 1 mm/10 min pretreatment with overnight peptide incubation; positive cytoplasmic expression was obtained also after 30 min incubation/room temperature. at 1.5 mm concentration for 10 min epitope retrieval, cytoplasmic and nuclear positivity raised over 75 % expression, independently of peptide exposure (overnight/room temperature). conclusion: for optimizing peptides/antibodies, we have to be aware that nuclear/cytoplasmic expressions depend on digestion and peptide concentration. histological morphology is also important and discordant results may be erroneously obtained. ps-10-013 kras testing in clinical laboratory: optimizing targeted therapy l. cheng * , l. miravalle * indiana university, dept. of pathology, indianapolis, usa objective: activating mutations in the kras gene are found in more than 30 % of colorectal tumors, where they are associated with a poor response to anti-epidermal growth factor receptor therapies. mutation testing techniques have therefore become an urgent concern. several methods for kras mutation detection have been described in the literature. most of these are laboratory developed tests and only a few commercial assays are currently available. method: we studied the performance characteristics of a kras mutation detection assay on the abi-3130xl genetic analyzer using a new commercial mutation detection kit. samples were analyzed in parallel by different reference laboratories using alternative methodologies. various sample types were used including formalin-fixed paraffin-embedded tissue, fine-needle aspirates, and cyst fluid specimens. results: a high level of agreement (100 % correlation for formalin-fixed paraffin-embedded tissue and fine-needle aspirate samples and 93 % correlation for cyst fluid specimens) was obtained despite the use of different methodologies. conclusion: shift termination assay is a simple, robust, and sensitive method for the identification of kras mutations in wide variety of specimen types. objective: micrornas (mirs) are small rnas that modulate protein expression via post-transcriptional regulation of mrna. they are related to malignancy in several tumors. deregulation of mirs expression has been described in high grade astrocytomas of adult patients, however there is scarce information in pediatric patients. in this work we quantified the expression profiles of mirs in pediatrics astrocytomas. method: total rna was extracted from 59 astrocytomas and 8 normal brain (nb) tissues, formalin fixed paraffin embedded. the expression levels of mir-124, mir-128-1 and mir-221 were quantified by using mirna-specific taqman mirna assays. results: the mir-128-1 was more abundant in nb against to mir-124 and -221 (p<0.05). mir-124 and mir-128-1 were significantly down-regulated in all grades compared to nb (p<0.05) but in grade iv was more decreased: 8000-and 1200-fold, respectively. in patients with recurrent tumor, the expression of mir-221 was lower (p<0.002). live patients expressed high levels of mir-128 (p< 0.02) and mir-221 (p < 0.0026) against deceased. conclusion: micrornas are differentially expressed between astrocytomas and nb. the low expression of mir-128 and -221 could be a potential marker in recurrent pediatric astrocytomas, and both are associated to less survival. embedded tissue is useful to describe and evaluate expression of molecular markers of malignancy. objective: isolation of genomic dna from formalin fixed paraffin embedded (ffpe) tissues is a critical step for molecular diagnostic assays. horizon diagnostics has generated ffpe cell line reference standards containing defined mutant allelic frequencies, enabling the quality control of both assay sensitivity and dna extraction. method: a panel of x-man™ (gene-x, mutant and normal) cell lines were developed using our patented gene editing technology (genesis™) including; b-raf v600e, v600k; egfr δe746-a750, t790m, l858r, l861q; k-ras g12a, g12c, g12d, g12r, g12s, g12v, g13d; pi3kα e542k, e545k, h1047r. ffpe blocks containing specific allelic frequencies including: 50 %, 33 %, 25 %, 20 % or 5 % were generated and sections cut. dna was extracted using five different extraction methods and analysed using droplet digital™ pcr. results: mutant alleles could be detected using droplet digital™ pcr at each defined allelic frequency and the reproducibility of each test was very high. the allelic frequency was consistent throughout each block. the total dna yield from each section was consistent using the same extraction method but varied between methods. conclusion: this study has demonstrated horizon diagnostic's ffpe reference standards have a highly accurate defined mutation specific allelic frequency together with a consistent dna quantity. transcript variants and isoforms of the phosphatase subunit ppp2ca and its regulatory binding partners in haematological malignancies g. grech * , c. saliba, b. shawn, r. avellino, p. j. m. valk, r. delwel * university of malta, dept. of pathology, msida, malta objective: the importance of feedback mechanisms involved in suppression of growth factor-induced signals is gaining importance both to understand molecular mechanisms of disease and also as potential therapeutic targets. our previous studies show that erythroid differentiation can be blocked by constitutive expression of the pp2a inhibiting subunit, alpha4. the aim of this study was to identify variants and transcript isoforms of ppp2ca and the inhibiting subunits alpha4 and set, using (1) cell lines derived from haematopoietic disease, and (2) objective: epithelial cells grow by proliferation and adhesion, which will form an organ/tissue. stat3 is involved in cell proliferation and various cellular events. in the present study, we studied the role of stat3 in cell adhesion in hepatocytes. method: stat3 knockout cells (s3ko cells) and liverspecific stat3ko (l-s3ko) mice were generated. cellular adhesion was analyzed by light/electron microscopy. expression of adhesion molecules was examined by immunohistochemistry and western blot analysis. protein expression and cellular adhesion were also examined in the pre-/post-hepatectomy liver tissue in control and l-s3ko mice. results: mrna and protein of e-cadherin were not expressed in s3ko cells. il-6 up-regulated e-cadherin in control cells but not in s3ko cells, and induction of constitutively active mutant of stat3 restored e-cadherin level in s3ko cells. interestingly, membrane-bound beta-catenin expression was not affected, but released to cytosol in s3ko cells. s3-ko cells showed almost normal proliferation but did not form cell cluster due to lack of e-cadherin. electron-micrograph confirmed lack of intercellular adhesion structures (desmosome) in s3ko cells. also in l-s3ko mice, hepatocytes lack desmosome structure and cell adhesion. objective: alk positive anaplastic large cell lymphoma (alcl) is characterized by anaplastic lymphoma kinase (alk) expression, most commonly associated with the t (2;5)(p23;q35), fusing the alk and nucleophosmin (npm) genes. however, in a significant proportion of cases the alk gene has a number of other than npm translocation partners. the aim of the study was to detect the npm/ alk fusion gene and the other possible fusion genes. method: npm/alk was detected by rt-pcr, npm/ alk negative lymphomas were analyzed by alk specific rapid amplification of 5´cdna ends (5´race). we prepared q-rt-pcr assay for quantification of 3á lk mrna. molecular findings were correlated with i-fish. results: we analysed 42 alk positive alcl. chromosomal breakpoints affecting the alk locus were detected by i-fish in all patients. the npm/alk was detected in 32/42 patients. 5´race identified atic/alk in 3, cltc/alk in 2 patients. other fusion genes (tpm4/alk, tpm3/alk, alo17/alk) were found in one patient. in all specimens, overexpression of 3´alk mrna suitable for the minimal residual disease (mrd) detection was found. objective: a 17 year-old girl, presented with an osteolytic epiphyseal lesion of the distal ulna. based on histopathological examinations, the lesion was classified as giant cell tumor of bone (gct). four years later, the girl developed a second lesion at the same site. the histopathological examination revealed aneurysmal bone cyst (abc) with solid spindle celled and giant celled areas which raised a question of a recurrent gct or a primary abc. the aim of this study was to analyze the clonality of these two processes. determination of maternal and paternal x chromosome activation status is useful in the diagnostic analysis of nonrandom x inactivation patterns. method: the human androgen receptor (humara) gene polymorphism assay was used to identify the clonality of these two processes. results: the patient was identified as heterozygous for the humara allele. the gct and abc samples exhibited a monoclonal pattern, but one of them was with maternal and the other with paternal x chromosome origin. conclusion: the findings of this case report demonstrate the clonal behavior of both lesions with different clonal patterns. the above investigation proved to be helpful in distinguishing between recurrent gct and de novo abc in the field of previous surgical intervention. . in all specimens gen her2/neu was quantifed agains reference gen by qrt-pcr method (lyghtcycler® 480 ii, roche). results: the results of cytogenetic analysis have been showed in 40 % that didn´t corespond with the results of qrt-pcr quantification. on the contrary the results of ihc was correlated with the results of qrt-pcr in 100 %. methods sish vs fish has in 25 % different of results. in regard to the quality of ish signals the sish is more preferable for determination her2/neu. conclusion: a pilot study conducted on a group of 20 patients showed that among the methods used in the process of determining her2/neu amplification are significant differences, even in evaluation and interpretation of test results. regardless of the histological type of tumors (diffuse and intestinal type) as problematic for the evaluation of samples proved to be endoscopic route. the use of molecular methods in diagnosis of malignant melanoma of biopsy s. libor * , p. dundr, s. lísová, c. povýšil * general faculty hospital, inst. of pathology, prague, czech republic objective: distinction between benign and malignant melanocytic lesions commonly represents a big challenge for the pathologist. in this case it appears as a useful auxiliary method of fluorescence in situ hybridization (fish). the aim of this study was by using fluorescently labeled probes detection of numerical changes occurring in malignant melanoma and thus supply or confirm the diagnosis. method: the retrospective study included 14 samples tissue with an established histological diagnosis (6x malignant melanoma, benign melanocytic lesions 8x). for detection was used probe vysis lsi rreb1/lsi myb/lsi ccnd1/ cep6 (abbott molecular, usa), olympus ax70 fluorescent microscope (immersion lens 1000x). in the area of the tumor were calculated signals of individual probes and evaluated according to the manufacturer's instructions. results: evaluable results were obtained in 10/14 cases (71 %) of the melanomas were 4 and 6 benign melanocytic lesions. in all four cases of malignant melanoma have been burdened with genetic changes, mainly in the ccnd1 (in 55 %) gene amplification and preb1 (in 40 %) and myb (in 5 %). in contrast, for all 8 benign melanocytic been demonstrated normal findings. conclusion: dna abnormalities detected by fish occurr in the vast majority of malignant melanomas but are not seen in benign nevi. this fact make the fish test an important extra step in the differential diagnosis melanocytic lesions with ambiguous or borderline histological findings. objective: egfr is a receptor on the cell membrane with tyrosine-kinase activity and which is a regulator of proliferation, apoptosis, angiogenesis, tumor invasion. he is found to be overexpressed in some lung cancer histological subtypes. men1 is a tumor suppressor gene, with a role in cellular growth and differentiation, dna reparation, and apoptosis. method: surgically resected specimen from 99 patients (men n=66; women n=33, age 57±11) with lung cancer were studied: carcinoid tumors (ct)-23, small cell lung carcinomas (sclc)-13, large cell neuroendocrine carcinomas (lcnec)-6, adenocarcinomas (ac)-29, and squamous cell carcinomas (scc)-28. the histological subtype, ptnm stage, and gene expression of men1 and egfr in tumor and normal lung tissue were evaluated. results: overexpression of egfr was observed in 34 %(scc-61 %, ac-31 %, sclc-23 %, lcnec-17 %, ct-13 %). decreased expression was observed in 22 %(lcnec-66 %, sclc-54 %, ct-22 %, ac-14 %, scc-7 %) (p<0.001). overexpresssion of men1 was observed in 57 % of all tumors (sclc-69 %, ct-65 %, scc-57 %, lcnec-50 %, ac-48 %) (p>0.05). significant correlation between overexpression of men1 and early stage scc was observed (p=0.03). conclusion: egfr is a target for therapy with monoclonal antibodies, so the tumors that overexpress egfr can be considered for treatment with these drugs. men1 can eventually be a marker for good prognosis and a potential target for therapy. objective: homeobox genes encode transcription factors controlling cellular proliferation and differentiation. altered expression of prox1 homeobox gene is related to many cancers, including breast, esophagus, lymphatic and oral. method: after overexpression of prox1 gene in scc9 cell line, total rna was extracted from three overexpressing-prox1 clones (oc) and one control-transfection cell clone (cc). microarray analyses were performed using the whole human genome 44 k according to the manufacturer's instructions. genes with a fold change of greater or lower than 2.0 in oc versus cc, were considered as increased or decreased, respectivelly. gene ontology (go) was used to assign biological process related to significantly differentially expressed genes. results: down-regulated genes mmp2, timp3 and notch1 were further validated by qrt-pcr. comparative gene analysis of oc and cc revealed 925 up-regulated and 789 down-regulates genes. pathways induced upon prox1 overexpression in go terms included vascular development control, cellular adhesion, regulation of proliferation, among others. mmp2, timp3 and notch1 expression by qrt-pcr showed reduced expression leves in oc. these genes are commonly overexpressed in oral squamous cell carcinoma and have been related with metastatic tumors and worse prognosis. objective: barrett´s esophagus is the unique known precursor for esophageal adenocarcinoma (eac), in a gradual progression to dysplasia. our objective is to determine the correlation between the hypermethylation in cpg islands of the promoter region of p16 tumoral suppressor gen in epithelial dna and the histopathological pattern, as possible biomarker for risk of progression. method: 1. a study was performed about the evolution of precursor lesions, in a group of 55 patients diagnosed following the vienna classification. 2. p16 hypermethylation is analyzed in paraffin embedding samples, through laser microdissection, dna extraction, pcr amplification, pyrosequencing and quantification. results: 1.14 of 20 cases with 2 to 5 biopsies remained as nd, id or lgd, and 6 cases progressed to hgd and/or eac. 2. within the control group the methylation grade is 6.53 % and in the diagnostic groups is 12.04 % (nd), 7 % (id), 17.05 % (lgd), 8.50 % (hgd) and 23.33 % (eac). conclusion: 1. 70 % remained stable, but those reaching hgd all progressed to ace (only in 50 % of the latter, hgd is recognized with h&e). 2. methylation´s grade is higher in all diagnostic groups comparing to the control group, progressively increased as the dysplasia grade found with h&e, so it may be a good biomarker for neoplastic progression. objective: in high risk hpvs the expression of oncoprotein e6 is responsible for the degradation of p53, while e7 inactivates prb and causes the progression to s phase of cell cycle, both sustaining the conversion to and the maintenance of malignancy. the aim of this study is to compare the performance in the detection of the e6 and e7 mrna expression of hr-hpv using the nuclisens easy q test (biomerieux) or hpv oncotect (incelldx) a test based on flow cytometry-fish method. method: we enrolled 50 patients positive for hr-hpv dna and/or pap smears. all subjects underwent a colposcopy histological evaluation and were tested both for the nuclisens easy q test and hpv oncotect. results: 31 out of 50 subjects resulted positive for rna expression. the patients were divided into two cohorts based on the histological diagnosis: low grade lesions (cin1) and high grade lesion (cin2+). patients with cin1 were 26 with nuclisens easy q test and 13 with hpv onco tect, those with cin2+ were 5 with either tests. conclusion: these preliminary results suggest that hpv oncotect have a better specificity than nuclisens easy q test, while more samples need to assess the difference in sensitivity. detection of pik3ca/akt mutations in human meningiomas a. saetta * , g. tomara, i. chatziandreou, p. tsioli, e. el-habr, i. sakalidou, g. vretakos, e. boviatsis, p. korkolopoulou, e. patsouris * university of athens, 1st dept. of pathology, greece objective: the pi3k/akt pathway is a major signaling pathway frequently activated in human cancer due to pik3ca and akt1 gene mutations thus representing a potential therapeutic target and prognostic biomarker. in this study, we examined the mutational status of pik3ca and akt1 in meningiomas. method: 91 meningiomas were screened for activating mutations in "hot spot" exons 9, 20 of pik3ca using real time pcr and high resolution melting analysis. pik3ca wild-type samples were analyzed for mutations in exon 4 of akt1. the mutations were verified by sequencing and/or pyrosequencing results: mutations were detected in 2 out of 91 samples (2 %) in exon 9 of pik3ca and were identified as p.e547k and p.s541f. regarding exon 20, 7 out of 91 samples (7,5 %) showed the following mutations: p.r1023q, p.t1025t, p.h1020v, p.m1043i, p.h1047r (2 cases), p.h1046t. finally, in exon 4 of akt1, 8 mutant cases were detected (9 %) all identified as p.e17k. in 19 % of the patients the activation of the pi3k/akt pathway is due to mutations in the two examined genes. conclusion: aberrant activation of the pi3k/akt pathway due to pik3ca and akt1 mutations is commonly observed in human meningiomas and these genes could be considered as potential targets in new therapies for cancer. study of pi3k/akt/mtor pathway in urothelial bladder carcinoma a. saetta * , n. prekete, e. trigka, g. levidou, m. karlou, p. pavlopoulos, p. korkolopoulou, e. patsouris * university of athens, 1st dept. of pathology, greece objective: deregulation of the pi3 kinase-akt/mtor pathway is a frequent event in tumorigenesis. we examined the possible significance of the components of this pathway in bladder urothelial carcinoma (uc). method: 108 cases with bladder uc were screened for mutations in exons 9, 20 of pik3ca gene and exon 4 of akt1 by pcr-sscp, hrma, sequencing and/or pyrosequencing. the expression of p-mtor, p-4e-bp1, p-p70s6k, p-akt, fgfr3 and p-erk was evaluated by immunohistochemistry. results: 4,6 % of the cases were mutant in pik3ca gene and 3 % in akt1. cases with wild type akt1 displayed higher fgfr3 receptor expression (p=0.0521). p-4e-bp1 expression was more frequent in low grade (p=0.018) and in pta-t1 tumors (p = 0.053). furthermore, superficial tumors presented higher levels of p-p70s6k expression (p = 0.035) and lower levels of p-akt expression (p = 0.048). in multivariate survival analysis, p-4e-bp1 immunoexpression emerged as an independent prognostic factor of adverse survival (hr =9.207, p=0.0039), along with tumor grade and t-category. conclusion: activation of pi3k/akt/mtor pathway in bladder uc is not exclusively related to the presence of akt1 and pik3ca mutations. expression of p-4e-bp1 could serve as an independent prognosticator. comparison of dna extraction methods of formalin fixed, paraffin-embedded archival tissues b. senguven * , e. baris, t. oygur * gazi university dental faculty, dept. of oral pathology, emek, turkey objective: formalin fixed, paraffin-embedded (ffpe) archival tissues are valuable resources for many molecular studies. the goal of this study is identify the optimal method for dna extraction from ffpe tissues. method: 32 human gingival tissues which has obtained from patients with gingival hyperplasia were used. serial sections of 10 μm thickness obtained using a standard microtome. half of the sections were deparaffinized on glass, the other half collected directly to a 1,5 ml tube. in order to identify the optimal method for dna extraction, we compared phenol-chloroform protocol and dna extraction mini kit. the duration of proteinase k digestion were also compared. spectrophotometric evaluation of the yield and purity of dna was conducted. to deteminate the amplifiablity of extracted dna, three different bp fragment of the beta-globin gene were amplified using pcr. results: the phenol-chloroform method had the lowest yield and purity. deparaffinized specimens on glass, digested for 72 h and isolated using mini kit had the highest yield. amplification of the 120 bp fragment of beta-globin gene was successful in all samples. conclusion: according to our results deparaffinization on glass, increasing the time of proteinase k digestion and using commercial kits for isolation seems the best method to obtain amplifiable dna from archival specimens. microrna signatures associate with fallopian tubal implantation in humans r. shao * * gothenburg university, physiology and endocrinology, sweden objective: micrornas are small non-coding rna molecules that regulate a large number of cellular pathways and deregulation or altered expression of mirnas is associated with many disease states. the function of the fallopian tubes appears to involve orchestrated spatiotemporal alterations in transcriptome profiles, the regulation of tubal gene expression and function by mirnas may thus be of primary importance in tubal ectopic pregnancy. method: both implantation sites and non-implantation sites of fallopian tubes from women with ep and decidual biopsies from women undergoing therapeutic surgical termination of pregnancy were collected and analyzed by mirna array. the unique mirna profiling results were validated by taqman qrt-pcr, and bioinformatics' analysis was employed to further predict the mirna targets. results: a total of 47 mirnas were differentially expressed in implantation sites compared with those in non-implantation sites of fallopian tubes after comparison of decidual mirnas, among which 19 were up-regulated while 28 are down-regulated. the mir-424 was significantly increased, whereas let-7i, mir-149, and mir-182 were significantly decreased. differentially expressed mirnas were predicted to be related with several signaling pathways in normal intrauterine implantation. conclusion: our findings establish an mirna signature associated with tubal implantation and provide the experimental basis for further understanding the molecular and cellular mechanisms of initiation and development of tubal ep in humans. lung is an organ that can sense any perturbation of the air, due to its constant interaction (long live) of the air. our hypothesis was that lung could be an organ sensor of climatic change observed today. method: this preliminary study analyzed the expression of 3 isoforms of the genes of hsp90. the cases were chosen from archive of autopsies from 1970 to 1979, with different diagnosis, and were matched in sex and age with a same number of cases chosen from 2000 to 2009. the expression was studied by rt-pcr from lung tissue embedded in paraffin, 20 samples of each decade. results: the expression levels of hsp90 were normalized to β-actine. we found expression in six cases of 2000-2009: five for hsp90aa1-2, and 1 for hsp90ab1, one case expressed both. their matched cases in 1970-1979 never expressed any isoform. the cause of dead in all cases doesn't fit with any pattern. conclusion: with these results we propose than the lung could be a sensor of global warming, and the expression of these genes could be molecular markers of this climatic change. apoptosis associated genes and their role in predicting responses to neoadjuvant breast cancer therapy d. tvrdík * , h. skálová, p. dundr, l. stanek, c. povýšil, l. petruželka * general university hospital, institute of pathology, prague, czech republic objective: neoadjuvant chemotherapy is used in the treatment of breast carcinoma because it substantially reduces the size of the primary tumor and lymph node metastases. this present study is aimed at the investigation of biomarkers that can predict a pathologic response to the therapy. method: the transcriptional profile of 84 key apoptosis genes was evaluated in both pre-therapeutically obtained tumor tissue by core needle biopsy and in specimens removed by final surgery, using a pathway-specific real-time pcr assay. results: on the basis of a hierarchical cluster analysis of 13 significantly changed genes, we divided patients into good and bad prognosis groups, which correlate well with progression-free survival. in the good prognosis group, we found a statistically significant downregulation of the expression of mcl1 and igf1r genes after neoadjuvant treatment. we also found a statistically significant overexpression of bcl2l10, bcl2af1, casp8, casp10, casp14, cideb, fadd, hrk, tnfrsf25, tnfsf8 and cd70 genes. in contrast, we found upregulation of igf1r after the treatment in the group with poor prognosis. conclusion: as we have shown, gene expression profiling after neoadjuvant chemotherapy is a valuable research tool for investigating molecular markers, which may better reflect tumor biology and treatment response than standard prognostic and predictive factors. objective: non-small cell lung carcinoma (nsclc) is one of the most serious cancers. identification of genetic changes (mutations, amplifications or rearrangements) within egfr, kras and alk oncogenes, associated with nsclc, allows choosing the patients, benefit from biological therapy with tyrosin kinase inhibitors. method: dna is isolated from formalin fixed paraffin embedded specimens or cytology specimens. mutation detection is performed by real-time pcr, fragment analysis, primerextension analysis and mutant-enriched pcr. wt-egfr patients (e.g. with no mutation detected) are tested for alk gene rearrangement and egfr gene polysomy or amplification using the fluorescence in situ hybridization (fish) method. results: since 10/2010 till 2/2012, 238 dna samples were analyzed. out of these, 17 patients (7,14 %) were found to be positive for activating mutations within egfr gene. since 07/2011 till 2/2012, 80 patients were analyzed for alk gene rearrangement, egfr gene polysomy or amplification. alk gene rearrangement has been proven in 5 (6,25 %) cases, alk gene amplification in 2 (2,5 %) cases and egfr gene polysomy or amplification in 35 (43,75 %) cases. conclusion: determination of genetics changes in tumor can provide powerful tool for setting up strategy and therapeutic protocols in nsclc patients. objective: a 61 year old gentleman, ex-smoker with a history of asbestos exposure presented to the emergency department with a 1 day attack of severe abdominal pain and bilious vomiting. history of abdominal pain, lethargy, and weight loss over the last 12 months and 4 years of recurrent pleural effusion. pleural aspirates and a videoassisted thoracic biopsy were negative. method: x-ray confirmed small bowel obstruction and an emergency laparotomy performed. the terminal ileum was intussuscepted into the caecum causing proximal obstruction. a right hemi-colectomy was performed. results: on gross examination there was intussusception of the terminal ileum secondary to a polypoid mass at the ileo-caecal valve. histology showed an oncocytic tumour with neuroendocrine features arising from the serosa of the small bowel. the tumour cells were positive for ck7, calretinin, and vimentin but negative for ck20, chromogranina, cd56 and ttf-1. the features were in keeping with a malignant peritoneal mesothelioma infiltrating the small bowel wall. conclusion: peritoneal malignant mesothelioma is an uncommon tumour which rarely causes mechanical small bowel obstruction. to our knowledge this is the first case of localised primary peritoneal mesothelioma presenting with intussusceptions. giant mesenteric cystic lymphangioma in adult; rare tumor, unusual location and uncommon age of occurrence: a tunisian case report s. attafi * , w. ajouli, n. bouchiba, a. chouchene, m. h. balti, k. bellil * fsi hospital, dept. of pathology, marsa, tunisia objective: cystic lymphangioma is a rare benign neoplasm arising from the lymphatic system. it occurs as a result of congenital malformations of the lymphatics, leading to the obstruction of local lymph flow and the development of lymphangiectasia. lymphangioma is common in pediatric patients, but it is extremely rare in adults, with only about 100 cases reported in literature. most lymphangiomas are found in the head and neck; intraabdominal and specially mesenteric locations are very unusual. the aim of this work was to study its clinical, histological and therapeutic features and their diagnostic difficulties. method: we report the case of a 46 years-old woman, who presented a painful syndrome of the right iliac fossae (rif). the physical examination found a mass of the rif. abdominal ultrasonography and magnetic resonance image showed a mesenteric cystic formation. at laparotomy, a large cystic tumor of the caecal mesentery, measuring 23 cm, was found. histopathology showed a fibrous and thin walled cyst, lined by flat low lying epithelium with surrounding tissue of scattered lymphoid cells. results: the diagnosis of cystic lymphangioma was retained. conclusion: mesenteric lymphangiomas are very rare, but they can cause acute abdomen that requires an emergent surgery. therefore, they should be included in the differential diagnosis of cystic intra-abdominal lesions raises several possibilities, including both malignant and benign soft tissue tumours. objective: the spleen metastases from colon cancer are rare conditions and usually associated with extensive disease. there are only eight reports in english-language literature of isolated splenic metastases from colorectal carcinoma, which generally metastasize to regional lymph nodes, liver and abdominal peritoneum. method: we report a case of multiple splenic metastases in a 60-year-old woman. results: in november 2009, the patient had undergone right hemicolectomy, lymphadenectomy and chemotherapy for stage iii tubular adenocarcinoma of the ascendant colon with positive pericolic lymph nodes. in december 2011, she was admitted in our hospital for diffuse abdominal pain and intestinal transit disorders. computed tomography of the abdomen revealed multiple metastases of different sizes in liver and spleen. during the surgery, which was performed with the goal of curing metastatic disease, there were found two nodular hepatic secondary tumors, and numerous spleen metastases, with diameters from 0.2 cm to 1.5 cm. the histopathological evaluation of splenectomy specimen revealed a metastatic tumor deposit, histologically similar to the primary tubular adenocarcinomas of colon. conclusion: this is the ninth documented case of splenic metastasis from colon cancer. previously reported cases of this type were isolated tumor, this one being the first reported with multiple splenic metastases. the role of fatty acid synthase in inflammatory bowel disease (ibs) e. bas bozkurtlar * , n. ozkan, a. e. kedrah, c. celikel * marmara university, dept. of pathology, istanbul, turkey objective: it was demostrated that fas expression increases not only in mucosa involved by active colitis but also in normal mucosa of ulcerative colitis (uc) patients. our aims were to evaluate the role of fas expression in differential diagnosis of ibd, to search for any possible change during the progression of the disease in patients with clinical and endoscopic follow-up. method: among 82 colonoscopic biopsy samples of 50 uc cases,13 samples were classified as remission,15 as resolution, and 54 as active period;35 samples of 30 crohn disease (cd) cases were classified as active period. fifteen cases with normal endoscopic and morphologic findings comprised the control group. results: between uc and cd active periods and control group, a significant difference was found (p=0,0001). there was a statistical correlation between fas expression of basal crypt of normal mucosa in uc active period and the disease duration (p=0,036). conclusion: the increasing in fas expression in patients with ibd can not be explained only by the inflammation. fas expression can not aid us in the differential diagnosis of uc and cd, but it seems that it is related to the extent of inflammation in uc. the relation between the disease duration and an increase in fas expression active uc, might have a significant role in carcinogenesis in uc. micropneumatosis -an (un) usual finding in gastrointestinal specimen? k. blaue * , m. plauth, j. knolle * städtisches klinikum dessau, pathology, dessau-roßlau, germany objective: a rather rare disease, micropneumatosis describes the findings of cyst-like cavities devoid of lining epithelium in the gastrointestinal wall. micropneumatosis is often secondary to intestinal bacterial infection or mechanical factors. this study evaluates the incidence of micropneumatosis in our institute. method: we analyzed all cases of micropneumatosis obtained in our institute between 2001 and 2011. available data were retrieved from patients' records. results: out of 300.000 specimens obtained between 2001 and 2011, 71 cases (0.02 %) presented with micropneumatosis (37 female, 34 male; mean age 61 years, age range 18 to 85 years). most common site was the colon (32 cases), followed by stomach (30), small intestine (8) and greater omentum (1). conclusion: micropneumatosis represents a rare but harmless diagnosis in gastrointestinal specimen. due to the histological picture, which may be confused with dilated lymphatic vessels, submucosal lipomas, and, to the untrained eye, even with signet ring cell carcinoma, incidence may even be higher. in unclear cases, where there seems to be a endothelial layer, immunohistochemistry may be necessary to confirm diagnosis of micropneumatosis. this study shows that micropneumatosis should be considered in differential diagnosis in every age and sex. predicting lymph node metastasis in pt1 colorectal cancer -a meta-analysis providing rationale for therapy decisions s. bosch * , i. nagtegaal * radboud university nijmegen, pathology, netherlands objective: we conducted a meta-analysis of published reports on the predictive value of risk factors for lymph node metastases (lnm) in pt1 colorectal cancer in order to provide a rationale for choosing follow-up or radical surgery after local excision. method: local excision is an attractive treatment option for colorectal cancer, but is only safe in the absence of lnm. several pathological factors have been associated with lnm, however it remains unclear how to integrate these in clinical decision making. results: a pubmed search revealed 17 studies totaling 3741 patients. strong predictors of lnm were lymphatic invasion (rr 5.2 [95 % ci 4.0-6.8]), budding (rr 5.1 [95 % ci 3.6-7.3]), and high grade histology (rr 4.8 [95 % ci 3.3-6.9]). deep submucosal invasion was also strongly associated with lnm, however in a risk stratification model this factor was of limited added value. conclusion: the absence of lymphatic invasion, budding and high grade histology may justify withholding radical surgery. the independent role of submucosal invasion depth is probably limited. models for risk stratification based on these factors need to be validated. objective: dendritic s100+ cells in intestines have been described in more studies. their connection to s100+ fibres of nervous system is lesser of an object of interest. method: we have processed the samples of intestinal mucosa of people with crohn's disease and colitis ulcerosa by the means of form-paraffine technique and anti s100 antibodies. results: in lamina propria and in submucosa at crohn's disease were s100+ the fibres of various width and orientation. outside of them the diffusely scattered round s100+ mononuclear cells could be seen. contrary to colitis ulcerosa the granulation tissue at crohn's disease contains in the area around abscesses a thick accumulation of s100+ cells and this is similar also in the formed lymphoid tissue. conclusion: the positive fibres can be seen in submucosa at the edge as well as in lpm between the bases of the glands. in between the plexus/ring and longitudinal muscle there are massive bands of connective tissue which contains bundles s100+ of ganglion cells and nerve fibres. the fibres are present in ring as well as in longitudinal muscle. in this area s100+ fibres can also be seen in vessel walls. numerous bundles of granulatory tissue do not contain positively reacting substance. in the basal part lamina propria there are visible s100+ cells as well as long, smooth, positively reacting fibres. ps-11-010 ncf1-deficient mice with impaired oxidative bursthave a more agressive progression of dextran sulfate sodium (dss) -induced colitis l. carvalho * , t. rodrigues-sousa, a. alarcão, a. f. ladeirinha, m. souto-carneiro * amadora, portugal objective: intestinal inflammatory disease (iid) as a primary immunodeficiency depends on mutations in the nadph oxidase complex, responsible for the production ofreactive oxygen species (ros). one of the most common clinical patterns in iid is chronic colitis. ncf1-mutation in mice leads to deficiency in ros, rendering them susceptible to autoimmunity. here we studies how ros-deficiency in ncf1-mutant mice influenced the immune response to dss colitis, its recovery and answer to a second induction. method: colitis was induced in wild type (wt) and ncf1mutant (ncf1) b10.q mice by administration of 3.5 % dss in the drinking water for 1 week. after 1 week recovery, dss was administered for another week. mice were sacrificed at days 0, 7, 14 and 21, the colon was removed and folded into a swiss roll. sections of the colon were stained with he, and monoclonal antibodies against b cells (b220), cd3+ t cells; and macrophages (mac1/cd11b) were applied. results: colitis was more severe in ncf1 than wt mice, with epithelial dysplasia, hyperplasia of peyer's patches and poor epithelium recovery (hyaline scars). at all time-points the amount and location of colonic b cells, t cells and cd11b + cells was distinct between groups. these results suggest that ros are crucial for leukocyte recruitment and tissue-repair in dss-induced colitis. histopathological pattern of polypoid lesions of colon in albanian population g. cekodhima * , a. cekodhima, a. beqiri, g. andrea * university hospital maria teresa, dept. of pathology, tirana, albania objective: our aim was to study histopathologic pattern of the polypoid lesions in albania. method: we studied 216 lesions, 184 polypectomies and 32 colorectal surgery in 139 patients in tirana. results: there were observed 216 polypoid lesions of large intestine in 122 males and 91 females.105(48.6 %) were adenomatous polyps, 31(14.35 %) were hyperplastic, 24 (11 %) were inflammatory polyps with ulcerative colitis, 10 (4.6 %) were inflammatory 6(2.77 %) were juvenile polyps, 2(0.92) non hodgkin's lymphoma and 1 (0.46) was fibrolipoma. tubular adenomatous polyps were the commonest polyps. they were more common 56 (25.9 %) in male population as compared to female 19(8.79 %). high grade dysplasia was present in 50 (23.14 %) and malignant change in 26 (12 %). the size of the polyps range from 0.5 to 4 cm. conclusion: adenomatous polyps were the most frequently found polyps in our study; the approximately 24 % were advanced lesions. the role of cancer stem cells in biology and prognosis of colon cancers a. f. çiçek * , ö. öngürü, m. gamsizkan, a. günal * gülhane military medical academy, dept. of pathology, ankara, turkey objective: this study was designed as a retrospective clinicopathological observation based on immunohistochemical and statistical findings to show the relationship between the disease progression and the intensity of the stem cell population within the tumor for colorectal cancers. method: for this purpose, we investigated 97 colorectal carcinoma cases, retrospectively. paraffin embedded blocks was obtained from pathology archive and demographical patients' data from the patient's files of gastroenterological surgery department. immunohistochemically, we used cd133 and musashi-1 antibody to determine the stem cells within the tumor. we noted the age of the patients, histopathological diagnose, tumor location, grade, tnm status, clinical stage, disease-free-survival and the outcome. then we statistically compared all prognostic data with the immunohistochemical findings results: all of the cases were immunoreactive for both antibodies, furthermore we found a significant statistical correlation (p=0.043) between cd133 expression value and patient outcome. when the value of cd133 expression was high, clinical outcome was poor. in addition, there was a relation between high musashi-1 expression and poor outcome. conclusion: based on these findings we reported that overexpression of both cd133 and musashi-1 antibodies may be a poor prognostic factor in colorectal cancers. immunohistochemical evaluation of vegf expression in colorectal carcinomas d. crisan * , m. florea, d. fodor * university of medicine and pharmacy, pathology, cluj-napoca, romania objective: vascular endothelial growth factor (vegf) is an important angiogenic glycoprotein secreted by the tumor cells and host cells which proved to be a powerful prognostic factor in various human cancers. the aim of our study was the evaluation of vegf expression in colorectal carcinomas using immunohistochemistry, in order to identify the relationship between the presence of the protein in the tumor cells and a series of morphological parameters. method: we analyzed 30 consecutive surgically removed colorectal carcinomas. immunohistochemistry was done on formalin-fixed and paraffin embeded tissue sections, using the anti-human vegf-a monoclonal antibody (clone vg1, dako). the extent and intensity of staining were graded and used to calculate the immunoreactive score for each case. statistical analysis was performed with spss (statistical package for the social sciences), using nonparametric tests. the level of statistical significance chosen was p <0.05. results: vegf was expressed in all tumors, with a heterogeneous distribution. the only parameters that correlated with high vegf positivity were the extent of the tumor necrosis and the presence of lymph node metastases (p =0,001). conclusion: high levels of the vegf expression may be an indicator of poor prognosis in colorectal carcinomas. collision tumour of the appendix: mucinous cystadenoma and carcinoid -report of a case c. dastamani * , a. paraskeva, e. carvounis, t. theodosopoulos, a. kondi-pafiti * aretaieion hospital, dept. of pathology, athens, greece objective: appendiceal carcinoids are usually located at the tip of the appendix and occur as incidental findings. epithelial neoplasms of the appendix are uncommon and consist of mucinous adenomas, carcinomas and neoplasms with features of both carcinoid and adenocarcinomas. method: a case of dual mucinous cystadenoma and carcinoid of the appendix is reported. results: the patient, a 57-year old-asymptomatic female presented with a cystic right iliac fossa mass. the excised specimen was appendix cystically dilated measuring up to 8,5 cm in diameter. the luminal content consisted of viscid mucin. the base of the appendix was thickened (up to 1,5 cm) and firm. microscopically the cystic part of the specimen had features of mucinous cystadenoma with adenomatous epithelium extending as well over the thickened appendiceal wall. at that area the wall was involved by a well differentiated neuroendocrine neoplasm who gr i (carcinoid). the neoplastic cells infiltrated the entire thickness of the appendiceal wall, the mesoappendix and reached the serosal surface. the two neoplasms where separate and there was no transitional zone between them. conclusion: dual carcinoid/epithelial neoplasia is a rare occurrence in the appendix. the prognosis appears to be no worse than for either of the two components alone. gastrointestinal stromal tumor (gist) of the anal canal: a case report l. de carvalho * , p. a. teixeira, v. g. siqueira, m. pereira, t. or, e. pereira * centro universitário lusíada, dept. of pathology, santos, brazil objective: gastrointestinal stromal tumors (gist) are mesenchymal tumors derived from interstitial cells of cajal. they are found more frequently in the stomach, small intestine while colon and rectum represent unusual sites. gist's of the anal canal are extremely rare. method: 43-years-old woman who presented bleeding, pain and constipation for several months. the rectal examination revealed a well defined mass located within 10 cm of the anal verge. the magnetic resonance imaging confirmed a well-circumscribed mural mass without adenopathy. the local excision of the tumor was performed for pathological study. results: gross examination showed a 4.5×3.0 cm fibrous mass. histological examination revealed a spindle cell tumor with moderate atypia and mitotic count of 6 mitosis/ 50hpf. neoplastic cells showed marked positivity for c-kit and cd34 and negativity for muscular markers. a diagnosis of gist with intermediate risk of aggressive behavior was made. conclusion: gists of the anal canal are extremely rare with only few cases reported in the literature. we described an additional case of gist of the anal canal with histological and immunohistochemical study. intestinal graft versus host disease with giant cells b. doganavsargil * , a. vink, e. j. petersen, g. j. arnold offerhaus * ege university, school of medicine, izmir, turkey objective: graft-versus-host disease (gvhd) is a common complication of allogeneic bone marrow transplantation in which immune cells in the transplanted marrow attack the immunocompromised host. it can occur in either an acute or in a chronic form and may affect various organs. intestinal gvhd is particularly important because of its frequency, severity and its effects on the general condition of the patient. method: here we report a 68-year-old man who developed gvhd, 8 months after receiving allogeneic non-myeloablative peripheral blood stem cell transplantation for refractory chronic lymphocytic leukemia. colonic biopsies revealed an edematous and congested lamina propria, dilated crypts lined by flattened epithelium, increased apoptotic figures and vanishing crypts. notably there were some giant cells in between crypts. however subsequent sections and special stains didn't show any granuloma formation or presence of an infectious agent. results: to our knowledge presence of giant cells in gvhd hasn't received proper attention before. although we don't know the actual importance or clinical consequences of the entity yet, we report this case to increase awareness of this feature within the context of gvhd. we have seen more cases with giant cell gvhd afterwards; comparison with other cases of gvhd without giant cells may help clarifying their significance. differential mutation patterns of kras and braf in adenomatous and serrated neoplastic sequences o. erdogan * , b. savas, a. ensari * ankara university, faculty of medicine, turkey objective: colorectal carcinomas develop through adenomatous or serrated neoplastic sequence. different molecular mechanisms underly these morphologic sequences which may be translated into different therapies. we aimed to assess adenomatous and serrated neoplastic sequences in terms of kras and braf mutations. method: the study group comprised of adenomatous polyps (aps, n=20, 13.6 %), conventional adenocarcinomas (conca, n = 30, 20.4 %), serrated polyps (sps, n = 67, 45.6 %) and mucinous adenocarcinoma (mucca, n=30, 20.4 %). kras mutation was assessed for exon 2 and exon 3 using pyrosequencing while braf v600e mutation was analysed using allele specific pcr. chi square test was used for statistics. results: kras mutations were observed in 41.5 % of carcinomas and 25.3 % of polyps. kras mutation rate was significantly higher (p<0.05) in conca (85 %) than in mucca (50 %), and in aps (55 %) than in sps (16.4 %). braf mutation was found in one mucca (1.7 %) while all sps were mutated. tsas had the highest kras mutation rate (36.8 %) in comparison to ssa/ps (17.6 %) (p<0.001) whereas ssa/ps and hps had significantly higher rates of braf mutation (64.7 %, 61.1 %, respectively) than tsas (26.3 %) (p<0.001). conclusion: kras seems to take part in adenomatous sequence while braf, seem to play a significant role in serrated neoplastic progression of the colorectum. objective: quiescent ulcerative colitis (q-uc) is morphologically characterized by typical architectural and cellular mucosal changes that define the process as chronic. it is not clear which of these changes are more ubiquitous in q-uc. our aim was to evaluate which classical histological findings of q-uc are more prevalent in rectal samples. method: rectal biopsies were collected from patients with clinical and endoscopical q-uc. the biopsies were evaluated for mixed inflammation in the lamina propria, crypt architectural abnormalities, basal plasmacytosis, fibrosis, and paneth cell metaplasia as well as for features of active disease (cryptitis, lamina propria neutrophils and erosion). results: forty-five patients (64 % female; median age 56 years) were included. thirteen (28,9 %) biopsies showed focal activity and four cases (8,9 %) had criteria for active disease. epithelial distortion, chronic inflammatory infiltrate and basal plasmacytosis emerged as features present in 95, 81 and 73 % of biopsies. overall, 4 biopsies were histologically normal (no chronic features, no active disease). conclusion: rectal mucosa from patients with clinical and endoscopical q-uc can show microscopic active disease, which illustrates that endoscopy alone may be insufficient to identify quiescent disease. in q-uc, the most frequent changes (present in over 75 % of the biopsies) were epithelial distortion and chronic inflammatory infiltrate. vacuum-based preservation of colorectal cancer specimens: a comparison with formalin fixation j.-f. fléjou * , s. el-naderi, p. cervera * hôpital saint-antoine, dept. de anatomie pathologique, paris, france objective: in pathology, an alternative to immediate fixation in neutral buffered formalin (nbf) is vacuum sealing and cooling (vsc) ("tissue safe" system). there have been few evaluations of vcs. method: we assessed msi, kras, braf in colorectal cancers, conserved with vcs before fixation (51 cases with surgery in a hospital distant from our centre), or immediately fixed in nbf (56 cases with surgery during the same period in our hospital). dna was extracted from paraffin embedded tissue. msi was assessed by mlh1 and msh2 immunohistochemistry and pcr; kras and braf were screened by multiwell-plate based real-time pcr (lightcy-cler® 480, roche), confirmed by sanger sequencing. results: there was no difference regarding morphological analysis. immunohistochemistry was interpretable in all cases, with 4 negative cases in the vcs group (4 mlh1), 11 negative cases in the nbf group (10 mlh1, 1 msh2), and a 100 % correlation between immunohistochemistry and pcr. dna extraction was possible in all cases. kras and braf mutations were detected in 21 and 1 cases in the vcs group and 16 and 7 cases in the nbf group, respectively. conclusion: we show that analysis of msi, kras and braf is feasible in surgical specimens after vcs. this procedure can be an alternative to formalin fixation. clinical significance of cd204-positive m2 macrophage in colorectal cancer s. fushimi * , m. matsumoto, s. takahashi, t. ogino, j. itakura, t. ito, a. matsukawa * okayama university, dept. of pathology, japan objective: tumor-associated macrophages are divided into two phenotypes, termed m1/m2 macrophages. m1 macrophages promote tumoricidal responses whereas m2 macrophages assist tumor progression and metastasis. in this study, we have analyzed a clinical significance of m2 macrophages in excised sections from patients with colorectal cancer. method: patients who had surgical resection for colorectal cancer between 2005 and 2006 were identified from a prospective database. tissue sections with adenocarcinoma in pt3 category were employed in this study. the sections were stained with anti-cd204, a marker of m2 macrophages. the relation between the staining pattern and tumor budding or patient prognosis was examined. results: fifty-two patients (30 male, 22 female) with a median age of 67 years (range 23-86) were studied. the tumors were classified into four subtypes according to the staining patterns of cd204-positive cells, i.e. sparse type (n=9), invading-tip type (n=6), peri-nest type (n=18), and dense diffuse type (n=19). only a dense diffuse type was related to the high degree of tumor budding. the prognosis with a dense diffuse type demonstrated a poor prognosis as compared to the other types. conclusion: even the case with comparable tumor depth, cd204-positive macrophages with dense diffuse distribution was related to the poor prognosis. objective: the treatment of locally advanced rectal cancer has shown a significant development over the last decade. the total mesorectal excision and the use of pre-operative radio (chemo)-therapy are leading the patients to a gradual improvement of local control of the disease. the aim of this study is to evaluate the pathological tumour response to pre-operative treatment through correlation between the clinical stage (ctnm) and the pathological stage (yptnm). method: since 2009, 35 patients with locally advanced rectal cancer, assessed by high resolution magnetic resonance imaging (hr-mri) and tnm staging system, have been evaluated by the colorrectal multidisciplinary team. pre-operative radio (chemo)-therapy was mandatory before undertaking standard surgery. surgical specimens were evaluated according to the vikingo project's protocol implemented by the spanish association of surgeons. results: 34 patients completed the pre-operatory treatment. after evaluation of clinical and surgical study specimens, we observed variable degree of downstaging in 27 (79,5 %) patients, 5 of whom showed complete response. the 7 (20,5 %) remainig patients were assessed as stable disease. no local progression of tumours was observed. conclusion: in this study, approximately 80 % of patients, who were staged by hr-mri and underwent preoperative radio (chemo)-therapy for locally advanced rectal cancer, showed some degree of downstaging. we remark the fact that nearly 15 % of patients reached complete response. the density of macrophages in colorectal cancer is inversely correlated to tgf-beta1 expression and patients' survival m. gulubova * , i. manolova, j. ananiev * trakia university, general and clinical pathology, stara zagora, bulgaria objective: the role of macrophages in colorectal cancer tumorogenesis is complex because they can both prevent and promote tumor development. we investigated cd68 infiltration in tumor tissue and its correlations with proteins ot tgf-beta1 signaling pathway. method: a nonselected panel of 206 primary tumors of colorectal origin was investigated immunohistochemically with antibodies against cd68, tgf-beta1, smad4, smad7, tgfrii and levels of tgf-beta1 were measured by elisa. results: lower cd68 infiltration in tumor nests was associated with expression of tgf-beta1 (chi2 = 9.236, р = 0.002) and smad4 (chi2=2.871, р=0.090) in tumor cells and with tgfrii expression (chi2=5.699, р=0.017) in tumor cells membranes. there was not correlation between cd68 cell numbers in tumor tissue and tgf-beta1 serum levels. we have found higher frequency of liver metastases in patients with lower infiltration with cd68 in invasive margin (chi2=11.364, р=0.001). the survival time was shorter for patients with low cd68 infiltration in tumor nests and invasive margin, compared with the survival time for patients, with higher cd68 infiltration in both tumor compartments (p<0.001). conclusion: the increased levels of tgf-beta1 in the tumor have an immunosuppressive effect on cd68 infiltration. inflammatory myofibroblastic tumor of the colon: the 25th reported case s. gurzu * , t. bara, i. jung * university of targu mures, dept. of pathology, romania objective: inflammatory myofibroblastic tumor (imt) is a very rare tumor of the colon in which the diagnosis is especially based on immunohistochemistry. in the colon, we report the 25th case of imt. method: case presentation results: we report the case of a 56-year old male who presented with symptoms suggesting colon cancer. a 20 mm protruded tumor was endoscopically described in the ascending colon. right hemicolectomy was performed. macroscopically the tumor was well defined, covered by normal mucosa with a central depressed area, suggesting a gist (gastrointestinal stromal tumor). microscopically spindle cells with fascicular arrangement admixed with mononuclear leukocytes were observed. the tumor cells expressed vimentin, sma, desmin and alk and were cd34 and cd117 negative. intense angiogenesis and cd117 expression in the endothelial cells were also observed. conclusion: in the imt of the colon, clinical endoscopic and gross feature can imitate a carcinoma or a gist. the ihc pattern of imt can offer informations about its histogenesis. based on the immunophenotype, we hypothesize that this tumor seems to occur from the the pluripotent stromal cells, cd34 positive which can be either differentiated in the interstitial cells of cajal (telocytes), which are also cd34 positive or, during differentiation, can loss cd34 positivity. objective: determining the fraction of tumor cells in colon carcinoma samples analyzed for kras mutation is important for choosing the proper testing modality. however, when asked to determine tumor cell fraction in tissue samples, different pathologists give considerably different estimations, possibly leading to erroneous interpretation of kras mutation analysis results and poor treatment choices. method: we developed a free, easy to use computer program that estimates tumor cell fraction on colon carcinoma slides that are immune-stained with anti-cytokeratin antibody. sixty samples were evaluated by the program and results were compared to actual measurement of tumor fraction. results: the tumor cell fraction estimated by the computer program showed highly significant correlation with the actual measurements (r=0.64, p<0.001). additionally, we found that a short calibration step prior to beginning the computer estimation increased the accuracy of the results. in four cases (7 %) there was some discrepancy between the computer estimation and the actual measurements, however, this was attributed to lower quality immunohistochemical staining. conclusion: in conclusion, we believe that this program can be used for standardizing the evaluation of tumor cell fraction in colon carcinoma, and that its use might aid in making better diagnosis and treatment choices for these patients. magnetic resonance imaging assisted tumour block selection in colorectal cancer c. hunter * , g. brown, l. temple, m. abulafi, a. arnaout * croydon university hospital, dept. of colorectal surgey, united kingdom objective: inadequate sampling may result in understaging in colorectal cancer. fat clearance techniques and whole mount sections are time consuming and costly. we hypothesise that high resolution magnetic resonance imaging (mri) of the colorectal cancer specimen may aid histological sampling and result in upstaging. method: patients undergoing resection are prospectively recruited, and randomised to "conventional histology" or "mri assisted histology". in addition to routinely selected tumour blocks, 4 additional blocks and any additional lymph nodes are selected with the aid of specimen mri or visual inspection and palpation according to group. t stage, depth of extramural invasion, distance to resection margin, lymph node (ln) number and involved ln are compared between the two groups. results: to date, 116 of 218 patients have been recruited. we present the methods and preliminary results. there is a trend towards greater t stage and ln yield. no difference in n stage, extra-mural vascular invasion or involved non-peritonealised resection margin has yet been observed. conclusion: early results suggest that specimen mri may upstage colorectal cancer specimens by aiding tumour block selection. we will complete recruitment of 218 colorectal cancer patients over the next 12 months to determine whether observed differences are significant. diverticular disease of transverse colon is rare and perforation may occur in crohns disease c. hunter * , a. arnaout * croydon university hospital, dept. of colorectal surgey, united kingdom objective: diverticular disease commonly affects the sigmoid colon in the west and the right colon in asia. perforation is uncommon affecting 4/100 000 per year. perforated diverticular disease of the transverse colon is therefore very rare. we present a case of crohns disease complicating transverse diverticular disease that lead to perforation and unfortunate death. method: radiological and histopathological features are presented with a review of the literature results: to the best of our knowledge, this is the first reported case of crohns disease complicating diverticular disease of the transverse colon. we suggest that distal stricturing leading to increased intraluminal pressure or increased weakening of the colonic wall due to coincident crohns fissure and diverticulum are possible pathogenic mechanisms. conclusion: crohns disease complicating transverse diverticular disease is uncommon, but may be one pathogenesis leading to the rare event of transverse colon diverticular perforation. results: there were 12 episodes in 9 patients. they start between day 7 and 499 (178±159 days). duration ranged from 3 to 87 days (27±22). the three moderate and the three mild ar reversed with treatment. of the 6 severe ar, 3 caused the failure of the graft. five episodes de ar began as moderate or severe directly. severity in the course of each episode was fluctuating. sixteen indeterminate rejections were seen in 9 grafts (did not reach definitive criteria). they lasted 1-31 days (mean: 5±8 days). conclusion: 1-acute rejection is the most common cause of morbidity in the intestinal transplant (56 % of the grafts). 2-it can develop at any time and relapse. 3-the duration and severity of the episodes are fluctuating. 4-in our series, 75 % reversed, but severe rejection often lead to graft loss (50 %). histopathological study of 7 intestinal grafts lost in a series of 16 adult transplants. hospital 12 octubre. madrid. method: we studied 4 explants and 2 autopsy. the autopsy was not authorized in one death. results: seven grafts were lost in 6 patients (3 multivisceral). two types of failure were seen. a) local: 3 severe acute rejections (ar) (days 77, 158, 511) and 1 lymphoproliferative syndrome (lps) in day 148; b) systemic: 3 sepsis (days 31,88,161). ar showed 3 types of morphology: ulcerative, pseudomembranous and with disappearance of the villi preserving the crypts. lps was a high-grade b lymphoma and affected the graft and the lymph mesenteric nodes with venous thrombosis that cause ischemic necrosis. the 3 sepsis were due to acinetobacter, to adenovirus in the graft and pulmonary aspergillosis and to pseudomona aeruginosa with necrohemorrhagic pancreatitis. conclusion: 1-the incidence of igl was 44 %. 2-the cause of igl was local (ar and lps) in 57 % and sepsis in 43 %. 3-severe ar was the most frequent cause of failure. 4-histological examination properly determine and document the causes of igl. cap polyposis: report of two cases r. ivanova * , d. kyoseva, g. trifonov, r. nikolov * hospital of endoclinology, laboratory of pathology, sofia, bulgaria objective: cap polyposis is a rare colorectal disease characterized by mucoid, bloody diarrhea associated with multiple inflammatory polyps covered by a cap of fibrinopurulent mucous. the disorder was first described in 1985 and up to date a small number of cases has been reported in the literature. method: we report two cases with multiple polyps of rectum and sigma, which were histologically diagnosed as cap polyposis. results: the cases were two males on age of 18 years and 64 years. in both cases there was a history of mucoid and bloody diarrhea. on the basis of colonoscopy findings, in the young patient there was a broad differential diagnosis including m. crohn, inflammatory pseudopolyps and cronkhite-canada syndrome. in the second case the clinical diagnosis was polyps of sigma. endoscopic biopsies and polypectomy were done. in both cases, the histology showed typical histological features of cap polyposis -polypoid lesions containing elongated, tortuous and often distended crypts covered by a cap of inflammatory granulation tissue. conclusion: the recognition of this rare disease is of practical significance because its clinical symptoms have some similarity with inflammatory bowel disease or irritable bowel syndrome. histopathological findings in 496 consecutive appendectomies: a retrospective analysis j. jeruc * , z. dolenc stražar, v. jovic, a. cerar, n. zidar * university of ljubljana, faculty of medicine, slovenia objective: appendectomy is one of the most common surgical procedures. histology usually confirms the clinical suspicion of acute inflammation, but sometimes other clinically relevant diagnoses are made. the aim of our study was to audit the appendectomies at our institution and summarise atypical pathological findings with emphasis on benign and malignant tumours. method: we reviewed the histopathology results of 496 consecutive appendectomies received in a 5-year period. results: excluding three cases of metastatic process, appendiceal tumour was found in 21 cases (4.2 %), which is a higher rate than reported in the literature. there were four endocrine tumours, all g1, three cases of classical adenomas, 10 cases of low-grade appendiceal mucinous neoplasm (one associated with pseudomyxoma peritonei) and three cases of carcinoma (one mucinous adenocarcinomas, one intestinal type, one undifferentiated carcinoma). we also diagnosed one case of multicystic mesothelioma. endometriosis was diagnosed in seven cases. in 18 cases appendicitis was associated with diverticula, in 6 cases changes were suggestive for crohn's disease and in one case for cystic fibrosis. negative appendectomy rate was 10.3 % that falls within the range reported in the literature. conclusion: histopathological examination of appendectomy specimens may reveal many different conditions not previously suspected; therefore, it should be performed in all cases. results: cdx2, muc1, muc2, muc5ac, and muc6 expression was observed in 39.7 % (75 cases), 35.4 % (67 cases), 27.5 % (52 cases), 28.6 % (54 cases), and 16.9 % (32 cases), respectively. while siac patients with cdx2 expression showed less nodal metastasis (p=0.01), those with muc1 expression tended to have siacs with nodular or infiltrative growth (p = 0.003), poor differentiation (p = 0.005), and more lymphatic invasion (p=0.04). muc5ac expression was associated with siacs with well differentiation (p=0.02) and frequent pancreatic invasion (p=0.04). patients with cdx2+/muc1-had more polypoid (p=0.02) and well differentiated (p=0.006) tumors and a significantly better survival (median, 80.8 months, p<0.0001) than those with other immunophenotypes (cdx2−/muc1+, cdx2+/ muc1+, and cdx2−/muc1−). patients with cdx2 expression (median, 71.2 months) had a significantly better survival than those without cdx2 expression (23.0 months) by univariate and multivariate analyses (p<0.0001). conclusion: cdx2 expression is an independent good predictor of survival in surgically resected siac patients. small neuroendocrine tumor of appendix with metastasis in the ileocecal lymphnode: a case report g. kalan * , s. tušar, n. zidar * general hospital jesenice, dept. of pathology, slovenia objective: neuroendocrine tumors (net) of appendix are believed to behave less aggressively than nets at other sites. separate staging criteria have therefore been proposed, and tumor size >2 cm appears to be the dominant criterion for aggressive behavior in appendiceal nets. however, it is controversial whether radical surgery is indicated in tumors measuring >2 cm. we report a case of a 13-year-old girl with acute appendicitis in whom appendectomy and right-sided hemicolectomy were performed. method: surgical specimens were sampled and processed according to standard histological and immunohistochemical (ihk) procedures. results: in the base of appendix, there was a tumor measuring 1.5 cm in diameter, focally invading mesoappendix, with free resection margins. microscopically, tumor exhibited characteristic features of net g1, with positive ihk for chromogranine and synaptophysin, and ki67 index <2 %. ihk for podoplanin revealed focal lymphovascular invasion. in the right-sided hemicolectomy specimen, there was no residual tumor, metastasis was found in one ilecoecal lymphnode. conclusion: our case shows that appendiceal net smaller than 2 cm can metastasize to regional lymphnodes. ihk against podoplanin might be helpful in searching for lymphovascular invasion, helping to separate it from the retraction clefts, thus providing additional information concerning risk factors for a more aggressive behavior. lipomatosis of ileocaecal valve causing small bowel obstruction mimicking crohn´s disease m. kalman * , p. szépe, j. marcinek, t. balhárek, l. plank * university hospital martin, dept. of pathology, slovakia objective: lipomatosis of ileocaecal valve is a rare cause of small bowel obstruction. method: we describe a 41-year-old male treated for crohnś disease and clinical and imaging presentation of chronic small bowel stenosis and obstruction. results: a right hemicolectomy was performed, and hypertrophy of bauhin´s valve causing intestinal obstruction was found. microscopical examination of the specimen revealed morphological changes consistent with lipomatosis of the ileocaecal valve. the ileum and colon were macroscopicaly unremarkable and histomorphological features of crohn´s disease were absent. conclusion: many patients with lipomatosis of the ileocaecal valve are asymptomatic, or the lipomatosis gives causes insignificant symptoms only or rarely obstruction may occur. then, as in the presented case, the surgical resection with ileo-colic anastomosis is the only effective treatment. overexpression of cxcr4 in tumor buds is a strong predictor of lymphatic invasion and lymph node metastasis in colorectal cancer e. karamitopoulou-diamantis * , d. kassahn, i. zlobec, v. koelzer, h. dawson, a. lugli * universität bern, inst. für pathologie, switzerland objective: cxc chemokines enhance tumor cell survival and proliferation. especially cxcr4 promotes tumor development by stimulating angiogenesis and favoring metastasis. in contrast, cxcr3 is angiostatic and may exert an anti-tumor effect. since tumor budding is linked to vascular/lymphatic invasion and lymph node (ln) and/or distant metastasis in colorectal cancer (crc), here we explored the expression of cxcr3+4 in relation to tumor budding. method: a multiple-punch tissue microarray of 220 crcs with full clinicopathological information including therapy underwent immunohistochemistry for cxcr3 and cxcr4. expression was evaluated in tumor-center, -front and -buds and correlated to clinical data. results: cxcr3-expression was homogeneous throughout tumor-center, -front and -buds (average 40 %) and was unrelated to clinicopathological features or survival. cxcr4-expression was high in tumor-center and -front but reduced (p<0.001) in buds (76 %, 77 %, and 20 %, respectively). maintenace of cxcr4-expression within buds was predictive of ln-positivity (p<0.0001) and lymphatic invasion (p<0.0001) but not of venous invasion or distant metastasis. cxcr4-positivity in buds was associated to poor outcome (p=0.0048) in ln-negative crcs (p=0.0191). conclusion: maintenance of cxcr4-expression in buds has a profound effect on tumor aggressiveness and prognosis in ln-negative crc-patients. this would help in establishing a tumor budding "profile" especially linked to lnmetastasis in crc. objective: cox-2 expression was investigated in colorectal cancer and colorectal precancerous lesions. its expression in adenomas is associated with increasing size and neoplastic potential. little is known about cox-2 expression in subtypes of serrated colonic polyps, which constitute substantial number of colon cancer precursors. the aim of the study was to assess cox-2 expression in serrated polyps of the colon and to investigate its potential discriminative role. method: 175 consecutive serrated polyps were analyzed. they included: 26 traditional serrated adenomas (tsa), 36 sessile serrated polyps (ssp) and 113 hyperplastic polyps (hp). cox-2 expression was assessed semi-quantitatively (0-3). kruskall-wallis test was used for the comparison of cox-2 expression between serrated categories (α=0.05). objective: in colorectal cancer (crc), tumor buds represent a more aggressive tumor cell type at the invasive front with apparently low proliferation. the aim of this study was to determine the proliferation potential of tumor buds by comparing ki67 staining across different tumor areas and adjacent normal tissue. method: whole tissue sections from 197 patients with crc underwent immunohistochemistry for ki67. 10 high-powerfields were evaluated for each of the following regions: normal mucosa, tumor center, tumor front and tumor buds. ki67-positivity was correlated to patient outcome. results: average ki67-positivity was 5.2 % in normal mucosa and significantly higher in the tumor center (38.2 %; p < 0.0001) and invasion front (34.9 %; p < 0.0001). strikingly, only 0.3 % of all tumor buds showed ki67-positivity (p<0.0001). although ki67-positivity in the tumor center or front was unassociated with clinicopathological features or patient survival, the greater the number of ki67-positive tumor buds, the worse the prognosis. this effect was also found after adjusting from tnm stage (hr (95 % ci): 2.25 (1.1-4.5); p=0.0193)). conclusion: a marked absence of ki67 staining is found in most tumor buds, suggesting a substantially decreased proliferation rate. however, the association of ki67-positivity with worse prognosis in 15 % of cases points towards a heterogeneous population of tumor buds. kayexalate and intestinal necrosis: an underrecognised entity p. luís * , a. alves, c. ferreira * hospital de santa maria, dept. de anatomia patológica, lisboa, portugal objective: there are only few cases of intestinal necrosis secondary to administration of kayexalate described in the literature, most of them from postoperative patients, having the largest study only 11 patients in 9 years. we report our experience in the last 4 years with a brief review of the pathophysiology and histologic aspects of the lesions induced by the kayexalate. method: between 2008 and march 2012 seven patients were diagnosed with ischemic necrosis related to kayexalate administration, five in colon biopsy and two in resection specimens. they're comorbidities and clinical evolution was extracted from the medical records. results: the patients' age ranged between 47 and 78 years. in one case we had the clinical information of chronic renal insufficiency. in three of them, there was no previous history of renal impairment. one patient developed symptoms after one single administration of kayexalate. the clinical presentation ranged from abdominal pain, gastrointestinal bleeding and three of them had a fatal outcome. the administration of kayexalate can have associated risks with different clinical impact in any patient. we must be aware of the related complications, as the histological identification of the kayexalate crystals indicates the etiology of the ischemic lesions to the clinicians and alerts them to the dangers of this therapeutic. objective: egfr plays an important role in colorectal cancer (crc) progression and represents a natural target for molecular anticancer drugs. in this study we evaluated 800 consecutive crc aimed to analyze k-ras mutational status in relationship to: different clinical-pathological parameters, egfr immunohistochemical (ihc) expression, response to cetuximab treatment. method: k-ras mutations and egfr expression were determined by direct cycle sequencing and ihc respectively in 565 surgical specimens and 235 biopsies. ihc findings were evaluated using four different score systems. results: the distribution of k-ras mutations did not significantly vary between surgical or bioptic specimens or with respect to different anatomic tumor localization. in contrast, k-ras is more frequently mutated in egfr negative/low score tumors than in positive ones (p<0.0001). focusing on the 419 surgical treated crc patients, we found a higher percentage of k-ras mutations in t4 crc (p=0.01) and in younger patients (p=0.002). finally, we observed that 6 % of primary crc, concomitantly evaluated with their paired metastases, changed k-ras mutational status during progression. conclusion: our data showed that tumor size, patient age and egfr ihc expression significantly influenced k-ras mutations. interestingly, we observed that cetuximab treated patients, had a better clinical outcome when egfr presented a high ihc score. multiple immunohistochemical investigation of signaling pathways in colorectal cancer t. micsik * , l. kopper, t. krenács, z. nagy, o. horváth * semmelweis university budapest, 1st department of pathology, hungary objective: despite the recent advances in therapeutic armamnet colorectal cancer (crc) remains one of the leading cancer deaths worlwide. crc has a heterogenous molecular background with known prognostic and predictive markers, but the whole picture is rather complex with many unexplored connections. method: we performed multiple immunohistochemical stainings on tissue microarrays (tma) made of 95 crc cases to study the correlations between egfr-ras, cell cycling, apoptosis, cell adhesion and tumor invasion pathways. we evaluated digitized slides using matrix scores considering both the frequency and positivity of cells and statistical analysis followed. results: we found strong linear correlation between positivity and (p-1068, p-1173) phosphorylation of the egfr receptor and p53 positivity detected with different clones. furthermore, phospho-nf-kb p65 positivity was strongly linked to survivin-expression. conclusion: activation of egfr-related pathways including nf-kb is a key factor in crc-growth. the strong link between cancer promoting egfr signaling and the mutational lack of the p53 driven apoptosis suggests a deadly cooperation, which can be further aggravated by forced tumor survival through survivin. correlation of these markers potentially predict poor disease outcome but can be specifically targeted by the upcoming new molecules of tailored therapy. objective: kras mutational status is important in colorectal carcinoma (crc). it is unclear, however, which is the most informative tissue source for study in crc cases showing more than one tumor mass. to explore this issue we determined kras status in a series of primary (pt) and metastatic crcs. method: a total of 68 tumors belonging to 25 patients (17 males and 8 females) with ages ranging between 46 and 80 years (average, 64 years) were studied. kras mutation hotspots in codon 12 and 13 were analyzed by polymerase chain reaction and sequentiation. wt-kras cases were confirmed with the kras stripassay tm. objective: lynch syndrome (ls), the most frequent form of hereditary colorectal cancer (crc), is caused by germline mutations in the mismatch repair system genes. a recently identified mechanism involving the epcam gene is responsible for 6.3 % of msh2-negative ls cases. we herein explore epcam protein expression in ls-associated, msh2-negative crcs to evaluate its potential value in the algorithmic approach to ls population screening. method: we studied a total of 19 msh2-negative crcs from 14 different patients in whom we were able to perform a complete germline analysis. expression of both msh2 (1:200 dilution, clone g219-1129, pharmingen) and epcam (1:100 dilution, clone ber-ep4, dako) was evaluated by immunohistochemistry (ihc). results: nine patients showed a deleterious germline mutation that involved the msh2 gene in three instances and the epcam gene exon 9 in six instances. all patients harbouring the epcam mutation belonged to the same family. of the19 crcs, epcam expression loss was seen in only five, all of them were from patients with a germline epcam deletion. conclusion: due to the high specificity of epcam protein loss for identifying ls patients with an epcam deletion, we recommend adding epcam ihc to the ls diagnostic algorithm in msh2-negative crc cases. objective: colorectal carcinoma with signet ring cell component (crc-src) is a rare and distinct subtype with little molecular information. we investigated the frequency of braf mutation in 28 crc-srcs and its relation with clinicopathologic parameters. method: we categorized tumors into groups 0-9 %, 10-24 %, 25-49 % and >50 % according to signet ring cell component. genomic dna was isolated from parafin blocks and analyzed for braf v600e mutation by polymerase chain reactionrestriction fragment length polymorphism (pcr-rflp). results: eleven of cases showed braf mutation (39.3 %). the results were also confirmed with sequence. no statistically significant differences were found in clinicopatologic parameters between braf wild-type and mutant crc-src. on the other hand, when we adjusted age, gender, percentage of signet ring compenent and stage, we found a statistically significant increased risk in braf mutant group compared to braf wild-type in cox regression analysis (hr=7.68, 95 % ci=1.06-55.78, p=0,044) conclusion: braf mutation is frequent in crc-srcs. this finding may support its diverse molecular pathogenesis and could have important therapeutic implications for those patients. to clarify the relation of braf mutation with clinicopathologic parameters and prognosis in crc-src, studies with multi-instutional larger series are needed. coexisting lipomas and adenocarcinoma of the colon: case report g. orgun sonmez * , u. bayol, o. akman, s. cumurcu, m. olmez * tepecik research and training hospital, izmir, turkey objective: lipomas of the large bowel are rare, they are invariably submucosal and therefore may intussuscept. infrequently lipomas may also present in the form of multiple polypoid masses in the colon. we present a 69-years old female with 3 submucosal lipomas and a coexisting adenocarcinoma of transverse colon. method: 69-years old female patient was admitted to hospital because of rectal bleeding for 3 months. colonoscopic examination revealed mucosaly intact polypoid tumoral masses and an ulcerated tumoral mass in transverse colon. biopsy of the ulcerated mass was diagnosed as adenocarcinoma. extended right hemicolectomy was performed. results: grossly, ulcerated tumor was 1,5×1×0,4 cm and there were 3 polypoid tumors in cecal area of the right colon which were 2.5-2.0-1.5 cm in their greatest dimensions. polypoid tumors were yellow, lipomatous on cut surfaces. microscopically ulcerated tumor was composed of adenocarcinoma morphology and the other polypoid tumors were composed of submucosal mature adipocytes with a thin capsule around them. the diagnosis was colonic adenocarcinoma (pt1n0m0) and submucosal lipomas of colon. the patient had no additional therapy and she is healthy with no evidence of recurrence. conclusion: this case is presented to remind that there may be multiple lipomas of colon with coexisting malignant epithelial tumors. objective: primary liposarcoma of the small intestine is exceedingly rare. to our knowledge, only 7 cases have been reported so far. we report a case of the primary myxoid liposarcoma of the ileum. method: the patient was 77-year-old woman. ct and mri revealed a 6-cm-intrapelvic mass, which was proved to be a tumor of the distal part of the ileum, and, therefore, partial ileectomy was performed. results: the tumor was 6.7×6.3×3.7 cm in size, relatively well circumscribed and located within the intestinal wall without invasion to the neighboring tissues. microscopically, the tumor had mixture of cellular and myxoid areas, showing proliferation of short spindle and stellate shaped cells in a delicate capillary vasculature. univacuolated tumor cells and a small number of lipoblast-like cells were also seen. immunohistochemically, these proliferating cells were positive for s-100 protein. according to these pathological findings, we diagnosed the tumor as myxoid liposarcoma. fish methods showed the tumor had chromosomal translocation t(12;16)(q13;p11). conclusion: the differential diagnosis of this includes other spindle cell tumors such as gist and myogenic tumors. but we could arrive at the proper pathological diansosis following the above morphological features and the specific chromosomal translocation. we will also discuss the clinico-pathological features of the cases including ours. prognostic significance of selected immunohistochemical markers in colorectal cancer e. paltseva * , o. samofalova, y. gorbacheva, p. tsarkov * first moscow state medical university, pathology, russia objective: the aim of our study was to identify a combination of markers whose expression is predictive of invasion and metastasis of colorectal carcinoma. method: the expression of e-cadherin, β-catenin, tenascin c, kai-1, epidermal growth factor receptor (egfr) and vascular endothelial growth factor (vegf) was studied immunohistochemically and correlated clinicopathologically in 72 primary colorectal carcinoma cases (26 with metastatic lymph nodes). 17 patients (24 %) developed haematogenous metastases at median 6,5 months postoperatively (3-11). results: expression of following markers significantly correlated with depth of tumour invasion: cytoplasmic accumulation of e-cadherin (p=0,035), nuclear staining for β-catenin (p=0,03), increased tenascin c expression (p=0,001), and loss of egfr and vegf expression (p=0,015 and 0,02, respectively). decreased tenascin c and kai-1 expression associated with the presence of lymph node metastases (p= 0,027 and 0,046, respectively). weak cytoplasmic e-cadherin expression correlated with distant metastases (p=0,045). conclusion: our results suggest that abnormal expression of proteins involved in cell adhesion and migration (e-cadherin, βcatenin, tenascin c, egfr) and angiogenesis (vegf) may be related to the invasion of colorectal carcinoma. detecting the expression of e-cadherin, tenascin c and kai-1 probably possesses clinical significance in evaluating lymph node and distant metastasis and predicting the prognosis of colorectal cancer. objective: in colorectal adenocarcinomas (crcs) microsatellite instability (msi) can be documented by immunohistochemical detection of mismatch repair proteins (mmr). the aim of the present study is to identify the percentage of msi-positive crcs, in the epirus region of greece and compare it to the international data. method: a total of 50 cases of sporadic crcs, 25 adenomas and 10 hyperplastic polyps, formalin-fixed paraffinembedded, were examined immunohistochemically using standard methods (en vision system), with the antibodies mlh1 (clone es05), msh6 (clone pu29) and pms2 (clone m0r4g) (novocastra-menarini). immunostained sections were evaluated semi-quantitatively, taking in account the percentage of neoplastic positive cells and estimating nuclear staining intensity, compared to the internal positive controls (normal epithelium, lymphocytes). results: all hyperplastic polyps and adenomas expressed the examined mmr. two out of 50 crcs were negative for msh6 and pms2, while one of them was also negative for mlh1. interestingly, one of these two crcs had an adjacent adenoma, which also showed negative staining for msh6 and pms2. conclusion: immunohistochemical staining for mmr is possible in archival material. loss of mmr varies, depending on the protein examined. in crcs from the epirus region the estimated loss of at least two mmr is 4 %. objective: matrix metalloproteinases (mmps) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all extracellular matrix (ecm) components. genetic alterations in mmp-2 and mmp-9 have been implicated to play an important role in colorectal carcinogenesis (crc). the aim of this study was to investigate the hypothetic correlations between the mmp-2 and 9 mrna expression and the clinicopathologic variables of the crc. method: the expression of mmp-2 and -9 mrna was assayed by rt-pcr for 53 tunisian sporadic colorectal adenocarcinomas. results: mmp-2 was highly expressed in the 53 cases. mmp-9 was not detected in 18 cases and moderately or highly expressed in 35 cases. absence of mmp-9 mrna expression was significantly related to an advanced tumoral invasion stage (pt3) (p=0.003). conclusion: our study suggests that the overexpression of mmp-2 could be the result of an imbalance in the system of transcriptional regulation of mmp-2, which would involve the loss of expression or activity of its inhibitor: timp-2. a variable expression of transcripts of mmp-9 could be explained by the hypothesis of hypermethylation of the promoter or degradation of its transcript after protein translation. relationship between matrix metalloproteinases 2 and 9 promoter polymorphisms and colorectal cancer risk objective: the carcinogenic schema of colorectal cancer (crc) follows a multistep process governed by mutational events that affect multiple micro-environmental factors, including matrix metalloproteinases (mmps). this present work aims to contribute to a better understanding of mmp-2 and -9 promoter polymorphisms involvements in the genesis and progression of sporadic crc in tunisian population. method: the mmp-2 and mmp-9 promoter genotypes were determined by pcr-rflp in 53 tunisian patients with sporadic colorectal cancer and 69 healthy control subjects. results: no significant associations were found regarding mmp-2 (-1306 c/t) polymorphism and crc susceptibility. there was a connection between the mmp-2-1306 promoter polymorphism and gender (p = 0.012). the mmp-9 (−1562 c/t) was significantly associated with crc (or= 2.39 [1.023 to 5.58]). no implication of mmp-9-1562 promoter polymorphism in the clinicopathological parameters of crc was evident. conclusion: our findings suggest that the mmp-2 promoter polymorphism was not a predictive factor for the crc occurrence while the mmp-9-1562 ct genotype was a risk factor for crc susceptibility. hodgkin's lymphoma in distal rectum in a patient with crohn´disease treated by biologic therapy -a case report r. sampaio * , f. costa, e. diane, j. palla garcia, j. r. vizcaino * anatomia patológica, valpaços, portugal objective: rare cases of hodgkin`s lymphoma have been reported in the setting of inflammatory bowel disease. method: we report a case of hodgkin`s lymphoma in a 37 years old male patient with corticosteroid-dependent crohn´disease (cd) who had been medicated with azathioprine for many years. in the last 4 years he began periodical therapy with infliximab, till the time when he complained of worsening of symptoms and the appearance of asthenia, tenesmus and weight loss. infliximab was withdrawn and replaced by adalimumab. a colonoscopy with several biopsies revealed the presence of a hodgkin lymphoma. results: the histological examination revealed granulation tissues where we identified atypical cells (reed-sternberg cells) with the following immunohistological staining: cd30+, cd15+, lmp1 + lca-, cd20-, cd3-, alk-. these results in conjunction with the morphological aspects and the clinical history of cd favored the diagnosis of hodgkin lymphoma in a patient with preceding cd. the patient was subjected to radical colectomy. postoperative pathological examination showed cd with involvement of the distal rectum by hodgkin's lymphoma. conclusion: the present report may serve as a reminder to clinicians of the possibility of occult lymphoma in patients with colonic cd treated with biological therapy. objective: dna mismatch repair (mmr) status in sporadic colonic cancers (ccs) had provided valuable prognostic and predictive information in the appropriate clinical setting; however there are no definitive guidelines for microsatellite instability (msi) testing. we evaluated the expression of mmr proteins and correlated them with outcome in a cohort of patients with sporadic colon cancer. method: we reviewed 352 cases of sporadic stage ii colonic cancer from abril 27th 1995 to december 12th 2009. clinical data including tumor grade, prognostic factors, therapy and outcome were searched for. tissue microarrays using a manual beecher system were constructed and stained with antibodies for mlh1, msh2, pms2 and msh6. results: 8 % (28) of 352 ccs had loss of expression of one or more mmr proteins, including defect in 1 protein 3.7 % (13), 2 proteins 3,7 % (13) and tree proteins 0,6 %(2). the mean overall survival was 144 months with no significant differences in patients between microsatellite stable and unstable tumors. conclusion: we detected a fairly low prevalence of the loss of immunohistochemical expression of mmr proteins. this result might have influence our mean overall survival between microsatellite stable and unstable tumors. the pathological effects of orchidectomy on intestine tissue and serum level of carcino embryogenic antigen s. sattari * , r. ahmadi * azad university of iran, science dept., hamedan, iran objective: carcino embryogenic antigen (cea) is a wellknown tumor marker influenced by carcinogenic changes in intestine tissue. the purpose of this study was to determine the effects of orchidectomy on serum level of cea and histological changes of intestine in male rats. method: male wistar rats were randomly divided to control, sham, uni-orchidetomised and biorchidectomised groups of 5 in each. 10 weeks after operation, blood samples were collected using cardiac puncture method. serum cea level was measured using elisa method. the effect of orchidectomy on intestine tissue was also histologically studied. the results were statistically analyzed using anova. results: serum level of cea was significantly increased in orchidectomised rats compared with control animals (p< 0.001). the increase in serum cea level was more in biorchidectomised than uniorchidectomised rats (p<0.001). there was also an enhancement in tissue lymphocytes, plasma cells and inflammation in intestine of orchidectomised animals. the results showed that orchidectomy results in pathological changes in intestine tissue leading to increased serum cea level. the plasma cells density in pararectal lymph nodes of patients with rectal cancer after neoadjuvant therapy m. sezak * , n. ozsan, b. pehlivanoglu, s. ozkok, t. yoldas, b. daganavsargil * ege university, dept. of pathology, izmir, turkey objective: neoadjuvant chemoradiotherapy (nt) is standard procedure for locally advanced rectal cancer. in this study, we compared the morphology of lymph nodes and the amount of plasma cells in the interfollicular region of lymph nodes in rectal tumour surgical excision material, between two groups who had received and not received nt prior to surgery. method: fifty cases with nt and forty cases without nt were included. the number and diameter of lymph nodes were recorded. the morphology of the lymph nodes were evaluated, and the percentages of interfollicular plasma cells were demonstrated immunohistochemically (cd138). tumor regression grade was assessed using mandard scoring system. results: average number and diameter of lymph nodes were greater in patients without nt (p<0,05). atrophy of germinal centers was noted as 54 % in nt group, versus 5 %. the amount of plasma cells in the interfollicular region was found to be higher than fifty-one percent in 54 % of nt group versus 5 % (p<0,05), as well. complete response rate (mandard's: 1) was 18 %. no significant association was found between the amount of plasma cells and tumor regression. conclusion: nt leads to a decrease in the volume of the lymph nodes and atrophy of germinal centers, conversely, causes stimulation of the proliferation of interfollicular plasma cells. adenomyoma of the ileum: pathohistological features with reference to pathogenesis b. snezana * , b. andrejic, n. solajic * clinical center of vojvodina, center for pathology, novi sad, serbia objective: adenomyoma is a rare benign non-neoplastic tumor-like lesion. it originates from abnormal embryonic buds but its pathogenesis has not been fully elucidated. method: a 63-year old man with severe pain, intussusception and ileus underwent a resection of a part of the intestine containing a tumor mass acting as a lead point. tissue was analysed using h&e, histochemical and immunohistochemical stains. results: lesion located in the mucosa, submucosa and muscularis propria consisted of glandular structures varying in size and morphology, the larger ones containing papillary projections and the smaller ones being similar to brunner's and peribiliary glands, both of them surrounded by smooth muscle bundles. the lesion was not accompanied by ectopic pancreas, and it indicated a diagnosis of adenomyoma of the ileum. glandular elements were ck7+ and ck20− and cdx-2−, opposite to intestinal mucosa. conclusion: adenomyoma is a mass lesion rarely found distal to the duodenum, which bares close clinical and morfological resemblance to a tumor and some non-neoplastic conditions. cytokeratin expression favors the heterotopic pancreas theory of pathogenesis, but abnormal interaction between the endoderm-and mesoderm-derived tissues can not be excluded. smoothelin in biopsies of colorectal carcinomas as marker of muscularis propria invasion p. stoemmer * , p. torres-galea * gemeinschaftspraxis pathologie, forschungslabor, augsburg, germany objective: staging of colorectal carcinomas is of eminent importance for prognostication and treatment; preoperative distinction between high grade dysplasia, invasion of submucosa (with permigration of the muscularis mucosae mm) and infiltration of muscularis propria (mp) is difficult in biopsies, due to similarities of smooth muscle cells in mm and mp. smoothelin is a robust marker of the mp in normal colon, not expressed in mm.we analysed it value for distinguishing mp and mm near carcinomas. objective: tuberculosis can affect any part of the gastrointestinal tract. anal localization, in particular, has a very low incidence (0,7 %). method: a 66 year-old male presented, complaining of perianal pain, constipation, weight loss of 5 kilos the last 4 months and night sweating. physical examination was normal, except for an ulcerated lesion in the anal region. the patient was submitted to computerized tomography (ct) and incisional biopsy of the lesion. results: the ct of the chest revealed multiple, scattered, calcified nodules in the upper zones of both lungs and histological examination showed confluent, non-necrotizing, epithelioid granulomas with the presence of langhans' multinucleated giant cells. acid-fast bacilli were detected both within the anal lesion and in the sputum. mantoux test was positive. the patient had a history of pulmonary tuberculosis 40 years ago. a diagnosis of anal tuberculosis was made and the patient was put on a four drug anti-tuberculous regimen. in 6 months the symptoms improved and the perianal lesion healed. conclusion: a tuberculous origin must be considered when the cause of anal and perianal lesions is unclear. therefore histological and bacteriological confirmation is essential in order to avoid undue delay in diagnosis and treatment. the relation of braf v600e mutation and microsatellite instability in colorectal carcinomas i. turkmen * , n. bassüllü, b. toptas, t. öztürk, r. yasar, p. korkmaz, n. saygili, o. öztürk, g. demir, g. dogusoy * istanbul bilim university, dept. of pathology, istanbul, turkey objective: colorectal carcinogenesis is associated with various morphological and molecular pathways. microsatellite instability (msi) pathway has familial and sporadic forms. among crc's %15 of the sporadic cases are associated with msi and these have hypermethylation of the mlh1 promoter associated with braf v600e gene mutation. familial forms do not show braf v600e mutation. method: we investigated the microsatellite instability (msi) by immunohistochemical study of mlh1, pms2, msh2 and msh6 together with the braf mutation presence and their correlation with standard histopathological parameters in 54 sporadic crc cases. results: the age distribution was 25-88 with an average of 67,9. immunohistochemical expression loss of mlh1, pms2, msh2 and msh6 was seen in 18,5 %, 22,2 %, 7,4 % and 11,1 % of the total 54 cases respectively. msi was seen in 27,8 % of the cases. the braf v600e gene mutation was seen in 18 cases and among these 8 cases (44,4 %) showed msi, while msi frequency was 19,4 in non-mutated group. and this was statistically significant (p < 0.05). msi was found significantly correlated with grade and lymph node number (p<0.05). conclusion: our study confirmed that sporadic cases of crc with msi are associated with the braf v600e gene mutation. msi is correlated with histologic grade and dissected lymph node number. colon biopsy diagnostics may reliably be performed using virtual microscopy objective: virtual microscopy using whole slide images (wsis) is a feasible alternative to optical microscopy, offering major advantages for pathology practice. the present study aims to prove reliability of this promising technique for colon biopsy diagnostics. method: colon biopsies (n=295) were assessed (into 7 main diagnostic groups) using both glass slides and wsis by 4 pathologists and 2 residents. two of the pathologists having ample experience using wsis, scored the biopsies in a primary diagnostic setting. for each case the criterion standard diagnosis was defined based on glass slide diagnoses. accuracy was defined as the percentage of concordance with the criterion standard. kappa statistics were calculated as a measure of observer agreement. results: the overall concordance rates were 89.5 % for wsis and 91.4 % for conventional microscopy. the intraobserver (wsis versus glass slides) agreement was good to excellent, with kappa values ranging from 0.73 to 0.87 (mean 0.78) and was higher than the interobserver agreement for glass slides (mean 0.71). concordance with the criterion standard varied less between wsis and glass slides in the diagnoses of pathologists with virtual experience. conclusion: this study showed good diagnostic accuracy and reproducibility for wsis, indicating this technology may be used for colon biopsy diagnostics. intestinal occlusion due to colonic lipoma: a case report f. vukmirovic * * clinical center of montenegro, dept. of pathology, podgorica, montenegro objective: lipomas of the digestive tract are rare benign tumor and most often found incidentally during a colonoscopy, computed tomography scan, surgery or autopsy. lipomas of the colon were first reported by bauer in 1757 and are most often located in the ascending colon. the incidence of this lesion is estimated between 0.2 and 4.4 % and represents 1.8 % of the colonic benign lesions. we report a case of patient with symptoms of ileus due to colonic lipoma. method: a 78-year-old man patient was urgently admitted with symptoms of ileus. right hemicolectomy was performed, and clinical impression was that it was a malignant tumor. results: examination of the cecum we found polypoid whitish yellow tumor, size 4×2,5 cm. on histology the tumor was composed of mature adipose tissue without cellular atypia. the tumor was located into submucosa. larger parts of the surface mucosa was eroded. the postoperative course was uneventful and intestinal passage was quickly established. conclusion: although colonic lipomas seldom cause severe symptoms in patients and are easily removed by endoscope while they are small, severe symptoms like abdominal fullness, intestinal obstruction, intestinal bleeding and intussusceptions may appear as a lipoma grows larger. in some cases lipoma may clinically mimic colonic carcinoma. prognostic impact of lymph node ratio outperforms positive lymph nodes and lymph nodes harvested: a time-dependent analysis in mismatch repair-proficient and -deficient colorectal cancers i. zlobec * , e. karamitopoulou, l. terracciano, d. inderbitzin, a. lugli * university of berne, inst. of pathology, bern, switzerland objective: we compare the prognostic strength of the lymph node ratio (lnr), positive lymph nodes (+lns) and collected lymph nodes (lncoll) using a time-dependent analysis in colorectal cancer patients stratified by mismatch repair (mmr) status. method: 580 stage iii-iv patients were included. multivariable cox regression analysis and time-dependent receiver operating characteristic (troc) curve analysis were performed. the area under the curve (auc) over time was compared for the three features. results were validated on a second cohort of 105 stage iii-iv patients. results: the auc for the lnr was 0.71 and outperformed + lns and lncoll by 10-15 % in both mmr-proficient and -deficient cancers. lnr and + lns were both significant (p <0.0001) in multivariable analysis but the effect was considerably stronger for the lnr [lnr: hr=5.18 (95 % ci: 3.5-7.6); +lns=1.06 (95 % ci: 1.04-1.08)]. similar results were obtained for patients with >12 lncoll. an optimal cutoff score for lnr = 0.231 was validated on the second cohort (p<0.001). conclusion: the lnr outperforms the + lns and lncoll even in patients with >12 lncoll. its clinical value is not confounded by mmr status. a cut-of score of 0.231 may best stratify patients into prognostic subgroups and could be a basis for the future prospective analysis of the lnr. prognostic value of tumor-stroma ratio in rectal adenocarcinomas s. zoidze * , r. scheer, j. klaase, m. elferink, a. baidoshvili * laboratorium pathologie oost, enschede, netherlands objective: recently, tumor-stroma ratio (tsr) has been identified as a strong predictor for survival in colorectal cancer. despite an identical biology clinical implications are quite different for colon and rectal cancer regarding to anatomical differences. method: tsr was estimated on h&e stained histological sections of 154 patients who underwent resection for rectal adenocarcinoma between 1996 and 2006. none of these patients had received neoadjuvant chemo-or radiotherapy. the tsr was determined, by two independent investigators, in different layers of the rectal wall at the point of highest tumor infiltration and at the border of the tumor. tsrs were categorized into three categories: tsr-low, tsr-moderate and tsr-high. results: patients with stage i and ii disease (t1-4 n0) and tsr-high showed significantly better 5 year survival rates for overall survival compared to tsr-low and tsr-moderate (p=0.010) and a trend to a better disease specific survival (p=0.067) and disease free survival (0.057). in a multivariate cox regression analysis the tsr remained an independent prognostic factor for overall survival, when adjusted for age, pt-status and grading. conclusion: tsr as a prognostic tumor characteristic can be used to identify patients with a good and a poor outcome in lymph node metastasis negative cases. objective: goblet cell carcinoid (gcc) of appendix vermiformis is a rare neoplasm that share histological features of both adenocarcinoma and carcinoid tumor. while its malignant potential remains unclear, gcc's particularly show transmural dissemination and are more aggressive than conventional carcinoids. patients usually present with acute appendicitis. they usually lack the formation of a well-defined tumor mass; thus, it is somewhat difficult to accurately assess their size. method: case presentation: fifty years old male patient applied to the emergency service with abdominal pain and laparatomy was performed with suspicion of acute appendicitis. grossly, conjestion and exudation on the distal edge of appendectomy material were seen. a 1×1 cm lesion which was spreading into peripheral adipose tissue was seen on the cut surface. microscopically, tumor was composed of goblet cell groups resembling "signet ring cells". tumor was infiltrated into muscularis propria and mesoappendix. immunohistochemically, chromogranin, synaptophysin, cytokeratin 20, mcea and p53 were stained positive. ki67 (mib 1) proliferation index was 18 %. conclusion: adenocarcinoid of the appendix is a rare tumor, which is very difficult to diagnose preoperatively and even macroscopically, making histological examination essential. immunohistochemical staining is required for definitive and differential diagnosis. here, we present this rare case with literature reviewed. normal colon tissue and colon carcinoma show no difference in heparanase promoter methylation d. hershkovitz * , y. peerless, e. simon, e. sabo, o. ben-izhak * rambam health care campus, dept. of pathology, haifa, israel objective: heparanase, the sole heparan sulfate degrading enzyme, has a role in cellular invasion. accordingly, a large number of studies have demonstrated an association between heparanse expression and tumor stage and patients' prognosis. in colon carcinoma, heparanase shows increased expression in tumor compared to normal tissue and its expression correlates with the presence of metastasis. one of the regulatory mechanisms of heparanase expression is methylation on its promoter. in the present study we evaluated the role of heparanse promoter methylation in colon carcinoma. method: analysis of heparanse promoter methylation was done on 32 samples of colon carcinoma as well as 30 sample of normal colonic mucosa. dna was extracted from ffpe tissue and subjected to bisulfite conversion. the relative fraction of methylated and unmethylated dna was evaluated with real-time pcr. results: the fraction of methylated dna was 1 %±0.6 in the colon carcinoma group, and 2.4 %±0.6 in the normal colon group (p=0.11). only one case in the normal group and one case in the tumor group showed more than 10 % methylation in the heparanase promoter. conclusion: we did not find any difference in heparanase promoter methylation between colon carcinoma and normal colonic mucosa, suggesting that heparanase overexpression in colon carcinoma is mediated by other mechanisms. objective: among b-cell non-hodgkin's lymphomas, neural cell adhesion molecule/cd56 expression is exceptional. the aim of this study is to report unusual cd56 (neural cell adhesion molecule, ncam) expression on diffuse large b cell lymphoma (dlbcl). method: the panel of antibodies included cd3, cd20, cd10, bcl-2, bcl-6, mum-1 and cd56. results: a total of 50 cases of dlbcl were identified and one case was also detected positive for cd56. the subject was female, 48 years old. the patient had presented generalized lymphadenopathy. she had not documented involvement of extranodal sites. the case is alive with disease 1 year after diagnosis and chemotherapy treatment. objective: a case of mediastinal t-cell lymphoblastic lymphoma with indolent clinical course in a 52-yearold man was studied by immunohistology and molecular biology. method: formalin-fixed sample. histology with ematox-ilin&eosin and giemsa; immunohistochemistry by apaap method. t-cell receptor gamma rearrangement by biomed2 protocols. results: after one fainting episode the patient was found to have pericarditis caused by a xx-cm-diameter mediastinal mass. the 2 first biopsies were unsuccessful and the third diagnostic one was done 6 months later. in this time-period the patient was well without symptoms and the mass stable. after two mediastinal biopsies with unspecific features (castleman disease?), the diagnostic one showed fibrosis, abundant small reactive lymphocytes (cd20+/cd3+) and areas with diffuse small-medium size lymphocytes positive for cd3, tdt, cd34, cd7, lmo2 and high ki67. t-cell receptor gamma genes were monoclonally rearranged. conclusion: indolent t-lbl is exceedingly rare: for this reason in our case lmo2 was a key marker for the diagnosis which was delayed for the presence of fibrosis and reactive b cells. objective: granulocytic sarcoma (gs), also termed myeloid sarcoma or chloroma, is a rare malignant solid tumor resulting from the extramedullary proliferation of myeloblasts or immature myeloid cells. gs most frequently occurs in patients with acute myeloid leukemia, myeloproliferative neoplasms or myelodysplasia. gs rarely presents in the absence of systemic myeloid disease. gs most commonly occurs in the soft tissues of the head and neck, bone, skin and less often in the central nervous system and spinal cord. results: a 40 year old woman presented with 1 month history of weakness of both legs. mri of the thoracal spine showed 4 cm diameter epidural mass compressing the dural sac in the spinal canal at the t9-10 levels. decompressive laminectomy and tumor removal were performed resulting in neurological improvement. histologically neoplastic cells have round nuclei with finely dispersed chromatin and scant cytoplasm proliferating in a diffuse pattern. immunohistochemically, tumor cells were positive for mpo, cd117, cd45, cd99. diagnosis was gs without bone marrow involvement. conclusion: we report the case of gs due to unusual localization and nonleukemic presentation. results: there were 5 males and 1 female with a median age of 40 years (22-79 years). physical examination shows splenomegaly in all cases and hepatomegaly in 2 cases, hematological parameters shows lymphocytosis with peripheral hairy cells in all cases. flow cytometric immunophenotyping was done in 2 cases, and confirmed the diagnosis of hcl with cd11c, cd25+, cd103+, cd123 +, and cd5-, cd10-, and cd23-. bone marrow biopsy was done in all cases, showing massive infiltration with villous cytoplasmic projection cells that were cd20 positive. the treatment consists of blood transfusion in all cases, associated with splenectomy in 3 cases, only 1 patient was treated with (2 cda: cladribine) with a complete remission after a follow up of 24 months. conclusion: hcl has consistent cytologic histologic and immunologic features that make classification reliable and reproducible; it remains problematic because of the variety of the disorders and the differential diagnosis of splenic lymphoma of the marginal zone. objective: primary cns lymphomas (pcnsl) are uncommon tumors in immunocompetent patients, they represent up to 1 % of non hodgkin lymphomas (nhl) and 3-5 % of all brain tumors. the majority of pcnsl are diffuse large b cell lymphomas, 2-5 % are t cell lymphomas, in rare instances low grade b cell lymphomas. we determine clinical characteristics, histological findings and treatment outcome of pcnsl. method: five cases of pcnsl occurring between 1993 and 2007 were retrospectively reviewed. results: they were 4 males, 1 female, median age 50 years, range (23-74). most common symptom was neurological deficits (4cases). mri of the brain revealed an expansive tumor affecting the parietal bone in 3 cases, the temporal bone in 1 cases and the frontal bone in 1 case. immunohistological finding showed large b-cell nhl in 2 cases, anaplastic lymphoma in 2 cases, and large-cell immunoblastic lymphoma in 1 case. treatment consists on exclusive tumoral resection in 3 cases, surgery and chemotherapy (1 case), complicated by death in all these cases. surgery followed by high dose chemotherapy and radiotherapy (1 case) with complete remission after a follow-up of 7 years. conclusion: chemotherapy followed by involved field irradiation appears to be an adapted therapy. objective: primary cutaneous non-hodgkin's lymphoma (pcnhl) is defined as lymphoma limited to skin without extra-cutaneous involvement at presentation. we describe here epidemiological and histo-pathological aspects of pcnhl. method: retrospective review of clinical data of pcnhl patients diagnosed in hematology department, frahet hached hospital, sousse (1993 . results: fourty three patients with pcnhl, median age: 48 years (11-84), sex-ratio was 2.3. the site of cutaneous involvement was upper lumb in 4 cases, lower lumb in 7 cases and disseminated in 23 cases. pcnhl was small t cell lymphoma in 13 cases, large b cell lymphoma in 16 cases, anaplastic, cd4+ cd56+ hematodermic neoplasm, and follicular type in 1 case respectively. according to tnm classification 21 cases were t3bnxmx, 12 cases were t1n0m0. after first line therapy, 26 patients in cr, pr was obtained in 1 case and failure in 6 cases, 10 patients relapsed within 1 to 72 months (median 6 months), all of them were disseminated cases. conclusion: our study indicate that the extend of cutaneous involvement at the time of diagnosis is a significant prognostic factor in pcnhl. objective: follicular lymphomas (fls) account for one third of non hodgkin lymphomas (nhls) in adults. the disease is characterized by a response to initial treatment, followed by relapses, sometimes associated with histologic transformation into high grade nhl. analyze epidemiological characteristics of patients with fl. method: retrospective study of 22 of adult fls diagnosed in hematology department (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) . results: median age was 59 years (29-77), sex-ratio: 1.7 the most frequent clinical symptom at diagnosis was lymphadenopathy in 13 cases. involvement of the spleen in 3 cases. b symptoms were present in 10 cases. performance status was more than 1 in 4 cases. according to the international prognostic index (flipi), 7 patients have low index, 5 have intermediate index, and 10 have high index. grade 2 fl was seen in the majority of cases. treatment modalities varied over time and according to the disponibility of drugs especially to anti cd20 monoclonal antibody. cr was obtained in 13 cases, pr in 6 cases; 3patients relapsed within 8-48 months. histologic transformation was seen in 3 cases, we have noted 10 deaths related to the disease in 7 cases. conclusion: treatment results in fl might not only be improved by more effective induction regimens but also by maintenance treatment. objective: cd99 and fli-1 are widely used for their diagnostic utility in ewing sarcoma/peripheral neuroectodermal tumor (es/pnet). cd99 expression has been documented in a variety of tumors, including lymphoid malignancies of t cell origin. also fli-1 expression is common in different tumor types. however, few studies have investigated the cd99 and fli-1 immunoreactivity in diffuse large b-cell lymphoma (dlbcl). we aimed to determine the frequencies of cd99 and fli-1 immunohistochemical expressions in dlbcl which may lead to a misdiagnosis. method: cd99 and fli-1 expressions are retrospectively investigated by immunoperoxidase staining in prechemoterapy primary tumors of 42 dlbcl cases. results: out of 42 cases, cd99 and fli-1 expressions were observed in 14 (33.3 %) and 7 (16.7 %) tumors, respectively. concomittant expressions of cd99 and fli-1 proteins were found in 5/42 (11.9 %) cases. the present study revealed that cd99 and fli-1 are frequently expressed in dlbcl, thus dlbcl should be considered in the differential diagnosis of cd99+ and fli-1+ neoplasms. primary cutaneous marginal zone lymphoma with biclonality e. beretouli * , t. koletsa, a. bouzakis, s. mavropoulou, g. karkavelas, i. kostopoulos * ahepa hospital, dept. of pathology, thessaloniki, greece objective: primary cutaneous marginal zone lymphoma (pcmzl) represents a b-cell lymphoma which presents with papules, plaques or nodes. the presence of a monotypical light chain b cell population supports the diagnosis of pcmzl. we describe a case of pcmzl which showed different light chain restriction in two heterochronous lesions. method: α 50 year-old male patient presented in 2004 with a cutaneous nodule in the anterior surface of the tibia, which was totally excised. the patient did not show other lesions since 2011, when a new cutaneous nodule located in the right ankle, was appeared. immunohistochemical analysis was performed in both biopsies. results: histological and immunohistochemical findings of both biopsies were consistent with marginal zone lymphoma of the skin with lambda light chain restriction in the biopsy of the first lesion and kappa light chain restriction in the biopsy of the second lesion. blood laboratory studies, chest and abdominal computerized tomography scans revealed no evidence of systemic involvement by lymphoma. conclusion: the finding of both kappa and lamda light chain restricted b cell populations in pcmzl is unusual. the monoclonal light chain switching in pcmzl is rare and its pathogenesis is discussed. objective: microvessel density (mvd) has prognostic significance in some malignancies. little information exists about angiogenesis in mantle cell lymphoma (mcl), although antiangiogenic drugs are used experimentally. prognostic factors of mcl are needed as outcome and therapy are heterogeneous and often unsatisfactory. the aim is to assess mvd in mcl and to evaluate its prognostic significance. method: 177 mcl specimens (ffpe) were examined by immunohistochemistry with anti-cd34 antibody. mvd was quantified using systematic uniform random sampling and unbiased counting frames. clinical data were analyzed in kaplan-meier survival curves and log-rank test. results: median survival (ms) of all patients was 46 months, median progression-free survival (mpfs) 22 months. mean mvd was 172,7 microvessels/mm2, median 158,2/mm2. dividing cases into quartiles: <117,4/mm2, <158,2/mm2, <206,6/mm2 and ≥206,6/mm2 (43, 44, 45, 45 cases), ms and mpfs did not differ statistically significantly between these groups (p=0,307; similar for therapy groups). visible differences are only between the fourth quartile and the rest: objective: plasmablastic lymphoma (pbl) is a rare aggressive subtype of diffuse large-b-cell lymphoma which occurs primarily in the oral cavity of hiv-positive patients. it is extremely rare in immunocompetent patients. method: a total of 5 cases of pb were retrospectively analyzed. all cases had tissue available for immunohistochemistry and in situ hybridization. results: the age range of the 5 patients was 35-81 years (median 68) and all were men. three patients were hiv negative. hiv + patients: tumors involved the oral cavity (n =1), and small intestine (n=1). hiv-patients: tumors were located in bone marrow (n=1) and lymph nodes (n=2). all tumors were composed of monomorphic plasmablastic cells. tumors were negative for cd45 and cd20, while they showed diffuse positivity for cd138, cd38, mum1 and ema. by ish, monoclonal light chain restriction was detected. ebv (eber) was positive in all cases. the median overall survival was 21,4 months regardless of the intensity of chemotherapy. only one patient is alive 1 month after diagnosis. conclusion: pbl may appear in non immunocompromised patients and other locations than oral cavity. any patient diagnosed with pbl should be tested for hiv. patients who had hiv-negative pbl have lower rates of oral involvement. spinal granulocytic sarcoma preceding clinical manifestation of acute myeloid leukemia v. cemerikic-martinovic * , n. drndarevic, n. andjelkovic, p. djordjevic, d. jovanovic, r. nikolic, t. martinovic * beo-lab, dept. of pathology, belgrade, serbia objective: granulocytic sarcoma is a rare malignant neoplasm of primitive myeloid cell origin, most commonly found in association with acute myeloid leukemia. granulocytic sarcomas generally occur in the soft tissues, bone, and skin. method: we report a case of a 55-year old man with spinal granulocytic sarcoma manifesting as rapidly progressive paraplegia preceding clinical manifestation of acute myeloid leukemia. results: magnetic resonance imaging revealed a tumor in lumbal spine (l1-l3). baseline laboratory data were normal. he underwent emergent laminectomy and the tumor was totaly resected. histological examination of lesion demonstrated diffuse proliferation of large atypical cells with round nuclei with delicate chromatin and one or more prominent nucleoli. immunohistochemical studies revealed positive staining for lca, cd33, cd43, cd163, lysozyme and cd15 while myeloperoxidase, lymphoid, neuroendocrine markers, s-100 and cd99 were negative. the final histological diagnosis was granulocitic (monoblastic) sarcoma. no chromosomal aberrations were detected by fish. a month later, acute myelomonocytic leukemia was diagnosed upon a peripheral blood and bone marrow examination showing an increased number of abnormal monoblasts. chemotherapy, with multiple regimens for leukemia in combination, did not affect the tumor and the patient died. conclusion: granulocytic sarcoma should be considered in the differential diagnosis of spinal tumors. objective: survivin, a member of the inhibitor of apoptosis family might play an important role in the pathogenesis of diffuse large b cell lymphoma (dlbcl). we investigate clinical and prognostic significance of survivin expression in nodal dlbcl. method: biopsy specimens obtained from 56 patients with newly diagnosed nodal dlbcl treated with immunochemotherapy (r-chop) were immunostained for survivin. results: survivin immunoexpression (>45 % positive tumor cells) has been found in 22 (39.28 %) patients. significant difference in immunoexpression was noticed between gcb and non-gcb subtype of dlbcl (p=0.031). there was no significant association with ipi, "bulky"disease, ki-67 immunoexpression or other clinico-pathological parameters. univariate analysis showed that survivin expression was unfavorable factor for therapy response and shorter survival in patients with dlbcl (p=0.048 and p=0.034, respectively). patients with survivin overexpression experienced relapse more often that the patients without expression of this apoptotic protein (27.3 % vs. 11.8 %), but this difference did not reach statistical significance (p=0.131). the results of this study showed that disregulation of survivin expression had the important role in determination of course of disease in pateints with nodal dlbcl treated with r-chop. therefore, survivin represent potential therapeutic target in dlbcl. langerhans cell histiocytosis of the jaws: a report of ten cases with an analysis of the mechanism of eosinophilic infiltration y. objective: langerhans cell histiocytosis (lch) frequently presents eosinophilic infiltration. here we report ten cases of lch that involved the jaws and our investigation of the mechanism underlying eosinophilic infiltration in lch. method: we evaluated the ccl-11/eotaxin-1 expression of lch cells via immunohistochemical staining. toluidine blue staining was used to inspect the densities of mast cells, with ten periapical granuloma specimens serving as a control group. results: every patient was classified as having single-system lch, even though acute lymphoblastic leukemia occurred in one patient during lch treatment. the ratio of mandible to maxilla lch was 5:1. the jaw lesions were the earliest manifestation in seven patients with multifocal lch. toluidine blue staining revealed that the number of mast cells in lch lesions was not significantly higher than in periapical granulomas. however, upon immunohistochemical examination, most of the patients showed diffuse positivity for eotaxin-1 in lch cells, while few eosinophils/ t cells were immunoreactive. conclusion: we surmise that the eotaxin-1 expression of lch cells may be relevant to eosinophilic infiltration in lch. further studies of the eotaxin-1 functions including its influence on the immature state of lch cells may be needed to understand the pathogenesis of lch as well as the role of tissue eosinophilia in lch. method: we report a case of sant in 53 years-old man presented with abdominal discomfort and pain in a left lumbal region. abdominal computed tomography scan revealed a massive haematoma extended from spleen to the enlarged left kidney and left retroperitoneal space. the patient also had a spontaneous rupture of right kidney 2 years ago. results: macroscopically, the cut surface of spleen showed multiple well circumscribed red brown nodules. microscopically, sant consists of multiple well-circumscribed angiomatoid nodules showing plump endothelial cells and extravasated erythrocytes. nodular formations are surrounded by a variable lymphoplasmacytic infiltrate, spindle cells and collagenous stroma. immunohistochemical staining displayed endothelial phenotype that resembled splenic capillaries (cd34+/cd31+/cd8−). expression of cd68 was also present. conclusion: the differential diagnosis of sant includes splenic hamartoma, inflammatory myofibroblastic tumor, littoral cell angioma and hemangioendothelioma. it has been postulated that sant represents a peculiar hamartomatous transformation of splenic red pulp in response to an exaggerated nonneoplastic stromal proliferation. sant has a benign clinical course and splenectomy has been supposed as curative. collision tumor of menningioma and non hodgin malignant lymphoma of cerebellum h. erdem * , a. k. uzunlar, u. yildirim, a. sav, m. dosoglu * duzce university, dept. of pathology, turkey primary central nervous system lymphoma (pcnsl) constitutes a rare group of extranodal non-hodgkin's lymphomas (nhls), primarily of b cell origin, whose incidence has markedly increased in the last three decades. immunodeficiency is the main risk factor, but the large majority of patients are immunocompetent. this report presents the case of a 71-year-old woman with a collision tumor of primary malignant lymphoma and meningioma in the cerebellum. collision tumor of primary malignant lymphoma and meningioma have not been described in the literature. the morphological aspect is interesting with regard to the problem of collision tumors. prognostic significance of immunohistochemical expression of the angiogenic molecules vegf-a, vegfr-1 and vegfr-2 in patients with classical hodgkin lymphoma e. georgiadi * , g. dimtsas, p. karakitsos, t. vassilakopoulos, i. thymara, p. korkolopoulou, e. patsouris, c. kittas, i. doussis-anagnostopoulou * larisa, greece objective: classical hodgkin lymphoma (chl) is characterized by the presence of a small percentage of malignant hodgkin and reed-sternberg (hrs) cells amongst a reactive background. the role of angiogenesis in chl is still unclear. the aim of the study was to evaluate the expression of vegf-a, vegfr-1 and vegfr-2 and their correlation with clinicopathological parameters and prognosis. method: the immunohistochemical expression of vegf-a (vg1, dako), vegfr-1 (rb-9049, neomarkers) and vegfr-2 (sc-6251, santa cruz) was studied in a large cohort of 199 patients with chl and the results were correlated with clinical characteristics and patient outcome. results: the neoplastic hrs cells expressed vegf-a, vegfr-1 and vegfr-2 in 90.3 %, 97.2 % and 94.1 % of the cases respectively and their expression levels were intercorrelated. expression of vegfr-1 and vegfr-2 was significantly and positively correlated with early disease stage, absence of b-symptoms, wbc <15.000, alb ≥4, ips ≤2. vegfr-2 was additionally positively correlated with male gender and esr <50. all three molecules were statistically correlated with ramifications of blood vessels but not with microvessel density. conclusion: based on our results, we could speculate that, in contrast to solid tumours, the process of angiogenesis is probably an early event in neoplastic progression in the context of hodgkin's lymphoma. ps-12-019 p53 upregulation is associated with proliferation and is not capable of inducing apoptosis in hodgkin's lymphoma e. georgiadi * , g. dimtsas, t. p. vassilakopoulos, v. g. gorgoulis, i. a. doussis-anagnostopoulou * larisa, greece objective: p53 is a tumor suppressor protein described as the "guardian of the genome". in a normal cell, p53 is inactivated by its negative regulator, mdm2. once activated, p53 induces cell cycle arrest and apoptosis by activating various cell cycle related genes, like waf1/ cip1 encoding for p21. although mutations that deactivate p53 are the most common genetic alteration found in cancer, they are rare in the context of hodgkin's lymphoma (hl). method: p53 immunohistochemical expression was investigated in a subset of 81 cases of primary hl. the study of the p53-downstream protein p21 was used as an indirect way of analyzing its functional status. p53 expression was correlated with proliferation, apoptosis, clinicopathological data and prognosis of the patients. results: p53 showed a median value of 32.4 %, suggesting an upregulation of the p53 gene in hl, and a significant correlation with p21 (p=0.043) and the proliferation marker mib1 expression (p=0.038). conclusion: although p53 overexpression is a frequent finding in hl and there is indication of its functionality, this upregulation does not lead to apoptosis. it can be speculated that this is possibly due to mdm2-p53 interactions. ps-12-020 simultaneous gastric adenocarcinoma and b cell lymphoma of the stomach f. gerin * , k. turkoz, b. kantarcioglu, c. ataizi celikel * marmara university, dept. of pathology, istanbul, turkey objective: the simultaneous association of gastric carcinoma with gastric lymphoma is a rare event. method: we describe a 52 years old man, who had been diagnosed as marginal zone lymphoma in a cervical lymph node biopsy 3 years previously.no endoscopic examination was evaluated then. there was no pathological appearance in the gastrointestinal system by ct scanning and patient was considered nodal marginal zone lymphoma. patient had taken 8 cycles of chemotherapy which consisted of rituximab, cyclophosphamide, vincristine, and prednisolone.2,5 years after completion of chemotherapy, a gastric wall thickening compatible with gastric lymphoma or linitis plastica was detected on ct and gastroscopic examination was indicated. endoscopic examination revealed an erosive, fragile, hemorrhagic, malignant-looking lesion with a diameter of 4.5 cm in the cardia. besides, rugae of fundus and corpus were severely rough and hyperemic suggesting a diffuse malignant infiltration. multiple biopsies were taken from cardia, fundus and corpus of the stomach. results: cardiac samples showed pancytokeratin positive signet ring cells which contain neutral and acidic mucine in microscopic examination. samples of corpus and fundus did not show similar carcinoma cells, instead, there was diffuse infiltration of atipical lymphocytic infiltration consistent with marginal cell lymphoma both morphologically and immunophenotypically. conclusion: co-occurrence of carcinoma and marginal zone lymphome is a rare event and dramatically exacerbates prognosis of a patient with such an indolent lymphoma. assessment of demographic data, staging and experesion of cd20, cd30, cd15, bcl-2 in reed-sternberg cells of hodgkin lymphoma cases in their first admission in urmia imam komini hospital b. ilkhanizadeh * , p. mazlomi * imam komini hospital, dept. of pathology, urmia, iran objective: immunohistochemistry plays an important role in diagnosis of hodgkin lymphoma. beyond diagnostic importance of immunohistochemistry, so far many studies have investigated the correlation between cellular markers and the prognosis of patients with hodgkin lymphoma. amongst other markers, cd20 is one of the most studied marker with a lot of controversy around it. method: retrospectively 66 eligible patients entered the study. clinical and laboratory data and immunohistochemistry findings analyzed for possible correlation between disease stage and other parameters. results: in our study 63.6 %, 90.9 % of cases respectively expressed cellular markers cd15 and cd30 while only 18.2 % of cases expressed cd20. furthermore statistical analysis revealed that cd15 was inversely correlated with disease staging. (p=0.027). in contrast we didn't find any relation between cd20 or cd30 positivity with disease staging. (p=0.482, p= 0.376). however in our study expression of cd20 was not related to stage or other parameters of poor prognosis which proposes that in our patients this marker possibly was not related to disease prognosis. conclusion: therefore we suggest that cellular marker cd20 is not beneficial beyond ips factors and its usage should be confined to diagnostic purposes. primary naturall killer cell lymphoblastic leukemia/ lymphoma of central nervous system g. karagkounis * , t. argyrakos, e. ronne, e. dimitriadis, d. papadopoulos, g. stranjzalis, d. rontogianni * athens, greece objective: a 25-year old woman with a 2-year history of a multifocal lesion occupying the cerebellum who periodically received cortisol and etoposide as it was considered tumor-forming multiple sclerosis. method: two years after initial clinical diagnosis a stereotactic biopsy was performed. immunohistochemistry and in situ hybridization were performed on the tissue sample. results: the perivascular (virchow-robin) space of microvessels was occupied by middle sized lymphocytes with blastic chromatin and scant cytoplasm. the neoplastic cells were negative for: 1. b-cell markers: cd19, cd22, cd20, cd79a, pax-5, 2. myelogenous differentiation antigens: mpo, cd13, cd33, 3. plasmacytoid dendritic differentiation antigens: cd123, tcl1, 4. blastic cells markers: cd34, cd117 5. t-cell markers: cd3, cd5 with restricted expression of cd2 and cd7 in a few cells, 6. cytotoxic enzymes tia-1, granzyme-b and perforin. all cells expressed intensely cd56 and tdt and some of them cd16. in situ hybridization for ebv virus (ebers) was negative. pcr analysis: t-cell receptor and immunoglobulin genes were in germ line. bone marrow was negative. objective: lymphomas constitute 33,65 cases on 100 000 inhabitants annually, its frequency grows. method: this work presents epidemiological, morphological analysis of nodal/exstranodal lymphomas. analysis of data from izhevsk cancer center during 2009-2011 was carried out. results: lymphoma appeared in 2.94 % among first diagnosed malignant tumors. the panel of monoclonal antibodies was used for tumor immunophenotype identification. nodal lymphomas presented 183 cases: 55 -hodgkin lymphoma (hl), 128non-hodgkin lymphoma (nhl). exstranodal lymphomas presented 111 cases, various variants of nhl were more frequent. small lymphocyte lymphoma -37, diffuse large b-cell lymphoma (dlbcl) -36, follicle center lymphoma -18, mantle cell lymphoma -8 were frequent among nodal lymphomas. t-cellular nhl presented 16 cases (6 -peripheral t-cell lymphoma, 7 -angioimmunoblastic tcell lymphoma). among other organs of lymphatic system lymphomas appeared in tonsils (19), thymus (15) and spleen (9). exstranodal nhl were registered in stomach (25), rarely in other organs -testis (7), central nervous system (3), nose (3), breast (3). as rule dlbcl was more frequent (36). conclusion: results coincide with data on lymphoma's frequency and morphological characteristic in other regions. in situ mantle cell lymphoma in nasopharynx t. koletsa * , f. tsiompanou, c. poulios, g. karkavelas, i. kostopoulos * auth, dept. of pathology, thessaloniki, greece objective: the nasopharynx normally contains abundant lymphoid tissue and it can be the site of both lymphoid hyperplasia and lymphomas. we present the first case of the recently described in situ mantle cell lymphoma (mcl) in nasopharynx. a 70 year-old woman presented with nasal obstruction in 2008. a mass located in nasopharynx was found. biopsy was sent for histological examination. a new biopsy was taken 3 years later. histological, immunohistochemical and fish studies were performed. results: histological examination of both biopsies revealed lymphoid hyperplasia, characterized by cd5 and cyclind1 positive cells of mantle zones, findings consistent with in situ mcl. the diagnosis of in situ mcl confirmed by fish analysis for t(11;14). the patient examined thoroughly and has remained free of an overt lymphoma to the present day. conclusion: pathologists and otorhinolaryngologists should be aware that in situ mcl may be observed in nasopharynx. it may be appropriate to perform cyclind1 immunostain, even in cases with clinical and histological findings compatible with lymphoid hyperpasia. the patients should be examined thoroughly, since in situ mcl may accompanied by an overt lymphoma in other sites or it may be a precursor of an overt mcl. multisystem langerhans' cell histiocytosis coexisting with metastasizing adenocarcinoma of the lung a. lovrenski * , m. panjkovic, z. eri * institute for lung diseases, dept. for pathology, sremska kamenica, serbia objective: langerhans' cell histiocytosis (lch) is an uncommon disease of unknown etiology characterized by uncontrolled proliferation and infiltration of various organs by langerhans' cells. to our knowledge, this is the first case reporting an association of multisystem lch with metastasizing adenocarcinoma of the lung. method: we present the case of a 54-year-old man, heavy smoker, with dispnea, cough, hemoptyses, headache and ataxia, who died shortly after admission to our hospital. on the autopsy, we found tumor in the posterior segment of the right upper pulmonary lobe as well as a right-sided occipito-parietal lesion which penetrated into the right ventricle resulting in internal and external hematocephalus. histologically and immunohistohemically, the diagnosis of primary lung adenocarcinoma with brain metastasis was made (tumor cells showed positivity for ck 7 and ttf-1 which confirmed the diagnosis). light microscopic examination of the other organs showed lch involving the pituitary gland, hypothalamus, spleen, mediastinal lymph nodes, and lungs. immunohistochemical studies revealed cd68, s-100 and cd1a immunoreactivity within the langerhans' cells. conclusion: multisystem form of lch with extensive organ involvement was an incidental finding, while the metastatic lung adenocarcinoma to the brain, that led to hematocephalus, was the cause of death. langerhans cell histiocytosis associated with extramedullary hematopoiesis in a 65-year-old male patient l. lungoci * , l. elena, b. flavia, m. rodica * city hospital, pathology, timisoara, romania objective: langerhans cell histiocytosis is a rare disease histologically characterized by the proliferation of langerhans cells. method: a 65-year-old male came to our attention presenting with erythematous-squamous lesions all over the body, axillary and inguinal adenopathies and hepatosplenomegaly. we have taken a cutaneous and an axillary lymph node biopsy. results: microscopic examination of the cutaneous fragment revealed the presence of a dermal infiltrate of large cells with an eosinophilic cytoplasm and vesicular, nucleolated nuclei with an irregular nuclear margin, raising suspicion of a cutaneous lymphoma. immunohistochemical stains for cd45, cd20, cd79a, cd3, cd5, cd15, cd30, and bcl-2 were negative, and the previous diagnosis was not confirmed. the microscopic examination of the axillary lymph nodes showed foci of extramedullary hematopoiesis, raising suspicion of a lymphoproliferative process, but the immunohistochemical stains and the paraclinical data didn't confirm it. based on the intense positive reaction for cd68, s100 and ki-67 showed by the tumoral cells from the prelevated tissues, correlated with the clinical and paraclinical findings, a diagnosis of langerhans cell histiocytosis was established. conclusion: the particularity of our case consists in the presence of extramedullary hematopoiesis in the lymph nodes of a patient diagnosed with langerhans cell histiocytosis. unusual primary cutaneous presentation of b-cell chronic lymphocytic leukaemia s. mavropoulou * , z. tatsiou, g. xanthopoulidis, a. goutzouvelidis * general hospital, laboratory of pathology, xanthi, greece objective: b-cell small lymphocytic lymphoma/chronic lymphocytic leukaemia (b-cll) is a low-grade lymphoproliferative disorder and cutaneous lesions are rarely the presenting findings. method: we report the case of a 62-year-old woman who presented in winter with erythematous plaque on her nose, of 3 months duration. laboratory data showed elevated white blood cell count (40.000/mm3) with small mature lymphocytes predominance (60 %). results: histopathologic examination of the skin lesion reveals infiltration of the reticular dermis and subcutis, consisting mostly of small lymphoid cells, without epidermotropism. immunohistochemical examination revealed positivity for cd20, cd79a and cd5 in the neoplastic cells. according to the laboratory and histopathologic findings the diagnosis was consistent with b-cll with a primary cutaneous presentation. a staging bone marrow aspiration and biopsy showed 80 % infiltration by a clonal b-cell proliferation with typical cll immunophenotyping. ct-imaging was normal, so stage i according to binnet classification was confirmed. the patient received 6 cycles of chemotherapy and remained in remission for 1 year, when the skin lesion recurred in winter, at the same location. conclusion: in conclusion we report an extremely rare case of subclinical b-cll with cutaneous presentation. it is important to maintain a high index of suspicion for a lymphoproliferative process in skin lesions with atypical lymphocytic infiltration. myeloid sarcoma mimicking nasal polyp s. mungan * , u. cobanoglu, s. ersoz, g. guvendi, m. yilmaz * karadeniz technical university, pathology, trabzon, turkey objective: a myeloid sarcoma is a tumour mass consisting of myeloid blasts with or without maturation occuring at an anatomical site other than the bone marrow. every site of the body can be involved, skin, lymph node, gastrointestinal tract, bone, soft tissue and testis. %10 cases presents at multiple anatomical sites. method: the current case is 75 year old woman presenting with nasal polyp and underwent polypectomy. the polyp was 2,5 cm in diameter and had a bloody appearance. by microscopic examination there was a diffuse infiltration of neoplastic cells under the cilliated epithelium. the mitotic index was high in neoplastic lymhoid cells. immunohistochemical study was helpful for the differential diagnosis. objective: the aim of this study was to describe the epidemiology and anatomo-clinical features of the ear, nose and throat (ent) lymphomas in the centre of tunisia. method: all ent lymphoma cases diagnosed in the pathology department, farhet hached hospital, sousse during a 15-year period were analyzed. results: a total of 34 cases of ent lymphoma were reported (21 males, 13 females) with a sex-ratio of 1.4. the age at diagnosis varied from 9 to 85 years with a median age of 56.5 years. the majority of ent lymphoma cases (81 %) were of b cell type including large b-cell lymphoma (58 %) and small-cell b lymphoma (23 %). nasopharynx and tonsils were the most involved areas with 44 % and 42 % of cases, respectively. according to the ann arbor staging system, these lymphomas are diagnosed in the stage ii (42 %) followed by stage i (30 %). conclusion: the most frequent lymphoma developed in ent region was b cell lymphoma, especially located in nasopharynx and tonsils. results: a total of 44 cases were reported (26 males, 18 females) with a sex-ratio of 1.4. the median age was 10 years (range: 3-15 years). lymph node lymphomas were the most frequent accounting for 63.6 %, followed by ent, mediastinum, and haematopoietic system (6.8 % each one); cutaneous and digestive location were less frequent (4.5 % each one). among the 44 patients, 23 (52.3 %) had had hodgkin's lymphoma and 21 (47.7 %) had had non-hodgkin's lymphoma (nhl). diffuse large b-cell lymphoma was the most diagnosed (57 %) followed by burkitt lymphoma (23.8 %) and t-cell lymphoma (9.5 %). according to the ann arbor staging system, 29.6 % of patients were diagnosed in an advanced stage. method: we present a case of composite ailt and diffuse large b-cell lymphoma (dlbcl) a 45-year-old woman with multiples lymph nodes. biopsy of inguinal node was performed. three months later the patient had enlarged cervical node which was removed. results: firstly, the inguinal lymph node was diffusely infiltrated by small-medium size lymphocytes with scanty cytoplasm. the nuclei were irregular. several large cells were observed in a background of prominent arborizing high endothelial venules. the small cells were cd3 positive. the large cells were cd79a, cd30 positive. theses cells showed nuclear positivity for eber by ish. secondly, the cervical lymph node showed large areas of necrosis admixed with pleomorphic and medium size t lymphocytes. there were a population of neoplastic ebv + pleomorphic b lymphocytes; some of them with rs-like morphology. we performed b and t-cell clonality studies. we detected tcr-gamma t-cell and igh b-cell gene clonal rearrangement. conclusion: occasionally igh gene rearrangement has been detected but only a few cases have demonstrated, morphologically, composite ailt and ebv-associated diffuse large b-cell lymphoma (dlbcl). typhlitis as initial manifestation of granulocytic sarcoma of the appendix: a case report p. rohani doost * , b. ilkhanizadeh, f. noroozinia, a. objective: myeloid sarcoma is a localized mass of myeloblasts or immature myeloid cells involving any extramedullary site. reports in the literature documenting leukemic infiltration of appendix are uncommon and extremely rare when symptoms mimicking typhlitis. method: a 30-year-old man with a history of relapsed acute myelogenous leukemia (fab classification, m2) admitted for receiving induction chemotherapy. after 10 days of chemotherapy, he presented with fever, nausea, vomiting, abdominal pain and severe neutropenia (0.026×109/l as absolute neutrophil count). this manifestation was attributed to typhlitis and he received antibiotic therapy, but he came back 50 days later with acute abdomen prompting exploratory laparotomy which revealed inflamed appendix. results: histologic examination showed diffuse appendiceal wall infiltration of mononuclear cells with medium-to-large vesicular nuclei, conspicuous nucleoli and pale eosinophilic cytoplasm. immunostains showed the diagnosis of granulocytic sarcoma with myeloperoxidase, leukocyte common antigen, cd15, cd117 and cd68 positivity and epithelial membrane antigen, keratin, cd3, cd5, cd10, cd79a and cd20 negativity. conclusion: our case declares that physicians and surgeons should be aware of granulocytic sarcoma in the differential diagnosis of mild tenderness on palpation of the abdomen as a complication of acute myelogenous leukemia. objective: angioimmunoblastic t-cell lymphoma (aitl), one of the most frequent entities among peripheral t-cell lymphoma, is characterized by lymphadenopathy, b-symptoms, and an aggressive behaviour. method: we reported 3 cases of aitl during a period of 4 years (2004) (2005) (2006) (2007) . results: the age of patients varied between 20 and 73 yearold. they hadn't any pathologic history. two patients presented with a multiple cervical lymphadenopathy, and weight loss. the other patient had fever and sweat. physical examination found multiple lymph nodes with hepatosplenomegaly in 2 patients. abdominal ultrasound and ct-scan showed diffuse lymphadenopathy with features raising the possibility of lymphoma. the diagnosis of aitl was confirmed by a microscopic and an immunhistochemical study of lymph node or liver biopsy. 2 patients received a cure of chemotherapy conclusion: angioimmunoblastic t-cell lymphoma (aitl) is an aggressive non-hodgkin t-cell lymphoma. aitl is frequently associated to immunological and hematologic diseases such as autoimmune hemolytic anemia, vasculitis, rheumatoid arthritis, and autoimmune thyroid disease. its natural history has been the subject of controversy, considered for many years to be a nonmalignant disorder or a dysimmune disease, until the clonal nature of aitl was proven by molecular studies. although patients are usually treated with corticosteroids, chemotherapy, and/or plasmapheresis, outcomes were dismal. objective: low grade lymphomas (indolent) are commonly located on extranodal sites. nodal location are relatively of low incidence. our aim is to describe the epidemiological status of nbcl and to assess their pathological features in the center of tunisia. method: we report 50 cases of indolent nbcl diagnosed in the department of pathology, during a period of 10 years (2000-2009) . clinical, pathological and immunohistochemical (large panel of antibodies), treatment and outcome data are collected. results: the mean age was 63 year old (33 and 89 years). the sex ratio was 1.27. nbcl was 6.5 % of all n h lymphomas and 42.73 % of indolent nbcl of any sites. nodal enlargement and hepatomegaly were the main symptoms. small lymphocytic lymphomas were the most frequent variant (56 %), followed by follicular lymphomas (26 %). mantle cell lymphoma and marginal zone b cell lymphoma were rare (12 % and 6 %, respectively). they were exclusively of b cell type. 76 % of cases were in stage iii-iv. 90 % of patients received chemotherapy. 2 % of recurrence and 48 % of death were found. conclusion: nbcl in the center of tunisia are relatively uncommon. small lymphocytic lymphoma is the most frequent in our population. they are still of relatively worse prognosis. objective: bone marrow metastases can be found in some malignant tumors, but diagnosing a nonhematologic malignancy from marrow is an unusual event. herein we present a case of metastatic signet ring cell carcinoma with an unknown primary site. method: a 39 year-old woman admitted to the hospital with weakness and bone pain. positron emission tomography/computed tomography (pet/ct) revealed disseminated bone lesions showing increased metabolic activity. bone marrow biopsy and aspiration were performed. results: bone marrow biopsy revealed extensive necrosis and nearly total replacement of normal bone marrow elements by atypical tumoral cells. tumoral cells were arranged individually or in small clusters, having hyperchromatic, eccentric located nuclei and abundant cytoplasm with signet ring cell morphology. even after thourough investigation of all the systems, a primary site of malignancy could not be detected. she received nine cycles of chemotherapy. craial mri revealed multiple metastatic lesions. 9 months after the initial diagnosis, she died of disseminated metastatic lesions. conclusion: even after thorough investigations, a primary site of malignancy could not be detected. in the literature, all the presented cases had very short survival varying from days to a few months. our patient lived for 9 months with multidiciplinary approach. objective: approximately 20 % of follicular lymphomas (fls) are bcl2 negative and 10-15 % of fls lack a translocation t(14;18). previously we have found that negative bcl2 staining in cases that do have a t(14;18) is caused by mutations in the epitope for the bcl-2 antibody, but this is never the case in bcl-2 negative, t (14;18) negative cases. now that translocation detection is more commonly performed we noticed that many of the cases that were referred to us as translocation negative fl are actually often nodal marginal zone lymphomas (nmzls) with extensive follicular colonization. method: t(14;18) negative cases that on h&e sections look like fl were analyzed by morphology and immunohistochemistry, including antibodies against cd10, bcl-2, ki-67, and cd23. results: careful observation of the bcl-2 staining pattern revealed that follicles that appeared positive at low power contained negative cells at high magnification. the amount of negative cells varied from follicle to follicle and was associated with the extent of ki-67 positivity. conclusion: based on morphology and immunohistochemistry we reclassified many cases of bcl-2 positive, t(14;18) negative fl into nmzl. although there is presently no positive marker for nmzl, we believe that such cases are responsible for most t(14;18) negative fl. objective: recent studies do not provide a definite answer on the origin and pathogenesis of lichen planus (lp). histologic findings are the same, regardless of the area involved. electron microscopy is useful to confirm the diagnosis in atypical cases and monitoring lp development. this study aimed an ultrastructural characterization of keratinocyte degeneration in pemphigoides, hypertrophic, and follicular lp types correlating this with levels of gelatinolytic activity caused by metalloproteinase-9 (mmp-9). method: skin biopsies were processed conventionally and examined in a jeol 1011 transmission electron microscope. mmp-9 expression was detected immunohistochemically. results: lp pemphigoides demonstrated intact basal keratinocyte cell membrane, and the lamina densa lining blister cavity correlating with moderate basal mmp-9 expression of weak intensity. hypertrophic type demonstrated good preservation of keratinocytes and their junctions along with strong basal and suprabasal mmp-9 expression of moderate intensity. both types demonstrated diffuse and strong mmp-9 immunostaining in dermal lymphohistiocytic infiltrate. a hypertrophic type revealed an increase of expression within dermal sweat glands comparing with lp pemphigoides. a diffuse and strong epidermal and follicular mmp-9 staining was noticed in follicular variant of lp. conclusion: ultrastructurally keratinocyte involvement displays deviations in various types of lp, and these changes correlate with levels of gelatinolytic activity caused by mmp-9. objective: clevudine is a recently introduced anti-viral agent that shows efficacy against hepatitis b virus related chronic liver diseases. however, it has been reported that certain patients who have received long-term administration of clevudine exhibit myopathy involving mitochondrial abnormalities. to make a differential diagnosis congenital mitochondrial myopathy and drug induced mitochondrial myopathy, we investigate ultrastructural findings of drug induced myopathy. method: we studied histopathological features of myopathy focused on the various ultrastructural mitochondrial abnormalities found in 18 patients with long-term clevudine therapy. results: in every case, ragged red fibers and multinucleated fibers with eosinophilic granules were observed. additionally, type 2 fiber atrophy was found in 6 cases. mainly concentrated in subsarcolemmal or inter-fibrillar areas, the abnormal mitochondria were enlarged and swollen, showing a variety of morphological types. most of the abnormal mitochondria indicated structural abnormalities in cristae, including the apparent decrease in the number, concentric lamellar pattern, and structure that is branched or lattice-like. conclusion: from the overall analysis, clevudine-induced myopathy is characterized by ragged fibers showing proliferation of abnormal mitochondria with various forms of inclusion bodies and abnormal cristae. another particular feature of interest is the presence of multinucleated fibers, which, in most cases, are filled with abnormal mitochondria. ultrastructural features of human mature oocytes subjected to cryopreservation s. a. nottola * * university la sapienza, dipt. die anatomia histol., rome, italy objective: oocyte cryopreservation protocols have not been fully optimized yet. our aims were to evaluate and compare the ultrastructure of human mature oocytes frozenthawed (f/t) after slow freezing and vitrification. method: the oocytes, fixed at sampling (fresh controls) and after freeze/thawing, were processed for light and transmission electron microscopy (lm and tem) observations. results: by lm, both fresh and f/t oocytes were rounded cells surrounded by an intact zona pellucida (zp) and containing uniformly distributed organelles. by tem, numerous vacuoles were found in f/t oocytes after slow freezing. on the contrary, vacuoles were only occasionally detected in f/t oocytes after vitrification, and in fresh controls as well. amount and density of cortical granules (cgs) appeared abnormally reduced in f/t oocytes, irrespective of the protocol applied. conclusion: in conclusion, a) cryopreservation currently ensures a good overall preservation of the oocyte; b) however, vacuolization appears as a recurrent form of cell damage during slow freezing, whereas the quasi absence of vacuoles seems the most relevant marker of quality in vitrified oocytes; c) premature cg exocytosis -and the consequent hardening of the zp -seems a non-specific, ubiquitous phenomenon occurring during freeze/thawing, suggesting the appropriateness of the use of icsi as the preferred insemination method after cryopreservation. tuesday, 11 september 2012, 09.30 -10.30 results: there were 3 women and 2 men with a median age of 60 (range 45-80 years). symptoms were dominated by abdominal pain, jaundice, vomiting, and alteration of the general condition. radiologic investigation showed multiloculated cystic mass of the pancreas, associated to splenic thrombosis and bone metastasis in one case. in gross, we received multilocular cysts of 3 to 16 cm in wall, their wall was fibrous, and thick, the internal surface showed many papillary projections and mural nodules. there was no communication with the duct system. on histologic examination, cysts had a fibrous wall lined by mucinous atypical cells showing frank anaplasia, wall's cyst was invaded by anaplastic glands; the stroma was of ovarian type in all cases. conclusion: pmc are uncommon neoplasia, their diagnosis is based on the histopathologic exam, their diagnosis is better than duct carcinoma and depends on the extent of tumour invasion. heterotopic pancreas of the gallbladder associated with acute cholecystitis o. akman * , e. e. pala, u. bayol, m. emiroglu * tepecik training research center, dept. of pathology, izmir, turkey objective: pancreatic heterotopia is a rare entity, which is commonly found in the stomach, duodenum, jejunum and meckel's diverticulum. heterotopic pancreas is extremely rare in the gallbladder. despite being a congenital condition, it takes years to become symptomatic. it can be associated with cholecystitis or cholecystolithiasis. method: a 21-year-old female patient was admitted to emergency service with abdominal pain. on physical examination, right upper abdomen was tender without defense or rebound. abdominal ultrasonography revealed small sized gallstones and pericholecystic fluid. magnetic resonance cholangiography showed dilated intrahepatic and extrahepatic bile ducts. laparoscopic cholecystectomy was performed. results: cholecystectomy specimen was 75×35×30 mm. on gross examination we noted a few milimetric yellow colored stones and 12 mm yellow solid intramural nodule. the mucosa was covered with fibrinopurulent exudate. microscopic examination revealed acute cholecystitis and aberrant pancreatic tissue consisting of acini and ductules. the phlegmonous inflammation of the gallbladder infiltrated the aberrant pancreatic tissue. the patient recovered completely after cholecystectomy. conclusion: we found this case worth reporting because, pancreatic heterotopia of the gallbladder is a rare, clinically silent entity unless complicated with gallstones and acute cholecystitis. expressions of c-erb-b2, egfr, p27, pten, mtor, pi3k in hepatocellular carcinomas and adjacent liver tissues method: fifty hcc cases were stained immunohistochemically with these markers. correlations between the markers and pathologic characteristics were analyzed. results: the cases had an average age of 56,72. male/female ratio was 44/6. hbv is more common in advanced stages and right lobe location (p<0.05). tumor size is significantly larger in patients older than 50 years (p<0,04). no membranous c erb b2 staining was seen while cytoplasmic positivity was present in 92 % and significant correlation was found with multiplicity (p<0.041) and p27 positivity (p<0,011). egfr membranous positivity was present in 90 % and significantly correlated with stage (p<0.05). p27 was negative in 92 % and pten is reduced or absent in 56 %. all markers were similarly expressed in adjacent noncancerous tissue. no significant correlations were found between pten, mtor, pi3k and the pathological parameters. conclusion: in our study, as a first step, none of the markers among c-erb-b2, egfr, p27, pten, mtor, pi3k was found significant in correlation with pathologic features. we look forward to obtain further results in the next study consisting of correlations with follow ups. the value of echo guided liver biopsy in the positive diagnosis of a rare primary liver tumorpathological approach g. becheanu * , v. herlea, v. serban-barbu, m. dumbrava, s. enache, g. smira, c. angelescu, a. pop, m. diculescu * bucharest, romania objective: hepatic angiomyolipomas are rare mesenchymal hepatic tumour. the most important problem about their diagnosis on biopsy specimens is to exclude a hepatocellular carcinoma. method: we present two cases of hepatic angiomyolipoma diagnosed in fundeni clinical institute, bucharest. both females patients, 39 and 43 years old, were imagistically diagnosed with a 13 cm hepatic left lobe tumor in the first patient, respectively a 10.5 cm hepatic right lobe tumor in the second one. both tumors were echo guided biopsied. the biopsy specimens measured about 8 mm, respectively 20 mm. the tumors were surgical removed. the biopsy and resection specimens were analyzed in light microscopy, including immunohistochemistry. results: initial diagnosis on he stain was cell variant hepatocellular carcinoma, in the 8 mm biopsy, respectively suspicious for angiomyolipoma in the second case. further immunohistochemical studies showed positivity for vimentin, hmb45, melan a, s100 and actin, and negativity for och1e5, ck7, ck8/18, cea, cd34, ki67 and factor viii in both biopsy specimens, consistent with hepatic angiomyolipoma diagnosis, confirmed on surgical specimens too. conclusion: diagnosing a hepatic angiomyolipoma is not easy to do especially on a biopsy. the size of biopsy specimens plays a very important role in a correct diagnosis. results: a 62-year-old woman presented with a 2-day history of nausea, fever and diffuse abdominal pain. ct scan revealed a well circumscribed small lesion of the pancreatic body. on arterial phase imaging, the mass was of low density relative to the pancreas without marked contrast enhancement. based on these findings, the patient underwent surgical resection of this mass and a distal spleno-pancreatectomy was performed. microscopic examination showed a well circumscribed but non encapsulated soft tissue proliferation consisted of blood vessels lined by a single layer of uniform flattened cells, with dilated lumen filled with red blood cells and inflammatory cells. immunohistochemical staining confirmed the vascular nature of the lesion. the diagnosis of pancreatic hemangioma was made. conclusion: adult pancreatic hemangioma is an extremely rare tumor. the review of all published cases showed that this tumor often do not demonstrate the contrast-enhanced ct features typical of an hemangioma, so a poor arterial phase enhancement cannot rule out pancreatic hemangioma and the histological examination is very important in these cases. objective: hepatocholangiocarcinoma (hcc-cc) is an uncommon form of primary liver cancer with features of hepatocellular and biliary epithelial differentiation. it accounts for 0.4-14.2 % of all primary liver carcinomas. many of the demographic and clinical features of this tumor remain unclear. it was previously suggested that coexisting chronic liver diseases were rarely seen in patients with hcc-cc. method: we report a case of hcc-cc diagnosed in patient with cirrhosis. results: a 48 year-old man presented with ascites, jaundice, vomiting and symptoms of portal hypertension. on abdominal ct scan the right hepatic segment was heterogeneous, hypervascular in the late arterial phase and low-attenuated on portal venous phase. based on a these clinicoradiological informations, diagnosis of infiltrating hepatocellular carcinoma was made and hepatic biopsy was performed. histopathologic examination showed, in addition to features of liver cirrhosis, the presence of malignant proliferation composed of tubular structures as well as microtrabecular and compact foci. on immunohistochemical stainings the tumor cells expressed cytokeratins 7 and 20. cells lining the tubular structures reacted with cytokeratin 19 allowing the diagnosis of hepatocholangiocarcinoma. conclusion: hepatocholangiocarcinoma is a rare primary liver cancer. preoperative noninvasive diagnosis with conventional radiography is often difficult especially when occurring in patients with liver cirrhosis or other hepatic chronic disease. the diagnosis is frequently made only after histological and immunohistochemical examination. morphological changes in different zones of the gallbladder in cholelithiasis m. bokhodir * , t. vervekina * tashkent medical academy, dept. of patological anatomy, uzbekistan objective: gallstone disease remains a serious problem of modern surgery. an important aspect of the problem is the question of the urgency of surgical intervention, depending on the condition of the gallbladder. method: it was carried out the analysis of 1130 case histories and excised gallbladder with different forms of cholelithiasis. 81.3 % were women in the age of 41-50 years. acute catarrh cholecystitis was found out in 20 % cases, acute phlegmonousin 21 % cases, acute gangrenous -in 11 % cases, chronicin 28 % and chronic relapsingin 21 % cases. results: all forms of cholecystitis characterized by a maximum thickness of the body wall, minimal at the bottom of the gallbladder. in all forms adipose tissue inflammation in the connective tissue layer was increased and it was found out the atrophy of the mucosa. in the inflammatory infiltrate in acute forms of inflammation were found neutrophilic and basophilic leukocytes: with simple acute cholecystitis -in the bottom, and with acute phlegmonous and gangrenous cholecystitis -in the area of the body of the gallbladder. in the cases with chronic cholecystitis were dominated mononuclear cells in all areas, and in the cases with chronic relapsing -neutrophilic and basophilic leukocytes in the bottom and the body, and mononuclear cells -in the neck of the gallbladder. ps-14-008 histological features of chronic hepatitis c in hemodialysis patients i. delladetsima * , a. kokkori, v. sypsa, m. psichogiou, s. sakellariou, j. boletis * athens university, 1st dept. of pathology, greece objective: chronic hepatitis c (chc) in hemodialysis patients has not been thoroughly investigated, despite its high frequency. the present comparative study aims to highlight the histological features of chc and its association with putative pathogenetic parameters in this specific group of patients. method: sixty-one biopsies of hemodialysis patients and 326 biopsies from the general population with chc were comparatively evaluated for the severity and specific histological features of chc. results were examined in relation to age, time of dialysis, viral load and genotype. results: patients on hemodialysis were older than patients of general population (p=0.031), showing a similar genotype distribution (p=0.328) and lower viral loads (p=0.001). chc on hemodialysis was significantly milder according to stage (p=0.033), activity and its parameters (p<0.001). significantly reduced was also the frequency of lymphoid aggregates (p< 0.001), bile duct damage (p<0.001) and steatosis. (p=0.033). severity of hepatits was not associated with time on hemodialysis. in multivariate analysis the differences were independent of age, which was associated with more severe disease. steatosis was associated with hemodialysis duration and age. conclusion: chc in hemodialysis patients is significantly milder than in general population. limited necroinflammatory activity and absence of immune mediated lesions are indications of defective immune response although involvement of low viral load cannot be overlooked. ps-14-009 inflammatory (myofibroblastic) pseudotumor of the liver: case report e. demiralay * , s. altaner, h. özdemir * baskent university, dept. of pathology, istanbul, turkey objective: inflammatory pseudotumor of the liver (ipt) is a rare, benign tumor-like lesion of which etiology is not fully known. it is confused with primary and metastatic malignant tumors as it seems as an occupying mass on radiologic examinations. method: thirtyeight-year-old male patient who had fatigue and dyspepsia was detected to have elevated liver enzymes and hepatomegaly. on computed tomography, two masses of which middle is mildly hypometabolic, measuring 34× 31 mm in right liver lobe and 45×39 mm in left liver lobe were detected. biochemical tests were normal except elevated ast and alt levels. ultrasound-guided needle biopsy was done for two masses. results: on microscopic examination, a lesion composed of abundant fusiform fibroblastic cells and vascular structures among inflammatory cells composed of lymphocytes, plasmocytes, histiocytes and eosinophillic leucocytes and including sharp margins between regenerated liver tissue was observed. on immunohistochemical examination, sma was stained focal positive in fibroblastic cells. staining with desmin, s-100, panck, cd34, ki-67 and cd117 was not seen. conclusion: ipt does not have a specific radiologic finding and definite diagnosis is made with pathologic examination. this case is presented as it is rarely seen, radiologically resembles to malignant tumors and for review of pathological differential diagnosis. enhancer of zeste homologue 2 (ezh2) expression in malignant and benign hepatic tumors k. dezso * , v. szabó, e. bugyik, k. schlachter, s. paku, z. schaff, p. nagy * semmelweis university, 1st dept. of pathology, budapest, hungary objective: the immunohistochemical demonstration of enhancer of zeste homologue 2 (ezh2) proved to be a useful marker in several tumor types, including hepatocellular carcinomas.in order to recognize the diagnostic value of this protein in hepatic tumors we have investigated the presence of ezh2 in the most common liver neoplasms. method: the presence of ezh2 has been studied by standard immunohistochemistry in several formalin-fixed paraffin-embedded tumor samples (hepatocellular adenoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, metastatic liver tumors and primitive childhood tumors). results: forty of 44 hepatocellular carcinomas, 22 of 23 cholangiocarcinomas, 29 of 31 hepatoblastomas and 14 of 17 metastatic liver tumors stained positively, but all the investigated hepatocellular adenomas (n = 24) and proliferating biliary structures were negative. the other primitive childhood tumors that were examined all expressed ezh2. conclusion: based on these results ezh2 is a sensitive marker of malignancy in hepatic tumors regardless of their histogenesis. in routine surgical pathology ezh2 could be helpful to diagnose hepatocellular carcinomas and it might be the first marker to distinguish transformed and reactive cholangiocytes. ps-14-011 nuclear ploidy as a marker of intraductal papillarymucinous pancreatic neoplasms malignancy e. dubova * , o. mishnev, a. shchegolev * v.i. kulakov scientific center, dept. of pathology, moscow, russia objective: intraductal papillary-mucynous pancreatic neoplasm (ipmn) represents 0,5 to 9.8 % of all tumors of exocrine pancreas. our aim was to study nuclear ploidy of malignant and benign ipmns cells. method: we studied 20 pancreatic surgical specimens from 5 females and 15 males. we performed histological, morphometrical and ploidy analysis of all specimens. results: ipmn cells nuclear perimeter and size was higher than in normal pancreatic duct cells. this parameters were higher in malignant noninvasive ipmn than in benign tumor. nuclear size and perimeter of invasive malignant ipmn were in intermediate position between benign and malignant noninvasive tumor. mean nuclear ploidy (mnp) of normal ductal pancreatic cells was 2.4c. mnp of benign ipmns was 2.5c, borderline tumors -3.0c. mnp of noninvasive malignant ipmns was 5.1c, of invasive ipmns -4.5c. we also revealed that aneuploidy coefficient (ac) of benign ipmns was 0 (there weren't aneuploid nuclei in its cells). ac of borderline ipmns was 0.11. maximal level of ac was in malignant noninvasive ipmns whereas ac of invasive ipmns was lesser. conclusion: nuclear morphometric parameters and mean nuclear ploidy level as well as aneuploidy coefficient could be used as additional criteria for determining malignant potential of ipmns. ps-14-012 an intraductal papillary mucinous neoplasm of the pancreas with concomitant neuroendocrine tumor: a case report j. garcia * , f. e. costa, r. sampaio, r. dias, a. duarte, j. ramon vizcaíno * lisbon, portugal objective: intraductal papillary mucinous neoplasms (ipmns) are a recently classified pancreatic neoplasm with an increasing incidence. ipmn of the pancreas is a lesion consisting of mucin-producing cells with neoplasic potential, characterized by papillary intraductal growth and varying degrees of cytologic atypia. neuroendocrine tumors of pancreas (pnet) are rare pancreatic neoplasms comprising 1-2 % of all pancreatic tumors. pnets express neuroendocrine markers, however the true cell or cells of origin are not fully understood. pnets are classified as functional or nonfunctional based on the presence or absence of a specific clinical syndrome associated with hormone oversecretion. intraductal papillary mucinous neoplasm of the pancreas with concomitant neuroendocrine tumor are a highly rare occurrence. method: a literature review of ipmns and pnet was made, as well as a review of the clinical data of a patient with ipmn and concomitant pnet. results: a 60-years-old caucasian female, underwent a laparoscopic distal pancreatectomy with splenectomy for ipmn. macroscopic examination revealed discrete dilatation of pancreatic ducts and nodule in the pancreas tail. hystologic examination revealed neoplasic lesion of intraductal papillary-mucinous in the body and tail of pancreas with moderate dysplasia (ipmn borderline) and neuroendocrine tumor grade 1 (who 2010). conclusion: this case reports a highly rare occurrence. glypican 3 and agrin expression in hepatocellular carcinoma and cholangiocellular carcinoma: a immunochemistry study n. gursan * , h. balta, b. gundogdu * ataturk university, medical faculty, erzurum, turkey objective: glypican 3 (gpc3), which plays a role cellular growth, differentiation and migration is one of members of glypican family. although gpc3 expression in the liver, glypican upregulation of the vast majority of tumors are available. agrin is found the surface on the cells and the extracellular matrix, the micro-vessels method: in this study, formalin-fixed, paraffin-embedded surgical specimens from 20 patients with hepotocellular carcinoma and 20 patients with cholangiocellular carcinoma diagnosed between 2000 and 2011 years in ataturk university, faculty of medicine, department of pathology were studied with immunohistochemistry agrin and gpc3. results: we detected agrin in around bile ducts and blood vessels within the portal areas in the normal liver. i̇n the malignant hepatocellular carcinoma (hcc) is seen in a dramatic increase in the quantity of agrin. gpc3 immunpositivity showed in %85 of hcc and %15 of cholengiocellular carcinoma. agrin immunpositivity showed in %95 hcc and %50 of cholangiocellular carcinoma. conclusion: gpc3 and agrin, that is useful in early diagnosis of hcc cases; cholangiocellular carcinoma, or not enough was understood to be very limited. value of glypican 3, hep par and alpha fetoprotein in diagnosis of hepatocellular carcinoma n. gursan * , h. balta, b. gundogdu * ataturk university, medical faculty, erzurum, turkey objective: glypican 3 showed high expression of the embryonic liver and intestine, is silenced in normal adult tissues. it is an oncofetal protein. i̇n general, oncofetal proteins, while having a critical role in tumor progression or immunotherapy is a potent tumor marker used as a target. alpha fetoprotein, clone c3 localized, normally produced by gestational age, fetal liver and yolc salc, glycoprotein structure emerging marker for patients with hcc. hep par 1 antibody was developed in 1993 using fixed liver as immunogen. method: i̇n this study formalin-fixed, paraffin-embedded surgical specimens from 20 patients with hepatocellular carcinoma diagnosed between 2000 and 2011 years in ataturk university, faculty of medicine department of pathology were studied with immunohistochemistry gpc3, heppar 1, and afp. gpc3, heppar 1 and afp expression was divided into 2 categories negative (negative or weak under %5 tumor cells cytoplasmic staining) and positive (and %5 over of tumour cells) moderate or strong cytoplasmic with membranous accentuation. results: immunpositivity at gpc3, hep par 1 and afp were 85 %, 95 %, 75 % respectively. objective: fibroblastic growth factor receptor (fgfr) family is known to be related to the development and progression of various types of cancers. the aim of this study is to determine the clinical implication of fgfr expressions in patients with hepatocellular carcinoma (hcc). method: immunohistochemical analysis was done in 842 cases of hcc using tissue microarray. diffuse cytoplasmic staining for fgfr1, 2, 3 and 4 in more than 10 % of tumor cells were designated as positive. the results were analyzed in terms of various clinicopathologic parameters. results: on univariate analysis, the overall survival rates of patients with fgfr2 expression (152 cases, 18.1 %) were significantly lower than those with no expression (hr 1.838, 95 % ci 1.452-2.328, p<0.001). however, the overall survival rates of patients with fgfr4 expression (446 cases, 59.6 %) were significantly higher than those with no expression (hr 0.636, 95 % ci 0.521-0.776, p<0.001). there was no statistical significance between patients' overall survival rates and expressions of fgfr1 (46 cases, 5.5 %) or fgfr3 (33cases, 4.0 %). on multivariate analysis, only fgfr2 expression is independently associated with reduced os (fgfr2: hr 1.790, 95 % ci 1.404-2.282, p<0.001). conclusion: the fgfr2 expression can be used as an independent prognostic factor in patients with surgically resected hcc. objective: hurp (hepatoma upregulated protein) is a putative oncogene, overexpressed in many human cancers, including hepatocellular carcinoma. it has also been shown recently that high hurp protein levels correlate with resistance to chemotherapeutic agents. in the present study we investigate the expression of hurp and its correlation with pancreatic adenocarcinoma, prognosis and patient survival. pancreatic adenocarcinoma is one of the most aggressive types of cancer and represents the fourth most common cause of death, either in the male or in the female population of the united states of america. method: hurp immunoreactivity was assessed by immunohistochemistry in a series of 28 primary pancreatic adenocarcinomas. in parallel, hurp expression was examined in 10 normal pancreatic tissues. statistical analysis related hurp expression levels with clinicopathological characteristics and survival. results: results showed a positive correlation between hurp overexpression and grade as well as lymphovascular invasion. all non malignant biopsies were negative. furthermore, positive expression of hurp appeared to be an important independent prognostic factor too, related with poor survival rates. conclusion: our results showed that hurp overexpression is associated with poor prognosis in pancreatic adenocarcinoma and indicated a diagnostic potential of this protein. its role in the carcinogenetic process awaits further elucidation. hepatocellular carcinoma in patient with hepatic porphyria: a case report j. marcinek * , p. szépe, t. balhárek, m. kalman, l. plank * university hospital martin, dept. of pathology, slovakia objective: hepatocellular carcinoma (hcc) is frequently associated with liver cirrhosis or chronic hepatitis of various etiology, less often with other chronic hepatopathies, including hepatic porphyria. we present a case report of hcc diagnosed in patient with clinically unrecognized porphyria cutanea tarda (pct). method: a 70-year old male was admitted to hospital complaining of a pressure below low costal margin, anamnesis of a trauma 1 year ago and clinical suspicion of a posttraumatic intrahepatic haematoma. by a surgery, a partialy necrotic and hemorrhagic tumor mass 10 cm in diameter was removed. results: the mass corresponded histologicaly to moderately differentiated clear cell hcc, resembling renal clear-cell carcinoma. the surrounding parenchyme showed steatohepatitis with fibrosis and distinct intracellular porphyrin crystals. conclusion: development of hcc is a well documented complication of pct, a disorder of porphyrin biosynthesis. pct can be inherited (autosomal dominant trait), the acquired form (sporadic pct) is more common. known etiological factors of pct include toxins, alcohol abuse, estrogens or chronic viral hepatitis, or association with other hepatic diseases like iron overload (including hereditary haemochromatosis) or chronic hepatitis c, increasing the risk of malignancy. however, in the presented case none of these associations was recognized and its etiology remains obscure. objective: signet ring cell carcinoma is an extremely rare type of gallbladder carcinoma composed overwhelmingly (90 %) of signet ring cells. only a few cases of this histologic type have been published and detailed knowledge of this disease is not available. method: we report a case of signet-ring cell carcinoma of the gallbladder in a 70-year-old woman who was admitted with epigastric pain. under the preoperative diagnosis of cholangiocarcinoma, which was based on findings of ct scan and ultrasonography. cholecystectomy was performed. results: microscopic examination revealed a signet ring cells tumor that arose from the sub epithelial layer of mucosa and involved all the layers of gallbladder. nuclear atypia and mitoses were present. periodic acid schiff (pas) stain highlighted the intracellular mucin in the tumor cells. lympho-vascular emboli were detected in the subserosal layer. the cystic duct surgical margins were invaded. conclusion: gallbladder adenocarcinomas are seen frequently, but signet-ring cell carcinoma is a rare entity. owing to the location of the gallbladder, dissemination of the tumour to the adjacent tissues is usually presented at the time of the diagnosis. it is necessary to exclude a gastric or colonic signet ring cell carcinoma secondarily involving the gallbladder. objective: gastrointestinal stromal tumors (gists) are mesenchyme neoplasms of the gastrointestinal tract which express cd117. gists can occur in the entire length of gastrointestinal tract, but very seldom can also arise in omentum, mesentery and retroperitoneal space, kidney, urinary bladder etc. there they are located adjacent to stomach or intestine, but not originated from the latter. these tumors are designated as "extra-gastrointestinal stromal tumors" (egists). there are only 14 description of the primary egists reported in the literature. method: we report a case of a pancreatic egist in a 38year-old female patient. results: ct scan showed a mass in the head of pancreas. grossly encapsulated tumor, 9 cm in diameter, was found in the head of the pancreas (figure 1) . histologically, tumor demonstrated spindle-cell pattern consisted of distinct fascicles and bands. the number of mitoses was 1-2/50 high power fields in "hot-spot" areas. ihc revealed strong positivity for cd117, cd 34 in neoplastic cells and negative for sma, desmin, s-100. ki67 labeling index was 3 %. conclusion: we presented a rare case of pancreatic gist. the tumor has very good prognosis. hepatoblastoma is classified as epithelial (56 %) or mixed epithelial/mesenchymal (44 %). epithelial hepatoblastoma is further divided to pure fetal (31 %), embryonal (19 %), macrotrabecular (3 %) and small cell undifferentiated (3 %). aim: to increase the awareness regarding possibility of hepatoblastoma in adult age. method: we review four cases of hepatoblastoma epithelial type diagnosed in pathology department of fundeni clinical institute between 2009 and 2012. immunohistochemistry was done in all cases: cea, vimentin, ck 8/18, ck 19, ck 20, ki 67, ttf1, cd 10, cd 34, cd 56, muc 5a, alpha-fetoprotein. results: two of the patients were males and two females with age ranging between 17 and 27 years. the tumors varying in size from 8 to 12 cm. on microscopic examination the tumor was composed mainly of epithelial elements. the pathological diagnosis was epithelial hepatoblastoma epithelial type. conclusion: hepatoblastoma is a rare tumor in adult age and epithelial type is the rarest from all type of hepatoblastoma in adult age. the pathological diagnostic is quite difficult even using immunohistochemistry because none of the markers are not specific for hepatoblastoma. submassive hepatic necrosis with regenerative nodules: a series of cases y. rodríguez-gil * , j. salazar, j. delgado sánchez, g. lópez alonso, c. ibarrola de andrés, f. colina ruizdelgado * hospital universitar 12 de octubre, dept. of surgical pathology, madrid, spain objective: confluent hepatic necrosis is the morphological correlate of fulminant liver failure. a group of these livers develop a combination of areas of necrosis and regenerative macronodules (rmn). method: sequential biochemical and clinical data and tissues from two groups of patients were compared. group 1: 10 cases of submassive necrosis associated with rmn; group 2: 19 cases with total confluent necrosis without nodular regeneration. results: group 1: mean age was 33,6 years (ranged 10 months-63 years); group 2 mean age was 42,6 years (ranged 18 months-63 years) (p>0,05). a longer course (42,8±34,6 days vs 12,6±6,0, p<0.05) and higher maximum serum bilirubin levels (23,5 mg/dl ±13,08 vs 14,9 mg/dl± 10,9, p<0 .05) were observed in group 1. serum ast, alt and gamma-gt were lower in group 1 (p>0.05). morphologically livers from group 1 showed well defined regenerative green-yellow nodules with large regenerative hepatocytes in acinar pattern without surrounding fibrosis. the remaining parenchyma showed confluent necrosis, haemorrhage, mixed inflammatory infiltrate and ductular proliferation. conclusion: a special anatomoclinical form of subacute liver failure was characterized by regenerative macronodules in a background of extensive confluent liver necrosis. illness duration was longer and had higher maximum bilirubin levels than those without rmn. immunohistochemical study of muc gene family in pancreatic cancer g. setdikova * , o. paklina * moscow, russia objective: investigation of the muc gene in pancreatic ductal adenocarcinomas (pda). method: in the present study, we examined the expression of muc1, 2 and 5 ac types by immunohistochemical analyses in pda from 74 patients. overall survival curves were drawn by the kaplan-meier method. for all analyses, p<0.05 was considered to be statistically significant. results: in our cases most of the pda was presented as muc1+/muc5ac + phenotype -42 % cases (31/74). the group with intestinal phenotype mucin, which characterized by positive expression of muc2 and cdx2, was only 7 % (5/74). the group which characterized by positive expression only muc5ac (gastric phenotype), was 15 % (11/74). the survival rate of patient better in group with muc2 expression. cumulative survival at 12 months after surgery was 1.0 and the median postoperative follow-up period was 17 months. the most aggressive behavior was pda with expression only muc1 ("true pancreatobilliary type"). cumulative survival at 12 months after surgery was 0.25 and the median postoperative follow-up period was 7 months. conclusion: the mucin profile as a prognostic factor is important not only for intraductal pancreatic mucinous neoplasm, but and for ductal pancreatic adenocarcinoma. ps-14-026 hepatocellular carcinoma microvessels density depends on tumor differentiation: radiologic-pathologic correlations a. shchegolev * , e. dubova, u. tumanova * v.i. kulakov scientific center, dept. of pathology, moscow, russia objective: prognosis and recurrence level in hepatocellular carcinoma (hcc) depends on microvessels density. method: we performed ct and pathological correlations in 22 cases of hc (5 cases of highly (h-hcc), 11 cases of moderate (m-hcc) and 4 cases of low differentiated hcc (l-hcc)). results: vessels number in w-hcc in compare to m-hcc was higher by 26.1 % (p<0.05). total vessel area was higher in m-hcc and l-hcc than in w-hcc by 11.7 % and 20.4 % accordingly. we observed positive correlation between ct density of h-hcc and number and total vessel area. correlation between h-hcc ct density and mean vessel area was negative. we also revealed modest negative correlation between m-hcc density and the number vessels in all phases of ct. negative correlation between total and mean vessel area and m-hcc density was revealed in native and arterial ct phases in compare with venous and delayed phases where positive correlation was revealed. in all phases of ct we revealed strong negative correlation between l-hcc density and the number of vessels whereas correlation between l-hcc density and mean vessel area was strongly negative. conclusion: we revealed decreasing in angiogenic activity in hccs neoplastic progression and growth. ct signs correlate with hcc histological differentiation. ps-14-027 primary sporadic liver schwannoma: an extremely rare diagnosis r. silva * , c. eloy, j. m. lopes * centro hospitalar de são joâo, serviço de anatomia patológica, porto, portugal objective: primary liver schwannoma (pls) is extremely rare. results: case report: a 69-year-old female with previous history of cholecystectomy, appendectomy, arterial hypertension and diabetes mellitus was admitted due to an intrahepatic nodular lesion identified in routine ultrasound examination. she was submitted to partial hepatectomy with postoperative uneventful evolution. macroscopically, the specimen disclosed a 2.1 cm nodular, well circumscribed, yellowish tumour. histologically, the tumour displayed an expansive growth pattern, comprising short bundles of spindle cells with mild atypia, without mitotic figures or necrosis. immunohistochemically, the tumor cells expressed diffusely vimentin, s-100 and gfap, in the absence of ae1/ae3, cam5.2, cd117, and hmb45; ki-67 index was 2 %. staging procedures did not disclose evidence of any other tumor. there was no personal or family evidence of neurofibromatosis. these features are consistent with sporadic pls. conclusion: schwannoma is a rare benign tumour in the gastrointestinal tract with few cases reported in the liver. the clinical presentation is usually an upper abdominal pain but they can be asymptomatic. secondary cystic degeneration and hemorrhage are common in large tumours. differential diagnosis by imaging evaluation includes several benign and malignant, namely metastatic, tumours. therefore, the pathological examination is crucial for the diagnosis of primary liver schwannoma. metastases of hepatocellular carcinoma to the costa and soft tissue: a very rare entity e. tastekin * , t. d. yalta, o. yalcin, t. ciftci * trakya university, dept. of pathology, edirne, turkey objective: hepatocellular carcinoma (hcc) is the most common primary tumor of the liver with a potential of metastasis primarily to the lung, abdominal lymph nodes and bone. however, metastases to the costa, soft tissue are rare. method: an 82-year-old man was admitted to hospital with weakness, anorexia. chest x-ray and ct-scan demonstrated a 5×5 cm sized, intrathoracic mass on the lateral-side of the left 2.costa and also infiltrating the axillary soft tissues.a secondary mass with the identical radiological features was also detected within the c7-vertebra. whole-body ct-scan was planned.a 2×2 cm sized nodular, hipodense mass was detected in the upper segment of the liver. interestingly, none of these masses demonstrated a significant pathological fdg-uptake in the pet.a trucut biopsy was taken from the axillar. microscopically, large tumoral nests were observed in connective tissues. polygonal shaped tumoral cells were found to have granular cytoplasm, pleomorphic nuclei. immunohistochemistry results: positive cytoplasmic reaction with heppar, ttf-1; canalicular staining with polyclonal-cea, cd10; weak cytoplasmic staining with pankeratin, b72.3; negative reaction with vimentin, afp, ck7, ck20. results: these histomorphological, immunohistochemical findings strongly supported to the diagnosis of hcc metastasis. conclusion: hcc that is, in general, morphologically similar to mesenchymal and epithelial tumors, should be considered among the differential diagnosis of unknown primary tumors. morphological justfication of using pulsed electric discharge in surgical treatment of echinococcosis m. tussupbekova * , d. kaliyeva, e. turgunov * medical university karaganda, dept. of pathological anatomy, kazakhstan objective: echinococcosis is characterized by lesions of internal organs, severe complications, often lead to disability and death. method: new method of processing of the residual cavity with impulsive electric discharge after echinococcectomy was experimentally developed and introduced into clinical practice. the effectiveness of the method is confirmed by morphological examination of operational bioptic of the liver tissue. results: 20 patients underwent surgical treatment with the new method (patent of the republic of kazakhstan ¹ 63558). parasitic cysts with an inner germinal layer and protoskoleks, outdoor layer -chitinous shell with a productive reaction with lymphocytes infiltration, single eosinophils, separate groups of hydatid bubbles with their invasion in the liver tissue were detected in the histological examination of material taken intraoperatively prior processing of residual cavity with electropulse discharge. fibrinoid necrosis, destruction of germinal elements, single lymphoid infiltration, hyperemia of blood vessels, absence o inflammatory reaction were marked in the histological material after processing by impulsive electric discharge. the results showed that echinococcectomy with processing of the residual cavity with impulsive electric discharge is an alternative for pericystectomy and liver resection. objective: congenital peribronchial myofibroblastic tumor is a rare, solid mesenchymal tumor of the fetus and neonate, usually associated with nonimmune hydrops fetalis. we report the case with clinical, radiological and pathologic findings and review the other cases in the english language literature. method: we present a case of cpmt, whom a right lung mass was detected in intrauterine life. 12 days after delivery by cesarean section, right lobectomy was performed. results: the tumor was limited to lung, and composed of spindle cells, proliferating around bronchial unit. central necrosis and 4-5/10 hpf was present. patient is well 15 months after surgery. conclusion: congenital peribronchial myofibroblastic tumor is a rare solid pediatric tumor of lung which was named differently in the past, such as congenital fibrosarcoma, congenital leiomyosarcoma, congenital mesenchymal malformation of the lung, neonatal pulmonary hamartoma. albeit it resembles sarcoma with high cellularity, mitosis and necrosis features microscopically, no additional therapy to tumor resection is needed. so it is important to keep in mind this scary looking, but innocent tumor both in prenatal and postnatal evaluation. morphologic alteration of metastatic neuroblastoma in bone marrow after chemotherapy g.-e. bae * * samsung medical center, dept. of pathology, seoul, republic of korea objective: the aims of our study are to evaluate the histologic features of metastatic neuroblastoma in bone marrow (bm) in comparison with those of primary neuroblastoma, and to compare the histologic characteristics of metastatic neuroblastoma in bm before vs. after chemotherapy. method: total 92 biopsies from 19 children diagnosed as stage 4 neuroblastoma with bm metastasis were examined histologically; 19 primary neoplasm biopsies, 19 bm biopsies before chemotherapy, 19 primary neoplasm excision biopsies after chemotherapy, and 35 bm biopsies after chemotherapy. results: 1) primary neoplasms were classified as neuroblastoma poorly differentiated (n=10), neuroblastoma differentiating (n=5), ganglioneuroblastoma intermixed (n=1) and neuroblastoma type unclassifiable (n=3). 2) metastatic foci in bm before chemotherapy were composed of undifferentiated and/or differentiating neuroblasts but not ganglion cells, in neuropil but not schwannian stroma. 3) metastatic foci of bm after chemotherapy showed differentiation such as ganglion cells and schwannian stroma, which was more prominent after more cycles of chemotherapy. 4) metastatic neuroblastomas in bm after chemotherapy were as mature as or less mature than those in primary neuroblastomas after chemotherapy. conclusion: metastatic neuroblastomas in bm initially consist of more immature components than primary neuroblastomas, whereas they become differentiated as primary neuroblastomas after multi-cycle chemotherapy. morphometric evaluation and clinical correlations in malignant small round cell tumors c. bansal * , a. gupta, a. kumar, a. srivastava * csmmu, dept. of pathology, lucknow, india objective: nuclear size increases in malignant tumors and reflects dna content, ploidy and proliferation index. present study investigated; could nuclear morphometery differentiate histomorphologically similar paediatric malignant small round cell tumors in h & e stained sections for using in a poor resource country? method: morphometric analysis was done in 26 confirmed but difficult to differentiate round cell tumors histomorphologically and were analyzed by cell images from 6 different areas in each section, using leica q win 500 images software. results: nuclear measurements were obtained for retinoblastoma (9), hodgkin lymphoma (6), wilms tumor (3), medulloblastoma (2), ewing's sarcoma (2), alveolar rhabdomyosarcoma (1), malignant hemangiopericytoma (1), non-hodgkin lymphoma (1) and neuroblastoma (1). amongst the retinoblastomas, maximum mean nuclear area percent (24.93) was seen in cases with nerve involvement and metastasis, followed by cases with only nerve involvement (21.60) and smallest area (16.57) was in non-nerve involving, non metastatic cases. wilms tumor cases with metastasis had higher nuclear area (21.25) than non metastatic (19.47). non-hodgkin lymphoma nuclear area (20.03) was more than hodgkin's (18.60). amongst all tumors, minimum value (14.93) was seen in malignant hemangiopericytoma. conclusion: morphometric evaluation in paediatric malignant round cell tumors have generated useful data, and needs further multicentric confirmation for implementation. objective: survivin, a bifunctional protein that regulates cell division and suppresses apoptosis, may play an important role in tumorigenesis. the aim of this study was to determine survivin expression patterns in wilms tumor (wt) and to analyze it in relation to stage, prognostic category and histological type. method: immunohistochemical expression of survivin was analysed in 59 cases of primary wt and in 10 normal kidney specimens uninvolved by the tumor. results: fifty one out of 59 cases of wt (86.44 %) showed decreased cytoplasmic survivin expression compared to the expression in normal kidney tissue. decreased cytoplasmic expression (in all components of wt) of survivin was found significantly more often in low stage compared to high stage wts (86.7 % vs. 27.3 %; p=0.002). tumors of intermediate risk group showed more often decreased cytoplasmic expression of survivin in comparison to high risk group, but the difference was not significant. decreased survivin expression was found more frequent in wts with diffuse anaplasia and in epithelial wts compared to other histological types, but without statistically significant difference. conclusion: decreased survivin cytoplasmic expression may be associated with the favorable prognosis wt. objective: hepatic tumors accounted 5 % of congenital neoplasms. mesenchymal hamartoma of the liver is a rare benign tumor of children. we report a case of hepatic mesenchymal hamartoma in a premature male neonate. method: a premature neonate born at 33 gestational weeks from 34 year-old mother, g3p3 with non-consanguineous marriage; prenatal ultrasonography showed a fetal macrosomia, a highly vascularized abdominal mass occupying two-third of the abdomen and displacing the bowel loops associated with poly-hydramnios. the newborn examination showed abdominal firm lump. ultrasound found and anterior highly vascularized mass displacing the left lobe of liver. the infant died few hours later. a complete autopsy was performed. results: external examination showed a male neonate anatomically of 34-35 week having an increased periombilical diameter and a macrosomia, ascitis, pulmonary hypoplasia, cardiomegaly, pleuropericardial effusion, liver tumor developed in left lobe and dilation of renal vessels. histologically the tumor showed a mixture of normal liver tissues with blood or lymphatic vessels, bile ducts within an abundant edematous and myxoid stroma. conclusion: in neonate and fetus, prenatal diagnosis is possible by ultrasonography. large tumors can affect the viability of the newborn. adequate excision is curative in most of cases. ps-15-007 ncam polysialylation as potential initiator of differentiation and proliferation of renal progenitors in human fetal tissue s. cirovic * , j. tadic, n. radunovic, c. müller, g. müller, j. markovic-lipkovski * inst. for pathology, nephropathology, belgrade, serbia objective: objective: neural cell adhesion molecule (ncam) is widely expressed on mesenchymal and early tubular epithelial cells during kidney development although with still undefined function. ncam can be polysialylated and as psa-ncam has been shown to be involved in proliferation and migration of neuronal cells during brain development. the aim of this study was to evaluate the presence of psa-ncam in nephron precursors in relation to the expression of renal progenitor and proliferation markers. method: human fetal and neonatal kidneys were analyzed using double-immunofluorescence (dif) staining and western blot (wb) analysis. specific antibodies against ncam, psa-ncam, epcam, cd24 and ki-67 were applied. results: on wb only fetal tissue samples have bands with ncam at 140 to 250 kda which suggest that ncam molecule is polysialylated; dif analysis of fetal tissue show psa-ncam + ki-67+ cells in all structures known as nephron precursors. while in neonatal tissue psa-ncam and ki-67 were positive only on rare single cells in intersticium. conclusion: psa-ncam expression appears to characterize a very early stage of induced nephron progenitors differentiating from ncam + epcam-mesenchymal cells. according to psa-ncam localization and coexpression with ki-67 during development and its practically absence in neonatal tissue, suggest that psa-ncam present potential initiator of proliferation and differentiation of renal progenitor. objective: cystic hygroma (ch) typically develops in utero, late in the first trimester to early in the second trimester of gestation. many of the fetuses with ch present additional malformations commonly associated with chromosomal anomalies. method: a review of 548 fetal autopsies performed over a 10 year period revealed 19 cases of ch (3,4 %). the results of cytogenetic analysis and the prenatal ultrasound findings were retrieved and compared to the autopsy findings. results: fetal death was due to therapeutic abortion in 9/19 cases, intrauterine death in 8/19 cases, and spontaneous abortion in 2/19 cases. cytogenetic analysis was available in 12 cases. the results showed an abnormal karyotype in 7 cases (5 cases of turner syndrome and 2 cases of trisomy 21). the mean size of ch was 5,4 cm. other findings suggestive of the cause of fetal death were diagnosed in 10/19 cases (52,6 %). the most common autopsy findings were hydrops and central nervous system anomalies. the autopsy findings were in agreement with the prenatal ultrasound findings in 14/19 cases, while in 5 cases (26,3 %) additional findings were detected during autopsy. conclusion: our study is confirms the strong correlation between ch and chromosomal anomalies of the fetus. immunohistochemical expression of e-cadherin in primary and metastatic nephroblastoma cases i. franckevicha * , r. kleina * university children´s hospital, dept. of pathology, riga, latvia objective: in nephroblastoma cases association between decreased e-cadherin expression and higher stage of tumor (safford sd, 2005) and lower expression in metastatic tumors (alami j, 2003) was described. some authors considered that e cadherin is not likely to play tumor suppressor role in nephroblastoma ( shulz s, 2000) . purpose of this study was to compare the expression of e-cadherin in metastatic and primary tumor cases in latvia. method: 26 cases of primary tumors, 8 metastases and 1 case of relapse were analyzed immunohistochemically using visualization system envision. the number of e-cadherin positive structures per field was assessed (magnification× 100). comparison between primary tumors and metastatic/ relapse tumors groups was made using mann-whitney test. results: in primary tumor group the number of positive structures in the field ranged from 0 to 86, average 27.19, sd 24.13. in the metastatic tumor group, the number of positive structures varied from 0 to 40, average 9.89, sd 14.87. comparison of expression observed in both primary and metastatic tumors groups showed the decreased e-cadherin expression in metastatic tumor group (z= −2.33, p=0.046). conclusion: expression of e-cadherin is lower in metastatic tumor group that may suggest about negative correlation between it and higher tumor grade. ps-15-010 testicular fibrous hamartoma: a case report a. kilitci * , f. yilmaz, s. yanik, h. ozturk * abant izzet baysal university, dept. of pathology, bolu, turkey objective: fibrous hamartoma (fh) of childhood is uncommon benign tumor. they are generally seen in the head and neck region, gastrointestinal system and lung. as in the case we report, they may also occur in other unusual sites such as groin and testis. the clinical presentation is almost always a mass or swelling, however our case was admitted to our hospital because of a left testicular atrophy. we report a fh in the testis, which has a rarely location and clinical presentation. method: a 7-year-old male presented to department of pediatric surgery because of left testicular atrophy. left orchiectomy was performed. results: in macroscopic examination, testis dimension was 1.5×1.3×0.4 cm. its cut surface was smooth and dirtywhite. in microscopic examination, the tumor showed disorganized matur tissue that composed of fibrocollagen stroma, vessels, muscular and adipose tissue. by this findings, it was diagnosed as testicular fibrous hamartoma (tfh). conclusion: in conclusion, tfh should be always kept in mind with testicular atrophy not only testicular mass or swelling. and knowledge of this particular type lesion is important to distinguish the fh of childhood from other situations in testis such as testicular torsion, incarcerated hernia, malign neoplasm, etc. in order to allow a correct diagnosis and avoid inadequate treatment. placental pathologic features in diabetes and hypertension p. luís * , a. costa-silva, a. alves * hospital de santa maria, dept. de anatomia patológica, lisboa, portugal objective: the aim of this study was to evaluate the most frequent placental findings in diabetes and hypertension and their main differences. method: retrospective study of 229 selected placentas from 19 to 41 weeks gestation, in a universe of 3231 placentas examined over the last 10 years (6 % associated with hypertension, 1,5 % with diabetes and 0,4 % with both diabetes and hypertension (dwh). results: in diabetes the most frequently found abnormalities were immature villi (35 %) and infarction (27 %). in dwh the most frequent lesions were the immature villi (29 %) and inflammatory lesions (either acute or chronic) (29 %). in the hypertension group the most frequently found lesions were infarction (54 %) and accelerated maturation of the villi (23 %). in 35 % of placentas in diabetes, 21 % in dwh and only 15 % in hypertension, no lesions were found. the incidence of fetal death was 8 % in diabetes, 0 % in dwh and 5,5 % in hypertension. when evaluating placental weight, small placentas were more frequent in the hypertension group (37 %), and large placentas were more frequent in diabetes (27 %). conclusion: our findings may contribute to evaluate the consequences of diabetes and hypertension in fetal outcome. as inflammatory conditions are usually not directly attributed to diabetes or hypertension, placental examination may help in diagnosis of associated pathology like infection. placental villi morphometry in preeclampsia k. pavlov * , e. dubova, r. shmakov, a. shchegolev * v.i. kulakov scientific center, dept. of pathology, moscow, russia objective: terminal villi structure abnormalities could play an important role in preeclampsia (pe) and its complications development. our aim was to perform comparative morphometric study of the placentas from mild and severe pe pregnancies. method: complex morphological and morphometric study of 9 term placentas from mild pe (mpe) cases (1st group), 6 term placentas from severe pe (spe) cases (2nd group) and 10 term placentas from uncomplicated pregnancies (control group) was performed. results: we revealed significant decreasing in terminal villi size in both preeclamptic groups in compare to control. this decreasing was much prominent in spe group. we also observed significant terminal villi perimeter decreasing in both pe groups without any difference between mpe and spe groups. morphometry of cd31 stained specimens revealed significant decreasing in mean capillary number in both pe groups. single capillary area and perimeter were significantly lower in mpe and spe groups and these changes were much prominent in spe group. total villous capillary area and perimeter were significantly lower in both pe groups with minimal values in spe group. degree of villous capillarisation was significantly lower in both pe groups. conclusion: revealed features reflect villous structure changes in preplacental hypoxia, caused by mild and severe preeclampsia. sudden intrauterine death: the usefulness of autopsy f. portelli * , e. orlando, e. di stefano, e. maresi * university of palermo, section of pathology, italy objective: we investigated in 1006 autopsies of stillbirths the usefulness of autopsy even in the absence of risk factors and/or apparent anatomical/clinical causes which could explain the death. method: from january 1990 to december 2010 (institute of pathology, paolo giaccone-palermo) 2643 autopsies on dead uterine foetuses were performed. 1637 cases were abortions (<25 weeks), 1006 were stillbirths (≥25 weeks). in all cases the autopsy included a macroscopic and microscopic examination both of the foetus and the placenta. the final diagnosis of death was based on both the morphological and clinical data. of the 1006 stillbirths, 715 cases were "risk factor pregnancies", 291 non. results: the stillbirth mortality was classified as follows: -sudden intrauterine unexplained death (siud,"sine materia" and absence of risk factor autopsies),4 cases; -explained intrauterine death ("cum materia" with or without risk factor autopsies),987 cases; -borderline intrauterine death ("sine materia" and presence of risk factor autopsies),15 cases. conclusion: in the absence of autopsy in 715/1006 cases it was possible to establish the cause of death based on the presence of risk factors. in the absence of risk factors the autopsy showed a certain anatomical cause in 287/1006, only in 4/1006 it was "sine materia". the role of "traditional" and "tomography" autopsy in foetal congenital heart disease (chd) f. portelli * , f. p. busardò, l. gutsul, l. averna, e. orlando, e. maresi * university of palermo, section of pathology, italy objective: the role of systematic autopsy in foetal chd is to identify the morphology of cardiac and possible associated extracardiac malformations (ecm) and the correlation between morphology, suspected clinical etiology and genetics. we evaluated the role of autopsy in 643 foetal chds with/without prenatal diagnosis. method: from january 1990 to december 2010 (institute of pathology, paolo giaccone -palermo), 2371 foetal autopsies were performed. the autopsy protocol used was "tomography" for abortions, the "traditional" technique was adopted for stillbirths. results: in 643 cases chds were identified. in 196 cases chd diagnosis was made only through autopsy, in 447 after a certain (244) or suspect (203) clinical/genetic diagnosis. the etiology of chd associated to ecm (621) was: chromosomic type (271), syndromic/sequence type (233), association type (117). the etiology of chd without ecm (22) was never syndromic. conclusion: "traditional" and "tomography" autopsy plays a key role in the diagnosis and counselling of chd, either when it represents the only diagnostic tool (30,48 %) or when it is preceded by a clinical/genetic study. in the latter cases, its value depends on the detection of ecm, useful to "consolidate" a suspect clinical diagnosis (31,57 %) and to "complete" a malformative picture etiologically known thanks to a clinical/genetic analysis (37,9 %). objective: deregulation of cell cycle control is a hallmark of cancer. we have examined protein expression and gene amplification of cyclin a in wilms tumor (wt) and to analyze it in relation to tumor stage, prognostic group and histological type. method: real-time quantitative pcr was used to detect gene amplification of cyclin a in tumor tissue from 36 patients with wt, while immunohistochemistry was applied to detect protein expression of the same cyclin. results: cyclin a gene amplification was found in 4 out of the 36 (11.8 %) cases of wt. cyclin a protein overexpression was detected in all four cases, but was also found in 84.7 % of cases without detectable gene amplification. so, there was no significant correlation between cyclin a gene amplification and protein overexpression. all cases with amplification of cyclin a were of favorable histological type, intermediate risk group and three out of four cases were low stage wts. on the other hand, overexpression of cyclin a was found significantly more often in high stage wts compared to low stage wts (p=0.04). conclusion: cyclin a gene amplification might be associated with the favorable prognosis of wt (low stage, intermediate risk group and non-anaplastic tumors). the gold standard to diagnose is lung biopsy or necropsy. up to 40 % of cases have mutations or deletions in the gene foxf1 (cr16q24.1) who plays a crucial role in the development of the lung vasculature. method: we report a case of a term newborn female affected of acd/mpv who was diagnosed by a lung biopsy. she died at 38 days of life. autopsy and molecular diagnosis were also performed results: in the biopsy and autopsy specimens a decline in the number of capillaries in the alveolar septa and detachment of the epithelial lining was observed. secondary proximal plexiform arteriopathy, muscularization of arterioles and venous-venular dilatation and proliferation were evident. a pathogenic mutation in the gene foxf1 (frame shift mutation in the first exon) confirmed our diagnosis. conclusion: acd is defined by a decrease in capillaries with alveolar septal thickening and hypertrophy of the middle muscular layer of arterioles. mpv suggests an imbalance of angiogenesis. the foxf1 mutation helps to prenatal diagnoses of high risk families and to give the diagnosis to patients who fail to perform biopsy or autopsy. objective: prognosis of rhabdomyosarcoma (rms) has improved significantly over the last 20 years. overall survival (os) is>80 % in the majority of patients; even though, children with metastatic tumors have a dismal prognosis with an os <30 %. method: we analyzed in 18 cases, the possible correlation of notch activation with histology, presence of metastasis and outcome. immunohistochemistry (ihc) was performed for the notch downstream effectors hes1 and hey1. results: hes 1 was strong or moderate in 95 % of the cases. hey1 was positive in 50 %. 3 out of 4 alveolar rms (arms) were positive for hes1 and 1 was positive for hey1. both stains were negative in the fusion-negative arms. in embryonal rms (erms), eight out the 9 were hey1 positive and 14 of the 17 were hes1 positive. the 2 patients whit metastases had staining for hes1 and hey1. 3 patients who died showed hes1(+). 14 of the 15 patients in remission showed positivity for hes1 and 7 for hey1. conclusion: hes1 expression was found in the majority of rms while hey1 was more erms specific. is no correlation between pathway activation, metastasis or outcome. the blockage of the pathway with specific inhibitors could offer a new therapeutic option for this patients. a renal epithelioid angiomyolipoma in a young woman with tuberous sclerosis complex, cortical tubers by neuroimaging, facial angiofribromas and lung lymphangioleiomyomatosis j. trillo-tinoco * * hospital general de mexico, dept. of surgical pathology, mexico city, mexico objective: tuberous sclerosis (ts) is a genetic disorder affecting cellular differentiation and proliferation, which results in hamartoma formation in many organs like skin, brain, lung, kidney and heart. method: a seventeen-year-old female with history of facial angiofibromas a 10 years before, right renal tumor diagnosed a year before as theratoid-rhabdoid tumor, with chemoradiation as adjuvant treatment, cortical tubers by neuroimaging a few months before, and diagnosis of lung lymphangioleiomyomatosis recently. after last diagnostics, we reviewed again the renal tumor, new orientation of tissue, with new histological sections were performed. (fig) results: at histological examination, the kidney showed an infiltrating tumor, very cellular, consisting mainly of polygonal cells with eosinophilic cytoplasm, other multinucleated similar to the ganglion cells, also small hamartomatous areas with smooth muscle, fat and blood vessels proliferation. the tumor cells expressed hmb45, mart-1, sma, vim and cd10, ckae1/3 were negative. (fig) conclusion: the majority of renal angiomyolipomas is sporadic and 50 to 80 % occurs as part of ts, and their partnership is more close with epithelioid variant, recently, a rare entity with aggressive behavior, difficult histological characterization and poor prognosis. pulmonary mast cells in sudden infant death syndrome (sids) c. zaharia * , c. loddo, p. schmidt, r. m. bohle * university of saarland, inst. of pathology, homburg, germany objective: several theories of the underlying mechanisms of sids have been proposed, one of them is focusing on shock including anaphylaxis. increased concentrations of mast cell tryptase in post mortem blood have been observed without increased mast cell numbers in lung tissue. the aim was to evaluate the age-related distribution of pulmonary mast cells in infants dying of sudden infant death syndrome and controls. method: 6 infants (up to 1 year of age) who died of sids and 23 controls who died of other non-pulmonary causes were examined. peribronchial mast cells exhibiting tryptase immunoreactivity were evaluated and quantified in high power fields in lung sections. results: the number of mast cells in peribronchial regions amounted to 18,9 (± 6,6)/mm2 in children aged 1 month up to 13 months. mast cells in sids cases were 21,2 (± 4,5)/mm2. the difference was not significant (p=0,113, student'st test). conclusion: it is unlikely that increased pulmonary mast cells are indicators of sids. the role of mast cells in sids remains controversial. trophoblast apoptosis in placentas from pregnancies complicated by preeclampsia s. zekic tomas * , i. kuzmic prusac, d. roje, i. tadin * clinical hospital centre split, pathology, croatia objective: to assess trophoblast apoptosis separately in cytotrophoblast, syncytiotrophoblast, total villous trophoblast and syncytial knots, as well as to investigate the expression of apoptotic factors fas ligand (fasl), bcl-2 and proliferation marker ki-67 in trophoblast of placentas from preeclamptic patients. method: the study included placental samples from 25 preeclamptic and 25 normal pregnancies. for the detection of apoptosis and proliferation antibody m30 and antibody against ki-67 antigen were used. expression of fas ligand and bcl-2 was assessed using semi quantitative hscore method. syncytial knots were expressed as the number of syncytial knots per individual villus and as the total number of syncytial knots in each placental sample. results: apoptosis in all stages of trophoblast differentiation, number of syncytial knots per individual villus and the total number of syncytial knots were significantly higher in preeclamptic placentas than in control group placentas. fas ligand expression was significantly less, and bcl-2 expression significantly greater in the villus trophoblast among the study subjects compared with controls. there was no difference in the trophoblast proliferation between groups. conclusion: our findings might suggests that increased apoptosis and syncytial knots formation combined with reduced fas ligand expression could be involved in pathophysiological mechanisms of preeclampsia. contribution of fetal autopsy for diagnosis of meckel-gruber syndrome m. jo * , i. guerra, g. perez de nanclares, p. morales, j. j. aguirre, z. s. quintero, c. gomez, n. t. villagra * hospital txagorritxu, pathology, vitoria, spain objective: meckel-gruber syndrome (mgs) is a lethal rare autosomal recessive malformation. the six implicated genes encode proteins involved in primary cilia function. groups of families in finland, india and north of africa have been identified. method: a fetus,46 xy, karyotype, therapeutically aborted at 21 weeks with alobar holoprosencephaly in ultrasound. fetal autopsy was performed. genetic counseling was proposed to the family revealing moroccan origin and consanguinity, the parents were first cousins. results: a male fetus showing cyclopia, proboscis and a single opening with two rudimentary eyes was the external morphology. histological examination confirmed holoprosencephaly and also showed bilateral corticomedullary renal multicystic and periportal hepatic fibrosis with bile duct dilatation. the main diagnosis was mgs although other ciliophaties and non-ciliopathies conditions were considered, such as bardet-bield, joubert, smith-lemli-opitz syndrome and trisomy 13. however, the pathological characterization, parent's consanguinity and their north african origin makes mgs the likely diagnosis.a genetic study on paraffin-embedded material was requested, the poor quality of dna stopped definitive genetic diagnosis. conclusion: the pathologist may encounter atypical cases that require morphologic diagnosis to determine the type of underlying mutation and provide genetic counseling to parents.a meticulous autopsy is necessary to establish the diagnosis of mgs. fresh material would have to be frozen in order to make current diagnostic techniques of molecular pathology. mesenteric cysts in the pediatric age group r. jankovic * , j. sopta, m. stojanovic, b. lekic, b. jovanovic, z. stojsic * university of belgrade, faculty of medicine, serbia objective: mesenteric cysts are extremely rare lesions arising with an incidence of 1/20,000 admissions in children. clinically, mesenteric cysts are generally comprehended as a unique diagnostic entity, although they exhibit histological diversity. the objective of this study was to determine the incidence and the histology of the mesenteric cysts in the pediatric age. method: all cases of mesenteric cysts operated at the university children's hospital belgrade over the 10-year period of 2002 to 2011 were reviewed using pathology reports from the files of the institute of pathology. histological slides were re-examined and immunohistochemistry was applied, when necessary. results: a total of 27 cases of mesenteric cysts were identified. cysts of lymphatic origin (cystic lymphangiomas) were recorded in 12 patients (44 %), cysts of enteric origin -in 14 patients (52 %): 13 duplication cysts and one isolated enteric cyst of the mesenterium. only one example of the cyst of mesothelial origin, i.e. benign cystic mesothelioma was diagnosed (4 %). the most frequent site was mesenterium and mesocolon (86 %), followed by omentum (7 %) and the retroperitoneum (7 %). conclusion: cysts of enteric origin are easily recognized. it is important to differentiate between cystic lymphangioma and cystic mesothelioma due to their different natural history. objective: myxoid liposarcoma belongs to the group of soft tissue sarcomas with lipomatous differentiation. breast is a rarely affected (only 0.3 % of breast sarcomas), and often misdiagnosed. method: a 67-years old woman presented with a painful timorous lump of the left breast. mammography showed oval, lobulated lesion between medial quadrants, while ultrasonography revealed hipoechogenic, inhomogeneous nodule. patient underwent core biopsy which was histologicaly inconclusive and followed by quadrantectomy with excision of both medial quadrants. results: a lump (6 cm) was visible on the skin surface. serial sectioning revealed solid gray-white tumor with cystic and prominent necrotic areas. the tumor was located in the deep mammary tissue an it infiltrated the overlying dermis without involving the epidermis. it consisted of atypical stelate and spindle cells with infrequent mitoses and low ki-67 proliferative index. on the periphery mature adipocytes and lipoblasts were present. stroma was abundant, myxoid with plexiform capillary pattern. the myxoid substance stained slightly alcian-blue positive and tumor cells showed cytoplasmic immunopositivity for s100. phylodes tumor was excluded because of absence of epithelial component. conclusion: myxoid liposarcoma has a distinct morphology, rarely confused with other soft tissue tumors, although on cytological smears or biopsy samples it may be unrecognized. objective: synovial sarcoma poses a difficult diagnostic challenge since it can be confused with other benign or malignant entities. method: four biphasic and one monophasic synovial sarcomas were studied. we used the break apart/split signal kit (vysis) to detect the t(x;18) translocation and we performed immunohistochemistry for tle-1, ini-1, d2-40, cd56, cd99, bcl-2, ema, ck7, cd34, s-100, desmin and claudin-1. results: immunohistochemically we observed: tle-1+(5/ 5), ini-1 (4/5) with reduced nuclear and (1/5) with no expression, d2-40+(1/5), cd56+(5/5), cd99+(5/5), bcl-2 +(5/5), ema focal expression (5/5), ck7−(5/5), cd34−(5/ 5), s-100−(5/5), desmin−(5/5), claudin-1 + (5/5). fish revealed the split signal between the centromeric and telomeric end of the syt gene. conclusion: t(x;18) translocation remains the diagnostic hallmark of synovial sarcoma. ini-1 which is typically negative in atypical teratoid/rhabdoid tumor, epithelioid sarcoma and myoepithelial carcinoma seems to be reduced or even negative in synovial sarcomas. the focal expression of d2-40 must be considered when the differential diagnosis includes mesothelioma. finally tle-1 is a very sensitive marker for synovial sarcoma. ps-16-004 "mixed aneurysmal bone cyst" and "simple bone cyst", represent a different group of cystic lesion e. ayhan cinar * , b. doganavsargil, m. sezak, f. oztop * ege university, pathology, izmir, turkey objective: there are some mixed cysts (mc) with overlapping histological features of aneurysmal bone cyst (abc) and simple bone cysts (sbc). method: we reviewed 69 pure abc, 40 sbc and 23 "mixed cysts (mc)", and compared them by nonparametric tests. results: mcs, mainly showed two paterns as "type 1: sbc with secondary abc foci (n=18)"and "type 2: cysts with fully developed sbc and abc areas (n=5)". the median age for abc, sbc, and mc were 16±18.2 (range: 3-53), 21±13.7 (range: 3-54) and 9±11.47 years old (range: 3-50) respectively. mcs were more frequent in males than abc and sbc (p=0.017) and the most frequently involved bones were humerus-femur-pelvic bones in descending order (p= 0,021). "type 1 mc" showed more frequent cementum-like amorph material (p<0.001), ectatic venules (p=0.040), calcifications (p=0.044), less cholesterol deposition, necrosis and no different fracture and osteoid matrix when compared with type 2 mcs and sbcs. conclusion: cysts with overlapping features of abc and sbc are not uncommon. though they usually represent "sbcs with secondary abc component", they have dissimilarites with ordinary sbcs which needs to be further clarified with larger series. objective: percent of tumor necrosis after neoadjuvant chemotherapy, determined by detailed specimen mapping, has a high prognostic value. however both the mapping and grading systems has some practical problems. method: 128 cases were reviewed for potential pitfalls in handling and reporting processes and correlated with radiological findings by nonparametric-tests. results: most of the tumors were osteosarcoma (74.2 %) and ewing sarcoma (16.4 %), located in femur (53 %), tibia (22,7 %) and humerus (10.2 %). cortex, soft tissue and joint invasion was observed in 94 %, 77.6 % and 30.3 % of the cases, respectively. a median of 27.5 tumor blocks (9-74± 14,64) were submitted for histology. tumors were totally necrotic in 30 cases (24 %) (huvos grade-hg-iv), hg iii in 20.3 %, hg ii in 28.1 % and hg i in 24.2 % of the cases. overall good (hg iii-iv) and poor responders (hg i-ii) correlated well with radiological findings (p<0.001). discrepant cases showed extensive oedema and congestion enhancing contrast medium, patchy necrosis unabling accurate histologic evalation, or failure to prove tibiofibuler joint involvement because of sagittal sectioning (p<0.05). conclusion: evaluating chemotherapy responce is a laborious work. radiologic orientation prior to grossing and applying morphometric technics may enhance more accurate evaluation. atypical ewing sarcoma/primitive neuroectodermal tumor with unusual melanocytic differentiation -a case report p. buzrla * , j. dvorackova, i. urbanovska, h. bielnikova * inst. of pathology, ostrava-marianske hory, czech republic objective: atypical ewing sarcoma/primitive neuroectodermal tumor with the melanocytic differentiation is a very rare, malignant tumor which occurs in infants and adolescents. it is localized mainly in soft tissues. its biological behavior is aggressive, but its response to the chemotherapy is prognostically good. method: a 4-month old male is presented with the 17×11 cm tumor of the soft tissue, localized in the right front over the cranial margin of the orbit. the tumor was surgically exstirpated and then sent to the histological investigation with the additional immunohistochemistry and the fish. results: microscopically, the tumor is hypercelullar and consisted of oval to polygonal cells with the vesicular nuclei, the prominent nuclei and the amphophilic cytoplasm. in other parts of the tumor, there are smaller neoplastic cells with hyperchromatic nuclei. some neoplastic cells contain the melanin pigment in the cytoplasm, which was positive in fontana-masson method. the immunohistochemical stains for vimentin, ae1/3, ck7, cd99, hmb-45, fli-1 were positive. the fish investigation demonstrated the translocations (t 11;22, q24;q12) in 95 % of neoplastic cells and (t 21;22, q12;12) of 5 % ones. conclusion: in literature, the ewing sarcoma/pnet showing the myogennic differentiation is usual, but the melanocytic differentiation is rare and can be confirmed not only immunohistochemically, but also by fish investigation. objective: poly(adp-ribose) polymerase-1 (parp-1) is a nuclear enzyme involved in the repair of dna single-strand breaks. parp-1 inhibitors may be efficient in therapy of malignancies. this report evaluates the expression of parp-1 in primary testicular germ cell tumors and correlates expression patterns with histological subtypes and patient/ tumor characteristics. method: group of 124 patients with testicular germ cell tumors were investigated for parp-1 expression by immunohistochemistry, scored by the multiplicative quickscore (qs) method and compared to parp-1 expression in normal testis. results: we observed higher expression of parp1 in testicular tumors compared to normal testis (mean qs=10.04 vs. 3.60, p< 0.0000001). the parp-1 overexpression (qs>9) was most often detected in intratubular germ cell neoplasia itgcn (100 % of specimen with parp-1 overexpression), compared to 1.9 % of normal testicular tissue specimen. there was no association between parp-1 expression and clinical variables. conclusion: parp-1 expression is higher in tumor tissue than in normal testis. parp-1 could represent a novel treatment target in tgcts and the assessment of parp-1 expression in tumor samples may lead to the consideration of tgcts patients for parp inhibitor therapy. supported by 2007/30-nou-01, vega 1/0724/11 and itms: 26240220052 cofinanced by european regional development fund. objective: gardner fibroma (gaf), a rare lesion, typically occurs in infants, children and young adults, with predilection for the trunk (particularly paraspinal region) and no gender predominance. gaf is an ill-defined plaque-like mass, with rubbery consistence, usually asymptomatic, ranging from 0.3 to 12 cm. although benign, 70-90 % of the cases may be associated with apc mutations, familial adenomatous polyposis (fap) and/or gardner syndrome (gs). we report 2 cases of gaf, which allowed the identification of 2 families with gs. results: case 1: 7-month-old boy, with 2 nodules in the left paraspinal region, the largest with 2.5 cm. case 2: 5month-old boy, presenting a 1.5 cm ill-defined deep right scapular mass. both lesions were infiltrative, paucicelular densely collagenized proliferations of bland spindle cells. a gaf diagnosis was made and clinical/genetic investigation advised. apc gene sequentiation revealed, in both cases, a frameshift germline mutation in exon 15. additionally, case 1 presented a del(5)(q11q35). heredogram showed typical manifestations of gs: case 1: mandibular osteomas, epidermoid cysts, "soft-tissue tumours", colo-rectal carcinoma. case 2: hepatoblastoma, colo-rectal carcinoma. conclusion: given the high association with gs and the fact that it can be its first manifestation, gaf is considered a sentinel lesion for this syndrome. therefore, an accurate diagnosis is of the utmost importance. prognostic significance of bcl-2, c-myc and survivin in synovial sarcoma d. demir * , b. yaman, y. anacak, b. keçeci, g. kandiloğlu, t. akalın * ege university, pathology, izmir, turkey objective: in this study, we evaluated 81 synovial sarcoma cases, who had been referred to our tertiary tumor center during the last 20 years. we applied bcl-2, c-myc and survivin as immunohistochemical markers and evaluated the relation with conventional prognostic findings and prognosis for those 64 patients who have follow-up. method: in this study; ten-year tumor free survival rate was 38 % reflecting the agressive behaviour of synovial sarcoma. tumor grade was the most valuable prognostic input. progression free survival (pfs) was 159 months for gradeii cases (40 cases) and 36 months for gradeiii cases (24 cases) (p=0,000). results: immunohistochemically, there was weak relation between bcl-2 staining intensity with prognosis. overall survival was 211 months for weak or negative cases (9cases), 132 months for focally intense cases (21cases) and 101 months for diffuse and intense cases (34 cases) (p= 0,042). there was also weak relation with c-myc staining pattern with prognosis. overall survival was 193 months for c-myc negative cases (25cases), 114 months for cytoplasmic positive cases (23cases) and 68 months for nuclear positive cases (16 cases) (p=0,043). there was no relation between survivin and prognosis. conclusion: in conclusion; tumor grade is the most valuable prognostic parameter in synovial sarcomas. immunohistochemically c-myc and bcl-2 staining have weak relation with synovial sarcoma prognosis. immunohistichemical profile of primary and recurrent desmoids e. dubova * , t. sidorenko, a. shchegolev, a. adamyan * v.i. kulakov scientific center, dept. of pathology, moscow, russia objective: desmoids is a locally recurrent and invasive but not metastatic tumor. our aim was to study immunohistochemical profile of primary and recurrent desmoids. method: complex morphological study of 16 abdominal desmoids. all the tumors were divided into 3 groups: primary, first recurrent and second and more recurrent cases. we used antibodies against β-cathenin, cox-2, apc, survivin and ki-67 for immunohistochemical study. results: nuclear and cytoplasmic β-cathenin expression levels were significantly higher in recurrent than in primary desmoids. apc cytoplasmic expression level was also significantly higher in recurrent tumors. we revealed only cytoplasmic cox-2 expression in desmoids, and its level was significantly higher in recurrent tumors with the activity increasing accordingly to the number of recurrences. survivin expressed both in nuclei and cytoplasm of the tumor cells and its expression levels were significantly lower in recurrent desmoids. conclusion: revealed immunohistochemical properties of primary and recurrent desmoids reflect tumor transformation and progression and could be used as additional prognostic markers in this disease. objective: many bone and soft tissue sarcomas (bsts) are aggressive tumors with fatal prognosis. the importance of angiogenesis for the growth and progression of solid bsts is now well recognized. a variety of chemokines have been described that either promote (angiogenic) or inhibit (angiostatic) angiogenesis. the aim of this study is to characterize the expression profile of some chemokines in a series of xenotransplanted human bsts. method: one ewing sarcoma (es), 1 grade 3 chondrosarcoma (chs), 1 osteosarcoma (os), 1 synovial sarcoma (ss), 1 fibrosarcoma and 1 gastrointestinal stromal tumor (gist) were xenotransplanted into the backs of nude mice (athymic balb-c nude mice). when the tumor size reached the 3 cm, animals were sacrificed and tumors analyzed for the expression of cxcl1/2/3, cxcl9 and cxcl10, using two-color staining fluorescence on each slide under a confocal microscope (olympus fv1000). results: we observed that angiostatic chemokines (cxcl9 and cxcl10) presented higher expression than angiogenic (cxcl1/2/3) chemokines in es, grade 3 chs, os and gist whereas fibrosarcoma and ss were more positive for angiogenic chemokines. conclusion: the expression profile of angiostatic and angiogenic chemokines depends on the type of bsts and could be related to their different biological behaviour. other elements such as angiogenic or pro-inflammatory markers should also be considered. objective: the wide range of differential diagnostic possibilities of rhabdomyosarcomas (rms) shows the need for more specific and sensitive markers. low survival rates of high risk rms patients require new prospective therapeutic targets. method: archival material ffpe samples of 15 rms (12 embryonal -erms, 3 alveolar -arms) and non-rms soft tissue tumors were evaluated by immunohistochemistry for myogenin, myod1, egfr, vegf, cox-2, p-akt and p-mtor expression. the presence of pax3/7-fkhr forming translocations, myogenin, myod1, gamma subunit of fetal acetylcholine receptor (achr) and k-ras mutational state were determined by rt-pcr. results: 10/12 erms and 3/3 arms showed expression of myogenin, myod1 and gamma achr. non-rms tumors were negative. translocations were only found in arms. egfr expression was characteristic for erms, without the presence of activating k-ras mutation. strong expression of vegf was detected in all samples. p-akt and p-mtor showed overlapping expressions in 86,7 % rms. in most of the samples weak cox-2 positivity was demonstrated. conclusion: myogenin, myod1 and the fetal achr are specific and sensitive diagnostic markers of rms. rms subtypes are identifiable by pax3/7-fkhr detection and probably egfr expression. the results indicate vegf, egfr, cox-2 and akt-mtor pathway directed therapy to be considered in rms. supported by itms 26240220052. immunohistochemical analysis of potential targets in desmoid tumor therapy a. janegova * , z. hlavata, p. babal, p. janega * comenius university, dept. of pathology, bratislava, slovakia objective: desmoid tumors (dts) are clonal fibroblastic/ myofibroblastic proliferations. although histologically benign, desmoids are locally invasive and often have an unpredictable clinical course. dts are infrequent lesions, but they have a high incidence in patients with familial adenomatous polyposis (fap). the treatment of dts needs to be individualized. method: to explore the molecular bases of potential pharmacologic targets in dts we evaluated the immunohistochemical expression of steroid hormone receptors (era, erb, pr) and cox-2 protein in sporadic (n=6) and fapassociated desmoid tumors (n=9) together with gi adenomas of fap patients (n=5). results: nuclear erb expression was found in 14/15 dts. all adenoma samples showed nuclear erb positivity, which was weaker than in the surrounding normal epithelial cell. era and pr expression were lacking in all samples. cox-2 was found in 14/15 dts. adenomatous polyps showed intense cox-2 expression compared to surrounding normal mucosa. conclusion: high incidence of erb positivity in dts supports the usage of hormonal therapy in these lesions. open question is the effect of anti-estrogen therapy in dt patients with adenomatous polyps, as in adenomas estrogen seems to have preventive potential. cox-2 expression suggests the benefit of anti-inflammatory treatment in dts adenomatous polyps. supported by itms: 26240220052. retroperitoneal sarcomas: clinicopathological features in a series of 68 cases e. kairi-vasilatou * , a. tsagkas, a. melloy, a. paraskeva, a. kondi-pafiti * aretaieio nosokomeio university, dept. of histopathology, athens, greece objective: retroperitoneum is the less common site of origin accounting for approximately 10 % of soft tissue sarcomas. method: between january 1990 and december 2010, our hospital's records of 68 patients with retroperitoneal sarcomas were retrospectively studied. results: the patient median age was 57 years and there was no sex predominance. median tumor size was 18.5 cm (ranging from 6 to 55 cm) with 60 % of them being larger than 10 cm. the most common histological type was liposarcoma (36/68-52,9 %), followed by leiomyosarcoma (16/68-23,5 %) and undifferentiated soft tissue sarcoma (7/68-10,2 %). the remaining 9 tumors (13,2 %) included 2 chondrosarcomas, 2 well-differentiated fibrosarcomas, 1 pecoma, 1 hemangiopericytoma, 1 ewing sarcoma and 1 malignant peripheral nerve sheath tumor. 44 of the sarcomas were high grade (65 %) and 24 (35 %) low grade. 1-year recurrence rate was 34,3 %. the 3-and 5-year overall survival rates were 56,2 % and 53,1 % respectively. seven patients received adjuvant chemotherapy. conclusion: the most commonly encountered histologic subtypes are liposarcoma and leiomyosarcoma, which are consistent with the results of the present study. complete tumor resection at first operation is the only treatment factor that consistently predicts improved survival. abdominal desmoid tumor y. lorenzo mahia * , m. san martin alonso, b. iglesias rodriguez * hospital maixoeiro, anatomia patologica, vigo, spain objective: abdominal desmoid tumours are rare benign neoplasms. they are commonly found in the mesentery, while they are rarely found in the intestinal wall. most cases are sporadic, although there is a link with colonic polyposis, trauma, and oestrogen. they predominate in 25-35 year-old women. this present case is notable due to its location in the jejunal wall, possible relation with previous surgery, and the age and sex of the patient. method: we present a 51-year-old male patient previously operated for umbilical hernia years before. he came to the consultation on noticing an abdominal mass. since the initial suspicion was of jejunal wall gist tumour, surgery was performed. results: the histopathological and immunohistochemical findings, support the diagnosis of desmoid tumour. conclusion: desmoid tumours consist of fibroblastic monoclonal proliferation developed from aponeurotic muscle structures. some authors consider them non-neoplastic processes given their limited aggressiveness while others classify them within distinct low-grade sarcomas. their origin is not well established, although there are known factors involved such as mutations in the apc gene or beta-catenin and trisomy 20 and 8. the originating cell, the myofibroblast, is involved in post-traumatic cellular regeneration. this explains why we find these tumours associated with previous surgery. treatment of advanced dermatofibrosarcoma protuberans with imatinib mesylate with or without surgical resection w. michej * * cancer centre institute of warsaw, dept. of pathology, poland objective: dermatofibrosarcoma protuberans (dfsp) is a rare soft tissue sarcoma of the skin characterized by the presence of specific col1a1-pdgfb fusion protein, which appears as a consequence of the t(17;22) (q22;q13) translocation. method: the aim of the study was to perform an analysis of patients with advanced dfsp treated with imatinib, with or without surgery. we examined 15 patients (6 male, 9 female; median age 56 years) with locally advanced/initially inoperable and/or metastatic dfsp treated with imatinib 400-800 mg daily between 12/2004 and 06/2009. all diagnoses were ascertained cytogenetically (fluorescent in situ hybridization). median follow-up time was 16 months (range: 4-81). results: metastases were present in 8 cases (two lungs, two soft tissue, two lymph nodes). fibrosarcomatous transformation was confirmed in 7 patients. after treatment with imatinib overall responses were: 10 partial responses, 2 stable diseases (13 %) and 3 progressive diseases (20 %). seven patients (47 %) after resection had residual disease confirmed by pathologic examination and remained free of disease. conclusion: we proved that anti-tumour effect of imatinib in dfsp with presence t(17;22) had in most cases good responses. imatinib therapy may in some cases leads to tumour resection because of lesser size. objective: at an estimated incidence of 2 cases per million per year, osteosarcoma is the most common malignant primary bone tumor. method: we conducted a retrospective study to identify the osteosarcoma cases diagnosed at major tertiary care hospitals in turkey. results: our study group was made up of 745 cases: 440 men, 305 women, aged 3-82 years (mean 23.7 year). all patients had been diagnosed with skeletal osteosarcoma between 2001 and 2011 at one of the 10 tertiary care referral center. tumor was most frequently located in femur (50.4 %) followed by tibia. apart from the long bones, pelvic and gnatic bones were the next in location, 4.4 % and 1.6 % respectively. there were 16 (2.1 %) secondary osteosarcomas, related to previous irradiation and various underlying diseases. for the histological types conventional osteblastic intramedullary tumors were most prevalent, making 71.8.% of the cases. chondroblastic and telengiectatic osteosarcoma are the next common histologic types. surface tumors were detected in 51 (6.8 %) cases. rare histological types like small cell, epitheloid, chondroblastoma like and fibrous dysplasia like were also reported. conclusion: this study is conducted as a preliminary work to form the basis of a pathologic database for the osteosarcoma cases diagnosed in our country. objective: kaposi's sarcoma is a rare disease likely associated with human herpes virus 8 infection, and occurs predominantly in jewish, mediterranean and middle eastern men. since there is a paucity of reports on the pattern of its occurrence in tunisia, we here analysed the epidemiological pattern and anatomoclinical features. method: we retrospectively studied 71 consecutive cases of kaposi's sarcoma diagnosed in the pathology department, farhet hached hospital, sousse during a 15-year period. results: kaposi's sarcoma represented 21 % of soft tissue sarcomas. there were 23 (32.4 %) females and 48 (67.6 %) males (male-to-female ratio: 2.1:1). median age at diagnosis was 69 (range: 10-98 years). the age distribution showed that elderly (≥60 years) were the most affected patients with a frequency of 66.2 %, followed by patients aged 31-59 years (25.3 %), and patients under 30 years (8.4 %). the most common location was the lower limbs, particularly the distal lower extremity (73.3 %), followed by contiguous location (7 %), and soft tissue, nos (7 %). conclusion: kaposi's sarcomas were more frequently diagnosed in elderly. the distal lower extremities were more involved. objective: rhabdomyosarcoma is the most common soft tissue sarcoma in the first two decades of life. in this study, we analysed the epidemiological pattern and antomoclinical features of rhabdomyosarcoma in central tunisia. method: we retrospectively studied all cases of rhabdomyosarcoma diagnosed in the pathology department, farhet hached hospital, sousse during a 15-year period. results: there were 15 (31.9 %) females and 32 (68.1 %) males (male-to-female ratio: 2.1:1). median age at diagnosis was 9 (range: 0-85 years). rhabdomyosarcoma was more frequently diagnosed in childhood (63 %) than in adults (37 %). in children, the tumour size was higher than 5 cm in 73 % of cases, the embryonal subtype was the most frequent (60 %) and the two most common sites of disease were the head and neck (50 %) and genito-urinary tract (23.3 %). in adults, the tumour size was higher than 5 cm in 90 % of cases, the pleomorphic subtype was the most diagnosed (41 %), and limbs were the most involved sites (41 %). conclusion: rhabdomyosarcomas is more frequently diagnosed in children than in adult. head and neck locations were the most involved and embryologic type was the most diagnosed. in adults, rhabdomyosarcomas were more frequently localized in limbs and diagnosed as pleomorphic type. microarray-based dna methylation study in ewing sarcoma of bone h.-r. park * , y.-k. park * hallym university, sacred heart hospital, anyang, republic of korea objective: alterations in the dna methylation pattern are a hallmark of malignancy and also of ewing sarcomas. however, most epigenetic studies in ewing sarcoma have focused on the analysis of few candidate genes and comprehensive studies are required. method: here, we report for the first time a microarraybased dna methylation study of 1505 cpg sites of cancerrelated 807 genes in 69 ewing sarcomas. we used illumina's goldengate methylation cancer panel i microarray. results: using appropriate controls (n=14), we identified a total of 104 cpg sites hypermethylated in ewing sarcoma. most of hypermethylated genes are related with cell adhesion, cell regulation, development, and signal transduction. we compared the methylation mean of each tumor according to the survival data. the methylation mean was significantly higher in the alive patient group (0.25±0.03) compared to the dead patient group (0.22±0.05) (p=0.0322). however, the methylation mean was not significantly correlated with age, sex, or tumor location. we selected the most popular hypermethylated genes, gdf10, osm, apc, and hoxa11, but, their methylation levels were not significantly correlated with the survival data. conclusion: we have characterized the dna methylation profile of ewing sarcomas and detected 104 cpg sites that were significantly hypermethylated in ewing sarcomas. these might therefore play an important role in the development of ewing sarcomas. pleomorphic and dedifferentiated leiomyosarcoma associated with lynch syndrome: a case report h. quiceno * , f. j. queipo, r. carías, j. j. sola, f. j. pardo * clinic university of navarra, anatomical pathology lab, pamplona, spain objective: the lynch syndrome or hereditary non-polyposis colorectal cancer (hnpcc) is a hereditary syndrome that predisposes to different types of colonic and extracolonic cancer, mainly in endometrium, stomach, ovary, and hepatobiliary and urinary tract. occasional sarcomas have been reported in hnpcc patients. method: we describe a pleomorphic and dedifferentiated leiomyosarcoma of the gluteus in a patient with lynch syndrome. we study her clinical, pathology, immunochemistry and molecular alterations. results: we present a 71 year old woman with previous colorectal, endometrium and breast cancers in a lynch syndrome with mutations in exons 1 and 2 of mlh2, which appeared with a right gluteus mass. grossly, it was encapsulated, whitish-yellowish with necrotic areas and measured 7,2 cm. microscopically, the tumor was composed by a diffuse and polymorph spindle cells with fascicular pattern, focally myxoid and zones of necrosis and large hyalinization. the cells had large nuclei, sometimes giant multinucleated, with a brisk atypical mitosis activity. there was ihq variability: positivity to desmine, msa, calponin and negative to caldesmon and sm. there was loss of msh2 and msh6 repair proteins. conclusion: we must suspect a lynch syndrome relational sarcomas if we found one in a patient with hnpcc in order to test to mismatch repair proteins. immunohistochemical review of 42 synovial sarcomas, including expression of tle1: a "new" marker, with molecular confirmation in 21 cases b. rekhi * , r. basak, s. desai, n. jambhekar * tata memorial hospital, dept. of pathology, mumbai, india objective: synovial sarcoma displays a wide clinicopathological spectrum and a specific translocation t(x; 18)(syt-ssx)(p11.2; q11.2). cost constraints limit molecular confirmation in every case. lately, tle1 has been recognized as a useful ihc marker. herein, we present ihc review of 42 synovial sarcomas, including tle1 expression. method: forty-two synovial sarcomas included monophasic spindle-cell type (26) (61.9 %), biphasic 13 (30.9 %), calcifying (2) (4.7 %) and poorly-differentiated type (1) (2.3 %). 21 tumors were confirmed with molecular analysis. tle1 immunostaining was graded from 0, 1+, 2+, 3+, with 2+ or 3+ grades interpreted as positive staining. results: on ihc, various synovial sarcomas were positive for ema (26/34)(76.4 %), ck7 (6/10) (60 %), ck/mnf116 (6/21)(28.6 %), bcl2 (36/37)(97.3 %), mic2 (23/ 31)(74.1 %) and tle1 (40/42)(95.2 %), while negative for cd34 (0/21). among 70 other tumors, tle1 was positive in schwannomas (5/5) (100 %), neurofibromas (2/2)(100 %), mpnsts (2/12)(17 %) and pnets (4/10)(40 %). sensitivity and specificity of tle1 for synovial sarcoma was 95.2 % and 72 %. 50 % of pnst (75 % of schwanomas, 50 % of mpnst and 25 % of neurofibromas). in contrast, her3 positivity was observed in 20 % of other mesenchymal tumors (28 % uterine and 9 % non-uterine leiomiomas, 18 % uterine and 25 % nonuterine leiomiosarcomas, 29 % ups). conclusion: her3 overexpression is frequently found in pnst, including 50 % of mpnst. due to the crucial role of her3 receptor in cell signalling as a main activator of the pi3k pathway, these results support the rationale of developing new therapeutic approaches in mpnst. objective: nonneural granular cell tumour (nngct) is a rare neoplasm described in 1991 by leboit et al. till now, no more than 35 cases have been presented in the literature, nearly all were limited to the skin. we report the first nngct of the breast and present its morphological and immunohistochemical characteristics. method: a 39 year-old female underwent lumpectomy because of tumour of the breast. after routine histological examination, the immunohistochemical studies were performed using commercially available antibodies against cytokeratins (ae1/ae3), cd68, cd1a, s100, cd34, smooth muscle actin, desmin, miogenin, inhibin, and ki67 antigen (mib1). results: microscopically, the tumour was composed of ovoid and polygonal cells with abundant granular, eosinophilic cytoplasm. the cells presented mild or moderate polymorphism and trace mitotic activity. they were arranged in diffuse sheets with no evidence of nesting or fasciculation. the histological texture of the tumour resembled that of classical granular cell tumour of abrikosoff. however, the tumour cells were strongly immunoreactive for cd68 and inhibin, whereas the reactions against s100 protein as well as other antigens studied gave negative results. the value of ki67 index did not exceed 2-3 %. conclusion: the differential diagnosis of granular cell myoblastoma (abrikossoff tumour) should include the nonneural granular cell tumour. low grade fibromyxoid sarcoma: a study of 10 cases k. shelekhova * , a. konstantinova * petrov´s research institute for oncology, st. petersburg, russia objective: low grade fibromyxoid sarcoma (lgfms) is a specific type of fibrosarcoma with deceptively banal appearance and malignant behavior. method: a 5 year retrospective study revealed 10 lgfms cases. morphological and immunohistochemical analysis was performed. follow-up information was obtained for 5 cases. results: all tumors occurred in adults (mean 40 year). they developed in neck (1), extremities (6) and trunk (3). beside classical morphology of lgfms following features were observed: necrosis (3), rosettes (3), areas of increased cellularity (5), foci of epithelioid cells (5), marked nuclear pleomorphism (1), prominent myxoid change (3), invasive border (6), bone formation (1), focal retiform pattern (1) and sclerosing epithelioid fibrosarcoma-like areas (1). immunoprofile was the follow: vimentin (100 %), ema (30 %), cd34 (10 % focally), s100 (0), sma(0), desmin (0), ae1/ae3 (0). two patients were without evidence of disease, one developed lung metastasis after 1 year. two patients developed secondary tumours in 6 and 12 years after initial diagnosis. there were differences in the location and histologic features compared with primary ones. secondary tumors were less cellular, without necrosis or epithelioid cells. behavior of secondary tumors did not fit to the classical concept of tumor progression that implies increase of malignancy. conclusion: lgfms is a distinctive but unusual type of soft tissue sarcoma demonstrating a wide histologic spectrum and behavioral range. immunohistochemical and mutational study of synovial sarcomas k. specht * , m. bettstetter, g. keller, h. rechl, r. von eisenhart-rothe, h. höfler, m. straub * technische universität münchen, inst. für pathologie, germany objective: synovial sarcomas are mesenchymal tumors of unknown histogenesis. their molecular signature is a specific t(x; 18)(p11.2;q11.2) translocation. no effective targeted therapies are currently available. the aim of this study was to evaluate the expression and mutational status of potential molecular therapeutic targets. method: 38 molecularly confirmed cases of synovial sarcomas were included in this study. immunohistochemical stainings of the egf-r family (egf-r, her2/neu, her3), and signaling molecules implicated in the mtor pathway (akt, mtor, pten), as well as e-cadherin and snail was performed. in addition, cases were screend for mutations in the egfr, pik3c, b-raf, k-ras, and n-ras genes. results: egf-receptor family members as well as e-cadherin and snail are important for defining the tumor phenotype by determining epithelial-mesenchymal transition of synovial sarcomas. activation oft the mtor pathway is seen in a significant number of cases. mutations of the genes studied are an overall rare event in synovial sarcomas and other types of sarcomas studied. conclusion: egf-r expression is found in many synovial sarcomas, however, mutations of egfr or downstream molecules appear to be rare. activation of mtor pathway is frequently seen in synovial sarcomas. the benefit of targeted therapy against these genes in synovial sarcomas remains to be determined. collagen v induces differentiation of rabbit adipose tissue-derived stem cells in chondrocyte-like phenotype w. teodoro * , i. brindo da cruz, a. p. velosa, s. carrasco, c. goldenstein-schainberg, r. fuller, e. parra, v. capelozzi * faculdade de medicina da usp, disciplina de reumatologia, são paulo, brazil objective: stimulated mesenchymal stem cells (mscs) have capacity of differentiation in many cell types. it is being used in degenerative diseases treatment protocols. we evaluated the collagen v (col v) and collagen xi (col xi) influence in the differentiation of rabbits adipose tissue-derived mscs in a chondrocyte-like cell phenotype. method: mscs isolated of new zealand rabbits adiposetissue were maintained in culture by 4 weeks. colv, colxi and colv/xi (10 μg/ml) were added to culture during 72 h. the cells aggregates were stained with toluidine blue, alcian blue and picrosirius. chondrocyte-like phenotype was confirmed by immunofluorescence to cd34, vimentin and collagens i, ii and iii. results: mscs stimulated with colv expressed proteoglicans and collagen, when compared with colxi and colv/ xi and control. in the presence of colv, mscs was capable to increase collagen ii expression confirming its chondrocyte-like cell phenotype. in contrast, mscs cultured with colxi and colv/xi express collagen i and iii. conclusion: the data suggest that colv may facilitate the differentiation of rabbit adipose tissue-derived stem cells into a chondrocyte-like phenotype. further studies are urged in order to evaluate the influence of colv in the ability of chondrocytes to remodel osteoarthritic joint surface at ultra structural and molecular levels. objective: xrcc1 is essential for dna base excision repair, single strand break repair and nucleotide excision repair. method: we evaluated xrcc1 immunohistochemically in early stage breast (n=2046), ovarian (n=157), gastric (n= 140), colorectal (n=250) and pancreaticobiliary cancers (n= 240). pre-clinically, we evaluated a panel of xrcc1 deficient and proficient chinese hamster ovary and human cancer cell lines. double strand break repair (dsb) inhibitors targeting atm (ku55933), dna-pkcs (nu7441) and atr (nu6027) were evaluated for synthetic lethality and cisplatin alone or in combination with dsb inhibitors for chemopotentiation. results: in breast cancer, xrcc1 loss (16 %) was associated with a 2-fold increase in risk of death and metastasis (p< 0.0001). in ovarian cancer, xrcc1 positive tumours (44 %) were more resistant to platinum chemotherapy (p=0.0001). xrcc1 positivity conferred a 2 fold increase of risk of death (p=0.002) and independently associated with poor survival (p= 0.002). pre-clinically, ku55933, nu7441 and nu6027 were synthetically lethal in xrcc1 deficient compared to proficient cells as evidenced by dsb accumulation, g2/m cell cycle arrest and apoptosis. xrcc1 deficient cells were hypersensitive to cisplatin which was enhanced by dsb repair inhibitors compared to in proficient cells. conclusion: conclusions: xrcc1 deficiency in human tumours may be suitable for synthetic lethality application and exploited for cisplatin chemotherapy potentiation. analysis of bcl2 oncoprotein expressing breast cancer by molecular subtype a. abolins * , i. strumfa, z. simtniece, a. vanags, g. trofimovics, j. gardovskis * riga stradinš university, inst. of oncology, latvia objective: breast cancer is the most common malignancy in western women. despite the progress in morphological investigation, active research is devoted to potentially important targets for prognosis and intervention. bcl2 oncoprotein represents such factor. method: consecutive breast cancer cases were examined by routine protocol approach. the bcl2 oncoprotein expression was detected immunohistochemically. expression was considered positive if it was in more than 25 % of tumour cells. the molecular subtype ( objective: breast cancer is the most common malignant tumour of latvian women (www.csb.lv). correct diagnosis, including identification of tumour histogenesis, is the prerequisite for appropriate treatment. method: consecutive breast tumour cases were selected by systematic retrospective archive search and were examined by breast cancer panel. if expression of oestrogen and progesterone receptors and her2 protein was negative, mammaglobin, cytokeratin ae1/ae3, vimentin, cd45, cdx2, cytokeratin 20, ttf-1 and melanosome protein hmb-45 were detected. results: five patients (0.89 %, 95 % ci=0.38-2.10 %) with secondary breast tumours were identified among 559 cases. breast was affected by metastatic small cell lung cancer (1), malignant neuroendocrine tumour of small intestine (1) as well as by epithelioid melanoma metastasis in breast tissues (2) or intramammary lymph node (1). analysing medical records, multiple synchronous and/or metachronous metastasis, involving brain, kidneys and ovaries, were found in all patients with haematogenous tumour metastases in the breast. in contrast, the lymphogenic tumour spread was isolated. conclusion: 1. lymphogenic or haematogenous metastasis rarely (0.89 % of malignant breast tumours by morphology) can develop in breast tissues. it should be taking into account when planning the differential diagnostic approach, especially immunohistochemistry. 2. haematogenous metastases in breast are associated with a wide synchronous or metachronous extramammary tumour spread. the prognostic significance of tumor-associated stroma in invasive breast carcinoma objective: fibroblasts in the stromal component of a tumor may influence tumor progression in various organs. the prognostic significance of tumor-infiltrating lymphocytes is also frequently reported. however, the prognostic significance of the stromal component in breast cancers, particularly those of high grade, has not been established. method: in this study, we analyzed surgically resected specimens from 545 patients with breast carcinoma, including 193 high grade tumors, for tumor-stroma ratio, dominant stroma type (collagen (c), fibroblast (f) or lymphocyte (l) dominant type), and central fibrosis on hematoxylin-eosin stained histological sections. we correlated these features with clinical prognosis. results: among the 533 specimens examined, 127 (23.3 %) were of c type, 292 (53.6 %) of f type, and 114 (20.9 %) of l type. central fibrosis was found in 99 tumors (18 %). the dominant stroma type was a significant prognostic factor on univariate and multivariate analyses, together with t classification, nodal status and bloom-richardson grade. tumorstroma ratio and central fibrosis did not predict survival on multivariate analysis. even in high-grade tumors, relapse-free intervals differed significantly according to dominant stroma type. conclusion: conventional hematoxylin-eosin stained tumor slides may contain more prognostic information than previously thought; in particular, the dominant stroma type in invasive breast cancer may potentially be used to predict outcome. ps-17-007 pathology in breast implants substitution l. alfaro * , j. serra, j. m. ibañez * valencia, spain objective: health alert concerning breast implants brand pip led to a review of patients harboring these prosthesis and in many cases replacement by new ones. anaplastic lymphoma described in these patients has been an additional problem to be faced by plastic-surgeons and pathologists. method: ninety two cases of women with breast implants were studied. most of them had pip implants (although no information of prosthesis type was available in all cases). eighty of them presented with ruptures of different size. after implant replacement, histopathologic analysis of fibrous capsules and liquid from seromas in periprosthetic cavities was carry out. following fda recommendations, immunohistochemical studies to rule out lymphoma was performed in 85 cases. results: fibrous capsules showed synovial metaplasia in all cases. morphology was practically identical that in real articular synovial cells, and occasionally intracavitary nodules were seen detached from the surface in a process similar to synovial chondromatosis. no cases of lymphoma were seen and no expression of cd30 or alk markers occurred. a case of pericapsular ductal invasive carcinoma was discovered. conclusion: synovial metaplasia seems to be very common in capsules around breast implants. development of conventional breast carcinoma is probably much more frequent than lymphomas independently of the possible implant influence. post-radiation angiosarcoma of the breast: report of a case a. apostolaki * , m. sofopoulos, s. tsitsiou, e. pigadioti, n. mylona, n. arnogiannaki * agios savvas hospital, dept. of anatomical pathology, athens, greece objective: post-irradiation angiosarcoma generally occurs after breast conservation and radiation therapy. it affects the dermis of the breast within the radiation field. the incidence of post-radiational angiosarcoma is about 0.14 %. method: an 82-year-old woman who had undergone conservative surgery and radiotherapy for breast cancer 10 years ago presented with multiple red-purplish papules on the skin of her right breast. excisional biopsy followed by simple mastectomy was performed. results: excisional biopsy revealed a neoplasm composed of highly pleomorphic cells with prominent nucleoli forming solid areas and neoplastic slit-like vascular channels. many mitotic figures and some individual apoptotic cells were also present. the tumor cells were positive for endotelial cell markers (factor viii, cd31, cd34) and negative for ck7. the gross inspection of the mastectomy specimen showed multiple reddish-purple papule-like lesions on the skin, spreaded in an area of 12×10 cm. sectioning showed numerous homogenous slightly hemorrhagic white nodules measuring from 2 to 12 mm located in the dermis. histopatology and immunohistochemistry findings were consistent to those of the excisional biopsy. infiltration of the subdermis was noted. conclusion: diagnosis was high grade post-irradiation angiosarcoma. simple mastectomy is the treatment of choice. adjuvant chemotherapy should be considered in high grade neoplasms like this one. prognostic factors in invasive lobular carcinoma of the breast g. askan * , g. ayranci, n. özkan, h. kaya, u. ugurlu * marmara university, dept. of pathology, istanbul, turkey objective: determination of molecular features in breast carcinomas, such as hormone receptor expression, can guide clinicians to the optimal choice of therapy. in this study, the relationship between the histologic grade, pathologic stage and the prognosis of invasive lobular carcinoma of the breast, and the tissue expression levels of er, pr, her-2/neu, p53, bcl-2, ki-67 and e-cadherin was investigated. method: 31 cases of invasive lobular carcinoma of the breast, from 2003 to 2011, were included in this study. a single best representative paraffin block was selected for each case and h&e staining and immunohistochemistry procedures were performed. ki-kare test was used for statistical method. results: 24 of 31 cases had classical lobular carcinoma, 7 cases had tubulolobular, pleomorphic, signet ring cell, or apocrine features. the median age of patients was 54 years. er and pr were positive in 28 cases. all cases were negative for e-cadherin. ki-67 was greater than %15 in 2 cases with pleomorphic lobular carcinoma. p53 positivity increased with grade. in 14 cases p53 was negative and bcl-2 was positive. bcl-2 and p53 were positive in 29 and 17 cases respectively. conclusion: in contrast to the literature, there was no correlation between bcl-2 status and other molecular markers, including p53. rapid immunohistochemistry in intraoperative sentinel axillary lymph node evaluation p. baldin * , m. c. cucchi, y. ishikawa, v. eusebi, m. p. foschini * university of bologna, dept. of anatomic pathology, italy objective: sentinel lymph node (sn) examination is the current procedure to establish the status of axillary lymph nodes in breast cancer. to avoid a two step delayed surgical procedure, a reliable and quick method of sn evaluation is advocated. rapid immunohistochemical technique (uich) for keratin has been only recently proposed. aim of this study is to apply keratin uich in frozen sections (fss) of sns. method: a consecutive series of 231(series a) sn cases was studied at fs level followed by two sections stained with keratin uihc. all procedure requires 23 min. for comparison 131 consecutive cases (series b) of sn were studied with fs only. all residual tissue from both series was paraffin embedded (ps). results: series a: sns showed tumour involvement in 41 cases (17.7 %). in only 8 cases (3.75 %), ps sections evidenced additional neoplastic cells (micometastases) not seen with keratin uihc, that led to a delayed axillary dissection. series b: ps sections revealed metastatic deposits (micrometastases) not seen in fs in 6.1 % of cases. conclusion: fs coupled with keratin uhic of all the entire lymph node accurately evidences carcinoma cells in sns. high concordance of 6 her2 in situ hybridization methods with abbott fish j. boers * , l. krol, c. netjes, h. meeuwissen, c. prinsen, c. van krimpen, e. van der logt, j. bart, e. schuuring * isala klinieken, dept. of pathlogy, zwolle, netherlands objective: we conducted a comprehensive concordance study of 6 ish methods with abbott fish in a large series of breast carcinomas. method: tissue micro arrays (tma) were constructed by taking 3 tissue-cores from praffin blocks of 402 primary breast carcinomas. up to 384 cases were analyzable in 7 ish assays. scoring was performed by two independent observers without knowledge of the other ish data according the asco-guidelines for her2-testing. cases were considered positive when the ratio was ≥2.0. discordant cases were reviewed and scores were reassigned on consensus of opinion. concordance and cohen's kappa score were calculated in relation to fish, abbott. results: in 372 cases analyzable with abbott her2 fish, 12.1 % were her2-positive. concordances (kappa-scores) of the 6 other assays were: dako fish 98.1 % (0.90), dako duocish 97.2 % (10.4 %), zytovision fish 99.1 % (0.96), zytovision duocish 99.1 % (0.96), single probe sish ventana 98.9 % (0.95), dual probe sish ventana 99.4 % (0.97). conclusion: conclusion: concordance of 6 her2 ish assays with abbott fish were shown to be 97.2 % or higher. in this study, dako assays had a lower kappa score with abbott fish than ventana or zytovision assays. which is the best method to measure multiple breast cancer? m. boros * , c. marian, o. pop, s. stolnicu * umph targu mures, pathology, romania the size of the breast tumor is relevant in a patient s management also affecting the prognosis. for unifocal lesions, tumor staging depends on the maximum diameter of the tumor, whereas in multiple lesions, this issue is not standardized. the aim of this paper is to study which is the best method in the assessment of the tumor size in multiple invazive carcinomas (multifocal and multicentric) in correlation with the lymph node metastases developement. two different assessments of the tumor size (diameter of the largest focus=ld, and combined, aggregate diameter of all the foci=ad) were used in 418 primary invasive breast lesions (91 multiple, 327 unifocal) and compared with the nodal status (chi-test). the use of combined tumor focus upstaged 23 (25.27 %) patients with multiple tumors (16 upstaged from pt1 to pt2 and 7 from pt2 to pt3). there was no difference in nodal positivity based on pt status between ld and ad. we observed a statistically significant difference in the mean diameter of the largest focus between the unifocal and multifocal group (31,47 vs 39,67 mm) (p=0,0013). aggregate diameter in multiple breast cancer is not correlated with an increase of axillary metasases and should not be used for staging. central nervous system metastases in women with invasive breast carcinoma, not otherwise specified, are associated with estrogen receptor status e. cambruzzi * , a. g. reginatto, c. g. zettler, k. l. pêgas, v. grings, j. m. venites, c. a. coelho * ulbra and ufrgs, dept. of pathology, porto alegre, brazil objective: central nervous system metastases (cnsm) from breast cancer (bc) are relatively common and can present as the first site of disease progression. lymph node status and tumor size are regarded as important prognostic indicators for disease-free and overall survival in bc. the aim of this study was to investigate prognostic/predictive pathological data in bc that could define a high-risk group to develop cnsm. method: the authors evaluated 97 female patients with invasive breast carcinoma, not otherwise specified, previously submitted to setorectomy/mastectomy, in order to determine lymph node status, tumor size, histologic grade, estrogen receptor status (er) and immunoexpression of her2/neu. of these cases, 10 patients developed cnsm. results: the patients who developed cnsm were younger (median age 54.7±8.152 years/p=0.251) and more likely to have t2n2 disease than patients with no cnsm. the presence of encephalic disease was associated with er (p= 0.031). lymph node status (p = 0.84), tumor size (p = 0.339), histologic grade (p=0.933), and her2/neu expression (p=0.31) were not significant risk factors. conclusion: although the literature data discriminate that her2/neu overexpression in bc is related with cnsm, the authors suggests that these lesions can be related to er too. efforts to determine other risk factor for development of cnsm may be warranted. androgen receptors and sex hormone serum levels in breast carcinoma: study of the ordet cohort l. cimetti * , s. sieri, a. m. chiaravalli, n. sahnane, f. sessa, c. riva, c. capella * university of insubria and ospedale di circolo, varese, italy objective: androgens and androgen receptors (ar) are involved in breast cancer (bc) pathogenesis. high testosterone serum levels increase the risk of developing mainly er + bc, especially after menopause, although androgen role in tumor progression is not clearly elucidated. method: correlations between serum sex hormones and clinico-pathological features of 131 bc arisen among 10,786 women previously recruited for ordet study were investigated. prediagnostic estradiol, testosterone (free/total) and shbg serum levels were available. immunohistochemistry for ar was evaluated along with er, pr, her-2 and mib-1. results: ar + was found in 90.8 % of bc. higher estradiol (p=0.0001), free testosterone (p=0.02) and shbg (p=0.02) were seen in premenopausal patients. in dead patients higher free testosterone was observed (p=0.0003). no correlation was found for hormone levels vs stage, histotype and grade. higher shbg was seen in pr-rich tumours (p=0.02). her-2+/er-cases showed a trend for a higher total testosterone (p=0.18) and shbg (p=0.2). testosterone, estradiol and shbg were similar in ar + and ar-tumors. among triple negative, ar + tumours showed higher free testosterone (p =0.04). bc with few ar + cells showed higher total testosterone (p=0.0004) and a worse outcome (p=0.29). conclusion: our results confirm the role of ar in bc and suggest the androgen involvement in tumour progression. mitotic count in metastatic breast carcinoma to lymph nodes: preoperative study a. córdoba * , l. gomez, f. vicente, i. amat, c. llanos, d. guerrero * hospital navarra, ap, pamplona, spain objective: axillary lymph node metastasis (alnm) is one of the most important prognostic factors in breast cancer. the reasons why tumours are capable to result in axillary metastasis remain unclear. the evaluation risk of alnm would improve the treatment planning. we study the metastatic breast carcinoma to the lymph node to obtain information about the metastatic risk. method: we study 65 patients with metastatic breast carcinoma to the lymph node diagnosed preoperatively by needle biopsy. we study the mitotic average (×10 hpf), metastasis size, positive lymph nodes, total lymph node studied, lymph node ratio (nº of positive node/total nodes nº), primary tumour size and grade. results: all the cases with high mitotic count were associated with macrometastasis. we didn't find any relation between the mitotic rate or the metastatic size with the number of lymph node affected, and the lymph node ratio. breast carcinomas g1, t1, <6 mitosis/hpf lymph nodes showed 25-50 % of positive lymph nodes. conclusion: we tried to obtain information from the metastatic breast carcinoma to the lymph node to predict the axillary status. in our cases we couldn't predict the lymph node involvement based in the tumour size, grade, metastatic size, and metastatic mitotic rate. the preoperative study of a breast tumour and their lymph node metastasis don't allow predicting the lymph node status in our series. ps-17-020 rassf1a hypermethylation is associated to the presence of tumoral cells detected by one-step nucleic acid amplification (osna) a. córdoba * , f. vicente, n. perez janices, j. perez vizcaino, e. gochi, n. torrea, d. guerrero * hospital navarra, ap, pamplona, spain objective: one-step nucleic acid amplification (osna) is used in routine clinical use for sentinel lymph node biopsy (slnb). it consists of the molecular quantitation of a tumoral marker (cytokeratin-ck 19 mrna). gene hypermethylation is one of the most common mechanisms of inactivation of suppressor genes. rassf1a gene, is a region frequently hypermethylated in breast cancer. method: 51 patients with breast cancer were included in the study, and a total of 87 lymph nodes were analysed. results: 41 %, 33.3 % and 21.6 % of the tumours were of low, intermediate and high-grade, respectively. in 40.8 % of the cases there are not tumoral cells, in contrast to 12.2 %, 4.1 %, 14.3 %, 28.6 % that showed low number of cells/itc (<250 copies/sample), micro-(<5000 copies/sample) and macro-mtx, respectively. there is a very clear association between rassf1a hypermethylation and the presence of tumoral cells (p= 0.004), being more frequent in macro-mtx compared to the rest of groups (p=0.007). rassf1a hypermethylation is also correlated to unfavorable histologic grade in the tumour (p=0.025) and lymph node involvement at the diagnosis (0.001). conclusion: the analysis of rassf1a hypermethylation could provide additional information to osna to detect tumoral cells in lymph nodes. the spare tissue material derived from osna could be good material to consider new molecular studies. results: of these, 15 patients (88,23 %) had palpable tumor at presentation, 12 (70,58 %) with palpable axillary adenopathy. in 10 cases (58,82 %) the tumor diameter was over 2 cm at the time of diagnosis. in 15 cases (88,23 %) the histological type was infiltrative ductal carcinoma, with 13 cases having high grade. most of the tumors expressed er and pr and were her-2 negative (none was triple negative). however, in 11 cases (64,70 %) the ki-67 index was more than 50 %. conclusion: young women with breast cancer have been shown to have a poorer prognosis because of the high grade and hormonal status. our study shows some similar aspects, but the hormonal status is totally different from the dates in the literature, revealing a possible better response to hormonal treatment and a better survival. immunohistochemical predictive markers for trastuzumab resistence a. cuesta diaz de rada * , e. honrado franco, m. baltasar moreira, f. m. izquierdo garcia * hospital de león, anatomía patológica, spain objective: her2 positivity defines a clinically challenging subgroup of patients with breast cancer with variable prognosis and response to therapy. the main aim of this study is to identify immunohistochemical markers to predict trastuzumab resistence. method: tumours from 57 patients with invasive ductal carcinoma, who were previously treated with trastuzumab, were included in a tissue microarray and stained for er, ki67, p53, cyclin d1, p16 and for her2 and sish (silver in situ hybridization). results: after 5 years of follow up, 80 % of patients with p53 negative tumours were disease free (p 0.029) and 90 % were alive (p 0,014) and the patients cyclin d1 positive, 90 % were alive (p 0.031) and 84 % were disease free (p 0.005). among her2 3+, 10 % did not amplify (polysomy 17). moreover, 12 % of treated patients were her2 0−2+ and did not amplify by sish. conclusion: the best immunohistochemical markers to predict a good response to trastuzumab treatment were p53 negative and/or cyclin d1 positive. all her2 positive 3+ tumours should be confirmed by hybridization, since polysomy 17 is found in 10 %, and these tumours should not be treated with trastuzumab. variations in the immunostaining techniques could induce trastuzumab treatment to patients with her2 non amplified tumours. ps-17-023 cd99 expression in breast carcinoma p. czapiewski * , j. szade, a. zaczek, m. welnicka-jaskiewicz, w. biernat * medical university of gdansk, dept. of pathology, poland objective: cd99 is a membraneous protein that is expressed widely among various soft tissue tumors. there is growing evidence that its expression in some carcinomas correlates with epithelial to mesenchymal transition (emt) and is a poor prognostic factor. its importance in the breast carcinoma remains unsettled. method: the analysis was performed on breast cancer samples from 122 patients. tumors were graded histologically according to the nottingham system. cd99 expression was scored by grading system used for her-2. only cases showing grade (3+) were regarded as positive. additionally, expression of estrogen (er) and progesterone receptor (pr), her-2, e-cadherin, vimentin, twist, ki-67, c-myc, cyclin d1 and topoisomerase 2alfa was performed. expression of the cd99 was correlated with all these markers and with clinical outcome. results: expression of cd99 was observed in 11 patients and correlated significantly with negative pr status (p=0,01), higher histological grade (p=0,05), positive twist (p=0,04) and topoisomerase2alfa expression (p=0,04). there was also a trend toward higher frequency of cyclin d1 positivity (p= 0.09). no impact on prognosis for cd99 expression was found. conclusion: expression of cd99 correlates with high histological grade, negative progesterone receptor status and expression of certain emt and proliferation markers, but it does not influence prognosis in breast carcinoma. primary plasmacytoma of the breast: a case report l. de carvalho * , g. g. monteiro, l. casagrande, t. ricci, t. lebrão, v. tarricone, p. dinamarco * centro universitário lusíada, dept. of pathology, santos, brazil objective: extramedullary plasmacytomas of the breast are extremely rare, especially those that are not associated with multiple myeloma and they can mimic mammary carcinoma. method: case report:69-year-old woman presented a palpable mass in the left breast previously diagnosed as a lobular carcinoma by core biopsy performed in other service. in our institution a conservative surgery with lymph node dissection was proposed. results: frozen section was required and the gross examination showed a 4.0×4.0×3.0 cm firm-elastic nodule with surgical margins free of tumor and the microscopic examination showed tumor cells with abundant cytoplasm with hyaline appearance. the paraffin sections showed a proliferation of plasma cells with moderate atypia. the immunohistochemical study confirmed the diagnosis of plasmacytoma. there was no axillary lymph node involvement. there were no bone lesions in the additional investigation. conclusion: primary plasmacytomas of the breast are extremely rare and have to be included in the differential diagnosis with breast carcinomas especially in material of core biopsy. sentinel lymph node in breast cancer: form morphology to molecular examination l. di tommaso * , b. fernandes, b. fiamengo, c. navligu, p. spaggiari, s. manara, c. a. garcia etienne, g. masci, a. testori, c. tinterri, m. roncalli * irccs istituto clinico humanitas, dept. of pathology, rozzano, italy objective: sentinel lymph node (sln) examination is a standard in breast cancer treatment. it can be performed on formalin-fixed paraffin-embedded material (ffpe) or on frozen sections (fs). ffpe or fs suffer two drawbacks: 1) partial examination; 2) operator's dependence. to avoid these limitations, a molecular technique (osna) targeted to quantify a tumoral fingerprint (ck19), has been introduced. our aim is to compare the performance of ffpe, fs and osna. ps-17-026 her2 assessment in invasive breast cancer using ihc and fish: results from 7479 consecutive cases p. drev * , b. gazic, j. contreras * inst. of oncology, dept. of pathology, ljubljana, slovenia objective: her2 in invasive breast cancer (ibc) should be assessed according to recommended algorithms employing ihc as screening tool and ish only in equivocal cases. therefore there is little data on possible missdiagnosis in discordant cases. method: her2 was assessed by both ihc and fish in 7479 consecutive ibc. distribution of ihc and fish scores, incidence of her2+ ibc, concordance and level of amplification in discordant cases were analysed. results: ihc distribution: neg(0) 49.6 %, neg(1+) 27.0 %, equivocal(2+) 11.3 %, pos(3+) 12.1 %. fish distribution: nonamplified 84.6 %, amplified 13.6 %, equivocal 1.8 % of which in 42.1 % ratio was ≥2.0. 15.0 % ibc were her2+ and 0.7 % were double-equivocal. 14.4 % of equivocal(2+) were amplified. discordance was infrequent (1.2 %) (p< 0.0001): among neg(0) and neg(1+) 0,3 % and 1,6 % were amplified, while among pos(3+) 4.7 % were nonamplified. 64 ihc negative tumors, representing 5.7 % of all her2+, were amplified, however in 89.1 % ratio was ≤4.0. conclusion: 15 % ibc are her2+. application of two standardised methods results in excellent concordance and enables detection of all her2+ ibc, while recommended algorithms lead to missdiagnosis in 6 % of her2+ ibc, but in these amplification is low-level. case report: unusual breast cystic lesion in a 46-yearold female p. farrajota * , p. cusati, d. esteves, c. dias, c. carvalho, g. falconieri * centro hospitalar do porto, dipt. do anatomia patologica, portugal objective: papillary carcinoma of the female breast may exhibit a broad phenotype. we present an unusual case of a pseudoencapsulated invasive papillary carcinoma featuring transitional cell features. method: a specimen of internal right female breast biquadrantectomy was routinely processed and a panel of antibodies applied on paraffin tissue section. results: grossly, the specimen showed a 12.5 cm cystic lesion with fibrino-hematic material and multiple pinkish-grey papillary structures, the largest measuring 4 cm. the histological examination revealed a malignant epithelial papillary proliferation reminiscent of transitional cell carcinoma, with high nuclear grade, frequent mitotic figures, multiple areas of invasion and rare necrotic foci. tumor cells were immunoreactive for p63, 34βe12 and ki-67 (90 %) and negative for other stains including her2/neu, estrogen and progesterone receptors. our differential diagnosis included a metastasis from a primary urothelial carcinoma, an intraductal papillary lesion, a metaplastic carcinoma and a papillary adnexal neoplasm, all reasonably excluded after careful clinico-pathologic evaluation. conclusion: metastatic involvement of the breast is uncommon but it should be considered if tumor phenotype is inconsistent within usual or "special type" breast carcinoma. yet, some rare primary misleading lesions are difficult to recognize. ps-17-028 volume measurement of female sprague-dawley mammary tumors induced by n-methyl-n-nitrosourea: comparing ultrasonography and caliper a. faustino-rocha * , c. teixeira-guedes, j. pinho-oliveira, r. soares-maia, r. arantes-rodrigues, b. colaço, r. ferreira, p. oliveira, m. ginja * utad, dept. of veterinary sciences, vila real, portugal objective: n-methyl-n-nitrosourea (mnu) is a chemical carcinogen frequently used to induce mammary tumors in female rats, which experimental evaluation requires the monitoring of tumor's volume. for this purpose several methods are described, namely: caliper and ultrasonography measurement. the aim of this work was to compare data obtained by caliper and ultrasonography. method: twelve female sprague-dawley rats with 187.7± 14.3 g body weight were intraperitoneally injected with mnu (50 mg/kg) at 50 days of age. thirty-six weeks after mnu administration forty-one tumors volume was determined by caliper (vito®) and ultrasonography (logiqp6®, general electric healthcare) measurement. the tumor volume (v) was calculated according to the following formula v=π.〖s_1^2〗^.s_2⁄12, being s_(1) and s_2 the tumor diameters (s_(1)<s_(2)). results: the means tumor caliper and ultrasonography measures were 4.116±5.801 cm3 and 2.578±4.302 cm3, respectively. the icc for scoring reproducibility was 0.75 (95 % confidence interval, 0.57 to 0.86) and paired-sample t-test was significant (p<0.01). conclusion: usually mammary tumors in rat are measured by caliper. however, our results showed that there are volume differences between both methods studied. ultrasonographic method is more accurate and more precise than caliper. therefore, we recommend the use of ultrasonography to rat tumor evaluation. objective: there is a significant percentage of patients with breast cancer and her2 receptor overexpression that do not respond to trastuzumab-directed therapy. the aim of this paper is to study the alterations of one of the her2 signalling pathways related to trastuzumab response in breast cancer. method: the study comprised 127 cases of breast carcinoma in different stages of evolution with her2 overexpression (herceptest 3+) or gene amplification (fish). all patients were treated with trastuzumab. the assessment of pten and pakt expression was based on staining intensity and percentage of stained cells. we also determined the molecular alterations in pik3ca. results: our series showed a statistically significant association between a positive expression of pten and the absence of pik3ca mutations with better overall survival from treatment onset with trastuzumab (p=0.022 and p= 0.003, respectively). the association with the expression of pakt was statistically significant if survival is evaluated from the pathological diagnosis of the tumor (p=0.043), pakt positive tumors had worse survival. objective: expression of mammaglobin a (mg-a) in breast cancer has been reported to be of prognostic value and to be associated to hormone receptor-positivity. mg-a is also useful in the diagnosis of metastatic breast cancer. the present study evaluates the differential mg-a expression in two subsets of primary breast cancer (metastasizing vs. non-metastasizing) and in their correspondent metastases. method: immunohistochemical analysis of mg-a (clone 304-1a5) was performed in 55 non-metastasizing primary breast tumors and 19 paired primary and metastatic breast cancers. a percentage of 1 % of stained tumor cells was considered positive. the expression of mg-a in the different groups, together with other clinicopathologic factors (grade, size, hormone receptors, her2, etc.) was analyzed by t-test. results: expression of mg-a was significantly higher in estrogen receptor-positive (er+) tumors (p<0.003). a trend was noted for er + metastasizing primary breast cancers to show a higher expression of mg-a, compared to those nonmetastasizing (p=0.064). no significant differences were noted in the expression of mg-a between metastasizing primary tumors and their correspondent metastases, with a good correlation (p=0.033). conclusion: these results suggest that mg-a might be of prognostic value in determining a subset of er + breast cancers prone to progression. larger series are needed to confirm these observations. concordance of st. gallen intrinsic subtypes in core biopsies and related surgical specimens of breast cancer c. m. focke * , t. decker * dietrich bonhoeffer klinikum, abt. pathologie, neubrandenburg, germany objective: to evaluate the relevance of intrinsic breast cancer subtyping (ibcs) on core biopsies (cb). results: 39384 samples were tested in 2009. score 0 was found in 45.43 % cases, 1+ in 28.86 %, 2+ in 12.97 %, 3+ in 14.73 %. the amplification was revealed in 32.12 % of 2+ cases, 15.82 % tumors were 3+/fish+. 43485 cases were tested in 2011: score 0 −38.42 %, 1+ 30.61 %, 2+ 15.65 %, 3+ 15.32 %. the amplification was seen in 28.17 % 2+ cases and 18.68 % tumors became 3+/fish+. there was no significant distinction using 10 % and 30 % cells score system for 3+ tumors, whereas for groups 1+ and 2+ it was. conclusion: asco/cap scoring criteria developed in 2007 have no effect on her2-positive tumors detection but increases number of cases with equivocal result, what require retesting with ish. microvessel density in triple-negative breast cancer: characteristics and prognostic implications h. gobbi * , m. de brot, r. rocha, f. soares * ufmg, dept. of anatomic pathology, belo horizonte, brazil objective: we assessed topography and characteristics of blood vessels in triple-negative breast cancer (tnc) and examined their association(s) with clinicopathological features, basal-like phenotype and influence on patient prognosis. method: blood vessels were assessed in tissue sections from 133 tncs stained for cd34. slides were scanned using scanscope® xt and analysed with aperio image-scopetm, microvascular density v1 algorithm. microvessel density (mvd) was assessed in three areas: intratumoural (it), peripheral tumoural (pp) and peritumoural (pt), and correlated with clinicopathological features and prognosis. results: the majority of blood vessels were located largely at the peripheral tumoural and peritumoural areas. tumours with higher total mvd and pt-mvd were significantly associated with the presence of recurrence (p = 0.05; 0.01), distant metastasis (p = 0.01; 0.005), hematogenous metastasis (p= 0.006; 0.01); and shorter overall survival (os; p= 0.006; 0.01) and disease free interval (dfi; p=0.04; 0.09). high pt-mvd correlated with a higher stage at diagnosis (iii and iv; p= 0.02), lymph node (ln) metastasis (p=0.04), basallike phenotype (p = 0.01), high histologic grade (p = 0.05), presence of distant metastasis (p = 0.02), and shorter os (p =0.01). conclusion: blood vessels are mainly located at the peritumoural and peripheral areas in triple negative breast cancers and seem to have a major role in tumour progression by representing means for disease dissemination. expression of cd44 and cd24 in primary ductal invasive carcinoma of breast and metastatic lymph nodes m. gudadze * , k. kankava, t. javakhishvili, m. sarishvili, g. burkadze * central university clinic, dept. of pathological anatomy, tbilisi, georgian, republic of georgia objective: cancer stem cells are believed to have certain role in progression, metastatic potential and outcome of ductal invasive carcinoma of breast. method: the archived materials of 2008-2012 years from the department of pathological anatomy, n. kipshidze central university clinic were used for the research. 196 cases of ductal invasive carcinoma were chosen for the study. the cd44, cd24 immunohistochemistry (biocare medical) was used for staining. evaluation was performed using spss software. results: the results showed: 68.5 % of grade ii cases and 90.5 % of grade iii cases were lymph node positive. cd24 positivity in grade ii tumors was seen in 29.6 % of primary site and 45.9 % of metastasis; in grade iii tumors-50 % of primary site and 55.2 % of metastasis. cd44 positive cases in grade ii-48.1 % for primary tumor and 27 % in metastasis, for grade iii-73.8 % for primary tumor and 39.4 % for metastasis. the results show that cd44 positivity is significantly less frequently seen in metastasis than in primary tumor; cd24 positivity is more frequently seen in metastasis for grade ii cases. this indicates, that cells with stem cell phenotype decrease in metastasis reflecting heterogeneity between primary and metastatic sites, which should be taken into consideration for assessing progression and treatment. ps-17-035 solid neuroendocrine breast carcinoma: three case reports b. ince alkan * , s. yuksel, e. kadan, s. percinel, s. dizbay sak * ankara university, turkey objective: it seems that neuroendocrine breast carcinoma (nebc) is underrecognized in routine practice. 2003 world health organization classification of tumors of the breast defined nebc as a subtype of invasive mammary carcinoma in which >50 % of the tumor cells express neuroendocrine markers. three cases compatible with solid nebc are discussed with an emphasis on identifying features useful in recognition of this tumor type. results: the patients were 35, 55 and 57 years old women. all patients presented with a palpable mass, two in the right and one in the left breast. microscopically, infiltrating, solid cohesive nesting pattern with delicate sinusoidal vasculature or peliosis and papillary pattern were observed. the tumor cells were round or polygonal with abundant granular, eosinophilic cytoplasm. the nuclei had hyperchromatic/vesicular or "salt and pepper" chromatin. two cases had ductal carcinoma in situ component. all cases were positive for synaptophysin and chromogranin a in >50 % of tumor cells. while a positive status for estrogen receptor was detected in all cases, progesterone receptor was observed in two cases. none of the tumors displayed her2 overexpression. conclusion: morphological clues suggestive of nebc must be recognized for immunohistochemical confirmation for appropriate classification of this clinically distinct subtype of invasive breast carcinoma. objective: automation of her2 fish may improve her2 gene testing. the aim of our study was to evaluate an automated her2 fish assay for assessing the her2 genomic status. method: core biopsies of 100 invasive breast carcinomas were analysed in parallel using the manual pathvysion™ her-2 dna probe kit and the automated leica her2 fish system for the bond™ instrument. to assess intermethod agreement, concordance analysis was performed for various numerical and categorical parameters. results: carcinomas with all her2 immunohistochemical scores were included (0+: 20; 1+: 20; 2+: 30; 3+: 30). using either her2/cep 17 ratio >2.2 or ≥2.0 as criterion for her2 amplification, high levels of concordance were observed between automated and manual fish (concordance rate 96 %, k coefficient 0.92). high levels of inter-method agreement were also found for her2 copy number, cep17 copy number, her2/cep17 ratio, the percentage of carcinoma cells with her2/cep17 ratio >2.2, and the presence of her2 genetic heterogeneity, her2 clusters and cep17 polyploidy. conclusion: her2 testing using automated fish is feasible on breast carcinoma core biopsies. automated her2 fish using the leica her2 fish system for bond is an alternative to manual her2 fish in evaluating the her2 status of primary invasive breast carcinomas. myofibroblastoma of the breast: presentation of three cases e. kairi-vasilatou * , c. dastamani, a. tsagkas, a. paraskeva, a. kondi-pafiti * aretaieio nosokomeio university, dept. of histopathology, athens, greece objective: myofibroblastoma is a rare, usually solitary, benign spindle cell tumour composed of myofibroblasts. it affects both genders equally. method: three cases diagnosed in our laboratory are presented. results: two patients were postmenopausal females (53 and 75 years old) and one was male (55 years old). the tumour was 1,6 cm in greatest diameter in the first case, 7 cm in the second and 2 cm in the third. microscopically, all neoplasms were circumscribed and consisted of uniform, bland spindle cells separated by broad bands of hyalinized collagen. the neoplasmatic cells had abundant eosinophilic cytoplasm, oval nuclei and were arranged in fascicles. cellular atypia was found only in one case and mitoses were scarce. immunohistochemically, all neoplasms were strongly positive for vimentin, cd34 and bcl-2, while the expression of desmin and sma varied. s-100 protein was negative. the neoplasms of the female patients were strongly positive for er and negative for pgr. the stains for estrogen and androgen receptors were not carried out in the case of the male patient. conclusion: differential diagnosis can be complex. fibromatosis of the breast, nodular fasciitis, myoepithelioma, myofibrosarcoma, solitary fibrous tumor, inflammatory myofibroblastic tumour should be excluded based on the histological and immunohistochemical findings. method: using light-and transmission electron microscopy fragments of mammary gland tumors, received intraoperative, from 58 patients 26-82 years with infiltrative ductal breast cancer were studied. results: most of the mast cells were localized in the areas of infiltrative growth, at a background of lymphohistiocyte infiltration and in peripheral part of the tumor. intratumourous mast cells were totally degranulated. the mast cells with intact structure at a background of cells and tissue destruction were also in mammary gland. in destructive mast cells the nucleus with electron-dense circle and chain of granules were observed. on transmission electron microscopy their deformations and the decrease of density were revealed. the mast cells were of elongated of rounded shape with large nucleus and invaginations of karyolemma. the nucleus occupies almost completely the cell's area. it has a ring of large but pale granules. small protuberances were observed on the surface of the cell membrane. their number decreased in direct proportion to the size of must cells and their remoteness from microvessels. such protuberances are necessary for mast cells in moving. destructively changed cells with the signs of swelling, vacuolization and clasmatosis were frequent in breast cancer. objective: determination of eligibility of breast cancer patients for treatment with anti-angiogenic drugs has been always considered as a challenge for oncologists. each mutation in the genes of proliferation phase enhances the angiogenesis of tumor. we aimed to determine the effect of concurrent mutations of her-2 and tp53 on angiogenesis. method: 32 women affected by invasive ductal carcinoma (idc) sporadic breast cancer were included. immunohistochemical study was performed with her-2, tp53, ki-67 and annexinv markers. angiogenesis index was semiquantitatively calculated by mvd-cd34 technique. statistical associations between parameters were evaluated. results: prevalence of her-2 positive and tp53 positive cases were 21.4 % and 20.0 %, respectively. 6.5 % of patients showed concurrent mutation of these genes. concurrent mutation led to significant increases in both angiogenesis and proliferation and a significant decrease in apoptosis. there was no statistically significant association between concurrent mutation and tumor grade. conclusion: this study demonstrates that most of the tissue prognostic factors are poor in concurrent mutation of both genes. also, our study illustrates that the concurrent mutation correlates with a higher angiogenesis and thus an increased risk of recurrence. we can conclude that in priority setting for administration of anti-angiogenic agents, patients with concurrent mutations are more eligible. adenoid cystic carcinoma in male breast: a case report e. kimiloglu sahan * , u. karinoglu, a. akyildiz igdem, n. erdogan * taksim´s hospital, dept. of pathology, istanbul, turkey objective: adenoid cystic carcinoma of the breast is a rare variant of breast cancer that accounts for 0.1 % of all breast carcinomas and occurs commonly in women between the ages of 25 to 80. in the literature, only a few examples have been reported in men. it is well-differentiated tumour with favorable prognosis and generally presents as a painful breast mass. method: here, we are presenting a 60 years old male patient with a 13×8 mm diameter solid mass on left breast retroareolar region. on fine needle aspiration biopsy, there were 'atipical proliferating ductal epitelial groups'. then, hookwire localization and excision have been performed. microscopically, there were tubulary and cribriform islands composed of basaloid type cells with eosinophilic cytoplasm and myxoid material in the cribriform spaces at the center of the islands. we used immunohistochemical markers such as p 63, s 100, ck 7, cd 117, ck 14 and smooth muscle actin for differential diagnosis. results: the diagnosis was 'adenoid cystic carcinoma'. the margins of the tumor were positive, so radical mastectomy was performed for treatment. conclusion: because adenoid cystic carcinoma of the breast in male is a very rare example, we present our case here. comparison between the bond oracle her2 immunohistochemical system, the polyclonal her2 dako antibody and chromogenic in situ hybridization in breast carcinoma h. kourea * , v. tzelepii, i. nikolatou, p. ravazoula, v. zolota * university of patras, dept. of pathology, greece objective: the sensitivity and specificity of immunohistochemical (ihc) methods for her2 testing are very important given the therapeutic implications. this study compares the concordance between oracle and the her2 dako polyclonal antibody (her2), in breast carcinomas (bcs) that were equivocal (2+ or not evaluable/ne), by her2 staining, considering as gold standard the chromogenic in situ hybridization (cish). method: bcs (n=34), problematic by her2 staining (1−2/ 2+ or ne), and studied by cish in our institution, and 24 additional consecutive bcs were stained with her2 and oracle, and scored separately by three pathologists. consensus scoring for each ihc method and cish results were recorded. descriptive statistics and measurement of the cohen's kappa coefficient were performed. results: the overall agreement between the 2 tests in a 3×3 analysis shows a concordance in 62 % of cases (κ=0.405). among the cases studied by cish, equivocal were 26 and 11 cases, for her2 and oracle, respectively. seventeen her2 equivocal cases were negative with oracle and cish. in problematic cases, using cish as gold standard, the sensitivity, specificity, positive and negative predictive values for her2 and oracle were 100, 20.7, 17.9, 100 and 100, 72.4, 38.5, 100, respectively. conclusion: in problematic cases, oracle testing shows higher specificity and positive predictive value. stromal p53 and ki67 expressions of the mammary phyllodes tumors: are they the clues in determination of tumor grade? u. kucuk * , u. bayol, e. e. pala, s. cumurcu * tepecik training hospital, dept. of pathology, izmir, turkey objective: conventionally growth pattern, stromal overgrowth, stromal cellularity, stromal mitotic activity are the main parameters in grading of phyllodes tumors. recent studies revealed that p53 and ki67 expressions are both correlated with grade of phyllodes tumors of the breast. method: we searched for p53 and ki67 expression rates of benign and malignant phyllodes tumors in our archival data and correlated them with conventional parameters such as stromal cellularity and mitotic activity rates. 17 benign, 9 malignant phyllodes tumors were reevaluated as regards stromal cellularity (low/moderate and high), mitotic activity (low and high), p53 expression (low, moderate, high), ki67 expression (low and high) rates. statistical correlation amongst the whole parameters were searched with chi-square test. results: stromal cellularity, mitotic rate, p53 and ki67 expression rates were all closely correlated (p = 0.000-0.001) for benign and malignant histologic subgroups. ki67 expression was significantly correlated with histologic subgroups, stromal cellularity and mitotic rate (p=0.000-0.001). similarly p53 expression was correlated with histologic subgroups, stromal cellularity and mitotic rate (p= 0.000-0.002). conclusion: both ki67 and p53 expression rates are statistically significantly correlated with grade of mammary phyllodes tumors, so they can be used in determination of tumor grade, especially for differential diagnosis of benign and malignant ones. quantitative measure proliferative markers by image analysis of invasive ductal carcinoma a. kudaybergenova * , s. kalantarli * rscrct, dept. of immunohistochemistry, st. petersburg, russia objective: we analyzed a total number of tumor cells) in invasive ductal breast carcinoma, proliferative activity (% ki67-positive cells) and mitotic index (% phh3positive cells) to establish absolute quantity tumor cells per sq.mm of histological slide and relations this measure with proliferation and mitosis. method: the study included 46 patients diagnosed with breast carcinoma from baku oncology hospital during the 2001-05. after whole slide scanning by mirax scanner (3dhistech, budapest) of he, ki67 and phh3 stained slides we juxtaposed all three slides in one screen to the found area with maximal ki67 level in tumor and the corresponding area in other slides. morphometric analysis was performed using the pannoramic viewer software (3dhistech, budapest). for each case we analyzed a total number of tumor cells in1 mm2, number of ki67 and phh3 positive cells. results: mean tumor cells in 1 mm2 of histology slide was 4180+\−251 cells, median -3976 cells, 1185+\−166 (31 %) were positive for ki67 and 124+\−23 (3 %) were in mitosis. there was moderate correlation between cell density and ki67 r=0,44 (p=0,0032) and phh3 r=0,42 (p=0,0018). conclusion: by analysis of breast cancer, was established a total tumor cell per mm2 and main proliferative characteristics for invasive ductal carcinoma. objective: adenomyoepithelioma of the breast is a very rare benign tumor with biphasic proliferations of epithelial and myoepithelial elements. it is morphologically and immunohistochemically identical to epithelial-myoepithelial cell carcinoma of the salivary gland. the histologic criteria of malignant ame is not well-established because of the rarity of ame. we report a case of malignant adenomyoepithelioma in a 65-year-old woman. method: on ultrasonography, a well-marginated and lobulated solid mass was found at liq of the right breast.. an ultrasono-guided core biopsy was performed. the diagnosis of core biopsy was myoepithelial lesion. she subsequently underwent a wide local excision of the lesion. results: microscopically the tumor mass was composed of biphasic patterns which showed formation of tubules lined by an inner layer of ductal epithelial cells surrounded by proliferation of myoepithelial cells that also formed solid nests. but this tumor had foci of infiltrating margins and proliferation of spindle cell components. also noted are numerous mitotic figures and increased mylepithelial ki-67 positivity (10-15 % of tumor cells). conclusion: breast lesions which have predominently myoepithelial cells can be divided into myoepithelial hyperplasia, adenomyoepithelioma and malignant adenomyoepithelioma. tavassoli divied ame into tubular, papillary, and solid subtypes. the criteria of malignant ame is not wellestablished but some criterias can apply to make a diagnosis of malignant ame. objective: neoadjuvant chemotherapy (nact) is available for patients with breast carcinoma. however, resistance to chemotherapy is still a main cause of mortality. method: differentially expressed genes were identified from previously published studies that examined chemoresistant and chemoresponsive cell lines or patients with breast carcinoma. the expression of 14 selected gene products was assessed in tissue microarray slides comprising 75 post-nact resection specimens from breast carcinoma patients using immunohistochemistry, and analyzed according to the molecular subtype and residual cancer burden (rcb) grade. results: most cases were positive for abcb1 (97.7 %) and myc (96.0 %), but negative for top2a (100 %). rcb-ii cases expressed much higher levels of muc1 (p=0.011) and clu (p=0.021) than rcb-iii cases. positive expression for calr (p=0.002) and lgals3 (p=0.018) was observed more often in triple negative types than in luminal types, and cytoplasmic cdkn1b expression was observed more often in luminal types (p=0.014). conclusion: positive expression of abcb1 and myc and negative expression of top2a in the residual carcinoma after nact implies general resistance to nact. expression of calr, lglas3 and cytoplasmic cdkn1b may be associated with resistance depending on the subtype. expression of muc1 and clu can be used to predict the rcb grade or response to nact. primary and metastatic melanoma of the breast-review of 4 cases i. liepniece-karele * , l. osipova, m. sperga, s. isajevs, a. grjunbergs, j. eglitis * riga ecuh, dept. of pathology, latvia objective: primary breast melanoma is a very rare tumour accounting for <5 % of all malignant melanomas. the malignant melanoma can be with different manifestation in the breast (primary breast tissue or primary breast skin melanoma as well as metastatic melanoma). method: in this study 4 cases of breast melanoma were identified from our records of the past 3 years (0.41 % of all breast cancer cases). a histological and an immunohistochemical (ihc) study was performed using antibodies against ckae1/3, hmb-45 and melan-a on both the biopsy and operation material. results: obtained results showed that in 3 cases pigmented, epitheloid and spindle cell melanoma, but in one case epitheloid cell amelanotic melanoma was found. by ihc the melanoma cells expressed hmb45 in three cases, melan-a and s100 in all cases. no expression of ckae1/3 was observed. conclusion: careful histological and immunohistochemical examination of malignant tumour is essential for adequate diagnosis, follow-up and treatment of breast melanoma. stem cell expansion in ductal carcinoma in situ of breast c. lopes * , a. paula, o. marques, a. rosa, a. rema, f. carvalho * icbas, dept. of pathology, porto, portugal objective: the aim is to study the ability of a cell marker panel -aldh1, cd44 and ki67to identify breast stem cells in no malignant and ductal carcinoma in situ. method: double-color triple-immunohistochemistry -to aldh1, cd44 and ki 67 -was done in 169 paraffin embedded tissue specimens from 111 patients arrayed in tissue microarray blocks. statistical was done using chisquare probability test: differences were considered significant when p<0.05. results: significantly higher immunoreactivity was seen in dcis than in benign lesions of breast (p<0.01) with used markers. in a total of 169 specimens, cd44+/aldh1+/ ki67-cells were identied in 110 cases. the distribution was as follows: dcis (79/57); fibroadenoma (45/29), atypical hyperplasia (23/13); other benign lesions ( objective: data from in vitro and clinical studies suggest that casr expression can be associated with the development of bone metastases. most probably casr stimulates production and secretion of pthrp via egfr pathway. the aim of our study was to assess expression of casr in the primary breast cancer and correlate it with the risk of bone metastases. method: we have analysed 170 patients with the breast cancer. bone metastases were diagnosed in 102 cases, 68 patients died without skeletal involvement (control group). casr expression was assessed in primary tumors using tissue microarray (tma) and immunohistochemical technique (polyclonal antibody pierce bio. pa1-37213). to evaluate cytoplasmatic casr expression we have used 0-1 point scale in which 1 was defined as uniformly strong or medium staining in more than 50 % of tumor cells. results: strong or medium staining in more than 50 % was identified in most studied cases, however it was more predominant in patients diagnosed with bone metastases than in the control group (93,14 % vs 83,82 %), p=0,053. conclusion: expression of casr is common in primary tumors of patients with disseminated breast cancer irrespectively of the metastatic site. however, the patients with bone involvement have higher rate of expression, that is borderline significant statistically. as a result, a group of patients with very high risk of bone dissemination might be separated. bcl2 expression is associated with centromere 17 alterations in luminal b breast cancer a. matsionis * , i. pavlenko, a. petrov * rostov refional institute, dept. of experimental pathology, rostov regional institute objective: bcl2 is an important established prognostic parameter in human breast cancer (bc) and chromosome 17 centromere (cep17) copy number is proposed to be the same too. we evaluated bcl2 expression in different bc's molecular subtypes in relation to increased cep17 level (cep17>3 per nucleus). method: immunohistochemistry for bcl2 assessment and fluorescence in situ hybridization for detection of cep17 alterations were used. statistical analysis was performed with fisher's exact test. results: a total of 226 cases of female invasive bc's were analyzed (2010) (2011) . the tumour subtypes were as follows: luminal a (er/pr+, her2/neu-, ki67<14 %) -118; luminal b (er/pr+, her2/neu-, ki67>14 % or er/pr+, her2/neu+) -62; her2/neu+(er/pr-, her2/neu+) -24; triple-negative -20. in our study group, bcl2-cases were preferentially er-in agreement with previous reports. however, 20,5 % tumours of luminal b subtype were bcl2-too and all of them had increased cep17 copy number (p= 0,0011, rφ=0,57). we therefore hypothesized that not only er had influenced bcl2 in luminal b bc but cep17 alterations also. conclusion: centromere 17 alterations are associated to bcl2 downregulation in luminal b bc's. the mechanisms responsible for that remain to be established but the underlying cause could be promoter methylation or transcriptional repression. outcome of excision of radial scar diagnosed on core biopsy: a single centre analysis f. menezes * , m. caldas, n. coimbra, c. leal * ipo porto, dept. de anatomia patologica, portugal objective: radial scar (rs) is a sclerosing lesion of the breast which may be associated with a spectrum of epithelial proliferative lesions and carcinoma. it is frequently subject to biopsy when presenting as a mammographic abnormality. the need to excise rs diagnosed on core biopsy (cb) remains controversial. the aim of our study is to determine the frequency of upgrade in diagnosis after excision of rs. method: a retrospective study of rs diagnosed on cb in our department between 01/01/2000 and 31/12/2011 was performed. results: 117 cases were retrieved, all women, with a median age of 53 years. of these, 101 pairs of cb/resection specimens were obtained. cb diagnosis was rs without atypia in 79 cases, rs with atypia in 19 cases and rs with in situ carcinoma in 3 cases. after excision, 21/79 (26,6 %) cases diagnosed as rs without atypia were upgraded: 17 to atypia, 3 to in situ carcinoma and 1 to invasive carcinoma. of those cases diagnosed on cb as rs with atypia, 9/19 (47, 4 %) were upgraded: 8 to in situ carcinoma and 1 to invasive carcinoma. conclusion: our results support the excision of all lesions diagnosed on biopsy as rs. *fm and mc are joint first authors. outcome of excision of papillary lesions diagnosed on core biopsy: a single centre analysis f. menezes * , m. caldas, n. coimbra, c. leal * ipo porto, dept. de anatomia patologica, portugal objective: papillary lesions of the breast (plb) comprehend a spectrum of entities with different morphologies and malignancy risk, which present a diagnosis challenge on core biopsy (cb). although diagnostic accuracy has improved with immunohistochemistry, the need to excise benign plb remains controversial. the aim of our study is to determine cb diagnosis accuracy in plb, and subsequent need to excise all lesions. method: a retrospective study of plb diagnosed by cb in our department between 01/01/2000 and 31/12/2011 was performed. results: 96 cases were identified, with a median age of 59 years. cb diagnosis was benign in 66 cases, some kind of atypia found in 10, and malignant lesions ('in situ' and encysted/invasive types of carcinoma) in 20. surgical excision was performed on 70 cases, including all cases with carcinoma. following excision, 6/42 and 4/42 benign cb results were respectively upgraded to atypical and malignant; 3/8 with atypia were upgraded to malignant. overall, 14 % (7/50) of benign and atypical plb cb diagnosis therefore missed malignancy. conclusion: our results show that, despite good correlation between cb and excision diagnosis, some cases of carcinoma are missed on cb, so the excision of all plb remains advisable. *fm and mc are joint first authors. myoepithelial carcinoma of the breast: a case report i. michalopoulou manoloutsiou * , b. christoforidou, p. xirou, v. bostani, e. goupou, i. themeli, f. patakiouta * theagenion hospital thessaloniki, dept. of pathology, greece objective: myoepithelial carcinoma of the breast is an extremely rare tumor, composed purely of myoepithelial cells, predominantly spindle, with identifiable mitotic activity. method: we report a case of a 50 year-old female patient with a palpable, well demarcated lump in her left breast, measuring 3,5 cm in its maximal diameter. surgical excision and axillary lymph node dissection were performed. results: histologically, the tumor displayed an infiltrating growth pattern and consisted of spindle cells, that appear to emanate from myoepithelial cells of ductules entrapped in the center of the lesion. mitotic activity did not exceed 5 mitotic figures/10 hpf. immunohistochemically, the tumor cells revealed positivity for p63, cytokeratin 5/6, cytokeratin 34βε12, smooth muscle actin, cd10 and s100 protein, whereas they were negative for desmin, cytokeratin 7, cd34, hmb45, estrogen, progesterone receptors and her-2 oncoprotein. approximatelly 25 % of tumor cells showed nuclear positivity for mib-1/ki-67. all axillary lymph nodes were free of metastases. conclusion: myoepithelial carcinoma of the breast is a potentially highly aggressive neoplasm and its differential diagnosis is fairly broad, including metaplastic spindle cell carcinoma and a variety of myofibroblastic lesions. objective: we investigated the differential expression of several biological markers between primary invasive breast carcinomas and their paired lymph node metastasis analyzing separately epithelial and stromal components. method: representative samples of 42 idc and paired compromised lymph nodes were arrayed in a tma and 15 selected markers: hormonal receptors, her-1, proliferation (p53, pakt, pmtor, tgfβ1) , motility (cd9 and cxcr) and basal markers (ck5, ck14, c-kit) were evaluated by ic. results: in the primary tumor, p53, mib-1, tgfβ1, cd9 and cxcr4 were more expressed in epithelial cells (p< 0.05), while pakt, pmtor, c-myc and c-kit showed a similar frequency in both components. hormone receptors, her-1/her-2 and cytokeratins were not expressed in stromal cells. the proliferative biomarkers were concordant in the epithelial component. cd9 frequency was similar but cytoplasmic cxcr4 as opposite to nuclear was predominantly expressed in lymph nodes (p=0.008). stromal cells from lymph node showed a reduced frequency of cd9 (p= 0.029) and c-myc (p=0.003) when compared to the stromal component of primary tumors. conclusion: epithelial cxcr4 expression may facilitate lymph node metastasis whereas the low frequency of cmyc and cd9 in the lymph node stromal component indicated decreased proliferation enhanced motility of stromal cells in this site. expression of hypoxia-inducible factor-1a and associations with vascular endothelial growth factor expression, high microvessel density and features of aggressive tumors in african breast cancer h. nalwoga * , j. b. arnes, h. wabinga, l. a. akslen * university of bergen, the gade institute, norway objective: breast cancer in africans is reported to have poor clinical outcome. whereas hypoxia-inducible factor-1α (hif-1α) expression has been linked to treatment failure and poor prognosis in breast cancer, there is a lack of reports about hif-1α expression in africans. the aim of this study was to evaluate hif-1α expression in relation to vascular endothelial growth factor (vegf) expression, angiogenesis, and other tumor characteristics in an african population. method: in total, we analyzed 192 breast cancers by immunohistochemical staining. we determined microvessel density (mvd), proliferating microvessel density (pmvd), and vascular proliferation index (vpi) in the most vascularized areas as well as expression of hif-1α and other biomarkers using tissue microarrays. results: expression of hif-1α (in 128/182 tumors; 70 %) was associated with vegf expression (p<0.0005), mvd (p =0.037), high tumor grade (p=0.001), high ki-67 proliferative rate (p<0.0005), and p53 expression (p=0.032). conclusion: there is a high expression of hif-1α in this series of breast cancer which is strongly associated with vegf expression and increased mvd. more studies are required to assess the therapeutic implications of hif-1α expression in this population. the patient underwent lumpectomy. the gross specimen had a tan grey firm nodule of 25×15×15 mm. histological examination revealed a proliferation made of signet ring cell and glandular structures, with islands of goblet cells. immunohistochemistry revealed strong positive staining with ck7 and ck20, and a sparse positive staining with synaptophysin and chromogranin.er, pr and her2 were negative. the patient underwent appendicectomy with a final diagnosis of breast localization of a primary occult appendiceal gcc. conclusion: the differential diagnosis between primary carcinoid tumor of the breast and signet ring cell carcinoma metastatic to the breast is often controversial in surgical pathology. diagnoses need to be made correlating clinical and histological examination in difficult cases in which there is not a diagnosis of carcinoid tumor elsewhere. their histological appearance may mimic ductal adenocarcinoma of the breast. the distinction is important due to differences in management and prognosis. determination of her2 gene amplification by chromogenic in situ hybridization (cish) in breast carcinoma m. neagu * , c. ardeleanu, g. butur, a. florin * national institute of pathology, bucharest, romania objective: detection of her2neu amplification is an integral part of breast carcinoma diagnostics to decide therapy. method: we study 55 cases of breast carcinoma embended paraffin tissues, both cish and fish were performed on each case using (spot-light her2) for cish and both her-2 and chromosome 17 probes for fish (vysis). sixty tumor cells were evaluated in each case. the scoring system and interpretation of cish -invitrogen. results: concordance between cish and fish was found in 94,8 % cases, considering fish as gold standard, sensitivity of cish was 97.5 % and specificity 94 %. cish is more practical alternative due to lower cost, no requirement of fluorescence microscope, use of existing bright-field microscopy and techniques it s similar to ihc, archivable and quantitative results, it s easy to observe both the tissue morphology and the gene amplification evaluation of the 55 cases analyzed, 53 showed similar results for both methods. two cases were discordant. in these cases, low-level amplification was suggested by cish but nonamplification by fish. conclusion: our results, suggest that cish is a useful technique to determine her-2/neu oncogene status, in breast carcinoma for paraffin embendded tissues, is a highly accurate, reproducible and practical technique, with a high sensitivity. usual ductal hyperplasia with central necrosis, microcalcifications and multiple foci of pseudoinvasion arising in a radial scar: a potential diagnostic pitfall a. nechifor-boila * , s. stolnicu * university of medicine and pharmacy, tirgu-mures, romania objective: the diagnosis of radial scar (rs) is often difficult, especially when associated with proliferative changes, pseudoinvasion and necrosis. method: a 41-year-old woman was referred to the surgery department for a palpable mass in her left breast, regarded as a possible fibroadenoma on ultrasonography. results: lumpectomy was performed and the macroscopy revealed a tan-white 11 mm diameter lesion, with irregular margins and firm consistency. on light microscopy, a sclerosing lesion, with a stellate arrangement of ducts surrounding a central fibro-collagenous zone was identified. haphazardly arranged, distorted ducts associated with massive intraductal proliferation, highly suspicious of infiltrative were also present in the center of the lesion. the proliferating ducts showed slit-like, irregular, secondary lumens, several containing central necrosis and a heterogenous cell population. the presence of central necrosis and pseudoinvasion were, however, worrisome. immunohistochemistry for p63 demonstrated the presence of myoepithelial cells surrounding all the areas with pseudoinfiltrating features, while ck 5/6 displayed a heterogenous, mosaic-like positivity, characteristic for usual ductal hyperplasia (udh). conclusion: because pseudoinvasion in benign rs can easily be misinterpreted as invasive carcinoma, immunohistochemistry is mandatory to establish the presence of myoepithelial cells. although rare, central necrosis may occur in udh and should not be used as a single diagnostic criterion of malignancy. phenotypical and morphological heterogenity of breast cancer: our experience j. nieslanik * , j. dvorácková, m. uvírová, š. laciok, r. ondruššek, d. žiak * cgb laboratory a.s., dept. of pathology, ostrava, czech republic objective: breast cancer is the most frequent malignant tumor in women with a rising incidence. about 4 500 new cases are diagnosed in czeck republic every year, up to 43 % die on it´s account. in cgb laboratory are investigated 500 of malignant breast tumors every year. we evaluate the morphology, grade of differentiation and phenotype properties-hormone receptors expression, overexpression and amplification of her-2/neu gene. 2 % of carcinomas posses significantly morphologically and phenotypically heterogenic tumor population in one and the same tumor leasion. results: the most often sign of phenotype heterogenity found is hormone receptors expression or overexpression of her-2/neu gene. often we see two morphologically different tumor populations coexisting. the most interesting cases from our practice are on the poster. conclusion: as for now, studies about the origin of heterogenity inside the same leasions, did not find a single theory to resolve it. a possible answer gives the theory of tumor stem cells and the model of clonal evolution. our observations demonstrate and may explain the practical reason of different phenotype properties found in core cut biopsy where one population may be held, against the heterogenic properties of the tumor shown when resected as a whole. high grade infiltrating carcinoma with squamous features -a case report d. r. novac * , c. ardeleanu, s. taban, f. cadariu, a. dema * municipal clinical hospital, anatomical pathology, timisoara, romania objective: apocrine carcinoma is a rare and histologically distinct type of invasive breast carcinoma. method: the patient, a 73 years-old woman was admitted to the hospital for a tumor mass in the upper internal quadrant of the left breast. tissue fragments from the quadrantectomy specimen were routinely processed by paraffin embedding. for the immunohistochemical (ihc) study we used the following antibodies: ck7, androgen receptors (ar), estrogen receptors (er), progesterone receptors (pr), high molecular weight cytokeratin (hmwck), p63, gross cystic disease fluid protein-15 (gcdfp-15), en vision system, visualization with diaminobenzidine. results: the gross examination revealed a nodular, well delineated mass of 2,5/2,7/3 cm, firm, gray-beige with areas of necrosis on cut section. microscopically, the tumor had a predominantly solid, partially cystic and less tubular growth pattern. the tumor cells had abundant eosinophilic granular cytoplasm and pleomorphic high-grade nuclei. a small tumor contingent presented squamoid features. ihc stains showed the following profile: ck7+, ar+, gcdfp-15+, er-, pr-, p63/hmwck + (focal/zonal). a diagnosis of high-grade infiltrating apocrine carcinoma with squamous differentiation was established. conclusion: there are only a few cases of apocrine carcinoma on record and our case is even more special because of his peculiar aspect, the squamous differentiation. the effects of treadmill exercise in the number and weight of mammary tumors chemically induced in female sprague-dawley rats: preliminary results j. oliveira * , c. teixeira-guedes, a. faustino-rocha, r. soares-maia, r. arantes-rodrigues, m. j. pires, m. ginja, p. oliveira, r. ferreira * utad, dept. of veterinary sciences, vila real, portugal objective: we hypothesized that moderate exercise in treadmill may affect the mammary tumor development. n-methyl-n-nitrosourea (mnu) is a commonly used carcinogen to induce mammary carcinomas. the aim of this study was to evaluate the influence of treadmill exercise in the development (number and weight) of female rat mammary tumors. method: in this experimental protocol were used 21 female sprague-dawley rats. mnu was intraperitoneally administered at 50 days of age in a dose of 50 mg/kg. animals were randomly divided in two groups: sedentary (n=11) and exercised (n=10). the exercise program was started after carcinogen administration. animals were exercised in a treadmill control le8710® after an initial period of familiarization. thirty-six weeks after mnu administration animals were sacrificed and tumors were counted and weighted. results: sedentary and exercised group presented 25 and 20 tumors, respectively. pearson chi-square value was not significant (p>0.05). the mean tumors weight of sedentary group (5.34±10.58 g) was lower than exercised group (8.64 ±13.01 g). the difference between groups was not significant (p>0.05). conclusion: we observed that exercised group showed minor number of tumors, however the lesions presented higher volume. future morphological and biochemical analysis of tumors will allow a better understanding of the relation between mammary cancer and physical exercise. objective: primary non-hodgkin's lymphomas is an uncommon disease representing approximately 0.15 % of all reported malignant mammary neoplasms. clinically they are mainly observed as solitary lesions but may also be seen as multiple foci. herein, we present a very rare case with the diagnosis of multifocal malignant lymphoma of the breast with a detailed clinicopathologic evaluation. method: a 56-year-old-female patient with a right palpable breast lump admitted to the hospital. mammography and ultrasonography findings showed 2 different foci of hypoechoic solid mass forming lesion in the right breast. excisional biopsy has been performed. results: in the histopathological evaluation, both lesions demonstrated diffuse infiltration of mammarian tissue with foci of necrosis; mitotically active tumor cells with large nucleus and prominent nucleoli. the immunohistochemical analysis revealed diffuse and strong lca, cd20, cd43, focal cd68 positivity while pancytokeratin, ema, cd34, sma were negative. the case was diagnosed as diffuse large b-cell lymphoma. conclusion: breast is an uncommon site for primary malignant lymphomas. we report a very rare case with a diagnosis of multifocal primary non-hodgkin's lymphoma of the breast. ps-17-065 cytological, histopathological and clinical correlation at differential diagnosis of granulomatous mastitismalignancy t. ozgur * , e. atik, s. toprak, h. gokce, n. sengul, m. temiz * mustafa kemal university, medical faculty, antakya, turkey objective: idiopathic granulomatous mastitis (igm) is a rare disease that is difficult to diagnose by only radiological methods and clinical findings. method: first case; a 30 years old woman with a well circumscribed mass of 5 cm diameter at right breast applied to our hospital general surgery outpatient clinic. in the examination of fine needle aspiration biopsy (fnab), palisaded epitheloid histiocytes and scattered atypical cells have been seen on a bloody and inflammatory background. we suggested excisional biopsy. second case; a 39 years old woman applied to our hospital general surgery outpatient clinic with a sore mass in her left breast. the mass has been excised for malignancy suspect. foci of abscesses, active chronical inflammation, areas of hemorrhage and numerous granulomas containing epitheloid histiocytes are observed microscopically. results: in microscobic evaluation of the excisional biopsy of the first case prediagnosed as granulomatous mastitis invasive ductal carcinoma has been our diagnosis. in the second case there have been numerous granulomas containing epitheloid histiocytes, microscopically. our diagnosis was granulomatous mastitis in this patient prediagnosed as malignancy. conclusion: especially fnab findings are easy to confuse with malignancy. we wanted to emphasize to clinician and pathologist to be more alert on distinction of granulomatous mastitis and malignancy. objective: triple-negative-breast-cancer (tnbc) that accounts for 10-20 % of all breast carcinomas is defined by the lack of estrogen receptor, progesterone receptor, her2 expression with agressive clinical behavior. tnbc is categorized into basal like subtype which is characterized by the expression of basal cell markers and normal breast subtypes. method: we studied on 41 immunohistochemically tnbc patients to determine egfr, cytokeratine5/6 (ck5/6), p53, ki67, gcdfp15 expression patterns by immunohistochemistry, her2/chromosome 17 gene status by fish. results: most of the tumors (90,2 %) were invasive ductal carcinomas. p53, ki67, gcdfp15 mean positivity rates were 55,6 %; 51,7 %; 3,2 % respectively. gcdfp15 positivity was noted in 8 cases of which 6 were ck5/6(−). the cut-off value for ck5/6 positivity was 5 %. egfr immunoreactivity was grouped into 0, 1+ as negative; 2+, 3+ as positive categories. ck5/6 was positive in 56,1 %, egfr was positive in 51,2 % of the patients. the relation between ck5/6 and egfr expression was statistically significant (p <0.01). her2 fish was negative in all cases. conclusion: as a result gcdfp15 alone is not a useful marker to detect the metastasis of basaloid type breast cancers. ck5/6 and egfr coexpression can be used to diagnose basaloid tumors with 5 % cut-off value. objective: lobular neoplasia (lns) of the breast include atypical lobular hyperplasia (alh) and lobular carcinoma in situ (lcis). because lns do not present typical clinical or radiologic findings, the diagnosis is incidental. many reports suggest that ln is not only a risk factor of invasive lobular carcinoma but also is as a precursor. it is important to know about the incidence of lns and associated disease in korea. method: a total 1551 cases of breast biopsy or excision from five major hospitals in daegu were reviewed independently by 5 pathologists of the daegu breast pathology study group. the incidence of alh and lcis, associated disease, the presence of microcalcification and necrosis were reviewed. all suspected cases of lns were confirmed on e-cadherin immunohistochemical staining. results: only 46 cases out of 1551 cases (2.9 %) revealed lns. associated disease were 17cases of invasive ductal carcinoma, 10cases of in situ ductal carcinoma, 6 cases of invasive lobular carcinoma, 5 cases o f other benign disease, and 3 cases of florid ductal hyperplasia or columnar cell change. conclusion: the incidence of lns was similar to western women. incidental lns were frequently associated with invasive ductal carcinoma and further excision is suggested if it was present on the needle biopsy specimen. silver-enhanced in situ hybridization (sish) detection assay for her2 gene status determination in breast carcinoma: a four year experience in our laboratory f. patakiouta * , g. c. balis, v. theodorou, e. triantafillidou, c. fotiou, i. boukovinas, c. andreadis, g. sibilidis * "theagenion" cancer hospital, dept. of pathology, thessaloniki, greece objective: assessment of her2 status in breast cancer is important in the clinical management of patients and can be identified by a number of methods. in this study we present the results of the sish technique used in our laboratory in the last 4 years. method: we analyzed 398 cases of invasive breast carcinoma, including 41 core biopsies, which were previously characterized immunohistochemically (cb11) as equivocal on a protein level (her2 2+). all cases were evaluated by bright-field sish using the automated ventana benchmark xt machine. evaluation was independently performed by two pathologists (fp, gb) based upon the algorithms of the manufacturers and asco/cap guidelines. results: out of 398 cases the her2 gene was amplified in 82 cases (20.6 %), while in 302 cases (75.9 %) there was no gene amplification. fourteen of the cases (3.5 %) were characterized as equivocal. the concordance between the two pathologists was high (97.6 %). conclusion: the sish method is new, fully automated and very rapid. furthermore the bright-field sish signal does not decay and can be visualized by any standard bright-field microscope even after long periods of time, making it particularly suitable for routine application in surgical pathology. objective: breast fibromatosis (bf) represents 0.2 % of breast tumours that simulate carcinoma. the aim of this study is to characterize the cases of bf diagnosed in our institution. method: bf cases diagnosed from 1999 to 2009 were evaluated with clinicopathological and immunohistochemical parameters. we also studied mutations in exon 3 of the gene for β-catenin in 4 cases. ultrastructural study (us) was performed in 1 case. results: there were 5 women between 33 and 69 years old. radiologically they showed deeply located hypoechoic irregular lesions. size ranged between 1 and 23 cm. histologically they were characterized by an ill-defined spindle cell proliferation without epithelial elements or atypia. immunohistochemical study was negative for ae1/ae3, ck5/ 6, p63, cd34 and bcl2. actin was diffusely positive in 4/ 5, and s100 and desmin focally positive in 4/5. β-catenin showed cytoplasmic(5/5) and nuclear(1/5) expression. the us showed double smooth muscle and myofibroblastic differentiation. three patients showed mutations in exon 3 of the β-catenin gene (2 with substitution at codon 41 (t41a) and 1 deletion of 33 base pairs (a43_e65del)). conclusion: focal desmin expression and us suggest smooth muscle differentiation in some bf. all cases showed cytoplasmatic positivity for β-catenin but nuclear expression was seen in only 20 % of cases. 75 % of the valuable cases showed genetic alterations of β-catenin. objective: breast cancer is a heterogenous disease consisting of distinct entities characterized by different gene expression patterns. gene expression profiling of breast cancer revealed 4 major subtypes, including hormone receptor (hr) positive luminal a and b, her-2 positive and basallike breast cancer. most of the basal-like carcinomas are triple negative (hr and her-2 negative). as most triple negative breast cancers express egfr, mutations in the signal transducing cascade of ras/raf/map kinase might play a role in tumor progression. the aim of this study was to determine the incidence of kras and braf v600 mutations in triple negative breast carcinomas. method: total genomic dna was obtained from 37 formalin-fixed, paraffin embedded, triple negative breast tumors. kras was analyzed by pyrosequencing of codon 12, 13, 61. braf (exon 15, v600) mutations were analyzed with autogenomics infiniti braf assay on the autogenomics infiniti analyzer. results: we found kras mutation in only one case (2.7 %), apocrine carcinoma which was gly12asp (ggt> gat) mutation. the braf v600 mutations were not detected in all analyzed tumors. conclusion: we concluded that kras mutation was rare in triple negative breast carcinomas and high frequency of egfr overexpression in this subtype might be related to other pathways in egfr signaling. the pre-lymphatic system and the lymphatic network of the breast in menstruating and in menopausal women: a comparative study s. popovska * , i. ivanov, t. betova, t. dineva, z. ivanova * medical university hospital, dept. of clinical pathology, pleven, bulgaria objective: the pre-lymphatic system was initially described in the 1960s by casley-smith and florey. it consists of cd34 positive fibroblasts lined stromal spaces communicating with the lymphatic capillaries. the aim of the following investigation was to compare the lympho-vascular density and pre-lymphatic network density of breast tissues in the context of the menstrual status of the patients. method: formalin-fixed, paraffin embedded tissue specimens from 105 patients with primary breast cancer were studied. tumor-free tissue materials were selected. tissue processing and immunohistochemical staining with d2-40 and cd34 antibody was performed in accordance to standard laboratory protocols. quantitative evaluation of the lymphatic and pre-lymphatic system was performed. the kruskal -wallies test was used to compare differences between the median values of the studied variables. statistical significance of the differences was considered if p<0.05. results: the pre-lymphatic network was found to be significantly more dense in menstruating, compared to menopausal women k-w=61.04; p<0.0001 n=105. lymphatic vascular density was higher in menstruating compared to menopausal women k-w=7.60882; p=0.006; n=105. conclusion: involution changes in the pre-lymphatic and lymphatic system of the breast were found to appear in postmenopausal women. this may be the explanation for the slow local progression of breast cancer observed often in elder patients. comperative study of herceptest and fluorescence in situ hybridization results in breast carcinoma: indeterminate (2+) group must not be a wide range uncertainty category y. saglican * , u. ince * acibadem maslak hospital, dept. of pathology, istanbul, turkey objective: herceptest is an exclusively used immunohistochemistry method for testing her2/neu overexpression in breast carcinomas. the purpose of this study is to select the proper patients candidate to in situ hybridization, with an indeterminate her2 score. method: a retrospective analysis of 163 cases with invasive breast carcinoma were evaluated for her2 status using immunohistochemistry and fluorescence in situ hybridization. i̇mmunohistochemistry of cases that had been previously evaluated by nine different pathologists in our department, were reviewed by two experienced pathologists. interpretation of the immunohistochemistry results was made without knowing the first results. results: compared to the first evaluation; cases with score 0, 1+ and 3+correlated with the review of immunhistochemistry and none of score 0 and 1+ tumors were positive with in situ hybridization. gene amplification was detected in 43 cases; 23 of them with 2+score. three of 3+ tumors and 106 of 2+ cases were negative with in situ hybridization. discordance was detected in 21 cases in 2+ score; 17 of them were reclassified as negative and 4 were technically unsatisfactory. conclusion: using a different cut-off value for indeterminate her2 results and training of pathologists to minimaze the interpretational differences, will maximize the accuracy, while not submitting to unnecessary molecular tests. objective: the excision repair cross-complementation 1 (ercc1) enzyme plays an essential role in the nucleotide excision repair pathway and is associated with resistance to platinum-based chemotherapy in different types of cancer. the aim of the present study was to evaluate the clinicopathological significance of ercc1 expression in breast cancer patients. method: we used immunohistochemical to analyze ercc1 expression in a tissue microarray from 135 breast carcinomas. ercc1 expression analysis was available for 109 cases and was correlated with clinicopathologic factors and outcome data. results: ercc1-positive was observed in 58 (53.2 %) cases and was correlated with smaller tumor size (p= 0.007) and with positivity for estrogen receptor (p=0.040). ercc1 expression did not correlate with overall and disease-free survival rates. the majority (72.7 %) of special histological types of invasive breast carcinomas was positive for ercc1 compared to invasive ductal carcinomas (ercc1-positive in 51.1 % of the cases). similarly, triple negative breast cancers (tnbc) were more frequently negative for ercc1 (61.5 % of the cases) compared to the non-tnbc (41.5 %). conclusion: ercc1 expression correlated significantly with favorable prognostic factors, such as smaller tumor size and er-positivity, suggesting a possible role for ercc1 as a predictive and/or prognostic marker in breast cancer. a study of cd10 positive basal/myoepithelial cells in a consecutive series of in situ lobular neoplasia of the breast s. shousha * , g. forbes * charing cross hospital, dept. of histopathology, london, united kingdom objective: we have previously reported a few sporadic cases of in situ lobular neoplasia (iln) associated with marked proliferation of cd10 positive basal/myoepithelial cells. method: twenty consecutive cases of pure iln and 9 dcis were studied. new sections were cut and stained for e-cadherin and cd10. results: one out of the 20 cases showed foci of invasive lobular carcinoma in the new cut sections. all 20 iln cases showed increased proliferation of cd10 positive basal cells, varying from a focal mild increase to a marked proliferation surrounding or mingling with the lobular cells. cd10 positive cell proliferation was least observed in a case of iln with central necrosis and in foci of iln seen adjacent to the invasive carcinoma. the invasive tumour cells were cd10 negative. all cases of dcis had a thin layer of cd10 positive myoepithelial cells, that was incomplete or absent in some foci. conclusion: our findings confirm the presence of a unique relationship between excess proliferation of cd10 positive basal cells and iln; an association which is not seen in dcis or invasive carcinoma. this is particularly interesting as recent evidence indicates that cd10 is involved in the regulation of mammary stem and 'sphere forming' cells. objective: primary neuroendocrine carcinomas (nec) of the breast are defined by the who classification as a group of neoplasms that express neuroendocrine markers in more than 50 % of the cells. these tumors can exhibit different morphologic appearances including solid, nested or alveolar pattern, papillary or mucinous differentiation or conventional ductal structures. the aim of this study is to identify histomorphological features useful in the recognition of this tumors. method: 160 invasive breast carcinomas were reviewed, none of them previously typified as nec. 154 were ductal carcinomas (26 grade i, 61 grade ii and 66 grade iii) and 6 lobullilar carcinomas. in all cases immunohistochemical staining for the neuroendocrine marker synaptophysin was performed. results: eight of the 160 cases showed intense immunoreactivity for synaptophysin. all of them were predominantly composed of solid, confluent nests and cords of cells with medium-to high grade nuclear atypia. two cases showed intense desmoplastic or sclerotic stromal response. a colloid component was identified in another two cases. conclusion: due to its morphologic variability, mammary nec is frequently underrecognized. a predominantly solid pattern of confluent nests and trabeculae associated with a prominent sclerotic stromal response or colloid differentiation should prompt to perform additional immunohistochemical staining in order to exclude neuroendocrine differentiation. objective: association between centrosome abnormalities and response to chemotherapy has not been fully elucidated in breast carcinomas (bc). here we analysed the association between aurora kinase a (aurka) and gamma-tubulin (gt) and response to neoadjuvant chemotherapy (pst) in patients treated with bc. method: the immunohistochemical expression of aurka and gt was analysed in 44 core biopsies of bcs taken before administration of pst. aurka expression was analysed using a modified allred-like scoring system (intensity and percentage of staining combined). cells containing one or two centrosomes as determined by gt were considered negative, whereas cells containing more than two centrosomes were regarded as positive. centrosome amplification was graded in each specimen as follows: negative (0-2 % of cells); weak (2-10 %); moderate (11-20 %); and strong (21 %<). pathological response rates were assessed using chevallier's classification. results: centrosome amplification was significantly higher in patients achieving complete response when compared with the cases where partial remission or no response was defined (p=0.03), whereas we found no significant correlation between the expression of aurka protein and response rate to neoadjuvant chemotherapy (p=0.24). conclusion: bcs showing centrosomal amplification as determined by gt present higher response rates to neoadjuvant chemotherapy, but need further validation. breast granulocytic sarcoma with aleukemic presentation: a case report o. tzaida * , p. giagkazogloy, p. repousis, m. kotsopoulou, i. kasselaki, c. valavanis, i. iacovidou * anticancer hospital metaxa, peiraias, greece objective: granulocytic sarcoma (gs), a rare solid tumor of immature myeloid cells in exrtamedullary sites, usually occurs during the natural course of acute myelogenic leukemia (aml). rarely is presented without overt hematological disease. the breast is an uncommon site of localization. method: we report a case of breast gs with aleukemic presentation, condition that is extremely rare and requires a high index of suspicion for diagnosis. a 46 year old woman, presented with a solitary, non-tender, palpable left breast mass. a lumpectomy was done. results: histopathology revealed breast parenchyma diffuse infiltration, in a targetoid pattern, by immature small cells intermingled with eosinophils. lobular carcinoma and lymphoma were considered in differential diagnosis. the myeloid origin of the neoplasm was established by immunohistochemical analysis that revealed lca, mpo, cd34, hladr, cd68 (kp1) positivity. there was no evidence of leukemia in the peripheral blood and bone marrow. the patient was treated as aml with systemic chemotherapy and 5 months after the diagnosis she is without evidence of disease. conclusion: breast gs is a challenging situation both for pathologists and hematologists. available evidence strongly favors the application of systemic chemotherapy despite the appeared localized nature of the disease. method: paraffin blocks of tumor tissue of 152 breast cancer cases collected at our cancer centre for 3 years. the immunohistochemical study was performed by standard techniques using following antibodies (dako): er, pr, her-2, egfr, ck5/6, ck17, ki67. results: all cases were classified into the 5 subtypes: luminal a (n = 91; 59.8 %); luminal b (n =13; 8.6 %); her-2-positive (n=19; 12.5 %); basal (n=19; 12.5 %); 5negative (n=10; 6.6 %). conclusion: luminal breast cancer phenotype predominates in our collection, predictive/prognostic value of molecular genetic subtypes is planned to be investigated. we recognized statistically significant differences (p<0,001) in five-year survival rates between tumors of luminal (a, b) and basal subtypes. proliferating trichilemmal cyst with atypical cytological features r. g. wright * , c. gallivan, t. molden-hauer, r. liang * gold coast hospital, pathology queensland, australia objective: proliferating trichilemmal cysts are relatively common occurring within the scalp of elderly women. proliferating trichilemmal cysts of the breast, however, are rare. these cysts are benign with very few demonstrating malignant transformation. method: we report a case of proliferating trichilemmal cyst of the breast with atypical cytology which prompted excisional biopsy. cytological assessment was useful in this case as it indicated excisional biopsy rather than a more extensive excision. results: smears produced from the fna showed sheets of atypical epithelial cells with foamy histiocytes, multinucleate cells and fibrotic material with evidence suggestive of fat necrosis. a cell block was acellular. a specific diagnosis was not made. an excision specimen was advised and a proliferating trichilemmal cyst was demonstrated. objective: a large clinical value has determination of molecular subtype of breast cancer (bc). to clarify morphological and molecular biological similarities and differences between bc depending on age, we compared features between tumors of younger and older women. method: 573 patients with bc are included in research: from 18 till 86 years, among them 254 patients were till 35 years. er, pr, her-2/neu, p53, p63, ki67, ck5/14, p21, bag1, mcl1, ps2, vegfr, her-1 were analyzed in all cases by immunohistochemistry. results: the younger patients with bc had higher expression of p53, p63, p21, bag1, mcl1, ki-67, vegfr, her-1 (p<0,001) than older patients. there was a basal-like molecular subtype of bc 3-fold more frequent compared with older patients (21,6 % versus 7,2 %; p<0,0001). threeyears overall survival in patients till 35 years was by 11,5 % and a five-year overall survival was by 15,5 % lower than in patients over 35 years (p<0,001). objective: breast her2 ish relies on correct enumeration of her2/cep 17 signal. however, as with immunohistochemistry, there are technical pitfalls which may render ish difficult/unreliable to interpret. the uk neqas has established a 'technical' ish module to provide feedback to laboratories using both chromogen and fluorescence based tehcniques. method: unstained slides consisting of 4 breast cancer cell lines were distributed. laboratories were asked to demonstrate her2 gene amplification using their routine assay, and then return the slides for assessment. chromogen based methods were assessed around a multi-header microscope with 4 assessors scoring each slides, whilst the fluorescent ish method was assessed by a single individual. assessors scored the 'readability' of each slide, without counting the probes, and provided feedback where the hybridisation technique could hinder interpretation. results: cish based methods showed acceptable staining in 50 % (n=32) of cases. staining problems ranged from loss/poor her2/cep17 signal, non-specific staining and morphological damage. the fish pilot assessment showed an acceptable rate of 32 % (n=21), with the low pass rate being attributed to poor preservation/quenching of fluorescence signal. conclusion: greater emphasis needs to be placed on the 'readability' of an ish slide, prior to carrying out the process of enumeration. tuesday, 11 september 2012, 09.30 -10.30 objective: intravenous leiomyomatosis is a very rare growth pattern variant of leiomyoma in which nodular masses of tumor grow within venous channels. occasionally the tumor can extend to vena cava and the right heart. method: we present a case of a 45-year-old woman, who was admitted in the emergency room with rapidly evolving exertional dyspnea. cardiac ultrasonography revealed a "big mass in the right chambers". she was submitted to a right atriotomy with resection of part of the tumor, which was sent for intraoperative consultation. results: grossly, the tumor was polypoid, firm, with a smooth surface. the frozen section showed a lesion composed of tortuous vessels and in some areas a fibrillar eosinophil extracellular matrix and others with spindle cells. no significant atypic or pleomorphic cells, mitosis or necrosis were observed. the diagnosis was deferred for definitive paraffin sections. in the definitive h&e and with immunohistochemical stains, the case was diagnosed as an intravenous leiomyomatosis, and the diagnosis was confirmed in the hysterectomy specimen. conclusion: intravenous leiomyomatosis with cardiac involvement is an extremely rare condition. clinical information is essential for the correct diagnosis in frozen section. characterization of a specific mechanism for late loss of cardiac allograft: the antibody mediated rejection (amr) as a major factor of cardiac allograft vasculopathy (cav) p. bruneval * , c. toquet, a. loupy, p. pouvier, a. cazes, j.-p. duong van huyen * france objective: the rational of this study was to investigate explanted failing grafts in the light of new concepts in rejection mechanisms, namely amr. method: this retrospective multicentric study collected 31 explanted cardiac grafts failing ≥1 year posttransplantation. the vasculature of the grafts was assessed from epicardial coronary arteries to myocardial microcirculation. immunohistochemistry was performed for c4d complement fraction deposition and cd68-positive macrophages in the explanted grafts and previous endomyocardial biopsies. donor specific antibodies (dsa) were retrospectively assessed using luminex sa technique. results: a pure classical coronary atherosclerosis pattern was observed in 6/31 (19 %). a pure pattern of cav was present in 12/31 (39 %) and a mixed pattern associating cav and atherosclerosis features in 9/31 (29 %). interestingly the cav pure and mixed patterns were associated with vascular inflammation within the arteries and/or the microcirculation with c4d deposits and macrophages (15/21) in the explanted grafts versus nil (0/6) in pure atherosclerosis pattern (p<0.002). furthermore they were associated with previous amr episodes on endomyocardial biopsies (11/ 21) and by positive dsa (10/14) versus nil in pure atherosclerosis pattern (p<0.05 and p=0.05, respectively). conclusion: in failing cardiac grafts cav lesions are associated with makers of amr. cav should be the consequence in coronary arteries of an ongoing amr process. results: in our cases increase in fibrous and adipose tissue concordant with age, indicating an age related nature, were detected. fibrous and fatty tissue infiltration appeared at the age of 35. fatty infiltration started at the age between 20 and 34 at sinoatrial node. in 4 cases calcification and in 19 cases inflammation was observed. amyloid accumulation was not present. in 7 cases myocardial infarction not involving ccs was seen. in 1 case fibroelastoma was detected. conclusion: in turkish population age related fibrosis and fatty infiltration in ccs appeared at the age of 35 and increased with age. fatty infiltration in sinoatrial node started at a younger age than that of reported in the literature. in cases whom the cause of death could not be determined we could not detect lethal pathologic features. however we think that examination of the ccs will improve the quality of autopsy diagnosis. periadventitial adipose tissue in human coronary atherosclerosis: a neurotrophin study p. ghenev * , p. panayotov, g. chaldakov, l. aloe * medical university varna, dept. of pathology, bulgaria objective: recent evidence demonstrates that epicardial adipose tissue, including coronary periadventitial adipose tissue (tunica adiposa), are paracrine sources of bioactive mediators (adipokines, no, h2s) which may be involved in coronary atherogenesis. because of the increasing interest for extra-neuronal effects (inflammation, wound healing, lipid and glucose metabolism) of the neurotrophins nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf), including in human coronary atherosclerosis in autopsy samples, the aim of the present study is to evaluate the expression of ngf and bdnf and their receptors (trka and trkb, respectively) in cardiosurgery biopsy samples of pericoronary adipose tissue. method: immunohistochemistry of ngf, bdnf and their trk receptors. results: ngf, bdnf, trk a and trkb expression was lower in periadventitial adipose tissue of atherosclerotic coronary arteries as compared to non-affected coronaries. conclusion: we provide the first evidence for a possible role of neurotrophins in the molecular remodeling of tunica adiposa in human coronary atherosclerosis. this study is dedicated to 103rd anniversary of rita levi-montalcini, the nobel laureate for the discovery of ngf. objective: in normal myocardium, apoptotic myocytes are usually absent, or if affected, with most of 28 positive cells per million. method: the apoptosis was measured by the tunel method in 60 patients with dilated cardiomyopathy (dcm) and myocarditis (mc), and expresed by apoptotic index (ai) in 30 patients within both groups, and to correlated with ejection fraction (ef) and with different morphological stages of these entities. statistical analisys was performed, and p values ≤0.05 were considered statistically significant. results: ai in mc cases was 4,23+12,16, and in dcm cases the value was significantly higher 5,41+9,33 (p= 0.005). in the group mc patients, we didn't found statistically significant correlation between ai and different values of ef (p=0.701). the analysis of the value of ai between patients with different morphological stages of mc didn't show statistically significant correlation (p=0.535), as well as in dcm (p=0.312). conclusion: apoptosis play a significant role in dcm and mc but its significance in the progression to heart failure has still to be established. objective: cardiac lesions following blunt chest trauma are multiple and of various degrees of severity. "cardiac contusion" is a difficult diagnosis, especially antemortem and if signs of thoracic trauma are not evident on external habitus and on the underlying internal plans of cardiac area. is the contusion a cause of death per se? method: the authors present two cases of road accident male victims, aged 43 and 50 year-old, submited to complete postmortem examination with ancillary diagnostic methods. results: no traumatic lesions or underlying pathology was found at the autopsy. epicardial and myocardial haemorrhagic foci were histologicaly documented. toxicology was negative. conclusion: cardiac contusions are caused by one of the following mechanisms: 1) heart compression between bone structures, 2) sudden acceleration-deceleration movements and 3) sudden thoracic/abdominal pressure increase. they may be asymptomatic or lead to rhythm disturbances responsible for unexpected death (10 %) . they seem to be underdiagnosed, thus suspecting the entity "contusio cordis" is crucial, not only antemortem for correct surveillance and treatment, but also postmortem to adequately establish causality nexus in cases of post-thoracic trauma deaths. the influence of bone marrow-derived multipotent stromal cells on myocardial scar healing in experimental myocardial infarction l. kakturskiy * , t. fatkhudinov * institute of human morphology, central laboratory of pathology, moscow, russia objective: previous studies have shown the benefits of intracoronary infusion of bone marrow-derived multipotent stromal cells (bm-mscs) in heart disease. our aim was to study engraftment, differentiation of bm-mscs and its role in myocardial reparation. method: acute myocardial ischemia was produced by transient occlusion (total of 20 min) of the proximal left anterior descending coronary artery and followed by reperfusion. bm-mscs were isolated, expanded by standard and labeled with pkh26 (sigma). cells were cd90 and cd105 positive. 30 days after occlusion during cross-clamping of aorta the cells were administered into the cavity of the left ventricle at the concentration 5×106 in saline solution. in 1 day, 2, 4 and 6 weeks after transplantation the labeled cells were detected in the cryosections and heart morphometry was performed. results: bm-mscs were detected only in the scar tissue and had a fibroblast-like phenotype. they neither differentiated into cardiomyocytes nor into the cells of blood vessels. in 4 weeks after transplantation the scar thickness was higher in the group with cell transplantation (p<0.001). conclusion: engrafted bm-mscs promote myocardial fibrosis only in the scar, but not in the perifocal myocardium, provided strengthening of the scar, remodelling of outflow tract, and improvements the myocardial function. objective: from january 2000 to december 2009 (institute of pathology and legal medicine, paolo giaccone -palermo) 57 autopsies were performed on 37 males and 20 females who died of a massive pte. method: systematic autopsy. results: saddle pulmonary thrombosis in 33 cases, in 24 isolated in both the main pulmonary arteries. in all cases the transverse heart diameter was 1,5-2 cm superior to the longitudinal one, the interventricular septum became rectilinear with a right ventricular dilation. the coronary artery anatomy was: critical stenosis through fibroatheromatous plaque of the dominant right coronary artery (rca, 2), non dominant left coronary artery (lca, 3); non critical stenosis of rca (14), lca (8); absence of stenosis (30). histological ventricular examination showed hypoxic-ischaemic and/or reperfusion and overload lesions. in all cases, the lungs showed plurifocal occlusive thrombosis of intramural pulmonary arterial (ipa) vessels associated with adjacent infarctual necrosis (49); acute partially haemorrhagic basal oedema (35) and desquamative macrophagic alveolitis (28). conclusion: in all cases of massive pte the thrombosis of ipa vessels associated with an adjacent infarction (86 %) is constant. this suggests that death is nearly always preceded or triggered by one or more episodes (clinically silent or with non specific symptoms) of thromboembolism of ipa vessels. cardiac findings in routine fetal autopsies: more than it meets the eye? e. rios * , c. bartosch, c. ramalho, j. monterroso, o. brandão * centro hospitalar s joão, pathology, porto, portugal objective: congenital heart disease (chd) is the most common malformation in newborns. our aim was to evaluate the spectrum of chd in consecutively performed fetal autopsies and to correlate prenatal and postmortem diagnoses. method: a retrospective study of 726 fetal autopsies was performed in a tertiary referral hospital. chd was classified in seven categories: left ventricular outflow tract obstruction-lvoto, right ventricular outflow tract obstruction-rvoto, septal defects, connection anomalies, conotruncal anomalies, complex anomalies and others. cardiac defects were also classified as isolated or associated with others anomalies. results: chd was identified in 99 (13.6 %) fetuses. most common categories were septal defects and complex anomalies. associated anomalies were found in 67 fetuses (67.7 %). in this group, septal defects were the most common chd, frequency being significantly higher than in the group of isolated cardiac anomalies (p=0.012). comparison of prenatal and postmortem diagnoses (50 cases) showed complete or partial agreement in 36 and 10 cases (72 % and 20 %, respectively) and complete disagreement in 4 cases (8 %). in the latter group, prenatal diagnosis had not been done by a pediatric cardiologist. conclusion: the high prevalence of chd in lost pregnancies highlights the importance of systematic fetal autopsy performed by a specialized pediatric pathologist. objective: the appearance of pathological calcification, which leads to significant changes of vascular wall, which underlie the development of atherosclerotic complications, has a great importance in the morphology of atherosclerosis development. purpose of the work is the study of pathological biomineralization in heart valves affected by atherosclerosis. method: the study was conducted on sectional material of mitral and aortic valves obtained during autopsy. the tissue of heart valves was studied by methods of histology, electron microscopy, x-ray diffraction, infrared spectroscopy. results: macroscopically mitral and aortic valves are thickened, opaque, dull whitish, sometimes with deformity and ulceration. histology observed the formation of atherosclerotic plaques, fibrous changes, and inflammatory infiltration. according to x-ray phase analysis biominerals in heart valves are represented by apatite crystalline phase. sizes of crystals have distinct age dependence. the results of infrared spectroscopy revealed absorption bands of carbonate apatite replacement; in all instances they correspond to the type of substitution b (co3 2-replaces po4 3-). conclusion: the study of pathological mineral formations on human heart valves by the range of morphological and physicochemical methods show that they are aggregates of interacting organic and mineral components, their ratio changes with "maturation": the organic component decreases and the mineral component, represented by carbonate containing hydroxyapatite doped with chlorine grows while improving, so the process is dynamic. relationship between myocardial injury, oxidative stress mechanism and sepsis/septic shock in infants submitted to surgery for congenital heart defects m. silva de oliveira * , e. medeiros floriano, p. henrique manso, r. nilsson sgabieri, j. guilherme klamt, e. zangiacomi martinez, w. vilella de andrade vicente, s. gusmão ramos * university of são paulo, dept. of pathology, ribeirão preto, brazil objective: a progressive ventricular dysfunction caused by ischemic myocardial injuries remains one of the leading causes of death during the postoperative course in congenital heart disease (chd). the aim of this study was to investigate the role of oxidative stress in these myocardial injuries. method: myocardial injuries and oxidative stress mechanisms were assessed by histopathology and immunohistochemistry and quantified by morphometrical analyses. results: myocardial injury was observed in pediatric patients submitted to surgery for chd with cardiopulmonary bypass, followed by lethal exit. oxidative stress mechanisms were directly related to these particular types of myocardial injuries. importantly, 4-hydroxynonenal (4-hne), a marker of lipid peroxidation, is strongly expressed, especially in irreversible myocardial lesions. although morphologically similar, myocardial injuries observed in patients who evolved with sepsis in the peri-operatory period exhibited a completely different set of oxidative stress mechanisms. increased concentrations of nitrotyrosine protein adducts were observed in these patients, suggesting that peroxynitrite-mediated protein nitration may be the predominant oxidative stress mechanism found in these situations. conclusion: the underlying mechanisms of these lesions seem to be related to the development of ischemia or ischemia/reperfusion followed by oxidative stress mechanisms that vary depending on whether sepsis was present. while the exact mechanism is not fully understood, it has been suggested that endogenous catecholamine release could have a role in this process. objective: a significant decrease of the mean cardiomyocyte dna content and increased numbers of diploid cardiomyocytes after ventricular unloading was demonstrated, suggesting a numerical increase of cardiomyocytes. the heart harbours several stem cells populations including c-kit (cd 117) + cells and side population cells (spc), that might proliferate after unloading and generate diploid cardiomyocytes. it was tested, whether there is an increase of abcg2+ spc and cd117+ cells after unloading. method: in paired myocardial samples (before and after lvad), the number of cells with immunoexpression of abcg-2, c-kit/cd 117 and mef-2 was assessed by immunohistochemistry and morphometrically determined. results: a significant increase of spc and cells with coexpression of c-kit and mef-2 after unloading was observed (p=0.001). a significant positive correlation between both spc and cells with coexpression of c-kit and mef-2 expression was observed (p=0.007 and 0.01). no correlation was found between the number of spc and the mean cardiomyocyte dna content. conclusion: spc are significantly increased in the myocardium after ventricular unloading, suggesting a role of stem cell proliferation during "reverse cardiac remodelling". these cells might proliferate and commit to different cell lineages such as cardiomyocytes or endothelium, and thus ameliorate cardiac function. role of elastin gene polymorphism in ascending aorta aneurysm development in patients with essential hypertension v. zakharova * , v. dosenko, m. kostiv, e. rudenko * cardiovascular surgery institute, pathology, kiyv, ukraine objective: there is supposition about genetic propensity in the ascending aorta aneurysm (aaa) development. aim: to investigate role of elastin gene polymorphism (rs2071307) in aaa development in essential hypertension (eh). method: dna samples from persons with aaa and eh (1 group), with eh only (2 group) and healthy subjects (3 group) were examined. polymorphism rs2071307 was studied with real-time pcr. histological examination of aaa walls was performed in 1 group. results: distribution on genotypes a/a, a/g, g/g was the following: 1 group -42.1 %, 42.1 %, 15.8 %; 2 group -68 %, 24 %, 8 %; 3 group -18.5 %, 45.6 %, 35.9 %. fragmentation of elastic membranes and media collagen component strengthening were revealed in persons with a/a, a/g, g/ g genotypes in 54.2 %, 70.8 %, 11.1 % respectively. persons with g/g genotype have more frequently (66.6 %) than others (a/a -16.6 %, ag -12.5 %) signs of inflammation, which promote aaa development parallel to eh. conclusion: we obtained evidence that elastin gene polymorphism (rs2071307) plays important role in eh and aaa development. in persons with allele a in rs2071307 risk of eh and aaa development is higher than in g/g genotype. mostly allele a in rs2071307 is accompanied with elastic membranes fragmentation and their substitution by collagen that decreases arterial wall elasticity. tuesday, 11 september 2012, 09.30 -10.30 objective: our aim is to inform on the frequency, clinical aspects, and histopathological features of the tumor metastatic to the orbit. in adults, the most common tumor metastatic to the orbit are carcinomas, breast carcinoma the most frequent. less common are the metastases from gastrointestinal tract, kidneys, and prostate. cutaneous malignant melanoma is the most common nonepithelial tumor metastatic to the orbit. method: orbital metastatic tumors may occur at any age, but most often between 40 and 70 years of age. most reported statistics reveal a slight predominance of women, accounted for by the fact that breast carcinoma is causing metastases most frequently. a frequent finding in the cases of metastatic carcinoma is enophthalmos that is associated with scirrhous carcinomas. we present two cases of orbital metastatic tumors in which the primary tumors were a basal cell carcinoma and a cutaneous malignant melanoma. results: as to the metastasis from a basal cell carcinoma, the primary tumor was located in the external angle of the eyeball and metastasized the external 1/3 of the orbit. in the case of malignant melanoma, the primary tumor was located in the upper lid and invaded the orbital floor and fat. in both patients eyeball enucleation was performed. conclusion: we chose to present these cases given the differential diagnostic challenge and rarity of their location. uveal melanoma a. dumitriu * , s. dumitriu * umf iasi, dept. of neurology, romania objective: uveal melanoma which arises from melanocytes residing in the stroma is the most common primary intraocular cancer in adults. we report a case of uveal melanoma presenting as complicated cataract in a 65 years-old male. results: the right eye was blind for 6 months. the patient underwent extended enucleation and histopathology was consistent with uveal melanoma. conclusion: we present this case in order to provide the medical community a basic reference that would help to make further progress in this rare disease, which remains difficult to treat. results: there were more women than men (52 % versus 48 %). metastases developed in 39 % (total group mean follow up of 7.14 year). mixed tumors were most common overall (43.5 %): for non metastasizing tumors there were 41.1 % mixed, 36.1 % fusiform and 17.2 % epithelioid types. for metastatic tumors there were 46.7 % mixed, 21.5 % epithelioid and 21.5 % fusiform types. the difference between cell type and metastatic development was significant in all but epithelioid compared with mixed tumors. epithelioid type had a relative risk of mortality of 3.51 and mixed had 2.18 compared to fusiform cell type. conclusion: our patients were younger, with larger tumors and different histological proportions from those of the collaborative ocular melanoma study. five year survival times were similar to those for europe. uveal melanoma between preserving vision and prolonging survival. pathological contributions to a better clinical management m. mera * , l. blaga * umf cluj-napoca, anatomic pathology, romania objective: uveal melanoma has a high metastatic potential which is invariably fatal. there is an ongoing debate on the impact of ocular treatment on metastatic disease and survival. improved prognostication is needed to identify cases that could avoid unnecessary loss of vision while avoiding unethical care. our study is aimed at contributing to the developing of reliable tools to assess the metastatic risk. method: an accurate prediction should include both clinical features and histologic parameters as biopsy techniques continuously advance. 120 cases of uveal melanoma, either reviewed or prospectively managed in our pathology department, were included in a log-logistic multivariate survival model that we have considered would best accomodate our data. the performance of the model was assessed by bootstrap re-sampling. results: extravascular matrix patterns, optic nerve invasion, tumorinfiltrating lymphocytes, tumor cytomorphology, nucleolar size and the mitotic count together with initial visual acuity have been found to be the most important predictors, ahead of the traditional tnm stage. the results are provisional but encouraging. conclusion: there is still need for multidisciplinary approach and multicenter collaboration in an effort to elucidate the uncertainty related to the best management of one of the most traumatising types of cancer. orbital hemangiopericytoma: a case report e. omoscharka * , k. lankachandra * university of missouri, dept. of pathology, kansas city, usa objective: hemangiopericytomas are rare vascular tumors arising from zimmerman's pericytes, which ubiquitously surround blood capillaries and post-capillary venules. they rarely occur in the orbit, accounting for only 1 % of all orbital neoplasms. in most cases the primary presentation is progressive proptosis. method: a 26-year-old female was first seen for the evaluation of a right orbital mass which has been causing proptosis. associated symptoms included peripheral vision loss in the right eye, persistent headache with occasional balance problems. a palpable, 3.2 × 2.5 cm well circumscribed mass with a smooth surface, resting on the inferior orbital wall was completely removed through right anterior orbitotomy. h&e stained histologic sections were studied. in addition, tissue sections were subjected to immunohistochemical stains for cd34, cd99, bcl2, sma, desmin, ema and s-100. results: the mass was composed of spindle cells with very low mitotic activity. staghorn vascular channels were evident, and in several areas the tumor cells invaded the pseudocapsule. the tumor cells were positive for cd34, cd99 and bcl-2 supporting the histopathologic diagnosis of benign hemangiopericytoma. conclusion: orbital hemangiopericytoma is a slow growing tumor with a potentially malignant behavior, and a high local recurrence rate if incompletely excised. complete, intact removal prevents tumor recurrence and provides a good outcome. expression of sod family in rat lacrimal gland after intermittent light exposure c. l. zamfir * , f. e. zugun, e. cojocaru, r. o. temneanu, r. folescu, m. spataru * university of medicine iasi, dept. of histology, romania objective: any disturbance of chronobiological rhytms represents a potential stressor, acting like a strong oxidant trigger. sod family (cuzn-sod, mn-sod and ec-sod), is one of the most significant enzymatic complexes involved in antioxidant defense. monitoring local sod distribution in lacrimal gland after intermittent light exposure can be able to provide insights into the distinct links of antioxidant adaptative mechanisms.so long as lacrimal gland morphology and functions are dependent to a normal light exposure, mobilisation of its own antioxidant resources, especially sod, is an usefull starting point in antioxidant ophtalmic therapy. method: immunohistochemistry was performed in microscopic analysis of glandular fragments prelevated from 30 male wistar rats, randomized in control/intermittent light exposed groups for 45 days. there have been used specific antibodies for each enzymatic member of sod family. blood samples were prelevated to control sod levels. the study respected all imposed ethical criteria. results: lacrimal glandular tissue exhibits a strong cuzn-sod immunoreactivity, a moderate one for mn-sod and only a small ec-sod reactivity, while the level of sanguin sod is high, as a marker of oxidative aggression. conclusion: oxidative stress may be engagged in a deep alteration of lacrimal gland. mobilisation of sod, depending on their specific glandular location and tissular receptivity, should be regarded as a promising alternative for antioxidant therapy. tuesday, 11 september 2012, 09.30 -10.30 objective: liposarcoma is the most commonly diagnosed soft tissue sarcoma in adults and occurs predominantly in the lower limbs and retroperitoneum. primary mediastinal liposarcomas are rare accounting for less than 1 % of all mediastinal tumors. method: we report a retrospective study of 4 cases of mediastinal liposarcoma during an 8-year period. diagnosis was made on mediastinal biopsy in 2 cases and on resected specimen in the 2 other cases. results: there were 3 men and a women ranging in age from 28 to 81 years, explored for respiratory symptoms. radiological findings showed an anterior inhomogeneous mediastinal mass in all cases with extension to left hemithorax in one case. a surgical resection was achieved in only 2 cases but was incomplete. histopathologically, tumors were classified into 2 well differentiated lipoma-like liposarcoma, one myxoid liposarcoma and one mixed liposarcoma (sclerosing liposarcoma with myxoid liposarcoma). all patients received adjuvant radio or chemotherapy. one of them presented lung metastasis during the follow up and another died from respiratory failure. the two other are still alive. conclusion: mediastinal liposarcomas include a heterogeneous group of bulky tumours, the progression of which depends on the histological type. histopathologic examination is always necessary as much for diagnosis as prognosis. mediastinal epithelioid hemangioendothelioma with a highgrade clinical course a. konstantinova * , k. shelekhova, v. f. klimashevsky * st. petersburg state university, russia objective: epithelioid hemangioendothelioma (eh) is a rare neoplasm usually presenting in soft tissues. about twenty cases of eh have been reported in the mediastinum. most of these mediastinal eh exhibited an indolent course. results: a 21-year-old woman presented with focally encapsulated mass, 5.5×4 cm in size, in the anterior mediastinum. the tumour of bone density was adhered to the lung tissue and occluded left subclavian vein. microscopically, the tumor consisted of foci of so-called blister cells typical for eh, anastomosing cords of small epithelioid cells embedded in myxohyaline matrix with haphazardly distributed metaplastic bone and hemorrhagic cellular stroma. spindling of the tumor cells was prominent. the tumor cells demonstrated moderate atypia, but mitotic figures were not found. immunohistochemically, cells of primary tumor were positive for fli1, vimentin, cd31 and cd34. focal staining with ema was observed. multiple metastases were subsequently found in the liver and lungs. metastatic deposits had the same morphology as a primary tumor. we present the case of rare mediastinum epithelioid hemangioendothelioma with peculiar histological features such as prominent spindling of neoplastic cells and abundant metaplastic bone formation in aggregate with aggressive clinical behavior that exhibited a profound metastatic poteintial. objective: thymic carcinomas comprise rare malignant epithelial neoplasms, which exhibit a disproportionately large variety of growth patterns. we present three cases of primary thymic carcinomas, including two squamous cell carcinomas and one papillary adenocarcinoma. method: all three patients were males, aged 51, 61 and 77 years, and presented with mediastinal masses measuring 17 cm, 6.5 cm, 3.7 cm in maximal diameter respectively. results: microscopically the first two tumors were poorly differentiated squamous cell carcinomas, immunohistochemically positive for cd5 and cd117, whereas the third tumor was a papillary adenocarcinoma, positive for cd5 and negative for cd117 and ttf-1. immunohistochemical features of the tumors were supportive of thymic derivation. conclusion: primary thymic carcinomas are rare malignant tumors with clear-cut atypia, largely lack of organotypic features and a very diverse differentiation. squamous cell carcinoma is the most frequent subtype. they are often a diagnosis of exclusion, since metastases to thymus and anterior mediastinum, mainly from the lung, are far more common. immunoreactivity to cd5, cd70 and cd117 may support the thymic origin of neoplastic squamous cells. treatment options include surgical excision, radiation and/ or chemotherapy depending on tumor stage and patient's condition. the prognosis is generally poor with squamous cell carcinomas having a slightly more favorable outcome. wednesday, 12 september 2012, 09.30 -10.30 objective: surgical blood loss can lead to ineffective tissue perfusion of vital organs. physiologic solution chosen for blood volume replacement may be determinant for preserving renal integrity. our aim is to study kidney histopathological changes in a hemorrhagic model, followed by intravascular volume replacement with ringer's lactate or hydroxyethylstarch (hes) 130/0.4 solutions. method: thirty one pigs under general anaesthesia with propofol and remifentanil underwent haemorrhage at a volume of 30 ml kg-1, over 20 min. after a waiting period of 20 min, intravascular volume was replaced using hes 130/ 0.4 (g1) and ringer's lactate (g2). one hour after, pigs were euthanized with iv potassium chloride and sixty two renal samples were taken for histopathological examination, using pas staining. renal damage was assessed for glomerular, tubulointerstitial and vascular lesions. results: mean arterial pressure reached 40 mmhg after bleeding, and recovered for values above 60 mmhg in both groups after volume replacement. histopathological lesions observed in g2 were more frequent than those in g1. conclusion: hes 130/0.4 may reduce the incidence of histopathological lesions secondary to renal hypoperfusion after severe bleeding when compared with ringer lactate. results suggest that the reestablishment of intravascular volume with hes 130/0.4 may preserve renal integrity secondary to blood loss. expression of aeg-1, p53 and its clinicopathological significances in the malignant lesions of renal cell carcinomas h. erdem * , m. oktay, u. yildirim, a. k. uzunlar, m. a. kayikci * duzce university, dept. of pathology, turkey objective: astrocyte elevated gene-1 (aeg-1 also known as metadherin) is associated with various aspects of tumor malignancy. the aim of this study was to investigate p53 relationship between aeg-1 and prognostic parameters. method: this study was made 50 paraffin blocks (tumoral samples), which were histopathologically diagnosed at department of pathology from 2005 to 2011. subtypes of the cases were 24 (48 %) clear cell renal cell carcinomas (rcc) and 26 (52 %), non-clear rcc respectively. by immunohistochemical analysis, we investigated aeg-1, p53 expression in carcinomas of kidney and we determined its relationship with clinicopathological parameters. results: there were significant relationship between increased aeg staining score and tumor capsule (p=0.01), lymphovascular invasion (p=0.015) and significant relationship between the increased diameter with the increase of p53 (p=0.028). there were significant correlation between increased diameter of tumor and degree of increase the fuhrman (p=0.02). conclusion: high aeg-1, p53 expression correlates with prognostic parameters in the rcc. in addition, aeg-1, p53 expression in rcc may be associated with tumor progression. relationship of cd95, cox-2 and p53 in renal cell carcinomas with survival and other prognostic parameters: a tissue microarray study h. erdem * , u. yildirim, a. k. uzunlar, m. a. kayikci * duzce university, dept. of pathology, turkey objective: renal cell carcinomas (rcc) is the seventh most common human malignancy. rcc is now recognized to be a complex neoplasm consisting of several different tumor subtypes, each with distinct genetic and clinical features. the aim of this study was to investigate the expressions of cyclooxygenase-2 (cox-2), p53 and cd95 in rcc that has different clinicopathologic characteristics. method: this study was conducted on a total of 49 paraffin embedded kidney samples (tumoral samples), which were histopathologically diagnosed at department of pathology from 2005 to 2011. ihc stains for cox-2, p53 and cd95 were performed on tissue microarray using standard procedures. results: there were significant correlations between cox-2 and subtype (p=0,044), cox-2 and diameter (p=0,026). significant relationships were found between p53 and age (p=0,050), p53 and diameter (p=0.050). besides, there were significant correlations between cd95 and furhman grade (p=0,050). conclusion: cd95, cox-2, p53 expression correlates with prognostic parameters in the rcc patients. in addition, cox-2, p53, cd95 expression in rcc may be associated with tumor progression. objective: ebv related malignancies (skin cancers, lymphomas, kaposi sarcomas) complicates organ transplantation ebv-associated smooth-muscle tumors are rare. method: among 1500 kidney grafted patients, three developed ebv-smt. case 1. female 51 y, with ebv-smt (generalized) case 2. female 46 y, breast phylodes sarcoma, reviewed diagnosis: ebv-smt. case 3. male 23 year, severe abdominal pain, 2 gut tumor-like nodules were excised suspection of tbc. ebv-smt was microscopic diagnosis. results: progressive weight-loss all 3patients, chronic cough in patient 1, chest x ray multiple pulmonary lesions, on ct-scan paravertebral lesions. transthoracic biopsy: spindle cells proliferation with mixed cellularity eosinophilic cells intermingled fascicles resembling smooth muscle, mononuclear inflamatory cells and capillarie evoqued myofibroblastic tumor. no mitosis, mild nuclear atypia. negative immunohistochemestry: cd21, cd34, cd99, ema, cytokeratin, s100, cd117. alpha-smooth muscle actin diffusely positive, evb-lmp negative. in situ hybridization : ebv + nuclei. ebv-smt was established. conclusion: smt in immunocompromised are ebv associated. primary target of ebv are b lymphocytes, may infect smooth-muscle through receptor for ebv cd21 mechanisms of ebv related-tumor genesis is integration of ebv-dna within alk locus in tumor cells. alkgene rearrangement and expression; associated with inflammatory myofibroblastic tumors, anaplastic large-cell lymphomas etc. clonally of multifocal ebv-smt using southern blot showed, multiple tumors constitute independent primary lesions. method: the degree of glomerulosclerosis was scored into 0-3, mesangial proliferation was scored into 0 (absent) and 1 (present), and the degree of crescents was scored into 0-2. glomerular injury score (gis) was obtained by adding the above 3 scores and grouped into 3 categories (group 1, score 0-1; group 2, 2-4; group 3, 5-7). results: serum creatinine level was significantly increased (1.3±0.4, 1.8±0.9, 2.2±0.8 mg/dl, p=0.007), estimated glomerular filtration rate (egfr) was significantly decreased (68.5±17.6, 52.1±22.5, 40 .9±20.4 ml/min, p= 0.001) and proteinuria was significantly increased as gis increased (1.3±2.7, 1.9±1.6, 4.6±7.1 g/24 h, p=0.030). interstitial fibrosis of more than 25 % of cortical area increased as gis increased (2.3, 26.0, and 57.1 %, p<0.001). when a multivarate analysis was done, gis group 3 was the most important predictive factor of egfr (p=0.009) and proteinuria (p=0.014). objective: the aim of the study is to evaluate the histopathological finding and the c4d staining patterns of the patients who were biopsied due to graft dysfunction (gd) after initial well-function within 1 month of deceased donor kidney transplantation (ddkt). method: histological analysis and c4d immunostaining were performed 34 on needle core biopsies. results: thirty-four patients (mean age: 37±8 years, male: 59 %) were included. histological analysis revealed acute rejection (ar) (n:7), acute tubular injury (ati) (n:8), allograft infection (n:1), borderline changes (n:8), normal morphology (n:3), and donor-related changes (n:7). c4d staining was detected in 44 % (15 of 34) biopsies; staining patterns were diffuse (n:5), focal (n:5) and minimal (n:5). diffuse (n:3) and focal (n:1) c4d positivity accompanied 4 of 7 cases of ar. in remaining 27 patients having non-ar histological picture, diffuse or focal c4d positivity were detected in 2 and 4 cases, respectively. peritubular capillaritis was detected in 41.1 % (14/34) of biopsies, of which four had c4d diffuse, one had c4d focal positive. conclusion: beyond cold ischemia time-induced ati, immunological causes including antibody mediated process may play an important role in early impairment of graft function after ddkt. immunohistochemical analysis of the renal interstitial fibroblasts s. kostadinova-kunovska * , r. jovanovic, m. bogdanovska, v. janevska, l. grchevska, g. petrushevska * faculty of medicine, inst. of pathology, skopje, macedonia objective: renal fibrogenesis is a process common to all progressive kidney diseases. the main executive cell of this process is the fibroblast, by secreting and remodeling the extracellular matrix. the number of fibroblasts is minor in healthy kidney interstitium, but it increases during the process of fibrosis. their morphology and immunophenotype vary due to different intrinsic and extrinsic factors, thus making their identification and visualization, as well as determination of their origin, very difficult. method: we performed morphological and immunohistochemical analyses on kidney biopsies with primary glomerulopathy and interstitial fibrosis, using the following antibodies: vimentin, α-sma, s100a4, cadherin 9 and cd34. results: interstitial fibrosis with focal, rather than diffuse distribution, was present in all analyzed cases. the total interstitial fibroblast population was positive for vimentin, majority of the cells were positive for s100a4, and a smaller proportion of cells were positive for α-sma, cadherin 9 and cd34. furthermore, different cells in the fibroblastic population showed positivity for different markers. conclusion: the above stated observations contribute to the theory that different subpopulations of fibroblasts, with different origin, take part in the renal fibrogenesis. soluble epoxide hydrolase inhibition reduce blood pressure and organ damage independently of nitric oxide (no) in mice with goldblatt two kidney, one clip model (2k1c) p. kujal * , l. kopkan, l. cervenka, z. vernerova * charles university, third faculty of medicine, prague, czech republic objective: investigate the role of no in the blood pressure (bp)-lowering effects of soluble epoxide hydrolase (seh) inhibition in 2k1c model. method: the endothelial no synthase gene knockout mice and their wild-type controls were used. renal concentrations of epoxyeicosatrienoic acids (eets) and dihydroxyeicosatrienoic acids (dhets) were measured in nonclipped kidney. renal no synthase activity was determined by measuring the rate of formation of l-[14 c]citruline. results: treatment with the seh inhibitor caused the same bp decrease that was associated with increase in daily sodium excretion in both types of mice. the ratio of eets/dhetes in the nonclipped kidney was increased and did not alter renal nos activity. seh inhibition reduced significantly glomerular and tubulointestitial injury. conclusion: bp-lowering effects of chronic seh inhibition in 2k1c mice are associated with normalization of the reduced availability of biologically active eets in the nonclipped kidney and their direct natriuretic actions. total inflammation in 6-month surveillance renal transplant biopsies is associated with decreased renal function and de novo class 2 donor specific antibody z. laszik * , s. chandran, f. vincenti * ucsf, dept. of pathology, san francisco, usa objective: the goal was to correlate inflammation and acute rejection (ar) with renal function and antibody status in 6-month renal transplant surveillance biopsies. method: relative risks of inflammation and ar in 380 6month biopsies was calculated by multivariable poisson regression. results: ar was seen in 7.3 % (n=28), borderline change in 19.7 % (n=75), and c4d positivity in 2.1 % (n=8) of all biopsies. total cortical inflammation (ti), present in 32.6 % (n=124) cases, was associated with 7 ml/min/1.73 m2 lower egfr at 6 months (95 % ci= −10.8, −3.2). de novo hla class 1 was identified in 17.4 % (n=44) and class 2 dsa in 26.1 % (n=66), with most having mfi values <1000. increased risk of ar was seen with higher levels of de novo class 1 (rr 1.49, 95 % ci=1.08, 2.0) or class 2 dsa (rr 1.34, 95 % ci=1.04, 1.72). class 2 (but not class 1) dsa was associated with a higher risk of ti (rr 1.40 for ti-score ≥1, 95 % ci=1.13, 1.72) at 6 months. conclusion: both ti and ar in 6-month surveillance kidney transplant biopsies are strongly associated with de novo hla class 2 dsa. total inflammation at 6 months correlates with decreased kidney function. objective: tenofovir disoproxil fumarate has been used in the treatment of hiv patients producing occasional renal dysfunction and fanconi syndrome. method: a 38-year-old patient was referred with severe acute renal failure and important metabolic acidosis and glycosuria with normoglycemia in urine analysis, altogether with urinary alkaline ph and proteinuria with a protein/ creatinine ratio of 4. he was hiv and b positive diagnosed 17 years earlier. his medication regimen consisted of lopinavir, ritonavir, tenofovir and lamivudine. the patient had a history of nausea, vomiting and some diarrhea with low fluid intake for a week. besides slight dehydration, the physical examination was unremarkable. renal ultrasound showed normal kidneys. with adequate fluid restoration, the renal function improved but other metabolic alterations like nephrogenic diabetes remained. results: renal biopsy revealed toxic acute tubular necrosis predominantly involving proximal tubules with prominent eosinophilic inclusions within proximal tubular cell cytoplasm, which represented giant, abnormal mitochondria, consistent with tenofovir toxicity. some histochemichal studies (cox and sdh) were performed. conclusion: although prospective clinical trials have demonstrated a low incidence of renal toxicity with tenofovir, there are several such reports in the literature. current recommendations suggest close monitoring of renal function after initiation of therapy with tenofovir, specially at the beginning and when used in combination with certain antiretroviral agents. objective: nutcracker syndrome (ns) is caused by compression of the left renal vein between the aorta and superior mesenteric artery. the main presenting symptom of this rare entity is haematuria, with various degrees of proteinuria. the diagnosis of ns syndrome is primarily by imaging and can sometimes be challenging. method: a 21-year-old male was referred to nephrology with a 3 months history of intermittent gross haematuria. all blood test including blood cell count, biochemistry, immunoglobulin electrophoresis, antinuclear antibody were normal. urinary analysis revealed numerous red blood cells and a protein/creatinine ratio of 0.3. a previous cystoscopic examination was normal. renal ultrasound revealed a diffuse increased echogenicity. results: renal biopsy showed a normal histology with no immunofluorescence deposits. ct scan revealed dilatation of the distal left renal vein with narrowing between the superior mesenteric artery and the aorta. conclusion: ns is a rare entity causing haematuria. there are few reported cases with histology, although it usually shows no abnormalities. the proposed mechanism for the haematuria is an abnormal communication between the submucosal venous plexus and the calyceal system presumably induced by renal venous hypertension. banff initiative for quality assurance in transplantation (bifquit): reproducibility for bkv immunohistochemistry in renal allografts m. mengel * , s. chan, j. climenhaga, h. regele, b. colvin, p. randhawa * university of alberta, laboratory of medicine, edmonton, canada objective: detection of bk virus associated large t-antigen is crucial for the diagnosis of polyomavirus nephropathy. method: in an international multi-centre trial we assessed the inter-observer and inter-laboratory variability for bk immunohistochemistry. a tissue microarray was constructed comprising 23 specimens representative of the whole analytical spectrum from negative over mild to strong sv40 positive cases. 81 participants at 60 centers stained the tma slides using local protocols. participants evaluated their slides following a provisional banff grading schema. details regarding local staining protocols and evaluation scores were collected online. stained slides were returned for centralized panel re-evaluation. weighted kappa statistics were used to determine the variability. results: the bk inter-observer reproducibility was substantial (mean kappa 0.64), but inter-laboratory reproducibility was below chance (kappa −0.22). separating components of bk evaluation schema into stain intensity and stain percentage showed no significant improvements in reproducibility. however, collapsing the proposed bk scoring schema into a simple positive/negative call improved bk inter-laboratory variability to 0.77. conclusion: these results indicate a significant variability between laboratories for detecting the sv40 large-t antigen by immunohistochemistry in paraffin sections. any proposed grading schema for bk nephropathy, which is dependent on percentage and intensity of nuclear staining, will essentially not be reproducible between laboratories. objective: cryopyrin-associated periodic syndrome (caps) is a rare hereditary inflammatory disorder with three differents phenotypes: familial cold autoinflamatory syndrome, muckle-wells syndrome and neonatal-onset multisysten inflamtory disease caps results from a mutation of the nlrp3 gene (1q44) coding for cryopyrin, which forms intracellular protein complexes (inflammasomes). results: case report: 41 year old women with chronic renal disease stage 3 (creatinin 2,2 mg/dl and proteinuria 180 mg/ 24 h) who developed urticarial episodies since 6 months old related to cold exposure. after 11 years old patient describe associated to these episodios bilateral arthritis (knes, ankles and elbows), conjunctival inflammation, shivers, and asthenia, more frequently in winter and precipitated by cold exposure, air conditioning, stress and menstruatión. aditionaly the patient developed in last years bilateral hypoacusis a kidney biopsy showed a amorphous, acellular and acidophilic material congo red positive deposits at interstition, arteries and arterioles and less frequent at glomeruli. this material displays apple green birefringence by polarizad light microscopy. diagnosis: secondary renal amyloidosis conclusion: the clinico-pathologial findings of this case are compatible with muckle-wells syndrome. secondary amyloidosis is a severe complication which occurs in 25 % of muckle-wells cases. amyloidosis is cause by increase of c-protein and a amyloid during the episodies before described and deposition of the a amyloid in differents tissues. glomerulocystic disease associated with thrombotic microangiopathy in two kidney allografts j. m. mosquera reboredo * , e. vazquez martul * complexo hospitalario universitario, dept. of pathology, la coruña, spain objective: glomerulocystic kidney disease (gckd) is a rare condition usually congenital and reported in infants and young children. only few cases of adquired gckd had been reported often following hemolytic uremic syndrome (hus). method: histological study of two kidney graft explants. results: we present two cases of kidney transplant who developed hus in allografts. both cases showed at the histological examination typical vascular and glomerular changes of thrombotic microangyopathy (tma). a cystic transformation with increase of urinary space and retraction of glomerular tuft was frequently observed. chronic transplant vasculopathy was found in the two cases. humoral active rejection was also demonstrate in one case with moderate peritubular capillaritis and glomerulitis and diffuse c4d deposition at peritubular capillaries. conclusion: in a few cases gckd appear to develop after another kidney disease includes single case reports of gckd associated with mesangial glomerulonephritis, wegener's granulomatosis, progressive systemic sclerosis, after hus (include some case in adult patient). our two cases represent a initial stage of adquired gckd. the ethiopathogenetic relationship is not clear but some authors propous that cystic dilatation of the bowman's capsule associated to tma/ hus may be secondary to ischemic mechanism. case report: cytomegalovirus gastritis in renal transplanted man f. noroozinia * , k. makhdoomi, a. esmaeili, a. saffarifard * emam khomeini hospital, dept. of pathology, urmia, iran objective: cmv is an important pathogen in immunocompromised hosts, including patients with aids, neonates and transplant recipients. this infection develops in 70-90 % of transplant patients. upper gi symptoms in solid organ recipient are common (20 %) and clinical signs are more serious in 10 % of cases. method: we report a 30 year old man with end stage renal disease underwent kidney transplantation from a cmv negative donor on june 2011. after 1.5 months he admitted with fever, generalized body pain, oral aphtous ulcers and epigastric pain accompanied by malaise. endoscopic examination revealed multiple antral erosions with surrounding erythema. clinicopathological investigations revealed cmv viremia with a ig-m antibody titer and cmv gastritis confirmed by histopathological examination. results: the patient was started on intravenous (iv) ganciclovir 5 mg/kg per day every 12 h initially for 3 weeks, afterwards the fever decreased; cell blood counts throwback to normal ranges and general condition of the patients improved. conclusion: cmv infection develops in 70-90 % of the transplant patients. the colon and stomach are the most common sites of gastrointestinal infection. though the rate of gi affliction by cmv is high, localization to the gastric antrum is not common. immunohistochemistry study of c-kit expression in renal cell carcinoma f. noroozinia * , f. abbasi, z. yekta, f. meisami, a. saffarifard * emam khomeini hospital, dept. of pathology, urmia, iran objective: renal cell carcinomas include about 2-3 % of adults neoplasms and 90-95 % of all renal tumors. in many cases, it is possible to distinguish. rcc subtypes on the basis of hematoxylin-eosin staining alone. however, overlapping morphologic features pose some difficulties in making a proper diagnosis. to render an accurate diagnosis, additional methods like immunohistochemichal staining against c-kit have been recommended. method: we reviewed 65 cases of rcc diagnosed during 9 years. formalin fixed, paraffin embedded specimens was available in 65 cases. the expression of c-kit was evaluated using immunohitochemistry. results: six cases of 39 clear cell type (15.4 %), 8 of 13 papillary type (61.5 %), and 11 of 11 chromophobe type (100 %) were positive for c-kit that considering chi-square test there is significant relevation between rcc's subtypes and c-kit expression (p: 0.001). from 8 cases with renal vein invasion, 3 showed positive expression of c-kit (37.5 %) and in 55 cases with no venous invasion, c-kit expression was detected in 22 (38.6), so no significant relation was found between renal vein invasion and c-kit expression. conclusion: the expression of c-kit in rcc may have diagnostic significance. objective: igg4-rsd shows abundant igg4-positive plasma cells, diffuse fibrosis, and increased serum igg4 levels. although igg4-rsd typically results in autoimmune pancreatitis (aip), any organ may be involved. thus, igg4-rsd tin often goes unrecognized in the absence of aip. this is an igg4-rsd tin case with mediastinal lymph node and pulmonary involvement. method: a 73-year-old male presented with rapidly progressive renal failure, hypergammaglobulinemia, hypocomplementemia, and enlarged mediastinal lymph nodes and bilateral pulmonary nodules on ct. renal and mediastinal lymph node biopsies were performed and igg4 ihc was done on both after standard techniques. results: the renal cortex and medulla showed diffuse interstitial fibrosis with tubular atrophy and abundant plasma cells (25 igg4-positive cells/hpf), numerous lymphocytes and some eosinophils. if showed igg, c3, kappa, and lambda granular deposits in tubular basal membranes and bowmann capsules. lymph node follicular and paracortical hyperplasia with abundance of mostly igg4positive plasma cells was seen. an igg4-rsd diagnosis was rendered and high serum igg4 levels were demonstrated. steroid therapy resulted in lymph node and lung nodule reduction. conclusion: tin with abundant plasma cells and diffuse interstitial fibrosis, especially if accompanied by hypergammaglobulinemia, hypocomplementemia, or extrarenal involvement, should suggest igg4-rsd and prompt serum igg4 level determination and renal igg4 ihc. objective: a 39-year-old woman was admitted for chronic renal failure. clinical examination was normal. biological explorations showed creatinin clearance around 55 ml/min, tubular proteinuria with bence jones κ protein. serum immunoelectrophoresis identified abnormal monoclonal immunoglobulin g and κ-light chains (lc). bone marrow histology was normal. method: kidney biopsy revealed diffuse intracytoplasmic vacuoles in the proximal tubules resembling osmotic nephrosis. distal tubules, glomeruli, vessels and interstitial compartment were normal. immunofluorescence (including anti-κ and -λ staining) was negative. electronic microscopy (em) revealed intracytoplasmic immunoglobulinic crystals containing κ lc inside the vacuoles, leading to the diagnosis of light chain proximal tubulopathy (lcpt). results: lcpt is a rare complication of dysglobulinemia. it may be associated with crystals within the cytoplasm of proximal, less frequently distal, tubular cells, consisting more frequently in κ lc. rarely, diffuse tubular vacuolization is present, often indistinguishable from osmotic nephrosis. in our case, there was no proximal tubular dysfunction and immunofluorescence was negative. the first evocated diagnosis by light microscopy was "osmotic nephrosis". however, we failed to identify any causal factor. finally, the diagnosis was performed by immunoem. conclusion: in conclusion, before a picture of osmotic nephrosis without obvious cause, em and immunoem may be helpful for the diagnosis of lcpt, often revealing a dysglobulinemia. opportunistic infections in renal transplantation -a case series r. sampaio * , r. dias, p. farrajota, a. coelho, t. almeida, a. duarte, j. r. vizcaino * anatomia patológica, valpaços, portugal objective: oporto's hospital centre is one of the portuguese hospitals with more renal transplantation activity (performed since 1982). although increasingly rare, opportunistic infections (oi) in transplanted patients remain a major diagnostic challenge and are associated with high mortality rate. method: in order to evaluate the incidence of oi in renal transplant patients and to identify the location of infection, the respective techniques of diagnosis used and the survival time after infection, we conducted a retrospective study using the nephrology department's database on renal transplants. we consulted the registries from 2004 to 2012. results: we investigated 2041 cases and found 82 cases of oi caused by herpes virus (n=7), cytomegalovirus (n=27), polyomavirus (n=16), aspergillosis (n=4), alternaria (n= 2), mucormycosis (n=1), candidiasis (n=12), tuberculosis (n=9), cryptococcus (n=2) and pneumocystis (n=2). the lung and urogenital system were the most affected systems. kidneys were affected in 3 cases respectively by mucormycosis, tuberculosis and cryptococcus. of all the cases of oi, 39 were diagnosed by the department of anatomic pathology (21 by biopsy; 16 by cytology; 2 by biopsy and cytology). conclusion: in many cases the diagnosis could only be performed through histologic/cytologic examination. prompt diagnosis and treatment are necessary to avoid life threatening complications and may greatly improve prognosis. papillary renal cell carcinoma with osseous metaplasia and bone marrow elements: a case report l. l. santos * , a. polónia, r. henrique, c. lobo * ipo porto, dept. of pathology, viana do castelo, portugal objective: renal cell carcinomas might display foci of calcification and even ossification. this rare event has been reported mostly in the clear cell variant. herein, we present the case of a 69 year-old man, previously diagnosed with colon cancer, incidentally found to have a calcified mass in the kidney, which was interpreted as non-characteristic for renal cell origin. results: radical nephrectomy was performed. a 5.5×5.0× 4.0 cm tumor was found, with a heterogeneous brown cut surface, containing areas of necrotic tissue and extensive calcifications. histopathologic examination disclosed papillary structures covered by small cells with scant cytoplasm, dispersed among mature bone tissue enclosing marrow elements. epithelial tumor cells were imunorreactive for cytokeratins, cd10 and pax2, and negative with hmb-45. hale`s colloidal iron was negative. trisomy of 3p, 7p and 17p was detected by fish. a diagnosis of prcc type 1, fuhrman grade 2, with extensive osseous metaplasia was rendered. conclusion: ossification of renal cell tumors is rare, occurring mostly in clear cell type and the underlying mechanism is unclear. these tumors show atypical radiological features and might be confused with non-renal cell tumors. to the best of our knowledge, this is the first report of prcc with osseous metaplasia and bone marrow elements. the effect of doxycycline on glomerulosclerosis in 5/6 renal ablation s. sarioglu * , d. sonmez, a. celik, f. saglam, o. yilmaz, e. koraltan, z. cavdar, g. oktay * dokuz eylul university, faculty of medicine, izmir, turkey objective: the effect of matrix metalloproteinase (mmp) inhibitors in segmental sclerosis is unknown. the aim of this study is to investigate the effect of a mmp inhibitor, doxycycline, on glomerulosclerosis (gs) in renal ablation nephropathy. method: fourteen of the 32 female wistar albinos were 5/6 nephrectomised. doxycyline was given to half of each group (40 mg/kg/day total 28 days). after sacrification, the gs, mmp-2, mmp-9, timp-2 expressions were analyzed histopathologically. pro and active mmp-2 and -9 were analyzed by gelatin zymography. timp-1 and timp-2 were measured with elisa assay. results: doxycycline administration to the 5/6 nephrectomy group improved gs, but did not inhibit glomerular mmp-9 or cortical pro-and active-mmp-2 and pro-mmp9 but increased timp-1 and timp-2 expression in all groups in cortical tissue. mmp-9 expression and gs were increased in all groups receiving doxycycline. conclusion: we have demonstrated improved gs in renal ablation model by doxycycline administration but also doxycycline has an unexpected adverse effect. the effect of doxycycline on the expression of mmp-2 and -9 cannot explain the improvement in gs, but increased cortical tipm-1 and -2 may be an important contributing factor for inhibition of mmps. digitally reinforced hematoxylin-eosine polarization in diagnosis of renal amyloidosis s. sen * , b. sarsik * ege university, faculty of medicine, izmir, turkey objective: systemic amyloidosis is a rare disorder, characterized by extracellular accumulation of congo-red (cr) positive fibrillar amyloid protein deposits. the kidney is the most commonly affected organ by systemic amyloidosis. cr staining which increases the positive birefringence of the weakly birefringent unstained amyloid. in this study we investigated potential power of digitally reinforced birefringence of routine hematoxylin-eosine (he) slides on the renal biopsies. method: we reviewed 130 he stained slides for polarization. sixty five amyloidosis cases were diagnosed by renal biopsy from 2008 to 2012 at our laboratory. all biopsies were evaluated by light and immunofluorescence microscopy. slides were reevaluated blindly using a microscope (olympus bx51) attached polarization filter and connected to a digital camera (olympus dp21, sal). depositions which show green birefringence on he with digitalized microscopy were considered as positive and results were confirmed using cr. results: of the 65 cr confirmed amyloid positive biopsies, 61 showed green birefringence with he. of the 65 cr confirmed amyloid negative biopsies, two were considered as false positive. the sensitivity, specificity, positive and negative predictive values were estimated as 94 %, 97 %, 97 % and 94 %, respectively. conclusion: we concluded digitally polarized he sections can be used as a fast and first step diagnostic method for renal amyloidosis. sirolimus ameliorates cyclosporin-induced nephrotoxicity in a rat modelfocus on renal lesions, oxidative stress, inflammation, proliferation and angiogenesis j. sereno * , a. m. romão, m. teixeira, b. parada, c. mega, h. vala, e. t. lemos, f. teixeir, f. reis * ibili, medicine faculty, laboratory of pharmacology and experimental therapeutics, coimbra, portugal objective: sirolimus (srl) have been pointed as a feasible option for minimize the use of cyclosporin a (csa), especially because of putatively less nephrotoxicity. this study aimed to characterize the histological lesions and the molecular pathways implicated in csa-induced nephropathy and prevention when converted to srl. method: the following 4 groups (n=6) were tested during 9 weeks: vehicle, csa, srl and conversion (csa 3 weeks + srl 6 weeks). bp and hr were monitored. blood was collected and kidney gene expression of markers of inflammation, proliferation, angiogenesis and oxidative stress were assessed. histology: h&e, pas and gordon & sweets staining. statistics: anova and post´hoc tests (p<0.05). results: after 9 week of csa treatment, there was important kidney lesions, including glomerular, tubulointerstitial and vascular: mesangial expansion, atrophy, bowman capsule enlargement, hyaline cylinders formation, tubular calcification and vascular congestion, as well as arteriolar vacuolization and arteriolosclerosis. conversion to srl, ht and tachycardia were reduced, accompanied by amelioration of kidney dysfunction and lesions (glomerulosclerosis and tubulointerstitial fibrosis), together with reduction of oxidative stress, proliferation and angiogenesis. conclusion: in conclusion, srl ameliorates csa-induced nephrotoxicity in a rat model, which might be due to protection against oxidative stress, proliferation and angiogenesis, but these mechanisms deserve better exploitation. acknowledgements: fct(sfrh/bd/63962/2009). non-lupus "full-house" nephropathy in serbian population in last six years j. vjestica * , s. cirovic, s. tatic, r. naumovic, s. simic-ogrizovic, j. markovic-lipkovski * inst. for pathology, medical faculty, belgrade, serbia diffuse glomerular and sometimes focal mesangial staining of immune complex depositas (iga, igg, igm, c3, and c1q), also known as "full-house" pattern commonly indicates lupus nephritis. however, some non-lupus nephropathy also can be present with a "full-house" immunofluorescence pattern, mimicking lupus nephritis. the aim of this study was to define the clinicopathological spectrum of originally non-lupus "fullhouse" nephropathy. biopsies from january 2005 till december 2011 were analyzed in order to identify all renal biopsies cases showing "full-house" pattern. the study included 192 "full-house" renal biopsy diagnosiss. from all analyzed cases 117 (60 %) had lupus nephritis expressing "full-house" pattern and 75 (40 %) cases had non-lupus "full-house" nephropathy. in the absence of clinical and/or serological evidence of systemic lupus erythematosus (sle), at the time of renal biopsy, in 36 cases diagnose was membranous glomerulonephritis (gn), than 11 mesangioproliferative gn, 9 membranoproliferative gn, 7 iga nephropathy, 6 rapidly-progressive gn and 6 membranous/membranoproliferative gn cases. nonlupus "full-house" nephropathy is present in broad spectrum of different types of gn, predominantly in cases of membranous gn. the possibility of "full-house" nephropathy preceding the emergence of overt sle remained to be clarified. objective: although the renoprotective effects of prostacyclin have been demonstrated in many studies, the protection mechanisms of prostacyclin in chronic kidney disease, especially at the terminal stage, are still remained unclear. in the present study, we performed pathological and pathphysiological analyses of prostacyclin renoprotective effects using a stable prostacyclin analogue, beraprost sodium, in the disease kidney of anti-gbm glomerulonephritis (gn) rats. method: beraprost was administrated from 2 weeks after induction of gn. the condition of renal microvascular network and localization of apoptotic cells were examined using renal vascular corrosion casts, immunostainings and tunel-staining. the intracellular apoptotic signaling pathway was analyzed by western blot and qpcr. results: in the kidney of beraprost-treated rat, significantly high density of renal microvascular network was maintained, and apoptosis of vascular endothelial cells was suppressed even at the terminal stage of anti-gbm gn. pathophysiological analyses revealed that transcriptional and post-translational modifications of bcl2 and xiap, which were anti-apoptotic proteins in mitochondria dependent apoptotic pathway, were occurred in the kidney of beraprost-treated rat. conclusion: these results suggested that prostacyclin protects renal vascular network by inhibiting mitochondria dependent endothelial apoptosis, and it play an important role for preservation of renal function in the chronic kidney disease. wednesday, 12 september 2012, 09.30 -10.30 objective: although villous adenomas commonly occur in the gastrointestinal tract villous adenomas of the urinary tract, including the bladder, are infrequently encountered. method: we report a case of urinary bladder villous adenoma in a 72-year-old man. the patient was undergone cystoprostatectomy because of diagnosis infiltrative urothelial carcinoma in the transurethral resection of bladder. results: histopathological examination of cystoprostatectomy specimen there was no urothelial carcinoma. a 2,5 cm polypoid mass was seen at the dome of the urinary bladder. histopathology confirmed that this tumor was a villous adenoma with a polypoid growth of the glandular epithelium consisting of small tubular glands, dilated cystic glands or papillary fronds lined by a columnar epithelium. the glandular epithelial cells displayed mild nuclear atypia and nuclear pseudostratification with some mucus cells admixed. the patient was diagnosed with a rare case of villous adenoma of the bladder. over the past 5 months of follow up, the patient is alive and no metastasis. conclusion: patients with isolated villous adenomas in the urinary bladder have an excellent prognosis and surgical resection is curative. however, it is uncertain whether an untreated lesion might eventually develop into an adenocarcinoma. therefore, close follow up is recommended because of the possibility that this condition might be premalignant. caprin 1 overexpression in urothelial carcinomas of bladder b. akkaya * , z. cetin, s. berker-karauzum, m. baykara * akdeniz university, school of medicine, antalya, turkey objective: caprin1 encoded by cytoplasmic activation/ protein-1 gene located in 11p13 chromosome region. it has been reported that caprin 1 is associated with cell proliferation in various types of cell lineages. method: we researched whether caprin 1 might be overexpressed or not in urothelial carcinoma of bladder and its overexpression could be correlated with clinicopathologic parameter (age, sex, invasion). fifty urothelial carcinoma of bladder (29 infiltrative; 21 non-infiltrative) were stained by immunohistochemically in tissue microarrays. results: the expression of caprin 1 was observed in 24infiltrative urotelial carcinoma cases (%82) and 18 non-invasive urothelial carcinoma cases (%85). age range was 32-85. eight patients were female, 42patients were male. conclusion: in the literature caprin 1 overexpression was reported in different types of tumors including esophageal, stomach, prostate, lung, liver. caprin 1 overexpression might be correlated with the cellular proliferation potential. to determine of importance of caprin -1 overexpression new studies are necessary. objective: pseudohyperplastic squamous cell carcinoma of the penis (pscc) is a low grade tumor with specific clinical and pathological features. this very uncommon tumor occurs in association with lichen sclerosus, the main location is foreskin, and the high degree of differentiation may difficult its discrimination with pseudoepitheliomatous hyperplasia. method: a 66-year-old male presented a penis lesion involving glans and foreskin, clinically suspicious of malignancy. size was 1,6×1,4 cm and a conservative resection was done. pathological study was performed, and hpv detection with a commercially available kit: pcr amplification and reverse hybridization with probes to 35 hpv types. results: the lesion showed a non-verrucous well differentiated squamous cell tumor. upper layers lacked any atypia but infiltrative growth was evident in basal layers, with atypical cells and mitosis. lichen slerosus changes were evident bordering the tumor. hpv was negative for all types studied. no further treatment was employed and after a 18 months follow-up no recurrence has been observed. conclusion: pscc should be taken in mind when handling penis tumors. a correct differentiation from benign lesions and a knowledge of its low grade to avoid overtreatment will benefit patients. lichen sclerosus and not hpv seems to play a precancerous role. evaluation of sunitinib malate and meloxicam as single agents or in combination in bladder cancer cell lines r. arantes * , r. pinto-leite, c. lopes, l. santos, a. colaço, p. oliveira * utad, dept. of veterinary sciences, vila real, portugal objective: currently accepted for the treatment of advanced renal cancer, sunitinib malate is a small molecule inhibitor of the vegfr family, with ability to regulate tumor growth, progression, angiogenesis and metastasis. several reports have suggested that encouraging effects can be achieved by combining cox-2 inhibitors with anticancer agents. the goal of this work was to evaluate the effects of sunitinib malate and meloxicam isolated and combined on three human bladder cancer cell lines. method: t24, 5637 and ht1376 cells were treated with several concentrations of sunitinib malate and meloxicam, as single agents or in combined schedule. their influence on cell proliferation was determined by mtt method after 72 h of treatment. control samples were processed in the same way as treated samples but in drug-free medium. absorbance values of each well were read at 492 nm using an elisa plate reader. results: a reduction in cell proliferation rate was observed when all cell lines were treated either with sunitinib malate or meloxicam isolated. simultaneous exposure to both agents enhances the inhibition of cell proliferation. statistical significances were obtained when treatment groups were compared with control group. conclusion: these results suggest a potential clinical application of sunitinib malate in combination with meloxicam on bladder cancer. bcl-2 expression in prostate carcinomas b. balinisteanu * , a. dema, s. taban, c. lazureanu, d. herman, s. ursoniu, a. loghin, a. vaduva * municipal hospital, pathology, timisoara, romania objective: the disturbance of apoptosis represents an important event in the genesis of tumors with different localization. the study of anti-apoptotic protein bcl-2 expression from the perspectives of the prognostic and predictive value in prostate cancer has led to inconsistent results, even contradictory. method: expression of bcl-2 was analyzed in 3 groups of prostatic carcinoma: localized, locally advanced and with distant metastases. for histological grading of carcinomas we used gleason score. classification of the tumors into prognostic subgroups was made according to nccn guidelines. for the immunohistochemical study we used anti-bcl-2 antibody (clone 174), envision system, visualization with diaminobenzidine. the results of immunohistochemical reaction were assessed by evaluating the extent and intensity of immunostaining. statistical analysis was performed using stata 9.2. results: 17 of the 59 analyzed cases of prostate carcinomas showed bcl-2 over-expression: 5,9 % localized carcinomas, 23,5 % locally advanced carcinomas and 70,6 % carcinomas with distant metastases (p<0.001). although most of the bcl-2 positive tumors were poorly and moderately differentiated, the correlation between bcl-2 over-expression and tumor grade did not show statistical significance (p=0,085). conclusion: bcl-2 overexpression in advanced prostate carcinomas suggests involvement of this marker in the progression of tumors in this location. urothelial carcinoma of the bladder: a clinicopathologic study of 92 cases g. benkhedda * , s. khalifa, y. lamouti * chu frantz fanon, dept. of pathology, blida, algeria objective: urothélial carcinoma (uc) accounts for nearly 90 % of urinary bladder tumors.a variety of histological variant of uc have been recently recognosed. some variants have prognostic and therapeutic implications. the aim to this study is to assess the pathological features from our series and to compare our results that of the literature. method: we retrospectively studied 92 patients who were diagnosed histopthologically with urothelial carcinoma using the who classification system. results: the mean age of patients at diagnosis was 60(range, 30-70 years). 86,67 % were male (80 h/12 f). all tumors were classified as urothelial carcinomas: 2,1 % urothelial neoplasm with squamous differentiation,2,1 % with glandular differentiation,2,1 % urothelial tumors nested and 1 % sarcomatoid. in this study most tumors were grade 2 (67 cases) and stage pt1. conclusion: adult urothelium has the capacity to undergo several pathways of phenotypic cellular and structural differentiation as a result of the embryological origin of the bladder from the multipotent tissues of the cloacal endoderm and the mesodermal wolffian ducts. the clinical course of bladder cancer varies depending on the histological type of neoplasm, grade and stage of the tumor. hight-grade muscle-invasive urothelial cancers and tumors schowing variant microscopic morphology have in general hight mortality and poor prognosis. comparison of insignificant cancer detection rates in prostatectomies performed following 6 and 12-core biopsy schemes u. berber * , a. haholu, i. yilmaz, z. kucukodaci, d. demirel * gata heh, dept. of pathology, istanbul, turkey objective: widespread use of extended biopsy protocols have increased the prostate cancer detection rates. besides this improvement, whether detection of clinically insignificant cancer detection rates are increased by extended biopsy protocols is not well documented. in the study, we aimed to compare the rates of insignificant cancers found in prostatectomy specimens performed following 6 and 12-core biopsy protocols. method: retrospectively, we investigated the low volume/ low grade (lv/lg) prostate cancers in 160 prostatectomy specimens. tumors volumes were calculated digitally as multiplying total tumor areas by 3 mm for average block thickness, and corrected for tissue shrinkage by multiplying a factor of 1.25. results: of the 160 prostatectomies, 54 were performed following sextant technique, and 106 were performed after 12-core protocol. review of the h&e stained sections revealed 21 insignificant cancers. number of lv/lg tumors found in 6 and 12-core groups were 3(5.6 %) and 18 (16.9 %), respectively. conclusion: when compared to sextant technique, detection of lv/lg tumors were significantly raised in prostatectomies performed following 12-core protocol, and this increase points out the need for new approaches in patient management to avoid overtreatment after extended biopsy protocols. ps-22-008 impact of total core length for cancer detection in a lateral zone targetted 12-core prostate biopsy scheme u. berber * , a. haholu, z. kucukodaci, i. yilmaz, d. demirel * gata heh, dept. of pathology, istanbul, turkey conclusion: total core length is significantly associated with cancer detection rates and may be used as a reliable adjunctive tool in deciding repeat biopsies for patients with negative biopsy result. objective: primitive neuroectodermal tumors (pnets) are highly malignant tumors of neuroectodermal origin. we report a case of renal pnet in a 52-year-old male with a 6-month history of intermittent hematuria.. he underwent a right radical nephrectomy. macroscopically, the inferior pole was replaced by a multinodular, grey, glistening tumour measuring 8.7/7/6 cm, with foci of necrosis and hemorrhage. method: serial histological sections have been assessed using hematoxylin-eosin and van gieson stain and the indirect immunohistochemical analysis for antibodies: mnf 116, vim, cd56, nse, mic2/cd99. results: histological examination revealed a uniform population of undifferentiated, small-to medium-sized tumor cells, arranged in alveolar-insular patterns, with round to oval nuclei, small nucleoli, numerous signs of mitosis and scattered apoptosis, geographic zones of necrosis. dispersed cells showed cytoplasmic vimentin positivity favouring the diagnosis of pnet. few tumoral cells appeared positive for mnf116. expression of cd56 was positive in a large number of tumoral cells and an area of the tumour exhibit a milder reaction of positive nse. mic2 was positive with moderate staining in almost all tumoral cells. conclusion: diagnosis is based on histology and immunohistochemistry but pathological evaluation can be challenging because of the differential diagnosis with other small round cell tumors. nonamyloid fibrillary glomerulonephritis: presentation of two cases e. beretouli * , g. dimas, g. karayannopoulou, t. koletsa, d. grekas, g. karkavelas * ahepa hospital, dept. of pathology, thessaloniki, greece objective: fibrillary glomerulonephritis (fgn) is a rare disease, characterized by fibrillar deposits in the mesangium and the glomerular capillary loops. these deposits do not have an amyloidlike cross-β structure and are readily distinguishable from amyloid by the larger thickness of fibrils and lack of congo red staining. method: we report two cases of a 52-and 64-year-old women, who presented with severe nephrotic syndrome, rapidly progressive chronic kidney disease and lymphoproliferative disorders. glomerular crescents were present in about 30 % of both renal biopsy specimens (18 of 54 and 9 of 27 glomeruli, respectively). immunohistochemical analysis, immunofluorescence and an electron microscopy (em) studies were performed. results: renal biopsy showed a deposition of an amyloidlike extraneous substance in the mesangium, as well as within the glomerular basement membranes. congo red staining was negative. the em examination revealed fgn. conclusion: fgn must be included in the differential diagnosis of rapidly progressive chronic renal disease. em confirm the diagnosis of fgn, which suggests a poor outcome. objective: urachal carcinomas represent less than 1 % of bladder-related cancers. most are adenocarcinomas, but urothelial, squamous and small cell carcinomas may occur. there are pathological criteria for assessing an urachal origin. a specific staging system is lacking for these tumors. method: a mass of the bladder dome was found in two 56 and 58 year-old men. a 76 year-old woman presented an urachal cyst. ct-scan revealed no other tumors and partial cystecomy was performed. pathological examination showed partially cystic adenocarcinomas, one enteric type g2, and two mucinous type g1. two tumors extended into the bladder mucosa and one was limited to the urachal cyst. surgical margins were negative. immunostainings were positive for ck7, ck20, cdx2, cytoplasmic beta-catenin, and negative for p63. results: with a follow-up of 1, 6 months and 6 years, all patients are free of disease. conclusion: no tnm classification exists for urachal carcinomas. specific staging systems have been proposed by sheldon, and more recently by the mayo clinic. two cases are sheldon iiia/mayo clinic ii, and one sheldon ii/mayo clinic i. since stage, grade and surgical margins are the main prognostic factors, a clear and relevant staging system is needed for these rare carcinomas. objective: renal cell carcinoma (rcc) with two different histologies must be included in the unclassified group (who classification). whether these cases should be included in this histological subgroup or be considered hybrid rcc is a matter of debate. we report one of such cases in which papillary and chromophobe phenotypes meet. method: a 5 cm in diameter asymptomatic left renal mass was discovered incidentally in the radiological follow-up of a breast carcinoma diagnosed 12 years before in a 62 yearold woman. follow-up showed right adrenal gland metastasis. patient died 12 months later. results: grossly, tumor well circumscribed, tan-yellow, with haemorrhagic and necrotic areas. the neoplasm showed two different histologies clearly defined: one showed solid nests of polyhedral cells, with eosinofilic cytoplasm, central and hyperchromatic nuclei, occasional mitosis recapitulating chromophobe rcc. the other component presented a well defined tubulopapillary growth pattern typical of papillary rcc. focally sarcomatoid transformation, with tumor necrosis and chronic inflammation. by immunohistochemistry, chromophobe and papillary areas retained their specific phenotypes. conclusion: hybrid renal carcinomas do exist, but they are most probably hidden in the unclassified group of renal tumors. however, the exact histological context for which a renal neoplasm deserves the name "hybrid" remains to be defined. objective: renal cell carcinoma with thyroid-like follicular pattern is a rare histological subtype of renal carcinomas that has been very recently described. method: a 3.3 cm in diameter asymptomatic left renal tumor was discovered during the study of a macroscopic hematuria in a 32 years old man. the lesion was organconfined. results: grossly, the tumor was a well circumscribed, solid, brown and homogenous intraparenchymatous nodule. proliferating cells were arranged in a microfollicular pattern with colloid-like material resembling thyroid adenoma. cells displayed low grade nuclear features and had eosinophilic cytoplasm. some areas showed a solid pattern of growth resembling an oncocytoid neoplasm. by inmunohistochemistry, the tumor was negative for thyroglobulin and ttf1, and positive for ema, ck7, ae1/ae3, and ecadherin. conclusion: renal cell carcinoma with microfollicular thyroid-like features has been very recently identified in the literature. there is no agreement on the exact nature of this neoplasm so far, and the 2004 who classification of renal tumors still does not consider this phenotype as a distinct histological subtype. anyway, the tumor must be distinguished from metastatic thryroid carcinoma, another quite unusual condition. evidence for clonal fibroblast proliferation and autoimmune process in idiopathic retroperitoneal fibrosis l. cheng * , j. clevenger, a. lopez-beltran * indiana university, dept. of pathology, indianapolis, usa objective: we sought to determine if idiopathic retroperitoneal fibrosis is clonal process and if it is an autoimmune, or igg4-driven, process. method: thirty cases of idiopathic retroperitoneal fibrosis, in whom known causes of retroperitoneal fibrosis were excluded and those for which paraffin blocks were available, were included in this study. we performed clonality analysis in 16 female patients. genomic dna samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser capture microdissection. results: eight of 15 information cases (53 %) showed nonrandom x-chromosome inactivation, or a clonal process. of the 26 patients for which igg4 analysis was performed, 14 (54 %) were positive for igg4-positive plasma cells and all were negative for alk. of the 12 patients, for which both clonality analysis and igg4 analysis were performed, 4 were clonal and igg4 negative (33 %), 2 were clonal and igg4 positive (17 %), 2 were nonclonal and igg4 positive (17 %), and 4 were nonclonal and igg4 negative (33 %). conclusion: our data indicate that a significant proportion (53 %) of idiopathic retroperitoneal fibrosis cases in females is associated with a clonal expansion of fibroblasts. in addition, a subset of idiopathic retroperitoneal fibrosis cases could be classified in the igg4-related sclerosing disease spectrum. although a conclusive association with malignancy, urologic disorder, or systemic disease has not been established, often the lesion carries a challenging clinical differential diagnosis that includes malignancy. we examined clinical and histopathologic characteristics in 41 patients. medical records were assessed for presentation, clinical diagnosis, associated urothelial carcinoma, radiation treatment, tobacco use, immunologic/urologic disorder, and treatment strategy/outcome. results: the mean age was 68 (range 28-87 years). presenting symptoms were: pain (37 %), hematuria (27 %), and dysuria (20 %), in contrast to asymptomatic (32 %). clinical diagnosis favored malignancy in 10 % of cases. concurrent or subsequent urothelial carcinoma was present for five patients (12 %), though none developed urethral carcinoma. histologic features included mixed hyperplastic urothelial and squamous lining, overlying a variably fibrotic, edematous, inflamed, and vascular stroma. invaginations of urothelium extending into the stroma were common (68 %), showing rounded nests with cystic or glandular luminal spaces, similar to urethritis cystica/glandularis, without intestinal metaplasia. two lesions included an organizing thrombus, one with intravascular papillary endothelial hyperplasia. twenty patients were treated with topical medications without resolution. three lesions recurred (7 %) after excision. conclusion: urethral caruncle is an uncommon lesion that may clinically mimic benign and malignant conditions, making tissue diagnosis critically important. human papillomavirus (hpv) is not involved in urothelial tumorigenesis l. cheng * , r. alexander, a. lopez-beltran * indiana university, dept. of pathology, indianapolis, usa objective: the purposes of this study were to investigate the possible role of human papillomavirus in the development of squamous cell carcinoma of the urinary bladder and to determine if p16 expression could serve as a surrogate marker for human papillomavirus in this malignancy. method: forty-two cases of squamous cell carcinoma of the urinary bladder and 27 cases of urothelial carcinoma with squamous differentiation were investigated. hpv infection was analyzed by both in situ hybridization at the dna level and immunohistochemistry at the protein level. p16 protein expression was analyzed by immunohistochemistry. results: human papillomavirus dna and protein were not detected in 42 cases of squamous cell carcinoma (0 %, 0/42) or 27 cases of urothelial carcinoma with squamous differentiation (0 %, 0/15). p16 expression was detected in ten cases (31 %, 13/42) of squamous cell carcinoma and nine cases (33 %, 9/27) method: a review of clinical data from an adpkd patient with tcc admitted in our hospital and a review of current literature regarding adpkd and tcc were made. results: the patient is a 77 year-old woman with chronic renal failure due to adpkd, on hemodialysis for 2 years. she was admitted with left lumbar pain, hematuria and fever. radiological exams revealed various complex cysts with dense material that did not enhance with contrast, compatible with hematic cysts in the context of adpkd. the whole clinical findings suggested cystic infection complicated with sepsis, so nefrectomy was performed. macroscopic examination of the ressected kidney revealed a white granulous nodular formation with 4.5 cm diameter in the renal pelvis. histological examination confirmed a highgrade papillary tcc with parenchymal infiltration (pt3) and extense scamous differentiation. conclusion: the present case illustrates that tcc can occur in adpkd, despite its rarity. it can be difficult to successfully diagnose tcc on adpkd based on clinical-radiological findings. objective: carcinoid tumor is a very uncommon neoplasm in the kidney. we report the histopathologic and immunohistochemical (ihq) study of two new cases. method: case 1: 48 year-old female with a 4 cm asymptom-atic¨cystic¨mass discovered in a routine exam. the patient underwent tumorectomy. case 2: 77 year-old female with a 3 cm renal mass who underwent left nephrectomy. two months later a needle biopsy confirmed carcinoid tumor metastasis in the liver. both patients currently free of disease. results: both tumors showed similar histologic features. neoplastic cells were grouped in nests, ribbons, and pseudoglands with rosette-like appearance and showed eosinophilic granular cytoplasm and chromatic nuclei. low mitotic index. ihc: diffuse/intense cytoplasmic staining for cd56, cd57, ae1/ae3, chromogranin and synaptophysin. conclusion: primary renal carcinoid tumor is rare in the clinical practice. the histological findings correspond to a well differentiated neoplasm and are similar to carcinoid tumors in other locations, which makes the diagnosis feasible even without previous personal experience. ihc confirms the diagnosis. it usually behaves as a low grade neoplasm, but recurrences and metastases do occur. only single cases and short series have been published so far. there is no accumulated experience to establish long term prognosis. prostatic adenocarcinoma occurring simultaneously with large cell neuroendocrine carcinoma of the urinary bladder: an extraordinary collision tumor p. czapiewski * , m. sieczkowski, m. matuszewski, k. krajka, w. biernat * medical university of gdansk, dept. of pathology, poland objective: radical cystoprostatectomy is a standard surgical procedure for male patients with muscle-invasive urinary bladder (ub) carcinoma. vast majority of these tumors are urothelial carcinomas, while large cell neuroendocrine carcinoma (lcnec) is a very uncommon tumor with less than 20 reported cases. invasive prostate carcinoma is incidentally detected in up to 20 % of cystoprostatectomy specimens. it is usually well differentiated and shows low propensity for dissemination and local recurrence. method: clinical and pathological description of an extremely rare collision tumor composed of lcnec of the urinary bladder and a high grade acinar prostate carcinoma. results: a 79-year-old male patient was admitted to the urology department due to severe gross haematuria. cystoscopy revealed large tumor of the posteriorinferior wall of the ub with involvement of the left uretheral orifice. in the radical cystoprostatectomy specimen lcnec of the ub, involving bilaterally prostate and spermatic vesicle (pt4a), was diagnosed. additionally both lobes of the prostate were involved by the acinar prostatic carcinoma (pt2c, gleason score 5+4=9). conclusion: high-grade prostate carcinoma may rarely coexist with ub tumors of uncommon histology. objective: despite of numerous researches in the field, data regarding immunohistochemical (ihc) expression of androgen receptor (ar) in prostate carcinomas, in terms of prognosis and therapy, are quite controversial. method: the ihc expression of ar was analyzed on 3 groups, each of 20 primary diagnosed prostate carcinomas: localized, locally advanced and distant metastasized. the anti-ar antibody (clone ar441) was used. for each tumor was determined the percentage of ar-positive nuclei, assigning a staining score (0 to 4). a value of 64 % was considered to be discriminat o r y b e t w e e n t u m o r s w i t h h i g h a n d l o w a r expression. results: all the analyzed carcinomas showed ar. 54.2 % of tumors had high ar expression and 45.8 % showed low ar expression. 7/19 localized tumors (one tumor vanished during processing), 15/20 locally advanced and 10/20 distant metastasized tumors showed high ar expression (p=0.051). 5/18 well-differentiated, 16 of 23 moderately differentiated and 11/18 poorly differentiated tumors showed high expression of ar (p=0.022). conclusion: although not reaching statistical significance, the ar expression tended to correlate with the stage of disease and with the degree of differentiation in terms of an increased expression of ar in advanced, moderately and poorly differentiated tumors. objective: prostate lesions with atypical stromal component are rare and poorly characterized in terms of evolution and prognosis. method: we retrospectively analyzed 3 cases of unusual prostate tumors diagnosed on prostate needle biopsy. the biopsies were performed due to an elevated serum psa and/ or phenomena of bladder outlet obstruction. for the immunohistochemical (ihc) diagnosis there were used the following antibodies: ck ae1/ae3, psa, psma, vimentin, sma, er, desmin, pgr, cd34. results: two of the three patients, one 68 and the other 66 years-old, with serum psa level of 34 ng/ml and 14.58 ng/ml respectively, were diagnosed with sarcomatoid carcinoma, the former presenting with liver metastases at diagnosis. in evolution, the death of the first patient occurred 3 months after the diagnosis and the second patient refused surgery. the third patient, 75 years-old, with a nodular prostatic mass of 3.3 cm, was clinically suspected to have an unusual malignant prostatic tumor considering that, despite his 7 ng/ml of serum psa, he presented multiple bone metastases. the histopathological diagnosis was stromal tumor of uncertain malignant potential. conclusion: the prostate lesions with atypical mesenchymal component are rarities in urologic pathology. an accurate diagnosis relies on meticulous pathological examination techniques and supplemented, for rare cases, with other clinical and laboratory data. immunohistochemistery study in a case of nephrogenic bladder adenoma m. r. farzaneh * , a. safaei, a. amin shareefi * shiraz, iran objective: nephrogenic adenoma is a rare benign lesion of bladder that may be confused with malignant lesions. there is strong relation with urinary tract irritation and intravesicl instrumentations. nephrogenic adenoma was initially thought to originate from urothelial metaplasia however no solid proof is available. method: we present this 55 years old lady with urinary problem. cystocopic examination show a sessile mass and biopsy show circumscribed proliferation of tubules, cysts, and papillae lined by cells with low cuboidal to columnar epithelial cell. nephrogenic adenoma can be a significant diagnostic pitfall as certain histological features, such as the presence of enlarged nuclei with prominent nucleoli results: immunohistochemistery study showed strong reactivity to ck7, p504s, cd10, and ema but negative for ck20, psa, and p63. conclusion: we recommend that any lesion in cystoscopic examination should be followed and immunohistochmistery examination some times is mandatory to differentiate them from malignant tumors. regulators of apoptosis and the cell cycle are overexpressed in bladder cancer metastases and may predict survival a. fleischmann * , r. seiler, a. perren, t. george * universität bern, institut für pathologie, switzerland objective: expression of biomarkers and their prognostic relevance may differ between primary tumours (pt) and its metastases (met). there is little information about this phenomenon concerning apoptosis and cellcycle associated biomarkers in urothelial bladder cancer (ucb). method: nodal positive patients (n=152) with ucb underwent cystectomy and lymphadenectomy. immunohistochemical expression of bcl-2, bcl-6, mdm-2, p53 and cyclind1 was quantified in tissue microarrays constructed from pt and corresponding nodal met. results: frequency of mdm-2 positivity increased from pt (17 %) to met (37 %, p < 0.001). this trend was not significant for bcl-2 (pt: 7 %; met: 10 %, p=0.4) and bcl-6 (pt: 8 %; met: 12 %, p=0.08). median percentage of p53 and cyclind1 immunostained cells increased significantly (p<0.05) from pt (15 %/30 %) to met (40 %/50 %). mdm-2 and cyclind1 expression were positively correlated in pt (p<0.004) and met (p<0.002). p53 and mdm-2 positivity were inversely correlated in pt (p<0.03). only high cyclind1 expression in the metastases predicted early death significantly and independently (p=0.017). conclusion: biomarkers of apoptosis and cell-cycle associated are up-regulated in the metastases indicating differences in survival and proliferation of cancer cell compared to primary tumours. both tumor components may harbor different prognostic information and are not necessarily surrogates for each other. evaluation of histopathologic and histomorphometric changes of testicular tissue and gonadotropins levels following consumption of methylphenidate in male mice z. ghahri saremi * , s. fazelipour, z. tootian, m. shafii * tehran, iran objective: one of the most common psychiatric disorders in children is adhd (attention deficit hyperactivity disorder), which is treated extensively by methylphenidate. this study investigates the assessment of the effects of methylfenidate on histopathologic and histomorphometric changes of testes and serum level of gonadotropin in adulthood which produces gametes and has importance for future generations. method: in this study 36 adult male mice (balb/c) were used. after determining the body weight, the animals were divided randomly into two experimental groups of and one control group. the experimental groups received ritalin via gavage as follow: the group 1 received 2 mg/kg/day and the group 2 received 10 mg/kg/ day for a period of 40 days. after evaluation of body weight, general anesthesia was used for taking blood samples from the heart in order to measure gonadothropins levels in serum. then for the purpose of body weighing and measuring of diameter of germinal epithelium the testes were removed and the possibility of any pathologic changes was considered. results: the results showed that methylphenidate with different doses could decrease germinal epithelium and also body weight significantly. besides some significant changes in serum gonadotropins, without any pathological changes were observed. conclusion: our findings demonstrated that methylphenidate administration in adulthood due to influence of enhanced computed tomography and magnetic resonance imaging was characterized as 8×8×7 cm sized welldemarcated left renal middle-superior polar mass with central necrosis and cystic changes. the contralateral kidney was normal. he underwent left radical nephrectomy by thoracoabdominal approach. gross examination of the specimen revealed yellowish white 9×9×7 cm sized mass, with multifocal necrozis and cystic changes. histopathology of these resection specimens; scattered spindle cells and foam cells, fine vascular network and necrosis. immunohistochemistry revealed cd68-positive xanthoma cells. the tumor also stained positive for vimentin, cd34, but negative for cytokeratin, ema, desmin, sma and myod1. a diagnosis of mfh was made. conclusion: mfh is a primitive mesenchymal tumor with some histiosytic and fibroblastic differentiation primary renal mfh is an extremely rare lesion. because this malign mesenchymal tumor is indistinguishable clinically and radiologically from renal cell carcinoma diagnosis and histopathology of this rare lesion are discussed. hyaline ring granulomas in the urinary bladder: a case report n. hammer * , n. gatt, j. de gaetano * swatar, malta objective: hyaline ring granulomas, or pulse granulomas, are rare inflammatory responses to vegetable matter, characterised by aggregates of hyaline rings and other inflammatory components around vegetable matter. the vast majority occur in the oral cavity and only a few specific extra-oral cases have been reported. we present a case of pulse granulomas in the wall of the urinary bladder, occurring in a 50 year-old gentleman with a concurrent history of diverticulitis and colo-vesical fistula. pulse granulomas were recently described in the bladder in association with interstitial cystitis, in mesocolonic fat and mesocolonic lymph nodes in association with inflammatory bowel disease and in enterocutaneous and recto-salpingeal fistulas in association with diverticulitis. to our knowledge this is the first case reporting the direct association of the occurrence of bladder pulse granulomas in relation to diverticulitis. method: the tissue sections were fixed with 10 % buffered formalin and stained with h&e. results: histopathological examination revealed several hyaline ring granulomas within the outer muscle layer and serosa of the urinary bladder, together with hyaline ring granulomata within the outer wall of the sigmoid colon associated with diverticula. conclusion: it is important to differentiate pulse granulomas from parasites, hyaline ring vasculopathy, granulomatous inflammatory disorders and even malignancy. objective: micropapillary carcinoma (mpc) is associated with poor prognosis, and may lead to immediate cystectomy irrespective of stage. we study mpc, with "classical" histologic features, to "stage-matched urothelial carcinoma (uca), not otherwise specified (nos)", and other uca with divergent differentiation to compare outcome. method: 89 cases from 80 patients with mpc, uca with squamous (sq) or glandular (gl) differentiation, small cell carcinoma (smca), and nested variant (neca) variant of uca were identified. histologic and clinical data including stage and outcome were collected. results: all groups show male predominance and similar age range. mpc showed the highest rate of nodal metastases (table 1) . patients with mpc tumor volume of >50 % showed the highest rate of nodal metastases of all other subgroups. the majority of patients with mpc died, a large percentage from unknown causes. compared to mpc, sq showed similar presentation at higher stages and similar survival, but a lower propensity for nodal metastases. in our cohort, gl presented at lower stages, showed slightly better survival, and a lower rate of nodal metastases, when compared to mpc. our cohort included fewer cases of smca and neca. however, both patients with neca died of disease. conclusion: mpc, particularly when it represents >50 % of tumor volume, shows higher rate of nodal metastases than remaining subgroups, and is associated with a larger percentage of patient deaths. objective: müllerian-like stroma with er/pr expression is typical of renal mixed epithelial and stromal tumor and has been described in kidneys with obstructive pathology. wt1 overexpression is not reported in this setting. method: immunohistochemical study for er, pr and wt1 in autosomal dominant polycystic kidney disease (adpkd) (n=5), acquired cystic kidney disease (ackd) (n=5), xanthogranulomatous pyelonephritis (xp) (n=5), and renal lithiasis (rl) (n=5). controls: fetal (<24 w, n=4; >24 w, n= 4) and adult autopsy kidneys (n=5). results: stromal er was found in 100 % of adpkd, ackd, xp and rl; pr expression in 100 % of adpkd, ackd and rl and 80 % of xp, and nuclear wt1 in 20 % of xp and rl. er were negative in 100 % of controls. we found pr expression in 100 % of fetal controls of second trimester. wt1 was negative in the stroma of adult controls (100 %). 80 % of fetal controls showed wt1 expression in the peripheral cortex. conclusion: er and pr expression is frequent in kidneys with chronic obstructive and inflammatory pathology. wt1 is expressed occasionally. more studies are needed to determine whether these findings are a consequence of renal obstruction or they are involved in its pathogenesis, as well as their potential therapeutic implications. telomerase expression in urothelial carcinomas of the urinary bladder: does it make sense for carcinogenesis or prognosis? d. kankaya * , s. kiremitci, k. zengin, c. tuygun, a. sertcelik * ankara university medical school, dept. of pathology, turkey objective: human telomerase reverse transcriptase (htert) has been reported as poor prognostic marker in several cancers. in the present study, we examined htert expression in urothelial carcinoma (uc) to investigate whether it has a role on the carcinogenesis or prognosis. method: immunohistochemistry was performed to detect htert protein expression in tisse microarray blocks consisting of cores of papillary uc (n = 63) and infiltrating uc (n = 13) cases. nucleolar staining was considered and staining scores (intensity × distribution) were determined. tumors were grouped as low or high in terms of histological grade, and as early (ta, t1), or late stage (t2-t4) in terms of pathological stage. results: htert expression was significantly higher in the papillary uc group (p=0.028). tumors in late stage were more likely to show low staining scores of htert (p= 0.013). no correlation was found with tumor grade and recurrence rate. no effect on survival.was detected. conclusion: these findings indicated an association of htert protein with early stage ucs as its expression significantly decreased with muscularis propria invasion. incidental multifocal renal cell carcinoma in graft nephrectomy with cellular rejection findings: concurrence of papillary renal cell carcinoma and renal carcinoma associated with xp11.2 translocation d. kankaya * , s. kiremitci, a. ensari, a. sertcelik * ankara university medical school, dept. of pathology, turkey objective: malignancy represents the second main cause of death in renal transplant patients and increase markedly by 20 years after transplantation. method: a 41 year old male who has been on hemodialysis for 8 months for chronic renal failure, underwent living kidney transplantation from his brother. he received immunosupressive therapy of siclosporin and prednisolon. twelve years later, due to the impairment of renal functions hemodialysis was started again and ultrasonography revealed chronic renal parenchymal disease. graft nephrectomy was performed with the pre-diagnosis of chronic rejection. results: macroscopic examination revealed two well-circumscribed tumor nodules with 12 mm and 6 mm in largest diameter. microscopically, both tumor nodules showed tubulopapillary organization. larger tumor consisted of clear cells with tfe3 positivity, whereas the small one showed eosinophilic cells with diffuse cytokeratin7 and amacr positivity, without any tfe3 expression. concurrence of papillary renal cell carcinoma and renal cell carcinoma associated with xp11.2 translocation was reported. non-tumoral renal paranchyme revealed interstitial inflammation, tubulitis and transmural arteritis with fibrinoid necrosis compatible with type iii cellular rejection. conclusion: as far as we know this is the first case in the english literature demonstrating concurrence of papillary renal cell carcinoma and renal cell carcinoma associated with xp11.2 translocation in a graft nephrectomy. the effect of postoperative intravesical bcg and mitomycin c therapy on recurrence in superficial bladder cancer d. kankaya * , k. zengin, a. sertcelik, c. tuygun, n. sertcelik * ankara university medical school, dept. of pathology, turkey objective: to evaluate the efficacy of two mostly used intravesical agent, bcg and mitomycin c, in case of tumor recurrence. method: between 2002 and 2008, we performed tur-bt to 127 patients whom pathology results were superficial bladder cancer. 41 patients were treated with intravesical bcg once for week 6 weeks then monthly up to one year beginning from 14. day postoperatively. 26 patients were treated with intravesical mitomycin c, beginning from first 6 h postoperatively and once a week for 8 weeks. 60 patients didn't get any further treatment, and excluded from study. results: for bcg group, expected disease free interval was 58.1 months, and 34.6 months for mitomycin c group. when two groups compared for expected disease free interval, statistically significant difference observed (p=0.017). for bcg and mitomycin c group, recurrence was seen in 13 and 9 patients, respectively. for patients treated with bcg, 1 and 3 years disease free survival rate was %92.7 and %75.7, respectively. in mitomycin c group 1 and 3 years disease free survival rate was %76.9 and %62.9. conclusion: intravesical adjuvant bcg maintenance therapy is more effective for tumor reccurence than intravesical mitomycin c therapy. papillary cystadenomas of the epididymis: case presentation p. katafygiotis * , l. abou-asabeh, a. nomikos, s. chranioti * hospital of voula, dept. of pathology, athens, greece objective: papillary cystadenomas of the epididymis are rare, first described by sherrick in 1956. they are considered hamartomatous lesions rather than neoplastic and may be seen as intrascrotal swellings in a wide age range. we report here the case of a 32-year-old male who presented with a swelling of the right testis over the past year. his past medical history was otherwise unremarkable. method: ultrasonography revealed the presence of an epididymal cyst. grossly the cyst had a 2 cm diameter with a cyst wall of 0.2 cm thick attached to a part of epididymis measuring 2×1 cm filled with clear fluid. results: histologically the cyst showed pseudopapillary structures lined by monotonous clear cells. there was mild nuclear pleomorphism, no mitotic activity and no necrosis. immunostaining for ae1/ae3, ema, were positive, as well as focally for ck5/6 whereas calretinin and cea was negative. similar pattern of staining was seen in the epididymal parenchyma. conclusion: papillary cystadenomas are benign neoplasms and there have been no reports of recurrence or metastasis in the recent literature. bilateral disease is often associated with von hippel-lindau syndrome. our patient received no further treatment and remains asymptomatic and well 10 months after the intervention. effect of neoadjuvant sorafenib treatment on histology of clear cell renal cell carcinoma and occurrence of circulating tumor fragments g. kats-ugurlu * * umc st. radboud, dept. of pathologie, nijmegen, netherlands objective: clear cell renal cell carcinoma (ccrcc) generally presents with a micronodular phenotype (mp) due to high expression levels of vascular endothelial growth factor (vegf-a). earlier we have shown that mp is associated with shedding of multicellular tumor fragments (mtf) into the circulation and pulmonary metastasis. we hypothesized that vegf inhibition will destroy mp resulting in less mtf. method: 8 ccrcc patients were treated for 4 weeks by daily administration of sorafenib (400 mg bid). three days after therapy, nephrectomy was performed and kidneys were perfused via the arteria renalis. venous perfundate was filtered and processed to agarcytoblocks for mtf. treatment effects were studied using immunohistochemistry. results: all tumors were ccrcc as demonstrated by high vegf-a expression. none of the tumors showed mp after sorafenib treatment. tumors showed large areas of necrosis and fibrinoid necrosis of the blood vessels, concomitant with profound perivascular inflammation. 6/8 ccrcc patients (75 %) had mft vs 33 % in a control group (p= 0.03 fisher's exact test). individual tumor cells in the mtfs showed increased mitotic activity. conclusion: sorafenib destroys mp in ccrcc, attacks tumor vasculature, causes extensive necrosis and inflammation. post-treatment mtf are increased in venous perfundate. care should therefore be taken with neo-adjuvant sorafenib treatment of ccrcc. objective: claudins are major transmembrane proteins of tight junctions. as the disruption of their function have important impact on tumorogenesis, invasion and metastasis. claudins became a focus of interest for targeting therapies. although their expression profiles have been studied in many organs, researches on claudin expression in bladder are in limited number. the aim of this study is to present the differential expression of claudins in invasive urothelial cell carcinoma (iucc), noninvasive papillary urothelial carcinoma (npuc), carcinoma in-situ (cis), papillary urothelial neoplasm of low malignant potentiall (punlmp) and control group (cg). results: 83 cases (31 iucc -further divided into:15 muscle invasive ucc, 16 ucc with lamina propria invasion-, 17 npuc, 13 punlmp, 7 cis and 15 cg) were evaluated with claudin-1, 3, 4. interestingly, high claudin-1 and -3 score in cg (100 %, 80 %), decreased significantly in all non-invasive lesions (mean 18 %, 22 %). claudin-4 expression appeared to decrease in iucc vs. others (%52 vs mean %82). conclusion: higher expression of claudin-4 in low-grade and non-invasive lesions may be used as a diagnostic tool. in terms of claudin-1 and -3, their decreased expression in non-invasive lesions when compared to cg and their trend to show more increased expression in iucc needs to be studied further in larger studies. can we rely on alternative sampling method of radical prostatectomy specimens? t. d. kokenek-unal * , a. s. oguz-erdogan, m. alper * yb research and training hospital, dept. of pathology, ankara, turkey objective: prostatic adenocarcinoma is the most common cancer and second leading cause of cancer death in men. the incidence of prostate cancer has been increasing because of efficiency of modern cancer scanning programs and residual awareness of the patients. for that reason, there is a considerable increase in the number of prostatectomy specimens in the university and research hospitals.it is costly and time-consuming procedure and causes in turn increased workload. the aim of this study is to compare the results of total and alternative sampling methods and to delineate the differences if there are any. method: totally embedded 50 radical prostatectomy specimens were randomly selected and evaluated for key pathologic parameters. these cases then were reevaluated with limited sampling. the limited sampling method was built to include similarly embedded apical margins, bladder neck margins and seminal vesicles. in addition to that new slices were selected by skipping every other slice as differently from totally sampling protocol. results: the %37 reduction was achieved in number of blocks. the concordance rates between two sampling methods were %40, %50.4, %70.3, and %60 respectively for gleason scores, perineural invasion, extraprostatic extension and pathologic stages. conclusion: although the limited sampling protocol provides statistically significant results, because of critical role of pathological assessment in treatment of prostatic adenocarcinoma, they can be found unsatisfactory for many pathologists. objective: initially described in embryonic neuroepithelium, nestin is an intermediate filament involved in cell differentiation and transiently related to vimentin, keratin and glial fibrillary acidic protein (gfap). in tumoral and vascular proliferation, nestin is recognized in stem/progenitor cells. the octamer-binding transcription factor 4 (oct-4) is a biomarker of this lineage. since there are few data concerning the presence of nestin in leydig cells and testicular tumors, our aim was to investigate a series of leydig cell hyperplasia and tumors. method: 31 cases (13 hyperplasia, 14 adenomas, 2 malignant leydig cell tumors and 2 lymph node metastases) and controls were immunostained with anti-nestin, vimentin, keratin, gfap and oct-4 antibodies. results: twenty-six (84 %) cases (tumors and hyperplasia) were nestin positive with mostly weak, finely granular cytoplasmic staining. four (13 %) were negative, 1 (3 %) not interpretable. vimentin was expressed in twenty-seven (87 %) cases. seventeen (55 %) were focally keratin positive. only three (10 %) showed oct-4 positivity. all were gafp negative. conclusion: in leydig cell hyperplasia, adenomas and malignant tumors nestin and vimentin are expressed frequently, sometimes with keratin. this would be a sign of epithelialmesenchymal transition. further investigations are needed to understand the relationship with the malignant potential of these tumors. morphological changes of testis in atherosclerosis n. krupnov * , a. astrakhantsev * bureau of forensic medical exp., ryazan, russia objective: up to now in the field of morphology of reproductive system of men the problems of testis involution and morphogenesis at atherosclerosis haven't been thoroughly investigated. we have investigated histologically testis of 63 died patients aged 36-89 with general atherosclerosis and testis of 38 men aged 36-89 with minimal manifestations of atherosclerosis died in an accident. results: the absolute volume rete testis of patients being ill with atherosclerosis decreases by 65-80 %. absolute volume of convoluted seminiferous tubules and interstitial tissue decrease by 24-27 %. in testis there are zones of focal sclerosis of seminiferous tubules, whose area goes as far as 12.7 % of shear section area. it was revealed that on the periphery of sclerosis zones there is a reduction of transaction area of seminiferous tubules by 20-33 % with sertolli cells quantity reduction by 17-24 %. it has been stated the downward changes in index of spermatogenesis in convoluted seminiferous tubules by 63-65.6 %, accounted for the quantity reduction of all kinds of cells. conclusion: morphological changes of testis at general atherosclerosis characterize atherosclerotic testiculopatie, caused by chronic ischemia of testis. a leydig-cell tumor in a cryptorchid testis: report of a case e. lambropoulou * , a. datsis, p. morfaki, g. charalambopoulou * general hospital of messologhi, dept. of pathology, greece objective: leydig cell tumor is a rare form of testicular neoplasm, representing only 1-3 % of all testicular tumors. according to accepted medical knowledge, this type of tumor isn't linked to cryptorchidism, unlike germ cell tumors of the testicles. herein, a case of leydig cell tumor in a cryptorchid testis is described. method: a 59-year-old, unmarried man was admitted for surgical repair of inguinal hernia. his past medical history included untreated bilateral cryptorchidism first noted in childhood, as well as poliomyelitis that presented at age two. the past surgical history and review of systems were noncontributory. a ct scan of the abdomen and pelvis revealed two 3×3 cm testicles at the level of the inguinal canal. he underwent bilateral inguinal orchiectomy combined with surgical repair of his inguinal hernia. results: the right testis contained a solid, well-circumscribed, round, tan-colored tumor 1.5 cm in diameter. the histological appearance was that of a leydig cell tumor exhibiting no mitotic figure. there was no clinical or radiological evidence of metastatic spread. conclusion: although cryptorchidism is considered to be a risk factor for developing germ cell tumors, there have been a few reported cases of leydig cell tumors with a history of cryptorchidism. our case adds to the evidence that there may be a link between the two conditions. objective: cd44, e-cadherin, β-catenin are "cell adhesion molecules" and appear to influence development, inflammation, cancer invasion and metastasis. we studied the expression of these cams in prostatic adenocarcinoma (pca), high grade prostatic intraepithelial neoplasia (hgpin) and nodular adenomatous hyperplasia (nh). method: 135 specimens of radical prostatectomies were assessed. these cams were determined by immunohistochemistry. all sections included pca, hgpin, and nh. the expression of these markers was evaluated with three scores. the correlation of immunopositivity with gleason score and tnm stage was investigated. results: cd44 was strongly expressed in 41.5 %, 46.7 % and 37.8 % of nh, hgpin and pca, respectively. e-cadherin immunostaining was higly detected in 71.1 %, 78.5 % and 63.0 % of nh, hgpin and pca areas while β-catenin immunostaining was exclusively membranous in 80.7 % of nh and nuclear/cytoplasmic in 70.4 % and 48.9 % of hgpin and pca areas. all markers were unrelated to gleason score (p=0.352). cd44 and e-cadherin immunopositivities were inversely associated with tnm stage (p=0.021 and p=0.042 respectively); such an association was not observed in β-catenin (p=0.556). conclusion: cd44 and e-cadherin decreased expression is probably associated with invasive potential of prostate cancer. β-catenin staining pattern in neoplastic lesions differs from that in non-neoplastic prostate lesions. results: the loss of ecad expression was significantly higher at the tif when comparing with tscp and nnm. the loss of ecad was correlated with histological grade, infiltrative pattern, lymph node metastasis, perineural and vascular invasion. the vimentin expression showed association with histological grade, infiltrative pattern, t stage, lymph node metastasis, perineural and vascular invasion. conclusion: the loss of ecad and the gain of vimentin expressions occur more frequently at the tumor invasion front and are associated with classic factors of poor prognosis and low survival rates. prognostic value of immunohistochemical markers in bladder cancer i. m´sakni * , a. chaabane, f. bougrine, b. laabidi, a. bouziani * rades meliane, tunisia objective: the tumor stage and grade of bladder tumors are the major elements to define the prognosis. however, it is sometimes difficult to identify an infiltration of chorion or detrusor muscle. moreover, the evaluation of tumor grade is subjective and not reproducible. method: our objective is to study the prognostic value of the expression of proliferating cell nuclear antigen (pcna), ki67 antigen, the tumor suppressor gene p53, the protooncogene c-erb b2, the receptor for epidermal growth factor (egf-r), the apoptosis suppressor gene bcl2, carcinoembryonic antigen (cea) and epithelial membrane antigen (ema). results: the study showed that the pcna expression was significantly associated with the early recurrence (p=0.010) and the tumor stage (p=0.003). the mib1 expression was correlated to the early recurrence (p=0.010), and tumor progression in stage and/or grade (p=0.007). the c-erbb2 expression showed significant association with the tumor grade (p=0.007). the prognostic value of other markers has not been proven. conclusion: these findings may be useful providing better classification of bladder tumors thus the better management of patients. the c-erbb2 expression contributes to refine the tumor grading. pcna and mib1 can predict the early tumor recurrence; they could be relevant for the determination of endoscopic controls rhythms of patients. objective: primary urinary bladder neuroendocrine carcinoma (pubnec) is a rare tumor characterised by an aggressive behaviour and poor prognosis. method: we report five cases of pubnec diagnosed in the departement pathology of farhat hached hospital between 1990 and 2011. results: our population is composed by four mens and a woman. all patient are heavy smokers. the most common presenting symptom is hematuria and dysuria in all cases. one patient presented a complete urinary retention. a cystoscopic examination with transurethral resection was performed in all cases. the pathological examination with use of immunohistochemical markers of neuroendocrine differentiation were consistent with a large cell neuroendocrine carcinoma in four cases and a small cell neuroendocrine carcinoma in a case. a cystoprostatectomy was made in two cases followed by chemotherapy. conclusion: the clinical presentation of pubnec is similar to other bladder cancers and is characterized by advanced stage at diagnosis and rapidly progressive disease. the diagnosis of poses several problems: a vesical metastasis has to be excluded and such lesions have to be differentiated from transitional cell carcinoma, lymphoma, paraganglioma and peripheral nerve neuroblastoma. there is no gold standard for the management of patients affected due to low disease frequency ps-22-052 igg4-associated inflammatory pseudo-tumor (ipt) of the ureter: a case report a. marando * , g. d´ambrosio, f. catanzaro, f. sessa * university of insubria, dept. of surgical, varese, italy objective: igg4-associated inflammatory pseudo-tumor (ipt) is a novel clinico-pathologic entity characterized by intensive infiltration of igg4-positive plasma cells, associated with systemic igg4-related sclerosing disease. many reports described igg4-related ipt in various locations such as pancreas, salivary glands, liver, breast, lung and recently also ureter. method: it is described a case of ureteral ipt with pathologic and immunohistochemical features of igg4-related ipt, fibrohistiocytic type. results: the study case is a 82-year-old female with severe stenosis of the left ureter and hydronephrosis, who underwent to nephroureterectomy and endoscopic resection of multiple lesions in the bladder. the histological examination showed transmural fibrosing inflammatory lesion of the affected ureteral wall, with abundant plasma cells intermixed with many histiocytes, lymphocytes, fibroblasts and scattered eosinophils. the majority of infiltrating plasma cells were positive for igg4. bladder lesions showed similar histological features. the diagnosis was igg4-related ipt, fibrohistiocytic type. conclusion: igg4-related ipt of ureter is extremely rare, with only few cases reported in literature. recognition of this entity is clinically relevant because this type of ipt can be treated with steroid therapy and may be associated with sclerosing autoimmune disease in other organs. a rare case of malignant fibrous histiocytoma of the urinary bladder s. mavropoulou * , z. tatsiou, i. amplianitis, p. nasos * general hospital, laboratory of pathology, xanthi, greece objective: malignant fibrous histiocytoma (mfh) is an extremely rare malignant mesenchymal neoplasm of the urinary bladder with only a few well-documented cases reported in the english literature. method: we report the case of an 88-year-old man who was brought to our hospital due to sudden massive haematuria. catheterization failed to obtain haemostasis so, a suprapubic incision and direct exploration of the bladder was performed. a large solid tumor was found with a shaggy haemorrhagic surface and biopsies were taken. results: histological examination revealed an infiltrating malignant neoplasm composed of variably pleomorphic ovoid neoplastic cells with eosinophilic cytoplasm, bizarre tumor giant cells and prominent stromal osteoclastic giant cell reaction. immunohistochemical examination was negative for cytokeratin, desmin, smooth muscle actin, psap, nse, s100protein and hmb45 whereas it was positive for cd68 in a large number of tumor cells. accordingly, the diagnosis of undifferentiated pleomorphic sarcoma with histological features compatible with giant cell mfh of the urinary bladder was made. the patient died 4 days after the diagnosis. conclusion: in conclusion, mfh of the bladder should be kept in mind when facing with an undifferentiated malignant tumor. despite the poor prognosis, early diagnosis and aggressive salvage therapy might offer the chance of long-term survival in selected cases. partial nephrectomy experience at a single tertiary-care oncology centre: a clinicopathologic study of 60 cases s. menon * , g. bakshi, h. tongaonkar, v. noronha, a. joshi, k. prabhash, s. desai * tata memorial hospital, dept. of pathology, mumbai, india objective: partial nephrectomy (pn) is replacing radical surgery as a gold standard in the treatment of small renal masses. intra-operative frozen consultation for margin status is aimed at achieving a disease free state in order to reduce chances of recurrence. method: a retrospective clinicopathologic analysis of consecutive pns performed at our institute from 2004 to 2011 was undertaken. results: sixty cases of pn were analysed. male to female ratio was 1.8:1. median age was 51 years. six cases were benign: oncocytomas (3), angiomyolipoma (3); while 54 cases were malignant: renal cell carcinoma (rcc) -conventional (43), papillary (8), chromophobe (1), mucinous tubular spindle cell carcinoma (1) and one case of primitive neuroectodermal tumour. in 4 patients, the renal tumour was a second malignancy. median tumour size was 3.5 cm with 42 cases of stage pt1. intra-operative margin was positive in 16 cases. mean margin for all cases was 2.3 mm. median follow-up was of 24 months. none of the tumours recurred or metastasized during follow-up. conclusion: conventional rcc is the commonest histology in pn cases. frozen section analysis has a definite role in achieving margin free status. pn is not associated with increased risk of local recurrence in small renal tumours. clear cell tubulopapillary renal cell carcinoma: a clinicopathologic study of two cases g. muñiz * , a. corominas, n. cerda, a. perez, v. caamaño, m. gonzalez, l. etxegarai, j. i. lópez * hospital universitario cruces, dept. de anatomía patológica, barakaldo, spain objective: cleal cell tubulopapillary renal cell carcinoma has been recently identified as a low grade renal cell tumor with distinct histological features. method: patient 1: 65 year old female with a history of fibromyalgia and persistent loin pain. a right renal mass, 2.8 cm in diameter, was discovered in the rheumatologist's follow up. right nephrectomy was performed and the patient is free of disease 7 months later. patient 2; 55 year-old female with a history of diabetes mellitus type 2, hypertension and renal failure grade 3 with a 2.5 cm in diameter renal tumor in the routine studies. she underwent tumorectomy. results: grossly, both were cystic tumors with gelatinous fluid and white-yellowish solid areas. tumors showed a tubular and papillary architecture. proliferating cells displayed clear cell cytoplasm and hyperchromatic nuclei placed in the luminal side. ihc showed positive staining with e-cadherin, ema and ck7. conversely, cd10, cd117 and amacr were negative. conclusion: clear cell tubulopapillary renal cell carcinoma should be considered a distinct subtype of renal cell carcinoma according to its unique morphologic and inmunohistochemical features. the few cases reported so far behave in an indolent course. survivin expression in renal epithelial tumors: its usage in the differential diagnosis of eosinophilic renal epithelial tumors a. ozcan * , n. yigit, o. onguru, b. a. firat, s. ozaydin * gulhane military medical academy, dept. of pathology, ankara, turkey objective: the differential diagnosis of renal tumors can be problematic due to overlapping morphologic features. the purpose of this study was to assess the potential contribution of survivin expression in the differential diagnosis and determination of therapy modalities of these tumors. method: this study consisted of 15 chromophobe (chrcc), 15 clear cell (ccrcc) and 9 papillary (prcc) renal cell carcinomas, and 13 oncocytomas. sections were stained against survivin antibody. results: prccs and ccrccs showed diffuse and strong survivin expression. survivin expression was strikingly prominent in type1 prccs and cystic ccrccs. in ccrccs, survivin expression was more pronounced in low grade areas than high grade and sarcomatoid areas. in chrcc, survivin expression was more limited and weaker than that of oncocytomas and other malignant renal tumors. in non-neoplastic renal tissue, survivin expression was more pronounced in podocytes and atrophic tubules than other nephron parts. conclusion: our findings suggest that survivin may contribute to the differential diagnosis of renal tumors because of the partially unique staining patterns. it was purposed that knockdown of survivin reduced growth, induce apoptosis and enhance in vitro radiosensitivity of rcc cells. taken together, to be known different survivin expression patterns in renal tumors may help to determine new therapeutic strategies for rccs. objective: there are several tumor-like lesions and miscellaneous neoplasms of rete testis. we present a case with adenomatous hyperplasia of rete testis (ahrt). method: the patient was 24 years old with undescended testis and referred to our hospital. there was no clinical or endocrin abnormalities. cryptoorchidism was unilateral and the other testis was normal. right orciectomy was performed and sent to pathology labarotory for examination. results: there was no tumoral lesion in gross examination but in microscobic examination there was gland like tubular structures. some of these were back to back position with little intervening stroma and mild to moderate atypia. ema and pancytokeratin immunohistochemistry findings with morphology confirmed the diagnosis of ahrt in this case. conclusion: ahrt is a rare proliferatif lesion and can be confused with malignancy. it is incidentally realised in microscobic investigation. it may present as a very small lesion detected in microscobic examination or solid-cystic mass lesion which is macroscobically evident. clinic history, localization, histologic features and immunohistochemistry are criterias for differentiating these lesions.we present this case for both surgeons and pathologists with its importance to be confused with malignancy. objective: prostate cancer is the second leading cause of death in men. the localized disease often responds to conventional therapies like androgen ablation via castration and/or administration of chemical inhibitors but advanced disease resistant to any curative therapies is still challenge for investigators. there are increasing efforts to enhance the possibility of finding positive and sensitive immune markers for diagnosing and treating prostate cancer. method: we applied immunohistochemical markers; amacr and inos. formalin-fixed parafin embedded tissues of 64 prostate needle biopsy specimens diagnosed as prostate adenocarcinoma between 2005 and 2010 years were enrolled in the study. results: amacr expression has been found in 58 (90.6 %) and inos expression in 54 (84.4 %) of 64 prostate adenocarcinomas.no significant relationship of amacr and inos has been obtained (p>0.05). there was no significant correlation of histopathologic grade of the tumors with amacr and inos expression (p>0.05). conclusion: the expression of amacr and inos might be important diagnostic immune markers for prostate adenocarcinomas especially in needle biopsies when the quantity and quality of tissue are limited. a tissue microarray study of napsin-a expression in renal tumors a. panizo * , f. j. queipo, j. j. sola, j. pardo * hospital de navarra, anatomia patologica, pamplona, spain objective: napsin-a is an aspartic protease present lung, renal, and thyroid cells. there are few studies evaluating napsin-a in renal neoplasms. therefore, we studied ihc expression of napsin-a in a wide spectrum of renal tumors. method: ihc for napsin-a (rabbit polyclonal antibody) was performed in a series of 334 cases of primary and metastatic renal tumors on tma. cytoplasmic immunoreactivity was scored: intensity (0-3+) and extent (% of tumor cells: 0-3). the 2 scores were added: positive case: combined ihc score>2; negative if combined score of 2 or less. objective: the diagnosis of prostatic carcinoma can be challenging on needle core biopsies. the aim of this study was to assess the utility of alpha-methylacyl-coa racemase (amacr/p63) antibody cocktail for prostate cancer diagnosis. a prospective analysis of 50 consecutive radical prostatectomy specimens and 50 prostate needle biopsy semples was performed to select histological sections showing foci of minimal prostatic carcinoma, high grade prostatic intraepithelial neoplasia (hgpin) and benign mimickers of prostatic carcinoma (atrophy, adenosis). method: serial histological sections were stained with hematoxylin and eosin, van gieson and immunomarkers: amacr and p63 using a prediluted antibody cocktail. results: the cocktail was very useful in highlighting prostatic carcinoma associated with hgpin (flat or cribriform) and distorted foci of minimal carcinoma. amacr was positive with moderate and strong staining in almost all cases for which the immunohistochemical result converted the atypical diagnosis to a final cancer diagnosis. the cases whose diagnosis was changed from "atypical" to cancer were all highly suspicious for cancer based on he histology and negative basal cell markers. conclusion: this cocktail would be of diagnosis utility when limited tissue is available for histopathological evaluation of small diagnostically difficult foci (prostate needle biopsy and surgical specimens). overexpression of cytokeratin 20, ki-67 and topoisomerase-ii-a can significantly stratify the recurrence risk in patients with bladder cancer after transurethral resection s. petrov * , k. malkhasyan, r. khasanov * kazan cancer center, dept. of pathology, russia objective: the current predictive models based on main clinical tumor features are not accurate for the biggest bladder cancer patient group, who underwent the transurethral resection (tur). method: overall 103 patients with primary urocarcinoma after tur were included in this retrospective study. the follow up plan in all cases included cystoscopy and biopsy every 3 months in the first 2 years. the recurrence criteria were cystoscopical and pathological confirmation of the tumor growth. in all cases using tma technique (tma master, 3dhistech) there was done the ihc expression study of p53, p63, ck20, e-cadherine, b-catenin, cd44v6, ki-67 (10 % cut-off), topo-ii-a (10 % cut-off) and her2, as well c-erb-b2 amplification study (cish). results: there were no association found between the her2 expression and c-erb-b2 amplification in urocarcinoma patients. in multivariate regression analysis only ck20, ki-67 and topo-ii-a showed the significant prognostic power in recurrence prediction. these markers were used to develop the powerful predictive index for bladder cancer patients after tur. conclusion: although the her2 overexpression is relatively common event in bladder cancer, c-erb-b2 gene amplification isn't main mechanism of its realization. the ck20, ki-67 and topo-ii-a are promising prognostic markers for recurrence and should be validated in further prospective study. solitary fibrous tumor of the urinary bladder associated with a high-grade urothelial invasive carcinoma. a case report a. pitino * , s. squillaci, c. spairani, m. ferrari, m. f. cosimi, w. fusco, c. rossi, f. montefiore, g. l. bigatti, v. la paglia * san giacomo hospital, division of anatomic pathology, novi ligure, italy objective: solitary fibrous tumor (sft) is an unusual spindle cell neoplasm, which can exceptionally occur in the urinary bladder. method: we present a case of urinary bladder sft in a 60 year-old man who complained of pelvic pain. cystoscopy revealed a large protruding, fleshy mass at the anterior wall of the bladder. a biopsy was first misclassified as inflammatory myofibroblastic tumor (imt). subsequent complete transurethral resection was performed. results: macroscopically, a 9×7×5 cm greyish lobulated firm polypoid tumor was seen. microscopically, it consisted of uniform spindle cells with elongated tapered ends nuclei forming a patternless growth in a collagenous background. mitotic figures were rare. immunohistochemically, the neoplastic cells were positive for cd34 and bcl-2, and negative for α-sma, desmin, ck ae1/ ae3 and alk-1. the bladder urothelium showed foci of high-grade transitional carcinoma with lamina propria invasion. conclusion: initially, sfts were thought to be of mesothelial origin. then, these tumors have also been observed in extrapleural and extraserosal sites, which suggests a mesenchymal cell origin. the differential diagnosis should always include other spindle cells lesions such as sarcomatoid carcinoma, leiomyosarcoma and imt. to date, this is the first reported case of association of urinary bladder sft and high-grade urothelial invasive carcinoma. renal oncocytomas with unusual features: clinicopathological study of 9 cases f. j. queipo * , h. d. quiceno, f. j. pardo, án. f. panizo, m. l. gómez-dorronsoro, g. aisa, f. j. monzón, e. mejía, c. del agua, j. alfaro * clínica universidad de navarra, pathology, pamplona, spain objective: oncocytoma (ro) is a benign renal neoplasm, with a wide morphologic spectrum and excellent prognosis. recently, it has been described worrisome morphological features. method: ro treated in our centres were reviewed, and we focused on identifying the worrisome and the atypical features. results: we identified 9 ro with at least one of the worrisome feature. patients: 5 m/4 f (mean age 72,89 year; range: 63-88). mean tumor size: 4,24 cm (range: 1,6-11,5 cm); right kidney: 5, and left: 4 cases. invasion into the perinephric or renal sinus fat was the most frequent worrisome feature: 7 tumors (77,8 %) and focal chromophobe carcinoma-like areas in 3 cases (33,3 %). lymphovascular invasion, entrapped renal tubules-glomeruli, necrosis and mitosis were found in 2 (22,2 %) respectively. all but two ros had at least 2 worrisome features. followup was available for all cases (median 11 months; range 3-108): all patients were alive without recurrence or metastasis. conclusion: ro is a tumor which often can show worrisome and atypical morphology. it is necessary to know and recognize the worrisome features to prevent diagnostic errors, if otherwise typical oncocytoma morphology is present. despite these atypical morphological data, the prognosis is excellent. ki67 and p53 expression in urothelial carcinomas and clinicopathologic correlation a. ribeiro * * chlc, epe, serviço de anatomia patológica, lisboa, portugal objective: the most important predictive parameter for the biological behaviour of urothelial carcinoma is histological grade, except for depth of invasion. the aim of this study was to investigate the expression of p53 oncoprotein and ki67 antigen in a series of transitional cell bladder carcinoma with papillary morphology (ptapapillary carcinoma and pt1) with histological grade and recurrence. method: this study included 59 cases diagnosed with urothelial carcinoma with papillary morphology (pta, pt1). immunohistochemical expression of ki67 and p53 were examined in each case, and were graded accordingly to the percentage of cells stained, in low, moderate and high-expression groups. results: as described previously in other publications the expression of p53 and ki67 has a relationship with histological grade. we also noted that of the 13 recurrences, 11 were associated with moderate to high expression of either p53 or ki67, or both. conclusion: we concluded that p53 and ki67 expression combined with histological grade and pathological stage may be helpful in assessing more accurately the biological behaviour of urothelial carcinoma. and the overexpression of p53 and ki67 are related with an unfavourable prognosis. objective: prostatic stromal hyperplasia with atypia is a rare lesion with fewer than 200 cases reported worldwide. it can be mistaken for sarcoma because of the presence of atypical, bizarre cells. its malignant potential is uncertain. the follow up study of every case is important. method: a 71-year-old man with previously diagnosed atypical prostatic adenostromal hyperplasia with atypia of stromal cells was hospitalized with urinary obstruction after transurethral resection of prostate made 6 years ago. repeat turp was performed and tissue specimens were investigated and compared with the initial biopsy. results: the second biopsy showed the benign hyperplastic prostatic glands with atypical, bizarre, frequently multinucleated giant stromal cells between them. they displayed intense immunoreactivity for actin, vimentin and androgen receptors. the microscopical picture was identical to that seen in the first specimens. the nuclear abnormalities looked like in an atypical symplastic leiomyoma of myometrium. no evidence of sarcomatous or carcinomatous transformation, mitotic index evaluation was noted. conclusion: this case maintains the viewpoint that the prostatic adenostromal hyperplasia with stromal cell atypia is a benign lesion. but it can recur and requires the repeat turp or radical surgery. cancer risk in patients with precancerous lesions of the prostate y. rogov * , v. zakharava, t. liatkouskaya, e. cherstvoy * belarusian medical academy, dept. of pathology, minsk, belarus objective: prostatic intraepitelial neoplasia (pin) and atypical small acinar proliferation (asap) has a high predictive value as markers for prostate cancer (pca). method: pca-risk in patients with precancerous lesions has been assessed on biopsy material in 172 patients having morphological suspicious to pca. suspicious foci were estimated with use of cocktail amacr + hwc + p63. results: according to our results revealing of precancerous lesions in biopsy specimens has been associated with pca identification in re-biopsies (f=0,04). within the first-5years the overall incidence of pca in re-biopsies made 27 % in the group of precancerous lesions and 36 %in the asap group. life-time-without-pca median made up 5-years with no reliable difference between pin and asap groups (ww=2,35; p=0,06). thus, the cumulative share of patients without pca in the asap group formed 86 %-82 %-82 %-73 %-60 % at the end of the first-second-thirdfourth-fifth year of supervision respectively. conclusion: within the first 5 years pca risk makes 27 % in the general group of precancerous lesions of the prostate and 36 % in asap group with no difference between asap and pin groups. сumulative share of patients without pca in rebiopsy specimens decreased from 86 % to 60 % during these 5 years. benign prostatic hyperplasia and prostate carcinogenesis after the chernobyl accident in ukraine a. romanenko * , a. chekalova, a. yurakh, p. harkonen, s. vozianov * institute of urology, kiev, ukraine objective: the prevalence as well as immunohistochemical (ihc) study of latent, incidentally found prostate cancer (lpc) as well as precancer lesions, in patients, who underwent surgery for bph were studied. method: bph samples were obtained by prostatectomy from 120 ukrainian patients consisting of 30 patients from areas without radio-contamination (control group 1) and of 90 patients living in 137cs contaminated areas of ukraine (group 2). ki-67, p53, p27kip-1, p63 and bcl-2 proteins were ihc investigated in bph from all patients. results: the incidences of lpc (gleason score 4), chronic prostatitis, pia and pin were 16.67, 53.34, 20, and 26.67 % in group 1; 12.23, 64.45, 43.45 and 36 .67 % in group 2, respectively. greatly elevated levels of p53, ki-67, bcl-2 associated with decreased levels of p27kip-1and p63 in areas of pia and less lpc and pin in group 2 to compare with group 1 patients were obtained with statistically significant differences. conclusion: our study suggests that chronic long-term lowdose radiation exposure might result in the increase of chronic inflammation and it is now found to be associated with increased incidences of pia and pin in bph accompanied by p53, p27kip-1 and bcl-2 alteration which in turn could lead to prostate carcinogenesis. eosinophilic globules in rete testis mimicking yolk sac tumor in a testicle with seminoma e. ronne * , t. argyrakos, d. rontogianni * evangelismos hospital, dept. of pathology, athens, greece objective: the recognition of a non-seminomatous component in an otherwise typical testicular seminoma changes the choice of adjuvant chemotherapy. method: a 37-year old male underwent orchiectomy for a testicular mass. serum tumor markers levels (β-hcg/afp/ ldh) were normal. results: macroscopical examination revealed a 2.1 cm white-yellow tumor. the tumor was composed of the characteristic for seminoma homogenous, clear cells arranged in nests and islands separated by septa and infiltrated by lymphocytes. immunohistochemically the tumor cells were positive for plap, oct3/4, cd117 and d2-40 and negative for cd30 and ck8.18. there was also a regular pattern of tubular structures infiltrated by the seminoma cells while retaining a low columnar type epithelium. the presence of sphaerical eosinophilic globules (pas+/dpas+/afp−) within the tubular lumina was strongly reminiscent of the hyaline globules of yolk sac tumor. the tubular structures were positive for ck8.18 and negative for afp, glypican-3 and oct3/4. conclusion: the presence of tubular structures with sphaerical eosinophilic globules creates a suspicion for a yolk sac tumor component. the absence of afp and glypican-3 expression, the regular pattern of the tubular structures and their continuity with rete testis excluded this suspicion. hyaline eosinophilic globules in rete testis should not be confused with the globules produced by yolk sac tumors. beta-catenin expression and ctnnb1 mutations in a series of wilms tumours r. santi * , p. pinzani, f. salvianti, g. baroni, m. pepi, g. nesi * university of florence, pathological anatomy section, italy objective: ctnnb1 mutations have been found in 15-30 % of wilms tumour (wt) cases. nuclear beta-catenin protein has been detected by immunohistochemistry in a higher proportion of wts, thus suggesting alternative genetic pathways leading to beta-catenin activation in these neoplasms. method: sixteen renal wts and 7 secondary wt localizations were retrospectively investigated. the series included 17 paediatric patients, 10 females and 7 males, with a mean age of 4.5 years and a 34 year-old female patient. immunohistochemical analysis of beta-catenin was performed and findings were reported for each neoplastic component (i.e. epithelium, stroma and blastema). tumour dna was extracted for direct sequencing analysis. results: the majority of wts showed moderate to strong membranous staining for beta-catenin in the epithelial (76.5 %) and the blastemal (60 %) components. nuclear betacatenin expression was observed in combination with cytoplasmic staining in the mesenchyma and/or the blastema of two primary renal tumours. in these cases the deletion of codon 45 p.s45del and the missense substitution p.t41a were detected. conclusion: preliminary results of this ongoing study highlight beta-catenin cytoplasmic and membranous expression in the tumour cells of primary and metastatic wts, with few cases demonstrating nuclear expression. in our series, betacatenin nuclear expression was invariably associated to ctnnb1 mutation. a preliminary study on o6-methylguanine-dna methyltransferase and type 2 transglutaminase expression profile of renal cell carcinomas b. sarsik * , b. pehlivanoglu, d. tunali, a. simsir, e. gokmen, s. sen * ege university, faculty of medicine, izmir, turkey objective: o6-methylguanine-dna methyltransferase (mgmt) repairs o6-methylguanine in dna, therefore provides a tumor supressor effect. type 2 transglutaminase (tgase-2) is a multifunctional enzyme involved in many biological processes. few data are available in the literature on their expression in kidney cancers. in this preliminary study, we evaluated immunohistochemical expression of mgmt and tgase-2 in renal carcinoma cases. method: forty cases of renal carcinoma including ten clear cell, ten chromophobe cell, ten papillary and ten urothelial carcinoma were randomly selected. thirty-one patients were male and average age was 61,5. staining intensity and percentage of staining tumor cells were scored. total score was categorized as weak, moderate and strong. results: strong mgmt positivity was demonstrated in 15 cases (38 %). eight cases (20 %) showed strong tgase-2 expression. fifty percent of clear cell carcinomas strongly expressed mgmt and tgase-2. no correlation was found between mgmt and/or tgase-2 expression, tumor size and grade. eighty percent of urothelial carcinomas strongly expressed mgmt. conclusion: the prognostic value of mgmt and tgase-2 as well as their potential role in treatment response have been investigated recently. mgmt and tgase-2 may be prognostic factors in renal carcinomas. further investigation is required to verify our findings. three dimensional topographic analysis of 904 cases of radical prostatectomy a. n. seo * , k. s. lee, g. choe * seoul national univ. bundang, pathology, seongnam-si, objective: prostate cancer is typically mutifocal, and there has been no report about the exact number of prostatic carcinomas in each case. we have performed topographic analysis using three dimensional mapping technique. method: we established data base including 904 cases of radical prostatectomy consisting of 2717 individual adenocarcinomas, and performed comprehensive pathologic analysis. objective: tmprss2-erg gene fusion is the most common genetic alternation in prostate cancer. it is associated with the expression of oncogene erg protein. recently, the immunohistochemical staining method that using anti-erg antibody was verified strong correlation with erg protein which is the product of genetic alteration. aim of this study is to declare that the relationship between erg expression and clinicopathological factor. method: otal 307 cases of radical prostatectomy specimen were assessed. all cases were constructed tissue microarray and immunohistochemical staining was performed. results: erg-positive rate was 24.1 % (74/307) and significantly higher expression of erg expression was observed in the subgroup that has lower gleason score. (p<0.05) analysis with the histologic pattern of prostate adenocarcinoma, tumors with discrete glandular unit (gleason pattern 3) is shown higher frequency of erg expression (p=0.007). conclusion: erg-positive case was smaller than that of western population (about 50 %) and other factors including age, tumor volume, initial psa level, pathological stage and margin status were not significantly related with erg expression. in conclusion, positive rate of erg immunohistochemical staining is meaningful higher in the tumors with wellformed gland that is represented by lower gleason score. solitary fibrous tumour of the kidney mimicking renal cystic neoplasm t. tichy * , j. skarda * university hospital olomouc, institute of pathology, czech republic objective: solitary fibrous tumour (sft) can develop at any anatomic site, but in the kidney is described rarely. in general, sft forms an unencapsulated solid mass. we report a case of a 57-year old woman with benign sft of the left kidney. the tumour showed extensive pseudocystic change and mimicked renal cystic neoplasm. method: a nephrectomy specimen showed cystic tumour beneath renal capsule and in peripelvic adipose tisssue. histological sections were used for hematoxylin-eosin and for immunohistochemistry (antibodies against vimentin, smooth muscle actin, desmin, s-100 protein, cd117, cd99, bcl-2, cd34, ck18, ae1-ae3, ema). results: microscopically the tumour showed uniform spindle cell proliferation with expansive tumour margins, without necrosis or hemorrhagies. mitotic activity was 1 per 10 high power fields. cystic spaces without epithelial lining contained eosinophilic proteinaceous fluid. imunohistochemically tumour showed difuse positivity for cd34, cd99, bcl-2, vimentin and stained negatively for s-100 protein, cytokeratins, ema, smooth muscle actin, desmin and cd117. conclusion: sft of the kidney is infrequent. some renal sft can undergo pseudocystic transformation and mimic renal cystic neoplasm both clinically and macroscopically. objective: renal angiomyolipomas are mesenchymal tumors that comprised of adipose tissue, smooth muscle like cells and abnormal thick walled blood vessels admixed in various proportions. epithelial renal angiomyolipomas are rare variants and may exhibit atypia. in the literature epithelioid renal angiomyolipomas with atypia are reported to have malignant potential. method: left radical nephrectomy was performed due to renal mass with flank pain and hematuria. results: in gross examination; 6.5×3.5×3 cm gray white mass with occasionally necrotic areas at the middle portion of left kidney renal sinus and perinephritic fatty tissue, macroscopically. microscopic eveluation reveals; tumoral lesion comprised of adipose tissue, smooth muscle and blood vessels, showing areas of epitheloid morphology and moderate to severe degree of nuclear atypia. in addition to histomorphology positivity hmb-45 immunohistocemistry elaborated to the diagnosis of "epitheloid renal angiomyolipoma with atypia". conclusion: this case has been presented due to its infrequent occurrence and malignancy potential. correlation of minute focus of prostate adenocarcinoma on random multifocal needle biopsy with radical prostatectomy specimen a. urbanskiy * * russian research centre for ra, pathology, st. petersburg, russia objective: this work attempts to determine the importance of small foci of prostatic cancer in random multifocal needle biopsy specimens. method: 64 patients with a microscopic focus confined to a single core specimen (which defined as tumor less than 1 mm with a gleason score of 6 or less) were identified from a retrospective review of 1206 needle biopsies of the prostate. twelve of these 64 subsequently under went radical retropubic prostatectomy at our centre. clinically significant tumors were defined as those with volume greater than 0.5 cc. results: average tumor volume was 0,9±0,6 cc (range 0,068-2,9 cc). in 41,66 % (5) of the cases was less 0,5 cc (range 0,068-0,458 cc, mean volume -0,22±0,15 cc). there was 1 (8,4 %) of patient with extraprostatic extension. conclusion: our data have shown high frequency of revealing insignificant tumours (mean 42±28 %; р=0,95). however, about 60 % of patients had clinically significant tumors warranting definitive therapy. the smallest focus of cancer on needle biopsy is not a guarantee of a clinically insignificant tumor. ps-22-085 e-cadherin in mice: expression in normal urothelium, pre-neoplastic and neoplastic urothelial lesions c. vasconcelos nóbrega * , c. costa, r. arantes-rodrigues, a. henriques, h. vala, a. colaço, l. santos, c. lopes, p. oliveira * escola sup. agrária viseu, dzerv, portugal objective: e-cadherin is an adhesion molecule that promotes the integrity and stability of the urothelium. a decrease in its expression is associated with more aggressive tumour phenotypes, with the ability to invade and metastasize. our aim was to describe the expression of e-cadherin in normal urothelium and in urothelium with pre-neoplastic and neoplastic lesions of icr male mice. method: urothelial lesions were chemically induced by nbutyl-n-(4-hydroxybutyl) nitrosamine in icr mice, and evaluated by immunohistochemistry in order to determine the staining pattern of e-cadherin. results: in normal urothelium, 87.5 % of e-cadherin expression was at the cellular membrane level. in simple hyperplasia, the same pattern was observed in 66.67 % of lesions. nodular hyperplasia exhibited a mixed pattern (50 % membrane and 50 % cytoplasmic pattern). in 86.67 % of dysplasia, a cytoplasmic pattern was seen. on invasive carcinoma the majority of invasive urothelial cells exhibited a pattern of membrane and cytoplasmic staining. on squamous metaplasia it was observed a membrane pattern on basal and intermediate layers, and a loss of immunoreactivity in the most superficial ones. conclusion: e-cadherin is a valuable tool for investigating the cellular adhesion status of the urothelium in mice. neoplastic lesions exhibited an abnormal, heterogeneous staining pattern. new potential prognostic and predictive factors in conventional clear cell renal carcinoma p. latalova * , p. flodr * fmd pu and fh olomouc, dept. of clinical and molecular pathology, czech republic objective: biological behavior of conventional clear cell renal carcinoma (ccrc) is associated with tumour stage and grade. cancer progression with metastasis is part of a process epithelial-mesenchymal transition (emt) and is joined with invasiveness due to adhesion and cytoskeleton change. the central role of the emt mechanism is attributed to snail factor. the intermediate filament expression is changed through the progression of many types of neoplasms. these changes are organ or tissue specific and depend also on the degree of malignant transformation (genome instability acceleration). the expression changes of cytokeratin 18 (ck18) are signs of aggressive biological behaviour in some tumour types -e.g. colorectal and breast carcinoma (downregulation of expression) or ccrc (upregulation of expression). the expression of snail and ck18 molecules and mrna ck18 levels correlate to stage and grade of ccrc (according messai et al. 2010) . objective: study was based on the expression of immunohistochemical markers (ck18, ck7, vimentin, snail, cd10) in 15 cases with evaluation of the primary tumour and its metastasis. results: the difference and similarity of intensity and percentage of positive neoplastic population between the primary and the secondary carcinoma will be presented. conclusion: snail and ck18 seem to be potential tumour progression factors and may be included in so called personalised medicine. objective: sarcomatoid carcinoma (sc) of the prostate is a rare variant of prostatic cancer representing less than 1 % of prostate tumors. tumors are most commonly composed of an admixture of both malignant glandular and spindle cell elements. the sarcomatoid component can vary from 5 % to 99 %. method: we report the case of a 63-years-old patient with an invasive tumor of the prostate for which he had a radical prostatectomy. histopathological examination showed that the tumor was responding to an undifferentiated proliferation made of beaches and clusters of pleomorphic cells usually spindle-shaped, with eosinophilic cytoplasm, and elongated or oval nuclei, which are very irregular, sometimes monstrous, multinucleated, basophils with numerous mitotic figures. in some places, there were areas of glandular differentiation and foci of comedocarcinoma. immunohistochemical study showed immunoreactivity of spindle and pleomorphic cells with cytokeratin and vimentin. results: the diagnosis of sarcomatoid carcinoma of the prostate-grade gleason 5+5 was confirmed. conclusion: sc of the prostate is an exceedingly rare tumor. retrospective analyses render prostate sc as one of the most aggressive prostate malignancies. the prognosis is dismal regardless of other histologic or clinical findings. extra-uterine extension (figo stage ii-iv) were significant parameters of poor prognosis. no multivariate analyses were performed due series limitation. conclusion: detailed pathology evaluation, including histological subtype, and figo stage are essential in the adequate management decision/prognosis evaluation of u-lms. objective: over 90 % of women with endometrial cancer show signs of uterine bleeding, so most cases are diagnosed at an initial stage. this study covers two patients who presented the first signs of this disease in an exceptional manner. method: case-1 a 71-year-old woman was afflicted with a six-month toxic syndrome. an omentum tumor was identified in the x-ray. eleven days after the excision, she demonstrated oliguria, dysuria, and scant uterine bleeding. the pelvic ultrasound revealed a uterine mass. case-2 a 56-year-old woman demonstrated disabling right inguinal pain. after an x-ray examination of her pelvis, a metastasis was suspected. a biopsy confirmed it. the immunochemistry pointed to a gynecologic, pancreatic or intestinal tumor. the computer tomography scan revealed a uterine mass. results: the first case only suggested an estromal gastrointestinal tumor or a mesothelioma, misdiagnosing and missing the endometrial carcinoma. the second case was uncommon as bone metastasis is found with solid tumors but seldom occurs with endometrial cancer and even less so as a first sign. conclusion: we must consider endometrial cancer at signs of uterine bleeding, however, further investigation is required in order to consider the 10 % who do not show the common signs. extrapelvic endometriosis presenting as a retroperitoneal tumor a. alves * , p. luís, a. ribeiro, m. ferreira * hospital santa maria, servico de anatomia patologica, lisboa, portugal objective: endometriosis is a benign disease characterized by the presence of functional endometrial tissue in ectopic locations. retroperitoneal, liver or kidney involvement are extremely rare. method: we present a case of a 52-year-old woman, which was referred to surgical consultation because of a retroperitoneal mass that was found on a routine abdominal ultrasonography (us). a subsequent ct scan showed a 12 cm tumor involving the liver, right kidney and adrenal gland. the gynecological us revealed ovaries with normal size and two probable uterine leiomyomas. she was submitted to a tumorectomy with segmental hepatectomy and nephrectomy. results: grossly, the tumor was 12×8×6 cm, invaded the kidney, the liver and a segment of diaphragm. it was white, fasciculate and with hemorrhagic focus. histologically the tumor was composed of a biphasic proliferation of endometrial glands and endometrial stroma, both without atypia. the case was diagnosed as deep infiltrating extrapelvic endometriosis. with a 5 years follow-up, the patient is doing well and without evidence of disease. conclusion: despite being an extremely rare presentation, endometriosis can involve retroperitoneal organs and simulate a malignant neoplasm, even without a previous history of pelvic involvement. objective: malignant melanoma of the female genital tract is a multifocal disease resulting from a disorder of melanocytes cotyledonoid dissecting leiomyoma is a benign smooth muscle tumor which can mimic a malignant lesion due to its alarming aspect. there are some variant forms of leiomyomas with an unusual infiltrative growth pattern. due to its worrying appearance of the gross specimen, it is often mistaken for malignant lesion and it is important to be aware of this entity not to over treat this benign smooth-muscle neoplasm. we report a case of a 55 years old woman who underwent hysterectomy because of the suspect of a tumor growing from the right lateral uterine wall. on gross study the tumor measured 10 mm in diameter and was nodular, brown in color and irregular on cut section. histological examination showed a nodular tumor composed of smooth muscle cells with a storiform pattern which dissects the uterine wall. the tumor was highly vascularized, included a myxoid component and showed some areas with invasion of vascular components. cellular atypia, mitosis and necrosis were absent. the patient has had a good clinical course, without relapses until the date, what supports the fact that these tumors have a benign clinical behavior, even though they may have a malignant appearance. uterine leiomyoma with lymphoid infiltration a. n. deger * , c. kocak * s.b. dpu. kec. ea hastanesi, patoloji bolumu, kutahya, turkey objective: although leimyomas of the uterus are common, lymphoid infiltration of leimyomas is a rare occurrence. we presented a case of a 45-year-old woman whose leiomyoma was diffusely infiltrated by lymphocytes. lymphoid infiltration was analyzed with immunohistochemical methods and infiltration was found to be polyclonal type. method: a 45-year-old woman with abnormal uterine bleeding was prediagnosed uterine leiomyoma and she was applied total abdominal hysterectomy and bilateral salphingo-oopherectomy. the analysis of the sample sent to pathology lab showed two leiomyomas. hematoxylin eosin -stained slides were prepared after sampling. ımmunohistochemical stains were applied. results: microscopic examination of leiomyoma in greater diameter revealed a lesion with well-circumscribed borders composed of interlacing fascicles of bland monomorphic spindle cells diffusely infiltrated by lymphoid cells. the infiltrate not extended into the surrounding myometrium. immunohistochemical analysis showed positive staining with cd 20, cd3, cd68. it was seen that inflammatory infiltration was polyclonal and involved t cells, b cells and histiocytes. leiomyoma with lymphoid infiltration was diagnosed. the postoperative course was uneventful within a 6 month follow up period. conclusion: leiomyoma with lymphoid infiltration first described in 1989 by ferry et all. from that date on, the literature involves few cases. because it is rare and differential diagnose with malignant lymphoma is crucial, we presented the case and reviewed the literature. objective: vulvar intraepithelial neoplasia is divided into two groups: usual type and differentiated type. the differentiated vulvar intraepithelial neoplasia, which is frequently seen with invasive squamous cell carcinoma, can be confused with some benign lesions. the aim of this study is to analyse p16, p53, and ki-67 expression characteristics of different histological types of vulvar intraepithelial neoplasia, invasive squamous cell carcinoma, and benign lesions of the vulva. method: in this study, immunohistochemical analysis of 18 vulvectomy cases with p16, p53, and ki-67 was performed. results: of 18 patients who underwent vulvectomy, nine had invasive squamous cell carcinoma and nine had vulvar intraepithelial neoplasia. four additional vulvar intraepithelial neoplasia lesions were found accompanying the invasive squamous cell carcinomas. nine benign lesions were found accompanying the invasive squamous cell carcinomas and vulvar intraepithelial neoplasia. mean ki-67 proliferation index was 32.3 % in the usual type of vulvar intraepithelial neoplasia cases and 26.4 % in the differentiated vulvar intraepithelial neoplasia cases. no p53 expression was present in benign lesions. conclusion: ki-67 pi does not recognize the usual type or differentiated type of vulvar intraepithelial neoplasia. p53 positivity can be of value in distinguishing differentiated type vulvar intraepithelial neoplasia from benign lesions. an audit of surgical pathology reports of endometrial carcinoma: experience from a referral centre in india k. deodhar * , b. rekhi, s. menon, b. ganesh * tata memorial hospital, dept. of pathology, mumbai, india objective: the aim was to see, compliance to minimum data information in carcinoma endometrium reports, in a team of 13 pathologists; and also to analyze these parameters e.g. tumor size, type, grade, depth of myometrial invasion, lymph node yield, ptnm stage etc. method: during the period of 2008-2010, from the files of pathology department of our hospital, reports of operated 114 carcinoma endometrium cases were retrieved and analyzed for various, above mentioned, parameters. results: the median age was 58.04 years and median tumor size was 4 cm. endometrioid adenocarcinoma was the commonest type (82.5 %); followed by mmmt (6.1 %) and serous carcinoma (3.5 %). grade 2 was the commonest tumor grade (42.1 %). less than half of myometrial invasion was seen in 50 % of the cases, =/> half myometrial invasion was seen in 46.5 % of cases. (information-not available in 4 cases). parametrial involvement was seen in 5.3 % cases. the ptnm stage was not mentioned in 71.9 % reports. the median lymph node yield was 15. conclusion: the compliance to adhere to minimum data information in carcinoma endometrium reports is generally good. lymph node yield is reasonable. parametrial involvement and mentioning of ptnm staging is to be done more meticulously. use of proformas/checklists is recommended. extraovarian granulosa cell tumor: a case report i. efstratiou * , s. pervana, e. pazarli, d. alataki * papageorgiou hospital, dept. of pathology, thessaloniki, greece objective: extraovarian granulosa cell tumor is a very rare neoplasm. we report a case of a tumor located in mesocolon. method: a 58-years-old woman presented with abdominal pain and fever. abdomen ct showed a mesocolic mass adherent to the left kidney and the spleen. she underwent resection of a 22 cm long segment of the left colon with a circumscribed mesocolic tumor measuring 7×10 cm. the tumor was infiltrating the subserosa of the large intestine. intraoperatively normal ovaries have been identified. results: histologically the tumor is composed by monomorphous cells in solid nests developing cavities which contain serous or hemmorhagic fluid. the tumor cells have scanty cytoplasm and often grooved nuclei. mitoses are very rare. the tumor cells were immunoreactive for vimentin, inhibin and progesterone receptors. the postoperative course was uncomplicated and the patient is free of disease 18 months later. conclusion: light microscopic and immunohistochemical features of the tumor are similar to those of ovarian granulosa cell tumors (gct). since the tumor was resected in toto and the mitotic activity of the cells is very low, we expect a favorable prognosis. extraovarian gct are very rare and only 7 cases have been published during the last 50 years. probably these tumors arise from residual tissue of the genital ridge (so-called secondary mullerian system). detection of amplification of terc and tert genes in cytology samples from cervical neoplasias by fluorescent in situ hybridization a. farkasova * , e. kudela, t. balharek, p. zubor, j. danko, l. plank * jfm cu, dept. of pathology, martin, slovakia objective: increased telomerase activity represents an early event in cervical carcinogenesis allowing cell immortality by recovering chromosomal telomeres. thus, detection of terc and tert gene amplification might represent a diagnostic and valuable prognostic biomarker of cervical neoplasias. method: cervical smears from 13 patients classified according to bethesda as nilm (n=3), asc-us (n=2), l-sil (n=2), h-sil (n=4) and scc (n=2) were analysed for terc (3q26) and tert (5p15) gene amplification by fluorescent in situ hybridization (fish) in 100 cells per slide using a four-color fish probe (fhact™). results: the numbers of terc and tert copies, average ratio of gene copies and average number of cells with ≥4 terc gene copies were highest in scc, followed by h-sil, l-sil, asc-us and nilm cases. correlation between terc/tert amplification intensity and oncocytological findings was statistically significant (p<0.0001). conclusion: fish analysis of terc and tert genes could be effective tool for the diagnosis of cervical neoplastic lesions. using together with cytology and hpv dna testing it can achieve higher sensitivity and specificity to discriminate h-sil and invasive carcinomas from l-sil lesions. objective: ovarian stromal hyperplasia (osh) is characterised by non-neoplastic overgrowth of the ovarian cortical. mild hyperplasia of the cortical and medullary stroma is found in the ovaries of about one-third of perimenopausal and postmenopausal women. it is nearly always diffusely bilateral. we report a case of osh associated with endometrial carcinoma (ec). method: a 40-years-old caucasian woman, obese, smoker, with fatty liver, gilbert´s syndrome and cholelithiasis. she had endometrial curettage biopsy for dysfunctional uterine bleeding for 5 month, which was diagnosed of complex atypical endometrial hiperplasia. a total abdominal hysterectomy whith bilateral salpingo-oophorectomy were performed. results: the diagnosis was well differentiated endometrioid ec and bilateral osh with focal stromal hypertecosis. conclusion: osh of moderate to severe degree may be found in women with disorders associated with androgenic and estrogenic manifestations including ec, obesity, hypertension, and glucose intolerance, but these findings are less frequent and less obtrusive than in stromal hyperthecosis. obese woman are at risk for developing endometrioid ec as a result of the increased capacity in adipose tissue to convert androstenedione to oestrone, and testosterone to oestradiol. a relationship in the origin of hormone-dependent endometrial pathology may exist between osh, blood androgen levels and ec. detection of micrometastases in para-aortic lymph nodes in patients with carcinoma of the uterine cervix after negative frozen section analysis l.-c. horn * , c. kellner, r. scherling, m. höckel, j. einenkel * medizin. universität leipzig, institut für pathologie, germany objective: previous studies considered the presence of micrometastases (mm) in pelvic lymph nodes as clinically relevant prognostic indicator. method: frozen section analysis was performed in all cases. after fs-examination nodes were examined by one h&e-stained slide. all nodes without metastatic disease after frozen section and permanent section examination were subject of the present study. 43 patients and 418 pan were enrolled and immunohistochemical staining using two cytokeratin-cocktail antibodies (ae 1/ae 3 and kl-) was performed. results: in one case, one single node showed micrometastasis, representing an incidence of 2.3 % of the studied cases and 0.23 % of the examined lymph nodes. in three cases benign endosalpingiosis was seen. the patient with mm is alive without evidence of disease 96 months after surgery. itc were not observed. conclusion: the frequency of mm in pan is very low. there are only limited data regarding their prognostic impact within the literature. after careful examination of all removed pan using h&e-staining (and step sectioning), immunohistochmeical ultrastaging cannot be recommend for routine use. serous tubal in situ carcinoma (stic) in tubal and primary peritoneal carcinomas l.-c. horn * , k. leonhardt, s. kafkova, j. einenkel * medizin. universität leipzig, institut für pathologie, germany objective: serous tubal in situ carcinoma (stic) has been defined as one important precursor of pelvic serous cancer. morphologically, stic is defined by are cytologic atypia, high proliferative index and strong staining for p53. method: the present study evaluates the presence of stic and p53-signature in consecutive cases of 12 prophylactic salpingo-oophorectomy in women with brca-1-mutaion (bso), 11 macroscopically inconspicuous tubes of patients with primary tubal cancer (tc) and 9 cases of primary peritoneal cancer (ppc) using immunohistochemistry against ki-67 and p53 (clone do-7). results: the frequency of p53-signature and stic was 8 % and 0 % in cases of prophylactic surgery, 9 % and 18 % in tc and 0 % and 33 % in ppc. conclusion: stic and p53-signature as precursor lesions of pelvic serous cancer is seen in macroscopically inconspicuous fallopian tubes in unilateral tc in patients with elective bso and patients affected by ppc. we propose that the sectioning and extensively examining the fimbria protocol be applied to all cases with ppc, tc and in women with prophylactic bso. objective: primary adenocarcinoma of the vulva is rare, and enteric differentiation is excepcional. only a few cases of neoplasms of pure intestinal-type in the low genital tract have been reported. they are considered to arise from cloacal remants or from intestinal heterotopia. method: we present the case of a 54 year-old woman, with a histerectomy performed 10 years before. on clinical examination a polipoid lesion in the vulvar vestibule was noticed. biopsy revealed a tubular adenoma of intestinal type with a focus of adenocarcinoma. the first histologic interpretation was a metastatic intestinal tumor. clinical examination, recto-colonoscopy and magnetic resonance imaging of the abdomen excluded this possibility. results: the immunohistochemical study showed reactivity with citokeratin 20 but didn't with citokeratin 7. all this led to conclude that it was a primary intestinaltype adenocarcinoma of the vulva arisen from an adenoma. conclusion: on our knowledge there are less than 100 reported cases of intestinal adenoma in the genital tract. they have been described also in the vagina and cervix but only a few developed an adenocarcinoma. it is important to be aware of this tumor type and to distinguish it from metastatic colorectal adenocarcinoma in order to plan appropriate treatment. ligneous cervicovaginitis and endometritis: case report m. koyuncuoglu * , e. dogan * dokuz eylul university, dept. of pathology, izmir, turkey objective: ligneous (wood-like) disease is a rare chronic pseudomembraneous inflammation of the mucous membranes which may also affect genital tract. the underlying pathogenesis is still unclear and an effective method of treatment has not yet emerged. method: here we present a 32 years old female patient presented to our clinic with 7 years of unexplained infertility. her diagnostic infertility work-up revealed no abnormality. however, at gynecologic examination there was a thick and hard granulation tissue at the cervix extending to the posterior vaginal wall. transvaginal ultrasound was unremarkable except for increased endometrial thickness. results: cervicovaginal and endometrial biopsy with diagnostic hysteroscopy was performed which demonstrated ligneous inflammation of both cervix and vagina. endometrial biopsy was reported as chronic non-specific endometritis with dense fibrin deposition. conclusion:. the disease may also affect other organs including oral cavity and eyes. this unusual condition is difficult to treat and lack of awareness makes the diagnosis also problematic. objective: differential diagnosis lm vs stump may be controversial, especially regarding necrosis evaluation; few studies address the relation between different treatments and their morphological effects. we aim to characterize a series lms and putative stumps harboring necrosis. method: consecutive putative stumps (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) and a series of lms with necrosis diagnosed at chsj & ipop-porto. clinical files, particularly previous hormonal treatment (ht), gross specimen and histology features. results: 18 putative stumps and 35 lms with necrosis. lms mean age at diagnosis: 43 years; mean size: 9.0 cm (2.3-18); 45 % with other lms; treatment: myomectomy (49 %) and hysterectomy (51 %); 23 % peri-partum, 8.5 % post-uterine artery embolization; 17 (49 %) previous ht: progestin (17 %), oral contraceptive-oc (20 %), hormonal-iud (8.6 %), and medical assisted reproduction (8.6 %). stumps mean age at diagnosis: 45 years; mean size: 10 cm (2.0-20); 44 % with other lms; low mitotic index-mi (mean 3.16; median 1/10hpf); slight cell atypia; variable ischemic/ tumor-type necrosis. treatment: myomectomy (22 %), hysterectomy (83 %) and radiotherapy (11 %); 12 (67 %) previous ht: progestin (22 %), oc (39 %), hormonal-iud (11 %); no recurrence in remaining cases (n=6): median follow-up: 97.5 months. conclusion: lms with necrosis and stump may display overlapping features. diagnosis of stump should consider previous ht, detailed morphology (necrosis type/atypia/mi), to prevent overdiagnosis of stump. positivity. plap and chromogranin were negative. ki-67 nuclear positivity was found in less than 1 % of sertoli cells and in about 1 % of leydig cells in hyperplastic areas. karyotype was 46xy. conclusion: although, surgical pathologists encounter tfs rare in a clinical practice, they should be aware of this condition, especially in lack of relevant clinical data, when it could be interpreted as a tumor. objective: frozen section (fs) diagnosis of ovarian mucinous tumors can be difficult due to the size of these tumors, heterogeneity and potential risk of metastasis from gastrointestinal (gi) neoplasms. method: we reported 79 ovarian mucinous tumors submitted for (fs) evaluation between january 2007 and avril 2012 was conducted. results: fs and final pathology results were collected. the average tumor size was 22,1 cm (1-55 cm). the fs and final pathology diagnosis were concordant in 84,8 % (67/79) of the cases. of the 12(15,1 %) discordant cases, one (1,2 %) was downgraded and 8 cases (13,9 %) were upgraded. of the 30 tumors interpreted as borderline mucinous tumors (bmt) on fs, 8(26,6 %) were malignant at final diagnosis (4 ovarian, 4 gi), 21 (70 %) remain as bmt and 1 (3,3 %) was benign. of the 18 benign tumors on fs, 3 cases (17, 6 %) were upgraded to bmt at final diagnosis. tumors with a malignant diagnosis on fs (30 cases) were 100 % concordant with final diagnosis. conclusion: our study showed a 15,1 % rate of discordance between fs and final diagnosis. given that gi origin is a possible finding, intraoperative assessment of the appendix should be performed in all mucinous ovarian tumors. uterine müllerian adenosarcoma: a clinicopathologic study of 31 cases a. nasfi * , l. charfi, k. mrad, s. sassi, r. sellami-dhouib, r. doghri, m. driss, i. abbes, s. nechi, w. jomaa, k. ben romdhane * salah azaiez institute, dept. of pathology, tunis, tunisia objective: müllerian adenosarcoma (ma) is a distinctive type of uterine tumor, traditionally regarded as a low-grade variant of mixed müllerian tumors. method: thirty one cases of adenosarcoma were examined during a period of 19 years (may 1993 -february 2012 . results: thirty tumors were of the uterine corpus and one of the cervix. the mean patient age was 54,2 years (range: 16 to 73 year). the main clinical manifestations were vaginal bleeding and pelvic pain. physical examination showed cervical/vaginal mass or pelvic mass. treatment was known in 25 cases: patients underwent hysterectomy with bilateral salpingo-oophorectomy in 17 cases and lymphadenectomy in 6 cases. tumor size ranged from 2,5 to 12 cm (mean: 7.1 cm). microscopically, sarcomatous overgrowth was found in 4 cases (13 %), heterologous elements in 5 (16 %). eleven cases (35 %) had myometrial invasion involving the internal half of the myometrial thickness in 9 cases, and more than 50 % in 3 cases (9 %). the cervical tumor presented as an endocervical polyp without invasion of the cervical wall. conclusion: uterine ma are low-grade neoplasms capable of local recurrence and much less commonly distant metastasis. surgical excision is the main treatment strategy with a good prognosis in the early stage disease. the 2 most important adverse prognostic factors are deep myometrial invasion and sarcomatous overgrowth. spectrum of epithelium changes in adenomyosis n. nizyaeva * , e. kogan, t. demura * scientific center of obstetric, dept. of pathology, moscow, russia objective: adenomyosis (am) is a very common gynecological disorder. despite high incidence of the disease epithelial changes precise developmental events leading to the condition remain controversial. the aim of the study was to investigate and compare epithelium morphologicaly and markers expression of proliferation, apoptosis, invasion and neoangigogenesis in am foci. method: this study was done on biopsy samples of uterus taken from 70 women with adenomyosis. immunohistochemical staining of tissues was performed with antibodies to apocas, ki-67, mmp-2, timp-1, claudins 3, 5 (cl3,5), e-cadherin, cox-2, egfr, vegf. results: four variants of epithelium changes were found in am foci such as proliferative type, hyperplastic type with and without atypia, and atrophic type. times during 3 years and investigated using routine light microscopy and immunohistochemistry (ihc) to control the treatment response. results: in the course of progestin treatment the complex endometrial hyperplasia with intraepithelial neoplasia became histologically less complicated and than normal. ihc profiles and ki-67 expression had the same dynamics. in the youngest patient the unusual gland cell atypia with focal calcification was observed one time and hysterectomy was considered. conclusion: the study confirms that the repeated curettages with conservative treatment and histological studying can be successfully used in some cases of complex endometrial hyperplasia with intraepithelial neoplasia. value of ki67, p16 and ck17 markers in differentiating cervical intraepithelial neoplasia and benign lesions a. safaei * , m. pourjabali, f. sari aslani, m. momtahan * shiraz university of medical sciences, iran objective: the cervical cancer is one of the most common cancers among women worldwide. diagnosis of cin affected by high rates of discordance among pathologists. therefore, we need to other adjunct methods for accurate diagnosis of cin versus benign lesions in equivocal cases. the aim of this study was evaluation of ki-67 (mib-1), ck17 and p16 ink 4a (p16) markers by immunohistochemical method in differentiating cin from benign cervical lesions. method: seventy-seven cervical biopsies that originally diagnosed as non-cin (n = 31) and cin (n = 46), were reviewed by three pathologists and re-classified as non-cin (n = 54) and cin (n = 23), based on agreement between at least two of three, to obtain a consensus diagnosis. consensus diagnosis was defined as the "gold standard". then immunostaining for ki67, p16 and ck17 was performed on all cases and their results were compared with original and consensus diagnosis. results: the overall agreement between original and consensus diagnosis was 67.5 % (kappa = 0.39, p-value < 0.001). the sensitivity and specificity of ki67 immunostaining were 95.6 % and 85.1 % and for p16 were 91.3 and 98.1 %, respectively. the sensitivity and specificity of ck17 negative staining for cin detection were 39.1 % and 40.7 % respectively. conclusion: we recommended using ki67 and p16 markers as complementary tests for differentiation between dysplastic and non-dysplastic lesions. ps-23-038 wt1 expression in ovarian borderline and malignant surface epithelial tumors m. tahamtan * , f. sari aslani, a. safaee * shiraz, iran objective: wilms tumor gene product, a tumor suppressor gene, now is considered to have oncogenic functions. there seems to be differences in wt1 expression among surface epithelial ovarian tumor subtypes. method: immunohistochemistry for wt1 was done on 35 serous & 3 mucinous cystadenocarcinomas, 9 borderline serous & 10 borderline mucinous tumors, 7 endometrioid ovarian carcinomas, 3 clear cell carcinomas, 1 malignant brenner tumor, 2 metastatic adenocarcinomas and 6 endometrial adenocarcinomas. a tumor was considered negative if<1 % of tumor cells were stained. positive reactions were graded :1+,1-24 %; 2+,25-49 %; 3+,50-74 %; 4+,75-100 %. results: of serous cystadenocarcinomaes, 30(85.7 %) were positive,4 showed reactivity of<50 % of the tumor cells and one were negative. all borderline serous tumors were positive. all mucinous tumors, endometrioid carcinomas, clear cell carcinomas, metastatic adenocarcinomas and primary endometrial carcinomas were negative. the single malignant brenner tumor were positive for wt1. conclusion: wt1 is a good marker to distinguish primary ovarian serous carcinomas from other surface epithelial tumors (especially endometrioid subtype) and metastatic carcinomas (especially endometrial serous carcinoma), other than malignant mesothelioma. degree of expression is not an indicator to separate high grade borderline serous tumors from low grade ones. objective: fatwo is a rare neoplasm originating from the mesonephric duct remnants that occurs predominantly in the broad ligament but also in the ovary. it is considered a lowmalignant potential tumor. however local recurrences and metastases have been reported. method: a 45 year-old female, on routine gynecological physical examination, followed by pelvic ultrasonography and computer tomography was diagnosed with a mass at the right ovary. a salpingo-oopherectomy was performed. on gross examination a well-circumscribed, solid and focally cystic tumor measuring 10,5×7×5,5 cm arose in the ovarian hilus. results: microscopically the tumor consisted of mediumsized, ovoid to polygonal cells arranged in a solid, tubular and sieve-like pattern. some cystic spaces were lined by low-cuboidal cells and contained amorphous, eosinophilic material. cellular atypia and mitoses were rare. the tumor cells were positive for vimentin, inhibin-a (focally), cd10, cytokeratins 8/18, 19 and 7 (focally) and negative for cytokeratin 20, epithelial membrane antigen, carcinoembryonic antigen and a-fetoprotein. conclusion: histopathological and imunohistochemical findings consistent with an ovarian fatwo. differential diagnosis includes endometrioid carcinoma, clear-cell carcinoma, sertoli-leydig cell tumors (retiform variant) and rete ovarii adenoma. surgical excision is the optimal treatment. radiation therapy, chemotherapy or even targeting molecular therapy is questionable. müllerian adenosarcoma is a rare mixed tumor of low malignant potential. usually presented as a large endometrial polyp in postmenopausal women. they are associated with tamoxifen or radiation therapy. microscopy shows a mixture of benign glandular epithelium and low-grade endometrial sarcoma is typically concentrated around the glands. the differential diagnosis is made with the adenofibroma, but now is doubted the existence of this tumor and is considered more of a distinct adenosarcoma. the treatment is total hysterectomy. sisters of 24 and 16 years with a history of dysmenorrhea. hysteroscopy was performed to the eldest one, in which multiple polyps are observed and partially resected. the pathological diagnosis was mixed mullerian tumor, adenofibroma/adenosarcoma. total abdominal hysterectomy was performed. histological examination showed a mixed tumor with glandular component and a low-grade sarcomatous, scarce mitosis, mild atypia and no necrosis. the diagnosis was: mullerian adenosarcoma. a year later, a hysteroscopy and endometrial biopsy was performed to the youngest sister, that showed endometrial polyps with stromal predominance, and no significant atypia. with the diagnosis of mullerian adenosarcoma, and taking into account family history, total hysterectomy was performed. adenosarcoma is a rare tumor whose family presentation is not described in the literature. gliomatosis peritonei is associated with frequent relapse but not affects overall survival in patients with ovarian immature teratoma n.-r. yoon * * samsung medical center, dept. of pathology, seoul, republic of korea objective: gliomatosis peritonei (gp) associated with ovarian teratoma has known to have no adverse prognostic effect. we investigated the clinicopathological features of ovarian teratoma associated with gp, and compared immature teratomas (its) with gp to its without gp. method: we investigated 16 patients with ovarian teratoma with gp and 27 patients with it without gp, who were diagnosed at samsung medical center (seoul) from january 1995 to august 2010. results: six patients with it with gp (37.5 % of 16 patients) had recurrence. when it with gp (n=15) was compared to it without gp (n=27), patient of it with gp showed larger tumor size (median, 19 cm vs. median 13 cm) (p<0.001), more frequent relapse (40 %, 6/15 vs. 3.7 %, 1/ 27) (p= 0.005), and frequently elevated pre-operative ca125 level (100 %, 12/12 vs. 50 %, 10/20) (p=0.004). survival curves showed significantly shorter relapse-free survival in patients of it with gp (p=0.002). two-year relapse-free survival rates were 59.3 % and 96.3 % in it with gp and it without gp, respectively. however, all patients except one case of it with gp alive. conclusion: ovarian it with gp was characterized by larger tumor size and frequent elevation of preoperative ca125 level, and gp was associated with frequent relapse in patients with ovarian it. corelation between histopathologic diagnosis with p16 and ki67 immunostaining, cytologic features in cervical neoplasia c. altunkaya * , s. yilmaz, a. barin, s. ekici, g. s. yalcin, m. caydere, h. ustun * s.b ankara e. a.h, pathology, turkey objective: frequency of cervical carcinoma decreases nowadays since cytologic screening methods have been using extensively. the incidance of cervical intraepithelial neoplasia increases. cytologic diagnosis og these lesions are substantially important. so we aim to identify the role of p16 and ki67 immunostainings for predictive factors of cervical neoplasia. method: the study was conducted january 2007 to january 2012. ninety-three cases (mean age 43,9; range 22-71 years) diagnosed as squamous intraepithelial lesions were included, objective: there are several prognostic factors in melanoma. it was suggested that her-3 expression may influence the tumor behavior. the aim of the study was to investigate the relationship of her-3 expression with various prognostic factors. method: her-3 expression was evaluated in 52 melanomas, 26 without metastases and 19 with lymph node and 7 with distant metastases. membranous, cytoplasmic and nuclear her-3 expression was separately analysed. the staining intensity and percentage of positive tumor cells were evaluated. results: the cytoplasmic staininig was seen in all cases, with average intensity of 2 and percentage of positive cells ranging from 5 % to 90 % (mean 48.8 %). the percentage of cytoplasmic her-3 positive cells was inversely correlated with clark and breslow stage (r=−0.29 and r=−0.43). cytoplasmic her-3 reaction was significantly stronger in cases with lymph node metastases (2.3 vs. 1.8 p<0.03). in 22 cases a dot-like cytoplasmic reaction was seen. membranous positivity was seen in 28 cases (54 %). conclusion: her-3 may play a role in melanoma progression. it may be involved in lymphatic dissemination. further studies of prognostic significance of her-expression in melanoma are needed. hypopigmented mycosis fungoides with unusual vitiligolike presentation in child h. erdem * , n. buyukbabani, h. turan * duzce university, dept. of pathology, turkey objective: mycosis fungoides (mf) is the cutaneous t cell lymphoma. classical, clinical and histopathological findings of mf are detected in most of the patients. however, some of the patients have defined atypical mf. hypopigmented mf (hmf) is one of the atypical forms. hmf could be misdiagnosed with clinical and histopathological examination. hmf is considered mistakenly vitiligo. therefore, it should be considered differential diagnosis. mf is usually seen in the middle aged and elderly. the occurrence of mycosis fungoides in children is very rare. method: an 7-year-old girl child attended dermatology clinic with complaints of pruritus and hipopigmented patches. lesions was performed punch biopsy and reported hmf. conclusion: herein, this case presented because it was considered vitiligo as clinically and diagnosed hmfas histopathologically. reducing block sampling in wide local excisions for melanoma c. fives * , c.c.b.b. heffron * cork university hospital, dept. of histopathology, ireland objective: it is established practice that wide local excision (wle) is undertaken for the further management of cutaneous melanoma, however, definitive guidelines for macroscopic sampling have not been established. our aim was to determine whether our sampling of wle specimens was adequate, inadequate or excessive and to establish guidelines for these specimens. method: 128 cases which underwent initial biopsy and subsequent wle in 2010 were identified. we recorded the specimen size, macroscopic appearance, number of blocks sampled and margins of the original biopsy. results: there was wide variation in the number of blocks sampled (range 1-27). residual melanoma was identified in 7 of the cases (9.4 %) which had clear margins on the original biopsy. of these, 4 had evidence of a pigmented lesion on macroscopic examination. the remaining 3 cases had margins of 1 mm or less on the original excision. no subsequent surgery was performed on these cases. conclusion: our study has shown that in wle specimens with no evidence of a macroscopic lesion and in which margins of the original biopsy were clear by greater than 1 mm, little is to be gained from extensive sampling. reduced sampling would result in saving laboratory resources with a predicted 49 % block reduction in our laboratory alone. functioning oxyphil adenoma of parathyroid gland: a case report m. genadieva -yordanova * , s. hristova, a. vlahova, g. todorov, b. miserliovska, s. yordanov * alexandrovska hospital, dept. of pathology, sofia, bulgaria objective: the most frequent cause of primary hyperparathyroidism are adenomas of the parathyroid gland, the majority of which are composed of chief cells. oxyphil adenomas are uncommon and account for 3 % of functioning parathyroid adenomas. up until 1978 year they have been considered non-functional. method: laboratory findings revealed calcium levels of 2.86 prior to operation. the serum parathyroid hormone level was 151.40 pg/ml. a ct scan of the neck showed normal size thyroid lobes with heterodense structure with an oval hypodense lesion in the lower right pole of the thyroid. results: at surgery a mahagony-brown mass measuring 10 mm in diameter was removed and reported as oxyphil adenoma on frozen section. histopathological examination revealed encapsulated adenoma composed of oxyphil cells with abundant, granular pink cytoplasm and a rim of normal parathyroid tissue. mitotic figures were absent. postoperatively, a decrease of the serum calcium down to 2.31 mmol/l was declared. the followup period was unremarkable. conclusion: there appear to be a growing evidence that a big part of oxyphil adenomas of parathyroid gland can produce parathyroid hormone and contribute to the cases of primary hyperthyroidism. effectiveness of uvb phototherapy on mycosis fungoides using histologic guitart criteria in iranian patients a. ghanadan * , a.-h. ehsani, h. seirafi, m. khiabani * tehran university, razi hospital, iran objective: mycosis fungoides (mf) is the most common primary cutaneous lymphoma. mycosis fungoides often develops slowly over many years, presenting with a generalized erythroderma, skin patches or plaques. the diagnosis of mf requires the integration of clinical and histopathologic findings. narrowband uvb (nbuvb) is widely used to treat mf. to evaluate the effectiveness of therapy the histopathologic findings before and after nbuvb reviewed using guitart criteria. method: we enrolled 20 patients (12 women, 8 men; age range, 10-80 years; mean age, 45.5 years) with clinically and histologically proven mf. the patients received nbuvb phototherapy three times a week. a biopsy was performed 3 months after onset of the treatment and guitart criteria used to scoring mf before and after therapy. results: phototherapy was reduced the primary intraepidermal atypical lymphocytes (p value=0.008), dermal atypical lymphocytes (p value=0.008), epidermotropism (p value= 0.001), density of infiltration (p value=0.002) and lymphocytic infiltrate without inflammatory features (p value = 0.046) in all patient. but the relationship between the reticular fibroplasia of papillary dermis and phototherapy was not proved (p value=0.18). overall score of patients was significantly lower after phototherapy (p value=0.000). conclusion: our data suggest that nbuvb therapy is reduces histiologic score of mf and is effective for the treatment of iranian patients. nodular colloid degeneration of the skin: report of three cases a. ghanadan * , k. kamyab-hesari, m. daneshpajouh, k. balighi, m. khosravi * tehran university, razi hospital, iran objective: nodular colloid degeneration (ncd) is a rare dermatological disorder and also a rare type of colloid milium. the degeneration may be related to sun exposure. method: three cases of ncd were enrolled from the archive of dermatopathology department of razi hospital during 2009-2011. results: in this report, three cases, all presented with multiple plaques and nodules in the nose and the face, are depicted. histologically, these nodular masses were homogeneous, with eosinophilic clefted materials expanding the papillary dermis and extending into the deep dermis. histochemical review showed the reactivity of the colloid materials via the pas, crystal violet and methyl violet staining. all the three cases were finally diagnosed as nodular colloid degeneration. conclusion: ncd is a rare disease but it should be considered in any cases with a history of long exposure to the light. we suggest the long term exposure to the sun as an etiologic factor thus, sun protection would be the most preventive and available treatment. glypican-3 protein expression in melanoma: a immunocytochemistry study n. gursan * , h. balta, b. gundogdu * ataturk university, medical faculty, erzurum, turkey objective: glypican-3 (gpc3) is a cell surface heparan sulfate proteoglycan. serum gpc3 was shown to be expressed in 40 % of melanomas (ms) but gpc3 expression in melanoma tissues had not been investigated.in this study, immunohistochemical analysis of gpc3 protein expression was investigated in histologic sections from melanoma tissues. method: 60 melanoma patients, twenty patients with insitu melanoma, twenty patients stage 0 and stage i melanoma, twenty patients stage ii and stage iii melanoma. all cases were stained with anti-gpc3 antibody. gpc3 expression was divided into 2 categories: negative (negative or weak cytoplasmic staining) and positive (moderate or strong cytoplasmic with membranous accentuation). results: gpc3 immunopositivity. showed in 40,3 % of melanomas. gpc3 expression at stage 0, i, ii, iii were 44.4 %, 40.0 %, 41.6 %, 35 % respectively. method: we report the case of a 77 year old man presenting with an erythematous plaque of the right hemithorax. results: macroscopically the lesion had irregular borders and was mildly infiltrative with a violet hue. microscopic examination revealed tumor cells within lymphatic spaces, without infiltration of the adjacent stroma. the histologic and immunophenotypic characteristics of these cells were compatible with a carcinoma of lung origin. ct scan revealed a lung mass as well as multiple liver and chest wall metastases. conclusion: malignancies originating from the breasts, lungs and large bowl are the most common to involve the lymphatic net of the skin. melanoma on the other hand is more frequently presenting with lymphangitic invasion, whereas inflammatory carcinomas may also affect lymphatic vessels and may be confused with erysipelas. the revelation of the primary origin is not always easy and differential diagnosis should also include gynecological malignancies, kidney and urinary bladder carcinomas. in our patient the diagnosis was established on the grounds of histology and immunohistochemistry, and was supported by the clinical and radiological findings. objective: inverted follicular keratosis (ifk) is almost always a solitary lesion occuring mainly in adult life. men are affected twice as often as women. pathologically, they can be confused with a variety of lesions, both benign and malignant. squamous carcinoma is the most serious differential diagnosis. method: we decribe a retrospective study about 7 cases of ifk diagnosed over a 18-year-period (1994-2012) . results: we collected 7 lesions, all treated by surgical excision. haemalum eosin sections were studied in all cases. all lesions were single. five, of them, were situated on the face and two on the scalp. all the patients were adults with a mean age of 34 years, average between 19 and 66 years. they presented mostly as asymptomatic papules and all were small lesions. the different sections of the lesion showed skin well delineated endophytic epithelial proliferations with inverted papillomatous and acanthotic components containing several circumscribed squamous eddies. they was no atypia, mitotic activity necrosis or stromal invasion. conclusion: ifk pose very real diagnostic problems unless one is aware of this entity. in fact, they may mimic malignant lesions especially squamous cell carcinoma, both clinically and pathologically. primary cutaneous follicular lymphoma with prominent spindle cell areas. so called spindle cell follicular lymphoma. case report g. ivády -szabó * , t. strausz, e. tóth * national institute of oncology, dept. of surgical oncology, budapest, hungary objective: spindle cell type variant of cutaneous follicular lymphoma is a rare histologic variant of primary cutaneous follicular lymphomas. it is caracterised by the presence of spindle and bizarr cells. we present a case of a 25 years old man who had a nodule on his scalp and two smaller nodules on his frontal region. we examined an excisional biopsy specimen of the forehead. method: he, stains all and immunohistochemical stains were used. results: histology revealed dense lymphoid infiltrate showing nodular pattern in the dermis and in the subcutis. the lymphoid infiltrate predominantly composed of large centrocytes. there were areas where the neoplastic cells show spindle cell morphology with bizarre nuclei and in these areas the stroma were myxoid, mucinous. with immunohistocemical stains the neoplastic cells were positive for cd20, bcl-6, and negative for cd5, cd10, vimentin, cd34, s100, actin, desmin, ae1/ae3. cd21 showed residual network of follicular dendritic cells in the background. conclusion: spindle cell lymphoma is a very rare variant of cutaneous follicle centre cell lymphoma and the presence of the spindled bizarre cells can cause differential diagnostic problems. the main differential diagnostic entities are primary or metastatic spindle cell sarcoma and spindle cell melanoma. careful morphological and immunohistochemical analysis are required to the correct diagnosis. the histopathologic and immunohistochemical features of mycosis fungoides b. a. karabork * , a. okcu heper, s. yuksel, i. kuzu * ankara university of medicine, dept. of pathology, turkey objective: the histopathologic diagnosis of early mycosis fungoides (mf) is often difficult. the lesions can mimic a variety of inflamatory dermatitis. we aimed to establish and draw attention to the most frequent histopathologic and immunohistochemical features of mf. method: we reviewed 115 skin biopsies of clinicopathologically diagnosed mf cases at medical university of ankara. we looked for a) epidermotropism, atypical lymphocytes, morphologic features in the epidermis, dermoepidermal junction and dermis b) immunohistochemical staining ratios of cd3, cd4, cd8, cd20, cd30 and expression loss of cd5, cd7. results: atypical lymphocytes) (96 %), dermal fibrosis (90 %), epidermotropism (86 % single cells, 44 % linear arrangement, 15 % pautrier's microabscesses, 83 % 'haloed' lymphocytes), epidermal acantosis (72 %), basal vacuolar degeneration (focally 43 %, marked 15 %) and a perivascular lymphocytic infiltrate were the most common and important features. dermal edema (46 %) extravasated erythrocytes (34 %), spongiosis (27 %), eosinophils (17 %) and necrotic keratinocytes (14 %) were the less seen and non-specific ones. immunohistochemical results correlated with a ratio of 78 % cd4, 22 % cd8 positive mf. expression loss of cd7 was seen in 73 %, cd5 in 31 % of cases. conclusion: diagnosis of early mf requires the correlation of morphologic, immunohistochemical and clinical features. also due to the disease's heterogenity, biopsies from different locations and rebiopsies will enhance the diagnosis. connexins of cutaneous melanocytic tumours g. kiszner * , z. buday, i. teleki, e. varga, i. b. nemeth, i. korom, t. krenacs * semmelweis university, 1st dept. of pathology, budapest, hungary objective: connexins (cx) form transmembrane channels that can transport ions and small regulatory molecules between adjacent cells. they also function as hemichannels and through protein interactions and are involved in the control of cell replication and maintenance of multicellular homeostasis. method: we have tested the expression of connexins in melanocytic tumours using immunohistochemistry in tissue microarrays of 21 common and 73 dysplastic nevi, and 61 primary and 23 metastatic malignant melanomas. results: cx23 was not found in melanocytic tumours despite expressed in the basal epidermis. cx30.3 reaction showed punctate cell membrane staining in 71 % of naevi in their superficial regions including atypical nests, and displayed cytoplasmic staining in 28 % of melanomas. low levels of cx32 were revealed in the cytoplasm of >80 % of naevi and melanomas but only 23 % and 8 % showed membranous positivity, respectively. cx36 perinuclear/cytoplasmic immunostaining was observed in 57 % of naevi and 24 % of melanomas and as cell membrane reaction in 24 % and 12 %, respectively. punctate cx43 reaction was detected in vertical tumour nests in 69 % of naevi, while only 11 % of melanomas proved positive and showed cytoplasmic cx43 delocalization. conclusion: therefore, most tested connexins were significantly down-regulated in malignant vs. benign melanocytic tumours that possibly contribute to their malignant phenotype. quantitative follow up study of cd 1a, cd 8 and cd 68 positive cells in multiple basal cell carcinoma cases after combined treatment r. kleina * , i. truksane, j. kisis, i. franckevica * riga stradins university, dept. of pathology, latvia objective: in last years appear more cases with multiple basal cell carcinomas (bcc). skin immune system reaction is especially important in nonsurgically treated cases. aim of study is to evaluate the dynamics of cd 1a, cd 68, cd8 marked cells in bcc and in skin adjacent to tumour (5 and 10 mm) before and after the treatment with cryotherapy and imiquimodum. method: from 68 bcc cases with multiple tumours investigated immunohistochemically, in dynamics we have characterized 8 patients. antibodies for cd1a, cd8, cd68+ cells in derma and epidermis were used. they were evaluated in 3 fields of vision (400×) before and after treatment. 5 % imiquimod cream and double freezing was used. control group: three normal skin samples. statistical analyses of results were done. results: after treatment were erythema, then crust and exfoliation. microscopically expressed fibrosis instead bcc was found. amount of immune cells before and after treatment were: cd8 25±3,1/34±4,8 (also in epidermis), langerhans cells 7±2,3/14±3,4, cd 68 19±3,8/10±2,9. conclusion: all immune cells of skin react to the combined treatment of bcc in radius of 5 mm. there is significant statistical difference between the numbers of langerhans cells in normal epidermis and in bcc cases treated with cryomethod and imiquimodum. regulatory t-cells in invasive and in situ squamous cell carcinoma of the skin and actinic keratosis h. kourea * , a. stravodimou, v. tzelepi, h. papaioannou, h. papadaki, c. scopa * university of patras, dept. of pathology, greece objective: regulatory τ-cells (τregs) participate in tumor tolerance and facilitate tumor growth and foxp3 transcription factor is necessary and sufficient for their development and function. we investigated the presence of t-regs in invasive (in) and in situ (is) squamous cell carcinoma (scc) of the skin and actinic keratosis (ak). method: tregs were identified using immunohistochemistry for foxp3 and recorded using image analysis in 55 cases of inscc (and their adjacent is, ak or benign tissue (bn), when present), in 18 cases of is and 46 cases of ak (and their adjacent bn tissue). statistical analysis was performed using the paired t-test. p-values <0.05 were considered statistically significant. results: in inscc cases, τregs in the tumor were more numerous than the adjacent βn (n=46) (mean 155 vs. 79, p <0,001). additionally, adjacent is had more tregs than adjacent ak (n=6) (160 vs. 86, p=0,045). significant differences between inscc, and is or ak were not observed. in is and ak cases, τregs were more numerous than the adjacent bn (206 vs. 42, p<0,001 and 148 vs. 47, p< 0,001), respectively. conclusion: treg infiltration of the skin increases early from the precancerous ak, indicating early involvement of tregs in the development of scc. objective: mastocytosis is a rare disorder and its true incidence is unknown. skin is most commonly involved, followed by the bones and the gastrointestinal tract. method: five men presented with many brown lesions in the trunk and one woman with multiple redish brown nonpruritic macules in the extremities. we used immunohistochemistry for c-kit and tryptase for the definite diagnosis. the expression of cd25 and cd2 were also evaluated. results: histology showed five cases of urticaria pigmentosa with severe diffuse infiltration of the papillary dermis by mast cells and one case of telangiectasia macularis eruptiva perstans (tmep) with scattered mast cells around dilated capillaries and venules of the papillary dermis. the mast cells were positive to c-kit and tryptase and only in one case there was a coexpression of cd25 and cd2 >50 % of the mast cells. in that case involvement of bone marrow was observed and the diagnosis of indolent systemic mastocytosis was established according to who 2008 criteria. conclusion: urticaria pigmentosa is the most common form seen in adults while tmep is an uncommon form that occurs exclusively in adults. the skin involvement in indolent systemic mastocytosis is a part of the spectrum of the disease. ps-24-025 subvisual nuclear characteristics are different between keratoacanthoma and keratoacanthoma-like squamous cell carcinoma k. metze * , m. tabai, r. adam, i. watanabe, a. de moraes, m. cintra * university of campinas, pathology, brazil objective: the differential diagnosis between keratoacanthorna and keratoacantoma-like squamous cell carcinoma may be extremely diffcult. our objective was to study whether computerized image analysis could be helpful. method: in 34 patients biopsies of keratoacantoma-like lesions were taken at admission. one month later surgical excision was performed in growing lesions, whereas regressing lesions were left untreated. a final diagnosis was established combining clinical and histological evaluation, digitalized images from ki-67 immunostained and hematoxylin counterstained sections of first biopsies were obtained. tumor nuclei were marked interactively. an inhouse computer program analyzed the geometric relations between the nuclei. nuclear gray values and their histogram entropy were calculated. results: 27 keratoacnthomas and 7 keratoacantomalike squamous cell carcinomas entered this investigation. basic variables of the geometric analysis did not differ between the two entities regardless whether all nuclei or only the ki67 positive ones had been examined. chromatin gray values were significantly lower and their histogram entropy higher in the keratoacantoma-like squamous cell carcinomas. conclusion: basic geometric variables do not seem to be different beteen both lesions, but a shift in the gray value histogram to lower values with increased entropy in carcinomas indicated important differences of the chromatin structure, supported by fapesp and cnpq. non-infectious erythematous papular and squamous lesions of the skin in our institute, with clinicopathologic correlation t. ozgur * , a. c. dogramaci, e. atik, s. hakverdi, m. yaldiz, z. a. tas * mustafa kemal university, medical faculty, antakya, turkey objective: non-infectious erythematous, papular and squamous lesions of the skin are basic lesions that pathologists differentiate in routine laboratory examinations. our aim has been to analyse these lesions by pathologic and clinical findings in our institute with determining clinicopathologic correlation. method: in our study 420 cases prediagnosed as erythematous, papular and squamous lesion by dermatologists and evaluated in pathology laboratory between 2004 and 2010 have been reviewed. results: the lesions comprised %14.3 of the total load of surgical pathology and 9.1 % of total number of skin biopsies. the highest percentage was in the 41-50 year age group (39.1 %) with a female predominance of 51.2 %. the limbs were most frequently involved (36.9 %). psoriatic lesions were the commonest (50.7 %), classic generalized plaque variant psoriasis (89 %) being the most frequent. correlation with the histopathologic diagnosis was positive in 75.3 % cases and negative in 24.7 % cases. conclusion: the contribution of histopathology to the final diagnosis was significant. it confirmed the diagnosis in 75.3 % cases and gave the diagnosis in 7.3 % cases. ps-24-027 vulvar lichen sclerosus: a misnomer for an entity with decreased fibrillar components and increased amorphous components in extracellular matrix remodeling e. parra * , c. a. pires de godoy, v. l. correia feitosa, w. teodoro, a. p. velosa, v. l. capelozzi * fmusp, dept. of pathology, são paulo, brazil objective: the hyalinization of subepidermal skin is one of the histopathological characteristics of the vulvar lichen sclerosus (vls). it was found that patients with vls present autoantibodies against the extracellular matrix protein 1 (ecm-1) and the deficiency of this protein is responsible for the development of a different disease, the lipoid proteinosis. this disease shows a similar histology with vls and has better characterized morphology. method: we analyzed 20 vls patients biopsies and the control group was composed by 20 vulva samples from authopsy. the biopsies and control samples were analysed by immunofluorescence for collagen i, iii and v and th total collagen fibers by picrosirius staining. the elastic fibers were stained with verhoeff and the proteoglycans and glycosaminoglycans with periodic acid-schiff and alcian blue. collagen quantification was performed through image analysis. results: it was observed a significant reduction in all studied collagens as well as in the elastic fibers. on the other hand, the proteoglycans were increased in the vls biopsies. conclusion: this study did not found an increase in collagen sclerosis that would justify the term used for the ls. it was observed a predominance of the edema area probably caused by the increase of glycoproteins as in lipoid proteinosis. combined high-grade basosquamous carcinoma and malignant melanoma of the scalp (malignant basomelanocytic tumor) metastasizing to the breast. a case report a. pitino * , s. squillaci, c. spairani, f. ottelli zoletti, m. f. cosimi, m. ferrari, a. tropiano, p. c. rassu, f. tuo, p. maiocchi * san giacomo hospital, division of anatomic pathology, novi ligure, italy objective: background. basal cell carcinoma (bcc) is a very low grade, usually not metastasizing skin malignancy, which needs to be radically excised. methods. an 80 year-old woman was treated for a 3×2,2 cm multinodular mass in the scalp. one month later an ultrasonography of the left breast showed a 5,5 cm mass which was excised with clean margins. axillary lymph nodes were free of metastasis. results: results. the dermal tumor presented well demarcated basaloid epithelial nests with squamous differentiation focally connected with the epidermis. they showed peripheral palisading and high nuclear grade with pleomorphism, brisk mitotic activity and multiple prominent nucleoli. atypical cells, arranged as strips, nests or isolated elements, were observed at the tumor edge, separated by a grey-zone from the overlying malpighian epithelium. immunostains were positive for cytokeratins, melanocytic (s-100/hmb-45/ mart-1) and neuroendocrine (cd56/nse/cromogranin) markers. the 4,8 cm breast tumor corresponded to a high grade metastatic bcc with reactivity for epithelial markers, s-100 and cd56. objective: pyoderma gangrenosum (pg) was described at first by brocq and named by brunsting et al. in 1930. superficial granulomatous pyoderma (sgp) was described as a variant of pg in 1988. we present three cases of this rare variant. method: there were two women and one man with age ranging from 40 to 78 years. all of them had a long history of a slowly enlarging, eritematous and ulcerative plaque in different areas including leg and breast skin. a biopsy was performed in all cases. results: the superficial dermis showed neutrophilic inflammation with an admixture of granulomatous inflammation and sinus formation. there wasn't vasculitis and fat tissue was not affected. conclusion: sgp is a rare variant of pg. diagnosis of this entity generally is made on the basis of skin biopsy results with all features mentioned previously and a typical clinical appearance. most of times, these lesions are diagnosed as granulomatous dermatitis, thinking in infectious pathology. this is the most important differential diagnosis, because sgp is responsive to corticosteroids. the pathogenesis of pg is unknown, though is now believed to altered neutrophil chemotaxis and some authors suggest that sgp may be a delayed-type hypersensitivity to an unknown antigen. pathergy is the inciting factor in several patients. high concordance in braf status between native-braf malignant melanoma and matched lymph node metastases a. santos briz * , e. godoy, l. arango, p. antúnez, m. yuste, c. roman, e. fernández, m. d. ludena * hospital universitario salamanca, dept. de anatomía patológica, spain objective: the discovery of selective v-raf murine sarcoma viral oncogene homolog b1 (braf) v600 mutation as an oncogenic mutation in cutaneous malignant melanoma (mm) has changed the treatment paradigm for melanoma. selective braf inhibitors have demonstrated response rates far higher than standard chemotherapeutic options. braf mutation analysis is usually performed on primary tumour tissue; however, no conclusive data are available on the concordance of test results between primary tumours and corresponding metastases. we assessed the concordance of braf mutation status in a study of 16 primary brafnative tumours and their corresponding lymph node metastases. method: 16 patients with histologically confirmed nonmutated braf mm who underwent surgical resection of the primary tumour and positive selective lymph node biopsy or lymphadenectomy were included. mutation status was determined by means of a realtime pcr assay (cobas 4800 braf v600 mutation test, roche molecular systems). results: there was a 100 % concordance of the braf results in primary tumor and corresponding metastases in 16 analyzed pairs. conclusion: our findings show total concordance in braf status between primary braf native tumors and their paired metastases, and suggest that acquisition of braf v600 mutation in metastases from primary native tumors is a rare event. dermatoscopic and histologic score correlation in atypical spitz/reed nevi m. saravia * , z. pellicer, j. m. martin, c. monteagudo * hospital clinic university valencia, dept. of pathology, spain objective: different dermatoscopic scores of pigmented skin lesions have provided a valuable tool in daily routine practice to differentiate malignant from benign lesions. however, these semiquatitative methods have a high rate of false positives in spitz/reed nevi. we have compared semiquantitative histopathologic and dermatoscopic findings in a series of spitz/reed nevi. method: we collected 19 cases (15 female and 4 men) of atypical spitz and/or reed nevi. all cases were microscopically confirmed. a histologic index (hi) was constructed scoring the following findings: symmetry, sharp demarcation, architectural dysplasia, melanocytic atipia and nest size, and compared with validated dermatoscopic scores (abcd score, 7-point checklist and menzies score). distributions of these scores for the different dermatoscopic patterns were also analyzed. results: median age at presentation was 15 years (range 1 to 36). the hi varied from 0 to 5 points. we found a strong positive correlation between the hi and menzies score (r=0,539, p=0,01). in addition, hi values differed significantly when the multicomponent dermatoscopic pattern group was compared with the other patterns (u =22, 5 z =−2,25, p =0.01). conclusion: menzies is the only dermatoscopic score which strongly correlates with histologic findings in spitz/reed nevi and may be useful in the diagnosis of atypical lesions. correlation of nonmolecular and molecular subtyping of inherited epidermolysis bullosa k. veselý * , h. bucková, l. fajkusová, m. veselá, b. jerábková * st. anne's hospital, dept. of pathology, brno, czech republic objective: inherited epidermolysis bullosa (eb) is a heterogeneous group of hereditary mechanobullous diseases with skin blister formation of variable severity and numerous extracutaneous manifestations. eb comprises three main groups and many subtypes and their recognition is important for prognosis and genetic counselling. method: we examined skin biopsy samples of 42 patients with eb using transmission electron microscopy, immunofluorescence antingen mapping (am) and their peripheral blood samples by mutational analysis. results: in the total number of 42 cases, concordant results of molecular and nonmolecular methods were found in 35 cases (83 %). in 7 patients (17 %) mutational analysis did not confirm the results of ultrastructural and am analysis. conclusion: strong correlation in the results obtained by different methods was observed. using combination of nonmolecular and molecular diagnostics methods high diagnostic accuracy can be achieved. granulomatous slack skin (gss): a case report with evidence of t cell clonality i. yilmaz * , h. baloglu, u. berber, z. kucukodaci, m. arcila * gata heh, dept. of pathology, istanbul, turkey objective: gss is a very rare variant of mf. we describe an additional case of this rare disorder in a 42 yo female that has been approved by tcrb&g tcell clonality assay. method: a 42 yo female with a single bulky skin lesion in the inguinal fold which had first appeared 6 y before and enlarged up to 20 cm, referred to plastic surgery for esthetics. h&e section of the deep skin biopsy of lesion showed a granulomatous infiltrate in the dermis. nuclear atypia and epidermotropism is not significant. but there is numerous multinucleated histiocytic giant cells. elastophagocytosis are present. tcrb&gt cell clonality assay showed a clonal rearrangement in tcr gamma gene. she was referred to dermatology for treatment and now she is getting uva1 phototherapy. results: the diagnosis of gss is difficult in early stages of the disease. clonal rearrangement of tcr genes can be a useful diagnostic tool in early stages of the disease. conclusion: 20-50 % of patients with gss carry a risk for the development of a second malignancy. gss may be associated with lymphoproliferative disorders. therefore, follow up of the patient is important. this case report supports that gss is an indolent variant of mf due to clinical, histological and t-cell gene rearrangement results. unusual occurrence of multicentric reticulohistiocytosis with systemic involvement in childhood s. zurac * , c. dobrea, a. diaconeasa, a. colita, c. solovan, c. arion, f. staniceanu * colentina university hospital, dept. of pathology, bucharest, romania objective: multicentric reticulohistiocytosis (mr) with systemic involvement is an extremely rare disease afflicting mainly adults. method: we report a case of sr arising in a 3 year 7 month old boy with concomitant celiac disease. results: since 6-7 months, he developed several brown to dark-red skin nodules (up to 0.8 cm) located mainly on face and arms; current episode include hemorrhagic syndrome, severe pancytopenia and massive hepato-splenomegaly. bone marrow aspirate ruled out acute leukemia and tezaurismoses; bone marrow biopsy and skin biopsy showed important infiltration with numerous large cells with abundant fine granular/ground glass eosinophilic cytoplasms in a reactive inflammatory stroma (lymphocytes, few plasma cells and few eosinophils); bone marrow cells were mononucleated, cutaneous cells were both mono&multinucleated; tumor cells immunophenotype was consistent with histiocytes (cd68+, s-100 protein + (faint), cd1a-, langerin-) both in bone marrow and skin biopsies; no hemophagocytosis was seen. mr with systemic involvement of skin, bone marrow, liver and spleen was diagnosed. cyclophosphamide therapy was instituted with initial diminishing of splenomegaly; latter on, despite adding cyclosporine and etoposide, no further impact on pancytopenia and hepatosplenomegaly was recorded. conclusion: complete hematologic examination is mandatory for patients diagnosed with cutaneous reticulohistiocitosis, irrespective of their age, in order to identify mr and institute proper treatment. primary cutaneous follicle centre lymphoma: a case report and review of literature k. diamantopoulou * , c. zorzos, c. eftychiadis, i. famellos, g. piagkos, g. karamanis, i. babalis, s. binder, h. mahera * general hospital kat, pathology, athens, greece objective: primary cutaneous follicle centre lymphoma (pcfcl) is an indolent, primary cutaneous b-lymphoma with an excellent prognosis but a high incidence of reccurence. apart from standard surgery and local radiotherapy, targeted strategies with anti-cd20 (rituximab) has introduced new treatment modalities. method: woman, aged 82, with solitary skin lesion on her face. after surgical excision, histo-and immunohistochemistry with the abc system on paraffin embedded tissue was performed. results: microscopically, a non-epidermotropic perivascular and periadnexal neoplastic infiltrate with a follicular growth pattern was observed. the lesion consisted of predominantly medium-sized centrocytes and several centroblasts enmeshed in a network of cd23(+) dendritic cells. immunohistochemically, the neoplastic lymphoid cells were cd20, cd79a, bcl2 and bcl6 positive. cd30, mum1 and ig were all negative. the patient received one cycle of rituximab and 6 months later is free of disease. conclusion: primary cutaneous b-cell lymphomas (cbcl) account for 25 % of cutaneous lymphomas. pcfcl is one of the three major categories of cbcl, recently classified by who, (the other two: pcmzl and pcdlbcl, leg-type). bcl-2, except for systemic follicular lymphoma, it can also occur in pcfcl, as in our case. pcfcl, usually locally treated with surgery and radiotherapy, could possibly offer a candidate for anti-cd20 targeted therapy, especially in multifocal lesions. objective: thirteen and ten years old male patients admitted with 4 months interval, having diffuse hemorrhagic papules on extremities and trunk. the first has not responded to methorexate previously. the second had varicella infection 3 months before and, his rushes spreaded out recently. method: multiple punch biopsies of the first patient revealed epidermal necrosis, basal vacuolisation and subepidermal diffuse infiltrate composed of cd3 and mostly cd8 positive t cells showing a striking angiocentric distribution. there were cd30 positive large cells with vesicular nuclei both in epidermis and dermis. some of the vessels exhibited lymphocytic vasculitis with fibrinoid necrosis. punch biopsy of the second patient revealed subcorneal pustule, dyskeratosis and basal vacuolisation, papillary dermal edema, erythrocyte extravasation and severe lymphocyte-rich cellular reaction infiltrating the interface. lymphocytes were cd3 and mostly cd8 positive. in between, there were large cells with vesicular nuclei exhibiting cd30 positivity. results: diagnoses were pleva based on the vascular and interface changes in accordance with clinical findings. both patients responded to oral antibiotics. conclusion: pleva is among the reactive conditions that can simulate cd30 positive lymphoproliferative diseases. although cd8 positive lymphomatoid papulosis is defined in pediatric patients, no response was seen to oral antibiotics. are hmb-45 and mib1 results reliable for safe diagnosis of nevi? z. yusifli * , o. kurtulan, ç. aydin, m. bugdayci, ö. gököz * hacettepe university, pathology, ankara, turkey objective: melanocytic lesions having subtle features suspicious for malignancy create considerable difficulty in decision-making.process. lack of deep hmb-45 positivity and low dermal mib-1 index can be reliable tools in association with morphology. method: twenty-seven acquired melanocytic lesions in which hmb-45 and mib-1 have been performed are reviewed to determine the importance of these markers in routine diagnostic practice. asymmetry, focal loss of maturation, presence of mitoses, epidermal consumption and lymphocytic inflammation were the morphological features that have created difficulty especially when seen alone in a bland appearing nevus. results: all cases with epidermal consumption and/or mitoses and nearly half of cases with inflammation, asymmetry and focal loss of maturation was associated with.focal deep dermal hmb-45 positivity. thirteen cases had an mib-1 index of ≤1 %. two of 3 cases with mib-1 index of >1 % had also hmb-45 positivity and/or inflammation. the case with a mib-1 index of 8 % was an acral nevus bearing 4 of the evaluated morphological features. conclusion: focal deep dermal hmb-45 positivity goes parallel with epidermal consumption and dermal mitoses, mostly used prevailing criteria of malignancy in melanocytic lesions but it's expression is not adequate for diagnosis of melanoma. low mib1 index can be a reliable marker of benignity. objective: leptospirosis in humans usually courses with hypokalemia and hypomagnesemia, and the putative mechanism may be related to nitric oxide production. methylene blue is a known inhibitor of inducible nitric oxide synthase, and have beneficial effects on clinical and experimental sepsis. method: serum creatinine and ionic changes were evaluated at various time points (4, 8, 16 and 28 days) in hamsters. we also determined the effect of methylene-blue treatment when used as adjuvant therapy combined with a late start of standard antibiotic (ampicillin) treatment. results: rather than k and mg depletion, hyperkalemia and hypermagnesemia were observed during acute infection. these findings are probably associated with an accelerated progression to renal failure, since this model is not feasible to mirror the supportive therapy (including fluid expansion) that retards progression to the oliguric/hyperkalemic state. infected hamsters at day 8 presented diffuse tubular cell swelling with mild or no nephritis. at days 16 and 28 they showed variable degrees of acute tubular changes, regeneration of tubular epithelia and nephritis. survival and renal pathology did not differ among different treatment groups. conclusion: adjuvant methylene blue had no effect on the survival, renal pathology or mg and k serum levels during acute-phase leptospirosis in hamsters. occupation of the striated muscle fibre by trichinella spiralis is associated with increased intracellular sialylation p. babal * , r. milcheva, d. ivanov, s. petkova * comenius university, dept. of pathology, bratislava, slovakia objective: the knowledge about glycoproteome in skeletal muscle is limited and most of the information come from studies on aberrant glycosylation in inherited muscle diseases. this work describes the intracellular changes in sialylation of skeletal muscle fiber during the process of its transformation into a nurse cell after occupation by the nematode trichinella spiralis. results: the study was performed at defined time points post infection. lectin histochemistry with tml, mal and sna detected increased production of sialylated glycoproteins within the affected fibers, and it was evaluated by acidic ninhydrin reaction in muscle homogenates. increase of total sialyltransferase activity was estimated by measurement of incorporated cmp-n-[14 c]-acetylneuraminic acid, and immunohistochemistry showed higher expression of α-2,3-sialyltransferases ii and iv within the affected fibers. sna lectin affino-blots showed at least four protein bands with approximate molecular weight between 126 and 159 kda, which were more reactive to sna compared to their counterparts from the control samples. conclusion: it is evident that skeletal muscle injury induced by trichinella activates biosynthesis of glycoproteins bearing sialic acids, which are not present in the normal muscle fiber. however, the protein identity, function and the biological significance of these sialoglycoproteins remain to be elucidated. bg051po001 objective: protozoal and helminthic infestations of the central nervous system (cns) are rare and their incidence is less than 1 %. they are more common among children, the elderly and immunocompromised individuals and tend to follow a fatal course. method: we report 2 cases of cerebral amebic abscess occurring in 2 immunocompetent men. results: they were 2 men aged of 43 and 18 years old. headache; altered mental status and fever were common presenting symptoms. in the 2 cases the diagnosis of cerebral abscess was suspected on cerebral ct scan. histopathologic analysis revealed extensive areas of necrosis and hemorrhage with granulomatous lymphoplasmacytic inflammatory infiltrate. other conditions such as malignancy and tuberculosis. it occurs normally in the mouth and tonsils, however, it can sometimes occur in the chest, abdomen, pelvis or other areas of the body. method: we describe a retrospective study about 56 cases of actinomycosis diagnosed over a 21-year-period (1990-2011) . results: the 56 cases were divided in 20 cervicofacial actinomycosis, 18 genito-urinary tract actinomycosis, 8 cutaneous actinomycosis, 7 abdominal actinomycosis and 3 cases occur in central nervous system. all patients were symptomatic. the most frequent symptom consisted in fever. the diagnosis was based on histologic study in all cases. tissue histologic studies show suppurative and granulomatous inflammatory changes, connective proliferation, in which bacteria form typical granular colonies composed of radiating gram positive filaments. conclusion: diagnosis of actinomycosis is usually made retrospectively by means of histologic examination of surgically obtained specimens, but rarely preoperatively. it is generally treated with long term antibiotics. expression of angiogenesis factors in placentas with intrauterine transmission of hiv a. kolobov * , e. musatova, v. karev, v. zinserling, d. niauri * st. petersburg, russia objective: placenta plays an important role in the prevention of the mother-to-child transmission of hiv during pregnancy. the purpose of this investigation was to study expression cd31, bfgf and tgfβ in human placentas in cases of intrauterine hiv infection. method: group a -11 placentas from women and children with hiv infection. in control -group b, were 16 placentas from women without hiv infection. on paraffin slices expression of cd31, bfgf, tgfβ and p24 was evaluated. we counted the area of positive cells related to the square of the slice. results: in all placentas with hiv infection p24 has been found in placental macrophages and villi's vessels. in group a expression of cd31 in endothelial cells was 4,22±0,85 % of the slice's square (in control -3,69± 0,53 %) (p>0,05). expression of bfgf in villi's stroma was 5,49±1,48 % (in control -4,01±0,6 %) (p>0,05). expression of tgfβ in villi's vessels was 11,2±3,6 % (in control -2,71±0,63 %) (p<0,05). conclusion: thus, placentas in cases with intrauterine transmission of hiv are characterized by increased expression of tgfβ the significance of this fact is still to be evaluated. objective: unilocular cystic echinococcosis which echinococcus granulosis causes is seen frequently in the animal husbandry practised underdeveloped countries. method: the 1709 unilocular cystic echinococcosis cases which were seen in the eastern part of turkey, in 30 years between 01.01.1981 and 01.01.2010, except faculty of medicine of atatürk university are presented. results: organ distribution: female: liver 557 (sd:15.614), lung 259 (sd 15.928), spleen 42 (sd 13.108), abdomen 25 (sd 11.55), kidney 21 (sd 11.92); male: liver 331(sd 15.406), lung 233 (sd 18.027), spleen 25 (sd 10.12), brain 23 (sd 11.55), soft tissue 15 (sd 14.197) . conclusion: the frequently seen organs in both gender are liver and lung. the rarely seen organs were spleen 67, brain 40, abdomen 39, kidney 36, vertebrae 29, soft tissue 27, pleura 26, bone 25, breast22. the others were very rare: eye 4, gallbladder 4, thyroid 4, neck 4, ovary 1, testis 1. relation of the length of appendix and parasitosis: preliminary report a. kurt * , s. a. özmen, i. erdem * bölge egitim ve arastirma hastanesi, dept. of pathology, erzurum, turkey objective: is there an effect of the length of appendix vermiformis on the settlement of the bowel parasites? we have decided to research. method: it was studied retrospectively for not to be under the influence. randomly chosen from the archives, 65 parasitic appendix (41 women + 24 men) and 65 appendix vermiformis (41 women and 24 men) lengths were compared. the last 65 appendix materials of the appendixes which include parasites (56 e.vermicularis, 8 a.lumbricoides,1 t.saginata) and operated any causes were examined. placeholder conditions such as fibrous obliteration and tumour were excluded. the premeasured length of the appendix in the series (65+65=130) were listed with the gender. results: appendix length in the control series: the length of appendix in men:7.15 the length of appendix in women:6.85 the length of appendix in both gender:6.95 cm. the length of parasitic appendix: the length of appendix in men:7.30 the length of appendix in women:6.76 the length of appendix in both gender:6.94. objective: on the basis of recently detected receptors for erythropoietin on the surface of mscs we hypothesized that introduction of epo together with mscs may enhance their effect and improve the results of sepsis treatment. method: 50 wistar male rats were randomized into 5 groups with 10 animals in each: group1 -the healthy controls, 2-5 groups were intraperitoneally introduced bacterial lps 20 mg/kg. group3-4 got × 105 allogeneic mscs, group4 -8.5 μg of recombinant epo-beta, group 5 -mscs and epo in the same doses. the morphological study of liver, spleen, thymus, lung and kidney was performed. histological specimens were evaluated by qualitative, semiquantitative and quantitative methods. results: in lung tissue the thickness of alveolar septums progressivly reduced from group 3 to 5.in kidney and liver tissue the dystrophy of hepatocytes and nefrocytes and a blood vessel's dilatation in studied groups also reduced.in lien tissue we observed hyperplasia of white pulp in groups 4 and 5. structural features of thymus were connected with patterns of t-lymphocyte proliferation and differentiation. conclusion: combined treatment with epo and mscs can reduce acute lung injury and kidney damage, cause hyperplasia of lymphoid tissue and enhance the immune response more than separate treatment in an experimental model of sepsis in rats. mycobacterial spindle cell pseudotumor of the liver a. alves * , a. costa, a. fernandes, g. do carmo, i. tavora * hospital santa maria, servico de anatomia patologica, lisboa, portugal objective: spindle cell pseudotumors are rare manifestations of mycobacterial infections. we describe a case in the liver in an immunocompromised patient with tuberculosis. method: a 28-year-old man, with active substance dependency, hiv-1 infection known for 7 years and acquired immunodeficiency syndrome for 3 years, was receiving tuberculostatic medication for disseminated tuberculosis and investigated for persistent fever. a liver ultrasonography confirmed a moderate hepatomegaly and showed multiple dispersed nodules in both lobes, with 1 to 3 cm of greater diameter. results: a needle biopsy from one of the nodules showed a proliferation of spindle cells without atypia, grouped in intersecting bundles, with immunoreactivity to vimentin, cd68 and lysozyme and negative for cd34, ps100, sma and desmin; a ziehl-neelsen stain revealed several acid fast bacilli in the spindle cells. they were pas negative. conclusion: pseudotumoral lesions in hepatic tuberculosis usually correspond to granulomatous inflammation. a spindle cell proliferation, like that seen in other organs are more frequently associated with non tuberculous mycobacteria, is very rare. we found only another case in the literature of liver spindle cell pseudotumor associated with tuberculosis. this lesion must be differentiated from mesenchymal neoplasms. sixteen months follow-up in a treated case of whipple disease (wd) c. popp * , m. petre, g. micu, a. bastian, r. chirculescu, r. mateescu, f. staniceanu * colentina university hospital, dept. of pathology, bucharest, romania objective: wd is a rare, multi-systemic infection caused by a ubiquitous environmental bacteria -tropheryma whippleithat affects the duodenum and small intestine but also the brain, endocardium, skin, lungs and joints. method: we are presenting a 49 years old man with a 2 years history of diarrhea, malabsorption and weight lost (about 30 kg). endoscopical examination revealed an unspecific, yet unusual pattern of lesions: small, yellow, multiple, slightly elevated patches involving duodenum and jejunum. histological examination revealed numerous foamy macrophages in lamina propria with pas (+), ziehl-neelsen (−) material in cytoplasm. results: treatment with trimethoprim/sulfamethoxazole was initiated with a spectacular response: after 2 days of therapy the patient had normal stool and in 6 months he already gained back 20 kg. after 1 year of treatment the biopsy confirmed a partial remission with fewer macrophages persisting in lamina propria of duodenum. after 16 months of treatment (march 2012), very few macrophages with pas (+) material are still identified on duodenal biopsy. conclusion: wd is a potentially fatal disease with unspecific clinical and endoscopical signs that can be diagnosed on duodenal biopsies. histological follow-up is a useful tool of managing the treatment and histopathological confirmation of the absence of foamy macrophages is mandatory in discontinuing therapy. small cell urothelial carcinoma (scuc) comtherapy. method: we report two cases of scuc. both of them refer to 50 year-old males, smokers. they presented with gross hematuria. transurethral resection bladder tumor (turbt) specimens showed scuc, followed by cystoprostatectomy and pelvic lymphadenectomy, confirming the initial diagnosis. immunohistochemistry was used on paraffin embedded tissue. results: microscopically, the tumor cells show a diffuse architectural pattern with occasional nesting, are small, uniform, with scant cytoplasm, nuclear crowding, nuclear molding, inconspicuous nucleoli and finely stippled chromatin. numerous mitoses, azzopardi phenomenon and foci of necrosis are present, as well as areas of urothelial carcinoma. tumor cells are strongly (+) with ker ae1/ae3, cam 5.2 (dot-like perinuclear pattern), cd56 (the most consistent neuroendocrine marker), p53, ki-67(60 %), focally (+) with synaptophysin and (−) with chromogranin, ker34βe12, ker7, ker 20, ttf-1, lca. conclusion: pathologic and immunohistochemical data identify scuc. differential diagnosis includes metastasis of a small cell carcinoma from another site rare variant: primary renal malignant fibrous histiocytoma n primary leiomyosarcoma of the kidney: three cases b renal pelvic and ureteral dilatation was seen in the first case because of the mass effect. microscopically the alternating spindle tumor cells were detected which were positive with actin, desmin, caldesmon and negative with s100, pan-ck, cd34 and cd 117 congress hall foyer 3rd floor ps-23 poster session gynaecological pathology although rare, u-lms is the most frequent malignant gynaecological mesenchymal tumor, usually disclosing unfavorable prognosis. u-lms diagnosis may be controversial. in this study we evaluated clinical-pathological features/prognostic factors of u-lms. method: all consecutive u-lms diagnosed (1987-2012) at chsj and ipo-porto and with available material were reevaluated. the clinical files were reviewed, including demographic, clinical, imaging, surgical, staging, and follow-up parameters. the pathology features, including immunohistochemistry evaluation, were re-evaluated and the tumors staged according to most recent consensus criteria. results: the mean age at diagnosis of the 38 cases was 57 years (39-83); 68 % were multiparous and 60 % postmenopausal women. the majority (82 %) had symptoms at diagnosis, mostly abnormal bleeding. histological classification displayed: 63 % spindle, 13 % epithelioid, 3 % myxoid, 10.5 % pleomorphic, and 10.5 % mixed u-lms. treatment included surgery (97 %), radiotherapy (67 %), and chemotherapy (63 %). overall median survival was 49 months portugal objective: u-lms is the most frequent malignant gynaecological mesenchymal tumor, disclosing dm results: our series: 35 (39.8 %) with metastasis; literature: 65 with metastasis. global mean age at diagnosis: 52 years (28-74); initial treatment: hysterectomy (96 %), radiotherapy (28 %), and chemotherapy (40 %) pancreas, vulva/vagina, salivary gland, oral cavity, heart, mediastinum, adrenal, and breast; 38 % with metastatic related symptoms. overall median time to first metastasis: 21 months (ci 95 % 15.5-26.5). pancreas, heart and mediastinum were the earlier metastatic sites microglandular adenocarcinoma (mga) of the endometrium, a rare subtype of endometrial mucinous adenocarcinoma, has histologic similarities to the microglandular hyperplasia (mgh) of the cervix. it should not be misdiagnosed as cervical mgh especially in a small endometrial biopsy specimen. method: eight cases of mga of the endometrium and 5 cases of control mgh of the cervix were immunostained for ki-67, cd34, p16, p53, p63, vimentin, estrogen receptor (er), progesterone receptor (pr) and pten. hpv dna analysis was performed using paraffin-embedded mga tissue by polymerase chain reaction amplification endometrial carcinoma (ec) displays variable outcomes. method: immunohistochemistry for p53, ki67, egfr, her-2 (%positive), pten, estrogen and progesterone receptors (er/pr), perk, pjnk (h-score results: ade: er/pr mean h-score from 70/190 to 11/ 110, from g1 to g3, 34 for pten, 13 and 67 for perk and pjnk, two adeg3 >75 % p53 and one 20 % ki67. ads: 55 % >75 % p53 and 42 % >10 % ki67, 14/61 for er/pr, 79 for pten, 18 and 116 for perk and pjnk. adcc: none er/pr; 90, 144 and 73 for perk -regulated) compared to ulm and myometrium, several known to be involved in cell-cycle and/or apoptosis pathways (p53, rb, e2f, cyclin-d1/e) and/or metastasis. conclusion: albeit some overlap, mirna expression profile of ulms is different from ulm but relatively similar to non-ulms. ulms and non-ulms share deregulation of several mirnas that may be linked to specific cell-cycle/ apoptosis pathways 50 %) included rhabdomyoblastic (5), cartilaginous (2), adipocytic (1) & sex-cord-type (1). four cases had leiomyomas; alivewith-disease (mean, 29.4 months) and 5 free-of-disease (mean, 15 months). conclusion: adenosarcomas are uncommon endometrial endometrioid carcinoma is the most common malignant tumor of the uterine corpus usually developing in postmenopausal women. more seldom it occurs in young reproductive age and sometimes develops from an endometrial intraepithelial neoplasia (ein). the ein diagnosis and treatment in such patients have some difficulties. method: we have studied curettage tissue specimens from three women with ages 27, 33 and 35, with abnormal uterine bleeding and obesity. the severe complex endometrial hyperplasia with low grade intraepithelial neoplasia was revealed in all cases polypoid glands with sarcatomatous stroma (phylloides-like pattern). b. periglandular condensation. c. sarcomatous overgrowth. d. decidualization. e. cartilagenous dedifferentiation. f. rhabdomyoblastic dedifferentiation (inset). g. vimentin positivity. h. desmin in rhabdomyoblasts. i. er positive glands. which were examined histopathologically, and in terms of cytologic features, p16 and ki67 immunoexpression. results: cervical smear samples from 93 patients were diagnosed as 6 agus, 9 ascus, 12 asc-h, 19 hgsil, 47 lgsil cases three types are generally acknowledged: circumscriptum (or capillary), cavernous, and cystic. 54-years-old female. method: case report: a 54-years-old woman presented to the department of obstetrics and gynecology at our hospital. for uterin myoma, she underwent total abdominal histerectomy and bilateral salpingo-oophorectomy congress hall foyer 3rd floor ps-24 poster session dermatopathology atypical spitz tumor or spitzoid melanoma: which morphological criteria ensure the diagnosis? method: thirty-one spitzoid lesions were reviewed by a scoring system based on seven selected criteria reported previously appreciating characteristics of spitzoid lesions. as a prevailing criteria, presence of deep mitoses had a score of 2; ulceration, asymmetry, epidermal consumption, solid growth pattern, loss of maturation, pigment heterogeneity were scored as 1 for each. final diagnosis was sm for scores 4-8, ast for 1-3, sn for 0. results: the most frequently seen criteria, loss of maturation was solid growth pattern and pigment heterogeneity were nearly equally seen in sm and ast. first diagnoses of 16 sn switched to ast, 8 to sm, and of 2 ast to sm. conclusion: presence of deep mitoses is the single criteria present only in sm. loss of maturation, epidermal consumption and ulceration can be used to differentiate ast from sm basal cell carcinoma (bcc) of the skin is the most common form of human cancer. sometimes the histological appearances of benign adnexial neoplasm of hair follicle apparatus tumors (trichoepithelioma, trichoblastoma) are diffucult to distinguish from bcc. the aim of this study is to investigate the usefulness of cd10 and p53 in distinguishing bcc and benign adnexial neoplasm of hair follicle apparatus tumor. method: this study included 237 bcc and 27 benign adnexial neoplasm of hair follicle apparatus tumor. we used immunohistochemical stains for p53 and cd10 in all cases. results: in bcc cases the expression of cd10 was noted in tumor cells in 222 cases objective: a widely accepted adequacy criteria for histopathologic evaluation of prostate core biopsy protocols has not been defined yet. selection of patients for repeat biopsies after a negative initial biopsy result is also a matter of debate. we aimed to evaluate the impact of total core length for detecting prostate cancer in a 12-core transrectal ultrasound guided biopsy scheme. method: retrospectively, we investigated the core lengths of 768 patients underwent initial prostate biopsy by a lateral zone targetted 12-core protocol between 2006 and 2011. results: cancer was identified in 292 (38.0 %) patients. mean total core length for positive and negative patients were 15.3 cm and 14.6 cm, respectively. cancer detection rates in groups for total core length <12 cm, 12-15 cm, and >15 cm were 30.9 % (55/178), 40.6 % (96/236), and 39.8 % (141/354), respectively. when a threshold of 10 cm was selected for total core length, overall cancer detection rates for <10 cm and ≥10 cm groups were 23.9 % (17/71) and 39.5 % (275/697), respectively. conclusion: although molecular confirmation remains its diagnostic gold standard, tle1, ema, bcl-2, mic2, cks and cd34 constitute an optimal for sy-novial sarcoma. awareness of tle1 expression in other tumors and its correct interpretation are necessary. objective: malignant peripheral nerve sheath tumors (mpnst) comprise 5-10 % of sarcomas. prognosis is poor and the search for new treatments is ongoing. her3 is a crucial receptor for neuregulin signalling in schwann cells but to date, has not been studied in sarcomas. our aim was to determine the prevalence of her3 receptor expression in soft tissue tumors, including mpnst. method: her3 expression was evaluated by immunohistochemistry in a total of 50 benign and 71 malignant mesenchymal tumors, including 16 neurofibromas, 16 schwanomas, 14 mpnst and 75 other lesion (uterine and non-uterine leiomiomas and leiomiosarcomas, synovial sarcomas, undifferentiated pleomorphic sarcomas (ups)). results: her3 overexpression was found in 31 % of cases. significantly, her3 positivity was present in ps-17-075 breast cancer in young czech women: an institutional review f. sobande * , a. ryška * dept. of pathology, hradec kralove, czech republic objective: breast cancer in young women poses unique diagnostic and management challenges however, very few large studies investigating clinico-pathologic and immunophenotypic characteristics within this group exist and varied, arbitrary age-limits are used to define 'young'. method: an institutional archive search was performed for breast cancer diagnosed in women <50 in [2006] [2007] . age at diagnosis, tumor histological type and grade, estrogen and progesterone receptor (er and pr), her2/neu, ki-67 and p53 status as well as clinical course during the follow-up period were recorded. patients were sub-classified into agegroups of 5-year intervals for statistical analysis. results: ninety of 516 carcinomas were diagnosed in women <50. they frequently displayed aggressive morphology, high proliferative activity, increased expression of p53 and high incidence of lymph node metastasis. we found a statistically significant decrease in her2/neu over-expression with increasing age using 40 as the cut-off (<40: 42 %, ≥40: 11 %; p=0.0032, chi-square test). no significant age-related differences in er or pr expression were observed within the group of women <50. conclusion: we show that 40 should be used as the cutoff for defining young women; in whom breast cancer is most likely to over-express her2/neu. the mechanisms behind this finding and its significance are yet to be elucidated. objective: osna is a molecular assay for lymph node in breast carcinoma (bc) based on m-rna detection of ck19. the aim of this study is to evaluate the rate of negative o heterogeneous immunohistochemical expression of ck19 in bc. method: ck19 was assessed in 345 consecutive samples. cases were categorized as positive, negative and heterogeneous. other parameters recorded where histological type and grade (nottingham) and molecular subtype (st. gallen2011). results: 319 cases (92.4 %) were ck19+. ten (2.8 %) were ck19-and 16(4,6 %) were heterogeneous. there were 2 intraductal carcinomas, 1 heterogeneous and 1ck19-. among 24 invasive carcinomas, there were 9ck19-: 6 ductal, 2 papillary and 1 metaplastic. three cases corresponded to g1, 4 g2, 1 g3 and 1t1mi not available. five cases were luminal a(la), 1 luminal b/her2-(lb-), 1 her2+ and 2 triple negative (tn). among 15 heterogeneous cases, 13 were ductal, 1 metaplastic, and 1 atypical medullary carcinoma. eleven were g3, and 4 g2. four were lb-and 11 cases were tn. conclusion: all lobular carcinomas expressed ck19 ck19 was negative in 2.8 % and heterogeneous in 4.6 % of bc. they represent a heterogeneous group but half of these cases are either g3 or tn. 74 % of heterogeneous cases were tn and 26 % lb-with no la cases in this subgroup. case report of an extremely rare tumor of the breast: mucoepidermoid carcinoma f. staniceanu * , c. socoliuc, a. birceanu, e. gramada, l. nichita, r. andrei, a. croitoru * colentina university hospital, dept. of pathology, bucharest, romaniaobjective: staniceanu and socoliuc are first authors in equal proportions mucoepidermoid breast carcinoma, classified in category of purely epithelial metaplastic carcinomas, is rarely encountered with only a few female cases described in literature. it is composed by mucous, intermediate and epidermoid cells, similar to its salivary gland counterpart. method: we present the case of a 67 years old man with a 2,2/1,6 cm breast tumor located under the nipple, poorly circumscribed, with increased consistency and central cystic area containing serous fluid. tissue samples were preserved using 10 % formalin. all sides were stained using hematoxylin-eosin. immunohistochemistry was performed using labeled streptavidin-biotin method. results: microscopic examination reveals islands and sheets of polygonal cell with clear/pale cytoplasm, "signet cells" figures, areas of squamous metaplasia and centrally located hyperchromatic nuclei, some with visible nucleoli and rare mitoses. tumoral parenchyma contains ducts and cysts of varying sizes, with pale eosinophilic luminal accumulation pas, d-pas and alcian blue positive (mucin). immunohistochemical tests were performed. differential diagnosis was made among several benign and malignant lesions, the final diagnostic being mucoepidermoid carcinoma, low grade. conclusion: mucoepidermoid carcinoma of the breast is an extremely rare entity; however its specific morphologic and prognostic features (similar to its salivary gland counterpart) allow proper diagnostic. endomyocardial biopsy value in differential diagnosis of hypertrophic cardiomyopathy j. markovic * , i. ranisavljevic katuca, s. glumac, r. jankovic, j. d. vasiljevic * clinical center of serbia, dept. of pathology, belgrade, serbiaobjective: endomyocardial biopsy (emb) may be used to diagnose different types of cardiomyopathies, to monitor transplant rejection, evaluation of myocarditis, heart failure of unknown origin, arrhythmias, drug toxicity and heart neoplasmas. the aim of this case is to show how noninvasive cardiac investigations are sometimes not sufficiently conclusive for distinguishing between hypertrophic obstructive cardiomyopathy (hocm) and the other cardiac diseases.method: thirty-one representative left ventricle biopsies were obtained following suspected hocm. samples underwent routine standard and special staining procedures. five histologic parameters were used for assesment (disarray and hypertrophy of myofibers, myocardial short runs, perinuclear halo and bizarre nuclei and fibrosis) and graded from 0 to 3, presenting in summary histological hocm index (hhi-ranging from 0 to 15). results: the histological findings in emb of thirty-one patients, in 8 (25,8 %) patients hocm diagnosis was confirmed, in 6 (19,35 %) patients hhi was under or equal to 50 % (8) so hocm diagnosis couldn't be confirmed, amyloidosis in one (3,22 %) patient, myocarditis in 4 (12,9 %) patients, dilatative cmp in 6 (19,35 %) patients and unspecific cardiac changes in 6 (19,35 %) patients. conclusion: a definitive diagnosis can be obtained by means of a multidisciplinary approach including emb findings. morphometric study of structural changes in the heart of people who abused alcohol for more than 5 years l. mitrofanova * , e. naumova * almazov federal heart, blood and cardiomorphology, st. objective: the information about structural changes in the heart of people abused alcohol (paa) is very contradictory. method: we studied 22 hearts of paa (>100 g per day), 10 hearts of patients with ischemic heart disease (ihd) and 10 hearts of healthy persons. at the morphometric study of heart we measured the mean cardiomyocyte diameter (mcd), the relative areas of fatty infiltration (fi), fibrosis, cardiomyocytes. immunohistochemistry was performed using antibodies against cd3-antigens to count the number of antigen-positive cells per 1 mm². the average heart weight, the mcd of the paa were not significantly different compared to the ones of the control group. the average relative area of fi of rv for the group of paa was 21±15 % and was significantly different compared to the one of both the control group and the group with ihd. the relative area of rv fibrosis in the group of paa was 9.9±6.5 % and was not significantly different compared to the ones of the other two groups. the mean number of cd3+ lymphocytes/mm² of 23 % of paa was more than 14. conclusion: the paa did not have myocardial hypertrophy. 23 % of them had chronic myocarditis. marked fi in paa was significantly higher than the other two groups. objective: the aim of the study was to evaluate the expression of angiotensin-converting enzyme 2 (ace-2) in left ventricular myocardium in the presence of chronic myocardial ischemia. method: heart specimens with persistent ischemia (n=43, pre-infarction ischemic heart disease (ihd) group) and postinfarction scar (n=32, post-infarction ihd group) from dissected males and heart explants (n=15, end-stage ischemic heart failure group) were studied. heart specimens (n=29) selected at autopsy from individuals who died from accidents were used as controls. the slides of myocardium were incubated with polyclonal antibody against the ace-2 (1:1000, ab15348, abcam). results: ace-2 expression in cardiomyocytes of the preinfaction ihd and post-infarction ihd groups did not differ from the controls and was increased only in the end-stage ischemic heart failure group (p<0.05). ace-2 expression in vascular endothelial cells, smoth muscle cells and macropages of the pre-infarction ihd group did not differ from the controls; but the number of ace-2 expressing cells in the post-infarction ihd and in the end-stage ischemic heart failure groups was greater, as compared with the pre-infarcion ihd group and the controls (p<0.05). conclusion: expression of ace-2 is increasing in progression of ischemia-induced myocardial dysfunction. thrombotic occlusion of the peripheral pulmonary arteries: anatomical "trigger" of the massive pulmonary thromboembolism (pte) f. portelli * , f. p. busardò, l. gutsul, p. fleres, p. pugnetti, e. orlando, e. maresi * university of palermo, section of pathology, italy objective: tubulocystic carcinoma of the kidney (tcrcc) is a rare variant of renal tumor showing unique gross and microscopic features. several recent studies favour its inclusion within the papillary renal cell carcinoma (prcc) group. method: patient 1 was a 55 year-old man with a left asymptomatic renal mass incidentally discovered by sonography. patient 2 was a 72 year-old man with aortic estenosis and coronary disease. a right renal mass was discovered in the work up for aortic valve replacement and coronary bypass. results: both tumors were well circumscribed masses with "sponge-like" appearance. on light microscopy, the tumors were composed of ectatic tubules and cysts of variable size, lined by a single layer of tumor cells with eoshinophilic cytoplasm, apocrine appearance, rounded nucleus sometimes overlapping, and prominent nucleoli. ihc showed positive staining with ck7, e-cadherin, cd10, and amacr. conclusion: tcrcc has been recently recognized as a distinct subtype of prcc by some authors. in fact, some few cases reported have histological transition between tubulocystic and papillary phenotypes on routine histological sections. additionally, tcrcc shows trisomies of chromosomes 7 and 17. the exact location of this tumor within the 2004 who classification is a matter of debate. objective: our purpose is to evaluate her2 protein expression in urothelial carcinoma of the bladder. her2/neu is a tyrosine kinase receptor (family of human epidermal receptors), showing overexpression in a large variety of tumor cells. method: 30 bladder biopsy and cystectomy specimens were included in this study. the age of the patients ranged from 25 to 86 (mean 71). among them 25 (83.33 %) were male and 5 (16.67 %) female. all cases were reevaluated histopathologically according to who 2004 grading system. her2 protein expression was detected by immunohistochemistry (cb11) using the same criteria as for breast cancer. results: all 30 bladder neoplasms were low-or high-grade urothelial carcinomas. her2 protein overexpression was observed in 10 (33.33 %) cases (2+: 5 cases and 3+: 5 cases), while 20 (66.67 %) cases were negative (0: 3 cases and +1: 17 cases). among her2 positive cases 9 (90 %) were high-grade carcinomas, while 1 (10 %) was low-grade carcinoma. among negative cases 14 (70 %) were lowgrade carcinomas, while 6 (30 %) were high-grade carcinomas. conclusion: these findings indicate that her2 protein overexpression is predominately correlated with high tumor grade in urothelial carcinoma of the bladder and patients with these characteristics could be potential candidates for targeted therapy. mucinous adenocarcinoma of the renal pelvis: a case report and review of the literature m. otani * , t. tsukamoto, h. serizawa * tokyo medical university, division of diagnostic pathology, japanobjective: primary mucinous adenocarcinoma of the renal pelvis is extremely rare and about 50 cases have been reported to date. we present a case of mucinous adenocarcinoma followed by renal cysts. method: the patient is a 52-year-old male. a renal cyst in the right upper region was found incidentally. he had no symptom at that time. after 4 years, he came to the hospital because of hematuria and back pain. the examination revealed that the renal cyst was enlarged accompanied with solid lesion. the high serum level of cea indicated malignancy and tumorectomy was performed. results: macroscopically, a renal cyst (15×10 mm) in the right upper portion was adjacent to a solid mass (22×17 mm). the tumor showed papillary growth (20× 15 mm) in renal pelvis. microscopically, the tumor was consisted of adenocarcinoma with tubular or papillary structure, spreading from cyst wall to pelvis. the tumor cells contained mucin and some showed signet-ring celllike feature. conclusion: clinically, high serum level of cea is a marker of mucinous adenocarcinoma originated from renal pelvis. in our case, the tumor has already from pelvis to cyst wall. we thought it originated form renal pelvis, however, the renal cyst is also a candidate for its origin. results: an 8 cm tumour was revealed in douglas cavity, histologically, deciduoid morphology was predominant with abundant atypical mitoses and massive neutrophyls infiltration. immunohistochemical positivity: calretinin, cytokera-tin5-6, cam5.2, cytokeratinae1ae3, e-cadherin, vimentin, hmbe1, ca125 and negativity: cd99, cd1a, melan-a, myod1, wt-1, cd10, hmb45, actin, thrombomodulin, desmin, alpha-fetoprotein, moc31, cerb-2, estrogen and progesterone. normal dna-repair proteins expression. conclusion: mdm is highly aggressive neoplasm, differential diagnosis includes: squamous carcinoma, gist, deciduosis pseudotumoral, anaplasic large cell lymphoma, trophoblastic tumour, oxyphilic variant of ovarian clear cell carcinoma. this case represent a rare variant of mesothelioma, in which, was possible a complete resection with a probably better pronostic unlike most of these cases. objective: we evaluated morphological features of male gonad in an adult patient with tfs with an accent on its immunohistochemical properties. method: our 47 years old patient had a primary amenorrhea. external genitals were female, without pubic and axillary hair. us confirmed cystic formation of the left gonad and lack of uterus. tumor markers were normal but testosterone was high. cystic formation of the left gonad was surgically removed, initially specified as a left ovary, as well as the other gonad. pathohistological examination and karyotyping was done. results: immunohistochemical analyzes of the gonad sample reveal that sertoli cells showed positivity for inhibin α, vimentin, cd99, ckae1/ae3 and calretinin and leydig cells showed positivity for inhibin α, vimentin, cd99, ckae1/ae3 and calretinin, and weak positivity for s-100, synaptophysin i nse. immunoreactivity for cd99 in hyperplastic areas of leydig cells showed the most intensive conclusion: we conclude that cd10 and p53 expression may be a useful adjunct marker in distinguishing bcc and benign adnexial neoplasm of hair follicle apparatus tumor. objective: pigmented purpuric dermatosis (ppd) comprises a group of vascular disorders of unknown etiology. histologically, it is characterized by lymphocytic capillaritis in the papillary dermis. method: a retrospective and monocentric study concerning the cases of ppd diagnosed in the department of anatomopathology la rabta over 6 years (from 2006 to 2011). these cases were studied clinically and histopathologically. hepatitis c and b profile was carried out to evaluate a possible relation. results: 6 cases of ppd have been diagnosed; 2 men and 4 women, aged from 20 to 80 years. only one patient tested positive for hepatitis c with an elevated viral load. she had a profuse eruption involving the trunk and the limbs of 4 years duration, contrary to the other patients in whom only the lower limbs were affected. she has been treated by alpha interferon and ribavirin with a disappearance of the eruption within 3 months. in the five other patients, the lesions resolved spontaneously within a period of 2 to 9 months. conclusion: hepatitis c may play a role in the pathogenesis of ppd especially when the lesions are profuse and longstanding. in our patient, the disappearance of lesions 3 months after the onset of interferon alpha therapy with a significative decrease of viral load is an argument supporting our hypothesis. objective: fibrolipomatous hamartoma of nerve is a benign lesion, very rare in medical practice, occurring in children or young adults. case report: a 44 year old woman, g. n., presented with a slowly growing plantar mass in the right foot that had been present for 7 years. neither the patient nor members of her family had a history of neurofibromatosis or other soft tissue tumors. she had no pain at rest, but she had shooting pain while running or walking. physical examination revealed a mobile, tender mass located in the plantar soft tissue. method: after surgical excision, the lesion was well circumscribed, 20/10/8 mm, white colour with yellow areas. tissues specimens were fixed in 10 % buffered formalin, routinely processed and embedded in paraffin, sectioned at 3 μm, then stained routinely by hematoxylin -eosin, than examined by light microscopy. results: the lession is characterized by proliferation of mature fatty and fibrous tissue within the epineurium of the nerve accompanied by prominent concentric perineurial fibrosis. the histopathological diagnosis was fibrolipomatous hamartoma of nerve. conclusion: the tumour usually affects nerves of the upper extremities, most commonly the median nerve. involvement of other nerves is rare. the lesion is benign and the recommended treatment is surgical excision. objective: leishmaniasis refers to the spectrum of diseases caused by leishmania species, which are protozoa of order kinetoplastida. clinically leishmaniasis is divided into visceral (kala-azar), cutaneous, mucosal, and mucocutaneous syndromes. the final diagnosis of the disease, lesions of the parasites, as well as shown smear'lerde factor confirmed by histopathological and clinical evaluation. method: in our study, 6 patients with the diagnosis of leishmania cutaneous between 2006 and 2012 in department of pathology, ataturk university faculty of medicine were presented. results: four (66.6 %) cases were females and 2(33.3 %) were males. age ranging of cases was found from 0 to 60 years and median age 31.85±12.15 years. the lesions were found on the face in patients. facial skin biopsy specimens were taken in all cases. giemsa-stained touch preparation of the skin biopsy revealed amastigotes inside macrophages, consistent with leishmaniasis. lesions of 2 patients who have long been different from the rest of them were those who migrated to the cukurova region. conclusion: leishmaniasis is public health problem in southeastern anatolia region of turkey, especially in sanliurfa and çukurova region. autoimmune blistering skin diseases in east anatolia n. gursan * , o. ceylan, h. bilen, n. akdeniz, m. atasoy * ataturk university, medical faculty, erzurum, turkeyobjective: autoimmune blistering skin diseases can be divided into pemphigus diseases and subepidermal bullous diseases. in pemphigus, the autoantibodies are directed against intercellular contact structures; in the pemphigoid diseases, the autoantibodies are directed against adhesion molecules of the basal membrane zone. method: a retrospective study of 102 cases of autoimmune blistering skin diseases obtained over a period of 7 years from 1st october 2005 to 31st december 2011 in the department of pathology, medical faculty of ataturk university was designed. results: in the present study, the mean age was 52 years and male: female ratio was 0,7. the most commonly affected sites were the buccal mucosa and the palate. %18 of cases, the oral cavity was the primary site of involvement. in histopathological examination we showed intraepithelial vesicle formation, subcorneal separation with acantholysis, and the presence of tzanck cells in biopsy specimens. conclusion: the incidence of autoimmune blistering skin diseases in our department increased in the past 7 years. pemphigus vulgaris is the most common of these diseases. %70 of autoimmune blistering skin diseases was pemphigus diseases. objective: acute febrile neutrophilic dermatosis (sweet syndrome) is the most common neutrophilic dermatosis. a dense dermal neutrophilic infiltrate is the usual histological findings. the absence of histological image of vasculitis has been considered a characteristic of the disease and a distinctive feature of the other neutrophilic dermatoses. however, recent reports suggest that vasculitis should not exclude the diagnosis. method: this retrospective study examines 32 cases collected over a period of 14 years (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) . we studied its clinical and histological characteristics. skin biopsy specimens were reviewed to determine the prevalence of vasculitis. results: sweet syndrome is significantly more common in women than in men (sex-ratio=1/15). the patient's mean age was 51 years. disease developed most often in autumn. atypical presentation was noted in 18 patients. skin biopsy revealed a sweet syndrome pattern in all cases. the prevalence of vasculitis (vessel wall damage with fibrinoid necrosis) was 28 % (9 of the 32 patients). conclusion: our series of patients is particular by the frequency of atypical clinical presentation and the presence of vasculitis in histologic examination, a feature that shouldn't exclude the diagnosis. it was suggested that vasculitis in sweet syndrome represents an epiphenomenon rather than a primary immune-mediated process. dermal lymphangitic carcinomatosis as a primary manifestation of lung carcinoma: report of an unusual case c. hadjileontis * , e. angelidou, a. kafanas * serres general hospital, dept. of pathology, greeceobjective: although dermal lymphangitic metastases are reported to range between 1 and 4,5 % of all visceral carcinomas, their presence as a primary manifestation of the tumor is rather unusual. different structures (isolated or forming small clusters) with the morphological characteristics of amoeba trophozoites were identified in the areas of necrosis with and without an inflammatory reaction. the diagnosis of cerebral amebic abscess was then made. the 2 patients received antibiotic treatment. conclusion: cerebral amebic abscess is rare but has poor prognosis. it should be considered as a diagnosis for any patient with subacute and/or chronic meningoencephalitis without evident bacterial etiology. more effective antibiotic drugs can be chosen for post-surgical treatment; furthermore, better survival may be achieved by diagnosing the disease in early stages. morphometric characteries of women placenta which can be different forms of syphilis m. bokhodir * , g. raimnazarova, z. parpiev * tashkent medical academy, dept. of patological anatomy, uzbekistanobjective: many literary information give us about getting down coefficient of fertitity -placenta at women which suffer from pregnancy with syphilis. method: verified data analysis about morphometric placentae and it's comparison with newly-born child at 50 women which suffer from different forms of syphilis. results: research placenta with syphilis show us the area of placenta surface is decreased, it was formed primary syphilis −297,+−42,1 sm, secondary −316,8±28.8sm and with latent form of syphilis 319,7±28.8(at control 331.4±234,8sm2 p> o.l), at that time as a mass, placentary cell was been bigger and it formed (at primary-589.1±32.7gr and at secondary-568,2±25,5gr and at latent form of syphilis 582.8±20.7gr, then in control (555,0±35,7) but all this different were been statistically unreliable (p>0,05). comporision mass of placenta and mass newly born-child show us corfficient of fertitity-placenta is make up (5,86±0,21) and actually it is not different at control fact group. conclusion: the change coefficient of fertitity -placenta was typifying for until penicillin era, that time. when actual condition, treatment syphilis with antibiotic to bring on development phathomorphism placentae with out different coefficient of fertitity -placenta. influenza b virus-associated pneumonia: report of one fatal case associated with sickle-cell trait l. de carvalho * , l. avila, h. camargo, c. torriso, r. penny, a. sementilli * centro universitário lusíada, dept. of pathology, santos, brazil objective: influenza virus pneumonia complicated with acute respiratory distress syndrome is rare and has a high mortality rate. patients with sickle cell trait are asymptomatic, however, complications with increased risk of thromboembolism can occur. method: case report: a 30-year-old woman with no preexisting medical conditions presented nonproductive cough and fever for 4 days. for the next 3 days, developed acute respiratory failure. she was treated for septic shock and acute respiratory distress syndrome without success with death. expectoration and blood cultures were negative and the real-time polymerase (rt-pcr) of the thracheal swab was positive for influenza b virus. results: the autopsy showed a diffuse necrotizing alveolitis and intra-alveolar hemorrhage with pulmonary infarction. there was no histological evidence of bacterial pneumonia. an evident blood cell sickling was observed associated with sickle cell trait. rt-pcr was also positive for influenza b virus in pulmonary tissue and the immunohistochemistry was negative for influenza a virus. conclusion: severe hypoxemia caused by the acute respiratory distress syndrome was a possible cause of the blood cell sickling and thrombotic events. objective: lupus vulgaris (also known as "tuberculosis luposa") are painful cutaneous tuberculosis skin lesions with nodular appearance, most often on the face around ears nose, eyelids and lips, cheeks. ıt is still a major health problem in developing countries. cutaneous tuberculosis is a rare form of extrapulmonary tuberculosis. lupus vulgaris (lv) is the most common form of cutaneous tuberculosis. lv should make differential diagnosis other granulomatous disease. all three cases of old female patient. conclusion: herein, three cases were presented which they treatment with correlation clinical and histopathological examination. objective: actinomycosis is a rare, chronic, and slowly progressive granulomatous disease caused by filamentous gram positive anaerobic bacteria from the actinomycetaceae family. it is often misdiagnosed because it can mimic conclusion: the mean length of appendix in both series was about 6.95 cm. there were no important difference of the length of either parasitic appendixes or all cause appendectomy materials. in this study, it is concluded that there isn't an effect of the length of appendix on the settlement of bowel parasites. objective: cystic hydatid disease (chd) is an infection produced by larvae of the parasite platyhelminth echinococcus granulosus. living in a rural area is an important risk factor for this disease. it is still an endemic disease in some regions of the world, particularly in many mediterranean countries. the organs most commonly affected are the liver and the lungs. method: we present a new case of multiple thoracic hydatidosis. results: we present the case of a 37-year-old caucasian man who presented chest pain and dyspnea. chest-x-ray showed a well limited cardiac opacity of the upper lobe and of the right lung. chest-ct-scan showed a mediastinal cyst evoking a hydatid cyst in the left ventricle associated to a right pleural effusion. the patient underwent surgery that consisted in the drainage of multiple pericardial cysts, cystectomy of a left ventricle cyst and right pleural cystectomy. microscopic examination showed a pleura-pulmonary and cardio-pericardial hydatidosis. the patient was treated with albendazole and did not present complications or recurrences after 1 year of follow up. conclusion: multiple thoracic hydatidosis is rare. cardiac location is the most challenging because it is difficult to manage and is life threatening. treatment modalities are mainly based on surgery. preventive measures are necessary to avoid disease recurrence. objective: lymphadenopathy is frequently present in hiv positive patients, especially in those with severe immune deficiency and consequently elevated risc of opportunistic infections and neoplasms. considering the large spectrum of possible differential diagnosis a thorough lymph node analyses should be performed. method: we examinated 30 peripheral lymph nodes from 27 patients from latero-cervical, axilar, inguinal and supraclavicular regions; 10 nodes (33,3 %) had diameter less then 1 cm. results: histopathological findings indicated mycobacterial infection in 15 patients (55,5 %), reactive hyperplasia in 7 patients (25,9 %), lymphoma in 3 patients, metastasis from kaposi sarcoma in 2 and supurative inflamation in 1. clinically suspected lymphoma was microscopically infirmed in all 4 cases. 4 cases of tuberculosis, 1 of lymphoma and 1 of kaposi sarcoma metastasis were identified, all of them without prior clinical suspicion we identified tuberculosis, lymphoma and metastasis in 33,3 % of nodes smaller then 1 cm. conclusion: in 74 % of cases lymph node biopsy identified tuberculosis or malignancy, and established a reliable diagnosis in various clinical suspicions. we consequently emphasize the importance of lymph node examination in the evaluation of lymphadenopathy in hiv positive patients, even is not prominent. stăniceanu and nichita are first authors in equal contribution. the importance of autopsy in hiv infected patients objective: nowadays possibilities of medical investigations offer miriads of accumulating data regarding hiv infected patients status. correlation and/or validation of these informations with pathological findings is mandatory to establish in order to ensure appropiate management of hiv infection. method: we performed a comparative study between clinical/paraclinical and autopsy findings in some of the most frequent afected organs in hiv infection; we studied in 31 hiv positive patients autopsied between 2000 and 2011; all patients had no or incipient (up to 1 year) antiretroviral treatment. results: 26 patients presented lung pathology, clinico-pathological correlations revealing 13 discrepancies, 13 total concordances and 5 partial concordances. most of these inconsistencies referred to opportunistic infections, clinically suspected and pathological refuted in 9 cases and clinically unsuspected and pathological identified in 7 cases. clinically suspected neurological alterations were microscopically infirmed in 33,3 % of cases, while 3 cases presented unsuspected lesions. opportunistic infections were key: cord-009997-oecpqf1j authors: nan title: 2018 aspho abstracts date: 2018-03-31 journal: pediatr blood cancer doi: 10.1002/pbc.27057 sha: doc_id: 9997 cord_uid: oecpqf1j nan myelodysplastic syndrome (mds) and frequently arise in the context of inherited bone marrow failure (bmf) syndromes, such as shwachman diamond syndrome (sds). monosomy 7/del(7q) is associated with high grade mds and propensity to progress to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited bmf. development of non-transplant strategies to treat bone marrow failure without simultaneously stimulating outgrowth of malignant clones remains a major challenge. objectives: the aim of this study is to investigate the molecular consequences of del(7q) in the context of bmf with the goal of developing more effective treatments. design/method: to study the biological and molecular consequences of monosomy/del(7q) in bmf, induced pluripotent stem cells were generated from sds patients (sds-ipsc) . a deletion of the mds-associated region of the long arm of chromosome 7 was then introduced using a previously published modified cre-lox approach. results: the sds ipsc phenocopied bone marrow failure with slow proliferation and impaired hematopoietic differentiation. we next explored whether deletion of 7q conferred a relative fitness advantage within the context of bone marrow failure. proliferation of the sds-del(7q) ipscs was reduced below that of both the isogenic sds ipscs and normal controls without an increase in cell death. sds-del(7q) demonstrated reduced hematopoietic differentiation compared with isogenic sds cells. these data demonstrate that deletion of 7q fails to confer a relative growth advantage relative to isogenic sds ipscs and results in further impairment of hematopoiesis. to gain insight into the mechanisms of del7q-associated clonal evolution in sds, we performed rna sequencing (rnaseq) of sds+/-del(7q) ipsc. expression of tgf pathways and their downstream targets were reduced in sds-del(7q) ipscs compared to isogenic sds ipsc. single cell rnaseq analysis of primary sds bone marrow cells confirmed that the tgf pathway is hyperactivated in sds. western blot analysis showed increased phospho-smad2 levels in sds ipscs compared to sds-del(7q) and normal controls, while total levels of smad2 were unchanged. pharmacological targeting of tfg with small molecule inhibitors resulted in selective improvement of sds hematopoietic colony formation and myeloid differentiation without stimulating outgrowth of the isogenic sds-del(7q) cells or normal controls. these results demonstrate that del(7q) reverses the tgf pathway hyperactivation of sds. furthermore, inhibition of tgf selectively rescues hematopoiesis in sds but not in isogenic del7q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del7q clonal outgrowth. background: standard therapy of medulloblastoma consists of treatment with alkylating agents and radiation after surgical resection. although a statistically significant increase in survival is reported with this regimen, 1/3rd recur and become resistant this class of agents ultimately leading to mortality. large numbers of somatic mutations were observed in recurrent medulloblastoma (rm) after alkylating agent and radiation treatment. high mutation rates in tumors can have twofold effect; 1) a large number of non-synonymous mutations that have no role as drivers can still cause functional tumor antigens increasing the neoantigen burden and immunogenicity. moreover, 2) such tumors can gain mutations in canonical or non-canonical dna repair pathways leading to a gain in the number of mutations as seen in case of glioblastoma, this can lead to even higher accelerated mutational rate. evidences suggest that high mutational load can cause higher neoantigen burden thereby making the tumor more susceptible to immune checkpoint inhibition. we propose that post therapy recurrent medulloblastoma gain mutational signature and immunophenotype of malignancies demonstrating clinical response to immune checkpoint therapy. objectives: 1) rm has molecular signatures identical to tumors with high immunogenicity and clinical response to immune check point inhibition. 2) rm has the immune inflammatory phenotype; harboring high percentage of tumor infiltrating lymphocytes (tils), macrophages and monocytes. design/method: to test our hypothesis, we downloaded the raw bam files of previously published data from international cancer genome consortium (icgc) . this set of about 30 matched primaries and recurrent medulloblastoma cases forms our discovery cohort. we have called somatic variants using the gatk pipeline by the broad institute. to validate our key findings, we have procured human medulloblastoma specimens and are conducting whole exome sequencing. the primary assays utilized to assess immunogenicity are immunohistochemical (ihc) staining of formalin fixed and embedded recurrent medulloblastoma tissue to identify tils, tumor associated macrophages and other markers. 300 mg/m2 had dlts of dyspnea (grade 4)/hypoxia (grade 3) but no dlts were observed in any other cohort. adverse events were generally mild to moderate, consistent with the safety profile observed in adults. across the desc cohorts, plasma concentrations were dose-proportional and steady state concentrations were lower on day 15 vs. day 1. mean systemic exposure in the 1200 mg/m2 cohort was ∼ 4-fold greater compared with the adult rp2d of 800 mg bid. a pk:pd relationship between tazemetostat exposure and h3k27me3 levels in peripheral blood monocytes and granulocytes was observed in the desc phase. consistent and significant post-dose reductions in h3k27me3 occurred at doses ≥900 mg/m2. further analysis of twelve patients treated at the rp2d confirmed that h3k27me3 inhibition was maximally inhibited. doses 520-900 mg/m2 showed confirmed objective responses (cr/pr) per recist/rano in patients with es (n = 1), chordoma (n = 2), and atrt (n = 1). background: previous studies established that the platelet/ fibrin(ogen) axis promotes metastatic potential by impeding the clearance of newly formed micrometastases by natural killer (nk) cells. however, multiple important questions remain, including the potential of fibrin(ogen) to promote metastasis through interactions with cells other than platelets (e.g., inflammatory cells), and the fundamental question of whether fibrin polymerization is required for metastasis. objectives: determine the role of fibrin polymerization and fibrin(ogen) engagement of integrins iib 3 and m 2 in metastasis. design/method: we performed experimental and spontaneous metastasis assays in immunocompetent mice carrying specific fibrinogen structure/function alterations. results: expression of a mutant fibrinogen lacking the binding motif for the leukocyte integrin m 2 (fib 390-396a) significantly decreased metastatic potential relative to wildtype fibrinogen, suggesting a role for fibrin(ogen)inflammatory cell interactions mediated by m 2 in metastasis. to directly determine the importance of thrombinmediated fibrin polymerization in metastasis, we analyzed metastatic potential in fibaek mice, which carry a form of fibrinogen essentially "locked" in the soluble state due to a mutation in the a chain thrombin cleavage site. metastatic potential in fibaek mice was diminished relative to control mice, speaking to the importance of thrombin-mediated fibrin polymerization in the metastatic process. however, the fibaek mice retained significant metastatic potential relative to complete fibrinogen deficiency, indicating that fibrinogen monomer retains significant prometastatic properties. in order to better define the role of fibrin(ogen)-platelet interactions in metastasis, we compared metastatic potential in control and fib δ5 mice, carrying a form of fibrinogen lacking the chain binding motif for the platelet integrin iib 3. surprisingly, this mutation had no impact on metastatic potential. together, these studies suggest fibrinogen plays a multifaceted role in metastasis. fibrin(ogen)-leukocyte interactions mediated by m 2 appear to have a role in metastasis. previous studies showed that macrophages promote the metastatic potential of circulating tumor cells, which may represent at least one important m 2 expressing cell type whose prometastatic behavior is influenced by fibrin(ogen) interactions. these studies show that thrombin-mediated fibrin polymerization promotes metastasis, but soluble fibrinogen retains some significant prometastatic capacity. surprisingly, loss of the fibrinogen chain iib 3 binding motif had no impact on metastasis. given the established importance of platelets in metastasis, these findings suggest that fibrin (ogen) is capable of platelet stabilization through mechanism(s) independent of this iib 3 binding motif. platelets may bind polymerized fibrin at other sites, and/or fibrin interactions with other matrix proteins capable of binding iib 3 are sufficient to support platelet functions required for metastasis. the role of platelets in hemostasis and thrombosis is well defined, but it is becoming increasingly evident that platelets also assist in host defense and inflammation. platelets participate in the innate immune system through direct antimicrobial activity and interactions with effector cells (chapman2012, garlanda2013, kapur 2016). in the adaptive immune system, platelets recruit and costimulate t-cells, and promote b-cell differentiation and antibody class switching (kapur2016, morrell2017). the question remains: which mechanisms influence platelet immune function and are they developmentally regulated? preliminary studies in the palis lab have revealed significant dif-ferences in embryonic versus adult platelet gene expression, including regulators of immune and inflammatory responses such as beta2-microglobulin (b2m) and major histocompatibility complex class i (mhc1). mhc1 is expressed on all cell surfaces except red blood cells and its molecular chaperone b2m is a marker of inflammation highly expressed in platelet alpha granules (zufferey2014). preliminary data from the morrell lab reveals a mass release of b2m during platelet activation, which drives monocyte differentiation to an inflammatory phenotype through tgfb receptor signaling. we therefore sought to determine whether developmental changes in platelet b2m expression mediate differences in platelet-mediated monocyte activation. with trilineage hematopoiesis with a predominance of early myeloid precursors, with full maturation. microarray, elane and sbds sequencing and deletion/duplication analyses were negative. immunologic evaluation was significant for agammaglobulinemia and an absence of memory (cd19+cd27+) b cells. a 207 gene primary immunodeficiency panel revealed two variants of unknown significance-c.1957g>a and c.2292g>t in dnmt3b; one previously reported in association with icf1. parental testing demonstrated parental heterozygosity. centromeric instability was confirmed in mitogen stimulated lymphocytes showing characteristic, multibranched chromosomes containing at least 3 arms of chromosome 1 and 16 joined near the centromere. decondensation of the 1qh and 16qh regions and triradial configuration of chromosome1 was noted, and a diagnosis of icf1 syndrome was made. the patient was started on monthly intravenous immunoglobulin (ivig). prophylaxis for pneumocystis jiroveci pneumonia and respiratory syncytial virus was initiated. a 10/10 matched sibling hsct is being planned. demonstrated the diagnosis of high grade osteosarcoma. the patient was started on multi-agent chemotherapy with planned a whole femur prosthesis at time of local control. 7 cases of osteosarcoma have been described in the literature in patients with nf1 (median age; 25 years, range 17-37 years) with slightly male predominance (4 cases). the femur was the most common site of involvement (5 cases). four patients died of metastatic disease despite surgery and multi-agent chemotherapy. conclusion: nf1 represents a major risk factor for development of malignancy and uncommonly osteosarcoma in adolescents and adults. we report a rare case of an extensive involvement of osteosarcoma of the left femur in a child with known diagnosis nf1. this presentation should alert the pediatric oncologists to monitor for bone tumors in patients with nf1 by physical exam and detailed medical history. hasbro children's hospital, providence, rhode island, united states background: dysautonomia is a paraneoplastic syndrome most commonly described in adult malignancies. despite current therapies aimed at symptoms management, it is often debilitating. we present a case of a 16-year-old girl who initially presented with autonomic dysfunction and was subsequently found to have hodgkin lymphoma. objectives: describe hodgkin lymphoma presenting with dysautonomia and discuss symptom management with rituximab design/method: case report a 16 year-old-girl presented with severe symptoms of orthostatic hypotension necessitating prone positioning to prevent syncopal episodes. additionally, she reported anhidrosis, xerostomia, urinary retention, and constipation. she had unmanageable peripheral neuropathic pain despite multiple analgesia medications. initially, it was suspected that her symptoms were caused by an atypical presentation of guillain-barre syndrome. she was treated with intravenous immunoglobulin g, without response. due to a suspicion of a paraneoplastic syndrome a positron emission test/cat scan (pet/ct) was performed and revealed widespread fdg-avid nodal and splenic disease. pathology from a thoracoscopic biopsy of a mediastinal lymph node demonstrated classical hodgkin lymphoma. she was classified as stage ivb. a paraneoplastic panel obtained during the first cycle of chemotherapy revealed elevated anti-amphiphysin antibodies and glutamic acid decarboxylase (gad) antibodies. therapy was initiated with abe-pc (doxorubicin, bleomycin, etoposide, prednisone, cyclophosphamide) ; vincristine was held given her significant neuropathy. due to persistence of autonomic symptoms following her first cycle and presence of antiamphiphysin and gad antibodies, rituximab was incorporated into her treatment. following two cycles abe-pc, she had a rapid early response by fdg-pet/ct. she completed an additional three cycles of abd-pc. end of therapy imaging demonstrated complete response with a single persistent mildly fdg-pet avid lymph node (deauville 2) and her antibodies were negative. she continues treatment of maintenance rituximab with significant improvement, but not resolution, of her orthostatic hypotension. at this time, the patient can ambulate with assistance. constipation and urinary retention have fully resolved and, her peripheral neuropathy, xerostomia, anhidrosis have improved. conclusion: this is rare case of a pediatric hodgkin lymphoma patient developing dysautonomia associated with antiamphiphysin and glutamic acid decarboxylase antibodies and subsequently managed with chemotherapy and rituximab. clinicians should be suspicious of a paraneoplastic syndrome when a neurologic disorder fails to improve with standard treatment. results: labs obtained at an outside hospital one month prior to presentation showed absolute neutrophil count (anc) 78 and hemoglobin 10.2 g/dl. she presented to our institution with 11 days of fever, hepatomegaly 1 cm below costal margin, a white plaque on her tongue, and circumferential perianal ulceration. labs were significant for anc 0 and hemoglobin 7.8 g/dl. anti-granulocyte antibody testing was positive. bone marrow biopsy showed arrest of neutrophil maturation. after initiation of filgrastim (2.7 mcg/kg/day), her anc increased to >500 and repeat bone marrow biopsy demonstrated left shifted myelopoiesis. biopsy of her oral lesion demonstrated invasive actinomyces prompting a prolonged course of antibiotics. biopsies of her oral and anal lesions were reported as myeloid sarcoma without mll rearrangement. chemotherapy was not initiated due to complete resolution of both lesions within 6 weeks of initiating filgrastim and appropriate antibiotic coverage. she has not developed any further lesions concerning for malignancy. testing for common genes associated with severe congenital neutropenia and autoimmune lymphoproliferative syndrome was negative. her immunoglobulin levels and the measurement of age-appropriate vaccine responses were normal. after her lymphocyte subpopulation analysis indicated a selective deficiency in cd8 positive t-lymphocytes (absolute cd8 cell count 185), the severe combined immunodeficiency panel from genedx showed compound heterozygous mutations in results: a male infant was born with a large thigh mass. the child was clinically well aside from restricted movement of affected leg. mri showed mass expanding into pelvis without other lesions. an interventional-radiology guided core biopsy of the mass was reported as high-grade spindle cell sarcoma without etv6 rearrangement. surgery was deferred because of concern that it would result in excessive morbidity. the mass was treated with vincristine and dactinomycin per infantile fibrosarcoma protocols. after 3 months of therapy, no significant change in size of the mass was noted on physical exam or imaging. repeat biopsy was obtained to confirm diagnosis and allow for expanded tumor testing. this biopsy showed triphasic distribution of adipose, fibrous and mesenchymal tissue consistent with fhi with rare sarcomatous foci. additional chemotherapy was deferred and the child was followed clinically. his tumor has remained approximately the same size and still unresectable. next generation sequencing of tumor utilizing panel based technology revealed braf-erc1 fusion consistent with braf activating mutation. this mutation was confirmed by fluorescent in situ hybridization (fish) probe for braf. braf and mek inhibitors have been pursued as treatments to decrease size of tumor and allow for resection. conclusion: braf mutations have been characterized in a variety of malignancies. inhibition of braf and downstream signaling components has produced promising results in a variety of patients. this is the first case report of a braf mutation in a fhi. although management of fhi is typically surgical, this does suggest a potential therapeutic target and may allow for improved surgical outcomes especially in cases where up-front surgery would result in unacceptable morbidity. genetic sequencing of fhi and other rare tumors is an important tool and has the potential to identify mutations amenable to targeted therapies. background: icf is a rare autosomal recessive disorder characterized by hypo-or agammaglobulinemia and often opportunistic infections suggesting t-cell dysfunction. it is further categorized into subtypes 1-4 based on mutations in dna methylation. mutations in the helicase-lymphoid specific (hells) gene, which is required for t-cell proliferation and participates in de novo dna methylation, are characteristic of icf type 4 (icf4). of approximately 70 reported cases of icf, less than 10 percent are characterized as icf4. while malignancy has been reported in icf1 (angiosarcoma, acute lymphoblastic leukemia), and icf2 (hodgkin lymphoma), here we describe the diagnosis and management of an icf4 patient with neuroblastoma and neutropenia, which has not been previously described. objectives: describe a novel phenotype and mutation of icf4 and its management to further expand our understanding of this disease. results: a 6 month ex-31 week premature male with bronchopulmonary disease and failure to thrive presented with acute respiratory failure in the setting of recent viral bronchiolitis with associated chronic diarrhea. he was subsequently diagnosed with multiple infections including pjp pneumonia, norovirus, parainfluenza, rhinovirus, and pseudemonal cellulitis. he presented with profound neutropenia and agammaglobulinemia with presence of b and t cells on lymphocyte phenotyping. ct revealed a paraspinal mass that was mibg-avid on further study, strongly suggesting neuroblastoma. bone marrow was normocellular and negative for malignancy, however revealed marked granulocytic hypoplasia and maturation arrest concerning for severe congenital or, less likely, immune-mediated neutropenia. metastatic workup was negative. whole exome sequencing revealed a homozygous variant of unknown significance (c. 668t>c) in the hells gene, portending a working diagnosis of icf4 syndrome. immunoglobulin supplementation, pentamidine prophylaxis, and g-csf were initiated. he was able discontinue g-csf after 4 months of treatment. his neuroblastoma, initially categorized as l1, met criteria for observation. however, followup mri revealed interval growth nearing the spinal canal. he underwent tumor resection, confirming mycn non-amplified, favorable histology neuroblastoma. after infectious prophylaxis and immunologic support were initiated, he incurred two other hospitalizations, the first for g-tube cellulitis and the second for parainfluenza respiratory illness. he now has stable neutrophil counts off g-csf and remains in remission from neuroblastoma. current plan is to proceed with bone marrow transplantation for immunodeficiency. conclusion: icf4 has not previously been described with neutropenia or neuroblastoma. this report not only describes a novel mutation and phenotype of icf4 and the management thereof, but also reveals the potential curative role of bone marrow transplantation in such disease. staten island university hospital -northwell health, staten island, new york, united states background: desmoid tumors are rare tumors that arise from highly differentiated fibroblasts. they occur in isolation or as part of the disease spectrum of familial adenomatous polyposis (fap) . fap mutations between codons 1445-1578 typically correlate with increased extraintestinal disease such as desmoid tumors and upper gastrointestinal polyps. we describe a patient with a large intra-abdominal desmoid tumor who is heterozygous for a c.8161c>t (p.arg2721cys) apc gene mutation. we are not aware of any other patients reported with this germline apc mutation presenting with a desmoid tumor. objectives: to discuss a novel apc mutation and the presentation of a rare case. design/method: review of clinical presentation, genetic analysis and management of a rare tumor. a 17-year-old female with no significant medical history presented with abdominal asymmetry and intermittent pain. she reported urinary urgency, shortness of breath, early satiety, decreased appetite and a 20-pound weight loss over the course of 7 months. ct scan of the abdomen demonstrated a 24 × 15cm abdominal tumor abutting the local organs but no presence of bowel obstruction. a biopsy revealed a spindle cell neoplasm favoring fibromatosis. there was no known family history of fap, colon cancer, or desmoid tumors. apc gene mutation analysis demonstrated a c.8161c>t (p.arg2721cys) heterozygous gene variant. due to size and location of the tumor, it was initially deemed unresectable. the patient was started on a course of monthly liposomal doxorubicin. she tolerated the initial cycles well and interval ct after 3 cycles of chemotherapy revealed a 40% decrease in tumor volume. variability exists in phenotypic presentation with regards to the location of the afp mutation locus. while fap mutations associated with desmoid tumors typically have changes in the 1445-1645 codon region, our patient presented with a heterozygous mutation resulting in a missense mutation at codon 2721. due to the change in polarity and size, the mutation is not considered to be of conservative nature. we are only aware of one other report of this mutation, which occurred in an individual with a personal and family history of colon cancer. we are not aware of any patients with desmoid tumors who also have this germline apc gene mutation. our case report highlights an apc gene mutation that is not well-described; we are not aware of any other cases of this mutation reported in patients with desmoid tumors. future evaluation and tracking of this mutation may lead to the determination of further clinical significance. background: over time, advanced care planning for location of death has been associated with increased deaths at home rather than in the hospital. in some cases, however, complex management and symptom control can prevent families from achieving their goal of keeping their child out of the hospital and at home at the end of life. ascites is a sequelae of many conditions including malignancy that might lead to significant morbidity. increasingly, interventional procedures are being utilized. peritoneovenous "denver" shunts are placed internally with one end in the peritoneal space and the other buried within a major vessel such as the svc. a one-way valve and pump buried under the skin allows the patient to pump fluid from the peritoneal to the vascular space. the shunt is used frequently in adults, but has not seen much use in pediatric oncology patients. objectives: to describe a case of a terminally ill patient with refractory wilms tumor with ivc involvement who received symptomatic relief with denver shunt placement. results: an 8-year-old female was diagnosed with relapsed, refractory, metastatic wilms tumor with pulmonary and hepatic involvement, with tumor extension to the hepatic veins and ivc. multiple chemotherapeutic regimens and palliative radiation to the ivc were administered, but her disease continued to progress, leading to pressure on the portal vein and portal hypertension. the resulting ascites was causing the patient significant pain and was difficult to manage. the patient's code status was changed to dnr/dni after discussion with her mother, who identified a desire to have the child die at home as comfortably as possible. a peritoneovenous shunt was placed in order to control the patient's pain and avoid frequent medical procedures and therapies. despite initial anxiety, the patient was able to utilize the pump and achieve significant improvement in her ascites and pain. she was able to spend the remaining six weeks of her life at home. ascites is a common phenomenon of end stage disease. peritoneovenous shunts are a treatment modality that may be considered to allow for pain control at the end of life for pediatric oncology patients with ascites. the procedure is relatively low risk, allows for self-control of the pump to maintain comfort, and is easy enough to use by the patient or family. background: extraneural metastases (enm) from pediatric glioblastoma multiforme (gbm) are rare, with an estimated frequency of 0.3%. etiologic factors include multiple neurosurgical procedures and sarcomatous dedifferentiation. their occurrence can seriously affect the patient's quality of life and survival. while enms have been well documented in adults, pediatric cases have not been previously summarized. a 15 year old male with a cerebral gbm developed extension of disease outside of the neuraxis approximately 18 months post initial presentation and at the time of disease progression. metastases included exracranial temporal lesions, cervical and mediastinal lymph nodes and s29 of s301 bilateral lung nodules. a large pleural-based soft tissue metastatic focus was identified on imaging when the patient presented with respiratory distress secondary to a right tension pneumothorax, which was recognized and managed promptly. we summarize the main reported cases in literature to better define risk factors for and evaluate the proposed mechanisms underlying these systemic metastases. design/method: we performed a literature review on the pubmed database using the terms gbm and enm. patients under 21 years of age who met the weiss criteria for the diagnosis of enm from primary cns tumors were included. results: our patient fulfilled two of the three weiss criteria with confirmed gbm at the primary site with all enm in the temporal soft tissue and cervical lymph nodes displaying histopathologic features similar to the primary cns tumor. the intrathoracic adenopathy and lung nodules detected upon chest imaging during workup for respiratory distress were assumed to represent additional metastatic foci. our literature review identified 22 pediatric patients with enm from gbm with a median age of 12 years (range 3.5 -21 years) and a slight female predominance (55% females vs. 45% males). the most common sites of metastases reported were pleura/lungs, bones, lymph nodes and liver. in 9 of 22 patients, metastases were associated with csf shunting. conclusion: pediatric oncologists should have an increased index of suspicion when caring for patients with gbm, particularly those who have undergone shunting procedures and present with systemic symptoms including bony pain, respiratory changes, transaminitis or cytopenias which should prompt timely investigation for enm. although enm of cns tumors carry very poor prognosis, their diagnosis has potential therapeutic importance because treatment of metastatic lesions may alleviate symptoms and improve the quality of life. additional studies may be warranted to evaluate the incidence of enm that can provide valuable insight into the pathogenesis and biology of high-grade gliomas. nicklaus children's hospital, miami, florida, united states background: sinusoidal obstruction syndrome (sos) has been reported in patients undergoing intensive chemotherapy and as a complication post-hematopoietic stem cell transplan-tation. sos may be complicated by portal hypertension, hepatorenal disease or multi-organ failure. however, despite treatment, there may be further potential complications that can be anticipated in patients with history sos. we report two patients with history of sos presented with post-procedural bleeding after gastric tube placement. we believe that their presentations may be associated to their previous diagnosis of sos. design/method: pubmed search was done with search for terminology including "sinusoidal obstruction syndrome" "defibrotide", and "bleeding". papers relevant to our cases were selected for literature review. results: case 1: a 4 year-old female with history of desmosplastic medulloblastoma status-post resection and intensive chemotherapy was diagnosed with sos one month after her second part of planned tandem transplant. she was managed with paracentesis and defibrotide. due to malnourishment, patient had a gastric tube placement 5 months after she completed therapy and had an episode of upper gastrointestinal bleeding postoperatively from the g tube site. case 2: similarly, a 4 year-old male diagnosed with anaplastic medulloblastoma status post resection and adjuvant multiagent chemotherapy. his treatment course was complicated with sos after the second cycle of induction chemotherapy which responded to 21-day course of defibrotide. likewise, the patient had a major bleeding event from the g-tube site approximately two months after sos diagnosis. defibrotide was discontinued in both cases before g-tube placement. both patients had no previous history of bleeding disorders or relevant family history. in addition, comprehensive laboratory evaluations were within normal limits before both procedures. in sos, there is blockage of fluid out of the liver that leads to congestion, ascites, ischemia of the liver, and post-sinusoidal portal hypertension. two related causes of sos should be considered as an explanation for g-tube bleeding. similar patients should have close monitoring postoperatively or if possible surgical intervention should be delayed until the sos process has been evolved. nicklaus children's hospital, miami, florida, united states background: the development of treatment related acute myeloid leukemia (t-aml) and myelodysplastic syndromes (t-mds) is a potential complication after cytotoxic chemotherapy or radiation therapy. the incidence of development of t-aml/t-mds varies from 1-20% depending on the treatment regimen used. cutaneous myeloid sarcoma (ms) is a common presentation of extramedullary leukemia and usually occurs in the setting of aml. we report a rare case of cutaneous ms in an adolescent female after successful treatment for ovarian yolk sac tumor (yst) stage i with bep (bleomycin, etoposide and cisplatin) therapy. the ms was managed only with biopsy and close observation. design/method: a pubmed search was conducted for queries including t-aml/t-mds, cytotoxic agents, cutaneous myeloid sarcoma, regression. relevant papers were selected for literature review. a 13 year-old female was diagnosed with a left ovarian yolk sac tumor, for which she underwent left salpingooophorectomy and successfully completed 4 cycles of bep over 4 months. during routine follow-up 8 months after initiation of treatment for ovarian yst, she was noted to have a small, non-tender, indurated nodule on the left side of her upper back approximately 1cm in diameter. punch biopsy of the skin nodule was performed and pathology was positive for cutaneous myeloid sarcoma. at the time of next follow-up less than one month later, the skin lesion had resolved. two subsequent bone marrow aspirates were performed one month apart and were negative for leukemic involvement or mds. examinations and work-up including whole body pet with ct scan were negative for evidence of disease. although cutaneous ms can be regarded as the herald of systemic myeloid disease rather than a localized process, our patient was monitored periodically with physical exam and laboratory evaluations. she remains free of disease more than four years after the presentation of cutaneous ms without any further treatment. spontaneous regression ms has been previously reported. the authors would like to stress that a conservative approach with close observation could be an option in cutaneous ms even with history of chemotherapy exposure. nesreen ali, iman sidhom, sonia soliman, sherine salem national cancer institute, cairouniversity, egypt children cancer hospital egypt, egypt background: acute leukemia is the commonest malignancy in childhood. the coincidental occurrence of leukemia with hemophilia is extremely rare. hemophilia is a congenital rare x linked bleeding disorder. the main complication of the two diseases is bleeding diathesis which may be lifethreatening due to many factors, deficiency of coagulation factors in hemophilic patients, thrombocytopenia from disease and chemotherapy in leukemic patients, certain cytotoxic drugs such as asparaginase which may result in coagulation disorders and infection which may lead to disseminated intravascular coagulation. objectives: reporting such a case is imperative to set up treatment guidelines for prevention of bleeding and to optimize the therapeutic approach for these patients. design/method: seventeen years old boy, presented to children cancer hospital egypt in june 2015 with pallor and multiple ecchymoses.he was diagnosed with precursor b acute lymphoblastic leukemia, cerebrospinal fluid (csf) was free, the chromosomal analysis revealed hypodiploidy 36, xy. he had moderate type of hemophilia a since birth, factor viii level was 1.5 % at time of diagnosis, coagulation profile revealed prolonged partial thromboplastin time 89 (normal 26-45), factor viii was low 1%, prothrombin concentration and prothrombin time were normal100% and 13 seconds, virology screening for hepatitis b core igg/igm, hbs ag, hiv and hc igg /igm were negative.the patient started induction total xv sjcrh protocol, factor viii 40 unit/kg was given at presentation before doing bone marrow aspiration(bma), csf and as a prophylactic before intramuscular asparaginase injection, intrathecal and bma. it was given immediately within 2 hours before the procedures and platelets transfusion was given regularly to maintain platelets count about 50,000. the minimal residual disease by flow cytometry was 0.81% and 0.11% at d15 and d42 induction. results: our patient received his induction and reintensification chemotherapy without any major bleeding event which reveals the success of our guidelines for the prevention of bleeding. he developed very early relapse at w7 maintenance by the same clone. he received salvage chemotherapy but didn't achieve remission and died out of disease and resistant clone. the development of leukemia on top of hemophilia is a major problem. bleeding complication during chemotherapy can be prevented by regular prophylactic factor viii and platelets concentrate transfusion with good supportive care. life threating bleeding complication may be correlated with the severity of hemophilia. we need to collect data about the biology of leukemic cells, complications, and cause of death to optimize care for these patients. background: mucoepidermoid carcinoma (mec) is a rare malignancy that arises from exocrine glands in the upper aerodigestive tract and tracheobronchial tree. conventionally, mec diagnosis is based on histology, with prognosis based on the extent of resection and detection of metastases. mec is characterized by a translocation of chromosomes 11q and 19p resulting in a fusion between the mect1 and maml2 genes, that occurs in 38-81% of cases. this fusion transcript has been recognized to have a favorable impact on disease features and prognosis of mec. however, recent studies indicate that high grade mec can have mect1-maml2 fusion positivity and multiple other genomic imbalances that have not been studied in much detail. owing to the rarity of mec tumors, more definitive data related to the clinical and prognostic significance of these molecular markers are limited. objectives: 1. identify the presence or absence of mect1-maml2 fusion in the tissue of our patient. 2. analyze the incidence of the fusion in 25 mec cases in children and young adults retrieved from the iowa cancer registry. 3. determine if fusion status correlates with clinical, pathological and outcome data in our cohort. design/method: we describe the case of a 12 year-old caucasian male who presented with recurrent pneumonia, persistent cough and radiographic evidence of right lobar collapse. bronchoscopy revealed an endobronchial lesion and the patient underwent right upper lobe sleeve resection. pathology report was consistent with low grade muco-epidermoid carcinoma. we retrieved 25 archived formalin-fixed paraffinembedded (ffpe) specimens of pediatric and young adult mec cases (ages 0-29) reported in iowa from 1973-2016 using the iowa cancer registry. testing for the mect1-maml2 fusion in the index case and 25 ffpe specimens will be done using a custom-designed laboratory validated next generation sequencing (ngs) assay with the ability to detect novel fusion partners. clinical, pathological and outcome data (age, sex, tumor site, tumor size, nodal metastases, clinical stage, histologic grade, treatment and follow up) will be analyzed to correlate with fusion status. the mect1-maml2 fusion tested positive in our index patient. we will obtain irb approval to test for the fusion in the 25 archived ffpe specimens and correlate clinical, pathological and outcome data. conclusion: mect1-maml2 fusion is a frequent event in mec that has prognostic and potential therapeutic applications in adults. the results of this study may enlighten the clinical management of mec in children and young adults. children 's mercy hospital, kansas city, missouri, united states background: mutations in the samd9 gene are associated with a rare syndrome comprising of myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (mirage syndrome). diagnosis is made through exome sequencing. in the largest reported case series, of eleven patients diagnosed with mirage syndrome, two developed loss of chromosome 7. given the potent growth restricting activity of samd9 mutants, the loss of chromosome 7 is considered the first documentation of adaptation by aneuploidy mechanisms in humans and led to myelodysplastic syndrome (mds), with deaths occurring from related complications at 2 and 5 years of age. objectives: to report a case of mirage syndrome with congenital thrombocytopenia progressing to bone marrow failure, managed uniquely with bone marrow transplantation. results: male born at 29 weeks gestation with prenatal diagnosis of iugr, two vessel cord, oligohydramnios was found to have ambiguous genitalia, adrenal insufficiency, partial panhypopituitarism and congenital thrombocytopenia with bone marrow showing absence of megakaryocytic precursors. severe thrombocytopenia was present from birth. bone marrow evaluation demonstrated a hypocellular marrow with markedly reduced megakaryocytic and myeloid precursors and no evidence of myelodysplasia. he required gastric tube placement for failure to thrive, had a laryngeal cleft repaired and developed focal segmental glomerulosclerosis. mpl gene testing for congenital amegakaryocytic thrombocytopenia was negative. testing for fanconi anemia, shwachman-diamond syndrome and dyskeratosis congenita was also negative. approximately 30% of cells had loss of heterozygosity on chromosome 7q. exome sequencing showed that he is heterozygous for a de novo gain of function variant, c.2471g>a (p.arg824gln), identified in the samd9 gene, confirmed by sanger sequencing and consistent with a diagnosis of mirage syndrome. at 6 years of age, he developed pancytopenia requiring frequent transfusions with platelets and packed red blood cells. he underwent a successful bone marrow transplant at 7 years of age without significant complications, and remains transfusion independent without cytopenias greater than 18 months from bone marrow transplantation. conclusion: it is imperative to pursue work up for persistent congenital thrombocytopenia in a stepwise multidisciplinary manner. to the best of our knowledge, this is the first case of mirage syndrome associated bone marrow failure treated with bone marrow transplant. due to the individual rarity of mirage syndrome and pediatric myelodysplastic syndrome, it is important to maintain an index of suspicion given their association and explore bone marrow transplant as a therapeutic option. results: the patient demonstrated disease regression, initially, and continued without disease progression for 36 months. the regimen has been well tolerated with only minimal side effects of dry skin (ctcae grade 1) and a transient episode of brief erythrodysesthesia (ctacae grade 2) that resolved spontaneously. the combination of sorafenib and capecitabine was effective and well tolerated in this adolescent patient with fl-hcc. our observations, although in a single patient, lend support for further testing of this novel oral chemotherapy regimen in patients with fl-hcc, a disease for which there is no effective standard chemotherapy approach. background: epstein-barr virus (ebv) is a ubiquitous virus associated with a broad range of malignancies due to its oncogenic potential. history of organ or bone marrow transplantation, immunosuppressive therapy, and primary or acquired immunodeficiency syndromes increases the risk of ebvassociated tumors. epstein-barr virus associated smooth muscle tumors (ebv-smt) are unique and rare neoplasms typically discovered in immunocompromised patients. most information related to pathogenesis and therapeutic options is limited to case reports and case series of adult patients. there are several gene expression pathways that ebv utilizes, the most notable of which is the mammalian target of rapamycin (mtor) pathway. the mtor pathway performs a key role through integrating various cell growth signals and factors to regulate protein synthesis and metabolism related to smooth muscle proliferation. sirolimus is an immune modulating therapy that targets the mtor pathway to block activation of lymphocytes. objectives: several case reports have demonstrated shortterm clinical remission of ebv-smt in adult patients with the use of sirolimus. we report the first case of long-term background: bilateral neuroblastoma is characterized as neuroblastoma arising in both adrenal glands, a rare presentation with little data on its genetic make-up. a two-monthold patient was diagnosed with bilateral neuroblastoma in our clinic. her risk assignment was based on biopsy of the left adrenal lesion, which showed mycn amplification, an unfavorable genetic marker. treatment regimen was intensified accordingly and after 5 courses of chemotherapy tumors were excised. patient went on to receive a stem cell transplant and immunotherapy. with no knowledge of genetic similarity between the two tumors it is unclear whether biopsy of the right lesion would have yielded similar results or whether bilateral biopsies are needed for risk assessment of bilateral neuroblastoma. objectives: utilize whole exome sequencing (wes) to characterize the genomic signature of bilateral adrenal neuroblastomas excised following chemotherapy treatment. design/method: paraffin-embedded samples from left (l) and right (r) tumors underwent wes at the broad institute. we analyzed resulting data including somatic variant calls, indel mutations, and copy number variants (cnvs) using ingenuity software to evaluate and compare differences between the two tumor samples. preliminary analysis of the data shows important descriptive information on the two tumor samples. out of 29 somatic mutations in the r tumor cells and 25 mutations in the l tumor cells, only two common somatic mutations were present. out of 40 cnv calls in the r tumor and 60 in the l tumor, 31 cnvs were common between the two tumors, or 73% of each tumor's cnv calls. there was a 14 fold higher frequency in gains versus losses. the median size of the common cnvs was 56,683 (range 220 to 3,323,064 bp). cancerrelated genes with increased copy numbers included transcription factors, receptors for signal transduction pathways, and histone methylation proteins. conclusion: preliminary analysis of the wes results of the two adrenal tumors show some genomic divergence. because the tumor tissue was exposed to chemotherapy prior to excision it is difficult to determine whether genomic divergence is a result of independently originated tumors or subsequent adaptation to chemotherapy of a clonal cell population. the high number of common cnvs in the two tumors points to a common cell of origin, however the low number of common somatic mutations does not fit that picture. a future study to help elucidate the question will be wes of the original biopsy tissue to provide information on tumor mutations prior to the effects of chemotherapy. baylor college of medicine, houston, texas, united states background: although there has been significant improvement in the overall survival rates of children with cancer many children will still die from their illness or complications secondary to treatment. research surrounding the deaths of children who succumb to their disease is warranted to ensure we are providing the best care possible for these patients. objectives: this case series aims to explore pediatric cancer deaths by focusing on perhaps the most extreme cases of high intensity end of life care. we explore those patients whom we know are dying or our very likely to die as evidence by their do not resuscitate (dnr) orders. in all of these cases despite the patients very grim prognosis, their great likelihood of death and limitations placed of resuscitation methods all patients continued end of life care in the pediatric intensive care unit (picu). the primary medical records of all children with a cancer diagnosis who died between february 1, 2011 and january 31, 2017 in the picu with a dnr order seven days or earlier prior to death. each medical history included disease-directed treatment history and response with particular attention to the events surrounding the terminal admission. results: eight patients met criteria for this study representing 1.9% of all cancer patients who died during this time period and 7.4% of those who died in the icu. the average time between dnr and death is 19.6 days (7 days -32 days). the average length of terminal admission was 43.5 days (1 day -153 days). the average time between diagnosis and dnr is 10.75 months (0 months -22 months). the average time between diagnosis and death is 11.25 months (0 months -23 months). conclusion: these cases highlight the journey that patients, families and providers endure leading up to death. medical care is complex, there are very few absolutes that are encountered when caring for patients and decisions around limiting or withdrawing medical care are made in a context of the prior journey. . these cases help to understand the complexity of death and how two seemingly opposite ideals can be congruent in the event of an anticipated death. most of these cases show the need for improved anticipatory guidance surrounding death and greater consideration for de-escalation of care when death is expected. the hospital for sick children, toronto, ontario, canada background: rhabdomyosarcoma (rms) is the most common soft tissue sarcoma in children, with embryonal (erms) and alveolar (arms) representing the most common subtypes. arms tumors are associated with inferior outcome when compared to erms, and they are characterized in about 80% of the cases by a t (2;13) or t(1;13) chromosomal translocation with creation of a pax3-foxo1 or pax7-foxo1 fusion gene, respectively. it is increasingly clear that the pax-foxo1 fusion status is an important poor prognostic factor, thus the histological classification tends to be replaced by the fusion status, particularly in terms of risk stratifica-tion in contrast to arms, there are no recurrent chromosome alterations in erms; however, there are multiple numerical chromosome changes that are frequent in these tumours: gain of chromosome 2,8, 12 and 13 have been found in 25 to 50 % of emrs karyotypes. moreover, erms tumors show frequently allelic loss, the 11.p15.5 chromosomal region being the most frequently involved. recently, novel gene fusions have been described also in erms tumours. these fusions involved mainly the ncoa2 and or the vggl2 genes. the rearrangement partners are variable, and include, i.e. pax 3 (2q35), srf (8q11) and tead 1 (11p15). objectives: to present a patient who died as a consequence of brain metastases while on therapy in the setting of an foxo1negative rms and the identification of a new translocation t(2;15)(q21;q22). design/method: case report and retrospective review of the literature. we report a case of pelvic embryonal rhabdomyosarcoma in a 3-month old boy. he was treated as per cog arst 0531 intermediate risk group, but unfortunately was found to have a large cerebellar tumour during the course of his chemotherapy treatment and he subsequently passed away. a novel translocation between chromosomes 2 and 15 was observed in 11 of 20 metaphase cells by g-band analysis in the autopsy sample of the brain lesion. breakpoints of the translocation were estimated to be at 2q21 and 15q22. there were no additional clonal chromosome abnormalities in the tumour cells. conclusion: erms tumors with fusion genes involved have been exclusively described in patients less than 12 months of age; they seem to be associated with spindle cell histology and, a favorable outcome. in our patient, a novel (2;15) translocation was found and clinically, the patient had a dismal outcome. further studies are indicated to inquire whether this finding is of significance in term of prognosis for these patients. children 's national medical center, washington, district of columbia, united states background: iatrogenic immunodeficiency-associated lymphoproliferative disorders (lpds) are a group of lymphoid s35 of s301 proliferations or lymphomas that are well known to be associated with an immunosuppressed state. these disorders most commonly occur following hematopoietic or solid organ transplantation (called post-transplant lymphoproliferative disorders or ptld), but cases have also been described during the treatment of autoimmune and rheumatologic disorders by immunosuppressive and immunomodulatory medications. these disorders are strongly associated with infection by the epstein-barr virus (ebv) as a result of impaired immune function in the immunosuppressed state. while this phenomenon has been well documented in autoimmune conditions, cases affecting pediatric patients while on antileukemia chemotherapy are lacking. background: atypical teratoid/rhabdoid tumor (at/rt) of the central nervous system (cns) in children younger than 3 years old has a prevalence of 1% to 2% and accounts for 1.6% of all pediatric cns tumors. only 15-30% of patients have leptomeningeal dissemination. rhabdomyosarcoma is the most common soft tissue tumor in childhood, but represent only 3-4% of all pediatric cancers. rarely, it can metastasize or even directly extend into the cns, but typically, cases of cns involvement arise either from parameningeal areas or other primary sites. primary spinal or meningeal rhabdomyosarcoma is extremely rare. objectives: our objective is to describe two unique cns malignancies presenting as rare, primary leptomeningeal disease. design/method: case 1 a 19-month-old female presented with vomiting, fatigue and listlessness, despite a normal head ct and brain mri. csf showed hypoglycorrhachia and mild pleocytosis. ceftriaxone was started, but she developed nuchal rigidity and cranial nerve vii palsy. repeat brain mri showed evolving leptomeningeal enhancement concerning for meningitis. she gradually developed worsening opisthotonus and ultimately a brain biopsy of the temporal lobe was consistent with at/rt. case 2 a 3-year-old male presented with new generalized tonic-clonic seizure activity and intermittent headaches with photophobia, phonophobia, and vomiting. brain mri was significant for enhancement of interpenducular and suprasellar cisterns extending to the optic nerves and chiasm most consistent with meningitis. neurosurgery ultimately placed a lumbar drain for hydrocephalus, and a tissue biopsy demonstrated primary meningeal rhabdomyosacroma. results: in case 1, our patient's temporal lobe biopsy demonstrated grade iv malignant tumor cells consistent with atypical teratoid/rhabdoid tumor. fish demonstrated a homozygous deletion of smarcb1 (22q11.23). she was started on chemotherapy per the dana farber at/rt protocol but ultimately was discharged home on hospice. in case 2, our patient's lumbar arachnoid biopsy demonstrated cellular tumor consistent with group iiia embryonal rhabdomyosarcoma. immunostaining was positive for cd99, desmin, myogenin, and myo-d1 with neural markers ema and gfap highlighting the meninges but without a neural component to the tumor. he completed craniospinal radiation to 36gy total with lumbar boost to 50.4gy total. he is currently receiving chemotherapy per arst0431 protocol. conclusion: these two cases are particularly instructive because of their similar initial presentations and neuroimaging, but with very different and unique diagnoses. university of iowa, iowa city, iowa, united states background: ebf1-pdgfrb fusion causes ph-like b-cell acute lymphoblastic leukemia (b-all), which has a philadelphia positive phenotype without the bcr-abl translocation. this is one of several mutations associated with ph-like b-all and leads to downstream overexpression of tyrosine kinase. ebf1-pdgfrb fusion accounts for about 8% of children with ph-like b-all. patients with ph-like b-all previously had poorer outcomes with conventional chemotherapy. the addition of tyrosine kinase inhibitors (tki), like imatinib, has improved the outcome for many patients predicted to have tki sensitive mutations. objectives: to review clinical characteristics and outcomes of two cases of ph-like b-all at the university of iowa stead family children's hospital and to compare these outcomes to similar cases reported in the literature. design/method: a retrospective chart review was performed for two cases of ph-like b-all diagnosed and treated at the university of iowa stead family children's hospital. results: both patients were males diagnosed at 8 years of age with high wbc count (110,700 and 347,200) and positive for ebf1-pdgfrb gene fusion. patient 1 (pt1) was cns 2b at presentation while patient 2 (pt2) was cns negative; neither had testicular involvement. both started treatment according to cog protocol aall1131. peripheral blasts cleared by induction day 22 for pt1 and induction day 14 for pt2. at end of induction, pt1 had m3 bone marrow and pt2 had m1 bone marrow but mrd 8%. dasatinib was started induction day 13 for pt1 and induction day 31 for pt2. pt1 was still not in remission at end of consolidation; bone marrow cell culture for tki resistance showed best response to dasatinib. pt1 proceeded to anti-cd19 car t-cell therapy followed by tbi-based matched unrelated donor bone marrow transplant. pt2 had negative mrd at the end of consolidation and continues chemotherapy according to aall1131, dasatinib arm. both patients are currently clinically well. our patients had the same tyrosine kinase gene fusion and similar initial clinical courses. while both patients had persistent disease at end of induction, pt1 had almost 80% blasts while pt2 had significant reduction of disease burden before starting tki. pt2 showed good response with the addition of dasatinib while pt1 did not. these findings suggest that response to conventional chemotherapy may potentiate the effect of tki and may predict overall outcome. there are likely additional factors which must be taken into account when determining response to tki for patients with ph-like b-all which have not yet been identified. background: medulloblastoma is the most common malignant brain tumor of childhood. classically, medulloblastoma presents as a well-defined mass lesion in the cerebellum, with a high rate of metastatic dissemination. primary leptomeningeal medulloblastoma (plmb) is an exceedingly rare type of medulloblastoma presentation with a dismal prognosis in which patients present with isolated leptomeningeal disease without an associated mass. to our knowledge, only three pediatric and three adult cases of plmb (ages 5 -30 years) have been reported, all of which died within 6 months of diagnosis. this is the first case of plmb to report a molecular classification. objectives: to report the case of a pediatric patient with plmb in which histopathologic and molecular characterization was performed and to describe the patient's treatment and clinical course. design/method: retrospective review of the patient's electronic medical record and review of the literature. a 9-year-old boy presented with headache, vomiting, diplopia, and fatigue. physical examination revealed upward gaze palsy, left-sided extremity and facial weakness, and ataxia. magnetic resonance imaging (mri) of the brain revealed diffuse cerebellar leptomeningeal enhancement and edema without an identifiable mass and moderate hydrocephalus. mri of the spine and cerebral spinal fluid analysis were normal. a diagnosis of cerebellitis was rendered, and the patient underwent placement of a ventriculoperitoneal shunt. an extensive infectious, neurologic, rheumatologic, and oncologic workup did not identify an etiology. empiric antibiotics, high-dose steroids, and intravenous immunoglobulin therapy yielded minimal improvement. two months later, repeat mri of the brain performed for declining mental status demonstrated progressive thickening of cerebellar leptomeningeal disease. a suboccipital craniectomy with decompression and cerebellar biopsy were performed. pathologic examination revealed a diagnosis of plmb, classic histology, non-wnt/non-shh, without gain/amplification of myc/mycn, and p53 wild type pattern. craniospinal radiation to 4140 cgy with a 1440 cgy boost to the posterior fossa was delivered with concurrent carboplatin/vincristine over six weeks. two months following chemoradiation, mri of s37 of s301 the brain demonstrates significantly reduced pathological leptomeningeal enhancement of the cerebellum, and the patient is awaiting initiation of systemic chemotherapy while recovering from a surgical wound infection. conclusion: plmb is extremely rare but should be considered in patients with cerebellitis and diffuse leptomeningeal involvement who are refractory to medical management or in whom an etiology has not been identified. cerebellar biopsy is recommended early to enable timely treatment and improved outcomes. molecular classification should be performed in cases of plmb to further characterize this disease, inform treatment decisions, and improve clinical outcomes. background: primary intracerebral osteosarcoma is extremely rare and limited to case reports. ptpn11 gain of function is associated with noonan syndrome, which has increased risk of multiple cancer types including brain tumors, but osteosarcoma has never been described. ptpn11 mutations have been reported in many cancers as both oncogenes and tumor suppressors, however no ptpn11 mutations have been described in osteosarcoma. pdgfr-a is a growth factor receptor whose activation is implicated in several malignancies. pdgfr-a and ptpn11 concurrent mutations are described in glioblastoma. there is no known link between holoprosencephaly, noonan syndrome, and osteosarcoma. we report a case of multifocal intracerebral osteosarcoma in a child with lobar holoprosencephaly and chronic subdural hemorrhage and discuss the genetic changes found in the tumor. design/method: a seven-year-old caucasian female, with a known diagnosis of lobar holoprosencephaly, chronic subdural hemorrhage and well controlled seizure disorder presented with status epilepticus shortly after completing antibiotic therapy for infection of subdural hematoma. mri showed diffuse dural thickening with mass lesions in the frontal lobe, temporal lobe, and the parasagittal region, the largest of which was contiguous with the subdural space but none of the lesions were associated with bone on mri or by direct neurosurgical visualization. tissue obtained for concern for recurrent infec-tion resulted in a diagnosis of high grade osteosarcoma. dna analysis was performed to help guide treatment choice. results: standard metastatic work-up was negative for skeletal primary tumor or metastatic lesions outside of the brain. she was treated with high dose methotrexate for two cycles per modified aost1331. despite maximal supportive care, she quickly developed rapid tumor growth as well as intratumoral hemorrhage with resultant herniation and death from respiratory failure just three months after diagnosis. tumor gene sequencing discovered three mutations with described roles in cancer: pdgfra d842>vr, kdm6a loss of exons 1-4, and ptpn11 a72v. conclusion: to our knowledge, primary multifocal extraosseus intracerebral osteosarcoma has not been previously described. despite known cns penetration of high dose methotrexate, this tumor proved resistant and aggressive. holoprosencephaly is associated with a multitude of known genetic drivers, but none are found in this case. furthermore, the genetic changes in this tumor are not typical for osteosarcoma. pdgfr-a over-expression is described in osteosarcoma, but is not clearly correlated with worse overall survival. further research is required to determine the role of ptpn11 in osteosarcoma. background: anaplastic lymphoma kinase (alk) encodes a receptor tyrosine kinase whose activation induces pathways associated with cell proliferation, angiogenesis, and cell survival. alk rearrangements are rare in neuroblastoma, while alk mutations and gene amplification occur more frequently. alk mutations have been found to be associated with increased alk protein expression that is associated with a worse prognosis. alk is commonly mutated in neuroblastoma at three hotspots (f1174, r1275, and f1245). the eml4-alk rearrangement has mostly been associated with lung adenocarcinomas, with only a few cases of non-lung cancers found. it has never been reported in neuroblastoma. multimodal therapy and to report the successful management of treatment related iron overload. results: a 10-year old male presented with abdominal swelling and ct showed a right kidney mass and bilateral lung nodules. he underwent right radical nephrectomy with lymph node sampling. pathology was reviewed centrally and revealed wilms tumor with diffuse anaplasia with rhabdomyosarcoma arising within the stromal component and 3 of 13 nodes positive. he received adjuvant intensive chemotherapy and radiation to the hemiabdomen and whole lungs. the 49-week chemotherapy regimen was vincristine, doxorubicin, cyclophosphamide (per cog arst0431) alternating with carboplatin and etoposide (per cog aren0321 revised uh-1). treatment was complicated by multiple episodes of fever and neutropenia and anorexia requiring g-tube placement. post-therapy, he had persistent neutropenia and thrombocytopenia without related complications. every 6 months for 3 evaluations he underwent a bone marrow which revealed normocellular marrow with maturing trilineage hematopoiesis. evaluation for a bone marrow failure syndrome was unrevealing. starting at 6 months into therapy and all posttherapy imaging showed splenomegaly. he received 29 units of packed red blood cells through the duration of therapy. he was diagnosed with iron overload based on serum ferritin and imaging, including t2*mri. he received therapeutic phlebotomy for 2 years with normalization of serum iron studies, t2* of the heart, and liver iron concentration. he is more than 6 years from completing therapy with no evidence of recurrent disease. asymptomatic cytopenias persist and he has no evidence of iron overload. conclusion: though a rare development, clonal sarcomatous transformation can occur in wilms tumor. our patient's tumor was successfully treated with intensive multimodal therapy targeting the diffusely anaplastic wilms and the rhabdomyosarcomatous component. treatment-related iron overload in a pediatric patient with a solid tumor was successfully treated with phlebotomy. consideration should be given to screen patients with solid tumors who receive multiple packed red cell transfusions for iron overload at the completion of cancer therapy. primary children's hospital, university of utah, salt lake city, utah, united states background: malignant solid tumors are less frequently encountered in infants. primitive myxoid mesenchymal tumors of infancy (pmmti) are a myofibroblastic malignancy and cases are rarely reported in the literature. cure is achieved in the majority of cases with surgical resection, however treatment for unresectable cases remains an enigma. recently published literature postulates that the newly discovered bcor duplication found in pmmti is tumorigenic via an epigenetic pathway. this molecular signature resembles that of clear cell sarcoma of the kidney (ccsk) and the growing number of bcor mutated sarcomas. a similar chemotherapeutic backbone and local control used for ccsk, has been proposed for the unresectable subset of pmmti. utilizing this approach a 19 month-old with relapsed disease has remained disease free for 12 months. however, given the rarity of this disease and the lack of published literature, there is no known standard of care treatment for unresectable and/or recurrent ppmti. we report a case of unresectable recurrent pmmti, a rare infant tumor, with less than 20 cases reported. design/method: medical record, radiological studies, pathology and literature was reviewed. results: our patient is a now 12 month-old female who presented with constipation and lower extremity weakness in the first weeks of life. an mri demonstrated a large lumbar epidural mass with spinal cord impingement. given prolonged (>14 days) neurological symptoms and location, emergent chemotherapy was initiated. biopsy showed a bcor positive, primitive myxoid mesenchymal tumor of infancy (pmmti). she was treated with ifosfamide, carboplatin and etoposide, and demonstrated clinical and radiographic response. we gave two additional cycles of cyclophosphamide, carboplatin and etoposide until surgical resection was feasible followed by two post-surgical cycles of chemotherapy. unfortunately, four month post-therapy mri demonstrated two new lesions; an unresectable paraspinal soft tissue mass and a left iliopsoas groove mass. given bcor association and reported successful therapy with vincristine, doxorubicin, cyclophosphamide alternating with ifosfamide and etoposide, we elected to incorporate vinca-alkaloid and anthracycline into her regimen. she is being treated with vdc/ie with plan for radiation consolidation. conclusion: pmmti is a locally aggressive tumor, for which surgical resection is curative. for those not amendable to resection, best care practices are still being determined. we report a case of pmmti initially responsive to chemotherapy, but not curative. this is the second case to conclusively demonstrate chemo-responsiveness. bcor mutation seems to be a common feature of this cancer; its role in the pathogenesis and as a target is an area of investigation. medical college of wisconsin, milwaukee, wisconsin, united states background: atypical teratoid/rhabdoid tumors (atrt) are central nervous system (cns) tumors that most commonly occur in very young children. there is no widely accepted standard of care for atrt patients, and while survival rates are improving they are historically poor. patients with metastatic disease to the spine at diagnosis have a worse prognosis, and for patients >3 years old, the presence of metastatic disease often results in the use of craniospinal radiation. the importance of correctly identifying metastatic disease at diagnosis aids in decision making and can have both prognostic and therapeutic implications. mr imaging at diagnosis is used to identify metastatic disease; however, here we present a case of diffuse leptomeningeal enhancement that spontaneously resolved after resection of a primary supratentorial atrt. objectives: to describe the resolution of diffuse leptomeningeal enhancement after resection of a primary atrt tumor in a 12-month-old prior to any adjuvant therapy. results: a 12-month-old male presented with a 2 month history of vomiting and weight loss, regression of gross motor developmental milestones, and left hemiparesis. a brain mri demonstrated a 7 × 5.8 × 5.7 cm solid and cystic right atrial mass with diffusion restriction and post-contrast enhancement. smooth diffuse enhancement was noted along the surface of the brainstem and within the interpeduncular fossa. a spine mri demonstrated diffuse circumferential post-contrast enhancement along the surface of the entire spinal cord. the patient underwent a successful near total surgical resection of the primary mass. pathology confirmed the loss of ini-1 staining in tumor cells, consistent with a diagnosis of atrt. no immediate adjuvant radiation or chemotherapy was given. repeat imaging was completed 15 days after resection. brain mr demonstrated expected post-operative changes within the surgical cavity without definitive residual mass or leptomeningeal enhancement. spine mr demonstrated complete resolution of the previously seen circumferential enhance-ment along the entire spinal cord. csf evaluation at that time was negative for tumor cells. after recovery from surgery, chemotherapy treatment was initiated. conclusion: leptomeningeal enhancement at the time of diagnosis of atrt has historically been considered clear evidence of metastatic disease. this case raises questions about the previously accepted etiology of these imaging changes and suggests that widespread leptomeningeal enhancement should be carefully interpreted in future patients with similar imaging findings. in this setting, clinicians should consider repeat imaging following primary surgical resection in order to provide appropriate prognostic information and inform therapeutic decisions. poster # 047 primary ewings sarcoma of cervical cord mimicking cauda equina syndrome sucharita bhaumik, joshua chan nyu winthrop hospital, mineola, new york, united states background: ewing's sarcoma (es) is a malignant primary bone tumor usually involving long bones. primary es of spine is quite uncommon (0.9%) and its location in the cervical spine is even more rare. cauda equina syndrome (ces) is symptoms due to damage to the bundle of nerves below the end of the spinal cord known as the cauda equina (low back pain, radiating shooting pain down the legs, paraplegia, and loss of bowel or bladder control). it often occurs with lesions of lumbosacral spine. treatment with high-dose steroids may provide pain relief and improved neurologic function (by reducing edema) while awaiting diagnostic studies objectives: to demonstrate an unusual clinical presentation and emergent management of cervical es presenting with ces like symptoms. : 15 year old male presented with a left sided posterior neck mass. soon after, he developed weakness of left arm, urinary and stool retention and inability to walk or bear weight in both legs. on physical exam a left tempero-occipital 6 × 4cm fixed, non-tender, non-fluctuant mass was noted as well as motor and sensory impairment of left upper extremity, bilateral spastic paraplegia and loss of sphincter control. mri cervical spine showed a left cervical tumor with moth eaten appearance involving the vertebral bodies of c2-c3, adjacent muscles, displacing vital structures of the neck and compressing the cervical spinal cord. the thoracic and lumbosacral spine had no disease involvement. due to rapidly worsening spinal cord compression he was emergently treated with high dose steroids. he gained back all function in his extremities and regained bowel and bladder control. this eliminated need for urgent neurosurgical intervention. results: biopsy of the neck mass showed small blue round cells consistent with es with ewsr1 gene rearrangement. staging work up revealed no additional metastatic involvement. he then initiated treatment for localized es with systemic chemotherapy and radiotherapy and has had excellent response to treatment so far. conclusion: this is the first known case of non metastatic primary cervical es mimicking ces where an acutely enlarging mass presented with rapidly progressive neurologic deficits due to compression of anterior spinothalamic tract. in these unusual presentations of ces without lumbodorsal involvement it is important to consider cervical lesions. early rapid steroid initiation should be considered while awaiting biopsy results to prevent worsening cord compression followed by es focused treatments. this increases the chance of a successful outcome. the initial improvement with steroids may confuse the tumor with being a lymphoma children 's mercy hospital, kansas city, missouri, united states background: von willebrand disease (vwd) is a relatively common bleeding disorder with a high degree of genotypic and phenotypic variation. bleeding is usually mucocutaneous but can be severe and include muscle and joint bleeds especially in type 3 vwd patients. most common bleeding management consists of desmopressin, anti-fibrinolytics, and/or plasma-derived antihemophilic factor/von willebrand factor (ahf/vwf) complex. a recombinant vwf has become available in the last few years. anaphylaxis and inhibitor development in vwd are rare. objectives: to describe the rare clinical manifestation of anaphylaxis to factor concentrate in a patient with severe type 1 vwd. results: a 13-year-old female with severe type 1 vwd [baseline vwag 10%, activity < 10%, factor viii (fviii) 26%] originally presented with heavy menstrual bleeding (hmb) leading to anemia requiring blood transfusion. she underwent placement of a levonorgestrel-releasing intrauterine device (lngiud) and began norethindrone. her hmb continued despite the lngiud and an increase in norethindrone dosing. plasma derived ahf/vwf complex was administered, which she had previously received. following the infusion, the patient developed anaphylaxis with hives, wheezing, tachycardia, and itching requiring 2 doses of diphenhydramine and 1 dose of hydrocortisone with resolution of symptoms. subsequently, she received recombinant vwf without incident. however, due to her low fviii level, she also required treatment with a full length recombinant fviii product. she again developed hives and itching after this infusion. she has since received recombinant vwf with recombinant fviii/fc fusion protein without further allergic reaction. there was no evidence of an inhibitor with her most recent post-infusion vwf level was 101%, factor viii 199%. conclusion: anaphylaxis to plasma derived factor products has been documented far less frequently within the vwd population compared to those with hemophilia and is typically seen in those with large gene deletions, usually with type 3 disease. therefore, similar type 1 vwd patients with severe disease may benefit from gene sequencing. it is unclear in this patient's case to which aspect of her treatment she is allergic, as she reacted to plasma-derived ahf/vwf and full length recombinant fviii, but not recombinant vwf or recombinant fviii/fc fusion protein. we hypothesize that she may be allergic to an epitope in the fviii b domain, or that the presence of fc fusion may have had a protective effect. further investigation including genetic analysis is planned. nodules. biopsies were consistent with neuroendocrine carcinoma, large cell type (g3). next generation sequencing revealed a khdrbs2-braf fusion. he received conventional cytotoxic chemotherapy regimens both with cisplatin/doxorubicin, capecitabine/temozolomide, and doxorubicin/etoposide, but achieved a minimal response followed by rapid disease progression, massive ascites, and renal failure secondary to bilateral ureteral obstruction. results: based on his prior genomic testing, therapy with single agent mek inhibitor (trametinib) was initiated. this produced a rapid, dramatic response with greatly reduced disease burden at all sites, resolution of ascites and return to completely normal activity within 2 months. this response lasted for approximately 6 months before the tumor again progressed. further therapy with an erk inhibitor was ineffective, and the patient expired from progressive disease. located on the chromosome 7q34, the braf oncogene, as part of the ras/mapk pathway, is involved in cellular proliferation, differentiation, migration, and apoptosis. braf mutations are recognized in a wide range of adult malignancies: thyroid cancers, non-small cell lung cancer, cholangiocarcinoma, ovarian cancers, and multiple myeloma. braf mutations have also been described in adult neuroendocrine carcinoma of the colon. trametinib is a highly specific inhibitor of mek1/mek2, a downstream mediator in the braf pathway. it has demonstrated activity in a number of tumors including advanced melanoma and gliomas. trametinib was chosen for this patient based on his atypical braf fusion. we believe this is the first documented case of its successful use in neuroendocrine carcinoma in the pediatric population. conclusion: this case demonstrates the presence of braf fusion in a case of pediatric neuroendocrine carcinoma and significant response to single agent mek inhibition in this context. this cases raises the question as to whether the combination of a targeted inhibitor, in addition to either conventional chemotherapy or other braf inhibitors, might offer a better approach to therapy than current treatment options. albany medical center, albany, new york, united states background: warm autoimmune hemolytic anemia (waiha) is characterized by autoantibody, and occasional complement binding of protein antigens, on the surface of red blood cells at temperatures ≥37 oc resulting in targeted destruction. we describe the case of a 17 year old male with a history of evan's syndrome, poor immune response to vaccines and lymphoid hyperplasia, presenting with altered mental status and severe anemia, found to have a warm igg pan agglutinin with evidence of both intra and extravascular hemolysis. his course was complicated by respiratory failure requiring intubation, pulmonary emboli, enterococcus bacteremia and hypertension. he received multiple transfusions with only transient increases in hemoglobin. the aiha was refractory to multiple rounds of treatment with high dose steroids, ivig, rituximab, cyclophosphamide, bortezomib, plasma exchange and mycophenolate mofetil (mmf). objectives: given the refractory nature of our patient's aiha the decision was made to trial eculizumab, a monoclonal antibody targeting c5 complement, preventing its cleavage and activation, and shown to be effective in treatment of atypical hemolytic uremic syndrome and hemolysis due to an igm cold agglutinin. prior to eculizumab infusion, ch50 and sc5b-9 assays were significantly elevated. design/method: the patient was given two doses of eculizimab 6 days apart. results: his hemoglobin steadily rose independent of red cell transfusions with a corresponding decrease in reticulocyte count, ldh and ch50 levels. the patient has remained stable with a normal hemoglobin (12-14 g/dl) on maintenance steroids and mmf. although we cannot definitively conclude that eculizumab directly caused his recovery, the clinical course post-eculizumab suggests this may be an efficacious treatment for aiha. genetic testing showed monoallelic frameshift mutation of the nfkb1 gene and monoallelic missense mutation of the dock2 gene. given the role of nfkb1 in both immunodeficiency and autoimmunity, it is thought that the patient's phenotype is due to nfkb1 haploinsufficiency and he is currently considering hematopoietic stem cell transplant. st. joseph's regional medical center, paterson, new jersey, united states background: heterozygous -thalassemia typically manifests as thalassemia minor, characterized by mild microcytic hypochromic anemia with minimal clinical ramifications. coinheritance of -globin gene triplication has been reported to exacerbate the clinical and hematological phenotype ofthalassemia trait, due to increase in the alpha/non-alpha-chain imbalance. reported phenotypes range from asymptomatic thalassemia minor to moderate thalassemia intermedia, usually diagnosed in adulthood without transfusion dependence. this combination has been described in mediterranean, european and asian populations, but rarely reported in hispanics. objectives: to report two cases of unusually severethalassemia intermedia in hispanic patients with heterozygosity for triplicated -globin gene and a (0)-thalassemia allele. results: case 1: sixteen-month-old male of mexican descent presented with persistent microcytic anemia and jaundice. peripheral smear showed nucleated rbcs with basophilic stippling and target cells. hemoglobin electrophoresis revealed: hba-79%, hbf-17%, hba2-4.3%. -globin gene testing revealed heterozygosity for (0) mutation (ivsi-i, g→a). given the unusually severe anemia, -gene testing was performed which showed -globin gene(anti 3.7) triplication ( / ). at four years, he had splenomegaly and bilateral maxillary prominence. head ct showed irregular contour of the parieto-occipital region due to medullary expansion. due to significant persistent anemia (6-8g/dl) and progressive bony deformities of the skull, patient began chronic transfusions at age eight after family declined splenectomy.case 2: fifteen-year-old female, of peruvian and honduran descent, presented for evaluation prior to cholecystectomy for gallstones and recurrent ruq pain. father had known thalassemia trait. her hb was 9.2 g/dl with hypochromia, microcytosis, and target cells. electrophoresis indicated -thalassemia trait (hba-94%, hba2-4.7%, hbf-1.3%), confirmed by gene testing (heterozygous for a (0) mutation in codon 39 c>t). given jaundice and gallstones, -globin gene analysis was ordered showing triplication ( / ). ruq pain resolved post-cholecystectomy, but she developed persistent painful splenomegaly. she began hydroxyurea to increase gamma-globin production and decrease excess alpha chains, but it was discontinued due to hematological toxicity. due to recurrent luq pain and progressive splenomegaly, she underwent laparoscopic splenectomy at age 22 with resolution of symptoms and improved hemoglobin. conclusion: -globin gene testing should be considered in -thalassemia carriers with an atypical clinical presentation including hispanic patients. the wide variability in the phenotypic expression of (anti 3.7) mutation andthalassemia trait suggest interplay of other genetic factors which remain undefined. the clinically significant presentation amongst certain subjects, as in our two cases, makes it imperative to identify these factors to aid in phenotype prediction and genetic counseling. ashley bonheur, shivakumar subramaniyam, jogarao vedula, sucharita bhaumik nyu winthrop hospital, mineola, new york, united states background: wilms tumor (wt) is one of the most common solid malignant neoplasms in children. a diverse range of genes and mechanisms are implicated in wt pathogenesis. predisposing syndromes result from a disruption of wt1 gene, crucial for renal and gonadal embryogenesis. another gene is wt2 gene locus at 11p15, an area of imprinting. the p53 tumor suppressor gene on chromosome 17p13.1 is seen in patients with anaplastic histology. in addition to these genes, whole and partial chromosome gains of 1q, 2, 7q, 8, 12, & 13 and losses of 1p, 7p, 16q, 22q, as well as loss of heterozygosity (loh) are commonly seen. some genetic markers appear to be predictive of outcome and are now incorporated into the assigning of risk-directed therapy. patients with loh at chromosome 1p and 16q are treated with more intensive chemotherapy, as they have been associated with increased risk of relapse and mortality. objectives: to describe a new complex translocation involving chromosome 2, 7, and 12 in a case of pediatric wt. design/method: a four-year old female presented with abdominal pain and emesis. on exam, patient had a firm and large abdominal mass. radiologic studies revealed a complex lobulated right renal mass. right radical nephrectomy was performed. histopathologic studies showed wt with triphasic histologic features with blastema predominance, invasion of the lymphovascular and perinephric adipose tissues, perinephric lymph node involvement and no anaplasia. chest ct scan showed bilateral lung metastases. tumor cytogenetics showed an abnormal karyotype, a complex translocation of 2, 7, and 12. the rearrangement occurred due to translocation between chromosomal bands 7q22 and 12q15, with an insertion of 7q22-32 on the 2q21 region. pcr based genotyping using microsatellite markers additionally identified loh for chromosome 1p36 and 16q22. the patient was treated for high risk stage iv wilms tumor with favorable histology and received intensive chemotherapy and radiation therapy to the flank and the lungs. she is now in remission 8 months after, with no evidence of recurrence on surveillance scans. complex translocations associated with wt have not been rigorously studied. a question for further study is whether there is any relationship between recurrence potential with a complex translocation compared to common chromosomal abnormalities. further knowledge of the molecular pathology and genetic changes in wt will help the development of new targeted therapies, as well as new biomarkers to aid diagnosis, risk stratification, and monitoring of treatment and relapse. results: a 4 week-old girl was referred for evaluation of an abnormal newborn screen. mother was a known carrier of hb khartoum trait while father was a known carrier of thalassemia trait. patient's hemoglobin quantification performed by capillary zone electrophoresis showed hbf 92%, hb variant 8%, and no detectable hba. the hb variant ran in the d zone, a pattern consistent with mother's hb. alkaline agarose gel electrophoresis banding pattern showed f/s. acid agarose gel electrophoresis pattern showed v/f. later testing revealed abnormal isopropanol stability with 3+ precipitation at 20 minutes. this electrophoresis pattern is consistent with the pattern previously reported of hb khartoum. clinically, the patient is a healthy, active child whom we have followed for two years. she has not had any significant anemia outside of her physiologic nadir. she has not had any hemolytic episodes, and her bilirubin levels have always been within the normal range conclusion: to the best of our knowledge, this is the only reported case of hb khartoum/ thalassemia. the proline to arginine substitution of hb khartoum introduces a charged group on the chain at the site of 1 1 contact. the resulting unstable 1 1 chains can dissociate into monomers and favor the formation of methemoglobin, leading to hemoglobin instability. we had wondered if this unstable hemoglobin might result in clinical hemolysis when challenged with oxidative stress, such as in periods of infection. however, in the two years we have followed this patient, she has never had a hemolytic episode. at two years of age, she has hbf 7.8%, hb khartoum 85.5%, and hba2 6.7%. whether hbf elevation is protective from oxidative stress remains to be determined as we continue to follow this child. university of puerto rico -medical science campus, san juan, puerto rico, united states background: gm1 gangliosidosis is a lysosomal disorder caused by -galactosidase deficiency due to mutations in the glb1 gene. it is a rare autosomal recessive neurodegenerative disorder with an incidence of about 1:100,000-1:200,000 live births worldwide. this neurological disorder has three clinical forms. gm1 type 1, or infantile form is characterized by psychomotor regression by the age of 6 months, visceromegaly (hepatosplenomegaly), macular cherry red spot, facial and skeletal abnormalities, seizures, and profound intellectual disability. we present a 4-year-old female with gm1 type 1 and acute lymphocytic leukemia (all). design/method: she was diagnosed with gm 1 type 1 at the 1st months of age and family history was remarkable for an older sister with gm 1 type 1. diagnostic studies reveal homozygous exon 7 of the glb1 gene for a sequence variant defined as c.622c>t, predicted to an amino acid substitution p.aarg208cs. results: patient presented to our hospital with petechiae in lower extremities, pallor and intermittent tracheal bleeding. physical examination shows a hemodynamically stable girl that is chronically ill dependent of mechanical ventilation, severe mental retardation and scatter petechiae at upper and lower extremities. laboratory workup revealed severe normocytic anemia (hgb: 5.7g/dl) with immature peripheral cells and thrombocytopenia (81 × 109/l). serum chemistry revealed increase ldh (695u/l), increase hepatic enzymes (ast: 63u/l), normal uric acid level. there was no evidence coagulopathy. chest x ray was unremarkable except for evidence of chronic pulmonary illness. abdominal sonogram hepatosplenomegaly. during hospitalization, bone marrow aspirate and biopsy was performed which was diagnostic of b cell acute lymphoblastic leukemia (all) with 26.5% lymphoblast and orderly myeloid/erythroid maturation. flow cytometry: 26% b lymphoblast with aberrant phenotype c/w b-acute lymphoblastic leukemia. karyotype revealed hyperdiploid female of favorable prognosis. cytogenetic by fish: hyperdiploid all with extra copies of runx1 and igh (no bcr-abl translocation). family was oriented about the new diagnosis and the dismal prognosis in conjunction to her primary condition. parents agree on no chemotherapy treatment for all with only supportive treatment. to this date, there is no evidence in literature that has previously described association of gm1 and leukemia. life expectancy of patient's primary condition is null therefore, correlation with leukemia might not be a coincidental finding. this patient opens the possibility of malignancy as part of gm1 type 1 thus, malignancy diagnosis should be considered as part of their medical lifetime course. university of south florida, tampa, florida, united states background: hematological manifestations related to hiv infection are not uncommon, with thrombocytopenia having an estimated prevalence of 5-15%. the pathophysiology is likely multifactorial. studies suggest that the primary mechanism may be immunologic resulting in accelerated platelet destruction. additional theories suggest that infection of megakaryocytes may also play a role causing inadequate platelet production. treatment of hiv-related thrombocytopenia is challenging. first-line treatments include initiation and optimization of antiretroviral therapies, immunoglobulin (ivig), and glucocorticoids. however, this approach is not effective in all patients and second line treatment options are less well studied, particularly in the pediatric population. objectives: we aim to present and discuss the case of a 13 year old patient with perinatally acquired hiv-1 infection and persistent thrombocytopenia who, after failing first line therapies, showed normalization of platelet count on the novel thrombopoietin receptor agonist, eltrombopag. design/method: a retrospective chart review of the case patient's medical record was conducted. additionally, a thorough literature review was performed on this topic including the pathophysiology of hiv related thrombocytopenia and its treatment modalities. the patient required monthly ivig infusions for about 1 year, but did not show a sustained response, often with platelet count dropping to less than 10,000 in between infusions. after initiation of 50 mg eltrombopag daily the patient showed a sustained increase in platelet count (range 32,000-88,000). during a brief 2 week lapse in eltrombopag treatment his platelet count dropped to 17,000. upon re-initiation of therapy his count increased to 84,000. the patient has remained asymptomatic, off of ivig for over one year, with undetectable hiv viral load and greater than 500 cd4 t cell counts. no side effects or grade 2 laboratory abnormalities were reported. conclusion: treatment of hiv-related thrombocytopenia can be challenging. first line therapies, including ivig and glucocorticoids, are not effective in all patients. several other treatment modalities have been utilized, including anti-d immunoglobulin, dapsone, danazol, interferon alfa, vincristine, thrombopoetic growth factors including romiplostim and eltrombopag, or splenectomy, but these are less well studied. this represents the first reported case of a pediatric patient with hiv who showed a positive response to eltrombopag with a sustained improvement in platelet count and no adverse effects from treatment. eltrombopag may be a safe alternative to first line therapies in those patients with hiv and refractory thrombocytopenia, however additional studies are needed. university of illinois college of medicine at peoria, peoria, illinois, united states background: achromobacter xylosoxidans is a gram negative rod with peritrichous flagella which causes rare opportunistic infections most commonly encountered by immunocompromised patients. it is primarily associated with uncomplicated bacteremia, cather-associated infections, and pneumonia. most reports of bacteremia associated with a. xylosoxidans are nosocomial, associated with neoplasm, and occurring mainly in adults. most reported infections with a. xylosoxidans in children are associated with cystic fibrosis. there are very few reported cases of septic shock from a. xylosoxidans bacteremia and pneumonia in the pediatric oncology population. objectives: to describe a rare case of a. xylosoxidans septic shock in a pediatric patient with relapsed neuroblastoma results: a 4-year old boy with history of stage iv highrisk neuroblastoma underwent standard frontline therapy with chemotherapy, hematopoietic stem cell transplant, radiation therapy, and immunotherapy, followed by a dfmo trial for maintenance. his 3-month follow-up scans demonstrated relapse and he was subsequently treated with additional chemotherapy, surgical resection, and mibg therapy, crizotinib for an eml4-alk fusion and finally ifosfamide, carboplatin and etoposide (ice). he developed neutropenic fevers and was started on cefepime, vancomycin and fluconazole. blood cultures were initially negative. on the 4th day of fever, his previously scheduled pet scan was performed during hospitalization and showed new pulmonary opacities. he did not have respiratory symptoms, but therapy was escalated to meropenem, vancomycin and amphotericin. emergent bronchoscopy was performed the same day, with all bacterial and fungal cultures remaining negative. overnight, he developed tachypnea and saturations in the upper 80s, requiring nasal cannula. ir-guided lung biopsy was performed the next day, a flexible bronchoscopy was done to remove blood clots in the airway, the patient was placed on a ventilator, femoral lines were placed, granulocytes ordered and pressors were started for deterioration to presumed septic shock. arterial and femoral lines were placed but patient continued to have hemodynamic instability on multiple pressors. the following day, blood and respiratory cultures returned positive for results: at 33 days after the start of iti, the inhibitor was <0.1 bu and continued undetectable 6 months after initiation of iti therapy. in this patient, iti with high-dose plasma-derived factor viii and von willebrand factor (vwf) complex was well tolerated and effective. genetic analysis confirmed a large factor viii gene duplication of exons 7 to 22. we believe our patient developed inhibitor so quickly (14 exposure days) due to the possibility of this mutation causing a frameshift that introduces a premature termination codon. this might be functionally similar to a deletion in the factor viii gene which poses the highest risk for inhibitor development in patients with severe hemophilia a. this variant has only been identified previously in two unrelated patients diagnosed with severe hemophilia a. this duplication is not listed in dbsnp variant database, nor observed in the general population database. our case proves the effectiveness of this method for patients with severe hemophilia a and an inhibitor. it also shows that more research is needed to identify patients at risk for inhibitor development. background: mercaptopurine (6-mp) is a prodrug that is a core component of maintenance chemotherapy for patients with a diagnosis of acute lymphoblastic leukemia (all). suppression of the neutrophil count is used to demonstrate adequate dosing of 6-mp during this phase of therapy. bone marrow suppression is mediated by the active metabolite 6-thioguanine (6-tgn), whereas the metabolite 6-methylmercaptopurine nucleotides (6-mmpn) has been shown to cause hepatotoxicity. allopurinol has been used infrequently in all maintenance therapy in the setting of skewed metabolism when adequate myelosuppression is difficult to achieve due to excessive hepatic toxicity. when given in combination with allopurinol a reduced dose of 6-mp may result in both increased 6-tgn levels and decreased 6-mmpn levels. objectives: describe the characteristics and clinical course of patients treated with allopurinol and reduced dose 6-mp during maintenance chemotherapy for all. we performed a retrospective chart review of patients at aflac cancer and blood disorders center of children's healthcare of atlanta with new diagnoses of b or t-cell all who received allopurinol during maintenance chemotherapy. we identified eleven patients with b-cell or tcell all who received allopurinol adjunctive therapy during maintenance chemotherapy at a single institution between 2014-2017. these 11 patients received adjunctive allopurinol for 2-120 weeks (median 52 weeks) with reduced 6-mp (25-65% of full dose). all ten patients with genetic testing for thiopurine s-methyltransferase (tpmt) had wildtype genotype associated with normal enzyme levels. indications for allopurinol use were most commonly unfavorable 6-mp metabolite levels, transaminitis (n = 8), pancreatitis (n = 3) and hyperbilirubinemia (n = 3). favorable metabolite shift was achieved in all patients. liver enzymes improved in 6 of 10 patients with transaminitis after initiation of allopurinol/reduced 6-mp. three patients who experienced pancreatitis during maintenance did not have recurrence after initiation of allopurinol (2 of these patients previously reported). six patients developed pancytopenia while on allopurinol, and two of those patients developed pancytopenia severe enough to require allopurinol cessation. four patients developed isolated anemia (hgb <11.0 g/dl) without thrombocytopenia or severe neutropenia. no patient has experienced a recurrence of leukemia. overall, treatment with allopurinol and reduced dose 6-mp was successful in producing a favorable 6-mp metabolite distribution and reducing toxicity. therapy was generally tolerated; however a major and notable side effect was pancytopenia, in two cases severe enough to stop allopurinol treatment. anemia may be more prominent with allopurinol usage. allopurinol effect is variable among individual patients despite normal tpmt genotypes. baylor college of medicine, houston, texas, united states background: congenital sideroblastic anemia, b-cell immunodeficiency, periodic fevers and developmental delay syndrome (sifd) is a rare inherited sideroblastic anemia syndrome, first described in 2013 with 12 clinically similar cases. genetic variations of trnt1 were identified as causative. objectives: to present an unusual presentation of a patient with sifd complicated by diagnosis of concomitant alpha thalassemia trait. design/method: retrospective chart review. a five month old male infant was referred to our hematology center for evaluation of elevated hemoglobin barts identified on newborn screen. despite numerous attempts, blood work was unable to be collected. at seven months of age he had microcytic anemia (hemoglobin 7.5 g/dl, mean corpuscular volume 44 fl) more severe than what would be expected with alpha thalassemia trait. no variant hemoglobin was identified with isoelectric focusing or high performance liquid chromatography. by nine months of age he developed growth failure, intermittent emesis with fevers, developmental delays (predominantly gross motor), hearing loss, a disproportionally large head and coarse, thinning hair. over the next ten months, he was seen by numerous specialists for seemingly unconnected problems including sensorineural hearing loss, elevated liver enzymes and growth hormone deficiency. alpha globin analysis revealed deletion of two alpha globin genes. at 20 months of age, he was admitted with one week of fevers, jaundice, and emesis. peripheral blood smear showed microcytic hypochromic anemia with marked anisopoikilocytosis including target cells, elliptocytes, tear drops, spherocytes, poikilocytes, marked polychromasia, and coarse basophilic stippling. given the inconsistency of his laboratory findings with the diagnosis of alpha thalassemia trait and clinical syndromic findings, bone marrow biopsy was performed which revealed rare ringed sideroblasts. one month later whole exome sequencing revealed trnt1 splicing variant c.1057-7c>g and novel missense variant c.1092a>t consistent with sifd. hemoglobin barts on newborn screen with moderate to severe microcytic anemia directed initial diagnostic work-up towards variant alpha thalassemia. as additional medical conditions developed the focus shifted to a unifying syndrome. compared to previously described cases, our patient was diagnosed at an older age, presented with anemia rather than episodes of febrile illnesses, and had rare sideroblasts on bone marrow examination. diagnosis in this case led to identification of the novel c.1092a>t variant in his sister who had similar, but milder, features. sifd is a rare disease with variable phenotypic severity making diagnosis challenging without high index of suspicion which is crucial for appropriate management. wiseman, blood, 2013 . chakraborty, blood, 2014 background: cholelithiasis is uncommon in childhood. cholelithiasis is known to occur more frequently in children with predispositions, including female sex, obesity, parenteral nutrition, previous abdominal surgery, use of oral contraceptives, family history of gallstones, chronic hemolytic anemias, hepatobiliary disease, or exposure to specific drugs. although there have been occasional case reports linking cholelithiasis to childhood leukemia or leukemia therapy, the prevalence and risk factors of cholelithiasis in patients with childhood leukemia remain unclear. objectives: to estimate the prevalence of cholelithiasis in patients diagnosed with childhood acute lymphoblastic leukemia (all), and to evaluate possible risk factors for the development of cholelithiasis in patients with childhood all. we performed a computer-assisted review of the electronic medical records of 503 patients diagnosed for b or t-cell all at children's healthcare of atlanta in the period from 2010 to 2016. patients with diagnoses of cholelithiasis, cholecystitis or who had a cholecystectomy were identified. possible risk factors of age, sex, bmi, history of abdominal surgery and parenteral nutrition use were abstracted. patients with underlying chronic hemolytic anemia or pre-existing gallbladder disease were excluded. results: seventeen cases of cholelithiasis and 2 cases of cholecystitis without documented cholelithiasis were identified. among patients with cholelithiasis, 8 were female. median age at diagnosis of cholelithiasis was 14.0 (range 2.7 -21.6) years. seven patients had no symptoms referable to cholelithiasis at the time of diagnosis. the median age of leukemia diagnosis among these patients was 12.1 (range 0.9 -18.8) years. the median interval from diagnosis of leukemia to gallbladder disease was 1.0 years. four patients had bmi over the 95th percentile for age. two patients had a prior history of intraabdominal surgery. no patient received oral contraceptive pills. six patients received parenteral nutrition for more than 30 days. there was no documented family history of cholelithiasis. seven patients did not receive any cholelithiasis directed therapy. two patients were managed with medical management only, 1 with endoscopic retrograde cholangiopancreatogram with stone extraction, and 7 with cholecystectomy. our study estimates the prevalence of cholelithiasis in childhood lymphoblastic leukemia to be 3.3%, higher than the reported prevalence in the general pediatric population of 0.13-0.22%. although our cohort size is small, it appears that all therapy and supportive care modalities associated with all are likely to play a larger role in the development of cholelithiasis than known predisposing factors in the general population. further studies are warranted. background: an uncommon side effect of intravenous immunoglobulin (ivig) administration is clinically apparent, sometimes severe hemolysis. we describe a severe case of coombs-positive hemolytic anemia secondary to ivig administration. ivig is a blood derivative manufactured from pools of 5,000 to 10,000 individual plasma donations. ivig is not abo-type restricted, so anti-a, anti-b and anti-a,b isoagglutinins are detectable. objectives: to describe a rare but serious type of transfusion reaction leading to gross hemolysis after ivig administration. results: a 16-year-old male with a past medical history of obstructive sleep apnea and obesity was admitted to the pediatric intensive care unit for adenoviral pneumonia and subsequent respiratory failure requiring mechanical ventilation. he had a complex hospital course with many complications including acute respiratory distress syndrome (ards), septic-shock, and coombs-positive hemolytic anemia. the patient was treated with commercial ivig (baxter/baxalta) 400-mg/kg daily for five days. he had two isolated episodes of severe hemolysis in relation to ivig administration requiring multiple transfusions of packed red blood cells (prbc). examination of pre-transfusion peripheral blood smear showed spherocytosis with rouleaux formation and large clumped rbc aggregates. the patient's blood type was classified as blood group a, rh-negative and his initial prbc transfusions were of this type. subsequently, the patient's coombs test was found to be positive using polyspecific and anti-igg typing sera. the patient's antibody screen against reagent group o screening cells was negative ruling out autoimmune hemolytic anemia. however, type specific anti-a antibodies were detected in his plasma as well as the acid eludate prepared from the coombs-positive red blood cells. it was concluded that the patient's hemolysis was due to anti-a antibodies presumed to arise from ivig. the patient's rbc transfusions were changed to o-negative blood and the hemolytic process resolved. the patient ultimately died due to complications of ards. although hemolysis is a known side effect of ivig, it is rarely considered when deciding to administer ivig. in addition, it has rarely been described in the pediatric population. ivig is used in the treatment of a growing number of medical conditions. due to the critical nature of many of these patients, hemolysis secondary to ivig may not be considered and continued blood transfusions with the patient's specific blood type may be used. it is crucial to remember that severe hemolysis can occur from ivig, and the importance of transfusing with blood group o, rh-negative blood when applicable. university of maryland medical center, children's hospital, baltimore, maryland, united states background: coagulopathy is a well-described complication of acute promyelocytic leukemia (apml), and remains a leading cause in induction failure. with treatment, coagulopathy associated with apml has been shown to rapidly improve. multiple organ dysfunction syndrome (mods) in apml, including acute respiratory distress syndrome (ards), has been associated with infection, traumatic injury, malignant infiltration, and cytokine release syndrome. when mechanical ventilation is no longer sufficient, extracorporeal membrane oxygenation (ecmo) can be considered; however, coagulopathy, severe end-organ damage, and malignancy are all relative contraindications to initiation of treatment. we report the case of a 17-year-old female presenting in respiratory failure, disseminated intravascular coagulopathy (dic), with intracranial hemorrhage, and mods, diagnosed with apml, successfully treated with ecmo therapy. design/method: retrospective case analysis and literature review. our patient, a 17-year-old female was admitted in respiratory failure and altered mental status, following a fall shortly prior to presentation. initial laboratory values were notable for pancytopenia, dic, and acute renal failure. a non-contrast head ct showed left temporal lobe intraparenchymal hemorrhage. she was diagnosed with apml by peripheral smear, later confirmed by fish for t(15:17), and was started immediately on high-risk induction chemotherapy as per cog protocol aaml1331, including all-trans retinoic acid, arsenic trioxide, idarubicin, and dexamethasone. cvvhd was required for acute renal failure. despite maximal respiratory support, she remained hypoxemic, with oxygenation index of 46, pao2/fio2 ratio of 70. ecmo was initiated 24 hours after start of induction, 48 hours after admission. coagulopathy resolved on day 6 of induction, ecmo was discontinued after 9 days, mechanical ventilation and cvvhd were stopped after 15 days and she continued to improve, eventually achieving remission with few neurologic side effects. despite relative contraindications to ecmo, this patient was successfully treated with ecmo without significant neurologic side effects. the correction of her coagulopathy was multifactorial: 1) restoration of adequate oxygen delivery via ecmo improving endothelial function; 2) successful organ support to allow sufficient response to induction chemotherapy with atra leading to the terminal differentiation of leukemic blasts; 3) complement and contact system activation through contact with ecmo circuitry. this case illustrates that ecmo can still be considered in patients despite coagulopathy and end organ damage. sinai hospital of baltimore, baltimore, maryland, united states background: primary polycythemia vera is an extremely rare diagnosis in the pediatric patient and is defined by a marked elevation of red blood cells due to erythropoietin-independent mechanisms. presentations of this disorder range from the asymptomatic person to severe thrombotic events, such as budd-chiari syndrome or cerebrovascular stroke. mutations in the jak2 gene are found in adult and pediatric patients with polycythemia vera; however, the jak2 v617f mutation is less commonly identified in pediatric patients. we describe an otherwise healthy 16-year-old female who presented with a significantly elevated total erythrocyte count, hemoglobin, and platelets, incidentally discovered upon routine annual blood work obtained by her pediatrician. design/method: this is a report and discussion of a rare case. demonstrated cellular marrow with trilineage hematopoiesis and no dysplasia. cytogenetics were not assessed. his hemoglobin and platelet count recovered but leukopenia and neutropenia persisted. follow-up evaluation at three months revealed fevers, ongoing cytopenias, a one-month of a nodular skin rash on the trunk and extremities resembling erythema nodosum, and hepatitis (peak alt and ast of 1,176 and 1,008, respectively). following clinical evaluation, a skin biopsy was performed and was remarkable for atypical lymphocytes within the subcutis with t-cell markers, a high ki-67, and positive tia-1, perforin, and -f1 immunoperoxidase stains. negative stains for cd56, cd30, and ebv were noted. these results are consistent with sptcl. additional evaluation did not support a diagnosis of hlh. a staging evaluation was performed. pet-ct showed widespread hypermetabolic subcutaneous activity in the legs, trunk and skull and diffuse marrow hyperplasia. bone marrow demonstrated involvement with precursor b-cell acute lymphoblastic leukemia, with a mll gene rearranagement. his skin biopsy was retrospectively stained with tdt, cd34, pax-5, cd79a, and cd20 with negative results, and a blood smear taken at the time of the skin biopsy did not demonstrate leukemic cells. conclusion: this is the first report of a patient with sptcl having a synchronous malignancy. the patient is doing well, currently in the maintenance phase of treatment for his all, and his skin disease has resolved on pet-ct. while it is possible that his presentation was a function of chance, the possibility of an underlying immune dysfunction or cancer predisposition warrants further investigation. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hereditary xerocytosis (hx) is a rare red blood cell (rbc) dehydration disorder, characterized by variable hemolysis and propensity to iron overload. hx is often misdiagnosed as hereditary spherocytosis (hs). while splenectomy is curative for hs, it is relatively contraindicated in hx due to a substantial thromboembolism risk, signifying the importance of delineating these diseases. blood smear abnormalities are variable and often insufficient to make an accurate diagnosis. osmotic-gradient ektacytometry and genetic confirmation are critical in distinguishing these overlapping disorders. objectives: describe a family with hx, initially misdiagnosed as hs. discuss the importance of distinguishing these disorders and the utility of ektacytometry in making this distinction. design/method: a 13-year-old caucasian male was diagnosed with hs after presenting with prolonged neonatal jaundice starting on the first day of life. he described mild scleral icterus and history of intermittent jaundice and dark urine, without need for transfusions. his father, paternal uncle and paternal grandmother were all diagnosed with hs during childhood and underwent cholecystectomy. additionally, his father underwent splenectomy for abdominal pain. the child's blood counts revealed compensated anemia (hb 12.9 gm/dl) and reticulocytosis (arc 347 × 103/mcl) with increased mcv (96.6 fl) and mchc (38.2 gm/dl). blood smear showed increased polychromasia and poikilocytosis with rare spherocytes and few stomatocytes. while the child had normal ferritin, his father had iron overload (ferritin 800 ng/ml) despite no prior transfusions. osmotic-gradient ektacytometry profile of the child and father's rbcs showed a characteristic left-shifted, bell-shaped curve with decreased omin and ohyp, diagnostic of hx. the family is currently undergoing genetic studies. despite clinical similarities between hs and hx, distinguishing these diseases has significant management implications. hx is a disorder of rbc permeability, causing shortened rbc survival. stomatocytes on blood smear can raise suspicion for hx, but are insufficient to make an accurate diagnosis. identifying characteristic biomechanical membrane properties using osmotic-gradient ektacytometry is the gold standard for clinical diagnosis, which can then be confirmed by molecular studies. hs and hx can be easily and reliably distinguished using ektacytometry, as both disorders have very distinctive curves representing different rbc deformability patterns. after hx diagnosis was made, we counseled the family against splenectomy, as the risk of thromboembolism is significantly increased in hx compared to hs, and the father was diagnosed with iron overload. conclusion: hx is commonly misdiagnosed as hs. this case highlights the importance of making this distinction, and the utility of osmotic-gradient ektacytometry in reliably distinguishing these conditions. penn state health children's hospital, hershey, pennsylvania, united states background: relapsed acute myeloid leukemia (aml) presenting as an isolated central nervous system myeloid sarcoma (cns ms) is very rare and its treatment is not well-defined. thiotepa, vinorelbine, topotecan and clofarabine (tvtc) has been successful for re-induction therapy to induce remission prior to hematopoietic stem cell transplant (hsct). objectives: to describe our experience in utilizing tvtc therapy in two children with no extramedullary disease at initial diagnosis who presented with relapsed aml as intracranial myeloid sarcomas. results: case 1: 34 month-old female was diagnosed with flt3 negative aml and completed treatment per the children's oncology group (cog) aaml1031 study on the low risk arm without bortezomib. cerebral spinal fluid (csf) negative at diagnosis. fish testing positive for tcf3 gene deletion of unknown significance. mrd was undetectable after induction i and remained undetectable after each cycle. nine months off therapy, recurrent headaches prompted mri imaging which revealed two posterior fossa masses. csf and bone marrow testing were negative. stereotactic biopsy of the larger mass confirmed recurrence of aml. patient underwent two cycles of tvtc with a total of seven doses of intrathecal cytarabine with almost near resolution of the cns ms. completed cranial radiation and proceeded to allogeneic stem cell transplant with unrelated cord marrow donor and is disease free at approximately day +200.case 2: 5 year-old female diagnosed with flt3 and mll negative aml and completed treatment per cog aaml1031 study on the low risk arm without bortezomib. csf negative at diagnosis. mrd was undetectable after induction i and completed therapy without complications. two months off therapy, a retrospective analysis of her diagnostic bone marrow by the cytogenetic laboratory to test a new panel identifying novel 11q partners revealed a cryptic insertional 10:11(mllt10/mll(kmt2a) translocation. at four months off therapy, acute mental status changes prompted mri imaging which revealed two intracranial ms and lumbar spine involvement. resection of the larger lesion for symptomatic relief confirmed the mllt10/mll(kmt2a) fusion. csf positive for blasts and marrow negative for relapsed disease. patient completed two cycles of tvtc with a total of seven doses of it cytarabine with near resolution of cns disease (only 3 mm contrast enhancement in the medulla). she received craniospinal radiation and is awaiting improvement in her cardiac function before proceeding to hsct. conclusion: tvtc is a successful reinduction regimen for relapsed aml with cns ms prior to hsct. background: acute severe anemia can be a life-threatening medical condition. the differential is quite broad for possible etiologies of acute severe anemia, including autoimmune hemolytic anemia (aiha) and atypical hemolytic uremic syndrome (ahus). autoimmune hemolytic anemia is an antibody-mediated process that targets the protein antigens located on the surface of red blood cells. treatment options for aiha include corticosteroids, with up to 80% of patients being responsive, with some requiring splenectomy. atypical hemolytic uremic syndrome is a medical urgency, defined as the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. the etiology is usually due to genetic causes, or less commonly, due to autoantibodies or idiopathic reasons. prognosis is very poor. objectives: differentiating between autoimmune hemolytic anemia and atypical hemolytic uremic syndrome can be a time-sensitive diagnostic dilemma while the patient is in critical condition, but this important delineation can vastly alter therapeutic options. design/method: here we discuss two cases highlighting the diagnostic workup involved in differentiating between atypical hemolytic uremic syndrome and autoimmune hemolytic anemia. patient a is a 3-year-old male who presented in extremis with severe anemia, uremic encephalopathy, and severe acute renal injury requiring hemodialysis and multiple blood transfusions. patient b is a 10-month-old male, who also presented in extremis with respiratory failure secondary to adenovirus/rhinovirus/enterovirus, with acute progressive renal failure and microangiopathic hemolytic anemia, requiring hemodialysis and cardiorespiratory support. : patient a underwent a full hematologic and infectious disease workup. subsequent laboratory studies confirmed enteropathogenic e.coli (epec) in the patient's stool; blood cultures remained negative. renal biopsy results were consistent pigment nephropathy. bloodwork indicated positive direct coombs. patient a was ultimately treated with steroids 2mg/kg/day, with significant improvement. patient b also included a full hematologic work-up, including adamts13 activity and ahus genetic panel, as well as full infectious disease work-up. subsequent laboratory test-ing revealed blood cultures growing streptococcus pneumoniae, with adamts13 activity at 41% (adult ref range: >/ = 70%), and normal complement levels. imaging findings also supported diagnosis of ahus. the management of a critically ill patient with acute severe anemia requires a thorough hematologic and infectious disease work-up. while molecular and genetic are helpful in definitive diagnosis of ahus, the utility of such results is limited by time. overlapping clinical presentation of a patient in extremis due to acute severe hemolytic anemia with progressive renal failure presents a rather broad differential, with time-sensitive treatment and prognostic implications. the favorable response to steroids delineates aiha from hus. background: d-2-hydroxyglutaric aciduria (d-2-hga) is a rare metabolic disorder characterized by developmental delay, hypotonia, and bi-allelic mutations in d-2hydroxyglutarate dehydrogenase (d2hgdh) or isocitrate dehydrogenase 2 (idh2). metaphyseal chondromatosis with d-2-hydroxyglutaric aciduria (mc-hga) is a type of d-2-hga that has been previously reported in seven patients (omim 614875; pmid 24049096), three of whom had somatic mosaicism for r132 variants in isocitrate dehydrogenase 1 (idh1). we describe a 3-year-old boy with mc-hga who subsequently developed acute myeloid leukemia (aml) and was found to have a r132 variant in idh1 in a leukemic bone marrow sample. we report the first case of aml with this metabolic disorder. design/method: a 1-year-old hispanic boy presented with short stature, developmental delay, abnormal skin pigmentation, and unilateral congenital cataract. workup revealed multiple skeletal enchondromatosis and elevated urine d-2-hydroxyglutaric acid levels. he was diagnosed with mc-hga. no pathogenic variants in d2hgdh, idh1 and idh2 were identified in peripheral blood. germline testing with biopsies of skin lesions was declined by the family. two years later, he presented with streptococcal sepsis and pancytopenia. blasts were noted on peripheral smear. bone marrow morphology was consistent with acute myelomonocytic leukemia (∼23% blasts). chromosome analysis showed normal 46 xy, and molecular testing by pyrosequencing idh1 and idh2 revealed a r132c variant in idh1 (25% mosaicism). the patient is being treated as per the cog study aaml1031. end of induction i bone marrow aspirate was hemodiluted, but there was no obvious residual disease by flow cytometry (0.01-0.01% sensitivity) or morphology. the previously identified idh1 variant was no longer detectable (limit of detection <10%). although targeted therapy for aml with idh1 mutation is currently in phase i clinical trials in adults, there is no safety or efficacy data for using idh1 inhibitors in children. treatment with ivosidenib is therefore not currently an option for our patient. conclusion: this is the first case of aml reported with this rare metabolic disorder. somatic r132 variants in idh1 have been identified in three other mc-hga cases. this same mutation leads to the accumulation of d-2-hydroxyglutarate in gliomas and aml. without any confirmed germline mutation or somatic mosaicism testing of multiple specimen sources, we can only speculate that the patient has an underlying somatic idh1 mutation associated with mc-hga which subsequently led to leukemogenesis. we present the first case of this association, to increase index of suspicion for development of aml in children with metabolic disorders associated with variants in idh1. background: congenital combined deficiency of the vitamin k-dependent coagulating factors (vkcfd) is a rare heterogeneous autosomal recessive bleeding disorder. vkcfd is caused by mutations in the genes of either gamma-glutamyl carboxylase (ggcx) or vitamin k epoxide reductase complex (vkorc), which are responsible for the gammacarboxylation of vitamin k dependent proteins (vkdps) allowing for their activation. the clinical presentation ranges from no bleeding to intracranial hemorrhage. to date, vkcfd has been reported in few patients worldwide. objectives: we report a case of a girl with novel homozygous mutation of the ggcx gene, highlighting her clinical and biochemical characteristics with a review of the literature. a 3-month-old girl of consanguineous emirati parents, presented to our hospital with a history of bleeding from puncture site after receiving her second-month vaccine. that was associated with episodes of mild mucosal bleeding. review of systems was negative for jaundice, steatorrhea and failure to thrive and physical exam was unremarkable. investigations revealed markedly prolonged pt and aptt with high inr. fibrinogen, hemoglobin and platelets were always normal. activities of vitamin k-dependent factors including fii, fvii, fix, fx, protein c and s were all low. a measurement of proteins induced by vitamin k absence (pivka-ii) was done and came very high. this was associated with a mild elevation in liver enzymes but normal liver function test. the picture was supporting vitamin k deficiency, and as a result, she was started on oral vitamin k supplements of 1 mg/day. she responded partially to vitamin k and required higher doses to stabilize her inr. after excluding acquired causes and due to her requirement of high doses of vitamin k, a mutation in either ggcx or vkorc genes was suspected. genetic analysis was conducted for her which revealed a novel missense homozygous mutation in the ggcx gene (c.548a>t) confirming the diagnosis of combined deficiency of vitamin k-dependent clotting factors type 1. the asymptomatic parents were both heterozygous for the same mutation. results: she is currently stable on 10 mg/day of vitamin k supplements. conclusion: vkcfd is a rare bleeding disorder with an overall good prognosis due to the availability of several effective therapeutic options. the function of the mutated gene is unknown. our patient demonstrated a partial response to vitamin k supplements suggesting presence of a residual carboxylation capacity and a possible role of this gene in the enzymesubstrate interactions. university of alabama at birmingham, birmingham, alabama, united states s55 of s301 background: gata2 is a zinc finger transcription factor that plays a critical role in the regulation of hematopoiesis and lymphatic angiogenesis. mutations leading to gata2 deficiency (gd) have been linked to a variety of clinical conditions. patients with gd have a striking predisposition to develop myelodysplastic syndrome (mds), acute myeloid leukemia (aml), or chronic myelomonocytic leukemia (cmml). acute lymphoblastic leukemia (all) has not been associated with gd, although the association of bcell all and gd has been previously reported. objectives: to describe a unique association of gata2 deficiency and t-cell all in a young child. results: an 8-year-old female presented with a one-week history of fever and malaise. she had a significant past medical history of verruca plantaris and self-resolving leukopenia associated with febrile illnesses. significant family history included sister with neutropenia and human papilloma virus (hpv) infection, and mother with neutropenia, monocytopenia, atypical mycobacterial infections, and hpv infection. peripheral blood revealed hemoglobin 9.3 g/dl, hematocrit 26.7%, platelets 176,000/ul, and white blood cell 1,740/ul (neutrophils 122/ul, lymphocytes 1601/ul, monocytes 17/ul). patient underwent a bone marrow biopsy demonstrating lymphoblast infiltration. flow cytometry analysis demonstrated monoclonal lymphoid blast population that co-expressed cd117, cd34, cd13, nuclear tdt, cd2, however, lacked expression of cd33, cd10, cd3, cd19, hla-dr, or myeloperoxidase. findings were consistent with tcell all with aberrant myeloid markers. cytogenetics analysis revealed 45,xx,dic(21;22)(p11.2;p11.2). patient began treatment as per children's oncology group aall0434 and achieved remission at the end of induction. course of therapy was complicated by episodes of fever, reciprocating junctional tachycardia, asparaginase-associated thrombosis, viral meningitis, recurrent episodes of verruca plantaris, and resistant streptococcus pneumoniae or haemophilus parainfluenza infections causing chronic cough. later, she was also found to have low igm levels; after completion of therapy, she developed monocytopenia. lymphocyte subset panel revealed absent b cells, decreased number of natural killer (nk) cells, and cd4/cd8 inversion. further work-up included gata2 sequence analysis that showed heterozygous nonsense mutation (c.58c > t/c; reference nm_001145661) likely resulting in gata2 haploinsufficiency. patient continues to be in remission, is receiving monthly immunoglobulin replacement and is on azithromycin for atypical mycobacterial prophylaxis. surveillance bone marrow biopsies have shown no evidence of mds or leukemia, however, have demonstrated persistent hypocellularity. the possibility of undergoing an allogeneic bone marrow transplant is actively being discussed given its curative potential. clinicians should be aware that t-cell all may be associated with gata2 deficiency. cincinnati children's hospital medical center, cincinnati, ohio, united states background: treatment for severe hemophilia a is centered on factor viii (fviii) replacement therapy. development of an alloantibody (inhibitor) against fviii is a significant treatment complication occurring in as many as 25-30% of patients. high titer inhibitors render treatment with factor viii ineffective, necessitating the use of bypass agents that may not achieve hemostasis with the same efficacy. considering the substantial ramifications of inhibitor development on treatment, eradication of inhibitors is of great importance to achieve adequate hemostasis in this patient population. desensitization by immune tolerance induction (iti) is the primary method of inhibitor elimination. however, not all patients respond to iti. immunomodulation may be considered as the next line of therapy, although controversy remains in regards to agent selection and use. objectives: there is incomplete data on the use of immunomodulation therapy for inhibitor eradication in severe hemophilia a. we present a case of a pediatric patient with severe hemophilia a and high titer inhibitor who failed initial iti therapy to better illustrate potential treatment options for the future. design/method: a retrospective chart review was performed on a patient with severe hemophilia a at cincinnati children's hospital medical center. results: an 8-year-old caucasian male with severe hemophilia a secondary to intron 22 inversion, was initially diagnosed following extensive bleeding after circumcision at birth. he was identified as having an inhibitor (312 bethesda units (bu)) at 12 months of age after 15 exposure days of treatment. he failed multiple attempts of iti, with recombinant and plasma-derived (pd) fviii. he was advanced to immunomodulation therapy in combination with pdfviii, however demonstrated anaphylaxis to rituximab and ofatumumab. he underwent tolerization to rituximab, and received a six month course with a partial response (nadir of 0.56 bu). 12 months following last dose of rituximab, a rising inhibitor titer (7.44 bu) was found. mycophenolate mofetil (mmf) was initiated with subsequent inhibitor stabilization and a decreasing titer (1.48 bu) over the course of the following year. mmf has been well tolerated without major side effects or infection throughout therapy. conclusion: development of an inhibitor against fviii is a considerable complication in patients with severe hemophilia a. use of immunomodulatory therapies following iti failure remains controversial. mmf has not been well studied in this patient population. we report a case of a patient who is being successfully treated with mmf with minimal side effects. further prospective studies should be considered to further define the role of mmf immunomodulation therapy. background: down syndrome (ds) children with aml (ds aml) have higher cure rates than their non-ds counterparts. outcomes for refractory/relapsed cases, however, remain dismal. somatic mutations of the gene encoding the transcription factor gata1 in ds aml patients are responsible for the observed hypersensitivity of ds aml blasts to cytosine arabinoside (ara-c). in view of excellent survival rates (approaching 90%) of ds aml patients, the ongoing children's oncology group (cog) aaml1531 study seeks to determine the feasibility of treating standard risk (minimal residual disease/mrd negative) ds aml patients using a reduced dose (7-fold decrease) ara-c backbone. although results from japanese trials with this approach are promising, north american and european data are conflicting. although chromosome 7 rearrangements in ds aml do not appear to carry the same adverse prognostic significance as in non-ds aml, monosomy 7 in ds aml patients has been associated with a moderately worse outcome. isochromosome 7q, however, is rare and has only been reported in 3 previous cases of ds aml. objectives: to report our institutional experience of very early relapse involving 2 cases of ds aml patients treated per the reduced dose ara-c arm (3.8 g/m2) of the aaml1531 study. design/method: we hereby report the disease course and cytogenetics of the above 2 ds aml patients. : patient 1 is a 20 month old caucasian female who had gata1 mutation negative aml. patient 2 is a 3-year old caucasian male whose chromosomal analysis revealed isochromosome 7q (3 copies of the long arm of chromosome 7). both patients achieved negative mrd (<0.05%) after induction i chemotherapy with thioguanine, low-dose ara-c and daunorubicin and proceeded per the reduced dose ara-c arm of aaml1531. patient 1 relapsed immediately after completion of chemotherapy. salvage chemotherapy with mitoxantrone/high dose ara-c (hidac) failed to induce a second remission and the patient subsequently died of disease. patient 2 relapsed within 4 months from end of therapy. the patient underwent salvage chemotherapy utilizing a hidac backbone and remains in disease remission. the noted very early relapse following a reduced dose ara-c regimen in our 2 above ds aml children suggests that testing for gata 1 mutation and chromosome 7 rearrangements may play a useful role in the development of future risk-stratified treatment strategies for ds aml. university of rochester, rochester, new york, united states background: in developed countries in the 21st century, severe nutritional deficiency is not an often considered differential diagnosis of unexplained childhood anemia. aside from iron deficiency anemia, vitamin deficiency severe enough to impact hematopoiesis is uncommon in the general pediatric population. here we present the unique case of a 10-monthold infant who presented with intermittent emesis, failure to thrive (ftt), developmental delay, macrocytic anemia, and neutropenia which was initially concerning for a congenital bone marrow failure syndrome. instead, she was discovered to have an underlying, potentially familial deficiency of b12. objectives: 1. to describe the unique case of an infant with b12 deficiency. 2. to outline the importance of including b12 deficiency in the differential diagnosis of unexplained megaloblastic anemia in children. a 10-month-old exclusively breastfed infant presented for gastroenterology evaluation due to persistent emesis and poor weight gain over the course of 2 months. her history was notable for delayed developmental s57 of s301 milestones and hypoactivity. marked pallor prompted hematologic evaluation, which revealed concern for macrocytic anemia (hemoglobin 7.1 g/dl, mcv 107), reticulocytopenia (48.1 × 10^3/ l), and neutropenia (anc 0.4 × 10^9/l). an otherwise reassuring physical examination and laboratory evaluation was notable only for the discovery of an undetectable b12 level and marked hyperhomocysteinemia (162 mol/l). her hemoglobin (hgb) continued to decline (to 5.9 g/dl) over the first few days after presentation, and she required red blood cell (rbc) transfusion. within only a few days of initiation, daily cyanocobalamin injections resulted in a robust reticulocytosis response, improved hgb, immediate normalization in the neutrophil count, and resolution of hyperhomocysteinemia. additional history and laboratory evaluation from the patient's mother revealed a concurrent, asymptomatic maternal b12 deficiency as well as a history of a need for b12 supplementation in the maternal grandfather, raising concern for an inherited etiology. despite the rarity of vitamin-deficient hematologic abnormalities in the general pediatric population, b12 deficiency should be considered as a potential cause of an otherwise unexplained megaloblastic anemia, especially in the setting of concurrent ftt and neurodevelopmental delay. a detailed family history should be obtained in such cases and may have helped to prevent this patient's clinical sequelae had the deficiency been discovered sooner. our patient has experienced a favorable clinical response to b12 supplementation, attesting to the importance of vitamin b12 in early childhood growth and development. background: peg-asparaginase is universally utilized in the treatment of pediatric acute lymphoblastic leukemia (all). despite its high efficacy in this disease, it is associated with hypersensitivity and allergy in 10 -20% of patients. protracted anaphylaxis has been described in circumstances such as severe food allergy with ongoing allergen exposure; however, it has not yet been described in relation to peg-asparaginase. we describe the first reported case of protracted anaphylaxis after peg-asparaginase administration, provide guidance as to time course and management of protracted anaphylaxis, as well as evidence that erwinia asparaginase may be safely administered even in this high risk population. objectives: to provide guidance regarding the duration, course and management of protracted, severe anaphylaxis after peg-asparaginase therapy. a 15 year old male with very high risk all presented for consolidation therapy with peg-asparaginase (intramuscular) and vincristine. one hour after administration, he developed generalized hives and angioedema, for which he was given diphenhydramine. he then quickly developed progressive hives, angioedema, subjective throat and chest tightness, and wheezing. he was treated with diphenhydramine, epinephrine, albuterol, and methylprednisolone with resolution of symptoms. one hour later, symptoms recurred and the patient became hypotensive; he was retreated with methylprednisolone and epinephrine, and was transferred to the pediatric intensive care unit (picu). in the picu, he was placed on an epinephrine drip, and continued on methylprednisolone, diphenhydramine, cetirizine, albuterol, and ranitidine. the epinephrine drip was successfully discontinued after 48 hours, and his other medications were gradually weaned over the course of two weeks. of note, the patient did have st segment changes in his electrocardiogram during the first 48 hours of anaphylaxis. these were associated with normal ventricular function as per echocardiogram, and resolved within one week. this patient has subsequently tolerated multiple doses of erwinia asparaginase (intramuscular) without premedication. this patient was acutely managed in the pediatric intensive care unit with steroids, anti-histamines, and continuous infusion epinephrine. symptoms consistent with severe anaphylaxis including hives, angioedema, throat and chest tightness, wheezing, and hypotension persisted for a total of four days before finally resolving. he has thus far tolerated multiple doses of erwinia asparaginase without any symptoms of allergy, hypersensitivity, or anaphylaxis. protracted severe anaphylaxis after peg-asparaginase therapy can be successfully managed with multi-agent therapy, including antihistamines, steroids, and continuous infusion epinephrine. re-challenge with an alternate form of asparaginase may be tolerated, even in a patient with protracted anaphylaxis to peg-asparaginase. ucsf benioff children's hospital oakland, oakland, california, united states background: vincristine (vcr) is widely used in pediatric cancers. unlike most cytotoxic agents, hematopoietic toxicity is uncommon. vcr-induced anemia has been observed but its mechanism has not been well studied. vinca alkaloid-induced membrane changes were seen in early studies of hereditary spherocytosis (hs) and anecdotal cases suggest vcr may increase hemolysis in such patients. here we describe a case involving severe vcr-induced anemia in a patient with hs and an explanation as to the mechanism. objectives: to describe the mechanism of vcr-induced anemia in hs. design/method: case report. a 6 year-old female with hs was diagnosed with t-lymphoblastic lymphoma. she had required 2 packed red blood cell (prbc) transfusions as a neonate and thereafter had done well without episodes of acute hemolysis or aplasia. complete blood counts (cbc's) demonstrated a compensated hemolysis, and she did not require further transfusions until she commenced chemotherapy. by the start of maintenance she had received many more prbc transfusions than the average patient. intermittent drops in hemoglobin (hb) did not correlate with any particular agent, and she had stable, mild splenomegaly. a clear pattern emerged during maintenance. her hb was 8-9 g/dl at monthly clinic visits, when she received vcr, intermittent intrathecal methotrexate, and corticosteroids. within 3-4 days, her hb dropped to 5.8±0.6 g/dl, and reticulocyte count decreased from 13.4 to 4.3±0.7%. transfusion at day 4 corrected hb, and the reticulocytes and hb returned to baseline. white blood cell and platelet counts did not change after vcr. blood samples from pre, immediately post, and 4 days post vcr were analyzed and rbc characteristics and markers of hemolysis were not significantly different. ektacytometry showed identical curves, indicating no change in rbc deformability. in vitro incubation of patient blood samples with vcr also did not affect the osmotic deformability, confirming that a change in rbc rigidity was unlikely the reason for the drop in hb. these data indicate that a dysregulation of erythropoiesis was responsible for the anemia after vcr, rather than damage of peripheral rbc's. in most patients, maintenance therapy for lymphoblastic lymphoma does not cause severe anemia, likely because a temporary reduction in erythropoiesis in patients with a normal rbc survival and low reticulocyte count is not noticed. however, in a patient with decreased rbc survival and a brisk reticulocytosis, a disruption in rbc generation is more apparent. in conclusion, vcr administration to patients with an rbc disorder warrants close observation for potentially severe vcr-induced anemia. background: the addition of tyrosine kinase inhibitors (tki) to conventional chemotherapy has improved outcomes for pediatric patients with philadelphia chromosome-positive (ph+) acute lymphoblastic leukemia (all), however there remains an increased risk of relapse compared to other types of childhood all. typically, in relapsed disease the philadelphia chromosome persists and several mechanisms of resistance involving acquired mutations of the bcr-abl1 chimeric oncoprotein have been reported. objectives: describe a unique case of a pediatric patient with ph+ b-precursor all relapsing with b-precursor all without the philadelphia chromosome. results: an 8-year-old boy was diagnosed with ph+ bprecursor all with the presence of the t(9;22)/bcr-abl1 translocation by cytogenetics and fluorescence in situ hybridization (fish), respectively. additional abnormalities included gains of runx1 and loss of one copy of etv6. a remission bone marrow with negative minimal residual disease (mrd) was achieved at the end of induction with dasatinib and the esphall chemotherapy backbone. duration of tki therapy was two years post diagnosis. nearly one year after the completion of therapy, cytopenias prompted a bone marrow investigation. relapsed b-precursor all was established by immunophenotyping, however fish analysis did not identify the bcr-abl1 rearrangement. moreover, quantitative reverse transcriptase pcr was negative for the bcr-abl1 fusion transcript. again fish analysis of the bone marrow revealed multiple additional copies of runx1 and mono-allelic loss of etv6, similar to the initial diagnostic sample. the patient was re-induced per aall0232 anticipating a ph+ all relapse. however, with confirmation of the loss of the ph+ clone, tki therapy was not re-initiated. due to positive mrd of 3.5% at the end of re-induction therapy, the patient was salvaged with blinatumomab therapy and subsequently underwent an allogenic stem cell transplant with a sibling donor. conclusion: this is the first known report of a pediatric patient with ph+ b-precursor all who developed recurrent b-precursor all without the philadelphia chromosome. the persistent findings of gain of runx1 and loss of etv6 makes it unlikely that a second unrelated b-precursor all developed following successful treatment of the original disease. this case highlights the possibility of a genetically distinct subclone present at the onset of disease that shared abnormalities of runx1 and etv6 but did not contain the philadelphia chromosome. nevertheless, the subclone harbored leukemogenic potential in the absence bcr-abl1 expression. it is plausible that the predominant clone present at diagnosis was effectively treated with dasatinib and extinguished, but the bcr-abl1-negative clone persisted in the face of tki therapy. background: ligneous conjunctivitis is a rare form of pseudomembranous conjunctivitis that develops specifically in patients with type 1 plasminogen deficiency. lack of plasmin activity in those patients result in defective fibrinolysis and formation of fibrin-rich membranous material/ masses that develops on the palpebral conjunctiva as well as other sites in the body.current management involve surgical excision of the masses that is usually complicated by multiple recurrences. recently, use of topical plasminogen concentrates helped delaying recurrence, but currently, those concentrates are not commercially available. we report on a 7-year-old omani girl, with hypoplasminogenemia who required optimization of plasminogen level at the time of surgery to delay/ prevent recurrence. objectives: case report on the peri-operative use of ffp versus cryopricipitate transfusion as an alternative replacement of plasminogen during surgical excision of ligneous conjunctivitis. design/method: pharmacokinetic study was performed to assess plasminogen recovery after ffp (15 ml/kg) and precipitate (1 bag/5kg) transfusion results: plasminogen levels remained subnormal after either ffp or cryoprecipitate administration. with ffp, the maximum concentration reached was almost 50% of normal. although half-life of plasminogen is known to be 2-2.5 days, the patient seemed to have a high catabolic rate after receiv-ing cryoprecipitate, with plasminogen levels reaching basal levels within 4 hours. because of the better recovery profile with ffp, we opted to give ffp before and after surgery. peri-operative management included ffp transfusion at 20 ml/kg/12 hours one day before and for 3 days post operatively, followed by 10 ml/kg once daily from day 4-6, then 20 ml/kg on 7th post-operative day. topical treatment was initiated using antibiotic and steroids ed on the day of surgery, followed by heparin ed on the second day. on follow up, she used topical heparin, cyclosporine, prednisolone, and topical lubricant eye drops for variable duration. clinical picture remained stable for almost 1 year post operatively, when she started to develop recurrence of ligneous lesions again. background: ponatinib (inclusig®, ariad pharmaceutical) is a 3rd generation multi-targeted tyrosine kinase inhibitor (tki) approved for treatment of adults with chronic myeloid leukemia (cml) and philadelphia chromosomepositive acute lymphoblastic leukemia (ph+ all) resistant to or intolerant of other tkis. ponatinib has numerous drug-drug interactions and a black box warning for associated serious adverse vascular events and hepatotoxicity. for this reason, ponatinib use has been confined to specific high-risk populations. however, in patients who prove refractory to other therapies, the potential benefits of ponatinib may outweigh risks. to date, ponatinib has not been studied in the pediatric/adolescent and young adult (aya) population. furthermore, literature describing the use of ponatinib alone or in combination with other agents in pediatric oncology patients is scarce. objectives: to describe a single institutional experience using ponatinib in the pediatric patients with ph+ all. design/method: two cases of ponatinib use in pediatric ph+ patients resistant to other tkis were identified at our institution and are described. peripheral blood samples obtained from both patients identified bcr-abl1 p190 fusion transcripts and sanger sequencing was used to identify resistant mutations. results: our first case is a 15-year-old female who received upfront multi-agent chemotherapy plus dasatinib for ph+ all. relapse was confirmed on end-of-therapy bone marrow evaluation, thus bcr-abl mutation testing was performed and revealed a t315i mutation. ponatinib was initiated then discontinued after one week due to clinically significant fluid retention with peripheral edema and bilateral pleural/pericardial effusions. the second case is a lateadolescent female with ph+ all who relapsed 4-years after stem cell transplant (sct). following relapse, tki therapy included both imatinib and dasatinib. due to persistence of bcr-abl fusion transcript despite tki therapy she was switched to ponatinib. shortly following initiation of ponatinib she developed a diffuse, maculopapular rash, which persisted despite dose reduction, resulting in ultimate discontinuation of the drug. bcr-abl mutation testing identified f317l and f357v resistance-conferring mutations. to date, there is scant existing literature detailing the use of ponatinib in pediatric patients. appropriate dosing is undefined and side effect profile not well described, particularly when used concurrently with other chemotherapeutic agents. thus, this case series reporting the response to and toxicity of ponatinib in pediatric ph+ all patients has important clinical implications. additionally, this is the first report of a pediatric ph+ all patient with documented t315i mutation underscoring the importance of bcr-abl mutational testing, particularly at the time of relapse. cooper university hospital, camden, new jersey, united states background: myh9-related disorder is a rare autosomal dominant disease, encompassing several subtypes: may hegglin anomaly, epstein syndrome, fechtner's syndrome, and sebastian syndrome. heterozygous mutations are seen in the gene encoding non-muscle myosin heavy chain iia (nmmhc-iia) which is involved in cell motility as well as functions to maintain cellular shape and integrity. the presentation of myh9-rd is mainly characterized by macrothrombocytopenia, but various related expressions exist: nephritis often leading to renal failure, cataracts and sensorineural deafness (1). a 4-year-old girl with history of extensive dental caries, hyperactivity, and speech delay due to suspected hearing loss was incidentally found to have thrombocytopenia at the time of genetic evaluation. she did not have any bruising or excessive bleeding. she did not respond to observation, immunoglobulins, or steroid therapy. her platelet count remained persistently low (4-23 k/ul). she underwent extensive evaluation to rule out platelet disorder vs. coagulation defect. her peripheral smear showed enlarged platelets by giemsa stain but no inclusion bodies were noted in granulocytes. her platelet aggregation and platelet surface glycoprotein by flow cytometry were negative. her coagulation profile was also normal. objectives: this case report summarizes the complexity in diagnosing myh9-rd in a pediatric patient. design/method: since a unifying diagnosis for her clinical presentation was not apparent, whole exome sequencing (wes) was undertaken. results: wes revealed the r702c heterozygous pathogenic variant, located in exon 17 in the myh9 gene. myh9 gene alteration explained the patient's clinical features of macrothrombocytopenia and hearing loss. this mutation was paternally inherited, and her father demonstrates mosaicism. he was asymptomatic with normal platelet count but his morphology showed enlarged platelets with no inclusion bodies in granulocytes. when dealing with patients who have mild or no symptoms of bleeding diathesis but evidence of persistent macrothrombocytopenia, considering a platelet disorder belonging to myh9-rd can help delineate certain predisposing syndromes and guide clinical management. patients are likely to benefit from early genetic testing while receiving supportive therapy. wes can highlight syndromes and provide information on recurrence risk for families. the renal and hearing abnormalities are indistinguishable between epstein and fechtner's syndromes, but the pathogenic variants differ (2). the genotype-phenotype correlation implies that our patient may have either syndrome, although clinical features compatible with nephritis have yet to manifest. patients should be monitored closely for long-term progression of myh9 disease, and treatments should be initiated accordingly. we present an 11-year old female evaluated by genetics at birth due to prenatal microcephaly. chromosomes and microarray were normal. at age 3 she developed standard risk pre-b-cell acute lymphoblastic leukemia (all). she completed treatment in 2012 and has been doing well in the interim, remaining in complete clinical remission. during and after treatment she exhibited developmental delay and neurocognitive deficits. at age 11 her height and weight were at or below the 5th centile and head circumference was below the 2nd centile (approximately 6 standard deviations below the mean and corresponding to the 50th centile for a 9-month-old girl). bone age was appropriate. she had a distinctive triangular face with micrognathia and a pointed nose resembling a seckel-like syndrome. the patient also had clinodactyly of the 4th toes, zygodactylous triradius involving the 2nd and 3rd left toes, tendency to sydney line in the right palm and a radial loop in the left middle finger. the patient's unique clinical presentation prompted a more thorough genetic evaluation, which led to a novel finding we feel is clinically significant with regard to the development of malignancy. design/method: whole exome sequencing (wes) was performed on the patient as well as her biological parents (trio). a de novo heterozygous mutation in the gene pcdh17 with potential relation to the phenotype was discovered. this c.716dupa variant causes a frameshift starting with codon asparagine239, changing this amino acid to a lysine residue and creating a premature stop codon at position 34 of the new reading frame denoted p.asn239lysfsx34. this variant is predicted to cause loss of normal protein function via protein truncation or nonsense-mediated mrna decay. conclusion: pcdh17 is a member of the protocadherins family which is important in cell-to-cell adhesion and synaptic function in the central nervous system and is highly expressed in areas of the brain involved in higher cortical function and speech. aberrant expression of protocadherins has been associated with the development of malignancies in many organ systems. with regards to leukemia, the methylation status of this gene at diagnosis has been implicated in the prognosis of all and could be used as a biomarker to predict relapse. this patient's de novo mutation and clinical presentation are unique to what has been previously presented in the literature. we feel that this mutation is a clinically significant finding that may shed light on the role of this gene in the development of hematopoeitic malignancies. background: acquired hemophilia a (aha) is an uncommon and potentially life-threatening hemorrhagic disease characterized by sudden onset of bleeding in patients with neither personal nor family history of bleeding dyscrasia. it is usually seen in adults with autoimmune diseases, solid tumors, lymphoproliferative diseases, pregnancy or during the postpartum period; occurrence in the pediatric population has rarely been reported. we report a case of an otherwise healthy teenager who was found to have aha when he presented with acute onset of atraumatic soft tissue hematoma. results: a 13-year old male of middle eastern descent with history of congenital absence of the right external ear, but otherwise in good general health, presented to our emergency department with a three day history of progressive worsening of right lower leg pain, swelling, and paresthesia, without preceding history of trauma. evaluation by the pediatric orthopedics service documented significantly elevated compartment pressures, necessitating immediate four-compartment fasciotomy. pre-operative labs were significant for prolonged activated partial thromboplastin time (aptt) of 67.7 (23.4-38.9) seconds with normal prothrombin time (pt) and international normalized ratio (inr). ptt did not correct on mixing studies, suggesting the presence of a circulating anticoagulant. factors xii and xi were in the normal range; factor ix was elevated, 247 (60-150). factor viii level was 4% and fviii inhibitor level was 5.3 bethesda units (<0.8), confirming the diagnosis of aha. work up for autoimmune disease was negative. his bleeding and surgical hemostasis were managed with recombinant factor vii (novoseven) 90 mcg/kg every 3 hours for 24 hours post operatively, with gradual interval prolongation. factor viii antibody eradication was managed with prednisone 1 mg/kg/day. factor viii and inhibitor levels normalized by day 5 of hospitalization. recombinant factor vii was discontinued; steroids were gradually tapered and discontinued at discharge (hospital day 15). conclusion: acquired hemophilia is likely an underdiagnosed condition in pediatrics. while it is typically seen in adults with underlying autoimmune disease, solid tumors, lymphoproliferative disease, or during pregnancy or the postpartum period, pediatric cases may have no identifiable etiology. this case highlights the importance of considering this diagnosis in any patient with unexplained bleeding regardless of their age, so as to intervene early and prevent adverse consequences. university of oklahoma, oklahoma city, oklahoma, united states background: myeloid neoplasms associated with eosinophilia is a rare subtype of chronic leukemia characterized by clonal eosinophilia. the true incidence is unknown due to its rarity and possible classification as idiopathic hypereosinophilia syndrome. the most common chromosomal aberrations involve platelet-derived growth factor receptors (pdgfrs). we report one such rare case in a pediatric patient. most of the pediatric management of this entity is derived from adult case reports and case series. objectives: to describe a case of chronic leukemia presenting as eosinophilia results: a previously healthy 15 year old caucasian male presented with a several week history of migrating joint pain, splenomegaly, and abnormal blood counts with leukocytosis, thrombocytopenia and absolute eosinophilia. white blood cell differential showed myeloid precursors suggestive of chronic myeloid leukemia. bone marrow evaluation showed 10% blasts and 18% eosinophils. bcr-abl testing was negative, ruling out cml. fish analysis for eosinophilic clonality revealed deletion of chic2 gene, resulting in fip1l1/pdgfra fusion gene, diagnostic for myeloid neoplasm with eosinophilia associated with pdgfr abnormalities. treatment was started with tyrosine kinase inhibitor (tki), imatinib 100mg daily. within 3 months, fish analysis for fusion gene was negative. after approximately 18 months of daily imatinib, he was switched to maintenance dose of 200mg weekly. he is approximately 36 months since diagnosis and doing well on maintenance imatinib. in 2008, the who revised its classification of some chronic eosinophilic leukemias to myeloid and lymphoid neoplasms associated with eosinophilia and rearrangement of pdgfra, pdgfrb, fgfr1. the most common abnormality is the fip1l1/pdgfra fusion gene. other less common abnormalities include fusion genes kif5b-pdgfra and etv6-pdgfrb and point mutations in pdgfra22. some features of chronic eosinophilic leukemia include absolute eosinophilia, splenomegaly, elevated vitamin b12 and tryptase levels, and organ damage from eosinophil infiltrates and cytokine release. patients with rearrangements or mutations involving pdgfra are usually very responsive to imatinib. starting doses have not been well studied or established. experts recommend co-administration of corticosteroids during the first few days of imatinib therapy in patients with a history of cardiac involvement and/or elevated serum troponin levels to prevent myocardial necrosis, a rare complication of imatinib therapy in eosinophilic patients. fortunately our patient did not have cardiac involvement and to date has not exhibited signs of chronic tki toxicity. conclusion: myeloid neoplasms with eosinophilia constitute a rare form of chronic leukemias. they are often associated with pdgfr abnormalities and are usually very responsive to tyrosine kinase inhibitor therapy. walter reed national military medical center, bethesda, maryland, united states background: germline samd9l mutation is a rare cause of constitutional bone marrow failure with a unique propensity for clonal evolution to monosomy 7 and mds. objectives: previous case series have demonstrated diverse clinical outcomes in patients with a germline samd9l mutation. our case presents a novel samd9l mutation (p.val1551leu). additionally, the case highlights the challenges in clinical decision making for a patient with a gene mutation that is known for clonal evolution towards monosomy 7 with risk of progression to myeloid malignancy, but also known for self-correction through uniparental disomy or inactivating mutations which results in disease remission. design/method: a retrospective chart review and review of the literature was performed. dna was isolated from peripheral blood and used for whole exome sequencing. a peripheral blood sample from the patient's mother and father showed no samd9l mutation. skin biopsies of the patient and parents were evaluated for uniparental disomy or new mutations. to determine the pathogenicity of this novel mutation, the specific samd9l mutant dna was transfected into the 293human embryonic kidney cell line to assess its role in inhibiting cell proliferation. our patient presented at 8 months of age with pancytopenia and hypocellular bone marrow in the setting of s63 of s301 sepsis. he had evidence of dysfunctional immune activation with hemophagocytosis and elevated soluble il2 with simultaneous severe hypogammaglobulinemia. analysis of the peripheral blood showed no increase in chromosomal breakage, normal telomere length, and normal flow cytometry. gene testing for primary hemophagocytic lymphohistiocytosis and inherited bone marrow failure were negative. after the patient recovered from his presenting illness, a repeat bone marrow biopsy demonstrated improved cellularity with myelodysplasia and cytogenetics significant for monsomy 7.whole exome testing demonstrated a novel samd9l mutation. the patient continued to require intermittent ivig and failed to demonstrate appropriate leukocytosis with intermittent infections. on repeat bone marrow evaluation over the course of 9 months, the patient demonstrated no evidence of evolution towards self-correction and had a persistent monosomy 7 clone. the patient is scheduled to undergo a matched unrelated donor bone marrow transplant. our case highlights the unique clinical picture associated with constitutional marrow failure and clonal evolution secondary to a novel samd9l mutation which is thought to cause pancytopenia by inhibiting cellular proliferation and often results in the development of monosomy 7 which rescues hematopoiesis but with a risk for malignancy. background: notable labs developed a flow cytometricbased assay with a custom robotic platform to test fdaapproved drugs for anti-cancer activity against individual patient's tumor cells. this personalized assay is a potential method for identifying novel agents and drug combinations to treat aml patients who have failed standard therapies. objectives: to present the case of a teen who underwent successful treatment of relapsed aml post-sct with bortezomib, panobinostat, and dexamethasone-a regimen selected based upon results of notable lab testing. results: a 15-year-old male with m4-aml had an isolated bone marrow relapse 8 months after completion of scheduled therapy. at relapse, his aml was flt3-itd positive. he achieved a second remission with negative mrd and underwent matched sibling donor bmt after busulfan/cyclophosphamide conditioning. bma performed on day +180 was mrd positive (0.13%). repeat bma done on day +204 showed 5.7% mrd. he started sorafenib on day +212. he received donor lymphocyte infusion (dli) on day +246, then received 2 cycles of azacitadine (aza) followed by dli. marrow mrd by flow after sorafenib alone, sorafenib with dli, and sorafenib with aza/dli were 16%, 15.7%, and 0.16%, respectively. treatment was complicated by varicella meningitis, grade i skin agvhd, febrile neutropenia and c. difficile colitis, and metapneumovirus pneumonia. despite extremely low levels of leukemia (marrow mrd 0.16%), notable lab testing performed on the patient's leukemia cells from marrow collected after aza/dli/sorafenib revealed sensitivity of his leukemic blasts to a combination of bortezomib, panobinostat, and dexamethasone. because of prolonged cytopenias, multiple infectious complications, and persistently positive mrd, he discontinued aza/dli/sorafenib and on day +368 started bortezomib 1.3 mg/m2 iv on days 1, 4, 8, and 9; panobinostat 20 mg po on days 1, 3, 5, 8, 10, 12; and dexamethasone 20 mg po on days 1, 2, 4, 5, 8, 9, 11, and 12 . chemotherapy cycle 2 started 21 days later. he tolerated treatment without side effects and with resolution of rash and cytopenias. he achieved full donor chimerism, negative flt3-itd, and complete remission by morphology and flow after two cycles. notable lab testing is a powerful tool for evaluating the sensitivity of small populations of leukemic blasts to novel drug therapy. results from notable lab testing may serve as a useful guide for treatment selection after failure of standard aml therapy. this patient achieved morphologic and mrd remission post-sct with bortezomib, panobinostat, and dexamethasone-a regimen predicted to be efficacious based upon notable lab results. maria ahmad-nabi, christine knoll, sanjay shah, esteban gomez, lori wagner phoenix children's hospital, phoenix, arizona, united states background: development of inhibitors in patients with factor ix deficiency (fixd) is a well-recognized complication occurring in 1-3% of patients. within this subset a small percentage can develop anaphylaxis to factor. desensitization with cyclophosphamide, an alkylating agent used in the management of various oncologic malignancies, and reported for use in factor viii desensitization has been previously unreported for use in desensitization in patients with fixd. rituximab, an anti-cd 20 antibody, however has been used. objectives: to induce immune tolerance (it) in patients with inhibitors to factor ix with either novel or under reported methods using cyclophosphamide and/or rituximab. we report a case series of 2 patients at phoenix children's hospital with fixd who achieved it with cyclophosphamide and/or rituximab. results: patient one was a 14 year old male with severe fixd, who at the time of desensitization had inhibitor levels of 12 bu. he was desensitized with cyclophosphamide, then admitted for infusion of recombinant factor ix. he experienced a few minor symptoms of intolerance including an urticarial rash which was self-limited, and hemarthrosis of the right elbow on day 1 which responded to novo 7. he tolerated the remainder of his infusion without issues. he continued recombinant factor ix daily, and returned to clinic for monthly cyclophosphamide for 6 months. he did develop urticaria with hemarthrosis and spontaneous muscle bleeds which were tempered with zantac, zyrtec, solumedrol, and benadryl. he remained without a recurrence of inhibitors, however did have intermittent hemarthrosis of his ankles thereafter requiring prophylactic twice daily dosing recombinant factor ix. patient two was a 10 year old male with severe fixd and a family history of anaphylaxis to factor causing early death in all male relatives with the disease. he had never received factor ix and did not have a detectable inhibitor prior to desensitization. he successfully underwent desensitization to recombinant factor ix with rituximab in the icu, and returned to clinic for weekly infusions x 4. he experienced no adverse reactions concerning for anaphylaxis. he continued to tolerate factor ix products without evidence of intolerance, development of inhibitors, and continues on as prophylactic dosing of recombinant factor ix every other day. our experience at a single institution proves cyclophosphamide as a novel agent for inducing it in those with fixd and anaphylaxis. it also provides further evidence that rituximab can desensitize patients with severe fixd. differences include longer duration for cyclophosphamide therapy (6 months vs 1 month). background: cartilage-hair hypoplasia (chh) is an autosomal recessive chondrodysplasia associated with defective cell-mediated immunity caused by mutations in the ribonuclease mitochondrial rna processing (rmrp) gene. cancer incidence is 7-fold higher in patients with chh than in the general population, especially non-hodgkin lymphoma. the use of rituximab, an anti cd20 antibody, results in decreased host b-cell number and impaired humoral function for 6-9 months. the safety of rituximab in pediatric patients with cancer and immunodeficiency is not well documented. a diagnosis of underlying immunodeficiency may discourage physicians from using rituximab due to the risk of severe bacterial infection or viral re-activation. objectives: to report a case of burkitt lymphoma in a young adult female with chh and defective cellular immunity successfully treated with rituximab. results: an 18-year old amish female with disproportionate short stature presented to our center for management of stage iv biopsy proven burkitt lymphoma with myc rearrangement. she had presented a week earlier with cervical, occipital, and submandibular lymphadenopathy, splenomegaly; fevers, night sweats, and weight loss for 2-4 weeks. on exam, her height was three feet associated with brachydactyly, mild bowing of the legs, normal size head without frontal bossing, fine and sparse hair. she had normal intelligence. her pattern of dysmorphisms was suggestive of chh (genetic testing not performed at time of diagnosis). pet-ct scan showed stage iv disease with involvement of cervical lymph nodes, spleen, iliac bone and bone marrow. treatment with standardintensity fab/lmb therapy (group c) with the addition of rituximab was initiated. she had an incomplete response to cop (∼80% reduction of tumoral masses) but achieved complete remission after copadam1. her course was complicated with severe varicella zoster but she completed therapy and remains in complete disease remission for 24 months after treatment completion. genetic testing subsequently performed proved homozygosity for chh with a n.71a>g variant. she had no other opportunistic infections during or after therapy. conclusion: the use of rituximab was both safe and beneficial in our patient despite defective cell mediated immunity secondary to chh suggesting that rituximab may be safe to use in patients with cellular immune deficiencies. background: hemophilia a and b are bleeding disorders characterized by deficiency in factor viii or ix, respectively. spontaneous or provoked hemarthrosis is a known complication of hemophilia. repetitive episodes of hemarthrosis can lead to debilitating hemophilic arthropathy. lyme disease is a tick-born infection which is endemic to increasing parts of the united states. chronic lyme disease, the phase in which lyme arthritis typically develops, occurs months to years after initial infection and is characterized by swelling of one or more large joints generally in the absence of systemic symptoms. objectives: review cases of hemophilia a and b patients with episodes of provoked hemarthrosis refractory to intensive recombinant factor replacement therapy found to have concurrent lyme arthritis. design/method: we report two clinical cases and review relevant literature. results: first, we report a 12 year-old male with moderate hemophilia a with a provoked knee hemarthrosis which failed to improve despite 3 months of intense factor replacement therapy requiring multiple hospitalizations. factor replacement regimens included twice daily standard half-life recombinant factor viii products or daily to every other day extended half-life recombinant factor viii products with trough levels aimed as high as 50-80%. factor viii pk studies were obtained for dosing, to confirm adherence, and to evaluate for subclinical inhibitors (inhibitor testing was negative). given protracted symptoms additional workup for hemarthrosis was pursed. lyme titers were positive for (8)igg, though negative for igm. he was treated with 28 days of doxycycline during which time hemarthrosis greatly improved on examination and imaging, and he was able to recover function through physical therapy. second, we report a 6 year-old male with moderate hemophilia b who required multiple hospital admissions for a provoked knee hemarthro-sis with no improvement in symptoms despite weeks of daily or twice daily factor replacement with standard halflife recombinant factor ix products aiming for 100% correction. we performed inhibitor testing (which was negative) and pk studies to assess for non-detectable inhibitors, dosing and adherence. lyme testing was positive for (6)igg, though negative for igm. he was treated with amoxicillin for 28 days during which time hemarthrosis significantly improved on examination and imaging. diagnosis and follow-up imaging studies for both patients included mri and serial bedside ultrasounds performed as per uc san diego school of medicine mskus guidelines. background: relapse/refractory aml following allogeneic hematopoietic stem cell transplant (hsct) holds a high mortality rate. current relapse/refractory therapy modalities for younger patients may include re-induction with a clofarabinebased regimen followed by second allogeneic hsct. even for patients who undergo second hsct, the five-year survival rate is dismal. new therapies, including small molecule inhibitors, are being studied in the post-hsct relapse setting or those unfit for hsct with promising results. venetoclax is a small molecule inhibitor that has received breakthrough designation for aml treatment in elderly patients objectives: to report a young adult aml patient with relapse post hsct who was successfully re-induced with topotecan, vinorelbine, thiotepa, clofarabine (tvtc) and has sustained remission with venetoclax maintenance therapy. this approach appears to be unique in terms of reported literature. results: our patient is now a 23-year-old female noted to have mll rearranged aml at initial diagnosis when she was 21 years old. she underwent chemotherapy consisting of cytarabine/daunorubicin according to standard 7+3. due to persistent disease, she was re-induced with g-csf, clofarabine, and high-dose cytarabine (gclac) which put her in cr. her course was complicated by sepsis, colitis, gastrointestinal bleed, deep venous thrombosis, and transfusionassociated circulatory overload. given her co-morbidities, she received another cycle of clofarabine/cytarabine, and then proceeded to reduced intensity allogeneic hsct, according to bmt ctn 1101. the patient tolerated hsct well and experienced no transplant-related complications, including no acute or chronic gvhd. unfortunately, she relapsed about 10 month's post-hsct. initial salvage therapy consisted of another course of g-clac, but due to persistent disease the decision was made to re-induce her with topotecan, vinorelbine, thiotepa, and clofarabine (tvtc). during this time however, she was found to have extensive infection with a fusarium species requiring a course of anti-fungal therapy. bone marrow evaluation showed no residual disease with an mrd of <0.1%. once the absolute neutrophil count recovered, the patient was started on single-agent venetoclax for maintenance therapy, which has been well-tolerated. she remains in morphologic remission for over 8 months. we describe herein a young adult with multiply relapsed aml wherein tvtc re-induction, followed by maintenance with venetoclax were safely used in the post-hsct setting. venetoclax therapy in the relapsed aml setting warrants further study. background: vitamin b12 deficiency is uncommon in children in developed countries, especially in the absence of risk factors like malabsorption or inadequate dietary intake. it often presents with non-specific symptoms and signs and can elude diagnosis. the recognition and treatment of vitamin b12 deficiency is critical as it can lead to bone marrow failure as well as severe neurological and developmental problems in children. to increase index of suspicion of vitamin b12 deficiency anemia in children. we report a rare case of vita-min b12 deficiency anemia in a child who presented with a severe macrocytic anemia, with signs of hemolysis and concern of malignancy. design/method: an almost three-year-old previously healthy girl presented with a few day history of fever, emesis, fatigue and pallor. she had no dysmorphic features, hepatosplenomegaly or lymphadenopathy on exam, growth and development were normal. laboratory findings showed severe macrocytic anemia (hemoglobin 4.4 grams/dl; mcv 104.1 fl) with reticulocytopenia. signs of intravascular hemolysis were present with elevated lactate dehydrogenase (3,842 units/l) and haptoglobin below assay limit. immune-mediated hemolysis was ruled out. initial picture of a hemolytic anemia was compounded by other findings of moderate neutropenia, mild thrombocytopenia and peripheral smear showing occasional blasts. further workup was done with a broad differential diagnosis that included leukemias, hemolytic anemias, bone marrow failure syndromes, and specific deficiencies. results: workup revealed abnormally low vitamin b12 levels along with significantly elevated homocysteine and methylmalonic acid levels indicating functional vitamin b12 deficiency. bone marrow evaluation showed megaloblastic anemia and dyserythropoiesis consistent with vitamin b12 deficiency, and ruled out leukemia. vitamin b12 deficiency can cause a hemolytic anemia like picture secondary to intramedullary hemolysis due to ineffective erythropoiesis. myeloid precursors are also affected which can lead to neutropenia, thrombocytopenia, and abnormal peripheral blood cells. in our patient, initial symptomatic anemia was treated with blood transfusion, followed by intramuscular vitamin b12 injections with normalizing lab values. so far, workup for an etiology for vitamin b12 deficiency is negative except for an equivocal range of anti-parietal cell antibodies raising concerns for pernicious anemia; however it is rare in this age group. another rare condition is an inborn error of the cobalamin transporter. she is currently on oral vitamin b12 supplementation and further workup will be planned based on response. conclusion: this case highlights the importance of early consideration and thorough evaluation of vitamin b12 deficiency in children with unclear etiology of anemia, so that prompt treatment can be initiated. memorial hospital/ university of miami, miami, florida, united states background: despite great success in the treatment of acute lymphoblastic leukemia (all), the outcomes for patients with relapsed all remain poor. prognostic indicators include timing and site of relapse. blinatumomab, is the first agent in its class that simultaneously binds cd3-positive cytotoxic t cells to cd19-positive b cells resulting in lysis of malignant cells. however, mechanisms of leukemia resistance to blinatumomab are unclear. objectives: to describe a case with multiple sites of extramedullary (em) relapse during blinatumomab therapy. results: a 9-year-old hispanic male with philadelphia positive, cd19-positive b-precursor cell all refractory to chemotherapy, had failed a bone marrow (bm) and was placed on blinatumomab and imatinib. he achieved minimal residual disease (mrd)-negative systemic remission, but during his fifth cycle developed bilateral periorbital masses. biopsies confirmed cd19-negative isolated em relapsed disease, which was treated with radiation therapy (rt). there was notable resolution of em disease and he continued systemic therapy. subsequently, he presented with a painful left scapular swelling. imaging showed muscle and lung parenchymal em relapse with cd19-positivity confirmed on histology. he continued on blinatumomab with localized rt while awaiting car-t cell therapy. his bm mrd remained negative until he developed systemic mrd-positivity with cd19-positive blasts following the sixth cycle. primary resistance to blinatumomab is poorly understood. it is proposed that expansion of cd19-negative clones or downregulation of cd19 following blinatumomab may play a role. this was observed in our patient's periorbital relapse; but subsequent em and systemic relapses were cd19-positive, consistent with the co-existence of multiple clones in relapsed all. it has also been postulated that em relapse could be linked to the failure of blinatumomab or t cells to migrate to em sites of disease or drug inactivation by the microenvironment. the second em relapse in our patient, with cd19-positive disease suggests this as a possible mechanism of relapse. this was reported in patients with cd19 positive non-hodgkin lymphoma (nhl), and higher doses of blinatumomab however, have shown promising results in this population. despite blinatumomab's effectiveness in inducing remissions in patients with refractory/relapsed all, it appears to have limitations in patients with em disease. these may arise either from the multiclonality associated with relapsed all or due to the emergence of resistance to blinatumomab, including failure to migrate to em sites. background: cyclic neutropenia is a rare hereditary disorder, characterized by recurrent neutropenia, cycling at about 3 week intervals, with variable associated symptoms including oral ulcers and fever. there are 4 reported cases of cyclic neutropenia associated with chronic inflammation leading to development of reactive aa amyloidosis. one patient also presented with amyloid goiter. we report a new case of cyclic neutropenia with associated renal and thyroid amyloid. design/method: a 12-year-old female presented with a 1 month history of thyromegaly, and recurrent aphthous ulcers associated with fevers. laboratory workup showed severe neutropenia, anemia, azotemia, and abnormal thyroid function, with an absolute neutrophil count -0/ l, hemoglobin -9.0 g/dl, serum creatinine -1.89 mg/dl, and uric acid -9.0 mg/dl. thyroid stimulating hormone was elevated -12.5 iu/ml, and normal free t4. urinalysis showed 2+ protein, 2+ blood, and 5-10 urine red blood cells/hpf. chest radiograph showed mild narrowing of the trachea from thyroid compression. bone marrow biopsy showed a hypocellular marrow, with tri-lineage hematopoiesis, left shifted myeloid maturation with very rare mature neutrophils. both renal biopsy and thyroid fine needle aspiration revealed abundant amyloid. of note, her father had aa amyloidosis, resulting in end-stage renal disease (esrd) requiring hemodialysis, and recurrent aphthous ulcers. the family history suggested a familial predisposition. genetic testing revealed a pathogenic elane c.358 a>t gene mutation with autosomal dominant inheritance confirming the diagnosis of cyclic neutropenia. we treated our patient with daily granulocyte colony stimulating factor to reduce the burden of chronic inflammation induced by cyclic neutropenia, and to preserve renal and other end organ function affected by further amyloid deposition. results: proband with elane gene mutation positive cyclic neutropenia, amyloidosis of thyroid and kidney, with a positive paternal history of aa amyloidosis resulting in esrd. cyclic neutropenia may result in chronic inflammatory states leading to secondary amyloidosis. university of kentucky, lexington, kentucky, united states background: overall survival of burkitt lymphoma (bl), regardless of stage, is greater than 85% in the pediatric population when treated with multi-agent chemotherapy. adenovirus is a common, usually self-limited infection within the pediatric population; however, findings can vary within an immunocompromised host. hepatitis is a rare complication, with very few reports of radiologic findings in this patient population. we discuss a three year old male with history of bl who presented with clinical and radiographic evidence of relapse but was found to have adenovirus hepatitis. design/method: a case report of a patient with bl in complete remission after completion of standard of care chemotherapy, who presented with return of high fever, elevated ldh, transaminitis and hepatic lesions. we describe the hepatic imaging and pathology consistent with adenovirus hepatitis in this immunocompromised host. our patient presented at three years old with a six week history of worsening abdominal pain and fevers. he was found to have a right sided pleural effusion, multiple lesions of the liver, and diffuse abdominal lymphadenopathy; biopsy of lymph tissue was consistent with bl. he completed therapy per anhl1131 arm b and was in a complete remission at the end of planned therapy. one month after completion of therapy, he returned with high fever, abdominal pain and transaminitis, similar to his initial presentation. ct scan showed multiple hypodense discrete lesions throughout the liver and re-accumulation of right sided pleural effusion. ldh peaked at 3580 u/l (uln 370 u/l). uric acid remained within normal limits. bilirubin peaked at 4.0 mg/dl, conjugated 3.2mg/dl. liver biopsy was performed, showing smudgy nuclei with immunohistochemical staining positive for adenovirus. there was no evidence of lymphomatous involvement. resolution of hepatic lesions and transaminitis, with normalization of ldh and fever, occurred with symptomatic treatment alone. adenovirus is known to cause systemic disease in immunocompromised patients and rarely hepatitis. no pediatric patients with discrete hepatic lesions secondary to adenovirus have been reported in the literature. three cases of discrete hepatic lesions have been reported in adult immunocompromised patients, two with fatal fulminant liver failure and one who required cidofovir. this case demonstrates that a common pediatric viral infection can present with lesions concerning for metastatic disease in a pediatric lymphoma patient. prompt diagnosis is vital in the management of these patients when recurrent lymphoma is in the differential. background: heparin induced thrombocytopenia (hit) is an immunologic process in which antibodies bind a heparin complex and cause a paradoxical hypercoagulable state. ramifications of this process may include a multitude of thrombotic events and bleeding complications secondary to platelet consumption. in our patient, hit manifested as increased bruising, an acute decrease in platelet count, and continual clotting of her crrt circuit. hit, although rare in pediatrics, should be included in the differential for children with thrombocytopenia who have received heparin products. to present a unique case report of a critically ill pediatric patient who developed hit in the presence of multiorgan system failure and to discuss the challenges encountered with identification of an alternative anti-coagulant. results: a 12yo obese, caucasian female child presented to our facility with bilateral pulmonary emboli (of unclear etiology). initially, she was started on a continuous heparin infusion, but was transitioned to enoxaparin within 3 days without issue. five days after enoxaparin was initiated, the patient developed acute kidney injury (evidenced by increasing creatinine) attributable to her biventricular heart failure. due to her need for continuous renal replacement therapy (crrt), she was transitioned back to a continuous heparin infusion. whereas her initial platelet count on transition was normal, she developed severe thrombocytopenia (15,000ul) within 48 hours. due to intermediate risk but low suspicion for hit, pf4 antibodies were sent which were positive. after much discussion, she was transitioned to an argatroban infusion which was titrated according to ptt levels. within 48 hours, her platelet count normalized. at discharge, she was prescribed apixaban for anti-coagulant management. conclusion: hit is an uncommon presentation in the pediatric population. given its rarity, there is often a delay in diagnosis which increases risk of complications such as bleeding, stroke, and limb ischemia. even if the diagnosis is suspected or proven, there may be challenges in initiating alternative agents as limited data exists on pediatric options. as argatroban remains the treatment of choice for patients with hit, experience in pediatric patients is limited, and dosing recommendations have been extrapolated from adult studies. anecdotal data exists for use of bivalirudin in children, although studies, primarily, focus on use in specific cardiac cases. in our patient's case, choice was further complicated by renal failure. this case study highlights the need for further research regarding the identification of a secondary anti-coagulant agent for use in pediatric patients with hit. background: subcutaneous panniculitis-like t-cell lymphoma (sptl) is a rare form of non-hodgkin's lymphoma characterized by infiltration of cytotoxic t-cells into subcutaneous tissue. sptl occurs in both adults and children and can present in both patient populations as either alpha/beta or gamma/delta subtypes. patients with the gamma-delta phenotype have an overall poorer survival, although the exact etiology is unclear. interestingly, both subtypes of sptl can present with secondary hemophagocytic lymphohistiocytosis (hlh), and this is associated with a worse prognosis. currently, there are no standardized treatment protocols for sptl, and clinical management includes watchful waiting, corticosteroids/immunosuppression, chemotherapy, and stem cell transplant. the primary objective was to compare how two patients with the same diagnosis responded acutely to therapy. we performed a retrospective chart review of two pediatric patients at our institution who were diagnosed with alpha/beta sptl and secondary hlh. we examined each presentation, treatment course, and outcome. we then completed a brief review of the current literature describing treatment of and outcomes for sptl with secondary hlh. results: these two patients presented in a similar manner with signs and symptoms of hlh. each was then subse-quently diagnosed with alpha/beta sptl after biopsy of cutaneous nodules and each had diffuse disease, as measured by pet. however, they demonstrated vastly different acute responses to therapy. one patient was pre-treated with systemic glucocorticoids before receiving definitive chemotherapy and tolerated therapy well as an outpatient. the other patient started systemic chemotherapy without steroid pretreatment and developed severe cytokine storm characterized by hypotension, cardiac dysfunction, multi-organ failure and cytokine elevation. both patients achieved complete remission (cr) after treatment with chop chemotherapy and remain disease-free 12-24 months off therapy. in patients presenting with sptl and secondary hlh, we propose that initial treatment with antiinflammatory or anti-cytokine therapy can decrease, or even prevent, the possibility of life threatening cytokine release as a result of cytotoxic chemotherapy. background: congenital dyserythropoietic anemia type ii (cda ii) is a rare autosomal recessive disorder, rarely presenting in the neonatal period. iron overload often occurs as a late sequela of ineffective erythropoiesis and intramedullary hemolysis. objectives: to report the novel use of iron chelation in an infant with cda ii associated with severe iron overload. the patient is a 3-month-old, former 27-week infant with prenatal non-immune hydrops and transfusion-dependent fetal anemia who presented with persistent anemia, reticulocytopenia, hyperbilirubinemia, liver dysfunction, and hyperferritinemia. his initial ferritin was 4822.3 ng/ml, tibc 185 ug/dl, and transferrin 116 mg/dl. his bone marrow biopsy showed trilineage hematopoiesis and erythroid dyspoiesis characterized by binucleation of late-stage precursors. genetic testing revealed a compound heterozygous missense mutation and splice site mutation in the sec23b gene, confirming the diagnosis of cda ii. initial liver biopsy revealed mild portal fibrous expansion, and abundant hepatic iron deposition. his ferritin continued to increase, peaking at 21,114 ng/ml, along with liver enzymes peaking at an alanine aminotransferase (alt) of 505 u/l and aspartate aminotransferase (ast) of 776 u/l. ferriscan showed an elevated estimated liver concentration of 2.8 mg/g dry tissue. repeat liver biopsy 3 months later showed giant cell hepatitis with worsening mild portal fibrosis and hemosiderosis. additionally, tissue liver iron concentration was 4755 mcg/g dry weight. cardiac t2* mri revealed mild cardiac iron deposition. given his significant degree of iron overload, deferoxamine was used to reduce hemosiderosis and liver morbidity in preparation for bone marrow transplantation. the patient received deferoxamine 15 mg/kg/day iv x 5 days/week for three months, without any clinically significant adverse events. blood counts and hepatic and renal function were monitored weekly without any abnormalities. growth parameters and liver enzymes significantly improved while receiving chelation therapy. as a noninvasive, cost-effective method, serum ferritin levels were monitored monthly to gauge response to treatment. despite receiving blood transfusions every 3-4 weeks, serum ferritin decreased to 344 ng/ml and liver enzymes decreased to alt 29 u/l and ast 26 u/l prior to bone marrow transplantation. we report the use of deferoxamine in a patient with cda ii less than 2 years of age, for treatment of iron overload. our patient tolerated deferoxamine well without significant adverse events or organ toxicity. deferoxamine may be a well-tolerated method of reducing iron burden in young patients with iron-loading pathologies. background: low grade gliomas with kiaa-1549-braf fusions typically have a favorable prognosis with infrequent rates of high grade transformation, low rates of metastasis and even lower rates of extra cns metastasis. while highgrade transformation has been reported for tumors with braf v600e mutations and cdkn2a deletions, it has not been pre-viously reported in gliomas with kiaa-1549-braf fusions. while there are case reports of high-grade cns malignancies metastasizing through a ventriculo-peritoneal (vp) shunt, low-grade gliomas metastasizing in this manner are extremely rare. objectives: to describe a unique case of peritoneal tumor dissemination of a braf fusion positive high grade neuroepithelial tumor in a child with a vp shunt placed for multifocal braf fusion positive low grade astrocytomas results: an eight-year-old male was initially diagnosed with multifocal low-grade astrocytomas of the hypothalamus and c2-c4 spinal cord. initial testing revealed the kiaa-1549-braf fusion, but no cdkn2a or braf v600e mutation. initial surgical management included a vp shunt and resection of the cervical spinal lesion. he received vincristine and carboplatin, followed by transition to vinblastine given new thoracic metastatic lesions after 10 months of therapy. at 15 months after diagnosis, scans were concerning for diffuse leptomeningeal progressive disease and new intracranial lesions, necessitating craniospinal radiation. following a near cr, he presented 13 months later with acute onset of abdominal pain. a ct scan revealed peri-renal and perirectal soft tissue masses, confirmed by exploratory laparotomy to be peritoneal tumor dissemination of high grade neuroepithelial tumor. a kiaa1549-braf fusion was noted and confirmed by rt-pcr, identical to that seen in the original cns tumors. additional findings included deletion of chromosome 1p (without 19q loss) and heterozygous and homozygous deletion of cdkn2a found by fish. brisk mitotic activity justified a high-grade designation. salvage chemotherapy consisted of 4 cycles of ice with subsequent resolution of pet-avid disease and only minimal peri-nephric tissue remaining. given the favorable response, surgical resection and multiple tissue biopsies were performed which documented no residual active disease. the shunt was revised and he started trametinib for maintenance. we present a unique case of peritoneal dissemination of high grade neuroepitheial tumors with the same kiaa-1549-braf fusion as multifocal low grade astrocytomas in a child with a vp shunt. this raises suspicion for tumor metastasis and transformation to a higher grade malignancy versus two distinct diseases, which may be indicative of an underlying cancer predisposition. texas children's hospital, houston, texas, united states background: polycythemia is a common referral to hematology. it is important to evaluate for a high oxygen affinity hemoglobinopathy, ensuring appropriate testing is performed for early diagnosis and avoidance of additional tests and procedures. a 17 year old mexican female presented with an elevated hemoglobin and hematocrit, symptoms of plethora of her hands and feet, chest pain, palpitations, and fatigue. further confounding the picture, she also had significant menorrhagia and iron deficiency. she was diagnosed with the rare high oxygen affinity hemoglobin new mexico variant, only previously described once in the literature in a 4 year old black boy. objectives: the patient initially presented at age 14 with a hemoglobin of 16.7g/dl and a hematocrit of 52.4%. initial work up consisted of a hemoglobin electrophoresis which diagnosed sickle cell trait, a co-oximetry panel which was normal, and erythropoietin level of 7mu/ml, also normal. she was then lost to follow up and re-referred at age 17. she is a competitive basketball athlete, and at that time, she presented with a hemoglobin of 17.1g/dl, and hematocrit of 50%. erythropoietin level continued to be normal at 13mu/ml. design/method: cardiology was consulted regarding chest pain and palpitations with a normal evaluation. chest x-ray was also normal. a bone marrow aspirate and biopsy was performed with results significant for mild erythroid hyperplasia and mild reticulin fibrosis. jak 2 mutation, von hippel lindau, bpgm, and hereditary erythrocytosis mutations including phd2, hif2a, and epor mutation analysis were sent, all of which were normal. testing to mayo clinic for p50 rbc oxygen dissociation returned low at 19mmhg (24-30mmhg normal range) and subsequently a hemoglobin electrophoresis identified a hemoglobin variant leading to beta globin gene sequencing. results: patient found to be heterozygous for hemoglobin new mexico, with 41.9% hb new mexico and 54.5% hba, and 3.6% hba2. there was no evidence of hbs. when evaluating patients with polycythemia, maintaining a high index of suspicion for high affinity hemoglobinopathies may eliminate further unnecessary and invasive testing for patients. caution should be used when using hemoglobin electrophoresis testing since hb new mexico is known to migrate similarly to hbs on hplc with minimal change that may not be detected in regular laboratories. most high affinity hemoglobinopathies are reported to not have significant symptoms. in this case, our patient complains of fatigue, occasional palpitations and plethora of hands and feet. we will need to further follow this patient for possible attributable symptomatology. divya keerthy, simone chang, warren alperstein, patricia delgado, claudia rojas, ofelia alvarez, matteo trucco university of miami jackson memorial hospital, miami, florida, united states background: improved technology is enabling detection of previously unidentified translocations and mutations in otherwise unclassified sarcomas. one such mutation is the bcl-6 co-repressor -internal tandem duplication (bcor-itd) allowing for the new classification of bcor positive undifferentiated round cell sarcomas (urcs). this sarcoma has a similar appearance to clear cell sarcoma of the kidney (ccsk), potentially representing an extra-renal manifestation of this tumor, but their clinical pathologic features are not identical. objectives: this case highlights how recombinant polymerase chain reaction (rt-pcr) and bcor immunohistochemical staining can ease the diagnosis of this rare sarcoma. results: a 5 month-old female presented for right sided pre-septal cellulitis and a temporal subcutaneous mass. the detection of multiple other subcutaneous nodules on exam raised the concern for malignancy and she was admitted for evaluation. she had two subcutaneous masses on her abdomen, with more cutaneous masses on her legs, back, shoulder, cheek and submandibular areas. she lacked spontaneous lower limb movement and had bilateral clonus. imaging confirmed multiple masses throughout the body including paravertebral area from t3 to l2, bilateral adrenal glands, left kidney and muscles of upper and lower extremities. initial differential included neuroblastoma, infantile myofibromatosis, rhabdomyosarcoma or atypical presentation of a renal tumor. however, synaptophysin and chromogranin stains were negative. with standard immunohistochemistry, the tumor could be only broadly classified as "undifferentiated sarcoma" maintaining the diagnostic challenge. using rt-pcr in the setting of a morphologically primitive round cell neoplasm with strong bcor expression, two external institutes simultaneously diagnosed the tumor as bcor-urcs. the primary lesion is unknown but potentially may have arose from the kidney. bcor-urcs has a heterogeneous histology with tumor cells appearing monomorphic in nests of 6-10 cells separated by septa with uniform nuclei. there is frequently an "orphan annie eye" appearance and sparse cytoplasm to the cells. diagnosis cannot be made solely on evaluation of this nonspecific histology. rt-pcr uses the genetic abnormality in undifferentiated sarcomas to narrow the differential and bcor immunohistochemical staining provides further context. bcor has significant diagnostic value given its sensitivity and specificity in urcs. another potential marker includes ywhae-nutm2b fusions, which occur in smaller subset of cases, but requires further study. rt-pcr has helped further classify tumors leading to the diagnosis of a rare undifferentiated sarcoma with bcor overexpression. while this technology is beneficial, its availability is limited. if accessibility improves, earlier identification and treatment may be possible maximizing the chance for a positive outcome. background: hematohidrosis is a rare condition that mimics bleeding disorders. cases present with oozing blood tinged fluid from various sites like eyes, ears, nose, skin, etc. reported causes of this condition were stress or fear, physical activity, psychological disorders. the condition is self-limited and don't affect the general condition of the patients, but it may contributes to psychosocial problems and may increases their stress and anxiety. so this condition needs to be promptly treated. to test the response of this disease and the associated headache to propranolol treatment. design/method: our case female patient 11 years old 1st offspring of non consanguineous marriage, was admitted with recurrent episodes of oozing blood tinged fluid from eyes, ears and nose 2 months before admission, about 0.5-1 ml from each orifice, lasted 5-10 minutes and subsided spontaneously. it could involve the 3 sites simultaneously or 1-2 sites. the number of attacks was 3-4 times per day then gradually increased to 15-20 times per day. later on the patient developed a bleeding attack from umbilicus. these attacks were aggravated by stress and physical activity and decreased with rest and sleep. the condition was associated with severe headache involving the whole head, throbbing in nature of gradual onset, increased by physical activity and relieved by analgesics. the condition was not associated with vomiting, blurring or diminution of vision, ocular pain, eye discoloration. no earache, tinnitus or diminution of hearing. there was no other form of discharge from eyes, ears or nose. no history of ecchymotic patches, bleeding from other orifices or blood product transfusion. no history of trauma, drug intake, fever or rash. no symptoms of other system affection. past history of recurrent attacks of epistaxis and two operations were done that passed without remarkable bleeding. no similar condition in the family physical examination was free, no evidence of psychological problems. complete blood count, coagulation profile, platelets function, factor 13 and c.t brain were normal. oozing fluid from the patient was analyzed showed the same components as blood. results: our case started oral propranolol 0.5mg/kg/day based on its use in similar cases in literature. the frequency of attacks and headache reduced then stopped after 2 months of treatment and didn't recur after stoppage of propranolol. propranolol can treat this condition successfully. further investigations are needed to determine the link between this condition and severe headache our case was suffering from. background: wilms tumor is the most common renal solid tumors of childhood and is derived from primitive metanephric cells located in the kidney. primary extra-renal wilms tumors (erwt) are extremely rare, estimated to comprise 0.5-1% of all wilms tumors. despite similar histologic appearance intrarenal and erwts differ in embryologic tissues of origin. erwts arise from the more primitive mesonephric or pronephric origin and, therefore, can develop anywhere along the craniocaudal migration pathway of these primitive tissues, most often retroperitoneal, inguinal/genital, lumbosacral/pelvic and mediastinal. these tumors are typically staged and treated per national wilms tumor study (nwts) guidelines, and, by definition, are stage ii or greater due to location beyond the kidney borders. based on the cases reported in the literature, outcomes for erwt are comparable to renal wilms tumors with an 11% local recurrence rate and an 85% two-year event-free survival. we report the first case of a stage iii testicular extrarenal wilms tumor in an 8-month-old male with an intrabdominal undescended testis who underwent complete surgical excision followed by chemotherapy and inguinal radiation. results: a full term 8-month old male underwent orchipexy for an undescended left testicle. the testicle was noted to be grossly abnormal with a pea-sized thickened tissue adherent to the upper pole and a separate mass outside of the scrotum on the superior epididymis. both masses were removed, and s73 of s301 pathology demonstrated wilms tumor with favorable histology and negative margins. ct imaging of the chest, abdomen and pelvis were negative for a primary renal tumor, local residual disease, pathologic lymph node enlargement or distant metastases. the tumor was classified per nwts as stage iii due to tumor removal in multiple pieces. the patient completed dd-4a treatment with vincristine, doxorubicin and dactinomycin per aren0534 with 10 cgy left inguinal radiation. he is currently 15 months off therapy without clinical or radiographic evidence of recurrent disease. primary erwt is an extremely rare malignant neoplasm associated with challenges in diagnosis, staging and treatment. based on the 80 cases reported in the literature, outcomes are similar to that of intrarenal wilms tumor. there are four pediatric paratesticular wilms tumors reported in the literature and, to the best of our knowledge, this is the first case of stage iii testicular wilms tumor successfully treated with dd-4a chemotherapy and radiation. in erwt, nwts guidelines for staging and treatment should be applied with evaluation of both kidneys to exclude an intrarenal primary tumor. background: patient is a 20 yo f, with esrd secondary to atypical hus versus ttp, who presented with thrombotic microangiopathy, aki, thrombocytopenia and anemia after a living unrelated donor kidney transplant. patient initially had downtrending creatinine. on post-op day 2, hematology was consulted for an increasing ldh and drop in platelets. peripheral smear was notable for an absence of schistocytes. yet, biopsy of the kidney revealed microthrombi. the patient was diagnosed with a thrombotic microangiopathy. plasmapharesis was initiated on day #3, at which time ms r was noted to have significantly elevated creatinine. plasmapharesis did not yield any correction in labs and significant bruising developed. patient was started on eculizimab; plasmapharesis was stopped. shortly after, creatinine, anemia and thrombocytopenia corrected to levels at which she was discharged. overall, patient was found to have progressive anemia, thrombocytopenia, an increasing creatinine and ldh (600s) concerning for atypical hus, despite absence of schistocytes on peripheral smear. she responded well to eculizimab, with correction of hematologic changes during induction. she was discharged on eculizimab and continued to respond with normalizing platelet counts and hemoglobin. the differential in light of patient's thrombotic microangiopathy and thrombocytope-nia also included ttp. yet, adamts13 remained normal. dic was unlikely given normal fibrinogen level and d-dimer. objectives: presentations of atypical hus vs ttp. discuss eculizumab as a treatment of atypical hus. highlight atypical presentations of illness in transplant patients. results: despite absence of schistocytes by smear, pt was diagnosed with atypical hus based on presentation and after failing plasmapharesis, she responded well to eculizumab. though her presentation was abnormal, her response to this antibody that blocks the complement cascade suggests that she was experiencing a complement-mediated process. there are rare documented cases in the literature of atypical hus without schistocytes. hemolytic uremic syndrome (hus) is characterized by hemolytic anemia, thrombocytopenia and acute kidney injury. atypical hus is a diagnosis of exclusion, not due common etiologies such as shiga toxin. among atypical causes are complement-mediated forms, caused by an antibody to complement factor. in addition to plasmapharesis, renal transplant and supportive care, the mainstay of treatment for atypical hus is eculizumab (an antibody that blocks the complement terminal cascade). this case describes a patient unique in that, she was diagnosed with atypical hus without any schistocytes by smear. secondly, she responded to eculizumab, with unremarkable gene studies. finally, this case highlights that transplant patients often have unique presentations. nicklaus children's hospital, miami, florida, united states background: synovial sarcoma is a spindle cell tumor categorized as a soft tissue sarcoma. the chromosomal translocation t(x;18) leading to the ss18-ssx fusion protein is unique to this sarcoma. it is a slow growing tumor with common recurrences and often, at presentation, with evidence of metastatic disease. if resection is not feasible, then neoadjuvant with adjuvant chemotherapy is recommended. metastasis carries an unfavorable prognosis given synovial sarcoma historically does not respond well to chemotherapy. trabectedin is a well-tolerated alkylating agent currently indicated for the treatment of liposarcoma and leiomyosarcoma. we present a 17-year-old male with metastatic synovial sarcoma to the lungs that progressed and was refractory to chemotherapy. he was administered trabectedin as a form of palliative chemotherapy, with significant clinical and radiographic response. design/method: pubmed search was done with search for terminology including "synovial sarcoma" and "trabectedin". papers relevant to our case were selected for literature review. a 17-year-old male patient presented with a large right axillary mass. initial imaging showed a heterogeneous multiseptated mass invading the subscapularis and teres major muscles along with innumerable lung nodules. biopsy confirmed diagnosis of monophasic synovial sarcoma. the patient was started on protocol arst 0332 with ifosfomide, mesna, doxorubicin. he completed 4 cycles followed by radical resection and 33 sessions of radiation. due to progression of disease multiple chemotherapy regimens were tried including topotecan and cyclophosphamide, protocol advl 1522 with lorvotuzumab, and pazopanib. imaging of the chest continued to show significant progression of metastasis. the patient's clinical status deteriorated with worsening respiratory status, requiring 10l of oxygen therapy, and inability to ambulate. he was started on trabectedin 1.2mg/m2 for palliative care. after 2 cycles of treatment patient was no longer requiring oxygen and was ambulating without assistance. radiological imaging showed significant reduction in number and size of lung nodules. trabectedin is a recently approved alkylating agent for the management of sarcomas resistant to first line treatment. response in synovial sarcoma is scarcely documented in the pediatric population. epidemiology places the most common age group in the young adults and children. our case opens the doors to further consideration of the use of trabectedin in the pediatric patient with metastatic synovial sarcoma. background: gata1 is an x-linked gene that plays critical role in hematopoiesis. mutations of gata1 gene can be associated to various blood disorders including diamond blackfan anemia, cytopenia, congenital dyserythropoietc anemia and acute megakaryoblastic leukemia. we report a patient with macrocytic anemia and platelet dysfunction who carries a novel gata1 mutation that has not been reported. results: a now 28-month-old male with complex medical history including prematurity at 33 weeks, dysmorphic features, global developmental delay, hyperinsulinism, hypogonadotropic hypogonadism, growth hormone deficiency, micropenis, failure to thrive, patent ductus arteriosus status post ligation, and severe hypotonia, was referred to hematology at 16 months old for resolved, transient thrombocytopenia and macrocytic anemia since 1 month of age. chromosomal microarray showed chromosome deletion of 14q21.3, which is the rps29 gene. he doesn't have a family history of diamond blackfan anemia (dba), despite mom having the same rps29 mutation. he was then diagnosed with dba. his lab workup showed mild macrocytic anemia (hgb 9.1 g/dl, mcv 95fl), normal to inappropriately low reticulocyte count, normal white blood cell and platelet counts, hgf 0%, erythroid ada 1.39 eu/gm hgb (elevated). he has abnormal pfa-100, with prolonged closure time of both adp and epinephrine. he had low von willebrand antigen and ristocetin cofactor activity. he has severe pancreatic insufficiency. bone marrow biopsy showed normocellular marrow with trilineage hematopoietic maturation, without ringed sideroblasts. since mother has the same rps29 gene mutation, maternal labs were done and showed no evidence of macroytosis or anemia. the diagnosis of dba was questioned. whole exome sequencing did not identify any pathogenic sequence changes in the coding regions of rps29 gene, but detected a gata1 mutation r140w, which was reported variant of uncertain significance. his mother shares the same mutation and is asymptomatic, but she may not be affected since gata1 iis xlinked. his father doesn't harbor the gata1 mutation. conclusion: gata1 gene encodes zinc finger dna binding hematopoietic transcription factor, which is important during erythroid differentiation. gata1 mutation r140w has not been reported in literature and is a novel variant of gata1 mutation, which might be contributing to this patient's clinical picture. further studies are warranted to confirm gata1 mutation r140w to be a pathogenic sequence change. alexander boucher, tomoyuki mizuno, alexander vinks, greg tiao, stuart goldstein, james geller cincinnati children's hospital medical center, cincinnati, ohio, united states background: hepatoblastoma (hb), the most common pediatric primary hepatic malignancy, can be associated with specific congenital syndromes. recently, chronic kidney disease and genitourinary anomalies have been linked to hb. cisplatin is a key chemotherapeutic agent in treating hb but its renal clearance and toxicity profile can limit its use for those with end-stage renal disease (esrd). objectives: using an institutional case series, we present data using cisplatin for hb in dialysis-dependent esrd and define recommended dosing for future use. design/method: a chart review of patients with concurrent hb and esrd on dialysis treated with cisplatin at our institution was undertaken. demographic data, diagnostic history, tumor pathology, alpha fetoprotein (afp), hearing assessments, dosing schema, treatment outcomes, and therapyrelated toxicities were reviewed. total cisplatin levels were collected at 5 time points within 10 days after each infusion. free cisplatin levels were also collected for 2 infusions, as were dialysate cisplatin levels. pk parameters were generated using bayesian estimation with a published population pk model as a priori information. results: three patients meeting these criteria were identified. each had "low risk" (non-metastatic resectable) disease at presentation and underwent upfront resections. all had congenital renal anomalies with esrd prior to their hb diagnosis. all cisplatin infusions were given over 3 hours, followed 3 hours later by hemodialysis. patients 1 and 3 received cisplatin at 50% of children's oncology group's ahep0731 weight-based dosing (1.67 mg/kg). patient 2 received 50% of ahep0731 body surface area-based dosing (50 mg/m2) during cycle 1 but required a second dose reduction (25 mg/m2) for cycle 2 due to prolonged cisplatin exposure (total area under the curve 342 mg⋅h/l; average for all seven evaluable cycles 238 mg⋅h/l) and early sensorineural hearing loss at 2000-4000 hz. no other hearing loss in any patient was identified; mild toxicities also included grade 1-2 emesis and grade 1 neutropenia and thrombocytopenia. the median (range) of clearance, volume of distribution at steady-state, and elimination half-life at terminal phase for total platinum were 0.19 (0.15-0.31) l/hour/70 kg, 69.1 (59.0-105.7) l/70 kg and 155 (102-185) hours, respectively. patients 1 and 2 received 2 cycles with rapid afp normalization. patient 3 required an additional 2 cycles, for a likely second primary hb 1 year after initial therapy. cisplatin can be used successfully in pediatric patients with esrd on hemodialysis to treat hb with minimal morbidity using 50% standard mg/kg-based dosing (1.67 mg/kg), achieving pharmacologically appropriate cisplatin exposures. background: treatment for immune thrombocytopenia (itp) has been grouped into rescue and maintenance therapy and often is reserved for patients with bleeding, severe thrombocytopenia, or for improvement in quality of life. splenectomy is considered one of the more invasive but definitive treatments with success rates of 70-80%. treatment of itp can be more difficult in the setting of previous treatment with immune modulation or when the patient is immunocompromised and not a candidate for splenectomy. objectives: present an interesting case of a patient with an autoimmune disease that presented with severe thrombocytopenia, un-responsive to rescue therapy, and requiring emergent splenectomy in the setting of acute intracranial hemorrhage (ich). a 13 year old female with a history of juvenile dermatomyositis presented with a fine purpuric rash on her extremities, wet purpura, and a platelet count of 1k/ l. bone marrow evaluation at that time was consistent with itp. she was on cyclosporine and plaquenil for dermatomyositis. platelets failed to increase after three doses of intravenous immunoglobulin and high dose steroids. following a two week course of oral prednisone and eltrombopag, she presented with persistent severe thrombocytopenia of 1k/ l, anemia of 6.4 g/dl, and a lower gi bleed. she was started on amicar, novo-seven, rituximab, and given platelet transfusions with no improvement in bleeding. subsequently, she developed a subdural hematoma with midline shift. surgery performed an emergent open splenectomy with concurrent continuous platelet transfusion. results: she was monitored closely post operatively and, due to ich, transfused to maintain platelets greater than 100k/ l. by 1 week post-op she had normal platelet counts off transfusions. all medications were stopped within three days of discharge. she represented eight days later with abdominal pain and thrombocytosis and was found to have a portal vein, splenic vein and mesenteric vein thrombosis. she was started on lovenox therapy and admitted for monitoring due to her history of ich. it is unknown whether our patient's underlying immune dysregulation and history of treatment with immunosuppressive medications may have contributed to her unresponsiveness to multiple therapeutic agents. in addition, her significant bleeding did not allow us to fully evaluate her response to second tier therapy. this adds to the scarcity of literature of itp response in pediatric patients with autoimmune disease, and may support more aggressive therapy upfront in these patients. background: multivisceral organ transplantation involves concurrent transplantation of the stomach, pancreas, liver, and intestine with splenectomy, and has been classically used in the pediatric population for infants with intestinal failure from disorders affecting foregut integrity. while there is some data demonstrating its efficacy in adults with low-grade abdominal malignancies, it has not been traditionally used for hepatocellular carcinoma treatment. to describe a unique pediatric case of multivisceral organ transplantation as definitive therapy for refractory fibrolamellar hepatocellular carcinoma in an adolescent male. a 16 year old male presents with a history of fibrolamellar hepatocellular carcinoma, tumor invasion of the portal vein, severe portal hypertension complicated by bleeding esophageal varices and hypersplenism. he had two treatments with yttrium-90 radioembolization, without significant response. he completed six cycles of traditional chemotherapy in combination with sorafenib with resolution of petavidity, but minimal decrease in tumor size and continued portal hypertension. since his disease remained relatively stable for over 2 years, he was evaluated and listed for multivisceral organ transplantation. at approximately 2 years and 7 months after diagnosis, he underwent en bloc liver, pancreas, stomach, small bowel, and colon transplant with splenectomy. a single lymph node was positive for malignancy at the time of resection. in addition to expected post-transplant complications, he also developed skin only acute graft versus host disease at 2 weeks after transplant, treated successfully with a thymoglobulin course. he clinically improved and was back to his baseline activity level, on full oral feedings within 3 months post-transplantation. at three and six month post-transplantation, there is no concern for relapsed hepatocellular carcinoma on comprehensive imaging and evaluation. he is maintained on protocol immunosuppression and posttransplant support. we present the first known case of successful multivisceral organ transplantation in the treatment of refractory pediatric fibrolamellar hepatocellular carcinoma. background: hematohidrosis is a rare disorder that presents with spontaneous excretion of whole blood from intact skin or mucosa. diagnosis is based on clinical observation of the occurrence with the proven presence of erythrocytes and other blood components, without other abnormalities to account for the phenomenon. the existing literature is scarce and consists of primarily case studies. most reports describe bleeding from facial sites around the eyes, ears, and nose. the available literature suggests anxiety and physical or emotional stress reactions as the most common inciting events. little evidence exists regarding the ideal therapeutic approach, however propranolol has been used successfully to reduce bleeding frequency and severity in multiple case reports. a specific genetic etiology has not been elucidated, and no familial cases have previously been reported. we present a pair of half-siblings, both of whom presented with spontaneous cutaneous and mucosal bleeding before two years of age, and report on preliminary results of propranolol therapy. tanzania. at 20 months of age, he became ill and developed spontaneous bleeding from his ears, nose, and scalp. he continued to have frequent bleeding episodes, usually related to illness or physical distress. a bleeding diathesis work-up was unremarkable, however some episodes were severe enough to require transfusions. the patient was subsequently diagnosed with hiv and hepatitis b, presumably acquired via unscreened blood product transfusions. patient b is an infant female born to the same mother as patient a, with a different father. she was healthy until two months of age when she developed spontaneous bleeding from the hairline, eyelids, ears and genital/rectal area. bleeding episodes were nearly always associated with irritability and crying. extensive coagulation workup was unremarkable. results: propranolol therapy was started in both patients, titrated to a goal of 2 mg/kg/day. in both patients, the frequency and duration of bleeding episodes significantly improved. patient b continues to have milder occasional bleeding episodes from her eyes, ears and scalp but has significantly less discomfort and irritability during the episodes. conclusion: to our knowledge, there are no prior reports involving two related patients with hematohidrosis. this case series suggests that there may be a genetic predisposition which has yet to be identified. propranolol has shown effectiveness in reducing symptom frequency and severity. background: gliomas are the most common central nervous system tumors in children. they are classified into different grades based on genotype (idh, braf, tsc, etc.). lowgrade gliomas such as oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas are classified as grades i and ii. of the molecular level alterations this case report focuses on the braf v600e mutation. braf is a member of the raf family of serine/threonine protein kinases and it plays an important role in cell survival, proliferation and terminal differentiation. objectives: here we discuss two cases where dabrafenib, a braf kinase inhibitor, was utilized in the management of gliomas. the cases focus on the use of dabrafenib late versus early in disease course. design/method: patient jl is a 20 year old female who was diagnosed with a low-grade glioneuronal tumor (c7-t1 with a metastatic lesion to the brain) in 2007. jl was treated with chemotherapy, radiation, and surgical resection. despite treatment, the patient's disease progressed. she developed lower extremity dysfunction, urinary incontinence, poor truncal control, and hydrocephalus. dabrafenib was started after the braf v600e mutation was confirmed. patient lg is a 12 year old female who presented in november 2016 with left facial and upper extremity weakness. ct and mri scans demonstrated a mixed solid and cystic lesion extending from the optic chiasm and hypothalamus to the right thalamus and posterior basal ganglia with additional involvement of the right cerebral peduncle. neurosurgical intervention was undertaken and dabrafenib was started after the braf v600e mutation was confirmed. results: patient jl's mri scans have demonstrated improvement of the spine with diminished areas of enhancement along thecal margins, decreased volume and enhancement within the trigeminal plate cistern and resolution of ependymal enhancement within the right ventricle. the patient's most recent mri exhibits no disease progression in head or spine. jl has shown improvement clinically since starting dabrafenib. patient lg has shown improvement in strength and recent mri of the brain has shown resolution of enhancement along surgical resection margins, decreased hyperintensity along the inferomedial aspect of the right basal ganglia and no new enhancements. conclusion: low grade gliomas can alter a person's quality of life and even lead to life threatening complications. often the standard chemotherapy, radiation and surgery don't prevent these complications. genetic analysis can help clinicians target therapy towards certain mutations such as braf v600e. dabrafenib has shown to decrease tumor burden, early utilization as therapy can help prevent morbidity and mortality. children's hospital of pittsburgh of upmc, pittsburgh, pennsylvania, united states background: copper is an essential cofactor in enzymatic reactions essential to proper hematologic, skeletal, neurologic and vascular function. copper requirements in children over the age of 4 are 15 mg/day, which is readily acquired in a typical diet. copper deficiency is known to occur in patients with the rare x-linked mutation and in older individuals with gastrointestinal bypass surgery; however, it is rarely reported in other conditions. objectives: to highlight individuals with autism spectrum disorders or developmental delay with a limited dietary repertoire are at risk for copper deficiency, thus a high index of suspicion must exist in order to diagnose the disorder. design/method: a 15 y/o boy with a prior diagnosis of global developmental delay and oral aversion presented with slowly progressive fatigue, weakness, gait instability, and weight loss. his longstanding feeding difficulties were refractory to intensive feeding programs. his daily diet consisted of 50-60oz of milk and 25-30 individual servings of butterscotch pudding (1680-1880calories/day, 0.7mg iron/day). initial complete blood count demonstrated white blood cell count of 3.3, absolute neutrophil count of 760, hemoglobin of 4.4, mean corpuscular volume of <50, reticulocyte count of 0.5, platelet count of 392. review of his peripheral blood smear revealed microcytic, hypochromic red cells without marked fragmentation, anisopoikilocytosis and ringed sideroblasts; there were no morphologic abnormalities of his leukocytes or platelets. iron studies demonstrated ferritin of 45, total iron binding capacity of 514, and 2% iron saturation. he had no evidence of b12, folate deficiency or blood loss. additional evaluation revealed a serum copper level of 6 (range 60-190), and cerulosplasmin of 2.1 (range 22-58). results: once a diagnosis of copper deficiency was made, the patient promptly began a course of parenteral copper repletion. he received iv copper 35mcg/kg/day x 3 days then weekly intravenous infusions. given his malnutrition, a gtube was placed to begin oral copper repletion and enteral nutrition. within 3 weeks his copper level improved as well as his blood counts. unfortunately, although his blood counts and copper levels normalized, his neurologic status remains below his old baseline although, he has made gains in his gross and fine motor abilities. conclusion: acquired copper deficiency in the pediatric population is a rare event but given the hematologic and neurologic consequences, prompt recognition and treatment is important. this patient's clinical course demonstrates the need to have a high index of suspicion of concomitant nutritional deficiencies other than those routinely evaluated such as iron, b12 and folate. background: lymphoepithelioma-like thymic carcinoma (lelc) is a rare, aggressive neoplasm with a high rate of invasion, metastasis and recurrence. there are no known curative therapies for metastatic lelc. we report the case of a 16-year-old male who presented with metastatic ebv positive lelc. sites of disease included a large primary anterior mediastinal mass and metastases to hilar lymph nodes, lungs and liver. he was initially treated with cisplatin and 5fluorouracil followed by mediastinal radiation. he had a partial response to therapy but his end of therapy scans showed disease progression in lungs, liver, and hilar, supraclavicular and axillary lymph nodes. objectives: molecularly targeted therapies tailored to the patient's genetic profile offer a novel approach to obtain improved survival outcomes. design/method: the patient enrolled on a precision medicine trial, nmtrc009: molecular-guided therapy for the treatment of patients with relapsed and refractory childhood cancer (nct02162732). in this study, tumor/normal whole exome sequencing and tumor rna sequencing were performed and a molecular report detailing the results of genomic and gene expression analysis was generated. a treatment plan was designed within a molecular tumor board comprising oncologists, pharmacists, genomicists, and molecular biologists with domain expertise. results: exome sequencing revealed 26 somatic coding point mutations and no structural mutations (focal copy number changes or translocations). candidate somatic driver mutations included tp53 s94x and r248w as well as kit n655k. both genes have been previously implicated in thymic carcinoma. rna expression analysis demonstrated aberrant activation of biological pathways, including overexpression of kit, hdac1, 2 and 9, tyms, and dhfr. the molecular tumor board selected the combination of pemetrexed (500 mg/m2) on day 1 of a 21 day cycle, imatinib (400 mg daily), and vorinostat (400 mg days 1-5, 8-12, and 15-19) . on day 8 of cycle 1, he was admitted with a herpes zoster infection and imatinib was discontinued in order to reduce risk of herpes zoster recurrence. imaging after 2 cycles showed a complete metabolic response on f-18 fdg pet and a partial response by ct size criteria. as of december 2017, the patient had received 15 cycles of pemetrexed and vorinostat. scans in december 2017 showed an increase in the size and metabolic activity of two right lower lobe pulmonary nodules. there were no new sites of disease and imatinib was re-started. background: systemic lupus erythematosus (sle) is a chronic autoimmune disease that affects multiple organ systems and is associated with many different autoantibodies. patients can present with vague constitutional symptoms including fever, rash, fatigue, and weight loss. some of the various hematologic manifestations of sle include anemia of chronic disease, leukopenia, autoimmune hemolytic anemia (aiha), and idiopathic thrombocytopenic purpura (itp). these can be the presenting signs of sle. evans syndrome (es), a disease characterized by itp and aiha, is a rare hematologic manifestation of sle. neurofibromatosis 1 (nf1) is a relatively common neurocutaneous disorder. these patients are at risk of developing benign and malignant tumors. its association with autoimmune disorders, including sle, remains rare. objectives: there are few cases in the literature that have patients with the combination of sle and nf1. this is the only case that has a patient with sle, nf1, and es. results: a 16-year-old caucasian female presents with two months of vaginal bleeding, weight loss and petechiae. her exam is remarkable for petechiae and café au lait macules. laboratory findings show severe anemia and thrombocytopenia. she receives blood and platelet transfusions during stabilization, and a bone marrow aspirate is performed to rule out a malignancy which is negative. based on the presence of thrombocytopenia and a positive coombs test, an autoimmune process such as es is considered. screening tests for sle reveal positive antinuclear and anti-double stranded dna antibodies as well as low complement. she receives intravenous immunoglobulin and methylprednisolone and eventually her vaginal bleeding slows and her counts recover. she begins sle therapy with hydroxychloroquine and azathioprine. due to the presence of café au lait macules on her exam, a genetics evaluation is performed and the patient is also diagnosed with nf1. to date, there are seven cases of sle with nf1 reported in the literature, only two of which are pediatric cases. there are no reports of the combination of sle, nf1, and es. conclusion: es is a rare hematologic manifestation of sle but can be the initial presentation of this disease. one large study estimates 2% of childhood-onset sle cases are observed to have es. screening for sle should be considered in all es patients even in the absence of typical clinical findings. association of nf1 and sle has been rarely described. whether this association reflects a causal relationship or is coincidental needs more investigation. (lube, ped blood & cancer, 2016) . university of california san diego, la jolla, california, united states background: high grade glioma (hgg) has poor outcomes in adults and children. extraneural metastases are very rare in hgg, and poorly characterized with only a few small case series in adults and only isolated case reports in pediatrics. no genomic data has previously been published for any children with hgg who develop extraneural metastases. objectives: our objective is to describe the natural history of two children with hgg and bony extraneural metastases, comparing their clinical characteristics as well as whole exome sequencing data for both tumors. this information would suggest similar patients should be monitored closely for extraneural metastasis and may benefit from more systemic therapy. design/method: we present a case series of two patients who presented with hgg and had development of bony metastases less than six months after initial diagnosis. both patients had molecular profiling with whole exome sequencing (wes). the first patient was an 11-year-old male with a tumor found in the left lateral ventricle invading into the fornices, hypothalamus, and left midbrain, who had subtotal resection. bony metastasis were found at 3.5 months after diagnosis, and he died 9 months after diagnosis. he initially received radiation, followed by nivolimuab. the second was a 12-year-old female with a tectal/pineal tumor and multiple spinal cord metastases, who had subtotal resection. she developed bony metastasis at 5.5 months after diagnosis and died 13 months after diagnosis. her histologic diagnosis was pineoblastoma, revised to hgg after whole exome sequencing. she received craniospinal radiation followed by chemotherapy per acns0332 (cisplatin, vincristine, and cyclophosphamide) for 2 cycles. when she failed to respond satisfactorily to this therapy, wes of tumor was performed and the findings were consistent with hgg. treatment was transitioned to temodar and lomustine after hgg diagnosis was given. she had ongoing progressive disease despite this therapy as well as trials of nivolumab, everolimus, and vorinostat. neither patient had extraneural metastasis at presentation. in both tumors, whole exome sequencing identified the h3f3a k27m mutation. both tumors also had additional known mutations associated with hgg but no other overlapping mutations. this case series represents the first description of the genetic alterations of pediatric hgg patients who developed extraneural metastases. while h3f3a k27m is a common mutation in pediatric midline hgg, especially dipg, and is associated with more aggressive disease, there has not been an association with extraneural metastasis prior to this series. background: deferiprone-induced agranulocytosis is a well -known albeit rare side effect of the drug. incidence of agranulocytosis varies from 0.5-3.6%, while milder neutropenia is reported in 8.5% of patients treated with deferiprone. deferasirox is unknown to cause such a complication. clinical trials and post marketing side effect monitoring studied possible correlations between different risk factors and development of agranulocytosis. unfortunately, no studies directly addressed a special risk in a community with background of ethnic neutropenia, like oman. objectives: to report on the incidence of neutropenia among omani children with b thalassemia using different iron chelators design/method: a retrospective study conducted on patients < 21 year-old with b thalassemia treated with different iron chelators. electronic patients records were reviewed to detect episodes of neutropenia either mild (anc 1.0-<1.5/cmm), moderate (anc 0.5-<1), severe < 0.5, or agranulocytosis anc = 0). data were collected including sex, age, personal or family history of ethnic neutropenia, iron chelating agent, infective complications, management and outcome. detailed clinical, laboratory ± radiological information were reported for patients who developed life-threatening agranulocytosis. among 179 young patients with b thalassemia, treated between 2007-2017 in squh, neutropenia, was reported in 78 patients (43.6%).severe neutropenia was encountered on 14 occasions in 11 patients (11/179: 6.1%) (8 on deferiprone including 5 episodes of agranulocytosis, 1 on defersirox, 1 on combined chelation, and 5 off chelation). moderate neutropenia was encountered in 29 patients (29/179: 16.2%), on 36 occasions: deferiprone (15), deferasirox (8), combined chelation (4), and 9 episodes off chelation. mild neutropenia was more prevalent, encountered in 59 patients (32.9%) on 124 occasions (30 on deferiprone, 44 on defersirox, 19 on combined chelation, and 31 off chelation) of 85 patients exposed to deferiprone, 35 patients had neutropenia (41%), higher than previously reported. deferiproneinduced agranulocytosis was encountered in 4 patients (4/85 = 4.7%). three of them had life threatening complications. one patient developed pneumonia complicated by rupture of pulmonary artery aneurysm-massive hemoptysis, who recovered fully after catheter embolization. the second had facial cellulitis and treatment with gcsf was complicated by frequent ventricular extrasystoles. the third had sepsis, disseminated herpes simplex and required admission to icu for inotropic support. in a community with background ethnic neutropenia, neutropenia is more common to be encountered among thalassemic patients, both on and off chelation therapy. careful monitoring of anc and rational choice/modification of chelating agents is required for optimal management of iron overload and to avoid life threatening complications. objectives: this case control study aimed to evaluate the systolic and diastolic cardiac function in 2 groups of children with ti: non transfused group and a group that received early regular blood transfusion comparing them to healthy controls. design/method: thirteen regularly transfused patients with ti with a mean age of 11.8+5.6 years were compared with eight patients who are non-transfused or minimally transfused (< 3 rbcs transfusion/year); mean age 11.8+9.4 years and 18 healthy controls with a mean age of 8.8 ± 3.9 years. clinical parameters and standard echocardiographic and tissue doppler imaging (tdi) were compared. results: young non-transfused ti patients had a statistically significant higher peak late diastolic velocity of the left ventricular inflow doppler, a mitral valve a wave duration over the pulmonary vein a wave duration ratio and the pulmonary s81 of s301 vein s/d velocities ratio compared to the transfused group with p values of 0.028, 0.01, 0.01 respectively. in addition, they have a lower e/a ratio of the mitral valve inflow and a larger left atrial to aortic diameter ratio compared to the control group with p values of 0.025 and 0.01 respectively. the diameters of the right and left outflow tract were significantly larger in the non transfused group with a trend to have a higher cardiac index compare to the transfused group. systolic function was similar in the 3 studied groups and none of the patients had evidence of pulmonary hypertension. young patients with ti who are receiving early regular blood transfusion have normal systolic function. diastolic function assessment revealed indicators of an abnormal relaxation of the left ventricle in the non transfused group which indicate diastolic dysfunction. the abnormalities affected multiple diastolic function parameters which give an indication that the changes are clinically significant. a statistically significant increase in the diameters of the outflow tracts are likely attributed to high cardiac output status in nontransfused ti patients as they had a trend to have a higher cardiac index. these findings support the early commencing of regular blood transfusion therapy for ti patients to prevent serious cardiac complications in adult life. background: in the 52-week sustain study, crizanlizumab 5.0 mg/kg significantly reduced the frequency of scpcs versus placebo (1.6 vs 3.0, p = 0.01) and increased the time to first on-treatment scpc (4.1 vs 1.4 months, p = 0.001) in patients with sickle cell disease (scd). to evaluate time to first scpc in sustain study subgroups and the likelihood of not experiencing scpc for the duration of the trial using post hoc analyses. design/method: sustain was a randomized, double-blind, placebo-controlled, phase 2 study (nct01895361). inclusion criteria were: scd patients aged 16-65 years; 2-10 scpcs in previous 12 months; concomitant hydroxyurea use permitted if ≥6 months and stable dose for ≥3 months. patients were randomized 1:1:1 to receive intravenous crizanlizumab 5.0 mg/kg, 2.5 mg/kg, or placebo. study treatments were administered on days 1 and 15, then every 4 weeks to week 50, with the final assessment at week 52. median time to first scpc after first dose was summarized for crizanlizumab 5.0 mg/kg or placebo in these subgroups: 2-4 or 5-10 scpcs in previous 12 months; scd genotype; and hydroxyurea use at baseline. hazard ratios (hrs) for crizanlizumab 5.0 mg/kg versus placebo were calculated based on cox regression analysis, with treatment as a covariate. descriptive statistics were used to summarize the frequency of patients who were scpc event-free for the duration of the study by prior scpc events, scd genotype, and hydroxyurea use at baseline. : 67 patients received crizanlizumab 5.0 mg/kg and 65 received placebo. there was a meaningful delay in time to first scpc with crizanlizumab 5.0 mg/kg versus placebo observed in the entire study population. the effect was present in both scpc subgroups, and the largest treatment difference was observed in hbss scd versus other genotypes (4.1 vs 1.1 months; hr: 0.50). in patients taking hydroxyurea who experienced 2-10 scpcs in the previous year, time to first onstudy scpc was longer with crizanlizumab 5.0 mg/kg versus placebo (2.4 vs 1.2 months; hr: 0.58). a greater proportion of patients treated with crizanlizumab 5.0 mg/kg were scpc event-free versus placebo in each of the analyzed subgroups. one third of patients who were taking hydroxyurea and treated with crizanlizumab 5.0 mg/kg were scpc event-free during the study versus 17.5% with placebo, possibly suggesting an additive effect. with crizanlizumab 5.0 mg/kg, there was a clinically meaningful delay in time to first scpc and an increased likelihood of being scpc-free versus placebo in all subgroups investigated. cincinnati children's hospital medical center, cincinnati, ohio, united states background: shwachman-diamond syndrome (sds) is an inherited marrow failure syndrome associated with increased risk of myelodysplasia (mds) and acute myeloid leukemia (aml). objectives: this multi-institutional retrospective study investigated clinical features, treatment, and outcomes of 38 sds patients who developed mds or aml by central pathology review. design/method: nine individuals presented with aml (4 male, 5 female), 5 mds-eb1/2 (3 males, 2 females, 23 with mds (11 male and 12 female), and one male with isolated persistent somatic tp53 mutation. one mds-eb1 and 1 mds patient progressed to aml. median age (years) at diagnosis of mds was 16 (range 0.5-30), mds-eb1/2 was 9 (range 0.7-20) and aml was 28.8 (range 5.5-47). complex cytogenetics were noted in 10/11 aml cases, with one having normal cytogenetics. complex clonal cytogenetic abnormalities were noted in 4 of 5 mds-eb1/eb2 patients and clonal abnormalities in 17 of 18 mds patients. follow up was available for 10 aml patients; 9 are deceased. 9 received chemotherapy with intent to proceed to hematopoietic stem cell transplant (hsct). four failed to achieve remission and died with disease without proceeding to transplant. one patient proceeded to hsct without prior chemotherapy. four of six transplanted subjects died with relapsed disease. treatment related mortality was largely infectious or gvhd. the sole surviving aml patient had normal cytogenetics, achieved remission with chemotherapy and underwent hscts with 3 separate stem cell infusions due to two primary graft failures. he remains alive in remission more than 4 years after diagnosis. of the 5 mds-eb1/2 patients, 4 underwent ric hsct, three of whom are alive, one died of infection. the fifth patient has stable disease on continued decitabine monotherapy for 4.75 years. of 19 mds patients with treatment data, 13 had upfront hsct therapy, 2 upfront chemotherapy and 4 had no therapy. three patients required ≥2 hscts all due to graft failure. follow up is available for 18, 11 of whom are deceased, 6 with relapsed disease. treatment related mortality was largely infectious or graft failure. one individual died of hepatic failure unrelated to mds. seven mds patients are alive in remission. in summary, prognosis is poor for patients with sds who develop aml due to resistant disease and treatment-related complications. better markers for risk stratification are needed to identify patients who would benefit from early transplant. novel therapeutic strategies are urgently needed to improve outcomes of sds patients with mds or aml. background: unlike primary myelofibrosis (pmf) in adults, which is associated with somatic mutations in jak2, mpl, or calr, myelofibrosis in children is rare and the underlying genetic mechanisms remain elusive. here we describe 3 families with autosomal recessive congenital macrothrombocytopenia with focal myelofibrosis (cmtfm) due to germline mutations in the megakaryocyte-specific immune receptor tyrosine-based inhibitory motif (itim) receptor g6b-b. objectives: to characterize the clinical phenotype, histological features and identify the causative gene for cmtfm. we performed affymetrix snp 6.0 genotyping on the index family to identify shared regions of homozygosity by descent. whole exome sequencing (ws) was performed on all three pedigrees to identify potentially causative mutations. we studied 6 affected children from 3 families, with macrothrombocytopenia, anemia, mild leukocytosis and a distinctive pattern of bone marrow (bm) fibrosis centered around clusters of atypical megakaryocytes. affected children had mild to moderate bleeding symptoms and required platelet and red cell transfusions. none showed evidence of extramedullary hematopoiesis, and all were negative for mutations in jak2, mpl, and calr. snp genotyping identified multiple statistically non-significant genomic loci, including the region of the major histocompatibility locus (mhc) on chromosome 6p (lod = 2.01). we focused on this region because affected individuals in two families shared a common homozygous human leukocyte antigen (hla) type and had congenital adrenal hyperplasia (cah) due to 21-hydroxylase (cyp21a2) mutation; the cyp21a2 and hla loci are located at 6p21.33 and 6p21.32-6p22.1. wes revealed homozygous frameshift mutations in the megakaryocyte and platelet inhibitory receptor g6b-b, encoded within the candidate linkage region. we identified two distinct g6b-b frameshift mutations (c.61_61+1dup; p.20fs and c.147inst; p.49fs) in 7 individuals within these three families. no other mutations that segregated with the phenotype were identified. to validate g6b-b as a potential disease-causing gene, we evaluated g6b-b expression in bm biopsy specimens from affected patient and control samples by immunohistochemical staining using a monoclonal antibody. g6b-b was strongly s83 of s301 and selectively expressed in megakaryocytes of control samples, but completely absent in clinically affected individuals. a murine knockout that lacks g6b-b has a strikingly similar phenotype with macrothrombocytopenia, myelofibrosis and aberrant platelet production and function, further affirming the causality of g6b-b mutations. we showed that autosomal recessive loss-offunction mutations in g6b-b cause cmtfm, uncovering the molecular basis of this rare disease. loss of g6b-b-dependent inhibition of megakaryocyte activation likely underlies the distinctive focal myelofibrotic phenotype and might be important in other forms of marrow fibrosis. cardinal glennon, saint louis, missouri, united states background: intrauterine transfusion is the method of choice for management of fetal anemia due to red blood cell alloimmunization. despite the decrease in prevalence of anemia due to rhesus d alloimmunization with prophylactic administration of anti-rhd immunoglobulin in rh d negative patients, maternal red red blood cell alloimmunization with other type of red blood cell antigens remains an important cause of fetal anemia. newborn who received intrauterine transfusion for hemolytic disease may have prolonged postnatal transfusion requirement. objectives: 1-to evaluate clinical outcome of fetuses and newborns who received intrauterine transfusions. 2-to determine the need of packed red blood cell transfusions until 6 months of age. we conducted a retrospective case series study of all intrauterine transfusions due to anemia secondary to red blood cell alloimmunization performed in our regional center ssm in st louis missouri, between april 2011 and january 2016. we evaluated the indications, diagnosis, gestational age, and frequency of intrauterine transfusions, along with the infant's gestational age at birth, duration of admission, timing of blood transfusion and monitoring of hemoglobin. results: 37 intrauterine transfusions were performed in 14 patients. the most common causes of alloimmunization were due to d antibodies (n = 7, 50%) and kell antibodies (n = 5, 35.7%). the median gestational age of the first intrauterine transfusion was 28.1 weeks, and the median pre-transfusion hemoglobin was 8.9 g/dl. the gestational age at the first intrauterine transfusions was found to be significantly correlated with the number of postnatal transfusions (r = 0.8. p = 0.001). the median gestational age at birth was found to be 35 weeks (28.6-36.9 weeks), with a hemoglobin of 13.1 (10.8-14.1). in our population, 6 patients (42%) received postnatal transfusions, of which 4 were during the first 3 weeks of life, and close monitoring follow up with a hematologist was established in 6 patients at their discharge from the nursery/nicu. one neonatal death occurred and severe morbidity due to severe anemia occurred in one infant. despite the continuing risk factor for persistent anemia, only 8 patients had follow up hemoglobin monitored by their primary care provider. conclusion: infants with anemia due to red blood cell alloimmunization treated with intrauterine transfusion should be monitored closely via regular complete blood count for persistent anemia due to suppression of fetal erythropoiesis. sebastian hesse, piotr grabowski, juri rappsilber, christoph klein dr. von hauner childen's hospital, lmu university hospital, munich, munich, germany background: neutrophil granulocytes are the most abundant leukocytes in the peripheral blood. validated diagnostic options for these cells are limited, leaving many patients with functional neutrophil defects without a defined diagnosis. objectives: here we evaluate proteomics as a new diagnostic tool to investigate defects of neutrophil granulocytes. we analyzed neutrophil granulocytes from 6 children with severe congenital neutropenia (scn) associated with elane mutations, 4 children with chronic granulomatous disease (cgd) with cyba (2) or cybb (2) mutations and 2 children with leukocyte adhesion deficiency (lad) due to itgb2 mutations. in addition we collected samples of children with genetically undetermined neutrophil defects. neutrophils from 68 healthy individuals served as controls. cells were isolated from fresh venous blood using negative selection (purity >99%). whole cell proteome analysis was done by data-independent acquisition. showed a correlation coefficient of ∼0.9. principal component analysis demonstrated unequivocal separation of the proteome of healthy and diseased cells. differential expression analysis showed minimal proteome aberrations in lad with deficiency in cell surface receptors and upregulation of alpl (total downregulated proteins: 7/ total upregulated proteins: 5). analysis of neutrophils from cgd patients also showed limited proteome aberration. cyba and cybb were both diminished independent of genotype, whereas protein clusters around a stat1/2 centered network were increased (total down: 11/ up: 23). neutrophils with elane mutations showed the gravest proteome disturbance (total down: 47/ up: 93) with an upregulated translational apparatus (srpdependent ribosomes and protein folding complexes) and increased mitochondrial proteins. proteins of each granule subset were dysregulated and metabolic pathways upregulated. a detailed analysis of the proteome from patients with genetically undefined diseases is currently ongoing. one patient with clinical phenotype of cgd was found to have no mutations of nadph oxidase members in whole exome sequencing but critically low levels of ncf1 on protein level. heterozygosity mapping showed autozytocity in the ncf1 region warranting current efforts to sequence promoters and intronic regions of the gene. mass spectrometry based proteomics promises exciting new insights into monogenic disease of neutrophil granulocytes and may offer new diagnostic options, in particular in synergy with genome sequencing. by virtue of our international care-for-rare alliance, open to new partners, we hope that our proteome focus may lead to better delineation of as yet unknown disease of neutrophil granulocytes. background: warm autoimmune hemolytic anemia (aiha) is an igg mediated disease. although it can be post-viral, it is often idiopathic and can also be a forme fruste for malignancy or an autoimmune disease. initial management includes steroids. it often relapses on steroid wean and can be refractory to the use of second line treatment such as rituximab. objectives: abatacept (ctla-4-ig fusion protein, ctla-4 mimetic) has been used to ameliorate autoimmune manifestation associated with ctla-4 haploinsufficiency. we used abatacept as a novel therapeutic agent to manage patients with refractory aiha. design/method: a retrospective case series of two patients at phoenix children's hospital with severe refractory aiha. results: patient 1, a previously healthy 12 year old female, presented with 8 weeks of icterus, fatigue, and hemoglobinuria. spleen was enlarged 8cm below the costal margin. laboratory evaluation demonstrated: hemoglobin 3.8 g/dl, mild leukopenia 3200/microliter, platelets 99,000/microliter, reticulocytosis 14.3%, positive direct coombs' test, mycoplasma igm and igg positive. bone marrow evaluation showed a hypercellular marrow. she continued to need packed red blood cell (prbc) transfusions despite receiving high dose steroids, ivig and rituximab from may-july 2017. in august, she started sirolimus decreasing her transfusion requirement. after starting abatacept (10mg/kg/dose bi-monthly for three doses and then monthly) in october, she maintained hemoglobin of 9-10 g/dl without transfusion. patient 2, a previously healthy 2 month old male, presented with one week of progressive fatigue, jaundice, and poor feeding. splenomegaly was absent. laboratory evaluation revealed hemoglobin 3.8g/dl, leukocytosis 20,200/microliter, platelets 324,000/microliter, reticulocytosis 16.1%, negative direct coombs' test, and non-specific reactivity on antibody screen. evaluation for inherited hemolytic anemia including a next generation sequencing panel was negative. further evaluation by blood bank showed 2+ positive coombs' for c3d due to a warm antibody. cold agglutinin disease was ruled out. bone marrow evaluation was normal. he received high dose ivig as a steroid sparing agent but continued to require prbc transfusions weekly. when prednisone did not seem to slow down hemolysis, treatment with abatacept was initiated and he has not required transfusions for two months. steroids are being weaned. we present successful treatment of two refractory aiha cases with abatacept. patient 1 is steroid and transfusion free and continues on monthly abatacept and sirolimus. patient 2 is also transfusion free and continues on a steroid taper. ctla-4 is crucial for suppressive function of treg cells. abatacept by binding to cd80/86 seems to enhance treg activity ameliorating autoimmune hemolysis. children's minnesota, minneapolis, minnesota, united states s85 of s301 background: transfusional iron overload is common in patients receiving chronic red cell transfusions. as a result, iron chelation is required to minimize toxicity from iron overload. chelation with a single agent can be inadequate at controlling or reducing iron burden. when combination therapy is required deferoxamine may be added to oral chelation. deferoxamine is generally given subcutaneous over 8-12 hours for 5-6 days a week at 25-60mg/kg/day. many patients struggle to remain compliant with this schedule which has prompted trials of intravenous high-dose (hd) deferoxamine. prior reports of short-term hd deferoxamine have shown minimal side effects however, prolonged use of hd deferoxamine has known toxicity. when compliance is a concern, our center has used hd deferoxamine infusions at 15mg/kg/hr x 48hours every 2 to 4 weeks. objectives: evaluate the safety and efficacy of hd deferoxamine at our institution to help guide future therapy. design/method: a retrospective review was completed of patients previously treated with hd deferoxamine between april 2011 and september 2017 at children's minnesota. final sample included 8 patients ages 3 to 14 years with underlying diagnosis of thalassemia (7) and diamond-blackfan anemia (1). deferoxamine infusions were given for 48 hours every 24-35 days with a mean length of treatment of 279 days. results: all patients were on combination therapy with deferasirox, however deferasirox was held during deferoxamine infusion. mean pre-deferoxamine liver iron concentration (lic) was 31.75mg/g and mean post lic was 12.11mg/g (p = 0.0008). ferritin mean pre-deferoxamine was 2677ng/ml compared with mean post 1594ng/ml (p = 0.0107). two patients had possible allergy, leading to deferoxamine discontinuation. one patient developed hives, eye swelling and cough while the other had emesis and cough. another patient experienced facial nerve palsy of unclear etiology, which did not recur with resumption of deferoxamine. no respiratory complications were seen. results showed significant decrease in iron burden following combination therapy with high dose deferoxamine and deferasirox. no significant pulmonary, liver, renal, vision, or hearing toxicities were observed. three patients reported reactions to deferoxamine infusions. however, one of these was able to successfully continue deferoxamine without further incident. short-term, hd deferoxamine was effective at reducing lic in combination with oral chelation but requires further evaluation to assess for potential increased risk of toxicity. short-term hd deferoxamine may be considered in the setting of poor compliance of subcutaneous administration or inadequate chelation with single agent therapy. further studies are needed to clarify ideal dosing, timing and risk of toxicity. background: immune thrombocytopenia (itp) is the most common cause of symptomatic thrombocytopenia in childhood but remains a diagnosis of exclusion warranting further evaluation if atypical findings are present. two male children (15 months and 3 years old) with newly diagnosed immune thrombocytopenia (itp) were found on initial evaluation to have persistent elevations of lactate dehydrogenase (ldh), alanine aminotransferase (alt), and aspartate aminotransferase (ast). these serum enzyme abnormalities cannot be attributed to itp. in the setting of thrombocytopenia, elevated transaminases and ldh create diagnostic complexity for the hematology/oncology provider as their elevation raises concern for malignancy, hemolytic disease, and other systemic diseases. to raise awareness about an unexpected pattern of duchenne muscular dystrophy in patients undergoing evaluation for itp. to expand the differential of a hematologist/oncologist when abnormal labs support a nonhematologic diagnosis design/method: this case-series of two patients with their clinical and laboratory findings were discovered with retrospective chart review. results: after a thorough evaluation for hemolytic anemias, liver disease and infectious etiologies was negative, bone marrow and liver biopsies were considered. eventually, both children were found to have severely elevated serum creatine kinase (ck). skeletal muscle has the highest concentration of ck of any tissue. thus, significant ck elevation is almost exclusively attributable to muscle injury and is the most sensitive and specific enzyme for diagnosis of muscle disease. referral to a neuromuscular specialist and further genetic testing confirmed the diagnosis of duchenne muscular dystrophy in both children allowing initiation of appropriate interventions. to date, there is no clear genetic predisposition to itp in patients with muscular dystrophy although further investigation may be needed. hematology/oncology providers should consider obtaining a serum ck to rule out muscle disease in any male child with unexplained elevations of serum ldh and/or aminotransferases, as it provides an easy and inexpensive, non-invasive approach to screening. additionally, clinical history and physical examination can aid in the diagnosis of muscular dystrophy, with gross motor delay, abnormal muscle bulk, gower's sign, and proximal muscle weakness all possible findings. objectives: to identify the range of cbcs in patients with ds without infections, hematologic or immune disorders and to create more accurate reference ranges for total white blood count; hemoglobin; hematocrit; mcv; platelet count and absolute neutrophils (anc), lymphocytes, monocytes, eosinophils, and basophils. design/method: a retrospective investigation of healthy pediatric patients with ds who received a cbc between 2011 and 2017 as part of their medical care at a single, large, pediatric teaching hospital. the study group consisted of 562 children with ds (male = 310, 55.2%; mean age = 2.10 years, sd = 3.17) at time of blood draw. initially 692 children were reviewed for possible participation in the study; however, 130 patients were excluded due to not meeting the study's inclusion criteria. descriptive statistics were performed on demographic and clinical characteristics. kruskal-wallis h tests, anova, and t-tests were run to determine the significant associations between independent means. results: a significant difference in absolute neutrophils between racial groups, f(2, 183.553) = 3.990, p = 0.020, was observed. there was an increase in anc from 2.5 +/-1.1 with african americans to 3.0 +/-1.6 in the other racial groups and to 2.9 +/-2.0 with caucasians. differences were also found in anc in hispanics/latinos versus non-hispanic/latinos. the results were higher in non-hispanics and latinos, a significant difference of -.584 (95% ci, -.791 to -.376), t(1255) = 2.572, p = 0.001. preliminary kruskal-wallis h tests run determined that there were significant differences between age groups for total white blood cell, hemoglobin, hematocrit, platelets, lymphocytes and anc. further studies are being run to evaluate in which age groups these differences lie and create reference ranges by age, race and sex. conclusion: among patients with ds, there are differences between racial groups and age groups. this data has been compared to previously established reference ranges for cbcs, but we are currently establishing healthy cbc controls which we will use to validate the reference ranges. these ranges will be published to help guide providers in workup and management of patients with ds. background: transfusion is a critical part of the care provided in the neonatal intensive care unit, but it is not without risks. low birth weight and premature infants can become anaemic from an immature haematopoietic system and frequent phlebotomy. these infants often receive multiple red blood cell transfusions. identifying infants more likely to require such intervention is important in ensuring the appropriate usage of this scarce resource. to determine whether birth weight, gestational age, gender, length of stay and mode of delivery can predict red cell concentrate (rcc) transfusion, units required, donor exposure and time to exposure. design/method: a retrospective chart review of all infants born below 32 weeks gestation and/or birth weight less than 1,500g who received a red blood cell transfusion between july 2009 and july 2012 in the cork university maternity hospital neonatal unit. results: 224 infants met the inclusion criteria, 105 (46.87%) received a rcc transfusion. our study showed lower gestational age (p<0.01) and lower birth weight (p<0.01) infants are more likely to be transfused. donor exposure increases with a lower birth weight (p = 0.016). multivariate analysis showed infants with a lower gestational age (or -0.019 per day; p<0.05); lower birth weight (or -0.002 per 1g; p<0.01) and a longer length of stay (or 0.016 per day; p<0.05) are more likely to receive a higher number of rcc transfusions. the time to first rcc transfusion is shorter in those with lower birth weight (or 0.013 per 1g; p<0.05) and lower gestational age (or 0.356 per day; p<0.01). gender and mode of delivery were not found to be predictors of red blood cell transfusion in this study. conclusion: low birth weight and premature infants are more likely to receive a rcc transfusion during admission to the neonatal unit. our study highlights predictors of rcc transfusion, donor exposure and time to transfusion. these can be used in identifying at risk infants, counselling parents and in anticipating transfusion requirements. emily southard, r. grant rowe, david williams, akiko shimamura, taizo nakano children's hospital colorado, aurora, colorado, united states background: the mecom locus encodes transcription factors that regulate hematopoietic stem cell self-renewal and maintenance. overexpression of mecom has been noted in 5-10% of acute myeloid leukemia, several solid tumors, and denotes a poor prognosis. mutations that reduce mecom expression or that disrupt protein function, however, have been implicated in the development of bone marrow failure (bmf) through undefined pathways. an association between mecom mutations and radioulnar synostosis with amegakaryocytic thrombocytopenia (rusat) syndrome has been reported, however further characterization of this phenotype has yet to be explored. to characterize the phenotypic spectrum of a cohort of pediatric patients with novel mecom mutations. we performed a retrospective review of five patients with mecom mutations who were referred to hematology at children's hospital colorado or boston children's hospital. clinical, laboratory, and genetic data was collected on subjects and available family members. results: four of 5 subjects were identified in infancy presenting with congenital cytopenias or physical dysmorphisms that prompted broad genetic screening. platforms for genetic detection included microarray, targeted genetic panels, and whole exome sequencing. three of 4 subjects with cytopenias presented with congenital thrombocytopenia, 1 of whom rapidly progressed to severe aplastic anemia. four of 5 subjects presented with congenital anomalies, 3 of whom demonstrated radioulnar synostosis. additional dysmorphic features identified include craniofacial (low set ears x1), cardiac (pda x1, vsd x1, aortic root dilation x1), pulmonary (pulmonary hypertension x1, arteriovenous malformations x1), and developmental delay. one subject presented at age 13 years with acute pancytopenia, hypocellular marrow, no dysmorphisms, and a mecom variant of unknown signif-icance. the identified mecom mutations include one 4.1 mb deletion involving several genes including mecom, one variant affecting a splice acceptor consensus sequence predicted to disrupt splicing, and three novel missense mutations, tyr949cys, arg950thr, and tyr1118cys, all of which were absent from public databases and were predicted in silico to be deleterious. we describe the phenotypic spectrum of 5 patients with novel mecom variants. a subset of patients lacked radio-ulnar synostosis and had presence of additional systemic anomalies, demonstrating a varied clinical phenotype that is not isolated to rusat syndrome. a centralized publically accessible database to share clinically annotated mecom variants, together with analysis by experts in mecom function would advance our understanding of the clinical interpretation of mecom variants. mecom should be considered in the differential diagnosis of bone marrow failure and we advocate for the inclusion of mecom in targeted sequencing panels. cairo university, cairo, egypt background: beta thalassemia is regarded as a serious public health problem in the mediterranean region, southeast asia, and the middle east. however, very few studies have been conducted to assess the quality of life (qol) among thalassemia major patients. objectives: to assess the quality of life among b-thalassemia major patients using short form (sf)-36 questionnaire and to determine the factors associated with their quality of life. design/method: a cross-sectional study was conducted among thalassemia major patients who were attending the hematology outpatient clinic at cairo university hospital, during the study period. data were collected between october 2016 and march 2017. the quality of life was assessed for patients aged ≥17 years. the mean age of the studied group was 18.32± 1.33 years. the majority (93.63%) had one monthly blood transfusion. the mean total score of sf-36 was 44.90±7.54. general health perception domain was the most affected domain with mean score, while vitality was the least affected one. there was no statistically significant difference between males and females regarding different quality domains except for vitality where the mean score was significantly higher in males than females (p = 0.05). age at onset of disease, and at first blood transfusion were the most documented factors positively correlated with the quality of life among the enrolled thalassemia patients. conclusion: the quality of life in thalassemia major patient was found to be compromised. all thalassemia patients should undergo assessment of the quality of life so that interventions focusing on the affected domains can be implemented. background: international adoption of children with special needs has become more prevalent in recent years leading to tremendous growth in the number of u.s. thalassemia patients adopted from foreign countries. currently 13% of the 1,119 thalassemia patients registered in the cooley's anemia foundation (caf) patient database have been adopted from foreign countries, primarily china. as this population continues to grow, further information is needed in order to provide these families with best supportive care. the primary goal of this study is to characterize the socio-demographics and health statuses of adopted children with thalassemia and their families. a secondary goal is to describe adoptive families' motivations, experiences, challenges, and support resources. design/method: a redcap survey was accessed by families of adopted children with thalassemia through the caf website and caf social media from january to august 2017. following a four-question screen, eligible subjects were directed to complete an adoption questionnaire. families who had at least one adopted child with thalassemia receiving care at a participating thalassemia treatment center or hematology office in the u.s. were considered eligible. descriptive statistics were analyzed using sas 9.4. respondents who were ineligible or who provided incomplete data were removed from the dataset prior to analysis. of 78 survey respondents, 67 qualified and completed the survey. these households had adopted a total of 74 children with thalassemia (33.8% male), most from china (93.2%), where they had been living in orphanages (73.0%). legal guardians identified primarily as christian (87.3%). the majority had completed post-secondary education (76.9%) with reported household incomes greater than $80,000 (76.1%). most adoptive families were connected to an adoption group or community including online groups, local support groups, and adoption networks (98.5%). commonly cited challenges were: 1) volume of frequent medical appointments, 2) insufficient support from their local care centers, and 3) financial burdens. the reality of care for the population of adopted patients with thalassemia in the u.s does not seem to match the expectations set by their providers. we are hopeful this data will be used to assist adoptive families navigating the complexities of thalassemia care. the findings suggest that this population would benefit from additional outreach, education, guidance, and advocacy resources -especially in the early stages of adoption and during initiation of post-adoption medical care. background: in many higher-income countries, thalassemia major has become a chronic disorder; many outcomes are different in emerging countries with more limited resources. most analyzes of health-related quality of life (qofl) in thalassemia have been conducted in high-income settings. objectives: to assess the impact of health status on qofl in thalassemia patients in an emerging country. we assessed qofl in 110 randomly-selected patients (72 thalassemia major; 33 with hemoglobin e thalassemia; five thalassemia intermedia) at the national thalassemia center in kurunegala, sri lanka where approximately 800 patients are managed. treatment is free, but compared to north america/europe, access to tertiary staff and other resources are limited. overall, control of body iron as estimated by serum ferritin concentration (mean± sem, 2906±267 g/l) was not optimal in many patients. to understand the impact of health status on qofl, we used the sf36v2 health survey, analyzing scores of physical function, pain, general health, social functioning, emotional and mental health, to generate overall physical and mental component scores. results: compared to reports from higher-income countries (american journal of hematology 2011; 86:92-5), physical function scores (mean±sd, 48.16±10.72) were similar in sri lankan patients; indeed, in three categories (physical role, social function, emotional role), sri lankan scores were slightly higher. by contrast, compared to scores from higherincome settings, those estimating bodily pain, general health, and mental health were significantly lower, resulting overall in a significantly lower physical component score in sri lankan patients. male sri lankan patients reported higher scores than females, and somewhat surprisingly, in four categories (physical function, physical role, social function and emotional role) reported higher scores than those obtained in higher-income settings. lower scores in physical functioning, leading to an overall lower physical component score, were recorded by females. patients with hemoglobin e thalassemia reported generally poorer qofl than those with thalassemia major. the lack of differences in qofl in patients with "high" and "low" hemoglobins was likely related to low pre-transfusion hbs (mean±sem, 8.31 ± 0.14 g/dl) in nearly all patients. these early data in a small cohort of thalassemia patients in an emerging setting suggest that in many patients bodily pain, reduced mental health, and poorer views of general health affect overall qofl. prospective studies in larger cohorts including evaluation of adequacy of transfusions and chelation therapy, complications, and overall accessibility of care may guide approaches to improve qofl in lower-income settings of thalassemia care. geetanjali bora, anand prakash dubey, tarun sekhri, mammen puliyel, aparna roy maulana azad medical college, new delhi, delhi, india background: in the last two decades, the presence of osteopenia has been described in optimally treated patients with transfusion dependent thalassemia, the pathogenesis of which seems to differ from osteopenia in non-transfused patients. the prevalence rate of low bone mineral density (bmd) in pediatric population is highly variable amongst studies done worldwide. furthermore, the role of metabolic and endocrine factors in determining bone mass in this population is not well understood. objectives: to assess bmd in subjects with transfusion dependent beta thalassemia by dual-energy-x rayabsorptiometry and find its co-relation with clinical, biochemical and hematological parameters. design/method: this is a comparative cross-sectional study and includes patients with transfusion dependent beta thalassemia between ages 6 to 16 years enrolled from a thalassemia day care center in the year 2012 -2013. at the time of enrollment age, sex, bmi z scores, pubertal staging, duration and type of chelation therapy were noted. enrolled subjects were scanned for bmd at lumbar spine l2-3 and left femoral neck using dexa scan. the bmd was expressed in mean values and z scores. age, bmi, ethnicity and gender matched historic controls were used to generate z scores. 5 ml of pre transfusion fasting venous blood samples were obtained to test for serum calcium, phosphate, alkaline phosphatase, pth, thyroid function panel, serum ferritin and serum igf-1 levels. mean values for pretransfusion hemoglobin and serum ferritin over last 12 months were calculated. results: total no of subjects 50, median age 11.6 years, male 32 (64%), female 18 (36%), ethnicity 100 % asian, bmi < 3rd centile 13 (26%), pre pubertal 50%, all receiving transfusion and chelation therapy. prevalence of low (z score < -1 sd) and very low (< -2.5 sd) bmd was 74%, 52 % at l1-l2 respectively and 66 %, 24% at left femoral neck respectively. there was trend of lower bmd z scores with advancing age. statistically significant co-relation (p value < 0.05) was found between low bmd and low mean pretransfusion hemoglobin, serum phosphate, igf -1 and vitamin d levels conclusion: a sizable proportion of children and adolescents with transfusion dependent thalassemia have suboptimal bone mineral density and this decline may start as early as 6-7 years of age despite being on transfusion regimen highlighting the importance of yearly dexa screening and optimization of pre-transfusion hemoglobin, vitamin d and igf 1 levels. vanderbilt university medical center, nashville, tennessee, united states background: it is well described that iron deficiency anemia (ida) can co-present with thrombocytosis or thrombocytopenia, though cases of thrombocytopenia are less frequent than thrombocytosis. prior reports of thrombocytopenia have included adult and pediatric patients with menorrhagia (1-2), menorrhagia due to uterine fibroids (3), or other gynecologic abnormalities (4). our cases highlight the pattern of ida, thrombocytopenia, and menorrhagia in the setting of significant menstrual clotting without observed gynecologic abnormalities in african-american adolescents. objectives: to describe the clinical course of three adolescent females with severe ida, menorrhagia, and thrombocytopenia. results: our cases included three female african-american patients ages 12-17 who presented with severe anemia and concurrent thrombocytopenia in the setting of menorrhagia. all three patients reported heavy and prolonged menstrual cycle bleeding with significant clots. two of the three were admitted for transfusions at presentation and noted to have significant menstrual bleeding with continued blood loss requiring additional transfusions until bleeding was controlled with estrogen therapy. these two patients were evaluated with pelvic ultrasounds revealing a prominent endometrium in both patients and hyperechoic material consistent with a clot in one patient. average hemoglobin on presentation was 4.3 gm/dl (2.8-6.5), average platelet count was 70,000/mcl (18,000-99,000), and average mcv was 63 (54-68). all had severe iron deficiency with an average ferritin of 4 ng/ml (2-7) subsequently treated with oral iron. one patient had a prior history of ida that required transfusion and had subsequent normalization of her complete blood count. two patients had subsequent thrombocytosis before normalization of their platelet counts. two patients received platelet transfusions: one due to recent neurosurgical intervention with a higher goal platelet count and the other to help control menstrual bleeding after a nadir platelet count of 9,000. a review of the clinical history and red cell indices pointed to ida and ongoing blood loss from menorrhagia as the reason for the bicytopenias. the thrombocytopenia in these cases may have been exacerbated by consumption of platelets in the significant clots all three patients reported. it is reasonable to treat with iron supplementation and supportive care which may include transfusions or management of menorrhagia with oral contraceptives or other hormonal methods. background: sickle cell disease is one of the most common inherited red blood cell disorders, yet many are not aware of their carrier status. the american college of obstetricians and gynecologists' guidelines recommend that pregnant women of african, mediterranean and southeast asian descent be screened for hemoglobinopathies with a cbc and hemoglobin electrophoresis1. however, adherence to this practice and frequency of improper screening with sickledex is unknown. proper screening and counseling can impact families' knowledge, allowing for establishing relationships with pediatric hematology providers earlier. objectives: we sought to assess prenatal hemoglobinopathy screening practice patterns and methods of obstetrics & gynecology (obgyn) and family medicine providers in the nyc regional area. design/method: a cross-sectional electronic survey was administered to obgyn and family medicine practitioners from four nyc institutions. questions focused on prenatal hemoglobinopathy screening practices using case scenarios with variations on parental trait status and ethnicities. chisquare analyses were used to compare the two provider groups on categorical variables. there were 98 total responses; 71 surveys were complete, of which 48 were obgyn and 23 family medicine providers. respondents were mainly from academic medical centers, with the majority being faculty (68% of the obgyns and 41% of family medicine). no significant difference was found in frequencies of screening patients with a positive family history of a hemoglobinopathy. when asked about screening practices for patients without a personal/family history of a hemoglobinopathy, 92% of obgyns versus 70% of family medicine providers "always" screened for hemoglobinpathies (p = 0.03). when analyzed by ethnic background, there were significant differences by group in screening patients of white (92% vs 70%), black (94% vs 74%), mediterranean (94% vs 74%), and asian descent (94% vs 70%) (p≤0.05 for all). however, in cases where the hemoglobinopathy carrier status of both parents was known, there was no difference in screening with a hemoglobin electrophoresis. furthermore, >20% of all respondents use sickledex for screening in the case scenarios. conclusion: this pilot survey highlights a difference in the methods and likelihood of prenatal hemoglobinopathy screening based on the type of prenatal care provider. screening differences can lead to variations in prenatal guidance, diagnostic procedures, informed decision-making and knowledge of families referred to pediatric hematology clinics. this is the first study analyzing prenatal screening for hemoglobinopathies in obgyn and family medicine. improving prenatal screening practices by collaborating with hematologists may decrease health care disparities and allow for earlier relationship building with pediatric hematology. 1. acog, opinion#691, 2017 poster # 220 hermansky-pudlak syndrome: spectrum in oman background: hermansky-pudlak syndrome (hps) is a rare autosomal recessive disorder, characterized by the triad of oculocutaneous albinism, a hemorrhagic diathesis resulting from storage pool-deficient platelets, and accumulation of ceroid/lipofuscin-like material in various tissues. before 2016, nine different types of hermansky-pudlak syndrome were identified, which can be distinguished by their signs and symptoms and underlying genetic cause. in 2016, a tenth type was defined based on mutations in the ap3d1 gene. hps type 2 is characterized in addition by severe neutropenia and recurrent sinopulmonary infection. the disease is more common in puerto rico, and this is the first report from oman. to describe the clinical, laboratory and genetic characteristics of hps sub-types in oman, including the first 2 cases of hps type 2. design/method: this is a retrospective study, including 7 cases with hps that had been suspected clinically and confirmed through genetic mutation analysis. clinical data included sex, age at presentation, initial clinical presentation (skin, eyes, development, neurological involvement, bleeding tendency, recurrent infections) and course of disease. laboratory data (complete blood counts, platelet and absolute neutrophil counts, coagulation screening, platelet function tests by platelet function analyzer, and platelet aggregation studies using different agonist had been recorded. pcr and next generation sequencing for genetic confirmation by testing mutations in hps1, ap3b1, hps3, hps4, hps5, hps6, dtnbp1,, bloc1s3, bloc1s6 genes had been done. results: seven omani cases with hps have been identified (4 males and 3 females). their age ranged between 0 (at birth) to 10 years. two patients had hps type 2, 1 patient had type 6, while the other 4 cases had hps type 3. no other sub-types were encountered in oman. all patients were products of consanguineous marriage. one patient had adrenal hge, while the others had mild hemorrhagic phenotype, characterized by recurrent bruising and mild epistaxis. laboratory testing confirmed variable platelet aggregation defects with different platelet agonists. all patients had characteristic hypopigmentation, iris transillumination, nystagmus, and foveal hypoplasia. both patients with hps type 2 had the same homozygous mutation in the ap3b1 gene (c.12_13delta), and presented with severe neutropenia. early diagnosis and initiation of gcsf on one of them improved outcome and prevented the development of complications. late diagnosis in the other patient resulted in the development of bronchiectasis as a result of recurrent sinopulmonary infections. background: sickle cell disease (scd), a genetic disorder characterized by defective sickle hemoglobin (hbs), triggers red blood cell sickling, hemolysis, vaso-occlusion, and inflammation. ischemic injury from scd starts in infancy and accumulates over a lifetime, causing pain, fatigue, and progressive end-organ damage that culminates in early mortality. voxelotor (gbt440) is an oral, once-daily therapy that modulates hemoglobin's oxygen affinity, thereby inhibiting hemoglobin polymerization. objectives: to assess the safety, pharmacokinetics, and efficacy of voxelotor in pediatric patients with scd. design/method: this ongoing study is being conducted in 2 parts: part a: a single dose of voxelotor 600 mg in pediatric and adolescent patients; part b: multiple doses of voxelotor 900 mg/d or 1500 mg/d for 24 weeks in adolescents. part b's primary objective is to assess the effect of voxelotor on modifying anemia. secondary objectives include measuring other markers of disease modification, such as hemolysis; daily scd symptoms, using a patient-reported outcome (pro) measure; and safety. results: as of november 6, 2017, 24 patients (10 females) had received voxelotor 900 mg and 12 patients (6 females) had received voxelotor for ≥16 weeks. the median age for the 12 patients was 13 years, 92% were receiving hydroxyurea (hu), and 41% had ≥1 painful crises in the past year. data for hemolysis measures are available for 11 patients who received voxelotor for 16 weeks. six of the 11 patients achieved a hemoglobin (hb) response of >1 g/dl increase. laboratory markers of hemolysis improved concordantly; the median reductions in reticulocytes and indirect bilirubin were 11% and 40%, respectively. ten of 12 patients showed reduction in total symptom scores (tss) at week 16, with a 94% median reduction in tss from baseline. there were no treatmentrelated serious adverse events (aes) or drug discontinuations due to aes. voxelotor 900 mg for 16 weeks in adolescents with scd, the majority receiving hu, demonstrated consistent, sustained efficacy on hb levels and measures of hemolysis; >50% of patients showed a >1 g/dl improvement in hb. improvement in tss in mildly symptomatic patients suggests that the pro is sensitive to treatment effect and supports use in the ongoing hope phase 3 study. voxelotor's reassuring safety profile is consistent with results in adults. these interim results support ongoing clinical evaluation of voxelotor as a potential disease-modifying therapy for adults and children with scd. supported by global blood therapeutics. background: acute kidney injury (aki) is a common complication in sickle cell disease (scd), and a potential risk factor for sickle nephropathy. aki is associated with acute decline in hemoglobin (hb) during vaso-occlusive pain crisis and acute chest syndrome (acs). it is unclear which pathologic factor plays a stronger role in aki development during hb drop: increase in free heme during vaso-occlusive events secondary to hemolysis or hb decline itself. objectives: to investigate if hb decline alone is associated with aki, we tested if the renal function of patients with scd worsened during parvovirus b19-induced transient aplastic crisis (tac), in the absence of accentuated hemolysis. design/method: with irb approval, a retrospective study of patients who had laboratory confirmed parvovirus-b19 was conducted. serum creatinine (scr), both during and within 12 months from the tac event, was collected. comparisons of the clinical and laboratory characteristics were analyzed using the wilcoxon test for continuous variables. aki was defined as an increase in scr by ≥0.3 mg/dl or a 50% increase in scr from baseline. to evaluate differences in change in hb on aki risk, changes in scr during tac were compared to those during pain crisis or acs admissions by fitting a generalized linear mixed model for binary outcome. a comparative sample of 149 acs events and 197 vaso-occlusive pain crisis were used to estimate rates of aki according to hb levels. results: three (9%) of the 34 patients with scd developed aki during tac. no association was identified between change in hb from baseline to tac event (p = 0.08). no cases of aki were identified until hb decreased <5.0 g/dl or the change in hb was ≥4.5 g/dl from baseline. next, we developed a model to evaluate the impact of change in hb from baseline for patients admitted with tac, pain crisis or acs on aki. with a 2 g/dl decrease in admission hb from baseline, patients with tac had a 3% probability of developing aki, while acute chest syndrome and pain crisis would have a 9% and 27% probability, respectively. our data suggest that aki is still prevalent during parvovirus b19-induced tac. however, the risk of aki during a tac event is 3 and 9 times lower than that from severe anemia induced by acute chest syndrome and vasoocclusive pain events, respectively. hemolysis-induced anemia during scd crisis appears to have a more significant role in the development of aki as compared to agenerative anemia. background: the natural history of hemoglobin e beta thalassemia (hbethal), the commonest form of severe beta thalassemia worldwide, has been examined in very few longterm studies. previously, we reported findings in 109 hbethal patients in sri lanka.1 objectives: to evaluate longterm requirements for transfusion and splenectomy, complications and death in hbethal patients. design/method: all available patients were reviewed 1-4 times annually over 12 years. results: 33 patients (30%) died, aged (mean ± sem) 29.3 ± 4.2 years; the (known) causes commonly included iron overload (9) and infection (12); 76 patients surviving patients are aged 33.4 ± 1.2 years. of 109 patients originally classified by severity (group 1 the mildest, and group 5 the most severe, phenotypes), 62 (57%) were assessed as mild (groups 1 and 2), of whom transfusions had been discontinued in 46. ultimately, 23/46 (50%) resumed transfusions, often following shifts to increasingly severe phenotypes including increasing intolerance to anemia. age at resumption of transfusions (following a transfusion-free interval of 14.9 ± 1.6 years) was 32.8 ± 2.7 years; in the more severe groups 4 and 5, regular transfusions were stopped in 27/33 patients and resumed in 22/27 (81%), at younger ages (19.9±1.8 years) and after shorter transfusion-free periods (10.1±1.4 years) than in "milder" patients. mid-parental height (mph) was ultimately achieved in 53%. 84 patients (77%) were splenectomized; updated analysis of responses to splenectomy (originally "group 3" patients), showed that splenectomy (at 9.0 ± 0.9 years) was followed by an extended, but impermanent, transfusion-free interval (11.1 ± 1.4 years); 50% patients resumed transfusions, usually related to exercise intolerance or poor growth. in groups 1 and 2, complications of anemia and ineffective erythropoiesis, including leg ulcers (in 38% and 50%) and gallstones (44% and 54%), were more frequent than in groups 4 and 5; fractures were observed (25-48%) across all groups, except for regularly-transfused group 5 patients (0%). pulmonary artery pressures >30 mm were recorded in 39% patients. evaluation of patients with hbethal requires observations over years, without which definition of patients as "mild" or "severe" may be misleading. while in many patients transfusions may be withheld or reduced in frequency, troublesome complications may surface with advancing age even in "milder" patients. although individual consideration of transfusion requirements is critical, the availability of effective chelation, where this can be provided without prohibitive cost, may alter the balance of risks and benefits of regular transfusions in hbethal. 1(premawardhena a. lancet 2005). background: social determinants of health (sdh) are environmental and socioeconomic factors, such as access to food and housing that affect health outcomes. pediatricians are increasingly screening for sdh as part of primary care visits, however less is known about screening for sdh in pediatric hematology. evidence suggests that sdh play a role in disease severity for children with scd, who face significant socio-economic and racial disparities. the goal of our quality improvement (qi) project was to increase the percentage of patients with scd who were connected to community resources for unmet social needs. design/method: we based our intervention on the successful implementation of wecare in our institution's pediatric primary care clinic. eligible patients were identified at the start of each clinic session. on arrival the parent was given a self-reported screening tool for six sdh (childcare, education, employment, food, utilities and housing). results were entered in the electronic health record by the physician or social worker who then printed a pre-existing resource list for patients with a positive screen. we used a series of plan-do-study-act (pdsa) cycles to study tests of change. we tracked process measures (percentage of patients screened, percentage of patients with an unmet social need who received a resource sheet), outcome measures (percentage of patients with an unmet social need who connected with a community resource) and balancing measures (staff, patient and provider satisfaction). run charts were reviewed weekly and then monthly to inform further tests of change. examples of pdsa cycles include who gave the paper survey to patients (social worker or physician versus medical assistant) and length of time between surveys (3 to 6 months). results: between august and december 2017 screening rates improved from 57% to 88%. of the patients screened, 67% report at least one unmet social need; of those 33% received a targeted list of community resources in the first month of the project, and 92% in the fifth month. finally, 44% of patients reached by phone had connected with a community resource within 2 weeks of the clinic visit. we have successfully implemented universal screening for sdh for patients with scd in our urban pediatric hematology clinic without requiring extra staff. next steps include further pdsa cycles to connect more patients to appropriate resources, and tracking improvement in health care utilization outcomes from addressing sdh in this vulnerable patient population. background: the clinical manifestations of sickle cell disease (scd), chronic hemolytic anemia, and vaso-occlusion occur as a direct result of sickle hemoglobin (hbs) polymerization. voxelotor (gbt440) is a first-in-class, oral, oncedaily investigational agent designed to modulate hemoglobin's oxygen affinity in a targeted approach to inhibit hbs polymerization. objectives: to examine the pharmacokinetics (pk), safety, and dosing of voxelotor in children (aged 6-11 years) and adolescents (aged 12-17 years) with scd from part a of the gbt440-007 study. design/method: gbt440-007 is an ongoing, open-label, phase 2a study in patients aged 6-17 years with scd (sickle cell anemia or sickle beta zero thalassemia). part a of this study (the focus of this abstract) is examining pk of singledose (600 mg) voxelotor. pk samples to measure whole blood and plasma voxelotor concentrations were collected up to 15 days following single-dose administration. separate population pk (ppk) models were developed to describe the concentration versus time profiles of voxelotor in whole blood and plasma using nonlinear mixed effects modeling (non-mem, version 7.3). ppk modeling and physiologically based pk (pbpk) modeling were used to simulate voxelotor pk parameters and support dose selection for future evaluation in younger children. : part a included 7 adolescents (4 females; median age 16 years [range 14-16]) and 6 children (3 females; median age 8.5 years [range 6-10]). mean weight was 52.8 kg (range 45-66 kg) and 21.1 kg (range 16-38 kg) in adolescents and children, respectively. voxelotor was well tolerated with no drugrelated grade ≥3 adverse events (ae) or serious aes. a 2compartment model with first-order absorption best described the pk of voxelotor (and was the same model structure used for adults with scd). voxelotor pk exposures in adolescents were comparable to those observed in adults, but higher exposures were observed in children. ppk and pbpk modeling support the use of a weight-based dosing strategy in younger children (aged <12 years) in future trials. adult voxelotor doses can be used in adolescents. however, based on higher pk exposures, a lower weight-based dosing strategy is recommended in children. ppk and pbpk modeling provides an innovative approach to minimize experimental dosing in children and accelerate dose selection of voxelotor in ongoing and future clinical studies. this abstract is supported by global blood therapeutics. background: hydroxyurea (hu) reduces rates of acute complications, and improves long term outcomes in patients with sickle cell disease (scd) and is now fda approved for children. through previous work we have increased the number of eligible patients on hu in our clinic, however accessing a compounding pharmacy remained a significant barrier to hu adherence for infants and children who cannot swallow capsules. objectives: the objective of our quality improvement project was to improve adherence to hu among pediatric patients with scd at our urban safety net hospital by addressing barriers to obtaining liquid hu. design/method: to begin we met with the leadership of our outpatient pharmacy which offers mail order delivery. however, like most retail pharmacies, they do not have the necessary protective equipment to compound liquid hu. through a series of discussions, we began a unique partnership with our institution's inpatient chemotherapy pharmacy who compounds the liquid hu and delivers it to the outpatient s95 of s301 pharmacy, who then dispenses liquid hu to families. using a series of plan-do-study-act (pdsa) cycles we tracked adherence by calculating the medication possession ratio (mpr), defined as the percentage of days in a given period of time that each patient had their medication on hand. the mpr for liquid hu mpr among enrolled patients was tracked by pharmacy staff and reviewed monthly. additional pdsa cycles included adding automatic refills and reminder calls by pharmacy staff and improving communication about delivery. we also tracked patient satisfaction. results: between march 2016 and december 2017, a total of thirty pediatric patients were enrolled in our program for on-site compounding and free mail order delivery of liquid hu. mpr for liquid hu is currently 93.8% among enrolled patients, significantly higher than the mpr of 60% reported in the literature, and has risen steadily since the beginning of the project. families are highly satisfied with the program, specifically appreciating the convenience of mail order delivery, saving on delivery fees, and reminder calls when refills were due. by compounding and dispensing liquid hu directly from our institution's outpatient pharmacy we have significantly improved adherence to this hu therapy in our high-risk population. next steps include analysis of change in clinical outcomes for patients enrolled in this program. as adherence to hydroxyurea is associated with decreased acute care utilization and cost, programs such as ours could play a crucial role in reducing the excessive costs and ed utilization among this patient population. background: experience with the iron-chelator deferasirox is reported widely in higher-income settings. by contrast, real-life experiences in emerging countries are infrequently reported. objectives: to evaluate, in a non-trial setting, the real-life response to deferasirox in an emerging country. design/method: in sri lanka's national thalassemia center which manages 800 patients without tertiary staff, quantitative evaluations of body iron or estimates of extra-hepatic iron, the records of 328 patients who began deferasirox in 2010/11 were retrospectively reviewed. results: baseline assessments (mean±sem) indicated substantial iron loading [serum ferritin (sf) 4,529±125 ug/l; serum alt 111±3.5 u/l (normal ≤ 40 u/l)]. deferasirox was introduced at low doses (21.2 ± 0.3 mg/kg/day); many patients started at <20 mg/kg and, after 12 months, doses remained ≤30 mg/kg/day in 60% patients. after 24 months, sf in 50% patients remained >2,500 ug/l; only by 48 months had (mean) sf declined to <2,500 ug/l (2475±344; p<0.001). similarly, mean alt normalized (to 35 ± 5 u/l) only by 60 months. death and complications were not systematically recorded by staff who had been charged, without provision of additional resources, with the introduction of this new drug in hundreds of patients. these results contrast to those in sri lanka's tertiary thalassemia center where, in 107 patients following the introduction of deferasirox 32 ± 0.1mg/kg/day, sf declined rapidly, even in relatively less ironloaded patients (from 3,231±278 to 2,153±218 g/l after 24 months; p = 0.0002). these findings underscore the importance, during the implementation of new drug regimens in lowerincome centers with marginal resources, for investments in methods to quantitate body iron burden, hands-on educational initiatives to guide day-to-day management by competent but non-expert staff, and data systems to record efficacy, effectiveness, toxicity and compliance. such investment is critical to optimising therapy and improving complications in thalassemia patients worldwide: even in sri lanka, where resources directed to thalassemia management are greater than in most of asia, results in the oldest living cohort (born 1980-1990) indicate under-treatment [elevated iron burdens (sf 3,565±323 ug/l) and high prevalences of diabetes (27%) and hypothyroidism (29%)]. even in a younger cohort (born 1990-2000) which has benefitted from improved treatments, the prevalence of many complications exceeds those reported from high-income settings. over the next decade, and two decades after the 2006 who declaration that the impact of thalassemia on global mortality and morbidity is underrecognized, increased investments by governmental and nongovernmental sources will be necessary to improve outcomes for asian patients with thalassemia. background: a major barrier to success in hydroxyurea (hu) treatment of patients with sickle cell disease (scd) is non-adherence. objectives: to optimize hu adherence in patients with scd. design/method: a care model was designed by the sickle cell (qi) team at children's hospital to improve hu adherence among scd patients. the original model included bimonthly family phone contact, monthly dispensing pharmacy phone contact and lab monitoring. adherence measures included obtaining hu from pharmacy monthly, completion of monthly labs, hb f percentage and mcv, and mtd achievement. from 6/2016 -6/2017, several pdsa cycles refined our care model. a one-year follow-up survey gathered feedback on the care model. the first-year data involved ∼ 30 patients. the biggest improvements resulted from making pharmacy calls before patient/family calls, shipping liquid hu to outlying patients, and tracking call time/content. the qi goal was 75 % hu adherence by 12/2017. the 33% baseline adherence rate increased to 80% by 12/2016, and has remained in that range. the completion rate of patient/parent phone calls increased from 33% the first month to 77% at six months. pharmacy prescription pick-up has increased from 50 % to 77% per month. lack of liquid hu availability was overcome by shipping the medication to the patient's home. parental hesitance to share information by phone, especially with qi team members with whom they had no established relationship, was overcome by having the longtime sickle cell nurse do many of the early calls. however, survey feedback showed families became comfortable with several clinic personnel calling. the calls gave families the opportunity to ask questions about their child and/or get additional information about scd. the calls also provided an opportunity for seasonal flu shot or tcd testing reminders. the surveys gave information on the optimal time of day to reach each family, providing individualization and further increasing the percentage of completed calls. two families surveyed said they no longer needed two calls a month because they were now able to remember to pick up hu, administer it, and get labs on their own. this qi project has not only improved hu adherence, but also fostered health education/counseling, increased patient/parent satisfaction, and enhanced service utilization. medical team member and patient/family comments demonstrate that it has helped build relationships and trust between families and the medical care system. based on survey feedback, we will further individualize care to increase adherence rate and sustain improvements. cincinnati children's hospital medical center, cincinnati, ohio, united states background: the thalassemias are a heterogeneous group of genetic blood disorders caused by mutations that decrease or eliminate the synthesis of the -and/or -globin subunits of hemoglobin. the phenotype of thalassemia depends on the interaction of the -and -globin gene clusters, because both loci determine the -/ -chain balance. for example, a -thalassemia phenotype can be more severe than expected when coinherited with -globin gene triplication (copy number gain), which exacerbates the -/ -globin imbalance. objectives: describe four individuals with an incorrect diagnosis of -thalassemia trait who were later properly diagnosed by comprehensive genetic testing to have -thalassemia intermedia caused by heterozygous -thalassemia mutations coinherited with triplicated -globin loci. design/method: sequence analysis of the -globin (hba1/hba2) and -globin (hbb) genes, and copy number variation analysis of the -and -globin gene clusters by multiplex ligand-dependent probe amplification. results: four unrelated individuals of northern european ancestry were evaluated for signs and symptoms not explained by a diagnosis of -thalassemia trait (previously made by a pediatric hematologist), including growth delay, splenomegaly, moderate anemia, marked elevation of hemoglobin f, thalassemic facies, reticulocytosis, and/or indirect hyperbilirubinemia. genetic testing revealed that all were heterozygous ( / 0) for the same, single -globin mutation [hbb.c.118c>t (p.q40*)] and also heterozygous for an -globin triplication ( / anti-3.7). their previous diagnoses of thalassemia trait had been made by complete blood counts, hemoglobin electrophoresis, and/or sequence analysis of the -globin genes only. these individuals' phenotypes ranged from moderate anemia only to multiple stigmata of thalassemia, demonstrating the phenotypic variation of a thalassemia genotype. correct diagnosis was made at an average age of 8.9 years. a trial of chronic transfusions was initiated for one patient for growth failure. all were educated about the potential for exacerbations of anemia, gallstones, osteoporosis, and iron overload (even without transfusions). parental genetic testing was recommended to assess reproductive risk, because inheritance of this complex genotype can be apparently autosomal dominant. conclusion: heterozygosity for a -thalassemia mutation does not necessarily indicate -thalassemia minor or "trait". when coinherited with -globin gene triplication, a symptomatic form of -thalassemia can occur. correct and timely diagnosis of thalassemia requires careful consideration of the degree of anemia and examination for organomegaly, bony changes, and jaundice. sequence analysis and copy number variation analysis of both the -and -globin gene clusters is key. hematologists need to be aware of this diagnostic possibility and how to test for it to prevent inaccurate or delayed diagnosis. background: the burden of healthcare costs for sickle cell disease (scd) is nationally estimated at over $488 billion. the major components of these costs are inpatient and emergency center (ec) visits, many of which are potentially avoidable. in several chronic conditions, a subset of patients account for most of the avoidable encounters. identifying these patients is the first step in targeted care delivery. objectives: to measure and analyze scd patient utilization patterns in the ec and inpatient at texas children's hospital (tch). we identified all individuals under 21 years old with any encounter at tch associated with an international classification of disease (icd)-9 or 10 code for scd, including hgb ss, hgb sc, and hgb s/beta thalassemia. for each patient, we identified all inpatient and ec encounters in the 365 days prior to their most recent encounter. finally, each encounter was classified as associated with pain, acute chest syndrome (acs), or "other" using an algorithm of discharge diagnosis codes and pharmaceutical delivery. the total number of scd-associated ec and inpatient encounters over the prior year was calculated for each patient. we stratified each patient according to their utilization patterns: low (0-1 encounters), intermediate (2-3 encounters), and high (≥4 encounters). we identified 952 unique patients with scd that had at least one encounter from july 2016 until june 2017. there were 1,100 scd-related encounters in the 365 days prior to their most recent encounter. most (74%, n = 701) patients exhibited low-utilization patterns and 18% (n = 174) were intermediate. finally, a small subset (8%, n = 77) demonstrated high-utilization patterns and accounted for 41% of all encounters. high-utilization was associated with older age and public payment mechanisms. pain encounters were predominantly in pre-adolescents and teenagers with high-and intermediate-utilization patterns. acs was most frequent in pre-teens and younger teens in the intermediate-utilization group. finally, the youngest-aged high and intermediate users presented for other reasons such as febrile episodes and splenic sequestration. our findings reflect national trends in that a significant portion of encounters are attributed to a small subset of patients exhibiting a high-or "super-" utilization pattern. at our institution, scd super-utilization is associated with older age and pain. we also identified a group of infants and toddlers with frequent encounters for fever. to comprehensively address this burden, it will be important to design interventions targeted toward age and specific medical needs. background: background: the rarity of diamond blackfan anemia (dba) has hindered describing the spectrum of disease, identifying predictive correlations, and guiding datadriven recommendations. long-term toxicities from steroid or transfusion therapy that start in childhood remain the major clinical problems in patients with dba who do not receive stem cell transplant. objectives: objective: to define the dba patient population at st. jude children's research hospital including treatment responses and toxicities to help inform recommendations on treatment and monitoring. design/method: method: medical records were reviewed for all patients with dba treated at st. jude between 1997 and 2017 for diagnostic testing, treatment types and regimens, and outcomes. two-sample t-test or wilcoxon rank sum test was used to compare continuous variables in two groups depending on the normality of the data tested by shapiro-wilk test. results: a total of 22 patients with dba were identified with a median age of 8.29 years (range 3 months -35 years) at last follow up. a ribosomal protein gene mutation was identified in 15/22 patients (68%) with an rps19 mutation 8/22 (36%). thirteen different congenital malformations were described in 9/22 patients (41%). fourteen of twenty (70%) patients treated with corticosteroids had an initial response and 3 of those achieved full remission. three patients became steroid-refractory and 2 were unable to wean to an acceptable dose. five of twenty patients continue on lower-dose steroids. five patients currently require no therapy. univariate analysis revealed no statistically significant genetic predictors of response or remission, however, 3/3 rpl11 patients responded to steroids with 2/3 (66%) in long-term remission. ten patients are maintained on chronic transfusions and 2 have undergone successful hematopoietic stem cell transplant. nineteen of 20 treated patients (95%) had a treatment-related toxicity. patients on steroids were more likely to have short stature than patients on transfusions or in remission (p = 0.005). severe bone mineral density deficit occurred in 4/20 (20%) patients, in 2 before age 7 years. eight patients had hepatic iron overload, in one documented by age 2 years. other severe toxicities included restrictive cardiomyopathy from iron overload, pathologic fracture, diabetes mellitus, and premature ovarian failure in one patient each. this genotypically and phenotypically heterogeneous dba cohort had a high rate of treatment-related toxicities, notably growth retardation, bone density loss, and hepatic iron overload even in very young children. these findings underscore the need for early standardized monitoring. background: patients with sickle cell disease (scd) face worsening morbidity and mortality between ages 18 and 30, when they must transition from pediatric to adult healthcare.(1) an effective curriculum addressing disease knowledge, educational and vocational skills, self-efficacy, and social supports is critical to a successful transition. traditional didactic approaches have not led to durable knowledge retention. (2) technology-based methods have been attempted, but the best educational approach remains unknown. objectives: 1. to understand how adolescent and young adult (aya) patients with scd view existing transition education. 2. to include patient preferences in improving our transition curriculum. we developed a qualitative survey to assess patient views of existing approaches for learning about scd and their opinions about preferred transition topics. thirty patients with scd aged 12 to 24 years old were recruited between january and december 2017. responses were managed using redcap electronic data tools hosted at the university of rochester.(3,4) qualitative and quantitative data analyses were performed, including independent t-testing to compare responses between age groups. results: approximately 68% of subjects were under 18 years of age, while 32% were 18 or older. seventy-one percent had a computer, and 93.5% had a cell phone, with most reporting daily use. subjects reported greatest satisfaction with learning from their doctor during clinic visits (83.9% agree or strongly agree) and websites on a cell phone (77.4% agree or strongly agree); the least popular methods were online chat rooms and microsoft® powerpoint presentations. satisfaction was similar across age groups. recommended transition topics were viewed positively, with subjects ranking highest understanding their bloodwork (87.1% agree or strongly agree) and understanding laws protecting students with chronic disease (93.6% agree or strongly agree). older subjects (18-24 years old) agreed more strongly with learning about opioid addiction and understanding differences between adult and pediatric doctors than did younger subjects (12-17 years old) (p < 0.05). this pilot study was successful in helping us to understand the educational needs of aya patients with scd. preliminary data underscore the importance of education provided by the pediatric hematologist. our results also suggest that the optimal use of technology-based methods requires further investigation and that tailoring transition education by age group may be useful. background: similar to patients with transfusion-dependent beta-thalassemias (tdt-beta), survivors of hemoglobin barts hydrops fetalis (homozygous alpha-0-thalassemia, tdtalpha) will require lifelong transfusions of erythrocytes. we have previously shown that a transfusion strategy that is based on the guidelines developed for tdt-beta (conventional transfusion) is suboptimal for these patients owing to the differences in the pathophysiology of anemia in the two conditions: in tdt-alpha, conventional transfusion strategy will lead to a gradual increase in non-functional hbh with subsequent tissue hypoxia and hemolysis. an aggressive transfusion strategy that was based on reduction of hbh and increase in "functional" hemoglobin level resulted in improvement of tissue oxygenation and reduction of hemolysis but was associated with significant increase in transfusional iron burden [amid et al, blood 2016] . objectives: to define the optimal chronic blood transfusion targets for hbh% and functional hemoglobin in patients with tdt-alpha. design/method: following research ethics board approval, longitudinal data of 6 patients with tdt-alpha (2 males, median age 11.5 (2.1-18.0) were retrospectively collected. variables of interest included total pre-transfusion hemoglobin, hbh%, and "functional" hemoglobin [measured as total hemoglobin x (1-hbh/100)]. outcome variables were lactate dehydrogenase (ldh, marker of hemolysis), and soluble transferrin receptor (str, marker of erythropoiesis). hemoglobin analysis was done using high-performance liquid chromatography and capillary zone electrophoresis. we examined the association of "functional" hemoglobin with str, and hbh% with ldh, using repeated-measures anova to adjust for the effect of multiple testing. we constructed receiver operating characteristic curve and calculated the area under the curve to define the best cut-off values for variables of interests. there was a strong association between functional hb and str, as well as hbh and ldh. the optimal cut-off for "functional" hemoglobin that was associated with str <2.0 mg/l was 98 g/l (auc = 0.94, sensitivity and specificity of 82.76% and 100% respectively). the optimal cut-off for hbh to supress ldh to <1000 u/l was 18% (auc = 0.94, sensitivity and specificity of 87.5% and 87% respectively). the optimal pre-transfusion hbh% for reduction of hemolysis was 18% and the optimal "functional" hemoglobin to adequately supress erythropoiesis was 98 g/l. to meet these hbh% and functional hb targets by simple blood transfusions, patients with tdt-alpha would require a hypertransfusion regimen with a minimum pre-transfusion total hb of 116 g/l and consequently high transfusional iron burden. an alternative approach using exchange transfusion to reduce hbh% and improve functional hemoglobin would be associated with less volume of transfusion and potentially better long-term outcome. hospital sacre coeur, milot, haiti background: initial results of work developing a pediatric sickle cell disease (scd) clinic at the hôpital sacré coeur (hsc) in milot, northern haiti were presented at aspho 2017. the purpose of this clinic is for a pediatrician with a special interest in scd to provide scd care, advising on trait and managing disease with penicillin prophylaxis (pcn) and hydroxyurea therapy (hu) for select patients. this clinic was started in collaboration with a us based hematologist and support from yale-new haven hospital. objectives: to describe the success and challenges of providing pcn and hu in the scd clinic at hsc through a review of patient records. design/method: since this clinic's inception, a database of patients, with basic clinical information has been kept and made accessible, through 'drop-box', to the us hematologist. the records of those that presented to the clinic were reviewed. the hemoglobin diagnosis was made either by clinical history and sickle cell prep or by hemoglobin electrophoresis through alpha laboratory, port-au-prince, haiti. results: ninety-nine individuals were seen in the first 2 years of the program. fifty-six underwent a hemoglobin electrophoresis. of these 99, 49 are ≤ 6 years old. thirty-two were started on pcn vk, of which 10/32 (31%) were ≤ 3 years old. eleven patients were started hu therapy. all patients on hu have shown progressive increases in hemoglobin. there have been no clinical complications of hu therapy. none of the patients taking hu have required hospitalization or transfusion in 2017. three patients (not on hu) were hospitalized in 2017 for complications of scd (osteomyelitis, pain). in 2016, with less than half the numbers in the program, there were 7 admissions for severe anemia, pain, stroke and splenic sequestration. with ongoing external support and a local reputation for excellence in sickle cell care, the clinic at hsc has been able to expand services and improve the health of a growing number of patients with scd. early data suggests that pcn and hu therapies are helping to reduce complications and improve quality of life. challenges to date have included lack of funding for transportation to clinics, for hospitalizations and to cover the cost of electrophoreses. at the same time as continuing providing excellent care and gathering data, it is crucial to explore opportunities for collaboration and cooperation in ways that will assure that the clinic can become independently sustainable while continuing to improve the quality of life for the individuals it serves. background: ykl-40 is an inflammatory glycoprotein expressed by infiltrating macrophages in various inflammatory conditions. it has been found to be elevated in patients with different pathological conditions like acute and chronic inflammations, increased remodeling of the extracellular matrix (ecm), development of fibrosis and cancer. several studies have found elevated ykl-40 concentrations in sera of patients with liver diseases such as hepatic fibrosis by hepatitis c virus. it has been suggested that ykl-40 concentrations reflect the degree of liver fibrosis. to evaluate serum ykl-40 levels in patients with -thalassemia and its relation to viral hepatitis, liver stiffness as assessed by transient elastography (fibroscan, fs) and hepatic iron concentration. design/method: a prospective study included 100 patients with -tm (43 males and 57 females) with mean age 13.8 ± 2.7 years (range: 5-18 years). serum ferritin level, liver enzymes (alt and ast), hbs ag, anti hcv ab and serum ykl-40 using elisa kit were evaluated. all patients were subjected to liver mri t2* to detect liver iron content by the sequence and transient elastography (fibroscan, fs) to assess degree of liver stiffness. results: mean fibroscan value was (10.99±11.5) kpa with a median 6.7 (range 1.3 to 47) kpa. 64 (64%) patients were categorized as f0-1 and 17 (17%) were stage f2-3, 19 (19%) patients had severe fibrosis. their median serum ferritin was 3100 ng∖ml, with 61 (61%) patients had values exceeding 2500 g/l. median cardiac t2* was 24.2 with 30 patients had values below 20 ms, and the median lic was 16.21 mg/g dw with 68 patients showed readings above 7 mg/g dw. nyl-40 was evaluated as a marker of inflammation and liver fibrosis and showed mean value 1505.1 (±960.9) pg/ml, and range from 500 to 3529 pg/ml. mean ykl-40 was significantly higher among males (p = 0.03), patients on chelation therapy (p = 0.002), patients on dfs (p≤0.001), in those with abnormal liver enzymes, splenectomised patients, patients with hbv sero-positivity, those with moderate elevation of t2* and patients with high grades of liver fibrosis (p<0.05). ykl-40 showed positive correlation with the rate of transfusion, lic, ferritin, alt and ast but negative correlation with weight, height and t2*. roc curve analysis revealed that the cutoff value of ykl-40 at 1500 pg/ml could differentiate -tm patients with and without viral hepatitis with 86.7% sensitivity and specificity of 91.4%, area under the curve (auc) 0.933, positive predictive value 81.2 and negative predictive value 94.1 (p<0.001). roc curve analysis revealed that the cutoff value of ykl-40 at 1600 pg/ml could detect -tm patients with liver cirrhosis with 93.4% sensitivity and specificity of 97.1%, area under the curve (auc) 0.972, positive predictive value 93.7 and negative predictive value 97.1 (p<0.001). conclusion: serum ykl-40 levels are elevated in patients with -thalassemia and can detect patients with active viral hepatitis and liver stiffness. background: the most common splenic complication in pediatric patients with sickle cell disease (scd) is acute splenic sequestration (ass), which has often been managed with splenectomy. although splenectomy has been a treatment of choice for years, long-term vascular complications have not been thoroughly evaluated. pulmonary hypertension (phtn) is a severe complication of scd. in adults with scd, phtn has been associated with a 40-month mortality rate of approximately 40%. it has been reported that splenectomized patients with hemolytic disorders are at even greater risk of phtn. several medications exist to treat phtn, but with few studies of their efficacy or toxicities in patients with scd. additionally, these patients are often treated with either chronic prbc transfusions or hydroxyurea (hu) to raise hemoglobin, reduce hemolysis, and prevent vaso-occlusive events. objectives: to evaluate effect of chronic prbc or hu vs. no intervention, on tricuspid regurgitant jet velocities (trv) in pediatric patients with scd and history of splenectomy. design/method: retrospective chart review of splenectomized patients with hbss followed at marian anderson center at st. christopher's hospital for children, philadelphia, between 1999 and 2017. we analyzed 73 trvs (16 hu, 40 prbc, and 17 from control group receiving neither treatment) from 35 patients (10 hu, 13 prbc, 12 neither). mean age at echo was 13.31 +/-5.1. data was analyzed with linear correlations and analysis of variance (anova), including the post hoc test of least significant difference (lsd) for all pairs of treatment groups. results: trv was not significantly correlated with age at time of assessment or with time between splenectomy and trv. univariate anova among groups yielded trv means of: 214.0 +/-36.0 cm/s (hu), 231.3 +/-28.1 (prbc), 231.3 +/-25.4 (neither). we found a notable difference as the mean of the hu group was almost 18 cm/s lower than the others, but no overall statistically significant association for any of the groups exists. however, when we performed post hoc tests to adjust for multiple comparisons and looked at all 3 pairings within the anova, we found that the lsd between the hu and the prbc groups was statistically significant (p = .051), and that a trend exists between the hu group and the neither treatment group (p = .095). our data suggests that treatment with hu is correlated with a reduction in trv in pediatric patients with scd who underwent splenectomy. given these promising results, we believe our data warrants further study with larger treatment groups. nancy olivieri, gaurav sharma, susmita nath, rajib de, tuphan kanti dolai, prakas kumar mandal, abhijit phukan, amir sabouhanian, robert yamashita, angela allen, david weatherall, prantar chakrabarti background: hemoglobin e thalassemia (hbethal), which accounts for 50% of all severe beta thalassemia worldwide, has an estimated prevalence of 1.4/10,000 in west bengal, from which little information about clinical findings has been reported. objectives: to document clinical and laboratory findings in patients with hbethal, ultimately to improve resources for clinical management. design/method: we reviewed records from: a database recording patient names; clinic charts; "special" charts containing additional details; and, in transfused patients, transfusion day-care records. additionally, because in india's public hospitals original lab/imaging reports are commonly retained at home, 20% of families were interviewed to provide additional information. we excluded records of patients aged <5 years and patients aged <30 years who had not been reviewed since 2014. results: while at least one visit had been recorded in 1,398 hbethal patients at nrs hospital, most patients are not regularly reviewed there. we examined 219 charts [84 (38%) aged ≥30 years; 135 (62%) aged 5-29 years; 61% male], representing approximately 70% of regularly-reviewed patients. most families (84.9%) reported monthly incomes (<5,000 indian rupees), below the monthly cost of living (70,000 rupees) in kolkata. mean (±sem) hemoglobin was 6.9±1.1 g/dl. 43% patients were receiving eight or more transfusions per year; from 2013, 40% had been treated with deferasirox, 26.5±8.5 mg/kg/day. iron control estimated by serum ferritin concentration (1357.2±1187 g/l) was highly variable. a total of 24% patients were splenectomized. a substantial obstacle to documenting complications was the lack of recording, in any of the five sources, of many relevant parameters: for example, the status of sexual maturation (normal, delayed, or absent) was documented in less than 60%, and measurements of fasting blood glucose in less than 50%, of records. where recorded, complication rates were high: delayed/abnormal sexual maturation was recorded in 15% patients aged >30 years; in the patients aged >30 years and those aged 5-29 years, respectively, hypothyroidism was recorded in 31% and 44%, and elevated serum alt in 30% and 35%. in most evaluable patients >30 years, height was measured between the 3rd-10th percentiles. cardiac findings, rarely documented, included pulmonary hypertension and reduced left ventricular ejection fractions in a few patients. despite dedicated attention to many aspects of thalassemia care, insufficient documentation limited a clear understanding of the current morbidity in hbethal patients. investment in personnel and technology will be critical to record relevant information, ultimately to improve clinical management, over the next decade. children's hospital of richmond at vcu health, richmond, virginia, united states background: sepsis is a common cause of death in children with sickle cell disease (scd). recommendations for care of fever in children with scd include immediate medical evaluation including blood culture and initiation of broad-spectrum antibiotic therapy. the increasing availability of pcr-based respiratory pathogen panels (rpp) provide the opportunity to rapidly identify viral causes of fever. the role for rpps in identifying the source of fever in children with scd and how it affects provider practice is not well studied. (1) to determine the epidemiology of respiratory virus-associated fever in children with scd and (2) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all children with scd seen in our emergency department (ed) with temperature ≥38.3oc at home or in the ed from january 1, 2016, through september 30, 2017, as well as, all febrile children for whom rpps were sent since the introduction of rpps april 2014. we reviewed the results of blood cultures, rpps, chest radiographs, and ed notes and discharge summaries to identify sources of infections. independent t test and chi-square analysis were used as appropriate to compare results using spss©. overall, the rate of bacteremia was 1%. there were no cases of bacteremia among children with positive rpps. 4% of children with negative rpps had true bacteremia. a positive rpp did not reduce the likelihood of bacteremia (p 0.11). patients with bacteremia had higher presenting temperatures than those without bacteremia (39.5oc vs 37.9oc, p 0.017). the most common rpp findings were rhinovirus/enterovirus (38%), human metapneumovirus (13%), and influenza a (10%). sending an rpp did not affect admission rate (29% and 26% respectively, p 0.70); however, likelihood of admission was lower in patients with positive rpps (21% vs 49%, or 0.27 [0.13-0.56], p 0.004). length of stay (los) was shorter in patients for whom an rpp was not sent (3.1 vs 4.5 days, p 0.036). as previously reported, bacteremia in febrile children with scd is very low, but remains a serious concern, particularly in the setting of high fever (>39oc). a positive rpp did not reduce the odds of bacteremia, but did have a sta-tistically significant impact on both admission rate and los. more work is needed to understand how rpp results impact provider decision-making and care for children with scd. cincinnati children's hospital medical center, cincinnati, ohio, united states background: diffuse myocardial fibrosis is a common, if not defining, feature of the heart in sickle cell anemia (sca) that is strongly associated with diastolic dysfunction. we found diffuse myocardial fibrosis in every patient in a sca cohort (n = 25) ranging in age from 6 to 61 years (niss 2017). the treatment and prevention of this complication of sca has not been studied before. objectives: because diffuse myocardial fibrosis must begin in early childhood, we hypothesized that early initiation and uninterrupted use of disease-modifying therapy for sca can prevent it. design/method: we use cardiac magnetic resonance imaging (cmr) to measure the myocardial extracellular volume fraction (ecv) to quantify diffuse myocardial fibrosis in individuals with sca who have been treated, uninterrupted, with hydroxyurea or chronic transfusion therapy since ≤4 years of age. two comparison groups were used: individuals with sca who have not been treated with disease-modifying therapy since ≤4 years of age (n = 25) and controls without sca (n = 16). results: we studied 7 individuals (3m/4f) with a mean age of 13.4 years (range 7 -24). mean age at the start of diseasemodifying therapy was 2.5 ± 0.4 years (range 1-4). only 1 had evidence of mild diffuse myocardial fibrosis (ecv 0.339); the other 6 had no detectable diffuse fibrosis (all had ecv <0.304, the upper limit of normal). mean ecv was 0.283 ± 0.012, which was significantly lower than the ecv of individuals with sca who have not received early uninterrupted therapy (0.441 ± 0.016; p = 0.009) and not statistically different from normal controls (0.257 ± 0.004; p = 0.898). none had macroscopic fibrosis by late gadolinium enhancement or evidence of myocardial hemosiderosis by t2* imaging. no patient had diastolic dysfunction by echocardiographic classification, right heart catheterization, or both. disease-modifying therapy for sca can prevent diffuse myocardial fibrosis, and possibly diastolic dysfunction, if started in early childhood. prospective trials of disease-modifying and anti-fibrotic therapy are planned to prevent diffuse myocardial fibrosis, which can be monitored noninvasively by cmr, and improve outcomes in sca. (niss, blood, 2017) . background: a statewide sickle cell surveillance system (sscss) was developed with the goal of determining the prevalence of sickle cell disease (scd) in indiana and the level of care that patients receive throughout the state. persons with scd are at high risk of infection, especially with encapsulated organisms, as well as at increased complications from influenza. utilizing sscss data, the relationship between vaccination status and mortality was explored. to determine if vaccination status is associated with mortality in persons with scd. the project was granted a waiver of consent by the st. vincent irb. death certificates were obtained to identify cause of death. deceased patients (cases) were matched by age, gender, and sickle genotype to living patients (controls). vaccination data were collected from the medical record and the children and hoosier immunization registry program (chirp) through the date of death for each case. cases and controls were assigned a point for completion of the pneumococcus, meningococcus and haemophilus influenza type b (hib) vaccine series and one point if the influenza vaccine was given within a year prior to death of the cases [max vaccine status score (vss): 4]. total points were compared between the cases and controls. two tailed t-tests to compare means of continuous data and wilcoxon signed-rank test to compare ordinal data. one thousand forty-eight individuals were included in the sscss. six hundred and seven (48.6%) were seen at one institution and included in this analysis (mean age = 21 years). thirty-three of the 607 (5.4%) were deceased at the time of analysis. six point one (6.1)% of controls and 12.1% of cases received a vss of 4. the mean vss for cases was 0.7±1.3 and 0.6±1.1 for controls. thirty point three (30.3) % of controls had a vss of one or more, compared to 27% of cases (p = 0.41). patients who died of infection [streptococ-cus (n = 1), pseudomonas (n = 1) and unidentified organisms (n = 4)] were not up to date on vaccination against encapsulated organisms, but two had received the influenza vaccine in the year prior to death. in this sample, mortality occurred exclusively among adult patients, which is consistent with current patterns in developed countries. among these adults, vss and mortality rates were not related. limitations to the study include small sample size and potential incompleteness of vaccine records. vaccination rates and other standard of care indicators should be explored in a larger cohort of patients to determine associations with mortality. background: sickle cell disease (scd) is a genetic disorder resulting in acute and chronic complications, including delayed puberty. delayed puberty can have adverse physical and psychosocial effects on affected children and families. there are no published reports from ghana on pubertal timing in children with scd. the aim of this cross-sectional study was to describe pubertal changes in children with scd at korle bu teaching hospital (kbth), accra, and compare these findings to those in a control group without scd. design/method: 178 children with scd and 174 children with hb aa, ages 8-19 years, were consecutively recruited and matched for age, sex and socioeconomic status. investigator-administered questionnaires were used to obtain demographic data for all participants and information on menarche (girls only). pubertal status was assessed by physical examination using tanner staging. testicular volumes were determined in boys using a prader orchidometer. body mass index (bmi) and socioeconomic status (ses) of participants were analyzed to determine if there were any associations with tanner stage. of the 178 with scd, 133 (74.7%) were hb ss and 45 (25.3%) hb sc. females comprised 51.1% (cases and controls). mean age at onset of breast development was significantly delayed in girls with scd (13.1 ± 1.9 years) compared to controls (10.8 ± 1.9 years) but there was no significant age difference at onset of pubic hair development. mean age at menarche was significantly delayed in girls with hb ss (14.0 ± 1.8 years) and hb sc (13.5 ± 1.5 years), compared to those with hb aa (12.5 ± 1.3 years). in boys, the mean ages at onset of puberty were significantly delayed in those with scd (13.6 ± 2.7 years, for genital development and 15.1 ± 2.2 years, for pubic hair development), compared to those without scd (11.3 ± 1.9 years and 11.4 ± 1.9 years, respectively). mean testicular volumes were significantly lower in cases compared to controls, across all age ranges (p<0.001). mean bmi in both cases and controls were similar at onset of breast development in girls. however, in boys with and without scd, mean bmi values were significantly different at pubertal onset. in univariate analysis, ses was not associated with tanner stage for both genital and breast development. mean ages at pubertal onset were significantly delayed in children with scd. longitudinal studies are needed to further characterize any associations with bmi and determine potentially modifiable risk factors affecting pubertal onset in scd. background: sickle-cell disease (scd) is a life-threatening genetic disorder associated with multiple chronic and acute complications. specific monitoring and treatment for children is a major part of the medical focus, but there remains a lack of real-world evidence of the disease burden and practice patterns among the pediatric scd population. objectives: to examine the clinical burden and management of scd among pediatric patients. design/method: a retrospective claims study was conducted using the medicaid analytic extracts database from 01jan2009-31dec2013. pediatric patients (aged <18 years) with scd were identified using icd-9-cm diagnosis codes (282.41-282.42, 282.60-282.69 ). the first observed scd diagnosis during the identification period was designated as the index date. patients were required to have continuous medical and pharmacy benefits for at least 6 months pre-and 12 months post-index period. patient data were assessed until the earliest occurrence of the following events: disenrollment, death, or the end of the study period. patient demographic and baseline clinical characteristics, clinical outcomes (mortality, incidence of pain crisis, complications), scd management, and healthcare utilization were examined. all variables were analyzed descriptively. results: a total of 12,388 patients met the study inclusion criteria, with a mean age of 7.7 years. most patients were black (59.9%) and had a charlson comorbidity index score of 0 (80.9%). mortality during follow-up was 0.1 in 100 personyears, and the event rate of pain crisis in the inpatient setting was 54.0 in 100 person-years. the three most common complications after pain crisis (highest rates in 100 person-years) were fever (31.9), infectious and parasitic diseases (27.7), and asthma (14.5). rates of life-threatening complications were also examined in 100 person-years, including acute chest syndrome (7.0), stroke (1.8), splenic sequestration (1.1), pulmonary hypertension (0.3), and pulmonary embolism (0.1). 83.9% of patients were prescribed antibiotics during the one-year post-index period. other frequent medications utilized among children were folic acid (39.2%), nonsteroidal anti-inflammatory drugs (37.4%), opioids (11.2%), and hydroxyurea (11.5%). 16.0% of patients had a blood transfusion within one year post-index date. patients had frequent health care utilizations in the inpatient (1 visit), emergency room (2 visits), office (7 visits), and pharmacy (11 visits) settings during the one-year follow-up period. pediatric scd patients are burdened with a high rate of complications including pain crisis. in addition, patients utilized a substantial amount of health care resources including outpatient office care and acute care visits. background: novel use of hydroxyurea in an african region with malaria (noharm, nct01976416) is a randomized controlled trial of hydroxyurea for very young children with sickle cell anemia living in uganda. during year 1, study participants received blinded study treatment of hydroxyurea or placebo; those receiving hydroxyurea had no increased risk of malaria, but had both laboratory and clinical benefits. during year 2, all study participants received openlabel hydroxyurea treatment. to assess the effects of open-label hydroxyurea treatment in a very young population of children with sickle s105 of s301 cell anemia living in uganda. study endpoints included the rates and severity of malaria infections, clinical sickle-related events, and laboratory effects. design/method: all children in the noharm trial were enrolled at mulago hospital sickle cell clinic in kampala uganda. during year 2, all children received open-label fixeddose hydroxyurea (20 mg/kg/day) for 12 months, after previously receiving either hydroxyurea or placebo for 12 months. results: a total of 198 children entered year 2 of the noharm trial and received fixed-dose hydroxyurea, including 107 males and 91 females, at an average age of 3.3 ± 0.9 years. among 99 children previously on placebo, there were 6 malaria events in 6 children, including 3 with severity grade ≥3, and three deaths (two acute chest syndrome, one sepsis). clinical adverse event rates dropped from 3.0 to 1.6 per patient year, and hospitalizations were reduced from 35 to 7. expected hematological benefits of increased hemoglobin, mcv, and fetal hemoglobin, along with decreased neutrophils and reticulocytes, were rapidly achieved. laboratory adverse events were infrequent at 0.2 events per patient-year, and only half of those were dose-limiting hematological toxicities. among 99 children previously on hydroxyurea, there were 7 malaria events in 5 children, including 2 with severity grade ≥3, and two deaths (one acute chest syndrome, one sepsis). clinical adverse event rates and hospitalizations were maintained at low rates, the hematological benefits of hydroxyurea continued throughout the extended treatment period, and dose-limiting toxicities remained infrequent. fixed-dose hydroxyurea treatment of young children with sickle cell anemia living in uganda is associated with no increased risk for malaria. clinical and laboratory benefits occur, including children previously on placebo who crossed-over to hydroxyurea treatment. future studies should focus on the optimal dosing and monitoring strategies, in an effort to determine the overall feasibility and safety of introducing hydroxyurea therapy across sub-saharan africa. background: acute chest syndrome (acs) is the second most common cause of hospitalization in patients with sickle cell disease and is a leading cause of morbidity and mortality. in mid-2009, an algorithm was implemented at cohen children's medical center to initiate transfusions within four hours of diagnosis of acs in order to improve patient outcomes. objectives: the aim of this project was to analyze the effect of early blood transfusion on the outcomes of patients with acs. we focused on the number of total transfusions, need for exchange transfusion, need for intensive care unit (icu) stay, and length of hospitalization. design/method: a retrospective chart review was completed on patients admitted to ccmc with a primary diagnosis of sickle cell disease and a secondary diagnosis of either acs or pneumonia during the years of 2006-2012. data from the three years directly prior to implementation of the algorithm was compared to data from the three years directly after implementation of the algorithm. a total of 118 patients were analyzed, of which 45 belonged to the pre-algorithm group and 73 to the postalgorithm group. patients from the post-algorithm group had a higher incidence of transfusions (78% with a mean transfusion number of 1.49 pre versus 86% with a mean of 1.83 post) as well as exchange transfusion (17% pre versus 27% post). the post-algorithm group had a shorter overall length of stay (mean of 6.0 days pre versus 5.0 days post). while the overall percentage of patients requiring an icu admission was similar in each group (27% pre versus 29% post), the post-protocol group had a lower likelihood of requiring an icu admission for reasons outside of line placement for exchange transfusion, most commonly for icu-level respiratory support (13% pre versus 4% post). despite a higher total number of transfusions, early recognition and transfusion for acs can lead to decreased lengths of hospitalization as well as decreased need for icu-level respiratory support. further studies comparing different center's clinical practice guidelines are necessary to improve the standard of care. background: novel use of hydroxyurea in an african region with malaria (noharm) was the first placebocontrolled randomized clinical trial of hydroxyurea in sub-saharan africa. in noharm, young children with sca received either hydroxyurea or placebo during year 1, followed by open-label hydroxyurea for all study participants during year 2. an ancillary noharm project was designed to determine if hydroxyurea treatment lowers transcranial doppler (tcd) velocities and possibly reduces stroke risk in this very young cohort. objectives: to perform tcd screening on the noharm cohort, measuring the time-averaged mean velocity (tamv) at the end of both year 1 and year 2. we hypothesized that the maximum tamv would be lower for noharm study participants receiving hydroxyurea compared to those receiving placebo, and that key clinical and laboratory parameters would also influence tcd velocities. design/method: all children enrolled in noharm were eligible to undergo tcd examination at two study time points: month 10-12 when they were completing the blinded treatment phase, and again at month 22-24 at the end of the open-label treatment phase. tcd measurements included tamv readings from the main intracranial arteries: middle cerebral artery, distal internal carotid artery, and bifurcation on tcd. all tcd examinations were scored and classified as normal (less than 170 cm/sec), conditional (170-199 cm/sec) or abnormal (greater than or equal to 200 cm/sec), with higher scores correlating to greater risk of stroke. results: at the end of year 1, 185 tcd exams were conducted of which 164 were suitable for analysis (81 hydroxyurea, 83 placebo). based on the maximum tamv, the median velocity was 138 cm/sec (iqr 120 -159) for children on hydroxyurea and 150 cm/sec (iqr 134 -168) on placebo, p = 0.0509. maximum tamv values had negative correlations with hemoglobin concentration (-0.47), fetal hemoglobin (-0.32), and oxygen saturation (-0.27); positive correlations were noted with age (0.27) and absolute neutrophil count (0.27). at the end of year 2, 187 tcd exams were conducted and all were suitable for analysis; the median velocity was 137 cm/sec on open-label hydroxyurea treatment, regardless of previous blinded treatment. all correlations with tamv were maintained except for age. conclusion: compared to placebo, hydroxyurea treatment for young children with sca living in uganda was associated with lower tcd velocities, which have been correlated in other studies with lower risk of primary stroke. tcd velocities were correlated with hematological and clinical parameters that can be improved by hydroxyurea therapy. children's hospital of richmond at virginia commonwealth university, richmond, virginia, united states background: acute chest syndrome (acs), defined by respiratory symptoms and a new pulmonary infiltrate, is a serious complication of sickle cell disease (scd). acs can occur during hospitalization for non-pulmonary conditions, such as a vaso-occlusive crisis or after surgery. nih clinical practice guidelines encourage incentive spirometry (is) which decreases the incidence of acs. it is additionally widely accepted that early, frequent ambulation in post-operative and pneumonia patients decreases the length of stay (los). to decrease acs events in children with scd at our children's hospital, we aimed for is use in 100% of ageappropriate pediatric sickle cell admissions. design/method: a multidisciplinary team examined inpatient acs prevention practices, including is, at children's hospital of richmond. key drivers were identified, including educational awareness of patients and healthcare staff, order placement, and documentation. we aimed for all scd patients ≥ 15 months of age hospitalized with any admission diagnosis to participate in is with the use of a traditional incentive spirometer or similar age-and ability-appropriate devices (e.g. positive expiratory pressure devices, bubbles, and pinwheels). we secondarily aimed to increase activity events, specifically ambulation and out of bed time. educational and outreach tools included patient informational brochure and incentive program, and staff informational sessions and reference materials at workstations. a disease-specific order set was implemented including desired is and activity orders. data were collected prospectively may through november 2017, during which 3 pdsa cycles were conducted. admissions during the corresponding months of the previous year were reviewed for comparison. independent t-test analysis was performed using graftpad prism 6 statistical analysis software. results: improvements reaching statistical significance included increase in is order placement from 44% to 89% of admissions (p < 0.01), and admissions with documented is use increased from 32% to 59% (p < 0.01). los decreased from a mean of 3.7 days to 2.7 days (p 0.02). post-admission development of acs also decreased from 12% to 4% of admissions, but did not reach statistical significance (p 0.18). there was an additional increase in appropriate activity order placement and documentation of activity events. conclusion: improving education and outreach to patients and staff, including implementation of a disease-specific order set, can improve is use and activity events. the decline seen in incidence of acs development during hospitalization, though not statistically significant, and the decreased los are encouraging, and efforts continue to improve on these trends. background: painful vaso-occlusive crises (voc) are a frequent and debilitating complication of sickle cell disease (scd) and are thought to occur due to progressive blockage of the microvasculature with rigid sickle shaped red blood cells. any trigger that decreases the microvascular blood flow (mbf) can promote entrapment of sickled cells in the microvasculature and progression to voc. exposure to cold wind and changes in weather are common triggers of voc and are associated with increased frequency of hospitalizations for pain in patients with scd. there is limited experimental data on the physiologic effects of these factors on peripheral perfusion in scd. to study the effect of graded thermal stimuli on the peripheral mbf in scd. design/method: 17 scd and 16 control (healthy or sickle trait) subjects aging 13 to 39 years were exposed to their individual threshold temperatures for heat and cold detection, heat and cold pain via tsa-ii thermode that was placed on the thenar eminence. mbf was measured on the contralateral thumb using photo-plethysmography (ppg). the vasoconstriction response within the complex ppg signal was detected using cross-correlation technique. mean mbf was derived from the ppg amplitude during each of these stimuli and compared to baseline mbf. cross correlation analysis showed that cold pain caused significant vasoconstriction response in 67% of the subjects, followed by heat pain (58%), cold detection (36%) and heat detection (18%).there was a significant drop in the mbf during cold pain (p <0.0001), heat pain (p <0.0001), heat detection (p = 0.0005) and cold detection (p = 0.02) when compared to baseline mbf, with cold pain causing the greatest drop in mbf. thermal sensitivity and mbf responses were comparable between scd and controls. conclusion: exposure to graded thermal stimuli causes a progressive drop in mbf with exposure to cold pain eliciting the strongest vasoconstriction response. vasoconstriction occurred in the contralateral hand at an average of 11 seconds after the stimuli, suggesting a neurally mediated mechanism. although there was no significant difference in vasoconstriction responses between scd and controls, the drop in mbf in patients with sickle cell disease can increase the likelihood of entrapment of the sickled red blood cells, leading to vaso-occlusion. these findings are consistent with extensive reports in literature that exposure to cold weather is associated with a higher frequency of voc. this suggests that neurally mediated vasoconstriction is likely an important factor in the pathophysiology behind cold exposure leading to voc in scd. background: vaso-occlusive crisis (voc) is a major cause of hospital admissions in children with sickle cell disease (scd). although the use of clinical biomarkers in voc has been studied, especially with regards to acute chest syndrome (acs), there is less data regarding overall voc severity prediction. in addition new biomarkers such as platelet to lymphocyte ratio (plr), neutrophil to lymphocyte ratio (nlr), and lymphocyte to monocyte ratio (lmr) have been little studied with regards to scd. objectives: to identify whether admission laboratory values, changes from well baseline laboratory values, and new biomarkers such as plr, nlr, and lmr could predict severity of vaso-occlusive crisis in children with sickle cell disease admitted with voc. design/method: this was a retrospective single center observational study of admissions of voc in children aged 1 -21 years with hbss or hbs-b0thal from september 2014 to november 2017 excluding those on hyper-transfusion protocol or having an admission diagnosis of acs. univariate analysis was done using student's t-test, mann-whitney non parametric test, or fischer's exact test as appropriate depending on the distribution between admission laboratory data of complete blood count (cbc), reticulocyte count, comprehensive metabolic panel, lactate dehydrogenase (ldh), change from well baseline cbc values within 6 months previously, plr, nlr, lmr, and the development of complicated voc. complicated voc was defined as the development of secondary acute chest syndrome, prolonged admission duration > 5 days (120 hours), requirement of blood transfusion, and readmission within 30 days. results: a total of 109 admissions were studied. fifty-nine (54.1%) were female. of the 109, 50 (45.9%) were complicated with no significant differences in sex (p 0.447) or age (p 0.435). univariate analysis revealed significant elevations in total bilirubin (p 0.017), ldh (p 0.010), and platelet count (p 0.019) in those with complicated voc. there is also significant difference in the percentage change of platelet count from baseline with greater decline in uncomplicated voc (p 0.014). there were no significant differences in plr (p 0.186), nlr (p 0.775), or lmr (p 0.445). conclusion: elevations in total bilirubin, ldh, and platelet count in admission laboratory values are associated with developing complicated voc. in addition, those with complicated voc present with significantly less decline in platelet count from baseline well cbc. plr, nlr, and lmr do not seem to be useful predictive biomarkers for severity of voc. background: sickle cell disease (scd) causes health problems of varying frequency and severity. the only validated biomarker for children with scd is transcranial doppler. if reliable predictors existed for scd severity, children with scd could be treated according to risk category. many patients with scd face psychosocial or economic hardships, but these factors have not been evaluated as risk markers for medical or functional severity of scd. objectives: the goal of this project was to develop and stratify a preliminary list of psychosocial risk factors for health outcomes that could be used as scd severity predictors. st. vincent institutional review board. a list of potential psychosocial risk factors for adverse health outcomes was compiled based on assessment materials utilized by the sickle safe program (indiana's hemoglobinopathy newborn screening follow-up program). this list of 39 items was distributed to child abuse prevention (12) and scd (17) experts, who ranked each item on a likert scale of 1 (least important) to 5 (most important). mean scores were calculated using spss version 24; 163 assessments were retrospectively analyzed to determine psychosocial risk factor frequency. risk factors occurring in ≥15% of homes were considered high frequency events. overall, there was high agreement among experts on the risk factors that were considered the most important predictors of severe scd outcomes. the risk factor with the highest frequency (92%) was eligibility for public assistance programs. fifteen risk factors were rated ≥4 by the experts. four (26.7%) were high frequency events occurring in ≥15% of homes: a child with hbss or hbs 0thalassemia not taking hydroxyurea (15%); parent report that they had treated a fever (>101®f) at home in the past 6 months (25%); tobacco use by someone in the household (23%); and the family reporting significant psychosocial stressors in the past year (30%). tobacco use in the home was significantly correlated with several other risk factors (smoking during pregnancy [r = 0.503], other health concerns in the child [r = 0.459], and child having health insurance [r = -0.459]), suggesting that it is part of a constellation of health risk. in general, the risk factors that were rated as most important for health outcomes occurred less frequently in the sample. this study represents important progress toward identifying a group of psychosocial risk factors for scd severity, which is a necessary first step for future investigation of empirical relationships between candidate risk factors and scd outcomes. unitversity of cartagena, cartagena, bolivar, colombia s109 of s301 background: sickle cell disease is an autosomal recessive disorder characterized by a mutation in the -globin chain, which produces hbs. acute and chronic complications as aplastic crisis, acute chest syndrome, priapism, stroke, leg ulcers and primary/secondary prevention of stroke can be treated with simple transfusion or exchange transfusion. the latter offers advantages as lower iron overload, post-treatment hbs goal control, lower viscosity and improved microvascular circulation. but it is not a widely-used option because is associated with technical difficulties. objectives: standardization of a new partial exchange transfusion protocol in a group of patients with sickle cell disease, within the framework of a chronic transfusion program. design/method: this is a prospective descriptive study, which included 25 patients under 18 years with sickle cell disease (20 hbss, 5 hbs-tal), with indication of partial exchange transfusion in a chronic transfusion program, according to the institutional protocol; patients who fulfilled the inclusion criteria were enrolled in the study between february 2016 and december 2017. a registry of the medical and technical complications was made in each of the procedures. a database was constructed in excel, and the graph-pad prism® version 6 oc software was used for statistical analysis. the sequence is as follows: isovolemic phlebotomy and transfusion of packed red cells. depending of the recent hemoglobin level (48 hrs), we do the phlebotomy there: hb:7-7.9: 10 cc/kg, hb: 8-8.9: 15 cc/kg, hb>9: 15 cc/kg; isovolemic solution (ns 0,9%) there: hb:7-7.9: 10 cc/kg, hb: 8-8.9: 15 cc/kg, hb>9: 15 cc/kg and packed red cell transfusion there: hb:7-7.9: 15 cc/kg, hb: 8-8.9: 15 cc/kg, hb>9: 10 cc/kg. the safety of this exchange transfusion protocol was analyzed in 25 patients with sickle cell disease (176 procedures). there were no differences in the sex distribution, and the median age was 8 years. 80% of the population was homozygous. the indication of transfusion was 52.27%(92/176) primary stroke prevention, 44.31%(78/176) secondary stroke prevention and 2.84%(5/176) was other reason. a low percentage of complications was found (7.3%); of which, those of medical origin (hypotension and nausea/vomiting) were only presented in 2.2% of the total procedures. the standardization of this protocol was safe and its use could be extended to other low-income centers that treat patients with sickle cell disease that need chronic transfusion program including patient with hemoglobin level until 7gr/dl. we suggest do studies for measure the security and efficacy of this protocol in patients with acute complications. background: clinical trials that aim to achieve pain reduction have challenges achieving clinical endpoints as pain has no quantifiable biomarkers and may be unrelated to scd. furthermore, the threshold of seeking medical care differs between patients and vocs that occur at home are missed. we present a non-interventional, longitudinal study to identify vocs in patients with scd. objectives: to examine the longitudinal relationship between pros and biomarkers in subjects with scd before, during, and after a self-reported voc event, in order to build a model of in-home and clinical voc and to collect longitudinal pros and biomarker data from subjects that span voc events in the home, clinic and the hospital. design/method: longitudinal measures of pain, fatigue, function, activity, and biomarkers from scd patients in steady state and voc were studied over a six month period. patients self-reported pain, fatigue, function, and medication use using a novel epro tool. voc was reported in real-time, triggering a mobile phlebotomy team. blood was collected sequentially after self-reported voc (at home or hospital). blood samples were drawn two days after resolution of voc, as reported by the patient. during non-voc periods, blood was drawn every 3 weeks to establish a baseline. biomarkers included leukocyte-platelet aggregates and circulating microparticles, cell and soluble adhesion molecules, cytokines, inflammatory mediators and coagulation factors. patients wore an actigraphy device to track sleep and activity and rest. results: twenty-seven of thirty-five patients experienced a total of 286 days with voc >4 hr, of which only 58 days resulted in healthcare utilization. voc days had significantly higher pain and fatigue scores. voc days were associated with significantly decreased functional scores, with significantly greater decreases during vocs requiring medical contact compared to at-home vocs. different activity profiles were identified for non-voc, at-home voc and medical contact voc days by actigraphy monitoring. at-home voc days exhibited increased daytime resting compared to non-voc days. medical contact vocs had decreased average and peak activity, and increased daytime resting compared to non-voc days. a sleep fragmentation index trended up for both at-home (16%) and medical contact voc days (18%). significant changes during voc days were observed in: c-reactive protein (54% increase), nucleated rbc (34% increase), monocyte-platelet aggregates (25% increase) and neutrophil-platelet aggregates (35% increase), interleukin-6 (112% increase), interleukin-10 (19% increase) and tnfalpha (14% increase). the identification and assessment of at-home vocs through use of epros, actigraphy and biomarkers is feasible as demonstrated by this innovative at-home study design. background: risk-stratifying sickle cell disease (scd) patients and demonstrating response to disease-modifying therapies is challenging due to the phenotypical heterogeneity of scd. a pathogenic role for procoagulant von willebrand factor (vwf) via excess vwf high molecular weight multimers (hmwm) has been proposed, with variable reports of increased vwf and hmwm in crisis vs. steady-state in adults, but less so for vwf in children with scd. moreover, vwf and multimers have not been studied in sickle trait. objectives: our pilot study evaluated the potential for vwf antigen (vwf:ag) and hmwm on densitometric tracings to serve as biomarkers for disease severity or treatment response in children and young adults with scd compared to sickle trait (hbas) siblings. design/method: we evaluated vwf:ag, vwf multimers and retrospective clinical data from 10 hbss, 3 hbsc and 5 hbas subjects at steady state. one hbsc subject also had a crisis sample. median scd age was 17 years (8.0-20.1 years). 46% were female. scd severity was judged by annual vasoocclusive and acute chest events, or stroke/elevated tcd. eight of 13 (6 hbss and 2 hbsc) took hydroxyurea. four hbss subjects had severe scd, all of whom were chronically transfused. results: mean vwf:ag (normal 50-160 iu/dl) was higher for hbss (175+/-17.4) and severe hbss (195+/-33.5) compared to hbsc (103+/-3.2, p = 0.049 and 0.044, respectively); however, lacked statistical significance when compared to hbas (152+/-34.5, p = 0.52 and 0.41, respectively). vwf:ag was elevated in 7/10 (70%) steady-state, including 3/4 (75%) with "severe" disease on chronic transfusion and 4/6 (67%) taking hydroxyurea, in 1 hbsc crisis but no hbsc 3/3 (100%) at baseline. vwf:ag was high in 2/5 (40%) hbas siblings. four (31%) had increased hmwm at baseline: 1 hbss/severe disease/chronic transfusion, 2 hbss/hydroxyurea and 1 hbsc untreated. hmwm were increased only during vaso-occlusive crisis in 1 hydroxyureatreated hbsc subject. no ultra-large hmwm were observed. in this preliminary study, in young scd subjects, vwf:ag trended higher in hbss vs. hbsc and in severe hbss participants at a single time-point, but serial evaluations at baseline, in crisis and with optimized diseasemodifying therapy are needed to determine the potential of vwf:ag and hmwm as biomarkers for severity or treatment response. surprisingly, vwf:ag was high in some sickle trait subjects. since hbas is associated with some health challenges such as increased thrombosis risk, further examination of vwf and endothelial dysfunction in sickle trait may provide novel insights into its role as a biomarker. background: the 2014 national heart lung & blood institute(nhlbi) guidelines for acute management of voe recommends rapid evaluation and treatment of pain, including administration of a parenteral opioid within 30-minutes of triage or 60-minutes from registration, pain reassessment & repeat opioid delivery within 15-30-minutes. inf use has been increasing in peds due to its rapid onset and ease of administration. objectives: to evaluate ped utilization of inf & its effect on intravenous (iv) opioid administration and pain control for the treatment of voe. design/method: a retrospective review of 250 emr was performed on children with scd±2years presenting to a ped with voe (pain scores 6 on a 0-10 scale) from jan-june 2017. variables studied were median time (iqr,95%ci) from ped arrival to first-parenteral-opioid-administration, time-to-first-iv-opioid, first & final pain score, disposition and readmission rate. time-to-first-iv-opioid was also compared to historical data (jan-dec2012,n = 231) prior to inf protocol initiation. . additionally, 15% patients received iv opioids within 60 minutes of ed arrival in the inf+iv opioid vs. 40% in the iv opioids alone group (p<0.01). no differences in 72-hour-returnrates were found in any of the groups, including inf alone group. conclusion: use of inf in the ped for voe is an excellent strategy to shorten time-to-first-parenteral-opioidadministration, improve pain scores & improve adherence to the nhlbi guidelines. however we had 2 distinct unexpected findings: (1) delays in iv opioid delivery after inf use & (2) inf alone appeared to provide sufficient pain control without iv opioids for disposition home in 17% of voe patients. whether the latter reflects insufficient pain management or that there is a milder subgroup for whom inf alone is sufficient, requires further investigation. this study illustrates our experience with a ped-based inf protocol in terms of unanticipated delays in iv opioids and also discharges after inf alone. efforts are underway to further improve use of inf in voe management. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: folate supplementation is commonly included as standard management in patients with sickle cell disease. however, clear evidence supporting the clinical benefits of this practice is lacking. a single study demonstrated improvement on the occurrence of repeat dactylitis at a higher dose of folic acid. to compare clinical outcomes in pediatric patients with sickle cell disease treated with folate supplementation versus those who were not. design/method: this study was a retrospective chart review that included patients 3 to 23 years old with sickle cell disease type ss and s 0 followed at st. christopher's hospital for children. data collected included information about folate supplementation, red cell indices and the presence or absence of clinical outcomes including vaso-occlusive crisis requiring hospitalization in the last six months, acute chest syndrome, infections, asthma, sleep apnea, nephropathy, cerebral vascular disease, stroke and avascular necrosis. analysis of variance (anova) was used to evaluate mean differences between age, number of infections, number of voc events, hemoglobin, reticulocyte count, and mean corpuscular volumes. additionally, chi square analysis was implemented to evaluate differences in folate and non-folate groups for left ventricular remodeling (lvr), sickle cell nephropathy, asthma, obstructive sleep apnea (osa), nocturnal hypoxia, and avascular necrosis (avn). mean differences between the folate and non-folate groups were compared for patients on and off hydroxyurea therapy. one hundred and seven patients met inclusion criteria following review of clinical data. of the patients included in the study, 45 patients were found to be taking folate (42%), while 62 patients were not (58%). statistical analysis showed that there were no significant differences in the incidence of clinical outcomes between patients on folate versus those who were not on folate. of the patients who were not on hydroxyurea, hemoglobin levels were significantly higher in patients on folate versus those who were not (p = 0.053), but not significantly different for the patients on hydroxyurea. this study suggests that folate supplementation makes no significant impact on the red blood cell indices of anemia nor on the incidence of adverse clinical outcomes in children with sickle cell disease. however, a larger prospective study is needed to guide future considerations for folate supplementation in sickle cell patients in the clinical setting. background: tanzania ranks 3rd globally for the number of infants born annually with sickle cell disease (scd) but lacks a national newborn screening program. the prevalence of sickle cell trait (sct) and scd is highest in the northwestern regions around lake victoria served by bugando medical centre (bmc) a teaching and consultancy hospital in mwanza. bmc also houses the hiv early infant diagnosis (eid) laboratory that tests dried blood spots (dbs) from hivexposed infants. dbs can be tested for hiv and then retested for sickle cell trait and disease. to determine the prevalence of sickle trait and disease by region and district in northwestern tanzania using existing public health infrastructure. secondary objectives explored associations between sct, scd, malaria and hiv. design/method: the tanzania sickle surveillance study (ts3) is a prospective year-long cross-sectional study of hivexposed infants born in northwestern tanzania, whose dbs collected by the eid program are tested at bmc and available for further testing of sct and scd. samples from children ≤24 months of age were tested by isoelectric focusing (ief) and scored independently by two tanzanian staff as normal, sct, scd, variant, or uninterpretable. dbs samples scored as disease or variant were repeated. over the course of 9 months, 157 ief gels have been run. a total of 10,019 dbs samples have been scored, including 9,567 from children less than 24-months old. the overall prevalence of sct is 20.65% and the prevalence of scd is 0.99%, along with 0.10% hemoglobin variants. quality of the laboratory results is extremely high, with only 0.15% dbs samples yielding an uninterpretable result. geospatial mapping of the first 5,900 samples revealed a regional scd prevalence ranging from 0.3% up to 2.0% among the 9 regions served by bmc. the prevalence of sct and scd is very high in northwestern tanzania. geospatial mapping will identify high prevalence areas where targeted newborn screening can be started using existing public health infrastructure with minimal start-up cost and training. further data will enhance the accuracy of the map to the district level. background: pediatric patients with sickle cell disease (scd) could develop obstructive, restrictive or mixed abnormalities of pulmonary function (pf). several publications report progressive worsening of pf over time, which could lead to severe morbidity in adult patients with sickle cell disease. in adults with sickle cell anemia up to 20-30 % of mortality is related to lung disease. early intervention aimed at improvement of lung function could significantly decrease morbidity and possibly improve life expectancy. among disease modifying approaches commonly used in scd are hydroxyurea (hu) and chronic prbc transfusions. both interventions lead to increase of hemoglobin, decrease of hbs fraction, leading to decreased hemolysis. reports of effect of hu on pulmonary function are conflicting with some suggesting no effect and others proposing a slower decline of pulmonary function. the goal of our study is to evaluate effect of disease modifying therapies, like hu and chronic prbc on change of pulmonary function in pediatric patients with sickle cell disease. design/method: this study utilized a retrospective chart review of children with scd who had multiple pfts. we analyzed 286 pfts from 80 patients done during clinic visits. scd patients were divided into three treatment groups: hydroxyurea, chronic transfusions or neither. data was analyzed with linear correlations and analysis of variance (anova). comparison were made between the three groups specifically observing the changes in absolute numbers on pfts over time using the first and last pft the patient had. results: there were a total of 80 patients with multiple pfts (ranging from 2-7); control (40), hydroxyurea (23) and chronic transfusion (10). the mean changes of the control, and hydroxyurea for the pft parameters fev1 (-5.53 the chronic transfusion group demonstrated a small improvement in pfts over time for fev1 (0.300), fvc (1.300), fef25-75 (0.400), however there was a decline in fev1/fvc (-0.013). however, there was no statistically significant (p-value <0.005) in the difference in any pfts parameters between any of the groups. in children with scd there is a decline of pf parameters over time. although no significant differences were seen between the three groups it appears chronic transfusion may improve or limit the decline in pfts. larger studies need to be done to evaluate difference in pf decline in patients with scd patients. background: the use of mobile technology in health care has been a growing trend. patients with chronic diseases such as sickle cell disease (scd) require close monitoring to provide appropriate treatment recommendations and avoid complications. we conducted a feasibility study for patients with scd hospitalized for pain using our self-developed mobile application (tru-pain: technology resources to better understand pain) and a wearable activity tracker. subjective symptoms such as pain and objective data such as heart rate (hr) were measured. we aimed to 1) correlate nursing recordings with mobile technology recordings; 2) get feedback from patients about usability. design/method: we enrolled patients with scd >8 years old and <36 hours from admission for uncomplicated vasoocclusive crisis, excluding patients admitted to icu. patients were given an ipad and a wearable device. they were instructed to record in the application at least once per day and to keep the wearable on, removing only to charge. prior to discharge, patients completed a feasibility questionnaire. we enrolled 20 patients, 40% females, median age 17.5 (range 13 to 54) who were admitted for a median 5 days (range 2 to 8) for uncomplicated pain crisis. patients used the application throughout hospitalization and made one entry/day (range 0 to 2). pain scores recorded via tru-pain correlated well (r = 0.74, p<0.005) with pain scores recorded in emr. there was an average of 10,930 data points recorded per day, by the wearable, with a maximum of 54,693 data points/day. the median amount of hours of wearable data per day was 4.21 (maximum of 18.05). the hr recorded via the wearable correlated significantly with the hr recorded in emr (r = 0.69, p-value < 0.005). as for usability, 70% of patients indicated never having a problem with the technology, 90% found tru-pain 'very easy' or 'somewhat easy' to use, and 60% were 'very satisfied' with their participation in the study, indicating that it helped them track their pain. our pilot study during hospitalization shows strong potential for using tru-pain for patients with scd. pain data from application and hr from wearable correlated well to the emr data. according to the feedback received, our application was easy to use and helped patients track their pain. despite limitations of battery life, the use of wearable technology is feasible, providing additional data such as activity. we are optimistic that we can continue to improve our tru-pain system to help improve care in patients with scd. background: hydroxyurea, chronic blood transfusion, and bone marrow transplantation can reduce complications, and improve survival in sickle cell disease (scd), but are associated with a significant decisional dilemma because of the inherent risk-benefit tradeoffs, and the lack of comparative studies. these treatments are underutilized leading to avoidable morbidity and premature mortality. there is a need for tools to provide patients high-quality information about their treatment options, the associated risks, and benefits, help them clarify their values, and allow them to share in the process of informed medical decision making. objectives: to develop a health literacy sensitive, web-based, decision aid (ptda) to help patients with scd make informed choices about treatments, and to estimate in a randomized clinical trial the acceptability and effectiveness of the ptda in improving patient knowledge, involvement in decisionmaking and decision-making quality. design/method: we conducted qualitative interviews of scd patients, caregivers, stakeholders, and healthcare providers for a decisional needs assessment to identify decisional conflict, knowledge, expectations, values, support, resources, decision types, timing, stages, and learning, and personal clinical characteristics, and to guide the development of a ptda. transcripts were coded using qsr nvivo 10. stakeholders completed alpha and beta testing of ptda. we conducted a randomized clinical trial of adults, and of caregivers of pediatric patients to evaluate the comparative efficacy of the ptda, vs. standard of care. results: ptda (www.sickleoptions.org) was developed per decisional needs described by 223 stakeholders and finalized following alpha testing, and beta testing by 68 and 87 stakeholders respectively. in a randomized trial of 120 subjects considering various treatment options, qualitative interviews revealed a high level of usability, acceptability, and utility in education, values clarification, and preparedness for decision making of the ptda. a median 68% rated the acceptability of ptda as good or excellent and provided narrative comments endorsing the acceptability, ease of use, and utility in preparation for decision making. the ptda met international standards for content, development process, and efficacy with the exception of having a full range of positive and negative experiences in patient stories. compared to baseline ptda group had statistically significant improvement in preparedness for decision making (p = 0.005) and informed subscale of decisional conflict (p = 0.02) but not for decisional self-efficacy, knowledge, choice predisposition, or stages of decision-making. a ptda for patients with scd developed following extensive engagement of key stakeholders was found to be acceptable, useful, easy to use, to improve preparedness for decision making, and decrease decisional conflict. background: painful vaso-occlusive crisis (voc) accounts for the majority of emergency department (ed) visits and hos-pitalizations in sickle cell disease (scd). we are interested in studying mental stress and associated autonomic nervous system (ans) imbalance that cause vaso-constriction as possible triggers of scd pain. to this end, we developed a mobile phone application (app) to record daily pain frequency and intensity as clinical endpoints that might be predicted by ans parameters measured in the laboratory. in particular, we think that the aura may represent ans instability that precedes or even triggers change in blood flow and voc. objectives: to assess the feasibility of using an app to evaluate frequency and severity of voc and its potential association with mental stress and presence of aura. design/method: an app was developed for both ios and android systems to allow patients to track pain, stress, and aura. the idea was to create an app that was easy to use with the intent to only capture pain episodes, rather than detailed description of the pain. all scd patients were eligible and a parent version was available for younger children. de-identified data was automatically transferred to a hipaa compliant database via a cloud-based server interfaced to the main research project database. a feedback questionnaire was implemented after at least a month of utilization to assess usability. of the 51 scd patients enrolled, 39 participants utilized the app and 21 of the 23 participants that provided feedback indicated the app was easy to navigate. the mean pain scale was 6 out of 10 (standard deviation 1.97) for those that entered they had pain that day. although the mean stress level was 3 out of 10, there was a statistically significant correlation between increasing stress levels and increasing pain scores (p < 0.05). aura was reported by 26 patients, with 5 patients reporting more than 10 episodes. moreover, on days aura was present there was greater incidence that pain was present as well (p < 0.05). however, there was no statistically significant association between pain intensity and presence of an aura (p = 0.14). conclusion: consistent with prior research, reported pain intensity is significantly associated with reported stress intensity. although there was an association between presence of aura and pain, it did not seem to correlate with pain intensity. this uniquely designed app can monitor scd pain clinically and help understand the role of sickle dysautonomia in the genesis of scd pain. university of florida college of medicine, gainesville, florida, united states background: evidenced-based guidelines recommend the emergent evaluation of fever in children with sickle cell disease (scd). as the prevalence of bacteremia has decreased, outpatient management has become more common. however, fever can sometimes herald other complications of scd, such as acute chest syndrome, vaso-occlusive pain crisis, splenic sequestration, or aplastic crisis. institutional practices regarding fever management in scd remain variable, and little is known about the clinical outcomes of children hospitalized for uncomplicated fever. objectives: the primary objective was to determine the rate of bacteremia or scd-related complications per febrile episode in children with scd admitted to a single institution between january 2014 and june 2017 for uncomplicated fever. this was a retrospective cohort study of febrile patients up to 21 years of age with scd, any genotype, admitted to the university of florida during the defined study period. eligible patients were identified by a database search using admitting diagnosis codes for scd and fever based on the international classification of diseases 9th and 10th revisions. encounters were manually reviewed to confirm eligibility. patients were excluded if they had other indications for hospitalization apparent at the time of admission, such as an acute vaso-occlusive episode requiring parental narcotics, asthma exacerbation, or additional complications of scd. the database search identified 211 encounters, of which 83 were excluded based on confounding indications for hospitalization. sixty-three eligible patients accounted for 128 hospitalizations. the median age was 2 years (range 5 weeks-17 years); 60.2% were male. mean duration of hospitalization was 2.6 days (range 1-13 days). eight positive blood cultures were identified; six of these were classified as contaminants. bacteremia or the development of a scd-related complication was identified in 18 (14.06%) admissions. these included acute chest syndrome (n = 4), bacteremia (n = 2), splenic sequestration (n = 1), and red cell transfusion (n = 11). exploratory analyses of potential predictors of bacteremia or scd-related complications showed no association with the presenting white blood cell count or degree of fever (p = 0.36). of the patients classified as having a scd-related complication, 94% had hemoglobin ss disease and 78% had at least one prior documented complication. 64% of the patients transfused had at least one prior transfusion. conclusion: while improvements in preventative care have substantially lowered rates of bacteremia in children with scd, fever warrants careful evaluation for other acute scdrelated complications. providers should consider inpatient observation in select cases. additional studies are warranted to define subsets of patients suitable for outpatient fever management. background: children with sickle cell disease (scd) exhibit lower neurocognitive functioning than healthy peers, even in the absence of stroke. among the domains commonly affected, working memory (wm) seems particularly affected by disease processes and wm deficits have significant implications for academic achievement and disease selfmanagement. few interventions to improve working memory in pediatric scd have been evaluated. to determine the effects of cogmed, a homebased computerized wm training intervention, in children with scd using a randomized controlled trial design. design/method: participants (ages 7-16) with scd completed a baseline neuropsychological assessment and those with wm deficits were randomized to either begin cogmed immediately or enter an 8-week waitlist. cogmed is a homebased intervention completed on an ipad that consists of 12 increasingly challenging exercises targeting visual-spatial and verbal wm, practiced over 25 sessions. at the end of training, participants completed a post-intervention neuropsychological assessment, including tests of visual-spatial and verbal wm from the wechsler intelligence scale for children-fifth edition (wisc-v). results: ninety-one participants (m age = 10.43, sd = 2.93; 59% female; 69% hbss) enrolled in the study; 52% (n = 47) exhibited wm deficits and were randomized to either begin cogmed immediately or wait 5-8 weeks before starting cogmed. among those that have received the intervention and reached the end of their training period (n = 42), 27 participants (59%) completed at least 5 cogmed sessions, 19 (41%) finished at least 10 sessions, and 7 finished at least 20 sessions (15%). the mean number of completed cogmed sessions was 9.10 (sd = 7.77). paired samples t-tests revealed significant improvements on the working memory index (t[38] = -2.44, p = 0.020) and on the digit span (t[40] = -3.02, p = 0.004), and spatial span-backward (t[39] = -2.83, p = 0.007) subtests. improvements were especially pronounced for participants completing at least 10 sessions. partial correlations controlling for respective baseline scores indicated that the number of cogmed sessions completed was positively correlated with post-test scores on digit span (r = .38, p = .017) and spatial span-backward (r = 0.45, p = 0.004) subtests. among participants who completed at least 10 cogmed sessions, 77% scored in the average range or higher on the working memory index at the post-intervention assessment, compared to 58% at baseline. results support the efficacy of cogmed in producing significant improvements in wm. a dose-effect was observed such that participants who completed more cogmed sessions had greater improvements in wm. home-based cognitive training programs may ameliorate scd-related wm deficits but methods for motivating and supporting patients as they complete home-based interventions are needed to enhance adherence and effectiveness. background: sickle cell disease is associated with myriad complications that lead to significant morbidity and early mortality. hydroxyurea has been used successfully to reduce the incidence of these complications and has led to significant improvements in quality and duration of life. at children's minnesota we recommend hydroxyurea in all patients with hb ss/s 0 thalassemia as early as 5 months of age with a goal of starting all patients before 12 months of age. objectives: the purpose of this study was to evaluate the use of hydroxyurea therapy in young patients with sickle cell disease, with particular attention to those children less than one year of age. design/method: a retrospective chart review was conducted on patients less than 5 years of age with sickle cell disease who began hydroxyurea therapy between january 1, 2008 and december 31, 2016. the study population was divided into three cohorts based upon age at hydroxyurea initiation: cohort 1 (0-1 year), cohort 2 (1-2 years), and cohort 3 (2-5 years). outcomes included laboratory data, clinical events (hospitalization, dactylitis, pain crisis, transfusion, splenic sequestration, acute chest syndrome), and toxicity occurring in the first 2 years of life. results: a total of 65 patients were included in cohorts 1 (n = 35, mean age 7.2 months), 2 (n = 13, mean age 19.5 months), and 3 (n = 17, mean age 35.5 months). patients in cohort 1 had higher hemoglobin (p = 0.0003) and mcv (p = 0.0199) and lower absolute reticulocyte count (p = 0.0304) when compared to cohort 3. the wbc (p = 0.0007, <0.0001) and anc (p = 0.0364, 0.0025) were significantly lower compared to both older cohorts. however, no patient had therapy held because of neutropenia. the mean baseline hemoglobin f in cohort 1 was 31.5% compared to 19.7% and 16.5% in cohorts 2 and 3 respectively (p = 0.002, p<0.0001). the mean duration of therapy in cohort 1 was 31.3 months, compared to 57.6 months in cohort 2 (p = 0.018) and 29.1 months in cohort 3 (p = 0.401). during this time, hb f levels remained higher in cohort 1 (mean 29.9%) compared to cohorts 2 and 3 (mean 20.4%, p = 0.007 and mean 20.6%, p = 0.003). patients in cohort 1 experienced fewer hospitalizations (p = 0.0025), pain crises (p = 0.0618), and transfusions (p = 0.0426). there was no difference in toxicity between groups. hydroxyurea was used safely in infants 5 to 12 months of age and resulted in more robust hematologic responses and a decrease in sickle-related complications when compared with patients starting hydroxyurea later in life. children's national health system, washington, district of columbia, united states background: children with sickle cell disease (scd) have a significantly greater risk of silent or overt cerebral infarction than the general population. infarcts are associated with declines in cognitive functioning and academic achievement. while infarcts are reliably identified using mri, scans are expensive and occasionally necessitate sedation. moreover, mri's are not recommended for routine monitoring of cerebral infarcts. additional tools are needed for discriminating the presence of a cerebral infarct that are brief, noninvasive, inexpensive, and repeatable. objectives: to evaluate differences in performance on cogstate, a computerized neurocognitive assessment, in patients with scd with and without history of cerebral infarct. design/method: participants included 112 children with scd ages 7-16 (m = 10.61, sd = 2.91; 58% female; 70% s117 of s301 hbss) enrolled in a cognitive intervention trial. participants completed the cogstate pediatric battery, which measures processing speed, sustained attention, verbal learning, working memory, and executive functioning. history of silent or overt infarct was determined via health record review. participants also completed measures of intelligence (iq) and math fluency. results: participants' standard scores across most neurocognitive measures were lower than expected compared to the standardization sample (mean iq = 91.03, sd = 13.34). thirty percent of participants (n = 33) had a documented history of cerebral infarct. participants with a history of cerebral infarct scored lower on cogstate tasks measuring sustained attention (t[108] = 2.93, p = 0.004) and executive functioning (t[98] = 2.46, p = 0.016), as well as on a measure of math fluency (t[88] = 2.16, p = 0.033). receiver operating characteristic (roc) analyses demonstrated that the cogstate task measuring sustained attention was a fair discriminant of patients with and without a history of infarct (auc = 0.75, ci95 = 0.65-0.84, p = 0.0001), whereas iq score was not (auc = 0.58, ci95 = 0.46-0.71, p = 0.19). cogstate processing speed and sustained attention tasks fairly discriminated between patients with at least average or below average intelligence (auc = 0.77, ci95 = 0.67-0.86, p = 0.00001 and auc = 0.74, ci95 = 0.64-0.84, p = 0.0001, respectively). finally, the cogstate processing speed task was good at discriminating between at least average or below average math fluency (auc = 0.80, ci95 = 0.70-0.90, p<0.00001). multiple tasks in the cogstate pediatric battery appear to adequately identify patients with a history of cerebral infarcts. in addition, cogstate tasks appear to be fair predictors of impairments in iq and academic achievement outcomes. cogstate is inexpensive and can be easily administered in a medical setting with minimal training in approximately 20 minutes. results support the potential for cogstate to be used as a screening tool for medical and neuropsychological abnormalities in children with scd. st. christopher's hospital for children, philadelphia, pennsylvania, united states background: cardiovascular disease contributes to the morbidity and mortality of patients with sickle cell disease (scd). hydroxyurea therapy in scd has known clinical efficacy including improving anemia, decreasing episodes of vasoocclusive crisis and acute chest syndrome, and decreasing mortality. effect of hydroxyurea on cardiac function in children with scd is not well studied. an earlier study suggested the protective effect of hydroxyurea on left ventricular (lv) hypertrophy in scd. we hypothesized that hydroxyurea use would be associated with decreased lv remodeling and improved cardiac function. we aimed to evaluate the association between hydroxyurea use and lv remodeling and cardiac dysfunction in children with scd. design/method: we completed a retrospective study of patients with scd who were 10 to 22 years old, followed at st. christopher's hospital for children and had an echocardiogram completed in the past 18 months. data collected included gender, bmi, scd genotype, hydroxyurea use, chronic transfusion use, and 2d and doppler echocardiographic parameters. cardiac structure, geometry, systolic function, and diastolic function echocardiogram parameters were included. analysis of variance (anova) tests were performed to assess for statistical significance of differences in cardiac parameters between patients with and without hydroxyurea use. analysis of covariance (ancova) tests were performed to control for age. results: demographic and echocardiogram data was collected on all 93 patients who met inclusion criteria. of the 93 patients included, 31 (33%) were on hydroxyurea therapy. patients on hydroxyurea had significantly lower mean relative wall thickness (p = 0.026) and significantly higher mean peak early lv filling velocities (p = 0.032) and peak early lv filling/septal annuli early peak (e/ea) velocities (p = 0.002); however, only the e/ea velocities remained significant when controlling for age (p = 0.001). mean peak early lv filling velocities approached significance when controlling for age (p = 0.052). hydroxyurea therapy resulted in a significantly higher e/ea velocity, suggesting that these patients had worse diastolic function. it is possible that the patients initiated on hydroxyurea already had worse disease manifestations than those not on hydroxyurea, possibly accounting for the decreased diastolic function. when controlling for age, hydroxyurea use did not result in significant differences in cardiac structure parameters, systolic function parameters or cardiac geometry. prospective studies and larger sample size are needed to validate our findings, examine for additional statistically significant differences, and develop preventive strategies for cardiovascular disease in children with scd. background: acute chest syndrome (acs) is now the leading cause of death in children with sickle cell disease; mortality in the u.s. is reported to be 1-2% and is mostly due to respiratory failure. early transfusion improves clinical outcomes. although patients with concurrent asthma are considered at increased risk for poor outcomes, risk factors for respiratory failure in pediatric acs have not been well-defined. to determine whether specific epidemiological and clinical features of children hospitalized with acs are predictive of the need for mechanical ventilation. design/method: data from the kids' inpatient database were reviewed to identify patients age < 20 years with a discharge diagnosis of acs for the years 2003, 2006, 2009, and 2012 . outcomes were defined by the international classification of diseases, ninth revision, clinical modification code. data were weighted to estimate total annual hospitalizations according to hospital characteristics in the united states. trends in healthcare costs, length of hospital stay, transfusion, and mechanical ventilation use were analyzed using multivariable linear regression. in addition, multivariable logistic regression was used to ascertain specific clinical or epidemiologic factors associated with mechanical ventilation use after adjusting for patient and hospital characteristics. the total hospitalizations for acs were 5,018 in 2003; 6,058 in 2006; 6,072 in 2009; and 6,360 in 2012. reported use of mechanical ventilation ranged from 2.8% to 5.6% and was associated with non-black compared to black children (or, 1.53; 95%ci, 1.02 to 2.31) and the fall season (or, 1.36; 95%ci, 1.05 to 1.74), but not with age, preexisting asthma or hb-genotype. comorbidities of obesity (or, 3.35; 95%ci, 1.94 to 5.78), obstructive sleep apnea (or, 3.72; 95%ci, 2.23 to 6.20) and heart disease (or, 2.19; 95%ci, 1.47 to 3.27) were associated with mechanical ventilation use. the use of simple and exchange transfusion during all acs admissions ranged from 30.1% to 40.5% and 2.6% to 2.9%, respectively. among pediatric acs patients, those with obesity, obstructive sleep apnea or heart disease were at increased risk for respiratory failure and might benefit from early intervention (e.g., transfusion). surprisingly, asthma in children with acs does not appear to be a distinct risk factor for respiratory failure, and further studies are needed to clarify whether differences in treatment approach (e.g., addition of corticosteroids, bronchodilators) might impact on acs progression and/or severity even in high risk patients without asthma. objectives: to compare pulmonary functions between aa and k children with scd and to assess if a high hb f level contributes to better function. design/method: a cross sectional study was done on children with scd (hb ss disease) followed in comprehensive sickle cell programs. aa patients were followed at brookdale hospital, ny and k patients were followed in mubarak hospital, kuwait. children between the ages of 6 and 22 years who had pulmonary function tests (pft) done as a routine screening were enrolled. pft was done using spirometer and plethysmography. patients with congenital or anatomical lung abnormality, heart disease, pulmonary disease such as acute chest syndrome, acute asthma or pneumonia within 4 weeks were excluded. results: there were 74 children (37 in each group) with scd,. restrictive pattern on pft was seen in 18/37 (49%) of aa vs. 10/37 (27%) of k (p>0.05). obstructive pattern was seen in 6/37 (16%) of aa vs. 13/37 (35%) of the k group (p>0.05). in both groups, 13 children (35%) had normal pft. three/13 (15%) in the aa group had a hb f>20% as compared to 11/13 (85%) in the k group (p<0.01). abnormal pft was noted in 24/37 children (65%) in each group. hbf was >20% in 3/24 (13%) in the aa group vs. 15/24 (63%) in the s119 of s301 k group (p<0.01). in patients with abnormal pft, mean hbf was 10.4±8.4 in aa group, compared to 22.4±8 in k group (p<0.01). conclusion: abnormal pft is highly prevalent among children with scd in both groups. aa children are more likely to have restrictive disease and k to have an obstructive pattern. level of hbf did not seem to protect k patients from abnormalities on pft. this finding should emphasize the importance of performing pft as part of the initial evaluation of all children with scd. background: sickle cell disease (scd) is a life-threatening disease with varied clinical spectrum and severity leading to premature death. there is a lack of validated prognostic marker in scd. recent evidence suggests that inflammation and platelet adhesion plays a critical role in the pathophysiology of vaso-occlusion in scd. elevated mean platelet volume(mpv) values are associated with a higher degree of inflammation in many disease states but it's effect on sickle cell disease or it's severity is unknown. objectives: to analyze the role of mpv in predicting disease severity/mortality in pediatric patients with scd. design/method: this is a single center retrospective study and included patients with sickle cell disease between 6 months and 18 years of age during a 10-year period (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . demographic information, lab data and clinical information including acute chest syndrome (acs), priapism, transfusions, sepsis, pain crisis, avascular necrosis were collected. all laboratory data were collected in steady state with no crisis in the recent past 3 months. the disease severity score/probability of death was calculated using a validated model to predict risk of death in sickle cell disease (sebastiani et al. blood 2007) . pearson test was used to analyze correlation between mpv and probability of death. results: total no. of patients = 92; male 45 (48.9%); female 47(51.1%). median age is 6.0 years. all patients were of african-american origin. disease severity, hb ss -58(63%); hb sc -25(27.2%) and sickle-beta thalassemia 9(9.8%). patients on hydroxyurea has significantly lower mpv, p = 0.023 and this is independent of hb f levels. mpv has a significant positive correlation with the probability of death, p = 0.016 and correlation coefficient, r = 0.254. on subgroup analysis, the correlation is even more significant in the age group between 6 and 18 years, p = 0.004, r = 0.405. using linear regression model, with probability of death as a dependent variable and hydroxyurea, mpv as independent variables, mpv maintains a significant association with probability of death (p = 0.016). conclusion: mpv is an independent biomarker predicting disease severity and probability of death in pediatric patients with sickle cell disease. hydroxyurea a known disease ameliorating agent is associated with lower mpv values. this effect is independent of the levels of fetal hemoglobin and may be due to anti-inflammatory effect of hydroxyurea or effect on the platelets. background: major success with initial qi projects by the sickle cell care team at children's hospital has precipitated ongoing inclusion of the qi approach to many other aspects of patient care. objectives: to optimize scd patient care utilizing qi processes. design/method: success of the scd qi team's initial project on transcranial doppler studies (tcds) and a second more complex project on hydroxyurea (hu) adherence, led to additional projects on completion of key immunizations, rbc phenotyping, and vitamin d level testing. using similar processes and principles from the hu adherence project, plan-do-study-act (pdsa) cycles were used to conduct smallscale tests of change. patient chart prep sheets, created for bi-monthly pre-appointment chart prep meetings, were significantly modified to include these focused care qi objectives. because of difficulty with emr database capability, data collected from the emr was tracked in excel spreadsheets or other unique tracking vehicles for the various parameters. for example, due to the clinic's diffuse, geographically scattered population, many separate non-shared primary care emrs, and lack of a mandatory state immunization registry; immunization records needed to be retrieved from pcps, outlying hospitals, public health departments, and fqhcs, and added to the emr and excel database. starting in 12/2016, all such data was collected and updated monthly. in one year's time (2016 -2017) , the average immunization completion rate for seven key immunizations (pcv 13, pcv 23, hepatitis a, hepatitis b, meningococcal a, meningococcal b, and hpv) has increased by 20%. the biggest improvements were a 57% and 44% increase in completion for meningococcal a and meningococcal b, respectively. completion rate for rbc phenotyping rose from 34.8% to 74.4%. patients with at least one vitamin d lab test increased from 27.8% to 67.9%. since starting the tcd project in 2013, the percent of patients who have completed their annual tcd has gone from a baseline of 50% to a sustained value of > 80%. conclusion: these qi projects have not only increased adherence to national recommendations for care of scd patients, they have helped establish a scd clinic methodology to create and implement sustainable processes. having the focused care initiatives prominently displayed on the patients' chart prep sheet serve as a reminder to medical team members to check the status of that item. this methodology is currently being used to formulate additional qi projects on annual renal function parameters and specialty visits, such as annual eye and dental exams. background: dominican republic has a high burden of sickle cell disease, and 5-10% of children with homozygous hbss (sickle cell anemia, sca) will develop primary stroke. transcranial doppler (tcd) ultrasonography is an effective screening tool for primary stroke risk, but is not routinely available in dominican republic. hydroxyurea and blood transfusions are available, but no prospective screening and treatment program for stroke prevention has been implemented to date. (1) to screen a large cohort of children with sca living in dominican republic, using tcd to identify elevated stroke risk; (2) to determine the effects of treatments for stroke prevention (hydroxyurea for conditional velocities and transfusions for abnormal velocities). we hypothesized that both hydroxyurea and blood transfusions will decrease elevated tcd velocities and help prevent primary stroke. design/method: stroke avoidance for children with república dominicana (sacred, nct02769845) features a research partnership between cincinnati children's hospital and robert reid cabral children's hospital in dominican republic. the protocol, consent forms, and redcap database were prepared collaboratively and translated into spanish, and then irb approval was obtained at both institutions. in the initial prospective phase, children receive tcd screening over a 12-month period; those with conditional tcd velocities (maximum time-averaged velocity 170-199 cm/sec) receive fixed-dose hydroxyurea at 20 mg/kg/day, followed by dose escalation to maximum tolerated dose, while those with abnormal tcd velocities (≥200 cm/sec) receive monthly transfusions for stroke prevention. results: a total of 283 children were enrolled in sacred, with an average age of 8.7 ± 3.4 years. initial tcd screening revealed 200 (70.7%) normal, 63 (22.3%) conditional, 11 (3.9%) abnormal, and 9 (3.1%) inadequate velocities. among 48 children (25 males, 23 females, average age 6.8 ± 2.8 years) who initiated hydroxyurea at 20 mg/kg/day for conditional tcd velocities, 42 completed six months of treatment with expected hematological benefits including significant increases in hemoglobin concentration (7.5 to 8.5 g/dl) and fetal hemoglobin (15.8 to 28.4%). no clinical strokes have occurred in the treatment group. repeat tcd examination after 6-months of hydroxyurea treatment revealed 69% (29/42) with previous conditional velocities had normal tcd velocities. the prevalence of conditional tcd velocities in the dominican republic is high, indicating an elevated stroke risk among children with sca. hydroxyurea treatment is associated with improved hematological parameters, lower tcd velocities, and probable decreased stroke risk. sacred is an important prospective and collaborative research trial providing epidemiological data regarding tcd screening, stroke risk, and hydroxyurea effects among children with sca. background: red blood cell aggregation is a rheologic property that explains the shear-thinning behavior of blood. at lower shear rate blood flow, red cells tend to aggregate, s121 of s301 whereas in higher shear rate blood flow, these aggregates are dispersed. this property is especially important in the venous system, where low shear rate blood flow predominates. there is inconsistent data in the literature concerning aggregation and aggregability in sickle cell disease (scd). objectives: because the lorrca and myrenne instruments have been shown to be similarly effective methodologies in red cell aggregation measurements, we aimed to determine whether the measurement of aggregation indices in scd, by myrenne and by lorrca, is consistent in our lab. design/method: we measured aggregation in blood samples corrected to 40% hematocrit. aggregability was measured using 70kda dextran in the myrenne but not the lor-rca. aggregation index using lorrca was measured in 26 patients with scd and 22 healthy subjects enrolled in a study of blood flow between 2014 and 2017. aggregation and aggregability using the myrenne was measured in 67 patients with scd and 15 healthy subjects enrolled in a separate study of blood flow between 2008 and 2013. results: using lorrca, we found that aggregation index in patients with scd was less than that of healthy subjects (p<0.001). in the myrenne, aggregation at stasis was slightly higher in patients with scd compared to healthy subjects (p = 0.05) but aggregation at low shear rotation was not different. aggregability was higher in the patients with scd compared to healthy subjects at both stasis and low shear rotation (p<0.0001). red cell aggregation is an important determinant of low shear blood flow. deoxygenated venous blood is particularly important to low shear blood flow in patients with sickle cell disease. we found that two different aggregometers predict different aggregation results for scd. it is unclear why there is a systematic difference between the two methods, but there are some possibilities. first, the syllectogram in the lorrca is generated by the backscatter of light from the laser, while the myrenne measures transmitted light. second, the distance between the bob and cup in the lorrca is 300 microns, while the gap between plates in the myrenne is 50 microns, which might affect the disaggregation of red cells. further work is needed to understand the differences in red cell aggregation and aggregability when using these instruments, particularly when using aggregation as a predictor of blood flow and tissue perfusion. background: children with sickle cell disease (scd) are at risk of acute splenic sequestration crisis (assc). assc is a life-threatening complication characterized by splenomegaly, pain and severe anemia. assc most often occurs in young children with the most severe forms of scd and one-third of patients will have more than one episode. treatment is based primarily on expert opinion and includes blood transfusion and surgical splenectomy. objectives: we plan to assess the clinical practice patterns of physicians treating children with assc. design/method: a survey study was performed. the survey included six scenarios of severe scd with variation in age, hydroxyurea-use, and episode number of assc; questions focused on the acute and chronic management of assc. the survey was disseminated on three occasions over a six-month period, using an online survey tool, surveymonkey, to pediatric hematologist-oncologists participating in the american society of pediatric hematology-oncology hemoglobinopathy special interest group. the survey had a response rate of 43% (28/65). most respondents were recent graduates (61%; 17/28) practicing in academic urban centers with greater than 100 sickle-cell patients. seventy-nine percent (22/28) recommended hydroxyurea initiation in 9-12 m/o with severe scd. prophylactic penicillin after surgical splenectomy was continued by 93% (26/28) after 5 years. for the acute management of assc results did not vary despite patient age, hydroxyurea use, and the number of previous assc episodes. simple transfusion was preferred by 89% (25/28), with 54% (15/28) recommending slow transfusion and 36% (10/28) recommending routine simple transfusion. for the chronic management of assc, results varied based on patient age and the number of previous assc episodes. for a 12 m/o after the first episode, 36% (10/28) recommended observation and 32% (9/28) hydroxyurea initiation. for a 12 m/o with any prior episode of assc, 39% (11/28) recommended chronic transfusion therapy and 36% (10/28) surgical referral for splenectomy. for a 3 y/o after the first episode, 39% (11/28) recommended surgical splenectomy and 32% (9/28) increasing hydroxyurea dose. for a 3 y/o with any prior assc episode, 64% (18/28) recommended referral for surgical splenectomy. in this survey, we found most providers continue to recommend simple transfusions for assc and surgical splenectomy after two episodes. the majority of providers continue to delay referral for surgical splenectomy until age two, but earlier referral in children under two and use of chronic transfusion therapy were also reported. variability in chronic management highlights the need for further research of splenic sequestration. background: developing therapies for sickle cell disease (scd) is challenging in part because the accepted endpoint, vaso-occlusive crisis (voc), occurs infrequently, does not measure full disease burden, and is a measure of healthcare utilization. in phase 1/2 studies of patients with scd, voxelotor (gbt440) has demonstrated increased hemoglobin (hb) levels and reduced hemolysis and has been safe and welltolerated. voxelotor is being evaluated in the ongoing hope phase 3 trial. objectives: to report the innovative phase 2/3 hope trial design with novel primary and secondary outcomes to accelerate drug development. design/method: hope (nct03036813) is a phase 3, randomized, placebo-controlled, multicenter study of oral voxelotor in patients with scd (aged 12-65 years) with baseline hb 5.5-10.5 g/dl and 1-10 episodes of voc in the prior year. to accelerate clinical trials to support drug development, the study combines a phase 2 exploratory, dose-selection phase (group 1) with a pivotal phase (groups 2/3). patients in group 1 will be randomized 1:1:1 to voxelotor 900 or 1500 mg/day or placebo. analysis for dose selection will occur when the final patient has received 12 weeks of treatment. group 2 will continue enrollment with randomization 1:1:1 until dose selection based on analysis of the group 1 cohort. group 2 will allow for a seamless transition into group 3, which will randomize patients 1:1 to the selected dose or placebo. the final data analysis set will include group 2 patients who received placebo or the selected dose and all group 3 patients. the primary endpoint is an objective laboratory measure and surrogate of clinical benefit, increase in hb >1 g/dl, from baseline to 24 weeks based on voxelotor mechanism of action (inhibition of hb polymerization). this trial is the first to use a patient-reported outcome (pro), the 9-item sickle cell disease severity measure, as a secondary endpoint. this novel electronic pro, developed specifically for the hope study following fda guidance, will evaluate changes in scd symptom exacerbation and total symptom score from baseline to 24 weeks. additional secondary endpoints include measures of hemolysis, rates of voc, transfusions, and opioid use. the study was designed to enable selection of pro-defined symptom exacerbations or traditionally defined voc as the key secondary endpoint after the group 1 analysis. results: this study is ongoing. the hope trial, expected to complete enrollment by late 2018, will evaluate the efficacy and safety of voxelotor compared with placebo in patients with scd. supported by global blood therapeutics. background: inflammation, coagulation activation, oxidative stress and blood cell adhesion are elements of sickle cell disease (scd) pathophysiology. patients with scd have low levels of the omega-3 fatty docosahexaenoic acid (dha) and eicosatetraenoic acid (epa) in plasma and blood cell membranes. dha is a bioactive fatty acid with anti-inflammatory, anti-blood cell adhesion and anti-oxidant properties. altemi-atm is a novel dha ethyl ester formulation with a proprietary delivery platform (advanced lipid technology® (alt®)) that enhances oral dha bioavailability. the scot trial investigated the effects of altemiatm in children with scd. objectives: to demonstrate the effects of altemiatm on blood cell membrane omega-3 index and selected biomarkers of inflammation, coagulation, adhesion and haemolysis associated with scd. s123 of s301 design/method: children with scd, aged 5-17 years (n = 67), were enrolled. subjects were randomized to receive either placebo or one of three daily oral doses of altemiatm (12-26, 26-48 or 51-72 mg/kg/day dha) for two months. the effects of altemiatm on red blood cell (rbc), white blood cell and platelet membrane omega-3 fatty acids index (total dha + epa levels) were assessed after four weeks of treatment. the effects of altemiatm on markers of inflammation, adhesion, coagulation, and hemolysis were assessed after eight weeks of treatment. cell membrane dha and epa concentration was determined by using lc-ms/ms method. the percent changes from baseline on blood cell membrane omega-3 index and select scd biomarkers were compared between the three dose groups and placebo using a mixed-model repeatedmeasures (mmrm) analysis with baseline blood cell membrane omega-3 index, hydroxyurea use, and treatment as fixed effects and patient as a random effect. after four weeks of treatment, blood cell membrane dha and epa levels were significantly increased in all altemiatm doses (p<0.01). after eight weeks of treatment, significant reductions were observed in se-selectin (p = 0.0219), and d-dimer (p = 0.025) in patients exposed to altemiatm dose level 2 vs. placebo. hemoglobin was significantly increased at altemiatm dose level 1 versus placebo. plasma high-sensitivity c-reactive protein, lactate dehydrogenase, soluble vascular cell adhesion molecule-1 and white blood cell count showed improvement after 8 weeks of treatment in all three altemiatm doses levels but did not reach significance. conclusion: treatment with altemiatm enriches dha and epa in blood cell membranes of patients with scd and improves select sickle cell disease biomarkers of blood cell adhesion and thrombin generation. these findings provide insight into the mechanisms of action of altemiatm in sickle cell disease. brown university -hasbro children's hospital, providence, rhode island, united states background: despite clinical advances in the treatment of sickle cell disease (scd) in pediatric and young adult patients, pain remains a significant source of disease-related morbidity. physical therapy has been shown to be useful for the treatment of pain in children and young adults with various chronic illnesses of which pain is a significant component, however no data exists regarding potential benefits of physical therapy in pediatric and young adult patients with scd. objectives: to query healthcare providers and others involved in the care of pediatric and young adult scd patients regarding possible benefits of and barriers to physical therapy as a potential treatment modality. design/method: we conducted a web-based survey of healthcare providers within the new england pediatric sickle cell consortium (nepscc) in an attempt to identify potential benefits of and barriers to outpatient physical therapy in this patient population. results: nearly 92% of survey participants felt that physical therapy had the potential to be "somewhat beneficial" or "very beneficial" in pediatric and young adult patients with scd. a majority of physicians reported having referred patients with scd for physical therapy in the past. the most frequently identified perceived potential benefits included improved functional mobility, improvement of chronic pain symptoms, decreased use of opiates, improved mood symptoms, improved acute pain symptoms, and improved adherence with medications and clinic visits. significant perceived barriers identified included lack of transportation, time constraints, patient lack of understanding, and difficulty with insurance coverage. our study indicates that healthcare providers have an overwhelmingly positive view of the use of physical therapy in the management of pediatric and young adult patients with scd. significant barriers exist which need to be addressed. future research should focus on patient and parent perspectives regarding physical therapy, as well as a randomized controlled trial of a physical therapy intervention in this patient population. background: vitamin-d deficiency is fast becoming increasingly recognized in patients with sickle cell disease (scd). while it is estimated that these patients are five times more likely to develop vitamin-d deficiency, the exact clinical significance of this is largely unknown. given that this deficiency can be inexpensively and easily treated, our study sought to establish the prevalence of vitamin-d deficiency in our patient population and its relationship with disease severity. objectives: to estimate the prevalence of vitamin-d deficiency in patients with scd in our institution and to analyze their disease severity in relation to their vitamin-d level. design/method: through retrospective chart review we analyzed subjects that represent a cohort of patients followed at the adult and pediatric hematology services at university of miami with known diagnosis of scd that had a vitamin-d level drawn between january 01st, 2013 and august 31st, 2016. we conducted a cross-sectional study and recorded the first vitamin-d level during this period. patient demographics, medical and social history information were collected along with laboratory data. the number of admissions for vaso-occlusive crisis (voc) and acute chest syndrome within one year preceding the collection the vitamin-d level was also recorded. results: a total of 476 charts were reviewed, 279 adult charts and 207 pediatric charts. after exclusion, 119 patients were enrolled. subclinical vitamin-d deficiency is only evident on laboratory blood testing of vitamin-d (25-hydroxy) and according to this laboratory result patients were classified as sufficient (≥32 ng/ml), insufficient (<32 to 20 ng/ml) and deficient (<20 ng/ml). out of the 119 cases, 61.7% (74/119) were deficient, 21.7% (26/119) were insufficient and 15.8 % (19/119) were optimal. after statistical analysis two negative correlations were identified, increasing vitamin-d levels with decreasing white blood cell count (ci 95%-0.1931133 (-0.36057544, -0.01359017)and decreasing incidence voc (ci 95%-0.3149722 (-0.4684118, -0.1430889). conclusion: this study confirms that there is a significant prevalence of vitamin-d deficiency in patients with scd. furthermore, the results of this investigation proved that vitamin-d deficiency is associated with acute pain and leukocytosis in patients with scd. given the multitude of confounding factors that affect vitamin-d absorption and intake, multivariate analyses are required to truly further investigate this relationship. texas children's hospital, houston, texas, united states background: hemophagocytic lymphohistiocytosis (hlh) is a rare but life-threatening condition of hyper-inflammation that is characterized by splenomegaly, cytopenias, hyperferritinemia, hypertriglyceridemia, hemophagocytosis and coagulopathy. although timely diagnosis is imperative, it is often challenging as these individual signs and symptoms may occur in a variety of clinical conditions. to report a case of undiagnosed sickle cell anemia presenting with severe ebv viremia and associated hemophagocytic lymphohistiocytosis results: a 21-month-old previously healthy male presented with respiratory distress, increased fatigue, and a focal seizure following a two-week history of cough and lowgrade fevers. physical exam was consistent with hypovolemic shock and revealed significant splenomegaly. laboratory testing revealed severe hypoglycemia, acidosis and electrolyte disturbances including hyperkalemia, hyperphosphatemia, and hyperuricemia. labs showed a leukocytosis (wbc 53,000), severely low hemoglobin (1.9), and platelets of 56,000. coagulation testing revealed prolonged pt/inr and ptt, hypofibrinogenemia and a highly elevated d-dimer. additional workup was completed to determine etiology of acute presentation, given broad differential diagnosis. infectious studies were consistent with an acute ebv infection (plasma ebv pcr >550,000). elevated levels of soluble il-2 and ferritin completed 6/8 criteria for the diagnosis of hlh. bone marrow evaluation showed trilineage hematopoiesis with no abnormal blast population or hemophagocytosis. results from hemoglobin electrophoresis sent from the initial cbc sample were notable for hbs 72.5%, hbf 25.0%, and hba of 0%, confirming the diagnosis of sickle cell disease. the patient was started on hydroxyurea and penicillin and splenomegaly resolved. with supportive care, he demonstrated gradual improvement in symptoms and laboratory abnormalities, including normalization of soluble il-2, ferritin, cd163, il-18 levels, immunoglobulins, and declining ebv titers. nk cell function has remained abnormally low, not eliminating the possibility of acquired hlh despite spontaneous improvement. conclusion: splenic sequestration associated with sickle cell disease in combination with acute infectious mononucleosis could have explained many of the presenting symptoms including anemia, thrombocytopenia, and splenomegaly. however, it does not explain the unusually high ebv titer and degree of inflammation meeting diagnostic criteria for hlh, which raises concern for an underlying immunologic abnormality such as x-linked lymphoproliferative disorder (xlp). although testing for xlp was negative, he will require s125 of s301 continued monitoring in the future for signs of relapse. this case illustrates the complexity of diagnosing lymphohistiocytic disorders and the significant overlap in presentation between these disorders and other medical conditions. background: vaso-occlusive crisis (voc) is one of the most distressing occurrences in patients with sickle cell disease (scd). patient controlled analgesia (pca) is recommended by nih and expert opinions favor its early use. we aim to review the use of pca in patients with voc and to evaluate if its early use is associated with faster pain control and reduced length of stay (los). design/method: this retrospective single center study included all pediatric patients admitted and treated with pca for a severe voc from 2010 to 2016. "early" use was defined as start of pca within 48 hours of arrival in the emergency department (ed) and "late" use after 48 hours. time to reach adequate analgesia was defined as oucher, verbal scale or faces pain scale < 5/10 obtained twice consecutively in a 4-hours interval. time to reach adequate analgesia and los were compared between early-pca and late-pca groups. results: a total of 46 patients presented 87 episodes of voc treated with pca during the study. sixty-one episodes (70%) were treated with early-pca and 26 (30%) with late-pca. both groups were comparable in terms of age (13.2 vs 12.8 years old), gender (55.8% female vs 57.7%), hemoglobin phenotype (80.3% hbss vs 76.9%), but median pain score at admission was higher in early-pca than in late-pca (9/10 vs 7/10, median difference 1 (95% ci 0, 2). early-pca was associated with a median reduction in los of 3.15 days (95% ci 1.65, 4.82) (median early-pca los 6.4 vs late-pca 10.0 days). time to reach analgesia could be evaluated only in a subset of patients (20 in early-pca and 12 in late-pca group). although time to reach adequate analgesia tended to be shorter in the early-pca group, it was not statistically different: median102.9 hours vs 123.5 hours, difference of 30.4 (95% ci -4.0, 72.5). side effects were observed during 29 (33.3%) pca treatments (19/61 (31.2%) episodes in early-pca, 10/26 (38.5%) in late-pca group) among which 16 (18.6%) were significant adverse events. these were observed in 15 patients who required interventions: 2 desaturations requiring oxygen without intubation, 8 neurologic abnormalities (hallucinations, visual abnormalities, no stroke), 6 urinary retentions. conclusion: early use of pca for severe voc was associated with a reduced length of hospital stay despite that these patients had higher pain score on admission. prospective studies are needed to support these positive outcomes. background: acute chest syndrome is one of the leading causes of death in children with sickle cell disease1-2. while the cause of acute chest syndrome most commonly is not identified, fat embolism and infectious causes are believed to be most common. with an extremely high mortality rate, rapid identification and initiation of therapy is essential for survival. case presentation: we describe the case of an 18-year-old female with sickle cell sc disease who was admitted for vasoocclusive pain crisis and quickly progressed to multi-system organ failure due to fat embolism syndrome and parvovirus b19 infection objectives: the case highlights the presentation and diagnosis so other providers can optimize outcomes for those with this under-recognized syndrome design/method: her parvovirus studies returned after 7 days which showed: parvovirus b19 dna pcr detected; parvo igg 1.99 (positive > 1.1); and igm 12.98 (positive > 1.1). the patient experienced an approximately 2.5 g/dl drop in hemoglobin(8.7 to 6.0 g/dl/24 hrs) with progressive thrombocytopenia (from 269,000 to 54,000/ul) and a peripheral smear showed microcytic,normochromic red cells with nucleated rbcs and occasional nuclear budding, slight polychromasia, schistocytes, and polymorphic cells with toxic granules that suggested leukoerythroblastosis. she was emergently transferred to the regional quaternary care hospital for ongoing ecmo therapy where she experienced a change in her pupillary exam prompting a stat ct scan that showed severe, diffuse cerebral edema with transtentorial herniation. the decision was made to withdraw life-sustaining therapies and her family refused a post-mortem autopsy examination. fat embolism syndrome is a severe and uncommonly recognized complication of sickle cell disease, seen most commonly in those with a non-ss phenotype and previous mild disease course who present with severe, unrelenting vaso-occlusive pain episode and/or acute chest syndrome that progresses to respiratory distress with altered mental status and cutaneous changes. rapid identification and initiation of exchange transfusion therapy should be initiated with clinical suspicion because of the extremely high mortality rate. although previously considered rare, it needs to be considered in the differential diagnosis of more commonly encountered complications of sickle cell disease. background: patients with sickle cell disease (scd) experience vaso-occlusive crisis (voc), which results in extreme pain, often requiring opioids and admission. genetic and environmental factors affect the frequency and severity of these episodes. previous research has born conflicting evidence on whether environmental temperature is contributory. edmonton, alberta is the northern most city with a population over a million in north america. there is an increasing sickle cell population which is exposed to extreme winter conditions. this provides a suitable population and atmosphere to study the influence on cold external temperatures in scd. this study sought to identify if pediatric patients with scd, experience greater morbidity in cold external temperatures. board approved retrospective case control series. patients were identified through a clinical database, and emergency visit, phone call and admission data was collected over a fiveyear period. the average, minimum and change in temperature on day of presentation, 24 and 48 hours prior, was collected from the government of alberta, and was statistically analyzed using descriptive statistics, to determine the relation to vaso-occlusive events. results: one-hundred and eighteen patients were identified, and 258 voc events reviewed. the mean patient age was 6.6 years of age with a range from 0.3-17 years old. the female to male ratio was equivalent with 133 female (51.6%) and 125 male (48.4%) voc events. eight records (3%) had docu-mented cold exposures. the analysis between the temperature and the frequency of events did not yield significant correlation. average and minimum temperature on day of admission had the largest percentage of voc events occur at mild temperatures, from -4.99 to 20 • c and -4.99 to 5 respectively. change in temperature on day of admission, 24 and 48 hours had the largest percentage of voc events at a mild to moderate change in temperature of 10-15 degrees. data at 24 & 48 hours prior to admission showed similar results. secondary data analysis accounting for the lower proportion of extreme weather days in comparison to moderate temperate days showed no significant impact. there was no correlation of average, minimum or change in temperature on day of admission, 24 or 48 hours prior. multiple cofounding factors likely contribute to these results. as it was a retrospective study many confounding and precipitant factors may not be recorded or identified. a prospective study to better record specific cold exposure is warranted. children's national health system, washington, district of columbia, united states background: achieving optimal anticoagulation with unfractionated heparin (ufh) in pediatric patients receiving extracorporeal membrane oxygenation (ecmo) is often challenging due to antithrombin (at)-mediated heparin resistance (hr). intermittent at dosing during pediatric ecmo support does not maintain adequate at levels. continuous at infusion (cati) presents an alternative strategy to achieving consistent goal at levels and optimizing heparinization. however, cati during pediatric ecmo has not been adequately studied. objectives: to describe our center's experience with an ecmo cati protocol. design/method: in 2014, we modified our ecmo anticoagulation protocols to include ufh titration according to anti-factor xa (anti-fxa) levels and cati in patients with at-mediated hr. the cati rate was calculated using baseline and goal at levels while accounting for the circuit volume. cati was administered with ufh into the circuit via a s127 of s301 y-infusion set. at and anti-fxa levels were monitored every 6 hours. recombinant at (r-at) concentrate was used at our center until 2015 with subsequent transition to a plasmaderived at (pd-at) concentrate. due to the longer half-life of pd-at concentrate, the protocol was modified so cati is stopped once target at and anti-fxa levels are achieved. we conducted a retrospective study of all patients who received cati during ecmo support at our center. data are reported as median and interquartile range and compared using the mann-whitney u test. two-tailed p-value <0.05 was considered statistically significant. since 2014, 24 patients [13 males, age 1 month (0.03-8)] on ecmo support received 27 catis (12 rat, 15 pd-at) per our protocol (3 patients received 2 pd-at infusions during one ecmo run). the duration of cati was 48 hours (23-72). cati administration led to significant increases in at and anti-fxa levels from baseline of 43% (39-53) and 0.11 units/ml (0.08-0.22) to the first level within goal of 64% (55-83) and 0.39 units/ml (0.35-0.5), respectively (p<0.00001). the respective times to achieve goal at and anti-fxa levels were 9 hours (5-21) and 13 hours (6-23). the respective peak at and anti-fxa levels were 83% (70-99) and 0.53 units/ml (0.35-0.63). during cati, no patient required circuit change, 1 patient developed cannula thrombosis and 5 patients experienced non-fatal major bleeding. conclusion: cati in pediatric patients receiving ecmo support with close monitoring of at and anti-fxa levels was associated with significant rapid increase in at, optimization of heparin effect, and reduction in thrombotic complications without increase in major bleeding compared to prior reports. a prospective study of this at dosing strategy is warranted. children's hospital of orange county, orange, california, united states background: inherited factor xiii (f13) deficiency is a rare bleeding disorder with wide heterogeneity in clinical manifestations ranging from mild bruising, and mucosal and umbilical stump bleeding to spontaneous, severe intracranial bleeding. the bleeding phenotype is influenced not just by zygosity of the fxiii mutation alone, but also by co-inheritance of variants in other clotting protein genes that also play a major role in clot formation and stability. we present a series of three siblings found with f13a1 gene variant and platelet dysfunction linked to bleeding phenotype. design/method: retrospective chart review of the index case, coagulation studies and whole gene sequencing. the index patient presented at two years of age with a subdural hematoma after a fall, requiring emergent craniotomy. a week after initial evacuation, she re-bled, prompting an extensive work-up for potential bleeding disorders, including f13 activity, von willebrand profile, comprehensive fibrinolysis panel, pai-1 antigen level, platelet mapping thromboelastogram (plt-teg), and f13 genetic analysis. the patient's identical twin and older sibling, who had symptoms of bruising, underwent a similar evaluation. the index patient demonstrated consistently low f13 activity (31-49%), and platelet function testing revealed decreased response to adp agonists. the twin and older sibling had normal f13 levels, and only slightly decreased response to adp in platelet studies. whole gene analysis of f13 and 21 other genes on our next generation panel, revealed several intronic deletions in the index patient that were not shared by her siblings, which likely account for her decrease in circulating f13 levels. her symptoms have responded well to monthly treatment with factor 13 concentrate. all three children shared the f13 variant, pro564leu, previously described as a risk factor for intracranial hemorrhage. the f13 mutation, pro564leu, has been associated with intracranial hemorrhage in young women, but the presence of the variant alone may not be enough to cause a severe bleeding phenotype. family studies identified novel deletions in the index patient which may account for her decreased f13 levels, which would have been overlooked with standard sequencing. future studies, including evaluation of 'platelet' f13 levels, should be performed when platelet dysfunction is detected. further laboratory and clinical evaluation is required to delineate the long term implications of the interaction of even mild f13 deficiency if present with additional clotting disorders such as the platelet function defect in these siblings. background: acquired hemolytic anemia can occur due to mechanical shearing of red blood cells and is classically seen in patients with prosthetic heart valves. there are reports of this same traumatic effect with other repairs, including annuloplasty. following valvular procedures flow disturbances can exist across the valve that lead to shear stress and hemolysis. although von willebrand disease (vwd) is typically seen due to an inherited disorder in the pediatric population, flow disturbances in the setting of valve abnormalities can lead to acquired von willebrand syndrome (avws). von willebrand factor multimers become unfolded and elongated in the setting of shear stress resulting in increased susceptibility to cleavage by adamsts-13. specifically, loss of high molecular weight multimers (hmwms) can lead to a syndrome akin to type 2a vwd. objectives: to describe a case of mechanical hemolysis with acquired type 2a vwd design/method: a 3-month-old girl with history of hypoplastic left heart syndrome and severe tricuspid valve insufficiency underwent norwood procedure, blalok-taussig shunt placement and subsequently a bidirectional glenn and tricuspid valve annuloplasty. during the following month she requires weekly red blood cell (rbc) transfusions due to intermittent anemia. she also experienced bloody stools and dark urine. laboratory evaluation was notable for normocytic anemia, reticulocytosis, elevated lactate dehydrogenase, and low haptoglobin consistent with hemolytic process. immune-mediated hemolysis from transfusion reaction or presence of autoimmune or alloimmune antibodies testing was negative. to investigate gi bleeding, work up for vwd revealed normal vw activity and antigen but with loss of high molecular weight multimers consistent with acquired type 2a vwd. in consultation with cardiology, it was felt her tricuspid valve insufficiency jet could be leading to mechanical hemolysis and avws. a repeat echo showed persistent moderate tricuspid insufficiency but no other significant changes. due to the patient's continued need for weekly rbc transfusions she was subsequently trialed on pentoxifylline which is used in adult patients to decrease blood viscosity and increase erythrocyte flexibility in patients with mechanical hemolysis. her transfusion needs remained the same and the medication was discontinued after two weeks. she required one transfusion a week later but no transfusions since that time. although not commonly seen in pediatric patients, the diagnosis of mechanical hemolysis accompanied by avws should be pursued in a patient with congenital heart disease with significant anemia and/or bleeding. the work up in these patients is difficult as echocardiograms can be inconclusive thus an extensive hematologic evaluation is usually necessary. objectives: our aim was to assess incidence of and potential risk factors for central line-related dvt at our institution between 2011-2016. additionally, our goal was to analyze if that incidence differed between the three central line types and identification of line-specific risks. design/method: a retrospective chart review of 377 central line placements in pediatric patients at cleveland clinic between 2011-2016 was conducted. data included demographics, potential risk factors, line characteristics and any related thrombotic events. the study cohort consisted of 377 lines in 326 pediatric patients aged 1-18 years of age. there were 1.5 thrombi (95% ci 1.0-2.3) per 10,000 line days. statistically significant risk factors for thrombus include diagnosis group (liquid tumor highest rate of 16%, solid tumor lowest at 2%), type of line (picc 5%, broviac 29%, and mediport 4%), location of line, greater number of lines per patient, peg asparaginase (23% vs 4%), sepsis, and history of procoagulant state. line characteristics such as lumen size and number of lumens were not identified as a significant risk. there was a significantly higher rate of thrombus in 2016 than in the previous years when pooled (12% in 2016 vs 4.3% from 2011-2015, p = 0.020). the incidence of dvt in pediatric patients at our institution was highest with broviac lines, and significant risk factors in our patient population included liquid tumor, femoral vein location, peg asparaginase, sepsis, and history of a procoagulant state. the incidence of thrombi was highest in 2016, and therefore highlights the urgent need for improvement in nationwide hospital practices to minimize risk of thrombi formation and early detection in the higher-risk s129 of s301 populations. there is still much to be learned regarding the characteristics specific to different central lines, which would influence thrombi formation. nyu winthrop hospital, mineola, new york, united states background: pediatric immune thrombocytopenic purpura (itp) is an autoimmune disorder with platelet counts <100000 causing increased risk for significant hemorrhage. there is increased immunologic platelet destruction due to production of specific autoantibodies along with inhibition of platelet production. few randomized trials exist to guide management and ultimately each patient requires an individualized treatment plan. itp may be acute (diagnosis to 3 m) or chronic (> 12 months). one of the treatments of chronic itp is laparoscopic splenectomy (ls), which is very well tolerated. a rare complication of ls is splenosis, an autotransplantation or implantation of ectopic splenic tissue within the abdominal cavity or in any other unusual body compartment. splenosis is sometimes associated with relapsed itp due to preserved immune activity. the usual management of symptomatic splenosis is surgical resection. objectives: to describe medical management in a young patient with itp relapsed due to extensive unresectable splenosis following ls design/method: our patient was originally diagnosed at 2 years with itp and was treated with ls at 5 years of age for chronic severe thrombocytopenia and persistent bleeding not responding to first line therapies. she tolerated it well and had a complete response (cr) defined as a platelet count of >100000 measured on 2 occasions >7 days apart and absence of bleeding. she maintained a normal platelet count for twelve years after which she relapsed (loss of response after cr) with severe thrombocytopenia and hematuria necessitating high dose steroids. ct scans showed multiple wellcircumscribed soft tissue masses in the left lower quadrant adjacent to uterus and left ovary, involving left omentum and the anterior abdominal wall partly. findings were confirmed by damaged rbc nuclear scan to be splenosis. during laparoscopy the splenosis lesions were deemed too extensive and were not resected completely to avoid postoperative morbidity. she was started on sirolimus around the same time for treatment of her relapsed itp and steroids were weaned off. results: eight months since beginning sirolimus with therapeutic levels she remains in cr with no bleeding and has not required any steroids, immunoglobulins or anti d immunoglobulin. conclusion: sirolimus is a safe and effective steroid-sparing agent in treatment of chronic itp. this is the first instance of a patient with poorly resectable splenosis responding well to medications for itp. more data is needed regarding the longterm efficacy of such an intervention and whether it will eliminate the need for a second surgery in relapsed itp patients with extensive splenosis. background: storage pool disorders affecting platelets result in bleeding symptoms related to a deficiency or defect in alpha granules or delta granules. in delta-storage pool disorders (dspd,) there is a deficiency of the delta granules and their constituents, which results in the inability of platelets to properly activate as well as lack of proper constriction of blood vessels during bleeding episodes. amongst patients with dspd, females most commonly present with menorrhagia, while males tend to present with epistaxis and easy bruising. the international society on thrombosis and hemostasis (isth) developed a screening bleeding assessment tool (bat) for mild bleeding disorders, shown to be a validated tool in children. diagnosis of dspd is classically made with a platelet electron microscopy (pem) value <3.69 delta granules per platelet (dg/pl), but recently lower diagnostic thresholds of 2 dg/pl or even 1.2 dg/pl have been suggested. objectives: evaluate the correlation between pem and bleeding scores, and also examine various cut-off values used to diagnose and risk stratify patients with dspd. design/method: retrospective chart review of 96 pediatric patients followed by hematology with a diagnosis of dspd was performed. clinicians obtained bleeding scores for each patient as standard of care in the hemostasis clinic. quartile ranges were established to appropriate three stages of severity based upon bleeding scores. statistical analysis was performed using software r and exploratory data analysis to evaluate for a correlation. results: amongst all patients, the average bat score was 6.17 and pem was 2.37 dg/pl. the average bleeding score for pem between 3.69 dg/pl and 2 dg/pl was 6.17, while the average bleeding score for pem below 2 dg/pl was 4.65. the correlation coefficient between pem and bleeding scores is 0.30. using a threshold of 2 dg/pl, 31% of patients would have met diagnostic criteria. quartile ranges for the bleeding scores are as follows: 1st quartile was 2-4, 2nd quartile was 5-7, and 3rd quartile was >8. conclusion: patients with a more marked granule deficiency do not exhibit a more severe bleeding phenotype, suggesting proper platelet function is not solely determined by granule quantity in these patients. bleeding severity may be more appropriately assessed with bleeding scores rather than pem values, and using quartile ranges may aide in risk stratification and therapeutic interventions for dspd patients. further work remains to determine the optimal diagnostic threshold of pem dspd in pediatric populations. texas children's hospital, houston, texas, united states background: warfarin management has many challenging aspects including pharmacogenomics, food and drug interactions, lack of standardized dosing, patient compliance, tracking lab results from multiple lab locations, and the potential for significant bleeding or thrombotic complications. a literature review revealed limited data highlighting anticoagulation monitoring workflow and emr documentation and specifically, no data in the pediatric population. historically, the texas children's hospital cardiology and hematology centers were each documenting anticoagulation data within the epic tm system differently. epic's tm original design for anticoagulation documenting resulted in the necessity to duplicate documentation in order to see at-a-glance critical anticoagulation monitoring information. objectives: the objective of this project was to standardize inr documentation across departments to reduce the risk of patient safety events and improve workflow. design/method: a workgroup assembled consisting of nurses from the cardiology and hematology departments, along with staff members from the epic tm is support group. the workgroup identified current documentation practices, available epic tm tools, and brainstormed ideas to streamline and improve both documentation with the current epic tm tools. physician partners were identified in cardiology, hematology and coagulation laboratory to gain their input. a new anti-coag (ac) encounter was developed and first made available in an epic tm practice environment, then once approved, epic tm written education and training session were completed by both departments' staff. results: surveys were sent to 19 health care providers in the cardiology and hematology centers prior to the new ac encounter, and also to 27 health care providers six months after implementing the ac encounter. six responses were received for each survey. the pre-implementation survey showed the most problematic part of the documentation system for anticoagulation was no single place in the emr to find a complete anticoagulation picture. post ac encounter implementation survey results revealed more health care providers using the epic tm inr reminder pool, less time needed to compile a report of three months of anticoagulation information, less time needed to document individual encounters, less locations needed to document ac information and decreased amount of types of documentation used. standardized ac encounters improves workflow with less time needed to document and compile information, less types of documentation utilized and easier access to patients ac information. next steps include retrospective review of patients' inr time in therapeutic range to determine if there was an impact on patient compliance and continue to evaluate and modify the ac encounter to enhance user friendliness. caitlin tydings, jennifer meldau, christine guelcher, carole hennessey, eena kapoor, michael guerrera, yaser diab s131 of s301 children's national health system, washington, district of columbia, united states background: venous anatomic abnormalities (vaas) are considered a risk factor for developing deep vein thromboses (dvts) that occur as a result of significant alterations in venous blood flow. identification of predisposing vaas can be challenging. hence, diagnosis can be delayed or overlooked especially in pediatric patients. dvts in children or adolescents with predisposing vaas have been only described in sporadic case reports and small case series. objectives: to describe characteristics and outcomes of dvts in pediatric patients with underlying vaa treated at our center. design/method: we conducted a retrospective chart review of all pediatric patients with objectively confirmed extremity dvt treated at our institution over a 6-year period from 2011 to 2017 and identified all patients with underlying vaas. patients were managed according to standardized institutional protocols based on published guidelines. post-thrombotic syndrome (pts) was assessed at our center using the manco-johnson instrument. relevant data were collected and summarized using descriptive statistics. during the study period, 20 of 227 pediatric patients (9%) [14 females, median age 17 years (range 11-20)] diagnosed with extremity dvt at our center were found to have an underlying vaa. vaas included may-thurner anomaly (13 patients), venous thoracic outlet obstruction (5 patients) and inferior vena cava (ivc) atresia (2 patients). additional provoking factors were identified in 14 patients at time of presentation. dvt locations included upper extremity veins (5 patients), lower extremity veins (9 patients) and lower extremity veins and ivc (6 patients). the majority of dvts [17 patients, (85%)] were completely occlusive. high risk thrombophilia (defined as inherited deficiency of antithrombin, protein c, or protein s, or antiphospholipid antibody syndrome) was present in 6 patients (30%). all patients were treated with therapeutic anticoagulation with 6 patients continuing indefinite anticoagulation. endovascular interventions were performed in 18 patients and included percutaneous pharmacomechanical thrombectomy and/or catheter-directed thrombolysis (15 patients), balloon angioplasty (11 patients) and stent angioplasty (9 patients). surgical interventions included thoracic decompressive surgery (5 patients) and surgical thrombectomy (1 patient vvas represent an important risk factor for developing extensive extremity dvt in adolescents. this special population is at risk for short-term and long-term com-plications. early identification and correction of vaas may improve outcomes. however, multicenter, prospective studies are needed for developing optimal evidence-based treatment approaches. alexander glaros, roland chu, sureyya savasan, meera chitlur, madhvi rajpurkar, yaddanapudi ravindranath children's hospital of michigan, detroit, michigan, united states background: acute budd-chiari syndrome (bcs) is a rare thrombotic emergency in children, and etiologies/treatment are less well-defined than in adults. in adults, a systematic approach including anticoagulation, relief of venous obstruction, and treatment of the underlying cause has proven successful. more recently treatment has tilted towards aggressive surgical interventions, which carry significant risk and are often not feasible. objectives: review our experience with three different patients with bcs and suggest a mechanistic based approach to treatment. the records of three patients with bcs were reviewed and their presentations, etiologies, treatment, and outcomes were reported. results: patient a was a 17-year-old female with paroxysmal nocturnal hemoglobinuria who presented with recurrent worsening abdominal pain over several months. narrowing of inferior vena cava (ivc) and hepatic veins was noted on imaging. liver transplant was not considered surgically feasible. she was treated with eculizumab, steroids, and anticoagulation with restoration of hepatic venous flow in 4 weeks. patient b was a 14-year-old male with several weeks of right upper quadrant pain, fatigue, and pre-syncopal episodes, with a history of blunt abdominal trauma from football scrimmage 4 weeks earlier. he was found to have near complete occlusion of the ivc and hepatic veins. liver transplant was not considered feasible. he was successfully treated with anticoagulation alone. patient c was a 3-yearold male with acute myeloid leukemia in induction cycle 2 who developed severe pancytopenia; typhlitis was diagnosed and managed medically. days later he acutely decompensated, arrested, and was placed on extra corporeal membrane oxygenation, and imaging showed complete occlusion of the portal vein, hepatic veins, and ivc to the level of the atrium, with bilateral pulmonary emboli. emergency liver transplant or catheter based interventions was deemed not feasible. treatment with eculizumab was considered for presumed inflammation induced complement activation (c3 59mg/dl [normal 77-171]; ch50 was 12u/ml [normal 42-91]) as a trigger for thrombosis, but the patient progressed quickly and died before it could be initiated. our experience with bcs shows that invasive interventional options and liver transplant may not be feasible in most patients for multiple reasons. rapid diagnosis and aggressive etiology-based medical management are paramount to successful treatment of this rare complication. eculizumab may be considered in treating bcs with complement activation not only due to innate disorders, but also secondary to acute inflammation when proper laboratory evidence is present. background: platelet aggregation studies are the gold standard for the diagnosis of platelet function defects during the evaluation of a patient with bleeding problems. the platelet aggregation test measures how well platelets clot in response to different concentrations of epinephrine, adenosine diphosphate (adp), collagen, arachidonic acid and ristocetin. because platelet function defects are often under-recognized and under-diagnosed in the pediatric patient, the true incidence is unknown. we report our experience in the diagnosis of platelet defects at our institution over a 5-year period in order to add some clarity to the limited pediatric data available. objectives: our primary objective is to document correlations/trends between less well-known platelet function abnormalities and clinically significant bleeding at our institution over a 5-year period. design/method: after appropriate irb approval obtained, we performed a retrospective chart review of all children who had platelet aggregation testing done from 2011 to 2015. data collected included demographics (age, sex, race), personal and family history of bleeding, screening for coagulation defects and platelet aggregation test results. symptoms examined in our data were limited to epistaxis and heavy menstrual periods. for each of these symptoms, results were further analyzed to those with abnormal responses to adp and epinephrine. patients with existing bleeding diagnoses and those with incomplete medical records were excluded. we identified 159 patients. of the patients with epistaxis, 70% had abnormal platelet aggregation testing while only 36% of those with heavy menstrual periods had abnormal results. within our population, abnormal platelet function assay (pfa-100) results or race did not appear to correlate with abnormal platelet aggregation testing. in the cases of epistaxis, sex was also noncontributory. our preliminary results suggest that platelet aggregation testing was more useful in predicting platelet defects in those with a clinical bleeding history of epistaxis as opposed to heavy menstrual periods. for other presenting symptoms, platelet aggregation testing did not offer diagnostic benefit. abnormal response to adp in the platelet aggregation test was the most common finding in our population; the clinical significance of which is not well understood. going forward, we plan to document whether abnormal results correlated significantly with the subsequent final diagnoses of our patients. background: decision making for severe hemophilia a in previously untreated patients (pups) has recently become a significant ethical debate. recombinant factor viii (rfviii) products previously were recommended to avoid transmission of blood borne pathogens associated with plasma-derived fviii (pdfviii) products. however, the increased incidence of fviii alloantibody inhibitors with rfviii products compared to pdfviii products has challenged this former standard of care. despite the support of the medical and scientific advisory council, recommendations considering pdfviii products for a pup remains controversial. design/method: we used a modified utilitarian approach involving clinical, public health, and research ethics. shared decision making permeates the framework to maximize understanding, minimize bias, respect informed consent or dissent, and provide care that aligns with patient and family values when medically and practically feasible. the framework has three tiers. first, it evaluates whether resources are scarce or abundant for equitable resource allocation. if fviii products are scarce, we s133 of s301 recommend developing a central supply for emergency use and then evaluating the needs of the severe hemophilia a patients. prioritization of who receives the factor products would be decided by a designated team based on the availability of the factor products and clinical scenarios, with no preference given to those on research trials. however, if resources are abundant, treatment for acute bleeding and standard of care prophylaxis measures, including primary prophylaxis, could continue. the second tier accounts for whether there is a new infectious epidemic or concern where a pathogen cannot be eliminated. if there is, healthcare and public health workers may limit the use of pdfviii products. if not, pdfviii and rfviii products are to be equally considered. the third tier evaluates whether the clinical scenario is emergent or not. if there is acute, emergent bleeding, the immediately available resource should be used, along with bypassing and/or adjuvant resources as needed until the bleeding has resolved or improved. to align with patient and family preferences, attempts to have both pdfviii and rfviii products available at similar costs in institutions would be ideal. this ethical framework endeavors to balance autonomy, beneficence, nonmaleficence and justice in helping guide discussions among providers, pups with severe hemophilia a, and their families. disclaimer: findings and conclusions are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention, emory university, or children's healthcare of atlanta. background: von willebrand disease (vwd) is a common bleeding disorder which affects up to 1% of the population without gender predilection. bleeding associated with this condition results from a deficiency or abnormality in von willebrand factor interfering with formation of primary hemostasis. ehlers-danlos syndrome (eds) is a group of rare inherited connective tissue disorders which may have an associated bleeding manifestation without abnormalities in coagulation testing. bleeding symptoms reported in eds result from capillary and tissue fragility. joint hypermobility syndrome (jhs) is an inherited condition which is nearly indistinguishable from eds iii. reports of coinheritance of vwd and eds or jhs are infrequent. the objective of this retrospective study was to review patients with coexisting vwd and eds or jhs at the indiana hemophilia and thrombosis center in order to describe the type and severity of bleeding symptoms, physical examination findings, and pertinent laboratory data. design/method: the electronic medical record database of the indiana hemophilia and thrombosis center was queried for patients with a diagnosis of vwd and one of the following descriptors: hypermobility syndrome, hypermobility, hypermobile joints, or ehlers-danlos syndrome. the records of identified patients were reviewed for demographics, type and severity of bleeding symptoms, beighton scores (bs), vwd antigen, ristocetin cofactor, factor viii levels, vwd multimer pattern, vwd subtype, genetic testing for eds, and family history of eds. results: a total of 6 patients with dual diagnoses of vwd and eds and 21 patients with vwd and hypermobility were identified with this query. two patients had completed genetic testing for eds, and one had a col1a1 gene mutation identified. significant bleeding symptoms in the vwd and eds group included hematuria and postoperative hemorrhage. two of these patients had delayed wound healing postoperatively. seven of the 19 patients identified to have type i vwd and jhs had moderately severe and somewhat unusual bleeding episodes reported including hematuria, hematemesis, and hemoptysis; 4 of these patients had significant perioperative bleeding. females composed 83% of the vwd and eds group and 76% of the vwd and jhs group. conclusion: coinheritance of vwd and eds is an uncommon phenomenon. patients with vwd and eds or jhs may have atypical and moderately severe bleeding, especially with procedural intervention. incorporation of bs into the assessment of patients with bleeding disorders is useful to identify potential inherited collagen disorders, as diagnosis of these conditions may impact clinical management. in the year-long phase ii study (ro1fd003712), 11/12 khe patients responded. patients were followed for 5 years after study completion, collecting data on growth and development, complications of therapy, unexpected toxicities, and need for continuing sirolimus. objectives: after study therapy treatment of one year, objectives include: 1. assess long term toxicity over the 4-5 year period after study therapy completion 2. assess unexpected toxicity 3. assess overall condition of the patient 4. assess need for restart or continuation of sirolimus therapy design/method: prospective follow-up of patients with a diagnosis of khe from 2 institutions. inclusion criteria: follow-up for 4-5 years post-study. results: follow-up included data at 5 year (n = 5) and 4-4.5 year (n = 4) time points. average age at the start of treatment was 12 months. 9 of 12 patients were available for follow up. four patients are no longer on sirolimus: one patient completed study therapy and remains off treatment (ot) (7 years), 1 required 2 years of treatment and is now 2.5 years ot and 2 required an additional treatment course prior to successful discontinuation now 17 and 22 months ot. of the 5 patients still on sirolimus, all restarted medication for symptoms of pain, swelling and/or edema interfering with quality of life and have made an average of 2.5 attempts to discontinue sirolimus. no patient had reoccurrence of kmp. all patients had improvement in clinical and radiologic appearance of khe but all have residual lesions noted on imaging and/or clinical exam. no unexpected toxicity, growth delay, developmental issues or other long term toxicity of sirolimus was noted. conclusion: this is the first prospective data on long-term follow up of khe patients treated with sirolimus. although numbers are small, sirolimus is well tolerated; however, over half the patients were still on medication at 4-5 year follow up. this stresses the need for continued long term follow up in these young patients and investigation of the mechanism of sirolimus effect. nationwide children's hospital, columbus, ohio, united states background: recent studies have identified that adult persons with hemophilia (pwh) have a higher prevalence of hypertension and renal disease than the general population. while hematuria is a known complication of hemophilia a and b (ha, hb), its long-term impact on pwh is not currently known. by annually screening our patients with urinalysis, our pediatric center identified that just under half of our patients demonstrated hematuria over a four-year period. motivated by a desire to identify early markers of hypertension and renal disease, we sought to determine if this finding is reflected in the pediatric hemophilia population as a whole. objectives: establish the population-wide prevalence of hematuria in pediatric pwh. design/method: we used the pediatric health information system (phis) database, which contains clinical and resource utilization data for inpatients from 45 hospitals nationwide, to analyze the prevalence of hematuria, hypertension, renal disease and related diagnosis codes in pediatric pwh who were admitted from january 2010 to september 2015. results: during the five-year period, 2,197 unique pediatric pwh accounted for 4,802 admissions. while the majority of admissions were for bleeding or infectious concerns, 96 (4.4%) patients had an affiliated admission code for hematuria. for admissions as a whole, the median age was 7 years with 12% of those admitted being infants, 32% toddlers, 27% children, 28% adolescents, 2% older than 21. we identified 83% of admissions were for ha with the remaining 17% were for hb. there were 1254 (26%) admits in which a bypassing agent was administered. the median length of stay for persons with hematuria was 2 days compared to 3 days for nonhematuria/other bleeding. there were 120 (2.5%) admissions with hypertension reported; though, only 3 patients received an antihypertensive medication during that admission. additionally, only 31 (0.6%) admissions reported a diagnosis code of renal disease. our study demonstrated that pediatric pwh are experiencing hematuria. in general, only patients with persistent hematuria require hospital admission so we suspect this data underrepresents the numbers of pwh experiencing hematuria that is managed in the outpatient setting. we also suspect that hypertension is grossly underreported and undertreated in pediatric pwh. additionally, there are a low number of patients experiencing renal disease requiring hospital admission among this cohort. given that there is little research into the long-term impact of hematuria in hemophilia, we feel these findings support the need for further vigilance of our pediatric pwh. background: gla and gsd can aggressively destroy bone, with significant impact on morbidity and mortality. the mtor inhibitor, sirolimus has been shown to be effective in the treatment of these diseases. based on the addition of mtor inhibition to bisphosphonate therapy in metastatic cancer therapy, regimens have been used for refractory or high risk gla and gsd but there is heterogeneity of diagnosis, and variability of drug regimens and assessment of effectiveness. objectives: 1. assess the variability of clinical features of gla and gsd 2. assess the heterogeneity of diagnosis 3. assess drug regimens and response assessment across multiple institutions design/method: we conducted a retrospective review from 5 institutions of 19 cases of gla and gsd treated with sirolimus and a bisphosphonate for at least 2 months with assessment of clinical features, treatment protocols, response regimens and side effects. results: patients included gla (n = 8) and gsd (n = 11). the average age at diagnosis was 10 years. clinical features included effusions: gla (n = 4), soft tissue lymphatic malformations: gla (n = 3), gsd (n = 1), multiple splenic lesions: gla (n = 3), and soft tissue swelling at the site of bony lesion: gsd (n = 3). the presenting symptom in 17 patients was pain with 2 patients (gla) presenting with shortness of breath. fracture was noted in 5 patients: gla (1), gsd (4). diagnostic and/or response imaging included mri, ct, bone scan, skeletal survey and dexa scan. treatment consisted of: initial sirolimus use with the addition of bisphosphonate secondary to worsening disease (n = 4), initial therapy with other agents (interferon, chemotherapeutic agents, radiation) and change to sirolimus and bisphosphonate secondary to toxicity (n = 6), sirolimus and bisphosphonates (n = 7) and sirolimus, bisphosphonates and interferon (n = 2). seventeen patients had stable disease and 8 patients had improvement of disease. sirolimus protocol was standard; however, bisphosphonate protocol varied in dosing and frequency. side effects were tolerable and expected with no grade iii or iv toxicity. sirolimus and bisphosphonates are a safe and effective therapy for gsd and gla. a consistent medication regimen, redefined response and an improved radiologic classification will be important for the development of a prospective clinical trial. background: hemophilia a is a bleeding disorder from the deficiency of clotting factor viii. the most significant sequelae of hemophilia a is the tendency to develop hemarthrosis that incites joint destruction. the prevalence of overweight and obesity has been increasing in the general and hemophilia population and leads to several morbidities including arthropathy. this is a particular concern for hemophilia a as arthropathy is a consequence of joint bleeding. objectives: the purpose of this study was to detect the relation between body mass index (bmi) and joint health endpoints in a pediatric hemophilia population. design/method: participants in this study included 64 patients from the hemostasis and thrombosis center at children's hospital los angeles. participants were pre-screened and approached for this study during routine follow-up appointments. patients aged 4-18 years old who have been diagnosed with hemophilia a, including mild, moderate, and severe, qualified for the study. informed consent was obtained from the patients or parents before enrollment. joint health was objectively measured by physical therapists from children's hospital los angeles using the hemophilia joint health score (hjhs). an hjhs total score is calculated by assessing: swelling, duration of swelling, muscle atrophy, crepitus on motion, flexion loss, extension loss, joint pain, and muscle strength in 6 major joints. subjective data was also obtained by patients recording their annual bleed rate within the past year. of the 64 patients, 28 (44%) were normal weight, 12 (19%) overweight, and 24 (38%) obese. we used chi-square analysis to compare joint scores across bmi classifications (chi square = 2.87, df = 2, p-value = 0.24). although, this did not approach statistical significance, the average hjhs score in patients who had a hjhs >0 shows an increasing trend among bmi classifications: 6.19 in normal bmi patients, 6.75 in overweight bmi patients, and 7.00 in obese bmi patients. the average number of annual bleeds in those with positive values show: 8 in normal bmi patients, 5 in overweight bmi patients, and 12 in obese bmi patients. although a positive effect of adiposity was found in the joints of hemophilia a pediatric patients, the effect shows there was not enough evidence to conclude a difference. future studies are needed to address whether obesity has an effect on hemophilia and to determine whether overweight/obesity can lead to further complications in hemophilic joints. background: stagnant blood flow in slow-flow vascular malformations (vm), particularly in their venous components, can lead to localized intravascular coagulation (lic) that is characterized by elevated d-dimer levels, low fibrinogen and decreased platelet count this coagulation derangement can lead to localized thrombosis or bleeding which can result in pain, functional limitations, and possible progression to disseminated intravascular coagulopathy (dic). the treatment of vm and their associated coagulopathy has proven difficult. patients with complex vm are frequently managed with sirolimus, an mtor inhibitor, and have clinical benefits, including reduction of pain and improvement in functional impairment. it is possible that some of these improvements from sirolimus could be secondary to improvement in the coexisting lic. objectives: this study assessed the use of sirolimus to manage the coagulopathy seen in slow-flow vm. design/method: we reviewed charts of patients with vm who are followed in the vascular anomalies center at arkansas children's hospital and were started on sirolimus. efficacy was objectively assessed through improvement of ddimer, fibrinogen and platelet count. three sets of lab values (pre-sirolimus, 1-3 months post-sirolimus, and most recent) were obtained for each patient when available. we identified a total of 35 patients who had been prescribed sirolimus. eighteen were excluded based on underlying condition other than slow-flow vascular malformation and 1 for inadequate medical records. a total of 16 patients (13 combined vascular, 3 venous) were included in the study. all 16 had elevated d-dimer levels (mean 4.64 mcg/ml feu, median 2.99 mcg/ml feu, range (0.83-14.65)) prior to treatment. two patients had an associated low fibrinogen (below 175 mg/dl), indicating severe lic. with treatment, 14 (87.5%) patients showed an overall decrease in d-dimer levels with an average decrease of 1.52 mcg/ml feu between pre-and post-sirolimus labs, and an average decrease of 1.03 mcg/ml feu between pre-sirolimus and most recent values. the two patients with low fibrinogen prior to treatment showed a decrease in d-dimer levels (mean decrease of 7.845 mcg/ml feu) and an increase and normalization in fibrinogen (mean increase 83.95 mg/dl) after beginning sirolimus. no patient had thrombocytopenia. we report that treatment with sirolimus was effective in improving coagulopathy associated with slowflow vm as evidenced by decreased d-dimer levels and increased fibrinogen and/or platelets. long-term use of this medication in this population may decrease the bleeding and thrombotic complications that these patients experience, especially following invasive vascular procedures. background: safety and efficacy of bay 94-9027, a sitespecifically pegylated b-domain-deleted recombinant factor viii, in previously treated adolescents and adults aged 12-65 years with severe hemophilia a was demonstrated in the phase 2/3 protect viii study and ongoing extension. objectives: this subanalysis examines the efficacy and safety of bay 94-9027 in adolescents in protect viii and the ongoing extension study (data cutoff, january 2015). design/method: in protect viii, 134 patients (including 12 adolescents) received bay 94-9027 on demand or as prophylaxis for 36 weeks. prophylaxis regimens for weeks 10-36 were twice-weekly (30-40 iu/kg), every-5-days (45-60 iu/kg), or once-weekly (60 iu/kg) infusions based on bleeding during a 10-week run-in period of 25 iu/kg twice-weekly prophylaxis. patients continued their prophylaxis regimens in the extension or changed regimens at any time. results: twelve patients aged 12-17 years were included in the protect viii intent-to-treat population; 1 s137 of s301 additional patient discontinued after 1 dose (included in safety population). for 11 patients receiving prophylaxis before study enrollment, median (range) number of total and joint bleeds in the 12 months before study entry was 8.0 (0-15) and 6.0 (0-10), respectively. ten patients (83.3%) had target joints at baseline (median [range],1 [0-4] per patient). during weeks 10-36 of protect viii for the entire time patients remained on their designated prophylaxis dosing frequency, the median (quartile [q]1; q3) annualized bleeding rate (abr) for patients receiving twice-weekly (n = 3), every-5-days (n = 6), and once-weekly prophylaxis (n = 3) was 0 (0; 2.0), 1.1 (0; 8.2), and 18.4 (0; 19.3), respectively (overall prophylaxis [n = 12], 1.0 [0.0; 10.1]). two patients switched from once-weekly to twice-weekly (n = 1) or every-5-days prophylaxis (n = 1), and number of bleeds decreased from 2 to 1 in one patient and 6 to 4 in the other. all 12 patients from the main study continued in the extension; mean abr in the extension was 3.2 and varied by dosing regimen (twice weekly [n = 3], 3.2; every 5 days [n = 5], 5.2; once weekly [n = 2], 0.9). two patients changed from every-5-days to once-weekly prophylaxis during extension (mean abr, 0.6). one patient had a nonneutralizing antibody to bay 94-9027 at baseline; end-of-study titers were negative. no patient developed anti-peg antibodies or factor viii inhibitors or experienced a serious adverse event related to bay 94-9027 during the main study or extension. in previously treated adolescents with severe hemophilia a, bay 94-9027 prophylaxis was effective in prevention of bleeds, with less bleeding overall versus prestudy, and was generally well tolerated. funded by bayer. cincinnati children's hospital medical center, cincinnati, ohio, united states background: vascular malformations (vms) consist of a heterogeneous group of congenital disorders characterized by the abnormal development of blood and/or lymphatic vessels, which cause a broad spectrum of clinical manifestations. although considered benign, vms are frequently associated with cutaneous complications that can cause significant morbidity such as nodular overgrowth, skin thickening, pruritus, oozing or bleeding of lymphatic blebs and secondary infection. oral sirolimus has shown to be effective in the treatment of complicated vascular malformations but has known side effects and need for frequent laboratory monitoring. currently, there are limited studies on the use of topical sirolimus for the treatment of cutaneous manifestations of vascular malformations. objectives: to evaluate the efficacy and safety of topical sirolimus in vms with cutaneous complications and propose indications for use. design/method: this is a retrospective review of medical records of patients with vascular malformations treated with topical sirolimus from january 2012 to december 2017. response was determined by subjective and objective improvement. results: twenty-four patients, 16 (66%) females and 8 (33%) males, with vascular malformations and cutaneous manifestations were treated with topical sirolimus. age ranged from 4-27 years. indications for treatment were: blebs (79%, n = 19) causing either leaking, bleeding, pain, pruritus, swelling or recurrent infection; nodular overgrowth 8% (n = 2); pyogenic granuloma 4% (n = 1); bleeding 4% (n = 1) and cosmetic 4% (n = 1). treatment course ranged from 1-18 months. no major side effects were reported. one patient reported burning and itching sensation. regarding clinical response: 83% (n = 20) patients had improvement in cutaneous lesions; 12% (n = 3) had a stable lesions; and 4% (n = 1) stopped treatment due to side effects. for prior/concomitant treatment: 83% (n = 20) had prior surgery, laser or sclerotherapy; 37% (n = 9) had concomitant oral sirolimus. of the 15 patients not receiving concomitant systemic sirolimus, only 13% (n = 2/15) had been on oral sirolimus. of these patients, 80% (n = 12/15) had a very good response to topical treatment. : topical sirolimus appears to be beneficial and well-tolerated with a minimal side effect profile for the treatment of cutaneous manifestations of vascular malformations as a single agent or as adjuvant therapy with systemic sirolimus when symptoms are not adequately controlled. further studies are needed to prospectively analyze efficacy and safety of topical sirolimus in this patient population. objectives: to evaluate the safety and efficacy of long-term romiplostim in children with itp. design/method: all patients received weekly sc romiplostim from 1-10 g/kg to target platelet counts of 50-200 × 10(9)/l. median (min-max) treatment for the 65 patients was 135 (5-363) weeks for a total of 182 patient-years, or 2.8 years per patient. at baseline, median (min-max) age was 11 (3-18) years; 56% were female; 9.1% had prior splenectomy. median (min-max) average weekly dose was 4.8 (0.1-10.0) g/kg, including escalation to a stable dose; 20 patients started on 1 g/kg. reasons for discontinuing romiplostim (n = 28, 42%) included consent withdrawn (n = 10), required other therapy (n = 6), and ae (n = 2) (asthenia, headache, dehydration, and vomiting in one patient and anxiety in the other; none treatment related). fifty four serious aes occurred in 19 patients but were treatment related in one (concurrent grade 4 thrombocytopenia, grade 3 epistaxis, and grade 2 anemia). anti-romiplostim neutralizing antibodies were detected in one patient who discontinued to receive other therapy; antibodies were absent on retesting. from week 2 on, median platelet counts remained >50 × 10(9)/l; median platelet counts were >100 × 10(9)/l from weeks 24-260. nearly all (94%, 61/65) patients had ≥1 platelet response (platelet counts ≥50 × 10(9)/l, excluding ≤4 weeks after rescue medication). most (72%, 47/65) patients had a platelet response ≥75% of the time and 58% (38/65) did ≥90% of the time. sixty (92%) patients (or caregivers) self-administered romiplostim. fifteen (23%) patients had treatment-free periods of platelet counts ≥50 × 10(9)/l for ≥24 weeks (ie, remission); these patients (9 girls, 6 boys) had had itp for a median (min-max) of 3.5 (1.3-13) years, none had prior splenectomy, and had received romiplostim for 2.1 (0.7-6) years. all 15 had platelet counts >100 × 10(9)/l for ≥3 months and 12/15 for ≥6 months; the median (min-max) duration of being ≥100 × 10(9)/l was 42 (13-109) weeks. of baseline characteristics such as sex, platelet counts, itp duration, and number of past itp treatments (1, 2, 3, >3), only age <6 years was predictive of developing treatment-free periods ≥24 weeks (p = 0.0035). in this seven-year open-label extension, >90% of children with itp achieved a platelet response and romiplostim was well tolerated. importantly, 23% of patients were able to discontinue all itp medications for ≥6 months. funded by amgen inc. background: sirolimus is an immunosuppressive drug that is widely used in solid organ and bone marrow transplantation, and more recently for the treatment of vascular and lymphatic anomalies. sirolimus has been associated with decreased immunity in the transplant setting in patients that have received other immunosuppressive drugs or were immunosuppressed from previous chemotherapy. the effects of sirolimus on the immune system in chemotherapy naïve children who have not received other immunosuppressive agents are not well understood, and there is variability in the approach to fever and pcp prophylaxis. to understand the effects of sirolimus on the immune system of patients with non-complicated vascular or lymphatic anomalies by evaluating anc, alc prior to and after sirolimus therapy. design/method: multi-institutional retrospective review was done to include patients with non-complicated vascular or lymphatic anomalies. those with effusions/ascites, multiorgan involvement, or history of vascular-anomaly-related infections prior to treatment were excluded. results: twenty patients with kaposiform hemangioendothelioma (n = 6), generalized lymphatic anomaly (n = 2), cloves syndrome (1), and simple vascular malformation (n = 11) were included. age at initiation of sirolimus treatment ranged from 0.5 -20 years. male to female ratio was 9:11. sirolimus was initiated due to extensive disease, lack of response to steroids or bisphosphonates, pain, dment, lymphatic drainage, and prevention of ongoing overgrowth. prior to the start of sirolimus (sir-0) the mean anc was 3850 and alc was 2875. the target level of sirolimus varied by indication and patient, and ranged from 6 to 10. after the 1st steady state level, 1 month after sirolimus (sir-1) the mean anc decreased to 2951 and alc was 2793. at 3 months after sirolimus (sir-3) the mean anc was 3108 and alc was 2874. the first sirolimus levels (sir-1) mean was 11.3; and sir-3 level was 7.8. nine patients were placed on pcp prophylaxis at the start of sirolimus. none of these patients had an infectious complication while on sirolimus at a median f/u of 13 months. one patient had mild neutropenia (anc >500) which normalized after discontinuation of pjp prophylaxis. conclusion: in this small cohort of patients we found that the anc and alc level in patients with non-complicated vascular or lymphatic anomalies at sir-0 was not different from the sir-1 or sir-3. prospective studies that specifically track anc, alc, igg, and lymphocyte function should be conducted to better understand the effects of sirolimus in the immune system. this data will allow for uniform recommendations regarding prophylaxis and management of febrile episodes. background: acute infections and the associated systemic inflammation can increase the risk of venous thromboembolism (vte) and in certain well-defined clinical scenarios may be the primary trigger of vte in pediatric patients. pediatric data on vte in the setting of acute infection are sparse. objectives: to describe characteristics and outcomes of vte in pediatric patients with acute infections. we conducted a retrospective chart review of all pediatric patients with objectively confirmed vte treated at our institution since 2011 and identified all patients in whom an acute infection was identified as a vte trigger. patients were managed according to standardized institutional protocols based on published guidelines. relevant demographic, clinical and laboratory data were collected and summarized using descriptive statistics. since 2011, acute infection was identified as a trigger in 147 of 429 vtes (34%) diagnosed at our center. the median age at time of vte diagnosis in this group was 2.3 years (interquartile range 0.3-16). males were more commonly affected than females, representing 56% of cases. neonatal vte events accounted for 14% of cases. sepsis was the most common acute infection to be identified as a vte trigger [59/147 cases (40%)]. most vte events (80%) associated with acute infections were considered hospital-associated vtes. at time of vte diagnosis, 61% of patients were critically ill. extensive vte (defined as completely occlusive thrombosis involving >1 venous segment) occurred in 16% of patients. acute infection was deemed to be the primary trigger for vte in 30/147 patients (20%). infection-associated vtes in this cohort included cerebral sinus venous thrombosis due to sinus or cns infection (13 patients, 43%), septic throm-bophlebitis (11 patients, 37%), lemierre's or lemierre's-like syndrome (4 patients, 13%) and osteomyelitis-associated deep vein thrombosis (2 patients, 7%). systemic anticoagulation was prescribed in 124/147 patients (84%). anticoagulationrelated major bleeding occurred in 10/124 patients (8%). vte complications included vte recurrence (16 patients, 10%), vte progression (1 patient), acute pulmonary embolism (2 patients) and arterial ischemic stroke (2 patients). our study indicates that acute infection is a common risk factor for pediatric vte, especially in critically ill children, and can be the primary trigger in a significant proportion of vte cases associated with acute infections. anticoagulation appeared to be overall safe in this population and was associated with low rates of serious vte-related acute complications. however, our study also suggests that this population may be at increased risk for vte recurrence and anticoagulation-related major bleeding. background: epithelioid hemangiomas (eh) are rare benign vascular tumors that occur in soft tissues and bone and present between the third and sixth decades of life. a subset (29%) of eh harbor fos rearrangement. eh has been described in children, but little is known about the long-term outcomes of pediatric eh. the main objective is to obtain data to be used for improved understanding of this rare disease in order to provide standardization of care and development of future research studies. board-approved retrospective review of clinical, pathologic, and radiographic characteristics, and treatment outcomes in 11 patients diagnosed with eh between 1999 and 2017. results: eight patients were male; mean age at diagnosis was 14.8 years (range: 6-23). lesions involved the lower extremities (n = 5), cranium (n = 3), pelvis (n = 2), and spine (n = 1). multifocal disease was identified in five patients. the most common presentations involved significant localized pain and neurologic symptoms: headache, cranial nerve injury, loss of consciousness. radiographic studies identified variable features, such as multifocal lytic bony lesions with sclerotic margins, enhancing soft tissue component, and surrounding inflammatory edema. histologically, all specimens were composed of vascular channels lined by epithelioid endothelial cells without significant cytologic atypia; solid cellular areas (n = 2). endothelial cells were positive for cd31 and egr, and negative for camta1. fos rearrangement was assessed in only one specimen and detected. mean follow-up time was 545 days (range: 23-2642). patients were treated with surgical resection, intravascular embolization, bisphosphonates, propranolol, interferon, and sirolimus. one patient treated with interferon and one with sirolimus exhibited partial response for mean follow-up of 1566.5 days. although eh is a benign neoplasm, it is difficult to manage without standard protocols and portends considerable morbidity. our findings suggest medical management, particularly sirolimus, may benefit these patients; however, long-term follow-up is needed in treated children. novel fos inhibitors are in development and may benefit patients with fos rearrangement. penn state health children's hospital, hershey, pennsylvania, united states background: central venous catheters (cvc) are often required in critical care settings in order to provide a secure point of access for life sustaining care. clinical studies identify cvc presence as the single most important risk factor for deep vein thrombosis (dvt) in children. venous thromboembolic event (vte) incidence rates in critically ill children with a cvc range from 0.3-18% and 0.06-32.5 per 1000 catheter days depending on the population studied. per institutional protocol, the penn state health children's hospital picu (hershey, pa) utilizes a low dose continuous infusion of unfractionated heparin (ldufh) at 10 units/kg/hr as prophylaxis against cvc-related vte and to maintain line patency. the efficacy of this approach has never been evaluated. to determine if ldufh for prophylaxis results in lower incidence of cvc-related vte, catheter dysfunction and central line associated blood stream infection (clabsi) without increasing morbidities. to determine if the incidence of catheter related vte is lower than historical published data, a retrospective chart review was conducted utilizing the institutional electronic medical record for all patients in 2015, aged 0-17.99 years, who had a cvc during a picu admission. secondary objectives such as the incidence of catheter dysfunction, clabsi, and any associated bleeding complications are also being analyzed. results: interim data analysis revealed 478 cvcs (400 nontunneled cvc, 18 totally implantable devices, 19 tunneled lines, 41 peripherally inserted central catheters [picc] ) in 374 total patients with a median age of 1.9 years. overall vte incidence was 1.88% (9/478) with 7 vtes associated with non-tunneled cvc and 2 with piccs. sixty one percent of non-tunneled cvcs received ldufh and 85% (6/7) of the patients with vtes associated with non-tunneled cvcs did receive ldufh prophylaxis. vte incidence rate of nontunneled cvcs with ldufh was 2.5% (6/243) and 2.56 per 1000 picu catheter days. the only other vte events identified within our study cohort were in the picc group where two patients experienced vte, one of which was receiving ldufh. clabsi incidence was 1.2% (4 non-tunneled cvc, 1 tunnel cvc, 1 picc). no major bleeding complications were associated with ldufh. preliminary data demonstrates ldufh is efficacious in preventing cvc-related vte in comparison to published reports. further analysis will compare another similar sized and acuity level picu which does not practice the same method. background: fibroadipose vascular anomaly (fava) is a rare, challenging disorder associated with pik3ca mutations. fava often causes painful replacement of muscle and soft tissues with fibrotic and adipose tissue and is associated with ectatic draining veins. treatments for focal lesions are surgical excision, cryoablation or sclerotherapy and the role of medical therapy is unclear. some fava lesions are too extensive or directly involve neurovascular structure, resulting in refractory pain. objectives: to retrospectively evaluate the efficacy of sirolimus in patient with residual symptoms after procedural therapies for fava design/method: retrospective review of individual 7 cases from 6 institutions of fava refractory to other therapies treated with sirolimus for at least 3 months. cases were s141 of s301 identified by polling member of the aspho vascular anomalies special interest group. results: all seven patients report improvement on sirolimus therapy. all patients had received prior procedures, including sclerotherapy (6 patients), cryoablation (2 patients) and/or resection (3 patients). mean age at sirolimus initiation was 16y (range 6-29y). mean length of therapy is 18.4 months (range 3-29 months). six patients were treated with bid dosing and one adult received daily dosing. goals of sirolimus were improvement in pain or musculoskeletal dysfunction. pain and function improved in all patients, including discontinuation of narcotic use and resumption of participation in sports. time to symptom improvement ranged from 1-4 weeks. in four patients for whom dose was lowered, pain recurred in all four and responded to restarting or increasing sirolimus dose. while all patients do not have pre-and postsirolimus imaging, decrease in fava lesion size is seen in cases with available imaging. sirolimus side effects are similar to prior reports, most commonly mouth sores, elevated lipids and acne. we report the first known data supporting a role of sirolimus in refractory fava cases. sirolimus is welltolerated and initial improvement is rapid, within 4 weeks of initiation. whether sirolimus has a role in upfront therapy to reduce lesion size prior to procedures deserves further study. objectives: to assess platelet responses in children with itp receiving romiplostim. design/method: eligible children had itp for ≥6 months, ≥1 prior therapy, and screening platelet counts ≤30 × 10(9)/l or uncontrolled bleeding. weekly dosing was from 1-10 g/kg to target platelet counts of 50-200 × 10(9)/l. bone marrow biopsies were evaluated in europe at baseline and after 1 or 2 years (cohorts 1 and 2). as of mar 2017, 203 patients received ≥1 dose. at baseline, median (min-max) age was 10 (1-17) years, itp duration was 1.8 (0.5-13.8) years, and platelet count was 14 (2-265) × 10(9)/l; 10 patients (5%) had had prior splenectomy. the median (q1, q3) % time with a platelet response (platelet count ≥50 × 10(9)/l, no rescue medications past 4 weeks) in months 0-6 was 50% (17%, 83%) (primary endpoint). over the course of the study, 88% (179/203) of patients had a platelet response. four patients maintained platelet counts ≥50 × 10(9)/l with no itp medications for ≥24 weeks. median (min-max) treatment duration was 53 (8-119) weeks for 226 patient-years in total. median (min-max) average weekly romiplostim dose over the course of the study was 6.9 (0.2-9.5) g/kg; the median dose was 9 g/kg at 1 year (n = 106) and 10 g/kg at 2 years (n = 17). most (63%) patients initiated self-administration. sixty-four patients (31%) discontinued treatment, most frequently for lack of efficacy (n = 38), patient request (n = 7), and adverse event (ae) (n = 7). fortyone (20%) patients had serious aes (saes) including epistaxis (5%) and decreased platelet count (3%). five patients had treatment-related saes: 2 headaches, 2 abdominal pain, and 1 each of presyncope and neutralizing antibodies (ab). there were 6 cases of neutralizing ab to romiplostim (of 201 patients tested), but none to tpo; 5/6 had continued elevated platelet counts and in 2/6 cases ab were not found on retesting. for cohort 1, of 30 patients with baseline bone marrow biopsies, 27 had evaluable on-study biopsies scheduled for 1 year; 1 patient had an increase from grade 0 to 2. there were no findings of collagen or abnormalities. in this interim datacut of a romiplostim openlabel study in children with itp, 88% of children had a platelet response. overall, the median dose was 6.9 g/kg; the median romiplostim dose over time reached 10 g/kg. no new safety signals were observed over 226 patient-years. funded by amgen inc. background: hepatic hemangiomas are benign vascular tumors without a medical home, managed by multiple specialties. the diagnosis has been assigned historically to various vascular lesions affecting the liver with completely different clinical presentations, resulting in difficult standardized management. objectives: the consensus steering committee identified an acute need of clear definitions and evaluation guidelines using the updated international society for the study of vascular anomalies (issva) classification. the goal was to formulate recommendations that will be adopted by all specialties involved in the care of children with hepatic hemangiomas. design/method: we used a rigorous, transparent consensus protocol, with input from multiple pediatric experts in vascular anomalies from hematology-oncology, surgery, pathology, radiology and gastroenterology. in the first section, we precisely define the subtypes of hepatic hemangiomas seen in children (congenital and infantile) using clinical course, histology and radiologic characteristics. inclusion and exclusion limits to the diagnosis are noted. the following two sections describe these subtypes in further detail, including complications to be considered during monitoring and respectively recommended screening evaluations. conclusion: while institutional variations may exist for specific clinical details, a clear understanding of the diagnosis of hepatic hemangiomas affecting the pediatric population and the possible complications that require screening during the monitoring period should be standard. as patients with hepatic hemangiomas are managed by different medical and surgical specialties, a multidisciplinary consensus based on current literature, on the data extracted from the liver hemangioma registry and on expert opinion was required and was accomplished by this manuscript. objectives: to investigate the association between routine prophylaxis with bay 81-8973 and bleeding outcomes after adjusting for key patient and pharmacokinetic (pk) characteristics. design/method: the leopold kids study evaluated safety and efficacy of bay 81-8973 prophylaxis in 51 previously treated boys aged ≤12 years with severe hemophilia a. patients received bay 81-8973 25-50 iu/kg 2x/wk (n = 21) or >2x/wk (n = 30) and were followed up for 6-8 months. prophylaxis dose and frequency were assigned by investigators. pk parameters, including area under the curve (auc), half-life, and clearance, were derived from a population pk model and reflect predicted pk values with a 50-iu/kg dose. patient characteristics were compared between the 2x/wk and >2x/wk groups using wilcoxon rank sum or chi-square tests. negative binomial regression was used to model the association between prophylaxis frequency and annualized bleeding rate (abr) for total bleeds, first without adjustment and then adjusting for age, pk parameters, and bleed history. results: mean ± sd age for patients in this analysis was 6.4±3.0 years. patients receiving prophylaxis 2x/wk had more bleeding episodes in the 12 months before study entry (mean ± sd, 13.0±16.6 [median, 6.0] for 2x/wk vs 4.3±5.7 [1.0] for >2x/wk; p = 0.027) and were more likely to have been treated on demand (38% vs 10%; p = 0.035). pk parameters were similar between the 2x/wk and >2x/wk groups. without adjustments, abr during the study was 12% higher in the 2x/wk group compared with the >2x/wk group (rate ratio [rr], 1.12; 95% ci, 0.44-2.90; p = 0.81). abr was 36% lower in the 2x/wk group (rr, 0.64; 95% ci, 0.24-1.70; p = 0.37) after adjusting for age, auc, and number of bleeds in the prior 12 months. conclusion: abr was numerically lower but not significantly different between the 2x/wk and >2x/wk groups after adjusting for age and pk parameters. these findings suggest that even among patient groups that are homogeneous with respect to age, pk, and bleed history, further individualization of bay 81-8973 prophylaxis based on other characteristics may help reduce bleeding episodes even at a lower treatment frequency. larger real-world studies are needed to verify these findings. funded by bayer. stanford, palo alto, california, united states s143 of s301 background: vascular malformations may be of lymphatic, arterial, venous or capillary endothelial origin. they may be simple or complex, with complex malformations being a combination soft tissue and skeletal overgrowth. although likely present at birth, these malformations often become symptomatic with puberty or infection, and range from little or no clinical impact to life threatening symptoms. in malformations primarily of venous origin, pain may be significant and hypothesized to be caused by phlebolith development (intra-malformation thrombi), inflammation, consumptive coagulopathy, vascular engorgement, and endothelial proliferation. anti-angiogenic and anti-platelet therapies have been reported to relieve pain. however, the use of anticoagulation for pain is not well described. objectives: to report clinical features and outcomes of patients with vascular malformations of venous origin treated with anticoagulation for pain. we performed a retrospective review of patients with vascular malformations followed by the hematology service between january 2010 and december 2017 who were treated for pain with anticoagulation. pain relief was determined both by wong-baker pain scales and patient report. clinical data were extracted from electronic medical records. we identified five patients with venous malformations (vm) who had received anticoagulation for pain. four patients were female and median age was 8 years old (range 4 to 29 years old) at time of initiation of anticoagulation. all five patients had vm of the extremity, two with vm of the lower extremity, and three patients had vm of the upper extremity. two patients had concomitant coagulopathy and demonstrated decreased d-dimer after initiation of anticoagulation. four patients received enoxaparin, and one adult patient received rivaroxaban. all patients reported improvement in pain after administration of anticoagulation. one patient exhibited mild epistaxis and bruising at the injection site. there was no significant bleeding or other complications. pain is a significant complication in patients with venous malformations. our case series suggests that anticoagulation is a safe and effective therapy for pain relief in this population. further investigation is indicated to compare the effect of anticoagulation to other therapeutic interventions such sclerotherapy, surgery, and sirolimus in the treatment of pain associated with venous malformation. maria ahmad-nabi, christine knoll, sanjay shah, lucia mirea phoenix children's hospital, phoenix, arizona, united states background: estimates of the incidence of dvt in patients with osteomyelitis range widely from 5%-30%, however risk factors and outcomes of dvt in this cohort have not been thoroughly established. objectives: this study aims to estimate the incidence of dvt in patients with osteomyelitis, and to assess risk factors and outcomes of dvt in this cohort. design/method: after irb approval, a retrospective chart review was conducted for patients aged 0-18 years seen at phoenix children's hospital between 2012-2016 with icd 9/10 codes for osteomyelitis. exclusion criteria included chronic recurrent multifocal osteomyelitis, and chronic dvt. demographics, clinical factors and outcomes were compared between osteomyelitis patients with and without dvt using the fisher-exact and wilcoxon-rank sum tests, as appropriate for the data distribution. results: a total of 179 study subjects with osteomyelitis had a mean (standard deviation) age of 8.4 (5.7) years. dvt was present in 14 (8% of 179) patients, and 4 (28%), 5 (36%) and 5 (36%) patients received anticoagulation for < 6, 6-12 and ≥12 weeks, respectively. patients with vs without dvt were more likely to be male (86% vs 59%; p-value = 0.05), and had significantly higher rates of bacteremia (64% vs 24%; p-value = 0.003). rates of central lines were comparable between dvt and non-dvt patients (71% vs 68%; p-value = 1.00); however patients with dvt vs without dvt had significantly longer mean length of stay (18 vs 9 days; p-value <0.0001) and higher rates of icu admission (71% vs 16%; p-value <0.0001). the incidence of dvt among osteomyelitis pediatric patients was estimated at 8%, with risk increased by male sex and bacteremia. patients with dvt had significantly higher rates of icu admission and longer length of hospital stay. many of these patients had standard practice management of their dvt with 6-12 weeks of anticoagulation. our data highlights the need for recognition of high risk patients, and the need for future efforts targeting dvt prophylaxis. baylor college of medicine, houston, texas, united states background: lymphatic malformations (lm) frequently occur in the head and neck and can often be disfiguring and even life-threatening. management options include observation, surgery, sclerotherapy, and sirolimus. the optimal sequence of therapeutic interventions has not been determined due to the lack of comparative clinical trials or established guidelines. thus, prenatal planning with a multidisciplinary team is beneficial. we present a case series of ten children with head and neck lms evaluated in 2017 at our multidisciplinary vascular anomalies center. a chart review was performed to assess treatment modalities and recent trends. results: seven of 10 patients (70%) with head and neck lms were diagnosed prenatally. six patients required an ex utero intrapartum treatment procedure. all patients were started on sirolimus at a median age of 12.5 months (range 12 days -18 years). four patients most recently started on sirolimus were less than 3 months of age at the time of initiation. six patients underwent partial excision of lm during the first year of life; none of whom received sirolimus prior to surgery. sirolimus was discontinued in one patient given chronic clostridium difficile infections, and non-compliance in another patient. five patients received sclerotherapy. tracheostomy was necessary in six patients; one patient was de-cannulated after 7 months on sirolimus. all patients have had radiographic and clinical improvement of lm with varying treatment modalities. current clinical observations show improved response with sirolimus and demonstrate tolerability of sirolimus at a young age. conclusion: treatment of pediatric head and neck lms is challenging and a multidisciplinary approach is necessary. as the majority of patients are diagnosed prenatally, prenatal planning and discussion of potential use of sirolimus is beneficial. availability of vascular anomalies experts in the prenatal/neonatal period offers the best management results, and early initiation of sirolimus should be considered for complex lesions. long-term follow up is warranted to investigate the efficacy and timing of treatment options. yale school of medicine, new haven, connecticut, united states background: to mitigate transfusion of pathogencontaminated platelets, amotosalen, a synthetic psoralen compound, is added to sdp components. exposure to uv-a light activates amotosalen and crosslinks dna/rna base pairs, preventing replication of a broad spectrum of viral, bacterial, and other pathogens that may contaminate platelets. pr-sdps were fda approved for clinical use with no age restrictions in 2014. we initiated use of pr-sdps in november of 2016 for all patients. we retrospectively analyzed usage of pr-sdp vs conventional (non-pr) platelets (cp) in neonatal and pediatric patients with thrombocytopenia to compare hemostatic efficacy and the incidence of transfusion reactions (tr) for these products, after one year of a dual platelet inventory. design/method: since pr-sdp were fda-licensed, no irb approval was required; pr-sdp and cp were both considered standard of care. we evaluated transfusions for all pediatric patients age 0-18 years who received any platelet transfusion between november 2016 and november 2017. we determined the volume (mean ml ± 1sd) of each type of platelet component transfused, the number of platelet transfusion episodes, and reported trs based on cdc hemovigilance guidelines. a subgroup analysis was performed for thrombocytopenic neonates (0-4 months). results: patients 0-18 years who received only cps (n = 46) received a total of 8,030 ml of platelets (175 ± 151 ml/patient) over 62 transfusions (1.3 ± 0.6 episodes/patient). for comparison, in 38 patients who received only pr-sdp, a total of 4,350 ml of platelets (115 ± 107 ml/patient, p = 0.04) were infused over 61 transfusions (1.6 ± 0.9 episodes/patient, p = 0.12). for neonates (0-4 months, n = 26) who received only cps, 2,195 ml of cps (84 ± 105 ml/ patient) were transfused over 36 episodes (1.4 ± 0.6 episodes/patient). for comparison, those who received only pr-sdp (n = 27), received 1,613 ml of pr-sdp (60 ± 41 ml/patient, p = 0.27), transfused over 48 episodes (1.8 ± 0.9 episodes/patient, p = 0.08). for all recipients 0-18 years (n = 162), including additional patients who received both cp and pr-sdp, there were three reported allergic trs over 757 transfusion episodes, while no allergic reactions were reported with 537 pr-sdp transfusions. one febrile tr was reported to cp transfusion, while three were reported for pr-sdp. in conclusion, pr-sdps, in our pediatric population age 0-18 years, were comparable to cp products in regards to volume and episodes of platelet transfusions, and incidence/type of transfusion reactions. pr-sdp were safe and effective for use in this pediatric patient population. background: vascular anomalies are classified as either vascular tumors or vascular malformations. fibro-adipose vascular anomaly (fava) is a newly described entity which presents with distinct clinical, radiographic and histopathologic findings. we present a case in which the diagnosis of fava was complicated by a persistent low platelet count secondary to immune thrombocytopenia (itp). to describe a challenging diagnosis of a novel vascular anomaly (fava) complicated by severe thrombocytopenia. a 17 year old male presented to hospital with bruising and left thigh pain related to a remote sports injury. blood work revealed a platelet count of 9 × 109/l, but with an otherwise normal complete blood count. the following were also normal: aptt and fibrinogen; d dimer levels were slightly increased. he was treated with one dose of ivig (0.8 mg/kg) for presumed itp and responded well with his platelet count increasing to 118 × 109/l. he returned to hospital 3 weeks later with recurrent thrombocytopenia and worsening leg pain. an ultrasound of the left thigh revealed a 7.7cm x 4.0cm x 2.9cm lesion within the vastus medialis. the diagnosis of an intramuscular hematoma secondary to persistent thrombocytopenia was made. the patient presented with multiple episodes of thrombocytopenia over the next several months. his itp did not respond to oral prednisone (150 mg/day for 4 days). he continued to have short-lived responses to ivig requiring infusions every other week as his platelet count would fall below 10 × 109/l. his leg pain progressed, restricting him to a wheelchair. further imaging by mri brought into question the diagnosis of a hematoma and a biopsy of the thigh lesion was performed. the results were consistent with a diagnosis of fava; this was subsequently excised. conclusion: this is a unique case where a vascular anomaly was misdiagnosed as a hematoma due to a patient's persistent thrombocytopenia and history of an injury. fava is a newer entity which, unlike other vascular anomalies, has not been linked to thrombocytopenia or a localized consumptive coagulopathy. after excision of the fava, the patient's chronic pain, and mobility resolved, though his itp persisted. objectives: this preliminary, exploratory analysis of realworld administrative data was conducted to determine units dispensed and factor replacement product-related direct expenditures associated with a currently marketed shl or ehl rfix product. design/method: de-identified claims data from the commercially available truven health marketscan® research u.s. claims database were used to identify direct expenditures and number of international units (ius) dispensed for all patients aged 0-17 years with a diagnosis code of icd-9 286.0/icd-10 d66 who used nonacog alfa or eftrenonacog alfa during the study period (june 1, 2014 to july 31, 2017). reference weight measurements from the centers for disease control and prevention national center for health statistics' (cdc nchs) anthropometric data were used to estimate product dispensation on an iu per kg basis. the nonacog alfa and eftrenonacog groups comprised 37 and 11 patients, respectively. the median [iqr] age in the two groups was 8.0 [9.0] and 13.0 [4.0] years, respectively. while 10 of the 11 patients in the eftrenonacog alfa group had >1 calendar quarter of available data, only 23 of the 37 patients in the nonacog alfa group had >1 available quarter. the median rfix product dispensation per quarter was 29,074ius (iqr, 13, 967 ius) in the nonacog alfa group and 62,268ius (iqr, 18, 882 ius) in the eftrenonacog alfa group. incorporating attributed weight values, the median rfix product iu dispensation per kg per week was 97.39 iu/kg/wk (iqr, 31.92 iu/kg/wk-153.92 iu/kg/wk) in the nonacog alfa group, and 96.27 iu/kg/wk (iqr, 53.05-154.03 iu/kg/wk) in the eftrenonacog alfa group. applying 2016 wac prices (eftrenonacog alfa = $2.97/iu; nonacog alfa = $1.37/iu), the calculated estimates of $/kg/week were $133 and $286 in the nonacog alfa and eftranonacog alfa groups, respectively. conclusion: preliminary real-world data derived from a large u.s. claims database revealed differences in product dispensation and factor product-related expenditures among pediatric patients with any severity of hemophilia b to whom an shl or ehl rfix product was prescribed. refinements of these data, potentially to exclude instances of sporadic usage, may shed light on real-world dispensation of rfix products among pediatric hemophilia b patients. background: vascular malformations can be classified as simple (including capillary, venous, lymphatic, arteriovenous), combined, malformations of major named vessels or associated with other anomalies. multiple modalities including laser treatments, sclerotherapy, embolization, surgery and pharmacological intervention (with mtor inhibitors like sirolimus) have been used for treatment of vascular malformations. these interventions have been used alone or in combination with varied outcomes. we present our institution's experience with a multimodal approach to simple and combined vascular malformations. design/method: we performed a retrospective chart review of patients with vascular malformations who were referred to our center for an interventional radiology evaluation from june 2015 -july 2017. we included 22 patients (age at presentation:4 months -25 years), referred initially for interventional radiology procedures (irp) for vascular malformations. all patients had symptoms of pain and/or swelling/deformity. diagnosis of was based on vascular imaging (doppler ultrasound, mri/a/v). nine patients had venous malformations (vm), five had macrocystic lymphatic malformations (lm), six had lymphatic-venous malformations (lvm), and two arteriovenous malformations (avm). 19 patients initially underwent interventional radiology procedures. all the vm patients responded to sclerotherapy alone. three patients with lm responded to sclerotherapy alone, remainder required surgical intervention. one avm patient responded well to embolization, the other needed surgical resection after embolization. four lvm patients underwent irp with minimal improvement in symptoms (3-8 procedures attempted), surgical resection was attempted in 3 patients with poor response and 5 patients were started on sirolimus (0.8mg/m2/dose twice a day). all lvm patients started on sirolimus have responded well (decreased pain and swelling); time to initial symptom response ranged from 2 weeks -1 month from starting medication. in this case series, patients with simple vm responded well to sclerotherapy alone, avm and lm patients needed irp and/or surgery for complete response. complex lvm did not respond well to surgery or irp; 83.3% had improvement in clinical symptoms with addition of sirolimus to the treatment regimen. response to various modalities of treatment varied based on the type of vascular malformation. a multidisciplinary approach to management of vascular malformations is essential to provide multimodal therapeutic options for rapid symptom relief and improve the quality of life of these fragile patients, especially those with complex malformations. background: von willebrand disease (vwd) is the most common bleeding disorder in humans, affecting ∼1% of the united states' population. desmopressin (ddavp) is a longacting vasopressin analog that induces vasoconstriction and release of vwf. ddavp is used in patients with vwd and as a surgical prophylaxis, but carries anti-diuretic properties. to avoid electrolyte imbalance and hyponatremia, fluid restrictions are recommended in the 24 hours post-ddavp administration. objectives: this study sought to examine perioperative practices and outcomes following ddavp administration and a fluid restriction protocol in a population of pediatric patients with von willebrand disease. design/method: a retrospective chart review was conducted for patients with von willebrand disease who underwent surgical procedures at children's hospital of pittsburgh of upmc between january 1, 2015 and december 31, 2016. patient age, sex, weight, diagnosis, surgical procedure, total fluids administered, and post-operative sodium level were recorded. the primary outcomes noted were the proportion of patients exceeding 50% of the recommended fluid consumption for the 12-and 24-hour periods post-ddavp s147 of s301 administration, as defined by local guidelines. secondary outcomes were the presence of any bleeding requiring an er visit or readmission or hyponatremic seizures within 72 hours of ddavp administration. results: data was compiled for 42 patients (23 females, 19 males). the mean age was 11.19 years (sd 5.13 years), median age was 12 years (range 3 to 19 years). procedures included dental (13), otolaryngology (9), orthopedics (7), gastrointestinal (5), plastics (3), neurosurgery (1), ophthalmology (1), dermatology (1), general surgery (1) and gynecology (1). 30% of patients exceeded 50% of the fluid volume recommended for the first 12-hour period post-ddavp administration while still in the surgical setting. no patients exceeded 50% of the fluid volume recommended for the total 24-hour period post-ddavp administration. post-operative sodium levels were obtained in only 7 of 42 patients. no patients returned to the er or were admitted for bleeding in the 72 hours post-ddavp administration. no patients returned to the er or were admitted for hyponatremia or seizures in the 72 hours post-ddavp administration. maintenance of a fluid restriction protocol effectively deterred negative outcomes in this cohort. however, a significant fluid volume was administered in nearly a third of patients despite the restrictions. given the risk of hyponatremia, and limited compliance with fluid restrictions, postoperative sodium levels should be recorded in following ddavp administration to assess the possibility of a hyponatremia and to reinforce the importance of fluid restrictions and their communication. results: a male fetus required in utero insertion of a pleuroamniotic shunt for bilateral pleural effusions diagnosed antenatally by ultrasound. shortly after delivery at term, he developed respiratory distress and was found to have reaccumulation of the pleural effusions. blood work on day 1 of life showed a platelet count of 157,000/ l, which then decreased precipitously. he demonstrated schistocytes on blood-smear, signs of consumptive coagulopathy with hypofibrinogenemia and high d-dimers, and compensatory reticulocytosis. he required multiple transfusions and admissions to the intensive care unit for respiratory support. investigations ruled out congenital ttp, neonatal alloimmune thrombocytopenia, and noonan syndrome. given high clinical suspicion for an underlying vascular lesion causing kmp, a full body mri without contrast was undertaken. this showed a focal area of suspicious signal intensity in the upper paraspinal musculature. an ultrasound and mri with contrast demonstrated an extensive infiltrative vascular lesion involving the paraspinal musculature, prevertebral space, posterior extrapleural space, mediastinum, and neck. the child was commenced on prednisone (2mg/kg/day) and rapamycin (0.9 mg/m2 twice/day). there was no clinical or laboratory improvement after one month. a biopsy was performed which confirmed khe. in the second month of rapamycin therapy, the platelet count gradually normalized and the patient was discharged from hospital at 3.5-months of life. prednisone was weaned off at 4.5 months of life. a repeat mri at 7 months showed significant reduction in the khe. he is now almost 2 years into therapy and doing well. conclusion: this is a unique case of khe with kmp that initially presented with extensive and recurrent pleural and pericardial effusions. this case demonstrates the importance of suspecting an underlying vascular malformation in the presence of kmp. our patient had a delayed but overall good response to rapamycin. further studies investigating duration of rapamycin therapy is key for the optimal management of these patients. rosa diaz, donald mahoney, lakshmi srivaths, donald yee texas children's hospital, houston, texas, united states background: since von willebrand disease (vwd) is the most common inherited bleeding disorder, it must co-exist with other less common bleeding disorders in some dually affected patients. however, reports of combined deficiencies in factor viii (fviii) and von willebrand factor (vwf) are rare. objectives: to study the prevalence and bleeding phenotype of combined deficiencies of fviii and vwf in males with hemophilia a in a hemophilia treatment center. design/method: we retrospectively reviewed the electronic medical records of 99 males with hemophilia a followed at our institution during the past 10 years. the primary and secondary outcomes for the study were (1) the prevalence of combined fviii and vwf deficiencies and (2) the bleeding phenotype of these patients. we identified vwf deficiencies in 9% (n = 9) of the patients with hemophilia a. most (n = 6, 67%) patients were tested for vwf deficiency as part of the initial hemostatic evaluation, but one-third were tested due to clinical concern for inadequate response to fviii concentrate. the median duration of follow up was 9.5 years (range 3.4 to 17.2). patients were referred to our clinic at a median age of 12 months (range 0 to 6 years) for evaluation of easy bruising (n = 4, 45%), mucosal (n = 3, 33%) and surgical bleeding (n = 2, 22%). primary diagnoses included 4 with severe, 3 moderate and 2 mild discrepant hemophilia a. secondary diagnoses included 6 with low vwf activity, 2 type 1 vwd and 1 with type 2 unclassified. patients experienced episodes of musculoskeletal (n = 7, 78%), mucocutaneous (n = 6, 67%) and cns bleeding (n = 1, 11%). a total of 8 patients received factor prophylaxis. half of the patients were initially treated with fviii concentrates but subsequently changed to combined fviii/vwf products due to the frequency of breakthrough bleeding despite good compliance. all patients are on combined fviii/vwf products at the time of this review. a total of 7 (78%) of this cohort developed chronic joint disease manifest as decreased range of motion and/or abnormal mri findings. combined deficiencies of fviii and vwf were present in 9% of our center's hemophilia patients. these patients exhibited a severe bleeding phenotype as evidenced by the high frequency of hemarthrosis, need for prophylaxis and high prevalence of chronic joint disease. while the optimal treatment strategy remains to be elucidated, early recognition of a combined deficiency may have important clinical implications, particularly in patients who demonstrate a suboptimal response to fviii concentrate alone. background: childhood neutropenia is heterogeneous and may be congenital or acquired. cerebral cavernous malformation 3 (ccm3) is a neurovascular malformation disorder where lesions consist of low flow, dilated capillary endothelial channels with increased permeability, predisposing to hemorrhage and thrombosis. programmed cell death protein 10 (pdcd10) activity has been implicated in glia and neuron migration, and recently linked to the dysregulation of the actin and microtubule cytoskeleton, thereby affecting cellular morphology and migration. variants of pdcd10 encoding pdcd10 have been associated with ccm3. ccm3 causes a greater and earlier disease burden than other ccms, with 26% presenting younger than 10 years. some patients have associated extra-neuronal manifestations, suggesting that pdcd10 plays a role in other tissues. we describe a patient with significant blood cytopenias associated with ccm3. design/method: retrospective chart review to obtain patient data. results: an 8-month old female presented with seizure and was found to have multiple intracranial cystic lesions and abscesses due to s. pneumonia serotype 33f. during her treatment, she developed anemia (hemoglobin 7.6-8.7 g/dl), thrombocytopenia (platelets 73,000-128,000 cells/l), and profound neutropenia (absolute neutrophil counts of zero). initial bone marrow evaluation revealed a normocellular marrow but with marked granulocytic hypoplasia and 38% hematogones on flow cytometry. florescent in situ hybridization excluded cytogenetic changes characteristic of myelodysplastic syndrome. further evaluation included testing for neutrophil antibodies, chromosome breakage, and telomere length and results were normal. whole exome sequencing excluded mutations affecting congenital neutropenia genes, but detected a de novo pdcd10 variant (c.474+5g>a), thereby diagnosing ccm3. the neutropenia has responded well to granulocyte colony stimulation factor (gcsf), which is still needed at 26 months of age. moreover, the thrombocytopenia has progressed, requiring periodic platelet transfusions. over time, the bone marrow hematogone population has decreased to 8% at 20 months of age, though the granulocytic hypoplasia persists. conclusion: our case describes the first patient with neutropenia and thrombocytopenia associated with ccm3. we hypothesize the pdcd10 variant is the etiology of bone marrow dysfunction due to its role in actin and microtubule cytoskeleton formation, akin to the pathophysiology of xlinked neutropenia. supportive features of an underlying genetic cause of marrow dysfunction include the persistence of cytopenias beyond infection resolution as well as presence of hematogones. hematogones were previously reported to occur in patients with other congenital neutropenia disorders, indicating they could be a feature of congenital neutropenia and may be reactive to surrounding cell apoptosis. further testing of pdcd10 role in hematopoiesis should be explored. background: 10-15% of adult women will suffer from heavy menstrual bleeding (hmb) during their lifetime. 90% of women with inherited bleeding disorders suffer from hmb. there is a paucity of data about hmb among adolescents and young adults (aya), a population in which hmb may have large social and educational effects. objectives: to study the social and academic implications of hmb in an aya population. design/method: this is a questionnaire based survey conducted in a medium-sized city in california. we recruited females 14-24 years of age from one high school and from local university. the questionnaire was set up in research electronic data capture (redcap) at our institute which allowed us to obtain objective data about the respondents' menstrual cycles. a link was sent to the high school students via their online portal schoolloop and to the university students via social media and word of mouth. data was collected over 12 weeks from may 2017 to august 2017. we received 145 replies, some were not complete. using regression analysis, data was analyzed from 115 respondents in the age group of 18-24 (with a mean age of 19) years. we developed a composite score for hmb based on factors including saturation levels, number of pads, duration of bleeding, soaking of a pad within two hours, passage of clots, size and number of clots, and gushing sensation. we conducted statistical analysis of the drivers and implications of hmb based on the composite score. results indicate that having a relative with hmb, having other bleeding problems, and having anemia are drivers of higher hmb score. the results also indicate that hmb adversely affects quality of life as measured by participation in sports, social activities, after-school activities, tiredness, absenteeism, and gpa. hmb is also associated with increased rates of anemia and use of anti-depressants. hmb-driven anemia further adversely affects gpa. under-represented minorities are more likely to have a higher hmb score, as well as an increased adverse effect of hmb on gpa. the results suggest that the social costs of hmb are pervasive in the aya population, and especially pronounced among minorities. a relative with hmb is a significant driver of heavy menstrual bleeding. a hemostatic screen should be included when assessing the aya population with hmb. johns hopkins all children 's hospital, st. petersburg, florida, united states background: propranolol is a non-cardioselective beta blocker medication frequently prescribed for hemangiomas and hyperthyroidism. propranolol inhibits types i and ii iodothyronine deiodinases, enzymes that convert bioinactive thyroxine (t4) into bioactive triiodothyronine (t3). hypothyroidism is a well-recognized complication of diffuse hepatic hemangiomas that produce type iii deiodinase, an enzyme that converts t4 into bioinactive reverse t3 and t3 into diiodothyronine. thyroxine is typically selected for replacement in this population, even though doses up to 60% above physiologic may be necessary. we hypothesized that low dose, nearly physiologic t3 would be safer and equally effective because it bypasses propranolol's impact on the pituitarythyroid axis. we report an infant with diffuse hepatic hemangiomatosis and acquired hypothyroidism successfully treated with propranolol, prednisone, and triiodothyronine. design/method: a 7mo healthy female presented with abdominal distension, poor oral intake, and hepatomegaly. mri confirmed diffuse hepatic hemangiomatosis, the largest lesion measuring 4.4 cm by 3.9 cm. thyrotropin (tsh) was elevated at 47.9 (reference range* 0.5-6 mcgiu/ml), total t3# 165 (rr 60-300 ng/dl), and total t4^18.8 (rr 6-14 mcg/dl). treatment was started with prednisone (2 mg/kg/day) for three weeks, propranolol (3 mg/kg/day) and t3 (0.64mcg/kg/day). the t3 dose was slowly titrated to a maximum of 1.72 mcg/kg/day. thyroid hormone levels rapidly improved on t3 replacement. after two weeks, the tsh was 14.4, tt3 92, and tt4 16.5. after eight months, the tsh was 2.9, tt3 161, and tt4 10.1. at twelve months, the tsh dropped to 0.6, tt3 294, and tt4 3.7, suggesting decreased tumor production of type iii iodothyronine deiodinase. liver mri confirmed fewer hemangiomas, largest being 1.3 cm by 1.6 cm. the patient's t3 dose was reduced. both propranolol and t3 were discontinued after twenty-four months of treatment. one year off all therapy, this child has normal growth and development, only two <1.3cm hepatic hemangiomas and no evidence of hypothyroidism (tsh 2.2; tt3 153; tt4 6.4). conclusion: t3 at near physiologic doses corrects the consumptive hypothyroidism associated with diffuse hepatic hemangiomas. t3 replacement is preferable to thyroxine due to its lower risk of rebound hyperthyroidism as the hemangiomas involute and type iii deiodinase production declines. there are two prior case reports describing t3 use without t4, one employing propranolol and the other utilizing steroids for hemangioma management. this is the first case report with long term follow-up of a child treated with multimodal therapy including propranolol, prednisone, and triiodothyronine. *rr = reference range; #tt3 = total t3;^tt4 = total t4 background: multifocal lymphangioendotheliomatosis with thrombocytopenia (mlt) is a rare congenital disorder first described in 2004 that is characterized by multiple vascular abnormalities commonly involving the skin and gastrointestinal tract as well as consumptive coagulopathy often resulting in gi bleeding in infancy(1). to describe an unusual presentation and successful management of mlt in a neonate. design/method: baby h was born at full term after a pregnancy complicated by maternal sinus venous thrombosis requiring anticoagulation beginning at 28 weeks. at birth, she was diagnosed with multiple hemangiomas based on clinical exam. at two weeks of age, she developed melena and hematemesis. cbc revealed platelet count of 70 and she was referred to the ed. abdominal ultrasound was concerning for abnormal hepatic waveform; cxr showed multiple pulmonary nodules. workup revealed no other lesions and no further hematologic abnormalities. biopsy of presumed hemangioma ultimately revealed a smooth muscle-lined vascular proliferation without glut-1 immunoreactivity, consistent with mlt. her early course was complicated by an acute hemodynamically significant gi bleed; esophagogastroduodenoscopy identified six bleeding vascular malformations within the stomach that were injected with epinephrine and sclerosed with successful hemostasis. she received multiple prbc and platelet transfusions. central access was obtained and she was started on oral sirolimus based on previous reports of successful use in management of vascular malformations given its antiangiogenic and immunosuppressive effects (2). she has tolerated it well with no evidence of toxicity and has achieved a partial response with stable of hemoglobin >8 and platelet count >90. cutaneous lesions have diminished in intensity and she has had no further signs of gi bleeding. she receives pentamidine for pcp prophylaxis. she continues to have appropriate growth and development. we describe here an unusual presentation of an already rare disease. while cutaneous and gi lesions are typical of mlt, pulmonary involvement is not well-described in the literature. early identification of tissue-based diagnosis enabled timely stabilization and treatment of the patient. five months later, she continues to tolerate sirolimus and has shown significant response with diminished coloration of cutaneous lesions, stable blood counts, and no further bleeding. mlt is a relatively newly-recognized disorder with significant phenotypic variability. given that bleeding secondary to a kasabach-merritt-type consumptive thrombocytopenia is the major cause of morbidity and mortality in the first year of life in children with mlt, it is essential to recognize the diagnosis and initiate appropriate treatment as early as possible. 1north, arch background: patients with generalized joint hypermobility (jhm) may experience easy bruising or bleeding given the association between these symptoms and abnormalities in collagen, a required component of primary hemostasis. heavy menstrual bleeding (hmb) is a common initial presentation for females with underlying hemostatic defects and may be the sole manifestation of a bleeding disorder. however, limited reports describe jhm as a cause of hmb, leading to under recognition. objectives: to describe the clinical characteristics and management of young women presenting with hmb in the setting of jhm. design/method: this study utilized our hmb research registry. we included subjects 11-18 years, seen in the nationwide children's young women's hematology clinic between february 2014 and november 2017 with both hmb and jhm. medical records were retrospectively reviewed for history of presentation, menorrhagia impact questionnaire (miq): a validated quality-of-life tool for females with hmb, medication profiles and relevant laboratory studies. results: twenty-five patients met inclusion criteria (median age 15 years, range 11-18) with an average beighton score of 6.2 (range 4 to 9). participants presented an average of 3.2 years (range 5 months to 6 years) after menarche despite 76% of patients reporting heavy to very heavy menses since menarche. according to the miq responses, most participants expressed hmb-associated limitations in physical activities (84%), social activities (68%), and work or school activities (64%). of the participants, 92% reported bleeding symptoms in addition to hmb, most commonly easy bruising (56%), epistaxis (48%) and cutaneous bleeding (44%). forty percent of young women presented with anemia due to chronic blood loss. results of hemostatic testing were unremarkable, with the exception of one patient who was also found to have type 1 von willebrand disease. additionally, 44% of females reported arthralgia, with knees and ankles the most commonly affected joints. at time of presentation, 32% of participants reported failure of initial therapies and most patients (88%) were managed long-term with oral hormone therapy. in a small population of young women found to have jhm who initially presented with hmb, patients were likely to have prior bleeding symptoms as well as substantial delays from menarche to timing of presentation at our young women's hematology clinic despite limitations in activities of daily life. greater awareness of the associations between bleeding symptoms and jhm, despite typically normal hemostatic laboratory results, is necessary so that patients can more easily be identified and receive appropriate therapy. the objective is to determine the impact of cl care practices involving the home environment on ambulatory clabsi rates. design/method: information for the pi was collected through a comprehensive survey that was completed annually by the ccbdn member hospitals. responses to the questions about cl care practices involving the home environment were selected from the pi for 2015. ambulatory clabsi rates and ambulatory total bloodstream infection (bsi) rates were obtained from another ccbdn database. the proportion of hospitals that did or did not employ a particular cl care practice was tallied. the mean ambulatory clabsi rate and mean ambulatory total bsi rate of the hospitals that did or did not employ a particular cl care practice were compared using generalized linear model techniques assuming an underlying negative binomial distribution. results: twenty-five hospitals submitted responses to the 8 questions about cl care practices involving the home environment. one hospital was excluded for lack of bsi data. sixty-three percent of the hospitals programmatically educated parents about all aspects of the cl care bundle. the mean ambulatory clabsi rate for the hospitals that educated parents was significantly lower than that of the hospitals that did not (0.20 infections/1000 cl days vs. 0.30 infections/1000 cl days; p = 0.02). the mean ambulatory total bsi rate was also significantly lower (0.35 infections/1000 cl days vs. 0.53 infections/1000 cl days; p = 0.01). the mean ambulatory clabsi rates and mean ambulatory total bsi rates were not significantly different for the other 7 cl care practices. conclusion: an analysis of cl care practices involving the home environment reveals that parental education of all aspects of the cl care bundle is associated with a lower ambulatory clabsi rate and lower ambulatory total bsi rate. this finding highlights the importance of systematically teaching family members the proper method of handling cl. background: children undergoing chemotherapy are at a high risk for developing nausea. dr. amy baxter in collaboration with pediatric oncology patients and nurses, developed and validated a pictorial nausea rating scale for children aged 7-18 years, called the baxter retching faces (barf) nausea scale. staff nurses at a large, academic, pediatric hospital located within washington, d.c., have identified variability in nursing assessment and documentation of chemotherapy induced nausea and vomiting (cinv) in pediatric oncology patients. the purpose of this quality improvement project was to utilize the barf scale to standardize assessment and documentation of nausea in pediatric oncology patients receiving chemotherapy. the primary aims of this project were to: assess feasibility of the barf scale in clinical practice; increase nursing knowledge about cinv through education sessions; increase documentation of nausea assessments through the use of the scale. the secondary aim of this project was to: increase the recognition of nausea through the use of a standardized assessment tool. design/method: the pdsa model was used to guide the design and implementation plan. in the first phase of the project data was collected to identify the prevalence of nausea in patients admitted for chemotherapy in the prior three months. education sessions discussing cinv and the utilization of the barf scale were conducted. pre and post assessment of nurses' knowledge of cinv and documentation were assessed. in the second phase the barf scale was implemented into practice. nurses were asked to utilize the barf scale to assess and document nausea scores in patients, aged 7 to 18 years, receiving chemotherapy. at the end of the implementation period nurses were surveyed about the feasibility of the scale. post data was collected to identify the prevalence of nausea documented in the electronic health record. this project was undertaken as a quality improvement initiative at children's national and it does not constitute as human subjects research. as such it was not under the oversight of the institutional review board. results: all data has been collected; however complete data analysis will be conducted in the upcoming weeks. background: sickle cell disease (scd) is the most common inherited blood disorder in the united states (us); however, there are few quality measurements to evaluate scd practice. in 2014, the nhlbi published guidelines that include two key interventions for children with sickle cell anemia (sca): the use of transcranial doppler (tcd) screening for stroke prevention and hydroxyurea (hu) to prevent scd pain crisis. we conducted a national survey of scd management sent to providers in over 20 institutions in the us to better assess knowledge of the guidelines and barriers to hu counseling and tcd screening guideline implementation. it was hypothesized that the barriers to tcd screening are different than barriers to hu counseling and prescribing. a 33-question anonymous survey was sent to 49 providers by mail (follow-up by email). survey themes included nhlbi guidelines knowledge and comfort with understanding and implementing both tcd screening and hu use. the response rate was 59% (29/49) however one survey was incomplete. thus, 28 were analyzed in the final data set. all of the respondents are in active practice, 96% s153 of s301 in academics and all care for children with scd. the majority of providers (96%) felt "very" or "extremely" confident in their knowledge of tcd screening and interpretation. similarly, 100% of providers felt "very" or "extremely" familiar with hu dosing and management. for tcd screening, 36% of providers estimated their screening rates were >90% and 64% providers felt their annual screening rates were 75-90%. the two biggest barriers to tcd screening noted by providers (of moderate to extreme significance) included: lack of support staff (36%) and lack of time during a patient visit (26%). regarding hu prescribing practices, 71% of providers offered hu to at least 90% of children with sca over nine months of age. the biggest barrier to hu prescribing noted by 46% of providers was concerns about patient adherence or access to the medication. only 7% providers felt that lack of support staff was a moderately significant barrier to hu prescribing. the pediatric scd providers surveyed all have access to the nhlbi guidelines. despite widespread guideline knowledge, there are different barriers for tcd screening versus hu prescribing, which prevent optimal implementation. as a result, although both recommendations are from the same nhlbi guideline, they likely will require different implementation strategies (systems-based interventions for tcd screening; interventions to improve patient adherence for hu counseling) to improve outcomes. background: invasive fungal disease (ifd) is a major cause of mortality and morbidity among pediatric immunocompromised patients such as those who receive chemotherapy or hematopoietic stem cell transplantation. the current diagnostic 'gold standard' of ifd remains culture of infected tissue obtained by biopsy. noninvasive biomarker testing for galactomannan or 1,3-beta-d-glucan (bg) can have low sensitivity and does not provide species-level identification. nextgeneration sequencing (ngs) of cell-free plasma is a promis-ing noninvasive approach to providing species-level identification of ifd via a blood test and can further guide specific treatment. objectives: describe the incidence of positivity for fungal specific pathogens on ngs analysis in a high-risk immunocompromised pediatric population and correlate results with other 'standard' infectious studies if performed. design/method: immunocompromised pediatric patients with suspected ifd were enrolled and plasma was collected at time of enrollment. ngs was performed on extracted dna in cell-free plasma (karius, redwood city, ca). after removing human reads, remaining sequences were aligned to a curated database including 1251 pathogens. organisms present at a significance-level above a predefined threshold were reported. results: twenty-seven samples from 33 enrolled patients have been processed thus far. of these 27 subjects, 14 were enrolled for prolonged febrile neutropenia (≥96 hours) despite broad-spectrum antibiotics, 5 for recrudescent febrile neutropenia, 5 for abnormal imaging, and 3 with other findings. after evaluation of routine studies performed, 4 patients met criteria for proven ifd, 2 for probable ifd, and 9 for possible ifd using eortc/msg guidelines. the ngs plasma test identified the same pathogen as cultured from infected tissue or blood in 100% (4/4) of the proven cases. in the probable cases, pneumocystis jirovecii was identified in a patient with a positive bg (389 pg/ml) and pneumonia. among the possible cases, toxoplasma gondii was detected in a patient with prolonged febrile neutropenia and lung imaging suggestive of ifd. additionally, candida glabrata was isolated in a patient with prolonged febrile neutropenia but no other criteria for ifd. numerous pathogens were also identified that could explain the above clinical parameters, including hsv1, cmv, vzv, hhv6, ebv, bk polyoma virus, and ureaplasma parvum. the cell-free plasma ngs test can detect invasive fungal infections from blood. the test identified fungi from proven ifd, detected pathogens in both probable and possible ifd cases, and is a useful diagnostic tool in the evaluation of ifd. supplies and sample shipment and processing supported by karius, inc. baylor college of medicine, texas children's hospital, houston, texas, united states background: practicing medicine is a lifelong learning process. as noted in the institute of medicine's seminal report, 'to err is human,' adverse outcomes do not typically result from individual recklessness; rather, they result from faulty systems, processes, or conditions that provide an environment conducive to making a mistake, or failing to prevent one. learning to systematically review errors and translate lessons learned into quality improvement (qi) initiatives is a critical component of practice-based learning and improvement for practitioners at all career levels. objectives: to develop a methodical, self-reflective and nonthreatening approach to incident analysis and translation of lessons learned into qi initiatives. design/method: we used a validated, structured case audit approach, modified from szostek et al: 1) review all documentation relating to the case and identify all health care providers involved; 2) interview stakeholders, including those who directly provided and supported care; 3) use a qi tool to conduct a root-cause analysis; 4) identify a systems issue that contributed to the outcome; and 5) propose systems-level interventions and prioritize initiatives based on effort-yield projections. results: pdsa cycle 1: plan: establish a committee to 1) identify potential cases, 2) triage cases for conference presentation, 3) determine timing and frequency of conferences, 4) develop a training manual, 5) record identified qi initiatives. do: we established a quarterly section-wide meeting to which all members of the pediatric hematology/oncology service are invited, including administrative and nursing leadership. we developed a training manual and structured presentation template. prioritized cases were discussed in advance during multidisciplinary case review sessions, and presented by senior fellows who were instructed to focus discussion on potential opportunities for qi. study: we identified 23 cases, 10 meeting criteria for mmi presentation. qi initiatives identified from this conference resulted in a number of systemic practice changes; however, we encountered challenges to sustaining these changes over time. act: objectives for the next pdsa cycle are to 1) establish a method for tracking the adherence to recommended changes in practice, 2) maximize sustainability by integrating qi initiatives into institutional qi leadership and practice standardization committees. we have successfully implemented an mmi conference that meets 5 out of 6 institute of medicine quality domains: safety, effectiveness, patient-centeredness, timeliness, and efficiency. a standardized, consistent approach to mmi presentations that includes identification of contributing factors and specific qi implications has the potential for improving both provider education and patient care/safety. johns hopkins university, baltimore, maryland, united states background: receiving a cancer diagnosis is a life-changing event for patients and caregivers, although little is known about the experience. while some oncologists receive dedicated training in delivering this bad news, the initial conversation is often with a primary pediatrician, and these providers often feel they do not receive adequate training in the communication of a cancer diagnosis. objectives: our objectives were two-fold: first, to better define the experiences of caregivers/patients when told of a cancer diagnosis, and to query how caregivers/patients believe providers can improve the disclosure of this bad news. secondly, to assess what, if any, training primary pediatricians received in this skill, and to assess how comfortable providers in various settings and stages of training are with communicating cancer diagnoses. design/method: from november 2016-2017, semistructured, in-depth interviews were conducted with pediatric oncology patients and caregivers of patients (n = 6) diagnosed in the past year regarding their experiences receiving the diagnosis at our institution. in addition, pediatric residents (n = 6), outpatient pediatric primary care physicians and pediatric emergency medicine physicians (n = 6) were interviewed regarding their experiences delivering cancer diagnoses. interviews were analyzed following principles of thematic analysis. interviewers with patients and caregivers had two common themes: 1) all emphasized their wish for direct and thorough information; 2) both patients and caregivers emphasized the gratitude they felt for physicians who gave them hope by emphasizing the good prognosis of their child's cancer. lack of training in this area, as well as lack of comfort delivering this news was common will all providers. additionally, providers report variable approaches to giving bad news, including 1) whether to tell caregivers separately or tell the child and parents together, and 2) whether to give favorable prognostic information. additionally, attending physicians also differed significantly in their approaches to teaching residents. while some believed residents should give the news to gain experience, others felt that this is not appropriate if residents are inexperienced. only one resident reported ever receiving feedback on his communication skills in this type of discussion. conclusion: we plan to build on these interviews to develop a national survey of patients, caregivers, and providers to better understand the issues surrounding this discussion. we will use the findings to develop a communication curriculum for pediatric residents, focusing on the discussions that occur in the outpatient setting by primary pediatricians. background: human papilloma virus (hpv), common in both females and males, is responsible for pathologies ranging from benign genital warts to cervical and penile cancer. hpv strains 16 and 18 are responsible for 21,000 malignancies each year in the united states, and one third of them arise in men. pharmaceutical companies have now developed a vaccine that will help prevent the virus-associated malignancies. the cdc initially recommended that females ages 11-26 years receive the vaccine series, then starting in 2011 they expanded the eligibility to males ages 11-21 years. despite being widely available and highly publicized, only 40% of eligible females receive the full vaccine series. objectives: this study aims to assess the knowledge of hpv, the attitudes towards the hpv vaccine, and identify barriers preventing its full utilization. once identified, we aim to overcome the barrier(s) in order to improve vaccination rates in eligible adolescents. we distributed a standardized questionnaire to the parents of eligible female and male patients in our pediatric hematology-oncology clinic. it assessed the parents' knowledge of hpv and the vaccine, their views of the vaccine, and reasons why they may oppose it. results: approximately 80% of parents claim they have been educated about hpv, mostly by their primary care physician. however, 35% did not know what disorders hpv caused; 35% felt the vaccine should not be added to the typical vaccine schedule; 25% of parents do not intend to vaccinate their child. of those that opposed the vaccine, one-third were concerned about potential side effects and nearly 35% feel they do not have enough information. additionally, 25% of parents are not aware that the vaccine is available at their child's doctor and only 30% of parents have discussed the hpv vaccine with their child's doctor. the largest barrier to the utilization of the hpv vaccine that we have identified appears to be lack of educa-tion. as a result, we have begun distributing the cdc's hpv and vaccine patient guide to our patients' families as an intervention. we are currently in the process of re-administering our survey to these families after implementing the intervention to assess its success in increasing both knowledge and utilization of the hpv vaccine. cancer institute, chennai, chennai, tamilnadu, india background: rasburicase is a recombinant urate oxidase enzyme approved for use in tumor lysis syndrome (tls) and it acts by reducing serum uric acid levels. using rasburicase at the recommended dose of 0.2mg/kg/day for 5 days is expensive and it is not known whether this extended schedule is clinically beneficial compared to a single fixed dose of 1.5 mg. the aim of the present study was to evaluate the efficacy of single dose rasburicase 1.5 mg in prevention and management of tls. design/method: rasburicase is available as single use 1.5 mg vial. at our institution a single dose of rasburicase 1.5 mg irrespective of bodyweight has been used in adults and in children a dose of 0.15 mg/kg (maximum 1.5 mg) has been used since 2012 for prevention and management of tls and subsequent doses are given based on biochemical response and clinical condition. we retrospectively analysed the case records of patients who had received rasburicase from january 2012 to january 2017. the study included 186 patients with hematological malignancies who received rasburicase. children accounted for 56.4% (n = 105) patients and males comprised 73% (n = 135). rasburicase was used prophylactically in 59 (31.7%) patients, for laboratory tls in 76 patients (40.8%) and for clinical tls in 51 (27.4%) patients. single fixed dose rasburicase prevented laboratory/clinical tls in 87% of the prophylactic group and prevented clinical tls in 72% of the laboratory tls group. none of the patients in prophylactic and laboratory tls group developed clinical tls. however, majority of the patients with clinical tls required more than one dose rasburicase. single dose of 1.5 mg (1 vial) rasburicase is efficient in preventing and managing laboratory tls and is economically viable in resource constrained settings. nicole wood, lauren amos, nicholas clark, chris klockau, karen lewing, alan gamis children's mercy kansas city, kansas city, missouri, united states background: medication reconciliation for newly diagnosed oncology patients is complicated and cumbersome. these patients are often admitted on no medications, and leave on multiple. chemotherapy and supportive medications are crucial. despite numerous individuals overseeing this process, prescribing errors or omissions still occur. when reviewing the literature, improvement occurs when there is an interprofessional and standardized process to medication reconciliation. objectives: this project's aim was to improve the accuracy of the discharge medication reconciliation process from 74% to 90% from february 2017-august 2017. the process measure was the percentage of patients discharged with an accurate checklist. additional time for staff spent in completing the checklist and avoiding an increased error rate by changing the prescribing process were followed as balancing measures. we created a discharge medication checklist which included a list of required home medications prescribed by the resident, ideally 24 hours prior to discharge. it required fellow or attending review and pharmacy to review the list and educate the family. checklists were collected monthly and reviewed against the electronic medical record (emr) for accuracy. results: six pdsa cycles were completed. there were 2 errors during the data collection time frame. in pdsa cycle 1, a patient received acetaminophen for pain control which is avoided at home. in addition, this patient received diphenhydramine instead of ondansetron, which is preferred as an antiemetic. in pdsa cycle 4, a patient with a pending diagnosis was sent home with acetaminophen. of note, this patient did not have a checklist completed upon discharge. this project provides a novel and important method to standardize the discharge medication reconciliation process in a complex patient population. it clarifies which types of medications these patients need, provides pharmacy teaching to families which was not done previously, and prescribes discharge medications to families sooner. after the first medication reconciliation error, the checklist was revised. no further errors were made following revision, with the exception of one patient without a completed checklist at dis-charge. our accuracy rate increased from 74% at baseline to 92% following implementation. we are in the process of making the checklist electronic and accessible in the emr. in the interim between the end of data collection and implementation into the emr, a leukemia patient was sent home without an epinephrine pen, further demonstrating the importance of this standardized discharge process. for this reason, we have re-instituted the checklist until the electronic version is available. background: survivors of pediatric cancer are at risk of losing pre-existing protective antibodies to vaccine preventable diseases. in a prior study, 35% of children < 7 years lost humoral immunity to measles as a result of chemotherapy induced alterations in immune system. measles in recipients of immunosuppressive chemotherapy has mortality rates up to 50%. because of volitional vaccine refusal, there has been a dramatic increase in measles infection from 63 cases in 2010 to 677 in 2014, including several statewide outbreaks. small pediatric oncology practices frequently share floor/clinic space with the general pediatric patients putting them at risk for measles since virulence starts 48 hours prior to symptoms. there is no standard protocol for revaccinating post-chemotherapy patients. to assess measles risk based on serial humoral immune status in a cohort of pediatric oncology patients receiving intensive chemotherapy design/method: patients < 21 years age with known vaccination status receiving intensive chemotherapy between july 2015-june 2017 at our institution's pediatric oncology practice were included in this prospective study. serial measles igg antibodies were measured at diagnosis, 6 months and 12 months after initiation of chemotherapy using elisa. measles immunity was defined per lab standards. a comparison of pre-chemotherapy and serial post-chemotherapy immunization titers was made for all patients by diagnosis. the study population consisted of 31 children (17 male); 8 patients had all, 7 non-hodgkin lymphoma, 11 sarcoma and 5 other solid tumors. two patients (6.4%), both unvaccinated had non-protective measles antibody levels at s157 of s301 baseline. of the remaining 29 patients, 13.7% patients (2 leukemia, 1 lymphoma and 1 sarcoma) lost protective antibody titers at 6 months after initiation of chemotherapy and 27.5% (4 leukemia, 1 lymphoma and 3 sarcoma) at 12 months after initiation of therapy. 60% of the remaining 21 patients who retained measles antibody titers within protective range at 12 months also demonstrated a steady decline in antibody titers at 6 and 12 months from therapy initiation. the loss of protective measles humoral immunity occurred significantly more often in patients with leukemia compared to other malignancies. oncology patients in our practice undergoing intensive chemotherapy demonstrated progressive waning of protective measles igg titers. our data suggests that it should be standard practice to check all patients for measles humoral immunity prior to starting chemotherapy and at completion. larger studies need to be performed to establish guidelines for revaccinating post-chemotherapy pediatric patients, an intervention that is easily applicable and of low cost. background: the accurate determination of glomerular filtration rate (gfr) is important to screen for acute kidney injury, to dose chemo-therapy, and to identify risk for chronic kidney disease.being correlated with inulin clearance, measured gfr by iohexol plasma disappearance (igfr) is a new gold standard for measurement of gfr in pediatric cohort studies. igfr is based on the clearance of an exogenous marker and is unaffected by endogenous compounds or a patient's muscle mass. we compared igfr with 24-hour urine creatinine clearance (24crcl) and gfr estimating equations using serum creatinine (scr) and serum cystatin c (cystc) in pediatric patients with cancer. we recruited participants who were ages 6 to 16 yrs, continent of urine, and diagnosed with a malignancy in the past 5 years. eligible subjects had stable kidney function for at least two weeks prior to the assessment of igfr. consented subjects had baseline assessments including height, weight and vital signs. blood samples were obtained for serum chemistry, and time zero iohexol. igfr determined by 5ml iohexol solution infused over 1-2 minutes followed by 10ml of sterile saline. blood was drawn at 10, 30, 120 and 300 minutes.at the same time of igfr, the 24crcl was collected. igfr was calculated using a two-compartment model and area under the curve. we compared igfr to published gfr equations (schwartz et al, kidney int 2012). results: ten subjects (7 female/3male) agreed to participate. the distribution of diagnoses for the subjects: all = 6, lymphoma = 1, brain tumors = 2 and hepatocellular carcinoma = 1. six patients were off therapy. the lower gfrs are noted in patients who had malignancies other than leukemia, likely due to the use of cisplatin based therapy. the average igfr was 85ml/min/1.73m^2 whereas 24crcl was 155.8 ml/min/1.73m^2; demonstrating the 24crcl overestimates gfr compared to igfr. comparing igfr to univariate equations using scr, cystc, and the multivariate equation with both, the univariate cystc equation correlated well with igfr; the others overestimated igfr. we found that 24crcl overestimated igfr. the univariate cystc equation better correlated to igfr than equations with scr. the poor performance of scr based methods to assess gfr might be due to decreased muscle mass and inadequate nutritional status. creatinine-based determinations of gfr alone, may not be accurate in this population. further study is needed to determine if igfr should be a standard of care to assess gfr in children with cancer particularly who are receiving nephrotoxic medications and incontinent of urine. background: pediatric oncology patients undergoing chemotherapy through indwelling venous catheters are at increased risk for severe sepsis especially when neutropenic due to chemotherapy. rapid triage and early recognition are essential because delayed initiation of antibiotics and fluids in these patients or delayed transfer to higher level of care after initial stabilization is associated with poor clinical outcome. our pediatric oncology out-patient clinic is designated as an article 28 unit whereby the providers can initiate and give treatment such as intravenous fluid, antibiotics, chemotherapy and blood products. objectives: global aim-optimize management of early sepsis and decreased morbidity, mortality and hospital length of stay in the high risk pediatric oncology patients. smart aim-improve timely management with initiation of fluids and antibiotics and transfer of septic patients to higher levels of care by 10% in 6 months in above patients design/method: multidisciplinary team with physicians and nurses was created. retropective chart review of sepsis patients treated at the clinic from april 2016 to october 2016 was done using an audit sheet to identify the barriers in the delivery of care. three patients were identified and data analyzed prior to intervention; two were analyzed post interventions. a key driver diagram was created by the group to drive intervention. a process map was designed to identify the different steps in the care of these patients to pinpoint areas needing improvement. different timed data points were used starting from time of arrival to clinic, time to antibiotics and fluids and time to transfer to higher level of care. rapid pdsa cycles were done to improve the processes and delivery of care. run charts were created. there was an improvement close to the goal of 10 % for all data points used. pdsa cycles for improvement included conducting frequent mock codes with appropriate feedback real time coaching and process planning with nursing staff. we partnered with pharmacy for close loop communication with clinic staff and we improved communication between physicans at different levels. conclusion: sepsis in neutropenic pediatric oncology patients is deadly and can be reversed with timely management at different levels. given the promising results of the above project, we want re-inforcement of the processes to be a part of the daily practice of first line clinical staff. eventually we will extend the principles learnt in management and triage of sepsis to other outpatient emergencies chemotherapy related anaphylaxis background: chemotherapy-induced nausea and vomiting (cinv) is a common side effect in children receiving antineoplastic chemotherapy. recommended prophylactic antiemetic medications are based on the classification of chemotherapy emetogenicity. however, despite appropriate use of these antiemetic agents, some patients will still experience nausea and/or vomiting. children's oncology group clinical practice guidelines recommend the addition of olanzapine to prophylactic regimens for management of breakthrough cinv. objectives: our pediatric hematology oncology center implemented a quality improvement (qi) project aimed to increase the use of olanzapine in pediatric cancer patients 7 years of age and older receiving moderately or highly emetogenic chemotherapy and experiencing breakthrough cinv over a 3 month period. design/method: this qi project was conducted utilizing plan-do-study-act (pdsa) cycles. for the first pdsa cycle, baseline data was collected through chart review to determine the rate of olanzapine use for breakthrough cinv over a 6 month period from july 2016 to december 2016. breakthrough cinv was defined as use of 2 or more doses of antiemetic agents other than those given for cinv prophylaxis. guidelines for treatment of breakthrough cinv were reviewed with pediatric hematology/oncology attending physicians and fellows. flyers were created that listed chemotherapy regimens considered moderately and highly emetogenic. if a patient experienced breakthrough cinv, a flyer was to be placed in the patient's roadmap binder to signal olanzapine should be added to the next chemotherapy block. data was collected over a 1 month period in september 2017 following this first intervention. the second pdsa cycle consisted of didactic education and training of pediatric oncology nurses as well as pediatric residents regarding the addition of olanzapine for breakthrough cinv. rates of olanzapine use were then collected from october 2017 through november 2017. results: olanzapine use increased from 3.8% at baseline to 58.3% after the first pdsa cycle ( 2 = 14.666, p = 0.000). after the second pdsa cycle, olanzapine use increased another 14.1% to 72.4% ( 2 = 0.777, p = 0.378). the administration of olanzapine was successfully increased by modifying patients' roadmaps after patients experienced breakthrough cinv as well as with education and training of pediatric oncology staff, fellows, residents, and nurses. background: venous thromboembolism (vte) is increasingly affecting children. according to an administrative database study, there was a 70% increase in the incidence of vte among children admitted to free-standing children's hospitals in the united states from 2001 to 2007. risk factors for hospital-acquired vte are well-known and well-studied in adults, with evidence-based preventative measures available. similar guidelines are lacking for children. objectives: there is an ongoing national-initiative to develop and institute methods for screening and preventing hospitalacquired vte in children. in 01/2014, nationwide children's hospital instituted an electronic screening form required for all patients admitted ≥24 hours. patients were scored and riskstratified based on eight risk-categories. a summated score was used to determine the vte risk level, and used to make prophylaxis recommendations for patients ≥18 years; as well as patients ≥14 years who were admitted to an intensive care (icu), surgical, or trauma unit. the purpose of this irb exempt, quality improvement initiative was to retrospectively review our experience with this risk-stratification tool. results: 262 hospital-acquired vte events occurred in 232 unique subjects. median age at vte diagnosis was 2 years. only 69 (26%) vte occurred in children ≥14 years of age. 237 (91%) vte were deep vein thrombosis (dvt), and 16 (6.1%) involved pulmonary embolism. vte was most common in subspecialty units including the pediatric and cardiac icus 87 (33.2%); neonatal icu, 43 (16.4%); and hematologyoncology, 31 (11.8%). 184 (70%) vte were associated with central venous catheters (cvc) and 144 events (55%) were associated with altered mobility. congenital heart disease/heart failure was the most common chronic medical condition associated with vte (69 (26.3%) events); whereas infection and trauma/surgery were the most common acute medical conditions associated with vte (137 (52.3%) and 89 (34%) events, respectively). during 249 (95%) events, subjects scored a summated score ≥3. in summary, in this single institution, prospectively maintained database, cvc remains the most common risk factor for vte, followed by cardiac disease, infection and trauma/surgery. most subjects who developed vte scored high (score ≥ 3) on our screening tool. only a small proportion of vte occurred in patients older than 14 years and thus eligible for thromboprophylaxis. our results indicate that future vte prevention endeavors should include these age groups in addition to exploring more aggressive prophylactic modalities including pharmacological prophylaxis. background: pediatric fellows are required to have active engagement in quality improvement (qi) activities, and yet a national acgme review found most trainees had "limited knowledge of qi methods" and "limited participation in interprofessional qi teams". the twenty fellows in our pediatric hematology/oncology training program identified blood culture utilization as their qi priority. our institution recently introduced a hospital-wide decision algorithm to guide providers regarding when to obtain blood cultures. there is often a low threshold to obtain blood cultures in immunocompromised pediatric oncology patients, but these are often low-yield or result in falsepositives. our fellows spearheaded a project to implement the algorithm in the inpatient pediatric oncology population and improve the proportion of appropriately drawn blood cultures. we investigated how appropriately the algorithm was being utilized on the inpatient pediatric oncology floor prior to and after several educational steps aimed at disseminating the algorithm to members of the care team. our primary endpoint was to quantify the proportion of culture episodes drawn "inappropriately", with a goal of reducing inappropriate episodes to ≤10%. the algorithm was initially introduced to the nursing staff and residents covering the twenty-bed inpatient unit in september 2016. qi project planning took place with upper level fellows in january 2017. fellows and faculty received intensive training on the algorithm in july-august 2017. we then conducted a retrospective chart review of blood culture episodes drawn between august 2016 and november 2017. upper level fellows scored ∼500 culture episodes as to whether the decision to culture and number of cultures drawn were "appropriate" or "inappropriate", and catalogued the indications for culture episodes and if applicable, why the episode was found to be inappropriate. additionally, fellows discussed inappropriate culture episodes with the team onservice, to provide direct feedback on where the algorithm failed. results: between august -december 2016 on average 337 cultures/1000 patient-days were drawn. forty-nine percent of culture episodes were inappropriate. from january -october 2017, following targeted education on the algorithm, the rate of blood cultures drawn decreased to 263 cultures/1000 patient-days. the average proportion of inappropriate culture episodes fell to 16.7%, representing a 66% decrease in inappropriate culture utilization. correct application of a decision algorithm for blood culture utilization can reduce total cultures drawn on an inpatient pediatric oncology unit. fellow-led education of the multi-disciplinary team decreases the rate of inappropriate culture episodes as well as provides active engagement in qi. background: inadequate understanding of sickle cell disease (scd) is common and can affect patients' compliance and therefore their morbidity and mortality, especially after transition to adult care. optimal clinical care for scd includes disease education, which can be difficult given the breadth of possible topics and limited time in clinic. it is unclear how best to provide personalized, efficient education for adolescents with scd. this quality improvement (qi) study aimed to implement a questionnaire-based system to improve patients' knowledge of their scd and documentation of education by the nurse or physician. the study objective was to improve provider documentation and patient knowledge about their scd by identifying patients' gaps in comprehension. by january 2017, the study aimed to increase education documentation from 50% to 75%. by april 2017, the study aimed to increase use of a smart phrase for education documentation from 0% to 50%. by june 2018, the study aimed to increase patients' knowledge about their disease by 20%. design/method: twenty-one scd patients enrolled on an irb approved qi study, with twenty active patients. our comprehensive team generated a questionnaire with knowledgebased questions for two age groups: 12-14 and 15-18 years old. at each comprehensive visit, a questionnaire was distributed, with at least 3-month intervals. the provider scored questionnaires and reviewed two educational topics, with wrong answers taking priority. plan-do-study-act (pdsa) cycles included pdsa#1: patients completed questionnaire. pdsa#2: a smart phrase addressing questionnaire topics was created and shared with providers. pdsa#3: patients received education handouts during clinic education. documentation in clinic notes was the process measure and questionnaire scores was the outcome measure. results: pdsa#1 is complete, pdsa#2 has four patients remaining, and pdsa#3 is ongoing. due to variable visit frequency, there are multiple concurrent cycles. after pdsa#1, free text documentation was completed an average of 61% over the course of 3 months. after pdsa #2 documentation increased to 100% within 3 months and questionnaire scores increased from an average of 59% to 78%. of the questions that patients got wrong on their first visit, they were significantly more likely to improve on retesting if the topic was taught to them than if it was not addressed (72% vs. 33%, p = 0.04). we are currently completing pdsa#3 and collection of post pdsa#3 data. questionnaire-based scd education coupled with standardized smart phrases improves patients' scd knowledge and documentation by providers. further improvement in knowledge is expected with the addition of handouts. background: exposure to suffering can have a profound impact on the wellness of caregivers, often referred to as the "cost of caring". this cost is especially high in pediatric hematology/oncology. repeated exposure to suffering has the potential to negatively impact resilience and increases the risk of burnout, thus impacting quality of care and patient satisfaction. we have developed a peer support team utilizing the critical incident stress management (cism) model. this model has been successfully used in other professions that frequently face traumatic events such as fire fighters, police and emergency medical technicians. the h.o.p.e.s. team (helping our peers endure stress) consists of 18 volunteer multidisciplinary staff members who have received training to provide both group and peer support following any 'critical incident' that may impact one or more staff members. we hypothesize that implementation of the h.o.p.e.s. team will improve staff resilience, decrease overall rates of burnout and improve compassion satisfaction. s161 of s301 design/method: we are using both empiric metrics and anecdotal reports to assess the impact of the h.o.p.e.s. team. prior to the activation of the team, all pediatric hematology/oncology clinical staff members were surveyed using validated tools to assess their levels of resilience, burnout, secondary trauma and compassion satisfaction (proqolv5 and brief resilience scale). they were also asked to rate the number of times they had experienced critical incidents, as well as their perceived level of distress after dealing with traumatic events. after the h.o.p.e.s. team has been functional for 6 months, we will send the same survey to staff members to measure changes, paying special attention to resilience and rates of burnout and compassion satisfaction. results: enthusiasm for development of the team has been high. 18 of 19 people approached to volunteer their time to participate in the multidisciplinary team agreed, including attending physicians, fellows, nurses, nurse practitioners, child life specialists, social workers, clergy and psychologists. all volunteers participated in a 3-day training conducted by an instructor from the international critical incident stress foundation. engagement in the first staff survey has been high, with 91 of 150 responding to date. data collection is ongoing. clinical staff in pediatric hematology/oncology may be particularly vulnerable to burnout and decreased resilience by repeatedly witnessing suffering and trauma. peer support interventions following critical incidents may lead to increased resilience and compassion satisfaction while decreasing rates of burnout. enthusiasm for the development of a peer support team has been high. background: monthly blood transfusions are an indicated therapy for pediatric patients with sickle cell disease with certain complications. maximizing transfusion efficiency in a busy infusion clinic requires: ensuring that appropriate blood units are available in the hospital blood bank; laboratory specimens are obtained from patients in advance; and coordination of clinic appointment and nursing availability. we sought to improve clinic efficiency through identifying ways to better communicate with patients/families regarding upcoming laboratory and transfusion appointments, and to assess the efficacy of implementing a web-based personalized text reminder (pinger.com). we measured the baseline frequency with which transfusion appointments were missed by families, moved to later within the week, or delayed due to late labs. a convenience sample of patients receiving monthly transfusions received a questionnaire about patient/parent preferences for appointment reminders and barriers to keeping appointments. those patients/parents who did not opt-out of an additional text reminder received personalized texts from their care team reminding them of lab and transfusion appointments. rates of missed/moved/delayed appointments were compared between the group receiving the additional text messages and the group only receiving standard, hospitalgenerated appointment reminders (telephone call). results: forty-one families (45 patients) responded to the survey, capturing information on 63% of patients receiving chronic transfusion therapy. thirteen families (32%) declined the additional text reminders. families reported a preference for text reminders (66%), more often than email (49%) or telephone (37%), and 80% of families wanted to receive reminders for both transfusion and laboratory appointments. the majority (43%) of families reported competing work/life priorities as the reason for missed/late appointments. other families noted transportation/travel (29%), fear/illness/pain (21%), and lack of reminders (21%) as the reason for missed appointments. at baseline (twelve weeks), 3.7% of appointments were missed on a weekly basis (range 0-3 of 20 available per week), 10.4% were moved, and 5% of appointments were delayed. during our intervention period (twelve weeks), 7% were missed, 9.2% were moved, and 8.7% were delayed (combined, both groups). there was no difference in missed (7.0% texted vs 7.1% standard), moved (8.0% texted vs 10.0% standard) or delayed (8.0% text vs 9.3% standard) appointments. though families at our center reported a preference for a text-based reminder, personalized text reminders for appointments did not improve clinic efficiency as measured by missed, moved or delayed transfusion appointments. there was no improvement in appointment adherence in the group receiving personalized texts in addition to standard hospital reminders. university of utah, salt lake city, utah, united states background: childhood cancer outcomes have improved significantly, in large part due to multi-institution collaborative clinical trials run by the children's oncology group (cog). approximately half of eligible children with cancer will enroll on a therapeutic trial, but little is known about the factors affecting caregiver decision-making regarding enrollment or how well the required elements of informed consent are conveyed during the consent process. objectives: 1. assess coverage of ten of the required elements of informed consent for cog therapeutic trials. 2. describe factors affecting caregiver decision-making regarding therapeutic trial enrollment. we surveyed families of children who were offered enrollment onto a phase 3 cog therapeutic study for an initial cancer diagnosis in the previous 18 months. fisher's exact or wilcoxon rank-sum tests were utilized to compare demographic and other motivating factors related to enrollment decision-making. results: seventy participants were surveyed. regarding 10 of the basic required elements of informed consent, 96% knew the trial involved research, 99% knew consent was required, 76% knew the enrollment length for the trial, 97% knew they could continue care independent of enrollment, 73% knew who to contact with questions, 71% knew there were options besides enrollment, 83% knew they could withdraw at any time, 93% knew the information was confidential, 34% knew there were risks associated with the trial, and 46% knew there were benefits. of all participants, 84% (n = 59/70) enrolled onto a therapeutic study. among enrollees, 37% (n = 22/59) of the primary caregivers had completed college compared to 18% (n = 2/11) of those not enrolled (p = 0.3). when asked about factors impacting their decision, 69% (n = 41/59) of those enrolled said they felt there were no risks or did not know if there were risks associated with the study compared to 45% (n = 5/11) of those choosing not to enroll (p = 0.17). of those enrolled, 61% (n = 36/59) reported the physician recommendation "somewhat" or "strongly" affected their decision to enroll compared to 0% (n = 0/11) of those not enrolling (p = 0.0001). of those who enrolled, 17% (n = 10/59) reported feeling pressured to enroll while 45% (n = 5/11) of those not enrolled reported pressure (p = 0.05). of enrollees, 10% (n = 6/59) reported they did not have enough time to decide compared to 36% (n = 4/11) of those not enrolled (p = 0.03). failure to convey all 10 required elements of informed consent highlights possible deficiencies in the consent process for cog therapeutic trials. caregivers' perception of being pressured and lack of time to make an informed decision may impact clinical trial enrollment. background: abnormal uterine bleeding (aub) is a frequent adolescent gynecologic complaint. however, limited research exists to guide management, and acute care varies. we sought to improve emergency care for adolescents with aub by developing a clinical effectiveness guideline (ceg) and assessing its impact on quality of care. design/method: a stakeholder engagement group consisting of members from the departments of hematology/oncology, adolescent medicine, general pediatrics, and emergency medicine designed a ceg algorithm for emergency aub management. pediatric residents received ceg training and their knowledge and attitudes were assessed using pre and post intervention surveys. icd-9 and 10 codes identified electronic health record data for patients presenting to the pediatric emergency department (ed) for aub 6 months before and after ceg implementation. pre-pubertal patients and those with vaginal bleeding from trauma were excluded. a weighted, 20-point scoring system consisting of prioritized aspects of history, laboratory studies and management was developed to quantify the quality of care provided. t-test, chi square test, wilcoxon rank sum test, and a run chart were used for analysis. of the 91 patients identified, 62 met inclusion criteria. there were 37% of patients currently using some form of contraception, while 12.9% had bleeding related to a current or recent pregnancy. median aub quality care scores were 14 pre-and 16 post-intervention (p = 0.064). run chart data showed no shifts or trends (overall median score, 14-points). both pre and post-implementation, points were deducted most frequently for not assessing personal/family clotting disorder history and inappropriate use/dosing of oral contraceptives. we successfully designed and implemented a ceg and educational intervention for aub management in a pediatric ed. these data suggest our ceg may be an effective tool to improve emergency aub care for adolescents, though additional cycles are needed. background: high-dose methotrexate (hd-mtx) is a common chemotherapy administered inpatient at most centers. its administration is particularly susceptible to error due to the need for frequent drug levels with resulting changes in supportive care. errors can prolong patient stay and cause patient harm. objectives: global aim-to reduce the length of stay (los) of hd-mtx admissions. smart aims-to increase the percentage of patients whose pre-hydration fluids are started by 10am from 0% to 20% by 1/31/18, and to increase the percentage of patients who receive hd-mtx by 5pm from 43% to 100% by 6/30/18. we used rapid process improvement methods to target earlier methotrexate administration. a key driver of prolonged los was hypothesized to be drug levels returning overnight rather than in the day time due to delayed hd-mtx start. changes implemented have included scheduling hd-mtx patients as the first patients of the day for their exam in clinic and scheduling labs to pass for hd-mtx on the day prior to admission. there are ongoing pdsa cycles to change the location of pre-hydration start from the inpatient room to the clinic exam room in order to meet hd-mtx administration time goals. we are piloting two different education materials to improve patient experience. one explains hd-mtx levels in a red/yellow/green stoplight format and the other reminds patients how to prepare for the admission. other interventions regarding how we test urine ph and safety checks in the ordering process for history of delayed clearance are in the planning stage. the project is ongoing, but as of 12/12/17, we start methotrexate by 5pm 50% of the time which is improved from a baseline of 43%. when the project was started, pre-hydration was never started before 10am. now, fluids are started by 10am 40% of the time. pdsa cycles are ongoing and we have yet to sustain reductions in los, but some months have shown decreased los by as much as 19 hours from baseline measurements. rapid cycle improvement can be utilized to decrease los hd-mtx admissions. this has important financial implications as well as the potential to reduce secondary harm from unnecessary time in the hospital. pediatric cancer centers should schedule hd-mtx admissions first thing in the morning so that data regarding kidney injury and drug clearance can be interpreted by the day team and children are not cleared for discharge in the middle of the night. background: education and training for interdisciplinary pediatric oncology providers requires training in principles of palliative and end-of-life (eol) care. the experiences of bereaved parents can inform and enhance palliative care educational curricula in uniquely powerful and valuable ways. the objective of this study is to present an innovative palliative care educational program for oncology providers facilitated by trained bereaved parents who serve as volunteer educators in local and national palliative care educational forums and to describe how incorporation of bereaved parents in these educational forums affects participant comfort with communication and management of children at the eol. design/method: survey tools were adapted to determine how bereaved parent educators affected participant experiences in 3 different educational forums: institutional seminars on pediatric palliative and eol care, role-play based communication training sessions, and an international symposium on pediatric palliative oncology. pre-and post-session surveys with incorporation of retrospective pre-program assessment item to control for response shift were used in the evaluation of institutional seminars and communication training sessions. results from feedback surveys sent to all attendees were used to appraise the participants experience in the international oncology symposium. results: involvement of trained parent educators across diverse, interdisciplinary educational forums improved attendee comfort in communicating with, and caring for, patients and families with serious illness. importantly, parent educators also derive benefit from educational with interdisciplinary clinicians. integration of bereaved parents into palliative and eol care education is an innovative and effective model that benefits both interdisciplinary clinicians and bereaved parents. background: poorly controlled chemotherapy-induced nausea and vomiting (cinv) significantly impairs patients' quality of life and contributes to ongoing medical costs through increased length of stay in the hospital or readmissions and outpatient visits for control of nausea, vomiting or dehydration. lack of adherence to national evidenced-based guidelines that dictate antiemetic prescribing for variably emetogenic chemotherapy leaves patients vulnerable to increased cinv and its ensuing complications. objectives: to review our institution's antiemetic prescribing practices and their consistency with the antiemesis guidelines from the national comprehensive cancer network (nccn) and children's oncology group (cog)-endorsed supportive care guidelines and to further develop tools to increase adherence to these national-based guidelines to improve control of cinv. we performed a retrospective chart review of inpatient chemotherapy encounters. we evaluated emetogenicty of chemotherapy (high, medium, low), initial antiemetic regimen ordered, number of as needed medications required and adherence to national evidenced based guidelines tailored to each level of emetogenicity in the prescription of antiemetics. results: fifty-five total inpatient chemotherapy encounters were reviewed over 8 months. eighteen of these encounters were considered to have been highly emetogenic chemotherapy (hec) with the remaining 37 of these considered to be moderately emetogenic. only 9 out of 18 hec encounters completely included all guideline-recommended agents. there was a demonstrable lack of consistency across providers with dosing of aprepitant and most as needed medications. there was significant variation in order of first, second and third line anti-emetics ordered -with lorazepam and promethazine being used most frequently. with an aim of improving antiemetic prescribing practices for our patients, we are currently rebuilding chemotherapy treatment plans in our electronic medical record to incorporate antiemetic drug order sets that follow evidenced-based guidelines for variably emetogenic chemotherapy. this will be used in conjunction with an education initiative about best practices in supportive care for all prescribers of antiemetics. review of our department's recent inpatient chemotherapy encounters show we are falling short in following nationally recommended standards for appropriate antiemetic coverage during chemotherapy. identification of these deficiencies allows for implementation of quality initiatives to improve prescriber adherence to evidenced-based guidelines for better control of cinv. background: there are currently no consensus guidelines for the management of pediatric oncology patients presenting with fever without neutropenia. historically, these patients had been treated similarly to neutropenic patients with empiric antibiotics. while there has been a shift towards reducing unnecessary empiric treatment, there has been limited research into the outcomes associated with withholding empiric iv antibiotics in this patient population. we assessed the safety and efficacy of our institution's current protocol of observing well-appearing patients who present with fever without neutropenia and compared the outcomes of the patients who did and did not receive empiric iv antibiotics. design/method: this was a prospective, single-institution cohort study. patients were included if they were currently undergoing chemotherapy for an oncologic diagnosis and presented initially as an outpatient with fever and nonneutropenia (defined as anc ≥ 500 cells/mm3). for each episode we recorded lab and blood culture results, signs and symptoms of initial presentation, and clinical outcomes, including antibiotic administration and hospital admission. results: a total of 242 episodes of well-appearing patients with fever without neutropenia were identified. compliance with the institutional protocol was high; 81.8% of patients were observed without receiving empiric iv antibiotics. the majority of patients were discharged home and there were no serious complications or infectious deaths. the incidence of positive blood cultures was low (3.7% including several likely contaminants), despite the presence of central venous catheters in the majority (84.7%) of patients. there were no significant differences in age, oncologic diagnosis, central s165 of s301 line access, anc value, or incidence of bacteremia between patients who did and did not receive empiric iv antibiotics. patients who were admitted to the hospital were significantly more likely to have received iv antibiotics (p <0.001) despite documentation of a reassuring exam. however, admitted patients who initially received iv antibiotics were just as likely to discharge within 48 hours compared to patients who were observed. we propose that empiric iv antibiotic administration in febrile, non-neutropenic, otherwise well-appearing patients is unnecessary. our study demonstrated no adverse consequences of observation and no significant differences in clinical outcomes between patients who did and did not receive iv antibiotics aside from rate of hospitalization. this supports the practice of observation without empiric antibiotics for such patients. background: children with hepatoblastoma (hb) undergo repetitive computed tomography (ct) scans to determine response to treatment and assess for relapse. this imaging exposes children to radiation, anesthesia, and imposes financial and emotional burden. objectives: review our institutional experience to determine if afp measurements are sufficient to assess response to treatment and detect relapse. we conducted a retrospective chart review of all patients diagnosed with hb at our institution between 1978-2017. data collected included serum afp, total number and type of imaging studies during and post treatment, and how relapse or progressive disease was detected. results: thirty-one patients were diagnosed with afp positive hb. during therapy, 173 ct scans were performed: 118 to assess for response to therapy or surgical planning (average 4 scans/patient) and 55 due to concern for progression with rising afp. off therapy, 213 surveillance ct scans were performed (average of 5.3 scans/patient) and 72 (33%) included the chest in patients with no lung metastasis at diagnosis. relapsed patients averaged 12.5 surveillance scans, 6.5 of which were done before relapse was noted on imaging. there were no cases of radiographic evidence of relapse without a prior increase in afp. during treatment, response to therapy based on imaging correlated with a decline in afp in all patients, arguing that repetitive scans are not needed in this setting unless required for surgical planning. only 3 of 213 scans performed during off therapy surveillance displayed evidence of relapse, all of which were preceded by rise in afp. our study represents the largest cohort of hb patients. prior studies suggest similar results, but included fewer patients, lower stage of disease and less than 10 years of surveillance monitoring. at our institution, the cost of a ct c/a/p is $15,169 with reimbursement varying from 30-50%. in comparison, the cost of an afp measurement is $101.50. many scans also require anesthesia and result in emotional toil for families concerned about this procedure as well as the results. thus, afp demonstrates greater sensitivity, with significant cost savings and decreased emotional burden, and should be used for monitoring both during and off therapy, replacing routine serial imaging. background: we observed that our practice of drawing daily blood cultures in hospitalized patients with fever and neutropenia was wasteful; it resulted in excessive negative cultures that did not add to patient care. the smart aim of this quality improvement project was to reduce the number of negative blood cultures drawn on hospitalized patients with fever and neutropenia by 25% in 6 months. design/method: after reviewing published evidence suggesting drawing daily blood cultures in febrile neutropenic patients was unnecessary, a new blood culture guideline was implemented: cultures were drawn at presentation for fever with neutropenia and, if negative at 24 hours, repeat cultures were not drawn except for clinical change, new fever after being afebrile >24 hours, or antimicrobials were being changed/broadened. to impact key drivers, we educated staff and changed blood culture order sets to require providers to select a reason for ordering the culture and to eliminate a nursing order to draw daily cultures with fever. we compared the number of blood cultures drawn per 1000 central linedays (/1000-cld) and the proportion of positive versus negative cultures pre-guideline (july 2015-may 2016) and postguideline (june 2016-december 2016). we calculated the cost savings from reducing cultures. to assess patient safety, potential septic events without a corresponding positive blood culture were reviewed. data were analyzed by service (oncology and stem cell transplant). a chi-square test was used to compare rates. in stem cell transplant patients, pre vs. postguideline, there were 492 vs. 258 total cultures drawn/1000-cld; 25 vs. 21 positive (16% decrease, p = 0.404) and 467 vs. 237 negative cultures/1000-cld (51% decrease, p<0.0000001). in oncology patients, pre vs. post-guideline, there were 266 vs. 181 total cultures drawn/1000-cld; 17 vs. 13 positive (24% decrease, p = 0.024) and 249 vs. 168 negative cultures/1000-cld (32% decrease, p<0.0000001). the decreased positive culture rate among oncology patients may be due to decreased culture contaminants and/or the effect of a concurrent initiative to decrease clabsi in that group. there were 2 safety concerns; however, chart review concluded that the guideline did not lead to missed infections in these patients. for the first 6 months of the guideline, the total cost savings in blood cultures was $31,454.31. the implementation of our new blood culture guideline successfully led to a substantial reduction in the collection of negative cultures and a cost savings without compromising the detection of bacteremia in hospitalized pediatric patients with fever and neutropenia. background: there are various evidence-based guidelines for treatment of adult cancers, such as the nccn guidelines. previously, care was standardized for most new diagnosis pediatric cancer patients through enrollment on a clinical trial. with decreasing clinical trial availability and enrollment and few, if any, evidence-based guidelines for pediatric cancer, care standardization is challenging for pediatric cancers. objectives: to assess consistency of care, as determined by plan of treatment by diagnosis, for pediatric patients receiving chemotherapy for newly diagnosed cancer at a single center. design/method: patients with a new cancer diagnosis at a large, tertiary care pediatric oncology center in calendar year 2016 were identified through reports from the chemotherapy order entry (coe) system. reports included diagnosis (recorded through standardized options) and the plan of treatment. chart review was used to exclude patients who started treatment elsewhere and patients being treated for relapse, to clarify diagnosis if the standardized options in coe were unclear, and to clarify treatment plan if needed. data was entered and analyzed in a redcap database. specific diagnoses were clustered into higher level disease groups and the distribution of treatment plans for patients within each was determined. this project was deemed exempt from irb approval for human subject research as a qualifying quality improvement project. of the 324 patients with a first chemotherapy order in 2016, 142 were excluded due to one or more reasons: stem cell transplant (62), transfer of care (54), relapse (25), and other (9). an additional 61 patients were excluded because <5 patients/year/diagnosis. there was no cns tumor disease group with >5 patients. thus, 121 patients with hematologic malignancies or non-cns solid tumors are the focus of this analysis. for patients with intermediate risk rhabdomyosarcoma, the plan of treatment was the standard arm of a cog protocol, arst0531 for 3 patients and arst 1431 for 1 subsequent patient after protocol activation. for all other diseases including lymphoblastic leukemia/lymphoma (excluding infants), classical hodgkin lymphoma, aml (excluding trisomy 21 and apml), stage iii/iv burkitt lymphoma/diffuse large b-cell lymphoma, posttransplant lymphoproliferative disease, wilms tumor, rhabdomyosarcoma, ewings sarcoma, osteosarcoma, neuroblastoma, and retinoblastoma, only one treatment plan per risk category was used. conclusion: this analysis demonstrates highly consistent chemotherapy treatment at a single center for patients with hematologic malignancies and non-cns solid tumors. next steps include exploring strategies to group diagnoses for cns tumors and assessing the quality of evidence supporting the treatments given. background: rapid initiation of empiric antibiotics in patients with fever and neutropenia has been shown to reduce morbidity and mortality. current practice guidelines call for the initiation of antibiotics in these patients within sixty minutes and time-to-antibiotic (tta) has been suggested as a quality-of-care measure. many institutions, including our own, face barriers to meeting this time limit. objectives: utilizing a quality improvement model, determine barriers and implement an intervention to reduce the time-to-antibiotics for pediatric febrile patients with suspected neutropenia who present to the emergency department (ed) at our institution. we have identified and implemented an intervention utilizing the plan-do-study-act model for quality improvement. a twelve-month retrospective review was conducted to evaluate the efficacy of the current practice algorithm at our large, academic tertiary-care hospital. subjects identified were pediatric oncology patients undergoing active chemotherapy who presented to the ed with febrile neutropenia. we identified two specific barriers, triage level assignments and delay in ordering antibiotics. to address these barriers, we have created a wallet sized "fever card" that patients were instructed to show upon arrive to the ed. in collaboration with the ed staff, efforts were also made to educate all pediatric staff on the use of the fever card. post-intervention data collection is currently underway and pre-and post-intervention antibiotic delivery times will be compared. the pre-intervention cohort consisted of thirty-three encounters with a mean time-to-antibiotic delivery of 135 minutes, or seventy-five minutes greater than the accepted standard of care. only one patient received antibiotics within sixty minutes of arrival. post-intervention data collection is currently underway. since identifying two barriers to meeting the standard of care at our institution, we have implemented a quality improvement measure that empowers patient families to direct appropriate triage in the ed as well as simplifying the treatment protocol for ed providers. we expect to identify an improvement in time-to-antibiotics from the pre-intervention to the post-intervention period. background: sickle cell disease (scd) is a genetic disorder in which sickle hemoglobin (hbs) triggers multiple downstream effects, including red cell sickling, hemolysis, vaso-occlusion, and inflammation. scd, a lifelong disease initiated at birth with injury that accumulates over time, causes significant end-organ damage and clinical complications that are undertreated and associated with early death. homozygous mutation (hbss) causes the severe form of scd. individuals with scd are at increased risk of infection, stroke, and retinopathy. clinical guidelines for pediatric patients with scd recommend prophylactic penicillin use (ages 2-5), annual screening for stroke with transcranial doppler (tcd) imaging (ages 2-16), and annual ophthalmology exams to assess for retinopathy (ages ≥10). there are limited real-world data on implementation of these nhlbi-based recommendations. objectives: to describe utilization of penicillin, tcd screening, and ophthalmology care in children with hbss disease. medicaid administrative claims databases were used to identify us patients aged 2-16 years at first indication of hbss recorded in each calendar year from 2009 to 2014. patients were required to have medical and pharmacy benefits for the calendar year in which they were identified and for 12 months prior to their first recorded hbss indication. prior year utilization of penicillin, tcds, and ophthalmologist visits was measured for each annual cohort. annual cohorts included 347-438 commercial (mean age 9.5 years, 52% female) and 1024-1557 medicaid (mean age 8.3 years, 48% female) patients with hbss disease. fewer than half of all patients had received a tcd scan in the previous year, with similar rates seen across all age groups for both payers. ophthalmologist visits increased as patients aged, and while patients aged 12-16 years had the highest proportion with an ophthalmologist visit in both payer populations, the overall implementation remained low. in contrast to the low use of tcd and ophthalmology visits, penicillin use was highest in the 1-5 year age group: >80% use in any given year for both payers. conclusion: although our data demonstrated high penicillin use in the 1-5 year age group, consistent with guidelines there is an opportunity to improve implementation of other guidelines-based recommended screening. for example, tcd screening can identify children at risk of scd-related stroke in order to initiate preventive therapies. further research to understand potential barriers to proper screening and to evaluate strategies to improve awareness, adherence, and implementation of recommended screenings in children with scd is warranted. supported by global blood therapeutics. background: childhood cancer therapy has improved where there are many long-term survivors. while psychosocial difficulties in pediatric cancer survivors are recognized, the prevalence of these problems at initial survivorship presentation is unclear. objectives: to examine the prevalence of overall internalizing symptoms (e.g., depression/anxiety) in pediatric cancer survivors presenting to a survivorship clinic and to examine how this is mitigated by receiving psychological services and by evidence of parental depression/anxiety. design/method: pediatric cancer survivors attending their first visit at the reach for survivorship clinic at vanderbilt (ages 3-18) were included. survivors' parents (93% female) completed the child behavior checklist (cbcl), beck depression inventory-ii, and beck anxiety inventory. survivors >12 years completed a self-report. the wilcoxon rank-sum and pearson's 2 test were used for univariate analyses. the effect size and 95% confidence intervals (ci) estimated from the multivariable linear regressions were reported. results: 142 childhood cancer survivors a median of 12 years old and 3.3 years off therapy were included. thirty one survivors (22%) showed at least borderline clinical internalizing problems (t score >60) on the cbcl, but only 8 of these patients (26%) reported receiving psychological services. nine other survivors with normal t score ≤60 also reported receiving psychological services. parental depressive and anxiety symptoms were correlated to the parental report of survivor overall internalizing symptoms (spearman = 0.415, p = <0.001 and = 0.476, p = <0.001 respectively), however they were not correlated to survivor selfreports. furthermore, parents with mild to severe depressive symptoms or mild to severe anxiety symptoms were more likely to rate their child as having higher overall internalizing symptoms (p = 0.001; p = 0.008, respectively). multivariable linear regression showed that when adjusted for age, gender, cancer diagnosis and time off treatment, reported utilization of psychological services ( = 8.58, 95% ci [3.54, 13.62],p = 0.001), and parent depressive symptoms ( = 0.43, [.22, .65 ],p<0.001) were significantly associated with child overall internalizing symptoms. in an otherwise identical alternate model substituting parental anxiety for parental depression, parental anxiety was also a significant risk factor ( = 0.70, [.38, 1.03], p<0.001). alternatively, parent anxiety/depressive symptoms were not significantly associated with child self-report of internalizing symptoms. childhood cancer survivors have an elevated prevalence of experiencing internalizing symptoms but seldom report receiving psychological services. childhood cancer survivors' parents with anxious/depressed symptoms are more likely to rate their children as having more internalizing problems, compared to patient self-reports. ongoing longitudinal analyses will help clarify the best timing for potential interventions. background: life expectancy for adults with sickle cell disease (scd) has remained unchanged over the past 30 years despite improvements in pediatric scd survival. at greatest risk are the adolescents and young adults (ayas) transitioning from pediatric to adult care. allen county ranks 3rd in scd incidence among the 92 counties in indiana, and has 2 board certified pediatric hematologist-oncologists. when children "age out" of the pediatric system, there are few providers knowledgeable about managing adults with scd in the region. a novel partnership between hematologists and the family medicine residency program in allen county was initiated to educate family medicine residents (fps) about scd, hydroxyurea (hu), and management of scd-related complications with the goal to increase the number of knowledgeable providers to care for adults with scd. to determine the effectiveness of online learning modules in educating fps about hu, best practices for aya scd care and transition. three online learning modules about scd (comprehensive care of ayas with scd, hu, best practices in aya transition) were developed and cme-accredited. electronic pre-and post-tests were distributed to 32 fps with five questions for each module covering: contraception; screening tests; hu indications, dosing and monitoring; developmental milestones and scd knowledge assessments. the st vincent irb reviewed the protocol and granted a waiver of consent. results: twenty-six fps (81%) completed the pre-and posttests. over two-thirds correctly identified the clinical benefits of hu on both assessments. knowledge about the rationale for hu therapy increased after the completion of the hu module (30% correct on pre-test vs. 62% on post-test, p = 0.009). the proportion of correct responses increased for all comprehensive aya scd care post-test questions, but only the leading cause of death and the priapism-related questions reached statistical significance (15% vs. 35%, p = 0.048; 0% vs. 23%, p = 0.003, respectively). the proportion of correct responses for 2 of the transition-focused questions was unchanged (89% for both), while the proportion of correct post-test responses on the self-care assessment question significantly increased (0% vs. 12%, p = 0.04). after module completion, fps were able to correctly identify common scd complications and why hu is an effective treatment for individuals with scd. the best practices of transition clinic module may need modification to improve physician understanding of the intricacies in establishing and maintaining a scd transition clinic. overall, online training is effective at educating fps and could be used to increase the number of providers knowledgeable about scd care. background: survival rates for pediatric hodgkin lymphoma (hl) exceed 95% with contemporary therapy. studies of pediatric hl survivors treated in the 1970s-1990s have shown increased risk for treatment-related chronic health conditions. risk-adapted therapy, including tailored radiotherapy, has been developed to reduce long-term morbidity while maintaining excellent survival. little is known about chronic conditions associated with contemporary therapy presenting during the first 5 years from therapy completion (early outcomes). objectives: to analyze survival and early outcomes of pediatric hl patients treated with contemporary therapy. we conducted a retrospective review of hl patients diagnosed <21 years of age at our institution from 2010-2014. three-year overall (os) and event-free (efs) survival were calculated with kaplan meier statistics using sas 9.4. results of standardized screening for targeted toxicities that developed between 2 -5 years from therapy completion were identified and graded per ctcae criteria. censoring occurred at date of death, 5 years from therapy completion, or december 31, 2016. data from the last collection point were used for prevalence calculations in cases with multiple evaluations. we identified 83 patients (51% male; 43% non-hispanic white; mean age at diagnosis 13.6 ± 3.7 years) with a median time since therapy completion of 3.3 years (range 0.1-5.0). initial treatment included: 56 (67%) chemotherapy only and 27 (32%) multimodality treatment. all patients received anthracyclines (median dose 200mg/m2) and 96% received alkylating agents (median cyclophosphamide equivalent dose [ced] 3600mg/m2). the 3-year os was 99% with an efs of 90% (90% chemotherapy only, 91% multimodality treatment; p = 0.76). patients with relapsed/refractory disease received salvage treatment including chemotherapy only (n = 1), multimodality therapy (n = 1), or multimodality treatment including stem cell transplant (autologous n = 4; autologous+allogeneic n = 1). no patients developed thyroid dysfunction, cardiac dysfunction, subsequent neoplasm, or male gonadal dysfunction during the study period. pulmonary dysfunction was limited to ctcae grade 1. anti-mullerian hormone (amh) below the normal range was found in 10/11 pubertal females who received ced ≥7000mg/m2 compared to 0/12 females who received ced <7000mg/m2. two of the females with low amh also had follicle stimulating hormone >30iu/ml. this study is the first to evaluate early outcomes in pediatric hl survivors. the results indicate contemporary chemotherapy and a lower rate of radiotherapy utilization lead to excellent 3-year survival rates with minimal early toxicities. females exposed to ced ≥7000mg/m2 are at increased risk for gonadal dysfunction and should be prioritized for fertility preservation approaches prior to initiation of cancer therapy. background: cancer is one of the leading disease-related causes of death among individuals aged <20 years in the united states. recent evaluations of national trends of pediatric cancer used data from before 2010, or covered ≤28% of the us population. objectives: this study describes pediatric cancer incidence rates and trends by using the most recent and comprehensive cancer registry data available in the us. design/method: data from us cancer statistics were used to evaluate cancer incidence rates and trends among individuals aged <20 years during 2001-2014. data were from 48 states and covered 98% of the us population. we assessed trends by calculating average annual percent change (aapc) in rates using joinpoint regression. rates and trends were stratified by sex, age, race/ethnicity, us census region, county-based economic status, and county-based rural/urban classification, and cancer type, as grouped by the international classification of childhood cancer (iccc). we identified 196,200 cases of pediatric cancer during 2001-2014. the overall cancer incidence rate was 173.0 per 1 million; incidence rates were highest for leukemia (45.6), brain tumors (30.8), and lymphoma (26.0). rates were highest among males, aged 0-4 years, non-hispanic whites, the northeast us census region, the top 25% of counties by economic status, and metropolitan counties. the overall pediatric cancer incidence rate increased (aapc = 0.7, 95% ci, 0.5-0.8) during 2001-2014 and contained no joinpoints. rates increased in each stratum of sex, age, race/ethnicity (except non-hispanic american indian/alaska native), region, economic status, and rural/urban classification. rates were stable for most individual cancer types, but increased for non-hodgkin lymphomas except burkitt lymphoma (iccc group ii(b), aapc = 1.2, 95% ci, 0.4-2.0), central nervous system neoplasms (group iii, aapc = 0.4, 95% ci, 0.1-0.8), renal tumors (group vi, aapc = 0.6, 95% ci, 0.1-1.1), hepatic tumors (group vii, aapc = 2.5, 95% ci, 1.0-4.0), and thyroid carcinomas (group xi(b), aapc = 4.8, 95% ci, 4.2-5.5). rates of malignant melanoma decreased (group xi(d), aapc = -2.6, 95% ci, -4.7--0.4). this study documents increased rates of pediatric cancer during 2001-2014, in each of the demographic variables examined. increased overall rates of hepatic cancer and decreased rates of melanoma are novel findings using data since 2010. next steps in addressing changing rates could include investigation of diagnostic and reporting standards, host biologic factors, environmental exposures, or potential interventions for reducing cancer risk. increasing pediatric cancer incidence rates may necessitate changes related to treatment and survivorship care capacity. background: while childhood cancer treatment modalities have improved, the delayed effects of cancer treatment continue to compromise the quality of life in survivors. metabolic syndrome (ms) is diagnosed based on the presence of three of the following findings -obesity, dyslipidemia, hypertension and insulin resistance per the world health organization (who) criteria. the increased risk of ms among childhood cancer survivors was first reported in the 1990's and is known to increase the incidence of cardiovascular disease in these individuals. objectives: assess the frequency of ms in childhood cancer survivors at our institution. . we conducted a retrospective chart review on pediatric cancer survivors, 4 -18 years of age, who had been treated at sri ramachandra medical institute and research foundation between august 2015 and august 2017. patients who received at least one year of treatment with s171 of s301 chemotherapy and/or radiation and surgery were included. medical history, family history of diabetes, cardiovascular diseases, and hypercholesterolemia, tanner staging, weight for height (<5y per who criteria), bmi (>5y per indian academy of pediatrics iap), blood pressure (nhlbi criteria), fasting blood sugar levels and lipid profile were obtained from the charts. statistical analysis of the data was done using ibm spss statistical software (version 22). results: 97 patients were studied, 63.9% were male. 22.68% were under 5 years of age, 38.14% between 6-10 years and 39.18% above 11 years. leukemia survivors comprised 39.18% of the sample and non-leukemic's were 60.82%. 51.5% were treated with chemotherapy alone, 19.5% with radiotherapy and chemotherapy, and 28.8% underwent surgery with radiotherapy and chemotherapy. hypertension was found in 19.5% of the study group, dyslipidemia in 68%, impaired fasting blood glucose in 4.12% and 36.08 % were found to be obese. 10% of the study group was diagnosed with ms based on who criteria. conclusion: 10 % of our study population was found to have ms per who criteria. individual metabolic complications were detected in 68% of the population. acute lymphoblastic leukemia (all) survivors appeared to be at high risk in our population. ms has been known to increase cardiovascular complications in cancer survivors. a multidisciplinary team approach to management of these patients is important to closely monitor and manage the long-term complications related to ms such as type 2 diabetes and atherosclerosis. such an approach is essential to decrease long term morbidity and mortality from ms in this vulnerable population. background: the 5-year survival rate for childhood cancer exceeds 80%. however, up to 38% of these children require admission to the pediatric intensive care unit (picu) within three years of diagnosis. these children account for approximately 10% of all picu deaths, with mortality being higher for those post-hematopoietic stem cell transplant (hsct). national guidelines recommend that providers share informa-tion regarding prognosis and treatment options within the first 48 hours of icu admission. these prognostic goals of care conversations (pgocc) are critical to the care of children with malignancies, a subpopulation at risk for increased mortality. to determine the frequency of pgocc as well as describe differences in patient characteristics and critical care therapies by pgocc status. design/method: a retrospective cohort study was conducted using the university of michigan virtual picu system database. picu admissions lasting longer than 24 hours for patients ages 0 to 25 years between july 1, 2011 and june 30, 2016 with an oncologic diagnosis and/or hsct were identified. data on pgocc, patient demographics, diagnoses, picu interventions, and outcomes were recorded and compared between children with pgocc and those without using chi square test for categorical variables and kruskal-wallis test for continuous data. of 128 picu admissions, 53% were male; the mean age was 10.5 years. the leading diagnoses were acute lymphoblastic leukemia (46%), acute myeloid leukemia (14%), lymphoma (14%), neuroblastoma (5%), and brain tumors (5%), and 43% of patients were post-hsct. pgocc was documented in 35 (27%) patients. in comparison with patients who did not have a pgocc, children with a pgocc were more likely to be readmitted to the picu (45% vs. 15%, p <0.01) and more likely to have had relapse of disease (41% vs. 18%, p<0.01). patients with a pgocc had higher severity of illness scores (p = 0.02), higher use of non-invasive (22.9% vs. 8.6%, p = 0.04) and invasive conventional ventilation (68.6% vs. 23.7%, p<0.01), and high frequency ventilation (25.7% vs. 4.3%, p <0.01). also, patients with pgocc were more likely to receive continuous renal replacement therapy (37.1% vs. 5.4%, p<0.01), arterial catheterization (54.3% vs. 23.7%, p<0.01), and cardiopulmonary resuscitation (22.9% vs.1.1%, p<0.01). in only 1 in 3 critically ill children with hematologic-oncologic disease is pgocc held. children with pgocc were sicker and received more critical care interventions. future research is needed to evaluate the content of pgocc. background: central nervous system (cns) tumors and autism spectrum disorder (asd) represent significant disease cohorts in the pediatric population. asd diagnoses in children have a prevalence of 2%, 1 in every 88 children in the united states. additionally, more than 4,000 cns tumors are reported in children age 0 to 19 years in the united states with brain tumors being the most common solid tumor and the leading cause of death among all childhood cancers. the genetic etiology of autism and cns tumors is complex. specific gene alterations present in certain cancers have similarly been described and suspected to play a role in asd subtypes. targeted therapy panels, like foundation one (fo), have been beneficial in guiding treatment for some cancers based on distinct gene alterations. given the genetic overlap, the potential for therapeutic benefit and crossover from such actionable gene target panels merit further exploration in asd and cns tumors. we aim to identify and describe genetic alterations with known actionable targets in cancer therapy from fo as potential diagnostic, therapeutic and research targets for neurodevelopmental diseases. we plan to discuss the common genetic alterations between our cancers and neurodevelopmental diseases described in the literature. fo data was extracted and compared to the literature. each reported gene alteration from fo plus the keywords "autism", "psych" were used on pubmed to search for a suspected association if any with a neurodevelopmental disorder. results: twenty-one patients representing a cohort of six unique (astrocytoma-five, ependymoma-six, gbm-four, glioma-three, nerve sheath tumor-one, etmr-two) cns tumors were investigated. fo produced eighty total with sixty unique gene alterations. thirty-one (52%) of these yielded at least one published, suspected association to a neurodevelopmental disorder. the most common gene alterations were tp53-four, cdkn2a/b-five and braf-four. the main functional categories were cellular: proliferation, structure, differentiation and degradation; chromatin modeling; histone transcriptional modification; dna methylation and repair; strna; and neural signaling. sixty unique gene alterations were found in our cns tumor set using foundation one. thirty-one (52%) of these discrete alterations paired with at least one description in the literature as having been similarly altered in an asd subtype. many of these alterations have actionable targeted therapies presented through foundation one for our cns tumors and may be a relevant guide in the future of targeted therapy and research in asd subtypes. monoclonal antibody therapy usage is associated with significantly improved survival in b-cell nhl aya patients. although the usage has increased in the aya population from 2006 to 2013, the magnitude of the increase is low. factors that affect the use of mab include race and insurance s173 of s301 type. further research is warranted to identify why privately insured patients are less likely to receive these drugs. background: prevention of chemotherapy-induced nausea and vomiting (cinv) remains a challenge despite advances in pharmacotherapy and the development of cinv clinical practice guidelines by the pediatric oncology group of ontario (pogo) that have been endorsed by the children's oncology group. achieving control of cinv in pediatrics further is complicated by the difficulty young children have vocalizing their symptoms. use of a validated nausea-assessment tool in conjunction with improved adherence to evidence-based guidelines may result in better quantification of symptoms and reduction of both nausea severity and vomiting frequency for pediatric patients undergoing chemotherapy. the pediatric nausea assessment tool (penat) has been validated for children ages 4-18, and its integration into clinical practice may help optimize cinv control. objectives: this single-institution study sought to improve control of cinv in patients admitted for chemotherapy by standardizing the antiemetic regimens prescribed by all providers according to an institutional cinv algorithm developed from the pogo guidelines. we hypothesized that treatment using a standardized guideline would improve cinv control in patients admitted for chemotherapy. a baseline cohort of 70 admissions for chemotherapy completed penat assessments and cinv diaries prior to receiving chemotherapy, four times daily during each admission, and daily for 7 days following completion of chemotherapy from may 1, 2013 to january 31, 2014. providers then were provided an institutional cinv treatment algorithm based on the pogo guidelines and received education at departmental meetings on appropriate implementation of this algorithm. a second cohort of 78 admissions completed penat assessments and cinv diaries in a similar fashion from july 1, 2014 to december 31, 2016. results: complete control of vomiting markedly improved following cinv guideline implementation (72% vs 52%, p <.0001) with treatment failure also significantly reduced (6% vs 23%, p <.0001). after controlling for the degree of emetogenicity of chemotherapy received, a patient was 3.33 times more likely to vomit prior to guideline implementation (or 3.33, ci 1.96-5.56). there was no difference in nausea control, even after adjusting for the emetogenicity of chemotherapy. conclusion: control of chemotherapy-induced vomiting (civ) improved following widespread implementation of an institutional cinv treatment algorithm at a single institution. the severity of nausea reported remained unchanged which may reflect the difficulty of assessing nausea or an inadequate sample size. future research may focus on cinv treatment management through the use of guidelines specifically for breakthrough cinv and delayed cinv. background: aspho's professional development committee (pdc) recognized pediatric hematologists-oncologists (phos) serving in the united states (us) military have unique professional development needs that may not be addressed by aspho or a similar professional society. these individuals may also encounter challenges when transitioning to a civilian career. however, barriers to professional development have not been systematically characterized. the objectives were to characterize the number of phos with current or prior military service (mphos) and to identify any unmet professional development needs. design/method: a working group consisting of pdc members and both senior and early career mphos was formed. initial comments were solicited by email from known mphos regarding potential gaps in professional development and interest in working with aspho to improve support of mphos. a survey was developed and piloted with four members of the advisory group, questions were revised based on their feedback, and a final version was distributed via the aspho website and online community forum. targeted emails were sent to mphos identified through aspho and military databases. eligibility to complete the survey included 1) completion of a fellowship in pediatric hematologyoncology, and 2) current or prior service as an active duty military provider. quantitative and qualitative information were collected, including demographic data and perceived barriers to professional development. responses were summarized using descriptive statistics. results: sixty-five mphos were identified and 34 surveys were completed for a 52% response rate. respondents were engaged in a variety of professional activities; 64% were male, 52% were serving active duty commitments, and 32% felt there were professional development gaps. areas of concern were categorized into nine themes with the most concerning being 1) limited civilian knowledge of mpho practices (76% of participants), 2) inability to attend professional society meetings (60%), and 3 possibility of deployment (56%). participants expressed a desire for educational products to meet their specific needs and for networking opportunities with civilian colleagues. qualitative analyses identified concerns about low patient numbers and practice size. a subset of mphos perceive significant gaps in professional development. additional research is needed to better define areas for intervention, but many of the concerns align with those of similarly sized civilian programs and may be addressed through professional society networking opportunities, such as an aspho special interest group. background: infertility is an established cause of distress and has a negative impact on quality of life among childhood cancer survivors. the american society of clinical oncology has established guidelines on fertility counseling for individuals of reproductive age diagnosed with cancer, with the goal of improving reproductive and psychosocial outcomes. studies have shown that instituting a fertility team that can provide counseling and discuss fertility preservation (fp) options results in improved patient satisfaction in patients with cancer. objectives: the goal of this study was to examine predictors of referrals to the multidisciplinary fertility team, and documented fp interventions among these patients. design/method: an irb-approved retrospective medical record review was performed at a large pediatric academic center. all patients with new cancer diagnoses receiving chemotherapy were included from january 2015 (when the fertility team was established) to present. a standardized abstraction form was used to collect information about: age at diagnosis, gender, cancer type, whether a fertility consult was placed, and documented fp interventions. data were summarized descriptively and comparisons were made using nonparametric statistical methods. results: 265 patients met inclusion criteria, of which 152 (57%) were male. cancer types were as follows: 126 leukemia/lymphoma, 51 cns tumors, 44 sarcomas, 37 embryonal tumors, and 7 langerhan's cell histiocytosis (lch). the mean age was 8.2 years, (range <1-31 years). overall, 27% of all patients had a consultation with the fertility team. patients were significantly less likely to have a fertility consult if they were younger (p<0.001). further, there were differences in the consultation rate between diagnoses, with 53% of sarcoma patients completing a consult, compared to 31% of those with cns tumors, 41% of those with embryonal tumor, 44% of those with leukemia/lymphoma and none of the patients with lch. our findings show that many children, adolescents, and young adults newly diagnosed with cancer are still not receiving fertility counseling despite: 1) an expanding body of literature supporting the need to provide this counseling, 2) guidelines published by several organizations recommending discussions about infertility risk and fp options, and 3) presence of a multidisciplinary fertility team. specific strategies need to be developed to improve access for younger children, and for disease groups in whom fertility consults are underutilized, such as youth with cns tumors, embryonal tumors, and leukemia/lymphoma. background: socioeconomic status (ses) has on impact on overall survival in the pediatric oncology population. unfortunately, data are insufficiently detailed to explain the mechanism behind this phenomenon. how parents handle the health management demands placed on them at the time of a child's cancer diagnosis may represent a point of differentiation in health outcomes. objectives: determine the association between socioeconomic factors, cancer literacy, and parents' understanding of home emergency management and their responses to instances of pain, nausea, and fever. in a prospective observational study of parents whose children were newly diagnosed with cancer, we obtained demographic information and, using a validated instrument, (dumenci, 2014) we evaluated cancer literacy. we tested understanding of the education parents received about home emergency management with a 6-item multiple-choice vignette-based questionnaire focused on actions needed in home scenarios. we then followed parents' actual behavior through periodic phone calls assessing instances of nausea, pain, and fever and their responses to these episodes. results: preliminary analysis of 24 participants showed an average score of 4 on the 6-item parental understanding questionnaire (range 0-6). variables associated with increased score were college-level education by 1.2 points (95% ci [.3 to 2.1]), private insurance by 0.9 points [.13 to 1.7] and adequate cancer literacy by 1.2 points [.2 to 2.2]. actual behavior reported by families indicated that married parents and those with income above $75,000 were less likely to treat instances of pain by 51% (95% ci [28 to 72]) and 43% [6.6 to 79], respectively. white parents, those with college-level education, and those with adequate cancer literacy were less likely to treat instances of nausea by 31% [6 to 57], 29% [5 to 53] and 29% [5 to 53], respectively. no associations were found between socioeconomic markers and parental responses to instances of fever. our findings suggest an association between demographic and socioeconomic markers and improved parental understanding of home emergency management. paradoxically, the same markers show a decrease in treatment response to pain and nausea. larger prospective studies are needed to link this behavior pattern to health outcomes, and help inform the extent of ses impact on home emergency management. emory university/children's heathcare of atlanta, atlanta, georgia, united states background: cardiovascular disease is a leading cause of morbidity and mortality in childhood cancer survivors (ccs). previous research showed wide practice variation in referral patterns to cardiology from the survivor clinic and in recommendations from cardiologists about the need for further testing or exercise restrictions. to develop a cardio-oncology algorithm in order to standardize referrals to cardiology and provide guidelines for cardiologists evaluating pediatric ccs. design/method: survivorship and cardiology experts developed a weighted scoring system for pediatric ccs who received cardiotoxic therapy based on time since treatment and risk factors identified by the children's oncology group (cog) and american heart association (aha). the cardiooncology algorithm assigned a score of 1-21. the score range was categorized to guide cardiology referral: screening echo only (1-3), consider cardiology referral (4-6), recommend cardiology referral (7-9), and regular cardiology follow-up (≥10). the algorithm also provides recommendations to cardiologists for screening and exercise modifications based on the score. after establishment of the algorithm, a convenience sample of institutional survivor clinic patient charts were retrospectively reviewed from the first month of each quarter from april 2015-march 2016 to validate the algorithm, evaluate referral patterns to cardiology, and assess cardiology recommendations. the retrospective chart review evaluated 243 patients (51% male; 61% non-hispanic white; 46% leukemia survivors; median age at diagnosis 4.0 years [range 0-19.7]; median time off-therapy 7.2 years [range 2.3-18.2]). 230 patients (95%) received anthracyclines (median dose 154mg/m2, range 30-512) and 73 (30%) received cardiac radiation. assigned cardio-oncology scores resulted in: 35% echo only, 47% consider cardiology referral, 16% recommend cardiology referral, and 3% regular cardiology followup. when evaluating detection rates of late effects by cardiooncology score, 12 survivors (5%) had an abnormal echo: 1/83 echo only, 4/115 consider referral, 4/38 recommend referral, and 3/6 regular cardiology follow-up. assessing referral patterns prior to initiation of the algorithm revealed forty-two survivors (17%) referred to cardiology: 8/83 echo only, 16/115 consider referral, 14/38 recommend referral, and 4/6 regular cardiology follow-up. of the 37 patients seen by a cardiologist at our institution, 4 had further diagnostic testing ordered (i.e., stress test) and 7 received exercise restrictions. a cardio-oncology algorithm and guidelines will standardize cardiac care for survivors by assigning a score to guide referral and cardiology practice after referral. prospective clinical use has begun and review will occur in one year to determine changes in detection rates of cardiac late effects, referrals, and recommendations from cardiologists. oregon health and science university, portland, oregon, united states background: delirium affects 10-30% of patients (pts) in pediatric intensive care units (picu) and is associated with increased length of stay, decreased attention in school, and post-traumatic stress disorder. the diagnostic and statistical manual of mental disorders (dsm v) defines delirium as a "disturbance of consciousness […] with reduced ability to focus, sustain or shift attention" due to an underlying medical condition. despite the medical complexity of the hospitalized pho population, there are no published prospective studies looking at delirium in these pts. hypothesizing that delirium is under recognized in the pho population, we designed a year-long prospective study using a validated screening tool to determine the frequency of delirium in hospitalized pho pts and to identify associated clinical factors. design/method: baseline frequency of pts with symptoms suggestive of delirium was determined through retrospective chart review using a data mining program of electronic medical records (emr). for the prospective study, pho and picu nurses were trained to use the cornell assessment for pediatric delirium and to record scores within the emr on all pho pts once every 12-hour shift. predetermined demographic and clinical variables were entered daily into a red-cap database on all hospitalized pho pts. results: baseline frequency of delirium, without active screening, was determined to be 4.5% of hospitalized pho pts. in the first 6 months of the prospective study, 405 consecutive admissions occurred among 152 unique pho pts: 347 oncology, 30 hematology, and 23 stem cell transplant pts. 25 pts had at least 1 positive delirium screen, for a prevalence per admission of 6.2%. statistically significant variables associated with delirium, at p < 0.0002 by univariate logistical regression, included prolonged length of stay, pt location (picu vs pho unit), and fever. adjusting for length of stay, administration of benzodiazepines and opiates were also significantly associated with delirium, p = 0.048 and 0.044, respectively. on average, nurses completed delirium screening in 70% of each pts' 12-hour shifts. study accrual ends in jan 2018 and final data analyses will be reported in the abstract presentation. conclusion: delirium does occur in the pho hospitalized population and screening by trained nursing staff is feasible. pts at highest risk appear to be pts with prolonged hospital stays, picu admissions, or frequent use of benzodiazepines/opioids. routine screening should improve our recognition of delirium and allow us to promptly intervene, or prevent delirium in an effort to avoid potential acute and long term consequences. background: with high survival rates for children and adolescents with hodgkin lymphoma (hl), treatment regimens are now designed to maximize cure while decreasing risk of long-term health outcomes associated with chemotherapy and radiation therapy. within contemporary treatment regimens, the comparison of toxicities experienced by patients receiving chemotherapy plus radiotherapy (crt) versus only chemotherapy (co) has not been studied extensively. objectives: this study examines select self-reported adverse health outcomes in survivors of contemporarily-treated pediatric hl to better understand the balance between efficacy and toxicity associated with chemotherapy and radiation therapy. (cog) ahod0031 that evaluated a response-based treatment paradigm in pediatric hl. patient who received initial chemotherapy were randomized based on early response to continued chemotherapy, chemotherapy plus radiotherapy or augmented chemotherapy plus radiotherapy. patients completed self-report questionnaires on health problems at 0, 1, 3, and 5 years following therapy. we examined selected patient-reported pulmonary, gastrointestinal (gi), cardiac and endocrine outcomes. kaplan-meier survival curves were used to determine probability of survival without the selected adverse health outcome. log-rank tests were used to compare the co versus the crt group. results: a total of 1,051 enrolled patients, 251 patients in the co group and 800 patients in the crt group, completed 2,134 questionnaires at a median of 1.3 years after s177 of s301 completion of therapy (q1, q3: 0.6, 3.3) which were analyzed. the cumulative 10-year incidence of endocrine dysfunction was significantly greater in the crt group versus those in the co group (17% versus 6%; p<0.001), driven by the incidence of hypothyroidism (11% versus 2%; p<0.001). there were no significant differences in cardiac (7% versus 9%; p = 0.226), pulmonary (11% versus 10% p = 0.068), and gastrointestinal dysfunction (20% versus 17%; p = 0.317) between the co and crt patients. conclusion: this study demonstrates low cumulative incidence overall of organ dysfunction early post completion of contemporary therapy for hl. the addition of radiation therapy significantly increased risk for hypothyroidism, but with no higher risk noted for cardiac, pulmonary or gi dysfunction. limitations include self-report status, potential selection bias, and relatively short latency period following end of therapy. longer follow-up is needed to determine more delayed risks for organ dysfunction in order to best define the balance between therapeutic efficacy and long-term adverse health outcomes related to chemotherapy and/or radiation therapy. background: identification of an organism via bronchoalveolar lavage (bal) or respiratory tract biopsy (rtb) has historically been considered the gold standard for diagnosis of invasive fungal infection (ifi); however, data previously published by our group showed that these procedures infrequently lead to a change in management in children with an oncological diagnosis or undergoing hematopoietic stem cell transplant (hsct). there is also a paucity of data on the cost of ifi in this population. to compare the costs of work-up and management of pulmonary ifi diagnosed based on ct scan alone versus ct scan or chest x-ray prompting a bal or rtb. design/method: we collected cost data on patients at ann & robert h. lurie children's hospital of chicago undergoing chemotherapy or within 6 months of hsct who were suspected of having an ifi between 2007 and 2012. in order to include sufficient time to account for post-procedure compli-cations but avoid including costs unrelated to ifi, data were included for 14 days from the day of their diagnostic scan or procedure. cost data was available for 76 of the 101 patients previously studied. thirty-six of these patients were diagnosed with suspected ifi based on ct only and 40 patients underwent bal or rtb. when evaluating specific costs, inpatient beds costs were higher in the bal and rtb group (median $1,555 versus $1,255, p = 0.01), yet there was only a trend towards higher costs for antifungal agents (median $1,635 versus $1,089, p = 0.26) and respiratory support (median $257 versus $0, p = 0.14). many of the initial ct scans were not captured in the 14-day evaluation period for the bal or rtb group based on the study design; however, even when accounting for ct scans up to a week prior these procedures, the total cost of ct scans was higher in the ct only group (median $963 versus $635, p = 0.0002), as they had more scans. despite this, total costs were significantly higher for patients who underwent bal or rtb versus ct scan only (median $14,087 versus $5,900, p <0.0001). combined with our previous data that bal and rtb infrequently leads to a change in management in children with an oncological diagnosis or undergoing hsct suspected to have an ifi, the significantly higher costs associated with these procedures makes these invasive diagnostic techniques even less desirable. batra, pediatr blood cancer, 2015. background: while infants >12 months of age with acute lymphoblastic leukemia (all) have a poor prognosis, infants with acute myeloid leukemia (aml) fare better despite more intensive therapy. there are limited data on this difference, particularly differences in supportive care requirements during induction therapy for infants. objectives: to compare induction mortality and resource utilization in infants relative to non-infants aged <10 years, separately for all and aml. design/method: we used previously established cohorts of children treated for new onset all or aml at children's hospitals in the us contributing to the pediatric health information system. patients with down syndrome were excluded. follow-up started on the first day of induction chemotherapy and continued until the earliest of: 35 days after commencement of chemotherapy, start of the subsequent course, or death. high acuity of presentation, defined as icu requirements involving 2 or more organ systems within the first 72 hours following initial admission were compared using log binomial regression. 35-day inpatient mortality was compared using cox regression. resource utilization rates (days of use per 100 inpatient days) were compared using poisson regression. results: a total of 10359 all (405 infants, 9954 non-infants) and 871 aml (189 infants, 682 non-infants) were included in the analyses. infants were more likely to present with high acuity compared to non-infants for both all (12% and 1%, rr = 12.2, 95% ci:8.6, 17.5; p<0.0001) and aml (6% vs 3%; rr = 2.0, 95% ci: 0.96, 4.3; p = 0.06). infants with all had higher inpatient mortality compared to non-infants even after accounting for differences in acuity of presentation (2.7% vs 0.5%, adjusted hr = 2.7 95% ci: 1.2, 6.1; p = 0.015). in contrast, inpatient mortality was more similar for infants and noninfants with aml (3.2% vs 2.1%, adjusted hr = 1.2 95% ci: 0.3, 3.9; p = 0.73) and comparable to rates among infants with all. infants with all and aml had higher rates of utilization of fresh frozen plasma, cryoprecipitate, diuretics, supplemental oxygen, and ventilation relative to non-infants. infants with all also had higher rates of total parenteral nutrition, ecmo, and patient controlled analgesics compared to noninfants. infants with all experienced significantly higher induction mortality compared to noninfants, a difference not entirely explained by acuity at presentation. differences in ru among infants may reflect higher presentation acuity and greater treatment related toxicity. further work is needed to elucidate the contribution of treatment related toxicity to early mortality in infants with all. background: fever in a child with cancer is a medical emergency due to the significant risk of a serious bacterial infection. many attempts have been made to risk stratify these patients. the respiratory pathogen panel (rpp) is a panel of polymerase chain reaction tests that identify seventeen common respiratory viruses and three bacterial infections. samples are taken via nasopharyngeal swab. rpps are frequently sent, but we do not have data to determine whether a positive result can lead to stratification to a lower risk of bacterial infection. (1) to determine the epidemiology of respiratory virus-associated fever in pediatric oncology patients (2) to determine whether a positive rpp is associated with reduced risk of bacteremia in this population. this was a single-center, retrospective cohort study. we identified and reviewed the medical records of all pediatric oncology patients seen in our emergency department (ed) with fever from the introduction of the rpp in april 2014 to september 30, 2017. we reviewed the results of blood cultures, rpp, chest radiographs, and discharge summaries to identify sources of infection. we also identified the patients' cancer diagnosis, age, absolute neutrophil count (anc), and absolute lymphocyte count (alc). results: 107 positive rpps were found among pediatric oncology patients who presented to the ed with fever. the most common positive rpp findings were rhinovirus/enterovirus (rev) (45%), parainfluenza (14%), influenza (11%), coronavirus (11%), and polyviral (10%). among patients with a positive rpp, 4% had bacteremia compared to 12% bacteremia among all pediatric oncology patients with fever (or 0.42 [0.18-0.99], p 0.048). all cases of bacteremia were associated with rev. there was no bacteremia identified in patients with rpps positive for other viruses (or 0.0596 [0.0037-0.9715], p 0.048). rev positivity did not confer a lower risk of bacteremia than rpp negative patients ], p 0.97). anc (p = 0.87) and alc (p = 0.89) less than 500, and number of patients with severe neutropenia (p = 0.27) were not statistically different between the rev and non-rev positive rpp groups. rpps positive for viruses other than rev reduced the likelihood of bacteremia in febrile pediatric oncology patients in the ed setting. patients with bacteremia may have concurrent infection with rev. a larger study is warranted to determine if positive rpp results can inform clinical management of a child with febrile neutropenia. emily mueller, anneli cochrane, seethal jacob, aaron carroll s179 of s301 background: the usage of mobile health (mhealth), which refers to the application of mobile or wireless communication technologies to health and healthcare, has grown exponentially in recent years. mhealth tools have been used by caregivers of other vulnerable populations, but little has been focused on caregivers of children with cancer. objectives: to conduct a survey to understand the mobile technology usage, barriers, and desired mhealth tools by caregivers of children with cancer. we conducted a mailed cross-sectional paper survey of caregivers of all children who were diagnosed with cancer at riley hospital for children between june, 2015 and june, 2017. the survey contained 13 questions, both fixed and open-ended, in both english and spanish. up to three rounds of surveys were sent to those who did not respond. of the 121 respondents, they were primarily parents (93.2%), median age was 40.7 years (range 23-63), and most were white (78.5%) and non-hispanic/latino (87.1%). the top three annual household income brackets included $50,000 to $74,999 (21.2%), $25,000 to $49,999 (20.3%) and under $25,000 (17.8%). the majority had an education: 35.6% college graduates, 22% graduate degree, and 18.6% high school education or ged. nearly all respondents owned a smart phone (99.2%) and 61.2% owned a tablet. the majority used an ios operating system (62.8%), while 49.6% reported use of a device with an android operating system. all caregivers reported use of at least one mobile website/app regularly for their personal use. while 35.5% of respondents reported no barriers to mobile technology use, the top barrier selected was "data limitations" (21.5%). overall, 84.5% wanted at least one medical managementrelated website/app: medical knowledge (58.7%), healthcare symptom tracking/management (47.1%), and medication reminders (43%). healthcare system-related desires were high, as 59.5% wanted access to their child's medical record and 56.2% wanted a website/app to facilitate better communication with medical providers. there were no significant associations between socioeconomic status (income or education) with barriers or types of websites/apps desired by caregivers. since the vast majority of caregivers use mobile technology with minimal barriers, future research should focus on designing an mhealth tool to address the medical management needs by caregivers of children with cancer. by supporting caregivers through this type of mhealth tool, it could positively impact patient clinical outcomes through greater adherence to medications and treatment protocols. background: in children with fever and neutropenia, early initiation of targeted antibiotic therapy improves outcomes, yet there are no standards for choice of empiric antibiotics. in 2013 our institution implemented an early empiric ceftriaxone (eec) protocol to reduce time to antibiotic administration in febrile hematology-oncology patients who are potentially neutropenic when the absolute neutrophil count is not yet know. ceftriaxone is given immediately after obtaining blood for culture and lab studies. in patients found to be neutropenic, ceftriaxone is discontinued and cefepime is initiated. the purpose of this retrospective study was to evaluate our eec protocol in neutropenic patients by assessing ceftriaxone sensitivity of positive blood cultures and comparing rates of adverse outcomes with a cohort of patients treated prior to implementation of the protocol. we are now conducting a prospective study to more thoroughly investigate antibiotic sensitivities of organisms isolated from blood cultures of neutropenic patients. design/method: hematology-oncology patients with at least one positive blood culture between january 2011 and december 2013 were identified. patient demographics, neutrophil count, antibiotic treatment, isolated organisms and sensitivities, and adverse outcomes (increased respiratory support, hypotension requiring intervention, and icu admission) were obtained by retrospective chart review. fisher exact test was used to compare dichotomous variables between patient groups. we are now prospectively identifying febrile neutropenic patients with positive blood cultures and performing antibiotic sensitivity testing to several antibiotics commonly used as empiric therapy for febrile neutropenia. results: retrospectively, we identified 58 neutropenic patients with a total of 127 bacterial isolates from blood cultures. of organisms isolated, 47 were tested for sensitivity to ceftriaxone and 23 (49%) were not sensitive, 6/18 (33%) of gram-positive cultures and 18/29 (62%) of gram-negative cultures. ten of 16 (63%) eec patients had an adverse outcome versus 13/26 (50 %) of non-eec patients (p = 0.277). notably, 31% of eec patients required icu admission versus 4% of non-eec patients (p = 0.049). thus far our data obtained prospectively is revealing similar rates of ceftriaxone resistance with 9/19 cultures not sensitive to ceftriaxone (47%, ci 24.9%-71.1%). in our retrospective study, no statistically significant difference was seen in overall adverse outcome rate between the two cohorts, though icu admission rates were significantly higher in eec patients. ceftriaxone resistance rates were high in tested isolates, which is further supported by preliminary data from our ongoing prospective study. given these data, eec may not be effective at improving outcomes in febrile neutropenic pediatric hematology-oncology patients. background: approximately 1 in 5 children diagnosed with cancer will die of their disease, despite advances in treatment. results: two focus groups of six parents each met in june 2017. the parents were predominantly female (11 female, 1 male) and had lost their children an average of 2.8 years prior (range 1-5.3 years). two parents were in the same family. nearly all patients were offered palliative care (10/11), all were offered hospice and most died at home (9 at home, 2 in the icu). parent discussion uncovered six broad themes: beneficial provider qualities, optimal communication, helpful systematic supports, struggles to feel like a good parent, struggles with a loss of control and unmet needs. parents appreciated providers who were consistent, reliable and honest. parents desired communication that was sensitive to the needs of the patient and family with a balance of hope and realism. parents appreciated the tangible supports pro-vided by social work and the emotional support of child life both for the patient and their siblings. some parents struggled to define and advocate for their child's quality of life, especially when it led to disagreeing with the medical team. several parents expressed frustration with unfamiliar caregivers in the hospital, especially trainees. they expressed a strong desire for more anticipatory guidance about the end of life including how to discuss it with their children. they also wished for a cancer-specific support group for bereaved parents. conclusion: bereaved parents of pediatric oncology patients in our focus groups appreciated consistent, reliable providers who communicated with a balance of realism and hope. they appreciated the tangible and emotional support they received and wanted more anticipatory guidance at the end of their child's life. these results can help guide clinical care, especially in communities without strong palliative care support. further research is needed to develop interventions to improve end of life care. background: clinical trials involving human subjects depend on informed consent (ic) to ensure ethical protections for participants. parents of children with cancer often lack full understanding of the basic elements of ic for clinical trials. additionally, the stress of their child's cancer diagnosis may affect their decision-making capabilities. this is especially problematic as these children rely on parents to fully comprehend clinical trials and weigh their benefits and risks. physician communication is critical for effective family-centered care. the acgme mandates that training programs teach and assess trainees' communication skills. however, there are currently no published curricula aimed at training pediatric hematology/oncology fellows to deliver ic effectively for cancer clinical trials. to develop and pilot-test a simulation-based curriculum to enhance communication skills of pediatric s181 of s301 hematology/oncology fellows in the delivery of ic for cancer clinical trials. we developed, tested, and implemented the curriculum from 2016 to 2017 in two phases. in phase-1, we reviewed literature on simulation-based curricula and completed a needs assessment to create a clinical scenario and full curriculum using standardized patients. using miller's pyramid model, fellows' assessments included: immediate de-brief, surveys to assess pre/post confidence and knowledge of the basic ic elements ("knows" and "knows how"), and 360-degree summative assessments compiled from fellow self-assessments, faculty, and standardized patients ("shows how"). after initial testing and refinements done with 1 fellow, in phase-2, we implemented the curriculum with our 8 fellows. likert scale (1 strongly disagree-5 strongly agree) and basic p values are reported. results: fellows gave high mean ratings for training relevance (4.7) and standardized patients' preparedness (5). almost all (4.9) reported they have used the knowledge gained in their clinical practice. increase in self-reported confidence (pre/post) was noted in all domains: general -describing possible benefits of the clinical trial 3.5/5 vs.4.1/5 (p = 0.025), risks and potential side effects 3.5/5 vs.4.3/5 (p = 0.004), and explaining alternatives 3.1/5 vs.3.6/5 (p = 0.016); research -discussing purpose of the clinical trial 3.1/5 vs.3.7/5 (p = 0.006), and randomization 3.3/5 vs.4.0/5 (p = 0.046); and family-centered -addressing emotions during ic 3.6/5 vs.4.5/5 (p = 0.004), and delivering bad news 3.1/5 vs.3.6/5 (p = 0.016). summative evaluation mean ratings for all fellows were 4.5 (range 4.1-4.9). our novel simulated-based ic curriculum, significantly increased fellows' self-reported confidence and skills during ic delivery. importantly, our ic curriculum addressed not just research-related content but also management of parental emotional needs during the ic discussion. next phase includes kirkpatrick model program evaluation and dissemination across other training programs in our institution. national kaohsiung normal university, kaohsiung, taiwan, province of china background: taiwan's childhood cancer foundation reported in 2016 that the 5-year survival rate of childhood cancer was 75%. as a result, many childhood cancer survivors were back in school after treatment. however, childhood cancer survivors' educational outcomes suffered because of their long-term absence from school and late effects of cancer and cancer treatment. a few school reentry protocols have been developed by the nursing professionals in taiwan to facilitate students' return to school but remained experimental in nature and hardly accessible. parents, students, and teachers were left to their own devices to make individual school reentry plans. objectives: this study aimed to examine and uncover the commonalities among three middle school students' successful school reentry experiences from their teachers' perspectives and to analyze the factors contributing to their success. design/method: this is a qualitative interview study. indepth semi-structured interviews were conducted with three middle school teachers in december 2017 about their perceptions, observations, and experiences working with adolescent childhood cancer survivors. the students were two boys with leukemia and one girl with bone cancer. they were diagnosed in the first year of middle school when they were 12-13 years old and returned to school for the third and the final year. these students met the following criteria for successful school reentry: regular school attendance, average/above average academic performance, friendship maintenance, and high school diploma. the theme -bring the class to the hospital was found to be the key to the adolescents' successful return to school. without a prescribed school reentry protocol and in the face of limited bedside education services, the homeroom teachers, as links between school, home, and hospital, brought the class to their hospitalized students. they doubled as bedside teachers conducting lessons at the hospital or students' homes, became friends with the parents, witnessed firsthand the students' pain and triumph during treatment, brought the students back to school for visits and celebrations, delivered the classmates' wishes and news to the students, encouraged and welcomed classmates' visits to the hospital, and, together with parents and other teachers, developed flexible school reentry schedules for the students. this on-going study demonstrated the critical roles and functions of homeroom teachers in successfully bringing the students back to school during and/or after cancer treatment. further analysis will be focused on how and why these three homeroom teachers were able to carry out this unexpected task on top of their already full workload. jennifer kesselheim, shicheng weng, victoria allen, collaborative group fellowship program directors dana-farber/boston children's cancer and blood disorders center, boston, massachusetts, united states background: a novel, 4-module, case-based curriculum entitled "humanism and professionalism for pediatric hematology-oncology" (hp-pho) aims to foster pho fellows' reflection on grief and loss, competing demands of fellowship, difficult relationships with patients and families, and physician well-being and burnout. in small group facilitated sessions, fellows work to identify coping strategies and explore how the challenges of fellowship influence both their own doctoring and the patient experience. objectives: to administer the hp-pho curriculum in a prospective, cluster-randomized trial, measuring whether exposure to this educational intervention, compared to standard conditions, fosters humanism and professionalism and improves satisfaction with training. design/method: pho fellowship programs (n = 20) were cluster-randomized to deliver usual training in humanism and professionalism (control) or the novel curriculum (intervention) during the 2016-2017 academic year. the primary outcome measure was the pediatric hematology-oncology self-assessment in humanism (phosah). secondary measures included a 5-point satisfaction scale, the maslach burnout inventory (mbi), the patient-provider orientation scale, and the empowerment at work scale. participating fellows were pre-tested in summer 2016 and post-tested in spring 2017. a change score was calculated for each study instrument. we compared each outcome between arms using mixed effect models adjusted for pre-test score as a fixed effect and site as a random effect. results: randomization yielded 59 intervention and 41 control fellows. the two arms did not significantly differ in distribution of fellow age, gender, or post-graduate year. the 9 intervention sites successfully administered 33 of 36 (92%) modules. change scores on the phosah were not significantly different between the control and intervention arms (adjusted mean difference = 0.5; 95% confidence interval [ci] -1.0, 2.0; p = 0.5). compared to the control arm, fellows' exposed to the curriculum gave significantly higher ratings on several items within the satisfaction scale including satisfaction with their training on "physician burnout" (adjusted mean difference = 0.8; 95% ci 0.4, 1.2; p<0.001), "physician depression" (adjusted mean difference = 0.9; 95% ci 0.4, 1.4; p<0.001), "balancing professional duties and personal life" (adjusted mean difference = 0.7; 95% ci 0.3, 1.1; p = 0.002), and "humanism overall" (adjusted mean difference = 0.4; 95% ci 0.03, 0.9; p = 0.03). change scores on other secondary measures were not significantly different between study arms. conclusion: exposure to the hp-pho curriculum did not alter fellows' self-assessed humanism and professionalism. however, the curriculum proved feasible to administer and intervention fellows expressed higher levels of satisfaction in their humanism training, indicating the curriculum's positive impact both for fellows and their learning environment. background: recent work has documented significant levels of unmet needs among adolescents and young adults with cancer, particularly psychosocial challenges during the transition to adulthood, (e.g., abrupt disruption to school and social life, and social isolation). given that adolescents and young adults drive mobile app use, a mobile-phone may be an ideal way to deliver a psychosocial intervention to adolescents and young adults with cancer. to use a patient-centered approach to inform a mobile-based mindfulness and social support intervention for adolescent and young adult patients with cancer. design/method: participants were ten aya with sarcoma (50% female; 50% adolescents); parents of the five adolescents, and six healthcare providers (n = 21). formative research involved three steps: (1) in-depth interviews were conducted with ten aya with sarcoma; parents of the five adolescents, and six healthcare providers (n = 21). (2) adaptations were made to an existing mindfulness app which offers a program for youth. modifications included creating a 4-week "mindfulness for resilience in illness" program, with 28 relaxation exercises, and the addition of videos featuring two sarcoma survivors as program hosts. content was informed by the mindfulness curriculum for adolescents, learning to breathe. (3) a private facebook usability group was organized to (i) elicit beliefs about the mindfulness app and potential future enhancements, and (ii) promote social support. results of the in-depth interviews revealed themes around adolescents' functioning and coping, including body image concerns; recurrence-related anxiety; anger over loss; and being overwhelmed by medical information. themes from the interviews were incorporated into a demonstration version of the mobile app. a patient-centered approach is widely recommended in the development of mobile-based health behavior change interventions and may be a useful way to inform development of a mobile-based mindfulness and social support intervention for adolescents and young adults with cancer. background: medical trainees consistently report suboptimal instruction and poor self-confidence in communication skills. despite these deficits, few training programs provide comprehensive pediatric-specific communication education, particularly in the provision of "bad news." an in-depth survey to examine the historical experience and communication needs of pediatric fellows was conducted at a large academic pediatric center as the first step towards the development of a comprehensive communication curriculum. to determine the previous educational and clinical experiences of pediatric subspecialty fellows, assess their levels of comfort in the context of various communication topics, and query potential modalities and topics for future communication training. design/method: the needs assessment survey was developed using previously developed and validated questions and review of the literature. the survey was reviewed by internal and external pediatric oncology and palliative experts and pre-tested with a subset of trainees to enhance content validity. results: thirty-two out of a total of 38 fellows completed the survey (84% completion rate), of which 81% were pediatric hematology-oncology or subspecialty fellows. most fellows had participated in previous teaching sessions (97%), including those involving role play or simulation (75%). however, few fellows had received feedback from senior clinicians on their communication skills (31% of fellows had received feedback ≤ 3times). on a scale of 1-x, with 1 indicating "not well prepared," the mean score for 12 of 23 communication items was <3. fellows felt least prepared to lead discussions around informed consent for experimental therapies, end of life care, and autopsy. fellows indicated that didactic educational sessions and additional coursework were less useful strategies for improving their communication skills, whereas small group role play sessions with faculty and/or bereaved parent educators were most useful. fellows' overall communication preparedness score was not correlated with post-graduate year but was positively associated with the number of times they previously had delivered bad news to patients and families. fellows requested additional training on many topics, with greatest interest in learning skills to optimize communication with an angry patient or family. additional topic requests included placing limitations on resuscitation, withdrawing/withholding further therapy, and ageappropriate inclusion of patients in difficult discussions. despite self-report of prior communication skills training, pediatric subspecialty fellows felt underprepared to participate in difficult discussions with patients and families. learners identified role-playing and coaching with real-time feedback from other physicians and bereaved parents as more useful training strategies as compared to didactic sessions. background: when children die of cancer, parents must adjust to their child's absence amidst the lingering turmoil of what preceded their death: witnessing their child undergo painful treatments, making difficult decisions, and anticipating a devastating loss, all the while hoping for a recovery. adjustment to a child's death, as depicted by current bereavement literature, necessitates making meaning of one's loss. professional care staff can help parents make sense of their child's illness, and in turn, of their own parental experience during treatment. however, the extent to which relationships with professional care team members influence parents' ability to make sense of, and successfully cope with, their loss has not been examined. objectives: to examine how bereaved parents' interactions with their deceased child's pediatric oncology professional care team have impacted their grief symptoms design/method: to better understand how interactions with professional care staff relate to parents' grief outcomes, we conducted a mixed-methods study examining staff impact on parental grief. thirty participants whose children died of cancer one to three years ago completed an in-depth interview and psychometrically validated surveys measuring meaningmaking, depression, and grief symptoms. results: correlational analyses of the measures found that an increase in meaning making was associated with lower depressive and grief symptoms. a content analysis of the interviews found that many participants regarded staff "like family," had on-going relationships with staff after their child died, and described various ways staff interactions during treatment and after the child's death helped them make sense of their loss. in particular, participants described how interactions with staff have helped them find benefits in their loss and learn to create a new relationship with their child despite their physical absence. quantifying the interview data and statistically analyzing it along with the measures found that participants' increased frequency of describing staff's positive impact on their grief correlated with higher meaning-making scores and lower grief symptom scores. our study found that bereaved parents who lost their children to cancer were articulate in sharing their experiences of staff engagement and communication during treatment, offering numerous examples of how staff aided them in making meaning of their loss that were reliably associated with their subsequent grief. we hope the results of this mixed methods research encourage further study of the importance of staff interaction with families during the critical period of their children's care, and the lasting impact this can have regardless of the treatment outcome. memorial sloan kettering cancer center, new york, new york, united states background: although resiliency has been recognized as necessary for healthcare professionals, trainees feel unprepared for the emotional challenges inherent in caring for sick and dying patients. compounded by long hours, challenging work environments, and lack of formal training on handling emotionally difficult situations, many institutions are recognizing the need for interventions to reduce trainee distress. the goals of this fellow-led quality improvement initiative were: 1) to determine whether there is a need for emotional support amongst pediatric hematology and oncology fellows, 2) to provide formal resiliency and debriefing sessions, and 3) to measure feasibility, acceptability and effectiveness of implemented curriculum. design/method: an anonymous survey to determine need for resiliency and debriefing sessions following a traumatic event was distributed to 24 active pediatric hematology & oncology fellows at memorial sloan kettering cancer center in january 2017. once need was established, an intervention consisting of a formal curriculum was developed and initiated in june 2017, involving: 1) scheduled and ad hoc debriefing sessions in response to traumatic events (including patient death, codes, interpersonal conflicts, end-of-life care); led by a psychiatrist and social worker with fellows and a pediatric oncologist mentor in attendance, and 2) a resiliency didactic curriculum, led by a palliative medicine specialist, focused on skills such as contesting cognitive distortions and mindfulness. the effectiveness of these sessions will be measured using follow-up anonymous surveys at 6 months (currently underway) and 12 months post-initiation of intervention. the initial survey demonstrated most trainees (19/24) were present at 3 or more deaths during their training, while less than half of respondents had attended a post-event debriefing session. 85% of respondents felt there was not sufficient emotional support from the institution for physicians caring for dying patients. a separate pre-intervention survey found all respondents (14/14) expressed a need for regular debriefings, and nearly all anticipated that they would benefit from such debriefings. concerns identified by trainees that would preclude participation in the curriculum included preference to deal with emotional situations privately and time constraints. trainees identified a need for formal debriefings and resiliency skill development. the program was easily implemented, and is both feasible and acceptable with good attendance. feedback received at the 6-month mark will determine deficits and possible improvements to the curriculum. the 12-month survey will measure effectiveness of the program and whether it should be continued. background: acute kidney injury (aki) is a common but under-recognized complication among patients with leukemia. it is associated with prolonged hospital stays, increased mortality, progression to chronic kidney disease, and delays or changes in cancer therapy which may affect a patient's prognosis. however, data on aki in pediatric patients with cancer is still lacking overall. we investigated the incidence of aki in patients who were newly diagnosed with all at our center from january 2009 to september 2017. we performed a retrospective chart review of all patients who were newly diagnosed with all from neonate to 18 years in our facility. we determined the incidence of aki in our population using the kidney disease: improving global outcomes (kdigo) diagnostic criteria. we also assessed for nephrotoxic exposures, nci all risk stratification and risk of aki, and tumor lysis syndrome (tls). we identified 62 patients diagnosed during the study period who met inclusion criteria. median follow-up time was 29.5 months (range 3.6-60.3). the cohort was predominantly male (54.8%) and hispanic (93.5%). our analysis showed 51.6% had aki by kdigo criteria (29% grade 1, 14.5% grade 2, and 8% grade 3), 62.5% had aki on presentation, and 75% had multiple aki episodes during the study period. older age and longer length of hospitalization were associated with aki (p = 0.019 and p = 0.009, respectively). there was no association between aki and nci all risk classification, contrast exposure, hyponatremia, elevated white blood cell count, uric acid levels, antimicrobial therapy, or diuretic use in this study. conclusion: aki was a common finding in our study population. the majority had grade 1 aki by kdigo criteria. however, aki was associated with older age and a longer length of stay. further study is needed to determine the short-and long-term impact of aki on pediatric patients with all. st. jude children's research hospital, memphis, tennessee, united states background: in some regions, the availability of trained pediatric oncologists is a limiting barrier for the care of children with cancer. in 2003, the unidad nacional de oncología pediátrica (unop) and the universidad francisco marroquín school of medicine in guatemala established a pediatric hematology/oncology fellowship program sponsored by st jude children's research hospital to provide central america and the caribbean with well-trained specialists. a systematic analysis of the impact of fellowship programs in pediatric oncology has never been done, especially in the context of a regional education program. objectives: this study sought to analyze the impact of the unop fellowship program based on the regional number of providers, pediatric cancer centers and patient volume. in addition, it sought to characterize the jobs and scientific output of the graduates. the impact will be evaluated in the context of a cost analysis. to define the volume of providers, pediatric cancer centers and patients, the directors of pediatric cancer centers in central america were sent an online survey to obtain these data. all the centers contacted maintain an updated hospital-based patient registry. in addition, the 22 graduates of the fellowship program were also sent an online survey, asking about their job at graduation, current role and scientific productivity. the cost analysis will include assessment of direct costs including salaries and stipends for away rotations, as well as the indirect costs of faculty time spent teaching. since the establishment of the unop fellowship program, the region has more providers for pediatric cancer (p<0.05) and centers treat a larger volume of patients (p<0.05). two new centers have opened with graduates of the program. all but one graduate practice pediatric oncology (21/22) and the majority do it in their country of origin (19/21). no graduate practices outside of this region. almost half of the graduates (44%) hold a leadership role at their institution. the majority of their time is spent in the public sector (>95%). the majority of graduates participate in clinical research (61%) and have participated in the creation or implementation of therapeutic protocols (67%). on average, the graduates have published 2 peer-reviewed articles since completion of training. the unop fellowship program has had a favorable impact on pediatric cancer care in the region, contributing to the capacity to treat a larger volume of patients. graduates practice pediatric oncology in the region in the public sector, frequently hold leadership roles and are scientifically productive. background: abandonment of treatment is a major cause of treatment failure and poor survival in children with cancer in low-and middle-income countries. the incidence of abandonment in peru has not been reported. objectives: the aim of this study was to examine the prevalence and associated factors of treatment abandonment in pediatric patients with cancer of peru. we retrospectively reviewed the sociodemographic and clinical data of children referred between january 2012 and december 2014 to the two main tertiary centers for childhood cancer, located in lima, peru. definition of treatment abandonment was used from the siop (international society of paediatric oncology) podc (paediatric oncology in developing countries) abandonment of treatment working group recommendation. results: data of 1135 children diagnosed with malignant solid tumors and lymphomas were analyzed, of which 209 (18.4%) abandoned treatment. univariate logistic regression analysis showed significant higher abandonment rates in children living outside the capital city, lima (p<0.001); prolonged travel time to a tertiary center (> 5 hours; or 2.75, p = 0.002); living in a rural setting (or 3.44; p<0.001) and lack of parental formal job (or 4.39; p = 0.001). according to cancer diagnosis, children with retinoblastoma were more likely to abandon compared with other solid tumors. in multivariate regression analyses, rural origin and lack of formal parental employment were independently predictive of abandonment. conclusion: treatment abandonment prevalence in our country is high and closely related to socio-demographical factors. treatment outcomes could be substantially improved by strategies that help prevent abandonment of therapy based on these results. st. jude children's research hospital, memphis, tennessee, united states background: to improve the quality of a pediatric hematology/oncology fellowship program, a systematic assessment must be performed that can evaluate its current state and identify areas of opportunity, as well as modifications over time. unfortunately, widely agreed-upon metrics of quality for pediatric hematology/oncology fellowship programs currently do not exist. this is particularly important in this field due to the global shortage of specialists. for this reason, an assessment instrument that is applicable throughout the world must be created. objectives: the st. jude global education program assessment tool (epat) is a novel instrument that seeks to evaluate pediatric hematology/oncology fellowship programs around the world in systematic and objective way. epat will help determine key performance indexes that are relevant for quality education in pediatric hematology/oncology fellowship programs and establish the framework for improvement. design/method: firstly, key domains to be evaluated for program assessment were identified a priori based on the continuum of pediatric hematology/oncology fellowship programs in the context of geography and educational structure. subsequently, questions were formulated to evaluate these key domains, seeking to assess elements involved in ensuring competence in clinical practice, academic productivity and regional impact. due to the novelty of this tool and the lack of defined metrics of quality, epat relies on expert opinion in a two-step process: internally in the department of global pediatric medicine at st. jude children's research hospital and, subsequently, from a panel of experts in global pediatric oncology and medical education from around the world. ten key domains were identified to evaluate all aspects relevant to training programs around the world, regardless of educational and geographic context. questions have been created to assess these domains and, to make epat quantitative, these have assigned weights with a value reflective of their relative importance. this grading system allows for a score in each key domain, permitting monitoring of changes over time. epat is currently at the stage of external expert review, and subsequently will be piloted in five fellowship programs around the world to provide different geographical and patient care contexts for its validation. once epat is finalized, it will be distributed to pediatric hematology/oncology fellowship programs around the world to be applied. epat proposes a novel strategy to assess training programs in a systematic way that includes all aspects relevant for a training program in a global context. this tool will help guide improvements in pediatric hematology/oncology fellowship programs and assure a well-trained workforce. background: with the improvement in pediatric oncology patient survival and outcomes in the past several decades, monitoring for recurrence and long-term effects of therapy has become even more important. the utilization of personalized treatment summaries and survivorship care plans (scps) is one way to communicate this information with patients and families. the american college of surgeons commission on cancer (coc) created a standard regarding provision of scps to 50% of eligible patients by december 31, 2017 as a metric for accreditation of all cancer centers. the standard applies to all patients with stage i, ii, and iii cancer diagnoses and requires creation of the scp within one year of diagnosis or six months of completing treatment. during implementation at our pediatric cancer center, we identified barriers to use of the guidelines in the childhood cancer setting. objectives: define eligibility for an scp for pediatric oncology patients to include all patients with curative intent and to deliver scps within six months of finishing therapy. design/method: using chart review and a cancer center registry query, we identified childhood cancer patients potentially eligible for an scp by collecting stage, goal of therapy, and dates of treatment. all patients with curative intent were deemed eligible for an scp regardless of stage i-iv. patients being followed in the oncology clinic for posttreatment surveillance and care were included even if they had received an scp in the survivorship program or were greater than six months off therapy at time of implementation. as expected in the pediatric oncology population, acute lymphoblastic leukemia (all) was the most common diagnosis comprising 31.5% of patients. all is stratified into risk groups instead of surgical staging categories, and treatment duration is greater than one year, unlike many adult-onset malignancies. these differences required interpretation of the guidelines to apply to our pediatric population for all and other pediatric diagnoses with non-surgically based staging. our pediatric oncology clinic has to date provided scps to 141 of 277 eligible patients by adapting the guidelines to focus on patients with curative intent to receive an scp by six months off therapy. cancer staging guidelines and goals for curative intent as well as lengths of treatment vary between the pediatric and adult populations. the coc guidelines require adaptation for optimal applicability to the pediatric oncology population. background: education in communication for fellows in fields that require difficult discussions with families are few in nature. adult learning pedagogies such as role play are under-utilized in medical education, and have been shown to be as effective as traditional teaching methods such as lecture. an 8-module course for fellows in hematology/oncology, hospice and palliative medicine, radiation oncology, and pediatric hematology/oncology was implemented in january/february 2017. 12 fellows participated in the program. topics covered including fundamentals of communication, coping and spirituality, delivery of bad news, communicating with families, sexual dysfunction during treatment, palliative care/death and dying, and burnout. objectives: overall goal of this course is to foster holistic physicians who views their patients as people with cancer, not cancer patients, and physicians that can communicate effectively with their patients throughout the disease continuum. by the end of the course, learners should be able to practice the fundamental principles of good communication. design/method: fellows initially participated in a pre-course osce to establish baseline skills. osce was facilitated by the center for learning and innovation at northwell, and included actors portraying a pediatric patient and family member to whom the fellow had to break bad news. two months later, the course was carried out over the span of eight weeks and included didactic sessions followed by 45 minutes of role play scenarios. five of the eight modules included role play, with faculty members serving as simulated patients. after the course, a second breaking bad news osce was held. both osces were filmed, and feedback was given by the on-site actors. additionally, faculty members were given access to the videos in an on-line format and were given an evaluation tool to assess the fellows' performance pre-and post-intervention. fellows were given subjective surveys pre-and post-course as well. results: subjective data from participants showed a noticeable increase in comfort level in all areas on the pre-and post-course survey. data obtained from osce videos showed improvement in communication skills as assessed by sps and faculty members using a new evaluation tool developed by faculty. initial first-run data shows that this course is successful in improving communication skills as well as increasing fellows' comfort level across several domains of communication. future directions for our course include improving and validating our assessment tool, expanding our topic base to include more aya and pediatric scenarios, faculty development for improved role play, and investigating impact on practice after course completion. background: acute lymphoblastic leukemia (all) is the most common form of childhood cancer with approximately 2900 children diagnosed each year. survival rates have improved significantly over the past several years. children with all are at risk for developing musculoskeletal complications during and after completion of treatment, which can contribute to impaired activity, elevated body mass index (bmi), and risk for complications. interventions involving physical activity could improve musculoskeletal strength as well as overall health in these children. the aims of this study are to examine the feasibility of a directed physical activity program for children with newly diagnosed all during the initial intensive phase of therapy and to evaluate the overall health and quality of life of children participating in the directed physical activity program. design/method: all subjects will receive education materials about the importance and safety of physical activity and a nutrition handout. all subjects will also participate in the directed physical activity program under the supervision of a trained physical therapist for at least 40 minutes every week for 12 weeks. the program will entail four stations including a cardiovascular, balance/proprioception, strength and flexibility, and coordination and cardio. feasibility will be assessed by tracking the participation rate throughout the study period. other assessments will be made at study entry, at the end of 12 weeks of physical activity initiative and 3 months after completion of the intervention. assessments include overall strength and flexibility, weight, height, bmi, blood pressure and performance scores. descriptive statistics will be used for this study. results: a total of 10 patients, 3 male and 7 female, enrolled in the study over a 9.5 month period. patient ages ranged from 5-16 years. half of the patients enrolled have completed the 12 week program and all 5 patients had stability or improvement of their physical functioning scores. further data collection and analysis is ongoing. patients in the early intensive phase of all therapy are at risk for complications that can affect their physical functioning. a directed physical activity protocol may improve their overall physical functioning. patients may not need specific physical therapy; however a directed physical activity program appears to be beneficial for these patients. the main roadblocks to successful completion of the program were difficulty with scheduling, strain on the parents and patient from treatment, unplanned admissions for fever, as well as nausea and fatigue at time of visit. albany medical center, albany, new york, united states background: communication skills are a core competency highlighted by the acgme. increasing resident confidence in delivering difficult news has been shown to lead to more s189 of s301 effective communication. currently, the majority of residency programs lack formal training in communication skills. our objective was to demonstrate feasibility and efficacy of integrating a standardized-patient based training program for communication skills into the curriculum of pediatric residents design/method: to date, 10 pediatric and 4 medicine/pediatric residents have participated in the program during the intern year. the program consists of three, two-hour long sessions, in which each resident is given several opportunities to act out case scenarios with a standardized patient. scenarios included informing a parent of their child's new cancer diagnosis and disclosure of a positive hiv test to a teenager. residents received post hoc peer to peer, and preceptor to learner feedback. pre and post-program surveys were completed by residents. results: following course completion residents reported an increase in confidence in multiple areas of communication including giving a difficult diagnosis (p<0.05), discussing a poor prognosis (p<0.02), responding to different patient/family member emotional responses i.e. crying or anger (p<0.01), and organizing vital information to be relayed (p<0.01). in conclusion, communication skills training of pediatric residents is feasible and provides a platform for developing valuable skills not taught elsewhere within the curriculum. background: for children with cancer, transitioning back to school during or after treatment can be challenging. literature supports the need for school re-entry programs to ease this transition. however, these programs vary widely among pediatric cancer institutions with little data addressing their program components. data from this study provides information on current school re-entry programs across these institutions. objectives: one objective of this study was to assess for correlation between the presence of a school re-entry program and other factors, such as geographic location and institution size. a second objective was to establish a list of differences between institutions' school re-entry program components. finally, we aimed to describe current school reentry practices, as well as program benefits and perceived areas for improvement. states with membership in the children's oncology group were offered enrollment in this study. a member of each institution was invited to participate in a survey established by the research team. this person was closely associated with the institution's school re-entry practices. each interview queried institution demographics, as well as program components (e.g., participants, target audience, resources). comment was also collected on program benefits and potential for improvements. analysis of transcripts was performed using pearson's correlation to assess for relationships between institution size, geographic location, and program presence. grounded theory was used for analysis of benefits and improvements. results: thirty-nine of forty-one pediatric institutions who were offered enrollment participated in this study. twentynine institutions (76%) indicated the presence of a school reentry program, and ten (24%) stated they had none. no correlation was found between institution size and the presence of a school re-entry program (p = 0.627, ns). there was also no correlation found between institution location and the presence of a school re-entry program (p = 0.921, ns). a major theme surrounding the benefits of having a program included education for the returning student's peers. for those with programs, perceived improvements included increasing staffing and the ability to offer more services. the results do not support the hypothesis that the presence of a school re-entry program is influenced by the size and geographic location of the treating institution. however, data seem to suggest that available staffing may influence the presence of a program. future studies are needed to address other potential influences, as well as to take an evidence-based approach to determine the effectiveness of the interventions present in these programs. cohen children's medical center/ zucker school of medicine at hofstra-northwell, new hyde park, new york, united states background: genetics/genomics is evolving at an extremely rapid pace. current advances lead to individual algorithms toward disease treatment for each disease with multiple branch points. fellows learn only a fraction of the knowledge and there is no formal approach to teaching critical analysis of information and application algorithms toward disease. additionally, as knowledge evolves extremely rapidly, any approach must teach self-acquisition and application of evolving discoveries. objectives: to create, implement and evaluate a novel curriculum for genetics/genomics targeted toward pediatric hematology/oncology fellows design/method: the curriculum includes four components: 1) genetic and genomic medical knowledge, with one initial team-based learning session and weekly online multiple choice questions; 2) essential pathways, which will teach molecular pathways common in oncogenesis and relevant to targeted therapy in microteaching sessions with using auditory, visual and tactile learning; 3) knowledge acquisition and clinical judgment, to allow learners to gain experience into researching data available, then developing and prioritizing potential treatment plans using problem-based learning sessions in which they will stage a patient, research treatment options, prioritize and present findings; and 4) synthesis to demonstrate independent ability to research and recommend therapy through an independent project in which the learner, given a case, will present the case and research findings, genetics/genomics, molecular pathways and make recommendations for therapy in molecular tumor board for faculty and fellows. to evaluate, we plan to recruit 12 to 16 institutions, match for size of programs and implement in half and evaluate 2nd and 3rd year fellows in both groups by mcq exam and satisfaction surveys. the creation of a multi-module, adult-learning based curriculum for genetics and genomics in pediatric oncology is feasible. implementation and evaluation are necessary to demonstrate efficacy. background: neuroblastoma is the most common extracranial solid tumor in children. chimeric anti-gd2 antibody ch14.18 (dinutuximab) therapy has improved the survival of children with newly diagnosed high-risk, neuroblastoma patients as well at the time of first relapse/progression. acute neuropathic pain is a well-documented side effect of dinutuximab administration. however, additional adverse effects including sensorimotor neuropathy, ocular symptoms, and behavioral changes have been described. the incidence and severity of these effects are currently not well-documented in pediatric patients. with improved long term survival of patients receiving this modality, it is important to look for the potential late effects of dinutuximab. objectives: to determine the incidence and severity of neurologic, ophthalmologic, or behavioral changes after dinutuximab administration at our institution. we performed a retrospective chart review using our electronic medical record. we included all patients with high-risk neuroblastoma between the ages of 1 and 21 years at our institution diagnosed between 1997 and 2017 who received dinutuximab. patients with history of opsoclonus-myoclonus syndrome or gross sensorimotor neuropathy prior to receiving dinutuximab were excluded. we examined clinical documentation for subjective reports and objective exam findings of neurologic, ophthalmologic, or behavioral changes. we also looked for referrals made to neurology, ophthalmology, physical medicine & rehabilitation (pm&r), and psychology. : twenty-two patients met inclusion criteria. at the time of chart review, 15 patients were alive and 7 were deceased. eighteen patients received dinutuximab per anbl0032; 5 patients received dinutuximab per anbl1221. of these 22 patients, 11 patients reported symptoms of interest and 5 reported multiple symptoms. six patients reported symptoms that began at least 12 months after completing dinutuximab. nine patients had objective findings on exam, including decreased deep tendon reflexes, abnormal pupils, and nearsightedness. for 10 patients, 15 referrals were made to ophthalmology, pm&r for neuropsychologic testing, or neurology. two patients who reported symptoms of interest were not referred to a specialist. conclusion: neurologic, ophthalmologic, and behavioral symptoms were commonly reported and demonstrated on exam among pediatric patients with high-risk neuroblastoma who received dinutuximab. it is important to identify these effects so that appropriate specialist referrals can be placed for adequate management of these changes. we recognize that these symptoms may not be solely due to dinutuximab as these patients receive other agents including opioids, so a prospective trial is needed to further evaluate the long-term effects of dinutuximab and to determine how best to screen for these effects. akron children's hospital, akron, ohio, united states background: pediatric cancer is the leading cause of diseaserelated death in children in the united states (u.s.). in 2014, over fifteen thousand children were diagnosed with cancer in the u.s. this population is at high risk for malnutrition due to the multimodal therapies they receive: surgery, chemotherapy, radiation therapy, antibody therapy, and/or bone marrow transplant. adverse effects of these therapies include taste changes, loss of appetite, diarrhea, vomiting, and/or mucositis, making it difficult for the children to be able to consume adequate amounts of nutrition during therapy. there is no "gold standard" measurement tool for identifying patients at risk for malnutrition. nutritional status is not frequently evaluated as a component of clinical trials. assessment of anthropometric measurements (weight, height, z-scores) at diagnosis, as well as over the duration of treatment, can assist in the early identification of malnutrition. the incidence and prevalence of malnutrition in this population is unknown at akron children's hospital. the purpose of this study is to describe the nutritional status and provision of nutritional support therapies in pediatric patients during their first year post new oncologic diagnosis. objectives: identify the incidence and prevalence of malnutrition across oncologic diagnostic categories over the first twelve months post diagnosis. we performed a retrospective records review of all patients newly diagnosed with cancer in 2015 at akron children's hospital. demographic and anthropometric data was collected at time of diagnosis and nutritional status categorized by z score. anthropometric and nutrition support data was then collected every two months for the first year after diagnosis along with incidence of unplanned inpatient admissions. results: a total of 65 patients were included in the analysis, with 6.2% malnourished at time of diagnosis; 12.3% developed malnutrition the first year. patients with solid tumors represented 50% of patients with pre-existing or acquired malnutrition. overall, 47% of patients received at least one nutritional support modality. patients with pre-existing or acquired malnutrition had a non-significant increase in unplanned admissions (p = 0.1196). our study demonstrated that patients with solid tumors were found to be at increased risk of pre-existing and acquired malnutrition, followed by leukemias, and experienced higher incidence of unplanned admissions in the time period observed. prospective, multi-center replication of this study, including detailed collection of nutrition therapies is recommended to guide development of diagnosis specific nutrition support guidelines. background: pediatric and young adult oncology patients treated with intense chemotherapy have a high incidence of transfusional iron overload. iron deposition can lead to heart failure/arrhythmias, liver abnormalities, endocrine dysfunction, ineffective erythropoiesis, and increased cancer and mortality risk. however, there is a paucity of data regarding recommendations for management of transfusional iron overload in these cancer survivors. consequently, long-term complications of transfusional iron overload specific to these patients have not been assessed. objectives: to assess screening and phlebotomy-based treatment algorithms for this population. design/method: a retrospective chart review of pediatric and young adults who completed oncology management, had iron overload, and initiated phlebotomy treatment was conducted. tiered screening occurred in patients that received at least 5 packed red blood cell (prbc) transfusions. patients were recommended for evaluation and possible phlebotomy if: (1) liver iron concentration (lic) >5 mg of iron/gram dry weight liver tissue by ferriscan and/or (2) cardiac mri t2* < 20 ms. during phlebotomy, iron status was assessed quarterly and phlebotomy discontinued with lic <5 or normalization of ferritin/imaging lic verification. descriptive statistics were employed to report the characteristics of the study population. spearman correlations were utilized to describe associations between transfusions, lic, ferritin, iron saturation and number of phlebotomy sessions. results: twenty five survivors underwent phlebotomy. the mean age was 11.6 years (sd 6.1) and 10 (40%) were female. oncologic diagnoses: all (36%), aml (8%), nhl (12%), ewing sarcoma (16%), osteosarcoma (4%), neuroblastoma (12%) and cns (12%). patients received a median of 25.0 (iqr 17 -34) transfusions. median number of phlebotomy sessions was 6 (iqr 4-8) over 0.36 years (iqr 0.28 -0.59). prior to phlebotomy, median lic was 7.5 mg/g (iqr 5.6-9.0) and ferritin was 1110.0 ng/ml (iqr 700 -2030) . no patients demonstrated abnormal cardiac t2* mri (n = 18). 23 (92%) patients completed phlebotomy. one discontinued due to poor vascular access. no patients developed iron deficiency. lic was reduced by a median of 2.4 mg/g (iqr 1.1 -3.6) and ferritin by 586 ng/ml . correlation between number of transfusions and phlebotomy sessions was poor (r2 = 0.017). conclusion: management guidelines are lacking for transfusional iron overload in pediatric and young adult survivors of cancer. we demonstrate a phlebotomy algorithm that is effective and tolerated. correlation between number of transfusions received and phlebotomy treatments was poor, necessitating serial assessments. using this management algorithm, prospective studies can evaluate the effect of iron removal on iron overload complications in this patient population. penn state children's hospital, hershey, pennsylvania, united states background: cancer therapy leads to an impaired immune system that takes time to recover. it is important to ensure that these survivors have adequate immunity to prevent common yet potentially severe childhood illnesses. no validated guidelines currently exist for surveillance testing or re-immunization in this population. retrospective analysis involving a small cohort of pediatric cancer patients treated at penn state children's hospital showed 46% of patients screened for varicella immunity after therapy completion did not have adequate disease titers. to determine the proportion of pediatric cancer survivors who have lost humoral immunity to previously received vaccines; to determine the rate of response to single dose boosters or full vaccine series in seronegative subjects after one booster. design/method: pediatric cancer survivors treated at the children's hospital who are at least 12 months from completion of cancer therapy are prospectively tested for antibody levels to hepatitis b, tetanus, varicella, measles, and 6 strains of pneumococcus (4, 6b, 9v, 18c, 19f, and 23f). samples are analyzed by the cdc for measles and varicella avidity. seronegative subjects by commercial studies, are eligible to receive booster vaccines. titers are rechecked at least 6 weeks after boosters to re-evaluate immunity; if still seronegative, subjects will receive the entire vaccine series. titers are finally tested at least 6 weeks after the final dose of the vaccine series. immunity analyzed after therapy, after boosters, and after vaccine series. results: of 37 pediatric cancers survivors who completed therapy, 78% were non-immune to hepatitis b, 92% nonimmune to >50% of pneumococcal strains tested, 24% nonimmune to measles, 54% non-immune to varicella, and 2% non-immune to tetanus. 1 of 13 subjects who received mmr vaccine after therapy and prior to study enrollment did not have protective antibodies to measles. of the 15 subjects who received varicella vaccine after end of therapy and prior to study enrollment, 6 did not maintain protective antibody levels. cdc results for measles and varicella are pending, as well as repeat studies after vaccine boosters and series. conclusion: a significant percentage of pediatric cancer survivors do not retain immunity to hepatitis b, pneumococcus, measles, and varicella. after one booster, a high percentage of subjects did not develop protective immunity to varicella. only 1 subject did not have immunity to tetanus, which is consistent with the high immunogenicity of tetanus toxoid. formal guidelines are needed to protect this population from vaccine-preventable illness post-therapy. children's hospital of richmond at virginia commonwealth university health system, richmond, virginia, united states background: childhood cancer survivors are at risk for being overweight. diet and physical exercise are important in maintaining a healthy lifestyle and weight; however, it has been reported that cancer survivors are less active than their peers. one reason for this may be that there are no clearly established risk-based exercise recommendations for cancer survivors. another reason may be that providers tend to focus s193 of s301 recommendations for exercise more towards patients who are overweight. objectives: to describe changes in physical fitness of childhood cancer survivors who exercise. design/method: 'moving forward' is a wellness and physical fitness program that the center for care beyond the cure at chor offers in partnership with the ask childhood cancer foundation and the ymca. the program is available for any childhood cancer survivor between 8y and 18y age, being seen at our center. survivors define their fitness or wellness goals and then work with a trainer once a week (at least) for 30 min sessions throughout the year to achieve these goals. baseline and ongoing measurements for core strength, endurance, overall strength and balance were collected. the average of each of the parameters of all participants were compared from the beginning to the end of the program. over the year, there was a 30% increase in endurance as measured by the average of the miles walked in 6 minutes, 40% increase in core strength as measured by the average number of sit-ups in 30 secs, an 80% and 44% increase in overall strength as measured by the average weight lifted by leg press and the average weight lifted by chest press, and a 10% increase in balance as measured by the average number of seconds balancing on a single leg. in addition, each child had actually gained weight in the process with an approximately 10% increase in the average of the weights of all children. there are benefits to regular exercise beyond weight control, and improvements in physical fitness can be seen even without weight loss. regular physical exercise results in improved physical fitness and should be universally advocated to all patients. determining insulin resistance, measuring changes in fatigue and wellness perception following exercise are future directions that we intend to explore. dana-farber cancer institute, boston, massachusetts, united states background: improvements in adolescent and young adult cancer patient (aya) survival rates and quality of life outcomes have lagged behind those of children and older adults, highlighting a need for research targeting this unique population. current literature supports the value of strong ayaclinician communication, notably in facilitating therapeutic alliance, however little is known about aya communication priorities during cancer care and barriers to optimal ayaclinician communication. objectives: to explore aya and oncology clinician communication priorities and to identify barriers and facilitators to aya-oncology clinician communication. design/method: semi-structured interviews were held with 21 aya cancer patients and survivors (ages 15-25 years) from a single large academic institution and 22 oncology clinicians (physicians and nurse practitioners) from 7 academic institutions in the northeastern united states. interviews were conducted in english by phone or in person. all interviews were audio-recorded and transcribed verbatim. analyses were aided by nvivo11 software. ayas identified a wide range of topics as important to discuss with clinicians. the most frequently identified topics were 1) side effects of treatment (with an emphasis on physical appearance and function, n = 16), 2) social issues (including friendship, family, and school, n = 13), 3) looking ahead to the future (n = 12), and 4) sexual & reproductive health (including future fertility, contraception, and romantic relationships, n = 8). clinicians prioritized 1) cancer treatment and side effects (n = 17), 2) emotional and psychological health (n = 11), and 3) sexual and reproductive health with a focus on fertility risk and fertility preservation (n = 8). aya reported facilitators to good communication including an open and long-established relationship with the clinician (n = 16) and clinician engagement in age-appropriate and patient-directed conversations (n = 7). barriers included parental presence during visits (n = 7). clinicians reported barriers including 1) clinician discomfort (not feeling wellequipped to discuss psychosocial topics such as sexual health, spirituality, and relationships with peers, n = 13), 2) presence of parents/family (n = 12), and 3) perceived patient discomfort discussing specific topics (such as sexual health, n = 10). clinicians acknowledged the need for collaborative efforts with additional team members (i.e. nurses, psychosocial providers) to assist in meeting aya communication needs. conclusion: aya and clinician-reported communication priorities are largely aligned. however, ayas emphasize some topics, such as social function, appearance, and sexual health that are not highly prioritized by clinicians, which may result in gaps in care for ayas in treatment and in survivorship. these data identify opportunities for intervention, including clinician education, patient and family education, clinic-based intervention, and systems-based changes that can be developed and tested. background: primary care physicians (pcps) cite lack of knowledge and inadequate communication with the oncology team as major barriers to providing recommended surveillance for late effects of treatment to childhood cancer survivors. a standardized telephone handoff to pcps posttherapy is a potential strategy to increase survivorship care by pcps through interactive communication. to determine the feasibility of a structured telephone communication using the situation, background, assessment, and recommendation (sbar) communication tool delivered by a trained oncology nurse to increase pcp knowledge and willingness to provide survivorship care. design/method: from 12/12/16 to 1/23/17, a registered nurse expert in childhood cancer survivorship attempted to contact by telephone the pcps of the 30 most recent patients attending yale's childhood cancer survivorship clinic that were <18 years old, english-speaking, and ≥2 years posttreatment. all pcps had been previously sent an individualized survivorship care plan (scp) that listed the patient's previous treatment history and recommended surveillance tests. upon successful contact and after confirming receipt of the scp, the nurse explained the definition of late effects, description of patient's diagnosis and treatment history, and associated potential late complications and schedule of recommended surveillance tests. the pcp was also asked about his/her ability and willingness to provide needed surveillance for late effects in the future. overall, 26 of 30 pcps were successfully contacted with a median of 1 phone call (range: 1-3) that lasted a median of 6 minutes (range: 3-10) after a median of 1 business day (range: 0-18). no pcps ended the call mid-conversation. all 26 pcps were receptive and expressed appreciation for the call. twenty-five of 26 (96%) pcps expressed an understand-ing of the material discussed and endorsed belief in their ability and willingness to provide late effects surveillance for their patients. no pcps questioned discussing their patient's care with a nurse versus a physician. interactive, structured communications between nurses and pcps by telephone are feasible and are associated with high-levels of pcp confidence in providing survivorship care. background: childhood cancer (cc) admissions account for 5% of non-newborn pediatric hospitalizations. these hospitalizations are longer and more expensive than other hospitalizations. admission payer (medicaid or commercial) reflects both health policy and sociodemographic status. the objective of this study was to determine if length of stay (los) or cost of cc admissions differed by payer. we used the 2012 kids inpatient database, a sampling of all pediatric hospital discharges in the united states. analysis for this study was limited to admissions containing a cancer diagnosis in any discharge icd-9 codes. admissions were further subcategorized by discharge codes according to diagnosis (leukemia, lymphoma, solid tumor and brain tumor) and reason for admission (chemotherapy, procedure, infection, non-infectious toxicity or "other"). charges were converted to costs using cost-to-charge ratios. multivariable linear regression models were performed to control for age, gender, race, reason for admission, and diagnosis. results: there were 105,752 weighted admissions for children with a cancer diagnosis in 2012. of these admissions, 40.5% had medicaid, 50.6% had commercial insurance, and less than 1% had other payers. the mean los for medicaid admissions was 7.37 days (95% ci 7.2-7.5), compared with 6.33 days (95% ci 6.2-6.4) for commercial insurance. surgical admissions accounted for the largest difference in length of stay with medicaid admissions being 2.77 days longer than those covered by commercial insurance (11.19 days vs 8.42 days), however, the difference was significantly different for all reasons for admission. in multivariable analysis admissions associated with commercial insurance were 6% shorter s195 of s301 (p<0.001), accounting for approximately one hospital day, than admissions associated with medicaid after controlling for other variables including race. the mean overall cost for medicaid admissions was $23,464 (95% ci 22840-24088), compared with $21,849 (95% ci 21343-22356) for commercial insurance. in the multivariable model, cost was collinear with race. conclusion: los and cost of admissions associated with medicaid differed from those associate with commercial payers. medicaid admissions were 6% longer on average than commercial insurance, accounting for a difference in length of stay of approximately one day although the difference varied with the reason for hospitalization (chemotherapy, surgical procedure, infection, other toxicity, other). costs of admissions were not independent of race. further investigation into potential explanations for this difference including differential access to home care needs, outpatient reimbursement differences, social indications for prolonged hospitalization, and provider biases, is warranted. background: pediatric cancer is a major cause of morbidity and mortality among children surpassed only by accidents. despite improved outcomes in high income countries (hic) survival rates remain poor in the developing word. there are various diagnostic and therapeutic limitations contributing significantly for the survival gap. the main objective of the study is to to evaluate the outcomes of pediatric cancer in armenia and identify diagnostic and therapeutic limitations in the country. we conducted a retrospective study among 97 (≤18 years old) children with cancer (solid tumors and hematological malignancies), who were diagnosed and treated at the clinic of chemotherapy of muratsan hospital complex of yerevan state medical university between 2008 and 2016. those patients, who didn't receive chemotherapy for any reason were not included in the study cohort. epidemiological, social, medical information was collected through the patient charts review. this included patient age at diagnosis, sex, place of residence (city vs village), the educational level and employment status of parents, type of cancer, stage, presentation of symptoms, first medical specialty consulted and the time consulted, initial work-up, the type of treatment received, information on the diagnosis/treatment received abroad. results: at our clinic during the mentioned period of time the majority of patients presented with hematologic malignancies-71%. 77 (74.6%) patients had information on diagnosis delay. average delay in diagnosis was about 42 days. in 33% of cases the first contact with "healthcare system" was through pediatrician, and in 20% with surgeon. out of 19 relapsed patients 10 received salvage treatment in armenia and 4 abroad. from those who stayed for treatment in armenia 4 patients survived. majority of relapsed patients had acute lymphoblastic leukemia. from 35 leukemia patients immunophenotyping and cytogenetics were available for 26 (74.3%) patients; the majority of missing cases were between 2008 and 2012, when these diagnostic modalities were not available or affordable in the country. 43 (45%) patients received part of diagnosis and/or treatment abroad. the most frequent reason for going abroad was bone marrow transplantation, otherwise none available in armenia. out of 97 patients 22 were lost to follow-up, 15 patients had a fatal outcome. 60 patients were in remission at a median follow up of 3.57 years. conclusion: unavailability of cancer registry and several essential diagnostic/treatment modalities, luck of multidisciplinary care and palliative support, high rate of out-of-pocket expenses were among the main challenges of pediatric cancer care in armenia. background: adverse drug reactions (adrs) are increasingly recognized as important and sometimes irreversible complications of cancer treatment. anthracyclines and cisplatin are effective chemotherapeutic agents, but their use can be limited by cardiotoxicity (anthracyclines) and ototoxicity (cisplatin) in up to 60% of patients. genetic variants that can be used to predict who is most at risk of developing these adrs have been discovered and replicated. objectives: to create pharmacogenetic risk prediction models for anthracycline and cisplatin toxicities and discuss results with oncologists to facilitate incorporation into treatment decision-making when appropriate. design/method: risk prediction models were developed from the linear regression of strongly-predictive genomic variants (odds ratios ≥ 3) discovered and replicated in at least three patient populations. these models were used to assess an individual patient's genomic risk of developing cardiotoxicity from anthracyclines or hearing loss from cisplatin. risk results were returned to oncologists showing where the specific patient's genetic risk of toxicity lies on a continuum between the lowest and highest risk groups across all studied patients using a multi-gene model. interviews were conducted with patients, families, and oncologists to determine how results were valued and utilized. results: 227 patients have been genotyped and had their genetic risk results returned to their oncologists. the first 140 patients have been characterized to determine the impact these test results have had on their clinical care. results were described as being useful in decision-making by patients and/or oncologists in 100% of cases. additionally, for patients in the most extreme risk groups (highest and lowest risk), a change in treatment plan was ordered 30% of the time for cisplatin patients and 35% of the time for anthracycline patients. this included increased cardiac and audiological monitoring, the addition of a protective agent, or choosing an alternative treatment protocol if the risk outweighed the benefits of remaining on the current treatment plan. in interviews, patients indicated that they felt more involved in decision making, and felt reassured by understanding their genetic risk of toxicities. genetic risk prediction models for anthracycline cardiotoxicity and cisplatin ototoxicity were highly utilized by patients and oncologists in decision-making. results were found to be an important tool for informing patients of the risk of adrs during cancer treatment, and resulted in patients and their families feeling more involved in decision-making. background: childhood cancer survivors are at increased risk of developing executive dysfunction, and low socioe-conomic status (ses) has been identified as one of the mediators of executive functioning. previous studies have used traditional measures of ses, such as parents' education level, family annual income and occupation. but more recently, area based socioeconomic measures like block group poverty status are deemed to be more useful in monitoring of social inequalities in health in the united states. block groups are statistical divisions of census tracts and generally contain between 600 and 3,000 people. the current study aims to understand the association of block group poverty status (percentage of households in family's block group of residence living below the federal poverty level) with executive functioning among cancer survivor children. design/method: we used a retrospective cohort of 67 childhood cancer survivors. relevant information was collected from the medical record, administrative data sets and parent-filled surveys. address information was geocoded using arcgis 10.2 to obtain data on the block group poverty status. a priori cut-points were set to represent block groups with families living below poverty level at 0%, 0.1% to 9.9%, and ≥10.0%. executive functioning were assessed through a parent-rated instrument, the behavior rating inventory of executive functions (brief). multiple linear regressions were used to determine the relationship between block group poverty status and the brief scores. results: data was examined from 67 families of childhood cancer survivors, ranging in age from 6 to 18 years. in this sample, 32.8% families reported an annual income <$60,000, 32.8% reported income between $60,000 and $100,000 while 34.3% reported annual income ≥$100,000. primary care giver of 85.1% of cancer survivors had more than more high school education, and 31.3%, 41.8% and 26.9%, of families were living in a block groups with 0%, 0.1-9.9% and ≥10% poor households respectively. block group poverty level was not significantly associated with annual income levels (spearman's rho = 0.14, p = 0.25), or parental education level (spearman's rho = -0.02, p = 0.84). in a step-wise multiple linear regression, there was no statistically significant association seen between block group poverty status and executive functioning after adjusting for co-variables in the final model. future prospective study with a bigger sample size, longer follow up period and more robust measures of the executive functioning like a clinician administered test are needed to understand the effect of block group poverty status on executive functioning. to d100 completion was 45.5 days (range 21-63). all parents strongly agreed/agreed that d100 was helpful and would recommend d100 participation to another family. ten parents (100%) reported time spent on d100 was "just right." no parent felt more worried due to the intervention, though 1 parent found d100 participation stressful. this interim analysis suggests that parents have a favorable d100 experience and recommend the intervention. to date, <20% of enrolled parents fail to participate. d100 shows promise as an acceptable interdisciplinary communication intervention targeted to the early treatment period for childhood cancer. children 's hospital and research center oakland, oakland, california, united states background: screening echocardiograms are recommended by children's oncology group (cog) guidelines to assess for anthracycline-induced left ventricular (lv) systolic dysfunction. the yield of screening echocardiograms during chemotherapy and in the immediate post-therapy period is uncertain. objectives: to assess the incidence of lv dysfunction detected by screening echocardiograms during chemotherapy and in the immediate post-therapy period, defined as 0-24 months off-therapy. design/method: children diagnosed with cancer between january 2013-march 2016 who received anthracycline chemotherapy were identified. echocardiograms were performed as per protocol, institutional and cog guidelines, and were reviewed retrospectively. lv dysfunction was defined as fractional shortening (fs) <28% or ejection fraction (ef) <55% (1) results: in this cohort (n = 195, median age 6 years), the most common diagnosis was all (54.8%), followed by aml (9.7%). of 357 echocardiograms, 224 (62.7%) were performed during treatment and 133 in the immediate posttreatment period. thirty-eight (19.3%) patients had a >10% decrease in fs compared to their pre-treatment echocardiograms. none of these patients required any treatment modification or cardiac medications. only 1 patient (0.5%) had echocardiogram-proven lv dysfunction discovered on a screening echocardiogram during her treatment course. she eventually died due to multi-organ failure following septic shock. this patient was receiving treatment for aml and had received 300 mg/m2 of doxorubicin-equivalent anthracyclines at the time of the abnormal echocardiogram. one patient with metastatic ewing sarcoma had borderline lv dysfunction with a fs of 30% detected a month before completion of therapy. she had received 375mg/m2 of doxorubicin equivalent anthracyclines at the time of the abnormal echocardiogram. she did not require any therapy modification or additional cardiac medications. serial echocardiograms done on this patient have shown stable ventricular function. no off-therapy screening echocardiograms identified lv dysfunction. in our experience, the yield of echocardiograms to detect anthracycline-related cardiac dysfunction during treatment and in the immediate post-therapy period is very low. one patient developed lv dysfunction during treatment and one had borderline fs, while no lv dysfunction was identified within 24 months of completing chemotherapy. though fs decreased in 19% of patients, none required intervention. further study is needed to optimize the use of echocardiography screening in children treated with anthracyclines. references: 1. landier w et al. jco 2012. background: platinum-based chemotherapy increases the risk of sensorineural hearing loss in children with cancer. little is known about the impact of hearing loss on cognitive and emotional functioning in survivors. to determine the association of severe/profound hearing loss after platinum-based chemotherapy with 1) cognitive impairment and 2) emotional distress (i.e. anxiety and/or depression). cross-sectional study of all patients attending yale's childhood cancer survivorship clinic ≥2 years off therapy for cancer diagnosed at <21 years and treated with cisplatin and/or carboplatin, but with no history of cns tumor, cranial radiation, congenital hearing loss, or developmental delay. hearing loss severity and hearing aid data were abstracted from audiograms and detailed clinical history. cognitive impairment was defined as behavior rating inventory of executive function t score ≥65, assessment by neuropsychologist, and/or history of special education. emotional distress was determined by brief symptom inventory t score ≥63 (global or two subscales) or behavioral and emotional screening system t score ≥61, psychologist interview, and/or history of psychotropic medication/psychotherapy. the most recent available patient data were used. logistic regression with sas software, version 9.4 was performed. results: overall, 37 patients (57% female, 78% white) met eligibility criteria with a median age of 9.0 years (iqr = 12.1) at diagnosis and 22.3 years at evaluation (iqr = 10.4) after a diagnosis of sarcoma (36%), neuroblastoma (32%), or other (32%) for which 84% received cisplatin and 30% received carboplatin. fifteen patients (41%) had severe/profound hearing loss in at least one ear. patients with severe/profound hearing loss had a significantly increased risk of cognitive impairment (or = 5.14; 95% ci = 1.17-22.69), but not emotional distress, compared to patients without severe/profound hearing loss. there was no significant association between age at diagnosis, current age, time since diagnosis, sex, race, ethnicity, or diagnosis with either cognitive impairment or emotional distress. similarly, there was no significant interaction between 1) age at diagnosis and hearing loss or 2) sex and hearing loss with either cognitive impairment or emotional distress. ten of the 15 (67%) patients with severe/profound hearing loss in at least one ear were recommended hearing aids, of which 3 (30%) reported compliance most of the time. we conclude that severe/profound hearing loss is significantly associated with cognitive impairment, but not emotional distress, in childhood cancer survivors. our data supports the need for interventions to improve hearing in these patients, including compliance with hearing aids. background: who grade 3 anaplastic astrocytoma is a high grade glioma dependent on vascular endothelial s199 of s301 growth factor (vegf) mediated angiogenesis for its growth and infiltration. bevacizumab is a recombinant humanized monoclonal antibody which binds vegf-a and inhibits angiogenesis. common adverse effects of bevacizumab are hypertension, proteinuria, thrombosis and bleeding. while animal model based studies have shown that bevacizumab may impair ovarian function the effects of bevacizumab therapy on human fertility are not clear. since the physiology of pregnancy involves neovascularization/angiogenesis it is recommended that conception be avoided for at least 6 months following exposure to bevacizumab. to describe the course of a young adult who became pregnant after receiving bevacizumab and radiation therapy for treatment of an anaplastic astrocytoma. a 20 year old woman diagnosed with a localized hemispheric who 3 anaplastic astrocytoma was treated with chemotherapy and radiation (temozolomide/59.4 gy) followed by 12 cycles of bi-weekly bevacizumab/temozolomide. patient opted not to pursue fertility preservation prior to initiation treatment. she experienced bevacizumab-associated proteinuria and hypertension during treatment but received all protocol mandated doses (cumulative doses: bevacizumab = 240 mg/kg; temozolomide = 15.78 gm/m2). she had a spontaneous unassisted pregnancy 18 months after completing treatment. her pregnancy was uneventful and she was normotensive throughout. fetal ultrasonography at 16, 20, 27, 33 weeks revealed no abnormality of the brain, heart, great vessels, kidney, extremities, placenta and umbilical cord. at 39 weeks she delivered a female infant via cesarean section (birth weight: 3890 grams, apgars: 95 and 1010) excessive post-partum hemorrhage was not reported. placenta was bi-lobed and weighed 604 g. histological analysis revealed normal placental villous development and maturation and two small infarcts. conclusion: exposure to bevacizumab in our patient had no detrimental effect on fertility and on placental/fetal vascular development. we hope this report will add to the existing data on the effects of bevacizumab therapy on fertility. children's healthcare of atlanta, emory university school of medicine, atlanta, georgia, united states background: reports of malnutrition incidence and prevalence in young cancer patients are variable and not well established. previous research suggests children, especially less than 3 years old, treated with intensive cancer-directed therapy are at higher risk for malnutrition. however, no standardized assessment has been used to evaluate risk in this population. objectives: we aim to assess the trends of weight-for-age for patients following cancer diagnosis. this study will be the first to use a standardized measure of treatment intensity (intensity treatment rating scale, itr-2) and will assist in targeting interventions for identification and treatment of malnutrition. design/method: this observational, retrospective study obtained data through the center's pediatric cancer registry and electronic medical record. patients were classified by tumor type (brain or non-brain tumor) and treatment intensity (itr-2). itr-2 incorporates diagnosis, chemotherapy, radiation, and surgery, beginning with lowest intensity (1) to highest intensity (4). inclusion criteria included new cancer diagnosis 2007-2015 at less than 3 years old, with weight obtained and available within 2 days of therapy start date. incomplete data, alternate growth charts, or treatment intensity of 1, were excluded. weight was obtained at start of therapy and through 2 years after treatment initiation (approximately 750 days) and converted to z-scores adjusted for age and sex. weight trajectories were modeled using generalized linear mixed models with subject-specific random intercepts and spline functions. separate functions were constructed for subgroups of interest (tumor type and itr). results: there were 402 patients included: 53 patients with brain tumors (13.2%) and 349 with non-brain tumors (86.8%). of included patients, 165 had treatment intensity of 2 (41.0%), 192 of 3 (47.8%) and 45 of 4 (11.2%). over the observation period, 34,593 valid weights were recorded. at initiation of treatment, no difference existed between z-score by tumor type (p = 0.880) or by intensity (2 vs. 3, p = 0.879; 2 vs. 4, p = 0.665; 3 vs. 4, p = 0.558). tumor type did not affect z-score through the follow up period. z-scores were higher for intensity rating 2 vs. 3 and 2 vs. 4 (p = <0.001 and p = 0.015 respectively) at 240 days after the start of treatment and persisted through 720 days (p = 0.003 and p<0.001 respectively). higher treatment intensity is associated with decline in z-score and failure to return to baseline. future directions include further analysis on specific risk factors and timing of weight loss, longer-term follow-up of weight trends, and targeted interventions for identification, prevention, and treatment of malnutrition. objectives: asses the pt requirements for bleeding episodes in a prospective cohort of pcp using a <10 × 10e9 threshold compared to a <20 × 10e9/l threshold in a historical cohort. we collected pt data in all pcps treated at our center between january/2013 through december/2017. diagnosis, prescription for pt (prophylaxis vs bleeding disorder), plt count and transfused units were assessed for each pt. pcps treated from january/2013 through june 2015 received prophylactic pt with a <20 × 10e9 threshold (cohort a), and pts treated from july/2015 through december/2017 received prophylactic pt with a <10 × 10e9 threshold. pts done for procedures and pts with concomitant hemorrhagic pathology were excluded. we compared the number of pts prescribed as prophylaxis vs bleeding episode between cohorts. data analyzed: graphpad prims 6.0®. statistical analysis: percentages with confidence interval (ci); t-student test (parametric variables) and mann-whitney test (nonparametric variables). statistical significance: p<0.05. we reviewed 2093 pts (871 in cohort a, 1222 cohort b) in 209 patients. 62% had acute leukemia, 33% received and auto or allo hsct. diagnoses and the proportion of patients undergoing hsct was comparable in both cohorts. the average number of pts per patient was 8,54 in cohort a and 8,24 in cohort b (p = ns), but a significant difference was found when hsct patients were excluded from this comparison (7,34 pt per patient in cohort a vs 6,07 in cohort b, p = 0,005), which resulted in an estimated 16,4% reduction in pts prescription. furthermore 61 (7,1%) pts were prescribed for bleeding episodes in cohort a versus 99 (8,2%) in cohort b (p = ns). patients receiving hsct in the entire group ver-sus those not receiving hsct had similar pt requirements for bleeding episodes (10% vs 8,5% p = ns) conclusion: a <10 × 10e9 plt count threshold for prophylactic pts is safe in pcp in chemotherapy and hsct. it can result in a significant reduction in pt usage. key words: platelets, transfusions, prophylaxis, cancer, childhood. ucsf benioff children's hospital oakland, oakland, california, united states background: transition of care for adolescent and young adult (aya) survivors of childhood cancer from pediatric to adult-oriented long-term follow-up (ltfu) is complex. loss to follow-up is common, and little is known about the success rates among different models. the survivors of childhood cancer program (sccp) at ucsf benioff children's hospital oakland employs a community-based model for transitional care. our multidisciplinary team provides aya survivors a comprehensive treatment summary and recommendations, then facilitates transition to primary care or adult oncology ltfu programs. evaluate the success rate for transition of care among aya survivors of childhood cancer in our ltfu program, and identify barriers to successful transition. design/method: aya patients seen from november 2010 to august 2017 in the sccp with intent to transition were asked by email or telephone if they had followed up with their designated provider. the primary outcome was successful transition, defined as establishing care within 18 months of their visit. patients were also asked about barriers to transition and to rate the new provider's familiarity with their cancer history and ltfu needs. results: transition was intended for 88 patients. eightyseven were contacted and 43 responded. of these, 29 (67%) successfully transitioned, while 14 (33%) were lost to followup. ages ranged from 19 to 48 years, at 2 to 32 years since completion of therapy. ten (34%) transitioned to a primary care provider, 20 (69%) to an adult oncology ltfu program, and 1 (3%) to a pediatrician. patients rated their new provider's knowledge above average (3.76) on a 5-point scale from poor (1) to excellent (5). survivors lost to follow up indicated the following barriers to transition: loss/change of insurance (3), inability to find a provider (1), too busy/forgot (4), problems with transportation (1), concerns about cost/copay (2), and s201 of s301 other (4). twelve patients requested further assistance with transition. conclusion: two-thirds of responding patients successfully transitioned. more work is needed to overcome various barriers to transition for one third of aya survivors. albany medical center, albany, new york, united states background: the transition from active treatment, to offtherapy follow-up, is a stressful event for parents of children with cancer. the psychosocial needs of parents after therapy have received limited attention in the united states with only 3 published quantitative studies, the largest with 35 parents. we have secured funding for and recruited a transition care coordinator (tcc) to investigate this further. objectives: our objective is to assess and screen parents at the end of their child's treatment, and to develop interventions to support parents during this time and thereafter. design/method: after informed consent, a standardized questionnaire, the psychosocial assessment tool (pat 2.0), was administered to parents at end of therapy (t1), 6 months later (t2) and 1 year later (t3). the tcc provided "universal" intervention to all families with an end of therapy binder containing a treatment summary, follow-up roadmaps, information on late effects, and survivor scholarships. based on their pat2.0 scores, some parents were provided intervention specific to symptoms (targeted intervention for scores 1-1.99) or referred to a behavioral health specialist through the clinic social worker for counseling (for scores >2). results: analysis of pat1 data showed that 45% of parents (n = 45) scored in the targeted or clinical ranges; 19% of parents scored in those ranges at pat2. significant gender differences were revealed with the mean score for men of 0.7 and for women of 1.13. this was confirmed by showing statistical significance (p = 0.017) when analysis was conducted for only a subgroup of data composed of couples (n = 24). analysis of pat2 data by couples (n = 10) showed the mean score for men was 0.64 and for women was 0.93 (p = 0.12). gender differences were most apparent in caregiver stress reaction questions that focused on ptsd symptoms. when the subgroup of couples' scores (n = 24) for caregiver stress reaction at pat1 was analyzed, there was a significant difference (p = 0.005) in caregiver stress reaction with a mean of 0.08 for men versus 0.3 for women. [note: subcategory scores range from 0 to 1]. this study was initiated in october 2013 using a tcc and the pat2.0 screening tool. the results suggest greater stress on mothers after therapy, with a substantial proportion of parents having symptoms of ptsd after therapy. background: hodgkin lymphoma (hl) is a common childhood cancer characterized by an inflammatory microenvironment. chemotherapy and radiation may exacerbate this inflammation and contribute to the development of late effects (pneumonitis or pulmonary fibrosis). in a heterogeneous cohort of childhood cancer survivors exposed to pulmonarytoxic therapy, no association between pro-inflammatory cytokines and late pulmonary dysfunction was observed. our objective was to test this association in a relatively uniform cohort of survivors of hl, given the well-recognized proinflammatory background of this disease. objectives: to characterize off-therapy pulmonary function in survivors of hl treated with contemporary therapy, and to investigate its association with persistent systemic inflammation. design/method: blood samples, clinical data, and pulmonary function tests were obtained from survivors of hl ≥6 months off therapy. lung function score (lfs), a validated method for assessing degree of pulmonary dysfunction on a scale of i to iv, was determined from diffusion capacity and forced expiratory volume in one second (fev1). for a control group, blood samples from patients with benign, noninflammatory hematologic conditions were used. plasma concentrations of 50 inflammatory cytokines were measured on a luminex platform (emd millipore). associations between clinical features or cytokine levels and lfs i (normal) vs. ii-iv were evaluated using logistic regression or wilcoxon rank sum tests, respectively. results: of 77 survivors (mean age at diagnosis: 14 years, range: 3-18; mean time off therapy: 3.3 years, range: 0.5-24), 70% were categorized as lfs ii (mild dysfunction), 8% as lfs iii (moderate dysfunction), and no survivors as lfs iv (severe dysfunction). higher lfs was associated with female sex (p = 0.01) but not other demographic, disease, or treatment factors. forty-eight survivors had blood samples collected at a mean age of 18.5 years (range: 10-32) with a mean time since treatment completion of 3.8 years (range: 0.6-6.1). of 31 controls, the mean age at time of blood collection was 12 years (range: 4-17). survivors did not have significantly elevated cytokine levels compared to controls. female survivors of hl ≥6 months off therapy are at increased risk of pulmonary dysfunction. neither evidence for pulmonary dysfunction, as measured by lfs, nor duration of time off therapy were related to systemic inflammation in this study. pulmonary function deterioration and clinical pulmonary symptoms are rarely observed immediately following therapy but increase over time. future studies may consider exploring the contribution of systemic inflammation to pulmonary late effects in survivors farther off therapy, when risk for this late effect is greater. background: thyroid carcinoma is a very rare tumor in pediatrics, accounting for 1.5-3% of childhood carcinomas in the united states and europe. we aim to detect the risk of second malignancies among pediatric thyroid cancer survivors. the cohort analysis consisted of pediatric cancer patients aged less than 20 years diagnosed with a primary thyroid cancer and identified by site code icd-0-3: c739, reported to a seer 9 database between 1973 and 2013. they were followed up by death or the end of the study period (december 31, 2013) . out of 1769 patients diagnosed primarily with thyroid carcinoma, there were 42 patients who had 45 incidences of subsequent malignancies. the mean age of patients at initial diagnosis of thyroid cancer was 16 years. females (90.5%) had significantly higher incidence of second malignancies (sm) than males (9.5%). the overall standardized incidence ratio (sir) of sm in thyroid pediatric patients was higher than expected (sir = 1.48). some specific sites showed significantly higher incidences: salivary gland (sir = 33.95), gum and other mouth (sir = 24.53) and kidney (sir = 5.72). the overall risk of sm in patients received radioactive iodine was higher than expected (sir = 4.41). the cumulative inci-dence of sms from the initial diagnosis of thyroid cancer was calculated with the survival methodology of competing risk, death treated as a competing event. cumulative incidence of sm was 2.7% [95 % ci (1.62, 3.83 %)] at 25 years and substantially expanded after 15 years, reaching 11.92% [95 % ci (4.9, 18.8%)] at 40 years. the cumulative incidence of each tumor type at 40 years was 0.452% [95 % ci (0.139, 0.765 %)] for breast cancer, 0.28% [95 % ci (0.034, 0.53 %)] for salivary gland, 0.22% [95 % ci (0.00034, 0.448 %)] for each one of kidney and cervix uteri and 0.169% [95 % ci (0, 0.361 %)] for each one of ovary and melanoma of the skin. cumulative incidence of sm was stratified based on race, gender and radiotherapy exposure, but there was no statistical difference in each of them. conclusion: race, gender, histological subtypes, and radioactive iodine may play an important role as prognostic factors for developing sm among pediatric thyroid cancer survivors. identification of underlying mechanisms that raise the risk of sm is important for both treatment and follow-up strategy. background: the ethical practice of informed consent requires it be both voluntary and understood by the research participant. in pediatric oncology, parents must undergo informed consent to enroll their child with cancer into clinical trials, but often it can be difficult to understand especially for parents with low english proficiency. previous research has shown that parents of children with cancer have difficulty understanding voluntariness, and that parental satisfaction with informed consent does not always correlate with adequate comprehension. objectives: to examine socio-demographic and contextual correlates of comprehension of informed consent, voluntariness, and satisfaction in parents who consented to participation of their child in a cancer clinical trial. we focused on characterizing differences between non-hispanics and hispanics, the fastest growing ethnic group in the u.s. design/method: parents/guardians (n = 121) of children aged 0-17 years with newly diagnosed cancer, who had consented to participation of their child in a clinical trial for cancer treatment at rady children's hospital-san diego were s203 of s301 prospectively recruited. parents completed questionnaires assessing comprehension, voluntariness, satisfaction, health literacy, socio-demographics, and acculturation level, if hispanic. comprehension was surveyed at baseline and longitudinally at 3 months. comprehension, voluntariness and satisfaction outcomes were analyzed by socio-demographics, health literacy, and acculturation level using logistic regression. results: of the 121 participants surveyed, 61 (50.4%) were hispanic and 60 (49.6%) were non-hispanic. we found that higher health literacy was associated with greater objective comprehension (p<0.001), voluntariness (p<0.001), socioeconomic status (p<0.001), and acculturation (p<0.001). hispanics reported lower objective comprehension (p = 0.025), voluntariness (p = 0.029), health literacy (p<0.001) and ses (p = 0.015) compared to non-hispanics. spanish-speakers reported lower voluntariness (p = 0.016), health literacy (p<0.001), and acculturation (p<0.001) compared to englishspeakers. at the 3-month follow-up, comprehension in hispanics significantly improved (p = 0.012) compared to their baseline comprehension. satisfaction was moderately high across all subgroups and was not significantly impacted by socio-demographics, health literacy, or acculturation. in this study, with equivalent numbers of hispanic and non-hispanic participants, we found that hispanic and spanish-speaking parents of children with newly diagnosed cancer had inadequate informed consent comprehension, voluntariness and health literacy despite high satisfaction. our study suggests that hispanics and individuals with limited english proficiency are not making truly informed decisions for their child with cancer. to ensure the ethical practice of research in pediatric oncology, the informed consent and decision-making process must be improved with culturally and linguistically interventions for these underserved populations. memorial sloan kettering cancer center, new york, new york, united states background: pediatric oncology patients undergo repeated bone marrow aspirations and biopsies (bma/bx). these potentially painful procedures can exacerbate anxiety and distress. standard practice at memorial sloan kettering (msk) department of pediatrics is to use propofol, which has amnestic but no analgesic properties. we sought to evaluate whether the addition of local anesthetic would improve patient experience with bma/bx. the purpose of reppair: reducing procedural pain and improving recovery of quality of life (qol) (nct02924324) is to evaluate the efficacy of local anesthesia with ropivacaine in reducing procedural pain and improving post-procedure qol in pediatric neuroblastoma patients undergoing bma/bx with general anesthesia. reppair is a prospective, randomized, crossover clinical trial that opened for enrollment october 2016. eligible patients were 3 -18 years old with neuroblastoma. participants were observed on trial for two sequential bm procedures; one procedure with intervention a: propofol alone (pa), and the other with intervention b: propofol plus ropivacaine (p+r). participants were randomized to intervention sequence ab or ba and were blinded to the order of interventions. participants and recovery room (rr) nurses, who were also blinded, followed a standardized postprocedure pain management algorithm. the primary endpoint was percentage of participants requiring opioid analgesia in the 24 hours post-procedure. secondary endpoints included total opioid in 24 hours, non-opioid analgesia use, pain scores, time to first opioid, and short-term qol. qol was assessed by a parent-proxy metric that evaluated pain interference with sleep, physical, emotional, and social recovery. as of january 2018, 105 patients were assessed for eligibility and 56 patients were randomized (47 have completed both procedures). for the primary endpoint, a slightly higher proportion of participants required opioid for pa than p+r (24% versus 21%, p = 0.6). pain scores in the rr were significantly higher for pa than p+r (median [25th, 75th percentile]: 2 [0,4] versus 0 [0,2], p = 0.004). there were no statistically significant differences in total opioid or non-opioid analgesia, 6-and 24-hour pain scores, median time to first opioid, or pain interference scores. there were no adverse events. conclusion: preliminary findings of the reppair trial suggest that local anesthesia does not reduce the need for opioid analgesia or improve short-term qol in pediatric patients undergoing bma/bx with general anesthesia. local anesthesia did improve pain scores in the immediate recovery period. final results of this study will help establish evidence-based guidelines and optimize the experience of pediatric patients with bone marrow procedures at our center. background: children with advanced cancer experience a range of symptoms throughout treatment or at end of life, some of which are poorly controlled. minimizing suffering, including effective symptom management, in children with advanced cancer is a central value for pediatric oncology clinicians. patient-reported outcomes have been used in symptomrelated research in pediatric oncology patients; however the majority of literature specific to symptoms during palliative care and end of life for children and adolescents with advanced cancer is based primarily upon medical record reviews and to a lesser extent, patient self-report. the purpose of this study was to prospectively describe symptom frequency, severity, and level of distress in children/adolescents with advanced cancer using patient selfreport and parent proxy. design/method: a prospective cohort design was used for this study. five pediatric oncology institutions from across the united states participated. children and adolescents were eligible to participate if they were 7-18 years of age, englishspeaking, and had a diagnosis of advanced cancer, defined as a 2-week history of progressive, recurrent, or non-responsive disease or a decision not to pursue curative-focused therapy. a modified version of the memorial symptom assessment scale (msas) was used to measure symptom frequency, severity, and level of distress and was administered to child/parent dyads electronically via smartphones every two weeks. information regarding disease status and cancer treatment was collected concurrently. data was analyzed using descriptive statistics and univariate logistic regression analysis. results: a total of 47 children and adolescents and 47 parents participated in the study. the median age of child participants was 13 years, with half being male. the median age of parents was 46 years. the child participants had a variety of primary diagnosis, including: leukemia/lymphoma (n = 11, 23%), solid tumor (n = 21, 45%), and brain tumor (n = 15, 32%). the most frequently reported symptoms by children with advanced cancer and parents were pain (n = 195/562, 34.70%), lack of energy (n = 186/561, 33.16%), and nausea (n = 156/560, 27.86%). presence of disease (p = <0.0001), recent disease progression (p = 0.0002), and receiving cancer therapy (p = 0.0004) were significant factors on the presence of pain. high intensity cancer therapy was a significant factor on pain frequency (p = 0.0445) and level of distress (p = 0.0224). it is feasible to collect data prospectively in children with advanced cancer regarding symptom frequency, severity, and level distress. clinicians' increased understanding of the symptom experience may promote communication with children and adolescents and timely intervention. more research is needed to understand symptom clusters in children with advanced cancer. vanderbilt children's hospital, nashville, tennessee, united states background: febrile neutropenia (fn) is a frequent occurrence in children undergoing chemotherapy. though guidelines recommend adding a second antibiotic to broad-spectrum antipseudomonal coverage in specific scenarios, augmenting empiric therapy with a second antibiotic is common practice. additional empiric antibiotic (aea) use increases the risk of antibiotic toxicity and future antimicrobial resistance. data clarifying the indications for aea are limited in pediatric patients. objectives: to identify risk factors for gram-positive (gp) and gram-negative (gn) bacteremia in patients presenting with fn to determine situations in which aea use is warranted. design/method: a retrospective chart review was conducted of pediatric severe fn with absolute neutrophil count <500/ l occurring at a single institution between 2006 and 2013. potential a priori risk factors based on clinical reasons for antibiotic expansion were chills, hypotension, mucositis, skin or soft tissue infections (sstis), recent administration of highdose cytarabine (hdac), and a diagnosis of acute myeloid leukemia (aml). potential factors for gn bacteremia were chills, hypotension, mucositis, and abdominal pain. the association between each potential risk factor and gp or gn s205 of s301 bacteremia was identified. logistic regression was used for multi-variable analysis. the review yielded 701 episodes. gp bacteremia was isolated in 75 cases (10.7%) and gn bacteremia in 37 episodes (5.3%). in multivariable analysis, hypotension (or 3.5 (95% ci 1.7, 7.2), p = 0.001) and sstis (or 3.1 (1.1, 8.7) , p = 0.036) were independently associated with increased risk of gp bacteremia, while mucositis (p = 0.376), recent administration of hdac (p = 0.34) and chills (p = 0.161) were not. ten patients with aml didn't receive hdac, thus the association between aml and gp bacteremia could not be reliably estimated. hypotension (or 4.9 (2.2, 11.0), p<0.001) and chills (or 5.0 (2.5, 10.1), p<0.001) were independently associated with a higher risk of gn bacteremia, while mucositis (p = 0.196) and abdominal pain (p = 0.509) were not. of the 37 gn infections, 6 (16%) were resistant to cefepime, the empiric agent of choice at our institution. patients with fn with sstis, hypotension, or recent hdac had increased risk of gp bacteremia indicating potential benefit of empiric vancomycin in these settings, while mucositis and chills were not associated with gp bacteremia. hypotension and chills were associated with gn bacteremia, potentially warranting empiric antibiotic expansion, while mucositis and abdominal pain were not. identifying specific indications for aea use in pediatric severe fn use may improve antimicrobial utilization, decrease unnecessary antibiotic use, and improve patient outcomes. background: for children/young adults with incurable high grade gliomas (hggs), like diffuse intrinsic pontine glioma (dipg) or glioblastoma multiforme (gbm), oncologists endeavor to align therapy with patient/family goals of care, but may be influenced by providers' preferences or limited resources. ethical challenges can arise around the perceived purpose, risks and benefits of therapy options, provider conflicts of interest, access to care, deciding decisional priority between patients and families, and conflicts around end-oflife care. objectives: evaluate factors that play into longitudinal decision making for children and young adults with hggs, their families and oncologists using a qualitative approach with ethnographic elements. design/method: eligible patients were aged 0-21 with dipg, gbm, or secondary hgg. patient exclusions included: non-english speaking, in state custody, death prior to diagnosis, seen by oncology once, or an oncologist declined participation. key decision making visits (e.g. mri reviews) were serially audio-recorded, along with subsequent 1:1 semistructured interviews with patients and/or parents about the decision making process. field notes from clinician meetings, chart notes, and oncologist questionnaires were obtained. discussions and interviews were transcribed and independently coded by three investigators. inter-rater reliability was assessed during code book development. discrepancies were discussed until consensus met. constant comparison analysis with maxqda software continued until thematic saturation. results: twenty-two of 34 eligible patients were approached; 15 agreed to participate. one withdrew upon transferring care. mean age was 9.9 years (sd 5.9); 71% male, 50% caucasian, 29% african american, 14% hispanic, and 7% asian. four encounters, (2.5 hours), were recorded on average per patient. parent/patient interview themes included: 1) hope (for a cure, prolonged life, and quality of life), 2) importance of physician recommendations, 3) importance of support systems (family, community, social media), 4) food (as cancer etiology, intervention) 5) finances (personal, research funding), 6) communication (with medical providers, family, community), 7) death, and 8) god (beliefs, prayer, existential questions). oncologists desired prolonged quality of life, while patients/families transitioned to that hope from hope for a cure. decisions made in the setting of hggs are multi-factorial, ultimately reflecting the competing values of decision makers. optimism about treatment efficacy is held in tension with poor prognosis, allowing for functional hope. acknowledging patients' and families' shifting hopes allows for changes in goals of care and shared decision making. future work is needed to 1) develop preference tools for pediatric patients and families to inform medical providers and 2) provide training in communication and shared decision making with oncologists. emory university, atlanta, georgia, united states background: bone marrow transplantation (bmt) is a potentially curative but underutilized treatment for scd. our previous work has shown that there is variation in physician philosophy and practice in considering bmt as a treatment option for patients with scd, and physicians may not discuss this with patients and families as a potential treatment option. in a randomized clinical trial to test the effectiveness of a decision aid for disease modifying therapies for sickle cell disease, adult patients with scd as well as caregivers of adult/pediatric patients were interviewed about how they seek or have sought information related to scd, made decisions about treatments for scd, and identified a treatment option they were interested in learning more about using the decision aid tool. we performed a secondary analysis of these baseline data to understand patient information needs and attitudes regarding bmt as a treatment option for scd. the goals of this analyses was to understand patient and caregivers' attitudes and perceived information needs regarding bmt as a treatment option for scd. we performed an analysis of baseline interviews from caregivers of patients with scd or adult patients from a randomized control trial for a decision aid tool for scd. 13 of the 38 interviews belonged to caregivers of patients with scd. in addition to reviewing interviews for discussion of bmt, we interrogated for mention of terms such as 'bone marrow transplant' or 'cure' or 'stem cell transplant'. interviews were coded using nvivo10 and analyzed for emerging themes. results: of the 98 baseline interviews, 38 interviews met selection criteria. thirteen of the 38 interviews were with caregivers of pediatric patients, and the remainder were with adult patients, including young adult patients with scd. the majority of participants want to learn about bmt or curative options. in many participants, this was expressed despite knowledge that they were not a likely candidate for transplant. desired information about bmt included eligibility, benefits, risks, long-term effects, quality of life and financial aspects related to bmt. of the patients who discussed how they learnt about bmt, approximately half mentioned that their healthcare provider had not previously mentioned this to them. we then examined knowledge of bmt and attitudes with demographic and clinical variables. patients and caregivers of pediatric patients with scd want to learn about bmt as a treatment option. healthcare providers should consider discussing bmt with their patients with scd. natasha frederick, anna revette, alexis michaud, jennifer mack, sharon bober dana-farber cancer institute, boston, massachusetts, united states background: adolescents and young adults (ayas) consistently identify the need for improved patient-clinician communication on sexual and reproductive health (srh) issues. however, oncology clinicians do not routinely integrate srh conversations with ayas through disease treatment and survivorship. little is known about why these conversations do not take place. objectives: explore aya perceptions of and receptiveness to srh communication with oncology clinicians and to identify barriers and facilitators to these conversations. design/method: semi-structured interviews were held with 21 aya cancer patients and survivors (ages 15-25 years, 6 men, 15 women). twelve participants were on active treatment and 9 were within 2 years of treatment completion. interviews were conducted in english by phone or in person. the interview transcript underwent pre-testing with ayas. all interviews were audio-recorded and transcribed verbatim. transcripts were analyzed and summarized by two trained qualitative researchers according to standard comprehensive thematic qualitative analysis methods. analyses were aided by nvivo11 software. results: ayas perceived existing srh communication between ayas and oncology providers as inadequate. all ayas reported a need for improved srh communication with oncology providers, and three key areas of need emerged: 1) general education; 2) addressing specific srh issues experienced during treatment and survivorship; and 3) understanding the long-term impact of cancer and treatment on srh. ayas felt that current srh discussions are limited and too narrow in scope and scale. ayas reported that most srh conversations focus exclusively on fertility (n = 17), usually taking place at the start of treatment. other additional yet limited communication reported was about sexual activity (n = 7), contraception (n = 7), sexual function (n = 1). no ayas reported conversations about potential treatment complications related to sexuality other than infertility. key barriers to srh conversations include patient discomfort initiating conversation (n = 14) and presence of family members (n = 10), with additional reported barriers including perceived provider discomfort (n = 4), lack of rapport with provider (n = 4), and age/gender differences (n = 4). ayas felt that s207 of s301 communication tools such as handouts, brochures, and websites would be helpful facilitators to direct communication from the oncology clinician, and wanted conversations to start before treatment initiation and to continue through treatment and survivorship conclusion: ayas identify a key role for pediatric oncology providers in srh care from diagnosis through survivorship, however multiple barriers interfere with discussions about srh on a regular basis. identified barriers suggest that future efforts should focus on provider education and training in srh and srh-related communication in order to optimize care provided to this unique patient population. background: peripherally inserted central venous catheters (picc) provide secure vascular access in pediatric patients for the delivery of necessary therapies. the ease of placement in the inpatient and outpatient settings has expanded their utilization. however, recent data analyses show a significant increase in venous thromboembolism (vte) risk with the use of picc lines. with its rising use, modifiable risk factors need to be understood for preventative measures. objectives: in this study we aim to understand patient and catheter specific characteristics in relation to the development of vte. design/method: with irb approval, a retrospective interrogation of the electronic medical record and a picc database, at rainbow babies and children's hospital, was completed. the study cohort contained patients < 18 years of age who had a picc line placed between january of 2004 and december of 2016. data collected included indication for line placement, line dwell time, location of insertion including blood vessel and extremity, number of attempts at line placement, lumen size and indwelling line length. in addition, we collected number of days to vte formation, associated symptoms and location of vte. chi-squared analyses and fischer's exact test were used where appropriate for statistical analysis. we analyzed 3729(1098 neonatal) newly placed picc lines. fifty line-associated vte events were found, for an incidence of 1.3%. all vte occurred with the placement of the first picc line. intravenous therapies were the most common reason for line placement. no statistical significance was found between various indications for placement. the most common symptom of vte manifestation was extremity swelling, follow by extremity pain. right extremity picc was found to have a higher incidence of vte. larger catheter lumen sizes (> 4 french) had a higher incidence of vte. we found a mean time of 20.07 days to vte detection. we were unable to find any clinical, patient or line specific factors leading to increased vte formation after statistical analysis. special consideration should be given to the duration of picc line use as this may reduce the incidence and comorbities associated with vte. there is still much to be understood about catheter associated vte formation as our analyses indicates the need for prospective data collection on a larger scale in hopes to create guidelines related to catheter use in pediatrics. background: the decision to transfuse a patient is a complex one and is never based solely on a number; however, certain hemoglobin or platelet count thresholds have been proposed in aiding physicians make transfusion decisions. in our hospital, the thresholds for packed red blood cell (prbc) and platelet transfusion in pediatric oncology patients are hemoglobin levels below 8.0 g/dl and platelet counts below 20,000/mm3 (< 35,000 for brain tumors), respectively. recently, these thresholds have been questioned and we were asked whether we could safely lower the thresholds to < 7.0 g/dl of hemoglobin and < 10,000 /mm3 platelet count objectives: to investigate platelet and hemoglobin transfusion thresholds for oncology patients at children hospital of michigan design/method: retrospective chart review over a 6-month period, examining platelet and hemoglobin pretransfusion levels for each prbc and platelet transfusion given to oncology patients results: over the course of 6 months, 60 eligible oncology patients (median age 6 years) received 584 transfusions (233 prbc transfusions and 351 platelet transfusions). the mean pretransfusion hemoglobin level was 7.6 ± 1.0 g/dl (range 2.3-11.9) (n = 233) for total prbc transfusions and this was not different among disease categories (p = 0.146). patients who had anemia symptoms and signs (n = 190) had a slightly lower hemoglobin level compared to those who did not (n = 43): 7.5 ± 1.0 vs 7.8 ± 0.9 g/dl (p = 0.058). the mean pretransfusion platelet count was 24,570 ± 15,151 /mm3 (range 2,000 -104,000) for total platelet transfusions (n = 351); 32,800 ± 14,586 /mm3 in patients with brain tumors (n = 84); 20,610 ± 15,270 in patients with leukemia (n = 119); and 23,090 ± 13,628 in patients with solid tumors (n = 148). the mean pretransfusion platelet count was significantly higher in transfusions for brain tumors compared to that in the other disease groups (p<0.001 for both). the mean pretransfusion platelet count was not different among those patients who had bleeding/bruising symptoms (25,150 ± 17,898, n = 115) versus those who did not (24,290 ± 13,648, n = 236) (p = 0.620). the bleeding/bruising rate was slightly but insignificantly higher in those who had platelet counts <10,000 vs those who had ≥ 10,000 (38.2% vs 32.2%, p = 0.564). since most patients develop symptoms of anemia at hemoglobin above 7 g/dl and about 1/3 of patients develop bleeding/bruising symptoms at platelet counts above 20,000 /mm3, our current policy so far reflects a safe threshold for transfusion, and further lowering of the thresholds should be investigated in prospective studies. background: renal impairment is an important complication of childhood cancer and its treatment. serum creatinine level is frequently used as a screening test to monitor renal function; however, patients can have significantly decreased glomerular filtration rate (gfr) with normal serum creatinine. to determine the prevalence of chronic kidney disease (ckd) among children with cancer diagnosis, based on calculated gfr. to compare the difference between using serum creatinine value alone versus gfr in detecting ckd. design/method: retrospective review of medical records of 150 patients, age 1-18 years, diagnosed between 1/2011-12/2016 with solid tumors were analyzed. serum creatinine and calculated gfr using schwartz formula were recorded. ckd as classified by the foundation of kidney disease and outcome quality initiative was used: ckd stage 2: gfr (60 to 89 ml/min per 1.73 m2) ckd stage 3: gfr (30 to 59 ml/min per 1.73 m2) statistical analysis using spss software v.23. chi-squared test for proportions within group, and pearson chi-squared and fisher exact tests for statistical differences between groups. p-value <0.05 was considered to indicate significance results: out of the 150 records reviewed, 81 (54%) were males and 69 (46%) females, with mean age of 9.2±5.6 years. 37 (24.6%) patients received one or more of nephrotoxic chemotherapy drugs; cisplatinum, carboplatinum, or ifosphamide mainly in the non-wilms solid tumors group (94.5%) compared to (5.5%) in the wilms tumor (wt) group. based on calculated gfr (by schwartz formula) ckd stage 2/or 3 was diagnosed in 66 (44%) patients with overwhelming majority (98%) were in the mild stage 2 ckd, only 3 (4.5%) of those patients had abnormally high serum creatinine levels (p = 0.006). 56.7% of patients who received nephrotoxic chemotherapy developed ckd, compared to 39.4% in those who did not receive it, (p = 0.01). despite that only 2/18 (11%) of wt group patients received nephrotoxic chemotherapy, yet this group had higher percentage of ckd (83.3 %) compared to non-wt group (34.8%) p = 0.02. significantly lower mean gfr 73.8±10 was noticed in the wt group compared to 99.8±29 in non-wt group (p = 0.001) conclusion: high prevalence of mild ckd was found among solid tumor patients. using serum creatinine alone as measure of renal function significantly under estimates renal impairment in those patients. early identification of ckd is easily achieved by using calculated gfr, which can helps providers and care givers to avoid potential nephrotoxic antibiotics, contrast media, nsaids and dehydration that may further deteriorate renal function the university of texas southwestern medical center, dallas, texas, united states background: children with down syndrome (ds) have increased risk of developing leukemia. pediatric patients with ds-associated acute lymphoblastic leukemia (ds-all) are known to have significant toxicities with reinduction chemotherapy and historically poor outcomes with stem cell transplant (sct). anti-cd19 chimeric antigen receptor (car) t-cell therapy, tisagenlecleucel, demonstrated high rates of durable complete remission (cr) and a manageable safety profile in children with r/r b-cell acute lymphoblastic leukemia (b-all). objectives: characterize the efficacy and safety of tisagenlecleucel in pediatric/young adults with ds-all. design/method: pooled data from 2 single-arm, multicenter, phase 2 trials of tisagenlecleucel in pediatric/young-adult patients with r/r b-all (eliana, nct02435849; ensign, nct02228096) were analyzed. eight patients with ds-all were enrolled (data cutoff: eliana, 23 november 2016; ensign, 1 february 2016). seven were infused with tisagenlecleucel; 1 patient died from all progression and intracranial hemorrhage before infusion. no manufacturing issues occurred during production. 5/7 infused patients were male, 2/7 had prior sct (age range, 6-16 years). 6/7 patients achieved cr or cr with incomplete blood count recovery (cri) by day (d) 28 (cr+cri, 86%); 1 died before d28 and was not evaluable. analysis of minimal residual disease was negative in bone marrow in responding patients. two patients had cd19negative relapses at 5 and 8 months. ongoing remissions in 4 patients without relapse ranged from 2 to 11 months. the safety profile (n = 7) appears similar to that in patients without ds in the same trials (n = 90). grade (g) 3/4 cytokine release syndrome occurred in 43% (3/7) of patients with ds and in 44% without ds. rates of other g3/4 adverse events of special interest did not appear to favor a consistent trend between patients with/without ds (febrile neutropenia: 43% vs 36%; neurological events: 14% vs 11%; tumor lysis syndrome: 14% vs 2%). g3/4 infections were not observed in patients with ds (0% vs 23%). one patient died after infusion due to intracranial parenchymal hemorrhage on d15 associated with ongoing coagulopathy. time and extent of tisagenlecleucel expansion and long-term persistence were similar between groups. conclusion: this is the first analysis of car t-cell therapy in pediatric patients with r/r b-all and ds. these data suggest that toxicities appear similar to those in patients with b-all without ds, remission rates in ds-all are high, and longterm outcomes with sustained persistence appear promising. further exploration of tisagenlecleucel as an alternative to sct in children with r/r ds-all is warranted. sponsored by novartis. background: hispanic adolescence and young adults are twice as likely to develop acute lymphoblastic leukemia (all) with high risk features as non-hispanic whites. they also have poor prognosis and 39% higher death rate. b-all with crlf2 overexpression caused by genetic alteration of the cytokine receptor, crlf2 is five times more common in this subgroup. approximately 80% of crlf2 b-all cases also have ikzf1 genetic alterations. ikaros is involved in transcriptional regulation of several important genes involved in leukemogenesis. overexpressed casein kinase ii (ck2) impairs functions of ikaros. objectives: understand the molecular mechanisms that regulate crlf2 expression in crlf2 b-all. here we present evidence that ikaros-mediated repression of crlf2 transcription in b-all in hispanic children is regulated by ck2. design/method: primary b-all patient samples from hispanic children were used. ikaros retroviral transduction, ikaros shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: ikaros binding to promoter of crlf2 was confirmed using quantitative chip. functional experiments such as overexpression of ikaros in b-all primary cells results in transcriptional repression of crlf2 whereas ikaros silencing using shrna resulted in increased transcription. these results suggest that ikaros negatively regulates crlf2 expression. molecular inhibition of ck2 with shrna targeting the ck2 catalytic subunit, as well as pharmacological targeting of ck2 with cx4945 resulted in transcriptional repression of crlf2. ck2 inhibition was associated with increased ikaros dnabinding to the promoter of crlf2. however, the ability of cx4945 to repress crlf2 is lost or severely reduced, in cells with shrna silencing of ikaros, as compared to cells with intact ikaros. moreover, similar results were noted following treatment with cx4945 in leukemia cells obtained from high risk b-all patients with deletion of one ikzf1 allele. ikaros binds poorly to promoters of crlf2 gene in these cells. treatment with cx4945 restores ikaros dnabinding to the promoters of crlf2, which is associated with its strong repression. serial qchip analysis of the epigenetic signature at the crlf2 promoter showed that increased ikaros binding to the crlf2 promoter, following ck2 inhibition, is associated with enrichment for the h3k9me3 histone modification, which is a marker of repressive chromatin. results demonstrate that crlf2 expression is epigenetically regulated by the ck2-ikaros axis .cx4945 show antileukemic effect via restoration of ikaros tumor suppressor function, resulting in crlf2 repression suggesting advantage of using ck2 inhibitors as potential therapeutic approach in crlf2 altered b-all. results: hypodiploid all (modal chromosome number <44 and/or di <0.81) was identified in 131 patients (1.6% of all patients; 0.9% of nci standard risk (sr) and 2.9% of nci high risk (hr)), who were removed from frontline protocol therapy post-induction. overall 5-year efs and os were 52.1%±4.9% and 58.9%±4.8%. transplant status was retrospectively available for 113/131 (86%), 61 of whom underwent hsct in cr1. five-year efs with hsct was 57.4%±7.0% vs. 47.8%±7.5% without (p = 0.48). 5-year os with and without hsct was 66.2%±6.6% vs. 53.8%±7.6% (p = 0.34). when corrected for the median time to hsct (137 days), there were no significant differences in 5-year efs or os rates with and without hsct: 53.1%±7.3% and 64.2%±6.9% vs. 48.8%±7.8% and 53.8%±7.8%. no nci risk group or mrd subset benefitted significantly from cr1 hsct. sr patients (n = 42) had 5-year efs and os of 68.8±10.3% and 77.3%±9.2% with hsct (n = 27) vs. 57.1%±13.2% and 64.3%±12.8% without. hr patients (n = 71) had 5-year efs and os of 48.3%±9.0% and 57.6%±8.8% with hsct (n = 34) vs. 44.4%±9.2% and 49.9%±9.4% without. for those with end-induction mrd <0.01% (n = 74), 5-year efs and os were 66.3%±7.9% and 79.5%±6.7% with hsct (n = 39) vs. 60.3%±9.2% and 66.7%±8.8% without. end-induction mrd-positive patients (n = 30) fared poorly with both 5year efs and os of 29.4%±14.3% with hsct (n = 18) vs. 16.7%±10.8% and 22.2%±13.9% without. multivariate regression analysis including nci risk group, mrd, and cr1 hsct, showed only mrd negativity was significantly associated with efs (hr 0.256, p<0.0001) and os (hr 0.216, p<0.0001). patients with hypodiploid all fare poorly, particularly those with end-induction mrd ≥0.01%. while cr1 hsct is a standard treatment approach, it does not confer significant benefit. we were unable to assess bridging therapy prior to hsct, and comparator groups are small. taken together, however, new strategies are urgently needed for these patients. background: ras-pathway mutations are known to play a pivotal role in a significant proportion of myeloid malignancies, including upwards of 20% of pediatric aml cases. ras-pathway mutations in myeloid malignancy commonly co-occur with mutations of epigenetic regulators, suggesting cooperative leukemogenesis. among the epigenetic modifiers most frequently mutated in myeloid malignancy are regulators of dna methylation. this indicates that the alteration of dna methylation contributes to leukemogenesis. the ten-eleven translocation 2 (tet2) is an epigenetic regulator that plays an important role in regulation of dna methylation through its action of hydroxylation of 5-methylcytosine, which ultimately leads to passive de-methylation of dna cytosines. in myeloid malignancy, loss of function tet2 mutation is one of the most frequently co-occurring lesions in ras mutated malignancy. how specifically the altered methylation patterns in ras-pathway driven diseases promotes leukemogenesis is unclear. objectives: we hypothesize in mice with a ras-pathway mutation, that when an epigenetic modifier co-occurs, such as loss of function of tet2, this primes stem cells and/or early differentiating progenitors for transformation by preventing the repression of stem cell self-renewal genes, inhibiting differentiation, enhancing ras signaling and leading to leukemogenesis. we have generated a novel murine model with constitutive deletion of tet2 (tet2-/-) combined with an inducible activating krasg12d mutation (krasg12d/wt). mice have been tracked for evidence of hematologic malignancies and compared to mice with corresponding single genetic lesions. cooperative leukemogenesis will be demonstrated by decreased latency to disease onset, impact on malignancy lineage, in addition to investigating mechanistically through which pathways leukemogenesis may be promoted. results: krasg12d/wt/ tet2-/-mice demonstrate statistically significant differences in peripheral white blood cell count, hemoglobin, and platelet levels as early as 4-weeks post ras-pathway activation. peripheral cell lineage analysis demonstrates early skewing toward myeloid differentiation and marked splenomegaly in mice harboring both genetic lesions compared to wild type or mice with single genetic lesions. phospho-flow cytometric analysis reveals increased perk and ps6 activation in krasg12d/wt/ tet2-/-sca-1 enriched bone marrow cells compared to either genetic lesion alone. our study utilizing a murine model to examine how in ras-pathway mutations the addition of a co-occurring epigenetic lesion demonstrates that these lesions appear to cooperate to promote early myeloid differentiation with attendant changes in signaling pathways. this exploration to elucidate the mechanics of ras-pathway mediated disease lay the foundation for identification of patients who may benefit from existing therapies, such as dmtis, or identify new signaling targets for therapeutic exploration. background: the humoral immunogenicity of car19, a chimeric antigen receptor (car) with a murine scfv domain developed for treatment with tisagenlecleucel in relapsed/refractory (r/r) pediatric/young-adult acute lymphoblastic leukemia (all), was evaluated in 2 studies. little is known about the presence/impact of preexisting/treatmentinduced anti-murine car19 (mcar19) antibodies in patients treated with car therapy. objectives: patients from eliana (nct02435849; n = 68) and ensign (nct02228096; n = 29) were evaluated before and after tisagenlecleucel infusion to determine the impact of anti-mcar19 antibodies on cellular kinetics, efficacy, and safety. design/method: anti-mcar19 antibodies were determined by flow cytometry and reported as median fluorescence intensity. assay validation included evaluation of the interferences of intravenous immunoglobulin (ivig) treatment with the anti-mcar19 antibody assay. impact of preexisting and treatment-induced immunogenicity on cellular kinetics, efficacy, and safety was determined. treatment-induced immunogenicity was defined by a positive increase in anti-mcar19 antibody levels over baseline and was assessed by calculating the fold-change between preexisting (ie, baseline) and postinfusion levels. results: 90% of patients displayed preexisting anti-mcar19 antibodies; a similar incidence was detected in healthy volunteer samples during method validation. 35% of patients developed treatment-induced anti-mcar19 antibodies. no relationship was identified between tisagenlecleucel expansion (auc0-28d) and preexisting/treatment-induced anti-mcar19 antibodies (r2<0.001 and r2 = 0.006, respectively); similar results were seen for cmax. presence of treatment-induced anti-mcar19 antibodies did not appear to impact transgene persistence or response. kaplan-meier estimates showed that preexisting/treatment-induced anti-mcar19 antibodies did not appear to impact duration of response or event-free survival. strip plots showed consistent levels of preexisting/treatment-induced anti-mcar19 antibodies across patients with safety events, including cytokine release syndrome, neutropenia, thrombocytopenia, and neurological events. there was no apparent relationship between treatment-induced anti-mcar19 antibodies and b-cell recovery categories (≤3months, >3 and ≤6months, >6months, and ongoing sustained aplasia). no association existed between time of b-cell recovery and presence of treatment-induced anti-mcar19 antibodies. b-cell aplasia requiring ivig occurred following tisagenlecleucel in the majority of patients. the tisagenlecleucel concentration-time profiles in patients with treatment-induced anti-mcar19 antibodies were categorized by time following ivig administration. time of ivig administration had no impact on in vivo transgene expansion and persistence. we report the first comprehensive assessment of the impact of anti-mcar19 antibodies on clinical endpoints with car therapy. pediatric/young-adult patients with r/r all had a high frequency of baseline anti-mcar19 antibodies, and preexisting/treatment-induced anti-mcar19 antibodies did not impact the cellular kinetics, safety, and efficacy of tisagenlecleucel. cell-mediated immunity studies are ongoing. sponsored by novartis. background: adoptive immunotherapy, using cd123 engager (cd123-eng) t-cells, has shown success in preclinical studies, recognizing and killing acute myeloid leukemia (aml) blasts in vitro and in vivo. cd123-eng t-cells secrete bispecific molecules that recognize cd3 (t-cells) and cd123 (aml blasts), and are able to direct transduced t-cells and recruit bystander t-cells to kill cd123-positive blasts. however, cd123-engs do not provide costimulation and have not shown the capability for sequential killing of targets in vitro. we are seeking to improve the expansion, persistence and sequential killing capabilities of cd123-engs by genetically modifying these cells with an inducible costimulatory molecule, which can be activated by a chemical inducer of dimerization (cid). we generated a retroviral vector encoding cd123-eng and the inducible costimulatory molecule myd88.cd40 linked by a 2a sequence (cd123-eng.2a.imc). cd123-eng and cd123-eng.imc t-cells were generated by retroviral transduction, and their effector function was compared with and without cid. we used flow cytometric analysis to assess transduction efficiency, chromium release assays to evaluate cytolytic activity, and elisa to determine cytokine production. we successfully generated cd123-eng.imc tcells and achieved a mean initial transduction efficiency of 63% that was maintained above 50% throughout our study period. cd123-eng.imc t-cells +/-cid and cd123-eng t-cells readily killed cd123-positive aml blasts (molm13 and kg1a) in cytotoxicity assays when compared to the cd123-negative control (k562). in co-culture assays, cd123-eng.imc t-cells secreted increased il-2 and ifn-gamma in the presence of cid and cd123-positive targets (kg1a and molm13) when compared to co-culture with cd123-positive targets in the absence of cid. in addition, cd123-eng.imc t-cells displayed enhanced sequential killing capabilities and ifn-gamma secretion when stimulated weekly with cid and tumor cells at a 1:1 ratio when compared to cd123-eng t-cells. conclusion: cd123-eng.imc t-cells are able to recognize and kill cd123-positive aml blasts in an antigen dependent manner. cd123-eng.imc t-cells have improved effector function in the presence of cid as judged by cytokine production and their ability to sequentially kill cd123-positive target cells. thus, inducible myd88 and cd40 costimulation is a promising strategy to improve the effector function of cd123-eng t-cells, and warrants further active exploration in preclinical studies. background: eliana (nct02435849; n = 75) is a pivotal multicenter study testing the efficacy of tisagenlecleucel, anti-cd19 car-t, in children/young adults with r/r b-all. tocilizumab (toci) has been used for management of moderate/severe (grade 3/4) crs in ≈38% of patients treated with tisagenlecleucel at equivalent doses used in approved nononcological pediatric indications (<30kg received 12mg/kg; ≥30kg received 8mg/kg [800mg max dose]).(1) crs onset, as graded by the penn grading scale, generally occurred at a median of 3 days (range, 1-22) after infusion, requiring administration of 1-3 toci doses in some patients via a protocol-specific treatment algorithm. toci is a humanized monoclonal antibody that inhibits il-6 receptor (il-6r) signaling. the pharmacokinetics (pk) and pharmacodynamics (pd) of toci in pediatric patients with b-all with carassociated crs have not previously been described. objectives: characterize toci pk/pd for crs management following tisagenlecleucel infusion and describe its impact on cellular kinetics. design/method: toci pk and levels of soluble il-6r (sil-6r) were determined from serum and quantified using validated assays. maximum toci concentration (cmax) was derived using noncompartmental methods. sil-6r, proinflammatory cytokines, and crs resolution time were characterized to describe toci pd. summary statistics and graphical analyses of tisagenlecleucel exposure by number of doses were performed to describe the impact of toci on tisagenlecleucel kinetics in patients responding to tisagenlecleucel infusion. : 28/58 patients with crs received the first toci dose at a median of 5 days (range, 1-18) after crs onset. seventeen patients received 1 dose (range, 6.9-12 mg/kg); 8 received 2 doses (8-12 mg/kg); 3 received 3 doses (8-12 mg/kg), per the crs treatment algorithm. first-dose mean cmax (sd) was ≈111(30.6) g/ml; second dose, ≈265(376) g/ml. individual patient pd concentration-time profiles showed increased sil-6r levels after the first toci dose which remained elevated following the second dose. following toci administration, median time to crs resolution (including fever resolution) was 5 days (range, 2-29). crs onset coincided with tisagenlecleucel expansion, followed by a peak in serum cytokines, including il-6. the geometric mean auc0-28day and cmax of tisagenlecleucel transgene (by pcr) were 358% and 216% higher in tisagenlecleucel-responding toci-treated patients. conclusion: crs symptoms resolved within a median of 5 days after toci administration. toci levels achieved in patients with b-all were similar to reported pediatric nononcological indications (tocilizumab label) and resulted in concentration/time-dependent sil-6r increases. transgene continued to expand and persist following toci administration. these data support treatment with toci for crs management. (1) buechner, eha, 2017. sponsored by novartis. background: in acute myeloid leukemia (aml), mesenchymal stem and stromal cells (mscs) in the bone marrow microenvironment contribute to extrinsically mediated chemo-resistance and are therefore important potential therapeutic targets. the study of patient-derived mscs is at a competitive disadvantage, however, because traditional means of isolating mscs from a bone marrow aspirate interferes with isolating the more highly prioritized leukemic cells. many opportunities to study mscs are therefore missed. objectives: to develop a novel method of isolating mscs using the otherwise discarded portion of a bone marrow aspirate, thereby de-coupling the isolation of primary mscs from the isolation of leukemia cells. design/method: aml patient bone marrow aspirates were obtained prospectively from the children's oncology group. healthy patient marrow was purchased. experimental mscs were isolated from the bottom-most layer (rbc-layer) produced by density-gradient separation of a bone marrow aspirate, which is typically discarded. control mscs were isolated from the buffy coat (mnc layer). non-adherent cells were removed after 72 hours, and adherent cells were cultured at 5% co2 with mem-alpha containing 20% fbs. growth curves were obtained by seeding 6-well plates with 10,000 cells per well. cells were stained using oil red o to observe adipocyte differentiation. results: rbc-layer mscs grow successfully following overnight shipment of the aspirate. identical to mnc-layer mscs, rbc-layer mscs exhibit a fibroblastic morphology and are adherent to plastic. rbc-layer mscs persist in culture up to 14 passages before senescence. they exhibit a slower growth curve relative to mnc-layer mscs, but their overall doubling time is similar at approximately 120 hours. surprisingly, mscs from the rbc-layer exhibit adipocyte differentiation on stimulation, revealing their stem-cell like qualities. we present a method of isolating mscs from the discarded portion of a bone marrow aspirate that does not interfere with the isolation of leukemia cells from the same patient. this portion of the aspirate can be shipped, or can sit for at least 24 hours, without sacrificing its mscs. rbclayer mscs are nearly identical to mscs obtained conventionally. perhaps most importantly, rbc-layer mscs retain a stem-cell like capacity, showing them to be a highly valuable cell population in aml research. future plans include investigating potential selective enrichment of stem-cell mscs in the rbc-layer, which could explain the unexpected difference in growth kinetics. aml researchers now have the opportunity to study this exciting component of the bone marrow microenvironment without sacrificing valuable leukemic cells in the process. background: neutropenia is one of the most frequent side effect of chemotherapy associated with an increase in the risk of infection, especially in the cases when the depth and duration of neutropenia are extended. some genes, as variations of darc, gsdma and cxcl2 are known to influence white blood cell and neutrophil counts. our previous study conducted in children with acute lymphoblastic leukemia (all), showed that polymorphisms in these genes might play a role in the onset of chemotherapy complications during consolidation and maintenance treatment. objectives: in order to support our previous finding, we have expanded the study to the induction period in a cohort of 233 all children treated at the sainte-justine university health center between july 1995 and july 2005. design/method: previous associated single nucleotide polymorphisms (snps) in darc, gsdma and cxcl2 genes were analyzed for an association with the complications occurring during induction including the duration of low neutrophil count (pnn) and low absolute phagocyte count (apc), proven infections and delay between induction and consolidation phases. results: significant effect was found for all studied polymorphims. minor alleles of darc rs3027012, cxcl2 rs1680408 and gsdma rs3859192 were all associated with higher risk of complications during induction treatment, whereas that of darc rs12075 (particularly gg genotype) had a protective effect. the gg genotype of rs12075 was associated with a lower risk of post-induction delay (p = 0.02 or = 0.1, 95%ci 0.02-1.0), less frequent febrile episodes (p = 0.02) and lower number of days with apc/pnn count reduction (p = 0.008 for apc<0.5 and p = 0.02 for pnn<0.5). in contrast, the minor t allele of another darc polymorphism (rs 3027012), was associated with longer apc/pnn count reduction (p = 0.01 for apc<0.5 and p = 0.02 for pnn <0.5), as it was the tt genotype of gsdma rs3859192 (p = 0.02 for apc<0.5 and p = 0.04 for pnn<0.5). the patients with the gsdma rs3859192 had also a higher risk of documented febrile episodes (p = 0.04 or = 2.4 95%ci 1-5.5). the aa genotype of rs1680408 cxcl2 was associated with a higher risk of post-induction delay due to infection (p = 0.04, or = 3.4, 95% ci 1.1-11.5). conclusion: this complementary study confirmed our previous results, showing overall that variations in darc, gsdma and cxcl2 genes influence the onset of chemotherapy complications in pediatric all, regardless of treatment phases. these polymorphisms might be useful pharmacogenetics markers possibly guiding an adjustment of chemotherapy intensity. background: pediatric acute myeloid leukemia (aml) has a poor survival rate of about 70% and there is an urgent need for newer targeted therapies. car t-cell based therapies are effective against all but similar therapies against aml are still under development. recent clinical trials have highlighted the concerns about toxicity and therapy related deaths from car t-cells. antigen selection is the key factor determining the specificity, efficacy and toxicity of car t-cells. while contemporary adoptive t-cell therapies use monoclonal antibodies against tumor associated antigens we employed the naturally occurring flt3 ligand (fl) to target aml cells expressing flt3 receptors. flt3 receptor is expressed on multipotent and myelomonocytic progenitors as well as myeloid leukemia cells. to generate fl containing chimeric tlymphocytes designated flcar t-cells and to evaluate their efficacy against aml cells. design/method: flcar was constructed by fusing the coding sequences of the human fl, cd28 costimulatory domain, and cd3-zeta chain (intracellular region) in series. it was then cloned into the phiv-egfp lentiviral vector for expression in cell lines and primary t cells obtained from healthy donors. the empty phiv-egfp vector was used as a negative control. flcar was expressed on both cd4+ and cd8+ t-lymphocytes, confirmed by western blot. cell cytoxicity was evaluated by co-culturing flcar t-cells and aml cells followed by flow cytometric analyses. cytokine production was assessed by analyzing expression of interleukin-2 using quantitative rt-pcr. results: flcar t-cells were generated from cd4+ jurkat and cd8+ tk-1 cell lines with up to 70% lentiviral transduction efficiency. the efficiency for primary t cells was lower (5-10%). flcar was expressed as a ∼42 kda protein in cells and was partially phosphorylated on tyrosine. the expression of flcar on lymphocytes lead to increased basal il-2 expression in the cells. this was further augmented (by > 5 folds) upon co-incubating flcar t-cells with flt3expressing target cells. jurkat cells, tk-1 cells and primary human t cells expressing flcar suppressed the growth of flt3-expressing aml cell lines and primary aml cells in vitro. notably, flcar t-cells generated from healthy donors caused strong inhibition of aml cells even at a lower transduction efficiency. in vivo experiments using nsg-sgm mice xenografted with human aml cells are underway. our data demonstrate that flcar can be effectively expressed on t-lymphocytes and mediate potent cytotoxicity against flt3-expressing aml cells in vitro. being a completely human derived chimeric protein, it represents a promising candidate for further therapeutic development. holly pacenta, kelly sullivan, ahwan pandey, kelly maloney, joaquin espinosa children's hospital colorado, denver, colorado, united states background: individuals with down syndrome (ds) have a 20-fold higher risk of developing acute lymphoblastic leukemia (all) than the typical population. there are several important differences between all in individuals with ds (ds-all) and all in individuals without ds (nds-all): first, patients with ds-all have a lower percentage of favorable cytogenetic features compared to nds-all. second, patients with ds-all are more likely to have activating mutations in jak2, crlf2 overexpression, and ikzf1 deletions. despite these clear genotypic differences, this knowledge has not yet been exploited for therapeutic purposes in ds-all. when outcomes for ds-all are compared to nds-all with similar cytogenetic features, the survival rates are similar. however, individuals with ds-all have an increased risk of treatment-related mortality (trm). current therapy for ds-all is similar to that for nds-all, with the exception of small changes to decrease toxicities that are more prevalent in ds-all. it was recently identified that interferon signaling is constitutively activated in healthy individuals with t21. we hypothesize that aberrant interferon signaling could play a role in the unique leukemias observed in ds patients. objectives: to identify differences in gene expression and intracellular signaling cascades that are unique to individuals with ds-all, relative to both nds-all and healthy individuals with ds that can be exploited for therapeutic use. design/method: bone marrow samples were obtained from ds-all patients and matched nds-all patients based on clinical characteristics and genetic features. rna sequencing of these samples was performed and a total of 19 samples were used for the transcriptome analysis (6 ds-all vs. 13 nds-all). the differential expression data was generated by deseq2 and analyzed using ingenuity pathway analysis. the analysis revealed that the chromosome 21 genes that have been implicated in leukemogenesis are not differentially expressed in the ds-all samples, relative to nds-all. an inflammatory signature was identified, which included interferon gamma as an upstream regulator with predicted activation in ds-all. this finding is consistent with prior observations from healthy individuals with ds. other examples of results with potentially actionable targets include the upregulation of several genes in the ras pathway and genes involved in histone methylation. the increased interferon signaling seen in healthy individuals with ds was also identified in ds-all. this may contribute to the development of mutations in inflammatory pathways such as jak2 and crlf2 in ds-all. targeting these common pathways with small molecule inhibitors may have a therapeutic benefit in ds-all. cincinnati children's hospital medical center, cincinnati, ohio, united states background: next-generation sequencing (ngs) guides precision medicine approaches in oncology using therapies targeting molecular alterations found within an individual cancer. increased availability of ngs coupled with a proliferation of targeted drugs in development heightens the need for reliable pre-clinical animal models. here we report a patientderived xenograft (pdx) system with integrated molecular profiling for pre-clinical testing of conventional cytotoxic and novel targeted agents. objectives: to utilize ngs from patients with pediatric leukemia to guide rational pre-clinical trials in pdx leukemia avatars, and to determine pdx mice tolerance of and response to cytotoxic and targeted therapies. pediatric acute lymphoblastic leukemia (all) samples were obtained in adherence to an irb-approved protocol and xenografted into nod/rag/interleukin-2 (il-2)rg (nrg) mice. ngs was performed clinically using the foundationone® heme panel. a de novo all sample bearing mutations involving jak2, crlf2, ntrk3, cdkn2a/b, ptpn11 and wt1 was used for pre-clinical testing. thirty-seven nrg mice were transplanted with 2 million patient cells/mouse via iv injection. standard 4-drug induction chemotherapy was administered consisting of vincristine, dexamethasone, pegaspargase, and daunorubicin [vxpd, n = 6 mice], in comparison to vehicle control [n = 8]. parallel pdx cohorts were treated with single agent targeted therapies based on ngs findings, including ruxolitinib [n = 7], crizotinib [n = 8] and loxo-101 [n = 8]. the four-week treatment period began on day +30 from transplant after confirmation of engraftment. following completion of therapy, residual disease burden was analyzed by flow cytometry (hcd19+, mcd45-cells) in the bone marrow [bm] . to date, pdx models have been established using over thirty ngs-profiled pediatric all samples, including six samples bearing philadelphia (ph) chromosome or phlike mutations. pre-clinical testing was performed in a repreconclusion: ngs reveals concomitant mutations in ph-like all that may represent additional targets for therapy, or predict tyrosine kinase inhibitor (tki) resistance. we show that all xenograft nrg mice can tolerate a 4-week multi-agent cytotoxic chemotherapy induction regimen, as well as rational targeted agents, and serve as a robust pre-clinical model for precision medicine trials. background: osteonecrosis is a well-characterized all therapeutic toxicity attributed to glucocorticoids, asparaginase, and methotrexate that disproportionately affects adolescents. in ccg-1961, alternate-week dexamethasone during double delayed intensification (di) reduced osteonecrosis vs continuous dexamethasone with single di in rapid early responders (rer) ≥10y. to compare efs and os between hr-all patients with vs without osteonecrosis. design/method: hr-all patients 1-30y on aall0232 (2004-11) received cog augmented therapy with a 2 × 2 randomization to: (1) induction dexamethasone (10 mg/m2 d1-14) vs prednisone (60 mg/m2 d1-28), and (2) interim maintenance (im) high-dose methotrexate (hdm) vs escalating-dose methotrexate/pegaspargase (ema). rer received single, and slow early responders (ser) double, im/di. initially, all received monthly dexamethasone maintenance pulses, patients ≥13y received di alternate-week dexamethasone, and patients ≤12y received di continuous s217 of s301 dexamethasone. there were 2 osteonecrosis-related amendments: after 10/2006 all patients ≥10y received di alternateweek dexamethasone; after 6/2008 all patients ≥10y were assigned to induction prednisone, and all patients received di alternate-week dexamethasone and maintenance prednisone pulses. results: osteonecrosis was confirmed in 315/2817 patients. the 5y cumulative incidence (ci) was 12.7% overall and increased with age: 1-9y 2.6%, 10-12y 15.3% (alternateweek dexamethasone 10.2% vs continuous dexamethasone 23.5%; p<0.0001), ≥13y 19.7% (p<0.0001). among randomized rer patients ≥10y, ci differed by glucocorticoid (dexamethasone 25.0% vs prednisone 15.0%; p = 0.0003) but not methotrexate assignment (hdm 18.8% vs ema 21.5%; p = 0.7). among randomized ser patients ≥10y, ci was 18.9% with no difference by regimen. results were similar for patients ≥13y. in the entire study population, patients with osteonecrosis had superior 5y efs (89.0% vs 76.1%; p<0.0001) and os (95.8% vs 85.9%; p<0.0001) than those without osteonecrosis. 5y efs was significantly higher among randomized patients ≥10y with vs without osteonecrosis (88.5% vs 72.9%; p<0.0001); this finding was present in different age ranges (≥10y, ≥13y, ≥16y) and rer/ser subsets within each, especially in the ≥10y rer (92.6% vs 81.8%; p = 0.0004) and ser (74.0% vs 43.7%; p<0.0001) cohorts. across groups, asparaginase allergy was significantly associated with reduced osteonecrosis risk (≥10y: hr 0.43; p = 0.0013). patients who develop osteonecrosis have significantly increased efs and os, suggesting host differences that increase sensitivity to develop osteonecrosis and render all cells more chemo-responsive. pennsylvania state university, hershey, pennsylvania, united states background: cdc42 (cell division cycle protein 42) belongs to rho family of small gtpases in ras-oncogene superfamily. pro-oncogenic role of overexpressed cdc42 in ras driven solid tumors are well known. however, role of cdc42 in leukemia is yet to be established. ikzf1 encodes ikaros protein which has important role in regulation of lymphoid development and tumor suppression in leukemia. casein kinase ii (ck2) oncogene is overexpressed in leukemia. ck2 impairs ikaros function which can be restored by using ck2 inhibitors. objectives: to investigate role of cdc42 in leukemia and regulation of cdc42 by ikaros and ck2 in b-cell acute lymphoblastic leukemia (b-all). shrna transfection, real time-pcr, luciferace assay, quantitative chromatin immunoprecipitation (qchip) coupled with the next-generation sequencing (chip-seq), cytotoxicity assay and western blot. results: cdc42 is identified as one of the ikaros target genes by analysis of genome-wide dna binding of ikaros using chip-seq and qchip in b-all primary cells. expression of cdc42 was also noted to be higher in all patient samples compared to normal bone marrow. functional experiments showed that ikaros overexpression via retroviral transduction results in transcriptional repression of cdc42. ikaros silencing using shrna resulted in increased expression of cdc42. these data suggest that ikaros negatively regulates transcription and expression of cdc42. ck2 directly phosphorylates ikaros and impairs its function as transcription factor. we noted that molecular inhibition of ck2 via sirna as well as treatment with specific ck2 inhibitor, cx4945 also decreases expression of cdc42. treatment with cx4945 of primary b-all with ikaros haploinsufficiency restores ikaros binding to cdc42 promoter and represses cdc42 expression. however, this effect is evident only in presence of ikaros. treatment with cx4945 in ikaros silenced (ikaros shrna) cells showed no change in expression of cdc42. these results emphasizes the importance of ikaros in regulating cdc42 expression. furthermore, we analyzed the changes in epigenetic signature at the cdc42 promoter following treatment with cx4945. results show that loss of histone marker of open chromatin (h3k9ac) and increased histone marker for repressive chromatin (h3k27me3), at the cdc42 promoter. these data suggest that ikaros transcriptionally represses cdc42 via chromatin remodeling. a specific cdc42 inhibitor, ml141 showed cytotoxic effects on primary b-all cells. conclusion: cdc42 may have important role in hematologic malignancies. expression of cdc42 in b cell all is regulated by ikaros and ck2. these results suggest that targeting cdc42 could be a potential therapeutic strategy in leukemia. caitlyn duffy, laura hall, justin godown, koyama tatsuki, scott borinstein monroe carell jr. children's hospital at vanderbilt, nashville, tennessee, united states background: systemic corticosteroids are widely used as treatment of acute lymphoblastic leukemia (all) and lymphoblastic lymphoma. there are anecdotal reports of bradycardia in pediatric patients receiving corticosteroids, but a more extensive analysis of this effect is needed. objectives: the aim of this study was to describe the incidence, severity, and timing of steroid-induced bradycardia and document any adverse events associated with bradycardia. design/method: we performed a retrospective review of all newly diagnosed patients at our center (2010-2016) with all/lymphoblastic lymphoma who received corticosteroids (dexamethasone 3-5mg/m 2 /dose or prednisone 30mg/m 2 /dose) during induction chemotherapy. patients were excluded if they had a pre-existing cardiac abnormality or if they received prior corticosteroids. the average 24 hour heart rate (hr) was assessed for the period prior to initiating steroid therapy and for the 24 hour period surrounding the nadir following steroid administration. the degree and time of steroid induced bradycardia was assessed. adverse patient events and concomitant medication use was documented to identify other contributing factors to bradycardia. a total of 153 children (80 females, 73 males, 16 months-27 years) were included in the analysis with 159 demonstrating a decrease in mean hr following steroid administration. median hr decrease was 22.9 beats per minute (quartiles 12.5-32) from prior to initiating steroids to surrounding nadir. sixty one percent developed bradycardia less than or equal to the 1st percentile for their age range. nadir occurred 7 doses (range 5-10) into treatment, which corresponded to 79 hours (55-109) after initiation of therapy. of 94 patients who experienced bradycardia, 78% were associated with dexamethasone rather than prednisone. hr nadir was not associated with other vital sign abnormalities. after completion of induction chemotherapy, 87% of patients had documented resolution of bradycardia with hr greater than the 5th percentile for age. it was observed that the children who continued to have relatively low hr were often younger (20 months-5 years old). examination of nadir hr during subsequent hospitalizations in which steroids were not being administered (excluding hr during procedural sedation) did not demonstrate a significant incidence of bradycardia. concomitant opioid, beta-blocker, or other medication exposure did not contribute to the incidence of bradycardia. corticosteroid-induced bradycardia is extremely common in children, teenagers, and young adults with all receiving induction chemotherapy. bradycardia was not associated with clinical adverse events and resolved after completion of corticosteroid treatment. therefore, further cardiac assessment may not be warranted in the presence of bradycardia suspected to be secondary to steroid administration. baylor college of medicine, houston, texas, united states background: survival in newly diagnosed pediatric acute myeloid leukemia (aml) is approximately 65%; however survival falls dramatically if a patient relapses. currently, approximately one-third of patients with pediatric aml relapse on standard chemotherapy regimens. aml cells are exposed to proteotoxic stress at baseline due to their rapid and inefficient metabolism; proteotoxic stress increases after chemotherapy due to accumulation of reactive oxygen species resulting in misfolded proteins. this leads to activation of cell stress pathways, such as the unfolded protein response (upr) in the endoplasmic reticulum. because an activated upr can make cells more sensitive to proteotoxic stress, we hypothesize that upr activation correlates with response to chemotherapy. objectives: determine the status of upr in pediatric aml and its correlation with chemosensitivity; design/method: peripheral blood samples from pediatric patients with aml were collected at the start of induction chemotherapy, 6-10 hours (h) and 24 h post initiation of systemic chemotherapy. tumor cells were sorted from peripheral blood mononuclear cells. expression of upr proteins was determined by chemiluminescence using an automated capillary electrophoresis system. clinical correlations were performed using an annotated database. we measured five upr proteins: grp78 (glucose regulated protein 78kda), phospho-eif2 , inositol-requiring enzyme 1 (ire1) and activating transcription factor (atf6). patients with aml had 2-5 times higher expression of upr proteins (except atf6) at baseline than normal controls. grp78-the key upr driver-had the highest level of protein expression in myeloid blasts. there was a wide variability in the level of baseline upr expression. eight out of 38 samples expressed >5 fold increase in grp78 above those with the lowest grp78 levels. similarly, 7 and 9 patients respectively, had a >5 fold increase in peif2 and ire1, compared to patients with low basal expression of these upr proteins. in our limited sample set, there was a trend towards lower overall survival (os) and event-free survival in patients with low baseline grp78 and ire1. conclusion: upr has a variable expression at baseline in pediatric aml, with a trend towards lower os in patients with a low basal grp78 and low ire1 expression, suggesting less chemosensitivity in this subgroup. conversely, it is possible that blasts with an upregulated upr prior to chemotherapy manage proteotoxic stress less effectively, having faster apoptosis and hence a better response to chemotherapy in patients with a high basal upr. we are currently expanding our findings in a larger cohort of patients enrolled in the children's oncology group aaml1031 protocol. background: children with newly diagnosed acute lymphoblastic leukemia (all) undergo chest x-ray (cxr) evaluation during initial diagnostic workup to ensure safe airway management. however, to our knowledge, no systematic assessment of cxr findings has been reported. objectives: to evaluate cxr findings at diagnosis of all and their associations with clinical characteristics. we reviewed the cxr findings at diagnosis of all in patients treated on the total xv and xvi protocols at st. jude children's research hospital. findings were evaluated for associations with clinical characteristics at presentation, and the clinical management of mediastinal masses was reviewed. mediastinal masses were seen in 107 (10.8%) of 990 patients evaluated and were more common in older patients (mean age, 8.51 years) than in younger patients (mean age, 7.14 years) (p = 0.005), in males than in females (p = 0.017), and in patients with t-all than in those with b-all (p<0.001). also associated with mediastinal masses were a higher white blood cell count (wbc) at diagnosis (mean, 107.02 × 109/l) (vs. a lower wbc; mean, 36.65 × 109/l) (p<0.001), cns involvement (vs. no involvement) (p = 0.028), and standard/high-risk disease (vs. low-risk disease) (p<0.001). other cxr findings included pulmonary opacity (160 patients [16.2%]), bronchial/perihilar thickening (187 patients [18.9%]), cardiomegaly (68 patients [6.9%]), and osteopenia/fracture/periosteal lesions (132 patients [13.3%]). pulmonary opacity was more common in younger patients (mean age, 6.51 years) than in older patients (mean age, 7.44 years) (p = 0.023) and in those with t-all (vs. b-all) (p = 0.010). bronchial/perihilar thickening, cardiomegaly, and osteopenia/fracture/periosteal lesions were also more common in younger patients than in older ones (p<0.001, p = 0.002, and p<0.001, respectively) and in those with low-risk disease (versus standard/high-risk disease) (p<0.001, p = 0.001, and p = 0.005, respectively). of the 107 patients with a mediastinal mass on cxr, 56 underwent a confirmatory chest ct scan, and 48 (85.7%) were confirmed to have a mediastinal mass. notably, 23 patients (41.1%) had airway compression, and compression of venous structures was identified in 18 of 48 patients (37.5%) who received iv contrast. the clinical course was evaluated for 107 patients with mediastinal masses detected by cxr. fifty patients (46.7%) required icu admission (mean stay, 3.0 days). general anesthesia was used for only 52 patients (48.6%), and 68 patients (63.6%) had a less invasive peripherally inserted central catheter. no deaths occurred in the acute phase. conclusion: cxr at the time of all diagnosis can detect various intrathoracic lesions and is helpful in planning initial diagnostic workup and management. background: mertk is a receptor tyrosine kinase that is aberrantly expressed in 80% of pediatric primary aml samples. mertk inhibition with the small molecule tyrosine kinase inhibitor (tki) mrx-2843 decreases tumor burden and prolongs survival in aml xenografts. while treatment with mrx-2843 reduces leukemia in the peripheral blood, it is less effective in the bone marrow, suggesting a role for the marrow microenvironment in therapeutic resistance. the jak/stat pathway has been implicated as a mediator of bone marrow derived resistance to tkis and inhibitors of this pathway are in clinical development for the treatment of aml. to determine the role of the bone marrow stromal niche in mediating resistance to mertk inhibition and to evaluate the efficacy of combined mertk and jak/stat inhibition. design/method: aml cell lines were cultured with or without the hs27 stromal cell line or hs27 conditioned medium, then treated with mrx-2843 +/-the jak/stat inhibitor ruxolitinib, or control. induction of apoptosis and cell cycle arrest in aml cells was measured by flow cytometry. expression of h2ax and total and phosphorylated stat5 were determined by immunoblot. results: co-culture with stromal cells significantly reduced aml cell death and g2/m phase arrest in response to treatment with 200nm mrx-2843 compared to no co-culture (cell death: 31.9% versus 61.2%, p<0.05; g2/m arrest: 8.9% versus 14.8%, p<0.01). g2/m arrest was accompanied by an increase in h2ax expression which was similarly abrogated in co-culture. conditioned medium did not provide protection from mrx-2843 induced apoptosis, g2/m arrest, or h2ax induction. mrx-2843 inhibited stat5 phosphorylation but direct co-culture and conditioned medium potently increased basal stat5 phosphorylation which was not inhibited by mrx-2843. to determine whether the observed induction of stat5 phosphorylation was functionally relevant, cocultures were treated with both mrx-2843 and ruxolitinib. while ruxolitinib potently inhibited the phosphorylation of stat5 in the presence of co-culture, combination treatment did not overcome stromal mediated protection from mrx-2843 induced apoptosis. similarly, the addition of exogenous gm-csf induced stat5 phosphorylation but did not yield protection from mrx-2843 functional effects in the absence of co-culture. together these data support a model whereby direct cell-cell contact with stromal cells in the bone marrow niche protects leukemia cells from mrx-2843 induced apoptosis, cell cycle alterations, and dna damage. while co-culture potently induces phosphorylation of stat5 in leukemia cells, this is neither necessary nor sufficient for stromal-cell mediated protection from mertk inhibition and combined treatment with jak/stat inhibitors is unlikely to be therapeutically efficacious. background: mercaptopurine (6-mp) is an immunosuppressive thiopurine drug that is a key component of acute lymphoblastic leukemia (all) treatment. 6-mp is metabolized into 6-thioguanine (6-tgn), which is responsible for anti-leukemic effects, as well as 6-methylmercaptopurine nucleotides (6-mmpn/6-mmp), which are associated with hepatotoxicity. some patients preferentially metabolize 6-mp to 6-mmpn/6-mmp, increasing their risk for hepatotoxicity and potentially reducing anti-leukemic effects. hepatotoxicity can cause interruptions or delays in therapy that may jeopardize cure rates. allopurinol has been increasingly used in patients with inflammatory bowel disease (ibd) to shunt 6-mp metabolism toward 6-tgn and away from 6-mmpn to minimize hepatotoxicity and preserve therapeutic effects. objectives: this retrospective chart review expands upon our previously published case series of three patients with all in whom allopurinol was successfully used to redirect 6-mp metabolism. twelve additional patients have subsequently received allopurinol and 6-mp combination therapy at texas children's hospital. data from this larger patient sample, with longer follow up, is being analyzed to increase knowledge of the effectiveness and longitudinal effects of adding allopurinol to 6-mp to reduce risk of hepatotoxicity. design/method: data were abstracted from the electronic medical records of 15 patients with all treated at texas children's hospital from 2012 to present, who had been found to have evidence of altered 6-mp metabolism and in whom allopurinol was added to 6-mp therapy due to concern for risk or recurrence of hepatotoxicity. metabolite levels, 6-mp dose, and alanine transaminase (alt) prior to initiation of allopurinol and approximately 8 weeks later were compared. wilcoxon signed-rank test was applied for statistical analysis. : after the addition of allopurinol, patients experienced a significant decrease in mean levels of 6-mmpn (p = 0.0007), correlating with a significant decrease in mean alt (p = 0.004). with the initiation of allopurinol, the mean 6-mp dose was decreased from 84 to 32 mg/m2/day over an 8-week period. mean 6-tgn levels increased (p = 0.14). in follow up beyond 8 weeks, no patients had further holds in 6-mp due to hepatotoxicity. addition of allopurinol appears to shift metabolism from 6-mmpn toward 6-tgn, with increases in mean 6-tgn levels despite a decrease in mean 6-mp dose. this may limit negative side effects, thus resulting in fewer gaps in therapy and possible improved outcomes. further analysis of 6-mp dose titration and effects on anc over time as well as effects on overall survival is ongoing. prospective background: alterations in epigenetic patterning are a fundamental feature in acute myeloid leukemia (aml). treatment with dna methyltransferase inhibitors (dnmti) yields responses in aml, but the molecular mechanisms underlying this effect are poorly understood. in prior work, we demonstrated induction of genes involved in the pirna rna (piwi) silencing pathway as a common gene feature of 4 aml cell lines treated with decitabine. the piwi pathway is an rna silencing system, distinct from classical small rna transcriptional silencing, responsible for transposon-silencing in gametogenesis; emerging data suggest a role for this system in somatic cells. based on these data, we postulate that piwi induction plays a crucial role in aml recovery following demethylation and that disruption of this pathway would modulate response and/or recovery from decitabine treatment. to assess the effect contribution of the pirna pathway response following dnmti treatment in aml. design/method: to choose target genes in the pirna pathway for disruption, molm13 cells were first treated with escalating doses of decitabine. using quantitative rt-pcr, the dose-dependent expression of several pirna-associated genes were analyzed. two genes, mael and piwil2, were selected for disruption experiments based on preliminary data suggesting decitabine dose-dependent responses. molm13 cells were transduced with shrna targeting these genes using a lentivirus delivery system with selection in puromycin. knockdown efficiency was assessed by rt-qpcr. to determine how gene disruption affected cell growth, knockdown cells were treated with decitabine 20nm. proliferation was assessed by celltiter glo assay following decitabine treatment. clonogenic potential was assessed by colony forming assays of transduced cells after treatment with decitabine at 5nm and 10nm. results: following decitabine exposure in molm13, there was a markedly increased expression of mael and piwil2 compared to untreated cells (2363:1 and 41:1, respectively) . thus, these were the candidate genes chosen for disruption. of 4 mael shrna constructs, two resulted in a 25% relative expression of mael compared to controls. of the 3 piwil2 shrna constructs, the best knockdown showed 75% relative expression. there were no significant differences in proliferation or clonogenicity of stably selected mael or piwil2 knock-down molm13 cells following decitabine treatment. using gene knockdown procedures, mael and piwl2 do not appear to have a marked effect on growth and response to decitabine treatment in molm13. however, these results may be limited by inefficient knockdown using shrna targeting methods. further work using a cas9/crispr based inactivation of these genes is ongoing. children cancer hospital cairo, egypt background: hypodiploidy <44 chromosomes is very uncommon and have particularly poor outcomes in childhood acute lymphoblastic leukemia (all). it is subdivided into: near-haploid (24-31chromosomes), lowhypodiploid (32-39chromosomes) and high-hypodiploid (40-43chromosomes). to determine if minimal residual disease (mrd) can identify a group of patients with better prognosis in the hypodploid population who can be treated with intensive chemotherapy alone. design/method: a retrospective study that included all patients under age of 18 diagnosed as hypodiploid b-precursor all during the period between january 2008-december 2015 and treated at children's cancer hospital egypt on sjcrh total study-xv for ir/hr all. sixteen patients had <44 chromosomes (9 nearhaploid and 7 low-hypodiploid), constituting 1% of all pediatric patients with b-precursor all during the study period. patients with near-haploid all had a median age of 6 years (range 2-17), initial leukocyte count (wbc) median of 7.5 × 109/l (range 1.3-86.6), 4 (44.4%) were males and 5/9 (55.6%) had hr-nci criteria. four patients (44.4%) are alive in complete remission(cr) (range 25-48 months, median 30), one died in induction and 4 (44.4%) had hematological relapse (range 6.8-33 months, median 15). patients with low-hypodiploid all had significantly older age (median 15 years, range 13-17), median wbc 4.3 × 109/l (range 3.5-13.5), 5/7 (71.4%) were males. one patient (14.3%) is alive in cr, one died in induction, one failed to achieve cr post-induction and 4 patients(57%) had hematological relapse (range 3.9-5.6 months, median 4.8). mrd<0.01% by flow-cytometry on day-15 and end of induction was achieved in 5/9 (55.6%) and 6/8 patients(75%) with near-haploid, compared with 1/6 (16.7%) and 3/6 patients(50%) with low-hypodiploid; respectively (p = 0.287, p = 0.58;respectively). allogeneic transplantation was performed during initial remission only in 2 mrd negative patients (one relapsed and one is in cr) and in the patient with induction failure (relapsed post-transplant). five of the total six patients who had negative mrd on day-15 and end of induction are alive in cr (4/5 with chemotherapy alone). all 3 patients with negative mrd at end of induction but with mrd levels≥0.01% on day-15 (range 0.02-0.33%) relapsed as well as all 4 patients with detectable mrd at the end of induction. the difference in relapse was statistically significant in relation to negative-mrd on day-15 (p = 0.005), but not at end of induction(p = 0.105). conclusion: children with hypodiploid all and negative mrd on day-15 of induction are highly curable with intensive chemotherapy alone, while patients with negative mrd at the end of induction and detectable mrd on day-15 had dismal outcome. background: overall survival in pediatric acute myeloid leukemia (aml) has plateaued between 60-70%, with death during induction chemotherapy seen in 4-11% of patients. respiratory complications contribute to morbidity and mortality in pediatric aml induction, however the incidence, patterns, and predictors of respiratory adverse events (aes) during this period are unknown. to estimate the incidence of respiratory aes during induction therapy for de novo pediatric aml, to characterize and grade these respiratory aes, and to identify predictors of respiratory ae development. we conducted a retrospective longitudinal study from presentation to day 42 in institutional de novo pediatric aml patients (≤ 21 years) between march 2009 and december 2016. outcomes included any nci ctcae grade 2-5 respiratory ae or death from another cause. demographic, disease, and treatment-related data were abstracted. the most specific, best-fitting ctcae category and grade for each ae was determined. descriptive statistics, survival analysis, multivariable logistic regression analysis, and time-toevent distributions were performed (sas v9.4, cary, nc) . among 113 eligible patients, 54.0% (n = 61) experienced 69 discrete respiratory aes. incidence of grade 3-5 aes was 46.0% (n = 52). a bimodal time-to-event distribution demonstrated peaks at treatment days 0 and 14. induction death occurred in 4.4% (n = 5) including 3 deaths from respiratory failure associated with disseminated fungal disease. in univariate analysis, those experiencing aes differed significantly in regards to older age at diagnosis (p<0.0001), higher initial wbc (p = 0.012), higher initial peripheral blast percentage (p = 0.002), coagulopathy at diagnosis (pt (p = 0.004), d-dimer (p = 0.002)), fluid overload status (p<0.0001), occurrence of infection (p = 0.01), and occurrence of tumor lysis syndrome (tls) (p = 0.016). patients with hyperleukocytosis (p = 0.005), fluid overload (p<0.0001), and fab m3 morphology (p = 0.0016) each had a significantly decreased probability of completing the follow up period without experiencing a respiratory ae. on multivariable analysis, fluid overload (aor 45.2 [95% ci: 5.4-376.0) and older age (aor 1.10 [95% ci: 1.01-1.20) were significantly associated with ae occurrence when gender, hyperleukocytosis, tls, and infection status were held constant. we describe a high incidence of respiratory aes during pediatric aml induction. fluid overload and older age at diagnosis are independently associated with ae development when controlling for other proposed risk factors. interventions focused on conservative fluid management and offset of fluid overload should be explored in newly diagnosed pediatric aml in an effort to reduce respiratory complications during induction. overall, all survival rates are outstanding and have continued to improve with risk-adapted therapy. the most striking improvement occurred in t-all where 5-year os rates now exceed 90% and parallel b-all. survival improvements, however, have not been observed uniformly across all subgroups. while the gap in outcome differences narrowed among blacks, outcomes for hispanics have remained static. further, no improvements in survival were observed in infants or ayas and new treatment approaches have been implemented for these populations. background: acute myeloid leukemia (aml) accounts for approximately 18% of new childhood leukemia cases. chest x-ray (cxr) is performed in all newly diagnosed aml cases to evaluate the safety of airway management for anesthesia during diagnostic procedures; however, cxr results in pediatric patients with aml have not been described. objectives: the primary objective was to evaluate cxr findings at diagnosis in patients with aml. the secondary objectives included assessing associations between cxr findings and clinical characteristics, with the overall goal of aiding in the evaluation of the use of cxrs as an initial diagnostic study in pediatric patients with aml. design/method: cxr findings and clinical characteristics were evaluated in patients with newly diagnosed aml who were enrolled in one of three protocols at st. jude children's research hospital (aml97, aml02, and aml08). the findings were categorized based on radiologic reports. further, the associations of these findings and clinical characteristics were evaluated. we evaluated cxr findings in a total of 302 patients: 85 from aml97; 101 from aml02; and 116 from aml08. common cxr findings were pulmonary opacity (n = 51, 16.9%), bronchial/perihilar thickening (n = 38, 12.6%), splenomegaly (n = 38, 12.6%), mediastinal mass and lymph nodes (n = 27, 8.9%), pleural effusion/thickening (n = 17, 5.6%), demineralization/fracture/periosteal lesions (n = 10, 3.3%), scoliosis (n = 10, 3.3%), and granulomatous disease (n = 7, 2.3%). three cxr findings were associated with younger age at diagnosis: pulmonary opacity (median age, 4.5 years in patients with positive findings vs. 10.1 years in those with negative findings, p<0.01), bronchial/perihilar thickening (median age, 2.7 years vs. 10.3 years, p<0.01), and demineralization/fracture/periosteal lesions (median age; 2.7 years vs. 9.3 years, p = 0.04). two cxr findings were associated with older age at diagnosis: scoliosis (median age, 16.5 years vs. 9.0 years, p<0.01) and granulomatous disease (median age, 15.1 years vs. 9.1 years, p = 0.02). higher white blood cell counts (wbcs) at diagnosis were associated with cxrs showing pulmonary opacity (median wbc; 33.7 × 10^9/l vs. 14.8 × 10^9/l, p = 0.01) or splenomegaly (median wbc; 36.4 × 10^9/l vs. 15.2 × 10^9/l, p = 0.02). french-american-british (fab) m4/m5 subtypes were more frequently associated with pulmonary opacity compared with others (p<0.01). we did not find significant differences between female and male patients. conclusion: cxr in patients with newly diagnosed aml showed a variety of thoracic, abdominal, and bony lesions that are important for the initial evaluation and management. pulmonary opacity was the most common finding and was frequently seen in patients who were younger or had higher wbcs at diagnosis or fab m4/m5. background: children diagnosed with acute lymphoblastic leukemia (all) require a central venous catheter (cvc) to administer chemotherapy safely. both external and internal cvcs carry risks of complications including thrombosis, infection, and possible replacement. internal catheters, such as a port, are generally used for the majority of patients for the duration of treatment since therapy lasts for several years. many institutions place a port at the time of diagnosis. other institutions prefer to start induction therapy via placement of a peripherally inserted central catheter (picc) and defer port placement until the completion of induction therapy due to concerns of increased risk of infectious complications with port placement. objectives: to compare rates of common cvc associated complications by type of cvc placed at start of induction therapy in children treated for newly diagnosed all at the jimmy everest center (jec) at the university of oklahoma health sciences center. design/method: a retrospective chart review analyzed data from newly diagnosed all patients treated at the jec between 2010-2017. data was collected on complications including thrombosis, bacteremia, insertion site infection, cvc malfunction and need for removal. data collection began at the start of induction and was completed at the end of induction therapy. statistical analysis used a univariate and multivariate logistic regression model to compare complication rates between those who had a port versus those who had a picc placed at start of induction. results: data was collected on 128 patients. fifty-six patients had a port placed at start of therapy while 72 had a picc placed. fourteen percent of patients had a cvc associated complication. univariate analysis showed no statistically significant difference in rates of cvc associated complications between the groups (port 16%, picc 12.5% p = 0.564). the rates of hospitalization for cvc associated complications were similar between both groups (port 14%, picc 11% p = 0.590). rates of cvc removal were also similar between both groups (port 4%, picc 4% p = 0.863). multivariate model that included baseline patient characteristics including type of all, patient body surface area, gender, ethnicity and age continued to demonstrate no significant difference in cvc associated complications between both groups. conclusion: this single institution study showed that there was no significant difference in cvc associated complications between port and picc line placement at the start of childhood all induction therapy. port placement can be considered as a safe option at the start of induction therapy. complete remission [cr] or cr with incomplete blood count recovery [cri]) within treatment cycles 1-4. interim data are reported (nct02538965). results: seventeen patients were enrolled and received ≥1 dose of lenalidomide; median age was 12 years (range 5-18); 7 patients were female. patients received median 5 prior regimens (range 2-13). nine patients had previously undergone bone marrow transplantation (bmt). four patients had relapsed aml and 13 were refractory to immediate prior treatment. median duration of study treatment was 4 weeks (range 1-12); patients completed a median of 1 treatment cycle (range 0-3). all patients were evaluable for primary outcome; 1 achieved morphologic cri after 2 cycles (no patients achieved cr). the responder was a 13-year-old male with history of r/r aml after first-and second-line treatment, bmt, and salvage chemotherapy. at baseline, he had a complex cytogenetic karyotype (monoallelic −21q22.12, −3q, −4q13.3, −8p12) with no identifiable molecular mutation; he was also positive for del(5q) (−5q11, −5q23). his post-treatment karyotype showed no abnormalities. sixteen patients experienced treatment failure; 12 due to resistant disease, 3 of indeterminate cause, and 1 had treatment failure before a post-baseline assessment was performed. all patients experienced ≥1 grade 3-4 treatment-emergent adverse event (teae). the most commonly reported were thrombocytopenia (n = 10), anemia (n = 7), febrile neutropenia (n = 7), and hypokalemia (n = 7). fifteen patients experienced ≥1 teae related to lenalidomide. all patients discontinued treatment; 3 remain in follow-up. the study is now closed to enrollment. ten patients died on study: 3 during treatment, 7 during follow-up. all deaths were attributed to aml or complications due to aml. conclusion: third-line lenalidomide monotherapy was associated with clinical response in 1 of 17 pediatric patients with r/r aml; however, treatment exposure was limited. safety data are consistent with the known profile of lenalidomide. lenalidomide was not an efficacious treatment for r/r pediatric aml. funding: celgene corporation, summit, nj, usa. cook children's medical center, fort worth, texas, united states background: it is well documented that pediatric patients with acute lymphoblastic leukemia (all) often experience significant weight gain during induction therapy and later struggle with obesity. however, some patients experience unintended weight loss during induction therapy; since this issue is not well reported, it often goes undertreated. although malnutrition is reported to be associated with decreased survival, increased risk of infection, and loss of lean body mass, there remains a scarcity of in-depth analysis of prevalence and risk factors that contribute to this problem. our study attempts to address this critical yet unmet need. objectives: our aim was to identify the clinical risk factors and outcomes associated with weight loss during induction therapy for pediatric all. design/method: this was a retrospective chart review of patients between 2 and 20 years of age diagnosed with all at cook children's medical center from 4/1/14 to 3/31/17. for each patient, we collected height, weight, age, body mass index (bmi) z-scores at diagnosis and end of induction therapy, risk stratification, and whether consolidation was delayed. patients with a bmi >85th percentile at diagnosis were categorized as being overweight or obese. using logistic regression analyses, we examined which variables predicted whether the patient had an increase or decrease in bmi z-score throughout induction. a critical alpha level of 0.05 indicated statistical significance. results: ninety-six patients met our inclusion criteria. of these, 40% experienced a decrease in bmi during induction therapy. compared to patients whose bmi increased during induction, patients with a decrease in bmi were more likely to be overweight or obese at diagnosis (55% vs. 22%; p<0.001), to be ≥10 years of age (53% vs. 16%; p<0.0001), to have a high-or very-high-risk stratification (87% vs. 31%; p<0.0001), and to experience a delay in the start of consolidation therapy (47% vs. 21%; p<0.01). conclusion: this research highlights a risk not previously identified in the literature that may impact outcomes. patients treated on high-or very-high-risk protocols, who are overweight or obese at diagnosis, and who are ≥10 years of age at diagnosis should be monitored closely for weight loss during induction therapy. patients who experience weight loss should receive prompt intervention. it is our hope that this information can be used for future prospective studies and to help develop evidence-based guidelines. background: 17p abnormalities have been observed in some patients with hematologic malignancies. loss of p53 function as a tumor suppressor gene in the chromosome 17 plays an important role for development of leukemia. these patients usually have poor outcome due to the chemotherapy and are associated with poor prognosis. objectives: this study aimed to identify frequency of 17p abnormalities between iranian children and adult patients with aml (acute myeloid leukemia) malignancy. design/method: the 17p abnormalities were analyzed via bone marrow karyotyping and fish method in 669 acute myeloid leukemia patients. in this study, 17p abnormalities were observed in 52 (8%) patients out of 669 diagnosed cases. a significant strong correlation between 17p abnormalities and other high risk factors (poor risk cytogenetic) were observed. from 52 patients with aml malignancy (17p abnormalities), 45 (86%) patients have complex karyotype, 35 (67%) patients monosomal karyotype and 34 (65%) patients have monosomal karyotype accompanied with a complex karyotype. overall, 17p abnormalities are independent risk factor in acute myeloid leukemia and evaluation of these abnormalities by fish or other complementary techniques prior to treatment, might help for better risk stratification of high risk aml patients. background: hepatotoxicity in treatment of acute lymphoblastic leukemia (all) is well studied and transiently affects most patients receiving antimetabolite therapy. rarely, patients develop liver injury severe or prolonged enough to undergo a liver biopsy. little is known about how these patients differ from patients that develop transient hepatotoxicity. we sought to describe disease and treatment characteristics for all patients that developed hepatotoxicity severe enough to undergo liver biopsy. we also looked for pre-dictive factors for liver biopsy, including signs of early hepatic injury from the initial treatment protocol. design/method: pathology reports of all patients from the liver biopsy database at children's healthcare of atlanta were collected. controls were matched 2:1 for age, all subtype, and treatment protocol. demographics, treatment protocols, and overall outcomes were collected through the electronic health record. hepatic lab results for transaminases, coagulation, and albumin were collected for induction, consolidation, interim maintenance, delayed intensification, and maintenance. results: sixteen patients diagnosed between 2003-2013 (median age at diagnosis 10 years, range 1-16; 50% male; 75% pre-b all) were included in the case series. the median time from diagnosis to liver biopsy was 1.5 years (range 1-11). eight patients (50%) were in maintenance at the time of biopsy; none had active disease. eight (50%) were postbone marrow transplant. biopsy results included: steatosis (3), acute inflammatory/infectious (3), liver infiltration (1), fibrosis (6) and graft-vs-host disease (gvhd) (2). six patients were deceased; 5-year all-cause mortality from diagnosis was 31%. thiopurine methyltransferase (tpmt) status was known in 44% cases and 80% controls. all cases had intermediate or wildtype status, which did not differ from controls (p > 0.05). patients requiring liver biopsy did not have evidence of acute hepatotoxicity (ast/alt > 10× normal values) during their initial treatment protocol. hepatotoxicity requiring liver biopsy is a rare outcome of all treatment. these patients had elevated rates of relapse, bmt, and 5-year all-cause mortality, suggestive of a more severe disease process. however, it is difficult to sort out the temporality of relapse, bmt, and hepatoxicity requiring biopsy in this limited sample. additionally, patients with bmt preceding liver biopsy have other confounding factors that makes them difficult to include in the analysis. finally, our limited descriptive data show no notable correlation between early hepatotoxicity and later indication for liver biopsy. future cohort or case-control studies with larger sample sizes are required to further explore early predictive factors for severe hepatotoxicity requiring liver biopsy. nathan gossai, joanna perkins, michael richards, yoav messinger, bruce bostrom background: the majority of chemotherapeutic agents used to treat hodgkin lymphoma are teratogenic. pregnancy screening prior to the start of chemotherapy is supported by clinical guidelines and baseline testing is a standard component in therapeutic trials. there is limited data available on the incidence of pregnancy screening prior to the start of hodgkin therapy but previous studies suggest that pregnancy screening, especially at pediatric institutions, is not consistently completed. objectives: the objective of this study is to evaluate the incidence of pregnancy screening and contraceptive counseling prior to the start of therapy in females diagnosed with hodgkin lymphoma. design/method: a retrospective chart review was performed for all female patients newly diagnosed with hodgkin lymphoma from 2000 to 2015 at the hospital for sick children in toronto, ontario. all patients who were intended to receive multi-agent chemotherapy were included, regardless of age. data collected included demographic and disease information, chemotherapy regimen and enrollment on clinical trial. all pregnancy testing within two weeks prior to the start of therapy was captured, as well as type of pregnancy test performed, documentation of menstrual status, contraceptive counseling and contraceptive provision. univariate and multivariate analyses were used to describe factors influencing the incidence of pregnancy testing. results: a total of 122 female patients with newly diagnosed hodgkin lymphoma between the ages of 5 and 17 years were identified. sixty patients (49%) had pregnancy testing done prior to the start of therapy. testing modalities included serum and urine screens as well as quantitative beta-hcg measures. older age (p = 0.0016), documentation of menstrual status at diagnosis (p = 0.019) and diagnosis between 2008 and 2015 (p = 0.004) were associated with higher incidence of screening. enrollment on a therapeutic trial was not associated with a higher incidence of screening (p = 0.374). contraceptive counseling was documented for 19 patients (16%) and 11 patients (9%) were prescribed contraceptive medications during therapy. pre-chemotherapy pregnancy testing was completed on 49% of females with newly diagnosed hodgkin lymphoma. improvement is required and interventions, including clarification of institutional standards, modification of chemotherapy order sets and staff education, are planned. (rao et al., cancer, 2016) . university of louisville, louisville, kentucky, united states background: granulocytic sarcomas (also known as chloromas or leukemia cutis) were first described by a. burns in 1811. they are solid tumors comprised of immature granulocytic cells and represent extramedullary manifestations of underlying leukemia. chloromas are most commonly associated with acute myeloid leukemia. they may arise in other myeloproliferative disorders but are rarely seen in b or t cell acute lymphoblastic leukemia (all). objectives: although patients with all rarely have chloromas, it should remain on the differential for patients with unusual swelling or masses. design/method: we present a case series of two patients from our institution diagnosed with b cell all who had a chloroma as the presenting symptom. the first patient is a 4yo who presented to his primary provider with nasal congestion and a one-week history of bilateral eye swelling and was referred to an allergist when the symptoms did not resolve with anti-histamines. his review of systems was otherwise negative. he was referred urgently to ent two months later for a 3 × 3cm mass palpated along the medial border of the left eye. an mri showed a left facial mass surrounding the zygoma and extending into the anterior inferior left orbit. biopsy revealed b cell acute lymphoblastic lymphoma, and bone marrow aspirate and biopsy confirmed the diagnosis as b cell all. the second patient is a 10yo who presented to his primary doctor for rapid growth of a scalp nodule that had been present for about 2 months. he was referred to dermatology and treated for a supposed kerion from tinea capitis. the lesion continued to grow and became more irritated with this treatment. punch biopsy revealed a complicated phenotype of lymphoblastic lymphoma. however, after a lymph node biopsy and bone marrow aspirate and biopsy, the diagnosis was confirmed as b all. his only other positive point on review of systems was a questionably pathologic 20-pound weight loss and an area of matted cervical lymph nodes. for both of our patients, the chloromas completely disappeared during induction therapy. it is worth noting that both of these patients presented with the chloroma as the only symptom of the underlying leukemia. this led to initial misdiagnosis and delay in identifying their leukemia. therefore, while it is very rare for a patient with b all to present with a chloroma, our experience shows that all should be on the differential for patients presenting with unusual swelling or masses. background: hodgkin lymphoma (hl) is a lymphoproliferative neoplasm that commonly presents with history of adenopathy and a predictable pattern of disease involvement with or without systemic symptoms of fever and/or weight loss. in the hands of an experienced oncologist the diagnosis of hl is usually not a challenge. occasionally a diagnostic challenge is presented by a patient who has an atypical presentation which is suggestive of an alternative diagnosis. we describe a case series of patients diagnosed with hl whose initial clinical presentations lead to a diagnosis different form hl. honduras, nicaragua and the united states. results: six pediatric oncology centers from the american continent conducted a retrospective review of patients diagnosed with hl since 2010. patients that had an initial presentation not suggestive of hl or who were initially diagnosed with a disease other that hl were included for a total of 25 patients. argentina n = 8, guatemala n = 7, honduras n = 1, nicaragua n = 2, united states n = 7. five patients were female and 20 male. patient's ages ranged from 2 to 18 years. most patients (n = 19) were older than 11 years. three patients (15%) presented with non-immune cytopenias without overt lymphadenopathy, of those one had active hemophagocytic syndrome. five patients (20%) were suspected to have localized solid tumors: ewing sarcoma n = 2, rhabdomyosarcoma n = 1, hepatocellular carcinoma n = 1, and soft tissue tumor of the cheek n = 1. two (8%) metastatic solid malignancy as they presented with disseminated pulmonary nodules. five (20%) with autoimmune disorders: hashimoto thyroiditis n = 1, autoimmune hemolytic anemia n = 1, nephrotic syndrome n = 3. ten (40%) with chronic infectious processes: brucella n = 1, tonsillar abscess n = 1, splenic abscess n = 1, and tuberculosis (tb) n = 7. patients with suspected tuberculosis were diagnosed outside of the united states. six of 7 patients were ultimately diagnosed as having both tb and hl. seventeen patients had ann-arbor stage iii or iv, seven patient had stage ii with either b symptoms or bulky disease. patients were treated with various chemotherapy regimens according to the treating center: abvd, abve-pc oepa-copdac, avpc, beacopp. two patients had recurrent disease, one died of disease progression and one died from causes not related to hl conclusion: a small proportion of hl patients have atypical or unusual presentations. hl should be included in the differential diagnosis of solid tumors, autoimmune disorders, infections or cytopenias. the most common atypical presentation is an infectious process. background: acute lymphoblastic leukemia (all) represents the largest group of pediatric malignancies. the high cure rate of childhood all represents one of the most remarkable success stories in the war on cancer. in a lower middle income country (lmic) like the philippines, we reviewed the five year survival in a tertiary referral center. objectives: this retrospective cohort study aims to determine the survival of children 1-18 years old with all treated at a tertiary referral center for childhood cancer in the philippines from january 2012 to december 2016. design/method: this is a retrospective cohort study that reviewed medical charts of newly diagnosed all ages 1 to 18 years old from january 2012 to december 2016. a total of 435 subjects were included in the study. the 5 year overall survival (os) and event free survival (efs) were 65.3% and 62.8%, respectively. the 5 year os for standard risk all was 68.8% and for high risk patients was 50%. the 5 year os for the patients on remission was 83.7% and for those who relapsed was 21.1%. univariate and multivariate by cox proportional hazards regression revealed wbc count at diagnosis, risk classification, immunophenotyping, and development of relapse showed significant prognostic impact for mortality. age and gender were reported with no prognostic significance. the 5-year os and efs were lower compared to developed countries but are comparable with other lmics. the prognostic factors for relapse and mortality were compatible with the literature. overall, the adopted treatment protocols for childhood all in this institution showed acceptable results. relapse has a significant prognostic impact for mortality. development of accessibility to care, increase awareness, early detection and resources at hand should be achieved. improvement in the follow up protocol to prevent delays in the treatment, patient education to prevent non-compliance and psychosocial support, to developed better supportive care, and expand facilities should be given emphasis to further improve survival and prevent relapse. objectives: here, we seek to further characterize this entity by describing the pathologic and clinical features of 4 pediatric cases of burkitt-like lymphoma with 11q aberration. we collected pathologic and clinical data from the medical record on all pediatric high grade b-cell lymphoma (hgbcl) cases diagnosed at our institution over a 5-year period (2012-2017) . for those cases classified as neither burkitt lymphoma nor diffuse large b-cell lymphoma (dlbcl), fish for myc, bcl-2 and bcl-6, as well as array comparative genomic hybridization (acgh), were performed. we identified 38 cases of hgbcl, including 5 cases of burkitt lymphoma presenting as purely leukemic phase. of the hgbcl cases, 20 had burkitt lymphoma as defined by myc rearrangements, and 13 had dlbcl. collectively, the majority of these 33 patients had primary disease outside of the head/neck, and most patients presented with advanced stage (iii-iv) disease. of the 5 remaining cases, 11q aberration was identified in 4 cases using acgh. all 4 cases histologically and immunophenotypically resembled burkitt lymphoma but lacked myc rearrangement, instead showing proximal gains in 11q13-q23 and telomeric losses in 11q24.1qter. all 4 cases involved primary disease in the cervical lymph node and/or tonsil. three of these cases were localized (stage ii), and the fourth case involved a few metabolically active but non-enlarged lymph nodes in the chest and abdomen (stage iii). all 4 patients achieved complete remission with standard therapy for mature b-cell lymphoma, and were alive with no clinical evidence of disease at a median follow-up of 23 months. although the number is small, our results suggest that the majority of non-burkitt, non-dlbcl cases of pediatric hgbcl carry 11q aberrations. in addition, patients with 11q aberrations appear to be more likely to present with lower stage disease, thus requiring less intensive therapy, and also tend to have primary disease in the head/neck. these findings further support the classification of burkitt-like lymphoma with 11q aberration as a distinct pathologic and clinical entity, and we propose that all pediatric non-burkitt, non-dlbcl cases of hgbcl regularly undergo further workup for possible 11q aberrations. marie claire milady auguste, joseph bernard st damien hospital, port-au-prince, port-au-prince, haiti background: hodgkin lymphoma (hl) and non-hodgkin lymphoma (nhl) account for 7% of cancers in the united states pediatric population (1, 2). in central america and the caribbean, they are in second position among all types of pediatric cancers (3). a previous study on pediatric cancers in haiti showed that the lymphomas were in fifth place after the leukemias, wilms tumor, retinoblastoma and the sarcomas (4). the main objective of this study is to present the epidemiological profile of lymphomas managed at a haitian pediatric hospital. design/method: this is a retrospective study conducted on the cases of lymphoma diagnosed and managed at st damien hospital from january 2006 to december 2016. key variables such as age, gender, stage at diagnosis, histopathological types and outcome were collected to present the characteristics of this retrospective cohort. of the 407 cases of cancer diagnosed during the study period, 23 (5.7%) had the diagnosis of lymphoma. the sex ratio was 2.8 (17 males for 6 females) and the average age was 8.9 years [0-19 years]. there were 11 cases of hl (47.8%) and 12 cases of nhl (52.2%). 69.6% of the patients were diagnosed at stages iii and iv. among the hl cases, 6 (54.5%) were nodular sclerosis lymphoma, 3 (27.3%) with mixed cellularity and 2 (18.2%) with lymphocytic predominance. for the nhl cases, 4 (36.4%) were burkitt's lymphoma and 3 (27.3%) lymphoblastic t-cell lymphoma. among the 12 patients for who immunohistochemistry was found, the 4 cases of hl were cd30-positive and 6 out of 8 cases of nhl were cd20-negative. only 1 patient was hiv-positive, and 4 patients had a confirmed exposure to epstein-barr virus. 8 patients (34.8%) were lost to follow-up, 7 (30.4%) were in remission, 3 (13 %) relapsed, 2 (8.7%) were still in treatment and 3 (13%) were deceased. university of chicago, chicago, illinois, united states background: due to the adoption of risk-adapted therapy, pediatric and adolescent acute lymphoblastic leukemia (all) is associated with high cure rates. despite excellent outcomes in most children, patients with certain blast cytogenetic features do not fare as well. furthermore, african american, native american, and hispanic patients have worse outcomes than caucasian patients. while the outcome discrepancies are certainly multifactorial, and blast cytogenetics are related to age, it remains unclear whether ethnicity and blast cytogenetics correlate. the diverse patient population at the university of chicago provides an opportunity to evaluate for such a correlation. objectives: to describe cytogenetic findings in a racially and ethnically diverse population of patients of all age groups diagnosed with all at university of chicago from 2006 to 2016 and determine if there is a correlation between race/ethnicity and blast cytogenetics. results: a total of 191 newly diagnosed patients with all between the ages of 1-100 from 2006-2016 were included in this study. of those, 167 patients (87.4%) had b-all, 22 had t-all (11.5%), one had early t-cell precursor all and one had mixed phenotype all (b/t). caucasians accounted for 46% of patients, african americans (aa) 22%, hispanics 24.6%, asians 5.24%, and 2% were of other races. age distribution had a bimodal pattern, with a peak in incidence at 5 and another at 58 years of age, consistent with published data. cytogenetic categories included: t(12;21)(p13;q22), 11q23 rearrangements (kmt2a), iamp21, t(1;19)(q23;p13.1), t(9;22) (q34;q11), hypodiploidy, hyperdiploidy and double trisomy of chromosomes 4 and 10. aa and hispanic patients with b-all presented more frequently between the ages of 10-18 years compared to caucasians (p = 0.002 and 0.02, respectively). in aa patients, t(1;19) (q23;p13.3) was overrepresented (p = 0.04 when compared to caucasians), and was mainly observed in patients between 10-18 years. caucasian patients were more likely than non-caucasians to have hyperdiploidy (p = 0.04), especially in patients aged 1-9 years. the rate of t(1;19)(q23;p13.2) was significantly higher in aa patients in our cohort, in particular in patients between the ages of 10-18 years. hyperdiploidy was more likely in caucasians aged 1-9 years. these findings may suggest that varying blast cytogenetics could contribute to outcome differences between races. ahmed elgammal, yasser elborai, mohamed fawzy, asmaa salama, eman d el-desouky, lobna shalaby national cancer institute, cairo, cairo, egypt background: hodgkin lymphoma (hl) in children is one of the malignancies that have a high chance of cure. stage iv hl remains a challenge for getting good clinical outcome as in other stages. many treatment protocols used to give combination chemotherapy while combined modality treatment is the mainstay in other treatment protocols. objectives: we aimed in to assess the outcome using consolidation radiotherapy to chemotherapy (combined modality treatment) versus combination chemotherapy alone in treatment of stage iv hl. design/method: we included patients with stage iv hl and whose data were retrieved from the medical records of the pediatric oncology department, national cancer institute, cairo university, egypt from 2005 till june 2013 and were followed till august 2015. treatment was either to give 8 cycles of abvd (adriamycin, bleomycin, vinblastine, dacarbazine) only or to give 6 cycles of abvd followed by consolidation radiotherapy. the study included 22 cases; 17 were males and 5 were females. mean age was 10.66 years ranging from 4 to 17 years. the histopathology subtype was nodular sclerosis in the majority of cases (15 cases) followed by mixed cellularity (6 cases) then only one case of lymphocyte rich. nine cases were initially bulky while 13 cases were not. constitutional manifestations were present in 10 cases while it was absent in 12 cases. bone marrow was involved in only 4 cases. radiotherapy was given after completion of chemotherapy to 10 cases while 12 cases received chemotherapy only. the 5-year overall survival for patients who received radiotherapy was superior to those who received chemotherapy alone; 100% versus 45.8% respectively with statistical significance (p = 0.02). the 5-year progression free survival was also higher with radiotherapy than others; 90% versus 44.4% (p = 0.095). patients with stage iv hl who received consolidation radiotherapy apparently had a better outcome than those who received chemotherapy only. this suggests that radiotherapy contributes significantly with chemotherapy to the cure rate for those patients. the feinstein institute for medical research, manhasset, new york, united states background: microrna (mirnas) are short non-coding rnas that play a decisive role in cancer biology, including leukemia. exosomes are microvesicles (30-100 nm) produced by most cells in biological fluids. exosomes represent the fingerprint of the parental tumor and are loaded with bioactive markers such as mirnas, which may regulate tumor growth. exosomal cargo can be transferred into target cells changing their biological properties. our study investigates a functional role for exosomal mir-181a in pediatric acute lymphoid leukemia (p-all). objectives: 1/ to demonstrate that p-all exosomes induce cell proliferation2/ to confirm that exosome-induced cell proliferation is disease-stage specific 3/ to analyze exosomal mir-181a expression profiles in p-all4/ to authenticate that inhibition of exosomal mir-181a reduces leukemia proliferation design/method: exosomes were isolated by ultracentrifugation from healthy donors (hd) & p-all serum and conditioned medium (cm) of sup-b15, jm1, and cl-01 (control) human cell lines. cell lines were exposed to different sources of leukemia-derived exosomes in a paracrine or autocrine fashion for 24hrs in triplicates. proliferation was assessed by microscopic cell counting and confirmed by gene expression for proliferation, pro-survival and pro-apoptotic genes. mirna profiling was performed with the human cancer pathway finder microarray (qiagen). silencing of exosomal mir181a was carried out by a mir-181a inhibitor (qiagen), utilizing exo-fecttm exosome transfection reagent (sbi, system biosciences). further, exosomal mir-181a silencing was confirmed by q-pcr. cellular uptake of texred-sirna (sbi, system biosciences) was confirmed by flow cytometry. transfer of exosomal mir181a to the target cells was evaluated by q-pcr. we elucidated that cm-derived exosomes from sup-b15 and jm1 cell lines induce cell proliferation in sup-b15, jm1 (autocrine and paracrine) and cl-01 cells (paracrine) (p<0.01). serum p-all exosomes promote paracrine cell proliferation in all cell lines compared to hdderived exosomes (p<0.0001). heatmap analysis of mirna profiles of leukemia exosomes (all cell lines and p-all) identified mir-181a significantly upregulated in leukemia exosomes compared to controls. mir-181a was also upregulated in all cell lines after exposure to leukemia exosomes that induced proliferation. moreover, exosomal mir-181a inhibition reduces leukemic proliferation in pediatric all. our data suggest that all exosomes induce cell proliferation of leukemic cell lines in both paracrine and autocrine fashion. exosomes regulate these phenomena in a highly orchestrated way, by transfer of functional exosomal mirnas such as mir-181a. the results of this study suggest s233 of s301 that exosomal mir-181a inhibition can act as a novel way for growth-suppression of pediatric leukemia. results: a total of 199 disease sites were detected at pet/ct, while 172 sites were detected at contrast-enhanced ct and bone marrow biopsy (bmb). pet/ct showed improved detection of nodal lesions (p <0.0001) (kappa value = 0.633), extranodal lesions (p <0.0001) (kappa value = 0.632) and bone marrow (p <0.0001) (kappa value = 0.728) compared to contrast enhanced ct and bmb. pet/ct had upstaged 15 cases (16%) and down-staged 4 cases (4.3%) (p <0.001) (kappa value = 0.649). among the upstaged 15 cases, 10 patients (10.9%) were upstaged from stage ii to iii, based on residual in pet/ct not seen in contrast enhanced ct after abdominal mass excision. four patients (4.3%) were upstaged from stage iii to iv based on bone marrow uptake in fdg-pet without positivity in bma or bmb.regarding response assessment, sensitivity was 60% for pet and 80% for contrastenhanced ct (p = 0.56). specificity was 100% for pet and 65% for ct (p< 0.0001). positive predictive value for pet was 100%, while was 12% for ct scan (p< 0.0001). negative predictive value for both pet and ct was 98% (p = 0.82). five patients had 2nd biopsy to confirm viability of the residual lesions, 4 lesions were negative in pathological examination (all of them were metabolic negative in pet/ct; deauville score below 4). one lesion was positive in pathological examination (was positive in pet/ct; deauville score of 4). conclusion: pet/ct detected additional sites compared with contrast-enhanced ct and resulted in changing stage of disease. pet scan is significantly more specific than ct in the management of children with burkitt lymphoma. background: deep sequencing of the immunoglobulin heavy chain (igh) locus indicates that each b all is composed of innumerable subclones. in many cases, subclones exhibit differing phenotypic qualities. however, it remains unclear whether subclones demonstrate distinct tissue distribution within a patient. objectives: 1. to quantify the extent of clonal heterogeneity in diagnostic b all specimens; 2. to identify variability in clonal composition between bone marrow (bm) and peripheral blood (pb) disease sites. design/method: igh sequencing was performed on purified dna from 10 pairs of matched bm and pb patient specimens. multiplex pcr was used to globally amplify the igh locus; next generation sequencing (ngs) was performed using illu-mina® miseq. index clones (defined as ≥ 5% of all sequence reads in a specimen) and their subclone progeny (defined by 6 shared nucleotide bases immediately upstream of a common jh, or 6n_jx) were identified using igblast-determined vh and jh alignments (http://www.ncbi.nlm.nih.gov/igblast/) and an established in-house computational pipeline. results: up to 3 index clones per specimen were discovered in 16 of the 20 samples. in the remaining 4 (2 bm/pb pairs), 1 pair did not reveal a clonal igh and was eliminated from analysis; in the other, clone frequency did not reach the 5% index threshold, but predominant clonal precursors were inferred by the prevalence of their subclone progeny. subclone counts ranged from 2 to 2,619 per index clone. a combined 2,900 subclones derived from 11 pb index clones were observed; in contrast, 12 bm index clones gave rise to only 400 subclones. subclone heterogeneity was observed between all paired specimens. in 6 bm/pb pairs, index clones existed in equivalent proportions between disease sites. in contrast, 1 bm/pb pair demonstrated 2 high-frequency index clones in the bm (32.6% & 12.5%) with limited representation of these clones in the pb (0.6% & 1.4%, respectively); in this case, the most prevalent clone in the pb (10.8%) matched the least frequent index clone in the bm (5.0%). similarly, another pair showed a predominant index clone in the pb (5.8%) which was below index threshold (3.6%) in the bm. in 2 paired patient specimens, index clone predominance was discovered to be overtly distinct between bm and pb. among all pairs, the extent of subclone progeny derived from each index clone showed marked variability, with far higher subclone frequency in the pb than in the bm. our data indicate that b all clonal composition differs between disease sites. valley children's healthcare, madera, california, united states background: tuberculosis (tb) presenting with hodgkin lymphoma (hl) is rare. their coexistence could lead to delay in diagnosis of both tb and hodgkin lymphoma due to the similarities in signs and symptoms of presentation. most cases have been reported in the adult literature. we describe a case series of 11 children that were suspected to have tb and were found to have coexisting tb and hl. results: a retrospective review of hl patients in guatemala and argentina over 6 six years, uncovered 11 patients with simultaneous diagnosis of tb and hl. eight patients were from guatemala (incidence of 4.5%) and 3 from argentina (incidence of 2.5%). there were 4 females and 7 males. age ranged from 4 -17 years (mean 10.5 years, media 9 years). nine patients were suspected to have tb at presentation by the referring physician. two patients were found to have tb at the time of relapse through routine tissue culture. initial systemic symptoms included fever (n = 5), weight loss (n = 2), and night sweats (n = 2). six patients had a second systemic symptom in addition to fever. time for referral to oncology center ranged from 2 weeks to 5 months. nine patients were diagnosed with tb and hl through a tissue cultures and 1 with serum quantiferon. one patient was found to have hl without tb. two patients had no systemic symptoms and the diagnosis of tb came to light through routine tissue culture. five patients had stage iiib and ivb, two stage iia and one iib at diagnosis. hl treatment was given according to the insti-tutional standards depending on stage and risk with abvd, oepa/copdac +/-radiation therapy, and ice for relapse. five patients started anti tb treatment (isoniazid, rifampin, pyrazinamide +/-ethambutol for 2 months followed by isoniazid and rifampin for 30-52 weeks) simultaneously with chemotherapy, and three others after completing 2 cycles. the two relapsed patients started tb treatment after 2 cycles of chemotherapy. seven patients are alive and have been followed for 5 months -6 years. one patient died during therapy, another died for causes not related to tb or hl and one is currently receiving treatment. conclusion: tuberculosis can coexist with hl. in areas were the prevalence of tb is high, microbiology investigations of biopsy specimen should be strongly considered. therapy for tb can be given simultaneously with chemotherapy. coexistence of tb and hl does not appear to affect outcomes. the children's hospital affiliated to the capital institute of pediatrics, united states background: the pi3k/akt signaling pathway plays a central role in cell growth, proliferation and survival in physiological conditions. this signal pathway is considered to be an innovative targeted therapy of cancer, and its abnormal activation has been proved to be related to t-cell acute lymphoblastic leukemia (t-all). despite improved treatment strategies, such as multi-drug combination, high-dose chemotherapy and all kinds of application and popularization of hematopoietic stem cell transplantation, children with drug resistance or relapse t-all are still rather worse and its overall outcome and prognosis are much poorer than the more common b-lineage all. objectives: to explore the relationship between the pi3k/akt pathway and the pediatric t-all, so as to probe the exact molecular mechanisms of t-all and provide more directions for its treatment. design/method: 7 cases of new or recurrent acute t lymphocyte leukemia children with clinical information were collected in the children's hospital affiliated to the capital institute of pediatrics from dec.2015 to oct.2017, with 7 age and gender matched healty children as control (all was informed consent). the expressions of key genes in pi3k pathway were s235 of s301 analyzed by western blot rt-pcr analysis, the pi3k enzyme activities were detected by elisa,and the ccrf -cem's proliferation and its apoptosis were tested by mtt and flow cytometry technology on t-all cell lines ccrf-cem in different treatment group. the results of t-all children in clinical showed that pi3k protein and gene expression level were higher apparent than the control group (p<0.05), and pi3k enzyme activity increased as well (p<0.05); pi3k inhibitor ly294002 made a significant inhibition of cell proliferation and promoted cell apoptosis. ly294002 also enhanced the effectiveness of clinical commonly used chemotherapeutic drug dnr. in combination ly294002 and dnr treatment group cell viability dramatically declined, apoptosis and the apoptosis relation protein casepase3 expression in t-all patients was obviously higher than the control and the single drug group; pi3k/akt signaling pathway related proteins and gene expression level, pi3k, akt, gsk3 transcription in ccrf-cem were significantly higher than the control (p< 0.05), while pten transcription was significantly lower than the control (p<0.05). the abnormal activation of pi3k/akt signaling pathway might play an important role in pediatric t-all patients, especially in the cell proliferation or apoptosis. the results might provide new train of thought and direction in targeted suppress this signal pathway or in combination with other chemotherapy drugs therapy in looking for the more effective and less cytotoxic treatment of pediatric t-all. cleveland clinic children's hospital, cleveland, ohio, united states background: non-hodgkin lymphomas (nhls) are a heterogeneous group of lymphoproliferative diseases which comprise 7% of all childhood malignancies. nhls can be divided in to b cell lymphomas and t cell/natural killer (nk) cell lymphomas depending on immunophenotype, molecular biology, and clinical response to treatment. although nk/t cell lymphomas occurring in childhood and adolescence comprise a small portion of all lymphomas, they present many diagnostic and therapeutic challenges. the role of angiogenesis in lymphoma pathogenesis is becoming more evident. high molecular weight kininogen (hk) is a central compo-nent of the kallikrein-kinin system. it has been previously reported that cleaved hk (hka) induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in matrigel plug and corneal angiogenesis models. however, the role of endogenous kininogen in regulation of angiogenesis is in tumor microenvironment is unknown. objectives: to elaborate the role of hk in lymphoma angiogenesis, we used a murine t-cell lymphoma model and compared angiogenesis and tumor growth between wild-type and kininogen deficient (mkng1-/-) mice. we also evaluated the effect of hka on lymphoma cell proliferation. design/method: el-4 murine t-cell lymphoma cells (5 × 10^5) were implanted into wild-type and mkng1-/-mice. tumor size was measured using calipers and tumor volume was calculated using the formula volume = length × width^2 × 0.52. seventeen days after cell implantation, tumors were harvested and processed by immunoblotting and immunofluorescent staining. cell proliferation assays (mts) were performed to investigate any possible inhibitory effect of hka on el-4 cell growth, with human umbilical vein endothelial cells (huvec) were used as a positive control. results: el-4 lymphomas grew more rapidly and to larger sizes in mkng1-/-mice compared to wild-type mice, with significant differences apparent by day 11 after tumor implantation (p<0.01). by day 17, the volume of tumors in mkng1-/-mice was approximately 1.4-fold larger than in wild-type mice (mean volume ± standard deviation; 2120 ± 536 vs. 1485 ± 272 mm3, respectively, p<0.01). mts assays showed that hka does not directly inhibit the proliferation of el-4 cells in vitro, though it does significantly impair the viability of ecs studied simultaneously. conclusion: these findings suggest that hk is an important endogenous regulator of angiogenesis and tumor growth in this t-cell lymphoma model, and suggests that hka specifically modulates endothelial proliferation in tumor microenvironment. further work is needed to understand the mechanisms underlying these findings and provide future anti-angiogenic approaches to increase the therapeutic options for patients with nhl. bruce bostrom, jack knudson, nathan gossai, joanna perkins, michael richards, jawhar rawwas, susan sencer, julie chu, nancy mcallister, yoav messinger children's minnesota, minneapolis, minnesota, united states background: osteonecrosis causes significant pain and morbidity in older patients treated for acute lymphoblastic leukemia. besides altering the schedule of dexamethasone in delayed intensification there is no other intervention known to reduce the incidence of symptomatic osteonecrosis. pamidronate has been shown to reduce bone pain from osteonecrosis but not to prevent joint collapse when advanced. objectives: to compare the incidence of symptomatic osteonecrosis in patients who received prophylactic pamidronate compared with concurrent controls. to describe any increase in side effects from the use of pamidronate. design/method: patients age 10 to 28 years at time of all diagnosis were given intravenous pamidronate monthly for one year at the discretion of the primary oncologist starting in the first year of therapy. concurrent controls were patients age 10 to 28 who did not receive pamidronate. all patients were treated according to the concurrent cog protocols and received intermittent dexamethasone during delayed intensification. patients with bcr-abl all were excluded as the use of imatinib may increase the risk of osteonecrosis. imaging was only done if osteonecrosis was suspect based on clinical symptoms. patients were censored at the time of relapse. data were analyzed as of 1/1/2018. this retrospective study was approved by the children's minnesota irb. of the 62 patients evaluated 58% were male and 42% female, 74% had b-cell and 26% t-cell. the median followup is 2.4 years with a range of 0.3 to 7 years. pamidronate was given to 23 patients with 2 developing symptomatic osteonecrosis. there were 39 concurrent controls with 14 developing osteonecrosis. there was no significant difference in the leukemia lineage, gender distribution or body mass index (bmi) at diagnosis between groups. for all patients the median bmi was 22 with a range of 15 to 47. the age at diagnosis was significantly higher in the pamidronate group with a median of 18.6 years vs. 15.7 in the controls (p = 0.014). by kaplan-meier analyses the incidence of symptomatic osteonecrosis was significantly lower in the pamidronate group at 14% vs. 43% in controls. the log-rank p-value was 0.049 and the breslow p-value, which is more sensitive to early events, was 0.039. there were no untoward side-effects from pamidronate. pamidronate infusions significantly reduced the incidence of symptomatic osteonecrosis in patients over the age of 10 compared to concurrent controls who did not receive pamidronate. arahana awasthi, dina edani, janet ayello, christian klein, mitchell cairo new york medical college, valhalla, new york, united states background: mature b-nhl, including bl and pmbl express cd20+/cd79b+ and have an excellent prognosis, however, subset of patients relapse secondary to chemoimmunotherapy resistant disease and have a dismal prognosis (≤ 20% 5 yr. efs, cairo et al. blood. 2007; gerrard/cairo et al., blood, 2013 , goldman/cairo et al. leukemia, 2013 . pv has been demonstrated to possess significant preclinical activity against indolent cd79b+nhl (polson et al. can. res.2009 ). we previously observed that obinutuzumab (anti-cd20 mab) significantly enhanced cell death and increased overall survival against bl (awasthi/cairo et al., bjh 2015) in xenografted nsg mice. however, additive/synergistic effects of pv with obinutuzumab against mature pmbl/bl are unknown. to determine the efficacy of the pv or obinutuzumab/rtx alone or in combination against pmbl and rituximab (rtx) sensitive/resistant bl cell lines. design/method: raji4rh (provided by m. barth, md, roswell park cancer institute) and raji/ karpas1106p (atcc, usa) were cultured in rpmi. tumor cells were incubated with pv, and/or anti-cd79b, mmae (generously supplied by genentech inc.) with obinutuzumab /rituximab (100ug/ml) for 4 hr with nk cells at 10:1 e: t ratio and cytotoxicity was determined by delfia cytotoxicity assay. six to 8 week old female nsg (nod.cg-prkdcscid il2rgtm1wjl/szj), were divided into 5 groups: pbs, isotype control, pv, anticd79b mab and mmae (5mg/kg). mice were xenografted with intravenous injections of luc+ bl and pmbl cells and tumor burden was monitored by ivis spectrum system. results: os of mice receiving pv alone was significantly increased compared to anticd79b ab or isotype control in raji (35.5 vs.17 vs.19.5 our preliminary data indicates that pv significantly increased survival in bl and pmbl nsg xenografts compared to anti-cd79b ab alone. furthermore, pv in combination with obinutuzumab significantly enhances in-vitro cytotoxicity in bl and pmbl compared to obinutuzumab or pv alone. results: maximal grades (g)1/2, 3, and 4 crs occurred in 36, 19, and 24 patients, respectively. median lowest fibrinogen levels were 3.5, 3.3, and 1.2g/l in patients with maximal g1-2, 3, and 4 crs, respectively. 3%, 11%, and 25% of patients with maximal g1-2, 3, and 4 crs had lowest reported fibrinogen levels of ≥1 to <1.5g/l. eight patients (all with g4 crs) had very low fibrinogen levels (<1g/l), which occurred before (n = 1) or during (n = 6) maximal crs grade or at time of improvement (n = 1). no patients with maximal g1-3 crs had <1g/l fibrinogen levels. at the onset of <1g/l fibrinogen levels, 1 patient had concurrent g3, and 7 had g0-2 increased international normalized ratio and activated partial thromboplastin. cryoprecipitate was the primary treatment in the us, and fibrinogen concentrate (fc) guidelines for tisagenlecleucel-associated coagulopathy were developed for other countries because administration of fresh frozen plasma can be problematic. fc was available at 7/25 sites for 20 infused patients: 3/7 (g4 crs) and 0/8 (g1-3 crs). cryoprecipitate was available at 18/25 sites for 77 infused patients: 12/17 (g4 crs), 2/15 (g3 crs), and 0/32 (g1-2 crs). risk of bleeding increases in pediatric patients with comorbid thrombocytopenia and anticoagulant treatments. 5/8 patients had g3/4 decreased platelets within 1 day of <1g/l fibrinogen levels. 1 fatal case of intraparenchymal cranial hemorrhage occurred during resolving crs with g3 hypofibrinogenemia, ongoing thrombocytopenia, and continuous veno-venous hemofiltration with citrate. hypofibrinogenemia was observed more frequently in patients with higher crs grades during/when crs was improving or resolving. fc and cryoprecipitate treatment guidelines were developed. frequent monitoring and fibrinogen replacement are needed in patients with g3/4 crs. sponsored by novartis. its prolonged cns half-life, may allow a reduction in the number of intrathecal injections. objectives: to safely reduce the burden of therapy by reducing the number of it injections and reducing the total dose of doxorubicin with the addition of liposomal cytarabine and rituximab. design/method: patients (3-31 years) with cd20+ b-nhl with fab group b good risk (=stage i/ii and stage iii with ldh < 2xuln), fab group b intermediate risk (=stage iii ldh ≥2xuln and stage iv {bm blasts < 25%}) and fab group c high risk were eligible. patients received fab backbone therapy with the addition of six rituximab (375mg/m2) doses; two doses prior to each of two induction courses and one dose prior to each of two consolidation courses. cumulative doxorubicin was reduced from 120 to 50 mg/m2 in gr patients. after systemic methotrexate clearance, patients received age based dosing of it liposomal cytarabine. it injections were reduced from nine to five. the primary outcome is safety and toxic deaths among 40 evaluable patients with an estimated 3-year survival above 90%, monitored by an independent dsmb. results: to date, 32 evaluable patients, 25 fab group b and 7 group c (6 cns positive), median age 12 years (range 3-23), 20 males, 16 burkitt/16 dlbcl with 18 gr, 7 ir and 7 hr have enrolled. there has been one grade 3 anaphylactic reaction to rituximab and one grade 3 facial nerve palsy. no other serious adverse events were attributable to protocol therapy. there has been 1 death from progressive disease and 1 relapse at a median follow up of 30 months. efs and os are 94% and 97%, respectively. our initial results show excellent efs and os, consistent with published standard of care outcomes, with the addition of rituximab and intrathecal liposomal cytarabine despite the reductions in therapy. further enrollment is ongoing and continued long term outcomes are needed to confirm early results. future randomized studies are needed to examine both short term (mucositis, infections, hospitalization days) and long term (late cardiac toxicity) endpoints. 1. goldman etal, leukemia, 2013 2. cairo etal, jco 2012 st. jude children's research hospital, memphis, tennessee, united states background: bereaved parents identify significant spiritual needs around time of death and throughout their bereavement journeys. spirituality has been identified as a primary means by which bereaved parents can find meaning in their losses, and this ability to find meaning is associated with lower maladaptive grief symptoms. the use of spiritual coping strategies has been associated with improved coping and mental health outcomes among bereaved parents. objectives: to better understand how bereaved parents' experiences with spirituality throughout bereavement effects objective measures of grief, depression, and meaning-making. design/method: thirty participants whose children died of progressive cancer or related complications one to three years prior to participation completed an in-depth semi-structured telephone interview about their experiences with grief. participants were prompted to describe the impact of their spirituality on their bereavement processes. additionally, participants completed surveys related to grief (prolonged grief disorder questionnaire, pg-13), depression (beck depression inventory, bdi), and meaning-making (integration of stressful life experiences scale, isles). results were analyzed using a mixed methods approach including semantic content analysis of qualitative content and kruskal-wallis h test and post-hoc analyses of quantitative data. results: correlation analyses demonstrated significant differences between participants with positive and negative spiritual experiences of bereavement. participants with negative experiences of bereavement had a statistically significant increase in scores on the pg-13 compared to those with positive spiritual experiences signifying greater symptoms of prolonged grief. participants with negative spiritual experiences with grief had significantly lower scores on the isles, suggesting a lesser degree of adaptive integration of their losses. there were no significant differences in depression scores between groups. conclusion: bereaved parents that have a negative spiritual experience of bereavement are at increased risk for prolonged grief symptoms and are less likely to find meaning in their children's deaths than bereaved parents that describe a positive spiritual experience of bereavement. providers should consider exploration of spiritual beliefs and provision of spiritual care for parents of children facing life-limiting illnesses during treatment and bereavement. background: langerhans cell histiocytosis (lch) is an inflammatory myeloid neoplasia characterized by frequent relapse, with treatment failure associated with higher risk of death and neurodegenerative disease (lch-nd). activating somatic mutations in mapk pathway genes have been identified in almost all cases, with braf-v600e in approximately 60% of lesions. targeted therapies have been successful in treating other refractory cancers with braf v600e mutations (such as melanoma). given the central role of mapk pathway activation in lch, mapk pathway inhibition may be an effective therapeutic strategy for children with lch. objectives: the purpose of this study was to report the efficacy and toxicity profile of a retrospective cohort of patients with lch treated with mapk pathway inhibitors. design/method: medical records from 12 pediatric patients with lch (systemic and/or lch-associated neurodegeneration) who were treated with a mapk pathway inhibitor were retrospectively reviewed from five institutions. all patients had failed at least one prior systemic therapy and had a proven mapk pathway mutation. results: all patients in this series were less than 21 years old (median = 10.1 years; range: 2-20 years) with a median of three prior treatments (range: 1-9). at the time of initial mapk inhibitor use, nine of the 12 patients had lch-nd diagnosed clinically and/or by radiographic imaging; the remaining three patients had systemic disease. patients were treated for a median of 9 months (range: 1-20 months) with various reasons for discontinuation. three patients received combination mapk inhibitor therapies and three patients received other concurrent lch-directed therapies. four of the twelve patients had a grade 3 or 4 toxicity reported and three of these patients required dose reduction in order to be able to successfully resume therapy. overall survival was 92% with median 20 month follow-up (range: 1-42 months) with only one patient achieving transient complete response. the remaining ten patients had partial response or stable disease and four of these patients developed progressive disease while on therapy. conclusion: mapk pathway inhibitors may be a relatively safe salvage therapy for refractory systemic lch and lch-nd but the efficacy and durability of this strategy remains to be defined. combination with cytotoxic chemotherapies may be required in order to eradicate the disease-causing cell. future prospective trials of mapk pathway inhibitors for patients with refractory lch are needed in order to directly compare their efficacy and toxicity relative to other current salvage strategies. cincinnati children's hospital medical center, cincinnati, ohio, united states background: medication adherence during maintenance therapy has been shown to have a direct relationship with disease relapse in pediatric leukemia. previous research determined that patients who are ≤ 95% adherent to 6mercaptopurine (6mp) have a greater risk for relapse. the primary aim of the present study is to examine the relationship between metabolite profiles of 6mp with behavioral adherence rates obtained via electronic monitoring at 5, 10, and 30 days. it is hypothesized that patients demonstrating low levels of thioguanine (tgn) and methylated mercaptopurine (mmp) will have lower behavioral adherence rates prior to the blood draw. design/method: in a multisite, prospective study of 139 patients ages 7-19 years diagnosed with acute lymphoblastic leukemia (all) or lymphoblastic lymphoma (lbl), 6mp adherence was measured across 15 months of maintenance therapy using behavioral adherence (electronic monitoring) and pharmacological (metabolites) measures of 6mp. 6mp is metabolized into mmp and tgn. cluster analysis was used to generate three mutually-exclusive profiles of 6mp adherence. behavioral adherence rates were calculated for 5, 10, and 30 days prior to the blood draw. results: this study identified three metabolite profiles of 6mp across 15 months. previous research indicated that low levels of both metabolites suggest nonadherence to medication. low levels of one metabolite with high levels of another metabolite indicate adherence to 6mp. in this study, 51.2% of the low tgn-low mmp group had 5-day behavioral adherence rates ≥ 95% (mean = 100%); 48.8% had adherence rates < 84% (mean = 48.5%). in the high tgn-low mmp group, 77.6% had a mean 5-day adherence of 100%; 22.4% had adherence rates < 84% (mean = 32.9%). the low tgn-high mmp group had 74% of patients with a mean 5-day adherence level of 100%; 26% had adherence rates < 84% (m = 54.6%). at 10 and 30-days, 62 to 66% of patients in the low tgn-low mmp group had adherence rates < 95%. conclusion: these findings suggest that electronic monitoring and metabolite concentrations can be used to monitor 6mp medication adherence during maintenance therapy. it is notable that there is a sub-sample of pediatric patients who are identified as being nonadherent to 6mp based on electronic monitoring, however, metabolite levels indicate adherence to 6mp. similarly, a sub-sample of patients were identified as being adherent based on electronic monitoring, but metabolite profiles indicated sub-therapeutic levels of 6mp. our findings underscore the clinical significance of using both objective measures of medication adherence to inform clinical decision making. cincinnati children's hospital medical center, cincinnati, ohio, united states background: hemophagocytic lymphohistiocytosis (hlh) is a life-threatening hyperinflammatory syndrome characterized by non-remitting fevers, rash, hepatosplenomegaly, cytopenias, liver dysfunction and coagulopathy, and can include central nervous system involvement. several genetic diseases cause hlh by impairing normal lymphocyte or macrophage function. the hlh panel at the cincinnati children's genetics laboratories includes 14 genes associated with hlh and other lymphoproliferative diseases, including the genes that cause primary hlh (prf1, unc13d, stxbp2, stx11, rab27a), x-linked lymphoproliferative diseases (sh2d1a, xiap), itk deficiency (itk), hermansky-pudlak syndrome types 2 and 9 (ap3b1 and bloc1s6), chediak-higashi syndrome (lyst), cd27 deficiency (cd27), xmen syndrome (magt1) and lysinuric protein intolerance (slc7a7). deletion/duplication analysis is available as a reflex test for all 14 genes, as copy number variations (cnvs) are not directly assessed by sequencing. objectives: the prevalence of cnvs among large groups of patients with hlh in north america is unknown. we assessed the frequency of cnvs in the genes on the hlh panel through a retrospective review of 522 orders for deletion/duplication analysis performed after next-generation or sanger sequencing: 397 orders for all 14 genes on the panel, and 125 orders of 1-5 genes from the panel. deletion/duplication analysis was performed on a custom 4 × 180k microarray annotated against ncbi build 37 (ucsc hg19, march 2006). deletion/duplication analysis resulted in a confirmatory diagnosis in 11 of 522 cases (2.1%). pathogenic or likely pathogenic cnvs were most common in the three x-linked genes: sh2d1a (3 deletions), xiap (3 deletions, 1 duplication), and magt1 (3 deletions). hemizygous deletions in xlinked genes in male patients were typically suspected after amplification failure during previous sequencing. of the autosomal recessive genes, pathogenic cnvs were observed once in each of three genes: rab27a (heterozygous), lyst (heterozygous), and stxbp2 (homozygous). in the two heterozygous cases, a second change was not identified by sequencing, so deletion/duplication analysis did not offer a confirmatory diagnosis. in 25 patients, deletion/duplication analysis was performed after a pathogenic or likely pathogenic variant was identified in an autosomal recessive gene during sequencing; however, in no case was a second mutation uncovered by cnv analysis. we recommend that deletion/duplication analysis be routinely performed in all male patients with hlh who lack a genetic diagnosis after sequencing of hlh-associated genes, especially if any regions failed to amplify. deletion/duplication analysis may be performed in female patients after sequencing if a genetic form of hlh is highly suspected, but the yield is expected to be low. cleveland clinic children's hospital, cleveland, ohio, united states background: the development of post-transplant neoplasia, typically from lymphoproliferative disease (ptld), is a severe complication in transplant recipients and affects approximately 12% of pediatric solid organ recipients. rates of lymphoma in adult heart transplantation patients are comparatively low, at less two percent at ten years. there are few published reports of the long-term outcomes of neoplasia after pediatric heart transplantation. we aimed to identify the subsequent malignancies that occurred in pediatric heart transplantation patients in a large single institution, and describe their treatment and subsequent clinical course. we performed a retrospective chart review of all pediatric heart transplant recipients followed at the cleveland clinic children's hospital from january 1985 to october 2012. we excluded patients who died within 30 days of heart transplantation. we reviewed in depth the history and clinical course of subjects who developed neoplasms. results: between 1985 and 2012, 101 patients underwent heart transplantation and survived at least 30 days post transplantation. nine patients (8.9%) developed a subsequent malignancy. in this case series, the median age at heart transplant was 3 years old and the median time to develop neoplasia was 88.6 months. primary neoplasia included monomorphic ptld (3), polymorphic ptld (1), burkitt lymphoma (2), hodgkin's lymphoma (1), plasmacytoma-like lymphoma (1) and epstein-barr virus-associated smooth muscle tumor (ebv-smt) (1). one patient with hodgkin lymphoma subsequently developed monomorphic ptld, one patient with polymorphic ptld subsequently developed ebv-smt and later, an undifferentiated gastric cancer. one patient with monomorphic ptld developed an ebv-smt. evidence of epstein-barr virus was present in six of nine patients at diagnosis of first malignancy. four of nine patients received reduction in immunosuppression as a primary intervention for the initial malignancy, with two complete responses (cr), one partial response, and one with progressive disease. five patients were treated with chemotherapy, with four cr and one with progressive disease. three patients died of malignancy (recurrent ebv-smt, undifferentiated gastric cancer, and monomorphic ptld post-hodgkin disease) and two patients died of other transplant related complications. conclusion: secondary malignancies represent a significant disease burden to survivors of cardiac transplantation. as expected, much of the malignancy burden is driven by ebv. despite aggressive histology, many malignancies can be successfully cured in this setting with a multidisciplinary approach. stanford university school of medicine, palo alto, california, united states background: current treatment of langerhans cell histiocytosis (lch) is based on extent of organ system involvement and if high risk systems are affected. gastrointestinal (gi) involvement is diagnosed in about 2% of lch patients, and classically presents in children under 2 years of age with malabsorption, failure to thrive, bloody diarrhea and anemia. although the gi system is considered standard risk, a mortality rate over 50% occurring within 2 years of diagnosis has been reported. this study was performed due to this discrepancy and the limited number of published cases. objectives: to review the clinical course and outcomes of patients diagnosed with gi lch. design/method: a retrospective chart review of patients with histologically confirmed gi lch diagnosed in the last 15 years identified from the bass center histiocytosis clinical database was performed. two other pediatric hematology/oncology centers (ucsf benioff children's hospital oakland and san francisco) were queried for additional cases. results: four patients with biopsy proven gi lch [3 subjects (2.9%) from 105 database records and l from center queries] were identified. failure to thrive, hypoalbuminemia, bloody diarrhea and rash were the most common presenting symptoms. lch of the skin was found in all patients. risk organ systems were involved in 2 patients. of note, 2 subjects were of african racial background. the median age at diagnosis was 3.5 months (1.5 months to 16 years), mean albumin 2.2 g/dl (1.2 -2.9 g/dl), mean esr of 56 mm/hr (37 -78 mm/hr). all patients initially received combination therapy per lchiii protocol (vinblastine, prednisone, and 6 mercaptopurine). two patients had recurrent disease and received second line therapy (cytarabine, 2cda, and local radiation therapy). all patients are alive without active disease at last follow-up (8 to 107 months after completion of therapy). a systematic approach to evaluate gi involvement should be performed in children diagnosed with lch. from our experience, combination chemotherapy for patients with lch involving the gi tract is an effective intervention for active disease. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bhatia indicated that rates of 6mp adherence ≥ 95% have better clinical outcomes. those with adherence rates ≤ 95% have an increased risk for disease relapse. the present study investigated patterns of 6mp medication adherence using group-based trajectory modeling in a large sample of pediatric patients. to describe patterns of behavioral adherence during the maintenance phase of therapy for a cohort of pediatric patients ages 7-19 years who were diagnosed with acute lymphoblastic leukemia or lymphoblastic lymphoma (n = 139). previous research has documented the relationship between optimal levels of medication adherence with positive health outcomes. it was hypothesized that three groups would be identified: optimal adherence, deteriorating adherence, and chronic nonadherence. it was hypothesized that patients in the optimal adherence group would have adherence rates ≥ 95%. those with poor adherence would have adherence rates ≤ 95%. design/method: the present study was a longitudinal, multisite study investigating adherence to 6-mercaptopurine in a pediatric cohort of patients using electronic monitoring devices. daily adherence rates (electronic monitoring of 6mp) were examined across 15-months. health outcomes were measured at quarterly intervals through medical chart reviews. results: unconditional growth curve modeling indicated that the mean percentage of behavioral adherence was 84.4% at baseline and declined to 75.2% at 15-months. three trajectories of 6mp behavioral adherence were identified: 1) optimal adherence (67% of patients): averaging 95% behavioral adherence across 15 months; 2) moderate adherence (20%): relatively stable nonadherence with rates of 67% across 15 months; and, 3) chronically nonadherent (13%): adherence decreased from 63% to 30%. with respect to patterns of medication adherence and relationship to clinically-relevant health outcomes, there were no significant differences in health outcomes between patients in the adherent versus nonadherent trajectories, including mean absolute neutrophil counts (anc), risk for infection as measured by anc, healthcare utilization, or risk for disease relapse. although longitudinal patterns of 6mp behavioral adherence were not related to health outcomes, it is notable that only 67% of the current sample had adherence rates ≥ 95%. in fact, 33% of the current sample demonstrated adherence rates ≤ 95%. our findings are important for development of future adherence promotion studies in pediatric cancer. our findings underscore the relative significance of tailoring adherence promotion interventions to subgroups of patients, including those with problematic patterns of adherence. patients who demonstrate adequate levels of adherence could still benefit from less intensive, preventative interventions to sustain and improve adherence. sophie gatineau-sailliant, pascale grimard, marie-claude miron, guy grimard, anne-sophie carret, jean-marie leclerc chu sainte-justine, montreal, quebec, canada background: vertebral involvement in langerhans cell histiocytosis (lch) is still a subject of interest, due to its low frequency and the absence of management's guidelines. objectives: to provide additional information on presentation, treatment and morbidity of pediatric lch vertebral lesions, we report cases of 11 children with vertebral lesion of biopsy-proven lch, between january 1st 2000 and december 31st 2015, at sainte-justine university health center (montreal, quebec, canada). we conducted a retrospective study by reviewing charts and imaging of vertebral lch in a population of 11 children (median age of 8.25 years at lch diagnosis), followed for a median duration of 34 months. symptoms at presentation, treatment modalities and morbidities were collected. results: vertebral lesions were present at lch diagnosis in 9 of 11 cases. they were usually diagnosed secondary to back pain in 10 of 11 cases and were asymptomatic in only one case. despite an epidural extension in 6 of 11 cases, no child developed neurological symptoms. lesions frequently involved vertebral body (10 of 11 cases) and were rarely unstable (2 of 11 cases). out of 29 vertebral lesions, most of them had a dorsal localization (15 of 29 lesions) and 8 of 11 patients had lch in multiple vertebrae. at diagnosis, median vertebral height loss was 37.5% compared to 25% at last imaging control. most used imaging modalities were pet-scan and plain x-rays. treatments were diverse and consisted in chemotherapy in all children but three and bisphosphonates in only 3 cases. radiation therapy was not used in any patient. six out 11 patients did benefit of an orthosis. a lch recurrence was observed in 6 patients and involved vertebrae in 4 cases. one patient with treatment-resistant lch disease had 5 relapses, and required multiple lines of treatment. all children were alive and disease-free at their last follow-up, 10 patients having radiological vertebral sequelae and only 3 had clinical sequelae. our study is consistent with the epidemiological data described in larger cohorts of children with vertebral lesions of lch and the favorable prognosis associated with such lesions. nevertheless, aggressive treatment and long term follow-up seemed to be essential as recurrences are s243 of s301 not rare and spontaneous bone regeneration often incomplete. plain x-rays appears to be a good follow-up tool for vertebral lesions as it allows reliable measures, less exposure to radiation at lower cost. national cancer institue, giza, giza, egypt background: acute lymphoblastic leukemia (all) is the most common type of childhood cancer and also the most complicated in the treatment, so it requires many interventions for both treatment and to alleviate suffer form side effects. pancreatitis is one of the toxicities, which is more common in all as it appears in about 16% of the patients. it occurs in many drug combinations which induce pre-pancreatitis and even direct destruction of pancreatic tissues. pancreatitis can be induced by many drugs used in the treatment such as chemotherapeutic agents or supportive treatment. lasparaginase is the backbone drug of the treatment of all in which 6 to 9 doses are required to achieve complete remission status in the induction phase of treatment and 12 to 19 doses in the maintenance phase.it is an enzyme that destructs the l-asparagine amino acid into aspartic acid and ammonia thus deplete the asparagine from the extracellular matrix . many drugs are investigated for their effect on treatment of induced pancreatitis such as interleukin-10, nsaid as antiinflammatory, glycerin tri nitrates as improvement of microcirculation, tnf-alpha antibody, paf inhibitor as specific anti-inflammatory and low molecular weight heparin .none of the drugs was investigated for their ability to prevent the occurrence of pancreatitis. objectives: this study was designed to evaluate the protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis design/method: acute pancreatitis was induced in rats by intra-muscular injection of l-asparaginase (1000 i.u/kg) given daily for five days. enoxaparin was given subcutaneous (100 i.u/kg) and diclofenac was given intra-peritoneal (2 mg/kg) daily for five days. then, markers of pancreatic injury, lipids, immune cell infiltration and oxidative stress were analyzed with histo-pathological examination of the pancreatic tissue results: during acute pancreatitis, oxidative stress markers were significantly changed as indicated by reduced tis-sue glutathione and increased malondialdehyde levels. this was accompanied with significant increase in immune cells infiltration as indicated by high levels of myeloperoxidase and pro-inflammatory cytokine tnf-alpha. triglyceride only showed increase level. treatment with enoxaparin and/or diclofenac restored levels of biochemical markers including serum alpha-amylase, reduced glutathione, malondialdehyde, pro-inflammatory cytokine tnf-alpha, myeloperoxidase and triglyceride. histological injuries of pancreatic tissues as vacuolation and necrosis of epithelial lining pancreatic acini, inflammatory cells infiltration and focal pancreatic hemorrhage were also reduced by treatment with enoxaparin and/or diclofenac. the present study emphasizes the potential protective effect of enoxaparin and diclofenac against l-asparaginase induced pancreatitis background: rosai dorfman disease (rdd), or sinus histiocytosis with massive lymphadenopathy (shml), is a rare condition of immune dysregulation of unknown etiology arising from the massive accumulation of non-langerhans type histiocytic cells inside lymph nodes. the disease classically presents as bulky, painless lymphadenopathy often associated with infection showing distension of lymph node sinuses by abundant histiocytic cells (cd1a(-), s-100(+)/cd68(+)). in some cases, the disease can be self-limiting, but in cases with a prolonged chronic course of exacerbations and remissions, those with extranodal involvement, or disease that threatens vitals structures, treatment may be necessary. there is no treatment consensus. to describe a case of life-threatening, unresectable, recurrent rdd successfully treated with langerhans cell histiocytosis (lch) 2009-inspired therapy. design/method: we compared this case to the current literature on chemotherapeutic treatments for rdd. we searched pubmed, ovid, and google scholar for similar cases. we believe this to be the first reported case of using lch therapy to successfully treat rdd. an 8-year-old male presented to an outside hospital with two years of massive neck swelling causing torticollis. biopsy confirmed rdd. he was intermittently treated with courses of antibiotics with partial response. surgical removal of the affected lymph nodes was unsuccessful due to proximity to the spinal cord. two years later, the patient presented to our institution. he was initially treated with prednisone with a fast tapering dose, but after a second relapse the decision was made to try chemotherapy following the lch-2009 protocol of weekly vinblastine (6 mg/m2), 6-mp (75 mg/m2), and high dose steroid bursts. he experienced two additional relapses off therapy at ages 12 and 14 years old, including cmv(+) associated septic shock and cytokine storm requiring rapid response, picu admission, and ionotropic support. this last episode was treated with a more prolonged induction and maintenance therapy. an extended and slowly tapered maintenance therapy regimen of 2.5 years of daily 6-mp, monthly vinblastine and steroids with a slowly tapered dose during his fourth remission has resulted in 38-months of continuous complete remission-the longest stretch of his life. no similar cases were found. literature search demonstrated no consensus regarding the most effective treatment of rdd, with no previous cases being successfully treated following lch chemotherapy protocols. we hypothesize that the multi-agent relatively mild lch-2009 therapy mitigates the immune dysregulation of rdd. this case suggests that lch-2009 therapy can be used to treat cases of rdd that is not amendable to surgery or observation. nicklaus children's hospital, miami, florida, united states background: central venous catheters (cvc) are necessary in the management of patients with malignancies, especially children. patients with acute leukemia (al) have higher rates of central line associated complications such as bloodstream infections compared with other malignancies. objectives: to examine the choice of placement of cvc and the differences in outcome between peripherally inserted central catheters (picc) and ports in patients with leukemia during induction. design/method: retrospective chart review of patients with newly diagnosed leukemia at nicklaus children's hospital between 2010 and 2016. results: ninety four patients with a new diagnosis of leukemia undergoing induction chemotherapy were identified. the average age was 6.9 years. overall, 51 (54.3%) patients had a port placed and 43 (45.7%) had a picc placed. the decision for picc or port was subjective and physician based. the main outcome measures were local inflammation/infection, bacteremia, thrombophlebitis, blocked catheter and premature removal. the most common complication was bacteremia (12. 8%). in a multiple logistic regression analysis for predicting whether patients had at least one complication, results showed that having at least one complication is 3.4 times the odds in patients with aml compared to patients with all (p = 0.032). when comparing picc vs. ports, patients with picc had more frequent episodes of blocked catheters (23.3%) and premature removal (20.9%) compared to the patients with ports (2.0% and 0.0%) (p = 0.002 and p = 0.001 respectively) during induction. local inflammation, bacteremia and thrombophlebitis were not statistically different (p = 1.0, p = 0.54 and p = 2.4 respectively). the most common place for port placement was the right subclavian vein (55%). there was no significant association between port location and having at least one complication (p = 0.112). acute lymphocytic leukemia subgroup analysis: fourteen patients (61%) in the picc group had at least one complication and 9 (39%) in the port group but that was not statistically significant (p = 0.128). our series showed a higher incidence of blocked catheters and premature removals with picc compared to ports in patients with leukemia during induction. the choice of placement of picc vs port was subjective and physician based. patients with all, despite receiving steroids and asparaginase during induction, did not show a statistically significant increase risk in thrombosis or infection but larger numbers may be needed in future studies. university of california, san francisco, san francisco, california, united states background: hemophagocytic lymphohistiocytosis (hlh) is classically a disorder of young children meeting systemic hyperinflammation criteria. presentation in late adolescence is uncommon. furthermore, though cns signs occur in 30-70% of cases, initial isolated neurologic presentation is rare, frequently resembling encephalitis or demyelinating disorders. these cns signs can be isolated or precede systemic disease, delaying hlh diagnosis. hlh declaring in adolescence with predominant psychiatric features has not been well documented. objectives: to describe a case of cns hlh presenting with neuropsychiatric features in absence of classic hlh criteria. design/method: retrospective review of clinical, radiologic, histologic, immunophenotypic, and molecular features of a patient with cns hlh. a 19-year-old female presented with acute-onset headaches following nine months of progressive anxiety, short-term memory loss, emotional lability, perceptual disturbances, and hypomania. brain mri demonstrated numerous enhancing t2 hyperintense supratentorial and infratentorial white matter lesions in the left thalamus and caudate head. brain biopsy showed histiocyte-rich inflammation and associated demyelination. extensive evaluation including universal microbial pcr failed to reveal underlying infection or malignancy. past medical history was notable for presumptive pulmonary sarcoidosis diagnosed 14 months prior with progressive respiratory failure with associated granulomatous pulmonary nodules which responded to systemic immunosuppression. at presentation of her neuropsychiatric symptoms, she had normal sil-2r, ferritin, fibrinogen, and triglycerides. there was no pancytopenia, coagulopathy, bone marrow hemophagocytosis, fevers, or splenomegaly. given the possibility of partial immune suppression of systemic symptoms and the prominent neurologic symptoms, hlh screening labs were sent and notable for decreased natural killer and cytotoxic t lymphocyte function, normal granzyme expression and cd107a mobilization, and absent perforin expression. genetic testing confirmed compound heterozygous mutations in prf1 (c.227g>a, c.626a>c) and familial hlh type 1. she was treated with low-dose dexamethasone and intrathecal chemotherapy per hlh-94. due to lack of evidence of systemic inflammation, vp-16 and high-dose steroids were held. within one week of initiating therapy, she had decreased anxiety and improved cognition, with sustained, incremental neuropsychiatric improvement with additional intrathecal treatments. she tolerated dexamethasone tapering without symptom flare. mri also demonstrated parenchymal lesion improvement. for definitive treatment, she underwent unrelated allogeneic hematopoietic cell transplantation and remains at neurologic baseline as of eight months post-transplant with ongoing imaging improvement. conclusion: this case of familial hlh with compound heterozygous perforin mutations in an adolescent with isolated neuropsychiatric symptoms illustrates that cns hlh may be an underrecognized phenomenon in absence of systemic signs. standard hlh therapy may effectively reverse these symptoms with associated radiologic responses. rush university children's hospital, chicago, illinois, united states background: posterior reversible encephalopathy syndrome (pres), a recognized complication of pediatric leukemia treatment has been reported in up to 5% patients in various series. hypertension, chemotherapy and cortical spreading depression have been implicated in the pathophysiology. due to the combinations used, it is difficult to identify the offending drug, several have been implicated. since delay of chemotherapeutic treatment in children with high risk leukemia is unfavorable, it is important to recognize the characteristic radiologic findings, manage appropriately and reintroduce the treatment as soon as possible. pharmacoethnicity is now recognized as an important factor for variation in neurotoxicity in children with all. ethnic differences in reported pres events in pediatric patients with all has not been well described in literature. to describe the factors associated with pres in a cohort of high risk pediatric all patients at a single institution. design/method: a total of 12 children with an average age of 9 years (1-20 years) diagnosed with all between 2013-2017 were retrospectively reviewed for the occurrence of pres. various demographic factors, therapy received, clinical features, radiology related findings and management were reviewed. a search for all published articles on pres in leukemia was conducted using pubmed databases. results: five (42%) children (average age 8.5 years) developed pres during days 10-29 of induction. 80% of the patients that developed and 45% of those that did not develop pres were hispanic. all the patients that developed pres and 43% of those that did not were diagnosed with high risk all. all patients received vincristine, 80% received daunomycin and intrathecal methotrexate and 20% received asparaginase in the 1 week prior to the event. mri findings confirmed pres in all 5 patients with no evidence of methotrexate related leukoencephalopathy or leukemia. at the time of pres all patients were in remission based on mrd and spinal fluid cytology. two-thirds of the patients had seizures and hypertension at the time of the event with no prior history of either. all patients had complete recovery of normal mental status after resolution of pres. a higher incidence of pres than previously reported was noted in our series. hispanic ethnicity, high-risk all and exposure to vincristine, daunomycin and intrathecal methotrexate in induction were associated with pres in our cohort. a new association that emerged was that of hispanic ethnicity with pres .larger studies to understand the importance of pharmacoethnicity in pres may help in individualization of chemotherapy based on ethnic differences. children's hospital of illinois, peoria, illinois, united states background: hyper ige syndrome is a primary immunodeficiency characterized by susceptibility to skin and lung infections as well as increased propensity for malignancy. hemophagocytic lymphohistiocytosis (hlh) is a syndrome characterized by overwhelming activation of t lymphocytes and macrophages occurring as either primary hlh caused by genetic abnormalities or secondary hlh associated with infectious, malignant, metabolic, or immunodeficiency causes. we describe the first case to our knowledge of hlh in a patient with hyper ige syndrome. to describe a case of hlh in a pediatric patient with hyper ige syndrome. results: a 7-year old caucasian male with known autosomal dominant hyper ige syndrome (stat3 mutation) was transferred to the pediatric intensive care unit secondary to concern for septic shock. the patient had persistent slow bleeding from oral lesions and central catheter sites despite the addition of aminocaproic acid and recombinant factor viia. he also required numerous blood product transfusions sec-ondary to anemia and thrombocytopenia. clinical suspicion was high for hlh and the patient met criteria for diagnosis of hlh with the following: ferritin > 40,000 ng/ml, triglycerides 264 mg/dl, decreased nk cell function with the sample only containing 1% nk cells, elevated soluble il-2 receptor at 4215 u/ml, splenomegaly, and fever. infectious workup was remarkable for a positive ebv qpcr with 80,700 copies/ml suggestive of ebv driven secondary hlh. familial hlh testing was unable to be completed. therapy was initiated based upon the hlh-94 study. the addition of ruxolitinib and anakinra were considered but the patient declined rapidly prior to treatment. ct of the head was concerning for a stroke with signs of edema and increased intracranial pressure likely leading to the development of symptoms consistent with brain stem herniation. the decision was then made to withdraw care. conclusion: to our knowledge, this is the first report of hlh in a patient with hyper ige syndrome. diagnosing hlh requires a high index of suspicion in critically ill patients, and prompt initiation of therapy is essential. this challenging case of hlh in a patient with hyper ige syndrome highlights the diagnostic challenge, variable presentation, and need for effective therapy in this vulnerable patient population. background: adolescents and young adults (ayas) with cancer are at risk for psycho-social as well as physical symptom burden during cancer therapy. the purpose of this study is to explore psychological and physical symptoms endorsed by aya while receiving therapy for cancer design/method: surveys were given in both inpatient and outpatient settings during cancer therapy. symptom screening in pediatrics tool (sspedi) and memorial symptom assessment scale (msas). symptoms severity was rated by teens on a 5 point likert scale. spss 22, used for statistical analysis. results: : a total of 39 aya on cancer therapy (age range 13-19.9 years) 35% female, 65% male, 43.6% acute leukemia, 48.7% solid tumors, and 7.7% diagnosis was not reported. 78% of aya on cancer therapy reported at least 1 or more symptoms, 45% reported >3 symptoms cluster. of the physical symptoms that were reported as most distressing to the teens, mouth sores and headaches were the top causes. of the physical symptoms that were most frequently endorsed; fatigue was on the top (58%), followed by change in appetite 45 %, vomiting 43%, and pain 40%., the least was bowel habit changes. aya rated sadness as the most frequent psychological symptom 38%, followed by feeling angry 32%, and scared 30%. statistically significant difference was noticed based on gender difference with more females reported symptoms (p = 0.01), while type of cancer (acute leukemia versus solid tumors) was not statistically different. conclusion: aya with cancer reported multiple physical and psychological symptoms with significant distress. females seem to report more symptoms compared to males. screening aya for cancer therapy related symptoms is feasible during routine visits and adds important information about the aya well-being. background: sinus histiocytosis with massive lymphadenopathy (shml), also known as rosai-dorfman disease, is a rare histiocytic proliferative disorder of unknown etiology. many treatment modalities have been employed; however, no uniform guidelines exist. objectives: literature review of treatment options for shml. design/method: chart review was performed on pediatric patients diagnosed with shml at the children's hospital at montefiore between 2010 and 2012 after irb approval. inclusion criteria included children between the ages of 0 and 21 years with shml. exclusion criteria included children with cutaneous shml. four cases of shml seen at montefiore are described. a comprehensive review of the literature identified 102 additional cases published between 1978 and 2018. manuscripts that did not include the treatment modality or outcome were excluded. results: many of the 106 patients with shml responded to observation alone. of 106 patients, 46 patients were observed, with 35 (76%) having resolution of disease, five having stable disease, and five being lost to follow-up. one patient received subsequent systemic therapy. surgical management was con-ducted upfront in 29 patients. of those, 18 (62%) had resolution of disease, one had stable disease, and one had recurrence with no further therapy noted. of the remaining nine patients, 77% were successfully treated with systemic therapy, consisting of either steroids (5) or steroids and chemotherapy (4). systemic therapy was used as first-line therapy in 31 patients. steroids alone or in conjunction with chemotherapy resulted in resolution of disease in 12/15 and 7/11 patients (19/26, 73%), respectively, with four patients having stable and three with progressive disease. chemotherapy without steroids resulted in resolution of or stable disease in 3/5 patients. radiation was ineffective. conclusion: shml is a rare disease with no published guidelines for treatment. from the results of the cases and a detailed review of the literature, it can be suggested that observation may be considered as first line management in patients providing there are no significant symptoms. for patients who are symptomatic or have significant progression, surgery may be considered. in patients with recurrence or refractory disease, steroids and/or chemotherapy may be used. the presence of nodal or extra-nodal disease did not seem to have a significant impact on the course of treatment. given the rarity of the disease, it is difficult to conduct a randomized control trial. further work, involving collaboration between centers and cooperation with the international rare histiocytic disorders registry would be helpful. boston children's hospital, boston, massechusettes, united states background: increasing census and intensified work compression on the inpatient oncology service at our institution was identified as leading to resident dissatisfaction, impaired resident learning and decreased perceived quality of patient care. objectives: to evaluate the impact of a redesign of a pediatric inpatient hematologic malignancy (ihm) service on resident perceptions of the educational value of the rotation and safety of patient care. design/method: during the 2016-2017 academic year, we initiated a bundled intervention on the ihm service. modifications included 1) decreased patient volume: the ihm service was divided into two teams, utilizing an extra attending -a teaching service consisting of residents and fellows and a team comprised of nurse practitioners. 2) intentional patient team assignment: patients were deliberately assigned to a care team based on educational opportunities and provider skill sets. 3) intentional attending faculty selection: attending faculty with deeper clinical and teaching experience were selected to supervise on the teaching team. 4) increased weekend staffing. after completing the service, junior residents completed an electronic survey to evaluate their perceptions of the educational value of the rotation, as well as their ability to deliver safe care while on the rotation. fisher's exact tests were used to compare responses from residents in 2017 who experienced the redesign to residents in 2016, whose experience results: survey completion rates were 70% (28/40) in 2016 and 57% (29/51) in 2017. intervention residents were significantly more likely than comparison group residents to choose the answers "very good" or "excellent" to describe both the overall quality of the rotation (76% intervention vs. 25% comparison, p<0.001) and the educational experience on rounds (52% intervention vs. 7% comparison, p<0.001). intervention residents also reported caring for fewer average primary patients daily on weekdays as compared to comparison residents (4.8 vs 8.7 patients, p<0.0002, 95% ci -5.14 to -2.64). furthermore, intervention residents were more likely than comparison residents to "agree" or "strongly agree" that they could provide safe patient care on weekend days (79% intervention vs. 14% comparison, p<0.001) and on nights (69% intervention vs. 25% comparison, p<0.01) while on the oncology service. a redesign initiative of an oncology service with the development of a new teaching service led to improved resident perceptions of the educational value of the rotation and ability to provide safe care to patients. this approach could be useful to other services and institutions to promote similar outcomes in resident education and patient care. background: alk-positive histiocytosis is a rare histiocytic proliferative disorder that has been reported in three infants presenting primarily with hepatosplenomegaly, anemia, and thrombocytopenia. given the rarity of this disease, there are no standard treatment algorithms for this diagnosis and the disease course and outcomes remain largely unknown. the published series describes treatment ranging from monitoring alone to multi-drug chemotherapy regimens. there was ulti-mately resolution of presenting symptoms in all three cases despite varying treatment strategies. objectives: to report a newly diagnosed case of alkpositive histiocytosis that was treated with a novel approach using cytarabine monotherapy. results: a full term male infant presented at birth with difficulty feeding and hyperbilirubinemia. over the first few weeks of his life, he subsequently developed thrombocytopenia, transaminitis, and profound hypoalbuminemia. by six weeks of life, he was experiencing significant abdominal ascites requiring repeat paracenteses, massive hepatosplenomegaly, respiratory distress secondary to abdominal distension, anemia, and coagulopathy. he underwent numerous diagnostic tests, including a liver biopsy followed by a bone marrow biopsy that showed alk-positive histiocytic infiltrates in both sites. treatment was initiated with cytarabine 170 mg/kg/day x 5 days, repeating every 4 weeks. throughout his course of five cycles of treatment, he experienced intermittent fevers and mild nausea with no other adverse events. by the end of five cycles, his hepatosplenomegaly resolved, his blood counts normalized, he demonstrated weight gain on oral feeds, and his liver enzymes normalized. he is currently 12 months post completion of therapy and remains well with a normal physical exam and laboratory values. conclusion: treatment of alk-positive histiocytosis with lose dose cytarabine resulted in complete resolution of our patient's symptoms with minimal treatment related adverse effects, and few long-term treatment related risks. given the rarity of the diagnosis, the reporting of effective novel treatment options is important for future patient care. background: adult patients with melanoma or lung cancer harboring braf v600e have benefitted from the development and subsequent approval of specific braf inhibitors. as such, delineating the subset of similarly targetable pediatric oncology patients may spur development and rational use of these inhibitors in children. importantly, other point mutations and fusions of braf may also be targetable in s249 of s301 children analogous to recent emerging data in adult cancer patients. objectives: to define the genomic landscape of known and novel braf alterations and raf1 fusions in pediatric malignancies and report index cases with clinical response to braf or mek inhibitors. design/method: dna was extracted from 40 microns of ffpe sections of 3,633 tumors from pediatric (<21 years of age) oncology patients, and cgp was performed on hybridization-captured, adaptor ligation based libraries to a mean coverage depth of 579x for up to 315 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. genomic alterations (ga) included base substitutions, indels, copy number alterations and fusions/rearrangements. a total of 221 (6.1%) braf-altered pediatric malignancies were identified. 172 (77.8%) harbored a single kinaseactivating braf short variant, indel, or fusion. an alteration resulting in reduced braf kinase activity was identified in 8 (3.6%) tumors while 7 (3.2%) tumors harbored multiple braf alterations, 3 of which contained at least a single activating short variant. the remaining 34 tumors (15.4%) contained functionally uncharacterized variants. kinaseactivating braf alterations were identified in diverse tumor spectra comprised of brain tumors (75.3%; 18 subtypes), carcinomas (10.6%; 6 subtypes, with melanoma constituting 50% of cases), hematological malignancies (8.8%; 5 subtypes), sarcomas (2.9%; 3 subtypes), and extracranial embryonal tumors (2.4%; 2 subtypes). seventy-two (32.6% of braf-altered cases) braf fusions were identified, 64 (88.9%) of which were kiaa1549-braf; 2 involved the novel fusion partners: stard3nl and khdrbs2. seven (0.2%) raf1 fusionpositive cases, predominantly brain tumors (5), were identified; 2 involved the novel fusion partners: tmf1 and sox6. index cases of response to therapy of intracranial tumors will be presented. we describe a population of pediatric patients with targetable braf alterations predominantly enriched in primary intracranial tumors, but spanning diverse solid tumor types and hematologic malignancies. we additionally report a cohort of raf1 fusion-positive patients. an index case and multiple previous reports suggest raf or mek inhibitors may benefit pediatric patients with either intracranial or extracranial disease, and development of such drugs in pediatric indications is strongly warranted. background: diffuse midline gliomas (dmg) with h3k27m mutation, including diffuse intrinsic pontine glioma (dipg), are the leading cause of brain tumor-related deaths in children. there are no effective therapeutic strategies and the median survival remains dismal. genomic studies have identified a recurrent mutation in the majority of dmgs involving a lysine to methionine substitution (k27m) in histones 3.1 and 3.3, resulting in changes in the epigenetic landscape that dysregulate gene expression and promote gliomagenesis. panobinostat, a multiple histone deacetylase (hdac) inhibitor, was found to be one of the most effective agents against dipg patient-derived cell cultures and xenograft models in previous studies and is presently in clinical trial for dipg. hdac inhibition with panobinostat may also exhibit activity against h3k27m+ diffuse midline gliomas of the thalamus and spinal cord. to evaluate the effect of panobinostat as a single agent against patient-derived thalamic and spinal cord h3k27m+ diffuse midline glioma cell cultures and in an orthotopic xenograft murine model of h3k27m+ spinal cord glioma. design/method: patient-derived thalamic and spinal cord h3k27m+ diffuse midline glioma cell cultures were treated with single agent panobinostat at a range of concentrations. cell viability was evaluated using the celltiter-glo assay. panobinostat was systemically administered to orthotopic xenograft murine models of luciferase-expressing spinal cord h3k27m+ diffuse midline glioma. response to panobinostat was evaluated with ivis in vivo imaging. results: hdac inhibition with panobinostat significantly decreases cell proliferation with an ic50 of 30 nm and 41 nm in the spinal cord and thalamic glioma patient-derived cell cultures respectively. panobinostat slowed tumor growth in murine models of spinal cord glioma by 1.5-fold in the brain (p = 0.0219, n = 5) and 2-fold in the spinal cord (p = 0.0176, n = 5) when compared to vehicle controls after 1 week of administration. panobinostat is in clinical trials for dipg. this study suggests that hdac inhibition with panobinostat may also be beneficial for patients with thalamic and spinal cord diffuse midline glioma h3k27m mutants. background: brain tumors are the most common solid tumor of childhood and the leading cause of childhood cancer deaths. while medulloblastoma is the most common malignant brain tumor of childhood with a 5-year survival 70-80%, children with high-grade gliomas (hggs) such as glioblastoma multiforme (gbm) fare much worse with a 5-year survival of 15-35%. implicated in this poor outcome is the presence of treatment resistant brain tumor stem-like cells. gbm stem-like cells (gscs) have been implicated in tumor growth, treatment resistance and patient relapse, making them a key therapeutic priority. antipsychotic drugs (apds) have been used for decades in various psychiatric clinical settings and are associated with a lower incidence of cancer, including malignant brain tumors. currently, atypical apds are being evaluated for their potential to alleviate cancer and treatment induced side effects. furthermore these drugs may have direct anti-tumor effects, potentially via inhibition of dopamine d2 receptors (drd2). objectives: determine the anti-cancer effects of atypical apds on gbm stem-like cells design/method: the anti-cancer effects of apds (quetiapine and risperidone) were evaluated on gbm stem-like cell lines developed in our laboratory (glio 3 and 38) and the group 3 medulloblastoma cell line hdmbo3. cell proliferation/viability was determined using trypan blue exclusion and mts assays. the effect of apds on cancer stem cell self-renewal was determined by neurosphere assay. receptor expression and apds effect on cell cycle proteins were examined by western blot analysis. results: western blot analysis of gscs and hdmbo3 demonstrated robust drd2 expression indicating a viable therapeutic target. both apds induced dose dependent cell death of all cell lines tested. treatment with only 2um of either apd for 10 days significantly reduced cell proliferation by 60% (hdmbo3) and 50-90% (gscs). consistent with these findings, we observed an increase in cell cycle inhibitors p21 and p27. furthermore at day 10 both apds induced a robust increase in gsc death, approximately 60% compared to only 10% in non-treated controls. lastly, 1um apds significantly reduced gsc neurosphere formation compared to untreated controls by up to 35% suggesting inhibition of gbm stem cell self-renewal. our data indicates that clinically relevant concentrations (low micromolar) of these apds induce anticancer effects in both gscs, which are enriched with tumor initiation/propagation properties, and in the group 3 (myc amplified) medulloblastoma cell line. these apds represent strong candidates as potential adjuvant therapies for the treatment of these brain tumors. background: while the poor prognosis for high risk neuroblastoma (hrnb) underscores the need for new treatment strategies, the elucidation of specific biologic subsets of neuroblastoma suggests a way to improve disease management. the identification of agents that target specific molecular pathways associated with the development or progression of diseases holds promise. dfmo, an inhibitor of odc, has been shown to decrease lin28 and mycn and target cancer stem cells in preclinical studies. currently 14% of patients undergoing immunotherapy relapse. dfmo is in studies to prevent relapse after immunotherapy and may be helpful during immunotherapy as well. the hypotheses for this study were that: 1) the incorporation of a targeted therapy, selected based upon upfront tumor genomic interrogation, into standard induction chemotherapy for hrnb is safe, feasible and may increase the pr/cr/vgpr response rate at the end of induction therapy; and 2) the addition of dfmo as maintenance during immunotherapy is safe and feasible and may decrease the relapse rate for hrnb. a multicenter feasibility pilot trial in subjects with newly diagnosed hrnb within the beat childhood cancer consortium. at diagnosis, patients' tumors underwent dna exome and rna sequencing which were analyzed within a molecular tumor board to identify the single best drug of 6 targeted agents to be added to cycles 3-6 of induction chemotherapy. after consolidation with asct and radiation, the patients received dfmo along with standard dinutuximab and retinoic acid and dfmo for 2 years after immunotherapy. patients were evaluated for additional toxicities with the addition of targeted agents and dfmo in addition to induction response. results: the pilot study of 20 eligible patients has shown this process to be feasible. all 20 patients have completed induction portions of the study. the combination of targeted agent with chemotherapy was shown to be safe without any unexpected toxicities. delays between induction cycles were < 2 weeks and related to surgery, infection, or thrombocytopenia. the induction response demonstrated 88% cr/vgpr/pr rate, which suggests improvement over historical 80%. in addition, 15 patients were eligible for the combination of dfmo with dinutuximab and retinoic acid was well tolerated and safe without additional toxicities due to dfmo. the pilot study of 20 patients has shown the process of genomic sequencing and addition of a targeted agent to upfront chemotherapy and addition of dfmo to dinutuximab and retinoic acid maintenance therapy in newly diagnosed hrnb patients and is feasible and safe without any unexpected toxicities. background: identifying sub-populations of medulloblastoma tumors with stem cell-like properties holds promise for reducing disease recurrence, but there is no known unifying marker of medulloblastoma cancer stem cells. the granulocyte stimulating factor receptor (gcsf-r or cd114) is well understood in the context of hematopoiesis, but its role in solid tumor pathogenesis is less clear. neuroblastoma and melanoma subpopulations expressing gcsf-r have cancer stem cell properties of chemoresistance and increased tumorigenicity, and are enriched in tumors after chemotherapy. gcsf-r activation leads to signaling through the jak-stat pathway, suggesting a potential therapeutic target. we hypothesized that a subpopulation of medulloblastoma cells would express the gcsf-r and that this subpopulation would demonstrate chemoresistance and response to inhibitors of the jak/stat pathway. objectives: our objective was to identify a subpopulation of medulloblastoma cells expressing the gcsf-r and determine their relative growth rates, tumorigenicity, and responses to chemotherapy and jak/stat inhibition. design/method: medulloblastoma cell lines were sorted via flow cytometry for gcsf-r surface expression. subpopulations of gcsf-r-positive and -negative medulloblastoma cells were then monitored for growth by continuous live cell imaging. responses to chemotherapy were measured in subpopulations of gcsf-r-positive and -negative medulloblastoma cells using continuous live cell imaging to measure percent cell confluence and cell viability assays. ic50 values were calculated for each cell line and each agent. parental medulloblastoma cell lines and isolated gcsf-r-positive and -negative subpopulations were also treated with the jak1/2 inhibitor ruxolitinib and growth rates, viability, and ic50 values were calculated. results: gcsf-r surface expression was identified on 0.2-1.3% of medulloblastoma cell lines. isolated gcsf-r positive cells demonstrate a slower growth rate compared to gcsf-rnegative or parental unsorted medulloblastoma cells. gcsf-r positive cells are more resistant in vitro to vincristine, etoposide, and carboplatin, when compared to the gcsf-r negative population and an unsorted population of the same cell line. ruxolitinib is cytotoxic to medulloblastoma cells in vitro, with higher ic50 values noted in gcsf-r positive cells compared to unsorted and gcsf-r negative cells. we show that a subpopulation of gcsf-r positive cells are present in multiple medulloblastoma cell lines via flow cytometry, and that isolated gcsf-r-positive cells have a slower growth rate than gcsf-r-negative or unsorted populations. we also show that ruxolitinib has in vitro activity against medulloblastoma cell lines. we propose that jak inhibition may represent an adjunct therapy targeting overall tumor burden and specifically targeting the gcsf-r-positive subpopulation of medulloblastoma cells that may drive tumor recurrence. we investigated the efficacy of intensified adjuvant chemotherapy in osteosarcoma patients. design/method: we retrospectively analyzed the medical records of 48 children with osteosarcoma treated at asan medical center between 2006 and 2015. all patients received a 3-drug induction consisting of 2 cycles of cisplatin and doxorubicin along with 4 cycles of methotrexate (map), and proceeded to surgical resection. adjuvant ct was map or map with the additional ifosfamide and etoposide (mapie), and mapie was mainly considered for poor responders (tumor necrosis below 90%) or patients with metastases. results: among 48 patients, 6 patients had metastases at diagnosis. surgery was conducted in 43 patients who responded to induction ct, and 17 showed over 90% tumor necrosis. among 43 patients who proceeded to adjuvant ct, 19 and 24 patients received to map and mapie protocols. with a median follow-up of 73 months, the 5-year overall survival (os) and event-free survival (efs) rates of all patients were 80% and 46.7%. of those 43 patients, 16 patients recurred, and 5 of them died of disease progression. relapsed patients received salvage ct and/or surgery, and 2 were rescued after autologous stem cell transplantation (sct). three patients developed treatment-related acute myeloid leukemia, and they are alive after allogeneic sct. according to the response to neoadjuvant ct, the os rates of good responders (n = 18) and poor responders (n = 30) were 100% and 68.1% (p = 0.011), and efs rates were 63.8% and 41.7% (p = 0.084). of the 30 poor responders, 9 patients received map as adjuvant ct, and the other 21 received mapie. the os rates of map and mapie group were 68.6% and 70.3% (p = 0.568), and efs rates were 30.0% and 48.3% (p = 0.247), respectively. when patients were classified into three groups: 1. localized disease & necrosis ≥ 90% (n = 17), 2. localized disease & necrosis < 90% (n = 24), 3. metastatic disease (n = 6), survival rates were in the order of group 1>2>3 (os = 100%:81.8%:16.7%, efs = 67.6%:43.6%:0%). in each group, intensified adjuvant ct by mapie did not improve survival outcomes. conclusion: initial metastatic disease and poor histological response to neoadjuvant ct were major risk factors for poor survival in osteosarcoma patients. we found that adding ifosfamide and etoposide to map did not improve survival outcomes of patients with adverse risk factors. more effective adjuvant therapy for these patients is needed. background: circulating cell-free dna (cfdna) that shed from tumors into circulation have been used for noninvasive molecular profiling in adult cancers but little is known about its utility in pediatric cancers. pediatric patients with metastatic and refractory solid tumors are known to have poor survival rates, and a key challenge in their management is obtaining biopsy samples especially at times when disease is widely spread or the patient is physically unfit for sampling. the development of a noninvasive profiling strategy is critical for optimizing molecularly guided therapy and assessing response to treatment. in this study, we want to determine the utility of cfdna to noninvasively analyze the molecular profiles of pediatric solid tumors such as neuroblastoma (nb), osteosarcoma (os), and wilms tumor (wt). design/method: tumor, plasma, and matched controls were collected from patients with nb, wt, and os, at diagnosis or time of disease progression. cfdna was extracted from the plasma and analyzed through multiple methodologies including a targeted next generation sequencing panels and shallow whole genome sequencing (swgs). results: fifteen nb patients, 10 os patients, and 2 wt patients had tumor molecular profiles known from different targeted next-generation sequencing platforms. in the cfdna of 7/15 nb patients, somatic mutations and copy number alterations previously reported in the tumors were detected, including recurrent nb drivers such as mycn amplification, alk, and atrx mutations. mutations not detected in the original tumor were also found in 6/15 nb patients including nras, mll2, arid1b, some of which are potentially actionable. in os, mutations known from the tumor were found in the cfdna of 5 of 10 patients, including atrx and notch3 mutations, as well as copy number alterations such as cdk4 amplification, which has targetable therapeutics available. of the two wt patients analyzed, cfdna revealed the same mutations as tumor in one patient, however in a cohort of patients where tumor was not available, cfdna revealed recurrent driver mutations such as amer1, dicer1. it is feasible to noninvasively identify somatic mutations and copy number alterations in cfdna of patients with pediatric solid tumors. establishing a platform using cfdna to identify molecular profiles of these tumors can serve as a powerful tool for guiding treatment and monitoring response to treatment. background: despite multi-modality therapy, the prognosis for patients with metastatic osteosarcoma remains poor necessitating development of novel targeted therapies. immunotherapy can be exploited to target osteosarcoma with exquisite specificity but remains limited by insufficient tumor specific targets. objectives: to overcome the dearth in tumor specific antigens, we have explored the use of tumor derived mrna (representing a tumor specific transcriptome) for development of personalized nanoparticle vaccines. design/method: rna-nanoparticles (rna-nps) can be amplified from limited amounts of biopsied tissue for induction of tumor specific t cells against osteosarcoma. since local vaccination strategies are mired by poor overall immunogenicity, we assessed the feasibility, immunogenicity and antitumor activity of intravenously administered rna-nps (tumor mrna complexed to dotap nanoliposomes) in pre-clinical murine and canine tumor models. we identified a clinically translatable np formulation for the delivery of rna to antigen presenting cells (apcs) that induces in vivo gene expression and preserves rna stability over time. tumor derived rna-nps induced antigen specific t cell immunity and mediated anti-tumor efficacy in several pre-clinical solid tumor models (i.e. b16f10, kr158b). when administered intravenously, rna-nps increased expression of co-stimulatory molecules (i.e. cd80, cd86, cd40, ccr7) and pd-l1 on cd11c+ cells throughout reticuloendothelial organs (i.e. spleen, liver, bone marrow) and within the tumor microenvironment; this phenotype was strictly dependent on type i interferon. targeted inhibition of type i interferon signaling (via infar1 mabs) abrogated anti-tumor efficacy mediated by rna-nps. we enhanced the immunogenicity of this platform by simply combining mrnas encoding for immunomodulatory molecules (i.e. hcv-pamps, gm-csf) or by combining rna-nps with immune checkpoint inhibitors. addition of checkpoint inhibitors (pd-l1 mabs) to rna-nps increased tumor infiltrating lymphocytes, and intratumoral mhc class i/ii expression, and mediated synergistic anti-tumor activity in settings where pd-1 or pd-l1 inhibition alone did not confer therapeutic benefit. we then explored the feasibility of rna-nps in a large animal osteosarcoma model. in ongoing studies for canines with osteosarcomas, we have shown that sufficient amounts of rna can be extracted, amplified, and manufactured into personalized rna-np vaccines. conclusion: rna-nps reprogram systemic immunity and mediate anti-tumor activity providing near immediate immune induction without the complexity of cellular immunotherapy. the immune correlate of preclinical response to rna-nps is hallmarked by interferon dependent pd-l1 expression on activated apcs (cd11c+ mhcii+ cd86+ cells). based on these findings, we are exploring the preclinical safety, efficacy and immunologic effects of rna-nps targeting canine osteosarcoma before first in-human evaluation. background: ewing sarcoma is an aggressive bone tumor affecting mainly adolescent and young adults. treatments are based on compressed schedule chemotherapy combined with local control (surgery and/or radiation). prognosis is poorer for patients with metastatic disease, older age and central primaries. survival when disease recurs within two years of diagnosis is <10%. the ews-fli1 fusion gene t(11; 22) (q24; q12) has been well characterized as a dominant ews driver-gene. the most common variation is ews exon 7 with fli1 exon 6 (60% of fusion positive patients). we designed a novel pbi-shrna tm ews/fli1 type 1 lpx which has demonstrated, safety and efficacy in animal model (rao et all). the pbi-shrna strategy silences target gene expression by concurrently inducing translational repression and p-body sequestration as well as post-transcriptional mrna cleavage. to determine the safety and maximum tolerated dose of intravenous administration of pbi-shrna tm ews/fli1 type 1 lipoplex in patients advanced ews. design/method: phase i study 3 × 3 escalation cohort. testing pbi-shrna tm ews/fli1 type 1 lpx (starting iv dose of 0.04 mg/kg) on patients (≥ age 8) with advanced ewing's sarcoma, all with a type 1 translocation. intravenous infusion was given twice a week for 4 weeks with the following escalation schema: 50% → 33% → 25% → 25% → 25%. required kps >80% and adequate organ function. cytokines induction pre and post-infusion was analyzed (il-12, il-6, tnf-alpha, il1ra). first cohort of patients has been enrolled (ages between 17-35 years). three relapsed patients had >3 lines of therapy and 1 patient had refractory disease, 3 patients received a complete cycle of pbi-shrna tm ews/fli1 type 1 lpx with twice a week infusions. a total of 69 doses were given. the most prominent related toxicity has been hematological, 1 patient developed transient g3 neutropenia, another patient developed g3 anemia that required prbc transfusion, and of note this patient had significant bone and bone marrow involvement. one patient only received two lpx infusions; she developed a fatal rsv pneumonia. other reported grade 2 toxicity includes fatigue and headache. evaluable patients (n3) had stable disease between 4 and 12 months before progression. one patient had sustained response for 12 month before progression, two patients are still alive. our preliminary experience supports the safety and potential efficacy of pbi-shrna tm ews/fli1 type 1 lpx as novel treatment for advanced ews with limited toxicity. il-6 increase correlates with higher bi-shrnai ews/fli1 lpx infusion rate and clinical symptoms. further clinical testing is indicated. background: as more children with cns malignancies (bt) are surviving, the late effects of the therapies they receive are better described. studies show that radiation therapy is particularly harmful to neurocognitive functioning, specifically processing speed, working memory, and attention span. these deficits have negative effects on quality of life, especially in academic and professional settings. a large proportion of s255 of s301 adult survivors of bt are unable to reach adult milestones such as living on their own, holding a steady job, and getting married. proton beam radiation therapy (pbrt), is touted for the potential to have fewer and less severe side effects than traditional photon radiation therapy (xrt). because of the properties of protons, the amount of damaging energy released in non-target healthy tissue is reduced when compared to xrt. although a study comparing iq testing between pbrt and xrt found no difference between the two therapies, no studies have compared the specific neurocognitive domains. it would be valuable to evaluate full neurocognitive testing scores (nct) since the specific domains, particularly processing speed (psi), appear to be most vulnerable to radiation therapy. objectives: our primary aim was to assess differences in psi for patients with bt who underwent pbrt versus xrt. a secondary aim was to assess differences in iq (fsiq) and working memory (wmi). we retrospectively evaluated all patients treated for bt at the jimmy everest cancer center within the past 20 years who received rt and had nct post radiation. we examined the full nct results for both subsets of participants to evaluate differences in the specific domains of processing speed, working memory, and iq by measuring percentiles scored in these domains. objectives: we report our experience on imaging children with mm treated uniformly on an institutional melanoma trial. we retrospectively reviewed the clinical and imaging findings of patients with ajcc stage iic-iv cutaneous mm treated on our institutional mel06 protocol. brain mri/ct, pet/ct, ct chest, abdomen, and pelvis (ctcap) were performed at diagnosis in all patients. on treatment, stratum a patients (peg-interferon; ajcc iic, iiia, iiib) (n = 16) had the same imaging repeated every 6 months; stratum b1 (peg-interferon and temozolomide; unresectable measurable disease metastatic, or recurrent) (n = 2) had pet scans every 2 months and brain imaging every 4 months; those in stratum b2 (peg-interferon and temozolomide; unresectable non-measurable, metastatic, or recurrent) (n = 3) had the same imaging performed every 4 months. off therapy all patients continued same imaging every 6 months for 3 years. results: there were 21 patients (11 female; median age 14 years). eleven had spitzoid and 10 conventional melanoma. primary sites included head/neck (n = 6), trunk (n = 7), and extremities (n = 8). patients with spitzoid melanoma had 236 imaging studies (86 pet, 81 ctcap, 11 ct chest, 10 ct brain, and 48 mri brain) with a median of 8, 7, 0, 4 and 0 studies/patient respectively. median cost per patient was $32,718. thirteen studies (5.8%) showed suspicious lesions with 28 additional scans and 2 diagnostic biopsies of which one only was positive stratum a with tert promoter mutation and died from disease). for conventional mm, 162 studies (61 pet, 57 ctcap, 8 ct chest, 7 ct brain, and 29 mri brain) were performed with a median of 7, 6.5, 0, 1, 3 studies/patient respectively. median cost per patient was $23,420. twenty (14%) showed suspicious lesions with 19 additional scans and 6 diagnostic biopsies; four were positive (two at diagnosis); both died of disease; the other two recurred locoregionally and were detected clinically; both are alive and disease free; one patient had diffuse metastases and died shortly after enrollment. after a median follow up of 6.3 years (range 0.4-9.2) 17 patients are alive and disease free. children with spitzoid melanoma should have minimal imaging at diagnosis and follow-up given the low risk of recurrence and low yield and high cost of aggressive imaging protocols. patients with conventional mm should be imaged according to the adult guidelines. nationwide children's hospital, columbus, ohio, united states background: the role of infections in the long term outcome of patients with bone tumors is controversial. two retrospective studies have shown increased survival in osteosarcoma patients who had a post-operative wound infection, while another showed no changes in overall survival. to determine the relationship between wound infections and/or bloodstream infection (bsi) on survival in pediatric and young adult patients with osteosarcoma and ewing sarcoma treated at a tertiary children's hospital. design/method: a retrospective chart review was performed for patients with diagnosis of osteosarcoma or ewing sarcoma from 2006-2016. patients received standard chemotherapy regimens for their disease type and stage. local control included surgical resection and/or radiation therapy. presence of infection was determined by bsi or wound cultures while receiving treatment for primary tumor. the median age of 85 patients was 14 (range 2-46 years) at diagnosis. 53% had a diagnosis of osteosarcoma and 47% had ewing sarcoma. of these, 46% of patients developed an infection during treatment; 25% had bsi, 26% had wound infections, and 5% had both. patients with bsi had a 5 year os of 63.3%, compared to 81% in those without bsi (p = 0.0015). those with both bsi and wound infections had the poorest overall survival of 50%, compared to 80.8% for patients without any infection. patients with wound infections alone had a 5 year os of 80.2%, compared to 75% of patients without a wound infection. our analysis revealed decreased os in patients with bsi; however, this could be due to other confounding factors in the presence of bsi. those with bsi or bsi and wound infections had the poorest survival. wound infections without bsi were associated with a slight increase in survival; however, this study was limited by the number of patients that had local wound infections. with the use of newer surgical techniques, availability of antimicrobials and routine use of prophylactic antibiotics, the incidence of infections while undergoing treatment is low. however, the importance of this clinical observation indicates a likely enhanced immune system associated with infection, supporting the role of immunotherapy for treatment of these aggressive tumors. background: hypoalbuminemia is a well-recognized effect of cancer and other chronic illnesses and is often regarded as a marker of malnutrition. in adults, hypoalbuminemia has been associated with adverse outcomes in patients with cancers of the lung, pelvis, head and neck, gastrointestinal tract, and bone marrow, as well as in some pediatric patients with ewing sarcoma and hodgkin lymphoma. hypoalbuminemia has not been well studied in children with cancer. to determine the incidence of hypoalbuminemia (using age-specific references) in children with cancer receiving chemotherapy at baseline (prior to starting chemotherapy) and to determine whether hypoalbuminemia is associated with inferior 5-year overall survival. design/method: we performed a single institution, irbapproved, retrospective review of pediatric oncology patients diagnosed between 1998 and 2012. five-year survival was estimated using the kaplan-meier method; groups were compared using cox regression. we identified 863 pediatric patients with a first diagnosis of cancer, brain tumor, or other condition possibly requiring chemotherapy. of these 863 patients, 204 were excluded for reasons including not receiving chemotherapy and missing data, leaving 659 patients who had a serum albumin level within 60 days prior to starting chemotherapy. the mean age was 8.1 years (sd 5.8 years); 62% were male; 92% were non-hispanic. the most common diagnosis was acute lymphoblastic leukemia (201 of 659; 31%). one hundred thirty nine of 659 (21%) had hypoalbuminemia prior to starting chemotherapy. there was no statistically significant difference in 5-year overall survival between those with and without hypoalbuminemia (78% vs. 82%, respectively; hazard ratio 1.27, 95% c.i. 0.85 -1.90). conclusion: hypoalbuminemia at baseline in pediatric oncology patients requiring chemotherapy is common (one in five), and was not associated with inferior 5-year overall survival in this cohort. leptomeningeal metastases at diagnosis. standard treatment for completely resected, non-anaplastic supratentorial ependymomas is close observation. treatment for anaplastic or incompletely resected non-anaplastic ependymomas is maximal safe surgical resection followed by focal radiation. however, up to 50% of localized ependymomas recur. the role of chemotherapy in treating ependymomas is under investigation. extraneural metastases of anaplastic ependymomas have rarely been reported and the outcome is dismal. objectives: to report extraneural cervical node metastases of a non-anaplastic ependymoma and successful treatment with surgical resection, radiation, and systemic chemotherapy. design/method: retrospective review of patient medical records, including radiographic imaging and tumor tissue pathology, and comprehensive literature review. results: a previously healthy 3-year-old girl underwent gross total resection (gtr) of an isolated right parietal lobe ependymoma (who grade ii). at age 4 years, magnetic resonance imaging (mri) revealed an isolated localized recurrence. she underwent gtr followed by observation. at age 6 years, she again experienced isolated localized recurrence and underwent gtr followed by 59.4 gy focal conformal photon radiation. at each recurrence, pathology revealed a non-anaplastic ependymoma, and cerebral spinal fluid (csf) cytopathology and spine mri were negative. at age 10 years, she developed an enlarged right posterior cervical chain lymph node. subsequent mri revealed a large rim-enhancing, t2 hyperintense lymph node and multiple abnormally enhancing regional nodes consistent with metastases. biopsy revealed a non-anaplastic ependymoma. mri of the brain and spine, computed tomography of the chest, abdomen, and pelvis, and csf and marrow evaluations were unremarkable. chemotherapy according to acns0831 was initiated. mri after course 3 demonstrated significant node size reduction. she underwent right neck node dissection. only one right level ii lymph node showed metastases. she was treated with 59.4 gy irradiation to the neck and 3 additional courses of chemotherapy. she remains in remission 24 months and 17 months after diagnosis of metastatic disease and end of therapy, respectively. literature review reveals rare reports of extraneural metastatic disease of anaplastic ependymomas to bone, lung, or liver, and only 2 involving lymph nodes, all associated with a poor outcome despite multimodal therapy. to our knowledge, this is the first report of extraneural metastases of a non-anaplastic ependymoma. extraneural metastases should be considered in children previously treated for non-anaplastic ependymomas who experience systemic symptoms, even in absence of cns relapse. multimodal treatment offers potential long-term disease control with acceptable toxicity. arun gurunathan, joel sorger, andrew trout, joseph pressey, rajaram nagarajan, brian turpin cincinnati children's hospital medical center, cincinnati, ohio, united states background: pigmented villonodular synovitis (pvns) is a benign neoplasm of the synovium. standard treatment is surgery, but post-operative recurrence rate is as high as 60%. radiation therapy can be used for local control, but is associated with late effects. while pvns is rarely fatal, aggressive disease and/or extensive surgery can result in substantial functional impairment. colony stimulating factor-1 (csf1) overexpression, often due to chromosomal translocation involving csf1, drives pvns through recruitment of synovial-like mononuclear cells expressing the csf1-receptor. tyrosine kinase inhibitors such as imatinib are active against the csf1-receptor, and have shown benefit in the post-surgical relapse setting. however, questions remain regarding the broader application of imatinib and regarding optimal response assessment. to present three patients with pvns, each with different clinical scenarios, who demonstrate clinical response to imatinib monitored by changes in metabolic activity (maximum suv) on pet/ct. results: three patients with pvns demonstrate pet/ct response to imatinib, guiding management of their challenging clinical scenarios. patient 1 is a 20 year-old female with left hip pvns and high grade articular cartilage loss, with decrease in metabolic activity (suvmax 8.3 to 4.7 in 3 months) on neoadjuvant imatinib, enabling total hip replacement surgery planning. patient 2 is a 16 year-old female with left knee pvns with recurrences after synovectomies, spared subsequent surgical control attempts after clinical improvement correlating with pet/ct response to imatinib (suvmax 10.2 to 4.3 in 2 months). patient 3 is a 27 year-old male with right knee pvns that recurred after total knee replacement, now with clinical improvement correlating with pet/ct response to imatinib (suvmax 11.2 to 5.2 in 3 months). all patients would have been characterized as stable disease by response evaluation criteria in solid tumors (recist). in each of these patients, imatinib has been tolerated well, with no therapy interruptions and absent or easily managed side effects (one patient takes dronabinol for decreased appetite, one patient takes prn immodium for diarrhea). all patients are currently still taking imatinib, with therapy length ranging from five to eleven months. in our series of three patients with pvns, imatinib shows promise for disease management in neoadjuvant and adjuvant settings with a tolerable side effect profile. imatinib should be considered in the treatment of pvns to spare surgical and radiotherapy related morbidity, and treatment effect can be monitored by pet/ct. background: metastatic rhabdomyosarcoma (rms) carries a poor prognosis with three-year event free survival rates ranging between 20%-69% (depending on oberlin risk factors) due to the lack of significantly effective breakthroughs in the recent past. there is an urgent and unmet need for new treatment strategies against this disease. metastatic rms cell lines exhibit increased expression of the erm family membrane-cytoskeleton linker protein ezrin. knockdown of ezrin expression using sirnas decreases the metastatic potential of these cells, whereas forced expression of ezrin results in increased degree of metastasis. the activity of ezrin is controlled by its phosphorylation at the threonine 567 (thr567) residue at the c-terminus of the protein, suggesting that alteration of ezrin phosphorylation may control rms growth and metastasis. our goal was to determine if pharmacological inhibition of thr567 phosphorylation in ezrin affects the growth, survival and metastasis in rms in vitro as well as in vivo. design/method: rms cell lines representative of the alveolar and embryonal histological subtypes were used. rms cells were treated with a small molecule inhibitor of ezrin, nsc668394, which specifically dephosphorylates ezrin at the thr567 residue. baseline expression of ezrin and perm levels as well as the effect of nsc668394 on perm levels in the rms cell lines was determined by western blotting of cell lysates. viability of cells was assessed by trypan blue exclusion, and morphology visualized by bright field microscopy. the extent of apoptosis was detected by imaging caspase 3/7 activation using fluorescent microscopy. motility of rms cells was examined by performing a wound-healing assay. subcutaneous and orthotopic xenografts were established in nsg mice using rd cells (embryonal rms). mice harbor-ing xenografts were treated with intraperitoneal injections of nsc668394 or dmso. results: ezrin is constitutively phosphorylated at the thr567 residue in a majority of the rms cell lines examined. nsc668394 dephosphorylates ezrin at the thr567 residue in these cell lines. treatment with nsc668394 inhibits growth, induces apoptosis and inhibits the migration of rms cell lines in vitro. further, treatment of nsg mice bearing subcutaneous or orthotopic embryonal rhabdomyosarcoma xenografts with nsc668394 significantly impedes tumor progression without any obvious adverse effects. our findings suggest that dephosphorylation of ezrin at the threonine 567 residue may have the potential to be a novel therapeutic strategy for rms patients. all india institute of medical sciences, new delhi, new delhi, delhi, india background: the role of laparoscopy in the management of pediatric intra-abdominal solid tumors is yet to be established. the safety of laparoscopic management of pediatric intra-abdominal tumors is still questionable. we study the results of the initial case series of pediatric intraabdominal tumors managed laparoscopically at our institute from july 2013 onwards. design/method: total 11 children (8-males, 3 females) who presented to us with pediatric intra-abdominal tumors were included. the tumors included wilms tumor (n = 7), neuroblastoma(n = 2), adrenal cortical tumor(n = 1), ovarian teratoma(n = 1).children were between 10months -7years and 4 received neo-adjuvant chemotherapy. a 4-port laparoscopic nephrectomy and lymph node sampling for wilms tumor and adrenalectomy for adrenal tumors was performed. the tumors were removed in-toto with no rupture (except in one). specimens were retrieved through a lumbar incision (n = 8) or an inguinal incision(n = 1). all the children are under regular follow up. two children with wilms tumor had recurrence. the neuroblastoma child underwent open surgery for recurrence later. conclusion: laparoscopy/laparoscopic assisted removal of pediatric intra abdominal tumor is a feasible and safe option. it has the advantage of less postoperative pain, shorter hospital stay and a better cosmetic result. proper patient selection, port placement and laparoscopic experience are contributory. background: targeting of proteins and cell surface antigens specific to cancer cells with monoclonal antibodies has proven to be an effective form of treatment in many forms of cancer. gd2 is a cell surface disialoganglioside that is expressed on the cell surface of some normal tissues including nerve cells, melanocytes, and mesenchymal stromal cells and is overexpressed in some pediatric cancers like neuroblastoma and osteosarcoma. dinutuiximab is a chimeric monoclonal antibody that is fda approved for the treatment of patients with high risk neuroblastoma and under investigation for the treatment of relapsed osteosarcoma. little is known about the patterns of gd2 expression in other pediatric malignancies. objectives: we sought to describe the patterns of gd2 expression in the following pediatric sarcomas: synovial sarcoma, rhabdomyosarcoma and ewing sarcoma. design/method: synovial sarcoma (n = 44), rhabdomyosarcoma (n = 35) and ewing's sarcomas (n = 12) formalin fixed, paraffin embedded cores were obtained from the seattle children's research institute tissue microarray (tma) biorepository. tma blocks consisting of melanoma cores stained with and without gd2 antibody were used as positive and negative controls, respectively. slides were incubated with anti-ganglioside gd2 antibody clone 2q594 (ab68456 from abcam) diluted 1:50 in 10% normal goat serum and 5% bsa in tbs overnight at 4˚c. the negative control of human melanoma section was incubated in 10% normal goat serum and 5% bsa in tbs without primary antibody. the expression of gd2 was indicated by characteristic brown diaminobenzidine staining. the intensity and location of tissue staining were assessed and compared to positive and negative controls. staining was considered positive (+++) if the intensity of the staining was consistent with that of the positive control with 67-100% of cells staining positive. classification of intermediate gd2 expression (++) was assigned to slides in which 34-66% of cells stained positive. slides were classified as sporadic staining (+) if 1-33% of cells stained positive. tissue was considered (-) if there was complete absence of staining, similar to the negative control. objectives: to evaluate the clinical presentation, management and treatment outcomes of children with malignant germ cell tumor at our institute design/method: a prospective study was conducted from june 1994 to dec 2016 in the department of pediatric surgery in a tertiary care institute in a developing country. all patients were evaluated for local disease and metastatic disease by imaging and tumor markers. risk stratified chemotherapy was used with low risk tumor receiving no chemotherapy, intermediate risk: 4 courses of peb chemotherapy and high risk: 4 courses of peb + 2 courses of pe. upfront resection of the primary or the residual disease after neoadjuvant chemotherapy if feasible was performed. follow up was done with monthly tumor markers for 6 months and imaging studies every 3-6 months for initial 3 years. five year overall survival and disease free survival was calculated. results: during the study we treated 152 children who formed the study group. of these 83 (55%) were gonadal (45;30% testicular and 30;25% ovarian) and the remaining 69 (45%) were extragonadal with sacrococcygeal (sct) being the most common site 48 (31%). one hundred and thirteen children (75%) presented to us primarily while the remaining 39 had received treatment elsewhere. stage 3 or stage 4 disease at presentation was present in 104 (68%) children. recurrence was noted in 50 (33%) patients. respectively. patients with testicular mgct and children with age 2-5 years and males had significantly poor rfs rates. conclusion: patients with mgct should be staged correctly and adjuvant chemotherapy is advisable to all patients except stage i endermal sinus tumor of testis. awareness regarding the same is still lacking in our country. meticulous follow up is needed as more than 30% of will recur. cure rates are dismal in children with recurrent mgct especially those who are not chemotherapy naïve. nemours children's specialty care, jacksonville, florida, united states background: radiotherapy for pediatric head and neck tumors often results in mucositis, limiting oral intake and compromising patients' nutritional status. this may be reduced through the improved conformality offered by proton therapy. despite widespread use of enteral tube feeding through a percutaneous gastrostomy (peg) or nasogastric tube (ngt), there is little data available regarding overall incidence of ngt/peg placement and perspectives of pediatric patients and caregivers. objectives: to (a) estimate the need for ngt/peg support and (b) characterize patient and caregiver perceptions surrounding enteral feeding in children with head and neck tumors undergoing proton therapy. design/method: dependent on development stage, patient (n = 16) or parents (n = 6) filled out a series of customized surveys according to a prospective irb approved study. seventythree percent of patients also received concurrent chemotherapy. questions addressed their current feeding route and perception, for example, "what aspect(s) of tube feedings are beneficial to you?" and "what aspect(s) of tube feeding worry or scare you?" fifty-five surveys were distributed before and after radiation, and with any change in feeding route. results: at the start of proton therapy, 1 patient had a ngt and 8 patients had peg. of these, 8 patients (36%) had a ngt/peg in place exclusively for the administration of medication; only 1 patient (4%) needed a ngt/peg for nutrition. in those patients without ngt/peg, 46% would "consider" enteral feeds. in patients without ngt/peg, the most commonly cited benefit was "maximizing my nutrition" (67%) and the most common negative aspect was "fear" of tube placement (100% of patients). all sub-populations (32% of patients) cited change in appearance as a negative aspect. in patients without ngt/peg at the start of proton therapy, 46% of patients/caregivers felt enteral feeding to be "unnecessary," and 83% of these patients would not "consider" ngt/peg even if their "physician advised it." over the course of proton therapy, the patients/caregivers who deemed enteral feeding "unnecessary" decreased from 46% to 18%. at completion of treatment, 7 patients (32%) were using a ngt/peg tube for nutritional support but only one (4%) patient relied exclusively on their enteral feeds. two patients (without ngt/peg) (9%) required parenteral support. our data does not support prophylactic placement of ngt/peg in of children with head and neck tumors undergoing proton therapy. ongoing research is needed to identify which patients will need ngt or peg to supplement their diet. in this cohort, anticipatory counseling should focus on pain, cosmesis, and utility. children's national medical center, washington, district of columbia, united states background: ovarian sex cord-stromal tumors (osct) are rare neoplasms that typically present with signs/symptoms of an adnexal mass and signs of hormonal production1 approximately 20% of ovarian sex cord-stromal tumors in children are sertoli-leydig cell tumors (slct) with median age of presentation 25 years overall.1 to our knowledge the youngest reported case in the literature describes a 9-month old female in china with a slct that was treated with oophorectomy alone.2 some studies have found an association in families between pleuopulmonary blastoma and osct with a germline mutation leading to dicer1 syndrome, which has been associated with a younger age at diagnosis.3,4 objectives: to describe an unusual case presentation of slct in an infant results: 3-month old, twin female, ex-32 week premature infant presented to the emergency department on multiple occasions for abdominal distention and feeding intolerance initially thought to be related to previous omphalocele repair and umbilical hernia. an ultrasound demonstrated an 8 × 6 cm mass arising from the right ovary with large volume ascites. she required admission to the intensive care unit due to s261 of s301 respiratory distress from her significant ascites. serum tumor marker including hcg, afp and ldh were negative. patient underwent right oophorectomy with tumor capsule noted to be open at time of surgery. further imaging post operatively demonstrated no other sites of disease. the patient was classified as figo stage ic due to the presence of her significant abdominal ascites that was presumed to be malignant pre-operative tumor rupture.5 the pathological diagnosis was challenging and eventually resulted as a mixed germ cell sex cord stromal tumor with pattern of sertoli cell tumor with neuroendocrine differentiation. based on the staging of figo ic with pre-operative rupture, the decision was made to treat with a standard platinum based regimen as there is a higher incidence of relapse in stage ic patients when compared to ia treated with observation alone.6 our patient tolerated four cycles of chemotherapy well and end of therapy scans showed no evidence of disease. interestingly, her dicer mutation genetics performed by ion torrent tm next generation sequencing was negative in germline and tumor studies. to our knowledge, our patient is the youngest described with slct. she will continue to be followed with serial imaging alone as she had no evidence of elevated tumor markers at diagnosis.6,7 due to young age and unusual diagnosis, she was referred to cancer genetics team. background: approximately 12% of patients with wilms tumor (wt) have metastatic disease at diagnosis and often have a grave prognosis. limited cell lines are available for the study of metastatic wt and long-term passaged cell lines do not always recapitulate the human condition. focal adhesion kinase (fak) is a non-receptor tyrosine kinase that controls cellular pathways involved in the tumorigenesis of pediatric renal tumors. using a novel patient-derived xenograft (pdx) model from a patient's primary wt (coa 25) and matched isogenic metastatic wt (coa 42), we previously demonstrated that fak is expressed and its inhibition led to decreased tumorigenicity of both the primary and metastatic pdxs. kinomic profiling is an innovative, high-throughput method used to investigate kinase signaling to identify potential therapeutic targets. to date, the kinomic profile of primary and metastatic wt has not been examined. objectives: investigate baseline kinomic differences between primary and metastatic wt and evaluate kinases upstream and downstream of fak as potential targetable therapies. design/method: cells from coa 25 and coa 42 were treated with pf-573,228 (pf), a small molecule fak inhibitor. protein from cell lysates of treated and untreated coa 25 and coa 42 were combined with kinase buffer, atp, and fluorescently labeled antibodies and loaded into a phosphotyrosine kinase or serine-threonine kinase pam-chip® per the uab kinome core protocol. phosphopeptide substrate analysis with the pamstation®12 kinomics workstation (pamgene® international), pamchip® protocol using evolve2 software, and bionavigator v. 6.0 were used to analyze kinases upstream and downstream of fak. the primary wt had increased epha8, ror1 sgk307 and decreased pdgfrb relative to the paired metastatic wt at baseline. treatment with pf increased ron, pdgfrb, p70s6kb, mak, camk2g, vacamkl, camk2d, ck1a1 and pskh1 in the primary wt. treatment with pf decreased tnk1, lmr1, cck4, epha5, pdk1, sgk196, lkb1 and increased pskh1 in the paired metastatic wt. primary wt displayed a different kinomic profile compared to metastatic wt in a matched isogenic pdx model. these data reveal that alternative therapies to specifically target metastases are needed. furthermore, fak inhibition resulted in diverse kinomic alterations between primary and metastatic wt. inhibitors targeting many of these pathways, such as pdgfrb inhibitors, are currently available and potentially could be combined with fak inhibitors in the treatment of wt. the results of the current study indicate that kinases upstream and downstream of fak in primary and metastatic wt warrant further investigation. background: use of high-dose methotrexate (hd-mtx, 12 g/m^2) is a mainstay of standard therapy for pediatric osteosarcoma (os) in north america. in pediatric os, there is a narrow therapeutic window for hd-mtx, with decreased tumor response rate with mtx concentrations <1000 m and decreased survival due to severe toxicity with concentrations >1500 m. risk factors for hd-mtx toxicity have been defined in adults, including body mass index (bmi) and male gender, but such studies have not been conducted in children. we sought to examine the relationship between mtx levels and toxicities during hd-mtx infusion for pedi-atric os, thereby identifying risk factors for increased toxicity and providing a framework for therapeutic drug monitoring. design/method: this retrospective chart review included patients treated at texas children's hospital with hd-mtx as first-line therapy for os from 2009-2015. data abstracted from electronic records included patient characteristics, bmi and body surface area (bsa), baseline and post-treatment laboratory values, mtx levels 4 and 24 hours after dose given (4h, 24h), hour mtx cleared (mtx <0.1 um), grade 3/4 mucositis, myleosuppression, persistent lft elevation (ctace v4.0), and % tumor necrosis. correlation between 4h mtx level and other covariates was summarized using descriptive statistics. we reviewed 128 hd-mtx infusions corresponding to 12 patients. bmi was found to significantly impact 4h mtx level (p<0.05). female gender was also significantly associated with higher 4h mtx level (p<0.001). percent necrosis (available in 9 patients) was associated with 4h mtx levels at near-statistical significance (p = 0.07). 4h mtx level was not found to contribute to toxicities or associate significantly with mtx clearance. analysis in a larger cohort is ongoing. we have identified at least one patient factor (bmi) that significantly impacts 4h mtx levels and is of potential use for future modeling, as current models incorporate bsa only. our findings concord with studies in adult os in that bmi significantly impacts 4h mtx level but diverge in that female gender is associated with higher 4h levels. importantly, these data support targeting 4h mtx levels to ensure that minimum concentration for adequate tumor necrosis is reached. these results do not suggest that monitoring 4h levels would prevent toxicities, thus necessitating further characterization of any intrinsic patient factors that associate with toxicity. overall, our definition of the clinical factors that associate with 4h mtx levels contributes to a framework for therapeutic drug monitoring in pediatric os. children 's mercy hospital kansas city, kansas city, missouri, united states background: post consolidation immunotherapy with dinutuximab, aldesleukin (il-2), granulocyte macrophage colony stimulating factor (gmcsf) and isotretinoin is standard of care for children with high risk neuroblastoma. dinutuximab is combined in 5 alternating cycles with s263 of s301 gmcsf or il2, followed by a 6th cycle with isotretinoin alone. il-2 is administered as a 96hour continuous infusion on days 0-4 at 3miu/m2/day followed by a higher infusion dose, 4.5miu/m2/day, in combination with dinutuximab on days 7-10 of cycles 2 and 4. the 3miu/m2/day dose may be administered inpatient or in the ambulatory setting. objectives: to retrospectively compare the incidence of inpatient and outpatient side effects and complications associated with low dose (3miu) il2 to provide the tolerability data necessary to evaluate these venues for future administration options. design/method: this study was a descriptive, singlecentered definitive study utilizing a retrospective convenience sample population of children with high risk neuroblastoma who received low dose il2 either as an inpatient or an outpatient without exclusion from may 2012 to june 2017. subjects were identified by a tumor registry query post irb approval. electronic and paper medical records were reviewed for the dates and location of the infusions, the home health company used if applicable and all documentation regarding clinical status, side effects and toxicity. demographics was limited to age and gender. results: infusion venue was chosen by provider preference. twenty-six infusions, 9 inpatient and 17 outpatient via 3 separate home health companies were all administered in entirety and without interruption. there were 10 males and 4 females ranging from 2-7 years of age. two children received a single outpatient infusion due to intolerance of il2 when combined with dinutuximab and 2 received therapy in both settings. fever, 3 inpatient and 1 outpatient was the only common side effect. no source of infection was ever identified. there was one incidence of diarrhea and one patient with pruritus in both the outpatient and inpatient settings respectively. no planned outpatient infusions required subsequent admission however the outpatient fever did necessitate an er evaluation. conclusion: low dose il 2 can successfully be administered outpatient. the medication has minimal side effects with fever occurring in 15%, none of which were associated with infection. no outpatient infusion required a subsequent admission. no patients who received cycle 2 infusions outpatient opted to receive the next cycle inpatient. baylor college of medicine, houston, texas, united states background: metastatic ewing sarcoma (es) has an extremely poor overall survival, necessitating investigations into molecular mechanisms to identify novel targets and develop new therapies. we previously performed an in vivo study, using our mouse model, designed to provide insights into transcriptomic and proteomic signatures for metastatic es to identify potential therapeutic targets. comparing profiles of primary tumors to corresponding metastatic lesions, we identified aberrant expression of integrin ß3 (itgb3) and downstream activation of integrin-linked kinase (ilk) in metastatic lesions compared to primary tumors, implicating this pathway as a key regulator in the ability of es to establish and enhance metastasis. our hypothesis is that upregulation of itgb3 and its downstream signaling events play a key role in es metastasis and are viable therapeutic targets. objectives: to investigate the role of itgb3 and its downstream signaling pathways in driving the establishment and enhancement of metastasis in es and to investigate this pathway as a potential therapeutic target. to investigate the role of itgb3 and ilk in es metastasis, we used sirna to knock down itgb3 and ilk expression in established es cell lines and then performed functional assays in vitro, including cell proliferation and invasion/migration assays. we also tested inhibition of this itgb3 signaling pathway using available small molecule inhibitors targeting itgb3, ilk and the downstream target ap-1, using cilengitide, compound 22 and sr11302, respectively. we are currently using these small molecule inhibitors as treatment in vivo and assessing rates of metastatic tumor formation. we generated stable itgb3 and ilk overexpression and knockdown cell lines, which we are using for similar in vitro and in vivo investigations. knockdown of itgb3 and ilk in our sirna cell lines resulted in decreased cell proliferation and decreased invasion and migration compared to controls. we also found significantly decreased cell proliferation using each of the small molecule inhibitors in vitro. our preliminary studies using compound 22 in vivo established a safety profile and dose escalation is underway to assess the effectiveness of inhibiting es metastasis. these results support our hypothesis that itgb3 and its downstream signaling events play a key role in the ability of es to establish metastatic foci and may serve as a potential therapeutic target. we continue to investigate this pathway in vitro. we are also using our small molecule inhibitors and itgb3 and ilk overexpression and knockdown approaches to study these effects on metastatic tumor development in vivo using our mouse model. background: neuroblastoma (nbl) is characterized by phenotypic heterogeneity. outcome is excellent for patients with low-(lr) and intermediate-risk (ir) disease, whereas only 50% of high-risk (hr) patients will survive. 5-hydroxymethylcytosine (5hmc) is an epigenetic marker of active gene transcription, and 5hmc profiles are prognostic in many types of adult cancers. we hypothesized that 5hmc profiles will serve as robust biomarkers in children with nbl tumors, refining current risk stratification. objectives: analyze genome-wide 5hmc in nbl tumors and correlate 5hmc deposition with chromosomal copy number and gene expression. design/method: 5hmc was quantified by nano-hmc-seal-seq from the dna extracted from 15 hr, 11 ir and 27 lr nbl tumors. read counts and clinical data were analyzed with deseq to identify genes with differential 5hmc patterns between risk groups. chromosomal copy number was assessed by chromosomal microarray analysis (cma) in a subset of samples (3lr and 9hr). expression of genes located on chromosome 1p was evaluated using publically available microarrays (e-mtab-1781) of 171 hr nbl tumors with known 1p loh status. results: globally, lr tumors had more 5hmc peaks (140,062) than ir (102,398, p = 0.36) tumors, or hr tumors (79,727, p = 0.01). 1,049 genes had different patterns of 5hmc deposition in hr versus lr tumors. 315 (30%) of these genes mapped to chromosome 1p and had decreased 5hmc in hr versus lr tumors (padj <0.05). in the cma analysis 1p deletion was detected in 5 of the 9 tumors tested. in the tumors with 1p loss, 322 genes that map to 1p showed decreased 5hmc deposition compared to the 4 hr tumors without 1p loss (p<0.05). further, compared to the tumors without 1p loss, the expression of 188 of the 322 1p genes was decreased (p<1 × 10-5), including chd5, camta1, and arid1a, known and proposed tumor suppressor genes in nbl. conclusion: different patterns of 5hmc accumulation are associated with neuroblastoma risk classification. nano-hmc-seal-seq is sensitive to copy number variations and has the potential to identify these changes in patient tumors. our results suggest that 5hmc deposition contributes to the silencing of tumor suppressor genes in 1p and may also regulate the transcription of other genes that drive tumor phenotype. background: metastatic osteosarcoma has a 5-year survival rate of 15-40%. pulmonary metastases remain a major treatment challenge in osteosarcoma. current treatment with conventional chemotherapy shows inadequate activity towards metastases and has toxic systemic side effects. chloroquine is a widely used anti-malarial drug and has been shown to have promising anti-cancer and anti-metastatic activity. polymeric drugs have been shown to have multiple advantages over their small molecular parent drugs, including enhancing the therapeutic efficacy, an improved pharmacokinetics profile and decreased systemic toxicity. we hypothesized that by developing chloroquine into a polymeric drug and combining it with conventional chemotherapy it will improve the treatment of metastatic osteosarcoma. objectives: to identify the optimal combination of polymeric chloroquine (pcq) with conventional chemotherapy active in osteosarcoma as a new means of treating metastatic disease in a murine osteosarcoma model. we synthesized and developed pcq and evaluated its anti-invasive activity using an osteosarcoma cell migration and invasion assay. we evaluated the efficacy of cell killing using combination drug therapies with pcq and a panel of conventional chemotherapy agents (doxorubicin, docetaxel, cisplatin and paclitaxel) using celltiter blue cell viability assay. to develop the murine osteosarcoma model, we intravenously injected luciferase-expressing human osteosarcoma cells 143b into nsg mice. we administered the drug combination that showed the strongest in vitro synergy to the mice and evaluated their anti-cancer and anti-metastatic effects in vivo. tumor growth and suppression were evaluated using whole body bioluminescence imaging. results: we successfully synthesized pcq that contains 16.7% chloroquine with a molecular weight of 18.9 kd. pcq was also found to decrease the toxicity of the parent chloroquine. pcq showed strong inhibition of osteosarcoma cell migration with 51% inhibition compared to 13% by chloroquine. we screened the combination drug therapies and found the combination of pcq and doxorubicin to show the strongest synergism. the pcq/doxorubicin combination is currently being evaluated in the murine model. combination drug therapy using pcq and doxorubicin showed synergistic cell killing and inhibition of cell migration in vitro. the combination represents a promising treatment strategy for pulmonary metastatic osteosarcoma. emory university/children's healthcare of atlanta, atlanta, georgia, united states background: survival for relapsed high-risk neuroblastoma (rnb) is < 5%, underscoring the critical need for novel therapies. rnbs have increased ras/raf/mapk mutations and increased yes-associated protein (yap) transcriptional activity. yap is a transcriptional co-activator that binds with tea-domain (tead) transcription factors to regulate cellular proliferation, self-renewal, and survival. we found that shrna inhibition of yap decreases nb cell proliferation and sensitizes ras-mutated nbs to mek inhibitors, supporting yap as a tractable therapeutic target. verteporfin (vp), a photodynamic drug used for macular degeneration, is the only drug found to inhibit yap expression or yap:tead binding to kill tumor-derived cells. peptide 17 is a 17mer yap peptidomimetic that also disrupts yap:tead interactions. we sought to determine whether these compounds are potent in nb via yap direct effects. design/method: yap expressing (nlf, sk-n-as) or yap null (ngp, lan5, sk-n-as-shyap) human-derived nbs were incubated with vp, with and without direct light exposure, or with peptide 17. celltiter-glo and immunoblots were used to assess for cell death and yap-downstream protein expression, respectively. results: without direct light exposure, vp inhibits yap expression at nm dosing, yet no nb cell death was observed at equal or higher concentrations. egfr and erk1/2 were inhibited along with yap, confirming yap/ras pathway coregulation. when vp was exposed to direct incandescent light for 30 minutes, > 80% nb cell death occurred in all nbs tested, even those lacking yap. peptide 17 caused no cell death or yap inhibition up to 75 um. neuroblastomas are resistant to vp at doses sufficient to inhibit yap expression. in macular degeneration, light-activated vp produces reactive oxygen species, which we hypothesize is the off target mechanism killing nbs independent of yap. given the off target effects and the need for light activation, vp is not an ideal preclinical or clinical yap inhibitor. accordingly, peptide 17 has poor cell permeability and low tead affinity, leading to its lack of efficacy. given the relevance of yap in rnb and other cancers, we are chemically optimizing a yap peptidomimetic with enhanced permeability, nuclear localization, and tead affinity to create a bonafide yap inhibitor for preclinical and clinical application. kayeleigh higgerson, aaron sugalski, rajiv rajani, josefine heim-hall, jaclyn hung, anne-marie langevin ut health san antonio, san antonio, texas, united states background: osteosarcoma is the most common bone malignancy in children, adolescents, and young adults. most study cohorts have 10 to 15% hispanic patients that encompass many different hispanic backgrounds. the university of texas health science center at san antonio (uthscsa) sarcoma team serves a latino population that is predominantly mexican american, thus providing a unique opportunity for evaluation this population. this study expands on previous data collected from january 2000 to december 2010 from the same institution, providing increased insight into outcomes of mexican american children, adolescents, and young adults with osteosarcoma. objectives: to further understanding of osteosarcoma in latino children, adolescents and young adults. design/method: a retrospective analysis of demographics, tumor characteristics, response to treatment, and survival outcome of all localized osteosarcoma of the extremity patients below 30 years of age diagnosed and treated by the uthscsa sarcoma team between january 2000 and june 2017 was performed. results: in our original cohort from january 2000 to december 2010, we observed a significantly decreased 5-year eventfree survival (efs) in patients diagnosed before age 12 (preadolescent) relative to patients diagnosed between ages 12 and 29 (11% vs. 57%, p<0.001). patients had a 5-year overall survival (os) and event-free survival of 65% and 48% respectively. in our expanded cohort from january 2000 to june 2017 we evaluated sixty-six patients with a median age of 14 (range, 2 to 28 y) with localized high-grade osteosarcoma of the extremity. the expanded cohort was 68% mexican american, with a median follow-up of 59 months (range, 5 to 192). the analysis of our expanded cohort is ongoing and we postulate that the findings will hold true, as we increase the cohort size and length of follow-up. conclusion: analysis of our previous cohort, predominantly of mexican american ethnicity, showed that preadolescent patients had an increased rate of relapse when compared with previous large studies. we also showed a trend towards decreased efs for the entire cohort. we hypothesize that we will further validate these findings with this expanded cohort and this will support further investigation into potential causes of poor outcome in this vulnerable latino population. background: neuroblastoma in infants has the potential to regress or mature spontaneously. growing literature showed that some cases subjected to initial observation didn't show inferior outcome compared to actively treated similar categories. objectives: we investigated whether early active treatment can be safely avoided/deferred in selected favorable cases at the children's cancer hospital-egypt (cche). design/method: patients enrolled on the watch and see strategy (w&s) at cche had small primary tumor; inss stage 1-2, uncomplicated stage 4s or stage 3 infants (< 365 days). tissue biopsy was not mandatory for infants below 6 months of age with localized adrenal mass (stage 1-2). on progression, immediate intervention took place according to stage and risk of disease after biological characterization. results: thirty four nbl patients were enrolled on w&s strategy; m/f:2.4/1. eighteen patients had stage 4s disease, 12 patients had stage 1-2 and 4 were stage 3. primary adrenal site was reported in 29 patients (85.3%), 21 patients (61.77%) had small mass measuring ≤5 cm in its largest diameter. the 5-year os & efs were 88.2±8.8% and 72.5±9%, respectively, with 43 months median follow-up (range: 1-106 months). spontaneous total/near total resolution of mass occurred in 16/34 patients (47%). median time to eliciting regression was 1.7 months (range: 0.4-14.7 months), and 20.7 months (range: 7-63 months) till complete resolution. only 8/34 patients (23.5%) witnessed progression (2 local, 2 distant and 4 combined local and distant progression); median time to progression was 8 months (range: 1-32 months) with 2/8 deaths after starting chemotherapy. watch and see strategy is a safe approach in localized and uncomplicated stage 4s neuroblastoma. progressive cases could be rescued. baylor college of medicine, houston, texas, united states background: ga-68 dotatate binds to somatostatin receptor 2 expressed in neuroendocrine tumors (nets). it was approved by fda in 2016 for use with pet/ct scan for localization of somatostatin receptor positive nets in adult and pediatric patients. pediatric approval was based mainly on extrapolation of data from adults. objectives: to describe the use of ga-68 dotatate pet/ct scan in children with neuroendocrine tumors and compare with other imaging modalities. design/method: patients with nets enrolled in texas children's rare tumor registry between february and october 2017 were reviewed and those patients who underwent ga-68 scan were included. results: four patients with nets underwent ga-68 scans without any adverse reactions. first patient was a 15-yearold female with small bowel net with multiple liver metastases. mri abdomen and fdg pet at diagnosis showed s267 of s301 multiple liver metastases but could not identify the primary lesion. ga-68 scan was able to accurately identify the enlarged lymph nodes in the small bowel and was better than fdg pet in delineating the liver metastases. second patient was a 13-year-old female with recurrent small bowel net with liver, lung and paraspinal metastases. the lesions were initially detected by ct scan. octreotide scan failed to show any uptake in the identified lesions while ga-68 was taken up by the liver lesions, lung lesions >1 cm in size and the paraspinal lesion. third patient is an 8year-old male with pancreatic net with peripancreatic lymphadenopathy, multiple liver metastases and cardiophrenic lymph node involvement. the primary lesion in the pancreas could not be identified by ct scan, ct angiogram, mibg scan, or octreotide scan. in addition, there was uncertainty about involvement of the enlarged cardiophrenic lymph node. in addition to clearly identifying the primary lesion, ga-68 scan was able to detect multiple peripancreatic lymph nodes not detected by other scans and revealed uptake in the cardiophrenic lymph node confirming its involvement by the tumor. fourth patient is a 15-year-old female with malignant abdominal paraganglioma with solitary lung metastasis. both mibg scan and ga-68 scan were able to identify the primary lesion. ga-68 scan was performed after the lung metastasis was removed and thus its ability to detect it could not be confirmed. background: neuroblastoma is the most common extracranial solid tumor of childhood, with overall survival for high-risk patients (hrnbl) near 50%. the outcomes of hrnbl have improved with high dose chemotherapy followed by autologous stem cell rescue (abmt). data about factors influencing the rate of hematopoietic recovery following abmt in hrnbl is lacking in the literature. our objective was to identify factors influencing the rate of hematopoietic recovery following abmt in hrnbl. design/method: this was a retrospective chart review of 55 patients with hrnbl treated at texas children's hospital from 2006 to 2016. neutrophil engraftment was considered the first of three consecutive days with post-transplant neutrophil count greater than 500 cells/ul. red blood cell and platelet engraftment were considered at a hemoglobin greater than 8g/dl and platelets greater than 20,000/ul three days after the last transfusion. race and conditioning regimen were analyzed using one-way anova; amount of infused cells was analyzed using pearson correlation coefficients; chemotherapy delay and bone marrow (bm) involvement after cycle 2 of induction chemotherapy were analyzed using independent sample t-tests. the study included 32 males and 23 females with a median age at diagnosis of 2.5 years. thirtyeight patients were caucasian, 6 african-american, 5 hispanic, 2 asian, and 4 did not have race documented. the mean dose of infused cd34+ cells was 3.36 × 10^8 cells/kg. forty-five patients received conditioning therapy with carboplatin/etoposide/melphalan (cem), 8 received busulfan/melphalan (bu/mel), and 2 received thiotepa/cyclophosphamide (thiotepa/cpm). the conditioning regimen administered was significant (p = 0.037) for time to engraftment of neutrophils, with bu/mel at 16.6 days, cem at 12.1 days, and thiotepa/cpm at 10 days. a delay of chemotherapy during induction (n = 25) was significant (p = 0.001) for time to platelet engraftment of greater than 75,000/ul and trended towards significance (p = 0.088) for time to neutrophil engraftment. bm involvement at diagnosis and after cycle 2 of induction was not significant for time to engraftment. dose of stem cells infused was the only variable significant for hemoglobin engraftment. background: osteosarcoma (os) is the most prevalent aggressive primary malignancy of the bone affecting children and young adults. approximately 10% to 20% of patients have metastatic disease at initial presentation, and 61% of those patients have isolated pulmonary metastases. although overall survival in patients with os has improved with advances in therapy, there have been no significant improvements in survival outcome in patients with metastatic disease. recent studies suggest that tumor-associated vascular cell adhesion molecule 1 (tvcam-1 or cd106) plays a critical role in the metastatic progression of various tumors. indirect evidence from these studies suggest that vcam-1/ 4 1 integrin signaling promotes tumor survival and metastatic progression by changing the tumor niche and associated immune response. to determine if interfering vcam-1/ 4 1 signaling between pulmonary metastatic osteosarcoma (pos) and macrophages (macs) by down-regulating vcam-1, depleting macs or blocking vcam-1/ 4 1 signaling will reduce pos and improve overall disease-free survival. design/method: we used a pair of spontaneous, high-grade murine os cell lines from balb/c mouse (h-2d), k7 and k7m2 (derived from in vivo k7 metastasis). we used lentiviral shrnas to knockdown vcam-1 mrna and protein expression in k7m2 (vcam-1kd). we introduced luciferase into k7, k7m2 and various k7m2 shrna cell lines to follow lung metastasis by bioluminescence (bli). we depleted macs by intranasal administration of liposomal clodronate formulation. we tested the ability of k7 and k7m2 supernatants to polarize m0 macs into m1 or m2 phenotype in vitro. we also administered anti-4 monoclonal antibody (anti-4 mab) intranasally to assess the outcome of functional blockade of vcam-1/ 4 1 signaling. results: k7m2 over-expressed vcam-1 compared to k7. mac depletion in k7m2-bearing animals exhibited reduced pos. weekly administration of anti-4 mab resulted in 80% tumor-free rescue among mice with established k7m2 pos. interestingly, supernatant from k7m2 but not k7 preferentially induced m2-like macs, suggesting a novel integrin-mediated mechanism of m2 differentiation. validation data with additional os cell lines will be presented. despite aggressive multimodal therapy, overall outcome for patients with pos remains dismal at 25-30%. for this reason, novel and directed therapy approaches are desperately needed. molecular targeted approaches for therapy are challenging, due to the complex genetic heterogeneity of os. immune-modifying therapy is a promising new alternative approach for pos. university of chicago, chicago, illinois, united states background: only half of all patients diagnosed with high-risk neuroblastoma achieve long-term survival. 123imetaiodobenzylguanidine (mibg) scans are routinely used to evaluate disease at diagnosis and following treatment, and the extent of disease is quantified using the curie scoring system. a previous study by yanik et al., has shown that for high-risk patients with mycn non-ampliified tumors, scores less than versus greater than 2 following 6 cycles chemotherapy are associated of superior survival, whereas scores less than versus greater than 0 were prognostic in patients with mycn-amplified tumors. however, the prognostic significance of specific sites of metastatic disease at diagnosis is not known. to determine if site of metastatic disease determined by 123i-metaiodobenzylguanidine (mibg) imaging in high-risk patients at the time of diagnosis was associated with outcome design/method: we performed a retrospective chart review of high-risk neuroblastoma patients treated at comer children's hospital and lurie children's hospital in chicago between 2006 and 2017 with positive mibg scans at the time of diagnosis. we collected imaging data as well as other clinical data including bone marrow status. sites of disease were defined as curie regions with any positive value. kaplan-meier analysis was performed to evaluate the association with disease sites and survival. pearson correlation coefficients were calculated to compare bone marrow disease to sites of positivity on mibg scan. the cohort consisted of 49 high-risk patients. 31 had skull disease, and 30 had pelvic disease. the presence of mibg positive disease in the skull and in the pelvis trended toward worse efs. efs at 3 years for patients with disease in the skull at diagnosis was 52 ± 10% and for patients without skull disease was 78 ± 10 % (p = 0.16). efs at 3 years for patients with and without pelvic disease was 49 ± 10% and 80 ± 9% (p = 0.10). consistent with prior data, we found that the presence of bone marrow disease was associated with worse survival with 3 year efs of 47 ± 10% and 88 ± 8% with and without marrow disease at diagnosis (p = 0.02). there is the highest correlation between pelvic disease on mibg scan and bone marrow disease with pearson coefficient 0.79. pelvic disease noted on mibg scan likely reflects underlying bone marrow disease. in patients with high-risk neuroblastoma, skull disease and pelvic disease on mibg scan at diagnosis may predict worse event free survival. background: osteosarcoma is one of the deadliest cancers in the pediatric population with little progress in morbidity and recurrence rates since the 1980's. oncolytic herpes simplex-1 virus (ohsv) is an attenuated virus that has shown encouraging results against certain solid tumors. programmed cell death protein (pd)-1-mediated t cell suppression via engagement of its ligand, pd-l1, is also of particular interest due to recent successes in selected cancers, especially those with high genetic mutational loads. most pediatric cancers do not have a wide variety of mutations; however, osteosarcoma has a chaotic genome, prone to genetic mutations. it has been shown through numerous other studies that pd-1 inhibition alone is not sufficient to result in statistically significant tumor growth delays in osteosarcoma models and patients. we hypothesize the addition of ohsv therapy as an immunologic stimulus to pd-1 inhibition is efficacious for osteosarcoma. (1) to determine whether ohsv therapy enhances response to pd-1 inhibition in immunocompetent murine models of osteosarcoma and (2) to quantify and characterize the anti-tumor t-cells infiltration after treatment with ohsv and pd-1 inhibition individually and in combination. we utilized an immunocompetent transplantable murine model using a cell line derived from a spontaneous metastatic osteosarcoma (k7m2, balb/c background). we transplanted established tumor wedges subcutaneously and monitored tumor volume by caliper measurement. once tumors reached 200-400mm3, we administered intratumoral injections of hsv1716 (1 × 108 plaque-forming units) every other day for a total of 3 injections. we then gave intraperitoneal injections of 250ug anti-pd-1 or control antibody twice weekly, up to 4 weeks, starting from the last dose of virus treatment. we monitored tumor growth via calipers twice weekly until tumors reached 2500mm3 or 2cm diameter. we quantified and characterized innate and adaptive immune cell infiltrates in tumors using flow analysis. we found significantly prolonged survival with our combination therapy group compared to all other groups. we found that anti-pd-1 by itself had little impact on t cell recruitment while the combination group had higher influx of cd8+ cells with a reduced amount of t-regulatory cells (cd4+foxp3+cd25+). we also found an increase in cd44+ effector memory cells. osteosarcoma is a deadly cancer with therapeutics remaining unchanged for the last 30 years. here, we describe prolonged murine survival after treatment with combination of pd-1 inhibition and ohsv injection. the combination treatment changed the microenvironment to be more inflammatory. our data support further preclinical and clinical studies. background: neuroblastoma is the second most common cause of cancer related death in children. treatment for high-risk neuroblastoma has improved significantly over the past twenty years, however cure rates remain below 50%. immunotherapy has emerged as an effective therapy for neuroblastoma, however new modalities and targets are needed to improve outcomes. objectives: our lab has developed a chimeric antigen receptor (car) that targets b7-h3 (cd276), an immune checkpoint molecule overexpressed on many cancers, including neuroblastoma. we hypothesized that b7-h3 would be a good target for car based immunotherapy for neuroblastoma. design/method: neuroblastoma tissue microarrays of primary patient samples were screened for b7-h3 expression by immunohistochemistry and cell lines were screened using flow cytometry. b7-h3 car t cells were tested in vitro by measuring tumor cell killing and cytokine production after coculture with tumor cell lines and in vivo in an orthotopic model of neuroblastoma. results: b7-h3 expression was detected by ihc on 82% of the 186 screened neuroblastoma patient samples. b7-h3 was expressed at high levels (2+ or 3+) in more than half of these samples (56%). almost all cell lines screened were homogeneously positive for b7-h3 by flow cytometry. retrovirally transduced b7-h3.4-1bb. car t cells were cocultured with three b7-h3 positive neuroblastoma cell lines (sk-n-be2, kcnr, and chla255) and robust tumor cell killing was demonstrated using an incucyte assay. supernatant from the co-cultures was harvested after 24 hours and both interferon gamma and il-2 production were detected by elisa.in an orthotopic subrenal capsule xenograft model of neuroblastoma, mice treated with b7-h3 car t cells show significant reductions in tumor growth and prolonged survival compared to those treated with untransduced control t cells. however, the treatment is not always curative.b7-h3 car t cells express high levels of exhaustion markers (pd1, tim3, and lag3) when compared to cd19 car controls. in order to overcome inhibition from exhaustion, b7-h3 car t cells were co-cultured with neuroblastoma cell lines and pd-1 blocking antibody. nivolumab significantly increased the production of il-2 and interferon-gamma by b7-h3 car t cells. further studies are underway to determine if b7-h3 car t cell activity is enhanced in vivo by treating animals with pd-1 blockade along with car t cells. conclusion: b7-h3 is expressed on a majority of neuroblastoma samples and appears to be a promising candidate for car t cell therapy. b7-h3 car t cells demonstrate activity against neuroblastoma xenografts that may be enhanced by the addition of pd1 inhibitors. helen devos children's hospital, michigan state university, grand rapids, michigan, united states background: osteosarcoma is the most common bone tumor in children. it is often metastatic at diagnosis and in this scenario less than 30% of children survive. polyamines, small molecules found in all cells, are involved in many cell processes including cell cycle regulation, immune modulation, cell signaling and apoptosis. they are also involved in tumor development, invasion and metastasis. in neuroblastoma, inhibition of the polyamine biosynthesis pathway with odc inhibitor alpha-difluoromethylornithine (dfmo) results in decreased cell proliferation and differentiation. these finding have led to multiple phase i and phase 2 ii multicenter clinical trials in pediatric neuroblastoma patients. dfmo is an attractive drug as it is oral, well-tolerated, can be given for prolonged periods and is already used in pediatric patients. the polyamine pathway has not been evaluated in osteosarcoma. objectives: evaluate effect of inhibition of polyamine biosynthesis with dfmo on osteosarcoma proliferation and cell differentiation. design/method: up to three osteosarcoma cell lines were used: mg-63, u-2 os and saos-2. cells were exposed to 5 mm dfmo for 6 days with replacement of media and dfmo on day 3. intracellular polyamine levels were measured by high performance liquid chromatography (hplc). cell numbers were obtained with a hemocytometer using trypan blue. flow cytometry cell cycle distribution (facs) and propidium iodide were used to evaluate for cell cycle arrest. the protein expression of several osteosarcoma differentiation markers was measured by sds-page and western blot using differentiation specific antibodies. a bioluminescent cell viability assay was used to measure cell recovery over several days after dfmo was removed and replaced with standard media. results: dfmo exposure resulted in significantly decreased cell proliferation in all cell lines. after treatment, intracellular spermidine levels were nearly eliminated in all cells. cell cycle arrest at g2 was observed in u-2 os. cell differentiation was most pronounced in mg-63 and u-2 os cells as determined by increased osteopontin levels. remarkably, cell proliferation continued to be suppressed for several days after removal of dfmo. conclusion: based on our findings dfmo is a promising new adjunct to the current osteosarcoma therapy for high risk patients. it is a well-tolerated oral drug that is currently in phase ii clinical trials in pediatric neuroblastoma patients as a maintenance therapy. the same type of regimen may also improve outcomes in metastatic or recurrent osteosarcoma patients for whom there have been essentially no medical advances in the last 30 years. background: recent studies demonstrate that lower levels of the ews-fli1 fusion oncoprotein are associated with enhanced metastatic capability in ewing sarcoma. the nf-kb transcription factor is a critical mediator of cxcr4 and cxcr7 -driven metastasis in multiple cancers, and increased cxcr4 and cxcr7 expression have each been associated with increased metastasis and poor prognosis in ewing sarcoma. we thus sought to investigate the impact of ews-fli1 on cxcr4/cxcr7-dependent nf-kb signaling in ewing sarcoma. objectives: the goals of this study are 1) to determine the impact of cxcr4/cxcr7 signaling on metastasis-associated nf-kb target gene expression in ewing sarcoma and then 2) to investigate how the ews-fli1 fusion oncoprotein modulates this response . design/method: we utilized multiple ewing sarcoma cells lines including a673, chla9, chla10, tc32 and tc71. cxcr4/cxcr7 cell surface expression was determined by flow cytometry. ews-fli1 level was modulated using sirna and expression levels were confirmed by western blot and rt-pcr. p65 dna binding was measured via elisa. nf-kb target gene expression was assessed via rt-pcr. results: consistent with ihc analysis of primary and metastatic patient tumor samples, the paired primary and metastatic ewing sarcoma cell lines chla9 and chla10 showed dramatic differences in cxcr4 and cxcr7 expression, with the metastatic chla10 line demonstrating much higher expression of both receptors. other cell lines (nonpaired) showed variable cxcr4/cxcr7 expression. genetic knock-out of cxcr4 lead to significant decrease in expression of both cxcl12/sdf-1 and il-6, two nf-kb transcriptional targets known to play a key role in tumor metastasis. knock-out of cxcr4 did not alter endogenous ews-fli1 mrna levels. conversely, lowering the level of ews-fli1 using sirna lead to enhanced nf-kb signaling, indicated by an increase in p65 dna binding. consistent with this observation, treating ewing cell lines with ews-fli1 sirna also resulted in significantly increased nf-kb target gene expression compared to control cells and target gene expression was then further enhanced upon cxcr4/cxcr7 receptor stimulation with the receptor ligand cxcl12/sdf-1. our findings indicate that the ews-fli1 oncoprotein negatively modulates cxcr4/cxcr7-dependent nf-kb signaling. this suggests that ews-fli1 low, cxcr4/cxcr7 high cells, which are associated with enhanced metastasis and poor prognosis, would be anticipated to exhibit enhanced expression of key nf-kb target genes. importantly, the nf-kb pathway is a druggable target that could potentially serve as an "achilles heel" in this subset of high risk tumors. current work is evaluating nf-kb inhibition as an approach to treating metastatic and refractory ewing sarcoma. background: acute graft versus host disease (agvhd) is a major cause of morbidity and mortality following allogeneic bone marrow transplant (bmt) in pediatric patients. gastrointestinal (gi) agvhd is the most serious manifestation. recently, decreased paneth cell (pc) in a predominantly adult cohort was shown to correlate with agvhd clinical grading and response to treatment. we aim to demonstrate the relationship between pc counts and gi agvhd stage and response to therapy. design/method: charts of patients who underwent endoscopy following bmt between 2004-2014 were reviewed. for repeated biopsies during the course of agvhd, only the first was included for analysis. one pathologist retrospectively reviewed the biopsies and counted pcs in 3 high powered fields; the average pc count was analyzed. twenty-six percent of biopsies were reviewed by a second blinded pathologist. statistical associations between pc counts and day 28 (d28) response, agvhd stage, and other study covariates of interest were gauged using general linear regression. agreement in pathologist pc counts was quantified by intraclass correlation (icc). the research was approved by the children's healthcare of atlanta irb. results: seventy-eight biopsies were included in the analysis. mean age at transplant was 10.5 years ± 5.8 (range: 2 months -20 years). most patients underwent transplant for hematologic malignancies (63, 74%). the majority of transplants used a matched unrelated donor graft -including cords (59, 69%) and myeloablative conditioning regimens (71, 82%) -52% received total body irradiation. of these, 64% were diagnosed clinically with gi agvhd (stage 1, 42%; stage 2, 14%; stage 3, 22%; stage 4, 22%). icc showed good agreement (0.833) between the pathologists. mean pc was 16.8 for patients with no gut agvhd, 21.3 for stage 1, 22.9 for stage 2, 9.4 for stage 3 and 5.9 for stage 4 (p = 0.001). on multivariate analysis pc was strongly associated with gi agvhd stage (p<0.001) after controlling for age, preparative regimen intensity, and diagnosis (malignant vs. non-malignant). mean pc counts were significantly lower in patients with no response to steroid therapy at d28 (complete response (mean 17.8) vs. persistent disease (4.2) vs. partial response (4.5) (p = 0.001)). patients diagnosed with gi agvhd with pc counts less than 10 had a higher risk of mortality (hr 3.1, 95% ci: 1.17, 8.09; p = 0.023). lower pc count correlated with stage 4 gi agvhd, refractory disease at d28, and mortality. incorporating pc count in pathology review during gi agvhd work-up may help in agvhd risk stratification. background: there have been increasing discussions pressuring health care teams and institutions for potentially bearing the cost of clostridium difficile infections (cdi) as a health care-associated infection in the recent years. the pediatric oncology patient population, though small, accounts for significant portion of all cdi with 10-15-fold increased risk. hematopoietic stem cell transplant (hsct) recipients constitute a unique subset with distinct risk factors, such as severe immune deficiency state and graft versus host disease (gvhd). although there is ample data on cdi in adult hsct recipients, reports on pediatric experience are limited. objectives: to evaluate the incidence and patterns of cdi among pediatric hematology, oncology and hsct inpatients at our institution. a retrospective review of all clostridium difficile (cd) stool tests performed using toxin enzyme immunoassay and later, polymerase chain reaction targeting toxin genes between 2007 and 2017 in a large, urban academic children's hospital was performed. the data were analyzed for hematology, oncology, hsct inpatient population and all the other cases separately and statistical comparisons were performed. results: a total of 5271 samples were submitted to the microbiology laboratory for cd testing during the study period. while hematology patients constituted 1.7%, oncology 5.9%, hsct 2.0% and others 90.4% of the cases on whom cd testing was done; per patient average test number was 2.0, 2.8, 6.2, and 1.5, respectively. of all the cd tests per-formed, 15.6% were positive. test positivity was higher in hsct (47.6%) and oncology (42.4%) cases tested compared with hematology (21.2%) and other cases (17.1%) with statistical significance (p<0.001). overall recurrence rate was 4.3%; hsct patients had the highest recurrence with a rate of 27% followed by oncology (13.6%), hematology (7.7%) and other (3.2%) cases, again reaching statistical significance (p<0.001). again, hsct patients had the highest average number of recurrences at 3.1 (2-6) followed by oncology 2.8 (2-10), general 2.52 (2-6) and hematology 2.25 (2-3) groups. there was no seasonal variability in the incidence of cdi among populations analyzed. prolonged hospital stay/antibiotic use and persistent diarrhea due to gvhd are the likely reasons for higher rate of cd testing in hsct as a result of increased monitoring and thus might have even caused underrepresentation of positive cd test frequency. higher incidence and frequencies of recurrence underscores the inevitable nature of cdi in hsct population as a consequence of the current therapies and may lead to future radical treatment approaches like fecal implantation. background: viral infections remain a challenge to treat post hct in children, and significantly contribute to morbidity and mortality. virus specific t cells (vsts) have shown tremendous clinical efficacy in treating viral infections post-hct, with minimal toxicity and long term efficacy. we have used donor-derived vsts in individual patients, however not all donors are agreeable to the process, and numerous patients may benefit from vsts who do not have an identified donor/have other disease indications objectives: we sought to actively build a third-party vst bank, for "off the shelf" use in eligible patients. design/method: vsts targeting cmv, adenovirus and ebv were manufactured using one of 2 techniques. initially ebv transformed b cells were genetically modified with an ad5f35pp65 vector and used as antigen presenting cells (apc) to stimulate and expand ebv, ad and cmvpp65 specific t cells. more recently, vsts were expanded using s273 of s301 apc pulsed with commercially available peptide pools (pep-mixes) to expand ebv/cmv/ad specific t cells. products were entered into the "bank" via two mechanisms: a) left over products from our "donor-derived" protocol when patients no longer required vsts or were not at risk of developing viral infections, or b) by targeting regular blood donors based on their hla typing to ensure an appropriate mix of high frequency hla types for optimal patient matching and antigen presentation based on current knowledge of antigen presentation. results: a total of 30 products are currently in the thirdparty vst bank ready for use. twenty seven of these are from our donor derived protocol, and three from targeted donors. all vst products met safety and in vitro efficacy testing. thirteen vst infusions have been given to 7 patients. eleven infusions have been given for cmv and two for adenovirus. five out of seven patients responded to thirdparty vst infusions, with a median of 2 vst infusions per patient (range 1-4). the median hla matching was 2 out of 10 per patient (range 1 to 4) no patients experienced adverse reactions, gvhd or other toxicity related to the vst infusion. a third-party vst bank is feasible and produces clinically appropriate vsts for use in patients with viral infections. hla typing and matching of vst products is essential to reduce toxicity and promote appropriate antigen presentation and expansion of vsts in vivo. further work is underway to further characterize the vsts using epitope mapping to better define the hla restriction and immunogenicity of each vst product. akron children's hospital, akron, ohio, united states background: acute graft-versus-host disease (agvhd) is a well-known complication of hematopoietic stem cell transplant (hsct) and a major cause of post-transplant related morbidity and mortality. first line therapy of agvhd involves corticosteroids and calcineurin inhibition. in patients with severe refractory gvhd, mortality can reach up to 90%. currently, there is no standard of care for the treatment of steroid refractory agvhd. many centers have looked at the use of antibody mediated control of agvhd to competitively inhibit the inflammatory cascade. basiliximab, a chimeric monoclonal antibody against the t-cell il-2 receptor, has been used in adults with steroid refractory agvhd. patients receiving this medication have demonstrated complete and partial responses to therapy with minimal toxicities. objectives: report the successful use of basiliximab in the treatment of agvhd in a 2-year-old following matched unrelated (mud) hsct. design/method: a 2-year-old male underwent mud transplant for high risk aml with monosomy 7. conditioning regimen included busulfan, fludarabine and equine atg. his clinical course was complicated by fever, mucositis and agvhd (stage 3 skin; stage 1 gi-biopsy proven). gvhd prophylaxis included tacrolimus and methotrexate, however with progressive skin rash, diarrhea, and early satiety, gvhd treatment with corticosteroids was initiated. as the patient continued to have worsening symptoms, basiliximab therapy was started. the patient received 2 doses (10mg) iv basiliximab on two consecutive days and then received weekly therapy for a total of 4 doses leading to initial improvement. the patient further developed acute on chronic gvhd on day +100, and subsequently received a second course of basiliximab. after initial administration of basiliximab, the patient had near complete resolution of symptoms. however, with a small wean in his tacrolimus dose, the patient experienced another skin gvhd flare prompting the second basiliximab course. the patient was subsequently weaned off all immunosuppression by day +376. the only acute complication the patient experienced while receiving basiliximab was right toe paronychia and asymptomatic low ebv titer. the patient is currently off all immunosuppression at the time of report without evidence of cgvhd. conclusion: this single case report, in a young pediatric patient, demonstrates the use of basiliximab may be a safe and efficacious treatment for pediatric patients with agvhd. university of california, san diego, la jolla, california, united states background: clinical outcomes after allogeneic hematopoietic stem cell transplantation (hsct) depend on restoration of t lymphocyte populations. association between recovery of cd4+foxp3+ regulatory t cells (tregs) and protection from chronic graft versus host disease (cgvhd) has been described in adult hsct. in adults, t cell recovery is driven by expansion of donor t cells and treg reconstitution is hypothesized to result from peripheral conversion. restoration of t cells in pediatric patients has a larger contribution from thymopoiesis, however, the relationship between thymopoiesis and treg recovery is undefined. objectives: we hypothesized that effective thymopoiesis is important for restoration of treg populations and protection from cgvhd in pediatric hsct patients. design/method: we performed longitudinal flow cytometry of peripheral blood t cells from 17 pediatric hsct patients and 9 age-matched healthy donors. laboratory data were correlated with clinical outcomes to evaluate impact. recovery of tregs occurred in 11/17 (64.7%) patients by post-transplant day 90. day 90 treg frequency in patients that developed cgvhd (1.7 ± 1.1% of cd4+ t cells) was reduced compared to cgvhd-free patients (3.2 ± 0.7%). failure to restore tregs to >2.0% of cd4+ cells by day 90 was associated with increased risk of cgvhd in the first year post-hsct (rr = 7.3, p = 0.05). a majority (60.8 ± 11.9%) of tregs from patients recovering the peripheral treg compartment expressed helios, a marker of thymic-derived tregs; only 21.5 ± 11.4% of tregs expressed helios in patients failing to restore adequate tregs. this prompted examining the relationship between defects in thymopoiesis and inability to restore tregs. we evaluated thymic function by flow cytometry quantification of cd45ra+cd31+ptk7+ recent thymic emigrant (rte) cd4+ cells (confirmed by qpcr for trec content). most (13/17, 76.5%) hsct patients had detectable rtes by day 30 post-hsct. thymic production of rtes was persistently absent in patients that developed cgvhd (<10/10^4 cd4+ cells in 5/5 patients), compared to cgvhd-free patients (7/12 patients >10 rte/10^4 cd4+ cells by day 30, average 22.3 ± 7.1/10^4 cd4+ cells). post-hsct thymic activity as measured by rte enumeration correlated with treg restoration; 6/8 (75%) rte+ patients restored tregs, compared to 3/9 (33%) of rte-patients. conclusion: failure to restore tregs after allogeneic hsct results in increased risk for cgvhd. in pediatric patients thymic generation of new t cells is an important contributor to restoration of the treg compartment. this data supports further investigation into mechanisms impairing post-hsct thymopoiesis and suggests peripheral blood tregs may be a prognostic biomarker for cgvhd. background: haploidentical stem cell transplantation (haplo sct) is riddled with unique challenges. objectives: we present our experience in the use of haplo sct with post-transplant cyclophosphamide (ptcy) and the adaptations required for each disorder for optimal outcome. design/method: we performed a retrospective study at the pediatric blood and marrow transplant unit, apollo cancer institutes, chennai, india. children up to 18 years of age, diagnosed to have benign disorders and underwent haplo sct with ptcy from 2002 to july 2017 were included. results: ptcy was used in 36 i.e. 73% haplo transplants for children with benign disorders. the underlying conditions included fanconi anemia 10, severe aplastic anemia 6, mds 1, jmml 1, hemoglobinopathy 3, prca 1, xld 1 and primary immunedeficiency disorders (pid) 13. source of stem cells was peripheral blood in 58%, bone marrow in 41%. conditioning included fludarabine with treosulphan or cyclophosphamide for pids and aplastic anemia respectively. neutrophil engraftment by day+16-21 with a durable graft was noted in 75% transplants with graft versus host disease in 20%, cmv reactivation in 35%. mortality rate was 45% with 2 infants less than 6 months of age developing severe fatal cytokine release syndrome. the median follow up is 1 year with 3 years being the longest. no significant late effects have been noted with chronic skin gvhd in 3 children. survival rate was superior among children with pids with survival of 70% in this group. haplo sct with ptcy is a feasible and costeffective option for cure in children with life-threatening benign disorders with no compatible family or matched unrelated donor. careful patient selection, reducing cyclophosphamide related free radical toxicity with the use of n acetylcysteine, limiting t cell numbers by capping cd34 at 5 × 106/kg, post-transplant viral monitoring protocols are required to reduce morbidity and mortality. we have been working on universal access to care for children from s275 of s301 all socioeconomic background and incorporating innovations to reduce the cost of hsct without compromising outcomes. haploidentical hsct using tcr / depletion costs 18000 usd as compared to ptcy priced at 25 usd. children with severe aplastic anemia and pids can be transplanted using reduced intensity conditioning and ptcy. in hemoglobinopathies, pretransplant immunosuppression is required to prevent graft rejection. graft versus host disease remains the main cause of mortality in children with fanconi anemia. mortality in infants less than 6 months after ptcy has been high, tcr / depletion would be superior in this cohort. cincinnati children's hospital medical center, cincinnati, ohio, united states background: fanconi anemia (fa) is a congenital bone marrow failure syndrome with hsct the only curative option for associated bone marrow failure. patients with fa undergoing hsct may experience increased toxicity related to either their underlying disease, or the effects of medications, resulting in the inability to tolerate prophylactic medications or sideeffects from anti-microbial therapy. objectives: we postulated that increased cd34 cell dose would be associated with a rapid immune reconstitution and therefore early withdrawal of anti-infective prophylactic medications. design/method: patients with fa transplanted at cchmc from an unrelated donor had peripheral blood stem cell grafts collected and cd34 selection performed. where possible, patients had serial measurements of their immune system performed at varying intervals post hsct. we defined immune reconstitution as normalization of lymphocyte subsets-cd3, cd4, cd8 and cd19 cells, as well as a normal response to mitogen stimulation including phytohemagglutinin, concanavalin a and pokeweed. the first measurement of either normal cell number or mitogen response was recorded for each patient. results: a total of 35 patients underwent hsct for fa at cchmc between 2012 and 2017. patient demographics included a median age of 8 years at hsct, the vast majority of patients having a fully matched or one anti-gen mismatched donor, and the majority of patients transplanted for bone marrow failure. there was a statistically significantly decreased time post-transplant to immune cell recovery in patients receiving >20 × 106/kg cd34 cells (median 25.7) compared to those receiving <20 × 106/kg cd34 cells (median 11.9). the median time to normalization of cd3 count was 224 days (cd34 count >20/kg) versus 371 days (cd34 count <20/kg), cd4 count 211 days (cd34 count >20/kg) versus 489 days (cd34 count <20/kg), cd8 count 193 days (cd34 count >20/kg) versus 344 days (cd34 count <20/kg) and cd19 count 93 days (cd34 count >20/kg) versus 109 days (cd34 count <20/kg). time to normalization of mitogen response was decreased posttransplant in those patients receiving increased cd34 cell dose at time of transplant, though this was not significant, reflecting low number of patients with evaluable responses. no patients in either group experienced gvhd or graft failure. patients with fa who are transplanted with higher cd34 cell doses have quicker immune reconstitution than those who receive lower cell doses. along with benefit to patients including less risk of infection and early termination of immune-prophylaxis medications, this supports the use of high dose cd34 selected grafts in this vulnerable population. background: parvovirus b19 (pvb19) infection after transplantation was first reported in 1986. since then, numerous cases of pvb19 infections after hematopoietic stem cell transplantation (hsct) and solid organ transplantation (sot) have been reported. most report anemia as the predominant clinical manifestation. however, pvb19 has been associated with pancytopenia, hepatitis, myocarditis, and allograft rejection. we present a patient with acute lymphoblastic leukemia who developed bone pain and pancytopenia following hsct in the setting of pvb19 infection. to describe an unusual presentation of pvb19 in a patient with acute lymphoblastic leukemia following hsct. design/method: a search of the english-language medical literature was performed using pubmed and medline databases. a review of the patient's medical history was performed. a 7 year old male with relapsed b-cell all and history of "fifth disease" in infancy presented four months after hsct with focal left arm pain and difficulties fully extending the arm. bone mri showed enhancement of the medullary space centered within incomplete transverse cortical fracture interpreted as pathologic fracture due to neoplastic involvement of the ulna with no history of inciting injury. subsequently, peripheral blood counts decreased from low normal values to wbc 1.9 k/microl, anc 310/microl, plt 57k/microl, and hemoglobin 8.6 g/dl. the patient's chimerism remained 100% donor. a bone marrow biopsy and aspirate were performed to assess for recurrent leukemia given persistence of bone pain and developing pancytopenia. marrow findings included morphologic cytopathic effects with erythroid precursors and strong parvovirus staining with no signs of red cell aplasia or recurrent b-cell disease by morphology or flow cytometry. pvb19 was detected in blood by pcr and immunoglobulins with resolution of cytopenia and bone pain. this case highlights an unusual constellation of symptoms following hsct in a child with all. unexplained bone pain and medullary infiltrates with pancytopenia suggestive of recurrent leukemia were likely triggered by pvb19 infection. the question remains if he had reactivation of pvb19, a primary infection by a new strain, or the virus was aquired through stem cells. bone biopsy could not be justified in light of clinical improvement. so far, bone lesions have only been described with congenital pvb19 infection. pvb19 appears to be uncommon after hsct, with a review of literature yielding 15 pediatric cases. however, it may be underestimated due to lack of routine screening. our patient's presentation supports that evaluating for pvb19 may be warranted in hsct patients presenting with symptoms suggestive of relapsed leukemia. background: cardiac injury may occur during hematopoietic stem cell transplant (hsct) in pediatric patients and can be asymptomatic for many years. recommendations for screening are available for patients who received anthracyclines or chest irradiation, but no guidelines exist for unexposed longterm survivors. we sought to define the prevalence of echocardiographic abnormalities in long-term survivors of pediatric hsct and determine the need for screening in asymptomatic patients. design/method: we analyzed echocardiograms performed on long-term survivors (≥ five years) who underwent hsct at cincinnati children's hospital between 1982 and 2006. we analyzed echocardiograms for left ventricular ejection fraction (ef), end-diastolic dimension (lvedd), septal thickness, posterior wall thickness, and global longitudinal strain (gls). we normalized linear measurements for age and patient body surface area. we included for further analysis patients who had echocardiogram obtained for routine surveillance. results: a total of 389 patients underwent hsct and were alive more than 5 years after transplant in 2017, with 114 having an echocardiogram obtained ≥ five years postinfusion. those with an echocardiogram were transplanted more recently (median 2003 vs. 1998 ). however, no difference between screened and unscreened individuals was noted for age at transplant, sex, transplant indication, anthracycline exposure, chest irradiation, or cyclophosphamide based preparative regimen. indications for echocardiograms included: cardiac symptoms 5 (4.4%), congenital cardiac anomalies 8 (7.0%), hypertension 2 (1.8%), known cardiac or pulmonary disease 2 (1.8%), routine post-hsct surveillance 95 (83.3%), and unknown 2 (1.8%). the mean time post-hsct was 11.7 years. among routine surveillance echocardiograms, the mean ef z-score was -0.97. mean lvedd zscore was -0.94, mean septal thickness z-score -1.00, mean posterior wall thickness z-score -0.98, and mean gls -21.96%. for patients that had echocardiogram performed for routine surveillance, 77/95 patients (82.1%) had ef measured, and 10/77 (13.0%) had ef z-scores ≤ -2.0 (abnormally low). patients exposed to anthracyclines had a mean z-score ef of -1.19 vs. unexposed patients -0.50 (p = 0.003). among individuals who received neither anthracyclines nor tbi only 1/31 (3.2%) was found to have an abnormal ef, 51.4% (z-score -2.73) or gls (-14.28%). only one patient who had a normal ejection fraction (z-score -0.39, ef 61.7%) had an abnormal gls, -15.9% (normal ≤ -16.0). long-term survivors of pediatric hsct who are asymptomatic and did not receive radiation or anthracyclines likely do not require surveillance echocardiograms, unless indicated by clinical symptoms. patients exposed to anthracyclines or tbi require close echocardiographic s277 of s301 screening and clinical monitoring for the development of cardiac complications. duke children's hospital, durham, north carolina, united states background: children undergoing pediatric blood and marrow transplants (pbmt) experience significant symptom distress. mobile health (mhealth) technologies can be leveraged to collect and monitor patient generated health data, and subsequently enhance our understanding of pbmt symptom clusters, patterns, and trajectories. better understanding of symptom complexity can foster development of precision health strategies to improve patient outcomes. however, limited research exists in integrating mhealth technology into pbmt management. we aimed to explore the feasibility, acceptability, and usability of using a pbmt specific mobile application to collect and monitor symptoms and wearable technology (apple watch) to measure objective data such as heart rate (hr) and activity. design/method: an exploratory mixed method design began in october 2017 to monitor pbmt symptoms for 20 patients using real-time data from: 1) a self-developed mhealth application (app) to collect subjective symptom data; and 2) apple watch to collect physiologic measures such as heart rate and number of daily steps. data is collected pre-transplant through 90 days. acceptability will be assessed through satisfaction surveys at study completion. we have enrolled 4 patients to date who are all currently using the app and watch. patients' average frequency of daily charting in the app 80%. the wearable average daily recorded measurements are 144 for hr and 29 for step count. most common symptoms recorded within the app include fatigue and pain. we have noted trends in data including a decrease in activity following transplant and gvhd and an increase following engraftment. patients have stated "the app is helpful to keep track of how my pain is doing day to day" and "i try to take more steps each day than the day before". patients often remove the watch for charging, then forget to put it back on, but consistently put it on upon reminder. finally, parents often were required to make app entries with patients too sick to record. we continue to enroll patients with enthusiasm from both patients and parents to use mhealth during pbmt. preliminary findings suggest feasibility of using the mhealth devices is strongly correlated to the patient's post-transplant stage and is facilitated by caregiver participation with device management (charging devices, reminders to wear watch and record in app). patients reported satisfaction and ease of use with devices, but found it difficult to keep up with charging and charting. these findings indicate using mobile devices may be useful methods to collect patient generated health data. cincinnati children's hospital medical center, cincinnati, ohio, united states background: bacterial bloodstream infections (bsi) are a common complication following hematopoietic stem cell transplantation (hsct) in both pediatric and adult populations, and are associated with poor outcomes. there is limited data describing the outcomes and characteristics of patients who develop three or more bsi after hsct. objectives: to describe the characteristics and outcomes of pediatric patients who develop three or more blood stream infections in the first-year post hsct. design/method: we performed a retrospective chart review of 373 consecutive patients who underwent hsct at our institution from 2011 through 2016 to compile this case series. data were collected through the first year post-hsct including: patient demographics, underlying disease and therapy characteristics; and transplant complications such as thrombotic microangiopathy (tma), graft versus host disease (gvhd) and overall survival. bsis were classified according to current center of disease control guidelines. results: of 373 patients, 18 (5%) developed 3 or more bsi in the first-year post transplant (total bsi cases = 77 including all patients). of the 18 cases, the majority underwent allogeneic hsct (n = 17/18; 94%). most cases were from unrelated donor (n = 15/18, 83%). more than half of patients had grade 2-4 gvhd (n = 11/18, 61%). sixteen (89%) had tma. of these 16 cases, tma preceded the first bsi in n = 10/16 (62%). the majority of bsis were classified as central line-associated bloodstream infections (clabsis, n = 37/77, 48%), followed by mucosal barrier injury laboratory-confirmed bloodstream infections (n = 29/77, 38%) and secondary bsi (n = 11/77, 14%). the majority of isolated organisms (45%) were associated with mucosal barrier injury pathogens. one-year overall survival in the cohort was 44% (n = 8/18). pediatric patients undergoing hsct who develop 3 or more bsis in the first-year post transplant demonstrated an increased rate of tma compared to the overall institutional incidence of roughly 30%. tma diagnosis preceded the first bsi in over half of patients, suggesting that tma may predispose to recurrent bsi. improved strategies for early detection and treatment of tma as well as prevention of clabsis may help reduce the number of bsis ultimately leading to decreased morbidity and mortality in this patient population. background: in neutropenic pediatric patients, infection remains a significant cause of morbidity and mortality. while granulocyte transfusions have been utilized for decades to treat infections, including in the pediatric population, the efficacy of this intervention remains poorly described. previous guidelines have primarily utilized information from adult populations. furthermore, recruitment of donors typically involves friends or relatives of the patient with periodic involvement of community donors. the use of a readily available local donor population to improve availability has yet to be well described. as the immunocompromised population is particularly susceptible to worsening infection and clinical deterioration, the ability to rapidly harvest and deliver granulocytes warrants further investigation. to investigate the efficacy, safety, and outcomes of severely immunocompromised patients receiving granulocyte transfusions from a local altruistic granulocyte program in a pediatric tertiary care center. design/method: a retrospective review was performed to evaluate the context for receiving a transfusion as well as primary outcomes including infection clearance, survival to discharge, and overall mortality. the indiana blood bank assisted with timing the interval from initial order placement to onset of first granulocyte infusion. results: among the patient population reviewed, 22 patients received 23 separate granulocyte regimens. ages ranged from 0-18 years with a mean neutrophil count of 77 at time of first transfusion. indications for transfusions included bacteremia (n = 11), fungal pneumonia (n = 6), and fungemia (n = 5). primary outcomes included clearing infection (70%) and surviving to discharge (57%). the median time from initial order placement to infusion was 46 hours, although there was no significant difference between responders who cleared the infection and non-responders who did not. however, additional investigation found that ward patients had a 75% chance of surviving to discharge while patients in the icu at time of initial transfusion had a 36% chance of survival to discharge. the readily available granulocyte transfusion program allows patients to quickly receive therapy in neutropenic settings. this is beneficial for patients as transfusion prior to clinical decompensation correlates with increased likelihood of infection clearance, and subsequently improved mortality. further investigation is needed, likely as a prospective study, to better explore circumstances that are beneficial for granulocyte transfusions. background: donor lymphocyte infusions (dli) are composed of immune cells to treat relapse after hematopoietic cell transplantation (hct). to date, data regarding its efficacy is limited in pediatric populations. furthermore, while outcomes related to cd3 content have been characterized, to our knowledge, the relationship between outcomes and other cellular content in dli has never been reported. objectives: determine whether the primary hematological malignancy, presence/absence of graft-versus-host disease s279 of s301 (gvhd), and unique phenotypic content of each dli impact overall survival (os) in pediatric patients with hematological malignancies. design/method: irb-approved, retrospective study investigating all consecutive dlis given to patients at the children's hospital of wisconsin. analyses were conducted using mann-whitney, fisher's exact, and chi-square. from 1980 from -2016 patients ≤20 years old with hematologic malignancies [myeloid (aml/ mds/cml/jmml),n = 23; lymphoid (all),n = 7] underwent 55 dlis (72%% ≥2 dlis). the median time between hct and dli was 0.6 (range, 0.1-5.8) years. there were significant differences between the lymphoid and myeloid groups, respectively, in regard to median age at hct (14.7 vs 7.5 yrs, p = 0.022) and at first dli (20 vs 8 years, p = 0.006). ultimately, there were no statistically significant differences in gvhd or os in products with either higher or lower cd3, cd4, cd8, cd56, or cd19 cellular content. however, the median cd3/kg content was more than double in the patients who developed gvhd as compared to patients who exhibited no gvhd after dli (29.99 × 106 vs 10.03 × 106, p = 0.346). patients receiving one dli had a 6-year os of 21 ± 9% vs those receiving 2+ dli of 52 ± 16% (p = 0.012). with a median follow-up of 0.74 (range, 0.04-16.61) years, the 6 year estimated os of patients in the lymphoid group was higher at 71 ± 17% vs 22 ±9% in the myeloid group, although not significant (p = 0.11). our results indicate a survival benefit when using dli in a subset of patients who relapse after hct. unlike adult studies demonstrating little effect of dli in lymphoid diseases, many children with all achieved durable remission. while our analysis did not demonstrate that dli cellular content had a statistically significant effect on gvhd or os, it is possible that differences could be found if a larger population and more targeted cell doses were studied. more data will be needed to further define these relationships and identify patients who stand to benefit most. cincinnati children's hospital medical center, cincinnati, ohio, united states background: many arabic speaking muslim parents of children requiring bone marrow transplantation (bmt) receive medical care in the united states. providers may not understand the impact of islamic parents' religious beliefs and practices on their health care experience. objectives: to explore how islamic parents used religion in decision making and to understand the impact of their religious beliefs and practices on their overall health care experience. design/method: we used grounded theory, an inductive method gathering data from interviews and analyzing text, to identify core themes. ten caregivers of bmt children from middle eastern countries were interviewed by an arabicspeaking provider; interviews were coded by an interdisciplinary team. we identified 5 key themes: 1. patience is a core belief in islam. patience results from the acceptance of allah's will. behaviors showing patience include praying rather than questioning and crying. 2. al qur'an provides comfort, healing, and protection. families listen to recitations of al qur'an in the patient's room because they feel that this practice not only comforts them but promotes healing as well. for some, certain portions of the qur'an were especially meaningful such as surat al-baqara, which explains that while we may think something is bad for us, allah will know it is good for us. 3. religious care in the medical center helped families feel respected. religious care in the medical center included interactions with chaplains, who were understood to be "religion experts," and provision of space for prayer and religious resources. 4. seeking religious consultation. religious consultation from imams or religious scholars (muftis or sheikhs) provides interpretations of the qur'an applied to the family's specific situation helps families make difficult decisions and follow allah's plan. 5. muslim beliefs guided decision making; muslim practices brought comfort, strength, and peace. drawn from the parents' understanding of islam. parents who addressed this topic said they would only do what islam allowed. they did indicate that most aspects of healthcare were understood to be allowed within islam. additionally, muslim practices of prayer, reading/listening to qur'an, and giving alms all provided comfort, strength and peace. we identified several recurring themes through our interviews that allowed us to understand how families use their muslim faith to deal with their children's illnesses and how it influences their decision making. we believe this better understanding will allow for more informed conversations about patients' health care and decision making, and shows respect for religious beliefs and practices. nemours/dupont hospital for children, wilmington, delaware, united states background: virtually all children will be infected with human herpesvirus 6 (hhv-6) by the age of two. hhv-6 reactivation after stem cell transplantation causes multiorgan toxicities, including encephalitis, with inflammation and destruction of the temporal lobes and hippocampi, memory loss, and seizures. catatonia is characterized by posturing, immobility, mutism, and autonomic instability, and it's associated with various psychiatric and medical conditions. we describe a patient with hhv-6 encephalitis and unusual neurologic sequelae, including cognitive and neurobehavioral dysfunction and catatonia, which may impact our understanding of the pathophysiology of hhv-6 reactivation encephalitis. objectives: describe a case of hhv6 encephalitis with practice implications for stem cell transplantation. results: our patient was diagnosed with acute myeloid leukemia at age 14. within 2 years, he relapsed and received two stem cell transplants. on the 29th day after his second transplant, he developed hyponatremia and refractory seizures. brain mri showed edema in the medial right temporal lobe with linear ischemic change. eeg showed diffuse encephalopathy. cerebrospinal fluid (csf) demonstrated 5 white blood cells, 2 red blood cells, and hhv-6 by pcr. his prophylactic antiviral was switched to foscarnet and ganciclovir. repeat mri showed abnormal signals in bilateral medial temporal lobes and the right insula. three months later he developed episodes of diaphoresis, hypothermia, agitation, mutism, and unusual posturing, recurring almost daily, recognized as catatonia. mri showed improvement of the abnormalities in the bilateral medial temporal lobes and hippocampi. eegs showed diffuse slowing. after 4 months of antiviral therapy, csf was negative for hhv-6. over the ensuing 3 years, he had numerous episodes of diaphoresis, hypertension, hypothermia, pruritis, confusion, agitation, cogwheel rigidity, and bizarre posturing. dopamine blocking agents did not help. clonazepam helped reduce their frequency, and hot showers helped break acute episodes. further mris showed generalized cortical volume loss. he suffered from depression and severely impaired sleep and cognitive function. we describe a novel, debilitating outcome of hhv6 encephalitis which may provide diagnostic considerations as we continue to improve our understanding of the breadth of possible neurologic sequelae in transplant patients. hhv-6 is understood to infect and destroy the temporal lobes and hippocampi, but our patient's autonomic dysfunction indicate involvement of the hypothalamus and basal ganglia. antidopaminergic agents may worsen catatonia, and they were not effective for our patient. treatment of catatonia includes benzodiazepines; electroconvulsive therapy was not attempted in this case but may also be useful. background: epstein-barr virus (ebv)-related posttransplant lymphoproliferative disorder (ptld) is a lifethreatening complication in patients following hematopoietic stem cell transplantation, with a frequency estimated at 3.2% and a cumulative incidence of mortality estimated as high as 31%. studies of ebv have hypothesized that the tonsils are critical for propagating this infection, as tonsillar epithelial cells have been shown to be the site of primary viral infection and continued viral shedding; however, to date no studies have been performed assessing the role of tonsillectomy in patients with ebv ptld. objectives: identify patients with localized ebv ptld treated with tonsillectomy to identify prognostic factors that may be able to help guide future treatment decisions. design/method: patients treated at memorial sloan kettering cancer center who had received hematopoietic stem cell transplantation and had billing codes for both ebv and tonsillectomy were eligible for inclusion in this study. a retrospective chart review was performed, assessing patient demographics, transplant characteristics, laboratory values, tonsillar pathology, and clinical course. any patient who did not have unilateral or bilateral tonsillectomy performed or who had non-localized disease (defined as disease involvement outside of the oropharynx and neck) was subsequently immunodeficiency; 17% (n = 17/100) fanconi anemia (fa); 17% (n = 17/100) hemoglobinopathy; 12% (n = 12/100) non-fa marrow failure and 3% (n = 3/100) a metabolic disorder. seventy one percent (n = 71/100) had normal amh for age pre-transplant, 29% (n = 29/100) had low amh for age pre-transplant; of these, 37% (n = 11/29) had an oncologic diagnosis; 37% (n = 11/29) had fa; 10% (n = 3/29) had previously treated hlh; 6% (n = 2/29) had non-fa marrow failure; one had a metabolic disorder and one a hemoglobinopathy. of the 33 patients with post-transplant amh measurement 72% (n = 24/33) had low levels. of the 25 patients with previously normal pre-transplant amh 52% (n = 13/25) underwent myeloablative conditioning (mac) regimen with a 100% (n = 13/13) having low amh levels post-transplant compared to 48 %(n = 12/25) who underwent reduced intensity conditioning (ric) regimen with 25% (n = 3/12) having low amh levels post-transplant (p 0.0002). fifteen percent (n = 5/33) had low levels pre-transplant and underwent mac regimen with 100% (n = 5/5) remaining low; 80% of these patients (n = 4/5) had fa. nine percent (n = 3/33) had low levels and underwent a ric regimen with 100% (n = 3/3) of amh levels remaining low; 66% (n = 2/3) of these patients had hlh treated prior to transplant. conclusion: amh levels can be used for detection of premature ovarian failure and fertility counseling. there is a higher risk of premature ovarian failure with mac regimens and prior chemotherapy vs ric regimens. follow up of this cohort will provide more information to understand the effects of hsct in ovarian function and the usefulness of amh as a predictor of fertility potential. background: there are no proven strategies to prevent blood stream infections (bsi) secondary to oral mucosal barrier injury after hematopoietic stem cell transplant (hsct). additionally, we recently reported progressive gingivitis and dental plaque accumulation in hsct recipients despite our current oral standard of care (three times daily oral rinse). xylitol is a non-fermentable sugar alcohol that reduces dental caries, plaque accumulation, and oral disease progression by inhibiting bacterial growth. we hypothesized that the addition of xylitol to standard oral care will decrease dental plaque accumulation, gingivitis and bacteremia from oral flora. objectives: identify a clinically effective strategy to improve oral health and prevent bsi secondary to bacterial translocation through the oral mucosa in patients undergoing hsct. we are conducting a prospective randomized control study to test our hypothesis. those in the intervention arm receive our current standard of care (three times daily oral rinse) in addition to daily xylitol wipes; controls receive oral standard of care alone. oral exams are performed at baseline and weekly for the first 28 days post hsct. metagenomic shotgun sequencing (mss) of gingival samples is performed at all time points to evaluate microbiome diversity and pathogenic bacterial load. finally, we performed whole genome sequencing of pathogenic bacterial isolates causing bacteremia to assess for genetic relatedness to corresponding strains present within the patient's oral microbiome preceding the infection. : preliminary interim analysis of 21 patients demonstrates improved oral health in patients receiving xylitol (n = 10) over those receiving standard of care (n = 11), measured by the oral hygiene index (p = 0.03) and gingivitis index (p = 0.02). in the nine patients having complete oral mss analysis, xylitol appeared to be associated with decreased streptococcus mitis/oralis domination in the oral microbiome. finally, patients receiving xylitol had no incidence of streptococcus mitis/oralis bacteremia through the first 21 days compared to three patients (27%) in standard of care arm. interestingly, streptococcus mitis/oralis comprised 70% of the oral microbiome in one child who subsequently developed a streptococcus mitis/oralis bsi. we expect to complete this study in the next 4 months (n = 50). the addition of xylitol to oral standard care appears to decrease dental plaque and gingivitis in patients undergoing hsct. xylitol may also impede streptococcus mitis/oralis dominance in the oral microbiome with potential reduction in blood stream infections. (range:4-159 days). twenty-one mdli (49%) were administered because of lymphopenia, fourteen of them (33%) in patients with concomitant viral/opportunistic infections. mixed chimerism/graft failure was the motive of 37% of the mdli (n = 16) and six (14%) were administered to accelerate immune reconstitution. all infusions were well tolerated without appearance or worsening of gvhd. an increase in t-cell counts was observed following six mdli (28.57%), although it was a transitory response (3-8 weeks) in five cases. viral/opportunistic infections were controlled in five cases (35.71%), requiring a median of 2 mdli to achieve this response. none of the mdli administered in cases of mixed chimerism/graft failure were effective in reverting this situation. our preliminary data suggests that mdli, is a safe adoptive immunotherapy strategy even with high dose of t-cells without infusion side effects or gvhd complications. some efficacy has been observed in patients with lymphopenia and opportunistic infections, with no positive results in patients with mixed chimerism/graft failure, up to date. however, to determine the real efficacy of this strategy, prospective studies are required. jun zhao, kristen beebe, lucia mirea, alexandra walsh, shane lipskind, alexander, ngwube phoenix children's hospital, phoenix, arizona, united states background: male adolescents undergoing myeloablative hematopoietic stem cell transplantation (hsct) develop infertility with impaired spermatogenesis with reported rates ranging from 17% to 80%. in nonmalignant diseases, myeloablative regimens have been replaced with reduced intensity conditioning (ric) with the hopes of better survival rate, less organ toxicity and improved quality of life. despite the increased use of ric regimens for hsct, the effects of ric on fertility remain unknown. objectives: to assess fertility following ric hsct in young adult males. we assessed gonadal function and semen characteristics in adolescent males (>14 years) who received a single ric hsct at phoenix children's hospital for nonmalignant diseases during 2006-2016. male patients who were a minimum of 1 year from ric hsct and had postpubertal development at tanner stage iii or above were eligible for this study. gonadal status was assessed by measuring fsh, lh, testosterone, and inhibin b levels, and semen anal-yses assessed fertility indicators (semen volume, sperm concentration, motility, viability, forward progression, morphology, and total count). results: hormone levels and semen analysis have been obtained for 3 patients thus far. the median time between transplant and semen analysis was 4 years. post hsct, 2 (67%) patients showed abnormally elevated lh levels, but fsh, testosterone (total and free), and inhibin b levels were within normal range for all patients. sperm morphology and viability testing were not able to be performed due to low concentrations and volumes. as a result, the total motile sperm count, the most useful estimate for fertile potential, is essentially 0 for all 3 patients. conclusion: recruitment is ongoing, but so far our limited results suggest that ric hsct may have detrimental longterm effects on male fertility. a multi-institutional trial may be appropriate due to small patient numbers at each institution. we are currently exploring options to expand to other centers. further consideration is warranted regarding decisions made by providers, ways to improve anticipatory counseling provided to patients and their families prior to transplant, and how to augment the preventive care of these patients in longterm follow-up. currently all male patients being considered for ric transplant should be counseled to sperm bank prior to transplant. background: a previous systematic literature review identified all published studies of defibrotide treatment for patients of all ages with vod/sos. to assess day+100 survival for defibrotidetreated pediatric patients (≤18 or ≤16 years, per study) all patients exhibited infectious complications with at least 1 viral infection. four patients also had bacterial infections. of note, no patient developed evidence of fungal infections. conclusion: early institution of ecp in patients with high risk acute gvhd (grade 3-4) was very effective at treating agvhd, allowed for an aggressive steroid taper and contributed to excellent overall survival rates (83%). infectious complications were primarily viral and bacterial, with no fungal infections in this very high risk population. background: vod/sos is a life-threatening complication of hsct conditioning. vod/sos with multi-organ dysfunction (mod) may be associated with >80% mortality. defibrotide is approved to treat hepatic vod/sos with renal/pulmonary dysfunction post-hsct in the us and severe hepatic vod/sos post-hsct patients aged >1 month in the eu. there are few published data on survival of neuroblastoma patients with vod/sos post-hsct. objectives: to report day+100 survival and safety post hoc for patients with neuroblastoma and vod/sos post-hsct in the defibrotide t-ind trial. design/method: vod/sos was diagnosed by baltimore or modified seattle criteria or biopsy, with/without mod, after hsct or chemotherapy. defibrotide treatment (25 mg/kg/day) was recommended for ≥21 days. this post hoc analysis is based on 1154 adult and pediatric patients receiving ≥1 dose of defibrotide, including 571 with mod. results: among 111 patients with neuroblastoma, 106 developed vod/sos after hsct. for these post-hsct patients, 60.4% were male and 39.6% were female, median age was 3 years (range 1-17 years): 7.6% aged 0-23 months, 90.6% 2-11 years, 0.9% 12-16 years, and 1 patient >16 years. day+100 survival data were available for 105/106 of these neuroblastoma patients (43 with mod and 62 without mod); 103 had autologous and 2 had allogeneic transplants. kaplan-meier estimated day+100 survival for the neuroblastoma group was 87.2% (95% confidence interval [ci] , 79.0%-92.4%). for the mod and no mod subgroups, kaplan-meier estimated day+100 survival was 78.4% (95% ci, 62.6%-88.2%) and 93.5% (95% ci, 83.6%-97.5%), respectively. in the overall t-ind hsct population aged ≤16 years (n = 570) and pediatric autologous hsct subgroup (n = 127), kaplan-meier estimated day+100 survival was 67.9% and 87.1%, respectively. treatment emergent adverse events (teaes) occurred in 45.3% (n = 48/106), with serious teaes in 23.6% (25/106; most common: multi-organ failure, 4.7% [5/106]). teaes lead to treatment discontinuation in 17.0% (n = 18; most common: pulmonary hemorrhage, n = 3); death occurred in 10.4% (n = 11; >2%: multi-organ failure, 4.7%; vod/sos, 2.8%). treatment-related adverse events, as assessed by investigators, occurred in 17.0% (n = 18; most common: pulmonary hemorrhage, 2.8%). this post hoc analysis found kaplan-meier estimated day+100 survival of 87.2% in patients with neuroblastoma and vod/sos post-hsct, which was consistent with outcomes in pediatric patients after autologous hsct. the safety profile of defibrotide in neuroblastoma patients was consistent with the overall hsct population in this study and other defibrotide studies in pediatric patients. cincinnati children's hospital medical center, cincinnati, ohio, united states background: blood stream infections occur in nearly 30% of patients undergoing hematopoietic stem cell transplant (hsct) and fever is often the first symptom. timely administration of antibiotics is associated with improved outcomes, thus, early recognition of fever is paramount. current standard of care (soc) includes episodic monitoring of temperature in hospitalized patients, which may delay fever detection. therefore, continuous real-time body temperature measurement may detect fever prior to the current soc. temptraq is a food and drug administration cleared class ii medical device and consists of a soft, comfortable, disposable patch that results: of 100 patients, 98 were started on a pca in the 30 days post hct. 65% were male with median age of 11y. 46% had all, and 37% aml. matched related donors were used in 21% and 79% received tbi. pca was initiated median d+2. oral mucositis alone was the most common indication (80%). a majority of patients were started on hydromorphone (79%); 20% started on morphine and 1% started on fentanyl. 49% started on continuous infusion. pca was used for a median of 20 days (range 4-144 days). median pain score was highest d+3 of pca use, however, there was inconsistency in charting of numerical pain scores. on d+3, 12 patients had insufficient data to determine efficacy of pain control; of the remaining 86 patients, 24% had good pain control while 60% had moderate and 15% had poor pain control using our devised scale. the most common toxicity observed was respiratory depression (∼30%), however, etiology was often multifactorial and not due to opiates alone. analysis is ongoing to assess variables predicting pca use as well as efficacy of pain control and correlation between current reporting scales and patient perception. conclusion: pca use is common in pediatric hct yet pain control remains inadequate. there's a need for better evaluation of pca management, especially uniform assessment of pain, thereby improving quality of life post hct. children's national health system, washington, district of columbia, united states background: actinomycosis is a rare invasive anaerobic gram-positive bacterial disease caused by actinomyces spp. that may colonize the oropathynx, gastrointestinal tract and urogenitial tract and can lead to abscesses. respiratory tract actinomycosis is characterized by pulmonary cavities, nodules, consolidations and pleural effusions. although actinomyces are nearly always sensitive to penicillin they are frequently resistant to cephalosporins and variable sensitives to fluoroquinolones. although rare in children, immunosuppressed patients are at increased risk for actinomycosis. to describe a case of next-generation sequencing identification of actinomycosis. a 13-year-old male with a history of very high risk b-cell acute lymphoblastic leukemia who was 5 months status post a 7/8 matched unrelated donor bone marrow transplant complicated by prolonged fevers, persistent weight loss, and splenic lesions, treated with posaconazole and levofloxacin developed fever and cough in the setting of neutropenia. blood cultures demonstrated staphylococcus epidermidis. ct showed micronodules and effusion not consistent with s. epi, prompting bronchoscopy. all bacterial cultures were negative. patient was prescribed a three-week course of vancomycin with rapid improvement. design/method: 16s next generation sequencing (ngs) from bronchoalveolar levage sample was performed at the university of washington laboratory results: ngs assay from bronchoalveolar lavage showed major abundance of actinomyces most closely related to meyeri or oodontolyticus. demonstrated actinomyces. the patient was started on a six month course of amoxicillin with continued clinical improvement. in retrospect, the splenic nodules that were presumed fungal disease were likely actinomycosis, partially treated with levofloxacin. this case highlights the potential utility of ngs in the diagnosis of rare diseases in immunocompromised patients. actinomycosis was only demonstrated through ngs and led to a change in treatment regimen and durable clinical improvement. because actinomyces often mimics malignancy, tuberculosis or nocardiosis, the use of this novel test both targeted appropriate therapy and reduced the exposure to unnecessary medications to treat the differential diagnosis. finally, we highlight that actinomyces should be considered in patients who present with unexplained fevers, weight loss, and night sweats. haneen shalabi, cynthia delbrook, maryalice stetler-stevenson, constance yuan, bonnie yates, terry j. fry, nirali n. shah center for cancer research, national cancer institute, national institute of health, bethesda, maryland, united states background: car-t therapy, while effective, may not be durable for all, and antigen negative escape is a growing problem. hct, in relapsed/refractory all, can be curative, particularly for those in an mrd negative remission. we demonstrated that cd19 directed car-t therapy effectively rendered patients into mrd negative remissions (by flow cytometry) and the leukemia free survival post-hct was high 1 . in pastorek, jesssica bruce, michael a. pulsipher, chloe anthias, peter bader, andre willasch, jennifer sees, jennifer hoag, wendy pelletier, brent logan, pintip chitphakdithai, lori wiener university of pittsburgh, pittsburgh, pennsylvania, united states background: more than 4,500 pediatric hscts are performed in north american and europe each year. the ethics of exposing a healthy child to donation procedures which have some risks and no direct medical benefits continue to be a topic of debate. pediatric donors may experience psychological distress and poorer quality-of-life during and after donation compared to healthy controls. although there are fact/jacie requirements related to the management of pediatric donors, it is unclear what standardized practices exist for psychosocial assessment/management of this group. objectives: to describe transplant center practices for psychosocial evaluation/ management of pediatric donors (<18 years) and to examine differences in practices by location (cibmtr/ebmt) and number of harvests (volume). design/method: data were collected via a single crosssectional survey distributed electronically to cibmtr and ebmt centers between 9/18/17 and 11/20/17. : 55/98 (56%) of cibmtr and 85/147 (58%) of ebmt centers completed the survey. most centers had written eligibility guidelines for pediatric donors (91%). most also had a process for ensuring that donors were freely assenting to donate (78%), managed by a transplant physician (61%). a single physician often jointly managed donor/recipient care (44%). half of centers had a pediatric donor advocate (51%), who was most often a physician (38%) or social worker (16%). cost was the largest barrier to having a donor advocate (82%). most centers performed psychosocial screening of donors (69%) but rarely declined donors based on psychosocial concerns (13%). less than half of centers provided post-donation psychosocial follow-up (41%). comparisons by center location indicated that ebmt centers were more likely to have a physician doing joint donor/recipient care (60% vs. 23%; p = .001), less likely to have a psychosocial assessment policy (53% vs. 79%; p = .011), less likely to have a donor advocate (37% vs. 69%; p = .002), but marginally more likely to do post-donation psychosocial follow-up (49% vs. 31%; p = .063). large volume centers were more likely to have a psychosocial assessment policy than their medium/smaller counterparts (69% vs. 23%, 27%; p = .003) â€"there were no other differences on key psychosocial management variables by volume. although most centers have written guidelines for pediatric donor eligibility and mechanisms for ensuring assent, substantial numbers of donors do not undergo psychosocial assessment, are jointly managed with the recipient by a single physician without an assigned donor advocate, and do not receive psychosocial follow-up. the field would benefit from guideline development for the psychosocial management of pediatric donors. background: germline mutations in samd9 and samd9l genes cause mirage (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy) and ataxia-pancytopenia syndromes, respectively, and are associated with chromosome 7 deletions, mds and bone marrow failure (bmf). there are limited data on outcomes of hct in these patients. to describe outcomes of allogeneic hct in patients with hematologic disorders associated with samd9/samd9l mutations. results: seven patients underwent allogeneic hct for primary mds (n = 5), congenital amegakaryocytic thrombocytopenia (camt)(n = 1), and dyskeratosis congenita (n = 1). retrospective exome sequencing revealed gain-of-function mutations in samd9 (n = 4) or samd9l (n = 3) genes. constitutional mosaic monosomy 7 was present in 6 cases. two samd9 patients had features of mirage syndrome. unusual findings of panhypopituitarism, laryngeal cleft, and glomerulosclerosis were noted in one case. in another case with a samd9 mutation hypospadias & bifid scrotum were the only findings. the remaining patients had no phenotypic abnormalities. median age at hct was 5y (range: 1.4 -12.8). patients received transplants from bone marrow (matched unrelated (n = 3) & hla identical sibling (n = 2)), or unrelated cord blood (ucb) (n = 2). five mds patients received myeloablative s295 of s301 conditioning (busulfan-based (n = 3) or tbi-based (n = 2)); 2 patients (mds (n = 1); camt (n = 1)) received reducedintensity conditioning (ric) (fludarabine, cyclophosphamide, with ratg or alemtuzumab). syndrome-related comorbidities (diarrhea, infections, malnutrition, electrolyte imbalance, lung disease and hypoxia) were present in both patients with mirage syndrome. one patient with a familial samd9l mutation, mds and morbid obesity failed to engraft following ric double ucbt. she died one year later from refractory aml. all other patients achieved neutrophil and platelet engraftment, at a median (range) of 15 (12-19) and 16 (12-31) days, respectively. posttransplant complications included severe hypertension (n = 1), pericardial effusions (n = 2), veno-occlusive disease of liver (n = 1), and recurrent aspiration pneumonias (n = 1). one patient developed grade iii agvhd which resolved with treatment. one patient developed mild skin cgvhd and suffers from chronic lung disease. all surviving patients had resolution of hematological disorder and sustained peripheral blood donor chimerism (98-100%). overall survival was 86% with a median follow-up of 3 years (range: 1.1 -14.7 y). patients with hematological disorders associated with germline samd9 /samd9l mutations tolerated transplant conditioning without unusual, or unexpectedly severe toxicities. allogeneic hct led to successful resolution of mds or bmf, with excellent overall survival. more data is needed to refine transplant approaches in samd9/samd9l patients with significant comorbidities, and develop guidelines for their long-term follow-up. shyamli singla, tiffany simms-waldrip, andrew y. koh, victor m. aquino background: steroid-refractory acute graft versus host disease (agvhd) is a potentially fatal complication of allogeneic hematopoietic stem cell transplantation (hsct). basiliximab (anti-il2-r monoclonal antibody) as a single agent or in combination infliximab (anti-tnf-monoclonal antibody) has demonstrated efficacy in adult cohorts with steroid-refractory agvhd, but has not been well studied in the pediatric population. we adopted the use of basiliximab and infliximab as our institutional standard of care for steroid-refractory agvhd in pediatric hsct patients. to determine the response and survival of hsct children who received basiliximab and infliximab for the treatment of steroid-refractory agvhd. design/method: we retrospectively reviewed children who received basiliximab and infliximab for steroid-refractory agvhd refractory between september 2011 and december 2017. complete response (cr) was defined as resolution of all clinical signs of agvhd. partial response (pr) was defined as at least one grade reduction in one target organ (e.g. skin, gut or liver) without increased grade in another target organ. no response was defined as either no improvement or progressive worsening of agvhd in at least one organ. baseline demographics, transplant details, laboratory findings, and treatment outcomes were also evaluated. results: of the 214 evaluable hsct patients, 15 children (median age 11 yrs, range 9 mo-19 yrs) with steroid-refractory agvhd received combination monoclonal antibody (mab) therapy. the median time from the start of steroid therapy to initiation of mab was 13 days. the overall glucksberg grade of agvhd at the time of initiating mab therapy was grade i (n = 1, 6.7%) ii (n = 3; 20%), iii (n = 8; 53%) or iv (n = 3; 20%). the overall response rate was 53%, with 3 (20%) patients achieving cr, 5 (33.3%) patients achieving pr, and 7 (46.7 %) patients with no response at 30 days following the start of mab therapy. the median overall survival was 613, 292, and 84 days for patients who exhibited cr, pr, and no response, respectively. the overall survival at 1 year following start of mab therapy was 40%. background: the role of high dose chemotherapy (hdc) and autologous stem cell rescue (ascr) in patients with high risk (advanced metastatic or relapsed) soft tissue sarcomas is controversial. despite multimodal chemotherapy, radiotherapy, and local control measure advancements, prognosis of patients with advanced metastatic or unresectable and relapsed sarcomas remains poor, with less than 10% 5 years disease free survival. objectives: to determine if consolidation with myeloablative hdc and ascr improves relapse free (rfs) and overall survival (os) outcomes in a high risk patient subgroup. we performed retrospective review of all high risk soft tissue sarcoma patients who underwent hdc and ascr at the children's hospital at montefiore, bronx, ny between october 2014 and january 2018. the protocol was approved by albert einstein college of medicine institutional review board. results: 7 patients (4 primary metastatic high risk disease, 3 relapsed or recurrent disease) received hdc with ascr. primary diagnoses were rhabdomyosarcoma (rms) (n = 2, alveolar histology), primary site nasopharynx (n = 1) and lower extremity (n = 1). ewing's sarcoma (ews) (n = 5), axial site (pelvic) in 3 patients (60%). median age 12 years (range 6-35 years), 5 (71%) were male. all patients were in complete metabolic remission before transplant. median pre transplant comorbidity index was 3 (range 0-4). 5 patients (2 rms and 3 ews) received conditioning with carboplatin, etoposide and melphalan. remaining 2 patients with ews received conditioning with busulfan, melphalan and topotecan. all patients received peripheral blood mobilized hematopoietic stem cell transplantation. stem cell mobilization achieved with high dose filgrastim in all patients except one who required addition of plerixafor. median cd34+/kg s297 of s301 recipient body weight cell dose infused was 6.34 × 10^6 (range 2.72-12.03 × 10^6). median times to neutrophil and platelet (>20,000/â l) engraftment were 9 (range 8-13) and 49 (20-76) days respectively. 2 patients (28%) developed bk viuria (one with grade iii hemorrhagic cystitis); 3 (43%) developed cmv viremia; and one patient (14%) had asymptomatic ebv viremia. there was no graft failure, sinusoidal obstruction syndrome or transplant related mortality. median follow up post-transplant was 452 days (range 155-1141 days). 3 year probability of os and rfs were 80% and 50% respectively. hdc with ascr is a promising therapeutic strategy to consolidate remission and improve survival in select high risk soft tissue sarcoma patient subgroups. prospective clinical trials will inform the impact of disease status prior to hdc and ascr on outcome, optimal conditioning and long term relapse free and overall survival. background: absence of minimal residual disease is paramount for cure of pediatric acute lymphoblastic leukemia (all). the testis may harbor occult leukemia and this disease may result in treatment failure. objectives: the purpose of this study was to assess the longterm outcomes of boys with or without testicular leukemia pre-hematopoietic stem cell transplantation (hsct). design/method: retrospective analysis of 16 boys with high-risk de novo (2 with hypodiploidy all) or recurrent/refractory all was conducted. flow cytometry of bone marrow mononuclear cells was used to determine remission status. testicular evaluations were performed by physical examination and wedge biopsy pre-hsct. the median age at time of transplant was 12.3 years. all patients were in remission by flow cytometry of bone marrow mononuclear cells at the time of transplant and none had evidence of clinically apparent testicular disease. testicular leukemia was detected in 1 patient and he underwent bilateral orchiectomy. he developed acute graft versus host disease (gvhd) of the duodenum and sigmoid colon which resolved, and the leukemia remains in second complete remission and he is free of hsct-related morbidity 33.6 months post-hsct. of the 15 patients without testicular leukemia 4 died a median of 8.2 months (range, 2.5 to 12.5) post-hsct (2 with adenovirus infection and 1 each with thrombotic microangiopathy and aspergillus pneumonia); 6 experienced infection (staphylococcus species, corynebacterium, enterococcus, klebsiella, citrobacter, e. coli, epstein barr virus, adenovirus, bk virus, human herpesvirus-6, candida albicans, fusarium, aspergillus, yeast, and other fungus); 11 experienced gvhd (8 of the gi tract, 6 of the skin, 5 of the liver, 3 of the eyes, 2 of the mouth, and 1 of the lungs); and 1 developed a second neoplasia (right lower leg leiomyosarcoma). one patient developed bone marrow minimal residual disease (2.7% phenotypically abnormal cells detected 9 months after 6/6 matched sibling hsct). reinduction therapy comprised 5 weekly doses of rituxan, 2 courses of blinatumomab and 2 donor lymphocyte infusions with il-2. two subsequent bone marrow evaluations were minimal residual disease negative. thirteen months post-hsct residual disease recurred (0.012%) and he will receive inotumumab. overall median survival post-transplant of the 16 boys is 32.1 months (range, 2.5 to 75.3) and of the 12 surviving boys is 37.3 months (range, 11 to 75.3). conclusion: testicular biopsy can detect occult leukemia pre-hsct. testicular leukemia pre-hsct does not appear to increase the risk of subsequent relapse or other hsct-related adverse events compared to those without it. yaya chu, nang kham su, sarah alter, emily k. jeng, peter r. rhode, mathew barth, dean a. lee, hing c. wong, mitchell s. cairo new york medical college, valhalla, new york, united states background: rituximab has been widely used in frontline treatment of b-nhl including burkitt lymphoma (bl), however, some patients retreated with rituximab relapse, which limit patient treatment options. novel therapies are desperately needed for relapsed/refractory b-nhl patients. several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering immune cells with chimeric antigen receptors (car), as well as second-generation anti-cd20 antibodies. nature killer (nk) cells play important roles in the rejection of tumors. however, nk therapy is limited by small numbers of active nk cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of nk cell immunosurveillance. our group has successfully expanded functional and active peripheral blood nk cells (expbnk). 2b8t2m was generated by fusing alt-803, an il-15 superagonist, to four single-chains of rituximab. 2b8t2m displayed tri-specific binding activity through its recognition of the cd20 molecule on tumor cells, activated nk cells to enhance adcc, and induced apoptosis of b-lymphoma cells. objectives: to examine if 2b8t2m significantly enhances the cytotoxicity of expbnk against rituximab-sensitive and -resistant bl cells. design/method: expbnks were expanded with lethally irradiated k562-mbil21-41bbl and isolated using miltenyi nk cell isolation kit. alt-803 and 2b8t2m were generously provided by altor bioscience. nk receptors expression and cytotoxicity were examined as we previous described. ifng and granzyme b levels were examined by elisa assays. equal doses of rituximab, alt-803, rituximab+alt-803, obinutuzumab (obinu) were used for comparison. igg was used as controls. anti-cd20 car expbnk cells were generated as we previously described by mrna electroporation. rituximab-sensitive raji andresistant bl cells raji-2r and raji-4rh, were used as target cells. results: 2b8t2m significantly enhanced expbnk cytotoxicity against rituximab-sensitive raji cells, rituximab-resistant raji-2r cells and resistant raji-4rh cells compared to the controls igg, rituximab, alt-803, rituximab+alt-803, obinu (p<0.001, e:t = 1:1). furthermore, we confirmed the enhanced cytotoxicity by measuring ifn-g and granzyme b production. 2b8t2m significantly enhanced ifn-g and granzyme b production from expbnk against raji, raji-2r and raji-4rh compared to igg (p<0.001), rituximab (p<0.001), alt-803 (p<0.001), rituximab+alt-803 (p<0.001), and obinutuzumab (p<0.001). when compared to anti-cd20 car expbnk cells, 2b8t2m + expbnk had the similar cytotoxicity against raji, raji-2r and raji-4rh as anti-cd20 car expbnk cells did (p>0.05). conclusion: 2b8t2m significantly enhanced expbnk activating receptor expression and in vitro cytotoxicity against rituximab-sensitive and -resistant bl cells. the in vivo functions of 2b8t2m with expbnk against rituximab-sensitive and -resistant bl cells using humanized nsg models are under investigation. background: cardiac dysfunction, including left ventricular systolic dysfunction (lvsd), is a known complication in stem cell transplant (sct) survivors. while detection of lvsd by echocardiography is important in this population, there has been minimal research to determine if subclinical cardiac dysfunction exists in sct patients. cardiopulmonary exercise testing (cpet) is a valuable tool to assess cardiac function, and to determine how the heart responds to the stress of exercise. no studies have been performed to determine if sct patients with normal lvsd on standard echocardiography may have abnormal cpet. to determine the feasibility of cpet, as well as additional echocardiographic parameters, to detect dysfunction in sct patients with a normal ejection fraction on echocardiogram. design/method: we performed a cross-sectional analysis of sct survivors who were at least 3 years post sct, 8 years of age or older and with an ejection fraction > 50% (low end of normal range) on echocardiogram. we assessed the exercise capacity of all patients with cpet, and sub-clinical cardiac dysfunction through tissue doppler and strain analysis from the echocardiogram. results: seven patients (6 male) have qualified and completed this study so far with an average age of 12.2±3.4 years. the median time from transplant is 4.4±0.8 years. all seven patients had a normal ejection fraction, however four patients had abnormalities on their cpet. these abnormalities included abnormal predicted peak oxygen consumption (vo2) (61%±9.8, normal > 70%) (the best predictor of functional capacity), predicted oxygen pulse (62%±10.1, normal > 70%) (measure of cardiac stroke volume) and ventilatory efficiency (ve/vco2 slope) (39±7.6, normal < 30). submaximal exercise data, used when patients are unable to complete a maximal effort test, demonstrated low-normal predicted vo2 at anaerobic threshold (45.8%±7.2%, normal >45% of was 19.4 days while patients who received autologous infusions had a mean number of days to engraftment of 13.3. engraftment after hsct needs to be prompt to minimize duration of neutropenia and maximize survival rates 6 . our data demonstrates that the infusion of hematopoietic stem cell products with a syringe or iv pump is an effective method of delivery for stem cell products and does not delay the time to engraftment. the median days to neutrophil engraftment was 14.5 days. this is comparable to data from the nmdp, which reports engraftment occurs within 14-20 days. the main limitation to this study was its small sample size due to the number of transplants done at our center. however, it does provide evidence to support that infusion of stem cell products via pump mechanism is a safe alternative to the infusion by gravity method in the process of the hematopoietic stem cell administration. johns hopkins all children 's hospital, st. petersburg, florida, united states background: leukemic relapse remains the most common cause of treatment failure after allogeneic hematopoietic cell transplant (allohct) for myeloid malignancies. most children who relapse post-allohct will die of their disease, making interventions to minimize this risk a high priority. objectives: to evaluate the safety and efficacy of posttransplant azacitidine for relapse prevention in children undergoing allohct for myeloid malignancy. design/method: we retrospectively reviewed the charts of children undergoing allohct for myeloid malignancies between february 2015 and november 2016 at johns hopkins all children's hospital. results: during the study period, 18 children (ages 2 to 20 years, median 12) underwent allohct for myeloid malignancies: de novo acute myeloid leukemia (aml), 11; mixed phenotype acute leukemia, 2; treatment-related aml, 2; juvenile myelomonocytic leukemia with aml transformation, 2; and myelodysplasia/aml, 1. thirteen were in first complete remission, 5 were in cr2 or greater. most patients (13/18) received fludarabine/melphalan/thiotepa conditioning; 11 received hla-identical related or unrelated donors, and 7 received haploidentical bone marrow grafts with post-transplant cyclophosphamide. three patients never received planned azacitidine (2 early relapse; 1 early trm), leaving 15 evaluable patients. azacitidine (32mg/m 2 /dose for 5 days, in 28-day cycles for up to 9 cycles) was started at a median of 66 days post-transplant (range 42-118). two-thirds (10/15) of patients received eight or more cycles. of five patients who stopped therapy early, only one was due to toxicity; other reasons included severe gvhd (1), parental preference (1), and relapse (2). cycle delays occurred in 9 patients, with a median 2 cycles delayed per patient, mostly for mild myelosuppression with early cycles. no patient required blood product transfusion during therapy, but g-csf was used in three patients to maintain anc>500/ l. dose-modifications were made in 3 patients (renal tubular acidosis, acute kidney injury, and myelosuppression). there were 3 relapses (20%), two of which occurred in patients in cr2, for a relapse incidence of 9% in patients in cr1, with a median follow-up of 20 months (range 12.5 to 28). no patients who received azacitidine died of transplant-related mortality. conclusion: administration of azacitidine in children undergoing allohct for myeloid malignancies is safe and feasible, with most patients successfully receiving all planned cycles. toxicity was acceptable and there was no trm or secondary graft failure. despite the limitations of a small cohort, relapse incidence-particularly in patients transplanted in cr1suggests a potential benefit in disease control that warrants investigation in follow-up studies. background: despite significant improvements in the success rate of hematopoietic cell transplantation (hct), graft failure remains an important complication in patients transplanted for severe aplastic anemia (saa). second allogeneic hct can salvage patients, but 5-year overall survival (os) rates have been reported as low as 60% 1 . objectives: identify patients who developed dropping donor chimerism, graft rejection, and/or graft failure after first hct for saa, necessitating additional hcts or cellular boosts (defined as stem cell products infused without preceding chemotherapy), and evaluate treatment-related complications and os. with vod/sos with and without multi-organ dysfunction (mod) pubmed and embase databases were searched for "defibrotide and retrospective chart reviews; excluded publication types were: case reports (<10 cases); meta-analyses; reviews; animal, modeling, pharmacokinetic, chromatography, and adult-only studies; guidelines; articles; and letters. resulting reports were screened for exclusion criteria. full-text articles were then reviewed for eligibility. study characteristics of selected publications were summarized, and publications were categorized by patients' mod status. when necessary, additional data tables were requested. a random effects model was used for pooling data for efficacy. interstudy heterogeneity was assessed with cochran's q-test. percentage of total variation across studies due to heterogeneity (i2) was evaluated we quantified ∼4300 proteins in each sample. reproducibility for one donor at 3 different time points children 's minnesota, minneapolis, minnesota, united states background: pediatric and young adult hodgkin lymphoma (hl) has five-year survival rates >90%. chemotherapy required to achieve this rate is associated with a lifetime risk of cardiac deaths, second malignancies, pulmonary disease and infertility. as effective salvage therapy exists, outcomes may be improved by de-intensifying initial therapy to lessen toxicity.objectives: we piloted a regimen in low and intermediate risk hl patients using agents without known association to significant late effects. this retrospective chart review was approved by children's minnesota irb.design/method: the bvg(p) regimen incorporated bortezomib (1.3 mg/m2 day 1,4,8,11); vinorelbine (25 mg/m2 day 1,8); gemcitabine (1000 mg/m2 day 1,8) every 21 days and prednisone (20 mg/m2/dose bid x 11 days). we treated 5 newly diagnosed patients, ages 10-18 years, with non-bulk stage iia (n = 4) or iib (n = 1) hl. two patients received bvg and 3 received bvgp with the addition of prednisone.results: newly diagnosed patients were all pet negative after the first or second cycle and remained pet negative at end of therapy, 4 cycles. nausea was well controlled with 5-ht3 antagonists and scopolamine. pegfilgrastim was not necessary due to the high absolute neutrophil count nadir [median 1.16 and minimum 0.56 × 109/l]. there were no episodes of febrile neutropenia, infection or transfusion need. no patients experienced alopecia. one patient developed sensory neuropathy after the eighth dose of bortezomib that was controlled with gabapentin and a switch to subcutaneous bortezomib administration. of the five newly diagnosed patients, four remain in remission at 227, 270, 557, 1191 days; 1 relapsed at previous disease sites at 861 days and subsequently achieved remission with bvgp with the addition of brentuximab. this series provides early evidence to stimulate expansion of this pilot experience and subsequent multiinstitutional study leading to a randomized trial of bvgp and current chemotherapy for low and intermediate hl. st jude affiliate clinic at st francis hospital, tulsa, oklahoma, united states background: symptoms suggestive of morning hypoglycemia has been noticed in children receiving all chemotherapy. only few small studies looked at this therapy related complication. factors increase risk of hypoglycemia in all patients include accelerated starvation, steroid induced adrenal suppression, mercaptopurine therapy and prolonged fasting for procedures.objectives: to study the prevalence and risk factors for hypoglycemia during all therapy design/method: medical records of of children (up to 18 years old) treated for all between 2011-2016 (86 patients) were studied for evidence of morning hypoglycemia defined as blood sugar (bs) < 60 mg/dl. statistical mean differences between the subgroups were analyzed with spss using a nonparametric mann-whitney u test.results: fifty two percent (52%) of patients developed hypoglycemia during all treatment, with an average of 2.2 episodes/patient. 59% were males and 41% females. almost 2/3 (65%) of patients with hypoglycemia were in maintenance phase of therapy. 34% of hypoglycemic episodes occurred in 10% of patients. majority of hypoglycemic episodes (78.2%) occurred on the day of procedure when patients were fasting overnight. 48.9% of hypoglycemic episodes occurred in children ≤3 years, with 75.8% in ≤6 years. patients who developed hypoglycemia were significantly younger (mean age at time of diagnosis of all was 4.93 ± 3.69 at the hypoglycemia group versus the non-hypoglycemia (7.27 ± 4.98) p<0.05. no statistically significant difference was found regarding sex, or tpmt genotype. 6% of hypoglycemic children-all <3years of age-presented with life threatening hypoglycemia symptoms including seizure and loss of consciousness. this study showed high prevalence of hypoglycemia during childhood all therapy. younger age, especially ≤ 3 years, is associated with higher risk of hypoglycemia as well as life-threatening episodes. to decrease fasting hypoglycemia during therapy for childhood all, we recommend that children under the age of 6 years receive bed time snack high in proteins and complex carbohydrates, and to get them up early the day of procedure to take clear sugary drink. hospital for sick children, toronto, ontario, canada ann & robert h. lurie children's hospital of chicago, chicago, illinois, united states background: childhood brain tumors are the most common solid malignancy and the leading cause of cancer-related mortality in children. the most aggressive type of pediatric central nervous system (cns) tumors is diffuse intrinsic pontine glioma (dipg). despite decades of clinical trials, there has been no substantial improvement with respect to therapeutic outcomes with most children eventually succumbing to the disease. research on adult high-grade gliomas has shown a targetable pathway through the inflammationinduced expression of indoleamine 2,3 dioxygenase 1 (ido1) and its recognized ability to suppress the anti-tumor immune response. a limited understanding into the role of ido1 in pediatric central nervous system tumors serves as the foundation of this research project. furthermore, the integration of nanotechnology is a fundamental step for the investigation and targeting of ido1. spherical nucleic acids (snas) composed of nanoparticles have been shown to transverse cellular membranes, exhibit stability in physiological environments, escape from degradation, and create precise targeting in brain tumors.objectives: the purpose of our project is to delineate the role of ido1 in pediatric dipg, and develop small inhibitory (si)rna oligonucleotides and snas aimed at therapeutically inhibiting the gene expression of immunosuppressive ido1. our specific aims are to: (1) confirm the gene expression ido1 in different human dipg cell lines; (2) generate and characterize sirna oligonucleotides targeting human ido1 in vitro; and (3) generate and characterize gold nanoparticles for targeted inhibition of ido1.design/method: unique patient-derived dipg cell lines were grown in culture, stimulated with increasing concentrations of the proinflammatory cytokine, ifn , and analyzed for mrna levels. sirna specific to ido1 was transfected into cells. sna generation is in progress.results: ido1 is expressed in multiple human pediatric dipg cell lines. sirna targeting ido1 among exons 9 and 10 results in a significant decrease in overall ido1 expression by dipg cells. sna generation for targeting ido1 with improved penetration & stability is ongoing, with preliminary results demonstrating a robust ability to inhibit ido1 expression. the grim prognosis of children with dipg, the lack of effective therapies, and the expression of ido1 by human dipg cells emphasize the importance of developing the treatment capability to inhibit ido1 gene expression, as a excluded from this study. the remaining patients were analyzed using descriptive statistics.results: a total of 17 patients meeting inclusion criteria were identified. of these, 4 patients (23.5%) received tonsillectomy alone, 1 (5.9%) underwent tonsillectomy and decreased immunosuppression, 6 (35.3%) received tonsillectomy and rituximab, and another 6 (35.3%) received tonsillectomy with additional therapy (including ebv-specific cytotoxic tlymphocytes, donor leukocyte infusion, and chemotherapy). of the 5 patients who received tonsillectomy with or without a decrease in immunosuppression, all were diagnosed with high-grade lymphoma and achieved clinical remission following tonsillectomy with no evidence of relapse to date. on further analysis looking at ptld risk factors, all patients were under 50 years of age, all received t-cell depleted grafts, and none had significant graft-versus-host disease (gvhd) at the time of ptld diagnosis. we have identified a population of patients with localized ebv ptld that achieved clinical remission with no evidence of recurrence following tonsillectomy, suggesting that tonsillectomy alone may be an adequate treatment for localized ebv ptld in a specific subgroup of patients. further analysis is needed to identify characteristics of this subgroup to determine which patients would be most likely to respond to this treatment. university of rochester, rochester, new york, united states background: malignant central nervous system (cns) tumors in young children have a poor prognosis and pose a significant therapeutic challenge. consolidation therapy with carboplatin and thiotepa was piloted in ccg-99703, cog acns0333, and cog acns0334 with the goals of intensifying therapy and omitting or delaying radiation.objectives: to document outcomes for patients undergoing carboplatin/thiotepa consolidation with autologous stem cell rescue (ascr) and to demonstrate the feasibility and toxicity of this regimen.design/method: patients up to 3 years old (median age: 12 months) with malignant cns tumors treated at the university of rochester from 2012-2017 with at least one cycle of carboplatin (17 mg/kg/day x 2 days) and thiotepa (10 mg/kg x 2 days) followed by peripheral blood ascr were included in retrospective analysis. data were recorded on time to engraftment (defined by absolute neutrophil count (anc) recovery to > 0.5 × 10^9/l), length of hospitalization, toxicity with each consolidation cycle, progression free survival (pfs) and overall survival (os). stem cell harvest data were also collected.results: eleven patients with malignant cns tumors (6 atypical teratoid/rhabdoid tumor, 3 primitive neuroectodermal tumor, 1 glioblastoma multiforme, and 1 pineoblastoma) received a total of 30 cycles of carboplatin/thiotepa. of these, 9 underwent stem cell harvest at our institution, with complications limited to procedure-related hypotension for 1 patient with known autonomic instability, and catheter-associated deep vein thrombosis (dvt) for 1 patient. four patients were in complete remission (cr) 1/status-post gross total resection, 1 was in cr2, and 6 had residual tumor at the time of consolidation. nine patients received 3 planned consolidation cycles, 1 patient 2 (of 2) planned cycles, and 1 patient 1 of an anticipated 3 cycles thus far. average time to engraftment for these 30 cycles was 10.2 (+/-1.4) days, with a mean hospital length of stay of 16 (+/-3.2) days. fever occurred in 17 of 30 cycles (57%); infectious toxicity included documented bacterial infection in 2 cases (enterococcus faecalis bacteremia in 1, klebsiella pneumoniae in 1). there were no regimenrelated deaths. with a mean follow-up of 23 months, 2 survivors have not yet completed all therapies, and 5 patients have relapsed (4 have died of disease). of the 7 survivors, 4 have been disease-free for >12 months. background: autologous hematopoietic stem cell transplantation (auto-hsct) has resulted in improved survival for patients with high-risk neuroblastoma. treatment intensification is however associated with greater complications. data on early infectious complications in low-and-middle income countries are limited.objectives: to review the early infectious complications following auto-hsct in patients with high-risk neuroblastoma.design/method: a retrospective chart review of pediatric patients with high-risk neuroblastoma who underwent auto-hsct at the american university of beirut medical center between 2003 and 2017 was conducted. infectious complications during the first 100 days post-transplant were reviewed.results: forty-three patients (27 males and 16 females) with a median age at diagnosis of 4.35 years [range: 0.5-13.9] years underwent auto-hsct during the above-mentioned period. conditioning regimen consisted of melphalan, etoposide and carboplatin. all patients received antiviral and antifungal prophylaxis. median time for neutrophil engraftment was 10 days [range: 8-19]. bacteremia and clostridium difficile infections occurred in 16 (37%) and 12 (28 %) patients respectively. seven (16%) patients developed enterocolitis diagnosed by imaging, 4 were adenovirus induced. cmv viremia was diagnosed in 7 (16%) patients, 5 of whom required treatment. varicella zoster reactivation, parvovirus viremia, toxoplasmosis encephalitis, bk virus cystitis (2 patients) and central nervous system ebv related post-transplant lymphoproliferative disorder were diagnosed in 6 different patients. there was no invasive fungal infection. sixteen (37%) patients have died, 6 of whom died in the early post-transplant period, 1 due to disease progression and 5 (11.6%) due to infectious complications. among the 5 patients who died due to infection, 1 developed toxoplasmosis encephalitis, 4 developed severe enterocolitis, 2 of which were adenovirus related. the mean igg level within one week post-transplant was lower in patients with clinically significant viral infection compared to others (4 vs 5.6 mg/dl, p: 0.08). the mean igg level at the time of clinically significant bacterial infection was lower in infected patients compared to others (4.4 vs 6.3 mg/dl, p: 0.03). neither absolute lymphocyte count nor absolute neutrophil count at day 20 post-transplant affected the incidence of clinically significant infections. our results show that the rate of infections during the early post auto-hsct period is higher than what has been described in developed countries and has a significant impact on mortality. prevention, early detection and improvement in the treatment is required to improve outcome. university of miami, miami, florida, united states background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative treatment for many malignant and non-malignant (bone marrow failure, immunodeficiency, or metabolic diseases) in pediatrics. despite advances in medicine, graft-versus-host-disease (gvhd) remains a significant cause of non-relapsed morbidity and mortality, specifically in those with malignant diseases.objectives: to highlight the complexity to acute gvhd management and seldom-described treatment approach. a 7 year male with a history of high risk acute myeloid leukemia (aml) due to failed induction therapy. he received a matched (10/10) unrelated donor hsctmarrow product-conditioned with busulfan, fludarabine, and anti-thymoglobulin (atg). his post-transplant course was complicated by hhv-6 viremia, pres (prompting a change from prograf to cyclosporine), mucositis, and grade iii acute gvhd (skin s3, gut s2, liver s0) around post transplant day 101, which later morphed to ocular involvement by d+120. he was started on 2 mg/kg steroids with good response but flared up with each attempt to taper steroid dose. a course of rituximab and later atg were tried without success in weaning off steroids. switching cyclosporine to sirolimus did not provide any additional benefit either. extracorporeal photopheresis (ecp) was started 3 times a week. he initially responded well, yet was not able to wean off steroids. in addition, he developed a flare when ecp session was reduced to 2 days per week. ecp was therefore increased to 5 days per week, which appeared to stabilize skin lesions. a trial of weekly methotrexate was attempted to wean off steroids and photopheresis, which provided no response. finally, a trial of bortezomib on days 1, 4, 8, and 11 of a 21 day cycle as published in a case series of multiple myeloma patients who developed post hsct gvhd. skin lesions improved remarkably however dose had to be reduced due to related pancytopenia. given the response to therapy, he was continued on a weekly dose of bortezomib, receiving a total 8 doses, which has permitted the slow taper of prednisone that has since been discontinued without a major flare. he however is currently maintained on ecp 3 times per week, which is now been slowly withdrawn.conclusion: management of acute gvhd in pediatric patients after hsct can be challenging with no definite options for those who fail steroids or become steroid dependent after initial response. in these situations, bortezomib could be a valid therapeutic option. background: neuroblastoma (nbl) is the second most common solid tumor in children and despite recent treatment advances, overall survival for high risk nbl remains <50%. the addition of immunotherapy has improved survival and includes anti-gd2 antibody therapy. the success of antibody therapy in neuroblastoma is primarily due to natural killer (nk) cell mediated antibody dependent cellular cytotoxicity. we previously demonstrated that nk cells from patients with high risk nbl can be successfully isolated and expanded to large numbers and exhibit potent anti-tumor effects against nbl (1). thus, infusions of autologous expanded nk cells in high risk nbl in combination with anti-gd2 antibody are being studied in clinical trials. toll-like receptors (tlr) present on the surface of leukocytes are responsible for pathogen recognition, and activation of these receptors stimulate the production of cytokines that critically link innate and adaptive immune responses. the tlr3 agonist, poly(ic) is a synthetic analog of dsrna that has previously been shown to directly stimulate cytokine production and improve cytotoxicity in primary nk cells through activation of genes regulated by interferon-response elements (ire) (2). we hypothesized that ex vivo activation of tlr pathways in nk cells during our normal 14-day expansion using k562 feeder cells expressing membrane bound il-21 would enhance their function.design/method: nk cells were isolated from peripheral blood mononuclear cells and expanded with our previously described expansion protocol in media containing il-2 and 50 ug/ml poly(ic) (3). at the end of the 14-day expansion, nk cells expanded with poly(ic) were compared to controls using a calcein cytotoxicity assay to measure cytotoxicity against high risk neuroblastoma and cytometric bead array to measure cytokine production. : surprisingly, the addition of poly(ic) during nk cell expansion did not improve proliferation, cytokine production or cytotoxicity compared to our standard expansion method. rnaseq demonstrated that our standard expansion method results in a modest decrease in tlr3 expression at the transcriptional level, but significant upregulation of several ireregulated genes. we conclude that either our standard approach interferes with tlr signaling or saturates the innate immune response pathway such that co-stimulation with poly ic does not produce an additive effect. we are performing expression analysis on nk cells receiving poly(ic) during expansion to further explore this hypothesis. background: gonadal dysfunction leading to infertility is a complication after hematopoietic stem cell transplant (hsct). anti-mã¼llerian hormone (amh) is a marker of ovarian reserve; it is not controlled by gonadotropins and has minimal inter-cycle variations, therefore, it can be used as a marker of ovarian reserve and aid in fertility counseling.objectives: assess ovarian reserve in hsct patients utilizing amh levels. background: tgf beta is an immune suppressive cytokine frequently elevated in the tumor microenvironment causing tumor immune evasion. acute tgf beta treatment potently inhibits nk cell cytotoxicity, cytokine secretion, and proliferation. however, tumor infiltrating nk cells receive chronic inhibitory tgf beta signals in conjunction with activating signals from tumor cells. objectives: to this end, we hypothesized that long-term tgf beta-cultured nk cells would induce functional and phenotypical changes on nk cells that differ from short-term tgf beta treatment.design/method: to explore this, primary human nk cells were cultured with the leukemia cell line, k562, alone or with exogenous tgf beta for 2 weeks. : surprisingly, nk cells cultured in tgf beta proliferated faster, and upon challenge with a variety of cell line targets they secreted much greater quantities of ifnî 3 (5-to 282-fold increase against 8/8 cell lines) and tnf (3-to 33-fold increase against 7/8 cell lines). further, the high cytokine secretion induced in these nk cells was no longer inhibited by adding additional tgf beta. degranulation was also increased (2/3 cell lines), however cytotoxicity was not enhanced in a 4-hour cytotoxicity assay. after resting in il-2, the cytokine hypersecretion of tgf betacultured nk cells was maintained for several weeks suggesting this functional change might involve cellular reprogramming. we investigated the mechanism behind these functional changes and profiled 92 genes involved in tgf beta signaling. we found significant reduction of smad3 transcription which corresponded to a striking decrease in smad3 chromatin accessibility. we also found significantly increased smad6 and decreased tgfbr3 expression. phenotypic analysis revealed that tgf beta also induced remodeling of the nk receptor repertoire with decreased nkp30, cd16, and klrg1 and upregulation of trail. the functional consequences of these tgf beta-induced changes on in vitro and in vivo nk cell function are currently under investigation. background: the use of t-cell depleted grafts in haploidentical stem cell transplantation (hsct) has been associated with a delay in early t-cell recovery which increases the risk of viral infections, relapse or graft rejection. conventional donor lymphocyte infusion (dli) after hsct transplantation is effective but conditioned because of a high prevalence of gvhd. the infusion of selected lymphocyte subpopulations with low aloreactivity is emerging as an effective strategy to rectify this issue. the depletion of cd45ra+ naive lymphocytes, preserving cd45ro+ memory t-cells, could provide a safe source of functional lymphocytes with anti-infection, antileukemic and anti-rejection properties, and lower rates of adverse effects. our objective is to present data of patients that have received cd45ro+ memory t-cells dli (mdli) and assess its safety and outcome. we present data of mdli performed after hsct in cases of mixed chimerism, persistent lymphopenia, viral/opportunistic infections or as a strategy to accelerate immune reconstitution.results: fifteen patients with diagnosis of all (n = 6), aml (n = 3), mds (n = 1), saa (n = 3), sideroblastic anemia (n = 1) and cgd (n = 1), received mdli after hsct. a total of forty-three mdli were infused. the median dose of cd45ro+ memory t-cells infused was 5.00 × 107/kg (range:4.8 × 104-4.25 × 108/kg), with a median dose of cd45ra+ naive t-cells of 3.90 × 102/kg (range:0-1.3 × 104/kg). the mdli were infused at a median of seventy-seven days after hsct (range:14-407 days), with a median interval between mdli of thirty-four days results: eight published studies reported survival outcomes for pediatric vod/sos patients (n = 1036), across all defibrotide doses. estimated day+100 survival (95% confidence interval) was 60% (53%-67%). for vod/sos with mod, 4 studies were identified (n = 402) with pooled estimated day+100 survival of 57% (52%-61%). only one openlabel expanded-access study, the treatment-ind, reported outcomes separately for pediatric vod/sos patients without mod (n = 289 patients aged ≤16 years). the day+100 kaplan-meier estimated survival for those patients was 78% (72%-82%). safety results were not pooled due to differences in reporting methodology; however, study results were consistent with the safety profile of the phase 3 historicallycontrolled trial in vod/sos patients with mod (43% pediatric), in which 101/102 defibrotide-treated patients and all 32 controls experienced ≥1 ae. hypotension was the most frequent ae (39%, defibrotide; 50%, controls); common hemorrhagic aes (ie, pulmonary alveolar and gastrointestinal hemorrhage) occurred in 64% of defibrotide-treated patients and 75% of controls. in this pooled analysis of studies with defibrotide-treated pediatric patients with vod/sos, estimated day +100 survival was 60% (without mod, 78%; with mod, 57%). safety results in individual studies were generally consistent with the known safety profile of defibrotide. taken together, these results show a largely consistent defibrotide treatment effect in pediatric patients treated with defibrotide for vod/sos, with or without mod. results: six patients met inclusion/exclusion criteria. all patients were started on ecp while concurrently receiving 1.5 to 2 mg/kg steroid therapy for agvhd plus a calcineurin inhibitor. patients had initiation of ecp within a maximum of 2 weeks from initial diagnosis of agvhd (range 3-12 days). patients had grade 2-4 agvhd (3/6 patients with grade 4) with skin, liver, and gi gvhd represented. patients received ei-ecp 2-3 times per week for the first 6 weeks and then had ei-ecp frequency tapered based on initial response.after 6 weeks of therapy 1 patient had a decrease in overall gvhd grade by 1 grade. all patients were able to have steroids tapered, with doses decreased by an average of 62% (33% -100% decrease).at 12 weeks of therapy, one patient with grade 4 agvhd died of mof associated with infections. three patients had complete resolution of agvhd and 2 patients decreased by 1 grade. steroid doses were decreased by an average of 91% (69% -100% decrease). continuously measures axillary temperature and wirelessly transmits real time-time data. the primary aim of the study was to evaluate the feasibility, safety and tolerability of continuous temperature monitoring in hsct patients using temptraq. we are performing a prospective observational study of pediatric patients (1-12 years of age) undergoing hsct at cincinnati children's hospital in cincinnati, ohio. enrolled patients wore a temptraq patch for 5 days. a 1-10 rating scale survey was completed by the parent/guardian at the end of the study to determine tolerability, ease of use, satisfaction and desire for future use in the inpatient and outpatient setting. temperature data from the temptraq patch was compared to the standard episodic temperature monitoring to determine detection of febrile episodes. seven of ten patients have completed screening. we anticipate completion of the study in early february. the temptraq patch was well tolerated by study subjects (mean tolerability rating of 8.7/10). one patient developed skin breakdown at the site of the temptraq patch attributed to recent thiotepa. the patch was easy to apply with an easy of application rating of 9.7/10. parents were overall satisfied (rating 8.4/10) and would like to use the temptraq patches in future hospitalizations (rating 8.4/10) and at home (rating 8.9/10). temptraq patch identified fever (≥ 100.4 • f) in 4 patients. the fever was never detected by episodic monitoring (soc) in 2 patients and significantly delayed in the other 2 patients (>12 hours). temptraq was well tolerated in pediatric hsct patients. timely fever detection was improved in temptraq over the current soc. background: serotherapy is commonly used in patients undergoing hematopoietic stem cell transplant (hsct) to reduce the incidences of engraftment failure and graft versus host disease. however, one well-known side effect is fever. as children undergoing hsct have compromised immune defenses, fever may also be an early indicator of bloodstream infection, which would warrant prompt use of broad-spectrum antibiotics. in a subset of patients with serotherapy-associated fever, antibiotics, which may induce antibiotic resistance and increase costs, may be unnecessary. we aimed to determine the incidence and characteristics of serotherapy-related fever, as well as the likelihood of concomitant bacteremia, in our institutional experience. a 5-year retrospective chart review was conducted of pediatric patients who received serotherapy as part of hsct conditioning at the university of minnesota. one-hundred sixty eight consecutive hsct patients who received serotherapy -either atg (n = 99) or alentuzumab (n = 69) -were identified. the median age at hsct was 6-years (range, 0.4-18 years). a total of 133 patients (79 %) developed fever while on serotherapy (atg = 79, alentuzumab = 54). one-hundred sixteen patients presented fever following the first infusion, and the median onset of fever was 7 hours after commencing infusion (range, 0.1-22 hours). fever resolved at a median 8 hours (range, 1-48 hours). one hundred and fourteen patients (98%) underwent blood cultures. only seven patient were not started on (6%) empiric antibiotics, while 14% (n = 17) were on antibiotic treatment prior to serotherapy for previously known or suspected infections. nine patients (7% of febrile patients, 4% of all patients) had positive blood cultures (atg = 6; alentuzumab = 3). no infection-associated deaths were observed.conclusion: while fever is common during serotherapy conditioning in children undergoing sct, episodes of concomitant bloodstream infection are rare. ongoing analysis identified potential risk factors for bacteremia as recent history of infection, first episode of fever following second or subsequent infusions, and previous central line placement. further analysis is being conducted to identify subgroups of patients for whom close monitoring alone may be safe. background: hsct is potentially curative for caya with high-risk leukemias; however, most lack an hla-matched 2018 aspho abstracts related donor. the risk of gvhd is increased with unrelated (urd) or partially matched related (pmrd) donors. selective t-cell depletion based on the elimination of t cells carrying and chains of the t-cell receptor may greatly reduce the gvhd risks, while allowing the maintenance of mature donor-derived alloreactive nk cells and / (+) t cells, which may augment the anti-leukemia effect.objectives: this is a prospective study of caya with acute leukemia who underwent hsct with mmrd or urds and tcr / /cd19 depletion. outcomes included engraftment, toxicities, viral reactivation, and relapse.design/method: this study included 36 caya with acute leukemia transplanted between october 2014 and may 2017. all received a myeloablative preparative regimen with targeted busulfan (n = 15) or tbi (1200 cgy/6 fractions) (n = 21), with thiotepa (10 mg/kg) and cyclophosphamide (120 mg/kg). atg (3 mg/kg x 3) was given to those receiving haploidentical grafts and to the first 17 who received urd grafts. immune suppression was not given post-hsct. the stem cell source was mobilized peripheral blood stem cells (pscs), which then underwent tcr / /cd19 depletion utilizing the clinimacs device under gmp conditions in the chop cellular immunotherapy lab.results: median age was 11 (range 1.3-21.7). diagnoses included all (9-b-cell, 3-t-cell) and aml (24; 3-secondary aml). urd were used for 28; 13 were 10/10 allele matched and 15 were 9/10 matched. haploidentical donors were used for 8. median cd34(+) dose -10.1 × 106, / (+) cd3(+) cells -5.63 × 106, and b cells -8.95 × 104. all patients achieved an anc at a median of d+13 (8-30), and 94% had platelet engraftment at median d+17 (12-40). nine patients (25%) developed acute gvhd (all skin, grades i-iv). five developed chronic gvhd (skin, gut, lung): limited in 4, extensive in 1. viral reactivations included: adenovirus (5, 14%), bk virus (8, 22%), cmv (10, 28%), and hhv6 (2, 6%). nine (25%) patients relapsed at a median of 147 days (range 57-625) post-hsct, including 7 aml patients (29.2%) and 2 all patients (16.7%). transplant-related mortality was 14%; causes included sepsis (6) and ards (2). os was 72%; efs was 58% (gvhd-free efs 39%, lfs 61%). hsct with tcr / /cd19 depletion demonstrates excellent engraftment kinetics with limited gvhd without immune suppression. elimination of post-hsct immunosuppression may offer an excellent platform to augment anti-leukemic immune therapy or to enhance immune reconstitution. background: hematopoietic cell transplantation (hct) is the only curative treatment available for patients with sickle cell disease (scd). low bone mineral density (bmd) has been described in scd, but little is known about the impact of curative hct on this outcome. to determine the prevalence of low bmd and variables associated with low bmd in scd patients after hct. we conducted a retrospective chart review of scd patients who underwent hct at children's healthcare of atlanta (choa) between 12/1993 and 12/2016 and survived ≥1 year post-hct. transplant characteristics, post-hct dual-energy x-ray absorptiometry (dexa) scan results, vitamin d levels, graft-versus-host-disease (gvhd) status, and fsh levels were reviewed. for patients 2-20 years of age, height corrected z-scores were calculated using a nihvalidated calculator, with t-scores used for older patients. bmd was categorized as low if between -1 and -2 sd below the mean and clinically significantly low if >-2 sd, in accordance with the children's oncology group long-term follow-up guidelines. vitamin d levels <20 ng/mol were considered deficient, and fsh levels >40miu/ml suggestive of premature ovarian failure. fisher's exact test was used to compare variables in those with normal versus abnormal dexa scan results, with p<0.05 considered significant.results: hct was performed on 71 patients with scd, with 67 surviving ≥1 year post-hct. dexa scans were obtained in 40 patients (55% female), with mean time from hct to dexa scan being 4 years (1.3-11.2 years) and mean age at time of dexa 13.1 years (6.1-22.3 years). patients with and without dexa scans did not differ by sex, donor source, age at transplant, or vitamin d status. low bmd was noted in 10 patients (25.0%), with these patients more likely to be >13 years (pubertal; 90.0 versus 40.0%, p = 0.009). acute gvhd was more common in patients with low bmd (50.0 versus 16.7%), but not statistically significant (p = 0.085). clinically significant low bmd was noted in 3 patients (7.5% of those with dexa scans). these patients were older (16.2 years at testing), were more likely to be male (66.6%), and all had acute and chronic gvhd, while none had evidence of gonadal failure.conclusion: clinically significant low bmd is uncommon after hsct for scd. patients at risk for low bmd include older patients and likely those with gvhd. this preliminary data suggests routine dexas may not be indicated for all patients who undergo hct for scd, but further data is needed. background: causes of renal dysfunction after hematopoietic cell transplantation (hct) include damage from radiation, nephrotoxic medications, graft vs. host disease (gvhd), hepatorenal syndrome, viral infections, or transplant associated microangiopathy. we sought to investigate the incidence of, and risk factors for, acute kidney injury in pediatric hct patients and associated risk with mortality.design/method: data from patients who underwent hct between 2013 and 2016 at a single institution were sequentially retrospectively captured on irb approved protocol. acute kidney injury (aki) was defined at multiple time points post-hct using the standardized criteria: kidney disease: improving global outcomes (kdigo). interval differences between values were analyzed using wilcoxon rank sum testing and categorical variables were analyzed using chi-square analysis.results: ninety-eight patients were included in the study: allogeneic (n = 96) and autologous (n = 2), mean age 8.7 years, of whom 50% were african american, 3% asian, 21% caucasian, 13% latino, and 13% mixed race. forty-seven percent of patients developed aki within the first 2 years of hct. increased risk for aki was associated with a lower pre-transplant creatinine level (p = 0.001), abnormal pretransplant bun (p = 0.019) and an unrelated donor (p = 0.022) while preparative regimen intensity, race, or primary disease were not. twenty-six percent of patients developed aki within 30 days of hct. of those with aki, 41% were exposed to either cidofovir, aminoglycosides, and/or ambisome for at least 5 days versus 18% without aki and 74% were exposed to vancomycin compared to 49% without aki. evaluating outcomes at 1 year after hct, of those with stage 1 aki: 10% had reduced gfr and 37% died, while 14% had reduced gfr and 43% had died for patients with aki stage 2 or 3. the absence of aki by day 30 was associated with 24% reduced gfr and 8% death at 1-year after hct. overall, those with aki at any time in the first year post-hct had a 3.7 fold increased risk of death compared to those without. for patients who required renal replacement therapy (rrt, n = 8), the risk of death was 19.5 fold greater compared to those who did not. in the 25% of patients who survived rrt, both recovered renal function within 2 years.conclusion: acute kidney injury is common after pediatric hct, and may be associated with low creatinine, abnormal bun, unrelated donor pre-hct, and renal toxic medications. early-onset aki post hct is associated with an increased risk of mortality. these data should be validated in a larger prospective study but may offer opportunities to intervene and enhance outcomes. background: myeloablative hematopoietic stem cell transplant (hct) for pediatric malignant disease is associated with significant morbidity with 90% patients experiencing mucositis. patient controlled analgesia (pca) utilizing opioids is an effective strategy for pain management. we sought to describe and analyze pca use in d+30 days post myeloablative hct for malignancies at lurie children's hospital of chicago from 2010-17.design/method: utilizing retrospective chart review, pca details were collected: indication, initiation day, pca duration, team managing pca (anesthesia or palliative), medication and dose in morphine equivalents, and pca toxicities. efficacy of pca was evaluated on pca day + 3, +7, +14, +21 using demands %, maximum pain score (rflacc, faces, vas) and subjective patient, parent and/or pain team perception of pain control. we devised a scale based on the above to designate pain control as good, moderate or poor. variables being analyzed include recipient age, sex, donor type, source, diagnosis, tbi use, gvhd/trm. this analysis, we analyze the depth of remission, car-t persistence, and post-transplant gvhd on our phase i anti-cd22 car-t protocol (nct02315612) to better understand the role of car-t in the peri-hct setting.design/method: children and young adults with relapsed/refractory cd22+ all treated on our phase i anti-cd22 car-t protocol were analyzed. mrd was assessed by flow cytometry (fc) in all, with pcr-based mrd analysis using igh or tcr testing assessed in select patients. hcts were performed at each patient's local institution based on standard of care and included varying conditioning regimens, donor types, stem cell source, and gvhd prophylaxis.results: on our cd22 car trial, 36 patients were treated, the majority of patients (n = 29) having relapsed following a prior hct. 23/36 patients (64%) attained a cr, 18 of whom were mrd negative by fc. concurrent pcr based mrd analysis available in 8 patients demonstrated that all patients achieved pcr based negativity. in 6, this was simultaneous with the 1 month mrd negative fc, and in 2, pcr negativity was achieved over time (fc remained negative). 4 patients proceeded to hct at a median time of 70 days (range: 54-117 days) post-car-t, which was a first hct in 2. these two patients remain in an mrd negative cr, 1 year post-car-t. no patients developed acute or chronic gvhd. car persistence was seen in 3 patients who had detectable car-t cells on the pre-hct marrow suggesting the possibility of ongoing anti-leukemia surveillance prior to initiation of the conditioning regimen.conclusion: by inducing pcr negativity, car-t therapy may have a synergistic role with hct to improve leukemia free survival, prior to emergence of antigen negative leukemia, without an increased risk of gvhd. while the sample size is small, car-t therapy may offer an effective bridge to hct, particularly for those who are pcr negative, and those who have not had a previous transplant. given the underlying risk of hct related trm, pre-hct car may potentially allow for hct conditioning de-intensification as it may not be needed to eradicate residual disease.1 lee dw, ash abstract 218, 2016 background: post-transplant lymphoproliferative disease (ptld) is a complication after solid organ transplantation (sot) that is frequently due to epstein -barr virus (ebv) as a decrease in ebv-specific t cell immunity due to immune suppression allows for uncontrolled proliferation of ebv-infected b cells. outcomes for ptld are suboptimal with relapse rates approaching 50%. however, ebv-infected b cells in ptld express the ebv antigens lmp1 and lmp2 that can be targeted with immune therapy.objectives: we hypothesize that third party "off the shelf" lmp-specific t cell products may improve outcomes and decrease associated co-morbidities for patients with ptld by not only target the lymphoproliferating ebv-infected b cells but also restoring ebv-specific immunity.design/method: lmp-specific t cells (lmp-tcs) are manufactured from eligible donors with a broad range of hla types in our gmp facility to be used in a children's oncology group (cog) trial (anhl1522) for patients with ptld after sot. lmp-tc products are manufactured from healthy donors using autologous monocytes and lymphoblastoid cell lines (lcl) transduced with an adenoviral vector expressing δlmp1 and lmp2 as antigen presenting cells. lmp-tc products undergo comprehensive characterization by ifn-elispot assay to determine lmpspecific epitopes, class i and/or ii response, and hla restriction to guide selection of lmp-tc product for each patient.results: thus far, 27 lmp-tc products have been manufactured. lmp-tcs were active against lmp2 (mean: 158 sfu/1 × 10^5 cells; range: 1-800), lmp1 (31; 0-355), and lcl (105; 0-424) as determined by ifn-elispot assay. at the time of cryopreservation, the lmp-tc products comprised a mean of 36% cd8+ t-cells, 45% cd4+ t-cells, and 8% nk cells. no b cells or monocytes were detected in the final products. thus far, we have identified 3 novel lmp epitopes (lmp1 specific: n = 1; lmp2 specific: n = 2). approximately 80% of the lmp-tc products have lmp-specific activity through multiple hla alleles, and 67% have a mixed class i and class ii response. conclusion: thus, lmp-specific t cell products can be expanded from healthy donors to creat a third party bank, and identifying epitopes and hla alleles with lmp activity will facilitate selecting the most appropriate product for patients. while lmp-specific t cells have previously demonstrated safety and efficacy in phase i studies, anhl1522 is the first trial using cellular therapy within a cooperative group setting. children's cancer hospital at the university of texas md anderson cancer center, houston, texas, united states background: in 2017, the united states food and drug administration (fda) approved the first chimeric antigen receptor t cell (car-t) therapy; tisagenlecleucel. this cd19-directed genetically modified autologous t cell immunotherapy has shown response rates of almost 90% among children and young adults with b-cell precursor acute lymphoblastic leukemia (all) that are refractory or in second or later relapse. cytokine release syndrome (crs) and car-t cell related encephalopathy syndrome (cres) are well described toxicities associated with car-t therapy. crs is a systemic inflammatory response and is typically characterized by fever, hypoxia, tachycardia, hypotension and multi-organ toxicity. cres may occur concurrently or following crs, or without any associated crs symptoms and is characterized by encephalopathy, delirium, seizures and rarely cerebral edema. almost half of patients who receive tisagenlecleucel may require pediatric intensive care unit (picu) support. crs and cres are generally reversible but may be associated with fatal outcomes. pediatric specific management guidelines, comprehensive training of multidisciplinary staff, effective communication and phased infrastructure ensure that adequate resources are available to facilitate early diagnosis and appropriate management of pediatric patients with crs and cres and allow for optimal patient outcomes and accreditation by the foundation for accreditation of cellular therapy (fact).objectives: develop a comprehensive program to ensure safe administration of immune effector cell (iec) therapy to pediatric patients.design/method: an inter-disciplinary pediatric cartox (car t cell therapy associated toxicity) committee consisting of cell therapy and picu physicians, neurologists, fellows, nursing leadership, advanced practice practitioners, pharmacists, registered nurses and social workers was created to monitor patient toxicity and establish specific clinical guidelines and diagnostic and treatments algorithms for pediatric patients receiving iec therapy. educational modules were developed as (i) live in-services and (ii) an online module with a competency based assessment. electronic medical record (emr) order sets and documentation and warning systems were also developed by the committee. the pediatric cartox committee developed a diagnostic and treatment algorithm for patients receiving iec therapy. emr orders and flowsheets were developed to support adherence to the algorithm. inter-disciplinary staff training and competency assessments were closely tracked. almost 97% of identified staff have completed training and achieved competency including, pediatric cell therapy staff, emergency center, picu, outpatient clinic/triage, neurology and sub-specialty staff and nocturnalists.conclusion: an inter-disciplinary approach can assist in institutional readiness for an iec program, promote quality assurance and perhaps fact iec accreditation. future directions include a program for ongoing staff competency assessments. predicted peak vo2) and abnormal oxygen uptake efficiency slope at the anaerobic threshold (1359.3±297.8.9, normal 1790±310). additionally, on echocardiogram three patients had evidence of diastolic dysfunction as evidenced by an elevated e/a ratio (1.9±0.4) on tissue doppler. three patients demonstrated depressed longitudinal peak systolic strain (-17.7±2.8), indicating dysfunction not captured by ejection fraction. in this feasibility study, sct patients without evidence of lvsd on standard measures by resting echocardiogram can demonstrate abnormal exercise capacity. additionally, they can demonstrate systolic and diastolic dysfunction by measures not always included in standard echocardiography. these data suggest the need for a more thorough screening of survivors, and will be further validated as additional patients are recruited for this study. background: in hematopoietic transplantation, the t lymphocytes of the inoculum play a determining role in promoting hematopoiesis, transferring immunity to pathogens and acting as mediators of the graft-versus-leukemia effect (gvl). however, they are also responsible for graft-versus-host disease (gvhd), the main cause of post-transplant morbidity and mortality. the depletion of cd45 ra lymphocytes, by eliminating naive t lymphocytes from the inoculum, aims to conserve the gvl without producing gvhd.design/method: since april 2016, 14 patients (8 boys and 6 girls), with a median age of 8 years, have undergone an allogeneic hematopoietic transplant from an hla donor identical with cd45 ra/cd19 depletion. the indication for transplant was: acute lymphoblastic leukemia (4), acute myeloblastic leukemia (4), myelodysplasia (5) and medullary aplasia (1). the donor was familiar in 4 cases and unrelated in 10. the conditioning regimen was with fludarabine, busulfan and thiotepa. the median of cd34 + cells infused was 6.82 × 10 6 / kg. on the day 0, +15 and +30 a programmed infusion of 1 × 106 / kg lymphocytes cd45ra-was performed.results: all the patients grafted with a median leukocyte (> 0.5 × 109 / l) and platelet (> 20 × 109 / l) engraftment time of 15 and 10 days, respectively. only one patient has developed acute gvhd grade i and no patient has developed chronic gvhd. immune reconstitution was early and rapid in all t cell subsets no patient has relapsed so far and only 1 patient with myelodysplasia has developed an aml. she has received a 2nd transplant and has died of relapse. there was no case of toxic mortality. the event-free survival (sle) was 90 â± 10% with a median follow-up of 10 months. at present, 13 patients are alive, out of immunosuppressive treatment and doing well. allogeneic transplantation with cd45 lymphocytes ra depletion resulted on very encouraging results, with a very low incidence of acute and chronic gvhd, but preserving the gvl effect by infusing cd45 ra-donor lymphocytes. miami children's health system, miami, florida, united states background: hematopoietic stem cell transplantation (hsct) using autologous or allogeneic progenitor cells is a potentially curative treatment for patients with high-risk malignancies and nonmalignant conditions. the american society for blood and marrow transplantation developed a task force to establish consensus guidelines for defining patient care in hsct and advocated for further studies to delineate safe procedural steps as an increasing amount of hsct are being offered to patients. there is limited evidence to support engraftment in recipients who receive their infusions via iv or syringe pump. we present novel data from patients who achieved neutrophil engraftment following hsct by a pump mechanism.objectives: to provide evidence supporting the use of pump (intravenous or syringe) infusion method in hematopoietic stem cell transplantations.design/method: a retrospective review was completed for 114 patients who underwent hsct between 2003 and 2012. inclusion criteria included patients who had received hematopoietic stem cell transplants between 2003 and 2012 and who were ages 6 months to 21 years old. the main outcome measure was days to neutrophil engraftment (defined as the first of three consecutive days with an anc > 5 × 10 9 /l).results: among 114 patients who received infusion of hematopoietic stem cell products via pump mechanism, 63 patients (55.3%) received autologous products and 51 (44.7%) received stem cells from allogeneic donors. neutrophil engraftment (anc > 5 × 10 9 /l) occurred in a median of 14.5 days after stem cell infusion. the mean number of days to engraftment for patients who received allogeneic infusions s301 of s301 design/method: a retrospective chart review was performed at the children's hospital of wisconsin. statistical analyses included kaplan-meier estimate for os, mann-whitney test for comparing outcomes between subjects, and descriptive analyses.results: from 2005-2016, 14 patients with a median age of 7.5 (4.3-23.0) years at 1 st hct were identified. patients were conditioned with cy/atg (n = 3), cy/flu/atg (n = 6), or cy/flu/atg/tbi (n = 5) and received marrow (n = 13) or cord blood (n = 1) with median cd34/kg dose of 6.82 (1.88-10.90) ×10 6 . two patients developed grade i acute graftversus-host disease (gvhd); none developed chronic gvhd. due to dropping chimerism, graft rejection, or graft failure, 2 nd hct (n = 9) or boost (n = 5) was offered. the median cd3 chimerism prior to hct/boost was 53 (0-100)%. median time between 1 st hct and 2 nd hct or boost was 134 days (42 days-5.1 years). in 9 patients receiving 2 nd hct, 5 used the same donor, of which 3 used the same stem cell source (marrow) and 2 switched to peripheral blood stem cells (pbsc). in 4 patients who switched donors, 3 used pbsc and 1 used cord blood. most patients receiving 2nd hct underwent a uniform conditioning regimen of cy200/flu150/equine atg/8 gy tli (n = 5) or cy120/flu120/rabbit atg/2 gy tbi (n = 3); one received cy/atg. acute and chronic gvhd (limited seen in 83%) developed in 50% and 43% of patients, respectively. four patients required 18 additional boosts and 1 additional hct. after final intervention, cd3 and whole-blood chimerism at last follow-up was between 95-100% (n = 9) and 90-100% (n = 5), respectively. with a median follow-up of 8.6 (3.8-12.8) years, 13 of 14 patients are alive with an estimated 10-year os of 92.3 ± 0.07%, having performance status ≥90% (n = 12) or 80% (n = 1). one patient developed chronic extensive gvhd and died of fungal infection 2.1 years after 2 nd hct. our single-center experience demonstrates excellent ability to salvage patients who develop graft failure after initial hct. transplant-related complications such as gvhd and infections remain significant concerns. key: cord-022888-dnsdg04n authors: nan title: poster sessions date: 2009-08-19 journal: eur j immunol doi: 10.1002/eji.200990224 sha: doc_id: 22888 cord_uid: dnsdg04n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx3, the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx3 facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th1-oriented adaptive response. actually, ptx3-deficient mice are highly susceptible to aspergillosis and develop th2 skewed responses; moreover, ptx3-deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx3, which does not show direct activity on fungal cells. finally, ptx3 alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx3-mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx3 amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx3-opsonised conidia, cd11b activation, internalization, recruitment to the phagocytic cup and cd11b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd11b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx3, fcgriia/cd32 mediates inside-out activation of cd11b and consequently phagocytosis of c3b-opsonised conidia. in vivo phagocytosis experiments performed with c1q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx3) and the cellular arm (fcgrs, cr3). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx3 is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx3 binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx3-/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd14, immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx3. we found high concentration of ptx3 in human colostrum (47.62 ± 13.5 ng/ml at day 1 post-delivery) compare to the one found in human serum ( x 2 ng/ml). the presence of ptx3 in human colostrum seems to be due to the secretion of ptx3 by human mammary gland since we report the production of ptx3 by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx3 production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx3 production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx3 supplying by breast feeding: (i) soluble ptx3 in colostrum (ii) hmc that can secrete ptx3 upon stimulation in the specific tissue, (iii) an increase of ptx3 production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx3 may participate to the beneficial role of breast feeding on the newborn health. a. m. baru 1 , j. stephani 2 , h. wagner 2 , t. sparwasser 1 1 twincore, institute for infection immunology, hannover, germany, 2 technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of 10 receptors in humans (tlr 1-10) and 12 in mice (tlr 1-9, 11-13). as transmembrane receptors, tlrs are expressed on the cell surface (tlr 1, 2, 4, 5, 6, (10) (11) (12) (13) and at endosomal membranes (tlr 3, 7, 8 and 9) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about 5 years later to this, toll-like receptor 9 (tlr9) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr9 as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr9 (hutlr9) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr9 (mutlr9) is also expressed on conventional dendritic cells (cdcs). consequently, tlr9 ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr9 (henceforth called as hut9 mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr9 knock-out background. we expect that hut9-mutlr -/mice mimic the human specific expression pattern of tlr9, i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr9. by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr9 ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c1q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin-12 is a heterodimeric cytokine consisting of the two subunits p35 and p40. the main inducers of il-12p40 are microbial components activating toll-like receptors with the magnitude of il-12p40 induction depending on the specific tlr engaged. differential induction of il-12p40 upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il-12p40 due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il-12p40 promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il-12p40 promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone 3 and 4 acetylation in specific regions of the proximal promoter region. acetylation of h4 showed a specific distribution pattern and occured mainly in regulatory elements within the il-12p40 promoter, whereas acetylation of h3 took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue 4 on h3 turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue 1 , w. ellmeier 1 1 medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi 1 , m. buxadé 1 , j. minguillón 1 , r. berga 1 , j. aramburu 1 , c. lópez-rodríguez 1 1 universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat5 is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat5 participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat5 is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat5, the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat5 could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat5 is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat5 is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat5. our work indicates that nfat5 is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr3, tlr4, tlr7 and tlr8 and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p50 and p65 nfxb subunits. interestingly, we observed that tlr3 stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr4, tlr7 and tlr8 stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl-2 expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr3, tlr4, tlr7 and tlr8. finally, in vivo tlr7 stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr4, tlr7 or tlr8 triggering can directly favor tumor development whereas tlr3 signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms-354825) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd8 + and cd4 + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr 4 ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd4 + and cd8 + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd4 + and cd8 + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: 1. innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr 4 stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . 2. adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni 1 , r. oatuni 1 , m. collini 2 , m. caccia 2 , p. castagnoli 3 , g. chirico 2 , f. granucci 1 1 university of milano-bicocca, biotechnology and bioscience, milan, italy, 2 university of milano-bicocca, physics, milan, italy, 3 singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin-2 (il-2) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il-2 production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca2+/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca2+ influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr4 engagement, depending instead exclusively on cd14. we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase-2 (cox-2) that, by generating prostaglandins (pgs), such as pge2, regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd14. given the essential involvement of cd14 in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd14 represents a major step towards the development of potential treatments with modes of action involving interference with cd14 functions. we have examined the interaction of cd55, a 80-kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd14. human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd14 antibody coupled to biotin visualised by qd-525-streptavidin and anti-cd55 antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd55 and cd14, suggesting a new functional role of cd55 as a member of a multimeric lps receptor complex. l. lundvall 1 , r.r. schumann 1 1 charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase-1 induction leads to an increased release of mature il-1b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d39) was established for assessing il-1b induction during this disease. the murine raw 264.7 cell line, the human astroglial u-87 mg and the murine microglial cell-line bv-2 were stimulated with the tlr4 ligand lps, the tlr2 ligand pam 2 cys, the nod2 ligand mdp, or the nod1 ligand c12-ie-dap, and, as control, atp alone or in combination. we assessed il-1b by elisa and caspase-1 and pro-il-1b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il-1b induction pathway. s. pneumoniae (d39) infected mice showed a significant increase in il-1b release after 24 hours. in vitro, an increase in il-1b levels after costimulation with lps or pam 2 cys, and mdp or c12-ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il-1b by tlr-and nlr-ligands was observed in raw cells, bv-2 cells, u-87 cells and primary astrocytes. active caspase-1 (p10) was induced by mdp or c12-ie-dap, corresponding with high il-1b responses when lps or pam 2 cys was added. sirna experiments show that a knock-down of nod2 leads to a diminished il-1b release after lps-and mdp-stimulation. the precursor forms of il-1b and caspase-1 seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il-1b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il-1b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il-1b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg 1 1 toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd3 and cd28 activation model system -tlr4 presence on cd4+ cells is found in mouse t cells, human t cells and jurkat cell lines. following cd3/cd28 activation for 48 hours we have identified a small but distinct populationof tlr4+ cells. further characterization indicates these cells to be cd4+cd25+ cells. further characterization of the expression and functional acitvity of the tlr4+ t cells indicates co-expression of tlr4 with md-2 indicating a functional tlr4 receptor. in addition lps activiation did not lead to upregulation of tlr4 expression in t-cells. the data indicate that tcr activation leads to tlr4 expression. the expression appears to be associated with cd4+cd25+ cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann 1 , d. kaul 1 , c. krüger 1 , f. zipp 1 , r. nitsch 2 , s. lehnardt 1 1 charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, 2 charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr7 plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna40) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr7 (and human tlr8) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr7 is expressed in these cells. incubation of microglia with all three of the above mentioned tlr7 ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il1-b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr7 knock out (ko) microglia by realbecause neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin-1 is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin-1-transfected plb985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd11b labeling. concerning apoptosis, increased coronin-1 expression in dmf-differentiated plb985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin-1 significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or bid truncation suggesting that coronin-1 interfered with mitochondria-related events. to validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin-1 immunolabeling. we concluded that coronin-1 could constitute a potential target in resolving inflammation. p.-n. hsu 1 1 national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw264.7 macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p38 map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf-6 sirna and traf-6 decoy peptide, indicating this pathway is traf-6 dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b7-h1 and b7-dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il-6 was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il-2 and ifn-g which could not be overcome by restimulation with acd3. this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd4 t cells into t helper 17 (th17) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th17 differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th17 cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il-1 receptor-related protein st2 could be implicated in lps tolerance. the natural ligand of st2 was recently identified as interleukin-33 (il-33), a new member of the il-1 family. in this study, we investigated whether il-33 triggering of st2 was able to induce lps desensitization of mouse macrophages. we found that il-33 actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il-33 enhancing effect of lps response is mediated by the st2 receptor since it is not found in st2 ko mice. the biochemical consequences of il-33 pretreatment of mouse macrophages were investigated. our results show that il-33 increases the expression of the lps receptor components myeloid differentiation protein-2 (md2), cd14 and tlr-4 and the myeloid differentiation factor 88 (myd88) adaptor molecule. in addition, il-33 pretreatment of macrophages enhances the cytokine response to tlr-2 but not to tlr-3 ligands. thus, il-33 treatment preferentially affects the myd88-dependent pathway activated by the tlr. c. klotz 1 , b. lenz 1 , r. lucius 1 , s. hartmann 1 1 humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., 2008) . the cystatin effect was blocked by the application of anti-il-10 receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin-10 (il-10). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il-10 in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il-10 production after avcystatin stimulation. application of specific inhibitors revealed that the il-10 induction was p38 and erk dependent, and inhibitor titration indicated a higher sensitivity towards p38. western blotting experiments confirmed the phosphorylation of p38 and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il-10 is also regulated by the phosphatidylinositol-3-kinase (pi3k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il-10 and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose-7-phosphate (s-7p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose-5p and xylulose-5p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p38/jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s-7p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts 1 , y. morias 1 , p. de baetselier 1 , a. beschin 1 1 vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m1) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m1 activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m1 activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m1 in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd88, ifng, il-10, ccr2 and nf-kb to the development and/or recruitment of pathogenic m1 in the liver was investigated using knock-out mice or by delivering il-10 in infected mice. results: we established that cd11b+ly6c+cd11c+ tnf and inos producing dcs (tip-dcs) represent the major m1 liver subpopulation. tip-dcs differentiated in an ifng/myd88-dependent manner from cd11b+ly6c+ inflammatory monocytes in the liver of infected mice. ccr2 promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr2 ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il-10 enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il-10 in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p50 was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p50 and il-10 play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m1, in particular tip-dcs. the inflammatory activity of liver m1 is controlled by il-10 and/or p50 nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov 1 , j. driesen 1 , z. abdullah 2 , a. niño castro 1 , t. chakraborty 3 , m. krönke 4 , o. utermöhlen 4 , c. wickenhauser 5 , j.l. schultze 1 1 limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, 2 institute of molecular medicine and experimental immunology, bonn, germany, 3 institute of medical microbiology, university of giessen, giessen, germany, 4 institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, 5 institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine 2,3-dioxygenase, cyclooxygenase-2 and cd25 and production of prostaglandin e2 (pge 2 ) and interleukin 10. all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd25, expressed by regulatory myeloid cells, acts as an il-2 scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge 2 or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd25 + ido + cox-2 + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin 12 (il-12) and il-18 in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il-12 and il-18 or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il-12 and il-18. in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il-12 and il-18. therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel 1 , m. rodriguez gomez 2 , m. niedermeier 2 , y. talke 2 , n. göbel 2 , k. schmidbauer 2 , m. mack 2 1 unversity hospital regensburg, internal medicine ii, regensburg, germany, 2 university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il-4 and il-6. activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to 24h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp-1 cells infected with m. bovis bcg, the avirulent m. tuberculosis h37ra strain and the virulent m. tuberculosis h37rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva 1 , l.d. miteva 1 , s.a. stanilova 1 1 trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il-23 is a heterodimeric cytokine composed of a p19 subunit associated with the il-12/23p40 subunit. like il-12, il-23 is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il-12p40-related cytokines controls the appearance of normal th 1 and pathological th 17 mediated immune responses. in this study, we examined the dynamics of inducible il-12p40 and il-23p19 mrna expression and protein production in purified human monocytes and how jnk and p38 mapks inhibitors influenced il-12p40 and il-23 production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il-23p19 gene expression was higher than those observed for il-12p40 gene at all time-points. the level of il-23p19 mrna increased after 6 th h and reaching a maximum level at 9 th h (43.4 fold for c3bgp and 22.7 fold for lps). c3bgp and lps triggered il-12p40 gene transcription were almost equal at the 3 rd h (4.4 and 4.1 fold) and at 9 th h (7.8 and 7.9 fold, respectively) after stimulation. the higher level of il-12p40 gene expression was detected at 6 th h in lps compared to c3bgp stimulated monocytes (8.1 vs. 4.9 fold). however, il-12p40 and il-23 protein production was increased in the highest level after c3bgp stimulation. the inhibition of p38 led to the statistical significant augmentation of c3bgp stimulated il-12p40 production. the inhibition of the same map kinase enhanced lps stimulated il-12p40 production without significant difference. the inhibition of jnk and p38 mapks significantly decreased c3bgp stimulated il-23 production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c3bgp and lps to induce the expression of il-12p40 and il-23p19 mrnas in purified human monocytes. we showed that inhibition of p38 mapk down regulated il-23 and upregulated il-12p40 production in stimulated monocytes. we concluded that in human monocytes p38 map kinase activation has an opposite effect on the il-12p40 and il-23p19 expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd83, cd86 and cd40, and secrete il-12, thus acquiring the ability to induce proliferation and th1 polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd1c, bdca-3, and cd16. by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd16+ subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd16+ dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd16+ dc, including their survival and their ability to produce il-12p70. besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il-6, il-8 and mcp-1 involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg 1 , s. wolke 2 , a. brakhage 2 , m. gunzer 1 1 otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, 2 hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in 2004 nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and 2-photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to 3 hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor 7 (irf7), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr9. in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr9 to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens 1 , s. vander beken 1 , p. bogaert 1 , j. grooten 1 1 ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th1-driven immunological counterpart of the th2-driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd45 bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th1-and th2-driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd45.1 + ram remained constant in cell number for the first 2 days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day 4 of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker 1 , a. stojanovic 1 , a. cerwenka 1 1 german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr-1 + cd11b + f4/80 + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas 1 , f. marchesi 1 , m. fabbri 1 , s. schiarea 2 , c. chiabrando 2 , a. mantovani 1,3 , p. allavena 1 1 istituto clinico humanitas, rozzano, italy, 2 istituto mario negri, milano, italy, 3 università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m1 and m2. m1 macrophages act as a first line of defence against pathogens whereas m2 cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about 50 % of the monocytes differentiated after 5 days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd16, cd68 and low levels of mhc class ii. tc-macro produced il-10, il-6, tnf but not il-12, even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl2, cxcl8, ccl17 and cxcl10. the transcriptional profile of tc-macro revealed that several genes in line with an m2 polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g 100.000 kda). il-3 and il-6 were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl-2h3 rat mast cell line. we demonstrate that gr nuclear translocation begins within 5 minutes and completes after 30 minutes in dx treated rbl-2h3 cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within 5 minutes as non-genomic. studying gc-caused apoptosis, rbl-2h3 cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl-2h3 cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. 5 minutes of dx treatment inhibited ca 2+ -signaling in antigen stimulated rbl-2h3 cells in the concentration range of 100nm -10mm. moreover, 5 minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl-2h3 cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box 1 (hmgb1) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb1 is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin (1) . extracellular hmgb1 can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb1 exerts antibacterial functions in human adenoid and testis (2) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity (3). we asked whether hmgb1 from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for 40 or 120 minutes with 25 nm phorbol ester (pma), 100 ng/ml interleukin 8 (il-8), or 100 ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h2a-histone h2b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb1 in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb1 is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb1 localizes on nets. we hypothesize that net-bound hmgb1 might exert a direct antimicrobial function, or that nets might concentrate hmgb1 locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation (37°c, 3 h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor-1 induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr-1 and anti-vegfr-2 antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il-18. altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd1 molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd1a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd1a and cholesterol-dependent lipid rafts impact on cd1a surface expression and cd1a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd1-restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd1 group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd1a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd1 molecules in multiple sclerosis. we first analyzed cd1 expression on monocytes from 16 ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd1a was not expressed on ms patient monocytes, while the other members of the cd1 family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd1 molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd1 molecules in this context. c. ohnmacht 1 , d. voehringer 1 1 ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il-4 reporter (4get) mice as well as in vivo depletion of basophils are used to uncover their role during type 2 immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th2 cells. these results demonstrate that basophils play a crucial role as effector cells in type 2 immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il-1beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water (10 mg and 100 mg daily) for 30 days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il-6 and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il-6 and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant 143038). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, mph1), and during resolution of inflammation (type-2 macrophages, mph2). methods: mph1 / 2 were generated by culturing cd14 + human monocytes for 6 days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph1 / 2 of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph1 / 2 were stimulated with lps and secreted il-6, il-8, il-10, il-12p40 and tnf were quantified by elisa. results: mph2 are relatively efficient phagocytes for apoptotic neutrophils whereas mph1 are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph1 and mph2 which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il-6, il-12 and tnf production is suppressed while il-10 secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp70 in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of 90 years old and older (long-livers). intracellular ros and hsp70 levels were registered by flow cytofluorimetry upon labeling with 2',7'-dichlorofluorescin diacetate (invitrogen) and anti-hsp70 antibody (brm-22, sigma), respectively. intracellular level of hsp70 was also estimated in neutrophils after heat shock (hs) performed at 43°c for 10 min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at 1-15 min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for 1 min at 42°c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp70 (hsp70 basal ) and ros level, both intracellular and extracellular. at the same time increased hsp70 level immediately after hs (hsp70 (0 min)) correlated negatively with intracellular ros (initial and after hs). the hsp70 increase value (hsp70 (0 min) -hsp70 basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp70 increase was registered in 60 min after hs (hsp70 (60 min) -hsp70 basal ). thus it was found that within this age group the alteration in hsp70 induced by hs in neutrophils but not basal hsp70 itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant #3303. d. goyeneche-patiño 1 , z. orinska 1 , f. mirghomizadeh 1 , s. bulfone-paus 1 1 forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd8 + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type 1 ifn. two novel genes, receptor transporter protein (rtp4) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp4 and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd88 and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd8 -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during 8 and 48 h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp4 and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine 2000. stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp4 in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd88 knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr 3 and 9, as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp4 and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd88 and trif and the pathways that they represent are not relevant in the expression of rtp4 and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano 1 , e. erba 2 , r. frapolli 2 , m. d'incalci 2 , a. anselmo 1 , s. pesce 1 , p. allavena 1 , a. mantovani 1, 3 1 humanitas clinical institute, rozzano, italy, 2 mario negri institute, milan, italy, 3 institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et-743) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl2, cxcl8 and the inflammatory protein pentraxin3 (ptx3) either at transcriptional and protein level, especially after tnfa/il1b stimulation. down-regulation of ccl2, cxcl8, ptx3 and also of il6 and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd31+ vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin-4 (il-4) is a key cytokine of the t helper 2 cell response. il-4 has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il-4 production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il-4 producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day 3-6 after birth, a small population of il-4 producing cells was observed in the absence of any stimuli. the il-4 + population was not detectable at 6 or 12 weeks of age. other cytokine producing cells (ifn-g, il-10) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il-4 + cell population. phenotyping of the neonatal il-4 + cells showed that they were ige + /mhcii -/cd4cells. the occurrence of cd4 + il-4 producing cells after pma stimulation increased slowly with age and did not reach adult levels by 12 weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il-4 at day 5 after birth. neonatal pbmc collected before colostrum uptake did not produce il-4 in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il-4 responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il-4 response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel 1 , d. voehringer 1 1 ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th1 cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat6-dependent manner when stimulated with the th2 cytokines il-4 or il-13. alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat6-dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat6-deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat6 in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat6 expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat6 expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat6-deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il-4 exposed macrophages from wild-type and stat6-deficient mice. candidate genes will be expressed using retroviral transfections of stat6-deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of 38 pleural effusions, including 15 malignant and 23 nonmalignant tumors. the following human malignant cell lines were tested: a549, ht29, hct116, sw60, mcf7, mda-mb231, jurkat and hl60. results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m1, and the tams isolated from the malignant effusions similar to m2. among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms4a4a and ms4a6a: highest trascriptional level after 18h of stimulation with 10-6m dexamethasone. ms4a murine genes are differently expressed respect to the human counterpart and only the homologs of ms4a6a (ms4a6b, 6c and 6d) have a similar regulation. finally, egfp-tagged ms4a4a, ms4a6a, and ms4a7 expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms4a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms4a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein-1 (mcp-1)/ccl2 and the gpcr ligand fmlp or the tlr4 agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il-1b or ifn-g, significantly and dose-dependently synergized with ccl2 in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin-8/cxcl8, but not of the ccl2 receptor ccr2 in monocytic cells. in turn, the induced cxcl8 synergized with ccl2 for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr1/cxcr2, because cxcl8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl2 intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, 2004) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for 20 min at 37°c with gentle shaking, followed by spinning down of pmns for 5 min, at 4°c and 500g. the supernatant was sedimented at 15000g for 10 min, 4°c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd56 has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd56 low cd33+ blood monocytes (mo) in healthy individuals and the increase in cd16+cd56+ blood mo in patients with inflammation has been reported. the functional activity of human cd56+ blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd56 expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd33 and activation markers such as hla-dr and trem-1. percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a 12.3 fold increase (p x 0.0001) in the total number of circulating cd56 low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd33 and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd56+ blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd33+ blood mo expressed increased levels of cd56 on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem-1+) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd56 on cd14 high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m1-and m2-polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m1 and m2 macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m1 type and were cd14 negative but cd80 and mhcii positive and produced high levels of il-8, tnfalpha and il-6 following lps stimulation. culture in the presence of mcsf resulted in induction of the m2 phenotype. these cells were cd14 positive with intermediate expression of cd80 and mhcii expression and produced high levels of il-10, il-6 and il-8 following lps stimulation. interestingly, already baseline il-8 production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m1-and m2-polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr4 ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl60 cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma (50 ng/ml) treatment for 48h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl60 cells expressed antigen receptors cd14, tlr2, tlr4 and cd68 , and displayed enhanced phagocytic activity and production of ros. expression of cd13, cd33 and cd15 was also maintained however the cells were hla-dr and cd1a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl60 cells to differentiate into macrophages in response to pma. hl60 may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta 1 , n. ferrer 2 , a. gómez 2 , j. gonzalo 2 , a. arbués 2 , a. anel 1 , c. martín 2 , j. pardo 1 , apoptosis, immunity and cancer 1 university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, 2 university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so2, a m. tuberculosis phop mutant strain that was shown (perez et al 2001) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al 2006) . in the present study, we compare the time course and phenotype of cell death induced by so2, bcg and wild type m. tuberculosis on the murine macrophage cell line j774 and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase-3 activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl2x4 deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type 1 receptor for vip (vipr1) gene is highly conserved through species and, in humans, is highly polymorphic. vipr1 has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from 53 blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr1 in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr1 after 3, 6, 9 and 12 h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g 50 %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from 53 donors that had been typed for the relevant vipr1 snps. the down-regulation of vipr1 correlates with the presence of a t at rs896 mapping in the 3'-end of the gene (p= 0.004). the vipr1 protein level was decreased about 40 % in monocytes of 3 subjects typed as t/t at rs896 whereas 3 subjects typed as c/c at rs896 maintained a high level of expression after 9h of lps treatment. the data show that different haplotypes of the vipr1 gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr1 vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska 1 , t. vavrochova 1 , d. filipp 1 , immunobiology 1 institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e7.5-13.5) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd14 in the early mouse embryo (me). facs analysis of cell suspension prepared from 10.5 day me showed that about 0.7-1 % of cells were positive for cd11b. these cells exclusively were also positive for cd14, tlr2, and cd45 antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days 7,5-13,5. reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e7,5), embryo-derived mhcii + macrophages start to appear in the embryo around day 13. multicolor facs analysis of cd11b, cd45, cd14, f4/80, tlr2, tlr4, c-kit and mhcii surface markers revealed differential expression of tlr2 and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd11b + tlr2 + cells isolated from the e10,5 embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il12/il23-dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies 16 presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and 11 have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in 9 out of these 11 patients mutations in several ifng pathway genes, other candidate genes are being investigated for the 2 other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp1. we did focus on the study of 2 genes which are considered as important pathogen recognition receptors of the innate immune system: tlr2 is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il-10 cytokine. using a case/household-contact cohort we did investigate polymorphisms of these 2 genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to 70-80 % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, 2009. hsp70 are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp70 have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp70 on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp70 (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp70 were used. exogenous hsp70 was used in concentration 1-10 ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio 1:1 and 2:1 directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp70 on their surface. results demonstrating effect of exogenous hsp70 on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp70 on amplitude of respiratory burst. the cells expressing surface hsp70 impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp70 might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp70 to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited 28 pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd14/cd86 and cd14/cd163 double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd14/86 subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd14/163 subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m2 polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević 1 , i. pilipović 1 , s. stanojević 1 , k. mitić 1 , k. radojević 1 , v. pešić 2 , g. leposavić 1,2 1 institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, 2 faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h 2 o 2 ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to 14-day-long propranolol treatment and measured both no and h 2 o 2 production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b 2 -and a 1 -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h 2 o 2 production. an increase in h 2 o 2 production in the presence of the a 1 -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h 2 o 2 production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b 2 -and a 1 -adrenoceptors on peritoneal macrophages (a stimulatory effect on b 2 -adrenoceptors and a suppressive effect on a 1 -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd14 ++ cd16 -('classical') and cd14 + cd16 + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd14 + cd16 + monocytes display higher tlr2 and -4 expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd14 ++ cd16monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr2 stimulation. methods: cord blood (n=8) and peripheral-blood (n=8) mononuclear cells were stimulated in vitro for 24hrs with peptidoglycan and subsequently analysed for cd14 and cd16 and intracellular il-12p70 and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il-12p70, both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il-12p70 was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il-12p70 (% positive cells and geomfi) was significantly higher for cd14 + cd16 + cells than for cd14 ++ cd16cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd14 ++ cd16cells were positive for tnf to a significantly higher extent than the cd14 + cd16 + cells. in particular the tnf response to tlr2 stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd14 ++ cd16monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a 16-year-old boy who admitted with complaints of seizures during the previous 2 months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g (50 mgr/m2/day, subcutaneously every other day) was started. after 2 months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for 3 months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf1 gene concerns the well-known gt deletion in the second exon of ncf1 gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = 45) subjected to moderate brain injury were randomized, and received either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after cci. levels of tnf-a, il-1b, il-6, il-10 and il-1 receptor antagonist were analyzed in brain tissue using real time rt-pcr at 4 and 72 hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at 24h and 7 days. data were analyzed with the mann-whitney u test and a two-tailed p x 0 .05 was considered significant. all cytokines were highly up-regulated 4 hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il-10 were significantly lower in the ubiquitin treated animals, whereas the levels of il-6 and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7. the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight (38) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while 18 were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p=0.02), tpp (p=0.00), osi (p = 0.00), mda (p = 0.00) were significantly raised but taa (p = 0.01) was significantly reduced in ptb patients compared with controls. the levels of mda (p = 0.04), neopterin (p=0.00) and tpp (p=0.00) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p=0.00), mda (p=0.00), neopterin (p=0.02), osi (p=0.00) were significantly reduced while taa (p=0.01) was significantly raised in c+m after 4 weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after 4 weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas 1 , e.m. cunha 1 , m.j. oliveira 1 1 icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles (20 nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to 5 minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage (70 %) of macrophages contained the metal particles than neutrophils (55 %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap-70 is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap-70 regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., 2006; sivalenka and jessberger, 2004) . swap-70-/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., 2002; sivalenka and jessberger, 2004; sivalenka et al., 2008) . crucial regulators of these processes are members of the rho family of small gtpases such as rac1 and rhoa. swap-70 interacts with rac1 in vitro and preferentially binds the active gtp-bound rac1. swap-70 supports the increase of active rac1 in vitro by a yet to be defined mechanism (shinohara et al., 2002) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap-70 and rac1. it was found that fulllength swap-70 preferentially binds to constitutively active rac1 (rac1q61l) but not to its dominant negative form (rac1t17n). binding assays with swap-70 truncated mutants showed interaction of swap-70's n-terminus with gtpgs rac1 or rac1 depleted of guanine nucleotide, whereas swap-70 central or c-terminal regions do not bind to any form of rac1. preliminary competitive-binding assays with overlapping 18mer peptides, spanning the entire swap-70 sequence, mapped the rac1 binding site near the n-terminus of swap-70. full-length swap-70 site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap-70 with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap-70. v. c. barbosa 1 , c. d. polli 1 , m.c. roque-barreira 1 , m.c. jamur 1 , c. oliver 1 , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl-2h3 cells were sensitized with ige anti-tnp and stimulated with dnp 48 -hsa or artinm. artinm binding to rbl-2h3 cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp-1 and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp-1 release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand 1 (psgl-1), b1 and b7-integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd44 (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il-1b, il-18 and tnf-a. herpes simplex virus 1 (hsv-1) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv1 infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il-18 and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv1. methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) (10ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv-1 for 18 h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot 2.0 software. results: in the first itraq experiment over 300 human proteins were identified in the hsv1 infected cell supernatants. from these proteins 119 had at least 3 fold increase after poly(i:c) + hsv1 infection compared to the uninfected cells. hsv1 infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of 2.7 fold increase in the protein amount in the poly(i:c) + hsv1 infected cell supernatant and a 2.6 fold increase in the hsv1 infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand 10, il-6, tnf-a induced protein 6, complement factor b, galectin-1 and mxa. at present, further experiments are on-going for more detailed analysis of the hsv1 infected macrophage secretome. h. p. prakash 1,2 1 german cancer research centre, translational immunology, heidelberg, germany, 2 max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein-1 (ciap-1) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap-1 and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal-3 (25 mg/ml) binding to monocytes, in the presence or absence of 10mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal3, laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal3 or rpmi and monocytes (1x10 5 ) were added into each insert. when necessary, hrgal3 was pre-incubated with 10mm lactose or sacarose. mcp-1 (100ng/ml) was used as positive control. we observed that hrgal-3 binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal-3 is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal-3, we observed that the association between these glycoproteins and hrgal-3 resulted in a 60 % increase in the number of migrating cells. both n-and c-terminal domains of hrgal-3 are involved in the association between laminin or fibronectin and hrgal-3, since the presence of lactose resulted in 50 % and 20 % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal-3 induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for 3 weeks in the presence of bmp4,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd14+cd45+ cells. the sorted cd14+cd45+ cells were further cultured for 7 -10 days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd14, m-csf receptor cd115 and the scavenger-receptor cd36, we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd14 and cd16 (fcgammariii) : the cd14lowcd16-and cd14+ cd16+. trancscriptional, phenotypic and functional assays suggest the alternative (m2) polarization of cd14+cd16+ embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m2 -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi 1 , p. kropf 1 1 imperial college london, immunology department, london, united kingdom the balance between t helper (th) 1 and th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th1 or th2 responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd4 + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg9vd2 t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f1-atpase) is expressed on many cell types and is a possible specific ligand for the vg9vd2 tcr. the present study aims at understanding the role of f1-atpase in antigen regognition. using video microscopy calcium imaging in single vg9vd2 t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f1-atpase. purified f1-atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg9vd2 cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f1-atpase. thus the f1-atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg9vd2 t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f1-atpase. by using purified f1-atpase and peptides derived from vg9vd2 tcr sequences, interaction sites between f1-atpase and tcr were identified on both ligands. based on these findings a generalized model for vg9vd2 t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg2d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg2d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg2d activation in vivo. by transiently overexpressing the nkg2d ligand rae-1-beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma5vdelta1 gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg2d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg2d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg2d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae1 induction. the primary systemic th2 response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg2d receptor suggest that different ones may play unique roles. a novel nkg2d-ligand, h60c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae-1 induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg2d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd1d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th1 (ifn-g, tnf) and th2 (il-4, il-13) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type 1 diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd4 -nk1.1 -inkt cells producing high levels of the pro-inflammatory cytokine il-17 has been identified (inkt17 cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt17 cells in type 1 diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il-17 as compared to c57bl/6 mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt17 cells present in nod mice were mainly cd4 -nk1.1 -, express the ror-g transcription factor and il-23 receptor, both molecules being usually associated with th17 commitment. we are currently analyzing, using co-transfer experiments, whether these inkt17 cells play a beneficial, a deleterious, or any role in the development of type 1 diabetes in nod mice. j. s. dodd 1 , r. muir 1 , s.s. affendi 1 , p.j. openshaw 1 1 imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd1d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd1d-deficient mice with poor nkt cell responses have inefficient induction of cd8 t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th2 immunity (increasing il-5 and il-10), promoting pulmonary eosinophilia and ablating cd8 t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd8 t cell activity (as measured by cd25 expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d4) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d7) weight loss by a cd8 t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd4 + ab t cells, are regarded as immature or t h 2 biased. vg9 + vd2 + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg9 + vd2 + t cells towards these activators. because il-23 is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg9 + vd2 + t cells. herein, we observed that zoledronate induced neonatal vg9 + vd2 + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h 1-like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h 2-like cytokines such as il-4 and il-5 were not induced. addition of il-23 to zoledronate selectively costimulated ifn-g production from neonatal vg9 + vd2 + t cells. furthermore, zoledronate/il-23 treatment resulted in neonatal vg9 + vd2 + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il-23 (il-23r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il-23 resulted in a further increase of t-bet expression in neonatal vg9 + vd2 + t cells. these changes in the expression of il-23r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il-23 treatment. of note, in contrast to adult peripheral blood vg9 + vd2 + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg9 + vd2 + t cells. altogether, these observations show that neonatal vg9 + vd2 + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il-23 is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e3 ligases such as hhv8 encoded k3, k5 or mhv68 encoded mk3. these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e3 ligases manipulate them. we discovered that both the cellular march and viral e3 ligases ubiquitinate cd1 molecules. however, whereas viral molecules inhibit cd1-antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd1 molecules. in contrast mhc class ii was only targeted by cellular and not by viral e3 ligases. furthermore cd1 molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e3 ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd2 negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht29 colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd2 neg clones in vivo, we generated hypodermal ht29 tumors in immunodeficient mice. concomitant injections of vd2 neg clones, in contrast to vd2 + cells, prevented the development of ht29 tumors. vd2 neg clones expressed chemokine c-c motif receptor 3 (ccr3) and migrated in vitro in response to chemokines secreted by ht29 cells, among which were the ccr3 ligands macrophage inflammatory protein (mip)-1d and monocyte chemoattractant protein (mcp)-4. more importantly, a systemic intraperitoneal (i. p.) treatment with vd2 neg clones delayed the growth of ht29 subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr3 antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd2 neg clones were not able to inhibit the growth of a431 hypodermal tumors. our findings suggest that cmv-specific vd2 neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht29 cells expressing the luciferase and realized orthotopic injection of ht29-luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint1, currently the only known determinant of a canonical iel compartment, that is selectively required for vg5vd1 + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint1 expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint1 function; for example, we demonstrate that the constitutive expression of wild-type skint1 fully restores detc development in a skint1 mutant mouse, but does not rescue normal detc function. thus, skint1 provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd1d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd19 + cd21 hi cd23 lo cd11c -) and splenic conventional dendritic cells (cdcs) (cd11c hi cd8a +/-cd11b +/-b220 -) from wt and cd1d -/mice as apcs for nkt cells from va14-ja18 transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd1d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd1d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il-4 by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il-4 producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h 2 response, whereas cdc-primed nkt cells rather favor a t h 1 response. objectives: il-18 is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il-18 give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd1d -/-) mice. we set out to investigate the activated b cells in il-18 injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il-18 (2 mg) for 10 days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day 14 was evaluated by flow cytometry and immunohistology. results: mice injected with il-18 developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd138 + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il-18 was increased in inkt cell deficient (cd1d -/-) mice, in contrast to published data. an increased response to il-18 was also observed in ja281 -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il-18 induced antibody responses. further characterization of the recruitment of b cells in il-18 injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il-18 induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd1d, are prone to autoantibody production and often involved in early immune responses. the il-18 induced antibody response in mzb deficient (cd19 -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il-18 induced antibody responses. we conclude that the role for inkt cells in il-18 induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c57bl/6 mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha 18-/-and cd1d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il-4. moreover, ifn-g production required the presentation of ehlppg by cd1d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il-12. similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd16 in cmv-infected individuals. cd16 is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that 71.9 ± 15.9 % of gamma-delta t cells from 21 cmv-infected ktr expressed cd16, when compared with only 19.8 ± 16.7% in 11 non cmv-infected ktr (p x 0.0005). similarly, 51.9 ± 25.7 % of gamma-delta t cells from 13 cmv-seropositive blood donors expressed cd16 compared to 27.1 ± 25.1 % in 15 cmv-seronegative donors (p x 0.01). cd16+ gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd16-dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il-12 and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd16 mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd107a on cd16+ gamma-delta t cell lines. cd16 is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd16+ gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a431 skin carcinoma cell line pre-incubated either with rituximab (anti-cd20) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd16-dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k5mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il-6, ifn-g, tnf-a, osm, ccl2, cxcl8, cxcl10, and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd40, cd86, hla-dr, and dc-sign, and lost cd14, ccr2, ccr5, and cxcr4. addition of further microbial stimuli (lps, peptidoglycan) induced ccr7 and enabled these inflammatory dcs to trigger antigenspecific cd4 + effector ab t cells expressing ifn-g and/or il-17. importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg9/vd2 t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg9/vd2 t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg9/vd2 t cells stimulated with hmb-pp in the presence of il-21 express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il-21 regulate expression of the b cell attracting chemokine cxcl13/bca-1, its receptor cxcr5, and co-stimulatory molecules involved in b cell help. purified peripheral vg9/vd2 t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to 4 days with and without hmb-pp, in the absence or presence of il-2 or il-21, or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl13 protein were detected in co-culture supernatants only when both il-21 and hmb-pp were provided, implying an il-21-dependent and tcr-dependent expression. vg9/vd2 t cells were confirmed as producers of cxcl13 by flow cytometry and immunofluorescence. under the same conditions, activated vg9/vd2 t cells expressed cd27, cd28, cd40l, cd70, icos and ox40. in contrast, neither cxcr5 nor ccr7 changed markedly by il-21 stimulation of peripheral vg9/vd2 t cells. conclusion: our findings confirm on the protein level that stimulation of vg9/vd2 t cells with hmb-pp and il-21 induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto 1 , m. emoto 1 1 gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)-12. although downmodulation of surface t cell receptor (tcr)/nkr-p1c (nk1.1) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il-12 stimulation. to determine whether failure to detect inkt cells after il-12 stimulation is caused by dissociation/internalization of tcr and/or nkr-p1c or by block of de-novo synthesis of these molecules, and to examine the role of il-12 in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p1c by inkt cells after stimulation with a-galcer or il-12, and influence of il-12 neutralization on down-modulation of stcr/snkr-p1c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il-12 neutralization. whereas s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of il-12, s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p1c remained unaffected by a-galcer or il-12 treatment, despite the down-modulation of ctcr and/or cnkr-p1c protein expression. in contrast, ctcr + cnkr-p1c + stcr -snkr-p1c -inkt cells and cnkr-p1c + snkr-p1c -inkt cells were detectable after in-vitro stimulation with a-galcer and il-12, respectively. our results indicate that tcr and nkr-p1c expression by inkt cells is differentially regulated by signaling through tcr and il-12r. they also suggest that il-12 participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p1c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg9-gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region 3 d1 (cdr3d1) and cdr3d2 repertoire, with a striking enrichment in a specific germline-encoded cdr3d1 sequence. differentiated gd t cells and the enriched cdr3d1 sequence were detected as early as after 21 weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd1-jd1 and vgi-jg1.3/2.3 rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd2-jd1 and vg9-jg1.2 selection was seen in pb tcrgd cells. detailed analysis of the cdr3 motifs revealed selection determinants in both vg9-jg1.2 (canonical length and cdr3 motif) and vd2-jd1 (minimal cdr3 length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚4) and tcrgd cb cells (6-7) to tcrgd pb cells (˚10 or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type 1 diabetes (t1d). we have previously shown that transgenic expression of a cd1d-restricted, va3.2-vb9 tcr in nod mice lead to an increase in cd1d-restricted type ii nkt cells (24abnkt cells), and prevention of the development of t1d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by 24abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc2.5 cd4+ t cells, in the presence or absence of selected cells from 24abnkt cell transgenic mice. results: in 24ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, 24abnkt cells expressed a high level of cxcr3 and a low level of ccr7 and cd62l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd4+ bdc2.5 t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from 24ab transgenic mice with bdc2.5 cd4+ cells resulted in the prevention of diabetes development. the protection from disease required a minor cd4+ subset of 24ab+ nkt cells, but was independent of cd25+ t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd1d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs44, induces in vitro inkt cell expansion and ifng and il-4 secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th2 profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd1d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il-10 with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr1 cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th17-type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th17 immune response is characterized by the secretion of il-17a and il-17f. the il-17 locus encodes the highly conserved il-17a and il-17f cytokines that are syntenic in 44kb distance to each other. besides cd4 + th17 and nkt cells, approximately 50 % of the il17a producers are gd t-cells. like cd4 + th17 cells, il-17 producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il-17 production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th17 cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il-17 or ifn-g. combined with the well known classification of il-17 producing gd t-cells along the markers cd27 and cd122, our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th17-type immune responses in vivo. in this context, the potential redundancy of il-17a and il-17f may complicate the analysis. so far, most studies were carried out with il-17a single-deficient or il-17f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il-17a and il-17f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg9vgd2 t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b1 and il-15 differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd2 tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp3) and, similarly to ab tregs, suppress the proliferation of anti-cd3/anti-cd28 stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd2 tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il-17 was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd11b in the liver following a-galcer treatment, numbers of cells lacking cd11b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma9/vdelta2 t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma9/vdelta2 t cells expansion upon stimulation with zoledronic acid (za) and interleukin-2 (il-2). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by 1) the bioinformatic analysis of gene expression profiling data 2) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in 43 patients (49 %) (responders, r), whereas 44 patients (51 %) were non-responders (nr). vgamma9/vdelta2 t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt2, whereas temra of r patients had an higher expression of the costimulatory molecule nkg2d. the proliferative response of vgamma9/vdelta2 t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, 82 % of r patients were m, whereas 77 % of um patients were nr (p x 0.001). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c57bl/6 mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il-12 p40 (clone r2-10f6; atcc) or anti-cd-1d (clone 1b1) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at 18 hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il-12p40, a component of both il-12 and il-23; pretreatment with anti-cd1d mab had no effect. il-12rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il-12 was not a critical factor. this suggestion is supported by the increased expression of il-23p19 and il-12p40 (but not il-12p35) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il-23 production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb1) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd27 expression. gd 27+ cells secrete interferon-g, while gd 27cells are capable of producing il-17. this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd27-defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine 123. cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to 40 % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚2% of gd 27+ cells but not at all in gd 27cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, 40-60 % of gd 27+ cells were positive for pgp activity, although their gd 27counterparts remained largely negative (p x 0.01). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd27. as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd 27+ and gd 27cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma9vdelta2 t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma9vdelta2 t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma9vdelta2 t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma9vdelta2 t cells from healthy donors were stimulated with different compounds (zol, ipp) for 24 hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma9vdelta2 t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma9vdelta2 t cells. moreover, mcp-2 depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma9vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma9vdelta2 tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma9vdelta2 t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg9vd2 t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg9vd2 t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg9vd2t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg9vd2 t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg9vd2 t cells and their effects on the viability of glioma cells, we expanded in vitro vg9vd2t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg9vd2t cell lines to kill three different glioma cell lines (t70, u251, u373) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg9vd2t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg9vd2 t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg9vd2 t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg9vd2 t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately 15 % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e7 protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd1d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e7-induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e7-expressing transplanted skin is dependent on interactions between populations of cd1d-expressing cd11c+/f480+ myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e7 graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il-2 and a-galcer. expression of cd1a, cd1d and the costimulatory molecules cd86 and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il-4) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd1d expression on the cell surface in comparison with control cells. in contrast cd1a expression was decreased. cd86 and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd1d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd1a, cd1b and cd1c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il-4 producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd1d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, 0.5-10 % of circulating lymphocytes express a vg9vd2 t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg9-enriched genes compared to conventional mhc-restricted cd8 + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf4 & crtam in gd and ab t cells respectively. because igsf4 binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd8 + ab t cells express crtam, and that engagement of igsf4 on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf4/crtam. we therefore sought to answer: 1. what is the function of igsf4/crtam on gd t cells? 2. how is the igsf4-crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf4 enrichment on resting gd t cells, with expression also detected on˚10 % of ab t cells. the properties of those cells are being examined. however, igsf4 generally correlates with markers of activation/antigen experience such as cd45ro. thus, igsf4 cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg9 + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf4 and crtam within 48 hours. however, engagement of igsf4 by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf4 may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg2d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf4 interactions are sufficient for cd8 t cells to kill gd t cells and/or vice versa. instead, crtam-igsf4 interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd3z is a 16 kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately 3-6 % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd2 tcr variable segment associated with the vg9 segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg9vd2 t cells without antigen presentation. in vitro stimulated vg9vd2 t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg9vd2 t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg9vd2 t cell anergy observed in hiv+ patients. to this aim, cd3z expression and ifn-g production by vg9vd2 t cells from hiv+ and hiv-subjects were analyzed. we show that vg9vd2 t cells from hiv-infected patients expressed lower level of cd3z compared with healthy donors. a direct correlation between cd3z expression and ifn-g production capability by vg9vd2 t cell was found. however, pkc activation by pma is able to restore cd3z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg9vd2 t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner 1 , m. swamy 1 , s.l. clarke 1 , a. hayday 1 1 king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd8aa or cd8 -cd4cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to 15 days. the cells are initially activated by plate-coated acd3 antibody and a cytokine cocktail and maintained further in medium containing low levels of il-2. after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg2d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il-6) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after 6 hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd94 in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg2 molecules in heterodimer with cd94, we screened the presence of these receptors on t cell subsets in bd. the expression of nkg2 a/c/d molecules on gd and cd8+ t cells were analyzed in 17 active and 9 inactive patients with bd and 21 healthy controls. expression of nkg2 molecules was evaluated on cd8+, gd t and cd56+ nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls (4.4 vs. 2.8 %, p=0.001). in addition to the increase of gd t cells, increased expression of activating nkg2c molecules was also observed on gd t cells (20 % vs. 11 %, p= 0.01). nkg2a expression on gd t cells was found to be higher than nkg2c expression in patients and controls; but nkg2a expression on the t cells was not statistically different in both groups (33.5 vs. 40 %). nkg2d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd8+ cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th1 and th2 cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg9/vd2+ lymphocytes. we have screened a panel of 26 lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg9/vd2+ cells, and selected two susceptible ("target") cell lines (over 60 % death in the assay) and two vg9/vd2+ resistant ("non-target") cell lines (under 20 % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs 697 (non-target), and validated the results by rt-qpcr quantification. results: we identified 37 commonly up-regulated and 50 commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp1, ifitm1 and prame, for example, are up-regulated, whereas cd274 and clec2d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg9/vd2+ target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd1d, in the rat. mice and rats have very similar cd1d and inkt tcr genes, with the exception of the va14 gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd1d revealed a very similar pattern of cd1d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il-4 production was 10-100 fold lower in the best responder rat strain (f344) compared to mouse (c57/bl6). since nkrp1a (rat homologue of mouse nk1.1) and tcr are not appropriate markers for rat inkt, cd1d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd1d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd1d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd1d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd1d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g9d2 t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g9d2 t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g9d2 t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g9d2 t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp-1. a significant (p x 0.01) reduction in intracellular bacterial numbers was observed (n=8) in the presence of pbmcs cultured with picostim+il-2 in comparison with pbmcs cultured with il-2 or media alone. picostim+il-2 caused significant expansion and activation of gd t cells following culture of pbmcs for 10-14 days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers 100-fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il-2, reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma9/vdelta2 (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than 5 % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: 1) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; 2) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); 3) to establish whether the same issues could be solved using a simplified protocol of dc generation. 1) dc were generated from cd14 + cells of healthy donors/mm patients; immaturedc on day 6 were induced to fully mature by incubation for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za. after 7 days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; 2) idc generated from cd14 + cells of hla-a*0201 + healthy donors/patients were pulsed with sv-peptide and stimulated for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za; after 2 rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd8 + t cells was determined by svpentamers staining; 3) the same experiments were performed both with dc generated following a standard protocol and a 48h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [6-3 h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd4 + cd25 + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin-2 on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp3 negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd1 subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg2d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd8+ have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n=8) and inactive (bdna) (n=20), versus healthy controls (hc) (n=30) and patients with recurrent oral ulcerations (ru) (n=14). to determine gd cytokine profile and surface markers treg-related in bd (n=9) and hc (n=11). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta1, vdelta2, cd8alpha, cd8beta, nkg2d, nkg2a and cd103. -intracellular expression of ctla-4, and foxp3. -intracellular expression of il-2, il-4, ifngamma, il-10 and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta2+ cells were significantly increased in ru. vdelta1+ and gdcd8+ lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg2d was slightly increased in gd from bda. -nkg2a expression by gdcd8+ was not different in bd versus hc. -most of gdcd8+ presented cd8alpha-alpha homodimers in bd and hc and were negative for cd103, foxp3 and ctla-4. gdcd8+ and gdcd8-subsets were (in bd and hc): -high ifngamma-producers without differences. -low il-2-producers: il2+ cells were lower in gdcd8+ than in gdcd8-. -low il-10-producers: il10+ cells were lower in gdcd8+ than in gdcd8-. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd8+ than in gdcd8--very low producers of il4 in most of cases. the hallmark in bd was the increase of gdcd8/vdelta1+. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg2a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd8+, except a lower percentage of il-2+ cells than in the gdcd8-subset. gdcd8+ from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd56 in humans. a subgroup, the invariant nkt cells (inkt), expresses the va24vb11 tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd3 + cd56 + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from 10 healthy umbilical cord blood samples and stimulated with ifn-g (50 ng/ml), anti-cd3 (25 ng/ml) and il-2 (500 ui/ml). these cells were cultured for 21 days and the expanded cd3 + cd56 + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd3 + c56 + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x 0,05 was considered significant. results: we could significantly expand cord blood cd3 + cd56 + nkt cells from 0,87±0,57 % to achieve an enrichment of 46,89±13,31 % (p=0,0002). table 1 shows the percentage (mean±sd,n=10) of phenotypic markers in cd3 + cd56 + cells at baseline (day 0) and after 21 days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb4 and vb8 families (p x 0,001). conclusion: our results show that cord blood-derived nkt cells are mainly cd8 + and cd94 + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb4 and vb8 families in these cells. l. marischen 1 , d. wesch 1 , p. rosenstiel 2 , a. till 2 , d. kabelitz 1 1 institute of immunology, kiel, germany, 2 institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod2. peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod2 gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp1 19 and pvama1 proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day 5 culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd69 + and cd25 + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day 3 after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd107a + (lysosomal associated membrane proteins: lamp-1) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th1 and th2 cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th1-like and th2-like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n=43) and healthy controls (n=13) were determined by flow cytometry using anti-cd3 and monoclonal antibody specific for the cdr3 loop of the invariant tcr a chain of inkt cells (clone:6b11). furthermore, after pma/ionomycin stimulation for 4 hours, intracellular ifng and il-4 cytokines were detected in cd4+cd8-, cd4-cd8-(dn), cd4-cd8+ and cd4+cd8+ subsets of inkt cells by five colour flow cytometry in patients with ad (n=10) and healthy controls (n=10). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x 0.01) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x 0.01). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r=0.726 and p x 0.001) and healthy controls (r=0.693 and p x 0.001). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il-4 level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x 0.05). the frequency, the number of inkt cells and the cytokine producing capacity of the cd4/cd8 inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il-4 that promotes the th2 differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells (1.8 %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd4 + nkt and cd4 -cd8 -nkt cells. nk1.1 + nkt cells may release large amounts of il-2, il-4, ifn-g and il-10 after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il-2 had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il-2. there was a significantly increase in the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd69 molecule existed in 68.95 % of the cells from large-scale selection. the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cell subsets in the giant lymphocytes were enhanced to 84.0 and 38.9 folds, respectively. under a light microscope at x400 magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than 1.0 and they are non-adherent cells. the differentiation pathway of the seb-activating cd8 + and tcrvb8 + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd8 + nkt cell and tcrvb8 + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl3 which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo-1am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h2begfp reporter mice. stimulation with anti-gd clone gl3 or anti-cd3 clone 2c11 elicited activation of gd t cells suggesting that tcr gd and cd3 molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl3 in vitro produced ccl4, ifng and tnfa. therefore, we were interested whether the ccl4 production of gd iiel influenced the homing of ccr5 cells such as lamina propria (lp) cd4 + foxp3 + cells (tregs). to test this, wt mice were i. p. treated with gl3 mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr5 + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr5 + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr5 expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il-4 production in g4 reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il-4 gene. in the absence of any manipulation gfp was expressed from the il-4 locus in populations of immature thymic nkt cells (predominantly cd4+cd44lotcrhi cells on a balb/c background, and cd4+cd44lonk1.1-on a c57bl/6 background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il-4 production was induced predominantly in cd4+ nkt cell subsets of the liver and spleen, and after i. n. administration, in cd4+ nkt cells of the airways. spontaneous and a-galcer-induced expression from the il-4 locus occurred in the absence of stat6 signalling, and did not require initial exposure to il-4 protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin-17 (il-17) plays an important role in neutrophil recruitment. herein, we investigated the role of il-17 receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c57bl/6 (wt) and il-17 receptor gene-deficient (il-17r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated 6 hours after surgery. the ability of il-17 mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il-17r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il-17 induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il-17r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il-17receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il-17 showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il-17 also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il-17 receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin-1 receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il-1r associated kinases to activate nfxb and p38 mitogen-activated protein kinase. the il-33-induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il-33 in different mast cell subsets. methods: different mast cells subsets (hmc-1, human cbmcs and murine bmmcs) were stimulated with il-33. the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk1/2, pakt, pnfxb, p38 and pjnk). additionally, we studied the signal transduction of il-33 in il-33r transfected hek293t cells. results: we found, that a tir family member, il-33r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il-33-induced cytokine production depends on c-kit transactivation. il-33r and il-1r accessory protein (il-1racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il-1r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il-33-induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il-33 in mast cells in absence of iger activation. (1) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase-3 activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease-1 (ho-1) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho-1 expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin 1 and 2. recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin 2 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap1 small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap1 activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr2 shows a bimodal kinetic, with the first peak at 30''/1' and the second at 5' after stimulation. rna interference-mediated depletion of beta-arrestin 2 specifically inhibits the occurence of the second wave of rap1 activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin 2 is involved in rap1 activation and that the oscillations in the formation of rap1-gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c3g (rap1gef) and spa1 (rap1gap): preliminary results suggest that spa-1 has probably a role in the early activation peak. since this oscillatory chemokine-induced rap1 activation is present on other myeloid cell lines (hl60, 32d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin 2, localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh3 containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of 153 different sh3 domains that revealed over 20 putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)-3 and il-5 stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il-3 and il-5 receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il-3 , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn 328 are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: (1) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna-146 expression is induced by activation of the toll-like/interleukin-1 receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna-146a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna-146a transfected cells showed significantly reduced levels of the active proapoptotic caspases 8 and 3 (casp8/3). in line with this, mirna-146a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna-146a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna-146a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna-146a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il-15, il-7r a-chain and il-15r a -chain genes in hiv disease progression. methods: we studied 70 antiretroviral treated patients (progressors) and 71 long term non progressors (ltnp). we analyzed 2 snps in the il-15 gene, 5 snps in the il-7r gene and 3 snps in the il-15r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il-15r aa 182 and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il-7r and il-15r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd4 and cd8 t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art1, and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art1 in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art1 transcripts by rt-pcr analysis in human blood leukocytes. soluble art1, released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek293 cells with art1 and tnf resulted in modification of tnf at at least 2 distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r32, a site that has previously been implicated in binding to tnfr2. binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit225 than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art1 resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik 1 , t.v.poroshina 1 1 institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: 23 patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and 11 control men were assessed for slpi, tnf-alpha, il-8 and free tgf-b1 in ejaculate by elisas using stat-fax 303 plus. a 3 to 5 day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at -20 degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients (5127.571 ± 971.731 pg/ml, p x 0.001) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid (125.49 ± 17.9 pg/ml; p x 0.05). the il-8 concentration was elevated in all patients (366.7± 49.5 pg/ml; p x 0.05) in seminal fluid. free tgf-b1 was present in normal seminal plasma in high concentrations (346.4 ± 29.2 pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b1 concentrations were 36.2 ± 6.2 pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il-8 and free tgf-b1 are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi 1 , e. a. elgaaeid 1 1 faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th1 or th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod2/card15 protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod2/card15 gene in cd. we performed a cases /controls study upon 75 cd patients and 90 healthy controls. this study suggests that in northen tunisian population, 3020insc mutation in nod2/card15 gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il-10 polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il-10 cytokine genetic profile in patients with ibd. we examined the contribution of il-10 gene promoter polymorphisms (-627 and -1117) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card15/nod2 gene mutations in cd presentation and location. in tunisian population, the 3020insc insertion in nod2/card15 gene is a marker of susceptibility to cd, while the a allele at position -627 in the il-10 promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il-10 gene polymorphisms and card15/nod2 gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il-2-dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il-2-driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il-2 molecule by ganglioside. but gangliosides apparently can also form complexes with il-2r; such complexes influence on the signal transduction through il-2r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il-2r subunits. in our work we use il-2-dependent cytotoxic t-cell murine line ctll-2. two different approaches for study of possible interaction types between exogenous gangliosides and il-2r subunits were applied: antibody staining of il-2r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with 125 i-dcp-gm1 followed by immunoprecipitation of il-2r subunits. the fluorescence intensity of the antibody-labeled il-2r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by 29.5 % after cells incubation with ganglioside gm1, and by 10.7 % after incubation with gm2. labeling of the cells with antibodies to the il-2r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm1 (2.8 %) or with gm2 (5.9 %). to determine the mode of this impressive masking influence of ganglioside gm1, photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm1 with subunits of il-2r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il-2r, but not for il-2r a-subunit. these results demonstrate that exogenous ganglioside gm1 can interact with a-and bsubunits of il-2r in different modes. interaction of il-2r b-subunit with ganglioside gm1 requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa 1 , j. bukur 1 , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak2 and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak2. results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak2 expression due to a gene deletion on chromosome 9, whereas the expression and functionality of other ifn-g signal transduction components like stat1 and jak1 were not affected. jak2 blockade by two jak2-specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak2 leading to a lack of jak2 expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak2 inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of 506 controls of the spanish population. methods: two il18 polymorphisms located on the il-18 promoter region, snps -607 a/c (rs1946518) and -137 g/c (rs187238) were genotyped using taqman snp genotyping assays. the functional il-18 gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il-18 -607 p=0,318. il18 -137 p=0,740) but may contribute to disease onset and aggressiveness: the genotype il-18 -607 cc genotype, which is associated with higher il-18 production, was significantly associated with higher tumor size (p=0,001), grade (p=0,030), t (p=0,001), m (p=0,012) and stage (p=0,002). the influence of the il-18 -103 gg genotype was less relevant, however was correlated with higher tumor size (p=0,036), grade (p=0,017), t (p=0,026) and stage (p=0,011). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il-18 polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il-18 gene (il-18 -607 and -137) may be associated with an worse prognosis of rcc. high levels of il-18 production can play an important role in grow, invasion and metastasis of renal cancer. interleukin-18 (il-18) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il-18 is its ability to induce the production of ifn-gamma in presence of il-12. moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l0. then it seems that il-18 has a crucial role in immunity against brucella infection. since the expression of il-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il-18 gene polymorph isms and brucellosis. methods: a total of 188 patients with brucellosis and 77 healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il-18 polymorphisms at positions -656, -607, 137, +113, +127 and codon 35/3 using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il-18 polymorphisms at position -137 g/+113t/+127c (correlated with high production of il-18), codon 35/3c and -656g/-607c (correlated with higher production of il-18) were significantly higher in the healthy controls than the patients (p=0.000022, p=0.00185 and p=0.0441, respectively). discussion: as data revealed genotypes that have correlation with higher production of il-18 are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il-18 at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor 2 (ifngr2) mouse mutant. we have generated a mouse line carrying a conditional ifngr2 allele using the 2 loxp 2-flp recognition target (frt) approach. a targeting vector with 2 loxp sites flanking exons 4-6 and 2 frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat1 following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat1 activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd4 cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr2 on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez 1 , r. carretero 2 , p. saenz-lopez 2 , j. cantón 2 , j. carretero 2 , f. ruiz-cabello 2 , l.m. torres 1 , cts-143 1 hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, 2 hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and 130 normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca)12 allele of ifgn is significant more frequent in healthy control than in cin patient (p=0,030) in contrast homozygosis for (ca)12 allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p=0,030 and p=0,045 respectively). conclusions: our study suggest that ifng (ca)12 allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin-15 (il-15), a th1-related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il-15 single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: 190 patients with brucellosis and 78 healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il-15 (c267t, g367a, c13687a and a14035t) polymorph isms using pcr-rflp. results: at position 267, the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p=0.025 and p=0.042, respectively). at position 13687, the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p=0.050, p=0.015). discussion: as shown the frequency of cc genotype and c allele at position 267 were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position 13687 is lower in patients than the healthy controls and the frequency of c allele at position 13687 is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf-7 and skbr-3, as well as their ability to respond to several cytokines and to the g-1 gpr30 agonist. the mcf-7 and skbr-3 human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il-2ra, il-2rg, il-3/il-5/gm-csfr, il-4/il-13r, il-5ra, il-6ra, il-6r (gp130), il-7ra, il-10r, tnfr i, tnfr ii, ifngra, cxcr1, cxcr2, cxcr3, cxcr4, cxcr5. cells were incubated with il-10, ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr30 agonist. cytosolic ca 2+ concentrations [ca 2+ ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr-3 cells were found positive for a larger number of receptors than the mcf-7 cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf-7 cells with il-10 reduced the calcium response to g-1, while pretreatment with ifn-g and tnf-a potentiated the calcium response to g-1. tnf-b had no effect on mcf-7 g-1 stimulation. no direct effect on basal [ca 2+ ] i stimulation could be noticed when administering the cytokines alone. in skbr-3 cells, pretreatment with il-10 or tnf-b had no effect on basal [ca 2+ ] i and did not significantly alter the calcium response to g-1, while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g-1. pretreatment with tnf-a produced calcium oscillations and reduced the response to g-1. conclusions: mcf-7 and skbr-3 cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr30 stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd8a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd8a -pdcs and cdcs were infected with mcmv, whereas after 24h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd8 t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag1-deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag1 and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd8 t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg 1668, respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd8a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt3-l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f4/80 + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd8 t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr7 and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd11c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd8 t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd4 t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd4 t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd8 t cells in response to an adova infection due to an impaired cd4 t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than 500 passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il-12. to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp33 and vacv-gp33 expressing the gp33 epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p14 t cell receptor recognizing the gp33 epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p14ifnar -/mice. upon adoptive co-transfer of p14ifnar -/and p14ifnar wt/wt t cells and subsequent challenge with mva-gp33, a massive expansion of p14ifnar wt/wt t cells was observed, whereas p14ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp33 induced a rather similar expansion of ifnar competent and ifnar deficient p14 t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h5n1-specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h5n1 induced an exceptionally nf-kb dependent antiviral response. irf3 is essential part of this interferon-response of human endothelia. furthermore, we identified hmga1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h5n1. finally, nfatc4 was found to be a transcriptional regulator for specifically h5n1-induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h5n1 which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns1, delta-ns1 influenza virus (a recombinant influenza virus lacking the ns1 gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns1 during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting 2 days post-infection, reached its maximum at day 4, and triggered t cell priming in vivo. a direct comparison of delta-ns1 virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns1 protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns1 protein. thus, we propose that the virally encoded ns1 protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin 18 is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin 18 is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that 14-3-3 proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of 14-3-3 target proteins. thus, these results suggest that 14-3-3 proteins have a regulatory role in host defence against viral infections. i. wessels 1 , d. fleischer 1 , l. rink 1 , p. uciechowski 1 1 institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)-1b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il-1b expression is not elucidated, yet. it is known that the il-1b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il-1b when stimulated. b-cells which are il-1b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il-1b promoter and the impact of methylation on il-1b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl-60 cells were differentiated into monocytic cells after dihydroxyvitamine d 3 treatment. the monocytic phenotype was confirmed by flow cytometry. the il-1b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with 5-aza-2-deoxycytodine (aza) and changes in il-1b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl-60 cells, differentiated cells displayed upregulation of cd14 antigen and acquired the ability to express il-1b. by comparing the accessibilities of il-1b promoter we detected that the il-1b promoter was not accessible in undifferentiated hl-60 cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl-60 cells, demonstrating that the chromatin remodeling of the il-1b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il-1b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il-1b expression were found. our data indicate that the il-1b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il-1b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg 1 , g. wetzel 1 , h. arnold 1 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p65 family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il-1b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp2, 3, 4, 5 and 6 mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd88 was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. (2)). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd8 + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. 2008 aug 1;181 (3)). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. 2008 dec; 82 (24) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il-1b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory 4t1 breast cancer cells over expressing il-1b (4t1/il-1b) tumor cells, but rarely in untransfected 4t1 cells. secretion of il-1b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg2d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il-4ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used 130 healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array 5.0 having p x 10 -4 in two independent cohorts each consisting of 200 blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for 24h and we measured the levels of il-6, il-8, il-10, tnf-alfa and il1-ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed 150 snps with p x 1,0*10 -4 . to identify/replicate the association of cytokine production for these 150 we reanalysed these on a 200 cohort. a combined analysis revealed 10 snps with p x 9,1*10 -5 .these results are not genome wide significant. the 10 snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find 10 nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp60 have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp60 on adipocytes. for the first time we could show a hsp60-stimulated release of the proinflammatory cytokines il-6, cxcl1 and mcp-1 in a time-and concentration-dependent manner from murine 3t3-l1 adipocytes. analyses of hsp60-signalling in these adipocytes revealed that members of the mapk-family (erk1/2, p38) and the transcription factor nfkb are involved in hsp60-mediated induction of the mediators il-6, cxcl1 and mcp-1. binding-studies with fluorescence-labelled hsp60 demonstrated that the interaction of hsp60 with adipocytes exhibits basic features of a receptor-mediated binding. hsp60-binding to adipocytes was saturable and reached its maximum at 3.5 mm. binding was inhibitable only by the unlabelled ligand (52 %), but not by unrelated proteins, thereby proving the specificity of hsp60-binding. further analyses to characterize hsp60-receptor structures on adipocytes revealed the presence of toll-like receptor (tlr)4 on adipocytes. tlr4 has been found to be expressed on macrophages and to interact with hsp60, therefore suggesting tlr4 as a potential receptor candidate for hsp60 on adipocytes. in order to identify the responsible binding-epitope of hsp60 we investigated the effect of specific antibodies directed against different epitopes of the hsp60-molecule. incubation with antibodies directed against the n-terminus of hsp60 (aa1-200; 5-25 mg/ml) were capable of inhibiting the hsp60-binding to adipocytes (47-80 %) indicating that the n-terminal region of hsp60 is involved in receptor binding. our experiments demonstrate that hsp60 stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp60-mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf3. we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf3 knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with 3 % of fetal calf serum at 37°c and 5 % co2. bone marrow nonadherent cells were removed after 24 h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd10, cd29, cd73, cd21 and stro-1 and were negative for cd45. intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost 100 % of msc. interleukin-2, ifn-gamma and tnf (th1 cytokines) increased msc contractility, whereas il-10 (a th2 cytokine) decreased msc contractility. by immunofluorescence, we observed that il-2, ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il-10 decreased that incorporation. our results suggest that th1 and th2 cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca 2+ -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as 40 nm and 400 nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents (10 mm) at the 24-h culture interval reached the values of approximately 3 ng/ml and 2 ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of 2-6 h in rat pecs. the 24-h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p38 and erk1/2. it was not suppressed by the calcium chelating agents bapta-am and tmb-8. the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr 305/07/0061. pin1 is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin1 substrates and targeting sites. recent data show that pin1 interacts with apo-bec3g (a3g). the pin1/a3g interaction results in a reduced a3g expression and a diminished a3g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin1 gene (-842 g/c and -667 t/c) modulate pin1 expression; in particular, the -842 gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, 28; 69-74, 2007) . the -842 c/g and -667 t/c polymorphisms in the promoter of pin1 gene as well as pin1 protein levels were analyzed in 30 exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; 40 hiv-infected patients (hiv) and 40 healthy controls (hc). the genotype and allele distributions of the -842 snp was skewed in esn (genotype: p= 0.008; allele: p= 0.013). in particular esn showed a significantly lower frequency of the -842 gg genotype compared to hiv and hc (p=0.017 and p=0.019, respectively) and consequently a lower g allele frequency (p=0.026 and p=0.028, respectively). no significant differences were found for the -667 snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, 9-(r)[2-(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, 9-[2-(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. secretion of cytokines was determined after the 5-h culture by elisa. production of no was assayed at the interval of 24 h using griess reagent. approximately 300 compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. 9-[2-(phosphonomethoxy)ethlyl]-2,6-diaminopurine derivatives, c) 9-[2-hydroxy-3-(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) 9-heteroalkyl substituted 2-amino-6-guanidinopurines, and f) 2-amino-3-(purin-9-yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately 30 compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip-1a and cytokines tnf-a and il-10. although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n 6 -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as 2 to 5 mm. the remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant 1m0508. host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and 10 million units of ifn-a treatment three times a week for 6 months was initiated. before and after treatment: percentages of the il-2 and ifn-g in cd4+ t cells were assessed to determine intracellular t helper cell 1 (th1) type cytokine expression. similarly, percentages of intracellular il-2 and ifn-g were detected to verify cytotoxic t cell 1 (tc1) type cytokine expression in cd8+ t cells. percentage of th2 and tc2 type cytokine expression, (il-4 and il-13) were determined in cd4+ and cd8+ t cells, respectively. six (50 %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th2 cells with respect to healthy controls before treatment. tc percentages, both tc1 and tc2, were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il-4 expression was higher and the percentages of th1 type cells were significantly low. il-13 expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency (29,8 %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects (6,6 % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt 1 , f. juengerkes 1 , b. schumak 1 , g. gielen 1 , j. kalff 2 , p. knolle 1 , b. holzmann 3 , a. limmer 1 1 university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, 2 university hospital bonn, department of surgery, bonn, germany, 3 department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il-12, the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh 1 1 rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il-17 and il-23 in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, 45 h. pylori-infected du patients (23 patients were positive for anti-caga antibody and 22 patients were negative for anti-caga antibody), 30 h. pylori-infected as carriers (15 subjects were positive for anti-caga antibody and 15 subjects were negative for anti-caga antibody) and 15 healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il-17 and il-23 was measured by elisa method. the mean serum levels of il-17 in total du patients (9.28 pg/ml ± 5.48) was significantly higher than those observed in total as subjects (5.19 pg/ml ± 3.75, p x 0.001) and healthy control group (3.55 pg/ml ± 3.76, p x 0.0001). in du group, it was found that the mean serum levels of il-17 in subjects with positive test for anti-caga (10.84 pg/ml ± 5.79) was significantly higher than those observed in subjects with negative test for anti-caga (7.65 pg/ml ± 4.74; p x 0.05). the mean serum levels of il-23 in du (8.66 pg/ml ± 8.41) and as groups (7.25 pg/ml ± 5.66) was significantly higher than those found in uninfected control group (3.64 pg/ml ± 3.36, p x 0.02 and p x 0.03, respectively). however, no significant difference was observed for mean serum levels of il-23 between du and as groups. moreover, in both du and as groups the mean serum levels of il-23 was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il-17 and il-23 in h. pylori-infected subjects as compared with control group. in du group the expression of il-17 influenced by the bacterial caga factor. a. aral 1,2 , a. atak 1 1 gazi university faculty of medicine, department of immunology, ankara, turkey, 2 gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il-6 levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il-6 elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il-6 levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the 48th hour and reached to maximum levels at the 1st week and decreased again at the 3rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the 1st week and started to decrease at the 3rd week. il-6 reached to its peak at the 3rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic 1 , m. jurisic 2 1 university of kragujevac, school of medicine, kragujevac, serbia, 2 university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in 43 radicular inflamed cysts and 15 odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd3, cd20 and cd68. tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x 0.05) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. 1) drawings blood from 20 patients and 20 healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. 2) detection of the gene expression of prolactin and tlr-2 in cd14+ peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p 0.05) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr-2 and peripheral prolactin expression in cd14+ monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u937 culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table-2 ). when tb household contacts and healthy controls were compared, cfp10 and esat6 seemed to be more useful than tst in tb contacts for displaying ltb (table-2) . although cfp10 spot numbers were much more than esat6 spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: 0,069)( table-3 ). both esat6 and cfp10 spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g 20 analytes immediately prior to liver transplantation and at sequential time points up to 90 days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip-10, il-6 and il-10. notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type 1 ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/1/68 (h3n2) (hk) differs from its putative avian precursor by 7 amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk-5aa-i62r, n81d, k92n, s193n, g144a, human (2-6); rhk-r2-l226q, s228g and rhk-7aa-i62r, n81d, k92n, s193n, g144a, l226q, s228g, avian (2-3)). among these variants, the double mutant rhk-r2 and the seven mutant (rhk-7aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l226q and s228g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about 50 pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip-10 and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r2 and rhk-7aa as compared to rhk and rhk-5aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il-8, shedded adhesion molecules (cd25, vcam-1, icam-1), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk-5aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of 98 newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il-10, and neutrophilic cd64 levels, procalcitonin and il-10 were measured by elisa technique while, neutrophilic cd64 by single colour flowcytometric technique. of these 49 "infected" infants, 16 had positive blood culture (subgroup ia: culture-positive sepsis), and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another 49 newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il-10, cd64, and crp. il-10 had the highest sensitivity and specificity, 92 % and 84 %, respectively, using cutoff n 17.3 pg/ml. for pct, the highest sensitivity and specificity, 65 % and 60 %, respectively, were at a cutoff value of n 36.4 pg/ml. neutrophilic cd64 had maximal sensitivity and specificity of 92 % and 71 %, respectively, at cutoff value of 2.6 %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il-10 and neutrophilic cd64, which together provided sensitivity and specificity of 95 % and 83 %, respectively, and npv 86 %. the combination of il-10 and crp had high sensitivity and moderate specificity, 93 % and 79 %, respectively. conclusions: il-10 and neutrophilic cd64 levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp3, naip5 or ipaf and the adaptor asc are involved in caspase-1 activation in response to bacterial infection, triggering the processing and secretion of il-1b and il-18. recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi3k, vps34 and can be inhibited with the pi3k inhibitors wortmannin and 3 methyladenine (3ma). in contrast, activation of akt, via class i pi3k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il-1b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with 3ma, wortmannin or an akt inhibitor. supernatants were analysed for il-1b by elisa. results: 3ma enhanced il-1b secretion by bmdc treated with the tlr3 and tlr4 ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il-1b secretion was greatly reduced in bmdc from nalp3 -/mice compared to wild type c57/bl6 controls. treatment with the akt inhibitor had no effect on lps-induced il-1b secretion by bmdc. tlr-dependent secretion of il-1a was also enhanced by treatment with 3ma. conclusions: these data demonstrate that il-1b secretion by bmdc in response to treatment with pi3k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp3 inflammasome. this response is limited to tlr3 and tlr4 agonists. inhibition of akt had no effect on lps-induced il-1b production, suggesting that the effect of wortmannin and 3ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi3k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr2 signaling by the inflammatory lipid mediator sphingosine 1-phosphate (s1p) through receptors 1 and 2 in human monocytes-macrophages, which could explain some of the s1p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s1p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s1p, and later analyzed by flow cytometry. a pharmacological analysis of the s1p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam-1 expression was increased following lps and s1p concomitant stimulation in both venous and arterial cells, suggesting that tlr4 and s1p receptors cooperate in the expression of icam-1. conversely, no cooperation was observed when tlr2 ligands were used. in order to elucidate which s1p receptor subtype was involved in the increase of icam-1 expression, we used a pharmacological approach with s1p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam-1 after lps and s1p challenge was significantly reduced by blocking s1p receptor 3 and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s1p receptors 2 and 3, which suggest that s1p receptor 1 might be involved in the effect. conclusions: altogether these data demonstrate that tlr4 and s1p receptors can interact to increase adhesion molecules such as icam-1 in human endothelial cells, and the s1p receptor subtype involved in the effect differs between arterial and venous cells. 15 with ssc without pah) and a pool of 12 sera of healthy controls (hc) were tested. results: in 1 dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with 7-10, 4-8 and 2-5 protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in 2 dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized 145±48, 127±26, 130±25 and 150 protein spots respectively. twenty one protein spots were recognized by more than 80 % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein 1 and peroxyredoxin 6 and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: 27 patients suffering from different diseases were enrolled in our study. 16 patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas 11 of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam-1 and vcam-1 were assesed by an enzyme immunoassay (elisa). results: 11 out of the 27 patients (40,7 %) had elevated levels of the intercellular adhesion molecule. 6 out of the 16 patients suffering from bone diseases (37,5%) had raised values (mean value 400 ng/ml) whereas 5 patients out of the 11 suffering from soft tissue diseases and diabetes (45,5 %) had raised values (mean value 735,5 ng/ml). reference value for icam-1 was 130-299 ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients (40 %) having increasd levels of icam-1. high icam-1 levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes (45,5 %) than in patients with bone diseases (37.5 %). mean values were found 735,5 ng/ml and 400 ng/ml accordingly. those findings verify the positive correlation between icam-1 and inflammatory diseases and tissue damage but not for vcam-1. colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: 16 cases of ulcerative colitis (urc), 16 adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, 33 tubular or tubulo-villous adenomas with low grade dysplasia, and 33 infiltrating adenocarcinomas. immunohistoobjectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd18 in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind 20 patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). 40 mg of pravastatina oral 2 hours they were administered before the procedure (group study, n=10) or placebo (group placebo, n=10), and control (group control, n=10). samples of outlying veined blood were extracted to the 24 hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd18 in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: 3 types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in 3 degrees: degree 0. without expression. degree 1. weak; degree 2. moderate; degree 3. intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree 0-1. the placebo group: mixed pattern, degree 1-2. group study (40 mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree 2-3. the percentage of cells that expressed cd 18 was greater in the group study (40 mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd18 answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam10 and adam17. we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam17 as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam17. these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam17 activity in the tissue. in the presence of the adam17 inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam17 as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam17-mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf-460/ vegf+405 is associated with rcc risk ( p= 0,017), metastases ( p=0,043), nuclear grade ( p=0,05), tumor stage ( p=0,029), and tumor size (p=0,04). on the other hand, the polymorphism vegf -2578 a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol 3,4,5-trisphosphate. therefore, pten is one of the main antagonists of the pi3-kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il-6 as well as il-8 levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il-6 as well as il-12 and il-23 mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th-17 t cells, we measured th-17 cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il-17 and a strong reduction of il-22 mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il-10 exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il-1ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il-10. based on our previous observation that support a direct role of il-10-activated stat3 in the enhancement of il-1ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il-1ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il-10. quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il-1ra promoter. crosslinked nuclear lysates were immunoprecipitated 30 and 60 min after il-10 addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il-1ra promoter. chip assays showed that the pol ii recruitment to the il-1ra promoter induced by lps is significantly increased by il-10, further strengthening the concept that the rapid enhancement of lpsinduced il-1ra gene expression by il-10 initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat3 immunoprecipitated dna showed statistically significant levels of stat3 binding to the il-1ra promoter only in cells stimulated with lps in the presence of il-10. surprisingly, anti-p65 and anti-p50 chip assays revealed enrichment of both p65 and p50 recruitment to the il-1ra promoter when il-10 was added to lps-stimulated cells, suggesting that il-10 enhances the recruitment of nf-kb to the il-1ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il-10-treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il-1ra promoter site is dependent on il-10-activated stat3, since it is greatly reduced when stat3 activation by il-10 is impaired. the molecular mechanism through which il-10-activated stat3 promotes the recruitment of nf-kb to the il-1ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il-15 acts as a specific negative transcriptional regulator of mouse mast cell protease-2 (mmcp-2). we examined the mechanisms underlying the repression of mmcp-2 gene expression. our data show that the "repressor" effects of il-15 on mmcp-2 promoter activity are still operating on the mmcp-2 591 bp long minimal promoter. moreover, il-15 deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy1. furthermore, chromatin immunoprecipitation revealed that il-15 promoted specific reciprocal recruitment of c/ebpb but not yy1 to the mmcp-2 promoter. finally, il-15 deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp-2. thus, we proposed that the expression of mmcp-2 and possibly other immunoregulatory genes may be regulated by il-15 through epigenetic modification and by balancing the content and binding of c/ ebpb and yy1 in mast cells. i. nagy 1 , k. filkor 1 , a. vörös 1 , l. kemény 2 , a. szász 1 1 bzaka, baygen, szeged, hungary, 2 university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir-203, mir-146a and mir-155, which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir-203 expression; in contrast, pgn re-stimulation had no further effect on mir-146a and mir-155 expression. next, we investigated the correlation between the expression of mir-203 and its two known direct targets: regulatory protein p63 and suppressor of cytokine signalling-3 (socs-3). although the gene-expression profile of neither p63 nor socs-3 changed, we found that the expression of mir-203 reversibly correlates with both p63 and socs-3 protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir-203 prior to pgn-treatment completely abolished both p63 and socs-3 down-regulation, revealing the involvement of mir-203 in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir-203 expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb1+/-had a lower incidence and burden of benign papillomas when compared to tgfb1+/+ animals. however, more scc developed in the tgfb1+/-mice. after acute and chronic promotion, tgfb1+/-skin showed a reduced proliferative response with no increase in epidermal tgfb1 or nuclear p-smad2 compared to tgfb1+/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb1 gene dosage. further, pharmacological inhibition of alk5 suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb1 are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb1+/-skin, tgfb1+/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb1+/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb1+/+ but not tgfb1+/-keratinocytes, indicating that tgfb1 switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb1 +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb1 acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of 132 patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp-1) and fibroblast (mrc-5) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of 132 ra patients and 82 controls the levels of survivin correlated to urokinase (upa) (r=0.46), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that 30/132 ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc-5 and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol 1,4,5-trisphosphate 3-kinase type b (itpkb) phosphorylates inositol (1, 4, 5) trisphosphate (ins(1,4,5)p 3 ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca 2+ responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h1 and h2 receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il-1b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il-1b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase-1 to generate the mature secreted active form. caspase-1 is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of 425 genes involved in splicing with an average 5-fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified 30 genes that significantly affect the production of il-1b by thp-1 cells after a 24h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il-1b secretion. tissue transglutaminase (tg2) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg2 in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg2 expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg2 can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg2 not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg2 inhibitor, in a transgenic mouse model cf and in the taz10 transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz10 tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg2 in generating inflammation in two very different pathologies. this work underlines the critical role of tg2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge 2 stimulated a-dc. preliminary results indicate no accumulation of hif-1alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif-1alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p38 but not erk1/2 or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt5a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il-12 and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt5a -but not wnt3a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt5a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt3a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp130 on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp130 in te. after infection with t. gondii, mice lacking neuronal gp130 (synapsin-cre gp130 fl/fl ) died significantly earlier in the chronic phase of infection than control gp130 fl/fl mice. death of synapsin-cre cre gp130 fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp130 fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp130-deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp130 expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp130 fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il-1b, il-6 and il-8 have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il-4, il-10 and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb1 further may generate more effective therapies. as tnfa mrna 3' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb1 suppresses tnfa protein production by upregulating the rnabinding protein fxr1, which can bind to tnfa mrna and inhibit translation. methods: using raw 264.7 cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb1, we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 3'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb1 and luciferase expression was quantified. cells treated with lps and tgfb1 were also examined for fxr1 expression using pcr and western blot. following fxr1 knockdown using sirna, the influence of tgfb1 and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb1. using the luciferase-tnf-3'utr vector we show that tgfb1 targets the 3'utr of tnfa. furthermore, tgfb1 and il-10 both upregulate fxr1 mrna and protein; and treatment with tgfb1 and lps can synergistically upregulate mrna expression, more than tgfb1 alone. following sirna inhibition of fxr1, tgfb1 can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp-1 and mmp-3 secretion from saecs, nhbes and fibroblasts to a peak of 2.5 +/-0.5 ng/ml, at 72 hours. interleukin-17 augmented comtb-stimulated up-regulation of mmp-1 and mmp-3 secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin-17 down-regulated mmp-9 secretion from saecs by 50 %. interleukin-22 up-regulated mmp-1 and mmp-3 secretion from fibroblasts but not from saecs. timp1 secretion from saecs was enhanced by interleukin-17 but there was no effect of interleukin-22. mmp up-regulation by interleukin-17 and comtb was inhibited by the pi3kinase inhibitor ly294002 and on western analysis akt (protein kinase b) was phosphorylated at 30 minutes. chemical inhibition of the p110d isoform of pi3kinase with ic87114 abrogated the il-17 and comtb driven secretion of mmp-3 from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome 10) accentuated mmp-3 secretion. these inhibitory effects were confirmed with sirna. mmp-3 up-regulation was secondary to increased gene expression with promoter activity peaking 24h after stimulation. in summary, interleukin-17 and interleukin-22 drive transcription dependent mmp-1 and mmp-3 secretion from airway epithelial cells and fibroblasts. interleukin-17 also increases timp but down-regulates mmp-9 gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi3kinase pathway is central in interleukin-17 driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto 1 , l.s. moreira 2 , e. gonzalez-rey 2 , m. delgado 1 1 institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, 2 university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within 15 years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper 1 response. cholesterol metabolism is regulated by factors such as pparg1 (proliferator activated receptor g), srb1 (a class b scavenger receptor), cd36 or abca1, that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th1 immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg1, srb1, cd36, and abca1. we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to 60 % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il-6 in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il-6 and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il-6, tnf-a, il-1b, il-8 and il-12, and the anti-inflammatory cytokine il-10, in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il-23 was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il-6 and il-10, and in some cases, of il-23. the maximal plasma levels of il-6 and il-10 were found at 24 hours and of il-23 between 48-72 hours. modest plasma levels of il-8 were also observed, with maximal production at various time points (4-24 hours). by contrast, production of tnf-a, il-1b and il-12 did not occur to a significant extent, while production of il-17 occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il-6, il-8, il-23 and il-10 also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il-6, il-10, il-8 and il-23, i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving 165 patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il-6, activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il-33 is a novel il-1 cytokine family member that is expressed as an intracellular precursor (pro-il-33) and is thought to be cleaved by caspase-1 to yield a mature bioactive form of the molecule (mat-il-33). to date however, evidence of cell-associated proteolytic processing and caspase-1 dependent secretion of mat-il-33 has not been reported. here we show that pro-il-33 but not mat-il-33 is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il-33 to the il-33r and also il-33r-dependent bioactivity of pro-il-33 on mast cells. we propose a previously unrecognized role for pro-il-33 as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il-23 p19 mrna expression in myeloid cells, and markedly increase secretion of il-23, but not il-12, by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il-23 promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop10. chromatin immunoprecipitation (chip) assays using anti-chop10 and isotype control mab were performed using nuclear lysates from u937 cells and il-23 promoter dna measured by qpcr. chop10 binding on the il-23 promoter was detected following stimulation of u937 cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il-23 promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop10 in il-23 gene transcription, u937 cells expressing shrna's specific for chop10 or non-specific gene target were tested for their ability to express il-23 following tlr and er stress stimulation. u937 expressing three independent shrna targets for chop10 exhibited significant reductions in il-23p19 mrna (up to 87 % reduction of the response to lps+tp) compared to u937 expressing a control shrna. chop10 shrna expression did not affect the expression of other lpsresponsive genes, including il-1, il-8, ccl3 and sod2. to identify if er stress induction of il-23 mediated by chop10 expression plays a role in a more physiological setting, we examined the role of chop10 in the induction of il-23p19 gene expression following chlamydia trachomatis (ct) infection. infection of u937 cells with live but not g-irradiated ct induced expression of er stress response genes, including chop10. u937 infected with live ct exhibited increased il-23p19 mrna expression compared to u937 infected with nonviable bacteria. chop10 silencing significantly reduced the ability of live ct to induce il-23p19mrna, confirming the important role of chop10 in this response. these data suggest that er stress induction of chop10 could contribute significantly to the pathogenesis of diseases in which il-23 plays an major role, through induction of il-17 and il-22 producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd45b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd45b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd45b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd45b maintained a sustained activation of p38 kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd45b-deficient mice showed only transient p38 activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd45b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd5 and cd69 were elevated on gadd45b-deficient thymocytes. thus, we provide evidence that gadd45b and a resulting persistent activation of p38 constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo 1 1 institut pasteur, paris, france, 2 monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il-7 is recognized as an essential factor for thymopoiesis, the nature of the thymic il-7 niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il-7 promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il7 transcripts (il-7 hi cells). il-7 hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl19, ccl25, cxcl12) and cytokines (il15) that are critical for normal thymopoiesis. in the adult thymus, il-7 hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il-7 levels. conversely, the frequency of il-7 hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il-7-expression by tecs. > together, our temporal-spatial analysis of il-7-expressing cells in the thymus suggests that thymic il-7 levels are dynamically regulated under distinct physiological conditions. this novel il-7 reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il-7 expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l5 and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl2-tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl2 and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl2-tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl2-tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg2 plasma cells and serum igg2 levels were˚5-10 fold increased. importantly, serum from young slc-tg;em-bcl2-tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in 2/3 and 6/6 cases, respectively. these values were increased when compared with control groups: 1/6 in fcgriib -/and 0/6 in bcl2-tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd27 + cd135 + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd117 hi cells, representing multipotent progenitors, or cd127 + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb2 and ephb3 expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b1 and/or ephrin b2, the ligands of ephb2 and ephb3 receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb1 or ephrinb2 genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb1/b2 double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd45+ cells in the cortex, increased proportion of k5+k8+ cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb1 and ephrinb2 in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink 1 , d. vanhecke 1 1 university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op9-dl1 cell culture system (1) . using this in vitro assay we obtain large numbers of human cycd3 + and cd4 + cd8 + double positive thymocytes starting from umbilical cord blood (ucb) derived cd34 + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il-7 and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd34 + cells upon notch signaling is cd7 followed by cd127. t cell specification is accompanied by the induction of cd1a and loss of cd34 on cd7 + cd127 + cells. these cd34 -cd1a + cd7 + cells become dependent on continuous il7 and notch signaling for sustained survival and further differentiation into cd4 + cd8ab + dp thymocytes. we found that flt3l is not essential for the differentiation of cd4 + cd8 + human thymocytes but that addition of exogenous flt3l in the co-cultures increases the number of cd34 + precursors and consequently result in higher yields of developing cd4 + cd8ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd3 + cd4 + cd8ab + dp subset in this in vitro assay suggesting that op9 stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn3 to dn4 stage, where bdnf and its receptor p75 are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga 1 , c. lópez-rodríguez 1 1 pompeu fabra university, barcelona, spain nfat5 is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat5 are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat5 participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat5 deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd8 + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat5-null mice are unable to mount cd4+-and cd8 + -immune responses. data from our laboratory indicate that nfat5-null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat5 during the development of t lymphocytes, we developed mouse models that delete nfat5 at early (lck-cre + /nfat5 flox/flox ) or late (cd4-cre + /nfat5 flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat5 is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat5 at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p53, is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues 58-88 of the proline-rich domain of p53. methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p53 null (p53-/-) and wild type (p53+/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p53-/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p53 has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p53 is not important for preventing thymic t-cell tumors. s. myrczek 1 , r. pardi 2 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, 2 vita-salute san raffaele university school of medicine, milano, italy jab1 is the catalytic subunit of the highly conserved cop9 signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab1 regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab1 regulates the activity of ap1 transciption factors. to date jab1 is thought to be essential for every cell type as jab1 knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab1. to investigate the function of jab1 in b cells we established a mouse strain deficient for jab1 selectively in b cells. mice with floxed alleles of jab1 kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb1-locus (m. reth, freiburg). ablation of jab1 expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b1 and b2 cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl2 under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab1-deficient, bcl2-transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab1 in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab1 when apoptosis is prevented. t. nitta 1 , s. murata 2 , k. tanaka 3 , y. takahama 1 1 university of tokushima, tokushima, japan, 2 university of tokyo, tokyo, japan, 3 rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd8 t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd8 t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd8 t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th1 and th2 antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd4+ t cells or functional deficits that selectively interfere with th1 or germinal centre responses. in this talk i will present data from some of the first 12 strains that have been identified including the first 5 strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn1 c-kit high (etp), dn2 and dn3 thymocyte populations was hybridised to affymetrix mouse 430a-2.0 genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors (85 out of 623 genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included 64 signal transducers (out of 590 genes) such as acvr1, bmpr1, fzd7, chemokine receptors cx3cr1, cxcr6 and integrins a2, a4, a9, ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata-3, tcf-1, notch-1, rag-1, rag-2 and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn2 stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn3 population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd8aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks 3 out of 4 of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e14 thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by 7-10 days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day 18 of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd34 + cd1ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged 1 month to 3 years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd34 + cd1precursors with recombinant wnt3a (100 ng/ml) or with licl (10 mm) for 12hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab (8e7) under conditions of phosphatase activity inhibition. wnt3a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il-15 and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st2 cells suplemented with il-7 and flt3l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt3a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt3a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il-7 and flt3l and modify the transcription factor profiles of cd34 + cd1thymocytes mainly increasing hes-1 and id3 expression levels. human th17 clones and circulating th17 cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th1 and th2 clones or circulating th1oriented t cells, respectively. accordingly, human th17 cells exhibited lower expression of clusterin, and higher bcl-2 expression and reduced apoptosis in the presence of tgf-beta, in comparison with th1 cells. umbilical cord blood naï ve cd161(+)cd4(+) t cells, which contain the precursors of human th17 cells, differentiated into il-17a-producing cells only in response to il-1beta plus il-23, even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd161(+) t cells but it increased the relative proportions of cd161(+) t cells differentiating into th17 cells in response to il-1beta plus il-23, whereas under the same conditions it inhibited both t-bet expression and th1 development. these data suggest that tgf-beta is not critical for the differentiation of human th17 cells, but indirectly favors their expansion because th17 cells are poorly susceptible to its suppressive effects. m. irla 1 , w. reith 1 1 university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd4(+) or cd8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd4(+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd40 on mtecs by cd40l induced on the positively selected cd4(+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd4(+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu 1 , i. ravens 1 , g. bernhardt 1 1 hannover medical school, institute of immunology, hannover, germany cd155 is originally identified as human poliovirus receptor (pvr) and as rodent tage4, which is overexpressed in rodent colon carcinoma. cd155 is also known as necl-5, a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd155 expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd155 is overexpressed in transformed cells and promotes the cell cycle. thus, cd155 seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd4 and cd8; the dn subset is further subdivided into four stages (dn1-4) by differential expression of cd44 and cd25. thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd4+ sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd8 lineage. here we show that the frequency of terminally matured cd8+ sp cells but not that of cd4+ sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd155, a selective deficiency of mature cd8+ sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd155 is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd8+ sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd8+ t cells. in cd155 deficient animals, a shift in the tcr repertoire displayed by the pool of cd8+ sp cells was found demonstrating that cd155 is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. 2005 , 202, 1599 . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht 33342, propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e9 (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd71 + ter119 + erythroid and cd45 + cd11b + myeloid cells are simultaneously cycling (s/g2/m) in the post-gastrulation mouse embryo (e10-12). the peak of lymphohematopoietic cell proliferation occurs at e13 in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence 24-48 hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e11-12 that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd40-cd154 interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd40, we hypothesised a role for cd40-cd154 interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd40 expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd154 message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd154-/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd154 on reconstitution of b cell numbers following depletion. we show that cd40 is expressed by pro-b cells, and these cells proliferate in response to cd40 signalling in vitro. pcr identified a source of cd154, negative for cd3eta, in the bm of wt mice showing this cd154 is not provided by activated re-circulating t cells. we have shown that when cd154 -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn1 (cd4 -/cd8 -/cd44 + /cd25 -) to the dp (cd4 + /cd8 + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn1 stage on. in the bone marrow we found yfp + /b220 + and yfp + /b220populations. thus these pta expression analyses show closely similar pattern to those observed with hucd25 preta-reporter transgenic mice (gounari f. et al. 2002 , martin et al. 2003 . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. 3, 489-496 (2002 ) martin c. h. et al., nat. immunol. 4, 866-873 (2003 . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz 1 , g. klein 1 1 zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm-332 which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x 70kda.mmp-19, a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm-332. in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp-19. by western blotting the zymogen and the activated form of mmp-19 can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm-332 and mmp-19 can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp-19 which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp-19, timp-2 and timp-3, are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp-19 plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of 15-20 % compared to only 5 % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd4/cd8 expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd4+cd8+ double positive (dp) tcrab low cells entering selection, and their cd4+cd8+ dp tcrab-immediate precedents followed by underrepresentation of the selected cd4+cd8+ dp tcrab high and the most mature cd4-cd8+ and, particularly, cd4+cd8-single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd4-cd8-double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd4+cd8-/cd4-cd8+ sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a 1 -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a 1 -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a 1 -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd4/ cd8/tcrab), in adult wistar rats subjected to 14-day-long treatment with a 1 -ar blocker urapidil (0.20mg/kg body weight/day s. c.). the a 1 -ar immunoreactivity was found in both thymocytes (mainly less mature cd3and cd3 low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed1-postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd4/ cd8/tcrab expression were observed. these changes comprised of an increase in the percentage of cd4+8+ tcrabthymocytes, which was accompanied by the reduction in that of cd4+8+ tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd4+8-tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd4-8+ tcrab high was reduced. in addition, the percentage of cd4+ t regulatory and cd161+tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a 1 -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a 1 -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around 4 weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c57bl/6 mice infected with m. avium (10 6 cfus, iv) were sacrificed at different time points after infection (5, 16 and 25 weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il-10) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at 16 weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at 24 weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il-10 in this organ. in the spleen, ifn-g reaches a peak of expression earlier (5 weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm29935 and ai067906 from nih and grant i-823 from the robert a. welch foundation to wtg, and by grants hl067256 and hl61897 from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. 2001) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c57bl/6 x balb/c)f1 mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f1 mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski 1 , s. lang 1 , m. stein 1 , t. winkler 1 1 friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h3ac) or methylation of h3 on lysine 4 (h3k4me2/3). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h3ac and h3k4me2/3) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar #3) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 5'rlm race at the ivar #3 element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo2 locus, is thought to be accessible only during the double-negative (dn) 1 and 2 thymocyte stages based on mrna expression, implying that translocations between lmo2 and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx1 (hox11), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed 1) to evaluate lmo2 and tlx1 breakpoint-site accessibility during thymocyte development; 2) to determine in which stage of development there is an increased chance for lmo2 or tlx1 translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo2 accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo2 and tlx1 loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn1 and dn2 development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn1, dn2, dn3 and immature single positive (isp) stages for both lmo2 and tlx1. conclusion: our findings show that both the lmo2 and tlx1 loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn1 and dn2 stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that 1 to 3 successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e10-12), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag2 -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under 6 months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd4 cell depletion. however, cd8 depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd8 t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd8 cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd8 t cells has important implications for vaccine development against neonatal infections. (2008) showed that irf8 knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf8 as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf8 in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf8 were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs17444416. conclusions: although recent findings indicated that irf8 function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf8 that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez 1 , t. boehm 1 1 max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn1 transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l1 expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd4 and cd8 t cells from tuberculosis patients highly express pd-1 when compared to healthy uninfected individuals. in addition, analysis of pd-1 expression in lung biopsies from tuberculosis patients revealed that pd-1 is expressed on cd4 and cd8 t cells confined to lung granulomatous lesions. finally, blocking of the pd-1/pd-l1 axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd-1/pd-l1 pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p100 to p52. among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt 1 aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly 1 wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th17 and th1 cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd4 + t cells, nfatc1 and c2 are predominantly expressed. nfatc1 is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc1/a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc1/c. as demonstrated by y2h screen and co-ips, nfatc1/c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc1/c -but not the unsumoylated nfatc1/a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc1 target gene interleukin-2. other lymphokines like ifng and il13 are reversely regulated. interestingly, ntreg cells which do not express il2 exerted only nfatc1/c, but no nfatc1/a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc1 function. therefore, especially ntreg cells and anergized cd4 + t cells might be regulated by the long sumoylatable isoform nfatc1/c. lnk/sh2b3 and aps/sh2b2, two members of the lnk/sh2b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b-1 cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il-7 signalling in pre-b cells overexpression of lnk dramatically inhibits il-7-dependent growth demostrating that lnk negatively regulates il-7 pathways. furthermore, we showed that il-7 stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e3 ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav1. to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd3 complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd3 complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd4 + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh2 or sh3 domains known to mediate protein-protein interactions are key players in these processes. sly2 (sh3 domain protein expressed in lymphocytes 2) was identified as a putative adaptor protein containing a sh3 and a sam domain as well as a bipartite nls. sly2 belongs to a family of three molecules: sly1, sly2 and sash1.in humans, the sly2 gene is located on chromosome 21, in mice on chromosome 16. sly2 is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly2 protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly2 we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin3-associated polypeptide p30 (sap30) as a putative interaction partner of sly2. sap30 is a conserved member of the sin3a-hdac corepressor complex that contains histone deacetylase 1 (hdac1) and histone deacetylase 2 (hdac2) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected 293t cells. in addition, we could show a direct interaction between sly2 and hdac1. to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly2 increases the activity of hdac1 in whole cell lysates and, more precisely, in nuclear extracts of 293t cells. the interaction of sly2 with sap30 and hdac1 indicates a transcriptional function of this protein. within the sin3a-hdac corepressor complex sly2 might act as a switch for the activity of hdac1. cd46-cyt1 and cyt2 are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd19 (b cell marker) fused to the transmembrane and intracellular domain of cd46-cyt1 or cyt2 in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt1 coengagement controlled il-10 secretion, while cyt2 coligation inhibited ifng production. moreover, our preliminary data suggest that cd46-cyt2 inhibits the phosphorylation of several molecules known to be activated by cd46 stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd46 cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh2 domain and three sh3 domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut78 cells with gst fusion proteins containing full length nck, the three sh3 domains or the individual sh3 and sh2 domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp76 and cd3epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam68) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip55 once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g4, results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g4) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g4) degranulation as measured by cd107a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi3k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose-6-phosphate receptor which exhibits structural and functional similarity to the vps10p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose-6-phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla-4 (sctla-4) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla-4 in immune responses. using a specific anti-human soluble ctla-4 monoclonal antibody, jmw-3b3 that selectively binds the soluble isoform but not membrane bound ctla-4, or cleaved fragments of it, we demonstrate that sctla-4 plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla-4, secreting increased amounts of cytokines including interferon-g, il-17 and tnf-a, but lower amounts of il-10. soluble ctla-4 was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla-4 induced secretion of the immunoregulatory cytokine il-10 by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine 2,3 dioxygenase enzyme cascade was also initiated by sctla-4. it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla-4 it is crucial to t cell inhibition. membrane-bound ctla-4 exists as a homo-dimer on t cells but sctla-4 is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b7 ligands on antigen presenting cells. a third important observation from this study is that sctla-4 exists both in serum and culture supernatants as a natural 46kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla-4, concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il-10 dependent regulation is most critical, boosting sctla-4 secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin-1/efhd-2, in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin-1/efhd2 is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin-1/efhd-2 in the immature murine b cell line wehi231 enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin-1/efhd-2 impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g1 cell cycle arrest. to understand how swiprosin-1/efhd2 enhances pro-apoptotic bcr signals, we analyzed whether swiprosin-1/efhd2 is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin-1/efhd2 enhanced bcr-induced calcium flux in wehi231 cells, whereas shrna-mediated down-regulation of swiprosin-1/ efhd2 impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin-1/efhd2 interacts with phospholipase cg2 (plcg2) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg2 and swiprosin-1/efhd2 was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin-1/efhd2 silenced wehi231 cells with swiprosin-1/efhd-2 was inhibited by the syk inhibitor bay 61-3606. in analogy, swiprosin-1/efhd2 regulated syk activity positively. moreover, swiprosin-1/efhd2 re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg2 and of syk tyrosine residue 352, which is involved in syk activation. finally, reconstitution of swiprosin-1/efhd2 knock-down cells with swiprosin-1/efhd2 mutants revealed that the n-terminal putative sh3-binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi231 cells. interestingly, swiprosin-1/efhd2 re-expression in swiprosin-1/efhd2-silenced cells induced already in unstimulated cells raft partitioning of syk, plcg2 and the bcr, which was reversed after 2 min of bcr stimulation. in summary, swiprosin-1/efhd2 is an accelerator of proximal bcr signalling and acts through syk and plcg2 by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e3 ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc1 tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd28 co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p14 mice die within hours after a second challenge with p33 peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e3 ligase dead mice can spontaneously reject tc1 tumors. conclusion: cbl-b e3 ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e3 ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e3 ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd8 t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd8 + cd45ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip10 and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal 1) and co-stimulatory signals (signal 2), provided by beads coated with anti-cd3/cd28 antibodies. gene expression patterns were compared for cells stimulated with anti-cd3/cd28 beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip10, itac). the enhanced expression of granzyme-b, ifn-g, trail and ip10 were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd3-coated p815 cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal-3 to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd3 + foxp3 + and cd4 + cd25 high t cells. moreover, t cell receptor activation with combined anti-cd3 and anti-cd28 stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk-506, but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv-1 infection leads to immune dysfunction owing to a successive loss of the cd4 + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv-1 virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd4 and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk1/2. this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk1/2 directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap-1, nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk1/2 is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal 1 , t. brdicka 1 , v. horejsi 1 1 institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd45 are key regulators of src-family kinases in leukocytes. while cd45 is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh2 domain of csk binds phosphotyrosine 317 of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd25 and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd69 upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd25-csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd8 t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd8 t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie-2kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd8 t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd8 t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd8 t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd8 t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa-1 integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git2 in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi 1 , s. parusso 1 , b. frossi 1 , g. gri 1 , c. pucillo 1 1 university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il-6 -/-mcs and anti-il-6 receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il-6 derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd40 on naï ve b cells and the interaction of cd40 on b cell surface and cd40l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd40l e cd40 on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves 1 , n. bercovici 1 , a. caignard 1 1 inserm u567, paris, france inhibitory killer ig-like receptors (kir2dl1-2/3) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd8 + t cell subsets. these receptors suppress cd8+ t cell activation through recruitment of the src homology 2 domain-containing protein tyrosine phosphatase 1 (shp-1). to further analyse the yet largely unclear role of inhibitory kir receptors on cd4+t cells, kir2dl1 transfectants were obtained from a cd4 + t cell line and primary cells. the transfection of cd4 + t cells with kir2dl1 dramatically increased the t cell receptor (tcr)-induced production of il-2 independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir2dl1-itim phosphorylation, shp-2 recruitment, zap-70 and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp-2 and p-pkc-v but not of shp-1. in contrast, the kir2dl1/hla-cw4 interaction led to a strong synaptic accumulation of kir2dl1 and the recruitment of shp-1/2, inhibiting tcr-induced il-2 production. kir2dl1 may induce two opposite signaling outputs in cd4 + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir2dl1 receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg1 is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg1 still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg1 antibody during these conditions. the observed igg1 switching behaviour mimics that of b cells responding to lps and il-4, but is mediated by a different, stat6-independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana 1 , c. schwindling 1 , m. pasche 2 , c. junker 1 , c. kummerow 1 , u. becherer 2 , e.c. schwarz 1 , j. rettig 2 , m. hoth 1 1 saarland university, biophysics, homburg, germany, 2 saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai1 channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than 150 nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd4 + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd4 + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of 21 days. under tolerogenic conditions (ova alone), cd4 + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, 4-1bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd4 + t cell expansion and contraction kinetics in the early phase of the t cell response (days 1-6). in the late phase of the primary response (days 7-21), under immunizing conditions, the large majority of transgenic cd4 + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il-2, ifn-g, and il-17a, and only few ova-specific foxp3 + regulatory t cells ( x 10 %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd4 + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells (30 %) was substantially increased. on day 14, both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells (50 %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd4 + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il-17a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd8 t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd8 tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin-2, interferon-g and macrophage inflammatory protein-1 by cd8 tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd8 tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd8 tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog 35-55 protein. our results provide direct evidence that mc contribute to cd8-specific priming in eae and show that the tc proliferation failure is specific for cd8 tc from mog 35-55 -immunized w/w sh mice. the role of mc-cd8 tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd8 tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd8 tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd8 tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd28 engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd28 costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd3complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla-4, but not via any conventional motifs in this region. overexpression of trim augments ctla-4 surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla-4 localisation, mainly restricted to the tgn. ctla-4 vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla-4 trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla-4 expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla-4 transport to the cell surface. it is imperative to reveal the mechanisms by which ctla-4 is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd1 molecules, nkt cells are activated and release cytokines, including ifn-g, il-2 and il-4. nkt cells are efficiently recruited to the liver via cxcr6-dependent chemotaxis toward cxcl16 and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd8 t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd8 t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd8 t cell tolerisation via interaction with lsec. to this end we analysed cd1d expression on lsec and their ability to activate nkt cells by presentation of the cd1d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd1d, as agalcerpresenting-lsec were capable to induce tnf-a, il-2, il-4 and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd54 and b7-h1 on lsec. as naï ve cd8 t cell tolerisation by lsec critically depends on b7-h1, we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb2 in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg9, jg1.2, and vd2 gene products. they recognize nonpeptide antigens like (e)-4hydroxy-3-methylbut-2-enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd-1. one of the inhibitory receptors, pd-1, is a member of cd28/ctla-4 family and contains a single ig v-like domain in its extracellular region. pd-1 can bind to two b7 homologue molecules, pd-l1 and pd-l2. it has been reported that interaction of pd-1 with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd-1 is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with 2-methyl-3-butenyl-1-pyrophosphate plus il-2 to obtain gd t cells. pd-l1+ and pd-l1-human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l1 mabs, the pd-l1 extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd-1 in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd-1/pd-l1 interaction. results: gd t cells expressed pd-1 upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l1. we first examined whether or not the engagement of pd-1 receptor could modulate the cytotoxic activity of gd t cells. pd-l1-expressing tumor cells tempered cytotoxic activity of pd-1+ gd t cells, and cytokine production such as tnf-a was down-regulated by pd-1 engagement. in addition, inclusion of anti-pd-l1 mab reversed cytotoxic activity and cytokine production when pd-l1-expressing tumor cells were challenged by pd-1-expressing gd t cells. conclusion: pd-1 delivers inhibitory signals in gd t cells upon engagement with pd-l1. peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il-2 production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e3 ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd4 + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova 323-339 peptide)-specific t cells from do11.10 tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e3 ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e3 ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler 1 , p. aichele 1 1 immh, university freiburg, immunology, freiburg, germany interleukin 12 (il-12) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il-12 has a direct influence on cd 8 + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal 1) and costimulation (signal 2). we analysed direct il-12 signaling to cd8 t -12 signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd8 t cells lacking il-12 signaling failed to up-regulate klrg1 and to down-regulate cd127 in the context of listeria but not viral infections. thus direct il-12 signaling to cd8 t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd8 + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd8 + t cells are tightly controlled in their effector functions by cd152 (ctla-4). we demonstrate that signals induced by cd152 reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd8 + t cells. for this novel function cd152 specifically represses the transcription factor eomes, but not t-bet. a cd152 mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd152-mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd8 + t cells differentiated in the absence of cd152 signaling could be demonstrated in vivo. the novel insights that cd152-mediated signal transduction in vivo indeed alters cd8 + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd8 + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p2 receptors. p2x 1-7 receptors open to non-selective ion channels, whereas p2y1, 2, 4, 6, 11-14 are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p2 receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr9 but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p2x7 antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca(2+)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek 1 , a. brouckova 1 , d. filipp 1 1 institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih3t3 cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y505flck. comparative 2-d gel analyses followed by ms/maldi identified rack1 as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih3t3 cells of ha-tagged rack1 with either a wild type lck or constitutively active y505flck revealed a significantly enhanced complex formation between y505flck and rack1 compared to that of wtlck. ectopic expression of y505flck with its domain-inactivating mutations showed that lck-rack1 interaction depends on functional sh2, sh3 and the c-terminal tail sequence of lck. lck-rack1 interaction is readily detectable also in primary cd4 + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack1 with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack1 co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack1 in the context of lck translocation to lr is further strengthen by the observation that rack1 is associated with elements of cytoskeleton. these results are the first to characterize rack1 as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin 21 (il-21) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd19+cd20+cd27-cd38-igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th2 response to alumova inducing large early germinal centres and massive plasma cell formation with more than 75 % of these switching to igg1. the plasma cells up-regulate cxcr4, but not cxcr3, a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th1-associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚5%), igg2a (˚30 %), igg2b (˚30 %) or igg1 (˚30 %). in addition to cxcr4, some 70 % of these plasma cells strongly express cxcr3. the induction of cxcr3 in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg2a switching during th1 responses. this is functionally significant for oti-dependent cxcr3 expression, as well as induction of switching to igg2a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg2b. t-bet is known to be induced in b cells exposed to ifng or tlr9 stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd88. objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd272) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd4+ t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd4+ t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla-4 pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa-1 and cd2 in the psmac of untransformed human t-cells. in marked contrast, tcr/cd3 accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl-1 regulates interclonal t cell competition during acute and chronic immune activation. we found p53-independent noxa gene induction and mcl-1 downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl-1, which was delayed in noxa -/cells. using ot-1 cells and altered peptide ligands we observed that the level of mcl-1 downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl-1 -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np366) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl-1 axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen 9 (ebag9) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag9 confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag9, which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g2-adaptin in t cells. both interactions suggested an involvement of ebag9 in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag9-/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag9-deficient ctls. these data imply a role for ebag9 in regulating the formation of mature ctl granules and identify ebag9 as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag9 defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag9-related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd8 t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd80/86 and il-12. in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd3/acd28 or pma/ionomycin could not significantly activate cd4 and cd8 t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca 2+ influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena 1 , s. carrasco 1 , i. merida 1 1 centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp1-ras-erk signal intensity is critical to determine the final cell outcome. rasgrp1 is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. 1. analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. 2. develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd8 cells expressing gfp-c1 domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c1 domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h4, cd278), a cd28-like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th2 biased responses and high production of the anti-inflammatory cytokine il-10, and was essential to the development of germinal centres. however, icos can also help in the il-21-dependent differentiation of inflammatory th17 cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd28 a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p85 subunit of class ia pi-3 kinases. these can complex with one of the three 110 kda catalytic subunits (p110a, p110b, and p110d) expressed by leukocytes that generate pip 3 affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi3k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi3 kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi3-k regulatory (p85a, p85b, p53a) and catalytic (p110a, p110b, and p110d) pi3-kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p85a g p53a g g p85b and p110a g p110d g g p110b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi3-kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd3-induced secretion of il-10 and il-4. during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap450, a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap450 construct that acts as a dominant negative, or sirna knockdown of endogenous akap450 expression in t cells prevents the correct organization of cd3z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa-1 localization was also analyzed to assess p-smac architecture and, interestingly, confocal 3d reconstruction revealed that lfa-1 ring was not clear in the akap450-disrupted cells. moreover, akap450 was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap450 decrease the phosphorylation of molecules such as lat, plcg1 and pkcv. these defective activation events as reflected in a reduction of il-2 production. together, our results underscore a key role for akap450 in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin85/cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh3 domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca2+ mobilization and cell activation involving the pi3k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc1d10c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr 1) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as 2) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. 3) we then studied the phenotype of carabin kd (shrna expressing) a20 b cells after bcr stimulation. 1) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t1 tot2 b cells and to follicular mature b cells. 2) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. 3) bcr simulation, but not lps stimulation, of carabin kd a20 b cells shows an acceleration of ras target erk1/2 phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk1/2 pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor 3 (tlr3) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd4 + t cells express tlr3 and respond to the well characterized synthetic tlr3 ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd4 + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr3. tlr3 stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd4 + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor 3 (irf3) as revealed by realtime-rcr analysis of ifn b and irf7, whose transcription depends on the activity of irf3. combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr3 signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd4 + t cells. this study was supported by dfg spp 1110 "innate immunity" (ka 502/8-3). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd3e cytoplasmic domain (cd3e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd3e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd3e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd3e cd and a fluorescent membrane dye (r18). with this assay, we show that the cd3e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd3e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd3e cd to lipid bicells. membrane binding by the cd3e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia 1 , y. ge 1 , u. quitsch 1 , p. beckhove 1 1 german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd4+ t cell help is believed to contribute to optimal cd8+ memory expansion via cd40l on cd4+ t cells binding cd40 on dendritic cells. however, a few reports suggest that cd40l-cd40 engagement may mediate direct cell-cell contacts between cd4+ and cd8+ t cells. in this study, we investigated the importance of cd4-cd8 co-operation and cd40l-cd40 interactions for t em proliferation. methods: we isolated human cd4+ and cd8+ t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd4+ and cd8+ populations were activated in vitro using anti-cd3/cd28 beads. proliferation was measured by [ 3 h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd40 or cd40l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd4+ or cd8+ t em cells, demonstrating that optimal t em expansion requires direct cd4-cd8 interactions. surprisingly, not only cd8+ but also cd4+ t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd4+ t em proliferation depends on signals from cd8+ t em cells. activation induced the expression of cd40 on both populations and cd40l on subsets of cd4+ and cd8+ t cells. blocking of cd40l on cd8+ t em cells impaired significantly cd4+ t em proliferation, which confirms that the improved expansive potential of cd4+ t em cells in mixed populations depends on cd40l co-stimulation by the cd8 t em subset. conclusions: our data demonstrate for the first time that activated cd8+ t em cells deliver help to the cd4+ t em subset via cd40l-cd40 signalling and may play an important role for cd4+ t em expansion upon stimulation. the t cell surface glycoprotein cd5, a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd5 localization upon t cell:apc conjugation. we have questioned which domains of cd5 mediate the localization within the is, and for this we have expressed cd5 mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd5 mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd5 by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd5 depends on sequences within the cytoplasmic domain, as a cd5 deletion mutant lacking most of the cytoplasmic tail, cd5.k384 stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd5 translocation was mapped within amino acids glu 418 and his 449 since the cd5.h449 stop mutant, just short of 22 aa is still able to translocate to the is, whereas cd5.e418 stop , that lacks 2 important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd5 translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu 418 -his 449 ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna-2 is missing, but lmp-1 is still expressed. using a hl derived cell line, we have shown that the cytokine il-4 can induce lmp-1 expression in vitro and can replace ebna-2. we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is 2, 3 or 4. a high affinity stat6 binding site is spaced by 4 nucleotides. we found three potential stat binding sites in the lmp-1 promoter, which we named lrs, tr and edl1. they were spaced by 4, 3 and 2 nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il-4-treated or non-treated kmh2-ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat6 binding site, or lrs-stat6. a stat6 complex binding to the gl-epsilon promoter and lrs-stat6 was induced by il-4. the specificity of the stat6 complex was shown by supershift experiments with anti-stat6, but not anti-stat5 antibodies. when gl-epsilon or lrs-stat6 was used as cold competitors in a 100-fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat6 contains a functional stat6 binding site. oligonucleotides, corresponding to lrs in which the stat6 site had been mutated, could not compete for stat6 binding. interestingly, the unlabeled lrs-tr with 3 nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by 2 nucleotides (lrs-edl1), it could not compete. thus, expression the transforming protein lmp-1 can be induced directly by the t cell derived cytokine il-4 in a stat6 dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp-1 and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd8 t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji 2007 (ji . 179:2250 . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd4 t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd4+ t cells primarily affect the proportion of cd4+ t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd40, cd80 and cd86 on parameters of cd4+ t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd40, a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd40l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd40 leads to an enhancement of ig and cytokine production. the current dogma postulates that these 2 signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd40l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd40l (rmcd40l) can increase proliferation induced by tlr3 (poly ic) and tlr4 (lps) agonists. by contrast, we never observed any synergy between rmcd40l and tlr1/2 (pam3csk4) or tlr2/6 (pam2csk4) agonists. to go further in the study of cd40l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd40l, named mini-cd40ls, based on a c 3 -symmetry core holding cd40-binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd40ls bind to immobilized human cd40 and compete with the binding of cd40l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd40l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd40l, mini-cd40ls synergize tlr4 (lps), tlr3 (poly ic) and tlr7/8 (r848) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd40ls and tlr 1/2 (pam3csk4), tlr 2/6 (pam2csk4) and tlr9 (odn 2395) agonists. synergy between cd40l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd40l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd8 + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd8 + t cell tolerance. we have defined a phenotypic profile for cd8 + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd25 and cd69, and high levels of expression of cd62l and ly6c. whereas, cd8 + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd25 and cd69, and loss of cd62l and ly6c expression. ly6c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly6c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly6c, expressed on naïve cd8 + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd8 + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for 3 weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to 60 days after infection. results: daily injection of paraoxon induced g 50 % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining 2 weeks of treatment. mice exposed to paraoxon exhibited g 80 % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with 80 % of mice surviving the infection compared to 20 % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th1 and th17 cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide 3-kinases (pi3k) constitute a family of enzymes that generate 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi3k are divided into two types: class ia p85/p110 heterodimers, which are activated by tyr kinases, and the class ib p110g (p110gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p110g deletion affects tcr-induced t cell stimulation. mice lacking p110g show a partial defect in t cell differentiation, activation and survival. p110g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd4+/cd8+ t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi3kg-specific inhibitor causes a reduction in the number of cd4+ memory t cells that mediate renal injury. similarly, pi3kg deletion in p65 pi3k transgenic mice also reduces the numbers of cd4+ memory t cells. there is therefore evidence that pi3kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi3kg regulates this process is not well understood. we studied the specific role of p110g in t cell activation. methods: we studied whether the tcr activates p110g and the consequences of interfering with p110g expression or function on t cell activation. results: we found that after tcr engagement, p110g interacts with and forms a complex with ga q/11 , lck and zap70. tcr stimulation activates p110g, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p110g controls rac1 activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p110g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p110g-/-t cells. our observations clarify the activation mechanism and mode of action of p110g in the control of t cell activation, confirming a crucial role for p110g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a4b2 and a7, expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c57bl/6j mice. they were stained with fluorescently labeled igm-, cd40-or cd23specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by 3 h-thymidine incorporation upon stimulation with anti-cd40 and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a7 nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a4 or b2 nachr subunits inhibited binding of igm-and cd23-specific antibodies but facilitated that of cd40-specific antibody. in contrast, antibody against a7 subunit prevented binding of anti-cd40 but not of anti-igm or anti-cd23 suggesting that a7 nachrs are located close to cd40, while a4b2 ones are close to bcr/cd23. consequently, anti-cd40-induced b lymphocyte proliferation was increased by mla much stronger than by a4b2-specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine-3. in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd40-mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a7 nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd4 + t h -lymphocytes to antigen presenting cells or of cd8 + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim1 and trpc3), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about 96 %. down-regulation of stim1 was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd4 + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, 2008) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be 300-600 pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein 70 (hsp70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd8 + cytotoxic t-lymphocyte (ctl) and cd4 + t-helper cell (th1) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k562 cells as well as targetindependent cytotoxicity. cd4 + cells exhibited a strong increase in proliferation after stimulation with hsp70, with rates of up to 29 %. in the presence of target cells, a 35-fold up-regulation of granzyme b mrna was observed after stimulation of cd4 + t-helper cells with hsp70 in combination with il-7, -12 and -15. the target cell-independent secretion of granzyme b by cd4 + cells was greatly augmented after stimulation with hsp70 plus il-2 or il-7, -12 and -15. in this study, we have shown that hsp70 is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd4 + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd3 + and cd8 + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp70 on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd40, which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd150 receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk1 overexpression in a model system was achieved by transfection. pjnk1/2 expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd150 on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk2, but not jnk1 activation. cd40 ligation on primary tonsillar b cells also resulted in jnk2 activation. however, bcr crosslinking did not affect the level of jnk1/2 phosphorylation. cd150-mediated jnk2 activation was independent from sh2d1a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd150 and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase 1 (hpk1) was associated with cd150 in primary b cells as well as in b cell lines. cd150-hpk1 association was independent from cd150 tyrosine phosphorylation and sh2d1a expression. overexpression of hpk1 in a model system significantly enhanced cd150mediated jnk2 phosphorylation. it is known that tnf family receptors such as cd30, cd40, rank trigger survival signals in hrs cells. we observed the expression of pjnk1/2 in hrs cells of primary classical hl. cd150 could be involved in sustained jnk2 activation in primary hrs cells, and this may reflect the role of cd150 receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk2 is activated via cd150 in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk1 is involved in cd150-mediated jnk2 activation. objectives: cd5 has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd5 upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd5 are involved in c-cbl phosphorylation and association. methods: el4 thymoma cell line was stably transfected with wild-type human cd5 or hcd5 cytoplasmic tail mutants: cd5.k384stop (maintaining only a pseudo itim); cd5.h449stop (lacking the distal s and y in the carboxy-terminal region); cd5. ¿ e418-l444stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y700 in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd3 in combination or not with anti-human cd5 biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py700) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x 0.05). in murine thymocytes, co-crosslinking of cd3 with cd5 induces an increase in c-cbl phosphorylation compared to cd3 alone. analysis of the el-4 transfectants showed that mutants cd5.k384stop and cd5.h449stop lost the ability to costimulate cd3-mediated phosphorylation of c-cbl. in contrast, cd5. ¿ e418-l444stop mutant, was able to efficiently costimulate cd3-mediated c-cbl phosphorylation, similarly to the hcd5wt. our results indicate that the absence of the pseudo itam in cd5 does not interfere with c-cbl phosphorylation in response to cd3 plus cd5 crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd5 appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd5 cytosplasmic tail, but rather, may indirectly associate with cd5 through the interaction with other sh2-sh3 domain-containing molecules, that may be recruited to cd5 through its carboxy-terminal region. l. kolly 1 , s. narayan 1 , j. tschopp 2 , a. so 1 , n. busso 1 1 chuv, rheumatology, lausanne, switzerland, 2 unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase-1 into inflammasomes and thus plays a key role in regulating capase-1-dependent il-1b and il-18 production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd4 + and cd8 + t cells were activated in vitro through anti-cd3 stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd3 ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd3 activated asc -/-t cells predominantly displayed a more th 2 phenotype, producing more il-10 (199 vs. 692 pg/ml; asc +/+ vs. asc -/-t cells respectively; p=0.0074) and less ifn-g (15,831 vs. 6 ,921 pg/ml; asc +/+ vs. asc -/-t cells respectively; p = 0.0021). when asc +/+ and asc -/-t cells were purified into cd4 + and cd8 + t cell fractions and activated individually using anti-cd3, no inhibition in proliferation was observed amongst activated asc -/-cd4 + and cd8 + t cells. interestingly, the activated asc -/-cd4 + t cell fraction produced significantly more il-10 when compared to activated asc -/-cd8 + t cells and asc +/+ cd4 + and cd8 + t cells (asc -/-cd4 + t cells = 380 pg/ml il-10; asc -/-cd8 + t cells = undetectable il-10; asc +/+ cd4 + t cells = 11 pg/ml il-10; asc +/+ cd8 + t cells = undetectable il-10). cd4 + and cd8 + t cell mixing experiments revealed that asc -/-cd4 + t cells are able to inhibit the proliferative ability of asc -/-cd8 + t cells, asc +/+ cd4 + and cd8 + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd4 + t cells. collectively, these results demonstrate that the absence of asc drives cd4 + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd4 + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge 1 and pge 2 suppress some t-cell functions including proliferation, activation and cytokine production. pge 2 signals through four types of gpcrs called the ep receptors. at low concentrations, pge 2 is believed to be necessary for t cell function, whereas at higher concentrations, pge 2 inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge 2 and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge 2 diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd4 + t cells with ep receptor antagonists was found to impair cell surface expression of cd71, cd69, cd25 and ox40 but not cd44. suppression of t cell proliferation by pge 2 has already been widely studied. however, blocking ep receptors in cd4 + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd4 + t cells to the chemokine sdf-1b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd44 + cd4 + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd4 + t cells. our results also suggest that considering pge2-mediated camp signaling in cd4 + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd4 + human t-cells was examined. oxidation affects several ca 2+ signalling pathways by altering the activity of ip 3 receptors, trp channels and store operated ca 2+ channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca 2+ signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd147 (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd147 knockout mouse possess enhanced mixed lymphocyte reactions and cd147 monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd147 signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd147 in jurkat t cells augments the secretion of the t cell growth-factor interleukin-2 (il-2) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il-2 promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd147, we identified the immunomodulatory sub-domain of cd147. supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd40 and il4 (simulates t cell-dependent activation). microarray assays identified about 104 mirnas in unstimulated b cells. 35 of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor-4 (irf-4). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf-4 transcripts differs from that observed for irf-4 protein abundance after b cell stimulation. further analysis identified the irf-4 transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk592 and the dfg forschergruppe for832. objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on 11 different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey 1 , f. hauck 2 , i. berberich 1 , f. berberich-siebelt 2 , gk 520 -immunomodulation 1 institute for virology and immunobiology, university of wuerzburg, würzburg, germany, 2 university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd4+ t lymphocytes, the transcription factor is predominantly expressed in t helper 2 (th2) compared to t helper 1 (th1) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g1 phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp-1 encoded by prdm1 is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp-1 is expressed in differentiated effector t cells where it is higher in th2 than th1 cells. the regulation of the blimp1 expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm1 promoter and activates blimp-1 expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp-1 in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp-1 in b cells using cre recombinase. moreover we found a new putative blimp-1 isoform lacking exon 2. currently, we analyze the expression of c/ebpb and blimp-1 in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening 53 cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd21low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd4 + t cells but not transitional b cells or cd8 + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein-1a (mip-1a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy2 revealed constitutively high background levels in cd21low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas 1 , v. courtois 1 , k. de luca 1 , r. sodoyer 1 1 sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il-2, il-4 or il-6 could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il-2, il-4 and il-6. furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a-136-0033-01835). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen-4 (ctla-4) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla-4 during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il3) in the tissue and the released eggs and first stage larvae (l1) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd3 and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla-4 during the infection, a neutralysing antibody (a-ctla-4; 4f10) was administered intraperitoneally (300 mg) two hours before subcutaneous infection with s. ratti il3. the in vivo neutralisation of ctla-4-signalling by applying a-ctla-4 during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th2 cytokines, such as il-4 and il-5 and a reduction of the proinflammatory cytokines ifn-g and il-17. the investigation of the humoral response showed a remarked increase of the igg1-titer in the serum during secondary infection in mice that had been treated with a-ctla-4 during primary infection. furthermore, the blockade of ctla-4 resulted in a diminished worm burden as indicated by reduced release of l1 in the faeces. these results suggest that the blockade of ctla-4 during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th2 response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg1 during secondary infection also reflects the induction of a potent th2 response. objectives: cd36 is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd36 is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd36 on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd36 expression. cd36 knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd36 ko compared to heterozygous mice. since reduced levels of cd36 are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd36 ko mice. after one injection of apoptotic cells, cd36 ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd36 on b-cells are involved in setting this threshold. conclusion: our data suggest that cd36 is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd152 acts as a major check-point of immune responses, but the mechanism by which cd152 controls peripheral t cell responses is unknown. the consequences of cd152 signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd152 in th1 cells reduced migration towards ccl4, cxcl12 and ccl19. crosslinking of cd152 together with cd3 and cd28 stimulation on activated th1 cells increased expression of the chemokine receptors ccr5 and ccr7, which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd152 expression and cd152-mediated migration-enhancing signals. importantly, migration of cd152 positive th1 lymphocytes in in vivo experiments increased, as compared to cd152 negative counterparts, showing that indeed cd152 orchestrates specific migration of selected th1 cells to sites of inflammation and antigenic challenge in vivo. these data show that cd152 signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd152 adds to the already significant role of cd152 in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd152 signaling on the longevity of cd28 null t cells. using a sensitive staining method for cd152, we show that human cd4 + cd28 null and cd8 + cd28 null t cells rapidly express surface cd152. serological inactivation of cd152 using specific fab fragments or blockade of cd152 ligands using ctla4ig in cd4 + cd28 null and cd8 + cd28 null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd152 crosslinking on activated cd28 null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd152 is mediated by pi3'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd152 act directly on activated cd28 null t lymphocytes and, due to its exclusive expression as a receptor for cd80/cd86 on cd28 null t cells, prevention of cd152 mediated signaling is likely a major target mechanism taking place during therapy with ctla4ig. objectives: cd45 is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd45 -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd148 (ptprj, dep-1) which acts as a positive regulator of sfk in cd45 -/-b cells and macrophages and can compensate for cd45 deficiency in these cells. indeed, combined deficiency of cd45 and cd148 in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd148 and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd148 -/mice were reported so far. however, in human t cells the role of cd148 may be different since naive human t cells express cd148 at a level comparable to b cells. using cd45 -/-/cd148 -/human t cell line (jurkat-derived js-7 cells) we tested the ability of cd148 to complement cd45 deficiency in t cells. we used retroviral transduction to express human cd45 or cd148 in js-7 cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd148 to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js-7 cells. expression of wild type cd45 or cd148 in js-7 cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd69 upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js-7 cells expressing cd148 when compared to control cells. finally, cd148 substrate trapping mutant expressed in js-7 cells interacted with lck in vivo suggesting that lck is a direct substrate of cd148 in js-7 cells. the results suggest a level of redundancy between cd45 and cd148 in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam1.6) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr 505 and tyr 394 we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native 2d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca2+ release-activated ca2+) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa-1 (leukocyte function-associated antigen 1) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa-1 prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al.2006) we describe a non-viral vector based approach (vockerodt et al. 2008) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in 9 independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd40 signaling cascade was conducted. after activating this particular signaling cascade (cd40) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. 2005 ). a rat thymic epithelial cell (tec) line (r-tnc.1) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc.1 cell line in vitro. it was found that a number of adhesion molecules, such as cd2, cd4, cd8, cd11a, cd18, cd54, cd90 was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc.1 line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il-2 activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc.1 cells. we found that both the adhesion (30 min and 3h) of activated thymoytes were partially blocked by mab to cd2 and cd8 molecules (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells). early adhesion was inhibited by mab to cd90, abtcr, mhc class i molecule (ifn-g stimulated r-tnc.1 cells) and cd4 molecule (ifn-g unstimulated r-tnc.1 cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc.1 cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay (6h), namely mab to cd2, cd4, cd8, cd90 molecule (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc.1 cells). our results also suggest the involvement of cd11a/cd18 dependent -cd54 independent pathway in adhesion and cd11a/cd18 dependent -cd54 dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc.1 cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin-27 (il-27), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il-27 favours naï ve cd4 t cell differentiation into th1 cells to the detriment of th17 or th2 differentiation. the il-27 receptor (il-27r) is a heterodimer composed of tccr, which confers ligand specificity, and gp130, a signal transducing chain that is utilized by several other cytokines. il-27 has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd8 t cells, but the potential impact of il-27 on human cd8 t cells has not been elucidated. our goal is to investigate the impact of il-27 on human cd8 t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il-27r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd8 than cd4 t cells expressing the complete surface il-27r (gp130+tccr). however, we detected high amounts of intracellular tccr in both, cd4 and cd8 t cells, but only polyclonal activation (anti-cd3) of cd8 t cells led to an actual increase of il-27r surface expression. purified cd8 t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il-27 activated stat1 and stat3 signalling with rapid kinetics in both cd8 and cd4 t cells, indicating the capacity of il-27 to signal through these molecules. addition of il-27 to anti-cd3 activated cd8 t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il-27 on human cd8 t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd-1/pd-l1 pathway is associated with production of the immunoregulatory cytokine il-10, the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l1 and pd1, as well as myelin basic protein (mbp)-stimulated il-10 production, pakt inhibition, and apoptosis (annexin v), in 50 ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); 24 had a diagnosis of stable disease (sms). results showed that: 1) pd-l1 -expressing cd14+ and cd19+ cells are reduced in ams compared to sms individuals (p=0.04); and 2) pd1 expression is increased in cd4+ t cells of sms individuals and is comparable on cd8+ t cells of ams and sms patients. this is associated with a significant decrease in il-10 production by mbp-stimulated cd14+ and cd19+ cells of ams patients (p=0.03). additionally, cd8+ anexin v+ (av+) cells were diminished and cd8+ pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd4+av+ and cd4+ pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l1/pd-1 pathway seen in ams patients result in a reduced mbp-specific il-10 production by cd14+ and cd19+ cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd8+ t. the pd1/pdl1 pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti-4-1bb in cd8 cells. this difference could be due to down regulation of cd28 by activated lymphocytes and possible preferential response of cd8 cells to anti-4-1bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin7 (stx7) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx7. rna interference technique was also used to down regulate stx7 expression in primary human ctls. results: we identified stx7 in ctls by pcr and immunocytochemistry. stx7 accumulates at the is after ctl/target cell contact. when stx7 function was blocked by overexpression of a dominant negative stx7 mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx7 is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the 20 years history of mouse t h 1 and t h 2 subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h 0, t h 1 or t h 2 phenotypes, based on their cytokine production (il-2, ifng or il-4). a comparative analysis of t-bet, ifng and il-4 mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca 2+response, membrane raft expression/organization, k + -and ca 2+ -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h 1 g t h 0 g t h 2, although the membrane cholesterol level (detected with anti-cholesterol ab, ac8) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h 1 cells upon activation than in t h 2 cells. t h 1 cells produced a more sustained calcium response with higher amplitude than t h 2 cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav1.2 and kv1.3 ion channels, major functional determinants of the sustained calcium influx. t h 2 cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd19-dynabeads. cd19 + isolated cells were facs sorted into cd19 + cd27naïve b or cd19 + cd27 + memory b cells. dna synthesis was measured by 3h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd19 + cd27naï ve b cells, of which bmp-6 and -7 were most efficient (40 % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd19 + cd27 + memory b cells by 40 -50 %. to induce differentiation, both naï ve and memory b cells were stimulated with cd40l and il-21. this increased the production of igm, iga and igg 10 -100-fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad1/5/8 in cd19 + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd40l/il-21-induced production of igs in mature human b cells. s. gutenberger 1 , k. warnatz 1 1 university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases 1 and 2 (erk 1/2). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd86 upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of 20 hd and 25 cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk1/2 phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk1/2. to increase the signal intracellular phosphatases were inhibited by h2o2. as markers of activation and initiation of proliferation, cd86 and ki67 expression were measured after 2 days of in vitro stimulation. k. theil 1 , p. aichele 1 1 immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd8 t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p14 cd8 t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b6-recipient mice and compared their expansion with wild-type (wt) p14 t cells after viral infection. we could demonstrate a severe impairment in the capacity of p14 t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p14 t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd8 t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p14 t cells into h8 mice. h8 mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp33-41. therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h8 mice are infected with lcmv8.7. this is a lcmv variant that has got a point mutation in gp33-41 and consequently cannot be recognized by the p14 t cells. s. frischbutter 1 , r. baumgrass 1 1 deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap1 which are important for expression of cytokines such as il-2, ifng and il17. it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl-10 was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl-10 phosphorylation but rather an inhibition of bcl-10 dephosphorylation. furthermore, calcineurin and bcl-10 co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl-10/malt1 signaling complex and dephosphorylates bcl-10 and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop 1 , j. lamoureux 1 , c. fortin 1 1 université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: 25 healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla-4 has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla-4 expression was higher after stimulation in t cells of elderly. there was differences between cd4 and cd8 t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd95l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh3 and ww domain proteins. since fasl surface expression is regulated by adam10-mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl2a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh3 domain proteins that potentially interact with the riped fasl prd, we used a sh3 domain phage display library containing all 288 sh3 domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx9 family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conseoptimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b7 family members on antigen-presenting cells with cd28 on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd28 co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound12, with classical gcs regarding their suppressive effect on cd28-costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd3 and anti-cd28, and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd28-costimulated human memory/effector cd4+ t cells by compound12 than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound12 was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound12 and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound12 than prednisolone. when evaluating possible mechanism for the increased activity of compound12 in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound12 which was not prevented by the partial gc receptor antagonist, ru-486, in vitro. moreover, in vivo we observed less induction of il-1beta and tnf-alpha by pre-treatment with compound12 than with prednisolone. our data indicate that the non-steroidal segra, compound12, may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b6 mice elicits robust cd8+ t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il-2+ cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa 224 -specific cells also express tnfa, but only about half of the ifng+ np 366 -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd8+ t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd8+ t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova 257-264 peptide shows a close correlation with division in vivo. early after antigen encounter (0-2 divisions) the vast majority of cells express only tnfa. after 3-4 divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions (4-6 divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd4+ t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd8+ t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd8+ t cells, expansion plays a very important role in the regulation of cd8+ t cell effector function. in addition to its chemo-attractant function, sdf-1a (stromal-cell derived factor-1a, cxcl12) has been described to costimulate cd4 + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd4 + t cells was increased by immobilized sdf-1a to a level similar to that obtained with the costimulatory molecule cd28. as visualized by real time confocal microscopy, t cells entering in contact with sdf-1a formed a tether and displayed an active scanning activity. since sdf-1a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd3 mabs, it is conceivable that the sdf-1a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf-1a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf-1a in the context of cd4 + t activation by antigen-presenting cells secreting sdf-1a. this study should help us to better define how sdf-1a modulates cd4 + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd4 + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd3 and anti-cd28 antibodies and cape for 2 days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by 3h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il-5 production and lymphoproliferation in cd4 + t cells stimulated by anti-cd3/cd28. cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd4 t cells express a variety of molecules on their surface, such as mhc-class ii, cd80, cd86, cd70, whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd4 t cells might induce t cell proliferation and differentiation from cd4 resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd4 t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after 5 days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd4 memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd25, cd30, and cd69. analysis of the cytokine profile of these cells revealed the differentiation of il-10-and ifn-g-double-producing cells in response to contact with th1 effector cells, and il-4-producing cells in response to contact with th2 effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd3/cd28 stimulation. whereas neutralization of ifn-g or il-4 during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd70, cd80, and cd86 could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to 64-85 %. conclusion: interaction of cd4 memory t cells with activated t cells resulted in the production of the cytokines il-4, il-10, and ifn-g. given the immunomodulatory capacity of il-4 and il-10, these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir 1 , s. thompson 1 , j.j. murphy 1 1 king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl1 leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il-2 and il-5 and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp36l1 by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp36l1 has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl1 differentiation was observed to be associated with downregulation of zfp36l1 protein. in an attempt to determine whether zfp36l1 downregulation was directly linked to bcl1 differentiation, a zfp36l1 shrna expressing lentivirus (psicor) was employed to knockdown zfp36l1 expression. this reagent downregulated zfp36l1 expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp36l1 shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp36l1 downregulation in promoting bcl1 plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of 32 patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all 32 isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd95 (fas, apo-1) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd3/cd28-triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo1 mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd95l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il-2 and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk1/2 phosphorylation, the expression of various activation markers, the il-2 production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb415) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb1,3galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd4+ and cd8+ t cells. in addition, mitogenic stimulus increase 3-fold the all binding to cd4+ t cells. previous studies in human pbmc showed that all binds to human cd4+ t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd4+ t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd3-dependent activation of purified cd4+ t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd25 and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd25 expression, but enhanced the anti-cd3-dependent proliferation and cd25 expression of purified cd4+ t cells. analisis of calcium influx showed that all enhanced anti-cd3 dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd4+ t cells by up-regulating t cell activation mediated by anti-cd3 stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in214609) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd3 complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd95 receptor. it has been previously demonstrated that cd95 ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp-76, slap-130 and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il-1r, il-2r, il-3r, il-7r, il-12r, il-18r, cd27, cd28, cdw137( 4-1bb), cd 95(fas) and cd178 (fasl) on hpbls in different cultured time, i. e. 0d, 1d, 3d, 5d, 7d, 9d, 11d and 14d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. 1. the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. 1) the expressions of membrane immune molecules before cultured. 2) expressions of the membrane molecules on hpbls during culturing. 5% fbs rpmi 1640 group (1640 group), il-2 group, pha group... (1) mtt assay. (2) proliferative times and growth curves of hpbls... 1. during cultured in vitro, there are expression changes of the il-1rs (il-1ra, il-2ra, il-2rg, il-3r, il-7r, il-12r, il-18r), co-stimulatory molecules (cd27, cd28, 4-1bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in 1640 group, il-2 group and pha group, but the rests are different. 2. our data also suggest that the hpbls cultured in cd3mcab+cd28mcab+il2+il1a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. 3. celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun 1 , r. tauber 1 1 zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p56lck and the ras/rac2 signalling pathway (1) followed by mitogen-activated protein kinases (2) and c-jun n-terminal kinase (1), which leads to an enhanced binding of l-selectin to soluble ligands (3). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues (4). here we show that the protein phosphatase 2a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp2a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp2a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer 1 , e. glasmacher 1 1 helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago1-4 genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm1 and rck or expression of dominant-negative gw182. interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd4 + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd8 + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd8 + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd8 + t-cell activation and differentiation. naïve cd62l hi cd44 lo cd8 + t cells were sorted and stimulated by anti-cd3 and anti-cd28 antibodies. results: firstly, we show that the activation and differentiation of cd8 + t cells require il-2 provided by activated cd4 + t cells at the initial priming stage after stimulation. secondly, this critical il-2 signal is delivered through il2rbg of cd8 + cells and is independent of il-2ra. besides promoting cell proliferation, il-2 stimulation increases the amount of ifng and granzyme b produced by cd8 + t cells. conclusion: therefore, our studies demonstrate that a full cd8 + t-cell response is elicited by a critical temporal function of il-2 released from cd4 + t cells, providing mechanistic insights into the regulation of cd8 + t cell activation and differentiation. most antigenic peptides recognized by cd8 t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a3168-176 is a tumor antigenic peptide presented by hla-a1 and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a3168-176 by tumor cells. by electroporating hla-a1 cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart-1/melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart-1/melan-a 26-35 specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart-1/melan-a 26-35 ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart-1 expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart-1/melan-a 26-35 epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart-1/melan-a 26-35 ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd8 + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd4 t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev 207: 293-313, 2005) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h2-m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity 11: 453-462, 1999) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide 84-103, whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from 84-103 peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the 84-103 epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, 2008), we found, after an initial induction at 8 hrs p. i., a strong inhibition of tapasin transcription at 24 hrs p. i. furthermore, also reduction of tap1 and tap2 transcription was observed contrasting to the elevated levels of erp57 and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß1, ß2 and ß5, which are replaced by their ifng-inducible counterparts ß1i, ß2i and ß5i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß5i is replaced by a thymus-specific subunit ß5t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß1i, ß2i, ß5i and ß5 in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß1,ß2 and ß5i (single intermediate proteasome), and the other comprises ß1i, ß2 and ß5i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent 10-20 % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about 50% of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a*0201) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a*0201 binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a*0201 from tapasin, but only ttp-ha dissociated hla-a*0201 from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl-721.220-a2 showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a*0201 from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a*0201. m. basler 1,2 , c. lauer 2 , m. groettrup 1,2 1 biotechnology institute thurgau, kreuzlingen, switzerland, 2 university of constance, division of immunology, department of biology, konstanz, germany two lmp7-dependent antigens have been described that relied on the 'structural presence' of lmp7 in the proteasome but not on the activity of lmp7. here we have investigated processing of the h-2d b -restricted uty 246-254 epitope of the male minor antigen uty reported to be lmp7-dependent. using splenocytes from lmp7 -/-, lmp2 -/and mecl-1 -/mice we found that the uty 246-254 epitope requires lmp7 and lmp2 but not mecl-1. curiously, a selective lmp2 inhibitor did not interfere with uty 246-254 presentation. objective: we investigated why the deletion but not the inhibition of lmp2 interferes with uty 246-254 presentation. we hypothesized that the 'structural' requirement for lmp2 is based on replacement of the caspase-like activity of b1 in the proteasome. methods: it was determined if t1a mutants of lmp2 and/or b1 can rescue the uty 246-254 epitope. we used a b1-selective inhibitor to determine if the inhibition of the caspase-like activity of b1 preserves the epitope. finally we determined by mass spectrometry if the uty 246-254 epitope embedded within a 25mer precursor peptide is differentially cleaved by lmp2-deficient and proficient immunoproteasomes in vitro. results: we found that t1a mutants of lmp2 and b1 rescue presentation of uty [246] [247] [248] [249] [250] [251] [252] [253] [254] . also inhibition of cells with a b1-selective inhibitor preserves uty [246] [247] [248] [249] [250] [251] [252] [253] [254] presentation. an aspartate in position 7 of the uty 246-254 sequence wmhhnmdli is preferentially used as a cleavage site by lmp2-deficient but not half as frequently by lmp2-proficient immunoproteasomes. the generation of the uty 246-254 epitope relies on the replacement of the caspase-like activity of b1 by lmp2 because the b1 activity destroys the uty 246-254 epitope. this is the first example for the 'structural' requirement of lmp2 for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp2 and perhaps also lmp7 and mecl-1 exert their function in antigen processing. a. linnemann 1 , a. musiol 2 , r. lindner 1 1 hannover medical school, cell biology, hannover, germany, 2 hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf6-regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf6) were overexpressed in 3t3 fibroblasts. we show that antibody-mediated oligomerization of mhc i in 3t3 fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf6: endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf6-regulated to a novel, arf6-independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv40 virus and since sv40 binding triggers mhc i oligomerization, this novel pathway may be involved in sv40 uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of 57 rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab3b, 3c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta2-microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab3b and rab3c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab3b or 3c positive vesicles. furthermore, the rab3b, 3c positive compartment were colocalizd with a fraction of rab27a at a juxtaposition of phagosomes. together these data demonstrate that rab3b and rab3c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over 90 % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over 80 viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd8+ t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf2a and bglf5 have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf1, has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens 2009). this represents a novel function for a virally-encoded gpcr. bilf1 is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf1 displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf1 is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf1 can hinder the recognition of virally-infected cells by cytotoxic cd8+ t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe 131 natural ligands, including two peptides derived from semenogelin-1, a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg1 is transcribed in thymus from both male and female individuals. finally, we detected the semg1 mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta1, beta2, beta5) and immuno-subunits (beta1i, beta2i, beta5i). deficiency in beta5i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta5i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta5i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta5i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta5i-deficient cells could be restored by treatment with d3t, which increases the amount of proteasomes independent of beta5i via induction of mixed proteasomes containing beta1i, beta2i and beta5. consequently, not the lack of the specific proteolytic activity of beta5i or immunoproteasomes, but the reduced proteasome quantity in beta5i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr2, 4 and 5, which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd4 + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd4 + t cells. the presence of exogenous tlr3 and tlr8 ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james 1 , i. bailey 1 , t. elliott 1 1 university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct26 colon carcinoma. this response is long lasting and mediated by both cd4 and cd8 t cells. importantly, the treg depleted ct26 specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a20, c26, bcl1, renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw11, which is h2-d d restricted. analysis of the generation of gsw11 in ct26 revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct26 cells substantially increased the amount of gsw11 present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct26 and immunised balb/c mice. greater than 80 % of mice injected with eraap kd ct26 were found to reject the tumour. analysis of t cell responses revealed the presence of gsw11-specific t cells, however, these responses were small (0.5-1 %). this compared to a much larger response to ct26 (˚5 %). preliminary results indicate that the majority of the t cell responses (non-gsw11-specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [1] . the di-allelic hla-a2 restricted minor histocompatibility antigen ha-1 locus codes for the highly immunogenic ha-1 his and the non-immunogenic ha-1 arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha-1 arg immunogenicity was the estimated numbers of cell surface presented copies i. e. 80/cell for ha-1 his and less than 5/ cell for ha-1 arg [2] . as ha-1 his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha-1 his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha-1 mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha-1 allelic peptides, we investigated the impact of the ha-1 his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha-1 his and ha-1 arg peptides were performed. moreover, the crystal structures of hla-a2 in complex with either ha-1 his , ha-1 arg or a ha-1 variant with a citrulline residue at position 3 were determined in order to obtain atomic level insights into the conformation of the hla-a2/ha-1 peptide complexes. our results exclude a role for antigen processing in preventing ha-1 arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha-1 arg allele in the hla-a2 peptide binding groove [3] . they provide a rationale for the lack of ha-1 arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd8 t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd8 t cell response. on the other hand, immunosubunit expression is not essential for development of cd8 t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp7 (ib5) and mecl-1 (ib2) develop a variety of autoimmune responses, including a latent form of t1d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd8 but not cd4 t cells. a cd8 t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose-6-phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd8 t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd8 t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, 162 autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr3 and dr4. analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type 1 (hsv-1) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd4 + and cd8 + t cells in hsv-1 immunity have yet to be clearly elucidated. to better understand the role of hsv-1-specific cd4 + t cells in immune control we have identified a 13 amino acid epitope derived from glycoprotein d of hsv-1. following flank infection, gd-specific cd4 + t cells were first detected in the draining brachial and axillary lymph nodes (ln) 5-days post-infection (pi), peaking at day 7 and declining thereafter. gd-specific cd4 + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day 6 pi and peaked at day 9. while hsv-specific t cells were first observed in the draining ln at day 5 pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as 2 days pi, with peak activity 4 days pi before declining to background by day 7. however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd4 + t cells was observed up to 23 days post-infection in the brachial ln. ex vivo analyses suggest that only cd11c + cells were involved in gd antigen presentation at days 2, 5 and 15 post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd8a + dcs can present the gd antigen to cd4 + t cells at day 2 pi, whereas by day 5 pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv-1 infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap1 and erap2 unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap1/erap2 in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp57. upon assembly of the heavy chain with b 2 m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp57. both crt and erp57 are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp57 to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp57 or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp57 must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp57 point mutant that fails to bind to cnx or crt was just as effective as wild type erp57 in normalizing rates of disulfide formation. we conclude that erp57 does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp57 point mutant, class i heavy chains, crt and the tapasin-erp57 disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp57 and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp57 have no effect on plc stability or class i surface expression, suggesting that erp57 plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies 5-11 % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb1*0405/dqb1*0401 haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr*0405 transgenic ab0 nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd4+ and cd8+ t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr*0405 transgenic mice (cd4+ t cell competent) develop aip even unprovoked, similar to ab0 nod mice (cd4+ t cell deficient), while hla-dr*0401, hla-dq8 or hla-dr*0405/dq8 (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr*0405 fails to protect from aip, likely due to defects in the induction of cd4+ regulatory t cells. our results identify hla-dr*0405 as a prominent risk factor for aip on the hla-drb1*0405/dqb1*0401 haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan 1 , c. britten 1 , h. overkleeft 2 , g. van der marel 2 , k. melief 1 , d. filippov 2 , f. ossendorp 1 1 leiden university medical center, section tumorimmunology, leiden, netherlands, 2 leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd8 and cd4 t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi 1,2 , x. hu 1,2 , a. kawanatachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping 8-mer and 10-mer epitopes (rypltfgwcf (nef138-10) and rypltfgw (nef138-8)) were found to be presented by hla-a*2402 and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y2f, y2w, t5c, f6l, w8r, f10r, f10y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef138-10 (wild type) and its four mostly common immune escape mutants (y2f, t5c, y2f&t5c, f6l), and also nef138-8 (wild type). we found that there was little difference between the nef138-10 (wild type) and nef138-10 (y2f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y2 or f2. interestingly the central bulge region of the peptide was becoming very flexible for the nef138-10 (t5c) and nef138-10 (y2f&t5c), which may affect the binding of peptide and the recognition of t cell receptor. for nef138-10 (f6l), the side chain of l6 was more flexible compared to the nef138-10 (wild type). alignment of the nef138-10 and nef138-8 showed that the nef138-8 became flat and the side chain of f6 was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef138-10 was featured, while the peptide nef138-8 was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef138-10 was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd8+ and cd4+ t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a2 restricted cmvpp65 derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a 2-3 log reduction of activation efficiency. this pattern was seen for 5/9 epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to 2-5 log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b7 restricted cmvpp65 rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t2 cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd8+ t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp100 and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp110, and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor-5 (fgf-5) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of 40 amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf-5 peptide was produced in vitro after incubation of proteasomes with a 49-amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf-5 spliced peptide by cells transfected with mutant constructs encoding fgf-5 proteins where the intervening segment was shortened from 40 amino acids to 30, 20 or 8 residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp100 peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b*4402, are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b*4405, appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b*4402 and b*4405 are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us3 protein. to address this hypothesis, us3 was stably coexpressed in b lymphoblastoid cell lines expressing hla-b*4402 or hla-b*4405. in the presence of us3, the surface expression of hla-b*4402 was substantially reduced whereas hla-b*4405 expression was relatively unaffected. although us3 was able to form complexes with both hla class i allotypes, only hla-b*4402 was retained intracellularly in an immature form whereas hla-b*4405 was transported to the cell surface. accordingly, in the presence of us3, hla-b*4405, but not hla-b*4402, constitutively presented a hla-b44 restricted alloantigen to reporter t cells, suggesting that us3 binds hla-b*4405 without interfering with peptide loading. us3 has been reported by others to bind the plc but surprisingly we have not detected such us3-plc complexes in our system. rather, in the presence of us3 we identified a pool of class i molecules distinct from the plc and only present in us3 expressing cells, implying that us3 may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa 1 , b. seliger 1 1 martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il12p70. since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for 24h with the gold standard of maturation (tnfa, il1b, il6 and pge2) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase-3 (lap3) had a more than 20-fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap1 and erap2 and of the immunoproteasome subunits lmp2 and lmp10 was enhanced between 2 and 5-fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr4 ligand mpla in comparison to the tlr-3 and 7/8 ligand polyi:c and r848. these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd4 + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il-4 induced surface expression of mature mhc class ii molecules and cd86. when ifn-g/il-4 primed pcmc were used as antigen presenting cells for cd4 + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd69 up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type 1-diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b15-23 to specific cd8+ t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd8 + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th1 immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd8+ t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd8+ tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd8+ t cell clones, faure, 2009, eur j immunol 39 (2): 380-90). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd8+ t cells after their in vitro differentiation into dc, 6 days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv-1 genome contains arfs within gag, pol and env genes encoding for a panel of hla-b*07 restricted epitopes. qprsdthvf (q9vf/5d) is one such epitope but its parental epitope qprsnthvf (q9vf/5n) has a significant higher frequency among hiv-1 isolates. strikingly, q9vf/5d-or q9vf/5n-specific ctls recognize apcs infected with hiv strains encoding for q9vf/5d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad8 ) encoding for q9vf/5n do not activate ctl responses raising the possibility that q9vf/5n epitope is not presented by infected cells. we asked whether introducing mutations within q9vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q9vf/5n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q9vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q9vf mhc-i presentation. we performed in vitro proteasomal digestions of 28mer peptides encompassing q9vf/5d or q9vf/5n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q9vf/5n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q9vf/5n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q9vf/5d or q9vf/5n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q9vf/5d peptide. we cloned and sequenced hiv-1 genomes from the three donors. surprisingly, out of 20 hiv proviral genomes isolated from pbmcs of q9vf/5d reactive donors, we could not find any virus bearing the q9vf/5d sequence. the isolated hiv sequences either encoded for q9vf/5n or had a stop codon within the epitope. in contrast, viruses encoding for q9vf/5d were isolated from pbmcs of the q9vf/ 5d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv-1 seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch 1 , y. wang 1 , d.c. tscharke 1 1 the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd8 + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd8 + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd8 + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h-2 bxd f 1 mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f 1 progeny, with some peptides being almost equally immunogenic in f 1 and inbred mice, while others elicit responses that are reduced by more than 90 % in f 1 mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f 1 mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f 1 mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd8 + t cells with a restricted vb usage in f 1 mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr2 agonist, s-[2,3-bispalmitoyiloxy-(2r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp-2), as an adjuvant for cross-priming against cellular and soluble antigens. malp-2 has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd8 + and cd8 -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr2/6 results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap1 expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap1) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to 90 % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, 50 and 10 % as compared to control rma cells, were injected s. c. in the flank of c57bl/6 syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to 45 days after injection. all mice injected with rma clone with a 50 % level of eraap expression developed a tumor and died within 23 days after injection. surprisingly, any animal injected with rma clone with a 10 % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd4 + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd4+ t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor (207 bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd20 therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor (2.5 bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg4 levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb1*0701-restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with 20-mer synthetic fviii peptides to stain epitope-specific cd4+ cells.cd4+ t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a2 domain: fviii 405-424 , fviii 421-440 and fviii 653-672 , as well as the c1 domain peptide fviii 2093-2112 , but not any c2 domain peptides. the c1 domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost.85:123-33, 2005 ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni 1 , s. albini 1 , m.z. limongi 1 , l. cifaldi 1 , d. fruci 1 1 ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p50 nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap1 and erap2, we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap1, erap2 and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap1 and erap2. this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p65 nf-kb subunit, we demonstrated that nf-kb is involved in erap1 and erap2 expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap1 and erap2 and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap1, erap2 and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch 1 , s. tenzer 1 , h. schild 1 , e. von stebut-borschitz 1 1 uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c57bl/6 mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th1 immunity, whereas ifng secretion of both cd4 + th1 and cd8 + tc1 cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il-12-dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd8 + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd8 + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm7 (lmp7 -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c57bl/6 mice. in addition, ex vivo co-cultures with infected dc from either lmp7 -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc1 cell ifng secretion and the dc restimulatory capacity of cd8 + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd8 + t cells, isolated from low dose infected c57bl/6 wildtype or lmp7 -/mice, were not detected. in summary, our data indicate that despite the fact that cd8 responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd8 + t cells against l. major is independent of the lmp7 subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a20, generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd4 + t cell epitope (84-103) from the g1 domain of aggrecan 10,000 times more efficiently than non-specific b cells and over 100 times more efficiently than the macrophage line j774. however, despite this highly efficient aggrecan capture, processing and presentation of the 84-103 epitope took at least 5 hours, comparable to the time required for presentation of aggrecan by j774. treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the 84-103 epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions 114, 115 and 116 at the peptide binding groove. position 116 also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b27 is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b27 subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b27 folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b*2705, peptide loading is relatively independent of this chaperone. we analyzed the effect of b27 subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b27 subtypes. the association of b27 heavy chain with tapasin was analyzed in c1r cells transfected with hla-b27 subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta-1, which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b27 peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me1 monoclonal antibody that recognizes b27properly folded b27/peptide complexes. the formation of fully assembled b27 molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc10, which recognizes mhc class i free heavy chains (hc), or with me1. maturation was analyzed by pulse-chase analysis, immunoprecipitation with me1 and treatment with endoglycosidase h (endo h). hla-b27 polymorphic positions other than 116, both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b27 subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd4 + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd8 + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c57bl/6 mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately 15 % of vacv global presentation in the context of h-2 b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd8+ and cd8-dc, which differ in their mhci antigen presentation capacities. cd8+ dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd8-dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd8+ and cd8-dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd8+ and cd8-dc was determined. surprisingly, cd8+ dc exhibit rapid surface mhci turnover compared to cd8-dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd8+ and cd8-dc were stabilized and no longer underwent rapid turnover. this suggests that cd8+ and cd8-dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd8+ and cd8-dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h-2kb loaded with the ova-derived peptide, siinfekl. cd8+ and cd8-dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd8+ dc, but not the cd8-dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd8+, and not cd8-dc. this data further validates the role for cd8+ dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp-1 cytosol using gelatinase b/mmp-9 as a model enzyme. in the first dimension, ion exchange chromatography separated the thp-1 proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp-9. in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp-1 cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp-9-cleaved or intact thp-1 cytosol. proliferation was assessed by measuring incorporated 3 hthymidine. results: 100-200 mmp-9 candidate substrates were isolated, of which 69 were identified, revealing many novel mmp-9 (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp-9 decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e3-19k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue 56 in hla-a11 and residue 93 in e3-19k are highly critical for association of both proteins. this defines a putative interaction surface between e3-19k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e3-19k and the class i antigen presentation pathway. moreover, because soluble e3-19k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap1 and tap2. tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap1 and tap2 were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap1 on tap2 was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap1 mutants suggested that the lack of tap1 protein expression is associated with a strong reduction of tap2 protein levels, which could be restored by tap1 gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap1 on tap2 different shrna plasmids specifically targeting tap1 or tap2, respectively, were stably transfected into constitutively tap1 and tap2 expressing hacat keratinocytes and colo794 melanoma cells. in both cell types the shrna-mediated tap1 and tap2 inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap1 caused not only an almost complete loss of tap1, but also a strong decrease of tap2 protein expression. in contrast, the tap2 knock down exhibited no influence on the tap1 expression. specific inhibition of the proteasome prevented the degradation of tap2 in the tap1 knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap1 on tap2 protein expression is not restricted to rare tap1 mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of 27 healthy volunteers was exposed twice to 1.5 minimal erythema dose of uv. blister roofs were then collected before and 1, 4 and 10 days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd14 + cd36 + and of a macrophagic cd14 -cd36 + cell subsets that emerge 1 and 4 days post exposition, respectively. more importantly whereas classical cd1a hi cd207 + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd1a low cd207and cd1a low cd207 + that emerged 1 and 4 days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd86 and hladr. finally, 10 days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey 1 , e. reeves 1 , t. elliott 1 , e. james 1 1 university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd4+ cd25+ regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct26, stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct26-derived cross-protective antigen, gsw11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. this protein has previously been shown to encode ct26-specific cd8 and cd4 antigens, implicating it as a 'hot-spot' for ct26 tumour antigens. interestingly, we have identified a truncated version of gp70 which may be responsible for generation of gsw11. expression studies have revealed increased gp70 expression in ct26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw11) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p1 pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p4, p6 and p9 tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp2 molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp85-99, bound to hla-dp2 with high affinity (10-30 nm). binding studies of mbp85-99 derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f91a, f92a and k93a) were affected, and only by a 20-50 fold reduction in affinity for hla-dp2. the observation that none of the substitutions resulted in a complete loss of binding to hla-dp2 indicates that 1) hla-dp2 binding to peptides does not depend on a large hydrophobic residue accomodated in p1, or 2) mbp85-99 can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp2 molecule binds the immunodominant epitope in ms, mbp85-99, possibly in more than one register. additional studies are required to resolve the hla-dp2 peptide binding properties, and to determine whether expression of hla-dp2 affects the disease course in ms patients. results: depletion of mncs for cd14+ cells abrogated the tg-induced cytokine production and proliferation of cd4+ t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g 98 % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd4+ t cell proliferation was significantly reduced in cd14/cd19-depleted mnc cultures, as compared to cultures depleted for cd14+ monocytes alone. the same applied to the production of il-2, il-6 and tnf-a. production of ifn-g and il-10 was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa 1,2 , p. pala 1 , g. miiro 3 , j. todd 3 , p. kaleebu 1,2 , n. imami 2 , f. gotch 2,3 1 mrc uganda, basic science, entebbe, uganda, 2 imperial college, london, united kingdom, 3 mrc uganda, entebbe, uganda background: hiv-1-specific t-cell responses are preserved in hiv-1 infected individuals with non-progressing hiv-1 disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv-1 for g 8 years, maintaining cd4+ t-cell counts g 500, and with minimal cd4+ decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: 17 art naï ve hiv-1 infected patients from the entebbe cohort in uganda were recruited and stratified by cd4+ t-cell count, cd4+ decline slopes, and time of enrolment, into 2 groups -10 ltnp and 7 rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd4 t-cell count, and ifn-g, il-2 and il-4 elispot responses to pools of 22 to 34 peptides (18-mers overlapping by 10aa). peptides were based on consensus sequences of gag and nef from hiv-1 clades a1, a2 and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il-4 responses were significantly higher in the ltnp than in rp (p=0.02, ifn-g responses to gaga2 pool 1; p=0.04, il-4 responses to gaga1 pool 2). il-2 responses were low and not significantly different between ltnp and rp. there was a positive correlation between il-4 responses to gaga1 pool 2 and cd4 t-cell counts (r 2 =0.502, p=0.04), but no correlation between either il-4 or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv-1 specific responses were higher in ltnps than in rps confirming previous results. non-specific il-4 responses were high possibly reflecting baseline th2 responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her2/neuand her2/neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using 7-aad for dna analysis and specific antibodies directed against the proliferation marker ki-67, the m-phase specific phistone h3 as well as for the h-2l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her2/neuversus her2/neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g 0 /g 1 -, s-and g 2 /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g 0 /g 1 -phase, which continuously raised and peaked within the s-phase. however, h-2l d surface antigen expression was not altered in her2/neucells during cell cycle progression. in contrast to her2/neufibroblasts the her2/neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h-2l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd207) and ccr6 were cultured from cd34+ stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd34+ derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il-4-dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mø). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to 45 min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mø than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mø have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, 1999) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of 30 healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, 1992, 1999) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x 0,0001), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd4+, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, 2252 patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for 2207 of them. for 538 of these patients hla-drb1 data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p=0.02). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb-1a preparations, whereas no differences were shown for ifnb-1b. an association between hla-drb1*11 and nab-negativity was detected (p=0.028). the known associations between hla-drb1*15 and csf-positive ms and hla-drb1*04 and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb1 potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb-1a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb-1b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart-1 and gp100 more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni 1 , s. lorenzi 1 , f. mattei 1 , f. spadaro 1 , l. gabriele 1 1 istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd8 t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd8a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd8 t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd8 t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg7 thymoma line, we show that type i ifn promote the ability of cd8a + dc to capture apoptotic eg7 cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd8a + dc and the persistence of apoptotic eg7-cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg7-derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd8a + dc. as a result, eg7-loaded dc become competent at inducing ot-i cd8 t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd8a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. (2). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd8+ dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam3csk4, and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd8+ dc but not cd8-dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm1, which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a549 human lung cancer cell line. methods: dm1 induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a549 cells. we examined the morphological change using optical microscope. and gfp-lc3 punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm1 was showed high cytotoxicity on various cancer cell line, especially a549 cells. dm1 treated a549 cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc3 and beclin-1 protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc3 was increased concentration and time-dependently. beclin-1 also was increased. and then, we examined gfp-lc3 punctation on a549/ gfp-lc3 stable cells using confocal. a549 cells were formed gfp punctuation after dm 1 treatment. to confirm these data, we measured cell death ratio using 3-methyladenine (3-ma), an autophagocytosis inhibitor. after 3-ma treatment, dm1 induced cell death was decreased comparing with dm1 treatment. conclusion: dm1 did not induce apoptotic cell death. but dm1 showed several characteristics of autophagic cell death. taken together, dm1 induced autophagocytosis on a549 cells. these finding indicated that therapeutic potential of dm1 by triggering autophagic cell death. s. b. rasmussen 1 , s. r. paludan 1 1 university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr)9, which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr9 initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr9 recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv-1 infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv-1, we found that tlr9 dependent ifn regulatory factor 3 activation was abrogated. in addition, inhibition by 3-methyladenine of phosphoinositol 3-kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr9 dependent recognition. in the ongoing project we are examining how hsv-1 triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko 1 , i. berberich 1 , gk 520 -immunomodulation 1 universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl-2 family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a1 and its human homologue bfl-1 are anti-apoptotic members of the bcl-2 family. a1 is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a1. unless the c-terminal ends of other bcl-2 proteins the tail of a1 does not function as a strong membrane anchor. nevertheless, the last amino acids of a1 are important for the protein. in fact, the c-terminus of a1 serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a1 undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl-2 factor bimel results in increased stability of a1. this is due to reduced ubiquitination of a1 after binding of bimel. we conclude that the cterminus of a1/bfl-1 serves as a docking site for e3 ubiquitin ligase(s) that control the stability of a1 by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e3 ubiquitin ligase that interacted with a1 in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs-1 in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs-1 is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs-1 significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs-1 was manifest not only by the suppression of jaks acting upstream of p38 mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak 1/2 activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs1 which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs-1 overexpressing cells. the results strongly suggest that socs-1 acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl12/sdf-1a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl12 effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u-87, and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p53 and fas-l expression was carried out by western-blot. results: cxcl12 induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p53 pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p53-ser46 levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after 24h of tnf-a treatment. conclusions: cxcl12 induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p53function/levels. s. y. demiroglu 1 , r. dressel 1 1 universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp70 is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp70 on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp70 does not protect against cell death mediated by ctl. acute overexpression of hsp70 can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res 63: 8212) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp70 on granzyme b-induced apoptosis. methods: hsp70 was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp70 induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type 5. results: hsp70 did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp70 in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g1 peak measurements (p=0.01). in contrast, a partial protection of ge-tet cells was observed after acute hsp70 overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp70 does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp70 effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood 102:1788) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk1034 and dr394/2-3. the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr1 and tnfr2, both belonging to the tnf receptor superfamily. tnfr1-mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr2-associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr2, resulting in depletion of traf2 from the cytoplasm. here we confirm that tnfr2 induced depletion of cytosolic traf2 by the use of tnfr1-and tnfr2-specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf2 in the alternative nfkb pathway, we show that tnfr2 induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr2 and membrane tnf. j. c. morales 1 , d. ruiz-magaña 1 , d. carranza 1 , c. ruiz-ruiz 1 1 universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase-8. in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r2 and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp36l1 is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp36l1 may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl-2 protein is an important cell survival protein at different stages of b cell development. bcl-2 mrna also contains are regions that could possibly be targeted by zfp36l1 protein. in the present study, we have tested the ability of zfp36l1 protein to bind to a bcl-2 mrna are probe and to degrade bcl-2 mrna. recombinant bacterially expressed zfp36l1 protein was shown to bind specifically to a bcl-2 are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp36l1 also provided evidence that endogenous zfp36l1 in b cells could bind to the bcl-2 are. in order to examine whether zfp36l1 binding to bcl-2 are resulted in bcl-2 mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp36l1 knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl-2 mrna was expressed in both wild-type and knockout mefs. the half-life of bcl-2 mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp36l1 does play a role in degradation of bcl-2 mrna. overall, our data are consistent with a role for zfp36l1 in degradation of bcl-2 mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp1. since lmp1 is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp1 function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi3-kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te1) and lymphocytic (nc5) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes1 or tes2 (transforming effectors site) derived from the c-terminal intracellular part of lmp1, in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp1's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase-8. using this nc5 tumorigenic model in scid mice, we showed that induction of the dn lmp1-tes2 prevent development of tumours and mouse death. these dominant negative derived from lmp1 could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs1 and socs3 differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs1 exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p38mapk activities, while socs3 promoted apoptosis with an increase in p38 activities. in contrast, both socs1 and socs3 display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs1 or socs3 induced a slight decrease in g1 or s phase cells and a prominent increase in g2/m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g1/s transition and g2/m arrest induced by socs1 or socs3. socs1 promoted dna damage-induced p21 induction and g2/m cyclin b expression, while socs3 induced decrease in g1 cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein 90 (hsp90) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp90 has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp90 can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp90 is released during necrotic cell death and proinflammatory effects of extracellular hsp90 have been observed. thus, we asked whether apoptozing cells cleave hsp90 during apoptosis or how hsp90 is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp90 protein content. we observed that hsp90 is degraded during apoptosis resulting in the formation of a fragment of about 50 kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp90 (hsp90 alpha and hsp90 beta) we could show that the 55 kda fragment is formed after degradation of the alpha isoform of hsp90. further, we were able to show, that hsp90 cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp90 and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp90 was inhibited or if exogenous hsp90 was added. these results demonstrate that cleavage of hsp90 represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc3-i is processed to lc3-ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf-7 gfp-lc3 cells were therefore incubated in starvation medium for 3 hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl-2/beclin-1 control of autophagy. recently, jnk was shown to be necessary for beclin-1 upregulation, and jnk-mediated phosphorylation of bcl-2 is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf-7 cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol 3-angelate (pep005), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep005 provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb4 turned their response to pep005 from an increased to decreased rate of apoptosis. furthermore, our data show that pep005 inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl-1 and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p75, trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch11 and analysed by flow cytometry. expression of the caspase 8 inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch11 treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase 8 in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase 8 by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases 9 and 3. diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor 1 (tnf-r1), and tumor necrosis factor (tnf)-a receptor 2 (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid 1-o-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine (et-18-och 3 ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd95 independent of its ligand fasl/cd95l. fas/cd95 is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin-1, an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl2-family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment (5-fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo 201, sw1116, lovo, caco-2, ht-29) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki-67, pcna, p53, bcl-2) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in 88 patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl-2, bax, caspase 3, 6, 9, nfkb, parp1/89kda, tnfr1/cd120a and cd95/fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl-2, bax, nfkb, parp1 was significantly lower and expression of caspases, tnfr1 as well as percentage of cd95 cells significantly higher as compared with control group. caspase 3 expression was significantly higher as compared with nfkb, parp1 and tnfr1. in comparison to the standard nutrition preoperative immunonutrition increased bcl-2 and nfkb expressions and decreased caspases and parp1 expressions. in addition, we found a significant down-regulation of bcl-2 expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega-3 fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x 0.01). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g1 phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x 0.007). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with 54 healthy volunteers receiving either la1 (10 9 cfu/day) or placebo, during 57 days prior to uv (2 × 1.5 med). blister roofs, liquid and skin biopsies were collected 1, 4 and 10 days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p53). while a similar decrease of lc for both groups was observed on day 1 after uv exposure compared to placebo, la-1 group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd1a low cd207 -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il-10 and a tendency to inhibit il-6 was observed in la-1 group compared to placebo. on day 4, la-1 group presented a greater recruitment of early lc precursors and a trend to increase cd1a low cd207 + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il-6, tnfa, il-8, and il-10) was observed in la1 group compared to placebo. we show that la1 limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la1 supplementation for photoprotection at a lower dose (1 log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn1 is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn1 appears to be expressed in all epithelial cells of the early thymic rudiment starting around e11.5. previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn1 in a nude (foxn1-deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn1. if true, it should be possible to target this cell type by use of foxn1-promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either 19 or 82 glutamine residues under the control of foxn1 promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn1 promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei 1,2 , y. toriumi 3 , k. kimura 4 1 european university viadrina, frankfurt (oder), germany, 2 shimane institute of health science, izumo, japan, 3 shimane university faculty of medicine, department of pediatrics, izumo, japan, 4 japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a 10-minute-rest period, followed by a 15-minute yoga exercise called asana, a 15-minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a 20-minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp2). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r=0.83, p x 0.02), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r=0.77, p x 0.05) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd4 (r=0.96, p=0.0001). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd4, within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper 2 response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd4 + cd25 + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox40l expressed on mcs and the constitutive expression of ox40 (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox40l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca 2+ responses and degranulation. the cross-talk between t reg cells and mcs through ox40-ox40l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th1 or th2 effector cells and the induction of antigen specific foxp3+ regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th2 cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th2 and treg subset, gata3 and foxp3 respectively, counter regulate each other (mantel y et al., 2008) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp3+ tregs and high gata3+ th2 cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost 100 million allergic patients are sensitized to the major birch pollen allergen bet v 1, which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v 1 recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v 1 molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g 90 %) of patients' ige binding to bet v 1 was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa 30-59 (p2) and aa 74-104 (p6) of bet v 1. cross-reactive ige epitopes between bet v 1 and related pollen allergens and plant food allergens involved primarily p2. p2 and p6 are not adjacent peptides in the bet v 1 sequence but define a surface-exposed patch on the three-dimensional structure of bet v 1. as determined by surface plasmon resonance, monoclonal antibody mab2 specific for p2 and mab12 specific for p6 showed high affinity, i. e., dissociation constants, k(d) = 8.35e-11 m and k(d) = 1.05e-9 m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p2 and anti-p6 antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v 1 allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th1 cells as well as tc1 cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th2 immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th2-mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th1/tc1 cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd4 + and cd8 + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd4 (gk1.5) or anti-cd8 (53.6.72) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr-1 monoclonal antibody rb6-8c5 or monoclonal antibody nimp-r14. one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd8 + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd4 + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd8 + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd8 + t cells, cd4 + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd4 + or cd8 + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th1 and tc1 cells operative in the elicitation of airway inflammation. conclusions: robust type 1 immune responses, although highly effective in the counter-regulation of local th2-mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h292. the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p 1, we were able to show that the major allergen of phleum pratense, phl p 1, although sharing molecular similarities with der p 1, does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p 1. chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il-6 and il-8 from nci-h292 cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p 1. in contrast to hdm extract grass pollen allergens induce the release of mcp-1 from respiratory epithelial cells, as well as moderate levels of il-12. detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr22) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, 3 distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): 7.2±5.7) and of il-13 (mean±sd: 1157±909 pg/ml), and reduced il-10 (mean±sd of il-10-spot forming units/2x10 5 cells (sfu): 912±510), compared to healthy subjects (3.4±2.7 si, p x 0.05; 355±396 pg/ml, p x 0.05; 1272±623 sfu, for proliferation, il-13 and il-10, respectively); children with non-ige mediated cma had a significant reduction of il-13 (192±362 pg/ml), compared to ige patients (p x 0.0004) and an increased, although not statistically significant, production of ifn-g (33.7±54.6 sfu) compared to control (9.5±9.5 sfu) and to ige-allergic patients (0.6±0.8 sfu). finally, tolerant patients showed reduced il-13 (636±1048 pg/ ml, p x 0.05) and proliferation (3.9±3.5 si), compared to acute ige-cma children. interestingly, the high level of il-10 observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il-4 was undetectable in all patients even blocking the il4-receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th2-like response with il-13 and proliferation is dominant in ige-mediated cma patients; by contrast a th1-skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang 1 , b. chua 1 , f.c. lew 1 , a. ho 1 , k. wong 1 , k.l. wong 1 , d. m. kemeny 1 1 national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd4 th2 t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th2 cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd8 t cells inhibits the induction of the th2 response. here we have investigated the effect of cd8 t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd8 t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of 3 airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd8 + t cells (36.7 %±4.1 % to 17.6 %±2.7 %). when ifn-gamma -/-ot-i cd8 t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced (39.6 %±5.1 %), suggesting an important role for cd8 t cell ifn-gamma. cd11c + cd103 + cd11blung dcs from cd8 transferred mice secreted higher levels (500pg/ml) of il-12p70 following ex-vivo stimulation as compared with animals that were not given cd8 t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd8 t cells can subsequently divert the local lung environment to one that favors th1 immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in 8 % of patients with hiv infection. this reaction is strongly associated with hla-b*5701 and appears to be mediated by cd8+ t cells producing inflammatory cytokines. we show that cd8+t cell responses can be primed in vitro, in normal blood donors who are hla-b*5701+, but not in non-b*5701+ donors. cd8 t cells, but not cd4 t cells, are expanded by abacavir pulsed autologous apc over a13-day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b*5701. similar responses were detected in abacavirhypersensitive hlab*5701+ patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b*5701 since they were undetectable using apc expressing closely related hla-b57 or b58 allotypes. responses to apc expressing mutants of hla-b*5701 demonstrated a crucial role for residue 116. isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b*5701 triggering cd8 t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b*5701 may be relevant to the role of other disease-associated class i allotypes such as hla-b27 and b51. a. jenckel 1 , s. bulfone-paus 1 , n. föger 1 1 research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin1a (coro1a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro1a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro1a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro1a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro1a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro1a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro1a during mast cell activation. cell fractionation experiments demonstrated that the association of coro1a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro1a phosphorylation. a functional correlation between coro1a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro1a. thus, coro1a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin1a (coro1a) deficient mice have demonstrated that coro1a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin1b (coro1b) is a closely related homolog of coro1a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro1b deficient mice and crossed them with coro1a deficient mice to obtain coro1a/coro1b double deficient mice. analysis of t lymphocytes from coro1a/coro1b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro1a/coro1b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro1a/1b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro1a/1b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn1 (also known as hacs1 or sly2) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology 3 (sh3) domain and a predicted bipartite nuclear localization signal. the samsn1 gene is located on mouse chromosome 16 and encodes a well conserved protein with 364 amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn1 in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn1 expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn1 in all tested hematopoietic cell types. the highest expression level of samsn1 mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd4+ and cd8+ t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly1 (hacs2) and sly3 (sash1) -were expressed only at a very low level in mast cells. the high level of samsn1 mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn1 in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn1 deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn1 deficient mast cells. in additional studies we are now investigating the effects of samsn1 deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn1 in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg1 antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., 2008). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg1 abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st8siai-v; st6galnac i-iv, st3gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg1 abs. results: we observed that the expression of st3gal i, iii and v coding genes was similar in both hybridomas, while the st3gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg1. in addiction, the expression levels of st8sia and st6galnac genes in the hybridoma producer of anaphylactic igg1 were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg1. conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg1 abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h 2 cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after 2 times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il-5 and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il-5 and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf1 mediates the differentiation into th1 cells by activating multiple genes which are independently crucial for the development of naive t cells into th1 cells. because irf1 is expressed in many different tissues, it can be considered as a master switch factor for th1 cell differentiation. methods: here, we tested mice deficient in irf1 in the murine acute asthma model to evaluate its importance in this th2 cell-mediated disease. the protocol setup was the following: 3 sensitizations s. c. with ova, followed by 3 challenges via ova inhalation and adoptively transferred wildtype cd8+ t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf1 deficient mice with the help of adoptively transferred cd8+ t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by 3.5 fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. 1) were also significantly higher in irf1 deficient mice. conclusion: interferon regulatory factor 1 plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd8+ t cells. mechanistically, a potential counterregulation of asthma by th1 cells is not available in irf1 knockout mice. together with our previous report that irf1 represents a susceptibility gene for allergy in the human, our data highlight irf1 as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c57bl/6 mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for 24hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk1-receptor and ppt1 mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd11c expressing apc substets characterized by cd4 and cd8 expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th-1 cytokine profile and increased levels of il-2 mrna. further the number of cd25 + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: 1) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase3, 2) asthma and ee are prototypic th2 diseases in which the cytokines il-4 and il-13 play a principal role through the activation of the transcription factor stat6, and 3) we have recently demonstrated that some chloromethyl ketones can downregulate stat6 by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat6, we analyzed its effect in the activation of stat6 by il-4 and il-13. we found that treatment of cells with ppis inhibited the ability of il-4 and il-13 to signal stat6 activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th2 mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied 12 asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd4 and cd25 high (treg)), that might inhibit the development of a th2 response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for 48 hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd69 or cd25 at 48 hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h4r. the level of intracellular ccl2 production in human lc was reduced after stimulation with h4r agonists and basal production could be restored when h4r was blocked with the specific antagonist jnj7777120. moreover histamine and a h4r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h4r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h4r in allergic skin diseases and encourage further exploration of the h4r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g1p3, an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g1p3 was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g1p3 which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd20 antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th2 to chronic th1-predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received 4 weekly intravenous infusions of rituximab at a dose 375 mg/m 2 body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g 5,000; g 6,000; g 19,000 mg/dl, respectively). all patients underwent prior treatment with omalizumab for 6 months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the 2 nd week after initiation of rituximab therapy up to 1 year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up 1 year), ige levels decreased from 5,512 to 1,500 mg/dl. the other two patients are in the 3 and 1-months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd20 antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) 1 and th2 cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either 1 %dncb, 25 %tma or to vehicle alone for 0.5-6h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il-2 (mean=131pg/ml, n=12, p x 0.001), il-17 (mean=69pg/ml, n=12, p x 0.001) and il-1a (683pg/ml, n=12, p x 0.001) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of 50ng/ear of murine recombinant il-2 or of the known regulator of lc migration; il-1b. interleukin-1b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after 4 (32 %) and 16h (31 %) (n=6, p x 0.001). in contrast, il-2 or control injections were without effect. however, il-2 administration caused an increase in cutaneous il-17 production (347pg/ml, n=12, p x 0.001) compared with control injection and naï ve tissue (19 and 63 pg/ml, n=12, ns). in addition, systemic treatment with anti-il-2 antibody failed to impact on lc migration provoked by dncb (33 % reduction; n=6, p x 0.001). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il-1a (987pg/ml, n=10, p x 0.001) and il-17 (248pg/ml, n=6, p x 0.01) expression was reduced to baseline levels by anti-il-2 treatment. these data demonstrate that il-2 is not involved in the regulation of lc migration, unlike il-1b and tnf-a. however, il-2 is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il-2 may influence lc and dermal dc maturation, via the expression of il-1a and il-17. allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey 2005), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p38-mapk-pathway related protein (p38prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p38 mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct-2005 -018681, www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, 15 % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il-1 receptor antagonist (il-1ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il-1b and of il-1-like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il-1-related genes by real-time pcr on mrna from blood cd14+ monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il-18, il-18bp, and caspase-1, both before and after therapy with kineret. il-1b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il-1b, tnfa, il-12) nor anti-inflammatory cytokines (il-10, tgfb) were detected. as expected, il-1ra was only detectable after therapy. il-18 was detectable in schnitzler's sera at higher levels than in controls (23.0 vs. 10.3 pm) and decreased after therapy (13.2 pm). the circulating il-18 inhibitor il-18bp was lower than in controls and not affected by therapy. thus, free il-18 levels were increased in schnitzler's patients as compared to controls (17.6 pm vs. 7.2 pm in controls) and decreased after therapy (9.9 pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il-1-related cytokines (il-1b, il-18), nor of the il-1/il-18-converting enzyme caspase-1 in blood monocytes. however, the high circulating levels of il-18 suggest an increased activity of caspase-1, as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around 20 % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr3 signal via myd88 to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd88 signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c57bl/6 myd88-deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd88-/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg1-titers in ova-treated myd88-/-mice compared to wildtype mice, although the nacl-treated myd88-/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over 15,000 species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies (6 mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = 4) or from non-infested contra-lateral sites. expression of mip-1a, igf-1, mcp-1 and ip-10 genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd3, cd4, cd8, cd21, mhc class ii, and p46 than that of holsteins. lymphocytes expressing wc1 and cd21 antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x 0.05). infested skin of nelores contained significantly more message for mip-1a, igf-1, mcp-1 and ip-10 than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th2 response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd3+ t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il-1 plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa-1 in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam-1/lfa-1 binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th1 than a th2 local response pattern by virtue of il-1 secretion and icam-1/lfa-1 interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th2 like micromilieu in acute towards a th1 dominated milieu in chronic lesions. the genotyping of ccl4l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi07/0329). psoriasis is an inflammatory dermatosis with 2 % prevalence among caucasians. hla-cw6 allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in -308 and -238 positions. strong association was found between polymorphisms in the -238 region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b27, and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to 40 years of age) were selected. hla-a -b -c -dr -dq alleles and tnf-238 and -308 snp were differentiated by pcr/ssp. analyzing the total group, 21 patients (30.5 %) were male, 48 (69.5 %) were female. in the male group, the mean age at disease onset was 16,3 years. severe forms were seen in this group (psoriatic arthritis in 4 cases and erythroderma in 2). seven patients (33,3 %) had a favorable evolution of the disease, but 14 (66,4 %) developed extensive psoriasis, covering over 30 % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw*06 allele was positive in 10 cases (47,62 %), tnf 238 g/a genotype was found in 5 (23,8 %) and tnf 308 g/a in 4 (19,1 %). in the female group, the mean age at disease onset was 14,6 years, one case of psoriatic arthritis and one of erythroderma. twenty-nine (60,4 %) had a favorable evolution of the disease and 19 (39,6 %) an unfavorable evolution. cw*06 allele was positive in 26 cases (54,2 %), tnf 238 g/a genotype was presented in 16 (33,3%) and tnf 308 g/a in 8 (16,6 %). severe disease was seen in male patients. there was no difference in frequency of cw*06 allele between male and female groups, but there was a tendency of significant difference in tnf 238 g/a genotype. we found that c57bl/6 mice were more susceptible than balb/c and dba/2 mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il-6 and mrp8/14, among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: 136 cases, which were studied retrospectively, included children (84 boys and 52 girls), aged 3-14 years, who had an anaphylactic reaction, out of the 915 that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food (44 %-particularly sea food and dried fruit), drugs (25 %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites (9 %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema (76 %), and gastrointestinal symptoms (37 %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g 100 was found in 26 out of the 47 severest cases (55,4 %), in which the adequate control was held, while in their vast majority (45 out of 47) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in 53,7 % (73 cases) there was a hereditary family history of atopy, while in 52 children (38 %) there was also a personal history of asthma. finally, at a great percentage (69 %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that 1)the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. 2) in particular, in many cases it is proved that there is a personal but also a family history of atopy. 3) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd86 we could observe dose dependent upregulation of programmed death ligand 1 (pdl-1) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a 60 year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since 35 years. in march 2006 a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters (120 mg) every second day. in april 2008 the patient presented himself in the consult with multiple livid papules with a diameter of 3 mm in the area of the auricle. the histological examination showed an hhv-8 positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th1 type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs1, socs3 and foxp3 in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n:4), the patients who showed recurrence or progression after treatment (non-responders, n:4) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n:7) were evaluated for socs1, socs3 ve foxp3 mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd3, cd4, cd25, foxp3, cd4 + cd25 high , cd4 + foxp3 + . expression of socs3 and foxp-3 mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs1 was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd25, foxp3, cd4+cd25 high , cd4 + foxp3 + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs1, socs3 and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs1 and socs3 molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of 62 silicosis patients, with mild (21), moderate (23) and severe (18) silicosis, 92 coal workers, exposed to inorganic dust containing fcs (exposed), and 43 healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than 0.05 was considered significant.neopterin level was significantly elevated in exposed (3,2±0,8ng/ml) compared to controls (1,8±1,3ng/ml; p=0,0001). moreover, the neopterin level in exposed was similar to silicosis patients (2,9±1,6ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls (81,9±22,7au vs 64,3±16,7au p=0,0001) and to silicosis patients (69,6±20,0au p=0,002). in contrast, igmcic was significantly elevated in silicosis than in exposed (70,2±18,8au vs 62,1±24,2au; p=0,03).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p=0,001 and 0,02 respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a*01, b*08, drb1*03 (these of good prognosis) *12/*13/*14/*15) or tbc (drb1*02/*05/*14/*16 and associations with dqa1*02/*03/*05) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a*01, b*08 and drb1*03 (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a*01, drb1*14. as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd4 + , cd25 ++ , foxp3 + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein 60 (hhsp60). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd4 + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il-17, il-6, il-8, tgf-b and ifn-g production prevailed, pointing to a th17/th1 weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp60. importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th17/th1 cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp60 might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices (1) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a 384 well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c3-complement, myeloid related protein 14 and two new proteins, integrin-ß4 and fibrinogen. data from more than 100 patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than 10 % and 16 %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least 3 mice per group with distinct cytokine pattern: th1 (c57bl/10, c57bl/6) and th2 (balb/c). muscular injury was performed by injection of bupivacaine. both th1-dominant strains presented more areas with regenerating myofibers and macrophages at 4 dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd3 bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at 4 dpi. balb/c mice showed increased collagen expression and decrease of mmp-9 activity associated with more mrna for tgf-b1. this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th1 cytokines) or myonecrosis and collagen deposition (th2 cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß1-adrenergic receptor (anti-ß1ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß1ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß1ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß1ec ii -specific rat monoclonal antibody (clone 13f6), and showed by elisa that it binds to the linearized ß1ec ii peptide. additionally, we confirmed with flow cytometry that 13f6 also binds the ß1ec ii in its native conformation, i. e. directly labeled circular ß1ec ii (dyl649-ß1ec ii ) peptide. moreover, we demonstrated activation of the ß1-adrenoreceptor by 13f6 using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß1ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß1ec ii -specific peptide-based therapy, by intravenously applying a circular ß1ec ii peptide in the dcm lewis rat model to neutralize the anti-ß1ec ii antibodies. while the peptide therapy strongly reduced the anti-ß1ec ii titers in the serum by up to 80 % and consecutively lead to clinical remission, elispot assays for the detection of ß1ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß1ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß1ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b3 or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca 614-629 epitope elicits autoreactive cd4 + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca 614-629 peptide. in a first step, hybridoma cells were generated by fusing bw5147 tcra -cd8lymphoma cells with myhca 614-629 -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca 614-629 -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle 1 , a. schwarting 1 , p.r. galle 1 1 university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase 3 (pr3), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr3 in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr3-positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il-8. pr3-mrna from the murine cancer cell line wehi-274 was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr3-promoter in the second intron of the mouse pr3 gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr3 transcript and its promoter indicates that the murine pr3 may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th1 mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd1d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th2 cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n=13) or pbs (n=11) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr4 activation, in the presence/ absence of och. subsequently we transferred 1.5x10 6 mdcs (n=11) or och-primed mdcs (n=11) (3 times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a 70.6 % reduction in plaque size compared to mice treated with mdcs (p x 0.05). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant 23.7 % (p x 0.05) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a 2 to 3-fold increase in these cells was detected 3 days after the last treatment with och-primed dcs (p x 0.05). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il-10. discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th2 phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c57bl/6 background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed 350 igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c57bl/6 and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr3 regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg2c were isolated only from fcgriib deficient mice, but not from c57bl/6 control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box 1 protein (hmgb1), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb1. we investigated if hmgb1-containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb1 remains associated with ncs released from late apoptotic cells in vitro. hmgb1-ncs complexes were detected also in the blood of patients with sle. hmgb1 containing ncs from apoptotic cells induced secretion of il-b, il-6, il-10, and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd88 and toll-like receptor 2. neither hmgb1-free ncs from living cells nor from apoptotic hmgb1-or hmgb1/2-deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb1 activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il-10 release by macrophages. immunizations with hmgb1-containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr2-dependent manner. in conclusion, hmgb1 in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher 1 , k.p. hoefig 1 , e. kremmer 1 , v. heissmeyer 1 stretches and helps in the selection of the correct splicing borders. a allele of (r61h) creates a strong binding site for a splicing enhancer protein srp40 according to bioinformatics. our findings indicate that, the putative branch point, r61h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank1. finally, we believe that bank1 delta 2 protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta 2 protein may contribute to the observed reduction in sle susceptibility. s. beermann 1 , r. seifert 1 , d. neumann 1 1 hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h 1 receptor (h 1 r), h 2 r, h 3 r, and h 4 r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd3 antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or 4-methylhistamine, a h 4 r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and 4-methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c57bl/6 mice induced by either a-cd3 antibodies or cpg-odn. this histamine effect was completely inhibited by the h 2 r-specific antagonist famotidine, while h 4 r-, h 1 r-, and h 3 /h 4 r-selective antagonists had no or only moderate effects. interestingly, the h 4 r-selective reagent jnj7777120, which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd3 induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h 4 r, jnj7777120 may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h 2 r and, to a much lesser extend, the h 4 r. using this assay system, cells obtained from control c57bl/6 mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega-3 fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of 20 ng/ml resolvin e1(rve1) on the release of tgfb, il-6 and il-17 in the culture of peripheral mononuclear cells (5x10 6 /ml) stimulated by phorbol ester (pma) (1nm), and the combination of pma and ionomycine (3 mg/ml) for 72 hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from 7 healthy subjects and from 10 patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = 0.010) and sjögren's (p=0.017) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve1 significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p=0.041) or pma+ionomycin (p=0.021), however rve1 was ineffective at reducing tgfb release in the sle and ss patients. rve1 caused a non-significant decrease in il-6 release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il-17 was not significantly modified by rve1 in any of the groups tested. the release of tgfb by 20 ng/ml of rve1 can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve1 tested, il-6 and il-17 release were not significantly affected in healthy or autoimmune patients. omega-3 fatty acid derived rve1 may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis 1 , a.k. tsirogianni 1 , m. herrmann 2 , h. m. moutsopoulos 1 , m.n. manoussakis 1 1 university of athens, dpt pathophysiology, athens, greece, 2 university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included 29 with primary ss (american-european criteria 2002) and 14 with sle (acr criteria 1997). age-and sex-matched healthy blood donors to the ss and sle groups (13 donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, 2007) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma (5 patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd68-staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p=0.0001). in ss patients, defective mb-phagocytosis involved only monocytes (p x 0.0001) and significantly correlated with the presence of extraglandular manifestations (p=0.02). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x 0.0001) and of ss (p=0.001). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: 30 healthy persons have been included in our study and 71 sle patients (66 women and 5 men) with various clinical signs (44 persons had 1 st degree of disease activity, 27 persons -2 nd degree of pathological process activity). 18 women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b 2glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in 36,6 %, aphl of igg class -in 45,1 % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x 2 =6,4; p x 0,02). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x 2 =3,9; p x 0,05). the increased levels of anti-ada were revealed in 11 of 18 women with hnp, and the combination of anti-ada and aphl (9/18) was found out more often than isolated anti-ada (2/18, x 2 =6,5; p x 0,02) or isolated aphl (3/18, x 2 =4,5; p x 0,05). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc1 and mdc2, and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc1, mdc2 and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from 31 pss patients fulfilling the american european consensus group criteria (aecc) and 28 gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p=0,0002) and mdc2 (p x 0,0001) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd5+ b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin 21 (il-21) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd5+ b cells and increased il-21 levels. we therefore hypothesized that il-21 may have similar biological effects on cd5+ b cells in autoimmune diseases. here we demonstrate that the amount of il-21 in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to 14 % of cd5+ b cells in sle individuals expressed grb. in-vitro experiments revealed that il-21 was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor 9. importantly, il-21 significantly decreased the cd5+/cd5-b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd5+ b cell death. these results suggest that il-21-induced grb may play a regulatory role for cd5+ b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il-21 and grb levels in sle and showing that il-21 reduces the cd5+/ cd5-b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il-21 may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il-21 in sle and other autoimmune diseases. r. de palma 1 , e. d'aiuto 1 , s. vettori 1 , g. abbate 1 , g. valentini 1 1 second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd4+ t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il-1beta, il-6 and il-8, cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed 169 igg antibodies from single facs purified cd19+cd27+cd38+cd138+ bone marrow plasma cells of 3 patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran 1 , e. moazemi godarzi 1 , e. kamali sarvestani 1 , e. aflaki 1 1 shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin 6 (il-6) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il-6 gene at positions -572 g/c and -174 g/c have been described that are key regulators of il-6 gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il-6 gene polymorphism at -174 position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the -572 position significantly differed in sle patients and controls. at this position gg genotype was observed in 77.9 % of patients compared to 68.9 % in the control group (p x 0.014). the frequency of -572 g allele in patients (87.3 %) was also higher than in controls (83.2 %) (p=0.034). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. -174 polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x 0.04). at -572 polymorphism, a significant difference with regard to photosensitivity in male patients (p=0.04) was found. in conclusion, results of this study showed that -572 polymorphism plays an important role in susceptibility to sle and that -174 polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained 161 patients with ibd (89 with cd, 41 with uc, 31 with gluten sensitive enteropathy, gse) and 33 healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of 7 asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd4-ve cd8b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd4(+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling-1 (rgs-1) protein, we have now found substantial over-expression (10-100 fold) of rgs-1 in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs-1 decreases primary t cell responses to cxcl12 and ccl19, strongly implying that it may regulate t cell localisation. thus, rgs-1 may be a novel target for modulating t cells in ibd, consistent with which snps in rgs-1 have been associated with both coeliac disease and type 1 diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c57bl/6j mice were divided in control (c) and experimental (e) groups. c1-c3 received peanut protein immunization, animals of the control groups c4 were sham immunized, and control group c5 received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a 30 day peanut containing cd, the experimental group was divided into 5 groups (e1-e5). ova feeding began 7 days prior cd (e1) on day 0 (e2), 7 (e3), 14 (e4) and 21 (e5) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e1) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio 2 as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase 1 and secretion of bioactive il1-b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il1-b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying 5 % destrane sulphate sodium (dss) in the drinking water for 8 days to wildtype mice. when ultrafine nanoparticles were administered on day 6 and 7 by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il10 -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl 1 , n. schneiderhan-marra 1 , m. blum 2 , g. stein 2 , m. schmolz 2 , t. joos 1 1 nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, 2 edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [1] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco-2 cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of 24-well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h 1-associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h 2-like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il-17 production and t h 17 polarization and decreases recruitment of cd4 + cd25 + foxp3 + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd11c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd11c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h 17 and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen 1,2 , g. van assche 3 , p. rutgeerts 3 , a. liston 1 , j. l. ceuppens 2 1 k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, 2 k. u. leuven, experimental immunology lab, leuven, belgium, 3 k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp-1 allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced 3 types of colitis dss (th1/th17), tnbs (th1) and oxazolone (th2) in hp ko mice. neutralizing anti-il-17 mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th17/th1 cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. 1) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; 2) in dss but not in oxa colitis mice, il-17, ifn-g, tgf-b and il-6 were significantly increased (p x 0.01, dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x 0.05, ko vs wt). in tnbs colitis, we found elevated il-12 and ifn-g (p x 0.01, tnbs vs control). although not significant, il-17 was also somewhat upregulated; 3) in dss colitis we observed that il-23 enhanced differentiated th17 cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il-6 and il-21, more mln-t cells from hpko mice differentiated into th17 cells; 4) anti-il-17 mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il-17 showed reduced il-6, il-17 and ifn-g in both mln and lp (p x 0.05, anti-il-17 vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il-17, supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann 1 , g. talabér 1 , g. süt" o 2 , p. németh 1 , t. berki 1 1 university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, 2 university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that 2.2 million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd4+cd25hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th1 and th2 cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd4+ and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd4+ inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells (74.5 ± 3.5 %, mean ± sem), while cd4+ inkt cells ratio was moderate (25.4 ± 3.5 %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: 38.0 ± 7.1 %; cd: 34.0 ± 5.2 %, mean ± sem), while the percentage of cd4+ inkt cells was elevated (uc: 62.0 ± 7.1 %; cd: 65.9 ± 5.2 %, mean ± sem) in both disease groups. proportions of foxp3+ treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b1 gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b1 adenovirus (tgf-b1-exo). the t cell inhibitory function of tgf-b1-exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b1-exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b1-exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd4 + foxp3 + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b1-exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b1-exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd4 + foxp3 + regulatory t cells (treg) revealed that the relative numbers of cd4 + foxp3 + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b1 gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd4 + foxp3 + treg. tgf-b1-exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd8+ t-cells was similar in the four vaccinees as observed by ifng, il-2 production-profiles. however, the killing capacity of expanded cd8+ t-cells was distinct as observed by the competence to kill ns3-peptide presenting transfectants in vitro. as depicted in figure 1 , cd8+ t-cells cells from both vac1 (cleared ) and vac2 (chronic) produced il-2 and ifng after stimulation with ns3-peptide59. however, specific killing of the peptide loaded transfectants was only observed in vac 1, who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure 1 ] killing of ns3 peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd8+ t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd8 t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns3 from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr4-expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns3 might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns3 or eda-ns3 under the control of cmv promoter were prepared. recombinant ns3 and the fusion protein eda-ns3 were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd40 agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns3 rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns3. results: immunization of mice with the plasmids expressing eda-ns3, but not ns3 alone, induced strong t cell responses against the main hla-a2 restricted cytotoxic t cell determinants from ns3. the recombinant eda-ns3 fusion protein, but not ns3, was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp-1 monocyte cell line. immunization of hhd mice with eda-ns3 fusion protein induced both cd4+ and cd8+ t cell responses against ns3 and, when immunized with poly(i:c) and anti-cd40 antibodies, was able to down-regulate the intrahepatic expression of hcv-ns3 rna. the recombinant eda-ns3 fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even 25 years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e2 protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd207+cd1a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd8+ responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to 3 years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd1a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd8+ t cells by mature dcs transduced with melan-a-recombinant human coronavirus 229e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin 2 (il2) and interleukin 15 (il15) will be involved, and fms-like tyrosin kinase 3 ligand (flt3l) will be expressed to modulate dendritic cells. il2 is known to enhance early t cell expansion and limits t cell overshoot, whereaes il15 guarantees survival of high affinity t cells during memory phase. on the other hand flt3l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik 1 , ag waisman 1 uniklinik mainz, 1. med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd8 t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il-10 production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd8 t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response 7 days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il-10 produced by b cells by conditional deletion of the il-10 gene in these cells. we found that b cell secreted il-10 has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb490 to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from 8 healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for 10 days in the presence of il-2 and il-7 with exposure to overlapping peptide pools of latent ebna-1 and lytic bzlf-1 antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd3, cd4, cd8, ccr7, cd45ra, il-2, tnfa, ifng and cd107a. the majority of ex vivo ebv-reactive cd4+ t cells as well as ebna-1-reactive cd8+ t cells were il-2 and tnfa-producing memory cells, the later being more frequent in bone marrow (cd4+, median, ebna-1: bm 0.69 %;pb 0.12 %; bzlf-1: bm 0.37 %;pb 0.01 %, p=0.039). after in vitro expansion a major subset of ebv-specific cd4+ and cd8+t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd4+ and cd8+ t cells retained, however, a tnfa single, tnfa/il-2 or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd4+ and cd8+ t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr7/cd45ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il-2 delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd4 + and cd8 + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd154 or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd4 + and cd8 + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd8 + and cd4 + t cells in peripheral blood with a hexon protein-spanning pool of synthetic 15-mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to 29-90 % in the t cell lines and the absolute numbers of both hexon-specific cd4+ and cd8+ t cells were 2 to 3 log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd8 + and cd4 + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype 5 (ad5) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad5 vaccine vectors expressing whv core protein was first examinated in c57bl6 mice. groups of mice were immunized with a dna prime-ad5 boost regimen or with dna and ad5 alone. ad5 was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd8+ t cell responses against peptide pools 1 and 3 in spleens of dna and dna-ad5 immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd8+ t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad5 very weak cd8+ t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad5 level of antibodies did not change. those antibodies were only igg2a subclass what indicates th1 t helper type of response. ad5-immunized mice had over 3-fold lower level of anti-whc: both igg2a and igg1 subtypes were detected. the weak response induced by ad5 may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m2e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal 2007) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m2e serum ab titers against peptide and native m2e. this correlated with a large number of m2-specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h1n1), which contains a variant m2e-sequence different in 3 amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x31 (h3n2) and the highly pathogenic pr/8 (h1n1) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m2e as a potential "universal" influenza vaccine. this research is supported by a nih t32 fellowship ca09171-32, the nih grant ai 46457 and a grant from the commonwealth of pa. l. yu 1 1 zhejiang university, zhangzhou, china interleukin-18 (il-18) is a cytokine produced by stimulated mononuclear macrophage system. in this report, 18-day-old chicken embryos were vaccined with the plasmid dna (pci-chil-18) encoding chicken interleukin-18 and the copy numbers of chil-18 in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil-18 in 18-day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil-18 could accelerate high concentrations of chil-18 in nonage peripheral blood, accelerate high expression of chil-18 in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil-18 could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil-18 (p x 0.05). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of 40nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd8 cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on 12 volunteers and a phase i study on 24 volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd8 cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv-1 neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) 120 and the transmembrane protein gp41. two sites on gp120 and gp41 are attractive targets for vaccine design: the epitope in the third hypervariable region (v3) is recognized by the human monoclonal antibody 447-52d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies 4e10 and 2f5. in order to elicit anti-hiv-1 neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp120-v3 region or the gp41-mper. the vlps are based on the acyltransferase component (e2 chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e2 chain self-assembles into a 24 nm protein scaffold resembling a vlp and that contains 60 copies of e2. efficient display and refolding of the v3 and mper regions in e2 vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the 5' of the gene coding for e2. the priming and boosting with a combination of vlps and specific hiv-1 envelope dna were used to immunize mice and rabbits. results: the v3-e2 and mper-e2 vlps were purified as stable 60mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of 447-52d, 4e10 and 2f5 antibodies to hiv-e2 monomers was confirmed by western blot. we obtained high titers of hiv-1 gp140-specific antibodies in mice immunized with a combination of vlps plus dna (hiv-1 sf162 gp160). these antibodies generated a low (20 %-27 %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e2 vlps plus dna elicited a low titer of hiv-1 gp140 specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp160 plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e2 vlps are able to display antigenic determinants of hiv and to induce high titers of hiv-1-specific antibodies. the e2 vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h5n1 whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h5n1 influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [1] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h5n1 whole virus wild-type vaccine, could induce an immune response and protect mice against h5n1 influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h5n1 antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h5n1 virus. the protective efficacy of the trivalent vaccine derived mainly from the h1n1 component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h5n1 virus, suggesting that antibodies are the main contributor to protection. h1n1 specific serum did not inhibit neuraminidase activity of h5n1 virus suggesting that protection was not mediated by neuraminidase n1-specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h5n1 whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h5n1 vaccine enhanced anti-h5n1 antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h5n1 vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h5n1 candidate vaccine. [1] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to 6 years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of 335 children in the first grade of primary school in the area of central and west macedonia. there were 234 greek and 101 foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following 1) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to 100%. 2) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). 3) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. 4) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type 1 diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om-85, a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd28 -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om-85. cytokine secretion, activation and proliferation of b cells and foxp3+ tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr2, tlr4 or the myd88 adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om-85. two synthetic tlr4 agonists used as adjuvant in human (om-174-dp and om-197-mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om-85-induced protection of diabetes required natural tregs, as nod-cd28 -/mice were not protected. remarkably, om-85 activated b cells and not t cells, promoting their proliferation and il-10 secretion, two phenomena that were tlr4-and myd88-dependent. om-174-dp and om-197-mp-ac two synthetic murine tlr4 agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd4 + cd25 + foxp3 + t cells and the proliferation of il-10 secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr4 signaling in the protective effect of om-85 on development of diabetes and show that two other tlr4 agonists induce proliferation of b cells and their secretion of il-10 as well as stimulation of regulatory cd4 + cd25 + foxp3 + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit 1 , a. legat 1 , s. thomas 1 , m. van mechelen 2 , p. hermand 2 , m. goldman 1 1 institute for medical immunology/université libre de bruxelles, gosselies, belgium, 2 glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide 4-phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor 4 (tlr4). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd14 (scd14) for its tlr4 ligand activity, we investigated the involvement of both forms of cd14 in the responses elicited by crx-527, a prototypical agp. first, we found that crx-527 efficiently induces nf-kb and irf3 activation in hek cells transfected with tlr4 and md-2 genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd14. likewise, crx-527 efficiently induces the synthesis of nf-kb and irf-3 dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd14 prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd14 respond to crx-527 but not to lps in serum-free medium. the addition of the soluble form of cd14 did not modify the levels of il12 and tnf produced by crx-527 stimulated dc but increased the levels of interferon-b (ifn-b). when scd14 was added to hek cells expressing tlr4/md-2, nf-kb activity was not modified but irf3 activity was increased in a dose-dependent manner in response to crx-527. we will further compare the responses induced by crx-527 in wild-type and cd14 deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h-2d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled 14 and 28 days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin-15 gene polymorph isms (c267t, g367a, c13687a and a14035t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [1] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n=14) have higher amount of both nonactivated (subtype infg+/cd69-, p=0.02) and activated (subtype infg+/cd69+, p=0.028) cbmc, producing ifn-g, as compared with newborns from urban mothers (n=79) exposure to pets and the risk of allergic symptoms during the first 2 years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc09/16 to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity (12-14 %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: 5 months -3 years (n=22), 4 -7 years (n=15) and 8 -12 (n=6) tb has the highest diagnostic value in children n 4 years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp-10 antigen is more sensitive for detection of tb-specific t cells compared to esat-6 antigen. 4. in children with tst 5-14 mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr2 and tlr4 in the development of spontaneous lupus disease by creating tlr2 or tlr4 deficient c57bl/6 lpr/lpr mice. methods: tlr2 and tlr4 deficient lupus prone mice have been generated by crossing c57/bl6-tlr2 -/-or c57/bl6-tlr4 -/-mice with c57/bl6 lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr2 or tlr4 deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr2 or tlr4 in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr4 deficient mice suggesting an important role of tlr4 in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr4 also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr4 pc14/13 expression of full length mcl-1 and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl-1, an anti-apoptotic protein. a splice variant of mcl-1 arises by removal of exon 2 and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl-1 (mcl-1l) and its splice variant (mcl-1s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl-1. method: neutrophils were isolated from children (diagnosed x 17 years) with jsle (n=14) and non-inflammatory conditions (control, n=14) and incubated with control serum, jsle serum alone or with jsle serum plus 20pg/ml gm-csf. quantitative real time pcr was used to assess mcl-1l and mcl-1s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl-1s to mcl-1l was also higher in jsle patients compared to controls (p x 0.05). the addition of gm-csf to jsle serum was associated with an increase in mcl-1l (1.66 ± 0.31) and a decrease in mcl-1s (2.56 ± 1.1) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl-1 compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl-1s, and less anti-apoptotic full length mcl-1 cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd4 + cyld ex7/8 t cells were transferred into rag1 -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex7/8 cd4 + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex7/8 mice are capable to control inflammatory responses. for this purpose cd4 + cd25 + cells were co-transferred with naïve wt cd4 + t cells into rag1 -/-recipients. interestingly, rag1 -/-recipients of cyld ex7/8 t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc17/16 the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a*03 in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb1*13, *01 and *0103 alleles, and a decreased allele frequency of drb1*15 in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb1*0103 is associated with the development of severe disease and positive association of cd with drb3*0301 and drb1*13. indeed, drb1*15 was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb1*07 frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb1*04 in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb1*15 were strongly increased with respect to cd and controls. however, the frequency of drb1*04 was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb1*15 is associated with uc in european, north american, japanese and korean populations methods: a total of 176 children were studied, (92 boys and 84 girls), up to 17 years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, 51 positive cases of children were found (29 %) with active infection : 28 boys (14 x 5 years of age, 12 5-10 years of age and 2 g 10 years of age) and 23 girls (10 x 5 years of age, 6 5-10 years of age and 7 g 10 years of age). pharyngitis was present in 47 children (92,2%), 39 had fever (76,5%) and 48 had lymphadenitis (94 %). the lab tests revealed leukocytosis up to 20.000 leukocytes in 29 cases (56,9 %) and leukocytosis g 20.000 in 9 cases (17,6 %). the most frequent complication documented was streptococcal superinfection in 13 children (25,5 %) and thrombocytopenia in 8 children (15,7 %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and 3) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il-13ra2, previously believed to be a decoy for il-13 only, is able to transmit a signal via il-13. our results support this and may suggest that il-13/ il-13ra2 signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il-4ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il-13 or il-13ra2 and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd4 + cd25 -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il-6 and il-1b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd4 + cd25 -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi-98, l. acidophilus ncfm tm and b. bifidum bi-504) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi-98 strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il-6 or il-1b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 1 1 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed 21 pediatric cd patients (13 active, 8 remission), 24 pediatric uc patients (17 active, 7 remission), and 37 age-matched non-ibd controls. nkg2d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x 0.05 was considered significant. results: nkg2d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd3+cd56+ nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg2d ligands on circulating monocytes is also observed. the dramatic increase of nkg2d+ lymphocytes, and the strong upregulation of nkg2d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg2d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc17/26 peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp3+ t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd4+ t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp7 knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß5i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp7 knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp7 knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp7 knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than 200 million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec-205 antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa 1-191) or hcv-ns3 (aa 1027-1218) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns3 and core we successfully optimized culture and protein purification conditions. briefly, ns3 was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec-205 to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec-205/ hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd01/2 mva-nef vaccination induces polyfunctional cd4 t-cells and increases the proliferative capacity of cd8 t-cells in hiv-1 infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv-1 protein nef (mva-nef) was safe in hiv-1 infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd4 t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il-2 and mip-1b, the expression of the activation marker cd154 and the differentiation marker cd45ra in nef-specific cd4 and cd8 t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il-2 and mip-1b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd4 t-cell response and a significant increase of polyfunctional nef-specific cd4 t-cells, simultaneously expressing ifn-g, il-2 and cd154. using the standard ics no increase of nef-specific cd8 t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd8 t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il-2 and cd154 expressing cd4 t-cells and the increase of proliferating cd8 t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd4 t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd8 t-cells in hiv-1 infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv-1 mediated membrane fusion and p24-detected replication. db81 was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with 25mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd4 + and cd8 + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns4 (1677-1756 aa) and rns5a (2061-2302 aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/2j mice were immunized intraperitoneally 2 times at a month interval with different doses (0.1 -2 mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns4 protein. immunization with rns5a-immunomax conjugate and rns5 in cfa (1.4 mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns5a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns5a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd01/21 degree of cross-genotype reactivity of hcv-specific cd8 t cells directed against ns3 the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd8 t cell response targeting hcv genotype 1 (gt1) and genotype 3 (gt3) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: 53 subjects (17 with gt1, 22 with gt3 and 14 anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns3 from gt1 or gt3. individual reactive peptides and the degree of cross-reactivity between the gt1 and gt3 variants were determined by ics. complete ns3 is sequenced from all viremic patients pd01/22 anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes 1, 4, 6 or 9 suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes 1, 4 or 9, but not 6 led to a significant reduction in viral loads. decreased splenic viral load after ifna1 treatment correlated with an expansion of activated fv-specific cd8 + t cells and nk cells in the spleen, whereas in ifna4-and ifna9-treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna2, 5 and 11 are under investigation pd01/23 elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd8 t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd8 t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd8 t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a*0201-or k b -restricted cd8 t cell responses by these dna vaccines differed. k b /ova 257-264 -and k b /s 190-197 -specific cd8 t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd4 + and cd8 + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least 16 healthy, randomly chosen blood donors were cultured for12 days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. 48 out of 72 tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than 50 % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of 5 peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, 3 hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd8 + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd8 + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il-12-exposed cd8 + t cells showed at least five-fold increase of survival rate in vivo than il-2-exposed cd8 + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd8 + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il-12-exposed cd8 + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd8 + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il-12 and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x 0.01) . there was a significant elevation of t ada and ada1 activities in iga deficient patients as compared to healthy individuals (p x 0.01) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd01/61 hiv-1 sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after 2-6 weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in 12 out of 17 patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, 10-day-old ross 308 broiler chickens divided randomly into 3 groups, 2 experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/79 strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/79 and h120 strains, via the drinking water at 10 days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, 7&14 days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd4 t cells from 25 patients with active tb and 28 patients with successfully treated tb were analysed for simultaneous expression of ifng and il2 at the single cell level using multi-colour flow-cytometry after 6 hours stimulation with ppd. moreover, cells were stimulated with esat-6 and cfp-10 receiver operator characteristics analysis revealed that a percentage of ifng /il2 dual positive cells x 56 % served as an accurate marker for active tb patients (specificity 100 %, sensitivity 65 %), while frequencies g 56 % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd03/7 necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in 1993 and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately 10 100 000 inhabitants) is 5.33 to 100 000. among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, 12 out of 14 children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently 6.12 to 100 000 inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of 1986 to 1993 led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd03/8 novel analogues of thalidomide inhibit cd80 expression and production of tnf-a, il-12, ifn-g, cxcl-9 this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta1-dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta1r7k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd80 and cd86. these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il-1a and il-1b, while il-6 levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il-1b production. using bmdc from nlrp3 -/-mice we examined il-1b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il-1b production is dependent on nlrp3. collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho-1 expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from 20 % in wild type (hmox1 +/+ ) mice to 87 % in ho 1 deficient (hmox1 -/-) mice. hmox1 -/-but not hmox1 +/+ mice developed end-stage multiorgan failure. mortality of hmox1 -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h 2 o 2 or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb1. similarly, circulating and cytoplasmic hmgb1 was increased in hmox1 -/-relative to hmox1 +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b-1,2-linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at 4-6 years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and 2-4 weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were 7.7 and 9.4 iu/ml and those of dtwp-pasteur were 8.2 and 8.6 iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were 30.2 eu/ml for dtwp-local and 47.9 eu/ml for dtwp-pasteur vaccines, respectively (p x 0.001). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd05/18 united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b:2 , a gram-negative coccobacillus. jrmt12, an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of 10 8 cfu of jrmt12. a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b:2 on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for 1 h before adding concanavalin-a (cona) pd06 -vaccination and immunotherapy against parasitic diseases pd06/1 evaluation of simian adenoviral vector adch63 expressing msp-1 as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype 5 (adhu5) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch63 is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp-1 and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm128 while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho-1 expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox1 locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho-1 is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp63 expression was confirmed by sds-page and elisa using monoclonal antibody against gp63. discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a5-180recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a5-180recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least 6 month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at -20°c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and 2 seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r=0.216, p=0.641) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il-17 also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il-17 on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il-17 to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il-17 and il-17-related inhibition of cfu-e growth were dependent on p38 mapk activity, since the p38 mapk inhibitor, sb203580, markedly downregulated il-17-induced activation of nos and reversed the growth inhibitory effects of il-17 on cfu-e. the in vivo stimulating effect of il-17 on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il-17 effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il-17 acts on bone marrow cells and also revealed a link between the il-17, no and hematopoiesis. further studies on il-17-mediated induction of both inos and enos methods: a total of 1785 blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of 68 samples (3,8 %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x 100 iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had 100 iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of 10 iu/l anti-hbs in all the blood units, which also goes for our study pd14/20 neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen 1,2 1 national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine 297 can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa-1a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa-1a iggs we monitored 12 children (9 boys and 3 girls) in ages from 3.2 to 17.2 years with average age of 8.9 years. in 11 of them all was diagnosed for the first time. 1 subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest (11 subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc3h1 gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. 2005, nature 435, 452-8). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 3'utr of icos, in which a 47bp sequence, containing a possible mir-101 binding site, was sufficient (yu et al. 2007, nature, 450, 299-303). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. 2000, j biol chem 275, 33655-62). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia-1-and ago2-deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r01 ai 42269. we have recently reported that 6-hydroxyl-1-methylindole-3-acetonitrile (6-hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw 264.7 macrophages. in this report, we investigated the effect of 6-hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin-8 (il-8), monocyte chemotactic protein-1 (mcp-1) in ht-29 human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed 6-hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated 6-hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. 6-hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p65. in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of 6-hma significantly reduced the mrna levels of il-8 and mcp-1 stimulated by tumor necrosis factor-a (tnf-a) in the ht-29 cells. pretreatment of 6-hma also significantly blocked the ixba degradation and nf-xb p65 nuclear translocation stimulated by tnf-a in the ht-29 cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of 6-hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, 6-hma could be new potential therapeutic agent for inflammatory bowel disease.cd4 serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd4 igg immune response in hiv-1-exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp120-binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd4 domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp120, reacted with the 2 external domains of soluble human cd4, in the absence of the target cells expressing the co-receptor ccr5. the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd4-gp120 complex and trigger the membrane fusion between effector (gp120 expressing) cells and target (ccr5 expressing) cells. thus, in the absence of ccr5 we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv-1 infections. a conventional protocol for the generation of monoclonal antibodies was used. db-81 (igg1, x), one of the anti-cd4 antibodies obtained, recognized preferentially cd4 complexed to gp120, as compared to cd4 alone, not competed for the gp120 binding site on cd4 and was specific for the second extracellular domain of cd4. g. röder 1 , l. geironson 2 , a. darabi 3 , m. harndahl 1 , c. schafer-nielsen 4 , k. skjödt 5 , s. buus 1 , k. paulsson 2 1 copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, 2 lund university, immunology bmc d14, lund, sweden, 3 lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, 4 schafer-n, copenhagen, denmark, 5 department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first 87 n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a*0201. to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn 1-87 /80 , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn /80 was demonstrated to be located on tapasin [40] [41] [42] [43] [44] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first 87 n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd8+ t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region 301-498 of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b*2705-restricted np-flu epitope (aa 383-391) was replaced by hla-b27 or hla-a2-restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard 51 cr release assay and through the ifn-g production. results: using hla-b27 or hla-a2 restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b27 molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a2 molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a2 restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b27 as a remarkable hla-class i molecule that, differently from hla-a2, can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b27 molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv-1)-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv1. consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p17-gag and p24 region was determined in vitro. 25mer peptides were digested with i20s and c20 proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv-1 p17-and p24-gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers 1 , g. koster 1 , o. landsverk 1 , f. skjeldal 1 , o. bakke 1 1 university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi3 kinases and thus ptdins(3)p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier 1 , l. visvabharathy 1 , j. fu 1 1 university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e3-19k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e3-19k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type 2 e3-19k and class i molecules.results: we showed that e3-19k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a11 molecules, with the mature form being more tightly associated. we also provided evidence that e3-19k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e3-19k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e3-19k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il-12, il-6, tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands 2 and 4, but also with the t-celldependent stimulus cd40l (cd154) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd40, cd83, and cd86 was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p38 and erk-1/2. strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd4+ t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th2 (il-4+) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th1 (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th1 / th2 balance and to identify potential t cell target antigens (ags), we analyzed circulating cd4+ t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il-4 expression in peripheral blood cd3+cd4+ lymphocytes were measured in 9 ccs patients and 7 healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il-4/ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il-4 and lower ifn-g intracellular expression were detected in cd4+ t cells from css patients, as compared to hs (il-4: 1.3±0.3 % vs. 0.50±0.21 %, p x 0.05; ifn-g: 14.2±4.5 % vs. 27.0±4.8 %, p x 0.025). similar results were obtained by elisa (il-4: 0.39±0.16 vs. 0.30±0.07 pg/ml, p x 0.05; ifn-g: 31.0±14.3 vs. 79.0±15.0 pg/ml, p x 0.05). elispot counts of hexavalent vaccine-stimulated cd4+ cells were positive for il-4 in 4/5 (80 %) css patients and in 5/5 (100 %) hs, and for ifn-g in 2/9 (22 %) css patients and 7/7 (100 %) hs. mpo stimulation determined significant ifn-g release in 5/8 (62 %) css patients, but not in hs (0/5) no il-4 response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd4+ cells from css patients show a th2-polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd4+ t cells, and responses towards it are instead th1-polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd4+ t cells in css. a. voigt 1 , e. opitz 1 , k. savvatis 2 , k. klingel 3 , k. stangl 1 , u. kuckelkorn 1 , p.-m. kloetzel 1 1 charité -universitätsmedizin berlin campus mitte, berlin, germany, 2 charite -campus benjamin franklin, berlin, germany, 3 universität tübingen, tübingen, germanymurine models of coxsackievirus b3 (cvb3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp2 of the stress-induced ip, were infected with 1x10e5 pfu cvb3 nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp7 as early as day 4 p. i. in lmp2-deficient mice. however, lmp2-deficiency was linked to less severe myocarditis at day 8 p. i. (he stain of cardiac tissue sections: wt 1.95 ± 0.20 vs. lmp2-deficiency 0.71 ± 0.06 (grade of myocarditis; scale 0-4; p x 0.001). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x 0.05), there was no difference in lmp2-deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb3 infection in lmp2-deficient mice. likewise, diastolic function was preserved in lmp2-deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp2-deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp2-immunosubunit function in regulatory processes of viral replication. absence of lmp2 confers host protection in enterovirus myocarditis. h. w. liao 1 , j. xu 1 , j.q. huang 1 1 sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype 2 (den-2) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr1 and tnfr1-2 were constantly very low whereas trailr2-4 decreased after den-2 infection. fasl was expressed at similar levels on huvecs throughout den-2 infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase 8, caspase 3 were also observed by western blot after by den-2 infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for 7 days into wis rat paw web tissue in saline containing 7.5x10 7 cells. group ii. s.epidermis was injected as in group 1 after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day 8.they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors 2,23 and 3,91 respectively (p x 0.05). immunohistochemical pictures showed increase in percentage of ox62 (migrating dendritic cell), mhc ii, his48 (granulocytes), ox7 (stem cells) and cd54 (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed1 (macrophages) and ox62 cells. popliteal lymph nodes contained evidently less of ox62, his48 and mhcii cells than in group i (p x 0.05). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega-3 fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega-3 fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega-3 fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega -3 fatty acids consumption. the results indicate that omega-3 fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega-3 fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega-3 fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density 1.24 and 202,000g, top 20 % layer contained lipoproteins only and 20-50 % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising 1 % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin-1. objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e2f2 mutation, and other affecting b cell apoptosis control, such as bcl-2 over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c57bl/6 (b6) genetic background. e2f2-/-bcl-2tg were obtained backcrossing e2f2-/-and e2f2+/+hbcl-2tg mice. e2f2-/-bcl-2tg, e2f2-/-, e2f2+/+hbcl-2tg and control mice (e2f2+/+) were followed up to 18mo-old. serologic studies: serum samples obtained at 3, 9 and 15 month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of 3, 9 and 15 mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h3]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl-2tg in b lymphocytes of e2f2-/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e2f2-/-bcl-2tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e2f2-/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e2f2 or the overexpression of a bcl-2 tg in the b6 genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor 9 (tlr9) abolishes the generation of antinucleosome igg2a and igg2b autoantibodies but exacerbates lupus disease. however, the tlr9-dependent tolerance mechanism is unknown. here we show that loss of tlr9 in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th1 and th17 t cells. transfer of a synthesized monoclonal polyreactive igm to tlr9 deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr9-dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n=115) (vitali et al. 2002) . ninety-seven of 115 (84 %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g 1 (fs+), while biopsies from 18/115 (16 %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in 27/115 (23 %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x 0.05). interleukin (il) 17, il-1ra, il-1beta, il-12p40, il-15, macrophage inflammatory protein (mip) 1alpha, mip-1beta, eotaxin, interferon (ifn) alpha, and il-4 levels were significantly increased in the gc+ patients (n=27) compared with the gc-patients (n=70). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il-4 found in these patients (gao et al. 2006) . increased titers of th17-associated cytokines, il-17, il-1beta and the il-23 subunit il-12p40, may indicate a higher activity of these cells in gc+ patients (nguyen et al. 2008) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il-6. the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg2. production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g 50 %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd88-dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd88-dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/-1 baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il-8. objectives: increased levels of il-18, an innate and inflammatory cytokine of the il-1 family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il-18 and other genes of the il-1/il-1r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from 48 patients and 32 healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il-18 and il-18bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il-18 are higher than in controls. serum il-18 correlates with disease activity and decreases upon remission. monocyte expression of the receptor il-18rb is increased and correlates with disease severity, while expression of tir8/sigirr (a down-regulatory receptor of the il-1r/il-18r family) is reduced. in mrl lpr/lpr mice, expression of il-18, caspase-1 and il-18rb genes precedes disease onset in lymph-nodes. in other organs, changes in il-1-related genes (il-33 and tigirr-1 up-regulation, tir8/sigirr down-regulation) occur after disease onset. free il-18 levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il-18 levels correlate with autoimmune lupus both in mice and humans. free il-18 may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il-18rb is a marker of pathology, suggesting increased il-18-dependent activation. both in mouse organs and human monocytes, tir8/sigirr expression decreases with disease, suggesting impaired control of il-18r activation. thus, il-18 may be involved in autoimmune lupus pathology, and il-18-related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f4 (amino acid (aa) 451-593) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f4 was recognized by all patients. fragment f1 (aa 5-30) was recognized by 9 of 34 dcssc patients. fragment f8 (aa 350-400) was recognized by 4 of 8 sle patients. analysis of clinical data revealed a significant difference between the f1 negative and f1 positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f1 and f8 could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd4 + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd4 + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril-2. by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd4 + t cells enriched in tregs were co-cultured with activated cd4 + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd4 + t cells anergy was also evaluated based on cbl-b, grail and foxp3 mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd4 + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd4 + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b-1 cells through targeting p110d by shrnas strategy. methods: we used the drugs, ly294002 and wortmannin, pan-specific inhibitors against pi3ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p110d. we then introduced either pan-specific inhibitors against all pi3ks or p110d-targeting shrnas into an sle-prone animal model, nzb/w f1 mice, for therapeutic purposes. the results suggested that pi3ks are not only important for the development of b-1 cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p110d. either pan-specific inhibitors against pi3ks or p110d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f1 mice. one inhibitor, ly294002, and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi3ks are critical for the maintenance of b-1 cell populations might shed light on future treating other diseases associated with b-1 cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool 1 , m. alarcón-riquelme 1 , s. kozyrev 1 1 institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank1 gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs10516487, r61h). we identified that bank 1 gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon 2 (delta 2). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank1 is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank1 isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs17266594), which is in complete ld with r61h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia1, which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a 57-year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti-2gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last 1year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow 1 1 cnrs 9021, strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed 18 patients, with minor clinical and/or biological manifestations of their disease, for at least 6 monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below 2. b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd19 surface protein for all patients. this cd19 lower expression is associated with cd45 lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf2 expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf2 lacking an exon in the c2-domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf2 in t-cells. smurf2 expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf2 expression was upregulated by tgf-beta stimulation in t-cells and smurf2 was markedly upregulated in tumor infiltrating cd4+ lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day 42, smurf2 transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf2 showed an upregulation of the tgfbrii and an activation of smad2 and 3 as compared to wild-type t-lymphocytes, which were previously described as typical smurf2 targets for degradation. in addition the transfection of smurf2 and a caga-luc plasmid into cos-cells for smad3-promotor studies yielded the same effect as shown by upregulation of the smad3 activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf2 splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf2 has beneficial effects on mucosal inflammation and tumor development. smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat3 has important functions in cytokine signalling in a variety of tissues. however, the role of stat3 in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat3 activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat3 activation in dss colitis is dependent on il-22 rather than il-6. il-22 was secreted by colonic cd11c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat3 activity were highly susceptible to experimental colitis, indicating that epithelial stat3 regulates gut homeostasis. stat3 iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat3 regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il-22 and epithelial stat3 was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat3 activation regulates immune homeostasis in the gut by promoting il-22-dependent mucosal wound healing. stat3 seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd40 and tlr4 expression at day 3 and day 7 after infection. mycobacterial infection did not result in differential tlr2 expression as compared to uninfected cells. cd40 is involved in stimulating th1 pro-inflammatory responses, although map may interfere with cd40 signalling (1) . tlr4 signalling elicits anti-inflammatory responses, which can contribute to bacterial replication (2) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd40 and tlr4 receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd8 t cells by exogenous antigen leads to colitis. methods: eight million naïve cd8 + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h2-kb, were transferred i. v. into b6 mice. at day 0 and 4, mice were treated intra-rectally (i. r.) with 50 % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day 2 after the injection of ot-i cells. the phenotype of effector cells was evaluated at day 5 by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd8, the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd8 + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd8 + t cells. our study demonstrates that the local activation of antigen-specific cd8 t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b6 mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of 535 patients, (248 male and 287 female), with age average 69,7 years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x 2 and t-test programmes. results: in 372 patients (69,5 %,with age average 62,7 years of age) no antibodies were detected. on the contrary, in the remaining 163, 52 male and 111 female, (30,5 %, with age average 76,4 years of age), antibodies were detected. out of them, in 106 cases the results were strong positive (32 male and 74 female) and weak positive in 57 cases (20 male and 37 female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: 1) helicobacter pylori infection is relatively common in the general population (30,5%).2) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women).3) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova 1 , t. ulmannova 1 , k. stechova 1 , k. tesarova-flajsmanova 1 , v. stavikova 1 , j. nevoral 1 1 charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of 20 mothers whose infants were diagnosed with allergic colitis and 20 mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis (2-27 weeks, average 16.8 weeks of infant's age) or at the age of 12 weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il-4, il-6, il-10, il-17, il-23, ifn-gamma, tgf-beta1, egf and eotaxin. man-whitney u test was used for statistical analysis, p x 0.05 was considered statistically significant.results: il-10 as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x 0.001) in breast milk of mothers whose infants were suffering from allergic colitis (range 0-8.45 pg/ml, mean 2.1 pg/ml) than in control group (range 0-3.41 pg/ml, mean 0.35 pg/ml). higher concentrations of il-4 and lower concentration of tgf-beta1 were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. 8310-5. background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il-8 (murine homologs kc and mip-2) and its receptor cxcr2 are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated 12 h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at 2, 4, 16 and 22 h post-transfer. anti-kc antibody or its isotype control was administered at 20mg/mouse one hour before transfer followed by whole body and organ imaging 4 hours post-transfer. results: facs analysis revealed 80 % neutrophil purity, 35 % of which were cxcr2 + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, 4 h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than 10 15 bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model (1.5 % dss in drinking water for 1 week, followed by pure drinking water for 1 week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, 2 % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell 2004), treatment of commensal-depleted mice with the tlr4 ligand lps in drinking water protected against the lethality of 2 % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of 1.5 % they may become pathogenic and drive an intestinal inflammation. at 2 % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by 1.5 % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type 2 immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il-13 production which is an important pathological factor. neutralizing il-4 or il-13 prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th2 cytokines in colitis remain undefined, we used mice deficient in il-4/il-13 or the key receptor through which they signal, il-4ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il-4ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il-4/il-13 double deficient mice which were protected from colitis. removing il-13 production from il-4ra -/mice, by using il-4ra/il-13 double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il-13 mediates susceptibility in an il-4ra independent manor. recent evidence pc17/33 introduction: the activation of cd4 + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th1-like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th1 cytokines, such as il-5,-6 and il-13. however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc2 was found in uc and cd colonic tissue compared to control specimen. transmitted to the th2-mediated oxazolone-induced colitis model, nfatc2-production is significantly increased in both diseases, too. nfatc2 deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc2 deficient mice compared to control mice, which can be observed by tunel assays, caspase3 and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl-2 and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il-6, ifn-gamma, il-13 and il-17 by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc2 regulates il-23/il-17 in an indirect way. last, administration of il-6 blocked the protective effects of the nfatc2 deficiency in experimental colitis, suggesting that nfatc through il-6 signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc2 in colitis by controlling mucosal t cell activation in an il-6 dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd4cd45rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd4cd45rb high t cells also regulatory cd4cd45rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at 3 different time points during colitis progression. after 3 weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at 9 weeks. microrna was isolated from colons of mice in different stages of colitis progression (3, 6 and 9 weeks) and control mice that do not induce colitis (n=3 for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from 11 micrornas that demonstrated an induction during the development of disease we selected 4 micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd4cd45rb high transfer model. objective: the purpose of this clinical trial (id: nct00287677 of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for 6 months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in 3 groups: group a (n=8) receiving haart+ gh (for 6 months) + tt+hva/b vaccines (at month 6 post gh adminsitration); group b (n= 6) receiving haart+gh but not vaccines; and group c (hiv control group, n=7) with haart+vaccines (at month 6) but without gh. all patients are followed up 6 months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after 24 weeks of administaring hormone the absolute numbers of cd4 incresase from 562 ± 93 to 704 ± 112 cells per mm 3 (mean and sem; p x 0.0025). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd4 absolute numbers from 550±97 to 470±103 cells per mm 3 (p x 0.02). viral load remained undetectable in all patients. despite the increase in cd4 counts the percentage of recent thymic emigrants (as assessed by the expression of cd31) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv (80,000-160,000), hepatitis c virus (hcv; 2.3-4.7 million) and hepatitis b virus (hbv; 8-16 million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv-1 infected patients with g 350 cd4 + t-cell counts and plasma hiv-rna levels of x 1.7 log 10 copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from 4 timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g 50 and when the responses were g 2 times the standard deviation above the mean of replicate negative controls (mock electroporated dc). 16/17 patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in 4/16 patients before and 11/16 patients after vaccination. for rev, 7/16 patients showed a pre-existing rev specific response and 14/16 patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already 11/16 patients responding before and 16/16 patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in 13 out of 16 patients before vaccination and 16/16 patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus 71 (ev71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev71 is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev71 inactivated virus. methods: mice were immunized intraperitoneally with 10 mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after 0, 2, and 4 weeks. blood samples were collected at 0, 21, 35, and 45 days. the total serum anti-ev71 igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il-5 from splenocytes was also measured. results: immunization with ev71/ps-g showed that the anti-ev71 igg levels were significantly increased compared with ev71 alone or ev71/cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev71/ps-g group compared to ev71 alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev71/ps-g-immunized mice compared to those of ev71 or ev71/cfa/ifa-immunized mice, indicating a th1 cells response elicited by heat-inactivated ev71 vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd8+ t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd8+ t-cell responses against hcv-ns3, studying induction of il-2, ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il-2 and ifng production by cd8+ t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv-1-recognizing cd8 + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv-1 replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd8 + t cells by electroporation, and chose tcrs which were able to recognize the hla-a2 restricted hivpol-peptide iv9, or the hivgag-peptide sl9. results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il-2, tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd25, after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd4 + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd8 + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd8 + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt3l pretreated balb/c mice were incubated for 3 hrs with hcv ns5-coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g 80 % dcs and was used for immunization. dc expression of the maturation markers cd40, cd80 and cd86 was determined before and after ingestion of ns5-coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns5 expressing sp2/0 cells). results: in immunized animals, the ctl activity was increased 3-fold compared to mock immunized mice. accordingly, tumor challenge with ns5 expressing tumor cells showed a significant reduction in tumor growth. the number of cd4 + ifn-g + cells was increased g 3-fold and the number of cd4 + il-2 + increased g 5fold in the dc-ns5-bead immunization group. these results paralleled the proliferative response of splenocytes to ns5 protein obtained from immunized animals with the most significant response in the cd4+ population of dc-ns5-bead immunized animals. the use of ns5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th1 biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec-205 antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec-205 induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec-205 antibody can be developed. we screened the anti-dec-205 sequence computationally for putative hla dr4-restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec-205 antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec-205 as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih 1r21ai078800 a live oral vaccine based on human adenovirus (ad)4 has proved safe and effective in us military recruits for nearly 50 years. in these experiments, we have investigated whether replication-deficient ad4 can be an efficient potential vaccine carrier for oral vaccination. ad5 vectors were used throughout to provide a benchmark for efficacy. we generated novel ad4 and ad5 vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad4 than ad5. ad5 routinely infected and provided transgene expression in˚10 % of human cd4+ and cd8+ t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to 25 -30 % of cd8+ t cells present, showing that ad5 infected a surprisingly large proportion of t cells. in comparison, ad4 provided egfp expression in x 2 % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad4 and ad5 vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad4 and ad5 induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day 3 after dosing, and transgene expression being reduced below detectable levels by day 8. interestingly, when delivered together ad4 and ad5 vectors targeted the same subset of cells. together, these data show that ad4 is a viable alternative to ad5-based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies (20 recently commercialized; g 300 in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the 667 mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that 667-treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g 8 months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, 2005; gros et al, 2008; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd8 + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp60) of rhdv at two different locations: 1) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp-2), and 2) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp-306). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd8 + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp-2) was more immunogenic than insertion at position 306 for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp-2 at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr-2 ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd8+ t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd8+ t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np 147-155 specific cd8+ t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr-2 targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l929cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during 7 days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd2+, cd4+, cd8+, cd16+ t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by 1,5-2-fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns4 and ns5a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h5n1 influenza virus inhibits the ifn-g synthesis (mibayashi et al., 2007) and causes a decrease in cd4 + and cd8 + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., 2000) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a 10 amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions 129-131 (tat'kov c. i. et al., 2000) . deltaferon was i. p. administered to male non-inbred mice in doses of 2-40*10 4 iu once or twice at an interval of 48 hours, alone or in combination with double-stranded yeast rna preparation (5 mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., 1983) . results: when deltaferon was administered in doses of 2-20*10 4 iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of 2*10 4 iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon (2*10 4 iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd8 responses (tetramer+ hiv-1 gag p24 cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd8 responses after pla-p24 immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p24 were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas 1 , c. prego 2 , s. vicente 2 , m.j. alonso 2 , a. gonzález-fernández 1 1 university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, 2 university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer 188, with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with 1 or 2 immunizations to balb/c female mice (4 weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day 15 to 260 post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease (100 miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigenbackground: infection with human immunodeficiency virus type 1 (hiv-1) is characterized by dysfunction of hiv-1-specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p24 antigen from hiv-1 and the tlr3 ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p24 microcapsules containing p24 and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for 10 days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p24 peptides. intracellular staining of interferon-gamma (ifn-g), interleukin-2 (il-2) and cd107a after p24 stimulation was also performed. results: mddc from hiv(-) subjects, incubated for 24 hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p24 containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il-2, upon p24 peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd16b between our model cytokine il-2 and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p30gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il-2 dependent proliferation assays of il-2 variants. results: murine il-2 attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il-2 dependent cell line ht-2. we found that the membrane anchors comprising one and four ig-like domains (il-2::1iggpi and il-2::4iggpi) resulted in severely reduced vlp production by 293 producer cells and despite of an increased targeting of il-2::4iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il-2 fused to the minimal gpi-anchor acceptor sequence of hcd16b (il-2::gpi). il-2::2iggpi, however, showed comparable particle production and biological activity in vitro when compared to il-2::gpi. furthermore, il-2 fused to 2ig::gpi showed an increased capacity to co-stimulate primary p14 tcr transgenic t-cells specific for lcmv-gp 33-41 in the context of h2-d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. 2ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il-2::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f1816-b13 of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant 812079 & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild 1 , interdisciplinary transplant laboratory 1 charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects 2-25 % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of 2-3 months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with 6 different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from 2,5 month to 16 days. t cell lines are composed of cd8 and cd4 cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker 1 , m. assenmacher 1 , a. richter 1 1 miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp65-specific cd4 + and cd8 + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd45ro -cd25 -) cd4 + and cd8 + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp65 peptide pool and cd3-depleted autologous pbmc as feeder cells in the presence of il-2, il-7, and il-15. already 9-13 days after primary activation pp65 495-503 /a2-tetramer + cd8 + t cells were detectable for 3 hla-a2 + blood donors. to analyze cd8 + t cells having other specificities than for the peptide pp65 495-503 as well as probably primed cd4 + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to 3.9% of the cd8 + t cells and up to 3.8% of the cd4 + t cells after restimulation with pp65 peptide pool, but not with either irrelevant ie-1 peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for 20 days led to a 10 -100 fold expansion of pp65-specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd154 and cd137 only if restimulated with the pp65 peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il-2, indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd4 + and cd8 + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf 1 , k. mozdzanowska 1 , l. otvos 2 , j. erikson 1 1 the wistar institute, philadelphia, united states, 2 temple university, philadelphia, united statesthe influenza virus a matrix protein 2 ectodomain (m2e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m2e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine 2003; virology journal 2007), we generated a novel peptide and investigated its efficacy in inducing an anti-m2e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd8 + t cells, have been considered. clinical data confirm a crucial role for antiviral cd8 + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a*0201 and hla-b*0702 previously. considering other frequent hla alleles cmv specific cd8 + t cells were monitored longitudinally in 30 hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd8 + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd8 + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a*2402/pp65 (341-349) (7.6x10 6 cells/l) and hla-b*3501/pp65 (123-131) (4.4x10 6 cells/l) specific cd8+ t cells are significantly lower than those for hla-a*0101/pp50 (245-253) (17.2x10 6 cells/l) and hla-a*0201/pp65 (495-503) (13.2x10 6 cells/l) specific cd8 + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. (2,5 -50 mcg) . no adverse effects were indicated during trials (up to 25 month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after 4th immunization with 50 mcg of vichrepol.no differences were detected in cd4+ and cd8+ t cell counts and cd4+/cd8+ ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp70 or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp70 or gp70 alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb6f1 hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb6f1 mice with adenoviral nanoparticles expressing fusion proteins containing gp70 resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd4 + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd4 + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf01 exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted 90-99 % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf01 did not show any increase in their levels of ifn-gamma. conclusion: caf01 enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from 13 children aged 1-2 years old (6 boys and 7 girls) -group 1, and 11 children (6 boys and 5 girls) 6-7 years old -group 2 were isolated on a gradient of density before vaccination ( or revaccination) with priorix, 1 week, and 2 months after and incubated with cfse. then 2 million/ ml pbl were incubated in rpmi-1640 supplemented with 10 % fcs (the negative control), at presence of 5 mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing 5 % cî 2 at 37°c within 7 day. intensity of a fluorescence estimated on fl1 by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control 90 % pbl in both groups did not enter mitosis. in the positive control 90 % of cells have passed one and more mitoses. in group 1 measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in 2 months 15-25 % of lymphocytes demonstrated antigen-specific proliferation. in group 2, on the contrary, before the vaccination the most part of cells (75-80 %) has not entered division, but 20-25% of cells have passed 2 and more mitoses. in a week specific lymphocyte proliferation decreased and in 2 months it was increased up to 40-50 %. production of the interleukin (il) 6, ifn-g, tnf-a, il-4, il-1 was more informative than il-5, il-7, il-8, il-12. measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp120 cd4-binding site (cd4bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv-1 neutralizing polyclonal iggs directed against the cd4bs. the mabs were shown to react with an anti-cd4bs human neutralizing mab (b12), to elicit antibodies that recognize the gp120 molecule and an anti-hiv-1 neutralizing response in rabbits, confirming them as cd4bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp120 antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd4bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd4bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp120 titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b12 in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p1 and p2) reacted in elisa only with the cd4bs-directed igg fraction. the clones were also recognized in elisa by b12. p1 and p2-immunized rabbit sera showed a strong anti-gp120 titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb2 strain glycoproteins. in particular, 3/5 rabbits in the p1 group and 1/5 in the p2 group showed an 80 % hiv neutralization at dilutions ranging from 1:20 to 1:150. the immunoenzymatic assay used, allowed to detect a p1 and p2 reactivity in hiv-positive sera and was able to detect a b12 concentration equal to 10 ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines (17d and 17dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a 23-year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following 17d-204 vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd16, cd32 and cd64) along with increased levels of nkt-cells (cd3 + cd16 +/-cd56 +/-/cd3 + ratio) and activated t-cells (cd4 + and cd8 + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il-6, il-17, il-4, il-5 and il-10) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il-4 + ), cd8 + t-cells (il-4 + and il-5 + ) and b-lymphocytes (tnf-a + , il-4 + and il-10 + ). the analysis of cd4 + t-cells revealed a complex profile with increased frequency of il-12 + and ifn-g + and decreased percentage of tnf-a + , il-4 + and il-5 + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester 1 , h.-g. rammensee 1 , s. stevanović 1 1 eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd8 t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd8 t cell epitopes for the frequent mhc class i alleles a*01, a*02, and a*24 from the proteins pii (hexon), pviii, and e1a of adv strains ad2 and ad5 by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a 12-day recall stimulation of pbmcs from at least 16 healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd8 t cells. we could identify 27 new peptides eliciting ifn-g responses, several of which were confirmed as novel cd8 t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle (5 ara criteria) was diagnosed in a 40-year-old african female patient with hiv-1 (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd4 t-cell count x 200 cells/mm 3 . initiation of 500 mg mmf bid was associated with biological and clinical remission of sle and cd4 t-cell increase. no opportunistic infections or cancers were noted during a 3-year follow-up and the patient remained always naive to art. hiv-1-specific cd4 and cd8 t-cell responses were analyzed after 18 months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il-2 production following stimulation with a panel of 192 hiv-1-derived optimal epitopes (9/10-mers) covering various hiv regions and a pool of 105 hiv-1-derived peptides (15-mers overlapping by 11 aminoacids) encompassing the entire gag protein. all peptides are derived from hiv-1 consensus strain iiib. results: highly polyfunctional hiv-1 specific cd4 and cd8 t-cell responses against gag were detected. 11 epitope-specific cd8 t-cell responses were identified: except for one response restricted by hla a*23 and another one by hla cw*07, all the others were restricted by hla-b alleles and mostly by b*58 (n = 5). seven out of 11 responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il-2+) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv-1 specific cd4 and cd8 t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv-1 t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of 10 6 dl50/ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf-1 of 12-16 g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the 7 th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt-46767, cofaa and cgpi-20090305. . state of vaccine-induced measles immunity was determined by means of elisa in 1-3, 4-6 and 7-9 years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in 4-6 years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p 0.05) measles immunity and antibody level much lower (p p 0.05) than among children with mt conversion. in 7-9 years the comparison group kept decreased (p p 0.05) measles immunity, the majority (92±5.54 %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p 0.05) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p 0.05), the majority (83.3±4.1 %) of persons lost protected antibody level; among children with mt conversion in 1-3 years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p 0.05) to low values; among children with hyperergic mt igg level decreased (p p 0.05) and reached low (in 1-3 years), minimal protected (in 4-6 years) and lower than protected (in 7-9 years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il-17 producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein 65 (dnahsp65). methods: balb/c female mice were infected by intra-tracheal route with 10 5 h37rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp65 genetic vaccine was done at days 30, 40, 50 and 60 post-infection. each dose consisted of 100 micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd4+, cd8+ and gamma-delta t cells from lungs were determined 10 and 50 days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x 0.05 were considered significant (t test). results: at day 10 after the end of the immunotherapy, dnahsp65 treated mice exhibit increased numbers of absolute cd8+ and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il-17 producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp65 treated mice showed more ifn-gamma producing cells in both cd8+ and cd4+ cell populations. at day 50 after the end of the therapy, the main observation in mice which received dnahsp65 treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd4+ and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd8+ cells, together with a more frequency of gamma-delta t cells producing il-17. finally, the immunotherapeutic effects of dnahsp65 vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd8+ cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il-17, are the main effects of dnahsp65 immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il-17 and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as 60 loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of 3-5 log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput (96-well format), requires only 100 ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in 5 different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that 5 candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls 71 % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all 5 m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards 4 of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to 100 % in pb, 75 % in rx and 93 % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd40l, a co-stimulatory molecule preferentially expressed on activated cd4+ t cells, is the ligand of cd40. cd40-cd40l interaction induces the production of il-12 and the initiation of a th1-type immune response. several studies show that cd40l is required for the activation of macrophages and the maturation of dcs. moreover, cd40l enhances the capacity of cd8+ t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd40l. we have constructed the recombinant bcg strain expressing cd40l (rbcg2) by electroporation of bcg with pgfm11/signalsequenceag85b-cd40lec and an another recombinant strain with empty vector pgfm11 (rbcg1) as a control. the expression of cd40l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th1 type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd40l 2 weeks after vaccination but not at 4 and 8 weeks. rbcg2 seems to be more protective against paratuberculosis than rbcg1, but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd40l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il-1a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x 0.05) of tnfa, ifng and il-1a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x 0.05) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd40 mab and ag85a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd40 binding on cd40 transfected l929 fibroblasts. the conjugates were tested in vivo in wild type and cd4 + cell-depleted mice for the induction of specific anti-ag85a serum antibodies. splenocytes were challenged ex vivo with ag85a and were examined for their ability to produce th1-related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il-2. we developed a method to successfully crosslink anti-cd40 mab to ag85a. serum antibodies against ag85a were detected after immunisation with this conjugate vaccine in both wild type and cd4 + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag85a alone. production of two other th1-related cytokines, tnfa and il2, was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd40 conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd4 + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer 1 , r. burger 2 1 robert koch-institute, cellular immunology, berlin, germany, 2 robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd4-positive and cd8-positive t-lymphocytes. cd8-positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd8+ t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for 5 days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to 30 % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd4+ t-lymphocytes in vivo. similarly, the cd4-positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd4+ and the cd8+ subpopulation depended on the presence of apc. stimulation oft cd8+ cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd8-positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target 6 (esat-6) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, 83 iranian and afghan adults (38 patients with sputum smear and culture positive tuberculosis, 24 recovered patients during 6 months after full course of chemical treatment and 21 healthy individuals) were recruited to quantify the frequency of esat-6 and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of 4 spot forming unit ( g 20 spots per million), we found detectable response to esat-6 in almost 80 % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and 77 % of these individuals had detectable esat-6 specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in 14 %, 57% and 62 % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th1 immune response, against esat-6, in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish 1331 in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal 1 , p.k. upadhyay 1 1 national institute of immunology, pdc-1, delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl 2 solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c57bl/6 mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: 2.4 % alginate, 0.012 % pva concentration gives particles with size of 2-10 micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with 5 % trehelose and 3.5 % pvp (poly vinyl pyrollidone). there was 6 fold increase in proliferation index of spleenocytes and releases 600 pico-gram of interferon gamma after 4 week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by 10 % increase in cd86 and 10.5 % in ccr7 expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b100 is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b100s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b100s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b100s. by cell cycle analysis we determined that b100s is able to induce apoptosis in up to 80 % of jurkat and molt-4 t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b100s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b100s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase 9 is the first to be cleaved, followed by caspases 3 and 8, thus suggesting that b100s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b100s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b100s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd40, cd80 and cd86, and of the inflammatory cytokines il-6, il-23 and mcp-1. using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd4 t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th2 and th1/th17 skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd4 t cell responses. microcapsule based vaccination resulted in a marked induction of il-17 secreting th17 cells, without inducing strong th1 (cfa) or th2 (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg1, igg2c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th17 responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd4 + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd80, cd86 and cd40 and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il-6 and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr4-dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr4-defective c3h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il-1b secretion by dc, through its effects on caspase-1 processing, which is also tlr4-independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr4 signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc2461 (st11) or lactobacillus rhamnosus ncc4007 (lpr), each at 5x10 8 cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week 1 to week 7 of life. fresh feces were collected at 3, 5 and 7 weeks of life for determination of iga levels (assessed by elisa). pups were bled 2 and 4 weeks after immunization for determination of measles-specific igg1 and igg2a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st11-fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from 200 kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at 3:1 ration and washed 3 times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw264.7 by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of 24 extracts from 10 euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc6/propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that 15 up to 24 euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd3+ cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on 35 th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il-5 and ige serum levels were increased on animals from group i when compared to control.the enhancement on th2 immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd8+ ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd4+t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg2a and igg2b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren 1,2 , n. almqvist 2 , a. lönnqvist 1 , s. östman 1 , c. rask 1 , e. telemo 2 , a.e. wold 1 1 university of göteborg, department of clinical bacteriology, göteborg, sweden, 2 university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; 0.8 mg/ml) in the drinking water for 5 days and, 3 days later, 50 mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin-5 and interleukin-13. examination of gut sections from sea treated donor mice revealed increased density of cd8a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp21, proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna 1826, a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp21, or groel together with cpg-dna 1826 reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp21 together with cpg-dna 1826. the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp21 microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp21 microspheres induced protective ifng-secreting cd4 + t-cells and raised pulmonary pmp21-specific iga levels in vivo. also, pmp21 microspheres caused lower il-6 serum levels upon administration than the injection of pmp21 together with cpg-dna 1826, indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in 44 cases of s. aureus bacteraemia in iv drug users and 44 cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised 44 iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen 2006).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day 0) and four weeks thereafter (day 28). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter 2007) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st59 (spa-type t172, agr 1, and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p=0.01).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st59 strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st59 strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at 4 and 9 years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in 15 and 7 ds children, respectively. samples were taken before and 3-4 weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg 1 -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at 4 years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg 1 , igg 2 and igg 4 ). at 9 years, ds children had lower postvaccination geometric mean igg 4 anti-tt-titers only. post-vaccination igg 1 -anti-tt avidity levels were decreased in 8/15 and 4/7 ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. 2 kda; 30, 6 kda; 23, 9 kda; 19, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 6 kda and 18, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 7 kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of 100 families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an 8-month period. sera were obtained from healthy subjects from the same community (2 months to 88 years of age). the highest incidence of salmonella-associated diarrhea, 74/1000, occurred in children under 5 years of age. the lowest incidence, 10/1000, was observed in the population aged 10 to 59. whereas serum from individuals ranging 15-70 years of age showed maximum igg1, igg3 and iga anti-s. typhimurium titres, children less than 5 years-old did not show detectable igg1 and igg3 titres and had weak igg2, iga and igm antibody levels; only their igg4 levels were comparable to those detected in adults. moreover, the levels of igg2 and igg3 antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs 07-18, a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses (67%), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a-1,6linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after 7-fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková 1 , s. bystrický 1 1 institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o135 using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd19, expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age 18-55 years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg1, igg2 and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present 5 years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after 5 to 7 days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg2 response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers 1:3200). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones (1:800 vs. 1:3200). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was 5. 85 fold of normal control). subcutaneous immunization gave a weaker stimulation: 1. 52 fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv 0032-06 and vega 2/7029/27 grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a-1,6-linked d-mannopyranose units and many branches composed of a-1,2 a-1,3 and/or b-1,2-linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant (6mg oligosaccharide per one conjugate dose) two times in 14 days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day 14 after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs (250g-350g) were treated orally with 50 mg respivax five consecutive days. after the last application, on days 3, 7, 10, 14, 28 and 42 six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on 4mm thick serial sections, stained with hemalauneosin. the populations of cd8, cd4 and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day 3 subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd8 positive cells, which number reached maximum at the end of the second week. on day 14 b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd4 and cd8 positive cells. on days 28 and 42 the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta 1 , s. majumdar 1 1 bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein-10 (ip-10) and interleukin-8 (il-8) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip-10 mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase2 (inos2) expression and was linked to the mapk signaling pathway via antagonistic regulation of p38mapk and erk1/2. further, ip-10 was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th1 cytokines and no. thus this study strongly demonstrates that ip-10, like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th1/th2 cytokines mediated by cd8+ t cells and activating cd4+ t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x 0.05) following immunization and after challenge with leishmania major. il-4 values were increased in all groups, but there was no statistical difference between the groups(p g 0.05) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x 0.05) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x 0.05) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x 0.05). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at 3 week intervals. three weeks after booster injection, 5×10 5 stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg2a/igg1was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type 1 immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin 10 (il-10), and cellular expression of cd4 and cd25. results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd4+ lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with 1,600 infective n. brasiliensis larvae on day 2 post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day 6 post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg1 and igg2a). this was independent of level of me supply. feeding regime did not affect levels of the type-2 cytokines il-4 and il-13. conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp63 is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp63 of l.major strain mrho/ir/75/er. methods: l.major promastigotes were grown in rpmi1640 supplemented with 10 % fcs. l.major rna extraction and cdna synthesis were carried out. gp63 gene segment was amplified by specific primers and cloned into ptz57r to construct ptz57r/gp63. the presence of gp63 into ptz57r was confirmed by pcr. then, ptz57r/gp63 was sent to determine the sequence of its nucleotides. after that the gp63 gene segment was sub-cloned into pet32 a (+) expression vector and transformed into e.coli bl21 (de3) plyss and gp63 protein was expressed in presence of 1mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of 100 % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, 2007) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including 50 volunteers. 10 male volunteers (18 and 45 years) for the adult group, and 40 children of both sexes (5-9 years) were enrolled. subjects received virosomal formulations containing 50 mg of ama 49-c1 (pev301t), an apical membrane antigen-1 derived synthetic phospatidylethanolamine (pe)-peptide conjugate and 10 ug of uk39 (pev302t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days 0, and 90. results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling 29 % of the treated mice to survive till 21 days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only 0.04 % of the amount fed and it remained in circulation in the blood only for 30 minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to 0.4 % ( of the amount fed but it's circulation was sustained till 6 hrs post feeding. under such conditions when 200mgm of curcumin bound to 300mgm of chitosan nano particles were fed one time daily for 10 days post infection to plasmodium yoelii infected mice 100 % of mice were cured and survived atleast for 100 days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma9vdelta2 t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il-10 responses, to epitopes on the dominant rbc autoantigen, anion exchanger-1 (ae-1, or band 3) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of 1 methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla-4 + regulatory t cells or soluble forms of ctla-4, may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =-0.81) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd8 + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b 1 (r medium =-0.74) and il-10 (r medium =-0.65) in isolated splenic hscs ex vivo. analysis of effector cd4 + t cells in spleen showed decrease of t h 1 cells quantity and simultaneous t h 2 cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd4 + cd25 + ctla-4 + -and cd4 + cd25 -il-10 + il-4regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h 1, t h 2 and regulatory t cells were correlated (r th1 =-0.74, r th2 =0.86 and r th3 =0.80 and r tr1 =0.68) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th3 =0.84 and r tr1 =0.76) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b1-and il-10-produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p38, jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin-17, t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il-17, as compared to non-infected controls. the infection also altered the effect of il-17 on mapk activation by preventing its stimulating effect on p38 mapk. moreover, in s.obvelata-infected animals il-17 markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il-17 induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. 2.-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from 1 ⁄2 to 1/64 with a normal serum pool. 3.-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r 2 g 0.98. the analytical range was from 1 g/l to 70 g/l. the coefficient of variation (cv) was x 10 % for [mc] n 1 g/dl, and x 20 % for [mc] x 1 g/dl. this procedure was successfully implemented to quantify the mc in 1100 serum samples between march 2008 and february, 2009. among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: 1. we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. 2. the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from 7 day and reached plateau level after 30 day of teratocarcinoma insertion. moreover, cd34 + cd38cells showed in spleen and main lymph nodes from 15 day and achieved plateau level after 60 day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at 22 day and had a maximum pick at 60 day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than 1,5 times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b 1 , il-10, pge 2 and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number =0.81 and r activity =0.92) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd40-ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd40l is cleaved to soluble (s)cd40l. we sought to examine the levels of scd40l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded 5 atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd40l -along with other products -were assayed by quantitative elisa. levels of il8, cd62p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd40l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l . to examine if scd40l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il-6 secretion. the il-6 concentration was consistently below 5-10 pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il-6. the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il-6 production over control (p x 0.05) at d2 after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l (p x 0.05). pre-incubation of b cells with cd40-blocking antibodies substantially abrogated il-6 secretion, unlike isotype-matched control. the partial blocking of cd40 binding on cd40 + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd40l (under investigation) and indicates a sustained role for pc-derived scd40l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd40l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule-1 (dnam-1) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from 30 aml patients before specific anti-leukemia therapy and 47 healthy donors. all results were analyzed statistically using spss version 15. results: aml patients under 65 years showed a significant reduction in the expression of dnam-1, nkp30 and nkp46 compared with age-matched controls. both healthy individuals and aml patients older than 65 years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp44 expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam-1 on nk cells and its ligand cd112 on aml blasts has been found in aml patients under 65 years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam-1 expression on aml patients and confirm previous reports showing a significant decrease of nkp30 and nkp46 on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel 1 , e. gorska 1 , u. demkow 1 , m. wasik 1 1 medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved 10 children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for 2 or 48 hours with or without dexamethasone in concentration 10 -6 m. analysis of: p-gp surface expression, p-gp function (rhodamine 123 test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was 14,52 %±11,32. after 48 hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine 123 efflux, which characterized p-gp activity, was 2,62 %±3,15. after 2 hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine 123 (4,31 %±4,03, p=0,015). average phi in lymphoblasts was 7,78±0,19. acidification of cells incubated 2 hours with dexamethasone was seen in 10-25 % percentage of cells 17) . rest of lymphoblasts showed alkalization (phi -8,00).the percentage of lymphoblasts in early stage of apoptosis after 48 hours incubation with dexamethasone (annexin-v test) was higher than in control cells (19,34 % vs 14,13 %; p=0,01). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd49d/cd29 integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd29 on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd38 and anti-cd29 (clon huts21) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures (18 hours). results: cd29 active form was expressed in the majority of normal and tumoral bm pc from healthy subjects (67.3±6.6, n=9) and mm patients in the early stages of the disease (32.6±7.5, n=17). in these cells, huts21 epitope was clearly upregulated by mn 2+ . in contrast, circulating pc were almost all huts21 negative, and levels did not significantly augment when these cells were exposed to mn 2+ . moreover, not only pb but also bm malignant cells from pcl patients were also huts21 negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index (2.35±0.9 brdu+ cells, n=3) in comparison to pc from mm patients in the first stages of disease (1.3±0.2 brdu+ cells, n=5). these results suggest that the active form of cd29 must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either 10 % fetal bovine serum (fbs) with and without fgf2,or 10 % human platelet lysate (hpl), 5 %hpl, (10 % fbs + 10 % hpl), (10 % fbs + 5 % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day 18). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p53 tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p53 dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: 1) ten cases with fa diagnosed on the basis of dna breakage analysis, 2) ten cases with aaa, and 3) ten normal control cases. the presence of p53 dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p53 dna was demonstrated in bone marrow of 90 % of children with fa, compared to 10 % in children with aaa (p x 0.001), while, no p53 dna was seen in normal control. a positive correlation between dna breakage and presence of p53 dna was seen in bone marrow from fa (p x 0.02, r0.81). the presence of p53 tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord-005453-4057qib7 authors: nan title: the 45th annual meeting of the european society for blood and marrow transplantation: physicians – poster session date: 2019-07-03 journal: bone marrow transplant doi: 10.1038/s41409-019-0559-4 sha: doc_id: 5453 cord_uid: 4057qib7 nan background: allogeneic hematopoietic stem cell transplantation is routinely offered to patients with high-risk or advanced all in the hopes of improving outcomes. use of truly non-myeloablative (nma) conditioning reduces toxicity in other contexts but outcome data for all patients after nma transplants is lacking. we report the outcomes of 31 patients with all transplanted using a nma conditioning without t cell depletion. methods: first transplant patients between october 2006 and june 2018 were reviewed. these were consecutive patients until 2015 then only those considered unfit for fmc conditioning as per the ukall 2014 protocol. all patients were conditioned with fludarabine 25mg/m 2 /day for 5 days and cyclophosphamide 1g/m 2 /day for 2 days. short course mtx and ciclosporin were used for gvhd prophylaxis. standard supportive care was employed. thirty-one patients with a median age of 43 (23-67) met the criteria for this case review. 30 had b-all and 10 were philadelphia chromosome positive. 24 patients (77%) had high risk disease by standard diagnostic criteria. 27 (87%) were in first complete remission (cr1). matched sibling donors were used in 13 instances with the remaining being fully matched unrelated donors. 58% of patients had a hct-ci score of 0, 32% a score of 1 or 2 with 3 patients having a score of 3 or higher. median cd34 dose was 5.3 x 10 6 /kg (0.93-34.12) with a median cd3 dose of 2.13 x 10 8 / kg (0.12-7.37) results: trm was low at 7% at 1 year and 11% at 2 and 3 years respectively. no factors included in a univariate analysis (which included age, diagnosis, disease status, hct-ci, donor type, cmv risk and cell dose) significantly impacted trm. the incidence of classical acute (a) gvhd grade 2-4 and 3-4 was 18% and 8% by day 100 and 29% and 13% by day 180 if late onset agvhd is included. 24 out of 30 eligible patients developed chronic gvhd of any stage. relapse incidence was low (22% at 3 years in all patients, 17% in cr1 patients) and was not impacted by any pre-transplant factors including positive mrd post phase 2 induction (present in 6 patients). notably, in univariate analysis relapse was significantly lower in patients who developed chronic gvhd. background: allogeneic stem cell transplantation (allosct) is the treatment of choice for many patients (pts) suffering from acute myeloid leukemia (aml). the graft vs. leukemia effect (gvl), applied by immunocompetent cells of donor origin, is the most important effector mechanism for the eradication of leukemia, the presentation of leukemic or allospecific antigens by malignant blasts is regarded as a crucial trigger for an effective allogeneic immune response. conversely, insufficient stimulatory capacity by the leukemic blasts is thought to be a relevant escape mechanism from cellular immunotherapy (allosct or donor-lymphocyte infusion (dli)). the purpose was to test, whether the ability of malignant blasts to differentiate in vitro towards dendritic cells of leukemic origin (dc leu ) is associated with response to allosct or outcome after immunotherapy (second allosct or dli) for post-transplant relapse in aml. methods: leukemic blasts were isolated from peripheral blood (pb) or bone marrow (bm) samples of aml patients before allosct (n=47) or at relapse after allosct (n=22). a panel of 6 different assays was used to generate dc leu in vitro (5 of them containing gm-csf). finally, in vitro results were correlated with clinical characteristics and outcome of patients treated with donor lymphocyte infusion and/or allosct. results: dc leu could be generated in vitro from all 69 samples. when correlating proportions of dc-subtypes generated ex vivo with clinical data, significantly higher mean proportions of dc leu in the dc-fraction were found in responders vs. non-responders to immunotherapy (76.8% vs 58.8%,p=0.006, range:13%-99%). vice versa, the chance for response to immunotherapy was significantly higher, if a dc leu /dc ratio of >=50% could be reached in vivo (p=0.004). those patientswere characterized by a longer time to relapse (p=0.04) and by a higher probability for leukemia-free survival (p=0.005). similarly, generation of higher amounts (>8%, p=0.04) of dc leu in the mnc-fraction, and generation of more mature dc (>47% cd83+, p=0.03 using the best gm-csf containing assay) were associated with a longer time to relapse in the respective patients. moreover, overall survival was improved, if >70% dc leu /dc could be generated with the best gm-csf containing assay (p=0.048). conclusions: in vitro generation of dc/dc leu from leukemic blasts obtained in active stages of aml before allosct or at relapse post transplant were associated with clinical outcome. this observation supports a role of antigen presentation by leukemic cells for an allogeneic immune response in aml. disclosure: nothing to declare background: the role of autologous hematopoetic cell transplantation (hct) in the treatment of aml is not clear. trials in the past have shown that autologous hct consolidation lowers the risk of relapse, however the magnitude of this effect is limited . autologous hct is advocated in patients with aml with lower genetic risk in cr1.many of these patients will eventually relapse and will undergo reinduction followed by allogeneic hct in cr2. methods: the aims of this study is to analyze outcome of allogeneic hct performed in cr2 comparing patients with prior consolidation by autologous hct vs. patients with chemotherapy consolidation. primary outcome is non relapse mortality (nrm) of allogeneic hct in cr2 in patients with, or without prior autologous hct in cr1. secondary outcomes include leukemia free survival (lfs), relapse rate (ri), graft versus host disease free relapse free survival (grfs), overall survival (os), and treatment related toxicities. results: 2619 adult patients reigstered with the alwp of the ebmt with de novo aml were included, receiving a first allogeneic hct in cr2, in 2000 cr2, in -2017 or without (n=2202) prior autologous hct. patient and transplant characteristics are shown in the table. patient groups were not entirely comparable, patients with prior autologous hct were younger, had less often a favorable cytogenetic profile, had more commonly donors other than matched siblings and more often received reduced intensity conditioning (ric) as compared to mac conditioning. univariate outcomes are shown in the table with slightly higher nrm risks in patients with prior autologous hct consolidation. in multivariate analysis nrm risks in patients with prior autologous hct were 1.34 (1.07-1.67), p=0.01 after adjustment for patient age, cytogenetic risk category, year of transplant, donor type, conditioning intensity, sex matching, time from diagnosis to relapse and time from relapse to allogeneic hct as compared to patients with chemotherapy consolidation. similarly, risks of events in lfs and grfs were higher with prior autologous hct, 1.17 (1.01-1.35 ), p=0.03 and 1.18 (1.03-1.35 ) p= 0.02, respectively, risk of death was also higher 1.13 (0.974-1.32) p=0.1 but this was not statistically significant. conclusions: we may conclude that some of the advantages of potentially higher anti-leukemic activity of high dose chemotherapy and autologous hct when given to patients with aml in cr1(as was shown in a randomized trial by vellenga e et al with lower relapse and higher lfs by approximately 10% but no significant differences in overall survival) may be lost by higher toxicity of allogeneic hct in cr2 in case of subsequent relapse. background: although relapse is a major cause of mortality in patients receiving allogeneic hematopoietic cell transplantation (hct) for acute leukemia, limited and conflicting data exist on extramedullary relapse (emr). we aimed to describe the incidence, risk factors, outcomes and prognosis in relapsed hct recipients. methods: we retrospectively reviewed charts of consecutive allogeneic hct recipients transplanted in our center with the indication of acute leukemia (7/1990-7/ 2018). we recorded: age, gender, disease, previous extramedullary involvement, phase at transplant, type of transplant, donor, conditioning, graft-versus-host-disease (gvhd), infections, treatment-related mortality and relapse mortality. in patients with extramedullary relapses, additional data on clinical manifestations, imaging, cerebrospinal fluid testing, histopathology and management were additionally documented. incidence of isolated emr (iemr) and bone marrow relapse (bmr) was calculated using cumulative incidence (ci) analysis, with each and treatment-related mortality considered a competing risk. results: among 554 allohct recipients followed for 1.8 (0.04-27.75 ) years, 61 (11%) patients presented with emr. the majority of emrs involved the central nervous system (cns, 56%). isolated emr was observed in 38 patients at 9.5 (1.8-67.3) months. 10-year cumulative incidence (ci) of 10.5% for iemr was associated only with pre-transplant advanced disease phase (p< 0.001). bmr was observed in 149 patients at 9 (0.3-276 months), with a 10-year ci of 34.8%. in the multivariate analysis, bmr ci was independently associated with fungal infections (p< 0.001), pre-transplant disease phase (p< 0.001) and lines of treatment (p=0.042). 10-year trm of our whole cohort was 33.2%. the majority of iemr and bmr (75% and 81%, respectively) patients received systemic treatment combined with local radiation for iemr (26%) and donor lymphocyte infusions (dlis, 16% and 28% respectively) when feasible. extensive chronic gvhd was recorded in 47% of iemr and 48% of bmr patients. outcomes were poor in iemr, with 10-year overall survival (os) of 18.3%. favorable os in iemr was associated only with sibling donors (p=0.049) and not with other factors, such as treatment with dlis or presence of chronic gvhd. similarly poor outcomes (10year os of 19.1%) were observed in bmr. favorable os was independently associated only with the diagnosis of aml (p=0.050) and absence of bacterial infections (p=0.049). in the whole cohort, both iemr and bmr were independent unfavorable predictors of os (p< 0.001) along with extensive chronic gvhd (p=0.012). conclusions: in a large population with long-term follow-up, incidence of iemr was relatively high, developed at the late post-transplant period and associated only with disease phase at transplant. furthermore, iemr and bmr conferred similarly poor outcomes despite systemic treatment or extensive chronic gvhd. these independent predictors of survival highlight the unmet clinical need of novel approaches either as maintenance or treatment to reduce extramedullary or systemic relapse post allohct for acute leukemia. disclosure: no competing financial interest. impact of t-cell depletion on outcome in patients undergoing allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia background: after a diagnosis of acute myeloid leukemia (aml) the majority of patients (pts) who achieve complete remission (cr) eventually relapse, with only approximately 30% of pts maintaining cr for 3 years or longer. late relapses (after 3 years in cr) occur rarely (6-10%) in pts receiving hsct in cr1 and late effects are followed up by routine surveillance as well as preventative measures. the purpose of this study was to investigate long-term outcomes in pts with diagnosis of aml undergoing hsct at our institution in cr1. methods: a standardized follow-up of hsct-survivors is applied at our center. we analyzed 116 adult pts with aml in cr1 consecutively transplanted between january 2004 and december 2016 at our institution. a written consent was given for the use of medical records for research. a landmark analysis was adopted for patients in cr at 2-y after hsct (ltcr -long-term cr). results: ltcr was achieved after hsct in 91/116 patients (male 55, female 36) transplanted in cr1. the median follow-up was 6 years and the median age at transplant 52 years (r 20-72). the selected donor was a family haploidentical relative in 29 cases, an hla identical relative in 21, a match unrelated donor in 39 and a cordblood in 2. in this cohort of ltcr, the 5-year overall survival was 92% (95% ci 83-96). cumulative incidence of relapseevaluated in competing risk with transplant related mortality (trm) -and trm -evaluated in competing risk with relapse -were respectively 7% (95% ci 1-23) and 2% for the cr1 cohort. the event-free-survival (efs) was 91% (95% ci 83-95). the causes of death were relapse (6/10 pts), second cancer (3/10 pts) and sepsis (1/10 pts). the 5-year incidence of dyslipidemia -defined as cholesterol >/= 200 mg/dl, and/or ldl >/= 115 mg/dl, and/or triglycerides >/= 150 mg/dl or need for specific treatment -was 24%. the 5-year incidence of osteopenia / osteoporosisdefined as t-score lower than -1 and greater than -2.5 and t-score lower than 2.5 respectively -was 38%. the 5-year incidence of second cancer was 11%: 10 nonmelanoma skin cancer, 2 lung carcinoma, 3 cervical intraepithelial neoplasm, 1 thyroid cancer, 1 gastric cancer and 1 colon cancer. the 2-year incidence of chronic moderate-severe gvhd was 27% (95% ci 13-38), with the latest diagnosis performed on day 570. of note, 4/24 pts are still on active treatment at last follow-up. conclusions: relapse incidence is low for patient that reached ltcr: patients in cr1 at transplant can obtain excellent os and efs once reached the target of ltcr. a proactive long-term follow-up and strategy of counseling are essential to keep at best quality the survival advantage offered by hsct in patients with aml in cr1. disclosure: chiara bonini has research contract with intellia therapeutics. the other authors declare that they have no conflicts of interest. background: relapse, graft-versus-host disease (gvhd) and gvhd-associated mortality are major obstacles to success of transplantation from unrelated (mud) donors in children with acute leukemia (al). negative depletion of αβ t cells and cd19+ b lymphocytes, conserves the mature donor-derived natural killer cells and γδ t cells in the graft, may improve gvhd control, immune reconstitution and prevent the relapse. we present a retrospect analyses of a cohort of pts with al in cr transplanted from mud with depletion. methods: a total of 59 children with acute leukemia (34 aml, 25 all, 21 female, 38 male, median age 8,5y) underwent allo hsct from matched unrelated donor between june 2012 and july 2017. all pts were in complete remission (cr1=34, cr2=23, cr>2=2). all pts, except one, received treosulfan-based conditioning. either melphalan (n=56) or thiophosphamide (n=2) or etoposide (n=1) were added as a second agent. fludarabine was used in all pts. two types of gvhd prophylaxis were used: type 1 (n=35): hatg 50 mg/kg and post-hsct tacro/mtx (n=30) or without prophylaxis (n=5); type 2 (n=24): thymoglobulin(ratg) 5mg/kg, rituximab 200mg/ m 2 with either bortezomib on days +2, +5 (n=21) or tacro/ mtx (n=3) . aβ t cell depletion with clinimacs was used in all cases. the median dose of cd34+ cells was 9 x10 6 / kg, aβ t cells -15 x10 3 /kg. median time of follow-up for survivors was 5,3 years (range, 2, 3 -6,5) . results: primary engraftment was achieved in 100% pts., the median time to neutrophil and platelet recovery was 15 and 14 days, respectively. all evaluable pts achieved sustained complete donor chimerism by day +30. early (100 day) mortality was 3,4% (1pt -bacterial sepsis, 1pt -adv fulminant hepatitis), 5-years overall ptrm at 4 years was 13,5% (95%ci:7-26). six late trm events were due to: viral infection in 2 pts (cmv=1, adv+cmv=1), bacterial sepsis in 2 pts and 2 pts had bacterial and viral infection, all late deaths were associated with cgvhd and prolonged corticosteroid therapy. ci of acute gvhd grades ii-iv was 36% (95% ci: 25-50), acute gvhd grades iii-iv 3,7% (95% ci :1,5-14,5) . ci of cgvhd was 27%(95%ci:18-41). regimen 2 was more effective in prevention of agvhd ii-iv in comparison with regimen 1: 8% (95% ci: 2,2-30) vs 45,7%, respectively, p=0,04. all events with acute gvhd grades iii-iv had pts with regimen 1. ratg was also effective in prevention of cgvhd: ci at 4 years after hsct was 12,5% vs. 37%, respectively, p=0,04. cumulative incidence of relapse was 25% (95%ci: 14-50) without difference between ratg and hatg. event-free survival (efs) (event=death or relapse) at 4 years was 61% (95%ci: 48-73), overall survival 59%(95% ci:47-72), there were no difference between age and diagnosis. conclusions: we confirm that the depletion of tcrαβ +/cd19+ t lymphocytes from the graft ensures high engraftment rate. transplant-related mortality is caused by infections, mostly associated with cases of chronic gvhd. gvhd prophylaxis including ratg/rituximab/ bortezomib improves gvhd control in recipients of tcrαβ+/cd19+depleted grafts in comparison to hatg/ tacro/mtx apparently without loss of anti-leukemic activity. disclosure results: at baseline, r/r all with emd and lbl were diagnosed in 7 and 11 ino patients and 5 and 6 sc patients. median (range) age of the ino and sc patients was 55.5 (20-78) and 47.0 (28-64) years, with 8/18 (44.4%) and 8/11 (72.7%) males, respectively. the rate of cr/cri was significantly higher in the ino group (12/18 [66.7%] , 95% confidence interval [ci] : 41.0-86.7) compared with sc (2/11 [18.2%], 95% ci: 2.3-51.8; p=0.0144) (table) . allogeneic hematopoietic stem cell transplantation was carried out in 6/ 18 (33.3%) ino and 2/11 (18.2%) sc patients prior to any post-study induction therapy. the pfs hazard ratio [hr] was 0.502 (97.5% ci: 0.203-1.240; p=0.0410), with median pfs of 4.4 (95% ci: 1.9-7.1) months among ino and 1.6 (95% ci: 0.8-3.7) months in sc patients. the os hr was 0.661 (97.5% ci: 0.269-1.621; p=0.1478), with median os of 5.9 (95% ci: 3.4-9.4) months in ino versus 5.5 (95% ci: 2.1-6.7) months in sc patients (figure) . all patients had adverse events (aes). serious aes occurred in 10/18 (55.6%) ino and 5/11 (45.5%) sc patients; 4 (22.2%) ino and 0 sc patients had grade 5 ae. one (1/15, 6 .7%) patient in the ino group died from veno occlusive disease. conclusions: among r/r all patients with emd and lbl, improvement in remission rates, transplant rates, and progression free survival was shown in the ino group versus the sc group. although patient numbers were small and limited the ability for a robust comparison, these results support the use of ino in patients in this difficult to treat population with r/r all and emd or lbl. background: bcr-abl-targeted tyrosine kinase inhibitors (tki) revolutionized the outcome of patients inflicted with ph+ b-all. moreover, addition of tki may be relevant strategy for ph-like all patients. methods: we hypothesized that overcoming the bm microenvironment-mediated protection of all cells from tki-mediated apoptosis may further enhance the responsiveness to tki therapy. results: in vitro treatment of bcr-abl-positive all cell lines nalm1 and nalm20) with dasatinib resulted in significant dose-dependent cell growth inhibition, with ic50 of 10-15 nm (p< 0.01). furthermore, dasatinib exhibited significant growth suppression of bcr-abl -negative all cells (nalm6 and reh), with ic50 of 250 nm and 185 nm, respectively. however, when cocultured with bone marrow stromal cells (bmscs), dasatinib-mediated effect was abrogated in both ph-and ph+ all cells. furthermore, dasatinib treatment promoted significant upregulation of chemokine receptor cxcr4, on both mrna and cell surface levels. elevated cxcr4 expression was accompanied by increased responsiveness of all cells to cxcl12 stimulation, resulting in strong and sustained phosphorylation of erk1/2 and akt and increased adhesion capacity to bmscs. therefore, dasatinib-induced upregulation of cxcr4 promotes stroma-mediated survival advantage of all cells upon tki therapy. next, in order to overcome the cxcr4-mediated stromal protection, we choose to combine dasatinib with the histone deacetylase inhibitor panobinostat, for its known ability to deplete cxcr4 in aml cells. single-agent treatment with panobinostat demonstrated significant inhibition of ph-and ph+ all cell growth at low nanomolar concentrations (p< 0.01). importantly, combination of panobinostat with dasatinib synergized (ci< 0.5), effectively overcoming the protection provided by bmscs and inducing the apoptosis of ph-and ph+ all cells, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization and dna fragmentation. furthermore, combining panobinostat with dasatinib significantly reduced cxcr4 surface levels in ph-and ph+ all cells. accordingly, cxcl12mediated responses, including erk1/2 and akt activation and adhesion to bmscs were significantly reduced upon combined panobinostat/dasatinib treatment. these data indicate that panobinostat effectively suppresses both basal and dasatinib-induced cxcr4 expression and function in all cells overcoming stroma-mediated resistance to dasatinib. to determine the molecular mechanism, we performed gene and protein expression analysis. panobinostat, alone or in combination with dasatinib, significantly down-regulated the protein levels of calcineurin, a serine-threonine protein phosphatase previously implicated in t-all and b-all pathogenesis, as well as of nfatc1, a critical effector of the calcineurin signaling cascade, and nfatc1-regulated target genes. it was previously found that calcineurin signaling positively regulates cxcr4 expression in t lymphocytes. additionally, cyclosporin a (csa) decreased both basal and dasatinib-induced cxcr4 surface levels in all cells, overcoming the protection of the bmscs which result in potentiation of the cytotoxic effect of dasatininb and panobinostat. combining csa with panobinostat resulted in deeper suppression of nfatc1-regulated target genes. we thus link the effect of panobinostat with calcineurin-dependent downregulation of cxcr4, blocking the ability of the leukemic cells to respond to cxcl12mediated stromal support. conclusions: taken together, our results identify calcineurin signaling pathway as a novel target of panobinostat in all cells and indicate that hdac inhibition with panobinostat may be effective strategy for facilitating the anti-leukemic activity of tki therapy. disclosure: nothing to disclose background: the treatment of relapsed/refractory acute lymphoblastic leukemia (rr-all) remains a clinical challenge with a generally dismal prognosis. allo-sct using a sequential conditioning ("flamsa"-like regimen) has shown promising results in relapsed/refractory aml, but little is known about the efficacy of this procedure in rr-all. methods: we identified 115 adult patients (45% females; median age: 38 y; range, 18-66) with all in primary refractory phase (26%) or in relapse (74%), allografted between 2000 and 2017 from a matched sibling (31%), matched unrelated (58%) or haploidentical donor (11%) at ebmt participating centers. almost half (49%) of the patients had t-all and 23% had a positive philadelphia chromosome. six patients (5%) underwent a previous autotransplant. karnofsky score was above 90 in 52% of patients. conditioning was myeloablative (mac) with high dose tbi in 30% of patients, reduced intensity (ric) including low dose tbi in 22%, or with chemotherapy alone in 48%. in vivo t cell depletion was performed in 77 cases (69%). most patients (74%) and about half of the donors (47%) were cmv positive. 14% of patients were males who received a graft from a female donor. the median follow-up was 37 (range, 13-111) months. results: overall, 14 patients (13%) failed to engraft, 18 (16%) died within 100 days after allo-sct without relapse, and 64 (56%) could achieve complete remission. at day 100, the cumulative incidences of grade ii-iv and grade iii-iv acute gvhd were 30% and 17%, respectively. the 2year cumulative incidences of chronic and extensive chronic gvhd were 25% and 11%, respectively. the 2-year relapse incidence (ri) and non-relapse mortality (nrm) were 45% and 41%, respectively. the 2-year leukemia free survival (lfs), overall survival (os) and gvhd relapsefree survival (grfs) were 14%, 17% and 12%, respectively. in a multivariable cox analysis, karnofosky score below 90 negatively affected ri, lfs, os and grfs. also, conditioning with chemotherapy alone, compared to tbibased conditioning, negatively affected relapse rates (hr=4.13; p=0.0006), lfs (hr=2.32; p=0.004) and os (hr=2.29; p=0.006). conclusions: allo-sct using a sequential conditioning regimen is proposed by different teams in rr-all, and could be an option, especially when considering a tbibased regimen. however, the overall 2-year lfs of 14% suggests that these patients still face extremely dismal outcomes, highlighting that other therapies (e.g. bite antibodies, inotuzumab, car t cells) need to be combined prior and/or after allo-sct in order to further improve outcome. disclosure: no conflict of interest, no funding received chemotherapy courses, only 2 pts were not treated: 1 pts for the worsening of the general status and the other for invasive fungal infection. results: forty-three pts (42%) were in complete remission (cr) and negative minimal residual disease (mrd) at the time of hsct; 16 pts were in active disease (16%), and 44 (42%) showed a morphological cr with positive mrd. 41pts (40%) developed chronic graft versus-host disease (cgvhd) as followed: 23pts (22%) mild, 17pts (16%) moderate, and only 1 sever grade respectfully. only 1 patient developed cgvhd after dli. the overall leukemia free survival (lfs) time was 16 months, the absence of cgvhd (hazard ratio -hr: 5,968; p = 0,01) and the pre-hsct disease status (hr 2,353; p = 0,028) were the most important factors on lfs. all pts treated with chemo-based regimens died due to progression or infective complications. 1 patient of aza/dli group is still alive with a extramedullary relapse; 2 pts treated with bl/dli are in cr. os was better for the dli group compared to the chemotherapy group (28 vs 2 months respectfully; p < 0,001). conclusions: dli after allo-hsct has exhibited definite anti-leukemic effects in post-transplant patients. bl and aza were reported to increase dli's graft vs-leukemia (gvl) effect. although cgvhd could be the most important protective factor against the relapse but it remains the main cause of morbidity. maximising the gvl effect without putting the patient at risk of gvhd still represents an unmet need. our data show that the combination of either bl or aza with dli infusion is safe and might represent an improvement in disease control in the early phase of relapse. disclosure: nothing to declare p018 increased detection of (leukemiaspecific) adaptive and innate immune-reactive cells under treatment of amldiseased rats and one therapy-refractory aml-patient with blastmodulating, clinically approved response modifiers (pg-e2,kit-k) or +pge1 (kit-m),patent 102014014993) convert myeloid blasts into dendritic cells of leukemic origin (dc leu ). after stimulation with dc leu , antileukemic tcells can be generated ex vivo. the compounds are approved for clinical use and are therefore attractive tools for immunotherapy in myeloid leukemia. methods: dc/dcleu-culture from rats'/patients' wholeblood (wb) with kits, mixed lymphocyte culture (mlc) of tcells with kit-treated blood, functional blast-cytotoxicity and leukemia-specificity assays (csa/elispot/degranulation/intracellular cytokine-assays). in addition flowcytometric evaluations of cellular and (leukemia-specific) lymphocyte compositions were performed from rats'/pts' blood in the course of the disease. results: 1) aml-diseased rats: each 3rats were treated with "i", "k" or "m" or were untreated (controls). a significant increase of dcleu could be detected in spleen/pb in kit-(esp. m) treated compared to untreated animals without induction of blasts' proliferation (ki67positivity): a significant reduction of blasts was seen with "m" (p=0.03/ 0.0001 in spleen/pb) and "i", but not "k". successful treatment correlated with an increase of cd62l+tcells, most likely representing tmem-cells, (p=0.07) and a reduction of cd4+treg (p=0.037). 2) 6 therapy-refractory aml-patients (during the course of decitabine/ld-aractreatment): kit-m was shown to ex vivo generate dcleu, activate immunereactive cells and mediate leukemia-specific/antileukemic response. activated or leukemia-specific lymphocytes were monitored in low proportions in active stages of the disease as well as of two patients during the further course of persisting disease. one of these patients (72 yo male), was offered an individual systemic salvage-treatment (kit-m, applied as continuous infusions) for refractory leukemia. after approval from the local ethical commitee,extensive information of the patient about the experimental nature of the treatment and obtaining his written informed consent. clinically the treatment was well tolerated and the patient improved clinically. neutrophils in wbc increased from 10% to 50%, thrombocytes reached 100 g/l after 24 days. after 4 weeks of treatment, the patient was discharged in good clinical conditions. 12 days later, progression of aml was seen with high blast counts in pb and bm. the patient developed severe sepsis and died few days later. immune monitoring showed (other than before treatment and in the patients without kit-m-treatment) a continuous increase of proliferating and non-naïve tcells, nk, cikand nkt-, th17 cells, bmem-cells and dc in pb. the production of ifnɣ producing t-, cik and nkt-cells was demonstrated, suggesting an in vivo production/activation of (potentially leukemia-specific) cells. immune stimulatory effects decreased after discontinuation of therapy. conclusions: treatment of wb as well as leukemically diseased organisms with blast-modulating kits (especially gm-csf and pge1) was well tolerated and induced clinical and immunological improvement (adaptive and innate immune system), whereas low counts of (leukemiaspecific) activated immune-reactive cells were found in non-kit-treated organisms. disclosure: nothing to declare p019 long-term outcomes after allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia with non-myeloablative and myeloablative conditioning: a single-center cohort study of 438 consecutive patients 1lars klingen gjaerde, niels smedegaard andersen 1 , lone smidstrup friis 1 , brian thomas kornblit 1 , søren lykke petersen 1 , ida schjødt 1 , henrik sengeløv 1 1 rigshospitalet, copenhagen, denmark background: since 2000, we have at our institution used a non-myeloablative (nma) conditioning regimen for older (>50 years) or significantly comorbid younger patients undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) for acute myeloid leukemia (aml). we aimed to compare the long-term outcomes of nma conditioned patients with myeloablative (ma) conditioned patients. methods: we studied 220 nma and 218 ma conditioned adult (>15 years) consecutive patients receiving their first allo-hsct for aml from 2000 to 2017 at rigshospitalet. nma conditioning consisted mainly of 2 gy total body irradiation (tbi) and fludarabine 90 mg/m 2 (95% of cases). ma conditioning consisted mainly of cyclophosphamide 120 mg/ kg and either 12 gy tbi (86% of cases) or busulfan 12.8 mg/ kg (5% of cases), or fludarabine 150 mg/m 2 and treosulfan 42 mg/m 2 (6% of cases). five percent and 19% of nma and ma conditioned patients, respectively, received anti-thymocyte globulin. patients were followed until death or end-of-followup on october 31 st , 2018. cumulative incidences with 95% confidence intervals (ci) of acute graft-versus-host disease (agvhd) grade ii-iv, chronic graft-versus-host disease (cgvhd), relapse and non-relapse mortality (nrm) were calculated and compared between nma and ma conditioned patients using gray's test with death as a competing risk (or relapse when comparing nrm). overall survival (os) was estimated by the kaplan-meier method. results: nma and ma conditioned patients were comparable when regarding sex (49% and 48% female, respectively) and donor (matched related donor in 34% and 36%, respectively), but differed, as expected by indication, with regards to age (median of 60 versus 42 years, respectively) and karnofsky score (< 90 in 18% and 11%, respectively). nma conditioned patients had generally a lower aml stage at transplant (1 st complete remission in 68% versus 49% of ma conditioned patients) and a lower aml cytogenetic risk (adverse risk in 17% versus 21% of ma conditioned patients). patients were followed for a total of 2090 person-years (median follow-up in surviving patients was 6.2 years). agvhd grade ii-iv occurred less frequently in nma conditioned patients (20% [ci: 15%-26%] versus 38% [ci: 32%-45%] in ma conditioned patients, p < 0.01), while cgvhd occurred in similar rates (50% [ci: 43%-56%] in nma conditioned patients and 51% [ci: 44%-58%] in ma conditioned patients, p = 0.77). there was a trend towards a higher relapse rate in nma conditioned patients (34% [ci: 28%-40%] versus 28% [ci: 22%-34%] in ma conditioned patients, p = 0.07), and nma conditioned patients had, however not with statistical significance, lower nrm (20% [ci: 14%-25%] versus 25% in ma conditioned patients, p = 0.27). os ( figure) was comparable, with 5-year os rates of 55% (ci: 48%-62%) in nma conditioned patients and 54% (ci: 47%-61%) in ma conditioned patients. conclusions: patients with aml undergoing allo-hsct with nma conditioning at our institution were older and frailer than ma conditioned patients, but their overall survival after transplantation was comparable. this might be explained by a generally lower aml stage and cytogenetic risk at transplant in nma conditioned patients. jedlickova 1 , saskia güller 1 , rosa toenges 1 , juliane steinmann 1 , hans martin 1 , hubert serve 1 , gesine bug 1 background: allogeneic hsct is urgently indicated in patients with aml in first complete hematologic remission (chr) after intensive chemotherapy with increasing or recurrent minimal residual disease (mrd). these patients are at high risk of hematologic relapse (hr) during preparation of their transplant and hsct with active aml was found associated with poor outcome. azacitidine has recently been shown to substantially delay or even prevent hr in >50% of patients (relaza2 trial, platzbecker et al., lancet oncology 2018) . we here present the outcome of a small cohort of consecutive patients with mrd-positive aml who received low dose cytarabine (ldarac) as bridging therapy prior to hsct. methods: mrd was assessed by quantitative polymerase chain reaction (qpcr) using mutated npm1 (n=5), runx1-runx1t1 (n=2), cbfb-myh11 (n=1) or kmt2a-ptd (n=1). mrd negativity was defined as ratio of oncogene to control gene (abl1) ≤0,01% while increased or recurrent mrd required a ratio >1% (shayegi et al., blood 2013) . primary endpoint of our retrospective analysis was progression to hr (≥5% bone marrow blasts or extramedullary disease); secondary endpoints were achievement of molecular remission prior to hsct, neutropenia g4 according to ctcae, thrombocytopenia g4, anemia ≥g3, admission to hospital, os and rfs. os and rfs were calculated from the first dose of ldarac. ldarac was self-administered subcutaneously by the patients at home at a flat dose of 20mg bid over 10 days and repeated after 4 weeks if necessary. results: between 12/2015 and 10/2018, nine patients (median age 55, range, 22-68 years) with low (n=7), intermediate (n=1) or high-risk cytogenetics (n=1) according to eln criteria 2017 were treated in continuous chr for increasing (n=2) or recurrent mrd (n=7) starting at a median of 260 (range, 154-651) days after the last consolidation therapy, i.e., duration of chr was >6 months in all pts. patients received one (n=4), two (n=2) or three cycles (n=3) of ldarac prior to hsct. in three patients, neutropenia g4 occurred and one patient needed platelet transfusion. all patients were managed in the outpatient setting. in eight out of nine patients (89%), hr was successfully prevented and 3 patients (33%) even became mrd negative prior to hsct. one patient (runx1-runx1t1 positive aml) progressed to hr after one cycle of ldarac and received salvage therapy with high-dose arac and mitoxantrone (ham) prior to hsct. all patients proceeded to hsct from a matched related (n=1), unrelated (n=7) or haploidentical donor (n=1) and are still alive (median follow-up of 666 days). conditioning regimens included fludarabine (flu)/melphalan (mel)/tbi (n=5), flu/mel (n=1), flu/tbi (n=1), flu/busulfan (bu)4 (n=1) and thiotepa/bu3/flu (n=1). after hsct, only the ldarac-refractory patient relapsed, resulting in a probability of rfs of 88% at 2 years. conclusions: our data suggest that a bridging therapy with up to three cycles of ldarac prior to hsct is feasible and was associated with favorable outcomes in patients with npm1-mutated or core binding factor aml and molecular relapse >6 months after achieving a first chr. the treatment has low costs, can be administered on an outpatient basis and is very well tolerated. clinical background: allogenic hematopoietic stem cell transplant (hsct) is the only curative treatment for all the patients with aml. high risk disease qualifies for upfront hsct irrespective of the presence of matched sibling donor (msd). in the absence of msd, haploidentical stem cell transplant is easier option with success rates as high as msd in a high volume transplant centre. we present our experience from a single centre. methods: we analyzed retrospective data of aml patients who have undergone hsct at our centre between january-2013 and august-2018. for msd transplant we used fludarabine + busulfan or fludarabine + melphalan conditioning regimen, in matched unrelated donor transplant (mud) regime used was fludarabine + busulfan + atg. we followed john hopkins's protocol for haploidentical hsct. cyclosporine + methotrexate was used as gvhd prophylaxis in msd and unrelated donor group and cyclophosphamide + tacrolimus + mycophenolate was used for haploidentical post-transplant. day 100 survival, overall survival (os), incidence of gvhd and cmv reactivation was computed. results: a total of 96 aml patients underwent hsct during the study period, the basic and clinical characteristics of the study patients are presented in table 1 . conditioning regime did not have significant impact on os. survival at day 100 was 78%. the os function and relapse free survival (rfs) function did not significantly differ between msd and haploidentical transplantation (68.3% vs 60.0%; p=0.225) and (68.3% vs 75.0%; p=0.760) (graph 1). disease status at latest follow up showed that 82% were in remission and 18% had relapsed. overall one year survival and five year survival in the entire cohort was 68% and 58% respectively. the average cost of msd transplant at our centre is inr 10,00,000 (€ 10000-12000), haploidentical transplant is inr 20,00,000 (€ 25000-27000) and mud transplant is inr 32,00,000 (€ 30,000 + 10000 for stem cell procurement). conclusions: our study showed comparable outcomes in msd and haploidentical transplant with respect to day100 survival, os, and rate of gvhd. in a developing country like india where patients are not covered under state health insurance, the additional cost of procurement of stem cells in a mud transplant would add to the financial burden to the patients. haploidentical transplant is a feasible option in case of non-availability of msd, due to ease of donor availability and strong motivation from the family donor to donate the stem cells. background: allogeneic stem cell transplantation (allo-hsct) is not indicated as consolidation of first complete remission (cr1) in favorable-risk acute myeloid leukemia (aml) bearing mutations in nucleophosmin (npm1) in the absence of flt3 internal tandem duplication (flt3-itd). nevertheless, a substantial proportion of patients eventually proceed to allo-hsct beyond cr1 or for chemoresistant minimal residual disease (mrd) while in cr1, which might compromise transplantation outcomes. the study aimed at examining the characteristics and results of allo-hsct in aml cases with mutated npm1 and wild-type flt3 (npm1mut/flt3wt), with special focus on molecular monitoring of mrd following transplantation. methods: from 11/2010 until 04/2018, 16 patients (women/men, 9/7) underwent allo-hsct for npm1mut/ flt3wt aml. at transplant, median age of patients was 44.5 years (range, 35-63) , and disease phase was cr1 (n=5), cr2 (n=9), or primary refractory (n=2). among the 13 patients who were transplanted in cr and had available molecular mrd assessments, 10 had detectable mutant npm1 transcripts by real-time quantitative pcr (rq-rcr). also, 4 patients fulfilled criteria of molecular relapse (increasing levels of npm1-mutated transcripts in two successive bone marrow samples), with mutant npm1 load of 386-4,900 transcripts/10,000 abl transcripts). the conditioning regimen was myeloablative in the majority of cases (n=14) or reduced-intensity (n=2). the type of donor varied, namely hla-identical sibling (n=6), matched unrelated (n=5), haploidentical relative (n=3), or double umbilical cord blood (n=2). results: engraftment was achieved in all cases, with a median time to absolute neutrophil count >500/ul of 16 days (range, 12-29) . among the 13 patients with posttransplant monitoring of mrd by rq-pcr, 9 exhibited a stable molecular remission whereas a rising level of npm1mutated transcripts was observed in 4 cases due to either hematologic (n=3) or molecular (n=1) relapse of disease. the cumulative incidences (cin) of hematologic relapse and non-relapse mortality (nrm) were 18.75% and 25% at 12 months, respectively. no events of relapse or nrm were encountered beyond 6 months from allo-hsct. out of 3 patients with hematologic relapse post transplant, 2 died of disease whereas one achieved a stable complete remission after withdrawal of immunosuppression. at a median follow-up time of 40 months (range, 14-89), 10/16 patients continue to be alive in cr. the estimated disease-free background: cpx-351 (vyxeos®) is an advanced liposomal encapsulation of cytarabine/daunorubicin at a synergistic 5:1 molar ratio. cpx-351 is approved by the us fda and ema for the treatment of adults with newly diagnosed, therapy-related aml or aml with myelodysplasia-related changes. methods: safety data were pooled from 5 studies of cpx-351 in adults aged 18-75 years with newly diagnosed or relapsed/refractory aml. cpx-351 induction consisted of 100 units/m 2 (cytarabine 100 mg/m 2 + daunorubicin 44 mg/m 2 ) on days 1, 3 , and 5 (second induction: days 1 and 3) . cpx-351 consolidation consisted of 65 or 100 units/m 2 (varying by study) on days 1 and 3. cpx-351 was evaluated against standard-of-care controls. results: baseline characteristics were generally balanced between cpx-351 (n=375) and controls (n=236); the majority of patients were aged ≥60 years (78%; 87%) and had secondary aml (55%; 72%). controls included 7+3 (n=192) and salvage therapy with mitoxantrone/etoposide/ cytarabine (n=23), idarubicin/cytarabine (n=8), other cytarabine-based chemotherapy (n=12), and mitoxantrone/ etoposide (n=1). the treatment-emergent adverse event (teae) profile of cpx-351 100 units/m 2 was comparable to induction controls, but associated with a greater proportion of patients with teaes, grade ≥3 teaes, and serious teaes during consolidation (table) . therefore, the cpx-351 consolidation dose was reduced to 65 units/m 2 in latter studies; this dose demonstrated an improved teae profile similar to consolidation controls. the most frequent system organ class was gastrointestinal disorders for both cpx-351 and controls; a lower incidence was reported for cpx-351 (90%) versus controls (95%), with this difference driven by the lower incidence of diarrhea for cpx-351 (46%) versus controls (66%). the most frequently reported grade ≥3 teaes were febrile neutropenia (cpx-351: 62%; controls: 59%), pneumonia (16%; 13%), hypoxia (10%; 11%), and bacteremia (10%; 3%). early mortality rates, both overall and by treatment period, appeared lower with cpx-351 versus controls at day 30 and day 60 ( table) ; the majority of early deaths were attributable to teaes. conclusions: across the 5 studies comprising the cpx-351 clinical development program, cpx-351 demonstrated a safety profile comparable to conventional chemotherapy in adults with newly diagnosed or relapsed/refractory aml. background: haploidentical hematopoietic stem cell transplantation (hsct) with post-transplantation cyclophosphamide (pgcy) marked improved clinical outcome. recent studies comparing allogeneic hsct using unrelated donors versus haplo donors in patients with acute leukemia have suggested equivalent outcomes. the depletion of tcells with pgcy was subsequently applied for unrelated hsct setting for patients with unrelated donor. methods: we performed a retrospective study on 90 patients with acute leukemia in order to compare the outcome after hla haploidentical (n=30) and unrelated hsct (n=60) with pgcy. the main characteristics of patients were similar in both groups. baseline disease were: 19 aml (63%) and 11 all (37%) for haplo group and 33 aml (55%) and 27 all (45%) for unrelated group. disease state at time of haplo and unrelated-hsct were following: 19 and 50 patients in cr1 (63% and 83%) and 11 and 10 non cr1 (37% and 17%). for aml recipients mainly received thiotepa, busulfan and fludarabine and for all recipients received tbi and etoposide conditioning. all patients who received pbsc graft were treated with rabbit antithymocyte globulin (atg) on days -2 and -1. results: at the time of analysis, the os and dfs did not differ between the haplo and unrelated groups (67% vs 63%, and 63% vs 56%). incidence of severe (grade [3] [4] acute gvhd was the same in two groups (10% versus 8%). recipients of haplo-hsct transplant were statistical significance less likely to experience disease relapse (3% vs 28%) and chronic gvhd (20% vs 47,5%). however, gvhd free relapse free survival (grfs) rate was slightly higher after haplo-hsct (77% vs 64%). addition, cumulative incidence of trm rate was higher after haplo-hsct (30% vs 15%).for haplo and unrelated groups who underwent hsct in cr1, the os were 84% and 67% versus 22% and 45% for those in non cr1. for aml, the os was same in two groups (haplo 63% versus unrelated 67%). however patients with all, the os was higher in haplo group compared with unrelated group (72% versus 59%). the impact of pretransplant disease state have a more powerfull effect on survival in the haplo-hsct setting (for aml cr1 77% versus non cr1 33% and for all cr1 100% versus non cr1 0%). viral reactivations were significant concern in both groups. conclusions: our retrospective analysis suggests largerly similar os and dfs with haplo versus unrelated transplants with pgcy for acute leukemia. our data indicate that haplo-hsct results in a lower incidence relapse and of chronic gvhd and higher grfs compared with unrelated hsct. in addtion, the pretransplant disease state have the important effect on the outcomes in both groups. allo-pbsc with atg can be used safely and effective as graft source in haplo-hsct with acceptable post-transplant outcomes and replaced bm in this settings. more statistical data for transplant related characteristics will be provided at the presentation.we emphasize that use the same pgcy gvhd prophylaxis for all types of allogeneic transplant. based on our results, we recommend haplo-hsct with pgcy against unrelated transplant for patients with acute leukemia. disclosure: disclosure of conflict of interest: none. excellent efficacy and tolerability of inotuzumab ozogamicin in b-cell all relapsed after allo-hsct background: donor lymphocyte infusion (dli) could be used prophylactically to reduce relapse after allogeneic hematopoietic stem cell transplantation for very high-risk leukemia/lymphoma without effective targeted therapy. to compare the safety and efficacy of prophylactic dli for prevention of relapse after allogeneic peripheral blood stem cell transplantation from haploidentical donors (hid-sct) and matched-sibling donors (msd-sct) in patients with very high-risk acute myeloid leukemia (aml), we performed a retrospective, observational cohort study enrolled in 21 hid-sct and 13 msd-sct recipients. methods: the very high-risk features were defined as: (i) in the non-remission (nr) state prior to transplantation, including primary induction failure, relapse untreated or refractory to reinduction chemotherapy, or untreated aml evolution from mds; (ii) achieving complete remission 1 with ≥3 cycles of induction of chemotherapy; (iii) carrying tp53, dnmt3a, tet2 or flt3-itd gene mutation. the scheduled time of the prophylactic dli was +30-60 days after transplantation for msd-sct recipients and +60-90 days for hid-sct recipients. the g-csfmobilized peripheral blood stem cells were infused to the recipient at a dose of 2×10 7 cd3 + cells/kg. csa was given at 2 mg/kg b.i.d from day -3 to day +90 (hid-sct) or to day +60 (msd-sct), and then tapered at 33% per month to be discontinued on day +150-180 (hid-sct) or on day +120-150 (msd-sct) unless graft-versus-host disease (gvhd) developed. if the patients received dli before day +90 (hid-sct) or day +60 (msd-sct), csa was given 8 weeks after dli in hid group and 4 weeks in msd group at a though concentration of 150-250 ng/ml for dliassociated gvhd prophylaxis, and then tapered and discontinued within 2 weeks unless gvhd developed. if gvhd occurred before the scheduled time of prophylactic dli, it would be delayed for 8 weeks when gvhd was well controlled. results: prophylactic dli was administered at a median of 71 (34-240) days for hid-sct recipients and 53 (35-97) days for msd-sct recipients (p=0.008), and both groups displayed similar baseline characteristics except for donor's gender distribution (table 1) . grade 2-4 acute graft-versushost disease (gvhd) at 100-day post-dli was higher in hid-sct group than that in msd-sct group (59.5% vs. 30.8%, p=0.05). grade 3-4 acute gvhd (17.5% vs. 7.7%), 1-year chronic gvhd (36.6% vs. 33.2%) and severe chronic gvhd (15.3% vs. 27.3%) were similar between two groups (p>0.05). one-year non-relapse mortality was higher in hid-sct group than that in msd-sct group with marginal significance (27.9% vs. 0.0%, p=0.061). one-year relapse rate was similar between hid-sct group and msd-sct group (21.6% vs. 36.5%, p>0.05). estimated 1-year overall survival (os, 55.1% vs. 83.9%) and relapsefree survival (rfs, 50.1% vs. 74.0%) rates were both similar between hid-sct group and msd-sct group (p>0.05). in multivariate analyses, non-remission status prior to transplant, poor-risk gene mutations and donor's age ≥ 48 years predicted a higher risk of relapse after dli. nonremission status prior to transplant predicted inferior os and rfs. patient's age ≥ 40 years also predicted an inferior os. conclusions: prophylactic dli after hid-sct demonstrated similar tolerance and efficacy for reducing relapse compared to that after msd-sct for very high-risk aml. disclosure: the authors declare no conflict of interest. prognostic impact of pre-transplant tim3 levels on transplant outcome in acute leukemia patients background: t cell immunoglobulin and mucin domaincontaining protein-3 (tim3), a negative regulator of t cells, is expressed on a variety of tumors including hematological malignancies like acute myeloid leukemia (aml) and some lymphoma types in which it was shown to be associated with an adverse prognosis. the aim of this study is to identify the prognostic impact of pre-transplant tim3 levels on early and late transplant related complications as well as post-transplant relapse and survival methods: a total of 177 hematopoietic stem cell transplantation (hsct) recipients with an initial diagnosis of acute leukemia [median age: 36(16-66) years; male/ female: 111/66] were included in the study. aml was the initial diagnosis in 99 patients (55.9%), acute lymphoblastic leukemia (all) in 74 patients (41.8%), mixed phenotype acute leukemia in 3 patients (1.7%) and blastic plasmacytoid dendritic cell neoplasm in 1 patient (0.6%). soluble tim-3 levels in pre-transplant serum samples were measured with enzyme linked immunosorbent assay (elisa). results: median pre-transplant tim3 level was 955.6 (65.8-3784.4) pg/ml in the whole cohort. pre-transplant tim3 levels were significantly higher in aml patients when compared to all [1063.7(409.5-3784.4) vs 831.4 (65.8-3254.4 ); p=0.01]. tim3 levels were significantly lower in patients with abnormal cytogenetics when compared to normal karyotype (p=0.017). cytogenetic abnormalities, including mainly a complex karyotype or chromosome 8 abnormalities, were more frequent in patients with low tim3 levels (p=0.053). pre-transplant tim3 levels were significantly higher in patients who developed post-transplant viral hemorrhagic cystitis (p=0.034). a positive correlation was demonstrated between tim3 levels and acute graft versus host disease (gvhd) grade (p=0.013; r=0.299). at a median follow-up of 14.6 (0.2-160.9) months, overall survival (os) was found to be better in low-tim3 group when compared to high-tim3 group, without statistical significance (%35.2 vs % 20.4; p>0.05) ( figure 1 ). probability of os was relatively better in both aml (42.6% vs 26.7%; p>0.05) and all patients (29.5% vs 19%; p>0.05) representing low pretransplant tim3 levels in the subgroup analysis conclusions: in this study, elevated levels of pretransplant tim3 levels in aml patients were compatible with the previous reports which had underlined an increased tim3 expression on aml stem cells. the possible association of tim3 expression with cytogenetic features should be confirmed with further studies as there is no adequate data except its relationship with flt3-itd mutational status. tim-3 is also expressed on exhausted t cells in patients with viral infections, including human immunodeficiency virus, hepatitis b and hepatitis c virus. it plays an essential role in the regulation of antiviral and antitumor immune responses which may be an explanation for the increased frequency of hemorrhagic cystitis in patients with higher tim-3 levels. the adverse prognostic impact of tim3 on gvhd and os was confirmed without statistical significance which may be related to small sample size. as tim3 has a wide spectrum of action in the tumor microenvironment including stimulatory and inhibitory activities, further clues are required to define the exact role of this molecule in the clinical course of allogeneic hsct in order to develop targeted therapeutic strategies clinical trial registry: n/a disclosure: nothing to declare p029 homozygous hla-c1 is associated with increased risk of relapse after hla-matched transplantation in recipients with acute lymphoid leukemia: a japanese national registry study background: after hematopoietic stem cell transplantation (hsct), the role of natural killer (nk) cells which express killer-cells immunoglobulin-like receptors (kirs) and recognize hla-class 1 ligands is important. kir2dl1 recognizes not hla-c asp80 (c1), but hla-c lys80 (c2) and has polymorphism based on the 245 th amino acid of the transmembrane domain. low frequency of c2 and high frequency of strong kir2dl1 are characteristics observed in japanese. by using large transplant database, we reported that homozygous hla-c1 (c1/c1) recipients displayed lower relapse rates than did c1/c2 recipients after hla-matched hsct for acute myeloid leukemia (aml; hr = .79, p = .006) or chronic myeloid leukemia (cml; hr = .48, p = .025). this effect seemed to be independent of acute graft-versus-host disease (agvhd) or cytomegalovirus reactivation occurrence (arima n et al bbmt 2018) . methods: relapse rates of japanese recipients who first underwent hla-matched hsct between 1996 and 2016 for the treatment of acute lymphoid leukemia (all) were compared between c1/c1 pairs and c1/c2 pairs, using data from japanese data center for hematopoietic cell transplantation and adjusting for transplant characteristics. cord blood transplantation was excluded. multivariable competing risk regression analyses were performed to evaluate relapses and relapse-free survival (rfs) was estimated using kaplan-meier method. results: after 61 recipients who did not achieve remission or experienced graft failure and 41 recipients not-expressing c1 were excluded, resting 2779 recipients aged 0-72 years (median, 31.2 years) were analyzed. the median follow-up period for survivors was 5.0 years. there were 2447 recipients expressing c1/c1 and 332 recipients expressing c1/c2, respectively. after hla-matched hsct, c1/c1 recipients had higher relapse rates than c1/ c2 recipients (hr = 1.55, p = .003), resulting in worse rfs among c1/c1 recipients (hr = 1.27, p = .034). the frequent relapse in c1/c1 recipients than in c1/c2 was noticeable among recipients with agvhd (hr = 1.89, p = .002), those without cytomegalovirus reactivation (hr = 1.84, p = .002), and those with ph-negative all (hr = 1.88, p = .001). conclusions: kir2dl1-positive nk cells may promote graft-versus-leukemia (gvl) in c1/c1 recipients with aml or cml but suppress gvl in c1/c1 recipients with all. one interpretation is that transplant-activated nk cells impair antigen-presenting cells or deprive cytotoxic tlymphocytes of their gvl effects on all cells. this hypothesis may be explained by the fact that agvhd was necessary for the recessive relapse in c1/c1 recipients with all. furthermore, ph-positive all cells sometimes mimic aml cells in terms of their frequent myeloid antigen expression and might be directly targeted by nk cells. it would be necessary to further clarify in vitro the character of nk cell-affecting in the transplant immunity against residual leukemia cells. disclosure: authors have nothing to declare. hematopoietic stem cell transplantation with sequential conditioning for children with relapsed/refractory acute leukemia nao yoshida 1 , kazuki matsumoto 1 , daiki yamashita 1 , yiqing zhu 1 , daichi sajiki 1 , ryo maemura 1 , hirotoshi sakaguchi 1 , asahito hama 1 1 children's medical center, japanese red cross nagoya first hospital, nagoya, japan background: patients with acute leukemia who fail to achieve complete remission show a dismal prognosis even with allogeneic hematopoietic stem cell transplantation (hsct). this study evaluated whether sequential conditioning approach that is cytoreductive chemotherapy applied shortly prior to the main conditioning followed by hsct can improve prognosis in such high-risk patients. methods: we retrospectively analyzed the outcomes of 90 children (median 8, range 0-18 years old) with primary refractory (n = 11) or refractory relapsed (n = 79) acute leukemia (aml n = 43, all n = 47) who received hsct in our department between 1990 and 2016. the stem cell source was related peripheral blood (pb) in 4 patients, related bone marrow in 31, unrelated bone marrow in 40, or unrelated cord blood in 15. the grafts were hla serologically matched (n = 63) or mismatched (n = 27) with the recipient. in total, 29 patients received the sequential conditioning approach. as cytoreductive chemotherapy, fludarabine/cytarabine/idarubicin/g-csf (flag-ida) was used in 12 patients, mitoxantrone or daunorubicin/cytarabin in 10, or other regimens in 7, and 6 of them were combined with gemtuzumab ozogamicin. without waiting for hematological recovery, the patients promptly underwent hsct; therefore, the median interval between cytoreductive chemotherapy and main conditioning was 11 days. the main conditioning regimens were total body irradiation-based myeloablative (n = 22), busulfan-based myeloablative (n = 4), or reduced intensity (n = 3). results: in 90 children with relapsed/refractory acute leukemia, the 5-year overall survival (os), leukemia-free survival (lfs), cumulative incidence of relapse (ri), and transplantation-related mortality (trm) were 24%, 21%, 53%, and 26%, respectively. in multivariate analysis, the use of sequential conditioning was identified as the most favorable factor for lfs (hazard ratio [hr] 0.37; p = 0.001), although there were no differences in the outcomes according to the types of cytoreductive chemotherapy or the main conditioning regimen. hla-matched donor (hr 0.46; p = 0.005) and pb blasts-negative at the beginning of conditioning (hr 0.49; p = 0.02) were also independently associated with better lfs. with sequential conditioning, leukemia burden prior to the hsct was significantly reduced; pb blasts became undetectable at the beginning of conditioning in 66% patients given the approach, while in 38% patients without the approach (p = 0.02). notably, the outcomes in the patients without pb blasts at the beginning of conditioning who received sequential conditioning were promising; the 5-year os and lfs reached 73% and 62% and the 5-year ri and trm were 33% and 5%, respectively. conclusions: our study reveals that hsct with sequential conditioning can be an effective and tolerable treatment option for children with relapsed/refractory acute leukemia. the treatment strategies that focus on the reduction of leukemia burden immediately prior to hsct may contribute to the induction of long-term remissions in patients with high-risk acute leukemia. disclosure: this research was funded by japanese red cross, nagoya 1st. hospital research grant nfrch18-0028. use of blinatumomab to achieve remission and consolidation with haploidentical transplant with cyclophosphamide post for the treatment of children with refractory acute lymphoblastic leukemia (all) background: most of patients with all in relapse or refractory to conventional treatment have only 30% possibilities to achieve long term remission. this report refers to the therapeutic efficacy and adverse events from the blinatumomab to achieve molecular remission in patients with pre-b cd19+ which lead to haploidentical with cyclophosphamide post transplant as a consolidation. methods: a pilot study was conducted in children with refractory all preb-cd19 +. as a strategy to achieved remission blinatumomab was used at a dose 10 μg/m2 for continous infusion of 48 hours, increasing the dose to 15 μg/m2 during 28 days, patients with a mrd of < 0.002 log, after 2 cycles received an haploidentical bone marrow transplant as a consolidation, the conditioning regimen was with total body irradiation scheme at 200 cgy/day/3 days, cyclophosphamide and etoposide. receiving prophylaxis for gvhd with cyclophosphamide. results: a total of 10 patients were included, seven of them achieved complete remission after 2 cycles of blinatumomab, one with partial remission (table 1) , these seven patients, six received an haploidentic transplant achieving graft in 6 of the transplanted patients. one patient had a bone marrow relapse in the first 6 months of the follow-up and 5 patients are free of disease with a follow-up to 20 months (figure 1). as a acute complication the 10 patients presented cytokine release syndrome, during the infusion of blinatumumab 10 patients presented tachycardia (table 3 ) and the 6 patients presented agvhd after hsct (5 grade i-ii and i grade iv). conclusions: allogeneic bone marrow transplant constitutes a treatment option on those patients that relapse or become refractory to treatment, one of the major problem is basically to identify a hla-identical donor, the alternative is an haploidentical donor. the most important factor to get these results is the disease status before transplant. the use of blinatumomab has proven to be effective in achieving remission in relapse acute linfoblastic leukemia pre-b cd 19+ or refractory to treatment. characteristics nº % male 4 40% median age at diagnosis, (range), years 9. 22 (7-12) status of disease 2 o + 3 relapse 6 60% refractory to primary or salvage therapy 4 40% complete remission after blinatumomab 7 70% partial remission after blinatumomab 1 10% active disease 2 20% [[p031 table] 1. table n°1 . demographic characteristics of patients undergoing blinatumomab (n=10)] disclosure: a. olaya-vargas, r. rivera-luna, y. melchor-vidal, h. salazar-rosales, g. lopez-hernandez, n. ramirez-uribe. we wish to confirm that there are no known conflicts of interest associated with this abstact, the only financial support was provided by mexican associations that helping children wiht cancer in a few patients. sequential high-dose chemotherapy reinduction followed by myeloablative allogeneic transplant for active acute myeloid leukemias methods: at our center, 27 relapsed/refractory aml patients were transplanted during chemo-induced neutropenia after high-dose salvage chemotherapy. median age at transplant was 52 years (range 21-62). patients suffered from de novo (n= 18/27, 67%) or secondary aml (n=9/ 27, 33%). genetic risk stratification was reported using stardardized groups proposed by the european leukemia net (eln) in 2010. favorable, intermediate i and ii and adverse risk category at diagnosis was observed in 1/27 (4%), 19/27 (70%), 7/27 patients (26%) respectively. all patients had active disease at the time of sequential therapy and median marrow blast count was 25% (range 7-88%). patients received a high-dose cytarabine based (mec in 17/ 27, 63%) regimen as salvage therapy. donors were haploidentical relatives for 15/27 (56%) patients, identical siblings and matched-unrelated for 6/27 patients (22%) and 6/27 (22%), respectively. a myeloablative conditioning was used to further implement anti-leukemic effects. conditioning, thiotepa-busulfan-fludarabine in 89% patients, was started at a median of 8 days (range [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] after the last day of chemotherapy. bone marrow and peripheral blood stem cells were used as graft source in 11/27 (41%) and 16/27 (59%) patients. graft-versus-host disease (gvhd) prophylaxis and supportive care were administered accordingly to each hsct platform. results: all patients engrafted. median day of neutrophil recovery was day +16 (range 12-23). median follow-up of survivors was 31 months (range 4-125). non relapse mortality and relapse incidences (nrm, ri) were 16% and 48% at 1 year and 16% and 58% at 3 years, respectively. overall cumulative incidences of acute and chronic gvhd were 48% and 43% at day +100 and + 400. one and 3 year overall survival (os) were 58% and 34%, while 1 and 3 year event-free survival (efs) were 35% and 26%. significant better os and efs were observed in patients with favorable-intermediate i-ii versus adverse risk score (1-3 years os 64% and 50% vs 43% and 0% p=0.022; 1-3 years efs 43% and 36% vs 14% and 0% p=0.013). adverse risk had a significant impact on os (hr 3.24, p=0.030) and efs (hr 3.33, p=0 .018) by univariate analysis and on ri (sdhr 3.02, p=0.031) by fine and gray test. conclusions: though small the patient cohort, our findings suggest that sequential therapy with a myeloablative hsct is feasible in treating relapsed/refractory aml. transplant-related toxicity was low (16%) and relapse was the major treatment-failure. however, even with this approach, patients with adverse cytogenetic features have a very dismal prognosis. for these patients, the use of new drugs before hsct and/or maintenance therapy after transplant is highly encouraged to improve outcomes. disclosure: alessandro busca: honoraria from gilead sciences, merck, pfizer pharmaceuticals and jazz background: in spite of satisfactory results of overall survival (os) after allohsct in 1 st and 2 nd cr aml, relapse free survival (rfs) and graft-versus-host-disease free/relapse free survival (grfs) require further improvement. the detection of mrd is one of the factors which influence on the outcome of allohsct in aml is unclear but identification is important to improve risk-adapted relapse prophylactic treatment after allohsct. aim. to evaluate outcomes of allohcst in 1 st and 2 nd cr pediatric aml depending on the level of mrd status before myeloablative (mac) or reduced intensity conditioning regimens (ric). methods: the data of 72 children with aml in 1 st and 2 nd cr underwent allohsct between 2008 and 2018 were analyzed. median age at the moment of allohcst was 8 years old (2-18). mrd negative status had 42 (58%) patients, 30 (42%) were mrd positive by flow cytometry. mac based on busulfan (16 mg/b.w.) received 27 (37%) patients, on treosulfan -7 (10%) patients. ric based on melphalan received 20 (28%) patients, based on busulfan (8 mg/b.w.) -18 (25%) patients. patients received prophylaxis of agvhd by atg 20 (28%) or ptcy -48 (66%) patients plus csa -23 (32%) or tacrolimus ± sirolimus -43 (60%) patients that depended on source of transplant (related, unrelated or haplo donor) . results: at the median follow up 3 years in the cohort of mrd positive patients os is 66% vs 72% in mrd negative (p>0,05). rfs is 56% vs 83% accordingly (p=0,01). graft-versus-host-disease free/relapse free survival (grfs) in mrd positive patients is 37% vs 51% in mrd negative (p>0,05). os, rfs, grfs in mrd positive patients after mac is 57%, 42%, 30% vs 75%, 68%, 43% after ric accordingly (p>0,05). os, rfs, grfs in mrd negative patients after mac is 75%, 85%, 55% vs 65%, 82%, 47% after ric accordingly (p>0,05). os, rfs in mrd negative patients with/without ptcy is 82%, 86% vs 42%, 78% (p>0,05); grfs is 62% vs 28% accordingly (p=0,042). os, rfs, grfs in mrd positive patients with/without ptcy is 66%, 55%, 54% vs 66%, 57%, 27% (p>0,05). conclusions: mrd status does not statistically significant affect on os that can be related to different approaches to the treatment of relapse after allohsct. mrd positive status statistically significant decreases rfs that underline the necessity of posttransplant therapy improvement. ric vs mac in all patients in first and second remission do not show statistically significant impact on os, rfs, grfs. ptcy significantly improves grfs in mrd negative patients. disclosure: none of the authors has anything to disclose. background: with increasing overall-survival (os) of lymphoma patients, higher incidences of therapy-related clonal bone marrow diseases, such as acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) are occuring. generally, the outcome is considered poor. allogeneic hematopoietic stem cell transplantation (allo hsct) often remains the only potentially curative treatment option. nonetheless, there is only little data available concerning this patient group. methods: we retrospectively collected data from 33 patients with therapy-related aml (taml) and mds (tmds) after treatment for hodgkin's lymphoma (hl; n=7 and n=2) or non-hodgkin's lymphoma (nhl; n=10 and n=14), who received an allo hsct between 2000 and 2018. median follow-up of surviving patients was 3.1 years (range 2.6 months-9.4 years). background: the prognosis of relapsed/refractory acute leukemia (r/r al) is poor and the treatment is challenging. in this setting, allogeneic stem cell transplantation (allo-sct) constitutes the only curative option although the high relapse rate and non-relapse mortality (nrm). the sequential conditioning regimen followed by allo-sct has been used for persistent disease and aims to improve disease control by intensified chemotherapy, thus conceding more time for the presumed graft-versus-leukemia effect to occur. methods: the clinical outcome of r/r al with the sequential conditioning regimen combining a chemotherapy rescue followed by ric allo-sct in our center is described. patients who underwent a sequential allo-sct from 2005 to 2017 are included. the primary endpoint was progression free survival (pfs) and overall survival (os) that were estimated by the kaplan-meier method. secondary endpoints were non-relapse mortality (nrm). background: recommendations of the 2017 european leukemia net (eln) for favorable-risk genetics (frg) acute myeloid leukemia (aml) favor consolidation over transplantation, although reviews suggest advantage of autologous stem cell transplant (asct) in event free survival. our objective was to compare the progression free survival (pfs) and overall survival (os) of normal karyotype npm1 mutated without flt3 itd or allelic ratio < 0.5 (npm1+) aml patients treated with consolidation chemotherapy alone (cc), asct or allogeneic stem cell transplant (allosct). methods: retrospective review of npm1+ frg-aml patients, treated in one institution (2008 to 2017) with the following induction regimens: cytarabine (ara-c) and vp-16 with daunorubicin (ade) or mitoxantrone (mice). consolidation regimens were ara-c with daunorubicin (ac-d), idarubicin, vp-16 and ara-c (mini-ice) or highdose ara-c (hidac). in asct, conditioning regimens were bucy or bvac and in allosct were bucy or flubu. pfs and os were calculated from the start of the last consolidation or stem cell infusion. results: a total of 39 patients were evaluated, with a median age of 53 years (y) (23-68y), 69% female, 95% with ecog performance status (ps) 0-1 and 36% with ageadjusted charlson comorbidity index (aacii) ≥2 at diagnosis. patients were treated with cc in 33% (n=13), asct in 36% (n=14) and allosct in 21% (n=12) of cases. there were no differences between groups for age, aacii, ps, leucocytes at diagnosis or extra-medullary disease. flt3-itd was more frequent in allosct group (64%) than cc (23%) or asct (8%; p=0.07). at induction, ade was used in 82% and mice in 18% of patients, with a complete remission (cr) rate of 95%. there were no differences between groups for induction regimen or cr. in cc group, consolidation regimens were 1 cycle (8%) and 2 cycles ac-d (61%) and 2 cycles mini-ice (31%). asct patients received consolidation with 1-2 cycles ac-d (78%) and 1 cycle mini-ice (22%), while allosct patients received 1-2 cycles ac-d (84%), 2 cycles hidac (8%) and no consolidation in 8%. [[p037 image] 1. figure 1 . pfs at 3y in cc, asct and allosct groups.] median follow-up was 39 months, pfs at 3y was 53% and os at 3y was 64%. pfs at 3y for asct group was superior then cc and allosct groups (61%, 51% and 44%, respectively; figure 1 ), although not statistically significant. os at 3y was statistically similar between groups, although inferior in allosct comparing to cc and asct (44%, 77% and 70%, respectively). conclusions: in this historical cohort review, although there was no advantage in os for asct in npm1+ frg aml, our data suggests that there might be a pfs improvement in asct over cc, which needs to be further addressed in prospective studies. disclosure: nothing to declare background: acute myeloid leukemia is a hematological malignant disease that motivates the persistent struggle in the scientific world to provide effective cure that can establish acceptable survival rates in this group of patients. autologous stem cell transplantation with myeloablative conditioning is still a powerful weapon that can be used against this entity methods: we have evaluated retrospectively patients with aml where autologous stem cell transplantation was performed in the period from 2000 till 2018. our group consisted of 94 patients; male patients 45 (47.8%), female patients 49 (52.2%). median age at diagnosis was 44 years (16-68). the average period from time of diagnosis to autologous sct was 7.05 months. results: in the majority of our group, we used myeloablative conditioning regimen with busulphan-cyclophosphamide, 60 patients (63.8%), in 2 patients (2.1%) we have added melphalan to bu-cy conditioning, in 22 (23.4%) patients we used beam conditioning and in the rest, 10 patients (10.6%) we used bam conditioning regimen. as auto graft we used peripheral blood stem cells (pbsc) in 78 patients (82.9%), and in 16 patients (17.1%) we used bone marrow. the main mobilising regimen for pbsc was g-csf + etoposide and it was performed in 44 patients (46.8%), and in the remaining 34 patients (36.1%) mobilising of pbsc was performed only with g-csf. the mean number od apheresis procedures done in our group was 1.55, and the mean number of collected mononuclear cells was 3.05x10 8 /kg tt. the mean time to engraftment was 12.8 days (9-23). the transplant related mortality (trm) was 2.1 %. the 5 year overall survival of our patients was 46.7 patients. the main reason for death was relapse of the primary disease(73%). 20 patients (21%)were treated with salvage chemotherapy regimen (flag-ida) because with the standard induction regimen 7+3 there was absence of adequate therapeutic response, or predominantly no complete remission was achieved. all patients were transplanted in complete remission conclusions: autologous stem cell transplantation could be an acceptable therapeutic solution for patients with aml as a consolidation therapy, where neither suitable compatible donor is available nor allogeneic stem cell transplantation could not be performed from various reasons depending on the bone marrow transplant unit disclosure: nothing to declare p040 prophylaxis dli alone may not prevent relapse of flt3-itd positive aml after allogeneic hct background: one of the most potent prognostic factors affecting outcomes in aml is the presence of cytogenetic and molecular markers which can guide the selection of post-remission therapies. recently, favorable outcomes of npm1 wt /flt3-itd neg /non-cebpa dm group after allogeneic hematopoietic cell transplantation (allo-hct) have been reported, that is similar to those of favorable risk by the eln risk classification. however, the role of allo-hct compared to consolidation chemotherapy has not yet been elucidated. methods: the data of 88 patients who were diagnosed with aml and received intensive induction therapy from 2015 march to 2017 july were included in the current study. to address the time dependence of the allo-hct, the simon and makuch method was used in the graphical representation and the mantel-byar test and andersen and gill methods for identifying risk factors for long-term survival. results: median age of the patients were 53 years (range 21-69), and 49 patients (56%) were male. npm1 mutation was detected in 14 patients (16%), and flt3-itd were none, low, and high ratio in 69 patients (78%), 9 (10%), and 10 (12%), respectively. the eln risk classification divided the patients into favorable, intermediate, and adverse risk group in 31 patients (35%), 38 (43%), and 19 (22%), respectively. npn1 and flt3-itd both negative group included 29 patients (33%). allo-hct was performed in 48 patients (55%). overall, complete response (cr) after induction therapy achieved in 63 patients (72%), and 7 patients (8%) were primary refractory disease. cr rates did not differ between npm1 wt /flt3-itd negative group (n=17/29, 58.6%) and other intermediate risk group (n=6/9, 66.7%; p=0.967) . with median follow-up duration of 12.9 months (range 1.3-39.0 months), one-year os rate were 100%, 83.5±6.9%, 56.1±12.8% in favorable, intermediate, and adverse risk group (p < 0.001). among intermediate risk group, os rate of npm1 wt /flt3-itd negative group was similar to other intermediate risk (p=0.403). allo-hct was performed in 11 patients of npm1 wt /flt3-itd negative group. one-year os rates did not differ between npm1 wt /flt3-itd negative and other for allogenic hematopoietic stem cell transplant (allo-hsct), as a strategy to prolong survival. methods: data from aml pts over 60 years, who underwent ric allo-hsct in our institution between september 2011 and september 2017, was retrospectively collected from clinical files to evaluate the overall survival (os) up to november 2018. we calculated the os using kaplan-meyer curves. results: we identified 15 pts, median age 62 y.o. (60-67) and median htc-i score 2. the median follow-up was 25 months. one patient (pt) had cml blast crisis and was on first major molecular remission. of the remaining 14 aml pts, 7 were in 1 st complete remission (cr), 4 in 2 nd cr and 1 with progressive disease (pd); the other 2 pts could be classified as mds according to 2016 who diagnostic criteria and were in cr1. donors (d) were: 3 matched unrelated (mud), 5 mismatched unrelated (mmud -9/10), 6 matched siblings and 1 haploidentical. thirteen pts were infused with peripheral blood hsc and 2 with bone marrow. conditionings were: flubcnumel in 5 unrelated donor (ud) pts and 4 siblings, flumel in 1 ud pt and 1 sibling, flubu in 2 ud pts and flutbi 2gy in 1 sibling and in the haploidentical. graft versus host disease (gvhd) prophylaxis was tacrolimus (tac) + mmf in 5 ud pts and 1 sibling, tac + mtx in 2 ud pts and cyclosporine (cya) + mmf in 1 ud pt and 5 siblings. all mmud pts had atg. ptcy was done in the haploidentical setting with tac + mmf. the median time to neutrophil and platelet engraftment for the whole cohort was 14 and 11 days, respectively. one pt with secondary engraftment failure required re-infusion of selected cd34+ cells. ten pts presented with mild acute skin gvhd. eleven pts had chronic gvhd, 2 classified as severe; 7 required systemic therapy, 5 of those beyond 1 year. the median time on immunosuppressants was 404 days. at 2 years the os was 63.5%. there were 6 deaths: 3 disease-related (2 relapses at 22 and 58 months and 1 pd at d+30), 2 infection complications (2 septic shock) and 1 to secondary neoplasia. other relevant complications were hypoxemic pneumonia in 5 pts, 1 urinary sepsis, cmv and ebv reactivation respectively in 8 and 4 pts; pulmonary and renal toxicity either in 2 pts. at end of follow-up, 9 pts were in remission, 8 without negative measurable residual disease (mrd), the other mrd negative pt died of septic shock and severe intestinal gvhd. conclusions: in this small cohort, chronic gvhd and infectious complications were major causes of morbidity but there were no treatment related deaths before d+100. pts maintaining or achieving mrd negativity after transplant had better survival. although with only 15 pts, these results suggest that allo-hsct is feasible as consolidation strategy in selected aml pts over 60 years. [[p043 image] 1. overall survival] disclosure: nothing to declare. background: hematopoietic stem cell transplantation (hsct) is the only curative option for fanconi anaemia (fa); an inherited disorder characterized by congenital anomalies, progressive bone marrow failure (bmf) and a predisposition to develop malignancies. methods: we retrospectively analysed the data of 27 consecutive patients that underwent hsct at this centre from 2001 till june 2018. the data was analysed for variables affecting the outcome in terms of overall survival (os). results: median age at diagnosis was 10 years (2-20 years). median age at transplant was 11.3 years (4-25 yrs). all patients at transplant were in aplastic phase. male to female ratio was 1.2:1. twenty-four (88.9%) patients had congenital anomalies along with bmf while 3 were phenotypically normal. twenty-three (85.2%) patients were 10/10 hla matched with siblings, 2 with parents and 2 with cousins. eleven (40.7%) patients had gender mismatch transplant. three patients had major and 6 had minor abo mismatch. background: paroxysmal nocturnal hemoglobinuria (pnh) is a rare clonal non-neoplastic hematopoietic stem cell disease whose incidence is 1.5-2.9 cases/million of individuals worldwide. disease characteristics and natural history have been mostly analyzed by multicenter, retrospective studies, with the limit of heterogeneous approaches. herein we report the incidence of severe complications and outcome in a real life setting scenario of pnh patients consecutively diagnosed and managed at our pnh referral center between january 1985 and june 2018. methods: patients received a homogeneous diagnostic and treatment approach according to the period of observation (availability of diagnostic tests and eculizumab). all patients treated with eculizumab received vaccination with conjugated anti-meningococcus acwyserotypes and, since 2016, conjugated anti-meningococcus b-serotype. in the event of any complication, patients could refer to dedicated hematology emergency rooms (er) 24 hours daily. the occurrence of renal failure and pulmonary hypertension was specifically evaluated. the renal function was studied according to the cockcroft-gault formula and the lung function was prospectively monitored by daytime-on exertion, nocturnal pulsoximetric profiles and complete spirometric tests, with dlco measurement. results: overall,48 pnh patients, median age 36 years (range 17-84), were analyzed. at diagnosis, 26 patients had classic pnh, 19 aplastic pnh and 3 an intermediate form. the cumulative incidences (ci) of thrombosis, and clonal hematologic neoplasm were 29%, and 6%, respectively. ci of pancytopenia in the 26 patients with classic pnh was 23%. one patient showed a spontaneous disappearance of the pnh clone. since 2005, eculizumab was administered in 28 patients. after eculizumab treatment 50% and 32% of patients reached hemoglobin level > 11g/dl and >8< 11g/ dl without transfusion, respectively, while 18% were nonresponsive. during eculizumab treatment no thrombotic event was observed while two severe infectious episodes (respiratory tract and urinary tract infection) were observed in only one of the 28 patients. extravascular hemolysis was demonstrated in 50% of patients. no patient showed a significant reduction of the renal function.out of 24 patients prospectively monitored for lung function no pathological alteration in any diurnal or nocturnal pulseoximetric test was observed. no patient showed obstructive or restrictive ventilatory deficiency, nor reduced dlco values. 30-years overall survival (os) was 90% (4 patients who died for non-pnh related reasons were censored at the last follow-up).a better os, even if not statistically significant,was associated to the absence of thrombotic events (90%vs70%), and the period of diagnosis (100% in 2006-2018, 91% in 1996-2005, 75% in 1985-1995) . conclusions: our study reports a better os and lower rate of severe complications in pnh compared to previous experiences. although renal failure and lung hypertension have been reported by other groups, we did not observe these complications along a prolonged follow-up. we can assume that the availability of a dedicated er service enabled an early diagnosis and prompt treatment in case of hemoglobinuria crises (reducing the risk or organ damage) or other complications. the use of eculizumab, together with improved supportive approaches, presumably accounts for the trend towards a better survival witnessed over the last decade in the management of pnh patients. disclosure: nothing to declare haploidentical and unrelated allogeneic stem cell transplantation in aplastic anemia:single center experience zafer gulbas 1 , elif birtas atesoglu 1 , meral sengezer 1 , i̇mran dora 1 , cigdem eren 1 , suat celik 1 , demet cekdemir 1 background: aplastic anemia is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia. allogeneic stem cell transplantation from hlamatched sibling is performed in the firstline setting in young aplastic anemia patients and in elderly patients who are refractory to immunosuppressive treatment. but if the patient does not have a hla-matched sibling, allogeneic stem cell transplantation is performed from unrelated and haploidentical donors. in this study, we analyzaed and compared the results of aplastic anemia patients who had undergone allogeneic stem cell transplantation either from matched unrelated or haploidentical donors. methods: we collected and analyzed data of aplastic anemia patients who had undergone allogeneic stem cell transplantation from matched unrelated or haploidentical donor between 2011 and 2018. results: there were 10 patients who had received allogeneic stem cell transplantation from unrelated donors and there were 10 patients who had undergone haploidentical transplantation. but in 4 patients who had undergone haploidentical transplantation, engraftment failure had occurred and they were transplanted from different haploidentical donors fort he second time. so a total of 10 unrelated and 14 haploidentical transplants were performed. the median age of patients who had undergone unrelated transplantation was 29(16-55) and the median age of patients who had undergone unrelated transplantation was 22(19-61) . the results of the haploidentical and unrelated transplantations are shown in table 1 . the neutrophil and platelet engraftment times were significantly longer in haploidentical transplantations (p=0,006 and p=0,005, respectively). however, vod, gvhd and 100 day mortality rates were not different in the 2 groups. similarly overall survival (os) of the patients who had undergone haploidentical or unrelated transplantation were not significantly different (p=0,38) ( figure 1) . conclusions: although the number of patients are low in this study, we can conclude that urelated and haploidentical transplantation in aplastic anemia have comparable toxicity and efficacy. background: autologous stem cells transplantation (ahsct) is an effective treatment for very aggressive autoimmune diseases such as multiple sclerosis (ms). however, toxicity remains the major concern to a wide application of this approach. post transplant viral reactivations may induce severe complications and a rigorous monitoring of peripheral blood viral load for a prompt and effective therapy is required. a higher rate of ebv and cmv reactivation has been observed in patients affected by ms and conditioned with beam plus atg compared with a controlled group of lymphoma patients without atg in the conditioning regimen [1] . we report here the policy of our center about both monitoring and treatment of such side effect. methods: a series of 37 consecutive patients with ms, transplanted between 2014 and 2018 is included in this analysis. all patients were mobilized with cyclophosphamide 4g/sqm + g-csf and conditioned with beam plus rabbit atg (thymoglobulin©, 7.5mg/kg). monitoring of cmv/ebv dna on whole blood by quantitative pcr was performed after the engraftment, weekly for at least one month, then at longer intervals. pre-emptive treatment with valgancyclovir was started in case of cmv viral load ³1x10^4 copies/ml. in case of ebv assay between 1x10^4 and 1x10^5 copies/ml further determinations were performed and rituximab-based treatment was started if the viral copies exceeded 1x10 5 copies. patients received treatment in case of symptomatic disease for any value of the pcr of both viruses or ebv titer ³1x10^5 copies/ml. results: detectable dna for cmv was observed in 15/ 37 (40,5%) patients at a median time from transplant of 23 days (range 12-36) and 6/37 (16%) required pre-emptive treatment. all patients promptly responded to treatment within 2 weeks. ebv viral load was detectable in 19/37 patients (51,3%) at a median time of 22 days (range 12-52). one patient out of 19 started the treatment on first determination for high viral load (>1x10^6/ml); nine presented an ebv viral load over 1x10^4 copies/ml, three of them were treated thereafter for the persistent increase of the viral load (> 10 5 /ml). six patients spontaneously recovered the ebv reactivation. previous treatments were not predictive of any higher risk of viral reactivation. no impact on engraftment related to the reactivation was observed. conclusions: this policy shows that, despite a high rate of cmv and ebv reactivation, no grade iii-iv adverse events were observed, suggesting the key role of viral monitoring in these patients and the efficacy of the preemptive treatment. ebv reactivation at low titers should be monitored to identify those cases that could achieve a spontaneous resolution and avoid the induction of further immunosuppression by rituximab. these data confirm that patients diagnosed with ad undergoing autologous hsct need a more intense pattern of care than hematological patients. background: autoimmune diseases are chronic serious conditions that are often refractory to standard therapies. since 1996, autologous haematopoietic stem cell transplantation (hsct) has been a very promising alternative that has shown satisfactory long-term results. the aim of this study is to evaluate immune reconstitution and mortality following hsct in patients with autoimmune disease. methods: a retrospective study was conducted on patients with diagnosis of autoimmune diseases that underwent autologous hsct between july 2012 and january 2018 at a tertiary referral center in colombia, south america. descriptive statistics were used to analyze patient's demographic and clinic characteristics. results: seven patients were included, with a mean age of 37 years (range 26-57). five patients were female (71%). the indications for hsct were systemic sclerosis (n=3) , multiple sclerosis (n=2), and myasthenia gravis (n=2). the conditioning regimen administered in patients with systemic sclerosis was cyclophosphamide + human anti-t lymphocyte immunoglobulin, beam (carmustine, etoposide, cytarabine (ara-c), and melphalan) + human anti-t lymphocyte immunoglobulin in patients with multiple sclerosis, and cyclophosphamide + human anti-t lymphocyte immunoglobulin in myasthenia gravis. median time to myeloid engraftment (neutrophils>0.5×109/l) was 12 days post-transplantation, and platelet engraftment, defined as >20,000 platelets/mm 3 untransfused, was 11 days post-hsct. median time of hospitalization was 21 days (range 11-66); longer in-patient management was due to infectious complications. infectious complications included bacteremia caused by e. coli and pneumocystis pneumonia that resulted in septic shock and acute respiratory failure, respectively. evaluating t-cell immune reconstitution, none of the patients had reached normal cd4+ cell value after one year of hsct follow-up. four patients (57.1%) reached normal cd8+ cells value at 3 months post-hsct. regarding bcell immune reconstitution, 85.7% of the patients (6/7) had reached both igg and iga normal levels at one-month post-hsct, and four patients (57.1%) had achieved normal igm background: multiple sclerosis(ms) is an inflammatory disease caused by autoimmune reactivity of t cells against myelin. there is accumulating evidence of the efficacy of highdose chemotherapy followed by autologous haematopoietic stem cell transplantation(ahsct) in ms patients who failed response to standard immunotherapy, despite a variability in eligibility criteria, conditioning regimens and outcome. methods: we retrospectively reviewed ms patients submitted to ahsct in our centre (2014) (2015) (2016) (2017) (2018) . patient eligibility criteria were active relapsing remitting(rrms) or secondary-progressive ms (spms), with prior failure to treatment with disease-modifying therapies and evidence of disease activity (clinical relapse or new active lesions in magnetic resonance [mr] ). mobilization of cd34+cells to peripheral blood was performed with granulocyte colony-stimulating factor(g-csf 10μg/kg/day) and conditioning regimen according to beam protocol. the severity of ms disability was classified according to the expanded disability-status scale (edss) and ahsct toxicity was evaluated by ctcaev5.0. results: seven ms patients had undergone ahsct (4 female/3 male), with a median age at ahsct of 39.9 years (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) . median age at ms diagnosis was 31.6 years(27-36) and median time from diagnosis until ahsct was 6.8 years (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) . four patients (57.1%) had spms and 3(42.9%) had rrms, with 4(57.1%) having mr active lesions. pre-ahsct relapse rate per year was 2 (1) (2) (3) (4) . median baseline edss was 6.5(5.5-7.5). median number of previous dmts was 6 (3) (4) (5) (6) (7) (8) (9) . all patients had been treated with corticosteroids and copaxone, 5(71.4%) received rituximab, 6 (85.7%) natalizumab, 1 (14.3%) alemtuzumab, 2(28.6%) fampridine and 2(28.6%) fingolimod. all patients collected enough cd34+ cells in a single apheresis session. median number of cd34+ cells infused was 7.2±3.8x10^6/kg, for a mean dmso volume of 50.4 ±14.8 ml. median inpatient stay during ahsct was 28 days(21-47). all patients developed febrile neutropenia, one was admitted to intensive care unit due to sepsis. one patient developed an anaphylactic reaction to transfusion and another a self-limited encephalopathy. two (28.7%) patients had grade≥2 oral mucositis, and all had gastrointestinal toxicity (grade 2-3). median time until neutrophils>500/μl was 11(6-12) days, to platelets>20,000/μl was 9 days(5-12), and to engraftment was 13 days (12) (13) (14) . patients were transfused with a median of 1(0-2) unit for erythrocytes and 3(1-8) for platelets. there was no treatment-related mortality and no long term side effects have been observed so far. for a median post-ahsct follow-up of 8.7 months, no patient developed new lesions in mr, but 2(28.6%) experienced symptoms consistent with a clinical relapse, at a median time of 20.5(5-35) months, effectively rescued with corticosteroids. the absence of evidence of disease activity at 6-months was 71.4%. although there was no variation concerning edss punctuation, 4(57.1%) patients self-reported significant benefits, especially concerning limb strength and sphincter continence improving. conclusions: our real life results claim a stabilization effect of ms patients with highly active/progressive disease after ahsct, with no significant toxicity. the failure in reporting benefits in edss punctuation is probably due to a small sample size and short follow-up. more studies are needed to establish the best patient selection criteria and define the ideal time to include these patients in transplantation programs, as well as to evaluate its long term outcome. disclosure: nothing to declare. elena poponina 1 , elena butina 1 , anna yovdiy 1 , galina zaytseva 1 , natalia minaeva 1 , igor paramonov 1 background: reactivation of epstein-barr virus (ebv) represents a potentially life-threatening condition in approximately 30% of patients after allogeneic stem cell transplantation, with no specific treatment available. methods: we have previously developed a manufacturing protocol for the expansion of cytomegalovirus (cmv) and ebv-specific t cells by stimulation of g-csfmobilized stem cell grafts with defined peptide pools (gary, 2018) . this advanced therapeutic medicinal product is currently investigated in an ongoing phase i/iia trial (eudract number: 2012-004240-30) . however, the expansion of virus-specific t cells relies on a pre-existing virusspecific memory compartment in the stem cell donor. in virus-seronegative donors, no expansion can be achieved. we therefore aim to identify ebv peptide-specific t cell receptors (tcrs) that can be translated into off-the-shelf cell products for the treatment and prophylaxis of ebv infection and ebv-associated malignancies. leftover cells from five allogeneic stem cell grafts were expanded in vitro in the presence of hla-b35*01-restricted peptides (hpvgeadyfey from ebna1, eplpqgql-tay from bzlf1) associated with latent and lytic ebv infection. after expansion, single ebv-specific t cells were facs sorted using peptide-mhc (pmhc) tetramers, and individual tcr αand β-chain pairs were determined with single cell sequencing (han 2014 , penter 2018 . to confirm peptide specificity, dominant tcr pairs were transfected into a murine 58αβreporter t hybridoma cell line with nfat-driven gfp expression (siewert, 2012) . functional tcrαβ candidates were transduced into human peripheral background: lymphoid and myeloid acute leukemia are the most frequent cause of cancer related death in children. interactions between nkg2d receptor, expressed in cytotoxic immune cells, and its ligands (nkg2dl), that are upregulated in many types of tumor cells including leukemic blasts, are important for anti-tumor immune surveillance. nevertheless, tumor cells may develop immune scape strategies like ligand shedding, which reduces nkg2dl expression and may cause nkg2d receptor downregulation. engineering t lymphocytes with nkg2d car may overcome immune evasion and become an effective therapeutic strategy. methods: cd45ra -t cells were obtained by depletion of non-mobilized apheresis with cd45ra magnetic beads using clinimacs. nkg2d-car t cells were generated by lentiviral (nkg2d-41bb-cd3z) transduction of cd45ra -t cells with moi=2. the expression of nkg2d ligands was analyzed in peripheral blood or bone marrow samples from a total of 97 leukemia patients (aml=13, b-all=52 and t-all=19), at different status of the disease (diagnosis, remission, relapse/refractory), and in 10 different leukemia cell lines by qpcr and flow cytometry. cytotoxicity of nkg2d-car t cells against leukemia cells was evaluated by performing conventional-4 hours europium-tda assays. soluble nkg2dl (snkg2dl) concentration was measured in the sera of leukemia patients by elisa. to evaluate the effect of snkg2dl on nkg2d-car t cells, those were cultured in the presence or absence of different concentrations of snkg2dl for 7 days. one week later, cell proliferation and car downregulation were measured by flow cytometry using cell trace violet and nkg2d labeling, respectively. the production of ifn-g and tnf-a was measured in the supernatants by elisa. the effect on cytotoxicity was evaluated in a 2 hoursdegranulation assay by co-culturing snkg2dl pretreated nkg2d-car t cells against k562 cell line. results: nkg2d ligands were expressed in leukemia cell lines and leukemic blasts. nkg2dl expression changed with disease status with a trend to decrease at diagnosis and relapse/refractory compared to remission. nkg2d-car t cells were cytotoxic against 8/10 leukemia cell lines with a percentage of specific lysis over 50%. myeloid and t-all cell lines were more susceptible to nkg2d-car t cells (specific lysis ranging from 50-78%) compared to b-all cell lines (19-52%). physiological concentrations of snkg2dl caused an increase in nkg2d-car expression. however, supra-physiological levels of snkg2dl decreased nkg2d-car expression up to 5 times and increased cell proliferation up to 4 times. cd4+ subpopulation was more affected by downregulation, while proliferation had more impact on cd8+ subset. the effects of snkg2dl were dose-dependent and attenuated by il-2. conclusions: nkg2d-car t cells are cytotoxic against leukemia cells, specially aml and t-all, and thus could be a novel therapeutic approach for non-b leukemia, or those b-all that relapse with undetectable cd19 after cd19-car treatment. nkg2d-car expression may be downregulated only by supra-physiological levels of snkg2dl, although antitumor activity is not affected. il-2 softens the negative effects of snkg2dl inducing nkg2d expression, cell proliferation and cytokines production. the changes observed in nkg2dl surface expression at the different stages of the disease could be related to ligands release and immune escape. disclosure: nothing to declare denis-claude roy 1,2 , ines adassi 1,2 , céline leboeuf 1,2 , vibhuti p. dave 1,2 1 hôpital maisonneuve-rosemont research center, montréal, canada, 2 university of montréal, montréal, canada background: for patients with high-risk leukaemia, allogeneic haematopoietic stem cell transplantation is the only curative treatment. the presence of alloreactive t cells in the donor graft, however, leads to a high probability of developing graft-versus-host disease (gvhd) . t-cell depletion minimises the presence of gvhd-causing alloreactive cells, but often results in an increased incidence of infections and disease relapse. photodepletion treatment (pdt) can specifically deplete activated alloreactive t cells while conserving resting t cells. pdt-treated cells have been utilised after t-cell-depleted haploidentical transplant to help reduce infection and relapse. the efficacy and safety of such pdt-treated cells is currently under clinical investigation in a phase iii trial (hatcy, nct02999854; kiadis pharma). here the reactivity of pdt-treated donor t cells was assessed toward tumour-associated and viral antigenic peptides derived from wilm's tumour protein 1 (wt1p), preferentially expressed antigen in melanoma (pramep), and from cytomegalovirus and epstein-barr virus (cmv/ ebvp). methods: healthy donor (hla-a*0201) peripheral blood mononuclear cells (pbmcs) were co-cultured with irradiated pbmcs from another mismatched donor (1:1) in a 4-day mixed lymphocyte reaction. th9402, a photoactive rhodamine derivative, was added and cells were exposed to visible light to deplete the th9402-containing activated alloreactive cells. elimination of alloreactive cells post-pdt was assessed using cd25 and hla-dr as activation markers. an ex vivo expansion protocol was exploited to evaluate the impact of pdt on reactivity to tumour and viral antigenic peptides. post-pdt t cells were co-cultured with irradiated autologous monocyte-derived dendritic cells (10:1) pulsed with wt1p, pramep or cmv/ebvp. antigen-specific t cells were re-stimulated on days 7 and 14 with wt1p-or pramep-pulsed autologous pbmcs or with cmv/ebvp added directly to the culture. mhctetramer staining was performed on days 14 and 21; ifn-γ elispot was conducted on day 21. [[p057 image] 1. functional wt1-specific and viralspecific t cells can be expanded post-pdt] results: pdt resulted in a drastic decrease of cd25 and/ or hla-dr activation marker-expressing cd4+ and cd8 + t cells. pdt-treated cells showed a significant increase in background: acute lymphoblastic leukemia (all) is the most common childhood cancer and relapsed or refractory all is still difficult to treat. engineered t cells equipped with a synthetic chimeric antigen receptor (car) targeting cd19 have demonstrated remarkable efficacy to treat all. however natural killer (nk) cells are known for their target-independent cytotoxic potential without induction of cytokine release syndrome (crs) or graft-versus-hostdisease (gvhd), car-nk cells can overcome the persisting problem in the therapy with car t cells. as the use of viral vector generated car nk cells is limited by theire genotoxicity, cost and regulatory demands, we are developing an innovative protocol using non-viral sleeping beauty (sb) transposition of third party nk cells as a source to produce 'off the shelf' car-engineered cell products. methods: nk cells are isolated from peripheral blood mononuclear cells (pbmcs) using cd56 selection kits. they are successfully expanded ex vivo with il-15 cytokine stimulation under feeder-cell free conditions. after few days of expansion nk cells are electroporated using pmaxgfp. transfection efficiency and percent of living cells after electroporation is analyzed by flow cytometry. transposition based nucleofection using an sb100x mrna and a minicircles (mc) dna vector is performed at different time points after nk cell isolation. the transient mc-venus longtime expansion and the viability after sb100x based nucleofection is measured over two weeks. furthermore, α-retroviral (α-rv) cd19-car transduction of nk cells with different viral amounts (moi) is conducted and the cytotoxicity of the engineered cd19-car-nk cells against the cd19 positiv cell line supb15 is addressed. results: for an α-rv cd19-car transduction of maximal 1x10 4 nk cells we could show transduction efficiency of 68,96% for moi10. the α-rv cd19-car modified nk cells had a high killing activity against cd19 positiv supb15 cells (e:t ration 1:1 90,85%) compared to cd19 negativ k562 cell lines (e:t ration 1:1 16,42%) and the non-transduced nk cells (e:t ratio 1:1 9,03%). in first experiments with pmaxgfp vector based nucleofection, we could show an increasing efficiency of 55,9% 48h post electroporation with a only slightly decreas of living cells (21,5%) comparing to the non-electroporated nk cell viability. using sb100x mrna with mc-venus dna we electrotransfected 1x10 6 nk cells after fews days of cultivation and we reached 36,6% of transfected nk cells 24h post electroporation and a transient expression of mc-venus positive nk cells up to 54,6% efficiency with an increasing rate of live cells over 14 days after electroporation. conclusions: the sleeping beauty based nucleofection of nk cells is a very promising non-viral method to generate more easy, safer and higher amounts of genetically modified third party nk cells for therapy of all and has also a broad range of clinical applications. disclosure: winfried s. wels is an inventor on a patent describing chimeric antigen receptors with an optimized hinge region. axel schambach is an inventor on a patent describing alpharetroviral sin vectors. michael hudecek and zoltan ivics are inventors on patents related to sleeping beaut gene transfer technology. the remaining authors have nothing to disclose. background: mature immune cells from the stem cell graft are essential for the graft-versus-tumor (gvt) effect to eliminate residual malignant cells after hematopoietic stem cell transplantation (hsct), but donor cells are also involved in complications such as graft-versus-host disease (gvhd). methods: we performed a prospective study of the detailed graft composition in 102 recipients of peripheral blood stem cells (pbsc) or bone marrow (bm) in order to identify correlations to clinical outcomes, table 1. grafts were characterized with concentrations of t cells and nk cells together with a multi-color flow cytometry panel with staining for tcrαβ, tcrγδ, vδ1, vδ2, cd3, cd4, cd8, hla-dr, cd196, cd45ro, cd45ra, cd16, cd56, cd337 and cd314 for detailed immune phenotyping. cell contents in stem cell grafts were analyzed both as fractions of cd45 positive lymphocytes and as absolute concentrations converted to transplanted cells/ kg. fractions were evaluated in patients receiving both bm and pbsc (n=102), while concentrations (cells/kg) were only analyzed in patients transplanted with pbsc (n=88). table] 1. table 1] [[p059 image] 1. figure 1 ] results: we found, that patients transplanted with graft nk cell doses above the median of 27x10 6 /kg and fractions of nk cells out of lymphocytes above the median of 8.1% had significantly increased relapse-free-survival compared to patients transplanted with grafts containing nk cell doses below these values, figure 1 ; results stayed significant in multivariate analyses. relapse incidence was significantly lower in uni-and multivariate analyses in patients receiving grafts with high nk cell fractions compared with low fractions, p=0.01, with 1-year relapse rates of 8% versus 27% in patients transplanted with high versus low fractions of nk cells, p=0.01. peripheral blood concentrations of nk cells obtained from samples from 17 pbsc donors before g-csf mobilization were significantly correlated to graft concentrations-and fractions of nk cells.. analyses of graft contents of nkt cells showed that the incidence of grade ii-iv acute gvhd were significantly lower in patients background: extracorporeal photopheresis (ecp) is an immunomodulatory treatment that has shown efficacy in steroid refractory acute gvhd, but the mechanism of action is only partially understood. there is no clear relationship between the ecp-treated mononuclear cells (mnc) or lymphocyte numbers and response to ecp. the objective of the study was to analyse the relationship between the infused subpopulation cellularity and response. methods: 65 patients from 7 different centers with a total of 1008 ecp procedures were retrospectively analized. ecp procedures were performed from january-2011 to june-2017. all ecp procedures were performed with the off-line system. the response was defined as responder (complete and partial response) and non-responder. infused cell numbers for lymphocytes, monocytes and mononuclear cells (lym+mon, mnc) were calculated. for analytic purposes, the median number of cells infused per procedure until response (or until the median number of procedures until response for non-responding patients) and the cumulative number of cells infused until response (or until the median in non-responders) were calculated for all the subgroups. same procedures were performed with the number cells infused until day 30 of ecp. finally, the response and survival impact of infusing a number of cells above or below the median and in different tertiles was assessed until the median number of procedures needed to achieve a response. [[p060 image] 1. results: the median number of procedures until response was 3. we observed a trend towards a higher median number of monocytes per procedure and cumulative infused monocytes in responding patients (median number infused 19.0 vs 13.5 x10 6 /kg p=0.071, cumulative infused median number 71.5 vs 41 x10 6 /kg p=0.067) that was lost in the day 30 of treatment. there was also a trend toward higher median infused mnc until response for responders (54 vs 37 x10 6 /kg p=0.087). we observed no differences in the number of lymphocytes infused, but patients who received a number of lymphocytes per procedure over the first tertile (31 x10 6 /kg) presented higher response rates (75% vs 45%, p=0.0317). none of the other analysed parameters showed a significant impact in overall survival. conclusions: patients with acute gvhd who responded to ecp received higher numbers of monocytes and mnc in the early phase of the treatment (a median of the first 3 processes). also the patients who received higher numbers of lymphocytes in the first procedures achieved a higher response rate. these findings suggest the possibility that higher number of treated and infused cells could influence the response to ecp, but specifically designed prospective studies are need to asses this possibility. disclosure: nothing to declare background: the field of kidney transplantation has made enormous progress over the last decades towards being a standard treatment for patients with end-stage renal disease. however, administration of immunosuppressive drugs is still one of the major limitations of long-term allograft survival. therefore, strategies for induction of donorspecific tolerance are highly desirable. to this aim, a clinical phase i study with donor-derived modulated immune cells (mics) was conducted. methods: donor-derived mics were manufactured under gmp conditions. potency of mics was tested by different in vitro bio-assays. mics were administered to patients with an escalation from 1.5 x 10 6 mics/kg on day -2 (n=3, group a), to 1.5 x 10 8 mics/kg on day -2 (n=3, group b) or on day -7 (n=4, group c) before kidney transplantation accompanied by standard immunosuppressive medication post-transplantation. frequency of adverse events (ae) was assessed from day 30 until day 360 post-transplant. dynamic changes of various lymphocyte subsets in patients after mic therapy were detected by multicolor flow cytometry. donor-specific immunosuppression was assessed by measuring anti-donor antibodies and mixed lymphocyte reaction (mlr) against donor and thirdparty cells. results: in all kidney transplant recipients, we observed a median serum creatinine of 1.4 mg/dl at day 30 which remained stable until day 360 (median creatinine of 1.4 mg/ dl) without significant proteinuria. none of patients experienced rejection episode. 69 aes were observed while three aes being severe. most importantly, none of them was associated with mics transfusion. besides two infectious complications, no post-transplant positive cross match results against the donor or titers of de novo donorspecific antibodies were recorded. notably, immunosuppressive therapy could be reduced without signs of rejection in group c. after infusion, we observed a dramatic increase of cd19 + b cells up to a median of 300 cells/μl until day 30, followed by a reduction to 35 cells/μl on day 180 in group c. notably, regulatory b cells significantly increased from a median of 2% on day 30 to 20% on day 180. in parallel, the plasma il-10/tnf-α ratio increased from a median of 0.05 before cell therapy to 0.11 on day 180. after mic cell therapy recipient lymphocytes showed no or only minimal reactivity against irradiated donor pbmcs in vitro, while reactivity against 3 rd -party-donor pbmcs was not impaired. moreover, in vitro mics product demonstrated their immunomodulatory potency by inducing tolerogenic dendritic cells (tdcs) characterized by low expression of costimulatory (cd80, cd86) and maturation (cd83) as well as high expression of inhibitory marker cd103. functionally, tdcs could inhibit not only the release of ifn-γ but also the proliferation of cmv specific cd8 + t cells. moreover, mic-induced tdcs showed the capacity to inhibit donor-specific allo-reactive cd4 + and cd8 + t cell proliferation. conclusions: mic cell therapy modulates the immune system of kidney transplant recipients by increasing the ratio of regulatory b cells and facilitates the reduction of conventional immunosuppressive therapy without allograft injury or rejection episodes. therefore, mic cell therapy represents a promising strategy in transplantation medicine. we currently prepare a phase ii trial with mic cell therapy. disclosure: nothing to declare p062 genome editing of graft-derived t cells for postbackground: immunotherapy using car t cells has shown promising results to fight cancer. however, car-t cell production requires specialized infrastructure and operators, which implies high cost and centralized production. automated production of car-t cells in clinimacs prodigy device allows clinical-grade manufacturing of car t cells. methods: 100 million cd45ramemory t cells from healthy donors were cultured in texmacs supplemented with 100 iu/ml il-2. at day 0 cells were activated with t cell transact for 24h. at day one, activated cd45ramemory t cells were transduced with nkg2d-cd8tm-41bb-cd3z lentiviral vector at moi = 2. then, nkg2d-car t cells were expanded for 10-13 days. nkg2d-car t cell products were next harvested, counted and analyzed for viability, nkg2d-car expression and anti-tumor cytotoxicity. different quality tests including sterility, vector copy number, genetic stability, quantification of viral particles in the supernatant, myc/tert expression and endotoxin detection were performed. spare cells were cryopreserved either in autologous plasma and 10% dmso, m199 35% albumin and 9%dmso or hypothermosol. after 12 months, cryopreserved nkg2d-car t cell products were analyzed for viability, nkg2d-car expression and cytotoxicity. results: nkg2d-car memory t cells expanded up to 2076 ± 697 million with 77,8 ± 20% nkg2d-car expression and 76 ± 10% viability. harvested car t cells showed 90 ± 14% of specific lysis against jurkat cells and 31 ± 16% against 531mii osteosarcoma cell line. no microbiological contamination was observed in final car t cells products. vector copy number was ≤5 in all validations except for one. cgh and karyotype showed no genetic alterations. free viral particles were undetectable in the supernatants. no overexpression of myc/tert was found except for one validation. endotoxins were ≤0.25eu/ml. all cryopreserved nkg2d-car t cell products kept nkg2d-car expression one year after freezing. however, viability and cytotoxicity was best preserved using autologous plasma 10%dmso. conclusions: automated production of large-scale clinical-grade nkg2d-car t cells using clinimacs prodigy is feasible and reproducible, allowing a decentralized protocol to generate car t cells for clinical use. background: immune reconstitution (ir) is essential to control severe infections after hematopoietic stem cell transplantation (hsct). reconstitution of adaptive immunity may take up to 2 years to recover t-lymphocytes (lt). delay in early lt recovery increases the risk of relapse, viral infections and transplant related mortality. adoptive transfer of selected t cell subset with low alloreactivity potential is emerging as a strategy to improve ir. methods: depletion of cd45ra+ naive t cells, preserving cd45ro+ memory t cells could provide functional lymphocytes to protect against infection and leukemia relapse with low risk of graft versus host disease (gvhd). we present our experience with high-dose donor cd45ro + memory t cell as donor lymphocyte infusions (dli) to assess safety and outcome. a total of 58 dli of cd45ro+ after hsct was performed in cases of cmv/ebv reactivation (50%), mixed chimerism (26%), persistent lymphopenia (8,5%), graft rejection (3,5%) , relapse (3%) or to boost ir (7%). dli product was obtained performing a cd45ra depletion on donor leukapheresis product using the clinimacs® device. results: twenty-two pediatric patients, median age 11 years (range 3-18), with malignant (n=15) and nonmalignant diseases (7), received cd45ra+ (n=14), tcr alpha/beta (n=2) depleted grafts from haploidentical and cd45ra+ depleted grafts from match unrelated (n=6) and match related (n=2) donors. at a median of 97 days (range 15-462) after transplantation, patients received a total of 58 dli of cd45ro+ cells, median 2 (range 1-6), containing a median of 2.87x10 7 /kg (range 4.8x10 4 -2x10 8 /kg), cd3 +cd45ro+ 1.05x10 7 /kg (range 4.8x10 4 -1.09x10 8 /kg) and cd45ra+ cells 5 x10 2 /kg (range 0-9.8x10 4 /kg). all infusions were well-tolerated and did not develop or worsen gvhd. a total of 11/29 episodes of cmv/ebv viral reactivations decreased viral load, 4/29 cleared viral load and 5/29 showed a clinical improvement. a total of 4/5 patients with persistent lymphopenia there was a slightly increase in total lymphocyte count, but not to normal levels. prophylactic dli of cd45ro+ to boost ir increased lymphocyte count in 2 of 3 cases. none of the dli administered in cases of mixed chimerism, graft failure or relapse were effective in reverting those situations. conclusions: our preliminary data suggest that infusions of high dose cd45ro+ memory t cells are a safe adoptive immunotherapy strategy. efficacy has been observed in patients with lymphopenia and cmv/ebv reactivation, with no positive results in patients with mixed chimerism, graft failure and relapse. however, to determine the real efficacy of this strategy, prospective studies are required. disclosure: nothing to declare. background: increasing clinical trials have confirmed that chimeric antigen receptor t cells (car-t) targeting cd19 antigen (car-t-19) is a promising effective approach for the treatment of relapsed/refractory(r/r) b-cell lineage malignancies. considering cd19 is frequently expressed in large part of t(8;21) acute myeloid leukemia (aml) cells, we suppose that car-t-19 may be used as an approach to rescuing r/r t(8;21) aml patients. methods: both patients received lymphodepletion chemotherapy with decitabine 20mg/m 2 ×5d, fludarabine 30mg/m 2 ×3d and cyclophosphamide 300mg/m 2 ×3d (dac +fc). two days after chemotherapy, autologous/allogeneic cart-19 cells provided by the unicar-therapy biomedicine technology co.(shanghai, china) at a total dose of 5-10×10 6 cells per kilogram(kg) were infused dose escalation within 2 to 3 days. the research protocol was approved by the institutional review boards of the first affiliated hospital of soochow university and both patients gave written informed consent. results: both cases responded well with transient and reversible toxicities. case 1 presented with grade 1 cytokine release syndrome (crs), manifested by intermittent fever and chill from day 4 after car-t-19 infusion for half months associated with neutropenia. car-t cells expansion were observed in blood without obvious increase of cytokines. after infusion, case 1 achieved and maintained molecular complete remission (cr) for more than 10 months. case 2 presented with grade 3 crs manifested by continuous high fever, hypotension and grade 1 liver disfunction from day 1 after car-t-19 cell infusion for 1 week. obvious cytokines releasing (peak il-6 serum concentration 1774.5 pg/ml, peak crp serum concentration 367pg/ml) were detected which were associated with car-t-19 cell expansion in blood and no severe off-tumor effect was observed. after infusion, case 2 achieved hematological cr and cytogenetic cr and got 3 months disease free survival. conclusions: our report implicates that car-t-19 is a safe and promising approach to managing r/r t(8;21)aml with cd19 expression, and may provide a salvage treatment approach for all aml patients with cd19 expression and benefit a certain population with aml besides b-linage malignancies. clinical trial registry: na disclosure: nothing to declare. this work was supported by research grants from the national key r&d program of china (2016yfc0902800), national natural science foundation of china (81873443, 81270645, 81400155, 81500146) background: chimeric antigen receptor engineered t (car-t) cells have emerged as a powerful cellular therapy to treat malignant disease, which is currently revolutionizing field of cancer immunotherapy. a cryopreservation step postmanufacture is not only a logistical necessity for large scale cell manufacturing processes but also a mandatory request by regulatory authorities. in case relapse after 1 st car-t cell transplantation, a second application, maybe at a higher dose constitutes a therapeutical option. however, data concerning clinical grade car-t cell stability and functionality after months of cryopreservation have not been released by companies so far. to investigate the effect of cryopreservation on car-t cells, we performed this study. methods: different batches of cd19 car-t cells were manufactured according to gmp requirements at our institution. final car-t products were frozen at concentrations of 1 x 10 7 cells/ml (high batch) and 2 x 10 6 cells/ml (low batch) by a controlled freezing process with the biofreeze bv40 device and stored in liquid nitrogen tanks below -150°c until release. quality control tests for sterility, endotoxin and mycoplasma were performed for each batch according to european pharmacopoeia and united states pharmacopoeia guidelines. stability of cd19 car-t cells in terms of viability, recovery, transduction efficiency and functional capacity were determined by microscopy, multi-parametric flow cytometry as well as chromium-51 release tests following our sops. results: all the results of quality controls fully met the requirements of the regulatory authorities. stability results were highly robust and reproducible over time for all our gmp car-t batches. duration of cryopreservation (up to 90 days) had no negative influence on cell viability, recovery of viable cd19 car-t cells and transduction efficiency. however, the cell concentration for cryopreservation has a significant impact on the post-thawing viability (low batches vs. high batches: 96.33 ± 2.17 vs. 74.87 ± 8.68, p < 0.05) and recovery (low batches vs. high batches: 89.67 ± 6.76 vs. 74.90 ± 9.19, p < 0.05) of cryopreserved cd19 car-t cells, but not the transduction efficiency. moreover, we observed four transient side-effects of cryopreservation on the amount of cytokines released by car-t cells, the cytokine release on a per-cell basis, the multifunctionality of car-t cells and the killing capacity. of note, functional capacity of cryopreserved car-t cells after overnight resting was comparable or even enhanced for inf-γ and tnf-α release by cd4 + and cd8 + cd19 car-t cells when compared to fresh car-t cells. the multi-functionality of car-t cells could be preserved. furthermore, the killing capacity of cryopreserved cd19 car-t cells after overnight resting could reach the level of non-cryopreserved/fresh car-t cells. conclusions: cryopreservation up to 90 days has no harmful effect on transduction efficiency and functionalities of car-t cells. however, the cell number per milliliter freezing medium matters. dose over 1 x 10 7 cells/ml should be avoided. for the conduction of in vitro bio-assays to determine the function of car-t cells, an overnight resting process could mimic the situation after clinical application and eliminate the transient side-effects of cryopreservation to fully regain the functional potency of car-t cells. disclosure: nothing to declare background: dc and specific t-cells are important mediators of ctl-responses. we could already show that allogeneic donor-or autologous t-cells obtained from amlpatients can be stimulated by dc leu , resulting in a very efficient lysis of naive blasts. methods: chemokine-release (cxcl8, -9, -10, ccl2, -5, and il-12) was analysed by cytometric bead array in serum of aml/mds-pts as well as in supernatants from 5 different dc-generating-methods and correlated with pts' clinical course, dc-and t-cell-interactions as well as specific t-cell-reactions. the lytic activity of dcleu/blast -stimulated t-cells in mlc against naive blasts was quantified in a cytotoxicity assay. results: minimal differences in median chemokine-levels in pts' serum subdivided in subtypes were seen, but higher release of cxcl8, -9, -10 and lower release of ccl2 and -5 tendentially correlated with more favourable subtypes (< 50 years of age, < 80% blasts in pb). in persisting disease, a higher serum-release of ccl5 and at relapse a significantly higher ccl2-release were found compared to first diagnosis -pointing to a change of 'disease activity' on a chemokine level. whereas chemokine-levels in dc-culture supernatants compared to serum were variable, clear correlations with lateron (after stimulating t-cells with dcleu in mlc) improved antileukemic t-cell activity were seen: higher values of all chemokines in dc-culture supernatants always correlated with improved t-cells' antileukemic activity (compared to stimulation with blast-containing mnc as control) -whereas with respect to the corresponding serum values higher release of cxcl8, -9, and -10 but lower values of ccl5 and -2 correlated with higher probabilities to improve antileukemic activity of dcleu-stimulated (vs. blaststimulated) t-cells. predictive significant cut-off-values could be evaluated separating the groups compared. moreover, correlations with lateron achieved response to immunotherapy and occurrence of gvhd were seen: higher serum values of cxcl8, -9, -10 and ccl2 and lower values of ccl5 correlated with achieved response to immunotherapy. predictive cut-off-values could be evaluated separating the groups compared in 'responders' and 'non-responders'. higher levels of ccl2 and -5 but lower levels of cxcl8, -9, -10 correlated with occurrence of gvhd. conclusions: we conclude, that in aml-pts' serum higher values of cxcl8, -9, -10 and lower values of ccl5 and in part of ccl2 correlate with more favorable subtypes and improved antitumor'-reactive function. since in dcculture supernatants higher values of all chemokines correlated with improved antileukemic t-cell reactivity we conclude a change of functionality of ccl5 and -2 from an 'inflammatory' or 'tumor-promoting' to an 'antitumor'reactive function. this knowledge can contribute to develop immune-modifying strategies that promote antileukemic adaptive immune-responses. disclosure: nothing to declare background: allogeneic hematopoietic cell transplantation (allo-hct) is a curative treatment option for patients suffering from hematologic malignancies. infusion of donor lymphocytes (dlis) can induce sustained remission in case of minimal residual disease or relapse through potent graftversus-leukemia (gvl) effects, although graft-versus-host disease (gvhd) represents a common dose-limiting toxicity. as invariant natural killer t (inkt) cells are known to prevent gvhd while promoting beneficial anti-tumor effects, we investigated the role of inkt cells for successful dlis. methods: we analyzed dli samples by flow cytometry. inkt cells were identified by staining with pbs57-loaded cd1d tetramers. culture-expanded and purified dli-inkts were then tested against tumor cell lines and primary leukemia cells in an ex vivo tumor control model. tumor cell viability after coincubation with dli-inkts was measured by flow cytometry using 7-aad. results: inkt cells represent 0.05% (range 0.001-0.55%) of donor lymphocytes and can be expanded 300fold following a two-week protocol with a preferential expansion of cd4+ inkt cells. tumor cell lines such as jurkat were efficiently lysed after coincubation with dli-inkts. cd107a as a marker of degranulation was significantly upregulated on dli-inkts after stimulation by jurkat. in addition, increased concentrations of tnfα, ifn-γ, sfasl and perforin were measured after coincubation of dli-inkts with jurkat. we observed that tumor cell lysis correlated with the expression of the mhc-i-like molecule cd1d. consequently, adding a cd1d antibody to the coculture abrogated the dli-inktmediated kill of tumor cells. dli-inkts also efficiently lysed primary leukemia cells such as aml blasts: expression of cd1d on these aml blasts significantly correlated with dli-inkt-mediated tumor cell lysis (r 2 =0.7, p=0.03). conclusions: ex vivo expansion of dli-inkts and subsequent dli enrichment is an immunotherapeutic approach that could improve leukemia control and thus, prevent relapse after allo-hct without exacerbating gvhd. disclosure: nothing to declare. generation of antigen-specific cytotoxic t lymphocytes targeting wt1 using activated b cells sun ok yun 1 , kyung won baek 1 , hee young shin 1 , hyoung jin kang 1 1 seoul national university, seoul, korea, republic of background: the wilms tumor antigen 1 (wt1) is highly expressed in many malignancies including leukemia and targeting wt1 as a tumor associated antigen (taa) in cancer immunotherapy is attractive. in this study, we generated wt1-specific cytotoxic t lymphocytes to confirm if activated b cells can act as a cancer antigen presenting cell and induce ctls. methods: for the induction of ctls against wt1, activated b cells were used as an antigen presenting cells. b cells were isolated from pbmcs of normal healthy donors and activated with α-galactosylceramide (α-galcer) and nucleofected with wt1-coding plasmid dna. activated b cells were the cultured with pbmcs for 17days in vitro and harvested for assay. results: cells expanded about 3 times after 17 days of culture. we examined characteristic of wt1-specific ctls by their surface markers. wt1-specific ctls had more than 90% cd3+ marker, and ratio of cd8 to cd4 was 1.7-5.8. we also examined nkt cell markers to see if nkt cells were activated by il-15, a cytokine used in the induction of ctls, and the portion of nkt cells was about 2%. the ctls showed a decrease in naïve cell (cd62l+cd45ra +) and an increase in effector memory (cd62l+cd45ra-) and central memory (cd62l-cd45ra-) compared with non-stimulated pbmcs. subsequently, the ifn-γ elispot (enzyme-linked immunospot) assay was performed to confirm the response of the induced wt1-specific ctls to the wt1 antigen. when wt1-specific ctls encounters a target that does not have a wt1 antigen, it did not produce ifn-γ, but when it encounters a target cells loaded wt1 antigen, it responded to secrete ifn-γ. killing assays were also performed to determine the immunogenicity of induced ctls. the induced wt1 ctls was found to be killing more than 90% when the e:t ratio was 10:1 when the autologous pbmc met the target with wt1 pepmix. in addition, we found that wt1 ctls has killing activity when it encounters leukemia cell lines that express wt1 and matched hla-a*0201. conclusions: in this study, we can induce antigenspecific ctls that specifically react to wt1 using activated b cells as antigen-presenting cells. these observations confirmed that b cells activated by α-galcer can act as a taa presenting cell to induce taa specific ctls as viral antigen, such as pp65 and ie1, and consequently wt1specific ctls could be induced. moreover, ctls induced activated b cells had ability to recognize and kill the target cells expressing wt1 correctly. our results demonstrate that these in vitro expanded wt1-specific ctls using activated b cells can be a promising candidate for adoptive immunotherapy against cancer. disclosure: nothing to declare judith böhringer 1 , michael schumm 1 , christiane braun 1 , marina schmidt 1 , patrick schlegel 1 , christian seitz 1 , murat aktas 2 , georg rauser 2 , sandra karitzky 2 , peter lang 1 , rupert handgretinger 1 1 university children's hospital tübingen, tübingen, germany, 2 miltenyi biotec gmbh, bergisch gladbach, germany background: t cells with chimeric antigen receptors (cars) on their surface facilitate to target specific surface expressed antigens. research and clinical trials with cd19-car t cells show impressive remission induction rates and increased survival in heavily pretreated patients. therefore, car t cells are introduced as new potent cellular therapeutics in the clinical routine. in order to establish the manufacture of cd19-car t cells, validation runs with the fully automated clinimacs prodigy t cell transduction process have been performed using the miltenyi anti-cd19-car lentiviral vector. methods: unmobilized leukaphereses from 3 donors (2 x healthy, 1 x all) were used for the clinimacs prodigy t cell transduction process. leukocytes undergo a cd4 + / cd8 + t cell enrichment via magnetic beads, followed by stimulation with macs ® gmp t cell transact™, transduction with an anti-cd19-car lentiviral vector, expansion with il7 and il15, and final formulation to the cellular product. during and after the manufacture, facs analyses were performed as well as cytotoxicity assays after cd19-car t cell production. results: total volumes of leukaphereses were between 60 and 280 ml with 2.6 -4.0 x 10 9 total mononuclear cells. after enrichment 100x10 6 cd4 + / cd8 + t cells were transduced with anti-cd19-car lentiviral vector and were further expanded. cells were harvested on day 12. the final cell counts of the cellular products were 6.1, 7.2 and 5.0 x 10 9 mononuclear cells from two healthy volunteers and the all-patient, respectively. the transduction efficiency of the cd19-car t cells was 48.6%, 43.4% and 32.0% among viable cd3 + cells. the final count of car t cells was therefore 2.9, 3.0 and 1.5 x 10 9 cells. the final products exerted excellent cytolytic activity against cd19 + bcp-all cell line nalm-6. importantly, cd19-car t cells generated from the all patient demonstrated complete eradication of autologous blasts at 0.3 to 1 e:t ratio after 24 hours incubation. conclusions: the clinimacs prodigy t cell transduction process has been shown to run a fully-automated manufacturing process over 12 days without any deviations in a clean room environment on a single device. the user interaction was reduced to activities at only 3 days to set up the system and provide fresh medium and reagents. the transduction process yielded a high number of t cells with a high frequency of cd19-car t transduced cells. the results were comparable for both unmobilized leukaphereses from healthy donors and showed expected slightly lower results in the patient. finally, these results demonstrate that the clinimacs prodigy t cell transduction process is well suited to provide the clinical mb-cart19.1 r/r cd19+ bcm study with appropriate investigational medical products. disclosure background: allogeneic hematopoietic stem cell transplantation (allohct) is an effective strategy in the long term control of several hematologic diseases, however, patients could experience complications, as graft versus host disease (gvhd) and disease relapse. recently, the introduction of post-transplant cyclophosphamide (ptcy) allowed to significantly reduce gvhd, but disease relapse remains an important issue. donor-lymphocyte infusion (dli) is an established adoptive cell therapy for disease relapse after allohsct, but, in order to be efficient and safe, patients have to be off immunosuppression treatments and gvdh-free. here we report our data about efficacy and safety of dli infusion as treatment for disease relapse in patients who received peripheral blood stem cell transplantation (allopbsct) from hla-matched unrelated/related plus ptcy as gvdh prophylaxis in our clinical trial (nct 02300571). methods: we collected data from 13 patients, treated with ptcy (50 mg/kg/die, days +3+4), mofetil mycophenolate (mmf) and tacrolimus (t) as gvhd prophylaxis after allo-pbsct, who received dli infusions. they were treated between january 2013 and october 2018. we report data about overall response rate (orr), disease control rate (dcr), and dli-related mortality and morbidity. diagnosis were as follow: 5 had multiple myeloma, 3 had acute myeloid leukemia, 3 had acute lymphoblastic leukemia and 2 had lymphomas. all patients but one, who had chimerism loss, received dli because of disease relapse. results: median time between transplant and dli was 9 (range 3-87) months. median number of dli infusions was 2 (range 1-13). 10 patients (77%) received cyclophosphamide 300 mg/m2 preparative regimen the day before the cryopreserved dli infusions, while in the other 3 cases dli were associated with lenalidomide, ponatinib and 5-azacitidine. the overall response rate (orr) was 50%, while disease control rate (dcr) was achieved in 75%. the patient who received dli because of loss of chimerism converted it in full donor after 2 infusions. after dli treatment the incidence of acute gvhd grade i-iii was 54%, while was 46% for grade ii-iii and patients were started on short course of systemic immunosuppression treatments . none of these patients died because of dli adverse events. estimated 1-year overall survival was 77% with a limited follow-up length (6 months). conclusions: the infusion of non-manipulated lymphocytes from allogeneic donors is a valuable and safe strategy of treatment for patients relapsing after allopbsct with ptcy. ptcy showed high efficacy in gvhd prevention, allowing early discontinuation of immunosuppression drugs. because of this, we can reach the goal to transform transplant in a platform where we could add early dli infusions as a new strategy for disease control. clinical trial registry: nct 02300571 disclosure: nothing to declare p074 extracorporeal photopheresis in the treatment of refractory chronic gvhd: analysis of mononuclear cell infusion gillen oarbeascoa 1 , maria luisa lozano 2,3,4 , luisa maria guerra 5 , cristina amunarriz 6 , nuria revilla 2,3,4 , pastora iniesta 2,3,4 , cynthia acosta fleitas 5 , jose luis arroyo 6 , eva martinez revuelta 7 , andrea galego 8 , dolores hernandez-maraver 9 , mi kwon 1,10 , aurora viejo 9 , jose maria garcia gala 7 , concepcion andon saavedra 8 , jose luis diez-martin 1, 10, 11 background: extracorporeal photopheresis (ecp) is an immunomodulatory treatment that has shown efficacy in steroid refractory chronic gvhd, but the mechanism of action is only partially understood. in some studies, a correlation has been suggested between treated mononuclear cells (mnc) or lymphocytes and response to ecp. the objective of the study was to analyze the relationship between the infused cellularity and response in chronic gvhd. methods: 48 patients from 7 different centers with a total of 930 ecp procedures were retrospectively analyzed. ecp procedures were performed from january-2011 to june-2017. all ecp procedures were performed with the off-line system. the response was defined as responder (complete and partial response) and non-responder. infused cell numbers for lymphocytes, monocytes and mononuclear cells (lym+mon, mnc) were calculated. for analytic purposes, the median number of cells infused per procedure until response (or until the median number of procedures until response for non-responding patients) and the cumulative number of cells infused until response (or until the median in non-responders) were calculated for all the subgroups. same procedures were performed with the number cells infused until day 30 of ecp. finally, the response and survival impact of infusing a number of cells over or below the median and in different tertiles (t1, t2 and t3) was assessed until the median number of procedures needed to achieve a response. results: the median number of procedures until response was 3. we observed no differences in the median number of lymphocytes, monocytes or mncs infused until response or until day 30 between responding and non-responding patients. there were no differences in response if patients received lymphocytes or monocytes above or below the median number. nevertheless, patients that received a total absolute number of mncs above the median (64x108 cells) showed a trend towards a higher response rate (75% vs 61%, p=0.09). the patients that received a cumulative number of lymphocytes in the 3 first ecp procedures above the median showed improved overall survival (os) (2y os 85% vs 55%, p=0.024). patients that received a number of monocytes above the median showed a trend towards better survival (p=0.09), that was significant when the number of monocytes infused surpassed the first tertile (2y os 38% for t1, 79% for t2, 92% for t3, p=0.003). finally, the patients that received a cumulative number of mncs above the first tertile also showed improved survival (2y os 47% for t1, 74% for t2, 94% for t3, p=0.015). conclusions: there were no differences in the infused cellularity between responding and non-responding patients with chronic gvhd. at the same time, we found that except for a trend toward better response with higher mncs infused, there was no relationship between lymphocytes and monocytes with the response rate as other previous studies have suggested. however, even if there is no relationship with the response rate, the patients receiving the highest numbers of lymphocytes, monocytes and mncs in the cohort showed an improved survival, suggesting that larger quantities of cells could exhibit a protective effect. nevertheless, prospective studies that address this relationship are needed. disclosure: nothing to declare comparative analysis of the cytotoxic potential of cytokine-induced killer and natural killer cells for neuroblastoma therapy annekathrin heinze 1 , beatrice grebe 1 , eva mudry 1 , jochen früh 1 , bushra rais 1 , claudia cappel 1 , sabine hünecke 1 , eva rettinger 1 , thomas klingebiel 1 , peter bader 1 , evelyn ullrich 1,2 1 university hospital frankfurt, frankfurt, germany, 2 german cancer consortium (dktk) partner site:, frankfurt, germany background: neuroblastoma (nb) is the most common solid extracranial tumor in childhood. despite therapeutic progress, prognosis for high-risk nb is poor and innovative therapies are of medical need. therefore, we investigated the cytotoxic potential of interleukin (il)-activated natural killer (nk) cells compared to activated cytokine-induced killer (cik) cells against different human nb cell lines in vitro. methods: nk cells were isolated from peripheral blood mononuclear cells (pbmcs) using cd56 enrichment or cd3/cd19 depletion kits. they were successfully expanded ex vivo with different cytokine combinations such as il-2, il-15, il-18 and/or il-21 under feeder-cell free conditions. in contrast, cik cells were generated from pbmcs by ex vivo stimulation with interferon-γ, il-2, okt-3 and il-15. a comparative analysis of expansion rate, purity, phenotype and cytotoxic activity against different nb cell lines following different culturing protocols was performed. results: cd56 enriched nk cells showed a median expansion rate of 4.3-fold after 10 to 12 days in culture with a final frequency up to 99.0% nk cells and a median frequency of 0.5% cd3 + cd56 -t cells. in contrast, the starting cell product after cd3/cd19 depletion consisted of a median frequency of 43.5% nk cells that expanded significantly faster with 7.5-fold and also reached up to 98.6% purity without any relevant t cell contamination. cik cells expanded with a median rate of 30.8-fold and contained 3.3% nk, 84.2% t and 6.2% nk-like t cells. interestingly, nk cells, particularly after cd3/cd19, showed a significantly higher median cytotoxic capacity against nb cells depletion (46.6% for cd56 enrichment, 53.7% for cd3/cd19 depletion) compared to cik cells that induced 7.2% killing of nb cells with e:t ratio 5:1 in a 3 hours' co-incubation assay. interestingly, prolonging the ex vivo stimulation after cd3/cd19 depletion to 15 days enhanced the median expansion rate to 12.3-fold with a slightly reduced cytotoxic potential (40.9% for 11 days' ex vivo expansion, 31.1% for 15 days' ex vivo expansion, comparison of the same donors). the addition of an il21boost prior harvesting increased the expansion rate to median 12.6-fold (compared to 11.7-fold for the same donors) with an improved cytotoxicity of 51.5% (compared to 45.8%) . fortunately, all nk cell products showed a high viability and no relevant t or b cell contamination (median < 0.2%). interestingly, further optimization of the culturing procedure with use of another cell culture medium led to an improved median 24.4-fold (compared to 9.6-fold) nk cell expansion rate in 15 days, also resulting in comparable cytotoxicity of 52.5%. conclusions: nk and cik cell products may offer an innovative immune therapeutic option for patients with high-risk nb after allogenic stem cell transplantation. our study revealed that nk cells have a significantly higher cytotoxic potential to combat nb. interestingly, the use of il-15 expanded and il-21 activated nk cells developed from a cd3/19 depleted apheresis product is highly promising as additional immunotherapy in combination with haploidentical stem cell transplantation of children with nb. disclosure: nothing to declare. quantitative determination of donor allo-reactive t-cells in haploidentical donor-recipient pairs by enzymelinked immunospot (elispot) and mixed lymphocyte culture (mlc) assays background: t-cell alloreactivity is responsible not only for graft versus host disease and morbidity, associated with hematopoietic stem cell transplantation (hsct) but also for graft-versus-leukemia (gvl) activity. in this regard, monitoring and quantitation of alloreactive t-cells (allo-t) may potentially provide valuable information for individualized clinical management of transplant recipients. the aim of this study was the optimization of allo-т detection and comparison of the elispot and mlc assays. methods: allo-t were determined in 20 haploidentical donor-recipient pairs before hsct. donor mononuclear cells (mnc) served as effector cells (ec) . patient cd3depleted mnc were used as stimulatory cells (sc).the ratio ec:sc were 5:1 and 10:1. the frequency of allo-t in donor peripheral blood was tested in elispot assay and mlc. elispot provides the detection and quantitation of activated t-cells on the basis of cytokine secreted by each cell. the co-incubation time was 24 h for ifn-gamma and 48 h for il-2 detection. in mcl assay donor mnc were labeled with cfse and allo-t, proliferating in response to stimulation with alloantigens, were determined by flow cytometry on day 5. results: the median number of ifn-gamma producing allo-t per 300 000 donor mnc was 143,5 (2-1469; ec:sc ratio -5:1) and 75,0 (1-1440; ec:sc ratio -10:1). the median frequency of allo-t was 0,056% (0,00076 -0,538; ec:sc ratio -5:1) and 0,019% (0,00019 -0,632; ec:sc ratio -10:1) among lymphocytes. il-2-producing allo-t were less frequent in donor mnc in comparison with ifngamma-producing allo-t. the median number per 300 000 mnc was 7,5 (0,5-356; ec:sc ratio -5:1) and 6,0 (0-169; ec:sc ratio -10:1). the median frequency of il-2-allo-t was 0,0028% (0,0002 -0,130; ec:sc ratio -5:1) and 0,0022% (0 -0,0619; ec:sc ratio -10:1) among lymphocytes. the ec:sc ratio 10:1 is enough for stimulation of il-2 producing by mnc in elispot assay, but for optimal stimulation of ifn-gamma producing cells ec:sc ratio 5:1 is preferable. this suggests that allo-t are predominantly ifn-gamma producing cells. alloreactive proliferating t-clones were detected in mlc in 10 of 14 donor-recipient pairs on 5 day of cocultivation. median percentage of proliferating t-clones were 9,5% (2,1 -32,5; ec:sc ratio -5:1) and 7,6% (3,0 -24,1 ; ec:sc ratio -10:1) among lymphocytes. however, mlc assay only permit a qualitative analysis that confirmed the presence of alloreactive t-clones, giving no information on their frequency within the culture. results of elispot and mcl assay directly correlated. conclusions: allo-t were detected in 100,0% of assayed haploidentical donor-recipient pairs by elispot and only in 71,4% by mlc. this difference in detection is due to the fact that elispot allows to detect single cytokine secreting cell whereas mlc can reveal proliferating аllo-t clones. the analysis of allo-t in haploidentical donor-recipient pairs may provide rationale to manipulate the allo-immune response and to exploit the powerful ability of allo-t to control hematologic malignancies. disclosure: nothing to declare allogeneic mesenchymal stromal cell as rescue therapy in an infant with life-threatening respiratory syndrome due to a filamin a mutation background: cell-based therapy has gained attention in the respiratory system diseases and encouraging results are reported following mesenchymal stromal cells (mscs) administration. due to their capacity to produce and secrete a variety of paracrine factors and bioactive macromolecules, mscs became a key player in lung tissue injuries and function, reducing fibrosis, promoting the normal development of alveoli and pulmonary vessels. for the first time we used the msc infusions as rescue therapy in a pediatric patient with flna gene mutation and life-threatening respiratory syndrome. methods: a child with a new pathogenic variant of the flna gene c.7391_7403del; (p.val2464alafster5) at the age of 18 months, due to the serious and irreversible chronic respiratory failure and dismal prognosis, was treated with 4 intravenous infusions of allogeneic bone marrow (bm)-mscs at the dose of 1×10 6 mscs/kg body weight. bm-mscs were produced at "cell factory", fondazione irccs policlinico s. matteo, pavia,isolated and expanded ex vivo from healthy donor bm, following a previously reported protocol. premedication with antistaminic drug, 30 min before every infusion to avoid any potential reaction was performed. the evolution of the respiratory condition was detected. peripheral blood were collected before each msc treatment for treg and th17 monitoring. treg, defined as cd4+ cd127neg cd25+ cells expressing the forkhead box p3 (foxp3) transcription factor, and th17, defined as cd4+ cells expressing intracellular il-17, evaluation was performed by flow cytometry (facscanto; bd biosciences, san diego, ca) as previously reported, following standard procedures. results: no acute adverse events related to mscs infusion was recorded. during follow-up, patient maintained a good general condition and showed a regular growth. no systemic or respiratory infections occurred. after the second infusion, the child experienced a progressive improvement of his clinical respiratory condition, with a good adaptation to mechanical ventilation, in the absence of episodes of respiratory exacerbations. the baby maintains adequate volumes of exchange with substantial reduction of the inspiratory support. a reduction of trigger sensitivity was also obtained. thorax ct scan showed a recovery of the basal parenchyma bilaterally and the improvement of the anatomical-functional alignment and aerial penetration. after the first msc administration, an enrichment of treg and th17 percentage in peripheral blood, was observed. while, after the second msc infusion a significant increase in treg/th17 ratio was noted. conclusions: this report suggest that msc serial infusions are a promising therapy in aiding the respiratory failure, even in a pediatric patient with flna mutation. intravenous administrations of allogeneic mscs are feasible and safe without toxicity. our results suggest that to mitigate lung injury, mscs may act as regulators of treg and th17 balance. further investigations are upcoming to establish the useful of this therapeutic proposal in interstitial lung diseases in children. disclosure: nothig to declare p078 feasibility of il-15 stimulated donor nk cells manufacturing for early infusion in patients with high risk acute myeloid leukemia undergoing haploidentical transplantation background: nk cells provide a potent antitumor effect in the setting of manipulated haploidentical hematopoietic stem cell transplant (haplo-hsct). we propose a novel strategy to enhance the antitumor effect of allogeneic transplant through the infusion of nk cells stimulated with il-15 exvivo in adult high-risk acute myeloid leukemia (aml) patients undergoing unmanipulated haplo-hsct. the objective of this study was to provide efficiency and productivity data obtained in the manufactured cellular products infused. methods: selection criteria included patients with highrisk aml undergoing unmanipulated haplo-hsct. lymphoapheresis of the haploidentical donor was performed using spectra optia (terumo® bct) on days +6 and +13 after transplant. from the obtained product a double immunomagnetic cellular selection with clinimacs system (miltenyi biotec®) was performed in two steps: cd3+ depletion followed by positive cd56+ selection. the obtained an enriched cellular product of cd3-cd56+ nk cells was incubated with il-15 (10 ng/ml) between 12 and 18 hours at 37ºc and 5% co2 in gmp conditions. quality and microbiological controls were performed at the end of each manufacturing step. dxh cellular counters (beckman coulter®) and multiparametric flow cytometry were used for lymphocyte subpopulations and viability analysis (navios cytometer; beckman coulter®, conjugated monoclonal antibodies; miltenyi biotec®). the final product was infused intravenously to the patient on days +8 and +15 if manufacturing conditions were met (range of 0.5-100x10 6 nk/kg, purity ≥ 80%, viability≥ 70% and < 1x10 4 cd3+ cells/kg). if not, it was discarded. nk cell activation in the product was measured by the expression of cd25 and cd69. results: between november 2017 and april 2018, 3 patients were included in this ongoing trial. two products were manufactured for 2 of the patients, and only one for the first patient, due to transplant complications between first and second infusion. one product did not meet minimum viability criteria and was discarded. in the infused final products mean and sem of nk cell purity, recovery and viability were 83.7%±4.4, 30.9%±4 and 76.3%±17.4, respectively. log cd3+ depletion ranged between -5.48 and -6.03. median infused doses of nk cells and cd3+ cells per kg were 3.78x10 6 (2.8x10 6 -4.57x10 6 ) and 114 (87-532). complete manufacturing data of all 5 procedures are shown in table 1 background: cytokine-induced killer (cik) cells are a promising immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (hsct) for acute leukemia or myelodysplastic syndrome. to show safety and efficacy, a multicenter clinical study with 20 pediatric and 20 adult patients including up to eight cik cell applications with escalating doses is ongoing. methods: we favor single large scale cik cell generation with the aim to apply fresh cik cells and cryopreserve ready-for-use doses according to the study protocol in contrast to recurrent manufacturing. therefore cryopreserved cik cells were tested against freshly generated cik cells to approve equivalence. furthermore, an alternative medium supplement for cik cell culturing was investigated to avoid supply bottlenecks in ab-serum. results: fresh frozen plasma (ffp), platelet lysate (pl) and ab-serum in cik cell culture showed median expansion rates of 10-fold, whereas cultivation without medium additive resulted in significantly lower proliferation (p< 0.01). cik cell composition including t cells, nk like t cells and a minor part of nk cells was not significantly influenced by changing the medium additive. moreover, neither cytotoxicity against thp-1 cells nor cd25 expression on nk like t cells were significantly influenced by the different medium additives. for cik cell generation either ficollized peripheral blood (pb) or unstimulated leukapheresis (lp) products were utilized. with regard to repeated manufacturing within the clinical study, also cryopreserved lp and pbsc as starting material came into the focus of interest. comparing cik cell expansion rates, no significant differences for the entire cik cells and the subgroup of t cells were detected between the four starting materials. cryopreservation of cik cells had no significant effect on cik cell composition, cytotoxicity and cd25 expression on nk like t cells. a small, albeit not significant effect of cryopreservation on viability was detected, which was 86.1% before and 79.4% after freezing and thawing. conclusions: the challenge was an efficient time-, personal-and cost saving production of cik cells within the clinical study. introducing ffp enabled cik cell manufacturing for an increased patient cohort by avoiding supply bottlenecks in ab-serum. furthermore, cryopreservation allows the storage of ready-for-use cik cell doses fulfilling the demands of the clinical study. clinical trial registry: eudract number 2013-005446-11 disclosure: nothing to declare. automated generation of cd45ra depleted donor lymphocyte infusion (dli) with the clinimacs prodigy® cd45ra system 1, 2 methods: the current clinimacs cd45ra system was developed for graft engineering. up to 20x10e9 magnetically labeled cd45ra+ cells from leukapheresis products can be depleted from up to 50x10e9 white blood cells (wbc). we developed a new clinimacs prodigy® process in order to ease the procedure for routine-use, to reduce the specifications according to reported cell numbers for dli applications, and to enable the use of peripheral blood products with high amounts of red blood cells (rbc). the new system was tested by performance runs. an new fluorescent flow analysis protocol was developed. results: the resulting clinimacs prodigy pb-45ra system is an automated procedure with integrated labeling and washing steps. the new application software pb-45ra depletion enables to deplete up to 1.8x10e9 cd45ra + cells from up to 5x10e9 total wbc from peripheral blood products. a major difference of this process is the rbc removal option based on an integrated camera for cell pellet detection. the final cell product is provided in physiologic saline. verification runs with peripheral blood products (n=6 in total, n=3 with whole blood, n=3 with leukapheresis products) resulted in a mean depletion of 5.0 log (range 3.9 -5.7) for cd45ra + t cells in the cd45ra depleted product. viability of the target products was always above 96%, and mean wbc recovery was 66%. the mean process time was 3h23min (range 3h to 3h50min) without including the manual steps, i.e. tubing set installation and downstream analysis of blood products by flow cytometry. this data were in line with preceding evaluation runs (n=6), and results obtained in cooperation with an external beta test site. 3 the performance results were furthermore in line with results obtained on clinimacs plus instrument runs. for quality control of cd45ra depleted products we developed a flow cytometric analysis strategy for fast, accurate, and convenient analysis of even rare counts of remaining unwanted cells. it allows to determine naïve t cells at two different levels of subset staining. the minimum requirement for the flow cytometric analysis includes 4 colors to define viable cd3+cd45ra+ cells. for further evaluation of the naïve t cell subsets 3 additional colors are used to define viable cd3+cd45ro-cd95-cd62l+cd197+ cells. conclusions: the automated clinimacs prodigy pb-45ra system process is capable to deplete cd45ra+ cells efficiently from peripheral blood products within 4 hours. the new process is a fast, convenient, and regulatory compliant method for the preparation of ready-to-use cd45ra-depleted cell products for clinical applications. the submission to an european notified body for ce certification is an important next step. myeloablative conditioning regimen was preferred for 24 patients out of 25. gvhd prophylaxis regimens are csa +mtx: 20(%80), csa+mmf: 2(%8), csa only: 1(%4). atg was given 20 patients. despite been given gvhd prophylaxis 47(%27,1) patients out of 173 transplanted patients had gvhd features. of 47 patients, 25 had experienced steroid resistant gvhd after transplantation, including 17 (%68) grade 3 and 8 (%32) grade 4. ecp treatment was started mean 11 days after diagnosis of steroid resistant gvhd and 2 (%8) patients had complete response while 12 (%48) patients had partial and 11 (%44) patients had no response to ecp treatment on day 28. sixteen out of 25 patients had also received mesenchymal stem cell therapy as salvage therapy. only one patient had experienced hypocalcemic tetany, a complication of ecp procedure. thirteen patients had died and 12 were directly related with steroid resistant gvhd. other conditions like relapse of primary disease or pres syndrome also played role in death. conclusions: extracorporeal photopheresis is a reliable and effective second line treatment modality in steroid resistant gvhd. starting ecp sessions as soon as gvhd symptoms occur increases its effectivity. mesenchymal stem cell administration with ecp for 16 (%64) patients limits our study to reach o conclusion for efficacy of ecp itself. need for hemodialysis catheters, the prolonged sessions while adequate flow is not possible and catheter related infections are the lmitations for feasibility of ecp. disclosure: nothing to declare donor lymphocyte infusion administrations after allogeneic stem cell transplantations in pediatrics: a single center experience background: loss of chimerism is one of the major problems after allogeneic stem cell transplantation(sct). donor-lymphocyte infusions(dli) are used as a treatment after taper or stopping immunosuppression. in this study, dli experience in 20 patients with loss of chimerism after sct due to various benign and malign hematological diseases was presented. methods: between july 2015-august 2018, twenty patients, detected chimerism loss and received dli after sct were evaluated retrospectively. patients received myeloablative or reduced intensity conditioning, atg, cyclosporine a and methotrexate for gvhd prophylaxis. chimerism analyses were performed with short tandem repeat(str) method from peripheral blood. results below 95% were considered as mixed chimeric and below %5 were nonchimeric. when patients considered as mixed chimeric, immunosuppression therapy was ceased immediately and treated with dli. donor lymphocyte infusions were performed at two-week intervals with chimerism follow-up. student t, mann whitney u, ki kare tests and kaplan-meier analysis were used. results: between 1-16 ages (median 4), 7 female, 13 male patients were evaluated. the initial diagnoses were thalassemia major(10), aplastic anemia (3),all(4), aml (3) . dli initiation time was 119.65+-92.71 days after sct, total number of dli administrations were 3.5+-2.19. dose of dli was 1x10 5 -63.6x10 6 /kg (mean 20.5x10 6 /kg). nine patients' chimerism out of 20, fell below 95% at first month after transplant; 4 patients were nonchimeric, 2 of them were complet chimeric and 3 were mixed chimeric. eleven patients´chimerism were below 95% between 1-6 months after sct, 5 patients were nonchimeric and 6 were mixed chimeric. early mixed chimerism was found relevant with graft rejections (p=0.04). patients were followed up for 91-645 days. eight patients' chimerism increased after dli infusion and continued to decrease in 12 patients. after dli, acute gvhd has been seen in both group.the group with decreased chimerism after dli, dose was mean 12x10 6 ±23x10 6 /kg while the group with increased chimerism had dli dose mean 22x10 6 ±19x10 6 /kg. although the difference was not statistically significant, numerical value revealed significantly different. eventually10 patients out of 20 were mixed chimeric, 6 patients were complete chimeric and 4 were none. in thalassemic patients, 7 patients with thalassemia-trait donor were mixed chimeric, in 3 patients whose donors were normal, 2 of them were complete chimeric and one of them was nonchimeric.the difference was significant (p=0.02). the cd34 infusion doses revealed mean 6.61 ±5.06x10 6 /kg in mixed chimeric patients, 7.16±3.31x10 6 /kg in complete chimeric patients and 6.625±1.37x10 6 /kg in the patients with loss of chimerism. cd34 amount was seen high as numerical value in complete chimerics but no statistical significance was found. overall survival was 85%, disease-free survival was 25%. conclusions: we evaluated the efficacy of dl for patients with mixed chimerism in our patient group. we concluded that chimerism loss in patients with early decreased chimerism is similar to those in literature in spite if dli practices. dose and application frequency were greater in patients with increased chimerism. the small number and the heterogeneity of the patients limited our study. in this regard, studies with larger series and homogeneous groups are acquired. disclosure: nothing to declare phase i clinical trial of repeated administrations of bone-marrow derived mesenchymal stem cells in steroid-refractory chronic graft-versus-host disease patients nayoun kim 1 , young-woo jeon 2 , jae-deog jang 1 , keon-il im 1 , nak-gyun chung 2 , young-sun nam 1 , yunejin song 1 , jun-seok lee 1 , seok-goo cho (cghvd) is the most common long-term complication of allogenic hematopoietic stem cell transplantation which is associated with poor quality of life and increased risk of morbidity and mortality. currently, there is no standardized treatment available for patients who do not respond to steroids. as an alternative to immunosuppressive drugs, mesenchymal stem cells (mscs) have been used to treat and prevent steroidrefractory acute gvhd patients. these studies and reports have also provided a basis for using mscs in steroid refractory cgvhd patients. methods: to evaluate the safety and efficacy of repeatedinfusions of mscs, we enrolled ten severe steroid-refractory cgvhds patients. steroid refractory was defined as either no response to steroids lasting at least 4 weeks or progression of disease during treatment or tapering lasting at least 2 weeks. patients were intravenously administered with mscs produced from third-party bone marrow donors at a 2-week interval for a total of four doses. each dose contained 1x10 6 cells per kg body weight and all four doses consisted of mscs from the same donor and same passage. results: we enrolled ten patients (3 female/ 7 male, with a median age of 41.5(range 17-68). median of cgvhd affected organs was 3 (range 2-4) including the skin (n=4), eyes (n=8), oral cavity (n=9), lung (n=1), liver (n=2) and joints (n=6). all ten patients received their planned four doses of mscs, administering a total of 40 infusions. median time from initial cgvhd diagnosis to first msc treatment was 709 days (range 222-4413). msc infusions were well tolerated with no immediate or delayed toxicities. after 8 weeks of the first msc infusion, all ten patients showed partial response showing alleviation in clinical symptoms and increased quality of life. organ responses were seen in skin (n=2), eyes (n=5), oral cavity (n=8), liver (n=1), and joint(n=5). however, one patient died of progressive gvhd and one patient relapsed from primary disease. conclusions: repeated infusions of mscs was feasible and safe and may be an effective salvage therapy in patients with steroid-refractory cgvhd. further large-scale clinical studies with long-term follow up is needed in the future to determine the role of mscs in cgvhd. background: the majority of pregnant polish women (84%) have heard of cord blood banking. however, most doctors do not have sufficient knowledge about the possibility of using cord blood in order to respond to their potential concerns. only 16.5% of healthcare professionals were aware that cord blood could be used to treat haematological diseases. in order to make doctors aware of this issue and provide patients with the information they expect, we would like to present data on the use of cord blood stored in our blood bank for haematological and nonhaematological therapies. methods: the table presented below has been created using data from the general database of the polish stem cell bank, warsaw, poland. no data regarding umbilical cord blood data have been excluded. all patients were planned to be assessed on day 1, day 28, on discharge, 100 days after transplantation and 1, 2, 3, 4, and 5 years after transplantation, but in some cases, patients were lost from follow-up due to a persistent lack of reports from transplantation centres. results: in 32 cases, the therapeutic use of cb was transplantation (replacement of patients' own tissue); in 13 cases it was administration (infusion without destruction of patients' own tissue). thirty-three were administered as standard therapy and 13 as experimental therapy. conclusions: the survey study cited above, indicated low awareness of cord blood use among healthcare professionals. on the other hand, one study indicated that the expectations placed in cord blood banking may be unreasonable. as a private cord blood bank, we support recommendations which underline the importance of patient education. in poland, cord blood has been approved as standard therapy in approx. 80 diseases; most of them are rare, but polish law allows the use of cord blood for the siblings, parents and grandparents of a donor. additionally, 168 active and planned clinical trials throughout the world evaluate the therapeutic efficacy of cord blood in such areas as haematology, neurology, cardiology, diabetology, congenital paediatric disorders, ophthalmology, dermatology, gastroenterology, hiv infection, and the quality of life during aging. therefore, further indications may be expected in the future. background: cytokine release syndrome (crs) has been identified as a clinically significant, on target, off tumour side effect of the chimeric antigen receptor (car) t-cell therapies. it is clinically increased in interkeukin 6 and elevations in other cytokines, lactate dehydrogenase (ldh), c-reactive protein (crp), and ferritin. these side effects can include fever, fatigue, headache, encephalopathy, hypertension, tachycardia, coagulopathy, nausea, capillary leak and multi organ dysfunction. crs symptoms can appear as early as one day after infusion and can resolve quickly or last for weeks. it´s severity to be related to the disease burden prior to car t-cell therapy. methods: the bristol oncology and haematology centre will be providing car t-cell therapy to patients in early 2019. on collating from the leading consultant on the ward and fellow nursing team members it was apparent that an effective way at managing patients post car t-cell therapy side effects is to provide an educational and informative poster depicting a flow chart that will aid the practitioner to recognise and effectively treat/manage a patient with crs symptoms. results: none as of yet as this is a prospective tool ready for our first patient in early 2019 conclusions: through continuing reading and study days prior to the ward receiving its first car t-cell patient it is increasingly important that the entire multi disciplinary team recognise crs and understand the importance of early detection, careful monitoring and early intervention. background: allogeneic stem cell transplantation (allosct) is the only curative procedure for primary and secondary myelofibrosis (pmf, smf). elderly people are mainly affected, limiting the feasibility of intensive myeloablative chemotherapy regimens. the introduction of reduced-intensity conditioning (ric) made allosct feasible and effective for old patients. nevertheless, the incidence of pmf and smf is not negligible in young patients, theoretically able to tolerate also high-intensity therapy. very few data are available about the efficacy of ric-allosct in the particular setting of young-aged mf patients. methods: this study includes 56 myelofibrosis young patients (age < 55y) who received allosct between 2002 and 2016 at the university hospital hamburg/germany. four patients were previously splenectomized. patients mostly fall into intermediate risk groups according to dipss. four patients belonged to the high-risk triple-negative category (jak2/calr/mpl-). asxl1-mut was tested in 50 patients (pos: 17). in 96% graft source was pbsc, 2 patients received bmsc. only 30% of patients had a 10/10 hla-matched sibling, the others were transplanted from fully-matched (36%) or partially-matched (34%) unrelated donor. all transplants were conditioned according the ebmt protocol with busulfan (10 mg/kg po or 8 mg/kg iv), fludarabine (150 mg/m2), atlg (grafalon® neovii, germany) administered in 3 days at a dose of 20 mg/kg die for mud, 10 mg/kg die for mrd transplants, followed by cylosporina, and mycophenolate in the first 28 days. results: engraftment rate was 98%, median neutrophil engraftment time 15 days. platelet engraftment was reached by 51 patients (91%, median 19 days). four patients (7%) developed poor graft function, successfully treated with cd34+ selected pbsc-boost. after a median follow up of 8.6 years, estimated 5y-pfs and os were 68% and 82% respectively. dipss-risk and donor hla-matching resulted the only significant impacting factors on os. neither cytogenetic nor molecular abnormalities were significantly related to os. twenty-five patients (44%) experienced agvhd grade >1. c-gvhd was observed in 34 patients (65%), mostly (82%) beginning in the first 300 days after transplantation. cumulative incidence of trm was 7% at 1 year, with a plateau after the first year (5y trm = 12%). trm was observed only in patients with maximal grade (3) of marrow fibrosis. furthermore, trm never occurred in previously splenectomized patients (p=0.00), but no significant impact from splenectomy on os was observed (p=0.32). after transplant, 11 patients (20%) relapsed: 1 died without any treatment because of infection, 9 received dli (3 durable cr), 7 patients (6 after dli) underwent a second allosct, with long-term survival in 5 cases. conclusions: ric followed by allogeneic sct is a curative treatment approach for younger patients with myelofibrosis with a low nrm. the most important outcome-determining factor is donor hla-matching. interestingly, marrow high grade fibrosis showed to significantly impact trm. biological markers such as asxl1 mutation and cytogenetics, largely known as highly predictive for poor prognosis in the disease natural course, did not show any impact on survival, suggesting that patients harboring these abnormalities could get the greatest benefit from allosct. further data collection, and a prospective randomized trial are needed to confirm our conclusion. disclosure: nothing to declare p087 abstract already published. splenectomy as a risk factor for relapse after allogeneic hematopoietic stem cell transplantation in patients with myelofibrosis -retrospective cohort study background: splenectomy is a common procedure in patients (pts) with myelofibrosis (mf) performed to achieve improvement in blood cell counts and reduce b-symptoms. however, it has also been shown that splenectomy may adversely predispose to leukemic transformation in this setting. aim: to evaluate in a single-center retrospective analysis the long-term impact of pre-or post-transplant splenectomy on transplant outcome regarding overall survival and relapse risk. methods: this retrospective analysis comprises the data of 163 pts (93 male and 70 female) with primary (n=108) or secondary (n=55) mf after allo-hsct from hla-matched sibling (n=48) or unrelated (n=115) donors in our center between 1994 and 2018. the median age was 56 years (range, 22 to 75 years). a myeloablative conditioning regimen was performed in 142 pts, while 21 pts where treated with a reduced intensity conditioning. peripheral blood stem cells (n=154) or bone marrow (n=9) with a median of 7.0 x 10 6 cd34 + cells/kg bodyweight (bw) (range, 1.0 to 30) were transplanted. splenectomy was performed in 41 of 163 pts: 21 pts were splenectomized prior to and 20 pts after allo-hsct. relapse was diagnosed in 22 (14%) of 163 pts. the median duration to relapse after transplantion was 13 months (range, 3-99 months). results: the median duration of follow-up of this cohort was 28 months (range, 2-120 months), the 10-year overall survival (os) was 46%. 74 pts died, including 7 pts who relapsed and 67 pts who died of treatment related causes. the observed probability of relapse was significantly higher in splenectomized pts than in non-splenectomized pts: 37% versus 10% (relative risk (rr) 3.7, 95% ci, 1.5-9.4, p=0.007). at 10 years, the os was 50% in nonsplenectomised and 39% in splenectomised pts (p=0.35) (fig.1) . the relapse rate in splenectomised pts was independent of pre-(5 of 21 pts, 24%) or post-transplant (6 of 20 pts, 30%) treatment (rr 1.3, 95% ci, p=0.73) . conclusions: on the basis of our cohort, we could assert that pre-and post-allo-hsct splenectomy was equally and significantly associated with an increased relapse ratio in patients with mf, which also tends to negatively affect overall survival. [[p088 image] 1. figure 1 : the overall survival after allo-hsct in patients with myelofibrosis.] background: b-cell prolymphocytic leukemia (b-pll) is a very rare lymphoproliferative disorder. although allogeneic stem cell transplantation (allosct) could be a curative option, patients often do not qualify for this consolidation treatment due to an aggressive course of disease. in this case study, we report on three patients who failed ibrutinib therapy but achieved complete remission and even mrd negativity after treatment with the chimeric cd20-antibody rituximab, enabling them to undergo allosct. methods: clinical data and follow-up data were collected by chart review. results: all three patients (pt#1: male, 42 years; pt#2: female, 66 years; pt#3: female, 64 years) were referred with b-pll harboring highly complex aberrant karyotypes, including 17p abnormalities in pt#1 and pt#2. a tp53 mutation could be detected in pt#2 and pt#3. all three patients had symptomatic disease with rapidly increasing hyperleukocytosis and massive splenomegaly. two of them were treatment-naive and one relapsed after chemoimmunotherapy. all patients were put on ibrutinib 420mg. despite initial response to treatment, two patients developed progressive disease after 4 (pt#1) and 9 months (pt#2) on ibrutinib, whereas pt#3 remained in partial remission with persisting leukocytosis, precluding consolidating allosct as originally intended. in pt#1, ibrutinib was replaced by venetoclax, but without response. in order to control rapid lymphoproliferation, rituximab was added to venetoclax. grade 4 infusion reaction / tumor lysis syndrome (tls) (fever, tachycardia and hypotonia requiring intravenous vasopressors) followed each rituximab administration despite fractionating rituximab to small doses. however, continuation of rituximab (100mg/d over 10d) led to complete and durable clearance of hyperleukocytosis (from 250/nl to mrd negativity) despite venetoclax cessation. a similar pattern was observed in pt#2, who received rituximab while showing rapidly increasing leukocytosis upon ibrutinib. again, complete clearance of b-pll cells in the peripheral blood (from 148/nl to mrd negativity) occurred after initial grade 4 tls despite only modest cd20 expression on tumor cells in this patient. also, pt#3 achieved profound b-pll cell depletion (from 38/nl to a mrd rate of 1.1%) upon addition of rituximab to ibrutinib (without tls in this case). subsequently, all three patients were able to undergo allosct after conditioning with fludarabine and total body irradiation with 8 gy. pt#1 received stem cells from a hla-ident sibling donor, whereas pt#2 and pt#3 had unrelated donors (hla-ident and hla-matched respectively). with follow-up times of 17 and 11 months post-transplant, pt#1 and pt#2 are currently in ongoing mrd-negative remission. pt#1 developed an acute graft-versus-host disease (gvhd) of the liver (grade 3), nevertheless the clinical course was well controlled by immunosuppression. in pt#2 a chronic gvhd of the skin occurred. pt#3, who achieved mrd negativity after allosct, developed acute and chronic steroidrefractory gvhd of the skin and gastrointestinal tract. nine months post-transplant, gvhd deteriorated and after further complications the patient died of pneumonia 11 months post-transplant. conclusions: supplementary treatment with rituximab can induce deep remissions in patients with ibrutinibresistant, genetically poor-risk b-pll, thereby enabling them to undergo successful consolidation with allosct. a high risk of life-threatening infusion reactions / tls associated with the addition of rituximab has to be taken into account. background: there is little experience on the use of the newer targeted therapies in cll patients relapsed after allogeneic stem cell transplantation (allo-sct). against this background, we evaluate the safety and efficacy of the bcr inhibitors (bcri), ibrutinib and idelalisib, administered after allo-sct for the purpose of treating the cll relapse. methods: data from 11 cll pts who relapsed after sct, and were subsequently treated with ibrutinib (n=6), idelalisib (n=3) or both (n=2),were retrospectively collected in collaboration with the spanish group of cll (gellc) and the spanish group of stem cell transplantation (geth). results: transplant characteristics are summarized in table 1. eight patients received the bcri as the first salvage treatment after sct relapse, whereas 3 patients had received ≥2 prior lines of treatment. at the time of the onset of the bcri, 7 patients had rai 4 stage and 6 patients had a lymph node size ≥5 cm. del17p was present in 4 patients and del11q and complex karyotype in 2 patients, respectively. tp53 gene mutation was detected in 3 patients (all with del17p13). median time from sct to bcri therapy was 53.9 months, being shorter in patients treated with ibrutinib (n=8, median 51 months) than in those treated with idealisib (n=3, median 111 months). median time on ibrutinib and on idelalisib was 7.3 months (4. 1-18.8 ) and 6 months (3.0-23.4 ), respectively. the best overall response rate (orr) obtained with ibrutinib was 75% (1 cr, 4 pr, 1 pr+l) whereas it was of 40% for patients receiving idelalisib (1 cr, 1 pr+l). among the 8 patients treated with ibrutinib, 7 (87.5%) presented an adverse event (ae), being diarrhea (n=3), asthenia (n=2) and infections (n=5) the most frequent. hypertension was seen in 1 patient and none patient developed atrial fibrillation. five patients stopped ibrutinib treatment, due to toxicity (n=4) or progression (n=1). after ibrutinib discontinuation, 4 patients were newly treated with idelalisib (n=2) or venetoclax (n=2). all patients treated with idelalisib developed at least one ae, being diarrhea (n=3), pneumonitis (n=2) and neutropenia (n=2) the most common. four patients discontinued idelalisib because progression (n=3) or toxicity (n=1). venetoclax was given after idelalisib in 3 patients. although acute and/or chronic gvhd before bcri was documented in 7 (63.6%) and 5 (45.5%) patients, respectively, only one patient (treated with idelalisib) reactivated a mild chronic gvhd. none patient received infusion lymphocyte from donor after bcri and one patient underwent a second sct. with a median follow-up of 14.3 months (5.9-33.9) after the onset of the bcri treatment, 4 patients had died, all of them due to cll progression (3 richter´s transformation), whereas 5 patients remained in response (3 cr, 2 pr). the overall survival probability of the whole series at 12 months was 77.8% ±13.9%. conclusions: in our study, ibrutinib and idelalisib, administered in cll patients relapsed after sct did not increase the risk of gvhd reactivation but they show high incidence of adverse events. nevertheless, bcri offers a possibility of disease control in these patients with poor prognosis. further studies are needed to confirm these data. background: prior to the introduction of tyrosine kinase inhibitors (tki), median survival of chronic phase chronic myeloid leukemia (cp-cml) patients was approximately 60 months and the standard treatment with interferon-alpha resulted in complete cytogenetic responses in about 30% of the patients. autologous stem cell transplantation (auto-sct) was first attempted for patients in transformation in order to restore a second cp and was introduced secondarily in cp to try to prolong the response. the main rational for autografting in cp resides on the reduction of the tumor burden and the number of leukemic cells at risk of developing blastic transformation. nevertheless, auto-sct alone was not able to maintain a long-term remission. nowadays, tkis represent the state-of-the-art therapy for cml and the concept of auto-sct has only little interest while long-term follow-up and outcome in this setting are currently unknown. the aim of our study is to evaluate at a first time the longterm outcome of cml patients who received auto-sct in chronic phase, and to evaluate at a second time in a subgroup analysis, the outcome of those who received tki after having been auto-transplanted, mainly for disease progression/loss of response and/or to enhance disease response. methods: we found a total of 969 patients who received auto-sct for cp-cml in europe between years 1989 and 2004, 578 (60%) were males, median age at auto-sct was 47 years (range: 19-67), the median time between diagnosis and auto-sct was 19 months, stem cells source was peripheral blood in 84% of patients, most frequent conditioning regimen was busulfan 4mg/kg/day 4 days + 1 day of melphalan 140 mg/m² one day prior to the cells reinfusion. information about receiving tki post auto-sct was available only for 103 patients, first tki was imatinib for 89 (86%) patients, dasatinib for 8 (8%), nilotinib for 6 (5%) and ponatinib for one (1%) patient. results: after a median follow-up of 9.5 years (range: 1-27) from time of auto-sct for the whole population, the probability of overall survival (os) at 10 years was 50% (95% ci: 46-53); there was 540 (56%) patients who relapsed after a median time of 16 months after auto-sct. there was a total of 530 patients transplanted before the tki era and survived until the availability of tkis. when we performed a landmark analysis evaluating the outcome of patients who received auto-sct, survived until the tki era and received tki (n=103), the 10 years os probability of these patients from tki treatment was 70% (95% ci: 58-78). additional data requests will be sent to centers querying about prognosis, molecular responses, treatment and disease details. conclusions: we demonstrate here with these preliminary results that the introduction of tki has improved survival of cml patients. in addition, patients who received auto-sct, survived until the tki era and also received tki, had encouraging rates of long-term survival. an extensive analysis will be performed when additional data will be available and the study will be updated with more results. disclosure: nothing to declare a 35 year single center transplant experience in chronic myeloid leukemia background: allogeneic hematopoietic stem cell transplantation (hsct) has been considered for decades the only curative approach for patients with chronic myeloid leukemia (cml). in the tyrosine kinase inhibitors (tkis) era, hsct for cml has been reserved only to patients not achieving a cytogenetic remission or showing progressive disease after multiple tki treatment lines. however, a progressive improvement in the long-term survival has been obtained in the overall hsct population. the present study aimed at evaluating whether in cml patients transplanted at our center over a long time period -from 1983 to 2018 -the outcome improved over time. methods: 136 consecutive patients who underwent a transplant between 1983 and 1999 were compared to 43 patients who received the transplant between 2000 and 2018. overall survival (os), leukemia-free survival (lfs) and graft-leukemia-free survival (glfs) were estimated using the kaplan-meier method and the log-rank test was used to compare risk factors categories. results: of the 179 patients [median age 35 years (range7-66)], 148 (82.7%) were in 1 st or 2 nd chronic phase, 25 (13.9%) in accelerated phase and six (3.4%) in blast crisis. matched related donors and alternative donors (matched unrelated donors, cord blood or mismatched related donors) were used in 156 and 23 cases, respectively. as stem cell source, bone marrow was used in 142 patients, peripheral blood in 33 and umbilical cord blood in 4. tbibased conditioning regimens were used in 89 patients, while in the other 90 cases irradiation-free conditioning regimens were used. both in univariate and multivariate analysis, irradiationfree conditioning regimens (hr 1.8; 95%ci 1.1-3.0, p=.0014) and transplants performed in 1 st chronic phase (accelerate phase hr 2.1; 95%ci 1.2-3.8, p=.008 -2 nd chronic phase hr 4.9; 95%ci 2.3-10.3, p=.0001 -blast crisis hr 2.5; 95%ci 1.0-6.4, p< .05) were associated with a better os. patients transplanted before 2000 had a worse os (hr 6.5; 95%ci 2.7-15.5, p < .0001) and dfs (hr 2.2; 95%ci 1.0-4.8, p=.045). a trend for a worse glfs was observed in univariate analysis (hr 1.6; 95%ci 1.0-2.7, p=0.05), in the first period of observation. conclusions: our single center experience confirms that higher os, dfs and glfs are observed in cml patients allografted in more recent years. improvement of conditioning regimens, use of tbi-free conditioning regimens and supportive therapy, have presumably contributed to these results, together with the more recent strategy of close monitoring of minimal residual disease, and prompt use of tki or donor lymphocyte infusion in case of relapse. hsct is nowadays a safer therapeutic procedure in cml patients that should be considered timely in tki-resistant patients to avoid progression into a more advanced disease phase. disclosure: the authors declare no conflict of interest. reduced-intensity transplantation (rit) in patients with high-risk or advanced chronic lymphocytic leukemia in last 5 years: improvement of transplant outcomessingle centre experience . hct-ci ≥ 3 was in 20% of pts. source of stem cells was peripheral blood in 80% and bone marrow in 20% of pts. the median of infused cd 34+ cells was 5,4x10^6/ kg. the conditioning regimen consisted of fludarabine and melphalan (+atg in unrelated donor). gvhd prophylaxis were cyclosporine and methotrexate. results: all pts engrafted. none of 3 pts in cr before rit progressed at day +30 after rit and among 17 pts beyond cr before rit all of them achieved at least pr at day +30 after rit. 13 pts (65%) developed acute gvhd (2 pts grade iii-iv) and among 19 evaluable pts 10 (53%) of them developed chronic gvhd (6 mild, 2 moderate, 2 severe). with median follow-up 50 months (range 6-63 months) 15 pts (75%) are alive in cr. 3 pts (15%) relapsed or progressed 5, 19 and 21 months after rit and 2 of them died. last relapsed patient achieved next cr after ibrutinib. 3 pts (15%) died due to nrm. nrm till day +100 after rit was 5%. the estimated probabilities of 2-years cgrfs, pfs and os are 55%, 73% and 83%. conclusions: in spite of relatively small number of evaluated pts and retrospective type of analysis our data show that rit in pts with high-risk or advanced cll has achieved promising results (2-and 5-years pfs and os 73% and 55% resp. 83 and 57%) in recent years and these results are better than outcomes of our historical patient cohort from period 2010-2012 (2-and 5-years pfs 41% and 26% resp. os 56% and 35%, p=0.009) or ebmt published data of pts transplanted for cll in period 2000-2010 (2and 5-years pfs 46% and 35% resp. os 62% and 45%). current results of transplantation should be taken into account in our future decision-making process on indications for transplantation in pts with high-risk cll, of course also in the context of new or updated results of targeted cll treatment and its complications. disclosure methods: retrospective data and treatment outcomes were collected from the singapore childhood cancer registry (sccr). most children with cancer in singapore receive therapy at one of the two public paediatric cancer centers (kkh or nuh). a total of thirty two cases were diagnosed with cml and received treatment in either of these centers over a twenty year period (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) . results: the age at diagnosis of the thirty two children ranged from 4 to 17 years (median 12.5years). six patients in the pre-tki era were treated with an upfront hsct. the remainder twenty six patients were initially started on a tki. of these 12/26 (46%) had a hsct at a median period of 22.5 months from diagnosis (range 5-43 months). the reason for hsct in ten out of the twelve children was due to high risk features i.e. accelerated/blastic phase/ no ccr/no cmr. the remaining two patients had a hsct due to parent and patient preference for attempt at upfront cure rather than the use of life-long and expensive tki therapy. non-compliance to tki therapy was a major finding in our teenage cohort. eleven of the eighteen transplants used a matched sibling donor. three patients had cord blood as their stem cell source. one patient had a single antigen mismatched related donor and three patients had a mismatched unrelated donor for their hsct. all patients except one had myeloablative conditioning with busulfan and cyclophosphamide. atg was added according to physician preference. one patient had cy/tbi conditioning because of pre-transplant lymphoid blast crisis. anti gvhd medications included cyclosporine/ methotrexate or tacrolimus and methylprednisolone in the cord transplant patients. six of the eighteen (33%) patients who had a hsct died. four died due to treatment related mortality (2 infections, 1 acute gvhd and 1 pulmonary fibrosis). one patient died due to an early relapse and one had a late relapse related mortality. for the pre-tki era, hsct related 5 and 10 year os was 83% and 67% respectively. post-tki era 5 and 10 year os was 75%. for the entire cohort, the 10 year os was 70%. conclusions: the post-tki era transplant outcomes from our two centers is comparable to leading centers in the world. outcomes for patients with mismatched unrelated donors was poor in our cohort. we recommend a haploidentical related donor transplant or an unrelated cord blood stem cell source for patients when a matched sibling or unrelated donor is not available. clinical trial registry: na disclosure: we have nothing to disclose. fludarabine, busulfan, and thiotepa may be a promising conditioning regimen for myelofibrosis patients undergoing allogeneic stem cell transplantation background: allogeneic stem-cell transplantation (sct) is a curative therapy for patients with myelofibrosis. however, recurrent disease and non-relapse mortality (nrm) are frequent causes of treatment failure. the optimal conditioning regimen for sct in this disease has not been defined. methods: we retrospectively analyzed transplantation outcomes of all adult patients given sct for myelofibrosis between 2003 and 2018 at a single large academic medical center. patients (n=59) were treated with several conditioning regimens that were grouped according to conditioning intensity. myeloablative conditioning (mac) included busulfan 12.8 mg/kg and cyclophosphamide 120 mg/kg (bucy, n=10), fludarabine and busulfan 12.8 mg/kg (flu/ bu4, n=9) and fludarabine and treosulfan 30-36 g/m 2 (flu/ treo, n=6). reduced-intensity conditioning included fludarabine and busulfan 6.4-8.0 mg/kg (flu/bu2, n=22). more recently we adopted the tbf regimen including fludarabine, busulfan 6.4-9.6 mg/kg and thiotepa 5-10 mg/ m2 (n=12). all patients were also given anti-thymocyte globulin during conditioning, irrespective of donor source. results: the median age was 59 years (interquartile range [iqr] 52-63). the majority of patients had documented splenomegaly (81%) and were not previously exposed to ruxolitinib (71%). donor type was an hla-matched sibling (41%), 10/10 (51%) or 9/10 (8%) matched unrelated donor. the dipps+ score distribution was intermediate-1 (16%), intermediate-2 (45%), or high (n=39%). the median followup was 3.2 years since the success of tyrosine kinase inhibitors (tkis), transplant-related mortality is considered too high to justify allogeneic hematopoietic stem cell transplantation (allohsct) as first-line treatment for chronic myeloid leukemia (cml) patients in chronic phase (cp). allohsct is currently considered for patients failing to at least 2 tkis or with disease in advanced phase. nevertheless, the optimal timing for transplant referral is still not well defined. methods: we performed a retrospective analysis on 23 consecutive patients with cml in cp receiving first transplants from an hla-identical sibling donor with partially t-cell depleted grafts from 1998 to 2016 at our center. partial t-cell depletion (ptd) consisted of in vitro alemtuzumab incubation of a part of the graft for infusion at day 0 while the rest, containing 100x10 6 cd3+ cells/kg was given as a t-cell add-back at day 1. donor lymphocyte infusions (dlis) were provided, in the absence of gvhd, in case of disease relapse or mixed chimerism. molecular monitoring was performed by 3-month bcr-abl1 rt-qpcr testing in peripheral blood during at least a 5-year period after hsct. thereafter, 3-month testing schedule was maintained where possible, or followed by a 6-month one. kaplan-meier method was employed to determine the probability of overall survival (os) and leukemia free survival (lfs) since allohsct. results: median age at hsct was 36 years (range, 18-58). all patients were in first cp but one who was in second cp. twelve patients were tki-naïve at hsct (1998 hsct ( -2001 , 4 patients had presented suboptimal response or/ and intolerance to imatinib (2002-2004 period) , while the last seven patients had presented suboptimal response or/ and intolerance to imatinib, dasatinib and nilotinib (2005-2016 period) . the time interval from diagnosis to transplant was < 12 months in 11/23 (48%) patients. 15 (65%) patients had an ebmt risk score of 0-2, while 8 (35%) patients of 3-4. the conditioning regimen was myeloablative for all but one patients. the stem cell source was pbsc for 22 patients and bone marrow for one. all patients engrafted. 14 patients presented molecular relapse and one patient hematological relapse with a median interval from transplant to relapse of 9 months (range, 5-70) . 17 patients received dlis (15 for relapse and 2 for mixed chimerism), while 3 patients in relapse also received tki. without prior administration of dli, 3(13%) patients presented grade ii agvhd and 2 patients moderate cgvhd. after dli, agvhd occurred in 3 and cgvhd in 4 patients. one patient died of disease progression 3 years after hsct and one of myocardial infarction 19 years after hsct. with a median follow-up of 14.4 years (range 2.3-20.6), 15-year os and lfs were 95%. at the time of the analysis 21/23 patients were alive and in major molecular response. conclusions: these results of excellent long-term survival and no transplant-related mortality suggest that ptcd improves the outcome of cp-cml patients transplanted from an identical sibling donor and they can be useful for deciding risk-adapted strategies. we believe that ptcd could allow earlier transplant referral of patients failing tkis and having an identical sibling donor. disclosure: nothing to declare single tertiary centre experience in allogeneic haematopoietic stem cell transplantation (allo-hsct) for primary and secondary myelofibrosis (mf) the only curative option for fit patients is allo-hsct. novel therapy is emerging but current recommendation is that eligible patients with life expectancy less than 5 years should be considered for allografting. methods: we retrospectively looked at the clinical features and outcomes of all allo-hsct for mf performed in our centre since 2010. results: 12 patients (10 male, 2 female) aged between 29-68 years old (median age 60) with intermediate-2 or high-risk mf as per the international prognostic scoring system (ipss) or dynamic ipps (dipps) were transplanted in our centre since 2010. 6 of them (50%) were diagnosed with pmf and the remaining 50% with secondary mf; 4 post-et and 2 post-pv mf. 2/12 of our patient group received a sibling allograft and 10/12 a matched unrelated donor allograft (70% received a 10/10 human leukocyte antigen (hla)-matched transplant, 20% a 9/10 hlamatched and 10% a 8/10 hla-matched graft). all patients received a reduced intensity conditioning (ric); 3/12 patients with fludarabine/ melphalan/ campath (fmc), 8/ 12 fludarabine/ busulphan/ atg (fbatg) and 1 fludarabine/ ara-c/ campath (flag/ campath); all received peripheral blood as source of hsc. engraftment occurred between day 13-48, with a median of d+17. one late graft rejection occurred. all patients were alive at d+100. 9 patients are currently alive; overall survival (os) is 75%. transplant related mortality (trm) was 16.6% at 1 year, 25% at 3 years. 1 patient died of graft versus host disease (gvhd) and 2 patients of septicaemia leading to multiorgan failure. acute gvhd grade ii skin occurred in 5 patients, grade iii and above in 2 patients. 5 patients have limited chronic gvhd. 2/12 patients received donor lymphocyte infusion (dli) for mixed chimerism (one of which had 2nd graft failure). out of these 2 patients 1 developed acute grade 4 gvhd and died. response rate: 6/9 alive patients i.e 66.6% exhibit no fibrosis in trephine biopsies, 1/9 alive patients had residual fibrosis but 100% donor chimerism, 1/9 alive patients had residual fibrosis with mixed donor chimerism, other patient non-assessable. conclusions: allo-hsct remains the only potentially curable option for myelofibrosis. in our centre which serves 1.1 million population, with 12 new cases per year, 12 patients were transplanted since 2010. our data suggest that close collaboration between mpn-treating haematologists and transplant physicians is required so that all suitable patients have a transplant assessment early in their disease course. novel molecular prognostic systems are likely to identify those best placed to benefit in future but this series currently supports allo-hsct survival and cure. (range, 105-4624) . dynamic international prognostic scoring system (dipss) score at the time of hct was intermediate-2 or high risk in 20 patients (95%), intermedate-1 in 1 patient. molecular evaluation was available in 14 out of 21: jak2 v617f mutation was detectable in 8 patients, mpl-w515k in 1 patient, carl in 1 patient. 4 patients were "triple negative" for driver mutations. cytogenetics information was available for 6 out of 21; among which 3 patients had complex karyotype, 1 trisomy 8 and 1 trisomy 9. 3 patients underwent splenectomy before hct. ruxolitinib was administered in 4 patients before hct. 10 (48%) patients received stem cells from an hla identical sibling, 9 (42%) from a matched unrelated donor and 2 (10%) from an haploidentical sibling. graft source was bone marrow in 7 patients (34%) and peripheral blood in 14 (66%). conditioning was myeloablative in 16 patients (76%), reduced intensity in 5 (24%). all patients engrafted. acute graft versus host disease was absent in 12 patients (57%), grade i-ii in 7 (34%), grade iii-iv in 2 (9%). in 18 evaluable patients chronic graft versus host disease was limited in 3 (17%), extensive in 4 (22%) and absent in 11 (61%). transplantrelated mortality at 180 days was 24%. main causes of death were: acute gvhd in 2 patients, chronic gvhd in 1, pancreatitis in 1, pulmonary aspergillosis in 1. relapse occurred in 7 patients and was the main cause of death in 4 of them. notably, 2 patients experienced late relapse after 6.6 and 17.6 years after hct. both of them are living while receiving ruxolitinib therapy. after a median follow up of 581 days (range, 38-11660), 10 out of 21 patients are alive. 7 of them (33%) are disease-free and 3 are living. the kaplan-meyer overall survival and disease-free survival at 10 years was 40% and 35%, respectively. conclusions: our experience confirms that hct is a valid option to achieve cure in one third of mf patients. two patients experienced very late (> 5 years) recurrence of mf. the rarity of this condition limits the amount of data and cases available for evaluation and study. life-long follow-up of all mf transplanted patients is warranted to better understand this rare event. disclosure: nothing to declare methods: а 37-years old female was diagnosed with jak2v617f-positive pmf, 46xx, ipss low risk, dipssplus intermediate -2 risk, subacute budd-chiari syndrome and portal vein thrombosis four years before allohsct. to reduce the splenomegaly and constitutional symptoms we performed pre-transplant ruxolitinib therapy 30 mg daily. after three months of therapy the patient achieved clinical improvement (eln criteria). contrast-enhanced computer tomography and magnetic resonance imaging showed enlarged intrahepatic collateral vessels and signs of portal vein thrombosis with cavernous transformation and multiple dilated collateral veins. gastroscopy documented enlarged esophageal veins. allogeneic stem cell transplantation was performed from 10/10 -hla matched unrelated donor with peripheral stem cells (6.1 x 10 9 сd34+ cells/kg). conditioning regimen consisted of fludarabine (180 mg/ m 2 ), busulfan (10 mg/kg p.o.). post-transplant cyclophosphamide was administered at 100 mg/kg at day +3, +4, and ruxolitinib 15 mg was used from d+5 till d+100 as graft versus host disease prophylaxis. results: starting d+1 the patient experienced eight episodes of ebv some of them with severe blood loss. to treat the bleeding episodes blackmore tube was placed six times with temporary effect. to place blackmore tube the patient was two times intubated and required mechanical ventilation. at d+18 leukocyte and neutrophil engraftment, full donor chimerism and molecular remission were achieved. platelet engraftment was documented only at d +42 and poor graft function was present due to cytomegalovirus reactivation (d+41) and parvovirus b19 reactivation (d+62). evb was stopped at d+95 only after two esophageal veins ligations, and two procedures of gastric veins sclerotherapy. soon (d+111) the patient achieved complete platelet recovery (more than 50x10 9 /l) and became red blood cells transfusion independent. at day + 180 complete remission was confirmed by splenomegaly resolution, regression of bone marrow fibrosis, full donor chimerism, jak2v617f-negative molecular status. cbc showed hb 130 g/l, platelets 73x10 9 /l, leucocytes 3,8x10 9 /l. ultrasound examination after transplant documented portal vein thrombosis recanalization. at day + 180 she developed mild (nih) chronic graft versus host disease with eyes and mouth involvement, which was managed with topical steroids. at d+958 after transplant the patient is alive in complete remission and has no recurrent bleedings. conclusions: splanchnic vein thrombosis can significantly complicate the course of allohsct in pmf. easy access to surgical, intensive care unit and endoscopic teams is required to make allohsct more feasible in this group of patients. disclosure all patients received treosulfan-based mac regimens, treosulfan(total dose, 36-42gms/m2) was given in combination with different conditioning drugs. the most commonly used regimen was treosulfan, fludarabine (150mgs/m2) and thiotepa(10mgs/kg) referred to as ftt that was used in 59%(n=55). serotherapy was given in 93% of patients(n=87), as either alemtuzumab or antithymocyte globulin in 82%(n=77) and 11%(n=10), respectively. post-transplant graft-versus-host disease (gvhd) prophylaxis was given in all patients, based mostly on ciclosporin. 46 patients(49%) received the transplant from identicalrelated donors, 46 patients(49%) received the transplant from matched-unrelated donors, and two patients(2%) had haploidentical transplants. 90% of the patients(n=85) were fully hla-matched. all stem cell sources were used as bone marrow in 59%(n=55), peripheral blood stem cells in 33%(n=31), and umbilical cord blood in 8%(n=8). this treosulfan-based conditioning was given as the 1 st transplant in 92%(n=85), and as the 2 nd transplant after the failure of a first procedure in 8%(n=9). two patients received treosulfan-based conditioned transplant twice. results: neutrophil engraftment and platelet engraftment occurred at a median of 13 days and 18 days respectively. chimerism was full donor in 55%(n=52), high donor in 18%(n=17), and mixed donor in 9%(n=8). gvhd developed in 43% of patients(n=40), with acute gvhd grade i/ii and grade iii/iv developed in 29%(n=27) and 2%(n=2), respectively. chronic gvhd grade i/ii and grade iii/iv developed in 13%(n=12) and 2%(n=2), respectively. all chronic gvhd were mild, limited, non-extensive, and resolved completely. none of our patients had persistent gvhd necessitating long-term systemic immunosuppression. mild vod occurred in 13%(n=12), and severe vod occurred in 2%(n=2). one of them died but was believed to be related to the underlying disease (wolman syndrome). viral reactivation occurred in 55% of patients(n=52), with cmv, ebv, and adenovirus reactivation was found in 34%, 21%, and 14%, respectively. five patients had invasive adenoviraemia that contributed to death in two of them. primary graft failure happened in two patients(2%) due to adenoviraemia. seven patients(7%) had secondary graft failure with autologous reconstitution. graft failure was significantly lower (p0.045) in the ftt group than other conditioning groups. at a median follow-up of 35 months (range, two-174 months), eleven patients(11.9%) died, with overall survival of 88.1%, and event-free survival of 80.9%. five patients died due to complications related to their original disease, while six patients died due to transplant-related causes (transplant-related mortality 6.5%). immune reconstitution in alive patients was achieved at a median of eight months. this time was significantly longer (p0.034) in ftt group. conclusions: this study demonstrates that treosulfan is a safe and effective conditioning drug that can achieve engraftment, with low rates of graft failure, transplantrelated mortality and morbidity, even if it is used twice in the same patient. disclosure: nothing to declare background: high-dose chemotherapy (hdc) followed by autologous stem cell transplantation (asct) is the treatment of choice for the patients with relapsed or high risk nhl. although the high-dose conditioning regimens commonly used in patients with non-hodgkin lymphoma (nhl) are beam (bcnu, etoposide, cytarabine, and melphalan), beac (bcnu, etoposide, cytarabine, and cyclophosphamide), survival of patients with nhl received above high-dose chemotherapy followed by asct was still unsatisfactory. methods: we prospectively evaluated the efficacy and toxicity of busulfan, etoposide, cytarabine and melphalan (bueam) including iv busulfan instead of bcnu of standard beam as a conditioning for asct in patients with nhl. the high-dose chemotherapy consisted of bu (3.2 mg/kg i.v. q.d. from day -6 to day -5), e (200 mg/m 2 i.v. b.i.d. on day -4 and day -3) a (1 g/m 2 i.v. q.d. on day -4 and day -3) and m (140 mg/m 2 i.v. q.d. on day -2) at 7 centers in korea. results: two hundred five patients were enrolled onto the study. main subgroup was diffuse large b cell lymphoma (n=104, 50.7%), t cell lymphomas (n=59, 29.8%), and nk/t cell lymphoma (n=22, 10.7%). upfront asct was performed in 160 patients (78.0%), and salvage asct in 45 patients (22.0%). the disease status of the patients before hdt/asct consisted of 133 patients (64.8%) with complete response and 72 patients (35.2%) with partial response. treatment related toxicities included nausea in 149 patients (72.7%), diarrhea in 127 patients (62.0%), anorexia in 107 patients (52.2%) and stomatitis in 97 patients (47.3%), which were grade i or ii in the majority of cases. the common grade iii toxicities were stomatitis (6.9%), diarrhea (5.9%), and anorexia (5.4%). there were no vod, and transplant-related mortality occurred in 4 patients (1.95 %), due to infection. one hundred fifty three patients (74.6%) achieved a complete response and 13 patients (6.3%) after asct, while 28 patients (13.7%) showed progressive disease. at a median follow-up duration of 38.6 months, the estimated 3-year overall survival and progression free survival for all patients was 74.5% and 56.6%, respectively. conclusions: the conditioning regimen of bueam for asct was well tolerated and seemed to be effective in patients with relapsed or high risk nhl. disclosure: none of declare background: allogeneic hematopoietic cell transplantation (hct) is potentially curative for high risk acute myeloid leukemia (aml) and myelodysplastic syndrome (mds), however both gvhd and disease relapse remain major challenges. we recently introduced a combination of posttransplant cyclophosphamide (ptcy) and atg (4.5 mg/kg) as graft-versus-host disease (gvhd) prophylaxis. the purpose of our study was to compare outcomes between ptcy/ atg and other gvhd prophylaxis regimens for high risk aml and mds. methods: we retrospectively investigated outcomes of 159 patients that underwent allogeneic hct between january 2014 and july 2017 for high risk aml (n=120, 75%) and mds (n=39, 25%). gvhd prophylaxis regimens were compared for overall survival (os), cumulative incidence of relapse (cir) and non-relapse mortality (nrm) in univariate and multivariable analysis. high risk aml was defined as secondary aml, therapy related aml, high risk cytogenetics (eln criteria) in cr1, good/ intermediate cytogenetic risk aml in cr2 and primary induction failure; high risk mds was defined as high/very high risk wpss score. results: median age of patients was 56 years (range 22-73 years). donors were matched related in 52 (33%) patients, matched unrelated in 89 (56%) patients and haploidentical in 18 (11%) patients. graft source was peripheral blood stem cells in 158 patients (99%). myeloablative conditioning was used in 54 patients (34%), reduced intensity regimens in 105 (66%) patients. ptcy combined with atg was used in 69 (43%) patients, other gvhd prophylaxis regimens were used in 90 (57%) patients. both donor and recipient were cmv negative in 18 (11%) patients. median follow-up of survivors was 29 months (range 14-56 months). univariate analysis demonstrated os of the entire cohort at 2 years was 49% (95%ci 41-57%), cir at 2 years was 22% (95%ci 16-29%) and nrm at 2 years was 32% (95%ci 25-39%). concerning gvhd prophylaxis regimen, 2-year os for ptcy/atg versus others was 46% (95%ci 33-58%) versus 51% (95%ci 40-61%) (p=0.87, figure) , 2-year cir for ptcy/atg versus other was 31% (95%ci 20-43%) versus 16% (95%ci 9-24%) (p=0.02) and 2-year nrm for ptcy versus other was 28% (95%ci 18-39%) versus 34% (95%ci 25-44%) (p=0.35). grade ii-iv acute gvhd was seen in 23% of ptcy/atg patients versus 59% using other regimens (p< 0.0001). chronic gvhd was observed in 20% of ptcy/atg patients versus 42% using other regimens (p=0.004). multivariable analysis for os confirmed that the gvhd prophylaxis regimen has no influence (p=0.19), while the predominant predictor of survival was age at hct (hr 1.03, 95%ci 1.01-1.05, p=0.01). for cir, the ptcy/atg combination had no influence compared to other gvhd prophylaxis regimens (p=0.6), while ric conditioning was the predominant predictor of relapse (hr 3.05 for ric, 95% p=0.01) . for nrm, the atg with ptcy combination demonstrated no significant difference (p=0.12), while age at hct was the predominant predictor (hr=1.04, 95%ci 1.01-1.07, p=0.02). conclusions: the ptcy/atg combination for gvhd prophylaxis has demonstrated on multivariable analysis similar os, cir and nrm with other previously used regimens at our center. a decrease in atg dose may potentially decrease the relapse rate while retaining the advantage of decreased gvhd. [ background: the combination of fludarabine with myeloablative doses of busulfan (fb4) represents a standard of care conditioning regimen before allogeneic transplantation in patients with myeloid malignancies (giralt, s.: the lancet oncology 2015). fb4 has potent antileukemic activity and is associated with low transplantrelated mortality and acute gvhd. however, early after transplantation (days 30-90), a proportion of patients may not convert to a full donor haemopoietic chimerism, particularly if anti-t lymphocyte globulin (atg) is used as gvhd prophylaxis (rambaldi a, et al.: the lancet oncology 2015) methods: we retrospective analyzed 104 patients who underwent an allogeneic stem cell transplantation after fb4 conditioning regimen at our hospital, from november 2007 to august 2018. the median age was 51 years (range 22-67) and diagnoses were aml 76%, mds 18% cml 5% mfi 1%). the disease status at transplantation was: cr1 in 59%, cr2 in 5% and active disease in 36% of patients. the stem cell source was represented by pbsc in more than 95% of cases and anti-t lymphocyte globulin (atg) was part of the conditioning regimen in more than 95% of cases at a dose of 5 mg/kg. the donor was a hla identical sibling (26%), a matched unrelated (65%) or mismatched (one allele or one antigen mismatched) unrelated, 9%. hematopoietic chimerism was molecularly evaluated by variable number of tandem repeats (vntr) on bone marrow (bm) mononuclear cells or peripheral blood (pb) t lymphocytes, purified by immunomagnetic positive selection (miltenyi, biotec). the analysis was performed at day 30, 60, 90, 180 and 360 after transplantation results: after 30, 60 and 90 days from transplantation, the proportion of patients with a full bm chimerism was 94%, 87% and 83%, respectively. at the same time points, the pb t cell chimerism was 47%, 65% and 69%. before day 100, 10 patients required the infusion of dli to treat a pending or overt hematologic relapse and 12 patients to convert the lymphoid chimerism from mixed (median 44%, range 0-76), to complete (successfully in 7 cases). after day 100, 13 additional patients required dli to treat disease relapse or progression and 8 patients to improve the chimeric status or the immune reconstitution. at 5 years, the overall survival is 64%, with a relapse and non-relapse mortality of 21 % and 11%, respectively ( figure 1 ). by uni and multivariable analysis, aml diagnosis and a mixed bm chimerism before day 100 were associated with relapse and overall survival while age > 50 was the only factor significantly associated with nrm. a mixed pb t-lymphoid chimerism before day 100 does not adversely impact on non-relapse mortality, cumulative incidence of relapse, leukemia-free and overall survival. conclusions: after fb4 and atg, a progressive increase of pb lymphoid donor chimerism develops gradually after transplantation, in most of cases without the need of dli. early mixed lymphoid chimerism does not compromise the main long-term clinical outcomes and may at least partially explain the low non-relapse mortality. an incomplete bm chimerism within the first 3 months strongly correlates with early disease progression or relapse. background: busulfan (bu) is widely used as a component of myeloablative conditioning regimen before hematopoietic stem cell transplantation (hsct) in children. bu has a narrow cumulative exposure window. the relation of bu exposure with toxicity is well established, but the link between the exposure and efs is not clear due to conflicting reports especially in pediatric patients. obtaining the ratio of bu to its metabolite i.e. metabolic ratio (mr) may serve as an indicator of bu gsh conjugating capacity of an individual, thus cumulative exposure of bu for a particular day that could be used along with auc as a marker to predict efs. the present investigation is aimed at evaluating the utility of bu mr to predict efs in children undergoing allogeneic hsct. methods: two different cohorts with children receiving bu in four times daily (qid, n=44) and once daily doses (qd, n=13) at st. justine's hospital, montreal were studied. bu and su levels were measured on day 3 of the conditioning regimen at the end of infusion (dose 9 in qid or dose 3 in qd dosing). efs was defined from the time of transplant until death, relapse, or rejection, whichever occurred first. a receiver-operator characteristic curve (roc) for bu mrs measured was plotted to show the trade-off in sensitivity vs. 1-specificity rates for efs, as the cut-off of the test was shifted from low to high. cutoff values were defined based on the youden´s j statistic (i.e. sensitivity+specificty-1). results: twenty-two males and 22 females aged from 0.1 to 19.9 years (mean±sd: 7.2 ± 5.7) from bu qid cohort had the mean mr of 5.9 (sd: 3.2). a cut off value of 4.9 in mr was chosen in roc analysis in this cohort, with better sensitivity (71 %) and specificity (70 %) for efs prediction (p=0.01, auc= 0.7 (95 % ci= 0.6-0.8). in qd cohort nine females, and four males aged between 0.4 and 15.8 years (6.7±5.1) had the mean mr of 29.3 (sd: 16.6). in roc analysis, a cut off value of 25.06 was chosen with better sensitivity (100 %) and specificity (100 %) for efs prediction (p=0.003; auc=1.0). conclusions background: treosulfan is an alkylating agent increasingly used prior to hematopoietic stem cell transplantation (hsct). the main objective of this study was to develop a population pharmacokinetic model of treosulfan in pediatric hsct recipients and to explore the effect of different covariates on treosulfan pharmacokinetics (pk). also, a limited sampling model (lsm) was developed. methods: in this multicentre study, 91 patients, receiving a dose of 10, 12 or 14 g/m 2 treosulfan a day, administered during 3 consecutive days, were enrolled. a population pharmacokinetic model was developed using nonlinear mixed effect modelling (nonmem version 7.3.0, using psn toolkit 4.7.0 and piraña version 2.9.7 as modelling environment). demographic factors, as well as laboratory parameters, were included as covariates. results: treosulfan pk was best described by a twocompartment model. a bodyweight-based allometric model improved the model more than a model incorporating body surface area (bsa). clearance (cl) and intercompartmental clearance parameters were 6.07 l/h/15.6kg (95%ci 5.46-6.68) and 2.15 l/h (95%ci 1.39-2.91). typical volumes of distribution of the central and peripheral compartments were 8.00 l/15.6kg (95%ci 6.88-9.12) and 2.05 l (95%ci 1.52-2.58). a model-based dosing table based on bodyweight is created to achieve a target exposure of 1540 mg*hr/l (table 1) , which was the median exposure of our population. estimated glomerular filtration rate (egfr) was shown to be the only parameter that significantly reduced interpatient variability in cl from 36.5% to 34.8%. a limited sampling model with 3 samples (taken at 1.5, 4 and 7 hours after start of infusion) accurately estimated pharmacokinetic parameters of treosulfan. conclusions: to the best of our knowledge, this is the largest cohort of pediatric patients treated with treosulfan used for a population pharmacokinetic study. we developed a two-compartment model with weight and egfr as covariates influencing treosulfan pk. recently we showed a relationship between treosulfan exposure and early toxicity. patients with an exposure >1650 mg*hr/l have an increased risk of developing grade 2 or higher mucositis and skin toxicity. another study in 87 pediatric patients with thalassemia major reported an association between treosulfan clearance (< 7.97 l/h/m 2 ) and poor overall survival. our model, together with the limited sampling strategy, can be used to adjust the dose, prior to or during treosulfan administration. ongoing studies conducted in different disease settings will determine if treosulfan exposure can influence patient outcome. subsequently, the optimal target exposure can then be established. background: autologous stem cell transplant (asct) is an effective treatment method for non-hodgkin lymphoma (nhl). until recently, carmustine, etoposide, cytarabine and melphalan (beam) was the most commonly used conditioning regimen. despite acceptable efficacy with beam, carmustine is associated with major pulmonary toxicity. for this reason, the aim of this study was to investigate the safety and efficacy of beb conditioning regimen for asct in nhl. methods: we conducted a prospective, multicenter, phase ii study for beb conditioning regimen for asct in nhl patients. a total of 33 patients were enrolled from 3 centers. they underwent asct with beb conditioning regimen (busulfan 3.2mg/kg for 3days, etoposide 400mg/ m 2 for 2days, bendamustine 200mg/m 2 for 2days) between 2016 and 2018. [[p111 image] 1. two year progression-free survival and overall survival.] results: the median age was 52 years (range 21-66) and 16 patients (48.5%) were men. the most common type was diffuse large b cell lymphoma (n=23, 69.7%) and more than half of patients (n=19, 57.6%) were classified as ipi score 3 or 4. eight patients (27.3%) had a history of relapse and 19 patients (57.6%) received more than 2 lines of chemotherapy before asct. most patients (n=27, 81.8%) were complete remission (cr) state at asct. a median number of 5.85x10 6 /kg cd34 cells were infused (range 2.0-18.6). all patients engrafted after a median time of 11 days (range 10-14). twelve patients (36.4%) experienced neutropenic fever and 16 patients (48.5%) had grade 3 toxicities during asct. however, no one had a documented infection, veno-occlusive disease, or treatment-related death. three months cr rate was 81.8%. during a median follow-up period of 10.2 month, 7 patients (21.2%) exhibited relapse or progression, while 1 patient (3.0%) died of the disease. the estimated 2-year pfs and os rate were 73.0% and 89.8%, respectively ( figure 1 ). conclusions: the beb conditioning regimens for asct is a feasible with tolerable toxicity in patients with nhl. disclosure: nothing to declare long-term report of total marrow or total lymphoid imrt in advanced leukemia, myeloma and lymphoma background: during the last three decades, total body irradiation (tbi) continues to play an important role in the conditioning regimens for patients undergoing stem-cell transplant (sct) for a wide variety of advanced hematological malignancies. however, tbi showed boundaries in dose limits for toxicity in allogenic and moreover in autologous stem cell transplantation. currently, the choice of conditioning regimen is based on the use of the least-toxic regimen to achieve the optimal therapeutic result. this report aims to assess the feasibility of a conditioning strategy based on high dose chemotherapy and whole-body radiotherapy focused on selective extensive tumor burden irradiation, both in allogeneic and autologous stem cell transplantation. methods: since december 2009, sixty-two patients (pts) have been irradiated by helical tomotherapy (ht) to extensive target before allogeneic or autologous transplantation. selected total marrow irradiation (tmi) schedules were planned to treat patients with high risk acute leukemia (all or aml) or multiple myeloma (mm) as a part of conditioning regimen. total lymphoid irradiation (tli) was planned for patients with refractory or relapsed (r/r) hodgkin (hd) or non-hodgkin lymphomas (nhl). results: tmi and tli allowed delivering therapeutic dose over extensive selected targets with wide reduction of toxicity to all the organs at risk (oars). the higher radiation doses rate to the oars is reduced from 30% to 70%. allogenic conditioning regimen was tli (4gy x 3fx) than fludarabine + endoxan for patients with hd (4 pts). tmi (4gy x 3fx) + fludarabine + melphalan for patients with mm (4 pts). tmi (4gy x 2 fx) + thiotepa + fludarabine + busulfan for advanced lam patients (4 pts). tmi as the boost (2-3gy) after conventional tbi was (12 gy in 6 bi-fractionated doses) by cyclophosphamide (18 pts). autologous preparation to sct consisted of tli (4gyx 3fx) followed by high-dose bendamustine and melphalan for patients older than 40 years and conventional feam (fotemustine, etoposide, cytarabine, and melphalan) for younger patients, in hd e nhl (20 pts). while tmi (4gy x 3 fx) plus melphalan was delivered for autologous sct in mm and lam (12 pts). no unexpected acute toxicity was found. in the allogenic setting, all the patients' engraftment was achieved in all patients. no acute graft versus host disease increasing was detected. within the autologous setting, only 33% developed grade 3/4 mucositis. none experienced grade 3/4 extra-hematological toxicity. outcomes of the specific disease will be reported. conclusions: the current report describes the clinical feasibility of using ht to deliver tmi or tli in the setting of autologous transplantation or during allogenic stem cell conditioning regimen, to allow all patients (old, fragile or with high tumor burden) to achieve an ablative regimen before sct. to our knowledge, this single institution experience describes data from one of the largest cohort of patients treated in europe since the development of this irradiation techniques. disclosure induction therapy in both groups of patients was based in polychemotherapy without the use of new drugs. case matching was performed according to age, clinical stage at diagnosis, and response to induction therapy. conditioning regimen consisted of iv bu at a dose of 3.2 mg/ kg once a day on days -5 to -3 followed by mel at a dose of -140 mg/m 2 on day -2 in the bumel group versus mel200 in the control group. maintenance therapy after transplant consisted of interferon and steroids in the majority of patients. results: the cut-off date for this update was june 30, 2018. after a median follow-up of 56 and 63 months in the bumel and mel200 groups respectively, 35 patients had relapsed in the bumel group and 82 patients in the control group. median pfs was 33 (95% ci, 25.4-48.3) months in the bumel and 24 (95% ci, 20.1-32.7) months in the mel200 group (p = 0.04) ( figure 1 ). in this update, 12 patients in the bumel group are in maintained response and 7 of them are in continuous cr (two with negative status for minimal residual disease) between 9 and 12 years after transplantation. ten-year os was not significantly different between both groups, being 41 (95% ci 30-58) months in the bumel and 29 (95% ci 18-47) months in the control group.transplant-related mortality was similar in both groups of patients (4% in the bumel and 2% in the mel200 group). regarding toxicity, bumel was associated with a higher incidence of mucositis and liver toxicity than the melphalan-only approach but no patient in our series developed sinusoidal occlusive syndrome and the hepatic toxicity observed was only grade i/ii. finally, no long-term side effects have been reported among bumel recipients. conclusions: this long-term follow-up analysis confirms that a therapeutic strategy including bumel as conditioning regimen beforeasct in patients with newly diagnosed mm is highly active and safe in these patients. [[p113 image] 1. figure 1 . progression free survival in the bumel (____) and control group (…… frequency of acute gvhd grade iii-iv [cc: 11%; ct: 17%; tt: 24%, p=0.057], and transplant-related mortality was higher in tt-carriers (cc:26%; ct:28%; tt:46%, p=0.02 cc&ct vs tt) . ta-tma, cmv infection/reactivation and cgvhd were also not different according to donor genotypes. fungal infections occurred more frequently as causes of death in carriers (cc: 9.7% vs. ct: 38.1% vs tt: 33.3%, p=0.022). conclusions: our results suggest that donor tgfb1 -1347c>t may exert an adverse influence on the outcome of myeloablative conditioning. our finding might be explained by the combination therapy of calcineurin and mtor inhibition in gvhd prophylaxis in myeloablative conditioning. disclosure: nothing to declare. treosulfan-based reduced intensity conditioning in hla-haploidentical transplantation using ptcy as gvhd prophylaxis in high-risk mds /aml of the elderly background: standard conditioning regimens prior to allogeneic hematopoietic stem cell transplantation (allo-hsct) are often associated with a considerable risk of severe adverse events, especially in elderly patients suffering from high-risk (hr) mds/aml. previous clinical studies have demonstrated feasibility of treosulfan-based reduced-intensity conditioning (ric) by stable engraftment, low non-relapse mortality (nrm), and favorable survival in elderly patients undergoing hla-matched related or unrelated allo-hsct (beelen et al, ash 2017 #0521). however, data for treosulfan-based conditioning in the t-cell-replete hlahaploidentical (haplo-hsct) setting in high-risk aml/mds patients are rare. here we report on the outcome of eleven patients treated with a treosulfan-based conditioning undergoing haplo-hsct using exclusively post-transplantation cyclophosphamide (ptcy) as gvhd prophylaxis. methods: eleven patients with high-risk (hr) aml (n=9)/mds (n=2) who underwent haplo-hsct using treosulfan for reduced intensity conditioning (ric) and ptcy as gvhd prophylaxis were retrospectively analyzed with respect to outcome and toxicity. all patients were >55 years old and transplanted between january 2016 and february 2018 at our institution. the majority of the patients (9/11) suffered from active disease at time of treatment initiation, only two patients presented in cr. all but one received sequential conditioning with cytoreductive chemotherapy using flamsa applied shortly prior to treosulfan-based ric (10g/m 2 over 3 days). a bone marrow graft was used in 9/11 patients. post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mmf. national cancer institute common terminology criteria for adverse events version 3.0 were used for nonhematologic toxicity assessment starting from sequential therapy initiation or conditioning until day +30. results: median age of the entire cohort was 63 years (range: 58-71). the hct-ci was ≥2 in eight pts (median hct-ci=2, range: 0-5). no graft rejection occurred. neutrophil and platelet engraftment were achieved in 100% and 91% of the patients at a median of 20 (16-23) and 26.5 (13-30) days, respectively. acute gvhd grade ii-iv occurred in 18% of the patients, exclusively involving the skin. no one developed severe (°iii-iv) acute gvhd. no patient died prior to haplo-hsct. severe nonhematologic regimen-related toxicities (°iii-iv) occurred in 2/11 patients, predominately affecting the gastrointestinal tract. no patient suffered from ≥two iii-iv°toxicities. all patients developed fever during treatment course, four with positive blood cultures. cmv reactivated in 6/7 patients at risk. no ebv reactivation or ptld occurred. six patients had clinical and radiological signs of pneumonia (probable invasive aspergillosis) without detection of aspergillus/antigen in the bronchoalveolar lavage. ci of nrm at day +180 was 0%. four patients relapsed within the first year after haplo-hsct, with two of them dying due to relapse. at last follow-up (dec 2018) 9/11 patients were alive. with a median follow-up of 5 months (2-31) estimated 1-year os and dfs were 80% and 59%, respectively. conclusions: treosulfan-based unmanipulated hlahaploidentical allo-grafting using ptcy as gvhd prophylaxis in hr mds and aml patients aged over 55 years is safe and well tolerated resulting in stable engraftment and a favorable toxicity profile. our preliminary data further show promising outcome with low nrm, no severe acute gvhd and favorable survival offering an attractive alternative in ric for haplo-hsct of the elderly. disclosure: nothing to declare comparison of outcomes of total body irradiation (tbi) vs non-tbi conditioning regimens in acute lymphoblastic leukemia for allogeneic transplantation background: in adult patients diagnosed acute lymphoblastic leukemia (all) long-term results are poor with intensive chemotherapy. allogeneic stem stem cell transplantation is the potential treatment that provides cure for these patients. myeloablative preparation regimens include total body irradiation (tbi̇)+ cyclophosphamide(cy) and busulfan + cyclophosphamide.in adult all patients wbi/cy widely used, but the toxicity rate is higher. the aim of this study is to compare the result and effect of the tbi/cy and busulfan/cy regimens in allogenic bone marrow transplantation in all patients. methods: between 1993 -2018 there were 137 all patients who underwent transplantation using myeloablative preparation regimen with or without addition tbi in the adult bone marrow transplantation units of medipol medical faculty, istanbul university istanbul medical faculty, sisli florence nightingale hospital, atakent acıbadem hospital adult bone marrow units . we analyzed overall survival (os), progression free survival (pfs), veno occlusive disease, acute and chronic graft versus disease development rates in these patients. results: demographic characteristics of patients summarized in table -1 there was no significant difference between groups in donor age, gender, stem cell source. it was observed that the relapse rate was not statistically significant in both group.there was no statistically significant difference between the patients who underwent myeloablative regimen and myeloablative regimen with tbi in relaps,death, os, pfs. (figure-1) [[p117 image] 1. figure 1 ] in terms of transplant complications there was also no respectable difference in development of vod and acute and chronic graft versus disease but vod was more common in the group that did not use tbi (p: 0.068) ( conclusions: although there are contradictory data in the literature, in our multicentre study, it was revealed that the addition of tbi in the myeloablative preparation regimen compared with myeloablative preparation regimen alone did not have a positive or negative effect on overall survival.we think that if we can prepare a good vod prophylaxis approches, we can give up tbi in future. disclosure (n=3) . for gvhd prophylaxis, cyclosporine a was given either alone (n=21), with mmf (n=13) or with methotrexate (n=1). the graft source was bone marrow (bm) in most cases (n=31), pbsc in seven cases, matched sibling cord +bm in two cases and one matched related cord. twentyfive of the donors were family donors and ten were unrelated. twenty-nine of the donors were 10/10 hla matched, six were 9/10 mismatched and one haploidentical. four patients had engraftment failure and required a second transplant, two of them were re-transplanted with cyclophosphamide and tbi, one with fludarabine, busulfan and campath, and one with no conditioning. thirty of the 35 patients are alive (86%). four patients died of transplant complications and one died of metastatic squamous cell carcinoma. eight survivors are mixed chimeras (81%-94% donor) and are all doing well, none of them developed any gvhd. nine patients developed acute gvhd, four of them with grade 3-4. seven of these patients later developed chronic gvhd, two of them have extensive disease. conclusions: our results show a high survival rate of 86%, with a low rate of engraftment failure and reasonable rates of gvhd. only one of our patients died of late effects of hsct for fa. mixed chimerism does not seem to present a problem. we conclude that reduced intensity fludarabine based conditioning regimens are a good treatment option for patients with fanconi anemia undergoing hsct. disclosure: nothing to declare total marrow irradiation + bendamustine as reducedtoxicity myeloablative conditioning prior to allohsct for younger patients with multiple myeloma background: the prognosis of patients with multiple myeloma (mm) has improved markedly over the last two decades. despite that, allohsct remains the only treatment option with curative potential. however, its use is limited due to high incidence of non-relapse mortality (nrm) after myeloablative conditioning while insufficient efficacy of reduced-intensity regimens. we developed a new protocol characterized by reduced toxicity while preserved myeloablative potential, based on the use of total marrow irradiation (tmi) in combination with bendamustine. the aim of this study was to evaluate its safety and efficacy in a singlecenter experience. methods: between years 2013-2018, mm patients below 55 years old were offered tandem auto-allohsct as part of first-line therapy. the decision was based on individual patient preferences after detailed description of potential risks. autohsct was preceded by melphalan 200 mg/m 2 iv. the conditioning prior to allohsct consisted of tmi performed using helical tomotherapy at the dose of 4 gy/d on days -3, -2, -1 (total 12 gy) and bendamustine 140-220 mg/m2/d iv. on days -5, -4 (total 280-440 mg/m2). the immunosuppressive therapy consisted of cyclosporine + methotrexate +/-atg. peripheral blood was used as a source of stem cells. results: the analysis included 18 patients (women -9, men -9). the median follow-up was 28 (4-68) months. the median age at allohsct was 44 (26 -53) years. the disease stage before allohsct was as follows: cr-6, vgpr-4, pr-6. patients were treated with hsct from either hlamatched siblings (n=7) or unrelated donors (n=11). the interval between autohsct and allohsct was 5 (4-23) months. all patients engrafted after allohsct with median time of neutrophil and platelet recovery of 14 and 12 days, respectively. one patient (6%) experienced grade 2 acute gvhd, while there were no cases of grade 3-4 acute gvhd. the incidence of mild, moderate and severe chronic gvhd was 17%, 0% and 6%, respectively. the rate of grade 3 non-hematological toxicities was 11%. one patient died of late bacterial infection. the incidence of trm was 5%. grade 4 adverse events were not reported. disease status 3 months after allohsct was: cr-10, vgpr-5, pr-3. the probability of os and pfs after 30 months was 94% (+/-6%) and 77% (+/-12%), respectively. the incidence of progression and trm was 17% and 6%, respectively. conclusions: allohsct using tmi 12gy + bendamustine conditioning protocol is characterized by good tolerance and low risk of gvhd. it may be used for younger patients with mm as part of tandem auto-allohsct strategy. encouraging results reported in this study should be confirmed in prospective clinical trials. disclosure: nothing to declare p120 comparison between two reduced intensity conditioning regimens in patients with a myeloid malignancy: a single center experience comparing fb2 with flumel background: hematopoietic stem cell transplantation (hsct) remains the only curative option for high-risk myeloid neoplasms. the optimal reduced-intensity conditioning (ric) is still debated. methods: a single-center retrospective analysis was conducted at our institution to compare two different ric regimens in adult patients transplanted for myeloid malignancy from 2001 to 2018. a total of 137 patients were analysed, 74 of them treated with busulfan-based (fludarabine 150 mg/m 2 , busulfan 6.4 mg/kg, fb2) and 63 with melphalan-based conditioning regimen (fludarabine 150 mg/m 2 ,melphalan 140 mg/m 2 , flumel). antithymocyte globulin (atg) was administered in all patients while no one received tbi. partial in vitro t-cell depletion was performed using alemtuzumab for low risk patients. results: the two groups were well balanced with a median age of 61 and 62 years in the fb2 and flumel group, respectively, and a median follow up of 46 months. the most frequent indication for transplant in both groups was aml (59.5 and 69.8% for fb2 group and flumel group, respectively) and the stem cell source was peripheral blood in 94.6 and 96.8% of patients. more patients in the first group had near to significant worst karnosfky status (< 90) at transplant compared to second (35. 1 vs 19%, p=0 .057) and more patients received a t-partial depleted graft (54.1 vs 33.3%, p= .028). the neutrophil engraftment was significantly shorter after flumel (15 vs 18 days, p < .01). the 3-year overall survival (os) and disease-free survival (dfs) were of 43.0 and 36.5%, respectively, after fb2 and 54.9 and 52.0% after flumel, respectively, and were not significantly different (p=.41 for os and .15 for dfs), with a karnofsky >= 90 being the only factor significantly associated in univariate analysis with better os and dfs (p=.02 for both). the cumulative incidence (ci) of grade 2 to 4 acute graft-versus-host disease (agvhd) was 16.2% after fb2 and 38.3% after flumel (p< .001) and was associated in multivariate analysis with both t depletion and ric type (p< .001 and .005, respectively). the ci of chronic gvhd at 3 years was 13.9% in fb2 and 22.1% in flumel group (p=.24) . the ci of non-relapse mortality at 3 years was 18.7% after fb2 and 29.6% after flumel (p=.11). the ci of relapse at 3 years was 44.8% for the first and 18.4% for the second group (p< .001) and was associated with conditioning regimen in multivariate analysis (p=.02). no difference in 3-years gvhd-free/ relapse-free survival (grfs) was observed between the two group (25.5% for fb2 and 37.6% for flumel, p=.48). conclusions: when comparing two ric regimens for myeloid neoplasms, we observed a higher incidence of agvhd after flumel whereas no statistical difference was noted for the cgvhd occurrence. while the toxicity appears to be higher after flumel, this result is counterbalanced by a higher proportion of relapse after fb2, accounting for no difference in os, dfs and grfs between the two groups. these findings could be partially explained by a larger proportion of patients receiving a partial t-depletion after fb2 ric, but a larger trial is needed to clarify this issue. disclosure: nothing to declare. once-daily vs 4-times daily intravenous busilvex in conditioning regimen before allogeneic stem cell transplantation for patients with myeloid malignancies: safety and efficacy background: busilvex (bu) is part of standard conditioning regimen before allogeneic stem cell transplantation (asct) for patients with myeloid malignancies and usually administered as an intravenous (iv) infusion 4-times daily. this study aimed to compare the saftey and efficacy of this schedule to a once-daily iv bu. we conducted a retrospective study in adult patients (≥18 years) with myeloid malignancies who received asct from hla-identical sibling donors between january 2011 and june 2018 following iv bu-based preparative regimens. graft-versus host disease (gvhd) prophylaxis consited of cyclosporine and short course of methotrexate. intravenous bu was administered 4-times daily (0.8 mg/kg every 6 hours x12 to 16 doses) or oncedaily in a 3-hour infusion (3.2 mg/kg x 3 to 4 days) since june 2015. results: ninty-nine patients were enrolled (54 men and 45 women). median age was 35 years (range, 18-50 y). the median time from diagnosis to asct was 5 months (range, 51days -7 years). diagnosis were acute myeloid leukemia (n=79, 80%), chronic myeloid leukemia (n=8, 8%), myelodysplasic syndrome (n=6, 6%), primitive myelofibrosis (n=4, 4%) and chronic myelomonocytic leukemia (n=2, 2%). thirty-seven (37.3%) patients had ebmt-score ≥2. sixty-five (65.6%) patients were transplanted in cr1, 5 (5%) beyond cr1 and 24 (24.2%) had active disease. conditioning regimens consisted of bu/cyclophosphamide in 86 patients (86.8%), bu/fludarabine in 13 patients (13.1%). four-times daily bu was given to 58 patients (58.5%, groupe1) and once-daily bu to 41 patients (41.5%, groupe 2). stem cell source were bm in 46 patients (46.5%) and pbsc in 53 patients (53.5%). globally, patients characteristics were well balanced between the two groups. the rates of severe complications were similar between the two groups with no statistically significant differences except oral mucositis (table1). non-relapse mortality (nrm) was comparable in the two groups (21% and 17% in groups 1 and 2, respectively, p=0.65). the relapse rate was 24% and 32%, respectively (p=0.4). after a median follow-up of 2 years (range, 5days -7years), the os was not significantly different between groups 1 and 2 : 64% vs 56% (p=0.09). however, the rfs was significantly better in the groupe 1 : 62% vs 56% (p=0.03). conclusions: once-daily iv bu regimen seems to be an efficient and safe alternative to the 4-times daily protocol. however, results should be interpreted with caution because the historical comparison and lack of bu pharmacokinetics studies. disclosure background: standard therapy of the most patients with juvenile myelomonocytic leukemia (jmml) is allogeneic hematopoietic stem cell transplantation (ahsct). the choice of optimal conditioning regimen for patients with jmml is crucial as well as long-term observation. we aimed to estimate the long-term follow-up and survival rates of patients with jmml after ahsct with the help of busulfan or treosulfan-based conditioning regimens. methods: thirty eight patients with jmml underwent ahsct in 2002-2018. we compared equal groups of patients received busulfan (n=19) and treosulfan-based (n=19) conditioning regimen. m:f=28:11. median of age at hsct was 2.5 (0.6-5). donor type: hla-related 10/10 -29% (n=11), hla-related 9/10 -2.6% (n=1), hlaunrelated 10/10 -39.4% (n=15), hla-unrelated 9/10 -15.8% (n=6), and haploidentical -13.2% (n=5). stem cell source: bm -55.3% (n=21), pbsc -26.3% (n=10), ucb -10,5% (n=4), and ucb+bm -7.9% (n=3) . disease status on hsct: cr -73.7% (n=28), refractory -26.3% (n=10). results: median follow-up 15.5 months (1-129 months) . the estimated 10-year overall survival (os) probability in patients received busulfan-based conditioning was 48,8 ±13,4% in comparison with 68,0±10,8% in patients with treosulfan-based regimen (р=0,458). event-free survival (efs) was 42,1±11,3% in group with busulfan-based regimen and 57,0±11,5% in patients with treosulfanbased conditioning (р=0,224). background: post-transplant relapse remains the leading cause of treatment failure in high risk (hr) acute myeloid leukemia (aml), myelodysplastic syndrome (mds), myeloproliferative neoplasia (mpns) receiving allogeneic hematopoietic cell transplantation (allo-hct), especially for patients with relapsed or refractory aml. recently, a sequential transplant approach, as developed by the munich group, comprising of intensive cytoreductive chemotherapy flamsa (fludarabine/amsacrine/cytarabine) to decrease leukemia cell burden shortly prior to conditioning regimen, has been successfully used for high-risk (hr) aml/mds with promising results. methods: we studied 48 patients (median age 53 years, range 26 -68) with hr aml (n=38), as defined by refractory, relapsed disease, secondary leukemia, or high/ very risk disease risk index risk, and hr mds (n=10) according to ipss-r, undergoing allo-hct using the sequential transplant approach in 2 institutions between january 2009 and october 2018. the sequential transplant approach combined a cytoreductive chemotherapy, which consisted of either flamsa (n=17), flag +/-ida (fludarabine/cytarabine/granulocyte colony stimulating factor /idarubicin) (n=23), or clo-arac (clofarabine/cytarabine) (n=8), followed by reduced (ric) (n=43) or myeloablative (mac) (n=5) conditioning regimen. all patients received peripheral blood stem cell from matched related donors (n=27) matched unrelated donors (n=14), or mismatched unrelated donors (n=7). post-grafting immunosuppression consisted of calcineurin inhibitor and mycophenolate mofetil in all patients. thymoglobulin was added for gvhd prophylaxis for unrelated donor transplant. results: the median time to neutrophil > 1000/μl was 10 days (range, 9-25) . with a median follow-up of 28.2 months (range, 1.4 to 103.1 months), the kaplan-meier estimate of leukemia-free (lfs) and overall survival (os) at 5 years were 45 % (95% ci, 8-30), 46% (95% ci, 8-30), respectively. patients receiving flag or clo-arac based sequential regimen showed a trend towards more favourable overall survival (os) as compared to patients given flamsa (5 year os: 53% vs 31%; p=0.236). at 2 years, the cumulative incidences of relapse and non-relapse mortality (nrm) were 46 % (95% ci, 31-60 %) and 15 % (95% ci, 7-21 %), respectively. in multivariate analysis, the type of sequential conditioning regimen did not show any significant impact on lfs, os, nrm or relapse. conclusions: sequential transplant conditioning with flamsa, flag or clo-arac followed by allo-hct is an effective strategy in overcoming the dismal prognosis of hr aml and mds, and enabling long-term disease free survival. more studies on effective strategies such as posttransplant maintenance therapy of prophylactic donor lymphocyte infusion, are needed to further eliminate the risk of relapse, without increasing risk of treatment related toxicity. disclosure: nothing to declare optimization of the blood sampling procedure for busulfan therapeutic drug monitoring (tdm) to optimize our sampling scheme (15 minutes, 1, 2, 3, 4, 6, 8 and 10 hours after the end of a 3-hour infusion), we reduced the number of blood samples collected, reducing nursing and laboratory staff time and increasing patient convenience. this study aims to show the performance of a simplified sampling protocol which includes the first 5 samples from the original protocol. methods: individual pk parameters were retrospectively estimated using 5 samples (simplified protocol) and were compared with those obtained after 8 samples (original protocol). individual pk parameter values for a one compartment model were estimated using a maximum likelihood estimation modelling algorithm (adapt 5.0) and the statistical analysis of the results was performed (statgraphics centurion xv). results based on the approved dosage recommendations, mean (sd) initial dose was 171.1(69.3) mg. after tdm, mean (sd) calculated dose at day 1 for the remaining days (to achieve the defined target cumulative auc) was 158,5 (72,6) mg obtained from the original protocol. according to the simplified protocol the result would be 159,8(73,7) mg. the median and the mean variation of the calculated dose were 0% and 1% (0-8%) between protocols. a strong relationship between the cl of the day 1-3 obtained from the original protocol and the simplified protocol is observed (r 2 =0.9985). this high correlation is also observed for patients with busulfan t 1/2 >3h (r 2 =0.9936), a population were the reduction of sampling could be more problematic. anova test for the log cl with the factors: patient, day of busulfan and type of sampling protocol was performed. sampling protocol was determined as non-statistically significant (p = 0.7248). conclusions: results suggest that both protocols are equivalent concerning to the busulfan cl estimation and calculated auc. variation between protocols regarding the calculated dose at day 1 for the remaining days to achieve the defined target cumulative auc is considered acceptable. we verified a strong relationship between busulfan cl obtained from both protocols and sampling protocol doesn't influence cl statistically. a reduced sampling collection of 5 determinations until 4 h after the end of the infusion is shown to be sufficient for the tdm of busulfan, so this was implemented in our centre in line with published data. disclosure: nothing to declare p125 impact of anti-thymocyte globulin doses in unrelated hematopoietic stem cell transplantation for patients with myeloid neoplasm background: anti-thymocyte globulin (atg) is widely used for the prophylaxis of graft-versus-host disease (gvhd) in hematopoietic stem cell transplantation (hsct). however, there is still controversy regarding the optimal dose of atg. therefore, we analyzed the impact of atg doses in unrelated hsct for patients with myeloid neoplasm. methods: this was a retrospective multi-center study that assessed the impact of atg doses on clinical outcomes in patients with acute myeloid leukemia (aml) or myelodysplastic syndrome (mds) undergoing an unrelated hsct. the patients who received peripheral blood stem cells (pbsc) transplantation after conditioning regimens containing i.v. busulfan (bu), fludarabine and rabbit atg between 2010 and 2017 were included in this study. results: a total of 96 patents, median age 45 years, with aml (n=74) or mds (n=22) were included in our analyses. 66 patients (69%) received a myeloablative regimen (i.v. bu>6.4 mg/kg). high-atg (atg 9 mg/kg), intermediate-atg (atg 4.5-5 mg/kg) and low-atg (atg 3 mg/kg) were given in 11, 49 and 36 patients, respectively. after a median follow-up of 23 months, the cumulative incidence of extensive chronic gvhd was 9.1% in the high-atg group, 13.8% in the intermediate-atg group and 29.7% in the low-atg group (p=0.31). conclusions: our study shows that the incidence of extensive chronic gvhd was similar regardless of the doses of atg after transplantation of pbsc from unrelated donor for patients with aml or mds. however, the rate of relapsefree survival and the rate of a composite end point chronic gvhd-free and relapse-free survival were significantly higher in the intermediate dose ( methods: we retrospectively retrieved data from the electronic medical records for consecutive patients aged 65 and older, who underwent an asct for lymphoma over the last 10 years at our institution. results: forty four patients ≥ 65 years old underwent asct between 1 aug 2008 and 31 aug 2018. twenty eight of them received a reduced-dose conditioning (median 25%, range 20%-33% dose reduction). the dose was reduced for 92% of patients ≥ 70 years old and for 53% of patients aged 65-69. the outcomes of the following three groups of patients were compared: a) age ≥ 65; without dose reduction, b) age 65-69; with dose reduction and c) age ≥ 70; with dose reduction (table 1). only one patient aged 70 received full-dose conditioning. there was no significant difference between the groups in the number of previous chemotherapy cycles (median 2, range 1-3). however, significantly more patients at the age of 65-69 were in complete remission (cr) pre-transplant in both full and reduced-dose conditioning groups (a and b). no significant intergroup differences were observed in the occurrence of complications (mucositis and infections), day 30 transplantrelated mortality (trm) or engraftment day. similarly, no significant differences were found either in the 1-year progression-free survival (pfs), which was 50%, 64% and 50%, or 1-year non-relapse mortality (nrm), which was 17%, 7% and 10%, respectively for groups a, b and c. the 1-year overall survival (os) tended to be higher in group b (85%), compared to groups a (66%) and c (70%). conclusions: beam/beac conditioning dose reduction was not found to adversely affect 1-year pfs and os rates. despite the fact that 2/3 of the patients in the age group ≥ 70 underwent asct in partial remission and had dose reduction, theier achieved trm, pfs and os rates were similar to those of patients aged 65-69. beam/beac conditioning at a 75%dose may be a suitable option for patients in their seventh decade requiring asct. this strategy should be further evaluated in prospective clinical trials. background: the transplant related mortality in autologous transplants for lymphoma and multiple myeloma, reported worldwide ranges from 0-5%. from 2004-2015, the trm at our center for these two diseases was approximately 20%. we introduced changes in mobilization schedule, conditioning regimens and drug dosages to determine whether these changes affected the transplant related mortality and overall survival. methods: from april 2004-december 2015, we used beam (bcnu: 300 mg/m 2 on day -6; etoposide 200 mg/ m 2 on days -5 to -2, cytarabine 200 mg/m 2 on days -5 to -2 and melphalan 140mg/m 2 on day -1 as conditioning chemotherapy for patients admitted in transplant unit for autologous transplants in hodgkin's and non-hodgkin's lymphoma. in patients with multiple myeloma high dose melphalan (200mg/m 2 ) was used. the mobilization protocol consisted of cyclophosphamide 1.5gm/m 2 followed by gcsf 5μgm/kg twice daily till stem cell collection was completed. from january 2016, we changed the beam protocol to bendaeam with dose modifications that included: bendamustine 150mg/m 2 on days -5 and -4, cytarabine 150mg/m 2 on days -5 to -2, etoposide 150mg/m 2 on days -5 to -2 and melphalan 100mg/m 2 on day -1. for multiple myeloma melphalan was reduced to 150mg/m 2 . we used only gcsf for mobilization of stem cells, which was continued till stem cell harvest was complete. response to treatment was evaluated by comparing trm and overall survival for two time periods: 2004-2015 and from 2016 till date. results: from april 2004 till december 2015, n=78 autologous transplants were performed. the male:female ratio was 2.4:1. fifty seven patients underwent transplant for lymphomas, n=16 for multiple myeloma and n=5 for other diagnosis. median age was 23±14.6 (2-64 years). the mean mnc was 4.7 × 10 8 ± 1.7/kg. engraftment was achieved in 80% of patients. the transplant related mortality was 19.5% and overall survival was 72% (follow up: 104 months). since january 2016 till march 2018 we have performed n=18 autologous transplants of which n=17 were males. fifteen transplants were performed for lymphomas (nhl:8, hd:7) and n=3 for multiple myeloma. median age was 24±15 (20 -64 years). the mean mononuclear cell count was 5.8 x 10 8 /kg and the mean cd34 count was 3.7 x 10 6 /kg. engraftment was achieved in all patients. the transplant related mortality was 0% and the overall survival was 83% (follow up 22 months). conclusions: we were able to reduce the autologous transplant related mortality to 0% by decreasing dosages of conditioning chemotherapy and changing the mobilization protocol. long follow-up is needed to determine late mortality and late relapse in comparison to standard chemotherapy dosages disclosure: nothing to declare risk and benefit of thiotepa based conditioning followed by autologous stem cell transplantation in high risk lymphomas 36 years (16-62) . stage (ann-arbor) at diagnosis of hd/dlbcl/pcnsl: stage ie n=0/ 0/10 (0/0/91%), stage ii n=7/2/0 (47/18/0%), stage iii n=2/ 0/1 (13/0/9%), stage iv n=6/9/0 (40/82/0%). median time from diagnosis to asct hd/dlbcl/pcnsl: 25/11/ 6 month (4-72). induction treatment in hd patients was abvd, in most dlbcl patients r-chop and in pcnsl patients high dose methotrexate and cytosin arabinoside. tumor status at asct hd/dlbcl/pcnsl: complete metabolic remission (cmr) n=4/1/11 (27/9/91%) and from pcnsl patient's n=8 (73%) were in first complete remission (cr1). type of stem cell graft was periferial blood stem cell in all case. conditioning: thiotepa (250mg/ m2 on days -9 to -7, busulphan 3,2mg/kg on days -6 to -4 and cyclophosphamide 60mg/kg on days -3 to -2 plus rituximab 500mg/m2 on day -10 in dlbcl and pcnsl. median follow up from asct 711 days . tumor stage at asct was defined with computer tomography with positron emission tomography (pet-ct). results: median time of engraftment was 10 days (9-14). thiotepa caused toxicoderma appeared at 9 (24%) patients. cytomegalovirus (cmv) reactivation was seen in 3 (8%) cases with low dna content (284,454,5500 copies/ml) and responded completely to oral valgancyclovir therapy. transplantation related mortality hd/dlbcl/pcnsl n= 2/3/1 (9/27/13%), in 4 cases bacterial sepsis and one systemic mycoses and one pulmonary fibrosis. incidence of long-lasting grade iii-iv thrombocytopenia and anaemia: n=15 (40,5%) and n=3 (8%), median time of duration from transplantation 71 days (31-720) and 35 days ( background: this study evaluated the efficacy and toxicity of intravenous busulfan and thiotepa as a conditioning regimen for autologous stem cell transplantation (asct) in patients with multiple myeloma (mm). methods: we retrospectively analyzed the data of 68 patients with mm who received the intravenous busulfan and thiotepa conditioning for asct between november 2016 and april 2018 in korea. results: the median time to transplant was 5.4 months, and 66 patients (97.1%) underwent asct within 12 months of the diagnosis. the overall response rate after asct was 95.6%, including 55.9% with complete response, 22.1% with very good partial response, and 17.6% with partial response. the most common severe non-hematologic toxicity (grade 3-4) was infection (44.1%). three patients (4.4%) developed venous-occlusive disease. one patient (1.5%) died due to severe pneumonia after asct. after a median follow-up of 13.0 months, the median progression-free survival (pfs) and overall survival (os) were not reached. conclusions: in conclusion, a conditioning regimen of intravenous busulfan and thiotepa was effective and tolerable. clinical trial registry: not applicable disclosure: the authors have declared no conflicts of interest. myeloablative haploidentical bone marrow transplantation with post-transplant cyclophosphamide in paediatric patients with haematological malignancies santanu sen 1 , sameer tulpule 1 background: haploidentical transplants have been shown to be safe and effective in treating haematological malignancies in the paediatric population. we have previously reported on our experience of using reduced intensity conditioning with post transplant cyclophosphamide in haploidentical patients. we herein report our experience of using a tbi based myeloablative conditioning to treat our first 8 patients with haematological malignancies. methods: 8 patients were enrolled in the study, 5 with relapsed acute lymphoblastic leukemia (all) and 3 with relapsed/resistant acute myeloid leukemia (aml). all aml patients had genetic markers of high risk disease and all all patients had very early relapses (either on therapy or within 6 months of stopping therapy). all patients were conditioned with an identical protocol using tbi-based myeloablative preparative regimen (fludarabine 30 mg/m 2 /d × 4 d and tbi 150 cgy bid on d −4 to −1 [total dose 1200 cgy]) followed by an infusion of unmanipulated peripheral blood stem cells from a haploidentical family donor. postgraft immunosuppression consisted of cyclophosphamide 50 mg/kg/day on days 3 and 4, mycophenolate mofetil through day 35, and tacrolimus through day 180. results: median time of neutrophil and platelet engraftment was 11 and 19 days, respectively. all patients achieved sustained complete donor chimerism by day +28. acute gvhd, grades ii-iv and iii-iv, was seen in 75% and 25%, respectively. disease progression occurred in 2 patients: 8 & 10 months after transplant and there was one death due to severe fungal infection. estimated twoyear survival and relapse were 75% and 24%, respectively. 2 patients had severe bk viremia and cmv reactivation occurred in 4 patients. all patients were successfully managed with appropriate supportive and antiviral therapy. conclusions: we report good outcome with a myeloablative conditioning in haploidentical transplants with excellent engraftment and hopefully a longer life expectancy. with small number of patients, it is difficult to state whether using a myeloablative conditioning would lead to better long term outcomes in this cohort of patients with very haematological malignancies, but we certainly showed that it is possible to achieve excellent early results. disclosure: nothing to declare fludarabine in combination with melphalan and atg can be the best conditioning for hematopoietic stem cell transplant of children with hemophagocytic lymphohistiocytosis methods: in this prospective study, we analyzed the outcome of two pediatric patients with hlh who had received hsct, using reduced-intensity conditioning (ric) regimen. they received the same ric regimen based on the use of fludarabine (30 mg/m 2 /day for 5 days) in combination with melphalan (70 mg/m 2 /day for 2 days) and horse antithymocyte globulin (atg 10 mg/kg/d for 4 days). cyclosporine and methotrexate were used as graft-vs.-host disease (gvhd) prophylaxis. results: a 2 months boy with primary hlh (fhl2) was transplanted from his mother and a 4 years girl with secondary hlh was transplanted from her brother. both of donors were hla match with their recipients. they were received 6 x 10 6 /kg and 10 x 10 6 /kg cd34 + cells from the harvested peripheral blood stem cells, respectively. they achieved full neutrophil and platelet recovery. the time to neutrophil recovery was 13 and 11 days, respectively. full chimerism was achieved for both of them. in addition, they was developed grade 3 and 2 of acute gvhd, respectively. gvhd was completely controlled with prednisolone. they are alive and in complete remission without any significant complications after 36 and 14 months, respectively. conclusions: it appears that fludarabine in combination with melphalan and atg may be the best conditioning regimen for hematopoietic stem cell transplant of children with hlh. due to a few number cases of this study, a study with sufficient sample size is required. disclosure background: hematopoietic cell transplant (hct) recipients often report depression and impaired quality of life (qol) before transplant. mixed evidence suggests depression may be a risk factor for greater mortality and worse qol. inconsistent findings may be due to the fact that previous studies have not evaluated antidepressant use. the aim of the study was to compare pre-transplant patientreported physical functioning and post-transplant overall survival (os) between four groups of hct recipients: 1) non-depressed/taking antidepressant (treated depression), 2) depressed/taking antidepressant (undertreated depression), 3) depressed/not taking an antidepressant (untreated depression), and 4) not depressed/not taking an antidepressant (control). it was hypothesized that physical functioning and os would be worse among patients with untreated and undertreated depression relative to those with treated depression and controls. methods: this retrospective case-control study included patients completing depression (phq-8) and quality of life (sf-12) questionnaires at pre-transplant. analyses were conducted separately for allogeneic and autologous recipients. results: participants (n=1,797) were 58% men, mean age 57 years (19-79), 39% allogeneic recipients. regarding depression and antidepressant use, 146 (21%) allogeneic patients were characterized as having treated depression, 47 (7%) as untreated depression, 49 (7%) as undertreated depression, and 461 (65%) as controls. hierarchical linear regression models indicated that after adjusting for significant univariate factors (performance status, disease status, and regimen intensity), allogeneic patients with treated depression (b=-2.58, 95% ci=-4.63, -0.54) reported better physical functioning than patients with undertreated depression (b=-6.06, 95% ci=-9.43, -2.70) and untreated depression ) but worse physical functioning than controls (p values <0.05). cox regression models indicated depression/antidepressant usage was not associated with os among allogeneic patients (p values>0.10).among autologous patients, 195 (17.82%) were characterized as having treated depression, 83 (7.59%) as untreated depression, 77 (7.04%) as undertreated depression, and 739 (67.55%) as controls. hierarchical linear regression models indicated that after controlling for significant univariate factors (gender, performance status, diagnosis, and disease status), autologous patients with treated depression (b=-2.97, 95% ci=-4.71, -1.23) reported better physical functioning than patients with undertreated depression (b=-8.63, 95% ci=-11.23, -6.03) and untreated depression (b=-8.62, 95% ci=11.15, -6.08), but worse physical functioning than controls (p values <0.05). cox regression models showed depression/antidepressant usage was associated with os (p values <0.05), with patients with treated depression demonstrating significantly worse os than other groups (p=0.05), but this association was no longer significant in multivariate analyses controlling for diagnosis and disease status (p=0.09). conclusions: patients with untreated or undertreated depression pre-transplant may benefit from depression screening and treatment to improve physical functioning. disclosure: hslj: consultant for redhill biopharma and janssen scientific affairs p133 eltrombopag (epag) induces a high percentage of responses in patients with post allo-hsct poor graft function (pgf) and no active gvhd lourdes aguirre 1 , aitziber lizardi 1 , pilar bachiller 1 , brigida esteban 1 , carmen gonzález 1 , nagore argoitia 1 , maría araiz 1 , aranzazu aguirre 1 , anunciación urquía 1 , carlos vallejo 1 background: persistent cytopenia is a life-threating complication after hsct. several causes can lead to this situation (viruses, gvhd, drugs, etc) . a specific entity is the one called "poor graft function (pgf)", which is diagnosed in pts with ≥2 cytopenias after day +30, in the presence of donor chimerism and the absence of gvhd or relapse. pgf is more frequent after alternative allo-hscts, such a haplo-identical, mismatched, or ucb. several therapeutic approaches for pgf, with poor results, have been tested. recently, epag has been shown to improve platelet counts in the post-allo-hsct setting. in this study, we analysed the efficacy of epag in pts with post-transplant persistent cytopenias. methods: the population analyzed includes all 175 pts who underwent allo-hsct from june 2015 through may 2018 in our unit. median age was 52 years (12-69). 102 were male (58.3%) and 73 female (41.7%). baseline diseases were: 69 aml, 39 lpd, 20 all, 18 mds, 15 mpd, 9 mm, and 5 bmf. donor was unrelated in 101 (54.3%) and was family in 74 (42.3%) (including 25 haplo-identical). conditioning was ric in 95 (54.3%) and intensive in 80 (45.7%). sc source was pb in 164 (93.7%) and bm in 11 (6.3%). median followup was 24 months (6-41). epag was initiated at some point during the first 6-month post-hsct period in 12 pts (6.9% of the series) due to thrombocytopenia (< 20000/mcl) plus, at least, one other cytopenia. patients characteristics shown in table 1. epag was started at 50 mg/day and escalated each 2 weeks to 75, 125 and 150 mg/day if platelet count was < 20000/mcl. global response was considered when, after epag, the patient needed no transfusions and reached the three of the following: platelets >50000/mcl, hgb >10 g/dl, and anc >1000/mcl. epag was tapered off in responders and discontinued if no response was reached after 16 weeks. results: at epag initiation, all the 12 pts had thrombocytopenia (< 20000/mcl), 10 had anemia (hgb < 10 g/dl), and 5 had neutropenia (anc < 1000/mcl). counts pre and post and response to epag are shown in table 2. among the 8 responders, all but one (who relapsed from thrombocytopenia and died from bleeding) were alive at analysis close (87.5%). among the 4 non-responders, three pts had gvhd-associated cytopenias, and finally died from infectious complications; the other patient relapsed from her aml, reached a new cr after treatment, and is alive and well 27 months afterwards. epag was tapered off and discontinued in 6/8 pts who responded; 2/8 responders are still on epag. epag was discontinued in the 4/4 pts who did not respond. rest of treatment details shown in table 2. conclusions: 1) epag worked striking well in subjects with pgf, an otherwise a life-threatening situation for patients. 2) epag induced impressive responses in platelets, but strong bilinear and trilinear responses were also seen. 3) epag did not improve gvhd-associated cytopenias. 4) to confirm these innovative and transcendent results, we have just initiated a multicenter prospective study on the role of epag for treatment of post-hsct pgf. 1232 * five out of the six urdt were mismatched ** the donor was a woman in the six cases: three sisters and three daughters background: hepatic vod/sos with multi-organ dysfunction (mod; typically, renal or pulmonary) may be associated with >80% mortality. defibrotide is approved for treating severe hepatic vod/sos post-hsct in patients aged >1 month in the eu, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the us. this analysis provides an overview of the safety results from 3 studies of patients with vod/sos, with or without mod, who received defibrotide 25 mg/kg/day. methods: safety data were pooled from patients with vod/sos post-hsct treated with defibrotide in a phase 3 trial (n=102) and a phase 2, randomized dose-finding trial (n=74 receiving 25 mg/kg/day). safety data for historical controls (hc) from the phase 3 study (n=32) also are provided. reported separately, due to differences in patient population and data monitoring protocol, are aes from the expanded-access program (t-ind) in patients with vod/ sos with and without mod (n=1000 post-hsct). vod/ sos was diagnosed by baltimore criteria/biopsy for the phase 2/3 studies; diagnosis by baltimore or modified seattle criteria was permitted in the t-ind. results: median patient age at hsct for the phase 2/ 3 studies was 24.0 years, 18.0 years for the hc, and 14.0 years for the t-ind. in the phase 2/3 studies defibrotide-treated group (n=176), 169 (96.0%) experienced aes; most common (>10%) were hypotension (36.9%), diarrhea (24.4%), and multi-organ failure (21.6%). treatment-related aes were at least possibly related to defibrotide (table) . any hemorrhage (an ae of special interest) occurred in 101 patients (57.4%); most commonly epistaxis (13.6%), gastrointestinal and pulmonary alveolar hemorrhage and hematuria (8.5% each), and conjunctival hemorrhage (6.3%). all 32 hc experienced an ae; most common (>25%) were hypotension (50.0%), tachycardia (43.8%), diarrhea (37.5%), nausea (31.3%), and pyrexia, agitation, and petechiae (28.1% each). any hemorrhage occurred in 24 patients (75.0%): most common (>10%) were petechiae (28.1%); hematuria, epistaxis, and pulmonary alveolar hemorrhage (15.6% each); and lip hemorrhage (12.5%). in the t-ind (n=1000), 385/512 patients with mod (75.2%) and 324/488 patients without mod (66.4%) had an ae; other than vod/sos and mod, most commonly (>10% in either subgroup) hypotension (15.2% and 8.4%, respectively). traes occurred in 210 patients (21.0%) ( table) . any treatment-emergent hemorrhage occurred in 166 patients with mod (32.4%) and 124 patients without mod (25.4%); most commonly (>5% in either subgroup) pulmonary hemorrhage (8.2% and 4.7%, respectively) and gastrointestinal hemorrhage (5.5% and 4.3%, respectively). conclusions: the incidence and type of aes were as expected in these critically ill patients. of the pooled patients, 96% had aes; 57.4% had a hemorrhage. all hcs had an ae, with 75.0% having a hemorrhage. in the t-ind, patients with mod had higher rates of aes. support: jazz pharmaceuticals event, n(%) phase 2/3 studies (n=176) disclosure: paul g. richardson has served on advisory committees and as a consultant, and has received research funding from jazz pharmaceuticals. angela r. smith and leslie lehmann have nothing to disclose. nancy a. kernan received grants from gentium during the conduct of the study, and her research was supported by the national cancer institute of the national institutes of health under award number p30 ca008748; the content is solely the responsibility of the author and does not necessarily represent the official views of the national institutes of health. she has a research grant from jazz pharmaceuticals. robert ryan and william tappe are employees of jazz pharmaceuticals and hold stock and/or stock options in jazz pharmaceuticals plc. stephan a. grupp has served on a steering committee and as a consultant to jazz pharmaceuticals. defibrotide for treatment of adults with hepatic vod/ sos with or without multiorgan failure after hematopoietic cell transplantation: results of a systematic review/meta-analysis background: although hematopoietic cell transplantation (hct), autologous or allogeneic, is potentially curable in various hematologic malignancies, the procedure is associated with serious and potentially life-threatening complications, among them veno-occlusive disease/sinusoidal obstructive syndrome (vod/sos) of the liver. several studies, prospective or retrospective, have reported outcomes of defibrotide, when used as prophylaxis or treatment, in a mixed population of adult and pediatric patients. in this systematic review/meta-analysis, we analyze outcomes of defibrotide when specifically used for treatment of adult patients with hepatic vod/sos with or without multiorgan failure. methods: a comprehensive search of 3 large databases (medline/pubmed, cochrane and embase) on november 2, 2018 identified 642 publications. analysis was restricted only to adult patients (defined as median age older than 16 years) who received defibrotide for treatment of vod/sos and were reported in prospective or retrospective (which included ≥ 5 patients) studies published in full manuscript form. there were no limitations based on language. data were extracted in relation to benefits [complete remission (cr) rate and overall survival (os)] and harms (hemorrhage, any site or organ-specific). a total of 15 studies (prospective=6; retrospective=9) with 1437 patients met inclusion criteria. results: the median year of publication of prospective studies was 2013 (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) and for retrospective ones 2016 (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) . the prescribed starting dose of defibrotide varied among studies ranging from 6.25 mg/kg/day to 80 mg/kg/day, mostly for a 21-day course. the pooled cr rate was 39% (95%ci=28-49%) for prospective and 54% (95%ci=39-69%) for retrospective studies. the pooled day +100 os rates were 43% (95%ci=37-48%) and 65% (95% ci=53-75%) for prospective and retrospective studies, respectively. the pooled rates of hemorrhage (any site) were 15% (95%ci=2-35%) for prospective and 21% (95% ci=4-43%) for retrospective studies. when analyzing organ-specific hemorrhage, 3 prospective studies (n=1091 patients) reported pooled rates of pulmonary alveolar (pa) hemorrhage of 2% (95%ci=1-3%) and of 5% (95%ci=3-7%) for gastrointestinal (gi) hemorrhage. only one retrospective study (n=14 patients) reported an incidence of pa hemorrhage of 7% (95%ci=0-34%) and a different study (n=14 patients) reported an incidence of gi hemorrhage of 14% (95%ci=2-43%). none of the 15 studies reported cerebral hemorrhage as a complication of defibrotide therapy. conclusions: this systematic review/meta-analysis confirms the efficacy of defibrotide for treatment of vod/sos with or without multiorgan failure, yielding cr rates of 39-54% and day +100 os rates of 43-65%. the purportedly higher pooled cr and os rates observed with retrospective (vs. prospective) studies are likely due to assignment-bias inherent to observational studies. moreover, although the pooled hemorrhage (any site) rates of 15-21% is considered proportionally significant, the pooled rates of pa and gi hemorrhage were ≤ 5%, in prospective studies. clinical trial registry: not applicable disclosure: m.a.k-d: consultancy for pharmacyclics m.m: received lectures honoraria and research support from jazz pharma efficacy and safety of defibrotide in the treatment of hepatic veno-occlusive disease/sinusoidal obstruction syndrome following hematopoietic stem cell transplantation: interim results from the defifrance study background: hepatic veno-occlusive disease/sinusoidal obstruction syndrome (vod/sos) is a potentially lifethreatening complication of conditioning for hematopoietic stem cell transplant (hsct) but may occur after nontransplant chemotherapy alone. vod/sos with multi-organ dysfunction (mod) may be associated with >80% mortality with supportive care alone. diagnosis of vod/sos was traditionally based on baltimore or modified seattle criteria; however, the ebmt recently published separate diagnostic criteria for adults and children. defibrotide is approved for treating severe hepatic vod/sos post-hsct in patients aged >1 month in the eu, and for hepatic vod/sos with renal or pulmonary dysfunction post-hsct in the usa. the goal of the defifrance study, requested by the french health authorities, is to collect real-world data on safety and efficacy in a broader patient population in france, including all indications. this is the first interim analysis of the largest current evaluation of defibrotide for the treatment of vod/sos in europe. methods: defifrance is an observational, multicenter, post-marketing study that includes any patient treated with defibrotide from hsct centers in france. this interim analysis is based on all patients treated with defibrotide, including those with severe and very severe post-hsct vod/sos. vod/sos was diagnosed using traditional criteria. day+100 survival, complete remission (cr; total serum bilirubin < 2 mg/dl and resolution of mod), and safety profile are reported. results: a total of 324 patients treated with defibrotide were included retrospectively and prospectively between july 2014 and october 2018 from 36 table] disclosure: mohamad mohty: has received honoraria and research funding from jazz pharmaceuticals, delphine lebon: nothing to disclose, ann berceanu: none, charlotte jubert: has received funding from jazz pharmaceuticals, ibrahim yakoub-agha: has received honoraria from jazz pharmaceuticals, stéphane girault: none, marie detrait: has received research funding from jazz pharmaceuticals, cécile pochon: none, fanny rialland: none, virginie gandemer: none, jean-hugues dalle: has received honoraria from jazz pharmaceuticals, régis peffault de latour: has received research grant / honoraria / board from pfizer, novartis, alexion; research grant amgen; and honoraria from jazz pharmaceuticals, david michonneau: has received honoraria from jazz pharmaceuticals, myriam labopin: has received honoraria from jazz pharmaceuticals, floriane delaval: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc, gerard michel: none, anne sirvent: none, laurence clement: none anne-lise menard: none, anne huynh: has received honoraria from jazz pharmaceuticals, virginie bouvatier: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc, raj hanvesakul: employee of jazz pharmaceuticals and holds stock and/ or stock options in jazz pharmaceuticals plc, zakaria medeghri: employee of jazz pharmaceuticals and holds stock and/or stock options in jazz pharmaceuticals plc p137 incidence and predictors of severe cardiotoxicity in patients with severe aplastic anemia after haploidentical hematopoietic stem cell transplantation zheng-li xu 1 , lan-ping xu 1 , yuan-yuan zhang 1 , yi-fei cheng 1 , xiao-dong mo 1 , feng-rong wang 1 , yu-hong chen 1 , wei han 1 , chen-hua yan 1 , yu-qian sun 1 , ting-ting han 1 , yu wang 1 , xiao-hui zhang 1 , xiao-jun huang 1 1 peking university institute of hematology, peking university people's hospital, beijing, china background: severe cardiotoxicity after hematopoietic stem cell transplantation (hsct) is a rare but fatal complication. the aim of this study was to evaluate the frequency of severe cardiac complications and to assess the ability of various factors to predict these complications in patients with aplastic anemia after haploidentical transplantation., this is the first study evaluating the values of both clinical and imaging factors in the prediction of severe cardiotoxicity among saa patients after haploidentical transplantation. methods: a retrospective study was conducted in 216 consecutive aplastic anemia patients who received haploidentical transplantation from 2006 to 2017. all patients received a unified regimen including busulfan, cyclophosphamide (ctx) and antithymocyte globulin at our single center. results: a total of 12 (5.6%) patients developed grade iii or iv cardiac toxicity. patients with cardiotoxicity had significantly poorer overall survival (os) than those without cardiotoxicity (12.5% vs. 89.6%, p< 0.001). our multivariable model identified four independent adverse predictors of severe cardiotoxicity, including pre-transplant ecog score (≥2), abnormal st-t wave on 12-lead electrocardiogram (ecg), hyperlipemia and recalculated ctx dose (≥1.8 g/m2/d). a predictive risk model was refined as low risk (0-1 factor), intermediate risk (2 factors) and high risk (3-4 factors) . the respective incidences of severe cardiotoxicity were 50.0%, 6.0%, and 1.3% in the high-, intermediate-and low-risk groups (p< 0.001). the corresponding os rates were 49.0%, 80.4%, and 90.3% in the three groups (p< 0.001) at the last follow-up. conclusions: patients with high risk scores had the poorest outcomes and should be monitored closely. a reduced intensity conditioning might be recommended for these patients. disclosure: there are no conflicts of interest to declare. background: allogeneic stem-cell transplantation (allo-sct) is associated with significant transplant-related mortality (trm). acute renal failure (arf) is a frequent complication and usually presents early after the procedure, compromising its feasibility. the aim of this study is to analyse the incidence of arf, its risk factors and its potential impact on trm after allo-sct. methods: 422 patients were included (244 males [58%]; median age 43 years, range 16-67) treated with allo-sct consecutively between january 2001 and april 2012 in a single institution. patient characteristics are detailed in table 1. median follow-up was 1.8 years (range, 1.0-2.7). renal function was evaluated using creatinine and data was collected pre-transplant (baseline) and at the point when arf was developed after allo-sct. arf was evaluated using akin criteria, being akin-1 an increase 1.5-to 1.9-fold from baseline, akin-2 an increase 2.0-to 2.0-fold and akin-3 an increase ≥3-fold. chronic renal disease was evaluated one year after the date of arf using kdigo criteria. results: cumulative incidence of arf at 1 year was 63% (akin-1, 25%; akin-2, 27%; akin-3, 15%). in the multivariate analysis, arf (akin-1/2) was associated with: non-use of antithymocyte globulin in conditioning chemotherapy, p=0.02 (hr 2.3, 0.2 to 0.9) and development of severe agvhd, p=0.04 (hr= 1.5, 1 to 2.3). in patients with arf akin-3, the most important variables in the multivariate analysis were: use of methotrexate (mtx) plus cyclosporine vs mycophenolate mofetil plus cyclosporine as gvhd prophylaxis, p=0.009 (hr=1.9, 1.2 to 3.1); myeloablative conditioning vs reduced intensity, p=0.03 (hr=1.7, 1 to 2.8) and use of total irradiation therapy in conditioning, p=0.02 (hr=1.7, 1.1 to 2.8). trm at 1 year increased significantly according to akin: akin-1, 25%; akin 2, 35%; akin 3, 51%; p=0,003; hr=11.2. overall survival at 3 years according to akin was: akin 1, 52%, akin 2, 45% and akin 3, 29%; p=0,004 (figure 1). the incidence of chronic renal disease at 1 year after allo-sct according to arf was: no arf (8%), akin-1 (11%), akin-2 (15%) and akin-3 (16%); p=0.006. conclusions: arf is a frequent complication during the first year after allo-sct and is associated with several factors. arf akin-3 was associated with more intensive strategies received during conditioning, meanwhile akin-1/2 were related to development of gvhd. there is an association of arf (akin-1, 2 or 3) with development of chronic renal disease. background: the introduction of cellular therapies such as car-t and modalities of gvhd-prophylaxis with posttransplant/cyclophosphamide (ptcy) that increase the number of admission days have boosted the pressure of available beds in the bm-units. in this sense, our centre started an at-home allogeneic stem cell transplantation (allo-sct) program to follow aplasia from the d+1 until independent ambulatory patient. to evaluate the feasibility and safety of allosct, we compared two groups: allohsct/athome (ah-group) vs. allohsct/in-patient (ip-group). methods: we included 78 patients receiving allosct (january 2014-november 2018) in a single centre: 39 patients, ah-group and 39, ip-group. all patients received conditioning at the hospital. gvhd-prophylaxis consisted in tacrolimus (tk) plus mycophenolate (mpm) or methotrexate, or ptcy (d+3, d+4) plus tk (d+5). all patients received prophylaxis with levofloxacin, fluconazole and acyclovir. besides that, ah-group patients received prophylaxis with ceftriaxone 1g/24h iv or ertapenem 1g/ 24h iv, and aspergillus-prophylaxis with inhaled liposomal amphotericin-b or posaconazole during neutropenia. patients of ah-group since d+1 or d+6 (in ptcyprophylaxis) received a nurse visit at-home once daily. the visits by the physician were performed at the hospital and only during complication events. first-line therapy of neutropenic fever was meropenem 1 g/8h in both groups, using a portable infusion pump in ah-group. in this group, the absence of focal infection or signs of severe sepsis allowed returning home after the initiation of antibiotics. the platelets support was performed at-home and the red blood support at hospital. results: the median (range) age (years) of the series was 54 . the median follow-up of the series has been not achieved. the source of the sct was peripheral blood in all cases. we didn't find statistical differences between two groups (ah vs ip) in terms of age, diagnosis, type of donor, intensity of conditioning, gvhd-prophylaxis, toxicity (mucositis, acute renal injury, neutropenia and thrombocytopenia), agvhd, aspergilosis and trm. interestingly, a significant reduction of neutropenic fever was observed resulting the lower use of meropenem in the ah-group than ip-group. the admission median days were similar in the both groups and it represented 21-23 days the reduction in the total economic cost of the ah-group. the whole analysis of the results are detailed in table: in-patient group, conclusions: in our experience, at home allosct, including ptcy-gvhd prophylaxis, is a feasible and safe procedure reflected in similar trm and aspergillosis incidence. at-home allo-sct is associated with a significant lower risk of neutropenic fever than in-patient group, as well as a very low readmission rate. disclosure: gonzalo gutiérrez-garcía: honoraria from gilead. grant from jazz pharmaceutical and janssen. laura rosiñol: honoraria from takeda, janssen, amgen and celgene. the others author do not have any disclosures to declare. background: renal complications in sickle cell disease (scd) include episodes of acute kidney injury (aki), progressive chronic kidney disease (ckd) and hyperfiltration, defined by abnormally high glomerular filtration rates (gfrs). hematopoietic stem cell transplant (hsct) from an hla identical sibling donor is a well-established curative treatment for scd, but traditional myeloablative conditioning (mac) regimens pose risks of kidney injury due to intensive use of chemotherapeutic agents, infectious risks, and use of calcineurin inhibitors (cnis). aki and subsequent fluid overload (fo) are common in pediatric hsct with reported aki incidence of 21%-50% (kyung-nam koh et. al., 2017). we report renal outcomes in pediatric patients with scd who received hsct following a non-myeloablative conditioning (nma) regimen without cni exposure. methods: retrospective chart review describing renal outcomes in pediatric patients (18 years of age or younger) with scd (hbss) who underwent nma hsct in alberta, canada from july 2013 to february 2018. the nma regimen is illustrated in figure 1 . reported renal outcomes: 1) measured gfr (dtpa) pre-hsct, 2) aki (kdigo definition) post-hsct by reviewing all serum creatinine levels from pre-hsct to one month post-hsct, 3) %fo calculated: (max post hsct weight -baseline weight)/ baseline weight x 100 for the two first weeks post-hsct, and 4) estimated gfr (egfr) using the pediatric schwartz formula at last follow-up post-hsct, ckd defined as egfr < 60 ml/min/1.73 m 2 , mildly reduced gfr: 60-90ml/min/1.73 m 2 , and hyperfiltration: gfr ≥ 150 ml/ min/1.73 m 2 . [[p140 image] 1. results: eighteen patients (33% male, 3-18 years old at transplant) were included. most common pre-morbid events: vaso-occlusive crisis (n=17), acute chest syndrome (n=8), splenic sequestration (n=6), and cholelithiasis (n=4). median follow-up time: 27 months (range: 7 -62 months). all patients engrafted successfully with no acute or chronic gvhd. baseline measured gfrs were all > 60 ml/min/1.73 m 2 (range: 79-227) with mildly reduced gfr and hyperfiltration seen in one (5.6%) and 12 (66.7%) patients respectively. at baseline (pre-hsct), the only aki event was one transplant related aki secondary to delayed hemolytic reaction after exchange transfusion in preparation for transplant. post-hsct, there were no aki events. additionally, no substantial %fo post-hsct was observed. average %fo week one post-hsct: +0.01% (range: -4.2% -+1.0%) and week two post-hsct: +0.04% (range: -4.24% -+1.5%). post-hsct egfr remained > 90 ml/min/1.73 m 2 at last follow-up in all patients. hyperfiltration was present in 5 (27.8%) of the patients. conclusions: this is the first study describing stable kidney function in children with scd after the present nma hsct regimen with alemtuzumab/300 cgy total body irradiation (tbi) with prolonged post-hsct sirolimus. no episodes of aki or significant fluid overload were observed during the first month post-hsct, and no patient developed ckd during follow-up. further prospective studies are needed to confirm our findings and to determine if stable renal function persists during longer-term followup. disclosure: nothing to declare. lung microbiota in patients with idiopathic pneumonia syndrome (ips) after hct background: idiopathic pneumonia syndrome (ips) is a non-infectious pulmonary complication after hematopoietic cell transplantation (hct) and the etiology remains unknown. recent studies have reported that various diseases are associated with changes of microbiota. the aim of this study was to evaluate the lung microbiota in hct recipients with ips and identify microorganisms potentially associated with ips. methods: frozen bronchoalveolar lavage (bal) samples from hct recipients with ips (n=18) and research bal samples from asymptomatic hct recipients as controls (n=12) were retrospectively analyzed. all samples were negative for common viruses by quantitative pcr. sequencing libraries were made with 1ng of input dna per sample (nextera xt, illumina). samples were pooled and sequenced by hiseq 2000 to obtain 100-bp paired end data. sequence data analysis and read classification were performed with sunbeam and the quality control and read classification were performed using komplexity and kraken, which classifies bacterial, archeal, and viral genomes. we used sequence data of bronchoscope prewashes from a separate cohort as controls for environmental sources (n=24). bray-curtiss dissimilarity among samples was calculated using the vegan r packages. permanova and a two-sided wilcoxon rank sum test were used to compare between the study groups. results: bal samples started at a median of 22x10 6 raw read pairs per sample and reduced to 21x10 3 reads assignable to microbial taxa following quality control. the bacterial phyla proteobacteria and firmicutes were most abundant followed by bacteroidetes and actinobacteria in both bal and bronchoscope prewash samples. separation of bal and prewash microbiota using bray-curtiss dissimilarity plots showed that bal samples were distinguished by sequences assigned to staphylococcus, acidovorax, and bradyrhizobium species, while prewash samples were distinguished mostly by pseudomonas and elizabethkingia species, consistent with environmental sources (figure) . within bal samples, staphylococcus species were the main drivers of separation between ips cases and the controls (p=0.002, permanova, figure) . consistent with this, a linear discriminant analysis to identify taxa best distinguishing cases and controls identified staphylococcus, especially s. epidermidis, in ips cases with lactobacillus and streptococcus species in controls. we then compared relative abundances of s. epidermidis between all study groups. ips case samples were significantly enriched in s. epidermidis compared to control (p< 0.001, two-sided wilcoxon rank sum test) and prewash samples (p< 0.001). viruses were classified by category as human pathogens, non-human pathogens, and bacteriophages. torque teno viruses (ttv) was the most commonly detected virus among viruses that replicate on human cells, and there was a trend towards higher abundance in ips case samples than controls. conclusions: lung microbial sequences in hct recipients predominantly consisted of proteobacteria and firmicutes, and had considerable overlap with environmental background. patients with ips had significantly more staphylococcus sequences detected than asymptomatic hct patients. these results suggest that patients with acute lung injury post-hct show distinct patterns of lung microbiota, although heterogeneity of sample collection and processing cannot be excluded and no singular organism was uniquely associated with ips. a prospective study is required to confirm these findings and define the clinical significance of differences in abundance patterns. disclosure: nothing to declare p142 abstract withdrawn. romiplostim for the treatment of thrombocytopenia after allogeneic stem cell transplantation background: thrombocytopenia is a common complication after allogeneic stem cell transplantation (allo-hct). with variable possible causes, such as drug side effects, infections, poor graft function, graft vs host disease (gvhd) and immune mediated. the purpose of this study was to evaluate the efficacy of romiplostim, a thrombopoietin receptor agonist, in patients with prolonged thrombocytopenia with no obvious cause after allogeneic transplantation. methods: retrospective analysis of allo-hct patients who received romiplostim at a single bmt unit between november 2015 and november 2018. romiplostim was given because of prolonged (>3 weeks) thrombocytopenia (< 60,000 μl) that couldn't be explained by obvious causes such as administration of drugs (antibiotics/antivirals), infection or gvhd. all patients were in complete remission and had complete chimerism. response to romiplostim treatment was considered transfusion independence or plt>80.000/μl. results: in total, 19 patients (median 45 years, 19-67) received romiplostim. patients (10 male, 9 females) had aml (10 pts), all (8), mds (2) or hodgkin (1), received a myeloblative (busiphex-based: 16, tbi-based:1) or ric (2) conditioning and were transplanted from a sibling (5), vud (11) or haploidentical (3) donor with pbsc (16) or bm (3) . all patients revealed primary neutrophil (median 14 days, range 10-19) and >20.000/μl platelet (13 days, 7-31) engraftment. romiplostim was started at median day +104 (range 58-419) with a median dose 5 μg/kg (1) (2) (3) (4) (5) . the median platelet count before commencement of treatment with romiplostim was 24.000/μl (range 12.000-57.000) and 10 them (59%) were transfusion-dependent. in total 14/17 (82%) patients responded to romiplostim treatment. eight out of the 10 (80%) transfusion dependent patients responded to the administration of romiplostim. six out of the 7 patients (86%) who were transfusion independent at romiplostin initiation (plt median 28.000/μl, range 19.000-56.000) responded. the median duration of treatment was 74 days (15-253) and the median follow up from the commencement of romiplostim was 177 days (15-1080). three out of 17 (18%) patients experienced relapse of thrombocytopenia after discontinuation of romiplostim and re-initiation of romiplostim was commenced in all of them, of which 2 responded and 1 didn't. the administration of romiplostim was done on an external basis and was well tolerated by the patients. two patients experienced gvhd during romiplostim treatment (both patients transplanted from 7/8 unrelated donor, 25 and 42 days after initiation treatment with romiplostim). 3/19 patients interrupted romiplostim due to disease relapse. 11/19 patients receiving romiplostim are alive in complete remission and 8 died (3 due to relapse, and 5 due to trm). conclusions: we present high response rates to romiplostim in patients with prolonged thrombocytopenia after allogeneic transplantation. in this retrospective study there were no side effects from the administration of romiplostim. however, the administration of romiplostim after allo-hct should be controlled in prospective trials. disclosure we report a single-center analysis of 29 adult patients (median age 22 years, range 18-57, m/f 13/16), receiving tpo agonists for isolated severe thrombocytopenia (n=7) and spgf (n=22) after allo-hsct. primary diagnoses were aml (10), all (6), mds (7), pmf (4), mds/mpn (4), saa (4), cml (1), nhl (1) . severe pgf was defined as cytopenia in ≥ 2 lineages (platelet < 20 × 10 9 /l, anc < 0.5 × 10 9 /l, hemoglobin < 70 g/l any time after sustained engraftment), full or stable mixed donor chimerism > 90 % and no signs of relapse. median dose of romiplostim was 5 (range, 3-5) mcg/kg weekly, eltrombopag -50 (range, 50-150) mg/day. overall response (or) included cr (platelet ≥ 100 × 10 9 /l, anc ≥ 1.5 × 10 9 /l, and hemoglobin ≥ 100 g/l) and pr (platelet > 20× 10 9 /l, anc ≥ 0,5 × 10 9 /l, hemoglobin > 70 g/l). results: median time from pgf diagnosis to treatment with tpo agonists was 14 days (0-119), median treatment duration was 3 weeks (1-43). tpo agonists were well tolerated with no cases of grade iii-iv toxicity. tpo agonists were combined with rituximab (n=4), rituximab and dli (n=3) and hsc boost (n=1) in 8 (28 %) patients. a total of 14 (48 %) patients met criteria of response (cr: n=4, 14 %; pr: n=10, 34 %). combination therapy showed no difference in or compared to tpo agonists alone. or was not depended on the tpo agonist used nor the time to therapy initiation. median increase in anc in responders was 3.4 × 10 9 /l (0.8-6.0), in platelet count -48×10 9 /l (21-205). a total of 15 patients died due to relapse (n=2), gvhd iii-iv grade (n=3) and infection (n=10). two-year os from the start of tpo agonist therapy was 44 % (95 % ci, 25-62) with a significant difference between responders and non-responders: 71 % (95 % ci, 33-90) vs. 18 % (95 % ci, 3-40) (p=0,002). conclusions: this study showed promising results of tpo agonists for management of spgf. further studies are warranted to specify optimal timing and dosing regimen, predictors of response. [[p144 image] 1. two-year os in responders and nonresponders to tpo agonist therapy] disclosure: there are conflicts of interest to disclose p145 cytomegalovirus reactivation kinetics and peak titers as novel predictors of survival and relapse after allogeneic cell transplantation for hematologic malignancies saskia leserer 1 , evren bayraktar 1 , nikolaos tsachakis-mück 1 , michael koldehoff 1 , lara kasperidus 1 , esteban arrieta-bolanos 1 , mirko trilling 1 , katharina fleischhauer 1 , dietrich w. beelen 1 , amin t. turki 1 1 university hospital essen, essen, germany, background: after allogeneic hematopoietic cell transplantation (hct), human cytomegalovirus (cmv) reactivation associates with non-relapse mortality (nrm) but also with reduced relapse in patients with leukemia, as shown by numerous studies that evaluated cmv reactivation as a qualitative yes/no parameter in the first months posttransplant. we hypothesized that longitudinal quantitative assessment of cmv reactivation kinetics and virus loads might improve patient-specific clinical outcome associations. methods: this retrospective study included 705 patients with hct for hematologic malignancies treated between 01/2012 and 12/2017 at university hospital essen, germany. cmv titers were monitored weekly by quantitative pcr (qpcr); cmv reactivation was defined by a cutoff of >500 genome copies per ml. patients were included for analysis, if at least 5 measurements were available during the first 200 days after hct. in total, 11,508 samples were analyzed. subgroup analyses were performed according to the time of cmv reactivation (before/after +30d) or the cmv viremia titer (>100,000, 20,000 -100,000 and 500 -20,000 copies/ml). results: cmv reactivation was detected in 350 (median age 58 years; range 17-76 years) out of 705 patients. baseline characteristics (age, gender, underlying disease, transplant) of patients without cmv reactivation were comparable. cmv reactivation kinetics followed a gaussian normal distribution with a median first reactivation at +33d and peak titers at +47d. all except 1 patient reactivated before 100d, 40 % before +30d. overall survival (os) of the cmv reactivation group as a whole did not significantly differ from the non-reactivation group (34 vs. 38 months). however, in subgroup analyses os was significantly reduced in patients with very early (< +30d) compared to later reactivation (17 vs. 59 months, p=0.040). moreover and importantly, os was significantly reduced in patients with cmv reactivation at high titers of >100,000 copies/ml compared to those with lower titers ((10 vs. 45 months) p< 0.0001).cox regression analyses confirmed significantly reduced os for patients with cmv reactivation >100,000 copies/ml and < day +30 as compared to the other cohorts (hr 2.03, 95%ci 1.45-2.86, p< 0.0001 and hr 1.36, 95% ci 1.01-1.83, p=0.041) respectively). the nrm was consistently higher (hr 2.59; 95%ci, 1.69-3.97, p< 0.0001) for patients with cmv copies >100,000/ml. the risk of hematologic relapse was exclusively reduced in patients with a peak cmv viremia between 20,000 and 100,000 copies/ml (hr 0.55, 95% ci 0.32-0.95; p=0.033) as compared to patients without cmv reactivation. for other levels of cmv reactivation this effect was not observed. conclusions: our data showed that cmv reactivations before +30d or with high titers of >100,000 copies/ml associated with significantly reduced os, while cmv reactivations at intermediate titers between 20,000 and 100,000 copies/ml had a positive impact on relapse incidence. these findings underline the complexity of cmv reactivations after hct outcome, and support longitudinal evaluation of cmv titers and individualized quantitative kinetics models for risk assessment after hct to distinguish the advantageous from the detrimental aspects of cmv reactivation. disclosure: att has received lecture fees from jazz pharmaceuticals and travel subsidies from neovii biotech outside the submitted work. the other authors declare no competing financial interests within the submitted work. association of serum ferritin levels before start of conditioning with mortality after allosct -a prospective, non-interventional study of the ebmt transplant complication working party background: elevated serum ferritin levels occur due to iron overload or during inflammation and macrophage activation. a correlation of high serum ferritin levels with increased mortality after allosct has been suggested by several retrospective analyses as well as by two smaller prospective studies. methods: this international multicentric study aimed to study the association of ferritin serum levels before start of conditioning with allosct outcome. patients with acute leukemia, lymphoma or mds receiving a matched sibling allosct for the first time were considered for inclusion, regardless of conditioning. data were prospectively collected between 8/2014 and 2/2018. a comparison of outcomes between patients with high and low ferritin level was performed using univariate analysis and multivariate analysis using cause-specific cox model. variables included in the multivariate analyses were age, sex mismatch, diagnosis, disease status, karnofsky score, number of cd34 cells given, intensity of conditioning, type of gvhd prophylaxis, atg use, time from diagnosis to transplant, year of transplant and cmv status. results: twenty centers from 10 european countries reported data on 385 allosct recipients. patient characteristics are given in table 1 . the ferritin cut off point was determined at 1500μg/l (median of measured ferritin levels). overall survival of allosct recipients with ferritin levels above cut off measured before start of conditioning was significantly shorter ( figure 1a , univariate hr=2.3 ci=1.4-3.6 p=0.00041; multivariate hr=2.5, ci=1.5-4.1, p=0.0005). progression-free survival was also shorter ( figure 1b , univariate hr=2.1 ci=1.4-3.2 p=0.00014; multivariate hr=2.4, ci=1.6-3.8, p< 0.0001). excess mortality in the high ferritin group was due to both higher relapse incidence (univariate hr=1.7 ci=1-2.8 p=0.03; multivariate hr=2.2, ci=1.2-3.8, p=0.007) and increased non-relapse mortality (univariate hr=3.1 ci=1.5-6.3 p=0.002; multivariate hr=3.1, ci=1.5-6.4, p=0.002). non-relapse mortality was driven by significantly higher infection-related mortality in the high ferritin group (univariate hr=3.9 ci=1.6-9.7 p = 0.003; multivariate hr = 3.9, ci = 1.6-9.7 p = 0.003). acute and chronic gvhd incidence or severity were not associated to serum ferritin levels. conclusions: ferritin levels before start of conditioning can serve as routine laboratory biomarker to predict mortality after allosct. disclosure: the authors declare no confict of interest related to this study p147 prediction of reduced lung function and acute gvhd by surfactant protein d in allogeneic stem cell transplantation transplantation (hsct) and may progress to bronchiolitis obliterans that has a high mortality rate. surfactant protein d (sp-d) is an innate defense molecule involved in immune regulation at the epithelial surfaces, particularly in the lungs, and elevated levels have been associated with exacerbation of chronic obstructive pulmonary disease (copd). the aim of this study was to investigate, whether sp-d plasma levels and variants in the gene encoding sp-d may predict the development of reduced lung function after allogenic hsct. methods: we performed a population-based, singlecenter study of children (aged 6-18 years) treated with allogeneic hsct. the study consisted of 1) a prospective study of serial plasma sp-d levels and rs721917 genotypes in 55 patients during the first 6 months after hsct, and 2) a retrospective study of rs721917 genotypes within the sp-d gene in 247 patients transplanted between 1990-2017. pulmonary function tests were performed regularly as part of the clinical monitoring. results: at the day of graft infusion (day 0) sp-d levels were reduced compared to levels before start of treatment with conditioning chemotherapy, defined as baseline (615 ng/ml (quartiles 441-1132) at day 0 vs 771 ng/ml (542-1348) at baseline, p< 0.01). from day +7 sp-d levels increased and remained increased during the whole study period (771 ng/ml (542-1348) at baseline vs 1287 ng/ml (713-2549) at 6 months, p< 0.01). acute gvhd (agvhd) occurred in 25 patients, of those 17 patients with grade 2-4. high sp-d levels at day +14 were associated with the development of agvhd (1402 ng/ml (1244-2023) vs 839 ng/ml (523-1630), p< 0.01) ( fig. 1 ). the c/c genotype was associated with generally low sp-d levels and low fev1/fvc at all time intervals compared to the other genotypes, significantly 24-36 months post-hsct (p=0.02). there was no overall correlation between sp-d levels and lung function, but stratifying for genotype, high baseline sp-d levels were predictive for reduced fev1/fvc at 8-24 months in cc and tt homozygous individuals. conclusions: patients with a genotype causing low capacity for sp-d production are at increased risk of developing pulmonary impairment after hsct. in addition, our data lend support to other studies indicating that spd production may increase during inflammatory pulmonary disease, acting as a reactive, protective mechanism. further research is warranted to define the role of sp-d levels and genotypes as a prognostic tool for lung function and agvhd. [[p147 image] 1. background: allogeneic hematopoietic cell transplantation (allohct) means a long period of restricted mobility and a range of therapy related side effects on muscle function. in this context patients demonstrated a huge decline of physical capacity and muscle mass in particular, accompanied with a decrease of quality of life (qol). resistance training could maintain muscle mass but is limited by patientsb lood values (platelet-count) and well-being. whole body vibration (wbv) was shown to maintain muscle mass during bed rest and has less impact on blood pressure than conventional resistance exercises. furthermore it was also shown to be feasible in patients during high dose chemotherapy. therefore the aim of our study was to examine the effects of wbv during allohct on patients physical and functional performance as well as qol. methods: 43 patients receiving allohct were randomly allocated to either a wbv exercise group (ig) or an active control group (cg) doing stretching and mobilization. both groups exercised during the whole time of hospitalization for 5 times per week and underwent pre-, post-and followup-assessment. physical capacity was determined by maximum oxygen consumption (vo 2peak ) and maximum power (p max ) during cardiorespiratory exercise test and by maximum strength of the knee extensors and flexors (ex max , flex max ) during isokinetic strength test. functional performance was assessed by jumping height during counter movement jump (cmj) and time of chair rising test (crt) as well as power output during both tests. qol was assessed by questionnaires of the eortc. results: during allohsct vo 2peak and p max decreased in both groups but till follow-up an increase is seen in the ig (p=0.035; p=0.011). at day +180/follow-up a vo 2peak group difference is seen (p=0.034). ex max (p=0.003) and flex max (p=0.044) were only reduced in the cg during hospitalization. jumping height and power output decreased in the cg during hospitalization (p=0.005, p=0.039) and a difference between groups were seen in changes of jumping height from pre-to follow-up-assessment (p=0.033): increase in the ig and decrease in the cg. the ig showed a decrease in time from baseline to follow-up (p=0.022) in the crt and an increase of power output (p=0.009). qol decreased only in the cg during hospitalization (p=0.015) while during follow-up qol increased in both groups (ig: p=0.013; cg: p=0.037). in the cg physical functioning decreased during intervention (p=0.001) whereas an increase was seen in the ig from pre-to follow-upassessment (p=0.035). body image was significant worse in the cg compared to the ig at hospital discharge (p=0.007) as well as at follow-up measurement (p=0.030) where it got worse over time (p=0.036). conclusions: wbv was shown to maintain maximum strength, jumping performance and qol during allohct. although cardiorespiratory fitness could not be maintained by wbv during hospitalization, it seems in the follow up period till day + 180 that recovery of the cardiorespiratory system is enhanced by wbv carried out during allohst. nevertheless reasons for this changes in recovery have to be analyzed in further studies as well as treatment effects of wbv compared to conventional resistance training. disclosure: supported by a grant of the faculty of medicine and comprehensive cancer center freiburg respiratory virus infection within 1 year after of allo-sct is the significant risk factor of obstructive ventilatory disturbance kosei kageyama 1 , michiho ebihara 1 , mitsuhiro yuasa 1 , daisuke kaji 1 , aya nishida 1 , shinsuke takagi 1 , hisashi yamamoto 1 , go yamamoto 1 , yuki asano-mori 1 , naoyuki uchida 1 , atsushi wake 1 , akiko yoneyama 1 , shigeyoshi makino 1 , shuichi taniguchi 1 1 toranomon hospital, hematology, tokyo, japan, background: obstructive ventilatory disturbance (ovd) is one of the major life-threading complication at the chronic phase of allogeneic stem cell transplantation (allo-sct). bronchiolitis obliterans has been the most established etiology as a part of chronic graft-versus-host disease and major cause of late non-relapse mortality of allo-sct. but other etiologies impact on respiratory function after allo-sct and risk factor of ovd have not been well understood. methods: to address these issues, we retrospectively reviewed the medical record of 747 consecutive patients who first allo-sct at toranomon hospital between 2009 and 2017. to detect ovd, forced expiratory volume in 1 second (fev1.0) showed less than 80% of predicted in spirometry test was defined as positive. in the recipients who showed fev1.0 less than 80% in pre-transplant test, more than 20% reduction of fev1.0 was regarded as positive. nasopharyngeal swab of those who had upper respiratory tract symptoms were tested for the presence of respiratory viral antigens (adv, piv, and rsv). patients with ecog performance status of 4, had active infection at transplant were excluded from this analysis. the cases of early death or relapse before 30 days post-transplant, and the cases of graft failure were also excluded. results: the median age was 55 years (range, 16-74). underlying diseases were aml in 403, mds/mpd in 73, cml in 26, all in 82, atl in 19, hl in 12, nhl in 104, and others in 28. five hundred twenty-nine (71%) were not in remission at the time of transplant. five hundred eightythree patients (78%) were conditioned with myeloablative regimens, whereas 164 patients received reduced-intensity regimens. donor sources consisted of related peripheral blood /bone marrow (bm) (n=85), unrelated bm (153) forty-six developed ovd on median of 198 (60-804) days post-transplant. cumulative incidence of ovd was 6.4% in total population. in 490 recipients those who could spirometry, overall survival at 5 years was 73.2% in patients who developed ovd and was comparable with those who did not develop it (64.3%, p=0.486). in univariate analysis, disease status (cr/aa or noncr), recipient age (age< 55 or ≥55), prior autologous stem cell transplantation (yes or no), intensity of conditioning regimen (mac or ric), tbi dose (< 8 gy or ≥8 gy), busulfan dose (< 9.6mg/kg or ≥9.6mg/kg), donor source (cord blood or non-cord) had no impact on the incidence of ovd. patients who developed respiratory virus infection showed significantly higher incidence of ovd compared to those who did not developed it (12.4% vs 5.3%, p< 0.01). in multivariate analysis, respiratory virus infection was the only significant risk factor for the development of ovd (hr=2.43, 95% ci 1.30-4.57, p< 0.01). conclusions: respiratory virus infection within 1 year after allo-sct is the significant risk factor of ovd. disclosure: nothing to declare. background: metabolic syndrome (mets) is related to increased risk of cardiovascular disease and type-2 diabetes (dm-2) and usually seen in overweight individuals in the general population. we investigated mets and clinical risk factors two decades after hsct. methods: all male survivors treated with myeloablative allo-hsct during childhood (< 17 years) between 1980-2010 in denmark were invited to a follow-up study. mets was defined as the presence of at least three ncep atp iii criteria: fasting plasma triglyceride (tg) ≥1.7 mmol/l, high density lipoprotein (hdl) < 1.03 mmol/l or medical treatment of hyperlipidemia; fasting plasma glucose (fpg) ≥5.6 mmol/l; abdominal circumference (ac) >102 cm; bp ≥130 mmhg (systolic) / ≥85 mmhg (diastolic) or medical treatment for hypertension. patients with overt dm-2 were included into the mets group. furthermore, patients were examined for chronic graft-versus-host disease (cgvhd) by the nih-criteria at the time of follow-up and high sensitivity c-reactive protein (hscrp) was measured. the prevalence of mets was compared to a nordic reference group (hildrum et al. 2009) . results: we included 49 out of 97 eligible males (participation rate 51%) aged 18-44 years, median 29 years. median (range) follow-up was 21 (8-32) years. of these 49 males, 74% had a malignant diagnosis and 65% were treated with tbi-based conditioning. donors were matched siblings (n=21), matched relatives (n=3) or matched unrelated donors (n=25). mets was more prevalent (33%) in the young adult survivors compared to the prevalence reported for 20-39year-olds in the nordic reference (16 %). instead the prevalence was comparable to that reported for the 50-69year-olds (32%). of the components of mets, elevated tg (51%), hypertension (47%), and decreased hdl (40%) were frequent, while fpg was elevated in 16%. importantly, only 4% of those with mets had increased ac and mean bmi (23.8 kg/m 2 ) of the hsct survivors was within normal range in contrast to features of mets observed in the background population. having mets was significantly associated with tbi (rr = 7.9, 95%ci (1.1-55.3), p=0.004) as was the following single components of mets (mean in tbi group vs. mean in non-tbi group): elevated tg (2.34 mmol/l vs. 0.93 mmol/ l, p= 0.006), lower hdl (1.04 mmol/l vs. 1.38 mmol/l, p=0.001) and higher diastolic bp (80 mmhg vs. 72 mmhg, p=0.03). mets was only demonstrated in one patient who received non-tbi based conditioning. sixteen of 49 patients had cgvhd of which nine were moderate to severe cases, but cgvhd was not associated with mets. however, low-grade inflammation measured by hscrp was related to increased ac (rho=0.41, p=0.004) and tg (rho=0.34, p=0.028). conclusions: our results indicate that male long-term survivors of allo-hsct during childhood have a high risk of mets at an earlier age than the general population. the presence of mets despite normal bmi in several patients suggests unconventional etiologies like the effect of tbi and low-grade inflammation. disclosure: nothing to declare. results: this survey was completed by transplant directors (46%), transplant consultants (41%), nonconsultant grade physicians (8%), hsct clinical nurses specialists (cns) (3%) and other (2%) from 114 centres in 24 countries. 58% of the centres are adult-only, 21% paediatric-only and 21% treat adult and paediatric patients (mixed centres). 46% are located higher than 50 degrees latitude (northern countries) and 54% lower than this latitude (southern countries). at the time of the survey 84% were members of the european union (eu). measurement of serum vd is routinely performed in 47% of the centres prior and in 70% after allogeneic hsct. the main clinical indications are known osteopaenia/osteoporosis (86%), previous fracture (71%), treatment with steroids (68%), premature menopause (46%) and established menopause (32%). monitoring occurs every 3 months (39%), every 6 months (24%), once a year (18%) or at other time-points (19%). in this regard, seasonality is not taken into account in the majority of the centres (94%). local and national/international guidelines (nice) are only followed by 19% and 18% of the centres, respectively. the most common cut-off value of serum vd for commencing on replacement is 50 nmol/l (32%). northern countries tend to use values of ≥75 nmol/l whereas southern countries ≤50 nmol/l. 15% do not use cut-off values. following hsct, 83% of centres prescribe vd supplements to maintain calcium metabolism and bone health (92%), enhance immune reconstitution post-hsct (24%), gvhd prevention (17%), enhance immune-suppression to treat gvhd (10%), treat depression/fatigue (3%) and reduce relapse risk 2%. a "loading" dose is administrated in 30% (54% adult, 25% mixed and 22% paediatric), with a mean duration of 4 weeks . the median daily loading dose is 2,000 iu (286-20,000). the median "maintenance" daily dose is 800 iu (67-10,000). there are not remarkable differences between adult and paediatric centres or northern and southern countries. vd replacement is prescribed by transplant physicians (75%), family physicians (10%), endocrinologists (3%), cns (3%), others (4%) and in 5% of the centres, patients are advised to buy it over-the-counter. vd is prescribed combined with calcium carbonate in 52% and alone in 48% of the centres. it is eventually discontinued by 69% of the centres when therapeutical levels of vd are reached (69%), dexa scan returns to normal (12%) and symptomatic improvement (9%). conclusions: this survey has demonstrated discrepancies in monitoring and replacement of vd across ebmt allogeneic hsct programmes. although awareness has arisen over the last decade, there is still lack of evidence about the optimal levels of vd required for immunemodulation post-hsct. this survey emphasises the need for specific guidelines to harmonise the current management of vd deficiency in adult and paediatric hsct setting. disclosure background: the use of unmanipulated haploidentical sct (haplo-sct) with post-transplant cyclophosphamide (pt-cy) as gvhd prophylaxis has widely extended. primary and secondary graft failure are relatively uncommon complications. however, poor graft function (pgf) after haplo-sct with pt-cy has not been described thoroughly. the objective of this study is to describe characteristics, treatments and outcomes of patients with pgf after haplo-sct with pt-cy. methods: we retrospectively analyzed 132 haplo-sct with pt-cy consecutively performed between 2011 and 2017 in our centre. pgf was defined as either occurring after initial engraftment: persistent neutropenia (anc < 500/ul) with the need of at least 3 doses of g-csf and/or thrombocytopenia (platelets < 20.000/ul) with platelet transfusion dependence, with complete donor chimerism and without concurrent severe gvhd or disease relapse. results: nineteen patients were excluded from the analysis due to early mortality (death before day +30), primary graft failure (absence of neutrophil engraftment by day +28, with mixed chimerism) or secondary graft failure (development of severe cytopenias and mixed chimerism after initial achievement of neutrophil engraftment). thirty one patients (27,5%) were diagnosed with pgf. main characteristics of these patients are summarized in table 1 . twenty six patients (84%) presented with neutropenia and were treated with g-csf, while 5 patients (16%) only developed severe thrombocytopenia without neutropenia, and were treated only with platelet transfusion. twenty four patients (77,5%) had at least 1 cmv reactivation, 15 patients (48%) had 2 or more cmv reactivations and 21 patients (67%) received valganciclovir for cmv reactivation treatment. although most patients achieved adequate peripheral blood counts (pbc) with initial salvage therapy, 6 patients (19%) had persistent cytopenias in spite of g-csf, platelet transfusion, cmv reactivation resolution and myelotoxic drugs withdrawal. four of them were treated with a boost of cd34+ selected peripheral blood donor cells at a median of 170 days after . median cd34+ cells infused was 3,42 x10 6 /kg. these 4 patients achieved adequate pbc after salvage therapy and two developed gvhd. the other 2 patients were treated with increasing doses of thrombopoietin (tpo) receptor agonist (tra) eltrombopag. one patient started treatment 160 days after hsct with 25mg daily and increased dose to 125mg daily, with complete recovery of pbc 6 months after initiating tra. the second patient started treatment 110 days after hsct with 25mg daily and increased dose to 100mg daily, with complete recovery of pbc 2 months after initiating tra. twenty one patients (67%) with pgf diagnosis had long term survival. conclusions: poor graft function is a frequent complication after haplo-sct with cy-post. cmv reactivation and myelotoxic drugs could be the most relevant factors associated with development of this entity. although most patients recover pbc without specific therapies beyond g-csf and platelets transfusion, there is a small group of patients with persistent cytopenias. boost of cd34+ selected cells is effective in reverting this condition, with gvhd as main complication of this procedure. use of tra seems to be an interesting option for these patients, although more experience is needed to draw definitive conclusions. disclosure: nothing to declare. were also frequently observed. the high risk patients for anxiety (hads-a score ≥ 8) and depression (hads-d score ≥ 8) was found in 14.9% and 13.6%, respectively. 10.4% of patients was in high distress status (nccn dt score ≥ 4). we found that younger age (< 60 years) was significantly associated with poor quality of life score (fact-bmt) (p=0.001) and high risk of fatigue (p=0.008), anxiety (hads-a) (p=0.001), and depression (hads-d) (p=0.025). female sex was significantly related to lower physical well-being score and higher distress score (p= 0.046 and p=0.05, respectively). acute lymphoblast leukemia (all) survivors after allo-hct showed significantly worse quality of life score (fact-bmt) (p=0.006) and higher depression score (hads-d) (p=0.028) compared to those with other disease. chronic graft versus host disease (gvhd) and continuous immunosuppressant usage also have significant adverse impact on lower fact-bmt score (p=0.024 and p=0.033, respectively) and higher hads-d score (p=0.015 and p=0.019, respectively). but there was no significant difference in fact bmt, hads-a, hads-d, nccn dt according to donor type, conditioning intensity, anti-thymocyte globulin use, acute gvhd. smoking and alcohol drinking was continued in 7.5% and 17.9% of allo-hct survivors. 20.9% of survivors did not exercise regularly. regular health screening tests have been done only in 40 patients (59.7%). conclusions: allo-hct survivors over 2 years following allo-hct still have many physical and psychological symptoms. younger patients (< 60 years), female, all, chronic gvhd, and sustained use of immunosuppressant were significant risk factors for poor quality of life and anxiety. we need to build more active survivorship care plan after allo-hct especially for those patients. disclosure: all authors have nothing to declare. evaluation of the new ebmt criteria for the diagnosis of vod/sos in 693 consecutive transplant patients using an electronic patient record analysis system asha aggarwal 1 , nicola gray 1 , oliver lomas 1 , katalin balassa 1 , nadjoua maouche 1 , robert danby 1,2 , andy peniket 1 , grant vallance 1 background: veno-occlusive disease (vod), or sinusoidal obstruction syndrome (sos), is a recognised complication of haematopoietic stem cell transplantation. hepatic vasculature endothelial cells are damaged by conditioning chemotherapy, leading to venous occlusion and centrilobar necrosis. the ebmt criteria for diagnosis of vod are bilirubin >=34 with two of painful hepatomegaly, >5% weight gain and ascites. vod is often under-diagnosed, and as a result, treatment may be delayed. integrated electronic patient record (epr) systems are now widely used, and provide an opportunity to retrospectively audit practice to identify patients in whom vod may have been un-diagnosed or in whom treatment was delayed. in addition these systems have potential for alerting clinicians to the potential diagnosis of vod. methods: we have developed software to analyse the data downloaded from epr to identify patients in whom vod was a possible diagnosis according to the new ebmt criteria. in order to identify patients who may have had vod we first screened for patients with a bilirubin of >= 34 mmol/l (which is an absolute requirement for the clinical diagnosis of vod) within the first 50 days of transplantation. epr data was then used to assess whether patients had >5% weight gain. radiology reports were reviewed for patients who had bilirubin >= 34 mmol/l to ascertain if they revealed ascites or painful hepatomegaly. results: 652 patients underwent 693 transplant procedures (january 1st 2013 to july 31st 2018). 162 of all transplant patients (23.4%) were found to have a bilirubin of >= 34 mmol/l. 39 of 403 (9.6%) autograft patients and 123 of 249 (49.4%) allograft patients had an elevated bilirubin at this level. these 162 patients were assessed for evidence of 5% weight gain. this was the case in 30 patients overall-1% of autograft patients, 10.5% of allograft patients. seven patients (2 autograft and 5 allograft) had radiological evidence of ascites. two patients had a recording of painful hepatomegaly (both post allograft). overall our analysis identified 5 patients (0.7% overall) fulfilling the ebmt diagnostic criteria for classic early vod all of whom received defibrotide. all patients had received allogeneic transplants. we failed to identify any cases of late onset vod or any undiagnosed patients over this period. conclusions: this analysis enabled us to efficiently perform a complete audit of our practice to identify patients with vod. we would recommend using electronic patient records to retrospectively audit practice in this way. the tool that we have created for this analysis will be made freely available for public use and the details will be presented at the ebmt meeting. we now plan to extend the function of our epr system to provide alerts to clinicians when vod is a possible diagnosis and may lead to more rapid treatment of these patients. our data suggests that elevation of bilirubin and weight gain of > 5% will be the most frequently occurring criteria on which to base these alerts. disclosure: g.vallance has performed consultancy work for jazz pharmaceuticals. endothelial activation and stress index in predicting outcome of allogeneic stem cell transplantation-a retrospective cohort analysis zinaida peric 1 , tomislav taborsak 1 , nadira durakovic 1 , lana desnica 1 , alen ostojic 1 , ranka serventi-seiwerth 1 , radovan vrhovac 1 1 university hospital centre zagreb, zagreb, croatia background: endothelial dysfunction is a common pathophysiology of major complications after allo-sct, such as graft-versus-host disease, veno-occlusive disease, thrombotic microangiopathy and sepsis. endothelial activation and stress index (easix) is a simple score comprised of standard laboratory parameters (creatinine, ldh and thrombocytes) developed as a potential tool to predict allo-sct mortality by luft and colleagues. a recent validation of easix included three retrospective cohorts and showed that easix taken before start of conditioning can be used as an independent predictor of survival after allo-sct. methods: the aim of our study was to retrospectively evaluate pre-transplant easix in our cohort of consecutive patients who underwent allo-sct in the university hospital centre zagreb between 2012 and 2017. with the use of a cut-off used in the validation cohorts, we compared two groups of patients for overall survival (os) and transplantrelated mortality (trm). group comparisons were done using the log-rank test or gray test for competing risks outcomes. a multivariate analysis evaluated the association of os with relevant variables by using a cox's proportionalhazard regression model. results: our study group included 313 patients and comprised 180 males (57%) and 133 females (43%, with a median age of 48 years (range, 18 to 67 years) at the time of transplantation. the most frequent malignancies in our population were acute leukemia (196 patients, 63%) and myelodysplastic/myeloproliferative neoplasm (451 patients; 16%). the donor was an identical sibling for 106 patients (34%), matched unrelated donor for 176 patients (56%) and haploidentical for 31 patients (10%). 104 patients (33%) received a myeloablative conditioning regimen while 209 patients (67%) received a reduced-intensity conditioning regimen. with a median follow-up of 16 months (range, 12-60) for the whole study group, the os at 24 months was 60%, (95%ci 54-68) in the group of patients with low easix score and 43% (95% ci 33-56) in the group of patients with high easix score (p=0.004). this difference was mainly attributed to higher trm in the group with high easix score (32%, 95%ci 22-45 at 12 months) compared to the group with low easix score (18%, 95%ci 13-23 at 12 months) (p=0.009). in the multivariate analysis which included easix, patients' age, intensity of conditioning, diagnosis (lymphoid vs myeloid), status of the disease at transplant and type of the donor, worse os was independently associated only with older age of patients (hr 1.66; 95% ci, 1.07-2.59, p=0.02) and high easix score (hr 1.51; 95% ci, 1.01-2.24, p=0.04). conclusions: our retrospective data support previous data and suggest that easix could potentially serve as a valid tool for prediction of allo-sct outcomes. as a simple biomarker panel, easix could easily be implemented in clinical decision making in the field of allo-sct. these retrospective data need validation in a prospective study which is currently being conducted. clinical background: veno-occlusive disease (vod) is a potentially devastating complication that can occur after hematopoietic stem cell transplant (hsct) and in severe cases can lead to multi-organ failure. (mohty 2016) defibrotide has been proven to be effective to prevent and treat vod, and it is critical that clinicians are aware of how to diagnose and treat this serious complication of hsct. this study was conducted to determine if an online, simulation-based continuing medical education (cme) intervention could improve performance of hematologists/oncologists (hem/ onc) and advanced practice providers (nurse practitioners and physician assistants, apps) in the diagnosis and treatment of patients with vod. ( methods: a cme certified virtual patient simulation (vps) was made available via a website dedicated to continuous professional development. the vps consisted of 2 cases presented in a platform that allows clinicians to assess the patients and make diagnostic and therapeutic decisions supported by an extensive database of diagnostic and treatment possibilities, matching the scope and depth of actual practice. clinical decisions were analyzed using a sophisticated decision engine, and tailored clinical guidance (cg) employing up-to-date evidence-based and faculty recommendations was provided after each decision. one case was about vod and the other case was about acute myeloid leukemia (aml). decisions were collected post-cg and compared with each user's baseline (pre-cg) decisions using a 2-tailed paired t-test to determine p-values (p < .05 indicates significance). data were collected between 9/21/2017 and 11/7/2018. results: at the time of assessment, 115 hem/oncs and 409 apps had fulfilled the participation criteria for completing the vod case simulation. conclusions: this study demonstrates that vps that immersed and engaged clinicians in an authentic and practical learning experience improved evidence-based clinical decisions related to the management of vod. this vps increased the percentage of clinicians who utilized standardized criteria to diagnose vod and who ordered defibrotide and iv fluids for vod management. however, further education is needed to increase the competence and performance of clinicians, particularly apps, in these areas in order to positively impact patients. disclosure: nothing to declare. a nationwide retrospective study of hematopoietic stem cell transplantation in solid organ transplant recipients: on behalf of jshct, transplant complications working group background: the outcome of hematopoietic stem cell transplantation (hsct) in solid organ transplant remain unclear. to address this issue, we conducted a retrospective survey of the 404 japan society for hematopoietic stem cell transplantation centers. methods: to address this issue, we conducted a nationwide retrospective survey of the japan society for hematopoietic stem cell transplantation (jshct) centers. a first questionnaire was emailed to jshct centers requesting information on cases of hsct in sot recipient. patients' data about sot were collected by sending a second questionnaire to the centers with the patient. based on these reports, patients' data about hsct was identified in the japan transplant outcomes registry database by the transplant registry unified management program (trump), confirmed in 2017. results: of the 404 jshct centers, 238 responded to the survey (58.9%). 14 of the responding centers reported a total of 19 patients who had undergone sot from living donor, and subsequent hsct. they consist of three autologous hsct (auto-hsct) and 13 allogeneic hsct (allo-hsct). in auto-hsct, all patients had received liver transplant for hapatoblastoma. they achieved neutrophil engraftment at 30 days after hsct, and two of three patients were alive at one year after hsct. in allo-hsct (n=16), seven patients had received liver transplants, and nine patients had received kidney transplants. five patients received hsct from unrelated donor, and 11 patients received hsct from related donor; two donors were identical in sot. their stem cell sources were seven peripheral blood stem cell, six bone marrow, and three cord blood. all but one patients achieved neutrophil engraftment at 30 days after hsct. five-year overall survival (5yos) was 37.5%. while 5yos in patients with bone marrow failure (n=4) was 100%, that in patients with malignant disease (n=12) was 16.7%; all but one patients with malignant disease received allo-hsct in non-remission. seven of nine kidney-transplant recipients experienced dialysis, and three patients experienced renal rejection after hsct. on the contrary, no liver-transplant recipient experienced hepatic rejection. conclusions: in sot recipients, the outcome of allo-hsct for malignant disease was poor, partly due to disease status before allo-hsct. severe renal complications were common in kidney-transplant recipients, suggesting renal care with caution during and after allo-hsct. disclosure: this work was supported in part by the practical research project for allergic diseases and immunology (research technology of medical transplantation) from japan agency for medical research and development, amed. high incidence but low mortality of ebv related ptld after t-cell replete allo-peripheral blood hct with aggressive monitoring and without pre-emptive rituximab background: the aim of the study is to report the incidence and outcome of post-transplant lymphoproliferative disorder (ptld) in the setting of allogeneic peripheral blood hematopoietic stem cell transplantation (allo-hsct) combining post-transplant cyclophosphamide (ptcy) and anti-thymocyte globulin (atg) as graft versus host disease (gvhd) prophylaxis. methods: between october 2015 and may 2018, 195 adult patients diagnosed with hematological malignancies underwent a first t-cell replete allo-hsct in our center. all patients received a reduced intensity conditioning regimen with fludarabine, busulfan, and 200cgy of total body irradiation, combined with rabbit-atg, ptcy and cyclosporine (csa). ebv titres were monitored weekly by quantitative pcr in plasma samples. the cut-off value for test positivity was >600 copies of ebv dna/ml of plasma. last follow up was november 2018. median follow up for patients known to be alive was 19 months (range 5-35). results: patient information is summarized in table 1 . ebv reactivation was documented in 117 (60%) patients. median time to ebv reactivation and the diagnosis of presumed/proven (p/p)-ptld were 75 (16-326) days and 97 (54-306) days [3 (0-10) months], respectively. median time between first ebv reactivation to p/p-ptld was 21 (0-175) days. seventeen (14%) of the 117 patients developed p/p-ptld. median age was 55 years . two (12%) received mrd, 9 (53%) 10/10 mud, 1 (6%) 9/10 mud, and 5 (29%) haploidentical donor grafts. twelve (71%) were on therapeutic cyclosporine at diagnosis. pre-emptive therapy was not given to any case and only probable or proven ptld were given rituximab. treatment was based on reduction of the immunosuppression in 3 patients and with the addition of weekly rituximab 375 mg/ m 2 in 15 cases. fifteen (88%) achieved complete clinical responses with pcr negativity. two (12%) patients died secondary to ptld. conclusions: atg based conditioning is associated with increased viral reactivations. frequent ebv monitoring and pre-emptive treatment may lead to rapid disease control. further research is required to optimize monitoring and management strategies in allo-hsct recipients. disclosure: nothing to declare p160 acoustically enriched extracellular vesicles as potential markers for allogeneic hematopoietic stem cell transplantation complications hooi-ching lim 1 , robert palmason 2 , stig lenhoff 2 , thomas laurell 1 , stefan scheding 1,2 background: extracellular vesicles (evs) contain a number of condition-specific proteins, dna and rna types and might therefore be used for the early detection of posttransplant complications. however, traditional ev isolation (ultracentrifugation) is time consuming and requires large sample volumes thus making it difficult to perform longitudinal studies on larger patient cohorts. we therefore investigated whether recently-developed acoustic trapping could be applied to isolate evs from patient plasma for biomarker development. methods: plasma samples were collected from 10 consecutive patients before and up to 3 months after allogeneic hematopoietic stem cell transplantation. patients (age: 22-58 years) with high-risk or refractory/relapsed diseases were transplanted with mobilized pbsc from related (n=2) and unrelated donors (n=8) after standard conditioning. gvhd prophylaxis was cyclosporine and methotrexate. plasma samples were frozen and thawed for ev enrichment using a novel acoustofluidic-based technology (acoustic trapping). acoustic trapping uses ultrasound as a local λ/2 acoustic standing wave produced by a piezoelectric transducer over a capillary. first, 12 μm polystyrene beads are captured which serve as seeding particles. after washing, target particles (evs) are then captured ("trapped") in the acoustic field. a semi-automatic trapping device (acoutrap) was used to isolate evs from diluted plasma (1:2 in pbs). the number of evs and size distribution were analyzed by nanoparticle tracking analysis. mirna analysis was performed by qpcr.evs were enriched in duplicate from 50μl and 300μl of diluted plasma for nanoparticle tracking analysis and qpcr analysis, respectively. results: evs were successfully isolated from all plasma samples. a total of 89 plasma samples were processed. numbers of trapped evs ranged from 3.7x10 8 -5.5x10 9 before conditioning to 4.4x10 8 -1.5x10 10 per 50μl diluted plasma after transplantation. the maximum change in ev numbers in individual patients compared to pretransplantation values ranged from 2-fold to 7-fold. most patients showed slight increases in ev size after transplantation. eight of the patients showed signs of infection and received i.v. antibiotics. increased levels of evs (> 2-fold) were recorded in three patients during these episodes. furthermore, increased ev numbers were observed in a patient who required i.v. antiviral therapy for cmv reactivation. acute grade i gvhd was observed in five patients of which two had increased ev numbers (> 2-fold). one patient developed grade iv gvhd which was accompanied by a 4-fold increase in ev numbers. interestingly, progressively increasing ev numbers preceded the detection of early relapse in a pre-b all patient by three weeks. rna isolation from trapped evs yielded sufficient material for mirna profiling. here, first mirna profiling data demonstrated that mirnas were detected in ev samples (mir-103a, -23a, -30c, -142 and -451a) , and that acoustically enriched evs were not affected by hemolysis in contrast to the corresponding whole plasma samples (dcq of mir-23a and mir-451a). conclusions: acoustic trapping allows for efficient and rapid enrichment of evs from small volume plasma samples. trapped ev samples contain sufficient amounts of mirna for downstream analysis and are thus promising candidates for biomarker development in transplantation. disclosure: laurell and scheding are founders and board members of acousort ab, a lund-based biotech sme that develops particle and cell sorting methods based on ultrasound. the incidence, risk factors and outcomes of primary poor graft function after allogeneic hematopoietic stem cell transplantation fei gao 1,2 , jimin shi 1,2 , yi luo 1,2 , yamin tan 1,2 , xiaoyu lai 1,2 , jian yu 1,2 , he huang 1,2 , yanmin zhao 1, 2 background: allogeneic hematopoietic stem cell transplantation (allo-hsct) is a curative therapy for both hematologic malignancy and many other blood disease. while, primary poor graft function (pgf) is still a severe complication following hsct which lead to poor prognosis. up to now, the incidence and risk factors of pgf have not been totally revealed. methods: from january 2013 to december 2017, a total of 647 patients who received allo-hsct in our center were analyzed retrospectively. there were 9 males (47.4%) and 10 females (52.6%) with a median age of 36.21 years (21-49 years) . pgf was defined as persistent neutropenia (≤0.5×10 9 /l), thrombocytopenia (platelets≤20×10 9 /l), and/ or hemoglobin≤70 g/l after engraftment with hypocellular bone marrow and full donor chimerism, without concurrent graft-versus-host disease or disease relapse. incidence was calculated from all patients. of the 647 total patients, nineteen (2.94%) developed primary pgf. a 1:4 ratio of nested case control study using the good graft function (ggf) subjects transplanted in the same year with the same sex and age of ±5 years was carried out. results: data was analyzed by univariate and multivariate logistic regression, and univariate analysis identified disease species, the time from diagnosis to transplantation, disease states, myelofibrosis, splenomegaly, serum ferritin (sf) level, cmv infection, mononuclear and cd34+ cells in graft as potential risk factors (p <0.1) for pgf. multivariate analysis identified 3 elements as the independent risk factors (p <0.05), including cd34+ cells <5×10 5 / background: transplant survivors affected by cgvhd usually take one or more immunosuppressants, as well as prophylactic antimicrobials; use of multiple medication classes concurrently poses a risk for drug-drug interactions or amplified side-effects. the use of medications other than cgvhd-direct immunosuppressive therapies has not been well-characterized. this study aims to evaluate patterns of opioid analgesic use in a cohort of patients severely affected by cgvhd. methods: patients (n=335) with cgvhd were consecutively enrolled in a cross-sectional natural history study (nct00092235) from 10/2004-12/2016 at the nih. participants underwent a comprehensive evaluation including a detailed history and physical examination (including current medications), multidisciplinary evaluations, and laboratory and diagnostic testing. for this analysis, respondents were classified as receiving or not receiving an opioid analgesic. following the initial screening by univariate methods (n=335), multivariable logistic regression analysis (mlr) was used to identify a set of factors which could jointly impact opioid use. for mlr data were divided into a training (n=167 patients) and a validation set (n=168). results: study participants´median age was 48.5 years (19-75), 44% were female, 74% had severe cgvhd per nih scoring criteria, and 77% were currently receiving high or moderate levels of systemic immunosuppression. approximately one third (33%) were taking opioid analgesics (oa). based on the univariate screening results (p< =0.05), a set of 24 parameters was evaluated by univariate logistic regression in the 167-patient training set, and the following parameters retained their significance and were included in the mlr model: nih average score per organ, total lss, patient impression of severity, nih cgvhd severity, presence of skin erythema, karnofsky performance score (kps,) clinician's therapeutic intent, nih joint score, and with the presence of several cgvhd symptoms including rashes, mouth sores, avoidance of food, vomiting, weight loss, joint and muscle aches, joint limitation, energy loss, need for naps, fevers, anxiety. multivariable logistic regression identified kps < 77% as predictive of oa use, or 0.92, 95% ci 0.89-0.95. in the training set 56.4% of pts using opioids were correctly identified, 78.2% of those not taking opioids were identified, an overall fraction of correctly identified pts was 70.9% (95% ci 63.3 -77.7%), while in the testing set, 45.5% of those using opioids were correctly identified, and 67.9% of those not taking opioids were correctly identified, with overall 60.5% (95% ci: 52.6-68.0%) classification accuracy. conclusions: this study showed the burden of oa in this cgvhd cohort. lower kps was significantly associated with oa use, as well as self-reported symptoms and a more severe cgvhd disease, which could be of interest in the development of non-pharmaceutical interventions in this patient population. additional, prospective studies are needed to explore the indications for and effectiveness of oa in this population of survivors. disclosure: no conflict of interest to declare. rcts that tested an internet-based program and patientcentered survivorship care plans for hct survivors. patient and caregiver input is essential to inform the design and features for the mobile app platform so that it is usable and engaging for those it targets. methods: using a qualitative research design, we conducted telephone focus groups of adult patients and caregivers in the united states. adult (age >18 years at the time of study entry) hct recipients had to be at least oneyear post-hct to participate. participants had to be able to communicate in english, and could have received a hct for any diagnosis, and from any donor source or stem cell type. those who had multiple transplants were included. participants were asked to review printed and online visual presentations of the mobile app before the focus groups so they were prepared to discuss their responses to the materials during the call. focus groups were conducted to saturation, when no new qualitative content was offered. results: three focus groups were conducted with 22 total participants (20 patients, two caregivers/patient advocates). all patients received an allogeneic hct; average time since hct was 8 years (range: 2-22 years).the majority of participants were female (77.3%). participants had differing perspectives on the usefulness of the app to track follow-up appointments, lab values, and other health care plans. there was high interest in having the app tailored to meet specific needs of patients, including tracking information over time (e.g. test results, medications), and having health information available specific to their needs. to minimize duplication of information and data entry, participants recommended syncing the app with their calendars and online patient portals they already use. reasons provided for not using the app included perception that the materials repeated information already received, side effects such as graft-versus-host disease that restricted vision or motor skills, and lack of comfort with apps for some older participants. conclusions: many health technology and mobile apps are being created to improve patients' health and survivorship care. in this study, hct survivors and caregivers identified a variety of features that they would want in an app or website, in particular, features tailored to individual needs. health technologies provide an opportunity to improve survivorship care, but patients and caregivers should be engaged in the process of developing these tools to assure the technology fits their needs and will be used. given the effort required to maintain these technologies, they require testing for health benefits in rigorous clinical trials. clinical background: thanks to allogeneic stem cell transplantation (allo-hsct) patients suffering from hematologic malignancies have seen an increase in there life duration expectancy, but they are many side effets including decreasing in physical performance and in quality of life. the intensity of physical performance decrease is variable between patients, and today we did not know why. the aim of our study was first to characterize the physical performance of subjects less than 1 year following allo-hsct by the use of a cardiopulmonary exercise testing (cpet), and then to determine the predictive factors of exercise performance. methods: we did a retrospective analysis from 59 patients who had an allo-hsct at hematology department of toulouse-oncopole and cpet from 01/2015 to 09/2017. the cpet was performed using a cycle ergometer with o2 and co2 analyzer breath by breath, (masterscreen cpx carefusion, san diego, usa), a continuous 12-lead electrocardiogram, and a blood pressure monitoring. the protocol included a 2-min rest period, a 2-min warm-up of 20w pedaling followed by a 10w/min incremental phase, up to exhaustion, then a 2-min active recovery of 20w pedaling, then a 2-min passive recovery. three exercise markers were analysed: the peak of oxygen uptake (peak vo 2 ), the ve/vco 2 slope and the first ventilatory threshold (vt 1 ). data relative to conditioning regimen, short-term complications, impairment at cpet day, and physical activity since allo-hsct were gathered. results: after allo-hsct, nearly 3 over 4 patients reported fatigue, a half reported dyspnea, and 1 over 4 or more reported pain, muscular, neurological or psychological impairment. more than 60% of patients suffer from moderate or severe physical intolerance, particularly when myeloablative conditioning regimen was used. only 37% of patients followed rehabilitation sessions supervised by a physiotherapist, and non-supervised physical activity has been performed by 87% of patients. despite normal lung function tests and echocardiography findings in most patients, 80% had exercise intolerance (ei), 85% exercise deconditioning, and 55% had abnormal ventilatory efficiency. patients with moderate and severe impaired exercise capacity were significantly younger at diagnosis and at allo-hsct, such as patients with severe deconditioning conclusions: based on a retrospective study, we reported for the first time complete results from cpet and detailed clinical evaluation concerning deficiency and disability following first year after allo-hsct. these results confirm that exercise impairment is very frequent with more than a half of patients suffering from alterations of one or more of the three performance markers, despite being active. disclosure: nothing to declare demyelinating disorders: a paradigm of immunity disorders after hematopoietic stem cell transplantation background: neurologic complications are a major problem in patients who undergone hematopoietic stem cell transplantation (hsct). given the higher survival of transplanted patients, the burden of neurological complications is increasing in the last years. a significant reduction in overall survival was demonstrated in patients who developed neurological complication after hsct, irrespectively of the hematopoietic stem cell (hsc) source. neurologic disorders in transplanting setting comprise a wide variety of ethiologies including demyelinating disease, which are caused by immune and non-immune mechanisms. here, we analyzed the clinical presentation and the underlie ethiologies of patients developing hsct-related demyelinating disorders in order to give diagnostic and prognostic clues useful to manage these severe but treatable complications in the transplant setting. methods: a total of 45 patients of our department which developed neurological complications after hsct were consecutively collected and 33 (73%) of them, namely those having a diagnosis of a demyelinating disorder, were grouped and described according to the ethiologies of their neurological disorder. results: in 14/33 (42%) patients, an immune-mediated process was found, while 10/33 (30%) were diagnosed as having an infective etiology and 2/33 (6%) were supposed to have a demyelinating disorder caused by toxic exposition. a definitive etiologic diagnosis was not formulated in the remaining 7/33 (21%) patients. when patients who developed an immune-mediated demyelinating disorder (10/33) were compared to those in which a clear immune pathogenic mechanism was not detected (23/33), a higher incidence of acute graft-versus-host disease (agvhd) was detected in the former than in the latter (20% vs 8%). moreover, comparison of these two groups revealed that those with no evidence of immune-mediated process have a slight higher prevalence of t-cell depleted hsct thanthose with an immune-mediated demyelinating disorder (60% vs 50%). finally, a lymphoproliferative disorder pre-existing the hsct was detected in 5/14 (36%) patients with immune-mediated demyelinating disorder but only in 4/19 (21%) of those without evidence of immune-mediated processes. conclusions: demyelinating disorders may be responsible of near 40% of neurologic complications in the posttransplant setting and, among them, an immune-mediated process is likely to be involved in more than 40% of cases. our results suggest that the immune mechanism that underliesthe agvhd may also be involved in developing demyelinating disease in transplanted patients. it also may be possible that the lymphoproliferative disorder preexisting the hsct is a risk factor able to increase the risk to develop an immune-mediated demyelinating disorder in the post-transplanting setting. using a t-cell depleted hsct can increase the risk of immune-mediated disorders in at least a small fraction of transplanted patients. despite our results should be validated on a larger cohort of patients, we can speculate on the possible connections between the wide range of complex and still poorly defined immunity disorders which can influence the prognosis and course of transplanted patients. disclosure background: injury to the mucosal barrier and subsequent development of oral mucositis (om) is among the most common toxicities of allogeneic stem cell transplantation (sct). despite the high prevalence of om and its debilitating nature, prospective studies evaluating determinants of om are scarce. we therefore prospectively evaluated the occurrence of om following sct. risk factors for om and its implications short and long-term outcomes were assessed. methods: om was prospectively evaluated on a weekly basis in patients undergoing allogeneic hsct. the grade of om was determined based on the national cancer institute common toxicity criteria for adverse events (ctcae) scale (v. 4.0). severe om was defined as grade ii to iv. conditioning regimens were evaluated individually and according to intensity; myeloablative (mac), reduced intensity (ric) or reduced toxicity (rtc). the latter category included only patients receiving fludarabine and treosulfan at dose of 30-42 g/m2 (flu/treo). risk factors for the development of severe om were initially identified by a univariate analysis and then analyzed in a multivariate logistic regression model. association of om with peritransplant infectious complications, iv morphine consumption, hospitalization length, neutrophil engraftment, acute and chronic graft-versus-host disease (gvhd), non-relapse mortality (nrm) and overall survival were assessed in a univariate analysis. competing events were considered in analyzing engraftment, gvhd, and nrm. results: 115 patients who underwent an allogeneic sct between 2016 and 2017 were included. median follow-up was 316 days. leading indications for transplantation were acute myeloid leukemia (49%), lymphoma (16%), and myelodysplastic syndrome (15%). the majority of patients received an allograft from a matched sibling or unrelated donor (88%) and methotrexate gvhd prophylaxis (74%). the median time to om onset was 7 (interquartile range [iqr] 5-9) days. prevalence of grade ii-iv om was 60%. the median duration om was 10 [6-13] days, and iv morphine was administrated for a median of 5 [6-12] days for patients with grades iii-iv om (45%). in a univariate analysis a younger age (p=0.023), lower bmi (p=0.01), recent smoking history (p=0.08), recent antibiotics exposure (p=0.018), mac (p< 0.001), and use of methotrexate (p=0.009) were associated with an increased risk for grade ii-iv om. in a multivariable model the risk for grade ii-iv om was lower with rtc (i.e., flu/treo) vs. mac (odds ratio [or] 27.31; p< 0.001) and rtc vs ric (or 7.25; p=0.001), mycophenolate mofetil vs. methotrexate (or 3.43; p=0.027) and recent smoking (or 4.7; p=0.075). compared to lower grades, grade ii-iv om was associated with a longer hospitalization duration (median 29 days vs. 27 days; p=0.006), delayed neutrophil engraftment (median 14 vs. 12 days; p=-0.004), and more gastrointestinal related infections (10% vs. 0%; p=0.045). grade ii-iv om was not associated with increased risk of bloodstream infections, acute or chronic gvhd, non-relapse mortality, and increased mortality. conclusions: oral mucositis is prevalent among allogeneic-sct recipients. importantly, fludarabine-treosulfan, which is considered a myeloablative is associated with a markedly reduced risk for om. consequences of om include prolongation of hospitalization, delay in neutrophil engraftment, and a tendency for gastrointestinal infections, but does not increase the risk for gvhd and mortality. disclosure: nothing to declare background: the advent of recent diagnostic techniques for the assessment of iron overload (t2*-mri) and their systematic use as screening tools in the setting of secondary hemochromatosis have led to an increased awareness that focal nodular hyperplasia (fnh) represents a possible incidental finding after hematopoietic stem cell transplantation (hsct). methods: clinical and radiological features of patients undergoing hsct in a single pediatric institution have been retrospectively reviewed for fnh. in order to provide an estimate of the prevalence of fnh after hsct, we analysed all the t2*-mri scans performed during the last 5 years in our centre and recorded the number of patients with fnh (group a). in addition, data about patients incidentally diagnosed with fnh at abdominal imaging performed for different clinical indications have been collected (group b). results: eight out of 118 (7%) transplanted patients who underwent at least one t2*-mri scan from september 2013 to september 2018 were incidentally diagnosed with fnh. group b included 3 subjects with fnh incidentally found at ultrasound or non-t2* mri scans performed before 2013. overall, 11 transplanted patients (5 males, 45%), transplanted for al (9 cases) or bone marrow failure (2 cases) at a median age of 13.1±3.6 years, were diagnosed with fnh between 0.6 and 12.8 years after hsct, namely 3.2±2.6 years in group a and 7.0±4.1 years in group b. a variable degree of iron overload was demonstrated in all patient (lic: 230-340±50 microg/g; baseline serum ferritin: 685-3189 ng/ml). the potential risk factors for fnh are reported in table 1. in 8/11 patients, the radiological finding was pathognomonic; in 1/11 the diagnosis of fnh was confirmed histologically, while 2/11 subjects were labelled as "fnhlike", although a potential diagnosis of hepatic adenoma could not be ruled out. in 3/11 patients, fnh presented with an isolated lesion, while 8/11 had 2 to more than 10 hepatic nodules at diagnosis. the size of nodules at diagnosis ranged from 3 to 41 mm. in unenhanced mri scans, lesions were predominantly hyperintense on both t1-and t2weighted sequences. in dynamic studies with contrast medium, all lesions strongly enhanced during the arterial phase, with a variable degree of wash-out in the late venous scans. hepatic function tests were normal in all the enrolled patients at diagnosis of fnh. among the 9/11 patients for whom at least a follow-up scan was available, 1 presented a complete regression, 3 a reduction and 3 an increase in the size and/or number of lesions, while in 2 patients the nodules remained substantially unchanged after a mean radiological follow-up of 4.5±3.3 years. no malignant transformations were observed. conclusions: fnh represents a relatively frequent incidental finding after hsct. although a malignant transformation is rare, given the demonstrated variable evolution of the hepatic nodules, a radiological follow-up is highly recommended. disclosure: nothing to disclose. incidence and risk factors for hepatic sinusoidal obstruction syndrome after allogeneic transplantation: retrospective multicenter study of turkish hematology research and education group (threg), updated data methods: ten centers from turkey were enrolled in the study. we retrospectively evaluated the medical records of patients who were treated with allo-sct between january 2012 and december 2015. a baltimore criterion was used for assessment of hsos. four hundred twenty six (97.2%) of 438 patients who were treated with prophylaxis with defibrotide alone or one or more of the n-acetylcysteine, diuretics and heparin used defibrotide (10-25 mg/kg/day). results: the study included 1153 patients (687 males/ 466 females) with median age of 38 (15-71) years. the demographic and clinical characteristics of patients were summarized in table 1 seventy-three (84.9%) of patients with hsos were treated with defibrotide after diagnosis. the median time of starting defibrotide in these patients was 13.5 (2-29) days. thirty-seven (43%) of patients with hsos recovered completely and forty-nine (57%) of them died as a result of multi organ failure. the incidence of hsos-related mortality in allo-hsct cohort was found to be 4.2%. in univariate analysis, statistically significant associations were not found between hsos incidence and age/sex of recipient, type of conditioning regimen, stem cell source and type of gvhd prophylaxis. on the other hand donor type, engraftment status and prophylaxis for hsos were significantly associated with hsos development. hsos prophylaxis was significantly decreased hsos-associated mortality (p=0.001). conclusions: hsos still remains a serious lifethreatening complication of allo-sct. although the incidence is low, hsos is associated with increased 100-day non-relapse mortality. hsos prophylaxis especially with defibrotide, seems to reduce hsos associated mortality in high risk patients. disclosure: nothing to declare prophylaxis with defibrotide in adults at very high risk of veno-occlusive disease: results in 11 patients background: hepatic sinusoidal obstruction syndrome/ veno-occlusive disease (sos/vod) is a life threatening complication that can occur after hematopoietic stem cell transplantation (hsct). severe sos/vod rapidly evolves in multiple organ dysfunction syndrome (mods), associated with a mortality rate exceding 80%. precocity of defibrotide (df) treatment is the leading factor for efficacy. prophylactic use of df is recommended in children, but its value has not been validated in the adult population, although factors for individual risk assessment for vod are debated. we here present a real-world experience of df prophylaxis in adult patients at very high risk of sos/vod receiving allogeneic hsct. methods: from 2016 to 2018 we treated with prophylactic defibrotide and ursodeoxycholic acid (udca) 11 patients, median age 30 years (range 21-59). nine patients received allogeneic hsct for acute lymphoblastic leukemia (8 b-all and 1 t-all), one patient for severe aplastic anemia, one patient for primary myelofibrosis. they were all at high risk for sos/vod because of previous hepatotoxicity (3 patients), previous hsct (6 patients), double alkylating agent (5 patients) or previous treatment with inotuzomab ozogamicin (io; 7 patients). of the 7 patients treated with io, 6 received 2 cycles of io, and 1 received 1 cycle, with the last io dose administered a median 41.5 days before hsct (range 34-61 d). defibrotide was administered in 4 daily doses for a total dose of 25 mg/ kg per day and udca at the dose of 300 mg twice per day, starting from day -6 prior transplant. all patients received treosulfan-fludarabine based conditioning. in 5 patients thiotepa was added to the conditioning and in 2 patients a low dose 4gy tbi. gvhd prophylaxis included posttransplant cyclophosphamide, rapamycin and mycophenolate in all patients, except one patient with aplastic anemia receiving atg, rapamycin and mycophenolate. donor source was pbsc in all cases. seven patients received family haploidentical (mmrd) transplant, 1 patient a mrd transplant and 3 patients a mud transplant. results: the median duration of defibrotide therapy was 34 days (range 32-64 days). documented non-severe gastrointestinal bleeding occurred in 2 patients requiring defibrotide temporarily discontinuation, no other significant bleedings were experienced. four patients developed grade ii-iv acute gvhd and no transplant-associated thrombotic microangiopathy were diagnosed. overall, sos/vod occurred in 3/11 cases within 21 days after hsct (days 9, 10 and 13) and no late-onset sos/vod were diagnosed. sos/vod was very severe, causing mods and death in all 3 cases. all 3 patients were characterized by a common pattern of very high risk factors for sos/vod by prior hsct and salvage treatment for b-all with 2 cycles of io close to hsct. furthermore, they all received a fully myeloablative conditioning regimen with treosulfan and thiotepa and a mmrd transplant. conclusions: defibrotide prophylaxis was safe and well tolerated with no severe related complications. sos/vod occurred despite continuous df prophylaxis in 3/7 patients treated with inotuzomab ozogamicin close before undergoing 2 nd transplant. to reduce the incidence of severe vod, pre allo-hsct treatment with inotuzomab ozogamicin should prompt avoidance of other cumulative risk factors for vod, such as use of double alkylating agents. disclosure background: busulfan is the backbone of many preparative regimens administered to children undergoing allogeneic and autologous hematopoietic stem cell transplantation (hsct). among its many long-term adverse effects, busulfan can cause various degrees of pulmonary injury. although well described in adults, there are few large series exploring pulmonary toxicity of busulfan in children. we describe long-term pulmonary follow-up in a large group of children treated at a single center who had received high-dose busulfan and examine the relationship of systemic drug exposure and lung function over time. methods: all surviving children who had received highdose busulfan between 1993-2013 in the context of hsct at the schneider children´s medical center, were referred for serial pulmonary function monitoring (including spirometry, plethysmography and diffusing capacity for carbon monoxide [dlco] . pre-transplant testing was available for children who were old enough to perform the procedure. spirometry results were adjusted according to the revised global lung initiative formulas for age, gender, and height. pulmonary injury was defined as a z score below -1.96 for spirometry, or < 80% of predicted for the other parameters. busulfan levels were monitored following the second drug dose. all patients received busulfan in four daily doses. area under the curve (auc) calculations were performed by bayesian calculations. results: between 1993-2013, 263 patients aged 0-18 years were diagnosed with malignant or non-malignant diseases and treated with high-dose busulfan. of 130 shortterm survivors, 75 had at least one post-transplant pulmonary function evaluation. the mean age at treatment with busulfan was 7.9 years (range, 0.4-27 years). of these 75 children, 22 children had undergone autologous transplantation and 53 children had an allogeneic transplant. 7 of these patients eventually relapsed and 3 died. 26 children had one or more pulmonary risk factors before hsct -chest or upper abdomen radiation (13), chest wall tumors or lung metastasis (8), chest surgery (5), prior administration of pulmonary-toxic drug (3) or asthma (2). during follow-up (up to 14 years, median 5.5 years), fev1 and fvc spirometry tests both decreased significantly (p=0.002 and 0.001, respectively), while the decrease in dlco was not statistically significant. 35% of patients had abnormal pulmonary function tests and seven children had symptomatic disease which in two may have been manifestations of gvhd. interestingly, no correlation was found between busulfan auc, busulfan peak levels, the number of busulfan doses administered, the type of transplantation (autologous vs. allogeneic) or primary disease to pulmonary injury. even after censoring of children with pre-transplant pulmonary risk factors we noted a decrease of fev1 and fvc. conclusions: as in adults, pulmonary injury is observed in children treated with high-dose busulfan prior to hsct. no correlation was observed between busulfan auc and pulmonary injury. follow-up of children who receive this drug should include regular pulmonary monitoring, referral to a pulmonologist when subclinical pulmonary compromise is found, and counseling regarding measures that might prevent or ameliorate pulmonary damage. continued follow-up of this cohort of patients should inform our pretransplant patient information sessions, and the future use of busulfan in children. disclosure: nothing to declare background: transplant-associated thrombotic microangiopathy (ta-tma) is a specific complication of allogeneic hematopoietic stem cell transplantation (hsct). post-hsct tma has been attributed to the vascular endothelial damage caused by high-dose chemotherapy, calcineurin inhibitors (cnis), graft-versus-host disease (gvhd), infections. there is a little evidence published regarding the efficacy and factors influencing the outcome of withdrawal of cnis. methods: the analysis comprised a total of 54 patients, with diagnosed hematologic malignancy (aml (16), all (12), mds (4), hodgkin lymphoma (5), cml (3) and neuroblastoma (1) received allo-sct, from a matched related, unrelated or haploidentical donor between 2007 and 2018. patients were diagnosed with ta-tma based on cho criteria. the median age of patients was 23 (37 adults, 17 children). gvhd prophylaxis was performed with tacrolimus (tac) in 42, cyclosporine a(csa) in 11, combination tacrolimus+sirolimus (sir) in 12. 24 patients received atg and 26 ptcy. withdrawal of cnis was accompanied by administration of systemic steroids (21 patients) or substitution with sir after reaching levels of csa< 100 ng/ml or tac < 3 ng/ml in 13. the target concentration of sir was 3-9 ng/ml. in pediatric patients who received combination tac+sir, the tac was discontinued in one step while sir continued. median time to development tma was 31,5 days after allo-sct (range 1-408). median follow-up of surviving patients was 395 days. the primary outcome was overall survival (os) up to 2 years after development of ta-tma. results: the following significant predictors of 2-year os were identified: tac replacement with sir (p< 0,001), ptcy in prophylaxis (p< 0,001), acute gvhd (agvhd) grade 2-4 (p=0,029), previous sepsis (p=0,003), level of ldh in debut (p=0,001), combination sir+tac in prophylaxis (p=0,05), major ab0-mismatch (p=0,022), severity of cns symptoms (p< 0,001). there was no significant difference in os according to patients' age, sex, "salvage" disease status at transplantation, previous vod, viral (hhv 1,2,6, cmv, ebv) reactivations, count of cd34+ cells transfused, ldh level, shizocytes and creatinine in the debut of ta-tma. in the multivariate analysis replacement of cnis with sir (hr 0.27, 95%ci 0.08-0.96, p=0.018) and baseline ldh level (hr 1.01, 95%hr 1.00-1.01, p=0.029) were associated with survival differences. the cut off for ldh was 4xunl. agvhd grade 2-4 (hr 1.96, p=0, 081) and use of ptcy (hr 0.535, p=0.317) were not significant in the multivariate analysis (figure 1). ta-tma cases after ptcy were significantly less frequently associated with clinically significant agvhd (19% vs 68%, p< 0,001). the survival was higher after ptcy (74% vs 15%), but not significant due to sample size and other ta-tma factors. leading causes of death were: gvhd progression (11%), bacterial infection (11%), tma (17%) and other (19%) . conclusions: replacing tac by sir is an effective therapeutic strategy in a group of patients with debut of ta-tma at least after ptcy, where it is less likely to be associated with agvhd. there is a significant overlap of populations with ptcy prophylaxis and substitution with sir, thus the study is not powered to provide guidance for patients on conventional prophylaxis with ta-tma. [[p172 image] 1. disclosure: none of the authors has anything to disclose. donor-recipient ab0 mismatch effect on the allogeneic hematopoietic stem cell transplantation outcome: a single-center retrospective study background: because transmission of major histocompatibility complex and blood group system genes is independent from each other, approximately 40-50% of all allogeneic hematopoietic stem cell transplantations (allo-hsct) are realized crosswise the ab0-blood group boundary. however, due to the widespread expression of ab0 antigens on a variety of human tissues other than erythrocytes, ab0 incompatibility may have an impact on the outcome of allogeneic hsct that goes beyond the wellknown immune-hematological complications such as immediate hemolysis due to the presence of isoagglutinins and delayed hemolysis due to passenger b lymphocytes. here we aimed to assess the donor-recipient ab0 mismatch effect on the allo-hsct outcome, comprising non-relapse mortality (nrm), overall and relapse-free survival, posttransplant prc transfusion requirement, as well as relapse rate, incidence of graft-failure and acute gvhd. methods: clinical and laboratory data from 180 consecutive patients undergoing allogeneic hsct between 01/2008 and 06/2018 at the fondazione irccs ca' granda maggiore policlinico hospital in milan, italy, were retrospectively collected. kaplan meier estimates were used for the analysis of survival outcomes while nrm, relapse and acute gvhd cumulative incidences were investigated by competing risk analysis. results: the patient series included 105 ab0-match, 31 major ab0-mismatch, 34 minor ab0-mismatched and 10 bidirectionally ab0-mismatch transplants. indication for allo-hsct were mainly aml/mds (77 pts), all (34 pts) and t-nhl/ctcl (32 pts). mean overall survival for groups of patients undergoing ab0-identical, major ab0 mismatch and minor ab0 mismatch hsct were 66 months (95% ci [55 ;77]), 47 months (95% ci [28; 65) and 46 months (95% ci [31; 61]), respectively. nrm in the three groups were significantly different, with point estimates of 12%, 29% and 26% at 5 years, respectively, whereas no significant differences were observed for relapse rate and graft failure incidence. although not statistically different, incidence of acute grade iii-iv gvhd was twice as high in patients transplanted from minor ab0mismatched donors than in the ab0 identical group (16% vs 8%). following transplantation, prc transfusion requirement was significantly higher in the major ab0 mismatch then in the ab0-match transplanted patients (median 14 vs 7, p=0.01), with a marginal positive correlation between the anti-donor a/b igg titers measured prior hsct and the total number of prc transfused during the first year following transplantation. we observed only one case of prca occurring in a 50year-old 0+ woman who was transplanted from a 32-yearold male a+ hla-identical sibling using peripheral blood as the stem cell source following a myeloablative conditioning for aml in first complete remission. anti-a igg isoagglutinin titers prior to transplantation were 1:256. during the first year post transplantation, the patient required a total of 46 prc transfusions, with gradual resolution occurring only after introduction of danazole treatment. conclusions: in our patient cohort, both major and minor ab0 mismatch associated to a significantly higher nrm. major ab0 mismatch associated to a higher prc transfusion requirement. a more frequently occurring severe acute gvhd was also suggested in minor ab0-mismatch transplants. altogether, our results suggest that allo-hsct outcome may be significantly affected by ab0 blood group mismatch. disclosure: nothing to declare background: at-tma is a severe endothelial injury complication and it may involve the intestinal vasculature. intestinal tma could be fatal and missdiagnosed. clinical and pathological criteria to differentiate from intestinal gvhd are needed. the aim of this study was to analyze the incidence and histological characteristics of intestinal tma in patients diagnosed of systemic tma. methods: we analyzed the incidence of tma in 555 patients who underwent allo-hsct in our institution between january 2010-august 2018. tma diagnosis was based on ho criteria. we do a pathological review in 103 biopsies from 25 out of 52 patients in whom an endoscopy have been performed 30 days before and 60 days after the diagnosis of tma for suspicious of gvhd. review was performed by a pathologist expert in gvhd, who examined the biopsies in search of hystopathological features of gvhd, tma or viral infection. diagnosis of gastrointestinal gvhd was stablished according to mcdonald and sales criteria, while intestinal tma diagnosis was stablished by warren et al criteria. results: 52 out of 555 patients (9,4%) were diagnosed of tma. transplant characteristics and tma data of patients with systemic tma are shown in image. 47 out of 52 patients with tma (90%) had been diagnosed with prior/ simultaneous acute gvhd, 20 of them grade iii-iv, and 80% with gastrointestinal gvhd. intestinal tma have been reported only in 7 out of 25 patients (28%) at diagnosis, whereas when review based on warren criteria was performed, in 19 patients (76%) the pathologist found at least 1 of the criteria of endothelial damage and 48 % of the patients 3 or more warren criteria were founded. the most frequent features were endothelial cell swelling (n=16, 64%) and perivascular mucosal hemorrhage (n=15, 60%). review hystological features of biopsies are shown in table 3 of the image. regarding gvhd, it was found in 21 patients (84%) at diagnosis and in 23 (92%) at pathological review. with a median follow-up of 10 months (1-73) 32 patients of the 52 with systemic tma (62%) are dead. 9 of the deaths (41%) were related to tma (3 tma, 3 tma +gvhd, and 3 tma+infection). patients with 3 or more warren criteria in pathological review had poor outcome compared with patients less than 3 criteria (30% alive vs 51% at 12 months, p=0.9). conclusions: intestinal tma is a life-threatening underdiagnosed entity. only 7 patients of 25 patients were diagnosed of intestinal tma. we found that most of our patients had endothelial damage in the gastrointestinal biopsy pathological reviews. gvhd histological criteria were present in most of the patients, mainly histological grade i-ii. prognosis of these patients is poor and pathologist effords in diagnosed the entity is guarranted. disclosure: nothing to disclosure p175 strategies to reduce neutropenic fever and hospital readmission in multiple myeloma patients managed at home after autologous stem cell transplantation background: neutropenic fever (nf) is the most frequent cause of readmission in the outpatient autologous stem cell transplantation (asct) programs. in our at home model for multiple myeloma patients, we added primary prophylaxis with ceftriaxone, decreasing the incidence of fever during aplasia phase from 85% to 57.6%. the aim of this study was to analyze the addition of two strategies to reduce the non-infectious nf: withdrawal of g-csf and the addition of primary prophylaxis for engraftment syndrome with corticosteroids after asct. methods: between january 2002 and august 2018 111 myeloma patients were managed at-home since day +1 of asct. all were conditioned with mel200. all patients received prophylaxis with quinolone, fluconazole, aerolized pentamidine, low-dose acyclovir (hvs+), and ceftriaxone (since day +4). the patients were classified into 3 groups: group a (n=33; g-csf since day +7 without corticosteroid), group b (n=32; no g-csf and no corticosteroid), group c (n=46; no g-csf with prednisone 0.5 mg/kg/day since day +7 until granulocyte recovery). first-line therapy at home of nf was piperacillin-tazobactam 4.5 g/6h i.v. using a portable intermittent infusion pump. fever was an indication of immediate medical consultation and those patients presenting signs of focal infection or severe sepsis were admitted. other indications for readmission were: willingness of the patient or caregiver, uncontrolled nausea, vomiting or diarrhoea, and mucositis requiring total parenteral nutrition or i.v. morphics. results: the main characteristics of the patients and outcomes are shown in table 1. there were no differences between groups regarding age, gender, immunological subtype, response before asct, hct-ci, and cd34 cell dose infused. there were more patients with advanced disease (iss iii) in group c compared to group a (19.5% vs. 6.1%; p=0.003). the duration of neutropenia was longer in those groups that did not receive g-csf (a: 8 days, b: 11 days, c: 10 days; p< 0.01). comparing group a with group c, we observed that the incidence of nf and the readmissions rates were lower in group c (nf: 57.6% vs. 23.9%; p=0.002; relative risk reduction: 0.41, and number needed to treat 2.97; readmissions: 12.1% vs. 2.2%; p=0.07, respectively). the 10-day cumulative incidence of nf were 54.5% in group a, 40.6% in group b, and 23.9% in group c; p=0.009. the non-administration of g-csf with the addition of prophylactic corticosteroid did not modify the incidence and grade of mucositis, the first day and duration of fever, nor the number of bacterial infections documented. in the multivariate analysis, this combination (no g-csf with corticosteroid) maintained its protective effect for the development of nf and hospital readmission (or 0.07; p=0.001 and or 0.07; p=0.05, respectively). conclusions: the non-use of g-csf and the addition of prophylactic corticosteroid in mm patients managed at home after asct minimize the incidence of non-infectious fever and optimize hospital resources by reducing hospital readmissions. disclosure: nothing to declare. background: antibody titers to vaccine-preventable diseases decline during the 1-10 years after allogeneic hematopoietic stem cell transplantation (hsct) if the recipient is not revaccinated. it is therefore considered best practice to try to offer hsct recipients the same level of protection against all vaccine preventable diseases as the general population. few data in the literature are available concerning vaccine-related problems in hsct recipients. we performed a farmacovigilance evaluation in a cohort of allotransplanted patients followed in our clinic during a 1 year period. methods: from october 2017 to november 2018 we administered a list of recommended vaccines to 49 hsct recipients attending our routine out patient clinic who fulfilled the following criteria: cd4 t cells>200/μl, cd19 b cells>20/μl, anti-cd20 antibody infusion>6 months, ivig therapy>2 months, no active and severe graft-versus-host-disease (gvhd), no chemotherapy or biological therapeutic agents on going. vaccines suggested were influenza, pneumococcal conjugate (pcv13), polio (inactivated polio vaccine), diphteria, tetanus, acellular pertussis, hepatitis b, hepatitis a, haemophilus influenzae type b, meningococcal quadrivalent (mcv4), human papillomavirus, meningococcal b, measles-mumps-rubella (mmr), varicella. live vaccines (mmr and varicella) were not recommended before 2 years after hsct and in patients with chronic gvhd. all the patients were asked to take the list to the local health facilities in order to have the vaccines injected and a vaccination table arranged with the doses already received and those to receive. we checked the vaccination tables at each visit and monitored potential side effects and gvhd status at 3, 6, and 12 months after the first vaccine injection. results: twenty-nine out of 49 patients were evaluable (table 1), 16 without gvhd and 13 with chronic gvhd (5 mild, 4 moderate, 4 severe). median time after hsct was 34 months (16-240). median number of vaccines received was 8 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) . as regards patients without chronic gvhd, 2 out of 16 experienced fever after vaccine injections; 1 out of 16 developed transient mild reduction of platelet count; 1 patient reported headache and otalgia after vaccine injection, while another one transient joint pain; 1 out of 16 patients presented signs of mouth chronic gvhd (score 1 nih) and transaminase increase (grade 1 according to world health organization toxicity scale) 3 months after the first vaccine dose, so that cyclosporine dose had to be augmented. as regards patients with chronic gvhd, 4 out of 13 experienced fever after vaccine injections; 2 patients with mild chronic gvhd of the mouth presented hepatic flare two and three months after the first vaccine dose, respectively. in both cases a new increase of cyclosporin and methylprednisolone doses determined progressive normalization of liver enzymes. conclusions: these data show that vaccines were globally well tolerated in hsct recipients, even when they suffered from chronic gvhd. however, close monitoring is warranted in order to better evaluate possible vaccine side effects in this setting of patients. background: allogeneic hsct improves survival for aml patients over the age of 60 years of age when compared to chemotherapy alone. the haematopoietic stem cell transplantation comorbidity index (hct-ci) and ebmt score predict for non-relapse mortality and overall survival, yet little is known about whether qol is preserved in this patient group and whether hct-ci and other performance scores pre-bmt correlate with qol post allo-hsct. methods: we conducted a retrospective analysis of patients 60 years and older who underwent ric allo-hsct at the university hospital of wales, cardiff between september 2011 and december 2017 (n=41). hct-ci, karnofsky performance score (kps) and ebmt scores were calculated prior to transplant and qol measured using the fact bmt (version 4) questionnaire, which was completed at 3, 6 and 12 months post transplant. patients were grouped at the 3-, 6-and 12-month time points for each of the different performance indices, allowing group comparison against compound sub scores using the mann-whitney u test. results: 41 patients were included in this study, with median age 65 years (range 60-74). patient characteristics, including conditioning, donor type, pre-transplant hct-ci and kps scores are summarised in table 1. the 2 year and 5 year overall survival (os) for the patient cohort was 65.4% and 48.7% respectively. hct-ci of ≥3 vs 0 was significantly associated with poorer bmt-related qol domains at 3 months (p=0.035) and general qol domains at 6 months (p=0.025) post-transplant. while ebmt score showed no correlation with qol parameters, patients with kps of 100 vs ≤90 showed significant differences in both general (p=0.01) and bmt-related qol (p=0.04) at 3 months and in all qol domains at 6 months (symptomrelated qol p=0.05, general qol p=0.01, bmt-related qol p=0.01). importantly neither the hct-ci nor the kps pre-transplant predicted for qol at 12 months post transplant. conclusions: patient selection is key to ensuring maximum benefit from allo-hsct both in terms of overall survival but also with regards to qol and survivorship. we note that while patients with hct-ci ³3 or kps ≤90 had significantly poorer qol at 6 months post allo-hsct, qol was recovered by 12 months post transplant, with this significant difference no longer seen. our data shows that in selected aml patients over the age of 60 years with good performance status and low comorbidity index, a favourable outcome can be achieved with good qol maintained throughout the post transplant period. background: advances in allosct technology, supportive care, and use of reduced intensity conditioning regimens for older patients have led to significant improvements in longterm survival after transplant. the survivors have an elevated probability of late morbidity and mortality, including abnormalities in phosphocalcic metabolism and bone disease. rapid and progressive bone loss occurs within the first 6-12 months after transplant, and this is followed by a slow process of recovery, with bone loss persisting for 48 to 120 months. bone fractures can worsen the quality of life of allosct survivors, but the real burden of the disease is unknown. the objective of the study is to ascertain the prevalence of bone pathology and vertebral fractures early after transplant in our center. methods: this is a retrospective and observational study. forty-nine patients (25 male/24 female, median age 54y, range 19-69) that underwent allosct were included in the study in the period of 6 to 24 months after transplant (may 2016-december 2017). pre-and post-transplant risk factors associated with bone disease were recorded: age >65 years, female sex, menopause, hormone replacement therapy, previous treatment with steroids, previous fractures, weight < 40 kg, bmi < 20-25, low physical activity, low calcium intake, smoking, alcohol intake, and history of femoral fractures in parents. in all patients laboratory data (including serum calcium, 25-hydroxyvitamin d, and pth), lumbar and femoral bmd (dxa), and spinal x-ray were also evaluated. a vertebral fracture was defined as a reduction of >20% in the anterior, middle or posterior high of the vertebral body. results: we identified vertebral fractures in 12 (24%) patients. five patients had fractures prior to transplantation, and 7 patients presented "de novo" vertebral fractures following transplantation; therefore, the prevalence of "de novo" postransplant fractures was 7/49 (14%). most (85%) of these fractures were asymptomatic at the time of diagnosis. most patients (64%) with vertebral fractures had >3 pre-sct risk factors (median risk factors pre-sct 3, range 2-6), the most frequent being low calcium intake, steroid exposure, presence of previous fractures, and menopause. those patients with fractures and less than 3 risk factors pre-tph, added new risk factors after transplant, mainly steroid treatment. forty-four patients (90%) had vitamin d insufficiency (< 30ng/ml), 15 (32%) had osteopenia and 9 (18%) had osteoporosis. vitamin d insufficiency and bone disease were more frequent in women than in men (98% vs. 84% for vitamin d, 37% vs. 28% for osteopenia, 29% vs. 8% for osteoporosis, and 25% vs. 20% for vertebral fractures, respectively). conclusions: the prevalence of post-transplant bone disease and vertebral fractures in our series is high. most fractures appearing "de novo" after allosct were asymptomatic and were diagnosed by x-ray. patients who presented vertebral fractures frequently had more than 3 risk factors identified pre-sct. patients undergoing allosct should have their bone health assessed early in their treatment and, if indicated, should start preventative therapy to avoid bone loss and fractures. other measures such as physical exercise, vitamin d and calcium supplementation, and dxa and spinal x-ray at baseline and following transplantation are also highly recommended. disclosure: maría suárez-lledó received a grant from dkms-spain foundation. other authors have nothing to declare the use of g-csf in selected patients after autologous stem cell transplantation is associated with low incidence of engraftment syndrome background: the use of g-csf after autologous stem cell transplantation (asct) accelerates neutrophil recovery, however it has been related to an increased risk of engraftment syndrome (es) development in some studies. for this reason, we do not routinely prescribe g-csf after asct and we only use it in patients with significant complications (enterocolitis, severe sepsis, atrial fibrillation) after stem cell infusion. the main objective of this study is to evaluate the incidence of es in patients who receive asct for monoclonal gammopathies (mg), non-hodgkin lymphoma (nhl) and hodgkin lymphoma (hl) and receive g-csf only if needed. as secondary objectives we evaluate differences in the engraftment day as well as the length of inpatient stay. methods: we retrospectively analyzed patients with mg or lymphoma, who underwent asct conditioned with high dose melphalan (140-200 mg/m 2 ) or beam, respectively, between 2015 and 2017 in our center. specific clinical features for es according to spitzer and maiolino criteria were evaluated between 3 days before and 7 days after the engraftment. statistical analysis was performed with spss v. 15.0. results: thirty-one patients with mg and 34 patients with lymphoma were analyzed. median age at transplant was 56.8 years (48.7-65.4 ) and 41 patients (63.1%) were male. median prior lines of treatment in patients with gm or lymphoma were 1 (1-3) and 2 (1-5), respectively. table 1 shows patients´characteristics. mobilization with g-csf ± plerixafor was performed in 27 patients (36%) and chemotherapy + g-csf ± plerixafor in 38 patients (64%). median cd34 x 10 6 /kg cells infused was 3.6 (2.7-5.3). eleven patients (16.9%) received g-csf, 5 due to infection (2 enterocolitis, 1 listeriosis, 1 acute hepatitis, 1 septic shock) and 6 because of atrial fibrillation or fibrilloflutter. median time from sct to first day of g-csf was 5 days (5-7) and median time on g-csf treatment was 5 days (4-7). patients who received g-csf showed a short time to neutrophil engraftment (≥0.5x10 6 /l), 10 days vs. 13 days, p< 0.001 but longer duration of hospitalization, 18 days vs. 15 days, p =0.050. non-relapse mortality at day +30, +100 and +180 was 0%. es was diagnosed in 4 (6.2%) patients, 1 amyloidosis, 2 multiple myeloma and 1 plasmablastic lymphoma. there was not statistical difference in the incidence of es between patients who received g-csf (9.1%) and patients who did not (5.6%), p=0.533. analyzed by disease, es appeared in 1 of 6 patients who received g-csf in the lymphoma group (16.6%) but none of the 7 patients with mg that received g-csf developed it. we did not find statistical differences between patients who developed es and those who did not in age (49 years vs. 56 years, p=0. 314), length of hospitalization (19 days vs. 15 days, p=0.185) and the number of cd34 x 10 6 /kg cells infused (3.65 vs. 4.62, p=0.408) . conclusions: the use of g-csf in selected patients is associated with low incidence of es. our study confirms that the use of g-csf accelerates neutrophil recovery but it is unclear if it can increase the incidence of es, especially in patients with lymphoma. [[p180 image] 1. background: graft failure is one of the top-3 problems of allo-hsct (after gvhd and relapse). the problem of graft failure becomes more significant due to increasing number of allo-hsct with ric conditioning regimen from haploidentical and hla-mismatched unrelated donors. role of t cells in graft failure is well known. here we report an impact of t-memory cell subsets count before antithymocyte globulin (atg) administration on primary graft failure after allo-hsct. methods: sixteen patients with acute leukemia transplanted in national research center for hematology were included on this prospective study. all patients received horse atg at dose 10 mg/kg/day from day -4 to -1 before allo-hsct as gvhd prophylaxis and were balanced by other factors that could affect engraftment. detailed patients characteristics are listed in table 1 . peripheral blood samples were collected on day -4 before allo-hsct (before atg injection) in edta-tubes. flow cytometry analysis was performed on bd facs canto ii (becton dickinson, usa) to define t-memory subsets: t-naive and t-stem cell memory (tnv+scm) -cd45r0-ccr7+cd28+; t-central memory (tcm) -cd45r0+ccr7+cd28+; t-transitional memory (ttm) -cd45r0+ccr7-cd28+; t-effector memory (tem) -cd45r0+ccr7-cd28-; t-terminal effector (tte) -cd45r0-ccr7-cd28-, among cd4+ and cd8+ t-cells . sysmex xe-2100 was used to calculate absolute count of different t-cell subsets. mann-whitney u test was used for nonparametric data analysis between two groups. fisher's exact test was used for 2x2 tables. p-value less than 0.05 was considered statistically significant. results: an influence of t-memory cell subsets count before atg administration on primary graft failure is shown in figure 1 . according to our data high absolute number of cd4+ttm and cd4+tte is associated with primary graft failure. conclusions: based on these findings high absolute number of cd4+ttm and cd4+tte could be one of the prognostic factors of primary graft failure after allo-hsct. optimizing atg dose due to recipient absolute t-memory cell subsets count before atg administering may prevent graft failure and improve posttransplant results. background: upper gastrointestinal graft-versus-host disease (gi gvhd) has been an increasingly recognised entity following allogeneic stem cell transplantation (sct). budesonide, widely used in inflammatory bowel conditions, has also been found beneficial in gi gvhd. the major benefit of budesonide is attributable to its poor absorption and extensive first-pass metabolism via cytochrome p450 (cyp) 3a4, which translates to less systemic steroid-related effects. however, transplant patients are often exposed to multiple drugs, among which some agents act as cyp3a4 inhibitors and therefore can increase budesonide bioavailability and might lead to systemic toxicity. azole antifungal drugs are probably the most common concomitantly used cyp3a4 inhibitors in transplant recipients. methods: we reviewed allogeneic sct records for patients treated with oral budesonide for gi gvhd at our transplant centre between 2015 and 2018 retrospectively. the aim of the work was to assess the development of adrenal suppression with or without clinical features of iatrogenic cushing`s syndrome. the standard dose of budesonide was 3 mg three times a day. patients receiving prednisolone or other glucocorticosteroids and those with no available serum cortisol level measurements were excluded. results: our analyses identified four allogeneic sct patients in whom adrenal suppression was diagnosed with undetectable serum cortisol levels during oral budesonide treatment. of these patients two developed iatrogenic cushing`s syndrome and both patients were treated with cyp3a4 inhibitors concomitantly: 1. clarithromycin and fluconazole; 2. clarithomycin and voriconazole. the development was rapid (within 3 and 4 weeks). symptoms included morphological features such as moon face, high blood pressure, weight gain, peripheral oedema and proximal myopathy. symptoms resolved gradually following cessation of azole antifungal agents and on gradual weaning of budesonide. conclusions: although single agent budesonide treatment given for gi gvhd is rarely associated with systemic side effects, patients on azole antifungal drugs and macrolide antibiotics are at higher risk of systemic toxicity due to drug interactions. patients who are allergic to penicillin and receive macrolide-based prophylaxis can be especially vulnerable. to our knowledge the number of cases reported in literature about systemic effects of oral budesonide in transplant recipients is less than 10. our observation supports previous reports on the potential of oral budesonide to induce systemic effects. we therefore advise careful monitoring of patients treated with budesonide in combination with cyp3a4 inhibitors, including antimicrobial agents routinely used in sct. disclosure: none implemented strategies to overcome barriers in the establishment of a consolidated hematopoietic stem cell transplantation program in a developing country background: the national institute of medical sciences and nutrition "salvador zubiran" is a national health institute located in mexico city. although mexico is considered an upper-middle income country, more than 50% of the population lives in poverty without health care coverage and patients within this social stratum are referred to our institution. the first hematopoietic stem cell transplantation (hsct) in mexico was performed at our institution in 1980. from this year until 1997, hsct were sporadically performed (n=33), showing a poor overall survival (os) and high non-relapse mortality (nrm). these outcomes resulted from an unstructured hsct program, limitedresources, patient low socioeconomic status, and paucity of population-adapted procedures. in 1998, according to these results, a decision to establish a hsct program was made. therefore, in order to set up a successful hsct program, implementation of financial and medical strategies were necessary. the objectives of this study were to describe the barriers and implemented strategies for the establishment of a hsct program in mexico along with the outcomes of patients undergoing this procedure throughout the reorganization of the program. methods: this study is a health services research. barriers were detected based on the results of the hsct program from 1980-1997 (not shown). table 1 shows the financial, medical, and research strategies that were implemented for each barrier. results: from november 1998 to november 2018, 363 hsct have been performed in 322 patients at our institution. most hsct were autologous (n=213, 59%). forty one patients underwent 2 hsct. from the 322 patients, most were males (n=196, 61%) and the median age was 33.5 years (range, 15-65). the most frequent underlying diseases for auto-hsct were lymphomas (n=68, 36%), non-seminomatous germ cell tumors (n=42, 22%), and multiple myeloma (n=42, 22%). acute leukemias (n=41, 34%), aplastic anemia (n=25, 21%), and myelodysplastic syndromes (n=20, 17%) were the most frequent diagnosis for patients undergoing allo-hsct; and acute leukemia was the most frequent diagnosis for patients undergoing haploidentical hsct (n=10, 83%). acute and chronic gvhd were present in 25% (grades i-ii 89%) and 35% (limited 76%), respectively. for allo-hsct, 30, 100day, and 1-year nrm was 2.5%, 8%, and 12%, respectively; 30 and 100-day nrm in auto-hsct was 1.5%; 10year os was 63% and 56% for auto and allo-hsct, respectively. conclusions: future perspectives of the hsct program include the acquisition of funds for unrelated donors; to improve outcomes of patients undergoing haploidentical hsct, and to increase the number of in-patient rooms. we conclude that despite paucity of resources and other limitations, the implementation of financial, medical, and research strategies have shown that barriers can be effectively overcome in a developing country in order to establish a consolidated and nationally renowned hsct program, providing good outcomes for patients. disclosure: none of the authors have any conflict of interest to disclose. the effect of protective buffering on daily stress and relationship quality in dyads following hematopoietic stem cell transplantation: results from daily process methodology malgorzata sobczyk-kruszelnicka 1 , aleksandra kroemeke 2 , zuzanna kwissa-gajewska 2 , sebastian giebel 1 background: cancer-related support communication (e.g., protective buffering) may impact the risk for psychological and relationship distress in patients following hematopoietic stem cell transplantation (hsct) and their caregivers. previous studies have revealed that protective buffering (i.e., hiding one's concerns and denying one's worries) has mixed effects: is beneficial (for "protected" person), costly (especially for the person using it), or unrelated to dyadic wellbeing. there has been, however, little evidence linking dyadic protective buffering with distress using daily process methodology. we assessed (1) the relationship between daily protective buffering, and same-and next-day stress and relationship quality in patient-caregiver dyads following hsct and (2) whether similarity or complementarity in protective buffering between dyads is adaptive. methods: two hundred patients (after first autologous or allogeneic hsct) and their caregivers (spouse or another relative) independently completed measures of daily protective buffering, daily relationship quality, and daily stress for 28 consecutive evenings after patients´hospital discharge. actor-partner-interdependence model (i.e., both partners' and caregivers' reports regarding support communication and distress were studied) was used to test study hypotheses. results: for both patients and caregivers, multilevel structural equation modeling showed a significant positive relationship between daily protective buffering and sameday relationship quality. association of protective buffering with same-day stress level was negative. in next-day analyses, patient-reported protective buffering was related to patient's higher relationship quality, whereas caregiverreported protective buffering increased patient's daily stress. complementarity in protective buffering was related to higher immediate same-day relationship quality for both patients and caregivers, while benefits from similarity have delayed effects, although only in patients. conclusions: contrary to previous studies, protective buffering rather has a beneficial effect in dyads following hsct. protection of the partner and relationship against revealing negative emotions and powerlessness was not related to costs in both parties. the findings suggest that the effect of daily protective buffering in dyads following hsct depends on support timing (same-or next-day effect) and differs for both parties. patients seem to benefit the most from the similarity in protective buffering, while caregivers from complementarity. the "fit" between patient and caregiver in support communication ought to be taken into consideration in the practical approach. disclosure: nothing to declare. virus reactivation and low dose of cd34+ cell were associatied with secondary poor graft function within the first 100 days after allogeneic stem cell transplantation yuqian sun 1 , xiao-jun huang 1 background: secondary poof graft function (spgf) was defined as the secondary cytopenia after initial engraftment of hsct. it was shown to be associated with poor prognosis, however there are very few reports on the incidence, risk factors and outcomes of spgf. methods: patients who received transplantation from peking university people's hospitial during january, 2015 to december, 2015 were retrospectively reviewed if they fulfilled the following conditions: (1) diagnosed with acute leukemia or myelodysplastic syndrome; (2) received allo-sct from either matched sibling donor (msd) or haploidentical related donor (hid). pgf was defined as persistent neutropenia (≤0.5×10 9 /l), thrombocytopenia (platelets ≤20×10 9 /l), and/or hemoglobin ≤70 g/l for at least 3 consecutive days, transfusion-dependence, associated with hypoplastic-aplastic bone marrow (bm), and complete donor chimerism without concurrent graftversus-host disease (gvhd) or disease relapse. primary pgf was defined as the failure to achieve initial engraftment by days 28 after transplantation, while secondary pgf was defined as the fulfillment of the criteria after initial engraftment hsct. results: during january, 2015 to december, 2015, 564 patients who received transplantation from peking university people's hospitial were retrospectively reviewed. among the 490 patients who achieved initial engraftment, 28 patients developed spgf. the cumulative incidence of spgf on day 100 was 5.7%. the median time of secondary pgf was 54.5 (34-91) days after transplantation. low (< median) cd34+ cell dose (p=0.019, hr 3.07(95%ci, 1.207-7.813)), ebv reactivation (p=0.009, hr 3.648(95%ci, 1.382-9.629)) and cmv reactivation (p=0.003, hr 7.827 (95%ci, 2.002-30.602)) were identified as independent risk factors with spgf. there is no significant difference of pgf incidence in msd group and hid patients (p=0.44). the overall survival of patients with spgf at 1 year after transplantation was significantly poor than patients with ggf (50.5% versus 87.2%, p< 0.001). conclusions: in conclusion, spgf develop in 5.7% patients after allo-sct, especially in patients with cmv, ebv reactivation or infused with low dose of cd34+ cell. the prognosis of spgf is still poor due to lack of standard treatment. disclosure: there is no conflict of interet thiotepa with treosulfan and busulfan based conditioning are significantly more gonadotoxic than treosulfan previous studies suggest that busulfan results in long-term gonadal toxicity. no previous studies have compared gonadal toxicity outcomes after treatment with busulfan with treosulfan, a newer agent with similar marrow toxicity to busulfan but with reduced non-marrow toxcitiy. our aim was to determine whether there are differences in pubertal and fertility outcomes in paediatric patients treated with treosulfan compared with busulfan. methods: inclusion criteria were patients who had received either busulfan or treosulfan or treosulfan with thiotepa, only one hct and were aged 14 years and above in august 2018. eligible patients were reviewed in clinic as part of their routine follow-up, thus research ethical approval was not required. follice stimulating hormone, luteinising hormone, oestradiol, and pubertal history were noted. ovarian reserve was estimated in female patients by measuring serum anti-mullerian hormone (amh). male patients had serum testosterone measured and were also offered semen analysis. results: thirty-five patients met the inclusion criteria, of which twenty-five wanted to be reviewed (71%); seventeen females and eight males. mean age at hct was 13 years, mean age at review was 19 years and mean years since hct was 5 years. female patients treated with busulfan or treosulfan with thiotepa (n=14) had minimal amh and none of these patients were having regular periods. females treated with treosulfan (n=3) had normal amh and regular periods without needing hormone replacement. only four male patients opted for a semen analysis and all had significantly reduced sperm counts. conclusions: our results suggest that females treated with treosulfan have minimal (if any) reduction in ovarian reserve compared to other conditioning regimens which casue significant compromise. although this was a small study, and thus not suitable for statistical analysis, the clinical findings are marked. future studies should further investigate optimal doses of treosulfan that could be used to achieve bone marrow engraftment and limit long-term effects on fertility. disclosure background: autologous and allogenic hematopoietic stem cell transplantation (hsct) are potentially curative treatments for hematological malignancies. patients with related complications may need admission to the intensive care unit (icu) for specific therapy and organ support. mortality risk factors, supportive care and principal causes of admission in icu are described in our cohort of patients (pts). methods: we retrospectively studied 326 pts, 185 male, with a median age of 56,63 years (range: 18-73) who underwent allo-hsct in our center between july 2014 and october 2018. two hundred and twenty-seven(69,6%) pts received autologous hsct (auto-hct) and 99 (30,4%) allogenic hsct (allo-hsct); 50 from unrelated donor, 38 from identical sibling, and the remainder, mismatched related donor 11. twenty-three (7,1%) out of 326 pts were admitted in the icu in the transplant procedure admission. results: fifteen (65,2%) out of 23 pts were male with a median age of 55 years (range: 28-69). patients' baseline diseases were: multiple myeloma (34,8%), non-hodgkin´s lymphoma (26,1%), hodgkin´s lymphoma (8,7%), acute lymphoblastic leukemia (8,7%), myelodisplasic syndrome (8,7%), solid tumor (8,7) and acute myeloblastic leukemia (4,3%). fifteen (65,2%) pts received auto-hsct, 5 (21,7%) allo-hsct from unrelated donor, 2 (8,7%) allo-hsct from identical sibling, and the remainder haploidentical hsct (1) (4,3%). so, 6,6% of auto-hsct pts and 8% of allo-hsct were admitted in the icu. the median stay in the icu was 5 days (range: 1-30) and reasons for admission were: respiratory insufficiency (60,8%), septic shock (30,4%), renal insufficiency (4,3%) and multi-organic failure (4,3%). twenty-one (91.3%) pts required respiratory support with: nasal cannula or oxygen mask (c/m) (19%), non-invasive mechanical ventilation (nimv) (66,7%) and invasive mechanical ventilation (imv) (14,3%). fourteen (60%) pts needed inotropic agents for shock treatment. finally, 4 (4,5%) pts required substitutive renal therapy with hemodialysis or haemofiltration (hd/hf). eleven (47,8%) out of 23 pts died, 7 (63,6%) were male with a median age of 55 years (range: 24-64). ten of them (90,9%) needed imv and were treated with inotropic agents. all patients who required hd/hf (n=4) died. imv and treatment with inotropic agents were associated with icu mortality (or 6,5; p=0,03, or 7; p=0,008; respectively). conclusions: in our series of pts, 7,1% needed admission in the icu, presenting a mortality rate of 48% approximately. there were no differences in the prevalence of icu admission regarding hsct donor. main reason for admission was respiratory failure with imv requirement in 14,3% of pts. imv and treatment with inotropic agents were associated with icu mortality. an early identification of pts at risk of icu admission could have a beneficial impact on survival improvement disclosure: nothing to declare is there any association between thrombotic risk factors and veno-oclusive disease in childhood allogeneic hematopoietic stem cell transplantation? background: veno-oclusive disease (vod) is a major complication of hematopoietic stem cell transplantation (hsct). in some studies levels of fibrinolytic factors especially plasminogen activator inhibitor-1 (pai-1) level were found associated with vod. however, little is known about the relationship between thrombophilia risk factors and vod. in this study we aimed to investigate association of major thrombophilic gene mutations on vod in pediatric hsct patients. methods: we reviewed retrospectively 35 patients with vod who underwent hsct between 2010-2018 in ankara pediatrics and pediatric hematology-oncology training and education hospital, bone marrow transplantation unit, turkey. fifty-one patients who did not develop vod and transplanted during the study period were accepted as control group. we evaluated plasma homocysteine and lipoprotein a level, protein s and c activity and antigen levels and factor v g1691a mutation, prothrombin g20210a mutation, methylenetetrahydrofolatereductase (mthfr) c677t and a1298c mutations, plasminogen activator inhibitor-1 -675 4g/5g polymorphism before hsct. we also evaluated the patients' hospital files and noted the demographic values and complications of hsct. statistical investigations were done with spss statistics 17.0 for windows and p< 0.05 has been accepted as significant. results: there was no difference between control and vod groups as regard to age, sex, diagnosis, donor type, conditioning regimen, hsc source, and hla typing . there was no difference between the groups according to homocysteine, lipoprotein a, protein s and c activity and antigen levels. we did not find any relation between the genetic variations of thrombophilia and vod (table 1 ). in vod group there were 6 patients (17.1%) with acute graft versus host disease (agvhd) and in control group there were 7 (15.9%) patients with agvhd (p=0.046). febrile episodes were more frequent in vod group compared to the controls (respectively; n=30, 85.7% vs. n=23, 54.8%, p=0.006). 8-year overall survival was %77.1 in vod group and 100% in control group (p=0.001). disease free survival was also different between vod and control groups (respectively; 74.3% vs. 97.3%, p=0.001). conclusions: in literature there are recent studies showing higher pai-1 levels in patients with vod. however, in our study we did not find any relationship between congenital thrombophilia factors and vod. new studies with larger sample groups is necessary to better evaluate the association of congenital thrombophilia factors and vod. disclosure: nothing to declare p189 different strategies of chemotherapy-induced nausea and vomiting (cinv) prevention in hematological patients receiving an autologous hematopoietic stem cell transplantation: a single center experience ilaria cutini 1 , riccardo boncompagni 1 , chiara nozzoli 1 , antonella gozzini 1 , stefano guidi 1 , chiara innocenti 1 , massimo di gioia 1 , lorenzo tofani 1 , riccardo saccardi 1 background: despite the improvements of pharmacological control, cinv still represents a major problem in patient undergoing hematopoietic stem cell transplantation (hsct). we present here a comparison of two pharmacological strategies for preventing cinv in multiple myeloma (mm), hodgkin (hl), and non-hodgkin lymphoma (nhl) patients who received an autologous hsct in our institution. methods: from january 2015 to july 2018, we retrospectively analyzed 250 consecutive patients, median age 58 years (22-71yo) , diagnosed with mm, hl, and nhl, who underwent an autologous hsct following a melphalan 200 mg/sqm and beam/feam condition regimens, respectively. the first 122 patients received cinv prophylaxis with palonosetron i.v and dexamethasone 8 mg die (regimen a), whilst the following 128 were administered with fosaprepitant iv, ondansetron iv and dexamethasone 8 mg die (regimen b) both cinv prophylaxis was administered the day of melphalan infusion (day -1 form transplant). emesis breakthroughs were treated with alizapride and metoclopramide. nausea and vomiting were assessed through the ctcae 4.0 score system. categorical variables were compared with pearson chi-square test. results: the overall incidence of nausea was 78%, (55% grade 1, 41 % grade 2, and 4% grade 3, respectively). in regimen a was shown to be 80%, (56% grade1, 41% grade 2, and 3% grade 3, respectively) while in regimen b was 77% (54% grade 1, 41% grade 2, and 5% grade 3, respectively). pearson chi-square test did not show any differences between the 2 groups (p=0.679). the overall observed vomit was 32% (83% grade 1, 16% grade 2, and 1% grade 3). in regimen a it was (47% (84% grade 14% grade 2, and 2% grade 3), and 17% in regimen b (77% grade 1 and 23% grade 2). conditioning regimens didn't' have any significant impact on either nausea or vomit. patientsyounger then median (58 yrs), were reported to have higher incidence of both nausea, (p=0.028) not related to cinv treatment, and vomit (40% vs 24%, p=0.012). in multivariate analysis the overall incidence of nausea is related to age (younger patients have higher probability to develop nausea (or 2,282; p= 0,024) whilst the higher incidence of vomit is related to: regimen a (or 3.958; p< 0,001), previously reported nausea (or 4,506; p< 0,001), and no smoking habits (or 2,761; p=0,02). conclusions: both regimens are equally effective for nausea control however regimen b evidenced a better vomiting control. this finding is particularly relevant when the center policies include an early discharge program, therefore improving both patient's quality of life and procedure cost-effectiveness. clinical background: patients who underwent an allogeneic hematopoietic cell transplantation (hct) are challenged by medical, psychological and social complications. support groups might help hct-survivors to cope with these challenges. however, the existing literature about post-hct support groups is scarce. moreover, data on professionallyfacilitated support groups do not exist. the aim of this project was (1) to establish a professionally-facilitated support group and (2) to assess the discussed topics. methods: from 11/2013 until 6/2017 all patients who received an allogeneic hct at the adult stem cell transplantation program of the university clinic mannheim were invited to participate in a professionally-facilitated support group. additionally, spouses and life partners were invited. a theologian who is also a physician served as facilitator. he had no further function within the transplant team. the format of the group was unstructured without any rules regarding regular attendance. the facilitator did not provide topics or a curriculum. during the first year the group met every 14 days followed by a monthly schedule. from the fifth until the 39th meeting the attendance and the discussed topics were minuted by the facilitator. the content of the minutes was analysed by a combination of an inductive and a deductive approach. all participants provided their informed consent for the study. results: altogether 23 patients (female: n=10; male: n=13) and 10 spouses/life partners (female: n=9; male: n=1) participated. 13 patients (57%) and 6 spouses (60%) attended more than one meeting. among those who participated in ≥2 meetings the median time of participation was 16 months. the median count of participations was eight. 30% of the participants attended the meetings longer than one year, 9% longer than three years. there was no sex difference with respect to the frequency and the duration of participation. however, the frequency of participation decreased significantly the longer a participant was attending the meetings. during 35 group meetings the facilitator recorded 5138 thematically different contributions to the discussions divided in 37 distinct topics. these topics were grouped into 5 main categories [(a) medical topics, (b) private life and environment, (c) human relationships, (d) physical and mental condition and (e) the support group itself] and eight further categories [(1) compliance, (2) economic issues, (3) religion, (4) sexuality, (5) death and dying, (6) support and coping, (7) objectives and needs and (8) not otherwise specified issues] which could not be grouped in one of the main categories. the most frequent issues were medical topics (34%), human relationships (16%), physical and mental condition (15%), private life and environment (14%), financial issues (5%), the support group itself (4%), support and coping (4%) and objectives and needs (4%). noteworthy, death and dying (0.5%) were rare topics and sexuality was never mentioned. conclusions: to our knowledge, this is the first prospective and systematic analysis of a professionallyfacilitated support group for hct-survivors. these data might help to establish support groups and to identify psychosocial needs of patients and targets for specific support. disclosure: nothing to declare background: endothelial damage is associated with inflammatory complications that appear early after hsct, such as sinusoidal obstruction syndrome or acute gvhd. engraftment syndrome (es) is an inflammatory condition diagnosed by maiolino clinical score. potentially, es can exhibit high morbidity and mortality, especially after autologous-hsct in multiple myeloma (mm) patients since the introduction of new drugs such proteasome inhibitors and immunomodulatory drugs (imids). the objective of the present study was to evaluate if es is associated with endothelial dysfunction in patients with mm who underwent auto-hsct. methods: we included six patients with mm who received induction treatment including new drugs and consolidated their response with an autologous-hsct. we analysed comparatively the effect of incubating endothelial cells in vitro with serum samples from patients with es vs. no es. serum samples were collected before (pre), and after 5, 7, and 10 days from the transplant. an additional sample was collected at the es onset and at the discharge day (no es group). endothelial cells (hmec) in culture were exposed to media containing 20% of serum from each patient for 24h. cell growth was controlled morphologically. expression of the adhesion receptor icam-1 on the cell surface was analysed by immunofluorescence, and activation of the inflammation related p-38 mapk signalling pathway was evaluated by sds-page and western blot. results: exposure of hmec monolayers to sera from patients who developed es (onset day, n=4) resulted in an increased icam-1 expression on the cell surface, higher that the observed with sera from patients who did not develop es (discharge day, n=4) (26.4% of labelled area vs. 6.4%, respectively). in addition, in experiments with sera from patients not developing es, icam-1 expression on cells exposed to sera from day +10 was reduced with respect to the observed with sera from day +5, probably due to the corticosteroid used as a prophylaxis in our centre. this reduction was not observed in es patients. regarding phosphorylation of p-38, it was significantly higher in cells exposed to sera from es patients than in response to sera from patients who did not develop es. conclusions: the increase in the expression of the adhesion receptor icam-1 on the surface and the intracellular activation of p38mapk in endothelial cells exposed to sera from patients developing es indicates the existence of endothelial activation in association with es. interestingly, the prophylaxis of es with corticosteroid seems to be less effective in patients who developed es than in patients who did not develop this complication. these results need to be validated in a higher number of patients and modifications in additional markers of endothelial dysfunction should be investigated. disclosure: gonzalo gutiérrez-garcía: honoraria from gilead. grant from jazz pharmaceutical the other authors do not have any disclosure to comment. p192 association between uric acid levels before and after allogeneic haematopoetic stem cell transplant and transplant outcomes: a single centre experience background: uric acid (ua) is a known endogenous danger signal which activates the nod-like receptor protein (nlrp)3 inflasome.ua is released from injured cells during conditioning in allogeneic stem cell transplantation (hsct). a pre-clinical study has demonstrated that nlrp3 inflasome-mediated il-1 production regulates graft-versushost disease (gvhd). the ua role in inflammation and gvhd is unclear. there are discordant reports in the literature about a potential protective role of ua on gvhd after a hsct. methods: we performed a retrospective study to assess the association between serum ua levels pre-and posttable] 1. table 1 ] results: the characteristics of the 142 patients are shown in table 1 . median age was 52 years (range 15-69), and 80 patients (56%) were male. twenty-seven patients (19%) received low doses atg as part of gvhd prophylaxis. allopurinol was from the day before start of conditioning therapy until day 0. the median levels of ua were 4,8 mg/ dl before conditioning, 2,85 mg/dl at day 0, 3,1 mg/dl at day +7 and 3,2 mg/dl at day +14. there was no impact between the ua levels and os at any time of the hsct. ua levels at day +7 were associated with a higher ci relapse at 5 years (34% [95% ci, 20-49%] for ua level > 3,1 mg/dl, and 17% [95% ci, 8%-30%] for ua level ≤ 3,1 mg/dl [p= 0,046]). there was a trend for a higher ci of grade ii-iv agvhd for the subgroup of patients not treated with atg with ua < 4,8 mg/dl (48% vs 30%; p= 0,083) on day -8 and a higher nrm with ua < 2,85 on day 0 (50% vs 30%; p=0,080). conclusions: in our study the ua levels showed no impact on os, and only a tendency for ci of grades ii-iv agvhd grades ii-iv and nrm for the subgroup of patients not treated with atg. surprisingly, high levels of ua at day +7 of hsct were associated with a significant higher incidence of relapse. disclosure: dkms foundation, pi14/01971 (instituto carlos iii) and sgr288 (grc), generalitat de catalunya. background: veno-occlusive disease (vod) is an early, uncommon but serious complication of stem cell transplantation (sct) that is associated with high morbidity and mortality. defibrotide is the only licensed treatment for vod, and time to start of treatment (tst) affects outcomes. minor differences exist between the seattle, baltimore and classical ebmt (2016) criteria, which may trigger different start points for treatment. late onset vod (>21 days) is less recognised and we hypothesize, may have worse outcomes with longer time to diagnosis, and more limited treatment options across different healthcare systems. methods: electronic patient records from sept.2013 -oct.2018 at king´s bmt centre and pharmacy databases were reviewed, timepoint to clinical and bio-chemical manifestation of vod, diagnosis, tst, survival and longterm outcomes were analysed. results: 30 of the 532 patients(5.6%) who underwent an allogeneic sct, developed vod, including 2 paediatric cases. none of the autologous sct patients developed vod. the paediatric and autologous sct patients were not analysed any further. 28 adult patients (male=22;78.5%) developed vod at a median age of 56 years(range 26-72), of whom 21 developed < 21 days and 7 patients had late-onset vod as per ebmt criteria(range 22-93 days). 24 cases classed as severe and 4 as moderate vod. 10 patients received defibrotide at diagnosis, 7 patients within 3 days, 5 patients between 4-7 days, and 6 patients received treatment after 7 days. overall mortality for this cohort was 50%(14/28). 12/21 (57.1%) of patients with early onset vod and 2/7(28.5%) patients with late-onset vod died. of the 14 deaths, 10 died of liver failure and a further 2 patients had vod as a likely contributing factor in their deaths. 1 patient died with subarachnoid haemorrhage and 1 with relapsed disease. patients that received defibrotide after 7 days, 5/6 patients(83.3%) died, as compared to 3/5(60%) for treatments between 4-7 days, 6/17(35.2%) for treatments within 3 days. the lone surviving patient who received treatment after 7 days has severe chronic liver disease and it's complications. of the 21 patients who fit seattle criteria for early-onset vod, only 6 fit the baltimore or ebmt criteria for classical vod. 4 of these 6 patients met the baltimore criteria later than the seattle criteria were met(range = 2-9 days). conclusions: vod carries high morbidity and mortality, and beyond the known risk factors and with the caveat of limited numbers in this study, we strongly suspect this is further increased when time to definitive treatment with defibrotide is delayed, particularly beyond 7 days. nearly a quarter of cases with vod are late-onset as per classical ebmt criteria. however contrary to our hypothesis, their overall outcomes and mortality do not appear worse, with time to treatment again emerging as a strong predictive factor. conditioning treatment related factors, which play a stronger role in endothelial dysfunction in the hepato-portal circulation, may not be as much at play, perhaps for late-onset disease. uniformity in the use of diagnostic criteria, and high degree of vigilance, even beyond 21 days, leading to early treatments may improve outcomes in vod. disclosure: nothing to declare background: hsct-associated thrombotic microangiopathy (ta-tma) affects 10-30% of patients receiving an allogenic sct, with a high mortality up to 80-90% in severe cases. endothelial injury mediated by complement activation has been atribuited a major role in the pathogenesis, and blockade of c5 with eculizumab offers promising results. methods: we present our experience with 6 pediatric cases of ta-tma treated with eculizumab. the diagnosis of ta-tma was stablished attending to jodele et al criteria. clinical data were collected retrospectively from medical records. results: all cases were diagnosed between august 2016 and april 2018, with a median age of 11 years (2.5 -17) at time of diagnosis. primary disease was acute leukemia in 2 cases (1 all and 1 aml), severe aplastic anemia in 3, and primary immunodeficiency in 1. they received their first sct in all cases, 3 from mud and 3 from mmrd (cd45ra+ depleted haploidentical grafts), with mac regimen in 2 cases, and ric in 4 cases. 3 of them received calcineurin inhibitors (cyclosporine) as gvhd prophylaxis. all patients developed agvhd (grade 2 or higher in 3 cases). and 5 patients presented viral reactivation. hypertension was present in 4 cases at tma diagnosis, requiring 2 or more antihypertensive drugs in 3 of them. all patients had renal injury consisting of less-than-normal glomerular filtration rate (median of 41 (20-59)) and proteinuria, with urine protein-to-creatinine ratio higher tan 2 mg/mg in 2 cases (data not available in 2 patients). serum haptoglobin was decreased in just 2 cases at diagnosis, and schistocytes were detected in 3 patients. cutaneos signs were present in all cases, digestive symptoms in 2, neurological affection in 2, and notoriously all of them developed polyserositis. c3 and c4 were normal in all cases, with sc5b9 higher than 244 ng/ml in 2 patients and lower in 1 (data not available in 3 cases). all patients received defibrotide as treatment, and 4 cases received also rituximab, associated to therapeutical plasma exchange in 3. all of them received eculizumab, as first line in 2 cases (median of 40 days between diagnosis and eculizumab start). treatment was correctly monitorized with ch50 levels in 3 cases (not available quick enough in other 3). median number of doses needed in induction therapy was 8, and median interval between doses was 7 days. 2 patients required reduced interval and higher doses to maintain ch50 supressed. 2 patients did not respond, and died because of tma. 4 patients had hematological response, with chronic renal injury in 3 of them and resolution of acute renal failure in 1 case. nevertheless 1 patient responding to eculizumab died because of tma related complications, and 1 because of an invasive fungal infection. 2 patients are alive, with a median follow up of 6 months from treatment start. conclusions: our experience supports promising results of eculizumab based treatment for ta-tma, highlighting the importance of an early treatment and a careful therapy monitoring by ch50 supression. prospective studies are needed to achieve a better knowledge of this pathology and its treatment. disclosure: nothing to declare background: approximately 40-50% of allogeneic hematopoietic stem cell transplant (allo-hsct) are made with some sort of abo blood group system incompatibility. an hsct abo donor-recipient incompatibility implies risks of complications during the process of infusion as acute hemolytic anemia (ah), delayed graft and other later complications due to the presence of isohemaglutinins (pure red cell aplasia or passenger lymphocyte syndrome). also, abo incompatibility could impact on graft versus host disease (gvhd) incidence, and could be associated with not relapse mortality (nrm) and overall survival (os). there are not concluded evidence about the abo incompatibility impact, so the aim of this study was to identify complications and response associated with abo incompatibility in patients undergoing allogeneic hematopoietic stem cell transplantation. methods: a retrospective study was performed on patients who receive an allo-hsct between january 2014 and august 2018. two groups were performed according to the presences or not of abo incompatibility. demographic and clinical information was collected from physical and electronic medical records, and information was analyzed in spss v21 results: sixty-eight patients were enrolled in the study, 54% male, the median age was 34 years (19-61) with the following diagnoses: acute lymphoblastic leukemia 44%, acute myeloblastic leukemia 26.5%, granulocytic chronic leukemia 17.6%, myelodysplastic syndrome 4.4%, dendritic cell neoplasm 4.4%, aplastic anemia 2.9%. ninety-one percent of the patients received a transplant from an identical hla donor and 8.8% received a haploidentical transplant. fifty-two patients (76%) were abocompatibility (g1) and 16 patients (24%) had aboincompatibility (g2). none patient with aboincompatibility received a haploidentical transplant. the contrast between groups didn't show differences in fever, infections, bacterial isolation, presence and degree of acute or chronic gvhd and relapse of the disease. graft failure was 6%(g1) vs 20%(g2) (p=0.27), intermediate risk cmv serostatus 79%(g1) vs 87(g2) (p=0.35). the most relevant characteristics and complications are described in table 1. contrast analysis between g1 vs g2 showed that within the whole group there were 29 deaths (40% vs 50% respectively) (p=0.69), the overall survival 1-year was 74% vs 66% (p=0.58) with a median of 29 vs 34 months respectively; mortality associated with relapse was 68% vs 87% respectively, and mortality related with transplantation was 35% vs 13 % respectively. conclusions: abo incompatibility did not show association with complications related with the infusion, but there was a higher tendency of graft failure in the abo incompatibility group. it has no statistical significance, but it is important to expand its study. disclosure: none declared methods: retrospective data for 142 nhl patients who underwent asct between 1999 and 2017 was analysed. patients were identified using the swbmt database and data on mets was collected using paper and electronic hospital records. forty-eight patients were excluded due to loss of follow-up, inaccessible/incomplete records, or death. cause of death was not determined. the ncep-atpiii definition of mets was used. this requires ≥3 of 5 criteria to be met. a bmi of ≥30kg/m 2 and hba1c of ≥42mmol/l were used to replace central obesity and impaired fasting glucose, respectively. other criteria include triglycerides (tgs) ≥1.7mmol/l or treatment, high density lipoprotein cholesterol (hdl-c) < 1.0mmol/l (male), < 1.3mmol/l (female) or treatment and blood pressure >130mmhg systolic or >85mmhg diastolic, or treatment. results: the prevalence of mets in the cohort was 33% (n=31). eighty-two percent of patients (n=77) met one or more criterion for mets. twenty-seven percent (n=25) fulfilled only one criterion, 23% (n=22) fulfilled two criteria, 20% (n=19) three criteria, 12% (n=11) four criteria, and 1% (n=1) five criteria. the greatest prevalence of mets was in the 60+ age group, accounting for 17 out of 31 (55%) patients with mets. overall prevalence decreased with declining age ( table 1 ). the number of patients aged < 20 years was too small to make any judgement on risk. raised triglycerides was the criterion most frequently met (61/94 patients), followed by hypertension (48), raised bmi (26), low hdl-c (23) and an increased hba1c (15). conclusions: the prevalence of mets in our cohort (33%) was higher than the estimated worldwide prevalence of 25%, with the majority in the 60+ age category. this is in keeping with other post-transplant studies, which show an increase in prevalence of mets after transplantation. moreover, the overall prevalence of mets was greater in the older population, which could be associated with the cumulative effect of ageing on the decline of normal metabolic homeostatic mechanisms. background: acute renal failure (arf) is a frequent complication in the early post-allogeneic hematopoietic stem cell transplant (allohsct) period with either myeloablative (ma) or non-myeloablative (nma) conditioning regimens. the aim of this study was to compare the incidence of arf in both types of hsct and to evaluate its impact on overall survival (os) and non-relapse mortality (nrm). methods: all allosct performed in one center between 2010 and 2018 were included in this study. allohsct from cord blood and from haploidentical donors were excluded. the renal function and the incidence of the main complications after allosct from day 0 to day +90 were evaluated. arf was defined according to kdigo (kidney disease improving global outcomes) classification; the relative increase of serum creatinine levels was considered a marker of kidney damage. results: seventy-seven patients received a ma allohsct and 72 a nma allohsct. recipients of nma allohsct had a higher median age (61 years [range: 18-69] vs. 41 years , p< 0.001), higher frequency of arterial hypertension (29% vs. 6%, p< 0.001) and showed most frequently active disease at allosct (42% vs. 18%, p=0.002). in both groups the most frequent graft-versushost disease (gvhd) prophylaxis regimen was cyclosporine a and methotrexate. the median follow-up time was 2.3 years for the nma group and 3.6 years for the ma group. patients from the ma group had higher incidence of grade 3-4 mucositis (60% vs. 22%, p< 0.001) and acute gvhd of any grade (70% vs. 53%, p=0.029) than patients from the nma allohsct. the incidence of arf was similar in both groups (72% in nma and 71% in ma). in the nma group arterial hypertension (hr 2.05, p=0.018), obesity (hr 4.16, p< 0.001) and prior pneumonia (hr 3.19, < 0.001) were predisposing factors for arf by multivariate analysis, whereas any factor was identified in the ma group. arf had no impact on 2-year os in both groups (28% vs. 35% p=0.406 for the nma group and 45% vs. 40% p=0.623 for the ma group). however, worse os were observed in patients with grade 2-3 arf in the nma group (18% vs. 43%, p=0.048) and in patients with grade 3 arf in the ma group (25% vs. 59%, p=0.011). in turn, arf had no influence on nrm in the ma group but was associated with a trend for higher nrm in the nma group (59% vs. 35%, p=0.084). conclusions: arf is a frequent complication in patients receiving allohsct irrespective of the intensity of the conditioning regimen. moderate and severe arf had negative impact on os. disclosure: supported by grants from: asociación española contra el cáncer, aecc (gc16173697biga), instituto carlos iii (pi14/01971 fi), 2017-sgr288 (grc), cerca program from generalitat de catalunya, and "la caixa" foundation. treatment and risk factors of hepatic veno-occlusive disease after pediatric hematopoietic stem cell transplantation: a single-center experience barbaros sahin karagün 1 , ilgen sasmaz 2 , ali bülent antmen 1 background: defibrotide emerged as a promising treatment option for hepatic veno-occlusive disease, a significant cause of mortality in recipients of hsct. as vod diagnosis is quite difficult even with the recently introduced ebmt 2017 criteria, studies which report treatment outcomes and response to prophylaxis are required. our aim was to evaluate the efficacy of defibrotide prophylaxis in hsct recipients at our center. methods: a total of 236 transplants in 210 patients from january 2013 to july 2018 were included in this study. all patients had factors that increased the risk of vod and all received 25 mg/kg/day prophylaxis. patients' coagulation, renal and liver function test were monitored daily and all clinical findings and complaints were recorded. diagnoses were made via the ebmt 2017 vod criteria and patients who developed vod received treatment with increased df dose (40 mg/kg/day) and supportive interventions. after complete remission of vod findings, patients were returned to the prophylaxis dose. close follow-up of patients was performed until 100 days. results: in total, 17 patients developed vod (7.2%), none of the cases were severe (13 mild, 4 moderate). median age was 8.5 years and the most common clinical findings were weight increase, hepatomegaly, right upper quadrant pain and ascites development. in those with vod, treatment with 40 mg/kg/day df was initiated and average duration of treatment with this dosage was 7.4 (5-11) days. no adverse events were reported in any of the patients. conclusions: our findings are consistent with previous studies on this topic, and we believe that the use of df as a prophylactic agent for vod is beneficial for pediatric patients with risk factors. disclosure: the authors report no conflicts of interest in this work. background: several factors might influence outcome of allo-hsct. analysis of the impact of donor-receptor blood group-incompatibility have been performed in different series not always showing the same results. as a consequence, its clinical impact remains controversial. minormismatch is characterized by the ability of donor b lymphocytes to produce anti-recipient antibodies. in majormismatch cases, antibodies against donor antigens are present in the recipient. methods: 343 pts underwent allo-hsct between may 2011 and august 2018 in our center. median age was 52 years (range: 7-69). 193 pts were male (56.3%) and 150 female (43.7%). baseline diseases were: 138 aml, 76 lpd, 44 mds, 41 all, 21 mpd, 16 mm, and 7 bmf. donor was unrelated in 191, and related in 152 cases (including 36 haplo-identical). donor-recipient abo compatibility was as follows: 69 (20.1%) majormismatched (including 11 bidirectional), and 274 (79.9%) nonmajor-mismatched (including 68 minormismatched and 206 matched). donor-recipient rh compatibility was as follows: 50 (15.6%) major-mismatched, and 293 (84.4%) nonmajor-mismatched (including 34 minor-mismatched and 259 matched). the impact of donor-recipient abo and rh compatibility on transfusion needs (prbc and platelet concentrates) and survival by day +100 was analyzed. results: for the global series the median number transfusions by day +100 was: 4 (0-81) prbc and 4 (0-92) platelets concentrates. day +100 overall mortality was 9.3%. rh-incompatible and nonmajor abo incompatible cases showed no different results. however, major abomismatched cases needed more prbc transfusions (median: 6; range: 0-49) and more platelet transfusions (median: 7; range: 0-60), and had higher day +100 mortality (18.8%) (p < 0.05) (see table) . conclusions: our analysis showed: 1) donor-recipient rh-incompatibility, as well as minor aboincompatibility had no impact on prbc and platelet concentrates transfusion needs nor on 100-day mortality; 2) contrarily, donor-recipient major abo-incompatibility had a significant adverse impact on prbc and platelet concentrates transfusion needs and 100-day mortality. 3) donor-recipient rh-incompatibility and minor aboincompatibility.might be considered of marginal importance at the time to choose a potential donor. 4) donorrecipient major abo-incompatibility should probably be a factor to be considered, along with other features, to choose the best donor background: survivors of haematopoietic stem cell transplantation (hsct) are at significant risk of developing treatment-related complications, including cardiovascular risk factors such as arterial hypertension, that could eventually lead to cardiovascular disease. the aim of this study is to evaluate the incidence and risk factors of hypertension following hsct in a colombian population. methods: a retrospective cohort study was conducted to assess the incidence and risk factors of hypertension in 220 consecutive adult hsct recipients who underwent transplantation between 2009 and 2017 at a tertiary referral center in colombia, south america. blood pressure data, from two different measures, were collected at 7 time points: day of mobilization for autologous hsct and day 0 before infusion for allogeneic transplantation, day 7, and months 1, 3, 6 and 12 post-transplantation. hypertension was defined as having a systolic blood pressure >=140mmhg and/or a diastolic blood pressure >=90 mmhg. patients with history of arterial hypertension were excluded. results: one hundred and seventy-five patients were included, with a mean age of 44 years (range 15-67). ninety-one patients (52%) were male. one hundred and sixteen patients (66.3%) underwent autologous hsct and 59 (33.7%) allogeneic hsct. the most common indication for hsct was acute leukemia (26.3%), followed by non-hodgkin lymphoma (23.4%) and multiple myeloma (22.9%). twelve patients (6.9%) had medical history of type 2 diabetes mellitus (dm), 11 (6.3%) dyslipidemia, 24 (13.7%) alcohol consumption, and 25 (14.3%) tobacco smoking. only two of the patients with history of tobacco smoking were active smokers at time of transplantation. twenty-four patients (13.7%) had developed hypertension by the end of the first year post-hsct follow-up. two patients (8.3%) had systolic and diastolic, 12 (50%) had only systolic, and 10 (41.7%) had only diastolic hypertension. only one patient was hypertensive at more than two time points. the incidences of hypertension at each time point were 2.3% on day 7 post-hsct, 6.9% at first month, 9.8% at three months, 12.1% at 6 months, and 13.8% at one-year post-transplantation. allogeneic hsct (p< 0.01), therapy with calcineurin inhibitors (p< 0.01), pre-hsct fasting glucose levels (p< 0.05), acute gvhd (p< 0.05), chronic gvhd (p< 0.05), and media of diastolic blood pressure (p< 0.05) were significantly associated with the development of arterial hypertension. however, age, history of type 2 dm, history of tobacco consumption, volume of infusion, prophylactic treatment for gvhd with mycophenolate, chronic gvhd, serum creatinine level on day of hsct, and being overweight or obese at time of transplantation were not significantly associated with the development of hypertension. conclusions: arterial hypertension is a fairly common complication in hsct recipients. similar to findings reported in previous studies, association between allogeneic stem cell transplantation, therapy with calcineurin inhibitors, and acute and chronic gvhd, and post-hsct hypertension was found in the present cohort. further studies are needed to assess the link between hsct and developing long-term cardiovascular complications. disclosure: nothing to declare tramadol-based pain management of oral and esophageal mucositis in pediatric hsct recipients background: mucositis is one of the most common early hsct complications seen in about 70% transplant recipients with 20% of patients developing gr iii-iv mucositis. mucositis is characterized by painful gastrointestinal mucosa lesions impairing the solid and liquid foods intake and increased risk of infections, bleeding, and intestinal paresis. thus, it greatly decreases the quality of life of a transplant recipient. according to who recommendations, the moderate pain control in pediatric patient is based on the use of low-dose morphine. however, there are some factors such as genetic polymorphisms causing variable morphine pharmacokinetics in children, side effects, and social factors (caregivers' general unwillingness to use narcotic analgesics), which cause the need for alternative pain relief options in pediatric practice. tramadol, which has both opioid and non-opioid mechanisms of action, may be a feasible option in mild to moderate pain. it may be delivered via patient-controlled analgesia (pca), although there is no consensus on its optimal parameters in pediatric practice. methods: a total of 69 pediatric patients with a median age of 8 (range 2 to 18) years receiving an autologous or allogeneic hsct in our clinic as part of the treatment regimen for solid tumor (n=40), leukemia (n=24), acquired aplastic anemia (n=3) or inherited condition (n=2) were included. conditioning regimens were myeloablative (mac) in 54 and reduced-intensity (ric) in 15 patients. all patients had oral and/or esophageal mucositis accompanied by moderate pain. the pain severity was assessed using the scales corresponding to patient's age and varied from 3 to 6 points. the pain control was based on intravenous tramadol administration using patientcontrolled analgesia (pca) approach. the following pca parameters were used: loading dose of 0.5 mg / kg (not exceeding 25 mg), basal infusion rate of 0.25 mg / kg (not exceeding 12.5 mg), a bolus of 0.25 mg / kg (not exceeding 12.5 mg), lockout interval of 25 min. the maximal daily dose was 8 mg/kg/day. the pain control was considered adequate if a patient was satisfied or the basic and breakthrough pain score values were not higher than 3 and, accordingly. in case of inadequate pain control nsaids were added. non-responders were switched to morphine. all patients were divided into 2 groups based on conditioning regimen intensity. results: as a whole, 46% of patients did not require pain control measures escalation. the tramadol pain control rate was slightly higher for ric (n=9, 60%) compared to mac (n=23, 47%) recipients. in most cases the inadequate pain control was due to progressive mucosal lesions. the pca regimen used was characterized by very few complications. drowsiness was observed in 4 (7%) of patients, in all cases the patients also had anemia. there was only 1 (7%) patients with severe nausea requiring switching to morphine. conclusions: tramadol is an effective pain control option in transplant recipients with mild to moderate pain due to oral and esophageal mucositis without progressive mucosal lesions. the pca allows achieving a very low complication rate. therefore, this option may be considered for both mac and ric recipients. disclosure: no immune reconstitution of lymphocyte subsets after allogenic stem cell transplant (sct) and vaccination background: infectious diseases are a major cause of morbidity and mortality after allogenic stem cell transplant (sct). vaccines constitute an effective strategy to prevent infections but the optimal timing to start vaccinating is not well stablished. in order to individualize the early vaccination schedule, we studied the lymphocyte subsets involved in generating enough response to produce protective serological levels. methods: we studied retrospectively 20 patients that had undergone allogenic sct at our hospital. patient distribution -age range: 21-68 years-old; diagnosis: acute leukaemia/myelodysplastic syndrome/ chronic myeloid leukemia (16 patients), lymphoma (4 patients). analytic parameters: tcd4+, tcd8+, nk, total b and functional b lymphocyte subsets (naïve igd+cd27-, memory igd+cd27+ and igd-cd27+, and effectors cd27++cd38++). immunoglobulin levels (igg, iga, igm) and specific igg for pneumococcus, tetanus, hbv, chickenpox, measles, rubella and mumps. clinical parameters were collected from medical records. results: we distributed patients in two groups, based on the timing of lymphocyte analyses: -less than 12 months since sct (5 patients) no patient showed complete immune reconstitution, although 2 had enough t and functional b lymphocytes to generate response to vaccination. in these patients, vaccination for pneumococcus was completed and they generated sufficient protection antibody levels, despite being under immunosuppressive treatment. -more than 12 months since sct (15 patients) before the beginning of vaccination, we collected specific antibodies of 7 patients. we compared the serological status before and after sct and observed that protection against tetanus was the most frequently preserved (6 patients) and hbv the least frequent (2 patients). other than one patient treated with alemtuzumab, all patients in this group had minimum absolute count of tcd4 + (>200 cells/microl), tcd8+ (>200 cells/microl), nk (>100 cells/microl) and b cells (>100 cells/microl). we also observed presence of b effector and b memory cells, with predominance of igd-cd27+ memory cells. immunoglobulin levels were within the normal range. in this group, we registered vaccination in 13 patients. all of them were vaccinated against flu, and 11 against pneumococcus and hbv. the rest of vaccines administered were heterogeneous in type and timing. 6 patients were under immunosuppressive treatment at the time of vaccination and were able to generate enough specific antibodies for pneumococcus. conclusions: immune reconstitution was not completed 12 months after sct, although minimal immunological reconstitution was observed tcd4+ and no-switching memory b lymphocytes were the last ones to reach minimum normal values according to patient age. some patients maintain serological protection after allogenic sct. immunoglobulin levels were normal, suggesting no need for immunoglobulin administration to prevent infections. flu, pneumococcus and hbv vaccines were the most frequently administered. pneumococcus vaccination generated a much larger serological response than hbv. this seroconversion occurred in patients under immunosuppressive treatment. the analysis of lymphocyte t, nk, b total and b functional subsets could be useful when programming an early vaccination schedule after sct. completion of the vaccination schedule was heterogeneous despite giving specific indications. therefore a more rigorous supervision of the process may be required. background: the significant advances that have been achieved in the allogeneic transplantation (allohct) field, have resulted in better post-transplant outcome and therefore complications other than the graft vs. host disease (gvhd) or disease recurrence become increasingly important. the post transplant metabolic syndrome (pt-ms), which caused by several factors (i.e. immunosuppressive agents, chemo-radiotherapy, anti-viral, and biologic therapies) is a well known post transplant complication in pediatric allografted long-term survivors however, only few studies have evaluated the prevalence of the pt-ms in adults. in this retrospective study, we sought to evaluate the incidence, the risk factors and the impact of the pt-ms on the allosct outcome. methods: since 2011, 42 patients (25 males and 17 females) with adequate clinical and laboratory data and a minimum follow-up of 6 months were included in the study. their median age was 35.5 (17-62) years and after a myeloablative (n=28) or a reduced intensity (n=14) regimen they received either mobilized peripheral blood stem cells (n=34) or marrow graft (n=8), originated from full-matched siblings (n=35) or haploidentical donors (n=7). calcineurin inhibitors plus either short-term methotrexate or mycophenolate mofetil were given as gvhd prophylaxis. the diagnosis of pt-ms was based on the ncep-atpiii criteria; for patients with unknown data for abdominal circumference the body mass index (bmi) ≥25kg/m 2 was consider as a criterion for pt-ms diagnosis. the independent t-test, logistic regression analysis and logrank tests were used for the statistical analysis. results: twenty (47.6%) patients (12 males, 8 females) assessed to have pt-ms within the first 6 months following the allograft. seventeen diagnosed after the 1 st trimester post allosct and additional 3 patients after 2 nd trimester. sixteen out of 20 patients had elevated glucose and bmi>25kg/m 2 , 13/20 elevated triglycerides levels, 12/20 low hdl levels and 10/20 hypertension. four (20%) had already known history of ms before allosct (for 10 patients no data were available for ms diagnosis before allosct). interestingly, for 7/20(35%) patients who had diagnosed with pt-ms either in the 1 st or in the 2 nd trimester the syndrome was reversible and did not fulfill the criteria for pt-ms beyond 6 months post allosct. patients' gender, age, bmi, the type of conditioning regimen and gvhd co-existence evaluated as potential predisposing factors for pt-ms diagnosis. in univariate and multivariate analysis only the: bmi>25kg/m 2 and age>35 years were detected as significant risk factors (p< 0.03). the pt-ms did not affected negatively the survival or the nrm incidence post allosct conclusions: in our study, in agreement with other publications, we demonstrated that the pt-ms is not an uncommon complication post in the early post transplant period however, for a significant number of patients the syndrome was a reversible. for patients with high risk features (bmi>25kg/m 2 , age> 35 years, known history of diabetes-mellitus, dyslipidemia, hypertension) apart of close monitoring, specific diet and encouragement for adequate exercise might help to reduce the incidence and the severity of pt-ms. nevertheless, prospective and well design trials are warranted to determine the accurate incidence, severity and the impact of pt-ms on the allosct outcome. disclosure: no conflict ofinterest experience of a single center in the humanization of the hospitalization process: technology and team training impact on the qol of the patient and family maria claudia moreira 1 , marcia rejane 1 , marcia garnica 1 , andrea ribeiro 1 , paulo cesar dias 1 , ilza fellows 1 background: hematopoietic stem cell transplantation (hsct) is one of the most aggressive therapeutic modalities of internal medicine, making it a highly stressful experience for the patient and his family. the duration of hospitalization can be prolonged by several intercurrences, frequently generating anxiety in the patient and their caregiver, which may lead to confinement and reactive depression. interventions in the hospital environment, in addition to the continuous training of the multidisciplinary team, can have a positive impact in this process with improvement in the process of discharge and quality of life of the patient and his / her family. methods: the objective of this research was to evaluate the impact of a reformulation in the unit, completed in may 2018, which modified the facilities with availability of hermetic balconies in each room, with a view of an internal garden. there was also the addition of a screen in the corridor of the floor with images -technology known as videoowall, interconnected to motion sensors (kinects) that allow interaction between patients and families, besides facilitating physiotherapy and physical exercise. there was re-training of the multidisciplinary team with emphasis on the practice of humanization. the methodology consisted in the application of questionnaires of satisfaction to patients and their families during the period of hospitalization in a bone marrow transplant unit in the third quarter of 2018. the items evaluated ranged from the quality of the information provided by the medical team and nursing, to the cordiality and agility with which the patient and his patient were treated by the global team. the results were compared with a similar period of the same unit in the previous year and with the indices collected simultaneously in another unit of the same hospital (cardio-intensive). results: overall and segmental satisfaction scores in the various items surveyed were higher when compared to the previous period of the same unit and were also higher in those obtained in a high complexity unit of the same hospital, composed of patients submitted to mental and psychological stress similar to onco-hematologicos.a reports of "free speech" were also obtained anonymously, in order to guarantee the authenticity and free expression of the subjects analyzed. conclusions: the results obtained allowed the validation of the technical and professional team initiatives, bringing indicators that will allow better monitoring and support of these patients and their relatives in this difficult time of treatment. they served as an initial tool in the continuous process of humanization and stimulated the multidisciplinary team to continuously improve this process. disclosure background: pure red cell anemia (prca) is a rare complication of abo-incopatible hematopoetic stem cell transplantation characterized by anemia, reticulocytopenia and absence of erythroid precursors in patient's bone marrow. most patients with prca resolve spontaneously within months, however a small number of patients requires continued red blood cell (rbc) transfusions. the treatment of this complication is difficult and not standardized. different approaches has been used such as rituximab, donor lymphocytes, plasma exchange with different outcome. recently, a remarkable response to treatment with bortezomib has been described in a case of prca. methods: we reviewed 146 patients who received an allogeneic hematopoetic stem cell transplant (hct) between januar 2012 and august 2018 at our institution. sixty eight patients received a major abo-mismached hct. prca was defined as a completely absence of erythroid precursors on day +30 bone marrow puncture, with absence of donor red cells and the recipient requiring rbc transfusion. results: only one patient developed prca (1.5%). a 18 years old male received a myeloablative hla-matched abomismatched sibling donor transplant (brother, 12 years) for acute myeloid leukemia (aml), with t(8;21) cr1,mrd positive (runx1-runx1t1). the donor was blood type a rh positive and the patient 0 rh positive. the patient had no complication after transplant. the day +30 bone marrow puncture has shown only few erythroid precursors and day +100 puncture and biopsy no erythroid precursors, he had transfusion dependent anemia requiring a rbc transfusion every two weeks and retukulocytopenia. parvo virus and cytomegalovirus were negative. due to very high ferritin level (>4.000u/l) and increased luiver enzymes without signs of gvhd, the treatment with deferasirox has been started. the patient has achived cr1, mrd negative, and has evidence of complete chimerism. high titers of anti-a and anti-b issohemagglutinin was present.we started the treatment with rituximab 375mg/m2 weekly, 4 weeks, however without response. the pathogenesis of the prca is thought to be due to the recipients plasma cells, bortezomib, a proteasome inhibitor inducing apoptosis of plasma cells has been given s. c. 1,3mg/m2 two times weekly, for two weeks. the patient responded to the treatment two weeks later with increase in hb, which was 12,9 g/dl and increase in retikulocyte number. the patient has continued to be well at the last control. conclusions: prca aplasia is a rare but serious complication after abo-incompatible hct. bortezomib is an effective treatment for this complication if mediated by residual host isohemeagglutinins after hct and should be recommended as standard of care. clinical methods: this work is retrospective, observational, cross-sectional and analytical. it included all patients who received hsct at stem cell transplantation unit (utmo, by its spanish acronym) at solca-guayaquil, between the years 2009 -2016.we use the kaplan-meier method to analyze the survival rate between the autologous and allogeneic transplant. the information collected for this study was obtained from the database of the solcay institute and the review of the files of the patients included. results: at least, 150 patients have been undergoing to hsct between 2009-2016 years. according to the type of hsct, 42.1% received an autologous transplant and 57.9% received an allogeneic transplant, from which 79.3% were from a related donor. the main source of transplant was peripheral blood in 86.67%, followed by 12% obtained from umbilical cord blood and 1.33% by bone marrow aspiration. the most frequently reported pathologies were acute lymphoblastic leukemia (all) (34%), multiple myeloma (mm) (22%) and acute myeloid leukemia (aml) (13.33%). the overall survival was 68% (ic: 95%). the 82.53% of patients that were undergoing to autologous transplant have survive, meanwhile the patients that were undergoing allogeneic transplant only the 57.47% have survived (p< 0.05). the highest death rate occurred during the first year after hsct, and decreased considerably after that period. the main cause of mortality related to transplant (mrt) was the graft-versus-host disease (gvhd) (8%); however, the main cause of mortality in the study population (n=150) was relapse in 12.66% of the patients, presented more frequently in all. conclusions: the results showed that 68% of patients undergoing to hsct have survived. a high rate of deceased patients in this study, have died in the first year before the transplant (26.6 %%), due to relapse. the main cause of deceased in the study is not related to hsct, and was the relapse in 12% of patients, in compare the gvhd was the main cause of mrt (8%). we consider that hsct is a technique that is still under development in ecuador, but despite the short time it has been taking and the institutional and medical limitations present in the health field, has presented excellent results comparable to studies conducted in developed countries. [ background: pigmented epithelioid melanocytoma (pem, early known as 'аnimal type' melanoma) is a rare tumor with unpredictable clinical behavior and metastatic potential. pem generally has favorable prognosis. involvement of regional lymph nodes is not rare. extranodal and distant nodal metastases are extremely rare. we report about patient with fanconi anemia (fa) and pem with developed distant metastases in the early term after allogeneic hematopoietic stem cell transplantation (hsct). methods: 10-years old boy with fa was hospitalized for hsct. the blue-black painless nodulus 15х15 mm was noted on the left cheek. this lesion was observed from early childhood and during life only slightly increased in size. there were no distant and regional metastases on computerized tomography (ct) and scintigraphy with 99m tc. the nodulus and regional lymph nodes were radically removed before hsct. the resection margin was within the normal tissue. microscopically the derma and subcutaneous fat were infiltrated with epithelioid and spindle cells with total expression of s100, melana, mhb45, cyclind1. ki-67 expression level was 15-20%. histological structure was specific for pem. hsct with tcrαβ+/cd19+ graft depletion from match unrelated donor was performed. the conditioning regimen included total lymphoid irradiation 2gy, fludarabin 150 mg/ m 2 , cyclophosphamide 40 mg/kg, rabbit atg 5 mg/kg and rituximab 100 mg/m 2 . results: at +45 day after hsct was detected the tumor on the left cheek and parotid region with a histological structure identical to the primary lesion. on ct in s6 segment of the left lung was detected focus 20x20 mm with a cavity. invasive aspergillosis was suspected and empirical antifungal treatment was started. but in 15 days the lung lesion increased in size to 52x30x32 mm and penetrated in the bronchus. after bronchoscopy with biopsy, pem metastasis was histologically confirmed. moreover, the tumor on the face continued to grow. therapy with cobimetinib and vemurafenib was not effective and patient died from progression of pem on +98 day after hsct. conclusions: pem was early described as indolent tumor with rare distant metastasis and favorable prognosis. we suspect that pem may acquire an aggressive course in the absence of immunological control, especially in high immunocompromised patients after hsct. disclosure: nothing to declare p208 abstract withdrawn lidia gartcheva 1 , antoaneta mihova 1 , penka ganeva 1 , margarita guenova 1 , branimir spassov 1 background: the main objective of the study is to assess the dynamics of quantitative and qualitative changes in the parameters of the b cell population and the production of immunoglobulins in patients after autologous transplantation of hematopoietic stem cells in the course of recovery of the immune system. methods: 56 patients with hematological neoplasms undergoing autologous transplantation were included in the study: 30 women and 26 men, with an average age of 31 years. patients were diagnosed with lymphoma (n = 30), multiple myeloma (n=7), leukemia (n = 7) and solid tumors (n = 12). at the time of transplantation, 16 patients were in complete clinical remission or at least with very good partial response, 30 patients were in partial remission and 10 patients -with progression. all patients were evaluated in nine time points through 356 examinations by clinical-laboratory, flow cytometric and immunochemical methods. results: the percentage of cd19 (+) b cells reached the minimum values one month after transplantation then began to increase in the second month reaching a plateau around the mean values in the period 6-12 months after transplantation. the absolute number remained low during the entire period of observation. the amounts of igg and igm serum immunoglobulins gradually increased within the reference range throughout the entire period, while the iga level varied around the lower reference range. conclusions: implementation of an adequate humoral immune response is hampered by the reduction of circulating b cells, suppressed proliferative potential and functional deficits. restoration of b-cell function occurs over a period of 6 months to 2 years after autologous transplantation. clinical trial registry: no clinical trials disclosure: nothing to declare justyna background: allogeneic hematopoietic stem cells transplantation (allo-hsct) is a life-saving and well established therapy for wide range of diseases. however, it is still uncommon treatment for infants less than 12 months of age. the data about indications and outcome of allo-hsct in the youngest group of patients is sparse. the primary objective of this study was to assess the incidence, indications, post-hsct complications and general outcome of allo-hsct among infants not older than 12 months. latter sequelae of hsct such as physical and cognitive development were secondary aim of this study. methods: we retrospectively analyzed data of 63 patients who underwent allo-hsct before 1 year of age in department of pediatric hematology, oncology and bone marrow transplantation in wrocław during years 1999-2017. clinical and epidemiological features as well as indications for transplantation, early and late complications and general outcome were assessed. results: infants who underwent hsct in our department comprise 8.2% of all patients undergoing hsct in analyzed period of time. thirty-one (49.2%) patients received stem cells from matched unrelated donor (mud), 26 (41.3%) from mismatched (haploidentical) related donor (mmrd) and 6 (9.5%) from a sibling donor (msd). non-malignant disorders were indication for transplant in 49 (77.8%) patients and malignant diseases in 14 (22.2%) . acute graft versus host disease (agvhd) occurred in 33 (52%) infants, chronic graft versus host disease (cgvhd) in 15 (24%). majority of graft rejections were seen in infants transplanted from mmrd 7(63.6%), whereas the rest 4 (36.4%) was associated with mud. median follow-up in study cohort was 860 days, 1148 days for alive patients (range 72 days-19.5 yrs) and 72 days for those deceased (range 3 days-341 days). overall survival (os) in study cohort was 0.682 and transplant related mortality (trm) was 0.317. in children with malignancy 5 (35.2%) patients died comparing to 15 (30.6%) patients in non-malignant group respectively. main cause of death in analyzed group of infants was infection (60%). conclusions: 1. allo-hsct is rarely performed in children less than 12 months of age. 2. majority of those patients receive stem cells due to non-malignant disorder. 3. among youngest hsct recipients, haploidentical transplant are more common than in general pediatric transplant population. 4. graft rejection is a significant problem in infants transplanted from mmrd. disclosure: nothing to declare unusual non-infectious lung complication after allogeneic haematopoietic stem cell transplantation claudia lucia sossa melo 1,2 , manuel rosales 2 , francisco fernando naranjo junoy 1,2 , sara inés jiménez 1,2 , luis antonio salazar 1,2 , angela maría peña 1,2 , maría angélica chacón manosalva 3 , maria luna-gonzález 1 , claudia marcela chalela 1 , manuel ardila-báez 1 jirovecii infections, viral infections or nocardia. we describe the case of a patient with acute lymphoblastic leukemia (all) diagnosis with pap associated to a hsct and pulmonary pneumocystis. methods: a 55-year-old colombian female patient diagnosed with b-precursor all of high-risk in january 2017, positive philadelphia chromosome, positive bcr / abl in february 2017, infiltration to the central nervous system (cns), 2.53% of lymphoblasts, and karyotype without legible metaphases. refractory to induction according to the pethema protocol (vincristine, daunorubicin, prednisone, l-asparaginase) with presence of 13.9% blasts at the end of the induction. re-induction was performed with the flag-ida protocol (idarubicin, fludarabine, cytarabine) achieving complete remission, obtaining minimum residual disease (mrd) < 0.01. dasatinib was initiated by bcr / abl expression and cns involvement at the time of diagnosis. an allogeneic hsct was performed, from a male brother donor, with low intensity conditioning tt buflu and prophylaxis of graft-versus-host disease (gvhd) with tacrolimus and sirolimus. patient showed early posttransplant complications, given the reactivation of cytomegalovirus and hemorrhagic cystitis grade i due to adenovirus. late complications such us gvhd at the cutaneous level and subsequent hepatic and gastrointestinal involvement were seen too, for which immunosuppressive therapy was administered with high doses of systemic corticosteroid. results: patient was hospitalized on day +270 posttransplantation due to febrile neutropenia and respiratory symptoms, with normal chest ct, and ct of paranasal sinuses with acute pansinusitis, for which she received meropenem 1gr intravenously every 8 hours plus vancomycin 1gr intravenously every 12 hours during 14 days with symptom resolution. she remained hospitalized for cytopenias with normal bone marrow and 100% chimerism. on day +291 posttransplant she presented fever and leukocytosis, with acute respiratory failure with chest ct that showed bilateral alveolar occupation, "crazy-paving" pattern and frosted glass (see image), so diagnostic fibro-bronchoscopy was performed, reporting postoperatively for pneumocystis jirovecii. she received 21 days of trimethoprim-sulfamethoxazole, with a torpid evolution requiring mechanical ventilation and tracheostomy, persisting with hypoxemia. the report of cultures for fungi, mycobacteria, and respiratory panel of filmarray were negative. a pathology report was obtained with 30% neutrophils, as well as pas staining with acellular pink material and elevated serum ldh, with a diagnosis of secondary pap. the patient continued with poor general condition, refractory hypoxemia, high ventilatory parameters and hemodynamic instability, due to which she was not able to be a candidate for treatment with total pulmonary lavage; leading to multi-organ failure and later death. [[p211 image] 1. high resolution chest ct. sample opacification in frosted glass (a) and pattern ''crazypaving'' (b)] conclusions: the importance of considering the diagnosis of pap as a noninfectious pulmonary complication in patients with allogenic hsct despite its low incidence is recognized. disclosure: nothing to declare methods: once the project was approved by the clinical trials and ethics committee, 154 pairs of blood samples were drawn (154 from picc line and 154 from venepuncture) from 33 voluntary allo-hsct recipients who were receiving continuous infusion tacrolimus from february through august 2018. the pts had inserted a double-lumen polyurethane picc. tacrolimus was always administered through the red line, and the blood draw always performed through the purple line. all of the patients signed the informed consent. 22 were male and 11 women. median age was 55 years (30-68). 24 of the venepunctures were carried out in the arm where the picc was set, and the other 130 from the contralateral arm. a limited group of nurses performed the extractions of the samples. results: as shown in the table, tacrolimus trough levels determined in blood from venepuncture were similar to those in blood drawn through the picc (median: 10.7 vs 11.1 ng/ml). when comparing one by one in the individual patients, the differences were not significant, and changed the dosing prescription in no cases. conclusions: in our experience, there are not significant differences in tacrolimus levels draw from the picc line, compared with a peripheral site. so, in our opinion, if the line for tacrolimus infusion is properly identified and the one used for the sample draw is the alternative one, venepunctures to obtain sample from peripheral sites are not justified for tacrolimus levels measurements. background: patients undergoing a hsct may require icu admission due to transplant-related toxicities. the aim of this study was to analyse a single centre experience with hsct patients requiring icu admission and the factors affecting outcome. methods: we included all adult patients (age >=18) who had an allogeneic or autologous hsct during 2017 (d0 between 1-1-2017 to 31-12-2017) at st. george's hospital. data was retrospectively collected from patients' notes. icu outcome and 100-day survival were analysed. for those patients who were admitted to icu more than once, outcome was analysed from their last icu admission. results: 20 allograft patients were included. 14 were male, with a median age 47 years (range 23-68 years). 6 were female, with median age 61 years (range 51-67 years). diagnosis n (%) includes all 3 (15%), aml 7 (35%), acml 1 (5%), cmml 3 (15%), hl 1 (5%), mds 2 (10%), mds/mpn 1 (5%), fl 1 (5%), scd 1 (5%). sixteen (80%) patients received their first transplant, 4 (20%) received second transplant. eight (40%) patients had sibling donor, 12 patients (60%) had unrelated donor. sixteen (80%) patients had 10/10 matched donor, 2 (10%) patients had 9/ 10 matched donor, 2 (10%) patients had 8/10 matched donor. nineteen (95%) received reduced intensity conditioning (ric), one (5%) received myeloablative (ma) conditioning. majority of ric allo-hsct patients were conditioned with fludarabine, mephalan, campath (fmc). a small number were conditioned with busulfan, fludarabine and atg. the ma allo-hsct patient was conditioned with tbi, cyclophosphamide. gvhd prophylaxis was ciclosporin alone starting on day -1 with a target level of 150-250 ug/l for all ric and ciclosporin and methotrexate for the ma patients. two (10%) allograft patients were admitted to icu on three occasions. both patients were male, 58 and 68 years old. one had mmud allograft for mds/mpn. the other had 2 nd mud allograft for relapsed aml. the reasons for icu admission include sepsis, cardiac arrest and respiratory failure. the median duration of icu admission was 5 days (range 2-9). there were 2 deaths within 100 days of transplant. one patient died on day +11 during his second icu admission with multi organ failure (mof). one patient died after icu discharge on day +23 with relapsed disease, bronchopneumonia with disseminated fungal infection. icu mortality rate was 50%, and 100-day mortality rate was 10%. nineteen autologous patients were included (median age 61 (range 36-71 years)), 16 (84%) were myeloma patients who were conditioned with melphalan, 3 (16%) were lymphoma patients who were conditioned with beam. the icu admission was 0%. the 100-day mortality rate was 0%. conclusions: our centre's icu admission rate, icu mortality rate, cause of icu admission in allo-hsct patients and autologous patients is comparable to literature reports. autologous transplant is safe with no deaths and icu admissions despite an older age. the mortality rate for allo-hsct patients requiring icu admission remain high. all patients were appropriately referred to icu and there was no one who was denied icu admission. this analysis is being extended to preceding years. disclosure: nothing to declare liposomal doxorubicin for the treatment of iatrogenic kaposi sarcoma following hematopoietic stem cell transplantation background: iatrogenic kaposi's sarcoma (iks) represent a rare complication after hematopoietic stem cell transplantation (hsct), related to hhv-8 infection in hivnegative immunocompromised patients (pts). methods: we describe a case of iks occurred after an allogeneic hsct and we provide a review of the literature using pub med. results: a 70-year-old man, hiv-negative, received full hla-matched related hsct after a reduced intensity conditioning regimen for relapsed aml. gvhd prophylaxis was based on atg (fresenius 30 mg/kg), cyclosporine (cya) and methotrexate. no severe complication occurred in the first 100 days after transplant. shortly after cya withdrawal, he developed grade i acute gvhd. gvhd resolved after restarting cya. at fifth month after transplant, the patient developed several red and purple angiomatous plaque and nodules involving the skin of both lower limbs, right arm and the nose (figure 1). skin biopsy revealed multiple localizations of iks and positive hhv-8 viremia was detected in the peripheral blood. a visceral involvement was excluded. patient was treated with cya tapering and nine courses of liposomal doxorubicin 20 mg/m2 every 15 days, obtaining a negativity of hhv-8 viremia and partial response of the skin lesions. at last follow up, at 15 months after transplant, the patient was in complete remission (cr) for aml, cya-free without signs of gvhd recurrence and with his single stable residual iks lesion on his left limb, currently waiting for local radiotherapy. we found additional 18 iks published cases after hsct. most of post-hsct iks were secondary to an allogeneic-hsct (16 out of 19, 84.2%) and occurred in adult (78,9%) and male (68,4%) pts. median age at the time of iks diagnosis was 47.5 years (range 7-70). thirteen pts (68.4%) had mediterranean origin. the most frequent underlying disease was aml (47.4%). gvhd prophylaxis was primary based on calcineurin inhibitor. half of the pts developed gvhd and were treated with steroid and other immune suppressive drugs. median time between the hsct and the occurrence of iks was 8.5 months (range . cutaneous iks was the prevalent form of manifestation, however visceral involvement was reported in 7 pts (36.8%). in four cases (21.1%) an hhv-8 associated bm failure was report. immune suppression drugs tapering (36.8%) and chemotherapy (26.3%) were the most frequent actions taken after the diagnosis of iks. in most cases, liposomal doxorubicin was used as chemotherapy. cr rate was high, 63.2%, whereas progression disease occurred in 5 out 19 pts (26.3%), all of which had visceral involvement. in 3 pts (15.8%), iks was the cause of death. conclusions: withdrawn of immune suppression drugs and anthracycline based chemotherapy can represent a feasible treatment option for pts with iks after hsct. clinical background: acquired haemophilia a (aha) is an autoimmune disease caused by the spontaneous production of neutralizing immunoglobulin g (igg) autoantibodies (inhibitors) targeting endogenous fviii. treatment of these inhibitors presents additional challenges in a hematopoietic stem cell transplantation (hsct) recipient, because preservation of the graft that restores a normal hematopoiesis is critical. here we describe the management of a case of aha in an acute myeloid leukemia patient following hsct. methods: the clinical, laboratory and molecular aspects of a 56-year-old italian male who developed aha after allogenic bone marrow transplantation were collected and presented in order to show how we diagnose and manage this severe but rare complication within the special setting of hsct. results: a 56-years-old man with a flt-3 itd, npm-1, runx1-runx1t1 and cbfb-myh11 negative, not differentiated, chromosomally normal acute myeloid leukemia (aml) in third complete remission (cr) was submitted to a hematopoietic stem cell transplantation (hsct) from his haploidentical son. the conditioning regimen consisted of oncothiotepa, busulfan and fludarabine and was followed by the infusion of a t-cell depleted bone marrow graft. gvhd prophylaxis consisted of cyclosporine a (csa) and mycophenolate mophetyl (mmf). neutrophil engraftment occurred on day +24. recipient's autoimmunity was negative. at 27 months post-transplantation the patient received an antipneumococcal vaccination. fifteen days post-vaccination the patient was admitted to our in-patient ward due to general malaise, diffuse muscle and joint pains, cutaneous bleedings, oedemas, hyperchromic urines and constipation. physical examination revealed diffuse ecchymosis, swelling of deep muscles with a progressive functional disability due to hematomas and hemorrhagic suffusions of the tongue frenulum. and anti-factor viii inhibitors 6.46 bu/ml (high titers >5 bu/ml). thus, a diagnosis of acquired autoimmune haemophilia a was made and treatment with feiba combined with prednisone was started. patient's clinical conditions dramatically improved as he referred an improvement of movements and the resolution of joint and muscle pains despite the persistence of deep hematomas just after one day of treatment that had determined an increase of fviii:c value to 1.32% and an improvement of aptt to 47.06 seconds. on the following medical checks physical examination showed the progressive disappearance of deep muscle hematomas, and normal values of fviii:c. conclusions: aha is a rare but severe complication following hsct and it could appear years afterengraftment. a prompt diagnosis and an early treatment with feiba and corticosteroid are necessary to avoid life-threatening sequelae. the inclusion of the coagulation panel in the laboratory exams performed during the follow-up is advisable in order to early detect this life-threatening complication. disclosure: nothing to declare background: splanchnic thrombosis is an uncommon complication of myelofibrosis and a controindication to proceed to hematopoietic stem cell transplantation (hsct) due to the risk of additional vascular and endothelial complications. we present a patient with myelofibrosis (mf) that proceeded to hsct from an unrelated donor, despite splanchnic thrombosis unresolved after heparin treatment and unable to proceed to surgical treatment due to severe thrombocytemia. methods: a 67-year woman with mf secondary to essential thrombocythemia, with intermediate-2 score according dynamic international prognostic staging system (dipss) and with extreme splenomegaly (maximum diameter 30 cm), refractory to ruxolitinib, showed an extensive thrombosis of the portal and splenic veins, unresolved after 4-week heparin therapy, at the time of availability of an hla (8/8) and abo matched unrelated donor. she received a conditioning regimen including fludarabine and thiotepa and a gvhd prophylaxis with atg thymoglobuline, cyclosporine and methotrexate, followed by the reinfusion of 5.8x10 6 /kg cd34+ pbsc. at the time of transplant we were aware of an high risk of developing sos, on the basis of the older age of the recipient, the unrelated donor, the advanced stage of myelofibrosis and the ferritin serum level of 2.223 ng/mg. results: on day +9 after hsct sos complicated the aplasia phase, characterized by jaundice, ascites, weight gain, progressive increase in creatinine and bilirubin serum levels. an ultrasound of abdomen confirmed an unchanged thrombosis extension and the development of ascites. on day +10 the patient was categorized as very severe sos stage, according to ebmt severity criteria, because of doubling of bilirubin serum level in 48 hours and a 20% increase in comparison with her baseline weight. therefore, defibrotide was promptly started in association with diuretic therapy. the treatment was continued for 3 weeks and allowed gradual restoration of the water balance and normalization of bilirubin serum level. at the last follow-up, 6 months after hsct, the patient shows the persistence of a non-transfusion dependent anemia, platelets 80.000x10^3/ul, palpable spleen 4 cm below the rib, >95% allogeneic chimerism in the granulocytic compartment and 90% in the t lymphocyte compartment. splanchnic thrombosis is partially recanalized and replaced by collateral circles with cavernous aspects. the patient is on treatment with fondaparinux and has shown neither significant infectious episodes or acute or chronic gvhd. conclusions: we conclude that defibrotide treatment allowed to perform a successfull allogeneic transplant in a patient with mf associated with an overt picture of splanchnic thrombosis. background: hematopoietic stem cell transplantation (hsct) is associated with an increased incidence of secondary malignancies including skin cancer. squamous cell carcinoma (scc) is the most common type in patients who are receiving immunosuppressive therapy and chronic graft-versus-host disease (cgvhd) appears to be an important risk factor for its development. recent studies describe voriconazole exposure as an independent factor that may contribute to this increased risk as well. in our best knowledge, no cases of scc have been reported in pediatric allogeneic hsct to date. methods: we present a case report of a 9 year-old boy who developed a scc with high-risk features six years after undergoing hematopopoietic stem cell transplant. results: a 9 year-old boy with acute lymphoblastic leukemia (all) underwent a matched unrelated bone marrow transplant 7 years ago. he developed grade iv agvhd followed by extensive cgvhd with generalized scleroderma. he required intensive and continued immunosuppressive therapy and was on prolonged antifungal prophylaxis with voriconazole. in march 2017, he developed scc involving left temporal region that was completely excised. two months later, more lesions in scalp and nose were noted and intralesion treatment with methotrexate was started. however, an unfavorable evolution was noted and he was put on systemic treatment including cisplatin and cetuximab receiving the whole scheme from january to march 2018 and continuing only with cetuximab, ten doses in total, until may, for unaceptable and severe tubulopathy that required admission at the hospital in several ocassions. he achieved a very good partial response but progression was noted shortly in follow up. at this point, non curative therapeutic options were found and he was put on intralesion methotrexate and photodynamic theraphy in a weekly basis with palliative intention. unfortunately, tumor growth was fast and patient passed away in august 2018, fifteen months after squamous cell carcinoma diagnosis, due to tumoral progression. conclusions: 1) scc is a rare, non-previously described, secondary malignancy in children undergoing hsct. 2) high-risk features scc constitutes an aggresive disease with a median overall survival below 1 year. 3) cgvhd appears to be an important risk factor for its development. 4) voriconazole induced-photosensitivity might have played a role. 5) cisplatin based regimens +/-cetuximab are a therapeutic option in disseminated and/or high risk cases. as outcomes are unsatisfactory in these cases, alternative therapeutic options need to be explored. disclosure background: pregnancy is a rare event after allogeneic stem cell transplantation (sct) for acute leukemia. here we report, to the best of our knowledge, for the first time on a successful pregnancy after treosulfan-based conditioning. methods: a 29-year old woman was diagnosed with acute myeloid leukemia (aml) secondary to chronic myelomonocytic leukemia in july 2015. ovarian preservation was performed by leuprolide acetate depot injection prior to cytostatic chemotherapy. of note, no cryopreservation of oocytes or ovarian tissue was conducted. she received two cycles of chemotherapy consisting of idarubicine (12mg/m² on day 1-3) and cytarabine (1000mg/m² b.i.d. on days 1, 3, 5 and 7). due to secondary origin of aml sct was performed in first complete remission of aml after conditioning with treosulfan (14g/ m² days 3-5) and fludarabine (30mg/m² days 1-5). she received 4.94×10 6 cd34-positive cells per kilogram body weight from a hla-matched unrelated donor. results: follow-up bone marrow aspirates showed continuous complete remission of aml. seven months after sct she became pregnant, but decided for induced abortion. in january 2018, 25 months after hsct she became pregnant again and desired the child. medical examinations were performed monthly on an outpatient basis in stringent cooperation with the maternity clinic. the course of pregnancy was unremarkable, although she was hospitalized due to premature labor in the 34 th week of pregnancy. however, gynecological examination showed no clinical significant findings, so that section was planned and she could be discharged again. in the 38 th week of pregnancy she gave birth to a healthy girl (50cm, 2810g) by cesarean section. peripartum she developed hypoethesia of the left body half. neurological examination showed no abnormalities and she recovered immediately. there were no other postpartum complications. breastfeeding was established but additional food was necessary for a sufficient nutrition of the child. conclusions: this case of successful pregnancy following sct demonstrates that fertility can recover after treosulfan-based conditioning. however, detailed studies of ovarian function and fertility are necessary to gain more insight into the risk of premature ovarian failure. disclosure: nothing to declare. experimental stem cell transplantation p219 cd19-cart therapy before allo-hsct in children and adolescents patients who diagnosed r/r b-all with e2a-pbx1 background: b-all with e2a-pbx1 in children and adolescents is described with favourable prognosis. but there are more than 10% patients with e2a-pbx1 diagnosed as relapsed or refractory. the results of allo-hsct in children and adolescents with this group leukemia in our center was analyzed in order to understand the therapeutic effect of cd19-cart on the patients. methods: retrospective analysis, from june 1st, 2012 to july 31,2018, all children and adolescents diagnosed relapse or refractory b-all with e2a-pbx1 who received allo-hsct, total 30 cases. all patients was divided into two groups depending on whether or not accepted cd19-cart before allo-hsct. according to fcm-mrd and e2a-pbx1 level before allo-hsct, os lfs and cumulative recurrence rate were analyzed. r 3.2.0 was used as statistical analysis software. results conclusions: 1. for r/r b-all with e2a-pbx1 in children and adolescents, fcm-mrd pre-transplant hasn't obvious effect on the outcome of allo-hsct, while the level of e2a-pbx1 has obvious effect. the out come of e2a-pbx1 negative group was obviously better than positive group. 2. cd19-cart can obviously improve the os and lfs, it is mainly because of cd19-cart can makes more patients fusion to zero. 3. for r/r b-all with e2a-pbx1 in children and adolescents, if chemotherapy can't make the fusion to zero. it is suggested to accept cd19-cart therapy to make the fusion zero. it can improve the outcome of os and lfs. disclosure background: currently, hematopoietic stem cell transplantation (hsct) represents the only curative treatment for numerous hematopoietic malignancies like leukemias, immune deficiencies or metabolic diseases. cd34 serves a quality marker for stem cell grafts, which is not solely expressed on stem cells but also on a variety of progenitors. the role and the impact of these subpopulations remains unknown. we made use of our genetic barcode system to analyze the influence and contribution during reconstitution on a clonal level. methods: fluorescence activated cell sorting (facs) was used to sort hematopoietic stem and progenitor populations, namely hscs, mpps, cmps and clps, which were lentivirally transduced with our previously established bc32 barcoding system. after mixing the marked cells with bone marrow support, lethally irradiated recipient animals were and transplanted and monitored over 16 weeks. we focused on bone marrow, blood, spleen and thymus, on chosen endpoints (1w, 3w, 8w, 16w) and samples were used to analyze the contribution of the subpopulations during the reconstitution process based on fluorescent protein (fp) expression. to investigate the clonal contribution in different organs, we performed next generation sequencing (ngs) and frequencies of unique barcodes in a sample were analyzed by bioinformatical approaches. results: a maximum of 15% of cells expressed the encoded fps, which were mostly derived from the hscs and mpps. cmp-derived cells were only detected 1 week after transplantation in the myeloid compartment. cells derived from the clps were not detected at any time point. we analyzed the barcode content of the differently marked cells after next-generation-sequencing. in accordance with the facs data, the majority of the clones during the 16 weeks of observation are derived from hscs and mpps. cmp-derived clones were only contributing during the first weeks and clp-derived clones are barely detectable. we did not observe any major differences with regard to age of donor or recipient, despite the total number of clones is higher in the group, which received the "aged" graft, independently from the transduced cell population. conclusions: here we show the suitability of our highly complex multi-color barcode system to study the clonal contribution of hscs and three progenitor populations after hsct. our results will contribute to a better understanding how these different populations interact to support the establishment of a new hematopoietic system. emphasized by the variability in data of graft and recipient age, this comprehensive analysis gives rise to an impression to the necessity of personalized graft composition, by which treatment success could be influenced. disclosure: nothing to declare survival and fate of adipose derived mesenchymal stem cells in a rat brain injury model background: mesenchymal stem cells have been identified as promising candidates in the treatment of central nervous system (cns) injury through neurotrophic support and immunomodulation. adipose tissue is an attractive source of mesenchymal stromal/stem cells (ascs) for regenerative therapeutic applications because they can be harvested from autologous donors with minimally invasive methods, can be rapidly expanded ex vivo, show low immunogenicity if allogeneic, and can be used in autologous or heterologous settings. the present study examines the fate and effects of intracerebroventricularly (icv) transplanted ascs in a traumatic brain injury (tbi) model. methods: ascs were isolated from inguinal fat pad of adult wistar rats under sterile conditions and cultured according to standard procedures. ascs at passage 2 (2x10 5 cells) were seeded and transfected with sleeping beauty transposase and pt2 venus-neo r plasmids. selection with g418 antibiotic resulted in the generation of a homogeneous asc population which expressed fluorescent venus protein for several passages, phenotypic characterization showed that these cells were 99.6% double positive for cd44 and cd90 stem cell markers, verifying their mesenchymal origin. tbi was induced by stereotactic surgery under deep anaesthesia and subsequently icv transplantation of venus+ ascs was performed on adult wistar rats. normal ascs-transplanted and tbi-saline transplanted rats were used as controls. the proliferation, migration, survival and fate of transplanted ascs and their effect on injury restoration were examined six weeks post transplantation (pt). results: six weeks pt ascs expressed the fluorescence venus protein and therefore were identified in brain parenchyma. their presence into brain was also confirmed by masson trichrome staining, which revealed their collagen depositions. ascs were found in lesser numbers compared to those transplanted and exhibited no proliferative activity. ascs were found scattered distributed in brain as individual cells, and there were no aggregates of ascs or mass formation into lateral ventricles. extensive migration of ascs was mainly performed through white matter tracks in the corpus callosum and fimbria of hippocampus. six weeks pt ascs retained the characteristics of mesenchymal cells and did not differentiate into cells of neural lineage. ascs exhibited limited long-term survival, which is restricted in perivascular areas probably contributing to vascular formation. homing of ascs into peri-injured area was detected in half of the animals and achieved through the corpus callosum, as revealed by the collagen depositions, in this white matter track. transplanted ascs reduced the area of tbi cavity and did not enhance the astroglial scarring in peri-injured area. in tbi +ascs transplanted animals, the cortical injury site, showed a significantly smaller volume and lower % tissue loss compared to that of tbi+vehicle animals (1.90 ±0.38mm 3 and 12.25±2.83% respectively, versus 1.12 ±0.34mm 3 and 6.57±1.67%, p=0.015 and p=0.019 respectively). conclusions: considering the effects of ascs on inflammation and regeneration, we suggest that their transplantation after brain injury may promote host brain repair mechanisms. ascs transplantation may be beneficial in tbi, however some of its effects need careful and indepth evaluation. disclosure: nothing to declare xie-na cao 1 , yuan kong 1 , zhong-shi lyu 1,2 , qi wen 1 , min-min shi 1,2 , qian-yu sun 1 , yu-hong chen 1 , yu wang 1 , lan-ping xu 1 , xiao-hui zhang 1 , xiao-jun huang 1,2 background: poor graft function (pgf) remains a serious complication after allogeneic hematopoietic stem cell transplantation (allo-hsct). our previous work reported that abnormal bone marrow (bm) endothelial cells (ecs) were involved in the pathogenesis of pgf patients after allo-hsct (bbmt2013; bmt 2016; blood2016), but the explicit mechanism requires further clarification. autophagy is a self-degradative process responsible for the elimination of cytosolic components including proteins and damaged organelles. recent findings demonstrated that stimulation of autophagy could reduce oxidative status and angiogenic potential in ecsafter high-glucose exposure, from diabetic patients.however, little is known regarding the autophagy of bm ecs in pgf patients. therefore, the current study was performed to evaluate whether autophagy in bm ecs play a role in the pathogenesis of pgf. moreover, to investigate the effects of autophagic regulation on ecs and thereby regulating hematopoietic stem cell (hscs). methods: in the prospective case-control study, the autophagy levels were compared in bm ecs from pgf patients, and their matched good graft function (ggf) patients.the expression levels of autophagy-related markers (lc3, beclin1, and p62), and intracellular autophagosomes were detected by immunohistochemical staining, flow cytometry, western blot and transmission electron microscopy. subsequently, rapamycin (the autophagy activators) or hydroxychloroquine (hcq, the autophagy inhibitor) were administrated tothe 7-day cultivated bm ecs and human umbilical vein endothelial cells (huvecs), respectively.the autophagic vacuoleswere detected by monodansylcadaverine (mdc) staining assay. the bm ecsand huvecs were evaluated by cell counting, dii-ac-ldl and fitc-lectin-uea-1 double staining, migration, cell proliferation, and levels of reactive oxygen species (ros). to explore whether autophagy would affect the ability of bm ecs to support hscs in vitro, bm cd34+ cells from healthy donors were co-cultured with cultivated bm ecs and huvecs. colony-forming unit (cfu) and the apoptosis of co-cultured hscs were analyzed. results: the defective autophagy in bm ecs, characterized by decreased intracellular autophagosomes and autophagic vacuoles, decreased expression of lc3-ii and beclin1, and high level of p62, were observed in pgf patients compared with ggf patients. moreover, the coculture of bm cd34+ cells with bm ecs showed significant deficient cfu plating efficiency, and increased apoptosis of cd34+ cells in pgf patients. in vitro upregulation of autophagy by rapamycin quantitatively and functionally improved bm ecsand huvecs, which manifested as more dii-ac-ldl and fitc-lectin-uea-1 double stained cells, increased capacities of migration, lower levels of ros and apoptosis via regulating beclin1 pathway, whereas inhibition of autophagy by hcq aggravated the huvecs and bm ecs from pgf patients. furthermore, in vitro upregulation of autophagy by rapamycin significant improved cfu plating efficiency, and decreased apoptosis in bm hscs co-cultured with huvecs and bm ecs from pgf patients. conclusions: these findings suggest that defective autophagy in bm ecs may be involved in the pathogenesis of pgf. the effect of rapamycin in pgfpatients is potentially mediated by improving the dysfunctional bm ecsto support hscs. therefore, it would be of value to investigate whether upregulating of cytoprotective autophagy of bm ecs may ameliorate pgf, thereby providing a novel clinical intervention for pgf in the future. clinical background: heparanase (hpse) in an endoβ-glucuronidase that specifically cleaves the saccaride chains of heparan sulphate proteoglycans (hs), leading to a loss of integrity of the extracellular matrix and to release of hs-bound cytokines, chemokines, angiogenic and growth factors. hpse gene is polymorphic and includes approximately 300 snps. the combination of two snps, rs4693608 and rs4364254, are involved in the regulation of hpse expression with an inverse correlation between mrna expression and protein levels: gg-cc, gg-ct, gg-tt, ga-cc (low group) expressed high hpse concentration; ga-ct and ga-tt (median group) expressed intermediate hpse levels; aa-tt and aa-ct expressed low hpse concentration (high group). we studied hpse snps in the allogeneic stem cell transplantation (hsct) setting to evaluate a possible association with post-hsct outcomes. methods: we enrolled 228 patients submitted to hsct in our department since 2005 to 2016. for each couple recipient-donor, rs4693608 snp was genotyped using restriction fragment lenght polymorphism assay, whereas for rs4364254 snp an allele-specific polimerase chain reaction was applied. hpse genotype distribution was compared in different groups according to post-hsct outcome: graft-versus-host disease (gvhd), transplantrelated mortality (trm), overall survival (os), infectious complication and disease-free survival (dfs). statistical analysis was performed using ncss 10. results: distribution of rs4693608 snp was as follows: gg 16.7%, ga 49.8% and aa 33.5% among recipients and 17.6%, 53.3% and 29.1% among donors, respectively. hardy-weinberg equilibrium (hwe) was respected. distribution of rs4364254 snp was as follows: cc 15.1%, ct 37.8% and tt 37.8% among recipients and 15.3%, 36% and 48.7% among donors, respectively. rs4364254 snp distribution did not respect the hwe. an association was found between recipient rs4364254 snp and the cumulative incidence of agvhd among patients submitted to a reduced intensity conditioning (ric): 37.3% for tt genotype and 69% for ct or cc genotype (p=0.03). on the other hand, an association was identified between donor rs4693608/rs4364254 snps combination and the cumulative incidence of agvhd: 81.5% for low group donor, 48% for median group donor and 40.8% for high group donor (p=0.04). conversely, aa genotype for donor rs4693608 resulted independent risk factor for cgvhd de novo development (p=0.049, od 2.1) together to donor-recipient sex mismatch (female donor to male recipient vs. others: p=0.005, od 3.56) . considering cmv reactivation rate after hsct, an association was observed according to recipient rs4364254 snp: 82% for cc genotype, 63.5% for ct genotype and 57.1% for tt genotype (p=0.049). multivariate analysis confirmed recipient rs4364254 snp as independent risk factor for cmv reactivation after hsct (p=0.04, od 2.62) together with recipient cmv serostatus at transplant (positive vs. negative: p< 0.01, od 8.49). conclusions: hpse role was widely studied in the setting of inflammation, autoimmune diseases, hematological disease and tumor. however, it still remains debated the inducing or protective activity of hpse in the setting of gvhd. obviously, our results need to be confirmed in a validation cohort. clinical trial registry: na disclosure: nothing to declare novel protocol for autologous hsct in patients with high risk of complications: ambulatory chemomobilization and transplantation of fresh hematopoietic stem cells with backup storage background: autologous hematopoietic stem cell transplantation (ahsct) is standard of treatment in many patients with high risk of complications: dialysed patients, patients with heart and kidney amyloidosis or patients with systemic sclerosis. we introduced recently a novel protocol for ahsct: combination of ambulatory mobilization with very low doses of ara-c and g-csf connected with direct ahsct with fresh cells. this protocol allowed us to reduce the transplant risk in various patient groups traditionally connected with high risk of complications. in this work we summarize the experience in such high risk patients. methods: the prospectively collected database of patients after ahsct was searched for patients who underwent ahsct after chemomobilization with ara-c and transplantation with fresh cells and who fulfilled at least one study inclusion criteria: a) dependence on dialysis b) amyloidosis c) systemic sclerosis d) disqualification from transplantation at other centre due to the high risk of complications. there were together 19 patients selected for this analysis -9 with amyloidosis (6 with ≥ 2 organs involved), 9 dialysed, 2 with systemic sclerosis, 2 unfit at other centre. the database included prospectively recorded serious adverse events during the mobilization and transplantation. results: there were 20 transplantations performed in this group of patients. mortality was 0% at 100 days. all patients underwent successful ambulatory mobilization. all patients received mephalan conditioning with single infusion with median dose of 200mg/m2 (min 140, max 200). mean engraftment was 10.5 days for white blood cells and 12.7 days for plt over 20 g/l. the rate of complications was low with 7 cases of neutropenic fewer, 1 single bacterial culture with staphylococcus epidermidis without clinical signs of infection, median mucositis grade of 0.4 and without patients on parenteral nutrition. the median time of hospitalization was 19 days (min 14, max 29). conclusions: we present here novel protocol of transplantation combining chemomobilization and ahsct with fresh cells with excellent safety profile among most severely ill patients allowing for safe and efficient transplants. with this protocol we were able to overcome multiple risk factors and perform full intensity transplantation in very fragile patients. disclosure: nothing to declare single umbilical cord blood transplantation provides durable disease remission of advanced hematological malignancies in elderly patients background: although allogeneic hematopoietic stem cell transplantation (allo hsct) is potentially curative therapy in a variety of hematological malignancies, little has been reported of the outcome for elderly patients who are not in remission at transplantation. but it has been pointed out that recipient age alone can not be regarded as contraindication for allo hsct in the literature recently, supported by suitable donor, conditioning regimens and appropriate management of complications. we conducted a retrospective study of elderly patients who had advanced hematologic malignancies to elucidate the outcome of single umbilical cord blood transplantation (sucbt) in toranomon hospital kajigaya, japan. methods: we retrospectively investigated the outcomes of 19 patients aged over 65 who underwent their first ucbt from june 2013 to december 2017 in our medical center. results: diseases included acute myelogenous leukemia (n=12), myelodysplastic syndrome (n=3), adult t-cell leukemia/lymphoma (n=2), myelofibrosis (n=1) and chronic lymphocytic leukemia (n=1). the median age at transplantation was 69 years (range, 65-75) and follow-up for survivor post transplantation was 642 day (range, 391-767). all patients were not in complete remission (cr) at the time of transplantation. reduced intensity conditioning (ric) regimens were used in 10 patients. all patients received tacrolimus and mycophenolate mofetil as graftversus-host disease (gvhd) prophylaxis. all cases except 4 early death achieved neutrophil recovery at median 18 days (range, 13-28). at 1 year, overall survival (os) rate and disease free survival (dfs) were 31,6% (95% confidence interval (ci), 12.9-52.2). we performed univariate analysis to identify the factor that influenced os at 1 year, but no statistical significance was demonstrated at the age of transplantation (aged 65 to 69 vs. ≧70, 33.3% (95% ci, 10.3-58.8) vs. 42.9% (95% ci, 9.8-73.4), p=0.68). the cumulative incidence of non-relapse mortality (nrm) at 100 days was 47.4% (95% ci, 23.6-67.9%) and relapse at 1 year was 9.1% (95% ci, 0.0-24.6%). only two patients developed acute gvhd(ii-iv) and one developed severe gvhd at 49 days after transplantation. the main causes of death was infection (n=10), including sepsis (n=8) and viral encephalitis (n=2), followed by idiopathic pneumonia syndrome (n=2) and thrombotic microangiopathy (n=1) during the early phase of transplantation. in contrast, no patients died of recurrence. conclusions: although our report consisted relapsed/ refractory disease of elderly patients at the time of sucbt, durable remission and lower incidence of gvhd could be noteworthy compared with previous reports. further strategies to reduce the rate of nrm and longer duration of follow up would be warranted. disclosure background: pearson syndrome and kearns-sayre syndrome are metabolic disorders caused by a de-novo deletion in the mitochondrial dna (mtdna). allogeneic stem cell transplantation has shown to improve metabolic function in distal organs in several metabolic disorders, but bears significant morbidity and mortality, especially for patients with mitochondrial disorders. novel gene therapies may correct diseases rising from genomic dna mutations, but targeting the mitochondrial dna is complex. mitochondria are able to transfer into cells and between cells, as seen in preclinical models of mitochondrial and other metabolic disorders. here, we introduce a novel concept of mitochondrial augmentation therapy (mat) of autologous cd34+ cells in 4 children with mitochondrial deletion syndromes. methods: patients were treated under a compassionateuse program, approved by the sheba medical center irb and the israeli ministry of health. briefly, mobilization was performed using gcsf alone (n=1) or in addition to plerixafor (n=3) . cd34+ cells were isolated via miltenyi clinimacs system and co-cultured with maternal mitochondria, drawn from peripheral blood and confirmed nondeleted, for 24 hours, and re-infused to the patient without any conditioning. patients were followed for clinical and metabolic parameters. results: all four patients presented with different deletions in mitochondrial dna, and different baseline characteristics, and were treated at the age of 6.5, 7, 11 and 14 years. despite normal cbc, significant bone marrow hypocellularity was seen in 3 evaluated patients (20%, 30% and 50% cellularity at age 7, 11 and 14), which correlated with low colony forming unit capacity of patients and low yield of cd34+ mobilization in the leukapheresis product. patients received on average 2x10 6 enriched cells/kg (range, 1.1 -2.8), and the median enrichment of cd34+ cells was 135% (range, 103-162%). no infusion reactions occurred, and the only severe adverse events of this cellular therapy were leukapheresis-related anemia, hypokalemia, hypocalcemia and alkalosis, all resolved promptly with proper supplementation. follow-up duration is variable, ranging 5-22 months. we were able to show improvement in mitochondrial heteroplasmy (proportion of deleted mtdna of total mitochondrial dna) and in normal mtdna content, starting 1-5 months from cell therapy, which correlated with improved atp production in peripheral blood derived mononuclear cells. clinically, patients showed improvement in aerobic function and endurance (measured by the half-bruce protocol, sit-to-stand test and 6-minute walk test), muscle strength (hand-held dynamometry), and in quality of life, measured by the international pediatric metabolic disability scale. no metabolic crises occurred following cell infusion. conclusions: patients with deletion in mtdna have metabolic dysfunction, including poor bone marrow cellularity and function. hematopoietic stem cells in patients with mtdna deletions can be enriched with normal mitochondria, via mat, as first shown in our patients. this novel process is safe and results in increase in the normal mtdna in peripheral blood of patients, and in improved metabolic and clinical function. clinical trial registry: clinicaltrials.gov nct03384420 disclosure: moria blumkin, noa sher and natalie yivgi ohana -minovia therapeutics, employment p227 high cytotoxic efficiency of alpharetrovirally engineered cd19-specific chimeric antigen receptor natural killer cells for treatment of acute lymphoblastic leukemia stephan müller 1 , tobias bexte 1 , annekathrin heinze 1 , franziska schenk 2 , axel schambach 3 , winfried s. wels 4,5 , ute modlich 2 , evelyn ullrich 1, 5 background: autologous chimeric antigen receptormodified (car) t cells with specificity for cd19 showed potent antitumor efficacy in clinical trials regarding relapsed and refractory acute lymphoblastic leukemia (all). natural killer (nk) cells are cytotoxic lymphocytes that are capable to kill their targets in a non-specific manner and additionally do not cause gvhd. therefore, using cd19-car-nk cells exhibits several advantages, such as safety in clinical use, possible allogenic settings and the potential to also attack heterologous leukemia cells which lost cd19. previous approaches used cd19-car-nk cells pre-stimulated by feeder cells, bearing potential risks. thus, we focused on the optimization of generating cd19-car-nk cells by viral transduction under feeder-cell free conditions. methods: human nk cells were isolated from healthy donor peripheral blood mononuclear cells via cd56 negative selection. after a feeder-cell free expansion phase with interleukin 15, transductions were performed with an egfp or a cd19-car encoding vector at different multiplicities of infection (moi). to optimize gene modification different transduction enhancers (retronectin and vectofusin-1) and viral vector systems (lentiviral and alpharetroviral) were compared. finally, generated cd19-car-nk cells were tested in their ability to kill cd19positive and cd19-negative cell lines. results: nk cells transduced with a lentiviral egfp encoding vector or a lentiviral cd19-car vector using retronectin and vectofusin-1 showed similar transduction efficiencies for both transduction enhancers (egfp: retronectin moi 10: 12.9%; vectofusin-1 moi 10: 12.8%; cd19-car: retronectin moi 5: 10.7%, moi 10: 9.2%; vectofusin-1 moi 5: 11.3%, moi 10: 14.4%). the generated cd19-car-nk cells showed increased cytotoxic capacity against cd19-positive cells compared to nontransduced (nt) nk cells (72.7% vs. 23.6%, effector to target (e:t) ratio 1:1). both nk cell populations were equally efficient in killing cd19-negative cells (30.5% vs. 25.7%). alpharetroviral transduction of nk cells with an egfp encoding vector showed higher transduction rates with vectofusin-1 than with retronectin (retronectin moi 1: 5.3%, moi 5: 9.7%; vectofusin-1 moi 1: 55.3%, moi 5: 51.6%). further using vectofusin-1, similar transduction efficiencies could be achieved with an alpharetroviral cd19-car encoding vector (moi 1: 11.1%, moi 5: 49.2%, moi 10: 68.9%), outperforming the efficiencies of lentivirally generated cd19-car-nk cells in the same experiments (moi 1: 1.5%, moi 5: 8.4%, moi 10: 14.9%). additionally, alpharetroviral cd19-car-nk cells showed a higher cell killing activity against cd19-positive cells than lentiviral cd19-car-nk cells or nt-nk cells (90.5% vs. 62.5% vs. 9%, e:t ratio 1:1). interestingly, similar killing activities were achieved with an e:t ratio of 0.5:1 (88.9% vs. 58.3% vs. 10.3%) and alpharetroviral cd19-car-nk cells remained a stable cytotoxicity level at lower cell concentrations down to an e:t ratio of 0.1:1. all three nk cell populations were equally efficient in killing cd19negative cells (16.4% vs. 23.6% vs. 12.3%, e:t ratio 1:1). conclusions: cd19-car-nk cells can be successfully generated under feeder-cell free conditions using different transduction enhancers and viral vector systems. these data suggest the usage of vectofusin-1 in combination with alpharetroviral vectors to genetically modify nk cells to achieve sufficient amounts of transduced cells. these cd19-car-nk cells mediate high cytotoxicity and therefore may offer a new therapeutic option in the treatment of all. disclosure: axel schambach is an inventor on a patent describing alpharetroviral sin vectors. winfried s. wels is an inventor on a patent describing chimeric antigen receptors with an optimized hinge region. the remaining authors have nothing to disclose. graft-versus-host diseaseclinical walter spindelböck 1 , bianca huber-krassnitzer 1 , barbara uhl 1 , gregor gorkiewicz 1 , hildegard greinix 1 , christoph högenauer 1 , peter neumeister 1 background: steroid-refractory acute gastrointestinal (gi) graft-versus-host disease (agvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-hsct) associated with a high mortality rate. loss of intestinal bacterial diversity is thought to be associated with severity of gi-agvhd and an impaired intestinal microbiota with reduced diversity is an independent predictor of mortality. methods: the fecal microbiota transplantation (fmt) procedures were performed according to a protocol approved by the local ethical committee (29-027ex 16/17) after obtaining informed consent. donors were healthy adult subjects screened for potential infections by serologic and microbiologic tests according to local standards. donor stool was diluted with saline and homogenized to a volume of~250 ml fecal solution for instillation into the terminal ileum and caecum via colonoscope. microbiota sequencing analysis of 16s rdna was performed before fmts and afterwards at predefined timepoints. results: we report the outcome of nine patients refractory to 3-6 lines of immunosuppressive therapies with lower gi-stage iii (n=1) or iv (n=8) agvhd following repetitive fmts from a single donor. all patients had received an allo-hsct for mds (n=3) , aml (n=4), pmf (n=1) and mm (n=1) following a reduced intensity (n=5) or mac (n=4) conditioning regimen using pbsc as stem cell source. after an onset of lower gi agvhd between 11-465 days after allo-hsct, nine patients refractory to several lines of immunosuppressive therapies received 1-6 fmts (6 patients were treated with more than 2 fmts, in 3 patients fmt was only administered once or twice) mostly in weekly intervals. five patients achieved a clinical complete response with resolved diarrhea and no gastrointestinal complaints, and four of these could be discharged without gvhd symptoms. two patients (pr, nc) were discontinued after 2 or 3 fmts in pr or nc due to concomitant infections (metapneumoviral pneumonia, cmv gastroenteritis), the 2 other non-responders succumbed to gvhdrelated infectious complications. the establishment of donors' microbiota with the emergence of new taxa, an increase in bacterial richness/diversity, and the disappearance of the "enterococcus signature" were associated with disease control and response to fmt. except the possible transmission of adenovirus by fmt in one patient, no other immediate procedure-related infections or other side effects were observed. conclusions: restoration of dysbiosis by fmt might represent a promising novel therapeutic approach for a subset of patients with refractory lower gi-agvhd. vigorous donor screening for infectious disease is mandatory. clinical background: migration of allo-activated donor effector tcells from lymphoid tissues to target organs is an important step in acute graft versus host disease (gvhd). the sphingosine-1-phosphate-1 (s1p1) receptor plays a crucial role in lymphocyte trafficking. data from animal models suggest that pharmacological modulation of the s1p1 receptor reduces gvhd and improves mortality. we investigated this mode of action by using the secondgeneration s1p1 modulator krp203 for the prophylaxis of gvhd in a pilot clinical trial in patients undergoing allogeneic hsct. methods: a multi-centric, phase 1b, prospective, open label, two-part study was conducted to evaluate the safety, tolerability and pharmacokinetics of krp203 in patients undergoing allogeneic hsct for hematological malignancies. primary endpoint was safety. initial efficacy was explored based on the incidence of gvhd, mortality and relapse. part 1 was a single arm open label study to investigate the safety of 3 mg/day krp203 added to standard of care gvhd prophylaxis (csa/mtx) in 10 patients. part 2 was a randomized two-arm open label study to compare the safety, efficacy and pk of 3 mg/day of krp203 in combination with tacrolimus/mtx to 1 mg/day of krp203 in combination with csa/mtx in 13 patients. in both parts, treatment with krp203 was initiated 10 days before hsct and continued for an additional 100 days. patients were followed up for up to 2 years. results: 23 patients were included in the study. 16 of 23 patients completed the 110-day treatment with krp203 at the assigned doses. median duration of follow-up was 264 days (range 153 to 271 days). krp203 was safe and well tolerated. 11 serious adverse events (saes) suspected to be related to krp203 were observed. macular edema (n=3) and peripheral edema (n=1) as s1p related adverse events occurred and resolved without sequelae. of note, the incidence of macular edema in hsct recipients is unknown. neutrophil engraftment was confirmed in all patients with a median of 16 days (range 12 to 45 days). 5 of 23 patients presented with grade iii or iv acute gvhd (on days 50, 56, 68, 99 and 102) . no gvhd or infection related death occurred during the first 100 days. 100-day survival was 96%, with no death occurring during krp203 treatment. 1 death occurred on study day 90 due to lymphoma relapse. a second death occurred on study day 121 due to liver gvhd. four patients died in the follow-up period due to gastrointestinal gvhd (day 265), aspiration pneumonia (day 327) and relapse (day 533 and day 877). the kaplan-meier estimate of overall survival at 1 year was 0.75. when comparing the data from the two dose groups (1 and 3 mg krp203), no major differences in safety, engraftment, gvhd rate or mortality were observed. conclusions: this clinical trial was the first to test s1p modulation in this population. our data suggest that krp203 had no negative impact on engraftment and overall, was safe, and well tolerated. based on exploratory data, when comparing to matched historical mortality data, krp203 may have favorable effects on overall survival ( figure 1 ). background: uric acid is a danger signal contributing to inflammation. relevance to allosct has been demonstrated in preclinical models: the depletion of uric acid led to improved survival and reduced gvhd (j exp med. 2013 sep 23;210(10):1899-910). results of a clinical pilot trial suggested that peri-transplant uric acid depletion reduce acute gvhd incidence (bbmt 2014 may;20(5):730-4). methods: this international multicentric study aimed to study the association of uric acid serum levels before start of conditioning with allosct outcome. patients with acute leukemia, lymphoma or mds receiving a matched sibling allosct for the first time were considered for inclusion, regardless of conditionning. data were prospectively collected between 8/2014 and 2/2018. a comparison of outcomes between patients with high and low uric acid level was performed using univariate analysis and multivariate analysis using cause-specific cox model. variables included in the multivariate analyses were age, sex mismatch, diagnosis, disease status, karnofsky score, number of cd34 cells given, intensity of conditioning, type of gvhd prophylaxis, atg use, time from diagnosis to transplant, year of transplant and cmv status. results: twenty centers from 10 european countries reported data on 385 allosct recipients. patient characteristics are given in table 1 . the uric acid cut off point was determined at 4.3mg/dl (median of measured uric acid levels). overall survival (os) and progression free survival (pfs) of allosct recipients with uric acid levels above cut off measured before start of conditioning were significantly shorter ( figure 1a , os univariate hr=2.4 ci=1.6-3.7 p< 0.001; multivariate hr=2.8, ci=1.7-4.7, p< 0.0001) ( figure 1b , pfs univariate hr=2 ci=1.1-3.7 p=0.02; multivariate hr=2.7, ci=1.4-5, p=0.003). nonrelapse mortality was significantly increased in allosct recipients with high uric acid levels prior to start of conditioning (univariate hr=2 ci=1.1-3.7 p=0.018; multivariate hr=2.65, ci=1.41-5.01, p=0.003). in addition, there was a non-significant trend towards higher acute gvhd incidence (gvhd grades ii-iv univariate hr=1.2 ci=0.8-1.9 p=0.4; multivariate hr=1.5 ci=1-2.4, p=0.08) in allosct recipients with uric acid levels above cut off before transplantation. finally, the incidence of relapse after allosct was moderately increased in the cohort with higher uric acid levels (univariate hr=1.6 ci=1-2.5 p=0.09; multivariate hr=1.59, ci=1.02-2.49, p=0.04). conclusions: high uric acid levels before start of conditioning correlate with high mortality after allosct. our results can serve as rationale for clinical trials on depletion of uric acid during allosct. results: we found significant correlation between donors' ctla-4 +49a>g polymorphism and hsct outcome. genotype aa was present in 170 donors, ag in 183 donors and 44 donors was homozygous for g allele. recipients who received graft from g allele carrier donors showed significantly increased cumulative incidence of relapse (at 24 months aa: 20.7%, ag: 23.6% and gg: 34.3%; p=0.04). on contrary, the frequency of the acute gvhd grades iii-iv and cytomegalovirus (cmv) reactivation/disease decreased according to the presence of the g allele in the donor ctla-4 genotype [agvhd: aa: 20%, ag: 12%, gg: 5%; p= 0.014; cmv: aa: 24%, ag: 16%, gg: 9%; p= 0.039]. cumulative incidence of agvhd was also markedly decreased among patients with g allele carrier donors (at 100 days aa: 19.9%, ag: 10.4%, gg: 6.4%; p=0.01). donor genotype similarly influenced hsct outcome in mud donor and mac conditioning subgroups. overall survival (os) was not different in patient subgroups according to donor genotypes [os at 24 months: aa: 55.5 ±3.8%, ag: 54.4±3.7%, gg: 49.4±7.6%; p= 0.68]. we did not find any correlation between recipients' ctla-4 +49a>g polymorphism and hsct outcome. conclusions: several ctla-4 snps have previously been described to be associated with relapse rate, incidence of agvhd and os, but results are often contradictory in the publications. in our study, ctla-4 +49a>g polymorphism of hsct donors influenced risk of relapse, agvhd, cmv and cause of death, but not overall survival. the genotyping of ctla-4 +49a>g polymorphism in donors may help in the risk assessment process and the choice of personalised therapy. disclosure: nothing to declare. background: although steroids remain first-line therapy for the treatment of acute graft versus host disease (agvhd), response rates in patients with grade iii-iv disease are poor, with no apparent improvement in survival over the past 15 years. we performed a prospective, multicenter trial to assess the efficacy and safety of the combination of ruxolitinib and etanercept as a novel approach to treat grades iii-iv sr-agvhd . methods: forty malignant hematologic disease patients with grades iii-iv sr-agvhd after allo-sct from three centers in east china were enrolled from january 2017 to june 2018. ruxolitinib was initiated at a dose of 5-10 mg bid for 2 months, and then tapered gradually for another one month. etanercept was administrated at 25mg biw for 2-8 weeks. results: the median age of patients was 25 (range 15-59) years. at day 30 after the combination treatment, the overall response rate (orr) was 90% including 30 crs (75%) and 6 prs (15%). the median time to the optimal response was 13 (range 3-34) days. the incidences of cr per organ were 95.7%, 80.8%, and 80% for skin, liver, and gut, respectively. the agvhd relapse rate was analyzed for the patients who had achieved cr or pr and survived beyond 60 days. relapses in agvhd occurred in 9.38% (3/ 32) of responsive patients. the patients who received ruxolitinib within 14 days after agvhd onset have a significant higher cr rate that those with delayed ruxolitinib therapy (96.2% vs. 42.9%, p=0.001). and the patients without gut infections have a significant higher cr rate than infected cohort (92.6% vs. 46.2%, p=0.002). by logistic regression analysis, the time from agvhd to ruxolitinib (rr=4.17, p=0.011) and gut infection (rr=3.31, p=0.031) were independent predictors for incomplete response. thirteen patients (13/40, 32.5%) suffered from at least 1 infectious episode after the start of the combination therapy, and pulmonary infectious diseases was a frequent complication (9/40, 22.5%). iii-iv cytopenia and cmvreactivation were observed in 30% and 47.5% of patients. the 1-year overall survival (os) after initiation of the combination therapy were 76.8%. the 1-year nrm and relapse incidence was 17.9% and 19.9%, respectively. patients with complete response on day 30 had significantly higher os probability than non-cr patients (1-year os: 86.1% vs 48.0%, p=0.01). compared with the historical cohort of basiliximab and etanercept for sr-agvhd in our center (n=31), no significant difference was found on the baseline. although the orr in patients treated with ruxolitinib and etanercept is identical with the historical cohort, ruxolitinib group achieved rapider remissions in liver agvhd and gut agvhd than the historical cohort (gut agvhd: 11 days vs. 17 days, p=0.026; liver agvhd: 21 days vs. 28 days, p=0.039), thus, with regard to hospital stay after agvhd onset, the ruxolitinib cohort stayed shorter (median: 18 days vs. 29 days, p=0.005) than basiliximab cohort. conclusions: combined treatment with ruxolitinib and etanercept resulted in a rapid cr to visceral agvhd and meanwhile reserve graft anti-leukemia (gvl) effect as the relapse rate of primary disease is relatively lower. the various infection complications associated with ruxolitinib merit more attention. disclosure: nothing to declare background: graft-versus-host disease (gvhd) remains one of the main life-threatening complications after allo-hsct, especially in patients with non-malignant diseases. the standard gvhd prophylaxis strategy is mostly based on the use of calcineurin inhibitors alone or in combination with other immunosuppressive (is) post-transplant cyclophosphamide (ptcy) is effective gvhd prophylaxis optiont for adult patients (pts), but has limited data in children. methods: the study aim was to evaluate ptcy as gvhd prophylaxis in pediatric pts with inherited disorders undergoing allo-hsct. 96 pts, the most of them are pediatric age (median age -3 y.o., range 7 month -30 y.o.) with different types inherited disorders (β-thalassemia -10, bone marrow failure syndromes -26, storage diseases -46, primary immunodeficiencydisorders -14) were inrolled in retrospective study. donor type was: matched/mismatched unrelated (mud/mmud) -69, matched related donor (mrd)-15, haploidentical (haplo) -12. conditioning regimen was: myeloablative (mac) -43, reduce-intensity (ric) -53. graft sourse was: bone marrow (bm) -68, peripheral blood stem cells (pbsc) -26, combintions bm +pbsc/bm+cord blood -2. ptcy 50 mg/kg days +3, +4 based gvhd prophylaxis recived 33 pts., standart gvhd prophylaxis based on calcineurin inhibitors -63 pts. results: cumulative incidence (ci) of agvhd was 48%. grade 2-4, 3-4 agvhd were 40% and 22% respectively. ptcy based gvhd prophylaxis reduced ci of agvhd (35% vs 56%, p=0,025). another reduce ci of agvhd factors were mac (31% vs 59% in ric pts group, p=0,044), mrd (13% vs 44% in haplo group vs 56% in mud/mmud group, p=0,024), bm as a transplant source (44% vs 62% in pbsc group, p=0,05). in a multivariate analysis mac (hr 2,6 95%ci 1,7-6,, p=0,02), time from diagnosis to allo-hsct less then 22 month (hr 2,6 95%ci 1,1-6,2, p=0,03) were predictive for reducing ci agvhd. for agvhd 2-4 st. significant factor increase ci was female donor both in univariate (51% vs 34%, p=0,04) and multivariate analysis (hr 0,7 95%ci 0,2-0,8, p=0,02).5 years overall survival (os) was 60%. improving os factors were: transplant age younger then 5 y.o. (80% vs 35%, p=0,000), time from diagnosis to allo-hsct less then 22 month (72% vs 38%, p=0,000), engraftment (72% vs 22%, p=0,000). in a multivariate analysis only transplant age younger then 5 y.o. (hr 3, 3 95%ci 1, (4) (5) (6) (7) (8) p=0, 006) and engraftment (hr 0,3 95%ci 0,1-0,7, p=0,005) were predictive for os. conclusions: ptcy-based gvhd prophylaxis can be effective options for reduce risk of acute gvhd. using unrelated donors, bone marrow as transplant source and mac can reduce ci of gvhd. performing allo-hscr earlier from diagnos and in earlier age can improve os patients with inherited disorders background: diarrhea is a frequent complication after allo-sct. at onset it is often difficult to differentiate gi gvhd from other causes of enterocolitis. recently, non-invasive tests, such as fecal calprotectin (fc), have been validated as markers of gut inflammation in patients with inflammatory bowel disease, but only a few studies have been published regarding its use as a diagnostic marker in gi gvhd. methods: our aim in this study was to explore the levels of fc in allo-sct recipients with new-onset diarrhea. so far we have included 43 allo-sct recipients who developed acute diarrhea ≥ stage 2-4 at a median of 75 days (range:12-328) post allo-sct. stool samples were analyzed as soon as possible after the onset of diarrhea. fc levels were determined in addition to an extensive microbiological panel for infectious enterocolitis (including norovirus pcr and c. difficile associated diarrhea). endoscopies for histologic analysis were performed according to the treating physicians' discretion (n=15). results: patients characteristics are summarized in table 1 . median follow-up for survivors was 524 days (range:151-1834). twenty-eight patients (65%) were diagnosed of gi-gvhd. the additional causes of diarrhea were: drug-related enterotoxicity (n=6), viral enteritis (n=2), food intolerance (n=2), c.jejuni-enteritis (n=1), and non-specific causes (n=4). the concentration of fc was higher in patients with gi gvhd vs. other causes of diarrhea (544μg/g +/-71 vs. 58 μg/g +/-33, p= 0.03). patients who did not develop severe enterocolitis had normal to slightly raised calprotectin at the onset of diarrhea [< 100-150 in 16 out of 19 (89%) cases], including 100% (6/6) of patients with enterotoxic drug-related diarrhea. among the 28 patients with gi-gvhd, 13 (30.2%) were later found to be steroid-resistant. as shown in figure 1 , we found a significant association between high fc (≥400μg/g) and severe-refractory gvhd (hr 5.7, p=0.01). of note, high values of fc were also found in 3 patients with severe infectious enteritis (norovirus, adenovirus and c.jejuni infections), with baseline fc>800 μg/g, respectively. overall survival was 78% (ic95%:65-91) at 12 months. hypoalbuminemia and thrombocytopenia were the only variables linked to 1-yr os in univariate analysis, regardless of the cause of enterocolitis. conclusions: in the absence of standarized (and expensive) biomarker panels for analyzing and predicting gvhd onset and outcomes, the fc test may be an useful tool in the allo-sct setting. our initial results show that fc is helpful in predicting mild causes of diarrhea and to identify patients with a high probability of developing severe (and potentially steroid-refractory) gi gvhd, although high levels are also found in severe infectious enteritis. background: there is an urgent need for effective therapy for severe acute gvhd. results of gvhd therapies beyond 6 months are rarely reported. we here report a median follow-up of 4 years. we introduced mesenchymal stromal cells as therapy for severe acute gvhd, with a dramatic response in some, but not all patients. the placenta protects the fetus from the mothers haploidentical immune system during pregnancy. we found that maternal stromal cells from the fetal membrane, so called decidua stromal cells (dscs) were more immunosuppressive than other sources of stromal cells. methods: we treated 21 patients, median 49 years of age (range 1.6-72) for severe acute gvhd. all had biopsy proven gastro-intestinal gvhd. all were steroid refractory,11 after >7days or with progression and 10 after >3 days. we used an improved protocol where dscs were thawed and infused in a buffer with 5% albumin. dscs were given at a median dose of 1.2 (0.9-2.9) x 10 6 cells/kg and 2(1-6) doses, given one week apart. viability of frozen and thawed dscs was 95% (89-100) and cell passage was 4 (2-4). results: complete resolution of gvhd was seen in 11 patients and 10 had a partial response. the cumulative incidence of chronic gvhd was 52%. six had mild, 4 moderate and one severe nih overall gvhd severity scoring. nine patients died, 3 from relapse, 1 acute gvhd and septicemia, 1 zygomycetes infection, 1 liver insufficiency, 1 cerebral hemorrhage, 1multiorgan failure and 1 chronic gvhd with obstructive bronchiolitis. four years transplant related mortalliy was 28.6% and overall survival was 57%. survival was not significantly worse (p=0.33) than 66% for all 293 patients undergoing allogeneic hematopoietic cell transplantation during the same period 2012-2015. conclusions: to conclude, dscs seems to be a promising therapy for severe acute gvhd. randomized trials are under way. disclosure: nothing to declare p237 anti-apoptotic protein bcl-2 is upregulated in graftversus-host disease stem cell transplantation (allo-hsct) with 30-80% developing either acute or chronic gvhd. recently, bcl-2 inhibitor venetoclax was approved for treatment of chronic lymphocytic leukemia. induction of apoptosis and depletion of lymphocyte subpopulations e.g. follicular b-cells or cd4 + and cd8+ t-cells led to further exploration in autoimmune disease. methods: to establish expression levels of genes in the bcl-2 pathway, low-input rna sequencing was performed on t cells isolated from non-inflamed skin and peripheral blood of hsct recipients at 5 different time points before until 1 year after transplantation. furthermore, we analyzed blood, lung, gut and skin samples of 105 patients post allo-hsct with and without previously untreated acute or chronic gvhd by rt-pcr, flow cytometry and tissue immunofluorescence. [[p237 image] 1. bcl-2 is up-regulated in t and b lymphocytes of acute and chronic gvhd lesions.] results: rna-sequencing revealed that t cells upregulated bcl-2 upon conditioning treatment (day 0) and cells of patients who later developed gvhd failed to downregulate bcl-2 after transplantation (day+14, day+100). bcl-2 protein levels were elevated in overall leukocytes and pathogenic cell subsets including monocytes, cd8+ t lymphocytes and nkt cells showed significantly higher expression of bcl-2 in peripheral blood of gvhd patients as compared to healthy controls. these results could be recapitulated in tissue samples, where disease-promoting lymphocytes (t, b, nk, nkt) were numerically expanded and expressed bcl-2 in acute and chronic gvhd skin lesions. notably, non-pathogenic cell types such as keratinocytes did not exhibit increased bcl-2 expression compared to control samples from hsct recipients and healthy donors. while bcl-2 rna expression did not depend on type of conditioning (mac vs. ric) or gvhd grade, it correlated to disease severity and was significantly elevated in biopsies of patients with steroidrefractory gvhd. conclusions: we could show exclusive upregulation of bcl-2 in gvhd-mediating cell types in peripheral blood and tissue samples affected by gvhd, correlating to gvhd severity and response to first-line therapy. thus, bcl-2 inhibition may present a novel and urgently needed targeted therapy in treatment of steroid-refractory acute and chronic gvhd. disclosure: supported by a docmed fellowship od the austrain academy of sciences background: graft-versus-host disease (gvhd) represents a major contributor to morbidity and mortality in recipients of allogeneic hematopoietic cell transplants (hct). several therapeutic strategies exist for gvhd prophylaxis and include post-transplant cyclophosphamide (ptcy) and antithymocyte globulin (atg). while several groups have described the use of ptcy in younger patients, there is a paucity of data about the efficacy of ptcy in older individuals, particularly when combined with atg. we investigated the combined effect of ptcy with atg on transplant outcomes in older patients at princess margaret cancer centre, toronto, canada. methods: this retrospective study included all patients age ≥60 who underwent allogeneic hct for any indication at our centre between december 2013 and july 2017. overall survival (os) was calculated using kaplan-meier analysis and multivariable cox proportional hazards regression. cumulative incidence of relapse (cir) and non-relapse mortality (nrm) were calculated using competing risk regression (fine and gray method). incidences of acute (agvhd) and chronic (cgvhd) were compared using the fisher's exact test. results: of 133 patients, 84 (63%) were male. median age was 65 (range 60-74) and median follow-up among survivors was 28 months (range 6-60). acute myeloid leukaemia (aml) was the most common indication for hct (57 patients, 43%), followed by myelodysplastic syndrome (37 patients, 28%) and myelofibrosis (17 patients, 13%). eightyfour (63%) patients had a matched unrelated donor, 37 (28%) had a matched related donor and 12 (9%) had a haploidentical donor. one hundred twenty-five (94%) patients received reduced intensity conditioning. sixty-two (47%) patients received ptcy combined with atg (4.5 mg/kg) while 71 (53%) received other forms of gvhd prophylaxis. os at 2 years was 46% (95% confidence interval (ci) 37-54) in the entire cohort. patients who received ptcy with atg had a superior 2-year os compared with other gvhd prophylaxis regimens ( figure 1a ): 57% (95% ci 44-69) vs. 37% (95% ci 26-49), respectively (hr=0.6, 95% ci 0.4-0.9, p=0.02). the 2-year nrm for the entire cohort was 37% (95% ci, 29-46). patients who received ptcy with atg had a lower 2-year nrm compared to those who did not ( figure 1b ): 23% (95% ci 13-34) vs. 49% (95% ci 37-60), respectively (hr=0.4, 95% ci 0.2-0.7, p=0.002). the 2-year cir in the whole group was 24% (95% ci 17-32). use of ptcy with atg was associated with a modest increase in cir at two years ( figure 1c ): 35% (95% ci 22-49) vs. 16% (95% 8-25), respectively (hr=2.1, 95% ci 1.0-4.0, p=0.04). there was a trend toward lower incidence of grade ii-iv agvhd among patients who received ptcy with atg compared to those who did not: 15% vs. 30 % (p=0.06). the incidence of grade ii-iv cgvhd was lower in individuals who received ptcy with atg compared to those who did not: 26% vs. 45% (p=0.03). conclusions: in older hct recipients, use of ptcy combined with atg is associated with improved os, lower nrm, decreased risk of both agvhd and cgvhd and a modest increase in relapse risk. therefore the ptcy with atg combination represents an effective strategy for gvhd prophylaxis in older allogeneic hct recipients. disclosure: the authors have no conflict of interest to declare. outcome of severe graft versus host disease in pediatric patients with nonmalignant diseases after allogeneic bone marrow transplantation. a single center experience irina zaidman 1 , sigal grisariu 1 , batia avni 1 , ehud even-or 1 , bella shadur 1 , adeeb nasereddin 1 , polina stepensky 1 background: hematopoietic stem cell transplantation (hsct) remained the only curative option for many nonmalignant diseases in pediatric patients. survival after hsct has improved the last few years due to significant advancement in human leukocyte antigens (hla) typing techniques, less toxic conditioning regimens and better supportive care and resulted to 90% survival and cure in some non malignant diseases. graft-versus-host disease (gvhd) remains a major complication of hsct and leading cause of morbidity and mortality. prognosis of patients with high grade gvhd is dismal and survival rate varies between 25% to 55% in pediatric patients. methods: the retrospective study included patients with non malignant diseases who underwent allogeneic hsct at hadassah medical center from 2008 to 2018. the collected data included patient´s clinical data and transplant characteristics. the study was approved by the institutional helsinki committee. results: 182 children with nonmalignant diseases underwent 194 allogeneic bone marrow transplantations in hadassah university hospital during ten years period. fifty seven patients (31%) developed agvhd grade 1-4, twenty five of them (13.7%) grade 3-4. median age was 6.34 (range 0.37-17.7), most patients were males (17 males, 8 females). 9 patients underwent bmt from fully matched family members, 6 children were transplanted from matched unrelated donors and 10 from mismatched donors. twenty one of 25 patients with severe gvhd (83%) survived. four patients (17%) died from severe gvhd and complications of immunosuppressive treatment. 3 of 4 deceased patients were transplanted from mismatched donor, in 3 of 4 cases the age of donor was advanced, 2 of 4 patients developed severe gvhd and died after second hsct. all 4 patients were refractory to different treatment modalities. three of 4 patients died in 2012 and one in 2015, it was no death from severe gvhd in 12 patients that were transplanted and developed high grade gvhd after 2015. conclusions: the results of this study show a high survival rate of 83% in pediatric patients with non malignant diseases and severe gvhd. significant risk factors for mortality in our group included mismatched donor, advanced age of donor and second transplant. trend to better survival was observed after 2015. additional multicentral studies analyzed the outcomes of agvhd in pediatric patients with nonmalignant diseases are urgently required. background: chronic graft-versus-host disease (cgvhd) is a serious late complication after allogeneic hematopoietic stem cell transplantation (allohsct) with heterogeneous presentation and still poorly understood pathophysiology including inflammation and endothelial dysfunction. factor viii (fviii) and von willebrand factor (vwf) are coagulation factors but also known indicators of endothelial dysfunction and inflammation in different settings, and therefore could serve as interesting candidate biomarkers of cgvhd. methods: since 2013 patients after allohsct were assessed by the multidisciplinary cgvhd team at the university hospital center zagreb, croatia, using established nih cgvhd-related measurements. an extensive history, physical and laboratory evaluations were performed, including fviii, vwf:ag and vwf:ac analysis. descriptive statistic and non-parametric analyses were performed. variables that showed significant univariate correlations were used in multivariate logistic regression (mlr) to identify the most predictive for fviii, vwf:ag and vwf:ac in cgvhd patients. results: 70 cgvhd patients and 41 controls (subjects after allohsct without cgvhd) were analysed. median age of cgvhd patients was 42 (9-65) years, 50% females, 91.5% underwent allohsct for hematologic malignancies, 55.7% had myeloablative conditioning and 52.9% matched related donor. median time from hsct to study was 450.5 days and from cgvhd diagnosis to study 82 days. there were no demographic neither transplant related significant differences between cgvhd patients and controls beside stem cell source (peripheral blood 71.4% vs 51.2%, p=0.041) and history of acute gvhd (70.0% vs 22.0%, p< 0.001). majority of patients had moderate (52.9%) or severe (42.6%) nih global cgvhd score, 57.2% active cgvhd by clinician´s impression. median number of organs involved by cgvhd was 3 (1-6), and the most frequently involved organs were mouth, skin and eyes (52.0% each). cgvhd patients compared to controls had higher fviii levels (median 206 (52-453)% vs 182 (51-406)%, p=0.044, reference range 50-149%) and higher vwf:ag (median 261.6 (76.6-601)% vs 203.2 (51.9-600)%, p=0.030, reference range 50-160%), while vwf: ac showed a trend toward higher levels among patients (median 253.4 (54-601)% vs 178 (48.6-601)%, p=0.084, reference range 50-150%). patients had higher ggt (p=0.002), lower anticardiolipin igg (p=0.001) and igm (p=0.003), and lower albumin (p=0.018) than controls, without differences between other laboratory parameters. univariate analysis showed that among cgvhd patients higher fviii was associated with worse karnofsky score (ks) (p=0.031) and performance score (ps) (p=0.030), higher leukocytes (p=0.031), cholesterol (p=0.003), triglycerides, ast, alt, ggt, ldh, and lower albumin. higher vwf:ag and vwf:ac in cgvhd patients were associated with worse ks and ps (p< 0.001), with more active cgvhd (p< 0.001), worse nih cgvhd liver (p=0.042; p=0.039) and nih cgvhd mouth (p=0.012; p=0.009), higher total nih score (p=0.044; p=0.005), higher number organs involved (p=0.013; p=0.003), higher esr, monocytes, ddimers, ast, alt, ggt, ldh, triglycerides, β-2-microglobulin, ferritin, total proteins, iga and lower albumin. mlr analysis showed leukocytes (p=0.018) and cholesterol (p=0.010) as the strongest predictor of fviii (r2=49.8%; p< 0.001), while strongest predictor of vwf: ac was number of organs involved by cgvhd (r2=71.7%; p=0.031). conclusions: results of this study detected high fviii and vwf levels in cgvhd patients with possible reflections to cgvhd manifestations, what needs to be further confirmed in larger longitudinal studies. disclosure: this work was supported, in part, by the unity through knowledge fund project entitled "clinical and biological factors determining severity and activity of chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation", and also, in part, by the croatian science foundation project entitled "new biomarkers for chronic graft-versus-host disease". antonela samardzic -work financed by the croatian science fondations`"young researchers`career development project -training of doctoral students" background: thrombotic microangiopathy (tma) is a severe complication of allogeneic hematopoietic cell transplantation (hct) with multisystem involvement. a few recent reports have recognized evidence of tma in the intestinal vasculature (intestinal tma/itma) of patients with graft-versus-host disease (gvhd) with or without tma. we aimed to identify patients with itma and describe histological, clinical and prognostic features. methods: we prospectively evaluated available endoscopic samples (stomach and/or colon) from consecutive adult hct recipients for previously described histopathologic signs of itma (january 2017-september 2018). systemic tma was diagnosed according to the international working group criteria. we compared findings among 3 clinical groups: gvhd/systemic tma, gvhd/no systemic tma and no gvhd/no tma. results: we studied 20 patients, 5 classified as gvhd/ systemic tma, 11 gvhd/no systemic tma and 4 no gvhd/no tma. baseline transplant characteristics (age, donor, hla matching, conditioning) did not differ significantly among groups. histological features of itma, including loss of glands, total denudation of mucosa, apoptosis and detachment of endothelial cells, intraluminal fibrin, intraluminal microthrombi and mucosal hemorrhage were found in 6 patients. previously described features of intraluminal schistocytes were not observed in our patients. interestingly, loss of glands, total denudation of mucosa, apoptosis and detachment of endothelial cells were also found in patients with gvhd and no itma, suggesting that these features are not pathognomonic of itma. among 6 itma patients, two patients were classified in the clinical group of acute gvhd/systemic tma, while the other 4 patients had clinical and histopathological features of itma and severe grade iii-iv steroid-refractory acute gvhd (3 patients) or extensive chronic gvhd (1 patient) but no evidence of systemic tma. in the majority of patients (5/6), itma occurred during the early posttransplant period at 1.7 (0.9-4) months. clinical features (gastrointestinal bleeding, diarrhea, pain, nausea) presented no differences between patients with or without itma. prognosis was poor for patients with itma who suffered from a significantly higher mortality rate of 83% compared to the rest patient population (p=0.014). with a median follow-up of 11.1 (2.1-67.5) months, 1year overall survival probability (os) was 22.2 for itma, 55% for gvhd and 60% for systemic tma. unfavorable predictive factors for os were itma (p=0.048), hla mismatched donors (p=0.008) and gastro-intestinal bleeding (p=0.021). conclusions: intestinal tma has emerged as a novel distinct entity in patients with gvhd and/or systemic tma. distinct histological features may be useful in differential diagnosis of these severe hct complications. mortality rates higher than those of systemic tma highlight the need of proper recognition of itma that needs to be further studied in terms of diagnostic and therapeutic potential. disclosure: e.g. was supported by the european hematology association clinical research grant. the remaining authors declare no competing financial interest. the beneficial effects of thrombomodulin gene polymorphisms after hematopoietic stem cell transplantation background: chronic graft-versus-host disease (cgvhd) remains the major cause of late morbidity and mortality after allogeneic blood and marrow transplantation. treatment options for cgvhd, particularly its sclerotic forms remain limited. active hedgehog (hh) signaling was shown as a therapeutic target in both mouse and human cgvhd, with limited efficacy and significant toxicities described in a published clinical trial (defilipp, 2017). methods: adult patients with steroid refractory sclerodermatous cgvhd, defined as requiring >0.5 mg/kg/day of prednisone dose equivalent (pde), or need for second-or third-line therapy beyond corticosteroids and calcineurin inhibitors or sirolimus were eligible for this open label study of vismodegib, a first generation hh pathway inhibitor. primary endpoint was failure free survival, defined as absence of non-relapse mortality, no recurrent malignancy, steroid dose at 6 months =< 0.2 mg/kg/day of pde, and no addition of new systemic treatment. vismodegib was administered orally for 6-12 months, with dose reductions at development of toxicities. peripheral blood mononuclear cells were isolated from samples collected at treatment initiation and every three months thereafter. the immune profile of circulating b cells was analyzed by flow cytometry and t helper polarization by qrt-pcr of sortpurified cd4+ t cells. results: at the time of interim analysis, 6 patients were evaluated. 3 patients completed 6 months of treatment and five patients completed 3 months of treatment. therapy was discontinued in 3 patients prior to 6 months due to treatment-related (n=2) and unrelated (n=1) side effects. most patients experienced grade 2 toxicities (muscle cramps and dysgeusia), with only a single grade 3 toxicity (weight loss). 5 patients who completed 3 months of therapy demonstrated partial response, and overall, the primary endpoint was reached in 50% (3/6) of patients. in 2 patients who discontinued vismodegib, cgvhd worsened acutely after discontinuation. correlative analysis of immune cellular subsets in peripheral blood in 4 paired samples (pre-treatment and month 3 of therapy) documented modulation of b cell subsets pathogenic in cgvhd (pregerminal center and plasmablast-like b cells) and diminished t helper 2 polarization in cd4 t cells. conclusions: overall, use of vismodegib was associated with potential clinical efficacy in sclerodermatous cgvhd with possible mechanistic evidence arising in correlative studies. while side effects were common, further studies of hh inhibition in cgvhd are warranted. future studies should employ adjusted dosing regimens, along with supportive care interventions to offset side effects, and testing of novel hh inhibitors with enhanced safety profiles. clinical background: graft-versus-host disease (gvhd) results from recognition of host antigens by donor t cells following allogeneic hematopoietic stem cell transplantation (sct). we tested the hypothesis that somatic neomutations occurring after sct from donor and/or recipient dna may trigger gvhd. methods: we longitudinally analyzed both constitutive and somatic mutations by whole exome sequencing (wes) in 2 patients who received sct from a sex-matched hlaidentical sibling for npm1 mutated acute myeloid leukemia (pt#1) and jak2 v617f mutated primary myelofibrosis (pt#2). both patients were initially refractory to alloreactivity, i.e. not displaying any signs of gvhd, even after several donor lymphocyte infusions. acute gut gvhd finally occurred after a further dli preceded by a lymphodepleting chemotherapy. in pt#2, gvhd correlated with a graft-versus-tumor effect. wes was performed on dna from recipient saliva and donor pbmcs (germline samples) and from sequential post-sct pbmcs samples on a hiseq2500 illumina with 2x100bp paired-end reads at a mean depth of coverage of 190-210x. germline and somatic mutations were determined using in-house bioinformatic pipelines (named ewok from the curie institute and smaug from the henri mondor hospital), using briefly gatk as variant caller for germline samples, and a combination of 3 variant callers for matched normaltumor pairs. we adjusted parameters to detect somatic mutations at a minimal variant allelic frequency (vaf) of 5% compared to recipient and donor germline for all variations (minimal coverage = 8x for germline and 5x for tumor sample). results: wes allowed detecting somatic driver mutations explaining aml and pmf for both patients in the initial timepoint and all these driver mutations disappeared at the following timepoints. as expected, the somatic variant rate was 10x higher in pt#1 with aml than in pt#2 with pmf at each timepoint, except for the final gvhd timepoint. indeed, at this final point, the somatic variant rate dramatically decreased by 80% as compared to previous timepoints. by subtracting variants detected pre-and post-sct from those identified at the ultimate time-point of gvhd occurrence, we created 2 sets of 5 and 7 variants respectively for each patient (keeping only variants with at least 4 reads of mutated dna). these variants can be classified in 2 categories: (i) those with only with a slight increase at time of gvhd, i.e. ≤ 2-fold compared to highest previous vaf (lrrc43, or8u1, or10g9, alpp, frg1, frg2b and lilrb3 genes), and (ii) those with a significant increase at that time, i.e. > 2-fold compared to highest previous vaf (phf2, smpd1, ercc8 and krtap9-1 genes). none of the variants or genes involved was common between the 2 patients. ontology classification of mutated genes showed the implication of some of them in cell death, regulation of map kinase activity, mrna splicing and immune system process, making them good candidates for further studies. identification of variants appearing pre-gvh and turning off at time of gvhd is ongoing to unveil putative neoantigens that could trigger the alloreactive response. conclusions: using a comprehensive, pre-and post-sct, wes of donor/recipient pairs, we identified several neomutations from donor and/or recipient dna correlating with gvh/gvt effect development. disclosure results: a total of 169 patients experienced cgvhd, and mild, moderate, and severe cgvhd were observed in 66, 67, and 36 patients, respectively. the 2-year cumulative incidence of total cgvhd was 60.5% (95% ci, 54.7-66.3%), and the 2-year cumulative incidence of moderate to severe and severe cgvhd was 36.6% (95% ci, 30.9-42.3%) and 12.7% (95% ci, 8.8-16.6%), respectively. the patients who had 3 loci mismatched had a higher 2-year cumulative incidence of total cgvhd (66.2% vs. 54.5%, p=0.025) and moderate to severe cgvhd (42.3% vs. 30.6%, p=0.028) compared to those of the patients who had 1-2 loci mismatched. the patients who had maternal donors had a higher 2-year cumulative incidence of moderate to severe cgvhd (50.8% vs. 32.7%, p=0.018) compared to that of the patients who had other donors. the patients who had grade iii to iv acute graft-versus-host (agvhd) had a higher 2-year cumulative incidence of total cgvhd (91.7% vs. 50.0%, p< 0.001) and moderate to severe cgvhd (70.8% vs. 26 .3%, p< 0.001) compared to those of the patients without agvhd. in multivariate analysis, grade iii to iv agvhd was the only independent risk factor for total cgvhd (hr=2.6, 95%ci, 1.6-4.2; p< 0.001) and moderate to severe cgvhd (hr=3.8, 95%ci, 2.1-6.7; p< 0.001). in the model excluding agvhd, maternal donor was the risk factor for moderate to severe cgvhd (hr=1.5, 95%ci, 1.1-2.3; p=0.030). conclusions: we observe that severe agvhd was the most important risk factors for cgvhd after haplo-hsct, and further interventions should be considered in these patients to prevent severe cgvhd. disclosure: none of the authors have any potential financial conflict of interest related to this manuscript. background: extracorporeal photopheresis (ecp) has been successfully used for the treatment of graft-versus-host disease (gvhd). ecp therapy might restore the balance between effector and regulatory cells which is severely impaired in gvhd. nk cells are the first lymphocyte subset to be reconstituted after allogeneic hematopoietic stem cell transplantation (allo-hsct). as an important innate immune cell population, nk cells can temporally bridge the transient period of t-cell deficiency post allo-hsct, by protection from opportunistic infections and prevention of leukemic relapse by graft-versus-leukemia (gvl) effect. nk cells not only preserve homeostasis through targeted killing of allo-reactive t cells and thereby control gvhd but also enhance inflammation by secretion of tnf-α and ifn-γ and thereby promote gvhd. therefore, we investigated here the role of nk cells in gvhd patients under ecp therapy. methods: thirty four patients with steroid-refractory/ resistant agvhd ≥ ii°and moderate to severe cgvhd received ecp therapy which performed according to the guidelines. glucksberg and nih criteria were used for clinical staging of agvhd and cgvhd under ecp therapy, respectively. the comprehensive phenotypical analysis of nk cells was evaluated by multicolor flow cytometry. nk activity in terms of killing function, cytokine release capacity and proliferation function was monitored by chromium-51 release assay, intracellular cytokine staining and cfse staining, respectively. results: five different nk cell subsets were defined based on cd56 and cd16 expression. cd56 bri nk cells displayed an immature and activation profile with high expression of cd62l and nkg2d. agvhd patients had a higher frequency of cd56 bri nk cells when compared with hds and cgvhd patients, who were characterized by significant increase of the cd56 dim cd16 + and cd56 -cd16 + nk cell subsets with high expression of differentiation markers cd11b and cd57. of note, cd56 bri cd16 -nk cells could serve as a novel predictive biomarker for the response of agvhd patients to ecp treatment. in responding agvhd patients, an increase of cd56 bri nk cells was observed already during the early ecp treatment phase, suggesting immune reconstitution. after priming of the progenitors, ecp could differentiate immature cd56 bri nk cells into mature cd56 dim nk cells with reduction of cd62l on cd56 bri nk cells. moreover, cd56 dim nk cells could further be matured through upregulation of cd57 expression by ecp. notably, ecp therapy could shift the nk cells from a cytotoxic to a regulatory phenotype within the cd56 bri nk cells. in spite the immunomodulatory effect of ecp on nk cells, nk activity could be kept intact under ecp therapy. the killing activity of nk cells was stable as confirmed by a 51 cr release assay. ecp therapy had no negative effect on the quantity and quality of cytokine release by nk cells upon k562 stimulation. especially, the polyfunctionality of nk cells was not altered significantly by the ecp therapy. conclusions: nk cells play an important role in gvhd and could serve as a predictive cell population for the clinical response to ecp therapy. in the current study, ecp influenced the differentiation, maturation and education of nk cells ameliorating gvhd without comprising the antiviral immune defense and gvl effect. disclosure: the authors declare no competing financial interests, except the following: therakos mallinckrodt gave a financial support to as and ms for the documentation of the clinical course and for the analysis of immune cells of the patients, pw has honoraria and membership on advisory boards for sanofi-aventis. abstract withdrawn. cyclosporine levels >200µg/l on day 10 post-transplant was associated with significantly reduced acute graftversus-host disease following allogeneic hematopoietic stem cell transplantation monica bianchi 1 , dominik heim 1 , claudia lengerke 1 , martina kleber 1 , dimitrios tsakiris 1 , jakob passweg 1 , alexandar tzankov 2 , michael medinger 1 background: acute graft-versus-host disease (agvhd) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). affected patients, especially with steroid-refractory agvhd, have a very poor prognosis. prophylaxis with cyclosporine a (csa) is the backbone of gvhd prevention in most conditioning regimens. methods: in a retrospective analysis of patients treated with allo-hsct, we correlated csa levels at the day of transplantation (day 0) and day +10 with the incidence of acute and chronic gvhd. we postulate that higher target csa levels >200μg/l will result in a lower incidence rate especially of agvhd after allo-hsct. results: we assessed 660 patients with either aml n=248, lymphoma/myeloma n=127, mds/mpn n=124, all n=79, cll n=36, cml n=23, or bone marrow failure n=22. in patients with clinically relevant agvhd grade ≥2, mean csa levels was lower on day 0 and day +10 (142±88 μg/l; and 183±64 μg/l; respectively) compared to patients without agvhd (156±81 μg/l; and 207±67 μg/l; respectively; day 0: p=0.003; day +10: p=7.57 x 10 -9 ). in patients with csa level < 200 μg/l, the incidence of agvhd was significantly more frequent compared to patients with csa levels >200 μg/l [(234/356; 66%) versus 91/248 (37%); p= 1.34 x 10 -12 ]. in patients with cgvhd, there was no significant difference between csa levels < 200 μg/l (128/330) compared to csa levels >200 μg/l (96/ 233; p=0.312). the optimal csa cut-off level for the prevention (i.e. roughly 50% incidence reduction) of agvhd was >201 μg/l at day 0 and >195 μg/l at day +10 ( figure 1 ) in a competing risk analysis, time to agvhd grade ≥2 (using death of other causes as competing risk) was associated with csa levels >200 μg/l on day 0 and on day 10, unrelated donors, myeloablative conditioning (mac), and for the diagnosis lymphoma/myeloma. conclusions: our data support close monitoring with active adjustments of csa dosing to maintain therapeutic csa levels above 195 μg/l in the first 10 days of allo-hcst to reduce agvhd. disclosure: noting to declare. liposomal cyclosporine a for inhalation (l-csa-i) to treat bronchiolitis obliterans syndrome: novel formulation with therapeutic potential for patients with bos following allo-hsct noreen roth henig 1 , emilie hofstetter 2 , dominik kappeler 3 , gerhard boerner 3 background: bronchiolitis obliterans syndrome (bos) is a rapidly progressive lung disease caused by t-cell mediated inflammation that leads to blockage of bronchioles, leading to respiratory failure and death shortly after diagnosis. approximately 4% to 10% of patients who undergo allogeneic hematopoietic stem cell transplant (allo-hsct) will develop bos, with 72-100% developing bos as a respiratory form chronic graft-vs-host disease (cgvhd) in addition to other signs of cgvhd. mean time to bos diagnosis ranges from 273 to 547 days post-transplant. the histopathology of bos after allo-hsct and lung transplantation is identical. early studies of l-csa-i for the prevention of bos in lung transplant recipients demonstrated therapeutic benefit. l-csa-i is a novel, liposomal formulation of cyclosporine administered via a pari investigational eflow â nebulizer which delivers a potent immunosuppressant to the site of disease. pharmacokinetics and tolerability of l-csa-i is presented. methods: retrospective review of two clinical studies of l-csa-i (isotonic, 4mg/ml) for bos associated with lung transplantation. both studies had a control arm and results reported here are for patients who received l-csa-i. subjects received 5mg (single lung transplant) and 10mg (double lung transplant) bid via inhalation. blood samples for pharmacokinetic analysis of cyclosporine a concentrations were collected before inhalation, immediately after inhalation, and thereafter in intervals of 15, 30, 60 min and 2, 4, 8 and 12 hours. local and general tolerability of l-csa-i was investigated. results: between the two studies, 85 subjects received either 5 or 10 mg bid of l-csa-i. pharmacokinetic models predict a constant drug level in the lung. maximum serum cyclosporine a concentration after inhalation was 57.42 ± 34.26 ng/ml. trough levels for up to 2-years of daily administration was 4-5 ng/ml with no evidence of accumulation following repeated exposure. tolerability data was assessed from 1068 patient-month exposure to l-csa-i. reported symptoms were: pharyngeal soreness 1%; cough 22%; dyspnoea 7%; and wheezing 1%. no subject discontinued due to intolerability. inhalation time is on average 9-13 min. conclusions: l-csa-i provides high and constant concentrations to the airways of the lungs and the site of bos. l-csa-i is well tolerated in lung transplant patients. use of l-csa-i instead of augmentation of systemic csa reduces the total drug exposure. a multicentre phase 2 safety and exploratory efficacy trial for the treatment of bos in allo-hsct recipients is underway. disclosure background: there are a number of biomarkers that predict non-relapse mortality (nrm), graft-versus-host disease (gvhd) and relapse incidence (ri) after conventional gvhd prophylaxis based on calcineurin inhibitors with or without antithymocyte globulin. currently there is limited data whether the conventional predictive biomarkers work with posttransplantation cyclophosphamide (ptcy) prophylaxis. methods: prospective single-center study in 2015-2016 enrolled 79 adult patients with acute leukemia in cr (34% with all, 66% with aml). 26 received matched related bone marrow (bm) graft with single-agent ptcy and 53 received unrelated peripheral blood stem cell graft (pbsc) with ptcy, tacrolimus and mmf. the grafts were studied by flow cytometry (facs aria ii, antibodies by miltenyi biotec). the following populations were analyzed: cd3, cd4, cd8, cd16cd56, nkt, inkt, treg, double-positive t-cells, double-negative t-cells, tcralpha/beta, tcr v11 memory cells. the crypreserved plasma from were analysed by elisa (commercial kits by ebioscience and critical diagnostics) for vegf a soluble tnf receptor (stnfr), il-8, il-6, soluble il-2 receptor, st2, il-17 and stnfr. the above mentioned biomarkers were tested in logistic regression with roc analysis, assays with auc>0.600 were selected for analysis in fyne-gray regression with competing risks. cut off levels were determined for significant parameters. results: median follow-up was 19 months (range 12-30). in the whole group overall survival (os) was 77%, eventfree survival (efs) 73%, grade ii-iv acute gvhd 13%, moderate and severe (m&s) chronic gvhd 20%, nrm 6%, mortality in patients with gvhd 0%, ri 20%. there was no difference between bm/related and pbsc/unrelated grafts in the incidence of gvhd, nrm and ri (p>0.11). the only significant predictor of acute gvhd were low levels of il-8 level on day+7 (p= 0.0490, 0% vs 15% with the cut off 40 pg/ml). m&s chronic gvhd was predicted only by the high percentage of inkt cells in the graft (p=0.0003, 31% vs 12% with the cut off 0.03%). there was a correlation between il-8 levels and number of nk cells in the graft (p=0.02). nrm was related to infectious complications, nonetheless high levels of vegf a on day 0 (p= 0.0458, 16% vs 0% with the cut off 100 ng/ml), st2 on day+30 (p= 0.0041, 11% vs 0% % with the cut off 40 ng/ml) and low percentage of cd16+cd56-cells in the graft (p=0.0215, 22% vs 2% with the cut off 1.3%). the identified biomarkers of nrm had no association with the pre-transplant crp and ferritin levels (p>0.4). the only significant parameter for ri was the level of cd34 cells in the graft (p=0.0434). none of the identified biomarkers significantly predicted overall survival (p>0.09). conclusions: in the related and unrelated grafts with ptcy the study of biomarkers has low clinical utility due to very low gvhd-related mortality. however st2 and vegf a can predict infection-related mortality. also the study verified previous observations that high level of il-8 is associated with reduced gvhd incidence after ptcy 1 and identified the importance of nk and inkt cells in the induction of tolerance with ptcy. references background: hematopoietic cell transplantation (hct) is the only curative approach for many hematological malignancies but life-threatening toxicities, such as graft-versushost disease (gvhd) and infections, still limit its fullpotential impact on the disease. strategies for keeping allohsct more effective and safe are needed in order to reduce morbidity while improving its immunological effect to control disease relapse. post-transplant cyclophosphamide (ptcy) has been demonstrated to improve acute gvhd (agvhd) and chronic gvhd (cgvhd) control in allogeneic bone-marrow hct from identical and haploidentical donor. the use of ptcy, after peripheral blood stem cell transplantation (allopbsct) from hla-matched unrelated/related donors, has been investigated by our group in a clinical trial (nct 02300571) and preliminary results were published last year. here we report updated efficacy and safety data about the expanded cohort of patients treated with ptcy followed by tacrolimus and mycophenolate mofetil (t/mmf). methods: we analysed data about 71 consecutive patients with high-risk hematologic malignancies received allopbsct from hla-matched unrelated/related donors between march 2011 and august 2018. gvhd prophylaxis was ptcy 50 mg/kg (days +3 +4), tacrolimus from day +5 and mmf from day +5 to day +28. primary objectives were cumulative incidence of agvhd and cgvhd. secondary objectives were event-free survival (efs), cgvhd-efs, overall survival (os) and non-relapse mortality (nrm). results: patients median age at transplant was 50 (range 19-74) years. 34 (48%) patients were transplanted in first complete response (cr), 16 (23%) patients in second/third cr, the others in disease control. a median dose of 7.0 (range 2-15) x 10^6 cd34/kg was infused. primary graft failure was observed in one patient. all patients were off mmf on day +28, the median day of tacrolimus discontinuation was 112 (range 39-467). eight out of 71 (11%) patients developed agvhd, 6 (8%) of them were grade ii-iii; median day of onset was day 68 (range . no grade iv was observed. no cases of late-onset agvhd were reported. cumulative incidence of cgvhd was 8% (6/ 71), median day of onset was 162 (range 140-268). systemic treatments were required, but all patients were able to discontinue immunosuppression (is). with a median follow-up of 22 (range 4-94) months, efs was 58%, cgvhd-efs was 52% and os was 72%. non-relapse mortality (nrm) was 3% (2/71): 2 patients died because of multidrug resistant bacteria septicemia. nowadays 41 patients are alive with no evidence of disease, being continuously off is and completely reintegrated in their normal daily life activities. conclusions: the updated reported results confirm, in a larger cohort of patients with a longer follow-up, that ptcy after pbsc-hct is highly active in agvhd and cgvhd prevention with extremely limited nrm. this strategy, not only allowed earlier discontinuation of immunosuppression, but also reduced the overall time of exposure to is for most of the patients. all these features might contribute, in the future, to transform hct into a safe immunologic platform that may be combined with advanced form of cellular therapies (car-tcells), aiming to increase safely the graftversus-tumor effect. clinical methods: pediatric patients (0-21 years) with nmd undergoing unrelated hct were eligible for this single center, phase i trial. following reduced intensity conditioning, abatacept (10 mg/kg iv on days -1, +5, +14, +28) was added to standard gvhd prophylaxis (cyclosporine, mycophenolate mofetil [mmf]). patients were followed for 2 years for standard hct outcomes. [[p257 image] 1. figure 1 results: since june 2014, 10 patients have been enrolled and transplanted (table 1 , excluding #7). donor source was bone marrow in all. with median follow-up of 3.5 years, 8 of 10 patients survive without disease. initial engraftment was successful in 9, at a median of 20 and 16 days, for neutrophils and platelets respectively. one patient (5) had secondary graft rejection in the setting of viral reactivation (cmv/ebv), with successful engraftment following a 2 nd unrelated hct. in 8 engrafted patients, myeloid (cd33) chimerism was 100% at all timepoints; t-lymphoid (cd3) chimerism was mixed but reached >/=95% (figure 1 ). one patient (7) with saa had primary graft rejection in the setting of inadequate tnc dose (0.5 x 10 8 /kg) and died from marrow aplasia/infection despite 2 nd hct. a second death from wilms' tumor occurred 17 months post successful hct, in a patient (1) with dba and constitutional chromosome abnormality. except patient 7, all patients received 4 doses of abatacept, which was well tolerated, with all severe adverse events expected for a hct population. cmv and ebv reactivation occurred in 3 patients each, with resolution using standard anti-viral therapy. one patient (6) was diagnosed with ebv-driven post-transplant lymphoproliferative disease, which responded to rituximab and immune suppression withdrawal. no patients developed severe acute (grade iii-iv) or chronic gvhd (table 1) , and no patients required systemic immune suppression at >1 year. conclusions: these preliminary data suggest that abatacept can be safely added to cyclosporine and mmf gvhd prophylaxis in pediatric patients with bone marrow failure undergoing unrelated donor hct, with encouraging rates of gvhd despite half of patients having a mismatched (7/8) donor. given the higher risk of graft rejection in this non-malignant cohort, rejection (in addition to gvhd) will be a primary focus in our subsequent multi-center, phase 2 trial. clinical trial registry: clinicaltrials. gvhd and may to have be separated from from toxicity to infectious complications in the early phase after allohsct. methods: from our files we identified 65 patients which had upper gastrointestinal tract endoscopy after allohsct in with biopsies were taken from the esophagus, stomach and duodenum simultaneously. of these patients 14 were excluded because of infection, reflux disease or drug toxity and the remaining 51 patients were included in our study. we evaluated the routine stained esophageal biopsies, applied a grading scheme and compared the histological findings with those within the stomach and duodenum, the endoscopic findings and the clinical course. results: in 32 of 51 biopsy samples of the esophagus, we identified histological features of acute gvhd, ranging from vacuolar degeneration (grade 1) and single-cell apoptosis (grade 2) to the formation of clefts (grade 3) and mucosa denudation in advanced cases (grade 4), resembling epithelial lesions in acute gvhd of the skin. these findings correlated with gvhd involving the stomach and duodenum and the clinical manifestations of gvhd in other organs. endoscopically patients with gvhd revealed signs of inflammation, ranging from erythema to ulceration in the more advanced cases, sometimes reminiscent of reflux or infection. clinically these patients had abdominal discomfort ranging from inappetence to nausea, accompanied by emesis or diarrhea and weight loss. conclusions: we have shown that acute esophageal gvhd occurs after allohsct and is correlated with acute gvhd in stomach and duodenum. it could be diagnosed and graded histologically. the endoscopic findings are signs of inflammation. our results may help to establish the histological diagnosis of acute gvhd using endoscopic biopsies from the esophagus and to explain the alterations observed in the esophageal mucosa in patients after allohsct. [ background: intestinal acute graft versus host disease (agvhd) is a major thread after allogenic hematological stem cell transplantation (allohsct), with a high mortality in patients which were refractory to steroid treatment in particular. recent papers point to a correlation of histological grading of intestinal gvhd and prognosis in patient after allohsct. however a comparison with clinical scores has not been performed so far. methods: in this analysis, retrospective data from 89 patients who underwent endoscopy due to clinical signs of agvhd (day +20 to +200 after allohsct) were evaluated. of each patient least 3 biopsies from different sites of the colon which were taken simultaneously. of each biopsy series the maximum histological grad of agvhd according to the lerner scheme was obtained and compared with the glucksberg stage of the lower gastro intestinal tract (gslgi) and the overall glucksberg grade (ogg). these three grades were compared for non-relaps related mortality using the log-rank test and for sensitivity to steroid treatment applying the receiver operating characteristic for the patients who received steroid treatment. for these patients the non-relaps related mortality for responder and non-responder were calculated using also log-rank test. results: the histological grade strongly correlated with the survival (p=0.009). a statistical significant correlation was also found for the gslgi (p=0.02), whereas the ogg revealed no significant correlation (p=0.09). non-relaps related mortality was mainly related to infection or sepsis (in 32/56 patients who died). -eighty-one of the patients received steroid therapy. the sensitivity to the steroid therapy correlated with each of the three scores (p< 0.0001) but was the strongest for gslgi (area under the curve (auc) 0.829), compared to ogg (auc 0.795) and the histological score (auc 0.691). the survival of the patients, which were sensitive to steroid treatment was significantly better than those of steroid refractory patients (p=0.005). conclusions: we found that histological and clinical grading in patients after allohsct with intestinal gvhd was correlated with survival and respond to steroid treatment. histological scoring may predict survival more precisely than ogg and gslgi but did not add substantial information to the prediction of treatment response. [ emerging evidences suggest that regulatory b cells (bregs) play essential roles in inflammation, autoimmune diseases and tumors. few data exist about the role of bregs in the contest of hematopoietic allogeneic stem cell transplantation (hsct). some authors have observed that bregs from patients with chronic graft-versus-host disease (cgvhd) were less frequent and less likely to produce il-10 than bregs as compared to healthy donors or patients without cgvhd. these findings suggest that bregs may be involved in cgvhd pathogenesis. the purpose of our study was to evaluate a possible role of b cell subsets on gvhd occurrence. methods: lymphocyte subset enumeration was performed by aquios cl flow cytometer (beckman coulter), a quantitative automated analyzer that performs two diagnostic panels: tetra-1 cd45-fitc/cd4-rd1/ cd8-ecd/cd3-pc5 and tetra-2 cd45-fitc/cd56 +cd16-rd1/cd19-ecd/cd3-pc5. b cell subsets (memory, mature and transitional b cells) on peripheral blood samples were analyzed by aquios designer software, a tool for the creation of user-defined applications. panel-1 cd19-fitc/cd10-pe/cd38-ecd/cd24-pc5/cd27-pc7 and panel-2 cd19-fitc/cd5-pe/cd38-ecd/cd24-pc5/ cd20-pc7 were specifically designed by beckman coulter for our center. the flow cytometric analysis was performed as follows: in donors and patients at basal level; on graft products and in patients at days +30, +60, +90, +120 after hsct. statistical significance was assessed with prism software (graphpad) by mann withney test. p < 0.05 was considered statistically significant. results: actually we enrolled 84 patients submitted to hsct in our center from november 2017. a preliminary statistical analysis was performed on 55 patients. stem cells source was peripheral blood (pb) in 27 cases and bone marrow (bm) in the others 28. the conditioning regimen was myeloablative in 26 patients and ric in 29 patients. agvhd was diagnosed in 22 patients (40%). no associations were found between b cell subsets in donors and patients at baseline and the occurrence of agvhd. however we found a higher median percentage of transitional b cells in graft products in patients without agvhd (9.6%, 4.5-26.7) compared to patients with agvhd (6.9%, 1.5-21.1) (p=0.02, fig 1a) . in addition, patients without agvhd showed a lower median percentage of memory b cells (24.2%, range 11.6-49) in graft product as compared to patients with agvhd (34.2%, range 10.3-68.15) (p = 0.03, fig.1b ). finally in the subgroup of patients receiving pb as stem cell source we observed a higher percentage of cd3+ lymphocytes in graft product in patients with agvhd (75%; range 68-80) compared to patients without agvhd (71%; range 62-78) (p=0.01). in the monitoring of b cells reconstitution we observed that cd19+ events did not appear before day +90 after hsct and these were b transitional immature events predominantly. conclusions: our data suggest a possible protective link between transitional b cells and agvhd development. these results data need to be confirmed in a larger cohort of patients. moreover, it will be interesting to evaluate the relationship between transitional b cells at day +90 and the occurrence of cgvhd. clinical background: agvhd is a major complication of allogeneic hematopoietic stem cell transplant (hsct) and a risk factor for post-hsct mortality. the objective of this analysis is to describe patients with agvhd who had a suboptimal response to corticosteroids. methods: patients who developed ibmtr severity index ii-iv agvhd after first hsct between 1/1/14 to 6/ 30/16 were included in an ongoing chart review at centers in the united states. patients who had ever participated in a gvhd prophylaxis trial or used jak inhibitors were excluded from the study. suboptimal response to corticosteroids was defined as use of additional systemic anti-gvhd therapy, inability to taper high-dose steroids (≥1 mg/ kg) by ≥25%, or tapered corticosteroids by ≥25% but not to < 10 mg/day. results: the analysis included 64 patients with suboptimal response to corticosteroids. mean age was 55 years; 66% were male. median time from transplant to agvhd diagnosis was 33 days. at the time of maximum agvhd grade, 33% of patients were grade ii and 66% were grade iii-iv; 58% had lower gi involvement, and 59% had ≥2 organs involved. from time of diagnosis to maximum agvhd grade, 52% of patients had new organ involvement or an increase in agvhd grade. median time from diagnosis to maximum grade was 5.5 days, and was 2.5 days for patients with lower gi involvement. systemic corticosteroids were initiated on the day of diagnosis for 73% of patients. average starting daily dose was 93 mg (1.1 mg/kg) for prednisone and 136 mg (1.6 mg/kg) for methylprednisolone. steroid dose was increased for 44% of patients during follow-up; 80% were unable to taper below 10 mg/day. among patients who received additional systemic anti-gvhd therapy (n=32), 41% increased their corticosteroid dose before initiation of additional anti-gvhd therapy. median time from initiation of corticosteroids to additional therapy was 16.5 days. frequently used therapies were mycophenyalate mofetil (25%), atg (16%), extracorporeal photophoresis (13%), tocilizumab (13%), etanercept (9%), and sirolimus (9%). agvhd recurred in 31% of patients and was managed by increasing corticosteroid dose in 70% of patients. 44% had any infection within first 100 days post-hsct. forty patients (63%) required hospital readmission(s); 40% had ≥2 readmissions within 100 days post-hsct, with a mean inpatient lengthof-stay of 28 days. relapse of underlying malignancy was reported for 13 (20%) patients. two-thirds (66%, n=40) patients died at a median of 87 (interquartile range (iqr): 44-180) days from agvhd diagnosis; a higher proportion (76%) of patients with maximum grade iii-iv agvhd died at a median of 63 (iqr: 41.5-186.5) days; majority (81%) of patients with lower gi agvhd died at a median of 68.5 (iqr: 44-137) days. conclusions: a majority of patients with agvhd who had suboptimal response to systemic corticosteroids had severe and rapidly progressing disease and resulted in a high mortality rate (66%); progression was more rapid and mortality increased for patients with lower gi involvement. most patients required readmission to the hospital with extended length-of-stay. an urgent need exists for effective and tolerable therapies that quickly resolve life-threatening agvhd in early stages of disease. disclosure results: median time to onset of bo from allohct was 6.9 months (range 0.6-31.2). previous acute gvhd in 52.2% (n = 24) [grades iii-iv 29.2% (n = 7)]. in 23.9% (n = 11) cgvhd had exclusive lung involvement, while the other 35 patients (76%) had other organs affected. at diagnosis of bo, 69.6% (n = 32) were under immunosuppressive treatment. 32.6% (n=15) of patients with bo received ecp as second-line treatment. median duration of treatment was 22 months (1.5-36.8 ) and time to response 9.9 months (1.9-30.2). median of sessions was 40 (4-108). evaluation of response was based on the evolution of fev1 measurement: 26.6% (n = 4) complete response; 40% (n = 6) partial response and 20% (n = 3) stable disease. one patient did not get any response and another was not evaluable. 66.6% of patients (n = 10) could reduce immunosuppression, and in one case it was completely discontinued. there is a trend for early separation between survival curves in favor of ecp ( figure 1 ). one patient had sepsis secondary to central venous catheter infection as complication related to ecp. conclusions: ecp has emerged as a promising treatment for bo after allohct. in our experience, ecp was effective to stabilize or improve the disease in many patients and allowed to taper esteroids with minimal associated complications. however, prospective studies and longer follow-up are needed to support these findings. disclosure: nothing to declare background: the key role of il-6 signaling in acute graft vs. host disease (agvhd) and cytokine release syndrome (crs) has evoked growing use of tocilizumab, an anti-il6 receptor (il6-r) antibody, in these settings. apart from regulation of t-and b-cell differentiation, immune cells migration to inflammatory sites and t-cell recruitment, il-6 complex with il6-r through gp130 upregulates production of fibrinogen (fg) and other acute phase proteins, including c-reactive protein (crp). methods: we retrospectively analyzed data of 7 patients treated with tocilizumab (8mg/kg) due to steroid-refractory (sr) agvhd and 4 patients because of crs. median age was 37 and 47 years, respectively. seven patients were transplanted from unrelated donors (mud/mmud) and 4 from sibling donors. eight patients received myeloablative and 3 reduced intensity conditioning regimen. analyzed data included concentrations of fg, crp, an incidence of infections at tocilizumab administration and in weeks following the infusion. results: stage ii agvhd was diagnosed in 1 patient, stage iii in 4, and stage iv in 2 patients. involvement of the gastrointestinal tract (gi) was observed in 85% of cases. the median fg concentration before tocilizumab administration was 2.56 g/l (range, 1.4-7) and crp 21 mg/dl (range, 1-260) and 60% of patients had an active infection. after infusion of the antibody, we observed a decline of fg and crp levels. the median level of fg was 1.02 g/l (range, 0.46-1.7) 7 -14 days after the tocilizumab infusion with no severe bleeding complications. a median crp value was 1.82 mg/dl (range, 0.1-36) despite confirmed infectious complications. three weeks after infusion of tocilizumab fg raised to the normal range in 85% of patients (fig 1) . five patients with sr agvhd achieved a complete response, and 2 had a partial response after tocilizumab therapy. [[p265 image] 1. fibrinogen levels in gvhd patients following tocilizumab infusion.] a group treated with tocilizumab due to crs had higher initial levels of fg 5.1 g/l (range, 4.1-6.6) and crp 143 mg/ dl (range, 80-227) before administration of the drug. reduced fg and crp levels from a baseline value were also observed in this group. however, concentrations were higher than in gvhd patients: fg 2.02 g/l (range 1.2-3.4) and crp 3.2 mg/dl (range 2.7-11). in all patients, a differential diagnosis of disseminated intravascular coagulation was excluded. conclusions: 1. fibrinogen declines after tocilizumab therapy due to its cytokine-regulated production in the liver. coagulation monitoring should be performed during the first 3 weeks after administration of the antibody to avoid serious bleeding complications. 2. crp concentrations remain low despite the presence of active infections following infusion of tocilizumab. crp fails as a marker of infection during 3 weeks following the therapy. 3. tocilizumab is an effective therapy in patients with agvhd, especially with the gi involvement. disclosure: nothing to declare vanishing bile ducts after allogenic hsct: is it really gvhd? antonio grasso 1 , lorenzo d'antiga 2 , aurelio sonzogni 2 , massimo gregori 3 , alessandra maestro 3 , roberto simeone 3 , natalia maximova 3 background: evaluation of liver gvhd was historically based by elevation of bilirubin levels and by reduction and degeneration of small bile ducts on histological samples of post-transplant liver biopsy. however, there is a lack of studies that compared histological finding of ductopenia between post-autologous hsct and post-allogenic hsct. studying severity of ductopenia following allogenic hsct, we aimed to demonstrate lack of correlation between ductopenia and clinical signs of liver gvhd. methods: we retrospectively collected a series of 72 allogeneic hsct performed from 2005 to 2017 in the institute burlo garofolo. all patients undergo percutaneous liver biopsy in most cases at three months, one year and three or more years after hsct. indications for biopsy were alteration noted at 2 weekly follow-up assessments of at least one clinical or laboratory marker of liver impairment or cholestasis. ductopenia was defined by number of portal tracts with no interlobular bile duct divided by the total number (severe if the ratio was less than 0.2). clinical gvhd was defined by nih consensus criteria results: our population involved 64% males and 36% females with oncological (70%) and non-oncological underlying disease (30%). clinical signs of liver gvhd were present in 18% of the patients (n=13), 8% with contextual intestinal involvement, 8% with cutaneous and intestinal involvement. 71 patients underwent biopsy at a mean time of 110 +/-32 days after hsct, 45 patients underwent a biopsy at 12 months after hsct and 35 patients after three or more years from hsct. results of biopsies are showed in table 1. no difference in incidence of ductopenia were found between liver gvhd group and no gvhd. table] 1. table 1 : incidence of ductopenia 3-6 months, 12 months and 3 or more years after hsct in total population, gvhd group and no-gvhd group] the group that not received chemotherapy prior the hsct had an overall incidence of ductopenia of 77% (severe ductopenia of 42%) statistically significative in comparison with the oncological underlying disease group (94% of ductopenia and 64% of severe ductopenia). furthermore, a little sub-group of 16 patients extrapolated from our population received liver biopsy before hsct for diagnostic assessments: of the 10 with an oncological underlying disease 70% already showed ductopenia, while no signs of ductopenia were found in the others with a nononcological disease. conclusions: there is no correlation between incidence of gvhd and histologically finding of ductopenia on liver biopsy. ductopenia may be caused in the first place by chemotherapy treatment received before hsct and myeloablative conditioning for hsct and it's not related with gvhd. this hypothesis is strengthened by the subgroup analysis of pre-hsct biopsy. background: second and third line therapies for steroid refractory acute graft versus host disease (agvhd) after allogeneic stem cell transplantation (asct) are still lacking. ruxolitinib, a selective januskinase 1/2 inhibitor could show high efficacy in agvhd, as well as extracorporeal photopheresis (ecp). here we report a single center experience of combining both therapeutic approaches in severe steroid refractory agvhd with additional analysis of immune status of these patients to elucidate direct effects of this treatment on immune response. methods: from june 2015 to february 2017, 18 patients (77.8% male, 22.2% female, median age: 58.5 years, r: 21-73) with steroid refractory agvhd of lower gi-tract after asct were treated with ruxolitinib and extracorporeal photopheresis as third, fourth or fifth line therapy. some patients showed additional agvhd of skin (n=7), liver (n=6) or upper gi-tract (n=2). all patients had an overall grade iii (50%) or iv agvhd (50%). steroid refractoriness was defined as no improvement in 7 days or aggravation after 5 days of steroid treatment.median start of ruxolitinib or ecp was day 86.5 after asct (r: 35-257). medianduration of ruxolitinib therapy was 59.8 days (r: 14-192) with a median start dosage of 20 mg per day (2 x 10 mg; r: 10-20 mg). all patients started with 2 ecp treatments per week with an individual reduction of treatment frequency. median number of ecp treatments was 20.5 (r: 2-71) with a median frequency of ecp therapy once a week (r: 0.5-2.1). cytomegalovirus (cmv) status of all patients and immune status of ten patients (lymphocyte count with cd4 + t helper lymphocyte and regulatory t cell count) were collected previously, after four weeks of starting combined treatment and four weeks after stopping the treatment. results: one-year estimated overall survival (os) of all patients was 50% with a median estimated os of 314 days. 3 patients died because of relapse of underlying disease, one of severe therapy refractory agvhd of lower gi tract and 5 due to infection complications in agvhd refractory setting. overall response was 55.5% (complete remission rate: 44.4%, partial remission rate: 11.1%). 72.2% (n=13) of the patients had cytopenia ctc i-iii during the treatment, no grade iv cytopenia was reported. cmv reactivation during ruxolitinib occured in 66.7% of cases (n=12). tapering of steroids could be performed rapidly with a medium reduction time of 1.75 days for reducing to half of the dosage.remarkably, regulatory t cells significantly increased during combined ruxolitinib/ecp treatment compared to regulatory t cell count before treatment (p=0.02) and after stopping treatment, regulatory t cell count decreased again (p=0.02, see figure) . significant changes in whole lymphocyte count or in cd4+ t helper cell count were not observed. conclusions: treatment of severe steroid refractory agvhd with ruxolitinib plus ecp could show a high complete remission rate of 44.4% with an one year os of 50%. detecting increased regulatory t cell count during the treatment underlines its direct effects on immune response and encourages to pursue this promising therapeutic approach. [ background: due to increased immunosuppression infections remain the main cause of death followed by higher risk of relapse in patients treated for acute graft versus host disease (agvhd) after allogeneic stem cell transplant (sct). here we report a single-centre experience with extracorporeal photopheresis (ecp) for acute gvhd that was introduced in order to reduce steroid treatment. comparison of overall survival (os) for patients on ecp and patients that received standard first line therapy for agvhd was performed. methods: we retrospectively analysed 62 patients (28 %) with acute gvhd grade ii-iv treated from january 2010 to october 2018 out of total 221 allogeneic sct in that period. all patients received calcineurin inhibitors or sirolimus while receiving steroid treatment for agvhd. twenty-five patients (40 %) received ecp with steroid lowering intent. we defined response as (1) reduction of steroid dose for at least 50% from baseline while not adding another immunosuppresive agent and (2) not repeating second steroid treatment if the ecp was started after lowering of steroids to prevent agvhd flare. we checked separately patient responsive and refractory/dependent to steroids. on average patients received ecp procedure once weekly. results: tapering of immunosuppressive therapy as defined was successful in 13 (52 %) out of 25 patients in ecp group. in a group of patients without ecp 14 (37 %) patients had steroid refractory or steroid dependent agvhd compared to 11 (44 %) patients in ecp group. four (16 %) patients with steroid refractory or dependent agvhd showed improvement in ecp group compared to only one (2,7%) in non ecp group. twenty (74 %) patients died due to infectious complication and 7 (26%) due to relapse in non ecp cohort. in ecp cohort 10 (77%) patients died due to infection and 3 (23%) due to relapse. median os was 5 months in non ecp group (r., 1-99) compared to 21 months (r., 2-76) in ecp group and os of 20 % at 5 years in non ecp compared to 35 % in ecp cohort was observed. patients with agvhd treated with ecp and faster steroid tapering had longer os compared to patients without ecp (p=0,03). conclusions: ecp enables successful tapering or withdrawal of steroid therapy in many patients, even in those who are steroid refractory or steroid dependent. reduction of immunosuppression leads to reduced incidence of infection and relapse which translates into a better overall survival. background: the curative potential of allogenic stem cell transplantation is hampered by graft-versus-host disease (gvhd). pre-clinical study showed an efficacity of jak1/2 inhibitor, ruxolitinib, in treatment of steroidrefractory gvhd. methods: we reported in this monocentric retrospective study, ruxolitinib response and follow up of 44 cases of chronic gvhd (cgvhd) not improved with standard immunosuppressive therapy. complete organ response (cr) was defined as the resolution of clinical manifestations of cgvhd in a specific organ. very good response partial (vgpr) was defined as an improvement of clinical manifestations of cgvhd with more than 50% decrease of corticosteroid, while a partial response (pr) was associated with less than 50% decrease of corticosteroid. treatment failure was defined by the absence of improvement of cgvhd, deterioration of cgvhd in any organ by at least one stage, the development of cgvhd manifestations in a previously unaffected organ, and the use of any additional agents to control the disease. results: median age at transplant was 52 years (range, 19-66). 59% of patients presented an acute myeloid leukemia. donor type was sibling (n=12), unrelated (n=24) or haploidentical (n=6).two patients benefited a cord blood transplant. patients received either myeloablative (64%) or reduced intensity (36%) conditioning regimens. stem cell source was peripheral blood for 75% of patients. patients presented mild (n=11), moderate (n=21) or severe (n=12) cgvhd according to nih score. median number of regimens prior to ruxolitinib was 1 (range, 0-6), among those corticosteroids (n=36). median follow-up after ruxolitinib was 18 months (range, 4-32). overall responses rate (orr) at 1 month was 84% with 34% cr, 6% vgpr and 43% pr ( figure 1 ). 13% of patients failed at 1 month after introduction of ruxolitinib. the rate of cr increased with time : 45% at 3 months (n=38), 53% at 6 months (n=30) and 65% at 12 months (n=23). but vgpr rate was rather stable at 3 months (24%), at 6 months (33%) and at 12 months (22%) vs 6% at 1 month. among the patients under steroids, 21 (58%) patients discontinued steroids. the 12-months overall survival (os) and diseasefree survival (dfs) after ruxolitinib was 89% (80%-99%, 95% ci) and 86% (76%-96%, 95% ci), respectively. severe cytopenia (grade 3 and 4) was observed in 12 patients. after introduction of ruxolitinib, 4 patients presented bacterial infections, 3 patients presented an invasive pulmonary aspergillosis and 1 patient developped a pneumocystis. cytomegalovirus reactivation requiring preemptive treatment was observed in 7 patients. no toxicities required withdrawal of ruxolitinib. [[p269 image] 1. figure 1 and partial response to mesenchymal stem cells (msc), as second-line therapy, varies from 15% to 82% in acute gvhd patients. we report our experience using mscs to treat refractory agvhd. methods: the study was a retrospective single center study. all data were collected from patients' files. twenty patients were enrolled (age ranging from 7 months to 19 years) between april 2014 and april 2018. results: five of these patients received reduced intensity conditioning and 15 patients received myeloablative regimens before hsct. one haploidentical, 1 autologous, 1 cord blood, 14 mud, 3 msd transplantations were performed. the patients were eligible if they developed grades ii-iv agvhd. all patients were treated with standard first-line treatment with corticosteroids and at least one second-line therapy. the definition of steroid resistant agvhd considered as either no response to steroid treatment lasting at least 7 days or progression during treatment of at least one grade within the first 72 hours. prophylactic treatment with calcineurin inhibitors continued at therapeutic dose level. totally, 77 doses of mscs were infused. the median dose of msc was 1.02 × 10 6 cells per kg body weight. the median duration between the diagnosis of agvhd and initiation of mscs therapy was 16 days (range: 4-241). the received msc doses ranged from one to seven. none of our patients had severe side-effects during infusions of mscs. overall, complete response (ocr) was obtained in 10 patients, partial response in 7 patients and no response (nr) was documented in 3 patients. in our study group, the complete response rates in liver, gastrointestinal, skin agvhd were 20%, 35%, 72% respectively. four patients (20%) died in 90 days after using mscs from complications of agvhd. eleven of 20 patients (55%) were still alive with a median follow-up of 558 days (range:171-989 days) after first mscs infusion. one year estimated probability of overall survival for patients achieving ocr and partial remission/no remission in 90th day of mscs were 87.5% and 20%, respectively. conclusions: in conclusion, mscs appears to be a safe and effective treatment option for pediatric patients with steroid refractory agvhd. disclosure: nothing to declare effect of extracorporeal photopheresis on production of serum elafin in chronic graft versus host disease arun alfred 1 , charlotte burton 1 , kathryn goddard 1 , nichloas matthews 1 background: extracorporeal photopheresis (ecp) is a second line therapy for steroid refractory, dependent or intolerant chronic gvhd (cgvhd). in order to guide ecp there is an unmet need for predictive and diagnostic biomarkers. elafin is a serine-protease inhibitor primarily produced by epithelial cells, particularly keratinocytes in inflammatory skin diseases and plasma and epidermal elafin have been identified as biomarkers of skin gvhd (1, 2) . since skin cgvhd is noted for a particularly high response rate to ecp, we conducted a study to investigate whether ecp affects the production of elafin. methods: serum samples were collected from 72 cgvhd patients (39 male /33 female; age range: 25-74) and 17 age-matched healthy controls (10 male / 7 female) before ecp and at 3 month intervals up to 1 year. patients had gvhd affecting skin (61/72), mucosal membranes (16/ 72), liver (14/72), joints (8/72), gut (17/72), eye (8/72), genital (4/72), and respiratory involvement (4/72). serum elafin was assessed by elisa (r&d systems). data were analysed using graphpad prism 6. statistical tests performed include 2-tailed mann-whitney, pearson's correlation test, and 2-way anova with repeat measures, as appropriate. results: chronic gvhd patients presenting for ecp had significantly elevated serum levels of elafin (p=0.0029; median of 22ng/ml, iqr 15-45ng/ml) compared to healthy controls (median of 14 ng/ml, iqr 9.6-18ng/ml).while 85% of patients had skin involvement, only 40% had elafin levels above the iqr of healthy controls. where disease scores were available (n=25) there were no significant correlations with modified rodnans (r=0.19) or nih bsa scores (r=0.37).sub-analysis was performed by grouping cgvhd patients according to quartiles of serum elafin at pre-ecp baseline. retrospective analysis of patients after 12 months of ecp (n=66) revealed that those with serum elafin levels in the upper quartile (elafin hi ) pre-ecp (min-max: 46-117ng/ml), showed a significant reduction after 3 months of therapy (p< 0.05; mean +/-sd :72ng/ml +/-26 ng/ml vs 53ng/ml +/-23ng/ml, respectively), which was sustained up to 12 months of ecp (p< 0.0001; mean +/-sd: 37 ng/ ml +/-20ng/ml). in contrast, patients with elafin levels below the upper quartile (elafin lo ) showed no significant change (mean +/-sd: 21ng/ml +/-11ng/ml vs 26ng/ml +/-20 ng/ml, respectively). of note, pre-ecp patients with elafin below the median received significantly more corticosteroid (cs) than those above, (p< 0.05; mean +-sd: 27+/-18mg/d vs 16+/-16mg/d, respectively), which was significantly reduced after 3 months of ecp (p< 0.001; to 13+/-10 mg/d), while cs dose was not significantly changed in elafin hi patients until 6 months (p< 0.05; mean +/-sd: 16mg/d +/-16mg/d vs 6mg/d +/-5.5mg/d, respectively). conclusions: consistent with recent data, we found that serum elafin is significantly elevated in a subset of cgvhd patients compared to healthy controls, but did not correlate with skin disease scores. ecp administration was associated with a reduction in serum elafin in the elafin hi subset. further, elafin lo and elafin hi patients tolerated different rates of ecp-mediated tapering of cs immunosuppression suggests pre-ecp elafin measurements may have predictive value. references : background: allogenic hematopoietic stem cell transplantation (hsct) is a potential curative treatment for many malignant and no malignant hematologic diseases, primary immunodeficiencies and some metabolic and deposit diseases in children. graft versus host disease (gvhd) is a major cause of morbidity and a leading cause of non-relapse mortality. corticosteroids are the standard first-line systemic treatment for both acute and chronic gvhd, whereas no second line option for corticosteroid-refractory patients is standardised. ruxolitinib is a potent inhibitor of jak 1/ 2 showing significant responses in refractory gvhd patients in recent reports. methods: we present two centres experience with ruxolitinib for gvhd treatment in pediatric patients. the study was conducted in two spanish pediatric hsct centres, hospital vall d'hebron (barcelona) and hospital universitario la paz (madrid). all patients receiving ruxolitinib since the drug was available were included for retrospective analysis. results: between march 2017 and december 2018 19 pediatric patients with acute or chronic gvhd with refractoriness to corticosteroids were treated with ruxolitinib, in 22 different episodes (one patient received it in 3 different moments, and one patient received it in 2). patient's sex at birth was female in 11 and male in 8 cases. median age at hsct was 10,94 years (0,79-18,11). primary disease was malignant in 11 patients and non malignant in 8. median time of gvhd diagnosis was 47,5 days (6-525). all gvhd episodes were treated with corticosteroids as first line, with maximum doses between 1-5 mg/kg/day (the main dose used was 2 mg/kg/day, 15/22 episodes). patients received a median number of 3,5 (2-7) previous lines of treatment including steroids before starting ruxolinib; they were extracorporeal photopheresis (19/22 episodes), sirolimus (7/22), mesenchymal cells (5/22) ruxolitinib initiation was indicated for acute gut refractory/steroid dependant gvhd in 9 episodes and chronic multisystemic in 7 episodes. other indications were chronic lung (3/22 episodes), chronic skin (2/22) and acute skin gvhd (1/22) . median post-hsct time of ruxolitinib start was 300 days. doses ranged between 2,5-10 mg/12h depending on age, weight, and tolerance (hematologic and liver toxicities). average duration of treatment was 186 days (11-616). complete response (cr) rate was 18,1%, global partial response (pr) 72,7%, and no response (nr) 9% (progression in one patient and recent treatment start in other patient). mean time to maximum response was 4 weeks. treatment stop cause was cr in 2 cases, infection in 4, liver toxicity in 1. no severe side effects directly related to ruxolitinib treatment were described. conclusions: ruxolitinib has been recently introduced as second line strategy for rescuing corticosteroid-refractory gvhd in pediatric patients. while results of randomized trials are lacking, we present our experience (two centres). the main indications for starting treatment were acute gut and chronic multisystemic gvhd. most patients achieved some grade of response (partial or complete), allowing stopping or tapering corticosteroids. toxicity profile appears to be acceptable. disclosure: nothing to declare. stability of tacrolimus concentration early after allogeneic hematopoietic stem cell transplantation reduces the risk of acute gvhd background: tacrolimus is used as an immunosuppressive drug after allogeneic hematopoietic stem cell transplantation (allo-hsct). it is well known that early concentration level of tacrolimus is correlated with the risk of acute graft versus host disease (agvhd), however, whether range of standard derivation (sd) of early tacrolimus concentration after allo-hsct also affect to the risk of agvhd still remains unknown. here, we investigate the correlation between the range of sd of early tacrolimus concentration after donor hematopoietic cells engraftment and the development of agvhd. methods: we retrospectively assessed 207 patients who underwent allo-hsct in our hospital from 2010-2017. all patients received standard gvhd prophylaxis by continuous intravenous (iv) tac with starting dose of 0.02 mg/ kg/day from 1 day before allo-hsct (day -1) and iv methotrexate on day 1, 3, 6 at dose of 10 mg/m 2 , 7 mg/m 2 , 7 mg/m 2 , respectively. tac dosage was adjusted to target the serum concentration of 8-12 ng/ml until at least day 30 and then tapered. to evaluate the sd of weekly tacrolimus concentration, the range of sd of tacrolimus concentration at day1-7 (week-1), day8-14(week-2), day15-21(week-3) and day22-28(week-4) were calculated. the difference of the range of sd between the 2 groups that develop or did not develop agvhd was compared by using mann-whitney u test. multivariate analysis was performed by using multiple logistic regression analysis. patients had given written consent allowing the use of medical records for research, in accordance with the declaration of helsinki, and the institutional review board approved the study. results: there were 114 males and 93 females and the median age was 45 years (range, 16 to 68 years). the risks of disease were low-standard in 141 and high in 66 pts. the number of donors were in 6 hla-identical sibling, 13 in hla-mismatched related donor, 81 in hla-matched unrelated donor and 107 in hla-mismatched unrelated donor. thirty-seven patients developed agvhd (grade i-ii; 28, gradeiii-iv; 9 patients). as a result, the wide range of sd at week-3 significantly increased the risk of agvhd (agvhd-group; 1.178±0.8159 ng/ml, agvhd+ group; 1.447±0.7359 ng/ml, p=0.02). multivariate analysis demonstrated that narrow range of sd of tacrolimus concentration at week-3 reduce the risk of agvhd (or=4.19; 95% ci: 1.59-14.80; p=0.005). there were no correlation between gender, age, disease status, hla with the development of agvhd. conclusions: the range of sd at week-3, an engraftment phase of donor hematopoietic cells, was significantly correlated with the development of agvhd. fine tuning of early tacrolimus concentration with narrow range of sd reduces the risk of agvhd, resulting in improvement of the overall survival after allo-hsct. disclosure: nothing to declair p274 ruxolitinib treatment for steroid-refractory graftversus-host disease han-seung park 1 , je-hwan lee 1 , jung-hee lee 1 , eun-ji choi 1 , miee seol 1 , young-shin lee 1 , young-ah kang 1 , mijin jeon 1 , kyoo-hyung lee 1 background: steroid-refractory graft versus-host disease (gvhd) is one of the most lethal complications after allogeneic hematopoietic cell transplantation. recent studies have shown that ruxolitinib, a janus kinase 1/2 inhibitor, is effective in patients suffering from gvhd. here, we report a retrospective result of ruxolitinib treatment for steroid-refractory gvhd. methods: all patients had received cyclosporine and a short course of methotrexate as gvhd prophylaxis. antithymocyte globulin was added for unrelated or mismatched familial donor hct. ruxolitinib 5 mg twice daily was added to immunosuppressive treatment in patients with steroid-refractory gvhd. results: a total of 27 patients with gvhd (acute, 8, including 3 patients with donor lymphocyte infusion [dli]related; and chronic, 19) were included in the analysis. all patients had grade 3/4 acute gvhd or severe chronic gvhd at the time of ruxolitinib treatment. six (75.0%) of 8 patients with acute gvhd responded to ruxolitinib, including 3 with complete response (cr). the median time to response was 13.5 days (range, 8-25) . nineteen patients received ruxolitinib for severe chronic gvhd, with the median of 3 involved organs (range 2-5). fourteen patients (73.7%) showed response to ruxolitinib, including 4 crs. the median time to response was 24 days (range, 12-138). five responders discontinued ruxolitinib and 9 patients are still on the agent. after a median follow-up duration of 8.7 months, 5 died (2 from relapse of disease, 3 from infection). the 1-year survival probability was 70.1%. eleven of 20 responders discontinued ruxolitinib. gvhd relapsed in 3 of 11 patients at 14, 35, and 149 days after ruxolitinib discontinuation. thrombocytopenia (12/27, grade3/4; 4) was the most common adverse event of ruxolitinib. during treatment, 4 with grade 3/4 infectious adverse events occurred; 2 pneumonias, 1 brain abscess, and 1 liver abscess. conclusions: ruxolitinib treatment seems to be effective for the treatment of steroid-refractory gvhd including long-standing chronic gvhd. the agent was well tolerated and relatively safe. disclosure: nothing to declare. il6-receptor antibody tocilizumab as salvage therapy in the treatment of severe chronic gvhd after stem cell transplantation: a retrospective analysis background: severe chronic graft-versus-host disease (cgvhd) remains the most relevant factor affecting survival and long-term quality of life after allogeneic hematopoietic stem cell transplantation (hct). besides corticosteroids there is no established therapy for cgvhd and many of the used immunosuppressive agents may lead to significant toxicity incl. infectious complications. tocilizumab (an il6-receptor antibody) has shown efficacy in acute gvhd and cgvhd. we retrospectively analyzed the efficacy and safety of patients having received tocilizumab for treatment of advanced cgvhd at our center between the years 2015 and 2018. methods: 9 patients with severe steroid refractory cgvhd and a median age of 50 years (range: 21-62 yrs) having received at least two prior lines of therapy for cgvhd (range: 2-8 regimens) were treated with tocilizumab for at least one cycle (q4w, dosage: 8 mg/kg iv, maximum: 800 mg) with a median number of 4 cycles (range: . nih consensus criteria grading for cgvhd and the immunosuppressive regimen were noted at the time of the first tocilizumab administration and after 3, 6 and 12 months of therapy. all patients received additional concomitant immunosuppressive agents already given at least 4 weeks without response before start of tocilizumab. no new immunosuppression (is) was added in parallel to tocilizumab and response assessment was stopped at start of any additional new is. all patients had received peripheral stem cell allografts. gvhd prophylaxis consisted of a calcineurin inhibitor in combination with methotrexate or mycophenolate and in case of unrelated donors atg was added. 8/9 patients had quiescent onset of cgvhd, one patient developed de novo cgvhd. the median number of days between hct and onset of cgvhd was 215 (89-545). the median number of days between hct and initiation of tocilizumab therapy was 1033 (510-1749) days. at cgvhd onset, 7/9 patients had mild cgvhd and 2/9 patients had moderate cgvhd. the thrombocyte count was < 100/nl in 5/9 patients. organs involved at initiation of tocilizumab therapy were skin (100%, all grade 3), eyes (78%), mouth (67%), fascia (67%), lungs (44%) and genitals (22%). 5/9 patients are still receiving tocilizumab at the time of analysis. results: as tocilizumab was given fairly recently in most patients, 6-and 12-month follow-up was only reached in 3/ 9 patients (33%). at three-month follow-up after initiation of tocilizumab therapy, 4/9 patients (44%) showed partial remission, 2/9 patients stable disease (22%), and 2/9 patients progressive disease (22%) of cgvhd. maximal response was partial remission (56%), stable disease (11%) and progressive disease (22%). 3 patients required subsequent new immunosuppressive treatment. one patient has not yet reached 3-month follow-up. during tocilizumab therapy none of the patients suffered recurrence of underlying malignancy. two patients developed significant respiratory infection and one patient developed soft tissue infection, all requiring antibiotic treatment and pausing of tocilizumab administration, hospital admission was not required. the os and rfs was 100% with median follow up of 8.5 months (range 2-12 months). conclusions: tocilizumab appears to be a promising treatment option in advanced cgvhd but further evaluation within a phase ii trial is required. disclosure: nothing to declare background: allogeneic hematopoietic stem cell transplantation (hsct) is for many patients suffering from aml the only curative treatment option. one major complication is graft versus host disease (gvhd), caused by donor immune cells attacking healthy tissue. regulatory t cells (treg) have been getting huge attention during the past years because of their important role in maintaining immune balance. here we collected peripheral blood samples from 11 patients at different time points after hsct to investigate immune-reconstitution of treg as predictive marker for the development of gvhd. methods: we collected blood samples from 11 patients in the course of allogeneic hsct prospectively once a week from d+7 up to d+200. all patients received conditioning regimen with fludarabine and melphalan, combined with alemtuzumab for t cell depletion. 9 patients developed acute gvhd in the later course. after isolation of pbmc`s we performed facs multicolor staining of t cell and nk cells. treg were identified as cd3 + cd4 + cd25 ++ foxp3 + , nk cells were characterized as cd3 neg cd56 + cd16 + and divided in nk cell subpopulation due to their expression of cd56 dim or cd56 high . results: 1. cd52 neg t cells: all patients developing acute gvhd in the later course showed significant elevated levels of cd4 + cd52 neg t cells, especially cd52 neg treg at d+50. 2. cd52 neg treg / cd8 + cd52 + t cells: one patient not developing acute gvhd showed lots of cd52 neg treg but missed cd8 + cd52 + effector t cells. we recently showed that cd8 + cd52 neg effector t cells are of impaired effector function. these data suggest that cd52 neg treg are only of relevance combined with functional cd8 + cd52 + effector t cells in the development of agvhd. 3. t cell marker: patients without agvhd showed elevated expression of garp on treg. garp was significantly higher expressed on cd52 + treg, indicating a better suppressive capacity of cd52 + treg. this was detected throughout from d+50 until d+200. tigit and ilt3 showed a heterogeneous expression profile without significant differences between the two groups. 4. nk cells: we detected a higher ratio of cd65 ++ /cd56 dim nk-cell population in patients without. we could also show that tigit is mainly expressed on cd56 dim nk cells. conclusions: we and others showed reconstitution of cd52 neg t cell subsets after alemtuzumab mediated t cell depletion -our data on effector t cells showed an impaired effector function for cd52 neg cd4 and cd8 t cells. recently we presented data on impaired suppressive capacity of cd52 neg treg and the association with acute gvhd retrospectively (wölfinger ebmt 2018, ash 2017). here we provide prospective data on patients after the use of alemtuzumab in the context of hsct: our preliminary data suggest that the total amount of cd52 neg-treg and the ratio of cd52 neg treg to cd8 + cd52 + treg on d+50 after allogenic hsct could predict agvhd. this data may be a basis for immune monitoring of patients at d+50 to evaluate their risk for agvhd and could lead to the use of prophylactic treg dli in the context of alemtuzumab mediated t cell depletion. disclosure: medac -travel support, novartis -consultancy fee, pfizer -consultancy fee, shireconsultancy fee background: multiple factors such as disease activity and severity, therapy and/or dietary habits can cause changes in nutritional status independently or by interacting with each other. presence of malnutrition or significant weight loss in chronic gvhd (cgvhd) patients was reported in literature up to 43%. the aim of this cross-sectional study was to identify factors that affect nutritional status in cgvhd patients. methods: nutritional status in patients with cgvhd treated at the university hospital center zagreb, croatia from 2015 to 2018 was assessed. anthropometric measurements (height, body weight (bw), body mass index (bmi)) and clinical validated tool patient-generated subjective global assessment (pg-sga) (where patients were categorized as well-nourished (pg-sga a), moderately malnourished (pg-sga b) or severely malnourished (pg-sga c)) were used. all patients were evaluated according to 2005 nih criteria for cgvhd diagnosis. descriptive and correlation analysis were preformed. results: in total, 44 adult cgvhd patients were included in the study, 23 women (52.3%), median age 47 (18-65) years, with mild cgvhd in 6 (13.64%), moderate in 24 (54.55%) and severe in 13 (29.55%) patients. according to the pg-sga rating 19 (43.18 %) patients had pg-sga a, 19 (43.18 %) pg-sga b and 6 (13.64%) had pg-sga c, giving a total malnutrition or risk of malnutrition prevalence of 56.82%. the mean bmi was 23.83±4.3 kg/m 2 with correlation to pg-sga rating (r=0.446, p=0.0024). malnutrition according to the bmi (defined as bmi< 21 kg/m 2 ) was found in 8 patients (18.18%). bw changes (10% or more in 6 months) were significant in 14 patients (31.82%). according to the pg-sga assessment tool, oral symptoms reported by 31 patient (70.45%) and decreased appetite reported by 12 patients (27.27%) were associated with oral cgvhd nih score (r=0.536, p=0.0002; r=0.441, p=0.0027) but not with bw or bmi. gastrointestinal (gi) symptoms assessed with sga, were generally mild with no correlation to gi cgvhd nih score. no significant association was found between nutritional status and other nih cgvhd scores. corticosteroid therapy present in 12 (27.27%) correlated with pg-sga rating (r=0.494, p=0.0006) but not with bw, bmi or appetite changes. in 23 patients (52.7%) with altered pg-sga rating, bmi, appetite and body weight changes, dietary counseling and oral nutritional supplementation were initiated. conclusions: oral symptoms, decreased appetite and corticosteroid therapy in our cgvhd patients were associated with altered nutritional status according to the pg-sga, but not with bmi. therefore, pg-sga might be a more sensitive tool in assessment of changes of the nutritional status and detection of patients at risk of malnutrition than bmi since it includes different factors like physical examination, presence of gi symptoms and corticosteroid therapy in its scoring system. nutritional counseling and support are important in cgvhd patients especially in presence of oral symptoms. disclosure: nothing to declare. background: sclerotic skin changes are common features in chronic graft versus host disease (cgvhd). one of the most challenging aspects in the diagnosis and management of sclerodermoid cgvhd (scgvhd) is the differentiation between reversible symptoms related to active cgvhd and nonreversible symptoms related to residual permanent damage such as long-standing fibrosis. although several candidate biomarkers of cgvhd inflammatory activity have been proposed, none of them are currently validated. therefore, there is a need for the development of more quantifiable and reproducible measurements tools to guide clinical decisions. we report our experience evaluating the usefulness of high-frequency ultrasonography (hfus) plus doppler ultrasound (doppler-us) and serum fibrosis biomarkers to determine the inflammatory activity of scgvhd. methods: we report 6 patients with scgvhd. hfus plus doppler-us were performed at diagnosis of scgvhd and at different time-points after treatment initiation. serum hyaluronic acid and pro-colagen-iii were measured as fibrosis biomarkers simultaneously with hfus and doppler-us. nih cgvhd 2014 consensus conference diagnosis criteria, scoring system, and response criteria were used to assess global and organ-specific cgvhd, and to measure overall response to therapy. abnormal ultrasound findings were defined as the presence of ≥ 1 of the following: hypoechogenic dermis, dermo-epidermal junction effacement, hypoechogenicity of septa and/or hyperechogenicity of lobules in hypodermis, hypoechogenic fascia, or myositis, for hfus; and, vessels thicker than 1mm in dermis and/or hypodermis, systolic pressure >10 cm/sec, and index of vascular resistance >0.75, for doppler-us. inflammatory activity was classified as mild, moderate and severe according to the severity of doppler-us findings. results: hfsu showed abnormal findings in all patients at diagnosis with no changes except in two patients along the treatment follow-up. inflammatory activity by doppler-us was observed in 5/6 patients at diagnosis (1 mild, 3 moderate, 1 severe). four patients responded to treatment (2 complete responses, cr, and 2 partial responses, pr), one presented clinical improvement less than pr, and one, progressive disease. all patients with clinical response had also a p-rom improvement or normalization. all patients achieving a response showed normalization (n=2) or improvement (n=2) of doppler-us findings. the patient with clinical improvement less than pr and the patient with progressive disease showed persistence of inflammatory doppler-us findings. most patients had normal or light increase of pro-collagen levels at diagnosis and no significant changes were observed during follow-up. levels of hyaluronic acid tended to be very high in patients with progressive scgvhd (patients 2 and 5) and tended to decrease or normalize in those who responded to therapy (patients 1, 2, and 3). conclusions: in this exploratory study, hfsu was a reliable method for evaluating sclerotic skin changes in scgvhd. doppler-us showed a good correlation with disease activity and response to treatment. serum hyaluronic acid levels might be a biomarker of disease activity that deserves further investigation. hfsu plus doppler-us is a useful, non-invasive, repeatable device in monitoring patients suffering from scgvhd. according to our results, doppler-us may be a more sensitive parameter than hfsu in assessment inflammatory activity of scgvhd. disclosure: maría suárez-lledó received a grant from dkms-spain foundation. other authors have nothing to declare post-transplant cyclophosphamide versus antithymocyte-globulin in hla-matched unrelated and haploidentical transplantation for hematologic malignancies background: post-transplant cyclophosphamide(ptcy) and antithymocyte-globulin(atg) are the most commonly used regimens for the prophylaxis of graft-versus host disease(gvhd). we compared these two regimens in hlamatched unrelated (mud) and haploidentical transplantation for hematologic malignancies. methods: we retrospectively analyzed the consecutive adult patients with hematologic malignancies who received mud and haploidentical transplantation at chungnam national university hospital between january 2013 and january 2018. patients who received second transplantation and had refractory disease were excluded. results: this study included 45 patients with median age of 54 (range, 18-71) years: 29 (64.4%) patients received mud transplant (8 and 21 patients in ptcy and atg group, respectively), and 16 (35.6%) patients received haploidentical transplant (11 and 5 patients in ptcy and atg group). graft source was peripheral blood stem cell in all patients. median follow-up duration was 15.5 months (range, 0.5-58.0). in mud transplant, the estimated 20-months survival rate were 85.7% in ptcy vs. 80.7% in atg (p=0.835), the 20-months relapse rate were 37.5% in ptcy vs. 35.0% in atg (p=0.663), the cumulative incidence of grade 2 to 4 acute gvhd were 25.0% in ptcy vs. 22.2% in atg (p=0.706), and the estimated 20-month extensive chronic gvhd rate were 25.0% in ptcy vs. 16.7% in atg (p=0.902). in haploidentical transplant, the estimated 20-months survival rate were 55.4% in ptcy vs. 40.0% in atg (p=0.936), the 20-months relapse rate were 42.9% in ptcy vs. 33.3% in atg (p=0.328), the cumulative incidence of grade 3 to 4 acute gvhd rate were 29.9% in ptcy vs. 33.3% in atg (p=0.686), and the estimated 12 month extensive chronic gvhd rate were 25.0% in ptcy vs. 50.0% in atg (p=0.575). patients receiving ptcy had significantly longer neutrophil engraftment time than those receiving atg in haploidentical transplant [median(range); 17.0(14.0-21.0) days vs. 14.0(13.0-16.0) days, p=0.005]. conclusions: ptcy might be a good option for the prophylaxis of gvhd in hla-matched unrelated transplant as well as haplo-identical transplant. disclosure: nothing to declare early fam therapy for post allo-hsct bronchiolitis obliterans syndrome background: bronchiolitis obliterans syndrome (bos) is a potential major complication after allogeneic hematopoietic stem cell transplantation (hsct). attributed to an allo-immune reaction against the small airways, bo is considered a pulmonary manifestation of chronic gvhd. reported incidence of bos ranges from 5 to 12%, and bos-attributed mortality as high as 30%-80%. a few years ago, a new therapeutic approach with fluticasone, azithromycin, and montelukast (fam) was described (norman bc, et al. bmt 2011) . our aim was to analyze the outcomes of pts who developed bos and were precociously treated with the fam scheme. methods: all the 209 allo-hsct performed in our center from january 2015 and july 2018 were included in the analysis. baseline diseases were: 69 aml, 49 lpd, 31 mds, 28 all, 16 mpd, 10 mm, and 6 bmf. day +100 and day +365 overall mortality were 9,1% and 24,4%, respectively. rest of characteristics of the series are shown in table. fam therapy was systematically started when any patient was first diagnosed with bo. results: eleven patients (5,3%) were diagnosed with bos. at diagnosis of bos, the pts exhibited a fev1 80% of predicted (median fev1: 74%; range; 54-86%) and/or a decline >10% from pre-hsct . at day +100, 6 pts had already the syndrome. two of them died before the end of the first year: one due to invasive zygomycosis (cns plus pulmonary) and the other to baseline disease progression. at day +365, 5 more pts had bos. two more pts with bos died at 15 and 17 months post-hsct due to baseline disease progression. at the close of the analysis, 7 of the 11 pts were alive. so, with a median follow-up of 29 months (range: 8-44), mortality and bos-attributable mortality of the pts with the complication were 36,4% and 9,1%, respectively. conclusions: 1) bos is an infrequent but very severe complication of allo-hsct; 2) bos seems to be less frequent in pts with prophylactic pre-transplant ratg or post-transplant cyclophosphamide, as well as in pts undergoing transplantation with bm (compared to pbsc). 3) early diagnosis and therapy are critical to minimize the bos-attributable mortality. disclosure background: donor lymphocyte infusion (dli) is an established treatment for patients with hematological malignancies relapsed after allogeneic hematopoietic stem cell transplantation (hsct). however, it is associated with an increased risk of graft-versus-host disease (gvhd) and modest anti-tumor activity. compared to the infusion of nonmobilized lymphocytes, granulocyte colony-stimulating factor (g-csf)-primed dli might induce a stronger anti-tumor effect and reduce the risk of infusion-induced gvhd. due to the limited experience of g-csf primed dli in patients relapsed after haploidentical hsct, we conducted a retrospective study of all patients at our hospital who received dli for the relapsed hematological diseases following related hla-matched or hla-haploidentical hsct. methods: the institutional research board approved the study. we identified 94 patients with hematological malignancies receiving dli following related allo-hsct at national taiwan university hospital between 1999 and 2018 aug. the infusate was obtained from the cryopreserved specimen, which had been collected and stored at multiple aliquots at the same time as the initial haploidentical peripheral stem cell graft. patients received dli for either hematological relapse, preemptive or prophylactic treatment. univariate and multivariate analysis was performed using cox proportional hazard regression model. results: for the 61 patients following related hlamatched and the 33 patients following hla-haploidentical hsct received 119 and 72 doses of dli, respectively. in comparison, the median cd3+ cell dosage of haplo-dli is significantly lower (p = 0.0224) than that of dli from sibling donors, with median cell dosage 0.45 × 10 7 /kg (range, 0.05-12 × 10 7 /kg) and 1.1 × 10 7 /kg (range, 0.04-9.78 × 10 7 /kg), respectively. the median time to dli from initial sibling hsct and haplo-hsct was 152 days (range, 13-3357 days) and 155 days (range, 13-946 days), respectively. overall, 12 (20%) of the 61 patients following sibling hsct developed grade 2-4 acute gvhd after dli, whereas 12 (36%) of the 33 patients receiving haplo-hsct developed grade 2-4 acute gvhd after dli (p=0.1460). importantly, for patients receiving dli with cd3+ cell dosage less than 1 × 10 7 /kg, there is no difference in the risk of developing grade 2-4 acute gvhd between patients receiving dli from sibling or haplo donors ( figure 1a) . interestingly, for patients receiving dli with cd3+ cell dosage more than or equal to 1× 10 7 /kg, 4 (50%) of the 8 patients following haplo-hsct developed grade 2-4 acute gvhd after dli, significantly more than 5 (14%) of the 37 patients following sibling hsct developed grade 2-4 acute gvhd after dli ( figure 1b) . the cumulative incidence of grade 2-4 acute gvhd at day 100 after haplo-hsct and sibling hsct were 50% (95% ci: 0.13 -0.79) and 13.5 % (95% ci: 0.05-0.27), respectively ( figure 1b , p = 0.0146). [[p282 image] 1. conclusions: our study shows that the administration of g-csf mobilized dli is feasible after haploidentical hsct for relapsed hematological malignancies. however, dli with cd3+ cell dosage more than or equal to 1× 10 7 /kg in patients receiving haplo-hsct is associated with significantly higher risk of developing acute gvhd than dli from the sibling donors. disclosure: the authors declare no competing financial interests. background: the fresenius phelix is a uva irradiation device used to photoactivate mnc collected on the amicus. the system is closed, utilizing a special mnc kit and modified instrument software. the preliminary results of a phase i safety trial involving three patients (12 treatments) with chronic graft vs. host disease are presented. methods: reasons for transplantation for the patients ages 37, 61 and 62 years were: acute myelogenous leukemia, myelodysplastic syndrome, and myelodysplastic syndrome with pnh. stem cell source was peripheral blood with a 10/10 match for all. each developed chronic skin gvhd. inclusion criteria included wbc and plt counts > 1000 and 25x10 9 /l, gfr > 30 ml/min/bsa, and ast 10-120 unit/l. exclusion criteria included active gi bleeding, nyha cardiac disease greater than grade iii, and the presence of light-sensitive diseases. amicus software 4.51 and phelix software 1.0 were used. settings included: 80 ml/min max draw rate, 2000 ml fixed cycle volume, 1.25 mg/kg/min citrate infusion rate, and 12:1 acd-a ratio. venous access was peripheral or subcutaneous port. target uva dose was 1.5 j/cm 2 and 8-methoxypsoralen dose was 3.4 ml. results: the following mean + sd procedure results were obtained: 2,341 + 14 ml whole blood with acd-a drawn, 191 + 3 ml acd-a used, 691 + 127 ml saline used, 91 + 5 minutes procedure time, and 4,881 + 419 ml total blood volume. minor alarms (n=4) on the amicus and no alarms on the phelix were encountered. all 14-day aerobic and anaerobic cultures were negative and mean endotoxin levels were 0.425 + 0.1752 eu/ml. mean pre/post cbc and plasma hemoglobin levels were: 12.5/11.8 wbc, 9.9/9.5 neutrophils, 0.06/0.05 basophils, 0.21/0.15 eosinophils, 1.06/1.03 lymphocytes, 1.23/1.06 monocytes, 314/293 platelets x 10 9 /l, 40/ 37% hct, 13.3/12.2 g/dl hgb, and 26.8/23.4 mg/dl plasma hemoglobin. plasma hemoglobin delta in the product was 0.00+0.001 grams and the subject was -0.15+0.70 grams. collected product hct. mean 1.95+0.255%. yields are in the table. adverse events included one each: acute respiratory failure, respiratory failure, muscular weakness, musculoskeletal discomfort, and peripheral swelling. three of four events occurred in one patient two weeks after the study procedure. none of the adverse events were considered related to the procedure or investigational product. the patient who experienced acute respiratory failure was removed from the study because of death due to pneumonia, felt to be unrelated to the procedure. conclusions: results indicate the new closed photopheresis system is capable of collecting sufficient mnc and irradiating the cells producing high lymphocyte apoptosis, with minimal alarms and adverse reactions. (21.4%)). 10 (23.8%) of the 42 patients also had acute gvhd of the skin or liver. 37 patients (88.09%) could be treated and controlled with methyl-prednislone monotherapy, 5 patients had steroid refractory gvhd of whom 3 patients (7.14%) could be salvaged with additional drugs (infliximab:2; tacrolimus:1); 2 patients (4.77%) had refractory acute gut gvhd and could not be salvaged despite more than three lines of therapy. at the time of reporting, 26 patients (61.9%) of the 42 are alive. 11 patients died due to transplant related mortality, while 5 patients developed relapsed disease. on binary logistic regression analysis, no baseline clinical or treatment related predictor (disease indication, disease status at transplant, transplant type, graft source, type of conditioning) could be identified for developing acute gvhd of the gastrointestinal system. conclusions: acute gvhd of the gastro-intestinal system is a significant cause for morbidity in allo-hct patients at our centre. further studies are warranted in our cohort, and a prospective analysis of gut microbiome analysis, faecal multi-drug resistance organism surveillance, conditioning related toxicity and antibiotic usage is ongoing. clinical trial registry: not applicable disclosure: the authors declare no potential conflicts of interest benefits and precautions of ruxolitinib in steroidrefractory acute gvhd background: corticosteroids are the standard first-line treatment option for patients with acute graft-versus host disease (gvhd), but approximately half of patients become refractory to steroids and require second-line treatment. ruxolitinib has the potential to treat gvhd in steroidrefractory (sr) patients based on retrospective clinical data. the ongoing prospective trials are currently enrolling patients to evaluate the therapeutic potential of ruxolitinib for gvhd. methods: we analyzed retrospectively clinical experience with ruxolitinib in patients (n=15) with grade 2~4 steroid-refractory acute gvhd patients compared with the control group not receiving ruxolitinib. in addition, immune status was evaluated about 6 weeks~8 weeks after the administration of ruxolitinib using flow-cytometry. ruxolitinib was used as a third option for sr gvhd, combined with previously used immunosuppressive drugs. and steroids were gradually decreased according to the symptoms and discontinued. patients received ruxolitinib 5 mg twice daily (bid), with increase to 10 mg bid if hematologic parameters are stable and no treatmentrelated toxicities. results: fifteen patients all were assessable for response. seven patients achieved a complete response, 5 had a partial response, and 3 had no response at 8 weeks after the first ruxolitinib dose. overall response rate was 75%. three were treatment failures. most adverse effects were manageable, except infectious complications. infectious complications were occurred in about 73% patients (n = 11), resulting in two deaths. common cause of infectious events included cytomegalovirus (n =5), herpes-zoster (n=2), epstein-barr virus (n=2), fungal infection (n = 2), pneumocystis jiroveci (n = 2), bacterial infections (n = 1), and pneumonia of unknown origin (n = 1). t cell counts tended to decreased in the group with ruxolitinib compared with the control group, especially cd4 cell counts. conclusions: ruxolitinib is effective in controlling sr gvhd and can lead to clinical benefits. however, we need to be aware of the infectious complications because ruxolitinib may lead to increased risk of opportunistic infections or reactivation of latent infections. in addition, common infectious complications are presumed to involve t cell dysfunction. clinical background: graft versus host disease (gvhd), being one of most common life-threatening complication post hsct, contributes significantly to morbidity and mortality. when affecting gastrointestinal tract (gi) it is the major cause of death in early period post hsct. due to widespread tissue involvement in most patients diagnosed with gi gvhd, surgical treatment is rarely considered. methods: among 972 allo-hsct performed in department of pediatric hematology, oncology and bone marrow transplantation in wroclaw, poland during years 1996-2018, 291 (29,9%) cases were diagnosed with gi gvhd. in this study we present 3 cases (1%) which were referred to and benefit from surgical approach. results: 1. male, 4 years old underwent hsct from matched unrelated donor (mud) due to chronic myelogenous leukemia (cml) and subsequent molecular relapse succesfully treated with donor lymphocyte infusion, followed by agvhd (skin and gut involvement, grade iv). extensive immunosuppression (steroids, mycofenolate mofetile, atg, okt3) resulted in significant resolution of agvhd symptoms. however aggravating severe abdominal pain and lack of gut movement suggesting bowel obstruction. due to presence of acute abdomen patient was immediately directed for laparotomy. resection of constricted bowel segment followed by 2 subsequent laparotomies for secondary obstruction provided complete resolution of abdominal symptoms. after 16 years of follow-up patient is alive and well. 2. eleven years old male was diagnosed with skin and gut grade iv agvhd on day +84 post mud-hsct performed due to acute myelogenous leukemia (aml). he received pronlonged immunosuppressive treatment including steroids, antibodies, msc and ecp which led to resolving of skin leasions and diarhoea. nevertheless patient was suffering from severe paroxysmal abdominal pain and incidentally vomiting. ct enterography showed partial small bowel constriction. after numerous surgical consultations, eventually on day+ 503 patient underwent laparotomy with constricted bowel resection. histopatological examination of resected tissue revealed moderate gvhd. immunosuppersion was tapered to low dose of steroids with ecp. for now, 2 years post hsct patient is alive, rarely experiencing mild abdominal cramps 3. fourteen years old female developed severe abdominal pain and high volume diarhoea on day +24 post mud-hsct performed for severe anaplastic anemia (saa). despite extensive immunosuppression (steroids, anti-tnf, anti-il2 antibodies) patient condition did not improved. through consistent stomach pain, suspected subileus confirmed by ct enterography, laparotomy was performed (day+158). resection of inflamated and obstructed bowel was made. microscopic evaluation confirmed prior gvhd diagnosis therefore immunosuppression including csa and tapered doses of steroids was continued. complete resolution of abdominal symptoms was almost immediately achieved post-surgery, however 2 months after recurrent abdominal cramps were observed and are now well controlled by pain killers. conclusions: commonly gi gvhd is diffused inflammatory process. however in some cases it may be localized and may lead to partial bowel constriction. in case of severe and prolonged stomach pain, despite of partial resolving of other gvhd symptoms, ileus should be considered. ct enterography may be useful for diagnosis confirmation. in those patients, surgical intervention may improve quality of life or even be a salvage approach. disclosure: nothing to disclose is there any impact of the uric acid levels during the preand early post-graft infusion period, on the gvhd occurence and allotransplant outcome? 36.8(16.7-61.6 ) years, who underwent allogeneic stem cell transplantation (allosct) from full-matched sibling donors for acute leukemia (n=31), very severe aplastic anemia/pnh (n=5), lymphoma (n=3), myelodysplastic/ myeloproliferative syndrome (n=3) . thirty-two patients were in remission at the time of allosct (cr1: 23, cr2: 7, beyond cr2: 2). for a better and more accurate assessment of the ua levels on the agvhd incidence, unlike to the other published studies which evaluated the ua levels only at day 0, we evaluated the ua levels in different time points during the the peri-transplant period (at the conditioning regimen initiation, and at days 0, +7 and +14). because the majority of our patients developed agvhd within the 15-30 days post-transplant, we did not incorporated in the study the of ua levels beyond the +14 day. we also investigated the effect of the ua on survival and the non-relapse mortality (nrm). the vast majority of patients received allopurinol from the 1 st day of conditioning regimen till day -1. the independent t-test, kaplan-meir method and logrank test were used in the statistical analysis. results: the median ua levels were 4.2, 2.5, 2.7 and 3.2 mg/dl at days -7, 0, +7 and +14 respectively. for the statistical analysis purposes, we grouped our patients as low-ua if they had values < 3 mg/dl or high-ua if they had >3 mg/dl. this threshold was chosen based on the ua values from all the collected samples (n=175). finally 16/ 42 (38%) patients developed agvhd; 14(33%) were assessed as gr ≥ii, while 7(16%) as gr iii-iv. the incidence of the agvhd gr ≥ii was similar (ranged from 30-35%) in both groups of patients (low-ua and high-ua) and for all the estimated time points (days -7, 0, +7, +14). we noticed a better 2-years overall survival for patients with low-ua (75% vs. 63%) however without any statistical significance. ten patients succumbed to nrm causes; 6/10 deaths attributed to gvhd complications. the nrm was assessed higher in the high-ua group (38% vs. 18%) but also this difference was not statistically significant. conclusions: though our study bears the limitations of the small number of patients and the retrospective origin, at least to our knowledge is the first which evaluates the impact of ua levels at different time points in the peritransplant period, on the agvhd incidence. in our study the ua levels did not influence the incidence or the severity of agvhd. the higher nrm rates for patients with ua>3.0 mg/dl merits further evaluation. definitely, the role of ua on the allosct outcome will be clarified through well designed prospective trials. disclosure results: five male patients (12%) had genital cgvhd manifestations presented by urethral stricture in 4/5 patients and phimosis requiring surgical treatment in one patient. all five patients had simultaneously cutaneous, oral, and/or ocular cgvhd manifestations. the first patient underwent urethroplasty of bulbomembranous part of urethra with termino-terminal anastomosis and urethroplasty of penile part of urethra with buccal mucosa autograft -bmg (dorsal onlay) that resulted in significant improvement of symptoms and normal miction afterwards. biopsy of the urethra showed mononuclear infiltration in lamina propria consistent with cgvhd. biopsy of the buccal mucosa was done prior to surgery and was negative for cgvhd involvement. the second patient underwent urethrotomy due to circular strictures, but symptoms reappeared again and he is now candidate for bmg. in two patients urethral dilatation was done, and the fifth patient presented with phimosis requiring circumcision, resulted in significant improvement of symptoms. conclusions: male genital cgvhd is an underrecognized and under-reported manifestation. patients after allo-hsct need to be actively asked about their genital symptoms and sexual function, especially if they are diagnosed with other mucocutaneous or ocular cgvhd. multidisciplinary approach, early recognition and frequent follow-up is necessary for timely start of treatment. new methods, such as bmg for cgvhd patients with urethral stricture seem promising and should be further investigated. disclosure: nothing to declare. p289 abstract withdrawn. heracles: a phase ii single-arm prospective study to assess the efficacy of fecal microbiota transfer in the treatment of steroid refractory gastro-intestinal agvhd post allo-hsct background: steroid-refractory acute graft-versus-host disease (sr-agvhd) is associated with an 80% mortality rate and reduced quality of life (qol). so far, there is no approved standard of care for agvhd second-line treatment. there is an urgent need to identify effective therapy for sr-agvhd to improve patients' outcomes. fecal microbiota transfer (fmt) might be beneficial to substantially improve the prognosis. higher gut microbial diversity is strongly associated with increased survival in gvhd patients. recent studies reported promising results of sr-agvhd patients treated with fmt. further evaluation to confirm the efficacy and safety of fmt for agvhd is warranted. the ongoing phase 2 study (heracles) investigates the efficacy of allogeneic fmt in the treatment of patients with sr-agvhd. heracles was launched after the odyssee study showed promising results in the reconstruction of gut microbiota diversity after induction chemotherapy with fmt in acute myeloid leukemia patients. we expect that fmt-based biotherapeutic drugs could be effective treatments to contain sr-agvhd, and thereby reduce the risk of life-threatening complications after allogeneic hsct. methods: heracles is a single-arm, multicenter prospective trial in 5 european countries. patients aged ≥18 years-old, who underwent allogeneic hematopoietic stem cell transplantation (allo-hsct) and developed a first episode of stage 3 or 4 agvhd with gut predominance resistant to a first-line steroid therapy are eligible for inclusion. main exclusion criteria comprise the use of other second-line gvhd therapy, patients with grade iv hyperacute gvhd, late onset agvhd, and overlap chronic gvhd and agvhd after donor lymphocyte infusion. patients receive a first maat013 enema within 5 days after sr diagnosis (v1) and 2 additional ones 1 week apart (v2/ v3) from each other. maat013 is a highly-diverse, microbiome-rich enema formulation obtained from pooled, rigorously screened faeces from healthy donors, manufactured with a standardized process using the signature maat microbiome restoration biotherapeutic (mmrb) platform. at inclusion (v1), before each dosing (v2,3), and 28 days post inclusion (v4), patients' faeces and blood are collected. safety monitoring will be performed with corresponding blood analyses. exploratory measures on faeces include characterization of gut microbiota composition and evolution, impact of maat013 on metabolism, and gut inflammation. immune system phenotyping will be performed by flow cytometry on peripheral blood mononuclear cells, and by elisa assay on plasma. patients' qol will be assessed using a standard, eq-5d-5l questionnaire. the primary objective is to assess the efficacy of maat013 by evaluating complete response (cr, according to modified glucksberg criteria) and very good partial response (vgpr, defined by martin et al., bbmt, 2009) 28 days post-inclusion (primary follow-up). secondary objectives include fmt safety assessment and evaluation of fmt impact on several endpoints, such as overall, relapsefree or gvhd-free survival and chronic gvhd evaluation, as well as multi-drug resistant bacteria carriage. patients will be followed-up until1 year after inclusion. overall, 32 patients are planned to be enrolled and treated, to assess overall response rates and maat013's safety profile. results background: anti-programmed cell death protein 1 (pd1) monoclonal antibodies can be used as "bridge to" a subsequent allogeneic hematopoietic stem cell transplantation (hsct) in patients with relapsed/refractory hodgkin´s lymphoma (hl). this strategy has been reported to be effective, but a frequent onset of steroid-refractory graft versus host disease (gvhd) was also reported. we report 3 clinical cases of patients affected by hl undergoing allogeneic hsct after having been treated with nivolumab. methods: the 3 patients of 48, 18 and 42 years respectively had advanced hl and had relapsed after a previous autologous (2) or allogeneic (1) hsct. they underwent a rescue therapy with 18, 12, 16 nivolumab cycles respectively, depending on the time of partial response achievement and the availability of a donor. two patients received a thiotepa-fludarabinecyclophosphamide conditioning, atg-based prophylaxis and pb cells from unrelated donors. the third patient received bm cells from an haploidentical donor using the "baltimora" nonmieloablative platform. results: at a follow-up of 11, 12, 15 months after hsct, respectively, all patients achieved and maintained a complete remission by pet-ct scans. all the 3 patients developed acute gvhd on day +32, +54 and +107, respectively. patient 1 progressed to grade iv acute gvhd with hepatic and intestinal involvement unresponsive to first line 2 mg/kg steroid therapy and second line etanercept plus extracorporeal photopheresis (ecp). third line therapy with ruxolitinib partially controlled the gvhd. gvhd onset in patients 2 and 3 was preceeded by a prolonged fever without microbiological findings. patient 2 developed hepatic grade ii gvhd with high transaminase levels, initially responsive to steroid therapy, then it progressed to gut requiring second line therapy with etanercept. patient 3 progressed to severe chronic gvhd with skin involvement and resulted unresponsive to steroids and ecp and it was partially controlled by ruxolitinib. immune reconstitution was delayed in all 3 patients: at 6 months post transplantation cd3 levels were 80/μl, 17/ μl and 127/μl and cd4 levels were 36/μl, 16/μl and 65/ μl respectively. only patient 2, that underwent haploidentical transplant and received post-trasplant cyclophosphamide (pt-cy), is off of immunosuppressive treatment at 11 months after hsct, without evidence of gvhd and no history of infections. out of the 2 patients receiving pbsc from unrelated donors and atg prophylaxis, patient 1 developed a disseminated fusariosis on day + 180 and died of cns fusarium localization 1 year after hsct, despite targeted antifungal therapy. patient 3 had pulmonary aspergillosis, sepsis by multidrug resistant psuedomonas aeruginosa and otomastoiditis: at +15 months after hsct, he is on ruxolitinib treatment with skin clinical partial response. conclusions: this case series confirms that nivolumab as "bridge to transplant" is effective in appropriately selected patients. however, risk of acute gvhd and delayed immune reconstitution may require a careful consideration at the moment of planning the transplant. a possible advantage of pt-cy gvhd platform and haploidentical donors should be addressed in larger studies. background: acute graft-versus-host disease (agvhd) is the most important complication after an allogeneic hematopoietic stem cell transplant (hsct). no standard secondline treatment has been established for the corticosteroid refractory agvhd. the anti-tnfα agents are a good option of treatment for these patients, especially when lower gi tract is involved. methods: from april 2010 to july 2017 we reviewed the outcome of 19 patients with steroid-refractory (sr) agvhd treated with etanercept as at least, second line treatment. etanercept dose was 25 mg twice a week for the first 4 weeks, followed by 4 weekly doses. results: median age was 52 years (range 15-69 years), and 12 patients (63%) were male. fourteen patients (74%) had a non-advanced disease status at hstc. eleven patients (59%) received a myeloablative conditioning, and the stem cell source was peripheral blood in 18 patients (95%). sixteen patients (84%) were 8/8 hla matched. the characteristics of the 19 patients, their agvhd stage previous to rescue treatment with etanercept and their outcome are shown in table 1 . seventeen patients (89%) had a classic agvhd while 2 had a late-onset agvhd. etanercept was given as a 2 nd , 3 rd and 4 th line in 3 (16%), 10 (53%) and 6 (31%) patients respectively. the median doses of etanercept administered were 7 (range 1-12), and just 4 patients (20%) completed the 12 doses planned treatment, of whom 3 were alive at 38, 24 and 18 months from the onset of rescue treatment. complications during etanercept treatment were: infection (n=9 [47%]: gram negative bacilli [n=6]), grade 2-4 neutropenia (n=8) and grade 3-4 thrombocytopenia (n=6). etanercept was indicated as a rescue treatment due to: progression after 3 days of agvhd treatment (n=1), no response after 7 days of treatment (n=11), no complete remission after 14 days of treatment (n=3) and relapse due to decrease corticosteroid doses (n=4). at the end of treatment 1 patient achieved a complete response and 3 patients a partial response, all of them are alive. these 4 patients received etanercept as a 2 nd (n=3) and 3th line (n=1) treatment, all of them had lower gi agvhd without any other organ significantly involved. causes of death were: agvhd with or without infection in 15 patients (78%) and leukemia relapse in 1 patient. conclusions: although if etanercept is an option for treatment of sr agvhd in some patients, their prognostic remains poor and more effective alternative strategies are needed. a prompt initiation of etanercept as a rescue treatment for sr agvhd is crucial to improve the prognosis. (58) 5(26) background: although both cyclosporine (csa) and tacrolimus are calcineurin inhibitors, csa is more widely used in pediatric hematopoetic stem cell transplantation (hsct) as a prophylactic drug for acute graft versus host disease (agvhd). there are some clinical experience but very few data about the clinical efficacy of conversion to tacrolimus. here, we present our single center data on this arguable topic. methods: this study involves the data of 71 pediatric hsct patients in medical park göztepe hospital between 2014-2018. all 71 patients had prophylactic csa therapy and for various reasons csa was converted to tacrolimus therapy. most of the patients had this conversion due to agvhd. as steroid is the first line therapy for agvhd, conversion to tacrolimus is done concurrently at the start of steroid therapy (within 72 hours after the start of steroid). and also, patients who had any other immunosupressive therapy for agvhd are excluded. response is defined as resolution of symptoms within 7 days after conversion. results: mean age of the study population is 130 months (5-266 months), male/female ratio is 1,3 (40/31), donor types are mud 48 patients (67%), mfd 18 patients (26%), haplo 5 patients (7%) and mean conversion time is 32 days (1-142 days) . the rationales for conversion are agvhd for 40 patients, unproper csa plasma levels for 7 patients, allergic reaction for 6 patients, nephrotoxicity for 6 patients, hepatotoxicity for 5 patients, severe headache for 2 patients, high arterial blood pressure for 2 patients and one each for refractory vomiting, autoimmune thyroiditis and visual disturbance. the subgroup analysis of agvhd patients reveals that mean conversion time for agvhd is 41 days (8-142 days) and there are only 11 responders whose agvhd resolve completely (%27) after conversion. all of the patients had proper tacrolimus levels after conversion due to unproper csa levels and also patients in allegic reaction, severe headache, visiual disturbance and refractory vomiting group responded to conversion completely but only one of the 6 patients in nephrotoxicity group responded and also 3 of the 5 patients in hepatoxicity group responded. the only one patient suffered from autoimmune thyroiditis did not respond to conversion. conclusions: in this study, it is obvious that there are response to conversion for some specific adverse effects of csa and tacrolimus is a good alternative for the patients who have unproper csa levels. conversely, the high percentage (%73) of non-responders shows that it is not feasible to make a conversion to tacrolimus for acute gvhd. disclosure: nothing to declare background: capillary leak syndrome is caused by the dysfunction of the vascular endothelial cells,and is characterized by weight gain,generalized edemas,unresponsive to diuretic treatment,and hypotension.it usually develops in the first 15 days post hsct.and it is of great difficuty to distinguish from other complications which are occured post the allo-hsct. to diagnose this complication at the early stage,it is very difficulty. methods: a 34-year-old man was admitted to ningbo first hospital for its abnormal in the peripheral blood .he was diagnosed with aml-m5 by the classical morphology and immunophenotype.cytogenetic evaluation showed a normal 46, xy(20).the patient achieved cr with induction therapy including idarubicin, cytarabine and etoposide. after consolidation therapy,an allo-hsct from hla identical related dornor(33-year-old male, donorrecipient matched by high resolution hla typing at hlaa, -b, -c, drb1, and dqb1, 10/10 matches) was performed.the recipient received conditioning with busulfan, 4 mg/kg/day injection for 3 days; cyclophosphamide, 50 mg/kg/day injection for 2 days; cytarabine, 2 g/m2/day injection for 1 day; semustine, 250 mg/m2/day orally for 1day; donor peripheral blood stem cells (pbsc:mnc: 6.43×109/l, cd34+:4.05×106/l) were mobilized, pheresed and administered to the recipient. gvhd prophylaxis consisted of traditional cyclosporine, short-course methotrexate (15 mg/ m2 at day +1, 10 mg/m2 at days +3, +6, and +11) and cyclosporin a injection 5mg/kg qer day was mot reduced untill the hematopoietic reconstitute sucessfully . on day +19, complete donor chimerism was acheieved. the csa was gradually reduced and tapered.on day +150,the patients was manifested with increasing in the time and volume of the faeces, he was diagnosed with ii°gvhd (gut).the standarded does of immunosuppressive drug including methylprednisolone and cyclosporin a was administrated. the immunosuppressive drug was gradually reduced when the gvhd was controlled.on day+271,the patient felt distress and the distress was not related to with the exercise,the temperature was normal,and he did not gain weight.there was no edema in the body.laboratory test including routine blood test,c-reactive protein,procalcitonin,blood gas analysis,cmvdna,ebvdna was normal. the ct scan shows that 1the pleural is filled up with water, and could not be enlarged promptly,2there is pericardial effusion in the body.pulmonary function test shows that 1 reduced function in ventilation and diffusion fuction.the laboratory test of the pleural effusion was normal,the blast cell was not detected in the pleural effusion,the cd34+ cell count was below the dectable level,the next generation sequencing for minimal residual disease shows that there was no gene mutation .thus, post capillary leak syndrome was considered .sirolimus was adopted and taken the place of cyclosporin a,immunoglobulin was adminstrated to reduce the edema. results: taking together comprehensively,the effusion in the pleural and cardiac was absorbed well. conclusions: occurance of capillary leak syndrome is rare,there is limited data about capillary leak syndrome. comprehensively,the mechanism of cls has not been totally identified.and there is no standard treatment to treat the complication.at present,the cls of this patient was absorbed well by administrating sirolimus,closely followup is needed. disclosure: nothing to declare graft-versus-host diseasepreclinical and animal models p295 short-term krp203 and posttransplant cyclophosphamide for graft-versus-host disease prophylaxis emi yokoyama 1 , daigo hashimoto 1 , takahide ara 1 , eko hayase 1 , takanori teshima 1 1 hokkaido university faculty of medicine, hematology, sapporo, japan background: post-transplant high-dose cyclophosphamide (ptcy) in combination with other immunosuppressants such as calcineurin-inhibitors (cis) has been increasingly used as gvhd prophylaxis after hla-haploidentical or matched hematopoietic stem cell transplantation (hsct). however,cis could hamper reconstitution of regulatory t cells (tregs) and tolerance induction after hsct, facilitating us to develop novel ci-free/ptcy-based gvhd prophylaxis. in the current study, we developed a novel gvhd prophylaxis in which ptcy was combined with short-term administration of krp203, a selective agonist of sphingosine-1-phosphate receptor type 1 (s1pr1), using murine models of mhc haploidentical bone marrow transplantation (bmt). methods: b6d2f1 (h-2 b/d ) recipients were lethally irradiated and transplanted with bone marrow cells and splenocytes from allogeneic b6 (h-2 b ) donors. cy at a dose of 50 mg/kg was intraperitoneally injected into the recipients on day +3, and krp at a dose of 1.0 mg/kg was orally administrated daily from day 0 to day +4 after bmt. donor t cells in the target organs and secondary lymphoid organs were evaluated by flow cytometric analysis. plasma levels of tnf-α were determined using cytometric beads array. to evaluate graft-versus-leukemia (gvl) effects, recipient mice were intravenously injected with luciferase-transduced p815 cells (p815-luc) on day 0, and in vivo bioluminescence imagingwas conducted weekly after bmt. results: severe gvhd was developed in allogeneic recipients and all mice died by day 50 after bmt.ptcy alone at a dose of 50 mg/kg significantly ameliorated gvhd and 30 % of ptcy-treated allogeneic recipients survived. oral administration of krp203 alone enhanced contraction of donor t cells in the lymph nodes and also ameliorated gvhd as has been previously shown with multi-s1pr agonist, fingolimod. next, we tested if shortterm krp203 on days 0 to +4 added to ptcy enhances anti-gvhd effects of ptcy. we found that survivals of ptcy+krp203 group were significantly prolonged compared to those of ptcy-alone group ( figure a) . plasma levels of tnf-a, clinical gvhd scores ( figure b) , and donor t-cell infiltration into the target organs such as the gut and skin were also significantly reduced in ptcy +krp203 group compared to ptcy-alone group (figure c and d) . unlike cis, addition of krp203 to ptcy promoted treg reconstitution after bmt. finally, bioluminescence imaging demonstrated that proliferation of p815-luc injected on day 0 was significantly delayed in ptcy +krp203-treated allogeneic recipients compared to control mice transplanted only with t-cell depleted bone marrow cells, suggesting that significant gvl effects persisted in ptcy+krp203-treated recipients. conclusions: a combination of short-term krp203 and ptcy is a promising novel calcineurin-free gvhd prophylaxis in mhc-haploidentical sct. we recently showed that donor inkt cells can be expanded ex vivo and that they are able to prevent activation and proliferation of alloreactive donor t cells while promoting efficient graft-versus-leukemia effects (schmid et al. 2018 ). however, the underlying mechanisms how human inkt cells induce immune tolerance after allogeneic hct are not fully understood. methods: monocyte-derived dendritic cells (dcs) were cultured in a mixed lymphocyte reaction with mhcmismatched t cells and culture-expanded inkt cells. tcell activation and proliferation was analyzed by multiparametric flow cytometry and released cytokines were measured via multiplex analysis. transwell assays and imaging flow cytometry were performed to elucidate cellcell interactions. bead-controlled flow cytometry-based cytotoxicity assays were used to evaluate dc apoptosis. apoptotic dcs were then purified by fluorescence-activated cell sorting to investigate their tolerogenic potential to prime regulatory t cells (tregs). results: the addition of inkt cells to mixed lymphocyte reactions resulted in a significantly reduced activation and proliferation of mhc-mismatched t cells. transwell assays and imaging flow cytometry revealed a cell contactdependent mechanism between inkt cells and dcs leading to apoptosis with increasing dna fragmentation of dcs over time. interestingly, various fluorescence-activated single cell sorted inkt-cell subsets were all able to induce apoptosis of host dcs. multiplex analysis revealed that dcs triggered inkt-cell release of cytotoxic factors like perforin, granzyme b and granulysin. blocking the inktcell receptor engagement with a cd1d antibody prevented inkt-cell degranulation as well as the subsequent induction of host dc apoptosis. inhibition of cytotoxic factors also abrogated apoptosis of dcs. in turn, sorted apoptotic dcs induced tolerogenic dcs characterized by a high expression of pd-l1 in mixed lymphocyte reactions. such tolerogenic dcs promoted the expansion of cd4 + cd25 + foxp3 + tregs and prevented activation and proliferation of mhcmismatched t cells. conclusions: we propose a novel mechanism how culture-expanded human inkt cells prevent gvhd after allogeneic hct. host dc apoptosis through donor inkt cells induces a tolerogenic immunoenvironment characterized by pd-l1 high dcs and expanding donor tregs inhibiting activation and expansion of alloreactive donor t cells. our findings pave the avenue for clinical translation of adoptively transferred culture-expanded inkt cells in humans. disclosure: nothing to declare results: vip-ko mice transplanted with allogeneic tcd bm alone had increased graft rejection with lower levels of donor chimerism and 33% day 75 survival compared with 73% survival of wt recipients. transplanting tcd bm plus 3 × 10e6 donor t cells from b10.br or balb/c donors in vip kio mice led to >95% donor chimerism and significantly increased gvhd-mortality compared with wt recipients, with 0% vs 40% survival in the b10.br-->b6 model (p< 0.01), and 0% vs 73% survival in the balb/c-->b6 model (p< 0.01). donor-derived t cells in vip-ko recipients had significantly higher th1 and th17 polarization, with higher rorγt in both cd4+ (p< 0.0001) and cd8+ (p< 0.0001) t cells, and higher frequencies of ifn-γ (p< 0.001), tnf-α (p< 0.05), and il2 (p< 0.01) in cd4+ and cd8+ t cells compared to wt recipients. b10.br-->b6 second allogeneic transplantation of radiation chimeras caused lethal gvhd mortality in vip-ko-->vip-ko and wt-->vip-ko mice, but not in wt-->wt or vip-ko-->wt b6 mice, demonstrating the protective effect of vip was due to synthesis by non-hematopoietic recipient cells. immunofluorescent imaging of allo-bmt recipients showed marked up-regulation of vip in lungs post-transplant and high vip production within neurons innervating the lungs. finally, we demonstrated that short-term administration of vip (10mcg/day) from day 0 to day 10 prevented gvhdmortality in vip-ko recipients transplanted with b10.br-->b6 mhc donor bm & t cells. conclusions: the absence of vip in recipient cells led to increased graft rejection in the absence of donor t cells and increased lethal gvhd when donor t cells were transplanted, indicating vip induced post-transplant regulates allo-reactivity of host graft-rejecting lymphocytes and donor gvhd-causing t cells. the protective effect of parenteral vip administration suggests vip-mimetics represent a novel approach to prevent and treat gvhd. these data also suggest a mechanism of action for the mitigation of gvhd by alpha-1 anti-trypsin (aat) whereby aat inhibits the proteolytic inactivation of endogenous vip. disclosure: dr. waller reports personal fees and other support from cambium medical technologies, grants from celldex, personal fees from kalytera, grants and personal fees from novartis, grants and non-financial support from pharmacyclics, and equity ownership in cerus corporation and chimerix outside the submitted work. in addition, dr. waller has intellectual property related to vip signaling that has been licensed to cambium oncology in which he holds equity. low-density neutrophils expansion is associated with acute graft versus host disease in allogeneic hematopoietic stem cell transplant patients background: low-density neutrophils (ldns) are distinguished from normal-density neutrophils (ndns) by their anomalous sedimentation within the mononuclear cell fraction after density gradient centrifugation of peripheral blood (pb). by analysing ldns and ndns from g-csfstimulated donors or lymphoma patients, we have previously demonstrated that, depending on physiopathological conditions, immature cd66b + cd10 -ldns can promote t cell survival and ifn-γ production, while mature cd66b + cd10 + ldns can exert immunosuppressive proprieties. aim of this study was to establish the frequency of cd66b + cd10and/or cd66b + cd10 + ldns in pb of allogeneic hematopoietic stem cell transplant (hsct) patients throughout immune reconstitution, and verify their potential correlation with acute graft versus host disease (agvhd). methods: patients undergoing hsct in our institution between december 2015 and june 2018 were prospectively enrolled in the study upon informed consent and after institutional board approval. criteria of inclusion were age ≥ 18 years and absence of rheumatologic or viral diseases. pb samples were collected at day +21, +42, +60, +90 and +180 after hsct and any time within day +180 in case of gvhd, before first-line therapy. eight healthy donors (hds) were enrolled as control. mononuclear, polymorphonuclear, and whole blood cells were analysed by flow cytometry after cd45 vioblue, cd16 apc-cy7, cd11b pe-cy7, cd10 pe, cd66b fitc staining. cd66b + ldns were expressed as percentage of cd45 + pb mononuclear cells (pbmcs) or cd45 + whole blood cells and were further characterized based on cd10 expression. cd66b + ndns, expressed as percentage of cd45 + whole blood cells, were also analysed for cd10 staining. results: 39 patients (m/f 25/14, median age 47) were enrolled in the study. patients received hsct from hlaidentical (13) or haploidentical (6) related and from hlaidentical unrelated (20) donors. after a median time of 33 (15-95) days, 13 patients developed grade ii-iv agvhd. no patients were receiving g-csf at agvhd onset. the scheduled assessments were interrupted in agvhd patients at the beginning of first-line treatment and in 4 patients relapsed of their primary malignancy. no patients developed de novo late-acute or chronic gvhd. starting from day +21 the frequency of ldns within cd45 + pbmcs was higher in all patients as compared to hds. the 25 patients that did not develop agvhd showed a decreasing frequency of cd66b + cd10 -ldns, with a progressive increase of cd66b + cd10 + ldns, from day +21 to +180. interestingly, patients with agvhd showed a significantly higher frequency of cd66b + cd10 -ldns as compared to patients without agvhd throughout the same time lapse (i.e. from day +21 to +90) (83.15 vs 50.8, p=0.027). consistently, patients with agvhd had a significantly lower frequency of cd66b + cd10 + ldns (12.1 vs 50.05, p=0.0014). the frequency of mature cd66b + cd10 + ndns was normal in all patients since day +21. conclusions: ldns are more represented in hsct patients than in hds, with a significant expansion of the cd66b + cd10subpopulation (with a parallel decrease of the cd66b + cd10 + subpopulation) in patients with agvhd as compared to those without agvhd. according to the previously demonstrated t cell activating function of cd66b + cd10 -ldns, it is tempting to speculate that the expansion of this subpopulation may contribute to agvhd development. disclosure: nothing to declare background: acute graft-versus-host disease (agvhd) is a major complication after allogeneic hematopoietic stem cell transplantation (allo-hsct) which has negative impact on the morbidity and mortality of the patients. accumulating evidences suggest that abnormalities of foxp3+ regulatory t (treg) cells contributed to the pathogenesis of gvhd, but the underlying molecular mechanisms still remain largely unknown. methods: in this study, we enrolled all the 40 patients treated with allogeneic hsct at the institute of hematology, chinese academy of medical sciences between 2016 and 2018,as well as 10 age-matched healthy adults as control samples. the ratio of tregs in pb and bm of healthy controls (hcs) and patients with and without agvhd was determined by flow cytometry. the transcription profile between tregs from patients with or without acute gvhd was measured,the pathway enrichment analyses were performed by the kyoto encyclopedia of genes and genomes (kegg) pathway database and geneset enrichment analysis (gsea).the expression of lkb1 at transcript levels and protein levels was measured by realtime pcr and analyzed by the nanopro1000tm system. a series of functional assays in vitro were performed to assess the function and stability of tregs from patients with and without agvhd.meanwhile, to assume the affect of lkb1 on gvhd outcome, we established a murine transplant model,which recipient balb/c animals were transplanted with the same amount of mixture made by bm, cd4 +cd25-tcon cells from c57bl/6 and cd4+ foxp3 yfp+ tregs from either foxp3crelkb1f/f or foxp3cre mice. results: in this study, we demonstrated that bm had decreased frequencies of tregs, accompanied with a reversed lower ratio of tregs frequencies between bm and pb in agvhd patients. meanwhile, the number and function of tregs in bone marrow also affected hematopoietic reconstitution. futhermore,to elucidate these mechanisms which regulate tregs homeostasis, we examined the role of lkb1 on tregs in patients with agvhd and in agvhd murine model. studies demonstrated that lkb1deficient tregs lost foxp3 expression and weaken suppressor function during agvhd. transcriptional profiling and pathway analysis revealed that nf-kb signaling activation and the impairment of a wide spectrum of immunosuppressive genes in agvhd tregs. further mice experiments suggested that cns2 methylation might lead to the instability of tregs in agvhd group. transplantation with marrow grafts from foxp3crelkb1f/fmice exacerbates gvhd lethality. conclusions: these studies indicate that lkb1 is a critical homeostatic regulator for tregs during agvhd. targeting of lkb1 therefore represents a novel therapeutic strategy that promote immune tolerance to mitigates the severity of agvhd. disclosure: national program on key basic research project (973 program) role of aryl hydrocarbon receptor in intestine after allogeneic hsct in mice won-sik lee 1 , soung-min lee 1 , sj-kil seo 1 1 inje university, busan paik hospital, hemato-oncology, busan, korea, republic of background: aryl hydrocarbon receptor (ahr) is a ligandactivated transcription factor that is activated by various small molecules from the diet, microorganisms, host metabolism, and xenobiotic toxic chemicals. the function of ahr has been demonstrated as a crucial regulator in intestinal homeostasis. here, we investigated the regulatory role of ahr in intestine of recipients after allogeneic hematopoietic cell transplantation in mice. methods: wild-type (wt) b6 (h-2 b ), ido -/-(h-2 b ) and ahr -/-(h-2 b ) mice were lethally irradiated and transplanted with 5 x 10 6 tcd-bm plus 2 x 10 6 t cells from balb/c donor mice. ahr activation in colon tissue of recipients was determined by the ahr target genes cyp1a1 and cyp1b1 expression using real-time pcr. the recipient mice were monitored every other day for survival and clinical score. histopathology and pathogenic effector cytokine levels in colon tissue were analyzed for evaluating ahr function. results: we observed that cyp1a1 was constitutively expressed in the colon tissue of naïve recipient mice. although the expression levels were increased by tbi conditioning, the additive up-regulation of its levels with donor t cell alloreactivity was not observed. in contrast, cyp1b1 expression was markedly induced in the colon tissue by donor t cell alloreactivity. we further observed that the cyp1b1 expression was significantly decreased in the colon of ido-/-recipients with donor t cell alloreactivity, but cyp1a1 was not changed. ido-/-and ahr-/recipient mice showed higher histopathological score for intestinal gvhd and increasing pathogenic cytokine levels in the colon compared with wt mice. conclusions: our results demonstrate that ahr-induced target gene profiles might be differently induced in intestine by ligand dependent manner after hsct, which affect intestinal gvhd. disclosure: nothing to declare. abstract already published. abstract withdrawn. in vitro platelet activation evaluation in allogeneic hematopoetic stem cell transplanted patients in response to haemostatic stimulation and cytomegalovirus stimulation (gvhd), complication of which one of the risk factor is cmv reactivation. the resultant inflammatory platelet response during the high-risk period of gvhd after allogeneic hsct remains unknown. our study aimed to characterize spontaneous platelet activation during the 2d and 3d months after allogeneic hsct, and in response to haemostatic stimulation and cmv stimulation. methods: we compared a group of healthy volunteers to a group of allogeneic hsct patients followed between the 30 th and the 90 th days after hsct. platelet activation was determined by the platelet surface expression of cd62p and cd63 using flow cytometer after stimulation by an haemostatic agent, thrombin-receptor activating peptid (trap) and after stimulation by cmv glycoprotein b. the inflammatory response was determined by the detection of immune mediators, rantes, cd62ps, pf4, cd40l and ccl3, using the elisa technique in the stimulated platelet supernatants. results: no platelet activation or molecules release were observed after stimulation by cmv glycoprotein b in both groups. rantes and cd62ps baseline levels are spontaneously higher in allogeneic hsc patients than in healthy volunteers. platelets from allogeneic hsct patients can be activated after haemostatic stimulation and release cd62ps and rantes. in this situation, platelets release more cd62ps, rantes and pf4 than platelets from healthy volunteers. conclusions: although no platelet activation was detected in response to cmv glycoprotein b stimulation, our study revealed a chronic platelet activation condition during the 2d and 3d months after allogeneic hsct with an haemostatic inducible hyper-responsiveness. this leads to the release of molecules with immune-modulating properties involved in the pathophysiology of gvhd. as we move further away from the hsct, that phenomenon seems to gradually weaken. clinical trial registry: nct03009708, fipalloc https://clinicaltrials.gov/ct2/show/nct03009708 disclosure: nothing to declare p305 efficient process and characteristics of umbilical cordderived mesenchymal stromal cells as a feasible source for anti-inflammatory therapy background: recently, umbilical cord (uc) has become attracted source of mesenchymal stromal cells (msc), because of abundant sources and ease of collection of fetal origin without invasive process for the donor and low immunogenicity with immunosuppressive ability and tissue repair potency. objectives of this study were to explorer the efficient and safe products and to evaluate the antiinflammatory potency of uc-mscs for the application of acute graft versus host disease (gvhd). methods: informed consent was obtained from mothers planning to have cesarean sections. uc tissue was cut and once cryopreserved. the safety assessment including infections and baby's health and development were done after 6 months of birth, and performed small-scale quality test of the frozen uc. then we initiated to isolate master uc-mscs from frozen-thawed uc by an improved explant method, which was passed for quality test. the master uc-mscs were cryopreserved once and thawed and expanded until p4. product cells were cryopreserved in original serum-free cryoprotectant dba-d solution. mixed lymphocyte reaction (mlr) assay co-cultured with uc-mscs was carried out using responder mononuclear cells (mnc) stained with cfse, and proliferation and cytokine secretion were analyzed by flowcytometry. results: uc-msc cultured showed significantly higher proliferation ability compared with those from bone marrow-derived mscs, and positive for cd105, cd73, cd90, and negative for cd45, hla-dr. cd80, and cd86 were negative even in the high concentration of ifn-γ, while bm-mscs became positive for hla-dr. pd-l2 was constitutively expressed in uc-msc, while pd-l1 was induced by the addition of ifn-γ. in mlr, responder t cell proliferation triggered by allogeneic dendritic cells was inhibited efficiently by 3rd party derived uc-mscs, in which was induced ido, pge2, hgf, and tgf-β analyzed by rt-pcr, and inhibited ifn-γ and tnf-α in the supernatant by cytokine beads array. uc-mscs migrated toward the tnf-α treated mnc and increased regulatory t cells incidence in peripheral mononuclear cells by the coculture. conclusions: these results demonstrated that cryopreserved uc are feasible and efficient source of mscs and frozen-thawed uc-mscs have high anti-inflammatory background: a new protocol is under development on the amicus separator that enables the device to perform ecp procedures. the amicus separator is used with a photoactivation device, disposable kit and 8-mop to provide ecp therapy in a closed system. the objective of this study was to evaluate the safety and performance of the investigational amicus ecp system in healthy human subjects. methods: an irb-approved written informed consent was obtained from 17 subjects (12 male, 5 female). the amicus ecp system processed either 500, 2000 or 4000 ml whole blood (n ≥ 5 per arm) using double-needle access and acd-a anticoagulation at a 12:1 wb:ac ratio. after mnc collection was completed, the subject was disconnected from the device. 8-mop (3.4 ml, 20 μg/ml) was injected directly into the collected mnc product and saline (approximately 200 ml total), which was photoactivated with 1-2 j/cm 2 uva light. post photoactivation, the amicus separator reinfused the treated mncs into a transfer pack. subject laboratory and safety parameters were evaluated; in vitro evaluations were performed on subject whole blood, collected mncs, treated mncs, and reinfused cells. lymphocyte and monocyte analysis were performed on samples purified using density gradient separation and cultured for up to 3 days post treatment. results: in 17 procedures, median (range) wb processed was 2016.0 (509 -4024) ml using 172.0 (51 -333) ml of acd-a. procedure time was 93.0 (66 -119) minutes, including photoactivation. no adverse events were reported. subjects' vital signs and hematology values were unremarkable and within expected values. the wbc count of the collected mncs was 13.50 (3.3 -30. 2) x10 3 /μl, comprised of 77.10 (47.9 -87.0) % lymphocytes, 15.50 (7.0 -36.8) % monocytes and 5.70 (2.9 -25.5) % granulocytes and platelet count was 94.0 (70 -169) background: transfusion of white blood cells (wbc) causes a number of transfusion reactions and complications, for example transfusion-associated graft versus host disease (tagvht), which still does not have effective treatment and is a fatal complication of transfusions. the only effective method of preventing tagvht is irradiation of blood components with ionizing radiation (x-ray or gamma radiation). but the use of ionizing radiation sources has a number of technical and material difficulties. the emergence of pathogen reduction technologies (prt) in blood components targeted by nucleic acids has opened the possibility of using these technologies as an alternative to irradiating of blood components. several prt demonstrated effective inactivation of wbc in platelet concentrates and blood plasma. so, determination of the influence of prt based on the combined effect of riboflavin (rf) and ultraviolet (uv) on the viability and proliferating potential of lymphocytes in whole blood is important. methods: samples of whole blood were obtained in 35 healthy volunteers. each sample was divided into three unequal parts: untreated control, gamma irradiated, and treated by rf and uv prt (mirasol, terumo bct inc.). mononuclear cells (mnc) were cfse stained, viability and proliferating activity were tested at intervals of 24 hours for 3 consecutive days by flow cytometry. statistical analysis was performed with xlstat 7.0. levels of significance were calculated by mann-whitney test, expressed as p-values (p< 0,05). results: the median viability of mnc after application of both methods of treatment was over 85,0% on day 0 and decreased to day 3 -median percentage of viable mnc were 84,0% (control group), 69,0% (after gamma irradiation) and 63,0% (rf/uv prt). the median of spontaneous proliferative activity on day 3 of untreated and gamma irradiated mnc did not differ (1,8% and 2,1% respectively, p< 0,05). phytohemaglutenin (pha) induced proliferation on day 3 in gamma-irradiated samples was significantly lower in comparison with control group (4,0% and 45,0% respectively, p< 0,01). in samples treated with rf/uv, spontaneous and stimulated proliferating cells was not detected. median percentage of proliferating mnc was less than 0,2%. the use of this prt on whole blood, as well as gamma irradiation, significantly reduces the viability of lymphocytes during storage for 3 days. conclusions: inactivation of wbc using rf/uv prt is a useful and very necessary bonus for a number of reasons. in one procedure two effects are achieved: infectious and immunological safety. the use of prt on whole blood gives the potential for obtaining pathogen-reduced and immunological safety components of blood, which reduces their material cost and staff loading. the use of rf/uv system does not have such complex security requirements and difficulties in servicing as the use of sources of ionizing radiation. the results demonstrate a promising potential for using this technology as an alternative to irradiation disclosure: nothing to declare p308 influence of patients´serum after allogeneic stem cell transplantation on t cell proliferation and treg function background: acute or chronic graft versus host disease (a/ cgvhd) is one of the major complications after allogeneic hematopoietic stem cell transplantation (ahsct). application of regulatory t cells (treg) as "immunosuppressive dli" to prevent or treat gvhd is investigated in clinical trials. here we ask the question, if there could be clinical conditions (e.g. cytokines or drug effects) limiting the efficacy of this approach. to face this problem we tested the influence of patients´serum on t cell proliferation and treg function. methods: lymphocytes from healthy donors were incubated with t cell medium (90% aim v + 10% serum + il2/okt3) containing serum from healthy donors or serum derived from patients after ahsct with or without gvhd (n=10). next we evaluated the suppressive function of treg by performing treg suppression assays, also comparing serum from patients suffering from gvhd versus serum obtained from healthy donors (n=8). proliferation of cfse stained t cells was measured after 5 days. to test the effect of immunosuppressive drugs on treg we performed treg suppression assays after incubation of treg with corticosteroids or tacrolimus or the combination of both drugs. results: serum of patients with acute or chronic gvhd had a negative effect on t cell proliferation. to avoid bias tests were performed with samples from patients without or only with low levels of immunosuppressive drugs. incubation with serum of patients without gvhd or with serum of healthy individuals showed no differences in t cell proliferation. treg from healthy donors showed a stronger antiproliferative capacity when incubated with serum derived from patients with gvhd. treg previously incubated with immunosuppressive drugs showed no decreased suppressive capacity. conclusions: components of serum from gvhd patients seem to have an antiproliferative effect on t lymphocytes itself. this fact might influence the clinical course of gvhd, but should not be a limiting factor for therapeutic application of treg dli. even the systemic treatment with immunosuppressive drugs e.g. corticosteroids or calcineurin-inhibitors should not diminish the treg application. in a next step we will analyze serum components responsible for this immunosuppressive effect with multi cytokine assays and proteomic analysis. the aim of our project is to develop new strategies to avoid gvhd and to optimize clinical settings for treg dli. disclosure background: hypercalcaemia can be very severe following stem cell transplant (sct) in some osteopetrosis patients. denosumab is a fully human monoclonal antibody that binds the cytokine rankl (receptor activator of nfκb ligand), an essential factor initiating bone turnover. rankl inhibition blocks osteoclast maturation, function and survival, thus reducing bone resorption. we describe the effective management of hypercalcaemia in a patient with rank mutation osteopetrosis who received a haploidentical sct. methods: our patient was diagnosed with osteopetrosis at 2 year of age with a defect in the tnfrsf11a gene which codes for rank and received a maternal haploidentical sct aged 4 years. the patients calcium levels were monitored regularly post sct. denosumab was administered for hypercalcaemia as per laboratory reports or clinical symptoms. the drug was diluted with water for injection to make 6mg/ml solution to facilitate subcutaneous administration. results: significant hypercalcaemia emerged on day +18 with a level of 3mmol/l and treated with hyper-hydration and diuretics. this was ineffective in reducing the hypercalcaemia; therefore denosumab was initiated on day +20 post-transplant. initial dosing was determined using the only available paediatric case report at 0.13 mg/kg. a repeated larger dose of 0.19mg/kg was given 4 days later due to an inadequate response (calcium decreased from 3.9mmol/l to 3.80mmol/l). the calcium decreased to 1.93mmol/l after this dose. four weeks later a third dose was required at 0.26mg/kg as the calcium level had increased to 4.1mmol/l. the dose was further increased to 0.32mg/kg for another four doses and then further increased to 0.65mg/kg for another 3 doses and repeated every 7 weeks. normalisation, but not excessive drop in calcium was achieved with these larger doses. over the 9 month follow up post-transplant there were three admissions lasting less than 24 hours for symptoms of hypercalcaemia. these were managed with denosumab administration and hyper-hydration. the remaining doses were given in an outpatient setting. conclusions: denosumab can be safely used as a first line agent in treating post stem cell transplant hypercalcemia in patients with osteopetrosis. a dose of 0.3mg/kg is required as an initial starting dose in order to control hypercalcemia. this is a new higher dose than previously suggested by the original report. denosumab can be effective even after dilution and safely given in children weighing less than 10kg. disclosure: nothing to declare methods: the clinical, laboratory and molecular aspects of this italian male patient who developed such a complication were collected and presented in order to discuss the origin, clinical outcome and management of this very rare post-transplant event. results: a 68-years-old man affected by a high-risk chromosomally abnormal, ph1-, mll-pro-b (egil b-i) all relapsed during maintenance treatment, nonresponsive to re-induction chemotherapy, in second complete remission (ii cr) after blinatumumab treatment received a female cb transplant. according to sorror's and ebmt scores he was considered a high-risk transplant. the patient and the cb unit were sex-mismatched, shared the same blood groups and were both cmv+/ebv+. he received a tbf conditioning regimen that was followed by the infusion of 0.54x10 5 /kg cd34+ cb cells. gvhd prophylaxis consisted of rabbit atg, cyclosporine a (csa) and mycophenolate mophetyl (mmf). neutrophil engraftment occurred on day +28, whereas platelets were never >20.000/μl. on day +67 a 2 cm bulged area became apparent on the left parietal region of the skull. an echotomography showed that the lesion adhered to the bone without infiltrating it and lacked blood vessels and suggested that it may be either a site of disease relapse or an area of infection. at the same time a bone marrow (bm) aspiration showed morphological cr confirmed by immune-phenotypic studies and x-y fish a complete chimera. since the patient was still febrile no biopsy was performed, but on day +94 the axial diameter of the lesion that on a ct scan showed the same appearance revealed by the previous echo-tomography increased to 4 cm. thus, the lesion was surgically removed and histological examination showed cd33+, cd14+/-, cd163+/-, cd45/lca+/-, cd21-, cd23-, cd35-, cd207-, and s100-neoplastic cells whose phenotype suggested a granulocytic sarcoma rather than a histiocytic sarcoma. immuno-chemistry confirmed this suggestion by showing a nuclear npm1 positivity. fish studies demonstrated that these neoplastic cells were of recipient's origin. a novel bm aspiration showed cr confirmed by immune-phenotypic studies and fish revealed a complete chimera. since the patient was still pancytopenic due to anti-cmv treatment, radiotherapy with 18gy in nine fractions were given and the lesion completely resolved. conclusions: a granulocytic sarcoma of recipient's origin occurring three months after a cb transplant is a very rare and unusual event. in order to explain such a complication we suggest that granulocytic sarcoma cells were dormant but already present at the time of pro-b all diagnosis and survived not only the initial all treatment but also the cb transplant conditioning regimen. we can't exclude that immune-suppressive treatments given early post-transplant might have promoted the outgrowth of these neoplastic cell population. disclosure: nothing to declare haemoglobinopathy and inborn errors of metabolism p311 abstract already published. addition of fludarabine on to anti-thymocyte globulin, busulfan and cyclophosphamide conditioning improves outcomes in low-risk matched-related bone marrow transplantation in children with severe thalassaemia flu-atg-bucy. atg dose was 4 mg/kg in all patients except patients with splenomegaly > 3 cm from costal margin and/or sex-mismatched/maternal donor in whom atg was increased to 7 mg/kg. all patients were younger than 15 years and had no hepatomegaly (liver ≤ 2 cm from costal margin) at bmt. results: actuarial overall survival (os) in the atg-bucy and flu-atg-bucy groups is 89% and 98%, thalassemia-free survival (tfs) 75% and 93%, gvhdfree and thalassaemia-free survival (gtfs) at a median follow up of 23.5 and 10.6 months was 72.6% and 93.3% months respectively, which is a significantly improved outcome by log-rank statistics (p=0.002) in the flu-atg-bucy group. there was no significant difference between the groups in pre-transplant characteristics and posttransplant complications except for the following: median cell dose more in 2 nd group with total nucleated cell dose of 8.7 vs 6.4 x 10 8 cell/kg with p< 0.0001; csa taper started later in the new protocol (184 day vs. 152 p=0.0005); median age at bmt (7.2 vs. 4.6 years, p=0.001); number of pre-bmt transfusions (p=0.01) and ferritin at bmt (2.214 vs. 1.599 ng/ml, p=0.002) were higher in the second group; day 30 and 60 chimerisms were also significantly higher in new protocol (p=0.02 and 0.03 respectively). there was a trend towards increased incidence of veno-occulsive disease (vod) and posterior reversible encephalopathy syndrome (pres) on the second group but this difference did not reach statistical significance. conclusions: adding fludarabine and targeted dose increase of atg in the standard bucy context seems to significantly improve outcomes of thalassaemia transplants without contributing to excessive gvhd or infectious complications. this protocol can be easily administered in low resource setting without major additional costs. clinical is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. however, there are still few reports on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) in patients with pnh compared to paroxysmal nocturnal hemoglobinuria-aplastic anemia (pnh-aa) syndrome. our study aimed to compare the outcomes of allo-hsct for pnh with pnh-aa syndrome. methods: the clinical data of 46 pnh patients received allo-hsct (pnh = 16, pnh-aa = 30) in our center from july 2007 to june 2018 were analyzed retrospectively to compare the outcomes of pnh group with pnh-aa group. the clinical data including 28 male patients and 18 female patients, the median age was 29 years (range 6-54). all patients had received various treatments before transplantation such as steroids, androgens, cyclosporine (csa), antithymocyte globulin, and growth factors. the median interval from pnh diagnosis to hsct was 6 months (range 3-240). the conditioning regimen was modified bu/cybased regimen in haploidentical donors and unrelated donors, csa, mycophenolate mofetil (mmf) and shortterm methotrexate (mtx) were administered for graftversus host disease (gvhd) prophylaxis. patients with matched sibling donors were treated with the flu/cybased regimen and csa were administered for gvhd prophylaxis. results: there were no differences of baseline between the 2 groups (p>0.05) except gender and haploidentical donors. the median values of absolute nucleated cell counts were 10.58 (3.83-13.83 ) ×10 8 /kg in the pnh group and 10.81 (3.96-33.40 ) ×10 8 /kg in the pnh-aa group (p = 0.668). the median doses of cd34 + cells infused were 5.00 (3.14-8.42)×10 6 /kg and 3.57 (1.97-6.17)×10 6 /kg (p = 0.002), respectively. all patients attained complete engraftment, no patient occurred graft failure. the median time for myeloid engraftment were 11 (range, 7-14) days in the pnh group and 12 (range, 10-26) days in the pnh-aa group (p = 0.003). the median time for platelet engraftment were 13 (range, 11-16) days and 18 (range, 12-75) days (p = 0.002), respectively. with a median follow-up of 36 (4-132) months in the pnh group and 26 (4-75) months in the pnh-aa group (p = 0.428). in pnh and pnh-aa groups the incidences of grade i-iv acute graft-versus-host disease (agvhd) were 12.50% and 33.30% (p = 0.121), grade ii-iv agvhd were 6.25% and 20.00% (p = 0.209); chronic gvhd were 12.50% and 35.67% (p = 0.274), moderatesevere chronic gvhd were 0.00% and 14.39% (p = 0.146). in haplo-hsct and msd groups the incidences of infection were 37.50% (6/16) and 33.33% (10/30) (p = 0.777). no patient occurred early death and relapse. 3-year estimated overall survival (os) of pnh and pnh-aa groups were 100.0% ± 0.0% and 85.7% ± 6.6% (p = 0.141), gvhd-free and failure-free survival (gffs) were 100.0% ± 0.0%、78.7% ± 7.7% (p = 0.067). conclusions: the preliminary results indicated that allo-hsct is a feasible choice for pnh with favorable outcomes, time for myeloid and platelet engraftment in pnh group were faster than pnh-aa group. there were no differences in os and gffs between pnh group and pnh-aa group. disclosure: no disclosure pattern of calcineurin inhibitor-associated neurotoxicity in sickle cell disease patients receiving a stem cell transplantation background: allogeneic hsct with a msd represents currently the only curative option for sickle cell disease (scd), limited by a donor availability < 20%. neurotoxicity (nt) contributes significantly to hsct-associated morbidity and mortality. calcineurin-inhibitor (cni) associated nt ranges from 4.2%-28.8% (severe nt 4%-11%). the elevated incidence of nt in scd (around 30%) might be triggered by the systemic vasculopathy of scd, with the brain being the primary target. although both cyclosporine a (csa) and tacrolimus (fk506) have a proinflammatory effect, it is more pronounced in csa. infusion modalities also might impact (10.3% after bolus injections versus 3.3% after continuous infusion). methods: in a pilot study, we compared t-cell depleted haploidentical hsct (t-haplo hsct) with msd hsct in patients (pts) with advanced stage scd, using almost identical conditioning regimens. 32 pts (3-31 years; yrs) with homozygous scd or hbs 0/+ ß-thal were treated between 2012 and 2018. nine pts received a msd bone marrow graft, 23 pts received 24 t-haplo-hsct (1 second t-haplo due to graft rejection). immunosuppression consisted of either csa (6 msd, 16 t-haplo) or fk506 (3 msd, , in combination with mycophenolate mofetil (mmf). fk506 was administered as a 20-hours continuous infusion, csa as 4-hours bolus injections; both target level adjusted (csa: 100-120 ng/ml; fk506: 5-8 ng/ ml). duration of immunosuppression was >6 months in thaplo-sct and < 6 months in msd, depending on chimerism. results: cni-related nt was observed in 36.4%, severe nt (pres, visual disturbance, aphasia) in 21.2%. nt was more prevalent in msd (n=5, 55.5%) than in t-haplo (n=7, 29.2%). the incidence of nt was identical under csa (8/22; 36.4%) and fk506 (4/11; 36.4%), however the majority of severe nt (all pres) occurred with csa. complete recovery of nt was achieved in all pts either spontaneously or after switching to fk506/everolimus or withdrawal of fk506. moreover, 66.6% of pts with nt were >18 yrs, and 91.6% >12 yrs, suggesting an increased risk with age. only 37.5% of pts with pre-existing cerebrovascular disease experienced post-hsct nt. of note, 57.1% of pts with severe nt also developed mild acute gvhd. the overall (os) and disease-free survival (dfs) with a median follow-up of 17 months in t-haplo-hsct and 22 months in msd hsct was 91% vs. 100%, respectively. conclusions: our data confirm an elevated nt risk in scd pts following allo-hsct. importantly, the incidence of nt seems to be related to age (91% of pts with nt were >10 yrs), donor source (msd 55.5% vs. t-haplo 29.2%) and type of cni inhibitor where almost all severe nt (71.4%, particularly all pres) was observed under csa. continuous infusion of fk506 vs. bolus injections of csa might have levelled concentration peaks. the nt observed with csa could be the consequence of predominantly csarelated vascular toxicity inflicting pre-damaged vessels in scd. the mechanism of action could be related to other systemic endotheliopathies such as vod, tam and agvhd, which was observed in 57.1% of pts with severe nt, compared to an overall agvhd rate of 30%. disclosure: nothing to declare background: matched-related bone marrow transplantation (bmt) may cure over 80% of low-riskchildren with severe thalassemia (st) defined as a thalassemia syndrome with inability to keep a spontaneous hemoglobin > 7 g/dl. it is well known that patient status at the time of transplant is critical in predicting transplant outcome. liver size > 2 cm is an established adverse prognostic factor in terms of transplant-related mortality and, in our own experience,a spleen size > 3 cm from costal marginis associated with increase rejection rates (blood 2017 vol. 130 no. suppl 1 1944) . optimising liver and spleen size prior to transplant is likely to improve transplant outcomes. methods: we retrospectively reviewed the effectiveness of our strategy to reduce liver and spleen size pre-transplant using hydroxyurea, super-transfusion and intensive iron chelation. we considered liver size < 2 cm and spleen size less than 3 cm below costal margins as good risk features. liver biopsies were not performed thus pesaro risk classification could not be assigned. all transplant candidates were started on hydroxyurea for a minimum of 3 months and pre-transfusion haemoglobin was maintained > 7 gm/dl while on hydroxyurea. if the child had hepatospenomegaly at enrollmentand no improvement in liver and spleen size after an adequate trial of hydroxyurea (minimum of 3 months of treatment achieving maximum dose of 50 mg/kg day or tolerable haematological toxicity, i.e. neutrophil count between 1000 and 1500/μl and/or platelet count between 100.000 and 150.000/μl) patients were given a trial of supertransfusion maintaining haemoglobin above 12 g/dl) for a minimum of 3 months prior to declaring the patient as having failed downstaging. results: out of 119 transplants across 3 collaborating centers in india, 85 patients had no hepatosplenomegaly at enrolment and hence were not actively downstaged. twelve patients were excluded due to inadequate information on their records. all of the remaining 22 patients with enlarged liver and/or spleen were downstaged to low-risk features. all patients received adequate hydroxyurea trial among which seven (32%) patients required super transfusion in addition to maximal hydroxyurea. out of the 22 patients 18 (82%) were successfully down-staged with the above strategy and proceeded to transplant as low-risk patients. among the remaining 4 (25%) patients 3 had liver > 2 cm and one had a spleen > 3 cm only. there was significant improvement in liver and spleen size from the time of enrollment to transplant (p value 0.0004 and 0.0002 respectively by wilcoxon test for paired samples -two tailed) with median duration of downstaging of 9 months (range 2-27 months). there was no significant difference in overall survival (os) and disease-free survival (dfs) by log rank test between the downstaged group and those who did not have hepatosplenomegaly at enrollment (p value 0.59 0.64 respectively). conclusions: in the majority of children with thalassaemia and high transplant risk features liver and spleen size can be reduced pre-transplant using hydroxyurea and supertransfusions thereby decreasing transplant risk. disclosure: nothing to declare p316 abstract already published. abstract withdrawn. longitudinal analysis of the effect of hematopoietic cell transplantation on ocular disease in children with mucopolysaccharidosis i shows ongoing disease progression background: corneal clouding is seen in nearly all patients with mucopolysaccharidosis-1 (mps-1) causing visual impairment. hematopoietic cell transplantation (hct) is able to stabilize disease in many organs including the brain. however, residual disease in peripheral tissues is often described. therefore, the aim of this study was to determine the long-term effect of hct on ocular disease in mps-1 patients. methods: corneal clouding (grade 0-4) and visual acuity (decimal scale) were prospectively collected from all consecutive mps-1 patients treated with hct between 2003 and 2018 at the umc utrecht. the primary outcomes of interest, the effect of time on corneal clouding and visual acuity, were analyzed using a linear mixed model. the correlation between corneal clouding and visual acuity was analyzed with pearson's rho. other parameters studied were clinical phenotype, age at time of transplantation and hematological enzyme level after transplantation. other outcomes of interest analyzed included intra-ocular pressure, refraction, and macula and lens abnormalities. [[p318 image] 1. results: 24 successfully engrafted mps-1 patients were included (92% with >95% chimerism and normal enzyme levels after hct). corneal clouding stabilized during the first years after hct, but increased rapidly beyond three years (figure 1). other predictors for increased corneal clouding were age at time of transplantation (0.74, 95%ci 0.34:1.15; p=0.0026) and clinical phenotype (-1.02, 95%ci -0.18:-1.86; p=0.0335). visual acuity also worsened significantly over time (-0.03, 95%ci -0.06:-0.007; p=0.01). corneal clouding was strongly negatively correlated with visual acuity (ρ -0.60, p = 7.12e-11). conclusions: after initial stabilization, ongoing ocular disease is seen in mps-1 patients despite successful hct. this hallmarks the shortcomings of current standard therapies. new therapies that overcome the weak spots of current therapies are necessary to improve the late outcomes of these patients. clinical trial registry: n.a. disclosure: b.t.a.v.d.b. was supported by a research grant from the sylvia toth charity foundation, the hague, the netherlands, while working on this study. the sponsors of this study are public or nonprofit organizations that support science in general. they had no role in gathering, analyzing, or interpreting the data. all authors would like to thank all parents and patients for participating in this study. all authors state they have no competitive (financial) interests in this study. background: paroxysmal nocturnal hemoglobinuria (pnh) is an acquired clonal disorder of the hemopoietic stem cells for which the only curative treatment is allogeneic hematopoietic stem cell transplantation. haploidentical donor hematopoietic stem cell transplantation (haplo-hsct) is now increasingly applied as a curative therapy for patients with hematologic diseases. however, there are still few reports on the use of haplo-hsct for the treatment of pnh. our study aimed to compare the outcomes of haplo-hsct with matched-sibling donor transplantation (msd-hsct) for pnh. methods: the clinical data of 40 pnh patients received hsct (haplo-hsct = 25, msd-hsct = 15) in our center from july 2007 to may 2018 were analyzed retrospectively to compare the outcomes of haplo-hsct group with msd-hsct group. the clinical data including 23 male patients and 17 female patients, 13 classical pnh and 27 pnh-aa syndrome, the median age was 29 years (range 6-54). all patients had received various treatments before transplantation such as steroids, androgens, cyclosporine (csa), antithymocyte globulin, and growth factors. the median interval from pnh diagnosis to sct was 6 months (range 3-240). the conditioning regimen was modified bucybased regimen in haplo-hsct group, csa, mycophenolate mofetil (mmf) and short-term methotrexate (mtx) were administered for graft-versus host disease (gvhd) prophylaxis. patients with msd-hsct were treated with the flucy-based regimen and csa were administered for gvhd prophylaxis. results: there were no differences of gender, age, patients of pnh-aa and median time from diagnosis to transplantation between the 2 groups (på 0.05). the median values of absolute nucleated cell counts were 10.74 (4.80-22.86) ×10 8 /kg in the haplo-hsct group and 12.19 (5.14-17.25) ×10 8 /kg in the msd-hsct group (p = 0.866). the median doses of cd34 + cells infused were 3.57 (0.68-7.80) ×10 6 /kg and 4.00 (3.02-8.42) ×10 6 /kg (p = 0.151), respectively. all patients attained complete engraftment, no patient occurred graft failure. the median time for myeloid engraftment were 12 (range, 9-26) days in the haplo-hsct group and 11 (range, 7-15) days in the msd-hsct group (p = 0.065). the median time for platelet engraftment were 19 (range, 11-75) days and 13 (range, 11-25) days (p = 0.027), respectively. with a median followup of 26 (4-65) months in the haplo-hsct group and 36 (4-132) months in the msd-hsct group (p = 0.294). in haplo-hsct and msd-hsct groups the incidences of grade i-iv acute graft-versus-host disease (agvhd) were 32.00% and 20.00% (p = 0.343), grade ii-iv agvhd were 16.00%、13.33% (p = 0.759). chronic gvhd were 30.69% and 24.62% (p = 0.418), moderate-severe chronic gvhd were 12.73% and 7.14% (p = 0.522). in haplo-hsct and msd groups the incidences of infection were 32.00% (8/25) and 26.67% (4/15) (p = 1.000). no patient occurred early death and relapse. 3-year estimated overall survival (os) of haplo-hsct and msd-hsct groups were 86.5% ± 7.3% and 93.3% ± 6.4% (p = 0.520), gvhd-free and failure-free survival (gffs) were 78.3% ± 8.6% and 92.9% ± 6.9% (p = 0.250). conclusions: the preliminary results indicated that haplo-hsct is a feasible choice for pnh with favorable outcomes, haplo-hsct and msd-hsct had similar therapeutic efficacy. disclosure: no disclosure p320 pres in bmt for thalassemia major in india: lower incidence and limited impact background: posterior reversible encephalopathy syndrome (pres) is a relatively common complication seen after blood or marrow transplantation (bmt) for hemoglobinopathies with a reported frequency of 10-19%. pres has also been associated with poorer survival rates. severe hemoglobinopathies are one of the most frequent indications for bmt in the developing world, particularly in india. given the risk of rejection in multiply transfused patients and the need to minimize gvhd risk, immunosuppression post-bmt for these non-malignant conditions can be particularly intense and prolonged. we sought to measure the incidence and impact of pres in developing countries. methods: we analysed 194 successive transplants for thalassemia using protocol 1 (atg-bucy+csa/mmf or csa/mtx) maintaining cyclosporine a (csa) blood levels 100-150 ng/ml for 74 patients and protocol 2 (flu-atg-bucy+csa/mtx) maintaining higher csa levels post, i.e. 150-250 ng/ml for 120 patients from fully matched donors with g-csf-primed bone marrow. for 3 patients this was the second transplant from a different matched related donor. pres was confirmed with brain ct/mri for all patients. results: all recipients who had pres had sibling donors, 5 males and 2 females. age median 7.4 (iqr 5.6-8 years). the frequency of pres was 3.6%; disease free survival for patients who had pres was 100%. pres resolved completely in all. csa was switched to mmf in 5 patients who had received mtx and were on csa only at the time of pres occurrence, while csa was stopped but mmf continued in 1 patients taking csa/mmf combination and csa was continued for 1 patient. three patients with pres had grade 2 acute gvhd, 1 had grade 1 gvhd and none developed chronic gvhd. csa levels at the time of pres were a median of 133 ng/ml (iqr: 89 to194) with 1 patient having 419 ng/ml. three patients had pres while they were thrombocytopenic. hypertension stage 2 was observed in four patients, stage 1 in one patient, one patient was not hypertensive and in one patient blood pressure values were not available. two patients were on methylprednisolone 1 and 1.3 mg/kg/day and one was on dexamethasone 10 mg/m2/day. one patient was started on csa again after the pres episode and within 2 weeks had another one while on csa (level 30 ng(ml), methylprednisolone 1.5 mg/kg/day and ruxolitinib for gvhd. protocol 2 had statistically significant improvement in disease free survival from 67% to 91% (p< 0.001) with probability of occurrence of pres increasing from 1.4% to 5.0% (p = 0.17, see figure 1 ), yet had a benign course in all patients. conclusions: not stopping immunosuppression may have been the key factor which could explain why we have better outcomes with pres than what is reported. intensifying immunosuppression pre-bmt did lead to more pres, albeit not significantly, and yet it was quite manageable. even with addition of fludarabine our pres incidence is lower than previously reported. [[p320 image] 1. background: sickle-cell diseases (scd) are a group of genetic hemoglobin disorders marked by brain vasculopathy. allogeneic hematopoietic stem cell transplantation (hsct) is a curative option able to stop vascular disease progression. diffusion-tensor imaging (dti) is a magnetic resonance imaging (mri) technique sensitive to the brownian motion of water molecules and cellular environment. this microscopic quantitative technique is able to detect white matter (wm) alterations before a conventional mri. the aim of this study was to use dti to evaluate axonal damage and structural connectivity in the brain of patients with scd submitted to hla-identical sibling allogeneic hsct. methods: sixteen scd patients with no extensive vasculopathy detected by conventional mri (11 male, age range: 9 -33 years) and 17 age-matched healthy controls (10 male, age range: 6 -31 years) participated in this prospective study. mri acquisitions were performed in a 3t scanner two times for patients (before and 1-5 years after hsct) and at a single moment for controls. from dti acquisitions, fractional anisotropy (fa), mean (md), radial (rd) and axial diffusibility (ad) were calculated in the wm of the whole brain. structural connectivity was also analyzed, based on graph theory, obtaining efficiency, length path and clustering coefficients of the brain network. an anova test was applied to analyze fa differences among controls and patients, before and after hsct. a paired two-tailed t-test was used to determine statistical significance of changes in the fa, diffusivity mean values and network parameters before and after hsct. results: mean fa was lower in patients before hsct than controls (p = 0,038) and increased after hsct being not statistically different when compared to controls (controls = 0,3504; patients before hsct = 0,3328; patients after hsct = 0,3422; post hoc dunnett's test -error 0,03; anova test). when patients were compared before and after hsct, md and rd decrease after hsct (p = 0,038 and 0,047, respectively). on the other hand, fa increased (p = 0,044). after hsct, efficiency was higher (p = 0,023) and path length index was lower (p=0,027) than at study entry (table 1) . conclusions: this study indicates that, before hsct, patients with scd present axonal damage not detectable by conventional mri, when compared to healthy controls. we also suggest that hsct is able to promote axonal recovery and reorganization. partial diffusivity recovery could be associate to a still unidentified mechanism of myelin regeneration. in the future, longer follow up and comparisons with other forms of treatment are required. background: bmt is a well-established treatment modality for haemoglobinopathies, limited by the availability of related donors. unrelated transplantation has historically shown variable outcomes driven by gvhd and toxicity, and usually restricted to 10/10 matches, but the impact of reduced toxicity conditioning regimens is yet to be known. methods: from 2011 to 2018 twenty-five consecutive unrelated bone marrow transplants were conditioned with fludarabine 160 mg/m 2 , treosulfan 42 g/m 2 , thiotepa 10 mg/ kg and atg (thymoglobulin) 11.25 mg/kg if the source of stem cells was marrow (n = 21) or ptcy if pbsc (n = 4). endogenous haemopoiesis was suppressed pretransplantation for a minimum of 8 weeks. gvhd prophylaxis was provided with ciclosporin/sirolimus and mmf. thirteen patients were transplanted for b thalassaemia major, one of a thalassaemia major and 11 sickle cell disease. the median age was 8 years (2 -19). ten patients were 10/10 matched (7 thalassemia and 3 sickle) and 15 patients had a 9/10 match (7 thalassaemia and 8 sickle). the median cell dose was 3.88 x 10 8 tnc/kg (range 1.38 -13.3) and 5.22 x 10 6 cd34+/kg (range 1.10 -27.41). the median survival was 13.4 months (0.7 -68.3). patients with thalassaemia were pesaro class i or ii (pesaro class iii patients were intensively chelated pretransplantation to return to class i or ii). patients with sickle cell disease were transplanted for stroke or recurrent vaso-oclusive crises and/or acute chest syndrome not responding to hydroxycarbamide. results: all patients engrafted and achieved evidence of donor haemopoiesis on day +28 and achieved transfusionindependence and donor haematological values, but subsequently one 9/10 patient with thalassaemia suffered secondary graft failure on day +75 after macrophage activation syndrome. median neutrophil engraftment was 13 days (range 9 to 19) and 12 days (9 -22) for 10/10 and 9/ 10 patients respectively. patient with sickle cell disease had the platelet count maintained >50 x 10 9 /l at all times. the median platelet engraftment >50 x 10 9 /l was 31 days (range 21 to 53) and 40 days (range 15 to 86) 10/10 and 9/10 patients respectively. there were three deaths, all in the 9/10 matched group: two with thalassaemia (day +257 due to idiopathic pneumonia syndrome and day +102 due to mas) and one with scd (day +43 due to ips). there were different trends of complications seen by degree of matching that did not segregate otherwise by disease. conclusions: in conclusion, unrelated bmt for haemoglobinopathies with reduced toxicity regimens is feasible. whilst gvhd caused significant morbidity during the transplant period, other alloreactive/endothelial complications (vod, macrophage activation syndrome, idiopathic pneumonia syndrome) were only seen in the 9/10 transplants. disease-free survival, dependent on transplantrelated mortality, and lack of long-term toxicity, including chronic gvhd, are determined by the degree of matching. 10/10 matched transplants have excellent long-term outcomes with no chronic gvhd >18 months and can be considered for patients without a related donor; whereas 9/ 10 transplant have significant toxicity and mortality, warranting a haploidentical approach. disclosure: no conflict. long-term safety and efficacy of lentiglobin gene therapy in patients with transfusion-dependent β-thalassemia following completion of the phase 1/2 northstar study patients with transfusion-dependent β-thalassemia (tdt) may benefit from gene therapy involving β-globin gene addition to hematopoietic stem cells (hscs) enabling production of functional hemoglobin (hb). lentiglobin gene therapy contains autologous cd34+ hscs transduced ex vivo with the bb305 lentiviral vector encoding β-globin with a t87q substitution under transcriptional control of the encoding β-globin locus control region. the safety and efficacy of lentiglobin was evaluated in adults and adolescents with tdt in the 2-year phase 1/2 northstar study (hgb-204; nct01745120). methods: patients with tdt (≥ 100 ml/kg/year of red blood cells [rbcs] or ≥ 8 rbc transfusions/year) received g-csf and plerixafor for hsc mobilization. to generate drug product (dp), cd34+ hscs were transduced with the bb305 lentiviral vector. patients underwent single-agent, myeloablative busulfan conditioning, were infused with the dp, and were followed for safety and efficacy. results: eighteen patients have been treated in the completed northstar study. as of 14 september 2018, patients had a median follow-up of 38.9 (min -max: 29.3 -48.1) months. the median age at consent was 20 (min -max: 12 -35) years including 15 patients ≥ 18 years old. patients received a median cell dose of 8.1 (min -max: 5.2 -18.1) cd34+ cells x10 6 /kg with a median dp vector copy number (vcn) of 0.7 (min -max: 0.3 -1.5) vector copies/ diploid genome. the median liver iron content (lic) at baseline was 5.7 (min -max: 0.4 -26.4) mg fe/g dw. outcomes by age and baseline iron status will be presented. the median time to neutrophil and platelet engraftment was 18.5 (min -max: 14 -30) and 39.5 (min -max: 19 -191) days, respectively. four patients had platelet engraftment ≥ day 60 and four patients had platelet counts of ≤ 100x10 9 /l at month 12. none of these patients had ≥ grade 3 bleeding events post-lentiglobin infusion. transfusion independence (ti, defined as weighted average hb ≥ 9 g/dl without rbc transfusions for ≥ 12 months) was achieved in 8/10 patients with non-β 0 /β 0 genotypes and 3/8 patients with β 0 /β 0 genotypes. in patients who achieved ti, total hb at last visit was 9.1 -14.1 g/dl. lic increased from baseline in patients who achieved ti by a median of 55.6% and 12.5% at month 12 and 24 then decreased from baseline by a median of 9.2% and 44.4% at month 36 and 48, respectively. non-hematologic grade ≥ 3 adverse events post-infusion in ≥ 3 patients included stomatitis, febrile neutropenia, pharyngeal inflammation, and irregular menstruation. there was no transplant-related mortality, vector-mediated replication competent lentivirus, or clonal dominance. two patients experienced grade 3 serious veno-occlusive liver disease (table 1) . events resolved following treatment with defibrotide and were attributed to myeloablative conditioning. conclusions: in the northstar study, 80% of patients with tdt and non-β 0 /β 0 genotypes and 38% of patients with β 0 / β 0 genotypes achieved transfusion independence. the safety profile of lentiglobin remains consistent with myeloablative busulfan conditioning. longer time to platelet engraftment was observed in some patients, but no graft failure was reported. clinical background: sickle cell disease (scd) is an inherited hemoglobin disorder associated with high morbidity and mortality. currently, allogeneic hematopoietic stem cell transplantation (hsct) is the only curative therapy for scd. transplant outcomes with thiotepa, treosulfan and fludarabine (ttf) preparative regimen are encouraging but this regimen has not been directly compared to other preparative regimens in scd. we therefore planned to compare the event free probability for death, rejection and high grade acute graft versus host disease (agvhd) between ttf and busulfan and fludarabine (bf) regimens. methods: in this retrospectively cohort study, we included all patients with scd who received allogeneic hsct at our center or who were transplanted in other centers and referred to ours for follow up before day 100. patients were transplanted between july 2007 and december 2017. we used kaplan-meier curve to estimate the event free probability for death, rejection and high grade agvhd (grades 3-4). cox regression was used to assess the impact of the preparative regimen on these outcomes. results: a total of 61 patients were included with a median age of 20 years (interquartile range [iqr]: 14-26) and a median hemoglobin of 10 g/dl (iqr: 9-10). sixtytwo percent were males. the proportion of patients who had splenectomy, stroke and acute chest syndrome was 34%, 23% and 57% respectively. all patients received peripherally collected hematopoietic stem cells from a matched sibling donor with a median stem cell dose of 6 x 10 6 /kg (iqr: 5. 1-8.8 ). most patients, 95%, received cyclosporine or tacrolimus based agvhd prophylaxis. most patients received ttf (41%) or bf (53%) preparative regimens. all patients in the bf group received atg. the median follow-up time was 44 months (range: 2-127). four patients died during the follow-up period with an os of 93% (95% confidence interval [ci]: 87%-100%) at 5 years. the os was not different (hr 1.3, p = 0.82) between the ttf (91%) and the bf (94%) regimens. the probability of high grade agvhd free survival at day 100 was 91% (95% ci: 84-99) for all patients. this probability was 85% in the ttf group and 93% in the bf group and the difference was not statistically significant (hr 2.2, p = 0.39). the rejection free survival at 12 months was 93% (95% ci: 87-100) for all patients. no patients in the ttf group rejected while the rejection free survival at 12 months for the bf group was 90%. this was not statistically significant (p = 0.07). conclusions: in patients with scd undergoing allogeneic hsct from a matched sibling donor, the ttf preparative regimen is not associated with improved os, rejection free or high grade free agvhd survival when compared to the bf preparative regimen. larger studies are needed to confirm these findings. disclosure: nothing to declare. novel strategy for haploidentical hematopoietic stem cell transplant in sickle cell disease methods: 9 consecutive patients suffering from scd who underwent hhsct between jan 2018 till date were enrolled in the study. all 9 underwent autologous backup (target dose>5x10 6 /kg) followed by pre-transplant immune suppression (ptis) 2 cycles at 3 weekly intervals using fludarabine @30mg/m2/day(d1-d5) + cyclophosphami-de@1000mg/m2/day(d1) + dexamethasone@20mg/m2/ day(d1-d5) along with hypertransfusion (target hb 11-13gm/dl), hydroxyurea (20mg/kg/day) and azathioprine (2mg/kg/day) from day -60. the graft was mobilized using gcsf@10mcg/kg/day(d1-d5) + plerixafor@0.24mg/kg s/ c on d5 6-8 hours before the pbsch. conditioning included thiotepa 10mg/kg in two divided doses (d-7), fludarabine 30mg/m2 (d-6 to d-2), cyclophosphamide 14.5 mg/kg (d-5, d-4), tbi 2gy with thymic shielding (d-1), ratg (genzyme thymoglobulin 1.5 mg/kg (d-9 to d-7). gvhd prophylaxis included ptcy 50 mg/kg/day on d3 and 4, sirolimus (target levels 10-15ng/ml) (till 9-12 months post hsct) and mmf (till d35) starting from d5. results: the median age of patient's was 7 years (range 3-22 years). before transplantation all patients had repeated episodes of one or other complication warranting a transplant, non-responsive to hydroxyurea. six had maternal donors, 2 paternal and 1 sibling. median age of the donor was 41 years (range 19-51 years). all were dsa negative with a cutoff mfi of >2000 iu. all patients received 10x10 6 /kg cd34 cells irrespective of harvested dose which ranged from (10.13-34.82 x10 6 /kg). median cd3 dose was 16.59 x10 7 /kg (range 10.44-41.9 x10 7 /kg). all patients engrafted with median time to neutrophil engraftment 13 days (range 12-15 days) and median time to platelet engraftment 13 days (range 11-16 days). median duration of hospital stay was 30 days (range 24-35 days). one patient had cytokine release syndrome needing tocilizumab. five had engraftment syndrome treated with short course of steroids. two had cmv reactivation needing treatment with ganciclovir/valganciclovir. acute gvhd grade ii was seen in one patient. till date of analysis none had features compatible with chronic gvhd. of the 9 patients, 8 are alive without sickle cell disease with lansky/ karnofsky scores of 100. at median follow up of 164 days (range 61-271) the probabilities of survival, sca-free survival, and transplant-related mortality after transplant were 88.9%, 88.9%, and 11.1%, respectively. one patient died due to mdr klebsiella sepsis after being discharged initially while he was receiving iv ganciclovir on day care basis. he had full donor chimerism. none of the patient had primary or secondary graft failure. conclusions: pre-transplant immune suppression and upfront use of plerixafor for graft mobilization decreases the risk of graft failure and graft versus host disease leading to overall better survival in hhsct for sickle cell disease. disclosure: none. combined haematopoietic stem cell transplant and enzyme replacement therapy in wolman disease: outcomes and challenges jane kinsella 1 , denise bonney 1 , helen campbell 1 , robert wynn 1 , simon jones 1 background: infantile lysosomal acid lipase deficiencymore commonly known as wolman disease -is an autosomal recessive lysosomal storage disease, characterised by storage of cholesterol esters in the liver, spleen and gastrointestinal tract. these children present under the age of 6 months and traditionally had a poor prognosis, with almost all being dead by the age of 12 months. bone marrow transplant has been used to correct disease manifestations, but limited by high procedure-related mortality with the significant co-morbidities. the survival has changed over the past few years due to pharmacological enzyme replacement therapy but still presents challenges for these patients and their clinicians. in these children haematopoietic stem cell transplant we have offered bmt with enzyme replacement therapy, in certain specific circumstances. methods: four children with wolman disease being treated with enzyme replacement therapy, limited by alloantibody, or poor venous access, received treosulfan-based, myeloablative conditioning with serotherapy followed by a matched haematopoietic stem cell transplant: two family donors, one sibling donor and one unrelated donor. results: three of the four children survived transplant. they have continued to receive enzyme replacement therapy but at reduced dose and frequency with improved tolerability. they have continues to grow and develop. growth and gastrointestinal histology is improved for children having received transplant compared to those receiving enzyme replacement alone. monitoring of peripheral blood chimerism has shown a disease-associated engraftment defect, with mixed chimerism in the 3 surviving patient. conclusions: haemopoietic stem cell transplant is a suitable treatment option in children with wolman disease in whom receiving enzyme replacement therapy is not possible because of venous access, sensitisation or cost reasons. it improves their tolerability of the enzyme treatment and allows for a reduction in enzyme dose and frequency. however, the results of engraftment are not as good as expected for a transplant with myeloablative conditioning and a matched donor. an engraftment defect has been observed in lysosomal acid lipase deficient animal models. a further understanding of this poor engraftment in children with wolman disease is required as to determine whether the risks of transplant is beneficial in these patients and for the consideration of future treatment options including gene therapy. background: thalassemia major is the most common transfusion dependent hemolytic anemia in the world. the absent or reduced production of the β-chain of hemoglobin causes severe ineffective erythropoiesis, massive erythroid hyperplasia in the bone marrow and extramedullary hematopoesis occurs. patients require regular transfusion therapy lifelong. currently, the only proven curative treatment of thalassemia is allogeneic stem cell transplantation (sct). methods: we evaluated the immune reconstitution results of 23 patients at 1 year after hematopoetic stem cell transplantation at our pediatric bone marrow transplantation center between january 2015 and december 2018. all patients were not receiving any immunosuppressive treatment at least for 3 months and they have normal lymphocyte counts, immunoglobulin levels and transfusion independent. lymphocyte subtypes and chimerism percentages and the relationship with the donor type were evaluated at 1 year of transplantation. results: ages of transplantation was ranged between 1-17 years (median: 5 years). seven (30%) of them was male. matched unrelated donor type was chosen in 7 patients while others (16 patients) were transplanted from family matched donor (matched sibling: 9 patients, matched family: 7 patients). all patients received myeloablative conditioning regimen containing busulfan/treosulfan, cyclophosphamide, thiotepa and fludarabine. follow up time was between 12-47 months (mean: 24 ± 10 months). in 9 patients, whole bone marrow product was used while peripheral stem cell harvest in remaining patients. cd3 levels were found low in only 4 patients, in normal patients mean was 59% ± 14%. cd4 levels were severely low in 18 patients while cd8 in only 1 patient. cd8 levels were increased in total 13 patients in as compensatory. cd4/cd8 ratios were very low in all patients (range: 0.2-0.7). b cells (cd19+) were low in 3 patients while immunoglobulin levels were normal. chimerism values between 55-99% (mean: 93 ± 11%). donor and product types did not differ in cd3+ lymphocyte reconstitution at 1 year (p=0.15, p=042 respectively). all patients were alive and well at 1 year after transplantation. conclusions: after 1 year of transplantation, although patients are in well condition regarding to infection frequency and transfussion dependency, it was seen that their lymphocyte subtypes reconstitution could not be achieved enough as in normal children. we can conclude that low cd4+ cell levels were an expected finding in almost all patients. so, these patients may have a tendency to suffer serious bacterial and viral infections, and close follow up be required in terms of infections as long as cd4 levels continue to be low. immunoglobulin replacement therapy did not required even in patients with low b cell levels. disclosure: nothing to declare p328 phase 2 international, multicentre trial to assess haploidentical aß t-cell depleted stem cell transplantation in patients with sickle cell disease with no available sibling donor background: sickle cell disease (scd) is an inherited disorder with an estimate of 300,000 affected newborns per year worldwide. allogeneic hematopoietic stem cell transplantation (hsct) with a matched sibling donor (msd) is currently the curative standard of care for scd patients (pts). however, msd availability is < 20%. a t-cell depleted haploidentical hsct (t-haplo-hsct) from a relative, mostly a parent, expands the donor availability while exhibiting low gvhd rates and thus could offer cure to the remaining 80% of scd patients. in a pilot study, comparing t-haplo-hsct with msd hsct in advanced stage scd, using almost identical transplant regimens for both. the overall (os) and disease-free survival (dfs) was 90% vs. 100%, respectively. methods: these results led to the design of a clinical trial to assess tcd-haplo-hsct prospectively which aims to demonstrate that a hsct from a haploidentical relative is not inferior to a msd hsct with regard to major outcome parameter. this phase 2, prospective, stratified, open-label study is targeting enrollment of 212 patients aged 1-35 years with homozygous hbs disease or heterozygous hbsc or hbs 0/+ ß-thal suffering from severe or moderate scd related complications. inclusion criteria are clinically significant scd related complications such as stroke, silent crisis, pathological angio-mri, transcranial doppler (tcd) velocity >200 cm/s, 2 or more episodes of acute chest syndrome (acs) in a lifetime, chronic transfusion dependency, transfusion-refractory allo-immunization and others. pts fulfilling inclusion criteria will be stratified according to donor availability. pts with a msd will receive a bone marrow graft, pts requiring an alternative donor will be transplanted with an aß/cd19 depleted graft from a haploidentical family donor. the conditioning regimen for both groups will be identical with the exception that antithymoglobulin (atg-neovii ® ) is given upfront in thaplo-hsct versus day -3 to -1 in msd. chemotherapy consists of thiotepa, fludarabine and treosulfan. posttransplant immunosuppression will consist of mofetil mycophenolate and tacrolimus for a duration >6 months in t-haplo-hsct and < 6 months in msd, depending on chimerism. (eudract number: 2018-002652-33) results: primary efficacy endpoint: event free survival (efs). event is defined as incidence of acute gvhd, grade iii -iv, chronic gvhd, rejection (graft failure) or death (for any reason). key secondary endpoint(s) are os, dfs, graft failure, hematological and immunological reconstitution, quality of life (qol) assessment and fertility. the primary null hypothesis is: efs of scd patients treated with t-haplo-hsct is non-relevantly inferior to efs in the msd arm. conclusions: results will help to determine if an a/ß depleted t-haplo-hsct can be considered equivalent to msd hsct with regard to dfs, adverse events and safety, in order to offer this form of cure to the majority of patients with scd. disclosure: nothing to declare hit three birds with one stone: successful stem cell transplantation from one family donor to three siblings methods: in august 2016, three thalassemic siblings were admitted to hospital for stem cell transplantation from a full match donor, their 17 years old sister. the patients' general health conditions and specific health issues due to thalassemia were checked extensively. it was decided to perform first transplant to older sister whom the disease and transplant complications are expected more intense due to prolonged transfusion and chelation therapy. the oldest daughter of family, healthy, was planned to accompany her sisters in transplantation unit so parents can take care the others and organize this period for whole family. results: the 13 years old sibling was first admitted to bone marrow transplantation unit in july 2017. the conditioning regimen was busulfan, fludarabine, cyclophosphamide and thiotepa with antithymocyteglobulin(atg) and defibrotide prophylaxis was given. the healthy donor was admitted to hospital and received g-csf for 5 continuous days before harvesting. stem cells were collected peripherally on day 0 and viable cd34 + cells were 2211/ul. patient received 5,5 x10e6/kg stem cell and the other cell products were divided into 4 parts according to other recipients´weight. no infusion problems were recorded in stem cell transfusion. gvhd prophylaxis was given with cyclosporin and methotrexate. severe sinusoidal obstruction syndrome was observed and successfully managed with supportive therapy. neutrophils were engrafted +11. day, and platelets were on day 64. full blood chimerism results were %98 in day 30, %99 in day 60 and %98 in day 180 consecutively. after 3 months from first transplant the 4 years old sister was admitted to hospital on october 2017. same conditioning with defibrotide prophylaxis and gvhd prophylaxis were given and 6,8 x10e6/kg peripherally derived and previously frost stem cell was infused without any complications. mild sinusoidal obstruction syndrome was observed and managed with supportive therapy successfully. neutrophils were engrafted +11. day, and platelets were on day 16. full blood chimerism results were %99 in day 30, %99 in day 60 and %99 in day 180 consecutively. the third transplant was performed on january 2018 with the same conditioning and prophylaxis regimen. although defibrotide was used mild sos was observed and treated with supportive therapy with success. neutrophils were engrafted +10. day, and platelets were on day 27. full blood chimerism results were %99 in day 30, %99 in day 60 and %99 in day 180 consecutively. conclusions: the patients are being followed for over a year after first transplat, neither adverse nor gvhd symptoms were observed. we presented this case for being a unique example for match family donor transplant and the first successful example from one donor to three recipients. disclosure: nothing to declare results: our female patient admitted for anemia at 3 rd month of birth and was transfused every 4-5 months from 6 th months to 3.5 years of age. since investigations directed towards hemoglobinopathies or membrane defects like hereditary spherocytosis were unremarkable, she was not transfused for 6 years after the age of 3.5-years because hemoglobin level was constant over 7 g/dl. her bm examination showed erythroid hyperplasia and feature of dyserythropoiesis with a few binucleated erythroblasts. it was decided to follow-up the patient with a diagnosis of cda ii. after the age of 10-years, the need for transfusion started again for every 4 to 5 months which led the parents of our patient to request for bone marrow transplantation, however, the diagnosis was not definite, and because of the insufficient data for the transplantations for cda ii patients, it was decided to go on to follow-up. nevertheless, after 2 years, the frequency of transfusion gradually increased to every 2-3 weeks, and bone marrow transplantation was brought into question again. at that time, genetic examination was started and sec23b gene was analyzed by direct sequencing. hsct decision from her hla 10/10 matched brother, carrying sec23b mutation in heterozygous state, was taken. in the preparation regimen, busulfan (bu) at a myeloablative weight adjusted dose (4 days), 200 mg/kg cyclophosphamide (cy) (4 days), and 30 mg/kg antithymocyte globulin (atg fresenius) were used. graftversus-host disease (gvhd) prophylaxis was with cyclosporin a started on day -1 and short-term methotrexate on day +1,+3 and +6. she was transplanted with bm with a dose of total nucleated cells=7.1x10 8 /kg and cd34=3x10 6 / kg. neutrophile and platelet engraftment were achieved at +20 and +38, respectively. indeed, grade 4 hemorrhagic cystitis due to bk virus and a moderate veno-occlusive disease prolonged platelet transfusion days which concealed the exact engraftment day of platelet. the patient was discharged on day 45 with no more need for any transfusion and followed up as a complete chimeric with no type of gvhd since then. now, she is 20 years old, under regular surveillance at our transplant centre without any symptoms. conclusions: hsct data in cda ii patients is still insufficient, however based on data from tm patients with similar treatment approaches in td cda ii patients, it is seen that the hsct is reliable and effective. disclosure: nothing to declare hematopoietic stem cells background: the prognosis after frontline therapy in b-all patients have improved due to monoclonal antibodies (cd20, cd19, cd22) and approximately 90% of patients achieve complete remission. in relapsed and refractory (r/ r) b-all and also in mrd + outcomes are relatively poor. disease-free survival (dfs) in this cohort is 10-20%. in this cohort allo-hsct is indicated and complete remission before transplantation is crucial for prognosis. conventional chemotherapy is associated with high failure rate and significant toxicity. immunotherapy with monoclonal antibodies and car-t are more promising approaches. the aim was to evaluate the efficacy (frequency of responses, os, dfs) and toxicity, especially neurotoxicity and cytokinerelease syndrome, of a bispecific monoclonal antibody blinatumomab in patients both children and adults with persistence of minimal residual disease (mrd + ) or r/r b-all as a bridge to allo-hsct. methods: this study included 120 patients with high risk b-all blinatumomab treated in 2013-2018, among them 14 pts (12%) with t(9;22), 10 (8%) with t(4;11), with mll 11(9%), 84 pts (70%) who were refractory to previous chemotherapy, 66 (30%) after allo-hsct from deferent type of donors. median age was 21 y.o. (range 5m-71y.o), 55 children 0-18 y.o. (46%) and 65 adults >18 y.o. (54%). r/r all had 63 pts (52%), mrd + -57 pts (48%), median days of follow up were 227 (18-720). blinatumomab was applied as 28-day cycles followed by a 14-day off-period before the start of the following cycle. majority pts received one cycle (n=94, 78%). in r/r all group dose was of 9 mcg/d during the first 7 days and afterwards 28mcg/d. patients with weight less than 45 kg received 5 mcg/m 2 /d and 15mkg/m 2 /d accordingly. in mrd group dose was 15 mcg/m 2 /d. results: the frequency of responses to blinatumomab was higher in mrd + pts in comparison r/r all pts (85% vs 62 % p=0.007). in mrd + pts cr mrdwas achieved in 47 pts (82.5%), 10 pts (17.5%) were mrd+ after blinatumomab. two-year os in this group was 61%. twenty pts (34%) received allo-hsct. in rr all pts cr mrdwas achieved in 30 pts (48%), 9 pts (14%) were mrd+ after blinatumomab, 24 pts (38%) had no hematological response . two-year os in r/r all was 43%. fifteen pts (24%) received allo-hsct. os in cr mrdpatients who received allo-hsct was not significantly different in comparison with patients who received blinatumomab as a monotherapy (84% vs 71%, p=0.08). no significant differences in dfs were observed at two years in cr mrdpts depending status of the disease before therapy-mrd vs r/r (66% vs 59%). of the reported adverse events, febrile fever was the most common 91pts (76%), neutropenia 43 (35%), thrombocytopenia 46 (38%), infection 32 (26%), neurotoxicity 29 (24%), cytokine-release syndrome 8 (7%). all complications were reversible. conclusions: blinatumomab is effective option in patients with high risk b-all especially in the group with mrd persistence after previous chemotherapy and facilitates effective bridging to hsct. blinatumomab therapy is generally well tolerated. disclosure here we address the transcriptional regulation of differentiated cells from human embryonic stem cells (escs) using self-assembling peptide hydrogel without stromal cells, and compare with embryoid body (eb) culture system. methods: esc differentiation was induced in eb culture system or three-dimensional (3d) hydrogel culture system. the engraftment potential of differentiated cells was evaluated by flow cytometry. cd34 + cells from mobilized peripheral mononuclear blood cells (mpbmcs) or differentiated from escs at different times (day 7, day 10, day 14) were purified by fluorescent-activated cell sorting. sorted cells were captured on medium-sized microfluidic chips using the fluidigm c1 single cell auto prep system. sequencing was performed by hiseq x ten. results: self-assembling peptide hydrogel formed a 3d scaffold for cell culture, the pore diameter of which ranged from 50 to 200 nm. compared to eb culture system, escs in 3d culture system differentiated more potently. the differentiated cells from 3d system were short-term engrafted in the nog mice, and myeloid cells, b cells and t cells could all be detected in peripheral blood after transplantation. however, the engraftment was not obtained in differentiated cells from eb culture system. we obtained and analyzed 301 escs, 554 cd34 + cells from eb culture system, 440 cd34 + cells from 3d culture system, and 218 cd34 + cells from mpbmcs. the cells were divided into 11 cluters ( figure 1a ). in both differentiation systems, the cd34 + cells from day 7 were more heterogeneous than cd34 + cells from day 10 and day 14 ( figure 1b) . however, cd34 + cells from mpbmcs were more homogeneous, probably because the differentiated cd34 + cells contained several cell lineages, including hematopoietic cells, endothelial cells and mesenchymal cells. there is transcriptional overlap between individual cd34 + cells from eb and 3d culture systems. however, we found that cluster 3, which is composed mainly of cd34 + cells from 3d at day 14 and day 10, expressed similar level of several hematopoietic regulator as hsc, such as tal1, lmo2, erg ( figure 1c ). the cluster 6, which is almost the cd34 + cells from 3d at day 7, also expressed the highest gata2 among the clusters from differentiated cells ( figure 1c) . conclusions: our study demonstrates that 3d hydrogel culture system facilitates hematopoietic specification of escs. disclosure: nothing to declare higher cd34+ cell dose increases overall survival in the setting of dual t-lymphocyte suppression with atg and ptcy in matched related and unrelated donor allosct background: there is no consensus on the cd34+ donor cell numbers required for optimal outcomes in allogeneic stem cell transplant (allosct). there is controversy on the benefits or harm in higher cell dose for allosct. this study aims to evaluate the impact of cd34+ cell dose in allosct patients receiving reduced intensity conditioning (ric) combined with anti-thymoglobulin (atg) and posttransplant cyclophosphamide (ptcy) using related (mrd) and 10/10 and 9/10 matched unrelated donors (mud). methods: this is a single-centre retrospective analysis of 140 adult patients who received allosct for hematologic malignancies between october 2015 and may 2018. all received ric using fludarabine (30mg/m 2 /day: day -5 to -2), busulfan (3.2kg/m 2 /day: day -3 and -2) and total body irradiation (200 cgy: day -1). all patients also received rabbit-atg (4.5 mg/kg: day -3 to -1), ptcy (50mg/kg/day: day +3,+4) and cyclosporine (from day +5). unmanipulated peripheral blood stem cells were infused on day 0. analyses were done using 2 thresholds: (1) an arbitrary cd34+ cell dose of 6 x10 6 /kg (as this was our target dose) and (2) cell dose according to quartiles (< 5.67, 5.67-7.98, 7.99-11.34 and ≥ 11.35 x10 6 /kg). results: median cd34+ cell dose was 7.98 x10 6 /kg. median follow up was 19 months (range 5-35). median neutrophil engraftment was 16 (range 13-31) days and platelet engraftment was 21 (range 11-83) days. a cell dose greater than 6 x10 6 /kg was associated with an increased overall survival (os) at 1 year (71.3%; 95% ci, 62.3-80.3 vs 46.7%; 95% ci 30.3-63.1; p=0.018, figure 1 ). the higher dose was also associated with shorter platelet engraftment time (p=0.016, figure 2 ). there was no significant difference in neutrophil engraftment, nonrelapse mortality (nrm), relapse free survival (rfs), grade ii-iv acute graft versus host disease (agvhd) and moderate to severe chronic graft versus host disease (cgvhd), (table 1) . analyses using quartile cell dose thresholds showed a trend towards decreased os with a cell dose of < 5.67x10^6/kg, however this was not statistically significant ( figure 1 ). higher cd34+ cell doses were associated with shorter platelet engraftment time (p=0.005, figure 2 ). there was no significant difference in neutrophil engraftment, nrm, rfs, agvhd and cgvhd (table 2) . conclusions: cd34+ cell dose greater than 6 x10 6 /kg significantly increases overall survival in the setting of ric and dual t-lymphocyte suppression with atg and ptcy in mrd and mud allohsct. further studies in a larger number of patients and longer follow up are recommended to validate these findings. disclosure methods: fifty two adult patients were included. median cd34+ cells requested for infusion were 6x10^6/kg. all patients received the same ric regimen including fludarabine (30mg/m2/day day -5 to -2), busulfan (3.2kg/m2/day day -3 and -2), and total body irradiation (200 cgy) (day -1) combined with rabbit-atg (4.5 mg/kg: day -3 to -1), ptcy (50mg/kg/day: day +3,+4), and cyclosporine. unmanipulated peripheral blood stem cells were infused. last followup was november 2018. median follow-up was 13 months (range 5-26). median cell dose count infused was 9.83 cd34+/kg. we arbitrarily divided the cohort in two groups with cd34+ dose of >8x10^6 cd34/kg as cut-off point. results: findings are summarized in figure1. the infusion of more than 8x10^6 cd34/kg dose had a significant worse impact on overall survival (os) (p=0.022), relapse-free survival (rfs) (p=0.042) and cumulative incidence of acute gvhd (p=0.000). chronic gvhd could not be compared between the two cohorts due to the different median follow-up. conclusions: the infusion of a cd34+ cell dose count higher than 8x10^6 cells/kg had a significant adverse impact in overall survival and grade ii-iv acute gvhd in the setting of ric and dual t-lymphocyte suppression with atg and ptcy for haplohsct. disclosure: nothing to declare p335 long-term thymic activity and immune-reconstitution after haplo-identical allografting with post-transplant cyclophosphamide background: the use of post-transplant cyclophosphamide (ptcy) has expanded the application of t repleted haploidentical stem cell transplantation (haplo-hsct). in this setting, to investigate thymus role in longterm clinical outcomes, evaluation of immune reconstitution kinetics was performed. methods: twenty-nine patients (median age 53) were enrolled. blood samples were collected before conditioning and at 1, 3, 6, 12, 18, 24 months after haplo-hsct. analyses of cd4 and cd8 t-cell subsets by flow-cytometry were correlated by generalized linear models with real-time pcr (rt-pcr) quantification of signal joint t-cell receptor excision dna circles (sjtrecs), specific marker of naive t-cells thymopoiesis. a) naive; b) central; c) memory; and d) revertant cd4 and cd8 t-cells were defined as follows: a) cd45ra+cd62l+; b) cd45ro +cd62l+; c) cd45ro+cd27-; and d) cd45ra+/45ro +, respectively. sjtrecs rt.pcr was performed on genomic dna (100 ng) extracted from sorted cd4 and cd8 t-cells. results: a gradual increase in absolute numbers of all cd4 and cd8 t cell subsets and of sjtrecs copies from the first month up to 2 years post-transplant was observed ( figure 1) . however, at 2 years, cd4 and cd8 t-cell levels and sjtrecs levels were lower than those observed in healthy donors. sjtrecs kinetics was associated with the increase in cd4 naive t-cells (overall, p < 0.002). this correlation suggests that most of cd4 naive t-cells derives from thymic re-education of donor precursor stem cells, whereas cd8 naive t-cells undergo peripheral expansion after thymic production. furthermore, an increase in cd4 revertant memory t-cells was also significantly correlated with sjtrecs kinetic (p 0,041). central and effector memory t-cells showed a faster thymic-independent expansion in both cd4 and cd8 tcells. interestingly, sjtrecs levels and thymic dependent immune-reconstitution were higher in a cohort of 63 patients undergoing hsct from hla identical donors (manuscript in preparation). clinical outcomes and thymic function were correlated starting at 6 months after hsct. lower thymic output was significantly associated by multivariate analysis with low pre-transplant trecs values (p 0,002 and p < 0,001 in cd4 and cd8, respectively), moderate-severe chronic graft-versus-host disease (gvhd; p < 0,001 in cd8), and age (≥50 years, p 0,006 in cd8). conclusions: the thymus, despite age-dependent involution, substantially contributes to t-cell reconstitution after haplo-hsct. chronic gvhd and older age were significantly correlated with reduced thymic function. overall, lower production of sjtrecs after haplo-hsct as compared after hla identical sibling hsct may partly be due to a higher degree of "mismatching" of mhc molecules during thymic re-education. [[p335 image] 1. figure 1 ] background: the use of allogenic hematopoietic stem cell transplantation (hsct) in the treatment of adolescents and young adults (aya) with philadelphia negative all is decreasing with the adoption of pediatric inspired protocols to treat this age group and the incorporation of minimal residual disease assessment in the routine care of all patients. previously, its use was defined mainly by disease risk features at presentation. methods: a study on 209 aya (age 14-39 years), who underwent allogenic hsct at our institute for philadelphia negative all, between february 2005 and december 2015. all the studied patients received calgb based adult chemotherapy protocol for induction, and underwent a matched related donor (mrd) transplant with cy/tbi conditioning and mtx/csa as gvhd prophylaxis. the patients were eligible for allogeneic hsct, if they have a mrd plus one or more of the following risk factors: (1) age ˃ 30 years, (2) high presenting wbc count (>30 for b-all, >100 for t-all), (3) high risk immuno-phenotyping (pro-b, pro-t, early t, and mature t), (4) bulky splenomegaly or bulky lymphadenopathy, (5) high risk cytogenetics (4;11, 1;19, low hypodiploidy/near triploidy, complex), (6) cns involvement, (7) relapsed or refractory disease at d28 of induction. in this study, we investigated the impact of those different risk factors on the long term outcome of allogeneic hsct. results: the median os of our studied patients was not reached at 12.5 years, with a median dfs of 8.3 years (figure 1 ). in a univariate analysis, relapsed or refractory disease prior to transplant was the only independent risk factor for os and dfs (p-value= 0.36, and 0.01 respectively) (figure 2 ). in addition, patients who had 3 or more risk factors (41, 19.6%) prior to transplant had a significantly lower long term outcome compared to patients, who had one (100, 47.9%) or two risk factors (68, 32.5%) with a median os of 23 months, and a median dfs of only 11 months (p-value=0.32, and 0.009 respectively) ( figure 3) . conclusions: our results show that the long term outcomes of hsct in aya with philadelphia negative all treated on an adult type chemotherapy regimen, were significantly better in patients who showed a good response to initial therapy and a limited poor prognostic factors at presentation, with worsening of dfs as the number of poor prognostic features increase. we can conclude that, using this risk score can be helpful in predicting the outcome of allogenic hsct in aya with philadelphia negative all treated with adult type chemotherapy protocol. disclosure: no conflict of interest a prospective single center survey on donor-specific anti-hla antibodies and desensitization strategy in patients undergoing an allogeneic stem cell transplant background: in the setting of hematopoietic stem cell transplantation (hsct), considering the risk of poor engraftment or graft failure (gf), the detection of antibodies (ab) directed against donor specific hla loci (dsa) represents a contraindication to proceed with the same donor, suggesting the search of other donors. in many cases, there is not sufficient time to search for alternative donors and it is necessary to plan an immunosuppressive strategy to decrease the dsa level, thus reducing the risk of gf. to date, there is no consensus on desensitization standards to manage dsas in hsct. the aim of this study was to determine the incidence of anti-hla ab and dsas in hematologic patients candidate to an allogeneic hsct, and the efficacy of our desensitization protocol. here, we present an update of the results obtained with our strategy. methods: between august 2014 and september 2018, we prospectively screened for dsa 140 consecutive patients candidates to an allogeneic hsct. anti-hla ab research was carried out using the luminex bead assay (lifecode screen and lsa i/ii-immucor). the results were expressed as mean fluorescence intensity (mfi); mfi >1000 was considered positive. in case of a mismatched related donor, a flow cytometric crossmatch test (fcxm) was performed. if the patient had dsas and only one available donor, a desensitization strategy was employed, scheduled with rituximab on day -15, single-volume plasmapheresis procedures (pp), usually on day -9 and -8, intravenous immunoglobulins on day -7, infusion of hla selected platelets for dsa absorption in case of persistent antibodies directed against class i hla antigens. the aim of this schedule was to avoid interferences with chemotherapy and anti-t-cell globulins, infused during condition regimen results: since august 2014, 140 patients have been prospectively screened. thirty-three patients (23.6%) showed anti-hla ab and 9 of them (6.4%) had dsas: 6 were treated with the desensitization strategy, applied according to the mfi score and the fcxm result, and all of them obtained an engraftment; in 2 cases, an alternative donor was selected and in 1 case the research for an alternative donor is still underway. dsa detection was performed every 7 days after hsct for the first month and 60, 180 and 365 days following hsct. neither a dsa rebound nor other complications were observed during the follow-up. conclusions: our prospective analysis underlines the high frequency of anti-hla antibodies detection in hematologic patients, confirming the necessity to routinely evaluate the presence of dsas before an allogeneic mismatched hsct. our desensitization schedules based on the combination of pp, rituximab, ivig and platelet absorption proved successful in reducing dsas. we confirm the necessity of a prospective multicenter collaboration to better define the role of dsas against each hla locus and the critical mfi cut-off level associated with a higher risk of gf. transplant and transfusion specialists should joint to define a consensus for a standard desensitization strategy. disclosure the most frequent technique used for counterbalance partial incompatible hsct is cd34+ selection that is associated with sustained engraftment and effective reduction of t cells that minimizes gvhd. on the other hand, this approach could delay immune reconstitution and increase risk of viral and fungal infection. in mud setting the use of pbsc is the procedure that most centers have recently adopted. this implies the infusion of a relevant higher number of t cells 5 to 10 times more as compared with bone marrow (bm). since in our centre most part of our patients are primary immunodeficiencies, we applied a procedure to minimize the risk of severe gvhd infusing a controlled number of cd3 positive cells. methods: we report data about 91 paediatric patients who received 92 mud hsct (1 patient received 2 hsct) between 2001 and 2018 in the bmt unit of the children's hospital of brescia. patients received conditioning, according to the european group for bone marrow transplantation (ebmt) and the european society for immunodeficiencies (esid) guidelines. cd34+ selection has been realized by a milteny column with an ideal addback of cd3 positive cells of 30x10 6 /kg. stem cell source was bm in 62 cases and pbsc in 30 cases. results: median patients age at transplant was 2 years (range 2.6 months-17 years). the mean number of infused cells were: 12x10 6 /kg cd34+ and 36x10 6 /kg cd3+ in bm product, while 20x10 6 /kg cd34+ and 39x10 6 /kg cd3 + in pbsc. mean time for engraftment was day 15 post-hsct. as concerns acute gvhd overall incidence 64.1 % (59/92) of the children presented this complication, but only 11% (7/ 59) presented gvhd grade iii and none gvhd grade iv, while chronic gvhd presented in 7.6% (6 limited, 1 extensive/92). while acute gvhd incidence and severity weren't significantly different between bm recipients and pbsc recipients, the cases of chronic gvhd were prevalently in the latters. no major infections presented in the post-transplant period and immunological reconstitution both cellular and humoral was completed by 12 months. overall survival at 10 years is 75% (23/92). the results obtained show how it is possible control severity of gvhd if an addback of a controlled number of cd3+ lymphocytes. acute gvhd wasn't severe and only few children presented with limited chronic gvhd. the method allows to graft primary immunodeficiencies patients even with pbsc without infusing too many t cells. in fact, especially in very young children, the number could be excessive and risky. nevertheless in case of an oncohaematological patient, gvl effect is preserved. disclosure background: dc is a rare genetic disorder that results from a defective telomere length maintenance and is characterized by mucocutaneous features, bone marrow failure (bmf) and a high predisposition to cancer and pulmonary fibrosis. bmf remains the major cause of mortality and the hsct is the only definitive treatment to restore hematopoiesis but is limited by a high incidence of treatmentrelated mortality. methods: a retrospective analysis of 28 patients (pts) with dc who underwent hsct at the bone marrow transplantation unit in the clinical hospital of federal university of paraná, brazil, between july-1993 and november-2017. results: 15 boys and 13 girls, with a median age of 14y (3 -30y) received a hsct from a mds (n=7), mud (n=17) or mmrd (haploidentical, n=4). 27pts received bone marrow (bm) and 1pt received a cord blood unit (cbu). the median of tnc infused was 4,76x10 8 /kg (range 2,26-10,12x10 8 /kg) and in the cbu was 6,5x10 7 / kg. two pts received a myeloablative preparatory regimen with busulfan (bu) 12mg/kg + cyclophosphamide (cy) 120mg/kg or fludarabine (flu) 150mg/m 2 + antithymocyte globulin (atg). the remaining pts received a ric regimen with cy200mg/kg (n=5), flu150mg/m 2 + cy60mg/kg + atg5mg/kg (n=17), and flu150mg/m 2 + cy30 + tbi200rads (n=4, haplo). graft versus host disease (gvhd) prophylaxis consisted of cyclosporin (csa) and methotrexate or steroids (cbu) and post-transplant cy + csa + mycophenolate mofetil in the haploidentical transplants. 26 of 27 evaluable pts engrafted with a median time to neutrophil recovery of 20 days (range:13-36 days). one patient experienced primary graft failure (haplo) while second graft failure occurred in other 3pts. all these 4pts went a second hsct and 3 survived. acute gvhd grade ii-iv occurred in 6 of 26 pts at risk. moderate to severe chronic gvhd occurred in 6 pts with 5 cases occurring in pts who had previously presented acute gvhd. overall survival (os) was 53,6% at a median follow-up of 6y. the 5y os was slightly better in msd transplants compared to the others (54,5% x 52,9% p=0,053). causes of early death include adenovirus sepsis (n=1), toxicity to preparatory regimen and sepsis (n=2), primary graft failure (n=1). 13pts remain alive between 1-16y after hsct with a median fu of 8y. among them only 1pt has developed organ involvement by the underlying disease: hepatopulmonary syndrome (hps). 7pts died due to pulmonary fibrosis (n=1), liver fibrosis(n=1), gi bleeding(n=1), hps (n=2); cgvhd and sepsis(n=1), infection (n=1), and 2pts were lost to fu. conclusions: early mortality from bmf can be reduced by hsct, but late outcomes remain a consequence of the underlying disease. long term fu is essential in order to detect late complications related to the hsct procedure or the underlying disease. disclosure: nothing to declare single intra-bone cord-blood transplantation with a treosulfan-based regimen, atg-free and sirolimusbased gvhd prophylaxis: fast hematopoietic engraftment and immune-reconstitution in 20 patients background: cord blood transplants (cbt) require less stringent hla-matching, compared to peripheral blood stem cell or bone marrow. however, cbt has been associated with delayed engraftment and immune reconstitution, especially if in vivo t-cell depletion, such as antithymoglobulin (atg), is used. methods: from 2010 to 2018, 20 patients with high-risk diseases received intra-bone infusion of unwashed single cb unit with an atg-free gvhd prophylaxis; 7 were in active disease at cbt and 8 had received prior allogeneic stem cell transplantation. median age was 44 y [range (r) . conditioning regimen was myeloablative, with treosulfan and fludarabine in all, intensified with melphalan in 15 or with 4gy tbi in 4. hla matches was 4/6, 5/6, 6/6 in 12, 5 and 3 cases, respectively. gvhd prophylaxis included sirolimus and mycophenolic acid (mmf). results: after thawing, median cd45+ cells was 1.39 x 10 7 /kg [r 0.69-5.9], median cd34+ cells 0.08 x 10 6 /kg [r 0.04-0.23], and median cd3+ cells 3.05 x 10 6 /kg [r 1.29-5.9 ]. of the 16 evaluable patients all engrafted with a sustained full donor chimerism at day 100. median time to neutrophils (16/16, anc> 500/μl for 3 consecutive days) and platelet engraftment (14/ immune-reconstitution of cbt patients (tables 1a-b ) was compared with two cohorts of patients transplanted at our center from any adult donor with (81) or without (126 patients, including post-transplant cyclophosphamide cohort) atg in association with sirolimus and mmf. profiles of immune-reconstitution at day 90 -180 -365 showed a better cd4+ recovery at any time-point in both cbt and no-atg versus atg cohort, with no statistic significant difference in the first 2 cohorts. moreover, cd4 +/cd8+ ratio at any time point was better in the cbt cohort vs the no-atg cohort. b cell recovery was faster in the cbt cohort; immunoglobulin recovery was superimposable across different platforms. focusing on late events (>180 days from cbt), 3/12 pts experienced ebv reactivation, median time 586 days [362-655] treated with rituximab, and 1 experienced late hhv6 and cmv reactivation, both solved at last visit. sirolimus was withdrawn after a median of 188 days [r 64-394]. only 1 patient developed severe chronic gvhd, solved at last visit. overall, after a median follow-up of 330 days [r 9-1311], 11 pts are alive and well. conclusions: our data confirm that intra-bone cbt without in-vivo t-cell depletion is associated with fast hematopoietic engraftment and immune-reconstitution, with very low rate of chronic gvhd and late infective events. background: a promising improvement of hematopoietic stem cell transplantation (hsct) may lie in the transplantation of high numbers of pluripotent stem cells to minimize the time span between transplantation and immunological reconstitution. hence, an ex vivo platform is needed that supports hsc proliferation before application and, at the same time, the maintenance of pluripotency by diminishing hsc differentiation into lineage-specific progenitor cells. methods: to artificially model the natural hsc niche in vitro, we used 3d bone marrow (bm)-like scaffolds made of polydimethylsiloxane (pdms). these structures are based on a human long bone cross section as a representative of the bm. human cryoconserved hscs were cultured in distinct cultivation systems for 14 days under different conditions. cell counting and facs analyses at day 14 were conducted. for characterization of the cultivated hscs, we used antibodies against cd34 alone or in combination with antibodies against cd38, cd90, cd45ra and cd49f. results: for optimization of culture conditions for human hscs, a commercially available medium was supplemented with a panel of cytokines and valproic acid. we found a significant increase in the number of cd34+ hscs by simultaneously increasing their vitality using the 3d system compared with conventional 2d culturing. a further improvement was achieved by introducing a silicon oxidecovering of the 3d pdms structures, suggesting that hydrophilic surface properties offer superior attachment for semi-adherent hscs. for a more precise characterization of the cultivated hscs, we introduced a panel of facs markers reflecting the immaturity of the amplified hsc. surprisingly, with increasing immaturity of the cultivated hsc, non-covered 3d pdms revealed to be best suited for amplification: cell number of vital immature hscs was increased after cultivation on non-covered 3d pdms compared with silicon oxide-covered 3d pdms and the 2d system. conclusions: by establishing a 3d scaffold according to the human bm, we found a platform mimicking the natural niche of human hsc which is suitable to amplify human hscs in vitro and support their vitality, pluripotency and ability for self-renewal. [[p341 image] 1. with the introduction of sion covering of 3d pdms structures the maintenance of cd34+ hscs could be further improved. despite the better conservation of cd34 + hscs by using silicon oxide-covered 3d pdms, we found that immature human hscs obviously prefer more hydrophobic conditions found on non-covered 3d pdms. disclosure: nothing to declare. abstract already published. improvements in neutrophil engraftment following changes in freezing method background: in the setting of autologous haematopoietic progenitor cell (hpc) transplants for haematological disorders, peripheral blood stem cells are routinely collected via apheresis and cryopreserved. leicester royal infirmary had been using a controlled rate freezer (crf) to cryopreserve cellular therapy products up until 2017. in 2017 a literature review of cryopreservation techniques was undertaken, since the crf required replacement. this review found consistent evidence that cryopreservation using minus 80 o c is comparable to crf, and engraftment times should not be negatively affected by changing to a more simplified method of freezing. there would also be cost saving benefits from switching from a crf to minus 80 o c freezers. methods: as a result two minus 80 o c freezers were purchased following the acceptance of a preparation process dosier (ppd) which was prepared for the human tissue authority. validation was carried out, and from january 2018 stem cell laboratory at the lri switched from the crf method to minus 80 o c for cryopreservation of cellular therapy products. briefly, cells are frozen using 10% dmso (wak-chemie) in 20g/l human albumin solution (grifols) in cyrobags (origen biomedical, inc). the cells are transferred on cold packs to the minus 80 o c freezer. cells are packaged in between stainless steel heattransfer plates. the plates are placed within a bubble wrap bag, and are placed in a rack within the minus 80 o c freezer, which allows air to circulate freely around each bag. in one plate a el-usb-tc thermocouple data logger (thermosense) is inserted between the bag and stainless steel plate, to record the freezing profile. this data is downloaded after each run. after an overnight freeze at minus 80 o c, the cells are subsequently removed from the bubble wrap and plates, and are transferred to minus 150 o c freezers the following morning. results: a total of 40 patients have had autologous stem cells frozen using this method so far this year. in addition to engraftment data for neutrophils, post-freeze trypan blue viabilities were also compared to the previous year. during 2017 a total of 51 patients, who had all their cells cryopreserved, underwent 72 collections. the post freeze median viability was 90% (75-98%). a total of median neutrophil engraftment was 12.0 days, with a median cd34 dose infused of 4.0 x 10 6 /kg. during 2018 so far, 40 patients have undergone 57 collections. median viability is 95.5% (82-98%). subsequent median neutrophil engraftment is 11 days, with a median cd34 dose infused of 4.1 x 10 6 /kg. conclusions: ongoing savings of approximately £6400 per annum have been made by changing our procedure. the benefit of changing to a simplified method of freezing has also resulted in a reduction in staff working overtime. more importantly, this simplified cryopreservation method has resulted in an improvement in neutrophil engraftment times since changing the cryopreservation technique from the previous method using crf to mechanical freezing using a minus 80 o c freezer. disclosure: nothing to declare low doses of granulocytes in the apheresis product predict a better outcome of autologous hematopoietic stem cell transplantation in multiple myeloma patients background: high-dose chemotherapy with autologous stem-cell transplantation (asct) remains the standard consolidation therapy for multiple myeloma (mm). peripheral blood stem cell collection may be contaminated with large quantities of granulocytes and its consequences on the outcome of asct are still unclear. on the other hand, the effect of performing apheresis with high levels of monoclonal component (mc) on outcome is unknown. the objective of this study was to analyze the effect of total nucleated cells (tnc) and granulocytes count (considered as contaminating components of apheresis products) as well as the influence of mc in the apheresis product on outcome of asct in mm. methods: eighty-two patients diagnosed with mm were mobilized with filgrastim 10 μg/kg/day (plus plerixafor if insufficient mobilization of cd34+ cells on day 4). apheresis collection was performed with cmnc program by spectra optia cell separator. cd34+ count was carried out according to ishage protocol (target: ≥2x10e6 cd34 +/kg). subsequent cryopreservation were performed according to the local protocol. results: the medians (range) of collected cd34+, tnc and granulocytes were 4.69x10 6 /kg (1.69-15.54), 9.35x10 8 / kg (2.16-19.66) and 13.7x10 9 /kg (1.4-90.6), respectively. the medians (range) of infused cd34+, tnc and granulocytes were 2.99x10 6 /kg (0.89-10.93), 6.46x10 8 /kg (1.93-18.60) and 8.30x10 9 /kg (1.1-86.0), respectively. a successful collection after first line therapy was performed in 94% of patients. treatment for mm was continued after carrying out apheresis in 40% of patients, as per protocol. a significant reduction of mc was observed prior to asct, indicating a further improvement in responses after apheresis (p=0.005). an optimal response (cr or vgpr) at the time of apheresis was achieved in 45% of patients and a suboptimal response (partial or minimal response) was observed in the remaining 55%. undergoing apheresis in optimal response did not result in lower number of tnc or granulocytes in the harvest. the subtype of mc did not influence on the number of tnc and granulocytes in the product of apheresis. no differences in collected tnc and granulocytes were observed when plerixafor was used as mobilizing agent. the type of chemotherapy given prior the apheresis did not have influence on the characteristics of the apheresis product. a significant improvement in overall survival (os) probability (95%ci) was observed when low tnc (< 6.46x10 8 figure 1a ). lower incidence of relapse (p=0.044) ( figure 1b ) and non-relapse mortality (p=0.040) was observed in patients who received low granulocyte count in the graft. no significant correlation was observed between the time of engraftment and the number of tnc or granulocytes infused. similarly, no increase in the frequency of the engraftment syndrome was observed when higher number of tnc or granulocytes were infused. conclusions: in our series, low doses of granulocytes in the product of apheresis predicted a better outcome of asct in mm patients. the amount of mc at the time of apheresis did not have influence in the characteristics of the harvest. efforts for avoiding contamination in grafts are important for its impact on outcome of asct in mm patients. disclosure: dkms foundation, pi14/01971 (instituto carlos iii) and sgr288 (grc), generalitat de catalunya. background: our initial experience (from 2012 to 2017 years) with hematopoietic stem cell transplantation (hsct) from mud with tcrαβ+/cd19+ graft depletion in patients with cgd showed high rate of secondary graft dysfunction, the incident of graft rejection was 29%. to improve the outcome, we hypothesized that the use of plerixafor and g-csf as additional agents in conditioning regimen would offers advantages and better outcomes. this trial was registered at www.clinicaltrials.gov (nct03547830) methods: between april 2018 and september 2018, 5 patients with cgd underwent allogeneic hscts from mached unrelated donors with tcrαβ + / cd19 + graft. the conditioning regimen included -treosulfan 42 g / m2, fludarabine 150 mg / m2, thiotepa 10 mg / kg, g-csf 50 mcg / kg and plerixafor 720 mcg / kg. all patients received rabbit atg -timoglobulin® ("genzyme europe b.v.", the netherlands) 5 mg/kg for serotherapy. in all cases, tcrab +/cd19+ graft depletion was used with the immunemagnetic method (miltenyi biotec, bergisch gladbach, germany) according to the manufacturer's instructions. stem cell source was g-csf mobilized peripheral blood stem cells in all cases. posttransplant gvhd prophylaxis was not performed. minimal follow up was 90 days after hsct. results: neutrophil and platelet engraftment occurred at 12-17 days post-hsct in all patients. 3 patients had full donor chimerism in whole blood and in 2 cases we observed predominantly donor mixed chimerism at last follow up. all patients had full donor chimerism in cd15+ compartment and mixed chimerism in cd3+ lineage, but it stable without any sign of graft dysfunction. acute gvhd is verified in 1 of 5 cases and was limited to skin grade 2. all patients are alive for periods from 3 to 8 months post-hsct with good graft function without severe clinical problems. conclusions: we presented first experience of g-csf and plerixafor addition to conditioning regimen before hsct with tcrαβ+/cd19+ graft depletion in cgd patients . we suppose, the preliminary results are encouraging, as the frequency of primary and secondary graft dysfunction in patients from this group is not observed today, there are no significant toxic complications, as well as clinically severe manifestations of gvhd. currently, the recruitment of patients is continuing, and estimation of the rates of immune reconstitution and a more detail analyses will be evaluated later. background: introduction -it is believed that aboincompatibility is of minor importance in allogeneic stem cell transplantation (allo-sct) and that the clinical outcome is equivalent to abo-compatible sct. therefore, we performed a single center retrospective study to characterize the impact of abo-incompatibility on the outcome of haploidentical stem cell transplantation (haplo-sct). methods: this analysis included 27 consecutive patients who underwent haplo-sct for various hematological malignancies at our center between october 2010 and may 2018. we used our institutional database to evaluate details and characteristics of patients and transplant outcomes. results: demographic features of the patients and donors have been summarized in table 1 . all of the patients had advanced hematological disease with a high risk of relapse (22 patients with acute leukemia). out of 24 patients, early transplant-related mortality was seen in this cohort of 5 patients. the remaining 19 patients were followed in 1 and 42 months. donor type abo group switch was observed in a median of 45 days (30-60 days) after transplant. we were not able to show any statistical difference in terms of blood group switch between minor and major abo incompatible transplant. the median red blood cell (rbc) transfusions in the first 30 days for the abo compatible and incompatible transplants were median 4 units (range, 0-11) and median 4 units (range, 3-12) (p=0.6). no statistical difference was also encountered for the rbc transfusion need for stem cell source, peripheral blood vs bone marrow. a total of 15 patients were followed up for reticulocyte engraftment. the median time for reticulocyte engraftment was 19 days (range, 15-60) for all patients. reticulocyte engraftment was tended to be faster in minor abo-mismatched group (p=0.05) than major or abo-compatible ones. nineteen patients achieved independence from rbc support after a median time of 44 days (range, 11-61 days) in abocompatible patients, 25 days (range, 7-52 days) in minor abo-incompatibilityand 34 days (range, 20-57 days) in major abo-incompatibilitygroup, respectively (p>0.5). the engraftman kinetics due to major and minor aboincompatibilitytransplants were presented in table2. pure red cell aplasia was not developed in our cohort. conclusions: the present single center study provides new evidence for the importance of the abo system for erythrocyte recovery in haploidentic stem cell transplantation. it's important to note that, randomize prospective and larger studies are warranted. disclosure: nothing to declare low dose of anti-t lymphocyte globulin protects against severe forms of graft versus host disease in patients undergoing allogenic stem cell transplantation background: graft versus host disease (gvhd) is the most important complication after allogeneic stem cell transplantation (allosct). optimal dose of different anti-tlymphocyte globulin (atg) formulations in this setting has not been established yet. the aim of this study was to analyze the impact of a low dose of atg-fresenius (atg-f) in allosct outcomes. methods: we analyzed 57 adult patients who received an allosct for hematologic malignancies from october 2012 to march 2018. the gvhd prophylaxis included a total dose of 21 mg/kg (7 mg/kg on day -3, -2 and -1) of atg-f for patients who received a graft from peripheral blood, an unrelated donor; with a mismatch, and/or were older than 55 years; associated to a calcineurin inhibitor and mycophenolate mofetil/short course-methotrexate. statistical analysis was performed using spss v.22 and r software. results: median age was 57 years (18-70) and 66.7% of patients were males. seventy-four percent of patients underwent myeloablative conditioning. the stem cell source was peripheral blood in 51 patients (89.5%), 80% were from unrelated donors (28% mismatched). seventeen (29.8%) patients had high risk cmv status (d-/r+) (see image 1b). engraftment was observed in 54 patients (94.7%). primary graft failure occurred in 3 patients (2 myelofibrosis, 1 aml). twenty (39.2%) out of 51 evaluable patients developed grade 2-4 acute gvhd. the cumulative incidence of severe agvhd and moderatesevere chronic gvhd were 8.8% (95% ci, 3.2-17.9%) and 35.2% (95% ci 22.7-47.9%), only 5 (8.7%) patients developed severe cgvhd. twenty-nine patients (56.8%) discontinued immunosuppression before the first year of transplant. the median duration of immunosuppression for patients with moderate-severe cgvhd was 488 days (207-2046). at 2 years non-relapse mortality (nrm) was 21.9% (95% ci, 12.0-33.7%). thirty-nine (68%) patients developed relevant infectious complications. two (3%) patients died within the first 30 days due to gram negative blood stream infection. eleven (19.2%) had at least two episodes of cytomegalovirus (cmv) reactivation between day 30 and 100. three (5%) patients developed cmv gastrointestinal disease, 2 (3%) had probable invasive fungal infection and 1 (1.7%), post-transplant lymphoproliferative disorder associated to epstein barr virus. with a median follow up of 28 months for alive patients , the gvhd and relapse free survival (grfs) at one year, overall survival (os) and progression free survival (pfs) at two years were 47.6% (95% ci, 42.8-52.2%), 59% (95% ci, 54.5-63.2%) and 52% (95% ci, 46.9-56.5%), respectively. the relapse incidence at two years was 26.3% (95% ci 14.7-39.4%). complete remission at transplant was associated with better long term survival (80% at 2 years, p < 0.01). hla disparity did not affect os (see image 1a). conclusions: the use of low doses of atg-f is protective against severe forms of acute and chronic gvhd in a cohort with high prevalence of unrelated donors and a high median age. this strategy showed good results in grfs, os and pfs in a population at high risk for developing gvhd or relapse. disclosure: nothing to declare performance parameters of a ngs-product for chimerism monitoring -applicable in patients after hematopoietic stem cell transplantation methods: for this purpose, samples from patients with mixed chimerism (mc) with increasing amounts of recipient dna were analyzed and compared using realtime pcr of insertions/deletions (indels), fragment analysis of short-tandem repeats (str) and ngs of indels. results: whereas real-time pcr displayed excellent sensitivity down to 0,01 % mc, but poor precision above 20 %, fragment analysis exhibited good precision with limited sensitivity (> 2,5%). in contrast, ngs chimerism demonstrated good sensitivity, with a limit of detection (lod) of 0,1 % mc, and precision throughout the whole spectrum of patient/donor mixed chimerism. the ngs chimerism product (devyser chimerism) exhibited at least three (average eight) and at least two (average 5) informative genetic markers (indels), suitable for monitoring mixed chimerism of patients with their corresponding matched unrelated (60) or related (56) donor samples. in order to establish the performance of the separate techniques for determination of mixed chimerism on retrospective patient samples, a cohort of 27 patient monitoring samples (3-7 weeks post-hsct) with low (< 5%), intermediate or high mixed chimerism (> 20 %) were included and analyzed. dna from all monitoring samples was extracted from sorted cell fractions. the results show that although all evaluated techniques are suitable for monitoring patient/donor chimerism after allogeneic hematopoietic stem cell transplantation (hsct), only the ngs chimerism product exhibits high sensitivity (lod 0,1 %) and a broad dynamic range (detection range 0,1-100%) with good precision and accuracy throughout the whole spectrum of mixed chimerism (% patient/donor). in addition, the ngs chimerism product employ 24 non-population dependent highly informative genetic markers providing stable resolution power and thus suitable for monitoring mixed chimerism. disclosure: dan hauzenberger is medical adviser at devyser ab and shareholder in devyser holding gender distribution: male -59% (n=58), female -41% (n=41). age median -5,8 years old (8 months -17). stem cell source: bone marrow -80% (n=81), peripheral blood stem cells -19% (n=19). 82 patients (83%) received 10/10 matched unrelated donors hematopoetic stem cells transplantation and 17 patients (17%) -9/10 matched unrelated donors hematopoetic stem cells transplantation. differences in the antigen blood system: single group 41% (n=40), minor 32% (n=31), major 20% (n=18), mixed 11% (n=9). age of donor: 18-25 years old -25% (n=16), 26-35 -42% (n=27), 36-45 -23% (n=15), 46 and more11% (n=7). gender differences in donor/recipient: male/female 25% (n=24), female/male 18% (n=18), one sex 57% (n=56). we also took into account the impact of gender difference and cytomegalovirus serostatus in the donor/recipient pair. results: in 10/10 group the estimated probability of overall 2-years survive was 76% and in the 9/10 group 2-years survive was 63%. the increase in donor age of 10 years reduces the 2-years survive by 9-11% (p=0,117), however, the 2-years survive from donors over 46 years old was 100%. we have found no difference between 2-years survive in transplants from donors that are compatible/ incompatible with the antigen blood system, cytomegalovirus serostatus, or the gender differences in donor/ recipient. in the study of donor-related factors, we found the negative impact of an human leucincompatibility (9/10) on the incidence of chronic gvhd -29% (p = 0.019). the combination of cytomegalovirus positive serostatus of the donor and the negative status of the recipient increases the risk of primary graft rejection up to 50%, in comparison with others (p = 0.001). conclusions: our study showed the role of genetic matching on the hla system between the patient and the unrelated donor, and the donors age value. 10/10 transplants have better outcome and lower incidence of severe a. gvhd and ch. gvhd. younger donor increases 2-years survive, but there is a significant increase in 2-years survive if the donor is over 46 years old. disclosure: nothing to declare allogeneic stem cell transplantation in chronic myelomonocytic leukemia. a single center experience nine patients (45%) relapsed with a median of 6,3 months (1-87) with different strategies at this point: in all cases we modulated immunosuppression, in 2 cases as the unique strategy, in 2 cases with donor lymphocyte infusion (dli), in 3 cases we employed hypometilating agents (hma) and in 2 cases with intensive chemotherapy, reaching cr only in two patients, one of them after dli and the other one after hma and consolidation with a second asct. eleven patients (55%) died being the relapse the main cause (64%). transplant related mortality (trm) at +100 were 5% and global trm were 15%. in the last follow-up, 9 patients (45%) are still alive, 8 (89%) in cr and 1 (11%) in relapse situation. with a median follow-up of 58 months (5-138), the event free survival (efs) were 19 months (5-32) and the overall survival (os) were 56 months (0-116). we observed advantage in terms of os in those patients that reach cr at +100 post asct and in those who develop chronic gvchd (p=0.065 and p=0.012 respectively) conclusions: asct is still the only curative option despite the high relapse rates. to reach cr at +100 post asct and the development of chronic gvhd seems that they confer advantage in terms of os. the importance of knowing the molecular profile of the entities that we consider for asct. disclosure this study documents a first experience of a cell processing lab seeking to integrate process automation technology to wash and volume reduce products which can account for the initial material source volume variability, product characteristics, and number of bags. methods: here we report the pre-clinical assessment of the lab's initial work with the lovo cell processing system for a 5 product experience over 2 days with 1 machine. this study used products intended for destruction. the workflow used parallel and sequential processing schedule. after water-bath thawing, bags were sampled, weighed to determine volume, and subsequently connected to lovo or pooled into a transfer pack and then connected to lovo. the bags were then diluted 1:1 at 50 ml/min with lovo at +4-8c using 6% hydroxyethylstarch 130/0.4 (voluven, fresenius kabi) and processed using a 3 cycle procedure. after processing the bags were weighed for volume, sampled, and stored in a 4-8c refrigerator in their lovo final product bags. samples were assessed from t=0 to t=24 hours. results: cd34+ viability and absolute counts were determined using flow cytometry. processing duration and solution volume consumed was determined by the lovo's sensors and confirmed by the operator. data is presented as a percentage relative to the post-thaw values. note, the values presented are not total process yields. the results focus on the lovo processing step. conclusions: the operators easily integrated into the software to drive the machine. the machine demonstrated it's flexibility with a wide-range of volumes, cell-inputs, and number of bags. the lovo produced products which meet our specifications in a quick and reliable manner. further work on this platform will be performed to validate and qualify this system for production use. properties. the aim of this study was to evaluate prospectively the efficacy of a fbm protocol for the prevention of om in patients undergoing a hsct. methods: all patients consecutively who underwent a hsct at our center from 201x onwards received five weekly fbm sessions with a bidiodic laser (lumix 2®, prodent, italy), which simultaneously emitted at 650nm and 910nm with a power of 89mw and energy of 4j per point. the procedure started the day before the beginning of the conditioning regimen up to the tenth day post-transplant. the laser was applied in a defocused mode on each of the mucosal surfaces (12 areas). at each session, the morphine dose, the om level (according to the who scale) and pain through a numerical rating scale (nrs) were recorded. results: 27 consecutive patients (19 male/8 female) submitted to a hsct were analyzed. the median age was 44 years (range 4-66). eighteen patients had acute leukemia, 3 myelodysplastic syndromes, 6 lymphoproliferative diseases. the median number of treatment lines before hsct was 2 (range 1-5). at transplant, 13 patients had advanced disease. the myeloablative conditioning regimen mac (thyotepa, busulphan, fludarabine) was employed in 17 patients; the same conditioning, with a reduced dose of busulphan (ric), was infused in 10 patients. seven patients (26%) had no evidence of om. the incidence of grade ii-iv om was 65% in the group of patients receiving mac and the median duration 12 days (range 3-28); grade 4 om was observed, for 1 day, in 1 patient. in the ric group the incidence of om was 50%, the median duration 11 days (range 7-16); no patient had evidence of grade iv om. in the whole population, the maximum nrs value was 4. morphine administration was required in 23 patients, due to the occurrence of non-oral complications. conclusions: in our experience, prophylaxis with fbm to prevent or reduce om was safe and effective, compared to results of previous experiences reported in the literature, which used no prevention against this complication that negatively affects the quality of life of transplanted patients. further studies on a large series of are necessary to confirm our results. disclosure: nothing to declare background: cytogenetic abnormalities are an essential part of prognostic systems in myeloid malignancies before hematopoietic stem cell transplantation (hsct), however, their role in posttransplantation prognosis is unknown. the aim of this study was to assess the prognostic impact of genetic risk stratification of aml and mds patients on posttransplantation course, which could be an additional tool in making decisions regarding preemptive therapy. methods: a retrospective analysis covering patients treated with allo-hsct between 2012 and 2018. cytogenetic studies included karyotyping (c-and gbanding) and fluorescence in situ hybridization (fish). the number of analyzed cells exceeded european cytogenetics association guidelines (for each fish at least 600 interphase nuclei were analyzed). cytogenetic risk group in aml was assessed based on the eln 2017 criteria. patients with mds were stratified into three groups; favorable (good and very good prognostic score), intermediate, and adverse (poor and very poor) prognostic score according to ipss-r 2012. interestingly, the poorest survival was in patients with monosomy of chromosome 7, which was present in 6 patients of whom 5 succumbed to refractory disease, while all patients who had deletion of long arm of chromosome 7 (del 7q)-are alive at the time of writing of this report after a median follow-up of 34 months (21-73). relapse was diagnosed in 31 patients (31%), including; 13 (42%) with adverse, 15 (48%) with intermediate and 3 (10%) with favorable cytogenetic risk. among 12 patients with a complex karyotype and/or cytogenetic evolution prior hct: 8 patients (67%)relapsed, including 6 (50%)-who died. follow-up cytogenetic studies in relapse after transplantation were performed for 24 patients; 4 of them (17%) had clonal evolutions of the original karyotype with additional abnormalities-(50% died) and 7 (29%) had new aberrations in cells without primary changes (all died). in 18 patients (18%) (8 unfavorable, 8 intermediate group) cytogenetic relapse was diagnosed by fish analysis and they were treated with azacitidine (+/-dli) achieving cr (n=9, 50%), stabilization-(n-=3, 16%), transient response (n=1), while 5 deceased). conclusions: cytogenetic studies in patients after transplantation may facilitate assessment of mortality. karyotype may undergo cytogenetic evolution after allo-hsct. patients with monosomy of chromosome 7 seem to have a particularly poor prognosis. transplanted patients are vulnerable to new cytogenetic alterations. disclosure: nothing to declare methods: the primary end-points were the rate of complete response (cr), defined as no emesis and no nausea without rescue medications, for both acute (cr-24) and delayed (cr 25-120) cinv and rate of post-transplant complications until discharge. we prospectively analyzed 61 patients undergoing autologous (85%) and allogeneic (15%) stem cell transplantation and receiving cinv prophylaxis with nepa and dexamethasone (schedules shown in fig.1 ). in our series, 30 patients (49%) were female. patients median age was 55 years (20-74). the most frequent diagnosis were myeloma (46%) and lymphoma (39%), while 15% of patients were diagnosed with aml or mds. myeloma patients received one day hd-ct with melphalan (75% mel200/25% mel140). lymphoma patients were conditioned with feam (87,5%) or tt_flu_edx (12,5%) hd-ct. busulfan-based mds-ct regimen was offered to aml/mds patients. results: the incidence of cr-24 and cr 25-120 observed was 82% (50/61) and 47,5% (29/61), respectively. more than grade 1 nausea and vomiting (according to ctae-4), was reported in 36% (22/61) and 5% (3/61) of patients, respectively. female sex was associated with an increased risk of acute (hr 19,6; p 0,037 95% ci 7.980-2.191) but not delayed (hr 1,58; p 0,06 95% ci 200-2.042) cinv. similar rate of cr24 and cr25-120 was observed in one-day hd-ct (82% and 50%) compared to mds-ct (89% and 42,5%) group (pns). median lenght of stay was 23 days (15-51). no case of cardiotoxicity and no exitus was observed. the incidence of febrile neutropenia was 61% (70% fuo; 22% sepsis; 18% pneumonia). only one patient experienced an agvhd on day +9. neutrophil (>1000/mcl) and platelet (>50000/mcl) recovery occurred in median on day 11 (9-26) and on day 15 (4-45) respectively. conclusions: nepa seems to be safe and effective in preventing acute and delayed cinv in patients receiving both one day hd-ct and mds-ct as conditioning regimen for hsct. more studies are needed to define the better 5-ht3ra and nk-1ra combination and the better schedule in transplant setting. disclosure: "nothing to declare background: vod and ta-tma represent two early endothelial complications occurring after allogeneic stem cell transplantation (sct) sharing many pre-transplant risk factors. the aim of our study is to evaluate the impact of donor graft composition, engraftment kinetics and infections on the development of these endothelial complications (ec). methods: we retrospectively reviewed 55 consecutive sct recipients at our institu-tion between january 2015 and june 2018. the median age was 48 years (range 17-65). acute leukemia was diagnosed in 33 patients (60%). complete remission was documented in 63% of patients at transplant. donor source was from hla mismatched donor in 53% and from unrelated donor in 56% of the patients. hbv positive patients were 13% of the sample. conditioning regimen was busulfan based in 63% of patients. ursodeoxycholic acid and unfractionated heparin were given to all patients as vod prophylaxis. cyclosporine was used as gvhd prophylaxis. lymphocytic subpopulation analysis (cd3+, cd4+, cd8+ and cd16+/cd56 +) and cd34+ cells count on the donor graft were performed using bd facs cantoll. all patients had routine monitoring for ebv and cmv pcr, hemolysis tests, creatinine and electrolyte panels, proteinuria, complete blood count, blood pressure and schistocytes by direct examination until day +100. the fisher's exact test was used to compare categorical variables, while continuous variables were analyzed with anova test. diagnosis of vod and ta-tma were carried out by using ebmt and cho criteria respectively. results: the incidence of very severe vod and tma was 13% (7/55) and 14,5% (8/55) respectively. cmv reactivations with viral load over 30.000 cv/ ml was 27% and 5% in patients with and without ec, respectively (p 0,041; hr 2,97 p0,0055 95%ci 1,38-6,4). the median day to neutrophils (ns) engraftment (500/ml and 1000/ml) was 14 vs 17 and 14,5 vs 18 in vod/tma group vs control group (p0,03 and 0,027, respectively). more rapid neutrophils engraftment (ns >500/ml and ns>1000/ml within 13 days) was related to a higher risk of ec with a hr of 2,67 (p 0,015; 95%ci 1,2-5,9) and 2,94 (p 0,006; 95% ci 1,4-6,3). patients with ec received a donor graft with a higher median numbers (x10e6/kg) of cd3+ and cd8+ (p>0,05) and a lower numbers (x10e6/kg) of nk cells (p>0,05). patients who received a cd8+ cells count >12x10e6/kg and nk cells count < 2,5x10e6/kg presented a relative risk of ec of 2,37 (p 0,036; 95% ci 1,06-5,3) and 2,35 (p 0,004; 95% ci 1,02-5,4), respectively. there were no differences with respect to the other analyzed variables between patients who developed vod/tma compared with those who did not. (pic_01) conclusions: cmv viremia, early neutrophils engraftment and donor nk and cd8+ cells infused are associated with the risk of vod and tma. very few studies evaluated the link between these variables and the risk of developing such two complications. it could be interesting to investigate these relationships on larger series. clinical trial registry: not applicable disclosure: nothing to declare p358 when the last hope turns out to be just as good as best: haplosct following tbf conditioning, pt cyclophosphamide and tacrolimus as gvhd prophylaxis background: hematopoietic stem cell transplantation is an effective therapy for a variety of severe hematological diseases. in last decades, haploidentical sct (haplosct) followed by ptcy as gvhd prophylaxis has been reported as a valid alternative for patients who lack an hla matched donor. we therefore analysed outcomes with this. methods: 38 patients without hla-matched donor received a haplosct between 04/13 and 08/18. thiotepa 5 mg/kg (2 days), fludarabine 50 mg/m2 (3 days) and oral busulfan 1 mg/kg/6 h (3 days,with pkd dose adjustments) was used as conditioning regiment; in patients >55 years busulfan administration was limited to two days. gvhd prophylaxis consisted of cyclophosphamide 50 mg/kg on day +3 and +4, and tacrolimus as a continuous iv infusion from day +5. all patients received pbsc as the stem cell source. outcomes analysed were overall survival (os), progression free survival (pfs); cumulative incidences (ci) of gvhd, relapse and non-relapsed mortality (nrm). results: median ages was 54 (range 25-71). 53% male and 47% female. diagnoses were: aml 20 (53%), mds 7 (18%), all 5 (13%), hl and nhl 5 (13%), and 1 mm (3%). 37% (n 14) were transplanted in early disease status, while most cases (63%) were in advanced status, including, second/third cr (29%, n 11), one (3%) in aplasia without progressive disease and 23% (n 9) had active/progression disease, 8% (n 3) had stable disease; four cases were second alosct. thus, 50% of patients had a high or very high rdri and 50% had intermediate. ebmt score was ≤ 4 was in 58% of patients (n 22) . the donor was as son/ daughter in 50%, 29% a sibling and 21% a patient's mother. median time to neutrophil (0.5x10e9/l) and platelet (>20x10e9/l) recoveries were +20 and +27 days, respectively (g-csf was not used). only one patient had a primary graft failure attributed to anti-hla donor specific antibodies. median follow up in survivor is 22 months (range 2-63). overall survival is 81±6.5% (at 12 months) and 61±9% (at 63 months). pfs is 67±8% and 52±9% respectively. the cumulative incidence of relapse was 20% and 35%, respectively, while nrm is 13% at 63 months. day +120, grade 2-4 acute gvhd were 13%, while mild/ moderate and severe chronic gvhd were 6% and 3% respectively. ebmt ≤ 4 and first alosct were the only variables to clearly impact 60-months os in univariate analysis. a combined covariate of ebmt ≤ 4-no prior alosct vs other patients showed a 60 month os 81±13% vs 38±12% (p 0.001), pfs 65±13% vs 35±11% (p 0.014) and nrm 0% vs 29% (p 0.008) but without impact on relapse 34% vs 35% (p 0.68). conclusions: haplosct with an age-adapted tbf conditioning regimen, pbsc and ptcy followed by tacrolimus, led to very encouraging results, mainly in patients with a low ebmt score and as a first alosct. although formerly considered as a last alosct strategy, we now agree that the time has come to compare this strategy with hla mud (and even elderly sibling donors) in ongoing prospective randomized multinational trials. disclosure: nothing to disclosure background: autologous hematopoietic stem cell transplantation (autosct) for acute myeloid leukaemia (aml) is increasing becoming a viable option for an increasing number of patients due to limited availability of matched sibling or unrelated donor for allogeneic hematopoietic stem cell transplantation (allosct). we examined the relevant long-term outcomes in our local patient cohort. methods: we retrospectively reviewed the data for all autosct done for aml in our centre over a 17-years period between 1 st january 2001 until 31 st dec 2017 from our electronic record. patients with acute promyelocytic leukaemia (apml) were excluded from this analysis. patients were further stratified based on the number of high risk features present; not achieving complete remission (cr) following induction chemotherapy, high presenting total white cell count (wbc > 100 x 10 6 /ml, adverse cytogenetics (example: complex cytogenetics) and adverse molecular mutations (example: flt3-itd & mll gene arrangement). outcome data including mortality (overall survival (os) and non-relapse mortality (nrm)) and morbidity (leukaemia free survival (lfs)) were recorded and analysed. results: a total of 64 patients were identified. median age at diagnosis is 34-years old. the cohort comprised of 34 males and 30 females. the overall median os and median lfs is 3.9 years and 2.2 years respectively. the nrm is 1.6% (1/64). there was no difference in the median os and median lfs for the patients achieving cr following induction chemotherapy and those not in cr following induction chemotherapy; 4.4 years versus 3.9 years (log-rank, p=0.9) and 3.4 years versus 2.1 years (log-rank, p=0.9) respectively. the median os were statistically significant for patients with zero versus one and two and more high risk features present; 10.2 years versus 3.7 years versus 2.2 years (log-rank, p=0.4) respectively. however, the median lfs were not statistically significant for these three patient cohorts; 3.6 years versus 1.9 years versus 1.4 years (log-rank, p=0.7) respectively. conclusions: in our patient cohort, autosct appeared to be a feasible option for patents with aml without matched sibling or unrelated donor available. disclosure: none to declare methods: between 2014-2018, 16 thalassemic patientsunderwent hisct. the median age of patients was5 (1-8)years with male preponderance (n=10, 62.5%). across the gender and abo mismatch transplants were done in 43.7% and 25% of patients. stem cell source was bone marrow in 5 (31%) while peripheral blood in 11 (69%) of patients. mean stem cell dose was 5.6 ± 2.9 x 10 6 cells / kg and mean volume of product was 188 ± 60.58 ml. preparative regimen included anti-thymocyte globulin, busulfan, fludarabine and cyclophosphamide.graft versus host disease (gvhd) prophylaxis comprised of posttransplant cyclophosphamide on day +3 & +4 followed by tacrolimus and mycophenolate mofetil. patients were observed for hematopoietic recovery (neutrophil and platelet engraftment) and transplant related mortality including acute and chronic gvhd for skin, gut, liverand lungs, primary and secondary graft failure and infectious complications. results: nine(56.25%) of sixteenpatients were engrafted with full donor chimerism. twelve (75%) patients belonged to pesaro class i and 4 (25%) toclass ii patients. median time to neutrophil and plateletengraftment were13(11-20) and16(12-36)days respectively. average number of packed red cell and platelet transfusions were 4.35±7.54 and 20.8 ±19.18 respectively.primary graft failure was observed in 3 (19%) and secondary graft failure was observed in 4 (25%) patients. two patients received a second dose of stem cells and they engrafted at 20and 32 days of infusion respectively.2 of 3 patients with primary graft failure died, one with sepsis (day +23) and the other because of intracranial bleeding (day +21).acute gvhdof gut and skin (grade ii-iii) was observed in 2 patients each, within first 100 days post-transplant. none of the patients had grade iv gvhd. cytomegalovirus reactivation occurred in 50% of patients, all of them received pre-emptive therapy with intravenous ganciclovir. none of them developed cmv disease. invasive fungal infection was not observed in any of the patient. culture proven bacterialinfection was documented in 62% of patients requiring intravenous antibiotics during first 100 days post-transplant.overall survival and relapse free survival were 81.25 % and 56.25% over a median follow-up of 500 (21-1757) days. conclusions: haploidentical transplant is a suitable modality for thalassemic patients lacking a full matched donor in pakistan. in view of our results, we suggest that thalassemia patients should be offered hisctas an option for cure. clinical background: gemtuzumab ozogamicin (go) is an anti-cd33 monoclonal antibody with significant activity in de novo and relapsed/refractory (r/r) acute myeloid leukemia (aml). a relevant side effect consists of hepatotoxicity and especially sinusoidal obstruction syndrome (sos). the objective of this study was to analyze tolerability of go during the induction and reinduction therapy in patients with aml, and its possible impact on subsequent hematopoietic stem cell transplantation (hsct). methods: from 2004 to 2017, 24 patients who had received go in three hospitals were collected and their medical records were retrospectively reviewed. results: fourteen patients diagnosed with de novo aml received go (3mg/m 2 ) on day +1 in combination with standard chemotherapy (idarubicin and cytarabine, 3x7 schedule) as induction therapy. hyperbilirubinemia (bilirubin >1.5 unl) was detected in 4 patients and increase of aspartate aminotransferase (ast) (>2.5 unl) in 1. twelve patients achieved complete remission (cr) and one was refractory (1 not evaluated). in the r/r setting, 10 patients diagnosed with aml (n=8), biphenotypic acute leukemia (n=1) and acute promyelocytic leukemia (n=1) received go as 3rd or subsequent rescue therapy either as monotherapy (n=4) or in combination with cytotoxic chemotherapy (n=5). prior hsct was performed in 5 patients (autologous [n=2], allogeneic [n=3] ). rescue therapy was indicated for refractoriness (n=2), relapse (n=5), partial response (n=1) or absence of donor (n=2). four patients received 3 doses of go (3 mg/m 2 ) and 3 patients, one dose. hyperbilirubinemia (>1.5 unl) was observed in 1 patient and increase in ast (>2.5 unl) in 2 patients. seven patients achieved cr, 1 was refractory, 1 obtained partial response and 1 died early during induction). thirteen patients received subsequent hsct (autologous [n=4], allogeneic [n=9]) after go therapy (10 in the de novo aml and 3 in the r/r group). the reasons for not performing hsct in the remaining 11 patients were: low cytogenetic risk (n=1), active chronic graft versus host disease (gvhd) in previous hsct (n=1), early death during treatment (n=3), relapse (n=1), severe complications in rescue treatments (n=1), and unknown (n=4). the conditioning regimen was myeloablative (n=8), non-myeloablative (n=4) and sequential (n=1), and the donors were matched sibling (n=6) or unrelated (n=3) . cyclosporine, methotrexate and thymoglobulin were administered as gvhd prophylaxis in 7 patients and cyclosporine, mycophenolate and thymoglobulin in 3. hyperbilirubinemia was observed in 2 patients belonging to the de novo aml group. death after hsct occurred in 10 patients due to infection (n=5), relapse (n=3), gvhd (n=1) and traffic accident (n=1). three patients are currently alive in remission. no sos was observed in any patient. conclusions: in both de novo and r/r aml the administration of low dose go is feasible and does not have impact on subsequent hsct outcome. although some degree of hepatotoxicity was observed, no cases of sos were observed, either before or after hsct. disclosure: supported by grants from: asociación española contra el cáncer, aecc (gc16173697biga), instituto carlos iii (pi14/01971 fi), 2017-sgr288 (grc), cerca program from generalitat de catalunya, and "la caixa" foundation. outcome of allogeneic hematopoietic stem cell transplantation in patients with benign hematological disorders saqib ansari 1 , tahir shamsi 1 , uzma zaidi 1 , saima siddiqui 1 , tasneem farzana 1 background: allogeneic hematopoietic stem cell transplantation is a potentially curative treatment modality for hematological disorders. we evaluated the outcome of patients suffering from benign hematological disorders, including aplastic anemia, fanconi's anemia and thalassemia after matched related allogeneic transplantation. methods: all patients having hematological disorders including aplastic anemia (aa), beta thalassemia (btm), fanconi's anemia (fa) and severe combined immune deficiency disorder (scid) with hla identical related donors who underwent allogeneic transplantation were included. donors were given g-csf at a dose of 10 μg/ kg/day daily for four days prior to harvest. the conditioning regimens for thalassemia included cyclophosphamide (cy) + busulfan (bu) in 21 (20%), bu + cy + thiotepa in 4 (4%) and bu + cy + antithymocyte globulin (atg) in 81(76%). conditioning regimens for aplastic anemia included, fludarabine (flu) + cy in 38 (33%), flu + atg in 28 (25%) and cy in 38 (33%), cy+ atg in 9 (8%) patients. for fanconi's anemia flu + atg in 12 (52%), flu in 2 (9%), cy + atg in 4 (17%) and flu+ cy + atg in 5 (22%). flu + atg in 2(25%) and cy given in 4 (50%) and no conditioning regimen was offered to 2 (25%) patients with scid. results: a total of 250 allogeneic transplants were performed for benign hematological disorders including aa (n=113), btm (n=106), fa (n=23) and scid (n=8) from 2011 to july 2018. median age was 7.5 years (range 0.5 -48). across the gender and abo blood group transplants were 109 (43.6%) and 80 (32%). the median time to neutrophil and platelet recovery was 13 days (range: 9-46) and 18 (range: 10-35). primary and secondary graft failure was observed in 34 (13.6%) and 29 (11.6%). overall survival in aplastic anemia (63/113, 56%), beta thalassemia (82/106, 77%),fanconi's anemia (15/23, 65%) and severe combined immune deficiency disorder (6/8, 75%). eighty four patients expired (33.6%) among them 41 patients expired within 100 days post transplant main cause of deaths included sepsis (31%), multi organ failure (6%) and gut gvhd (5%) conclusions: in developing world scenario where non malignant disorders are leading cause of morbidity and mortality. bone marrow transplantation has been successfully implemented with better long term diseases free survival and quality of life. clinical background: hematopoietic cell transplantation (hct) remains the only curative therapy for many diseases, yet transplant survivors carry an unusually high burden of morbidities, primarily because of exposure to intense chemotherapy, radiation and /or gvhd. this study aimed to evaluate the burden of chronic diseases at the end of life after allogeneic-hct and to identify the disability-adjusted life years (daly). methods: the pubmed, medline, and ovid databases were queried utilizing specific mesh terminology (post, allo stem cell, hematopoietic, bone marrow, transplantation).we collected data on the impact of the hct on the variables affecting survivor's health in all aspects .the rates of late complications were compared to the risks in the general population (united states). results: a total of 7 studies fulfilled the selection criteria totaling to 6619 patients (table 1) . median os at 5-year mark varied widely between studies from 19% to 92%. majority of the patients at 10-year mark were found to have new comorbidities thereby indicating a huge burden of late effects at the end of life, though exact dalys could not be calculated due to incomplete data. hct survivors were found to have higher risk of premature arterial disease (pad) at 6.8% compared to the general population, however gvhd or the addition of tbi to the conditioning regimen were not found to be significantly associated with pad. regarding the risk of new cancers, the cumulative incidence of their development at 5 and 10 years was 1.71, and 3.61, respectively. increased risks compared with the general population were seen for some solid cancer including cancers of the lip: p=0.02, tonsils: p=0.05, oropharynx: p< 0.01, bone: p< 0.01, soft tissue: p< 0.01, and vulva: p=0.01. with respect to mental health, depression was prevalent in (10.8%) survivors, in whom (82.5%) were still on antidepressants at the last follow-up. cognitive impairment and other psychiatric disorders were found in (2.4%) and (2.7%) survivors, respectively. the most common cause of nrm in the first 5 years was gvhd. however, after 10 years, the leading cause of death in those conditioned with mac regimen was secondary cancer, but in the ric group, new cancers and gvhd contributed equally. conclusions: hct survivors remain at risk of significant complications which lead to premature death and their burden of comorbidities at the end of life is significantly more than that of general population. background: late onset hemorrhagic cystitis (hc) is a common complication of hematopoietic stem cell transplantation (hsct) frequently associated with reactivation of bk virus (bk-hc).there is no consensus as to the best therapy for bk-hc, and many different treatments have been reported. hyper baric oxygen therapy (hbot) is used as primary or adjuvant therapy in diverse clinical situations involving hypoxic injury to tissues and has been explored as a useful tool in treating bk -hc. we report our experience with hbot in combination with non-invasive supportive care in children and adolescents suffering from bk-hc following allogeneic hsct. methods: the computerized database of schneider children´s medical center of israel was reviewed for all patients aged 0 to 21 years who underwent hsct between january 2000 and june 2018 and developed bk-hc. hbot therapy consisted of 2 hours sessions at 2 atmospheres, with patients breathing oxygen by mask. parents accompanied patients during the treatment. results: fourteen patients with a variety of underlying diseases received (hbot) for treatment of bk-hc following hsct. the initial treatment for children with bk-hc at our center prior to 2008 included continuous bladder irrigation and intravesicular instillation of various medications. beginning in 2008, we adopted a non-invasive strategy that included the administration of oral anticholinergics (oxybutinin), systemic pain management, hyperhydration and the administration of weekly cidofovir with probenecid. hbot was administered to patients who failed the above regimen. with this protocol, the average time of starting hbot dropped from 13 (prior to 2008) to 9.8 days. the median onset of hc was 33 days post hsct. all patients were receiving immunosuppressive treatment at the onset of bk-hc. all patients suffered from macrohematuria with blood clots (grade iii cystitis), 12 (92.3%) experienced severe dysuria and 11 (84.6%) urgency. bk viruria was present in all patients, and concurrent bk viremia was detected in 80% of those who were tested. patients reported symptomatic improvement at a mean of 3.6 days following the initiation of hbot. no patient experienced serious adverse effects due to hbot, but two patients required insertion of tympanic ventilation tubes. eleven of our 13 patients (84.6%) experienced complete remission of bk-hc following hbot, with an overall response rate of 92.3% (12/13 patients).eight of our patients (61.5%) eventually succumbed due to either hsct complications or disease relapse. conclusions: hyperbaric oxygen therapy is a safe, effective, non-invasive and well tolerated treatment modality for bk -hc and should be considered for first line therapy for this complication of hsct. clinical background: every year, almost one thousand cases of hematological malignancies in pediatric population are reported in peru. allogeneic hematopoietic stem cell transplantation (allo-hsct) is an alternative strategy in many of these cases. only between 20-30% of the pediatric population that requires a hsct has a compatible human leukocyte antigen (hla) donor. the remaining 70% have to access international donor registries, extending the awaiting time and conditioning the progress of the disease. allo-hsct has the potential to help children with several hematological disorders with non-compatible hla donor. hla genotypically identical sibling donors are the best option when pursuing an hsct. nevertheless, patients' alternative sources of stem cells could be obtained from an haploidentical donor like one of their parents. the haploidentical transplantation program with macs was implemented in peru in 2016 to reduce the risk of graft-versus-host disease (gvhd), support the immune system reconstitution and to expand pool of donors. it allows patients to access a treatment that is efficient and safe, as shown in the depletion of positive tcrα/β+ and b cells procedures for allo-hsct. methods: the mobilized leukapheresis products (n=19) of haploidentical healthy donor was washed to remove platelets and preparations were performed according to miltenyi's clinimacs® manual for tcrα/β+ and cd19+ cell depletion. analysis of the initial leukapheresis product and tcrα/β + and cd19+ depleted graft (target and non-target product) was performed using flow cytometer. cells were analyzed for cd3+, cd45+, 7-aad, cd20+, tcrα/β+, tcrγ/δ +, cd34+ and cd133+ with fluorochrome-labeled antibodies from miltenyi biotec using a novocyte cytometer. the results obtained with the ex vivo t-cell depleted allo-hsct procedures show an overall survival (os) over 70% with an ic95% at the end of the first year with low incidence of gvhd. macs of tcrα/β+ and cd19+ cells are effective (logp 3.9 and logp 3.3, respectively) obtaining minimum levels of depleted lymphocytes within clinical established parameters for diverse pathologies. in addition, tcrα/β+ and cd19+ cell depletion does not significantly affect hematopoietic stem cell populations such as cd34+ and cd133+ cells or tcrγ/δ+ cell population. cd34+ and tcr γ/δ cells are highly recovered (93.15% and 88.76, respectively), which contributes with a better engraftment after allo-hsct. conclusions: peruvian results oscillated within european ranges with an os over 70%. our data suggests that the macs method is an efficient, effective and safe strategy for haploidentical hsct which has a remarkable cost-benefit ratio and makes it viable in countries of the latin american region with peruvian socio-economic characteristics. evaluation of genoresistance for viral reactivation treatment has been implemented as a strategy to improve os. better results are achieved in patients after allo-hsct with the validation of these tests in peru. preliminary pharmacoeconomic evaluations allow us to establish magnetic activated cell sorting (macs) as a promissory strategy compared to other alternatives for haploidentical hsct. it is necessary to increase the number of procedures in order to confirm efficacy and safety of macs in a larger population. disclosure: all authors have no conflicts of interest. abstract already published. micro-costing study of hematopoietic stem cell transplantation in two hospital institutions from southern of brazil background: hematopoietic stem cell transplantation (hsct) is a potentially curative treatment indicated for patients with onco-hematological, hereditary and immunological diseases. considering the increase of patients indicated to the hsct and the lack of knowledge about the costs resulting from this treatment, is important to identify and detail the resources consumed in each phase of hsct and provide knowledge to the public health brazilian system. we aimed measure the total cost of related hsct, based on micro-costing study of patients assisted in two hospitals in the south region of brazil. methods: hsct costs were estimated using the timedriven activity based costing method (tdabc), which measured cost of services / products based on actual consumption of resources. we collected data from medical records of 12 patients submitted to allogeneic hsct in 2017 from public and private (philanthropic) hospitals. we interviewed professionals involved in the tcth activities, we performed chrono-analysis and, we consulted financial and administrative systems reports of hospitals. in order to compare costs according to clinical complications observed in patients, we grouped into two ranges of complexity: low/ medium and high. the study was divided into stages: hsct processes mapping; costs measurement; and analysis of results. finally, the costs were compared: by activity, by resource and by hospital. this study was financed by psid-uhs, by an agreement signed between ministry of health and moinhos de vento hospital, through adjustment term number: 04 / 2014, and approved by research ethics committees. results: from the hsct processes mapping, the following steps were defined: (i) hospitalization; (ii) conditioning; (iii) transplantation; (iv) period of aplasia; (v) engraftment; (vi) observation; (vii) pre-and medical discharge. seven patients were classified in low / medium complexity level, with hospitalization median time of 41 days and an median cost of usd 66,278.29, whereas the other five patients, classified as high complexity, presented median time of 101 days and median cost of usd 300,367.13. the hsct costs evaluation identified that steps ii and iv presented greatest cost in high complexity patients. lower complexity patients presented, in steps ii and iv, median costs of usd 44,274.52 while in higher complexity usd 100,226.26. in addition, median costs of materials and drugs were usd 13,926.02 and usd 181,094.15 in lower and higher complexity patients. conclusions: tdabc method allowed the identification of the moment when patients consume the most resources. of all the hsct stages, periods of conditioning and aplasia presented higher costs, representing 76.90% of the total hospitalization value. in these stages, higher complexity patients presented three times higher the median cost. the resources that had the greatest impact were medicines and medical materials, costing 120 times more than lower complexity patients. conclusion: this study allowed a detailed identification of the hsct costs in patients with different complexity ranges in two hospitals from southern brazil. therefore, the identification of service demand regarding the clinical complexity, allows the generation of important information for the management of the best care in the health service. disclosure background: immunodeficiency due to lrba deficiency is characterized by hypogammaglobulinemia and autoimmunity. hemolytic anemia, lymphadenopathy, autoimmune hepatitis and, above all, autoimmune enteropathy are the fundamental characteristics of these patients together with the history of recurrent invasive infections.the therapy includes immunosuppressants, endovenous immunoglobulins. bone marrow transplantation is the final therapy of these patients, especially in the most serious cases and in recent years, also on the light of increasingly targeted conditioning regimes, it is associated with ever better prognosis. methods: we present the case of caterina, an 8-year-old patient with lrba deficiency, diagnosed at 5 years of age for a history of hypogammaglobulinemia, recurring invasive, bacterial and fungal infections and a picture of autoimmunity represented by ahia, myelitis (c3-c5), autoimmune hepatitis and enteropathy in treatment with abatacept and sirolimus. it also presents leptin deficiency lipodystrophy.she presented to our observation for ostemielitis in multiple outbreaks (tibia, femur and left knee) secondary to sepsis from mrsa. broad spectrum antibiotic therapy and curettage surgery were performed during hospitalization.after 5 months of broad-spectrum antibiotic therapy (daptomycin, rifampicin, ceftaroline, cotrimoxazole, levofloxacin, dalbavancin) there was resolution of the infections.because of the seriousness of the disease it is therefore decided to subject catherine to hematopoietic stem cell transplantation. results: therefore were performed bone marrow transplant from mud after conditioning at reduced intensity, delayed in 9 days, with treosulfan, fludarabine and thiotepa. prophylaxis for gvhd was performed with atg (5mg / kg / day), sirolimus (already practiced for enteropathy) and mmf. because of the hepatic picture, we also performed prophylaxis for vod with defibrotide for 21 days. transplantation was performed by peripheral stem cells with 18 x 10^6 cd34 / kg and 30 x 10^6 cd3 / kg.the patient had always presented good general conditions with engrafment of the pmn to the d + 13 and the plt to the d + 11. the chimerism at d + 20 was 100% donor both on pmn and on pbl. prophylaxis for gvhd was changed on d + 7 by replacing the sirolimus with tacrolimus for the appearance of grade i cutaneous gvhd . conclusions: we have successfully performed bone marrow transplantation in patients with lrba deficiency. the new antibiotic molecules, used to induce infectious remission, the new low-intensity regimens, the prevention of the most fearful complications (vod) have been the key to success in such complicated case. the high number of cd34 cells infused with a controlled number of cd3 were the key then of rapid engraftment with minimal gvhd readily controlled by the immunosuppressant. disclosure background: in the absence of hla-matched related donor, allogeneic stem cell transplantation from haploidentical donors are potential alternatives for patients with hematological malignencies with an indication to allogeneic stem cell transplantation. herein, we retrospectively assessed the outcome of haplo-sct for patients with refractory hematological malignancies. methods: this analysis included 27 consecutive patients who underwent haplo-sct for various hematological malignancies at our center between october 2010 and may 2018. we used our institutional database to evaluate details and characteristics of patients and transplant outcomes. results: demographic features of the patients and donors have been summarized in table 1 . all of the patients had advanced disease with a high risk of relapse. the majority of patients underwent haplo-sct from their parents. out of 24 patients, early transplant-related mortality was seen in this cohort of 5 patients. four patients treated with second haplo-sct and recovered hematopoiesis after second transplant. the remaining 19 patients were followed in a median of 4 months. donor type abo group switch was observed in a median of 45 days (30-60 days) after transplant. the median time for engraftment was 19 days (range, 15-60) for all patients. after the first transplant, 9 patients developed acute gvhd (37.5%) with 7 patients having grade ii-iii acute gvhd. five (18.5%) had chronic gvhd, none of them with extensive manifestation. the prepative regimen was relatively well tolerated with limited regimen-related toxicity. cmv reactivation occurred in 11 patients (40.7%) during the follow-up of the study. eight patients (29.6%) relapsed after a median of 132 days post transplant (range, 45-588 days). cr was achieved in 17 (63%) patients after haplo-sct. mean estimated 5-year os and pfs are 66.7%±0.9% and 92.3%±0.7%, respectively. conclusions: given the growing data on the similarity of outcomes after hla-matched and haploidentical sct, further studies are required to determine whether factors may be more important for donor selection than hlamatching. clinical trial registry: -disclosure: nothing to declare outcome of allogeneic stem cell transplantation for hodgkin and non-hodgkin lymphoma: single center experince from turkey ayşe uysal 1 , hale bülbül 2 , nur akad soyer 2 , mahmut tobu 2 , murat tombuloglu 2 , guray saydam 2 , filiz vural 2 background: allogeneic sct (allosct) is generally optionally treatment choice for young and fit patients with relapsed/refractory lymphoma who were heavily pre-treated and after the failure of autologous stem cell transplantation (asct). relapse after asct is associated with a poor prognosis and allosct is a potentially curative therapy for lymphomas which have relapsed after asct. methods: in this study, we evaluated 19 patients with hl and nhl who had treated with allo-hsct between november 2014 and december 2018 in ege university adult hematology transplantation unit. results: patients, disease and transplant characteristics were illustrated in table. histologic subtype of nhl was evaluated as t cell lymphoma (n=8;61,5%), mantle cell (n=2;15,4%), diffuse large b-cell lymphoma (n=2;15,4%) and b-cell lymphoma, unclassifiable, with features intermediate between dlbcl and classical hodgkin lymphoma (n=1;7,7%). all histologic subtype of hl was determined as nodular sclerosing. the median number of prior treatments before allo-hsct was 3 (range, 1-4). twelve (63,2%) patients had refractory disease, 3 (15,4%) patients were in complete remission and 4 (21,1%) patients were in partial remission before allo-hsct. the median time from diagnosis to allosct was 24 (range, 8-144) months. peripheral stem cell was used for stem cell source in all of them. total body irritation plus fludarabine plus cyclophosphamide and busulfan plus cyclophosphamide were preferred most frequently for conditioning as non-myeloablative and myeloablative, respectively. neutrophil engraftment was occurred median of 17 (range, 10-21) days. graft versus host disease (gvhd) prophylaxis was applied all of them and cyclosporine plus methotrexate was preferred most frequently (n=16;84,2%). gvhd was occurred in 68,4% of them (42,1% acute gvhd, 31,6% chronic gvhd and 7,7% both). veno-occlusive disease (vod) was occurred in 2 (10,5%) patients. transplant related death was observed in 5 (26,3%) patients. overall survival (os) and disease-free-survival (dfs) were evaluated as median 9 (range, 0-45) and 7 (range, 0-45) months, respectively. analyze of os and dfs was illustrated in figure. six patients are alive without disease. after a rapid tapering of immunosuopressive therapy was underwent a therapy with ponatinibat dose of 45mg/die results: afther a month of therapy we observed a rapid decrease in minimal residual disease on molecular assessment with an mmr of p190-bcr-abl/abl non detectable confirmed by bone marrow revaluations at days + 190, +222nd +244 after the salvage therapy. the patient has not had experienced of graft-versus-host disease, ponatinib treatment was well tolerated and considered safe with easily manageable side. conclusions: maybe in the era of tyrosine kinase inhibitors (tkis), philadelphia chromosome positive acute lymphoblastic leukemia (ph+ all) it could benefit from a combined treatment between transpalnt and tkis however more studies are needed to confirm these hypotheses. disclosure: nothing to declare immunodeficiency diseases and macrophage background: although a number of patients with hiv infection and hematological disease have successfully undergone allogeneic hsct together with combination anti-retroviral therapy (cart), short and long-term outcomes remain not well known. we report the largest spanish experience of hiv-infected adult patients with high-risk hematological malignancies with allogeneic hsct. methods: we retrospectively reviewed 22 hiv-positive patients who received allogeneic hsct between 1999 and 2018 in 5 spanish centers within geth (grupo español de trasplante hematopoyético y terapia celular). results: baseline and transplant characteristics of patients are shown in table 1 . median age was 44 years and 77% of the patients were men. the most frequent underlying malignancies were non-hodgkin lymphoma (9, 41%) and aml (7, 31%). in half of the patients an hlaidentical sibling was the donor; and in the other half, an alternative donor was used. peripheral blood was used as graft source in 86% of the transplants. at the time of hsct, all patients had been receiving suppressive cart for a median of 6 years and only 2 of them showed detectable plasma hiv rna, one of them because of poor adherence to cart together with the accumulation of multiple resistance mutations; and the other patient had detectable hiv rna at low levels (< 150 copies/ml). all patients received cart throughout the transplant procedure, being temporally stopped in two patients due to significant mucositis. after a median follow-up of 65 months (8-112), 5-year overall survival (os) and event-free survival (efs) were 46%. nrm was 14% at 12 months and relapse was 24% at 24 months. grade ii-iv agvhd rate was 40%, and moderate/severe cgvhd rate was 41% at 24 months. a significant proportion of patients (68%) showed infectious complications with viral infections as the most frequent cause. two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. causes of death included infections (50%), relapse (43%) and toxicity (7%). among the 6 patients who died due to infections, 3 had severe chronic gvhd and were under immunosuppressive therapy. two patients showed severe toxicity related to drug interaction with anti-retroviral therapy. all survivors except one showed undetectable hiv load at last follow-up after hsct. conclusions: allogeneic hsct is an effective therapy for high-risk hematological malignancies in patients with hiv infection, providing long-term disease free survival together to long-term hiv suppression with cart. however, drug interactions with anti-retroviral agents, occurrence of gvhd, and frequent infectious complications account for a complex procedure in this population. selected hiv-infected patients with hematological malignancies should be considered for allo-hsct when indicated, in experienced centers, with a multidisciplinary care. disclosure background: primary immunodeficiencies (pid) are rare diseases often associated with genetic defects in the immune system, predisposing individuals to recurrent infections and increased risk of allergy, autoimmunity and malignancy. allogeneic haematopoietic stem cell transplantation (hsct) has been successfully used as a curative therapy for most severe forms of pid. because pid is a genetic disease, < 25% of these children will have a healthy, human leukocyte antigen (hla) matched sibling donor available, and umbilical cord blood grafts from unrelated donors are a suitable alternative cell source. we report the results of umbilical cord blood transplantation (ucbt) performed in 112 patients with pid between 2014 and 2018 at children's hospital of fudan university in china. methods: 112 patients included chronic granulomatous disease (cgd, n=39), severe combined immunodeficiency (scid, n=23), interleukin-10 receptor-a deficiency (il-10rad, n=27), wiskott-aldrich syndrome (was, n=7), leukocyte adhesion deficiency (lad, n=5), severe congenital neutropaenia (scn, n=3) and other immunodeficiencies (n=8). all patients were assessed by clinical immunologist to confirm clinical phenotype and genetic diagnosis. median age of 112 patients was 13 months (range, 2 to 144 months), and median body weight was 8.75 kg (range, 3.2 to 36 kg). all patients received a ≤3/10 hla alleles-mismatched cord blood unit, 16 were hla fully matched, 28 were 9/10 matched, 49 were 8/10 matched and 19 were 7/10 matched. median nucleated cells of the cord blood were 14.23x10 7 /kg (range, 3.55 to 51.5 x10 7 /kg), and median cd34+ cells were 3.86 x10 5 /kg (range, 0.73 to 30.27 x10 5 /kg). results: median follow-up time was 13 months (range, 1 to 57 months), the overall survival rate at 1 year for all patients was 74.6%, and was 83.7%, 78.3% and 60% for cgd, scid and il-10rad, respectively. 27 patients died, most deaths (19/27, 70.4%) occurred in +100 days after transplantation, the main cause of death was infection (24/ 27, 88.9%). 87/112 (77.7%) patients engrafted, median time of neutrophil engraftment was 24 days (range, 11 to 60 d), and median time of platelet engraftment was 36 days (range, 9 to 59 d). the cumulative incidence of grade 3-4 acute gvhd was 8.9%, and that of chronic gvhd was 10.7%. conclusions: unrelated ucbt should be considered for pid patients without an hla -matched sibling donor. effective control of infection before and after transplantation is important for improving survival. disclosure background: dedicator of cytokinesis (dock 8) deficiency causes a combined immune deficiency characterised by recurrent bacterial infections, susceptibility to viral infection, eczema, food allergies, vasculitis and increased risk of malignancy. due to the high morbidity and mortality of the disease hsct has been increasingly offered to patients as a potentially curative therapy 1 . methods: we retrospectively reviewed the outcomes of hsct for patients with dock 8 deficiency at great north children's hospital newcastle upon tyne between 2011 and 2018 (5 in published reference). results: ten patients with dock 8 deficiency were treated with hsct (median age 7.5y range 4.5-16.8y). median duration of follow up was 4.5years (range 1.3-6.5y). there were a range of donor sources (3 msd, 3 mud and 4 tcr ab/cd19+ depleted haploidentical), conditioning regimens (6 treo-flu, 4 treo-flu-thiotepa) and serotherapy (5 alemtuzumab, 3 atg+rituximab, 2 none). one patient who received a cd3+ tcr alpha beta/cd19+ depleted haploidentical transplant received add back t-cells with caspacide molecular safety switch (bellicum pharmaceuticals). skin only agvhd occurred in 3/10 patients (1x stage 1, 2x stage 3). no patients had cgvhd. overall survival was 60% (6/10). survival was comparable regardless of donor source. all deaths occurred within 13 months of transplant. the 4 patients who died had significant burden of disease pre-transplant: 1 patient had chronic liver failure secondary to cryptosporidial sclerosing cholangitis,1 had a cirrhotic liver secondary to cryptosporidium, cerebral vasculitis, an axillary aneurysm and aortic vasculitis requiring grafting of an ascending aortic aneurysm,1 was pn dependent for failure to thrive with a history of cryptosporidium infection and 1 had candida in a bal pre-transplant. causes of death in these patients were: respiratory failure (n=1), progressive encephalopathy (n=1), multi-organ failure with septic shock and encephalopathy (n=1) and multiorgan failure and septic shock after treatment for tma (n=1). two of these patients had reactivation of cryptosporidium prior to their death. pretransplant cryptosporidium was associated with mortality (graph 1). one patient who survived had suffered from stroke pretransplant. one suffered from a basilar artery aneurysm 7 years post-transplant at 19yo. at the time of latest follow up donor chimerism was 100% in 5/6 survivors and high level mixed in the other(100% cd3, 89% cd15 and 92% cd19). conclusions: this single centre study of hsct for patients is consistent with literature indicating that hsct is a potentially curative therapy for patients with dock 8 deficiency. the increased morbidity associated with cryptosporidial infection is likely to be a consequence of overall disease burden rather than an infection specific effect. this does however highlight the improved outcomes of transplant prior to development of multiple comorbidities and suggests that hsct should be considered early. it is unclear whether the late occurrence of vascular complications after transplant were caused by a manifestation of disease which is not corrected by transplant or a result of vascular injury sustained pre-transplant. reference methods: referred infants underwent testing for: immune phenotype (ab, gd, naïve and memory t cell, b cell and nk cell numbers); functional activity of t and nk cells; maternal engraftment; adenosine deaminase (ada) and purine nucleoside phosphorylase (pnp) enzyme activity; and genetic testing. those with a confirmed diagnosis of scid underwent either allogeneic hematopoietic stem cell transplant (hct) or (if eligible) gene therapy (gt). infants identified as having ada deficiency as the etiology of their scid received enzyme replacement therapy prior to proceeding to definitive therapy. results: twenty-three (66%) infants were confirmed to have scid. three (13%) of these infants had a family history of scid but would not have been identified without nbs. in addition, one infant, born prematurely at 28 weeks, was diagnosed as having pnp deficiency only after developing infections. this infant was identified by nbs but repeat testing at 32 weeks gestation was normal likely due to support of transient t cell production from exogenous enzyme provided by red cell transfusion. twelve infants with confirmed low trecs had a non-scid diagnosis: 5 with transient lymphopenia of infancy who normalized trec, immune phenotype and function, 1 with prenatal exposure to 6-mecaptopurine (6-mp), 3 genetically confirmed with digeorge syndrome, and 3 with prolonged lymphopenia. of the three with prolonged lymphopenia, two had recurrent infections: one ultimately diagnosed with ataxia telangiectasia and one with absent trec but near normal number of t cells, normal pha but no specific antigen responses, and absent b cells who will be undergoing transplant in the near future. the third continues with absent trec, short telomeres, low numbers of a/b t cells, presence of g/d t cells, vaccine responses and freedom from infection with no identified genetic etiology. in summary, 30% of the patients referred to msk with confirmed abnormal nbs for scid have a non-scid diagnosis. there is no uniform collection of data for these infants and the threshold trigger for repeat testing varies from state to state, so the incidence of significant non-scid disorders identified will also likely vary from state to state. although our institution specific experience is biased, as most infants had confirmation of a low number of trec prior to referral, the significant number of disorders in the non-scid cohort emphasizes the importance of full evaluations and follow-up for these infants. disclosure: none of these relate to the work being presented. susan prockop -research funding mesoblast and atara biotherapeutics. nancy kernan -research funding jaz pharmaceuticals. richard o´reilly research funding and royalties atara biotherapeutics. kevin curran consulting juno pharmaceuticals, novartis. j.j. boelens avrobio, magenta, chimerix and bluebird bio background: post-transplant autoimmune cytopenia (aic) is challenging and associated with substantial morbidity and mortality. we aimed to study the cumulative incidence (ci) of post-hct autoimmune cytopenia (aic) and its predictors in a cohort of children with primary immunodeficiency (pid). methods: in this retrospective study, we included 199 children with pid who underwent their first hsct with fludarabine(f)-treosulfan(t)±thiotepa(thio) at great north children's hospital from 2007-2017. main outcomes of interest were the ci of aic and its predictors. fine-and-grey regression models were used to analyse predictors of aic, considering death as a competing event. variables included were age at transplant (< 2.5 years vs >2.5 years), gender, diagnosis (scid vs immune-dysregulatory disorders vs other pids), pre-transplant aic, pre-and posttransplant respiratory virus, donor (mfd vs mud vs mmfd/mmud vs haploidentical donor), abo incompatibility, conditioning (ft vs ftthio), serotherapy (none vs alemtuzumab 0.3-0.6mg/kg vs alemtuzumab 0.9-1.0mg/kg vs atg), stem cell source (marrow vs pbsc vs cord vs exvivo t depleted pbsc), infused stem cell doses (tnc, cd34 and cd3), agvhd (none vs any agvhd), cgvhd (none vs any cgvhd), viral infections (cmv/adenovirus/ ebv/hhv6 viraemia), chimerism (full vs mixed chimerism (wb < 95%) within first year post-hct). impact of thymopoiesis using naïve t cell recovery was studied. results: median age at transplant was 2.4 years (range, 0.11-18.3 years). primary diagnoses were scid (22%), immune-dysregulatory disorders (28%) and other pids (50%). donors were mfd (21%), mud (48%), mmfd/ mmud (16%) and haploidentical parents (15%). stem cell sources were marrow (30%), unmanipulated pbsc (38%), ex-vivo t-depleted pbsc (23%) and cb (9%). 16% received additional thiotepa and 87% had csa/mmf as gvhd prophylaxis. median duration of follow-up of survivors was 2.9 years (range 0.2 to 10.2 years). 5-year os for the entire cohort was 81%. 6-month and 1-year ci of aic were 5% and 13%. of 21 developed aic, 17 (80%) had aiha, 3 (15%) had aiha±itp and 1 (5%) had aiha ±itp±ain. median onset of aic was 6.2 months post-hct (range 0.2-12.8 months). patients were treated with a median of 3 treatment modalities (range, 1-4). one (5%) had steroid, 8 (38%) had steroid+high-dose-ivig, 8 (38%) had steroid+high-dose-ivig+rituximab, 1 (5%) had steroid +high-dose-ivig+sirolimus and 3 (14%) had steroid +high-dose-ivig+rituximab+sirolimus. the median time to resolution in 18 (85%) who achieved remission after first aic was 6.5 months (range 1.4-28.6 months). 1 had one relapse and 2 had two relapses. 2 died after development of aic (1 aspergillus pneumonia; 1 multi-organ failure). of 19 (91%) surviving patients after aic, 4 had on-going aiha at median of follow-up 2.5 years post-hct (range 0.9-4.8 years). on univariate completing-risk analysis, age at transplant >2.5years (p=0.02) and pre-transplant aic (p=0.02) were associated with higher incidence of aic (figure 1a -ic). on fine-and-grey models, only age at transplant (hr 3.08, 95%ci 1.12-8.49, p=0.03) was independently associated with aic. of 157 with complete immune reconstitution data, naïve t cells >100cells/ml at 6 months post-hct was associated lower incidence of aic (hr 0.36, 95%ci 0.15-0.91, p=0.03) (figure 1d) conclusions: younger age and thymopoiesis were associated lower incidence of aic in children with pid after hct clinical background: human heme-oxygenase-1 (ho-1) deficiency has been reported to present with tetrad of anemia, nephritis, inflammation and asplenia and is fatal if not treated. its an auto-inflammatory disorder. macrophages/ monocytes express ho-1 and are engaged in recycling of red cells. human ho-1 deficiency results in intravascular hemolysis and severe damage to the endothelial system, kidneys, and other organs. transplantation of either healthy wild type macrophages or new macrophages produced by sct from healthy donor has been proven to be curative for ho-1 deficiency in mice. in 2018, we had reported first successful allogeneic sct for human ho-1 deficiency. here we report second successful non-myeloablative msd hsct for a child with ho-1 deficiency. methods: a 9-yr-old-girl presented with complaints of fever, anemia and severe hypertension. in the past, at the age of 7 years she was admitted for high fever for 1 month and needed blood transfusion for the first time for severe anemia and had high platelets and high ferritin. she was treated as macrophage activation syndrome with prednisolone alone and later cyclosporine was added. she had short stature, abnormal facies but normal development. hemoglobin 9 g/dl, urine for haemoglobin was positive, platelets 1055, 000/ul, ferritin 2568 mcg/l and urine albumin 4+ and urine rbc 20-30/hpf. ultrasound and ct scan abdomen showed asplenia. a diagnosis of ho-1 deficiency was suspected. mutation analysis showed homozygous missense mutations in exon2 (r44x) on chromosome 22q12, which would result in the absence of the functional ho-1 protein. both parents were carriers of this mutation. we managed her over next 6-years with prednisolone, hydroxyurea and mycophenolate mofetil (mmf). however she remained steroid dependent. hla-typing confirmed her healthy unaffected 13-year-old brother to be a fully matched donor. at the age of 16 years she was taken up for msd sct after taking informed consent. she weighed just 16 kg. we conditioned her with alemtuzumab-0.8mg/kg, fludarabine-160mg/m2, cyclophospamide-29mg/kg and total body irradiation 2 gray. we infused 9 million/kg peripheral blood stem cells from her brother. graft-vs.-host disease (gvhd) prophylaxis consisted of tacrolimus & mmf. results: she tolerated procedure very well. her entire hospital stay was uneventful and lowest platelet count recorded was 30,000/ul. her neutrophils engrafted on day +13 and she was discharged on day+19. his urine albumin was nil by day+7. she had no gvhd. her chimerism on day+22 showed 97 % donor cells, on day+60 was 98% and on day+180 was 98% donor. now he is day+210 post-sct and doing well. she has no evidence of hemolysis, proteinuria, hypertension, fever. she has normal ferritin and platelets. she has gained 3 cm height and 5 kg weight in last 7 months. she had no viral reactivation and her immune recovery at 6 months post sct is good. conclusions: non-myeoablative allogeneic msd sct is a curative treatment option for human ho-1 deficiency. disclosure: nil two decades of excellent transplant survival in children with chronic granulomatous disease: a report from a supraregional immunology transplant centre in europe background: haematopoietic stem cell transplantation (hsct) confers life-long curative therapy for chronic granulomatous disease (cgd). the ability of donor-derived neutrophils to replace recipient's defective neutrophils makes hsct a superior therapy compared to conventional standard of care using antimicrobial therapy. methods: we examined the outcome of children with cgd who received a first hsct at great north children's hospital from 1998 to 2017. outcomes included overall survival (os), event-free survival (es), toxicity endpoints, autoimmune disease, long-term survival and graft function. cox proportional-hazard models were used to analyse predictors of os and es. variables included for predictor analysis were age at transplant, donor, stem cell source, stem cell doses and conditioning. results: =55 children were included in this analysis. median age of transplant was 5.3 years (range, 0.6-18.0 years). 45 (82%) had x-linked and 10 (18%) autosomal recessive cgd. twenty (36%) had matched family donor, 31 (56%) had unrelated donor and 4 (8%) had parental haploidentical donor. prior to 2007, various conditioning regimens were used, with 21 (38%) patients undergoing conditioning with pharmacokinetic guided intravenous (iv) busulfan (bu) and iv cyclophosphamide with or without serotherapy. from 2007, the conditioning regimen was switched to flu-treosulfan-alemtuzumab with gvhd prophylaxis using ciclosporin (csa) and mycophenolate mofetil (mmf) for family and unrelated donors (n=24, 44%). flu-treosulfan-thiotepa-atg-rituximab was used for cd3 tcr alpha-beta cd19 depleted haploidentical grafts (n=4, 7%). ten (20%) patients had grade ii-iv acute gvhd while 5 had (9%) had grade iii-iv acute gvhd. none had chronic gvhd. the 5-year os for the entire cohort was 89% (95% ci, 67-95%) (figure 1 ). analysis by age at transplant revealed a 5-year os of 100% for children transplanted at < = 5 years of age and 81% (95%ci, 60-92%) for the children >5 years of age (p< 0.04) (figure 2) . the os was comparable between match family donor (88%, 95% ci, 61-97%) and unrelated donor transplant (89%, 71-95%) ( figure 3 ). all four haploidentical transplants were successful. the 5-year es for the entire cohort was 77% (95% ci 62-87%). none of the variables was associated with es. all seven patients with slipping chimerism received a successful second transplant. the five deaths were all due to transplantrelated complications (2 multi-organ failures; 1 pulmonary haemorrhage; 1 graft iv acute gvhd; 1 post-transplant lymphoproliferative disease). the median age at transplant of deceased patients was 10.0 years (range 8.4 to 18 years). the 1-year and 5-year cumulative incidence of autoimmune diseases were 9% and 12% respectively. three (5%) had immune cytopenia while 3 (5%) had autoimmune endocrinopathy (2 thyroid dysfunction; 1 type 1 diabetes mellitus). the median age of long-term survivors was 14 years (range, 2 to 36 years) with the median duration of follow-up of 6.5 years (range, 0.32 to 19.5 years). there was no late death in the entire cohort. the median donor myeloid chimerism was 100% (range 23 to 100%) conclusions: despite the limitations of a single centre study, our findings confirm that hsct is a safe and longlasting curative therapy for children with cgd disclosure: none non -medical challenges in the diagnosis and transplantation of patients with primary immune deficiency: an experience from a tertiary care center in india sagar bhattad 1 , stalin ramprakash 1 , raghuram cp 1 , chetan ginigeri 1 , fulvio porta 1,2 background: primary immune deficiencies (pid) are increasingly being recognized in several parts of india. despite being diagnosed, many patients fail to receive optimal care due to financial and social constraints. methods: case records of patients diagnosed and treated (including hematopoietic stem cell transplants) for pid diseases during feb 2018 -nov 2019 at aster cmi hospital, bangalore, india were analysed. factors leading to deferred or suboptimal care were assessed in detail. results: 65 patients with various pids were diagnosed during the study period (details in table). 35 of them warranted a hematopoietic stem cell transplant (hsct) as definitive curative treatment. a total of 7 children received 9 hsct. 2 of them died while 5 of them are alive and well. 17 children (13 with severe combined immune deficiency) died before a hsct could be carried out. 12 of them were critically ill at presentation, while 5 were stable but deferred further treatment citing financial and social constraints. 11 children needing transplant continue to remain on follow-up and have not been transplanted to date (4 of them have significant financial constraints, 3 families are not convinced about the need for transplant and 4 of them are being prepared for transplant). table: (scid -severe combined immune deficiency, vodi-veno-occlusive disease with immunodeficiency, cgd -chronic granulomatous disease, hlh -hemophagolymphohistiocytosis, was -wiskott aldrich syndrome, xlt -x linked thrombocytopenia, lad-leukocyte adhesion deficiency, msmd -mendelian susceptibility to mycobacterial disease, xla -x linked agammaglobulinemia, cvid -common variable immune deficiency, apeced -autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, cmcc -chronic mucocutaneous candidiasis, at -ataxia telangiectasia). conclusions: we present our experience from a developing country and discuss non-medical factors leading to suboptimal care in children with pid. only 20% children warranting hsct could be transplanted in our cohort. among those where hsct is potentially curative 48% of children died before hsct could be offered. transplants in developing countries pose unique challenges due to the absence of government funding and/or universal insurance coverage. in addition to delay in diagnosis and critical state of patients at admission, financial and social factors significantly contributed to poor outcome. disclosure: none the outcome of hematopoietic stem cell transplantation (hsct) in pediatric patients with hemophagocytic lymphohistiocytosis (hlh) in korea methods: the korea histiocytosis working party retrospectively collected nation-wide data from the patients diagnosed with hlh and underwent allogeneic hsct between 2009 and 2017. the clinical characteristics and treatment outcomes of the patients were analyzed. results: a total of 44 patients were enrolled. there were 31 patients with fhl (4 fhl2, 26 fhl3, and 1 fhl4), 7 infection associated hlh, and 6 secondary hlh of unknown cause. all the patients were treated with hlh-2004 protocol, and 30 patients achieved complete response (cr) after treatment for 8 weeks, while 14 did not. the main reasons for receiving transplantation were fhl in 26, reactivation in 17, and refractory disease in 1. the conditioning regimens were busulfan-based in 16 patients, fludarabine-based in 4, treosulfan-based in 7, and busulfan/fludarabine-based in 17. stem cell sources used for hsct were from peripheral blood in 36 patient, cord blood in 7, and bone marrow in 1. the donor types of hsct were unrelated donor in 33 patients and related in 11 (7 matched sibling donor, 4 haploidentical donor, 1 partially matched donor). the causes of death of 7 patients were disease reactivation/ progression in 3, acute gvhd with/without vod in 3, and graft failure in 1. five year overall survival rates were 82.4%, respectively. the disease status at the time of hsct was cr in 37 patients, and non-cr in 7. the 5-year survival rate of patients who received hsct in cr was 87% and 63% for patients transplanted while in non-cr status (p=0.046). patients who received hsct using peripheral blood stem cells had a better 5-year survival rate of 86% compared to 75% of patients who received cord blood stem cells, significantly. the presence of neurologic symptoms, disease status after intial 8 week therapy, conditioning regimen, and cd 34 positive cell count did not have statistically significant impact on survival. conclusions: hsct improved the survival of patients who had familial, or relapsed, or refractory hlh in the korean nation-wide hlh registry. these results are similar to other reports in the literature. the disease status at the time of hsct and the stem cell source of the transplant were the important prognostic factors that affected the survivals of the hlh patients who underwent hsct. clinical trial registry: no registry number. disclosure: to the best of our knowledge, the named authors have no conflict of interest, financial or otherwise p382 hematopoietic cell transplantation with reduced intensity conditioning regimen using fludarabine/ busulfan and fludarabine/melphalan for primary immunodeficiency diseases background: primary immunodeficiency disease (pid) is congenital disorders of innate or acquired immune system. hematopoietic cell transplantation (hct) was a treatment option for pids with life-threatening infections or immune dysregulations. reduced intensity conditioning (ric) was increasingly used to prevent complications in hct, but optimized regimens have not been established. we performed hct for pids with ric using fludarabine and busulfan (flubu) or melphalan (flumel) according to the guidelines of european society for immunodeficiencies (esid) / european society for blood and marrow transplantation (ebmt), and assessed the efficacy and safety of these ric. methods: from april 2004 to december 2017, 42 pid patients underwent ric-hct using flubu or flumel in tmdu were analyzed retrospectively. the auc of bu was set to 30 mg*hour/l for severe combined immunodeficiency disease (scid) and 60 mg*hour/l for non-scid. overall survival (os) was analyzed. results: the median age at hct was 2.0 (0.3-21) years old (35 male and 7 female patients). flubu was used for 23 patients (7 scid, 9 combined immunodeficiency disease (cid), 3 ectodermal dysplasia (eda), and 4 severe congenital neutropenia (scn)) and flumel was used for 19 patients (8 scid, 6 cid, 4 xiap deficiency, and 1 eda). anti-thymocyte globulin was used in 8 patients of flubu group and 7 patients of flumel group. cord blood in 21 and bone marrow in 21 was used for donor sources. matched donor was used for 7 and 8 patients in flubu and flumel groups (30.4 % and 42.1 %), respectively. median follow up period was 2.2 years. two years-os of all patients, flubu group patients and flumel group patients was 79.6 %, 89.8 % and 64.1 %, respectively. neutrophil engraftment was 92.9 %, 91.3 % and 94.7 % (all patients, flubu group and flumel group). in scid, all 7 patients in flubu group achieved engraftment and survived. seven out of 8 in flumel group achieved engraftment, but 1 patient had secondary graft failure and 3 patients died. in non-scid, 14 out of 16 in flubu group achieved engraftment, but 6 patients had secondary graft failure. all 11 non-scid patients in flumel group were engrafted and survived. two and 7 patients in flubu group and flumel group suffered from severe acute graft-versus-host disease (grade iii-iv). ten patients had hemophagocytic lymphohistiocytosis (hlh). viral reactivation or infection was observed in 20 patients, and resolved in all but one patient. conclusions: the ric-hct using flubu or flumel was advantagous for neutrophil engraftment, and flubu for scid and flumel for cid with immune dysregulation may be an effective opinion. flubu regimen needs to be improved for secondary graft failure in non-scid. prevention of hlh after transplantation using dexamethasone palmitate will be considered. disclosure: nothing to declare. survival after hematopoietic stem cell transplantation with tcrαβ/cd19 graft depletion in older children with primary immunodeficiencies background: tcrαβ/cd19 depletion is a graft engineering method that proved valuable in increasing the survival rate after hematopoietic stem cell transplantation (hsct) in patients with primary immunodeficiencies (pid). decreased survival rate in older patients with pid was previously reported after transplantations with nonmanipulated grafts. methods: 148 patients with various pid (excluding classic scid) who received allogenic hsct with tcrαβ/ cd19 graft depletion from 2012 to september 2018 in our center were analyzed. the median age at hsct was 3,5 years (range 0,43-17,63). patients were divided into 3 age groups: 0-6 years -95 patients, 6-12 years -30, 12-18 years -23. 112 patients received hsct from matched unrelated, 31 -haploidentical donors, 5 -siblings. conditioning regimens with 1-2 alkylating agents and antithymocyte globulin were used in all patients. 113 patient received short courses of various posttransplant immunosuppressants. median follow up after hsct is 1,95 years (range 0,04-6,3 years). results: overall survival (os) in 148 patients was 0,85 (95% ci 0,79-0,91). we observed similar os in the younger age groups: 0,89 (95% ci 0,83-0,96) in 0-6 years and 0,89 (95% ci 0,78-1,0) in 6-12 years of age. seven of 23 patients in older group (12-18 years of age) died, the os was only 0,65 (95% ci 0,43-0,86), p=0,065. all patients from older age group who died had combined pid: 1 -wiscott-aldrich syndrome, 1 -undefined pid, 1 -il2rg deficiency, 1 -dclre1c deficiency, 1 -nijmegen breakage syndrome (nbs), 1 -kabuki syndrome, 1 -icf syndrome. the median time of death after hsct was 0,64 years (range 0,26 -1,58). six of those had transplant-related mortality (trm). five patients had hsct-associated viral infections: 2 -cmv pneumonias, 3 -adv infections (1 -fulminant hepatitis, 2 -multiorgan). interestingly, 2 of them had prolonged history of disseminated viral infections (adv), with the reduction of viral load in blood and other fluids and tissues upon treatment. one patient with kabuki syndrome after hsct developed hhv8 associated kaposi sarcoma, was successfully treated. eventually all 3 patients with reduction of viral infection and sarcoma symptoms developed multiorgan failure with some clinical and laboratory evidences of endothelium cell damage syndrome. one patient with nbs died of high grade lymphoma progression. conclusions: hsct with tcrαβ/cd19 depletion demonstrates high survival rate in 148 patients with various pid. in our group patients' age older than 12 years predisposes to decreased posttransplant survival. patients with combined pid are at higher risks of posttransplant mortality. we conclude that at least in some of our patients with prolonged history of viral infections after hsct the cause of death could be multiorgan failure due to endothelium cell damage syndrome resulting from persistent inflammation and drug toxicity effects. disclosure background: chronic granulomatous disease (cgd) is a primary immunodeficiency (pid) caused by a mutation in 1 of the 5 subunits of the nicotinamide dinucleotide phosphate (nadph) oxidase, which leads to a reduction in the microbicidal activity of phagocytic cell. starting at an early age, cgd patients suffer from severe recurrent infections, as well as inflammatory events. allogeneic hematopoietic stem cell transplantation (hsct) is a curative option for cgd in patients with insufficient benefit from supportive care and prophylactic antibiotics. we reported a series of 39 patients with cgd who underwent unrelated umbilical cord blood transplantation (ucbt) at our center. methods: in this retrospective study, we observed a series of 39 consecutive ucbt performed at our center in children with cgd between 2015 and 2018.median age at transplantation was 17 months (range, 4 to 144 months), median body weight was 10 kg (range, 6 to 34 kg). 32/39 patients received a myeloablative conditioning regimen consisting of busulfan, fludarabine, cytarabine, cyclophosphamide and g-csf, 7/39 patients received another myeloablative conditioning regimen consisting of busulfan, fludarabine, cyclophosphamide and atg. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. results: engraftment occurred in 29/39 (74.4%) patients. 10/39 (25.6%) patients occurred graft failure, all of them received a myeloablative conditioning regimen without atg. median time to neutrophil and platelet engraftment were 24 (rang, 14-46) and 36.5 (rang, 15-51) days. 22/39 (56.4%) patients developed acute gvhd, with 4/39 (10.3%) episodes of grade iii-iv agvhd. chronic gvhd occurred in 3/29 patients (10.3%). at a median follow-up of 17 months (rang, 1 to 44 months), the overall survival rate was 83.7%, and event-free survival rate was 66.7%. conclusions: unrelated ucbt should be considered as potential curative methods in children with cgd. cgd patients who used myeloablative conditioning regimen with atg shows better graft and survival. disclosure: nothing to declare background: invasive fungal infections (ifi) remain a major cause of treatment-related morbidity and mortality in aml patients. although not uncommon, the presentation of unusually severe clinical features might be indicative for an underlying immunodeficiency. caspase-associated recruitment domain 9 (card9) is recognized to have a crucial role in effective antifungal response, leading to th1 and th17 differentiation and to the initiation of the inflammatory cytokine cascade. particularly interferon-gamma (ifng) increases macrophage activity. patients with homozygous card9 mutations are known to have a significantly increased susceptibility to life-threatening systemic candidiasis. however, some sequence variants may lead to increased ifi-susceptibility even in heterozygosity, e.g. under immunosuppression. ifn-γ has been described as an additive treatment option because of its immune stimulating effect on the leukocyte immune response in a situation of immunological "blindness". methods: here, we report the case of an 11-year old male with aml m6 with a severe systemic candida tropicalis infection, unresponsive to triple-antimycotic regimen, leading to multi-organ failure. he was discovered to bear a heterozygous card9 mutation, and ifn-γ immunotherapy leaded to complete response of all disseminated infections. results: the patient developed septic fever immediately after the first chemotherapy cycle. unexpectedly, candida tropicalis was confirmed in the blood culture within 24 hours. liposomal amphotericin b (ambisome ® ) was started immediately, however candida rapidly disseminated to lungs, liver, spleen, kidneys and cns despite extended antimycotic therapy with caspofungin, voriconazole and fluconazole. the patient was splenectomized due to massive infiltration ( figure 1 ). genetic testing for mycosis predisposition revealed a heterozygous mutation in the card9 gene, inherited from the father (c.809a>t(p.glu270val)). ifn-γ treatment was started (100 μg subcutaneously, 3 times per week), leading to an almost complete response of disseminated infections. due to the severe infection, chemotherapy had to be interrupted after one course. however, bone marrow remained in complete remission for almost one year. the patient experienced altogether two relapses requiring an unrelated allogeneic and a haploidentical hsct. under combined ifn-γ and ambisome/ fluconazole prophylaxis no further mycosis was observed despite extensive and prolonged immunosuppression. conclusions: ifi in aml patients are common, however an unusual presentation in presumably immune competent individuals should raise the suspicion for immunodeficiencies. in our case, an unexpected early candida-sepsis was completely unresponsive to an adequate multi-agent treatment. while ifn-γ is used in adults as an immune stimulatory cytokine, little data are available for children. to our knowledge, this is the first case of successful ifn-γ treatment of a pediatric aml patient with disseminated candida sepsis, bearing a card9 mutation. given the elevated mortality risk for ifi, and the apparently safe and well-tolerated application of ifn-γ, an adjuvant immunotherapy might be considered. further studies are needed to define the indication and duration of this kind of adjunctive immunotherapy. moreover, considering the wide heterogeneity of genetic mutations involved in ifi-susceptibility, genome-wide expression profiling might be useful for pediatric cancer patients, as the identification of specific immune pathways might help to identify individual ifisusceptibility in order to improve the outcome of those high-risk patients. [[p385 image] 1. background: chronic granulomatous disease (cgd) is curable by allogeneic hematopoetic stem cell transplant (hsct). recent reports of haploidentical donor hsct with with post transplant cyclophosphamide (ptcy) from family donors in pediatric primary immune deficiencies have shown encouraging results. however, it has not been reported in cgd. here we describe successful haploidentical hsct in a child with cgd with myeloablative conditioning and ptcy. a 3 year-old, male child diagnosed with cgd showed oxidative activity 0.3 % by dihydrorhodamine (dhr) test. he had no matched related or unrelated donor available so underwent haploidentical hsct after taking informed consent of the parents in may 2018. donor was his 5/10 hla matched healthy elder sister (oxidative activity 55 % by dhr). he has had multiple admissions for recurrent pneumonia prior to hsct. the conditioning was with rituximab 100 mg/m2 iv on day -8, thiotepa 10 mg/ kg/dose intravenous (iv) for 1 day (day-7), busulfan 3.2 mg/kg/dose daily iv for 4 days (day -6 to -3) and fludarabine 40 mg/m2/dose daily iv for 4 days (day -6 to -3) and rabbit anti-thymoglubulin (thymoglobulin) 1.5 mg/ kg/dose daily for 3 days (day-4 to -2). peripheral blood stem cells (8 million/kg cd34+ cells) were harvested from his sister and transfused to the patient on day 0. graft vs. host disease (gvhd) prophylaxis was with ptcy 50 mg/ kg on day+3 & 4, intravenous cyclosporine from day-3 (targeting levels 150-250 ng/ml) and mmf from day+5. results: his neutrophils engrafted on day+15 and platelets on day+17. chimerism on day+30, 100 and 6 months was fully donor. he developed no acute or chronic gvhd. at 6 months his lymphocyte counts showed cd4-280/ul, cd8-670/ul, cd19-15/ul and cd16/56-120/ul. his had no viral reactivation. he is disease free and gvhd free on day+190 post hsct and is on tapering doses of cyclosporine. his dhr test showed oxidative activity of 42 % on day +100. background: primary immune deficiencies (pid) are a functional disorder of inheritance immune system that increase predisposition to infectious disease in number and severity. the incidence is 1: 10,000 birth live; its immunological dysregulation may increase the predisposition of autoimmune diseases and malignancy, the latter being more frequent (4-25%). at present, the only curative treatment is hematopoietic stem cells transplant (hsct). methods: we describe all patients transplanted with primary immune deficiencies at instituto nacional de pediatria. the conditioning regimen depended on the type donor and pathology: myeloablative (41.07%), reduced intensity (37.5%) and non myeloablative (21.43%) without modification statistically significantly in overall survival. results: a total of 71 patients were included from 1998 to january/2017. severe combined immunodeficiency (scid) is the pathology most frequently transplanted ( figure 1) . seventy three percent have molecular diagnosis, and 44.83% have cases of family pid. the most used sources were umbilical cord blood (ucb) with 42.6% and peripheral blood (39.3%), however the trend of the source of obtaining it has been modified a few years ago (figure 2) . the median graft was 14 days for ucb, 16 days for bone marrow (bm) and 12 days for peripheral blood (pb) (figure 3 ) the main complications are infectious (bacterial 48.3% and viral 39.7%) and non-infectious as pre-graft syndrome (23.1%). conclusions: the overall survival was 54.1% survival according to pathology was: 100% chediak higashi syndrome, 68% scid, 66.7% griselli syndrome, 66.7% hyper igm syndrome, 60% was, 57% cgd, 50% hemophagocytic lymphohistiocytosis. disclosure: ramírez-uribe rosa maria nideshda, salazar-rosales haydeé, olaya-vargas alberto, lópez-hernández gerardo, del campo-martínez maria de los àngeles we wish to confirm that there are no known conflicts of interest associated with this abstact, the only financial support was provided by mexican associations that helping children wiht cancer in a few patients. long-term outcome following hematopoietic stem cell transplantation of wiskott-aldrich syndrome in a single institute mamoru honda 1,2 , yukayo terashita 1 , minako sugiyama 1 , yuko cho 1 , akihiro iguchi 1 background: wiskott-aldrich syndrome (was) is an xlinked disorder of hematopoietic cells, characterized by thrombocytopenia with small platelets, eczema, and immunodeficiency. hematopoietic stem cell transplantation (hsct) is the only curative treatment, and it is recommended to be performed as soon as was is diagnosed. myeloablative conditioning before hsct is recommended because there is a high risk of development of autoimmune disease in patients with mixed chimera after hsct. however, there are few reports about late complications such as pubertal development and eruption of teeth in patients with was receiving hsct. thus, we evaluated late complications in patients with was receiving hsct at hokkaido university hospital. methods: we reviewed medical records of 8 male patients with was who received hsct between 1995 and 2017. results: mean age at hsct was 1.4 (range, 0.6-6.8) years, and median follow-up time after hsct was 13.8 (range 2.9-23.4) years. conditioning regimen in all patients comprised busulfan at 4 mg/kg for 4 days and cyclophosphamide at 60 mg/kg for 2 days or 50 mg/kg for 4 days. additionally, anti-thymocyte globulin at 2.5 mg/kg/day for 1-2 days was administered in 6 patients. engraftment, normal platelet count, and complete chimera were confirmed in all patients. no patients showed complications such as severe chronic graft-versus-host disease, autoimmune disease, short stature (≤ -2.0 sd) and second malignancy. however, high ige level was observed in 4 patients. pubertal development has been confirmed in 6 patients. lack of complete eruption of permanent teeth has been observed in 3 patients who received hsct at age of < 2 years. conclusions: although this was a small-cohort study in a single institute, complete chimera has been achieved in all patients who received hsct with busulfan-based myeloablative conditioning. however, late complications such as male infertility and incomplete eruption of permanent teeth remain major problems. disclosure: nothing to declare methods: we performed unrelated umbilical cord blood transplantation (ucbt) in 5 consecutive children with lad-i. median age of 5 children was 13 months (range, 8 to 131 months), and median body weight was 13 kg (range, 8 to 24.3 kg). all patients received myeloablative conditioning regimen consisting of busulfan, fludarabine and cytarabine. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. all patients received a ≤2 hla alleles-mismatched cord unit, 1 was hla fully matched, 3 were 9/10 matched, 1 was 8/10 matched. median nucleated cells of the cord blood were 14.23x10 7 /kg (range, 4.6 to 20.62 x10 7 /kg), and median cd34+ cells were 3.87 x10 5 / kg (range, 1.95 to 5.77 x10 5 /kg). results: all patients engrafted, median time of neutrophil engraftment was 24 days (range, 13 to 28d), and median time of platelet engraftment was 33 days (range, 32 to 56d). median follow-up time was 13 months (range, 2 to 25 months), all 5 patients were alive with continuous completely donor engraftment, and achieved complete clinical remissions. 4/5 patients developed grade ii/iii acute graft-versus-host disease (gvhd), and 1/5 patients developed chronic gvhd with skin. conclusions: it is the first successful unrelated ubct for lad-i children in china. our data shows ucbt provided excellent outcome for patients with lad-i. disclosure: nothing to declare p390 excellent outcome using 'nktm' enriched hematopoietic stem cell transplants for patients with inborn errors of immunity results: majority of patients in the 2 cohorts had significant infective co-morbidities at the time of hsct with patients in the later cohort entering hsct earlier. patients in the later cohort were sicker at hsct. final engraftment occurred in all except 1 patient who received a hla mis-matched cord blood hsct. graft failures occurred in 6 patients (2 in earlier and 4 in later cohort); 3 of these patients received unmanipulated hscts from hla mis-matched unrelated donors (1 cb, 2 bm). second hsct were with same donors in 3 and different donors in 2 patients. no grade ii to iv acute gvhd or extensive gvhd occurred. one patient (cbt) died of infections/ non-engraftment. all 9 patients in the later cohort compared to 7 of 9 patients in earlier cohort are alive, engrafted and cured. performance status were 100% in all alive patients. of the 9 patients in the later cohort, 7 (6 hla mis-matched related and 1 hla matched related donors) received 'nktm' enriched hsct. in the 6 hla mis-matched 'nktm' enriched hscts, patients received high cd34+, cd3+cd45ro+ and nk cell doses, with median of 16.88 (range, 5.1 -36.5), 113.9 (range, 3.3 -232.6) and 89.0 (range, 22 -654) x10 6 /kg, respectively. no invasive infections occurred in these patients and immune reconstitution in t, b, nk compartments were complete at 1 year after hsct with cd4 > 200 by 6 months and tcrαb > 1500 by 1 year after hsct. background: viral infections contribute to significant morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-hsct), increasing both the human and the financial cost. antiviral agents are often ineffective or/ and associated with toxicity. methods: in view of t-cell anti-viral immunotherapy in greece, we evaluated the actual cost of conventional pharmacotherapy for cmv, ebv and bkv reactivations after allo-hsct, by calculating the costs of (i) the antiviral agents, (ii) the treatment (excluding transfusions) of antiviral drug primary toxicity (e.g. graft failure, cytopenias, renal or hepatic dysfunction) and secondary toxicity (e.g. leukopenia-associated bacterial infections), iii) the treatment (excluding transfusions, ie for bk cystitis) of infectionrelated complications, iv) the transfusions due to treatmentrelated toxicity (ie cytopenias) or infection-related complications (ie, bk cystitis), v) the inpatient or outpatient daily care. notwithstanding that blood and its products, as a common natural good, are provided free in our country, the costs related to blood and platelet collection, processing, storage, laboratory testing and infusions were included in our model. results: the treatment cost of cmv, ebv and bkv reactivations/infections for the first six months post allo-hsct was evaluated in 38/51 patients who reactivated viruses and were transplanted between 10/2015-11/2016 from matched related (17/51), matched unrelated (20/51), mismatched unrelated (8/51), haploidentical (5/51) and mismatched related donors (1/51). we detected 29 cmv, 35 ebv and 18 bkv infections/reactivations in 20, 32 and 17 patients respectively, with a mean of 2±0.4 infection per patient from all three viruses (1-7/patient). of note, 22/38 patients experienced reactivations from more than one virus, requiring repeated treatments with antiviral agents and/or rituximab. the cost of antiviral agents for all cmv, ebv and bkv reactivations/infections was 78,656€, 58,504€ and 11,331€ respectively (3,146€, 2,089€ and 1,416 €/patient, respectively). the treatment cost of toxicity related to antiviral drugs and infection-related complications was 70,358€ (4,309€/patient) excluding transfusions and 676,107€ (28,171€/patient) including transfusions. in particular, the cost of transfusions for bkv hemorrhagic cystitis reached 22,751€/patient. repeated (1-5) and/or prolonged (δm 38d, range 6-150d) hospitalizations were needed, up to a total of 745 and 159 days of inpatient hospitalization and short-term outpatient treatment, respectively. hospitalizations further increased the cost of inpatient and outpatient post-transplant care by 81,640€ and 8,000€ respectively (2,634€ and 500€/patient, respectively), onthe basis of a, rather underestimating the true cost, fixed, unified hospitalization fee (60€/day and 200 €/day). conclusions: overall, in a six-month study period, the treatment of cmv, ebv and bkv infections substantially increased the cost of post-transplant care by 956,283€ (27,322€/patient). the actual cost is undoubtedly higher as the hospitalization fee for transplant recipients is largely underestimated in greece. considering not only the hematopoietic but also the solid organ transplant recipients, the financial burden of antiviral treatment for national economies is enormous. given that antiviral pharmacotherapy is often associated with suboptimal efficacy, toxicity, development of drug resistance, reactivation recurrences and repeated hospitalizations, it is expected that a one-time treatment with multi-virus-specific t cells, able to expand in vivo and provide a long-lasting protection without significant toxicity, will serve as a powerful and costeffective treatment over conventional pharmacotherapy. disclosure: nothing to declare methods: this is a single-centre retrospective analysis of 293 consecutive patients who underwent tcd allo-hscts for myeloid malignancies between january 2012-june 2016. ebv-dna was monitored frequently on whole blood samples with standardised quantitative real-time pcr. serum protein electrophoresis was routinely tested with immunoglobulin subclasses identified by immunofixation electrophoresis. histological confirmation of ptld was based on standard who diagnostic criteria ('proven'), while those without biopsy were classed as 'probable' based on clinical & radiological criteria as defined by ecil-6 guidelines. results: majority of patients had aml(n-152/293) and mds(n-107/293) with a median age of 58 years(range . median follow up of survivors was 32 months(range 4-65). majority of patients(n-220/293;75%) developed ebv-r with a median time of 79 days[inter quartile range(iqr) 27-160 days] &higher cumulative incidence with atg(n-132) versus alemtuzumab(n-161)(p< 0.001). figure-1a shows schematic representation of ebv and ptld events (cumulative incidence of 6.8%(95%ci-4.0%-10.6%) at 12 months). significantly higher peak ebv dna viral load(evl) were noted in patients with ptld(p-< 0.001). development of post-hsct mg was observed in 29%(n-85/292). roc curve identified peak blood evl>150,000 copies/ml significantly correlated with risk of developing ptld (sensitivity-70.6%,specificity-79.4%;auc-0.82,p< 0.001). based on these estimates, subgroup of patients with no ebv-r(n-72/292), peak evl < 150,000(< 150k)copies/ ml(n-165/292) & >150,000(>150k)copies/ml(n-55/292) were categorised in 6 groups along patients with/without mg accordingly (groups 1-6;figure-1b). patients with ebv-r had significantly better os [5-year os of 52% vs 35%(no ebv-r);log-rank p< 0.001],with this survival benefit mainly driven by subgroup of patients with lower evl(< 150k)(p< 0.001). ptld patients had trend towards inferior 3-year os(15% vs 54%;p-0.051). patients with mg had a significantly better os irrespective of degree of evl (group 1-3,p< 0.001).we report a 'sweet spot' of low evl & presence of mg in these patients, with a clear survival advantage compared to those with no ebv-r and/or no mprotein (group-2 5-year os 62% vs 27% in group-6; hr-0.15;95%ci:0.06-0.34;p< 0.001;figure1b). overall cumulative incidence of relapse (cir) was 28%(95%ci:23-37) and non-relapse-mortality(nrm) of 24%(95%ci:18.6-30) at 5 years. multivariate analysis(mva) revealed absence of m-protein,high evl (>150k copies/ml) or no ebv-r and absence of any gvhd as significant factors for high cir. similarly, high evl or no ebv-r, absence of m-protein and itu admission were significant predictors of high nrm. conclusions: this study adds to our understanding of role of ebv viraemia & associated mg in tcd-hscts while highlighting its significant impact on risk of ptld, os, nrm & cir. low ebv burden and development of mg is protective with significantly better survival outcomes and we recommend pre-emptive approach of using rituximab for ebv-r /ptld is best employed at higher ebv burden (e.g. >150k copies/ml dna) in high risk patients and be prospectively evaluated in future studies. clinical trial registry: n/a disclosure: nothing to declare p394 impact of early candidemia on the long-term outcome of allogeneic hematopoietic stem cell transplant in non leukemic patients: an outcome analysis on behalf of idwp background: to assess the incidence of, and risk factors for, candida infection in the first 100 days post-allogeneic hematopoietic stem cell transplantation (hsct) and the impact on long-term survival. methods: outcome analysis of 50,188 patients, 61% male, median age 46 years (range 0-80), with diagnosis of hemoglobinopathies in 3176 (6.3%), bone marrow failure in 7626 (15.2%), lymphoma in 17743 (35.4%) and myelodysplastic/myeloproliferative disesases in 21643 (43.1%) patients who underwent hsct from 2000 to 2015: 420 with candidemia by day + 100, and 49,768 without candidemia. the incidence of 100-day candidemia was estimated by using the cumulative incidence method. the univariate and multivariate risk factor analysis for 100-day candidemia was performed with the cause-specific cox regression model. the occurrence of candidemia was analyzed as a timedependent covariate. the overall survival and non-relapse mortality after day +100 were assessed in a land-mark setting, this analysis was restricted to patients surviving to day +100 post transplant. [[p393 image] 1. figure 1a -1b] results: the incidence of candidemia by day +100 was 0.85% (95% c.i. 0.77-0.93) (420/50,188) and occurred at a median of 17 days post-hsct (range -7-100). considering the candidemia within 100-day from hsct as a time dependent covariate, a higher 100-day non-relapsemortality (nrm) (hr 3.47 (2.75-4.38), p < 0.0001), and a lower 100-day overall-survival (os) (hr 3.21, 95% ci 2.67-3.85), p< 0.0001) were obtained from the cox model for patients with candidemia. factors significantly associated with candidemia occurrence in the multivariate analysis were: gender female, increased age at hsct, bone marrow failure, lymphoma or myelodysplastic/myeloproliferative diagnosis, bone marrow or cord blood stem cell source, t-cell depletion, less recent year of hsct. among patients alive at day + 100, the 5-year nrm and os with and without candidemia were 28.2% vs. 18.8%, p < 0.0001, and 50.5% vs. 60.7%, p< 0.0001, respectively, after a median follow-up of 4.3 years (95% ci 4.3-4.4) (figure 1 ). in multivariate analysis, the occurrence of a candidemia episode within day + 100 was an independent risk factor for higher nrm, hr 1.52 (1.18-1.97), p=0.0013, and lower os, hr 1.30 (1.08-1.57), p=0.0061. conclusions: despite the general improvements in prophylaxis and treatment, the occurrence of early post-hsct candidemia had a negative impact on transplant outcome as showed previously in leukemic patients. abstract already published. carbapenem-resistant enterobacteriaceae colonizationimportance in the risk of cre bacteremia and mortality in stem cell transplant (hsct) and acute leukemia patients marcia garnica 1,2 , marco a f bellizze 1 , priscila g a de jesus 1 , rafaela r c gomes 1 , filipe m akamine 1 , alan j marçal 1 , luzinete co rangel 2 , andreia assis 2 , marcia rejane valentim 2 , angelo maiolino 1 background: spread of infections due to carbapenemresistant enterobacteriaceae (cre) is a worldwide phenomenon and has been associated with high mortality and clinical complications. gut translocation is the most important portal of entry of bacteria during neutropenia, and cre gut colonization is a possible risk factor for bacteremia during neutropenia. goals: in the present study, we describe the frequencies of cre colonization and analyzed its relationship with development of cre bacteremia and mortality in two different scenarios: stem cell transplant patients (hsct) and leukemia patients. methods: prospective cohorts of hsct (from 2012 to 2017) and leukemia patients (from 2016 to 2018). hsct patients were analyzed from conditioning until discharge (pre-engraphment phase) and leukemia patients from first induction chemotherapy until last intensification. if an hsct was performed in leukemia patient the patient was censored in leukemia cohort and included in hsct cohort. all patients had rectal swabs performed weekly during hospitalization for the identification of cre colonization. patients with at least one positive swab (cre colonization group) were compared to patients with no documentation of colonization (controls). the outcomes analyzed were bacteremia due to cre, and overall mortality. results: there were 493 hsct performed during the study (408 [83%] autologous and 85 [17%] allogeneic). multiple myeloma and non-hodgkin lymphoma were the most frequent baseline diseases (n=251; 51%, and n = 88; 18%), respectively. cre colonization was documented in 10% (n=50), and it was more frequent among allogeneic hsct and leukemia patients (p< 0.001 for both). cre colonized patients had longer hospitalization (25 vs. 20 days, p< 0,001), higher frequency of cre bacteremia (6% vs. 0.2%; p=0.004), and mortality (16% vs. 2.4%, p< 0,001) compared to non-colonized hsct. negative and positive predicted values for cre bacteremia were 6% and 99%, respectively. thirty-one patients were analyzed in leukemia cohort, accounting to 92 hospitalizations (median 3 hospitalizations per patient, ranging from 1 to 6). the median age was 58 years, and 90% aml vs. 10% all. cre colonization was documented in eight (26%), with a median time from leukemia diagnosis and colonization of 93 days (9 -503 days). cre bacteremia was documented only in colonized patients (25% vs. zero; p=0,046). all eight colonized patients were submitted to other cycles of chemotherapy after colonization, in one of them cre bacteremia relapsed. conclusions: a routine surveillance of cre colonization showed colonization frequencies from 10% to 25% in hsct and leukemia patients respectively and was effective to stratify cre bacteremia risk as the predictive negative value was over 95%. colonization had association with cre bacteremia and overall mortality. efforts to minimize risks for colonization and mortality are necessary. the information of surveillance can be a tool to improve adequacy in empirical febrile neutropenia therapy in hsct and leukemia patients. disclosure: nothing to declare background: the incidence of hepatitis b virus infection is high to 6.2% in asian population, so there is more and more attention to the risk of hepatitis b virus(hbv) reactivation in the hepatitis b core antibody positive patients during chemotherapy, anti-cd20 monoclonal antibody, hsct, or other intense immunosuppressive drug therapy (isdt). hepatitis b core antibody is associated with a significant risk of hbv reactivation in patients undergoing hsct. however, there are remain uncertain that the effect of anti-hbsag antibodies in hepatitis b virus reactivation among the hepatitis b core antibody positive patients undergo hsct. we aim to investigate the role of the anti-hbs and the necessity of anti virus in hepatitis b surface antigen(hbsag) negative, hepatitis b core antibody positive patients during hsct. methods: we enrolled 791 hematological malignant patients received hsct in our center from 2008 to 2016. we classified 665 hbsag negative and undetectable hbv dna patients into 4 groups as anti-hbc(-)anti-hbs(-) (n=189), anti-hbc(-)anti-hbs(+) (n=176), anti-hbc(+) anti-hbs(-) (n=49), and anti-hbc(+)anti-hbs(+) (n=251). results: hbv reactivation was identified in 16 patients (2.4%) after hsct. there was a significant difference in hbv reactivation rate in anti-hbc(+)anti-hbs(-) (12.2%) vs anti-hbc(+)anti-hbs(+) (2.8%) (p=0.01) and anti-hbc (+)anti-hbs(-) (12.2%) vs anti-hbc(-)anti-hbs(-) (0.0%) (p=0.000), anti-hbc(+)anti-hbs(-) (12.2%) vs anti-hbc(-) anti-hbs(+) (1.7%) (p=0.004), but not among anti-hbc(+) anti-hbs(+) (2.8%) and anti-hbc(-)anti-hbs(-) (0%) and anti-hbc(-)anti-hbs(+) (1.7%). whereas there were no difference according to the donor viral profile(p=0.774). the median time of hbv reactivation in hbsag negative patients accepted hsct was 645 (455-1957) days after hsct. all of the patients with hbv reactivation have been controlled with nucleos(t)ide analogues drugs, and 5 of them achieved reverse seroconversion which detect persistent anti-hbsag antibodies in their bodies. conclusions: the anti-hbsag antibodies negative and anti-hbc positive patients have the highest risk of hbv reactivation after hsct in resolved hbv patients. the anti-hbsag antibodies play a protective role in resolved hbv patients receiving hsct. we recommend not prophylactic anti hepatitis b virus in hbsag negativity and anti-hbsag antibodies positive patients following hematopoietic stem cell transplantation. disclosure: nothing to declare methods: 2230 allo-hscts performed between 1997 and 2016 for acquired bone marrow failure (70.6%) or hemoglobinopathies (29.4%), with bm±cb (75.8%) or pb±cb+bm (24.2%) as a stem cell source were included in this retrospective registry megafile idwp ebmt study. results: demographics: the median age of recipient was 17.7 years (range: 0.1-78), and 50.8% were children. 79.0% recipients and 75.4% donors were ebv-seropositive. 67.8% had hsct from a matched family donor, 4.6% from a mismatched family donor, and 27.6% from an unrelated donor. t-cell depletion was performed in vivo in 82.2%, and ex vivo in 6.6% patients. conditioning regimen was myeloablative in 63.7%, ric in 36.3%. median follow-up was 4.7 years (95% c.i. 4.3-5.0). transplant out-comes: ebv-seropositive recipients in comparison to ebv-seronegative recipients had lower os (85.4% vs 88.4%, p=0.035), and higher nrm (10.0% vs 6.4%, p=0.018). no other significant differences were found for: ri, rfs, and acute or chronic gvhd with respect to ebv pretransplant serostatus donor and/or recipient. multi-variate analysis: ebv serostatus as a risk factor did not reach significance, while a trend towards higher risk of development of cgvhd (hr=1.31; 95%ci 0.97-1.78; p=0.081) and better survival (hr=0.78; 95%ci 0.59-1.04; p=0.087) in allo-hsct from ebv-seropositive donors. allo-hsct in ebv-seropositive recipients had a trend towards lower risk of development of cgvhd (hr=0.75; 95%ci 0.56-1.02; p=0.066). when 4 subgroups (r-/d-, r-/ d+, r+/d-, r+/d+ ebv serology) were analyzed, the ebv serostatus had no significant impact on os, rfs, ri, trm and development of acute or chronic gvhd. conclusions: allo-hsct from ebv-seropositive vs ebv-seronegative donors are at 31% higher risk of chronic gvhd in patients with non-malignant hematological disorders undergoing allo-hsct, however this difference is non-significant in multivariate analysis. disclosure: nothing to declare. results: twenty-eight (30%) pts (19 male, 9 female) were tested positive (group 1) for subtype a (n=4, 14%), b (n=18, 64%) or a (h1n1) (n=6, 21%) while 67 (70%) pts (32 male, 35 female) were negative (group 2). vaccination rate in group 1 (32%) was significantly lower compared to group 2 (51%, p=0.002). the median time after transplantation (790 vs 565 days), t-cell counts (349 vs 296/ μl), bcell counts (162 vs 135/ μl), igg-level (8,3 vs 7,6 g/ l), proportion of immunosuppressed pts (75% vs 63%), male/ female ratio was not significantly different between groups 1 and 2. within group 1 influenza subtypes were similarly distributed in vaccinated and not vaccinated pts (a 11% vs 16%, b 67% vs 63%, a (h1n1) 22% vs 21%). pts. with subtype b infection had higher levels of t-(482 vs 274/ μl) and b-cells (232 vs 108/ μl) and a longer follow up from sct (1610 vs 470 days) compared to subtype a / a (h1n1) infection but differences were not significant. conclusions: influenza could be proven in one third of all tested pts. dominance of b and a (h1n1) pdm0e9 subtype occurrence corresponded to the flu epidemic dissemination in the german population. the most important protective factor for outpatient sct recipients was influenza vaccination. disclosure: nothing to declare background: cmv infection is one of the most frequent complications after haplo. some risk factors are well known but the best strategy (prophylactic or preemptive treatment) to mitigate this complication is not still well defined. the primary endpoint in our study is to describe incidence and risk factors to develop cmv infection or disease in haplo. as secondary objective we analyzed efficacytoxicity of treatment and cmv related mortality. methods: we analyzed 60 patients who underwent haplo in our center between may 2012 and may 2018. all of them received ptcy (d+3 and d+4), tacrolimus and mycophenolate as graft versus host disease prophylaxis. a preemptive therapy based on viral load was applied. treatment was started when >1000 ui/ml of cmv were detected in one determination or >500 ui/ml in two consecutive determinations. cmv analyses were made in plasma using cobas pcr technique® and positive viral load cut-off point was 137 ui/ml. the cmv viremia was determined weekly until d+100 and then every two weeks until immune reconstitution. results: the cmv infection and disease incidence at d +100 was 61.3% (38 episodes) and 19.4% (12 episodes), respectively. cmv disease was digestive (n=8), pulmonar (n=2), neurologic (n=1) and disseminated (n=1). the median time to first cmv infection was 39.5 days (20-151). thirty-six patients had at least one episode of cmv infection: 24 of them (66.7%) had one episode, 7 (19.4%) had two episodes and 5 (13.9%) had 3 or more episodes, respectively. only pre-transplantation cmv status was significantly associated with cmv infection (p< 0.001). risk factors are shown in image 1. the median viral load in first cmv infection and disease was 8314 ui/ml (542-51158) and 24842 ui/ml (271-126279), respectively (p=0.02).the median counts of cd4 lymphocytes at d+100 in cmv infection and disease were 262/mm3 and 120/mm3, respectively (p=0.09). preemptive therapy for the first 2 episodes of cmv infections (n=48) was valganciclovir (58,3%), ganciclovir (35.4%) or foscarnet (6.3%), reaching a complete viral load clearance in 77%, with a median time to response of 19.2 days (6-34) and a median treatment duration of 21 days (2-39). grade iii-iv toxicity (mainly hematologic) was observed in 55.6% (n=28), 30% (n=17) and 16.7% (n=3), respectively. three patients had an ul54 mutation, one of them with clinical and microbiological resistance to the 3 mentioned drugs. three patients (5%) had a graft failure secondary to cmv infections. five patients (8.3%) died as consequence of cmv infection: 3 before d+100 secondary to cmv disease (2 pulmonar, 1 disseminated) and 2 after d+365 due to graft failure and infectious complications. with a median follow up of 12.5 months, overall survival at 18 months for patients who had cmv infection was 58.9% compared to 66.5% for those who had no infection (p=0.08). conclusions: a high incidence of cmv infection in haplo with ptcy was shown in our series and it contributed to mortality in 8.3% of patients. only cmv status (d-/r+ and d+/r+) was significantly associated with higher risk of infection. identification of high risk patients and new prophylactic and treatment strategies may improve these results. disclosure: nothing to declare. methods: consecutive patients admitted at the sct unit between january-18 to november-18 were reviewed. only first admission was analysed. screening consisted of rectal and perineal swap on admission and weekly until discharge. in case of detection of mdro, patients were isolated and infection control strategies were applied. results: 67 patients were analysed, median age 53 years (18-70). 65% were male (n=44). median duration of hospitalization was 30 days(16-133). 293 swabs were performed, with a median of 4 swaps/patient (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) . patient characteristics are shown in table 1 . 60 patients (90%) spiked fever in a median of 9 days after admission . 13% (n=9) had previous documented mdro colonization. median neutrophil engraftment was 15 days (95%ci 14-15), in 24% patients (n=16) of patients had a positive screen: in 8 (50%) patients at baseline and in 8 (50%) patients were detected for the first time beyond baseline screen. cumulative incidence of colonization at 7 days was 10.4% (95%ci 3.2-17.6), at 15 days 18% (95%ci 8.2-27.8), and at 30 days 19.5% (9.9-29.1%) (figure 1 ). mdro identified were: 8 with extended-spectrum beta-lactamases producing e. coli (esbl-ec), 4 multidrug-resistant pseudomonas aeruginosa (mr-ps), 3 vancomycin resistant enterococci (vre) and 1 patient with carbapenemaseproducing (cp) citrobacter freundii. 7/16 colonized patients developed mdro infection (44%): 4 patients mr-ps, site of infection was 2 urinary tract infection (uti), 1 urethritis, 1 genital ulcer. two patients were treated with ceftolozane/ tazobactam, 1 with meropenem+amikacin;2 patients esbl-ec both uti treated with meropenem; 1 patient cpcitrobacter freundii uti treated with ceftazidime/avibactam. in 75% patients (12/16) antibiotic treatment at febrile episode was guided by positive screening. no mdro related icu admission or mortality was observed. in 76% patients (n=51) background: hepatitis e virus (hev) can cause chronic infection and liver cirrhosis in immunocompromised individuals. there is limited data on hev infections in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). the aim of this study was to investigate the frequency and clinical importance of hev in a swedish cohort of hsct recipients. methods: we analyzed serum samples from 262 hsct patients (241 adults and 21 children), collected 6 months after hsct. hev igg and igm were detected by elisa (dia.pro®), hev rna by reverse transcriptase pcr, and quantification of hev rna was performed by digital pcr. in all patients, who were positive for hev-rna and/or serology at 6 months, also samples collected at the time of hsct from both the patients and their donors were analyzed. in the hev rna positive patients, additional samples were analyzed to determine the duration of viremia. three hev rna negative controls were selected for each case of hev infection, matched for age, diagnosis, conditioning regimen and donor type. results: hev rna was detected in 8/262 (3.1%) patients. in three of the patients hev rna was positive during a period of 3-8 months, and two of these patients were infected already at the time of hsct. in five patients hev-rna was positive, at a low level, only at 6 months. 11/262 (4.2%) patients had detectable hev igg and/or igm, whereof eight patients were hev rna negative. in 4/8 (50%) patients with hev infection (hev rna positive) alanine aminotransferase (alt) was > 3 upper limit of normal (uln), in 1/8 (12.5%) patients > 1.5 uln, and in 3/8 (37.5%) patients alt was normal, at 6 months after hsct. bilirubin was elevated > 1.5 uln in 1/8 (12.5%) patients, and > 3 uln in no patient at 6 months after hsct. two patients died with ongoing signs of hepatitis and hev rna detected in blood. one of them developed acute liver failure, at the time interpreted as drug toxicity, and died of multi-organ failure. the other patient died of unrelated causes. the remaining six patients had cleared the infection at 7-24 (median 8.5) months after hsct. active gvhd was present at 6 months after hsct in 5/8 (62.5%) patients with hev infection, involving the liver in 3 of these patients. corticosteroid treatment was ongoing in 6/8 (75%) patients; the mean dose during the 14 preceding days was > 0.5 mg/kg in 1/8 (12.5%) patient, 0.25-0.5 mg/ kg in 3/8 (37.5%) patients, and < 0.25 mg/kg in 4/8 (50%) patients. hev infection correlated to elevated alt > 1.5 uln, or 8.3 p=0 .02) and > 3 uln, p=0 .02) at 6 months, but not at 3 months, after hsct, compared to hev rna negative controls. conclusions: hev infection was detected in 3.1% of patients tested at 6 months after hsct and was correlated to abnormal alt. spontaneous clearance was common but one patient died in acute liver failure, where hev may have contributed. hev infection is a differential diagnosis in patients with elevated alt 6 months after hsct. disclosure: nothing to declare monitoring of t-cell responses to viral-coded antigens in pediatric patients receiving tcrαβ-depleted haplo-hsct followed by bpx-501 cell administration background: αβ t-cell-depleted haplo-hsct is an effective option for children with hematological disorders in need of an allograft. however, recovery of adaptive immunity is impaired in these patients. thus, in order to accelerate immune reconstitution, we developed a novel approach based on post-transplant infusion of a titrated number of donor t cells, transduced with the suicide gene inducible-caspase-9, ic9 (bpx-501 cells, sponsor bellicum pharmaceuticals®; nct02065869). we previously reported on immune recovery of 108 children transplanted at our institution, showing that bpx-501 cells infused after αβ t-cell-depleted haplo-hsct expand in-vivo and persist over time, contributing to fasten adaptive immunity recovery (merli, ash2017). here, we report the results of lymphoproliferation assay to viral-encoded antigens to assess tcell function in patients transplanted with this approach. methods: we evaluated 142 children, 78 male and 64 female. median age at transplant was 5.7 years (range 0. [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] . patients had either malignant (69 children) and nonmalignant (73) disorders. no patient was given any posttransplant graft-versus-host disease prophylaxis. nine children were enrolled in the phase i portion of the trial consisting of 3 cohorts receiving escalating doses of bpx-501 cells. the remaining 133 patients (phase ii portion) received the recommended dose of 1x10 6 bpx-501 cells/kg identified in phase i. bpx-501 cells were infused at a median of 17 days post-hsct (range 10-82). antigendriven activation of peripheral mononuclear cells was evaluated by lymphoproliferation assay with 3 h-thymidine pulsing at d+4 and harvesting at d+5. stimuli included pha or cmv, ebv and adv whole viral lysate. results are given as stimulation indexes (si, cpm stimulated sample/cpm unstimulated control). thresholds for positive response were arbitrarily set at si>3 for viral-encoded antigens and at si>5 for mitogenic stimulation with pha. fractions of responders are indicated in the figure. results: patients were analyzed from d+30 to d+720 post-hsct. pha responders (a) increased to 80%, while cmv (b), ebv (c) and adv (d) responders were 61%, 52% and 61% at 2 years after haplo-hsct. responses to ebv and adv antigens were slightly delayed but improved over time. responses to pha and to cmv (e,f) were analyzed in the cmv-reactivating and cmv-non reactivating groups (cmv-yes/cmv-no). significant differences in pha response were observed at d+60 and d+270. moreover, increased cmv responses were observed in cmvreactivators at d+270, d+360 and d+720, with approximately 100% of responders at d+720, as opposed to cmvnon reactivators which comprised 50% responders. neither primary disease, age nor tbi during the conditioning regimen influenced proliferative capacity of the two subgroups (not shown). conclusions: we showed a rapid recovery over time of t-cell function after αβ t-cell-depleted haplo-hsct followed by bpx-501 cells administration. when patients were grouped according to cmv reactivation (previously demonstrated as a strong driver of immune reconstitution), a significant difference in the number of responders among the patients experiencing viral reactivation was observed using the cmv lysate only but not the immunodominant pp65 protein (not shown), suggesting that other viral antigens account for increased t-cell responses. results of t-cell function after bpx administration complements the phenotypic data we already reported. clinical trial registry: nct02065869 disclosure: nothing to declare. background: cytomegalovirus (cmv) is associated with significant morbidity and mortality in allogeneic hematopoietic cell transplantation (allo-hct) patients (pts). cumulative incidence of cmv infection in high-risk patients such as cd34-selected or haploidentical hct have been reported as high as 61.8-84.5% and 53-81%, respectively. letermovir (ltv) was approved in 11/2017 for prophylaxis (ppx) in cmv-seropositive recipients (r+) of allo-hct. since 12/2017, ltv ppx was implemented at our center for both primary and secondary ppx. we report our real-world experience. methods: adult cmv r+ allo-hct pts who initiated ltv as primary and/or secondary prophylaxis were identified between 1/1/2018 and 6/30/18. cord blood transplants were excluded. the primary outcome was the incidence of clinically significant cmv infection (cmv viremia requiring preemptive treatment or cmv disease). pts were followed through 9/2018. results: 53 pts initiated ltv. 69.8% pts were at high risk for cmv reactivation and disease (primarily ex vivo t-cell depleted hct [n = 18; 34%] or haploidentical t-replete hct [n = 12; 22.6%]). the most common indication for hct was acute myeloid leukemia (n = 16; 30.1%) and the majority of patients received myeloablative conditioning (n = 34; 64.2%). 39 pts (73.5%) received ltv as primary ppx after hct, with a median day of ltv initiation of d+7 (range d+7─d+40). at ltv initiation, 34 pts had an undetectable cmv dna, and 4 had cmv < 137 iu/ml. clinically significant cmv infection requiring preemptive treatment occurred in 2 of 39 pts (5.1%). one patient was treated with valganciclovir (vgv) for persistent cmv < 137 iu/ml and received ltv as secondary ppx. a 2nd patient developed persistently detectable cmv (< 137 iu/ ml) and breakthrough cmv viremia with a mutation in ul56 at site c325yltv successfully treated with vgv. the median duration of primary ltv ppx was 116 days (54-221), with primary ppx continuing beyond 14 weeks post hct in 29 pts. the median additional follow-up in patients who discontinued ltv was 40 days (0-154), without clinically significant cmv infection to date. an additional 14pts (15 pts overall; 28.3%) received ltv as secondary ppx after cmv pre-emptive therapy. the median duration of secondary ltv ppx was 127 days (18-270), with no reactivation. ltv was not discontinued due to toxicity or intolerance in any patient. cmv outcomes are summarized in figure 1 . all-cause mortality for the 53 pts over the observational period was 11.3%. conclusions: primary ltv ppx significantly reduced cmv reactivation, and high-risk patients may benefit from extended prophylaxis. in patients who received preemptive therapy for cmv, use of secondary ppx showed no recurrent cmv reactivation. ltv is well tolerated. additional studies are needed to determine optimal ppx duration and to clarify role of secondary cmv ppx in high-risk allo-hct. the future standard of care will likely include extended primary ppx and secondary ppx and result in decreased morbidity and mortality associated with cmv. disclosure: andrew lin -nothing to declare, molly a. maloy -nothing to declare, valkal bhatt -nothing to declare, lauren derespiris -nothing to declare, meagan griffin -nothing to declare carmen lau -nothing to declare, anthony j. proli -nothing to declare, juliet barker -angiocrine bioscience , letermovir primary prophylaxis (pp) has been shown to reduce clinically significant cmv infection with a favorable safety profile. letermovir pp will improve the outcome of seropositive patients. however, patients who did not benefit from pp and experienced > 1 cmv episode (infection or disease) after hct may be candidate to secondary prophylaxis (sp). indeed half of them will have >1 recurrent episode after pre-emptive treatment (pet). letermovir is available since november 2017 as part of the french early access program for pp and sp. we report the outcome of patients who benefited from letermovir sp in the context of this program. methods: letermovir is granted, in a restrictive manner, by the french drug agency (ansm) on a case-by-case basis for prophylaxis of cmv episode, in cmv-seropositive adult allogeneic hct recipients. sp patients should have a negative baseline cmv pcr, have already experienced >1 cmv episode, in the context of a potentially harmful pet according to physicians. planned letermovir daily dose was 240 mg in case of concomitant cyclosporine and 480 mg otherwise. all patients were routinely screened by blood or plasma cmv pcr. results: between november 2017-july 2018, 57 patients received letermovir in the early access program, 22 for pp, and 35 for sp. among the 35 sp patients, 6 had previous cmv disease (gut: 5; cns: 1). mean age was 55±13 years, m/f ratio was 22/13. the sp cohort included one cord blood and 10 haplo-identical hct. main diagnoses were acute leukemia (46%) and myelodysplastic syndrome (26%). the conditioning regimen was myeloablative in 47% and included atg in 61%. based on available data (6 missing data, md), previous gvhd was present in 22 (76%) patients, and active at letermovir initiation in 17 (59%). thirty two (91%) patients were planned to receive immunosuppressants. donor's cmv serology was negative in 12/15 (80%) (20 md). at baseline, cmv pcr was detectable in 1/35 patients. letermovir was initiated a median of 195 days (iqr: 154-308) after transplant for a mean duration of 112 ± 47 days. only one (3%) patient developed cmv breakthrough. the median follow-up from letermovir initiation was 103 days. among the 57 patients exposed to letermovir prophylaxis, two patients permanently discontinued because of letermovir-related adverse events (acute gvhd and nephropathy for one, loss of appetite, pruritus, diarrhoea and weight loss for the other); two deaths occurred with no causal relationship to letermovir. data were consistent with the known safety profile of letermovir. conclusions: letermovir is or will be soonly available in most european countries for cmv prophylaxis in hct recipients. pending its routine use, letermovir used as sp was well tolerated and effective, with only 1/35 patients developing a breakthrough infection. in this high-risk population for cmv recurrence, letermovir may provide a safe bridge between pet and specific immune reconstitution, pending tapering or discontinuation of immunosuppressants. whether sp may improve survival deserves further studies. disclosure: thierry allavoine is a former employee of msd france, nathalie benard and amir guidoum are employees of msd france, marion masure is an employee of icta pm, sophie alain and catherine cordonnier have participated in advisory boards and have been members of the speaker bureau of msd. ibrahim yacoub-agha has received honoraria from msd, other authors: nothing to declare p407 real-world data on letermovir prophylaxis for cytomegalovirus reactivation after allogeneic hematopoietic cell transplantation: a single center experience patrick derigs 1 , maria-luisa schubert 1 , paul schnitzler 1 , carsten müller-tidow 1 , peter dreger 1 , michael schmitt 1 1 heidelberg university hospital, heidelberg, germany, background: reactivation of cytomegalovirus (cmv) still contributes substantially to morbidity and mortality after allogeneic hematopoietic cell transplantation (allohct). recently, letermovir became available as the first drug approved in europe for prophylaxis of cmv reactivation in seropositive patients who have undergone allohct. letermovir is neither myelo-nor nephrotoxic, and significantly reduced the incidence of cmv reactivation in a pivotal phase iii trial (nejm 2017; 377:2433) . therefore we adopted letermovir prophylaxis according to the label as standard policy in our institution: in seropositive recipients letermovir was initiated after engraftment and continued until day +100 or cmv reactivation. the aim of the present study was to investigate if the favorable trial results could be reproduced under real-world conditions. methods: the study cohort consisted of the first seropositive 35 patients who received letermovir prophylaxis at our institution (between march and august 2018). these were compared with a control cohort transplanted between august 2017 and march 2018 before the advent of letermovir. study and control cohorts were matched for cmv donor/recipient sero-status, underlying disease and donor type source of stem cells and application of atg. cmv viremia was monitored by a quantitative pcr twice a week during the inpatient period and weekly thereafter. patients reactivating cmv prior to engraftment were not considered as event in both groups. results: no major side effects of letermovir intake were observed. with altogether 5 reactivation events, the cumulative rate of cmv reactivation on day +100 was 14% (95%ci 1-45%) in the letermovir cohort and thus significantly lower than in the control group (20 events, 58% (95%ci 42-71%); hr 0.23 (0.10-0.51); p=0.0003). the median time to reactivation was 53 days for the control group and not reached for the letermovir group. the cumulative number of days on valganciclovir before d +100 was 151d for the 35 letermovir patients vs 689d for the 35 control patients. there were no hospitalizations for foscavir administration in the letermovir group compared to 5 hospitalizations in the control group. there were 2 deaths before d +100 in the letermovir group (one pd, one nrm) and 3 deaths in the control group (all pd). conclusions: this observational study confirms the safety and efficacy of letermovir for the prophylaxis of cmv reactivation in seropositive patients after allohct in a real-world setting. our results are in good concordance with the phase iii trial. although letermovir appeared to reduce the need for therapeutic valganciclovir and foscavir tremendously, larger samples with longer follow-up are needed to assess the impact of letermovir prophylaxis on non-relapse and overall mortality as well as on resource consumption. background: cmv viremia occurs in 40%-80% of cmv r + hct recipients. pet use has reduced the risk of cmv end-organ disease (eod) and associated mortality; however, pet use may lead to substantial antiviral use and healthcare resource utilization. limited real-world data are available on the outcomes of pet. therefore, we aimed to examine cmv outcomes (eod, resistance), cmv-related mortality by day (d)180 and healthcare resource utilization between pet and no-pet groups among cmv r+ recipients undergoing first hct. methods: we conducted a retrospective cohort study of adults, cmv r+ recipients of first peripheral blood or marrow allograft at mskcc identified from march 2013 through december 2017. data was extracted from electronic medical records and hct databases. cmv+ recipients were monitored weekly by quantitative pcr assay starting on d14 through d180 post hct. use of antiviral therapy for cmv viremia defined pet. high cmv risk (hr) comprised t-cell depleted (tcd) hct by cd34+-selection regardless of donor hla match or conventional hct from mismatched or haploidentical donors; low risk (lr) included conventional hct from matched related donors. cmv eod was scored by standard criteria. cmv resistance mutations were confirmed by sequencing (viracor-eurofins). length of stay (los) for hct admissions and readmissions were identified through d180. stratified analyses were performed to examine outcomes by pet use and cmv risk. background: in a phase iii randomized, double-blind, placebo-controlled study of cmv-seropositive post-hsct recipients, letermovir prophylaxis significantly reduced the incidence of clinically significant cmv infection through week 24. the objective of this research was to assess the impact of cmv prophylaxis on rates of rehospitalization in adult cmv seropositive allogeneic hsct recipients from the letermovir phase 3 clinical trial. methods: rehospitalization was recorded as an exploratory endpoint in the clinical trial at end of treatment (week14), time of primary endpoint (week24) and through an extended follow-up period (week48). cmv-related rehospitalization was assessed in the trial. prespecified analyses describe the observed rates of rehospitalization for the letermovir and placebo groups at the specified times. fine-gray cumulative incidence function(cif) regression models were used to explore the rate of all-cause, and cmv-related rehospitalization accounting for the competing risk of mortality. a multiple linear regression model was used to describe the cumulative length of stay (los) for all-cause rehospitalizations that occurred through week48 (excluding time of initial transplant stay). results: observed rates of all-cause rehospitalization were lower for the letermovir group compared to placebo at end of treatment (36.6%vs.47 conclusions: letermovir was shown to significantly reduce the rate of clinically significant cmv infection in a placebo-controlled randomized clinical trial. these analyses suggest that there is also a reduction in the rate and cumulative days of rehospitalization. this trial was not sufficiently powered to detect differences in this exploratory endpoint. nonetheless, these data provide valuable insights into the economic burden of cmv. real world data and findings from future clinical trials are needed to better understand the nature of the association between cmv and rehospitalizations. clinical methods: all consecutive patients with hematologic disorders who received hsct at our center between january 2013 and august 2018 were included. among the 278 evaluable patients, 172 received levofloxacin as antibacterial prophylaxis (group a) while 106 did not receive any fq prophylaxis (group b). baseline characteristics were similar in the two groups, except for the number of patients with advanced disease (34% in group a and 47% in group b, p 0,042). median duration of neutropenia was 16 days (range 9-44) in group a and 14 days (range 4-31) in group b. a positive rectal swab for carbapenem-resistant enterobacteriaceae (cre) was detected in 3 patients in group a and 8 patients in group b. results: overall, bsi was detected in 58 patients (20,9%), 29 (16,9%) in group a and 29 (27,4%) in group b (p=0,048). the median onset of bsi was 8 days post transplant (range 0-28), without significant differences between the two groups. in univariate analysis, fq prophylaxis (or 0,54; 95% ic 0,30-0,97) and bone marrow stem cell source (or 2,08; 95% ic 1,05-4,12) were significant factors associated with the risk of bsi. gramnegative bacteria accounted for 44,8% (n=13) of bsi in group a and 65,5% (n= 19) in group b, and gram-positive bacteria for 48,3% (n= 14) of bsi in group a versus 27,5% (n= 8) in group b, without statistically significant differences (p = 0,16). polymicrobic bsi were 6,9% (n=2) in group a and 6,9% (n=2) in group b. mdrgram negative bsi were detected in 4 patients (14%) in group a and in 6 patients (20,7%) in group b (overall, 2 cre, 7 esbl producing enterobacteriaceae and 1 mdr-pseudomonas). death attributable to bsi occurred in 6 of 58 patients (10,3%); 5 of these patients did not receive fq prophylaxis, but 2 of them had both a pre transplant kpc colonization and active disease at transplant. neither antibacterial prophylaxis (p = 0,98) nor bsi (p = 0,4) had a significant impact on overall survival (os). conclusions: the preliminary data of our study show that fq prophylaxis is associated with a reduced incidence of bsi, in particular gram-negative infections, with no impact on os. the limitations of our study may be the different group sizes and the retrospective nauture of the study. whether antibacterial prophylaxis should be avoided in the pre-engraftment period in still a matter of debate and needs to be evaluated in larger prospective studies. disclosure: nothing to disclose. gillen oarbeascoa 1 , nieves dorado 1,2 , laura solan 1,2 , rebeca bailen 1,2 , pascual balsalobre 1,2 , carolina martinez-laperche 1,2 , ismael buño 1,2 , javier anguita 1,2 , jose luis diez-martin 1,2,3 , mi kwon 1,2 1 hospital general universitario gregorio marañón, hematology, madrid, spain, 2 instituto de investigación sanitaria gregorio marañón, madrid, spain, 3 universidad complutense de madrid, madrid, spain background: incidence and outcome of invasive fungal infection (ifi) are not well characterized in the setting of peripheral blood, non-manipulated haploidentical stem cell transplantation with postransplant cyclophosphamide (haplosct). the aim of the study was to analyze incidence and risk factors of ifi in patients who underwent haplosct at our institution. methods: 132 consecutive patients who underwent peripheral blood haplosct with postransplant cyclophosphamide between 2011 and 2017 at our centre were reviewed. ifi was classified according to eortc definitions. proven and probable ifi were included. results: patients´characteristics are shown in table 1 . primary antifungal prophylaxis was performed with micafungin from day -1 until oral intake, followed by posaconazole until day +35. patients on steroid treatment for gvhd received prophylaxis with micafungin or posaconazole. 92% of patients obtained neutrophil engraftment. twenty-two episodes of ifi were observed in 20 patients: 10 proven and 12 probable, with a cumulative incidence of ifi of 17% at 500 days. most commonly isolated organism was aspergillus spp (n=5), followed by candida spp (n=4: 1 c. kruseii and 3 c. parapsilosis), and fusarium spp (n=2). isolated cases of inonotus spp, mucor spp and trichosporon ashii were observed. pulmonary involvement was the most frequent clinical presentation (n=10), followed by fungemia (n=5: 4 candidemia, 1 trichosporon ashii) and skin-pulmonary involvement (n=2). among patients with lung involvement, 10 showed probable ifi: 5 with elevated serum galactomannan and 3 positive galactomannan in bronchoalveolar lavage (bal). there were 2 patients without galactomannan, one with a positive bal culture for penicillum spp and the other with an aspergillus spp. median time to ifi diagnosis was 21 days. thirteen cases were diagnosed in the pre-engraftment period, 4 after engraftment and 5 cases after day +30. among patients with late ifi, median time to development was 220 days. all of them were associated with gvhd (3 grade iii-iv acute gvhd and 2 moderate/severe chronic gvhd). ifi outcome was favorable in 13 out of the 22 ifi. treatment was liposomal amphotericin b in 7 cases, voriconazole in 4 and combined treatment (amphotericin b and azole) in 6. there were 7 ifi related deaths, with a cumulative incidence of ifi related death of 6.4%. prior transplant (or 4.5, p< 0.01), particularly allohsct was associated to ifi development (or 8.2, p< 0.01). patients with previous allohsct presented ifi mainly from molds: 3 aspergillus, 2 fusarium, inonotus, trichosporon and mucor. there were also 2 candidemia episodes. no other factors were significantly associated to ifi occurrence. conclusions: in our experience, cumulative incidence of ifi in the setting of haplosct with posttransplant cyclophosphamide was similar than observed in previous studies in allosct. having received a previous sct, especially allosct, was the most significant factor related to ifi development. this high risk population should be closely monitored and could benefit from prophylaxis with azoles. disclosure: nothing to declare. methods: rsv infection was diagnosed in nasal wash (nw) or bronchoalveolar fluid (bal) by dfa (millipore, usa) or pcr (seeplex, seegene, kor). urti and lrti were defined according to ecil-4 guidelines. death from all causes was assessed within 90 days after rsv infection and was attributed to rsv if the patient had persistent or progressive rsv infection with respiratory failure at the time of death. neutropenia and lymphocytopenia were defined as an absolute neutrophil count (anc) < 500/ul and absolute lymphocyte count (alc) < 200/ul, respectively. results: median number of confirmed rsv infections per year was 12, ranging from 5 to 34. an outbreak of rsv was detected in 2017, possibly due to a lack of compliance with contact precautions in the unit. median patients' age was 26 years and time to rsv infection was day 80 (-11 to 1837). twenty-three patients (pts) had received an autologous transplantation (17.2%) and 111 were allogeneic hsct recipients (82,8%). median time to engraftment was 15 days, ranging from 10 to 27 days. at rsv diagnosis, 108 pts presented with urti (80.6%) and 26 with lrti (19.4) . surprisingly, around 18% of the auto hsct recipients had rsv pneumonia at diagnosis. variables significantly associated with lrti at diagnosis were mud hsct (no/ yes, or 0.42; ci95 0.20-0.89); anc < 500/ul (or 2.75; ci95 1.01-7.45); alc < 200/ul (or 3.25; ci95 1.12-9.45); and recent or pre-engraftment hsct (no/yes, or 0.38 ci9 0.14-0.98). among the 108 pts with urti at diagnosis, 19 progressed to lrti (17.6%). forty-four of the 134 pts died (32.8%) and mortality rate was significantly higher in pts with lrti in comparison with pts with urti (53.8% versus 27.8%, p=0.011). death was attributed to rsv in 11 of the 44 pts who died (25%). conclusions: autologous hsct recipients are also at risk of lrti caused by rsv. risk of rsv lrti is higher in mud hsct, infection acquired pre-engraftment or early after hsct, and low neutrophil and lymphocyte counts. continued education is necessary to sustain compliance to contact precautions in hsct units. disclosure background: measles is a life-threatening infection after allogeneic hct. due to the decreased coverage of vaccination in many countries, the disease reappears, increasing the risk of outbreaks worldwide. allogeneic hct recipients have been shown to be seropositive for measles in roughly 40-50% of the cases 3 years after transplant. however, these data were obtained before the 2000's from hct populations mainly conditioned with myeloablative (ma) regimens. our aim was to assess measles immunity before considering vaccination in a cohort of hct survivors including patients conditioned with reduced intensity (ric) or non-ma regimens. methods: allogeneic hct adult recipients who had not been vaccinated for measles since hct were routinely screened for measles immunity. measles igg titers were determined with a chemiluminescence immunoassay (liaison measles igg kit, liaison xl analyser, diasorin, italy). patients were considered to be seropositive if the igg titer was > 16.5 ua/ml. risk factors for seropositivity were analyzed. qualitative variables were described as numbers (%) and compared using the chi-2 test or fisher exact test as appropriate. quantitative variables were described as median or mean (range) and compared using the kruskall-wallis test. ors were estimated separately for factor yielding a p-value < 0.20 in the univariate analysis using logistic regression models. results: eighty-six patients, transplanted 1.5 to 38 years (mean: 13,5 years) ago, were included. the mean age was 53 years (range: 21-79), the sex ratio m/f: 0,5. the underlying diseases were acute leukemia: 49 (57%), myelodysplastic syndrome: 5 (6%), lymphoproliferative diseases: 17 (19.5%), myeloproliferative neoplasms: 11 (13%) and non-malignant diseases : 4 (4.5%). the hct was performed from an hla-identical donor in 52, an unrelated donor in 30, and a cord-blood in 4. conditioning regimen were ma in 48 (56%), ric in 20 (23%) and non-ma in 18 (21%) patients no patient had experienced measles or had received measles vaccination since transplant. fifty-seven of the 86 (66%) patients were seropositive for measles. measles seropositivity was not associated with conditioning regimen, patient age at transplant, patient age at time of assessment, donor age at transplant, lymphocyte count or gammaglobulin levels, or type of transplant (hlaid. vs others) measles vaccination before transplant or previous measles before transplant. the only parameters significantly associated to seropositivity were absence of previous gvhd (any type or severity, p=0,033 or 0,31 [0,10-0,94]), and absence of previous extensive chronic gvhd (or 0, 28 [0, 87] p0,027). conclusions: sixty-seven percent of allogeneic hct are seropositive for measles at a median of 7 years after hct before vaccination. the only risk factor strongly associated with seronegativity is extensive chronic gvhd. in patients background: cytomegalovirus (cmv) reactivation is a frequent complication after hematopoietic stem cell transplantation (hsct). extracellular vesicles (evs) have emerged as a promising new category of biological biomarkers in different scenarios, including inflammation, tissue damage, cancer and viral infections. we recently reported on the potential use of serum evs as biomarkers of agvhd (lia g. et al. leukemia (2018) 32, 765) . here, we investigated the potential correlation of cmv reactivation with plasma evs in post-transplant cyclophosphamide (ptcy) haploidentical-hsct (haplo-hsct). methods: plasma samples were collected after mononuclear cell separation at given time-points (pre-transplant, on day 0, 3, 7, 14, 21, 28, 35, 45, 60, 75 and 90 after haplo-hsct) and evs were extracted by a protamine-based precipitation method and their concentration and dimension were characterized by nano-tracking particle analysis (nanosight). after extraction, evs were analyzed by flowcytometry (guava easycyte flow cytometer) with a panel of 14 antibodies (cd44, cd138, cd146, krt18, cd120a, cd8, cd30, cd106, cd25, cd26, cd31, cd144, cd86, and cd140a). results: thirty-two patients with hematological malignancies underwent haplo-hsct between 2011 and 2015. cmv reactivation was observed in 20/32 (62,5%) and occurred at a median of 50 (range: 10-275) days after transplant. preliminary analysis (17/32 patients) showed that cd140a fluorescence (platelet-derived growth factor receptor-α or pdgfr-α), cd30 fluorescence (ki-1 antigen) and cd144 fluorescence (ve-cadherin) were associated with an increased risk of cmv reactivation (or 2.67 p=0.045; or 3.11 p=0.011; or 2.37 p=0.08), whereas cd31 (platelet endothelial cell adhesion molecule, pecam-1) concentration level was associated with a decreased risk of cmv reactivation (or 0.26, p=0.032). all these biomarkers showed a signal change before cmv reactivation (an increase with cd140a, cd30 and cd144, a reduction with cd31). (figure 1 ). conclusions: we observed a potential association of 4 evs membrane proteins with cmv reactivation: cd140a, cd30, cd144 and cd31. these proteins are crucial for endothelium and immune cells interaction. cmv can infect different cell types including endothelial cells (bentz gl. pnas 105 (14) 2008). moreover, cd140a (pdgfr-α) has been shown to function as an entry receptor for cmv expressing gh/gl/go complex (wu y. et al. plos pathog 13 (4) 2017). we plan to implement our analysis characterizing evs contents (mirnas) and will be applied to investigate other viral reactivations (e.g. epstein barr virus and human herpes virus 6). [[p414 image] 1. methods: to explore the value of cmv dna extracted from gi tissue for the diagnosis of cmv gastroenteritis, we retrospectively evaluated 71 patients, aged 17-67 (median 44.8 years) who received allo-hct from sibling(26), matched unrelated(40) or haploidentical donors(5), after receiving myeloablative (56) or reduced intensity conditioning(15). they all underwent endoscopy for gastrointestinal symptoms between 2012-2018. cmv dna from tissue samples and parallel blood samples were measured by q-pcr. positive cmv dna on the tissue was considered cmv gi infection.cmv gi disease was proven with the identification of cmv inclusion bodies or positive immunehistochemical staining using anti-cmv antibodies. results: overall, 91 endoscopic tests were performed (55 gastro-,36 colonoscopies) at a median of 73 days (iqr:145) post transplantation. symptoms included nausea, vomiting, diarrhea, abdominal pain and weight loss. cmv dna was positive in 41/91 tissue samples: median 536 copies/ml, range: 11-131x10^6. only half patients (22/41) had concurrent cmv viremia (plasma viral load>100c/ml). cmv gi infection was not correlated to the type of transplant, acute or chronic gvhd. gi cmv disease was documented by biopsy in 13 patients. cmv dna of the tissue, but not the plasma viral load, was a predictor of biopsy positivity (or: 1.6, 95%ci: 1.1-1.8, p=0.006). thirty-six out of 41 cmv dna positive patients received specific treatment for at least 10 days. symptoms resolved in 21/36 patients (60%) and the gi viral load was not a significant factor to predict cure. gi gvhd was diagnosed in 42/91 patients, among which 45%(19/42) with cmv dna positivity. median os was 453 days (95%ci: 297-608) for patients with cmv infection, similar to those without (median os: 890, 95%ci: 80-1699 days, p=ns). we studied separately endoscopies of the upper (55/91) or lower gi tract (36/91) . there was no significant relationship between cmv gastritis proven by biopsy and cmv dna levels in gastric tissue. however, the viral load of the colon was a predictor of cmv enteritis (or: 1.9, 95%ci: 1.9-3, p=0.007). the auroc of the q-pcr was 0.849 (95%ci: 0.659 to 1), the sensitivity was 85.7% and the specificity was 78.6% with a cutoff value of 370 copies/ml dna. conclusions: pathognomonic findings in the biopsy remain the gold standard for the diagnosis, especially for the upper gi tract. however, when the lower gi tract is involved, quantification of cmv viral load in the tissue may be a valuable tool to support the diagnosis. positivity of cmv dna of the gi tissue, in linearity to the cmv viremia, may guide to preemptive treatment for prevention of cmv disease . disclosure: nothing to declare background: clostridium difficile infection (cdi) is caused by cd overgrowth in antibiotic-disturbed intestinal microbiota. antibiotics targeting unselectively beneficial for t-regulatory cell formation strains of clostridiales may increase pro-inflammatory processes in the guts promoting or augmenting the development of graft vs. host disease (gvhd). the efficacy of cdi treatment has impact on the persistence of inflammation which might influence the alloreactive reactions. methods: we retrospectively and, from 2016, prospectively analyzed the data from 5 transplant centers concerning cdi occurrence, treatment efficacy, and gvhd development. the study included 77 patients with hematological malignancies who underwent allogeneic hematopoietic cell transplantation (allohct) between 2012-2018. results: median time to cdi was 14 days post-allohct with detection of both toxins a and b in 57% of cases. disturbance of intestinal microbiome was confirmed by a 59% rate of colonization with multidrug-resistant bacteria (mdrb). the cdi symptoms resolved with the negative toxins after the first line treatment in 76.6% of patients. the median time to remission and therapy duration was 8 and 10 days, respectively. fifteen therapeutic failures were observed after treatment with metronidazole (10), vancomycin (2) and a combination therapy (3) . eleven patients responded to second line treatment. thirty-seven (48%) patients died due to infections (17), relapses (10) and gvhd/infections (10). we noted recurrent cdi in 6 cases. eight patients died with active cdi. we observed occurrence or exacerbation of gvhd in 35 (44%) patients following cdi, including 25 cases with gut involvement (gi-gvhd). treatment with metronidazole and failure of the first line therapy increased the development or escalation of gi-gvhd (p= 0.03 and p< 0.001, respectively). the duration of cdi exceeding 10 days also had impact on the gi-gvhd incidence (p= 0.002). conclusions: 1. patients colonized with mdrb are at high risk of cdi. 2. high mortality due to infections and/or gvhd in patients with cdi. 3. due to lower efficacy and harmful immunomodulatory impact, metronidazole should not be the first line treatment in cdi post-hct. 4. emphasis must be put on fast cdi resolution to interrupt a vicious circle of the intestinal inflammatory processes. disclosure: nothing to declare establishing optimal preemptive cytomegalovirus therapy threshold post allogeneic hct in a patient population with high prevalence of seropositive status background: preemptive therapy (pet) for cytomegalovirus (cmv) reactivation post allogeneic hematopoietic stem cell transplantation (hct) was shown to decrease the incidence of cmv disease. however, the optimal pet threshold is unknown and there are significant toxicities associated with anti-cmv therapy. at our institution, we initiate pet at cmv dna titer above 1000 copies/ml (1560 iu/ml). our aim was to examine the efficacy of this approach including the incidence of spontaneous clearance in a population with high prevalence of cmv seropositive status. methods: after due irb approval, patients that underwent allogeneic hct were identified and records retrospectively extracted.cmv reactivation was defined as the first detectable viral titer post hct from plasma samples whereas clearance of viremia as the first date of two negative pcr values obtained at least 1 week apart. cmv monitoring was initiated post hct performed at least weekly during the first 100 days and every 2-4 weeks thereafter. a high sensitivity assay abbott realtime cmv was used with detection threshold of 20 copies/ml (31.2 iu/ml). analysis was computed using jmp v. 14.0.1 results: a. baseline characteristics: a total of 195 patients were identified and included with a median follow up of 18.1 (0.7-95.6) months. median age was 26 (14-63) years and 58% were male. indication for hct was for a malignant disorder in 77% of cases. the majority had a matched related donor (87%) and cmv igg was positive in both donor and recipient in 98% of cases. myeloablative conditioning was given to 117 (60%) and 109 (56%) received tbi. in vivo t-cell depletion was given to 76 (39%); atg in 39 (20%) and alemtuzumab in 37 (19%). b. cmv reactivation and pet: a total of 178 (91%) patients had a positive cmv pcr with median days to reactivation post hct of 17 ; 129 (66%) patients had peak cmv titer < 1000 copies/ml (low titer) whereas the remaining 49 (25%) had a peak titer ≥ 1000 copies/ml (high titer). patients with high titer were more likely to be older (p = 0.019), have malignant disease (p = 0.019), haploidentical or unrelated donor (p < 0.0001) and higher incidence of agvhd grade ii-iv (p = 0.003) as shown in the table. median peak titers for the low and high groups were 111 vs. 4638, respectively (p < 0.0001).120 (93%) patients with low titers cleared spontaneously with median time to clearance of 40 days (4-188), 1 (1%) received anti cmv therapy and the remaining died with active viremia (range 49-561 copies /ml) with active disease. one patient in the high titer group developed cmv disease. 2-year os and ci-nrm was 67.9% vs. 55.4% (p = 0.1) and 8% vs. 19.1% (p = 0.034) in the low and high titer groups, respectively. conclusions: cmv reactivation was high in this cohort however of low titer viremia in over 70%. a pet threshold of 1000 copies/ml (1560 iu/ml) appears desirable as it was associated with spontaneous clearance in almost all patients while minimizing treatment related toxicity. validation of these observations is warranted. background: the risk of pneumocystis pneumonia often warrants antifungal prophylaxis for recipients of blood and marrow or solid organ transplantation. however, complications such as myelosuppression, nephrotoxicity, and intolerance with the existing standard, trimethoprim/sulfamethoxazole (tmp/smx), may hinder or interrupt prophylaxis. rezafungin (rzf) is a novel echinocandin in development for prevention of invasive fungal disease caused by candida, aspergillus, and pneumocystis species in blood and marrow transplant patients. rzf has a favorable safety and tolerability profile and a low risk of drug-drug interactions. furthermore, the stability and pharmacokinetics of rzf allow for once-weekly dosing and broad distribution to the lung and other target organs. rzf was shown to prevent in vitro pneumocystis biofilm formation and to reduce the viability of mature biofilms. a previous prophylactic study was conducted using a broader range of rzf doses. in the current study, the efficacy of rzf was evaluated to better understand the minimum doses necessary to prevent pneumocystis growth in a mouse model. methods: c3h/hen mice were immunosuppressed (dexamethasone 4 mg/l in acidified drinking water) and then infected intranasally with p. murina (2 x 10 6 /50 μl). given the slow growth of p. murina, test agents were administered at the same time mice were inoculated to test for prophylactic efficacy. mice received intraperitoneal injections of either vehicle (control/steroid [c/s]), tmp/ smx 50/250 mg/kg/3x/week (wk), caspofungin 5 mg/kg once daily, or rzf 5 mg/kg or 0.5 mg/kg once daily, 1x, or 2x/wk. after a 6-week dosing period, mice were sacrificed and lung homogenates were processed for analysis to quantify the nuclei (trophic) and asci (cyst) forms of pneumocystis. prophylaxis efficacy was based on reduction of organism burden compared with c/s. nuclei and asci counts were log transformed and analyzed by anova; individual groups were compared by the student-newman-keuls t test. survival rates were compared using graphpad prism v5. results: all mice in the rzf groups had significantly reduced nuclei and asci burdens compared with the c/s group, and all but the lowest doses of rzf (0.5 mg/kg 1x or 2x/wk) worked as well as tmp/smx at reducing nuclei levels. similarly, all rzf groups except for the 0.5 mg/kg 1x/wk group showed reductions in asci levels comparable to that of tmp/smx. the survival rates were not statistically different between treatment groups. conclusions: rzf demonstrated potent in vivo efficacy for prophylaxis against pneumocystis in an in vivo mouse infection model at dose regimens much lower than the human equivalent phase 3 regimen. these data support the development of rzf for the prevention of invasive fungal infections including pneumocystis pneumonia. disclosure: melanie t. cushion: research funding (cidara therapeutics) taylor sandison: employee, stockholder (cidara therapeutics) alan ashbaugh: nothing to declare. yuhua ru 1,2 , ziling zhu 1,2 , yang xu 1,2 , suning chen 1,2 , xiaowen tang background: immunocompromising period following allogeneic hematopoietic stem cell transplantation (allo-hsct) may allow opportunistic pathogens to thrive and result in fatal complications. epstein-barr virus (ebv) infects more than 90% of chinese population, and its reactivation after hsct is one of the major concerns due to the increased risk of ebv diseases and post-transplant lymphoproliferative disease. with the development of infection prophylaxis and supportive care after hsct, demographic data on ebv reactivation post-hsct needs to be updated. methods: we retrospectively analyzed the data of patients who received allo-hsct between july 2011 and july 2014 in the first affiliated hospital of soochow university. quantitative pcr (q-pcr) was used to monitor ebv-dna load in peripheral blood dynamically. ganciclovir (pre-hsct) followed by acyclovir was given as viral prophylaxis. the treatment protocol for ebv reactivation consisted of tapering of immunosuppressive agents, antiviral agents (including ganciclovir and sodium phosphonatel), and rituximab for persistent positive patients. results: totally 890 cases from most of the provinces in china were enrolled (characterized in table 1 ), among whom ebv reactivation developed in 175 recipients. most reactivation events (95.4%) occurred in the first year post-hsct, with a peak of 113.8 incidence rates per 100 personyears at the second month. besides, more episodes of lateonset reactivation occurred in patients receiving grafts from haploidientical donors ( figure 1a ) . multivariate analyses revealed that the major impactors of ebv reactivation included atg as gvhd prophylaxis (p< 0.001), hlamismatched donor (p=0.001) and the appearance of chornic gvhd (p=0.042). cumulative incidence of ebv reactivation was low (2.9%) among 890 patients with no major risk factors, but increased to 11.7%, 27.3% or 41.9% with 1, 2, and 3 major risk factors, respectively ( figure 1b) . there was no statistical difference of overall survival between people with or without ebv reactivation (p=0.871). conclusions: we concluded that there are similar ebv reactivation impactors in chinese population compared to literatures, including atg use, hla-mismatched donor and the appearance of chronic gvhd. additionally, incidences of ebv reactivation increased significantly with the accumulation of risk factors. however, ebv reactivation had no impact on overall survival in current virus management protocol. disclosure: nothing to declare background: several studies have shown loss of diversity of the gut microbiome in association with significant gut injury following hematopoietic stem cell transplantation (hsct). prolonged broad spectrum antibiotic use further promotes loss of microbiome diversity and increases the risk of intestinal colonization by multi-drug-resistant (mdr) bacteria. aims of this study were to prospectively evaluate the overall changes in gut microbiome composition after hsct and differences in patients colonized by mdr bacteria and treated with carbapenems. methods: we performed a prospective observational study evaluating the gut microbiota of 20 hematological patients undergoing hsct, from admission (t0) through day +28 (t5). fecal microbiota was assessed by 16s amplicon-based sequencing. clinical, and microbiological data as well as fecal samples were collected every 7 th day from admission. results: one-hundred fecal samples were analyzed. overall, we found a progressive decrease of bacterial richness from t0 to t5, with a significant reduction of blautia, ruminococcus and dorea species, which are strictly associated with the production of short chain fatty acids (sca) (fig.1) . moreover, in the 30% (no.6) of patients who were colonized by esbl bacteria, we observed a significant reduction of clostridium spp and bifidobacterium species. as for antibiotic therapies, carbapenems were used as second line treatment of febrile neutropenia in 50% (no 9) of cases, usually associated with aminoglycosides. in patients treated with meropenem, a strong decline of blautia and ruminococcus species was observed. this finding suggests a correlation between carbapenem regimens and increase of pro-inflammatory bacterial strains in the gut. conclusions: our data support the hypothesis that loss of intestinal commensals that produce short-chain fatty acids may increase dysbiosis. moreover, for the first time we report significant and progressive alterations in the composition of blautia, ruminococcus and bifidobacterium species in patients treated with meropenem and colonized by esbl bacteria, respectively. our findings offer potential modifiable targets to reduce risk of colonization by mdr bacteria and to promote a carbapenem-sparing approach in the hsct setting. clinical background: cmv is associated with significant morbidity after allogeneic hematopoietic stem cell transplantation. strategies to prevent cmv-related complications include universal prophylaxis and preemptive therapy, more widely spread. antivirals used for cmv reactivation (cmv-r) produces major toxicities and costs. rate and characterization of cmv-r after haploidentical transplantation with post-transplant cyclophosphamide (haplo pt-cy) is scarce. our goal was to analyze cmv-r rate after haplo pt-cy, outcome, complications associated to therapy, and to identify risk factors. methods: one hundred haplo pt-cy transplants using peripheral blood as stem cell source performed between 2011 and 2016 in our center have been retrospectively reviewed. gvhd prophylaxis consisted of pt-cy 50 mg/ kg/day on days +3 and +4, mmf and csa from day +5 for all cases. cmv pcr was performed in a biweekly basis during admission for transplant and treatment, and weekly thereafter. cmv-r was considered with any cmv dna level by pcr assay above 100 copies/ml. prior four consecutive negative weekly pcrs were needed to consider a new reactivation episode. preemptive strategy was applied in all cases. data collected in relation to cmv-r included: cmv serostatus of donor/recipient (d/r), number of cmv reactivations, length of each reactivation, antiviral treatment used, need for admission to receive treatment and adverse events related to cmv reactivation and/or antiviral treatment. results: patients characteristics are summarized in table 1 . among 100 patients, 78 of them with positive cmv serology, 128 episodes of cmv-r were detected. seventysix patients (76%) had at least one cmv-r in a median of 30 days after transplant. none of them had cmv disease or die as a consequence of cmv-r. median duration was 22 days (15-37). valganciclovir or ganciclovir was used in 101 episodes (79%). foscarnet was used in 43 episodes (34%). six of the episodes occurred after initial discharge, and required re-admission for treatment, with a median length of hospitalization of 16 days (6-27). cytopenias requiring transfusion or g-csf support occurred in 36 episodes (36%) treated with ganciclovir or valganciclovir. three of them needed further cd34+ cells booster for graft rescue. mild acute renal failure and genital ulcers were found in 21 (49%) and 5 (11,6%) events treated with foscarnet, respectively. no cases of severe renal failure were observed. serological status different than negative/negative (n/n) (p 0.001) and older age (46 vs 38 years, p 0.02) were significantly associated with cmv-r. no relationship was observed with gender, disease, donor relationship, conditioning, gvhd or cells infused. more than 2 reactivations were more frequent among patients with grade ii-iv acute gvhd (agvhd) and moderate-severe chronic gvhd (cgvhd). conclusions: in our experience, rate of cmv-r after unmanipulated haplo pt-cy, using pbsc as stem cell source, is considerably high. a significant proportion of patients presented complications associated with cmv-r and its treatment. cmv serological status other than n/n and older age are associated with high risk of cmv-r. patients with grade ii-iv agvhd are at higher risk of multiple reactivations. this population could be benefited from primary prophylaxis, in order to decrease treatment´s complications, re-admissions and costs. disclosure: nothing to declare. impact of infectious events occurring during the first hundred days after hsct for hematological malignancy: a monocentric retrospective study over a five-year period marie-pierre ledoux 1 , célestine simand 1,2 , karin bilger 1 , annegret laplace 1 , bruno lioure 1 background: patients undergoing hematopoietic stem cells transplantation (hsct) for hematological malignancy often present with infectious events in the early stages of the procedure, some of which having a documented impact on the outcome of the graft. for instance, cytomegalovirus (cmv) has been shown by some authors to have a protecting effect against relapse, whose features remain to be elucidated. we conducted a retrospective monocentric study regarding the outcome in terms of graft versus host (gvhd), relapse and survival of 224 consecutive patients over a period of 5 years, whether they presented or not with an infectious event by day 100 among the following: cmv viremia, epstein-barr virus (ebv) viremia, human herpes virus 6 (hhv6) viremia, bk virus (bkv) viruria, bacterial bloodstream infection (bsi) or invasive fungal infection. results: a high proportion of cmv seropositive recipients underwent a viral reactivation of cmv by day 100 of the hsct: 81% if the donor is seronegative and 74% if the donor is seropositive. we observed that cmv wasn't associated with a lower relapse rate in our cohort, and data weren't sufficient to conclude firmly, but showed a trend towards a worse acute gvhd (hazard ratio hr 2.08, pvalue 0.06). no significant correlation was found for ebv viremia. occurring in 26% of our patients and mostly with an early timing, hhv6 strongly correlated with worse acute gvhd (hr 3.25, p-value < 0.001) but its impact on survival was not significant. bkv (27% of our patients) and bsi (46% of our patients) both correlated with poorer outcome in terms of overall survival (logrank < 0.001 and 0.014 respectively) although not significantly associated with relapse or acute gvhd. fungal infections were too rare events to draw any conclusion. conclusions: thus, contrary to many studies, we found no protection against relapse induced by cmv, although the trend for worse acute gvhd was obvious. the mechanisms behind this discordance could include early treatment, but remain to be studied. whether hhv6 is a cause rather than a consequence of acute gvhd or its treatment is debated, but the correlation is strong and the sequence of events suggests hhv6 might act as a trigger for gvhd. the association between bkv viruria and a higher mortality is in contrast with previous observations, and the lack for association with gvhd and relapse could suggest bkv is a surrogate for poor immune recovery and therefore other causes of non-relapse mortality. in addition to the direct lethal risk of bacteriemia, bsi also are a promoter of late non-relapse mortality through indirect toxicity. through the expansion of immune effectors they promote, one could assume that infectious events play a role in gvhd and gvl, and therefore have an interference with relapse. however, the association between each infectious event and outcome remains to be clarified to guide our prophylactic and therapeutic choices by a better understanding of the bright and dark sides of infectious events. disclosure background: rezafungin (rzf) is a novel echinocandin in phase 3 development for treatment of candidaemia and invasive candidiasis and for antifungal prophylaxis against invasive fungal diseases caused by candida, aspergillus, and pneumocystis in blood and marrow transplant patients. rzf is differentiated by stable, prolonged pharmacokinetics (pk) that allow for once-weekly dosing and a pk-pharmacodynamic (pd) profile correlating with efficacy. clinical in vivo evaluations of drug interaction potential were performed proactively to assess the risk of drug-drug interactions (ddis) with respect to the phase 3 dose of 400 mg once weekly and known pk exposure in healthy individuals. methods: this open-label study of 26 healthy inpatients assessed ddis between rzf (as perpetrator) and drugs known to have interactions with cyp enzymes and transporters (probe drugs): repaglinide (cyp2c8), metformin (oct/mate), rosuvastatin (bcrp/oatp), pitavastatin (oatp), caffeine (cyp1a2), efavirenz (cyp2b6), midazolam (cyp3a4), and digoxin (p-gp), as well as tacrolimus, a drug likely to be coadministered with rzf. an initial dose of rzf 600 mg was administered on the first dosing day, to approximate a steady state plasma concentration of multiple once-weekly 400-mg doses, followed by 2 once-weekly 400-mg doses on days 10 and 15. probe drug cocktails containing ≥2 drugs were administered, once before and once after rzf administration, on a schedule designed to allow for washout between doses and to limit interactions with other probe and test drugs. samples were analysed to determine respective drug concentrations in plasma (except for tacrolimus which was in whole blood) to characterize the pk profile of each analyte. area under curve (auc) and maximum concentration (c max ) were calculated from the plasma/blood concentration-time profiles by noncompartmental analysis. ln-transformed pk parameters were statistically analysed using an analysis of variance model. the ratio of geometric least squared means between each substrate drug when administered with and without rzf and corresponding 90% confidence intervals (cis) were calculated for lntransformed c max and auc. results: when rzf was given concomitantly with the probe drugs, six of nine substrates (metformin, pitavastatin, caffeine, efavirenz, midazolam, and digoxin) statistically demonstrated the absence of drug-drug interaction, as their 90% ci were all included within the default 80-125% noeffect boundary. three substrates had the upper (repaglinide and rosuvastatin) or lower (tacrolimus) bounds of their ci falling just outside of this range (figure 1 ), and these changes are considered unlikely to be clinically significant. conclusions: no meaningful pk interactions were observed between rzf and 9 drugs known to have ddis and/or likely to be coadministered with rzf. these findings provide evidence that no dose adjustment is expected when rzf is co-administered with these commonly used drugs, which stand in contrast with the ddi complications widely associated with azole antifungals. disclosure: voon ong: employee, stockholder (cidara therapeutics), michael boily: employee (altasciences), hong wong: employee (altasciences), taylor sandison: employee, stockholder (cidara therapeutics), shawn flanagan: employee, stockholder (cidara therapeutics) abstract withdrawn. background: cytomegalovirus (cmv) continues to cause morbidity following allogeneic hematopoietic stem cell transplantation (hsct). letermovir is a newly approved drug for cmv prophylaxis in cmv-seropositive allogeneic hsct recipients. however, there is a paucity of data for its efficacy in patients receiving in-vivo t-cell depletion (tcd). at weill cornell medical center, we perform in-vivo tcd with alemtuzumab for related and hla-identical unrelated transplants, and anti-thymocyte globulin for umbilical cord blood transplant supported by third party accessory cells (haplo-cord transplant).although these drugs reduce the frequency of graft-versus-host-disease (gvhd), they significantly delay t-cell immune reconstitution post hsct, and may cause higher rates of cmv reactivation. our historical rate of cmv reactivation in cmv seropositive recipients receiving high dose valacyclovir prophylaxis is approximately 35%. therefore, we implemented letermovir for cmv prophylaxis in february 2018. the primary aim of this study is to determine the incidence of cmv infection (defined as cmv viremia warranting treatment or development of end-organ disease) in tcd cmv seropositive allogeneic hsct patients who received letermovir prophylaxis. methods: this is a single center, retrospective cohort study to determine the incidence of cmv infection in adult, cmv-seropositive recipients receiving letermovir prophylaxis after in vivo tcd hsct with atg or alemtuzumab for gvhd prophylaxis. all included subjects were at least 100 days post-transplant. results: 31 allogeneic hsct transplant recipients met inclusion criteria. median age was 60 years, iqr [47, 67] and 48% were male. eight (26%) had a matched related donor, six (19%) had a matched unrelated donor, and 17 (55%) were haplo-cord transplants. their underlying malignancy and conditioning regimens are summarized in table 1 . 17 (55%) received atg and 14 (45%) received alemtuzumab for gvhd prophylaxis. median follow up time for survivors is 141 days, iqr [107, 187] . the incidence of cmv infection in the first 100 days post-transplant was 3% as only one patient reactivated with detectable cmv viremia. this same patient developed cmv pneumonitis with documented ul 56 resistance, and was successfully treated with ganciclovir. the incidence of cmv infection within the first 150 days post-transplant was 5% (1/19 patients) . six patients (19%) developed acute gvhd in the first 100 days, and one (3%) had relapse of their malignancy. five patients (16%) died within 100 days post-transplant, but none of these deaths were cmv related. background: infectious complications caused by endogenous adenovirus (adv) are common and associated with morbidity and mortality rates in patients after hematopoietic stem cell transplantation (hsct). adv infections occur in about 3% to 20% of hsct recipients, with significantly higher rates in pediatric patients. a better understanding of adenoviral-specific t-cells (advt) response in donors can serve as a basis to develop more effective strategies for antiviral therapy. methods: frequencies of cytomegalovirus (cmv)-and adv-specific t cells were determined by enzyme-linked immunospot (elispot) assays with adv5 hexon and cmvpp65 respectively in 80 health donors. we used 3x10 5 of mononuclear cells (mnc) per well in elispot assays. all donors were divided into 3 groups according to the number of spots per well (spw) as follows: high responders (hr) (≥50 spots; n=32), low responders (lr) (>10 and < 50 spots; n=39), nonresponders (nr) (≤10 spots; n=9). the average spot area of adv-and cmv-specific lymphocytes was calculated by immunospot® multiplate autocount™. cd45ra+ and cd45ro+ t-cells were generated by immunomagnetic negative selection. hla typing for class i and ii was performed by sequence specific oligonucleotides technology. statistical analysis was performed using graphpad prism v7.00 software. levels of significance were calculated by mann-whitney rank-sum test, expressed as p-values (p< 0.05). results: the median frequency per well of advts were 72 in hr group, 24 in lr group, 4 in nr group. the median spw of cmv-specific t cells in donors mnc were 147 and didn´t differ between 3 groups. antiviral activity may depend not only on the amount of advt but also on their ability to produce ifnγ. the average spot area for advt did not differ between hr, lr and nr groups and were 22,4, 23,1 and 19,1 mm 2 respectively. the median of the average spot area for anti-cmv t-lymphocytes was equal to 13,5 mm 2 . thus, the frequency of advt was lower than cmv-specific t-cells, but advt have the ability to produce more ifnγ per cell (p< 0.0001). in order to evaluate the distribution of the advt between naive and memory t cell compartments, we evaluated response to adv in preselected cd45ra+ and cd45ro+ fractions of t-cells in a group of 17 donors. the median frequency of advt in unfractionated mnc was 87; the median frequency of advt in cd45ra and cd45ro fractions were 2 and 73, respectively. the amounts of cd45ra and cd45ro tcells were normalized to their amounts in mnc. we evaluated the impact of hla-alleles on the anti-adv response of t-cells in different groups and found 2 association: hla-a*1 with hr group (p-value=0,0043; rr=1,954; 95% ci: 1,285 to 2,604) and hla-a*31 with lr group 0251; rr=1, 889; 95% ci: 1, 135 to 4, 426) . conclusions: in this study the frequency of donors with advt is 88,75% which corresponds to the reported frequency of adv-seropositive people (95%) in population of russia. advt are exclusively cd45ro-positive cells. the analysis of advt in potential hsct donors will allow to determine more accurately the amounts and functional activity of specific antiviral t-lymphocytes administered to patient and optimize antiviral therapy. disclosure: nothing to declare p429 abstract withdrawn. chemotaxis and exhaustion of γδ t cells in the allografts are associated with cmv reactivation after hematopoietic transplantation background: cytomegalovirus (cmv) reactivation and its related diseases remain the most common and serious complications in patients who underwentallogeneic hematopoietic stem cell transplantation (allohsct). we previously reported that the incidences of total and refractory cmv reactivation reached approximately 90% and 50% after haploidentical hsct. while majority of studies in the literatures focused on the adaptive cd8 + αβ t cellsand nk cells in anti-cmv immunity, increasing evidences highlighted the important role of γδt cells in this context. a progressive and prolonged expansion of vδ1 + t cells in response to cmv reactivation was observed after allohsct. the effect of vδ1 + t cells associated with cmv clearance has been reported in vitro and in vivo. in contrast to the reconstituted γδt cells post transplantation, whether the phenotypes of γδt subsets in allografts correlate to cmv reactivation in hsct recipients have not been documented. methods: the proportions and phenotypes of γδ t cells were detected inallografts those were unmanipulated g-csf-mobilized bone marrow (bm) and peripheral blood (pb) harvests from 20 donors for haplohsct. bm grafts were collected by aspiration on the fourth day of g-csf treatment (filgrastim, 5 μg/kg/day), and pb grafts were obtained on the fifth day by leukapheresis. immunophenotyping for γδ t-cell subpopulations, including the expression of cd3, cd4, cd8, tcrγδ, tcrvδ1, tcrvδ2, hla-dr, nkg2d, cxcr4, ccr5, pd1, ki67, ifnγ, tnfα, and il-17, was performed using flow cytometry. for detection of the intracellular cytokines, bm and pb grafts were pre-stimulated with 1x cell stimulation cocktail (500x, ebioscience). cmv dna in the peripheral blood of recipients was routinely monitored by quantitative pcr. the association of γδ t-cell contents in allografts with cmv reactivation in haplohsct recipients was analyzed using the mann-whitney u test and spearman test. all calculations were performed using spss 22.0 statistical software. results: we found that the proportions of total γδ t cells, and vδ1 and vδ2 subsets in both bm and pb grafts for cmv+ and cmv-recipients were comparable. neither the expression of hla-dr nor nkg2d in the allografts were significantly different in correlation to cmv reactivation after hsct. the productions of intracellular cytokines of γδ t subsets did not varied in bm and pb grafts for cmv+ and cmv-recipients. interestingly, the proportions of cxcr4+vδ1 and ccr5+vδ1 cells in bm grafts for cmv + recipients were significantly higher than those for cmvrecipients (p = 0.012 and 0.045, respectively). meanwhile, pma-stimulated ki67+vδ1 cells in bm grafts for cmv+ recipients were less than those for cmv-recipients (p = 0.039). in parallel, the concentration of pd1+vδ1 cells in pb grafts for cmv+ recipients were significantly higher than those for cmv-recipients (p = 0.023). conclusions: this study is the first to connect the chemotaxis and exhaustion of γδ t cells in grafts to the risk of cmv infection after allogeneic hsct. future studies should explore how the expressions of chemokines and exhaustion marker on the effector γδ t cells in allografts facilitate cmv replication and/or dissemination in the setting of hematopoietic transplantation. disclosure: all authors do not have conflicts of interest. this study is supported by the national natural science foundation of china (grants no.81770191 and no.81670167) results: incidence of ic was 2,9%: allo-hsct -3% (n=15), auto-hsct -2,7% (n=7). the etiology: c. parapsilosis 50%, c. albicans 27%, c. krusei 14%, candida tropicalis 5%, candida dubliniensis 4%. the most frequent underlying diseases was acute leukemia -45% (n=10). the median age was 8 y.o. [3 month -18 years] . the median day of onset of ic after allo-hsct was 63 , auto-hsct -12 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . febrile fever was the main clinical symptom; septic syndrome develops in 32% cases. antifungal therapy was with echinocandins -23%, lipid ampho b -27%, azoles (fluconazole, voriconazole) -32%, without therapy (the early mortality) -18%. overall survival (os) at 30 days from diagnosis of invasive candidiasis was 50%. the central venous catheter (cvc) removal was the only factor significantly improved os (70% vs 33%, p=0,035). conclusions: incidence of invasive candidiasis in children after hematopoietic stem cell transplantation was 2.9%. the main etiology agent was c. parapsilosis. invasive candidiasis infections most often affect leukemia patients, developed later after allo-hsct than auto-hsct. overall survival at 30 days from the diagnosis was 50%. removing of cvc improved overall survival in children with invasive candida infections after hsct. disclosure: nothing to declare background: graft versus host disease (gvhd) and virusassociated enteropathy in allogeneic hematopoietic stem cell transplantation (allo-hsct) may cause severe quantitative and qualitative composition changes of intestinal microbiota, leading to the development of small intestinal bacterial overgrowth (sibo) on the background of immunodeficiency, which can have a negative impact on treatment effectiveness. the gold standard for diagnosis and the main criterion for sibo is the detection in the jejunum aspirate >10 5 /ml bacteria and/or the appearance of colonlike microbiota in small intestine. it is also acceptable to use an alternative non-invasive technique -hydrogen breath test, which could be especially important in patients with severe mucositis, grade iii-iv and thrombocytopenia grade iv. sibo diagnosis in the setting of the gastrointestinal tract damage and dysfunction in patients treated with allo-hsct is insufficiently studied. methods: the study included 7 patients with acute myeloid leukemia (n=3), acute lymphoblastic leukemia (n=1), myelodysplastic syndrome (n=1), non-hodgkin´s lymphoma (n=1), hurler syndrome (n=1), who underwent allo-hsct from an unrelated (n=4) and haploidentical donor (n=3) , and which were complicated by enteropathy development. in 2 cases, the enteropathy reason was a combination of intestinal gvhd and viral colitis (hhv-6), in 5 cases -viral colitis (hhv-6). all patients had esophagogastroduodenoscopy with species aspiration from descending part of the duodenum and feces collection, with further bacteria pcr identification. hydrogen breath test was performed also in which patients were treated with oral lactose 2 g/kg with subsequent hydrogen assessment after 30 and 60 minutes. the study was performed in the period from 20 to 741 days after allo-hsct. results: according to feces analysis data, colon microbiota composition significantly differed from the reference values. at the same time total bacterial mass of the duodenum was less in comparison with colon microbiota: 2e+08 (2e+7/8e+9) and 9e+10 (9e+7/3e+12), respectively (p< 0.004). quantitative composition of the duodenal microbiota was comparable to that of colon: lactobacillus spp. 4e+5 (1e+5/2e+7) > 1.5 e+5 (1e+5/4e+11), (p=0.48); bifidobacterium spp. 6e+6 (1e+05/3e+7) < 7.5 e+6 (2e+5/1e+9), (p=0.423); escherichia coli 7e+5 (1e+5/2e+6) > 5e+5 (4e+5/9e+5), (p=0.2); bacteroides fragilis group 6e+6 (0e+0/8e+9) > 5e+6 (2e+5/3e +12), (p=1.0); faecalibacterium prausnitzii 1e+6 (0e+0/ 1e+7) < 1.5 e+6 (1e+5/3e+10), (p=0.54), indicating the presence of sibo. in this case, the hydrogen breath test was completely uninformative: basal values -0.035 (0.01/0.06) ppm, hydrogen concentration in 30 minutes -0.04 (0.01/ 0.06) ppm, in 60 minutes -0.02 (0.01/0.07) ppm, which is less than in healthy volunteers. conclusions: quantitative composition of the duodenal and colon microbiota is similar in the case of intestinal gvhd and/or virus-associated enteropathy in allo-hsct patients, which may be of diagnostic value for sibo confirmation. the hydrogen breath test is an uninformative method for sibo identification in patients after allo-hsct. disclosure: nothing to declare johannes schulte 1 , patrick hundsdörfer 1 , sebastian voigt 1 background: adenovirus (adv) infections or reactivations frequently occur in the pediatric hematopoietic stem cell transplant (sct) setting and these infections contribute to increased morbidity and mortality. the nucleotide analog cidofovir might be effective in reducing adv load, however, nephrotoxicity is a considerable side effect. the new antiviral compound brincidofovir (bcv, cmx-001), a lipid-conjugate nucleotide analog with broad-spectrum antiviral activity in vitro, has been reported to be effective in cases where cidofovir treatment was unsuccessful. methods: data of eight pediatric patients undergoing sct for malignant and nonmalignant indications were analyzed. all patients were weekly monitored for adv viremia by pcr. in case two consecutive positive adv pcr results indicating a viral copy number > 1000/ml were documented, patients received a weekly dose of cidofovir. if no reduction of adv load was seen within two weeks after the commencement of treatment or side effects demanded cidofovir discontinuation, bcv was obtained through an emergency expanded access programme. results: eight pediatric patients developed adv viremia with maximum viral loads ranging between 9350 and 4430000 copies/ml. six patients had c type adv and two patients had non c type adv infections. five patients had viral co-infections: two had an additional cmv infection, one had an epstein-barr virus (ebv) and herpes simplex virus co-infection, one patient had an ebv co-infection and one patient had a bk virus co-infection. all eight patients initially received cidofovir, however, a substantial decrease in adv load could not be observed in any patient after a two-week administration course. except in one patient who had extensive intestinal graft-versus host disease (gvhd), adv infection was cleared in all patients within three weeks after the beginning of bcv treatment. in addition, all coinfections were cleared. no nephrotoxicity or other side effects were observed. conclusions: bcv was effective in all but one patient. oral bcv might not be effective in advanced upper gut gvhd, especially when applied via a gastric tube, yet this was observed in only one patient. eventually, without nephrotoxic side effects, bcv could be an useful alternative to cidofovir. disclosure: nothing to declare. background: the use of post-transplant high-dose cyclophosphamide (ptcy) has overcome the need for extensive depletion of t lymphocytes from haploidentical donor grafts, which traditionally resulted in severe and prolonged immunosuppression. however, reconstitution of cellular immunity may be delayed even after t cell replete haploidentical stem cell transplantation (haplo-sct) with ptcy. the study of the incidence and severity of viral reactivation is therefore relevant to the outcomes of haplo-sct with ptcy. methods: our study enrolled 42 patients (women/men, 19/23), who underwent t cell replete haplo-sct from 12/ 2013 to 10/2018 and achieved hematopoietic engraftment. median age at transplant was 53.5 years (range, 19-70) . the underlying disease was aml (n=17), all (n=9), mds (n=9), myelofibrosis (n=4), cml (n=2), or cll (n=1). the conditioning regimen was myeloablative (n=31), reduced-intensity (n=10) or non-myeloablative (n=1). peripheral blood was the graft source in the majority of cases (n=29) and bone marrow in the remaining (n=13). recipient/donor cytomegalovirus (cmv) serostatus was -/-(n=2), -/+ (n= 4), +/-(n=8), or +/+ (n=28). the combination of tacrolimus and mycophenolate mofetil was administered in addition to ptcy for prevention of graftversus-host disease. cmv, epstein-barr virus (ebv), and human herpesvirus-6 (hhv-6) reactivation was monitored by real-time quantitative pcr (rq-pcr) in blood twice weekly post haplo-sct. bk virus (bkv) reactivation was assessed by rq-pcr in urine and/or blood specimens in cases with symptoms suggestive of bkv-associated hemorrhagic cystitis (hc). results: with a median follow-up time of 25 months (range, , the cumulative incidences (cin) of relapse and non-relapse mortality (nrm) were 15.5% (95% ci, 6.2-28.6%) and 33.3% (95% ci, 18.9-48.2%) at 2 years, respectively. median disease-free (dfs) and overall survival (os) were 53.3% (95% ci, 39.5-71.9%) and 58.4% (95% ci, 44.6-76.5%) at 2 years, respectively. the cin of cmv reactivation/infection (>100 copies/ml) reached 75.7% (95% ci, 58.8-86.4%) at 3 months. cmv infection developed in 30 out of 40 patients who were at risk, whereas recurrent cmv reactivation was observed in 15 patients with a median number of 2 episodes (range, 2-6) per patient. the median total duration of antiviral therapy for cmv infection was 27 days (range, 14-199) . cmv disease (pneumonia) was documented in 2 patients. the cin of ebv reactivation (>1,000 copies/ml) was 50.9% (95% ci, 34.2-65.3%) at 12 months. no case of ebv-related post-transplant lymphoproliferative disorder was observed, however preemptive therapy with rituximab was required in 2 patients with rapidly increasing ebv viral load. hhv-6 reactivation (>1,000 copies/ml) was observed in 5 patients (cin, 12.3% at 6 months; 95% ci, 4.4-24.5%), with none of them requiring specific therapy. bkv-related hc occurred at a cin of 23.9% (95% ci, 12.3-37.7%) at 6 months. cystoscopy for bladder hemostasis was required in 4/11 and nephrostomy in 1/11 patients with hc. conclusions: despite preservation of non-alloreactive memory t cells, haplo-sct with ptcy is associated with substantial rates of viral reactivation (especially cmv and bkv) resulting in the need for prolonged antiviral therapy and considerable morbidity as well. therefore, strategies to prevent viral reactivation and disease are still warranted in haploidentical stem cell transplantation. disclosure: nothing to declare. background: adenovirus(adv) infections are a wellrecognised cause of morbidity and mortality in children and adults receiving an allogenic stem cell transplant(hsct).the reported incidence of adv infection is higher(6%-42%) in paediatric hsct than in adults(3-27%),but we currently lack accurate data of adv infection burden among adults.cidofovir has been extensively used as a pre-emptive anti-adenoviral therapy and is current standard of care.we present our single centre experience of adv incidence and outcomes with pre-emptive approach in adult patients receiving t-cell depleted(tcd) hscts for myeloid disorders. methods: this is a single-centre retrospective analysis of 332 consecutive hsct patients for myeloid disorders including aml, mds, mpn & aplastic anaemia between january 2012-june 2016 using atg or alemtuzumab based tcd.adv screening was performed in all patients with standardised real time quantitative pcr on weekly basis during standard risk period. figure 1a -1b] results: baseline characteristics (table1) of patients were similar across both cohorts with or without adv infection. overall 12.6%(n-42/332) patients were positive for adv dna on atleast one of the sanctuary sites(upper respiratory airway,blood,faeces,urine) and 45%(n-19/42) of these experienced disseminated infection(defined by adv in ≥2 sanctuary sites or rising adv dna copies in blood), while 2 developed typical adenoviral disease (pulmonary). among patients with disseminated infection,majority had adv in gastro-intestinal(42%),10.5% in genitourinary and 21% as both sanctuary site of infection,in addition to blood viraemia(85% of all cases).cumulative incidence of adv infection was 10.6%(95%ci:7.6-14.2%) at 12 months with median time of 108 days(iqr:19-304 days) to detect adv-dna post hsct.overall survival(os) at 5 years for whole cohort was 41%(95%ci:31-50;median os-57months) with no statistical difference between patients with disseminated adv infection vs those with none(log rank; p-0.68; fig-1a ). overall cumulative incidence of non-relapse mortality (nrm) was 23%(95%ci:18-28%) and relapse(cir) was 25.5%(95%ci:19-32%) at 5 years,but no statistical difference noted between patients with disseminated adv infection & those with none(nrm:p-0.31;cir: p-0.50;gray test).pre-emptive therapy with cidofovir (3mg/kg weekly iv infusion for 2 weeks and fortnightly thereafter until infection free) was required in 33%(14/42) of symptomatic adv infection patients and 52%(10/19) with disseminated infections.one patient required brincidofovir therapy for refractory disease,but one patient died due to severe sepsis, before adv specific therapy could be given.remaining patients were monitored and all self-recovered on cessation of immunosuppression.all patients treated with cidofovir developed renal impairment(defined by atleast >25% increase in baseline creatinine),however majority(73%) recovered their renal function near their baseline (fig-1b) . conclusions: adv infection remains a significant cause of morbidity in adult hsct patients, however pre-emptive management with cidofovir has improved os and nrm despite use of tcd conditioning.renal toxicity remains common with cidofovir but with use of intermediate doses, majority do recover their renal functions. clinical trial registry: n/a disclosure: nothing to declare background: publications on invasive fungal disease (ifd) in lymphoma patients are limited especially after allo-hsct. there are no data on outcome of allo-hsct in lymphoma patients with prior ifd. this study focuses on epidemiology of ifd before and after allo-hsct in children and adults with hodgkin's lymphoma (hl). methods: single center prospective study included 86 patients with classical r/r hl who received allo-hsct from 2002 to 2018. the median age was 27 (13-49) y.o., children (< 18 yo) -13%. allo-hsct from mud was performed in 45,4% (n=39), mrd -24,4% (n=21), mmud -15,1% (n=13), haplo -15,1% (n=13), with ric (100%) and predominantly ptcy-based gvhd prophylaxis (71%). primary antifungal prophylaxis was fluconazole in 85%, secondary -voriconazole (100%). eortc/msg 2008 criteria for diagnosis and bronchoscopy before allo-hsct in pts with ct-scan lung lesions were used. "active ifd" means ifd diagnosed just before hsct. median follow-up time was 12 months . results: incidence of ifd before allo-hsct was 12,8% (n=11). ifd prior to hsct were invasive aspergillosis (ia) with lungs involvement. antifungal therapy before allo-hsct was used in 81,8% pts with median duration -2 months. complete response to antifungal therapy was in 45,4% pts, partial response or stabilization -36,4%, and 18,2% pts had an "active ifd". after allo-hsct all pts received voriconazole as an antifungal therapy or secondary prophylaxis. cumulative incidence of relapse or progression of ia after allo-hsct was 18,2% with the median 49 day after hsct, which were successfully treated with voriconazole in post hsct period. incidence of ifd after allo-hsct for naïve patients was 17,6% (n=13/74). etiology of ifd after allo-hsct was ia -69%, invasive candidiasis (ic) -15%, mucormycosis -8% and 8% combined ifd caused by aspergillus fumigatus + rhizopus stolonifer. the median day of onset of ifd after allo-hsct was day+ 114 and was associated with post-hsct relapse of hl (p=0,04). the main site of infection were lungs (88%), the main clinical symptom -febrile fever (100%). antifungal therapy was used in all patients: voriconazole -59%, micafungin -17%, posaconazole -8%, lipid amphotericin b -8% and combination lipid amphotericin b with caspofungin -8%. overall survival (os) at 12 weeks from the diagnosis of ifd after allo-hsct was 80%. the 2-year os in children and adult with hl after allo-hsct was 73,3%. development of ifd after allo-hsct do not decrease the 2-year os rate (69,2% vs 74%, p=0,77). the impact of prior ifd on 2-year os in allo-hsct recipients was not statistically significant in all group (63,6% vs 74,7%, p=0,47) , and separately in children and adults. conclusions: incidence of ifd in children and adults with hodgkin's lymphoma before allo-hsct was 12,8%. incidence of ifd after allo-hsct in patients with hodgkin's lymphoma was 17,6%. the major etiology agents as before as after allo-hsct were aspergillus spp. ifd was a late complication after allo-hsct and associated with post-hsct relapse. despite the high incidence ifd before or after allo-hsct didn't influence the outcome in children and adults with hodgkin lymphoma. disclosure: nothing to declare our community has high cmv positive serostatus, which is a known risk for cmv infection or reactivation. we conducted a study to explore the incidence and outcome of cmv infection among post-hsct children. methods: medical records of pediatric patients (age ≤ 14 years) undergoing single allogeneic hsct from january 2014 to december 2016, at king faisal specialist hospital and research centre, riyadh, saudi arabia, were reviewed. all patients with active cmv infection or disease before and during transplant were excluded. a total of 307 patients were included in the study; 160 were female. median age at hsct was 5 years. recipient cmv serostatus was positive in 213 patients before hsct, and 232 donors were cmvpositive. the recipient-donor (r/d) serology was 63.7% r +/d+, 15.3% r+/d-, 13.7% r-/d+, and 7.3% r-/d-. indication for hsct was immune disorders 24.1%, hemoglobinopathies 20.8%, bone marrow failure 20.5%, malignant disorders 20.5%, histiocytic 8.1%, and metabolic disorders 5.9%. source of stem cells was bone marrow in 223, cord blood in 74 and peripheral blood stem cell in 10 cases. donor was matched related among 198, unrelated matched/mismatched in 74, haploidentical 24, and related with 1-antigen mismatch in 11. total body irradiation (tbi) based conditioning was used for 47 patients, while atg was used in 169 patients. results: out of a total of 307 patients, 101 patients developed cmv infection post-hsct (32.9%). incidence in female recipients was high (38.1% versus 27.2%, p-value 0.042). both recipient and donor cmv serology positive (42.5%) developed cmv infection (p-value < 0.001). however, no cmv infection in both recipient and donor negative group (r-/d-). the incidence of cmv infection post-hsct was high in patients received tbi based conditioning (22 out of 47, 46 .8%, p-value 0.027), and in haploidentical transplant with 66.7% (p-value 0.008). source of stem cells, myeloablative versus nonmyeloablative conditioning, atg use in conditioning and agvhd, did not exhibit significant association with cmv infection. in multivariable setting, when adjusted for primary indication for transplant, donor hla type, tbi based conditioning and recipient and donor cmv serology at transplant, haploidentical donor (odds ratio: 3.491, p-value: 0.041) and donor-recipient cmv sero-positivity (odds ratio: 2.228, p-value: 0.007) were found to be significant risk factors. cmv infection resolution rate was 83.2% (84). with a median follow-up time of 32.9 ±1.5 months from infusion, five-year overall survival of cmv infected group was lower (0.658±0.087) as compared to non-cmv infected (0.706±0.032, p-value: 0.285). conclusions: incidence of cmv infection post-hsct in our center is comparable to other centers. our data suggest that donor-recipient cmv positive serostatus, haploidentical donor, and use of tbi based conditioning necessitate close attention and surveillance. background: toxoplasmosis is a rare and underestimated complication following allogeneic stem cell transplantation (allo-sct) with an often fatal course. this is in part due to limited diagnostics relying mainly on imaging and detection of parasite dna by pcr. we present here eleven cases of toxoplasma disease following allo-sct. methods: we retrospectively analyzed 534 consecutive adult patients who received an allo-sct in our bone marrow transplant unit between july 2008 and july 2018. eleven (2%) of these patients were diagnosed of toxoplasma disease. the main characteristics of the patients are shown in table 1. all patients, except two cord blood, have received peripheral blood stem cells. fludarabine-based conditioning regimes were used in all patients. only the two cord blood patients received thymoglobulin in the conditioning. graft-versus-host disease (gvhd) prophylaxis consisted on tacrolimus plus mycophenolate mofetil in 5 (46%) patients and post-transplant cyclophosphamide followed by tacrolimus in 4 (36%). before the allo-sct was performed the igg/igm toxoplasma serology of the recipient and donor. we reviewed the absolute lymphocyte count (alc) and cd4 + lymphocyte count within four weeks prior to the diagnosis of toxoplasmosis, and if the patients took effective primary prophylaxis for this parasite. toxoplasma disease was defined as the presence of toxoplasma infection plus clinical, radiological or pathological evidence. toxoplasma disease was considered the main cause of death when no other major life-threatening infection or other potential fatal complication occurred immediately before death. results: median (range) age (years) of the eleven patients diagnosed with toxoplasma disease was 54 (19-69). for pancytopenia, no patient received trimethoprimsufmethoxazole (tmp-smz) but pentamidine for pneumocystis jirovecii-pneumonia (pcp) prophylaxis in 10 cases and atovaquone in one. toxoplasma serology pretransplant was positive (igg+/igm-) in ten of the eleven patients. all donors were seronegative (igg-/igm-) except two. toxoplasmosis was diagnosed a median (range) of 85 days (35-330) post allo-stc. the clinical presentations were as cerebral-encephalitis (n=4), chorioretinitis (n=3), pneumonitis (n=2) and disseminated toxoplasmosis (n=2). one case, patient and donor seronegative pre-transplant, was presented as a primary infection in form of chorioretinitis. all three patients with chorioretinitis were diagnosed after day +100 of allo-sct. at the time of toxoplasma disease, 8 of 11 (73%) of patients had an alc < 500 cells/μl and all of them with immunosuppressive therapy and corticosteroids for acute or chronic gvhd. we had cd4 + lymphocyte count only in four patients and in three of them was < 200 cells/μl. eight of the eleven (73%) patients died, with a median (range) of 21 days (9-65) since diagnosis of toxoplasmosis, and in 6 of them the toxoplasma disease was the main cause of death. conclusions: in our series, the incidence of toxoplasma disease after allo-sct is low and is related to high mortality, in accordance with what has been reported by other groups. positive pre-transplant serology and gvhd and its treatment were factors strongly related with toxoplasmosis. we encourage the use of tmp-smz instead of pentamidine for pcp-pneumonia prophylaxis in patients seropositive for toxoplasma gondii pre-transplant. clinical trial registry: data about its epidemiology in children are scarce. we retrospectively analyzed the incidence, the severity and the risk factors that contribute to the manifestation of this complication in a pediatric population. methods: during a 10-year period (january 2008 -june 2018) we performed in our center 376 allogeneic transplantations, 237 for malignant hematological diseases and 139 for non-malignant. the majority of our patients received myeloablative conditioning regimens. diagnostic criteria of hemorrhagic cystitis were the detection of the virus with pcr in urine samples and/or in blood samples, in combination with hematuria and lower urinary tract symptoms (dysuria, urinary frequency, urgency, suprapubic pain) that couldn't be attributed to any other reason. we defined the hemorrhagic cystitis as severe when one of the following factors was present: formation of clots and continuous bladder irrigation, obstructive uropathy with creatinine elevation or need for urological intervention. results: a total of 376 patients with median age 7,2 years (0,5-20) were studied. 37 children (10%, 95% ci, 7,2-13,3) manifested bk virus associated hemorrhagic cystitis with median age 12,9 years (5, [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 5) . onset of cystitis occurred at a median time of 27 days (day +-day +52) after transplantation. in 17 children cystitis was severe. the median duration of symptoms was 27 days (8-200). the median time of hospitalization for children with severe cystitis was 70 days (29-242) whereas for those who didn't manifest cystitis was 45 (25-184) . in 25 of the 37 patients we examined the presence of the virus not only in urine but also in blood samples. in 11 of them the test was positive and almost half of them (6) manifested severe cystitis. the risk factors that were examined were age, administration of antithymocyte globulin, type of disease, graft source, type of donor and the presence of acute graft versus host disease (agvhd). in multivariable analysis, independent risk factors for the manifestation of hemorrhagic cystitis were age > 6 years old (hr: 8,46, 95% ci, 2,86-25, p< 0,001), transplantation for malignant disease (hr: 2,26, 95% ci, 0,97-5,27, p=0,05) and the presence of agvhd (hr: 4,04, 95% ci, 2,01-8,1, p< 0,001). the overall survival of children with hemorrhagic cystitis was 46,3% vs 69,3% of those who didn't manifest this complication, but in multivariable analysis for survival cystitis wasn't a statistically significant risk factor. conclusions: according to our results, stem cell transplantation in children > 6 years old who suffer from a malignant disease and the presence of agvhd consist independent risk factors for the manifestation of bk virus associated hemorrhagic cystitis. the identification of the risk factors of this serious complication will contribute to better management of transplanted patients. further research through prospective trials can contribute to the better understanding of the pathophysiology of hemorrhagic cystitis and to the establishment of appropriate diagnostic and therapeutic guidelines. disclosure: nothing to declare p442 impact of natural killer cell reconstitution on outcomes in patients with early cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation background: cytomegalovirus (cmv) reactivation influences survival after allogeneic hematopoietic stem cell transplantation (sct) and induces natural killer (nk) cell expansion. we evaluated nk cell reconstitution and clinical outcomes following early cmv reactivation after sct. methods: lymphocyte subsets were measured by flow cytometry on day 100 in patients with hematologic malignancies undergoing sct between january 2009 and december 2017 at kanagawa cancer center, excluding patients with graft failure or death within 100 days. cmv reactivation was defined as initiation of preemptive cmv therapy following pp65 antigenemia surveillance. results: the subjects were 152 males and 94 females with a median age of 51 years (range: 18-69 years). the median follow-up period for survivors was 3.2 years (range: 0.8-9.6 years). there were 128 patients with acute myeloid leukemia, 64 with acute lymphoblastic leukemia, 32 with myelodysplastic syndromes, and 22 with other diseases. at transplantation, 166 patients were standard risk and 80 were high risk. myeloablative conditioning and reduced-intensity conditioning were employed in 99 and 147 patients, respectively. bone marrow transplantation, peripheral blood stem cell transplantation, and cord blood transplantation was performed in 98, 36, and 112 patients, respectively. cmv reactivation occurred in 141 patients (57%) at a median of 45 days (range: 15-93 days) after sct. grade ii-iv acute gvhd and chronic gvhd affected 89 patients (36%) and 80 patients (33%), respectively. among all patients, 5-year overall survival (os), cumulative nonrelapse mortality (nrm), and cumulative relapse (cir) rates were 66%, 10%, and 33%, respectively. in patients without cmv reactivation (cmvr-) versus patients with cmv reactivation (cmvr+), 5-year os, nrm, and cir were 79% vs. 55% (p < 0.001), 3% vs. 16% (p = 0.005), and 27% vs. 38% (p = 0.142), respectively. among all patients, the median level of cd3-cd56+ cells, cd16 +cd57cells, and cd16+cd57+ cells on day 100 was 236/μl (range: 19-2150/μl), 275/μl (9-2660/μl), and 98/μl (3-1767/μl), respectively. nk cell subsets showed no significant differences between cmvr-and cmvr+ patients. when patients were divided into low and high groups at the median level of each nk cell subset, cmvr+ patients with high cd3-cd56+, cd16+cd57-, or cd16+cd57+ cells showed significantly better 5-year os than those with low cells (72% vs. 35%, p < 0.001; 69% vs. 40%, p < 0.001; 66% vs. 45%, p = 0.001, respectively). high cd3-cd56+ cells were significantly associated with lower nrm (7% vs. 26%, p = 0.002), while high cd16+cd57-cells were significantly associated with lower cir (26% vs. 50%, p = 0.007). multivariate analysis confirmed these nk cell subsets as prognostic factors in cmvr+ patients. conclusions: nk cell reconstitution may contribute to improved transplantation outcomes in subgroups of cmvr + patients. disclosure: nothing to declare background: rezafungin (rzf) is a novel echinocandin in development for prevention of invasive fungal infections caused by candida, aspergillus, and pneumocystis spp. in patients at high risk of infection. rzf has demonstrated in vivo prophylaxis efficacy and low risk of drug-drug interactions. furthermore, the stability and pk profile of rzf allow for once-weekly dosing. rzf is also in development for treatment of candidemia and invasive candidiasis using a dosing regimen of rzf 400 mg followed by 200 mg once-weekly, which achieved >90% target attainment against candida. while lower doses might be useful to prevent candida and pneumocystis, invasive aspergillosis is a different challenge. we evaluated rzf dosing for prophylaxis against aspergillus fumigatus in blood and marrow transplant (bmt) patients using pk/pd simulations of the treatment dosing regimen. methods: a previous population pk model was refined using data from phase 1 and phase 2 trials of iv rzf (nonmem vers 7.2). stepwise forward selection (α = 0.01) and backward elimination (α = 0.001) were used to assess for relationships between interindividual pk variability and covariates, such as age, sex, bsa, albumin, liver and renal function markers, and infection status. the final model was validated by comparing model-based predictions to observed data. the model and demographic data from 100 bmt recipients at stanford medical center were used for monte carlo simulation (n=2,000) of expected rzf concentrationtime profiles in bmt patients receiving iv rzf 400 mg on week 1 followed by 200 mg weekly x 11. of the 100 patients included in the demographic dataset, 39 were female (mean values at baseline: age, 58 years [19-75 years]; weight, 69.8 kg [42-123 kg] ). the median (range) bsa in the demographic dataset was 1.84 m 2 (1.27-2.61), and albumin was 3.35 g/dl (1.8-4.43 g/dl) . free-drug concentration-time profiles were evaluated (97.4% human protein-binding) relative to the a. fumigatus minimal effective concentration required to inhibit 100% of isolates tested (mec 100 ; jmi 2014-2017 sentry international surveillance data). results: the population pk model was a linear, 4compartment model with zero order iv input. albumin, sex, infection status, and body surface area were statistically significant predictors of interindividual variability; clinical significance of these factors was not determined. the model provided precise, unbiased fits to the observed data (r 2 =0.993 observed vs individual-predicted concentrations). rzf plasma free-drug concentrations at weeks 1, 2, and 12 were above the a. fumigatus mec 100 (0.03 mg/l) for the entire dosing interval in 98.0%, 92.0%, and 90.2% of simulated patients, respectively, and in ≥99.8% for all 12 weeks based on the mec 90 (0.015 mg/l). conclusions: these data modelled from bmt patients support the rzf dosing regimen of 400 mg iv followed by 200 mg once-weekly for prophylaxis against a. fumigatus. current antifungal prophylaxis may be limited by toxicity, ddis, or patient factors such as mucositis. the pk of rzf and its spectrum, safety, tolerability, and lack of ddis may address current unmet needs in ifi prophylaxis for bmt and other immunocompromised patients. disclosure: janice brown: research funding, cidara therapeutics, elizabeth lakota: research funding, cidara therapeutics, shawn flanagan: employment, cidara therapeutics, taylor sandison: employment, cidara therapeutics, voon ong: employment, cidara therapeutics, christopher rubino: research funding, cidara therapeutics p444 is fungal prophylaxis necessary in non myeloablative peripheral blood stem cell allogeneic transplantation in the pre-engrafment period? julien vaidie 1 , jean-baptiste woillard 1 , stéphane girault 1 , marie-laure dardé 1 , arnaud jaccard 1 , daniel ajzenberg 1 , bernard bouteille 1 , pascal turlure 1 background: non myeloablative peripheral blood stem cell transplantation (pbsc), by limiting toxicity, can be proposed to elderly patients or patients with comorbidities. however, fungal infections remain a key issue that can negatively impact outcome, and increase duration and cost of hospitalization. systematic fungal prophylaxis have demonstrated benefits in outcome in the context of myeloablative conditioning but are not currently in reduced intensity conditioning allograft with pbsc. fluconazole prophylaxis is currently recommended in this situation (ecil). methods: primary objective of this retrospective study was to evaluate fungal infection incidence after allograft procedure in patients who received a non myeloablative allograft with pbsc in limoges university hospital between june 2009 and june 2018. patients received fludarabine 30 mg/m2/day between d-6 and d-2 before allograft and busulfan 3.2 mg/kg/day at d-4 and d-3. gvh prophylaxis consisted in rabbit anti-lymphocyte serum at the dose of 2.5 mg/kg at d-2 and d-1, and ciclosporin at the beginning dose of 3mg/kg per os twice a day. mycophenolate mofetil was adding for patients with hla-matched or mismatched unrelated donors. patients did not systematically receive antifungal prophylaxis during the neutropenic pre-engraftment period. when patients had fever during more than 72 hours, an empirical fungal treatment (caspofungine) was added to empirical antibiotics. as soon as neutropenic recovered and in the case of apyrexia without microbiologic documentation, antimicrobial treatments were stopped while in the case of microbiologic documentation, treatments were adjusted to germ in term of dosing and time of administration following recommendations. however, some patients received antifungal azole prophylaxis during the neutropenic pre-engraftment period in case of history of previous invasive aspergillosis (ia), or a nasal colonization by aspergillus. in post-engraftment period, posaconasole prophylaxis was administered for patients with systemic corticotherapy for acute graft-versus-host disease. results: 91 patients were evaluated (median [min-max] age of 60 [33-71] years). 20% of patients received an hlaidentical related donor, 69% an hla-matched related donor and 11% an hla-mismatched unrelated donor. the five years overall survival and survival without relapse or gvhd were 70% ic95 [59%-81%] and 62% respectively ic 95 [52%-73%]. the median time for neutrophil recovery was 15 days. 79 patients did not receive prophylaxis and only 12 patients received systematic fungal azole prophylaxis in the pre-engraftment period. two patients received an empirical treatment by caspofungine. only 1 ifi was documented during the neutropenic period : candida krusei in blood culture. in the post engraftment period, 37 patients with acute gvhd treated by corticotherapy received an antifungal prophylaxis by posaconazole and only 1 patient had a probable ia at day 100 despite prophylaxis by posaconasole. conclusions: except for patients with previous history of ifi, our results provide additional arguments against systematic fungal prophylaxis after reduced intensity conditioning with pbsc allogenic transplantation in the pre-engraftment period with a very low incidence of invasive fungal infections. in post-engrafment period, posaconazole prophylaxis is required for patient with gvhd treated by corticotherapy. disclosure methods: a simple, rapid and sensitive method using hplc with a diode-array detector (dad) was developed and validated for the quantification of letermovir in human serum using sorafenib as internal standard. after pretreating serum samples by liquid-liquid extraction with tert-butyl methyl ether, separation was achieved on a x-terra rp-18 column (dimension 150 x 2.1mm, 5μm) at 30 0 c using gradient elution with a mobile phase of 20 mm ammonium bicarbonate ph 7.8 (mobile phase solvent-a) and acetonitrile:20mm ammonium bicarbonate ph 9.2 (mobile phase solvent-b). samples were eluted at a flow rate of 0.3ml / min throughout the 20-minute run. uv wavelength mode was used, detection was at 260 nm. results: the calibration curve was linear (r > 0.99) in a concentration range of 25 -5000 ng / ml for letermovir. the hplc assay established for letermovir determination showed a high rate of accuracy and precision with an intraday variability of -9.21 to 12 % (accuracy) and 0.31 to 3.75 % (precision) and an interday variability of -2.6 to 6.9 % (accuracy) and 3.74 to 7.41 % (precision), respectively. 16 letermovir serum concentrations of 9 patients (8 male / 1 female, mean age 66.3 years) were determined in daily clinical practice. the mean concentration was 4752 ng / ml (median 4163 ng / ml, standard deviation 3925 ng / ml, range 89 -11452 ng / ml). conclusions: the newly developed hplc method is useful for the determination of letermovir concentrations. patient samples analyzed in a routine clinical setting demonstrated considerable interindividual variability. all measured concentrations were above the ec50 of letermovir. monitoring the concentration of letermovir could help to prevent over-or underexposure, especially in patients with polypharmacy which is frequent in allogeneic hematopoietic stem cell transplant recipients. disclosure background: the use of preemptive strategy (pet) has lowered the incidence of cmv disease in allo-sct to 5-10%. nonetheless the use of this strategy implies that more than 50% of seropositive patients will replicate cmv. several studies have shown that cmv replication is detrimental for patient survival although the viral load related to this bad outcome variates among studies. objective: to analyse thel impact of cmv replication in overall survival (os) in allo-hct patients. methods: to analyse the impact of cmv replication in os we perform a unicentric, retrospective study on 117 consecutive first allo-hsct patients transplanted between jan-2015 and oct-2018 with a median follow-up of 398 days (5-1266). all patients were monitored post-hct with real time pcr cobas-taqman® /cobas6800® (rtpcr) in plasma. the cut-off for inception of pet was 150 iu/ml. cmv mutations (ul54/ul97gene), were studied in plasma samples by sanger sequencing, median cmv viral load 3130 iu/ml (565-23900). results: patients (117): women/men (49%/51%), median age was 55 years (range 18-71). 101 identical allogeneic scts (86%), 16 haploidentical scts (14%). donors were related in 60 cases (51%) and 57 (49%) unrelated. progenitors source was 98% peripheral blood and 2% bone marrow. cmv status was (d+/r+) in 51%(n=60), (d+/r-) in 9%(n=11), (d-/r+) in 26% (n=31) and (d-/r-) in 11% (n=13), unknown 2 cases. positive pcrs were detected in 63 patients: one episode in 41 (65%); 2 episodes in 15(24%) and 3 to 5 episodes in 7 patients (11%). fifty-five patients (87%) received preemptive therapy. fourteen episodes (25%) were refractory/ probable refractory cmv infections (according to the criteria of chemaly r. cid 2018). a resistant mutation (ul54 gene) was detected in one patient with refractory infection patients that developed cmv infection had an inferior non-significant os at 3 years (61,3% vs 76,8% log-rank p 0,346). those patients that received pet for cmv had a significant inferior os compared with those that replicate cmv but didn't receive preemptive therapy (55,6% vs 100%, log rank p=0.04). os of patients that received pet was inferior compared with those without pet (with or without infection) (55,6% vs 79,9%, logrank p 0,05). no difference in survival was found for those patients treated pre-emptively that were refractory vs no refractory (53% vs 57,1%, log-rank p 0,51). conclusions: patients that received preemptive therapy had a significant inferior overall survival compared with those that didn´t replicate and those that replicate cmv but didn't receive preemptive therapy. this reinforce the relevance of prophylactic strategies for cmv with drugs with good safety profile like letermovir that in a randomised trial proved to decrease the need for preemptive therapy. disclosure: rafael, de la camara: has received grants from astellas, gilead, janssen, merck, novartis and pfizer clinical evaluation of stenotrophomonas maltophilia infection in allogeneic hematopoietic stem cell transplant recipients -retrospective single-center data analysis negative bacillus that causes severe infections associated with high morbidity and mortality in immunocompromised patients. the aim of our study was to determine incidence, characteristics and outcome of s. maltophilia infection in patients (pts) who underwent allogeneic hematopoietic stem cell transplantations (allo-hsct) in institute of hematology and transfusion medicine between october 2010 and november 2018. methods: we retrospectively evaluated incidence, clinical features and outcome of s. maltophilia infections in 338 consecutive patients with median age-42 years (range 19-71), who underwent allo-hsct from unrelated donors -238 (70.4%), matched sibling donors -87 (25.7%) and haploidentical donors -13 (3.9%) in our center. s. maltophilia was detected by culture-based microbiological tests. invasive infection was defined by isolation s. maltophilia from cultures in the presence of both clinical symptoms and signs of infection -blood stream infection (bsi), pneumonia with or without pulmonary haemorrhage. the only colonization status was defined as s. maltophilia culture-positive samples in the absence of infection symptoms. in vitro susceptibility tests to antibiotics were performed. results: 21 pts (6.2%) with median age-46 years (range 19-66) with s.maltophilia culture positive samples were identified. 19 (90.5%) underwent allo-hsct from unrelated donors, 1-from matched sibling donor and 1-from haploidentical donor. among them bsi developed in 7 pts (33.3%), pneumonia in 4 pts (19%) -with fulminant and fatal pulmonary hemorrhage in 3 pts (14.3%). all patients with pneumonia demonstrated bsi. positive sputum cultures were detected in 6 pts, in 4 pts hemoptysis was observed. the rest of isolated strains were identified as colonization (throat -in 3 pts, stool -in 9 pts). all 7 patients with invasive s. maltophilia infection before pathogen identification demonstrated persistent fever despite of the use of broadspectrum antibiotics (carbapenems, glycopeptides, aminoglycosides, colistin), prophylactic antifungals and antivirals. all of them received fluoroquinolone (ciprofloxacin) as a standard antibacterial prophylaxis before neutropenic fever occurred. all 3 patients (100%) with bsi, pneumonia and pulmonary hemorrhage died before engraftment (anc -0.0 g/l) -2 of them during 48-72 hours from the onset of a positive blood culture for s. maltophilia. the c-reactive protein (crp) concentration before identification of s. maltophilia invasive infection was > 26x-69x upper normal limits (unl). susceptibility to antibiotics of isolated strains from blood and sputum was respectively: 86% and 100% for ceftazidime, 100% and 100% for trimethoprim-sulfamethoxazole, 64% and 50% for levofloxacin; while 56% and 50% strains were resistant to ciprofloxacin. 1-year overal survival (os) and 2-y os for this group was 52.4 % and 47.6% respectively compared with 77.2% and 68.3% for group without s. maltophilia infection. conclusions: s. maltophilia invasive infections are associated with high morbidity and mortality in allo-hsct recipients especially in the period from conditioning therapy to engraftment. an exposure to broad-spectrum antibiotics in the treatment of neutropenic fever or confirmed bacteremia of other etiology is one of risk factors of breakthrough s. maltophilia infections. empiric therapy against s. maltophilia in selected patients in risk of such infection before pathogen identification may be lifesaving procedure. disclosure: nothing to declare. role of cmv reactivation following allogeneic stem cell transplantation in preventing relapses in patients with acute myeloid leukemia background: cytomegalovirus(cmv) reactivation is common in patients undergoing allogeneic stem cell transplantation. it has been shown recently that cmv reactivation is associated with reduced risks of relapse in patients undergoing allogeneic stem cell transplantation for aml. however the analysis of cibmtr data did not show any effect of cmv reactivation on relapse. with this background we conducted an analysis of patients suffering from aml who are undergoing allo-sct for their long term disease free survival with respect to cmv reactivation. methods: after obtaining permission from hospital medical records committee, we retrospectively analysed data from electronic medical records of patients undergoing allo-sct for aml at our center between january 2013 to august 2018. patients who underwent matched sibling, matched unrelated and partially matched allo sct were included. all patients underwent cmv monitoring with weekly pcr starting from the time of engraftment till d+100 following allo sct. value of ≥ 1000 copies/mcl was considered as cut off for initiation of treatment in matched sibling donor transplant but in unrelated donor or partially matched donor transplants, ≥ 500 copies/mcl was used as cut off for initiation of pre emptive therapy. results: total of 96 patients were included in study. median age was 28.01 ± 17.09years (2-69yrs). 60(62.5%), 30(31.3%) and 6(6.3%) patients underwent matched sibling, haplo (partially matched) and mud transplantation respectively. median follow up was 15 months(1-73months). (table 1) acute gvhd (grade2-4) was observed in 50(52.1%) of patients. cmv reactivation occurred in 70(72.9%) of patients. overall survival at last follow up was 63.5% (61/ 96patients). 17(17.7%) patients relapsed during follow up. relapse free survival at at last follow up was 61.5%. 64(90.1%) of 70 patients who had cmv reactivation didń t relapse, whereas 10(40%) of 25 patients who didn´t have cmv reactivation relapsed which was statistically strongly significant p < 0.001. (figure 1 ) similar results were seen in recently published paper from japanese society for hematopoietic cell transplantation (jshct) transplantation-related complication working group. conclusions: 1. cmv reactivation following allo sct had beneficial effect on preventing relapse in patients with aml. 2. probable immune activation resulting due to cmv reactivation may result in better graft versus leukemia effect preventing subsequent relapses. [ background: human herpesvirus 6 (hhv-6) causes lifethreating central nervous system disorders such as encephalitis after allogeneic hematopoietic stem cell transplantation (hsct). recent studies showed that cd134, a member of the tumor necrosis factor receptor superfamily, has been implicated as a specific receptor of hhv-6b, and that its expression levels in cd4-positive t cells after hsct could be related to the reactivation of hhv-6. real-time quantitative polymerase chain reaction analysis (qpcr) is the most commonly used method for detecting and evaluating hhv-6 reactivation after hsct, but more sensitive detection method is required. we recently developed a new monitoring method for hhv-6 reactivation using digital pcr (dpcr) which provides high sensitivity of detecting hhv-6 dna in clinical samples. in this prospective study, we evaluated the relationship between hhv-6 reactivation monitored by dpcr and expression of cd134 on cd4 + t cells before and after allogeneic hsct. methods: thirty-four patients who underwent allogeneic hsct for hematological diseases at keio university hospital (tokyo, japan) between january 2017 and march 2018 were consecutively enrolled into this study. peripheral blood samples of the patients were obtained before the conditioning (pre), the day of transplant (day 0), and weekly during the first month after transplantation (days 7, 14, 21, and 28) . hhv-6 viral load in plasma was quantitatively measured by dpcr. the primers and a probe of dpcr for hhv-6b were selected from immediate-early 1 (ie-1) protein transactivator region (u90). we evaluated the relationship between hhv-6 reactivation and the serial expression rates of cd134 in cd4 + t cells (cd134/cd4 ratio) measured by flow cytometry before and after hsct. results: median age of the patients was 51.5 years. onethird of patients received cord blood as a stem cell source. hhv-6 reactivation was detected in 23 patients (68%) with dpcr. a comparison of cd134/cd4 ratio between the patients with and without hhv-6 reactivation after hsct revealed that cd134/cd4 ratio was significantly higher in patients with hhv-6 reactivation than those without before conditioning ( in contrast, there was no such significant difference after transplant (days 7 to 28) . in multivariate analysis, higher cd134/cd4 ratio before conditioning (odds ratio (or) = 10.5, 95% confidence interval (ci): 1.3-85.1, p = 0.03) and stem cell source from human leukocyte antigen mismatched donor (including all cord blood transplantation cases) (or = 15.4, 95%ci: 2.0-121.0, p = 0.04) remained to be significantly associated with the incidence of hhv-6 reactivation. conclusions: higher cd134 expression rate in cd4 + t cells before hsct was associated with higher risk of hhv-6 reactivation, which could be a promising marker for predicting hhv-6 reactivation after allogeneic hsct. careful observation and monitoring may be needed in cd134 highly expressed patients. it is a subject of further research to clarify the role of cd134 + cd4 + t cell in hhv-6 reactivation. disclosure: nothing to declare. methods: criteria for the administration of ici (vistide) were grade iii-iv (clinically significant hematuria with clots) bk-related hemorrhagic cystitis after allo-hct which showed no improvement after symptomatic therapy with hyperhydration and bladder irrigation. cidofovir was diluted in 100 ml of normal saline and installed via a foley catheter which was blocked for 1 hour. not knowing the level of absorption of the drug we decided to give probenecid prophylaxis in all patients. ici was repeated weekly according to severity of symptoms. urine and plasma bkv viral loads were quantified by rq-pcr results: six patients (median 38 years, 29-51) received ici after allo-hct. patients had haematological malignancies (aml 2, all 3, mds 1), received busilfex-based myeloablative conditioning and a graft (pbsc 5, bm 1) from a 7/8 hla-matched (4pts), 8/8 (1pt) or haploidentical (1pt) donor. median time for the onset of bkv-hc after allo-hct were 53.5 days (range 32-117). all patients were under standard cyclosporine prophylaxis and none of the patients had any signs of acute gvhd at the time of onset of hc. the median pcr-bkv viral load at the onset of bkv-hc in urine and plasma were 5.75x10 8 (range 0.15x10 8 -59x10 8 ) and 141.5 (range 0-255), respectively. the median maximum pcr-bkv viral load in urine and plasma were 37.5x10 8 (range 4.7x10 8 -450x10 8 ) and 1.100 (range 0-400.000), respectively. five patients had impaired renal function (median egfr 49 ml/min, range 33-53) at first ici which was probably multifactorial. the median dose of intravesical cidofovir was 5 mg/kg (range 2.5-5 mg/kg) and a median number of 3.5 instillations (range 2-7) were given. in 5/6 cases symptoms of cystitis improved dramatically and hematuria resolved. virological response (at least 1 log reduction) was observed in all 5 cases. two patients experienced relapse of hemorrhagic cystitis and were retreated with ici which resulted in resolution of the symptoms and the hematuria. no deterioration of renal function of other systemic adverse effects were observed. after a median follow up of 190.5 days after transplantation (range 105-360), 3/6 patients are alive without cystitis symptomatology and 3 died (1 due to relapse and 2 due to trm). conclusions: in this retrospective study we propose that local therapy of bkv-hc with ici is safe and has high clinical and virological response rates. the administration of ici after allo-hct should be controlled in prospective randomized trials. disclosure: nothing to declare background: since cmv-preemptive therapy approach was implemented, cmv disease frequency is very low. however, cmv reactivation and the need of using nephrotoxic plus/less myelotoxic drugs is very frequent. in addition to the toxicity of the medications to avoid cmv disease, other potential adverse effects of cmv have been mentioned in medical literature. in this study, we wanted to estimate how recipient/donor serologic status influences the outcome of allo-hsct in our most recent series of patients. methods: the population analyzed for this report is the all 224 patients who underwent allo-hsct during the 4-year period from october 2014 september 2018 in our unit. median age at transplant was 52 years (range: 7-69). one hundred and thirty were male (58%) and 94 were female (42%). baseline diseases were: 83 aml, 51 lpd, 29 all, 29 mds, 14 mpd, 12 mm, and 6 bmf. donor was unrelated in 120 transplants (54,5%) and was family in 104 (45,5%) (including 34 haplo-identical). conditioning regimen was ric in 121 procedures (54%) and intensive in 103 (46%). stem cell source was pb in 210 (93,7%) and bm in 14 cases (6,3%). median follow-up was 23 months (range: 3-50). patient's and donor's cmv igg were positive in 188 (82,6%) and 92 (58,5%), respectively. recipient/donor serology was +/-(risk group 1) in 62 (27,7%), +/+ (risk group 2) in 126 (56,3%) , -/+ (risk group 3) in 6 (2,6%) y -/-(risk group 4) in 30 (13,4%). results: two pts underwent a second transplant before day +100 due to graft failure. overall mortalities (om) at days +100 and +365 of the rest of the series (222 pts) are shown in table. the highest risk group (recipient cmv + / donor cmv -) exhibited more than double om at day +100 and more than four times om at day +365, when compared with pts at lowest risk (recipient cmv -). those striking differences were mainly due to nrm. om for risk group ii (recipient cmv + / donor cmv +) was intermediate. conclusions: in our studied population, mainly adult patients, the combination of cmv-seropositive patient with a cmv-seronegative donor had a very clear adverse impact on hsct outcome. as a result, we considered that the election of a cmv-positive donor for a cmv-positive patient continues to be strongly advisable, whenever is possible. on the other hand, once letermovir has proved to be efficient and well-tolerated and has been licensed for prophylaxis of cmv in high risk recipients, this approach appears to be very attractive to try to avoid the adverse impact of recipient cmv-seropositivity, particularly when finally chosen donor is cmv negative. disclosure: nothing to declare an active surveillance and an early and individualized management is critical to avoid mortality from respiratory viral infections in allo-hsct recipients background: respiratory viral infections (rvis) are frequent among the general population. in transplant recipients, rvis are known to cause an important morbidity and potential mortality. for this reason and several others, as the need of preventing other pts from contagious or avoiding misdiagnosis with other infections processes, a high index of suspicion of vris is necessary. during the last few years, we have implemented an active and systematic surveillance policy orientedto early detection and management of rvis in the hsct recipients. methods: the population analyzed for this report is the 175 patients who underwent allo-hsct from january 2015 through march 2018 in our unit. median age at transplant was 51 years (range: 7-69). one hundred and four were male (59.4%) and 71 were female (40,6%). baseline diseases were: 64 aml, 38 lpd, 23 all, 19 mds, 15 mpd, 10 mm, and 6 bmf. donor was unrelated in 92 transplants (52.6%) and was family in 83 (47.4%) (including 29 haplo-identical). conditioning regimen was reduced in 91 procedures (52%) and intensive in 84 (48%).stem cell source was pb in 163 (93.1%) and bm in 12 pts (6.9%).median follow-up was 27 months (range: 8-46); at the close of the analysis, majority of the series (89.1%) had a follow-up superior to one year from hsct. a throat swab(ts) was taken from every patient with any, even minor, respiratory symptoms. the respiratorysamples were tested whith a complete pcr panel of human respiratory viruses: rhinovirus (rv), influenza a and b virus (iv-a, iv-b), parainfluenza virus (pivs 1-4), respiratory syncytial virus (rsv), metapneumovirus (mpv), coronavirus (cov), adenovirus (adv), and bocavirus (bov). results: day +100 overall mortality of the series was 8,6%. day +365 overall mortality was 25,1% (13,7% nonrelapse mortality -nrm-, and 11,4% progression/relapse mortality). causes of nrm reflected in table 1. no patients died due to rvis. from 1 st july 2016 through 30 th june 2018 (a 24-month period), 581 ts samples were obtained from 129 pts (73,7%).the median number of samples/patient was 3 (range: 1-17).a total of 162 (1-8) rvis episodes were diagnosed in 87 pts (49,7%).the median presentation of the first rvi was at the day + 226 (3-924) post-hsct. the viral distribution was: 47 rv (26.6%), 36 iv (20.3%), 33 piv (18.6%), 28 rsv (15.8%), 12 mpv (6.8%), 11 cov (6.2%), 9 adv (5.1%), and 1 bov (0.6%).there were 13 mixed (two or more viruses) rvi episodes. the temporary distribution of vri episodes is shown in figure 1 . conclusions: 1) symptomatic infections due to respiratory viruses are very frequent among the allo-hsct recipients. 2) a high level of suspicion, as well as an early and systematic screening and management policy, are critical to avoid potential attributable mortality and the nosocomial spread of rvis among the transplant recipients. 3) in our series, rhinovirus, parainfluenza and adenovirus might be detected at any moment of the year; the rest of the viruses showed a clear seasonal pattern (november to april). [[p452 image] 1. background: trimethoprim-sulfamethoxazole (tmp-smx) is the most suitable drug for prophylaxis against pneumocystis pneumonia and infections with toxoplasma after allogeneic haematopoietic stem cell transplantation (allo-hsct). allergic reactions or hypersensitivities, mainly exanthemas, occur in about 3-5 % of the patients, usually resulting in the use of alternative prophylactic drugs (e.g. pentamidine or atovaquone). it has been hypothesised that allergies might be cured with allo-hsct. methods: we conducted a retrospective chart review of patients with tmp-smx re-exposition after allo-hsct from december 2017 to september 2018. follow-up is current as of december 2018. results: six patients (f/m: 4/2, median age: 45 years, range: 25 -66 years) with a history of tmp-smx hypersensitivity prior to allo-hsct were re-exposed to tmp-smx after engraftment of a matched related (mrd, n=1) or matched unrelated (mud, n=5) donor. median time to re-exposition was 18.5 (range: 11-341) days after allo-hsct with one oral dose of tmp-smx. in four patients, tmp-smx was tolerated without any signs of hypersensitivity reactions and has been continued for a median of 231 days (range 87-315) until last followup. one patient (mud, re-exposition at d+341) experienced pruritus and erythema some hours after tablet intake. another patient (mud, re-exposition at d+18) developed an exanthema one day after re-exposition which was later diagnosed as a cutaneous gvhd. conclusions: re-exposition of tmp-smx in patients with prior hypersensitivity is feasible after allo-hsct. after successful re-exposition, patients can be treated with the best-studied drug for prophylaxis of infections with pneumocystis and toxoplasma. disclosure: nothing to declare brincidofovir for adenoviremia in paediatric hsct for primary immune deficiency background: reactivation of adenovirus is a severe complication of hsct associated with significant morbidity and mortality, particularly for children with primary immune deficiency (pid). the only drug currently licensed to treat adenovirus infection is cidofovir. brincidofovir is a lipidlinked derivative of cidofovir which has been shown to be a safe and effective alternative treatment to cidofovir. there is limited data describing the use of brincidofovir in patients undergoing hsct for primary immune deficiency. we reviewed all patients who received brincidofovir after undergoing hsct for primary immune deficiencies between 2016 and 2018 at the great north children's hospital, newcastle upon tyne, uk. results: of 78 patients transplanted for pid, 11 developed significant adenoviraemia (14%). all were treated with cidofovir initially but 6 were switched to brincidofovir because of a failure to respond or because of renal toxicity. of these, 4 resolved their adenoviraemia within 21 days of commencing treatment (figure 1). donor sources were tcr alpha/beta/cd19 depleted haplo-identical (n=4), tcr alpha/beta/cd19 depleted mmud (n=1) and 12/12 mud (n=1). patients were conditioned with treosulphan/fludarabine/thiotepa/atg/ rituximab (n=4), treosulphan/fludarabine/atg/rituximab (n=1) or treosulphan/fludarabine/alemtuzumab/ gcsf/plerixafor (n=1). occurrence of agvhd and treatment of agvhd are outlined in table 1. patient 5 died +111 days post-transplant of multi-organ failure, severe thrombotic microangiopathy and sepsis. although patient 2 initially responded to brincidofovir, reactivation occurred after cessation of treatment; severe diarrhoea precluded the reintroduction of brincidofovir and the adenoviraemia persisted with poor immune reconstitution. treatment with addback t cells was attempted however the patient died 194 days post-transplant after a cerebral haemorrhage. patient 3 had long-standing chronic diarrhoea which was thought not severe enough to warrant cessation of brincidofovir treatment. conclusions: the complete resolution of adenoviraemia in 4/6 patients who had previously failed to respond to prior therapy with cidofovir suggests that brincidofovir may be an effective treatment option for adenoviral reactivation post-hsct for pid. however, resolution of adenoviraemia is influenced by many other factors, including the adequacy of immune reconstitution, the degree of induced immune suppression and the presence of comorbidities such as gvhd. due to the small sample size it was difficult to assess the relative importance of these factors in this cohort. brincidofovir was well tolerated however its effectiveness may have been limited by poor gastrointestinal function in one patient (patient 3) and could not be used after a viral reactivation in another for the same reason. further studies of the use of brincidofovir in this specific cohort are needed to clarify the role and effectiveness of this treatment. background: there is a high prevalence of cmv seropositivity in algerian population. because of high morbidity and mortality in pts who underwent allo sct with cmv reactivation, effective surveillance and timely treatment using anti-viral therapy s required. the risk of cmv reactivation depends on the type of stem cell source, immunosuppression (is) and serological status of the donor/ recipient pair. methods: over a 24 months period (from 01/01/2016 to 31/12/2017), 254 pts underwent allo-hsct for malignant or non-malignant hematology diseases of which 246 pts are evaluated for this study. cmv reactivation was observed in 66 pts (26.82%) (aml: 33 pts, all: 8 pts, cml: 4 pts, multiple myeloma: 2 pt, nhl skin: 1 pt, primary myelofibrosis: 1 pt, aplastic anemia: 14 pts, fanconi anemia: 2 pts, β-thalassemia: 1 pt), with a median age of 30 years (4-54), sex ratio (m/f) of 1.75. allo-hsct done with sibling donors: 51 pts, haplo-identical donors: 14 pts and pheno-identical donor: 1 pt. all pts were treated by chemotherapy alone with myéloablative conditioning (mac) in 41 pts and reduced intensity (ric) in 25 pts. all pts received peripheral blood stem cells with an average rate of cd34 + cells: 7, 8.10 6 /kg (3.4-12.7). additional bone marrow graft was used in 7 pts that received a haploidentical graft without pt-cy. gvh prophylaxis associated cyclosporine (csa) and methotrexate (sibling and phenoidentical); csa-mtx-mmf or csa-mtx-cyclophosphamid (haplo-identical). before transplantation, donor/recipient pair is at high risk reactivation in 65 pts (98.4%). detection of cmv reactivation done by antigenaemia pp65 or by quantitative pcr weekly for the first 3 months and during an is treatment for acute or chronic gvhd. pre-emptive therapy is initiated by ganciclovir as soon as positivity of antigenaemia or increased viral load in pcr. results: a first reactivation occurred on average day 44 (16-83) in 66 pts (26.82%) of which 32 pts under corticotherapy for acute gvhd (27 pts), thrombotic micro-angiopathy (1 pt) and renal failure (4 pts) or due to a reinforced is for haplo-identical transplantation (5 pts). one pt with chronic gvhd presented a late reactivation 18 months after transplant. twenty pts presented a 2 nd reactivation on average day 135 (106-180) and 8 pts a 3 rd reactivation on average day 136 (130-143). pre-emptive treatment is introduced in the first episode by a viral dna polymerase inhibitor (ganciclovir: 22 pts; valganciclovir: 43 pts, foscarnet: 1 pt). the negativity of antigenaemia is observed on average at 5 days of treatment (3) (4) (5) (6) (7) . second line treatment was required in 14 pts (22%) due to resistance (12 pts), severe cytopenia (1pt) or renal failure (1 pt). the onset of severe cytopenia imposed a dose reduction (4 pts) or a therapeutic stop (6 pts) before 15 days. two pts received additional maintenance treatment for negativation delay. three pts (4.5%) died from cmv infections resistant to antiviral treatment (pneumonia: 2, colitis: 1). conclusions: cmv infection is a serious complication after allo-hsct. in the absence of vaccination, the systematic monitoring for cmv reactivation is strongly recommended for the establishment of a rapid and effective preemptive treatment. disclosure: nothing to declare p456 abstract withdrawn. results: in transplanted group, episodes of bkv reactivation occurred in 54 patients (42 %). in 26 cases only urine colonization (c) found before hsct. in this group in 10 patients (39 %) virus was transmitted from urine to the blood (b) . dysuria and/or hc were observed in 4/26 (15%) patients . all of them (100%) had urine and serum involvement. in 28 cases bkv replication was found after hsct (3 -cases detected in urine, 25 cases-bothserum and urine). dysuric syndromes and/or hc were found in 5/28 of cases (18%)-all in patients with serum and urine involvement. urinary tract was always first location of the virus. there was no case of isolated serum reactivation. the incidence of bk infection was higher in patients older than > 5 yrs (p< 0.05), transplanted from family donor (msd) (p< 0.05). mud recipients had more often both serum and urine reactivation (p< 0.05) than isolated urine involvement. sex, day of neutrophil recovery, conditioning regimen, or use of total body irradiation were not significant risk factors for bkv infection, or hc . six patients were treated with cidofovir (range 1-4 doses) with good response. there was no death due to evident bkv infection. conclusions: bkv reactivation remains one of the most frequent infectious complication in children undergoing allogeneic hsct. most of patients experienced mild infection and age < 5 years was the positive prognostic factor influencing its incidence. bkv monitoring and prompt treatment of hc resulted in excellent outcome. we observed surprisingly high rate of new bkv replication after hsct. disclosure: nothing to declare background: high-dose chemotherapy (hd-ct) and auto pbsct have been the standard therapy for multiple myeloma (mm) for more than two decades, despite a wide range of new therapeutic options. recurrent/refractory malignant lymphomas and recurrent/metastatic germ cell tumors (gct) also benefit from this intensive therapy. in comparison to allogeneic transplantation, this treatment is known for lower complication rates, e.g. infections. however previous studies have schown that treatment related toxicity may not be underestimated and depending on the conditioning regimen used. methods: we retrospectively analyzed 193 patients (225 cases) who underwent hd-ct plus auto pbsct between 2011 and 2017 in a single-center study. to anlyze the incidence of infections depending on the conditioning regimen, we formed the following categories based on the agiho: no infections, neutropenic fever, sepsis and severe sepsis. results: the median age in this analysis was 59 years; 74.2% were male. the most frequent diagnosis was mm (62.7%) receiving high dose melphalan (mel), followed by malignant lymphoma (26.7%) receiving beam (bcnu, etoposide cytarabine, melphalan) and relapsed/metastatic germ cell tumours (gct) (9.8%) receiving high dose carboplatin/etoposide (ce). 8% of all patients developed severe sepsis, 5 patients had to be ventilated and 2 patients died. sepsis was documented in 25.3% of all cases (57 cases). the majority of patients (58.2%, 131 cases) developed neutropenic fever and 8.4% (19 cases) didn´t have any infection complications. the beam conditioning regimen showed the highest tendency to result in a septic course (43.3%), followed by ce (27.3%) and mel (24.1%). the most commonly documented pathogen in 153 blood cultures was s. epidermidis (41.7%), followed by e. coli (18.5%) and s. mitis (9.9%). only in one blood culture we detected a multi-resistant pathogen (3mrgn e. coli). p. aeruginosa was detected in 4 blood cultures (2.6%), l. monocytogenes in 2 (1.3%) and s. aureus in 6 (4%). 77.8% of all patients developed diarrhea, only in 10.9% of these cases we could detect c. difficile. the conditioning regimen shows no significant effect on the incidence of c. difficile. the mean neutropenic period was 13.5 days in malignant lymphoma patients, followed by 10.8 in mm patients and 8.7 days in gct patients. the hospital discharge, calculated from the day of transplantation, was significantly different: for malignant lymphoma the mean was 20.8 days, for mm 19.6 days and for gct 14.5 days. conclusions: our data correspond to former published results by many groups. the beam regimen shows the highest infectious complication rate followed by ce and mel. the duration of neutropenia and hospital stay depends on the conditioning regimen. the type of infectious complication doesn't effect the progression free-and overall survival in our analysis. disclosure: nothing to declare. impact of donor and recipient cytomegalovirus serostatus on outcomes of unrelated allogeneic haematopoietic stem cell transplantation background: cytomegalovirus (cmv) is an important cause of morbidity and mortality in allogeneic haematopoietic stem cell transplant (hsct) patients. the aim of our study is to evaluate the outcomes of our cmv seropositive recipients who received grafts from seropositive unrelated donors (d+r+) compared with grafts from seronegative unrelated donors (d-r+). methods: this is a retrospective single center study on a series of cmv seropositive recipients who underwent hsct from unrelated donors between febuary 2012 to july 2018. a total of 96 patients were analyzed. their clinical course and laboratory results were reviewed for evidence of cmv reactivation and/or cmv disease. we defined cmv infection as detection of cmv reactivation or primary infection by antigenaemia or polymerase chain reaction (pcr) assays, but was not accompanied by signs and/or symptoms suggestive of a systemic disease. cmv disease occurred when cmv was isolated from any site in association with organspecific signs and/or symptoms. monitoring for cmv infection commenced upon engraftment (approximately day +14). peripheral blood samples were sent twice a week for cmv antigenaemia or cmv quantitative pcr. the duration of twice weekly monitoring was at least about 100 days. longer monitoring was performed in patients who experienced cmv infection after hsct. results: all patients received graft-versus-host-disease (gvhd) prophylaxis using anti-thymocyte globulin (atg) at 4.5mg/kg in addition to cyclosporin or tacrolimus. among the entire cohort of 96 patients, 72 (75%) had cmv infection, including 23 (82.1%) out of 28 patients from the d-r+ group and 49 (72%) out of 68 patients from the d+r + group. 16 patients (57.1%) from the d-r+ group and 19 patients (27.5%) from the d+r+ group had ≧2 cmv reactivation above the threshold for preemptive therapy respectively; p=0.007. 11 patients developed cmv disease, 6 (21.4%) from the d-r+ group and 5 (7.4%) from the d +r+ group. cmv resistance to both foscarnet and ganciclovir was detected in 3 patients (10.7%) from the d-r+ group but none from the d+r+ group. 2 patients died due to cmv disease, both were from d-r+ group. 2 year overall survival (os) were 60% versus 55% for d-r+ group and d+r+ group respectively; p=0.706. median survival was not reached at 2 years. 2 year non-relapse mortality (nrm) were 35% for d-r+ group and 24% for d +r+ group respectively; p=0.37. conclusions: the incidence of recurrent cmv infection was higher in the d-r+ group compared to the d+r+ group. there were no statistically significant differences between the 2 groups in terms of os and nrm. however, there was a trend towards higher nrm in the d-r+ group compared to d+r+ group. our findings suggest that for matched unrelated hsct, it may still be important to select a seropositive donor for a seropositive recipient. disclosure: none background: it´s known that some patients submitted to allogeneic stem cell transplantation (asct) could present a greater susceptibility to infection even when they are in long term complete remission or potentially cured. this fact is related to the dynamic of immunological recovery that is variable in every single patients and it is dependent from many factors: the haematological disease, the conditioning regimen, the age of patient and donor, the number of stem cell and lymphocytes infused, the anti-gvhd prophylaxis, the use of anti-thimoglobulins and others. in clinical practise we can observe patients who are potentially cured, who tapered and stopped the immunosuppressive treatment months or years ago and who are suddenly graved from opportunistic infections. the largest part of these infections is represented from varicella-zoster virus (vzv) cutaneous eruption. methods: in this report we retrospectively analysed a monocentric cohort of 38 patients submitted to asct for haematological malignancies from a median time of 30 months. all of them were free of disease. they stopped the immuno-suppressive treatment in a median time of 270 days after asct (range: 160-1642) and did not present later chronic gvhd needing treatment neither other moderate or severe chronic post transplant complications nor other diseases. prophylactic treatment with anti viral agents (acyclovir or valacyclovir) has been conducted simultaneously to immuno-suppressive treatment and for a period ranging between 1 to 6 months after its suspension. in this cohort of patients we considered the incidence of vzv eruption occurred after the suspension of the immunosuppressive treatment, and we analysed the immunological recovery in terms of lymphocytes sub-population after 6, 12, 18 and 24 months from asct. results: of these 38 patients considered, 10 developed at least one vzv manifestation. all the vzv presentation were cutaneous, we did not observe neurological, ophthalmic or visceral presentation. all the vzv manifestation occurred in patients who ended the anti-viral prophylaxis. median time of presentation was 575 days after asct (range: 174-1642) the remaining 28 patients did not present vzv manifestation nor other kind of opportunistic infection despite the absence of anti-viral prophylaxis. the analysis of lymphocyte sub-population after 6-12-18 and 24 months did not show a significant difference in b, t, t4, t8 and nk lymphocytes in the different post transplant period. conclusions: vzv reactivation seems not to be correlated with the number of the different lymphocyte subpopulations in the post transplant period. actually it is not possible to distinguish patients more suitable of vzv reactivation on the basis of lymphocyte sub-populations analysis, so anti-viral prophylaxis should be prolonged for a medium period after suspension of immuno-suppressive drugs. in absence of anti viral prophylaxis a careful clinical surveillance should be performed in order to treat early eventual vzv manifestations. disclosure background: infection and disease cytomegalovirus (cmv) are common problems in patients undergoing hematopoietic stem cell transplantation (hsct). cmv infection has a high overall seroprevalence, therefore, during the first 100days post-hsct, it is important to prevent reactivation of cmv. the international clinical recommendation is the use of ganciclovir as prophylaxis in hsct patients; however, the cost of this treatment is not accessible for our population. in this respect it has been used as an alternative valganciclovir because of its lower cost and oral administration. our study´s aim was to assess the response and safety of valganciclovir in comparison with ganciclovir to prevent viremia and cytomegalovirus disease in patients undergoing allogeneic hsct methods: a retrospective study was performed on patients who receive an hsct-allo between january 2014 and august 2018. participants were enrolled in two groups according to prophylaxis treatment: (a) ganciclovir 5mg/k once daily and (b) valganciclovir 450mg twice daily for 7 days pretransplant, at day +100; viremia was measured by pcr. demographic and clinical information was collected from medical records and furthermore analyzed in spss v21. results: sixty-eight patients were enrolled in the study, 54% male, the median age was 34 years (19-61) with the following diagnoses: acute lymphoblastic leukemia 44%, acute myeloblastic leukemia 26.5%, granulocytic chronic leukemia 17.6%, myelodysplastic syndrome 4.4%, dendritic cell neoplasia 4.4%, and aplastic anemia 2.9%. ninety-one percet of the patients received a transplant from an identical hla donor and 8.8% received a haploidentical transplant. thirty-four patients received ganciclovir (g1) and thirtyfour valganciclovir (g2). median age was 39 vs28 years (p=0.022), intermediate risk cmv 85% vs 76 (p=0.048), associated bacterial infections was 15%vs 32% (p=0.15), and fungal infections 6% vs9% respectively (p=0.5). the reactivation by cmv was presented in 21% vs 15% respectively (p=0.70). there were no significant differences in fever, bacterial isolation, dysfunction or graft failure, presence and degree of acute or chronic gvhd and relapse of the disease. the most relevant characteristics and complications are described in table 1. within the whole group there were 28 deaths, 53% in the ganciclovir group and 32% in valganciclovir group (p=0.14), overall survival 1-year was 66% vs 80% (p=0.58) respectively; in both groups 72% was associated with relapse and 28% associated with transplantation. conclusions: ganciclovir and valganciclovir were effective in preventing the reactivation of cmv, the only statistically significant difference was that the presentation of the disease appeared earlier in the valganciclovir group. no difference in toxicity between the groups was identified. disclosure: none declared background: invasive pulmonary aspergillosis (ipa) is a severe and serious complication that occurs in the immediate post-transplant period due to severe neutropenia or late usually following prolonged corticosteroid therapy during treatment of graft-versus-host disease (gvhd). the objective of this study is to analyze the epidemiological, diagnostic and evolutionary characteristics of this major complication over a period of 3 years. methods: from january 2015 to december 2017, 392 patients (pts) received an allogeneic hematopoietic stem cell transplantation (allo hsct) for malignant and nonmalignant haematological diseases. during the transplant procedure, anti-infectious prophylaxis consisted of pts isolation, digestive decontamination, fluconazole and aciclovir. secondary prophylaxis done for pts with prior history aspergillosis. during the follow-up, a standard chest x-ray is performed systematically at each control or in case of clinical signs a thoracic ct scan is requested from suspicion. the diagnosis of ipa is made according to the criteria of the eortc-msg based on the predisposing criteria of the host and clinico-radiological criteria (possible infection). galactomannan antigen and histopathology criteria are not common practice. results: a total of 29 ipa episodes (7%) were identified in 26 pts (aml: 15, aa: 5, all 3, cml 2, mm 1) of median age 36 (8-56) , sex ratio: 0.44. all of them had transplantation from a family donor (geno-identical: 22, haplo-identical: 4) with conditioning by chemotherapy alone and a graft of csp (24 pts) and peripheral stem cells-bone marrow (2 pts). all pts had at least one predisposing risk factor: antecedent of aspergillosis (3 pts), prolonged neutropenia> 10 d (5 pts), acute gvhd (8 pts), chronic gvhd (4 pts), prolonged corticosteroid therapy ≥ 0,3 mg /kg/day exceeding 21 days (12 pts). the diagnosis of api was possible on average at j172 (20-930) after appearance of clinical signs (in all cases) and evocative radiological in 25 cases (in 4 cases, the standard chest x-ray was normal). at the time of thoracic ct scan, 11 pts (37%) had characteristic signs: halo sign (6 pts), crescent sign (1 pt) and cavity (4 pts). other minor radiological signs are found in the other pts. empirical first-line antifungal therapy was started as monotherapy in 14 pts (voriconazole: 8, caspofungin: 6 pts) or in combination in 12 pts. a secondline treatment was required in 10 pts for failure after an average duration of 7 days . three pts presented a second episode after an average delay of 5 months (3) (4) (5) (6) with a favorable evolution of resumption of thetreatment. fourteen pts (54%) are alive with complete resolution after a median treatment time of 10 months (2-19). twelve pts (46%) died rapidly on average 14 days after diagnosis (ipa 11, relapse of his disease: 1) conclusions: ipa occurring after an allograft of allo-hsct is a severe complication with high mortality. it is essential, in each case, to identify the pts with risk factors, perform a thoracic ct-scan, send serum serology for apergillus galactomannan antigenand start specific treatment as soon as possible while waiting to be able to reinforce the diagnosis by direct examination or sputum or brochoalveolar lavage with aspiration. disclosure: nothing to declare background: cmv (cytomegalovirus) has a prevalence varying between 45-100%. its pathogenicity is relatively low in the general population, usually resulting in a selflimiting viral illness. in an immunosuppressed host, infection can lead to life threatening illness. disseminated cmv infection can manifest in a number of organs and is diagnosed using internationally accepted criteria. in the post solid organ and stem cell transplant (sct) setting, it is postulated that it is viral reactivation, rather than primary reinfection that leads to cmv viraemia. prevention of reactivation requires the presence of a competent immune system, mediated by t-cells. this accounts for the increased incidence in intensive and t-cell depleting sct conditioning regimens. despite improved outcomes following the introduction of cmv monitoring by pcr and pre-emptive treatments (current uk guidance), cmv pneumonitis still carries a high mortality. the use of cmv specific immunoglobulins (cmvig) for the treatment of this complication is generally not recommended post chemotherapy or sct in haematological cancers due to lack of evidence. however, cmvigs are widely used in the setting of cmv reactivation post solid organ transplants. we report the use of cmvig in patients with suspected cmv pneumonitis at a single uk centre. the aims of this retrospective study were to establish safety and review efficacy in this highly immunocompromised group of patients. methods: data was collected retrospectively on the use of cmvig in patients with haematological cancers post sct or chemotherapy alone between 2007 and 2017 at manchester royal infirmary, uk. all patients included had cmv positive pcr in blood (and or from bronchoscopy), as well as high resolution ct imaging evidence of cmv infection. the data was sourced from pharmacy database and crossreferenced with a departmental list. for each patient identified, case notes and prescriptions were sourced. data collected included patient baseline characteristics, timing of treatment, number of doses of cmvig and outcome. results: eight patients received cmvig for suspected cmv pneumonitis. seven patients were post sct and one patient was severely immunosuppressed with chemotherapy alone. median age was 40 years (range 16-68). the cmvig regimen used was 4ml/kg of cytotect ® on days 0, 2, 4 and 6, followed by 2ml/kg every four days until resolution of symptoms. there were no infusion related reactions observed. patients received a median of 4 doses of cmvig. four out of 8 patients responded to the treatment and showed full recovery but only 3 are alive and well to date. conclusions: this study shows that the use of cmvig is safe in the post-sct setting of acutely unwell patients with multi-organ failure. despite limitations of retrospective studies, there appears to be benefit for the use of cmvig in our patient population, with 50% of patients showing a full recovery from that episode. allogeneic sct plays a confounding role in the outcome of patients although the numbers in our study are small. there is clearly a need for better treatments of cmv pneumonitis. cmvig is a promising treatment but further studies are needed to identify the optimal dosing regimen and provide evidence of efficacy. disclosure: biotest-honaria p464 abstract withdrawn. background: the risk of fungal infection related to allogeneic transplantation is a well-known cause of morbidity and mortality. the main agents implicated are yeast during the neutropenic period and filamentous fungi after this period. methods: we decided to evaluate the effectiveness of a prophylactic regimen containing fluconazole since day -3. after discharge fluconazole was kept until day or 75 or switched to posaconazole in high-risk patients. patients with gvhd under steroids were kept under prophylaxis.the group of high risk patients was defined by one of the following variables: 1-non related donors 2-atg, campath or fludarabine in the conditioning 3-presence of gvhd with need of steroids above 0.5 mg/kg we have analyzed the patients submitted to allobmt during 2016 and 2017. all patients were first admitted to an isolation room with hepa filters.patients under secondary prophylaxis were excluded. breakthrough fungal infections during the first year and toxicity leading to discontinuation was evaluated. results: sixty six patients were included with 67 transplants. male/female ratio was 36/30. the age range was 0.6-64 yo with a median of 37. malignant (53) and nonmalignant (13)diagnosis were included. donor type was related (16) haploidentical (9) and non-related (42). the conditioning regimen includes atg in 21, campath in 5 and fludarabine in 41. fourteen patients were treated after discharge with fluconazole and 52 with posaconazole. three patients fluconazole were switched to micafungin for hepatic toxicity, two cases to amphotericin due to persistent fever and in one case to caspofungin for a proven fungal infection (candida parapsilosis in blood stream in day +18). after discharge and during the first year of follow-up a single case of possible fungal infection was diagnosed, in a patient with gvhd with a lung nodule. conclusions: during the neutropenic period after transplantation the main risk of fungal infection is associated with candidiasis. the greatest risk of aspergillosis occurs later and have a significant relation with gvhd. except for candida parapsilosis the main source of yeasts are the gi tract. the main source of aspergillus are aerosolized particles retained by hepa filters. in patients without a previous episode of fungal infection the main risk of filamentous fungi occurs only after discharge. we conclude that fluconazole alone or followed by posaconazole in high risk patients is a feasible and effective regimen for primary prophylaxis, in allogeneic transplantation. disclosure background: bk virus-associated hemorrhagic cystitis (bkv-hc) has emerged as a serious infection after hematopoietic stem cell transplantation (hsct). it is characterized by painful hematuria due to hemorrhagic inflammation of the urinary bladder mucosa, this causes significant morbidity, prolonged hospital care with extensive nursing requirements and increases in healthcare costs. the purpose of this study is to determine the incidence, risk factors, and duration of treatment in our center. methods: we performed a retrospective review of hsct patients at luis calvo mackenna children´s hospital in santiago, chile diagnosed with bkv-hc, from 1st january 2016 to 30th november 2018. we investigated the incidence, risk factors and duration of treatment of bkv-hc in paediatric patients undergoing hsct over a 35 months period. bkv-hc was defined as bk virus (bkv) detection in urine by pcr testing in association with clinical symptoms and hematuria grade 2 or higher. sixty-seven patients were trasplanted during this period. results: eleven patients were diagnosed with bkv-hc at our institution, only one with bk viremia. the cumulative incidence of bkv-hc in our series was 16%. all of them were treated with cidofovir. the median age at diagnosis was 9 years old (range: 3-13 y.o.). the median time from hsct to hemorrhagic cystitis (hc) was 40 days (range: 25-240 days), the median length of treatment was 9 weeks (range: 2-36). all patients received myeloablative conditioning regimens and used cyclophosphamide (100%); ten (90%) were unrelated cord blood transplant recipients and nine (81%) used antithymocyte globulin. a concomitant viral reactivation (cmv/vh6) was demonstrated in six (56%) patients. no patient died due to bkv-hc or its complications, but in the follow up three patients died, one in relapse and two of other post transplant´s complications. conclusions: bkv-hc is the result of a complex interaction between patient characteristics, donor type and conditioning regimen intensity. these patients experienced significant morbidity and prolonged treatment. in our cohort bkv-hc of all patients but one were transplanted with an unrelated umbilical cord blood unit, all of them received myeloablative conditioning regimen with cyclophosphamide and most of them received anti-thymocyte globulin. we also observed frequently co-existence of viral infections from herpes family as cmv and vh6. the main limitations of this work are its retrospective nature and it´s from a single center. more studies are necessary to better understand the epidemiology and risk factor associated with bkv-hc and the morbidities associated with its treatment. disclosure: nothing to declare how we manage hhv-6 reactivation in the posttransplant setting oscar borsani 1 , anna amelia colombo 1 , daniela caldera 1 , paolo bernasconi 1 1 university of pavia, san matteo hospital, pavia, italy background: hhv-6 encephalitis is a life-threatening complication in the post-transplant setting and it develops in about 1% of patients receiving traditional hsct. several risk factors were described. a differential diagnosis between hhv-6 encephalitis and other neurological complications is extremely important but often not-easy to achieve because of the highly heterogeneous clinical and radiological features and complexity of interpretation, especially in transplanted patients. here we described vignettes that represent and highlight distinct problems in the diagnosis and management of transplanted patients with suspected hhv-6 reactivation. methods: we collected the clinical, laboratory and radiological (electroencephalogram, brain mri and brain ct) data of transplanted patients who developed a neurological syndrome suspected for hhv-6 reactivation. hhv-6 was detected on serum and csf using rt-qpcr. results: 1) a 61-years-old patient developed a diffuse erythema and subsequent encephalitic syndrome following hsct. the brain mri revealed clear signs of limbic encephalitic and searching for hhv-6 on serum and csf revealed 150.000 copies/ml and 220.000 copies/ml respectively. an antiviral therapy was started but no clinical benefit was achieved. 2) a 59-years-old patient developed a typical neurological syndrome without brain mri findings of encephalitis and with no evidence of skin involvement. the lumbar puncture and csf analysis showed a total of 49.200 hhv-6 dna copies/ml. antiviral therapy with ganciclovir and foscarnet was promptly started with clinical improvement and a drastically reduction of hhv-6 dna on both csf and serum. a new brain mri revealed an acute limbic encephalitis. 3) a slight neurological syndrome consisting of confusion and amnesia developed in a 49-years-old-patient. brain mri findings were compatible with a wernicke syndrome, but no improvement of neurologic symptoms were obtained with thiamine supplementation. csf analysis did not revealed hhv-6 dna, which was detected at low copies number on serum analysis. a second brain mri was conclusive for limbic encephalitis, so an antiviral therapy with foscavir was started and radiological but not clinical improvement was noted. the patient died after few days. 4) in the last case we present a 52-years-old patient who developed a clinical picture of encephalopathy (i.e. amnesia, ataxia, drowsiness, weakness, depression) with rapid progression to coma after seventy-eight days from hsct. a brain mri showed a slight contrast enhancement in parietal-occipital regions. during the recovery phase from conditioning-induced cytopenia, an increasing in serum hhv-6 dna was detected. searching for hhv-6 dna on donor's follicles showed a chromosomally integrated hhv-6 (cihhv-6). cyclosporin a (csa) was interrupted and neurological improvement was observed in the following hours: a diagnosis of pres was made. conclusions: hhv-6 encephalitis should be suspected in transplanted patients with a clinical syndrome of encephalopathy. pcr detection of hhv-6 dna in csf associated with either typical brain mri abnormalities or a clinical diagnosis of nonspecific encephalopathy must lead to the urgent initiation of systemic antiviral treatment. if an increase of both serum hhv-6 dna and wbc is detected, a cihhv-6 should be confirmed. pres is an important differential diagnosis in transplanted patients which developed an encephalitic syndrome. disclosure: nothing to declare background: cytomegalovirus (cmv) infection is a major cause of morbidity and mortality after hematopoietic stem cell transplantation (hsct). it causes end-organ disease, multi-organ dysfunction syndrome, graft failure, increased susceptibility to infections and gvhd. greatest risk of cmv infection in a seropositive host is the reactivation of latent virus. methods: a prospective descriptive study performed at armed forces bone marrow transplant centre, rawalpindi, pakistan from dec 2016 to sep 2018. hundred consecutive patients who underwent hsct were followed with weekly cmv dna quantitative pcr from engraftment till day 100 for cmv reactivation. patients in whom cmv pcr showed more than 2000 copies/ml were treated with antiviral therapy. factors associated with cmv reactivation, outcome of antiviral therapy and effect of cmv on transplant outcome is studied. results: out of 100 cases, 82 were hla matched siblings, 15 were matched family donors and 3 were haploidentical transplants there were 66 males and 34 females. mean age was 11.9 ±8.9 years. fourty-two transplants were done in thalassemia, 35 in aplasia, 09 in leukemias and 14 in other hematological disorders and immune deficiencies. ninety-eight recipients and all the donors were cmv seropositive before hsct. cmv reactivation was seen in 81 patients and 42 of them had cmv viral load more than 2000 copies/ml and 39 patients had cmv viral load less than 2000 copies/ml. nineteen patients had no cmv reactivation. mean time to reactivation since transplant was 36±19 days. valganciclovir was given in 36 patients due to ease of administration and six patients were treated with ganciclovir during their hospital stay. only one patient had resistant disease. mean time to clear viremia was 20±12.8 days. the patients having viral load less than 2000 copies/ml, subsequently cleared cmv without any treatment. antiviral agents; ganciclovir and valganciclovir were equally effective for treating cmv infection with 99% efficacy, however, more adverse effects were seen with ganciclovir. myelosuppression i-iii was seen in 24% patients treated with valganciclovir and in 46% treated with valganciclovir. renal impairment i-ii was seen in 14% of valganciclovir and 20% of ganciclovir treated patients. steroid administration was strongly associated with cmv reactivation (p = 0.003). no statistically significant association was found with the use of atg, gvhd, underlying disease, abo or gender mismatch. os was 81.0 % and 93.1% in with and without cmv reactivation (p=0.1) and dfs was 83.3 % and 96.6 % in with and without cmv reactivation (p=0.06) conclusions: cmv reactivation was seen in 81% of the transplant recipients, this is higher compared to the western world due to high cmv seropositivity is this region. steroids administration in post-transplant period significantly increase the risk of cmv reactivation. preemptive therapy with valganciclovir effectively treats cmv reactivation with acceptable side effects. viral threshold for treatment should be decided considering the regional endemicity. cmv adversely affects the transplant outcome in terms of dfs and os. disclosure: no conflict of interest. acute nephritis requiring nephrectomy caused by adenovirus (hadv) and human polyomavirus bk (bkpyv) following allogeneic hematopoietic stem-cell transplantation in a patient with ph+ all background: adenovirus infection represents an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-hsct), with no established therapy. although different organs may be affected by disseminated hadv infections, kidney involvement has been rarely reported. co-infection of hadv and bkpyv are common complication in patients undergoing allo-hsct, but recent studies demonstrate that bkpyv may facilitate the replication of hadv and lead to elevated viremia with increased virulence and serious clinical consequences. here we report a case of an adult patient who required a monolateral nephrectomy due to hadv pyelonephritis as an early complication of allo-hsct for philadelphia-positive acute lymphoblastic leukemia (ph+ all). methods: in september 2014, an ethiopian gentleman was diagnosed with ph+ all at the age of 24 years. he was treated with polychemotherapy in association with the tyrosin kinase inhibitor imatinib mesylate achieving a complete remission (cr). one year later, due to disease relapse with cns involvement, he was started on vincristine and dexamethasone plus imatinib treatment and in april 2017 he was referred to our bmt center from ethiopia. upon confirmation of the p210 ph+ b-all diagnosis, therapy with the scr/abl dual inhibitor dasatinib associated to intrathecal chemotherapy was started and a salvage treatment with inotuzumab ozogamicin followed by an allogeneic hsct from a hla-identical brother was planned. having achieved a documented molecular cr disease status, in june 2017 the patient underwent allo-hsct following the fludarabine-melphalan reduced-intensity conditioning regimen. graft-versus-host prophylaxis included anti-thymocyte globulin, cyclosporine and mycophenolate mofetil results: on day +24 post-transplantation the patient developed macro-hematuria due to hemorrhagic cystitis and a ct scan unveiled a left pyelonephritis with marked kidney enlargement. kidney microbial investigations were all negative. at the same time, hadv viremia with very high copy number (>25000000 cp/ml) was documented and also elevated bkpyv (>25000000 cp/ml) viruria and viremia (6400 cp/ml). the genotyping of hadv evidenced serotype b11 mainly involved in infections of the urinary tract. treatment with cidofovir was immediately started; nonetheless, due to rapid clinical worsening despite maximal antibiotic therapy, on day +50 a left nephrectomy was performed, which led to a subsequent progressive resolution of the clinical symptoms and negativization of hadv and bkpyv viremia and viruria. pcr real time performed on the kidney tissue unveiled very high concentration of hadv copy number. conclusions: acute pyelonephritis due to disseminated hadv infection may represent a possible cause of severe complication following allo-hsct. monitoring of hadv copy number is helpful to evaluate infection severity and response to treatment. co-infection of hadv and bkpyv in immunocompromised patients should be always considered likely to worsen clinical course and outcome. disclosure: nothing to declare background: infection is a major cause of morbidity and mortality in patients (pts) receiving an allo-hsct. its severity is related primarily to the depth and duration of neutropenia. febrile neutropenia (fn) is defined as a neutrophil count below 500 cells/mm 3 and a fever ≥ 38.3°c at a single measurement or≥ 38°c 2 times at one hour intervals. the objective of our study is to analyze the epidemiological, clinical, biological characteristics of febrile episodes (fe) occurred in 136 pts who benefited an allo-csh over a period of 2 years. methods: from january 2016 to december 2017, 136 allo-hsct were performed in 136 pts including 106 sibling-hla identical, 26 haplo-identical and 04 phenoidentical for essentially acute leukemia (68 pts, 50%), acquired and congenital aplasia (45 pts, 33%). the median age is 22 years (3-59) and sex-ratio (m/f): 1.72. prophylaxis consisted on isolation sterile room with laminar flow, digestive decontamination, fluconazole and aciclovir. nine pts (6.6%) were infected at the time of hospitalization (cellulitis 03, pneumoniae 02, bacterial angina 01, veinitis 01, bronchial pneumonia 01, furuncle cutaneous 01) requiring treatment with antibiotics. conditioning regimen is myeloablative in all pts. anti-thymocyte globulin is used in 61 pts (44.8 %). peripheral blood stem cells (pbsc) are used in 117 pts (86%) with an average level of cd34+ cells: 7,5.10 6 /kg (2. 1-19.8) , bone marrow (bm) in 5 pts with a mean level of nucleated cells: 3.4 x 10 8 /kg (0.8-3.6) and the association of pbsc-bm in 14 pts (haplo-identical). at each fe, are practiced: chest x-ray, procalcitonin test, blood culture, microbiological study of urine and stool (if diarrhea). results: all pts showed aplasia with an average duration of 16 days (6-49), neutrophil engraftment was observed at day 18 (9-31). one hundred and twenty-nine pts (94.8%) presented 205 fe with an average of 1.5 per pt. eleven pts (8%) had 3 fe or more. forty nine (23,9 %) fe are clinically documented (digestive: 19, skin: 14, pulmonary: 11, urinary: 03, oto-rhino-laryngology: 02). the blood cultures are made at 195 fe, 136 fe are microbiologically documented (66 %): gram-positive bacteremia in 77% (mainly coagulase negative staphylococci) and gramnegative bacilli in 23% of cases. procalcitonin test performed during 157 fe: normal (32 cases), probable infection (90 cases), probable sepsis (15 cases), severe sepsis (19 cases) and septic shock (one case). empirical double antibiotic therapy is initiated in 120 pts without waiting for the results of the microbiological study. this association was sufficient in 15 pts (11 %). the transition to a second line was needed in 114 pts (83.8%) and third line in 82 pts (60%). antifungal is added in 36 cases (27%). eight pts benefited from g-csf. the evolution is favorable in 202 fe (98.5%), apyrexia obtained after an average of 3.3 days . three pts died (2%) by severe sepsis on a durable aplasia, 2 of which had a cellulitis before the conditioning. conclusions: fe increase morbidity and mortality in allo-hsct so prophylactic measures are essential. empirical antibiotics treatment has to be instituted very quickly in the absence of documentation. disclosure: nothing to declare p472 abstract withdrawn. atsushi satake 1 , masaaki hotta 1 , ryo saito 1 , akiko konishi 1 , hideaki yoshimura 1 , takahisa nakanishi 1 , shinya fujita 1 , tomoki ito 1 , kazuyoshi ishii 1 , shosaku nomura 1 1 kansai medical university, osaka, japan background: cytomegalovirus (cmv) infection remains a common complication after allogeneic hematopoietic stem cell transplantation (ahsct), which results in increased morbidity and mortality. letermovir is a novel anti cmv drug that inhibits the cmv-terminase complex. the purpose of this retrospective study is to elucidate the efficacy and safety of cmv prophylaxis with letermovir early after ahsct in clinical practice. methods: we retrospectively analyzed the incidence of cmv infection, cmv disease, preemptive therapy, adverse events through week 14 after ahsct, the rates of engraftment and overall survival. all patients underwent ahsct in our institution for hematopoietic malignancies between may 2018 and nov 2018. data collected in this study included patient's characteristics such as age, sex, disease status, donor source and cmv disease risk. cmv infection was evaluated by cmv antigenemia. this study was approved by the research ethics committee of the faculty of medicine, kansai medical university. results: thirteen patients (male 9, female 4) underwent ahsct and received cmv prophylaxis with letermovir. the median age was 54 years (range, 18-65 years). overall, 10 of 13 patients (76.9 %) were considered to be at high risk for cmv, including 5 patients (38.5%) with haploidentical donors, and 1 (7.7 %)with mismatched, unrelated donors. all patients began letermovir from day 0 after ahsct, and achieved engraftment (median 17, 13-22 days). no patient developed cmv disease and required preemptive therapy. one patient died of treatment-related mortality, and 2 patients died of acute gvhd. although one patient discontinued letermovir before day 100 after ahsct because letermovir was suspected to be a cause of persistent nausea, severe adverse events were not observed. conclusions: it is still unknown whether cmv prophylaxis with letermovir improves os and reduces trm; however, our data suggests that cmv infection is considerably inhibited by administration of letermovir early after ahsct. clinical trial registry: not applicable. disclosure: the authors declare noconflicts of interest for this study. background: cytomegalovirus (cmv) is cause of increased morbidity and mortality after transplantation of hematopoietic cells. the pathogenesis of cmv disease or infection is complex with multiple interactions with the immune system, mainly in acute and chronic graft-versus-host disease (gvhd). the aim of this study is to analyze the risk factors for the reactivation of cmv in patients undergoing allogeneic hematopoietic cell transplantation (hct). methods: prospective descriptive study of the risk factors for the reactivation of cmv in the described population. univariate and multivariate analysis of the predisposing factors were performed: donor graft, treatment with corticosteroids, use of antithymoglobin, serologic status, conditioning regimen and the presence of gvhd. results: during the period between august 2014 until january 2017, 71 patients were evaluated. 42.25% (n:30) had reactivation of cmv. average reactivation was 58 days post transplant. both (recipient and the donor) had positive cmv igg in 78.9%. in the univariate analysis, the reactivation of cmv was associated with haploidentical transplantation (p: <0.01), with the use of corticosteroids (p: <0.01) and gvhd (p: <0.01). in the multivariate analysis, the haploidentical transplant maintained its statistical significance in comparison with the related allogeneic transplant (p: 0.0012, or:7.07; ic95%:2.4-20.6) as well as the use of corticosteroids (p: 0.0014, or:9.25; ic95%:2.6-32.4). 100% of patients receiving corticosteroid treatment had grade ii / iii gvhd. the serologicac status, myeloablative conditioning regimen and the use of atg showed no statistically significant association. conclusions: in patients undergoing allogeneic transplantation, were found as risk factor to reactivation, those who received haploidentic transplantation and treatment with corticosteroids. another risk factor that showed greater reactivation was the presence of gvhd. disclosure: nothing to declare methods: a 37 y/o male was referred for allogeneic transplant following 3 cycles of induction therapy for aml with complex karyotype and axsl1 mutation having achieved complete remission following the first cycle of chemotherapy. his first induction cycle was complicated by a perianal myeloid sarcoma which became infected and required surgical drainage and formation of a defunctioning colostomy. results: following 2 allogeneic transplants, the first complicated by secondary graft failure and the second by primary graft failure he presented with two skin lesions, with a third lesion adjacent to his stoma developing shortly after admission. all lesions were erythematous with central necrosis and progressed rapidly in size over 48 hours. biopsy of the skin and para-stomal lesions revealed fungal mycelia, with culture subsequently identifying rhizopus oryzae. initial treatment was with liposomal amphotericin b 5mg/kg/day followed by dose escalation to 10mg/kg/day due to the development of new skin lesions. the patient had been taking posaconazole (tablet) prophylaxis since his first allogeneic transplant and peripheral blood drug levels checked at the time of admission were therapeutic confirming that this was a breakthrough fungal infection. consequently posaconazole was stopped and isavuconazole added to the treatment regimen. surgical assessment was undertaken but surgery was deferred on the basis of high risk due to the extent of the infection and the patient´s profound pancytopenia. the organism was tested for in vitro susceptibilitiy and found to be resistant to posaconazole (mic >16mg/l), with borderline resistance to isavuconazole (mic 16mg/l) and sensitive to amphotericin b (mic 0.25mg/l) (phe mycology reference laboratory, england). isavuconazole was therefore stopped and the patient was managed with liposomal amphotericin b along with daily granulocyte infusions. he underwent a third allogeneic transplant using a different unrelated donor and stable engraftment was achieved. post transplant there was initially an increase in the size of the para-stomal lesion, but no new skin lesions developed. following engraftment he underwent resection of the stomal lesion, with primary closure and re-siting of his stoma. amphotericin b was replaced by isavuconazole prophylaxis on discharge and he continues to make an excellent recovery. conclusions: whilst aspergillus species remain the most common cause of invasive fungal infections in allogeneic transplant patients, other species including the mucorales are seen, and generally associated with poorer outcomes. whilst there are standardised methodologies for susceptibility testing, fungi specific cut offs based on clinical outcomes are only available for a limited number of species/ antifungal agents. in this case, susceptibility testing demonstrated resistance to posaconazole which was consistent with the clinical presentation of invasive infection despite therapeutic levels of posaconazole. it is also worth noting that an estimated 45% of r. oryzae isolates in the uk are resistant to posaconazole. treatment with high dose amphotericin b resulted in improvement in small skin lesions with stabilisation of the larger stomal lesion until count recovery allowed surgical resection. background: total depletion of innate and adaptive immune cell populations occurs after intensive chemotherapy and hematopoietic stem cell transplantation (hsct). both t and b lymphocyte pools are restored slower that myelomonocytic populations. hsct patients are at high risk for bacterial and viral infections at early terms (< 100 days) post-transplant. the reconstitution of the immune system depends on the time required for stem cell recruitment, differentiation, expansion, maturation and release into the bloodstream. restoration terms for myeloid cells after hsct are usually defined as the 1st day with neutrophil count of ≥ 0.5 x 10^9/l with mean recovery terms of 12 to 20 days. high occurrence of cytomegalovirus (cmv) in hsct patients mostly result from reactivation of a latent virus acquired in early childhood. however, delayed immune reconstitution and subsequent infections such as cmv, adenovirus (adv) or herpes 6 (hhv-6) diseases are not unusual and still constitute a major cause of death in peru. methods: peruvian pediatric patients (n=22) diagnosed with aplastic anemia, mds, aml or all underwent a haploidentical hsct performed with the clinimacs device. patients treated were separated in two groups. the group of patients who received viral prophylaxis (ganciclovir) was compared to the group that did not receive any prophylaxis treatment. viral reactivation was confirmed by pcr test twice a week and clinical signs within 15 days after hsct. results: in the group that didn´t received prophylactic treatment, engraftment occurred close to day 10 post haplo-hsct and none of the patients developed gvhd (graft versus host disease). nevertheless, incidences of cmv, hhv-6 and bkv infections before day 15 post haplo-hsct were still high. an overall survival (os) over 65% with an ic 95% was reached at the end of the first year. on the other hand, the group of patients that received prophylaxis with ganciclovir did not developed gvhd and reached the engraftment close to day 10 with a very low viremia incidence after the first month post haplo-hsct. all viral reactivations were caused by cmv and the os was over 91% with an ic 95% at the end of the first year. previous prophylaxis to both the donor and the receptor with ganciclovir (5mg/kg) every 12 hours before and during the conditioning regimen has allowed a better control of viral reactivation. conclusions: the attempts to improve immune function and reduce nonrelapse mortality from infectious complications without increasing gvhd have focused on a partial t cell depleted graft, such as t cell depletion (tcr α/β). this graft retains a large numbers of effector cells, such as tcr γ/δ and natural killer cells. however, delayed immune reconstitution and subsequent infections are a big issue. a novel partial t cell depletion strategy such as depleted naïve t cells (cd45ra+ t cells) could enhance the recovery of immune function after haplo-hsct because donor pathogen memory t cells from the donor are retained. it is necessary to increase the studies and the database to set the scheme of previous prophylaxis to the recipient to contain the viral reactivation and to help a rapid immune reconstitution. disclosure: no conflict of interest is declared information was recovered from the medical records. results: thirty-four patients were included, of them with the following diagnoses: acute leukemia (27), granulocytic chronic leukemia (4), dendritic cell neoplasia (2), aplastic anemia (1). 80% of the patients received a transplant from an identical hla donor and 20% received a haploidentical transplant. mean age's patients was 32 years (19-57). prophylaxis with posaconazole was performed on 44% of the patients with identical hla and 86% on haploidentical group; the rest of the patients received fluconazole. the posaconazole group presented: fever 61%, mucositis gi-ii 94%, gastrointestinal toxicity gi-ii 67% (p= 0.006), hepatic toxicity 11%, kidney toxicity 17%, oral candidiasis 100%. during this period none of the patients presented invasive fungal infection in any group. there were 2 deceases, one on each group and none related to a fungal infection. the overall survival was of the 92% versus 93% on the posaconazole group and the fluconazole group respectively. conclusions: the prophylaxis with posaconazole and fluconazole is effective on the prevention of invasive fungal infection on the first 100 days. the toxicity was similar on both groups. posaconazole can be effective on the prevention of the haploidentical type. is necessary to continue following the patients with infection risk on a long-term period associated with the chronic gvhd. disclosure: none declared lymphoma these results open the question whether allo-sct should still be offered to these patients. methods: we aimed to define the role of allo-sct in refractory or relapsing after two lines de novo or transformed dlbcl patients, and its comparison with zuma-1 car-t cells trial (neelapu et al nejm 2017). we analyse long-term allo-sct results in 40 de novo (n=23) or transformed dlbcl (n=17) out of the 1000 allo-sct performed in our institution between october 1995 to october 2018. results: patients and transplant characteristics are summarized in table 1 . complete response (cr) at 100 days was 67,5% and 83% of them remain in cr at 12 months. with a median follow-up of 46 months, 5-year progression-free survival (pfs) was 54% and 5-year overall survival (os) 48%, with a 3-year transplant-related mortality of 18%. refractoriness at the time of the transplant was associated with a poorer prognosis, with only 2 out of 9 refractory patients being long term survivors (figure 1 ). similar results were reported for zuma-1 trial, with a best response of 55% cr retained in 79% of them at 12 months. with a median follow-up of 15 months, 18-months pfs was 41% and 18-months os 53%. patients characteristics did not differ in our series and zuma-1, except that all the patients in zuma-1 were refractory prior to therapy (table 1) . conclusions: although very few patients with de novo or transformed dlbcl are offering an allo-sct (4% of all allo-sct), this is a curative option in chemosensitive patients and with more mature data and longer follow-up than with car-t therapy; for these reasons, it should still be offer to these poor prognosis patients. moreover, almost all patients have now available donor, better graft-versus-host disease prophylaxis will decrease trm and morbidity, and new therapies will make more patients in sensitive disease before allo-sct. therefore, allo-sct and car-t cells are strategies to be discussed in every young patient with available donor. disclosure: honoraria as advisor or speaker from gilead ( methods: consecutive patients transplanted for hgbl (excluding burkitts lymphoma) between 2007-2017 in the ebmt database were included. data collected included age, sex, pathology subtype (hgbl (including subtypes), tfl, dhl), disease status at sct, conditioning (ma vs beam cam vs flu-mel-cam/atg), engraftment, day 100 outcome, trm, os and pfs and eligibility for emea licensed indication of car-t therapy. results: fifty patients (29m, 21f) with a median age of 47 at diagnosis and 50 at sct were included. the subtypes included hgbl (n=29), tfl (n=11) and dhl (n=10). indications for sct were: primary refractory (n=17), relapse < 12 months after primary treatment (n=12), previous autologous-sct (n=10) and dhl (n=11). the median lines of therapy was 4 (range 1 to 6). conditioning used was cytbi n=21, bu/cy n=1, etop/tbi n=1, flubucy n=1, beamcam n=7, fmc/t, n= 19. all patients engrafted with neutrophil >1.0 10 9 /l at median 15 days and platelets >20 10 9 /l at median 17days. the day 100 mortality was 8% (progressive disease 4%, nrm 4%) with a 5 year os of 56% and mortality due to progressive disease 22% and nrm 16%. disease subtype influenced outcome with an os for primary refractory hgbl, relapsed hgbl, tfl and dhl respectively of 53%, 58%, 57% and 89%. 23 patients were eligible for a licensed car-t product. conclusions: the outcome of these high risk hgbl patients have an acceptable os of 56%, with relapsed disease being the commonest cause of mortality. patients with dhl have a particularly good outcome in this series; recent evidence indicates that some of these patients with a non-immunoglobulin gene associated myc translocation could be managed more conservatively (ash sehn). the outcomes achieved with allogeneic-sct in this series will provide a baseline for outcome assessment with a cart program. disclosure: nothing to declare background: immune checkpoint inhibitors (ici) allow to achieve a durable remission in patients with resistant or refractory (r/r) classical hodgkin lymphoma. however, the information about optimal duration of therapy and the prognosis of the patients after ici cessation is limited (manson, blood 2018). therefore, the optimal role of hematopoietic sct (hsct) in this patient group is not defined. our aim was to determine remission duration in patients who discontinued ici monotherapy after achieving complete remission (cr). methods: this analysis included 20 patients (5 male/15 female) aged 19 to 47 (median 32 years) with r/r classical hodgkin lymphoma who were treated with nivolumab (3 mg/kg every 14 days) and achieved cr. response was assessed by positron-emission tomography/computed tomography (pet/ct) using lyric criteria every 3 month. after nivolumab therapy had been stopped the patients received no other treatment and disease was assessed every 3 months by pet/ct. median follow-up after therapy discontinuation was 20 (10-21) months. results: at the moment of therapy initiation 14 (70%) patients had stage 4 disease, 11 (55%) patients had progressive disease (pd), 4 (20%) patients had stable disease, 3 (15%) patients had partial remission and 2 (10%)complete remission; 12 (60%) patients had b-symptoms and ecog score >1. the median number of previous therapy lines was 5 (3) (4) (5) (6) (7) (8) (9) (10) . before nivolumab initiation high dose chemotherapy with autologous sct was performed in 9 patients (45%) and 8 (40%) received brentuximab vedotin. the median number of nivolumab cycles was 25 (18-30). cr was achieved after median of 6 (6-18) cycles. the median duration of therapy after achievement of cr was 7 (1-15) months. at the time of analysis, all patients were alive, 8 (40%) out of 20 patients relapsed after therapy discontinuation. the median progression-free survival (pfs) for the total group was not achieved. among patients with relapse, the median time before pd was 11 (5-20) months. after relapse all patients were retreated with nivolumab monotherapy or with chemotherapy combination. one patient achieved complete remission; 1 -partial remission; 1 -indeterminate response type 2. other patients are continuing the therapy and their response has not yet been evaluated. conclusions: while complete response was maintained in some patients at median follow up of 20 months after nivolumab therapy cessation, the pfs plateau was not reached. we report that patients with relapse after nivolumab discontinuation sustained sensitivity to nivolumab and achieved a response during retreatment with nivolumab monotherapy or with chemotherapy combination. in patients with unsatisfactory response to nivolumab retreatment, hsct option should be considered. disclosure: nothing to declare high dose chemotherapy with autologous stem cell transplantation in primary central nervous system lymphoma: data from the japan society for hematopoietic cell transplantation (jshct) registry center hospital, tokyo, japan, 4 national cancer institute, bethesda, md, united states, 5 okayama university hospital, okayama, japan, 6 kanazawa medical university, uchinada, japan, 7 kyoto university, kyoto, japan, 8 aomori prefectural central hospital, aomori, japan, 9 yamagata unversity school of medicine, yamagata, japan, 10 tenri hospital, tenri, japan, 11 hiroshima university, hiroshima, japan, 12 japanese data center for hematopoietic cell transplantation, nagoya, japan, 13 nagoya university graduate school of medicine, nagoya, japan, 14 shimane university hospital, izumo, japan background: high-dose chemotherapy (hdt) with autologous stem cell transplantation (asct) has been shown to improve prognosis of patients with central nervous system (cns) lymphoma. whereas the common regimen of hdt for pcnsl in the europe and the us is thiotepa-based regimen, e.g. bcnu-thiotepa, tbc (thiotepa-busulfan-cyclophosphamide), thiotepa-based regimen was only available before discontinuation of thiotepa in 2009 in japan. we report the results of asct for pcnsl from the japan society for hematopoietic cell transplantation (jshct) registry. methods: data from the jshct registry were retrospectively analyzed. 102 patients with pcnsl who received first hdt/asct between 2006 and 2015 were evaluated. distribution differences of clinical characteristics between groups were analyzed with fisher´s exact or mann-whitney u tests. overall survival (os) and progression free survival (pfs) were calculated using kaplan-meier method. two-group analysis of the cumulative incidence of relapse was conducted using the grey test. factors were analyzed in univariable analysis, and all factors with p≤.1 were retained in the multivariable model. all p values were 2 sided, and values were regarded statistically significant if p< .05. results: median age was 54 months (range 20-74) with 15 patients over 64 years of age; 38 males and 64 females. ecog-performance status (ps) at diagnosis was better (ps0-1) in 86 patients and poor (ps2-4) in 16 patients. serum lactate dehydrogenase (ldh) levels at diagnosis were elevated in 17 patients. karnofsky ps and cerebrospinal fluid (csf) protein concentration at diagnosis were not collected in the registry. 71 patients were in complete remission (cr), 21 patients were in partial response (pr), and 7 patients were stable disease (sd) or progressive disease (pd) at the time of hdt/asct. after hdt/asct, additional 20 patients achieved cr. with median follow-up period of 44 months, the 5-year os and pfs were 54.9% and 38.4%, respectively. the was no significant difference in os and pfs between upfront and salvage hdt/asct. since thiotepa, a key agent in hdt/asct for pcnsl, has been unavailable after the discontinuation in japan, the hdt regimens used were not uniform. thiotepa-containing hdt was received by 16 out of 32 patients before 2010, but by 2 out of 70 patients after 2011. thiotepa-containing hdt showed improved pfs (p=.019), lower relapse (p=.042) and a trend toward a survival benefit. in the multivariate analysis, non-complete remission at hdt/asct was an independent predictor for os (hr=2.40, 95%ci:1.25-4.58, p=.008) and thiotepacontaining hdt remained significant for pfs (hr=0.42, 95%ci:0.19-0.95, p=.038). [[p481 image] 1. os(a),pfs(b) in all patients (n=102) and cumulative incidence of relapse in cr patients (c; n=91)] conclusions: our results confirm the activity of thiotepacontaining regimen for hdt/asct in pcnsl patients. currently a pharmaceutical company re-develops thiotepa for new approval of hdt/asct in pediatric solid cancer and adult lymphoma in japan (japiccti-163433). further evaluation with the thiotepa by prospective clinical trials is warranted. disclosure background: t-cell non-hodgkin lymphomas (t-nhl) are rare diseases and they are associated with worse prognosis when compared to their b-cell counterparts. allogeneic stem cell transplantation (allo-sct) may have a curative potential for these patients due to the graft versus lymphoma effect. however, data is limited on the efficacy of allo-sct for these diseases. methods: we identified 53 patients (32% females; median age: 43 y; range,4-67) with t-nhl that underwent allo-sct at university hospital eppendorf between 1992 and 2017. twenty-one patients (underwent allo-sct from a matched sibling donor (msd) and 32 (60%) from a matched unrelated donor (mud). sixteen patients had ptcl (30%), n=8 (15%) anaplastic large-cell lymphoma (alcl), n=8 (15%) angioimmunoblastic large cell lymphoma, n=8 (15%) adult t-cell leukemia/lymphoma, n=5 (9%) hepatosplenic gamma/delta t-cell lymphoma, n=4 (8%) enteropathy associated t-cell lymphoma, n=2 (4%) tcell-prolymphocytic leukemia, and n=1 (2%) each extranodal t/nk-cell lymphoma, cutaneous t-cell lymphoma as underlying diagnosis. the median ann arbour stage at diagnosis was 4 (range, 1-4). ten patients (19%) had bone marrow involvement at diagnosis. all patients were heavily pretreated, 17 (32%) patients relapsed post autologous stem cell transplant (apsct) and one patient post allo-sct. fifteen patients (28%) were transplanted in complete remission (cr) (10 in 1 st cr, 2 in 2 nd cr), n=13 (25%) in partial remission (pr), and n=21 (40%) with advanced disease. most of the patients received myeloablative conditioning (91%). thirty-eight (72%) patients received total body irradiation based regimens and 15 (28%) received chemotherapy based regimens. twenty patients (38%) received anti-t-lymphocyte globulin (atlg neovii), and most patients (87%) received g-csf mobilized peripheral stem cells. results: overall, 49 patients (92%) had neutrophil engraftment (median days: 13; range,9-36) . at day 100, the cumulative incidences of grade ii-iv and grade iii-iv acute gvhd were 42% and 15%, respectively. after a median follow up of 12 months (range, 1-171) the cumulative incidences of chronic gvhd was 12% distributed evenly between limited and extensive. twenty nine patients (55%) achieved cr after allo-sct. median overall survival (os) and disease free (pfs) survival were 44 months and 12 months respectively. the 3 year os and pfs were 50% and 43% respectively. fourteen (26%; 95% ci [0.15-0.40]) deaths were due to non relapse mortality (nrm) and 15 patients (28%; 95% ci [0.17-0.42]) died due to disease progression. patients with a male donor had improved os compared to those with a female donor (3 year os male 56%, female 34%; p=0.038). patient gender, disease subtype, bone marrow involvement, type of allo-sct, donor, patient cmv status, abo incompatibility, disease stage at diagnosis, previous transplant, disease status at transplant, conditioning regimen, atg and stem cell source had no effect on os, pfs, nrm, and post transplant complications. conclusions: acknowledging the retrospective nature, our study shows that allo-sct induces high rates of complete remission, and may have a curative potential even in diseases relapsing post asct. however our findings need to be confirmed in larger prospective studies. disclosure: no funding, no conflict of interest p483 abstract already published. at-home autologous stem cell transplantation in lymphoma patients: clinical impact of non-g-csf administration post-transplant background: severe neutropenia remains the main cause of morbidity and mortality after autologous stem cell transplantation (asct). g-csf administration after asct is a common practice, performed to reduce the duration of neutropenia and its complications. in a previous work in patients with multiple myeloma managed at home after asct, we did not observe a deleterious clinical impact in those patients that did not receive g-csf post-transplant (martinez-cibrian n. et al, bmt 2016) . despite the fact that lymphoma patients receive a more intensive conditioning regimen that multiple myeloma patients, we hypothesized that the use of g-csf in lymphoma patients managed at home during the aplasia phase of asct does not provide a significant clinical benefit. methods: 93 lymphoma patients were managed at-home since day +1 of asct. between february 2010 and july 2016, 68 patients received at-home g-csf 5 μg/kg per day since day +7 until their anc reached 1x10 9 /l (g-csf group) and, since august 2016, 25 patients did not receive g-csf (non-g-csf group). all patients were conditioned with beam and received prophylaxis with a quinolone, fluconazole, aerosolized pentamidine and low-dose acyclovir (hvs+). in all cases we added primary prophylaxis with piperacillin-tazobactam 4.5 g/8h i.v., using a portable intermittent infusion pump (iip), from an absolute neutrophil count (anc) < 0.5x10 9 /l until the first day of fever or until attaining an anc of 1x10 9 /l. first-line therapy at home of neutropenic fever (nf) was refrigerated meropenem 1 g/8h i.v using a portable iip. fever was an indication of immediate visit to the hospital, and those patients presenting with focal infection or signs of severe sepsis were admitted. other indications for readmission were: willingness of the patient or caregiver; uncontrolled nausea, vomiting or diarrhea and mucositis requiring total parenteral nutrition or i.v. morphics. results: the main characteristics of the patients are shown in table 1. there were no differences between groups with respect to gender, diagnosis, stage of disease, comorbidity index (hct-ci), source of stem cells (peripheral blood) and cd34 + cell dose infused. the median (range) age (years) was 44 (19-71) in g-csf group and 52 in non-g-csf group (p=0.03). duration of neutropenia less than 0.5 x10 9 /l was significantly longer in non-g-csf group, with a median of 11 days (range 6-19), compared with 8 (range 6-17) in g-csf group (p < 0.0001). severe neutropenia, less than 0.1x10 9 /l, was also longer in the non-g-csf group (8 days (4-11) vs. 7 (5-13); p=0.014). no differences were observed in the time to platelet engraftment. g-csf post-transplant avoidance did not influence the incidence of neutropenic fever, the first day and duration of fever, the incidence and severity of oral mucositis, bacterial infections documented and number of readmissions. the median duration of the whole procedure at-home was 1 day shorter in the g-csf group (14 vs. 15 days; p=0.12). conclusions: the policy of not administering g-csf post-asct in our home-based program for lymphoma patients, that include intensive bacterial prophylaxis, did not have a deleterious impact on the main results reviewed, which suggests that elimination of its use can be achieved. disclosure the aim of this study was to analyze the spanish experience with patients diagnosed of nhl who received haplosct with pt-cy. methods: sixty patients who received haplosct with pt-cy in 17 spanish centers from 2012 to 2016 were analyzed. patients were followed-up until 2017. gvhd prophylaxis consisted in cyclophosphamide 50 mg/kg/d on days +3 and +4, and mmf and a calcineurin inhibitor from day +5. results: patients' characteristics are summarized on table 1 . median age of patients was 50, 75% male, and diagnosed from t cell lymphoma (37%). most of them didn´t achieve complete response prior to transplant (55%), but only 15% with active disease. up to 63% of patients had received previous transplant, from which 5% was an allogeneic transplantation. source of stem cells was mainly peripheral blood (90%), and reduced intensity conditioning was the preferred (82%) regimen. donors were 43% siblings (26), 38% offspring (23), and 18% parents (11). median neutrophil and platelet engraftment was 18 (16-21) and 28 (20-42) days, respectively. acute gvhd grade ii-iv rate was 55%, with only 6 patients developing grade iii-iv (10%). chronic gvhd rate was 17%, and only in 3 (5%) was extensive. median follow-up was 14 months. the 2-year overall survival and event free survival was 43% and 39%, respectively. the 2-year cumulative incidence of relapse was 14% and 2-year cumulative incidence of nrm was 22%. conclusions: relapsed/refractory nhl are aggressive entities with a fatal course in a short period of time. haplosct with pt-cy permit a new treatment option among these patients, with acceptable outcomes. more studies are needed with a larger cohort of patients and longer follow-up to confirm these results. disclosure: nothing to disclose. higher suv at pre-transplant and day 100 posttransplant pet scan both independently predict inferior survival in patients with diffuse large b cell lymphoma background: autologous stem cell transplant (auto-hct) can cure some patients with relapsed diffuse large b-cell lymphoma (dlbcl) but relapse occurs in about 50% of patients. while our center and others utilize routine surveillance imaging post-transplant, the utility in this setting is unclear. imaging is costly and exposes patients to radiation. novel interventions are now available for patients relapsing after auto-hct making early disease recognition crucial to intervene prior to clinical progression. hence, we studied impact of post-auto-hct surveillance (18)f-fdg-pet ct at day 100 on transplant outcomes. methods: we analyzed a cohort of 131 consecutive auto-hct recipients with relapsed/refractory dlbcl who then underwent pre-transplant pet/ct and surveillance pet ct at day 100 (interquartile range (iqr): 97-103 days) post-hct at the university of minnesota medical center. univariate analysis was performed to analyze pet parameters including deauville score (d), standardized uptake values (suv), total lesion glycolysis (tlg) and total metabolic tumor volume (tmtv) as predictors of relapse and survival after auto-hct. in addition, we assessed outcomes of patients with clinically versus radiographically detected relapsed dlbcl after auto-hct. other pre-hct factors analyzed included age, gender, conditioning regimen, performance status, consolidation radiation therapy, tmtv, suv, tlg. results: five-year cumulative incidence of relapse after auto-hct was 50% (95%ci 39 to 59) and overall survival (os) was 51% (95% ci 41 to 63). twelve (9%) relapsed prior to day 100. d-score for 91 patients with d100 pet/ct were d1 (22%), d2 (55%), d3 (0%), d4 (10%), d5 (13%) with median survival in years for d1, d2, d4 and d5 of 6.0, 6.8, 4.7, and 1.2, respectively. mean suv varied from 1.53 (d1) to 17.9 (d5). suv was predictive of relapse and os. risk of relapse increased with doubling of suv; 2-fold higher suv increased hr by 1.77 (95%ci 1.34-2.33; p= 0.01). mortality increased with doubling of suv in both pre-hct (2-fold increase in suv associated with hr 1.46 [95% ci 1.1 to 1.8]; p=0.01) as well as post-hct pet (hr 1.7 [95% ci 1.3 to 2.3]; p=0) irrespective of the bulk of tumor. in addition, risk of death was 4 times higher in d5 patients relative to d1 (hr 4.10 [95% ci =1.56 to 10.77]; p≤0.01). patients with d5 (n=12) had higher tmtv (137 cm 3 ) compared to d4 (n=9, tmtv 12.5 cm 3 ). the hazard ratio for death following relapse was 2-fold higher (hr 1.8 [95% ci 0.9 to 3.4]; p=0.08) if relapse was detected clinically versus only radiographically over a median follow-up time period of 3.3 years. other pretransplant patient and disease characteristics did not significantly influenced the outcomes. conclusions: in patients with r/r dlbcl undergoing auto-hct, surveillance pet/ct at day 100 identified patients with poor survival~1 year. higher suv in both pre-transplant as well as post-hct pet was predictive of increased mortality. these patients may benefit from novel treatments. [ there are concerns about the risks of nivolumab treatment before and after allo-hsct, due to the risk of heavy gvhd, thus the place of immune checkpoints inhibitors is not yet defined. this report include analysis of our center experience of nivolumab treatment in patients with r/r hl before and after allohsct. methods: we retrospectively evaluated the results of allohsct in 86 patients with r/r chl who had undergone transplant from 2002 to 2018. the analysis included patients received the flube conditioning and ptcy gvhd prophylaxis. in group a patients (n=20) received bridge therapy with nivоlumab and in group b patients (n=34) received bridge therapy with brentuximab vedotin or chemotherapy-based bridges. time from the last nivolumab administration to allohsct was at least 2 months. results: at the time of analysis, median follow-up was 12 (1-20) months for group a, and 15 (1-64) months for group b. there was no difference in two-year os (p=0,39) with significantly better efs (p=0,025) for group a versus group b: 95% and 95% vs 85,3% and 62% respectively. relapse incidence was 0% for group a versus 26,5% in group b (p=0,025). cumulative incidence of non-relapse mortality at 2 years was 5,0% and 13,8% in group a and group b, respectively (p=0,631). there was no difference in grade ii-iv (44% vs 27%, p=0.23) and grade iii-iv (22% vs 13%, p=0.3) agvhd, as well as extensive chronic gvhd (21% vs 28%, p=0,83) in groups a and b, respectively. ten patients with relapse after allohsct were treated with different doses (0,5-3 mg/kg) of nivolumab in cic725 center. at the median follow up of 16 mo (0,6-28) all patients remain alive. the objective response to therapy was assessed in 7 patients noted in all patients (100%), disregard the dose of the nivolumab: cr in 29%, and pr in 71%. the response was lost in four patients, which required nivolumab retreatment. none of the patients developed gvhd after nivolumab administration. in this analysis, there was also no correlation between dose of nivolumab and incidence and severity of adverse events. conclusions: allohsct in combination with immune checkpoints inhibitors is a good option for patients with r/r chl. consideration for immune-mediated toxicities and the potential for increased graft-versus-host disease remain important. early data suggest that nivolumab may be an efficient therapy in patients with r/r chl relapse after allo-hsct. further research needed. disclosure: the authors declare no conflicts of interest. background: transformation to diffuse large b-cell lymphoma (dlbcl) is considered to be one of the most unfavourable events of lymphoma natural history with poorer outcome as compared to de novo dlbcl (alonso-álvarez et al, bjh 2017). in patients suitable for salvage therapy, hematopoietic stem-cell transplantation (sct) could be an option, although its role is not well stablished. we analyse indication and outcome after transplant in transformed dlbcl at a single reference transplant unit. methods: out of 2565 total of transplants performed at our unit between 1995 and 2018 -1564 autologous and 1001 allogeneic-51 were dlbcl transformed from an indolent nhl. of them, 36 received an autologous sct (asct) and 15 an allogeneic sct (allo-sct). results: median age was 60 years old (range 40-69) and 52 (range 35-65) for patients receiving asct and allo-sct, respectively. all asct received beam as a conditioning regimen and most of the patients in the allo-sct group received a fludarabine/melphalan combination (73%). gvhd prophylaxis consisted on tacrolimus/sirolimus combination in 87% and calcioneurin plus methotrexate in 13%. regarding transplant disease status, 28 (78%) of the asct patients were transplanted in complete response (cr). in the allo-sct group, 11 (73%) patients had received three or more treatment lines before transplant and 13 patients (87%) had received a previous asct, being 11 (73%) in cr, 3 in partial response (pr) and 1 in progressive disease. transplant related mortality (trm) was 5.6% in the asct and 27% in the allo-sct group. overall survival (os) and progression-free-survival (pfs) at 25 months were 94% (os), 76% (pfs) for patients receiving asct and 63% (os) and 56% (pfs) for allo-sct. with a median follow up of 57 months for patients receiving an asct, 21 (58%) remain in cr. in the allo-sct group median follow up is 24 months for the whole group and 50 months for alive patients; 9 patients are alive and disease free and 6 have died, 4 due to trm (28%). regarding progression, 12 (33%) have progressed after autologous transplant and 2 after allo-sct. conclusions: indication for hematopoietic sct in transformed dlbcl is stablished in few patients. only 2% of the patients in our transplant unit receive a transplant due to transformed lymphoma, corresponding to a 2.3% of autologous activity and 1.5% of allogeneic activity. according to our results transplant should be considered a curative option. most of our patients were transplanted in cr, so new agents trying to reach best response before transplant should be considered. [[p488 image] 1. eva konirova 1 , antonin vitek 2 , marta krejci 3 , edgar faber 4 , katerina steinerova 5 , david belada 6 , jan novak 7 , juraj duras 8 , petr sedlacek 9 , veronika valkova 2 , andrea janikova 3 , ludek raida 4 , pavel jindra 5 , pavel zak 6 , tomas kozak 10 , marie trnkova 1 , michal karas 5 , marek trneny 1 management. however, differences in patient's characteristics as well as frequency of hsct indication in different lymphoma subtypes have been observed in the last decade. the aim of this study was retrospective analysis of hsct for lymphomas in czech republic. methods: data of adult patients transplanted between years 1993-2016 were retrospectively analyzed using ebmt database. results: between 1993 and 2016, 2816 autologous hsct (asct) were performed in 2651 patients (1511 men, 57%) with different lymphoma subtypes. the median age was 49 years (range 18-75). out of these, 2078 (78%) were patients with non-hodgkin lymphoma (nhl), 569 (21%) with hodgkin lymphoma (hl). the nhl group comprised of diffuse large b-cell lymphoma (dlbcl, 36%), follicular lymphoma (fl, 18%), mantle cell lymphoma (mcl, 16%) and t-nhl (9%). the frequency of asct in lymphomas increased from 1993 to 2000 and has been constant since 2000 (120-130 transplants per year). differences in frequency of asct were observed among lymphoma subtypes -decreasing numbers of dlbcl and fl and increasing numbers of t-nhl and mcl, with asct as part of the induction therapy. between 1996 and 2016 a total of 329 allogeneic hsct (allosct) were performed in 319 patients (200 men, 63%). median age was 46 years (range 19-66). out of these 257 (81%) were patients with nhl, 61 (18%) hl. the most common nhl subtypes were fl (27%), mcl (22%), t-nhl (22%) and dlbcl (16%). in the last 10 years the number of allosct for lymphoma is fluctuating around 20 per year. the median age at asct was significantly higher in the years 2010-2016 vs 1993-2000 [54.5 (18.1-74.8) vs. 40.9 (18.6-72.5), p < 0.0001, fig 1] , while the increase at allosct [46.5 (21.3-65.5) vs 41.6(20.3-64.5)] did not reach statistical significance (p=0.07). with median follow up for allosct, 5 y probability os for patient transplanted in the later period 2010-2016 was in relapsed dlbcl 25.7%, in fl 77.4%, in hl 51.0% and in mcl 45.7%, 5 y os for asct as part of first line therapy in the same period was in mcl 78.1 % and in t-nhl 48.2%. os was significantly better in all patients who underwent asct in the years 2010-2016 vs 1993-2000 (70.% vs. 57 .5%, p < 0.0001) and there was a trend towards better os in patients after allosct (with 44.7% vs 23.5%, p=0.1084) (fig 2) . conclusions: hsct remains important treatment modality for lymphomas in the era of targeted antibody and molecular therapy and we can transplant older patients due to better supportive treatment. acknowledgment: progress q28-9 uk from the czech ministry of education youth and sports disclosure: nothing to declare background: disease chemosensitivity to salvage treatment has been proven to be a major predictive factor for a favorable outcome after autologous stem cell transplantation (asct) for patients with refractory lymphomas. therefore the importance of effective and safe salvageregimens is indisputable. methods: we retrospectively compared the outcomes in terms of safety and efficacy, in 67 (hl:36, nhl:31) patients, with a median age of 34.5(16-75) years, who received as 1 st salvage either dicep [cyclophoshamide (1750 mg/m 2 ), etoposide (350mg/m 2 ), cisplatin (35 mg/ m 2 ), days 1-3, (n=23)] or the widely used regimen eshap (n=44). rituximab was additionally given to all cd-20 positive lymphoma patients. the statistical analysis based on the independent t-test, kaplan meir method and logrank test. results: the reason for salvage treatment was primary induction failure (pif, n=34), early relapse (< 12 months post induction-remission therapy n=14) and late relapsed disease (n=19). more specifically, 19/23 patients (83%) in the dicep-group, and 29/44 patients (65%) in the eshapgroup were assessed with pif or early relapsed disease, however this difference was not statistically significant. both regimens were well tolerated and no major organ toxicities were noticed. eleven patients (48%) from the dicep-group, while only 4(10%) from the eshap-group developed febrile infections. all patients were successfully managed with the appropriate treatment and only one, from the eshap-group, required for short period admission to the intensive care unit. after 1 cycle of dicep and 2 cycles of eshap the disease response was re-assessed by pet/ct scan. the overall response rate (>50% tumor reduction) was significantly superior for the dicep-regimen, reaching 92% (21/23 patients) vs. 64% (27/44 patients) for eshapregimen (p=0,003). eleven patients (48%) from the dicep-group and 14(30%) from the eshap-group achieved complete metabolic remission according to pet/ ct criteria (p=ns). the median hospitalization period was 20(5-25) days for the dicep-group compared to 10(10-19) days for the eshap-group. however, for the eshapgroup, an additional median of 20(6-33) hospitalization days were required, since 12 of the non-responders patients received a 2 nd salvage before asct. the mobilization and stem cell collection was successful for both groups, though significant higher number of cd34+ cells were collected in the dicep-group (17.2x10 3 /kg vs. 5.4x10 3 /kg, p=0,001). all but two patients (due to refractory disease) underwent asct. noticeably, the median period from 1 st salvage treatment to asct was significantly shorter for the dicepgroup (64 vs. 128 days, p=0,013), apparently because 12 non-responders patients from eshap-group treated with a 2 nd salvage. the 3-years overall and progression free survival were similar for dicep-and eshap-groups (95% vs. 88% and 70% vs 80% respectively). two heavily pretreated patients from the eshap-group developed secondary myelodysplastic syndrome post asct conclusions: in our series of patients both regimens proved to be safe. interestingly, despite the fact that more patients in dicep-group had poor risk disease the dicepregiment was significantly more effective, resulting thus in an earlier asct, less exposure to chemotherapeutic agents, that might led in less long-term toxicity. nevertheless, prospective trials with large series of patients are needed to define the role of dicep in the salvage treatment setting. disclosure: no conflict of interest background: although autologous hematopoietic stem cell transplantation (auto-hsct) is one of the best curative strategies for patients with chemosensitive t-cell lymphoma, major limitation remains a tumor contaminated graft-related relapse or residual disease after chemotherapy. several purging methods were introduced in auto-hsct for these limitations, however there are few studies of ex vivo purging of the autograft in lymphomas, especially t-cell lymphoma. therefore, we retrospectively analyzed 59 consecutive t-cell lymphoma patients receiving auto-hsct with/without ex vivo purging. methods: among them, 33 patients underwent autograft manipulation with ex vivo purging by cd34+ cells selection using a clinimacs device. results: with median follow-up duration of 42 months (range, 6-121 months), 3-year overall survival (os; 73.8% vs. 49.0%, p=0.017) and 3-year progression-free survival (pfs; 75.8% vs. 52.4%, p=0.039) in a purged and unpurged group, respectively. transplant-related mortality was observed in both groups (2 patients of a purged group and 1 patient of an unpurged group). neutrophil (10 vs. 9 days, p=0.240) and platelet (30 vs. 24 days, p=0.055) recovery were similar in both group and there was no engraftment failure. on subgroup analysis according to upfront and salvage auto-hsct, while survival outcomes were improved by stem cell purging in the upfront auto-hsct (os with p=0.039 and pfs with p=0.047), there were no different survival outcomes in salvage auto-hsct. the unmanageable late-infectious complications were few in both groups except for predominantly cytomegalovirus reactivation in a purged group (3 vs. 1 patient). conclusions: although cohort was a small number, ex vivo graft-purging method was feasible and safe in t-cell lymphomas. and this purging strategy observed the more favorable survival outcomes in the upfront auto-hsct than salvage setting. therefore, further randomized studies are needed to determine the firm efficacy of cd34+ purification with the large number of patients in auto-hsct for t cell-lymphomas. disclosure: nothing to declare nivolumab-based regimens in relapsed or refractory non hodgkin lymphomas: the role of hematopoietic stem cells transplantation methods: we analyzed data of 18 patients with r/r nhl, among them n11 with diffuse large b-cell lymphoma (dlbcl), n5 with primary mediastinal b-cell lymphoma (pmbcl), n1 with gray zone lymphoma (gzl) and n1 with gamma-delta peripheral t-cell lymphoma (ptcl), who received nivolumab-based regimens. the median age was 37 years (range, 18 -64 years). most of the patients n14 (78%) had a primary chemoresistant disease, the rest patients n4 (22%) had a relapse. the median of lines of prior therapy was 3 lines (range, 2-6 lines). all sixteen patients with dlbcl and pmbcl received 1 -3 cycles of nivolumab in combination with bendamustine, gemcitabine and rituximab (begern). the patient with gzl received 5 cycles of nivolumab in combination with brentuximab vedotin and epoch. and the patient with ptcl received 10 cycles of nivolumab monotherapy. results: at median follow up 8 months (3-16) objective response (or) after nivolumab-based regimens was noted in n10 (56%) patients, complete response (cr) and partial response (pr) in n9 (50%) and n1 (6%) patients, respectively. cr observed in n4 patients with dlbcl, n3 with pmbcl, n1 with gzl, n1 with ptcl. and pr observed in 1 patient with dlbcl. two responding patients with dlbcl underwent auto-hsct. and four responding patients (n1 dlbcl, n1 pmbcl, n1 gzl, n1 ptcl) received allogeneic hematopoietic stem cells transplantation (allo-hsct). the median duration of response for all n10 patients with or was 5 (range: 3-16 +) months. among n4 patients who achieved or without hsct, only n1 remain in cr. two patients who received auto-hsct had a relapse. one patient with dlbcl improved the response after allo-hsct from pr to cr, and all four patients with allo-hsct remain in cr. the probabilities of 1-year os and pfs rates were 49% and 31%, respectively. conclusions: nivolumab-based regimens can lead to an objective response in 56% patients with r/r nhl. however, the durability of response to therapy is not long. nivolumab-based regimens can be used as bridge to allo-hsct disclosure: there are no conflicts of interest to disclose background: patients with aggressive non-hodgkin lymphoma (nhl) who relapse after autologous stem cell transplantation have a dismal outcome and could benefit from radiotherapy, allogeneic stem cell transplantation or experimental treatments. systemic inflammatory parameters at diagnosis have demonstrated to be useful to predict lymphoma evolution. methods: we conducted a retrospective review of patients with aggressive nhl who underwent autologous stem cell transplantation (astc) to evaluate the relationship between ldh, β2-microglobulin, inflammatory parameters (lymphocyte (alc) and monocyte count (amc), ferritin or c-reactive protein) and imaging techniques before and on day +100 post-astc and progression free survival (pfs), as well as the role of residual disease directed radiotherapy (rt). results: one hundred and sixty patients with aggressive nhl received asct as consolidation treatment in our center between 2000 and 2017. the most common diagnosis was diffuse large b-cell lymphoma (dlbcl). one hundred and nine patients received upfront asct for high risk dlbcl (defined as age-adjusted ipi 2-3)(n=28) or for having received two or more lines to obtain first complete remission (n=28), for t-cell lymphoma (n=27) and for mantle cell lymphoma (n=26). the rest was performed in relapsed lymphomas. forty-seven patients (29%) relapsed and pfs was 110 months. pretransplant response was evaluated with ct scan in 66 patients (11 of this with partial remission (ct-pr) and 94 patients were evaluated with fdgpet/ct (27 were pretransplant positive (pet1); of these, 18 patients maintained positivity at day 100 after astc (pet2). pfs in patients with ct-pr was 46 months, in pet1 positive ones 99 months and in pet2 positive ones 21 months. univariate analysis showed pet2 positivity as the most accurate predictor of relapse (hr 3,13, p=0,004) followed by amc at day +100 (hr 1,002, p=0,13), albumin at day +100 (hr 0,93, p=0,03), ldh at day +100 (hr 1,002, p< 0,001) and pretransplant alc/amc ratio (hr 1,068 p=0,008). multivariate analysis only demonstrated an association with pet2 positivity (hr 7,78) p< 001 and ldh in day +100 (hr 1,008) p=0,003 with pfs. five and ten years overall survival were 61% and 37% in pet2 negative patients vs 25 and 5% in pet2 positive ones (p< 0,01). eight out of 19 patients with pet2 positivity did not relapse. salvage radiation therapy was used in 7 patients with positive residual mass and 5 of them did not relapse. two patients relapsed: one patient had residual mass and another had remote affectation from primary site and could be considered as progression before day +100. conclusions: post asct fdgpet/ ct is superior to conventional ct in predicting outcome in aggressive lymphoma after astc. pre and post asct systemic inflammatory parameters didn't help to improve the relapse risk prediction. addition of consolidative rt after astc has demonstrated improvement in pfs in patients with pet positivity. it would be neccesary to develop randomized trials to assess the role of rt in residual disease in advanced aggressive nhl with insufficient response to systemic treatment with pet response evaluation. disclosure: nothing to declare long term outcome of patients with lymphoid malignancy who underwent high dose chemotherapy followed autologous hematopoietic cell transplantation at a single institution over 20 years joanna romejko-jarosinska 1 , ewa paszkiewicz-kozik 1 , lukasz targonski 1 , lidia popławska 1 , jan walewski 1 background: high dose chemotherapy (hdt) and autologous hematopoietic cell transplantation (auto-hct) is a standard of care for relapsed/refractory lymphoma patients (pts) or it is used as a consolidation for myeloma and high risk lymphoma patients in first line treatment. we retrospectively evaluated long-term outcome including late effects and risk factors in patients with lymphoid malignancy who underwent auto-hct. methods: we collected data from 926 consecutive patients with hodgkin lymphoma (hl) (n=326), aggressive b lymphoma (dlbcl) (n=186), myeloma (n=199), indolent lymphoma (n=30), mantle cell lymphoma (n=122) and peripheral t cell lymphoma (n=63) who underwent auto-hct at our institution between 1997 and 2017. at transplant median (range) age was 46 (17-71) years, clinical stage iii/iv was found in 499 of lymphoma pts, complete remission, partial remission and stable/ progressive disease occurred in 462(50%), 397 (43%), 67 (7%) pts, respectively. beam regimen was used in 549 pts (59%), mel200 in 200 pts (23%) and other myeloablative regimens in 177 pts (18%). results 62%) ], respectively. partial remission or stable disease at transplant, clinical stage iii or iv, and age more than 60, were identified as risk factors associated with inferior os and pfs in univariate and multivariate analysis. histopathologic diagnosis was not a risk factor for os and pfs (p=ns). the outcome of patients who underwent auto-hct between 1997-2003 was inferior to the outcome of patients treated in 2004-2010 or 2011-2017. 5year os was 53%, 68%, 73%(p< 0.001) and 5 year pfs was 43%, 54%, 55% (p< 0.01), respectively. we recorded 37 (4%) cases of second primary cancer (18 solid tumors and 19 hematologic cancers). acute cardiotoxicity occurred in 5 patients from 1 to 15 years after transplant, and required heart transplant in 2 patients. 326 patients (35%) died. the main causes of death were progressive disease in 271 pts (89%), second primary malignancy in 22 pts (6.7%) treatment related mortality was 0.8% (8 pts), and mortality within 100 days was 22 (2,4%). [[p494 image] 1. pfs and os in patients underwent hdt and auto-hct 1997 -2003 , 2004 -2010 , 2011 -2017 conclusions: more than 45% of patients who underwent hdt and auto-hct had long term survival without progressive disease. older age, non-complete remission at transplant, advanced stage are associated with poor outcome. patients recently transplanted had a better outcome than patients transplanted before 2004. disclosure: nothing to declare outcomes after haploidentical and matched related hsct in lymphoma do not differ significantly: a single center study nadira durakovic 1,2 , zinaida perić 1,2 , lana desnica 2 , ranka serventi-seiwerth 2 , sandra bašić kinda 2 , ivo radman-livaja 2 , alen ostojić 2 , ante vulić 2 , dražen pulanić 1,2 , pavle rončević 2 , zorana grubić 2 , igor aurer 1,2 , radovan vrhovac 1,2 1 university of zagreb, school of medicine, internal medicine, zagreb, croatia, 2 uhc zagreb, zagreb, croatia background: allogeneic hsct still offers patients with relapsed/refractory lymphoma the best chance of long-term survival. in most such patients timing of hsct is crucial, therefore a related donor is preferred. we analyzed acute and chronic gvhd incidence, relapse and overall survival, but also time to immunosuppression (is) discontinuation and hematopoietic recovery comparing transplantation using haploidentical (haplo) and matched related donors (mrd) in single center in this indication. methods: in the time period between 5/2011 and 5/2018 at uhc zagreb, croatia, 10 mrd and 13 haplo transplantations in lymphoma were done, 15 for hodgkin and 8 for nhl. all patients transplanted from haploidentical donors received ptcy. data were computed using the r package. the probability of gvhd was calculated using the cumulative incidence method and subgroups were compared using the gray test. results: median age was 38 (19-62) years; 36 (19-62) in haplo and 41 (25-56) in mrd group. four patients were in pr and 9 in cr in haplo group, while in mrd group 5 patients were in cr and 5 in pr. in haplo group 12 patients (92%) received bone marrow (bm) and only 1 (8%) peripheral blood stem cells (pbsc). in mrd group all patients received pbsc. all patients in haplo group received nma ("baltimore") conditioning with ptcy while in mrd group 9 patients (90%) received flu-bu2atg, and only one received flutbi as conditioning protocol. in haplo group 85% patients were previously treated with autologus transplantation, 80% in mrd group. there was no significant difference in time to is discontinuation, 149 and 155 days in haplo and mrd group, respectively. patients after haplo recovered slower, recovering anc after 22.6 days (95% ci, 17.7-27.4) and 16.5 (95% ci, 13.9-19) (p=0.04) and recovering platelets after 34.6 days (95% ci, 15.5-53.7) and 10.8 (95% ci, 9.2-12.4) (p< 0.01) in haplo and mrd group. with a median follow up of 469 days, overall survival was 83% (95% ci, 63-100) in haplo and 80% (95% ci, 59-100) in mrd group. trm was 8% in haplo and 20% in mrd group. cumulative incidence of agvhd ii-iv was 31% (95% ci, 9-56) and 32% (95% ci, in haplo and mrd group, respectively (p=0.84). cumulative incidence of cgvhd requiring treatment was 22% (95% ci, and mrd 20% (95% ci, 0-62) in haplo and mrd group, respectively (p=0.46). all cases of cgvhd developed after dli. cumulative incidence of relapse was 32% (95% ci, 9-58) and 38% (95% ci, 7-72) for haplo and mrd group, respectively (p=0.77). conclusions: we found no significant difference in overall survival, relapse incidence, agvhd and cgvhd incidence between these two groups. hematopoietic recovery was slower after haploidentical transplantation, but it did not influence trm as it was higher after mrd. even though limited in number, this data contribute to the growing body of evidence that use of haploidentical donors, particularly in lymphoma setting, is as worthy as using matched related donors and should be at least second choice in donor selection, and in older patients (with older donors) probably the first one. disclosure: nothing to declare. adjuvant involved field radiotherapy post autologous stem cell transplantation for refractory/relapsed lymphomas results in favorable outcome with low toxicity: a single center experience background: involved field radiotherapy (ifrt) to previous bulky or localized residual disease, is a widely used treatment approach to minimize the risk of relapse post autologous stem cell transplantation (asct). however, the proper time for irradiation treatment remains controversial. adjuvant ifrt (adj-ifrt) in pre-asct period could cause undesirable toxicity which might delays or even cancel the asct resulting in increased risk of relapse, or could affect the marrow environmental and marrow niche resulting thus in impaired engraftment. on the other hand, the ajd-iftr in the early post-asct period, upon marrow recovery, offers a potential advantage by delivering irradiation after sufficient disease response, without affecting the engraftment. in this retrospective study we evaluated the safety and efficacy of the ifrt as adjuvant treatment in patients who had previously treated with asct for relapsed or refractory lymphomas. methods: twenty-three patients (hodgkin=14, non-hodgkin=9), aged of 34(16-76) years, underwent asct, for primary refractory (n=15) or relapsed (n=8) disease. patients who had bulky disease at the time of relapse or those with residual mass post salvage treatment, were considered as candidates for adj-ifrt, early (within 2-3 months) after documentation of autologous stem cells engraftment. all patients proceeded to asct with chemosensitive disease after a median of 2 lines of salvage therapy. at the time of asct 20 patients (80%) had residual disease while 4(20%) evaluated to be in complete remission. the preparative regimens were: single-agent melphalan (n=9), busulfan-etoposide-melphalan (n=7), beam (n=5) and bendamustin-etoposide-cytarabine-melphalan (n=3). filgrastim was given till neutrophills recovery, while prophylaxis against bacteria, fungus, viruses and pcp were administered till the completion of adj-ifrt. results: all patients engrafted promptly and successfully. no patient experienced any severe toxicity or active infection before adj-iftr. though our plan was to proceed with adj-ifrt within 3 months post asct, finally it was delivered after a median of 4.5 (2-7) months; the median radiation dose was 30(24-36) gy. ten patients received radiotherapy in the mediastinum, 9 in the abdomen/pelvis/ inguinal area 3 in the neck, and 1 in the left leg. the adj-ifrt was well tolerated. no patient experienced toxicity grade >3 and none required hospitalization. currently, after a median follow-up of 2(2-5) years, 19/23 patients are alive and well; the 5-years overall and progression free survival rates are 75% and 55% respectively. four patients died; 2 due to relapsed disease and 2 heavily pretreated patients due to secondary myelodyspalstic syndrome conclusions: in our study, the adj-ifrt in the early post transplant period demonstrated a safe and well-tolerated profile. taking into consideration the poor risk status of our patients (residual disease post salvage regimen or bulky disease at the time of relapse), the promising overall and progression free survival rates suggested that adj-ifrt post asct is also an effective approach. well designed trials are needed to clarify the role and the appropriate time of radiotherapy in the asct setting disclosure: no conflict of interest adverse prognostic impact of pre-transplant neutrophil/ lymphocyte ratio in lymphoproliferative disorders background: brentuximabvedotin(bv) is a chimeric anti cd30 igg1 antibody, conjugated to synthetic antitubulinmomomethylauristatin. bv is approved for the treatment of classical hodgkin lymphoma (hl) in relapse either after autologous stem cell transplantation (asct) or after two lines of combination chemotherapy in transplant ineligible patients. the aethera trial revealed increased pfs when bv is used as maintenance therapy for 16 cycles in high risk patients after asct. however, this schedule is associated with a high cost and significant toxicity particularly in term of peripheral neuropathy. our primary objective is to assess the efficacy of 4 cycles brentuximab as consolidation therapy after asct for relapsed/refractory (r/r) hl. secondary objectives include side effects, progression free survival (pfs), and overall survival (os). methods: this is a retrospective single center analysis approved by the irb of the american university of beirut medical center. we included in this study consecutive patients with r/r hl who underwent asct between 2014 and 2018, and received bv consolidation post-asct. results: we identified 18 consecutive adult patients with r/r hl treated with bv 1.8mg/kg iv every 3 weeks as consolidation therapy after asct. the indications for bv consolidation was primary refractory disease in 11 patients (61%), early relapse in 6 patients (33%) (after a median time of 10 months; range, 3-11) andextranodalinvolvement in one patient (6%). the median number of lines of therapy pre-asct was 3 (range, 2-5). the median time to bv initiation post-asct was 76 days (range, 35-188). patients received a median of 4 cycles (range, 3-4) of bv post-asct. after a median follow up of 30 months (range, 8-50), five (28%) patients relapsed after asct. the median time to relapse was 7 months (range, 4-21). median pfs and os were not reached. we did not observe any significant toxicities during or after therapy. conclusions: 4 cycles of bv consolidation after asct seem to be safe and effective in preventing relapse, however our findings need to be confirmed with larger prospective studies. chemotherapy or who progress after autohsct is poor. despite introduction of novel agents like brentuximab vedotin (bv) or nivolumab, allohsct appears the most effective treatment option with curative potential. the goal of this study was to evaluate efficacy of allohsct for hl, including patients pre-treated with novel agents. methods: between years 2012-2018, 45 patients (including 23 males) with hl were treated with allohsct in msc institute of oncology in gliwice, poland. median age was 32 (19-57) years. median lines of preceding chemotherapy was 4 (2-8); 34 (76%) patients had been pre-treated with autohsct, 24 (53%) with radiotherapy, 17 (38%) with bv, 7 (16%) -with nivolumab. disease status at allohsct was as follows: cr-19, pr-10, nr-16. patients were treated with hsct from either hla-matched sibling donor (msd, n=21), unrelated donor (urd, n=18) or haploidentical donor (n=6). conditioning was myeloablative in 21 (47%) cases. peripheral blood was used as a source of stem cells. results: all but one patient engrafted with median time of neutrophil recovery of 14 (9-20) days. the incidence of grade 2-4 and grade 3-4 acute gvhd was 16% and 11%, respectively, while the incidence of chronic gvhd was 43%. the probabilities of os and pfs at 2 years were 54% (+/-12%) and 55% (+/-8%), respectively. the incidences of progression and transplant-related mortality were 31% and 14%, respectively. the 2y pfs rates were 42% for msd, 72% for urd and 62% for haploidentical donors. in a univariate analysis pfs was affected by recipient gender (female -75%, male -38%, p=0.009) and disease status at allohsct (cr -78%, pr -47%, nr -38%, p=0.09). in a multivariate model the disease status other than cr was the only factor associated with increased risk of treatment failure (reverse pfs) -hr=3.13, 95%ci 1.45-6.76, p=0.004. neither donor type nor conditioning affected long-term outcome. conclusions: results of allohsct for patients with relapsed/refractory hl are determined by disease status at transplantation. efforts should be done to reduce tumour burden before transplantation, optimally to achieve cr. disclosure: nothing to declare background: brentuximab vedotin (bv), nivolumab and pembrolizumab have been assigned to chemorefractory hodgkin lymphoma treatment. impact of these agents on disease-free-survival after autologous stem cell transplantation (asct) remains under investigation. aim of the study is to compare bv-and nivolumab-treated patients with a control group. methods: clinical characteristics and outcomes of chemo refractory hodgkin lymphoma patients who underwent asct during 2011-2017. results: a total of 56 patients (33 men; 21 women, median age 37 years old, 19-65) were treated with bv: 24 pre-transplant, 30 post-transplant and 7 pre-and posttransplant. pre-transplant bv patients had primary refractory disease or early relapse in the majority (92%). post-transplant treatment occurred in the context of relapsed/refractory disease in 27 patients; 12 (40%) had an allogeneic stem cell transplant. among them, 6 had additional chemotherapy and 2 nivolumab, gaining a complete metabolic response. in the 15 rest of patients, change of treatment due to eventual bv failure occurred. bv was administered as a maintenance treatment in 10 patients. in six of them bv had already been administered pre-transplant as well. out of maintenance treatment patients, 2 relapsed and subsequently received nivolumab. two patients died due to prior chemotherapy complications, whereas 13 are currently on nivolumab treatment. pet-based response was available in 6 patients, 4 having a complete metabolic response (cmr) and 2 a partial metabolic response. stable disease was achieved by ctbased response in the rest patients. no major toxicities were observed. one patient presented with grade 2 asymptomatic hypothyroidism and one with grade 3 anemia attributed to non-inflammatory upper gastrointestinal blood loss. in total, 20 patients received anti-pd1 treatment, all post bv failure. with a median follow-up of 34.3 (1.5-202 .2) months, 5-year overall survival (os) was 65.9% in patients treated only with bv compared to 78.2% in patients treated with additional anti-pd1 treatment (p=0.356, figure) . median os for patients treated only with bv was 113.5 months, whereas median os has not been reached for patients that received anti-pd1 treatment. conclusions: bv pre or post-transplant and anti-pd1 treatment post-transplant after bv failure have outstanding results in chemo refractory lymphoma patients. treatment sequence in allogeneic transplantation eligible patients remains to be further studied. disclosure: nothing to declare background: allogeneic hematopoietic cell transplantation (allo-hct) with reduced-intensity conditioning (ric) has been used in heavily pretreated lymphoma patients with the promise of decreased treatment-related mortality. despite improvements in outcomes of patients with lymphoid neoplasms, several new agents emerge as potential therapies. therefore, we aimed to describe our long-term experience in patients with hodgkin (hl), non-hodgkin lymphomas (nhl) and chronic lymphocytic leukemia (cll) post allo-hct. methods: in this retrospective study, we enrolled consecutive patients who underwent allo-hct for lymphoid neoplasms in our institution (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) (2016) (2017) (2018) . results: in total, 50 patients (male:female=35:25) aged 36 (15-64) years, underwent allo-hct for hl (n=24), nhl (n=21) and cll (n=5). the majority of patients were diagnosed at stage iv (48%); 34% had bone marrow involvement and 66% had undergone autologous hct. most patients were heavily pretreated (median lines=4, range 1-11), 21 of them had received more than 4 treatment lines and at the time of transplantation only 14 had complete response, while 9 had partial response and 27 were refractory. according to disease-risk index (dri), patients were stratified at low (n=11, 23.4%), intermediate (n=12, 25.5%), high (n=20, 42.6%) or very high (n=4, 8.5%) category. among patients with hodgkin lymphoma, brentuximab vedotin was administrated in 7 and 4 of them were effectively bridged to allo-hct. all patients received ric, mainly fludarabine (150mg/ m 2 )-cyclophosphamide (2g/ m 2 ) in cll and nhl and thiotepa (10mg/kg)-fludarabine (120 mg/m 2 )-cyclophosphamide (60mg/kg) in hl from matched sibling (n=27), matched (n=15) or mismatched unrelated (n=8) donors. graft-versus-host disease (gvhd) prophylaxis consisted of cyclosporine or tacrolimus and mycophenolate mofetil or short-term methotrexate and additional low-dose antithymocyte globulin (5mg/kg) in unrelated donors. peripheral blood was the main cell source (48/50) and median number of cd34+ cells infused was 6.37x10 6 /kg (1.33-14.5) . two patients succumbed to advanced underlying disease before engraftment; while the other engrafted successfully. median time until neutrophil and platelet engraftment was 10 and 12 days respectively. eighteen patients (36.7%) developed acute gvhd (grade iii-iv, n=5), steroid sensitive in 10 (62.5%) and 11 relapsed. one-year cumulative incidence (ci) of extensive chronic gvhd was 78.2%, and 13 patients required more than one additional line of immunosuppression (range 1-5). ten patients presented cmv reactivation successfully treated with antiviral medication and 1 patient died from hsv7 encephalitis. with a median follow of 3 years (1-16 years), 10-year os was 40.4%, 10-year non-relapse mortality ci 23.4% and 10year dfs 32%. there was no difference in survival according to original disease (5-year os for nhl=60.2%, hl=46.7%, cll=31%%, p=0.67). multivariate analysis revealed high and very high dri as the single predicting factor of os (hr 9.69, ci 1.55-60.55, p=0.015), when assessing impact of disease, dri, prior treatment lines, gender and bone marrow infiltration at diagnosis. conclusions: our data suggest that ric allo-hct provides encouraging survival rates, potentially offering the chance of cure, with acceptable long-term mortality in selected high-risk patients with lymphoid neoplasms. dri that is mainly associated with disease stage at transplant independently affects survival. therefore, continued efforts are necessary for clinical application of novel agents aiming to lower disease stage pre-transplant. disclosure: nothing to declare results: six pts were identified, with a median age of 30 years at diagnosis: five with hl nodular sclerosis and 1 with lymphocyte depletion. the median number of therapeutic lines prior to allo-hsct was 5 [4-10]; four pts were previously treated with brentuximaband two pts had been submitted to high dose chemotherapy with autologous bone marrow support. at the time of allo-hsct, 4 pts had progressive disease (dp), 1 was in partial response and 1 in complete response (cr). five allo-hsct were performed with a related donor, 4 of wich were haploidentical (2 parents, 1 sibling and 1 descendant) and 1 with an unrelated donor (10/10). prophylaxis for gvhd was performed with tacrolimus and mycophenolate mofetil (with post-transplant cyclophosphamide in haploidentical allo-hsct). on day +100 evaluations, 5 pts had a cr and 1 patient (pt) had dp. the median time to relapse after allo-hsct was of 12 months. at the time of initiation of nivolumab, 5 pts were under steroid therapy for disease control, without other immunosuppressive therapy. the median time between allo-hsct and the beginning of nivolumab was 16 months. the initial dose was 1 mg / kg (associated with corticosteroid therapy), escalated up to 3 mg/kg biweekly, according to patient's tolerance. after the start of nivolumab, 3 patients, with previous gvhd manifestations, presented a worsening of the cutaneous gvhd, which required an escalation of immunosuppressive therapy. as toxicity, 1 pt had a grade 4 pneumonitis, 1 pt had a grade 4 encephalitis/hypophysitis, 1 pt had a grade 4 pancreatitis, 2 pts had headache (grade 1 and 3), 2 pts had a grade 2-3 cutaneous reation. with a median follow-up of 13 months since nivolumab treatment, the overall response rate was 100%: 1 pt obtained cr and 5 pts partial remission. nevertheless, there were 2 deaths after the onset of nivolumab: 1 pt at 4 monts with dp and another one due to acute myocardial infarction at 16 months. at the time of analysis, 3 pts maintained response under nivolumab treatment (median cycles 26) and 1 pt had therapy suspended because of toxicity. conclusions: these results demonstrate the high probability of achieving response with nivolumab treatment in patients with rr-hl relapsing after allo-hsct, but adverse events of grade 4 were frequent in this small group, and the treatment toxicity was significant. disclosure: nothing to declare background: intravascular large b-cell lymphoma (ivlbcl) is a rare form of large b-cell lymphoma with pathological findings of intravascular proliferation and/or sinusoidal involvement of lymphoma cells. according to their geographic distribution, ivlbcl could be dichotomized into asian and western variants. compared with the western variant, where skin involvement was common, the asian variant was reported to involve more frequently the liver, spleen and bone marrow, and hemophagocytic lymphohistiocytosis is more common in asian variant. diagnosis of ivlbl is still difficult because of the lack of overt lymphadenopathy and peripheral blood involvement. thus, timely diagnosis and immediate treatment remain as a challenge to improve outcomes for patients with the asian variant. therefore, we analyzed the clinical features and treatment outcomes of patients with the asian variant of ivlbcl. methods: we analyzed 46 patients who were diagnosed with ivlbcl between 2001 and 2018. all patients were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (r-chop). results: forty-six patients were diagnosed with ivlbcl, and their median age at diagnosis was 62 years (range: 34-82 years). male patients predominated (n=26, 57%), and b symptoms were present in 31 patients (78%). hepatomegaly and/or splenomegaly were observed in 26 patients (57%), whereas lymphadenopathy was less common (n = 22, 48%). bone marrow and liver were the most commonly involved extranodal organs (54%, and 48%, respectively) and were the most common sites of biopsy for diagnosis in this study. all patients received r-chop as a first-line treatment after diagnosis with a median number of six cycles (range one to eight). at the end of treatment, 31 patients achieved a complete response (cr), whereas eight patients showed progression. six patients died after the first or second cycle of r-chop, and the causes of death were treatment-related adverse events including cytopenia, infectious complications, and pulmonary hemorrhage. upfront asct was done for two patients including one patient with cns involvement at diagnosis, and these patients were still alive at the time of analysis without evidence of relapse. on the other hand, the outcome of six patients undergoing salvage asct after relapse was poor; thus, only one patient was alive. likewise, patients with disease progression at the end of treatment with r-chop showed dismal prognoses even after salvage chemotherapy except for one. at a median follow-up of 47.0 months (95% confidence interval, ci 25.0-69.0), the median overall survival was 45.0 months (95% ci 25.8-64.2). the treatment outcome of patients with the asian variant of ivlbcl is still not satisfactory. although upfront autologous stem cell transplantation might be effective for selected patients at high-risk of relapse, its role is still not clear, either. thus, further study should be warranted to develop more effective strategies for diagnosis and treatment. clinical trial registry: not applicable disclosure: nothing to disclare background: peripheral t-cell lymphomas (ptcls) are about 10% of non-hodgkin´s lymphomas usually with an aggressive clinical course and unfavorable prognosis.given their heterogeneity, consensus on the best first-line treatment and the role of autologous/allogeneic (asct/allosct) stem cell transplantation as consolidation is controversial. methods: we evaluated the overall survival (os), progression-free survival (pfs) and toxicities of a cohort of patients with ptcls submitted to asct/allosct intensification at our institution between january 2000 and july 2018. os was calculated from the date of diagnosis until death. pfs was measured from transplant until relapse, progressive disease or last follow-up. os and pfs rates were estimated using the kaplan-meier method and compared with the log-rank test. results: twenty-six patients were identified, 16 female (61%), median age was 46 years (range: 22 to 62). ninetytwo percent of patients presented with advanced-stage disease at diagnosis (ann arbor stage iii or iv), 38% with b symptoms. according to the 2016 who classification, histologic ptcl subtypes included angioimmunoblastic tcell lymphoma (n =12); ptcl not otherwise specified (n =6); anaplastic large cell lymphoma, alk-negative (n =5); anaplastic large cell lymphoma, alk-positive (n =1); nodal peripheral t-cell lymphoma with tfh phenotype (n =2). extranodal nk/t-cell lymphoma, nasal type and primary cutaneous subtypes were excluded. the ageadjusted ipi (aaipi) was low/intermediate low in 15 patients (57%) and intermediate high/high in 11 patients (43%). twenty-seven transplants were performed (19 asct, 8 allosct); 18 were consolidation in 1st response (16 asct and 2 allosct) with 16 in complete remission (cr) and 2 in partial remission (pr). nine transplants were performed as consolidation of 2 nd response (3 asct and 6 allosct) with 4 in cr and 5 pr. in 1 patient allosct was performed after asct, due to early relapse (< 12 months). beam regimen was used in asct as conditioning and flumel in allosct. all patients engrafted, the median time to leukocyte recovery > 1,000/μl was 14 days (range, 11 to 25). four of the 8 pts (50%), submitted to allosct had chronic graft versus host disease which was the most relevant complication of this analysis. considering the whole cohort, the median follow-up was 50.5 months (range, 2 to 217). the estimated 5-year os and pfs were 77% and 39%, respectively. seven patients relapsed (6 early) all after asct, there were no relapses after allosct, however, the results were not statistically significant between the allosct and asct groups; the 5-year os rates were 87% and 62% (p =0,16) and the 5year pfs rates were 85% and 59% (p =0,26) respectively. for the all patients treatment-related mortality (trm) was 3,7%; 7 patients died, 6 with progressive disease (asct) and 1 for hepatic toxicity (allosct) before d+100. conclusions: the results of this retrospective study, taking into account the adverse risk profile of the population, suggest that autologous/allogeneic stem cell transplantation as an effective and safe option for the consolidation of patients with ptcls. these results need to be validated in prospective studies, including a larger number of patients. disclosure background: autologous stem cell transplantation is used as consolidation therapy in relapsed lymphoma patients. however, outcome of lymphoma patients relapsing after autologous stem cell transplantation is poor and allogeneic stem cell transplantation which can be curative is used in the transplant eligible patients in this setting. besides, allogeneic stem cell transplantation can be an option before autologous stem cell transplantation in some high risk patients. in this study, we aimed to compare the survival rates of lymphoma patients older than 60 years of age and patients aged 50-60 who had undergone allogeneic transplantation in our center. methods: we collected the data of lymphoma patients older than 50 years of age who had undergone allogeneic transplantation in our center and analyzed the results by grouping them into 2, namely the ones between 50-60 years of age and the ones over 60 years of age. [[p506 image] 1. figure 1 results: there were 42 patients over the age of 50 who had undergone allogeneic stem cell trasplantation with the diagnosis of lmphoma between 2011 and 2018. 18 of these patients were over 60 years of age. 37 patients had non-hodgkin lymphoma and 5 patients had hodgkin lymphoma. the characteristics of the patients are summarized in table 1 . patients' comorbidity indexes were calculated with augmented hct-ci which includes patients' pretransplant ferritin, albümin and thrombocyte counts as a variable. no difference could be found between groups regarding neutrophil and platelet engraftment times and comorbidity indexes. however, acute graft versus rate and documented bacterial infection rate during the hospitalization period were higher in the 50-60 years age group (p=0,01). 100 day mortality rate and non-relapse mortality rate were not different between groups. more importantly, progression free survival(pfs) and overall survival(os) of patients in the 50-60 years age group and over 60 years of age group were not different (p=0,45) (figure 1) conclusions: in the present study, although the number of patients is low, we showed that lymphoma patients over 60 years of age have similar outcomes and transplant related toxicity as the patients between 50 to 60 years of age. pfs and os were very close in this study. we think that this may be due to low relapse rate in the patients and high mortality rate in relapsing patients. in conclusion, allogeneic stem cell transplantation which has a curative potential may be employed in transplant eligible elderly lymphoma patients disclosure: nothing to declare background: follicular lymphoma (fl) histologic transformation consist on the development of an aggressive lymphoma, usually a diffuse large b cell lymphoma (dlbcl). histological transformation has been considered to have poor prognosis. in pre-rituximab era median os ranged between 1 and 2 years, however, in recent series of patients treated with chemotherapy plus rituximab, the outcome of transformed fl has improved, especially in those that receive autologous stem cell transplantation (asct), who reach 5-year os up to 75% in some series. methods: we have retrospectively studied 19 consecutive patients undergoing asct for transformed fl between 2006 and 2015 in a tertiary center in the basque country, spain. patients were considered to have a transformed fl if they were diagnosed of a dlbcl and they have previous history of fl or histological evidence of a fl in another location. these patients were compared to a retrospective cohort of 38 dlbcl patients with high ipi or stage that received asct in first remission according to our institution strategy. pfs and os were calculated from the time of the asct. in the case of transformed fl, both relapses of the aggressive or indolent lymphoma were considered. survival analysis was performed with kaplan-meyer estimator results: a total of 19 transformed fl and 38 dlbcl patients were studied, with a median follow up of 41.4 and 51.7 months respectively. patient characteristics are described in table 1. 3-year pfs was 68% in transformed fl and 81% in dlbcl, and 3-year os was 81% and 84%, respectively (picture 1). there were no significant differences in pfs or os between this two groups (p = 0.44). in both groups all relapses occurred in the first three years after asct. among the patients with transformed fl 6 relapses were observed. five of them (83%) were aggressive relapses, while only one patient presented relapse as an indolent lymphoma (fl histological grade 3a with an aggressive clinical course). [[p507 image] 1. image 1: transformed fl and dlbcl pfs after asct] conclusions: in our experience, asct in transformed fl offers good results, similar to those in dlbcl. fl presents a natural course akin to that of dlbcl, with relapses occurring early and survival reaching a plateau. this data suggests that some patients with transformed fl can be cured after asct. disclosure: nothing to declare. safety and efficacy of intensive preconditioning regimen containing cladribine in autologous peripheral blood stem cell transplantation of refractory and relapsed young highly invasive lymphoma background: autologous peripheral blood stem cell transplantation (apbsct) is one of the main treatments for patients with non-hodgkin's lymphoma (nhl). effective and safe conditioning regimens can improve the cure rate of nhl. beam is the most common pretreatment scheme, but for refractory and relapsed young highly invasive lymphoma, especially for dual-expression dlbcl, pretreatment needs to be strengthened. studies have shown that the cladribine (clad)+gemcitabine (gem)+busulfan (bu) combination provides synergistic cytotoxicity in lymphoma cell lines.we evaluated the the safety and short-term efficacy of intensive preconditioning regimen containing cladribine (clad+gem+bu) for refractory and relapsed young highly invasive lymphoma undergoing apbsct. methods: ten patients with nhl received apbsct. ca)ctx+ ara-c) therapy followed by g-csf was used for pbsc mobilization. sevenr patients received conditioning regimens of beam(beam group): bcnu 300mg/ m 2 ·d -1 ×1d (-7d), vp16 100mg/m 2 · q12h×4d (-6d--3d), ara-c 200mg/m 2 ·q12h×4 d (-6d--3d), mel 140mg/m 2 ·d -1 ×1d (-2d). three patients received intensive preconditioning regimen containing cladribine (clgb group): clad 5mg/m 2 ·d -1 ×5d (-6d--2d), gem2500mg/m 2 ·d -1 ×2d (-6d, -2d), bu0.8mg/kg q6h×4d (-6d--3d). follow-up date expires on december 1, 2018. results: the age of 3 patients in clgb group was 41, 12 and 50 years, respectively. two patients were diagnosed as diffuse large b-cell lymphoma with double expression and one was diffuse large b-cell lymphoma with two recurrences. the patients of beam group were all high-risk, relapsed and refractory nhl.all patients were successfully engrafted after infusing apbsc. the average lowest leukocyte in clgb group and beam group were (0.023 ±0.023) ×10 9 /l vs (0.237±0.2033)×10 9 /l, respectively. the average lowest leukocyte in clgb group was lower than that in beam group. the average time to anc < 0.5×109/l in clgb group and beam group were 1.33d ±1.53d vs 3.57d±2.07d. the average time to anc≥0.5×10 9 /l in clgb group and beam group were 9.0d±1.0d vs 10.0d±2.6d; the average time to plt≥20×10 9 /l of clgb group was not different to that of beam group (8.0d±1.0d vs 9.6d±2.2d) the average time of neutropenia wasn't significantly different in two groups (8.33d±3.1d vs 8.0d±2.1d). the adverse reactions of gastrointestinal tract and oral mucosa were close in tow groups.vod, hemorrhagic cystitis, pretreatment-related interstitial pneumonia, liver and kidney dysfunction were not happened in tow groups. the rate of infectious fever was close in two groups (2/3 vs 4/7). the median followup period in beam group was 9 (1~19) months. in the beam group, a patient died 20 days after transplantation, because he was diagnosed with recurrent nkt cell lymphoma and intracranial infection caused by severe sinus infection. another case of beam group was diagnosed as double-expressed dlbcl, which relapsed 3 months after transplantation. the remaining patients in ebmt group survived disease-free. the follow-up time of 3 patients in clgb group were 2 months, 3 months and 9 months respectively. all patients survived without disease.however, the follow-up time is short and needs long-term follow-up. conclusions: the treatment of intensive preconditioning regimen containing cladribine (clgb) for refractory and relapsed young highly invasive lymphoma undergoing apbsct is safe. the time of hematopoietic reconstruction is short, and the adverse effects is tolerable for patients with refractory and relapsed young highly invasive lymphoma. the current short-term outcome is good, but the long-term effect need a longer time to follow-up. disclosure: this work was supported by national nature sciences found of china (81300417). there is no disclosure of conflict of interest.the all authors name: xiang-li chen, yu-zhu zang, wen-hui zhang, yin zhang, zhong-wen liu, ping-chong lei, jing yang, yu-qing chen, kai sun. background: small part of children with hodgkin disease (hd) demonstrate initial resistance to the standard and even "salvage" chemotherapy and need innovative drugs for the treatment. methods: a 15-year girl was diagnosed with classical hd (nodular sclerosis)corresponding to stage ii e b (fever >38°c) in april 2016.after two cycles of oepa (vincristine, etoposide, prednisone and doxorubicin) and next two cycles copp (cyclophosphamide, vincristine, prednisone and procarbazine) the patient again had progressive disease. as the patient achieved a partial response (pr) after "salvage"therapy with two cycles of igev (ifosfamide, gemcitabine, vinorelbine, and prednisone), she received auto-sct in february 2017 (patient status before auto-sct was pr). we used ccnu-containing conditioning regimen cem: lomustine (ccnu) 300 mg/m 2 + etoposide 1200 mg/m 2 + melphalan 180 mg/m 2 . at day +90 after auto-sct, the patient again demonstrated progression of the disease: pet/ct-examination showed mediastinal tumor mass enlargement with increased left lung nodule simultaneously to short metabolic activity. patient was under observation. at day +140 the disease had relapsed and progressed -examination by pet/ct demonstrated multifocal progressive disease with multiple pulmonary lesions and increased metabolism in comparison with the previous pet/ct scan. in july-october 2017, the patient had salvage chemotherapy with a combination of brentuximab vedotin (bv) (bv dhap (dexamethasone, cytarabine, cisplatin) + bv (without chemotherapy due to suspected invasive mycosis) + bv dhap), however, only partial pet-positive remission was achieved. because of many times relapsed and progressed disease pembrolizumab therapy was started in october 2017: 2 mg / kg every three weeks four cycles totally. toxic effects and serious complications during and after therapy by pembrolizumab were not observed. in february 2018, after pembrolizumab # 4, the patient showed complete metabolic remission of the disease by control pet-ct. in april 2018, the patient received haplo-sct with post-transplant hd-cyclophosphamide. we used conditioning modes with reduced toxicity (fludarabin 150mg/m 2 + treosulfan 42g/m 2 ), high doses of cy (50 mg / kg) on days +3 and +4. tacrolimus and mycophenolate mofetil started on day +5. mmf was terminated on day 35, tacrolimus -on day 180. patient did not have acute and chronic gvhd. results: at the moment the patient is alive and still in pet-negative cr with duration more than 9 mo. conclusions: pembrolizumab has demonstrated high activity against resistant hd even after post-auto-sct progression with good tolerability for the sick child. disclosure: nothing to declare p510 high dose chemotherapy followed by autolougous peripheral blood stem cell transplantation (asct) in diffuse large b cell lymphoma (dlbcl) median age is 38,9 years (14 to 67) and sex ratio (m/f) 1.14; ann arbor stage iii-iv: 104 pts. before hdt induction chemotherapy (chop, c2h2opa) was instituted and associated with rituximab in 100 pts (66,6%), 49 pts (32,7%) received more than 2 treatment lines and 16 pts (10,7%) received complementary radiotherapy. transplant disease status before hdt was complete remission (cr) in 93 pts, partial remission in 55 pts (rp) and disease progression in 2 pts. the delay from diagnosis to hdt is 11,7 months (4-103). the hdt protocols used are: tutshka : 87 pts, tutshka+vp16 : 42 pts, bam 12 (busulfan +cytarabine+melphalan) :16 pts et beam : 5 pts. all pts received, after thawing, mobilized pbsc obtained by g-csf mobilization (15μg/kg/d, 5 days) alone and froze in liquid nitrogen. the median rate cd34+ cells infused is 3,6 x 10 6 /kg (0. 87-17.36) . the median follow-up at 12/31/2017 is 67 months . results: the median time to graft (pnc > 0.5 x 10 9 /l) was 13 days (9-18). ten early deaths were observed including 8 infection (trm: 6,6%) and 3 in disease progression at 3 months. after 3 months of hdt 137 pts are assessable including 128 pts in cr (93,4%) and 9 pts in pr (6,6%). relapse was observed in 31 pts (22.6%) and it was earlier relapse in a period of 24 months in 15 pts (48%). deaths were among 39/150 pts (26%). persistent cr was achieved in 112/137 pts (81,8%) including 23/30 (76,6%) mlcl and 89/107 (83,1%) others dlbcl. the overall survival (os) and event free survival (efs) at 10 years are respectively 68% and 64%. the os and efs are better in patients who received rituximab in initial therapy : os (79% vs 54%; p< 0,001) et efs (79% vs 46%; p< 0,001). herein, we present one patient with refractory mcl, who were insensitive to chemotherapy and then experienced a dramatic improvement with ibrutinib mono-therapy but later developed ibrutinib resistance,ultimately resulting in the deterioration of disease and death. methods: we give the patient several examinations including ultrasound, bone marrow biopsy, lymph node biopsy, exome sequencing, sanger sequencing, and so on. for the treatment of lymphoma, the patient received chemotherapy, including 1 course of chop(cyclophosphamide 1.3g day 1, doxorubicin 40mg day 1,vinorelbine 40mg day 1, and dexamethasone 15mg from day 1 to 5) and 1 course of r-dhap (rituximab 600mg day0, cytarabine 2g q12 day 1, cisplatin 100mg day 1,dexamethasone 20mg from day 1 to 5)in succession.because of the failure to control disease progression, ibrutinib 560mg qd was used until the patient died. results: the 64-year-old man initially referred to our hospital for complaints of abdominal pain and distention over 2 months. ultrasound showed splenomegaly and multiple enlarged retroperitoneal lymph nodes.excisional biopsy conducted on the right neck lymph node revealed the presence of malignant cells.immunohistochemically, the neoplastic cells were positive for bcl2, bcl6,cd20, cd5, cd79a, cd43, ki-67(50%), sox11, cd21 (fdc) and cyclin d1 and negative for cd10, cd23 and cd3; fluorescence in situ hybridization(fish) showed igh/ ccnd1,t(11;14) 90%.thus a diagnosis of mcl was confirmed. 2 course of therapeutic chemotherapy were applied to the patient but he did not respond well.he suffered recurrent fever, thrombocytopenia, left abdominal pain, splenomegaly and multiple enlarged lymph nodes. then he received ibrutinib mono-therapy, and experienced a dramatic improvement as his body temperature was controlled, his hemogram became normal and his spleen and lymph node tapered.after about 6 months response of ibrutinib, the disease deteriorated rapidly and he died very soon. exome sequencing from the patient peripheral blood at this time detected one missense mutation in exon 5 of tp53 at nucleotide 524g>a, resulting in an argnine to histidine change at amino acid 175 (p.arg175his). but sanger sequencing of the patient bone marrow ffpe sample at the time of original diagnosis did not detect this mutation. conclusions: thus, our study reported a tp53 r175h mutation mcl patient who developed ibrutinib resistance and progressed aggresively, which may open new insight for future effort for alternative therapeutic strategies in ibrutinib-refractory mcl. disclosure: nothing to declare. minimal residual disease, tolerance, chimerism and immune reconstitution peripheral blood samples were obtained for routine analysis at several time points after hsct. all available blood samples between 0.5 and 2 years were used in the analysis. to assess changes in the cd19+b and cd19 +cd27+memory b cell counts over time while accounting for the correlation between the repeated measurements of each patient, we used linear mixed-effects models. wilcoxon rank test, kruskal-wallis test and linear regression were used for univariate analysis. results: at one year after hsct, univariate analysis showed that patients transplanted with a cb graft compared to bm and pbsc had a significantly higher absolute number of b cells (median bm= 501, median cb=1402, median pbsc=502 cells/μl, p=1.4e-5) and memory b cells (median bm= 26, median cb=75, median pbsc=23 cells/μl, . recipients with age under 5 years had significantly higher absolute numbers of b (median=919, median=473 cells/μl, p=2.9e-4) and memory b cells (median=54, median=22 cells/μl, p=2.7e-5) than above 5 years. increase in donor age was associated with a decreasing effect on b cell (r 2 = 0.35, p=5.9e-14) and memory b cell (r 2 = 0.25, p=2.1e-8) reconstitution as determined in regression analysis. following univariate analysis, we analysed these factors in a mixed effects model to assess the relation with differences in b cell or memory b cell numbers 0.5-2 years after hsct. in our analysis we found significant decreasing b cell and memory b cell numbers with increasing donor age corrected for recipient age and source (both p< 0.001). increasing recipient age also showed a significant decrease in b cell and memory b cell numbers (both p< 0.001) but there was no significant influence of donor source ( figure 1) . conclusions: b cell and memory b cell numbers after hsct are influenced by donor and recipient age but not by donor source. older donors and recipients show a decrease in b cells and memory b cells numbers 0.5-2 years after hsct. [[p512 image] 1. figure 1 . b cell development and donor age. green shows cb, red bm, blue pbsc at mean donor age.] 1 copenhagen university hospital rigshospitalet, copenhagen, denmark, 2 leiden university medical center, leiden, netherlands background: the outcome of allogeneic hsct is challenged by a delayed and long-lasting imbalanced t-cell reconstitution increasing the risk of acute gvhd, infections and disease progression. although the role of differentially and functionally distinct t-cell subsets in the development of complications has been addressed, little is known about the factors controlling their recovery. in this study, we investigated the impact of immuneregulating and homeostatic cytokines on the reconstitution of functionally distinct t-cell subsets and associated clinical outcomes. methods: we included 80 children undergoing allogeneic hsct for all (n=46) or aml (n=34) with a median age of 8.3 years (range: 0.8-17.8). donors were either mrd (n=21), mud (n=45) or mmud (n=14). bm (n=70) or pb (n=10) were used as stem cell source. conditioning regimens were based on tbi (n=25) or highdose chemotherapy alone (n=55) and included atg in 58 patients. thirty age-matched healthy children were included as controls. cytokines (il-7, il-15, il-18, scf, il-6, il-2 and tnfα) and active atg in plasma were longitudinally measured from before conditioning until 3 months after hsct (n=80) along with an extended phenotyping of t-cell maturation and differentiation by flow cytometry (n=41). results: the homeostatic cytokines il-7 and il-15 increased from pre-conditioning to peak 1-2 weeks post-hsct and gradually declined thereafter. il-6 levels were shortly elevated, while il-18 and scf remained relatively stable, and il-2 and tnf-α levels were below threshold of detection at all time points. the early rise of il-7 and il-15 was strongly associated with the degree of t-cell depletion by atg, while il-15 also correlated with markers of systemic inflammation. il-7 and il-15 levels were significantly higher in children treated with atg (p< 0.001) and correlated with both longer exposure to atg (p< 0.001) and increased levels of active atg (day +21: il-7: r=0.71, p< 0.0001; il-15: r=0.61, p< 0.0001), indicating that high levels of these cytokines reflected more pronounced t-cell depletion during lymphopenia. higher circulating levels of il-7 and il-15 were associated with a slow recovery of cd3+, cd4+ and cd8+ t-cell counts at day +30 and +60 post-hsct (p< 0.05), while the remaining cytokines did not correlate with immune reconstitution. looking into t-cell subpopulations, increased levels of il-7 and il-15 during the first month post-transplant were associated with lower numbers of naïve t cells and correlated with an increased proportion of cd4+ and cd8+ effector memory cells ( figure) . no differential effect of cytokines on polarization of cd4+ t cells into th1, th2, th17 cells or treg cells was found. in atg-treated patients, il-7 and il-15 levels at day +14 were significantly lower in patients developing acute gvhd grade ii-iv (p=0.0007 and p=0.0007, respectively). in the total cohort, increased il-7 levels were associated with a reactivation of ebv (p=0.040). conclusions: these findings suggest that quantification of il-7 and il-15 can be indicative for the degree of t-cell depletion during the first weeks after hsct and predictive of complications. overall, these results indicate that the lymphopenia-induced elevation of il-7 and il-15 is a major driver of the initial expansion of donor t-cells. background: mathematical kinetic models were adopted to study immune cell reconstitution after allogeneic hematopoietic stem cell transplantation (allo-hsct). the associations between acute graft-versus-host disease (agvhd), relapse and the immune cell reconstitution kinetic models were explored. methods: from june 1, 2011 to may 31, 2015, sixty-five patients with hematological malignancies after allo-hsct were recruited. peripheral blood was collected on +14 day, +28 day, +42 day and in +2 month, +3 month, +6 month, +9 month, +12 month, +18 month, +24 month. lymphocyte subsets were determined by flow cytometry, including in total t lymphocytes (cd3 + ), helper t cells (cd3 + cd4 + ), cytotoxic t cells (cd3 + cd8 + ), cd4/cd8 ratio, nature killer (nk) cells (cd3 -cd56 + ), nkt cells (cd3 + cd56 + ), b lymphocyte (cd19 + ), naive t cells (cd3 + hla-dr + ), static t cells (cd3 + hla-dr -), and regulatory t cells (cd4 + cd25 high foxp3 + ). mathematical kinetic models were calculated for immune cell reconstitution with spss. results: after allo-hsct, a logarithmic curve model was observed for cd3 + t cells. cubic curve models were observed for cd3 + cd4 + t cells, cd4 + cd25 high+ foxp3 + t cell, cd3 + hla-dr -t cells, cd3 + cd56 + nkt cells, cd19 + b cells. cd3 + cd8 + t cells, cd3 + hla-dr + t cells, and cd3 -cd56 + nk cells showed s type curve models. considering t cells were the major mediators for agvhd and graft-versusleukemia effect after allo-hsct. with established immune cell kinetic models, we found that different curve models were observed between patients with and without agvhd after allo-hsct. although the kinetic models were almost the same for leukemia-free and relapsed patients in the first 3 months after allo-hsct, significantly different kinetic curves could be observed thereafter. conclusions: the immune cell reconstitution showed different mathematical curve models after allo-hsct. kinetic reconstitution model of certain immune cell was associated with agvhd and relapse. hence, mathematical kinetic models of immune cell reconstitution may be potential indictor for predicting agvhd and relapse after allo-hsct. disclosure: nothing to declare lineage specific chimerism analysis in pediatric patients following allogeneic hematopoietic cell transplantation (hct background: the outcome of allogeneic hct is dependent on several variables that include patient age, disease and stage, cytoreduction, graft, graft manipulation, and graft versus host disease (gvhd) prophylaxis. one aspect of hct that remains poorly defined and studied is the donor/ host (d/h) chimerism post hct. since 2010, we followed patients with d/h lineage specific chimerism post hct. analyses were performed by short tandem repeat (str) polymorphism analysis at the american red cross blood services (philadelphia, pa). studies were performed on blood total leukocytes, myeloid/neutrophil cells, t-cells, bcells, and nk-cells. methods: in this retrospective study, the charts of 154 consecutive patients who underwent allogeneic hct between january 2010 to june 2015 on the pediatric bone marrow transplant service at mskcc were retrospectively reviewed. lineage specific donor chimerism post hct was studied including d/h chimerism trend, and factors with potential impact on chimerism including: age, disease, graft source, and t-cell depletion (tcd). preliminary analyzes performed on this cohort included wilcoxon rank test and cox proportional hazard analyses. results: 137 patients were selected based on the number of analyses. the median age was 11.3 years. patients had hematologic malignancies (n=95) or non-malignant hematologic diseases (n=27), or immune disorders (n=15). cytoreduction included tbi-(n=42), or chemotherapybased regiments (n=98). patients were recipients of t-cell depleted marrow or peripheral blood grafts (n=101), unmodified marrow or peripheral blood grafts (n=28) or cord blood grafts (n=8). full donor chimerism of myeloid cells, b-cells and nkcells, but not t-cells occurred early post-transplant. there was no difference in the percentage of total donor leukocytes at 3 months vs. 12 months post hsct (n=30), while the median of donor t-cell chimerism was 51% at 3 months and 91% at 12 months post hsct (p< 0.0001, n=42). figure 1 shows the impact of different factors including: (a) the use of tbi-or chemotherapy-based regimens, (b) age (< or > 3 years), and (c) type of graft (t-cell depleted vs unmodified vs cord blood). donor total leukocytes chimerism was significantly lower at 12 months as compared to 3 months for patients < 3 years of age (p=0.012). for most grafts, full donor chimerism of t-cells occurred early, while for t-cell depleted transplants, it took up to one year to complete. cord blood grafts were associated with high t-cell donor chimerism throughout the post-transplant period. there was a significant difference in the % donor t-cells at 3 and 12 months post hct when comparing t-cell depleted and unmodified grafts (p=0.015). conclusions: this preliminary analysis of lineage specific chimerism post-transplant showed that donor tcells may take one year to fully recover post-transplant, mostly following t-cell depleted grafts, without intervention. cord blood grafts were associated with high donor chimerism throughout the post-transplant period. lastly, factors associated with loss of donor chimerism posttransplant were younger age and non-malignant disorders. more in-depth analyses are being performed including the relation of chimerism and hct outcome. disclosure: eileen nicoletti -employee rocket pharmaceuticals, susan prockop -investigator atara biotherapeutics -research funding, susan prockop -mesoblast -research funding, nancy kernan -gentium -support; jazz pharmaceuticals -support, richard o´reilly -atara biotherapeutics -royalty, consultancy and research, jaap jan boelens -bluebird bio -consultancy, avrobio -consultancy; jaap jan boelens -chimerix -consultancy; magenta -consultancy background: the success of hematopoietic stem cell transplantation (hsct) lies with the ability of the engrafted immune system to remove residual leukemia cells via a graft-versus-leukemia effect. despite this, relapse remains the major cause of mortality among patients receiving hsct. one of the immune evasion mechanisms of leukemic cells to escape from donor t cell recognition in haplo-hsct is the genomic loss of the patient specific hla. it has been described in 20-30% of acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) relapses after haplo-hsct. the aim of this study was to analyze hla loss in a large cohort of patients who relapsed after t-cell replete haploidentical transplantation with posttransplant cyclophosphamide. methods: from december 2007 to september 2018, 180 patients with hematological malignancies who received a haplo-hsct were recruited. among them, 31 patients presented a relapse after haplo-hsct. hla typing was performed by real-time pcr using hla-kmr kit (gendx, netherlands).nine patients were excluded from the analysis because the kit employed did not include the recipientspecific hla. thus, a total of 22 relapse cases were analyzed. the analysis of chimerism was carried out using short tandem repeat pcr amplification (ampflstr sgm plus, thermo fisher, usa) with a sensitivity of 1%. results: genomic loss of the patient hla occurred in 6/ 22 patients (27%) ( table 1) . these patients presented different hematological neoplasms. interestingly, 4 patients presented lymphoid neoplasm (1 acute lymphoblastic leukemia (all-t), 1 dentritic cell leukemia (dcl) and 2 hodking´s lymphoma (hl)). hla loss relapses occurred later than classical relapses (370 vs.166 days). regarding the treatment received (table 1) , four patients were studied retrospectively. three of them were treated with donor lymphocyte infusions (dlis) + chemotherapy or other drugs at the time of the relapse. the other patient did not receive any treatment. in the end, all 4 patients died from disease progression. prospectively, we detected hla loss at relapse in other two patients. at the moment of relapse, the first case received brentuximab + haplo-hsct from alternative donor and the other case received daratumumab + haplo-hsct (pending). both patients are alive, the first one in complete remission (cr) and the second one in partial remission (pr). conclusions: the frequency of hla loss at relapse is similar in our cohort to what is described in the literature. hla loss has been identified in patients with lymphoid neoplasms, while this mechanism has not been previously described in such diseases. the analysis of this immune evasion mechanism should be implemented in the routine screening of patients transplanted from haploidentical donors in order to design effective rescue strategies. these treatments should not be based on dlis or second transplantation with the same donor, instead, alternative donors should be used. background: an adequate immune reconstitution (ir) is crucial to reduce transplant toxicity, relapse rate and mortality after allogeneic stem cell transplantation (allohsct). the aim of this, single center retrospective study was to investigate the correlation between the recovery of different lymphocyte subpopulations with the main transplant outcomes, including overall survival (os), disease free survival (dfs) and non-relapse mortality (nrm). methods: we analyzed the ir of 177 adult patients (aml n=62, all n=41, mds n= 24, nhl n=9, hd n=2, cll n=4, cml n=7, mm, n=11, mpn n=10) who underwent (allohsct) between january 2013 and march 2017. median age at transplant was 52 years (range 17. 3-71.3) with male/female ratio of 62%. donors were hlaidentical siblings (n=39, 22%), family haploidentical (n=11, 6%), matched unrelated (109, 62%), mismatched unrelated (n=11, 6%) and cord blood units (n=7, 4%). the stem cell source was the bone marrow (bm) in 30 patients (17%), the cord blood in 7 (4%) and g-csf mobilized peripheral blood in 140 (79%). the conditioning regimen was myeloablative in 99(56%) transplant, reduced intensity in 75 (42%) and immunosuppressive in 3 (2%). gvhd prophylaxis was based on calcineurin inhibitors in combination with methotrexate or mofetil mycophenolate. antilymphocytes immunoglobulins was used in 145 patients (81%) (anti thymocytes globulin, atg sanofi-genzyme in 114 or anti t-lymphocyte globulin, atlg -neovii biotech, in 31). the peripheral blood lymphocyte subsets (cd3+, cd3+cd4+, cd3+cd8+, cd19+ (b cells) and cd16+cd56+ (nk) were analyzed by flow cytometry at 1, 2, 3, 6, 12 and 24 months after hsct. post-transplant engraftment was molecularly determined by vntr analysis. results: as detailed in table 1 the proportion of full donor chimerism analyzed in the peripheral blood t lymphocytes improved progressively after transplantation and the same pattern was observed when the chimeric status was measured in bone marrow mononuclear cells. to favor the achievement of a full donor chimerism, dli were performed in 26 patients starting at the median of 96 days after transplant (range:34-447). with a median follow-up observation of 25 months (range 7-61), the one year os and nrm was 88% and 5%, respectively. at 6 months after allohsct, the achievement of values higher than 75, 175 and 65 /μl for cd4+, cd8+ and nk cells, respectively was significantly associated to a better os (figure 1 ), dfs (p = 0.05), and to a lower nrm (p< 0.001 for cd4+ and cd8+, p= 0.0034 for nk). a better lymphoid reconstitution was observed after the use of either a sibling or a haplo donor than a matched unrelated or cord blood donors. the use of atg was significantly associated with a delayed cd4+ recovery but with a faster nk cells reconstitution. conclusions: at six months after allohsct, recovery of cd4+ and nk cells predicts survival. monitoring of immune recovery may help to guide pre and post-transplant treatment strategies. days infections and disease control. several groups have demonstrated the independent prognostic value of different lymphocyte subpopulations in hsct outcomes. posttransplant cyclophosphamide (pt-cy) effectively prevents gvhd after hla-haploidentical hematopoietic stem cell transplantation (haplo). the use of pt-cy in hla matched related (mrd) or unrelated (mud) donors hsct, although less explored, has also been introduced. the aim of this study was to compare the early immune reconstitution after allogeneic hsct from haploidentical and hla-matched donors using pt-cy. methods: one hundred and sixty-four hsct performed in our center were analyzed: 125 haplo performed between 2011 and 2017 and 39 hsct from hla-identical donors (19 consecutive mrd sct performed with pt-cy between 2016 and 2017 and 20 mud sct with pt-cy between 2014 and 2017). pt-cy was administered at 50mg/kg/d in days +3 and +4 postransplant, followed by mmf 10mg/kg/ d and a calcineurin inhibitor (ciclosporina a or tacrolimus) from day +5 ahead. we retrospectively compared early immune reconstitution at day +30 and day +90 among these three populations. early ir was assessed through the analysis of lymphocyte subpopulations including total t lymphocytes cd3+, cd4 + and cd8+subpopulations, nk cd3-cd56+ cells, cd56 + bright immature subpopulation and total b cd19+ lymphocytes.. lymphocytes subpopulations were determined by multiparametric flow cytometry (fc500 and navios, beckman coulter®). results: all patients, but 1 mud and 1 haplo, received pb as stem cell source. 46 patients received prior transplant in haplo group. patient´s characteristics are shown in table 1. patients who received hsct from mrd showed the fastest ir, with statistically significant differences compared to haplo in almost all lymphocyte populations at day +30 (cd3+, cd4+, cd8+ and nk cells), and also in cd3+, cd8+ and b lymphocytes at day +90. comparison between haplo and mud hsct showed better ir among haplo, demonstrated by higher counts in cd3+,cd4+, cd8+ and nk cell counts at day +30. no differences were seen at day +90. (figure 1 ). percentage of immature cd56 bright nk cells was higher in mud hsct at +30, with no differences between haplo and mrd hsct. conclusions: in our cohort of patients with pt-cy based gvhd prophylaxis regimen, those who received hsct from mrd showed the earliest immune reconstitution compared to haplo and mud at day +30 and +90. haplo showed better ir compared to mud at day +30. nk maturation at day +30 was a little better among haplo and mrd hsct recipients than mud hsct patients. in our experience, using mostly pbsc as graft source, type of donor influenced early ir in pt-cy based hsct, background: cell-free dna (cfdna) isolated from plasma or serum has received increasing interest for diagnostic applications. however, the reported clinical usefulness of cfdna in patients undergoing allogeneic cell transplantation (hsct) is scarce. methods: the chimeric status both in peripheral blood and in cfdna obtained from plasma was investigated in 110 patients undergoing hsct. dna and rna were isolated from plasma within four hours of blood draw. patients were evaluated for chimerism at day +30, +100 and +365 post-transplant. a panel of seven microsatellites was amplified by pcr for chimerism detection and pcr products were analysed by capillary electrophoresis. for further cfdna characterization the micro rna (mirna) 200c was analysed using digital pcr. mutations frequently used for minimal residual disease assessment such as flt3-itd, npm1 and jak2 were also investigated in cfdna. results: the mean cfdna concentration in transplanted patients was 469 ng/ml, while in healthy donors used as control group (n=20) was 119 ng/ml. the mean cfdna concentration difference between both groups reached statistical significance (p= 0.0197). when analysing cfdna from transplanted patients and in the control group we could not detect dna fragments larger than 400 bp and the size range of the analysed fragments was between 80 and 200 bp. in 41 out of 110 patients a mixture of donor and recipient cfdna (mc) was detected. with the exception of three patients relapsing after transplant in which mc was detected both in peripheral blood and plasma in the rest of the patients (n=38) mc was detected only in plasma. the mean percentage of recipient cfdna in the plasma samples was 18% (range: 1-81%). all the patients with acute gvhd (agvhd) (grade: i-iv) (n=15) showed mc in plasma at least in one of the time-point tested. no significant difference was found in the mean recipient cfdna percentage in patients with agvhd grade i-ii when compared with grade iii-iv. meanwhile in the group of patients with chronic gvhd (n=42) mc in plasma was detected in 13 patients. in those patients with clinical improvement of agvhd (n=6) a decrease in the percentage of recipient cfdna was observed during treatment. in patients without improvement or even agvhd worsening (n=5) stable or increasing recipient cfdna percentage was detected. since recipient cfdna can be detected in patients without transplant-related complications we analysed the mirna 200c expression in all patients with recipient cfdna. a significant difference was found in the mirna 200c expression in patients with agvhd (mean mirna 200c: 9.7 mirna 200c copies/10 4 u6 copies) when compared with patients without gvhd (mean mirna 200c: 35.4 mirna copies/10 4 u6 copies). in those patients with extramedullary aml relapse (n=3) frequent mutations (flt3-itd, npm1) were only detected in the cfdna fraction. conclusions: longitudinal analysis of cfdna represents a useful complementary tool in particular for those patients with clinical complications after hsct. disclosure: nothing to declare comparison of the impact of atg/pt-cy-based and tcr αβ-depletion as gvhd prophylaxis regimens on the recovery of memory t-cell compartment background: over recent years haploidentical and hlamismatched donors have been increasingly adopted as a valid donor source. modern graft-versus-host disease (gvhd) prophylaxis regimens such as drug-based (antithymocyte globulin (atg), post-transplant cyclophosphamide (pt-cy)) or graft-manipulated (tcr αβ-depletion) demonstrate effective prevention of gvhd. here we report our data about an influence of different gvhd prophylaxis regimens after allo-hsct with pbsc as a graft source on cd8+ memory t-cells recovery. methods: our study comprised 32 leukemia patients who underwent allo-hsct with pbsc as a graft source in national research center for hematology, moscow, russia. detailed patients characteristics are presented in table 1 . peripheral blood samples were collected on day +30, +60 and +90 after allo-hsct. flow cytometry analysis was performed on bd facs canto ii (becton dickinson, usa) to define t-memory subsets: t-naive and t-stem cell memory (tnv+tscm) -cd8+cd45r0-ccr7 +cd28+; t-central memory (tcm) -cd8+cd45r0 +ccr7+cd28+; t-transitional memory (ttm) -cd8 +cd45r0+ccr7-cd28+; t-effector memory (tem) -cd8+cd45r0+ccr7-cd28-; t-terminal effector (tte) -cd8+cd45r0-ccr7-cd28-. sysmex xe-2100 was used to calculate absolute count of different t-memory cell subsets. mann-whitney u test was used for nonparametric data analysis. a p-value less than 0.05 was considered as significant. results: results of mann-whitney u test (calculated pvalues) to compare absolute number of t-memory cell subsets in terms of different gvhd prophylaxis regimens are presented in figure 1 . during all follow-up period the number of tnv+scm and tcm remains significantly reduced after atg+pt-cy or tcr αβ-depletion compared to atg-based immunosuppressive regimen. on day +30 we observe no difference in the number of tnv+scm and tcm cells after atg+pt-cy or tcr αβ-depletion. terminally differentiated cd8+ cells (ttm, tem, tte) count is significantly lowered in tcr αβ-depletion patients group in comparison to atg+pt-cy. nevertheless recovery of tnv+scm and tcm after pt-cy is faster than after tcr αβ-depletion. conclusions: according to our data the mechanism of pt-cy is seems to be more selective compared to tcr αβ-depletion due to its transient impact just on tnv+scm and tcm without affecting on the effector pool. through this it may lead to delayed reconstitution of adaptive immunity after tcr αβ-depletion compared to using pt-cy. clinical relevance of the quantitative characteristics of immune recovery in the context of different approaches to gvhd prevention remains to be established. background: immune effector cells, belonging to either innate or acquired immunity, play a key role on preventing disease relapse after allogeneic haematopoietic stem cell transplantation (hsct). most of known immune effector are cd3 + cd8 + t-cells and cd3 -cd56 + natural killer lymphocytes, while cd3 + cd4 + cells act as modulatory and regulatory cells. the early post-hsct ratio between these cellular subsets may be an indicator of graft vs-tumor (gvt) effect. methods: we retrospectively revised the immune recovery of 117 allogeneic hsct performed at our institution from 2013 to 2017, analysed on peripheral blood by multiparametric flow cytometry lymphocyte subpopulations panel. diagnosis were acute leukemias (69%), chronic myeloproliferative neoplasms (10%), lymphomas (10%), myelodysplastic syndromes (5%), multiple myeloma (3%) and severe aplastic anemia (3%). we established 2 early time-points of evaluation, 30 and 60 days from the graft infusion, to analyse the differences in disease free survival (dfs) and overall survival (os) between patients according to the cd3 + cd8 + x cd3 -cd56 + / cd3 + cd4 + ratio. results: median ratio at +60 days was of 0,5667. at this time point, patients who showed the ratio higher than the median had both a better dfs (median dfs time not reached vs 12 months; p = 0,018) ( figure 1 ) and os (median os time not reached vs 13 months; p = 0,014). likewise, ratio at +30 showed an advantage on dfs (p = 0,027), and not on os (p = 0,06). other factors possibly affecting both dfs and os were analysed in univariate analysis, such as the use of antithymocyte globulin (atg), conditioning regimen intensity, graft source, hla-matching and disease status at hsct, the latter being the only variable with a significantly detrimental impact on both os and dfs. disease status was confirmed an independent valriable associated with both dfs and os as well as +60 ratio both on dfs (hazard ratio [hr] -2,721; p = 0,015) and os (hr 2,627; p = 0,022). conclusions: our data show that cd3 + cd8 + x cd3 -cd56 + / cd3 + cd4 + ratio assessed at +60 is and independent predictor of transplant outcome, possibly representing a row indicator of anti-leukemic immune surveillance. the integration of this index with other known outcome predictors may help in improving the management of post-transplant phase. [[p524 image] 1. figure 1 background: allogeneic stem cell transplantation (alo-hsct) is a curative treatment but it is associated with lifethreatening complications. most deaths are due to relapse, graft versus host disease (gvhd) and infection. the pattern and quality of the immune reconstitution (ir) after transplantation may affect these outcomes. however, there are limited data on the association of the quality of the ir and either the development of gvhd and survival. methods: eighty-five patients who received a non t-cell depleted alo-hsct in our center from 2011 to 2014 were prospectively studied. most patients received hla-identical grafts. total cd4+ and cd8+ t cells, ccr7+cd4+ and ccr7+cd8+ (which include both naïve and central memory t cells) and naïve ccr7+cd62l+ t lymphocytes were quantified by flow cytometry. data were collected at days +30, +60, +90, +180 and +360 after alo-hsct. the association between ir and the gvhd was studied through an anova. for the multivariate analysis, a logistic regression was performed including those confusing clinical variables that were significant in the univariate analysis (p≤0.10). the study of overall survival (os) versus ir was performed with a cox regression model. results: total cd3+ t lymphocytes reached normal numbers within the first two months. median t cd8+ count was 263 cells/ul after one month, which is within the normal range. conversely, it took nearly one year to get normal counts of cd4+ t cells (542 cells/ul). the only two clinical parameters conditioning a worse recovery of the cd4+ t cells were the previous alosensitization of the donor and the sex, being female donor and male recipient the worst combination for the ir. no parameters influenced the quality of the reconstitution of cd8+ t cells. of note, the age or the hla status did not influence the quality of the ir. when the patients were divided into gvhd and no gvhd, we found no differences in the recovery of either the proportion or absolute count of every t cell subpopulation, including total t cells as well as naïve/central memory t cells, both cd4+ and cd8+. finally, a multivariant analysis confirmed that the absolute counts of cd4+ccr7+ t cells at day +90 as well as the absolute counts of both cd4+ccr7+ t cells and naïve cd4+ccr7+cd62l+ at day +180 were associated with better os. conclusions: in conclusion, neither the development of gvhd nor other relevant parameters seem to play a determinant role in the quality of the ir. to our knowledge this is the first study which demonstrate a clear association between the recovery of naïve cd4+ t cells measured by flow cytometry and the os. disclosure: nothing to declare p526 abstract already published. azacitidine (aza) for prophylaxis or pre-emptive therapy for myeloid neoplasms after allogeneic stem cell transplantation whom 21 were treated prophylactically and 11 preemptively. median age was 55 years [range, 15-69] and all patients had a diagnosis of aml or high-risk mds. prophylactic treatment consisted of aza 32 mg/m 2 for 5 days in cycles of 28 days. in the pre-emptive setting, 5 patients received 75 mg/m 2 for 7 days per cycle and 6 patients 75 mg/m 2 for 5 days per cycle. a median of 6 cycles [range, [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] was administered in the prophylactic group and of 4 cycles [range, 4-22] in the pre-emptive group. ten patients also received at least one dli after the third aza cycle: 8 patients in the prophylactic group and 2 patients in the pre-emptive one. results: during follow-up, 10 patients had significant delays in treatment plan due to transitory mild complications. however, 15% of patients (n=5) presented infectious complications requiring hospitalisation and 40% of patients (n=10 in the prophylaxis group and 3 in the pre-emptive group) presented some form of gvhd. in patients who developed gvhd, 3 had to discontinue treatment (all in the prophylaxis group); also 8 patients discontinued treatment due to disease progression. the overall drop-out rate was 37.5% (n=12). survival was analysed from initiation of treatment with aza and median follow-up was 16 months. one-year efs was 95% in the prophylaxis group, with only one patient relapsing and no deaths. in the pre-emptive group, the 1-year efs was 45% and the median efs was 6 months; 1-year os was 70% and median os was 24 months. conclusions: we conclude that post-transplant aza treatment is a well-tolerated therapy, but the incidence of side effects remains discordant in the literature. results in the prophylaxis group are excellent, but patients with positive minimal residual disease treated pre-emptively had a lower outcome with only stabilisation of the disease. randomised prospective trials are needed to define patients who would benefit the most from this treatment and at what timing, dosage and duration of treatment. disclosure: nothing to declare. abstract already published. results: all subjects experienced hematopoietic engraftment at a median of 17 days (range 14-20) and demonstrated full donor myeloid chimerism. m-mdsc and pmn-mdsc recovery peaked at a median of 22 days posttransplant. the median peak absolute m-mdsc count was 9,828 cell/ml (range 9,180-41,120/ml) representing a range of 1.5% to 4.8% of pbmcs. the pmn-mdsc peak was more robust, with a median absolute peak of 103,730 cells/ml (range 11,330-676,800/ml) representing a median of 25.3% of pbmcs (range 10. 3-44.2%) . of note, the one patient who developed severe, life-threatening gvhd had the lowest absolute and relative pmn-mdsc recovery (11,330 cells/ml and 10.3% of total pbmcs). recovery of m-and pmn-mdscs occurred at a similar tempo and magnitude in two recipients of standard gvhd prophylaxis (tacrolimus/methotrexate). however, while mdscs isolated from ptcy recipients exhibited clear t-cell suppressive capacity, those from the comparison patients did not (see figure) . conclusions: mdscs recover rapidly and robustly after allohct using ptcy as gvhd prophylaxis, and may play a role in mitigating gvhd risk by mediating t-cell suppression. this may be a mechanism by which ptcy results in donor-recipient tolerance. background: high dose chemotherapy followed by autologous stem cell transplantation (asct) offers a cure in the upfront and relapsed setting in both hodgkin (hl) and non-hodgkin lymphoma (nhl). asct also remains standard of care in previously untreated multiple myeloma (mm) patients after induction therapy, if eligible. the availability of new cellular or other immune therapies that can be used after asct underscores the potential importance of monitoring immune reconstitution after asct. methods: immune reconstitution panels (irp) were evaluated retrospectively in all lymphoma and mm patients over a 5-year span (2012-2017) whom underwent asct at our institution. patients were included if they had a pre-asct measured within 30 days of asct and two other irp at any of the following timepoints (1) day 30-45, (2) day 60-90, and (3) at 1-year post-asct. patients in the lymphoma cohort had their irp excluded if they had additional treatment within the first year post-asct (other than maintenance rituximab). mononuclear cells from peripheral blood were analyzed by flow cytometry for assessment of lymphocyte phenotype and numbers. absolute values were compared using the mann-whitney u test. results: the data on 78 patients were available for analysis (50 mm, 24 nhl, 4 hl) . all lymphoma patients were conditioned with beam. all mm patients were conditioned with a standard high dose melphalan regimen. the median pre-asct absolute cd3 counts in the lymphoma cohort were significantly lower than the mm cohort at 1709 cells/μl vs 3309 cells/μl, respectively (p=0.049). however, the mm cohort exhibited a greater percent reduction in cd3 cells on day 30 at 85.3% vs 63.5%, respectively which continued through day 365 at 79.8% vs 70.3%, respectively. this led to nonsignificant changes in absolute cd3 count by day 365 at 667 cells/μl vs 507 cells/μl, respectively (p=0.39) (figure 1). the median absolute cd4 count pre-asct for mm and lymphoma cohorts were 2101 cells/μl and 863 cells/μl, respectively (p=0.007). similarly, a greater percent reduction in cd4 cells led to comparable absolute counts on day 365 at 310 cells/μl vs 298 cells/μl, respectively (p=0.322). the failure of post-asct cd3 reconstitution to pre-asct levels was driven by lack of cd4+ cell recovery, namely cd4+cd45ra+ cells with a median of 21 cells/μl and 20 cells/μl in the mm and lymphoma cohorts, respectively at day 365 (figures 2 and 3). this led to markedly diminished cd4:cd8 ratios through day 365 (figure 4). [[p531 image] 1. conclusions: impaired t-cell reconstitution in both lymphoma and mm continues through 1-year post-asct. as shown, a larger percent reduction in median cd3 and cd4 counts through day 365 was appreciated in mm compared to lymphoma leading to the nonsignificant differences in the post-asct absolute counts despite significantly higher pre-asct counts in the mm cohort. impaired recovery of cd4 t-cells may increase the risk of opportunistic infections, decrease the response to vaccination and lead to ineffective anti-tumor response. further prospective and larger retrospective studies like this should continue in the modern-era as they may help predict responses to further interventions requiring a robust t-cell repertoire for maximal efficacy such as car-t cell and bite therapies. disclosure: nothing to declare p532 peri-transplant detection of measurable residual disease by multicolor flow cytometry is highly predictive for relapse in acute myeloid leukemia patients background: presence of measurable residual disease (mrd) prior to allo-sct has been shown to be predictive for survival in patients in hematological cr of aml. in this study we analyzed the impact of mrd in such patients measured by 8-color multiparameter flow cytometry (mfc) prior to and on day +100 post-transplant. methods: the bone marrow samples immediately prior to allo-sct and on day +100 post-transplant were retrospectively analyzed. mrd evaluation was carried out with antibodies against: (1) results: a number of 55 aml patients (male, n=35) with median age of 57 years (23-77) in hematological cr prior to allo-sct were enrolled in the study. we observed lower survival in patients with mrd by mfc pre-transplant (2y os: 61% (41-81%) vs. 92% (82-100%), p=0.018) due to increased relapse incidence (35% (17-53%) vs. 8% (0-18%), p=0.009). in multivariate analysis, mrd pos prior to allo-sct has strong significant impact on os (hr 6.5 (1.5 -30) , p=0.015). of 55 patients, a sample both before and on day +100 after transplantation was available in 33 patients. of those 33 patients, 15 (46%) were mrd negative prior to transplant and on day +100 (mrd neg/neg ); 14 (42%) patients were mrd positive prior to transplant and negative at day +100 (mrd pos/neg ); and 4 (12%) patients were mrd positive at both timepoints (mrd pos/pos ). dfs and os for these three groups were as follow: 2y dfs: mrd neg/neg : 93% (80-100%), mrd pos/neg : 78% (56-100%); mrd pos/pos : 25% (0-75%, p=0.006); 2y os: mrd neg/neg : 100%; mrd pos/neg : 93% (79-100%); mrd pos/pos : 25% (0-75%, p< 0.001). upon multivariate analysis, the mrd status prior to transplant and on day +100 showed strong significant impact on dfs (hr 3.6 (1.3 -9.9), p=0.01) and os ), p=0.017). we did not observe any significant impact of other factors included in the multivariate analysis (patient's age, patient's sex, and recipient/ donor sex constellation). conclusions: mrd positivity prior to allotransplant and at day +100 by mfc is highly predictive for survival after allo-sct. disclosure: nothing to declaire background: immune reconstitution is a critical factor for risk assessment of life threatening infections and long-term survival in patients undergoing allogeneic cell transplantation (hsct). methods: immune cell subsets (cd19, cd4, cd8, cd56, cd25+cd127-) were quantified by flow cytometry. trec and krec were quantified simultaneously using droplet digital pcr (dpcr). a total of 31 patients were evaluated. mean age at transplant was 56 years (range: 18-74 years) samples were obtained before hsct and at day 30, 100 and 365 after hsct. results: absolute numbers of cd3 and cd19 cells remained below pre-transplant levels until day 100, increasing further and eventually reaching pre-transplant levels one year after hsct. absolute counts of cd4 and cd25+cd127-cells remained below pre-transplant levels beyond one year after hsct. cd56 cells were characterized by fast reconstitution kinetics, reaching pre-transplant levels already at day 30. b cells correlated with krec levels at all time-points tested, whereas t cells correlated with trec levels only one year after transplantation. when we compared cell subsets, trec, krec levels and the reconstitution kinetics thereof between patients with reduced intensity conditioning (n=26) or full conditioning (n=5) no significant differences were observed. patients with pre-transplant trec levels above the mean (200 trec copies/ml blood) showed higher trec levels and a faster t-cell reconstitution after hsct suggesting that tcell reconstitution can be predicted by analysing thymic functionality before transplantation. indeed, in patients with a pre-transplant trec above 200 trec copies/ml blood, the positive predictive value for an efficient t-cell reconstitution was 0.889 (p=0.012). we analysed the recovery kinetics of the cell subsets, trec and krec levels in patients with and without transplant-related complications. patients with either acute graft-versus-host disease or severe infections showed a slower trec reconstitution when compared with patients without complications. conclusions: our data suggest that the analysis of immune cell subsets together with trec and krec quantification can be used to evaluate the immune reconstitution process after hsct. pre-transplant trec levels allow t-cell reconstitution efficiency prediction after hsct. disclosure: nothing to declare background: falling donor / mixed chimerism after allogeneic haematopoetic stem cell transplant (sct) is associated with an increased risk of relapse and the potential for graft rejection. donor lymphocyte infusions (dli) are often administered in patients with mixed chimerism to achieve full donor chimerism but there is little data on long term outcomes for dli given for persistent mixed chimerism. methods: a retrospective analysis of all patients administered dli for mixed chimerism between 2008 to january 2017 was performed. all patients were transplanted at the university hospital of wales within the south wales blood and marrow transplant (swbmt) programme. patients were identified by the swbmt database and additional outcome data gathered by review of patients' medical records. results: 58 patients were treated with 111 donor lymphocyte infusions between 2008 and january 2017. thirty one patients treated for relapse (with or without mixed chimerism) were excluded as was a further patient with a mismatched donor. the rest were 10/10 match. twenty six patients received a total of 54 donor lymphocyte infusions for mixed chimerism alone. the median age was 63 years (range: 27-71) with 65% women. fourteen (54%) of the patients had sibling donor transplants and twelve (46%) from matched unrelated donors. indications for transplant were: for aml or saml (n=16), myelofibrosis (n=4), mds (n=3), hodgkin lymphoma (n=1), non-hodgkin lymphoma (n=1) and all (n=1). escalating doses of donor cd3+ t cells were administered commencing at 5×10 5 /kg to 5×10 6 /kg then increased at half log increments according to chimerism results until full donor chimerism was achieved. the median number of doses administered was 2 (range 1-5). the median interval was 94 days (range 48-408). the median dose was 1×10 6 / kg (range 5×10 5 -6×10 7 ). seventeen patients (65%) achieved full donor chimerism and remained so until most recent follow up (median 25 months, range 12-64). one patient continued to receive dli after the study period and later reverted to full donor. two patients had ongoing mixed chimerism with no evidence of relapse. two patients relapsed; one of whom later achieved remission. there were six cases of gvhd; acute gvhd (grade ii n=2, grade iii n=1) and 2 cases of chronic extensive gvhd. one patient had gvhd features consistent with overlap syndrome. a total of five patients died, four due to infection (one in a patient with gvhd) and one due to cardiac toxicity from previous treatment (confirmed on post-mortem). conclusions: the results of our single centre study help reinforce the evidence for dli in establishing full donor chimerism when mixed chimerism is detected in the absence of relapse. incremental dli dosing is an effective strategy and associated with a low relapse rate. caution should still be given to the risk of gvhd following dli, however the risk appears to be low in this study. larger prospective studies are ongoing to address the optimal dosing strategy for dli post-transplant. disclosure: nothing to declare hypomethylating agents for the treatment of relapsed acute myeloid leukemia after allogeneic blood stem cell transplantation: a single center experience mariarita sciume 1 , giorgia saporiti 1 , elena tagliaferri 1 , nicola fracchiolla 1 , federica grifoni 1 , giorgia levati 1 , luca baldini 1 , francesco onida 1 the post-transplant period with well-balanced profile of good efficacy and moderate toxicity. we retrospectively evaluated the safety and efficacy of hma +/-dli in a reallife cohort of aml patients relapsing after allo-sct. methods: data from all patients with aml who underwent allo-sct at our institution in the last 6 years and subsequently received hma as a salvage treatment for disease recurrence or preemptively for loss of complete donor chimerism were collected. results: eleven patients with a median age of 64 years (range 41-66) were identified; median time between allo-sct and time to hma therapy was 10 months (range 4-42). according to eln genetic risk stratification, 2 patients were classified in the favorable group, 3 in the intermediate-i, 2 in the intermediate-ii and 4 in the adverse one. six patients were treated with aza, whereas the remaining 5 patients with dac. the cycles were repeated every 28 days. ten patients (91%) started hma for morphological aml relapse, while one patient received aza as a sequential treatment after dli administered for loss of complete donor chimerism. median number of cycles was 3 (range 1 -20). treatment strategy included combination with dli in 5 patients (2 in the dac cohort, 3 in the aza cohort), while in one case of flt3-itd + aml sorafenib was also associated to dac and dli. no grade 3/4 toxicities and no acute gvhd occurred. a clinically significant response was observed in four patients (36%), all receiving at least 4 cycles of hma therapy; in particular, a complete remission (cr) was achieved in 3/10 patients treated for morphological relapse, including the one who received the dac/dli/sorafenib combination and one (favorable eln risk) who received aza alone (not eligible for dli due to a concomitant lateonset cutaneous grade 2 gvhd). of interest, the latter patient also displayed a resolution of the cutaneous gvhd. full donor chimerism recovery with no gvhd was observed in the patient who received aza for the progressive donor chimerism loss not responding to dli alone. with a median follow-up of 7 months (range 4-29), the median os from hma treatment in responding patients was 16 months (range 4-29); at the time of data collection responses were maintained in all four patients. seven patients had died, six from aml progression and one for severe intestinal gvhd occurring after failure of dli+aza and a following salvage induction chemotherapy treatment. conclusions: although arising from a limited number of patients, our real-life experience of treatment with hmas +/-dli in aml patients relapsing after allo-sct showed a general very good safety profile and promising antileukemic activity, altogether suggesting a facilitation of the graftversus-leukemia effect (gvl) associated to a possible suppression of the gvh reaction. disclosure: nothing to declare conclusions: in this study, cd4-positive cell count and igg value had recovered about 15 months after sct. in our institute, we have achieved a low incidence of infection by education and medication for patients until recovery of cd4-positive cell count and igg. however, we found a higher incidence of infection after recovery of cd4-positive cell count and igg. at 12-15 months after sct, administration of prophylactic medications such as sulfamethoxazole-trimethoprim were terminated and social comeback such as return to school or work were achieved in most patients. it is possible that the high incidence of community-acquired infection was associated with their comeback. thus, we should consider additional prevention of infection for patients in this period and further evaluation of immunological markers is needed. disclosure: no potential conflicts of interest were disclosed. effect of minimal residual disease before transplantation on the outcome of haplo-identical hematopoietic stem cell transplantation for high-risk acute lymphoblastic leukemia yehui tan 1 , sujun gao 1 , xiaoliang liu 1 , long su 1 , wei han 1 , yu liu 1 , yangzhi zhao 1 background: to analyze the effect of haploid hematopoietic stem cell transplantation (hid-hsct) on high-risk acute lymphoblastic leukemia (all), and to explore the effect of minimal residual disease (mrd) before transplant on the prognosis. methods: a retrospective analysis was made on 39 high risk all patients accepted hid-hsct in our hospital from january 2013 to january 2018. the clinical features, stem cell implantation, complications, survival and recurrence were compared between pre-transplant mrd + and mrdpatients. results: all the 39 patients got successfully implanted. the overall survival (os) was 54.67%, the disease free survival (dfs) was 40.96%, the incidence of acute graft versus host disease (agvhd) was 53.8%, including 23.1% ii~iv degree agvhd and 2.6% iii~iv degree agvhd. there was no significant difference in stem cell implantation, gvhd, cytomegalovirus and hemorrhagic cystitis between mrd + and mrdpatients. dfs and os in mrd + patients were significantly lower than those in mrd -patients, and the cumulative rr rate increased significantly, there was no significant difference in cumulative trm. conclusions: hid-hsct was an effective method to treat high risk all, but mrd + patients had high recurrence rate and poor prognosis. strategy adjustment should be considered to reduce tumor residual and the transplantation strategy should be optimized for these kind of high risk patients, so as to improve survival rate. disclosure: nothing to declare background: lymphocytes are responsible for the cellular and humoral immunity and, consequently, its recovery after allo-hsct might be linked with the survival after the procedure. the aim of this study was to analyze this hypothesis in our series of patients. methods: all the 209 allo-hsct performed in our center from january 2015 through july 2018 were included in the analysis. median age was 52 years (range: 7-69). 122 pts were male (58,4%) and 87 were female (41,6%). baseline diseases were: 69 aml, 49 lpd, 31 mds, 28 all, 16 mpd, 10 mm, and 6 bmf. donor was unrelated in 113 (54,1%), and was family in 96 cases (45,9%) (including 31 haplo-identical). stem cell source was pb in 195 (93, 3%) and bm in 14 pts (6,7%). conditioning regimen was reduced in 111 procedures (53,1%) and intensive in 98 (46,9%) (including just one non-myeloablative). overall mortalities at days +100 and +365 (the latter in patients with follow-up superior to 1 year) were 9,1% and 24,9%, respectively. median follow-up was 25 months (range: 4-47). evolution of absolute lymphocyte counts (alc) and subpopulations at pre-hsct and during the first year after allo-hsct were analyzed. results: as shown in table 1, alc and cd4+ lymphocytes decreased after conditioning therapy, and recovered progressively during the post-hsct period. at day +365, majority of patients had >1000 alc/mcl, clearly improved compared to admission values. cd4+ lymphocytes at day +100 was still very low, but at day +365 around half of the series had 200-500/mcl. we found a strong link between alc, cd4+ lymphocytes, and cd19 + lymphocytes at days +30 and day +100 with overall survival at day +365 of the series (table 2) . conclusions: in our series, immunity recovery was a late event for majority of patients undergoing allo-hsct. in addition, in our experience, the precocity and quality of the alc, cd4+, and cd19+ cells recovery was clearly linked with long-term survival. background: the reconstitution of t and natural killer (nk) cells after hematopoietic stem cell transplantation (hsct) strongly influences the outcome of hsct including viral infection and graft versus-host disease (gvhd). the purpose of this study was to investigate the clinical efficacy of immune reconstitution including t and nk cells after hsct in children. methods: we reviewed the records of 30 patients who undergoing allogeneic hsct in department of pediatrics, pusan national university children's hospital, from january 2013 to july 2017. the counts of t lymphocyte subsets and nk cells was monitored in peripheral blood by flow cytometric technique during 1, 3, 6, and 12 months post-hsct. blood samples for cytomegalovirus (cmv) and epstein-barr virus (ebv) monitoring were tested by real-time pcr assay. results: for total of 30 patients, the mean age was 9.1 years (range, 9 months-19 years), 16 of the patients were boys and 14 was girl. out of a total 30 patients without pre-hsct cmv viremia or cmv infection, 10 (33.3%) recipients experienced cmv infection. the number of cd8 + t cells in 3 and 6 months post-hsct was significantly higher in patients with cmv reactivation compared to patients without (median 1066.11/μl vs. 979.0/μl, p=0.004, and 1047.45/μl vs. 551.44/μl, p=0.002) . in 6 (20%) recipients presented acute gvhd, the number of cd4 + t cells in 1 and 3 months post-hsct was significantly lower in patients with acute gvhd compared to patients without (median 239.13/μl vs. 365.0/μl, p=0.045, and 165.2/μl vs. 344.27/μl, p=0.035) . the number of nk cells in 1 months post-hsct was significantly lower in patients with cmv reactivation and acute gvhd compared to patients without (258.5/μl vs. 501.67/μl, p=0.004, and 162.5/μl vs. 464.88/μl, p=0.027, respectively) . in multivariable analysis, acute gvhd was shown to be the decisive factor influencing total t cells (p=0.028) and cmv reactivation was independently associated with cd8 + t cells (p=0.026). the cd4 + t cells counts were associated with prior hsct history and acute gvhd (p=0.042 and p=0.038), and the cd8 + t cells counts were also significantly associated with donor type (p=0.016). conclusions: overall, our study documents that immune reconstitution of cd4 + , cd8 + t cells and nk cells is strongly associated with cmv reactivation and acute gvhd. additionally, we show that acute gvhd is influenced by lack of sufficient numbers of nk cells as well as cd4 + t cells early after sct. cd8 + t cells, on the other hand, significantly increase after cmv-reactivation and most likely play an important role in reactivation. disclosure: nothing to declare background: curative effect of allogeneic hematopoietic stem cell transplantation (allo-hsct) depends on the alloreactive t-cell immune response toward residual malignant cells -graft-versus-leukemia reaction. however, alloreactive population has not been phenotypically defined. recent studies suggest that alloreactive t cells express both costimulatory and inhibitory receptors simultaneously. exhaustion caused by the inhibitory signaling dampens tcell functionality, which could lead to the disease relapse. here we aimed to investigate the expression of costimulatory and inhibitory receptors on antigen-experienced t cells after transplantation, to isolate subpopulation specific for allo-hsct patients and analyze their t-cell receptor (tcr) repertoire. methods: expression of coinhibitory and costimulatory molecules on pbmcs patients at various time points after allo-hsct was analyzed for expression of: cd3, cd8, cd4, cd45ra, ccr7, cd95, cd27, cd28, klrg1, tigit, pd1, cd137 and ox40 by flow cytometry and compared to healthy donors. cd3+cd8+cd95-cd27 +cd28+pd1+tigit+ fraction and cd3+ cd8+ control fractions were separated on facs aria ii cell sorter. double barcoded cdna libraries of tcr beta-chains for both fractions were prepared and analyzed by sequencing on illumina platform. sequencing results were processed by migec, mixcr and vdjtools software. enriched clones were identified by fisher's exact test (p>10 -10 ). results: we did not find any significant differences between patients after allo-hsct and healthy donors in single marker's expression, but, when considering coexpression of co-stimulatory and inhibitory molecules on t cells we found that cd3+cd8+cd95-cd27+cd28 +pd1+tigit+ subpopulation was significantly increased in allo-hsct patients. moreover it increased with the time since the transplantation (fig. 1 ). this population was isolated by cell sorting and alongside with total cd8+ fraction subjected to tcr beta-chain repertoire sequencing. the population contained clones significantly enriched compared with cd8+ fraction representing potentially alloreactive cells. this hypothesis is further supported by the notion that the level of expression of cd27 and cd28 co-stimulatory molecules is lower in the group of patients who subsequently relapsed, compared with the patients with complete remission, while the expression of inhibitory receptors was high in both groups. conclusions: according to our data patients after allo-hsct have a phenotypically distinct t-cell population characterized by simultaneous expression of costimulatory and inhibitory markers. this population contains specifically enriched clones, which may be specific for alloantigens. further functional assays are needed to confirm the alloreactive potential of this subpopulation. besides low expression of costimulatory molecules combined with high expression of inhibitory receptors on antigen-experienced t-cells of patients after allo-hsct might be associated with a disease relapse. fondazione mbbm, monza, italy, 3 ospedale san gerardo, laboratorio stefano verri, monza, italy background: poor graft function (pgf) is a severe complication after hsct, with a high risk of morbidity and mortality, mainly due to infections. donor cd34+ scb seems to offer high chances of haematological recovery, not jeopardized by gvhd. however, pediatric reports remain scarce. methods: 19 out of 215 patients undergoing transplantation in our pediatric unit from 2012 to 2018 have been retrospectively evaluated for at least 2 line persistent cytopenia (hb< 9.5g/dl, plt< 30000/mmc, n< 1000/ mmc) and/or transfusion-dependency beyond 3 months after hsct in the presence of full donor chimerism. bone marrow cellularity was evaluated through biopsy as further indicator of pgf. (1/8) to donate or medical decision (7/8). bone marrow cellularity was < 5% in 50% of the patients who underwent scb for which the histology was available (8 cases), and 25% in those who have not been treated (2/8). at 30 days after scb 10/11 (91%) patients had hematological response, which was complete in 46% and partial in 45% of the patients. only 1 patient had no response. the infusion was always well tolerated with no adverse events, and no worsening of gvhd. haematological recovery occurred spontaneously at 30 days after bone marrow biopsy in a significantly lower proportion of patients (2/8, 25%, p< 0.05) within the non-scb group. in two cases platelets engraftment was significantly delayed, up to one year after bone marrow biopsy and in one case thrombocytopenia persists and the patient is still receiving thrombopoietin agonists and red blood cells transfusions at 9 months after bone marrow biopsy. conclusions: a stem cell boost matched the goal to yield count recovery in our cohort. viral infections and gvhd may be possible risk factors for pgf.bilinear or trilinear cytopenia with transfusion dependency and bom cellularity < 5% and full donor chimerism are good indications for scb, that can provide a significantly earlier hematological reconstitution, without risks of gvhd. due to the proved early efficacy and safety of cd34+ stem cell infusion, we suggest that this procedure should be taken in consideration in children with severe bone marrow hypoplasia and persistent cytopenia after hsct. disclosure background: as allogeneic hematopoietic stem cell transplantation (hsct) is sometimes performed despite erythrocyte's antigens incompatibility and mismatch, it is essential to carefully track patients' genotypes after it. methods: for the study we used erythrocytes (n=189) and dna (n=20) from patients undergoing abo-or rhesus-mismatch hsct and their donors. we used posttransplant no transfused patients on the periods according transplant protocol by hemagglutination methods in plate and tube using monoclonal antibodies to abo and rhesus antigens (hematolog, russia). we extracted dna with dna kit (bag, germany) and conducted pcr-ssp with kits abo-type, rh-type (bag, germany). chimerism was assessed by the str-pcr analysis with cordis plus multiplex kit for amplification of 19 polymorphic strmarkers and amelogenin loci. the fragment analysis was performed on a 3130 genetic analyzer. informative loci were chosen by comparison of pretransplant patient's and donor's dna. the percentage of donor chimerism was calculated using standard formula. precise rhce and abo genotypes were determined by direct sanger sequencing. we revealed 3 patients with unexpected erythrocyte abo, rhesus phenotypes and genotypes after hsct on +30 (2 patients) and +160 days (all patients). chimerism analyses on str showed in a.e.kh. and g.l.v. patients 99% of donor's dna and less than 1% of recipient's one. b.n.a. patient was relapsed and chimerism analysis revealed 95% of recipient's dna and 5% of donor's one. using serological methods and pcr-ssp we revealed genotypes abo * a1b1; rhd+; rhce * ccee in patient a. e.kh. before hsct, abo * a2o1; rhd+; rhce * ccee in her donor, and abo * a1a2; rhd+; rhce * ccee on +30d after hsct. genotype a1a2 was no recipient's neither donor's origin. direct sequencing did not prove this genotype, but revealed donor's one.on +160d serological methods and pcr-ssp also revealed donor's genotype in this patient. patient b.n.a. had genotypes abo * o01/o01; rhd+; rhce * c w cee before hsct, her donor -abo * b1o1; rhd +; rhce * ccee. on +160d this patient relapsed, but rhesus genotype has been detected as rhd+; rhce * c wcee (lack e gene). direct sequencing revealed gene rhce*ee. abo genotype was recipient's origin -o1o1. in patient g.l.v. using serological and pcr-ssp methods we determined genotypes abo * o01/o01; rhd +; rhce * ccee genotype before hsct, and abo * a1/ o01; rhd+; rhce * ccee genotype in her hsc donor. on +30d patient had unexpected genotype abo * o01/o01 (a lack of a antigen); rhce * ccee (a lack of e antigen). in order to explain unexpected patient's genotypes after hsct we sequenced her rhce and abo genes and found donor's genotype ccee; a1o1 that was in agreement with results of str analysis. to resolve discrepancies between serological, pcr-ssp and sequencing analysis data we sequenced patient's rhce cdna and observed only ce allele. at present time the molecular basis of selective inactivation one of the two rhce alleles is not clear. on +160d patient had donor's genotype. conclusions: what kind of mechanisms led to discrepancies between results obtained by different laboratory methods are still not clear. an interesting case of expression of only one rhce allele in patient g.l.v. allows us to suggest involvement of some epigenetic mechanisms like dna methylation or histone modification in this process. clinical background: in relapsed patients with acute b -lymphoblastic leukemia (all-b) who achieved complete remission (cr) after re-induction therapy, minimal residual disease (mrd; ≥10 -3 all-b cells/ul) is often detected. according to available data, such condition varies from 30% to even 50% of cases, as assessed by pcr or flow cytometry (fc), while the presence of mrd is the most important risk factor for all recurrence. in this abstract, we describe our experience with bridging therapy using blinatumomab infusion after re-induction regimens and before the planned allogeneic stem cell transplantation (allo-sct). the procedure was performed in three young men suffering from relapsed ph (-) all-b at the age of 19, 22 and 34 years. in the first case (19yo), relapse with previous mrd accounting for 2.7% occurred 15 months after cr1 mrd neg . in the next patient (22yo) the second relapse with central nervous system (cns) involvement occurred 5 months after allo-sct performed in cr2 (26 months after cr1, mrd neg ), while in the third patient (34yo), recurrence with cns and testis involvement occurred 12 years after cr1 (mrd neg ). all patients underwent chemotherapy (flam, hypercvad and dnr/vcr/pegasp/dexa regimens respectively) followed by one cycle of blinatumomab (at a dose of 9mcg/d on days 1-7, followed by 28mcg/d on days 8-28 in a continuous infusion) and allo-sct (using eto/cy/tbi/atg/ conditioning regimen for ist and iiird patient and bucy2 for iind patient; using matched unrelated donor (mud, ist and iiird patient) or matched related donor (iind patient)). mrd status was assessed after each cycle of blinatumomab by fc. results: all patients achieved cr mrd pos after reinduction therapy followed by clearance of mrd after blinatumomab course (tab. 1). the second patient, due to positive mrd 6 months after allo-sct received 4 donor lymphocyte infusions additionally. during the administration of blinatumomab, no adverse events (aes) were observed in grade 3 or 4. one patient developed cytokine release syndrome in grade 1. the progression free survival, time to positive mrd and follow up are presented in tab 1. conclusions: the use of blinatumomab as a bridging therapy between re-induction regimens and allo-sct in patients with all-b and mrd pos appears to be safe and leads to the clearance of mrd which may be crucial in os and pfs prolongation after following allo-sct. future studies on larger groups of patients are necessary to confirm this thesis. background: haploidentical hematopoietic stem cell transplantation (hsct) is considered an alternative treatment for hematologic malignancies in patients who do not have an hla-identical sibling donor [1] . since infections and disease relapse resulting from delayed immune reconstitution (ir) are the most common causes of mortality among patients undergoing haploidentical-hsct [2], timely ir is essential in the recovery and survival of these patients. the aim of this study is to describe the evolution of ir after haploidentical-hsct and to estimate survival rates in patients with delayed vs. adequate reconstitution in a single center in colombia, south america. methods: a retrospective cohort study was conducted on 26 consecutive adult haploidentical-hsct recipients at a tertiary referral center. cd4+cells, cd8+cells, cd3+cells, and immunoglobulins levels were monitored before hsct, at first month, and then every three months for the first two years post-transplantation. descriptive statistics were used to analyze patient's clinical characteristics. the kaplan-meier method was used to assess overall survival (os) and relapse-free survival (rfs) rates. results: twenty-six patients were included (50% were male), with a median age of 25.5 years (range 16-59). the most common indication for haploidentical hsct was acute lymphoblastic leukemia (n=21, 80.8%), followed by non-hodgkin lymphoma (n=3, 11 .5%) and myelodysplastic syndrome (n=2, 7.7%). all patients received gvhd prophylaxis therapy with cyclophosphamide, tacrolimus, and mycophenolate mofetil. fifteen patients (57.7%) presented cytomegalovirus reactivation (25/26 at risk), 5 patients (19.2%) epstein-barr virus reactivation, and 4 patients (15.4%) developed adenovirus infection. median time to neutrophil engraftment (neutrophils>0.5×10 9 /l) was 15 days (range12-29) for the 23 patients recipients of peripheral blood progenitor cells (pbpcs) and 21 days (range20-27) for the three remaining bone marrow recipients. platelet engraftment, defined as >20,000 platelets/ mm 3 background: daratumumab is a human monoclonal antibody directed against the glycoprotein cd38 that is overexpressed on the surface of plasma cells in multiple myeloma patients. it is approved as second line therapy either as single agent therapy or in combination with lenalidomide or bortezomib for the treatment of patients with relapsed/refractory multiple myeloma. despite the curative potential of an allo-sct, the high relapse rate remains a clinical problem. data addressing the choice of an optimal salvage therapy regime for these heavily pre-treated patients is missing. methods: from april 2016 till november 2018 a total of 22 patients (male, n=10) with the median age of 64 years (40-72) received daratumumab as a salvage therapy for relapse of multiple myeloma after allo-sct at the university of hamburg. prior to allo-sct all but one patient had received an autograft, 9 patients even ≥2 autografts and 4 patients also a 1. allograft. the median number of salvage lines post-transplant and prior to first daratumumab infusion was 2 (0-4). these salvage regimens included cyclophosphamide, etoposide, bortezomib, lenalidomide, pomalidomide and carfilzomib. daratumumab was started at a median of 19 months (0-43) after relapse/ progress and initiated as single agent therapy in all patients. concomitantly, 14 patients received either an immunomodulatory drug (lenalidomid, n=10; pomalidomid, n=1) or a proteasome inhibitor (bortezomib, n=3) during a later course of daratumumab infusions. combination therapy was initiated when a slow rise of paraprotein and/or free light chains or no response to monotherapy was observed (median at the 11 th infusion). results: the median number of infusions was 14 . twenty adverse reactions were observed in 13 of 22 (59%) patients: dyspnea (n=5), bronchospasm (n=2) shivering (n=3) , cough (n=2), musculoskeletal pain (n=4), acute coronary syndrome (n=1), skin rush (n=1), facial edema (n=1), pressure on eyes (n=1). all adverse reactions appeared during the first infusion and were mostly mild or moderate (ctc 1-2, n=19). tolerance of the following infusions improved and in none of the cases therapy had to be stopped due to adverse events. three patients developed late onset infections (pneumonia, n=2; urinary tract infection, n=1) followed by temporarily therapy interruption. with a median follow-up of 14 months after the first administration 20 of 22 patients remain alive .9%). one patient died due to progress of myeloma and another died due to severe infection/sepsis. 15 of 22 patients responded (68%; pr, n=5; vgpr, n=8; cr, n=2) to the therapy with daratumumab. the responses (decrease of paraprotein and/or free light chains ≥50%) occurred at a median of 7 days (4-372) after the first administration and lasted for 5.0 months (0.5-26.6). conclusions: daratumumab shows an encouraging efficacy and acceptable toxicity profile in patients with relapsed/refractory myeloma after allo-sct. further studies are needed to investigate the role of the combination therapy with immunomodulatory drugs or proteasome inhibitors in this setting. disclosure: nothing to declare p546 clonal plasma cell detection by high sensitive flow cytometry in aphaeresis product is poor prognostic and not increased by use of plerixafor alone background: in an earlier from our center we have demonstrated that residual clonal plasma cells (cpc) decrease both overall survival (os) and disease free survival (dfs) (ash 2018).plerixafor is a selective antagonist of cxc4 chemokine receptor (cxcr4) and able to mobilize human peripheral blood stem cell (pbscs) by acting synergistically with g-csf.the purpose of this study was to evaluate the safety and efficacy of plerixafor in myeloma patients who were proven poor mobilizers and specifically to assess the flow cytometric measurement of residual clonal plasma cells in the apheresis products. methods: patients with a diagnosis of mm who underwent auto hsct at our center between january 2008-november 2018 were retrospectively analyzed.out of 164 patients, 16 patients received plerixafor as mobilization regimen due to poor mobilization with g-csf.pbsc grafts were tested for the presence of clonal pcs (cpc) and the number of normal pcs (npc) by multi-parameter flow cytometry (fcm).the acquisition of the cells was performed using the navios flow cytometer beckmancoulter) .upon the daily checks of the instrument, 3x10 6 cells for each sample were acquired and the collected data was analyzed using the kaluza software (beckmancoulter,usa). results: patient demographics are shown in table 1 .the majority of patients were male and median age was 62 years in the plerixafor group.the median interval from time of diagnosis to mobilization and follow-up from mobilization were 3.3 months and 14.3 months in plerixafor group, respectively. cpc contamination in the pbsc grafts was detectable in 32 and 7 patients with counts ranging between 0-8.7x10 -5 and 0-0.1x10 -5 in g-csf alone and g-csf+plerixafor groups, respectively (p=0.116).there were no significant differences in the proportion of the patients with graft contamination between subtypes of mm in both groups. one hundred (gcsf/plerixafor;93/7) patients had pre-asct pet-ct imaging done with 71 (gcsf/plerixafor;64/7) have active lesion at the time of mobilization. statistically significant association could not be demonstrated between the disease < cr status at mobilization and the number of apc in the apheresis product in both groups (p>0.05).twelve of 16 patients from plerixafor treatment arm proceeded to transplantation within median 9.5 months.the best overall response to induction treatment is shown in table 1 .thirtyfour patients from the g-csf alone arm and 2 patients from the g-csf+plerixafor arm died during the follow-up (p=0.52).disease progression was seen in 48 patients from g-csf alone group and 6 patients from g-csf+plerixafor group of the study(p=0.56).estimated mean os was better among patients w/o apc contamination in plerixafor group, respectively (38.9±5.3mos vs 16.8mos; p=0.52). conclusions: our results on 164 and few plerixafor used patients show that clonal plasma cells are detectable by multiparametric flow more frequently when patients are poor mobilizers and require plerixafor.the clonal pc contamination can be attributed to the myeloma biology as manifested by higher number of lines induction regimens and pet positivity among the plerixafor-required patients. the overall and disease survival was impaired by residual clonal pcs in the graft but not by plerixafor per se. neither was the content of clonal pcs differed from others.thus the cxcr4 shared by hsc and myeloma cells do not cause a myeloma mobilization. clinical trial registry: -disclosure: nothing to declare prognostic factors for overall survival after allogeneic hematopoietic cell transplantation in multiple myeloma patients all factors with significant influence on pts survival were included multivariate analysis (cox regression model) but only re-admission in the first 365 days demonstrated impact on os (hr 4, 082; p=0, 005) . conclusions: we analyzed risk factors for survival in mm pts who received allo-hct. our study identified disease-related risk factors like iss and transplantationrelated factors such as hct-ci and pam, hospital readmission, days of hospitalization and cmv reactivation that were associated with worse long-term survival. in our series, the most frequent death and re-admission cause was infection, so focusing the efforts in reduction of infection could have a beneficial impact on improvement of survival in mm undergoing allo-hct. [[p547 image] 1. figure 1 ] disclosure: there is no disclosure. novel protocol for autologous hsct in multiple myeloma: ambulatory chemomobilization and transplantation of fresh hematopoietic stem cells with backup storage background: autologous hematopoietic stem cell transplantation (ahsct) after melphalan conditioning is still a part of standard treatment of multiple myeloma patients. traditional transplantation of frozen stem cells poses additional risk for the patients connected with dmso and central venous catheter. the transplantation of fresh cells is an option -however, most mobilization protocols are either low-efficient (g-csf), expensive (g-csf + plerixafor) or toxic (standard dose chemomobilization) to directly proceed to transplantation in this fragile group of patients. we describe here the novel combination of ambulatory mobilization with very low doses of ara-c and g-csf connected with direct ahsct with fresh cells. methods: the prospectively collected database of patients after ahsct was searched for patients who underwent ahsct after chemomobilization with ara-c and transplantation with fresh cells (fc) and compared with control group of consecutive patients transplanted with standard protocol (sp) (transplantation with frozen cells) between july 2016 and october 2018. protocol of ambulatory mobilization was: 400mg/m² of arac on days +1 and +2 and g-csf at the dose 5 μg/kg/day from day +5 and escalated to 10 μg/kg/day split into two doses +10 to +13, apheresis started on day +14 (or later) and finished when at least 7.5 x 10e6 cd34+ positive cells were collected. the collected cells were split in three equal parts:1) for use as fresh transplant 2) frozen for possible 2 nd transplant 3) frozen as backup. results: there were 48 transplantations with fresh cells and 49 transplantations with frozen cells compared. both groups had same mobilization protocol -ambulatory low dose ara-c. the median age and number of transplanted cells was similar in both groups (56 vs 57, p=0.99; 5.2 vs 5.3 cd34+/kg, p=0.97 conclusions: we present novel approach that allows direct ahsct after chemo mobilization in all patients who are treated with melphalan. we show that it is not only feasible to do ahsct directly after chemomobilization but also the results may favour this approach when compared with current standard. disclosure: nothing to declare background: high dose chemotherapy followed by autologous hematopoietic cell transplantation (hsct) is considered, since the nineties, the standard of care for patients aged less than 70-75 years old with newly diagnosed multiple myeloma (mm). however, the optimal induction treatment prior to hsct to reduce the tumor burden has changed during the last few years. improved regimens have shown to be able to increase the quality of the pre-hsct response, which might subsequently impact on the post-hsct response, which has been proved to be associated with a longer pfs. we recently changed the induction therapy for pts candidates to hsct. in this analysis, we aimed to check if newer regimens impacted on pretransplant responses, and how auto-hsct changed the pre-hsct status. methods: all the 117 auto-hsct for 99 mm patients performed in our center from january 2014 through august 2018 were included in the analysis. median age was 60 years (range: 38-76). 54 pts were male and 45 were female. durie-salmon stage was distributed as follows: i (9.4%), ii (37.6%) and iii (53%); 13% had creatinine > 2 mg/dl. iss was: 1 (41%), 2 (28.2%), and 3 (30.8%). type of monoclonal component was: igg (51.3%), light chains (23.9%), iga (22.2%) , and non-secretory (2.6%). 51.3% had bence jones proteinuria. conditioning regimen was melphalan 200 mg/m 2 in 106 (90.6%), melphalan 100-140 mg/m 2 in 9 (7.7%), and other in 2 (1.7%). results: pre-transplant therapy was: vcd in 60 (mostly in 2014-6), vtd/vrd/krd in 34 (mostly in 2017-8), and others in 23 cases. status of the disease at transplant was: cr/vgpr in 75, pr in 35, and sd in 7. distribution of pretransplant response based on the type of induction is shown in table 1. peri-transplant mortality was 0%. day +100 mortality was 1.7% (2 pts), due to progressive disease. as shown in table 2, all patients (14/14) who obtained cr pre-hsct, maintained the response at day +100 post-hsct. among the patients in vgpr at hsct, 20 (32.8%) became into cr, and 37 (60.7%) maintained the response. the cr rate at post-hsct increased 264% compared to pre-hsct (37 versus 14 pts). altogether, after hsct 44 pts (37.6%) improved and 65 (55.6%) maintained the pre-hsct response. during the last period of time, 37 pts started on post-hsct maintenance/consolidation, mainly with lenalidomide. conclusions: 1) with the new chemotherapeutic schemes, 76.5% of patients underwent hsct in cr or vgpr; 2) majority of pts (93.2%) consolidated or improved the pre-hsct response; 3) cr increased substantially (2.64 times) after transplant; 4) optimized induction regimens, along with auto-hsct followed by the recently licensed use of maintenance therapy with lenalidomide, might result in a better pfs of patients with mm. background: autologous stem cell transplantation (asct) is commonly used in treatment of patients over 65 years with multiple myeloma (mm), however the safety and efficacy of this procedure is debatable. methods: we conducted a retrospective review of mm patients who underwent asct from 2013 to 2018 at our institution. the purpose of this retrospective study was to compare the 100-day mortality, time to engraftment, and incidence of grade 1-4 toxicities in elderly mm patients with younger patients taking into account comorbidity information. other secondary end points measured were overall survival (os) and progression-free survival (pfs). results: a total of 95 patients were analysed and categorized by age as young patients (37 to 64 y; n=70) or elderly (65 to 71 y; n=25). the compared groups did not differ in terms of gender, ecog, hct-specific comorbidity index (hct-ci), and disease status at asct. melphalan in a dose of 200 mg/m 2 was used as preparative regimen in 65% of younger patients, and in 43% of the elderly (p= 0.18). the remaining patients received 140 mg/m2 of melphalan or lower dose (range, 100-140 mg/m 2 ) due to hct-ci >2 or age, on the physician discretion. in the whole study group there were no transplant related deaths within the first 100 days of asct. stratifying by age, there was no statistically significant difference concerning febrile neutropenia (fn) incidence, which was observed in 14% of younger patients, and 17% of elderly. in contrast, fn was observed more frequently in patients with hct-ci > 2 (38% vs 12%, p=0.05). grade 1-2 infections were more frequent in older patients (35% vs 5%, p= 0.005), but no difference was found in grade 3-4 infections incidence rate such as pneumonia, uroinfections and neutropenic enterocolitis (25% vs 35%, p=0.52), nor grade 1-2 and 3-4 noninfectious toxicities (50% vs 38%, p=0.31, and 4% vs 21%, p=0.06, respectively) . the median time to granulocyte engraftment was 11 days (range, 9-19 days) in elderly and was comparable with younger patients. the time to platelet recovery was also similar. after the median follow-up of 12 months for survivors, os at 18 months was 94% in both groups. pfs at 18 moths was 65% for younger patients, and 55% for elderly (p=0.86).however, the association between pfs and the dose of melphalan used in conditioning was observed. pfs probability at 18 months for patients conditioned with the dose of 200 mg/m 2 , 140 mg/m 2 and 100 mg/m 2 was 71%, 58% and 21%, respectively (p=0.012). conclusions: our data show that asct in transplant eligible mm patients ≥ 65 years of age is safe and provides similar outcomes as seen in younger patients. disclosure: nothing to declare is mobilization with chemotherapy effect response in the multiple myeloma? background: high dose melphalan therapy with autologous stem cell support is a standart approach in symptomatic multiple myeloma patients. response rates increased with the novel anti myeloma agents and the use of chemotherapy for stem cell mobilization should be questioned. the purpose of this study is to determine the effect of cyclophosphamide used during stem cell collection on disease response and transplantation results. methods: we retrospectively collect data from 270 myeloma patients who underwent autologous stem cell transplantation (asct) in ankara university medicine faculty, blood and bone marrow transplantation unit between january 2012 and november 2018. 51 patients who received cyclophosphamide protocol for stem cell mobilization were included in the study. disease response were determined according to international myeloma study group (imwg) criteria before and after the cyclophosphamide. transplant responses and their effects on survival were also indicated. results: after the diagnosis of mm, 51 patients (male/ female: 32/19; median age: 59 years (between 40-74 years)) with median follow-up of 59.1 months (between 7,6 -185,8 months) underwent asct at a mean of 21,7 ±6,2 months.. forty-one patients were evaluated before and after cyclophosphamide (table 1). in 69% of the patients, the disease response was not altered by cyclophosphamide therapy, and 22% of the patients improved their response status. post-transplant response rates of patients who underwent stem cell mobilization with cyclophosphamide are also shown in table-1. the mean survival of the patients was 52,7 ±4,8 months. when patients were grouped according to changes in response status before and after cyclophosphamide; there was no statistical difference between mean calculated survival (improved response, disease progression and stable disease; 59,3±9,0 months, 22,7±14,7 months and 46,4±5,0 months respectively, p=0.425) (figure-1) . the rates of 1-year and 3-year overall survival (os) of the patients with no response to cyclophosphamide treatment were as follows; 74,2%±7,9% and 70,5%±8,3% respectively. thirteen patients who were followed up median 27 months after transplantation died at an average of 3,4±2,5 months; 6 of these deaths were caused by the infection after transplantation. conclusions: in our study, it was observed that the use of cyclophosphamide for cd34+ stem cell mobilization did not change the disease response rates by 69%. the posttransplant survival rates of mm patients who had progressive disease after cyclophosphamide use were lower. however, these results warranted confirmed by randomized controlled trials. clinical trial registry: -disclosure: nothing to declare results of a single center experience: an attempt to augment conditioning regimen in first autologous stem cell transplantation treatment of multiple myeloma (mm) continues to evolve in the era of novel agents. the addition of bortezomib to highdose melphalan (bor-hdm) has been reported by several groups, and it has been outcome and toxicity profile is comparable to high dose melphalan (hdm) alone. the aim of this retrospective study was to evaluate the outcome of the bor-hdm conditioning regimen on overall response for patients with mm undergoing first single asct at our institution. methods: this retrospective single center study reviewed 136 consecutive myeloma patients who had received the first asct either with bor-hdm (n=15, m/f= 11/4) or single agent hdm (n=121, m/f= 76/45) conditioning regimen. in the single agent hdm conditioning regimen, melphalan was administered intravenously at a total dose of 200 mg/m2 on day -3 and -2 and stem cells were infused on day 0. in the bor-hdm group, melphalan 200mg/m 2 was administered on day -2. bortezomib was administered intravenously at a dose of 1 mg/m 2 on day's -6, -3, +1, and +4 as described in a phase 2 study by intergroupe francophone du myeĺome (ifm). results: all consecutive patients who underwent single asct from january 2010 to march 2018 using bor-hdm as conditioning or hdm were evaluated. conditioning regimen was hdm in 121 patients and bor-hdm in 15 patients. median age was significantly lower in bor-hdm conditioned asct compared to hdm group (62 years vs 52 years, p=000). there was no significant difference for mm subtype, iss stage at diagnosis, prior treatment line among hdm vs bor-hdm cohorts (p>0.5). after a median of 4 cycles of induction chemotherapy, patients in the bor-hd exhibited ≥vgpr of 53.8% (n=7) compared to 56.7% (n=51) in the hdm group (p= p>0.5). pre-asct immune response (if (-)) was reported in 22.3% of patients treated with hdm, higher than that seen in the bor-hdm group (6.7%) (p=0.3). nine (69.2%) patients achieved post-asct immune response (if (-)) ≥vgpr compared to 92 (78.6%) in the hdm group. at the time of this analysis, ten patients in the bor-hdm group and 87 in the hdm group had already died, respectively (p>0.5). a total of 5 (45.5%) patients in the bor-hdm group and 50 (44.2%) patients in hdm group had already progressed (p>0.5). estimated mean os and pfs was shorter for group treated with bor-hdm; 48.2±6.9 mos and 40.2±8.5 mos vs. 76.7±3.9 mos and 67.3±3.8 mos, respectively (p>0.5) (figure-1) . we could not demonstrate the impact of pre or post transplant ≥vgpr immune response on survival and disease free survival. there was no engraftment failure observed on either treatment group and no worsening peripheral neuropathy was developed in the bortezomib arm. conclusions: this single center experience on a small patient pool was able to repeat the prospective randomized study results of ifm. further studies are warranted to explore this regimen, especially when induction treatment with novel agents are employed, with special emphasis on the high-risk myeloma patients where response rates are good but sustainability remains an issue. disclosure: nothing to disclosure the efficacy and safety of bortezomib plus busulfan/ melphalan as conditioning regimen in multiple myeloma undergoing autologous stem cell transplantation: phase 1/2 study background: bortezomib have a powerful antimyeloma activity and was regarded as backbone of therapy in the past decade but its safety and efficacy as a part of busulfan/ melphalan conditioning regimen of autologous stem cell transplantation is yet to be shown. methods: a phase 1/2 trial to explore the safety and activity of a bortezomib on days -6, -3, and +1 added to a conditioning regimen with busulfan and melphalan (bumel, 3.2 mg/kg/day and busulfan during day -5 and -3, 140 mg/ m 2 /day of melphalan on the day -2), in multiple myeloma (mm) patients who received autologous stem cell transplantation following bortezomib-based induction chemotherapy. in phase 1, escalating doses (0.7, 1.0, and 1.3 mg/m 2 ) of bortezomib with bumel were administered in each group with three patients. with determined maximum tolerated dose of bortzomib at a 1.3mg/m 2 /day, cohort with 41 patients were analyzed for phase 2. results: in phase 1, no dose limiting toxicity was observed at a 1.3mg/m 2 /day of bortezomib. in phase 2, overall responses at 3 months was shown as 75% of very good partial response (vgpr) or better and 55% of complete response (cr), whereas post-transplant overall best response included 83% of vgpr or better, and 68% of cr, respectively. with median follow-up duration of 31.4 months, median progression-free survival (pfs) was 26.8 months. the probabilities of 2 years-pfs and overall survival (os) were 56.5% and not estimable, respectively. especially, high-risk cytogenetics were associated adverse survival outcome compared to standard-risk cytogenetics, respectively (pfs, 12.2 vs. 35.7 months, p=0.039; os, 26.7 vs. 73.3 months, p=0.086) . with median 11 days and 10 days for neutrophil and platelet engraftments, any graft failure or delayed engraft was not observed. the common grade 3 or severe non-hematological adverse events included neutropenic fever (73.2%) and stomatitis (14.6%). except three cases with transplant-related mortality due to sepsis, other adverse events were manageable. conclusions: these results demonstrate that bortezomib is safe and can be a part of conditioning regimen in combination with bumel, for patients with transplanteligible multiple myeloma. clinical background: allogeneic stem-cell transplantation (allo-sct) is one of treatment option for patients with multiple myeloma (mm) refractory to novel agents. the reports on allo-sct for mm are limited and it is an important issue to argue appropriate conditioning regimens and stem-cell sources, and patient population who will benefit from allo-sct. methods: we retrospectively analyzed 25 consecutive patients who received allo-sct for relapsed and refractory multiple myeloma (rrmm) between oct 2009 and july 2018 at japanese red cross medical center. characteristics of patients, progression-free survival (pfs), and overall survival (os) were analyzed. results: median age at allo-sct was 47 (range 31-61). twelve patients were male and 13 were female. myeloma type were igg:14, iga: 3, igd: 2, and bence-jones: 6. stem-cell sources were peripheral blood from hlamatched related donor (rpbsct): 6, bone mallow from hla-matched unrelated donor (mud): 4, bone marrow from hla-mismatched donor (mmud): 7, and cord blood (cb): 8. twenty-three of 25 patients received flu/mel-base, one patient received bu/mel-based, and one patient received etoposide/cyclophosphamide-based conditioning regimens. twenty-two patients who transplanted after 2012 received 8gy of total body irradiation (tbi). responses before allo-sct were cr: 8, vgpr: 6, pr: 6, sd: 5. five-year pfs was 22% (95%ci: 6-45) and 5-year os was 42% (95%ci: 16-66). ten patients died during observation period and causes of death were primary disease: 8 and treatment-related mortality: 2. patients with vgpr or better before allo-sct showed significantly better pfs (p=0.04) and os (p=0.011) as compared with others. female recipients showed significantly better pfs (p=0.0034) and os (p=0.035) as compared with male recipients. recipients of mmud showed significantly better pfs (p=0.0034). among 15 patients surviving, 10 patients received treatments including maintenance therapy. conclusions: the reason for better pfs and os in female recipients is unknown. it is interesting that recipients of mmud showed better pfs, suggesting graft-versusmyeloma effects. allo-sct can be an effective treatment option if patients and stem-cell sources are appropriately selected. disclosure: authors declare that there are no conflicts of interest. second autologous hematopoietic stem cell tranpslant versus chemoimmunotherapy in relapsed multiple myeloma after first transplantation: single center data background: combination therapy, mostly triple, followed by autologous hematopoietic stem cell transplantation (auto-hct) is widely accepted as the first-line standard therapy for multiple myeloma (mm). despite the availability of agents such as new immunomodulatory drugs (imids), proteasome inhibitors (pis), histone-deacetylase inhibitors and antibodies, it is still possible to achieve longer and deeper responses, however, multiple myeloma is still not cured and relapse is inevitable. the availability of these novel agents has increased questions for determining optimal treatment of patients with relapse after the first auto-hct. methods: we retrospectively analyzed 60 patients who relapsed according to international myeloma working group (imwg) criteria after 1st auto-hct. first group [salvage chemotherapy(ct)] (n=27) was treated with only chemoimmunotherapy because of early relapse or refractory first auto-sct (within 6 months), ineligible to second transplantation because of co-morbidity, unwillingness to transplant. second group (n=33) (salvage transplantion) was treated with second auto-hct as a salvage therapy. consolidation and long term maintenance treatments were used in both groups. results: there was no difference in sex and age between salvage ct and auto-sct groups [female/male: 11 vs 13/ 16 vs 20; ]. the best response after salvage auto-sct was complete remission (cr) in 62,5%, partial remission (pr) in 15,6% patients, while cr in 44%, pr in 14,8% patients treated with salvage ct. progression free survival (pfs) were significantly better in second transplant group (pfs; 71 % on the first year; 46,9 % on the second year after transplant vs 59 % on the first year; 17% on the second year after the salvage therapy in chemotherapy patients)[p: 0,001]. overall survival (os) in salvage auto-sct group was longer than salvage ct (42,3 % 23.7%), although it did not reach a statistical significance (p>0.05). time to achieving the best response after salvage auto-sct and salvage ct was 1 (0-9) month versus 6,5(2-15) months [p:0,02]. grade 3 or 4 nonhematological toxicities were similar (auto-sct 19%, salvage ct 13%) in both groups. conclusions: salvage auto-hct may provides longer progression free survival with similar toxicity profile according to chemoimmunotherapy especially in patients with sensitive to first auto-sct. it is suggested that earlier and better responses, long-term progression free survival can be achieved with salvage auto-sct. we believe that there will be statistical significance in os such as pfs by increasing the number of patients. the authors believe that large scale randomized clinical trials are needed for optimal treatment of relapsing multiple myeloma after first auto-sct. disclosure: nothing to declare background: one of the conditions for successful transplantation of autologous hematopoietic stem cells (auto-hsct) in patients with multiple myeloma (mm) is the timely recovery of hematopoiesis, which is associated with the quantitative and qualitative characteristics of the graft. one of the key indicators is the content of cd34+ cells in the autograft, which depends on many factors. some of them are due to previous treatment, others are directly related to the patient: age, stage of the disease, features of the hematopoietic stem cells (hsc) microenvironment. the aim of the study was to assess the influence of the immune response genes on the autograft cellularity in patients with mm. methods: а retrospective analysis of the genotyping results was performed. evaluation of 21 loci in 15 genes immune response and harvesting of autologous hsc in 78 patients with mm has been made. hematopoietic stem cell mobilization regimen included cyclophosphamide 4 g/m 2 with granulocyte colony-stimulating factor. genotyping of the immune response genes polymorphic regions was carried out by the polymerase chain reaction with allelespecific primers. the number of cd34+ cells was counted on a 6-color facs canto ii flow cytometer. results: according to the results of the autologous transplant harvesting, two groups of patients were identified. first included 65 patients with an autograft cellularity of more than 2×10 6 /kg body weight. the second group consisted of 13 patients examined with the number of cd34 + cells in the autograft ≤2×10 6 /kg of the patient's body weight. comparing the identified haplotypes of the immune response genes with the cellularity of the transplantation material, it was found that the presence of the mutant allele in the homo-and heterozygous haplotypes of the il1β gene (t-511c) increased the chances of harvesting cellular material with a higher content of cd34+ cells in 4 times (χ2=5.04, p=0.02), and the carriage of the wild type allele in the homo-and heterozygous state of the tlr2 (arg753gln) gene is more than in 15 times (χ2=5.06, p=0.02). currently, it has been shown that single nucleotide or amino acid substitutions in genes can lead to changes in the expression pattern of their final products: increased secretion of interleukin 1β (il-1β) or changes in the spatial configuration and functionality of the receptors (tlr2). thus, in the presence of mutations in the il1β gene, the enhanced synthesis of il-1β influences on fibroblasts, immunocompetent, endothelial, epithelial and other cells, by activating hemopoiesis. in turn, the mutational status of the arg753gln locus located within the tir domain of the tlr2 receptor in the cytosol, determines the spatial configuration of the tlr2 acting as a co-stimulatory receptor of cd4+ cells, which ensure the engraftment of the graft. conclusions: identified haplotypical features of the il1β and tlr2 genes in patients with mm may act as predictors of the response effectiveness to mobilization of hscs in their carriers, which may contribute to the mobilization regimen optimization and will contribute to harvesting the optimal cellularity of an autologous graft. clinical trial registry: none. disclosure: authors declare no conflict of interest. differentiating diffuse from focal pattern on computed tomography: added values of a radiomics approach background: focal pattern in multiple myeloma (mm) seems to be related to poorer survival and differentiation from diffuse to focal pattern on computed tomography (ct) has inter-reader variability. therefore the purpose of this study is to assess if a radiomic approach could help radiologists in differentiating diffuse from focal patterns. methods: we retrospectively reviewed imaging data of 70 patients with mm between january 2013 and september 2018 of whom 61(27 men and 34 women; mean age 54.2 ±3.7) with ct, pet-ct or mri available before bone marrow transplant. two general radiologist evaluated in consensus only ct images to define a focal (at least one lytic lesion >5mm in diameter) or a diffuse (lesions < 5 mm, not osteoporosis) pattern. radiomic analysis on ct thinslice images was then applied with regions of interest (rois) done by one researcher not expert in medical imaging or mm blindly to the condition of the patients. the reference standard to differentiate diffuse from focal pattern was done by radiological evaluation of two expert musculosketal radiologists blinded to the clinical data reviewing ct, mri and pet-ct images. n=104 radiomics features were extracted and evaluated with an open source software. mann-whitney u test for unpaired data with 1000 bootstraps samples was used to compare radiomics features of diffuse and focal patterns and then feature reduction was done to avoid over-fitting. receiver operator characteristic (roc) analysis with area under the curve was done to compare radiologists and radiomics evaluation against reference standard. reading time to perform radiomic analysis was also estimated. results: the pathological group included: 22 diffuse and 39 focal patterns. after feature reduction, 9 features were different (p< 0.05) in the diffuse and focal patterns (n=2/9 features were shape-based: majoraxislength and sphericity; n=7/9 were gray level run length matrix (glrlm)). 150mg/kg). a number of eleven patients did not receive any additional immunosuppression except of post-cy. results: after a median follow up of 11.4 months (range 3.3 -34.5) 28 patients were alive. the 2-year probabilities of pfs and os were 42% (21-63%) and 65% (47-83%).the cumulative incidences (cis) of relapse and nrm at 2 years were 28% (11-45%) and 22% (8-36%), respectively. lower serum albumin level at transplantation (≤36 g/dl) was associated with increased relapses (hr 3.8 (1.2-12.7), p=0.028) and nrm (hr 7.9 (2-30), p=0.0026) and resulted in poorer pfs ), p=0.001) and os ), p=0.048). mmud and haploidentical donors were associated with poorer nrm (hr 6.2 (1.9-20.3), p=0.0027), and resulted in decreased pfs ), p=0.001). the high-risk cytogenetic at diagnosis showed no impact on survival. the cis of acute (grade ii-iv) at day +100 and chronic gvhd at 2 years were 26% (10-42%) and 48% (26-70%), respectively. absence of immunosuppressive medication beside post-cy was associated with poorer os ), p=0.01). conclusions: the conditioning with bu, tt and post-cy leads to a favorable pfs and os due to low incidences of relapse and nrm for patients with multiple myeloma relapsing after autografting. disclosure: nothing to declare methods: between january 2011 and may 2017, we included 51 patients with mm who underwent asct and received bortezomib/lenalidomide/dexamethasone (vrd) consolidation and maintenance therapy, mainly lenalidomide(r) 10mg/day for 21 days every 28 days. results: the median age at transplant was 55 years (46-75). forty-six (90%) of patients received r maintenance, 3 patients received vrd maintenance for higher risk features. median duration of r maintenance was 22 months . r dose was changed for toxicity (grade i-ii) in 17 (33%) patients. twenty-nine (57%) patients relapsed: 13 (25%) patients were shifted to different treatment protocols (treatment change). 6 patients (11%) were kept on the same r maintenance (observation group) and 10 (20%) patients had increased lenalidomide dose with dexamethasone (r/ d group). 6 patients (37%) of the last 2 groups required change of treatment later. the median follow up was 38 months . median tnt was 32 months (7-62). at 2 years, the estimated pfs and os were 39% and 97.5% respectively. the median os and pfs2 (from change of therapy) were 54 and 29 months for patients in the observation group, versus 52 and 27 months in the r/d group, and 45 and 33 months with treatment change, respectively. no statistically significant difference was noted. conclusions: our small monocentric study is limited by its retrospective design and small sample size. however, it suggests that increasing lenalidomide dose as well as adding dexamethasone in selected patients can postpone change to different lines of treatment without affecting survival. disclosure: nothing to declare can the drugs used before autologous hematopoietic stem cell transplantation have impact on cmv reactivation that results in decreased os in myeloma patients after asct? more intensive treatment regimens, such as proteasome inhibitors (pi) and/or immunomodulatory (imid) agents. we performed a retrospective, single center study to evaluate the incidence, risk factors, and outcomes of cmv infection in patients with mm who underwent asct with a high-dose melphalan-based regimen. methods: this study involved a retrospective review of all patients with who underwent asct between january 2015 and november 2018 at our stem cell transplantation center. a total of 144 consecutive adult patients with a diagnosis of mm (median age at diagnosis: 58, range: 35-77) underwent asct following induction treatment with novel agents (pis and/or imids). all patients received antiviral prophylaxis with acyclovir 600 mg/day (n=104) or valaganciclovir 1000 mg/day (n=36). results: baseline patient characteristics, according to induction treatment, are summarized in table1. one hundred-five of the 144 patients (97.2%) were cmv iggpositive before asct. overall, 14.6% (n=21) of cmvseropositive patients developed at least one episode of cmv viremia (cmv dna >100 copies/ml) after a median 24 months (range; 3-48 mos) follow-up. persistent cmv viremia (detectable cmv dna load in more than 2 sequential plasma specimens) occurred in 4.9% (7 of 144) of the seropositive asct recipients and all of them were preventive treated with ganciclovir (n=5) or valganciclovir (n=2). the time from stem cell infusion to the development of cmv viremia ranged from 3 days to 48 days. none of the patients with untreated viremia developed identifiable cmv sequelae. no case of primary infection in seronegative patients at transplant was observed. adding to that none of the patients developed cmv disease post asct. if we analyzed the subgroups of patients according to induction therapy (pi-based, imids, pi+imid), the incidence of post-asct cmv reactivation was higher but not statistically significant, in patients who received only pi vs pi+imid (13 (61.9%) vs 8 (38.1%); p=1.00). in univariate analysis, we could not demonstrate the importance of induction therapy with novel agents the occurrence of a post-asct cmv reactivation requiring antiviral treatment. however, statistically significant association found between the disease < vgpr status at asct and cmv reactivation (61.1% vs. 38.9%; p=0.035). after a median follow-up 14.3 months (range; 1-45.9 months), there was no significant impact on pfs, however there was significant decrease in estimated mean os who had cmv reactivation when compared to those without cmv reactivation (34.1±4.5 vs. 41.9±1.3; p=0.002) (figure-1) . conclusions: cmv establishes lifelong latency within host cells and in the setting of impaired cellular immunity; cmv may reactivate from latency, disseminate, and directly cause life-threatening disease. our data suggests that mm patients treated with pi-based induction regimens and immunological response < vgpr at time of asct seem to have higher risk of developing symptomatic cmv reactivation. however, further studies on a large number of patients are warranted to clarify these findings. clinical background: high-dose therapy followed by autologous stem cell transplantation (asct) has been shown to prolong survival in patients with multiple myeloma (mm) in randomized trials. however, these trials only include patients aged < 65 years. data regarding safety and outcomes in this patient population is lacking. methods: the aim of this study was to compare safety profile and outcomes in mm patients younger and older than 65 years-old who underwent asct in our unit from july 2014 to october 2018. patient's demographics, clinical characteristics, transplant related variables and probability of admission to the intensive care unit (icu) were analyzed. patients aged < 65 and ≥65 years-old would be called m1 and m2, respectively, from now on. sorror index was used to estimate risk of mortality in the two cohorts. results: a hundred and eleven patients with mm underwent asct in the study period. median age was 60.9 years-old (range 35-73) and 53.2% were male. thirtythree (28,8%) patients were ≥ 65 years. the probability of having a high risk comorbidity index was similar in both groups (m1 11,7 vsm2 12,1%). the median cells obtained in the apheresis procedure was 5.35 x10 6 (1,70-15.92) in m1 compared to 3.77 x10 6 (1.96-10-48) in m2. there were no differences in median admission lenght between the 2 cohorts (m1:19 days vs m2: 20 days). median days for neutrophil recovery above 1000 was 12 days in both groups with a wider range in m1 (11-23) compared to m2 (11-15) . no differences were found in platelet recovery above 100.000 (m1 20 days vs m2 21 days). median packed red blood cells and platelets transfusions were 1 (0-6) and 3 (0-16), respectively, in m1. in m2 cohort, they were 2 (0-5) and 4 (0-14), respectively. the incidence of grade 3-4 mucositis in m1 and m2 was 50.6% and 44.1%, respectively. there were no statistically significant differences in terms of using morphine for pain control between the two cohorts (m1, 63,4% vsm2, 58,8%). none patient requiered total parenteral nutrition (tpn) in group m2 and only one in group m1. the incidence of icu admission was 1.5 times higher in patients aged ≥65 than in patients < 65 years-old 6,1% vs3,9%), but differences were not statistically significant (p = 0.52). there were no deaths during the transplant procedure in any of the 2 cohorts conclusions: 1) in our series, high-dose therapy followed by autologous hematopoietic cell transplantation in mm patients aged ≥65 was feasible. 2) transplant procedure in older patients was as safe as in patients < 65 years-old. 3) no differences were found in terms of graft, transfusion support, transplant related complications and length of admission. 4) age should not be a limiting factor in considering the modality of asct in this patient population disclosure: nothing to declare the correlation between the kinetics of peripheral blood counts and the response to treatment after high-dose melphalan with stem cell support in multiple myeloma patients background: the long-term survival of mm patients has dramatically increased in the last 20 years, particularly for younger patients. this is attributable in part to the introduction and development of high dose chemotherapy with melphalan with stem cell support (hdm-asct). currently, frontline asct is still considered the standard of care for all eligible patients. many prognostic factors pre and post transplantation have been identified, e.g.: age, comorbidities, cytogenitcs, response to treatment and disease status prior to and post transplantation. to our knowledge there is no data correlating between kinetics of counts response to melphalan and prognosis. our aim was to assess the prognostic significance of the neutrophil and platelets decaying counts after high dose melphalan. methods -we retrospectively analyzed our cohort of 159 multiple myeloma patients who underwent hdm-asct at the hadassah medical center bone marrow transplant department, between the years 2007-2015. the kinetics of neutrophil and platelet decay during the first two weeks after melphalan administration was fitted using linear and exponential mathematical models. methods: we retrospectively analyzed our cohort of 159 multiple myeloma patients who underwent hdm-asct at the hadassah medical center bone marrow transplant department, between the years 2007-2015. the kinetics of neutrophil and platelet decay during the first two weeks after melphalan administration was fitted using linear and exponential mathematical models. results: factors associated with prolonged os in univariate analysis were: iss stage 1(p=0.024), ≤2 lines of treatment prior to asct(p< 0.001), favorable cytogenetics(p=0.004), response to treatment (pr or better, p=0.046) and rapid linear neutrophil decay (p = 0.046). in multivariate analysis, only ≤2 lines of treatment before hdm-asct and rapid linear neutrophils count decay remained statistically significant for os prolongation. no predictive threshold value of the neutrophil decay incline was found. improved pfs was associated with ≤2 lines of treatment prior to asct, and the response status after hdm-asct (p=0.003, p=0.019). additionally, toxicity evaluation showed prolonged neutropenia to be associated with inferior os (hr = 1.139, p=0.03) and rapid exponential decay of neutrophil counts to correlate with higher incidence of mucositis (p = 0.013). fast platelet decay was associated with delayed platelet engraftment (p< 0.01) conclusions: we have shown that rapid linear decay in neutrophil counts predicts better os without a significant benefit in pfs in mm patients undergoing hdm-asct. this discrepancy might reflect the problematic estimation in a retrospective analysis of pfs. rapid decrease in neutrophils and platelet counts was associated with more toxicity: higher mucositis rate and delayed engraftment, respectively. therefore a rapid decay of blood counts after hdm-asct appears to be an in-vivo phamacodynamic marker of higher efficacy and toxicity of melphalan. disclosure: nothing to declare p564 do we need to freeze hematopoietic cells for autotransplants in patients with myeloma conditioned with melphalan? daniel garcia belmonte 1 , beatriz aguado bueno 1 , miguel herrero coderch 1 , rafael de la camara 1 background: multiple myeloma (mm) is the most frequent indication of auto-hsct, representing 51% of all auto-hsct in 2016 (passweg jr. bmt 2018; 53:1139-48) . nearly all are performed with peripheral blood progenitor cells (pbpc), and melphalan 200 mg/m2 is considered the gold standard conditioning regimen. the standard procedure consists in obtaining progenitors, cryopreserved with dimethyl sulfoxide (dmso) and stored and subsequently thawed and re-infused in the patient on day 0. the procedure of cryopreservation is expensive and has some inherent toxicities (dmso) and loss of cells during the procedure. several groups have used non-cryopreserved progenitors showing some benefits compared with cryopreserved transplants, mainly a faster engraftment and a shorter length of hospitalization. objective: to compare noncryopreserved vs cryopreserved auto-hsct in mm methods: we perform an unicentric, retrospective study on 23 consecutive first auto-hsct mm patients transplanted with pbpc between nov-2011 and oct-2018, and conditioned with high dose melphalan (200 mg/m2). the median follow-up was 1620 days (range: 44-2413). 9 patients received non-cryopreserved and 14 cryopreserved auto-hsct. patients characteristics, without differences between non-cryopreserved vs cryopreserved: women/men (15/8); median age was 65 years (range 49-72); in the majority auto-hsct was done as consolidation after first line therapy (78%); year of transplant ≤2014 (61%), ≥2015 (39%). the number of infused cd34 cells were not different: median 3.5 x10 /kg (range 1.7-11.5) in noncryopreserved patients and 3.75 x10 /kg (range 2.1-17) in cryopreserved patients. results: we didn´t observe significant differences in the day of engraftment between non-cryopreserved vs cryopreserved although always was a little bit faster in the noncryopreserved group with a tendency to faster platelet engraftment (>50000/mm3): >20000 platelets/mm3 (median day: 12 vs 12.5, p 0.2), >50000 platelets/mm3 (median: 13 vs 16 days, p 0.09); >500 neutrophils/mm3 (median: 13 vs 13.5 days, p 0.3). the media of days of hospitalization was shorter in non-cryopreserved patients (18 vs 21 days) although not statistically significant (p 0.14). transplantrelated mortality at day +100 was 0% in both groups. overall survival at 5 years was not different: 83.3% in in non-cryopreserved vs 61.5% in cryopreserved patients (kaplan-meier, log-rank p 0.27). the accumulative incidence of relapse at the median follow up (1600 days) was similar: 33.3% in non-cryopreserved vs 35.9% in cryopreserved patients. conclusions: in our short experience, auto-hsct with non-cryopreserved pbpc in myeloma patients conditioned with high dose melphalan obtain similar results to those performed with classical cryopreserved pbpc and might has a faster platelet engraftment and shorter length of hospitalization. if no advantages are associated with cryopreservation, the simplicity of using fresh product is appealing. disclosure: nothing to declare p565 abstract withdrawn. . results: a early death is observed in one pt (group 1) and 5 pts(group 2). the median delay of aplasia is 14 days (10-22) and 12 days (9-17) respectively. in group 1, among the evaluable pts, 30/37 (81%) are in cr, 5 pts in pr and 2 refractory. in group 2, cr: 35/43 (81%), pr: 6 and 2 refractory. a relapse is observed in 32 pts/37 (86,5%) in group 1 and 35 pts/43 (81%) in group 2 with a frequency of 54% and 41% respectively in the first 24 months. at 36 months: 13 pts/38 (34%) in group 1 and 19 pts/49 (38%) in group 2 are dead. at 60 months: 20 pts (52%) and 29 pts (59%). at 120 months: 31 pts (81,5%) and 37 pts (75,5%). the overall survival (os) of the group 1 and group 2 pts were 66% and 62% at 36 months; 47.5% and 38.5% at 60 months; 18% and 24,5% at 120 months respectively (without significant difference). the event free survival (efs) of group 1 and 2 pts were 37% and 36,5% at 36 months, 25% and 13% at 60 months and 8% and 15% at 120 months respectively (without significant difference). conclusions: these 2 protocols with equivalent toxicity allow obtaining of long-term equivalent results on the response rate early transplant, on the rate of relapse and on the overall survival. these results are identical to those of fermand (1999) . disclosure: nothing to declare background: dimethylsulfoxide (dmso) is a major intracellular cryoprotectant, used for cryopreservation of stem cells. it is toxic to both cells and patients at temperatures above 0 o c. reduction of this effect is achieved by either washing of cells after thawing or by reduction of dmso during freezing and storage. the latter requires addition of extracellular cryoprotectants to the freezing media. we assessed the effect of low dmso concentration and different hematocrits of the frozen cells on cell viability and hematologic recovery in patients, transplanted for multiple myeloma. methods: cells were non-programmed frozen and stored at -80 o c in a cryoprotectant solution achieving final concentrations of 5% dmso, 3.6% of hydroxyethyl starch (hes, weight average molecular weight 450 000 da) and 3% of human serum albumin. the cell concentration in the frozen product for the first 77 patients (84 transplantations) varied between 100x10 6 and 250x10 6 cells/ml. in an attempt to reduce the amount of dmso infused, for the rest of the patients (n=172; 192 transplantations) we further decreased the volume of the freezing suspension by removal of the entire plasma. the average age of the transplanted patients was 55 (35 -71). the cells were bedside thawed at 37 o c water bath. the average cell dose was 2,95x10 6 /kg (1,3 -9,2x10 6 /kg). results: viability of the stem cells following thawing assessed by trypan blue exclusion was 95,34% . the hematocrit of the frozen cells had no effect on cell viability (94,80%(low) vs 95,52%(high)). the major complaints, if any, during stem cell infusion were coughing and an increase in nausea and vomiting induced by the prior conditioning. the average time for hematological recovery was 11,66 days (between 9 and 19) for the neutrophils, and 12,17 (between 9 and 20) days for the platelets. there was no significant difference in viability and hematologic recovery (11,86 and 12,60 vs 11,59 and 12,02) between patients receiving cells frozen with low or high hematocrit. conclusions: dimethylsulfoxide, despite its cryoprotective properties, is toxic for stem cells at temperatures above zero c and induces many side effects (cardiac, neurologic, respiratory, etc.) in the patients. to reduce those side effects we use lower dmso concentration, high hematocrit resulting in lower volume of the frozen cell suspension, thus reducing the final quantity of dmso to be infused to the patients. this does not affect the cell viability or the hematologic recovery of patients after transplantation. our easily performed method for unprogrammed freezing of stem cells with final dmso concentration 5% at -80 o c is safe, well tolerated, and provides cryopreservation, which allows high viability and stable cell engraftment, while reducing the undesired side effects of dmso. disclosure: nothing to disclose the conditioning regimen consisted of melphalan for most of the patients. the average age at the time of transplantation was 55 years (35 -71). patients were transplanted with an average cell dose of 2,95x10 6 /kg (1,30 -9,20x10 6 /kg) for the first transplantation and 2,36x10 6 /kg (1,64 -3,19x10 6 /kg) for the second one (every patient received the same cell dose as for the first) with average cell viability 95,34 % (70 -99%), with little difference between first and second transplantation. results: the average time for hematological recovery was 11,60 (between 9 and 19) days for the neutrophils, and 12.11 (between 9 and 20) days for the platelets. we found no correlation between the cell dose and the hematological recovery. there was no difference in the hematopoietic recovery between the first and the second transplantation in the patients, who underwent tandem or two transplantations. conclusions: recovery time is considered by some to be a function of the effective stem cell number. we did not find such correlation, probably because in the analyzed group all the patients, except four of them, received a dose greater than 2 x10 6 /kg cell, which is accepted as a safe dose for autologous stem cell transplantation. disclosure: nothing to disclose p569 plerixafor-mobilized patients have a high risk of noninfectious fever during engraftment after autologous peripheral blood stem cell transplantation background: plerixafor enables rapid and efficient mobilization of hematopoietic stem cells. however, its impact on adverse clinical events after autologous peripheral blood stem cell transplantation (pbsct) is not fully understood. fever is one of the major complications in the preengraftment phase of pbsct. in this research, we focused on non-infectious fever around the time of bone marrow recovery and investigated whether plerixafor as mobilization therapy plays a role in engraftment fever. methods: we reviewed 80 autologous pbscts for treatment of multiple myeloma at the japanese red cross medical center between 2012-2018. non-infectious fever was defined as temperature ≥37°c with onset between two days prior to and two days after engraftment without clinical or microbiological documentation of infection. results: patients were mobilized by cyclophosphamide and filgrastim in 57.5% (n = 46) and filgrastim and plerixafor in 42.5% (n = 34). the median number of transfused cd34+ cells were 2.61×10 6 /kg and 3.45×10 6 / kg, respectively (p=0.002). patients transfused with plerixafor-mobilized grafts had a higher risk of noninfectious fever (85.3% vs 63.0%, p< 0.05). cd34+ cell number or cyclophosphamide pretreatment had no relationship to non-infectious fever. the recovery of lymphocytes was more rapid in plerixafor-mobilized patients (p=0.001). however, the number of lymphocytes was not associated with non-infectious fever. conclusions: combination of filgrastim and plerixafor as mobilization therapy resulted in an increased risk of noninfectious fever during engraftment comparing to mobilization with cyclophosphamide and filgrastim. while the mechanism remains unclear and requires further studies, plerixafor-mobilized grafts may result in an unintended increase in engraftment fever. clinicians should be aware of this possibility if patients are transplanted with those grafts. disclosure: ks received honorarium outside the submitted work from janssen, novartis, celgene, ono pharmaceuticals, takeda, fujimoto pharmaceuticals and srl. ti received honorarium outside the submitted work from janssen, celgene, ono pharmaceuticals and takeda. we assessed the efficacy of a new conditioning regimen consisted of decitabine (dec), busulfan (bu), cyclophosphamide (cy), fludarabine (flud) and cytarabine (ara-c) for allo-hsct in patients with mds and mds/ mpn. fifty patients were enrolled, including 46 with mds and 4 with cmml. patients received dec 20 mg/m 2 /day on days -9 to -5, combining bu/cy/ flu/ ara-c modified preparative regimen. results: at a median follow-up of 522 (15-1313) days, the overall survival (os) was 86%, disease-free survival (dfs) was 78%, and relapse incidence was 11%. the incidence of severe acute (grade iii/iv) graft-versus-host disease (gvhd) was 22%, and that of (predominantly mild) chronic gvhd was 40%. os at 2 years was 74% for mds patients with high risk, 86% for mds patients with very high risk, respectively. the survival was delightful in patients with poor-risk mutations, such as tp53 and asxl1, (86%) and with three or more gene mutations (77%). among the total 12 patients with poor-risk mutations in our research, only one patient (8%) with tp53 relapsed and one (8%) with asxl1and tet2 died. result of continuous observation after transplantation, the percentage of nk cells in the peripheral blood of all patients who had received dec/flu/bu/cy/ara-c conditioning increased at day 28, which may essentially contribute to disease control post-transplantation. conclusions: in summary, the addition of a 5-day schedule of decitabine to a flu/bu/cy/ara-c conditioning regimen has proven feasible, with a low level of toxicity and promising early disease control especially in patients with high risk mds. disclosure: there are no conflicts of interest. the sfgm-tc mds score at day 180 is associated with post-transplant outcomes in patients with myelodysplastic syndrome who underwent cd34+ selected allogeneic stem cell transplant conclusions: in patients with mds undergoing tcd-hct, the sfgm-tc score at day 180 is significantly associated with survival. the lower incidence of acute gvhd in recipients of cd34-selected transplants and the use of myeloablative condition regimens, with lower relapse, may explain the difference with the original finding that the sfgm-tc was predictive at day 100 in unmodified grafts. disclosure the most frequent grade 3, 4 toxicities were thromobocytopenia and neutropenia. infections developed in 12 patients (33.3%), neutropenic fever in 8 (19.4%). five patients (13.9%) either developed or experienced exacerbation of acute graft versus host disease (gvhd), nonechronic gvhd. conclusions: azacitidine use is associated with only modest activity in patients who relapse after allo-hsct. however, in patients who respond to treatment it may allow for a durable disease control. disclosure: the authors declare no competing conflicts of interest background: somatic mutations in mds patients are closely related with clinical phenotypes and prognosis in mds patients. but whether mutations are prognostic for outcomes after allogeneic hematopoietic stem-cell transplantation (allo-hsct) remains to be elaborated. methods: targeted mutational analysis were performed on samples obtained before transplantation from 134 patients underwent hsct. we analyzed the relationship of mutations and clinical outcomes. results: all 134 patients carried more than one somatic mutations, most frequently in kmt2d(67.16%), arid1b (61.94%), ccdc168(52.24%), pclo(47.01%), asxl1/2 (46.27%), srcap(44.78%), u2af1(42.54%), dnah2 (41.79%), ush2a(41.04%) and tet2(34.33%). tp53 mutations were associated with higher ipss-r risk, complex karyotype and monosomal karyotype. dnah2 were more frequent in pediatric patients. in univariable analyses, tp53 mutations were related with decreased disease-free survival (p=0.047); dnah2 mutations were related with increased disease-free survival (dfs) (p=0.038). in multivariable analysis including ipss-r stratification, gvhd, hct-ci and candidate genes, dnah2 mutations were independently associated with better dfs(p=0.027). conclusions: dnah2 mutations is independently associated with better outcomes in mds patients treated with allo-hsct while tp53 may predict unfavorable outcomes. accounting for these somatic mutations may help better selection of candidates for allo-hsct among mds patients. disclosure background: there is a controversy among experts if and how patients with mds and saml should receive cytoreductive therapy before transplant. while aiming to reduce disease burden in order to lower the risk of relapse after transplant cytoreductive therapy is associated with several drawbacks. besides a considerable risk for toxicity and mortality preventing patients to proceed to transplant cytoreductive therapy may also favour the selection of resistant clones which may be difficult to treat at relapse. methods: to address this hypothesis we retrospectively analysed the response and survival following salvage therapy in 73 patients with mds and saml who had relapsed in median 5.6 months (1 to 110 months) after allo-sct according to their pre-transplant strategy (upfront transplantation n=32 44%; induction chemotherapy [ctx] n=26 36%; hypomethylating agents [hma] n=15 20%). results: the majority of these 73 patients received salvage therapy with hma (n=58, 79%; aza n=57, dac n=1) mostly in combination with dli, while the remaining received other salvage treatments (intensive chemotherapy n=1, dli alone n=1, 2 nd transplant n=3, bsc n=5, miscellaneous n=2, missing information n=3). when focussing on those patients treated with hma and dli it became apparent that a significantly higher proportion of patients in the upfront group (58%) achieved cr after salvage therapy in comparison to pre-treated patients (10% cr, p=0.0005; ctx group 5% cr; hma group 18% cr). accordingly, overall survival (os) calculated from the time of relapse was significantly longer in patients in the upfront group than in the group of pre-treated patients (2-year os 59% vs. 19%, p=0.0001). conclusions: overall, these findings imply that pretransplant therapy may favour the iatrogenic selection of resistant clones, which poorly respond to salvage therapy with hma and dli in case of relapse after allo-sct. furthermore, the results support the concept that an upfront transplant strategy is a promising alternative for patients with mds and saml that can be augmented by salvage therapy with hma and dli. disclosure: ts and gk received travel support, lecture fees and research funding from celgene gmbh conclusions: in our country, this procedure has shown to be feasible and we hope to improve it, with better infection control and by acquiring more experience related to the management of these patients. background: extramedullar relapse of mds is a rare complication after allogeneic stem cell transplantation. we present the case of a 69-year-old woman who was admitted into hospital because of insecure walking. paresis of both legs, hypaesthesia of the inner thighs, increased effort at urinating, reduced sphincter tonus, central paresis of the right arm and discreet paresis of the right facial nerve were documented at neurological exam. mri showed a large tumour of the dorsal thorax that immured the adjacent ribs and spine, affected the processus transversus of t9-10 and invaded the spinal canal. the patient had undergone ric allogeneic stem cell transplantation five years ago for mds-eb 2 with complex aberrant karyotype. following an uneventful course and no signs of gvhd, she had been off immunosuppression since 4,5 years. at the time of the admission the patient had slightly lowered wbc (2,9 gpt/l) and plt (111 gpt/l) and clearly increased ldh (793 u/l). methods: histology of a ct-based biopsy of the paravertebral tumour showed an infiltration of the muscles by blastous cells that were cd45-, cd34-, pax5-positive, tdt and cd79a were questionably positive. provisonal diagnosis therefore was lymphoblastic lymphoma, pox tested negative. the bone marrow was hypocellular with increased numbers of mature lymphocytes, but no definite signs of malignancy. cerebrospinal fluid revealed 1574 cells/μl with 90% blasts. immunotype was cd34, cd117, cd13, cd33, hla-dr positive, pox and lymphatic markers were negative. because of this we finally suspected meningeosis leucaemica. we completed the diagnostic workup with genetical and chimaerism tests and compared the result to the patients' mds before allogeneic stem cell transplantation. [[p580 image] 1. mri scan of the large thoracic tumour] results: cerebrospinal fluid (csf) cells consisted of 92% recipient cells, whereas peripheral blood cells were 100% donor. high risk mds at transplant displayed a complex caryotype including trisomy 8 and tetrasomy 8, now 10% of the cells in csf showed trisomy 8 and 70% tetrasomy 8. chimerism and fish of the solid tumour could not be performed, coexpression of myeloid markers within the tumour is pending. conclusions: in conclusion the patient has meningeosis as a result of exclusively extramedullary relapse of myeloid blasts originating from the initial high risk mds with blast excess and complex aberrant caryotype. the evolution of a trisomy 8 clone to tetrasomy 8 clone in relapse is linked to extramedullar manifestations. whether the solid tumour represents myeloid sarcoma with coexpression of lymphoid markers, extramedullary relapse of mds with lymphoid differentiation or, less likely, a separate lymphobastic lymphoma, is not yet clear. disclosure background: adoptive t cell therapy with genetically engineered t cells is a potent innovative immunotherapeutic approach for cancer treatment. unfortunately, the use of t cells redirected against tumor antigens, is severely limited by 1) the difficulty in identifying appropriate cell surface antigens, that could be targeted by car t cells and 2) the paucity of tumor-specific t cell receptors (tcrs) against shared, oncogenic antigens. methods: focusing on wilms´tumor 1 (wt1), a tumorassociated antigen overexpressed by acute myeloid leukemia and several solid tumors, we designed and implemented an innovative protocol for the rapid isolation of wt1-specific t cells and for the generation and characterization of a library of wt1-specific tcrs displaying different human leukocyte antigen (hla) restrictions, to be exploited by tcr gene transfer and tcr gene editing. to this aim, we repetitively stimulated t cells with autologous antigen-presenting cells, including immortalized b cells, pulsed with overlapping peptides spanning the entire wt1 protein. t cell recognition was assessed by flow cytometry in terms of cd107a expression and ifnγ production. recognized peptides were mapped by a deconvoluting grid and t cell clonotypes were longitudinally tracked by tcrαβ sequencing. results: we successfully expanded tumor-specific t cells from 14 consecutive healthy donors, in an average of 4 rounds of in vitro stimulations. the ability of wt1specific t cells to recognize naturally processed epitopes and their on-target specificity was demonstrated upon coculture with antigen-expressing targets including primary leukemic blasts. tracking of the tcrαβ repertoire during culture led to the identification of 20 clonotypes that recognize several tumor-associated peptides and are restricted by more than 5 hla alleles, including hla-a*02:01. tcrs were then expressed via genome editing. briefly, simultaneous editing of endogenous tcr α and β chain genes was achieved using crispr/cas9 technology (efficiency >90%), followed by transduction of t cells with lentiviral vectors encoding wt1-specific tcrs (efficiency >95% of cd8 + t cells). phenotypic characterization of edited t lymphocytes showed a major enrichment of cells harboring t stem cell memory properties. functional validation of the edited t cells is currently ongoing. preliminary results of a 6 hours coculture experiment show that tcr edited t cells kill fresh wt1 + leukemic blasts, harvested from hla-matched patients, with an efficiency up to 70% at an effector to target ratio of 5 to 1, while no killing of controls is observed. conclusions: we set up a protocol enabling consistent and efficient hunting for tumor-specific tcrs with no need for labor intensive t cell cloning. tcr genes can be easily and rapidly used to redirect t cell specificity against cancer cells by tcr gene editing. disclosure: chiara bonini: research funding from intellia therapeutics p583 car t cell therapy targeting relapsed or refractory cd19+ lymphoid disease with third-generation vector rv-sfg.cd19.cd28.4-1bbzeta maria-luisa schubert 1 , anita schmitt 1 , leopold sellner 1,2 , brigitte neuber 1 , angela hückelhoven-krauss 1 , kunz alexander 1 , lei wang 1 , gern ulrike 1 , birgit michels 1 , susanne hofmann 1 , carsten mueller-tidow 1,2 , dreger peter 1,2 , michael schmitt 1, 2 background: t cells genetically engineered to express chimeric antigen receptors (carts) directed against cd19 have demonstrated significant efficacy in patients with iymphoid malignancies including relapsed or refractory (r/r) b-lineage acute lymphoblastic leukemia (all) or r/r b-cell non-hodgkin's lymphoma (nhl). access to cart treatment for patients in europe has been limited so far given that the vast majority of cart trials have been performed in the united states and the p. r. of china. here we present the preliminary results of the first investigator-initiated trial (iit) cart trial in germany. hd-car-1 (eudract no. 2016-00 4808-60; nct03676504) is a phase i/ii trial with in-house cart manufacturing which was initiated in september 2018 at the university hospital heidelberg. methods: adult as well as pediatric patients with r/r all and patients with chronic lymphocytic leukemia (cll) or nhl including diffuse large b-cell lymphoma (dlbcl), follicular lymphoma (fl) or mantle cell lymphoma (mcl) are treated with autologous t lymphocytes transduced with a third-generation car retroviral vector (rv-sfg.cd19.cd28.4-1bbzeta) targeting cd19. the main purpose of hd-car-1 is to evaluate safety and feasibility of escalating third-generation car t cell doses (1-20×10 6 transduced cells/m 2 ) after lymphodepletion with fludarabine and cyclophosphamide. patients are monitored for cytokine release syndrome (crs), car-t-cell related encephalopathy syndrome (cres) and/or other toxicities. in vivo function, survival and anti-tumor efficacy of carts are assessed. results: to date, three patients (cll, dlbcl and mcl, respectively) have been enrolled and subjected to leukapheresis. high numbers of transduced carts were harvested on day 10 of culture (82-123x10 6 carts). transduction efficiency ranged between 33 and 42%. cart products were sterile and free from mycoplasms and endotoxins. no production failure occurred and all patients received the cart product. no signs of crs or cres > grade 2 have been observed. assessments of clinical responses are pending and will be presented at the conference along with updated technical results. conclusions: for hd-car-1, gmp-conform leukapheresis as well as cart manufacturing was effective. administration, patient monitoring and follow-up were performed in-house providing independency from transport or production sites outside the university hospital heidelberg, altogether suggesting that academic cart iits are feasible in germany. clinical background: the prognosis of adult patients (pts) with relapsed/refractory (r/r) precursor b-acute lymphoblastic leukemia (all) is dismal, including with allogeneic hematopoietic stem cell transplantation (allo-hsct). blinatumomab, a bispecific cd19-directed cd3 t-cell engager and inotuzumab ozogamycin (io), a cd22-directed antibody-drug conjugate revolutionized the field, improving their outcomes. anti-cd19 chimeric antigen receptor t (cart) cell therapy has led to further progress and improved outcome (jacoby e; am j hematol, 2018). nowadays, patients with r/r b-all can be offered both therapies, but there are limited data on the safety and efficacy of cart -cell therapy post antibody treatment. we detailed our single center experience in this regard. methods: this report is a part of a single center, phase 1b/2 study on therapy of b-cell malignancies with locally produced cart-cells (nct02772198). the approach uses autologous t cells with car construct that is composed of an anti-cd19 single-chain fv, cd28 co-stimulatory and cd3-zeta intracellular domains. cd19 expression on the blasts was documented prior treatment in all pts by flow cytometry. all pts received 1 x 10 6 /kg cart-cells after lymphodepletion with fludarabin and cyclophosphamide. results: six pts (2 males and 4 females) with r/r b-all were enrolled, including one with ph-positive b-all. the median age was 42 years (25-59). median number of prior therapy was 4 (3) (4) (5) . five pts had prior allo-hsct. four pts were given antibodies as the last therapy prior to cart cells. two pts received blinatumomab resulting in pr in one of them. two additional pts received io (1 after failing blinatumomab) achieving mrdpositive cr. cytokine release syndrome occurred in all pts and was severe in only one patient who required tocilizumab treatment. this patient was also the only patient who experienced neurotoxicity (grade 3), and was treated with dexamethasone. this patient eventually died 18 days post infusion of cart cells due to severe pseudomembranous colitis, toxic megacolon and sepsis. all pts had prolonged neutropenia for a median of 14 days (12-18) after the infusion of cart cells. at day 28 after infusion of cart-cells the cr for the entire cohort was 67%: three pts with mrd-negative and one with mrd-positive response. among the four pts who received antibodies prior the cart-cells, one patient had mrd-positive and two pts had mrd-negative response. the patient with ph positive b-all had progressive disease during the treatment. two pts were referred to second allo-hsct from other donors. one patient with mrd-negative response relapsed after the second transplant and was treated by salvage therapy. the second patient with mrdnegative response demonstrated prolonged remission (20 months) even without second transplantation. with a median follow-up of 11 months (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) the median progression-free and overall survival for the entire cohort were 7.5 and 9 months, respectively. conclusions: autologous anti-cd19 car t-cell therapy after debulking treatment with antibodies, including blinatumomab and/or io, is feasible and results in high response rates in pts with r/r b-all. patients may respond to anti-cd19 car t-cell therapy even after failure to their last salvage therapy with blinatumomab, which demonstrates similar mechanism of action. clinical background: genetically engineered t cells expressing a chimeric antigen receptor (car-t) targeting specific antigens present on acute lymphoblastic leukemia (all) blasts have generated promising results in children and adults with relapsed and refractory disease. the below report provides an insight of lineage switch occuring as a result of intense immunological selection after car-t cell therapy, even with a tumor clone that has no potential for this switch. methods: an eight year old caucasian male with precursor b (pb) cell lymphocytic leukemia was treated with cd 19 directed car-t cell therapy in third remission, and after relapse after previous bone marrow transplantation (bmt) . he was diagnosed with t(12;21) pb cell all at 2 years of age and treated with bfm protocol. he relapsed 8 months after completion of maintenance therapy, and had a 9/10 mmud bone marrow transplant after etoposide, tbi and alemtuzumab conditioning therapy. he had cutaneous acute and chronic gvhd but 8 months post-transplant, he relapsed again with pb cell all, with the same cytogenetic and immunophenotypic disease characteristics.. he was treated with lymphodepleting chemotherapy with fludarabine, cyclophosphamide and alemtuzumab followed by infusion of cd19 directed car t cells. he developed cytokine release syndrome of grade 1 severity manifested as persistent fever, associated with car t cell expansion in the blood. after car-t infusion,there was no detectable b cell all clone in the marrow by pcr and the cytogenetics were negative for t(12:21) translocation. 8 months after the car t cell therapy, he was found to have a mrd positive disease which was monitored closely. results: we document clonal evolution from cd19 negative, mrd positive disease to aml, with the same ig rearrangement (the same clonal disease) but with complete myeloid phenotype mpo, cd33, cd13 positive disease. there was cytogenetic evolution of the underlying clone but the original t(12;21) was retained within the evolved karyotype. sadly, our patient developed fludarabineneurotoxcity during an attempt to induce aml remission, and further curative-intent chemotherapy was not possible. conclusions: there are two case reports of mll rearranged b-all acquiring a clonally related myeloid phenotype associated with cd19-negative escape after cd19 directed car t cell therapy,so far. but, this is the first post car-t cell therapy transformation of all to aml with etv6-runx1 mutation, which is not recognised to have such lineage-switch potential. unlike mll, etv6-runx1 translocation in the pathogenesis of acute myeloid leukemia is not been reported in the literature. the theory behind such transformation is an intense immunological selection of the tumor, driving it to myeloid differentiation with additional clonal cytogenetic events. disclosure: nothing to declare p586 ivac-all-1: interim analysis of a phase i/ii clinical study on personalized peptide vaccination based on patient-individual tumor-specific variants in relapsed pediatric acute lymphoblastic leukemia armin rabsteyn 1,2 , christopher mohr 3 , olaf witt 2,4 , roland meisel 2,5 , cristiane chen-santel 6 , tobias feuchtinger 2,7 , christopher schroeder 8 , jakob matthes 8 , background: acute lymphoblastic leukemia (all) is the most common pediatric malignancy. standard chemotherapy is a successful treatment in 80% of patients, only about 20% develop a relapse, however these patients have a dismal prognosis. prevention of relapse after firstline chemotherapy or stem cell transplantation (sct) is therefore an urgent clinical need. we established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient hla types. we started clinical translation of this approach by starting a phase i/ii clinical trial in 2016 (nct03559413). besides feasibility and toxicity assessments, we aim to assess the capability of the peptide vaccination to induce neoantigen-specific t cell responses in high-risk all patients to target residual tumor cells and prevent leukemic relapses. methods: key inclusion criteria are: pediatric patients with all who suffered from second relapse after standard therapy or first relapse after sct. hematological remission has to be reached prior to vaccination. nonsynonymous mutations are identified by whole exome and transcriptome sequencing of patient leukemic blasts and healthy reference tissue. hla binding peptides harboring the altered amino acids are subsequently predicted in silico by algorithms syfpeithi, netmhc and netmhcpan for the patients' individual hla type. vaccine cocktails consisting of 3-5 individual peptides are produced and formulated under gmp conditions. the vaccination schedule is 16 vaccinations over 33 weeks using gm-csf and imiquimod as adjuvants. response to the vaccination is monitored by detection of t cells recognizing the vaccinated peptides occurring over time in peripheral blood of patients by prestimulation and intracellular cytokine staining. results: until now, 23 patients were recruited, for 17 of those, whole exome sequencing was performed to identify all-specific snvs using a comparative bioinformatics pipeline. we found an average of 118.8 mutations per patient on dna level. based on these data, an average of 178 hla binders derived from neoantigens could be predicted per patient. an average expression of 34.4% of mutations was assessed by rna sequencing. in all cases validated mutations could be identified and cocktail design was feasible. until now, 4 patients received vaccinations. the vaccine was generally well tolerated and no or only mild side effects were observed. immune monitoring was performed for 2 patients until now. in the first patient, we observed a transient cd4+ response against one vaccinated mhc class ii ligand and a sustained cd4+ response against the included wildtype control peptide derived from the antigen survivin. in the second patient, immune monitoring was performed for the first 8 vaccination timepoints, a t cell response was not measurable at this timepoint of vaccination. conclusions: whole exome sequencing of pediatric all patients is feasible and yields small amounts of expressed, tumor-specific mutations. these few mutations are sufficient to predict hla-binding peptides that can be used to formulate individualized peptide vaccine cocktails. we currently conduct a clinical phase i/ii trial in a small cohort of high-risk all patients to assess safety, toxicity and immunogenicity. clinical background: chimeric antigen receptor t cells (cart) are considered as gene therapy medicinal products (gtmp) and genetically modified organisms (gmo). hence, carts manufacturing for clinical application is strictly regulated. appropriate methods assessing car transgene copy number (cn) in a cart product and definition of the frequency of carts in treated cart patient are mandatory. although quantitative real-time pcr-based (qpcr) analysis has been used for this purpose, no standardized procedure to minimize systematic errors and enable comparability has been established yet. here, we report on a single copy genebased (scg) duplex (dp) pcr (scg-dp-pcr) for the determination of the vector copy number (vcn) in cart products as well as patient samples following cart administration. scg-dp-pcr was validated and compared to the broadly used absolute copy number qpcr (acn) approach within the framework of a clinical trial treating patients with good manufacturing practice (gmp)-grade carts (hd-car-1). methods: for conventional acn, primers and probe targeting the car vector rv-sfg.cd19.cd28.4-1bbzeta were designed. standard curves were established via serial dilutions of the sfg.car plasmid. amplification of the standard curve as well as target genomic dna for vcndetermination was performed as singleplex (sp) pcr (sp-car) (method a). on the same qpcr plate, duplex (dp) qpcr reactions were carried out. additionally to the components comprised within method a, the experimental setup contained haploid human genomes as well as primers and probe targeting ribonuclease (rnase) p as human scg. the amplifications for the sfg.car plasmid (dp-car) and the rnasep gene (dp-rnasep) were performed simultaneously (scg-dp-pcr; method b). scg-dp-pcr was performed for standard curves and target samples. target-sample dna was extracted from carts prepared from leukapheresis products of three healthy donors (hd). results: for method validation, efficiency and linearity (correlation coefficient) of the qpcr reactions of method a (sp-car) and method b (dp-car, dp-rnasep) were assessed by linear regression of the pcr signal to the reference standard curve. overall, standard curves were only considered valid if a correlation coefficient (r 2 ) of above 0.98 and efficiencies of 100% ± 10% were achieved. vcns applying method a and b to the same target sample were compared. sp-car pcr reaction displayed efficiency of 103.5 ± 7.1%; 104.2% ± 2.1% and 99.3 ± 1.6% efficiencies were achieved for dp-car and dp-rnase pcr reaction, respectively (table 1) . applying scg-dp-pcr using formula for relative cn assessment (2 -δct (dp-car -dp-rnasep) ) on hd samples resulted in an average of 0.8 ± 0.2 increased cn when compared to method a (table 2) . conclusions: in terms of efficiency and linearity by linear regression qpcr reactions were comparable. validation of scg-dp-pcr was achieved and represents an exact and less error-prone method to fulfil regulatory safety release criteria of cart products. besides of accurately assessing vcn of transduced cells, scg-dp-pcr is also a highly robust method to follow-up carts in treated patients. applying this approach, no standard curve is needed, hence significantly economizing required material as well as time. disclosure: nothing to declare background: t-cells' antileukemic responses in aml-pts need to be improved. dc leu effectively activate t-cells against leukemic blasts, resulting in blast-lysis ex-vivo. factors influencing these activities are not known. methods: we generated dc/dc leu from aml-blastcontaining mononuclear cells (n=74) using standard methods (mcm-mimic/ca-ionophore/picibanil/ifn-α, "mnc-methods") and from blast-containing heparinized whole blood (n=35) using modulatory kits (various combinations of 2-3 clinically approved response-modifiers, "wb-kits", patent 102014014993) and correlated statistically (t-/u-test, pearsons correlation, # means significant) proportions of dc-/t-cell-subtypes/cytokine-profiles with t-cells' antileukemic cytotoxicity (ctx), achieved after mixed lymphocyte culture (mlc) with/without mncmethod-("t*dc mnc "/"t*bla mnc ") or wb-kit-treated cultures ("t*dc wb "/t*bla wb "). ctx was given as proportions of cases with achieved or "improved" blast-lysis compared to control and as frequencies of viable blasts (bla via ) after effector-cell-influence. pooled data and data obtained with single methods in different cohorts are given. results: 1. generation of dc/dc leu : with a) mncmethods: ø18±15% dc and ø11±10% dc leu and b) wb-kits: ø13±8% dc and ø11±13% dc leu without induction of blasts' proliferation. t-cell-proliferation increased (vs uncultured t-cells) after mlc with a) mnc-methods: ø34±38% vs ø7±9% and b) wb-kits: ø63±25% vs ø23±20%. 2. antileukemic reactivity (t-effector-cell-cytotoxicity after mlc): pooling all data: a) mnc-methods ("t*dc mnc " vs "t*bla mnc "): we found a1) blast-cytotoxicity in ø70%(vs50%) of cases with ø59%(vs117%) bla via , a2) blast-cytotoxicity was improved (vs control) in 86% with ø decrease of bla via of 78%; b) wb-kits ("t*dc wb " vs "t*bla wb "): we found b1) blastcytotoxicity in ø80%(vs71%) of cases with ø68%(vs56%) bla via , b2) blast-cytotoxicity was improved (vs control) in 67% of cases with ø decrease of bla via of 43%. in general, these results could be confirmed with single methods: best mnc-methods were picibanil and mcm-mimic, best wbkits were kits containing gm-csf+picibanil or prostaglandins. 3. correlations: pooling all data: cases with "improved" lysis (vs "not improved" lysis) were characterized by a) mnc-methods: increased proportions of mature dc/cells (ø46±29% vs ø35±20%), dc leu /cells (ø10±10% vs ø5±4%) and proliferating t-cells (ø35±40% vs ø19 ±14%), b) wb-methods: dc/cells (r=0.781 # ), dc leu /cells (r=0.815 # ), dc leu /bla (ø40±23% vs ø29±16%), dc leu /dc (ø59±22% vs ø51±25%), cd8 + t-cells (ø58±20% vs ø50 ±24%), ifn-γ (r=0.868 #) , mcp-1 (593±242 vs 547±223 pg/ml). conclusions: blasts are regularly converted to dc leu in the presence of mnc-methods and wb-kits (simulating the in vivo microenvironment). t-cells' coculture with dc/ dc leu after mlc induces and improves antileukemic t-cell activation compared to controls. blast-cytotoxicity correlates with proportions of dc/dc leu -and t-cell subtypes and released cytokines. these data support a role of antigen presentation by leukemic cells (dc leu ) for the stimulation of an immune response in aml in vitro and possibly in vivo. disclosure: nothing to declare evaluation 1 year after the launch of the motion comic immuno-t, explaining patients and their caregivers how immunotherapy strategies work background: one year ago, the first version of immuno-t, a motion comic explaining to patients and caregivers how immunotherapy strategies work, was released. people were informed on and inspired to use the application during (inter)national meetings and events for the general public. meanwhile, the motion comic was further refined and adapted into a second version, based on the evaluations we've collected on the first version. adaptations included a multi-language tool (currently 6 languages), increased user friendliness, and a new supporting musical score. also, a website was launched from which the second version could be downloaded on tablet or smartphone (both android and apple) and a new online evaluation form could be filled in. in 10 months, 40 people have evaluated the motion comic online, and these results are presented here, as well as our future plans within the immuno-t program. methods: through an online questionnaire, 41 participants from belgium (n=38) and the netherlands (n=3) have evaluated the dutch version, and 4 belgian participants evaluated the french version of the motion comic. results: the total group (n=45) consisted of patients (n=15) and their families (n=2), general public (n=10), students (n=7), health care professionals (n=4), researchers (n=5) and kindergarten teachers (n=2). participants' age ranged from 19 to 80 years, with an even distribution amongst the different generations. the majority of the evaluators (97,8%, n=44) thought the motion comic is a good way to explain immunotherapy to patients. 23 individuals (51,1%) felt mainly interested after watching immuno-t, and a total of 32 participants (71,1%) felt hopeful or motivated. focussing on the patient group (n=15), all of the responders think the immuno-t motion comic is a good tool to use in a patient-doctor consultation. 11 patients (73,3%) felt hopeful and/or motivated after watching the whole motion comic, while 3 of them (20%) felt combative and 5 (33,3%) felt gripped and intrigued. as for the new musical score, 35 participants (77,7%) think the music is suitable for the app, while 6 evaluators (13,3%) think the new music is not or not at all fitted to support the motion comic. conclusions: the detailed evaluations allow us to further improve immuno-t, and aid us in the development of other motion comics we plan to release under the cancer-t in motion umbrella. with the current version of immuno-t, a single-center pilot study is being set up, to test the efficacy and usability of immuno-t, based on qualitative research during the experience of the tool, and using validated questionnaires. with this study we want to evaluate the impact of immuno-t on patient empowerment, and the decision making process. the study protocol will be presented at ebmt. disclosure: the development of immuno-t was partly financially supported by celyad, calgene, novartis, roche, amgen, bms, but these companies did not by any means influence the contents and development of the motion comic. a therapeutic strategy to trespass the blood brain barrier for adoptive nk cell therapy in glioblastoma multiforme induced rat: a preclinical study background: glioblastoma multiforme (gbm) is among the most common and aggressive primary brain tumors with very poor prognosis. according to the central brain tumor registry of the united states, central nervous system (cns) tumors in pediatric patients (ages between 0-14 years old) are the second most common malignancies after blood-born malignancies, and the first amongst solid tumors, and known the most common cause of cancer-related deaths. although hematopoietic stem cell transplantation has been exploited to treat many kinds of malignancies, currently its success rate in gbm is limited. therefore, the gbm treatment paradigm needs shifting towards more effective treatments such as immune cell therapy. natural killer (nk) cells have been recognized as potential anti-cancer effector cells, as they can recognize and target tumor cells. since a small percentage of blood cells are differentiated as nk cells, the number of this group of cells is hardly enough to fight tumors, and so their multiplication and activation would be a potential effective cancer treatment. methods: this preclinical study was focused on setting up an optimal protocol for expansion and activation of naïve nk cells and assessing their efficacy towards induced gbm in rat models. ex-vivo expanded and interlukin-2 (il-2)and heat shock protein-70 (hsp-70)-treated nk cells have been exploited. after in vitro study and confirming the efficacy of treated cells through cytotoxicity assays, we induced gbm in 6 male wistar rats (weighted 275-300 gr) using c6 tumor cells injection in rat brain through stereotactic surgery. the tumor formation was proven by mri imaging. following tumor establishment, we analyzed the effect of single injection of il-2-and hsp-70-treated nk cells compared with single injection of non-treated nk cells in two groups of rats. results: systemic intravenous delivery of il-2-and hsp-70-treated nk cells through tail's vein resulted in tumor shrinkage in different time intervals and complete remission in the first group of gbm-induced rat models, whereas in the other group of gbm rats receiving untreated nk cells, the tumor progressed. therapeutic efficacy of the treated nk cells was ascertained compared with non-treated nk cells considering tumor shrinkage observed in mri and survival rates between the two model groups. conclusions: the amelioration of tumor which has been confirmed by mri, proved the migration of activated nk cells through blood brain barrier and homing to cns, and finally targeting gbm tumor cells. our data suggest that nk-cells treated with il2/hsp70 may be a promising immune cell-based therapeutic approach towards treating the fatal gbm. disclosure: nothing to declare p592 abstract withdrawn. long term sorafenib response for extramedullary flt3 + aml relapse after allogeneic stem cell transplantation since june 2017, sorafenib dose has been tapered to 200 mg/day, due to mild skin and gi toxicity. after 3 years of treatment, she maintains cr at medullary and extramedulary levels, with no evidence of a disease that had escaped the mechanisms of action of chemo, hsct and dli. conclusions: in our patient, treatment with sorafenib has provided long-term control of this refractory extramedullary disease, even at adjusted doses. further studies are needed to confirm the efficacy of flt3 inhibitors in the control of relapses after allo-hsct, extramedullary disease and its potential role as maintenance agent. disclosure: nothing to declare background: although chemotherapeutic(ct) agents that used in the treatment of acute lymphoblastic leukemia (all) increase survival, the results are still weak. longterm survival with ct's in relapse all cases is difficult and the prognosis is very weak. inotuzumab ozogamicin is an anti-cd 22 monoclonal antibody and it has the potential to reduce the overall toxicity of intensive regimens for all, as well as to possibly increase the number of patients who may achieve a state of minimal residual disease. methods: 26-year-old male patient was diagnosed with b-cell all in december 2017.after the hoelzer kt protocol was started, maintenance treatment was continued. in the fifth month of treatment,flag ct protocol was started cause of recurrence was seen on 5% blast detection in peripheral blood smear. in august 2018, inotuzumab ozogamicin treatment was started and six cures were completed because the patient was not in remission. in september 2018, he had gone haploidentical bone marrow transplantation from his sibling donor(8/10)with defibrotid prophylaxis for veno-occlusive disease(vod)s. he engrafted succesfully and chimerizm was 99.85% in 30th days of transplantation. he is 60th day of transplantation and in a remission. results: bone marrow transplantation cannot be performed since the complete response cannot be achieved in patients with relapse and resistant b-all.in these patients, new therapies targeting malignant lymphoblasts are needed. inotuzumab ozogamicinis a monoclonal antibody drug conjugate that targets cd22 antigen on malignant lymphoblasts.in many studies, it has been shown that inotuzumab ozogamycin is effective and reliable anti-tumor activity in adults with recurrent and resistant cd22 positive all. however, monoclonal antibody drug conjugates have been shown to be associated with vod's.for this purpose, we used defibrotid to protect our patient from vod. conclusions: treatment with combination ct regimens in b-all is suboptimal and long-term survival is achieved in only 30-40% of patients.targeted molecular therapy and new regimens are needed in relapse and resistant patients.at this point, inotuzumab ozogamycin is an anti-cd-22 monoclonal antibody, as in our case, it provides remission in recurrent and resistant b-all patients and allows patients to complete their treatment with an allogeneic transplant from a fully compatible donor. disclosure: nothing to declare background: mesenchymal stem cells (mscs) are an attractive consideration for therapeutic cures of many difficult diseases on the cellular-level. due to the trophic effects of the cytokines and chemokines that they produce, mscs have shown multiple beneficial properties in the field of oncology. in this study, we will be investigating the effect of mscs derived from human bone marrow (bm), adipose tissue (at), and umbilical cord derived mscs (uc-mscs) on ovarian cancer. to differentiate the mscs, we performed a comparative analysis between the various sources for proliferative capacity, surface antigen expression, differentiation ability, tumor marker and paracrine activity, and their influence on ovarian cancer cell proliferation. methods: measurements of ovarian tumor marker proteins were computed by elisa. proliferative effects, immunomodulatory effects, and apoptosis of the mscs were measured by the culture and counting of colony formations. flow cytometry (fcm) was used to measure the variation of the immunophenotyping and cytokine secretions in co-culture, as well as gene expression. results: cells noticeably proliferated without any modifications to their immunophenotype during the third subculture. the colony-forming unit fibroblast (cfu-f) test showed a proliferation of the mscs along with healthy cells and cancer cell lines with no changes in their phenotype. the supernatant of mscs showed an increase in cellular death of the ovcar3 in ovarian cancer cell lines. a reduction in the level of ca-125 (75-90%; p=0.769) with ovcar3 in co-culture, and a decline of ldh (10-20%; p=0.03) and beta-hcg (10-20%; p=0.04) were observed in co-culture in caov3, skov3 and igrov3 cell lines. a decrease in cd24 of the cancer cell lines in co-culture with the msc supernatant showed a reduction of the cancer tumorigenicity and aggressiveness, while the rate of the cd24 and cd44 asserted their stem state. msc supernatant decreased cell proliferation and mmp-2, mmp-9, and ca-125 mrna expression, while increasing timp 1, 2, and 3. this suggests that mscs have a role in cell death and inhibition of ovarian cancer cell proliferation. an increase of anti-inflammatory il-4 and il-10 cytokines, and a decrease in growth factor gm-csf along with their proinflammatory inf-a, tnf-a, il-9, and il-17a cytokines were also noted. conclusions: the gene and cytokine activity indicate a potential therapeutic anti-inflammatory and antiproliferative role of mscs on ovarian cancer despite their sources. the reduction of ca-125, ldh, and beta-hcg in co-culture, along with the decrease in cd24 and amplified cellular apoptosis demonstrate the beneficial effects of stem cells in ovarian cancer cell lines. disclosure background: hematopoietic stem cell transplant (hsct) is the only cure in sickle cell disease (scd) so far. because of the risk of toxicity, its indication in france is restricted to severe patients with match sibling donor. this study compares the incidence of severe acute toxicity after hsct, between children aged less than 13 years and teenagers aged 13 to 18 years old, treated for scd. methods: all patients suffering from scd, aged less than 18 years at transplant, who received hsct in chu robert debré and necker, between 01/01/2005 and 12/31/2017, were included. severe acute toxicity, defined by onset of severe acute gvhd, organ toxicity or infection, was compared between the two groups of age. results: 73 patients (59 children and 14 teenagers) were included. all patients received a myeloablative conditioning regimen. bone marrow from a sibling donor was the main stem cell source (n=65; 89%). neither death nor rejection was observed with a median follow-up of 29.6 months (range, . the incidence of grade iii-iv acute gvhd was 12.3% and was similar between the two groups; no risk factor was identified in univariate analysis. teenagers had more frequently acute skin toxicity (21.4% vs 0%, p=0.006). in univariate analysis, patients presenting severe organ toxicity were significantly older than others (9.3 vs 7.5 years old, p=0.027). teenagers were more frequently treated for bacterial (64.3% vs 32.2%, p=0.035) or bk virus (42.9% vs 6.8%, p=0.002) infections. in univariate analysis, patients who developed infection were also significantly older at time of transplant (respectively 9.7 vs 7.5 years old, p=0.016). no severe sinusoidal obstruction syndrome was observed. regarding long-term toxicity, 2 patients presented an extensive chronic gvhd, they were both aged less than 13 years old. no cut-off of age could have been defined. conclusions: this study confirms the excellent results of hsct in scd, with a 5-year event-free survival and overall survival of 100%. an older age at transplant seems to be associated with more frequent severe acute toxicity. these results are consistent with previous studies and suggest that hsct should be performed as soon as possible, without any defined "best age". prospective studies are needed, in order to define the place of each therapeutic in scd, with the aim of reducing treatment-related toxicity and developing alternative strategies for patients without match sibling donor. disclosure: nothing to declare p599 new insights into risk factors for transplant-associated thrombotic microangiopathy (ta-tma) in paediatric hsct (n=170) was associated with ta-tma in 1% vs 5.8% vs 7.7% respectively (p=0.067). the presence of comorbidities at d0 (n=86) was significantly associated with an increased risk of ta-tma 10.4% vs3.7% in absence of co-morbidity (n=320); p=0.032. use of csa/tac-based gvhd prophylaxis was associated with less ta-tma with an incidence of 4% vs 13% if these agents were not included (p=0.01). in univariate analysis ta-tma was significantly higher among patients with agvhd grade ii-iv (12/138; 8.6%) vs grade 0-i (6/227;2.6%) (p=0.01). pres was recorded among 10 cases and 50% of them developed ta-tma. two out of the 21 patients with ta-tma had pathological gene mutations in their complement pathway. on multivariate analysis the presence of active comorbidity was a risk factor for ta-tma (or:6.6; 95% ci:1.1-37.6; p=0.032) while the use of csa/tac-based gvhd prophylaxis did not increase the risk for ta-tma (or:0.04; ci:0.004-0.39; p=0.005). in the presence of comorbidities the use of defibrotide as prophylaxis or therapy for vod (n=86) was associated with a drop in the incidence of ta-tma from 12% (9/79) in absence of defibrotide to 0% (0/7). 2-year overall survival was significantly lower among ta-tma cases (58%) in comparison to 81.5% in absence of ta-tma (p=0.001) (figure 1). conclusions: active co-morbidity is a significant risk factor for ta-tma. use of defibrotide prophylaxis in patients with co-morbidities at the time of hsct might offer protection against ta-tma. surprisingly the use of csa/tac based gvhd prophylaxis is not a risk factor for ta-tma probably through limiting the development of high grades agvhd. the association between pres and ta-tma suggests a common pathway of endothelial damage background: gonadal impairment is a severe late effect of myeloablative conditioning regimes with significant impact on quality of life of cancer survivors. the aim of this study was to analyze gonadal function after busulfan (bu) or treosulfan (treo) containing regimens with regard to pubertal stage. methods: this was a retrospective, multicenter study involving patients treated in pediatric ebmt centers between 1992-2012. patients receiving myeloablative doses of bu or treo as part of hsct conditioning were eligible for inclusion. analysis was conducted in two groups according to pubertal status at time of hsct. results: 137 patients (pts) were treated in 25 pediatric ebmt with bu or treo before allogeneic hsct. the median age at transplant was 11.04 years (range 5-18); 89/ 137 (65%) were males (m), 48/137 (35%) were females (f). 89/137 (65%) pts were pre-pubertal at hsct (f= 27;m=62) and 48/137(35%) were post pubertal (f=21;m=27). 118/ 137 (86%) patients received bu (f=38;m=80),77/118 (65%) were pre-pubertal. 19/137 (14%)(f=10;m=9) received treo,12/19 (64%) were pre-pubertal ( figure 1 ). females who received treo in pre-pubertal stage (n=6/6) reached more often spontaneous puberty (100% vs 38%; p=0.016) compared to pre-pubertal bu group (n=8/21) and occurrence of menarche was higher in treo group (p< .01) hormonal replacement therapy was given in 13/27 (48%) females transplanted in pre-pubertal stage and in 15/21 (71%) of those transplanted in post-pubertal stage. 77/89 (86%) males were pubertal at last follow-up and 8 of them (10%) performed sperm analysis (5 oligo-azoospermic,3 unknown). three pregnancies were reported in the population group, all received bu. regarding the evaluation of hormonal levels in pubertal patients at time of hormonal dosage (median 19.54 yrs) (66 bu and 15 treo), males treated with treo had significant lower lh levels (p = 0.045) compared to bu group. females treated with treo had significant lower levels of lh and fsh (p= 0.02 and p= 0.0035 respectively). conclusions: gonadal damage related to treo was significantly lower compared to bu. we observed that: females transplanted during pre-pubertal period had spontaneous puberty more frequently after treo compared to bu and that hypogonadism hypergonadotropic was more frequent after bu than treo. these results must be further confirmed on a larger population. background: viral infections significantly contribute to both morbidity and mortality in patients undergoing hematopoietic stem cell transplantation. traditional antiviral therapy is associated with lack of efficacy, potential toxicity, prolonged hospitalization and increased patient costs. viral specific t cells can be manufactured from donor blood to treat viral infections post-transplant, and are associated with increased clinical efficacy and low toxicity. we postulated that direct costs of vst therapy are lower compared to traditional anti-viral medications methods: vsts were manufactured according to local protocols and fda requirements. total drug cost (as per institutional charges per drug) was calculated for patients who required treatment for viral infections post-hsct. manufacturing costs of vsts are fixed per fda requirements. patients who were treated with investigational antiviral medications (including brincidofovir) were excluded from analysis. patients treated with vsts +/anti-viral medications over a 2 year period were compared to patients treated only using traditional anti-viral medications, including cidofovir, ganciclovir, valganciclovir, foscarnet and rituximab. results: demographics are shown in table 1 . there were no major differences between the two groups treated. the number of anti-viral medications used in the vst group was lower compared to the anti-viral treatment group. median cost of vst treatment was significantly lower compared to those threated with traditional anti-viral therapy ($12,757 vs $16,392, p-value=0.03) . conclusions: treatment with vsts post-hsct for viral infections was lower in cost compared to anti-viral medical therapy. it is likely that overall costs are further reduced with vsts due to reduced inpatient hospital time, less monitoring of labs associated with anti-viral medication side-effects and reduced ancillary costs including nursing and pharmacy. more studies are needed to examine these indirect costs further. background: dock8 deficiency is an autosomal recessive primary immunodeficiency (pid) disease caused by loss-offunction mutations in the dock8 gene (1) . patients with dock8 deficiency present with multiple abnormalities of the immune system, including defective t cell function and impaired production of antigen-specific antibodies. these lead to persistent viral infections of the skin, mucocutaneous candidiasis, recurrent sinopulmonary infections, atopic dermatitis, and other allergic disease, malignancies, and sometimes autoimmunity (2). hematopoietic stem cell transplantation (hsct) is currently the only curative treatment option available (3) . methods: we retrospectively evaluated our patients who underwent allogeneic hematopoietic stem cell transplantation due to dock8 deficiency in ege university pediatric stem cell transplantation unit between 2013 and 2018. results: we identified 16 patients transplanted at a median age of 7.8 years (range: 2-15.5 years). of 16 patients; 4 (%25) received hsct from matched sibling, 11 (%68) from unrelated donors and 1 patient from haploidentical donor. we used busulfan-based myeloablative conditioning regimen to 12 patients (%75), reduced toxicity myeloablative regimen with treosulfan to 2 patients (%12.5) and nonmyeloablative regimen to 2 patients (%12.5). eight of the recipients received bone marrow, 6 of the patients received peripheral blood stem cells, 2 of the recipients received cord blood as stem cell source. fifteen of 16 patients (%93) had achieved engraftment and median follow-up of patients was 29 months (1-64). grade iii-iv acute graft versus host disease (gvhd) occurred in 13% of patients and chronic graft versus host disease was seen 18 % of patients. one patient received cord blood from unrelated donor did not engraft and died from septic shock. four patients died from transplant related toxicity. our patient's survival was %68 ; 11/16 patients alive. conclusions: hsct is the only curative treatment option for dock8 deficiency. in particular, patients with high comorbidity scores have a high risk of toxicity and toxic death. therefore, reduced toxicity conditioning regimens should be used for these patients. references : background: eltrombopag, a low-molecular-weight synthetic nonpeptide thrombopoietin receptor agonist (tpo-r), is a second-generation tpo. it is an oral thrombopoietin mimetic licensed in chronic immune thrombocytopenic purpura that induces platelet maturation and release by binding to c-mpl receptors on megakaryocytes. in a recent study; for patients with refractory saa, eltrombopag induced a response (at 12 weeks) in at least one hematologic lineage in 44% patients and 36% no longer required platelet transfusions. and also 24% patients became rbc transfusion independent and 36% had a neutrophil response. trilineage responses were seen in 24% of patients; although surprising, this might indicate stimulation of c-mpl receptors on remaining stem cells. delayed recovery from thrombocytopenia is common after stem cell transplantation. in a study including 12 adult patients, eltrombopag was used to enhance platelet recovery for post-hsct thrombocytopenia. it is well tolerated and efficacious offering transfusion independence. methods: in our retrospective study, eltrombopag (50mg/day) was started in 19 pediatric patients (age ranging from 4 to 16 years with a median age 12.3 years) for posthematopoietic stem cell transplantation (hsct) thrombocytopenia. all patients fulfilled the following 3 criteria: (1) undergone hematopoietic stem cell transplantation (hsct), (2) had improved total leucocyte counts after leucocyte engraftment, (3) had prolonged thrombocytopenia (< 30.000) needing platelet transfusion. results: four of the patients have received ric while 15 patients ma conditioning regimens before hsct. two haploidentic, 2 autologous, 7 mud, 8 msd transplantations were performed. et (50mg/day) was started in 19 patients who had thrombocytopenia despite neutrophil engraftment on the +30th day of hsct a reduction in platelet transfusions and a platelet count of more than 30,000 were the primary endpoints. before et treatment, bone marrow biopsy was checked in 15/19 patients, 10/15 patients had decreased number of megacocyocytes. none of the patients had active bleeding at the start of eltrombopag but they were all at high risk of bleeding. according to the platelet monitoring, 12 patients had a dose increase starting from the second week. the number of patients in need of platelet transfusions was 7 at the end of the first month; and only 2 at the end of the 2nd month. all patients had a thrombocyte count of more than 30.000 in the third month. in 11 patients, et was discontinued after 2-11 months. no dose limiting toxicities have been observed. conclusions: as a conclusion, et was found highly effective for posthsct thrombocytopenia, with no drug related adverse effects. there was a gradual increase in platelet count, and none of the patients had any complication due to thrombocytopenia. disclosure background: isavuconazole (isa) is a new triazole approved for ifi treatment in the adult population. advantages are activity against both moulds and yeasts spp, excellent bioavailability after oral administration without relevant food or gastric ph effect, a water-soluble prodrug developed to facilitate intravenous administration without nephrotoxic excipients such as β-cyclodextrin, potentially poor drug-drug interactions. isa does not currently appear to require tdm. isa safety and efficacy have not been yet established in children and, in particular, no data are available in the pediatric hsct setting. methods: italian association pediatric hematology oncology (aieop) multicentric analysis of a cohort of allogeneic hsct pediatric patients who received isa as ifi treatment or prophylaxis. due to the lack of recommended dosing in pediatric patients and a clear target isa plasma trough-level range, the therapeutic monitoring (tdm) of isa concentrations was applied by a validated liquid chromatography-tandem mass spectrometry (hlpc-ms/ ms) assay technique. isa trough plasma concentrations (c0) and 3 hours after drug intake (c3h) were measured. results: a total of 16 allo-hsct recipients were included, (m/f 12/4); median age: 13,1 years, range 5-19, median body-weight 48,7 kg (range 17-80). isa was used as ifi treatment in 14 cases and as prophylaxis in 2 patients. donors were haploidentical in 9 patients, matched-sibling in 4, allogenic-unrelated in 4 cases. according to eortc criteria, ifi was proven in 3 patients (1 penicilum, 1 mucor, 1 aspergillus fumigatus), probable in 5 and possible in 7 patients. lungs were the main localization (12 cases), associated with cns involvement in 4 cases and paranasal sinuses in 3; 1 patient had possible hepatic candidiasis. all patients, but one, received isa as rescue treatment for previous therapeutic failure (6 ambisome, 3 voriconazole, 1 combination therapy, 1 posaconazole). seven patients received only iv isa, 3 received only oral isa whereas 6 patients received both iv and oral isa. patients under 20 kg body weight received half isa dose (100 mg tid loading dose on days 1 and 2, 100 mg/die manteinance). the others received adult schedule; only 5 patients received loading dose. isa was administered for a median of 83 days (range: 24-240). in 3 patients isa was administered in combination with caspofungin. tdm was applied to 5 patients including 1 patient with severe vod and 1 with renal failure secondary to ta-tma. the median isa concentrations were 3.94 (1.57-5.85) mg/l and 4.7 (1.29-8.37 ) mg/l for c0 and c3h, respectively. ifi complete remission was achieved in 5 cases, partial remission in 3; treatment failure was experienced by 2 patients. in 3 cases fungal lesions remained stable. ctae grade ii-iii toxicity was observed during treatment in 3 patients, with increased transaminase and/or creatinine levels which resolved after temporary isa withdrawal. no drug-drug interactions were observed in 3 patients receiving csa as gvhd prophylaxis and no modification of csa daily dose was needed. conclusions: isavuconazole use may be considered in the pediatric population, even in the hsct setting, for its safety, efficacy, tolerability, no drug-drug interaction. of course these data deserve further evaluation. disclosure: nothing to declare p608 new treosulfan-based conditioning regimens including epigenetic agents in patients with very high-risk neuroblastoma background: the pts aged 18 mos. or older with disseminated nb involving bone and bone marrow constitute a group of pts with very poor prognosis. although the majority of them are responsive to intensive conventional chemotherapy, most eventually relapse with efs at 5 years of < 10%. at the beginning of the year 2018 we came up with a protocol for this very unfavorable group including epigenetic therapy (5-azacitidine) in the phases of consolidation. methods: seven pts with a median age of 4 (2,6-8) years completed the protocol and received hdct with autologous sct as a consolidation. hdct included two different epigenetic agent containing regimes according to tumor response to the induction therapy assessed by 123 i-mibg and mri (ct-scan). three pts revealing large active residual tumor assessed by 123 i-mibg scan or multiple active bone metastases received a conditioning regimen (regimen a) including 131 i-mibg therapy at a dose 4.0-4.4 mbe/kg on d-14, treosulfan 10000 mg/m 2 /d, on d-6,-5 and -4 (total 30000 mg/m 2 ), melphalan 70 mg/m 2 /d, on d-3,-2 (total 140 mg/m 2 ), 5-azacitidine 75 mg/m 2 /d on d-9 to d-5 (total 375 mg/m 2 ). four pts with cr or vgpr received a «split» conditioning regimen (regimen b) including treosulfan 10000 mg/m 2 /d, on d 11 and -10, and on d-4 and d-3 (total 40000 mg/m 2 ), melphalan 70 mg/m 2 /d, on d-3 and -2 (total 140 mg/m 2 ), and 5-azacitidine 75 mg/m 2 /d, on d -14 to d -12 and on d-9 to -d-5 (total 600 mg/m 2 ). a median number of 5.3 (3.7-7) cd34+/kg was infused on d0. results: the median recovery times to wbc>1.0x10 9 /l and to an unsupported plt>20x10 11 /l were 10 (7-12) and 13 (9-17) days, respectively. all pts experienced grade 4 hematological as well as infectious toxicity assessed by nci-ctc score. there were 7 episodes of severe organ toxicity of grade 3 occurring in 4 pts. in 4 cases we observed a severe mucositis, in 2 cases gi toxicity and 1 episode of the erythema multiforme occurred. one pt revealed a lifethreatening episode of hypotension of grade 4. no transplant-related death occurred. the median number of transfused rbc and plt doses was 3 (1-4) and 6 (3-8), respectively. all pts are alive and well without signs of disease progression in complete hematological recovery with a limited follow-up of 5.3 (4-7) mos. from day 0 of hdct. conclusions: although it is rather early to evaluate the efficacy of the epigenetic agent's inclusion in the induction and/or consolidation phases of a very high-risk nb treatment, we can assume that, first, the hdct combining mibg i 131 and/or high dose of treosulfan with epigenetic agent such a 5-azacitidine was feasible and had an acceptable toxicity. second, the "split" modality of the treosulfan use in conditioning regimen would permit to increase the total dose of the alkylating agent with no inacceptable toxicity. disclosure: nothing to declare pre-and-post magnetic resonance imaging of hips and knees for detecting osteonecrosis in children undergoing hematopoietic cell transplantation: in whom is it necessary? ali y suliman 1 , sue c kaste 2 , ying li 1 , dinesh keerthi 1 , guolian kang 1 , brandon m triplett 1 , ashok srinivasan 1 between april 2016 and august 2017 at the university medical center hamburg-eppendorf, germany, were included. total and active ratg plasma levels were analyzed by elisa and flow cytometry, respectively. primary endpoint of the study was exposure to ratg. secondary endpoints included transplant-related mortality, incidence of acute and chronic gvhd, immune reconstitution, chimerism, rejection and viral infections. patients were monitored at least 100 days post transplantation. statistical analyses were performed using ibm spss statistics 24 software, or graphpad prism 5 software. results: median total grafalon™ and thymoglobuline™ peak plasma levels were 419.0 μg/ml and 60.4 μg/ml, respectively; median active grafalon™ and thymoglobu-line™ peak plasma levels (appl) were 77.9 μg/ml and 8.11 μg/ml, respectively. active thymoglobuline™ plasma levels showed highly variable pharmacokinetics compared to grafalon™. neither grafalon™ nor thymoglobuline™ exposure correlated with lymphocyte count prior to transplantation, cell count in the graft (wbc, mnc, t cells), age, body weight or body surface area (bsa). this is indicative for a saturation effect in both groups. to correlate high or low ratg exposure with clinical outcome parameters, we built two groups within each patient cohort by median appl. the incidence of gvhd was not dependent on high or low ratg exposure. until day 100 post hsct, 49 viral infections or reactivations (ebv, cmv, adv, hhv6, bkv) occurred in the 32 patients. interestingly, adv infections affected only children with high ratg exposure. the median time to leukocyte engraftment was not significantly longer in the high ratg groups compared to the low ratg groups (17 to 16 days for grafalon™, and 14 to 16 days for thymoglobuline™). there was a decreased and/or delayed recovery of cd3 + , cd4 + and cd8 + t cell reconstitution, but not of b cells and nk cells in the high thymoglobuline™ exposure group compared to the low thymoglobuline™ exposure group. overall survival was not statistically significant with 80% in the grafalon™ and 95.5% in the thymoglobuline™ group without influence of ratg exposure. conclusions: high and low exposure to grafalon™ or thymoglobuline™ did not result in significant differences in outcome parameters as incidence of survival, agvhd, cgvhd, rejection, or mixed chimerism in this limited cohort. delayed and decreased immune reconstitution in the high ratg exposure groups did not translate into different clinical outcome parameters. adv infections only occurred in the high ratg exposure group. grafalon™ and thymoglobuline™ showed distinct pharmacological and immunological differences in children and larger cohorts are needed to detect clinically significant differences and adjust dosing regimens individually. disclosure: nothing to declare background: the optimal conditioning regimen for allogeneic hematopoietic cell transplantation (allohct) in children with myeloid malignancies remains undefined, particularly when reduced-intensity conditioning (ric) regimens are utilized. methods: we performed a retrospective review of children undergoing allohct for acute myeloid leukemia (aml) and myelodysplasia-related aml (mdr-aml) over a 10 year period (2008-2018) at our institution, comparing the outcomes of those who received either a busulfan (bu)-or melphalan/thiotepa (mel/thio)-based conditioning regimen. results: a total of 49 patients were analyzed. twentyone received fludarabine/melphalan/thiotepa and 28 received myeloablative busulfan-based conditioning, either in combination with cyclophosphamide (n=19) or fludarabine (n=9). atg was used in 27 patients depending on donor. recipients of mel/thio were selected for ric regimens due to pre-transplant comorbidities (cardiac dysfunction, n=9, 3 requiring peri-transplant milrinone), transplant during chemotherapy-induced aplasia (n=5), underlying diagnosis of treatment-related aml (t-aml) and significant pre-allohct chemotherapy exposure (n=5). proportions of patients with de novo aml (mel/ thio, 57%; bu, 78%) and mdr-aml (19% and 21%) were similar between groups; however, recipients of mel/thio were more likely to have t-aml (24% vs 0%). cytogenetic and molecular risk factors were similar between groups. the majority of patients were transplanted in cr1 (mel/thio, 71% vs bu, 68%) or cr2 (24% vs 38%). more recipients of bu conditioning (71% vs 43%) were mrd-negative at the time of allohct; both groups had comparable proportions of patients with ≥m2 marrow (~10%). donor types and stem cell sources were similar between groups, except unrelated umbilical cord blood which was more common in bu recipients (21% vs 10%). there were no graft failures in mel/thio recipients, compared to 14% (n=4) in those receiving bu-based regimens. engraftment kinetics and immune reconstitution were similar. overall acute and chronic gvhd incidence was higher in recipients of mel/ thio compared to bu (39% vs 21%, and 29% vs 18%, respectively), but rates of grade iii-iv acute or extensive chronic gvhd were comparable. vod requiring treatment was diagnosed in 6 (21%) recipients of bu conditioning and no mel/thio recipients. median duration of follow-up was 30 months (range 2-70) in the mel/thio group, and 18 months (range 1-121) in the bu group. transplantrelated mortality (trm) was similar in both groups (1 patient), occurring before day 100. relapse incidence was comparable (mel/thio, 29% vs bu, 32%); however, relapse occurred at a later time in mel/thio recipients (median d +396 vs d+137). overall survival at 1 and 3 years was superior in mel/thio recipients (95% vs 74%, and 75% vs 50%, respectively). conclusions: in our single institution experience, use of a melphalan/thiotepa-based ric regimen was associated with similar outcomes compared to full-intensity bu-based conditioning, despite higher risk patient and disease characteristics. the majority of recipients of mel/thio conditioning had significant pre-transplant comorbidities, which did not translate into higher trm. while mel/thio recipients had less optimal leukemia control at the time of transplant and high-risk leukemia features (e.g. t-aml), relapse was similar between groups, occurring later in mel/ thio recipients, which may have contributed to better overall survival. disclosure: nothing to disclose methods: pubertal development and biological gonadal parameters were assessed in a retrospective monocentric cohort of pre-pubertal patients who underwent hsct after myeloablative conditioning with total body irradiation (tbi) or busulfan between 1981 and 2017. results: seventy-four patients (28 girls and 46 boys) were included. no spontaneous pubertal development was found in 50% of girls and 10% of boys (p < 0.001) and delayed puberty or no spontaneous pubertal development was found in 57% of girls and 24% of boys (p=0.009). hormone replacement therapy was used in 82% of girls and 24% of boys (p < 0.001). in univariate analysis, tbi conditioning (p=0.05), female sex (p < 0.001), acute gvhd (p=0.05), extensive chronic gvhd (p=0.021), steroid treatment >6 months (p=0.016), and malignant diseases (p=0.016) were associated with no spontaneous pubertal development, whereas tbi conditioning (p=0.003) and extensive chronic gvhd (p=0.005) were associated with delayed puberty. in multivariate analysis, factors independently associated with no spontaneous puberty onset were female sex (p=0.001) and age >10 years (p=0.033). factors independently associated with delayed puberty were extensive chronic gvhd (p=0.041) and age >10 years (p=0.031). tbi was not an independent risk factor for pubertal complications. conclusions: this study confirms the toxicity of myeloablative conditioning on pubertal development and the role of older age and female sex in increased pubertal issues, and suggests a possible role of gvhd in delayed puberty. disclosure: nothing to declare p613 abstract already published. neutrophil elastase activity may serve as a marker for neutrophil extracellular traps formation following stem cell transplantation ronit elhasid 1 , sivan berger-achituv 1 , hila rosenfeld-keidar 1 , szilvia baron 1 1 tel aviv sourasky medical center -tel aviv university, tel aviv, israel background: post-transplant infections rise dramatically in patients with quantitative or qualitative neutrophil defects and constitute a major source of morbidity and mortality following hematopoietic stem cell transplantation (hsct). neutrophils protect the host from microorganisms via multiple processes including phagocytosis and formation of neutrophil extracellular traps (nets). although reactive oxygen species (ros) production seems to be essential for nets formation, the key enzymes of the process are neutrophil elastase (ne) and myeloperoxidase (mpo). methods: ne and mpo activity as well as nets formation were investigated following hsct in 11 patients at week 1 to 6 and 30 after neutrophil engraftment. neutrophils were isolated using easysep direct human neutrophil isolation kit (stemcell technologies inc.) by immunomagnetic negative selection. enzymatic activity of ne and mpo were measured using colorimetric assays. nets formation of phorbol 12-myristate 13-acetate (pma)activated neutrophils was investigated by confocal fluorescence microscopy. all results were compared to those of healthy volunteers. statistical significance was calculated using one way-anova with bonferroni post hoc test. results: 11 patients (median age of 6.8 years [range 2-22 years]) were investigated, 6 following allogeneic hsct (2 acute lymphoblastic leukemia, 1 acute myeloblastic leukemia, 2 epidermolysis bullosa, 1 rhabdomyosarcoma) and 5 following autologous hsct (4 ewing sarcoma, 1 desmoplastic small round cell tumor). all patients experienced fever and neutropenia. at engraftment, average ne activity was significantly decreased compared to the average value of 50 healthy individuals. ne activity improved week by week in patients, reached the lower reference range at 4 weeks following transplantation (fig. 1a) and continued to increase. the enzymatic activity of mpo was comparable to the average value of 50 healthy individuals (fig. 1b) and showed no significant difference between the distinct time points. at neutrophil engraftment, nets formation was absent and comparable to those of non-activated neutrophils (fig. 1c) . although nets formation increased week by week, it did not reach the average of 7 normal controls during the monitored time period. also linear correlation between ne activity and nets formation (r 2 =0.978) was demonstrated. conclusions: impaired ne activity following hsct corresponds to decreased nets formation and could serve as a marker for netosis. strategies to accelerate the recovery of ne function post transplantation might improve nets formation and thereby induce better infection control. a) the average of ne activity (n=11) during 30 weeks following hsct. reference range was measured and calculated from measurements of 50 healthy volunteers using. b) the average of mpo activity (n=11) during 30 weeks following hsct. reference ranges were measured and calculated from measurements of 50 healthy volunteers using the quartile method. c) the average of netosis activity after 100 nm pma activation for 3h (n=11 background: to have a better understanding of incidence, treatment, outcome and risk factors of immune cytopenia in children after allogeneic hsct. methods: between january 2010 and september 2018, 105 pediatric allogeneic hsct have been performed in 99 patients at the ghent university hospital (ghent, belgium). autoimmune hemolytic anemia was defined by a positive direct agglutinin test (dat). dat was performed at moment of engraftment and in case of hemolysis or unexplained anemia. platelets antibodies were evaluated in case of no otherwise explained thrombocytopenia. results: the cumulative incidence of post allo sct autoimmune cytopenia is 9.5% (10/105). in 9 cases there were positive antibodies against red blood cells, and one patient against had antibodies against platelets. of these 10 cases, only 4 (3.8%) were clinically relevant and needed treatment. the median observation period post sct for the whole cohort was 36 months (3 -105) . the clinically significant immune cytopenia started at a median time of day+158 and day +113 in the group without symptoms. the patient who presented the autoimmune thrombopenia developed antibodies against anti-gpiib/iiia, this was resolved after 130 days, the treatment consisted intravenous immunoglobulins (ivig). two of the 3 patients with autoimmune hemolytic anemia had igg mediated antibodies, and 1 had complementmediated dat. these 3 patients were treated with ivig, steroids, rapamune and rituximab. one patient has still dat positive after 36 months, but clinical stable. the other two are also dat positive and have some hemolysis, but the follow up is much shorter (2 months). treosulfan-contained conditioning regimens were more frequently used in patients with significant immune cytopenia. conclusions: immune cytopenia is an infrequent complication after allogeneic hsct. however, its treatment can be challenging, and the hemolysis can persist for years. the association of rapamune and rituximab was adequate to treat this problem in our patients. background: approaches to the management of refractory and relapsed classical hodgkin´s lymphoma (r-r chl) are changing and become more effective. the role of anti-cd30 targeted immunochemotherapy with brentuximab vedotin (bv) has been extensively investigated in adults with r-r chl and is only to be elucidated in children. the study included 14 children and adolescents with r-r chl that were sucessfully treated with bv-based therapy prior to hematopoetic stem cell transplantation (hsct). median age of patients was 16 years (9-19), main histological variant -nodular sclerosis (93%, n=13), advanced stage at diagnosis -86% (n=12). most were heavily pre-treated (median number of previous therapies -4) and progression after autologous hsct was documented in 4 (29%). refractory disease was diagnosed in 8 (57%) and relapsed in 6 (43%). among relapsed patients 4 (67%) were with multiple episodes, 1 (16.5%) -early and 1 (16.5%) -late relapse. treatment regimens consisted of bv in monotherapy 1.8 mg/kg triweekly (n=7) or combination of bv 1.8 mg/kg on day 1 with bendamustine 90-120 mg/ m2 on days 1 and 2 of 3-week cycles (n=5) or combination of bv 1.8 mg/kg on day 1 with dhap (n=2). median number of bv infusions was 3.5 (2-7). all selected patients achieved complete (n=5, 36%) or partial remission (n=9, 64%) prior to hsct. consolidation with autologous hsct was performed in 9 (64%) and with allogeneic hsct -in 5 (36%). primary end points were overall (os) and progression free survival (pfs). response to bv was not assessed in the study as only responders to the bv-based treatment were included. results: with median follow-up of 497 days (105-1368) os and pfs for all patients are 68% and 66%, respectively. pfs after autologous hsct and allogeneic hsct are 70% and 60%, respectively (p=0.7) at present moment 10 (71%) patients are alive and are in remission. three patients died (21%): disease progression (n=1), postransplant idiopathic pneumonia syndrome (n=1) and posttransplant pneumonia (n=1). bv was generally well tolerated with only mild polyneuropathy in 3 patients (21%) as the main reversable documented adverse event. conclusions: in prognostically unfavourable heavily pretreated children and adolescents with r-r chl achievement of response to bv-based therapy prior to hsct is assosiated with promising rates of os and pfs. disclosure provides a treatment by restoring thymidine phosphorylase function and improving disease manifestations. here we report the outcomes of 4 affected siblings who underwent transplantation using an unaffected sibling donor to highlight important experiences in the transplant of such a rare condition. methods: four siblings of consanguineous pakistani descent aged 16, 15, 13 and 5 years underwent myeloablative hsct using fully hla-matched (10/10) peripheral blood stem cells harvested in a single apheresis from an unaffected 9 year old sibling. the oldest sibling, a 16 year old male, first presented in 2016 having emigrated from pakistan with a history of growth failure and abnormal movements. biochemical, nerve conduction and imaging studies confirmed a diagnosis of mngie. testing on three other siblings identified similar biochemical abnormalities, though the 2 youngest children had minimal clinical manifestations of the disease. based on the progressive nature of the disease and the availability of a fully matched donor, a decision was made to pursue transplant for all affected siblings. results: due to the severity of their disease, the 2 oldest siblings were transplanted first using a myeloablative conditioning regime of fludarabine, thiotepa and treosulfan with alemtuzumab. neutrophil engraftment occurred on day + 11 for both, with 100% donor chimerism achieved. there were no significant transplant related complications. the post-transplant course of the 15 year old sibling was complicated by a major stroke-like event characterised by dramatic imaging changes and requiring ventilation, though no cause was identified and the patient's neurologic deficits have since resolved. gastrointestinal symptoms have persisted and both remain tpn dependent, though symptomatically have shown gradual improvement. following the neurologic complications in their older sibling, the 2 younger siblings were conditioned with auc-targeted busulfan and fludarabine plus alemtuzumab. neutrophil engraftment occurred on day + 11, with full donor chimerism achieved. progression to enteral feeding has been much more rapid, with nutrition now fully enteral for both. there were no significant transplant related complications. conclusions: stem cell transplantation represents the only curative option for mngie. due to its rarity and relative infancy as a condition, little is known of the expected course following transplant or the best approach to transplantation itself. despite previous challenges with graft failure in mngie recipients, we were able to gain rapid and sustained donor engraftment using 2 different myeloablative conditioning regimes with minimal transplant-related morbidity and no mortality. in keeping with previous reports, resolution of established gastrointestinal symptoms has been slow, though the 2 siblings transplanted earlier in their disease course have shown more rapid improvement supporting the role of early recognition and access to transplant. it is essential moving forward that specialised transplantation centres collaborate so as to guide clinicians in the management of such a challenging condition. disclosure: there are no conflicts of interest to disclose. g6pc3 congenital neutropenia -biology of inflammatory colitis associated with gcsf use, and disease response to allogeneic transplant, a report of 4 cases background: an autosomal recessive disease, glucose-6phosphatase catalytic subunit 3 (g6pc3) deficiency is a relatively recently identified cause of chronic severe neutropenia. there can be a spectrum to the disease and patients may also present with non-haematological features including prominent chest veins, cardiac, endocrine or urogenital abnormalities. we describe in our patient cohort a response to gcsf but an inflammatory, incapacitating, biopsyproven colitis associated with that g-csf response. we have transplanted 3 children with such colitis, and describe a similar colitis with intestinal failure in a fourth. methods: we investigate the biology of the neutropenic colitis, and demonstrate necrosis of the stimulated neutrophils. in vitro studies demonstrated that unstimulated neutrophils from patients with g6pc3d exhibited significantly increased production of il8, reactive oxygen species (ros) and neutrophil extracellular traps (nets) alongside significantly higher expression of cd11b, cd66b and cd14. in contrast, neutrophils from patients with g6pc3d produced significantly less ros, mmp-9, neutrophil elastase and nets upon stimulation. neutrophils from patients with g6pc3d also exhibited significantly accelerated apoptosis and secondary necrosis which was exaggerated upon stimulation with live escherichia coli bacteria but could only be partially rescued with supplemental exogenous glucose. results: 3 patients have undergone hsct for g6pc3 neutropenic enterocolitis (1 unrelated donor and 2 msd) after fludarabine treosulfan and thiotepa conditioning therapy. alemtuzumab was given as as serotherapy. all 3 patients are alive and well, immune suppression has been discotniuned and there is no gvhd with normal organ function, and resolution of colitis. we describe a 4 th patient with no good donor who has continuing intestinal failure with g-csf use. conclusions: we describe the aetiology of intestinal inflammation and failure with an extensive study of neutrophil biology in this metabolic neutropenia. we describe a novel indication for hsct in this "g-csfresponsive neutropenia". disclosure: nothing to declare p620 does body mass index (bmi) pose a risk to outcome for pediatric non-infantile patients undergoing hematopoietic cell transplantation (hsct)? mona al-saleh 1 , khawar siddiqui 1 , amal al-seraihy 1 , abdullah al-jefri 1 , ali al-ahmari 1 , hawazen al-saedi 1 , awatif al-anazi 1 , mouhab ayas 1 , ibrahim al-ghemlas 1 with no evidence of toxicity. as benefits of stoss therapy in hsct remain unknown, and safety has yet to have been studied extensively in the pediatric population, we hypothesize that stoss therapy is an effective and safe method to reach and attain sufficient levels of vd in pediatric patients undergoing hsct. methods: this is an ongoing prospective, randomized clinical control trial at phoenix children's hospital that commenced december 1 st , 2017. following consent, subjects are randomized to the intervention (stoss) or control arm prior to hsct. stoss therapy consists of a single oral dose of vd (ranging 100,000iu -600,000iu), given based on baseline 25-hydroxyvitamin d [25(oh)d] level and age, followed by standard weekly supplementation. subjects enrolled on the control arm receive standard of care based on endocrine society guidelines of weekly vd supplementation. data collection includes demographics, 25(oh)d levels at baseline, day +30, and day +100, vd toxicity (hyperphosphatemia, hyperkalemia and renal calculi), as well comorbidities were collected. at each time point and for each trial arm, the mean 25(oh)d level and changes from baseline were computed with corresponding 95% confidence intervals (cis) to indicate variability. results: presently, 12 subjects have completed baseline assessment, with day +30 and day +100 follow-up completed for 11 and 9 of these, respectively. at baseline, the mean (95% ci) 25(oh)d was 19.8 ng/dl (10.9, 28.7) among stoss patients and 18.9 ng/dl (10. 3 results: total hrqol scores of transplanted patients were significantly improved compared to those on supportive care and also compared to healthy siblings (p < 0.0001 and 0.0002 respectively), the same was true for physical (p < 0.0001 and 0.0003 respectively) and emotional functioning (p < 0.0001 and 0.0073 respectively). social and school functioning of transplanted children were not different from healthy siblings (p 0.5893 and 0.7603 respectively) while were very significantly improved compared to children with st on supportive care (p < 0.0001 in both cases). conclusions: bmt in a lower-middle income setting may be even more impactful compared to high-income regions. our analysis clearly indicates normalization of hrqol in all major areas of children transplanted for st. a possible resilience effect was noted for physical and emotional scores which were improved compared to healthy sibling controls. we could not however quantify the effect of longer-term issues like fertility impairment after bmt which may eventually adversely impact hrqol, particularly in the indian culture. disclosure: none allogeneic hematopoietic stem cell transplantation in ataxia telangiectasia patients without malignancy background: ataxia telangiectasia (a-t) is a primary immunodeficiency with mutations in atm-gene. besides a slowly progressive neurodegenerative course, a-t leads to increased susceptibility to malignancies which affects 25% of patient (median:12.5 years) with a high mortality mainly due tocomplications of conventional radio-chemotherapy. the incidence of cancer correlates with the extent of immunodeficiency. patients often develop severe progressive granulomatous skin disease with evidence of vaccine-strain rubella-virus in the lesions. prolonged survival, neurologic improvement and malignancy prevention was observed in atm-deficient mice after treatment by syngeneic hsct. nevertheless, pre-emptive hsct is not routinely performed in a-t patients due to concerns about neurodegeneration and toxicity. methods: we present three a-t patients with severe immunodeficiency phenotype, undergoing successful hsct as an individual treatment strategy intending to restore immunodeficiency for long-term malignancyprevention (patient-1) and to treat progressive skin/joint granulomas (patients-2 and -3). results: patient-1 underwent a reduced intensity conditioning (ric) regimen at 4 years of age including fludarabine (150 mg/m 2 ), cyclophosphamide (80 mg/kg), and atg-fresenius (20 mg/kg/d) which was tolerated well. hematopoietic engraftment occurred by day +15. there was an expansion of naïve and memory cd4 + t-cells and cd19 + cells. while initially a mixed donor chimerism in patient's pbmcs (10-20% donor) was observed, patient's tcells (cd3 + ) reached over 90% of donor origin over time. at last follow-up (6 years) he is well, without signs of gvhd and organ toxicity, off immunosuppression with normal levels of atm-protein; his granulomas resolved. patient-2 is a 6 year-old male who was transplanted from his hla-identical sibling, conditioned with fludarabine (150 mg/m²), cyclophosphamide (120 mg/kg), and atg-fresenius (20 mg/kg). hematopoietic engraftment was observed by day +10. t-cell reconstitution started by day +40 with >200μl cd3+ t-cells. his mixed chimerism rapidly turned to donor origin (95% donor cd3+) over time. there was no acute toxicity, however, he developed lumbosacral pain episodes with evidence of urine bk-virus with spontaneous remission. an intermittent metapneumovirus associated pulmonary hypertension was observed with pericardial effusion. treatment included sildenafil and oxygen. at last follow-up (9 months) patient is well without immunosuppression. patient-3 suffered from recurrent chest infections, failure to thrive and progressive and debilitating rubella positive progressive granulomas of the skin. she received allohsct from an hla-identical family donor at 6 years of age. conditioning included busulfan (2.2 mg/kg), fludarabine (150 mg/m²), cyclophosphamide (40 mg/kg), and alemtuzumab (1 x 5mg/m², 3 x 10mg/m²). hsct was complicated by intermittent acute renal failure, cmv reactivation and tma. hematopoietic recovery was observed by day +22. t-cell chimerism increased rapidly over time (> 90% donor). at last follow-up (7 months) patient is well, off immunosuppression and ivig. her skin granuloma resolved with scarring residues. conclusions: pre-emptive allohsct is feasible in a-t when reduced intensity conditioning is used and can correct the immunodeficiency. it might be a treatment option for some a-t patients at high risk of hematological malignancy and severe granulomatous skin disease. to what extent the restored immune system and the increase of atm-protein in these patients could prevent the development of other malignancies needs to be evaluated further. disclosure: nothing to disclose p625 abstract withdrawn. hematopoietic stem cell transplantation in diamond blackfan anemia: brazilian experience background: diamond-blackfan anemia (dba) is a rare inherited red cell aplasia caused by an intrinsic defect of erythropoietic progenitors. the main therapeutic approach is based on repeated red blood cell transfusions and/or corticosteroid therapy. hematopoietic stem cell transplantation (hsct), a potentially curative treatment for dba, is indicated for patients that do not respond to first-line therapy. methods: the aim of our retrospective study is to report the outcomes of 30 brazilian dba patients transplanted between 1990 and 2017 in 9 bmt centers. the median age of the patients was 5 ys (range 1-15) and 60% were male. seventeen patients (57%) were transplanted with matched related donors (mrd) and thirteen (43%) from matched and mismatched unrelated donors (mud/mmud). in the mrd group all patients received bone marrow as hsc source, while in the mud/mmud, eight patients received bone marrow and five received cord blood. all patients with incompatibilities (mismatched) were ucb (5/5). nineteen recipients were conditioned with busulfan plus cyclophosphamide, while the remaining 11 received fludarabine and busulfan, which has been the preferred regimen in brazil in the recent years. after transplant, most (n=24) of the mrd and mud recipients received cyclosporine and short course methotrexate as graft versus host disease (gvhd) prophylaxis. results: twenty-two out of the 30 patients were alive and disease-free at a median follow-up of 24 months (range 1 to 213 months). the 3-year overall survival (os) was 71% (ci 51-91%) (fig 1) . similar results have been demonstrated in studies from europe and from the united states. when analyzed according to donor type, os was 73% (ci 55-100%) and 66% (ci 43-100%) in mrd and mud/mmud respectively (fig 2) . three out of the 5 patients who were transplanted with ucb died. these results are in agreement with those of previously published data showing worse results in unrelated ucb transplants. twenty-nine out of the 30 patients engrafted successfully. in 25 of the evaluated patients, the median time to neutrophil engraftment was 20 days (range 10-27). one patient experienced an early death from hemorrhagic shock on day 12, before neutrophil recovery, and another two patients experienced primary graft failure. post-transplant chimerism was available for 22 patients. sixteen had complete chimera (>90% chimerism), while 6 patients presented with mixed chimerism. acute gvhd was observed in 9 patients (32%), 6 of which classified as grade iv. five patients developed chronic gvhd, considered severe in three of them. eight patients died at a mean of 155 days (range 12-728 days) after hsct and the main causes of death were infections and hemorrhagic disorders. conclusions: hsct is a potentially curative treatment option for dba. in the present study, we report the outcomes of 30 patients with dba transplanted in brazil with a os of 71%, with better results in mrd compared to mud, as expected. despite the small numbers, we observed lower survival after mud/mmud ucb transplantation. since dba is a rare disease, international collaborative studies are essential to better understand the benefits of the hsct in the treatment of these patients. disclosure: nothing to declare p627 treatment of the obliterant bronchiolitis in pediatric allogeneic recipients: two periods compared results: in group 1, the therapies administered for bo included prolonged treatment with steroids in all patients, anti-tnf in 1, azatioprine in 4; while in the group 2, all patients received ima, montelukast and azitromicin, and 4 received i.v. mpd. the median duration of imatinib therapy was 4 years (0.3-7.1 years). after a median follow-up of 4.4 years (range 0.5-12.1 yrs), 9/11 patients of group 1 (82%) died with bo in progress for transplant-related causes. while in the group 2, 2/14 (14%) died in presence of worsening bo. the estimated os at 1 year after hsct was 75% (95% ci; 50-89) in group 1 and 83% (95% ci, 27-97) in group 2 (p=0.103) (figure 1 ), while the os after 4 year decreased at 42% (95% ci; 16-66) in the group 1 while remained stable in the group 2. conclusions: this experience shows a relevant improving in prognosis of children with bo with the use of this protocol including ima, since the significant improving of survival obtained, confirming as reported in adult populations. disclosure results: we presented 20 patients with pres, age ranging from 2 months to 19 years with a average of 9.5 years. there were ten patients with thalassemia major, two patient with acute lymphoblastic leukemia, three patients with sickle cell disease and one patient with myelodysplastic syndrome, one patient with immune deficiency, two patients with acute miyeloid leukemia, one patient with aplastic anemia. ten patients were males, ten were female. all patients were treated with csa or tacrolimus and metilprednisolone for the prophylasix of gvhd. pres occurred at a median of 90 days (range 5-625). clinical findings at onset of leukoencephalopathy were hypertension, headache, seizures, visual disturbance, and altered mental function. eighteen patients alive with normal neurological status. mri showed abnormalities in all patients including patchy bilateral cortical and subcortical lesions, especially in parieto-occipital lobes. conclusions: bmt is associated with several neurological complications that may be underlying diseases, bmt procedure, and severe immunosupression. pres is an uncommon but serious complication after bmt. we report 20 cases of pres who received allogeneic bmt for thalassemia major to emphasize the importance of early recognition and institution of appropriate management of pres during bmt. disclosure: nothing to declare p631 continuous complete molecular remission using three different monoclonal antibodies followed by allogeneic bone marrow transplantation in an infant with chemotherapy-refractory acute lymphoblastic leukemia bernd gruhn 1 , susan wittig 1 , thomas ernst 1 , jana ernst 1 1 university of jena, jena, germany, background: a 10-week-old infant was diagnosed with very immature acute lymphoblastic leukemia (all) with myeloid markers in a foreign university hospital. at the end of induction therapy according to the current lal/shop protocol 10% leukemic cells were detectable in the bone marrow. treatment was changed to fludarabine, cytarabine and granulocyte colony-stimulating factor (flag) in combination with liposomal doxorubicin. after this re-induction still 5% leukemic cells were detected in bone marrow, so the bispecific t-cell engager antibody blinatumomab was given. due to an increasing portion of leukemic cells during the continuous infusion, antibody therapy was stopped and a cycle of clofarabine, cyclophosphamide and etoposide was administered. unfortunately, still 31% leukemic cells were detectable afterwards. because of chemotherapy-refractory leukemia a palliative oral treatment with mercaptopurine was started. however, the parents did not accept the palliative situation and searched for alternative therapeutic options in other university hospitals in europe. after plenty of refusals the infant was admitted to our hospital five months after diagnosis. methods: for molecular characterization genomic dna was isolated from leukemic cells. a mll-mllt3/af9 rearrangement as a consequence of the translocation t(9;11) (p22;q23) was detected and used as a marker for minimal residual disease. for further molecular characterization targeted deep next-generation sequencing was performed for a panel of 54 leukemia-associated genes. interestingly, no mutation was found. to allow precise immunophenotyping of the leukemic cells treatment with mercaptopurine was stopped. results: as in the first immunophenotyping the cd33 antigen was found, we administered the anti-cd33 monoclonal antibody gemtuzumab ozogamicin twice within two weeks. because of the detection of cd38+ leukemic cells after infusion of gemtuzumab ozogamicin, the anti-cd38 antibody daratumumab was given alternating twice within two weeks. unfortunately afterwards, leukemic cells reappeared being negative for cd33 und cd38, but positive for cd22. therefore, we administered the third antibody, the anti-cd22 monoclonal antibody inotuzumab ozogamicin, whereupon our patient developed a tumor lysis syndrome and a severe bone marrow aplasia. shortly after, allogeneic bone marrow transplantation from an unrelated donor using a special conditioning regimen consisting of thymoglobulin, busulfan, fludarabine and clofarabine was conducted. clofarabine was added because an additional antileukemic effect especially in infant all with mll rearrangement was described. after transplantation the patient suffered from a severe hepatic sinusoidal obstruction syndrome with massive ascites, renal and pulmonary dysfunction, but finally the patient recovered completely. the first bone marrow examination 30 days after transplantation revealed a donor chimerism of 100% and a complete molecular remission using the mll-mllt3/af9 rearrangement as marker for minimal residual disease. in all follow-up bone marrow samples we observed a complete donor chimerism and a complete molecular remission. currently, eight months after transplantation the patient is in a very good physical condition with normal development according to the age. background: paediatric chronic graft versus host disease (cgvhd) is a debilitating condition associated with substantial morbidity and mortality. to date, there are no approved therapies for paediatric patients with cgvhd, and current treatments often lack sufficient efficacy or lead to severe/life-threatening toxicities that limit their effectiveness. ibrutinib, a first-in-class, once-daily inhibitor of bruton's tyrosine kinase (btk), is approved in the us for the treatment of adult patients with cgvhd after failure of ≥1 lines of systemic therapy. this phase 1/2 study will evaluate the use of ibrutinib in paediatric patients with moderate or severe cgvhd. methods: this open-label, multicenter, international phase 1/2 study (pcyc-1146) includes patients with moderate or severe cgvhd as defined by the 2014 nih consensus development project criteria. it is divided into two parts: part a will determine the recommended paediatric equivalent dose (rped) of ibrutinib in patients aged ≥1 to < 12 years, and part b aims to evaluate the safety and efficacy of ibrutinib in patients age ≥1 to < 22 years. for part a, patients with cgvhd aged ≥1 to < 12 years who have failed ≥1 lines of systemic therapy will receive once daily oral ibrutinib at a starting dose of 120 mg/m 2 to be escalated up to 240 mg/m 2 after 14 days, if no grade ≥3 toxicities occur, until the rped is determined. for part b, patients aged ≥12 to < 22 years with cgvhd who have failed ≥1 lines of systemic therapy or have newly diagnosed cgvhd will receive once daily ibrutinib (420 mg) until one of the following criteria is met: treatment is no longer required; new systemic treatment for cgvhd is initiated; progression of cgvhd; recurrence of underlying disease; or unacceptable toxicity. patients with newly diagnosed cgvhd will receive ibrutinib in addition to daily corticosteroids (0.5-1 mg/kg prednisone). patients < 12 years of age may be enrolled in part b and treated at the rped after it is determined in part a. key exclusion criteria include uncontrolled active systemic infection or active infection requiring systemic treatment; progressive underlying malignant disease or any post-transplant lymphoproliferative disease; or active hepatitis c/hepatitis b virus. patients must have adequate renal, hepatic, and hematologic function to be enrolled. the primary endpoint of part a is the rped of ibrutinib, as based on pharmacokinetic (pk) data; secondary endpoints include safety and pharmacodynamics (btk occupancy). the primary endpoints of part b are pk and safety of ibrutinib in paediatric patients with cgvhd. secondary endpoints for part b include response rate at 24 weeks as defined by the 2014 nih consensus development project criteria; duration of response; overall survival; and late effects on growth, development, and immune reconstitution. results: this global study is currently enrolling. conclusions: this phase 1/2 study will explore the use of ibrutinib in paediatric patients with cgvhd to potentially meet the high unmet need for proven effective therapies for this population. disclosure enzyme replacement therapy (ert) is the treatment of choice in non-neuropathic hunter syndrome, but as the recombinant enzyme does not cross the blood brain barrier and neuropathic hunter syndrome is left untreated. hematopoietic stem cell transplantation (hsct) is the standard of care in patients with severe mucopolysaccharidosis (mps) type i (mpsih, hurler syndrome) as early transplantation halts cognitive decline in these patients and significantly improves survival. only few case studies have been published on the potential benefit of hsct in mps ii and mostly used busulfan-based conditioning regimens. in one comparative non-randomised multicenter study, hsct might to be superior compared to ert. here, we present our experience in hsct in three children with hunter syndrome using a treosulfan-based conditioning regimen. methods: a retrospective chart review was carried out in patients, who underwent hsct for hunter syndrome. the conditioning chemotherapy regimen included fludarabine, treosulfan, thiotepa and atg. all patients received bone marrow of either related and or matched unrelated donors. gvhd prophylaxis was performed with csa and methotrexat. results: three patients with hunter syndrome were transplanted in our department in 2010. the age was six months, two years and four years, respectively. bone marrow donors were related in one patient and matched unrelated in two patients. the conditioning therapy was generally well tolerated. major complications were fever of unknown origin with need for antibiotic therapy and a mucositis. one patient developed a cmv reactivation. all patients engrafted successfully and recovered well from the hsct. there was no case of acute or chronic gvhd. in 2018 all three patients are alive. donor chimerism is complete in one patient; two patients have a mixed donor chimerism. after application of donor lymphocyte infusions in one patient, donor chimerism is stable at a low level of 16%. the donor chimerism of the other patient still slowly declines to currently 50%. after stem cell transplantation, two patients did not show further progression of the disease and even achieved psycho-motor improvements. interestingly, one of these patients is the one with the low donor chimerism of 16%. one patient suffers from a further progression of the underlying disease with psycho-motoric agitation, aggressive behavior and loss of speech, that occurred within the first year following hsct, but neurocognition stabilized thereafter. conclusions: we found a beneficial effect of hsct on the neuropsychological outcome or at least stabilization of neurocognitive function in our patients with a follow-up of eight years. despite low toxicity of the conditioning regimen, increased donor chimerism may further improve the neurological outcome. disclosure: nothing to declare. tandem sct in pediatric solid tumors, other than brain tumors, has no advantage in terms of efs over single procedure-single center experience , germ-cell tumour (gct), ewing sarcoma (es), nefroblastoma. patients were divided into 2 groups according to the number of procedures: 1st group-single sct procedure, 2nd group-multiple procedures. regimens used for stem cell mobilisation were: topo-cy for nbl and epi-tax for gct, followed by g-csf±plerixafor. conditioning regimens: bu-mel and thiotepa-cy for pts with nbl, thio-tax and ice for pts with gct. patients received antibiotic, antiviral and antifungal prophylaxis, parenteral nutrition and supportive treatment. patients received consolidation treatment, followed up monthly in the first year, then yearly. patients were evaluated for residual disease by imaging tests. parents signed informed consent forms. results: we performed 67 sct procedures to 52 patients: 65.3% nbl, 17.3% es, 11.6% gct and 5.8% nefroblastoma. for this study only patients with nbl and gct were considered. in 1st group were 79% of pts, 21% in 2nd group. patients were diagnosed, staged and treated according to international protocols. sex ratio was 18f/34m. age distribution was 1-4 y 38% (20 pts), 4-10 y 35% (18 pts), > 10 y 27% (14 pts). peripheral stem cell (pbsc) mobilisation was more difficult in patients with multiple courses of chemotherapy±radiotherapy. we found no difference in the period of engraftment following a 2nd or 3rd procedure. hospitalization and supportive measures increased in 2nd and 3rd procedures (26 to 29 days). patients with multiple courses of chemotherapy and multiple hospitalizations had increased infectious risk and during the 2nd or 3rd procedures developed various infectious complications.incidence of severe oral mucositis after the first hsct was 17%, after tandem hsct was 69%. nbl patients : 1st group-6/23 patients alive and efs, 1/23 receives anti g2 treatment; 2nd group-1/7 patients-alive, 2/7 patients-not reached timepoint for mibg scan; 4/7 patients-mibg negative at first, relapsed after 6 mo; 1/7 patient deceased due to pulmonary toxicity. gct patients: 1st group-1/5 patients alive and ef, 1/5 high values in afp levels and receives metronomic therapy, 3/5 patients deceased due to progressive disease, but only had 2 sct. only 1/5 patient had one procedure and died due to progressive disease. conclusions: in our study, tandem hsct in children with solid tumours lead to an increase in survival rates, at least in the first 6 months after sct. most patients (90%) had progressive or relapsed/refractory disease when referred to our department. multiple procedures require a higher number of cd34 cells, very hard to achieve in patients with multiple courses of chemo± radiotherapy. new approaches have to be considered in these diseases, especially in high risk group. disclosure: nothing to declare background: antimicrobial prophylaxis for prolonged neutropenia occurred during the pre-engraftment period is a common practice in allogeneic hsct recipients. data on its effectiveness are few and generally from cases series and not from randomized clinical trials, especially in children. methods: all clinical records of allo-hsct performed from january 1 st 2007 to november 30 th 2018 at hsct-unit of istituto g.gaslini, genoa-italy, were retrospectively reviewed. collected data were underlying diseases, type of donor, antibiotic prophylaxis administration and type, development of fever and pathogen isolated from blood culture, if any, during pre-engraftment neutropenia. antibiotic prophylaxis, usually starting together with the conditioning regimen, was categorized in "standard" (with amoxicillin/clavulanate or ampicillin/sulbactam) or "tailored" (when based on previous bacterial isolations or colonizations). results: 246 allo-hscts were performed in 217 pediatric patients (59% males) with a median age at hsct of 8 years (iqr: 4-13; range: 0-22). hscts were performed from alternative donor (ad) in 55% patients, from relative donor (rd) in 25%, and from haploidentical donor in 20%. table 1 shows the pre-engraftment febrile neutropenia episodes according to type of antibiotic prophylaxis. 224 (91%) hscts received standard prophylaxis, while 20 (8%) the tailored one; only in 2 (1%) did not receive any prophylaxis. fever occurred in 194 (87%) of episodes in patients receiving standard prophylaxis, in 19 (95%) of those treated with tailored prophylaxis and 1 (50%) in the group without prophylaxis; only 13% of patients who received prophylaxis did not develop fever.in 38% of patients, the febrile episodes were diagnosed as bloodstream infections: staphylococcus aureus in 2%; cons in 19%; enterococcus spp in 15%; enterobacteriaceae in 25%; pseudomonas aeruginosa in 11%; other non-fermenting gram negatives in 2% and fungi in10%. conclusions: the occurrence of fever in patients who received antibiotic prophylaxis suggested that it could not be effective in prevention of fever related to neutropenia after allo-hsct. the personalization of prophylaxis could be a possible path to follow these patients. disclosure methods: a total of 15 patients with leukemia or neuroblastoma were included in the study. patients' mothers signed an informed consent for participation in the study. six of study participants were boys and 9 girls, all aged 3 to 6 years. the control group consisted of 18 healthy preschool children (2 groups of 9 children aged 5 to 6 years), 8 boys and 10 girls. results: in most of games the role of a doctor was played by a child. only one child declined to impersonate both a patient and a doctor. younger children mostly agreed to have for a "patient" a toy (proposed by psychologist or one of child's own), child's mother or a medical psychologist. the game lasted for 15 -20 minutes. most patients preferred using real medical consumables and instruments (syringes, adhesive tape, winged infusion sets or, more rarely, pills). most often a syringe or an adhesive tape was chosen. as known from their mothers, among medical manipulations most unpleasant for children are injections and changing implanted catheter dressing. also, most healthy preschool children preferred using real medical instruments over toy ones. group 1 more often used a syringe, a winged infusion set, adhesive tape, gauze or pills. group 2 most often chose syringe or gauze. among medical instruments both groups more often chose a phonendoscope or thermometer.one patient refused to cause pain to a "toy patient". other children sympathized with a "toy patient", stroke injection or dressing location sites or used soothing terms ("wait a little", "it's going to be all right"), wished prompt recovery and hugged their "patients". one child was angry over his "patient" wishing him to "get hurt too". first preschool group children were mostly scolding a toy "patient" for "being guilty of getting sick". second group children were mostly compassionate, encouraged a "toy patient" telling that "all the procedures are needed to get healthy". from children's schoolmasters we know that all first group children received vaccination about a week before a test. children from second group had no injections. overall attitude towards toy "patients" was more mild in the second group. conclusions: 1. during a play children mostly use the medical devices which cause them most discomfort and/or pain. 2. manipulating the items children illustrate their own impression of medical procedures, which are most unpleasant. 3. children may express their negative emotions directed towards medical manipulations via their play actions, these negative reactions may be suppressed in different ways by parents or medical staff. 4. the intensity of child's own traumatic experience and an attitude of nearby adults may influence the child's attitude towards other patients. 5. the mother's wish for a child to tolerate all medical procedures with ease exceeds real capabilities of a small child. disclosure: nothing to declare. allogeneic stem cell transplantation in patients with mucopolysaccharidosis type iiia (sanfilippo): a case series methods: allogeneic sct was performed at the ages of 2, 5 and 4 years, respectively. all three patients received intrathecal enzyme replacement therapy within a clinical trial setting prior to hsct. the conditioning regimen consisted of treosulfan, fludarabine, thiotepa and thymoglobuline. gvhd-prophylaxis was carried out with csa and mtx in two patients and csa and mmf in one patient. stem cell source was bone marrow in two patients and peripheral blood stem cells in one patient. results: the conditioning regimen was well tolerated and all three patients successfully engrafted. two of three patients had an uncomplicated course without occurrence of acute or chronic graft-versus-host-disease (gvhd). at last follow-up 12 and 15 months after hsct, both patients are in good condition and show constant progress of psychomotor development. the third patient experienced severe steroid refractory acute gvhd of intestines (stage 4) and skin (stage 3), which resolved under intensive immunosuppression with cyclosporine, mycophenolate and ruxolitinib. around day 110 after hsct, this patient showed clinical and biochemical signs of transplant-associated microangiopathy (tma) with cerebral seizures and acute renal failure. the cerebral mri showed progressive cerebral atrophy and leukoencephalopathy, also consistent with a progress of the mps iiia. at last follow-up 15 months after hsct, this patient had recovered from tma and was in a stable clinical condition. conclusions: in consideration of the small case number and the short follow-up period in our cohort, allogeneic hsct might be considered as a salvage therapy for patients with mps iiia if other therapeutic options are unavailable for children with this otherwise unfavourable prognosis. however, the early psychoneurological course after transplant seems promising compared to the literature and hsct could become a treatment option for this rare disease. disclosure: nothing to declare methods: for the identification of underlying molecular mechanisms leading to the increased sensibility of rms cells, the activation status of different nf-kb signaling pathways were analyzed using western blot analysis and quantitative real time pcr (qpcr). further, flow cytometry was used to analyze the surface expression of death receptors on either sm treated or untreated rms cells. the overall effect on cell death induction was measured by pi/hoechst staining using a fluorescent microscope. results: treatment with sm led to the suspected degradation of iaps. followed by the activation of both the canonical nf-κb signaling pathway, indicated by the phosphorylation of iκbα and p65, and the non-canonical nf-κb signaling pathway, as indicated by the accumulation of nik and the degradation of p100 to p52. determination of selected target gene transcription revealed an upregulation of the inhibitor iκbα, nik, p100, il-8 and at later time points the death receptors trail-r1 and trail-r2. analysis of gene transcription also led to the finding of neither up-nor downregulation of ciap1 and p65. to evaluate the involvement of trail-r1 and trail-r2 in the sm induced sensitization towards nk cell-mediated killing, surface expression of both death receptors was analyzed. treatment with sm led not only to an induced transcription of trail-r1 and trail-r2, but also to an increased surface presentation of trail-r2. subsequent ligation of trail-r2 by either wt-trail or a specific agonistic antibody (etr-2) resulted in a significant increase in cell death induction. the aforementioned analysis of gene transcription hints towards a bimodal feedback mechanism regulating both, the canonical and non-canonical nf-κb signaling pathway. on the one side, the canonical pathway is negatively regulated by the induced transcription of the inhibitor iκbα. on the other side, the induced transcription of nik, p100 and relb points towards a positive feedback loop of the non-canonical pathway. one mechanism of the increased rms cell sensitivity might be the induced transcription and surface presentation of the death receptor trail-r2. the involvement of trail receptors is further validated by the cytotoxicity data, illustrating a sm mediated sensitization towards a trail induced cell death induction. this mode of cell death fits to the previous research, were trail transcription could be induced in nk cells by sm treatment. the graphical abstract shows the transcriptional upregulation of target genes leading to a putative bimodal nf-kb regulation and increased surface presentation of trail-r2 by treatment with smac mimetics. aim: to investigate the outcome of ucb transplantation in pediatric patients with malignant and non malignant diseases methods: data from 30 patients underwent first allogeneic bone marrow transplantation with ucb from 2/1999 until 6/2013 were retrospectively analyzed. eighteen had malignant disease (md), of whom 15 in complete hematologic response, and 12 non malignant disease (nmd) (scid 5, chronic granulomatous disease 1, severe aplastic anemia 2, s.kostmann 1, osteopetrosis 1, wiskott-aldrich1, amegakaryocytic thrombocytopenia 1). the majority of the patients were male, for md and nmd, as well (m:10/f:8, m:8/f:4, respectively), of median age 6.5 years (range 0.8-11.8 years) and 0.8 years (range 0.4-6.5 years), respectively results: all patients but one, received 1 ucb unit. hla compatibility in antigen/allele level was at least 5/6 and only in 3 patients with md was 4/6. conditioning regimens were myeloablative and tbi 12 gy was given in 4/30. gvhd prophylaxis consisted of cyclosporine and atg was given in all patients pre-transplantation. median value of nucleated cells for md was 3.75χ10 7 /kg (range 2-6.3χ10 7 / kg) and for nmd was 11.05χ10 7 /kg (range 2-27.2χ10 7 /kg). neutrophil and platelet engraftment was achieved in 13/18 and 12/18 patients with md respectively, in a median time of 31 days (range 17-44) και 43 days (range 20-60). in patients with nmd, neutrophil and platelet engraftment was achieved in 7/12 and 6/12 with median day of engraftment 21 days (range 18-28) και 29 days (range respectively. acute gvhd grade ιι-ιv presented in 9/18 patients with md and 4/12 with nmd, although none had cgvhd. the incidence of viral infections was 22 cases in 11 patients with md and 9 cases in 6 patients with nmd. disease relapse occured in 9/18 patients with md. after a median time of 10 years follow up, overall survival (os) and event free survival (efs) for children with md were 22% and 18.5% respectively, while for nmd, os and efs were 33%.treatment related mortality at d+100 was 8% for md and 50% for nmd. among 18 patients with md, 4 are still alive, while the rest died from relapse (n: 8), viral infections (n: 4), septicemia (n: 1) and agvhd(n: 1). among 12 patients with nmd, 4 are alive, while the rest died from viral infections (n: 2), septicemia (n:4) and multiple organ failure (n=2). the median time of hospitalization for patients with md was 76 days (range 32-168), whilst for nmd was 63 days (range 28-114). conclusions: transplantation of unrelated ucb in our unit was combined with high trm in children with nmd and higher probability of relapse for md. disclosure: nothing to declare p643 serum levels of 5-s cysteinyldopa is associated with stem cell transplantation related complications yukayo terashita 1 , mamoru honda 1 , minako sugiyama 1 , yuko cho 1 , akihiro iguchi 1 1 hokkaido university hospital, pediatrics, sapporo, japan background: diffuse hyperpigmentation is common in patients who received chemotherapy or stem cell transplantation (sct). however, there are few reports of the relationship between skin reaction such as pigmentation and the other complications. pigmentation of the skin is thought to be the result of melanin stagnating in the dermic layer due to increased synthesis of melanin and destruction of the basement membrane due to inflammation induced chemoradiotherapy. melanin pigments are classified into two types: brown to black eumelanin and yellow to reddishbrown pheomelanin. 5-s cysteinyldopa (5scd) is precursors of pheomelanin, and its serum level has been used specific biochemical marker for malignant melanoma. here, we examined serially 5scd during the course of sct to determine association with sct related complications, because visual evaluation of skin color is difficult, and there have been no reports about 5scd as sct related biomarker. methods: we prospectively analyzed 41(27 males, 14 females) patients who received sct between may 2011 and march 2015 in hokkaido university hospital. the median age at transplantation of the patients was 7.9 years (range, 0-22). indication for sct were acute myelogenous leukemia in 10 patients, acute lymphoblastic leukemia in 9 patients, and other disease in 22 patients; juvenile myelomonocytic leukemia(2), malignant solid tumor(11), immunodeficiency(6), anaplastic anemia(2), and diamond blackfan anemia (1) . 34 patients received allogeneic sct and 7 received autologous sct. myeloablative conditioning (mac) was used for 30 patients and reduced intensity conditioning (ric) was used for 11 patients. sera were obtained from patients before conditioning therapy, on day 0, +5, +10, +15, +25 and +40. all blood samples were centrifuged at 3,000 rpm for 15 min, and stored at -80˚c until used. we also examined sct related complications such as graft-versus-host disease (gvhd), viral infection, and pre-engraft syndrome (pes). statistical analyses were completed using the mann-whitney u test for unpaired samples, and kruskal-wallis test for three samples. each test was performed with a 5% level of significance. results: the average value of 5scd reached two peaks, day0 (21.6 nmol/l) and day5 (21.7 nmol/l), regardless of stem cell source and intensity of conditioning. in all patients, we found that the level of 5scd on day0 was associated with viral reactivation (p=0.049), 5scd on day5 was associated with pes (p=0.034), and 5scd on day40 was associated with malignant disease (p=0.04). similarly, in patient who received allogeneic sct (n=34), the level of 5scd on day0 was associated with viral reactivation (p=0.048), 5scd on day5 was was associated with pes (p=0.034), 5scd on day 40 was associated with malignant disease (p=0.04). in addition, the level of 5scd on day5 was associated with gvhd of skin (p=0.027), the peak level of 5scd was associated with acute gvhd (p=0.04). conclusions: we found that 5scd can be a biomarker for sct-related complications such as aute gvhd. it is presumed that the production of pheomelanin could be induced by inflammatory procedure in sct. disclosure: nothing to declare p644 hsct in children with bone marrow failure: outcomes from a single singapore centre prasad iyer 1 , michaela seng 1 , vijayakumari k 1 , ah moy tan 1 , mei yoke chan 1 , rajat bhattacharyya 1 1 kk women's and children's hospital, paediatric haematology-oncology, singapore, singapore background: children presenting with pancytopenia often present a challenge to the paediatric haematologist. the underlying diagnosis can be hard to establish as many of the inherited bone marrow failure syndromes (ibmfs) can present with protean manifestations. the large majority of patients with bone marrow failure are often diagnosed with idiopathic severe aplastic anaemia (saa) despite extensive testing. we report our experience of hsct in patients treated with primary and acquired bone marrow failure. methods: we reviewed case notes of all the children who underwent hsct for bone marrow failure in our centre. results: a total of fifteen patients underwent eighteen stem cell transplants in our centre between 2003 and 2016. three patients were diagnosed with fanconi anaemia, one with hoyeraal-hreidarsson syndrome, one with paroxysmal nocturnal haemoglobinuria and the remaining ten children had idiopathic saa. eight children had matched sibling donor transplant, 1 had a matched related donor, 1 had a matched unrelated donor, 3 had umbilical cord blood transplants and the remainder 5 were haploidentical transplants. four of the haploidentical transplants were t-cell depleted and one was t-cell replete. one child with fanconi anaemia had primary graft rejection with cord blood transplant and was successfully rescued with a haploidentical transplant. one child with saa had primary graft rejection twice (t-cell depleted graft) and then was rescued with an alternate haploidentical donor with a t-cell replete graft. of the two patients who died, one had a fatal fungal infection ten months after transplant, and the other died due to a severe influenza pneumonitis three and a half years after bmt. conclusions: haematopoietic stem cell transplant outcomes from our centre are comparable to leading centres in the world. the understanding of underlying conditions that present with bone marrow failure has improved our approach and the way we treat bone marrow failure syndromes. clinical trial registry: not applicable. disclosure: nothing to declare. methods: a retrospective study was performed in children treated with hsct who received pos or flu during early neutropenic period until engraftment from january 2000 to december 2017 at siriraj hospital in thailand. the efficacy, safety and tolerability of pos were compared to flu. results: there were 66 hsct recipients (allo-hsct 62.1%, auto-hsct 37.9%) with mean age of 7.6+4.3 years. most of the patients were thalassemia (34.4%) followed by hematologic malignancy (32.2%) and solid tumor (16.7%). seventeen and 49 cases received pos and flu, respectively. all of patients in pos group were allo-hsct whereas 48.9% in flu group were allo-hsct. in pos group, 2 cases were diagnosed with suspected ifi and 2 cases were probable ifi with total 4 cases (23.5%). in flu group, 10 cases were diagnosed with suspected ifi and 2 cases were probable ifi with total 12 cases (24.5%) which compared 2 groups were not statistically significant (p=0.937). no possible and proven ifi in both groups. in flu group patients received empirical antifungal treatment more than pos group but no statistical significance (20.4% vs.11.76%, p=0.498). both groups had similar rate of elevated liver function test (p=0.567). no early discontinuation of antifungal prophylaxis for intolerance was found in both groups. only 26.7% of patients achieved pos target trough level of 0.7 mg/l after 7 days of treatment with started dose 4 mg/kg three times a day. conclusions: pos and flu are comparably effective, safety and tolerability in ifi prophylaxis in neutropenic children treated with hsct. defining dose recommendation of pos in this setting requires larger studies. disclosure background: severe congenital neutropenia (scn) is typically characterized by anc of <500/μl, maturation arrest of bone marrow myeloid precursors at the promyelocyte-myelocyte stage, and susceptibility to lethal pyogenic bacterial and fungal infections. scn is a rare group of disorders resulting from intrinsic defects in myeloid cell proliferation and maturation caused by mutations in several genes; elane, hax1, gfi1, was, and g6pc3 are among the most common ones. almost 10% of patients are refractory to g-csf, and the only definitive curative approach for such patients is allogeneic hsct. the current absolute indications for hsct is failure to respond to g-csf treatment, or the development of mds/leukemia in patients with scn. here, we present the result of 10 children with scn who received allogeneic hsct . methods: we retrospectively assessed 10 allogeneic hsct in children with severe congenital neutropenia. all patients received busulphan (bu) based myeloablative conditioning regimen. busulphan was used according to weight adjusted dose. in addition, all patients received fludarabine 150 mg/m2 in five days or cyclophosphamide 200 mg/kg in 4 days and atg 30 mg/kg in 3 days. cyclosporin-a and mtx were used for graft versus host disease (gvhd) prophylaxis. donor chimerism was evaluated in either bone marrow or peripheral blood on days +30, +100 and +180. results: the median transplantation age of the patients was 49 months (range 11-167 months). six of them are male. two of the donors were matched siblings and 8 were unrelated two of which were 1 ag 1 ag mismatched. stem cell source was bone marrow in 6 patients, peripheral blood in 2 and cord blood in 2 patients. all patients engrafted. the median time of neutrophil and platelet engratment to was 15 (13-34) days and 16(9-90) days, respectively. graft rejection was experienced in 2 patients, one of them had received unrelated cord blood. all patients are alive, eight of which are with full donor chimerism (between 95-100 %) without any complication (no infection, no gvhd) with a median 40 months (range 24-83 months) follow up. probability of disease free and overall survival were found 80% and 100%, respectively. conclusions: we concluded that hsct is a useful treatment for scn patients, especially those who are unresponsive to gcsf treatment and at high risk for leukemic transformation. however, a larger number of scn patients and longer follow-up are necessary to identify appropriate conditioning regimens and long-term prognosis. disclosure: nothing to declare background: prolonged thrombocytopenia (pt) or secondary failure of platelet recovery (sfpr) are a lifethreatening complications that occurs in 20-40% and 12-20% respectively of the patients following allogeneic hematopoietic stem cell transplantation (allo-hsct). management strategies, including the use of growth factors, cd34+-selected stem cell boost, mesenchymal stem cell (msc) transfusion, and second allo-hsct, are not effective or possible for all patients. eltrombopag, is an oral non-peptide thrombopoietin receptor agonist, that leads to signal transduction and results in promoting the proliferation and differentiation of megakaryocytes. some recently studies show that also can promote haematopoiesis along all three lineages. methods: we described our experience in 4 paediatric patients with poor graft function or secondary failure of platelet recovery after allogeneic stem cell transplantation treated with eltrombopag. results: patients characteristics are detailed in table 1 . all the patients received and allo-hst. the median dose of cd34+ cells infusion was 6.43x10e6/kg (3.95-8.29 ). neutrophils engraftment occurred in +15 day (10-21d) and platelets in +26 day (16-42d). all the patients had an hypoplastic bone marrow with complete chimerism. the median duration from transplantation to spcf diagnosis was 10 months (1.5-24m) . one of the patients received a stem cell boost prior to eltrombopag, without response. the time onset from spgf/sfpr diagnosis to initiating eltrombopag was 16 days (8-32d). eltrombopag was started at a dose of 1mg/kg/d, requiring an increase dose in all cases. the median dose was 50 mg/d (25-100mg). the overall response rate was 50% (2/4). two patients achieved complete response (cr), as defined by platelet ≥ 50 × 109/l. both patients already got neutrophil ≥ 1.0 × 109/l without g-csf. the time from eltrombopag initiation to achieving cr was 21 (10-49d) days. the treatment was given for a median of 81 days (8-203). it was discontinued after 96 and 203 days respectively in the two responder patients. both patients maintain stable blood counts after discontinuing the treatment. the non-responders patients had to stop the treatment because of other reasons not related to eltrombopag. patient 4 had to be rescued with a cd34+ cells boost with a good response. two patients that were in treatment with voriconazole for a fungal infection developed hyperbilirubinemia. there were no grade 3-4 toxicities related to eltrombopag. conclusions: in our experience, according to recently published studies, eltrombopag is a safe and efficacy drug in the treatment of secondary failure of platelet recovery post-hsct. it may be used successfully in children. sometimes higher doses may be considered if no response is achieved. further prospective trials are needed to increase the level of evidence and to identify predictors of response. disclosure: nothing to declare very slow clearance of busulfan in a child with infant leukemia background: busulfan is a drug with a high interindividual variability between dose and exposition. therefore, it is recommended to perform therapeutic drug monitoring (tdm) in the context of myeloablative conditioning, especially in children. methods: we report on a 7-month old boy (7.2 kg, 66 cm) of caucasian decent born to non-consanguine parents with mll-rearranged prob-lymphoblastic leukemia. diagnosis was established one month after birth from peripheral blood and csf tap showed cns involvement. primary chemotherapy was commenced according to the interfant-06 protocol. however, mrd remained positive two months under treatment, leading to an indication for allogeneic stem cell transplantation. in the interfant-06 protocol, we opted for a conditioning regimen comprised of fludarabine (1.2 mg/kg for 5 days), busulfan and thiotepa (2x5 mg/kg). in our institution, busulfan is applied once daily with a target auc of 85-95 h*mg/l in this very high risk situation. according to body weight, busulfan was given with 5.1 mg/ kg as a three-hour infusion on the first day. busulfan concentrations in plasma were measured with gas chromatography-mass spectrometry (gc-ms) and auc was calculated using bayesian curve fitting. results: exact busulfan quantification was not possible after the first dose due to technical reasons. as the levels were estimated to be very high, we decided to reduce the second dose of busulfan by 25%. this resulted in a very high auc of 44 h*mg/l for the second dose, so that busulfan was discontinued after two days, because it was calculated that the patient already received busulfan with a cumulative auc of 90 h*mg/ml. trough levels after the first and second dose were 547 and 572 μg/l, respectively. the patient showed a very slow clearance of 2.1 l/h/sqm, while the volume of distribution was in the usual range (0.86 l/kg). bilirubin and liver transaminases were in the normal range at the time of conditioning, while albumin and quick were decreased on day +18 after transplantation the patient developed clinical und biochemical signs of venoocclusive disease (vod). vod symptoms completely resolved under therapy with defibrotide. leukocyte engraftment was established on day +14. unfortunately, the patients suffered from an early relapse of the leukemia from day +62. attempts to induce a second remission with blinatumomab failed. the patient is currently under palliative chemotherapy. conclusions: busulfan tdm is very important especially in infants receiving myeloablative doses of busulfan to prevent under-or over-exposure. there is evidence that high busulfan trough levels contribute to the development of vod, but anti-leukemic activity of busulfan and cns permeability make it a valuable drug for very high risk patients in childhood leukemia. larger patient cohorts are needed to assess the exposure dependent risks of toxicity versus relapse in infants and toddlers. disclosure: nothing to declare blood (ucb) obtained at delivery from three children who received a diagnosis of cerebral palsy. methods: immunophenotyping of the ucb leukocyte fraction was performed using multicolor flow cytometry. the procedure was performed according to the protocol by shatorje and colleagues (1) . briefly, the ucb samples were labeled with specific antibodies and incubated in the dark for 30 minutes. afterwards, the samples were treated with 5ml of bd facs lysing solution for 10 minutes to preserve the leukocyte fraction only. the cells were washed using pbs (roche) and then centrifuged twice (1500 rpm, 4°c, 4 minutes). the results were analyzed using the facsdiva software (becton dickinson). results: we found an increased white blood cell (wbc) count, lymphocyte count, and cd4:cd8 ratio in all ucb samples. one patient had a low nk cell count and percentage, and another had a low b-cell count and percentage. one sample displayed high t (cd3+) and th cell (cd4+) counts, but with percentages within the limits of the reference values. conclusions: we detected elevated wbc and lymphocyte counts in all ucb samples, despite a lack of intrauterine infection symptoms. many authors have described the pathogenesis of hypoxic-ischemic encephalopathy. briefly, after an acute hypoxia-ischemia insult, activated resting microglia show macrophage-like activity. this leads to a break-down of the blood-brain barrier, infiltration by peripheral leukocytes, and brain exposure, which further exacerbates inflammation. the role of systemic inflammation is being evaluated in the animal model. it is known that systemic inflammation plays a role in traumatic brain injury (tbi) and is an independent risk factor for poor outcome in isolated tbi patients. on the other hand, m2-phenotype microglia inhibit inflammation and protect neurons from secondary damage and death. however, anti-inflammatory mechanisms in neonates are immature and expose them to extremely intensive inflammation. therefore, anti-inflammatory agents, including stem cells, may be beneficial in these patients. disclosure: three of four authors are employees of the polish stem cell bank, warsaw, poland reference: background: under the hypothesis that early natural killer cell infusion (nki) following haploidentical stem cell transplantation (haplo-sct) will reduce relapse in the early post-transplant period, we conducted a pilot study to evaluate the safety and feasibility of nki following haplo-sct in children with recurrent neuroblastoma who failed previous tandem high-dose chemotherapy and autologous sct. methods: we used the high-dose 131 i-metaiodo benzylguanidine and cyclophosphamide/fludarabine/antithymocyte globulin regimen for conditioning and infused 3×10 7 /kg of ex-vivo expanded nk cells derived from a haploidentical parent donor on days 2, 9, and 16 posttransplant. results: seven children received a total of 19 nkis, and nki-related acute toxicities were fever (n = 4) followed by chills (n = 3) and hypertension (n = 3); all toxicities were tolerable. grade ≥ii acute gvhd and chronic gvhd developed in two and five patients, respectively. higher amount of nk cell population were detected in peripheral blood until 60 days post-transplant compared with reference cohort. cytomegalovirus and bk virus reactivation occurred in all patients and epstein-barr virus in six patients. six patients died of relapse/progression (n = 5) or treatment-related mortality (n = 1), and one patient remained alive. conclusions: nki following haplo-sct was relatively safe and feasible in patients with recurrent neuroblastoma. further studies to enhance the graft-versus-tumor effect without increasing gvhd are needed. disclosure: nothing to declare regenerative medicine p652 repeated administration of g-csf using stem cell mobilization protocol could induce improvement of cognitive functions of children with cerebral palsy: phase ii randomized placebo-controlled study background: we performed phase ii randomized placebocontrolled clinical study to reveal the safety and feasibility of repeated granulocyte colony-stimulating factor (g-csf) administration for improvement of cognitive functions of children with cerebral palsy. methods: forty-four children with non-severe type of cerebral palsy were enrolled, and their age were 2-10 years old. g-csf (5μg/kg) was administered for 5 days subcutaneously every 3months during 18 months. we compared their cognitive functions with the magnetic resonance imaging (mri) findings and the following tools between before and 18 months after treatment; zoo location and picture memory as working memory index (wmi) in wechsler preschool and primary scale of intelligence (wpssi), receptive and expressive vocabulary test (revt), and visual motor integration (vmi)/visual perception (vp) test. mobilized stem cell count and cytokine levels were measured before (d+0) and after (d+5) g-csf administration for 5 days every 3 months. results: no significant findings in demography was noticed between g-csf (g-) and placebo (p-) groups. no serious adverse events were observed during the whole study period. the non-severe adverse events such as urticaria (n=1), itching sense (n=3), bone pain (n=1), headache (n=1), fever (n=2), and stomatitis (n=1) were tolerable. the parents felt the clinical improvements of cognition in 10 cases of g-group and 4 cases of p-group (p=0.0367), of language in 8 cases of g-group and 3 cases of p-group (p=0.0632). in zoo location test, we can not find out the significant score (expressed as age equivalent) differences between g-and p-groups. however, in picture memory test, there were significant improvement of age equivalent of 10 months (45.60±19.66→55.60±23.27) during 18 months of study period in g-group compared to 3 months in p-group (p=0.0242). in revt, there were significant improvement of 18 months of age equivalent in expressive tests of g-group (57.87±33.66→76.00±43.38, p=0.0198) compared to 8 months in p-group. no significant findings were noted in receptive test. vmi test showed the increasing tendency of 14 months of age equivalent in g-group (47.75±13.22→61.13±17.78, p=0.0746) compared to p-group. the increment of cd34 + cell counts in peripheral blood were significant in ggroup compared to p-group. the changed levels of interleukin (il)-6, il-10, vascular endothelial growth factor (vegf) as well as g-csf were noted in g-group. we also observed the correlation of cognitive function tests and white matter connectoms of several networks using functionally-defined white matter atlases. conclusions: the repeated administration of g-csf using stem cell mobilization protocol is safe and feasible to improve the language and cognitive functions in children with cerebral palsy. further studies for cellular and paracrine effect of g-csf and/or mobilized peripheral blood stem cells would be needed. background: while high-dose chemotherapy (hdct) with autologous hematopoietic stem cell transplantation (auto-hsct) is an integral part of multimodal therapy for highrisk neuroblastoma (hr nb), there are still subgroups, in which the results are extremely poor. for these patients allografting (allo-hsct) may offer some hope. methods: we summarize the experience of 83 consecutive hr nb patients receiving therapy in our pediatric transplant department in 2008-2017. the median age was 4 years (8 months to 22 years). a total of 78 auto-hscts and 20 allo-hscts were performed. all auto-hsct recipients were characterized by one or several high-risk features: age of more than 18 months at disease (onset n=56), primary disseminated disease (n=59), unfavorable biologic variant (n=29), poor 1 st -line therapy response (n=16) or systemic relapse (n=9). most patients (n=75) received bu-mel hdct (in younger patients oral busulfan was replaced by busilvex), in 3 primary resistant cases a 5d/5d regimen was used. a total of 20 patients with 1st (n=11) or 2nd (n=2) chemosensitive relapse, resistant relapse (n=4) or poor mobilizers with locally advanced resistant tumor (n=3) received allo-hsct from haploidentical donor with fludarabine-based ric. in 12 cases the transplant was modified via immunomagnetic positive or negative selection, 8 patients received post-transplant cyclophosphamide (post-cy)-based gvhd prophylaxis. gvhd prophylaxis also consisted of calceneurin inhibitors and sirolimus. thirteen of 20 allo-hsct recipients received posttransplant immunoadoptive (n=6) or targeted (n=7) therapy. results: the 5-year os and efs in auto-hsct recipients was 48% and 36%, accordingly. all but one patient engrafted with a median time of 17 (11-51) days. bu-mel regimen was characterized by acceptable toxicity with most common toxicities being oral mucositis and infectious complications. the vod/sos incidence was only 3%. four patients dies due to infection (n=2), cns hemorrhage (n=1), and secondary leukemia (n=1). according to multivariate analysis the most important prognostic factors were response to 1 st line therapy and post auto-hsct mibg scan results. the prognosis in initially resistant patients with good response to 2 nd or 3 rd -line therapy was still very poor (all patients relapsed with the median efs of 12 months). all patients receiving a second auto-hsct after relapse died due to disease progression. with a median follow up of 6 (1-80) months 9 allo-hsct recipients are alive, 6 of them with no signs of disease progression. all long-term responders received post-transplant therapy. one patient died due to transplant complications, other deaths were caused by disease progression. there was no obvious difference between outcomes in post-cy based and transplant modification-based transplantations. agvhd more often developed in modified transplant recipients (8 patients vs 2 in post-cy group, 4 of these cases gr iii-iv), 3 patients in post-cy group had grade iii-iv hemorrhagic cystitis. the median time to engraftment was longer for ptcm group compared to transplant modification group (d +23 vs. d+17, accordingly). conclusions: while single hsct with auto-hsct is a golden standard in hr nb patients, the relapse rate is still high and the prognosis in relapsed/refractory patients is dismal. the allografting has some limited effectiveness in these cases and post-transplant therapy has a potential for further improvement. disclosure: nothing to declare p654 abstract already published. veno-occlusive liver disease (vod) is frequent but well treatable with early defibrotide administration in children with neuroblastoma receiving high-dose busulfan and melphalan background: using high-dose intravenous busulfan and melphalan (bumel) prior to autologous stem cell transplantation (sct) in children with high-risk neuroblastoma, seems to decrease toxicity of the myeloablative regimen, except for vod. in this multicenter retrospective study we aimed to assess the outcome of bumel-associated vod with early defibrotide treatment intervention. methods: we retrospectively analyzed 64 children with high-risk neuroblastoma who underwent autologous sct with i.v. bumel regimen in slovakia and prague, czech republic in the period 1/2008 -10/2018. busulfan was administered in q6 hour schedule, with therapeutic drug level monitoring in 84% of patients. all vod patients except one were treated with defibrotide starting at a standard dose of 25 mg/kg/day, given in 4 doses per day. 1 patient was treated with supportive therapy only. ursodeoxycholic acid was used as prophylaxis in all patients. vod was established using the modified seattle clinical criteria (corbacioglu, lancet 2012). results: the incidence of vod was 23% (15/64) in patients treated with intravenous busulfan and melphalan. there was no significant difference in busulfan total dose/kg between patients with (19.2 mg/kg (sd=1,8)) and without (18.0 mg/kg (sd=2,4)) vod manifestation. vod developed at a median of 17 days after sct (range 11 -22 days). anicteric forms of vod were documented, although 73% patients with vod (11/15) presented with increased bilirubin. 73% patients with vod (11/15) developed ascites but only 4 patients (27%) required ascites drainage. no vod patient received renal replacement therapy and only one needed mechanical ventilation. importantly, we successfully treated vod in all patients. relapse or progression of neuroblastoma was the cause of death in 4 vod patients (27%) who died. conclusions: despite targeting busulfan levels to decrease toxicity of the regimen, vod is common (we observed vod incidence exactly in the range of the siopen hr nbl-1 multicenter study (ladenstein, 2017) ). early recognition and early treatment with defibrotide seems to be effective in vod associated with bumel regimen -none of our 15 patients died due to vod. disclosure: nothing to declare results of high-dose chemotherapy (hdct) with autologous hematopoietic stem cell transplantation (auto-hsct) in the treatment of ewing sarcoma family tumors (esft) background: while current dose-intense treatment protocols allow achieving 70-80% survival in localized esft patients, the long-term survival in high-risk cases is still unsatisfactory. although there is a considerable body of data on high-dose consolidation the real effectiveness and optimal indications for this option are still not completely clarified. therefore, a large prospective cohort analysis may still yield useful data. methods: the whole cohort includes 73 consecutive highrisk esft patients with median age of 12 (range 1-23) years receiving hdct with auto-hsct in 2007 to 2018 after obtaining 1 st (n=25) or 2 nd (n=3) cr, pr (n=35), or stable disease (n=10). the high-risk features included lung (n=52), bone (n=25) or bone marrow (n=11) involvement, inadequate local control in primary axial tumors (n=52), large lesions volume or poor treatment response (n=52), and chemosensitive relapse (n=9). most patients had several risk factors. disseminated disease patients were also evaluated according to prognostic score by r.ladenstein et al. highdose busulfan-melphalan followed by autologous stem-cell transplantation (hdt/sct) was used. results: the median observation time was 60 (range 3-130) months. the 5-year overall (os) and event-free (efs) survival were 40% and 37%, accordingly. most important outcome predictors were inadequate local control in chemoresistant cases, a primary tumor volume more than 200 ml, more than one bone metastatic site, bone marrow involvement and additional lung metastases. according to prognostic risk score in disseminated disease esft patients identified three groups with 5-year os rates of 49% for score ≤3 (19 patients), 36% for score 3 to 5 (27 patients), and 8% for score ≥5 (12 patients), (p< 0.01). conclusions: while bu-mel hdct with auto-hsct may still be a feasible option with acceptable toxicity for chemosensitive patients with inadequate local control and some primarily disseminated cases it is ineffective in primary resistant or very high-risk metastatic patients. disclosure: nothing to declare efficacy of tandem high-dose chemotherapy with autologous hematopoietic stem cell transplantation in the treatment of infant embryonal brain tumors day +15 (range, 8-83), after the second auto-hsct was day +17 (range, 9-86). two-year overall survival (os) was 76% and disease free survival (dfs) was 68%. dfs was significantly better among patients with mb (95%) and pnet (75%) in compared to children with etmr (55%), pb (33%) and atrt (0%), (p=0,01). dfs in patients who received tandem auto-hsct was 75% in compare to infants who received only one auto-hsct (44%), (p=0,000). complications grade 4 (according to common toxicity criteria 2014) were observed in 14% of cases. conclusions: employment of tandem hdct with auto-hsct in primary infant embryonal brain tumors may be a feasible option for patients after induction treatment. both conditioning regimens had acceptable toxicity. all patients who had tandem hdct with auto-hsct had better os (75%) in compare with single hdct (44%). patients with mb and pnet had better prognosis with os 95% and 75%, respectively, in compare with other embryonal tumors. disclosure: nothing to declare background: metastatic extra ocular retinoblastoma is carrying a poor prognosis. therapeutic intensification with high-dose, marrow-ablative chemotherapy and autologous hsct has been explored, but its role is not yet clear. this study aimed to evaluate the survival outcome of patients with extraocular retinoblastoma post autologous stem cell transplant, treated at single center methods: this is a retrospective study included all patients with metastatic extraocular retinoblastoma (stages 4a and 4b) that underwent autologous hsct at the children cancer hospital egypt (cche) 57357 from november 2010 to july 2017, the treatment protocol was adopted from cog protocol (aret0321) as all patients received 4 cycles induction chemotherapy followed by consolidation myloablative conditioning, cem (vp16 416.6 mg/m2 x3, melphalan: 40 mg/m2 x4, carboplatin: 500 mg/m2 x3) and stem cell rescue. patients data including initial disease characteristics, transplant data, and survival outcomes were collected and analyzed results: a total of 11 cases were included with median age of 1.7 years, and male to female ratio 2.66. nine patients (81%) were initially presented by extra ocular disease, while 2 patients were presented by intra ocular disease and progressed to metastatic disease. according to cog staging of extra ocular disease, 4 patients had stage 4a, and 7 were stage 4b (5 of them had trilateral disease). after induction therapy, 7(63%) showed complete response and 4 (36%) had ≥ partial response. with average cd34 count of 4x10 6 / kg, the median time to anc and platelet engraftment were 10 days and 19 days respectively, and there was no transplant related mortality. post-transplant radiotherapy was given only to 2 patients. with median duration of follow up of 32 months, the overall and event free survival rates of whole patients were 88.9% and 87.5% respectively conclusions: high dose chemotherapy and stem cell transplantation is a promising potential curative option for patients with metastatic extra ocular only two primary gf (1.4%) occurred, both without dsa. 20 patients developed a primary pgf (15%). 3-years os, 3years pfs and 1-year nrm were analyzed according to the presence of dsa in comparison with negative population. no statistically difference was found. no impact of the presence of dsa on the risk of developing gf and pgf was revealed. major outcomes of transplant was analyzed separately in patients with pgf and good graft function (ggf). 3-years os, 3-years pfs and 1year-nrm in ggf and primary pgf populations were 62% vs 20% (p< 0.0001); 53% vs 20% (p< 0.001), 12% vs 40% (p=0.009), respectively. conclusions: the presence of low level of dsa in the absence of desensitization doesn't correlate with the risk of developing gf and pgf. patients who experienced a pgf had worse outcomes in comparison with patients with ggf. disclosure: nothing to declare. the impact of hla-dpb1 mismatch in t-cell replete unrelated donor allogeneic stem cell transplantation background: high resolution matching of donor-recipient hla improves outcome in allogeneic stem cell transplants. matching for hla-a, -b, -c, -drb1 and -dq is mandatory in our transplant centre, to identify 10/10 or 9/10 matched unrelated donors. high resolution matching for dpb1 has been added over the last 10-15 years. however, the role of dpb1 matching is not yet clearly defined. methods: in this study, we retrospectively analyzed the impact of hla-dpb1 matching on the outcome of t-cell replete allogeneic hematopoietic stem cell transplants with cya/mtx-and without atg as gvhd prophylaxis in patients with hematological malignancies at oslo university hospital between 2005 and 2017. 301 patients with an unrelated donor fully matched (10/10) at hla-a, -b, -c, -drb1 and -dqb1 loci were included. further, 87 patientrecipient pairs were also fully matched on dpb1 (12/12); 118 had permissive and 96 had non-permissive mimatches of one or two dpb1 alleles. the three groups were comparable with respect to diagnosis, gender, age, cytomegalovirus serostatus and conditioning regimen. results: cumulative incidence of relapse at 5 years were significantly higher in the dpb1 matched pairs compared with the permissive and non-permissive mimatched ones, at 40% vs 22% and 13% (p< 0.001) respectively. relpase free survival and overall survival were superior in the nonpermissive and permissive dbp1 mismatched groups vs the fully matched, at 60% and 49% vs 29% (p=0.01) and 59% and 53% vs 40% (p=0.09) respectively. no difference in frequency of acute gvhd grade ii-iv between the three groups were found; dp match 43%, permissive mismatch 40% and non-permissive mismatched 48% (p=0.49). neither was there a difference seen in gvhd grade iii-iv; 12% vs 17% vs 11%, respectively. finally, there were similar outcomes between the three groups regarding chronic gvhd and trm. in corrected multivariate analysis, only dp matching had significant influence on mortality and survival. conclusions: our results show a favorable relapse free and overall survival following a mud allotransplant with a dpb1 permissive or non-permissive mismatched donor compared to a fully dpb1 matched. this is likely due to an increased gvl-effect in dpb1 mismatched groups without the counterbalance of increased acute gvhd and trm. disclosure: nothing to declare p662 a haploidentical may be a better choice than a female genoidentical donor to transplant a patient with high risk acute myelogenous leukemia in first remission norbert gorin 1 , myriam labopin 1 , didier blaise 2 , goda choi 3 , gerard socie 4 , jean henri bourhis 5 , fabio ciceri 6 , emmanuelle polge 7 , arnon nagler 8 , mohamad mohty 1 china, 3 the first affiliated hospital of soochow university, hematology, suzhou, china background: despite the incidence of leukemia increases with age, currently the geriatric population is poorly represented in the standards of care concerning that older adults undergoing hematopoietic cell transplant (hct) may experience higher transplant-related mortality (trm). previous studies have demonstrated that donor age is vital for older patients by affecting trm and survival. accordingly a relevant question is whether outcomes can be improved with a younger hla-haploidentical offspring donor rather than an older hla-matched sibling (msd). in our previous multi-center report under atg+g-csf based protocol for haplo hct, offspring donor is correlated with lower trm and higher leukemia free survival (lfs) as compared with older msd in subgroup analysis for recipients >50years although it did not reach statistical significance. on the contrary, in a recent report from ebmt and cibmtr under ptcy modality for haplo hct, among patients aged 55 to 76 years, despite lower chronic graft-versus-host-disease (gvhd), graft failure, trm, and overall mortality were higher after transplant from offspring compared with an msd although there were differences in transplant platforms between the 2 groups. methods: we extended our multi-center dataset and a matched pair analysis was performed. outcomes of 142 acute leukemia patients (>=50 years) transplanted in cr1/ cr2 who received hct from offspring (n=57) or msd (n=85) between jan, 2013 and june, 2017 present in the multi-center database were analyzed. because the patient population was small, a 1:1 ratio matched pair analysis was implemented with the following matching factors: underlying disease (acute myeloid leukemia, acute lymphoblastic leukemia), disease status (cr1/cr2), age and sex of patients, year of transplant, blood group incompatibilities, and sex of donor. results: we were able to match 41 offspring with 41 msd patients. the two matched groups were comparable in baseline characteristics except for donor age due to the family relationship. all patients achieved myeloid recovery with a median time of 14d and 12d for msd cohort and offspring group (p=0.002). the 100d platelet recovery rate was 95% in both groups. the cumulative incidence of grade ii-iv acute gvhd in msd cohort was significantly lower than in offspring group (12% vs 37%, p=0.009) while the incidence of chronic gvhd in msd cohort was significantly higher than in offspring group (51% vs 27%, p=0.013). the 3-year trm (10% vs 31%, p=0.028) were significantly lower in offspring-hct compared with in msd-hct and relapse incidence was comparable (8% vs 14%, p=0.56). as a result, the 3-year overall survival (57% vs 82%, p=0.033) and lfs (55% vs 82%, p=0.024) ( figure 1 ) were significantly higher in offspring-hct compared with in msd-hct. in a multivariate analysis, msd-hct remained a significant factor for decreased overall survival (hr 2.791(1.141-6.824), p=0.024) by increased trm ), p= 0.032) in comparison with offspring-hct. conclusions: these data favor a young offspring over an older msd in patients >50 years. the current analyses confirm non-hla donor characteristics, rather than hla disparity, predominantly influence survival in older acute leukemia patients. validation of these findings requires a prospective trial wherein the transplant platforms can be closely matched. [[p664 image] 1. figure1. lfs in offspring-hct compared with in msd-hct (55% vs 82%, p=0.024)] disclosure: nothing to declare. impact of sibling donor-recipient sex combinations on rejection after hla-matched bone marrow transplantation for severe thalassemia 1 cure2children foundation, florence, italy, 2 sankalp india foundation, bangalore, india, 3 people tree hospitals, bangalore, india, 4 south east asia institute for thalassemia, jaipur, india, 5 pakistan institute of medical sciences, islamabad, pakistan, 6 central asiri hospital, colombo, sri lanka, 7 nawaloka hospital, colombo, sri lanka, 8 kokilaben dhirubhani ambani hospital, mumbai, india background: severe thalassemia (st), i.e. a thalassemia syndrome with inability to keep spontaneous hemoglobin > 7 g/dl, is a common indication for bone marrow transplantation (bmt) in children in the middle east and south east asia. sex mismatch has been associated with increased risk of solid organ rejection but is not generally considered an important transplant-associated risk factor in the context of fully matched sibling bmt for st. methods: a total of 154 consecutive sibling bone marrow transplants carried out between january 2009 and april 2017 after conditioning with busulfan (14 mg/kg oral, not adjusted to serum levels) and cyclophosphamide (200 mg/kg) (2 patients) in addition to either thiotepa (10 mg/kg) (49 patients), or anti-thymocyte globulin (genzyme 4 mg/ kg or fresenius 16 mg/kg on days -12 to -10) (92 patients) and fludarabine 150 mg/m 2 (11 patients) were analysed. all cases received cyclosporine and methotrexate or mycophenolate mofetil as gvhd/rejection prophylaxis. in the thiotepa group methylprednisolone at 0.5 mg/kg/day was also used during the first 30 days after bmt (lucarelli protocol 6i). bone marrow was the source of hematopoietic stem cells in all cases, in the atg group it was g-csfprimed (5 μg/kg/dose twice daily for 3 to 5 days prior to harvest). all patients were considered low risk based on liver size < 2 cm from costal margin and age less than 15 years (median 4.2 years, range 0.9 to 14.5), all sibling pairs where hla-compatible. results: [[p665 image] 1. sibling donor-recipient sex combinations.] the lowest rejection rate (5%) was observed in the sister to sister (s2s) group of 24 cases, followed by brother to brother (b2b) group of 40 cases with 11%. in the sister to brother (s2b) group of 59 cases, rejection rate was 21%, and 26% in the brother to sister (b2s) group of 31 cases. on univariate analysis the only significant difference at the p 0.05 level by log rank test was b2s vs. s2s groups (rejection proportions of 26% and 5% respectively). interestingly, all 3 patients with rejection and persistent pancytopenia were female recipients of male grafts. conclusions: even though several preparative regimens were employed over an 8-year period, our data suggests that sex mismatch among compatible siblings should be considered as a relevant variable related to bmt decisionmaking. we also recommend to consider autologous back up hematopoietic stem cell collection and storage in sibling sex mismatched transplants, particularly in brother to sister bmts. same-sex fully matched related bmt for severe thalassemia might be the best scenario in which reducedintensity preparation strategies aiming at maximizing fertility preservation might be explored. disclosure: nothing to declare. outcomes of t-cell replete hematopoietic cell transplantation from mismatched related or unrelated donors using high dose post-transplant cyclophosphamide based gvhd prophylaxis background: high dose post-transplant cyclophosphamide (ptcy) based gvhd prophylaxis overcomes immunological barriers in hla mismatched donor transplantation. ptcy has been adopted in many centers as de facto standard for hct from haploidentical donors (haplo hct). it's use in mismatched unrelated donor transplant (mmud hct) is less well established. methods: we analyzed retrospectively outcomes of contemporary cohorts of patients who underwent haplo hct or mmud hct using ptcy + cyclosporine (csa) and mycophenolate mofetil (mmf) at our center. we compared these outcomes with outcomes of cohorts of patients who underwent hct from matched unrelated donors (mud) using atg based gvhd prophylaxis or matched sibling donor (msd) with csa and mmf. patients and donors were considered matched if they background: hla-alloantibodies are a major risk factor for engraftment failure in allogeneic hematopoietic stem cell transplantation (hsct). particularly, complement fixing, donor specific antibodies were shown to be associated with early engraftment failure. prospective antibody-screening, although not currently required for donor search, could permit early identification of high risk patients for positive crossmatch. aim of this study is to set the basis for future applicability of antibody-screening-based definition of acceptable mismatches in donor selection, by creating a large prospective antibody-screening database of patients due to receive an hla-mismatched allogeneic hsct. methods: patients (n=2106) diagnosed with mds/mps, nhl, mm, cll, cml, anaemia (aplastic anemia, hemoglobinopathies, pnh) and hl were prospectively screened for hla-antibodies whenever initial donor search indicated that no completely matched donor would be available. screening was performed with an elisa class i +ii screening assay. all positive screening cases were tested for antigen-specific antibody identification with luminex sab, and acceptable mismatches were defined. the results were subsequently considered in donor search and selection. we now report the frequencies of alloimmunization observed in these patients. results: the highest rate of alloimmunization was observed in patients from the anaemia disease group (overall 40.5%) followed by those from the mds/mpn group (overall 24.8%). the lowest immunization rates were observed in cll (overall 6.9%) and hl (7.0%) patients. alloimmunization rates for hla-class i antigens (p< 0.001) were significantly higher compared to hla-class ii antigens. overall hla-class i immunization rates ranged from 2.3% to 33.3%. hla-class ii immunization rates ranged from 4.7% to 19.8% (table 1) . conclusions: our findings suggest that patients with high transfusion burden like anaemia and mds/mpn patients have the highest risk of hla-alloimmunization with 40.5% and 24.8% anti-hla prevalence rates, respectively. analysis of follow-up data, will enable us to confirm whether prospective definition and consideration of acceptable mismatches in donor selection may lead to similar engraftment failure rates between immunized and non-immunized patients undergoing hlamismatched hsct. background: mothers displaying a persistent fetal microchimerism (fm) proved to be the most suitable donor in t cell-depleted haploidentical stem cell transplantation (hhsct) in children. we presumed that fetal cells leave an imprint in the mothers' immune system which positively affects recognition and elimination of malignant cells in the child by maternal effector cells. distinct killer cell immunoglobulin-like receptors (kir)/hla constellations are not only associated with reduced relapse rates after hsct in children, but also supposedly influence the establishment of an fm. methods: after approval by the local irb and obtaining informed consent, we initiated a protocol to elucidate the factors that influence the establishment, persistence and effect of fm. we established a digital droplet pcr (ddpcr) protocol to determine the fetal microchimerism. for differentiation between maternal and fetal cells, biallelic short insertion/deletion polymorphisms were used. kir and hla-c genotyping was performed by ssp-pcr. parental nk cell alloreactivity against the respective leukemic blasts and kir phenotyping were analyzed by flow cytometry. results: we analyzed 45 parents, whose children were treated for hematological diseases at the university medical center hamburg-eppendorf. a fetal microchimerism was detected in 25% of the mothers. the amount of fetal cells varies between individuals (8x 10 -6 -9x 10 -4 ). we observed a positive correlation between a persisting fm and hla-c1 homo-and heterozygous mothers along with a maternal cen a/b and cen b/b genotype. additionally, fm positive mothers showed a higher surface expression of the hla-c1 respective receptors kir2dl2/s2. the percentage of alloreactive maternal nk cells against fetal cells was higher compared to paternal nk cells; while alloreactivity of fm positive maternal nk cells was similar to nk cells from fm negative mothers. conclusions: persistence of fm was more frequent in mothers carrying at least one hla-c1 allele and a centromeric b/x motif. phenotypically, fm positive mothers had higher expression of kir2dl2/s2 indicating a role of these receptors on the persistence of an fm. in vitro, maternal nk cells showed a higher alloreactivity compared to paternal nk cells. there was no difference in alloreactivity whether the mothers were fm positive or negative, suggesting other mechanisms are responsible for the superior outcome in transplantation from fm positive mothers. disclosure: nothing to declare background: although there have been significant improvements with conventional therapies in beta thalassemia major, hematopoietic stem cell transplantation is only curative therapy. related donors are preferred to diminish transplant risks. in lack of identical related donor, identical unrelated donors are second best choice. in this study, thalassemia major patients transplanted from unrelated donors (mud) were compared with thalassemic patients transplanted from relative donor (mrd) retrospectively. methods: 45 patients who were transplanted between june 2016 and december 2017 in bahçelievler medical park hospital pediatric bone marrow transplantation unit were evaluated retrospectively. all patients were classified according to pesaro risk classification. thirty four of 45 received busulfan, fludarabine, cyclophosphamide, thioteopa for conditioning, 11patients received myeloablative preparation regimen with treosulfan, fludarabine, thiotepa, cyclophosphamide. all patients were given atg, cyclosporine and methoteraxate for gvhd prophylaxis. the patients were compared in terms of acute complications in first 100 days, engraftment, chimerism, acute and chronic gvhd after transplantation. results were evaluated with ibm spss statistics 22 (ibm spss) program. results: a total of 45 patients, 26 (57.8%) male and 19 (42.2%) female, aged between 1 and 18 years (median 5 years) were evaluated. patients were evaluated in two groups as "mud" (n = 15) and "mrd" (n = 30) groups. there was no difference between groups about given stem cells (mud 6,16±1,07x10 6 /kg and mrd 6,25±1,24x10 6 / kg). neither significant difference between different pesaro risk groups in terms of developing acute and chronic gvhd and nor decreased chimerism were detected. neutrophil engraftment time (16,40 days) in mrd group was significantly longer than mud group (13,71 days) (p = 0.006) but no difference between platelet engraftments were observed. gvhd ratio was 33.3% in mud donor group and 13.3% in mrd group and no statistically significant difference was found(p> 0.005). the incidence of engraftment loss in mud group was 13.3% and 36.7% in the mrd group, and there was no statistically significant difference (p>0.05). the rate of decreased chimerism was found to be significantly higher in the mrd group (50%) than in the mud group (6.7%) (p:0.010; p< 0.05). the survival rate was 92.9% in the mud group and 96.7% in the mrd group. the disease-free survival rate was 90.9% in the mud group and 50% in the mrd group. the disease-free survival of mud group was significantly higher than mrd group (p:0.010). conclusions: in our study, transplant related complications and success of transplantation with both mud and mrds were found to be similar. it is promising for mud transplantations to found lower decreased chimerism and similar os and dfss. based on these results, it was concluded that hsct from non-family donors, especially for patients incompatible with chelation therapy and had organ damage, transplantation from unrelated identical donors can be a good choice. although the results of our study seem promising, larger patient groups and prospective clinical trials are required. disclosure: nothing to declare background: use of g-csf stimulation of bone marrow (bm) donors is beneficial in many aspects; it can enhance tnc yield, but also have an immunomodulatory effect on donor t cell function, particularly invariant natural killer t (inkt) cells expansion as well as apcs. we analyzed outcomes of 34 consecutive patients receiving bone marrow from hla-haploidentical donors that were stimulated with g-csf prior to harvest. methods: in the time period between 05/2012 and 05/ 2018, 34 patients received bone marrow from donors stimulated with 10 ug/kg bw of g-csf on days -2, -1 and day of bm collection. four patients (12%) received myeloablative (bucy) conditioning, one (3%) received tec ric conditioning while 29 (85%) received nma ("baltimore") conditioning. all patients received posttransplantation cyclophosphamide (ptcy) on days +3 and +4, tacrolimus and mmf were started on day +5. for 2 patients donors were fathers, 11 mothers, 9 siblings and 12 children. results: median age was 43 years (20-63), there were 14 female and 20 male patients. twelve patients had aml, 10 hodgkin lymphoma, 5 all, 3 mds, 3 nhl and 1 cml. median number of infused tnc in graft was 4.7x10 8 /kg bw (1.8-8.2) and cd34+ cells 1.9 x10 6 /kg bw (1-4.5) . after median follow up of 397 days (range 26-2139), overall survival was 57%, with median survival of 71 months. engraftment was established in 29 (85%) patients, 2 (6%) had primary rejection and 3 patients (9%) died in sepsis prior to engraftment. of 29 patients that engrafted, further 3 (9%) patients had secondary rejection, two of them were transplanted again from a haploidentical donor, both using pbsc as a source of graft. median time to neutrophil recovery (anc>500) was 23 days (12-36), while median time to platelet recovery (plt>20x10 9 /l) was 30 days (12-72) in evaluable patients. cumulative incidence of agvhd ii-iv was 27.7% (95% ci, 13-44);of note is that of 9 patients that developed agvhd only one had grade iii, while remaining 8 patients had grade ii. cumulative incidence of cgvhd requiring treatment was 6.9% (95% ci, [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . cumulative incidence of relapse was 36.1% (95% ci, 20-52) and trm was 25.2% (95% ci, 11-41). conclusions: the use of g-csf mobilized bm graft in the hla-haploidentical setting with ptcy has proven to be useful to us, not only in terms of tnc yield which was more than satisfactory and contributed to adequate hematological recovery, but also in the excellent control of both acute and chronic gvhd, with most patients developing agvhd of grade ii and only one grade iii (actually developed only after dli given for decreasing chimerism). comparative studies are of course warranted to prove benefit, but this data contributes to the growing body of evidence that indeed donor stem cell stimulation with g-csf has potentially powerful immunomodulatory effect. disclosure: nothing to disclose p674 other-relative donors as a reliable bank for allogeneic hsct in countries with culturally accepted cousin-cousin marriages: a two-year report from a pediatric center in iran background: although the optimal donors for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-hsct) are fully-matched siblings, the cousincousin (consanguineous) marriages in some countries have extended the chance to find a matched donor for the hsctrecipient. in this study, an outcome analysis of transplanted patients receiving stem cells from their relatives other than siblings (other-relatives or non-sibling donors) is provided. methods: in this retrospective cross-sectional study, a two-year report of patients who received allo-hsct from their other-relative donors during september 2016 to september 2018 at the department of stem cell transplantation of children's medical center in tehran, iran is presented. the patients were followed up until 1 st december 2018. results: during this time period, 178 patients underwent hsct (both autologous and allogeneic) at this center, of which 159 cases received allo-hsct. out of allo-hsct recipients, the donors of stem cells for 51 cases (32.1%) were their other-relatives. the median (range) age at hsct was 6 (1-11) years and the majority of patients were boys (31/51, 60.8%). according to disease class, the patients were most commonly involved with non-malignant hematologic diseases (18/51 patients, 35.3%) (figure) . the source of hscs for most patients (48 cases, 94.1%) was peripheral blood and for only 3 patients the source was bone marrow. the donors for 50 patients were fully matched and only one patient received the hscs from a one-locus mismatched donor. hsct was successful in 50 patients with most of them achieving full chimerism (44 patients, 86.2%) followed by those developing mixed chimerism (6 patients, 11.8%) and only one patient (2%) experienced graft failure. post-hsct complications included cmv infection in 34 patients (66.7%), other infections in 7 (13.7%), hemorrhagic cystitis in 3 (5.9%) and pres in 2 (3.9%). acute gvhd occurred in 22 patients (43.1%) and chronic gvhd in 3 (5.9%). death occurred in 7 cases and 5 of them were transplant-related, while 1 was due to disease relapse and 1 due to graft failure. the median of overall survival was 469 (40-792) days. conclusions: the likelihood of receiving hscs from an hla-matched other-relative donor in one-thirds of children undergoing allo-hsct, with comparable outcomes to sibling and unrelated donors (as evidenced in this study compared with other studies), introduces family bank as a reliable source for pediatric allo-hsct in countries with culturally accepted cousin-cousin marriages. hence, for transplant physicians, parental consanguinity would be an indication of an extended search for a potential matched donor among the patient's family. [[p674 image] 1. distribution of patients according to disease and disease class] disclosure: nothing to declare. abstract already published. update on the hla frequency distribution of the portuguese bone marrow donor registry eduardo espada 1 , dário ligeiro 2 , hélder trindade 2 , joão forjaz de lacerda 1 frequency distribution varied throughout the country, allowing for analyses of molecular variance and generation of relatively geographically accurate graphical representations of genetic distances between regions and districts. conclusions: with the most recent hla analysis of the portuguese bone marrow donor registry we were able to extrapolate high-resolution haplotype frequencies from the most common low-resolution hla-a/-b/-drb1 haplotypes (corresponding to 50% of the estimated haplotypes at that level), which will lead to an optimization of its use, hopefully limiting the time between donor search and allogeneic hematopoietic stem cell transplant. disclosure: nothing to declare. abstract already published. abstract already published. unmanipulated haploidentical donor transplantation compared to identical sibling donor had better antileukemia effect for refractory/relapsed acute myeloid leukemia in not remission status background: patients diagnosed with saa with no sibling donors and who are refractory to immunosupression are candidates to hematopoietic stem cell transplant using alternative donors. haploidentical donor transplants has been reported using cyclophosphamide (cy) post stem cell infusion as immunephrophylaxis. the present study has the objective of evaluating overall survival and engraftment rates after haploidentical stem cell transplant for saa in a reference center. methods: 15 saa adult patients (≥15 yo) received hsct from haploidentical donors from de january/2010 to august/2018. median age was 19 y (15-28); donor was the father in six, mother in five and a brother in four cases. stem cell source was marrow in 13 cases (87%). conditioning: 10 patients (67%) received cy 29mg/kg, fludarabine 30mg/m² e tbi 200cgy. the remaining received the same drugs but radiotherapy dose varied from 300-400cgy, all them received immunephrophylaxis with post transplant cy 100mg/kg, cyclosporine and mmf. median of infused cells (tcn) was 4,94x10 8 /kg (2,07-12,66). results: eight patients engrafted (53%). among seven graft failures four received a second haploidentical transplant and one received an unrelated donor transplant as salvage regimen. two patients were successfully rescued after the second haplo and the others died from infectious complications. three years overall survival was 55%. death causes included: five infections and two lung hemorrhage. median survival was 93 days (17-1065). no patient had acute graft-versus-host-disease (gvhd) and one patient had mild c-gvhd. conclusions: haploidentical transplant was feasible as therapy for saa refractory to immunessupression with an overall survival of 55% in this cohort. graft failure however is still a problem to be addressed in this setting. disclosure: no disclosure stem cell mobilization, collection and engineering p681 abstract already published. key performance and quality indicators for a successful bone marrow collection marco sampaio 1,2 , ana salselas 2 , fátima amado 2 , filipa bordalo 2 , sérgio lopes 2 , catarina pinho 2 , susana roncon 2 and one from ecc (staphylococcus spp.)presented positive microbiological results. conclusions: bm collection is a challenging strategy because it is a one-time procedure and manually operatordependent technique; simultaneously it is more difficult to control the final cellular content of the bm, which is a risk for donor volume depletion. bm collection is feasible even with donor and recipient weight difference. poorer performance may be found when higher tnc are requested. we respond efficaciously when the request is between 200 and 400*10 8 tnc but we fail to accomplish higher tnc values. we must emphasise that icc tnc demanded was generally lower than ecc. deciding the appropriate tnc for each patient remains a dare and an art. disclosure: nothing to declare. impact of adding plerixafor to mobilization protocol in the immune reconstitution of vδt cells after autologous hematopoietic stem cell transplantation efrat luttwak 1,2 , yael chava cohen 1,2 , odelia amit 1,2 , irit avivi 1,2 , svetlana trestman 1,2 , esti rom 1,2 , rinat eshel 1,2 , ram ron 1,2 1 tel aviv medical center, tel aviv, israel, 2 sackler faculty of medicine, tel aviv university, tel aviv, israel background: multiple myeloma has remained an incurable disease even in the era of novel therapies. front line treatment typically comprises of induction chemotherapy with 4-6 cycles of a bortezomib-based regimen, stem cell mobilization, and harvesting of peripheral blood stem cells (pbsc) by apharesis, followed by high dose melphalan with hct. while brotezomib-based induction regimens have demonstrated no adverse impact on hematopoietic cell harvest number and quality, no study analyzed the impact of timing of the last brotezomib dose prior to collection. in this study we aimed to determine the effect of the timing of the last dose of brotezomib before hematopoietic cell collection and the collection yield. methods: this was a single center historical prospective study, including all sequential newly diagnosed patients with myeloma between 2012 and 2017 that were given a bortezomib-based induction therapy (≤6 cycles) followed by pbsc collection. we excluded patients who either received 1 st line vtd-pace or lenalidomide-containing regimens. peripheral blood cd34+ cells were measured on the day of collection. patients with cd34+ levels of >10 cells/ microliter started collection on the same day, while those with lower levels were given plerixafor. we performed regression analyses to analyze the impact of a variety of precollection factors, including days from last bortezomib therapy on the collection yield. results: we identified 75 patients who fulfilled the inclusion criteria, table. median time from last dose of brotezomib to first leukapheresis was 11 (range, 2-27) days. a statistically significant correlation was found between the days from last dose of brotezomib and both the first collection day-cd34+ cells/kg (r=0.466, p< 0.001), and the total collected cd34+ cells/kg(r=0.341, p=0.03), figure. the optimal cutoff point as indicated by the roc curve was 8.5 days according to collection success with sensitivity of 79% and specificity of 74%, youden´s index 0.52. in multivariate analysis included other factors affecting collection yield (age, gender, status of disease at collection, and prior radiation) -timing of last dose of brotizomib remained significantly associated with the total collected cd34+ cells/kg (p=0.01). increasing age, female gender, and prior radiation were associated with lower collection yield (p=0.006, 0.012, 0.043, respectively). based on this, we developed a model to predict the total collected cd34pos cells = 11.76+ 0.13 (timing in days of last dose of brotezomib) -0.1 (age) -1.39 (if female) -0.01 (≥pr) -1.35 (if prior radation). conclusions: timing of last dose of brotezomib is an important factor for predicting a successful collection. a washout period of 9 days is associated with a better collection yield. these results should be further validated in a prospective study. age (median, range) 63 (37-78) gender -male (%) 44 (59) prior radiation treatment (%) 18 (24) disease status at collection (%) ≥pr -51 ( disclosure: nothing to declare p685 mobilization with plerixafor in "poor mobilizer" related and unrelated donors of hpc-a in case of failed mobilization with g-csf background: in the allogeneic hpc transplantation, both from related and unrelated donors, the most commonly used source is peripheral blood after mobilization with g-csf. it is however known that about 2% of donors are "poor mobilizers"; the rescue strategies are: a third apheretic collection; bone marrow donation. methods: in 2016-2017 in italy a procedure to be adopted in case of failed mobilization of peripheral blood stem cells has been defined and shared between ibmdr, cnt (transplant national center) and cns (blood national center) and the scientific societies simti, sidem and gitmo, using plerixafor, a selective reversible antagonist of the cxcr4 receptor with its binder (the stromal derived factor sdf-1), in combination with standard g-csf dose. moreover since 2017, in accordance with this protocol, the competent authority (aifa) has extended the registration of plerixafor (law no.648/96), also for the mobilization in "poor mobilizer" healthy donors. finally, in 2018 the protocol was extended to "poor mobilizer" family donors, making management equivalent in related and unrelated donors. at the time of the donor´s informed consent for the donation of hpc, the hypothesis of the lack of mobilization or inadequate collection was illustrated and the possible actions proposed as "back-up donation" were anticipated. failed mobilization of cse has been defined as the presence of one of the two criteria: a number of cd34+ circulating on peripheral blood lower than 20/μl on the 5th day of stimulation (5d), or the collection of cd34+ < 1.0x106/kg weight of the recipient at first apheresis. in these cases, a single dose of plerixafor is administered subcutaneously by health professionals under medical supervision, 0.24mg/kg of body weight, 6-11 hours before the start of apheresis. in case of use plerixafor due to failed mobilization of hpc-a or collection of an inappropriate number of cd34+, the notification is made by the collection center to ibmdr (for both family and non-family donors), and to the recipient transplant center : both donor and recipient express their consent; finally once the collection is completed, the collection center informs ibmdr, which in turn notifies cnt/cns/simti/sidem/gitmo. any adverse reactions/events are notified in real time, based on the sop specifications and current regulations. results: since the introduction of the national protocol, 7 donors (4 unrelated donors and 3 related donors) were treated, presenting at least one of the two inclusion criteria (cd34 < 20/μl at 5d or cd34 < 1x10e6/kg after first collection)after use of plerixafor in all donors, the required dose of cd34 was obtained to ensure successful transplantation, with a sufficient increase in the cd34+ cells. no side effects or adverse reactions related to the administration of plerixafor occurred. conclusions: in cases of failed mobilization in the related and unrelated donor, the use of plerixafor according to the methods described in the shared protocol between ibmdr, cns, cnt, simti, sidem, gitmo, proved to be safe and effective. this protocol emphasizes the great value of the sharing of procedures between the register, institutions and scientific societies, ensuring the supervision of the process and the protection of the donor and recipient. disclosure: nothing to declare background: autologous stem cells transplantation (asct) is an effective treatment option for young patients with multiple myeloma (mm). a minority of patients may still experience untoward toxicity due to delayed engraftment. thus, the current policy in many centers is aimed to increase the target dose of collected cd34 + cells up to an "optimal" level of 4x10 6 /kg per procedure. therefore, an ideal mobilization, aimed to collect 4 to 8 cd34 + cells/kg in one apheresis, should achieve a number of circulating cd34 + cells >40/mcl (very good mobilizers). plerixafor may help to maximize the cd34 + collection but its use is limited by high cost. we carried out a retrospective analysis aimed to predict the quality of mobilization and develop an algorithm to optimize both timing of collection and use of plerixafor. methods: we retrospectively collected data from 91 mobilization procedure performed in our center between 2006 and 2016 for mm. all received the same mobilization protocol with cyclophosphamide (range 3-4 gr/sqm) and g-csf 10mcg/kg from +5. cd34 + cell count was started when white blood cells (wbc) count exceeded 1x10 9 /l. patients were excluded from this analysis if 1) showed a cd34+ count >40/mcl (target achieved at first day count) and/or 2) cd34 + count on second day was missing and/or 3) plerixafor was administered on first day according to previous policies. sixty-eight patients were evaluable for the study. univariate and multivariate logistic regression analysis to study ccd34 + kinetics and assess predictors impact on mobilization was carried out. ratio cd34 +/wbc in first day count, gender, disease category and time from mobilization chemotherapy were also included results: among the 68 patients included in the analysis, the threshold of 40 cd34+/mcl cells on the second day was reached by 35 (51,47%) of patients (groupa) whilst the remaining 33 (24,7%) failed the target (groupb). median (range) wbc x10 9 /l and cd34 + /mcl counts in group a and b were 2,01 (1-5,01) and 21,33 (7,56 -39,91), 1,08 (1) (2) (3) (4) (5) (6) 46 ) and 3,31 (0,06-31,14) respectively, with a statistically significant differences among group (mann-whitney p= 0,01 and p=0,02 respectively). only cd34 + /wbc ratio and cd34 + /mcl on first day count had an impact on kinetics and optimal mobilization. logistic regression model highlight cd34+/mcl (or=1,270; 95% ci: 1,060 -1,522) on first count as an independent predictor of second day optimal mobilizer, with auc of 0.9% (0, 99) in roc analysis. two cd34 + thresholds were then calculated: < 7,56/mcl (ppv 0,81; npv 1,0) that identified poor mobilizer, and ≥ 24,06/mcl (ppv 1,0; npv0,59) that exclude probability to fail on second day. for those with a cd34 + count between 7,56-24,06 the cd34+/wbc ratio (or=1,768, 95% ci: 1,307-2,391) was a predictor of optimal mobilization (auc 0,94; 0,885-0,996); cut-off value was 5,63 (sensibility 94,28; specificity 84,84) conclusions: assessment of circulating wbc, cd34+ and their ratio at wbc recovery in a chemo-based mobilization is a valid tool to manage the collection strategy and the on-demand use of plerixafor. we have developed an algorithm aimed to the use of plerixafor to both rescue poor mobilizers and boost cd34 + count in intermediate mobilizers. background: successful autologous stem cell transplantation (asct) requires the infusion of a sufficient number of hematopoietic stem cells (hscs). peripheral blood (pb) is the most commonly used source of hscs, therefore, it is important to optimize methods used to mobilize the hscs. the most clinically used chemotherapeutic agents for effective mobilization are cyclophosphamide and etoposide. recent published studies suggest that etoposide has a better mobilization effect than cyclophosphamide even at lower doses, but it is not clear why this difference occurs. in this study, we tried to determine whether there is a difference in the mechanism of mobilization between cyclophosphamide and etoposide. methods: first, in order to confirm the clinical data for efficacy and toxicity of mobilization, we retrospectively analyzed the data of patients who were diagnosed with lymphoma and performed mobilization using cyclophosphamide or etoposide from january 2011 to december 2018. second, mesenchymal stem cells (msc) were primarily cultured from the healthy controls, then treated cyclophosphamide or etoposide at a concentration of 10% inhibition of cell growth, and cytokine analysis was performed to identify cytokines known to be associated with mobilization. third, mobilization mouse model using cyclophosphamide or etoposide was generated, total blood was collected at the time of hscs collection, and cytokine and network analysis (using ingenuity pathway analysis) was performed. results: the mobilization yields for cyclophosphamide or etoposide were analyzed. etoposide miblized a significantly higher median number of cd34+cells than cyclophosphamide. the rate of successful or adequate mobilization was also significanctly higher for etoposide in univariate and multivariate analysis (table 1 ). in the analysis of toxicity during mobilization, the incidence of neutropenic fever was higher in the cyclophosphamide group (p = 0.007). during mobilization, cyclophosphamide maintained lower wbc counts than etoposide and showed a large increase in wbc counts at the start of collection ( figure 1 ). the cumulative dose of cyclophosphamide or etoposide in patients who underwent autologous stem cell transplantation did not affect leukocyte (anc>1000/microl or platelet (plt >100k/microl) engraftment. in msc treated with etoposide at a concentration of 10% inhibition of cell growth, il-8, which is a cytokine that promotes hematopoietic stem cell mobilization, were shown a statistically significant increase (figure 2 ). in the mouse model of mobilization (figure 3 ), the levels of kc, one of the il-8 homologues in mice, had significantly increased in the etoposide-treated group compared with the levels in the cyclophosphamide-treated group. the levels of other il-8 homologues, mip-2 and lix, also showed increases in the etoposide-treated group compared with those in the cyclophosphamide-treated group; these differences, however, were not statistically significant (figure 4 ). network analysis based on in vivo cytokine results identified that etoposide could promote mobilization in association with matrix metalloproteinase as compared to cyclophosphamide ( figure 5) . conclusions: etoposide has a higher mobilization efficacy when compared to cyclophosphamide, which could due to the different mechanisms of mobilization through the elevation of il8 and the activation of matrix metalloproteinase associated therewith. background: high-dose chemotherapy followed by autologous blood stem cell transplantation (asct) is a standard therapy for wide range of hematologic and solid malignancies. although various methods have been introduced to improve the peripheral blood stem cell (pbsc) mobilization, autologous stem cell collection (ascc) is not successful in every patient. furthermore, even if the ascc is complete, not all of them lead to asct. we evaluated the result of ascc and actual use of pbsc grafts in current practical setting. methods: we retrospectively reviewed the all consecutive ascc procedures performed at the department of oncology in asan medical center, seoul, korea, between january 2000 and october 2018. the targeted number of background: fanconi anemia (fa) is a rare inherited genetic bone marrow (bm) failure syndrome. while abnormal bm cells production occurs very early in life, the usual age of diagnosis is 5-10 years old. gene therapy (gt) might be an alternative to hematopoietic stem cells (hsc) transplantation, but harvest a large number of autologous hsc remains a challenge. we started a mobilization assay, fancomob, to evaluate the safety and the efficacy of fa patients' mobilization with granulocyte-colony stimulating factor (g-csf) and plerixafor. this study is part of the fa's european gt project "eurofancolen". methods: four patients with fanca mutations following the inclusion criteria were selected before pancytopenia. to note, fa4 was diagnosed before clinical manifestation through family screening. they received subcutaneous injection of g-csf (12 μg/kg twice a day) from d-1 and plerixafor (0.24 mg/kg/day) from d-4. the collection protocol targeted 3x10e6/kg of cd34+ cells, based on a predicted future weight in 5 years. cd34+ cells and white blood cells (wbc) blood count were monitored tightly along the mobilization. patients with more than 10 cd34 +/μl or between 5 and 10 cd34/μl with a clustered aspect detected by flow cytometry after plerixafor injection underwent apheresis. cd34+ cells were immunoselected from the collection with clinimacs purification system (miltenyi) and cryopreserved for further gt manipulation. results: the mobilization target was not achieved for the first two included patients (fa1 12 years old and fa2 8 years old). the minimum value of cd34+/μl required wasn't obtained for fa1 and the flow cytometry cd34+ aspect was not clustered for fa2. cd34+ cells were mobilized quickly but transitionally after plerixafor injection for the last two patients, fa3 and fa4, 5 and 2 years old respectively. both patients underwent apheresis procedures. no cd34+ cell rebound was observed after the apheresis was stopped.collection target was not achieved after four days of collection for fa3. it was obtained the first day for fa4 (figure 1 ). back-up for hsc transplantation could not be cryopreserved because of the limited number of cd34+ cells collected in patients fa3 and fa4. no short-term adverse events were observed. following cd34+ immunoselection, cd34+ cell purity and recovery were poor but in the normal range described in the literature for fanconi patients (8-20%)( table i) . one month after the collection hemograms were unchanged. conclusions: our clinical study offer new data showing that mobilization of fa patients with g-csf and plerixafor is safe and more efficient for younger patients, especially before clinical manifestations of bm failure. further efforts are required to establish an effective technic to purify the cd34+ cells after harvesting. basal cd34+ cell and platelets count are a strong predictor for mobilized peripheral blood stem cells on the 4th day of g-csf treatment in donors cryopreserved pbscs. this was associated with considerable efforts for the patients and caused additional treatment costs. on the one hand, having the therapeutic option of an autologous transplantation in the future may represent a clinically relevant advantage. however, a huge number of stem cell products are kept in storage for many years without ever been used for transplantation. our study provides cause for a careful reevaluation of the current clinical practice, which may help to focus more precisely on patients who actually benefit from a cryostored autologous stem cell graft. [[p693 image] 1. fig. 1 : absolute numbers and relative distribution of stem cell grafts] disclosure: the authors confirm that there are no potential conflicts of interest to disclose, except the following: katharina kriegsmann: research funding from bms, celgene, and sanofi. patrick wuchter: membership in advisory boards for sanofi-aventis. reduction of dimethylsulfoxide (dmso) concentration from 10% to 5% in criopreservation of stem cells. influence on the kinetics of engraftment and tolerance to infusion patricia lopez-pereira 1 , beatriz aguado 1 , elena sola 1 , carmen cámara 1 , isabel vicuña 1 , lorena vega 1 , adrian alegre 1 1 hospital universitario de la princesa, madrid, spain background: dmso is the cryoprotectant most used in the cryopreservation of stem cells. it is associated with adverse effects during the infusion of the product, its toxicity being proportional to the volume infused. the most common concentration used has been 10%, although recent publications report that reducing it to 5% leads to lower rate of side effects without impact on the product or the graft. we retrospectively analyzed 207 patients recipients of autologous peripheral blood stem cell transplantation (hct) in our hospital from january 2009 to september 2017. they are divided into two groups according to the concentration of dmso used in the freezing (10% until september 2014 or 5 % since october 2014). the baseline characteristics of the patients, the infused product and the graft are shown in table 1 . the cd34 count was performed by flow cytometry. all freezings were performed with a biological freezer for programmed controlled rate cryopreservation and stored in ultrafreezers at -80ºc. results: both population groups are homogeneous. the t-test was used for statistical analysis. regarding the cd34 + variable, no statistically significant differences were observed (p = 0.636). neither for the variables leukocyte recovery and platelet recovery (p = 0.178, p = 0.991 respectively). the difference in the variable viability is 5.61 units (ci95%: [0.61-10.62]) and is statistically significant (p = 0.028) in favor of dmso 5%. regarding adverse effects, 100% (n = 4) of the serious adverse reactions occurred in the 10% dmso group (hypotension and seizures). the mild and moderate ones were similar in both groups, most were mild nausea, vomiting and flushing. overall, no statistically significant differences were observed due to the low rate of adverse effects found. patients starting with 2014 until october 2018, total of 414 patients attempted collection of autologous pbscs, and 38 poor mobilizers recieved plerixafor during first mobilization cycle. in total, 9 patients required repeated mobilization cycles (2,2%) of which 6 were from the plerixafor group. in total 42 patients recieved pleriksafor; 23 females and 19 males, median age 58 (8-71) with following diagnoses: 28 nhl, 5 mh, 5 multiple myeloma, 1 neuroblastoma, 1 nephroblastoma, 1 sarcoma ewing and 1 seminoma. of 6 repeated mobilizations with plerixafor, 2 patients (33,3%) still failed to collect adequate transplant. in this period we had altogether 3 unsuccessful mobilizations (33,3% in repeated cycles, 0,72% in total). this group of patients consisted of 1 male and 2 female patients, median age 42 (33-58), diagnosis of nhl and failure to collect after 4 leukapheresis procedures each. median number of leukapheresis needed for adequate collection was 2 with preemtive plerixafor use, and 4 in repeated mobilizations. conclusions: our expirience shows that preemtive use of plerixafor in poor mobilizers is efficient and has enhached success of the pbsc collections. due to drug high cost each institution needs to develop its own algorythm in management of poor mobilizers. the factors contributing to plerixafor mobilization failure still need to be elucidated. disclosure: nothing to declare. platelets recovered from mobilized leukapheresis units obtained from hla-haploidentical donors fulfill the criteria of a conventional hemocomponent and can be used for transfusion background: central venous catheter (cvc) related complications may lead to high morbidity and mortality. unlike cvc, peripheral cannulation offers a quick and inexpensive method for safe and non-traumatic vascular access (va) thus its utilization is strongly recommended whenever possible. the ultrasound (us) guidance for acquiring peripheral va is a useful tool for reduction or elimination of the need of using cvc for stem cells collection. we have made an attempt to introduce us method in our apheresis unit having no previous experience with us devices. the aim of the study was to measure the decrease of cvc insertions after introducing us and evaluate the quality of va by comparing average flow rate and confirming that the desired blood volume could be processed. methods: the theoretical education involved a free elearning course in peripheral ultrasound-guided va (pugva, usabcd, aarhus, denmark). subsequently, the personnel have implemented knowledge in practical training on gelatine and silicone phantoms and healthy volunteers. the practical activities also included a fiveday course in an apheresis centre with us-guided cannulation experience. the details concerning va were recorded, including va site, cannula size, average inlet flow rate, number of inlet pressure alarms reported by the apheresis device. the procedure details where traditional approach was applied i.e. palpable cannulation and cvcs have been collected. similarly, the necessary data for procedures where veins were assessed with ultrasound prior to apheresis were recorded. results: before introducing ultrasonography, 58 stem cell collections have been performed in 46 patients. of all these procedures, 18 were accomplished with cvc (31%) and 40 with peripheral va (69%). median cubital vein was the vessel of choice. out of the 40 peripheral va procedures, 14 (35%) were problematic, with 10 or more inlet pressure alarms during every procedure. after the training stage, 34 collection procedures were performed in 25 patients. after introducing us we have observed a significant reduction of the number of cvc insertion required for successful apheresis from 31% to 9% (p=0.01; chi-square test with fisher's exact). thirty one procedures were completed with peripheral va (91%). ultrasound device enabled cannulation not only the superficial veins but also for the deeper veins. cannulation sites included upper arm cephalic vein (39%), median cubital vein (35%), upper arm basilic vein (13%), median antebrachial vein (13%). out of the 34 collections, 13 were considered problematic (42%). no difference in an average flow rate was observed between procedures performed peripherally with and without ultrasound usage (p=0.34; u mann-whitney test). conclusions: despite no previous experience in us guidance, we have successfully managed to introduce the new method in our apheresis unit. within 5 months, we have reduced cvc usage threefold and as the personnel is gaining more experience, we suppose that the cvc usage may be reduced to episodic cases. despite slightly higher number of pressure alarms, all procedures with ultrasound guidance were completed as planned. ultrasound guidance is the most important tool for significant increase in peripheral va usage and may become the only option for patients with difficult va. disclosure: nothing to declare. abstract already published. donor blood management in healthy bone marrow donors: a retrospective single institution analysis background: over the last two decades mobilized peripheral blood stem cells (pbsc) have been established as the main source of stem cells because of improved engraftment and no necessity for hospitalization for the donors. nevertheless, due to the introduction of promising new transplant regimens, especially in the haploidentical transplantation setting bone marrow (bm) donations are regaining importance. although for both donation methods severe side effects are rarely described, bm collection is associated with considerable blood loss and hence symptoms of acute blood loss are commonly observed. therefore autologous blood are collected routinely in some institutions before donation. since the collected bone marrow amount depends on the target dose, the wbc yield in the product influences the required bone marrow volume. therefore we sought to investigate the relationship between collection volume, rbc volume removal, drop in hb and indications for blood transfusion. furthermore, we assessed wbc and cd34+yields in relationship to various donor parameters and to product volume, in order to find prediction tools for collection volumes. methods: 489 allogeneic bone marrow harvests from adult donors were performed at our institution and retrospectively analyzed. complete blood counts, serum iron and ferritin were assessed at work-up and 4 weeks after donation. the bone marrow product quality including wbc, hematocrit (hct) and cd34+ cells were assessed by automatic hemocytometry and single-platform flow cytometry with ishage gating. results: besides local pain most of the side effects were related to blood loss. none of the donors received blood transfusions. the mean reduction of hemoglobin levels was 2.5 g/dl with a minimum hemoglobin level of 8.9g/dl and a persistent anemia according to who criteria after 4 weeks in 7.9% and pathologically low ferritin levels in 29%. no donor presented symptoms with indication for blood transfusion. the median wbc concentration of the bm product was 26.5/nl (5-95% percentile:16.77-38.75/ nl) the cd34+cell concentration 186.5/μl (5-95% percentile: 73.92-346.9/μl). in the linear regression analysis leukocyte counts of the donor before donation correlated significantly with wbc concentration in the product. thus in order to collect with 90% certainty the 300 mio wbc which are a typical per-kg dose for an allogeneic recipient, 16.24 ml of bone marrow must be collected. collection volume did not systematically affect wbc or cd34+ cell concentration. conclusions: achieving high wbc yields in the bone marrow product allowed for collection of relatively modest bm volumes, thus protecting donors from excessive blood loss. acute adverse events were acceptable. optimization of perioperative management in healthy bone marrow donors may be achieved by good collection technique and reevaluation of wbc yields of each institution to calculate required bone marrow amount. the collection of autologous blood is not indicated. furthermore stringent pre-and postoperative hemoglobin management is predicted to limit adverse effects. disclosure: nothing to declare. donor-recipient weight ratio predicts successful stem cell mobilization on day four of gcsf mobilization results: in group 1 median age of donor was 41 years (range 19 to 51 years). in group 2 median age of donor was 31years (range21 to 48 years). table] 1. table 1 ] elaborates other parameters analyzed between the two groups. one patient in group 1 developed grade ii acute gvhd whereas 3 patients in group 2 developed acute gvhd grade ii-iv. at the last follow up no (0/9) patient in group 1 has any symptoms of chronic gvhd whereas (2/5) patients in group 2 have features of chronic gvhd (one extensive, one limited). conclusions: our observation suggests that upfront use of plerixafor in combination with gcsf modifies the graft favorably decreasing the risk of graft failure and graft versus host disease both acute and chronic. it also helps the donor by decreasing the total volume processed, amount of acd exposure and the duration of harvest. disclosure: none. impact of vitamin d levels on peripheral stem cell mobilization in autologous hematopoietic stem cell transplant recipients ferda can 1 , zeynep arzu yegin 1 , zubeyde nur ozkurt 1 , orhun akdogan 1 , lale aydın kaynar 1 and total product cd34 + cell count [6.7(02.8-15) vs 5.9 (2.9-17.2); p=0.047] were significantly higher in patients receiving chemotherapy+g-csf than g-csf only. the study group was divided into two groups based on peripheral cd34 (cut-off level: 20x10 6 /kg) as well as product cd34 levels (cut-off level: 5x10 6 /kg). vitamin d levels were found to be similar among these groups (p>0.05). total product cd34 + cell count was found to be relatively lower in patients with vitamin d levels below 10 μg/l [5.6(3.3-10) vs 6.7(2.8-17.2); p=0,09]. (figure 1 ) conclusions: based on its effect on stem cells in in vitro studies, it may be considered that vitamin d may have a favourable impact on stem cell mobilization. statistically insignificant but relatively lower total product cd34 + cell count in patients who had lower vitamin d levels, which may indicate a role for vitamin d in stem cell mobilization, needs to be confirmed with larger studies. considering the high prevalence of vitamin d deficiency in the general population, the possible role of vitamin d in hematopoietic stem cell mobilization deserves further consideration. disclosure: nothing to declare background: one of the factors, affecting efficiency of autologous hematopoietic stem cell transplantation (autohsct) in hodgkin lymphoma (hl) patients is early recovery of graft, depending on cd 34+ cell count and conditions of cell product cryopreservation and storage. it is well known, that dimethylsulfoxide (dmso), used for cryopreservation, can be cardiotoxic and cause diverse gastrointestinal, pulmonary, kidney, liver side effects and acute hemolysis. lethal for animals dose 30-40 mg/kg leads to life threatening arrhythmias and respiratory arrest. in order to improve dmso toxicity different ways of alternative cryoconservation modes are studied -lower dmso concentration (5% vs 10%), temperature -80˚c instead of ultra-low and washing of cell product. aim of the study is to evaluate the influence of dmso washing on hematopoietic recovery after autohsct. methods: retrospective analysis of hematopoietic recovery of 37 relapse/refractory hl patients after autohsct was performed. mobilization regimen included second line chemotherapy for hl (dhap, begev, igev, ice) with consecutive g-csf administration. cd 34+ cells were assessed, using 6-colour flow cytometer facs canto ii while cell collection, thawing and washing. cells with 10% dmso were stored at -196˚and washed in 14 cases of transplantation with human albumin-dextran (reopolyglukin) and centrifugation. statistical data processing was performed by the χ2 method -pearson criterion; p -the level of significance of differences. results: patient groups had no difference in age, disease stage, gender, time from treatment start to autohsct and cd34+ cell count (p>0,05). time to wbc recovery >1х10 9 /л was 9-29 (median 13,6) days vs 10-34 (median 13,7) days, time to platelet>30х10 9 /л recovery was 11-34 (median 16,9) days vs 11-58 (median 17,9) days in groups without and with cell washing respectively (p=0,507). no difference in blood component consumption was observed (p=0,546). in 18 out of 23 (78%) patients during cell reinfusion without washing nausea, vomiting, arterial hypertension was observed, no reactions were detected after cell washing (p = 0,03). conclusions: washing autologous mononuclear cells from cryopreservant dmso does not lead to low hematopoietic recovery rate after autohsct and can avoid toxicity, thus making autohsct more safe. disclosure: authors declare no conflict of interests. quality assesment of hematopoietic stem cells autografts after cryostorage, harvested using plerixafor background: the introduction of high-dose chemotherapy followed by transplantation of autologous hemopoietic stem cells (hscs) into the treatment program for multiple myeloma (mm) has significantly increased the frequency of achieving complete remissions and overall survival in patients. to obtain a sufficient amount of hscs, hematopoiesis is stimulated with granulocyte-macrophage factors (gm-csf) both in mono mode and after the administration of cytostatics followed by cytapheresis sessions (alone or after the cytostatics followed by cytapheresis sessions) . cryopreservation protocols are used to preserve cells in a viable state, followed by long-term storage of transplants in liquid nitrogen. however, in some patients it is not possible to obtain the necessary amount of hscs. the inclusion of plerixafor in standard mobilization schemes allows you to prepare the sufficient quantity of hscs in most patients with mm. methods: the study included 18 samples of autografts from 18 patients with mm from 2015 to 2017 (median 2.1 ± 0.3). hscs mobilization was performed on the background of unstable blood formation after high doses of cyclophosphamide 6 g/m 2 with the subsequent administration of g-csf at a dose of 12-24 μg / kg (9 samples from 9 patients) and with the addition of plerixaphor at a dose of 0.24 μg / kg (9 samples from 9 patients). immunophenotype viability of hscs in autotransplants after cryopreservation were determined by flow cytometry using the ishage protocol on a flow cytometer (facs cantoii, becton dickinson) by expressing surface markers of antibodies against cd34, cd45, cd90, cd38 and staining with 7aminoactinomycin (7-aad). the colony-forming activity of hscs (cfu-cfu-mix, cfu-gm, cfu-g, cfu-m) was evaluated in methylcellulose (methocult h4435, stemcell technologies, canada) for 1x10 5 transplanted cells for 14 days. results: the viability of hscs in autografts (cd45 + / cd34 + / 7add-) after cryopreservation in both groups was 98 ± 0.7%. in the group of samples using plerixaphor, a higher content of primitive hemopoiesis precursors (primitive cells) (cd34 + cd90 + cd38-) was detected compared with the control group (29.2 ± 2.7% and 13.2 ± 12%, respectively). the cfu count (cfu-cfu-mix, cfu-gm, cfu-g, cfu-m) in the plerixafor group was 100 ± 2.5 per 1x105 explanted cells, in the control group -70 ± 1.8 ( figure 1a-d) . conclusions: the use of plerixafor against the background of standard protocols for the mobilization of hscs allows to obtain high-quality graft with a higher content of primitive cells and proliferative activity. disclosure: no conflict of interest. nothing to declare. comparison of effectiveness of plerixafor plus g-csf in poor and very poor-movilizers: efficacy of the combination of plerixafor and g-csf in poor-movilizer background: healthy donors ocassionally show a poor response to mobilization agents. plerixafor+g-csf can be a salvage strategy in poor mobilizers. some series describe the use of plerixafor to collect greater doses of cd34+ cells in hematopoietic stem cell transplantation (hsct) with tcell depletion. plerixafor use in the mobilization protocol could help collecting higher cd34+ dose in indirect t-cell depletion (cd34+ selection) for ex-vivo manipulated haploidentical transplantation, with less number of apheresis and a rapid engraftment. methods: data of fourteen healthy peripheral-blood donors was retrospectively collected. they received 4 days 10 mcg/kg/day g-csf and 0,24 mg/kg/day plerixafor on 4º day as mobilization treatment. fourteen pediatric patients (median age 13 years, range 8-15) diagnosed with malignant and no malignant hematological diseases received haploidentical hsct with cd34+ selection and cd45ra+ depletion between february 2015 and july 2017 . results: one leukoapheresis procedure was performed in all cases. median processed volume was 11 liters (range 6-14). median of cd34+ cells obtained was 13,73x10 6 /kg (range 7,8-26,47) . after positive selection, >4x10 6 /kg cd34+ cells were infused in all cases (figure 1 ). neutrophil engraftment was achieved after a median of 15 days (range 10-17). few donors presented only plerixafor mild secondary effects. conclusions: our experience showed that a mobilization protocol using g-csf and standard dose of plerixafor (compasive use) is a safe strategy that allows collecting great cd34+ dose in one apheresis procedure. this could be useful for haploidentical transplantation with ex-vivo t depletion, especially if there´s a weight disproportion between donor and patient. background: mesenchymal stem cells (mscs) are selfrenewing multipotent progenitor cells with wide differentiation potential. their ease of isolation and expansion in vitro as well as their unique regenerative therapeutic properties suggest the use of msc as an approach for treating several disorders. extra-embryonic tissues as placenta have been proposed as potential sources of mscs due to the absence of ethical problems neither risks for the patients. furthermore, only protocols using fresh placental tissue have been described so far. a protocol for isolating mscs from delayed-manipulated tissue was designed and tested in order to optimize the use of placental mscs (mscs-p) in an advanced therapies context. methods: full term placentas (n=10) were obtained from healthy mothers in hospital universitario central de asturias (spain). informed consent was obtained from each mother prior to delivery. after dissection of 12 gr decidual tissue it was washing with saline (b. braun, germany) and cut into small pieces. these biopsies were conserved 24 hours in dmem media with 1% antibiotic solution 100x (gibco, usausa) until processing. the day after, tissue was mechanically minced and then enzimatically digested with a combination of 80ui/ml dnase i (sigma aldrich, usa) and 0.25% tripsin-edta solution (w/v) (biochrom, germany) at 37ºc for 1 hour. then, the mixture was filtered with 40μm cell strainer (bd bioscience, usa) and centrifuge at 300xg for 5 minutes. finally, cells were resuspended in 5ml of dmem media suplemented with 10% fbs and antibiotic, seeded in 24-cm flask and incubated in forma stericult co 2 incubator (thermo fisher scientific, usa) at 37ºc, 5%co 2 . culture-expanded mscs cells were phenotipically characterized by flow cytometry (facs aria iiu, bd) with antibodies against cd29, cd44, cd73, cd90, cd105, cd166, cd34, cd45, hla-dr cd14 and cd19 using mesenchymal cell kit (immunostep, spain). afterwards, these cells were differentiated to adipogenic, osteogenic and chondrogenic lineages using stemmacs adipodiff media, stemmacs osteodiff media and nh chondrodiff medium (miltenyi biotec, germany) respectively. after three weeks of differentiation cells were fixed in 4% paraformaldehide (merck, usa) and analyzed. adipogenic, osteogenic and chondrogenic differentiation was visualized after staining with oil red o, alkaline phosphatase and hematoxilin-eosin (sigma-aldrich, usa). results: mscs-p isolated cells were characterized according to the isct criteria for mesenchymal stem cells. they were positive for cd29, cd44, cd73, cd90 and cd105 and negative for cd14, cd34, cd45 and hla-dr, indicative of a typical msc phenotype ( figure 1 ). all the markers showed a high percentage of expression between 84.6 and 99.9%, meaning that msc population obtained with the designed method was very homogenous. similarly, staining for the three studied lineages was positive ( figure 1 ). conclusions: the described protocol allows us to obtain mscs from decidual placental tissue stored and processed 24 hours after the biopsy extraction using a unique enzymatic digestion. this circumstance permits to take advantage of placentas that are discarded after delivery giving us the option to obtain mesenchymal cells that could be used in clinical trials. disclosure: nothing to declare outcomes of umbilical cord transplant in high risk relapsed or refractory acute myeloid leukaemia background: high-risk relapsed/refractory acute myeloid leukaemia (aml) is a fatal disease. allogeneic haematopoietic stem cell transplantation represents the only chance of cure. as the transplant relies on the graft-versusleukaemia (gvl) effect, and if different donors exert different gvl effects, then choosing the right donor assumes great importance. in manchester, a large bmt centre in the north of england, our practice in such aml has been to choose unrelated cord blood (ucbt), without serotherapy in the conditioning therapy, as our preferred donor cell source. methods: we report the results of 16 unrelated ucbt in 15 patients (five boys and ten girls) with high-risk aml, defined as relapsed or refractory disease. thirteen patients (81%) received this as a 1 st transplant, two patients (13%) received this as a 2 nd transplant for relapsed aml post matched unrelated donor transplant, and one (6%) received ucbt twice, once in cr1 and once in cr2. nine patients (56%) had mismatched ucbt, and the rest were fully matched at class-i (hla-a, -b, and-c) and class-ii (hla-drb1). conditioning was given as treosulfan, fludarabine and thiotepa in half of the patients (n = 8), other treosulfanbased regimens were used in two patients (12%), and busulfan-based regimens were used in six patients (38%). no serotherapy was given. results: the median age at transplant was 5 years (range, 5months -15years). neutrophil and platelet engraftment were achieved in 15 and 12 patients at a median of 16 and 35 days, respectively. 13 patients (81%) had engraftment syndrome. all engrafted patients achieved 100% donor chimerism, except one patient who had mixed lymphoid chimerism initially, that was corrected spontaneously to 100% at three months after transplant. acute gvhd grade i-ii developed in six patients (38%), and grade iii-iv developed in three patients (19%). all cases resolved, except two patients where acute gvhd evolved into chronic gvhd (one with grade i skin gvhd which fully resolved, and one with grade iii gvhd gut colitis who was parenteral nutrition dependent till death). two more patients developed chronic grade i skin gvhd and resolved (chronic gvhd developed in 25% in total). three patients (17%) developed veno-occlusive disease (vod), that completely resolved with defibrotide treatment and necessitated ascitic drainage in one of them. viral reactivations occurred in five patients (30%) and were successfully treated. at a median follow-up of 20 months (range, seven months -four years), eight patients (50%) died at a median of 79 (range, 24 to 230 days), with a transplantrelated mortality of 25% and relapse-related mortality of 25%. five patients (31%) relapsed post-ucbt; four died and one had a successful second ucbt (event-free survival was 44%). immune reconstitution in alive patients was achieved at a median of eight months. conclusions: very high-risk patients treated with ucbt with good overall survival and event-free survival, similar to aml treatment rate with low-risk disease. disclosure: nothing to declare in haploidentical transplants is the incidence of acute and chronic gvhd strictly related to the stem cell source? results: the odds ratio was 1.78 with a 95% confidence interval of 0.94 -3.37 (p=0.07). conclusions: the risk of infection of the uc is not related to the microbiological status of the ucb. a possible explanation for this is the presence of antibiotics in the medium used for uc, but not ucb, transport. this means that cryopreservation of ucs from which contaminated cord blood has been obtained is justified. comparison of turkish stem cell coordination center (turkok) with istanbul university bone marrow bank (tris); a single center experience in match unrelated donors azize mergen 1 , selime aydoğdu 2 , başak aksoy 3 , yunus emre savcı 2 , gürcan dikme 2 , funda çipe 2 , ceyhun bozkurt 3 , tunç fışgın 1 older patients are increasingly being transplanted, thanks to improvement in allogeneic hematopoietic stem cell transplantation (allo-hsct) techniques. increasing donor age is associated with greater risk for mortality and graftversus-host disease (gvhd). since sibling donors are of similar age to recipients, we hypothesized that, in older patients, a young matched unrelated donor (mud) would be comparable to an hla-matched sibling donor (msd). methods: we retrospectively compared outcomes of allo-hsct from msd (n=1797) and 10/10 hla mud (n=2212) in patients aged ≥ 60 years with hematological malignancies transplanted between 2006-2015. all patients received reduced-intensity conditioning and graft source was peripheral blood. the primary outcome was overall survival. msds served as the reference category and were compared to muds split into three age groups (≤25 [n=524], 25-40 [n=1072], >40 [n=616] years) using univariable analyses and multivariable cox regression models adjusted for patient, disease, and transplantation features. results: the median age of hsct recipients was 64 years and was similar across groups. median donor age for msd was 60 years and 22, 33, and 45 for the mud age groups ≤25, 25-40, and >40 years. acute leukemia was the leading transplant indication (46%) followed by myelodysplastic syndrome, myeloproliferative neoplasms and indolent non-hodgkin lymphoma. disease risk distribution was similar across donor groups (low [48%], intermediate [42%] , and high [10%] in the complete population; p=0.771). time from diagnosis to hsct was longer with mud compared to msd and increased with an older age of mud. in a univariate analysis, overall survival was 49% (msd), 50% (mud≤25), 45% (mud 25-40), and 43% (mud≥40, p=0.002). corresponding non-relapse mortality (nrm) cumulative incidence was 22%, 25%.,31%, and 32.2% (p< 0.001) (figure) . gvhd-relapse-free (grfs) was 29%, 30%, 26%, and 24% (p=0.012). in a multivariable cox model, young mud (≤25) had a similar risk for mortality compared to msd (hr 1.00, p=0.97), while a monotonic increase in risk was observed with an older donor age (mud 25-40y: hr 1.17,p=0.014; mud≥40y: hr 1.26, p=0.001) (table) . findings were confirmed in a propensity score analysis, matched for key covariates. nrm and grade 2-4 acute gvhd were consistently higher with mud, with the greatest risk associated with older muds. the hazard for grfs was higher with mud aged 25 or higher compared to msd; risk was not higher with younger mud. conclusions: in older patients receiving reduced intensity conditioning, msd remain the optimal choice. however, when not available, young mud provide comparable results. disclosure: nothing to declare background: there is growing evidence that community acquired respiratory virus (carv) increase the risk of pulmonary invasive fungal disease (ifd) in recipients of allogeneic hematopoietic stem cell transplantation (allo-hsct). to date, there is a lack of knowledge regarding the rate of ifd, risk factors (rfs) as well as the most critical period for the development of a later ifd after carv infections in allo-hsct recipients. methods: in this prospective observational study, we retrospectively analyzed the effect of carv on the development of a later ifd in a consecutive cohort of 287 allo-hsct adult recipients who developed 597 carv infectious episodes from december 2013 to december 2018. respiratory virus in upper and/or lower respiratory tract specimens were tested using multiplex pcr panel assays. results: overall, 29 out of 287 allo-hsct recipients (10%) developed ifd within 2 months after a carv episode at median of 21 days (range 0-158 days) from the day of carv detection. all the ifds involved the lungs and in 28 cases (97%) the diagnostic was ia accomplishing criteria of probable (n= 26) or proven (n=2). of note, 26 out of 29 ifd (91%) occurred within the first year after transplantation. the overall rate of ifd after carv episodes was 5% whereas this rate was higher in recipients developing carv during the first year of transplant (7%). ifd was diagnosed in 25 out of 203 with carv lower respiratory tract disease (lrtd) episodes (12%) compared to 4 out of 394 carv upper respiratory tract disease (urtd) (1%) (p= 0.0001). twenty-three out of 133 carv episodes involving the lrtd during the first year after transplant (17%) developed ifd. we did not found differences in ifd rates according to the type of carv identified. multivariate analysis identified 4 rfs for ifd: the use of atg as a part of conditioning [odds ratio (or) 2.7, 95% confidence interval (c.i.) 1.2-3.4, p= 0.01], carv lrtd (or 11.8, 95% c.i. 3.8-36, p= 0.0001), carv infection during the first year of transplant (or 5.9, p731 natural killer cell alloreactive haploidentical stem cell transplantation for multiple myeloma patients catharina elssen 1 , lotte wieten 1 , peter von dem borne 2 , ellen meijer 3 , gerard bos 1 1 maastricht university medical center, maastricht, netherlands, 2 leiden university medical center, leiden, netherlands, 3 amsterdam university medical center, location vumc, cancer center, amsterdam, netherlands background: in the past years many new drugs for multiple myeloma (mm) have been developed and are responsible for a increase in survival. notwithstanding such progress, mm remains incurable. results from allogeneic stem cell transplantation (sct), including haploidentical transplantation, in mm has shown clinical results. however, these responses are only observed in a minority of patients. we hypothesize that this observation might be due to differences in natural killer (nk) cell alloreacitvity, since we have shown in in vivo and in vitro models that mismatched alloreactive nk cells hold the capacity to kill mm cells. the aim of this prospective phase 2 study is to evaluate if kir-ligand mismatched haploindentical bone marrow transplantation (bmt) with post-transplant cyclophosphamide will improve progression free survival (pfs) in poor risk mm patients. methods: poor risk mm patients, aged < 66 years were enrolled if they were responsive to their last line of therapy. poor risk was defined as, high-risk cytogenetics, or relapse within a year after autologous sct, or treated with three or more previous lines of therapy. a prerequisite of enrolment was the possibility of an nk cell mismatch and availability of a mismatched family donor. patients were excluded if donor-specific hla-antibodies were present. patients received a haploidentical bmt with a non-myeloablative conditioning regimen and post-transplant cyclophosphamide. primary endpoint is pfs at 1,5 years. secondary endpoints are engraftment, bone marrow reconstitution, nk cell reconstitution and repertoire, graft versus host disease (gvhd), infections and non-relapse mortality (nrm) at 1,5 years. results: in total 12 poor risk patients were included in the study of which 10 could be evaluated for the primary end point. graft failure and disease progression before transplant rendered the remaining two patients not evaluable. at this interim analysis 7 patients have already reached the 1,5 years of follow up, 5 relapsed within 1,5 years and 2 died due to treatment related infections, without showing progression of disease (20% nrm). average time of progression is 105 days (60-270 days). two of the remaining patients at follow up, still show responsive disease (days 210 en 120). the average time to neutrophil reconstitution is 19 days (14-28 days). all evaluated patients (6/10) show nk cell reconstitution with a mature phenotype in the bone marrow and peripheral blood by day 60. three patients developed acute gvhd (25%) of which 2/12 grade i-ii agvhd and 1/12 patient showed a grade iv agvhd. treatment related mortality was 3/12 (25%), which was in all cases due to infectious disease. conclusions: our interim analysis of mismatched haploidentical bmt in mm showed that the treatment is feasible and forms a possible platform for immunotherapeutic strategies. the majority of patients showed an early disease progression. we predefined that with a pfs of 25% at 1,5 years we would qualify this treatment option successful. with only two patients still in remission this goal will not be achieved and we hypothesize that the late nk cell reconstitution (day 60) is responsible for the lack of response. clinical background: mscs are known to have immune modulatory capacity and may be effective in the treatment of patients with acute gvhd. however clinical studies yielded inconclusive results which was in part due to the great heterogeneity of the msc used. the off-the-shelf msc preparation "msc-ffm", generated by a proprietary pooling process, selection by plastic-adherence, expansion for an aggregate four weeks followed by cryopreservation until use, is available in germany through a national marketing authorization. "msc-ffm" is indicated in steroidrefractory agvhd, dosed at 1-2 x10 6 /kg bw i.v. in four doses one week apart. methods: we report seven consecutive pediatric patients (median age 7.49 y), who received "msc-ffm" from unrelated hla disparate donors between december 2017 and november 2018 in our institution. we gave msc infusions to 6 patients with steroid-refractory grades iii-iv agvhd and one patient who had therapy-refractory background: regulatory t cells (treg) are known for their immunosuppressive function and have proven successful as graft-versus-host disease (gvhd) prophylaxis after allogeneic bone marrow transplantation in a number of preclinical as well as first clinical studies without compromising graft-versus leukemia (gvl) effects. in murine models of acute gvhd lymph node homing capacity via cd62l (l-selectin) proved to be essential for disease prevention. yet, treg recruitment from lymph nodes to peripheral sites of ongoing gvhd also seems necessary to achieve maximum protective as well as therapeutic effects. the chemokine receptor ccr4 directs activated t cells to sites of inflammation, thus high ccr4 expression should facilitate treg homing to affected gvhd target organs. with this project we lay the foundation for future in vivo studies of treg therapy for gvhd by upregulation of ccr4 expression. methods: we performed systematic ex vivo analysis of ccr4 expression on murine naive and memory conventional (tconv) and regulatory t cells isolated from spleen, blood, bone marrow, lymph nodes, liver and lung. cells were stained for characteristic surface and intracellular markers and characterized by multiparametric flowcytometric analysis. ccr4 expression kinetics following stimulation were analysed in tconv and treg isolated from murine splenocytes by facs and polyclonally activated by anti-cd3/cd28-coated beads in the presence of exogenous il-2. expression was monitored by daily flow cytometric analysis. ccr4 overexpression was induced by transduction of expanded treg with ccr4 mrna via electroporation. expression kinetics were monitored by facs, receptor function was tested in transwell migration assays using ccr4 ligands ccl-17 and ccl-22. results: systematic analyses showed higher ccr4 expression on memory treg than on their naive counterpart in all examined organs with bone marrow samples displaying the greatest disparity. memory treg showed higher ccr4 expression than memory tconv in all analysed organs, except for lymph nodes where both memory populations revealed equal expression levels. stimulation of in vitro expanded treg and tconv lead to a strong increase in ccr4 expression with maximum levels on d3 and d2 respectively, whereas restimulation (d7) resulted in no further relevant ccr4 expression on treg. we performed systematic optimization of stimulation and mrna-electroporation conditions to reliably achieve highlevel short-term ccr4 expression. transduction of treg on d11 of in vitro expansion resulted in a strong ccr4 expression, with maximum levels 2h after electroporation and strong ccr4 expression being detectable for at least 8h. transwell migration assays showed enhanced migrational properties of mrna-electroporated treg towards ccr4 ligands. analyses performed 2h and 16h after electroporation showed persistent migration even though measured ccr4 surface expression had already declined significantly. conclusions: we showed that high ccr4 expression can be detected on memory treg in all analysed organs. since in vitro stimulation of murine treg did not reliably induce ccr4 expression, we established a protocol for ccr4 mrna-electroporation. electroporated cells showed stable short-term ccr4 expression and enhanced migrational properties towards ccr4 ligands in vitro. future studies will show whether the induction of short-term ccr4 expression will facilitate in vivo homing of adoptively transferred treg to sites of ongoing gvhd and thus mediate long-term inflammation suppression. disclosure: the authors have no conflict to disclose. survival and immune reconstitution of syngeneic, haploidentical and allogeneic hematopoietic stem cell transplantation in atm-deficient mice ruth pia duecker 1 , patrick c. baer 2 , stefan zielen 1 , ralf schubert 1 from allo-hsct. it´s not necessary to do chemotherapy before transplantation for patients with bone marrow blast cells more than 10% at the time of diagnosis. [[p738 image] 1. figure 1 the hci-ct of patients before transplantation and occurance of grade iii-iv agvhd on overall surviv] background: 1. allogeneic haematopoietic stem cell transplantation (sct) offers the chance of cure for patients with transfusion-dependent thalassemia (tdt). based on the non-neoplastic nature of this condition sct approaches urgently require to prove both efficacious and safe. methods: 2. we report on 13 children, adolescents and young adults (median age: 6 years; range 2-28 years) with tdt receiving sct from an hla-matched donor (mud n=8, msd n=3, mfd n=2) in our center from 2011-2017. all patients received the same treosulfan-based conditioning regimen (treosulfan 3x14g/m2, fludarabine 4x40mg/m2, thiotepa 1x8mg/kg). gvhd prophylaxis was based on atg-fresenius™ (3x10mg/kg, if mud or mfd as donor), csa (with taper from day +270) as well as mtx (day 1,3,6,11) in 9/13 and mmf in 4/13 patients with mtx toxicity, respectively. stem cell source was bone marrow in 8, peripheral blood stem cells in 4 and cord blood in 1 patient. prior to transplantation 6 children received cytoreductive treatment with azathioprine, hydroxycarbamide and intensified erythrocyte transfusion. iron elimination therapy was carried out in 12/13 children with deferasirox. among the 11 patients with available ferris-can™ analysis 4 patients showed substantial liver iron overload (liver iron > 8mg/g) despite intensive chelation prior to sct. results: 3. all patients achieved leukocyte engraftment at median day +23 (range 15-39), however two patients required a cd34-selected pbsc boost on day + 36 and day +50 based on delayed platelet and/or erythrocyte engraftment. nine patients exhibited full donor chimerism in the bm at day +30, the other 4 showed mixed chimerism with < 5% autologous cells. on day +360 peripheral blood chimerism was complete in 12/13 patients with the remaining patient exhibiting stable split chimerism with 100% donor-derived erythrocytes and 60-80% autologous myeloid cells. acute gvhd was observed in three patients (grade 2: n=1, grade 3: n=2). however, all patients responded to immunosuppressive therapy with steroids ± ecp and re-initiation of cni (n=1). one patient suffered background: patients with relapsed or refractory acute myeloid leukaemia (aml) have a poor prognosis. allogeneic hematopoietic stem cell transplantation is the only curative option. however, allogeneic transplantation with active leukemia failed to improve significantly the longterm outcome. to improve the outcome of allo-hsct in such high-risk and refractory patients, sequential schedule of cytoreduction therapy followed by nonmyeloablative conditioning has been developed. methods: to evaluate the outcome of sequential intensified conditioning regimen followed by allogeneic hematopoietic stem cell transplantation (allo-hsct) for refractory acute myeloid leukemia (aml). results: a total of 20 patients with primary or secondary refractory aml transplanted between june 2011 to july 2018 were included. refractoriness was defined as primary induction failure, relapse within 6 months from induction/ consolidation chemotherapy or second relapse. median age is 39 years (1 to 61). the salvage chemotherapy administered was flag-ida. two patients did not receive intensive chemotherapy because of no recovery after induction chemotherapy. seven days after the end of flag-ida, a reduced intensity conditioning consisting of fludarabine,60 mg/m2, thiotepa, 5mg/kg and busulfan 6,4 mg/kg i.v. (n=12) for haploidentical donors or fludarabine plus busulfan (n=8) for hla identical sibling or unrelated donors was administered. graft-versus-host disease (gvhd) prophylaxis consisted of tacrolimus and mycophenolate mofetil. the mycophenolate was withdrawn at day +35 post-transplantation and tacrolimus at day +100. donor lymphocytes (dli) were infused in patients without agvhd at day +120 post-transplantation. seventeen patients achieved complete donor chimerism, 3 patients progressed early and 1 patient died before engraftment. one of the patients which recovery was with persistent leukemia reached donor chimerism after immunosupression discontinuation. ten patients are alive in complete remission. median follow-up of survivors is 28 months (range: 6-43). five patients died of leukemic progression, 3 as result of gvhd and 1 suffered intracranial hemorrhage. five patients received prophylactic dli. the incidence of acute moderate-severe gvhd and moderate-severe chronic gvhd were 70% (n=14) and 50% (n=7), respectively. the non-relapse mortality was 21% (n=4), mainly due to acute gvhd (n=3) . the 2-year cumulative incidence of relapse posttransplantation was 36.8%. the probability of relapse was 57%±13%. the 2-year os and dfs were 46% ± 12% and 38% ± 12% conclusions: the strategy of sequential chemotherapy followed by allohsct ± prophylactic dli has an acceptable toxicity profile and improves both the relapse rate and the survival for refractory aml patients. disclosure: nothing to declare background: the concept of immunological intervention to prevent relapse after hematopoietic stem cell transplantation is associated with the assessment of the chimerism status. distinguishing patient and donor hematopoiesis is usually performed by str-pcr, a powerful method developed for forensic purposes. however, this method shows restrictions with respect to detection limit, preciseness, and the possibility of automated read out. digital pcr could circumvent some of these limitations. methods: recently, validated for the bio-rad droplet digital platforms, the biotype mentype digitalquant assay was released. the assay uses indel polymorphisms on chromosomal dna to distinguish patient and donor hematopoiesis on a fret hydrolysis assay basis ("taqman assays"). thus the assay in principle is applicable on the chamber based 3 d digital pcr system (thermo fisher). due to different reaction chemistry and physical properties of thermal transfer between the digital pcr platforms protocols are reasonably not fully compatible which would lead to lower fluorescence intensities and poor signal resolution on the solid chip based thermo fisher 3d platform. an adjusted pcr protocol was established and optimized using representative markers, followed by determination of tolerable and optimal amount of input dna. specificity, sensitivity and reproducibility testing with artificial mixed samples preceded the extensive verification by comparative measurement of clinical samples (n=59) and ring-trial samples (n=20). source to allogenic bm or pbsc. ucb units are immediately available for transplantation as they are frozen and banked with defined hla typing and it has an advantage for patients who need urgent transplantation. in addition, a higher degree of hla mismatch appears to be acceptable with a comparatively lower risk of acute and chronic gvhd. meanwhile, a higher incidence of engraftment failure, delayed neutrophil and platelet recovery, and posttransplant immune disorders including pre-engraftment immune reactions (pir) are major problems in unrelated ucbt. methods: in our institute, gvhd prophylaxis in ucbt was changed after march 2013. between january 2007 and march 2013, thirty-two patients received tacrolimus plus methylprednisolone (tac/mpsl) and between april 2013 and january 2018, thirty-one patients received tac plus methotrexate (tac/mtx) for gvhd prophylaxis. to investigate better gvhd prophylaxis after ucbt, we compared transplant outcomes after ucbt using gvhd prophylaxis with tac/mpsl (n=32) and tac/mtx (n=31) in single-pediatric transplantation center. results: the cumulative incidence of neutrophil engraftment at day 30 in tac/mpsl group was 70.1 % and 90.3 % in tac/mtx group (p=0.09). median time of neutrophil engraftment was 2 days earlier in tac/mtx group (17 days) than tac/mpsl group (19 days). according to pir, and acute gvhd, tac/mtx group showed superior outcomes; the incidence of pir (p=0.020) and the cumulative incidences of acute gvhd at day 100 (38.7 vs 68.8 %, p =0.045 for grade ii-iv, 9.7 vs 34.4 %, p=0.021 for grade iii-iv) was significantly lower in tac/mtx group than in tac/mpsl. however, the incidences of relapse (p=0.921) and cytomegalovirus viremia (p=0.908), and estimated overall survival (p=0.87) and event-free survival (p=0.88) were comparable between two groups. conclusions: our results indicated that gvhd prophylaxis with tac/mtx had favorable effects; reduced incidence of rip and acute gvhd after ucbt without any negative influences. disclosure: nothing to declare transfer of donor regulatory t-cells after atg reconditioning cures severe refractory gvhd and leads to long term persistence of regulatory t-cells in the recipient cells on a clinimacs® plus device (miltenyi biotec). the cell product contained 83% foxp3 + t-cells. the patient received 3,4x 10 5 /kg t reg on day +177. subsequently intestinal gvhd decreased and finally resolved. three months after the first t reg transfer the patient got a second t reg transfer (3,4x 10 5 /kg) on day +281 due to decreasing t reg levels. thereafter t reg persisted and there was no recurrence of gvhd. the patient is well with low dose sirolimus and prednisone as the only immunosuppressants and is particularly recovering intestinal function. conclusions: this case illustrates an unusually severe acute gvhd after matched sibling sct. transfer of donorderived t reg was able to cure severe and refractory gvhd after t-cell ablation by atg. transferred t reg persisted in the recipient for a long period and did not lead to any adverse events. disclosure: no disclosures to declaim allogeneic hsct for patients with transfusion dependent anemia from matched and mismatched donors julia fekadu 1 , andrea jarisch 1 , jan sörensen 1 , emilia salzmann 1 , eva rettinger 1 , andré willasch 1 , shahrzad bakhtiar 1 , thomas klingebiel 1 , peter bader 1 after first asct. the mean harvest for patients receiving dhap was 10,02x10 6 cd34(+) cells/kg, 3,04 x10 6 cd34 (+) cells/kg for cy, 8.56 x10 6 cd34(+) cells/kg for igev, 6,76 x10 6 cd34(+) cells/kg for ice, 10,52 x10 6 cd34(+) cells/kg for choep. the patient mobilized with vtd-pace achieved 2,2 x10 6 cd34(+) cells/kg after 2 apheresis. 19 of the patients achieved the target number of > 2x10 6 /kg cd34+ cells after 1 apheresis, 15 after two, and 2 after three apheresis. the median time to apheresis was 13 days (8-18) without significant difference between the regimens. the mean wbc count at the time of apheresis was 42,8x10 9 /l after dhap, 34,5 x10 9 /l after cy, 21,2 x10 9 /l after igev, 23,1 x10 9 /l after ice, 45,8 x10 9 /l after choep. there was correlation between wbc and cd34 harvested cells (p=0.005). grate 3-4 thrombocytopenia was found in 8 patient (5 dhap, 1 ice, 1igev, 1 vtd-pace). grate 3-4 anemia was registered in 3 patients (2 dhap and 1 vtd-pace). no correlation was found between the cd 34+ harvest and the age, number of previous lines chemotherapy, the response before mobilization, the type of the lymphoma and the clinical stage. conclusions: our results demonstrate that the chemo-g-csf protocols have comparable effectiveness with accepbackground: cytomegalovirus (cmv) may cause severe complications in recipients of allogeneic haematologic stem cell transplantation (allohsct). letermovir (ltv, 480/ 240mg daily without/with co-administration of cyclosporine) was recently licenced only for cmv prophylaxis in adult allohsct-recipients. paediatric data as well as data on cmv therapy are missing so far. methods: we administered letermovir 240mg orally once daily (with no co-administration of cyclosporine a) to 2 paediatric patients after allohsct. edta-plasma were occasionally obtained at different time points and frozen for determination of letermovir levels using liquid chromatography/mass spectrometry (lc-ms/ms). results: for details on patients, treatment and cmv load see table 1. in short periods of letermovir administration, cmv blood levels became negative in both patients. considering the lacking safety data in paediatric patients, we stopped letermovir treatment in both patients, when liver parameters increased. in patient 1 hepatopathy turned out to represent histologically proven graft versus host disease (gvhd). in patient 2 liver parameters further increased despite withdrawal for another 4 weeks, however, hepatopathy was only mild and self-limiting. both patients additionally received other possibly hepatotoxic substances (mycophenolate mofetil and trimethoprim/ sulfamethoxazole). letermovir plasma levels were 11.900ng/ml (2h), 11.600ng/ml (12h), 1.640-2.190ng/ml (median 1.990ng/ ml, n=3, 24h) and 1.050 ng/ml (36h after administration). conclusions: during short letermovir treatment, we observed fast resolution of cmv viraemia as well as rising liver parameters in both patients. while elevated liver parameters represented gvhd in 1 patient, a causal relationship with letermovir might be considered in the other patient. letermovir peak levels after administration of 240mg were within ranges reported in adults after administration of 480mg while trough levels were higher indicating differences in pharmacokinetics in terms of delayed clearance. inguinal lymphadenomegaly. after failure to respond to seven conventional treatment lines: methotrexate, cop (cyclophosphamide, vincristin and prednisone); gemcitabine; puva; interferon; acitretin and extracorporeal photopheresis), allogeneic hsct from an identical hla male donor was indicated. the non-myeloablative conditioning consisted of fludarabine (200mg / m2), cyclophosphamide (50mg / kg) and total body irradiation(tbi) (400cgy). prophylaxis of graft versus host disease (gvhd) was performed with cyclosporine (3mg / kg) and mycophenolate mofetil (30mg/kg). after conditioning, there was improvement of pruritus and involution of the skin. bone marrow infusion occurred on 2/9/2018 (d0). on d + 83 he presented recurrence of skin lesions of fmf. donor lymphocyte infusion (dli) was performed (1 x 10 7 cd3 + cells / kg / recipient). he presented oral lichen and diarrhea respectively as manifestations of gvhd on d + 104 and d + 112. as infectious intercurrence, hemorrhagic cystitis occurred by bk virus 3 months after the first dli and he received conservative treatment and remained without systemic immunosuppression. nine months after hsct, a second dli (5 x 10 7 cd3 + cells / kg / receptor) was performed and at this time the patient is without clinical manifestations of fmf or gvhd. conclusions: the clinical response of the presented case confirms what has been reported in the literature. ctcls appear to be particularly susceptible to gvl effect, which makes hsct a potential cure for advanced ctcls in eligible patients. the timing to perform hsct in the clinical course of the disease remains a matter to be settled clinical trial registry: not applicable disclosure: no conflict of interest p755 abstract already published. methods: we applied next generation sequencing (pgm, ion torrent/ fischer lifetechnologies) to an unselected cohort of 414 patients (176 female, 238 male, median age 59 (2-80) years) who had been referred for allogeneic stem cell transplantation due to the presence of a high-risk myeloid disorder dnmt3a r882h (3.1%) .98), cebpa (18.1%; ceb-pap198s (16.9%) vus) kras (6.5%; krasr161r (3.1%) vus), and kit (3.9%; kitm541l (3.9%) .74). patients displayed a median of 3 sequence variants (aml, mpn and cmml patients each 3; mds, saa and patients with other, non-malignant hematologic diseases each 4 sequence variants found most frequently in aml were cebpap198s (13.2% of all sequence variants in patients with aml, p< 10 -3 , chi² test) in patients suffering cmml, dnmt3ar882h was particularly frequent (4.8% of all sequence variants in cmml, p=.064). asxl1e1102d (2.1% of all sequence variants in other, non-malignant hematologic diseases, p=.699), idh2r140q (2.1%, p=.677) and krasr161r (4.2%, p=.010) were frequent in other, non-malignant hematologic diseases. in saa, nrasg12d (4% of all sequence variants in saa patients; p=.005) was frequently found, as were dnmt3ak456fs* (8%, p=.007), tet2l1721w (8%, p=.080) and tet2i1762v (8%, p=.001). conclusions: taken together, these data show that vus occur with high abundancy in this high-risk cohort of patients, and that they differ in frequency between various myeloid disorders methods: retrospective analysis of patientswho experienced either hematological relapse or progressed to aml after allo-hsct and were treated with azacitidine for this indication at 7 hematological centers in poland. the primary end-point was overall survival (os), the secondary -response rate. results: 36 patients, 22 males (61.1%), median age 52 (range, 15-66), were enrolled. the primary indication for allo-hsct was aml median time from allo-(95%ci: 4.9-11); with 2-year os of 17.2% (95%ci: 5.1-35.2). for patients stratified according to ebmt aza relapse prognostic score 2-year os was: 9 prophylaxis of agvhd: with atg -atgam 60mg/b.w.-39pts(36,8%), posttransplant cyclophosphomide (ptcy) 50mg/b.w. on d+3;d+4-67pts (63,2%) conclusions: unmanipulated haplo-hsct in 1 st -2 nd cr in children and adolescents with high risk al allows achieving the long-term survival in 64,7%. the use of g-csf stimulated unmanipulated haplo-bm is associated with a satisfactory rate of engraftment. the main cause of death in our study was relapse after allo-hsct. the frequency of acute and chronic gvhd was acceptable, 10-years grfs rate of 35,3% in 1 st -2 nd cr represents good quality of life following unmanipulated haplo-hsct and therefore may be recommended as option for use in children and adolescents with high-risk al. disclosure: nothing to declare background: b7-h3 (cd276) is thought to act as an immune checkpoint and regulates t and nk cell responses. it is highly overexpressed on many solid tumors while on healthy tissue protein expression is limited. this makes b7-h3 an interesting target for cancer immunotherapy. in highrisk neuroblastoma patients, targeting disialoganglioside gd2 with the recently approved monoclonal antibody (mab) ch14.18 after autologous or allogeneic sct, significantly improves survival. however, gd2 expression is heterogeneous and ch14.18 causes severe adverse effects. thus, we evaluated b7-h3 as an alternative or additional target antigen. we investigated different anti-b7-h3 mab constructs and mab-cytokine fusions (immunocytokines) for their ability to elicit antibody-dependent cellular cytotoxicity (adcc) using expanded γ/δ t cells of healthy donors, chex5lf-il2 was confirmed to be the most effective mab construct. interestingly, chek5-il2 showed comparable target cell lysis -however, lysis was only transient while chex5lf-il2 mediated permanent target cell lysis. using patient pbmcs after receiving allogeneic sct, chex5lf-il2 and ch14.18 mediated comparable lysis. calculated specific lysis of lan-1 (after 96 hrs.; in ascending order): targets + effectors w/o mab (22 %) conclusions: b7-h3 is a suitable target antigen in case gd2 expression is low or absent. immunocytokines and fcoptimized mabs targeting b7-h3 might increase the efficacy of immunotherapy in gd2-negative tumors and in combinatory approaches. until now, the low-fucose immunocytokine chex5lf-il2 seems to be the most promising anti-b7 great ormond street hospital, nhs foundation trust petersburg/ raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, rehabilitation medicine, saint petersburg, russian federation, 2 first i. pavlov state medical university of st. petersburg/raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, bone marrow transplantation for pediatric solid tumor petersburg/raisa gorbacheva memorial institute of children's oncology, hematology and transplantation, pediatric hsct outpatients, saint petersburg, russian federation 1 university hospital carl gustav carus james`s hospital diagnosis was aml in 52%, all in 36% and mds in 12% of patients. 90% of patients received pbsc grafts, 10% received unmanipulated bone marrow grafts. os at 2 years was 70% in msd/mud-atg, 76% in haplo-ptcy, 80% in mmud-ptcy and 43% in mmud-atg groups (p=0.0027). in a multivariate cox model non-relapse mortality was 17%, 17%, 4.5% and 42% in msd/mud-atg, haplo-ptcy, mmud-ptcy and mmud-atg groups, respectively. cumulative incidence of acute gvhd grade 3 or 4 was 6%, 9.5%, 3% and 12% after in msd/mud-atg, haplo-ptcy cumulative incidence of chronic gvhd was 26% in the msd/mud-atg group uwe platzbecker 3 , verena wais 4 republic of china background: there are two most noteworthy strategies of haploidentical stem cell transplantations (haplo-hsct), the baltimore post-transplantation cyclophosphamide (ptcy) with or without anti-thymoglobulin (atg), and the beijing g-csf primed bone marrow (bm) plus peripheral blood stem cells (pbsc) (giac). however, the comparison of these two modalities is scarce. in this study, we aim to compare these two approaches for hematological malignancies based on the taiwan blood and marrow transplantation registry (tbmtr) with the comparable cd34 infusion amounts, the neutrophil engraftment time were statistically distinct among these three groups [d+12 (group 1) vs. d+15 (group 2) vs. d+17 (group 3), respectively as to the graft-versus-host disease (gvhd), the patients in group 1 had more grade ii-iv but similar grade iii-iv acute gvhd compared with others (grade ii-iv: 60.0% vs. 37.5% vs. 21.6%, respectively conclusions: haplo-hsct with different strategies is a feasible treatment modality for hematologic malignancies in taiwan. regarding the retrospective nature and limited patient numbers of this study republic of china background: we previously presented a low-resolution hla analysis of cedace, the voluntary portuguese bone marrow donor registry (ebmt 2011, poster p1127) and, more recently, its epidemiological characterization (ebmt 2018, poster b348). currently, cedace is one of the largest bone marrow donor registries in the world, including nearly 4% of the country's population, twice the number of donors present in 2011. the current work is an update on the most common hla haplotypes found in cedace results: of the 396545 donors in the cedace registry, 99.51% were typed in at least 3 loci (hla-a/-b/-drb1), 43.91% in 4 (including hla-cw), and 1.28% in 5 (including hla-dqb1)the 5, 25 and 150 most common haplotypes accounted for, respectively, 8.9%, 23.5% and 50% of the haplotypes found in the entire registry. the five most common haplotypes at the low-resolution at the 3 loci, low-resolution level, out of 394621 donors, 167505 individual genotypes were identified, leading to an hla matching probability at this level of 57.6% hid-hsct) have a stronger anti-leukemia effect compared to identical sibling donor hsct(isd-hsct) in high-risk features .but in refractory/relapsed(r/r) aml patients who not in remission status, it is unclear whether it also augments the gvl effect antithymocyte globulin was used in haploidentical hsct. unmanipulated bone marrow and peripheral blood stem cells for all patients. cyclosporine, short-term methotrexate were employed for gvhd prophylaxis. mycophenolate mofetil included in hid-hsct. performed multivariate analysis for all patients of pretransplantation variables and developed a predictive scoring system for survival according to adverse factors. results: the total survivor median period of follow up was 46 (20-73) months. hid -cohort had higher 5-year actuarial of os multivariate analysis showed isd-hsct,standard conditioning regimen and less than 50% proportional reduction in blast percentage pre-≥2. conclusions: haploidentical donor compared to identical sibling donor had better anti-leukemia effect in allo-hsct for r/r aml in nr status conditioning protocol was melphalan 200mg/m2 for mm and beam for nhl. the quantification and characterization of γδt cells in peripheral blood samples were performed by flow cytometry based on the expression of cd3/cd45/vδ1/vδ2/vγ9/cd27 at 30, 60 and 100 days after ahsct. percentage (%) of γδt cells represented the proportion of these cells among all t cells. results: median age at ahsct was 61 (40-70) years, 63% male. median time from diagnosis to mobilization was 8 (4-52) months, after a median number of therapeutic lines of 1 (1-3); 7pts (13.0%) received radiotherapy. seventeen pts (31.5%) were re-mobilized with plerixafor±g-csf (25% mm vs 60% nhl there was no difference in febrile neutropenia incidence (p=0.766), timeto-engraftment (p=0.093), time-to-neutrophils>500/μl (p=0.174) or erythrocyte transfusions (p=0.076). however, there was more time-to-platelets>20,000/μl (4 vs 8 days; subpopulations did not affected pfs. conclusions: our results showed that pts mobilized with plerixafor need more collection volume (less cd34+cells, higher dmso). plerixafor negatively affected platelet recovery, with similar hematologic and immune recovery for the remaining variables. its use was associated with higher %vδ1+, suggesting that it induces an antineoplastic phenotype. more studies with larger samples and follow-up period are needed to evaluate plerixafor results: total 1156 ascc procedures were carried out in 1072 patients over 18 years, once in 985 patients, twice in 83 patients, three times in 3 patients, and four times in 1 patient. non-hodgkin lymphoma (nhl) comprised 56.7% of all cases (n = 655) 001), total number of chemotherapy cycles before ascc (or 1.06, 95% ci 1.004-1.13, p = 0.035), failure to achieve at least partial response before ascc (or 2.97, 95% ci 1.01-8.73, p = 0.048), total number of days receiving g-csf for mobilization (or 1.10, 95% ci 1.002-1.23, p = 0.045), and salvage use of plerixafor (or 3.78, 95% ci 1.92-7.47, p < 0.001) were found to be independent factors associated with failure of ascc. at the end of the study period, 90.9% of successful collections (n = 962/1058) were used for asct, 4.5% (n=48/1056) were in storage awaiting transplantation hôpital necker-enfants malades melf 140 methods: a total 103 ahsct candidates [median age: 57(18-75) years; male/female: 54/49] were included in this study. twenty-seven patients (26.2%) were diagnosed as non hodgkin's lymphoma, 8 patients (7.8%) hodgkin's lymphoma, 52 patients (50.5%) multiple myeloma, 12 patients (11.6%) acute myeloid leukemia, 3 patients (2.9%) plasmocytoma and 1 patient (1%) testis cancer. premobilization serum 25-hydroxy vitamin d (25-oh d) levels were measured with immunoassay method peripheral cd34 + cell count background: some published data suggest a positive effect of iron chelators on the risk of post-transplant relapse, with an improvement in overall survival after allogeneic hematopoietic stem cell transplantation (hsct) conditioning regimen consisted to intravenous bu 130 mg/m 2 and, flu 40 mg/m 2 d-6 to d-3. gvhd prophylaxis included atg 2.5 mg/kg on d-2 and d-1, ciclosporin and methotrexate. all patients received peripheral blood stem cell transplant from an identical hla-related donor. iron chelation consisted on deferasirox (exjade ® ) 10-20 mg/kg/day, started at day 100, if serum ferritin level ≥ 800 -1000 ug/l and stopped when the level decreased below 600 ug/l or normalized at day 100 after transplant, 35/ 40 patients were evaluable (g1= 20 pts), (g2 =15 pts). patients were abo compatible (66%), had major incompatibility (29%), and 6% had minor incompatibility. median serum ferritin level at day 100, were 1200 ug/l (833-4449) and 1200 ug/l (123-1853) in g1 and g2 respectively with a median follow-up of 34 months, (12-68), disease relapse incidence was higher in patients who did not received iron chelation treatment (g1: 39.8%) versus those who received oral deferasirox (g2:15.6%) (figure 1), but the difference was not statistically significant conclusions: these results deserve more investigation choep (n=5), and 1patient received vtd-pace 10 μg/kg depending on the protocol. the aim was to collect at least 2x10 6 cd34(+) cells/kg body weight. results: forty patients all patients were stage iii and iv at diagnosis. 29 of the patients were mobilized after one line of treatment, six after two lines of treatment and five after 3 and more lines of treatment alexandra martínez-roca 1 , gerardo rodriguez-lobato 1 , gonzalo gutierrez-garcia 1,2 , maria suárez-lledó 1 background: hematopoietic stem cell transplantation (hsct) is an established procedure in lymphoma background: the role of inflammatory cascade in tumor microenvironment has been demonstrated in several studies. as a part of this issue, elevated neutrophil/lymphocyte ratio (nlr) was shown to be associated with an adverse prognosis, particularly in solid tumors. the aim of this study is to determine the impact of pre-transplant nlr on early transplant complications, as well as post-transplant relapse and survival.methods: a total of 119 lymphoma patients [median age: 49(18-71) years; male/female: 64/55] who underwent autologous hematopoietic stem cell transplantation (hsct) were included in this retrospective study.results: the initial diagnosis was hodgkin lymphoma (hl) in 35 (29.4%), b-cell non-hodgkin lymphoma (nhl) in 67 (56.3%) and t-cell nhl in 17 (14.3%) patients. of 116 patients who were evaluated for pretransplant disease status, 52 patients (44.8%) were in complete remission, 45 patients (38.8%) were in partial remission and 19 (16.4%) patients had refractory disease. median pre-transplant nlr was found to be 3.42(0-97) . when the study population was divided into two subgroups as "low-" and "high-nlr", based on median nlr value, number of febrile days were found to be relatively higher in the low-nlr group (p=0.051). a positive correlation was demonstrated between nlr and lactate dehydrogenase levels (r=0.218; p=0.03); and nlr and ferritin levels (r=0.277; p=0.003). at a median follow-up of 24 (1-264) months, overall survival (os) was found to be better in the low-nlr group without statistical significance [25 vs 13(1-91) months; p=0.069]. in univariate analysis, pre-transplant nlr represented a significant impact on os (p=0.021). other prognostic factors were age (p=0.041), platelet engraftment (p=0.018), post-transplant relapse (p=0.009) and pre-transplant ferritin level (p< 0.001). the permanent impact of ferritin on os was confirmed in multivariate analysis (p=0.008).conclusions: in this study, an adverse impact of elevated pre-transplant nlr on os was demonstrated in autologous hsct recipients with lymphoma. as a predictor of prognosis, nlr may be considered as a safe and cost-effective parameter. further studies are required in order to use this predictor in routine clinical practice.background: high cure rates in childhood diseases have been achieved by stem cell transplantation (sct). however, there is little knowledge concerning recovery of the immune system and community-acquired infection after sct. here we studied the long-term reconstitution of the immune system and incidences of community-acquired infection after sct.methods: we reviewed medical records for 44 patients (m/f: 31/13, median age: 5 years (range: 1-22years) who were treated in the department of pediatrics, hokkaido university hospital. we analyzed cd4-positive cell counts, serum immunoglobulin g (igg) levels, and incidences of community-acquired infection until 2 years after sct. indications for sct were all in 8 patients, aml in 4, aa in 6, nb in 5, rms in 2, jmml in 1, nhl in 1, cgd in 5, was in 3, xscid in 2, apds in 2, cd40ld in 2, and other solid tumor in 3 patients. stem cell sources were autologous pb/bm in 6, allogenic bm in 23 (9 related and 14 unrelated) and allogenic cb in 15 patients. in this study, we excluded patients who relapsed after sct.results: the duration of cd4-positive cell counts < 500/ ml after sct was 14.7±11.4 months in all patients. the durations were 12.6±7.1 months in patients with hematologic malignancies, 11.5±7.3 months in patients with hematologic disorders such as aplastic anemia and pid, 28.8±25.9 months in patients with solid tumor, 16.0 ±10.4 months in patients who received autologous sct, 25.1±22.2 months in patients who received related bmt/ pbsct, 13.8±6.7 months in patients who received unrelated bmt, and 10.5±5.8 months in patients who received cbt. the durations of igg < 500mg/dl after sct were 14.9±11.5 months in all patients, 15.8±9.7 months in patients with hematologic malignancies, 11.6±5.1 months in patients with hematologic disorders, 11.0±8.7 months in patients with solid tumor, 19.0±18.6 months in patients who received autologous sct, 12.0±8.3 months in patients who received related bmt/pbsct, 11.5±5.7 months in patients who received unrelated bmt, and 15.9±8.7 months in patients who received cbt. there was a significantly higher incidence of community-acquired infection from 6 months after sct. there were significant differences in the incidence of community-acquired infections between patients with cd4-positive cell counts of < 500/ml and background: allogeneic hematopoietic cell transplantation (allo-hct) has the potential to cure subgroups of patients with multiple myeloma (mm) but its role is controversial due to high transplant-related mortality. while autologous hct is well established as consolidation after induction therapy using novel agents, allo-hct is still considered experimental due to excessive early toxicity.methods: we retrospectively studied 45 mm patients (pts) ; 25 (55,6%) were males with a median age of 52 years (range: 32-60), who underwent allo-hsct in our center between 2007 and 2018.median time between diagnosis and allo-hct was 40 months (range: 20-143).results: the international staging system (iss) was iss i (33,3%), iss ii (28,9%), and iss iii (37,8%). twentyeight (62,2%) pts received cells from unrelated donor and 17 pts from identical siblings. stem cell source were: peripheral blood (n=25) and bone marrow (n=20). grouprisk distribution using hct-ci and pam index can be seen in figure 1. response was evaluated at day +100: cr (53,3%), vgpr (11,1%), pr (22,2%) and sd (2,2%); response was not evaluated in 5 pts. regarding hospitalization, 11 (24,4%) pts and 22 (48,9%) pts needed readmission in the first 100 days and 365 days post-allohct, respectively, and median days of hospitalization was 27 days (range: 4-92). reasons for first re-admission were: infection (68,2%), gvhd (27,3%) and renal insufficiency (4,5%). twenty-seven (60%) died and death causes were: infection (35,6%), progression (15,6%), secondary tumor (4,4%) and the remainder, gvhd and intracranial hemorrhage. in addition, 26 pts (57,8%) suffered cytomegalovirus (cmv) reactivation.median overall survival (os) was 44 months (range: 11-76). univariate analysis showed that re-admission in first 100 days (hr 4, 19; p=0, 001) , re-admission in first 365 days (hr 3, 6; p=0, 001) , cmv reactivation (hr 2,38; p=0,038), iss (iss-i vs iss-ii and iii; hr 0,347; p=0,033), days of hospitalization in the first 100 days and response at +100 day (cr plus vgpr vs the remainder; hr 0,143; p=0,04) were predictor factors for os. other factors like karyotype, response before allo-hct, hla-mismatch or baseline cmv serostatus, among others, do not impact on os.median os in pts with low hct-ci were 56 months (range: 0-156) vs 6 months in intermediate-high hct-ci background: allo-sct is a potentially curative therapy for patients with multiple myeloma as it provides a graftversus-myeloma effect and a myeloma-free graft. due to increased nrm and unclear os benefit the recent guidelines suggested allo-sct to be used in context of clinical trials focusing on the high-risk patients and those who relapsed early after autograft. reduced-intensity conditioning regimens may improve rate of nrm; however, optimal conditioning regimen is still to be determined. here we studied conditioning regimen with 3 alkylating agents consisting of thiotepa (tt), busulfan (bu) and gvhd prophylaxis with post-transplant cyclophosphamide (post-cy) in high risk myeloma patients relapsing after autograft.methods: a total of 40 patients (m, n=23) with median age of 55 years (range 40-67) underwent an allo-sct (mud, n=19; mrd, n=12; mmud, n=6, haploidentical, n=3) during a period from 2014 to 2018 in university of hamburg. the majority of patients had advanced disease (stage iiiab, 73%) and high-risk cytogenetics (56%). the median response durartion after autograft was 1.8 years (range 0.5-8. 3 ). the conditioning included tt (cum. dosage 10mg/kg), bu (median cum. dosage 9.6mg/kg i.v. or 6.4 mg/kg in elderly patients) and post-cy (cum. dosage 100mg/kg, day +3 and +4). eight patients (all of them received allografts from mmud or haploidentical donors) received additionally fludarabine (flu, cum. dosage of background: the overall incidence of active cmv infection in patients with multiple myeloma (mm) receiving background: several scoring systems have been developed to estimate outcomes in mds patients who undergo allohct. however, none of them have been specifically validated in t cell depleted grafts. the aim of this study is to investigate the prognostic ability of a recently published scoring system (sfgm-tc) in a cohort of patients with mds who underwent tcd transplants.methods: 109 patients underwent a first tcd allohct for mds from 2007 to 2018. the sfgm-tc score (caulier et al. curr res transl med. 2018 ) is performed at day 100 and ranges from 0-8, discriminating low (0), intermediate (1) (2) (3) , and high risk (4-8). additional analyses were performed at day 180 and day 365. a landmark analysis was done at each time point for the day 100, 180, and 365 analyses, respectively. background: hematopoietic stem cell transplantation (hsct) is the only curative procedure for the treatment of myelodysplastic syndrome (mds), but among several limiting factors for its accomplishment, such as the patient´s performance status, is a very relevant issue, i.e., the availability of a compatible hla donor and, when available, very often the donor´s age and comorbidities also constitute factors that hinder this medical conduct. considering this scenario, the possibility of a haploidentical transplantation (ht) has emerged as an option. in latin america, ht has been included as a treatment option since 2014. since then, these patients have been included in the latin american registry of transplantation in myelodysplastic syndrome, making it possible to analyze the viability and results of these transplants.methods: from october 2012 to october 2018, seventeen (17) patients were transplanted with a haploidentical donor and included in the latin american registry. none of these patients had an identical hla (8/8 match) related or unrelated donor. data were obtained from the latin american registry of hsct in mds. the statistical analyses were performed using the software spss, version 23.1 and graphpad prism version 5.0, with significance being set at p < 0.05.results: table 1 shows the patients and their characteristics. all donors were haploidentical. there was a predominance of reduced intensity conditioning, which was performed in 13 patients (76.5%), whereas the others received the myeloablative conditioning. cell source was peripheral blood in 10 (58.8%) and bone marrow in 7 (41.2%) of the patients. graft-versus-host disease (gvhd) prophylaxis in post-hsct was carried out with cyclophosphamide 50mg / kg on d+3 and d+5, cyclosporin from d0 and mycophenolate from d+1. complete hematologic recovery was achieved in 16 (94.1%) patients. the incidence of grade ii-iv acute gvhd was 11.7%, whereas chronic gvhd was 5.8%. one death occurred due to graft failure and none of the patients showed autologous recovery. three other patients died. one on d+210 due to a fungal infection, the second on d+90 due to sinusoidal obstruction syndrome and a third on d+61 due to pneumonia caused by pseudomonas. regarding overall complications, there was a predominance of mucositis (47%), overall infections (35.2%) and reactivated cmv in 23.5% of cases. of the total number of living patients, 8 (47%) achieved complete remission and 5 (29.4%) showed disease relapse. the mean follow-up was 39 months (ranging from 5 to 72 months). the lowest probability of disease-free survival at 3 years was 79% (95% ci: 71.48 -88.51).background: relapse is the most important cause of failure after allogeneic hematopoietic stem cell transplantation (hsct) for flt3-itd-positive acute myeloid leukemia (aml). treatment with flt3 tyrosine kinase inhibitors (tki) constitutes a promising clinical approach to induce remission without conventional chemotherapy.methods: a 50 year-old woman diagnosed with aml secondary to myelodisplastic syndrome (mds) with npm1 mutation and internal tandem duplications of the flt3 gene (flt3-itd) in october 2013. after achieving complete remission (cr) with conventional chemotherapy, she received a hla sibling allogenic-hsct in february 2014, with bucy. four months later, aml relapsed only at medullary level (flt3 ratio: 0,67 %), treated with chemotherapy and donor lymphocytes infusions (dli). she achieved 2 nd cr and developed limited chronic graft-versushost disease (cgvhd). nine months later (april 2015), she suffered the first extramedullary relapse only, breast and skin. disappearance of the lesions at all levels was achieved with chemo and radiotherapy. she always had full hematologic donor chimerism.in december 2015, she referred atypical precordial pain irradiated to the back. cardiac mri was performed and several masses were visualized in the pericardial sac, up to 5 cm in diameter. bm remained in cr with full donor chimerism.pericardial fluid showed massive infiltration by leukemic-flt3 positive cells (ratio: 0,7%). she was not considered background: ta-tma is a severe complication that can reduce survival after hsct. risk factors have been variably reported in adults although data on children remains scarce. we aimed to identify a risk profile for development of ta-tma in children undergoing hsct.methods: we retrospectively reviewed clinical charts of 398 children who underwent 406 hscts between 2013-16: at great ormond street hospital (gosh) and the great north children's hospital (gnch). ta-tma was defined according to revised criteria (jodele et al. 2013) . risk factors were categorized into patient derived [age, gender, active co-morbidity at d0 of hsct (uncontrolled viral/ bacterial or fungal infection, pulmonary, cardiovascular instability, steroid therapy >0.3mg/kg beyond d0, bcgiosis or autoimmune disease), transplant related factors (conditioning intensity, stem cell source, hla-matching, use of ciclosporin a (csa) or tacrolimus (tac), cd34+ dose, ex-vivo t cell depletion, use of defibrotide) and post-hsct factors (agvhd, post-hsct viral reactivation, venoocclusive disease (vod) and occurrence of posterior reversible leukoencephalopathy (pres).results: at a median of 7 months post-hsct, ta-tma occurred among 21/406 transplants (5.1%). there was no reported centre variation (5.7% vs 4.6% in gosh vs gnch; p=0.6). gender, underlying disease -primary immune deficiency (pid) (n=273) vs haematological disease (n=133), use of myeloablative (n=153) vs reducedor minimal intensity conditioning (n=229), use of serotherapy or mega doses of cd34 ≥10 x10*6/kg did not influence the development of ta-tma. donor type: msd/ mfd(n=100) vs mud (n=129) vs mmud/haplo-hsct background: we evaluated the outcome of haploidentical hct (hhct) using ex vivo αβ t cell-depleted (tcd) grafts after reduced-intensity conditioning (ric) containing low-dose tbi (ld-tbi) in pediatric patients with acute leukemia (al) in complete remission (cr).methods: between may 2012 and october 2018, 36 patients with acute leukemia (17 all and 19 aml) in cr received haploidentical hematopoietic cell transplantation (hhct) using tcrαβ-depleted graft at asan medical center children's hospital. eighteen patients received hhct between 2012 and 2015 (earlier time period) and the remaining 18 between 2016 and 2018 (recent study period). the conditioning regimens, the dose of αβ+ t cells and gvhd prophylaxis are summarized in table. results: all 36 patients achieved a sustained neutrophil engraftment at a median of 10 days (range, 9-13) . of 36 patients, 11 patients (8 all & 4 aml) relapsed at a median of 6 months (range, 3-16) after transplant. of the 11 patients, 10 patients died of disease. one patient died of disseminated tuberculosis at 11 months after transplant, leading to the trm of 4% at 1 year. as of december 2018, 25 of the 36 patients survive free of disease at a median follow-up of 21 months (range, . at a median followup of 35 months (range, 1-80), efs and os at 2 years for all patients were 59% and 67%, respectively. outcome of hhct in the recent study cohort was significantly better than that in the earlier study period (efs of 85% vs efs of 44%, p=0.05). among the 17 patients with all, the efs of 8 patients, who received hhct in early time period after conditioning with tbi of 600 cgy, was significantly worse than that of 9 patients, who received in recent study period after a higher dose of tbi at 800cgy (13% vs 83%, p< 0.05). the efss of aml were similar between the two study groups (70% for earlier cohort vs 86% for recent study, p>0.05).conclusions: in pediatric patients with acute leukemia in cr, our current haploidentical hct using ex vivo αβ tcd graft after ric containing ld-tbi without gvhd prophylaxis is feasible approach with a low trm. the background: anti-hla antibodies (ahab) have been recently recognized as an important risk factor for graft failure (gf), especially in hla-haploidentical stem cell transplantation (haplo-hsct). although, recently, ebmt consensus guidelines have been published [ciurea, bone marrow transplant 2018] , experience in pediatric t-cell depleted (tcd, another well-known risk factor for gf) haplo-hsct is lacking. in the present study, we report our experience on the use of a desensitization approach based on ebmt guidelines.methods: between june 2017 and august 2018, all patients affected by non-malignant diseases and scheduled for a transplant from an hla-haploidentical donor after negative depletion of αβ t and b cells as previously described [li pira, biol blood marrow transplant. 2016 ], were tested for ahab with luminex® solid-phase immunoassay (one lambda, thermo fisher scientific) 1 month before the hsct. all patients with a mfi higher than 5000, which is considered a cutoff predicting for gf, were treated with a desensitization protocol based on the use of anti-cd20 rituximab (375 mg/m 2 before and immediately after the end of plasma-exchange cycle) and plasma-exchange (pe) ± infusion of irradiated buffy coat (bc) (if after pe ahab mfi was still > 5000 mfi). this latter was obtained by the non-target fraction of the αβ t-cell/b-cell-depletion procedure and consisted of 5 * 10 7 irradiated nucleated cells/kg of the recipient; this was infused 2-4 hours before the infusion of the tcd graft. pe was performed with miltenyi life 18 tm apheresis unit (miltenyi, bergish-gladback) .results: in the study period, 37 patients received αβ tand b-cell depleted haplo-hsct. eighteen (48%) resulted positive for ahab (mfi> 1000); 14 (77%) of them had an mfi > 5000 for either anti-class i or ii ahab. these patients (see table i background: osteonecrosis (on) is a debilitating complication in survivors of allogeneic hematopoietic cell transplantation (hct). limited data is available on its course post-transplantation in children. the purpose of our study was to identify recipients of hct in whom pre-and post-magnetic resonance imaging (mri) is indicated.methods: the retrospective cohort consisted of 436 patients who underwent first allogeneic hct from 1998-2014, and prospectively underwent a total of 1092 pre-and post-transplant mri studies of the hips and knees done annually for 3 years regardless of symptoms. surviving patients were followed for a median time of 8.33 (range 3.93-14.10) years. cases of on were compared to controls matched for age, sex, transplant type, and follow up in a 1:4 ratio for the following variables: ethnicity, underlying disease, on pre-hct, conditioning regimen, graft source, bone mineral density z-scores, body mass index, presence or absence of graft-versus-host disease, steroid use and dosage, and survival status.results: thirty (6.9%) patients had mri findings confirming on post-hct. all patients with on except one were more than 10 years of age. twenty (67%) patients were male. on pre-hct (p < 0.0001) was the only factor associated with presence of on post-hct. epiphyseal on was seen in 9 (30%) patients pre-hct, and 26 (87%) post-hct. eighteen (60%) patients had involvement of more than 30% of articular surface, and were more likely to undergo surgery (p = 0.009).conclusions: the incidence of on in this large pediatric cohort was 7%. the only risk factor for on post-hct was pre-existing on. mri evaluation for on pre-hct is indicated in all patients. mri evaluation for on post-hct is only indicated for patients with on pre-hct and symptomatic patients. this will entail cost savings of usd 500,000 per 100 surviving allogeneic hct patients per year. patients with more than 30% involvement of the articular surface need close follow up.clinical trial registry: none disclosure: none impact of rabbit anti thymocyte globulin exposure on immune reconstitution and outcome after stem cell transplantation in children background: rabbit anti thymocyte globulin (ratg) has been frequently used for many years as gvhd prophylaxis in pediatric stem cell transplantation. precise dosing regimens are crucial but remain challenging due to several pharmacological characteristics in children.methods: ratg levels were measured in pediatric patients undergoing allogeneic stem cell transplantation after obtaining approval by the local irb and informed consent by legal guardians. 32 pediatric patients who received either thymoglobuline™ (n=22) or grafalon™ (atg-f) (n=10) as part of their conditioning regimen background: obesity among children is a growing health problem. malnutrition or being over-weight can be of prognostic impact among children who need hsct.scientific literature shows a lot of controversy in terms of effect of bmi at the time of infusion on the outcome of hsct.methods: we reviewed data of patients who underwent hsct at king faisal specialist hospital & research centre between 2010-2016 to correlate bmi with the outcome and complications of hsct. transplant naïve recipients with age at infusion between 2-14 years who received hsct from matched related donor or autologous hsct, were included in the dataset for analysis. a total of 423 patients' profiles were reviewed of whom 51.1% were boys. median age at transplant was 7.5 years. primary indication of disease was malignancy in 41.6% followed by hemoglobinopathies 26.7%, bone marrow failures 17.3% and immune disorders 12.5%. solid tumors accounted for 29.5% among malignant disorders. myeloablative conditioning was used in 97.2% transplants with 11.8% regimens containing total body irradiation. majority of the patients 81.3% underwent allogeneic transplantation using bm as the source in 80.4% and pbsc in the remaining 19.6% cases. donor was hla-identical sibling in 89%, parents in 10.2% and others in the remaining 0.9% patients. median tnc dose was 2.55 x 10^9 and cd34 was 5.63 x 10^6 per kg of the body weight at the time of infusion. age and gender specific bmi percentiles were obtained and classified according to the definition of centers for disease control and prevention (cdc); 24.8% (105) recipients were categorized as under-weight, 61.2% (259) normal, 5.9% (25) over-weight and remaining 8% (34) as obese.results: based on chimeric studies at day-100, our engraftment rate was 98.3% (400) out of 407 evaluable cases. median time to neutrophil recovery was 14-days from infusion and 26-days for platelets. no statistically significant difference was found for engraftment rate on d-100 as it was 100% (25) among 98.8% (247) in normal, 97% (97) in under-weight and 96.9% (31) in the obese (p-value: 0.467). median time to neutrophil recovery from the infusion date was 15-days in over-weight patients and 14 in the remaining three groups (p-value: 0.841). acute graft vs. host disease (agvhd) of any grade at day-100 was recorded in 19.9% (84). any-grade agvhd was more common in over-weight 24% (6), followed by obese with 23.5% (8), 21% (22) in under-weight and 18.6% (48) in normal bmi-category (p-value: 0.770). chronic gvhd was more frequent in over-weight (14.3%, 3), compared to 9) background: hematopoietic stem cell transplantation (hsct) is the standard treatment for children with severe aplastic anemia (saa) who have hla-identical related donor. there is no standard conditioning regimen for children with saa secondary to non-fanconi anemia (fa) constitutional bone marrow failure syndromes such as telomeropathies. we report the outcome of a consistent reduced intensity conditioning regimen in patients with idiopathic saa or inherited bone marrow failure syndromes other than fa who underwent hla matched related hsct methods: children with saa underwent hsct using the following conditioning regimen: fludarabine 175mg/m2, cyclophosphamide 80 mg/m2, and atg (thymoglobulin) (10 mg/kg) . gvhd prophylaxis included cyclosporin and mycophenolate mofetil. donors were all matched related and bone marrow was the stem cell source. all patients had normal chromosomal fragility testresults: a total of 18 children with saa underwent hsct, 12 females and 6 males. average age was 8.1 (range 0.8-13.8 years). all nine patients who were tested for telomere length had short telomeres. pathogenic or likely pathogenic mutations were reported in 5 patients (2 ercc6l2, 1 ankrd26, 1 tinf2, 1 lztfl1). all donors had normal physical examination, normal cbc, and negative genetic testing if patient mutation is known. all 18 patients engrafted successfully, median time to neutrophil engraftment was 16 (range, 11-29 days) and platelet engraftment 19 (range, 13-45 days). median infused nucleated cell dose was 3.3 (range,0.9-7.3 x10 8 /kg) and cd34 cell dose was 6.7 (range, 1.1-13.1 x10 6 /kg). none of our patients had acute gvhd and one patient had mild classic chronic gvhd of the skin that was controlled with topical therapy for a short period. three patients had secondary graft failure in the first-year post transplant. first patient had pancytopenia with loss of donor chimerism and underwent successful second transplant using fludarabine, atg, and melphalan. the second patient had a nonfunctioning graft despite full donor chimerism suggesting that the related donor might be affected and had silent phenotype. the third patient had homozygous ercc6l2 mutation and developed progressive cytopenia with myelodysplastic features few months post-transplant and subsequently underwent myeloablative matched unrelated transplant using busulfan, fludarabine, thiotepa and atg. however, the patient progressed to have acute myeloid leukemia six months post hsct. fifteen patients (83%) have normal cbc and stable donor chimerism. median follow-up duration of 1140 days (range 330 -2595 days). one patient with lztfl1 mutation developed chronic renal impairment five years post hsct.conclusions: hsct using lower dose cyclophosphamide (80mg/kg) as part of fludarabine based regimen was safe and effective in saa patients with shorter telomeres and described genetic abnormalities. optimal conditioning regimen in ercc6l2-associated bone marrow failure needs to be defined. larger study is needed to confirm our results.clinical trial registry: not applicable disclosure: nothing to declare late effects in patients with hemophagocytic lymphohistiocytosis treated with hematopoietic stem cell transplantation: a review of the literature background:hemophagocytic lymphohistiocytosis (hlh) is an inherited or acquired disorder of immunedysregulation. early diagnosis and immunosuppressive treatment can prevent significant organ-failure. the inherited forms, and some acquired cases can only be cured by hematopoietic stem cell transplantation (hsct). with modern transplant practices, a significant number of patients survive. the purpose of this literature review was to collect data on the frequency and type of late effects in hlh patients surviving after hsct and to examine the association with pre-existing hlh conditions (eg. involvement of the central nervous system (cns) before transplant) and with the pre-transplant conditioning regimens.methods: the medline, embase, web of science and pubmed databases were searched, by two librarians at the karolinska institutet, between may and september 2016 according to the preferred reporting items for systematic review and meta-analysis (prisma) statement. the search terms included "hlh", "fhl", "mas", multiple terms for "hsct" and late-effect conditions. inclusion criteria were publications in english that included children between january 1995 and may 2016. authors of this review screened all the abstracts of studies against the inclusion criteria.results: only nine papers published between 2006 and 2016, with information on late effects in hlh patients who had undergone hsct, were identified. three reports include only small numbers of hlh patients. the remaining 6 papers contain data on 261 long-term survivors with a median follow-up of 5.4 years. five papers address neurological sequelae with a reported incidence from 7-57%. the highest incidence was found after a thorough neurological assessment of 21 hlh patients compared to matched sibling controls. however, the association with cns disease before transplant and age at transplant was not clear. patients with ebvassociated hlh seem to have fewer long-term neurological problems. non-neurological late effects are described in 4 papers only, with endocrinological problems, namely short stature, being the most frequent. one paper specifically analyzed poor growth, thyroid dysfunction and vitamin d deficiency in a cohort of patients with non-malignant disorders including hlh who had undergone hsct after a reduced intensity conditioning regimen and found significant abnormalities in all groups.conclusions: data on late effects in hlh patients is scarce and is mostly based on the retrospective evaluation of small national cohorts. the available information indicates that a significant number of patients suffers from problems which affect their daily life, but lack of information does not allow to analyze the association between pre-transplant conditions and long-term sequelae. therefore, a retrospective comprehensive analysis of patients registered in the ebmt and cibmtr registries is currently performed. it will be crucial to better define the frequency and type of late effects in a large cohort. this knowledge will aid counselling prior to hsct, provide guidance for long-term monitoring of these patients, and potentially identify specific risk factors for late effects in this rare patient population.disclosure: nothing to declare. allogeneic stem cell transplantation in patients with mucopolysaccharidosis type ii (morbus hunter)bernd hartz 1 , nicole muschol 2 , matthias bleeke 1 , johanna schrum 1 , ingo müller 1background: the transplantation of hematopoietic stem cells (hsct) is one of the leading methods of treatment in patients with blood system diseases, primary immunodeficiency syndromes and genetic diseases. at the same time, the quality of life in patients in the long-term after hsct significantly differs from the quality of life of healthy people of the same age. deformations in psychosexual development including problems in the gender identity formation cause social isolation of adolescents, which makes their sexual selfrealization impossible and significantly reduces the quality of their life. the purpose of our study was an assessment of the level of gender identity formation of adolescents and psychosexual development correlation to the normal adolescents of the same age.methods: in a prospective single-center study in 2018, on the base of the department of rehabilitation medicine raisa gorbacheva memorial institute of children´s oncology, hematology and transplantation, we conducted a study of 17 families. the respondents were: 1) parents / guardians of patients accompanying them in the process of examination; 2) adolescents who underwent hsct treatment and undergo planned examinations at the clinic in the posttransplant period (after d + 100), (n = 17, of which 8 girls and 9 boys, age 12 -17 years, from the date of hsct 1 -5 years).the following methods were used to assess gender identity: specially developed questionnaires for teenagers and parents; questionnaire by sandra l. bem (sandra l. bem, 1974) ; projective techniques "the human picture", "the non-existent animal"; max lüscher´s color choices test.results: the traditional type of gender identity, which characterizes high masculinity among male respondents and high female gender indicators in 100% of cases, was not revealed. both among girls and among boys, the androgynous type prevails with a tendency toward femininity.on average, adolescents see themselves as a bit more courageous than their mothers, with rare exceptions, regardless of gender. this confirms the thesis that we received in a previous study that parents tend to see and encourage complacency of adolescents of both sexes, passivity instead of leadership, dedication and independence. all 100% of adolescents who participated in the test demonstrate a shift in the theme of aggression, 77% have some signs of preventing sexual self-determination, abandoning their body, gender, and age. 88% of patients do not communicate with their peers. in 47% of them, negative emotions prevail over positive ones. one third of the test participants demonstrate strong support for rest and minimizing their efforts.conclusions: the characteristics of family upbringing of adolescents who have undergone hsct often contribute significantly to limiting their social experience and lead to specific deformities of individuality, including in the sphere of gender identity. we consider advisable to introduce thematic group counseling of parents within the framework of the "patient's school" in psychological treatment support in the clinic. early diagnosis of the personal aspects of the psychosexual development of adolescents after hsct allows for timely identification of individual problems in this area and identification of general trends in the long term after hsct.disclosure: all authors -nothing to disclose. abnormalities in the morphology of the umbilical cord blood obtained at delivery from children who received a diagnosis of cerebral palsy maciej boruczkowski 1 , izabela zdolińska-malinowska 2 , maciej rojek 2 , dariusz boruczkowski 2background: embryonal brain tumors are the most common malignancies in infants less than 48 months of age. histologically characterized as undifferentiated small round cell tumors, all are similarly aggressive, have a tendency to disseminate throughout central nervous system and very poor prognosis. we tried to assess the effectiveness of tandem highdose chemotherapy (hdct) with autologous hematopoietic stem-cell transplantation (auto-hsct) in this patient group. methods: from 2010 to 2018, 52 infants under 48 months with different primary embryonal brain tumors such as medulloblastoma (n=28), different pnet nos (n=10), pineoblastoma (n=3), atypical teratoid rhabdoid tumor (n=3), etmr (n=8) after surgical resection and induction chemotherapy were planned to receive tandem hdct with auto-hsct. nine patients were conducted only single transplantation because of the development of lifethreatening complications after the first hdct (n=4) or the emergence of early disease progression (n=5). at the moment of hdct 31 patients were in complete remission (cr), 20 patients were in partial remission (pr) and 1 patient had stable disease (sd). the conditioning regimen for tandem auto-hsct were: the first hdct was carboplatin and etoposide, the second was thiotepa and cyclophosphamide, both with intraventricular methotrexate.results: the median follow-up is 24 months (range, 6-85). the median time to engraftment after the first auto-hsct was background: a series of findings suggest that optimizing natural killer (nk) cell reactivity could further improve outcome after allogeneic hematopoietic cell transplantation (allohct). this could be achieved by killer cell immunoglobulin-like receptor (kir) genotype informed donor selection. an enhanced receptor-ligand model which used kir2ds1 and kir3dl1 donor genotype information to augment nk cell activation and minimize inhibition demonstrated improved survival in one large aml study (boudreau et al, jco 2017) . likewise, a second model built on the classification of centromeric and telomeric kir haplotype motifs, also predicted mortality after allohct for aml (cooley et al, blood 2010) . this joint ebmt and cibmtr study aimed at validating the two approaches in an independent cohort of patients with mds or secondary aml.methods: donor samples were retrieved from the collaborative biobank (dresden, germany) and mapped to patient outcome data extracted from the ebmt and cibmtr. genotyping of all kir genes by sequencing exons 3, 4, 5, 7, 8, and 9 was performed by high resolution amplicon-based next generation sequencing. the impact of the classifiers on time-to-event outcomes was tested in cause-specific cox regression models adjusted for patient age, a modified disease risk index, performance status, donor age, hla-match, sex match, cmv match, conditioning intensity, type of t-cell depletion and graft type.results: clinical data from 1704 patients and corresponding donor genotype information were analyzed. the median age at allohct was 59.4 years (range, 18.1 to 79.6 years). the indication for allohct was mds for 72% and saml for 28% of patients. disease risk was low/intermediate and high/very high in 41% and 59%, respectively. donors were 10/10 matched for 79% of patients. myeloablative, reducedintensity and non-myeloablative conditioning regimens were used in 31%, 57%, and 12% of patients, respectively. peripheral blood stem cells were the predominant graft source (93% of patients). atg was administered in 56% and alemtuzumab in 9% of patients. during follow-up after allohct 776 patients died. in univariable and multivariable analyses of the whole cohort, overall survival and the cumulative incidence of relapse of patients with kiradvantageous versus disadvantageous donors were not statistically significantly different. we could not replicate the pattern of outcomes predicted by the kir3dl1/ conclusions: relapse incidence and overall survival after unrelated donor allohct could not be predicted using the kir3dl1/kir2ds1-receptor-ligand model and centromeric/telomeric kir-motif model in this large cohort of patients with mds or secondary aml. this points at the possibility of interactions between nk-cell mediated alloreactivity and disease type or procedural variations of allohct. available information on kir-genes, which have been sequenced but not yet analysed, will be investigated in exploratory analyses.disclosure: the authors have nothing to disclose in the absence of an alternative donor, it is recommended that patients undergo desensitization therapy, especially with high dsa levels (>5,000 mfi). the aim of this study is to analyze the impact of dsa on risk of gf and poor graft function (pgf), and on major outcomes in a consecutive cohort of patients who were systematically screened for dsa before haplo-sct. methods: 141 consecutive patients were candidates for unmanipulated haplo-sct with post-transplant cyclophosphamide (pt-cy) at our center from january 2012 to january 2018 and 135 were analyzed for the presence of hla antibodies.results: 134 patients underwent haplo-sct. hla antibodies were detected in 40 patients,19 of them were dsa, while 21 were non-dsa (ndsa). 10 patients out of 19 with dsa were transplanted using the same donor; 2 underwent a desensitization program before transplant.background: a recent study from ebmt comparing matched sibling (msd) versus haploidentical donors transplantations, showed better outcome with msd in adult patients with intermediate risk aml in first remission (cr1). however, a female donor to a male recipient transplant combination is a poor prognostic factor and this study did not address the question whether in this situation, a haploidentical donor transplant might do better. the present study compared the outcomes of allografted male patients according to whether they received stem cells from a female msd or a haploidentical donor, in the intermediate and high risk cytogenetics groups (mrc classification).methods: the study included 1066 male patients with cytogenetics transplanted between january 2007 and june 2017 and reported to ebmt. 834 received stem cells from a msd female donor and 232 from a haploidentical donor (133 male and 99 female). the follow up was 25 months (12-62). we studied separately intermediate and high risk patients. multivariate analysis was adjusted on factors differing significantly between the 2 groups.results: 1-intermediate risk group: 638 male patients received a female msd and 160 a haploidentical transplant. the distribution of group characteristics was even except that in the haploidentical transplant group, donors were younger (39 y versus 51; p< 0.0001), marrow was more frequently used (45% versus 17%, p< 0.0001) and the interval from diagnosis to transplant was longer (5.4 versus 4.5 months, p< 0.0001). by univariate analysis at two years post transplant, cumulative incidence (ci) of nrm post haplo was higher (26% versus 15%, p=0.002) and ci of extensive chronic gvh lower (14% versus 27%; p=0.002). lfs post msd and post haplo were 64% and 51% (p=0.03), os 69% and 60% (ns), grfs (43% and 43%). by multivariate analyses the only significant poor risk factors were the haplo-identical transplant for nrm (hr: 1.7 (1.1-2-61)) and the patient age for os (hr: 1.15 (1.02-1.28; p=0.02). haploidentical transplantation resulted in less chronic gvhd (hr: 0.43 (0.29-0.64); p < 10 -4 ), but a lower lfs (hr: 1.7 (1.1-2.61); p=0.04). 2-high risk group: 196 male patients received a female msd and 72 a haploidentical transplant. in the haploidentical group, donors were younger (38 y versus 54; p< 0.0001), marrow was more frequently used (42% versus 11%, p< 0.0001) and the interval from diagnosis to transplant was longer (5.1 versus 4.3 months, p= 0.003). by multivariate analysis, haploidentical transplants were associated with a lower relapse incidence (hr: 0,40 (0.21-0.75; p = 0.004),a better lfs (hr: 0,46 (0.28-0.77; p = 0.003),os (hr: 0,43 (0.25-0.75; p = 0.003), and grfs (hr: 0,53 (0.34-0.84; p = 0.006)(see figure) . the only other significant prognostic factor was patient age.conclusions: this study shows that in a male patient with intermediate risk aml, a genoidentical sister donor remains associated with a better lfs. in contrast, in a male patient with high risk aml in cr1, a haploidentical donor may be a better choice than an hla genoidentical sister.disclosure: nothing to declare p663 abstract already published. hematopoietic transplant for older acute leukemia patients: improved survival with offspring donor in comparison with older-aged matched siblingsyu wang 1 , sheng-ye lu 1 , qi-fa liu 2 , de-pei wu 3 , xiao-jun huang 1background: post-transplant relapse remains the major cause of death of treatment failure. therapeutic options for relapse after first allogeneic stem cell transplant (1st hsct) include chemotherapy followed by donor lymphocyte infusion or second allo-hsct (2nd hsct) from the original donor or change to another donor. however, there is unclear outcome for different treatment approach. in this retrospective cohort study, we aim to compare the clinical outcome after different treatment strategy for relapse after first allo-hsct.methods: between 1992 jan and 2018 oct, 1493 consecutive patients receiving 1 st hsct registered to the bmt database in national taiwan university hospital were analyzed. among them, 580 cases had relapsed after first allo-hsct. one hundred and three patients who received no treatment after relapse or with incomplete data were excluded. their transplant data was collected following the ebmt registry data collection forms and manuals. overall survival rate and progression free survival rate were performed by the kaplan-meier method. univariate and multivariate analysis were performed using cox proportional hazard regression model.results: of the 477 patients who experienced relapse after 1 st hsct, total 244 patients (51%) received chemotherapy followed by dli or 2 nd hsct from the same donor (no change group), 36 patient (8%) received chemotherapy followed by 2 nd hsct from different donors (change group), and 197 (41%) had conventional chemotherapy alone. the patients in "change group" were younger (median age 29 vs 37, p = .02), and had more patients achieving complete remission (cr) prior to 2 nd hsct (44% vs 10%, p = < .01) than patients in "no change group". after the 2 nd hsct, the cr was 68% for "no change group" and 91% for "change group". the progression-free survival at 3-year and 5-year were 10.5% and 5.9% (fig 1a, p = .0438%), respectively, for "no change group" and 9.4% and 9.4%, respectively, for "change group". while the overall survival (os) at 3-year and 5-year were 15.8% and 9% (fig 1b, p = .3459%), respectively, for "no change group" and 13.5% and 9%, respectively, for "change group". those who achieved cr prior to 2 nd hsct had a trend of better os than those without cr (17.5% vs 8.7% at 3-year; 8.7% vs 9.1% at 5year, p =.0365)( fig 1c) . there were 1 cases survived for more than 10 years in "change donor group" and 6 cases survived more than 10 years in "no change group". only one had developed relapse after 2 nd hsct but achieved subsequent remission again.conclusions: our study shows that change donor had similar poor outcome comparing to those using the same donor after the 1 st hsct. patients who achieved cr before 2nd hsct had a trend of better os than those without remission and the long-term survivors were only those who achieved cr prior to 2 nd hsct. novel therapy for cr induction would be warrant for this poor prognostic population.disclosure: nothing to declare functional relevance of fetal microchimerism in nk cell cytotoxicity against leukemic blasts in children: a role for hla-c1 and kir2dl2/s2?background: allogeneic stem cell transplantation (allo-sct) remains the most effective curative intent therapy for patients with unfavorable risk acute leukemia. various donor options are available for the patient who lacks an hla-matched sibling donor, such as unrelated donors (urd) and hla-mismatched family (haploidentical) donors. in order to discover the exact role of transplantation type, there are many retrospective analysis, which compared these donor sources, have been reported. recent studies showed some promising results of haploidentical donor transplantation (hidt) using post-transplant cyclophosphamide in comparison with unrelated donor. the goal of this study was to compare the outcome of allo-sct from haploidentical versus matched unrelated (mud 10/10) or mismatched unrelated donor at a single hla-locus (mmud 9/10) for patients with acute leukemia in remission.methods: ninety-six adult (18-65 years) patients with acute leukemia in first or second remission who underwent allogeneic transplantation with a minimum 100 days follow-up at florence nightingale hospital hematopoietic stem cell transplantation center between 2011 and 2017 were included in this study. patient characteristics and medical records of all patients were reviewed retrospectively. thirty-eight patients who received haploidentical donor transplantation were compared with 23 patients receiving a mud 10/10 and 35 receiving a mmud 9/10. patients who completed minimum 100 days post-transplantation follow-up were identified as eligible for survival analysis.results: the characteristics of the patients and transplant donors in this study are summarized in table 1 . median age of patients was 39.4±14 years. proportion of male patients was 39.1%, 74.2% and 57.8% for mud 10/ 10, mmud 9/10 and hidt groups, respectively, which is significantly different (p=0.02). the other baseline factors were similar, including patient age, donor age, recipient cytomegalovirus (cmv) status, donor cmv status, graft versus host disease incidence, median neutrophil and platelet engraftment times and disease status at post-transplant 100 th day. no significant difference was identified in survival analysis among the mud 10/10, mmud 9/10 and hidt groups, even if they were classified according to primary disease (aml vs all) and pre-transplant disease status (cr1 vs cr2). also, donor cmv status (cmv igg positivity or negativity) was not an important factor on survival analysis when compared between these three groups (p=0.406).conclusions: in our study population, clinical outcomes of hidt patients were inferior to mud 10/10 and mmud 9/10 groups. when choosing an alternative donor for patients without an available hla-matched sibling, urgency of transplantation and host/donor features should be considered. we believe that hidt might be a feasible alternative choice in this subset of patients.disclosure: nothing to declare p673 g-csf primed bone marrow in hla-haploidentical transplantation using post-transplantation cyclophosphamide (ptcy) could promote tolerance and further reduce risk of gvhd nadira durakovic 1,2 , zinaida perić 1,2 , lana desnica 2 , ranka serventi-seiwerth 2 , mirta mikulić 2 , brian melamed 3 , alen ostojić 2 , dražen pulanić 1,2 , pavle rončević 2 , zorana grubić 2 , radovan vrhovac 1,2 implementation, development, and coordination of unique quality management systems with evaluation audits, intrahospital and international accreditation and certification processes. quality of health care is a major focus for providers, patients, and accreditors; so, in this study, we aim to compare the quality of bm harvested at ipo-collection centre (icc) with the quality of bm received from external collection centres (ecc) during these 6 last years.methods: this retrospective evaluation included the number of total nucleated cells (tnc) requested by the transplant centre, the tnc collected, and the results of bm microbiological analysis performed; donor age, weight and infectious disease markers (idm); patient demographics and diagnosis. bm collection technique in use at icc was validated according to jacie standards.we consider successful a collection (sc) with tnc between 75 and 125% of the requested value, unsuccessful (uc) if lower than 75% and outstanding (oc) if over 125%.results: a total of 106 bm was collected, 99 for allogenic (75 unrelated) and seven for autologous transplant; 21 unrelated bm were received from ecc (nine from germany, seven from usa and five from portugal). patient main diagnosis were severe aplastic anaemia (n=28), acute myeloblastic leukaemia (n=20), and acute lymphoblastic leukaemia (n=15). donors idm were all negative and nonreactive.mean age (±standard deviation, sd) was 33 (±13.2) and 31 (±9.3) years for icc and ecc donors, respectively. at icc, we were asked to collect an average (±sd) of 221.7*10 8 (±136.0) tnc while ecc were asked for 184.7*10 8 (±105.7). we collected 169.7*10 8 (±82.3) and received 204.5*10 8 (±109.2) tnc. correlation between requested and collected tnc was 0.69 at icc and 0.56 for ecc.we had 41.2% sc and 26.8% oc meaning an accomplishment of 68.0%. we failed to collect required tnc in 32.0%. although 85% of received bm fulfil tnc requirements, bm processing lowered this value to 55% due to erythrocyte removal (seven patients with major abo incompatibility) and plasma reduction (two patients with abo minor incompatibility). these steps reduce final tnc available for transplant. weight difference between donor and patient had no significant impact on final tnc collection performance.sixteen bm from icc (seven staphylococcus spp., five propionibacterium acnesand fourcorynebacterium spp.) background: success of peripheral blood stem cell (pbsc) collections depends on patient biological parameters and stable apheresis device performance. peripheral blood cd34+ cell enumeration is the most reliable predictive factor of apheresis yield however, there are some unexpectedly poor cd34+ cell harvests despite successful mobilization. the aim of the study was assess total collections cd34+ yields and factors influencing main apheresis procedure outcomes including collection efficiency (ce).methods: of 2233 consecutive donors covering the period 1-1-2016 to 30-9-2018 were analyzed for the following parameters: pre cd34 count, cd34 yield per procedure, total cd34 dose collected per patient, cd34 collection targets requested by clinical teams. the efficiency of pbsc procedures was determined by calculating the ce and the correlation coefficient between pre cd34 count and yield per procedure. ce was correlated to preprocedure wbc, platelet count, pre cd34 count and blood volume processed. all pbsc collections were performed by optia spectra across 7 units in uk.results: of the 2233 donors, 1611 were autologous and 622 allogeneic. the autologous donors underwent in total 2543 procedures. the median cd34 target dose for these donors was 4x10 6 /kg.799 (50%) achieved the target dose with 1 procedure and 566 (35%) with 2 procedures. the median pre cd34 count was 30/μl. the median cd34 yield per procedure was 2.54x10 6 /kg and the median total cd34 dose collected per donor was 5.38x10 6 /kg. 92 (5.7%) of autologous donors collected a total cd34 dose < 2x10 6 /kg, of those 27 (1.7%) had a pre cd34 count < 10/μl and 65 (4%) >10/μl.the allogeneic donors underwent in total 878 procedures. the median cd34 target dose for these donors was 5x10 6 / kg. 381 allogeneic donors (61%) achieved the target dose with 1 procedure and 221 (36%) with 2 procedures. the median pre cd34 count was 51/μl. the median cd34 yield per procedure was 4.07x10 6 /kg and the median of total cd34 dose collected per donor was 6.70x10 6 /kg. 17 (2.7) % of allogeneic donors collected a total cd34 dose < 2x10 6 / kg, of those 3 (0.5%) had a pre cd34 count < 10/μl and 14 (2.2 %) >10/μl. the median ce for autologous donors was 55% (range 20-166) and for allogeneic donors was 47% (range12-116). the ce was negatively correlated to wbc (r= -0.29 and -0.37) and platelet count (r=-0.14 and -0.06) for auto and allogeneic donors respectively, but did not correlate to the pre cd34 and blood volume processed. the correlation coefficient between pre cd34 count and cd34 yield per procedure was r 2 =0.67 for the autologous and r 2 =0.34 for the allogeneic collections.conclusions: the majority of autologous and allogeneic donors achieved the target cd34 dose with one procedure. 94.3 % of autologous and 97.3 % allogeneic donors collected a transplantable cd34 dose of > 2x10 6 /kg. 4% of autologous and 2.7% of allogeneic donors did not collect a transplantable dose despite a precd34 count of >10/μl indicating suboptimal procedure performance. the ce was variable and was negatively correlated to the preprocedure wbc and platelet count. the ce and correlation coefficient are lower in allogeneic donors compared to autologous donors.disclosure: nothing to declare the outcome of autologous blood stem cell collection and its actual use in real world: the 21st century experiencekyoungmin lee 1 , jung yong hong 1 , dok hyun yoon 1 , jae-lyun lee 1 , shin kim 1 , kyoung min lee 1 , jung sun park 1 , cheolwon suh 1background: mobilized peripheral blood stem cells (pbscs) have largely replaced bone marrow as the graft source for allogeneic stem cell transplantation. pbscs mobilization with g-csf is highly effective even on the 4th day in order to collect enough number of stem cells. a longitudinal, prospective, observational, single-center, cohort study on healthy donors (hds) was designed to identify predictors of cd34+ cells on the 4th day. methods: as potential predictors of mobilization, age, sex, body weight, height, blood volume as well as white blood cell count, peripheral blood (pb) mononuclear cells, platelet count, hematocrit, and hemoglobin levels were considered. two different evaluations of cd34+ cell counts were determined for each donor: baseline (before granulocyte colony-stimulating factor [g-csf] administration) and in pb after g-csf administration on day 4. a total of 122 consecutive hds with a median age of 47.5 years were enrolled.results: the median value of cd34+ on day 4 was 43 cells/μl (iq 23-68). basal wbc, plt and basal cd34+, are significantly higher for group with cd34+ on the 4th day over the median than below. a multivariate quartile regression analysis, adjusted by gender, age, basal cd34 + and basal plt, shows a, progressively steeper, relationship between baseline cd34+, basal plt and cd34+ on the 4th day. the basal cd34+ cut-off for prevision of cd34+ on the 4th day was < =2 cells/μl and >=3 cells/μl whereas basal platelets count was < =229 x 109/l and >=230 x 109/l.conclusions: g-csf can be highly effective in hds on the 4th day in order to collect enough number of stem cells and we have developed a model for predicting the probability to perform pbsc collection after a short course of g-csf.disclosure: nothing to declare p692 pre-apheresis peripheral blood cd34+ cell counts highly correlates to actual stem cells collected background: prediction of stem cell yield on the basis of pre-apheresis cd34+ cell count and the processed blood volume is essential for the planning and executing of the apheresis process.methods: data analyzed included donor weight, complete blood count and cd34+ count on day of collection, total processed blood volume, cd34 + cell dose collected in the apheresis product and the number of aphereses performed. using the method described by pierelli et al, predicted cd34 + yields were calculated: predicted cd34 + yield x 106/kg = (benchmark ce x volume of blood to be processed x peripheral cd34+ count per μl) / (patient's weight in kg x metric conversion factor).results: in 2017 we established the method described by pierelli to predict the cd34+ cell yield. 323 allogenic aphereses were performed in 2017 with this approach. mean processed volume was 13.71 liters. the mean cd34+ peripheral count before apheresis was 68/ul, the mean collected cd34+count per kg bodyweight recipient was 6.35. pearson´s correlation coefficient (r) between predicted yield using pre-apheresis cd34+count and actually collected cd34+ cells per kg bodyweight recipient was 0.967. the mean difference between predicted and collected cell dose was +4.45%.with knowledge of the predicted stem cell count, we were able to adjust apheresis procedure. in case of marginal predicted yield compared to the requested cell dose, we increased the blood volume to be processed. this proceeding led to a significant reduction of second day donations in 2017 by 38% compared to 2016. in only 3 cases we saw more than -40% lower cd34+ doses collected than initially predicted. all 3 donors showed mild iron deficiency with rbc microcytosis, a factor known to affect apheresis procedure.conclusions: pierellis method of calculating the stem cell yield shows a good correlation between pre-apheresis cd34 + count and actual collected stem cells, making planning and adjusting of the apheresis procedure more feasible and reliable. this proceeding led to significant reduction of second day donations. attention should be paid to iron deficiency anemia, leading to lower than estimated cd34+ dose.[ background: for more than a decade many transplant centers routinely collect and cryopreserve two or more peripheral blood stem cell (pbsc) grafts for a tandem and/ or salvage autologous blood stem cell transplantation (absct) in patients with hemato-oncological diseases. however, subsequent high-dose chemotherapy (hd-cht) and absct is in many cases not performed for a variety of reasons, specifically in patients with aml, all, mpn and burkitt lymphoma. data about the actual utilization rate of the cryostored stem cell products are lacking.methods: we retrospectively analyzed the collection, storage and disposal practices of pbsc products from a large cohort of patients who were treated at the university hospital heidelberg or at the university medical center mannheim during a 12-year period. disease entities included acute myeloid leukemia (aml, n=74), acute lymphoblastic leukemia (all, n=22), mpn (n=18; primary myelofibrosis [pmf], n=9; chronic myeloid leukemia [cml], n=7; secondary fibrosis/essential thrombocythemia [et], n=1; not specified, n=1) and burkitt lymphoma (n=18). patients between 2001 and 2012 were included and followed until 2016.results: an adequate stem cell graft was defined as ≥2.0 x10exp6 cd34+ cells /kg body weight. 97% of the patients were able to collect at least one stem cell graft and the median number of grafts per patient was 1 (range 0-5). we could demonstrate that only 31% of all patients who had collected sufficient pbscs for transplant subsequently underwent an absct. among the disease entities the actual use of the stored pbsc grafts varied considerably from 0% to 56% (figure 1) .conclusions: we could identify striking discrepancies between the collection/storage and actual utilization of background: biosimilars (bio) of granulocyte colony stimulating factors (gcsf) were approved several years ago on the basis of some studies that indicated similar efficacy to the already patented gcsf (neupogen®, neu) both in terms of shortening the neutropenic period after chemotherapy as well as peripheral blood stem cell mobilization in patients with lymphoma and multiple myeloma (mm) treated with autologous stem cell transplantation (auto-hct). however, all these studies are retrospective and there are still concerns about the real efficacy of bio and even more about the real benefit on final costs.methods: we have retrospectively compared the characteristics of the mobilization procedure in both patients with mm and lymphomas, and healthy donors that received either neu or bio (with no chemotherapy) in 4 university hospitals in catalunya from december / 2013 to november 2017. bio replaced neu in june 2016 in all 4 institutions. primary objectives were the mobilization rate (defined as the percentage of patients that achieved ≥ 10 x 10 3 /ml cd34 + cells in peripheral blood on day 4) and the use of plerixafor (plex) in each group as pre-emptive strategy. a multivariable analysis of risk factors influencing the use of plex and mobilization failure (defined as collection of < 2 x 10 6 /kg cd34+ cells) was also performed.results: we treated 216 patients (102 lymphomas and 114 mm) and 55 healthy donors. both groups of patients (138 neu and 78 bio) and donors (33 neu and 22 bio) were comparable regarding pre-mobilization general characteristics. there was a trend for a lower median cd34+ peak on day 4 for bio patients (17 vs 20, p value = 0.1). a total of 53 patients received plex, although 7 of them (13.2%) out of strict theoretical indication, cd34+ cells > 10 x 10 3 /ml (range 10.5-13.5) and were removed for further analysis (n = 46, 31 in the neu group and 15 in the bio group). median number of cd34+ cells on day 4 was significantly lower in the group bio who needed plex (2.4 vs 4.8 for neu+plex, p=002), as well as cd34+ cells finally harvested (2.5 vs 3.3x 10 6 /kg, p=0.03). mobilization failure rate was higher in bio group (0 vs 20%, p=0.01). regarding no plex patients, median number of cd34 +cells on day 4 was also significantly lower for bio patients (33.4 vs 23.7, p=0.03). risk factors for plex use were age, basal disease (lymphoma) and number of prior mobilization therapies. the use of bio was the only risk factor for mobilization failure patients receiving plex [hr 10.3 (95%ci 1.3-77.8), p=0.02]. with respect to healthy donor mobilization, none of them needed plex but 2 cases from the bio group (9%) needed more than one apheresis procedures (2 and 3, respectively).conclusions: we found a lower efficacy of bio in the setting of stem cell mobilization of patients with only gcsf both in terms of a lower cd34+ cells peak on day 4 and a lower number of cd34+ cells in final apheresis product. bio gcsf also seems to be less effective in healthy donors.disclosure: no conflict of interest to be declaredbackground: auto-sct is a common treatment in patients with mm or nhl. the aim of the prospective multicenter goa (graft and outcome in autologous transplantation) study was to investigate the impact of mobilization method used on the cellular composition of collected blood grafts and eventually hematological and immune recovery as well as long-term outcome post-transplant. altogether 282 patients with mm or nhl transplanted between 5/2012 and 12/2016 at four university hospitals were included. the long-term goal of the study is to evaluate characteristics of optimal blood grafts in regard to post-transplant recovery and outcome. methods: altogether 147 patients with mm undergoing first auto-sct were compared with 135 patients with nhl. all nhl patients were mobilized with chemotherapy + g-csf, whereas 39 % of mm patients were mobilized with g-csf only (p < 0.001). mobilization data, graft cellular composition including cd34 + cell subsets and lymphocyte subsets of the blood grafts, post-transplant hematological recovery and outcome were evaluated. the median followup time was 46 months in mm patients and 35 months in nhl patients.results: mm patients mobilized cd34 + cells better (median peak blood cd34 + 65 vs. 40 x 10 6 /l, p < 0.001). the median number of aphereses was 2 in both groups (p = 0.09). altogether 26 % of the nhl patients received plerixafor compared to 12 % in mm patients (p = 0.002). the median number of cd34 + cells collected was higher in mm patients (6.4 vs. 3.9 x 10 6 /kg, p < 0.001).the median amount of cd34 + cells (with 7-aminoactinomycin) in the infused graft was 2.35 x 10 6 /kg in mm group and 2.5 x 10 6 /kg in nhl group (p = 0.02). the grafts contained more nk cells (median 10.1 vs. 6.1 x 10 6 /kg, p = 0.01) and cd19 + cells (median 1.69 vs. 0.00 x 10 6 /kg, p < 0.001) in mm patients. neutropenic fever tended to be more common in nhl patients (88 % vs. 79 %, p = 0.06) but mm patients had significantly more bloodstream infections (31 % vs. 15 %, p = 0.003). the median duration of hospitalization was longer in the nhl patients (22 d vs. 18 d, p < 0.001) and the nhl patients had significantly more often icu admissions (4 % vs. 0 %, p = 0.02).post-transplant neutrophil engraftment was faster in the nhl group (median 9 d vs. 12 d, p < 0.001). the median time to platelet engraftment was 11 days in both groups. platelet count was higher in the mm group from day 15 until 1 year after auto-sct. there were significantly more early deaths (< 100 d from the graft infusion) (4 % vs. 0 %, p = 0.02) and non-relapse deaths (6% vs. 0%, p = 0.003) in the nhl group.conclusions: mm and nhl patients differ in terms of cd34 + cell mobilization, graft cellular composition and post-transplant recovery as well as risk of non-relapse death. thus, the optimal graft may be different in nhl and mm patients.disclosure: the study was supported by vtr fund from north savo hospital district and study grant from sanofi. abstract already published. single-center experience in use of plerixafor for autologous stem cell mobilization: change in practice over 10 years background: plerixafor has been proven to mobilize human periferal blood stem cells (pbscs) alone or acting synergistically with granulocyte-colony stimulating factor (g-csf). it has mainly been used for rescue mobilization after failed regimen of chemotherapy plus g-csf, but lately preemptive use in poor mobilizers has been established as cost-effective. we aim to show ten years of experience and change in practice with plerixafor use in our center.methods: we retrospectively evaluated the outcome of mobilization procedures and leukapheresis collections in our center in the period from january 2009 to october 2018. practice from the first 5 years, when plerixafor was mainly used as rescue agent after failed attempt, was compared to period from 2014 till present when preemptive use in poor mobilizers (defined as cd34+ cell counts <10 x 10 6 /l blood) was established.results: in the period from 2009 to 2013, total of 434 patients underwent collection of autologous pbscs, and 55 patients required repeated mobilization cycles (12,7%). g-csf alone was used in 39 patients and 16 patients (29%) recieved combination g-csf with plerixafor. this cohort consisted of 10 males and 6 females with non-hodgkin (nhl) and hodgkin lymphoma (mh); 13 and 3 respectively. we noted 37 unsuccessful mobilizations (67,3% in repeated mobilization, 8,5% in total) of which one patient was from plerixafor group (6,3%).background: transplantation of hla-haploidentical hematopoietic stem cells (haplo-hsct) is an established procedure for the treatment of several different hematological diseases. one possible strategy to reduce the risk of graft-versus-host disease is represented by the selective depletion of ab t-lymphocytes (coupled with the depletion of cd19+ b-cells in order to reduce the risk of ptld) using the clinimacs device (miltenyi, bergish-gladback). before depletion, leukapheresis units are washed by lowspeed centrifugation, resulting in a platelet (plt) rich supernatant (prs) as a by-product generally discarded. we studied the possibility of recovering plt from prs of the haplo donor for transfusion to the recipient during the aplasia period occurring after hsct.methods: hsc donors were mobilized with g-csf (plus plerixafor in 6 out of 24 donors) as previously described [locatelli et al, blood 2017] . leukapheresis units, obtained with a spectra optia device, were washed twice (producing 2 prs bags) at low speed to remove plt before starting the ab t-cell/b-cell depletion. the two prs were leukodepleted by filtration, centrifuged, resuspended and pooled in a total volume ranging from 95 to 360 ml intersol (is-plt) for overnight incubation at 22°c with agitation (60 cycles/min). the is-plt samples were analyzed for the criteria established by the italian transfusion law. is-plt bags were examined for the following parameters: volume >40ml, plt after leukodepletion >2x10 11 , residual leukocytes < 1x10 6 , ph >6.4 at 22°c at the end of the 5-day storage period. the sterility was tested using bd bactec culture vials. the evaluation of the residual leukocytes was performed with the bd leucocount kit. the absolute plt counts were determined using hemocytometer sysmex xn2000.results: prs bags from 24 donors were processed to produce is-plt units. median resuspension volume in intersol was 220 ml (range 95-360). the absolute mean value of plt counts measured at the end of the storage period was 2.1 x10 11 (range 1.0-6.1 x10 11 ). this value was found below the threshold fixed by italian regulation in 8 cases (33.3%). mean value of residual leukocytes was 1.3x10 5 (range 0-9x10 5 ); the ph value was always > 6.4. sterility was observed in all cases. according to the work of slichter et al. conclusions: we demonstrated that plts recovered from leukapheresis bags can be accepted as a conventional hemocomponent according to the parameters fixed by italian transfusion law and thus can be administered to the haplo-hsct recipients early after transplantation. this strategy carries several advantages. indeed, apart from the obvious advantages in terms of reduced costs, is-plt can be used to desensitize the recipient by absorption of anti-hla class i antibodies, if present in the recipient. moreover, this strategy can avoid the risk of sensitizing the transplanted patients with hla alleles that differ from the donor's ones. finally, the is-plt unit is readily available. a clinical study aimed at testing the use of is-plt units in transplant recipients will be performed to confirm the clinical efficacy of the approach.disclosure: nothing to declare p700 ultrasound guidance as a powerful tool in increasing background: peripheral blood stem cells are generally the preferred graft source for allogeneic stem cell transplantation for malignant disease. in most centers first apheresis is performed on day 5 after 8 to 9 doses of granulocyte colony stimulating factor (gcsf) up to 5ug/kg twice daily. the dose of gcsf and the number of apheresis procedures required contribute to symptom, travel and time burden donors are put through during the process. we hypothesized that taking donor-recipient weight differences into consideration may help reduce this burden methods: a total of 261 healthy donors who donated peripheral blood stem cells on day 4 of gcsf mobilization in the period between january 2015 and august 2018 at the university medical center hamburg-eppendorf were included in this quality control evaluation. the donors were divided into two cohorts. the impact of donorrecipient weight ratio on stem cell harvest was tested in the training cohort (2015-2016) and validated in the second cohort (2017) (2018) . for the training cohort, donors were grouped according to donor-recipient weight ratio < 1.0 vs. 1.0-1.2 vs. > 1.2. for the purpose of this analysis a stem cells dose of 5 x 10 6 cd34+/kg recipient weight was set for successful apheresis.results: in the training cohort including 142 donors, 52 (37%), 36 (25%) and 54 (38%) had a donor-recipient weight ratio of < 1.0, 1.0 -1.2 and > 1.2 respectively. the target stem cells count of 5 x 10 6 cd34+/kg recipient weight was achieved in 36 of 52 (69%), 26 of 36 (72%) and 45 of 54 (83%) donors with donor:recipient weight ratio < 1.0, 1.0 -1.2 and > 1.2 respectively. the cut-off for the validation cohort was therefore set at a weight ratio of 1.2.in der validation cohort including 119 donors, 75 (48%) had a weight ratio > 1.2 while 62 (52%) had a weight ratio ≤ 1.2. overall in this cohort target cell count of 5 x 10 6 cd34 +/kg recipient weight was reached in 92 (77%) cases. this target was reached in 43 of 62 (69%) of donors with weight ratio ≤ 1.2 and in 49 of 57 (86%) donors with weight ratio > 1.2, p = 0.03.conclusions: a donor-recipient weight ratio of > 1.2 is seen in about 40% of peripheral blood stem cell donations for allogeneic stem cell transplantation. in these cases apheresis on day 4 after 7 doses of gcsf is reasonable. donors with lower weight ratios should preferentially donate on day 5 after 8 to 9 doses of gcsf.disclosure: all authors declare no conflicts of interest background: the effect of a second mobilization and collection of peripheral blood stem cells (pbsc) on the cell yield is low, as we previously demonstrated. however, donor safety has been poorly addressed with no changes in the clinical practices.methods: second donations of unrelated and related donors performed between 2001 and 2017 were evaluated (n=24), including pbsc+pbsc (n=18), bone marrow (bm)+pbsc (n=5) and pbsc+bm (n=1). analytical parameters including leukocyte, lymphocyte, hemoglobin and ldh quantification, obtained on the pre-harvest evaluation of first and second donation, were retrospectively analyzed and compared for all donors. the portuguese bone marrow donors registry (cedace) recommends a time between donations no lesser than 6 months. it also states that in very urgent situations like graft failure, donor should be clinical and analytical cleared and its safety ensured.in order to evaluate the impact of time between donations, donor population was divided in 3 groups: < 2 months, 2-6 months, >6 months; to determine the influence of donor age, donors were divided in 2 groups: < 40 and ≥40 years.results: among the total of 24 donors, 13 were volunteer donors of cedace and 11 were familiar. fifteen second donations were performed because of recipient graft failure and 9 due to disease progression or relapse. at the time of second collection, median donor age was 37 years (range 23-57). the median delay between both collections was 262 days (37-1519). time between donations did not seem to substantially impact the analytical donor evaluation: leukocytes, lymphocytes, hemoglobin and ldh results are kept within the reference values. however, donors with less than 2 months between donations showed a slight decrease on leukocyte counts (35%) and hemoglobin values (12%), from the first to the second pre-harvest evaluation. donor age showed no significant influence on the analytical evaluation. nevertheless, when considering only the pbsc+pbsc donations, donors with ≥40 years showed a small decrease on lymphocyte counts (19%) .conclusions: this study demonstrated that the analytical parameters, chosen based on literature, had no significant changes between first and second donation. however, particular attention should be paid when time between donations is lesser than 2 months or donor age is ≥40 years.as we concluded that no significant changes were observed in the group of 2-6 months, it is our opinion that the minimum of 6-12 months established by the registries can be shortened to 2 months ensuring donor safety. an accurate donor risk assessment with a larger population should be accomplished in order to strengthen this recommendation.disclosure: nothing to declare. gaurav kharya 1 , atish bakane 1 , pratibha dhiman 1 , anil khetrapal 1 , vikrant bhar 1 background: t cell replete haploidentical stem cell transplant (hhsct) is complicated mainly by increased risk of graft failure (gf) and graft versus host disease (gvhd). conventionally gcsf has been used to mobilize hematopoietic stem cells (hsc). in tcr hhsct gcsf mobilized graft with megadose of cd34+ cells expose the patient to higher doses of alloreactive t cells increasing the risk of gvhd. plerixafor based mobilization gives an advantage of giving high cd34 cell dose limiting exposure to high alloreactive t cell dose. we share our experience of gcsf + plerixafor based mobilization for tcr hhsct. methods: 9 consecutive patients suffering from scd who underwent hhsct between jan 2018 till date along with the respective donors were enrolled in the study (group 1). all 9 underwent pre-transplant immune suppression (ptis) 2 cycles at 3 weekly intervals using fludarabine+cyclophosphamide+dexamethasone.the graft was mobilized using gcsf@10mcg/kg/day(d1-d5)+plerixafor@0.24mg/kg(d5) 6-8 hours before the pbsch. gvhd prophylaxis included ptcy 50 mg/kg/ day on d3 and 4, sirolimus and mmf starting from d5. group 2 included 5 historical controls where graft was mobilized using gcsf@10mcg/kg/day(d1-d5). various parameters pertaining to mobilization, harvest, engraftment, gf and gvhd were assessed between the two groups.background: poor mobilizers (pm) defined as those with a peripheral blood cd34 + count ≤10cells/μl on day+4 is a significant risk factor for mobilization failure. within these, patients with < 5cells/μl are considered as very poor mobilizers (vpm). use of plerixafor in vpm patient is controversial. the aim of our study is to compare mobilizing and engraftment between pm and vpm who received plerixafor plus g-csf (p+g-csf).methods: in our center, mobilization with g-csf at dose of 10μg/kg/day was used in all pts. apheresis were scheduled on day+5. plerixafor (0.24 mg/kg) was added if the number of cd34 + cells on day +4 was < 10/ul for 2x10 6 cd34 + /kg requested (or < 20/ul for 4x10 6 cd34 + /kg), or if the number of cd34 + cells collected in the first apheresis was < 50% of cd34 + cells requested.between january 2016 and september 2018, 37 out of 157pts (23,6%) received plerixafor for mobilization. we retrospectively studied 30 pts who mobilized with p+g-csfdue to the number of cd34 + cells on day +4 was < 10/ul.results: twelve out of 30 pts were pm, 7 were females, median age 55,5 years (range:32-70). patients' baseline diseases were: 10 non-hodgkin lymphoma (nhl) (83,3%), 1 multiple myeloma (mm) and 1 hodgkin lymphoma. median cd34 + cell count on day +4 was 8/ ul (range:6-10).there was no mobilization failure. eighteen out of 30 pts (60%) were vpm, 9 were females, median age 62,5 years (range:34-69). patients' baseline diseases were: 10 nhl (55,5%), 7 mm and 1 solid tumor. median cd34 + cell count on day+4 was 2,5/ul (range:2-5). two out of 18 pts (16,6%) were considered mobilization failure, in 2 of them did not realized apheresis due to cd34 + cell count on day +5 was 2/ul. no difference was seen between both groups regarding gender, age, patients baseline disease or median cd34 + cells count on day +4.vpm needed more apheresis sessions, 5/16 pts required 2 sessions against 1/12 pts in pm (p=not significant (ns). we obtained enough cells to carry asct in 90% pts, although mean number of cd34 + cells obtained in vpm was lower than in pm (4,89x10 6 /kg vs 6,38x10 6 /kg, respectively) (p=ns).twenty-six pts underwent asct and mean number of cd34 + cells infused were 4,27x10 6 /kg in vpm vs have been the only route used for chpc administration. the appearance of other catheters types made us to reconsider the exclusive use of the cvc for the infusion of chpc. we analyzed the use of a peripheral iv cannula (pivc) as an alternative to cvc for the infusion of chpc in patients with cardiovascular diseases.methods: medical records of 65 patients who received an asct for hematological malignant diseases at the hospital clínic of barcelona from january 2017 to february 2018 were reviewed. of those, eight were infused through a pivc due to cardiac impairment related to previous treatments, ischemic cardiomyopathy or amyloid deposition.hpc were obtained from peripheral blood by apheresis after mobilization with g-csf using acd-a as an anticoagulant. cryopreservation was performed with autologous plasma and dmso 10% by mechanical means and stored in liquid nitrogen. analytical controls were performed including hematocrit, total nucleated cells, total polymorphonuclear neutrophils and platelets using the advia 120 analyzer. the cd34 + / cd45 + population was analyzed by flow cytometry following the ishage single-platform protocol. viability of total nucleated cellularity was carried out by vital staining with acridine orange and the specific viability of the cd34 + population through the technique of 7-aminoactinomycin d. thawing was performed bag to bag by immersion in a water bath at 37ºc and transferred to the bedside of the patient for gravity infusion using an infusion set without filter through pivc of 22 gauche. vital sings monitoring performed before, during and the end of the every infusion bag including: blood pressure, heart rate, oxygen saturation, body temperature and central venous pressure. other aspects assessed during the infusion were pain, cold sensation and signs of extravasation in the area of pivc insertion. after the infusion, the recovery time of the granulocyte series and platelet were evaluated.results: median volume and bags administered was 480 (360-600) ml and 4 (3) (4) (5) . the median of total nucleated cells, total nucleated cells / ml and total cd34+ cells/kg was 462.6 x 10 8 (329.5-657.8), 107.3 x 10 8 (64.66-156.7) and 3.24 (2.46-4.6) respectively. vital signs were within the normal range and allowed to perform the infusion in an average of 20-30 minutes/bag. no patient required stopping the infusion due to pain in the area of peripheral catheter insertion and no extravasations were detected. all patients referred some cold sensation in the insertion vein and its path. median hematopoietic recovery was 14 (11-19) days for neutrophils and 11 (0-19) days for platelets, similar to the recovery experienced from patients who received chpc through cvc.conclusions: based on our data, we conclude that the administration of chpc, through pivc and by gravity is safe for the product and for the patient, being the preferred choice for patients suffering from some type of cardiovascular disease.disclosure: nothing to declare background: by selective depletion of potentially alloreactive cd45ra + cells, t memory cells might be retained in the graft and could mediate pathogen specific immunity. however, cd45ra expression is not restricted to naïve t cells, but also available on b cells, nk cells and cd34 + stem cells to some extent. methods: within this project we aim to analyze cd45ra expression on stem-and nk cells by flow cytometric analysis to estimate the eventual loss of these cells during cd45ra-depletion. furthermore, clinimacs depletion following a one-step approach of direct cd45radepletion and a two-step approach with primary cd34selection followed by cd45ra-depletion of the negative fraction was investigated.results: cd45ra expression on cd34 + stem cells was in median 16.9%. with a median of 99.4% cd45ra expression was measurable on nearly all b cells, which obviates depletion via cd19. a comparably high cd45ra expression of in median 96.0% was detected on nk cells. unfortunately, the amount of nk cells in the cd45ra-depleted product was 0.24%. clinimacs depletion following one-step approach resulted in a stem cell recovery of 52.0%. memory t cell recovery was 24.2% following one-step and 42.0% applying two-step approach. depletion quality measured by log-depletion was 3.9 and 3.8 for cd45ra + t cells and 3.2 and 3.6 for cd19 + b cells for one-and two-step approaches, respectively.conclusions: with regard to stem cell recovery, a previous cd34-selection before cd45ra-depletion is recommendable.background: current clinical practice of routine use of filters for infusion of autologous hematopoietic cell transplantation (ahct) at bone marrow transplant centers across north america and europe is not known. the use of "y" administration tubing without a filter could possibly increase the risk of infusion of macro-aggregates and cellular debris, which may result in increased side effects.methods: we carried out a retrospective chart review of patients (pts) at spectrum health who underwent ahct. group a (gp a) pts received ahct using a "y" administration tubing with 170-micron filter from 3/2013 -3/2017. these patients were compared to a control group (gp b) that received ahct without filter administration tubing from 3/2014-4/2016.this change in clinical practice occurred due to a change in policy at our transplant center as a result of inorganic particles noticed during cryopreservation. we compared the neutrophil and platelet engraftment duration between these groups. we also studied the length of hospital stay and the effect of filter use on any immediate side effects after infusion. due to the retrospective nature of the study it was not feasible to evaluate the difference in duration of infusion between these groups results: the two groups were similar in their age, gender, primary disease distribution and median number of cd34 stem cells infused (table) . there was no difference in median neutrophil (11 vs 11 days) or platelet engraftment (19 vs 19 days) duration for the filter group and the nonfilter group respectively. the median length of hospital stay was also comparable (17 days). there was no statistically significant difference in the immediate side effects (fever, cough, dyspnea, fluid overload, flushing, nausea, vomiting, hypertension, hypotension and anaphylaxis) or confirmed post-transplant infections (viral, bacterial, fungal) experienced by these two groups.conclusions: our results show that the routine use of filter does not prolong hospital stay, and neutrophil/ platelet engraftment duration, thereby, suggesting that viable stem cells are not affected. on the other hand, filter use failed to demonstrate any appreciable decline in the immediate side effects experienced after ahct. gp background: allo-hsct from related haplo-identical donors (haplo-hsct) with post-transplant high-dose cyclophosphamide is increasingly employed in patients who lack a matched related or unrelated donor. the current standard is to use bone marrow grafts (bm) as peripheral blood stem cell grafts (pbsc) have been associated with an increased risk of acute and chronic gvhd. thus, the aim of our study was to compare the main transplant outcomes and especially the incidence of acute and chronic gvhd in recipients of bm and pbsc grafts. methods: thirty-five unselected patients with hematologic malignancy who underwent an haploidentical transplant at our unit between 2011 and 2018 and received bm (n = 17) or pbsc (n = 18) grafts after the same tbf conditioning regimen were analysed in order to assess differences in transplant outcomes.our gvhd prophylaxis consisted in cyclosporine a (csa) from day -1 to +180, a methotrexate "short course" and mycophenolate mofetil (mmf) from day +1 to +28.results: no statistically-significant differences were observed between patients who received bm grafts and those who received pbsc grafts. at transplant fourteen patients were in first complete remission (cr), twelve in advanced cr and 9 had active disease. according to sorror's risk, nine patients were low-risk, nine intermediate-risk and seventeen high-risk. twenty-eight cmv+ patients received the graft from twenty-three cmv+ and five cmv-donors, seven cmv-patients received the graft from five cmv+ and two cmvdonors. mean age at transplant was 51 years (range 23-72), mean donor's age 38 years (range18-39) and mean follow-up 16.9 months (range 1.9-56.7). median cd 34+ cell dose was 5.2x10 6 /kg (range1.4-10.4), 3.6 x10 6 /kg (range 1.4-7.7) in bm recipients and 14.0 x10 6 /kg (range 4.2-10.4) in pbsc recipients. median time to neutrophil recovery (>500/μl) was 22 days (range 16-39) posttransplant, 23 days (range 18-27) for bm recipients and 20 (range 16-39) for pbsc recipients. platelet recovery (>20.000/μl) occurred in all patients except one at a median of 17 days (range 10-151) post-transplant, at a median of 20 days (11-151) post-transplant for bm recipients and at a median of 14 days (range (10-26) for pbsc recipients. seven patients never reached platelets >50.000/μl. three patients developed a poor graft function. acute and chronic gvhd incidence was 28.5% and 22.8% and the risks of acute (hazard ratio [hr], 1.04; p = .955) and chronic (hr, 0.85; p = .816) graft-versus-host disease were similar in the two patient groups. in addition, there were no differences in relapse risks post-transplant (hr, 0.90; p = .898); relapse-free survival was better with pbsc grafts but this difference did not reach any statistical significance. finally, no significant differences were noted in overall mortality by graft type (hr, 0.62; p = .441).conclusions: despite in haplo-hsct the incidence of acute and chronic gvhd is reported to be higher with pbsc than with bm our small patient series does not confirm this assumption that should be clarified by additional studies. instead, our data suggest that pbsc background: since 1988, cord blood (cbu) has become an alternative source of stem cells for transplantation, with approximately 35,000 procedures currently performed. with a 2-year gs of 50%.objective: analyze the outcomes from all the patients transplanted with cbu in our hospital unit.methods: retrospective, longitudinal study. all patients transplanted with cbu in our hospital between 2007 and 2017 were included. we analyzed 40 patients with ages from 7 months to 14 years.results: two of them received doubled cord transplantation the ratio male: female was 1.3: 1. the transplanted pathologies were: bone marrow failure 35%, immunodeficiency 25%, aml 20%, all 17.5%, osteopetrosis 2.5%. ric regimens were used in patients with bone marrow failure and immunodeficiency and myeloablative conditioning regimens were used in patients with malignant hematology diseases. antithymocyte rabbit globulin (atg) based serotherapy was used. one case received cbu from a related donor (sister), the rest received unrelated cbu obtained from centro nacional de la trasfusión sanguínea. the infusion of cd34 + was in a range of 0.11 to of 2.7 x10 6 /kg with and average of 0.43 x 10 6 / kg. compatibility was 4/6 in 70%, 5/6 in 20% and 6/6 in 10%. post-thawing cellularity was not measured. the hla-c was not analyzed. forty two point five percent of the patients had a successful engraftment; the average time of engraftment was 30 days. primary graft failure was detected in 57.5% and secondary graft failure in 10%, for a total success of 32.5%. gvhd was detected in 20% of patients, of which 90% was grade i-ii and 10% grade iii-iv. the overall mortality was 52.5%. causes of death were: infection 45% relapse 30%, hemorrhage 20% and gvhd 5%. the cbsct continues to be an essential alternative in our patients who required transplantation knowing that this stem cell source allows the procedure to be done with less histocompatibility requirements and it is available immediately, which facilitates the process considering the great diversity that exists within our population. however, in our experience, the cbsct has shown a higher mortality risk, which can be improved by analyzing the hla c, choosing in this way the units with better compatibility, and improving cellular dosage since this is key in success.disclosure: nothing to declare the risk of infection of the umbilical cord is not related to the microbiological status of the umbilical cord blood methods: the statistical analysis was carried out on data obtained from samples taken in poland between 01-jan-2017 and 31-dec-2017. the samples were collected in hospitals by external midwifes and sent to the pbkm in accordance with the requirements of the american association of blood banks. after arrival in the laboratory, the blood samples were cultured and the ucs were assessed immediately for visual signs of infection, such as odor, altered color, or visible bloom. the status of both kinds of samples was introduced into the pbkm general database. for the purpose of this analysis, the ucs were considered as microbiologically pure if stored, destroyed after storage, or handed over to the pbkm. samples marked as infected or disqualified for unknown reasons (other than termination of the contract with the customer, viral infection of the mother, and lack of cell growth) were considered as infected. at the time of the statistical analysis, the samples of unknown ucb microbiological culture status were removed from the generated report. the data was summarized as percentages and the odds ratio was calculated. statistical significance was considered at p˂0.05.background: match family donors are the preferable options in allogenic stem cell transplant. however, in the absence of donor relatives match unrelated donors have been an option. in this study, the donor screening, transplant preparation phases of turkish stem cell coordination center (turkok) and the i̇stanbul university bone marrow bank (tris), were compared.methods: the unrelated donor scanning data between march 2015 and november 2018 in pediatric stem cell transplantation unit of altınbas university bahcelievler medical park hospital were evaluated. 93 unrelated transplants were performed in total. 65 % of these transplants (n=61) were included from the donors of turkok registration system and 34,4 % of these transplants (n=32) were included by means of tris from donors outside of turkey. 8 patients (5 tris, 3 turkok) were excluded from the study in consequence of screening update and postpone of transplantation. the statistics were carried out on a total of 85 patients, 67,1 % of whom were in turkok (n=57) and 32,9 % were transplanted via tris (n=28). the day of application to stem cell transplantation unit, reply dates and the transplantation dates were examined for the transplant patients.results: in the current study, the average response time of turkok was found as 0,91±2,96 day (median: 0), the average transplant time after receiving a reply was found as 98,24±63,89 (median: 85) day, the average number of days from date of application to date of transplantation of patients was found as 101,12±63,17 (median: 89). the average response time of tris was 18,78±18,36 (median: 15) day, the average transplant time after receiving a reply from tris was 136,82±63,84 (median: 119) day, average number of days from date of application of tris to date of transplantation of patients 155,61±72,23 (median: 140) day.the average response time of turkok, the average transplant time after receiving a reply from turkok and the average number of days from date of application of turkok to date of transplantation of patients was shorter than tris. the difference between them was found statistically significant (p< 0.05).conclusions: in this study, it was determined that the transplantation processes with turkok were progressing more rapidly. the rapid progress of the process was attributed to the fact that all donor hla tissue groups in the turkok database were studied in high resolution. in international scans carried out through tris, it was thought that the examination of the donor castings coming from bone marrow banks and the time differences between the countries prolong the process. it was thought that hla tests of the registered donors in the tris database and some international bone marrow banks were studied in low resolution but not studied the all hla loci, the centers wanted high-resolution hla, and therefore the involvement of social security institutions and payment procedures were among the factors extending this process.disclosure: nothing to declare background: allogeneic hematopoietic stem cell transplantation (ahsct) is being performed for a group of hematologic diseases with a curative intent. outcomes after ahsct are influenced by the type of donor used. haploidentical transplantation is an emerging option when a fullmatched donor is unavailable. methods: we retrospectively analyzed our transplants performed between january 2015 and november 2018, investigating outcomes and complications among haploidentical stem cell recipients.results: one hundred and nineteen patients underwent ahsct, 9 of them (7.5%) were recipient of a haploidentical stem cell and included in this study. one patient diagnosed with acute lymphoblastic leukemia (all) were performed a haploidentical ahsct for two times due to relapse. among those 9 transplants, 4 of them were diagnosed with acute myeloid leukemia, 2 with all, 1 with chronic lymphocytic leukemia, 1 with myelodysplastic syndrome and 1 with hodgkin lymphoma. the mean age of group was 35.7±15.2 years. three patients (2 aml, 1 all) were in remission at the time of transplantation. patients were given a conditioning regimen based mostly on busulfan, fludarabin and total body irradiation with a myeloablative intent. patients were also given a various combinations of post-transplant cyclophosphamide, calcineurin inhibitors, mycophenolate mofetil and antithymocyte globulin for graft versus host disease (gvhd) prophylaxis; post-transplant cyclophosphamide administered on 6 (67%) of those transplantations. peripheral blood was the source of stem cells in all patients. patients were infused with mean 6.15±0.67x10 6 /kg of cd34+ cells. hematological recovery was achieved with neutrophil engraftment at a mean of 19.2±2.1 days and platelet engraftment at a mean of 17.3±2.7 days. after a median 8month (0.2-40.6 months) follow up, the cumulative rates of grade 3-4 gvhd, relapse and non-relapse mortality were 11%, 28% (n=7) and 71%, respectively. one patient died due to relapse, at the end of the follow up two were still alive with remission. only one patient has died due to chronic gvhd affecting serosa and resulting with a fatal tracheoesophageal fistula. the mean overall survival was 6.9±2.6 months in our study.conclusions: haploidentical transplant is a feasible option in hematologic malignancies with novel gvhd prophylaxis approaches, especially post-transplantation cyclophosphamide. however, these results need to be supported with further investigations with a larger patient group.disclosure: nothing to declare results: a total of 25 patients between 19 and 58 years (median age: 33 years). transplant was done for following disease: acute leukemia (n=9, 36%), aplastic anemia (n=5, 20%), lymphoma (n=4, 16%), myelofibrosis (n=3, 12%), myelodisplastic syndrome (n=2, 8%), chronic myeloid leukemia (n=2, 8%). ten donors were from turkey and fifteen donors were from different countries of europe and america. two of 25 donors were 9/10 and the other 23 was 10/10 hla matched. the conditioning regimen was mostly non myeloablative (n=17, 68%) while eight patients were treated with myeloablative regimen. other than two patients who took tacrolimus and mycophenolate mofetile all of them got cyclophosphamide and methotrexate for graft versus host disease (gvhd) prophylaxis. the median time of neutrophil and platelets engrafman were 18 days (range 11-31) and 13,5 days (range 9-36) respectively. acute gvhd was seen nearly half of the patients (47,8%).overall survival was 40% for all patients and 15 of 25 patiens (60%) died within first month to 18 months (median 3 months). the mortality rate was more higher for the recipients who had donor source from countries other than turkey (30% vs 80% p=0,018). transplant related mortality was the most common reason of mortality (n=7/ 15, 46,7%) and other reasons were gvhd (33,3%), infections and cirrhosis respectively.conclusions: we found the mortality rate more higher in the patients whose donors were from out of our country. however, we need to further multicentric and prospective investigations to confirm our hypothesis, it would be related with impact of ethnicity.disclosure: nothing to disclose late-breaking abstracts p725 targeted twice daily busulfan-based ric-conditioning for allogeneic hematopoietic stem cell transplantation in pediatric patients with chronic granulomatous disease: a 10-year experience with the zurich protocol matthias felber 1 , mathias hauri-hohl 1 , ulrike zeilhofer 1 , federica achini 1 , jana pachlopnik-schmid 2 , janine reichenbach 2 , seraina prader 2 , tayfun güngör 1background: chronic granulomatous disease (cgd) needs sufficient myeloablation to avoid graft failure. for this purpose the ebmt inborn errors working party currently recommends treosulfan or busulfan-based conditioning regimens for cgd-sct. we analyzed the last 10 years of targeted busulfan-based ric-conditioning including engraftment, gvhd rates, chimerism and late term effects in our pediatric sct center in zurich. methods: between 2008 and 2018, n=34 consecutive pediatric cgd patients (median age 6 years, range 1-16 years, n=4 female, n=9 autosomal recessive inheritance) have been transplanted. all patients received therapeutic drug monitoring of twice daily administered iv busulfan (3 or 4 hour infusions; d-5 to -d2) to achieve a targeted cumulative auc of 45-70 mg/l*h. fludarabine (180 mg/ sqm; d-8 to -d3) and serotherapy (thymoglobuline 7.5 mg/ kg total, d-5 to d-3) or alemtuzumab (0.5-0.6 mg/kg total; d-8-to d-6) were used for immunoablation. donors were matched unrelated (10/10 hla; n= 19), mismatched unrelated (9/10 hla; n= 7), mismatched unrelated (hla 8/10; n=1), matched sibling (hla 6/6; n= 5) and haploidentical parental (hla 5/10; n=2). for 2 patients with haploidentical donor post-transplant cyclophosphamide (d-3 and d-4 with 50 mg/kg iv each) and upfront atg-grafalon (40 mg/kg -12 to-d-9) was used. stem cell sources were bm (n=29) and pbsc (n=5). gvhdprophylaxis included iv csa (d-3; continuous infusion) and iv. mmf (d-0, in 2-3 doses).results: follow-up was 6 to 118 months. good overall engraftment was noted, with n=1 secondary graft failure followed by successful retransplantation. in one patient a stem cell boost/dli was necessary due to decreasing myeloid donor chimerism during ebv reactivation, resulting in rapid myeloid donor reconstitution after intervention. low rates of gvhd were documented with n=2 agvhd grade iv and n=2mild/limited cgvhd (nih criteria). with exception of one patient, myeloid donor chimerism at last follow-up was over 82%, mostly over 95%. overall survival was 31/34 (91%). deaths were due to gi-gvhd (n=1), autoimmune hemolytic anemia/sepsis (n=1) and thrombotic microangiopathy (n=1).conclusions: precision dosing of iv busulfan in combination with fludarabine and serotherapy results in excellent outcome of hsct for pediatric cgd-patients with good engraftment, low overall cgvhd rates and stable, mostly excellent donor chimerism. graft failure rate was as low as 3%. low dose alemtuzumab prevented gvhd in the majority of patients. this analysis demonstrates that targeted busulfan-based conditioning is a valid option for pediatric cgd-patients. serum alemtuzumab or atg monitoring could further improve gf and gvhd rates in the future.disclosure: the authors declare no conflict of interest. abstract already published. young hla-matched unrelated donors are comparable to matched sibling donors in elderly patients receiving reduced-intensity conditioning: an analysis on behalf of the ebmt scientific council 95% c.i. 1.7-20.6, p= 0.001) and voriconazol prophylaxis during carv (or 4.2, 95% c.i. 1.1-115.6, p= 0.03). conclusions: we provide evidence that ifd after carv infection. allo-hsct recipients developing a carv lrtd during the first year after transplant may benefit from an adequate antifungal prophylaxis and a close monitoring for the development of a later ifd.disclosure: jose luis piñana has received both, advisory for preclinical/clinical research and financial support to assist to the spanish society of hematology annual meeting 2018 from msd. favorable outcome and engraftment following reducedintensity conditioned allo-hsct in children with primary haemophagocytic lymphohistiocytosis (hlh) and high-risk langerhans cell histiocytosis (lch) laura m. moser, emilia salzmann-manrique, andrea jarisch, jan sörensen, shahrzad bakhtiar, peter bader background: primary haemophagocytic lymphohistiocytosis (hlh) and high-risk langerhans cell histiocytosis (lch) represent two major entities of childhood histiocytoses, which are -although only of rare occurrence -severe in their clinical manifestations. patients present with multisystemic uncontrolled inflammation and multi-organ involvement requiring diverse courses of immunosuppressive and chemotherapy regimens. allogeneic haematopoietic stem cell transplantation (allohsct) is the only available curative option; however, the cumulative treatment toxicity and the underlying inflammatory disease often result in high organ toxicity and inflammatory complications of transplantation, such as graft versus host disease (gvhd) and/or graft failure. especially patients with unrelated donors often deal with high transplant-related mortality (trm) in the setting of conventional intensity conditioning.herein, we present the clinical course of the disease and transplant outcome of 14 children diagnosed with primary hlh (n=12) and high-risk lch (n=3) who underwent allohsct at our centre from 02/2003 to 08/2018.methods: the hlh cohort consisted of 7 cases of familial hlh (fhlh), 4 cases of griscelli syndrome, one xiap-deficient patient and one hlh-patient with inconclusive genetic testing. all hlh patients had developed clinical symptoms prior to transplantation and had been treated according to hlh-protocols 94, 2004 hlh-protocols 94, or 2015 median age at transplantation was 10 months (4 to 128 months). stem cells were derived from hla-matched siblings (msd, n= 4), matched unrelated donors (mud, n = 9) or haploidentical donors (n=1).the majority of patients (9/14) received a ric regimen containing fludarabine, melphalan and thiotepa (n=8) and fludarabine plus cyclophosphamide (n=1). myeloablative treatment (5/14) included a treosulfan-based regimen (n=3) and busulfan-containing treatment (n=2). the entire cohort received serotherapy using either muromonab (n=3), atg-fresenius® (n=9) or alemtuzumab (n=2).results: the overall survival of the entire cohort was 78.6% (11/14) on a median follow-up of 9.9 years ( figure 1 a+b) .all lch patients, being treated with fludarabine, melphalan and thiotepa, survived transplantation and showed complete remission (3/3) . within the hlh cohort the overall survival was 72.7% (8/11). fatalities (n=3) included two patients from the myeloablative group and one ric-treated patient. the cause of death were progressive disease activity during the conditioning phase, leading to multi-organ failure on day +15 despite immunosuppressive treatment (n=1) and complicated cerebral seizures followed by lung haemorrhage, possibly due to aspiration pneumonia with evidence of enterococcus faecium, resulting in septic multi-organ failure on day +4 (n=1). a third hlh patient developed a sudden cerebral edema and ensuing respiratory insufficiency on day +4. whether this was caused by acute neurotoxic damage by fludarabine or a consequence of relapsed hlh could not be conclusively specified. none of our patients suffered from transplant failure or nonengraftment. there was neither severe acute gvhd (iii-iv) nor chronic gvhd observed in this cohort.conclusions: primary hlh and high-risk lch are lifethreatening medical conditions needing rapid allohsct. ric regimens are well-tolerated and sufficient for proper engraftment and disease clearance. disclosure: the authors have no conflicts of interest to disclose. abstract withdrawn. thrombocytopenia following allo-sct concomitant to stem cell boosts. steroid refractoriness was defined as: progression after three days or no response after 5 days of steroid treatment. the median time from sct to the onset of agvhd was 22.5 d (range 9-122 d), and 11 d (range 3-40 d) from the onset of agvhd to the first msc infusion, respectively.the majority of our patients (n=5) suffered from a malignant disease and received a graft from a matched unrelated donor (n=6), while one patient had a haploidentical donor. gvhd prophylaxis was performed in all patients except the patient with the haplo-identical graft. all patients with agvhd were treated with steroids and the patient with thrombocytopenia required regularly transfusions and romiplostin therapy. the median msc dose was 1.7x10 6 cells/kg bw (minimum 1.1 x10 6 ; maximum 3 x10 6 ). three patients received 2 msc doses, two patients 4, one patient 5 and another 7 doses.results: at the time point of agvhd manifestation and msc application, two patients had cmv reactivation, one patient adenovirus infection and one patient ebvreactivation. by day 28, 5/6 agvhd patients responded to msc administration: 3 with complete response and 2 with partial response. at the last follow-up (median: 4.42 months, range 0,82-12.06 months), 4 of 6 patients were alive without acute or chronic gvhd. one patient died soon after msc treatment with no obvious response in the course of a systemic hyperinflammation syndrome. the other patient although complete responder to msc-ffm developed fatal adenovirus sepsis. this based on the profound tcell depletion induced by concomitant application of steroids. the overall survival probability at six month was 66.7%. no acute side effects occurred after msc infusions. the previously mentioned patient suffering from thrombocytopenia did not need any further transfusions after receiving 2 doses mscs combined with stem cell boosts while continuing romiplostin application.conclusions: our data confirm excellent tolerability and high efficacy of the licensed off-the-shelf msc preparation "msc-ffm" in pediatric steroid-refractory agvhd. in our center, current treatment algorithms have escalated "msc-ffm" to the second line, i.e. immediately after steroid refractoriness has been established. besides immunoregulatory properties, this product might facilitate hematopoietic stem cell engraftment.disclosure: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) expression of ccr4 modulates migrational properties of in vitro expanded murine regulatory t cells laura m. moser 1,2 , ulrike tischler 1 , christin riegel 1 , julia minderjahn 1 , rüdiger eder 1 , jaqueline dirmeier 1 , isabel zimmermann 1 , evelyn röseler 1 , petra hoffmann 1,3 , matthias edinger 1, 3 background: hematopoietic stem cell transplantation (hsct) as it is carried out successfully at other genetic instability syndromes seems to be an encouraging opportunity for a curative therapy to restore immunity and prevent the development of hematologic malignancies in ataxiatelangiectasia (a-t). however, experience in the conditioning regimen is limited and no transplantation strategy for a-t patients exists, especially in an allogeneic setting. conditioning regimen and donor selection are critical factors in the clinical setting of hsct and incur substantial risks, especially in a-t. the aim of this study was (1) to evaluate whether different approaches of hsct including allogeneic hematopoietic hsct are feasible in regard to graft versus host response (gvhd) and sufficient concerning immune reconstitution (2) and to de-escalate the toxic effects of the conditioning regimen by reducing the dose of cyclophosphamide (cp).methods: t cells from syngeneic, allogeneic and haploidentical donor mice were used to determine gvhd induced t cell proliferation in a mixed lymphocyte reaction (mlr). atm-deficient mice were treated with cp or reduced cp in combination with fludarabine (flu) and transplanted with 5x10 6 cd90.2 depleted bone marrow donor cells from 129/svev gfp-transfected wildtype mice (syngeneic) or from mice of the f1 generation of 129/svev wildtype mice and c57bl/6 mice (haploidentical), or from c57bl/6 mice (allogeneic). tracking of gfp-positive donor derived cells was performed using flow cytometry and atm pcr. oxidative stress and damage were detected by a rt profiler pcr array and 8-hydroxy-2′deoxyguanosine.results: mlr resulted in an increased proliferation of allogeneic donor t cells compared to syngeneic and haploidentical donor cells. response was lower on dendritic cells isolated from atm-deficient mice compared to wildtype controls. in vivo results showed the restoration of t cells in atm-deficient mice accompanied by a prolonged life span and through reduction of thymic tumors. however, allogeneic stem cell transplantation was accompanied with a higher mortality rate, compared to the haploidentical and syngeneic setting. decreased antioxidative capacity and a higher dna-damage were seen in cp treated atmdeficient mice.conclusions: haploidentical hsct seems to be a feasible strategy for a-t. our data provided further evidence for the high sensitivity against ros-inducing agents in a-t and this fact needs to be taken into consideration in the choice of the host-conditioning strategy.disclosure: nothing to declare this research received funding from action for a-t charity (14gou01) background: prognosis of pediatric patients and young adults suffering from refractory or high-risk soft tissue sarcomas remains poor with limited improvement over the last decades despite multimodal treatment strategies. replacing the immune system by an allogeneic hematopoietic stem cell transplantation (hsct) in refractory solid malignancies has been proposed as a potentially curative therapy due to its presumable graft versus tumor effect. based on this concept we additionally performed consecutive donor-derived lymphocyte infusions in allogeneic hsct-patients with refractory or relapsed solid malignancy to further increase anti-tumor efficacy post-transplant.methods: pediatric patients with relapsed and/or refractory cancers or with delayed responses to the respective induction therapies were offered donorderived cellular therapies after immune system replacement by an allogeneic hsct. cellular immunotherapies comprised of donor lymphocyte infusions (dli), natural killer (nk) cell or cytokine-induced killer (cik) cell infusions generated from the original stem cell donors. allogeneic nk cells were generated from unstimulated leukapheresis by a two-step purification procedure using immunomagnetic cd3 t cell depletion, followed by nk cell enrichment (cd56+) with or without in vitro il-2 stimulation and expansion for 9-14 days. for cik cell generation peripheral blood mononuclear cells were isolated and activated by in vitro cytokine stimulation (inf-γ, anti-cd3, il-2 and il-15) an expanded over 10-12 days. expanded cik cells represented a heterogeneous population of polyclonal t cells with in part shared phenotypic and functional properties of nk cells.results: between october 1 st 2003 and january 1 st 2014 a total of 18 patients (eight patients with rhabdomyosarcoma, one patient with synovial sarcoma, two patients with ewing sarcoma, five patients with neuroblastoma, one patient with hepatoblastoma, and one patient with nasopharynx carcinoma) were enrolled. seven of 18 (39%) patients in this study had achieved complete remission (cr) before hsct while another 7 of 18 (39%) patients had obtained at least very good partial or partial response (vgpr or pr). dli was applied in 5 patients, nk cell treatment was offered to another 6 patients, while cik cell therapy was given to 7 patients.1.5-year probabilities of overall survival (os) and progression-free survival (pfs) were 16.2% and 15.9% for all patients with a median follow up of 8.5 months (range, 1.5-115.1 months). patients in cr at the time of immune cell therapy (it) showed estimated 1.5-year os and pfs of 49.6% and 42.7%, respectively. the majority of patients relapsed and ultimately succumbed to their diseases with two of 18 (11%) patients still being alive 9.6 and 9.3 years after it. cumulative incidence of relapse was 79.8% at 1.5 years. t cell engraftment and immune reconstitution (ir) was improved by it, and correlated with treatment response. however, two of 18 heavily pretreated patients (11%) died due to cumulative treatment-related mortality (trm). furthermore, acute graft-versus-host-disease (agvhd) occurred in 9 of 18 patients (50%) with agvhd grade i-ii observed in 6 (33%) and agvhd grade iii seen in three (17%) patients.conclusions: altogether, the results of this study indicate that allogeneic donor-derived cellular therapy at its current state offers curative benefit in selected refractory childhood cancers but warrants further improvement. background: allogeneic stem cell transplantation (allo-sct) is the only curative treatment option for a variety of nonmalignant diseases. the success of allo-sct is strongly associated with rapid and sustained immune reconstitution (ir). we analyzed the ir in patients who received an allo-sct for nonmalignant diseases.ir was assessed on days +30, +60, +90, +180 and +365 after allo-sct analyzing leukocytes, lymphocytes, monocytes, cd3 + t cells, cd3 + cd4 + t helper cells, cd3 + cd8 + cytotoxic t cells, cd3 -cd56 + natural killer (nk) cells and cd19 + b cells.methods: we analyzed ir-data of 116 consecutive patients receiving allo-scts between september 2001 and november 2018. indications of allo-sct were hereditary anemias (thalassemia, sickle cell disease, diamond blackfan anemia; ha, n=35, 30%), inherited bone marrow failure syndrome (fanconi anemia, severe aplastic anemia, others; bmfs, n=34, 29%), hemophagocytic lymphohistiocytosis (hlh, n=12, 10%), immunodeficiency (id, n=28, 24%) and metabolic disorders (n=7, 6%). the median age at allo-sct was 7 years (range, 0.2 -26) and at diagnosis 0.8 years (range, -0.5 -17.8).patients received 1 st allo-sct from msd/mfd (n=53, 46%), mud (n=42, 36%), haploidentical mismatch family donors (n=15, 13%) and mmud (n=6, 5%). conditioning regimens were busulphan-based (n=14, 12%), treosulphan-based (n=45, 39%), flu-mel-thio (n=35, 30%) or others (n=22, 19%). graft sources were bm (n=84, 72%) and pbsc (n=32, 28%).in order to consider the age-dependency of ir we normalized each absolute cell count with its corresponding age-specific expected mean values. (huenecke et al.; eur j haematol; 2008) results: the ir pattern was similar between the ha and bmfs groups. the cd4 + t cells were recovering slightly faster in ha patients compared to the recovery of bmfs patients.monocytes and nk cells proliferate very fast. at day +60 half of the patients already reached their respective monocytes reference value except for id patients, who reached 80% of the reference value at the end of the first year.cd3 + cd8 + cytotoxic t cells recovered significantly faster in patients with hematologic diseases compared to patients with hlh (p< 0.001) and id (p=0.047). half of the patients reached the reference value of cytotoxic t cells in the hematologic diseases group at day +365. by far inferior was the ir for the hlh patients. in this group only 50% of the patients reached the 45 th percentile of the healthy age-matched reference. in the id group 50% of all patients reached the 87 th percentile of the age-matched reference group at day +365.b cells are profoundly decreased at day +30 in all groups. however, the longitudinal expansion of b cells was significantly lower both in the id group and hlh group compared to the hematologic diseases group. at day +365 fifty percent of patients with id, hlh and hematologic diseases reached the 37 th percentile, 52 th percentile and the 100 th percentile, respectively (p< 0.001; p=0.027).conclusions: allo-sct is the only curative option for patients with nonmalignant diseases. ir is dynamic and revealed a complex diversity pattern with regard to the original disease. to investigate factors influencing ir after allo-sct is crucial to improve outcome of these patients.disclosure: nothing to declare. allogeneic hematopoietic stem cell transplantation in patients with myelodysplastic syndrome of relatively high-risk groups: curative effect analysis and optimal timing selection results: among the 135 patients, 133 patients were transplanted successfully. the 3-year overall survival (os) rate and disease-free survival (dfs) rate was 76.8% ±4.2% and 76.7%±4.3% respectively. the 3-year cumulative relapse rate (rr) and the non-relapse mortality (nrm) rate was 13.9%±0.1% and 18.4%±0.1% respectively. the incidence of grade ii-iv acute graft versus host disease (agvhd) was 25.2%±0.1%. for the patients who survived more than 100 days after allo-hct, the 2 years cumulative incidence of chronic graft versus host disease (cgvhd) was 33.4%±0.2%. univariate analysis showed that the hematopoietic cell transplantation comorbidity index(hct-ci) and grade iii-iv agvhd are the high risk factors for os(81.2±4.5% vs 62.8±10%, p=0.027 and 81.9±4.4% vs 51.1±11.8%,p <0.001). multivariate analysis demonstrated that grade iii-iv agvhd and hct-ci are independent risk factors for os(hr=6.206, p <0.001, 95% ci:2.529~15.288 and hr=2.651,p=0.025, 95%ci:1.127~6.232). chemotherapy before transplantation did not improve os or dfs for patients with bone marrow blast cells more than 10% at the time of diagnosis.conclusions: allo-hsct is an effective treatment for mds patients of relatively high-risk groups. the physical condition of the patients and occurrence of agvhd are independent risk factors. for intermediate and high risk ipss-r mds patients, transplantation before the disease progressed into very high risk can achieve better prognosis, high-risk group can still benefit from rebound gvhd after cni tapering which was promptly responsive to treatment steroids, fk506 and ecp. aa one year after sct 12/13 patients were without gvhd and off all immunosuppression while one single patient was still on taper of immunosuppressant after rebound acute gvhd. no chronic gvhd occurred. sctrelated toxicity was common with mucositis in all patients (who grade 4: n=5), elevated liver enzymes (≥grade 3: n=9) and impaired renal function (gfr 40-60ml/min: n=8). five patients developed neurologic symptoms (seizures n=2, pres n=1, pseudotumor cerebri n=2) which all resolved without sequelae. overall survival and transfusion-free survival was 100% with a median observation time of 5 (2-8) years.conclusions: 4. treosulfan-based conditioning followed by sct from hla-matched related or unrelated donors represents a highly efficacious treatment approach for children, adolescents and young adults with tdt and exhibits an acceptable but not negligible safety profile. an individual risk-benefit assessment incorporating hazards such as secondary graft failure, gvhd and long-term toxicity including infertility and 2 nd malignancy has to be executed in the informed consent process for every patient and his/her guardians.disclosure: "nothing to declare" p740 abstract already published. early iron chelation with deferasirox might prevent relapse after busulfan plus fludarabine and atg as a myeloablative conditioning for hla-identical sibling allogeneic hct in aml results: we show an excellent concordance between chimerism assessment on bio-rad and 3 d platforms over the complete range of mixture ratios (r 2 >0,9) and proof the lower detection limit (0,1 %) compared to str-pcr.conclusions: our results promote the transfer of the established mentype assay to a more diverse instrument portfolio. that will allow to implement the analysis of patient and donor hematopoiesis by digital pcr methods in our lab.disclosure background: with the immense progress in therapeutic regimens in pediatric oncology and stem cell transplantation the survivor rates increased up to 80%. at the same time the field of reproductive medicine has achieved substantial advances to offer potential options for fertility preservation. therefore it is of great importance to implement fertility counseling and fertility preserving (fp) procedures for patients facing gonadotoxic therapy. in the report on the expert meeting of the paediatric diseases working party (pdwp) of the ebmt in 2017 counseling related to fp opportunities should be offered to each patient receiving stem cell transplantation (sct), as part of the pre-sct workup by a dedicated and trained team. yet there many medical, ethical, structural and financial issues to consider and overcome. we describe the setting up process to enable fertility counseling for all children with newly diagnosed cancer or those facing stem cell transplantation for malignant and nonmalignant diseases in our department of pediatric oncology and immunology/stem cell transplantation.methods: at our tertiary care center we assembled a multidisciplinary team involved in fertility preservation (pediatric hematology/oncology, pediatric immunology/ stem cell transplantation, reproductive medicine, andrology, psychology and pediatric surgery). we developed an internal grading system for recommendations regarding fertility preservation based on the current recommendations for fertility preservation of leading societies in this field like ebmt, gpoh and dggg. it is important to find a consensus within the team for the counseling to ensure reliable counseling. the third step is to implement structures needed for fertility counseling and performance of invasive procedures including legal aspects (amg). a detailed description of this process is given.results: after setting up structures for the counseling process we counseled 140 oncology and stem cell transplant patients (0-18 years) between january 2017 and may 2018. we analysed data of the patients including age subgroups and disease entities and the results of the counseling process. for those patients undergoing stem cell transplantation the risk of gonadotoxicity is very high, therefore even the very young children underwent fertility preserving procedures in alignment with our recommendations if they suffered from a nonmalignant disease. currently we discourage tissue preserving in malignant systemic disease due to possible contamination with malignant cells. postpubertal female patients were more likely to undergo invasive procedures such as ovarian tissue cryopreservation in the case of oncological diseases, while sperm cryopreservation was recommended in all postpubertal male patients. overall a high percentage of the patients and their family followed our recommendations.conclusions: fertility preservation should be considered as a very important part of the treatment plan for newly diagnosed children and young adults with cancer and those facing stem cell transplantation. unfortunately there is still a great need for setting up structures in institutions taking care of these patients. in addition fertility preservation sadly lacks funding by health insurance in some countries. with the presentation of our experience and data we want to facilitate incorporation of fertility counseling in other pediatric care centers to provide counseling for pediatric patients in need for fertility preservation.disclosure: no conflict of interest regulatory t-cells (t reg ) have been shown to play a role not only in autoimmune diseases and solid organ transplantation but also in gvhd. several mouse models showed a decrease of gvhd incidence after t reg administration. the few clinical trials regarding the application of t reg for the treatment of gvhd are encouraging, however the data is limited. methods: patient: a 14-year-old boy underwent allogeneic sct for chronic myeloid leukemia refractory to imatinib, dasatinib and nilotinib treatment from his 10/10 hla identical brother. freshly derived unmanipulated bone marrow was transplanted after conditioning with of fludarabine (40 mg/m²/d, day -7 to -4), thiotepa (2x 5mg/ kg, day -3) and melphalan (160 mg/m², day -2). cyclosporin a (csa) and mycophenolatmofetil (mmf) were used for gvhd prophylaxis. leukocyte regeneration (>1000/μl) was seen on day +15, granulocyte regeneration (>500/μl) on day +16 and thrombocyte regeneration (>50.000/μl) on day +21. on day +30 after sct he developed acute intestinal gvhd that exacerbated to grade iv°(bloody diarrhea, ileus) and did neither respond to steroids, nor to different immunosuppressive drugs such as cyclosporin, tacrolimus, sirolimus, mycophenolatemofetil and ruxolitinib. extracorporal photopheresis and the administration of immunmodulatory antibodies (adalimumab and tocilizumab) did not succeed either.results: by administration of low-dose interleukin-2 (il-2) in vivo induction of t reg was expected but did not succeed. finally antithymocyte globuline (atg, 20 mg/kg/ d) was administered on day +150 to +152 to eliminate the gvhd-triggering cells. hence, the gvhd declined to grade iii. finally, a decision was made to manufacture t reg from his stem cell donor. from an unstimulated leukapheresis t reg were selected by magnetic depletion of cd8 + t-cells and cd19 + b-cells followed by positive selection of cd25 + background: treatment of patients with transfusion dependent anemia like thalassemia major (tm), sickle cell disease (scd) and diamond-blackfan anemia (dba) has improved over the last decades. for the vast majority of patients, allogeneic hematopoietic stem cell transplantation (hsct) is the only available curative therapy. for a long time, hsct has only been performed from hla-identical sibling donors (msd) or matched family donors (mfd). however, approximately only 25-30 % of affected patients do have a matched sibling donor, therefore hsct from 10/ 10 (mud) and even 9/10 (mmud) matched unrelated donors has gained importance in recent years.methods: 36 patients (age range: 2-27 years) with scd (n=7), dba (n=6) or tm (n=23), receiving hsct from a msd, mfd, mud or mmud between 2010 and 2019 were included in our analysis. 23 patients received transplants from msd/mfd, 13 patients from mud/ mmud. 7 patients were identical for hla-a, b, cw, drb1 and dqb1, 6 patients shared only 9/10 genes. we analyzed extended haplotypes including drb3, drb4, drb5 and dpb1 for all patients with thalassemia. 7 pairs showed non-permissive dpb1 mismatch and 1 pair mismatch for dpb1 and drb4.results: median time for granulocyte recovery was 20 days in patients transplanted from msd/mfd and 22 days in patients transplanted from mud/mmud. platelet recovery was reached after 22 days after hsct from msd/mfd and 24 days after hsct from mud/ mmud. 28/36 (80%) patients showed complete donor chimerism in all controls. 5/36 (13%) patients showed low level mixed chimerism up to 20% during follow up. 1 patient died shortly after hsct, 1 patient showed slowly increasing mixed chimerism and finally developed autologous recovery and one patient rejected the graft.cumulative incidence of grade ii-iv acute graft-versushost disease (agvhd) of mud/mmud was 15,2%, whereas only 2 cases of agvhd grade i occurred in patients transplanted from msd/mfd. as 1 patient rejected the graft from a hla-identical parent, 1 patient transplanted from a hla-identical grandparent developed autologous recovery after 1 year and 1 patient transplanted from a mud lost the graft due to hemophagocytosis, the probability of event-free survival was 89,5 % after hsct from msd/mfd and 83,9 % from mud/mmud.altogether 34/36 patients (94,4 %) are alive and transfusion-independent with complete donor chimerism two years after hsct; resulting in an overall survival probability of 93,1%. in contrast, overall survival probability was 100% in the group of patients transplanted from msd/mfd and 80,1 % in patients transplanted from mud/ mmud after 2 years.there were 2 patients with thalassemia (6,9%) who died from transplantation-related causes. the first patient died 13 days after hsct from a mmud due to candida sepsis with pulseless electrical activity resulting from cardiac iron overload. the second patient died 5 months after hsct from a mud due to graft failure.conclusions: hsct from mud and mmud is a feasible therapy option for patients with transfusion dependent anemia. nevertheless, it should be noted that iron overload can cause severe complications; therefore, measurement of liver and heart iron concentration through mri prior to hsct as well as phlebotomy after transplantation are advisable.disclosure: novartis (consultancy: included expert testimony, speaker bureau, honoraria), medac (research funding, patents and royalties), riemser (research funding), neovii (research funding), amgen (honoraria) background: the therapeutic options for patients with hodgkin´s disease who relapse after first high-dose chemotherapy with autologous stem cell (1 st asct) support are limited. allogeneic stem cell transplantation in this setting is associated with a high level of transplant-dependent mortality rates in excess of 50-65%. new agent, such as brentuximab vedotin, have been approved for the treatment of these patients, however, their efficacy to provide longterm control or cure is still unknown. a second autologous stem cell (2 nd asct) has historically been considered as an option only in a small group of patients so the published experience is scarce. we report our institution´s experience with second autologous transplants in this patient population.methods: we evaluated the outcome of 16 adult patients (7 (44%) female and 9 (56%) male), who received an 2 nd asct between 10/2013 and 08/2018. planned tandem asct were excluded. the median age at 2 nd asct was 32 years (range 21-54), 15 (94%) patients had a karnofsky performance score ≥80%. 12 (75%) patients were in complete remission (cr) and 4 (25%) patients were in partial remission (pr) at day 100 after 1 st asct. seven (44%) relapses within 12 months after 1 st asct. patients received a median of 1 (0-3) treatment lines between 1 st asct and 2 nd asct. only 4 (25%) patients received brentuximab vedotin and none of the patients in our series received checkpoint inhibitors as salvage after 1 st asct. the median interval from 1 st asct to relapse/progression was12,9 months (range 2,9-133,3). the median interval from relapse/progression to 2 nd asct was 9,2 months (range 1,4-46,6). all patients received beam as the conditioning regimen for 1 st asct, and beeam as the conditioning regimen for 2 nd asct.results: the median time to neutrophil recovery (>0.5x10 9 /l) after 2 nd asct were 10 days (range 8-17). best response at day 100 following 2 nd asct included cr in 12 (75%) patients and pr in 3 (19%); 1 (6%) had stable disease. 3 (19%) patients received brentuximab vedotin and none of the patients received checkpoint inhibitors after 2 nd asct. 14 (87,5%) patients are currently alive, with a median follow-up 49,8 months (range 4,2-154,2).2 patient died after 2 nd asct. causes of death were hl progression. the 5-year overall survival was 86%.conclusions: the second asct in patients with a longterm response after the first asct may be the optimal therapeutic option, the effectiveness of which can be enhanced by using new drugs, such as brentuximab vedotin, at all stages of treatment.disclosure: nothing to declare effectiveness of chemo-g-csf protocols for mobilization of peripheral stem cells in patients with non-hodgkin lymphomas and hodgkin disease-single center experienceilina micheva 1 , stela dimitrova 1 , vladimir gerov 1 , trifon chervenkov 2 , liana gercheva 1 , igor reznik 1background: high-dose chemotherapy and autologous stem cell transplantation (asct) play an important role in achieving long-term remission in patients with non-hodgkin lymphoma (nhl) and hodgkin disease (hd). granulocyte colony stimulating factor (g-csf) combined with high-dose chemotherapy is a frequently used mobilization approach; however, the optimal mobilization strategy has not been determined.the objective of the study was to analyze the mobilizing potential of different regimens used for the collection of peripheral stem cells in patients with relapsed or refractory (r/r) nhl and hd. methods: we retrospectively analyzed 40 patients with r/r nhl and hd undergoing stem cell collection after chemo-mobilization in the transplant unit at the university hospital, varna. patients were mobilized after dhap letermovir is promising, even as a therapeutic agent. more paediatric data are urgently needed.disclosure: nothing to declare p752 development of paroxysmal nocturnal hemoglobinuria in a patient after mudallohsct due to jak2v617fpositive myelofibrosis-a case of successful treatment with the second transplantation from another donor agnieszka tomaszewska 1 , barbara nasiłowska-adamska 2 , iwona solarska 2 , kazimierz hałaburda 2background: paroxysmal nocturnal hemoglobinuria (pnh) is a very rare disease associated with pig-a gene mutations in hematopoietic stem cells. there are only single case reports on evolving myeloproliferative diseases to pnh in the literature. there is no data concerning development of pnh de novo after allogeneic hematopoietic stem cell transplantation. in our report we describe a patient with recurrence of jak2v617-positive myelofibrosis 5 years after matched unrelated donor allogeneic hematopoietic stem cell transplantation (mudallohsct) with simultaneous development of clinically significant pnh. a 51 year-old-man with a history of mudal-lohsct in may 2011 due to jak2v617-positive myelofibrosis secondary to essential thrombocythemia was admitted to our department 5 years later with mild anemia (hb-11.0 g/dl) and elevated lactate dehydrogenase (1400 u/l). during last 5 years he remained in complete remission of myelofibrosis with jak2v617 mutation negativisation and 100% donor chimerism. suspecting disease recurrence we performed trephine biopsy confirming myelofibrosis (mf2/mf3) with heterozygous jak2v617 mutation and in flow cytometry analysis of bone marrow we identified cell membrane defect in myeloid line (loss of cd66c). we decided to perform detailed diagnostic tests on pnh -multiparametric flow cytometry of peripheral blood revealed 89% granulocytes and 15% red blood cells with loss of gpi-anchored proteins -pnh clone. these results corresponded with donor chimerism -it was only 20% of donor dna in bone marrow and 33% in blood tests. molecular analysis didn't revealed any mutations in genes: calr, asxl1 and mpl. finally the diagnosis of myelofibrosis recurrence after mudallohsct with presence of pnh clone was established. the therapy with eculizumab was unreachable. so the second allohsct from another matched unrelated donor after fludarabinemelphalan-thymoglobuline-tbi 200 cgy conditioning was performed on 21.10.2016. we didn't observe any complications of this procedure, engraftment was slightly delayed: anc>0.5g/l on the 29 day and plt>50g/l on the 50 day.results: at present, more than 2 years after the second mudallopbsct, the patient remains in a very good condition with 100% of the second donor chimerism and without any features of pnh (clone is undetectable) and myelofibrosis.conclusions: presented case is the first in the literature well documented myelofibrosis recurrence after mudal-lohsct with concurrently development of clinically significant paroxysmal nocturnal hemoglobinuria. the second mudallohsct from another donor was safe and successful treatment strategy in this situation.disclosure: nothing to declare. abstract already published. cutaneous refractory t-cell lymphoma treated with allogeneic hematopoietic stem cell transplantationmarcia silva 1 , ercole orlando 1 , maria claudia moreira 1 , simone lermontov 1 , simone maradei 1 , yung gonzaga 1 , leonardo arcuri 1 , renato araujo 1 , decio lerner 1 1 instituto nacional de cancer, cemo, rio de janeiro, brazilbackground: folliculotropic mycosis fungoides (fmf) is an aggressive clinical course variant of cutaneous t-cell lymphoma (ctcl) -classic mycosis fungoides (mf) 1 , with distinct clinical and pathological characteristics, and it is less responsive to skin-directed therapies. for diseases in advanced stages, chemotherapy, autologous hematopoietic stem cell transplantation (hsct) or immunomodulator drugs may provide remissions with limited duration and the treatment remains substantially palliative 2,3 . these dismal results have induced to explore the therapeutical approach with allogeneic hematopoietic stem cell transplantation (hsct) in such patients. early studies have shown encouraging results also in patients with advanced disease, suggesting a major therapeutical role played by the graft versus lymphoma (gvl) effect 4,5,6 . methods: this is a case report of the use of allogeneic hsct as a potential cure for cutaneous refractory t-cell lymphoma type folliculotropic mycosis fungoides .results: case presentation : a 31-year-old male patient with refractory subtype b fmf t-cell lymphoma 7 , diagnosed in 2007, clinically characterized by exfoliative erythroderma, widespread plaques on the trunk and limbs, solitary tumor on the right shoulder, pruritus and bilateral key: cord-005460-ezrn8cva authors: nan title: physicians – poster session date: 2017-07-28 journal: bone marrow transplant doi: 10.1038/bmt.2017.134 sha: doc_id: 5460 cord_uid: ezrn8cva nan hematopoietic stem cell transplant unit, hematology department, hospital universitario de donostia, donostia/san sebastián and 2 informatics and automatics department, university of salamanca, spain the immature platelet fraction (% ipf) is a relatively new parameter that measures young (reticulated) platelets in peripheral blood (pb). ips rise as bone-marrow (bm) production of platelets increases. several clinical utilities of the %ipf have been already proved, as the treatment response monitoring in aplastic anemia or immune thrombocytopenic purpura. in this study, we aimed to found if ip measurement might be useful during the grafting phase of hsct. this study includes 141 patients who underwent allo-hsct in our center during the last 2.5 years. 79 were male (56%) and 62 female (44%). median age was 52 years (range: 7-69). baseline diseases were: acute leukemias (78), lymphoproliferative disorders (22), myelodysplastic syndromes (15), chronic myeloproliferative diseases (12), multiple myeloma (8) and bone marrow failures (6) . donor was unrelated in 79 cases, and related in 62 (including 23 haplo-identical). conditioning regimen was: busulphan-based (93), melphalan-based (17), tbi-based (17) and others (14) . progenitors source was pb in 128, and bm in 13. platelet count, %ipf and absolute ip count (aipc) from day +1 to the day of stable graft were analyzed. 52.4% patients reached plat ⩾ 20 000/mcl at day +14, 82.1% at day +21 and 86.9% at day +28. median first day of plat ⩾ 20 000/mcl was day +14 (range: . median %ipf was 2.6% (range: 0-15.4), 2.5% (range: 0-28.4) and 3.65% (range: 0-15.3) at days +9, +10 and +11, respectively. median aipc was 292/mcl (range: 0-2835), 336 (range: 0-2840) and 504 (range: 0-3660) at days +9, +10 and +11, respectively. among the time points analyzed, aipc at day +11 showed the best positive correlation with platelets counts at day +14 (r = 0.72). interestingly, patients with lower aipc at day +11 showed a delayed platelet graft (see table 1 ). contrarily, patients with higher aipc at day +11 had an earlier platelet graft. absolute immature platelet count before the graft seems to predict the precocity of the platelet graft for the majority of patients undergoing allo-hsct. this finding might help physicians for the patient management (anticipation of hospital discharge and so on). disclosure of conflict of interest: none. [p001] p002 analysis of genetic polymorphism for cardiovascular diseases (cvd) in placental and maternal blood in hypertension and hypercholesterolemia c khalil 1 , a azar 1 and a ibrahim 1,2 1 reviva stem cell research and application center, lebanese university, middle east institute of health hospital and 2 faculty of medical sciences, lebanese university, lebanon cardiovascular diseases are the world's leading cause of death representing 30% of the total global mortality. the genetic polymorphism of the 12 cvd genes, especially the ace: angiotensin converting enzyme gene risky alleles (ins/del) which are associated with a high and inappropriate level of ace can be considered as a genetic model in the development of hypertension and its complications in cvd. we evaluated the mutation impact of the 12 cvd genes in the lebanese population, based on 40 samples derived from placental blood (pb) and 40 samples derived from peripheral blood of postpartum mothers. adult females (age ⩽ 35 years) were divided (n = 20 per group) into group1 (normotensive, normocholesterolemia: nn), and group 2 (hypertension, hypercholesterolemia: hh). buffy coat were extracted from the 40 pb. all tests on pb and maternal blood were done by using the test strip assay to identify the most relevant genetic variations to estimate the risk for cvd. the presence of a double mutation (ins+/del+) related to the ace gene in the hh group was 75%. the presence of a single mutation (ins − /del+) was only associated to the hh by 25%. (ins − /del − ) was absent in 100% of the pb and nn. despite the presence of double mutation ins/del for cvd in maternal blood, pb was free of this mutation. therefore, beyond genetic mutations, other factors can play a major role in the occurrence of cvd. disclosure of conflict of interest: none. s124 b e mt automated red blood cell depletion in abo incompatible grafts in the pediatric setting c del fante, l scudeller, s recupero, g viarengo, f compagno, m zecca and c perotti fondazione irccs policlinico san matteo red blood cell (rbc) depletion by apheresis is employed to reduce the rbc content from abo major or bidirectional mismatch bone marrow (bm) grafts mainly to avoid severe haemolysis 1. rbc depletion results in a significant volume reduction (due to both rbc and plasma depletion) and buffy coat concentration 2.3. in pediatric setting, both rbc depletion and volume reduction before transplantation or cryopreservation can avoid fluid overload and renal impairment, especially in low/very low body weight recipients. the aim of this study was to evaluate the quality of the graft and immediate post infusion complications in rbc depleted bm in major and minor abo mismatch recipients using an automated device. patients and methods: bm aspirates for transplantation in pediatric setting were processed at our centre using the spectra optia (terumo bct) automated device. the initial collection preference was set at level 30 and then was adjusted in order to maintain a haematocrit of 5% (colorgram) in the collection bag. flow speed was set at 120ml/min for 10 cycles. mean recipients' body weight was 31 kg (range:11-72). pre and post procedure bm bag volume, hct%, mononuclear cells (mncs) count, (including b and t lymphocytes), cd34+ cell and cell viability were calculated. moreover, post procedure rbc volume and procedure time were registered. on the patient's side, post infusion complications (renal impairment, fluid overload, fever and haemolitic reactions) and time to engraftment were evaluated. results: a total of 20 rbc depletion procedures were consecutively performed on 20 bm grafts (13 major and 7 minor abo incompatibility, 16 mud and 4 related donors). data about pre and post procedure graft composition are reported in table 1. mean time to engraftment for pmn was 22.6 days (range:17-34) and for plt was 33.5 (range: 21-43). pre and post-procedure cell viability were always 497%. mean procedure time was 80.6 minutes (range:59-115). no bacterial or fungal contamination was detected. no infusion complications were recorded. one graft failure was observed. conclusions the spectra optia automated system is efficient in rbc depletion of abo mismatched grafts, permitting an effective volume reduction and an excellent mncs and cd34+ cell recovery in pediatric setting. automated rbc depletion may be proposed in low/very low body weight recipients both in abo major and minor incompatibility setting to minimize graft infusion side effects. building up a stem cell transplantation program in an emergent country, in the public setting, with limited economic resources, is not an easy work to do. international cooperation may be essential for the development of the program, in training, technological support and implementation of international guidelines. after 20 years, we show an experience of international cooperation between a highly developed center in france (institut paoli calmettes, marseille) and the stem cell transplantation department of hospital maciel, a public assistance service in montevideo, uruguay. fourteen persons between doctors and nurses have been trained in france in stem cell collection and processing, patient's clinical handling, nursing, outpatients care and quality management. french missions of experts have been also received in hospital maciel every year since 2002 for in situ human resources training. in last 5 years we developed a program for optimizing transplant results and reducing transplant related mortality (trm), based on several measures: improvement of patients selection, applying the sorror comorbidy index; adjustment of conditioning regimen doses, in order to reduce toxicity; development of a program to improve interaction with the intensive care unit; protocolization of the standard proceedings treatments; and initiating a program of quality and safety at the national institute of quality of uruguay inacal. 456 adult patients have been treated with autologous (asct) (347) or allogeneic (allosct) (109) sct, with hematological malignancies. different modalities of allosct have been included progressively, becoming the only center accredited by the national regulation authorities (fnr) to perform unrelated donor sct and the haploidentical donor sct. this increased the proportion of allogeneic transplants from the historical 20% until 33% in last 2 years. regarding patients health coverage, 45% comes from the private assistance system and 55% from the public health system. the major indications are lymphoid malignancies and acute leukemia, for asct and allosct, respectively, showing the same trend than cibmtr. three-year overall survival (os) for acute myeloid leukemia after allosct is 61%. considering asct for diffuse large b cell lymphoma, 3 years os after autologous sct is 82% and 52% for chemo sensitive and resistant disease, respectively. threeyears os after asct for hodgkin disease is 87 and 67% for sensitive and resistant disease, respectively. asct in multiple myeloma shows an os of 69 and 50% at 3 and 5 years, respectively. in trm, results during the last 5 years (after the described strategy) are shown in figure 1 . the development of the-program of continuous improvement in quality-and the impact of results was locally recognized by two annual prices from inacal in 2013 (bronze) and 2014 (silver) in the category ‛commitment to public service.' a successful mirna-223 and the level of proangiogenic cytokines: angiopoietin-1 (angpt1), matrix metalloproteinase-9 (mmp-9) and vascular endothelial growth factor (vegf) in patients with lymphoproliferative malignancies prior to autologous hematopoietic stem cell transplantation (hsct) and in early posttransplant period. twenty-four patients were enrolled to the study (11 f,13 m). the median (me) age was 57 years. the investigated group consisted of 19 multiple myeloma and 5 lymphoma patients. the plasma samples were collected on 4 time points: before chemotherapy-‛bc', on the day of hsct -‛0', 7 days after hsct-‛+7' and 14 days after hsct-‛+14. ' the cytokines were evaluated using elisa method, while mirna levels were estimated by qpcr method. the wilcoxon matched-pairs test was used to compare groups of dependent continuous variables: mirna's relative quantification (rq) levels or cytokines expression at two different time points. spearman rank correlation coefficient (r) was used to compare independent variables. we observed continuous decline of cytokines and mirnas level after conditioning treatment. the deepest decrease of expression was marked on ‛+7' day ( table 1) . we noticed a positive correlation between mirna-16, mirnacells in pbsc product. among the autologous transplanted patients between march 2011 and october 2016, we have selected 170 according to diagnosis, conditioning regimen and number of infused bags of cryopreserved pbsc. this group included 77 females and 93 males with median age of 56 (range: . most of them, 105 (61.76%), had multiple myeloma (mm), 41 (24.12%) had non-hodgkin's lymphoma (nhl) and 24 (14.12%) had hodgkin's disease (hd). after harvesting, cd34+ cell and leukocyte number in pbsc product were enumerated on flow cytometer and blood cell counter, respectively. pbsc were cryopreserved with 10% dymethil sulfoxide (dmso) and cell viability was measured with trypan blue exclusion test before and after adding dmso, and as well after thawing in water bath on 37°c. as a conditioning regimen for the mm patients, melphalan was used and for the nhl and hd patients we used beam regimen. all received one bag of cryopreserved pbsc and pegfilgrastim 6 mg on the first or the second post-transplant day. time to hematopoietic recovery was measured; for neutrophils 40.5 × 10 9 /l, leukocytes 41 × 10 9 /l and platelets 420 × 10 9 /l with at least 2 days without platelet transfusion. the median number of total leucocytes infused was 91.88 × 10 9 /l (range: 29.27-284.87 × 10 9 /l) of which cd34+ cells were 2-24.09 × 10 6 /kg of patient's body mass (median 4.75 × 10 6 /kg). pre-freezing cell viability before and after adding dmso was with a median of 100% (74.1-100) and 81, 75% (26.7-100), respectively, and post-thaw viability 57.37% (16.7-100) . the average time to engraftment was 9.8 days (6-26) for neutrophils, 10 days (6-27) for leucocytes and 10.8 days (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) for platelets. our results confirmed the known correlation between the number of infused cd34+ cells and engraftment of neutrophils (po 0.0001), leukocytes (p o0.0001) and platelets (p = 0.0005). we found inverse correlation between the infused leukocytes and cell viability with dmso (p = 0.0035) and after thawing (p = 0.0019). no correlation was found between prefreezing and post-thaw viability with hematopoietic recovery, and also between the cd34+ number and these viabilities. no differences were found considering patients' age, gender, diagnosis, conditioning regimen or day of applying pegfilgrastim. we can indirectly infer good survival of cd34+ cells and higher sensitivity of other nucleated cells to preparation of pbsc product. trypan blue exclusion assay, due to its inability to distinguish type of stained cells, is not relevant for cd34+ cells survival determination. disclosure of conflict of interest: none. chronic granulomatous disease (cgd) is a kind of primary immunodeficiency disorder of phagocytic cells which resulting in failure to kill a defined spectrum of bacteria and fungi and in concomitant chronic granulomatous inflammation. allogeneic hematopoietic stem cell transplantation is the only treatment proved to be potentially curative in cgd. unrelated umbilical cord blood (ucb) is increasingly used as an alternative to bone marrow. methods: unrelated ucbt was performed 14 consecutive cgd children at our center between 2015 and 2016. median age was 18.5 months (range: 5-143 months), median body weight was 10.3 kg (range: 8-34 kg). all patients received myeloablative conditioning regimen consisting of busulfan, fludarabine, cytarabine, cyclophosphamide and g-csf. all patients received tacrolimus as prophylaxis for graft-versus-host disease (gvhd). median nucleated cells were 9.2 × 10 7 /kg (range: 4.5-15.9 × 10 7 /kg), and median cd34+ cells were 3.0 × 10 5 /kg (range: 0.9-7.0 × 10 5 /kg). median follow-up time was 9.5 months (range: 6-23 months) results: 10 of 14 patients engrafted. median time to neutrophil engraftment was 30 days, and median time to platelet engraftment was 33.5 days. 13/14 patients were alive, and 10/14 had full donor engraftment. overall survival rate was 92.8%. disease-free survival was 71.4%. 2 of 14 patients had grades iii-iv acute gvhd. no patients developed chronic gvhd. only one patient died from multi-organ failure related to adenovirus infection. conclusion: unrelated ucbt should be considered as potential curative methods in children with cgd. myeloablative conditioning regimen has improved the engraftments of the ucb. disclosure of conflict of interest: none. reduced muscular mass and excess visceral fat in patients undergoing hsct are associated with higher mortality, longer hospitalization, longer use of immunosuppressive drugs, graftversus-host disease (gvhd) and comorbidities leading to shorter survival time. a recent study of patients undergoing allogeneic hsct showed that occurrence of enlarged areas of visceral and peripheral fat is inversely associated with the disease-free interval after the transplant. reduced muscle mass has also been associated with higher prevalence of chronic gvhd and low rates of success following allogeneic hsct. objectives: to investigate whether amount muscle mass and muscle strength (ms) as well as the amount of visceral fat (vf) of patients undergoing hsct would influence the duration of the engraftment time (en). we evaluated 14 hsct patients (⩾18 years) at hospital israelita albert einstein, são paulo, brazil, on their first day of hospitalization, before hsct. the thickness of the right femoral quadriceps muscle (rfq), measured at 6 cm from the top edge of the patella was measured using ultrasound (us) in b-mode. the dominant upper limb strength of the patients was evaluated by the hand grip test. the vf was measured in the abdominal region, by the thickness of the fat layer between the linea alba and the anterior wall of the aorta. most patients were women (57%) with a mean age of 50 years (±16 years) and 50% of our patients were elderly (⩾60 years). the haploidentical (57%) was the predominant hsct, autologous (36%) and allogeneic (7%). most patients were overweight, with body mass index (bmi) of 27 kg/m 2 (±4 kg/m 2 ). the average time en was 16 days (±6 days). rfq was 1.5 cm (±0.3 cm), ms was 31 kgf (±7.0 kgf) and the vf was 5.3 cm (±1.4 cm). patients with lower rfq had a longer engraftment time that was statistically significant as the negative correlation between rfq and en was rs = 0.8, p o0.05), independent of the age and the hsct type as analyzed by linear regression. no significant correlation between vf or ms with en was found. in this cohort of patients we found that longer engraftment times were significantly correlated to reduced muscle mass but no positive or negative correlation was found with superior limb muscular force or with the amount of visceral fat. disclosure of conflict of interest: none. 1 hematopoietic stem cell transplant unit, hematology department and 2 pharmacy department. university hospital of donostia. donostia/san sebastiań introduction: lymphocytes are the cells responsible for the cellular and humoral immunity and, consequently, critical for hematological patients. the aim of this study was to analyze the eventual conexion between lymphocyte recovery and survival (srv) after allogeneic hematopoietic stem cell transplantation (allo-hsct). patients and methods: we retrospectively analyzed data from 223 consecutive patients who underwent allo-transplants in our unit. in total, 126 patients were male (56.5%) and 97 female (43.5%). median age was 53 years old (range: . baseline disease was: acute leukemia (56.9%), lymphoma (11.2%), myelodysplastic syndrome (10.3%), chronic myelogenous leukemia (8.9%), multiple myeloma (4%), aplastic anemia (3.58%), chronic lymphocytic leukemia (3.13%) and others (1.79%). 55.1% of allo-hscts were from an unrelated donor, and 44.9% from a family donor (25% of them haplo-identical). the sc source was pbsc in 89.6%, and bm in 10,4%. a variety of conditioning regimens were employed, including: busulphan-based (69.5%), melphalan based (10.4%), tbi-based (9.86%) and others (9.86%). evolution of absolute lymphocyte counts (alc) and subpopulations during the first year after allo-hsct were analyzed. results: as shown in table 1 , alc decreased abruptly during conditioning therapy and recovered up to baseline at days +30 and +100; at day +365 median alc had clearly improved compared with admission values. median cd4+ cells were lower than 500/mcl in two thirds of pts at day +100 and in only one third at day +365. as shown in table 2 , we found a significant link between alc at day +30 and srv, as well as between cd4+ cells at day +100 and srv. in our series, immunity recovery was a late event for the majority of patients undergoing allo-hsct. in addition, in our experience, the precocity and quality of the alc and cd4+ recovery was clearly linked with long-term survival. disclosure of conflict of interest: none. although there is experimental evidence suggesting the presence of a common mesoderm cell as origin of both hematopoietic (hsc) and mesenchymal progenitor cells (msc) in an animal model, it is still controversial if durable engraftment of native donor-derived mscs without ex vivo treatment can occur in the recipient of allogeneic hsct. to assess the presence of donor-derived msc following hsct. between july 2015 and july 2016, a total of 33 recipients of hsct were analyzed for hsc and msc chimerism. eighteen patients received bm grafts (54%), 11 patients had peripheral blood as stem cell rescue (33%) and finally 3 patients had a cord blood transplantation (9%). patients received myeloablative (91%) or reduced intensity conditioning (9%) for malignant (91%) or nonmalignant disease (9%). bm aspirate cells were plated and expanded in α-mem with 10% human platelet lysate at 10 000 cells/cm 2 . after 5-7 days, nonadherent cells were removed, while the adherent cells were expanded until they reached confluence. after 2 weeks we quantified msc precursors as colony forming unit fibroblast (cfu-f). finally the amplified sequences were resolved by capillary electrophoresis (3500 ruo genetic analyzer, applied biosystems) and analyzed by comparing genotypes of bmt recipell detachment, nuclear dna was extracted (dneasy blood and tissue kit-applied biosystems) and specific polymorphic tandemly repeated regions (strs) were amplified by means of the polymerase chain reaction(pcr) following the specific manufacturers' instructions. (ampfℓstr identifile kit, applied biosystems following hsct (hsc and msc) to those of donors. we cultured 54 whole bm aspirates from patients following hsct with a median time of 244 day (range: 41-1606). cfu-f/1 × 10 6 growth was observed in a majority of bm the prevalence of human pegivirus in recipients of allogeneic hematopoietic stem cell olga koroleva 1 , e parovichnikova 1 , l kuzmina 1 , m drokov 1 , v vasilyeva 1 , z konova 1 , ekaterina mikhalcova 1 , d dubnyak 1 , n popova 1 , tamara romanova 2 , d tikhomirov 2 , t tupoleva 2 and v savchenko 1 1 bone marrow transplant department, national research center for hematology and 2 virology department, national research center for hematology human pegivirus (hpgv; previously named as gb virus c/hepatitis g virus) was discovered more than 20 years ago. it is an rna virus referred within the genus pegivirus of the family flaviviridae. hpgv rna is found in liver, spleen, bone marrow and peripheral blood mononuclear cell, including t-and b-lymphocytes, nk-cells and monocytes. despite of the fact that it is a molecular structure, mechanism of replication and transmission routes are very well understood but the clinical significance of hpgv is still not determined. recipients of allogeneic hematopoietic stem cell have a high risk infection of hpgv. it is known, that hpgv is a nonpathogenic virus, however, it may play a role in immunocompromised individuals. to investigate the frequency of occurrence of hpgv and its clinical significance in recipients of allogeneic hematopoietic stem cell. blood samples were obtained from 101 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-hsct): all n = 21, aml n = 53, mpn n = 7, cll n = 1, mm n = 1, lpd n = 4, aa n = 6, mds n = 8. a median of age was 33 years (19-64 years) . forty five patients were males and 56 patients were females. conditioning regimen was ric in 75 cases, mac in 26. bone marrow as a graft source was used in 68, pbsc-33. all patients received multiple transfusions of blood components at the previous stages of treatment. hpgv rna had been assayed by polymerase chain reaction real time (rt-pcr) on plasma samples before started pre-transplantation conditioning. despite the diagnosis incidence of hpgv was high 53.5% (rna-hpgv was positively in 54 patients). patients with piercings and tattoos had incidence of hpgv in 64% that was not statistically significant (p-0.5). hpgv is known as nonhepatotropic virus. in our study there was also no statistical reliability of specific changes in liver function test such as elevating the levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin due to the rna-hpgv. liver enlargement was also not statistically significant according to ultrasound scan results in patients infected with hpgv. we also analyzed the co-infection with hepatitis b and c virus. results are presented in table 1 . coinfection was not statistically significant. however, only one patient with hepatitis c was coinfected hpgv. leukocytes recovery median was 22 days (14-55). thrombocytes recovery median was 23 (11-82). the presence of rna-hpgv did not affect the recovery of peripheral blood cells in patients after allo-hsct. according to our study the frequency of hpgv infection in recipients of allogeneic bone marrow was quite high (53.5%), and it did not depend on the presence of any other hepatotropic viruses. clinical significance of hpgv infection in recipients of allogeneic hematopoietic stem cell has not been revealed, it is possible due to the short follow-up. it needs further clinical research. disclosure of conflict of interest: none. quantification of cd31+ recent thymic emigrants and t cell receptor excision circles (trecs) in umbilical cord blood transplanted patients v devlia 1,2 , j gridlestone 3 , m raymond 3 , s tulpule 1 , d tewari 1,2 , r hough 4 , c navarrete 3 , a madrigal 1,2 , b shaw 1,2 , r danby 1 and a saudemont 1,2 1 anthony nolan research institute, london, uk; 2 ucl cancer institute, london, uk; 3 nhsbt colindale, london, uk and 4 ucl, london, uk reconstitution of t lymphocytes is a limiting factor in the regeneration of an effective immune system in adult patients following hematopoietic stem cell transplantation. cd31 (pecam-1) is a transmembrane glycoprotein expressed on naive t-cells that have recently emigrated from the thymus into the periphery. in peripheral blood, cd31 + t lymphocytes also contain high numbers of t-cell receptor excision circles (trecs); excision loops of dna excised during t-cell receptor gene rearrangement during t cell maturation within the thymus (1) (2) (3) . however, quantification and correlation of cd31 and trec has not been formally investigated in patients following umbilical cord blood (cb) transplantation. quantification of cd31 and trecs post cb transplant will provide an insight into the immune reconstitution of t cells from the thymus. we therefore sought to measure cd31 and trecs in patients after cb transplant and assess whether these markers provided evidence of thymic recovery. we followed 67 adult patients (median age 52.9 years) who underwent cb transplant in the uk. patient samples were collected 28, 60, 100, 180, 365 and 730 days post transplant. using flow cytometry, we determined absolute counts of cd4+cd31 +cd45ra+ and cd8+cd31+cd45ra+, and quantified the copy numbers of trec genes in peripheral blood mononuclear cells (pbmcs) via real time pcr. results: at the six time points, the number of samples collected were the following: 44, 39, 37, 22, 24 and 14. in all of the samples, the overall median number of cd4+cd31+cd45ra+ was 29 cells/μl (range: 0-827 cells/μl). the median level of cd4+cd31+cd45ra+ cells increases from 16 to 50 cells/μl from day 28 to day 365. absolute counts of cd4+cd31+cd45ra+ at all of the six time points is 10-fold lower compared to healthy controls (median: 279 cells/μl, range: 105-523 cells/μl). the overall median number of cd8+cd31+cd45ra+ cells is 30 cells/μl (range: 0-2222 cells/μl). there is an increase in the median number of cd8+cd31+cd45ra+ cells between days 28 and 720 posttransplant from 2 to 92 cells/μl. however, the absolute median counts of cd8+cd31+cd45ra+ cells in patients are twofold lower, 2 years post transplant, compared to healthy controls (median: 252 cells/μl, range: 133-503 cells/μl). in the majority of the patient samples throughout all time points the trec gene copy numbers were undetected (n = 132). in a few patient samples (n = 9) trec gene copy numbers were quantified but with this limited sample size no correlations can be made between the absolute counts and trec gene copy numbers. our data suggests that cord blood transplant patients within the uk have reduced levels of cd4+cd31 introduction: common variable immunodeficiency (cvid) is a highly heterogeneous group of primary immunodeficiency characterized by defective antibody production, recurrent infections, lymphoproliferation and autoimmunity. autosomal recessive mutations in lrba, encoding lps-responsive beigelike anchor protein were first described as a cause of cvid-like disease in 2012. although hsct is accepted as a standard treatment modality for long-term resolution of severe primary immunodeficiencies, its role is less established in patients with lrba deficiency. patients and methods: whole exome sequencing of patient's genomic dna obtained prior to the hsct revealed a homozygous deletion in lrba (c.5527delt:p. c1843fs). immunological analyses including serum immunoglobulin levels, flow cytometry analyses of lymphocyte subsets, cytotoxicity/proliferation assays, vaccine responses were studied at several time points throughout the disease course, prior to and after hsct. a 14-year-old boy, born to consanguineous healthy parents of turkish origin became symptomatic at the age of 6 months. he hospitalized several times due to recurrent pulmonary infections. he developed pancytopenia, lymphadenopathy, hepatosplenomegaly and autoimmunity (autoimmune hemolytic anemia and thyroiditis) with low serum immunoglobulin levels at the age of 4. as a result, he received several courses of steroid and prophylactic immunoglobulin and wide-spectrum antibiotics. over time he manifested growth failure and diagnosed with ibd-like colitis. due to the cumulating severe cvid-related complications, a hsct was performed at the age of 14 years with the bone marrow stem cells from his hla identical brother after a conditioning regimen including fludarabine, busulfan and atg. severe intractable colitis with hypoalbuminemia continued till the engraftment despite vigorous fluid-electrolyte replacement therapy and accompanied with severe episodes of acute gastrointestinal bleeding. after the achievement of full donor chimerism, diarrhea episodes resolved. he received three doses of abatasept because of persistent cytopenia thinking about unresolved immune dysregulation. he is in complete remission at 1-year post-hsct with no signs of graft versus host disease. allogeneic hsct should be considered in patients with lrba deficiency prior to the development of disease-related severe cumulative manifestations. disclosure of conflict of interest: none. inflammatory bowel disease (ibd) is a chronic disorder of the gastrointestinal tract. very early onset ibd (veo-ibd) represents those severe children with disease onset occurring before 6-years-old. interleukin-10 receptors (il-10ra, il-10rb) mutation are considered to be one of the very important genes for veo-ibd. currently variant treatment, such as steroid medication, immunosuppressive agents and biological agents could not get complete remission. allogeneic hematopoietic stem cell transplantation (allo-hsct) was reported to induce remission in those with veo-ibd. we performed unrelated umbilical cord blood transplantation (ucbt) in five consecutive children with veo-ibd due to il-10 receptor mutation between 2015 and 2016. median age of five children was 15 months (range: 6-46 months), and median body weight was 7 kg (range: 3.2-9.5 kg). all patients received reduced intensity conditioning (ric) regimen consisting of busulfan, fludarabine and cytarabine. prophylaxis for graft-versus-host disease (gvhd) was tacrolimus. most patients (80%) received a 1 or 2 hla alleles-mismatched cord unit. median nucleated cells of the cord blood were 14.3 × 10 7 /kg (range: 11.2-51.5 × 10 7 /kg), and median cd34+ cells were 4.5 × 10 5 /kg (range: 3.6-14.9 × 10 5 /kg). median follow-up time was 10 months (range: 6-24 months). all patients engrafted, median time of neutrophil engraftment was 22 days, and median time of platelet engraftment was 27 days. four of five patients were alive with continuous donor engraftment, and achieved complete clinical remissions. colonoscopy at 6 months after transplantation in two children revealed the mucosa healing. two children had grade iii acute graft-versushost disease (gvhd). one child developed severe chronic gvhd of both lungs and died of ards at 6 months after transplantation. it is the first clinical trial that unrelated ucbt was performed in veo-ibd children in china. our data should unrelated ucbt with ric should be considered as a potentially curative therapeutic option in children with veo-ibd. disclosure of conflict of interest: none. patients with refractory primary induction failure and resistant relapse are poor candidates for hematopoetic stem cell transplantation (hsct) . additional attempts at remission induction with various combinations of chemotherapy will unlikely improve the outcome and will contribute to excess toxicity. a major goal of sct has been to develop strategies to reduce the risk of gvhd while maintaining or enhancing gvl. tcrαβ+/cd19+lymphocytes depletion is a technology of graft manipulation with a potential to increase gvl effect and improve gvhd control and immune reconstitution in this group of patients. a total of 31 pts with refractory aml (primary induction failure (n = 10), refractory relapse (n = 21)), 17 female/14 male, median age 9.7 years (1. [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] , underwent allogeneic sct between may 2012 and august 2016, median fu 1.5 years (0.3-3.8). 26 pts were transplanted from haploidentical donors and 5 from mud. all pts had active disease (ad) at the moment of sct and received treosulfanbased high-intensity conditioning regimen. three regimens of gvhd prophylaxis were used. regimen 1 (n = 7): atgam 50 mg/kg with (n = 5) or without (n = 2) post-transplant tacro/ mtx; regimen 2 (n = 9): thymoglobulin 5 mg/kg, rituximab 200 mg/m 2 and post-transplant bortezomib on day+2,+5 (n = 9); regimen 3 (n = 15): tocilizumab 8 mg/kg on day-1 and post-transplant bortezomib (n = 15), 4 pts receive additional abatacept 10 mg/kg on day+2, +7, +14, +28. tcrαβ+/cd19 +-depletion of sct with clinimacs technology was implemented in all cases. the median dose of infused cd34+ cells was 8 × 10 6 /kg (range: 4.2-17), tcra/b-14 × 10 3 /kg (range: 2-52). all engrafted pts received additional post-transplant courses of low-dose chemotherapy, including hypomethylating agents and dli. primary engraftment was achieved in 27 of 31 pts(three pts had disease progression, one died at the moment of engraftment), the median time to neutrophil and platelet recovery was 12 days (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) . early mortality within 100 days was 3.2% (one pt with aml had acute lung injury after engraftment on day +14), 1.5-years ptrm-6.7% (95%ci: 1.7-25) . there were no allergic or infusion-related adverse events associated with tocilizumab or abatacept. ci of gvhd grades ii-iv and iii-iv was 25.8% (95% ci:14-47), and 9.7% (95%ci:3.3-28), respectively. ci of cgvhd was 23% (95% ci:12-42). ci of acute gvhd was lower in a group with prophylaxis regimen without serotherapy: 20% (95% ci:2-28) vs 31.3% (95% ci:7-54) in atg group. no correlation between graft composition, donor type with the incidence of agvhd and cgvhd was noted at 1.5 years ppfs (event = death or relapse or progression) was 37% (95% ci:19-54), 1.5-years pos -48% (95% ci:30-67). median time of fu for survivors is 1.5 years (range: 0. [3] [4] . we confirm that the depletion of tcrαβ +/cd19+lymphocytes from the graft ensures high engraftment rate and low transplant-related mortality in pediatric pts with refractory aml. we suggest that tocilizumab and abatacept can be safety administered to children with acute leukemia in the context of treosulfan-based conditioning regimens. long-term follow-up will demonstrate if the gvhd prophylaxis without serotherapy and combined administration of tocilizumab, abatacept and bortezomib post-tcrαβ+/cd19 +depleted grafting will improve gvl effects without extensive gvhd-related morbidity and mortality in pts with refractory aml. disclosure of conflict of interest: none. the jacie experience at the university hospital of amiens l marie-noelle 1 , w brigitte 2 , f isabelle 3 , g bérengère 3 , h muriel 4 , h anne 3 , v elsa 5 , m jean-pierre 3 and c amandine 3 1 lacassagne; 2 oncopôle, chu amiens picardie; 3 hématologie clinique et thérapie cellulaire-chu amiens picardie; 4 oncopôle-chu amiens picardie and 5 the jacie (joint accreditation committee of isct and ebmt) accreditation aims to improve the management of patients benefiting from autologous or allogenic hematopoietic stem cell (hsc) transplants. usually, candidates' centers for jacie accreditation have already existing clinical activity when they have willingness to comply with jacie standards. here, we present our new experience in the implementation of jacie quality process, at the same time as allograft clinical activity. implementing process autograft clinical activity existed at amiens hospital, since 1991, but in lack of center cellular therapy laboratory and hsc collection that were outsourced. the collection activity for autologous transplant was set up in 2009, the cellular therapy laboratory in november 2011 and then the allogenic transplant was started in july 2012. as early as march 2011, we set up a steering committee with hematological clinicians, managers of each sector, a transplant coordinator nurse, the head of the processing laboratory and a part-time quality engineer recruited part-time. each actor had to become familiar with standards to obtain information from accredited centers in order to evaluate objectives and their prioritization. steering committee decided on deadlines and established a roadmap including the following: the list of jacie required standard operating procedures and their writing; assignment of the tasks for each actors in order to evaluate, writing and approval each document; organization of documents diffusion; information to all staff on the approach; creation of feedback committee for adverse events management; establishment of morbidity and mortality review; formalization of initial and continuing training for medical and paramedical staff; and organization of cross audits with external teams. at the same time, we assessed requirements for starting activity: training for medical and paramedical staff; training for the transplant coordinator nurse; circuits for taking care of donors; the organization of the in-patient department; the organization of follow-up of post-transplant patients; and authorizations of the national regulation agencies for processing facility on manipulations and cellular qualifications and for regulatory collection and transplant. allogenic transplant clinical activity started in july 2012. accreditations jacie visit occurred on 8 and 9 june 2015 and our center has been officially accredited since 16 march 2016. neither quality approach, nor clinical activities were easy to implement. medical and paramedical staff had to get acquainted with a new organization and restrictions. despite difficulties, implementing jacie quality process, concomitantly with allograft activity allowed to create a true team dynamics with a common reflection on the means to be implemented. moreover, quality approach has assured us best ensure to care graft patients. the result is true satisfaction, which be credited to all. disclosure of conflict of interest: none. previously published p024 three-dimensional co-culture of peripheral blood monocytes supports and expands functional hematopoietic stem/progenitor cell without immobilization y xu 1 , x li 1 , b wang 1 , w shan 1 , h chen 1 , s liu 1 , r tie 1 , y long 1 , s cai 1 , h xu 1 , x yu 1 and h huang 1 1 bone marrow transplantation center, the first affiliated hospital, school of medicine, zhejiang university very low numbers of circulating hematopoietic stem/progenitor cells (chspcs) are found in normal human peripheral blood (pb) without mobilization. here, we developed a three dimension co-culture system to seize and expansion chspcs from pb monocytes without mobilization. flow cytometry analysis was carried out to identify chspc phenotypes. multipotential properties of chspcs were determined using colonyforming unit assay in methylcellulose and reconstitution ability in the compromised animals. the critical regulation mechanism underlying chspcs was identified with transcriptome analysis based on next-generation sequencing technology at total or single cell levels. loose cobble stone colonies (lcs), round or vessel-like compact colonies (rccs or vccs) were presented in three dimension co-culture system after about 2 weeks. the colonies lasted for at least six passages with no obvious apoptosis sign, and expanded more than~10 000fold during the period. we studied the niche-mediated regulation mechanism of chspc fate at molecular level compared to the conventional method of two dimension culture. furthermore, chspcs were capable of forming all types of hematopoietic colonies, including cfu-gemm, and especially held short term engraftment capacity for compromised nogs by radiotherapy. transcriptome analysis by deepsage identified 167 genes significantly associated with regulating the function of chspcs. figure 1 : the cellular morphology in three dimension culture system for peripheral blood monocytes without mobilization during the culture for 2-3 weeks. figure 4 : short transplantable potential analysis of chspcs. figure 5 : (a) static of differentially expressed genes between three-and two-dimensional culture systems for peripheral blood cells. (b) go functional analysis classifies those genes by biological process, cellular component and molecular function. (c) the significant differences between the molecular phenotypes of three-and two-dimension chspcs indicating that chspcs from three dimension culture hold stem properties. our system may provide a more ideal and balanced approach which not only seizes circulating chspcs, promotes selfrenewal and expansion of chspcs, but also holds phenotypic and functional attributes of chspcs. 6. to wash or not to wash? comparison of neutrophil and platelet engraftment after infusion of cryopreserved autologous stem cells before and after the implementation of bedside thawing am halldorsdottir 1 , s atladottir 2 , m thorsteinsdottir, na arnason 1 , g runarsson 2 , t jonsson 1 , oe sigurjonsson 1,3 and s reykdal 2 1 the blood bank, landspítali, the national university hospital of iceland; 2 department of hematology, landspítali, the national university hospital of iceland and 3 school of science and engineering, reykjavik university cryopreserved autologous peripheral blood stem cell (pbsc) grafts are widely used after high-dose chemotherapy in the treatment of patients with myeloma or lymphoma. prior to infusion, cryopreserved grafts can be thawed at the bedside, or thawed and washed at the cell therapy laboratory. at our institution the practice of routine washing of stem cell grafts in the laboratory was discontinued in april 2012 and bedside thawing implemented instead. this was done to minimize the time thawed cells are exposed to toxic dmso. this study was performed at a single center, at landspítali-the national university hospital of iceland, which is the only transplant center in iceland. autologous pbsc transplants have been performed in iceland since 2004. the study compares outcome for two groups of patients, who received either; (a) thawed and washed autologous pbsc cell grafts from january 2008 to [p024] april 2012, or (b) autologous pbsc grafts thawed at the bedside from april 2012 to november 2016. the following outcomes were compared; days to neutrophil engraftment (absolute neutrophil count (anc) 40.5 per μl), and platelet engraftment (20 and 50 × 10e9/l). data on mean cd34+ cell content/kg of the infused grafts, measured prior to cryopreservation, were also compared. all patients have received premedication with solucortef, clemastine and ondansetron prior to infusion of the graft. from january 2008 to april 2012 a total of 84 patients received thawed and washed autologous pbsc grafts, and between april 2012 and november 2016 83 patients received autologous pbsc grafts thawed at the bedside. majority of the patients were diagnosed with either multiple myeloma or related disorders (n = 86) or lymphoma (n = 67) whereas the remaining patients (n = 14) had miscellaneous diagnoses. days to engraftment and the dose of cd34+ cells infused are compared in table 1 . there was no significant difference in the mean cd34 content of infused autologous stem cells in the two groups (6.9 vs 6.5 × 10e6 cd34+cells/kg, p = 0.41). there was also no difference in the mean number of days to engraftment of neutrophils (12.8 vs 13.3 days, p = 0.14), platelets at 20 days (19.2 vs 18.1 days, p = 0.64) or platelets at 50 days (33.1 vs 28.1 days, p = 0.62) after transplant. one hundred day mortality was comparable in the two groups or 2.4%. additional data on transfusion requirements, infections and use of granulocyte-colony stimulating factor will be presented. [p025] there was no difference in neutrophil or platelet engraftment after changing the autologous stem cell graft thawing procedure from post-thaw washing in the laboratory to bedside thawing. bedside thawing of stem cells is a safe procedure that results in acceptable cellular engraftment. disclosure of conflict of interest: none. the procedure of autologous hematopoietic stem cell (hsc) transplantation requires cryopreservation of hscs. addition of dmso (dimethyl sulfoxide) is necessary to secure the viability of such cells, but this cryoprotectant causes adverse reaction during infusion into patient. the concentrations of dmso in cryopreservation mixture vary strongly between different transplant centers. usually, the hscs are stored in mixtures containing 10% dmso, however, many centers successfully use lower concentrations. the main aim of the study was to evaluate the clinical impact of different dmso concentrations in cryopreservation mixture (5%, 7.5%, 10%) on reconstitution of hematopoiesis after autologous hsc transplantation. the project was approved by the local bioethics committee. written informed consent obtained from all of patients. the study is registered to clinicaltrials.gov (identifier: nct02452099). between january 2014 and july 2016, 150 consecutive patients with hematological malignancies or solid tumors, referred for autologous hsc transplantation, were recruited in the study. the patients were randomly assigned to one of three study arms (50 patients each). hscs obtained by leukapheresis were cryopreserved in three concentrations of dmso: 5%, 7.5%, 10%, respectively. study groups did not differ significantly with regard to the diagnosis (mostly mm, nhl or hl), age or conditioning regimen (chemo-or radiotherapybased). all patients received granulocyte-colony stimulating factor (g-csf, filgrastim) starting from day +4 after transplantation to support neutrophil recovery. in case of 7 patients, the transplantation was cancelled due to progression or other medical reasons. four patients died shortly after transplantation, due to refractory infections. data for 139 patients were subjected to statistical analysis. the viability of nucleated cells on the day of transplantation was similar in all groups (median 96%, range: 85-99% for 10% dmso group; 97%, range: 85-99% for 7.5% dmso; 97%, range: 90-99% for 5% dmso; p = 0.52). the dose of transplanted cd34+ cells was comparable in all group: (median 4.70 × 10 6 /kg of recipient body weight for 10% dmso, 4.35 × 10 6 /kg for 7.5% dmso and 3.97 × 10 6 for 5% dmso, p = 0.44). the median time to leukocyte recovery, defined as the first day with wbc count exceeding 1.0 × 10 9 /l was 10 days in all groups (ranges: 9-12 for 10% dmso; 7-11 for 7.5% dmso; and 9-12 for 5% dmso; p = 0.36). similar results were obtained in case of neutrophil recovery-the median day, when the anc exceeded 0.5 × 10 9 /l, was 10 in all arms (ranges: 9-12; 8-11 and 9-12, respectively; p = 0.20). the day when the platelets level were greater than or equal to 20 × 10 9 /l (sustained without transfusion within 7 days) was similar in all groups: medians were 15 days in 10%, 7.5% and 5% dmso (ranges: 8-20; 0-19; 0-24; p = 0.61). no serious adverse effects were observed during hscs infusion and during 24 h after transplantation. reduction of dmso concentration from in cryoprotective mixture 10% to 7.5% and 5% has no negative impact on cell viability during cryopreservation and engraftment after auto-hsc transplantation. disclosure of conflict of interest: none. a real-world cost-effectiveness analysis demonstrates that introducing plerixafor to improve mobilization in multiple myeloma patients who behave as poor mobilizers is cost-effective considering the whole mobilization and transplant procedure r touzani 1,2 , a-m stoppa 3 plerixafor, a cxcr4-antagonist, is efficient to improve cd34 + cell mobilization and collection in candidates for autologous transplantation who behave as poor-mobilizers. the cost of the drug is however of concern. published medico-economics studies were mostly conducted in the us, and few including detailed and comprehensive micro-costing of the collection and transplantation process; conclusions may thus not apply to european countries where cost structures are different. to compare costs and effectiveness of plerixafor-free and plerixafor-replete management strategies for multiple myeloma patients who behaved as poor-mobilizers after adequate administration of a standard rhg-csf mobilization regimen. sixty patients diagnosed with multiple myeloma were consecutively identified during years 2009-2011, immediately before and after ema granted marketing authorization for plerixafor. poor-mobilizers were defined as having circulating cd34+ cell counts below 20/μl. plerixafor was introduced or not as a result of the attending physician's decision, reflecting progressive changes in medical practices over this transitional period. the historical and study groups were matched over four criteria: disease stage at diagnosis, age, gender and number of chemotherapy treatments received before mobilization. two cost-effectiveness analyses (cea) were conducted; the primary cea looked at the criterion ‛collecting at least 2 × 10 6 cd34+ cells'; a secondary cea looked at the criterion ‛successful autologous transplant administered'. detailed micro-costing evaluations (2015 figures) did not or did include transplantation costs for the first and second cea, respectively. the two groups were similar in terms of age, sex distribution, disease characteristics or previous treatments. 27/30 and 26/30 patients proceeded to high-dose melphalan and autologous transplantation in the study and historical groups, respectively. there was a trend to a higher number of collected cd34 + cells in the control group; however, the proportion of patients who met the minimal target number of 2 × 10 6 collected cd34 + cells/kg was identical (28/30). length of hospitalization, times to neutrophil and platelet recoveries, numbers of prbc and platelet transfusions were identical in the two groups. mobilization and collection costs per patients were more important in the plerixafor group that in the historical group (8.757 vs 5.460 €, p o0.0001), and proportionally higher in patients who received plerixafor as part of a remobilization treatment rather than pre-emptively (10.401 vs 8.162€, respectively). the main cea concluded to a 3.237€ increase in costs for the same number of patients achieving a minimal target number of 2 × 10 6 collected cd34 + cells/kg. the second cea found a decrease in the cost of transplant, with 12.724€ in the study group vs 13.634€ in the historical group (ns). in total, the 2.035€ increase for the complete procedure cost (22.866€ per successfully autografted patient in the study group vs 20.831€ in the historical group) was not statistically different. cost-effectiveness arguments should not been used against the administration of plerixafor in multiple myeloma patients in the european context. future prospective researches looking at patients reported outcome criteria and labour organization in apheresis facilities are needed. disclosure of conflict of interest: this work was supported by a grant from sanofi s.a.; cc: research support, honorarium & hospitality from sanofi s.a. administration of plerixafor for peripheral blood cd34+ stem cell content of o30 × 10 6 /l for autologous stem cell mobilization leads to decreased apheresis days and increased total yield m kamdar 1 , s abebe 1 , gr gonzalez fontal, l gates 1 , a hammes 2 , d abbott 2 , j gutman 1 , b haverkos 1 , d sherbenou 1 and c smith 1 1 division of hematology and transplantation and 2 department of biostatistics and informatics, university of colorado, denver, colorado, usa autologous stem cell transplantation (asct) is an effective treatment for lymphoma and plasma cell neoplasm (pcn) (multiple myeloma and amyloidosis). granulocyte-colony stimulating factor (g-csf) is the most commonly used upfront mobilizing agent with plerixafor-based higher cost approaches reserved for poor/unsuccessful mobilizers. several mobilization algorithms utilizing g-csf and plerixafor have been published however the most efficient and cost effective strategy is yet to be determined. most transplant centers administer plerixafor for peripheral blood (pb) cd34+ stem cell content of o10 × 10 6 /l on day (d) 4 of g-csf mobilization. at the university of colorado (uch) we changed our programmatic approach in 2015 and administered plerixafor for pb cd34+ count of o30 × 10 6 /l on d4 of g-csf mobilization. in this study we evaluate the impact of this novel mobilization algorithm on apheresis days and total stem cell yield. patients (pts) with lymphoma and pcn who underwent asct at uch until 3/2015 received plerixafor if pb cd34+ cells on d4 of g-csf mobilization was o10 × 10 6 /l. based on our institutional review of poor/unsuccessful mobilizers and using logistic regression analysis this algorithm was revised in 4/2015. in the new algorithm all pts received plerixafor if pb cd34+ cells on d4 of gcsf mobilization was o30 × 10 6 /l. demographics were compared between pts with lymphoma and pcn before (group 1: 9/2013-3/2015) and after (group 2: 4/2015-4/2016) the new algorithm was implemented. the primary goal of this analysis was to assess the total days of apheresis and total stem cell yield between the two groups. we also sought to analyze days to wbc engraftment and platelet engraftment. a total of 131 pts were included in this analysis. group 1 consisted of 77 pts (26 pts had lymphoma and 51 pts had pcn). group 2 consisted of 54 pts (20 pts had lymphoma and 34 pts had pcn). we found that there was a significant increase in total yield (p = 0.0017) in group 2 as compared to group 1. on further disease subtype assessment we noted that pts with pcn in group 2 had a significant increase in total yield (p = 0.0014). in lymphoma pts on univariate analysis group 2 showed a significant decrease in apheresis days (0.468 days, p = 0.044, 95% ci: (−0.91, − 0.026)). on multivariate analysis there was still a marginally significant decrease in group 2 (0.47 days, p = 0.052, 95% ci: (−0.916, − 0.024)) compared to group 1. in pcn pts on univariate analysis group 2 showed a significant decrease in apheresis days (0.387 days, p = 0.025, 95% ci: (−0.718, − 0.056)). on multivariate analysis group 2 continued to show a significant decrease in apheresis days (0.426 days, p = 0.02, 95% ci: (−0.772, − 0.081) compared to group 1. we found no significant difference between the two groups in days to neutrophil engraftment and platelet engraftment. our analysis showed that a mobilization algorithm of administering plerixafor for a pb cd34+ stem cell count of o30 × 10 6 /l on d4 of g-csf mobilization led to a decrease of roughly 0.46 days in the lymphoma cohort and a significant decrease of 0.43 days in the plasma cell neoplasm cohort. we also noted a significantly increased total yield of stem cell collection in group 2. overall our programmatic approach led to decreased chair-time for apheresis and better resource utilization. pharmacoeconomic impact of this approach will be updated at the meeting. disclosure of conflict of interest: mk: speakers bureau, seattle genetics; remaining authors declare no conflict of interest. administration of stem cell boosts (scbs) from the original donor offers a therapeutic option. we report on 50 pediatric patients with pgf who received a total of 61 boosts with cd34 + selected peripheral blood stem cells (pbsc) after transplantation from matched unrelated (n = 25) or mismatched related (n = 25) donors. median time between hsct and infusion of the 61 scbs was 94 days (13-519). boosts contained a median number of 3.15 × 10 6 cd34+ progenitor cells/kg body weight (range: 0.71-27.9 × 10 6 ) with a median number of 2417/kg (range: 100-23 630) residual cd3+ t cells. within 8 weeks after application, a significant increase in median neutrophil counts (600 vs 1516/mm 3 , po 0.05) and a decrease in erythrocytes and thrombocytes transfusion requirement (median frequencies 1 and 7 vs 0, p o0.0001 and o 0.001), were observed, and 78.8% of the patients resolved one or two of their initial cytopenias whereas 36.5% had a complete hematological response. additionally median lymphocyte counts for cd3+, cd3+cd4+, cd19+ and cd56+ increased 8.3 fold, 14.2 fold, 22.3 fold and 1.6 fold, respectively. the rate of de novo acute gvhd grade i-iii was only 6% and resolved completely after treatment. no gvhd iv or chronic gvhd occurred. patients who showed a response to scb displayed a trend toward better overall survival (os) (p = 0.07). administration of cd34+ selected scbs from alternative donors is a safe and effective procedure. we hypothesize that the cd34+ progenitor boosts may have an enhancing effect on maturation of committed lymphoid precursors already present in the host or generate another wave of thymic seeding with accelerated t-cell differentiation process in the absence of any immune suppression. further studies are warranted to better define the impact on immune reconstitution and survival. disclosure of conflict of interest: none. plerixafor plus granulocyte-colony stimulating factor (g-csf) has been shown to mobilize more cd34+ cells than g-csf alone for autologous hematopoietic stem cell transplantation (hsct). however, there are few studies that analyze the impact of this strategy in engraftment. the aim of our study is to compare mobilization and engraftment between patients who received a combination of plerixafor plus g-csf and patients (pts) who mobilized with g-csf alone. a retrospective casecontrol analysis was performed in 24 pts with myeloma who mobilized with plerixafor plus g-csf (group p/g-csf) and was compared with 24matched for sex and age controls who mobilized with g-csf alone (group g-csf). all pts underwent hsct between 2009 and 2015. mobilization with g-csf at dose of 10 μg/kg/day was used in all pts. the aphaeresis was scheduled on day +5. plerixafor (0.24 mg/kg) was added if the number of cd34+cells on day +4 was o10/μl for 2 × 10 6 cd34+/kg requested (or o20/μl for 4 × 10 6 cd34+/kg), or if the number of cd34+cells collected in the first apheresis was o50% of cd34+ requested. conditioning and supportive care were similar in both groups. in p/g-csf group, 13 were male and 11 female. median age was 60.92 years (range: 49-71). in group g-csf, 15 were men and 9 female. median age was 60.67 years (range: 50-73 years). there were no differences between both groups. disease status at time of mobilization was different between groups (p = 0.023). in p/g-csf group: 11 (45.83%) pts were in complete remission (cr), 4 (16.66%) very good partial responses (vgpr), 7 (29.16%) partial response (pr) and 2 (0.08%) had no response to treatment. in g-csf group: 7 (29.16%) pts had reached cr, 13 (51.16%) vgpr and the remainder in pr. sixteen (66.67%) pts in p/g-csf group had received ⩾ 2 lines of treatment vs 9 (37.5%) pts in g-csf group (p = 0.046). no difference was seen on mean day-dose of g-csf (14 μg/kg/24 h in p/g-csf group vs 12 μg/kg/24 h) (p = 0.067). there was no difference on cd34+/kg requested (19/24 pts in p/g-csf were requested 2 × 10 6 /kg vs 18/24 in g-csf group) (p = 0.73). p/g-csf group needed more apheresis sessions, 17 (70.83%) pts required ⩾ 2 sessions against 4 (16.67%) pts in group g-csf (p o0.001). we obtained enough cd34+ cells to carry out hsct in all patients, although mean number of cd34 + cells obtained in p/g-csf group was lower than in g-csf group (2.92 × 10 6 /kg vs 4.98 × 10 6 /kg, respectively) (p o0.001). also, mean number of cd34+ infused in p/g-csf group was lower (2.92 × 10 6 /kg vs 3.55 × 10 6 /kg) (po 0.001). however, engraftment results were similar in both groups, as represented in table 1 . patients who required mobilization with plerixafor plus g-csf got an engraftment as good as patients who do not require the combination despite of worse baseline parameters. given that the number of cd34+ infused in the p/g-csf group has been lower than g-csf group, these results might suggest that the different composition of graft cell with plerixafor plus g-csf mobilization, described in some studies, could impact on engraftment outcomes. high-dose chemotherapy following autologous hematopoietic stem-cell transplantation (autohsct) is an effective method of treatment both recurrent and primary refractory lymphoma patients. however, some patients have mobilization failure (‛poor mobilizers') with inadequate collection of peripheral blood stem cell (pbsc). aim: to evaluate the efficacy and factors influencing pbsc mobilization and collection for the autohsct in patients with lymphomas. thirty patients were included in this study: 17-with hodgkin lymphoma, 7-with non-hodgkin lymphoma, 6-with multiple myeloma; 17 women and 13 men of them. the median age of patients was 36 years (24-64 years). the mobilization of pbsc with only colony-stimulating factors (csf) was carried out for 17 patients, chemotherapy (cyclophosphamide, etoposide) in combination with csf-for 13 patients. only one patient had plerixafor mobilization. the concentration of cd34+ in peripheral blood (pb) was studied on the day of the intended cytapheresis. cytapheresis was commenced when cd34+ concentration had been greater than 0.01 × 10 6 cells/ml. twenty-four patients (80%) from 30 had collection of pbsc. the collection was not performed in six patients (20%) because the concentration of cd34+ in pb on the day of the intended cytapheresis was lower than 0.01 × 10 6 cells/ml. there was no possibility to use plerixafor in these cases for economic reasons. the median concentration of cd34+ in pb on the first day of the intended cytapheresis in the group of patients that had cytapheresis was 0.013 × 10 6 cells/ml whereas in the group of failed-0.005 × 10 6 cells/ml (p o0.05). fifty-nine tests of cd34+ in pb were done. distribution and test results by days from the first day of the intended cytapheresis are presented in table 1 . the total number of the cytapheresis was 36. the majority of patients had 1 procedure of pbsc collection (n = 22), 13 patients had 2 procedures and only 1 had 3. the last patient had had two previous failed cytapheresis procedures and the adding of plerixafor helped him to collect necessary number of cells. the median of cd34+ cells on patient's kilo was 2.85 × 10 6 cells/kg. sex, age, mobilizing regimen, previous radiation therapy, the count of lines of chemotherapy before autohsct were not significantly associated with poor pbsc mobilization and collection. only tumor response before autohsct (complete/ partial response or stabilization) was significantly associated with cd34+ cell count in the product of cytapheresis. patients with complete or partial response had significantly better cd34+ count. [p034] disclosure of conflict of interest: none. factors associated with failure in mobilization of peripheral blood hematopoietic progenitor cells in autologous transplantation je dulon-tarqui, bl acosta-maldonado, l rivera-fong, sa sánchez-guerrero, jf zazueta-pozos, ja padilla-ortega, wj ladines-castro and lm valero-saldaña high dose therapy followed by autologous stem cell transplantation (asct) obtained from peripheral blood is currently the standard model for treatment consolidation in various hematologic malignancies. a global incidence of 5-40% of failure to mobilization is reported, and some factors associated with poor mobilizers in hodgkin's lymphoma (hl), non-hodgkin's lymphoma (nhl) and multiple myeloma (mm) when the yield in peripheral blood stem cells (pbsc) collection is unsatisfactory, the effects for the recipient can be serious. the donor's age, gender, body surface area (bsa), processed blood volume and the method of g-csf dose calculation may affect the cd34+ yield. as g-csf has a low distribution volume in the peripheral blood (pb), it might be appropriate to calculate the doses by using the bsa instead of per kg body weight. 175 consecutive allogeneic pbsc donations performed in 170 healthy donors at the karolinska university hospital in stockholm were included. a complete medical history, physical examination, electrocardiogram, chest x-ray and laboratory testing were done before pbsc donation. relevant data for analysis were collected from the institutional quality database for a retrospective review. the total blood volume was calculated using the formula by nadler et al. the bsa was calculated using the formula by du bois and du bois. the concentration of cd34+ cells in the pb and the processed volume of blood were significantly correlated to cd34+ cells yield (po 0.00005 and po0.001, respectively, see table 1 ). the g-csf dose per m 2 was significantly correlated to the concentration of cd34+ cells in the pb (p = 0.0003) and in the product (p = 0.01, see table 1 ). smaller bsa (p o0.001) and less processed volume (p o0.001) were found among female donors, who were given lesser g-csf dose per m 2 (p o0.001) and showed lower yield compared to men (po0.05). however, multivariate analysis of the yield showed that only the concentration of cd34+ cells in the pb and the processed volume remained independent significant (see table 1 ). [p036] in this study, we found the concentration of cd34+ cells in the pb and the processed volume of blood to be independent predictors of yield. we recommend to get a high concentration of cd34+ cells in the pb, and to process adjusted volumes of blood when needed. an evaluation if the calculation of g-csf dose per m 2 is more appropriate than per kg body weight should be done in future studies. autologous stem cell transplantation (asct) has been widely used in the treatment of hematological malignancies over the last two decades. despite its broad use, some characteristics that might influence engraftment have not been exhaustively investigated, particularly graft purity with respect to contamination by platelets (plts) and white blood cells (wbc). here we report collection characteristics and engraftment kinetics of a single center consecutive series of 510 asct. we retrospectively collected clinical records of 481 patients who underwent leucapheresis procedures (la; followed or not by asct) and data on 510 asct at our institution over 16 years (2000-2016) ( table 1 ). the impact on engraftment kinetics of conditioning chemotherapies, amount of infused cd34+ cells and wbc/plts graft contamination were analyzed. absolute neutrophil count (anc) engraftment was defined as the duration of neutropenia (from day 0 to the first of 3 consecutive days of anc4500/μl post asct). regarding cd34+ cell collection, no impact of mobilizing regimens and wbc count during la was observed. on the other hand, we observed a difference in the number of total cd34+ cells collected among different diagnoses: the median overall collection was 7.2 (0.65-64.06) × 10 6 /kg cd34+ cells for nhl patients, 5.66 (0.71-23.31) × 10 6 /kg for mm patients, 6 .15 (0.51-23.24) × 10 6 /kg for hl patients and 3.56 (0.64-20.3) × 10 6 /kg for aml patients) (p = 0.001). considering cd34 + cells/kg harvested on the first day of la, 59.2% of nhl and hl, 57.5% of mm patients and 34% of aml patients harvested ⩾ 5 × 10 6 /kg cd34+ cells. of note, among aml patients, 40.6% collected o 2.5 × 10 6 /kg. the differences were statistically significant (p = 0.003). moreover, an inverse correlation between collected cd34+ cells and age was shown (p = 0.001). anc recovery after asct was not influenced by conditioning regimen whereas diagnosis impacted on the duration of neutropenia (aml patients displayed a longer aplasia, po0.01). we observed that the median days with anco 500/μl were 10, 11 and 12 in patients who received 45.3 × 10 6 /kg, 3.5-5.3 × 10 6 /kg and o3.5 × 10 6 /kg cd34+ cells, respectively (po0.0001). furthermore, the same finding was observed considering the duration of thrombocytopenia (median number of days with plts o50 000/μl: 15, 18 and 20 in patients who received 45.3 × 10 6 /kg, 3.5-5.3 × 10 6 /kg and o 3.5 × 10 6 cd34+ cells, p o0.0001). looking at the apheresis product, we analyzed the impact of harvest contaminating wbc and plts on engraftment kinetics. notably, when the asct collection contained 4100 × 10 3 /μl wbc, anc engraftment (days with anc o 500/μl) lasted longer (median days 11) compared to patients who received a graft with lower wbc count (po 0.0001). a faster anc engraftment was also observed in patients receiving harvests with plts levels 4600 × 10 3 /μl compared to those who infused a collection bag with plts o600 × 10 3 /μl (p = 0.005). herein, we confirmed that the disease and the amount of infused cd34+ cells significantly influence time of anc and plts engraftment; furthermore, we observed for the first time that quality and purity of the graft have a substantial impact on engraftment kinetics. a combination of chemotherapy with growth factor is a commonly used strategy for hematopoietic stem cell (hsc) mobilization. the collection of timely and adequate numbers of hscs is a prerequisite for proceeding to transplantation. a variety of mobilization strategies are currently used. the knowledge of efficacy, safety and predictability of different hsc mobilization strategies might help blood and marrow transplantation (bmt) programs to effectively schedule patients for mobilization. given the many variables associated with the mobilization of hsc, collecting an adequate stem cell dose in a timely and effective manner is an art and science. factors that might affect the process includes type of disease and mobilization protocol, financial clearance, availability of chemotherapy beds, scheduling various diagnostic procedures and transplant urgency. to evaluate the effectiveness and related coordination efforts of ‛just-in-time' strategy of hsc mobilization and collection, we performed a retrospective study comparing all patients in whom peripheral hsc mobilization was attempted at khcc from january 2005 through november 2016. data collected included the disease type, mobilization protocol, days to and number of collections, cd34+ cell dose, calendar of the mobilization and collection. the records of a total of 1042 mobilizations were reviewed. 364 were of healthy allogeneic donors, and the remaining 678 were of patients undergoing autologous transplantation. table 1 depicts the overall summary of number of days and collection procedures per each protocol. detailed mobilization kinetics per disease type and mobilization protocol were also captured and evaluated. [p037] s140 [p038] detailed analysis of mobilization kinetics comparing different mobilization strategies aids in prediction of number of days of mobilization and anticipated number of collections. this helps in proactively scheduling patients based on collection predictability. a seamless communication through a shared calendar between key parties, primarily bmt physicians and nurse coordinators, bmt and flow cytometry laboratories and chemotherapy unit can be achieved. autologous stem cell transplantation is still a standard of care in the treatment of multiple myeloma. lenalidomide-based regimens are commonly used in both transplant-ineligible as well as -eligible patients. prolonged lenalidomide-exposure is known to affect mobilization of cd34 + cells, although the basic mechanisms are poorly understood. limited prospectively collected data is available on the effect of lenalidomide in the capacity to mobilize cd34 + cells for transplantation as well as graft cellular composition and post-transplant hematological recovery compared to the lenalidomide-naive patients. this prospective study included 60 newly diagnosed myeloma patients who received mobilization with low-dose cyclophosphamide + g-csf, were successfully apheresed and transplanted before the end of 2014. twenty-six patients had received a median of three cycles of lenalidomide-based induction (43 %), whereas 34 patients were lenalidomide-naive and served as the control group. both baseline characteristics and collection targets were similar between the groups. cd34 + mobilization and apheresis yields were analyzed and compared between the groups. blood graft cellular composition was analyzed from the thawed cryopreserved samples with a flow cytometry. graft function was evaluated by collecting engraftment data as well as by total blood counts at day +15 and at 1, 3, 6 and 12 months after post-transplant. the patients in the lenalidomide group had both lower median peak b-cd34 + counts and about 40% lower cd34 + yields of the first apheresis but without statistical significance ( table 1 ). the median number apheresis was significantly higher in the lenalidomide arm (2.0 vs 1.5, p = 0.039). the number of cd34 +cd133+cd38-, cd3+cd4+, cd3+cd8+ cells and nk cells in the cryopreserved grafts were comparable between the arms. time to neutrophil engraftment was 12 days in the both groups. the median time to platelet engraftment was 12 d in the lenalidomide group and 13 d in the control group. hematological recovery was comparable between the groups within 12 months post-transplant. lenalidomide-based induction therapy seems to have an impact in the number of apheresis needed, but not in the total yield of cd34 + cells in the graft. neither the graft cellular composition nor posttransplant recovery in myeloma patients was affected by the limited duration of lenalidomide used before mobilization and collection of blood grafts. between september 2015 and november 2016. siemens hematek 3000 system was used for luc count. luc numbers and percentage was measured before leukapheresis. we used pearson test for the correlation and roc curve for cut off value. patients' characteristics were shown in table1. there was not a correlation between luc number and mobilized cd34 positive stem cell number. but luc percentage was positively correlated with mobilized stem cell number (p:0.01). a count of 5 × 10 6 /kg collected stem cells are optimal for autologous stem cell transplantation. we found 2% luc percentage as a cut-off value for prediction of collecting optimal number of stem cells with 61% sensitivity and 60% specificity. as expected luc percentage was negatively correlated with white blood cell count. there was no correlation between mobilized cd34 positive stem cell number and age. both luc percentage and mobilized cd34 positive stem cell number did not differ with underlying disease. we found only one study in the literature that evaluated luc percentage as a tool for the prediction of successful stem cell collection. they found that baseline luc numbers negatively correlated with stem cell mobilization in healthy donors (1) . but we measure luc on apheresis day and found a positive correlation between luc percentage and stem cell mobilization. and we found a cut-off value for optimal stem cell mobilization with acceptable sensitivity and specificity. in our study we demonstrate that luc percentage measurement on apheresis day may be a very simple and cheap tool for the prediction of optimal stem cell mobilization. the spectra optia (so) apheresis system performs a wide range of therapeutic procedures, including peripheral blood stem cell (pbsc) collection in mobilized donors and patients (pts). the device was studied to evaluate the cellular composition of pbscs harvested in pts with multiple myeloma (mm), non hodgkin's lymphoma (nhl) and hodgkin's lymphoma (hl) planed for autologous peripheral stem cell transplantation (apbsct), and to optimize the collection of pbscs using the cd34+ precount and collection efficiency (ce2) of apheresis device which is calculated as follows: ce2 = total cd34 + cells collected × 10 6 /kg; cd34+ precount/ μl × blood processed (liters). the blood volume processed is calculated as follows: desired cd34+ × 10 6 /kg × recipient weight (kg): ce2 × cd34+ precount/μl in our study enrolled pts undergoing pbsc mobilization and planed for apbsct. we evaluated so system's mononuclear cell (mnc) collection performance, with respect to cd34+ cells and mnc collection efficiency, platelet reduction pre to post apheresis, and product purity in view of using prediction algorithms to optimize the procedure and predict the cd34+ yield, blood volume processed and platelets loss. we also evaluated neutrophil and platelet recovery in pts who underwent apbsct. results: between 30/3/2015 and 30/11/2016, 45 pts underwent pbsc harvesting by so device. median age was 46 years (20-71). there were 19 females and 26 males. diagnosis was mm in 21 pts, hl in 17 pts and nhl in 7 pts. the number of ahereses procedures was 59. mobilization consisted in g-csf alone in36 pts, chemotherapy and g-csf in 8 pts, and g-csf + cxcr4 inhibitor in one patient. median count of cd34 + cells pre-collection was 58/μl (16.5-372) . median total blood volume processed was 12.4l (6.3-19.9 ). median count of cd34 + cells collected was 4.1 × 10 6 /kg (1-23.6). median mnc collection efficacy was 48% .median cd34+ cell collection efficacy was 45.5% (15-95%). median platelet reduction pre to post apheresis was 30% (0-50%). median product hematocrit and granulocytes product was 5% (3-9) and 52% (5-93), respectively. twenty-six of the 45 pts underwent myeloablative high dose chemotherapy followed by apbsct which was performed for mm in 18 pts, hl in 6 pts, and nhl in 2 pts. the median count of cd34+ cells infused was 2.5 × 10 6 / kg (1.15-10.6). all the pts received g-csf post-apsct until neutrophil recovery. the median day for neutrophil recovery was 10 (8) (9) (10) (11) (12) (13) (14) . median duration of severe neutropenia (anc o0.5 × 10 9 /l) was 7 days (4-10). the median day for platelet recovery was 10 (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) . median duration of severe thrombocytopenia (platelets o20 × 10 9 /l) was 5.5 days (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) . conclusion: the study results confirm that the so apheresis system's mnc collection protocol is safe and effective. the neutrophils and platelets recovery in pts auto-transplanted was not inferior compared to historical controls. in addition, this system help to use prediction algorithms for whole blood processing to achieve a desirable and optimal yield based on cd34+ precounts and ce2 of the apheresis device. disclosure of conflict of interest: none. peripheral blood stem cell apheresis in small children is difficult! aa hedayati-asl 1 , m emam-jome, p dinarooni, v fallah, a mehrvar and r zangooei 1 in low-weight children with cancer and healthy donor children, peripheral blood progenitor cells (pbpcs) have largely replaced bone marrow as source of autologous and allogeneic stem cells in part because of their relatively easy collection. however, there is a concern regarding medical, psychosocial and technical difficulties in small children. we retrospectively analyzed peripheral blood stem cell apheresis in 38 collections. 30 patients were with cancer (17 patients = neuroblastoma, 4 patients = retinoblastoma, 5 patients = germ cell tumor, 1 patient = hepatoblastoma, 3 patient = wilm's tumor) and 8 healthy children donors. the study was conducted between 2012 and 2016. peripheral stem cell apheresis was performed in the mahak cancer children's hospital in a nice room for children where the patients stayed with their families. patients s142 were not routinely sedated. pbpc were collected by a cobe spectra cell separator (cobe, denver, co, usa). harvesting was performed after 5 days mobilization. mean body weight was 11.6 kg (range: 8-15 kg) for a median age of 3 years (range: 10 months-5 years). mean duration of harvesting was 205 min (range: 164-270 min). mean volume of stem cell collection was 135 ml (range: 110-240 ml). the mean number of total nucleated cells collected was 5.4 × 10 8 /kg (range: 3.2-9.9 × 10 8 /kg recipients). no side effects occurred. children didn't require an additional haematopoietic progenitor mobilization or additional apheresis in other day. pbsc collection was without transfusion in healthy donor children. pbsc collection may be difficult in small children owing to the large volume apheresis compared to the child's weight. various problems, such as metabolic or haemodynamic disorders may be were seen. peripheral stem cell harvest can be performed in low-weight children under safe and effective conditions even when systematic priming by blood is avoided. processing with increase of blood volume may to increase in the yield by recruiting progenitor cells. disclosure of conflict of interest: none. peripheral blood stem cell collection in low body weight children: a single centre experience g del principe*, g leone, s lazzaro, a meschini, k feri, p marchitelli, d carasso, f locatelli and m montanari department of pediatric hematology/oncology and transfusion medicine, irccs bambino gesù children's hospital, rome, italy pbsc became preferred source for autologous transplantation because of easier collection and faster engraftment. however apheresis for low body weight children ( o12.5 kg) is affected by some issues: venous access, extracorporeal volume, metabolic and hemodynamic complications, citrate toxicity, so is crucial to standardize harvesting procedure both maximizing stem cells collection and reducing adverse events. a dual lumen central venous catheter was used to obtain a minimal blood flow of 10-15 ml /min and pbsc collection was performed with spectra optia mnc v6.1 apheresis system, starting with cd34+ cell ⩾ 20 μl in peripheral blood. the priming of extracorporeal circuit was made with compatible, irradiated, leucodepleted packed red cell to avoid hypovolemic state. citrate dextrose solution a(acd-a), with a ratio of 1:24 to whole blood, and a bolus of heparin 50 ui/kg were used as anticoagulants. all patients, treated without sedation, were monitored by ecg, pulse oximetry and non invasive blood pressure; electrolytes panel (na, k, ca) and act (activated coagulation time) were assessed at the beginning, 30 minutes after and then every hour during apheresis. hypocalcemia was managed by 350 mg calcium gluconate slow infusion. we report our experience of pbsc collection in low body weight children ( o12.5 kg) treated in our apheresis department between january 2015 and november 2016. a total of 37 pbsc collections were performed in 17 children (8 m/9 f, median age 21 months, median weight 10.5 kg) affected by medulloblastoma (n = 4), germ cell tumor (n = 2), neuroblastoma (n = 8), retinoblastoma (n = 2), brain cancer (n = 1). total blood volume processed ranged from 2.0 to 4.55 tbv (median 3.05) and median count of cd34+ collected was 5.5 × 10 6 /kg(range: 1.5-54). all procedures were performed with a median duration time of 199 minutes (range: 114-293 min) and no serious adverse events occurred. in our experience pbsc collection is safe and feasible also in low body weight children using a tailored apheresis procedure. disclosure of conflict of interest: none. plerixafor on demand in the first or in the second attempt of cd34 mobilization j romejko-jarosinska, e paszkiewicz-kozik, l targonski, m szymanski, z pojda and j walewski 1 1 high-dose chemotherapy and autologous hematopoietic cell transplantation (auto-hct) is a recommended strategy for patients with relapsed, refractory or high risk lymphoma. mobilization failure of cd34+ cells after granulocyte colonystimulating factor (g-scf) with or without chemotherapy is a factor limiting patient access to this potentially curative procedure. the use of plerixafor with g-csf may improve cd34+ cell harvest in poor mobilizing patients. we evaluated the clinical effectiveness of plerixafor and g-csf ± chemotherapy administered on demand in the first and second attempt of mobilization in lymphoma or myeloma patients who were eligible for auto-hct. we evaluated data on 59 consecutive patients with hodgkin lymphoma (17), dlbcl (17), mantle cell lymphoma (10) , myeloma (8) and other lymphoma subtypes (7) who were mobilized with plerixafor between january 2011 and october 2016. median (range) age of patients was 47 (27-69). patients received a median of 2 (1-4) chemotherapy lines. radiotherapy was applied in 13 patients. all patients received g-csf (10 μg/kg/day) ± chemotherapy and plerixafor (240 μg/kg/day) on demand in the absence of increase in the number of cd34+ cells in peripheral blood above 10/μl on the day of the scheduled apheresis (within 20 days following the chemotherapy and after at least 4 days of g-csf). plerixafor was given to 36 patients in the first attempt of mobilization and to 23 patients during the second mobilization. the mobilization was considered effective if the harvest cell dose was 2 × 10 6 /kg cd 34 or more. after plerixafor administration circulating cd34+ cells increased to 410/μl in 21 patients (58%) and in 11 patients (43%) in the first and in the second mobilization, respectively (p = 0.26). the cd34+ cell collection was performed in 52/59 patients (88%): in 32/36 (89%) patients in the first and in 20/23 patients (87%) during the second mobilization cycle. the median number of apheresis was 3 (range: 1-6), for both mobilizing cycles. the median (range) cd34 cell dose collected in the first and second cycle was 3.03 (range: 0.77-16.87) × 10 6 /kg and 1.89 (range: 18-11.53) × 10 6 /kg, respectively (p = 0.007). the harvest was successful in 27/32 patients (84%) in the first and in 10/20 patients (50%) in the second cycle (p = 0.007). three patients (9%) who failed the collection with plerixafor in the first attempt, succeeded in the second cycle. additional second mobilization with plerixafor was successful in five patients (25%) who failed the first mobilization. in total, 30/32 (93%) and 15/20 (75%) of patients given plerixafor in the first or in the second mobilizing cycle harvested at least a minimum cd34 cell dose for auto-hct (p = 0.05). these results show that plerixafor administered on demand is an effective rescue strategy for poor mobilizing patients. each mobilization cycle with plerixafor resulted in the increase of circulating cd34 cell count. successful harvest is more frequent if plerixafor is administered in the first than in the second mobilization attempt. the evaluation of the prognostic factors for mobilizing failure with plerixafor is necessary to identify the poor mobilizers precisely. disclosure of conflict of interest: jr-j, ep-k, lt and jw: sanofi (travel grants); ms and zp: none; jw: lecture, honoraria cryopreserved stem cell grafts are still widely used both in the autologous or allogeneic settings. cryopreserved grafts can be thawed at the bedside or thawed and washed at the cell therapy laboratory. we recently reported that post-thaw washing did not impair hematopoietic engraftment, in a cohort of 2930 autologous transplanted patients receiving either unwashed or washed grafts (calmels b et al, bone marrow transplant. 2014). post-thaw washing can be implemented using various methods such as manual centrifugation, automated centrifuge-based (sepax 2, biosafe) or spinningmembrane devices such as lovo (fresenius kabi). we here report a step by step implementation of the lovo biomedical device (bmd) for washing thawed stem cell grafts. having defined a washing program, we aim to compare this protocol to our routine process, using the sepax 2 bmd. we took advantage of 11 apheresis products intended for destruction and cryopreserved in 2 identical bags; after dry-thawing (plasmatherm, barkey), bags were connected to the sepax 2 or to the lovo bmd, diluted volume to volume with +4-8°c 6% hydroxyethylstarch 130/0.4 (voluven, fresenius kabi) and processed using the smartwash program (sepax 2) or a 2cycles standard wash protocol on lovo (a cycle referring to one pass through the spinning membrane). the lovo settings were customized for this application: reduction retentate pump rate 75 ml/min, desired inlet pcv 10%, and automated volume to volume dilution. after processing, cd34 and cd45 absolute counts and viability were evaluated by single platform flow cytometry (stem-kit, beckman coulter) and dmso was quantified by capillary zone electrophoresis (p/ace, beckman coulter). post-wash data show comparable cd34+ cell recovery, viability and effective dmso depletion. we conclude that lovo enables high efficiency dmso depletion while preserving optimal cd34 viability and recovery. comparison with sepax 2, a widely used automated centrifugebased device, reveals comparable efficiency. moreover, the length of the procedure when using the lovo does not significantly delay the process as compared to bedside thawing. we are currently evaluating lovo for the processing of multiple bags and higher cell contents, due to its ability to concentrate large volumes of cells suspension. post-thaw washing using automated cell processing systems have thus to be preferred over bedside thawing, since they provide multiple benefits including a short processing time, efficient dmso and cell debris removal, precise determination of infused cd34+ cell dose, and improved cellular stability. [p047] disclosure of conflict of interest: none. using bone marrow (bm) as the graft source results in lower graft-versus-host disease incidence, which is particularity important in haploidentical (haplo) stem cell transplantations (sct). nonetheless achieving adequate cd34+ cell count might be complicated in cases of donor-recipient weight differences. priming with g-csf may partly solve this problem. also there are reports of immunomodulatory effect of bm priming. in the retrospective study we have evaluate the effect of priming on stem cell yield and the outcomes of sct. patients and methods: 50 patients with primed bm graft were matched in the ratio 1:1 to non-primed grafts. the criteria for matching were type of the donor, age of the recipient, underlying disease and disease status at the time of sct. priming was performed with three injections of filgrastim 5-7 mcg/kg daily for 3 days prior to bm harvesting. median recipient age was 18 years (range: 1-58). 60% of patients received the graft from haplo donor, 40% from matched related donor (mrd). 39% had acute lymphoblastic leukemia, 38% had acute myeloid leukemia, 10% had aplastic anemia, 13% had other malignancies. 68% were classified as salvage patients. 27% received myeloablative conditioning, 73% received reduced intensity. post-transplantation cyclophosphamide (ptcy) was used as graft-versus-host disease prophylaxis in 83% of patients. results: the yield of cd34+ × 10 6 cells /kg of recipient weight was only non-significantly higher in the priming group: 6.0 ± 3.4 vs 5.0 ± 2.5, p = 0.12. the yield of cd34+ cells per kg of donor weight was also not different: 4.3 ± 4.0 vs 4.3 ± 5.8, p = 0.55. there was no difference in the incidence of primary graft failure (14% vs 20%, p = 0.42). median time to neutrophil (21 vs 23 days, p = 0.02) and platelet (19 vs 23 days, p = 0.05) engraftment was shorter in nonpriming group. there was no differences between priming and non-priming groups in the incidence of acute grade ii-iv gvhd (14% vs 4%, p = 0.11), moderate and severe chronic gvhd (12% vs 11%, p = 0.88), 1-year non-relapse mortality (12% vs 18%, p = 0.46), relapse incidence (36% vs 38%, p = 0.91), overall survival (64% vs 54%, p = 0.52), event-free survival (52% vs 44%, p = 0.62) and gvhd-relapsefree survival (38% vs 36%, p = 0.62). conclusions: priming of the bone marrow with reported schedule did not result in higher cd34+ cell yield and was not associated with any differences in the outcomes of sct. nonetheless, these results should be interpreted with caution, because our study included large proportion of pediatric patients, patients with active disease and ptcy as gvhd prophylaxis, and they may not translate to the other groups of patients. disclosure of conflict of interest: none. priming with granulocyte-colony stimulating factor preserves the contents and abundant ifn-γ production capacity of γδ t cells z bian 1 , q fu 1 , m huo 1 , xj huang 1 and j liu 1 1 peking university people's hospital the increasing evidences indicate that removal of αβ t-cell and b-cell from grafts was efficient and reproducible in allogeneic hematopoietic stem cell transplantation (allohsct). γδ t cell is one of the functional subpopulations preserved by this graft manipulation and supposed to play a role in improving the transplant outcomes. thus, comprehensive understanding the subsets and functional capacities of γδ t cells in graft becomes important. although there is increased attention paid on this special t-lymphocyte subpopulation, the contents and cytokine production capacities of peripheral γδ t cells before and after granulocyte-colony stimulating factor (g-csf) mobilization for allohsct have not been reported. peripheral blood (pb) before g-csf treatment, g-csf-primed pb and bone marrow (bm) grafts were obtained from 19 healthy donors. the proportions of total γδ t cells and various γδ t-cell subsets were detected by flow cytometry. furthermore, effects of g-csf on the contents and cytokines production by γδ t-cell subsets were also determined. the percentages of most γδ t-cell subsets including cd27+, cd27-, vδ1+, vδ1+cd27+, vδ1+cd27-, vδ2+, vδ2+cd27+, vδ2 +cd27-, and non-vδ1/δ2 were preserved in the g-csf-primed pb grafts compared with those before g-csf mobilization. interestingly, we found that peripheral γδ t cells and various subsets all predominantly expressed ifn-γ in response to stimulation. this abundant ifn-γ production capacity of peripheral γδ t cells were maintained after g-csf treatment. in contrast, production of il-17 by γδ t cell and its subsets were decreased in the same context. priming with g-csf preserved the contents and abundant ifn-γ production capacity of γδ t cells. our data suggests a reasonable role of γδ t cells in preventing from allohsct associated complications and may help establish an effective γδ t cell-based immunotherapeutic approach to improve the overall survival of allohsct. disclosure of conflict of interest: none. processing of hematopoietic stem cells grafts: towards automation of cryopreservation/thawing steps a-l chateau 1 , j gaude 1 , c malenfant 1 , a autret 2 , c lemarie 1 , c chabannon 1 and b calmels 1 1 centre de thérapie cellulaire-institut paoli-calmettes and 2 unité de biostatistiques-institut paoli-calmettes autologous hematopoietic stem cells (hsc) support is still widely used to allow for high-dose chemotherapy in the context of myeloma and lymphoma treatment. in the autologous setting, mobilized aphereses are systematically cryopreserved. currently, cryopreservation and subsequent thawing rely on manual and largely operator-dependent processes such as manual addition of dmso for cryopreservation or thawing in standard water baths. these operations are thus hampered by significant intra-and inter-facility variability and have to be replaced whenever possible with automated and harmonized processes. the aim of our study was to evaluate a recent, versatile device: smartmax (biosafe, eysins, switzerland), based on the peltier-seebeck effect, for its ability to automatically add the dmso-containing solution to the cell product and to thaw hsc bags. we thus compared three different cryopreservation/thawing protocols ( figure 1 ). we first evaluated the use of the smartmax at the thawing step by comparing 110 cryopreserved apheresis products thawed using our routine device: the plasmatherm (barkey), an automated dry-thawing device that contains water (protocol a), with 51 products thawed with the smartmax (protocol b); after thawing, all products were washed using the smartwash program of the sepax2 (biosafe). we then evaluated the smartmax for its ability to automatically add the dmso solution: 9 autologous grafts were processed with the smartmax, both for cryopreservation and thawing (protocol c); we compared these 9 ‛fully automated processes' to 51 apheresis processed with protocol b. absolute cd34+ and cd45+ cell counts and viability were measured before cryopreservation and after washing using single platform flow cytometry. for all three protocols, the quality of the collected product was comparable in terms of median cd45+ cell and neutrophil contents. when comparing protocols a and b, viable cd34+ cell recovery after thawing and washing was slightly lower in the smartmax group (77%) as compared to the plasmatherm group (82%, p = 0.009). when comparing protocols b and c, viable cd34+ recovery was comparable (p = 0.8) when the cryopreservation solution was automatically added by the smartmax (81%), as compared to the manual technique (77%). these preliminary data need to be validated on larger numbers of procedures, however suggest that smartmax use can safely be substituted both to the manual addition of the cryoprotectant and to the traditional thawing step in water baths; potential advantages include complete water removal from sensitive clean rooms and gmp environments. full automation of previously manual and operatordependent technical processes will ultimately allow for improved standardization and reproducibility across cell processing facilities. [p050] disclosure of conflict of interest: none. reduced efficacy of mobilisation using gdp compared to ive a hunter, w merrison, am martin, k hodgson, f miall, r moore and r lewin university hospitals of leicester, nhs trust the use of ive ± rituximab for relapsed/refractory disease in lymphoma is well established. stem cell mobilisation using g-csf post ive administration has been the standard of care in our unit for 20 years. recent interest in cisplatnin-based treatments has seen a change in practice with the use of gdp ± rituximab increasingly common. we have assessed the success of stem cell mobilisation post gdp and compared it to ive using g-csf. patients were eligible for augmentation with plerixafor if their peripheral blood cd34 levels were between 5-10 × 10 6 cells/l at the time of collection. from sept 15 to oct 16 21 patients with progressive or relapsed lymphoma underwent stem cell collection. patients characteristics: 12 dlbcl, 4 follicular and 5 t-cell nhl. had a median age 65 (37-74 years). 13 received gdp, 8 ive. overall 23% patients failed to mobilise a sufficient cd34 cell dose to proceed to hdt. all the patients who received ive mobilised successfully but 5/13 (38%) patients receiving gdp failed to mobilise. of the patients who did mobilise the average cd 34 collection was higher in the patients who received ive 7.1 (4.5-25.4) and the number of apheresis procedures was lower, median 1 (1-2) compared to 3.4 (2.6-9.1) and 2 (13), respectively, in the gdp group. patients in the gdp group who failed to mobilise were not eligible for plerixafor because cd34 levels were below 5 × 10 6 /l. taking age into account the median age in the ive group was higher 65(48-74) than the gdp group 62 (37-74) and the lines of previous therapy were not different. patients who had successful stem cell collections went on to receive hdt with leam and all patients engrafted. in this small collection of patients we have experienced a higher failure of mobilisation post a cisplatnin-based protocol compared both to our historical controls pre plerixafor usage (data not shown) but also to current patients. further investigation is needed to ascertain the impact of cisplatnin on stem cell mobilisation and its impact of treatment strategies. disclosure of conflict of interest: none. single centre experience of zarziotm biosimilar granulocyte-colony stimulating factor (gcsf) for the mobilisation of healthy donors demonstrates good leukapheresis yields and safety profile at 24 month median follow-up jg taylor 1,2 , t seddon 2 , k alizadeh 2 , c agrawal 2 , l kempster 2 , jg gribben 1,2 and sg agrawal 2,3 1 centre for haemato-oncology, barts cancer institute, 2 dept. haemato-oncology, st bartholomew's hospital, london, uk and 3 experimental pathology, blizard institute, queen mary university of london, uk biosimilars have led to significant improvements in the affordability of growth factors such as granulocyte-colony stimulating factor (gcsf). data has shown similar performance and efficiency to parent drugs but concern has been raised about their use in healthy donors due to lack of data examining adverse effects in this setting. we conducted a retrospective analysis investigating mobilisation and adverse effects in 51 healthy sibling donors of adults undergoing an allogeneic haematopoietic stem cell transplant at st bartholomew's hospital from 2011 to 2014. harvest data were gathered from hospital records. adverse effects data were gathered from hospital records and telephone follow up. 58% of donors were male with a median age at harvest of 46 (13-65). all donors were mobilised using zarziotm biosimilar gcsf at a dose of 10 μg/kg/day. median number of apheresis required was 1 (1) (2) (3) . median cd34+ cell count was 5.6 × 10 6 /kg bodyweight (1.3-13.9) with 1664 × 10 6 cd34+/μl (168-3779) in peripheral blood. the target cd34+ count (45 × 10 6 /kg) was achieved in 59% of donors and an adequate yield (2-5 × 10 6 /kg) in 33%. in four donors (8%), the harvest was deemed to have been unsuccessful as the cd34+ count was o2 × 10 6 /kg. the patients with donor harvest yields o2 × 10 6 /kg proceeded to transplant; all four patients engrafted and one patient had mixed chimerism at day 28 but was fully donor by day 75. median cd3+ cell count was 2.7 × 10 6 /kg bodyweight (0.8-6.4) . median days to neutrophil engraftment (40.5 × 10 9 /l) was 16 . median days to platelet engraftment (420 × 10 9 /l) was 5 (0-23) with one patient never engrafting. forty (80%) of 51 donors were contacted at a median of 24 months (1-54) post mobilisation to establish incidence of adverse effects. three donors were uncontactable as they had moved overseas. eight donors were not contacted to avoid distress as their sibling had died since transplant. among contacted donors 42.5% reported side effects including bone and lower back pain controlled with analgesia, constipation and low mood. other side effects included chest pain which was considered to be musculoskeletal in origin on day 3 of gcsf administration associated with taking an increased dose due to patient error (n = 1) and abdominal contractions like labour while receiving gcsf (n = 1). three (7.5%) reported side effects lasting beyond one month post mobilisation: lower back pain lasting 2 months (n = 1), fatigue of 3 months duration (n = 1), and cough of 8 months duration (n = 1). our data demonstrates good mobilisation using 10 μg/kg/day zarziotm biosimilar gcsf without significant adverse effects at 2 years median follow up. this supports its ongoing use for the mobilisation of healthy donors. disclosure of conflict of interest: sga has received honoraria from sandoz and grant support from sandoz and amgen. stem cell mobilization in poor mobilizers with multiple myeloma (mm) or non-hodgkin lymphoma (nhl) before and after introduction of plerixafor: single center comparative analysis using a cost-efficient single fixed-dose schedule r wäsch 1 , c greil 1 , c kiote-schmidt 1 , s hildenbeutel 1 , k kühbach 1 , r bosse 1 , j duyster 1 and m engelhardt 1 1 department of hematology, oncology and stem cell transplantation, university medical center, freiburg, germany collection of hematopoietic stem cells (hsc) from the peripheral blood (pb) is routinely conducted prior to highdose chemotherapy and autologous transplantation. despite safety and efficiency of current apheresis procedures including mobilizing chemotherapy and granulocyte colony-stimulating factor (g-csf), there is a significant rate of mobilization failures due to different patient-dependent factors necessitating additional agents like plerixafor. while plerixafor is approved for patients with mm or nhl based on prospective studies using steady state mobilization with g-csf − /+ plerixafor, prospective studies using chemo-mobilization are lacking. here we compared the outcome of poor mobilizer from the pre-plerixafor era with poor mobilizers who received additional plerixafor in a real world analysis. we analyzed 50 consecutive patients with mm or nhl who were mobilized at our academic center between 2011 and 2016 and received plerixafor, because they were expected to be poor mobilizers, due to 1. low counts of cd34+ cells in pb samples prior to apheresis, 2. after a first apheresis day with insufficient yield or 3. as a rescue strategy after insufficient harvest with previous mobilizing chemotherapy (greil c,…engelhardt m, wäsch r. leukemia & lymphoma 2017, in press). we examined cd34+ cell counts in pb and in apheresis products to identify those patients who were able to collect a sufficient cd34+ cell count for transplantation after application of plerixafor. we compared these data with 100 consecutive poor mobilizers from the pre-plerixafor era, who were mobilized between 2000 and 2011 without plerixafor. the median pb cd34+/μl count at first apheresis was significantly higher after the first dose of plerixafor when compared to the pre-plerixafor group with 19.9 vs 9.8 (p o0.001). accordingly, the median collected cd34+ cells/d (×10 6 /kg bw) and total cd34+ cells (×10 6 /kg bw) were significantly increased with 1.67 vs 0.88 (p o0.001) and 4.13 vs 2.66 (p o0.001), respectively. the rate of 42 × 10 6 cd34+ cells/kg bw in first apheresis (%) increased from 11% in the pre-plerixafor era group to 38% after the first dose of plerixafor in the plerixafor group. consistently, the successful transplantation rate increased from 58% in the preplerixafor group to 90% in the plerixafor group. successful stem cell mobilization could be achieved with only a single fixed-dose of plerixafor in 62% of poor mobilizers as previously reported by our group. the addition of plerixafor to chemomobilization in poor mobilizers with mm or nhl significantly increased pb cd34+/μl counts, apheresis yields and transplantation rates when compared to poor mobilizers from the pre-plerixafor era. these favorable apheresis results can be obtained using our cost-efficient, single fixed-dose plerixafor schedule in the majority of the patients leading to a 90% transplantation rate in poor mobilizer. disclosure of conflict of interest: rw received research funding, advisory and speaker's honoraria from sanofi-aventis. high-dose chemotherapy followed by autologous peripheral blood stem cells transplantation (pbsct) is the standard of treatment for patients with hematological malignancies. recombinant granulocyte colony-stimulating factors (g-csfs) are widely used alone or in combination with chemotherapy, in order to mobilize patient's stem cells (cd34+) for autologous and allogeneic peripheral blood stem cells transplantation. aim: the aim of our study was to compare effectiveness and safety of different biosimilar products of filgrastim used in autologous pbsc mobilization in patients with hematological malignancies. our retrospective analysis included 282 patients (118 women and 164 men) with median age 54 years ( range: ,who underwent the procedure of autologous pbsct in years 2012-2014 in the haematology, blood neoplasms, and bone marrow transplantation clinic of medical university in wrocław. there were three different biosimilar products of filgrastim used: tevagrastim (teva) in 95 patients, nivestim (hospira) in 92 patients and zarzio (sandoz) in 95 patients. 90 (32%) patients were diagnosed with plasma cell neoplasms, 145 (51%) with hodgkin's and non-hodgkin's lymphomas, 20 (7%) patients had acute myeloid leukemia and 27 (10%) had other hematological malignancies. statistical analysis was conducted using statistica 12 (statsoft polska) statistical software. for quantitative variables arithmetic means and standard deviations were calculated for the estimated parameters in the studied groups. distribution of variables was tested using w-shapiro-wilk test. p0.05). there were also small variations in the mean number of leukapheresis necessary to obtain the minimum cd34+ cell count: 1.32 in zarzio group, 1.37 in nivestim group and 1.66 in tevagrastim group. however, there were no difference between biosimilar g-csfs. the highest rate of successful mobilizations (defined as 42 × 10 6 /kg cd34+ cells collected) was observed in 88.2% patients received zarzio, in 86.2% received nivestim and in 84.9% patients received tevagrastim. the safety profile was comparable between the biosimilar g-csf and included bone pain in 30 (10%) patients and headache in 25 (9%) patients. the results are shown in table 1 . all three used biosimilar g-csfs demonstrated similar efficacy and safety in stem cell mobilization in patients with hematological malignancies. therefore, it seems that all the analyzed products can be used interchangeably. presented observations should be verified with wider prospective research. [p055] disclosure of conflict of interest: none. use of g-csf stimulation of bmt donors might prove to be beneficial in many respects, improving tnc yield but also through immunomodulatory effect on donor t cell function and apcs1. we analyzed outcomes of 14 consecutive patients receiving bone marrow transplants from hla-haploidentical related donors that received g-csf stimulation prior to harvest. fourteen patients received hla-haploidentical bmt with pt-cy between 5/2012 and 10/2016. five donors were siblings, 4 children, 4 mothers and 1 father. donors received g-csf at the dose of 10 mcg/kg bw sc. on days − 2, − 1 and 0 before bm collection. twelve patients received nonmyeloablative conditioning according to baltimore protocol2, while two patients received myeloablative conditioning (bucy). along with post-transplantation cyclophosphamide, all patients received tacrolimus and mmf form day +5, as described earlier 2. median age was 41 years (range: 19-63), 7 female and 7 male patients. eight patients had aml, 1 cml, 4 mh and one all. ten of them were in remission, while 2 mh patients were in pr, and 2 aml patients had residual disease as evident by immunophenotyping. median number of infused tnc was 5.17 × 10 8 /kg bw (range: 1.84-8.21); cd34+ cells 1.88 × 10 6 /kg bw (range: 1-4.47 ) and cd3+ cells 1.35 × 10 7 /kg bw (range: 0.37-6.04). median follow up was 362 days (range: 26-1654). eleven patients engrafted (79%), one patient had primary rejection, one had overt disease relapse at day +35 and one patient died in aplasia due to sepsis. median day to neutrophil recovery (anc 40.5 × 10 9 /l) was 21 (range: 15-29), median days to platelet recovery (plt 420 × 10 9 /l) was 31 (range: 12-45). in all patients mmf was discontinued at d +35. two patients developed acute gvhd in our cohort (18%), one after receiving dli for falling chimerism at day +169. one patient (9%) developed chronic gvhd, after having received dli due to disease progression. at the time of analysis 10 patients are evaluable; 4 patients had disease relapse/progression (40%), 6 patients are alive and in remission. one patient died due to sepsis in aplasia (accounting for 7% non-relapse mortality). one patient that rejected the graft was transplanted again from the same donor, using myeloablative conditioning and peripheral stem cells as graft source and engrafted. overall survival median is 2.7 years, with significantly shorter survival if patient was not in complete remission at time of transplant (p o0.01). even though the experience with g-csf mobilized bm graft in the hlahaploidentical setting with pt-cy is relatively small, in our series it has been beneficial in terms of tnc yield. also, the incidence of acute and chronic gvhd in our patients has been low, particularly agvhd with one case developing only after dli. whether the observation is the result of limited number of patients, or it reflects the immunomodulatory effect of g-csf on bm graft as previously suggested1 remains to be seen as further studies are warranted. autologous transplantation of haematopoietic stem cells (ahsct) is usually perceived as a fully standardized and safe procedure; however, a minority of patients experience a delayed engraftment and seldom even an engraftment failure, possibly related to a poor quality of the graft. therefore the current policy in many centers is aimed to increase the target dose of collected cd34+ cells up to an ‛optimal' level of 4 × 10 6 /kg. plerixafor was introduced in the clinical practice to maximize the mobilization of hsc, in order to collect an optimal number of cd34+ cells in a limited number of collections also in poor and slow mobilisers. we carried out a retrospective analysis of our case series aimed to individuate mobilization predictors optimize the ‛on demand' use of plerixafor. we analyzed 162 patients who underwent mobilization with cyclophophamide (4 g/sqm) and filgrastim 10 mcg/kg from +5 in our unit from 2009 and 2016. diagnosis were multiple myeloma (mm) 74 (45.7%), non-hodgkin lymphoma (nhl) 46 (28.4%), hodgkin lymphoma (hdg) 14 (8.6%) and 28 (17.3%) autoimmune disease (ms 14.8%; ssc 2.5%). median age (range) was 53 years ; male/female ratio 82/80. circulating cd34+ cell count was started at white blood cells (wbc) recovery, which was defined as the first day when their count exceeded 1 × 10 9 /l. the primary goal was to identify at wbc recovery one or more factors predicting a suboptimal mobilization, which was defined as the failure to exceed 40 cd34+/mcl circulating cells in the day after the wbc recovery. patients were excluded from this analysis if 1) showed a cd34+ count 440/mcl at wbc recovery (very good mobilizers) and/or 2) had received plerixafor and/or 3) did not proceed to another cd34+ count the day after wbc recovery. binary logistic regression was used to obtain the factors that increased the odds for an optimal mobilization. overall 80 out 162 (49.4%) patients were shown as very good mobilisers as their cd34+ count exceeded 40/mcl at wbc recovery. on the remaining 82, 7 were excluded for the lack of a second assessment and 2 for the lack of data. among the remaining 73 patients, the threshold of 40 cd34+/mcl cells on the second day was reached by 55 (75.3%) of patients (group a) while the remaining 18 (24.7%) failed the goal (group b). median (range) wbc × 10 9 /l and cd34+/mcl counts in group a and b at wbc recovery were 2.01 (1-6.43) and14.65 (0.70-39.91) and 2.84 (1-20) and 7.39 (0-33), respectively, with a statistically significant differences among group (mann-whitney u test with p = 0.01 and p = 0.02, respectively). wbc (or = 2.193; 95% ci: 1.197-4.019) and cd34+/mcl (or = 0.858; 95% ci: 0.77-0.955) in first day count, but not gender, disease category and time from mobilization chemotherapy to first cd34+ count, were predictors of optimal mobilization. combining these two predictors we found that wbc/cd34+ ratio has a sensitivity of 82.4% with an auc 83.7 in roc analysis. assessment of circulating wbc, cd34+ and their ratio at wbc recovery in a chemo-based mobilization strategy can predict sub-optimal mobilization of hsc and support the decision of adding plerixafor. these data will be prospectively validated in a broader set of patients. disclosure of conflict of interest: none. human platelet lysate (hpl) is rich in growth factors (gf) and nutritive elements and represents a powerful xeno-free alternative to fetal bovine serum (fbs) notably for mesenchymal stem cell (hmsc) proliferation. however, there is a large variability in hpl preparations (various sources, use of different and non-standardized production protocols, with variable and limited number of donors), resulting in discrepancies in product quality, low management of product safety and poor batch-to-batch standardization. we describe here the development and the characterization of a standardized hpl prepared from outdated transfusional grade screened normal human donor platelet concentrates (pcs), manufactured on an industrial scale (batch size of 250 donors) and following a highly qualified process (clean room, trained operators, validated aseptic filtration). pcs were frozen at − 80°c and thawed at +4°c to lyse platelets. cell debris were removed by centrifugation and the supernatant (hpl) was recovered. clinical grade 10l batches of aseptic filtered hpl were characterized. first, we showed that hpl prepared from a limited number of donors displayed a variability in terms of gf contents. on the contrary, we observed a robust standardization between industrial batches of hpl (250 donors) in terms of gf contents (bfgf, egf, vegf, pdgf-ab, tgf-beta1 and igf-1), biochemical analyses (total proteins, albumin, fibrinogen, vitamins and iron) and efficacy on bone marrow (bm)-hmsc proliferation. secondly, we compared expansion and functional characteristics of bm-hmscs grown in clinical grade hpl vs msc-screened fbs batches. we showed a reproducible increase in cell growth kinetics using hpl, a maintenance of bm-hmsc clonogenic potential and membrane marker expression (with however a strong overexpression of cd90). we observed a similar adipogenic and osteogenic differentiation potential and finally that immunosuppressive properties of bm-hmscs (inhibition of t-cell proliferation) cultivated in parallel in both conditions also remained identical. finally, we demonstrated the stability over time of hpl stored at − 80°c and − 20°c. in conclusion, we demonstrated the feasibility to use a standardized, characterized, efficient and clinical grade hpl for research and cell therapy applications. disclosure of conflict of interest: sv, se, lc, pb, tb, al, fg and bd are employees of macopharma. previously published p061 alpha/beta t cell depleted donor lymphocyte infusion m karakukcu, e ünal, l kaynar, s özcan, g tezcan karasu and mb acar the main objective of this project is to improve a safe and efficient new donor lymphocyte infusion (dli) with depletion of αβ+ t cells which cause graft versus host disease (gvhd), and enrichment of anti-leukemic γδ+ t cells, nk cells and dendritic cells to build an effective and permanent anti-tumor effects for patients relapsed hematological cancers after allogeneic hematopoietic stem cell (hsc) transplantation who have blasts and mixed chimerism. this study is conducted with collaboration of erciyes university pediatric and adult hsct units, and bahcesehir university, medical park hospital pediatric hsct unit. the tcr αβ+ t cell depleted dli product that is used in the study was collected and separated at erciyes university apheresis unit. the cell contents obtained for tcr αβ+ t cell depleted dli used for patients were cd3 cells were reduced to 1.4-23.5 × 10 6 cells/kg, γδ+ t cells were reduced to 2.58-23.48 × 10 6 cells/kg, αβ+ t cells were reduced by 99.99%, and were obtained at 5-4100 cells/kg. a total of 10 patients (4 female, 6 male) were included in the study, consisting of an adult and 9 children. nine patients had hematological malignancies. five patients were referred for all, three for aml, one for mds and one for griscelli syndrome. efficiency: the clinical response to the αβ+ t cell depleted dli treatment was achieved in 7/10 patients (70%). in these patients, although the increase of chimerism was limited in 2 patients, no recurrence was occurred. one of the two patients who previously responded to the treatment but experience of decreasing chimerism had relapsed after 2 months, and 9 months later. one of these two patients died after relapse. the other was managed by the second transplant. the most important objective of this study was to show that αβ + t cell depleted dli treatment is reliable. none of the patient showed severe gvhd except one patient with mild grade ii gvhd. despite the presence of severe gvhd after hsct in two patients, reactivation for gvhd was not observed after treatment with αβ+ t cell depleted dli. none of the patients had a bone marrow aplasia. as a result, αβ + t cell depleted dli treatment seems to be highly safe, and effective in selected patients. disclosure of conflict of interest: none. hematopoetic stem cell transplantation (hsct) is associated with several potentially lethal complications; for example, relapse of the malignant disease, graft rejection, infectious complications and graft versus host disease (gvhd). higher levels of cd3+ cells in the graft have clearly been associated with increased risk of gvhd, but also superior gvl effect and less infectious complications. to tackle post-transplant complications such as graft failure and relapse, donor lymphocyte infusion (dli) have successfully been used for decades but with an associated risk of gvhd. to decrease the risk of gvhd but still use facilitating cells in the cell product we performed αβ depletion of grafts for use as stem cell booster after allogeneic hsct to treat infections or poor immune reconstitution. in this study, 11 patients were infused post-hsct with αβ t-cell depleted grafts. the indication for infusion of αβ t-cell depleted graft in all patients was poor immune reconstitution with associated infectious complications. for all 11 patients, the original hsct donor was used for the αβ t-cell depleted boost. to characterize the αβ-depleted stem cell grafts, samples were stained for various cellular subsets and analyzed by flow cytometry. we could show a median log depletion of αβ cells of 4.1 and a median yield of γδ t-cells (%) of 61. 4 . the median cd34+ cell dose (×10 6 /kg) was 5.5. all 11 patients were alive 3 months after infusion. after 1 year only one patient succumbed. despite that the majority of patients suffered from agvhd grade 2 or 3 before infusion of αβ t-cell depleted graft none showed increased symptoms afterwards. in more than 70 % of the patients there was a increase in granulocytes, thrombocytes and white blood cells 3 months after infusion. in conclusion, we describe the use of αβ t-cell depleted grafts as stem cell booster in 11 patients suffering infectious complications due to graft failure after hsct with encouraging results. disclosure of conflict of interest: none. delayed engraftment or graft failure still remains a concern in bone marrow transplantation (bmt). graft composition may predict engraftment after infusion. this study aims to determine which quality control parameters used for the characterization of bone marrow grafts are the most predictive in order to minimize the risk of engraftment delay or graft failure. we conducted a multicenter retrospective study in pediatric patients who underwent first allogenic bmt at two centers in barcelona (catalonia, spain) between 2011 and 2015. quantitative variables considered for the study were: total nucleated cells (tnc), mononucleated cells (mnc), cd34 + cells, cd3+ cells and granulocyte-monocyte (gm) colonies enumeration. qualitative variables considered for the study were viability assessed by flow cytometry and clonogenic efficiency of the cd34+ cells (clonegm) which is the ratio between gm colonies and cd34+ cells. 85 patients were included (median age (range) were 7 years old (0) (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) ). the median tnc(range) was 4.26e8/kg (0.51-29.18e8/kg) and 1.1e8/kg (0.48-5.88e8/kg) for mononuclear cells (mnc). on the other hand, the median (range) cd34+ cell dose was 5.03e6/kg (0.85-24.91e6/kg) and t-cell dose (cd3+) was 0.41e8/kg (0.10-44.86e8/kg). the median (range) colonyforming unit granulocyte macrophage (gm/kg) dose was 4.35e5/kg (0.33-48.62e5/kg). the median (range) of cd34+ cell viability, was 95% (63-99%) and the median(range) of the clonogenic potential of cd34+ cells (clonegm) was 10.35% (1.22-76.17%). the median (range) of engraftment was 22 days for neutrophils and 20(11-38) days for platelets. 1 patient was considered as primary graft failure. in the univariate analysis, cd34+ (p = .049) and mnc (p = .045) cell dose predicted a faster neutrophil engraftment and female donor a slower neutrophil and platelet engraftment (p = .012 and p = .040). cell viability also correlated to a better platelet engraftment (p = .030). in the multivariate analysis we observed a trend for a faster neutrophil recovery according cd34+ cells infused. again, female donor was associated with slower engraftment. in order to establish a safety threshold, we did a quartile analysis of cd34 dose and found 3.18e6/kg (quartile 25) discriminates a faster neutrophil engraftment [median 25 days vs 21 days for those with higher cd34+ cells (p = .026)]. in conclusion, we found an association between mnc and cd34+ cell dose and time to engraft, and established a safety threshold of 3.18e6 cd34+/kg. also, bm grafts from female donors were associated with slower engraftment. no other qualitative parameters were predictive of engraftment. disclosure of conflict of interest: none. plasma cell myeloma (pcm) is currently treated with chemotherapy and autologous stem cell transplantation (asct) but relapse rates remain high. adoptive transfer of mature haploidentical natural killer (nk) cells is a promising approach to provide pcm patients with highly immunocompetent effector cells with anti-myeloma function early post transplantation. here we report on the current clinical phase i/ii trial of multiple preemptive infusions of good manufacturing practice (gmp) expanded nk cells to pcm patients (clinicaltrials.gov nct01040026). ten pcm patients were recruited (seven males, three females, median age: 59y). all patients received four cycles of vtd chemotherapy (reaching cr: 4 × , vgpr: 5 × and pr: 1 × ) before high dose therapy with melphalan 200 mg/m 2 and asct. after successful stem cell mobilization and cryopreservation of patients' stem cells after the third vtd cycle, nk cells from haploidentical family donors were purified from unstimulated leukapheresis by t cell depletion and nk cell selection using clinimacs. highly pure nk cells (mean: 4.8 × 10 8 cells) were obtained with a minimal t cell contamination corresponding to a 6.1 log t cell depletion. nk cells were expanded ex vivo for 19 days in gmp-medium containing autologous irradiated feeder and interleukin-2 and -15. nk cell numbers increased 54-fold (range: 38-76-fold). in three nk cell products t cell contents were 11 × 10 5 cells/kg body weight (bw: 10 × above limit of clinical trial) and were successfully reduced by 2°cd3-depletion to 0.3 × 10 5 cells/kg bw. nk cell products were cryopreserved in escalating doses (1.3 × 10 6 , 1.3 × 10 7 and rest as multiple doses of maximal 1.0 × 10 8 cells/ kg bw). the pcm patients received 65-460 × 10 8 expanded nk cells (median: 3.8 × 10 8 cells/kg bw, range: 0.9-5.7 × 10 8 cells/ kg bw) as 3-8 infusions (median. 6 dlis). the nk-dlis were administered between day 2 and 21 after asct and were well tolerated without any acute adverse events. no signs of acute or chronic graft-versus-host disease were observed in any of the patients after a total of 57 nk-dlis. engraftment occurred between days 13-24 (median: 16 days). infused donor nk cells were monitored by short-tandem repeats pcr. donor nk cells were detected in peripheral blood one and 20 h post infusion (% donor nk of enriched blood nk cells: mean: 30%, range: 9-90%, and mean: 17%, range: 0-33%, respectively) indicating significant nk cell survival in recipients in the absence of il-2 support in vivo. clinical responses at last follow-up compared to a retrospective cohort of matched control patients will be presented. these results demonstrate the feasibility of large-scale gmp expansion and safety and immunotherapy with third-party leukemia-specific t cells (leuk-sts) represents an attractive approach for acute leukemia (al) patients lacking a fully matched donor or relapsing after allogeneic hematopoietic cell transplantation (hct). its application however, is limited by the demand for high numbers of antigen presenting cells (apcs), capable to produce clinically relevant numbers of leuk-sts. low volume, non-transplantable cord blood units (cbus) could theoretically serve as an easily accessible source to generate high numbers of dendritic cells (dcs) and subsequently leuk-sts, providing also the advantage of reduced alloreactivity, even in cases of partial matching. our goal was to generate clinically relevant doses of leuk-sts targeting al-related antigens, the wilms tumor protein (wt1) and the preferentially expressed antigen in melanoma (prame), through the exploitation of non-transplantable cbus. to generate dcs, immunomagnetically enriched cd34+ cells from cbus ⩽ 70ml were cultured in g-rex devices in the presence of scf, gm-csf and il-4. dcs matured by toll-like receptor ligand 3 and 7/8 were immunophenotypically characterized by flow cytometry (fcm). secreted cytokines were measured with elisa. matured dcs were activated with a peptide-mix of wt1 and prame and used as apcs to repeatedly stimulate naive t-cells (derived from the cd34fraction of the same cbu). the phenotype and the specificity of generated leuk-sts were determined by fcm and ifn-γ/ elispot, respectively. starting from mean 4.2 × 10 5 ± 1.1 × 10 5 cd34+ cells, from 4 non-usable cbus, we generated 3.3 × 10 9 (range:1.9-5.7 × 10 9 ) myeloid dcs (cd33+/cd11c+:76.8 ± 5.5%) in 35 days (fold change~11.000). the produced cells highly expressed maturation markers (cd11+/cd40+:79 ± 12%; cd11c+/hla-dr+:78 ± 10%) and secreted high levels of th1cytokines (ιl-12:224 ± 185 pg/ml; il-6:1.9 ± 0.1 × 10 5 pg/ml, tnf-α:5268 ± 1316 pg/ml) and low levels of the th2-cytokine, il-10. the average number of cd34-cell-derived leuk-sts after 4 week-culture was 7.5 ± 3.4 × 10 7 (~2 logs above clinical doses). the produced cells were enriched in cd3+ polyclonal cells (80 ± 7%), comprising of cd4+ (28 ± 10%) and predominantly cd8+ cells (52 ± 17%), expressing effector memory (cd45ra − /cd62l − :52.8 ± 5%) and effector memory ra markers (temra: cd45ra+/cd62l − :46 ± 4%), while containing insignificant numbers of cd4+/cd25+cells (1 ± 0.5%). specificity was seen after the second stimulation at the earliest and was increasing after each stimulation [mean spot forming cells (sfc)/2 × 10 5 cells at second, third, fourth stimulation: 106 ± 33; 422 ± 111; 1335 ± 314; respectively]. in particular, produced cells were highly specific for both targeted antigens (prame:1019 ± 275, wt1:316 ± 55), while they expressed low the programmed cell death protein-1 (cd3+/pd-1+:9 ± 4%), implicating absence of cell exhaustion after repeated stimulations. we report a paradigm of ‛circular economy' in science, by the exploitation of non-usable cbus, towards scalable generation of cb-cd34+-cell-derived dcs and cb-cd34-cellderived leuk-sts from the same cbu and establishment of leuk-sts banks. whether similarly produced leuk-sts could significantly advance the treatment of al or leukemic relapse after hct, will be ultimately determined in vivo. disclosure of conflict of interest: none. comparison of two different methods to generate antifungal-specific t-cells under pre-clinical-scale conditions r geyeregger 1 , s tischer 2, 3 invasive infections with aspergillus fumigatus constitute a major cause of morbidity and mortality in immunocompromised patients after haematopoietic stem cell transplantation. although adoptive immunotherapies against viral pathogens are already in phase i/ii trials, clinical-grade methods for the generation of aspergillus-specific t-cells (asp-t-cells) from healthy transplant donors or even related or unrelated thirdparty donors are still under development. in this study, two different strategies interferon-gamma (ifn-g) cytokine capture system (ccs) vs short-term in vitro expansion (ste) were performed from the same healthy volunteers in order to evaluate the most suitable approaches for the in-time generation of clinical applicable asp-t-cells. pbmcs from leukapheresis of healthy donors (n = 6) were first prepared in hannover for the ifn-g-ccs and then sent to vienna to prepare the ste. all donors belong to the allocell registry (www.allocell.com) of hannover medical school and the frequency of asp-t-cells was pretested by high-throughput ifn-g elispot assay. for the ifn-g-ccs, 1 × 10 7 cells were stimulated for 16 h with gmp-conform aspergillus lysate followed by magnetic selection of ifn-g-producing t cells. cells were characterized for phenotype and function by multicolour flow cytometry. for the ste, 20 × 10 7 cells were cultured in g-rex devices and stimulated for 12 days with either the aspergillus lysate alone or with pooled overlapping pepmixes (catb, crf1, f22, gel1, pmp20, shmt and sot) and il-15. to further characterize the final cell products, multicolour flow cytometry, ifn-g elispot and ifn-g/granzyme b flurospot analyses were performed. ifn-g-ccs: frequency of ifn-g positive asp-t-cells pre-magnetic enrichment ranged between 0.07 and 0.16%. recently we defined t-cell donors as eligible if ⩾ 0.03% specific ifn-g+ t cells are detectable. the purity of asp-t-cells among cd3+ cells, obtained from three donors after magnetic selection was in mean 64% ± 3 (range: 58-69%). the absolute number of selected ifn-g+ cd3+ t-cells was 706 ± 194. this could be approximately multiplied by a factor of 100, if 41 × 10 9 pbmcs are used for the generation of clinically applicable t cells using the ccs and the prodigy device. ste: after 12 days, asp-t-cells (n = 3) showed highly specific activity against the lysate (in mean 1339 ± 79 spot forming colonies (sfc)/105 cells) and pooled pepmixes (in mean 892 ± 276 sfc/105 cells). in both methods (lysate vs pooled pepmixes), predominantly cd4+ t-cells were expanded (84% ± 2.3 vs 82% ± 5.3 of cd3+) compared to cd8+ t-cells (12.6% ± 2,9 vs 14.7% ± 5.3). interestingly, whereas after ste, cd4+ t-cells include mainly central memory t-cells (mean 40%; cd62l+cd45ra − ), cd8+ t-cells include mainly effector memory t-cells (27%; cd62l − cd45ra − ). generated t cells were highly functional and cytotoxic as determined by the secretion of effector molecules granzyme b and ifn-g. based on the purity of up to 69% after the ifn-g-ccs and the high number of sfc received after ste with lysate and pepmixes, both methods seem to be suitable for clinicalscale productions. for patients who are in need for high asp-tcell numbers the application of first in-time ccs-purified asp-t-cells followed by the administration of ste cells might be a promising way to boost antigen-specific t-cell response. disclosure of conflict of interest: none. complete computerization of cell therapy product files (‛zero paper') in the qap 10 software o christéle 1 , r catherine 2 , r aline 3 , k mathias 4 , m lavinia 3 , d vincent 5 , m jean-pierre 6 and l marie-noelle 7, 8 1 hematologie clinique et thérapie cellulaire-chu amiens picardie, 2 simedia-ver, 3 hématologie clinique et thérapie cellulaire-chu amiens picardie, 4 simédia-ver, 5 direction système informatique-chu amiens picardie, 6 hématologie clinique et thérapie cellulaire, 7 lacassagne and 8 the computerized management of cell therapy products (ctp) is an obligation for processing laboratories to meet regulatory requirements. the software used is often independent of institutional systems in view of the specificity of cellular therapies and do not always allow the implementation of the ‛zero paper' policies that are being put in place. we report here our experience with the qap10 software (quality assurance partner) developed by the company simédia (www. qap10.com) in open source (mit license) allowing the management of fully computerized ctp files. the qap10 software has been developed to ensure the traceability of ctp for both preparation and quality control by combining the product preparation environment (personnel, premises, reagents, consumables, equipment). initially, with the help of the company simédia, we parameterized the software in accordance with our procedures for the preparation and quality control of ctp. we built a file that we printed out for archiving on paper. it soon seemed necessary to reverse this mode of operation to add to the software the documents papers to obtain a file completely computerized and to avoid paper archiving. the close collaboration between the cell therapy laboratory staff, the software referent within the information system department of the amiens hospital and the company simédia enabled: set up a document backup server sufficiently proportionate in memory. have simedia carry out the necessary developments so that all documents can be integrated into the software, set up a coherent working circuit, organize the registration of documents, put in place a rigorous verification of the mandatory elements of the file. the reflection on the computer file made it possible to evolve the software to widen its use to all documents of management of the laboratory: maintenance of equipment, control of premises, housekeeping, staff training, quality control of automatons, reagents and consumables, process, reception, distribution. rigorous formalization was mandatory to ensure that the record was organized in a uniform manner. an intermediate paper record is still necessary for a period of about 1 month: from the programming of the graft to the final validity of the injected product. this folder consists only of transient elements that cannot be integrated into the qap 10 software immediately. the transition from the paper file to a computer file took place in several stages, calling into question our functioning. the difficulties of this implementation are of several natures: the heterogeneity of the documents components a cell therapy product file, the impossibility of benefiting from an interface between all computer software used on the hospital, the psychological barrier prompting us to keep a paper copy, work habits, the guarantee of computer backup quality as well as its verification. but the complete computerization of the ctp file has the following advantages: easy and secure accessibility of information, resolution problems archiving paper files, a single backup media folder. disclosure of conflict of interest: none. conditioned media from allogenic mesenchymal stem cell culture (msc-cm) enhances wound healing in an allogenic 3d skin model moyasasr al-shaibani, x wang 1 , p lovat, a tulah and a dickinson 1 newcastle university migration of the epidermal layer towards the wound centre is an important step in the healing process. full thickness in vitro skin models can be used to investigate epidermal migration towards an injury site. since wound healing therapies often require allogenic transplantation of primary keratinocytes, an allogenic 3d skin model was developed to investigate epidermal migration. the effect of mesenchymal stem cell conditioned media (msc-cm) was assessed for wound healing using this in vitro human 3d skin model. human mscs were derived from human hip joints, and characterised using standard protocols. at 80% confluence, msc secretions were collected in serum free medium and referred to as msc-cm which were then analysed for protein content using elisa. fully humanised allogenic 3d skin models were developed (n = 3) and a 3 mm punch was induced into each model followed by daily treatment with msc-cm to investigate the migration of the epidermal layer towards the punch centre over the dermal layer at different time points (1 week, 2 weeks, and 4 weeks). intact and wounded models were characterised structurally by haematoxylin/eosin (h&e) staining and immunofluorescence (if) was used to validate the dermal and epidermal biomarkers such as collagen 3 (col3), cytokeratin 14 (k14), keratin 10 (k10), loricrin and involucrin. mscs were characterised as stipulated by the international society for cell therapy, that is, fibroblast like cells with the ability to differentiate into tri-lineages (adipocyte, chondroblast and osteoblast). phenotypically, over 95% of the cells were able to express phenotypic markers for variant stem cells such as cd73, cd90 and cd105. over 95% of the cells were negative for the expression of cd14, cd19, cd34, cd45 and hla-dr (p = 0.025). msc-cm contained different concentrations of a variety of growth factors such as keratinocyte growth factor (kgf), hepatocyte growth factor (hgf), platelet-derived growth factor (pdgf), stromal-derived factor-1 (sdf-1) and macrophage stimulating protein-1 (msp-1). h&e staining showed that the models had distinct dermal and epidermal layers similar to that of real skin. additionally, if showed that the models expressed dermal and epidermal biomarkers, for example, col3, k14, k10, loricrin and involucrin. after treatment with msc-cm, the epidermal multilayers of the punched models started to migrate towards the punch centre and covered the whole punched area after 4 weeks of treatment with recovered expression of the epidermal biomarkers, for example, k14, k10, loricrin and involucrin. a fully humanised allogenic 3d skin model is a useful tool to mimic the in vivo environment and evaluate the wound healing process. it could also be used as a screening method to test candidate wound healing drugs. allogenic keratinocytes could be used as a cellular sheet to cover the wound area with the ability to migrate towards the wound centre and promote wound healing. a possible explanation for promoting epidermal migration at the injury site is that msc-cm contains cytokines which accelerate cell migration such as kfg, sdf-1 and msp-1, in addition to other cytokines which promote both migration and proliferation of epidermal cells, for example, hgf and pdgf. disclosure of conflict of interest: none. before each freezing and after each thawing, a quality control is performed including a minima: (i) cd34+ quantification; (ii) estimation of the percentage of hsc cd34+ viability, via 7aminoactinomycin-d (7-aad) staining and (iii) evaluation of hsc functional ability to form colony ‛cfu-gm' (colony forming unit-granulocyte macrophage). apoptosis, or programmed cell death, involves complex pathways in part the path fas-fas ligand (fasl), mitochondrial components and caspase enzymes. the involvement of apoptosis dependent on caspases activation pathway in hsc cd34+ after thawing remains unknown. here, we assess the extent of apoptosis caspase-dependent before and after cryoconservation of hsc cd34+, using a fluorescent labeled inhibitor of caspases ‛flica. ' we tested the induction of apoptosis caspasedependent, before and after hsc cd34+ cryoconservation from patients with different hematological malignances: multiple myeloma (n = 21), lymphoma (n = 19). caspases pathway activation status was evaluated by flow cytometry, using a fluorescent labelled inhibitor of caspases ‛flica' staining test, in hsc cd34+, lymphocytes cd3+, monocytes cd14+ and natural killer cells cd56+. in order to assess cell viability, cells were stained in parallel with 7-aad. we determined positive cells %, that is, showing caspase activation in viable cells (flica+ cells), before and after cryoconservation. caspase pathway activation level was then correlated with hsc functional ability to form colony ‛cfu-gm,' and day's number of clinical aplasia. in our cohort, we showed a significant caspases pathway activation, with 18.9% cd34+ flica+ cells after thawing, compared with the 2.4% described in fresh cd34+ cells (p o0.0001). moreover, caspases pathway was significantly activated in thawing cd3+, cd56+ and cd14 + cells: flica+ cells % in thawing cells were, respectively, 16.8%, 31.1% and 6.2% vs 3%, 9.7% and o1% in fresh cells. we also report a significant increase of apoptosis caspasedependent in lymphoma patients (22.6% of cd34+ flica+) in comparison to myeloma patients studied (18.6% of cd34+ flica+) (po 0.0001). in contrast, no correlation has been established between observed caspases pathway activation and hsc cd34+ capacity to form cfu-gm, or still day's number of clinical aplasia. our results show substantial cell death, induced by the increase in caspases pathway activation, secondary to the thawing process, and across all study cell types. this advance of apoptosis caspase-dependent may affect the immune response quality during recipient aplasia, without detecting a clinical impact. moreover, caspases pathway activation through cd3+ and cd56+ subpopulations could modify the therapeutic result of donor lymphocytes infusion dli, though yet untested. thawing process in autologous graft induces apoptosis caspase-dependent in all apheresis product cells, particularly in hsc cd34+, without clinical impact in graft fate. disclosure of conflict of interest: none. donor-derived nk cell infusion combined with hla halpoidentcial blood stem cell transplantation to decrease leukemia relapse for high risk acute myeloid leukemia patients b wu, y huang, j xu, y he, jxm zhang*, z wu* hematology department, zhujiang hospital of southern medical university, guangzhou, china 510280 *shenzheng hank biologoical engineering co.ltd. hla halpoidentcial blood stem cell transplantation have solved the donor deficiency for patient who need to treat by transplantation. the high relapse of leukemia especially for high risk patient post transplantation affect the outcome of haploidentical stem cell transplantation. natural killer (nk) cells are part of the innate immune system and play a scavenger role to detect targets marked by ‛missing self' induced by viral infection or malignant transformation. infusion nk cells into receipt prior to stem cell transplantation could decrease the gvhd in mouse bone marrow transplantation model. in an effort to decrease the leukemia relapse and gvhd after halpoidentical stem cell transplantation for high risk acute myeloid leukemia patients, we evaluated the addition of donor-derived nk killer cells before halpoidentical stem cell transplantation in high risk acute myeloid leukemia patient. here we report interim results for five patients enrolled last year. five high risk acute patients received halpoidentcial stem cell transplantation combined with donor-derived nk cells infusion. all patients received an fbca conditioning regimen, which consisted offludarabine (25 mg/m 2 /day, intravenous) on days − 9 to − 5, busulfan (3.2 mg/kg/day, intravenous) on days − 8 to − 5, cyclophosphamide(60 mg/kg/day, intravenous) on days − 3 to − 2 and rabbit antilymphocyte globulin (atg 2.5 mg/kg/day, intravenous) on days − 5 to − 1. donor-derived nk cells were infused into patient prior to stem cell transplantation. gvhd prophylaxis was a combination of cyclosporine a (csa) and short term methotrexate. five high risk patients (2 patients with aml m5 cr2, 1 patient with aml m5 nr, 1 patient with aml m0 cr2 and 1 patient with aml m2 cr2) enrolled from jan 2015 to nov. 2015; the donors are parents and sibling. hla were mismatched between donor and patients. median cd34+ dose infused was 5.06/kg (range: 2.3-106/kg) and the nk cell dose infused was 1 × 10 8 /kg (0.8-1.2 × 10 8 /kg). all five patients got hematology recovery and achieved hematology cr. only one patient occurred grade ii agvhd post transplantation and controlled by methylprednisolone. at a median time of 12 months (range: 9-16 months) post peripheral blood stem cell transplantation, the incidence of acute gvhd grade ii is 20% (1/5) . no chronic gvhd observed. four patients are still cr and survival with event free survival with median 1 year follow up. one patient with aml m5 who had not achieved remission before transplant relapsed after 6 months and got cr with second nk infusion and still survival. nk infusion prior to transplantation was found to be safe and feasible. there was no increase acute gvhd or chronic gvhd risk. there was a trend towards increased 1-year survival for high risk leukemia patient. the potential benefit on overall survival remains to be further evaluated with additional patient enrollment and longer follow up. however, given the favorable safety profile of nk cells, future strategies to enhance efficacy such as repeat dosing or modification of nk cells are worth potential exploration. disclosure of conflict of interest: none. donor lymphocyte infusion (dli) is a therapeutic option in the treatment or prevention of relapse after allogeneic stem cell transplantation (allohsct).of note, the risk of graft-versus-host disease (gvhd) associated with the graft-versus-tumor (gvt) effect may be influenced by the level of hla disparity between donor and recipient. data on use of dli after unmanipulated haploidentical hsct (haplohsct) with post-transplant cyclophosphamide (pt-cy) are still currently limited. we report 7 patients (pts) receiving dli between 2014 and 2016 for prevention or treatment of relapse after haplohsct. seven pts were given 11 haplodli doses, as treatment for relapsed disease (n = 4) or as preventive therapy of relapse for high risk disease (n = 3). four pts had acute myeloid leukemia (aml), 1 had acute lymphoblastic leukemia and 2 lymphomas -1 hodgkin (hl) and 1 non-hodgkin dlbcl. 1 pt had intermediate risk disease features, 1 adverse risk and 5 pts had refractory disease at time of haplohsct. 4 pts had a previous hsct (2 allogeneic and 2 autologous). 6 of the 7 pts received a ric regimen and the source of stem cells was peripheral blood s153 (n = 5) and bone marrow (n = 2). gvhd prophylaxis was cyclosporine and mycophenolate mofetil (mmf), atg and pt-cy. median follow-up after haplohsct was 27 (range: months. median time to neutrophil and platelet (450g/l) recovery were 16 and 24 days, respectively. after haplohsct, 3 pts developed acute gvhd (agvhd) of grade i (n = 1) or ii (n = 2), at a median of 26 days after haplohsct. the median time from haplohsct to first dli was 204 days (range: 71-624). all pts had full donor chimerism at time of dli. before dli 3 pts relapsed at a median time of 149 days (range:86-177), of whom 2 pts had aml and received salvage chemotherapy and 1 pt with hl being treated by dli alone. of the 3 relapsed pts,1 showed progressive disease after first dli dose and 2 achieved a sustained cr (with duration of cr of 6 and 9 months at last follow-up). the remaining 4 pts were given dli in cr, in 1 case (of aml) associated with azacytidine. 5 pts received 1 dli dose and 2 pts were given 3 dli injections with escalating doses. the first dose of dli was 1 × 10 6 cd3/kg in 4 pts, 5 × 10 5 in 1 pt and 1 × 10 5 in 2 pts. the 2 pts who received 3 dli doses (lymphomas) were given: (1) 5 × 10 5 -1 × 10 6 -5 × 10 6 ; (2) 1 × 10 5 for 2 doses followed by 1 dose of 5 × 10 5 . four pts developed chronic gvhd (cgvhd, 57%) in a median time of 23 days (range:11-42) after dli (3 of them had presented previously agvhd grade i-ii). cgvhd was limited in 1 case, moderate in 1 pt and severe in 2 pts. 3 of these pts presented features of an overlap syndrome (acute/chronic gvhd) with signs of agvhd de grade i,ii and iii in 1 pt each. involved organs were skin/mucosal (n = 4), liver (n = 3), gastrointestinal tract (n = 2), lung (n = 1) and joints (n = 1). all patients experiencing gvhd after dli were treated by systemic corticotherapy, extracorporeal photopheresis and cyclosporine or weekly low dose methotrexate. median follow-up after first dli was 10 months (range: . none of the 4 pts receiving prophylactic dli relapsed during the follow-up period. 2 pts died,1 of relapse and 1 of severe cgvhd. 5 pts were in cr at last follow-up,3 with no signs of gvhd and 2 with limited cgvhd. despite the limited cohort, dli after haplohsct appears to be a therapeutic option in high risk pts allowing enhancement of gvt in the setting of haplohsct with post-cy infusion. disclosure of conflict of interest: none. previously published p075 early and sequential ctla4ig primed donor lymphocyte infusions (dli) following post-transplantation cyclophosphamide (ptcy)-based haploidentical pbsc transplantation for advanced hematological malignancies promote proliferation of mature natural killer (nk) cells with cytotoxic potential and markedly reduces relapse-risk without increase in gvhd sr jaiswal, s zaman, p bhakuni, s bansal, s deb, s bhargava and s chakrbarti 1 we have earlier shown that cd56 enriched cell infusion following ptcy resulted in rapid proliferation of mature nk cells with attenuation of gvhd and early use of prophylactic g-csf mobilized dli resulted in improved disease-free survival. ctla4ig has been shown to be effective in attenuating t cell activation and induce transplantation tolerance in preclinical models. it has recently been employed to induce transplantation tolerance and reduce early alloreactivity in patients with nonmalignant disorders undergoing ptcy-based haploidentical hsct. nk cells on the other hand are resistant to ctla4ig and in fact might demonstrate better anti-tumour effect in presence of ctla4ig as cd86 is a putative activation receptor. to explore this phenomenon, we employed sequential ctla4ig primed dli following ptcy-based haploidentical hsct in patients with relapsed/refractory hematological malignancies.15 patients (12-60 years; aml-6, all-5, nhl-4) received abatacept (ctla4ig) as a part of gvhd prophylaxis at 10 mg/kg on day − 1 followed by pbsc and sequentially on days +6, +20 and +35 followed 12 h later by dli of 5 × 10 6 cd3 cells/kg containing 0.03-1 × 10 6 /kg cd56+ cells. ptcy was administered on days +3 and +4 with cyclosporine from day +5 to day +60 and subsequent rapid tapering. the immune reconstitution of the study group (ctla4ig-dli) was compared with the cohort of patients with both malignant and nonmalignant diseases who received abatacept but not dli (n = 12; ctla4ig group) and those receiving cd56 enriched donor cell infusion on day +7 (n = 10; nki group). results: there were no acute infusion related toxicities. all patients engrafted at a median of 15 days (12-20 days). the incidences of acute and chronic gvhd (all mild) were 20% and 25%, respectively. three patients reactivated cmv and there was only one non-relapse mortality (6.9%). only 4 patients relapsed (27.8%) with a disease-free survival of 72.6% at 1 year. these cells had greater expression of cd107a compared to normal healthy donors. the recovery of cd56+, cd56+16+ and cd56 +16 − cells were similar in the ctla4ig-dli and nki groups at days 28, 60 and 90 post-transplant and this was significantly higher than the ctla4ig group ( figure 1 ). in contrast to ctla4ig group, nk cells recovered at day +28 with predominantly cd56dim cd16+ phenotype with significant population of cells expressing kir+nkg2a phenotype in both ctla4ig and nki groups with higher expression of cd107a. interestingly, the 4 patients who relapsed had attenuated recovery of cd56+16+ cells at 28 and 60 days(21/μl and 15 cells/μl) without cd107a expression, in contrast to the rapid and sustained recovery of this population of nk cells in those not experiencing relapse (cd56+16+ cells 181/μl and 103/μl). however, the recovery of tregs was prompt and sustained in the comparator groups, which remained low in the ctla4ig-dli group until day +90. there were no differences in the recovery of other t cell subsets between the three groups. the study demonstrates the unique ability of ctla4ig to augment nk cell proliferation, maturation and cytotoxicity and reduce relapse with attenuation of t cell activation and gvhd in the context of the early use of ctla4ig primed dli following ptcy-based haploidentical hsct without ex vivo selection or expansion. we hope this novel strategy might offer less expensive and yet a viable alternative in the field of nk cell therapy. [p075] disclosure of conflict of interest: none. enhanced cytotoxicity of γδ-cytokine induced killer cells against hematologic malignancies n bloom, s eldror, s caspi, s teihuman,h vernitsky, e jacoby, b bielorai and a toren cik cells are ex vivo expanded by scheduled addition of anti-cd3 mabs and a cytokine cocktail that contains ifn-γ, il-2 or il-15. cells represent an in vitro generated heterogeneous population consisting of different effector cells-cd3poscd56pos, cd3negcd56pos and cd3poscd56neg-t cells that mainly (495%) express α/β t-cell receptor (tcr) s154 and to a lesser extent (o 5%), tcr γδ phenotype. these nklike t cells product show a dual functional activity, retaining their original t cell specificity and nk cytotoxic capacity via marked up regulation of the nk cell receptor, nkg2d. pre and clinical studies showed that the optimal cytotoxic effect of cik cells against different malignancies (target cells) is achieved at 40:1 e:t ratio, which means high numbers of αβ t-cells that might increase the risk of gvhd. here we produced ciks from αβ tcr depleted cellular products (defined as γδcik) and tested their phenotype expression and in vitro cytotoxic activity against hematological malignancies. fresh apheresis products were processed using the clinimacs depletion reagent, according to manufacturer instructions. target product was cultured with rpmi1640 supplemented with 10% fcs and ex vivo expanded by scheduled addition of cytokine cocktail that contains ifn-γ (1000 iu/ml), anti-cd3 mabs (50 ng/ml) and 500 iu/ml il-2. the cells were cultured for 14 days. cytotoxic activity of the γδcik was evaluated against various target hematological malignant cell lines (k562, reh, jurkat, and u937). after 14 days, the αβ depleted cik cultures resulted in 97.5% γδ t-cells (41 folds expansion) compared to 1.0% of γδ t-cells immediately after depletion, and compared to only 1.6% in non-selected cik cells. the percentage of αβ t cells in γδcik cell cultures started from 0.002% (immediately after depletion) to 0.5% compared to 95.1% αβ t cells were found in non-selected cik cells cultures. γδcik cells produced robust cytotoxic activity at a 10:1 e:t ratio against reh cells (22.6 ± 5.3%), jurkat cells (51 ± 7.9%); u937 (62.5 ± 8.5%) and k562 (43.4 ± 2.0%), compared to nonmanipulated cik cell activity against the same targets (5 ± 1.0%; 8.3 ± 1.4%; 12.4 ± 6.1%; 7.3 ± 2.9%, respectively). we found higher degranulation capacity of γδcik cells compared to non-selected cik cells against reh (45.3 ± 16.1% vs 17.6 ± 3.8%), jurkat (42.3 ± 18.4% vs 6.8 ± 3.5%), u937 (37.6 ± 15.5% vs 17.1 ± 3.1%) and k562 (29.2 ± 16. centre de thérapie cellulaire, institut paoli-calmettes; 2 unité de transplantation et de thérapie cellulaire, institut paoli-calmettes; 3 centre d'immunologie de marseille-luminy and 4 laboratoire d'immunomonitoring, institut paoli-calmettes during the past 15 years, the major improvements in the field of allogeneic hematopoietic stem cell transplantation (hsct) (reduced intensity conditioning regimen, high level hla typing, alternative donors, gvhd prophylaxis…) significantly extended the feasibility of this procedure. in contrast, disease recurrence after hsct remains a main issue. thus, many post-hsct prophylactic interventions are under investigation. unmanipulated donor lymphocyte infusion (dli) remains one of the most frequently used post-hsct treatment, but its potential benefit in increasing gvl effect may be counterbalanced by the induction of gvhd. in this setting, the use of adoptive transfer of ex vivo enriched and activated nk cell infusions from the same donor (dli-nk) may induce gvl effect without causing gvhd. we therefore report on a single-center phase 1 clinical trial (nct01853358) evaluating the safety of ex vivo activated allogeneic nk cells infused between days 60 and 90 after hsct. the aim was to determine the maximum tolerated dose (mtd) of ex vivo highly purified and activated dli-nk after matched related donor hsct. the schedule plan a first phase of 3+3 dose escalation method using 3 dose levels (1.10e6/kg, 5.10e6/kg and 45.10e6/kg). grade 3-4 secondary adverse events according to nctci classification and severe gvhd occurring within 30 days after dli-nk were considered as dose-limiting toxicities (dlt). a second step allowed enrolling patients at the mtd. over a period of 3.5 years, 14 patients with various hematological malignancies (aml, all, hl, nhl, mds) were infused with activated nk cells at a median time of 91 days (range: 61-106) post-hsct. apheresis products were collected from the hsc donor, cd3-depleted and cd56-selected by immunomagnetic separation using clinimacs. selected nk cells were cultured for 7 days in medium supplemented with 10% fetal calf serum in the presence of 1000 u/ml of il-2 in air-permeable cell culture bags. after immunomagnetic separation, cd56enriched products had a median cd56+ cell purity of 94% (range: 77-100) and viability of 96% (93-99). after il-2 activation, the median cd56+ cell dose was 4.8 × 10e6/kg (1.2-21.4) , with a viability of 81% (71-94) and a residual cd3 + cell content of 0.4 × 10 4 /kg (0-1.5 × 10 4 /kg). all release criteria to be met were fulfilled for the 14 preparations infused : viability 490%, negative microbiological testing, cd56 + cell count ⩾ 1 × 10 6 /kg, and cd3 + cell content o5 × 10 4 /kg. standardized quality controls were employed at all steps of the manufacturing process, adding a level of consistency to the product testing before release. activated-nk cells were well tolerated in all 14 patients, with no occurrence of dlt. thus, mtd was not reached. two patients presented with a moderate chronic gvhd, both of them during cyclosporine a dose reduction. relapse occurred in 2 patients with aml. one patient died from idiopathic pneumoniae, without evidence of relapse, gvhd or infectious disease. with a median follow up of 30 months (1-41), 2 year os was 83% ( figure 1 ). therefore, infusion of highly purified, activated-nk cells of donor origin as a substitute to standard dli does not induce gvhd nor other side effects after hsct: the demonstration that modulation of nk cell activity can achieve disease control after hsct deserves to be investigated in larger trials. [p077] disclosure of conflict of interest: none. feasibility, safety, rapid production and efficacy of institution-produced cd19 car staff were trained on site for collection, processing and cryopreservation by regional experts. a total of 101 units were collected and processed as part of the initial validation of the project. ucb units were processed on either axp or sepax systems, and all cryopreserved in bioarchive (an automated, robotic cryopreservation system that can archive up to 3623 units). the characteristics of which as well as the post processing data are depicted in table 1 . [p079] we shared a successful story of establishing the first public cord blood bank in jordan. the first 101 units collected showed excellent sterility, viability, collection volume and total nucleated cells. a very good recovery of both nucleated and cd34+ cells were obtained using axp and sepax cell separation systems. the process of validation of equipments and methodology is complete. we anticipate moving to permeant facility of the cord blood bank in the new expansion in early 2017. we look forward for steady progress in ucb recruitments, hla typing, cryopreservation and adherence to netcord-fact standards as well as participation in international registries. functionally active ifn-gamma secreting cmv pp65 specific t cell therapy as an alternative for clinically urgent cmv related diseases n kim, y-s nam 1 , k-i im 1 , j-y lim, y-w jeon 1 , y song and s-g cho 1 the catholic university of korea, seoul cytomegalovirus (cmv) related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (hsct). it has been reported over the last two decades that cmv-specific cytotoxic lymphocytes (cmv-ctls) can provide long-term cmv-specific immunity without major side effects as an alternative to antiviral drugs. however, its application has been limited by prolonged manufacturing process of cell therapy. in this study, we apply the ifn-γ cytokine capture system (ccs) using the fully automated clinimacs prodigy device to rapidly produce cmv-ctls that may be applicable in clinically urgent cmv-related diseases. five validation runs were performed using apheresis samples from randomly selected cmv-seropositive healthy blood donors. then, clinimacs prodigy automatically performed successive processes including antigen stimulation, anti-ifn-γ labelling, magnetic enrichment, and elution which took~13 h. the original apheresis samples consisted of 0.3% ifn-γ secreting cd3+ t cells in response to cmv pp65 antigen (cd3+ifn-γ+ cells) which were mainly cd45ra+cd62l+ naive t cells. following ifn-γ enrichment, the target fraction contained 51.3% cd3+ifn-γ+ cells with reduction in naive t cells and the selection of cd45ra − cd62l − and cd45ra +cd62l − memory t cells. furthermore, extended culture of these isolated cells revealed functional activity including efficient proliferation, sustained antigen-specific ifn-γ secretion and cytotoxicity effect against pp65 pulsed target cells. therefore, we suggest ifn-γ ccs by clinimacs prodigy as a simple and robust approach to produce cmv-ctls, which may be highly feasible and applicable in clinically urgent cmvrelated diseases. disclosure of conflict of interest: none. in vitro generation of tumor antigen-specific t cells from patient and healthy donor stem cells s bonte 1 , s snauwaert 1 , g goetgeluk 2 , b vandekerckhove 2 and t kerre 1 1 hematology, ghent university hospital, ghent, belgium and 2 department of clinical chemistry, microbiology and immunology, ghent university, ghent, belgium acute myeloid leukemia remains a therapeutical challenge, as many patients relapse after chemotherapy. allogeneic stem cell transplantation is in most of these patients the only option for cure, but carries a high risk of morbidity and mortality and a suitable donor may be lacking. recently, advances are being made in the field of t cell immunotherapy. the classical protocol, in which peripheral blood lymphocytes (pbl) are transduced with a tumor antigen-specific t cell receptor (tcr), can generate t cells with low and possibly hazardous specificities (due to mispairing of the endogenous and introduced tcr α and β chains). therefore, we have developed a novel protocol in which we generate tumor antigen-specific t cells from cd34+ hematopoietic stem cells. we have already succeeded in generating large numbers of tumor-specific, naive and resting t cells that only carry the introduced tcr, starting from postnatal thymus and cord blood cd34+ cells. now we are optimizing this protocol for clinically more relevant samples, such as mobilized peripheral blood from healthy stem cell donors and from patients in remission after chemotherapy and/ or other treatments, and leukapheresis samples from patients at diagnosis. in our protocol, cd34+ cells were isolated from hla-a2+ fresh patient and healthy donor samples and cultured on op9-dl1 in the presence of scf, flt3l and il-7, until t cell commitment. subsequently, the cells were transduced with a tumor antigen-specific tcr and again co-cultured until cd4+ cd8+ double positive cells were abundantly present. at that point, agonist peptide was added, which induces maturation. finally, cells were polyclonally expanded on feeder cells. for hla-a2 negative samples, cd4+ cd8+ double positive cells were co-cultured with a cell line (t2 pulsed with the agonist peptide or a cell line with endogenous expression of the agonist peptide) which can present the agonist peptide to the maturing t cells. using the above protocol, we were able to generate tumor antigen-specific t cells from 3 out of 3 healthy donor samples, 1/1 sample from a patient in remission and 2/4 samples from patients at diagnosis, who were all hla-a2+. for most samples, multiple rounds of agonist peptide stimulation were necessary to obtain further maturation. in contrast, generation of mature t cells from cd4+ cd8+ double positive cells in postnatal thymus or cord blood co-cultures, requires only 1 round of agonist peptide stimulation. for the hla-a2 negative samples, we were able to generate an adequate cd4+ cd8+ double positive population from 1/1 healthy donor sample, 3/3 samples from patients in remission and 0/1 sample from a patient at diagnosis. agonist selection using a cell line seems inefficient as cd27 is not upregulated and cells did not mature to cd4+ or cd8+ single positive mature t cells. we are currently co-culturing more samples using our protocol. furthermore, we are investigating the effect of freezing and thawing on the in vitro t cell generation process (cell numbers and efficiency). finally, we are also working on optimizing the protocol for generation of tumor antigen-specific t cells from hla-a2 negative patient and healthy donor samples. disclosure of conflict of interest: none. increase of polyspecific immune responses against leukemia-associated-antigens (laa) and reduction of regulatory cytotoxic t-cell (ctl) responses against malignant cells play a major role in maintaining remission and prolonging overall survival in patients with hematologic malignancies after allogeneic stem cell transplantation (allo-sct) and/or donor lymphocyte infusions (dli). graft versus leukemia (gvl) effects after allogeneic stem cell transplantation and/or dli are considered to be t cell-mediated. many groups described specific t-cell responses against several leukemia associated antigens (laa) in different hematological malignancies. however, t cell responses after allo-sct and dli are not well characterized. in this study, we analyzed laa-specific t cell responses after allo-sct and dli. to this end, we assessed the frequency and diversity of laa-specific cd8+ t cells using elispot analysis and tetramer assays in 12 patients (5 patients (pts) with acute myeloid leukemia, 2 pts with chronic myeloid leukemia, 3 pts with multiple myeloma and 2 pts with chronic lymphatic leukemia) before and after dli. epitopes derived from prame, npm1mut, rhamm, wt-1 and other laa were tested. moreover, the frequency of regulatory t (treg) cells was measured and the course of cytokine profiles before and after dli was analyzed. these immunological findings were correlated to the clinical course in the respective patients. in elispot and tetramer assays, an increase in frequency and diversity of laaspecific t cells was observed in all patients. importantly, there was a significant increase from a median of 1 to 4 laa-derived t cell epitopes (p = 0.03) in clinical responders (r) when compared to non-responders (nr). these positive results in r vs nr where s157 confirmed by tetramer-based flow cytometry assays, where an increase in frequency from 0.5 to 2.3% in the r group of laaspecific t cell/all cd8+ t cells was observed. interestingly, the frequency of tregs in clinical responders decreased significantly from a median 72.9 % to 54.6 % (p = 0.008) while the frequency of tregs kept stable over time in non-responding patients. t cell subset analysis did not reveal significant differences before vs after dli administration. in cytokine assays using elisa for the detection of more than 10 cytokines before and after dli we found a shift towards proinflammatory and t cell stimulating cytokines. taken immunologic surveillance of leukemia is employed for the prevention and treatment of relapse post allohsct. to augment this effect donor lymphocytes are infused (dli) in patients at risk. this procedure is associated with a high risk of agvhd and we believe that this route of administration may not make the direct contact between infused cells and blasts the optimal one. to address these issues, we started delivering donor lymphocytes directly to the bone marrow cavity (ib-dli) in patients post allohsct at relapse. three with aml and one with cll, all relapsed post allohsct: 3 allosib: 50-year-old female aml patient (relapsed 2 years post hsct), 22-year-old aml male 7q31 del (relapsed in 3 years, traumatic brain injury), 25-year-old male aml flt3 itd+ received mud hsct (relapsed 9 months) and 64-year-old cll male, tp53 del, ebv reactivation (progressed 7 years). two patients (26% and 12% blasts in the marrow) received ib-dli up-front and two others due to higher proportions of leukemic cells received either flag (aml case) or anti-cd20 moab (cll case) followed by ib-dli. tcr clonotyping revealed in all 4 patients the presence of the prevailing oligoclonal response on the polyclonal background (characteristic for each individual) which was identified in the marrow and in the blood. however, in two out of 4 patients a distinct oligoclonal peak was seen at first in the marrow and then in the blood. microarray analysis of the transcriptome in the marrows of patients who received three ib-dli courses revealed in all patients preferential use of genes associated with lymphocyte or lymphocyte activation pathways. the patients who responded favorably (cr or pr) clustered with the transcriptomes of normal individuals, but those who failed to respond clustered separately. ib-dli was safe and not associated with gvhd. selective accumulation of cd8+cd279+ as well as the presence of a distinct oligoclonal peak in the marrow suggest that tcrbeta clonotypes may be private to leukemia cells recognition. the response may result in cr or pr and the patients were in a good physical shape during the treatment, which makes it possible to deliver the salvage chemotherapy if required. broad spectrum antibiotics were started. after the orthopedic consultation, the fourth finger was amputated and amputation from the left ankle was recommended. a stem cell transplantation option was offered to patients and their relatives as one of the therapeutic approaches. upon acceptance by patient, 10 μgr/kg of colony stimulating agent was started to patient. when the stem cell was 20/μl, the stem cells were collected. the obtained stem cell product was injected intra-lesionally (picture b). granulation tissue began to develop from the second week in the foot floor of the patient. after from 8th week, the necrotic tissue was disappeared and the granulation tissue was appeared. at 24 weeks, 50% of the lesion healed. at 48th week, there was normal tissue instead of necrotic tissue on plantar surface at left leg (picture c). this case report suggests that diabetic foot/ulcer can be healed with intralesional application of stem cells in patients with diabetes mellitus. [p085] disclosure of conflict of interest: none. and third (n = 5) cell infusions were cryopreserved. cells were infused following conventional chemotherapy (ia, mec, hdac) in 15 cases (44%), chemotherapy plus hypomethylating agents in 8 cases (24%) and hypomethylating agents alone in 11 cases (8 azacytidine, 3 decytabine; 32%). the procedure was well tolerated, with mild and transient ‛haploimmunostorm syndrome' (fever 84%, rash 28%, diarrhea 14%). only the two patients with cmml received corticosteroid. one patient suffered early infusional reaction that was resolved with support treatment. none of the patients showed acute or chronic gvhd or persistent donor engraftment in chimerism tests. four patients had bacterial infections, but no other significant invasive fungal or viral infections were observed. all aml/raeb patients treated achieved complete remission with microhct treatment (13; 87%). only one patient, with cmml, died during microhct induction (7%). four patients relapsed at 7, 9, 10 and 15 months after the infusion; two of them achieved a second sustained complete remission with another micro-hct from a different donor (one of them had developed anti-hla antibodies). as described in figure 1 , median overall survival is 16 months and overall survival at 2 years is 40%. microhct is a well tolerated procedure in elderly aml/mds patients who are not candidates to allogeneic hct. infectious complications are insignificant and the remission rates are very encouraging in very high risk cases, with no evidence of gvhd. patients can undergo a second microhct from a different donor. in addition to the experience by ai et al, we have also shown that microhct can be safely administered following a hypomethylant agent course instead of conventional chemotherapy. a large, international, randomized clinical trial will address the safety and efficacy of microhct for elderly aml/ mds patients (nct02171117). [p086] disclosure of conflict of interest: none. wilms tumor protein 1 (wt1) is expressed in a variety of solid tumors and is found in more than 80% of patients with acute myeloid leukemia which makes it an attractive target for immunotherapy. previously it was shown that t cells recognizing wt1 are suitable for adoptive t-cell therapy by increasing the graft versus leukemia effect. however, the efficiency of this therapeutic strategy is still limited due to the low precursor frequency and specificity of wt1-specific t cells in the peripheral blood of healthy donors. the ubiquitous antioxidant inducible enzyme heme oxygenase-1 (ho-1) and its products have immunomodulatory effects, which render it as a potential target for the modification of t-cell responses. recently, we found that inhibition of ho-1 enzyme activity via tin-mesoporphyrin (snmp) results in activation and proliferation of antiviral t cells from healthy donors. in this study we aimed (1) to identify the mechanism of ho-1 modification in the generation of wt1-specific t cells and (2) to develop strategies for the sufficient generation of wt1specific t cells from healthy donors to augment effective t-cell immunity in leukemia patients and to broaden the applicability of adoptive t-cell therapy to the majority of patients. the frequency of wt1-specific t cells in peripheral blood of healthy donors (n = 50) was examined before and after snmp treatment via ifn-γ elispot using the wt1-overlapping peptide pool (ppwt1). enrichment efficiency of wt1-specific t cells after ho-1 inhibition was verified in response to ppwt1 and the hla-a*02:01-restricted wt1 peptides 37 (vldfappga, wt137) and 126 (rmfpnapyl, wt1126) by ifn-g secretion assay and expression analysis of the t-cell activation marker cd137. phenotypic and functional characterization of wt1specific t cells were further assessed by multicolor flow cytometry, luminex assays and elisa with respect to t-cell subsets, cytotoxicity, proliferative capacity and secretion of effector molecules. in 24% of donors we found specific t cells against ppwt1 by ifn-γ elispot (10 spots/250.000 pbmcs). the frequency of wt1-specific t cells in these donors could be increased fivefold after inhibition of the enzymatic activity of ho-1 via snmp. to assess the possibility that ho-1 modulation might be clinically applicable in conformity with good manufacturing practice, enrichment of snmp-treated wt1-s159 specific t cells was evaluated based on ifn-g secretion and cd137 expression. compared to snmp-untreated cells there was a 3.74-fold higher response of ho-1 modified wt1-specific t cells pre-enrichment and an up to 16-fold higher enrichment efficacy, while snmp treatment did not affected the t-cell functionality. in conclusion, modification of the enzymatic activity of ho-1 resulted in a more effective generation of functionally active wt1-specific t cells suitable for adoptive t-cell therapy. this makes ho-1 a promising therapeutic target to boost antigen-specific t-cell responses for treatment we recently developed and characterized a standardized and clinical grade human platelet lysate (hpl) that constitutes an advantageous substitute for fetal bovine serum (fbs) for human mesenchymal stem cell (hmsc) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issue. because of the progressive use of pathogen reduced (pr) labile blood components, we evaluated the impact of the novel procedure theraflex uv-platelets for pathogen reduction on hpl quality (growth factors content) and efficacy (as a medium supplement for hmsc expansion). this technology is based on short-wave ultraviolet light (uv-c) and has the main advantage not to need the addition of any photosensitizing additive compounds (that might secondary interfere with hmscs). we applied theraflex uv-platelets procedure on fresh platelet concentrates (pcs) suspended in platelet additive solution and prepared hpl from these treated pcs. we compared the quality of pr-hpl with the corresponding non-pr ones, in terms of growth factor contents. then, we evaluated the efficacy of pr-hpl, in comparison with hpl and msc-screened fbs. we performed large scale culture of hmscs during 3 passages and evaluated the proliferation of cells and the maintenance of their properties: profile of membrane marker expression, clonogenic potential, immunosuppressive properties (inhibition of t-cell proliferation) and potential to differentiate in adipocytes and osteoblasts. we showed no impact on the content in 5 growth factors tested (egf, bfgf, pdgf-ab, vegf and igf) and a significant decrease in tgf-b1 (−21%, n = 16, p o0.01). a large scale culture of hmscs during 3 passages showed that hpl or pr-hpl at 8% triggered comparable hmsc proliferation than fbs at 10% plus bfgf (n = 3). moreover, after proliferation of hmscs in hpl or pr-hpl containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties (inhibition of t-cell proliferation) were maintained, in comparison with hmscs cultured in fbs conditions. the potential to differentiate in adipogenic lineage of hmscs cultured in parallel in the 3 conditions, evaluated using oil red o and nile red stainings and the measurement of triglyceride accumulation, remained quantitatively identical. we also showed that the potential to differentiate in osteoblasts (quantified using alizarin red s and von kossa stainings and alp activity measurement) of hmscs grown in hpl or pr-hpl was not impaired, in comparison with fbs. in conclusion, we demonstrated the feasibility to use uv-c treatment to subsequently obtain pathogen reduced hpl, while preserving its optimal quality and efficacy for hmsc expansion for cell therapy applications. although it is still not used widely in clinical practice. in this paper, we demonstrated a case of ada-scid who received hsct as an adolescent from matched unrelated donor (mudd) after termination of her peg-ada treatment due to severe intractable thrombocytopenia induced by peg-ada. patient showed good engraftment and incremental clinical improvement. her post transplantation course was complicated with multiple complications including: grade i gut gvhd as well as hemorrhagic cystitis (btk related) and ebv infection, additionally, she developed several cns complaints like headache, vomiting and dizziness which were found to be due to increased intracranial pressure with multiple enhancing cerebral lesions found on brain imaging. further investigations for the brain lesions confirmed the diagnosis of malignant diffuse large b cell lymphoma (dlbcl) involving the brain. the lymphoma was highly suggested to be originated from donor cells giving the timing relationship between transplant and establishment of the diagnosis. this lymphoma was successfully treated with full recovery and good final immune reconstitution but with lack of b cell engraftment and need for monthly ivig. we conclude that, peg-ada can rarely induce thrombocytopenia in an autoimmune manner by forming antibodies against platelets and good recovery of thrombocytopenia can be achieved after discontinuation of peg-ada. hsct can be considered as modality of treatment even in older patients with scid due to ada deficiency keeping in mind high possibility of complications including, autoimmunity and malignancy. disclosure of conflict of interest: none. (1) (2) (3) (4) . based on the pre and post-apheresis cd34+ cell counts, the collection efficiency of the apheresis amicus device was median 89.5% (54-170) and of the comtec median 82% (32-95). in mm the apheretic collections were started on median day 5 (4-6), while in lymphoma patients, due to chemotherapy, the day of apheresis start was 12 (9-18). after cryopreservation and thawing, viability (7-aad, bd) was median 87.5% (43-100). with these cell products, up to now we engrafted 9 patients following high-dose chemotherapy (5 mm autografted after mel200, 2 hl and 2 nhl autografted after beam). engraftment was prompt and stable in all with anc 0.5 and 1.0 × 10 9 /l on median day 11 (10-12) and 12.5 (11-15), respectively, and with platelet count 20 and 50 × 10 9 /l on median day 14 (11-17) and 17.5 (13-44), respectively. these results are similar to those obtained by most experienced centers in europe and us, and confirm the fact that autologous transplantation may be implemented also in developing countries when appropriate technology and application of standard procedures are employed. with this experience our center is also developing allogeneic transplantation, and the initial results in thalassemia will be reported in a separate abstract. disclosure of conflict of interest: none. extracorporeal photopheresis (ecp) is a safe and effective immunoregulatory therapy for steroid-refractory graft-versushost disease (gvhd) but its mechanism of action is poorly understood. ecp is a non-immunosupressive therapy whose modulating mechanism is thought to result in an increase in t-regs in the patient and in inversion of the cd4/cd8 ratio at the end of treatment. in this study, we evaluated the effect of ecp on t cell response in a cohort of steroid-refractory gvhd patients. from november 2009 to november 2016, 40 patients (28 con acute gvhd and 12 with chronic gvhd) treated with ecp in our unit, were retrospectively evaluated. patient characteristics are shown in table 1 . we performed an ‛off-line' system ecp using a cell separator (spectra optia, teruno bct) for the cmn apheresis; after 8-methoxypsoralen was added, the product was photoinactivated in the ultraviolet a irradiator (uvamatic-g1, macopharma). ecp procedures were performed for two consecutive days, initially weekly (agvhd), or every two weeks (cgvhd) and afterwards monthly according to clinical response. anthracycline-induced cardiotoxicity (aic) is irreversible, which has limited the use of this anthracycline in cancer chemotherapy. to explore the therapeutic effect and its possible mechanism of bone marrow derived mesenchymal stem cells (bmscs) on cardiac damage induced by anthracyclines in a rat model. study selects sd rats aged 2-3 weeks to isolate and culture bmscs, and flow cytometry was used for phenotypic identification of bmscs. 48 female sd rats were first randomly divided into 6 groups: the sham control, bmscs control, 4.0 mg/kg daunorubicin (dnr), dnr with bmscs, dnr with dexrazoxane (dzr), dnr with bmscs and dzr. left ventricular (lv) function before, during and after chemotherapy were assessed by echocardiography. at the end of 4 weeks, animals were euthanized and organs were collected in 10% buffered formalin for histopathology using hematoxylin and eosin staining and immunohistochemical analysis was used to identify the cellular subpopulations that infiltrate the cardiac tissues. after the construction of microrna-21 (mir-21)modified bmscs with lentiviral vector, 50 sd rats were randomly assigned into 5 groups: the normal control, the empty vector control, dnr, dnr with bmscs, dnr with mir-21-modified bmscs. the density of new blood vessels of rats in each group was detected by immunohistochemical method. mir-21, bcl-2, bax and vegf mrna expressions were detected by qrt-pcr. bcl-2, bax and vegf, cx43, troponin t and bnp protein expressions were detected by western blotting. all procedures performed in studies involving animals were in accordance with the ethical standards of the institutional. an animal model of drug-induced cardiomyopathy was built in the dnr treated rats.lv ejection fraction (lvef) and lv fractional shortening (lvfs) were significantly decreased compared to that of the sham control (p o0.001), and the signs of the myocyte injury (myocytolysis, vacuolization and disruption) in paralleled with the inflammatory infiltrates, marked by cd3 and hla-dr, were observed in the dnr group, while bmscs alone or synergistic with dzr facilitate the anthracycline-induced lv dysfunction returning to the baseline values and the recovery of myocarditis (p o0.001). in the mir-21-modified bmscs transplant group, mir-21 expression, cell migration and proliferation ability were higher than that in the bmscs and empty vector groups (p o0.05). the cardiac regenerative capacity of bmscs following significant myocardial injury were further enhanced by mir-21 compared to that of the dnr group and the control groups (all po 0.05), revealed by the significantly higher density of new blood vessels and upregulation of vegf expressions, during which the pro-apoptotic protein bax were down-regulated and the anti-apoptotic protein bcl-2 function were upregulated in the mir-21 overexpression group compared to that with the bmscs, dnr group and the control groups (all po0.05). western blotting demonstrated that the expression of c × 43 were significantly decreased, while expressions of troponin t and bnp were significantly increased in the mir-21 overexpression group in contrast to that with the dnr group (all p o0.05). these results showed that bmscs could reverse cardiac damage induced by anthracycline, and the cardioprotective efficacy was further enhanced by mirna-21-mediated regulation of apoptosis and angiogenesis. disclosure of conflict of interest: none. effective adoptive t cell therapy against cancer is dependent on long-lived tumor-specific stem cell-like t cells with the ability to self-renew and differentiate into potent effector cells. however, current protocols for ex vivo generation of tumorspecific cd8+ t cells result in terminally differentiated effector t cells. it was found that minor histocompatability antigen (miha)-specific cd8+ t cells with an early memory-like phenotype and long-lived memory transcription profile could be expanded from naive precursors using akt-inhibitor viii1. importantly, these akt-inhibited tumor-specific cd8+ t cells showed a superior expansion capacity and anti-tumor effect multiple myeloma bearing mice. for the clinical exploitation of ex vivo generated akt-inhibited tumor-specific cd8+ t cells, we tested the effect of potential clinical grade akt-inhibitors azd5363, gdc0068, gsk2110183, gsk2141795, mk2206 and triciribine in polyclonal stimulations, allogeneic mixed lymphocyte reactions (mlr), and antigen-specific t cell assays. polyclonal stimulation with anti-cd3/cd28 beads on cd8 naive t cells was used for a first screening of the akt-inhibitors. for all inhibitors, a dose dependent effect on the naiveassociated receptors ccr7, cd62l and cxcr4 was observed. this had limited effect on viability, activation and proliferation except for triciribine, which was therefore excluded for further assays. moreover, in the mlr, treatment of naive cd8+ t cells with remaining akt-inhibitors resulted in a dose dependent effect associated with higher ccr7, cd62l, cxcr4 and cd28 expression. furthermore, the akt-inhibited cd8+ t cell products showed a 10-25 fold increased expansion capacity upon restimulation in vitro. when expanding miha-specific cd8+ t cells from the naive repertoire in the presence of one of the akt-inhibitors, the miha-specific cd8+ t cells showed a more early memory phenotype compared to controls. this was displayed in higher levels of the naive-associated receptor cd62l ( figure 1 ). in addition, these miha-specific cd8+ t cells were shown to be functional, as antigen-specific restimultation resulted in degranulation (cd107a) and ifn-γ production. based on this ifn-γ production, the akt-inhibited antigenspecific cd8+ t cells can be selected using the cytokine capture assay (miltenyi, for enriched infusion in patients suffering from hematological malignancies. using aktinhibition in the generation of tumor-reactive t cells results in a more early memory tumor-specific cd8+ t cell product. this adoptive immunotherapy product retains superior proliferation capacity upon infusion, and its potential selfrenewal capacity could result in a long-term anti-tumor effect in patients suffering from a hematological malignancy. chimeric antigen receptors (cars) are composed of an extracellular domain-derived from a tumour-reactive monoclonal antibody, linked to one or more signalling endodomains. in early clinical trials, cd19 car-t cells have demonstrated impressive anti-tumour activity against different b-cell malignancies, including chronic lymphocytic leukaemia, acute lymphoblastic leukaemia (all) and non-hodgkin lymphoma. conventional alpha-beta car-t cells are however hla-restricted and could cause graft-versus-host disease (gvhd) when used across major mismatches, as expected in the highly anticipated setting of off-the-shelf car-t cells from third-party donors. besides being non-hla restricted, gammadelta t cells possess intrinsic anti-tumour reactivity, making them attractive effectors for next-generation car-t cell therapies. so far, however, attempts at exploiting gammadelta t cells in patients have been largely disappointing, possibly because of sub-optimal ex vivo culture conditions. the aim of our study was to optimise the generation of gammadelta car-t cells and to test their anti-tumour potency both in vitro and in vivo. starting from peripheral blood mononuclear cells of healthy donors, we stimulated gamma-delta t cells with zoledronate and il-2/il-15, and transduced them with retroviral vectors encoding for cd19 cars carrying either cd28.z or 4-1bb.z signalling endodomains. we assessed antitumour activity in vitro by measuring killing, secondary expansion and cytokine production after co-culturing gamma-delta car-t cells with different cd19+ all cell lines, and in vivo in nsg previously engrafted with a b-all semi-cell line. although allogeneic hematopoietic stem cell transplantation (allosct) is a curative option to treat hematologic malignancies, disease recurrence remains a concern in the setting of high risk diseases. thus, post allosct therapeutic strategies are needed to treat and/or prevent disease progression. in this setting, donor lymphocytes infusion (dli) is an option as post allosct immunotherapy aiming to enhance graft versus leukemia (gvl) effect. although dli may induce persistent remission, graft versus host disease (gvhd) is a potential complication following dli. because of the suspected higher incidence of gvhd in the presence of hla mismatches, few series focused on dli following haploidentical stem cell transplantation (haplosct) so far. we therefore report our experience of dli following haplosct using post-transplantation cyclophosphamide (pt-cy) platform. we included in this single center study all consecutive adult patients with hematological malignancies who received dli after haplosct with pt-cy as part of gvhd prophylaxis from 2013 to 2016 (n = 21). conditioning regimens were non-myeloablative (low dose tbi-based) or with reduced toxicity (various dose of busulfan according to disease and patient characteristics). ciclosporine a and mycophenolate mofetil were given as additional gvhd prophylaxis in all cases. dli were given at escalating doses, expressed as cd3+cells/kg, without gvhd prophylaxis, and ranged from 1 × 10 5 our study suggests that dli following haplosct with pt-cy is feasible. gvhd is frequent but with a relatively low incidence of severe forms. no response rate was achieved in the context of hematological relapse, underlining that preemptive or prophylactic strategy might be preferred. indeed, the overall good outcome in patients receiving prophylactic dli is promising taking into account the poor prognostic of the diseases indicated for alternative donor transplantation. further prospective studies are needed in specific disease settings to assess the benefit for using such post allohsct immune-intervention. [p103] disclosure of conflict of interest: none. dual specific cytokine-induced killer cell therapy as a treatment option for life-threatening ptld-a case report of the frankfurt experience l-m pfeffermann 1 infection with epstein-barr virus (ebv) is a frequent complication after allogeneic hematopoietic stem cell transplantation (hsct) and besides relapse remains a significant cause of morbidity. prolonged immunosuppression or delayed t-cell recovery may favor ebv reactivation after transplantation, which under these circumstances can lead to life-threatening lymphoproliferative disease (ptld). consensus is lacking on the optimal treatment of ptld. adoptive immunotherapies with both anti-tumor capacity and restored virus-specific cellular immunity may represent optimal treatment options especially when considered in the context of ptld. in this case report we applied in vitro activated t-cells namely cytokineinduced killer (cik) cells with dual specific cytotoxic capacities transferring both anti-cancer potential and donor t-cell memory against ebv infection for the treatment of ebvassociated ptld which progressed to highly proliferative large b cell lymphoma during delayed t-cell recovery after allogeneic hsct. the reported patient had received an allogeneic hsct for secondary myelodysplastic syndrome following acute myeloid leukemia, and due to delayed t-cell recovery had developed ebv-related ptld two months after transplantation. treatment with rituximab, conventional ebvspecific t-cells and wildtype cik cells failed, therefore the patient was offered ebv-specific cik cells on a compassionate use basis. ebv-specific cik cells were generated from peripheral blood mononuclear donor cells. cells were activated and expanded in the presence of ifn-γ, il-2, anti-cd3 antibody and il-15. on day 0 and 2 of culture an ebv peptide pool was added for additional priming. follow-up analysis included in vitro and in vivo monitoring of ebv-specific cik cells. with above mentioned protocol we were able to generate cik cells containing 1 × 10 4 cd8 + ebv-specific t-cells/kg body weight of the patient. infusion of ebv-specific cik cells resulted in rapid clearance of plasma ebv dna level and sustained disappearance of large (vol. 27cm 3 ) ptld-malignant lymphoma. during one-year follow-up analysis we were able to detect ebv-specific cik cells (cd4 + and cd8 + ) in vivo by flow cytometry using specific mhcdextramers. facs-monitoring of the patient´s blood revealed besides cd8 bright t-cells also an increasing cd8 dim t-cell population with a remarkable percentage of t emra cells within this compartment (up to 95%) indicating virus-specific t-cells. no cytokine release syndrome appeared after ebv-specific cik cell treatment, but cytokine secretion patterns, analyzing serum of the patient, reflected cytotoxic and anti-virus capacity provided by this treatment. cytotoxic potential, as well as t h 1 cell differentiation and function offered by ebvspecific cik cell treatment were further confirmed by in vitro analysis. ebv-specific cik cells revealed an 1. s168 hematologic disease. the global survival ratio in the follow-up was 56.8% (with 66.6%, 60.6% and 42.8% survivals in 3, 6 and 12 months, respectively). the variables significantly associated with greater survival were: type of gvhd (cgvhd), number of affected organs (an organ had to be moderately or severely affected to be included in this category) and steroid dependence as the main reason to initiate ecp (see figure 1 ). there was a trend towards significance for the degree of gvhd and cutaneous involvement to be factors associated to enhanced survival ratios. extracorporeal photopheresis is a safe treatment option for patients with gvhd, generating a response and decreasing immunosuppression in an important percentage of them. the presence of cgvhd rather than agvhd, a lower severity degree of the condition, having a lower number of affected organs, skin as main affected organ and steroid dependence as the reason to start the ecp treatment were all factors associated with greater survival in our sample. disclosure of conflict of interest: none. tx) . we report the case of a patient refractory to chemotherapy treated with ibrutinib as debulking therapy before allo-tx. in june 2014, a 62 years old woman was diagnosed with mcl. the staging performed by whole body ct scan, colonoscopy, egds and bone marrow (bm) biopsy was conclusive for stage iva with bulky lymph node over and below the diaphragm, bm, enteric and peripheral blood (pb) localization. the planned treatment included 3 cycles of r-chop, 1 cycle of high dose (hd) cy, 2 cycles of hd arac and autologous sct. after completion of hd cy, the restaging showed progressive disease, with a thyroid involvement and histologic switch in a blastoid variant. disease continued to progress even after 2 cycles hd arac, so we tried to control the disease with r-bendamustine (90 mg/mq on days 1-2 of 21-d cycle), but after the first cycle the neck circumference increased. we shift to lenalidomide (25 mg on days 1-21 of 28-d cycle) without any response after two cycles. we excluded patient from autologous sct programme because of chemo-refractoriness and we searched matched unrelated donor because no hla identical sibling was available. we started a therapy with ibrutinib (560 mg/die on days 1-21 of 21d cycle). after the first cycle we observed a rapid response with decrease of neck size and the disappearance of superficial lymphnode; we performed 6 cycles of ibrutinib, and we reached a good partial remission with lymphnode of max 4 cm, and a bm and pb involvement of 20%. meantime an unrelated donor with 7/8 hla matching was identified, so in december 2015 we performed allo-tx with reduced-intensity conditioning (thiotepa 5 mg/kg-fludarabine 90 mg/m 2 -melphalan 100 mg/m 2 ) and cyclosporine and short term methotrexate as gvhd prophylaxis. engraftment was at day +17. in the first 100 days after allo tx she experienced a clostridium enteritis, transient cmv reactivation and acute gastrointestinal gvhd on day +60 with rapid response to steroid therapy. main complication happened on day 100, when sudden fever and stupor, progressive to coma, occurred; subsequently pneumococcal encephalitis was diagnosed, with positive csf microbiological exam and two signal alteration in the right cerebral hemisphere at mri. the patient was treated with ampicillin and ceftriaxone with a favourable outcome. the mcl revaluations performed at 2, 5 and 9 months showed complete remission with disappearance of all pathological lymph node and pb involvement. currently the patient is at 1 year post asct, she is enrolled in a rehabilitation program and mcl is in complete remission. our experience seems to indicate that ibrutinib is safe and can be used as bridge to allo-tx therapy in refractory mcl. we will investigate side effects of this platform of therapy, and, given the early occurrence of pneumococcal infection, we will consider to perform capsulated bacteria vaccination before allo-tx. disclosure of conflict of interest: none. [p107] pt 2 was diagnosed with c-all in 2014 and received a mud-hsct (tbi 8gy, cyclosphosphamide) in 1/15 due to persistend mrd. following early rel 6/15, 2 cycles of blina led to mrd+ cr, for which a 2nd hsct from a haploidentical family donor (busulfan, thiotepa, fludarabine) was performed in 10/15. molcr lasted 3 months and rel3 was treated with 3 cycles of weekly io followed by one dli (5x10-4 cd3+ cells/kg), resulting in mrd+ cr and complete donor chimerism. five weeks after the last io cycle, the pt was admitted with ascites, hyperbilirubinemia and reduced general condition. vod was suspected, but diagnostic paracentesis revealed malignant ascites demonstrating fatal progressive disease. conclusion. our data suggest that the sequential use of io and dli is feasible even for heavily pretreated patients with r/r all after hsct and can induce molecular remissions. we observed an unusual case of late onset, severe vod responding to defibrotide and one all relapse manifesting itself with ascites in our patients. we therefore suggest close monitoring of liver function tests in the setting of this therapy and extensive diagnostic work-up for any developing liver abnormalities or ascites. disclosure of conflict of interest: ng: advisory board (pfizer, amgen); research support (amgen); gb: honoraria (amgen). [p108] p108 while allogeneic hematopoietic stem cell transplantation from matched related and unrelated donors has become a standard of care treatment for patients with hematological malignancies, transplantations from mismatched or haploidentical family donors remain challenging. currently t-replete and t-deplete transplantation strategies are applied aiming to improve the outcome after haploidentical transplantation. despite high rates of relapse many centers regard post-transplant cyclophosphamide, a t-replete strategy, as a standard of care approach. we have developed a t-depleted transplant approach where donor lymphocytes selectively depleted of alloreactive t-cells (atir101) using th9402, arhodamide-like dye, are infused after cd34-selected haploidentical hsct, to overcome the challenges of infectious complications, gvhd and relapse. in phase i (cr-air-001) we have demonstrated safe infusion of these lymphocytes at doses up to 2 × 10 6 viable t-cells/kg. recently, we reported a promising 1-year grfs was 57% from a phase ii trial (ash 2016),that is awaiting final results soon. here, we introduce a randomized, multicenter phase 3 study (cr-air-009), where 200 patients with acute myeloid leukemia (aml), acute lymphoblastic leukemia (all) or myelodysplastic syndrome (mds) are planned to undergo a haploidentical hsct with either a t-cell depleted graft and adjunctive treatment with atir101, or with a t-cell repleted graft and use of posttransplant cyclophosphamide. inclusion and exclusion criteria are listed in table 1 . all patients will undergo myeloablative conditioning consisting of either tbi (12 gy) or melphalan/ busulfan, in combination with thiotepa and fludarabine. patients in the atir101 study group will receive atg (2.5 mg/ kg once daily for 4 days) during conditioning and atir101 infusion at a dose of 2 × 10 6 viable t-cells/kg is given between 28 and 32 days after the hsct. patients in the ptcy group will receive cyclophosphamide (50/mg/kg) on day 3 and 4 (or 5) with subsequent use of immune suppression up to 6 months post-hsct. the primary endpoint of the study is gvhd-free, relapse-free survival (grfs). grfs is defined as time from randomization until grade iii/iv acute graft-versus-host disease (gvhd), chronic gvhd requiring systemic immunosuppressive treatment, disease relapse, or death, whichever occurs first. this endpoint captures both safety and efficacy. additional secondary endpoints are overall survival (os), progression-free survival (pfs), relapse-related mortality (rrm) and transplantrelated mortality (trm). patients are planned to be randomized in study centers in europe and north america. a number of 40-50 sites are planned to participate in the study. enrolment is expected to continue until mid-2018 with initial results being available first half 2019. results of this study will determine which transplant regimen provides most clinical benefit in haploidentical donor transplantation, with the promise of an effective regimen without the use of post-transplant immune suppression. disclosure of conflict of interest: jr is an employee of kiadis pharma. multipotent mesenchymal stromal cells (mscs) are used for prevention and treatment of graft versus host disease after allogeneic hematopoietic stem cells transplantation due to their immunomodulatory properties. mscs fate in vivo after infusion is unknown. the aim of this study was to analyze the changes in mscs and allogeneic lymphocytes properties when co-cultured in vitro to simulate their interactions in vivo. the bone marrow from 13 donors (7 male and 6 female aged 22-62 years, median 27 years) was used. mscs were cocultured with allogeneic lymphocytes in a ratio of about 1:10 for 4 days and their basic properties were analyzed over time. lymphocytes were activated by adding to the culture medium 5 mg/ml of pha (pha-lymphocytes). some mscs were treated for 4 h with 500 u/ml ifnγ (γmscs). determination of gene expression levels was performed by reverse transcription polymerase chain reaction in real time (modification of the taq-man) and of antigen expression on mscs and lymphocytes by flow cytometry. significant reduction in the proportion of viable cells was observed in mscs co-cultured with pha-lymphocytes. in γmsc co-cultured with pha-lymphocytes no reduction in the proportion of living cells was revealed. this indicates the sensitization of mscs by ifnγ to factors secreted by pha-lymphocytes. in mscs co-cultured with pha-lymphocytes and lymphocytes mean fluorescent signal intensity level (mfi) of cd90 gradually decreased. ifnγ treatment and co-cultivation with lymphocytes led to significant increase of hla-dr mfi on mscs. co-cultivation with lymphocytes increase the hla-dr mfi on mscs much stronger than ifnγ treatment. relative expression level (rel) of ido1 gene increased dramatically in both mscs and γmscs when co-cultured with lymphocytes. at a day 1 in γmscs rel of ido1 increased 500 fold, and then gradually declined. in mscs cocultured with lymphocytes il-6 rel increased almost 20-fold and then decreased 2-fold at the fourth day. the csf1 rel in γmscs showed twofold increases, upon incubation mscs and γmscs with lymphocytes csf1 rel increased fourfold and sevenfold, respectively. co-culture of msc and γmscs with lymphocytes led to decrease in the proportion of cd25, cd38, cd69, hla-dr, and pd-1 positive cells (both cd4 and cd8) after one day, compared with pha-lymphocytes without mscs. proportion of cd25+, hla-dr+ and pd-1+ cells also decreased after 4 days of co-culturing with msc or γmscs (compared with pha-lymphocytes without mscs), but anyway number of activated cells increased 1.2-3 folds compared with first day. number of cd69+ lymphocytes after 4 days of co-culturing with mscs or γmscs did not vary significantly from control and decreased in comparison with first day. main inhibition of activated lymphocytes by co-culturing with mscs occurs during the first day of their interaction, and then the inhibition became less effective, moreover in mscs decreased the rel of the main immunomodulating factors, and most of them were eliminated. mscs treatment with ifnγ resulted in improved survival and resistance of these cells to lymphocytes action. the results indicate that the effect of mscs injected intravenously to patients is limited to several days. disclosure of conflict of interest: none. autologous adipose-derived mesenchymal stem cells (admscs) embedded in platelet-rich fibrin (prf) promote healing in different types of wounds. by avoiding the needlerelated complications, prf-embedded autologous admscs graft provides a new effective stem cell-based therapeutic strategy for wound healing. adult male (age ⩽ 75yo) were equally divided (n = 5 per group) into group 1 (prf only), and group 2 (prf+admscs). regular dressing (without any agent) was used for both groups with a frequency of changing every 3 days. rpf with or without admscs was patched on the wound (maximum surface area 77 cm 2 ). all patients were followed up until complete healing. a complete healing was noted in both groups; however, the healing in group 1was very slow (after 10 weeks), compared to a quicker one in group 2 (after 6 weeks). control of the moisture was very well noted in group 2, less in group 1. group1 showed a lot of exudates on the wound; less exudate in group2 were noted. infections were absent. both groups had a colonized wound. signs of inflammation were very well noted in group 1; no signs of inflammation in group2. admscs embedded in prf offered rapid wound healing responses then prf alone. keywords: mesenchymal stem cells, platelet-rich fibrin, engineered tissue wound healing disclosure of conflict of interest: none. stem cell source p112 additionally cryopreserved g-csf primed pbsc can substitute the second transplantation for the patients with acute leukemia who lately relapsed after hematopoietic stem cell transplantation y lee 1 , j moon 2 , ih lee 3 and s sohn 2 1 department of hematology, kyungpook national university hospital; 2 department of hematology,kyungpook national university hospital and 3 kyungpook national university hospital although allogeneic hematopoietic cell transplantation (allo-hct) is a potentially curative therapy for acute leukemia, survival outcomes of the patients relapsed after transplantation remains poor with high early mortality and a small percentage of second remission. this study evaluated the efficacy of dli using g-csf primed pbsc additionally cryopreserved for the patients who relapsed after allo-hct. we retrospectively reviewed the medical records of the 255 patients who received allo-hct for acute myelogenous leukemia (aml), myelodysplastic syndrome (mds), and acute lymphoblastic leukemia (all) between 1998 and 2013 in kyungpook national university hospital. among the 93 patients who had relapsed after allo-hct, the 39 patients received dli using the additionally harvested cells. at the time of harvest for the first hct, collecting targeted pbscs (greater than 6 × 10 6 /kg cd34+ cells) allowed us to cryopreserve surplus pbscs, including cd3+ cells with dimethylsulfoxide in a nitrogen tank. then, we analyzed the efficacy of dli for the patients who were classified into early relapse or late relapse group by the median time of relapse after transplant. the median age at transplant was 38.5 years (range 15-68 years) and male was 111 patients (44.4%). primary diseases for allo-hct were aml/mds (n = 175, 70%) and all (n = 75, 30%). one hundred forty three patients (57.2%) were in cr1 (complete remission), 25 (10%) in further cr, and 87 (33.2%) in relapsed and refractory status. one hundred seventy one patients (68.4%) received myeloablative conditioning regimen. the median dose of cd34+ cell was 5.21 × 10 6 /kg (range: 1~20.6 × 10 6 /kg). almost 95% of patients achieved the neutrophil engraftment with a median time of 13 days (range: 9-24days). the 1-year overall survival (os), relapse free survival (rfs), non-relapse mortality (nrm) and graft-versus-host disease (gvhd)-free/relapse-free survival (grfs) since hct was 55.3 ± 3.1%, 66.0 ± 3.2%, 28.2 ± 0.3%, and 32.9 ± 3.1%, respectively. there was no significant difference according to s172 the infused cd34+ cell dose (lower o6 × 10 6 /kg vs higher ⩾ 6 × 10 6 /kg). the incidence of chronic gvhd was more frequent in higher cd34+ group (32.9% vs 48.2%, p = 0.042). median time from hct to relapse was 139 days (range: 21~1801 days). after relapse, 46 patients (49.4%) were treated with salvage chemotherapy, 9 patients (9.6%) with second allo-hct, and 38 patients (40.8%) with dli. the median number of cd3+t cell was 2.95 × 10 7 /kg (range: 0.1~5.43 × 10 7 /kg). fourteen patients (23.6%) achieved dli induced cr, 20 patients progressed, and 6 patients were not evaluable for response. dli induced acute gvhd was observed in 24 patients (63.1%) and chronic gvhd developed in 4 patients (10.5%). in late relapse group, the 1-year os since post-transplant relapse was significantly higher in dli group than non-dli group (figure 1 , 53.4 ± 7.4% and 26.7 ± 7.4%, p = 0.039) but, early relapse group had no difference. the patients treated with dli showed significantly survival benefit in late relapse group (median 286 days vs 103 days, p = 0.002). the incidence of dli-induced gvhd does not differ between two groups. dli for the patients who lately relapsed after allo-hct can be a feasible and an effective option in terms of response, donor convenience and it's cost. in the late relapse group, g-csf primed dli may replace second transplantation. disclosure of conflict of interest: none. cord blood transplantation-19 years of experience c alves 1 , f amado 2 , f bordalo 2 , s ferreira 2 , s lopes 2 , c pinho 2 , t rodrigues 2 , l antunes 3 and s roncon 2 1 serviço de imuno-hemoterapia; 2 serviço de terapia celular and 3 registo oncológico do norte, instituto português de oncologia do porto francisco gentil, epe allogeneic haematopoietic stem cell transplantation (hsct) is a well-established treatment for patients with malignant and non-malignant haematological disorders. cord blood transplantation (cbt) has extended the availability of hsct to patients that would not otherwise be eligible for this curative approach because of the lack of human leucocyte antigen (hla) identical donor. the aim of this retrospective study was to analyse and characterize 19 years of cbt activity in our institution (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) . we examined patient electronic files and created a database in excel to register cord blood unit (cbu) parameters and patient characteristics. after thawing, cbu was washed/diluted with validated procedures; the cellular content was evaluated by immunophenotyping and followed ishage recommendations; sterility was assessed by bacterial/fungal cultures, viability by trypan blue exclusion assay and functionality by clonogenic capacity. the transfusion of blood products after transplant was quantified and the hematological recovery (hr) established using cibmtr criteria. correlation between continuous variables was assessed with spearman coefficient. overall survival (os) was determined according to cellular content and hla-disparity by the kaplan-meier estimator. survival between groups was compared using the log-rank test. a total of 59 cbu were administered to 57 patients (table 1) : 30/57 female, the main diagnosis was acute leukaemia (35/57). a sibling cbu was used for 3 patients; the unrelated were imported from europe (66%), usa (32%) and oceania (2%). hla-matching was 6/6, 5/6, 4/6 and 3/6 for 8, 21, 15 and 6 patients, respectively; 47% were abo-identical. after thawing, 95% were washed and presented no microbial growth. the majority of patients were submitted to a bussulfan-based myeloablative conditioning regimen; graft versus host disease prophylaxis was performed with a calmodulin inhibitor+mycophenolate mofetil. complete and mixed chimerism was verified in 42% and 7% of patients; 5% had graft failure; the rest were unknown results (46%). at the moment, we reported 27 patients alive (22 in complete remission, 5 with evidence of disease relapse) and 30 dead at a median of 6 (0.4-198) months after cbt; the most frequent cause (43%) was recurrence of the initial clinical condition. the correlation between nucleated cells (nc) and cd34+ cells per kg/hr (p = 0.277; p = 0.123) and number of cd34+ cells per kg/os (p = 0.123) was not statistically significant. however, the engraftment and os was associated with hla-mismatch (p = 0.025; p = 0.012) and os was related to nc per kg (p = 0.012). our clinical results suggest that despite increased hla disparity, ucb offers promising results. ucbt is feasible in patients when the unit contains a high number of cells. there are several strategies for the future, related to cbu expansion and homing techniques, nurturing procedures, selection of optimal cbu unit and enhancement of immune recovery, in order to improve the application of cbu. s173 received sirolimus and mmf. median time to neutrophil and platelet engraftment was 10 days (9-13) and 18 days (17-124), respectively. one patient died from parainfluenza pneumonia (d111), one patient from ptld (d 203), one patient from late pulmonary vod (d379), and one patients from relapse (d89). with a median follow up for survivors of 17 months, one year survival is 64%. three patients had grade 2-3 gvhd and none of the survivors have chronic gvhd. though unrelated donor chimerism was dominant early after transplant and contributed to early count recovery, definitive engraftment was dominated by ucb chimerism in all but one patient. conclusion: among older adult patients with hematological malignancies,~20% lack haplo-identical relatives. for these patients, double or single ucb transplant is challenging because of delayed engraftment. cd34 selected partially matched grafts from unrelated donors hasten hematopoietic recovery and are over time outcompeted by ucb grafts which provide robust hematopoiesis with low risk of chronic gvhd. the combination of mismatched unrelated hematopoietic progenitors and ucb grafts provides an attractive alternative for older patients lacking hla-idental donors or haploidentical relatives. in planned trials, mismatched donors may be selected based on kir type to further enhance gvl effects. disclosure of conflict of interest: partially supported by miltenyi biotec. haploidentical stem cell transplantation (haplo-sct) is an attractive option for patients who do not have an hlamatched donor. historically it has been associated with high rates of graft rejection, relapse and low incidence of graft versus host disease (gvhd). to decrease these issues we have considered the use of primed bone marrow as stem cell source, early withdrawal of immunosuppressive therapy and the use of donor lymphocytes infusions (dli) in haplo-sct with high-dose post-transplantation cyclophosphamide (ptcy) as main gvhd prophylaxis. to analyse our incidence of acute and chronic gvhd and overall survival (os) in patients with haplo-sct with short course of inmunosupressive therapy. we retrospectively analyzed a cohort of 28 patients who underwent haplo-sct with primed bone marrow as stem cell source, between years 2012 and 2016 in our centre. gvhd prophylaxis consisted in ptcy (50 mgr/kg on days +3 and +4) and tacrolimus plus mycophenolate (mmf) from day +5 as recommended by baltimore group. mmf was stopped on day +28. tacrolimus was tapered off from day +50 with withdrawal on day +120 in patients without gvhd or with active disease. dli were considered if mixed chimerism, relapse or disease progression appeared. the characteristics of the patients are shown in table 1 . results: there was no primary graft failure. eight of 27 patients (29.6%) developed agvhd (grade ii-iv) and it was severe (grade iii-iv) in 2 patients (7.4%). cutaneous agvhd location was the most common presentation (9 patients (33%)) and it was associated with intestinal gvhd in 2 patients. twenty two patients were evaluable for cgvhd. thirteen patients (59%) developed chronic gvhd that was mild, moderate and severe in: 8 (36.3%), 3 (13.6%) and 2(9%) patients, respectively. the median time of onset cgvhd was 6 months (range: 3-33) and it was related with previous withdrawal of the immunosuppression in 5 (22.7%) patients, tapered off immunosuppression in 6 (27.2%) patients and dli in 2 (9%) patients. systemic treatment was required in 8/13 patients but only 2 patients were treated with high doses of steroids (41 mg/kg/day). the median days of is therapy in patients who developed gvhd was 172 days (range: 81-244). dli were used in 7 (25%) patients because of: relapse/disease progression in 5(17.8%) and secondary graft failure in 2(7.1%). two patients achieved complete remission and 2 patients developed cghvd. the median number of dli per patients were 2 (1-3) with a median cd3+cell of 3 × 10 6 /kg (range: 1 × 10 5 -1 × 10 7 /kg). with a median follow-up of 12 months (range: 1-56), the estimated os at 1 and 2 years after haplo-sct were 74% and 52%, respectively. at the moment of this study 17 patients (60.7%) were alive, 14 patients in complete remission, 2 in partial response and 1 in progression. eleven (39.3%) patients died due to: disease (4), infections (4), pleuropericarditis (1), hepatic veno-occlusive disease (1) and refractory gvhd. five patients (17.8%) are without is therapy and without gvhd symptoms. in our experience, early withdrawal of immunosuppression following haplo-sct with primed bone marrow and posttransplantation cyclophosphamide facilitates the development of chronic gvhd and can decrease the relapses in patients with high-risk hematological malignancies. it is necessary more follow up and more studies to confirm this preliminary results. [p115] disclosure of conflict of interest: none. s174 myeloid malignancy (n = 20 except 1 patient with saa) received fludarabine (flu)/busulfan ± tbi 4gy, while lymphoid malignancies (n = 9) received flu/tbi 8gy or cy/ tbi 12gy. all patients received g-csf-mobilized t-cell replete pbsc from a haplo donor. gvhd prophylaxis was ptcy 50 mg/ kg on day +3/+4, tacrolimus (d+5 to +180), and mycophenolate (d+5 to 35). the median duration of follow up of surviving patients is 21 months. median age was 45 (17-66) years, 17 patients (59%) were male, 14 (48%) were african american, and 14 patients (48%) had comorbidity index (hct-ci) ⩾ 3. all patients had hematological malignancy (except 1 patient with saa) including 12 patients (43%) not in cr. disease risk index was high/very high in 12 (42%) and intermediate in 14 (48%). on the day of transplant, 14 patients (48%) did not receive steroid premedication ( = no-steroid group), while 15 patients (52%) received 125 mg of methylprednisolone 30 minutes prior to pbsc product infusion ( = yes-steroid group). all the following outcomes are described in the ‛no-steroid' vs ‛yessteroid' group, respectively. cumulative rate (ci) of anc engraftment (⩾500/μl) on day +28 was 79% (95% ci 60-100%) and 93% (95% ci 81-100) (p = 0.07). ci of platelet engraftment (⩾20 000/μl) on day +56 was 71% (95% ci 51-99%) and 90% (95% ci 74-100%) (p = 0.2). primary engraftment failure was observed in 3 patients; 1 in yes-steroid and 2 in no-steroid. no primary engraftment failure was observed with myeloablative tbi (8-12 gy) (n = 9). ci of agvhd gii-iv (day+100) was 21% (95% ci: 8-58%) and 28% (95% ci: 12-64%) (p = 0.77). ci cgvhd (1 year) was 42% (95% ci 20-90%) and 49% (95% ci 27-87%) (p = 0.48). ci of relapse (1 year) was 22% (95% ci 7-75%) and 7% (95% ci 1-47%) (p = 0.71). ci of non-relapse mortality (nrm) (1 year) was 51% (95% ci 29-90%) and 38% (95% ci 19-77%) (p = 0.48). post-infusion noninfectious fever (d0 to +3) was observed in 12/14 (86%) and 12/15 patients (80%). median tmax was 103f and 102f (p = 0.38). only 1 patient in the no-steroid group developed life-threatening cytokine release syndrome and survived. no difference of viral reactivation was noted between groups. cmv reactivation occurred in 7 (50%) and 13 patients (87%), bk reactivation in 20% (in both groups), hhv6 in 36% and 40%, ebv in 14% and 7%. the 18-month overall survival was 46% (95% ci 17-74%) and 60% (95% ci 35-85%) (p = 0.53). the 18-month disease-free survival was 41% (95% ci 15-68%) and 53% (28-79%) (p = 0.46). t-cell replete haplo pbsc transplant is effective therapy for patients with high-risk hematological malignancies. high-dose steroid premedication with pbsc infusion neither influences transplant outcome nor prevents post-infusion febrile reaction. disclosure of conflict of interest: as discloses grant support (american porphyria foundation), consultation (medpace inc), research support (astellas and fate therapeutics), honoraria (alxion, and spectrum), and royalty for licensing of intellectual property (incysus biomedical). intrabone transplant of unwashed cb in hematological malignancies: engraftment and safety f giglio 1 , s marktel 1 , r greco 1 , m morelli 1 , mt lupo-stanghellini 1 , e xue 1 , l lazzari 1 , m marcatti 1 , m zambelli 2 , c parisi 2 , r milani 2 , s piemontese 1 , a assanelli 1 , c corti 1 , m bernardi 1 , f ciceri 1 and j peccatori 1 1 unit of hematology and bone marrow transplantation, irccs san raffaele scientific institute, milano, italy and 2 immunohematology and transfusion medicine unit, irccs san raffaele scientific institute, milano, italy cord blood transplant (cbt) in adult patients (pts) is limited by the risk of graft failure or delayed engraftment due to low cell counts. to improve the capacity and speed to engraft, intrabone (ib) cbt technique has been investigated, showing high rate of engraftment and low acute gvhd, also when compared with double cb transplant. cb units washing procedure has been suggested to remove dmso toxic potential effect. we report our experience in 15 adult pts with hematological malignancies receiving ib unwashed cb in an attempt to reduce the loss of progenitor cells and the risks associated with cell-washing procedure. between 2009 and 2016 we performed 15 allogeneic hematopoietic stem cell transplant (hsct) using unwashed cbu as a source and infusing them ib. all pts were adult and suffering from hematological malignancies. this population was characterized by very high-risk and advanced phase disease. all pts received a cb hsct because of unavailability of sibling or matched unrelated donors. eleven pts received a treosulfanbased myeloablative conditioning regimen and a sirolimusbased ghvd prophylaxis; four pts received busulfan-based myeloablative conditioning and a cyclosporine-based ghvd prophylaxis. cb units were thawed and diluted with albumindextran solution immediately before the transplant. this ‛nowash' dilution was implemented to reduce product manipulation that may results in cell loss. furthermore, graft manipulation risks potential contamination, requires increased technologist time, and delays time to infusion. the ib infusion was performed under local anesthesia and with short conscious sedation, at bedsite in the bmt ward. the infusion was preformed monolaterally or bilaterally according to the volume to be infused. starting from a 10% dmso concentration in the cb units before the dilution, the graft products contained a median of 3.6% dmso (range: 2.3-6.9) at ib-hsct. the median cd34+ cells infused were 0.09 × 10 6 /kg b.w. (range: 0.03-0.82). the median mono-nucleated cells were 15.66 × 10 6 /kg b.w. (range: 4.7-36.8). the median cd3+ t-cells were 2.6 × 10 6 /kg b.w. (range: 0-5.54). the median infused volume was 75 ml (range: 60-215). no procedure-related adverse events were observed, nor related to the ib technique, neither to the sedation. of the 15 transplanted pts, 9 were evaluable for engraftment (1 patient rejected the graft and 5 patients died before day +30 because of severe infections). all 9 achieved anc 40.5 × 10 9 /l after a median of 24 days (range: 16-45) and 8 achieved plt 420 × 10 9 /l at a median of 45 days (range: 25-141). three patients developed grade iii-iv acute gvhd grade. according to extreme heterogeneity of the population no correlations with relapse incidence and diseasefree survival could be evaluated. ib infusion of unwashed cb is feasible, safe, easy to perform. no adverse events related to the procedure were documented. no dmso toxicity was documented. engraftment was obtained in all evaluable pts. our data confirm that direct ib cbt overcomes the problem of graft failure even when low numbers of cb cells were transplanted, thus leading to the possibility of using of this technique in a large number of adult pts, for whom this approach represents the sole possibility of long-term survival. the ‛no wash' cb dilution can also help the implementation of ib transplant thanks to the easier graft manipulation. [p117] disclosure of conflict of interest: none. lower incidence of cgvhd after cord blood transplantation for hematological malignancies in comparison with hematopoietic stem cell transplantation from other donors: 20 years' experience in a single institute m yoshino, m obiki, m osakie, s ikeno, t sato, m nasashima, y kagaya, n kawashima, t morishita, y ozawa and k miyamura department of hematology, japanese red cross nagoya first hospital the outcome of cord blood transplantation (cbt) for hematologic malignancy was investigated. however the incidence of gvhd is not accurately known. the goal of this study was to compare the outcome of cbt with allogenic hematopoietic stem cell transplantation (allo-hsct) from other sources, mainly unrelated bone marrow (urbm). patients' characteristics: 755 patients who underwent allo-hsct, between 1990 and 2015 in our hospital were retrospectively analyzed. donor sources were cord blood cell (n = 112), urbm (n = 372), hla matched sibling bone marrow (sibling bm) (n = 166), and hla matched sibling peripheral blood stem cell (sibling pbsc) (n = 105). in cbt, the median age was 38.5 (17-68), and the diagnosis included aml (69), all (15), mds (13), cml (4) and other (11). the disease risk was good in 58 and poor in 54. disease risk was slightly higher in comparison with other sources. prophylaxis of acute gvhd was tacrolimus, short-term methotrexate (88), cyclosporine, short-term methotrexate (22) and others (2) . the 5-year overall survival (os) rate after cbt (1990 cbt ( -2004 10 .0%, engraftment failure 6.7%, acute gvhd 3.3%), relapse 10.0% and other 6.7%. in cbt cases, engraftment failure after allo-sct was observed in 24 cases (21.4%) which is higher than that among urbmt (8.6%), 32 out of 372. 10 cbt cases underwent the second allo-hsct and 9 patients achieved engraftment and 7 patients were alive at 100 days after allo-hsct. 6 of them survived at 2 years after allo-hsct. our results suggest that the outcome of cbt has improved in recent years. moreover, cbt has an advantage in the least cumulative incidence of acute/chronic gvhd. cbt may well create the best outcome in the future. disclosure of conflict of interest: none. chronic active epstein-barr virus (ebv) infection is a major type of ebv-associated t/nk-cell lymphoproliferative disorders (lpd) in childhood. however, young adults rarely develop chronic active ebv infection (caebv), and shows more aggressive features than that of childhood. umbilical cord blood transplant (ucbt) is a possible treatment option for caebv patients who have no hla-matched donor, but there is little information available about the efficacy and safety of ucbt for adult patient with caebv. we analyzed six adult patients with caebv who underwent a single-unit ucbt between 2010 and 2016 at our institute (including a case reported in 2014 [1] ). the diagnosis of caebv was made according to the criteria proposed in 2005 [2] ; persistent infectious mononucleosis (im)-like symptom and detection of increased ebv genomes in peripheral blood mononuclear cells (pbmc). ebv-dna load was measured using real-time quantitative pcr. median patient age at diagnosis was 27 (23-39) years. target cells of ebv-infection were cd4+t cells (n = 4) or nk cells (n = 2). median ebv-dna load was 2.6 × 10 4 copies per microgram of dna in pbmc (range: 1.6 × 10 3 -1.3 × 10 5 ) at the diagnosis. all patients were given prednisolone and cyclosporine, and then etoposide (n = 2) or combination chemotherapy (n = 4) before transplant. ebv load has slightly decreased to a median of 1.3 × 10 4 copies (range: 4.0 × 10 2 -7.1 × 10 4 ), but disease status was active at ucbt in all. median time from the diagnosis to ucbt was 101 days (range: 62-440). one patient received total body irradiation (tbi) 12gy + cyclophosphamide (cy) 120 mg/kg + cytosine arabinoside 8 g/m 2 , and the other five patients received fludarabine (flu) + melphalan (lpam) 80-140 mg/m 2 or cy 120 mg/kg with tbi 4gy before ucbt. umbilical cord blood (ucb) was 4/6 hla-matched to the recipients. median number of infused ucb cd34+ cells was 0.87 × 10 5 /kg (range: 0.76-1.93 × 10 5 ). gvhd prophylaxis was consisted of tacrolimus + methotrexate or mycophenolate mofetil. neutrophil engraftment and complete donor chimerism were achieved in four patients, but two of them developed secondary graft failure (gf) early after engraftment. the other two patients developed primary gf. second ucbt was successfully performed in the 4 patients with gf a median of 31.5 days (range: 28-58) after the first ucbt. ebv genomes in pbmc became undetectable immediately after ucbt. at a median follow-up of 873 days (range: 54-2446), ebv-dna was undetectable or very low, and im-like symptoms were resolved in all cases. however, at 7-9 months after ucbt, two patients developed ebv+ b-cell lpd derived from donor cells, that was successfully treated with rituximab therapy. this study suggested that ucbt could eradicate ebv-infected cd4+ t cell-or nk cell-clones. ucbt can be a treatment option for adults with caebv. rituximab monotherapy was effective for post-transplant lpd from donor b cells. however, a high incidence of gf was observed in patients receiving reduced-s176 intensity conditioning of flu/lpam or cy /low-dose tbi. further studies are needed to find more optimal regimens for stable engraftment of ucb in adult patients with caebv. there is an increased incidence of ab0 incompatibility-50-60%, in allogeneic hematopoietic stem cell transplantation (allohsct) in patients who are russian citizens as a result of the variability of genetic polymorphism in the multi-ethnic population and a significant number of unrelated donors from international bone marrow registries. ab0 incompatibility in different types of allohsct may be an additional aggravating factor for the development of immunological complications and decrease effectiveness of treatment, but the data is still controversial [1] . from may 1999 to december 2015 in raisa gorbacheva memorial institute for children oncology, hematology and transplantation 1131 patients with leukemia, malignancies and hereditary diseases were included to the study, who were performed 1428 hsct: allogeneic unrelated -814 (57%); allogeneic related-344 (24.1%); haploidentical -267 (18.7%); umbilical cord blood in 3 patients (0.2%). age was 0-76, median-25 years. patients were predominantly with acute myeloid leukemia-37% (n = 602), acute lymphoblastic leukemia-30% (n = 501) and chronic myeloid leukemia -6% (n = 94). results: in 54.6% of cases (n = 780) ав0 incompatibility was determined: major-37.8% (n = 295); minor-45.4% (n = 354); combined-16.8% (n = 131). ав0 incompatibility in allohsct did not influence overall survival (p = 0.56) and frequency of acute graft versus host disease (gvhd) (p = 0.2). also there was no difference in overall survival depending on combination of condition regimen and ab0 incompatibility: reduced intensity (ric) or myeloablative (mac) (p = 0.7). an increased frequency of acute gvhd was observed in ric and ав0 incompatibility (30.8%) compared to mac (15.3%, p = 0.002). ab0 incompatibility was not a major factor (log worth 0.87) which influenced the fact and speed of donor's transplant engraftment in comparison to level of hlacompatibility (15.1), hematopoietic stem cell source (7.05) and type of hsct. but the presence of major ab0 incompatibility increase the period of erythroid recovery (p = 0.01) as reflected in the higher amount of blood transfusions. complications caused by ab0 incompatibility were identified in 2.4% of all cases (n = 34) including acute and delayed hemolysis, partial red cell aplasia and immune thrombocytopenia. conclusion. the presence of ав0 incompatibility is not a limiting factor to perform allohsct, however, it demands high quality prophylaxis and accurate transfusion therapy depending on ab0 incompatibility type to prevent immune complications. keywords: allogeneic hsct, ab0-incompatibility. poor graft function or graft failure have become common indications for infusion of immune-selected cd34 + cells (‛boost') or second unprocessed allo-hsct, creating the need for remobilization of the same related or unrelated. we retrospectively compared the results of two consecutive cycles of rh-g-csf treatment and peripheral blood progenitor cell collections in 20 related donors cared for at our institution between 2008 and 2016. mobilization consisted of the administration of rh-g-csf at a dose of 10 μg/kg per day injected in the evening, and apheresis was started in the morning of the fifth day after the fourth dose of rh-g-csf. collection was performed with a spectra or spectra optia cell separator (terumo bct). eleven out of 20 were haplo-mismatched donors and 9 were hla matched donors. four donors were re-collected because of recipient graft failure and 16 because of poor graft function; in the latter situation, immunomagnetic selection of cd34+ cells was performed on the collected cell product prior to infusion into the recipient, using the clinimacs medical device, as previously published. median donor age was 43 years (range: 18-66) at time of first donation, median weight 70kg (45-112) and bmi 24 (18-34). median delay between mobilizations 1 and 2 was 192 days (28-1530). interestingly, the median delay between collections was 119 days (43-701) in the haplomismatched setting and 231 (28-1530) in the matched setting. median number of circulating cd34+ cells/μl after the first 4 injections of rhg-csf was 61 vs 37 at the first and second mobilization cycles (po0.001, table 1 ). seven out of 20 donors (35%) requested more than one apheresis session to obtain the target number of collected cd34+ cells during the first cycle, as compared to 12 out of 20 (55%) for the second cycle: this is largely due to the higher target of cd34 + cells for the second collection, expecting that the median cd34 recovery after immunomagnetic selection is 60% in our experience. our study shows that a second cycle of mobilized peripheral blood progenitor cell collection from related donors is associated with a significant reduction in response to hematopoietic growth factors and mobilization capacity. this information allows planning the number of aphereses at the second cycle-and subsequently the number of immunoselection procedures to be carried out-taking into account the higher cd34+ cell dose target needed for subsequent immunomagnetic selection. cmv reactivation remains one of the main complications after allogeneic stem cell transplantation (hsct), requiring antiviral therapy, causing myelosuppression, prolonged hospitalization, higher treatment costs and mortality. cmv seronegative donors are recommended for cmv seronegative recipients. however data about donor selection for cmv positive (cmv-pos) recipients is not conclusive. some studies showed that selecting cmv-pos donors for cmv-pos patients might be beneficial. cmv-seropositivity is very high in lithuania among healthy blood and bone marrow donors (65%) and even higher among hsct recipients (up to 90%), so donor selection for cmv-pos recipients is an object of interest. retrospective analysis of cmv reactivations in cmv-pos allogeneic hsct recipients (transplanted during 2005-2014 year in vilnius university hospital) who survived at least 6 months post hsct was performed. data about cmv reactivation frequency, time post hsct, duration, maximal cmv dna copy number at each reactivation collected. cmv reactivation was considered when cmv dna copies detected 4500/ml in patient's blood. statistical analysis conducted using sas 9.2; student's tests for statistical significance; kaplan-meier methods for overall survival. among 316 allogeneic hsct recipients 286 (90.5%) were cmv-pos. 286 cmv-pos allo-hsct recipients were further analysed. 150 of them received graft from cmv-pos (pos/pos group) and 136-from cmv-seronegative donors (pos/neg group). more patients in pos/neg group experienced cmv reactivation in first 6 months post hsct in comparison to pos/pos group (83.1% vs 65.3%, p o0.05). pos/neg group patients had more cmv reactivations (2.5 vs 2 times in 6 months post transplant period, po 0.05), reactivations were diagnosed earlier post transplant (50.7 vs 123 days post hsct, p o0.0001), had longer duration (60.9 vs 40.3 days, po0.0001) and larger maximal cmv dna copy number (285107,9 vs 24339.4 copy/ml, p o0.0001) in comparison to pos/pos group patients. pos/pos group patients showed tendency for better survival than pos/neg group patients, however did not reach statistical significance. in a univariate analysis only hla mis-match and donor cmv seronegativity were factors statistically significantly associated with cmv reactivation. donor cmv serostatus is significant factor selecting donor for allo-hsct recipients. according to our findings, selecting cmv-pos donor for cmv-pos recipient may reduce cmv reactivation frequency and duration. disclosure of conflict of interest: none. selection of the best hsc donor when a matched donor is not available is still a matter of debate, and reports in pediatric population are scarce. this is a retrospective study conducted by brazilian society of hsct (sbtmo), including 11 centers, aimed to compare matched unrelated (matched-urd), mismatched unrelated (mm-urd) and unrelated cord blood (ucb) hsct. all or aml/mds patients o 18 y/o who have received first unrelated hsct between 2010-2014 were included. hla 4-digit typing was available for urd; for ucb, hla class-i 2-digit typing. overall survival (os), and cumulative incidence (ci) of agvhd, cgvhd, nrm and relapse were analyzed. on an unplanned analysis, we fitted a lognormal bayesian survival model with random effects, imputing the probabilities of ucb matching at 8 loci. a total of 212 patients were included (93 matched-urd, 47 mm-urd and 72 ucb). median age was 8.7 y/o. most patients had all (61%). proportion of early disease in matched-urd was higher (37%, against 19 and 21%). matched-urd were 10/10 4-digit hla matched (except one, for whom dq was not available), while most of mm-urd (89%) were 9/10 hla matched. ucb were 6loci (58%) or 8-loci typed (42%). based on previous report 1 on extending 6-loci hla typing to 8-loci ucb, we estimated that 23 ucb were 0-1 mismatched at 8 loci, 26 had 2 mismatches and 23 had 3 or more mismatches. conditioning regimens were mainly myeloablative, tbi-(59%) or bu-(33%) based. grafts were in vivo t-cell depleted in 67% of the patients, not balanced between groups (p = 0.01). with median follow-up of 3.3 years, 3y os was 56%, 52% and 39% for matched-urd, mm-urd and ucb, respectively (p = 0.02, log rank test). for matched-urd, mm-urd and ucb, ci of grades iii-iv agvhd at 6 months were 18%, 13% and 17% (p = ns); moderate/severe 3y-cgvhd, 8%, 20% and 4% (po 0.05); 3y-relapse, 26%, 21% and 14% (p = ns); and 1y-nrm, 26%, 24% and 49% (p = 0.06). we found out that primary graft failure occurred in 14 (19%) of ucb, compared to 9% in mm-urd and 3% in matched-urd. when ucb matching probabilities at 8 loci were imputed and analyzed in a bayesian model (controlled for age, gender, disease status and diagnosis, and t-cell depletion), survival was inferior in ucb with 3+ mismatches (9.8-times lower median survival, 95 ci 2.6-39.4), but not with up to 2 mismatches (1.3-time higher median survival, 95 ci 0.4-3.8). of note, in vivo t-cell depletion marginally impaired survival (2.4-times decrease, 95 ci 1.0-6.0). discussion: in our population, overall survival achieved with mm-urd was not different to 10/10 matched-urd, despite higher incidence of moderate/ severe cgvhd. on the other hand, survival with ucb was significantly lower. recent report 2 have shown excellent os with ucb compared to 8/8 hla-matched urd. ucb cohort inferior results may have been due to hla disparity degree, since survival in ucb with up to 2 mismatches (out of 8) was not worse. one limitation of our study is that tnc and cd34 from ucb units were not available, impairing primary graft failure analysis. we have also found that in vivo t-cell depletion might have a detrimental effect on survival and should be studied further in prospective trials. in conclusion, mm-urd, especially hla 9/10, is a suitable option when a fully hla 10/10 matched-urd is not available. ucb matched at least 6/8 may also be a good option. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is a proven treatment for patients with high risk or relapsed hematological malignancy. the probability of having a hla matched family donor is about 30%. in populations with high consanguinity rates, the probability of having non-sibling matched family donor(mfd) is much higher. to explore the impact of msd vs non-sibling mfd on outcome of hsct recipients, we undertook a single center retrospective analysis of pediatric patients transplanted with the diagnosis of hematological malignancy at our center in the last five years. a retrospective cohort from 2011 to current included 113 pediatric patients with hematological malignancies transplanted from family donors, of which 98 were from msd and 15 from non-sibling mfd. hla matched family donors were identified by high resolution allelic typing and were matched 10 of 10 hla loci. diseases were all (n = 58), aml (n = 37), mds (n = 7), jmml (n = 5), kml (n = 2), nhl (n = 1) and hodgkin's disease (n = 3). conditioning regimens were tbi or busulphan-based myeloablative in all patients. the median age of the patients was 9.4 years (range: 5 month-18.9 years). although peripheral stem cell seemed to be used more commonly in non-sibling mfd recipients (64% vs 42%), the difference was not statistically significant. the median follow up time for alive patients 24 months (1-67 months) . two year overall survival and leukemia free survival did not differ between patients with transplantations from msd or nonsibling mfd ( 67% ± 5.0 vs 60 ± 5.1, p = 0.31) similarly, leukemia free survival was not different between msd and non-sibling mfd transplants (58.2% ± 13.1 vs 58.1% ± 13.2, respectively). the incidences of grade ii-iv acute gvhd in msd and nonsibling mfd transplants were % 18 and %13, respectively. the incidences of relapses were 28% in msd transplants and 20% in mfd transplants and the difference was not significant (p = 0.49). these data show that the results of hsct from nonsibling mfd is comparable to hsct of msd in children with hematological malignancy. our data emphasize the need for extended high resolution family typing for patients in regions where there is high rate of consanguinity. disclosure of conflict of interest: none. [p125] p127 donor-recipient rh incompatibility is a risk factor for mortality after pediatric matched related allogeneic hematopoietic stem cell transplantation k ghanem 1 , n hariss 1 , z merabi, n kreidieh 2 , n tarek, r saab 1 , s muwakkit 1 , h el-solh 1 and m abboud 1 1 american university of beirut, department of pediatrics and adolescent medicine and 2 american university of beirut, optimal donor selection is critical to achieve the best outcome after allogeneic hematopoietic stem cell transplantation (allo-hsct). there is no consensus regarding the effect of donor-recipient rh incompatibility on survival after matchedrelated donor (mrd) allo-hsct in children and adolescents. this abstract aims to study this effect in a single-institution cohort over a period of 11 years. this is a retrospective chart review for all patients aged o21 years who underwent allo-hsct at the american university of beirut medical center between august 2005 and june 2016. a total of 70 patients with a median age of 11 years (range: 0.6-21 years) underwent allo-hsct from mrd for the following diseases: leukemia (n = 36), bone marrow failure (n = 15), thalassemia (n = 8), scid (n = 5), metabolic diseases (n = 4) and lymphoma (n = 2). the stem cell source was bone marrow for 56 patients (80%) and mobilized peripheral blood stem cells for 14 patients (20%). the grafts contained a median of 5.3 × 10 6 /kg total cd34 cells. tbi was used in 13 patients (18%). all but 4 patients achieved sustained neutrophil and platelet engraftment. after a median follow-up of 47 months (range: 4-135 months), the 2-year overall survival rate was 70% (95% ci: 57-79%). by multivariate analysis using cox proportional hazard regression model looking at the following factors for overall mortality: diagnosis, recipient's age, donor's age, the use of tbi, stem cell source, cd34 count, donor-recipient abo incompatibility, donorrecipient rh incompatibility, and donor-recipient sex-mismatch, the only statistically significant risk factor for mortality was donor-recipient rh incompatibility (hr: 3.26, p: 0.04). this risk was not statistically significant when looking at transplantrelated mortality (hr: 3.9, p: 017) and relapse-related mortality for malignant diseases (hr: 3, p: 0.12). there was no association between the incidence of acute or chronic gvhd and rh incompatibility. donor-recipient rh incompatibility was associated with an increased risk of mortality in children and adolescents undergoing mrd allo-hsct. further studies with larger number of patients are needed to confirm this finding. disclosure of conflict of interest: none. effect of iron or vitamin b12 deficiencies on in vitro colony forming capacity of peripheral blood-derived hematopoietic stem cells in children ny özbek, mm zabun, y köksal and m özgüner iron deficiency (id), id anemia (ida)and vitamin b12 deficiency (vit-b12d) are common disorders in developing countries. in urgent situations, children with these disorders could be donors before treatment. in this study, we investigated capacity of peripheral blood-derived hematopoietic stem cells to develop colony-forming units (cfu) in children with id and vit-b12, in vitro. patients and methods: we included 102 children (age 6 months-18 years) in the study in 5 groups: children with id (n = 15); children with ida (n = 20); children with vit-b12d (n = 12); children with both id and vit-b12d (i/ vit-b12d; n = 18); and control children (n = 37) who has normal peripheral blood findings, and normal ferritin and vit-b12 levels. from each child complete blood counts (cbc), and levels of ferritin, vit-b12, and cero-reactive protein (crp) have been obtained. who criteria, adjusted for age and sex, have been used for definition of anemia, id, ida and vit-b12d. four ml peripheral blood drawn into tubes with edta has been used for cfu analysis. mononuclear cell suspension (1.5 × 10 6 cell/ml), obtained from peripheral blood by ficoll-hypaque density gradient separation method, has been cultured in dishes containing semi-solid agar culture medium (methocult, h4434 classic, stem cell technologies, canada) in appropriate conditions. after 2 weeks, number of cfu colonies [burst forming erythroid (bfu-e); colony forming unitgranulocyte macrophage (cfu-gm); colony forming unitgranulocyte-erythrocyte-monocyte-megakaryocyte (cfu-gemm)] have been investigated by an inverted microscope. results: statistical analysis showed no difference between groups for age, sex, crp levels, and cfu-e, cfu-gm and cfu-gemm numbers. however, expected differences between groups were present concerning mean values of hemoglobin, ferritin and vit-b12 levels, mean corpuscular volume (mcv), and red cell distribution width (rdw) ( table 1) . discussion: this study shows in vitro proliferation capacity of peripheral stem cells has not been influenced by id, ida, vit-b12d, or i/vit-b12d. our results may indicate normal grafting ability of peripheral stem cells obtained from donors with iron, vit-b12 or i/vit-b12 deficiencies for hematopoietic stem cell transplantation. however, in vivo analysis should also be performed in order to reach a definite conclusion. [p128] disclosure of conflict of interest: none. efficiency of day 4 compared to day 6 stem cell mobilization in allogeneic donors h al-gaithi 1 , s al-mamar 2 , m al-huneini 2 , d dennison 2 , s al-kindi 2 , k al-farsi 2 and m al-khabori 2 1 hematology residency training program, oman medical specialty board; 2 hematology department, sultan qaboos university hospital granulocyte colony stimulating factor (g-csf) given for 4-6 days is commonly used for mobilization of allogeneic stem cell donors. the optimal days of g-csf administration is still debatable. the primary objective of this study is to compare the yield of stem cell mobilization, assessed using cd34+ cell count, between day 4 and day 6. secondary objectives include the assessment of the impact of donor's age, weight, mean corpuscular volume and blood group on the difference in the cd34+ cell count. in this retrospective study we included all allogeneic stem cell donors mobilized with g-csf for 6 days from january 2003 till october 2015 in the bone marrow transplantation unit at sultan qaboos university hospital. of 106 donor records reviewed, 84 were with available data and selected for the study. descriptive and analytical statistics were performed using stata 13.1. we included 84 donors with median age and weight of 19 years and 60 kg, respectively. the median day 4 wbc and cd34+ cell count were 37.4 × 10 9 /l and 54 × 10 6 /l respectively; while the median day 6 wbc and cd34+ cell count were 44.4 × 10 9 /l and 86 × 10 6 /l, respectively, (figure) with a statistically significant difference from day 4 (p o0.001). in the multivariable model, there were no significant impact of donor's age (p = 0.215), weight (p = 0.108), height (p = 0.428) and mean corpuscular volume (p = 0.263) on the difference in cd34+ cell yield. however, donor's blood group ab predicated a significantly higher difference (p = 0.036). six days of g-csf mobilization achieves higher cd34+ cell count than 4 days in allogeneic stem cell donors especially in donors with blood group ab. however, cd34+ cell count on day 4 is high enough to allow for successful mobilization. appropriately designed prospective trial is needed to confirm these results. disclosure of conflict of interest: none. there are known differences between individuals on an unrelated hsc donor register who decide to proceed with verification typing (vt) vs those who choose not to. in the anthony nolan registry, white british donors are more than twice as likely as other ethnic groups to continue with testing at vt (or 2.44; po 0.001 (unpublished data)). the purpose of this study was to explore differences in key characteristics between white british donors and british donors from other ethnic groups with a view to developing interventions to reduce vt stage attrition. study recruitment occurred april 2013-may 2016. all donors not proceeding at vt were invited to participate, and a stratified random sample of those proceeding at vt were recruited to meet pre-determined targets for each ethnic group. data were collected via structured interview (telephone or online). 4 broad categories of participant characteristics were assessed: demographic, culturally related, psychosocial, and donation-related. measures were previously validated scales with established psychometric properties either created for, or used in other donation-related settings. for analyses donors were divided into two groups based on ethnicity: white british (wb), and non-white british (nwb). results: 170 wb donors and 187 nwb donors completed interviews donors proceeding at vt were more likely than their counterparts to participate in the study (66% vs 25%, p o0.001). mean donor age was 33.8 with no difference between ethnic groups and 43% of donors in both groups were female. nwb were statistically more likely to have completed higher education, and have a stronger religious affiliation. in contrast they were less likely to be blood or organ donors. nwb also described greater mistrust of the medical system and of hsc allocation. nwb donors were more likely to have joined the register at a recruitment event (p=0.012) or a place of worship (p=0.012), while wb donors were more likely to have joined online (p=0.013). wb donors reported significantly higher scores regarding feeling well informed about donation both at the point of joining, and at the point of vt and were more likely to remember joining the register and the two donation methods. this study highlights important differences in demographics, culturally related variables and donor interaction with the register between white british donors and donors from other ethnic backgrounds. given the higher rate of vt attrition in nwb donors, these findings could be used to tailor interactions/information given to donors on the register to ensure their priorities are addressed. disclosure of conflict of interest: none. data on mismatched family donor transplants for myelofibrosis are scarce due to the risk of poor engraftment, gvhd and exclusion from trials. outcomes from such transplants performed between 2001 and 2015 reported to the ebmt are presented. sixty-nine patients, median age 58 (27-71) years; 44 (64%) male, 50 (74%) had primary, 18 (27%) had secondary myelofibrosis (6 from et, 5 from prv and 7 others) and unknown 1(2%). jak2 v617f was mutated in 15/25. karnofsky performance status was 470% in 98 %; median time from diagnosis to allograft was 41.4 (range: 0.72-213) months. the donors were predominantly male 47 (68%), median age 42 (22-75) years, hla mismatched at 1 locus in 12 (17%) and 2 or more loci in 48 (70%). donor-recipient serology was cmv − / − in 8 (12%) ± in 4 (6%), − /+ in 15 (22%) and +/+ in 34 (49 %) missing 8 (12%) . bone marrow was used in 34 (49%) and peripheral blood in 35 (51%). the median total nucleated cell count (tnc) was 7.5 × 10 8 /kg (range: 2.3-21 × 10 8 /kg) (n = 17). the median cd34+ cell dose was 6.9 × 10 6 /kg [p130] s182 (range: 1.9-18.18 × 10 6 /kg)(n = 19). patients. conditioning was myeloablative in 48 (70%) and ric in 21 (30%). predominant conditioning regimes were fludarabine, busulphan, atg (fbatg) and thiotepa, busulphan, fludarabine (tbf n = 33). tbi was administered in 8 (12%) and t cell depletion in vivo in 22 (32%) and ex vivo in 5 (7%) patients. gvhd prophylaxis varied with post transplant cyclophosphamide administered in 33/67 (48%) and atg in 19/67 patients (28%).neutrophil engraftment occurred in 53 (82%) patients at a median of 20 days (range: 11-83). primary graft failure ensued in 8 (12%) and secondary graft failure in 4 (6%) patients at a median of 12 (range: 4.5-35) months. eleven patients had a second allograft at a median interval of 4 (1-20) months. responses to the first allograft censoring for a second allograft, data available in 45 patients, showed that complete remission was achieved in 35 patients (78 %), 6 (13 %) were never in cr and 4 (9 %) were not evaluable. relapse occurred in 8 (12%) of patients at a median interval of 3 (2.8-21.8) months. the cumulative incidence (ci) of grade ii-iv acute gvhd (agvhd)was 12% (95% ci 4-21%) and for grade iii-iv agvhd at was 5% (95% ci 3-11%). data for chronic gvhd (cgvhd) was valid in 49 patients of whom 47% developed cgvhd. the ci of cgvhd at 2 years was 58% (95% ci 43-72%):ci of limited cgvhd was 45% (95% ci 31-59%) whereas the ci of extensive cgvhd was 10% (95% ci 2-19%). median follow-up was 24 (95% ci 13-35) months. the 2 and 5 year os was 53% (95% ci 40-66%) and was 40% (95% ci 23-57%). the 2 and 5 year rfs was 43% (95% ci 30-56%) and 31% (95% ci 15-47%). the 2-year ci of relapse was 20% (95% ci 10-30%). the 2 year nrm was 37% (95% ci 25-49%), which increased to 49% (95% ci 32-65%) at 5 years. thirty patients died due to infection (16, 53%), gvhd (7, 23%), organ damage or failure (3, 10%), relapse/disease progression (1, 3%) and secondary malignancy or ptld (1, 3%) unknown 2. there was no significant effect (univariate analysis) of recipient or donor gender, degree of hla mismatch, cmv matching, primary or secondary mf, chronic vs advanced disease at transplant, conditioning intensity or regimen, gvhd prophylaxis with atg or post transplant cyclophosphamide or stem cell source on overall survival. the data are encouraging for patients with myelofibrosis, with engraftment, pfs and os being attained with limited severe chronic gvhd from family mismatched donors. disclosure of conflict of interest: none for all other authors, fc consulting with molmed. feasibility of salvage second allogeneic stem cell transplantation for disease relapse or graft failure: a single centre experience g battipaglia 1,2 , d salvatore 3,1 , r dulery 3 , f giannotti 3 , f malard 2 , e brissot 2 , s sestili 2 , f isnard 2 , s lapusan 2 , a-c mamez 2 despite high rates of toxicity and mortality, a second salvage allogeneic stem cell transplantation (second allohsct) might be an option to consider in patients experiencing disease relapse or graft failure after first allohsct. we retrospectively analyzed outcomes after second allohsct in a cohort of 30 patients (18 males and 12 females) transplanted either for disease relapse (group 1, n = 19) or graft failure (group 2, n = 11) between 2007 and 2015 in a single centre in france. median age at second allohsct was 38 (range: 17-64) years. diagnoses were acute myeloid leukemia (group 1: n = 12; group 2: n = 3), acute lymphoblastic leukemia (group 1: n = 2; group 2: n = 4), myelodysplastic syndrome (group 1: n = 2; group 2: n = 1), myeloproliferative neoplasm (group 1: n = 3; group 2: n = 1), bone marrow failure (group 1: n = 0; group 2: n = 2). median time from first allohsct to second allohsct was 38 (range: 2.5-230) months in group 1 and 1.5 (range: 1-34) months in group 2. graft source for the second allohsct were: haploidentical bone marrow (group 1: n = 3; group 2: n = 1), haploidentical pbscs (group 1: n = 5; group 2: n = 1), cord blood (group 1: n = 8; group 2: n = 8), matched unrelated pbsc (group 1: n = 3; group 2: n = 1). at time of second allohsct, 11 patients were in cr and 8 presented active disease in group 1. conditioning regimen was myeloablative in 5 patients (group 1: n = 3; group 2: n = 2), reduced intensity (ric) in 20 cases (group 1: n = 11; group 2: n = 9). a sequential schema consisting of a combination of thiotepa, etoposide and cyclophosphamide followed by a fludarabine and busulfanbased ric was used in 5 out of 8 patients with active disease in group 1. sixteen patients received atg as part of the conditioning regimen for second allohsct (group 1: n = 10; group 2: n = 6). all but one patient engrafted, at a median time of 18 (range: 6-51) days. cumulative incidence of acute and chronic gvhd were 27 ± 17% and 42 ± 19%, respectively, 2-107) months, non-relapse-mortality (nrm) and relapse incidence (ri) were 24 ± 15% and 27 ± 15%, respectively, while disease-free (dfs) and overall survival (os) were 49 ± 19% and 48 ± 15%, respectively, for the entire cohort. in all, 15 patients died of infections (n = 8), hematological disease (n = 4), gvhd (n = 1), hemorrhage (n = 1) and for unknown causes (n = 1). main outcomes of patients in group 1 were: ri 16 ± 15%, nrm 28 ± 18%, agvhd 26 ± 15%, cgvhd 56 ± 20%, dfs 56 ± 20%, os 55 ± 20%, respectively. main outcomes of patients in group 2 were: ri 45 ± 22%, nrm 18 ± 24%, dfs 36 ± 29%, os 36 ± 29%, agvhd 27 ± 23%, cgvhd 18 ± 23%. historically, a second allohsct was hampered by significant morbidity and mortality. however, the advent of reduced-toxicity conditioning regimens and improved supportive care allowed to significantly improve the results of patients receiving a second allohsct as suggested from the above results. therefore, a second allohsct could be considered as an option to rescue a certain number of patients experiencing disease relapse or graft failure, for which prognosis is very poor. decision is to be discussed on a case-by-case basis. disclosure of conflict of interest: none. haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide for patients with high-risk hematologic malignancies am carella department of oncology and hematology, irccs casa sollievo della sofferenza, san giovanni rotondo allogenic hematopoietic stem cell transplantation (sct) has been increasingly used for treatment of adult with high risk hematologic malignancies. for patients lacking an hlamatched related or unrelated donor, unmanipulated haploidentical (haplo)-sct is a potential alternative. haploidentical transplantation performed with post-transplantation cyclophosphamide (ptcy)-based graft-versus-host disease (gvhd) prophylaxis has been associated with favorable outcomes for patients with acute leukemia and lymphomas we analyzed outcomes of 45 patients with hematologic malignancies who received t-cell-replete haematopoietic stem cells and posttransplantation cyclophosphamide after myeloablative or nonmyeloablative hla-haploidentical donor transplantation. the median age was 37 years (14-68); twelve patients were in first remission (cr1), 4 in second remission (cr2) and 29 had an active disease. ). the diagnosis was acute leukemia (n = 32), myelodisplastic syndrome (n = 3), hodgkin disease (n = 7) non hodgkin lymphoma (n = 2) and multiple myeloma (n = 1). median follow-up was 260 days. stem cell source was bone marrow (bm) for 42 patients, and peripheral blood (pb) for 3. myeloablative conditioning (mac) was used in 37 patients and reduced intensity regimen (ric) in 8 patients. thirty one patients were first grafts, the others underwent previous autologous sct (n = 11) or mud (n = 3). gvhd prophylaxis s183 consisted in pt-cy on days +3 and +4, cyclosporine (from day +5), and mycophenolate (from day +5). the median day for neutrophil engraftment was day +20 (14-29). no graft failure was observed. chimerism was evaluable in 39 patient; on day + 30 all patients had 100% donor chimerism on marrow cells median follow-up was 260 days. the cumulative incidence of acute gvhd grade ii-iv was 22%, grade iii-iv 9% and chronic gvhd 15%. one-and 2-years os was 56.53% and 53.39 %, respectively. with a median follow-up for the surviving patients of 752 days (130-2207), the cumulative incidence of transplant-related mortality (trm) is 22%, and the relapserelated death is 26%. thus, we demonstrate excellent rates of engraftment, gvhd, and trm in adult patients treated with haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide. this approach is a widely available, safe, and feasible option for adult patients with high risk hematologic malignancies, including those with a prior history of myeloablative bmt and/or those with co-morbidities or organ dysfunction, also for patients with active disease at the time of transplant. disclosure of conflict of interest: none. it has recently been shown that t-replete allogeneichematopoietic stem-cell transplantation (allo-hsct) from a haploidentical donor (haplo-id) could be a valid option when a matched donor is not available. unfortunately, the worldwide donor registries comprise mainly donors of caucasian origin and patients of non-caucasian origin have a much lower chance of finding a matched unrelated donor (mud). the lengthy period of international search when required and the financial burden of this process are considered as additional significant limitations. at the american university of beirut medical center (aubmc) in lebanon, we started the mud program in 2011 and haplo-id hsct program in 2014. we report here our experience in this two groups of patients. patients and methods: we have transplanted 21 patients from a haplo-id donor since 2014 and compare their outcome with the 6 patients transplanted from a mud since 2011. the patients and transplant characteristics are listed in the table 1 . the 2 groups were comparable except for conditioning. patients in haplo-id group received two days of posttransplant high-dose cyclophosphamide (pt-hdcy) followed by cyclosporine a (csa) and mycophenolate-mofetil while patients in the mud group received pre-transplant antithymocyte-globulins and csa starting on day-3. all patients engrafted in the mud group, while one patient did not engraft in the haplo-id group, the patient had refractory all transplanted with progressive disease, and died on day +47. the median of anc 4500/mm 3 was 14 days (12-20) vs 17 days (12-29) in the haplo-id and mud groups, respectively. fourteen patients from the haplo-id group developed grade 2 acute graft-versus-host disease (agvhd) vs one after mud-hsct. two patients haplo-id group developed limited cgvhd and none after mud grafts. six patients relapsed in the haplo-id group vs three patients in the mud group. two and three patients died from non-relapse mortality in the haplo-id and mud group, respectively. at the last follow-up, 13 patients are still alive in the haplo group vs 2 patients in mud group and all of them are in cr. we conclude that t-replete haplo-id hsct followed by pt-hd cy is associated with promising results or at least comparable to patients transplanted from mud. haplo-id hsct seemed to be safe and feasible in patients with high risk hematological malignancies. finally, because of the obvious advantage in rapidly finding a donor (21 haplo transplants in three years vs 6 mud transplants in 5 years), development of haplo-id hsct is warranted to satisfy the regional needs. [p134] disclosure of conflict of interest: none. haploidentical hematopoietic stem cell transplantation (haplo-hsct) using t-cell-replete (tcr) grafts and posttransplantation cyclophosphamide (ptcy) provides a curative approach for patients with high-risk mds/aml lacking a conventional hla-matched donor. in children and adults haplo-hsct using ptcy as gvhd prophylaxis seems to be safe with low treatment related morbidity and mortality (trm). however, few data are available for elderly patients with advanced disease. we retrospectively analyzed the outcome of 49 patients with mds (n = 5)/aml (n = 44) age 50-74 years (median age 60 years; 24 patients 50-59 years, 25 patients ⩾ 60 years; 21 male), who underwent tcr haplo-hsct with high-dose ptcy at our institution between january 2009 and november 2016. disease was active in 41 patients while 8 had achieved cr. 12 patients failed previous allo-hsct. pretransplantation risk factors were scored using the hematopoietic cell transplantation-specific comorbidity index (hct-ci) which was ⩾ 3 in 19 patients (median hct-ci = 2, range: 0-8). a sequential therapeutic concept using either flamsa (n = 28) or clofarabine (n = 18) as cytoreduction was used prior to reduced intensity conditioning (ric) in all but 3 patients. ric consisted of fludarabine/cyclophosphamide combined with either melphalan (n = 32), busulfan (n = 1) or 4 gy tbi (n = 12). post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mmf in all patients. 57% received a bone marrow graft. one graft rejection occurred. neutrophil and platelet engraftment was achieved in 95% and 77% of evaluable patients, respectively at a median of 19 (13-89) and 33 (11-103) days. acute gvhd grade i-iii occurred in 29% of the patients whereas no grade iv agvhd was observed. chronic gvhd presented in 33%. it was most frequently assessed as mild to moderate (13 pts). only 3 patients developed severe cgvhd; no gvhd related death was observed. cmv reactivated in 22 of 36 patients at risk, one patient developed cmv disease (pneumonia). no ebv reactivation or ptld occurred. one-year trm was 24%. 12/49 (24%) patients relapsed, three within the first 100 days after haplo-hsct. at a median follow up of 27 months (range: 4-74 months) estimated one-and two-year overall survival (os) was 55/46 %, respectively. when stratified by age, estimated one-and two-year os was 65/42% in patients o60 years and 47/47 % in patients ⩾ 60 years (p = 0.771/p = 0.794). one-and two-year progression-free survival (pfs) was 50/45%, respectively. stratified by age estimated one-and two-year pfs was 53/41% in patients o 60 years and 47/47% in the elderly (p = 0.836/p = 0.887). unmanipulated haploidentical allografting using ptcy-based gvhd prophylaxis in high-risk mds and aml patients aged over 50 years is safe and well tolerated resulting in acceptable trm. a remarkable survival outcome can be achieved in elderly high-risk aml/mds patients with significant comorbidities. disclosure of conflict of interest: none. key performance indicators to assess the quality of a collection facility: experience of a single center s roncon*, c pinho 1 , f bordalo 1 , s lopes 1 , s ferreira 1 and f amado 1 1 allogeneic hematopoietic stem cell transplantation (allo-hsct) has evolved into an effective immunotherapy for the treatment of a variety of disorders. when patients do not have a familiar matching donor, transplant centers (tc) search for an unrelated and volunteer donor. this one must be previously evaluated by the collection center (cc) to donate peripheral blood stem cells (pbsc) or bone marrow (bm); lymphocytes can also be asked after allo-hsct. this work aims to evaluate our performance as cc, ensuring donor safety, quality of cell therapy products (ctp) and the accomplishment of tc requirements. we retrospectively analyzed all the requests of ctp collections sent by the portuguese registry from 2012 to 2016. countries of destination, number and type of ctp were determined. we established eight key performance indicators (kpi) classified into four categories: response time; product quality; satisfaction of patients and donors; and on-site donor motivation. the intended target was defined by the mean result obtained in the first half of 2012 (excluding kpi-7). written comments from donor center (dc) and tc were received by email or written in the local notebook. the donor's answers were obtained through a survey given on the collection day. a total of 349 requests were assessed: 231 pbsc, 61 bm, 16 lymphocytes and 41 cancellations; 84% were sent to europe (98/259 to portugal), 14% to america and 2% to oceania; 30/41 were withdrawn by tc (14 patients died, 14 presented progressive disease and 2 had a better hlamatched donor) and 11/41 by dc (7 donors not cleared and 4 refused). the results obtained with kpi-1, -2, -4 and -7 exceeded the intended target (table 1) . after the first kpi-1 results, we verified a positive evolution. we took an average of 3 days of delay in sending donor clearance. however, there is no holdup in the ctp delivery, as demonstrated by kpi-2. regarding kpi-3 it is important to notice that 60% of ctp with a cell number less than requested were bm and lymphocytes; when pbsc was considered separately, the result increased (87% vs 75%). analyzing kpi-4, 85% (n = 11/13) of the contaminated ctp were bm. concerning kpi-5, -6 acknowledgments and -8 commitment, we recognize that our initial targets were too ambitious (100%). the kpi-6 shows a low number of complaints (n = 4): one due to a misreading of the request and three to communication failures; all were properly examined and rectified. a good general status was guaranteed in almost all the donors (kpi-7). the decrease of kpi-8 is due to the fact that one donor refused to proceed after three postponements of the collection date by tc. table 1 -key performance indicators of the quality of our activity as a cc. the overall good level of our results reflects an extremely professional performance as a cc. we consider that these kpi should be continuously monitored with the purpose of earlier detect any deviation of the stated goals and assess the progress against settled strategies. we further suggest the establishment of universal indicators in order to standardize [p136] practices, share expertise and improve the quality of services and products provided to patients and donors. two year later, the patient had a genoidentical allogenic stem cell transplant (from the bone marrow stem cell of his sister, who was 35 years old, hla compatible). he had a reduced intensity conditioning, associating busulfan, fludarbine and antilymphocyte serum. 6 months from the asct, he was in complete remission with 100% donor chimerism. 4 years after the asct, the patient presented a progressive thrombocytopenia without any other peripheral causes. the bone marrow aspiration initially showed a refactory cytopenia with multilineage dysplasia. the patient was followed up during 12 months, and then a second bone marrow aspiration has shown a refractory anemia with excess blasts2 raeb2. a cytogenetic study has every time demonstrated a female karyotype (44,xx) on 20 mitoses out is 20, and chimerism was 100% donor. the diagnosis of the myelodsplastic syndrome of the donor cells was approved. the patient was treated by azacitidine (75 mg/m 2 , from j1 to j7, j1 = j28). after 6 cycles, the patient was in complete hematologic response (normalization of the platelet count) and a partial bone marrow response (normalization of the blasts rate but persistence of the signs of dysplasia). he received 6 more cycles, and presented hematologic relapse (reemerging of thrombopenia). a phenoidentical allogenic stem cell transplantation was suggested. conclusion the occurrence of mds on the donor cells is rare. these anomalies are secondary to intrinsic factors (of donor) or extrinsic factors )of the transplant recipient). the treatment is not definitely determined. disclosure of conflict of interest: none. nk-cell alloreactivity based on kir/ligand mismatch in the donor vs recipient direction provides better graft-versustumor effect in patients with active hematological malignancies undergoing allogeneic t-replete haploidentical transplantation followed by post-transplant cyclophosphamide a wanquet 1,2 , s bramanti 1,3 , s harbi 1 , s fürst 1 , f legrand 1 , c faucher 1 , a granata 1 , p-j weiller 1,2 , c picard 4 , b calmels 5 , c lemarie 5 , c chabannon 2,5,6 , l castagna 1,3 , d blaise 1,2,6 and r devillier 1, haplo-sct have been developed in the past years with very interesting results in high risk patients. gvhd prophylaxis using post-transplant cyclophosphamide (pt-cy) recently allowed extending the use of unmanipulated haplo-sct. it was shown that nk alloreactivity, triggered by donor-recipient inhibitory kir gene-gene mismatches, could lead to better outcomes and survival in the setting of in t-cell-depleted haplo-sct. however, few data is available on the impact of kir-ligand mismatch on the outcome after t-replete haplo-sct with pt-cy. we thus assessed the impact of nk alloreactivity on the outcome of patients who received haplo-sct followed by pt-cy. we retrospectively collected the data from patients from two centers who were treated for various high risk hematological diseases and underwent a haplo-sct with pt-cy from december 2009 to december 2014. we assessed the kir-binding epitope in hla-c and hla-b molecules for all patients, and we predicted nk cell alloreactivity in the donor vs recipient direction via the immune polymorphism database kir ligand calculator, based on the kir-ligand mismatch between donors and patients. because disease status at the time of haplo-sct is one of the most important predictor of outcome, we separately analyzed two cohorts of patients: those transplanted in complete remission (cr group) and those transplanted with active disease (no cr group). using a multivariate cox model (adjusted by disease type, age and conditioning), we therefore evaluated the impact of nk alloreactivity on outcome in both cr and no cr groups. we analyzed 144 patients with a median age of 54y (20-74). they were mostly transplanted for lymphoma (n = 72, 50%) or aml/ mds (n = 47, 33%). patients mostly received a tbi-based nonmyeloablative conditioning regimen (n = 94, 65%) and pbsc as graft source (n = 91, 63%). eighty one and 63 patients were transplanted in cr and in no cr, respectively. nk alloreactivity was found in 30/81 cr patients (37%) and 22/63 no cr patients (35%). with a median follow up of 30 months (12-77), cr patients had a significantly better outcome than those in the no cr group (2-year pfs 65% vs 32%, respectively, p o0.001). in no cr patients, multivariate analysis showed that nk alloreactivity was significantly associated with reduced the risk of relapse (hr = 0.25, p = 0.019, figure 1a ) with no increase of both acute (hr = 1.30, p = 0.648) and chronic gvhd (hr = 2.61, p = 0.232), and nrm (hr = 0.60, p = 0.277). this led to significantly better pfs (hr = 0.41, p = 0.014, figure 1b ) and a trend for better os (hr = 0.52, p = 0.069). in contrast, in cr patients, we found no difference in outcome according to nk alloreactivity for all end points (acute gvhd: hr = 1.78, p = 0.204; chronic gvhd: hr = 2.11, p = 0.321, nrm: hr = 1.69, p = 0.313, relapse: hr = 0.85, p = 0.762, figure 1c ; pfs: hr = 1.19, p = 0.637, figure 1d ; os: hr = 0.83, p = 0.672). our results suggest that nk alloreactivity provides better disease control with no increase of gvhd, especially in patients transplanted with active disease. thus, donor selection should rely on the prediction of nk alloreactivity. this may contribute to improve outcome of these patients with high risk of relapse after transplantation, underlining the need of a specific strategy of donor search, and the promising perspective of early post-transplant nk-cell-based immunotherapy. haploidentical bone marrow transplantation (haplo-bmt) with post-transplant cyclophosphamide (pt-cy) is being increasingly used, in the last five years, for patients lacking a suitable hla-matched donor. genoa study (eudract number: 2012-000703-32) provides for a modified gvhd prophylaxis platform compared to the original baltimora protocol. aim of the study: in this study we assessed outcomes in 282 consecutive patients transplanted from a haploidentical donor for haematological malignancies. all patients received a uniform gvhd prophylaxis: cyclosporine (csa) starting on day 0, mycophenolate (mmf) starting on day +1, and post transplant cyclophosphamide (pt-cy) 50 mg/kg, on days +3 and +5. all patients received a myeloablative conditioning consisting of thiotepa, fludarabine, busulfan (three doses n = 116 or two doses n = 111), or tbi, fludarabine (n = 55). the median age was 48 years (17-74); at transplant 145 (51%) patients were in remission of disease (cr1 and cr2), and 137 had an active disease (49%); all patients were first grafts. the diagnosis were acute myeloid leukemia (n = 111), myelodisplastic syndrome (n = 31), acute lymphoblastic leukemia (n = 56), myelofibrosis and myeloproliferative diseases (n = 43), non hodgkin lymphoma (n = 19), chronic lymphocytic leukemia (n = 9) and multiple myeloma (n = 13). the median follow up was 562 days (range: 6-2241 days). the median infused mononucleated cells was 3.4 × 10e8/kg (range: 1.1-7.7). seven patients died before engraftment, and 21 (7%) had autologous recovery: 15 (5%) after conditioning with 2 doses of busulfan. full-donor chimerism on day +30 was reached in 254 (90%) patients. the median day for neutrophil engraftment was day +18 (range: 13-60 days). the cumulative incidence of grade ii-iv and iii-iv acute gvhd (agvhd) was 17% (n = 49) and 5% (n = 15), respectively. two years cumulative incidence of moderate-severe chronic gvhd (cgvhd) was 13% (n = 39).sixty one (21%) patients experienced haemorragic cystitis. at 3 years the cumulative incidence of non relapse mortality (nrm), relapse and relapse related death was 17% (n = 47), 32% (n = 91) and 25% (n = 69), respectively. causes of death were infections (n = 34), hemorrhage (n = 7), gvhd (n = 5), secondary neoplasia (n = 1) and relapse (n = 69). at 4 years of follow up overall survival and disease free survival was 55.7% and 47%, respectively. at the same time overall survival rate was 73% for patients in remission and 29% for patients with active disease at transplant(p o0.001). in conclusion, a modified pt-cy as gvhd prophylaxis and ma conditioning regimen followed by haploidentical bmt results in a low risk of agvhd and cgvhd and encouraging rates of trm and dfs. disclosure of conflict of interest: none. the italian bone marrow donor registry (ibmdr), in collaboration with admo (associazione donatori di midollo osseo) since 2009 has implemented, as part of the donor enrollment strategy, public enrollment events (pe). our donor center (dc) has taken part to those events since the first years. one or more clinician (or trained biologist) has been present to pe to inform the potential donors, evaluate the candidates and supervise the collection of biological fluids. all the local permission where obtained. aim: aim of this study was to compare the compliance of the donor enrolled in pe with donors enrolled at our dc institutional site. we prospectively evaluated all the donors recalled for further evaluation and/or for requalification in the years 2014 and 2015 at our dc itmi07. we defined 3 possible results for the call: ‛success' (the donor was eligible and accepted to be evaluated, or only temporarily ineligible) ‛not eligible' (the donor was definitively ineligible) and ‛consent denied'. results: a total of 286 donors were called back in the years 2014 and 2015 (16 not found). eightyfour recalled donors had been enrolled after 2009. among them 53 (63.1%) had been enrolled at the dc and 31 during pe (36.9%). the two populations were not different for age at the call, age at enrollment and gender (table 1) . [p140] when evaluating the probability of obtaining a "success", no significant difference was found between the two populations: 86.8% vs. 83.9% (chi square p=0.23). no significant difference was also found for the "not eligible" and the "consent denied" categories. of note, when we turned to the whole 286 donor population we had called back (median age 34, range 21-55), the probability of "success" and "consent denied" were not related to donor age, and time from enrollment to recall, whereas donor ineligibility was (spearman test p=0.02 and 0.002). public events with the presence of an adequate trained medical team represent a valid option for the enrollment of new unrelated donors. disclosure of conflict of interest: none. the search for hematopoietic stem cell unrelated donors in patients with malignant hemopathies with not-sibling matched family donor: the experience of a center a pérez 1 , r goterris 1 , m gómez 1 , s blanco 1 , a segado 1 , c arbona 1 , jch boluda 1 , m poch 1 and c solano 1 1 hematology department, hospital clínico universitario, valencia unfortunately, as few as 30-35% of patients will have an hlaidentical matched sibling donor available for hematopoietic stem cell (hst) donation. the search for an unrelated donor (urd) (adult or cord blood) is often the best option for those patients lacking a suitable matched donor. below we describe the experience with the search for an unrelated donor in our center. between september 1995 and march 2016 the search for urd was activated for 263 patients. the median age of the patients was 46 years (range: 0.4-69), 10% were under 18 years and 60% were males. acute myeloid leukemia (n = 67), acute lymphoblastic leukemia (n = 43), non-hodgkin's lymphoma (n = 60), chronic/prolymphocytic lymphocytic leukemia (n = 23), hodgkin's lymphoma ((n = 13), multiple myeloma (n = 14), chronic myeloid leukemia (n = 15), philadelphianegative myeloproliferative neoplasms (n = 9), myelodysplastic syndrome (n = 8), aplastic anemia/paroxysmal nocturnal hemoglobinuria (n = 6), others (n = 5). the disease status in hematological malignancies was: first cr (n = 77), 4 second cr (n = 49), pr (n = 46) and refractoriness (n = 82). the donor type requested at the activation of the search was an adult (n = 110), umbilical cord blood (n = 7) and two options (n = 146). results: a compatible donor was found in 197 patients (76% of the series) after a median of 44 days (range: 1-847) from the activation of the search. the degree of adult donor compatibility (not available in 7 cases) was: complete hla identity (8/8: n = 49, 10/10: n = 37); an hla difference (7/8: n = 12, 9/10: n = 31); lower degree of compatibility (n = 13). the degree of umbilical cord blood compatibility: identity ⩾ 4/6 (n = 48). a total of 151 patients (57%) were transplanted, 103 from adult donor and 48 from umbilical cord blood. the median time between the activation of the search and the hst transplantation was 4 months (range: 0.7-29), being 3.2 months for acute leukemia and 5.1 months for other pathologies, and between the location of the donor and the hst transplantation 70 days (range: 5-412), being 45 days for umbilical cord blood and 76 days for an adult donor. there were 108 cancellations of the urd search (41% of the total) for the following reasons: clinical status of the patient (n = 63), performing a haploidentical transplant (n = 20), transplant center does not consider (n = 9), norms of the registry (n = 8) and loss of indication of transplantation (n = 8). the median time from the beginning of the search to its cancellation was 4.5 months (range: 0.3-53). at the time of analysis, the median follow-up of the 263 patients is 17 months. the survival of the series in the 5 years is 37% and 43% for patients transplanted from urd. 76% of the searches activated in our center allowed the localization of a urd with an adequate degree of hla compatibility. however, only 57% of the patients for whom the search was activated were finally transplanted. the most frequent cause of cancellation of the procedure was the clinical deterioration of the patient. disclosure of conflict of interest: none. the leukemic transformation of otherwise healthy donor stem cells provides a useful in vivo model to study the mechanisms involved in leukemogenesis. we report two cases of donor cell-derived haematological malignancy in which wholeexome sequencing (wes) was performed in bone marrow (bm) samples from recipient at different times after allogeneic hematopoietic stem cell transplantation (allo-hsct) in order to study the dynamics of emergence of mutations that precede the development of donor cell leukemia (dcl) and donor cell myelodysplastic syndrome (dc-mds). case 1: a 43-year-old female diagnosed with lymphoblastic leukemia-b t(1;19), who developed acute myeloid leukemia (aml) with normal karyotype, npm1+of donor origin 16 months after unrelated cord blood transplantation (ucbt). case 2: a 65-year-old male diagnosed with mantle cell lymphoma, who developed mds 45,xx,-7,del(12)(p12) of donor origin, 57 months after allogeneic bm transplantation from his hla-identical brother. the donor also developed mds several months later. wes (sureselect-xt human-exon 50mb) was performed by next generation sequencing (hiseq) on donor stem cells (scs) infused as well as on bm samples from recipient after allo-hsct. the exome of donor scs and 5 bm samples, from case 1, were aligned to the human reference genome (grch 37/hg19) and donor scs and 9 bm samples were aligned to grch 38/ hg38 in the second case. in both cases non-synonymous variants in the coding regions or synonymous variants in splice regions of genes related to leukemia were selected. in addition, bm samples were matched to their scs and to prior bm samples to identify the acquired variants. variants meeting such criteria were evaluated with 3 functional predictor software's (sift, polyphen2 and mutation taster). wes analysis revealed progressive emergence of multiple somatic mutations probably related to the development of leukemia in bone marrow samples post allo-hsct ( figure 1 ). both scs showed alterations that may be involved in leukemogenesis. (case 1: sh2b3 and case 2: kmt2c, kmt2a, arhgap26 and monosomy 7). somatic mutations, acquired over time, fall into genes that play well-established roles in signalling pathways (ras-mapk, pre-mrna splicing factor, apoptosis, dna doublestrand break repair, dna replication and so on). mutations in leukemic subclones that disappear after chemotherapy were indentified, as well as the acquisition of new mutations in resistant subclones. we propose a possible model of leukemogenesis in these cases ( figure 2 ). the present study reveals a process of sequential clonal expansions, promoted by the acquisition of additional somatic mutations in donor hematopoietic cells. detection of heritable or acquired gene mutations in donor associated with predisposition to haematological malignancies could have clinical implications for the patients undergoing to allo-hsct. although the cause of donor cell-derived haematological malignancy onset seems to be multifactorial, the infusion of a scu with pre-leukemic potential in a context of residual toxicity in recipient as a result of pre-transplant chemotherapy, a post-transplant environment characterized by a decreased immune surveillance may well have played role in these cases. the study of a greater number of dcl cases by next generation sequencing could help to understand this process and to detect new mutations involved in the emergence of aml. disclosure of conflict of interest: none. the impact of donor and recipient sex in allogeneic stem cell transplantation-single center experience (cic 859) y petrov 1 , p ganeva 1 , g arnaudov 1 , s lozenov 1 , y davidkova 1 , v stoeva 1 , i tonev 1 , m guenova 1 and g mihaylov 1 1 national hospital for active treatment of hematological diseases allogeneic hematopoietic stem cell transplantation (hsct) has been one of the most effective therapeutic modalities for patients with hematological malignancies and bone marrow failure syndromes. optimal donor selection is one of the key factors to enhance the success rate of this procedure. we [p142] s189 retrospectively investigated whether and how donor-recipient sex affects transplantation outcomes of 73 patients transplanted between 2010 and 2015 in our center. the median age of the patients was 37 years (range: 23-51). thirty-nine of the patients (53%) received a pbsc from a hla-identical sibling, and 34 patients (46.5%) received pbsc from matched unrelated donor. forty-six percent were male recipients with male donors (m-m), 11.9% were female recipients with male donors (m-f), 23.8% male recipients with female donors (f-m), and 17.8% female recipients with female donors (f-f). we performed a crosstab analysis and χ 2 tests to observe whether the donor sex affects our study population. patients with male donor had superior overall survival and progression-free survival compared to those with female donor (66.7% vs 29.0% p = 0.001 for os, and 52.3% vs 34.2% p = 0.003 for pfs; cramer`s v = 0.372). we further investigated how the disparity of the donor in the four groups (m-m, m-f, f-m and f-f) affects the os, pfs and nrm. the f-m group had a worse overall and progression-free survival comparing the other groups (11% 4-year os and 17% pfs; p o0.0001).this group had 27% relative increase in the non-relapse mortality compared with m-m group (p = 0.009). for m-m group there was a 2% relative increase in the subdistribution hazard of nrm compared with m-f group (p = 0.02). the f-f group and m-f group had similar subdistribution hazard of nrm (39% vs 40% p = 0.009). the incidence of acute gvhd and chronic gvhd for the groups was: 34% and 41% (m-m), 37% and 32% (m-f), 41% and 40% (f-m), 32% and 7% for the (f-f) group. the appearance of either acute or chronic gvhd did not show statistical significance regarding the os and pfs in the groups (p = 0.07). we examined the effect of donor-recipient sex incompatibility on the outcome of hsct in out center. our results showed inferior os and pfs for f-m group and a higher incidence of nrm compared with other groups. these effects might be associated with allogeneic immune responses against h-y antigens. key words: stem cell transplantation, donor sex, recipient sex, overall and progression-free survival [p143] disclosure of conflict of interest: none. from 2011 to 2014, 66% of the 3834 patients affected by hematological malignancy searching for an unrelated donor through the italian registry successfully identified a suitable donor. this proportion increases up to 71% when searching for a cord blood unit was considered, corresponding to total transplant efficiency of 62%. from april 2006, the rome transplant network adopted a unique policy for the identification of a potential alternative donor, following a hierarchical selection that considered as first choice a volunteer unrelated donor, secondly a cord blood unit and last a haploidentical related donor. before starting the unrelated donor search, a preliminary query through the bone marrow donor worldwide database was performed for all the patients referred to the rome transplant network. based on the low resolution hla typing (a, b and drb1) it was possible to arbitrary assign a good or poor score that might predict the identification of a full matched (8/8 a, b, c and drb1) donor. therefore, aims of the present study were to assess the utility of the preliminary query and the impact of the use of high resolution hla typing since the starting of donor search on the timing for the unrelated donor identification. moreover, the final aim was of comparing donor identification and transplant efficiency between the national registry, that considers only the unrelated donor and the rome transplant network, whose policy includes also haploidentical donor as third choice in the donor search process. at rome transplant network 79% out of 417 adult patients met criteria of a good preliminary query corresponding to a matched unrelated donor identification in 50% of cases vs only 12.5% for patients with poor preliminary query. our policy led to 78% and 74%, respectively, of alternative donor identification and transplant efficiency, significantly higher than the corresponding data of 71% (p = 0.007) and 62% (p o0.0001) reported by the national registry. moreover, the median duration of search process for mud identification has been significantly reduced by the use of hr hla typing patient at the start of the formal search activation from 88 (range: 1-1016) to 66 (range: 8-905) days at ibmdr (po 0.001) and from 61 to 41 days (20-321) at rtn (po 0.001). in conclusion, the preliminary query represents a useful tool to address the search towards the best donor choice and to perform transplant in adequate time. moreover, the timing of donor identification has been significantly reduced with the use of high resolution typing at the start of donor search. a search and selection donor policy should be basically established and should include the haploidentical donor to improve the transplant efficiency. disclosure of conflict of interest: none. the long term prognosis of elderly acute myeloid leukemia (aml) patients remains poor. advances in the uses of alternative donors and reduced intensity conditioning regimens have extended the use of allogeneic hematopoietic stem cell transplantation (hsct) to a wider number of patients. however, few studies have reported data on the efficacy of hsct from alternative donors in elderly aml patients. we retrospectively analyzed the transplantation outcome in 93 consecutive elderly aml patients aged 460 years who received hsct (2005 hsct ( -2015 at the catholic blood and marrow transplantation center. donor types were autologous (n = 18) or hla matched related (mrd, n = 28), unrelated (mud, n = 22), or haploidentical (n = 25). for graft-versus-host disease (gvhd) prophylaxis, methotrexate and cyclosporine (mrd) or tacrolimus (mud/haploidentical donor) were used. mud and haploidentical donors were given antithymocyte globulin. the median age was 63 years, with 23 patients (25%) 465 years. intermediate-or adverse cytogenetic risk was observed in 91% of patients. with a median follow-up of 44.7 months, overall survival (os) and disease-free survival (dfs) at 3 years after transplantation were 37% and 38% for autologous, 40% and 35% for mrd, 67% and 62% for mud, and 67% and 67% for haploidentical hsct, respectively. the 3-year relapse was significantly higher for autologous hsct compared to allogeneic hsct (40% vs 14%, p = 0.012), while it was similar among allogeneic donors: mrd, 13%; mud, 14%; haploidentical, 15% (p = 0.925). the 3-year non-relapse mortality (nrm) for mud (24%) or haploidentical donor (18%) hsct was comparable to that of autologous hsct (22%), while it was relatively higher for mrd hsct (52%, p = 0.056). of the 75 patients receiving allogeneic hsct, the 1-year cumulative incidence of moderate to severe chronic gvhd was significantly increased for mrd (64%) compared to alternative donor hsct (35%, p = 0.001). in multivariate analysis, patient age (hr 0.8, 95% ci 0.8-1.0, p = 0.005) and donor type (hr 3.5 95% ci 1.0-13.0, p = 0.056 for mud; hr 6.2, 95% ci 1.7-22.6, p = 0.006 for mrd compared to haploidentical donor) were significantly associated with the cumulative incidence of moderate to severe chronic gvhd, while female-to-male hsct showed a borderline significance (hr 2.1, 95% ci 0.9-4.7, p = 0.075). incidence of acute gvhd was similar according to donor type. in the multivariate analysis for nrm, patient age (hr 1.4, 95% ci 1.1-1.6, p = 0.001), mrd (hr 4.5, 95% ci 1.4-14.4, p = 0.011), and hematopoietic cell transplantation-comorbidity index high risk (hr 6.4, 95% ci 2.3-17.5, p = 0.001) were significantly associated. in conclusion, our results showed significantly higher relapse rate for elderly aml patients receiving autologous hsct compared to allogeneic hsct, responsible for the lower survival rate in autologous hsct. we observed that nrm rate for mud and haploidentical donors for elderly aml patients were lower than expected and similar to autologous hsct. relatively higher incidence of nrm for mrd hsct seemed responsible for the low long term dfs. these results suggest a need for strengthening of gvhd prophylaxis in mrd hsct for elderly aml patients. our results suggest a potential role of alternative donor hsct to improve long term survival rates in elderly patients with aml. disclosure of conflict of interest: none. for patients with saa, transplantation from an unrelated donor (ud) is usually considered after failure of at least one course of immunosuppression. this strategy is based on a relatively high risk of complications for ud transplant recipients, such as graft rejection, graft-versus-host disease (gvhd) and infections. however, the outcome of unrelated donor transplants has significantly improved in recent years, due to better donor selection, conditioning regimen optimization and better supportive care. the authors describe results from 51 patients with saa who receive unrelated allogeneic transplants in a single reference institution from 1997 to 2014. data was retrieved from the center databasis and there were 30 females and 21 males. median age was 15 years old . median total number of cells infused was 3.4 × 10 8 /kg.61% of the patients have received more than 50 transfusions previously. conditioning regimen were: cy 120 + tbi 1320 ± atg in 16 (31%) patients, bu 12 mg/kg+ cy 120+ atg in 18 (35%), and fludarabine + cy+atg in 8 (16%), fludarabine, cy+tbi 200 in 9 (18%) patients. stem cell source was marrow in 84%, cord blood in 13% and peripheral blood in 3% of patients. transplants were full matched in 32 (62%) patients, had one mismatch (out of 12) in 12 (24%) and 2 mismatches in 7 (14%) patients. engraftment was complete as evaluated by donor chimerism at day 30 and 100 post transplant in 36 patients (71%), partial in 4 (8%) and graft failure was observed in 9 (18%) patients. acute gvhd grade ii-iv was seen in 9 patients ( 18%) and nih moderate to severe chronic gvhd was seen in 8 (16%) patients. median overall survival was 328 days (4-4287) and estimated 5 years overall survival was 55%. risk factors for survival identified were: hla mismatch and stem cell sources other than marrow. unrelated transplants are a feasible salvage therapy for patients with saa refractory to immunosuppression, being hla compatibility and marrow stem cell source factors with a positive impact on survival. disclosure of conflict of interest: none. use of haploidentical stem cell transplantation continues to increase, the 2015 european society for blood and marrow transplant activity survey report jr passweg 1 , h baldomero 1 , p bader 2 c bonini 3 , rf duarte 4 , c dufour 5, , a gennery 6 , n kröger 7 , j kuball 8 , f lanza 9 , s montoto 10 , a nagler 11 , ja snowden 12 , j styczynski 13 and m mohty 14 for the european society for blood and marrow transplantation (ebmt) 1 hematopoietic stem cell transplantation (hsct) is an established procedure for many acquired and congenital disorders of the hematopoietic system, including disorders of the immune system, and as enzyme replacement in metabolic disorders. the annual activity survey of the ebmt describes the status of hsct in europe and affiliated countries and has become an instrument used to observe trends and to monitor changes in technology use. teams were invited to report their transplant activity for 2015 by indication, stem cell source and donor type using a single paged survey. a record number of 42 '171 hsct in 37 '626 patients (16 '030 allogeneic (43%), 21 '596 autologous (57%)) were reported by 655 centers in 48 countries in 2015. trends include continued growth in transplant activity during the period 2005 and 2015, with the highest percentage increase seen in middle income countries (allo 209%, auto 215%), and the lowest in very high income countries (allo 64%, auto 28%), for both allogeneic and autologous hsct. in contrast the absolute growth is highest in the very high income countries (growth allo rates 114 transplants per 10 × 10 6 inhabitants, auto rates 85 for very high income countries; allo rates 35, auto rates 38 for middle income). main indications for hsct were myeloid malignancies 9 '413 (25%; 96 % allogeneic); lymphoid malignancies 24 '304 (67%; 20% allogeneic); solid tumors; 1 '516 (4%; 3% allogeneic); and non-malignant disorders; 2 '208 (6%; 90% allogeneic). remarkable is a decreasing use of allogeneic hsct in cll from 504 patients in 2011 to 255 in 2015 and is most likely due to the development of potentially very effective cll drugs. use of haploidentical donors for allogeneic hsct continues to increase 2 '012 in 2015; a 291% increase since 2005. the highest growth is seen in myeloid malignancies 1 '008, with lymphoid malignancies 636, nonmalignant disorders 316 and 52 others. in aml, haploidentical hsct increases similarly for patients with both advanced disease and those in cr1. both marrow and peripheral blood is used as stem cell source for haploidentical hsct with higher numbers reported for the latter. this year's activity survey shows continued increase in the use of haploidentical hsct across europe within the main indication groups and cell source. it reflects in a timely manner current trends in stem cell transplantation and is an essential tool for health care planning and health policy makers. human bone marrow mesenchymal stromal cells derived exosomes (hbmmdes) are small membrane vesicles secreted from mesenchymal stromal cells that may serve as a vehicle for protein, mrna and microrna (mirna) transfer to distant cells; affecting gene expression, proliferation, and differentiation of the recipient cells. therefore, mdes may possess some of the immunoregulatory properties of their parental cells. in the present study we aim to explore the immunomodulatory function of mdes and understand the molecular mechanisms enabling it. for this purpose, we co-cultured hbmmdes with activated human lymphocytes. using ultracentrifugation, hbmmdes were isolated from expanded human bone marrow derived mesenchymal stromal cells (hbmmscs). using em and zeta sizer, particles were shown to be in the range of 80-120 nm. pha activated human peripheral blood lymphocytes (pbls), r-848/il2 activated b cells and anti cd3/cd28 activated t cells were co cultured with purified mdes. cell proliferation was tested using thymidine incorporation assay. we found that exosomes derived from 1 × 10 3 to 1 × 10 6 mscs exhibited a dose-dependent inhibition of lymphocyte proliferation. exosomes derived from 1 × 10 6 mesenchymal stromal cells co cultured with pha activated pbls, activated b cells and activated t cells showed proliferation inhibition of 53%( p ⩽ 0.001), 34.37% (p ⩽ 0.05) and 47.41 % (p ⩽ 0.01), respectively. in order to understand the molecular mechanism behind the immunomodulatory effect of mdes, we have profiled mde's mir content using illumina hiseq 2500 platform and we are currently profiling co cultured activated lymphocytes mrna content using next-generation sequencing system, illumina. preliminary results demonstrate some higher abundance of specific mscs derived mirs in the mdes. hbmmscs have been shown to serve as immune modulators in patients with acute and chronic graft versus host (gvhd). in the future, mdes may provide an alternative therapy for gvhd. compared with bmmscs, mdes are more stable, have no risk of aneuploidity or ectopic proliferation and have less probability of immune rejection. additional studies are needed to explore the applicability of mdes to serve as modulators of the immune response. disclosure of conflict of interest: none. graft-versus-host disease (gvhd) is the major complication after allogenic haematopoietic stem-cell transplantation s192 (hsct). extra virgin olive oil (evoo) is a source of phenolic compounds such as glycoside oleuropein, hydroxytyrosol and tyrosol. olive oil polyphenols have shown antioxidant, immunomodulatory, antiproliferative, anti-apoptotic and antiinflammatory properties that might be useful in the prophylaxis and treatment of gvhd. polyphenolic extract (pe) of evoo was obtained by the method described by vazquez roncero et al. with some modifications. briefly, fifty grams of evoo (oleoestepa, seville, spain) was extracted with methanol/water (80:20, vol/vol, 125 ml ). the mixture of evoo, methanol and water was decanted and the methanolic extract was concentrated and lyophilized. then, the effect of pe in cell viability and activation of t lymphocytes from healthy donor's buffy coats either resting or activated with anticd3 plus anticd28 was analyzed by flow cytometry after staining with 7aad, anexin-v and cd25. proliferation assays were performed with pkh and the quantification of il-2, il-4, il-6, il-10, tnf-α and ifn-γ cytokines in cell culture supernants with bd cytometric bead array (cba). signaling pathways were analyzed by western blot. finally, in a mouse model of acute gvhd (c57bl/6 in balb/c), mice were randomized into two experimental diet groups: standard diet (2014s harlan laboratories) and standard diet (2014s harlan laboratories) supplemented with 600 ppm of pe obtained of evoo. the severity of gvhd was assessed by a scoring system described by cooke et al. that incorporates five clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity. pe did not affect t cell viability. by contrast, pe decreased t-cell activation and proliferation of t-lymphocytes stimulated with anticd3 plus anticd28. in addition, there was a decreased production of th1 (ifnγ, il-2 and tnf) and th2 cytokines (il-4, il-6 and il-10) in the presence of pe. regarding the signaling pathways analyzed, pe inhibited phosphorylation of akt and nuclear translocation of nfkb in activated t cells. in the mouse model of acute gvhd, animals which received the pe supplemented diet had an increased survival as compared to mice receiving a standard diet. also, gvhd incidence was significantly lower among mice receiving the pe supplemented diet as assessed by both the presence of gvhd signs as well as pathological examination. polyphenols obtained from evoo are an important immunomodulatory agent capable to reduce the proliferation and activation of activated t cells and the production of proinflammatory cytokines. in a mouse model of acute gvhd, pe supplemented diet reduced the incidence and severity of the disease and increased the survival of mice. disclosure of conflict of interest: none. graft-versus-host disease (gvhd) is a leading cause of postallogeneic haematopoietic stem cell transplantation (hsct) morbidity and mortality (1) . extracorporeal photopheresis (ecp) is an alternative therapeutic strategy that appears to act in an immunomodulatory fashion, potentially involving regulatory t lymphocytes and dendritic cells in patients who are refractory to steroids. dendritic cells (dcs) are the most important antigen-presenting cells, playing a pivotal role in t-cell function and in the link between innate and adaptive immunity. moreover, dcs are also critical mediators of immune tolerance and energy. they can be divided into two major subsets, plasmacytoid dcs (pdcs) and myeloid dcs (mdcs) which have distinct functions. pdcs play a pivotal role in peripheral tolerance through generation of regulatory t (treg). on the other side mdcs promote, as well as pdcs, th2 and th0/tr1 responses (1-4). our study was performed to understand the mechanism of action involved in immunomodulatory effect of ecp. as the modulation of dcs and tregs number and function (7, 8) may be a central mechanism of ecp in maintaining self-tolerance, down-regulating immune responses, and limiting inflammation (9). eight patients affected by gvhd were included in this pilot study. in ecp apheresed mononuclear cells are exposed to 8methoxypsoralen and uva radiation. after this photoactivation, which induces dna damage and apoptosis, the cells exposed are re-infused into the patient inducing an immunomodulatory effect. all patients or their legal guardians gave their consent for this study. a sample of peripheral blood (pb) (basal condition), a sample of apheresis pre-uva photoactivation (pre-pa) and a sample of photoactivated apheresis (pa) were collected at the first day of ecp and every week for the first month of treatment. circulating dcs, mdcs (cd14/16-cd85+cd33+), pdcs (cd14/16-cd85+cd123+) and tregs (cd4 +cd25+foxp3+) were directly enumerated and phenotypically characterized. the assays were performed at day+1,+8, +15,+21,+30 data are expressed as mean ± s.d. of absolute number of cells/μl. at day +1 there were no differences in the absolute number of both mdcs and pdcs between pre-pa and pa. consequently there were no differences between pb and pa. from day +8 till +30 we observed an increase of these two cellular populations at every date of treatment. comparing the basal pb of day +1 vs day +30 we observed an increment of 40% and 120%, respectively for mdcs and pdcs (mdc from 11247 cell/μl to 15742 cell/μl; pdc from 6983 cell/μl to 15263 cell/μl). comparing the basal pb of day +1 vs day +30 we observed an increment of 115% of tregs (from 4257 cell/μl to 9142 cell/μl) while we observed a median increment of 34% calculated between pre-pa and pa of each day of treatment from day 1 to day +30. no firm conclusions can be drawn from a clinical point of view, however a biological effect has certainly highlighted. in particular no substantial differences in basal pb mdc or pdc emerged during the first month of treatment while a significant increase of mdc and pdc can be observed since day +15 following uva photoactivation. regarding tregs we observed an increment of 115% of tregs between pb from day +1 to day+30 and a median increment of 34% calculated between pre-pa and pa of each day of treatment. disclosure of conflict of interest: none. [p151] p152 impact of th17 cells on xenogeneic graft-versus-host disease l delens, s servais 1 , g ehx 1 , l vrancken 1 , g fransolet 1 , c gregoire 1 , m hannon 1 , s dubois 1 , c daulne 1 , f baron 1 and y beguin 1 1 giga i3 : hematology, university of liege acute graft-versus-host disease (gvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation. its pathophysiology is complex and not yet fully understood. in particular, the impact of th17 cells on murine acute gvhd has yielded conflicting results, while demonstration of increased levels of th17 cells at the site of acute gvhd provided only indirect evidence of their involvement in humans. here, we assessed the potential implication of th17 cells in a humanized mouse model of xenogeneic gvhd (x-gvhd). methods: x-gvhd was induced by infusing human peripheral blood mononuclear cells (pbmcs) into nod-scid il-2rγnull (nsg) mice given 2.5 gy total body irradiation 1 day prior transplantation. th17 cells were generated by culturing naive cd4+ t cells with anti-cd3/anti-cd28 coated beads under th17-skewing cytokines (tgf-β1, il1-β, il-6, il-21, il-23, neutralizing anti-il-2 and anti-ifnγ antibodies) in hypernatremic conditions (nacl 40 mm). results: after 8 days of culture, a median of 21.75% of il-17a+ cells was obtained. we confirmed the expression of il-17a, rorc and il-23r by these cells by rt-qpcr. we next assessed the co-injection of human pbmcs (1.106) with in vitro differentiated cells under th17 skewing conditions (1 × 10 6 ) (co-injection group, n = 20), in comparison with the injection of pbmcs alone (2 × 10 6 cells, pbmcs group, n = 17). we observed higher x-gvhd score (p60%) of cells expressing both il-17a+ and ifnγ+ cells (th17/ th1-like phenotype) among cd4+ il-17a+ cells while coinjected mice had higher blood concentration of il-17a (p = 0.026) than pbmc mice. these results demonstrate that addition of th17 cells worsened x-gvhd confirming their role in acute gvhd pathogenesis. disclosure of conflict of interest: none. although survival from allogeneic stem cell transplantation (hsct) has significantly improved, acute graft-versus-host disease (gvhd) remains a major cause of death. intestinal dysbiosis has been associated with acute gastrointestinal gvhd and poor outcome after hsct. we reported a correlation between microbiota (gm) composition and short chain fatty acid (scfa) production and gvhd in transplanted children. 1 to assess how the metabolic pathways of gm change during transplantation and identify modulators of immune response, we perform first longitudinal metagenomic analysis in children undergoing hsct. 8 patients (pts) (6male; mean age: 10y) with hematologic malignancies (7 all, 1 aml), who received busulphan-based myeloablative conditioning and t-cell replete bone marrow graft were enrolled. pts were prospectively enrolled in a protocol with at least 3 specimens fecal samples collected: one before and two after hsct, in order to build a proper trajectory. gvhd prophylaxis was cyclosporine for 3 pts receiving a matched related donor and cyclosporine, short-term mtx and atg for 5 pts receiving a matched unrelated donor. non-gvhd and gvhd patients had similar exposures to antibiotics during the stool collection. of these pts, 50% developed gvhd within the first 100 days. we applied shotgun metagenome sequencing to total fecal dna from samples collected. functionalities were assigned by reads mapping at different levels of the kegg database. 2 relative abundance was calculated and statistical analysis was performed. according to our findings, core functional profiles were overall conserved through the time-points in all patients ( figure 1a ), in contrast to the phylogenetic profiles behavior, this finding confirming the overall redundancy of gut microbiome core functionalities. analyzing the single metabolic pathways in subjects who developed gvhd, we found in the pre-hsct period a higher relative abundance of nucleobasis (purine and pyrimidine) metabolism (p o0.05) and branched-chain amino acids biosynthesis (p o0.05). functions related to the production of branched-chain amino acids are involved in the biosynthesis of the cell wall of gram-negative bacteria, microorganisms including subgroups with well know opportunistic pro-inflammatory. in addition, post-hsct samples of gvhd patients showed a lower abundance of genes involved in polysaccharides metabolism, as glycan biosynthesis and glycosaminoglycan degradation (p o0.05) ( figure 1b ). glycosaminoglycan degradation activity gets bacteria able to survive during extreme situations, as fasting using mucus polysaccharides as energy source, contributing to maintain a mutualistic composition of gm and scfa production by the saccharolytic functions of the endogenous mucus polysaccharides. this study detects functional peculiarities in the gm of non-gvhd pts. the gut metagenome configuration of non-gvhd patients is structured to derive scfa after hsct. the production of these metabolites promotes peripheral regulatory t-cell generation 3 , potentially explaining the protective role of gm from gvhd. although intestinal epithelial cells (iecs) are crucial regulators of barrier function and immune homeostasis, they also facilitate inflammation in exaggerate responses to proinflammatory mediators by pretransplant conditioning regimen, which plays a critical role in amplifying graft-versus-host disease (gvhd). thus inhibition of the converting to pathogenic iecs by conditioning may represent a novel approach to inhibit gvhd. aryl hydrocarbon receptor (ahr) is the ligandactivated transcription factor which has the ability to mediate the biochemical, metabolic, and toxic effects of environmental chemicals. recently, it has been demonstrated that ahr is an important regulator of cell development, differentiation, and function of both innate and adaptive immune cells. the ability of ahr is induced by respond to endogenous ligands generated from the host cell, diet, and microbiota. here, we investigated the regulatory role of ahr in iecs under inflammatory responses and its therapeutic activity for modulation of gvhd. ahr and cyp1a1 expression in mouse iecs were determined by real-time pcr. mouse iecs were pretreated with endogenous ahr ligands l-kynurenine (l-kyn, 300 mm) or pbs for 6 h and then stimulated with lps or il-1b for 24 h. cytokine levels were measured using the mouse flex-set cytokine bead array or real-time pcr. b6d2f1 (h-2b/d) recipients were administrated l-kyn daily by i.p. injection for 3 days. then the recipients were lethally irradiated and transplanted with 5 × 10 6 tcd-bm plus 2 × 10 6 t cells from b6 (h-2b) donor. mice were monitored every other day for survival and clinical score. colons were collected and stained with hematoxylin and eosin (h&e) for histopathological scoring. we found that ahr was constitutively expressed in the mouse iecs. cyp1a1 (an ahr target gene) was significantly increased by treatment of l-kyn under un-stimulatory condition. we further observed that l-kyn completely abrogated il-1β-mediated il-6 or lps-mediated tnf-a expression in iecs. administration of bdf1 recipient mice with l-kyn before transplantation significantly reduced the lethality and severity of gvhd. histopathology clearly revealed that treatment of l-kyn inhibited intestinal gvhd. our results demonstrate that 1) ahr is constitutively expressed in iecs, 2) treatment of endogenous ligand l-kyn induce ahr activation in the steady status, 3) ahr activation blocks conversation of the epithelial cells into pathogenic cell type, and 4) pre-administration of ahr ligand reduces gvhd. our study suggests that activation of ahr pathway in iecs before allogeneic hematopoietic stem cell transplantation (hsct) is a possible strategy to reduce intestinal gvhd. disclosure of conflict of interest: none. [p156] s196 relating with acute graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct), protecting endothelial cells (ecs) from damage may be a potent prophylaxis and therapeutic strategy of acute gvhd (agvhd). conventional agvhd therapies may cause many adverse side effects because of their multiple targets. therefore, we explored the therapeutic efficacy of simvastatin, a lipid-lowering drug, which has been demonstrated endothelial protection. our previous clinical observation has found patients with agvhd had lower angiopoietin-1 (ang-1) level at day 7 but higher ang-2 level at day 21 than those without agvhd. in this study, we explored changes in ang-1 and ang-2 expression in an agvhd mouse model and determined whether simvastatin prevents gvhd through regulating ang-1 and ang-2 expression. we preincubated ea.hy926 ecs with simvastatin (1mmol/l) 12 h before stimulated with tnf-a, then ang-1 and ang-2 concentration in the cell supernatant was measured by elisa. ang-1 and ang-2 mrna and protein level of treated and untreated cells were examined simultaneously. in vitro simvastatin increased ang-1 production and release but conversely inhibited ang-2 release from ea.hy926 ecs. donor mice spleen cells were injected along with bone marrow cells into recipient mice after lethal irradiation to induce agvhd. simvastatin was administered orally once daily to mice (10 mg/kg) for 7 days after allo-hsct and started −1 day after allo-hsct. then mice survival time was monitored and organ damage was evaluated. the plasma level of ang-1 and ang-2 was measured by elisa, expressions of ang-1 and ang-2 in aortic endothelium were assessed by immunohistochemistry. simvastatin improved the survival and attenuated the histopathological gvhd grades of agvhd mice. plasma levels of ang-1 were significantly decreased, while plasma levels of ang-2 obviously increased in agvhd mice after transplantation. simvastatin reduced plasma levels of ang-2, elevated the plasma levels of ang-1 as well as the aortic endothelial levels of ang-1 and ang-2. in summary, simvastatin represents a novel approach to combat gvhd by increasing ang-1 production while suppressing ang-2 release to stabilize endothelial cells. there is a growing evidence of safety and efficacy of posttransplantation cyclophosphamide (ptcy) in stem cell transplantations (sct) from different donors and graft sources. still the optimal combination of immunosuppressive agents with ptcy should be elucidated for different types of scts. we report the 2-year update of the prospective nct02294552 single-center trial that evaluated risk-adapted graft-versushost disease (gvhd) prophylaxis with ptcy in related, unrelated and haploidentical scts. 200 adult patients (median age 32 y.o., range: 18-62) with hematologic malignancies, including aml (47.5%), all (26.5%), cml (10.5%), mds (4%), and lymphomas (11.5%), were enrolled in the study. 23% of patients were classified as salvage. 26% received the graft from matched related (mrd), 65% from matched/mismatched unrelated (mud/mmud), and 9% from haploidedntical (haplo) donor. 43% received bone marrow graft (bm) and 57%peripheral blood stem cell (pbsc) graft. 18.5% had myeloablative conditioning and 81.5%-reduced-intensity conditioning. gvhd prophylaxis for matched bm grafts consisted of single-agent ptcy 50 mg/kg days+3,+4, for matched pbsc graft-ptcy+ tacrolimus+ mycophenolate mofetil (mmf) 30 mg/kg days 5-35, and for any mismatched graft-ptcy+ tacrolimus+ mmf 45 mg/kg days 5-35. median follow-up was 20 months (range: 4-40). grade ii-iv (10% vs 18% vs 11%, p = 0.37) and grade iii-iv acute gvhd (4% vs 6% vs 0%, [p157] p = 0.59) were not different in mrd, mud/mmud and haplo groups, respectively. moderate and severe chronic gvhd was infrequent in all groups with slightly lower incidence after mud/mmud graft: and 22% vs 9% vs 21%, p = 0.046. nonrelapse mortality (nrm) was not different after mrd, mud/ mmud and haplo sct (8% vs 14% vs 24%, respectively, p = 0.19), while relapse incidence was higher after mrd and haplo grafts: (45% vs 22% vs 52%, p = 0.0017). 2-year overall survival (os), event-free-survival (efs), and gvhd-relapse free survival (gfrs) were 73% vs 71% vs 44% (p = 0.0015); 48% vs 65% vs 33% (p = 0.0008); 29% vs 56% vs 22% (p = 0.0002) for mrd, mud/mmud and haplo groups, respectively. in the multivariate analysis only disease risk index (hr 2.2 95%ci 1.6-3.0, p = 0.0001), severe sepsis (hr 3.8 95%ci 1.8-8.0, p = 0.0004) and chronic gvhd (hr 0.53 95%ci 0.30-0.93, p = 0.02) were predictive for efs, while type of donor was not a significant factor (hr 1.0 95%ci 0.6-1.7, p = 0.99) (figure 1 ). the incidences of complications were: hemorrhagic cystitis-23%, sepsis-24%, severe sepsis-8%, invasive mycosis-8%, cmv reactivation-45%, veno-occlusive disease-2.5%, transplant-associated microangiopathy-3.5%, grade 3-4 liver toxicity-14%, grade 3-4 kidney toxicity-1%. more than one third of patients experienced poor graft function during 100 days after sct, and in 83% of them cmv, hhv and bk virus reactivations were identified as the cause. the reported risk adapted strategy alleviates the risk of gvhd and nrm after mmud and haplo grafts. the observed differences in the relapse incidence, os and efs were predominantly due to unbalanced disease risks in the groups. the relapse of underlying malignancy with this prophylaxis still significantly influences the outcome. substantial number of patients experience poor graft function, which doesn't translate into nrm. disclosure of conflict of interest: none. a high migratory capacity of donor t-cells in response to the lymph node homing receptor ccr7 increases the incidence and severity of graft-versus-host disease vg garcía de soria 1 , ip sainz 2 , e jiménez 1 , a arriero 1 , c fernández-arandojo 1 , c cuesta 2 , b colom 2 , a marcos 2 , a rosendo 2 and cecilia muñoz calleja 2 1 department of hematology hospital universitario de la princesa and 2 department of inmunology. hospital universitario de la princesa graft-versus-host disease (gvhd) pathogenesis involves migration of the donor t-cells into the secondary lymphoid organs (slo) in the recipient, which is steered by two homing molecules: cd62l and ccr7. therefore we investigated whether the migratory capacity of donor t-cells is associated with gvhd. this single center prospective study included 85 donor-recipient pairs. in vitro chemotaxis assays of the lymphocytes of the apheresis product were performed in parallel with the analysis of cd62l and ccr7 by flow cytometry. the potential of activation of ccr7+ t-cells was assessed through ex vivo activation assays with peripheral blood monuclear cells (pbmc) from healthy donors using anti-cd3 and anti-cd28mabs. the migratory index to the ccr7 ligands, ccl19 and ccl21, was higher in t-cells from donors whose recipients will develop gvhd. these data indicated that the migratory capacity of the donor t-cells is clearly related to the development of gvhd. this prompted us to study the relationship between gvhd and the expression of two of the most relevant molecules in the trafficking of lymphocytes towards slo, cd62l and ccr7,as a subrogate index of the migratory potential of t-cells. consequently, we quantified the numbers of cd62l+ and ccr7+ t-cells in the graft. the initial transversal analysis of our data revealed that the percentage of cd62l+ lymphocytes in the apheresis product was very low compared to healthy lymphocytes. the analysis also confirmed that cd62l undergoes plasma membrane shedding after g-csf mobilization thus making it a non-valid biomarker. the analysis of ccr7 molecule revealed that the acute gvhd group received higher percentage of cd4+ccr7+ t-cells, whereas chronic gvhd patients were transplanted with higher percentage of cd8+ccr7+ t-cells compared to the non gvhd group. these results were confirmed when patients were subdivided into degrees of severity. a multivariate analysis was performed to investigate the real value of ccr7 to predict the development and severity of gvhd, and confirmed that ccr7 expression is a risk factor for the development of gvhd. thus, the percentage of ccr7+cd4+ t-cells increases the probability of developing acute gvhd (or = 1.08, c.i (95%) = 1.01-1.16, p = 0.019) and suffering a higher degree (or = 1.08, c.i (95%) = 1.01-1.15, p = 0.014). similarly, the or of the percentage of ccr7+cd8+ t-cells was 1.17 (c.i (95%) = 1.01-1.36, p = 0.0031) and 1.21 (c.i (95%) = 1.05-1.39, p = 0.006) for the development of chronic gvhd and its degrees, respectively. finally, to study the potential of activation of ccr7+ t-cells, we carried out ex vivo activation assays with pbmc from healthy donors using anti-cd3 and anti-cd28mabs and the expression of cd40l on cd4+ t-cells and of cd69 on cd8+ t-cells as markers of activation, demonstrating that ccr7+ t-cells exhibited higher potential of activation than ccr7-t-cells. to our knowledge this is the first analysis of the influence of the migratory capacity of the donor t-cells on clinical outcome following allogeneic hsct. our data show that ccr7 could be considered a subrogate biomarker of the migratory capacity of the donor lymphocytes for predicting the risk of suffering gvhd. based on the previous findings, we propose that the selective depletion of ccr7 expressing cells could be an effective preventive therapy for gvhd. disclosure of conflict of interest: none. previously published p160 a single center research for outcome in patients receiving imatinib for steroid-refractory chronic gvhd after allogeneic stem cell transplantation l ni, y luo, y tan, y hu, y zhao, j shi and h huang despite of major progress in allogeneic stem cell transplantation over the last decades, steroid-refractory chronic graftversus-host disease (sr-cgvhd) remains a leading cause of late morbidity and mortality. pre-clinical evidence confirms cgvhd has antibodies activating the platelet-derived growth factor receptor (pdgf-r) pathway. since this pathway can be inhibited by imatinib, we performed a study including 16 patients with sr-cgvhd given imatinib at a dose of 300 mg per day. all patients with a median age of 25 years (range: 16-53) underwent allogeneic hematopoietic stem cell transplantation in our single center between 2008 and 2015, and chronic gvhd occurred at a median time of 10 months (range: 3-29) after transplantation. patients had active cgvhd with measurable involvement of skin, lung or other districts and had previously failed in first-line immunosuppressive therapy. the major organs involved were lung (n = 11), skin (n = 10) and mouth (n = 1), including 5 cases involving both lung and skin, 8 cases involving 3 or more organs. according to the 2014 national institutes of health (nih) criteria and nih global severity, 13 patients were evaluated as severe cgvhd, and the other three were moderate. meanwhile, the 2014 nih working group had updated its recommendations for overall responses, consisting of complete remission (cr), partial remission (pr), and lack of response (unchanged, mixed response, progression). cr was defined as resolution of all manifestations in each organ or site, and pr was defined as improvement in at least 1 organ or site without progression in any other organ. after 3 months treatment, 14 patients receiving sufficient dose of imatinib revealed overall response rate (orr) at 78.6%, and orr remained unchanged at 6 months assessment, but with cr rate increased to 28.6%. two patients couldn't meet the response of cr or pr were considered as a lack of response, including one evaluated as unchanged and one mixed response because of pr in lung accompanied by progression in eyes. with a median follow-up of 9 months, 14 patients were alive, with a 1 year estimated overall survival was 87.1%. 2 patients eventually died of pneumonia. except 1 patient discontinued imatinib because of grade 2 toxicity as gastrointestinal discomfort at the first month, no one had imatinib-related grade 3 to 4 toxicity. this study suggests that imatinib is a promising and better tolerated treatment for patients with sr-cgvhd. disclosure of conflict of interest: none. acute graft-versus-host-disease (agvhd) is a major complication after allogenic hematopoietic transplantation (allo-sct). in recent years, a number of tissue-specific proteins have been described as biomarkers that could contribute to anticipate and/or diagnose this complication earlier and more accurately. reg3α (regenerating-islet-derived-3-alpha) has been directly related to gastrointestinal (gi) agvhd. our objective was to analyze plasma levels of reg3α at days +15 and +30 in patients who underwent unmanipulated haploidentical transplantation with reduced conditioning regimen (haplo-ric), and to correlate the results with the development of agvhd. we retrospectively analyzed 63 consecutive patients (2009-2016) who underwent haplo-ric with post-transplant cyclophosfamide (days +3, +4), mmf and csa as gvhd prophylaxis. seven cases were excluded due to early death (before day +30) and 4 cases due lack plasma sample. characteristics of the 52 patients included in the analysis are described in table 1 . reg3α detection was performed by elisa (mbl international corp, woburn, ma) according to manufacturer's instructions on 200 μl of plasma obtained at day +15 and +30. the association of the incidence of agvhd with known clinical variables and plasma reg3a levels were performed by cox regression and mann-whitney u-test, respectively. the determination of the best cut-off of reg3α levels to stratify patients with gi agvhd was performed with roc curves. the stadistical program used was r v2.15.0. the cumulative incidence of grade ii-iv and grade iii-iv agvhd was 52% and 17%, respectively. characteristics of agvhd are shown in table 2 . no association was found between agvhd and usual clinical variables (stem cells source, age, sex, conditioning regimen, donor/recipient sex and number of infused cd34 + cells), and with plasma reg3α levels at day +15.plasma reg3α levels at day +30 were higher in patients who devolved gi agvhd compared to patients who did not showed gi agvhd (median [p161] s199 and range: 2483 (2022-5904) vs 1110 (0-7797) pg/ml, p = 0.14, figure 1 ).the best cut-off selected on day +30 was 1989 pg/ml (s85%, e71%). patients with levels higher than 1989 pg/ml at day +30 had a significantly higher incidence of gi agvhd grade ii-iv (hr 6.9, p = 0.008, figure 2 ). plasma levels of reg3α at day +30 after haplo-ric correlated with the occurrence of gi agvhd grade ii-iv. therefore, plasma levels of reg3α could be use for the prediction and/or diagnosis of gi agvhd. disclosure of conflict of interest: none. anti-fibrotic treatment with pirfenidone in patients with gvhd-associated bronchiolitis obliterans syndrome ke hostettler, s gerull, g nair 1 , j passweg 1 , m tamm 2 and j halter 1 1 hematology, university hospital basel, switzerland and 2 pneumology, university hospital basel, switzerland prognosis of lung gvhd remains poor due to progressive decrease of lung function and repeated infections. pirfenidone exhibits anti-fibrotic effects and has been shown to reduce disease progression in patients with idiopathic pulmonary fibrosis. five patients with established bos (nih criteria 2014) and stable or deteriorating lung function under standard immunosuppressive treatment without active infection were treated with pirfenidone (2403 mg/d) in addition to their current therapy. clinical assessments and pulmonary function tests were performed every three months. five patients (4m, 1f), median age 60y (range: 29-65y) that were diagnosed with bos at a median time of 13.5 months post-transplant started pirfenidone at a median time of 51months (8-102) after diagnosis of bos. two patients are currently still under treatment after 611 and 638 days. two patients had to stop treatment due to financial reasons after 189 and 206 days of therapy. one patient never reached more than 20% of the planned dose due to gastro-intestinal symptoms and was excluded from further analysis. at the start of treatment median fev1 was 0.94l (0.72-1.34); 34.5% predicted (range: 21-44%) and median fvc 2.59 l (1.62-3.24); 46-84 % predicted. median fev1 trajectory was − 0.65 % predicted/ month during median 6months before start of pirfenidone (median − 19ml/month) and +0.33% predicted/month (+9.8ml/month) during treatment with pirfenidon. the treatment was well tolerated except in one patient with gastrointestinal complaints, no phototoxic reactions or serious drugrelated adverse events occurred. in our small number of patients pirfenidone was rather well tolerated and generally safe. the observed, albeit small trend in change of fev1 trajectory justifies further studies of anti-fibrotic therapy as a new therapeutic option in bos after allogeneic hsct. disclosure of conflict of interest: none. anti-thymocyte globulin has been widely used for the prevention of severe graft versus host disease in patients undergoing hsct from unrelated donor. however, the optimal dose remains to be defined. in samsung medical center (seoul, korea), institutional strategy for the atg use has been changed since april 2013, and we hypothesized that the incidence of chronic gvhd may differ by atg strategy. before april 2013, atg 4.5 mg/kg was routinely used in allogeneic hsct from unrelated donor, whereas, the dose of atg was escalated to 7.5 mg/kg since april 2013. in this study, a total of 170 patients who underwent allogeneic hsct from matched or unmatched unrelated donor between jan 2010 and dec 2015 were retrospectively analyzed. peripheral blood was used as the source of stem cells in all patients. after a median follow up of 30.9 months, the cumulative incidence of moderate to severe chronic gvhd was 30.2% (95% confidential interval [ci], 18.3 to 43.0) in the low-atg group and 23.7% (95% ci, 14.5 to 34.1) in the non-atg group (p = 0.655). the rate of 2year overall survival (os) was not significantly different between the groups (49.3% in low-atg group vs 48.1% in high-atg group, p = 0.841), as was the rate of disease free survival (dfs) (40.8% in non-atg group vs 42.3% in atg group, p = 0.867) and cumulative incidence of relapse (cir) (23.9% in non-atg group vs 24.5% in atg group, p = 0.776). in allogeneic hsct from unrelated donor, larger atg dose (7.5 mg/kg) did not reduce the incidence of chronic gvhd when compared to lower atg dose (4.5 mg/kg). disclosure of conflict of interest: none. allogeneic hsct provides a curative chance for patients with hematological fatal disease. however, substantial risks remain for morbidity and mortality caused by disease relapse and graft-versus-host disease. in samsung medical center (seoul, korea), institutional strategy for the atg use has been changed since april 2013, and we hypothesized that the incidence of chronic gvhd may differ by atg strategy. before april 2013, atg was not routinely used in matched sibling donor (msd) transplantation, whereas, atg 5 mg/kg has incorporated into hsct process in transplantation from msd thereafter. in this study, a total of 182 patients who underwent allogeneic hsct from msd between jan 2010 and dec 2015 were retrospectively analyzed. peripheral blood was used as the source of stem cells in all patients. after a median follow up of 40.5 months, the cumulative incidence of moderate to severe chronic gvhd was 22.0% (95% confidential interval [ci], 13.5 to 31.8) in the atg group and 55.2% (95% ci, 42.9 to 65.8) in the non-atg group (p = 0.0018). the rate of 2-year overall survival (os) was not significantly different between the groups (62.5% in non-atg group vs 58.7% in atg group, p = 0.624), as was the rate of disease free survival (dfs) (61.1% in non-atg group vs 53.3% in atg group, p = 0.377) and cumulative incidence of relapse (cir) (23.4% in non-atg group vs 28.3% in atg group, p = 0.463). in allogeneic hsct from msd, atg use was significantly associated with less occurrence of chronic gvhd, but not linked to increasing risk of relapse, with showing similar os and dfs between atg and non-atg group. disclosure of conflict of interest: none. long-term follow-up from the prospective randomized phase iii multicenter trial comparing a standard gvhd prophylaxis with cyclosporine a and methotrexate with or without additional pretransplant atlg (grafalon, previously atg-fresenius s) (given 20 mg/kg/day, days − 3 to − 1) in unrelated donor hematopoietic cell transplantation after myeloablative conditioning resulted in a significant reduction of acute and chronic gvhd without compromising relapse rate and survival [1, 2, 3] . here we report on a subsequent prospective non interventional observational study evaluating the outcome of patients receiving atlg in unrelated donor transplantation in day to day clinical practice without the selective measures of a clinical trial (german clinical trials register drks00004581). thirteen transplant centers included 165 patients with haematological malignancies (median age 54 years, iqr 45-61 years, range: 18-77 years) in early (n = 75, 45%), intermediate (n = 29; 18%) or advanced (n = 61; 37%) disease status receiving marrow (n = 6) or pbsc (n = 159) from 10/10 matched (128; 78%) or mismatched (37; 22%) unrelated donors (n = 4 related) after myeloablative (n = 100, 61%) or ric (n = 65, 39%) conditioning. gvhd prophylaxis consisted of calcineurin inhibitors, mainly csa (n = 154, 93%) with mtx or mmf and atlg. different dosing regimens were allowed according to current practise of centers. median total atlg dose was 46 mg/kg (iqr 32-60 mg/kg, range: 15-91 mg/kg). median follow-up was 12 months (range: 8-14 months). as compared to patients in our randomized phase iii multicenter trial [1, 2, 3] , patients in this study were older; advanced disease status, 10/10 match, pbsc transplantation were more frequent, and given median atlg dose was lower. acute and chronic gvhd, nrm, relapse risk, dfs and os at one year were similar to the results obtained in our randomized trial: incidence of°ii-iv agvhd: 27%, iii-iv agvhd: 13%; moderate/severe cgvhd: 24%; nrm: 17%; risk of relapse: 23%; relapse mortality: 10%; os: 73%. the experience in day to day clinical practice confirms the results shown in our randomized trial, namely the gvhd protective effect of atlg without compromising nrm or relapse rates. baseline calprotectin as a predictor for acute gastrointestinal graft versus host disease (gvhd)-a prospective study n schmidlin 1 , a holbro 1,2 , jp halter 1 , d heim 1 , l infanti 1,2 , a plattner 2 , r plattner 2 , c rothen 3 , a buser 1,2 , c bucher 1 and jr passweg 1 1 division of hematology, university hospital basel, switzerland; 2 blood transfusion center, swiss red cross, basel, switzerland and 3 rothen medical laboratories, basel, switzerland graft versus host disease (gvhd) is a major complication after allogeneic stem cell transplantation. so far there is no good validated predictor for the incidence and severity of gvhd. fecal calprotectin (cpt) is a protein in leukocytes with antibacterial properties. it has been shown to be elevated in acute gastrointestinal gvhd. additionally, cpt may be predictive for treatment response. the aim of the current prospective study was to investigate the role of baseline cpt in predicting incidence and severity of intestinal gvhd. in this prospective study conducted at the university hospital basel, switzerland, we included all adult patients undergoing hsct. the institutional review board approved the study. data were collected prospectively. cpt was measured twice before conditioning and at transplantation. fecal samples for cpt were obtained before conditioning and on the day of transplantation and assessed twice by standard elisa. between march 2012 and april 2016 a total of 194 patients (55% males, 154 patients with both baseline and transplant cpt values) were included. patient, disease and transplant characteristics are described in table 1 . median age at transplant was 55 years (range: 21-73 years). most patients had myeloid neoplasia and 46% received myeloablative conditioning. gvhd prophylaxis consisted mainly of cyclosporine containing regimens (98%). cpt levels ranged from 19 to 1500 μg/g both at baseline (median: 100 μg/g) and at transplantation (median: 101 μg/g), with a good consistency between the two measurements performed (internal quality control). on the other hand, cpt did not correlate with c-reactive protein. the two measurements were taken in median 7 days apart, depending on the conditioning regimen. eighty-five patients had an increase of at least 50 μg/g between baseline and transplantation. overall 61 (31.4%) patients developed acute intestinal gvhd (grade 1: 19; grade 2: 18; grade 3: 16, and grade 4: 8 patients, respectively). cpt both at baseline and at transplantation was not predictive for the incidence of gvhd, acute intestinal gvhd, and for acute intestinal gvhd grade 3-4 ( figure) . additionally, we did not find a significant association between cpt levels and the above mentioned endpoints for patients showing an increase of cpt of at least 50 μg/g between baseline and transplantation. in the current prospective study, we didn't find any correlation between baseline cpt values and the incidence and severity of gvhd and intestinal gvhd. further studies identifying early markers and predictors of gvhd are urgently needed. [p166] disclosure of conflict of interest: none. calcinuerin inhibitor (ci) free graft-versus-host disease (gvhd) prophylaxis: its effects on resource utilization, renal function, and the cost of care m muilenburg 1 , k cole, m abidi 1,2 , s williams and as al-homsi 1,2 1 spectrum health blood and marrow transplantation and 2 michigan state university, college of human medicine effective gvhd prevention following allogeneic hematopoietic stem cell transplantation (ahsct) is vital to reducing transplant morbidity and mortality and improving overall outcomes. several strategies are currently utilized for gvhd prophylaxis including mtx, mmf, cis, post-transplant cyclophosphamide (cy), and proteasome inhibitors. recently, we described the results of a phase i-ii trial of cyclophosphamide (cy) and bortezomib (bor) where patients (pts) received cy (50 mg/kg) on days (d) +3 & +4 and bor on d 0 & +3. the incidences of grade ii-iv and grade iii-iv acute gvhd were 35% and 11%. the incidence of chronic gvhd was 28%. in addition to gvhd, there are other factors that affect patients' quality of life and cost of care and that should be considered. it is well documented that cis have an unfavorable toxicity profile. this includes nephrotoxicity and electrolyte disturbances. furthermore, the cis need serial level monitoring. thus, we endeavored to compare the effects of cybor combination against ci-based regimens by focusing on electrolyte requirements, specifically mg, and renal function. we also sought to better understand financial considerations surrounding the need for ci drug level monitoring. sixteen pts were randomly selected from the cybor group and 16 patients from an internal control group of patients who received mmf and cyclosporine or tacrolimus following reduced-intensity ahsct. the groups were well matched in regards to age, sex, disease status, pam score, and baseline renal function. on each pt, mg results from d 0 to +90 were compiled. based on institutional protocol, a mg replacement value was assigned as well as the corresponding drug and infusion charges. next, the number of immunosuppressant (is) trough levels from d 0 to +90 was tallied and the internal lab charges calculated. to compare renal function, gfr was calculated at baseline, d 0, and d +30. χ 2 tests and wilcoxon rank square tests were used to analyze the data. for the cybor group, median mg value was 1.9 mg/dl (iqr 0.3) vs 1.7 (0.3) in the control group (po0.0001). cybor pts required a median of 7 grams (16) vs 49 grams (55) in the control group (p = 0.001). the cost of mg replacement and infusion was significant (p = 0.001) ( table 1) . for is checks, drug levels were checked a mean of 0.625 times per patient in the cybor group compared to 18.75 times in the control group (po0.0001), which also translates to significant savings ( table 2) . considering these costs, the cybor group saved~$6000. for gfr, 3 cybor pts and 1 control pt had reduced gfr at baseline. on d +30, cybor pts had better renal function in comparison to the control group (p = 0.018) ( figure 1 ). in summary, cybor significantly reduced the use of resources post-transplant and thereby the associated cost related to mg replacement and need for drug level monitoring. furthermore, cybor preserved renal function at d +30. these findings could also impact patient's quality of life. although our cost analysis was restricted to certain aspects of care and did not take into account other factors, it highlights specific important benefits of ci-free gvhd prophylaxis and supplicates further study. a formal prospective comparison of cost and qol is warranted. . related donor n = 11 and unrelated donor n = 5 (compatibility 9/10 n = 1), with conditioning regimen: myeloablative n = 5 and non-myeloablative n = 11. median interval between transplantation and diagnosis of cgvhd of 11 months(5.1-40.2); and between cgvhd diagnosis and sativex 23 months (9.1-105.3), with a median of 4 prior treatment lines (2-9). at the time of beginning, the cgvhd was extensive in all patients, severe cgvhd n = 5 and moderate cghvd n = 11. all patients except one had cutaneous involvement (n = 13 with sclerodermal features). in addition, other organs were affected: digestive n = 2, pulmonary n = 7, hepatic n = 4, ocular n = 8, oral n = 9, genital n = 2 and muscular n = 7. drug was started because of pulmonary affectation in 3 patients and due to sclerodermal/muscular involvement in 13 patients. concomitant therapies during treatment were: topical cutaneous treatment n = 11, topical ocular treatment n = 10, pulmonary n = 7, sirolimus n = 8, tacrolimus n = 3, oral corticosteroids n = 9, extracorporeal photopheresis s n = 4, ruxolitinib n = 2, imatinib n = 1, mesenchymal stem cells n = 1. the mean dose were three puff/day (2) (3) (4) (5) , with good tolerance, only two discontinuations of treatment because of adverse effects. median time of treatment 156 days (45 to 561). at the time of the analysis 11 patients were still under treatment. responses mainly occurred within the first 60 days, s203 with a median time of duration of 106 days (20 in 450). responses after two months of treatment were: 6 partial organ response, 4 mixed responses, 4 unchanged and 2 organ progressions; at 120th day (14/16) only two patients maintained their responses (one pr and one mixed response). it must be pointed out that one patient who reached pr with sativex in monotherapy maintain response after 18 months of treatment. in addition, cramps were resolved in 5 patients. sativex appears to be an effective treatment option in patients with chronic gvhd, particularly in those having cramps, sclerodermal features and pulmonary affectation. as seen in multiple sclerosis context, the main issue with its use is the loss of response in the long-term follow up. the median dose is inferior to the one described in ms, leaving the question if higher doses can deepen the response. these results should be confirmed in prospective trials. disclosure of conflict of interest: none. several risk factors associated with acute and chronic graftversus-host-disease (gvhd) have been identified in multiple studies. most commonly associated factors are human leukocyte antigen (hla), mismatch between recipient and donor, as well as several other characteristics such as age, conditioning regimen and prior acute gvhd. objective: the aim of this study was to evaluate the characteristics of acute and chronic gvhd in patients who underwent an allogenic hematopoetic stem cell transplantation (hsct), identify differences in the profile risk factors for acute and chronic gvhd and their impact in post-transplant morbidity and mortality. this retrospective study included 90 mexican adult patients who received an allogenic hsct between january 2010 and march 2016, at instituto nacional de cancerologia. we analyzed 90 patients with a median age of 30 years (15-64), from which, 60% were male patients. among the participants with hematologic malignancies, 39 were previously diagnosed with acute lymphoblastic leukemia, 20 acute myeloid leukemia, 11 chronic myeloid leukemia, 8 lymphoblastic lymphoma and 3 with myelodysplastic syndrome. because bone marrow transplants are not performed at this institution, all transplants were from peripheral blood stem cell harvest. acute gvhd prophylaxis consisted in a triple immunosuppressive drug regimen for all patients. 86.7% of the patients had high risk disease prior to hsct. myeloablative conditioning represented 82% of the applied regimens, which consisted of iv busulfan in 63.3% of the cases. 44.9% of patients, were transplanted within 12 months from diagnosis. the cumulative incidence of acute gvhd at 100 days was 21.1% (19 patients). patients with acute gvhd had 42% grade a, 15% grade b and 42% grade c, according to the ibmtr grading system. 12 patients had skin involvement, with grade 1-2 acute gvhd in 83% of the cases, 4 patients developed liver involvement and 6 patients had gastrointestinal tract disease. 19% of the patients developed chronic gvhd, from which, 57% were classified as severe, 10.5% as moderate and 21.6% as mild. 36% of the patients who developed chronic gvhd had a single organ involvement, while 26.3% had 3 or more organs/sites. prior acute gvhd was associated with de development of chronic gvhd. the multivariate analysis identified hla unrelated donor as the only risk factor associated with the development of acute gvhd (hr, 5.1; 95% ci, 3.3-7.9, p = 0.043). the overall survival at 5 years was of 69% poor patients who developed acute gvhd and of 34% for those who didn´t (p = 0.065). our analysis showed that the incidence of acute and chronic gvhd at our center is lower than the reported at other centers, but we were not able to identify risk factors usually associated with the development of gvhd, perhaps due to the small population that we evaluated. graft versus host disease (gvhd) remains one of the main obstacles to broader application of allogeneic stem cell transplantation (sct). despite the routine use of prophylactic therapies, chronic gvhd (cgvhd) occurs in 10 to 80% of patients undergoing allogeneic sct. ciclosporin a (csa) remains the backbone for gvhd prophylaxis in both myeloablative (mac) and reduced intensity conditioning (ric) sct. however, in a significant proportion of patients, csa causes important side effects and needs to be discontinued. in this study we have evaluated the impact of substituting csa for mycophenolate mofetil (mmf) as immunosuppression (is), on the incidence of cgvhd. we have compared the outcome of 87 consecutive patients that underwent allogeneic sct from march 2011 to november 2015 at the bmt unit of the hammersmith hospital and received csa as part of the gvhd prophylaxis. of them, 54 patients (62%) remained on csa prophylaxis for the duration of the planned post sct immunosuppression period and 33 patients (38%) required a switch to mmf before day +100. the reason for changing the is was nephrotoxicity in the majority of cases (n = 25, 70%), neurological toxicity (n = 2, 6%), disease relapse (n = 1, 3%), intolerance (n = 2, 6%) or not determined (n = 3, 9%) . we excluded from the analysis those patients whose is was changed due to the presence of acute gvhd. both groups had similar patient and transplant characteristics (see table 1 ). [p171] however, distribution according to diagnosis showed a predominance of aml (43%) in patients that remained on csa and mds (2%) for those that switched to mmf. the mean survival rate of the entire cohort was 902.897 days (±87) . the mean survival of each group was: csa 996.86 days (±109.076) and mmf 602.474 (±94.779). this difference in survival reached statistical significance (p:0.04). we graded cgvhd using the nih scoring system as mild, moderate and severe. out the 54 patients that continued with csa, 55.6 % (n = 30) had no cgvhd; 16.7 % (n = 9) had mild cgvhd; 20.4 % (n = 11) had moderate and 7.4 % (n = 4) had severe cgvhd. in patients that switched to mmf 45.5 % (n = 15) did not develop any cgvhd; 9.1 % (n = 3) developed mild; 24.4 % (n = 8) moderate cgvhd and 21.25 % (n = 7) developed severe cgvhd. (p: 0.093). the cumulative incidence of any cgvhd at 2 years post sct was 58% for the csa/mmf group and 42% for the csa only group (p = 0.04). csa is one of the standards of care for gvhd prophylaxis in both ric and mac sct. in our cohort of patients, those who remained on csa had a better overall survival and a reduced incidence of chronic gvhd compared with those patients that stopped csa and replaced it by mmf. csa toxicity should be prevented to avoid gvhd-related complications. disclosure of conflict of interest: none. although the outcome of allogeneic stem cell transplantation (sct) from an unrelated donor (ud) has considerably improved over the recent years, graft versus host disease (gvhd) still represents a severe and potentially lethal complication. in vivo t-cell depletion with anti-thymocyte globulin (atg) has been shown to significantly decrease the risk of both acute and chronic gvhd without compromising survival, however the optimal dose has not been defined yet. aim of present retrospective study was to evaluate the impact of two different doses of rabbit atg (thymoglobulin) on gvhd incidence, infectious complications and outcome of 156 patients undergoing sct from ud. between february 2004 and september 2015, 40 patients received thymoglobulin 5 mg/kg (atg-5 group) and 116 received thymoglobulin 7 mg/kg (atg-7 group) in addition to cyclosporin and short course mtx as gvhd prophylaxis. the two groups were comparable regarding sex, age, diagnosis and disease phase at transplant, comorbidity index, stem cell source and antimicrobial prophylaxis. conditioning treatment was myeloablative in 90% of atg-5 group patients and in 78% of atg-7 group patients. donor and recipient pairs were 10/10 hla matched in 75% of the cases of the atg-5 group and in 39% of the cases of the atg-7 group (p 0.001). netrophil engraftment occurred in 150 (96%) patients at a median of 17 days post transplant (range: 11-41 days); six patients (2 in the atg-5 group and 4 in the atg-7 group) died before engraftment. overall, 48 patients (31%) developed grade ii-iv acute gvhd, without significant differences between the two groups (atg-5 32% and atg-7 30%, p 0.939). similarly, chronic gvhd was not significantly different between the two groups: moderate to severe chronic gvhd occurred in 30% of the patients in the atg-5 group and in 27% of the patients in the atg-7 group (p 0.846). univariate logistic regression analysis didn't show any significant differences between the two groups respect the incidence of bacteremia, invasive fungal infections acute and chronic gvhd. with a median follow-up of 66.6 months, 84 patients (54%) are alive, 79 in complete remission and 5 after disease relapse. transplant related mortality was superimposable in the two groups (atg-5 17% vs atg-7 20%). kaplan-meier estimates of overall survival and event free survival were 54% and 52%, respectively, without statistically significant differences between the two groups and between hla matched and hla mismatched sct. the results of our study suggest that different doses of atg tailored on hla compatibility might be effective for preventing gvhd with any detrimental effect on overall survival and incidence of infectious complications. a prospective randomized study is mandatory to confirm our preliminary results. disclosure of conflict of interest: none. c-reactive protein levels at acute gvhd diagnosis predict steroid-resistance, treatment related mortality and overall survival after allogeneic hematopoietic stem cell transplantation l minculescu 1 , ls friis 1 , bt kornblit 1 , sl petersen 1 , i schjødt 1 , ns andersen 1 and h sengeløv 1 department of hematology, rigshospitalet, copenhagen, denmark acute graft versus host disease (agvhd) remains an excessive cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (hsct). primary treatment consists of high-dose corticosteroids, but a small group of patients are steroid-resistant and their prognosis is especially poor. a predictor of patients at risk of steroid-failure would aid the decision of additional immunosuppressive treatment at an early stage. there is experimental evidence that co-existing inflammation aggravates agvhd. since c-reactive protein (crp) is a systemic inflammatory marker, we aimed to investigate whether crp levels at agvhd diagnosis could predict the risk of failing first-line therapy and developing steroid-resistance. we retrospectively studied 461 patients transplanted between 2010 and 2015, table 1. acute gvhd was diagnosed in 204 patients, 149 of whom had grade ii-iv. crp, total white blood cell-, lymphocyte-and neutrophil counts were available for all patients at the time of agvhd diagnosis. according to local protocol, patients with failed response to high-dose steroid (2 mg/kg) were treated with the tumor necrosis factor (tnf) alpha inhibitor infliximab and categorized as steroid-resistant. of 149 grade ii-iv agvhd patients 28 (19%) developed steroid resistant disease. crp levels at diagnosis among these where between o1 and 263 mg/l. crp levels where significantly higher in patients who developed steroid resistance compared to patients responding to high-dose corticosteroids, p = 0.001, hr 1.011 (95% ci 1.005, 1.018). this translated into significantly increased transplant-related mortality (trm) and decreased overall survival in patients with high crp levels, figure 1 . total white blood cell-, lymphocyte-and neutrophil counts were not associated with steroid resistance in agvhd patients. cxcr3 is chemokine receptor expressed on activated t lymphocytes, in particular on th1 cells, nk cells, dendritic cells, and subsets of epithelial and endothelial cells. cxcr3 ligands attract th1 cells into inflamed tissues and concomitantly block the migration of th2 cells. furthermore, inhibitory functional autoantibodies against cxcr3 occur in humans which play an important role in cxcr3-dependent immune regulation. in addition, cxcr3 regulates endothelial cell homeostasis. there are two variants of cxcr3: cxcr3-a and cxcr3-b. overexpression of cxcr3-a on endothelial cells is associated with an increase in cell survival, whereas overexpression of cxcr3-b dramatically reduced dna synthesis and up-regulated apoptotic endothelial death. here we have studied if a dysfunctional cxcr3 axis might be involved in gvhd pathogenesis and could link endothelial and t cell pathology in acute gvhd. we assessed concentrations of the cxcr ligands cxcl9, cxcl10 and cxcl11 as well as anti-cxcr3 autoantibodies in 98 patients with high grade (3) (4) acute intestinal gvhd for whom serum was available at gvhd onset. furthermore, anti-cxcr3 autoantibodies and cxcl9 levels were measured in sera stored before conditioning therapy. all variables were tested for influence on post-gvhd survival using cause-specific cox regression analysis. at gvhd onset, we observed a strong inter-correlation of cxcr3 ligands, but no correlation with anti-cxcr3 auto-antibodies. compared with pre-conditioning probes, cxcl9 levels strongly increased (median 303 to 721 pg/ml, p o0.001), whereas antithese results suggest crp levels at diagnosis as a valid predictor of developing steroid resistant disease in agvhd grade ii-iv and survival in allogeneic hematopoietic transplant recipients. [p173] s206 cxcr3 decreased (median 4.4 to 2.6 u/ml, po 0.001). anti-cxcr3 levels before conditioning and at gvhd onset correlated (coeff. 0.497, p o0.001), whereas cxcl9 levels did not. in multivariable analyses, low anti-cxcr3 and high cxcl9 measured at disease onset were strongest predictors of survival after acute gvhd. notably, high levels of the proinflammatory chemokine cxcl9 were particularly prognostic of an adverse outcome of gvhd in the presence of a high endothelial risk as assessed by the previously published easix score, while high anti-cxcr3 levels were most protective in patients with low easix score (that is, low endothelial risk). a score based on cxcl9, anti-cxcr3, and easix allowed an effective prediction of acute gvhd outcome ranging from mortality 490% (high cxcl9 + high easix) to mortality o20% (low cxcl9, low easix, high anti-cxcr3. our data suggest a strong role for the cxcr3 axis in the pathology of acute high grade gvhd. the opposing effects of cxcl9 and anti-cxcr3 indicate a functional, attenuating role for these auto-antibodies. the overall prognostic impact of the immunemodulating cxcr3 axis appears to depend on the underlying integrity of the patients' endothelial homeostasis. despite some progress in acute lymphoblastic leukemia (all) treatment including modern chemotherapy modalities, monoclonal antibodies and newer tyrosine kinase inhibitors (tki) for ph positive cases, the final success is still difficult to reach. allogeneic hematopoietic stem cells transplantation (allohsct) has remained an essential approach in attempts to cure all. tki routinely used for all ph(+) pre-and post-transplant treatment are also described as an alternative and adjunctive approach for chronic gvhd especially with fibrotic features due to their antifibrotic activity targeting the platelet-derived growth factor receptor (pdgfr) pathways. in this study we have tried to estimate the potential influence of pretransplant tki treatment on gvhd occurrence comparing all ph(+) and all ph(− ) cases treated with allohsct. a cohort of 119 all patients consisted of 93 all ph( − ) and 26 all ph(+) cases treated with allohsct was retrospectively analyzed. all patients were transplanted from sibling or unrelated donor (no haploidentical procedures were included). all ph(+) patients achieved pretransplant treatment with imatinib and chemotherapy, and ph( − ) patients with chemotherapy alone. the median age in ph( − ) and ph(+) group was 28 vs 35 (p = 0.04), the percentage of hla mismatched transplantations -4,.3 vs 19.2 (p = 0.00004), the percentage of acute gvhd cases-48.4 vs 76.9 (p = 0.01) and extensive chronic gvhd cases-80.5 vs 50.0, respectively. there were no significant difference between groups in patients sex (f/m-41/52 vs 14/12 respectively), ric/mac conditioning, unrelated/sibling donor, donors age, bm/pbpc transplantation, number of cd34 cells and chronic gvhd incidence. all patients received cyclosporine-and methotrexate-based gvhd prophylaxis. gvhd occurrence was analyzed in subgroups as previously described: all ph( − ) and all ph(+). as mentioned above the incidence of acute gvhd was higher in ph(+) group (higher number of hla mismatched transplantations in this group) but the incidence of extensive chronic gvhd was higher in ph (-) group. cox proportional hazard model analysis revealed death risk caused by gvhd higher in ph negative group (hazard ratio = 2.3; ci 95% = 1.02-5.18; p = 0.04). the analysis of competing events was performed to estimate the probability of death caused by gvhd vs other complications (transplant related mortality, infections and relapse). the impact of conditioning was not significant on gvhd related deaths vs other complications (p = 0.234 vs 0.009, respectivelyfigure 1 ). the same results were achieved with donor cmv status (p = 0.09 vs 0.04figure 2 ). we have not found any significant difference either in gvhd or other complications related deaths taking into account patient s sex/age, donor sex/age, patients cmv status, number of cd34 cells transplanted. on the other hand, the influence of agvhd and chgvhd on deaths related to other complications was not significant (p = 0.242 vs 0.147). cumulative probability of overall survival was higher in ph(+) group but the difference was not significant. the impact of pretransplant treatment with imatinib on gvhd occurrence has not been estimated so far. we are aware of our results to be preliminary and variety of data is still to be evaluated. however our results, if confirmed, may suggest the influence of imatinib on decreasing the extensiveness of chronic gvhd. disclosure of conflict of interest: none. seta-tsukinowa, otsu, 520-2192, japan; 3 department of pathology and laboratory medicine, emory university hospital, atlanta, ga, usa; 4 department of biostatistics and bioinformatics, rollins school of public health, emory university, atlanta, ga, usa; 5 pathology and pediatrics, emory university school of medicine, atlanta, ga, usa, aflac cancer center and blood disorders service, children's healthcare of atlanta, atlanta, ga, usa and 6 bloodworks northwest research institute, seattle, wa, usa more than 90% of allogeneic hematopoietic stem cell transplant (allo-hsct) patients receive red blood cell (rbc) and platelet (plt) transfusions in the peritransplant period. preclinical models indicate that rbc and plt transfusions trigger inflammation, raising the question of whether such transfusions are associated with development of severe acute graft-versus-host disease (grade iii/iv agvhd) and mortality in allo-hsct recipients. we conducted a retrospective analysis of rbc and plt transfusions, agvhd incidence, and mortality among 322 consecutive adult patients receiving non-t celldepleted allogeneic bone marrow (11%) or g-csf-mobilized blood stem cell grafts (89%). common underlying diseases were acute myeloblastic leukemia (41%), myelodysplastic syndrome (14%), and acute lymphoblastic leukemia (12%) . underlying disease risk was ranked as low (41%), intermediate (26%) or high (32%). allografts were obtained from 10/10 hlamatched sibling donors (35%), unrelated donors (43%), or from donors mismatched at 1-2 hla alleles (22%). graft sources were bone marrow (11%) or mobilized pbsc (89%). the cumulative incidences of grade iii-iv agvhd and mortality prior to day 150 without developing grade iii/iv agvhd were estimated using the cumulative incidence function and a cox proportional hazards regression model. covariates included in multivariable analysis was limited to baseline covariates associated with grade iii/iv agvhd at the p median number of rbc or plt transfusions ( figure 1 ). univariate analysis showed a lower hematocrit on admission (median of 5 rbc units transfused (p = 0.001) were significantly associated with the risk of developing grade iii/iv agvhd, while a longer time to neutrophil engraftment was inversely associated with grade iii/iv agvhd ( ⩾ median of 15 days, hr 0.58, p = 0.03). multivariate cox regression analysis showed only larger numbers of rbc units transfused and hla mismatch independently associated with severe agvhd (p = 0.02 and p = 0.008, respectively), while underlying disease risk and larger numbers of transfused rbc units were independently associated with overall survival in a multivariate analysis that excluded agvhd grade. overall mortality rate was lowest for the group with fewer rbc and plt transfusions (43%), and greatest for the group with more rbc and plt transfusions (67%). groups that received more rbc units had higher rates of mortality due to gvhd, while patients who received more plt transfusions and fewer rbc transfusions had greater mortality from relapse ( figure 2 ). these data support the hypothesis that peritransplant rbc transfusions are associated with the risk of developing severe agvhd and worse overall survival following allo-hsct. prospective studies are warranted to whether rbc transfusions promote t-cell activation and inflammation in allo-hsct recipients, leading to increased severe agvhd. disclosure of conflict of interest: none. early high umbilical cord blood cd3 chimerism associated with acute gvhd at time of onset in haplo-cord transplantation h choe 1 , j hsu 1 , s mayer 1 , u gergis 1 , a phillips 1 , t shore 1 and k van besien 1 1 department of hematology/oncology, weill cornell medicine, new york, ny 10065, usa introduction: haplo-cord transplantation is a combined haploidentical and cord blood transplant that allows for more rapid engraftment by the haplo with eventual loss of the haplo graft upon engraftment of the cord. haplo-cord transplants are associated with an approximate 25-43% incidence of agvhd. reported, using chimerism assessments at approximately day 56 after transplant for aml and mds, that lower umbilical cord blood (ucb) chimerism in the cd3 or cd33 lineages were associated with increased rates of relapse. we did not find a statistically significant association between day 56 chimerism and risk for acute gvhd.(2) here we report our analysis of chimerisms at the onset of agvhd. patients and methods we retrospectively reviewed all patients who underwent haplocord sct for all hematologic malignancies between july 2012 and march 2016. ucb for haplo-cord transplants were selected based on hla-typing and cell count. grafts were matched for at least 4 of 8 hla loci by the standard criteria and contained a minimum cell count of 1 ×10 7 nucleated cells per kilogram (kg) of the recipient's body weight before freezing. the haploidentical donor was a relative in the large majority of cases. we identified 90 patients evaluable for agvhd (onset before day 100) without preceding relapse or early death. of the total 90 patients, 31 patients were diagnosed with agvhd of any stage and grade. fractionated chimerisms including cd3 and cd33 components were routinely sent to evaluate for engraftment of the recipient vs haplo vs ucb. chimerism data was collected for both agvhd and no agvhd patients. the two-sided student's t-test was used to compare the agvhd cohort to the no agvhd cohort. chimerisms collected on patients with agvhd were within median ± 4 days of onset of agvhd. the median time to onset of agvhd was 47 days (range: 15-99 days). the median post-transplant chimerism recorded for comparison with the no agvhd patients was 57 days. the agvhd cohort had significantly lower cd3 recipient (p = 0.005) and higher cd3 ucb engraftment (p = 0.006). all other fractions, including the cd33 chimerisms, were not significantly different between the two cohorts. the agvhd vs no agvhd cohorts were further compared by degree of hla mismatch (4-6 out of 8 vs 7-8 out of 8). the frequency of agvhd was similar in the 4-6 out of 8 (23/62, 39%) and the 7-8 out of 8 (8/28, 28%) groups. within these subgroups, cd3 ucb chimerism was higher for those with agvhd (p = 0.03 and p = 0.03, respectively). conclusion the onset of agvhd in haplo-cord transplantation is associated with a significantly higher cd3 ucb chimerism and lower cd3 recipient chimerism. higher ucb chimerism may indicate that full ucb chimerism poses a higher risk of agvhd development. vice-versa persistent recipient chimerism may protect from acute gvhd. il-6 is a pleiotropic cytokine with both pro-and antiinflammatory properties (scheller 2014). the proinflammatory properties are mediated through trans-signaling that depends on the soluble il-6 receptor. il-6 trans-signaling is involved in several autoimmune diseases and in regulation of tissue regeneration of the gi-tract. specific snps in the il-6 receptor have been associated with increased baseline crp levels, severity of autoimmune diseases and response to interleukin-6 inhibition in rheumatoid arthritis. so fare little is known about the role of trans-signaling in graft-versus-host-disease (gvhd). in this study we investigated how 4 specific snps in the il-6 receptor influence pretransplant levels of crp, il-6 sil-6r and the risk of grade ii-iv acute gvhd in allogeneic stem cell transplantation (asct) in patients with family donor. dna was available for 103 patients (65 male, 40 female median age 48, range: 15-70) and 101 donors, that underwent asct with a matched related donor (97 sibling) at haukeland university hospital in the period 2006-2016. the majority received conditioning with either bycy (79) or flubu (17) and only 2 patients were transplanted with tbi-based conditioning. four different snps in the il-6r gene (rs2228145, rs4845617, rs4845618, rs4845374) were chosen on the basis of (i) documented or suspected roles in autoimmune disorders; and (ii) allele frequency between 0.10-0.49 and r 2 o0.5 between the different snps. genotyping was done using kaspar assays with viia7 instrument (life technologies). the overall genotype call rate was 98%. no departures (p-values o0.01) from hardy-weinberg equilibrium were observed. pretransplant serum levels of il-6, sil-6r and were analyzed with bio-plex kits (bio-rad, hercules, usa). both serum and dna analyses were performed in duplicates. patients being homozygous for the rs4845618 minor allele had significantly higher pretransplant serum sil6r levels but lower crp levels compared with patients homozygous for the major allele. the overall incidence of agvhd requiring high-dose steroid treatment (grade ii gastrointestinal, grade iii-iv liver and skin) in the cohort was 48%. when analyzing the conventional clinical and laboratory parameters only transplantation with a non-sibling donor was associated with increased risk of agvhd (p-value o0.01 hr 3,20 confidence interval 1.42-7.17). the presence of the rs4845617 in donor or recipient was associate with a significant increase in the rate of aghvd (p-value 0.027 hr 1.93 confidence interval 1.08-3.47). the snp rs4845617 (p-value 0.04 hr 1.86, confidence interval 1.04-3.35) was also significant in an adjusted model including both donor type and rs4845617. none of the evaluated snps were associated with an increase in early or late trm and did not influence os either. this study suggests that snps in the il-6r influence pretransplant biochemical characteristics and clinical outcomes after asct. future studies investigating the effect of il-6 inhibition as gvhd prophylaxis or treatment should include analyses of il-6 receptor snps to investigate their possible influence on treatment outcomes. graft-versus-host disease (gvhd) continues to be the major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant (allo-hsct). the prophylaxis scheme varies according to the center and the country. in ours institution we use triple-prophylaxis based on cyclosporin a (cya), metrotexate (mtx), and mycophenolate mofetil (mmf). this scheme has been used for more than one decade in asian centers where it has proven adequate effective and safe to prevent gvhd. we evaluated 90 patients undergoing allogeneic hematopoietic stem cell transplantation treated at the national cancer institute from january 2010 to december 2015. the triple-prophylaxis scheme consists in cya (adjusted serum levels, mtx (5 mg/m 2 days +1, +6, +11, +18) and we evaluated different doses of mmf, one of them includes 500 mg bid × 35 days and the other has high doses (15 mg/kg bid × 180 days), as gvhd prophylaxis. the response characteristic was analyzed using the pearson test, fisher's exact test on categorical variables and student's t-test, mann-whitney u on continuous tests. kaplan-meier method was used to estimate the probabilities of os, sle with the differences compared by the log-rank test. we analyzed 90 patients with median of age of 30 years (range: 15-64), 60% male gender, all were transplant with peripheral blood progenitor cells as a source. 52.2% were acute lymphoid leukemia and 25.5% acute myeloid leukemia, 12.2% chronic myeloid leukemia, 3.3% myelodysplastic syndromes, 3.3% aplastic anemias, 2.2% non-hodgkin's lymphomas and 1.1% hodgkin's lymphomas. myeloablative conditioning was used in 82% (bucy, cfm-gat) and 18% reduce intense conditioning (flubu, flucy, flucy-gat), 94.4% related hla compatibility. mmf 500 mg twice daily (bid) for 35 days (group 1) and of mmf 15 mg/kg bid for 180 days (group 2), in the group 2 the 85.3% developed febrile neutropenia vs 64.3% in group 1(p = 0.03). the frequency of gvhd was 19.6% group 1 vs 23.5% group 2 (p = 0.6), chronic gvhd was 23.5% vs 19.6% respectively (p = 0.7). at the moment of analysis 58.9% vs 26.5% were free of disease (p = 0.01). there no difference at 5-year overall survival was 37% (group 1) vs 49% (group 2) (p = 0.85), neither free-survival disease (p = 0.85). the mmf regimen shows noninferiority scheme for gvhd. the low doses and for shorter administration did not show differences in the incidence and severity of acute or chronic gvhd, os, dfs compared to the mmf regimen at 180 days with high doses. the high doses shows higher incidence of febrile neutropenia, but there were no differences in documented infections. disclosure of conflict of interest: none. a protein-losing enteropathy can develop due to conditioning regimen related gut toxicity and can cause albumin decline during peritransplant period in allogeneic stem cell transplantation (allohct). damaged intestinal mucosal barrier results in alloactivation of donor t cells and this situation is considered a primary event in the pathogenesis of acute graft-versus-host disease (agvhd). peritransplant albumin decline, as a result of conditioning regimen related protein-losing enteropathy, may predict agvhd (1) . in this retrospective study we tested this hypothesis. we evaluated 249 patients who received allohct between 2011 and 2016. albumin decline from the day of conditioning initiation until its nadir in the first 2 weeks of post-transplant period was calculated as delta albumin. acute gvhd was proven by biopsy in all patients. chi square and mann-whitney test were used for statistical analysis. patients' characteristics were shown in table-1. acute gvhd was developed in 78 patients and severe agvhd was developed in 15 patients. delta albumin was not different between agvhd patients and no agvhd patients. delta albumin was not related with severe agvhd. delta albumin was not different between patients who received myeloablative and reduced intensity conditioning regimens. when we used a cutoff value of 0.9 gr/dl for delta albumin, we could not find a relation between delta albumin and development of both agvhd and severe agvhd. we repeated the analysis for acute myeloid leukemia (aml) and myelodysplastic syndrome (mds) patients who receive myeloablative conditioning regimen and we found the same results, there was no difference between agvhd patients and no agvhd patients in terms of delta albumin. there was a number of studies that used albumin as a predictive and prognostic marker in the setting of agvhd. but albumin may decrease in patients due to many reasons like malnutrition, proteinuria, enteropathy, liver disease or being negative acute phase reactant. because of albumin value can show variability between patients, albumin decline may be a more objective criterion. rashidi et al. showed that 0.9 gr/dl decline in albumin may be a predictor of severe agvhd in 88 patients who was diagnosed with aml and mds and received myeloablative conditioning regimen. we repeat this analysis in our mds and aml (n = 98) patients but we couldn't find this relation. when we evaluated all our 249 patients, again there was no relation between delta albumin and development of both agvhd and severe agvhd. in conclusion, our study did not support rashidi et al.'s findings. because serum albumin level shows variability due to many reasons, it is hard to use albumin as a predictor of agvhd. sclerodermatous chronic graft-versus-host disease (scl-cgvhd) in its severe manifestation affects the patient quality of life and, due to complex pathomechanism, does not respond to standard immunosuppressive therapy-calcineurin inhibitors (cni) with corticosteroid. methotrexate (mtx) and rituximab appeared to be effective in some patients but the novel strategies, including extracorporeal photopheresis (ecp), imatinib, m-tor inhibitors (for example, sirolimus) and ruxolitinib seem to become the real breakthrough. we retrospectively analysed data of 33 patients with scl-cgvhd, who underwent allogeneic hematopoietic cell transplantation (hct) between 2009-2015 in 5 transplant centres. the study group consisted of 32 patients with haematological malignancies and one with aplastic anaemia, 14 female and 19 male, with the median age 36 (18-64). donors' median age was 40, with predominance of matched sibling donors (21 donors) and even distribution of the donors' gender. in 22 patients (67%) acute gvhd (agvhd) was diagnosed with skin involvement observed in 19 ones. acute gvhd directly progressed to cgvhd in 13 cases. in 11 patients (33%) cgvhd developed ‛de novo' and in 2 cases cgvhd was induced by dli. median time from hct to cgvhd diagnosis was 8 months and to scl-cgvhd diagnosis-32 months. seven patients (21%) were scored as moderate cgvhd and 26 patients (79%) as severe cgvhd according to nih-2014 cgvhd activity classification. in 14 patients sclerotic features had superficial form and in 19 ones deep sclerosis was observed. chronic gvhd manifestation in other organs includes: mouth (94%), joints and fascia (77%), liver (74%), eyes (64%), gi tract (33%) and lungs (21%). 26 patients were treated with ecp and/or sirolimus and /or imatinib with 80% response rate (complete-cr, partial-pr or minimal-mr). median duration of ecp therapy, sirolimus and imatinib treatment was 20 months (2-40), 3 months (1-30), and 5 months (1-18), respectively. sirolimus was added more likely (9 patients) as the first in case of suboptimal response to ecp after median 15 weeks and in 4 patients was subsequently replaced by imatinib with no favourable outcome in 3 cases. in 3 patients imatinib was initially used in combination with ecp therapy, leading to pr or mr. mtx without novel therapies was effective in 4 patients with limited skin involvement, 3 patients responded to mtx plus imatinib and 1 patient to mtx plus sirolimus. two patients, after failure of other therapies, have been receiving ruxolitinib with improvement. only 3 patient (15%) were nonresponsive to ecp (progressive or stable disease), 4 patients (36%) to sirolimus and 4 patients (33%) to imatinib. toxicity incidence was equally observed in case of sirolimus and imatinib and lead to the therapy discontinuation in altogether 4 patients. infectious complications were observed in 20 patients (60%). ecp confirms to be the most effective therapeutic strategy in severe forms of scl-cgvhd with favourable safety profile. imatinib and sirolimus, targeting different fibrotic pathways, both play important role in nonresponsive patients, improving the outcome in ecp and non-ecp group. in case of limited access to ecp, mtx remains to be beneficial in combination therapy of moderate scl-cgvhd and an alternative to cni. disclosure of conflict of interest: none. post-transplant morbidity and mortality are majorly determined by gvl effect counter-balanced by gvhd. treatment with systemic steroids represents the first-line therapy for gvhd, but is associated with increased incidence of infection and relapse. ecp can reduce the extent of gvhd while preserving anti-virus/-tumor activity. to elucidate this clinical phenomenon on an immunological level, we correlated clinical data with immunological findings in 20 patients under ecp treatment. nine patients with acute gvhd (agvhd) of the gut ii-iv suffering from severe diarrhea were treated by ecp in addition to triple-drug immunosuppressive therapy. furthermore, 11 patients with chronic gvhd (cgvhd) of the skin or lung despite triple-drug received ecp treatment. patients were evaluated according to their individual response and clinical condition. phenotypical analysis of different cellular subsets of patients and healthy donors was performed by multicolor flow cytometry. functional properties of virus-specific cd8+ t and nk cells were evaluated by inf-γ-elispot and 51cr-release assay. about 20 patients were treated by ecp in this study. however, two agvhd and two cgvhd patients had to be withdrawn from ecp treatment after a few ecp cycles due to pancytopenia or poor clinical condition. for patients with agvhd 8 up to 25 ecp cycles were needed for response. all patients achieving a complete response (cr) were still alive 1 year after initiating ecp therapy. overall response, that is, cr or partial response (pr) according to nih criteria, was obtained in 5 of 7 patients with agvhd (71.4%) including cr in 3 of 7 (42.8%). out of 9 cgvhd patients 7 (77.8%) reached pr, and 2 (22.2%) remained stable under ecp treatment. after 1 year, overall survival (os) was 60% for agvhd patients responding to ecp, while only 25% for non-responders. os for cgvhd patients was 91%. during intensive ecp treatment for patients with agvhd of the gut, the average stool volume and frequency decreased and consistency changed from loose to formed stool. steroids could be tapered down to a mean of 22% of the initial dosage. cgvhd patients were stabilized under ecp treatment and steroid dosage could be reduced to a mean of 38%. clinically responding patients showed increased numbers of regulatory cells including mdscs, foxp3+cd8+ and foxp3+cd25+cd4+ tregs, as well as cd4 − cd8 − cd3+ t, vδ2+ t cells and regulatory b lymphocytes. furthermore, loss of cd62l expression on effector cells like cd4+ te, cd8+ te, nk and nkt cells was observed under ecp treatment. interestingly, ecp treatment did not dramatically influence the frequency of cd4+cd8+cd3+ t, γδ t cells and nkt cells, which possess anti-virus/-tumor function. elispot and 51cr-release assays revealed stable anti-viral activity of cd8+ t cells as well as functional cytotoxicity of nk cells. moreover, cd8+ t, cd8+ tem, cd62l+cd4+ temra, cd56 +cd3 − nk and cd56brightcd16 − nk cells could serve as reliable biomarkers for prediction of response to ecp. conclusion: ecp treatment might stabilize or even improve clinical situation of patients suffering from gvhd. in clinically responding patients an immunomodulation was observed in terms of increasing numbers of regulatory cells with loss of migratory capacity of effector cells while anti-virus/-leukemia t-cell function was preserved. disclosure of conflict of interest: none. extracorporeal photopheresis affects dendritic cells by reducing total numbers and blunting cytokine production in patients with graft versus host disease tj altmann 1,2 , m bickerton 1 , am flinn 1 , u cytlak 1 , p milne 1 , s pagan 1 , m collin 1,2 , v bigley 1,2 and ar gennery 1,2 1 institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom and 2 newcastle upon tyne hospitals nhs foundation trust, newcastle upon tyne, uk graft versus host disease (gvhd) and concomitant immunosuppression is a leading cause of morbidity and mortality post hematopoietic stem cell transplantation (hsct). the pathophysiology of gvhd is complex, involving presentation of histo-incompatible antigen by activated recipient dendritic cells (dcs), activation and proliferation of donor t cells and resultant tissue damage. extracorporeal photopheresis (ecp) is a second-line treatment for steroid refractory or dependent gvhd that facilitates the reduction of immunosuppression. ecp's mechanism of action is unclear and is likely to be multifaceted. apoptosis of lymphocytes, induction of a th2 favoured environment and increased numbers of regulatory lymphocytes have been implicated 1 . although ecp has been shown to modify the function of in vitro monocyte-derived dcs 2 , its effect on primary (non monocyte-derived) dcs has not been studied. our aim was to determine whether ecp had immediate or long-term affects on primary dc numbers or function. we enumerated monocyte and dc subsets (cdc1, cdc2 myeloid dcs and plasmacytoid dcs) in whole blood before, during and after ecp cycles, and developed a novel dc function assay, suitable for use on clinical samples. four adults with immunosuppression withdrawal gvhd and four children with acute gvhd, received ecp during the study. all received ciclosporin gvhd prophylaxis and corticosteroid treatment at onset of gvhd. children received ⩾ 1 dose of infliximab prior to starting ecp. adults received two ecp treatments (one cycle) every 2 weeks and children received two ecp treatments (one cycle) weekly. whole blood was taken before and after each cycle of ecp. trucount flow cytometry analysis of whole blood was used to enumerate mononuclear leukocytes. to assess function, peripheral blood mononuclear cells were isolated by density centrifugation and stimulated with toll-like receptor agonists. cell-specific cytokine production was then analyzed by flow cytometry. samples were compared to healthy controls and pre-ecp samples. median time to first ecp treatment from gvhd diagnosis was 33.5 days. no gvhd flares were experienced during study period. (1) adult had a cycle of treatment delayed due to intercurrent pneumonia. numbers of cdc2, pdcs and classical monocytes were significantly reduced after each ecp treatment in the adult group. dc numbers followed the same trend after ecp in the paediatric group but were not significantly different before and after ecp. this is perhaps due to initial lower dc count compared to adults in the children before the first ecp cycle. functional analysis showed a reduction in cytokine production in dcs and monocytes in both groups over the course of ecp treatment. our data support a cell-intrinsic effect of ecp on monocytes and dcs, with numerical and functional consequences. this may contribute to the beneficial effect of ecp both through reduction of inflammatory effector function and through modulation of interactions with other immune cells. correlation with immunosuppression withdrawal and clinical events during treatment may provide further insight into the role of monocytes and dcs in gvhd and ecp, which may aid in the development of novel targeted therapies for gvhd. extracorporeal photopheresis as early second-line treatment for patients with steroid-dependent or refractory acute graft-versus-host disease: a single-centre experience i sakellari 1 , i batsis 1 , e gavriilaki, a-k panteliadou, a lazaridou, k leontopoulos, d mallouri, a bouinta, v constantinou, e yannaki, c smias and a anagnostopoulos 1 1 department of hematology bmt unit, g. papanicolaou hospital, thessaloniki, greece acute graft-versus-host disease (agvhd) remains a severe complication of allogeneic haematopoietic cell transplantation (allohct). corticosteroids as the backbone of initial therapy for agvhd result in varied complete responses (25-69%). traditional secondary treatments lead to profound immunosuppression without improved survival. on the basis of our experience in chronic gvhd, we aimed to prospectively assess the role of extracorporeal photopheresis (ecp) as early secondline treatment in steroid-dependent and refractory agvhd. we enrolled consecutive patients with steroid-dependent or refractory grade (gr) ii-iv agvhd post allohct from january 2013 to august 2016. all patients with unrelated or haploidentical donors received thymoglobulin (atg) 5 mg/kg as prophylaxis. post-transplant gvhd prophylaxis included cyclosporine-methotrexate in myeloablative and cyclosporine-mycophenolate mofetil in reduced toxicity or intensity regimens. ecp was commenced after assessment of response to 5 days of steroid treatment according to our protocol: two sessions per week for 1 month, one session per 2 weeks for 3 months, evaluation of response and one session per month for 6 months. we studied 20 patients, aged 35 (18-65), post allohct with myeloablative (14), reduced toxicity (4) and intensity (4) conditioning, from sibling (3), matched (8) or one locus mismatched (8) volunteer unrelated and haploidentical (1) donors. disease risk index was high (10), intermediate (9) and low (1). acute gvhd was observed at day +17 (8-50) in 15 patients, late onset at + 130 (110-160) in 4 patients and induced at +38 post donor lymphocyte infusion in a relapsed aml patient. skin, intestine and liver involvement was evident in 6 patients, skin and intestine in 10 and skin only in 4 patients. nine patients (2 with grii, 7 with griii agvhd) were steroid-dependent and 11 (8 with griii, 3 with griv) steroidrefractory. atg was administered simultaneously with ecp initiation in six refractory patients that further developed ebv reactivation (p = 0.032) treated pre-emptively with rituximab. ecp was commenced at day +51 for 15 (4-20) sessions. the majority of patients (16/20) presented partial (6), very good (9) or complete (1) response to ecp. with 8.3 (1.7-51) months of follow-up, immunosuppression was reduced in 10/20 and ceased in 1 patient. clinically significant bacterial infections were found in 17 patients, fungal in 2, cmv and ebv reactivation in 14 and 13, respectively, and other viral in 5 patients. cumulative incidence (ci) of chronic gvhd was 77.4 at 1 year. one-year ci of agvhd-related mortality was 21%. one-year overall survival (os) was 53% and significantly increased in steroid-dependent vs refractory patients (80% vs 36%, p = 0.039). reduction of immunosuppression (p = 0.008) and response to ecp (p = 0.034) were associated with improved os, irrespectively of other factors. our data indicate that ecp should be considered early in the course of steroiddependent or refractory agvhd, before irreversible end organ damage has been established. optimal timing of intervention, frequency, duration and tapering schedule of ecp remain important unanswered questions. [p186] disclosure of conflict of interest: none. extracorporeal photopheresis for treatment of chronic graft versus host disease m lanska, a zavrelova, j radocha and p zak faculty of medicine, 4th department of internal medicine-hematology, university hospital hradec kralove, czech republic allogeneic stem cell transplantation represents a curative approach to many hematologic disorders. graft versus host disease (gvhd) is a1 complication with significant morbidity, mortality and decreased quality of life. extracorporeal photopheresis (ecp) represents possible treatment approach. mononuclear cells (mnc) collected by apheresis are photosensibilized with 8-methoxypsoralenem ex vivo, irradiated with uva and transfused back to the patient. aim of the study: evaluation of patients treated with ecp for gvhd at our center. thirteen patients (8 females and 5 males, median age 44 years) were treated with ecp. about 12 patients (pts) had matched sibling donor and 1 patient had unrelated donor. about 10 pts had sclerodermic form of gvhd, 2 had concomitant pulmonary gvhd, 2 had pulmonary gvhd alone. one pts had mild, seven moderate and five severe gvhd according to nih. about 11 patients were treated with steroids. mnc separation was prepared on cobe spectra and spectra optia (terumo bct, usa). 8-methoxypsoralen was added, irradiation was done on macogenic g2 (macopharma, mouvaux, france). about 696 procedures in 13 pts were performed (median 41 procedures, 12-120 procedures). the schema was as follows: ecp on 2 consecutive days every 2-3 weeks first 3 months with subsequent increase in interval. median follow up was 35 months. in sclerosing form two pts reached cr, six pts pr, one is stable and one patients progressed. in pulmonary gvhd one reached cr, two partial improvement, one is stable. seven pts are still alive, six died (two due to relapse, one secondary malignancy and three infections). it was possible to withdraw steroids in 10 pts. adverse events were clinically negligible. ecp is an effective treatment for chronic gvhd. especially sclerodermic form responds to ecp very well. it is safe and well tolerated procedure with minimal toxicity. supported by prvouk p-37. disclosure of conflict of interest: none. allogeneic haematopoietic stem cell transplantation (hsct) is a potentially curative treatment option for children with a variety of haematological, oncological and immunological diseases. graft versus host disease (gvhd) represents a major cause of post-transplantation mortality and morbidity affecting multiple organs including skin, gut, liver and lungs. gvhd is considered a succession of inflammation and donor t-cell activation initiated by translocation of gastro-intestinal microorganisms through impaired mucosal barriers after chemotherapeutic conditioning and/or infection. diagnosis of gvhd is based on clinical symptoms and histological findings, necessitating invasive and potentially harmful procedures including endoscopy and biopsy. as yet, no non-invasive markers are available for diagnosis or treatment monitoring in children with gvhd. faecal calprotectin (fc) reflects intestinal mucosal inflammation of any origin. in the setting of allogeneic hsct in adults, fc has shown to be a marker for acute (steroid-resistant) gvhd. we aimed to evaluate the feasibility of prospective fc measurement as a non-invasive marker for diagnosis and treatment in children with gvhd. a prospective, observational, single centre study was started in july 2015. by december 2016, 21 paediatric allogeneic hsct patients (age 0-17 years) were included after informed consent. faecal samples were collected from 2 weeks before to 6 months after hsct. fc levels were measured by elia, according to manufacturer's instructions. clinical symptoms were prospectively evaluated and managed according to local guidelines. if gvhd was suspected on clinical grounds, histological confirmation was obtained. first-line therapy for gvhd consisted of corticosteroids. in case of steroid-resistant disease, more advanced immune modulation was applied. a total of five patients developed histologically confirmed gvhd: acute gvhd of skin and gut (n = 2, one patient with steroidresistant disease), acute gvhd of skin only (n = 1); chronic gvhd of lung only (n = 1) and acute gvhd of skin followed by chronic oromucosal gvhd (n = 1). without exception and regardless of gut involvement, gvhd occurrence was accompanied by rises in fc levels to values 4100 μg/g (range: 108-1600 μg/g). fc levels correlated with clinical and histological grading. moreover, adequate response to therapy was consistently reflected by return of fc levels to values o100 μg/g. sensitivity of fc levels to diagnose gvhd was poor due to increased fc levels in patients with posttransplant complications other than gvhd such as viral reactivation and pulmonary or gastro-intestinal infections. fc levels reflect gvhd occurrence and correlate with clinical and histological grading in paediatric allogeneic hsct patients. fc levels increase in case of gvhd regardless of gut involvement, supporting a central role for (subclinical) intestinal inflammation in gvhd initiation. although, in this interim analysis, fc lacks sensitivity to diagnose gvhd, fc may serve as a noninvasive marker for monitoring therapy response and, thereby, reduce the need for repeated invasive procedures including endoscopy and biopsy. acute graft-versus-host disease (agvhd) is a major complication of allogeneic hematopoietic cell transplantation (hct), and glucocorticoids are typically used as first-line treatment. the aim of our study was to evaluate the effect of first-line ecp +/ − steroid therapy in order to reduce the incidence of infections and toxicity. from december 2010 to january 2016, 48 of 180 pts (27%), were diagnosed with agvhd grade ⩾ 2 following allosct. 40 pts were treated with ecp +/ − steroid as first-line therapy. about 8 (20%) pts were treated with ecp only and 32 (80%) with ecp + steroid 1-2 mg/kg/day. we compare this cohort with an historical group of patients, transplanted between 2001 and 2011, who were treated with steroid only for grade 2-4 agvhd (n = 23 out of 130). the two cohorts were well balanced in terms of median age (p = 0.4), disease type (p = 0.9), disease status (p = 0.09), graft source (p = 0.2), conditioning regimen (p = 0.1) and hct-ci (p = 0.3). there were more female patients (p = 0.03) and more haploidentical transplant (haplo-sct) (p = 0.0005) in the cohort treated with ecp+/ − steroid. ecp was performed using the offline technique, and was started as soon as possible with a treatment schedule consisting of four rounds of two procedures per week, three rounds of two procedures every other week and finally two procedures every month. steroid was tapered as soon as possible after starting ecp. the clinical response was evaluated at day +28. median follow-up for alive patients was 28 months for ecp group and 97 months for control group. there was no difference in terms of median time of agvhd onset (38 vs 39 days) and number of pts with grade 2 or 3-4 agvhd ( figure 1 ). ecp was started after a median of 4 (0-30) days from agvhd diagnosis. every patient underwent a median of 19 (2-83) ecp procedures, during a median time of 6 months. on day 28 after starting agvhd treatment with ecp+/ − steroid, 24 pts (70%) achieved cr or pr, 10 pts did not respond and 2 experienced agvhd relapse to front-line therapy. one year cumulative incidence (ci) of agvhd relapsed/refractory was 28.5% for ecp+/ − steroid. these percentages were not different from the cohort receiving steroid alone. ci of moderate-severe cgvhd was lower in the ecp group, probably due to the higher frequency of haplo-sct with pt-cy in the ecp group. about 100 days after agvhd onset, ci of infection (49% vs 74%), especially cmv reactivation (34% vs 67%), was lower in the ecp group, but was not statistical significant. ecp allowed a faster taper of steroid: 17 (3-98) vs 75 days (23-338) (p o0.0001). overall survival, progression-free survival, non-relapse mortality and ci of relapse rates did not differ in the two groups. in multivariate analysis, visceral involvement by agvhd was associated with an increased risk of failure to front-line therapy (hr: 5.5; range: 0.7-41; p = 0.09). this observational study suggests that the overall response rate of ecp +/ − steroids is similar to steroid alone for front-line treatment of grade 2-4 agvhd, but is potentially associated with lower incidence of infection, and in particular of cmv reactivation. a prospective phase 2 clinical trial is warranted to address whether augmentation with ecp may be beneficial for agvhd frontline treatment. [p189] a 4-year-old girl with neuroblastoma received autologous stem cell transplant (asct), followed by antibiotic prophylaxis and filgrastim. her transplant preparative regimen consisted of busulfan and melphalan. engraftment of neutrophil took place on day 11 after asct. twentieth day after asct, she experienced nausea and diarrhea. there was neither skin rash nor elevation in liver enzymes. the diarrhea continued to worsen day by day and reached to a daily volume of 1500 ml/ m 2 . infectious studies for stool and blood including testing for influenza a and b, parainfluenza, adenovirus, epstein-barr virus, amebiasis, cryptosporidium parvum, cytomegalovirus, clostridium difficile, salmonella, campylobacter, yersinia and shigella were all negative. colonoscopy and endoscopy were performed by an experienced pediatric gastroenterologist and findings were suspicious for severe graft versus host disease (gvhd). colonoscopy and rectoscopy revealed severe inflammatory changes, friability and patchy dark exudates on the mucosa of rectum. endoscopy revealed erosions, ulcers in the esophagus and a pale mucosal surface with reticulated submucosal vessels accompanied with erosion and erythema in the antrum of stomach. grade 3 gvhd was confirmed by pathologic analysis that revealed diffuse crypt dropout and mucosal erosion on rectal mucosal biopsy. mucosal erosions, apoptosis of epithelial cells and small lymphocytic infiltration of the lamina propria were found on duodenal biopsy. after these results, we started methylprednisolone intravenously at a dosage of 2 mg/kg/day. on the fourth day of treatment we increased the dosage to 5 mg/kg/day and added cyclosporine to treatment. because of unresponsiveness to treatment we decided to administer thirdparty mesenchymal stem cells (msc) (1 × 10 6 cd73+/cd105+ cells per kg). these were given intravenously at day +49 asct as single infusion. the second dose was given at day +56. within 5 days after first application of mscs, the frequency of diarrhea decreased to one-third. at day +16 after second dose of mscs, the patient's stool became nearly normal. we tapered the steroids first and stopped cyclosporine at +92th days after asct. discussion and conclusion: to our knowledge, this is the second case report of spontaneous severe autologous gvhd in a child with a solid tumor malignancy. regarding the pathogenesis of autologous graft-versus-host disease, there may have multiple causes for the loss of tolerance to self because of disrupted immune system. alteration of t regulatory cells by previous chemotherapy may be key point. endogenous cells that survive conditioning and assist in post-transplant maintenance of self-tolerance may be affected. microchimerism due to maternal cells transmitted during fetal development and persisting throughout adult life has also been postulated as a cause. however it is not very clear for factors that may contribute to the pathogenesis of this rare disease. autologous gvhd has the potential to cause critical illness in the hematopoietic stem cell transplantation patient population. in patients with multiple myeloma some experts report pathologically verified gastrointestinal gvhd as high as 6%. responses to steroids are variable. however, a significant proportion improve dramatically after early therapeutic intervention. so clinicians and pathologists should be aware in suspecting and recognizing gvhd in patients with diarrhea to guide therapy as soon as possible. disclosure of conflict of interest: none. haplosct patients did not receive additional gvhd prophylaxis aside from the ex vivo t-cell depletion (tcd) with clinimacs system. of the bud bmts 36 out of 39 patients engrafted, 92% of which had gvhd. the 8% who did not have any gvhd, relapsed. eighty one percent had agvhd, of which majority (51%) were grade 2 ( table 1) three patients did not have agvhd (10/10 mud and two cord blood grafts), but developed cgvhd. one of the patients who had grade 4 agvhd died and one still has intermittent cgvhd 12 years post bmt. there were 55% who had cgvhd. currently, all of our haplosct receive a cd3/cd45ra tcd grafts (n = 14). the depletion techniques for the 14 others were either cd3tcd, cd3/ cd45ra /tcrab tcd+cd34 +/cd45ra tcd; tcrab tcd. all 28 patients who received haplosct engrafted and 57% had agvhd (56% grade 1) ( table 1 ). we noted that some of the patients presented with nonclassical agvhd signs (upper gut gvhd, oral gvhd, blood). there were no patients who presented with grade 4 agvhd. cgvhd in the cohort was 35%. of note, 35% of patients did not have any gvhd and did not receive any form of immunosuppression post bmt. only eight patients received further immunosuppression for agvhd, median duration 131 (range: 91-344) days. at the time of this report, there are four patients with agvhd still receiving immunosuppression all o100 days and all on tapering doses. haplosct using ex vivo tcd techniques has a lower risk of gvhd with comparable, if not superior outcome to bud. the degree and duration of immunosuppression is also much less. this may translate to earlier immune reconstitution and less viral reactivation. [p191] disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (allo-hsct) offers a potential cure for several hematological diseases, but it is burdened by severe life-threatening complications, being gvhd the major cause of morbility and mortality. recently, more have been understood of the physio-pathologic relationships between endothelium and graft-versus-host disease (gvhd), showing that vascular endothelium is an early phase target of gvhd. in recent years, the direct count of circulating endothelial cells (cec) has emerged as a valuable biomarker of endothelial damage in a variety of disorders. however, due to their rareness and complex phenotype, different published techniques have showed variable degrees of uncertainty, reporting a wide range of cec values in healthy subjects. by means of the commercially available rare cell isolation platform cellsearch system, for cec identification and count, we correlated cec count changes to gvhd onset and response to treatment in allo-hsct patients. cec were analysed in 90 allo-hsct patients (37 aml, 15 all, 11 hd, 4 nhl, 4 cll, 5 mds, 4 cms, 8 mm, 2 saa) at the following time points: t1 (pre-conditioning), t2 (pre-transplant), t3 (engraftment), t4 (day+28 or onset of gvhd), t5 (1 week after steroid treatment). the median cec/ml at t1 was 24 (range: 2-786), in comparison to a value of 2 (range: 1-14) in healthy controls (p 0%: or 4.2, 95% ci 1.6-10.8; p = 0.002). we confirm that cec count represent a valid biomarker to monitor endothelial damage in patients undergoing allo-hsct and can be a valuable tool in supporting the diagnostic definition of gvhd and in monitoring responsiveness to treatment. moreover, the use of the cellsearch system can be crucial in order to move routinely cec monitoring into clinical practice of allo-hsct. reference clinicaltrials.gov nct02064972. disclosure of conflict of interest: this research was conducted with the support of the investigator-initiated study program of janssen diagnostics, llc to ca. kb is employee of janssen diagnostics. results of hla mismatched unrelated donor (mmud) hematopoietic cell transplants (hct) are worse than results of fully matched hct due to higher risk of gvhd, infection and graft failure. atg during conditioning reduces incidence of gvhd but can increase risk of infection and relapse. high doses posttransplant cyclophosphamide (2 × 50 mg/kg) prevent gvhd in haploidentical hct. we initiated this approach instead of atg in hct from one alelle or antigen mismatched unrelated (7/8)mmud-hct in 2014. here we present outcome of 21 patients (cy-group) transplanted between 2014 and 2016, comparing it with outcome of 54 patients transplanted between 2010 and 2016 from 7/8 mmud with atg-f (fresenius) 40 mg/kg given during conditioning. 21 patients in cy-group (12 males, 9 females) were transplanted from mmud mismatched for hla ( a-8, b-3, c-5, dr-5). about 14 patients had aml, 2 mds, 2 cll, 1 cml, 1 all and 1 mps. med. age of patients was 43 years (25-60). about 16 patients received myeloablative (flu-175 mg/m + iv bu 12.8 mg/kg) and 5 nonmyeloablative(flu-175 mg/m+mel 100-140 mg/kg +-tt 5 mg/kg) conditioning. about 18 patients received pbpc and 3 bm as a graft. graft versus host prophylaxis consisted of cyclophosphamide (50 mg/kg aibw) on d+3 and +5, cyclosporine a from d 0 and mmf from d+1. all patients received antibacterial, antifungal, hsv and pcp prophylaxis. historical control (atg) group consisted of 54 patients (32 males, 22 females), med. age 54 y (19-65) who had mmud-hct for aml-21, mds-9, nhl-5, mf-5, all-4, cll-4, cml-2, h.d-1, saa-1, mps-1 and mm-1. there were 13 mismatches for a, 11 for b, 20 for c and 10 for dr. myeloblative conditioning was used in 32 and nonmyeloablative in 22 patients. all patients received atg-f 20 mg/kg × 2 given d-2 and-1. cyclosporin was initiated d-2 and mmf d-1. all patients received anti-infectious prophylaxis as described previously. three of 21 patients from cy group died so far. two of them due to relapse and one due to toxicity and infection during aplasia. five patients relapsed . two achieved cr after dli and one is alive in relapse expecting second hct. about 18 patients are alive, 17 of them in cr. eight patients experienced agvhd (gr.i-3, gr.ii-5,gr.iii-0, gr. iv-0) and eight developed clinically mild cgvhd). about 22 patients from atg group are alive 11-80 m (med. 32 m) posthct. about 32 patients died 1-74 m postransplant (med. 16 m) due to vod, gvhd, infections and relapse. 100-day mortality is 5% (1/21) in cy group and 19% (10/54) in atg group. one year mortality is 14% (2/14) in cy and 30% (13/44) in atg group. patients from cy group have 78% probability of os at 24 months posthct vs 47% from atg group. cyclophosphamide 2 × 50 mg/kg instead of atg fresenius(40 mg/kg) for gvhd prophylaxis reduced 100-day and 1-year mortality, and improved probability of 24 m os significantly in our cohort of patients. this approach seems to be safe and effective in 7/8 mmud-hct. disclosure of conflict of interest: none. high transplanted cd34+ cells are not associated with beneficial effect on graft-versus-host disease-free, relapse-free survival (grfs) after allogeneic hematopoietic cell transplantation y lee 1 and ih lee 2 1 department of hematology, kyungpook national university hospital and 2 inhee lee the success of allogeneic hematopoietic cell transplantation (allo-hct) is comprehensively assessed by individual comorbidity, relapse, graft-versus-host disease (gvhd) and death. besides, inconsistent results have been reported regarding the dose of cd34+ cells. in the current study we have addressed the issue of the potential effect of stem cell dose on the of gvhd-free/relapse-free (grfs) associated with cd34+ cells doses. we retrospectively reviewed the medical records of the 255 patients who received allo-hct for acute myelogenous leukemia (aml), myelodysplastic syndrome (mds) and acute lymphoblastic leukemia (all) between 1998 and 2013 in kyungpook national university hospital. the grfs included grade 3-4 acute gvhd, systemic therapy-requiring chronic gvhd, relapse or death. the patients were reclassified into two groups according to the targeted cd34+ cell doses (6 × 10 6 per kg) by knuh protocol. a lower cd34+ group (n = 165, 64.7%), patients who underwent allo-hct with cd34+ cell dose o6 × 10 6 per kg; and a higher cd34+ group (n = 90, 35.3%) patients who underwent allo-hct with cd34+ cell ⩾ 6 × 10 per kg. the median age at transplant was 38.5 years (range: 15-68 years) and male was 111 patients (44.4%). primary diseases for allo-hct were aml/mds (n = 175, 70%) and all (n = 75, 30%). one hundred forty-three patients (57.2%) were in cr1 (complete remission), 25 (10%) in further cr and 87 (33.2%) in relapsed and refractory status. one hundred seventy-one patients (68.4%) received myeloablative conditioning regimen. gvhd prophylaxis consisted of methotrexate and cyclosporine a or mtx and tacrolimus. the median dose of cd34+ cell was 3.94 × 10 6 per kg (range: 0.46-6 × 10 6 per kg) in lower cd34+ group and 7.54 × 10 6 per kg (range: 6.01-20.6 × 10 6 per kg) in higher cd34+ group. there was no significant difference in neutrophil, platelet engraftment between two groups. the incidence of chronic gvhd was more frequent in higher cd34 + group (32.9% vs 48.2%, p = 0.042). the median follow-up duration was 18.1 months, with a range of 0.2-209.7 months. the 1-year overall survival (os), relapse free survival (rfs), nonrelapse mortality (nrm) and graft-versus-host disease (gvhd)free/relapse-free survival (grfs) since hct was 55.3 ± 3.1%, 66.0 ± 3.2%, 28.2 ± 0.3% and 32.9 ± 3.1%, respectively. there was no significant difference according to the infused cd34+ cell dose (figure1). the relapse rate was not proportionally affected by the cell dose (22.4% vs 26.7%, p = 0.712). and there was no significant correlation between the number of cd3+ and cd34+ cells infused (spearman correlation coefficient: p = 0.307). in a univariate analysis, patients transplanted with the higher cd34+ cell doses and higher cd3+ cell doses had no increased grfs (p = 0.623 and p = 0.158). an independent factor associated with worse grfs was risk status at transplant (hr = 1.782, 95% ci:1.267-2.509, p = 0.001). these results suggest that careful assessing the cd3+ and cd34+ graft content and tailoring the cell dose infused may help in reducing cgvhd risk without negative impact on grfs. a large and prospective study in a homogenous population will be needed to confirm the effect of stem cell dose. disclosure of conflict of interest: none. imatinib associated with extracorporeal photopheresis can fully reverse severe sclerotic-type lesions in patients with chronic graft-versus-host disease: the lille university hospital experience l magro 1 , j gauthier, b catteau 1 , l mannone 2 , a lionet 1 , v coiteux and i yakoub-agha 1 1 lille university hospital and 2 nice university hospital severe sclerotic-type chronic graft-versus-host disease (cgvhd) is difficult to reverse and can dramatically alter the quality of life of patients after allogeneic hematopoietic cell transplantation (allo-hct). imatinib or extracorporeal photopheresis (ecp) used separately yield sustained responses in s218 only about 30% of patients with steroid-refractory cgvhd. given their respective modest efficacy we hypothesized that the combination of imatinib with ecp could lead to higher response rates. we are reporting here on seven patients with severe steroid-refractory sclerotic-type cgvhd treated at our institution using this combination. we retrospectively analysed all patients treated at our institution (n = 7) with the combination of imatinib with ecp for severe steroidrefractory scgvhd. imatinib was started at 200 mg/day and increased to 400 mg/day if well tolerated. the cellex closed system was used for ecp. ecp was initiated twice weekly during 4 weeks. after this « induction » period, ecp sessions were scheduled less frequently according to the response to treatment. additional immunosuppressants were tapered gradually in responding patients. initial grading and response evaluation was determined according to the nih 2014 criteria. steroid-refractoriness was defined as progression of gvhd on high-dose steroids (⩾1 mg/kg) or progression during corticosteroid tapering. patient characteristics are displayed in table 1 . patients received an allo-hct between may 2004 and april 2011. median age at allo-hct was 47 (range: 23-57). a variety of myeloablative (n = 2) and non-myeloablative conditioning regimens were used (n = 5). antithymocyte globulin was used before allo-hct in one patient. gvhd prophylaxis consisted of ciclosporine and methotrexate in six patients. one patient received tacrolimus and methotrexate. five patients had prior history of acute gvhd. nih global severity grade was severe in all patients (n = 7) due to severe sclerotic features. the median number of previous therapies was 3 (range: 2-5). all patients were steroid-refractory. after a median follow-up of 54 months (range: 13-76 months) the overall response rate was 100%. the complete response rate was 57%. median time on ecp associated with imatinib was 36 months (range: 4-60 months). median time to best response was 7 months (range: 3-22 months). corticosteroids could be discontinued in all patients after a median time of 8 months (range: 6-44 months). patients #1, #5 and #6 received maintenance therapy with ecp upon discontinuation of imatinib. in four patients, both ecp and imatinib led to complete response and could be discontinued after 38, 74, 4 and 53 months for patients #3, #4, #5 and #7, respectively. patient #4 and #6 passed away after due to a myocardial infarction and the development of a solid tumour, respectively. patient #4 was off therapy while patient #6 remained on maintenance with ecp. both remained in complete response. patient #2 remained in response during 25 months before progression of cgvhd while on imatinib and ecp. none of our patients experienced adverse events related to either imatinib or ecp. despite the limited number of patients in this report, we observed that the combination of imatinib and ecp can lead to complete and sustained reversal of severe steroid-refractory sclerotic-type cgvhd. these encouraging results should be confirmed in a larger cohort. disclosure of conflict of interest: lm: therakos (honorarium). allogeneic hematopoietic cell transplantation (allo-hsct) is an established treatment modality that is potentially curative for many patients (pts) with acute myeloid leukemia (aml). aml itself is the most common indication for pts undergoing hsct nowadays. for pts with high-risk disease, allo-hsct is, perhaps, the most effective curative treatment and is considered the standard post-remission therapy in first complete remission (first cr). this is a retrospective study to analyze those variables which were associated with patients' overall survival (os) after allo-hsct. the study population consisted of 31 pts who were diagnosed of aml from january 2010 to july 2016 at the hospital universitario central asturias, and submitted to allo-hsct in first cr. risk status based on validated cytogenetics and molecular abnormalities following recommendations of european leukemianet was performed. sixteen (51.6%) were male. median age was 42 years old (range: 1-64). clinical characteristics at transplantation are represented in table 1 . median follow-up was 27 months (5-75). considering the donor type, os at 1 year was higher in pts receiving sd (91.8%) compared to 66% in those who received urd (p = 0.012). regarding graft source, os at 1 year was 88.9% who received pbsc compared to 48% in pts receiving bmsc (p = 0.012). gender also showed significant association with os, which was higher among men, os at 1 year was 100%, compared to 47.4% for women) (p = 0.002). the presence of minimal residual disease (mrd) detected using multiparametric flow cytometry was performed prospectively after induction and consolidation, and before transplantation. thirteen pts had negative mrd before transplantation. median os was greater in pts with negative mrd before transplantation compared to the group with positive mrd (67 vs 27 months, respectively) (p = 0.24). this difference did not reach statistical significance probably because the low number of the sample. thirteen pts developed agvhd. only 4 (28.6%) pts receiving sd developed agvhd compared to 8 (50%) pts among those who had an urd; however this association was not statistically significant (p = 0.23). also, we observed higher incidence of agvhd in bmsc group (6 pts; 60%) whereas only 7 (36.8%) in pbsc group developed agvhd. this tender did not reach significant association (p = 0.2). one year os was 59.8% in pts who developed agvhd and 87.1% who did not (p = 0.05). all factors that had a significant influence on pts survival were included in a multivariate analysis (cox regression model): graft source, donor type, pts gender and agvhd development. developing agvhd kept an independent association with mortality (or 6.12, 95% ci 1.39-27.29, po0.001) and male gender also persisted as an independent protective factor (or 0.12, 95% ci 0.02-0.06, p = 0.003). in our series, agvhd has shown a significant and independent association with os over other parameters such as graft source, type of donor or mrd before transplantation. identifying reliable predictors for agvhd development, controlling well known risk factors for this disease, as well as improving management of immunosupressors should still be the key to potentiate longer os in our patients. larger studies are needed to confirm our results. acute and chronic graft-versus-host diseases (gvhd) are associated with increased morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-hsct). older patients undergoing allo-hsct may experience a high degree of transplant-related complications and this concern has historically limited the use of allo-hsct for some older patients. in many studies, age has been shown to be a negative prognostic factor for survival and associated with higher transplant-related mortality (trm). however, in others, age was not shown to be a significant factor if appropriate adjustments for other comorbidities are incorporated in the analyses. there are very few studies that evaluated the relationship between patient's age, the presence of gvhd and long-term transplantation outcome. the aim of this study is to evaluate the impact of age in patients who develop acute and/or chronic gvhd after allo-hsct for hematological malignancies on the trm incidence. we included in the study 595 patients with hematological malignancies who received allo-hsct and were followed in our center between january 2008 and january 2016. for the purpose of this study, only patients who developed grade ii-iv acute gvhd and/or limited or extensive chronic gvhd where considered for analysis (n = 306). patients were split into three homogeneous groups according to age at transplantation taking into consideration the underlying disease, type of conditioning and disease response at transplantation. group 1 (younger) included patients aged o40 years (n = 103), group 2 (intermediate) included patients aged between 40 and 55 years (n = 102) and group 3 (older) included patients older than 55 years (n = 101). gvhd evolution over time was followed as well as the cumulative incidence of trm was calculated in case of acute or chronic gvhd in each group. thirty seven percent of grade ii gvhd occurred in the younger group (n = 29), 32% (n = 25) in the intermediate group and 31% (n = 24) in the older group; majority (69%) resolved in the younger group as well as in 24 and 46% in the latter two groups, respectively, while trm rates at 1 year were 10%, 30% and 30%, respectively, sdhr = 4.8, p = 0.01. among patients who had acute gvhd grade ii, 51, 36 and 59% in the three respective groups developed chronic gvhd later. grade iii-iv gvhd occurred in 21% (n = 20) in the younger group, 38% (n = 38) in the intermediate group and 38% (n = 35) in the older group; with a respective resolution in 20%, 26% and 17% of patients and were associated with comparable trm rates at 1 year of 39%, 40% and 47%, respectively, p = 0.5. among patients who had acute gvhd grade iii-iv, 40, 37 and 38% in the three respective groups developed chronic gvhd later. de novo chronic gvhd was observed with a higher rate in the intermediate and in the older group (table) while patients with extensive chronic gvhd older than 55 years had significantly higher trm at 2 years (47%) compared to 32% in those younger than 55 years, sdhr = 1.9, p = 0.04. patients who develop acute gvhd grade iii-iv could incur over 40% of trm at 1 year independently of age. resolution of acute gvhd grade ii was significantly better in younger patients while older patients with grade ii acute gvhd or with extensive gvhd had higher mortality compared to younger ones. in addition to an adapted prophylaxis, a better preemptive gvhd strategy should be warranted in older patients. [p197] disclosure of conflict of interest: none. in vivo effects of nilotinib on lymphocyte subpopulation and function following allogeneic stem cell transplantation e marinelli busilacchi 1 , a costantini 2 , j olivieri 1 , n viola 2 , s coluzzi 3 , e pirro 4 , g mancini 5 chronic graft versus host disease (cgvhd) is a major complication of allogeneic stem cell transplantation and is characterized by frequent multiorgan involvement resembling autoimmune diseases; its pathogenesis is still incompletely defined and a standard treatment is lacking. donor-derived cd4+ and cd8+ t lymphocytes have been considered the main effector cells mediating cgvhd pathogenesis; however, recent studies suggest that b cells might also play an important role. in vitro data indicate that tyrosine kinase inhibitors (tkis) such as imatinib and nilotinib affects both innate and adaptive immune response by interacting with different cell populations (t cells, b cells, dendritic cells, mast cells and macrophages). we sought to evaluate the impact of different doses of nilotinib on the distribution and function of lymphocyte subpopulations. we analyzed 44 samples obtained from 15 patient with steroiddependent/refractory cgvhd enrolled in a phase 1-2 study with nilotinib in steroid-refractory cgvhd (nct01810718): triplets of patient were treated with escalating doses starting from 200 mg/die (5), 300 mg/die (5), up to 400 mg/die (5) . blood and plasma were collected at baseline and at day 90 and 180 of therapy. trough plasma nilotinib concentrations had been previously determined by hplc (abstract c039, haematologica: evaluation of nilotinib safety in patients with steroid-refractory chronic graft-versus-host disease: a phase i-ii gitmo study). peripheral blood mononuclear cells were isolated by density gradient centrifugation using ficoll biocoll. six color flow cytometry analysis (facs canto ii) was performed using conjugated antibodies (anti-cd3, cd4, cd25, cd16, cd56, cd19). inflammatory cytokine analysis was performed on plasma samples according to the instruction of bioplex pro human cytokine 17plex assay (bio-rad). statistical analysis was performed by 2-tailed student's t-test; differences were considered statistically significant for po0.05. flow cytometry analysis showed that nilotinib did not exert any significant impact neither on the proportion of t lymphocytes subpopulation (cd3+cd4+ t helper, cd3+cd4+cd25+ t regulatory, cd3 +cd4 − t cytotoxic), nor on b lymphocytes and nk cells. on the contrary, a statistically significant and dose-independent decrease of pro-inflammatory and th-17 cytokine production was observed ( figure 1 ): reduction of il2 (po0.02), il10 (po0.05) and ifnγ (po0.02) were already significant after 90 days; decreases of il17 (po0.05) and tnfα (po0.02) become significant after 180 days. interestingly, after 180 days of therapy, among the 21 patients enrolled (according to the itt criteria) ten patients showed cgvhd improvement and the other five remained stable. this study shows that therapeutic doses of nilotinib can reduce plasma levels of inflammatory cytokines without affecting the proportions of lymphocyte subpopulations. these findings correlate with clinical response and suggest that besides the previously demonstrated anti-fibrotic effects, nilotinib has also potent anti-inflammatory and immune regulatory properties, supporting its role in patients with cgvhd. disclosure of conflict of interest: none. [p198] previously published p200 infectious gastro-enteritis after allogeneic hematopoietic transplantation after reduced intensity conditioning (allo-ric): incidence and possible role in gastro-intestinal acute gvhd i garcía-cadenas 1 , r martino 1 , a esquirol 1 , a bosch 1 , n rabella 2 , s saavedra 1 , c muñoz 2 , j briones 1 , s brunet 1 and j sierra 1 1 hematology and microbiology departments and 2 hospital de la santa creu i sant pau, autonomous university of barcelona, spain enterotoxigenic c. difficile-associated associated disease or infection (cdi) is a common cause of diarrhea after hematopoietic stem cell transplantation (sct). recent studies have suggested the relationship of cdi with gastro-intestinal (gi) graft-versus-host disease (gvhd). the possible role of other types of infectious gastro-enterocolitis (g-ec) in gvhd development has not been studied. as a prior investigation to a national prospective observational study on this issue, we conducted a single-center retrospective analysis including all adult patients who received an allo-ric sct between january 2010 and march 2016. the aim was describing the cause(s) (if known), timing and outcomes of recipients with possible g-ec (defined as new onset acute diarrhea grade ⩾ 2) in the first year after sct. of the 123 patients studied (median age: 54 years, 62% male, 49% aml or mds as underlying disease), 97 (79%) had a total of 148 episodes of acute diarrhea, with 35 (28%) developing more than one event. these acute diarrheas occurred at a median of 39 days (range: 1-363) after sct. overall, a g-ec causing pathogen was identified in 33 of 148 stool specimens (22%) and included: cdi (7), c. jejuni (7), rotavirus (7), adenovirus (2), norovirus (2), b. hominis (3), s. stercoralis, g. lamblia, a. caviae, salmonella enterica and cryptococcus (one in each case). most postransplant diarrheas (68/148; 46%) occurred during the 4 weeks after infusion and were attributable to mucosal damage caused by the ric (negative microbial screening and no evidence of gvhd).the rate of infectious g-ec among the diarrheas occurring after day +30 was 41% (33/80). the overall incidences of enteric infection were 12.7% (95% ci: 6.5-18.9) and 17.6% (95% ci:10.4-24.8) at +6 and +12 months after sct, respectively. all the infected patients had mild to moderate disease, and no deaths were attributable to this complication. there were no differences in 2 year-os and nrm between the infected and uninfected patients (81% vs 73%, p = 0.6 and 16% vs 19%, p = 0.7, respectively). in univariate analysis age o50 years, prior sct, donor type, atg administration and prior grade 2-4 agvhd were associated with development of infectious gastro-enteritis. in multivariate analysis, unrelated donor and grade 2-4 agvhd were the only factors significantly associated with gastrointestinal infection (hr 2.7; 95% ci: 1.1-6.5, p = 0.02 and hr 3.6, 95% ci 1.5-8.5, p = 0.004; respectively). acute gvhd occurred in 46% of patients (n = 56), with a median onset of 54 days (range: 4-231). the cumulative incidences of 2-4 acute gvhd at 100 days and 6 months post-sct were 21% (95% ci: 15.3-32%) and 32.6% (95% ci: 23.4-42%), respectively, and there was a trend toward a higher risk of 2-4 gvhd in the group of patients with an enteric pathogen (48.2% vs 27% at 1 year, p = 0.06). more importantly, an enteric infection occurred just before the onset or aggravation of gvhd in 12/33 infected patients in our study (36%) at a median interval of 8 days after the infection (range: 0-24). in summary, our results confirm that enteric infections are a common complication after allo-ric, representing at least 20% of the episodes of acute diarrhea during the first year post-sct. a possible interplay between infectious g-ec and gvhd was observed in this study. disclosure of conflict of interest: none. long-term efficacy of extracorporeal photopheresis in chronic graft versus host disease m nygaard 1 , t karlsmark 1 , n smedegaard andersen 2 , i schjødt 2 , s lykke petersen 2 , l smidstrup friis 2 , b kornblit 2 and h sengeløv 2 1 department of dermatology, bispebjerg hospital, copenhagen, denmark and 2 department of hematology, rigshospitalet, copenhagen, denmark chronic graft versus host disease (cgvhd) activity is known to fluctuate over time, so we evaluated cgvhd continuously throughout the extracorporeal photopheresis (ecp) treatment course and after stopping ecp. patients with at least 1 year follow-up, who were treated with ecp at department of dermatology, bispebjerg hospital between 2009 and 2015 were evaluated. a single investigator retrospectively evaluated response to ecp monthly for 6 months, every 3 months until 2 years and every 6 months until 3 years. prednisolone doses were recorded every 3 months. responses were defined as complete remission (cr) if no symptoms of cgvhd were present, partial remission (pr) as improvement in cgvhd or stationary cgvhd with more than 50% reduction in prednisolone, no change (nc) as no difference in symptom burden and o50% reduction in prednisolone. progressive disease (pd) was defined as worsening of symptoms with unchanged or intensified immunosuppressive medication. ecp was performed with therakos uvar xts or cellex. there were 45 evaluable patients with moderate (n = 20) or severe (n = 25) steroid-refractory, dependent or -intolerant cgvhd. the median age was 58 years (range: 19-71) and there were 22 females and 23 males. conditioning regimen was myeloablative (n = 10) and non-myeloablative (n = 35). seventeen had related donors and 28 had unrelated. stem cell source was peripheral blood (n = 36), bone marrow (n = 7) or umbilical cord blood (n = 2). number of organs affected by cgvhd was one (n = 8), two (n = 16), three (n = 11), four (n = 9) or five (n = 1) and involved organs were skin (n = 36), eyes (n = 26), mouth (n = 24), lungs (n = 8), genitals (n = 6), liver (n = 6), musculoskeletal system (n = 5) or gastrointestinal tract (n = 2). time from diagnosis of cgvhd to first ecp was median 444 days (range: 11-2760) and time from referral to ecp and the first ecp procedure was median 52 days (range: 11-178). at the time of the first ecp procedure patients were also treated with prednisolone (n = 43), sirolimus (n = 21), calcineurininhibitor (n = 18), mycophenolate mofetil (n = 5), imatinib (n = 4), methotrexate (n = 1) or rituximab (n = 1). one patient received no immunosuppression. total number of ecp cycles was median 20 (range: 1-61). responses over time are shown in figure 1 . overall response to ecp was seen in 25 (56%) of the patients. most responses were seen after more than 3 months ecp treatment. in univariate analysis of possible baseline predictors of response, no significant associations were found. prednisolone dose was significantly reduced at every 3 months after start of ecp (p o0.01). additional cgvhd treatment was administered to 14 (31%) patients during ecp treatment (sirolimus n = 6, calcineurininhibitor n = 6, uva1 n = 4, methotrexate n = 3, rituximab n = 3, mycophenolate mofetil n = 2). about 6 (13%) patients had more than 1 additional treatment. prednisolone dose was increased at least once in 20 (44%) patients during ecp treatment. overall survival at 5 years was 80%. follow up was median 694 days (range: 62-2416). more than half the patients with cgvhd (56%) improve overall after treatment with ecp, but flares in cgvhd activity still occur. prednisolone dose is significantly reduced at all time points after starting ecp, but short term increased doses or additional immunosuppression was necessary in more than one-third of the patients. larger prospective studies with long-term end points are warranted. disclosure of conflict of interest: marietta nygaard has received a travel grant and speaker's fee from therakos/ malinckrodt. chronic graft versus host disease (cgvhd) remains a major cause of morbidity and mortality after hematopoietic stem cells transplantation despite the improvement of the immunosuppressive prophylaxis. skin, buccal, lacrymal and hepatic disorders are the most frequent. sclerotic gvhd remains a severe form and often refractory to standard treatment lines such as corticosteroids and calcineurin inhibitors. the antifibrotic activity of imatinib by the inhibition of pdgf-r and tgfb-β pathways has been used in the treatment of refractory gvhc with sclerotic features and systemic scleroderma. here, we report the results of imatinib treatment in 28 patients (pts) with refractory cgvhd. over a period of 13 years (january 2000-december 2012), 1308 pts received allogeneic stem cells transplantation from related donors, 28 of whom were treated with imatinib for refractory cgvhd: 24 pts for malignant diseases (14 cml, 9 aml, 1 nhl) and 4 pts for aplastic anemia. the median age is 31 years (6-55), the sex ratio m/f: 2.1. conditioning regimen used with chemotherapy alone: myeloablative (14 pts with gvhd prophylaxis combining ciclosporin and methotrexate), reduced intensity (14 pts with prophylaxis combining ciclosporin-mycophenolate mofetil). all pts received peripheral blood stem cell transplant with an average of cd34 cell count: 7.1 × 10 6 /kg (1.23-21.8). the median duration of the cgvhd is 8 months (3-27). the firstline treatment consisted of the combination of steroidsciclosporin with or without mycophelonate mofetil. imatinib was administered to these pts after median treatment duration of 48 months (9-108) for moderate (2 pts) and severe (26 pts) cgvhd according to the nih classification. treatment with imatinib, at doses ranging from 100 to 400 mg/d, was introduced in the second line for all pts. the evaluation is conducted in october 2016 after a median follow-up of 128 months (76-189). tolerance was good except in a one pt with severe thrombocytopenia that led to a transient cessation of treatment. after 6 months, analysis of pts who received imatinib according to couriel criteria and nih criteria: complete remission (cr): 1 pt (18%), partial remission (pr): 20 pts (71%), stable disease (sd): 5 pts, failure: 2 pts (7%). a long-term evaluation performed after a mean duration of treatment 62 months (6-91) finds similar results with a cr: 4 pts (14%), pr: 18 pts (64%), sd: 2 pts (7%) and failure: 4 pts (14%). corticosteroids were tapered or discontinued in 12 pts (cr or pr). at october 2016, 26 pts (93%) were alive and 2 pts (7%) died of severe infections. treatment with imatinib seems to be a good therapeutic option in the treatment of cgvhd in its moderate or severe form refractory to a minimum of two immunosuppressive agents according to the nih criteria as shown by our results in terms of response and survival with good tolerance. disclosure of conflict of interest: none. atg significantly reduces the risk of cgvhd both in unrelated and in hla identical sibling. the finke's study 1 randomised pts undergoing an allogeneic unrelated stem cell transplant (sct) after a myeloablative regimen to receive or not 60 mg/kg atg-grafalon reporting a significant reduction of cgvhd without increase of relapse and no os and dfs effect. however a successive study 2 didn't confirm those results (significant reduction of acute and chronic gvhd but poorer survival mainly due to higher relapse probability in the atg arm). the conflicting data reported on urd sct have several explanations, one is about the dose and the timing of atg. the timing of atg infusion has been demonstrated to be crucial for cb transplant 3 : an earlier administration is still active in preventing gvhd while ensuring engraftment and low hampering of immune reconstitution. here we report a large (193 sct) retrospective monocentric analysis on low atg doses (and 15-25 mg/kg for bm according to the degree of hla matching and 30 mg/kg for all pbsc sct) given early (from day − 6 to − 2). pts in the study were aml (n = 112, 58%), all (n = 57, 30%), hr mds (n = 21, 11%), cr1 (n = 111, 66%) cr2 or 4(n = 31, 18%), active disease (n = 27, 16%) for al; median age was 46 (range: 18-66). myeloablative conditioning were bu-cy120 (n = 72, 37%), bu-flu (n = 61, 32%), edx-tbi (n = 20, 10%), other (n = 40, 21%); pbsc was used in 41% (n = 80); sct were performed between 2005 and 2015 at the bologna transplant center. sct were performed from hla 10/10 identical urd (n = 63, 33%), or from 9/10 (n = 93, 48%), 8/10 (n = 30, 16%) and o8/10 (n = 7, 4%). median follow up was 55 months. overall, grade 2-4 agvhd was 26%, grade 3-4 agvhd 9%; cumulative incidence (ci) of cgvhd of any severity was 25%, for moderatesevere cgvhd 18%. ci of relapse and nrm was 28% and 21%, respectively. the 3-year overall and disease-free survival were 60% (95% ci: 52-67%) and 60% (95% ci: 51-68%). the gvhd (agvhd grade 3-4 and moderate-severe cgvhd) and relapse free survival (grfs) of the entire population ( figure 1 ) was 45% at 3 years (95% ci: 37-52%). restricting the analysis to patients in cr1-2, we found that cgvhd (any severity), gfrs and os at 3 years were 23%, 50% and 64%, respectively. comparing transplants with 10/10 urd to mismatched ones (9/10 or less) we found a trend for increased mod/sev cgvhd in pts undergoing transplant with mismatched urd (shr 2.32, 95% ci: 0.95-5.65, p = 0.06); agvhd grade 3-4 and cgvhd overall were not significantly increased; relapse incidence according to hla mismatches resulted 33% and 26% in 10/10 and ⩽ 9/10, respectively; grfs was 52% in 10/10 and 41% in ⩽ 9/10. the data reported show that low and early administration of atg is able to effectively prevent acute and chronic gvhd without increasing relapse thus ensuring really convincing grfs, even for o10/10 matched urd transplants. graft versus host disease (gvhd) is one of serious complications in patients after allogeneic hematopoietic stem cell transplantation. the application of mesenchymal stem cells (msc) represents a promising method for the treatment of severe steroid refractory gvhd. we present the data from an interim analysis of clinical trial, within which we applied msc in 28 patients with acute or chronic gvhd after allogeneic transplantation. the diagnoses included aml (12 pts), mds (5 pts), all (2 pts), cll/nhl (5 pts), mpn (4 pts). the patients underwent sibling hla-compatible (7), haploidentical (4), unrelated hla-compatible (13) or hla-mismatched (4) transplants. the median interval between the transplantation and msc was 6 months (1-95). the indications for msc infusion were steroid resistant acute gvhd (11 pts), steroid-dependent gvhd (agvhd 3 pts, cgvhd 11 pts) or chronic gvhd with the need for long-term immunosuppression and corticosteroid intolerance (3 pts). msc were applied as a single infusion at a median dose of 3.45 (0.9-5.0) × 10 6 /kg. response to treatment was assessed on day 14, 30, 60 and 100. the severity of gvhd prior to msc was graded as clinical stage 3 (2-3) in acute and stage 2 (1-3) in chronic gvhd, respectively. the median dose of corticosteroids was 0.92 (0.3-1.2) mg/kg/day in agvhd and s224 0.25 (0.1-0.5) mg/kg/day in cgvhd patients. on day +14 the partial response (pr) was achieved in 85% of patients with agvhd, the stabilisation of gvhd (sd) was found in 85% of patients with cgvhd. the dose of corticosteroids was reduced in most patients with agvhd (to 62% of the starting dose; 30-97%), while the early reduction was possible only in 36% of cgvhd patients. on day+100 only 19 patients were evaluable. the agvhd patients (7 pts) achieved a significant clinical response: 4 pr, cr 3 and dose of corticosteroids was reduced in all of them (to 17%; 10-60%). the minor responses were achieved in cgvhd patient (12 pts.) with 3 pr and 9 sd. however the dose of corticosteroid was reduced in 83% of these cases (to 56% of the initial dose; 21-71%). a total of 10 patients died because of infectious complications. most of them (8/10) were agvhd patients who expired early up to day +60. there were observed no side effects of msc application neither during the infusion nor later during the follow-up of 16 (2-45) months. the analysis of lymphocyte reconstitution revealed the changes of kinetics of some subsets as compared to the day +0 benchmark. the b-lymphocyte count tended to decrease in 82% of patients from chronic gvhd subgroup (vs 60% in agvhd). conversely nk cells declined in most agvhd patients (80% vs 36% in cgvhd). also the pro-inflammatory th17 cell was affected especially in agvhd (decrease in 63% pts vs 50% pts in cgvhd). the counts of myeloid/plasmocytoid dendritic cell increased in 80%/80% agvhd and 50%/91% ccvhd patients. the screening testing of cytokines (raybiotech, 42 cytokines, 6 pts, day +0 to +60) revealed changes of some analytes after msc infusion, including a decrease of proinflammatory cytokines such as ifn-γ, tnf-α, il-6. our experience with the treatment of gvhd using msc confirmed the safety of this immunotherapy. the favourable clinical effect with reduction of severity of gvhd and steroids dose was observed, especially in patients with acute form of gvhd. methotrexate day +1 omission is not associated with higher incidence of acute graft-versus-host-disease mm rivera franco, e leon rodriguez 1 and a campos castro 1 1 allogeneic hematopoietic stem cell transplantation (allo-hsct) remains a high-risk procedure due to its related morbimortality, limiting the broader application of this important treatment modality. despite extensive research over the years, acute graft-versus-host-disease (agvhd) affects the majority of patients undergoing allo-hsct, and up to 50% will develop clinically significant grades (ii). over the years, several methods for gvhd prophylaxis have been implemented, including immunosuppresive agents. methotrexate (mtx) is one of the earliest drugs used for gvhd prophylaxis. frequently, a short course of intravenous methotrexate (given on days +1, +3, +6 and +11 after hsct) is combined with a 6-month tapered course of cyclosporine. there is no consensus on which drugs and schedules for prevention of gvhd are best and clinical practice varies by institution. further, it is not clear whether omission of the day +1 dose of mtx has a negative effect on outcome in terms of morbidity. to describe the frequency of acute and chronic gvhd, mucositis and engraftment in patients receiving methotrexate (plus csa) as prophylaxis, omitting day +1. ninety-five consecutive patients who underwent allo-hsct from 1999 to september 2016, and received mtx as immunosuppressive prophylaxis were included. all patients received three doses of mtx, always excluding day +1. mtx was administered iv, either 10 mg/m 2 day +3, +6, +11 or 15 mg/m 2 day +3, and 10 mg/m 2 during days +6 and +11. we included 95 patients (55% male). the most frequent underlying diseases were aplastic anemia (21%) and acute lymphocytic leukemia (21%). ninety-nine percent of patients had a matched related donor. forty patients (42%) had gender disparity with their donor, and 13% presented abo incompatibility (major in 75%). most of the patients received myeloablative conditioning regimens (n = 73, 77%). the median of cd34+ infused cells were 2 × 10 6 (range: 0.8-6.8). the median neutrophil and platelet engraftment was 20 (11-43) and 15 (range: 5-46) days, respectively. from all the cohort, only 15 patients (16%) developed acute gvhd (53% grades i-ii) ( figure 1 ). thirty patients (32%) developed chronic gvhd, which was limited in 73%. most of the patients, 83% (n = 79), presented acute toxicity after the conditioning regimen, from which 76% (n = 60) corresponded to superior mucositis (50% grade i-ii and 50% grade iii-iv. the 10-year overall survival was 59% and the 10-year relapse free survival was 68%. our results showed a low incidence of acute gvhd, mostly grades i-ii, and similar survivals compared to previously reported studies, proposing that the administration of day +1 mtx as gvhd prophylaxis is not mandatory, however, prospective studies might be necessary to test our results. [p205] disclosure of conflict of interest: none. outcome of refractory graft versus host disease (gvhd) treated with extracorporeal photopheresis (ecp) as second line: a single-center experience j cornago navascues, b aguado bueno, av arriero garcía, edc jimenez barral, i vicuña andres and a alegre amor hematology department, university hospital la princesa, madrid, spain gvhd is a common and, sometimes, life-threatening entity related to hematopoietic stem cell transplantation (hsct). steroids remain the first-line therapy but they are not always enough to control it, or their side effects are simply unacceptable. both acute and chronic gvhd are responsible of impairment occurred in different organs that can lead to increase morbidity and mortality in our patients. different options are available as second line, but it is a well known fact that ecp, due to its inmunomodulatory mechanism, yields satisfactory response rates and presents excellent safety profile. from may 2012 to october 2016, 30 patients with steroid-dependent or refractory gvhd have been treated in our centre with ecp. we have performed 305 ecp procedures with the therakos cellex device, an integrated 'on line' system. the transplant was from a sibling donor in 13 cases and 17 from an unrelated donor. the median of cd3+ infused was 247.35 × 10 6 cd3/kg. eight patients (16.7%) presented agvhd, 17 (56.7%) cgvhd and finally, 5 (16.7%) had an overlap gvhd syndrome. most of patients (87.5%) with agvhd had a severe intestinal involvement as the main manifestation of the disease. however, all patients with cgvhd had a multiorgan involvement with a median of four organs affected, being skin, mouth, eyes and lungs the most common implicated. ten patients in our series have died, 7 for gvhd complications or infections and 3 due to relapse of aml. as firstline treatment they all received steroids and cyclosporine or mycophenolate mofetil. median ecp per patient has been 18 (2-31). ecp procedures were performed for 2 consecutive days, in initial phase weekly (in those with agvhd), or every 2 weeks (cgvhd) and then monthly according to clinical response, evaluated by clinical assessment and reduction in immunosuppression. about 75% of patients with agvhd had a significant clinical response to ecp so that steroid doses could be tapered and even in 37.5% of them withdrawal was possible. in the cgvhd group overall response rate (orr) to ecp was 94.1%. in 35% of these patients steroids could be suspended after a median of 8.5 ecp procedures. all patients who responded to ecp in cgvhd are still alive. independently of gvhd type, 81.4% of patients responded to ecp and 37% of them even could stop steroid therapy. those who had no response are dead. in cgvhd, 82.35% of patients remain alive, in contrast with agvhd or overlap syndrome patients whom survival is around 40%. about adverse events, 60% of patients did not present any complication associated with ecp. complications were mostly related to central venous catheter, with 12 cases of bacteremia and 2 thrombosis, easily recovered. in our experience, ecp is effective as second line treatment in gvhd, obtaining the best results in the chronic gvhd group. in fact, cgvhd patients with a good clinical response to ecp, specially when steroid doses can be tapered, have the better outcomes and longer survival. the tolerance to the procedure is excellent without severe adverse events. more experience is required to determine the best scheme of ecp and its role as prophylactic treatment. mesenchymal stromal cells (mscs) possess immunomodulatory properties and may play important roles in graft-versushost disease (gvhd) and engraftment. this study examined co-transplantation of mscs and hscs (hematopoietic stem cells). we investigated co-administration of ex vivo expanded mscs along with hla-identical sibling-matched hscs in β thalassemia major patients. we recruited 70 patients from january 2010 to january 2015 in our study. all participants received cyclophosphamide-based or fludarabine-based conditioning regimens and short-course methotrexate and cyclosporine as gvhd prophylaxis. mscs were administered intravenously (1.0-2.0 × 10 6 /kg) into patients (n = 41) 4 h before infusion of hscs. the outcomes were then compared to those of 29 patients transplanted with hscs alone. the median follow-up in the msc and non-msc group was 2.98 and 2.62 years, respectively. median time to wbc engraftment 40.5 × 10 9 /l was17.7 days (range: 15-20 days) in both groups (p-value = 0.83) and median time to platelet engraftment 420 × 10 9 /l was 27.2 days (range: 22-31 days) in the msc group, while it was 36.6 days (range: 22-50 days) in the non-msc group (p-value = 0.26). fifty-six percent of patients had acute gvhd in the msc group compared to the non-msc group where 65.5% developed acute gvhd (p-value = 0.42). meanwhile, chronic gvhd was 21% in the msc group and 37% in the non-msc group (p-value = 0.14). although the incidence of acute and chronic gvhd was lower in co-transplantation of hscs and mscs, no statistically significant difference was noted between the two groups. three-year overall survival rate was 70% and 61% in the msc and non-msc group, respectively (p-value = 0.78). three-year thalassemia-free survival rate was 54% in the msc group and 61% in the non-mscs group, showing no statistically significant difference (p-value = 0.35). the 3-year rejection incidence in the msc and non-msc group was 19% and 3 %, respectively (p-value = 0.07). there was no statistically significant difference between the two groups in terms of 3-year transplant-related mortality (pvalue = 0.79). this study indicates that co-transplantation of hla-identical sibling hscs with mscs does not inflict harm on bone marrow transplantation procedure and seems to be safe and secure. on the other hand, differences between the two groups in acute and chronic gvhd, engraftment, overall survival, thalassemia-free survival and rejection incidence did not reach statistical significance. therefore, despite the immunomodulatory activity of mscs and their role in gvhd amelioration and engraftment improvement resulted from in vitro studies, their efficacy in the clinical setting has not been conclusively proven which indicates further multicenter randomized clinical trials are required. keywords: β-thalassemia major, co-transplantation of mesenchymal and hematopoietic stem cells, engraftment, graft-versus-host disease. hematology-oncology and stem cell transplantation research center, tehran university of medical sciences, shariati hospital, tehran, iran. disclosure of conflict of interest: none. a number of studies were published with contradictory results comparing tacrolimus (tac) and cyclosporine a (csa) for graftversus-host disease (gvhd) prophylaxis, but there are only few that accounted for pharmacokinetic (pk) parameters. in this retrospective study we have identified pk parameters that affected gvhd incidence and incorporated them in the s226 multivariate comparison of tac-and csa-based prophylaxis. the retrospective study included 95 consecutive patients with csa and 239 consecutive patients with tac prophylaxis. 36% were grafted from matched related donor (mrd) and 64% from unrelated donor (ud). about 30% received busulfanbased myeloablative conditioning (mac) and 70% reducedintensity conditioning (ric). second agent for gvhd prophylaxis was short-course methotrexate (mtx) 10-15 mg/m 2 on days +1, 3, 6, 11 in 66% of patients and mycophenolate mofetil 30 mg/kg days − 1 to +30 in 34%. unrelated graft recipients also received antithymocyte globulin (atgam, pfizer, ny, usa) 60 mg/kg. the pk parameters analyzed were mean and median concentrations, pk variability parameters and number of concentrations below the targeted limit (nlow) within 21, 30 and 50 days after hsct. for tac the highest predictive value for acute gvhd was observed for median concentration during first 21 days (auc = 0.575), and for absolute skewness (auc = 0.567) of concentration data. for csa parameters with highest predictive value were median concentration (auc = 0.547) and variability coefficient (auc = 0.736) during first 30 days. nlow was also a significant parameter for tac current gvhd prevention regimens are partially effective, delay immune reconstitution, impair graft versus tumor effect and are cumbersome to use. therefore, there is a pressing need to develop innovative approaches for the prevention of gvhd. we completed a phase i-ii study employing a calcineurin and mtor inhibitor-free regimen based on a combination of post-transplant cyclophosphamide and bortezomib (cybor) in patients receiving fludarabine and busulfanbased reduced-intensity conditioning followed by peripheral blood, matched related or unrelated transplant. patients receiving grafts from unrelated donors also received ratg. we reported that the regimen was feasible and safe and yielded promising outcomes. (1,2) herein, we compare the results to those of a quasi-contemporaneous matched group of patients receiving a calcineurin-based gvhd prophylaxis. the experimental and control groups were well-matched in terms of age, sex, donor type, disease status, renal function and pam score. the cybor group (n = 28) was treated during a timeframe spanning from 2012 to 2016 and the control group (n = 15) from 2013 to 2016. gvhd prophylaxis for the control group was mmf and csa (n = 2) or tacrolimus (n = 13). both groups received supportive care according to standard institutional protocols. median follow-up for the cybor group was 28.7 months as opposed to 11.2 for the control group. median times to neutrophil engraftment for the cybor and control groups were 16 days (13-23) and 12 (10-21), respectively (p = 0.001). two patients from the cybor arm died before achieving platelet engraftment. for the remaining s227 patients, median time to platelet engraftment was 27 days (15-38). for the control group, five patients never dropped their platelet count below 20 × 109/l. for the remaining patients, median time to platelet engraftment was 17 days (10-29) (p = 0.002). there was no primary or secondary graft failure in either of the two groups. the incidences of acute grade ii-iv and grade iii-iv for the cybor group were 35.7 and 10.7%. for the control group, the incidences were 60 (p = 0.12) and 20% (p = 0.4). the incidence of chronic gvhd for the cybor and control groups were 28% and 14.3%, respectively (p = 0.62). treatment-related mortality was 14.3% and 20% for the cybor and control groups, respectively (p = 0.13). the incidences of cmv, ebv and bk reactivation for the cybor group were 57.1%, 32.1% and 17.9%, respectively. for the control group, the incidences were 46.7% (p = 0.49), 26.7% (p = 0.68), 0% (p = 0.09). the 2-year progression free survival and overall survival were 49.0% and 49.9% for cybor group and 18.8% and 31.3% for the control group ( figure 1 ). the 2year gvhd and disease-free survival (grfs) were 45.6% and 20%, respectively. despite the limitations of our study that include its size and its design and the delayed neutrophil and platelet engraftment associated with the cybor regimen in comparison to calcineurin-based prophylaxis, our data confirm the promising outcomes previously reported with the cybor combination and reaffirm the need for a large randomized study comparing cybor to a standard calcineurin-based regimen. clostridium difficile infection (cdi) causing enterocolitis may represent a serious clinical problem in patients undergoing allogeneic hematopoietic cell transplantation (allo hct). the reported prevalence varies substantially among heterogeneous patient cohorts. although cdi has been proposed as a risk factor for the development of gastrointestinal (gi) acute graft-versus-host-disease (agvhd), limited knowledge on the prevalence of cdi, occurrence of gi agvhd in cdi patients, relapse incidence and mortality of cdi patients in large patient cohorts is available. the aim of this analysis was to study the implications of cdi in a homogenous cohort of patients with either aml or mds undergoing allo hct. at our center all patients undergo stool test once a week for clostridium difficile antigen while in aplasia until discharge, irrespective of clinical symptoms for enterocolitis. patients with positive stool antigen tests (that is, toxin test) in the absence of clinical symptoms were referred to as cd+, in contrast to patients without a positive test and without clinical symptoms which were referred to as cd − . we retrospectively analyzed the data of a total of n = 727 patients with either aml or mds undergoing allo hct in our institution between 2004 and 2015. overall survival (os) was measured from allo hct to the date of death or last follow-up. after hsct, relapse and nonrelapse mortality were considered as competing events. eventprobabilities were calculated according to kaplan-meier for os and using competing event statistics for the cumulative incidence of relapse (cir), non-relapse mortality (nrm) and agvhd. 95% confidence intervals (ci) were provided for major endpoints. statistical analyses were performed using the r environment for statistical computing version 3.1.3 (r core team 2015, vienna, austria, www.r-project.org). from a total of n = 727 patients with either aml or mds who underwent allo hct, we identified n = 528 (73%) who were cd − , n = 103 (14%) who were cd+, and n = 96 (13%) who had cdi. interestingly, n = 33 (34%) of patients with cdi were diagnosed having gi agvhd as compared to n = 13 (13%) of patients who were cd+ and compared to n = 95 (18%) of patients who were cd − , p = 0.001. the three groups harbored no differences when comparing incidences of liver and skin agvhd or chronic gvhd, respectively. when dissecting gi agvhd according to ctcae criteria, only n = 8 (24 %) of cdi patients vs n = 7 (54 %) of cd+ patients, and n = 53 (56 %) of cd − patients had grade 3-4 gi agvhd, p = 0.007. with regard to os and trm, no statistical differences were observed between the three groups. the cir was 13% for patients with cdi, 15% for cd+ patients and 9% for cd − patients, p = 0.02, respectively. this analysis represents the largest published analysis of clostridium difficile in patients with aml or mds who underwent allo hct. the prevalence of cdi in this patient cohort was 13%. patients with cdi developed significantly more often gi agvhd as compared to patients who were either cd+ or cd − , respectively. however, this did not translate into differences in os or trm. disclosure of conflict of interest: friedrich stölzel has received research funding from astellas. the majority of studies on cytokines in allogeneic hsct were performed with classical gvhd prophylaxis, consisting of nonspecific immunosuppressive agents. with this type of prophylaxis almost in all studies published, higher levels of proinflammatory cytokines are associated with development of acute gvhd, while lower levels indicate the success of immunosuppressive agents in abrogation of alloreactive response. currently, there is no data, whether the dynamics of cytokines after ptcy is similar to the situation of classical gvhd prophylaxis. out of 192 adult patients transplanted at first state medical university with ptcy between 2014 and 2015 we have identified 20 cases with acute gvhd and plasma samples available. these patients were matched in the ratio 1:2 to patients who did not develop acute gvhd. the study group was comprised of 60 adult patients with hematological malignancies who underwent hsct. all patients received ptcy-based gvhd prophylaxis. five plasma biomarkers were studied by elisa: il-17a, il-6, il-8, tnf-α and ifn-γ. blood samples were obtained from patients on days − 7, 0, +7, +14. the fifth time point varied between day +21 and +28 to represent the sample after engraftment, but before onset of acute gvhd. about 10 (50%) out of 20 gvhd patients had a grade i, 7 (35%) grade ii, 5 (25%) grade iii agvhd, 6 (30%) patients developed multiorgan agvhd. about 13 patients (21.6%) had chronic gvhd. there was no difference between gvhd + and gvhd − groups in any of the clinical parameters. the median of engraftment for all patients was 21 (9-43: range). the median agvhd was 30 days (23-92: range). neither of the cytokine levels was significantly different in patients with agvhd grades i − iv and without gvhd. however, for patients with agvhd grade ii − iv we found that low levels of il-8 on day +7 (126.83 ± 43.794 vs 276.89 ± 310.51 pg/ml, p = 0.04) and ifn-γ on day +21-28 (34.70 ± 23.71 vs 60.96 ± 41.37 pg/ml, p = 0.03) were associated with increased risk of gvhd. the roc analysis was performed to determine the cut off values for il-8-133.56 pg/ml (auc = 0.714) and ifn-γ -35.94 pg/ml (auc = 0.720). the incidence of agvhd grade ii-iv was significantly higher in patients with levels of cytokines lower than cut off (40% vs 5.7%, p = 0.008 and 43.7%, p = 0.012 for il-8 and ifn-γ, respectively). the same pattern was observed for patients with agvhd grade iii-iv. low levels of il-8 (96.12 ± 39.79 vs 303.52 ± 346.19 pg/ml, p = 0.008) and ifn-γ (21.69 ± 14.78 vs 58.80 ± 39.92 pg/ml, p = 0.012) on day +28 were especially predictive. the cut off values for il-8 was 147.09 pg/ml (auc = 0.869) and for ifn-γ-25.71 pg/ml (auc = 0.858). the incidence of agvhd grade ii-iv was also significantly higher in patients with levels of cytokines lower than cut off (p = 0.004 and p = 0.0006 for il-8 and ifn-γ, respectively). for chronic gvhd only higher level of il-17 at day +28 (209.17 ± 329.59 vs 106.06 ± 210.65 pg/ml, p = 0.037 for patient with and without gvhd, respectively) was significantly predictive. in this pilot trial we have demonstrated that dynamics of cytokines after gvhd prophylaxis with ptcy may be different from conventional one, and well-known predictive biomarkers might not work after ptcy. further large prospective trials are warranted to elucidate reliable biomarkers for gvhd after this type of prophylaxis. disclosure of conflict of interest: none. graft versus host disease (gvhd) remains one of the main obstacles to broader application of allogeneic transplantation. gvhd prevention and treatment techniques are poorly standardized. the 1st-line treatment of newly diagnosed chronic (c) gvhd is corticosteroid. there is no standard 2ndline treatment for cgvhd. approximately 50-60% of patients (pts) with cgvhd require secondary treatment within 2 y after initial systemic treatment. recently the jak1/2 inhibitor ruxolitinib emerged as an efficacious treatment for corticosteroid-refractory (sr) acute and c-gvhd with a 24% of sr-cgvhd patients reporting a long lasting immunosuppression-free complete response. the current study seeks to analyse the efficacy and safety of ruxolitinib in highly pre-treated sr-cgvhd pts in our centre. ruxolitinib treatment was given off label after provision of an informed signed consent and in the absence of alternative therapeutic options including clinical trials. we analysed data prospectively collected at our long-term follow-up clinic between 2015 and 2016. a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. overall 5 pts (median age 57 y-range: 39-67 years; mean karnofsky score 70%) with sr-cgvhd were treated s229 with ruxolitinib. median time from transplant was 46 months (range: 9-68). ruxolitinib was initiated at a starting dose of 5 mg twice daily-median time on ruxolitinib 4 months (range: 2-15)-4/5 pts increased the dose up to 10 mg twice daily. four pts had a classic and 1 an overlap sr-cgvhd. all of them had skin sclerodermatous involvement and 4/5 joint and fascia involvement with significant decrease of range of motion and limitation of adl. all pts were previously treated with several lines of immunosuppression (3-11) including high-dose prednisone in 1st line (5/5), rapamycin (5/4), tk-inhibitor imatinib (4/5), extracorporeal photopheresis (4/5). all pts were pre-screened for risk of infection and regularly checked on a fortnightly basis. all pts were under active prophylaxis according to recommendation for gvhd pts and ruxolitinib therapy. after a cumulative follow-up of 867 days we reported only one serious adverse event represented by a cmv pneumonia requiring hospitalization with complete recovery. early time point evaluation (5/5 pts evaluable) at +1 month underlined how all pts were reporting subjective improvement at the patient global ratings according to nih 2014. data were confirmed at the health care provider global ratings. month 3 evaluation (3/5) confirmed meaningful responses (partial responses 3/3) according to nih 2014, with both patient and health care provider global ratings improvement and concomitant enhancement in lee skin symptoms score and sf-36 health-related qol. at last followup no evidence of myelosuppression, infections, pml, nonmelanoma skin cancer was registered. considering the concomitant treatment (with reference to azoles and rapamycin or cyclosporine) no cases of toxicity due to drug-druginteraction was reported. ruxolitinib is well tolerated in highly pre-treated sr-cgvhd. its safety profile seems to be reassuring. the efficacy data observed also at this early time point is preliminary but promising in this subset of pts with a long history (⩾3 lines) of treatment for cgvhd. confirmatory study in a larger number of patients is underway on a multicentre basis. disclosure of conflict of interest: none. severe acute enteral graft-versus-host-disease (gvhd) is a lifethreatening complication of allogeneic bone marrow transplantation. in case of resistance to corticosteroids as the firstline treatment severe enteral gvhd harbors a high morbidity and mortality. retrospective analyses indicate efficacy of the jak1/2-inhibitor ruxolitinib in the treatment of acute or chronic gvhd in adults, but experience in paediatric patients is limited. here, we report a small cohort of paediatric patients with stage 4 steroid-refractory gvhd of the gut who received ruxolitinib as salvage therapy within a multimodal immunosuppressive regimen. we retrospectively analysed four patients aged 8-16 years with severe, steroid-resistant acute gvhd of the gut who were treated with ruxolitinib in our institution. all patients were transplanted for non-malignant haematologic disorders, graft source was 2 × mmud, 1 × mud, 1 × msd. the conditioning regimen consisted of treosulfan, fludarabine and thiotepa. serotherapy with thymoglobuline was administered in all patients transplanted from unrelated donors. all patients received mtx and cyclosporine as gvhdprophylaxis. gvhd was staged according to the glucksberg-scale. ruxolitinib was added to the immunosuppressive regimen when acute stage 4 gvhd was reached and became resistant to treatment with methylprednisolone (2 mg/kg/day) as well as infliximab and mycophenolate (mmf) as second-line immunosuppressants. acute stage 4 enteral gvhd developed at a median of 38 days after transplant (30-58 days) and ruxolitinib was started at a median of 53 days post-transplant (48-85 days). the starting dose varied between 10 mg/day and 40 mg/day, that is, 0.25-0.5 mg/kg/day, taking into account the expectedly low bioavailability of the oral drug during severe diarrhea. upon improvement of gvhd symptoms and/ or increasing side effects the dose was gradually tapered and ruxolitinib was discontinued after a median of 39 treatmentdays (19-83 days) . after addition of ruxolitinib to the immunosuppressive regimen, the symptoms of acute gut gvhd gradually improved in all four patients with decreasing abdominal pain and stool volumes. immunosuppression with steroids and mmf could slowly be tapered. all patients are alive after a median follow-up of 392 days (95-571 days) from diagnosis of acute stage 4 gut gvhd. the most prominent side effect attributable to ruxolitinib was thrombocytopenia with a nadir in platelet counts after 30 days of ruxolitinib treatment in 3/4 patients. platelets recovered within 2 weeks after ruxolitinib was discontinued. neutropenia was observed in one patient with anc dropping o0.5/nl after 30 days of ruxolitinib treatment. mild to moderate elevation of liver transaminases was observed in all four patients during ruxolitinib treatment. one patient developed imminent acute renal failure, another patient showed symptoms of hemolytic uraemic syndrome. however, due to the multimodal treatment of these critically ill patients, these complications could not clearly be attributed to ruxolitinib. ruxolitinib is potentially beneficial in severe acute enteral gvhd in children refractory to corticosteroids as well as second-line immunosuppressants. however, randomized trials are warranted to verify safety and efficacy of ruxolitinib in this patient cohort. disclosure of conflict of interest: none. steroid refractory acute gvhd is a major cause of mortality after allogeneic stem cell transplantation. until date, no agent or treatment strategy has demonstrated superior efficacy in this patient group. the dose and duration of steroid treatment is associated with several short and long-term side effects, therefore concepts facilitating rapid steroid taper may be beneficial. both ruxolitinib and ecp have been reported to be effective in treatment of steroid refractory (sr) agvhd. we analyzed data from consecutive adult patients who received ruxolitinib for sr agvhd between march 2015 and august 2016 in our institution overall, 19 patients (male n = 12; female n = 7) with a median age of 58 years (range: 18-74) were included. donors for allogeneic sct were msd (n = 3), mud (n = 12) and mmud (n = 3). median time to gvhd onset after stem cell transplantation was 29 days (range: 7-154 days). about 14 patients had agvhd grade iii or iv (all with gi involvement), while 5 patients had skin grade 3 involvement. sr agvhd was diagnosed if agvhd manifestations were progressive after 3 days or persistent and without improvement after 7 days or no partial remission after 14 days of treatment with 2 mg/kg bw of systemic steroids. patients received additional ecp (n = 11), if response to ruxolitinib was lacking or slow (n = 9) or instead of ruxolitinib due to cytopenias (n = 2). ruxolitinib was first-line treatment for sragvhd in 11 patients (58%). median initial dose of ruxolitinib was 10 mg (range: 5-15 mg) twice daily. steroids were tapered and stopped, even if agvhd was still active. primary end point was non-relapse mortality at 6 months. secondary end point was response on day 28 after initiation of ruxolitinib. response occurred relatively slowly, resulting in a day 28 overall response rate of 58% (cr = 6, pr = 5). however, a total nine patients (47%) attained a complete response (cr), five with ruxolitinib alone and four others in combination with ecp. about 12 patients (63%) required dose reduction or interruption of ruxolitinib mainly due to cytopenias. after a median follow-up of 210 days, 8 patients are alive. causes of death were relapse of malignant disease (n = 1), gvhd (n = 2), infections (n = 7) and other (n = 1). median survival from diagnosis of sr agvhd was 61 days for non-responders and 252 days for responders ( figure 1 , p = 0.0096). in univariate analysis, non-response was associated with higher risk of nonrelapse mortality (rr; 5.6, 95% ci: 1.51-20.6, p = 0.01). ruxolitinib and ecp are two effective promising treatment options, which may be complementary in patients with sr agvhd. cytopenia is the most frequent side effect of ruxolitinib while infections remain the major cause of death. [p215] disclosure of conflict of interest: ayuk-therakos: honoraria; kröger: novartis: honoraria, research funding. steroid-refractory acute graft-versus-host disease (sr-agvhd) is associated with a dismal outcome. janus kinase (jak) 1/2 signaling has been shown to be instrumental in multiple steps leading to inflammation and tissue damage in gvhd. jak1/2 inhibitor ruxolitinib was studied in the treatment of sr-gvhd by zeiser et al. (leukemia 2015) , and the overall response rate was reported to be 81.5%. we have now studied ruxolitinib in the treatment of six adult patients with steroid-refractory, grade iii-iv, intestinal agvhd. all the patients were male. the median age of the patients was 50 (range: 19-59) years. three of the patients were transplanted for aml, one for all, mds and mm each. all the patients had been given a myeloablative conditioning treatment (cytbi 2, treosulfan+fludarabine 4). two patients had a sibling donor and four a matched unrelated donor. the graft was from peripheral blood in all the patients. gvhd prophylaxis consisted of cyclosporine and a short course of methotrexate, and in addition antithymocyte globulin in the unrelated donor setting and methylprednisolone in one sibling recipient. agvhd of the intestine manifested on days + 17, +25, +39, +60, +63 and +136 with diarrhea. in two patients it was preceded by agvhd of the skin by 7 and 49 days, respectively. gi-biopsy showed acute gvhd of grade iii and of grade iv in three patients each. treatment of intestinal gvhd was started with methylprednisolone 10 mg/ kg/day, tapering the dose to 5 and 2 mg/kg after 12 doses each. gastroduodenoscopy and colonoscopy were performed at the onset of symptoms indicating intestinal gvhd. biopsy confirmed the diagnosis in all cases. because the diarrhea continued in spite of methylprednisolone treatment, ruxolitinib was started 7, 9, 10, 10, 20 and 90 days from the first day of diarrhea. the dose of ruxolitinib was 10 mg × 2 per day orally. four patients showed a clear response to ruxolinitib, normalization of bowel function, after 3, 4, 16 and 27 days from the start of ruxolitinib treatment. the healing of the intestinal lesions was verified by biopsy. two of these patients had received extracorporeal photopheresis simultaneously. two patients did not benefit from ruxolinitib treatment. one of them had continuous infectious complications and therefore ruxolitinib was only started after 90 days from the start of diarrhea. the other patient died of fulminant diarrhea after 3 weeks of ruxolitinib treatment. cmv reactivation was detected in three of the responders, and two of them had also polyoma virus cystitis. one patient developed a pulmonary aspergilloma, which is under control with drugs. corticosteroid-resistant gastrointestinal acute gvhd was treated in six patients, out of whom four showed a good response. disclosure of conflict of interest: none. although methotrexate (mtx) is commonly used in the prophylaxis of graft-versus-host disease (gvhd) after allogeneic hematopoietic stem cell transplantation (allo-hsct), some small studies have also reported its use in the treatment of chronic gvhd. the aim of this study was to evaluate the efficacy and safety profile of low-dose mtx for treatment of sclerodermatous chronic gvhd (sgvhd) after the failure of first and second line treatments. we retrospectively evaluated 23 adult patients who received low-dose mtx as salvage treatment of sgvhd during the period elapsed between june 2006 and june 2016 in a tertiary referral university hospital in spain. there were 17 (73%) males and 6 (27%) females. the median age was 54 years (range: 28-69). all had received an allo-hsct for hematologic malignancies. the median time from allo-hsct to sgvhd was 666 days (range: 334-2679). thirteen patients (56%) had presented previous acute skin gvdh. superficial skin lesions mimicking lichen planus (lichenoid gvhd) were diagnosed in 19 (82%) patients, while lesions resembling lichen sclerosus, morphea or fasciitis (sgvhd) where seen in all 23 (100%) patients. the total body surface area was affected by more than 50% in 15 patients (65%). besides the skin, other organs/tissues involved were the eyes (65%), mouth (52%), nails (34%), lungs (17%), liver (8%) and gastrointestinal tract (4%). treatment lines prior to mtx administration were: prednisone (pdn) in 23 patients (100%), phototherapy (pht) in 4 (17 %), cyclosporine (cya) in 2 (8%), mycophenolate mofetil (mfm) in 2 (8%), pht + pdn + cya in 3 (13%), pdn + mfm + cya in 3 (13%), extracorporeal photopheresis + pdn in 1 (4%). the median time from sgvhd onset to mtx treatment was 308 days (range: 19-937 days). mtx was administered subcutaneously in 21 patients (91%) and orally in 2 patients (9%). median dose of mtx was 13.74 mg/week (range: 7.73-18.48 mg/week) and median length of treatment was 61 weeks (range: 2-148 weeks). in two patients (8%) early withdrawal of mtx occurred (one due to early death secondary to septic shock and other due to rapid disease progression). mtx-related toxicity occurred in three patients (13%): megaloblastic anemia, asymptomatic increase of liver enzymes and mucositis, respectively. response to mtx was evaluated in the 21 patients (91%) s231 who did not suffer early mtx discontinuation. seventeen patients (73%) presented a partial response; of them, two are still under mtx treatment for 26 and 59 weeks, respectively. fourteen patients (60%) received pdn concomitantly to mtx (median dose 20 mg/day, range: 5-60); 1 year after mtx treatment, only four patients were receiving pdn (median dose 5 mg/day, range 5-15). seven patients have finished mtx treatment without reappearance of symptoms, receiving only topical treatment with emollients, tacrolimus or corticoids for short periods. in four patients (26%) sgvhd progressed despite mtx administration. our data suggest that mtx is a safe, inexpensive and effective alternative for refractory sgvhd. its potential used in earlier phases of sgvhd deserves further investigation. disclosure of conflict of interest: none. severe chronic gvhd has a major influence on late morbidity and mortality after hematopoietic stem cells transplantation (hsct). ecp is a good approach to treat refractory-gvhd: leucocytes are obtained from peripheral blood by apheresis, incubated with 8-mop, irradiated and then infused to the patient where they undergo apoptosis and induce tolerance. it is a promising alternative that reduces doses of immunosuppressive therapy and their side effects in the treatment of gvhd. this study shows its efficacy in persistent refractory cgvhd. the procedure was applied to three patients (pts) aged 24, 28 and 32 years (two aml and one cml), sex ratio (1m/2f) who underwent allogeneic-hsct with myeloablative conditioning regimen based on chemotherapy alone from a peripheral blood stem cells with cd34 levels: 1.24, 7.6 and 8.23 × 10 6 /kg respectively. prophylaxis of gvhd combined ciclosporin and methotrexate in short cycle. severe extensive cgvhd (according to nih criteria) was observed in the three cases after an average delay of 5.3 months (3-7) with involvement of 1-6 organs (mouth, eyes, skin, liver, joints and lungs). all pts are refractory to three lines of immunosuppressive agents (ciclosporin-corticosteroids, mmf and imatinib), with an average of 3 thrusts/pt (2-4) over an average period of 65 months (09-103). ecp was performed under the open system or dissociated system (macopharma) for two sessions per week for 4 weeks, one session per week for 8 weeks, one session every 2 weeks for 12 weeks and one session per month for 3 months. after a median period of 6 months (3-10), an average of 22 sessions/pt (15-26) was performed. in terms of tolerance, a red blood cell transfusion was required in one pt, spontaneously resolved lymphopenia was observed in another pt, and a poor venous approach led to the pause of a central catheterization in one pt. the 3month and 6-month evaluation according to the couriel response criteria shows a partial response observed as of the first month with net improvement especially on skin sclerotic features and joints retractions initially refractory to all therapeutic lines. this allowed gradual reduction doses of corticosteroids. pce is recommended in the curative treatment for refractory chronic gvhd from the second line. this encouraging study on a small series shows its efficacy in persistent and late refractory forms. it is nevertheless necessary to evaluate it on a larger number of pts. disclosure of conflict of interest: none. successful treatment of steroid-refractory acute gastrointestinal graft-versus-host-disease by fecal microbiota transplantation p neumeister 3 steroid-refractory acute gastrointestinal (gi) graft-versus-host disease (agvhd) is a severe complication of allogeneic hematopoietic stem cell transplantation (allo-hsct) associated with a high mortality rate. loss of intestinal bacterial diversity is thought to be associated with severity of gi-agvhd and an impaired intestinal microbiota with reduced diversity is an independent predictive factor for mortality. fecal microbiota transplantation (fmt) is the application of a fecal suspension derived from a healthy donor into a patient's gi tract. it has been successfully applied in recurrent clostridium difficile associated diarrhea including patients who underwent allo-hsct. we report the complete resolution of lower gi-agvhd following colonoscopic fmts in three patients that had been refractory to 4-6 lines of immunosuppressive therapies. microbiota analysis by 16s rdna before fmts revealed a severely depleted microbiota in all patients. donors (different persons for each patient) were healthy adult subjects. repetitive (1-6) colonoscopic fmts were necessary to permanently establish the donor's microbiome. all patients responded clinically by reduction/normalisation of stoll volumes, stopping total parenteral nutrition and tapering of steroids. a possible causative relationship of fmt in the reversal of severe intestinal dysbiosis and subsequent resolution of gi-agvhd can therefore be hypothesized. the establishment of donors' microbiota and increase in bacterial richness was associated with disease control. no immediate procedure-related infections or other side effects were observed. besides restoration of an initially severely reduced microbial richness by fmts, response of gi-agvhd was sustained despite reduction and discontinuation of concomitant immunosuppressive treatments. restoration of dysbiosis by fmt might represent a promising novel therapeutic approach for refractory lower gi-agvhd. disclosure of conflict of interest: none. tear film proteomics reveals important differences between patients with chronic ocular gvhd and healthy controls k plattner 1 , n gerber-hollbach 1 , s moes 2 , p jenoe 2 , d goldblum and j halter 1 ophthalmology, university hospital basel, ch-4031 basel and 2 proteomics core facility of the biozentrum, university of basel, ch-4056, basel chronic gvhd frequently involves the eyes, leads to important decrease of quality of life and may threaten visual capacity. sicca syndrome is one of the hallmark of ocular cgvhd. analysis of tear protein composition may help to identify biomarkers for early diagnosis and prognosis of ocular cgvhd. tear fluid of 42 patients with ocular cgvhd were compared with 10 healthy individuals in this single center study. results of the first 10 patients are reported here. tryptic digests from schirmer strips were analyzed on an orbitrap mass analyzer. clinical examinations included slit lamp examination, fluorescein staining, schirmer test, break-up-time (but) and a quality of life questionnaire (osdi). outcome measures were differences and consistency of proteins in human tear fluid s232 between patients with ocular gvhd and healthy controls. statistical analysis was performed by one sample wilcoxon-tests, p-values o 0.01 was considered significant. ten patients (eight males, two females) with a median age of 47 years (range: 24-69) were analyzed. all underwent pbsct, eight from an unrelated donor. cgvhd overall score was moderate in three and severe in seven. eye organ score was 2 in six and 3 in four patients. all patients had more than one organ manifestation of cgvhd. eight were under systemic immunosuppressive therapy at the time of analysis, two had topical treatments only. in total 306 different proteins were detected in tears analyzed. compared to controls, 172 were differentially expressed in ocular cgvhd. expression was highly significantly different in 75 proteins. compared to controls, expression of 41 proteins was at least 10-fold increased, representing 11 different categories. among them, more than 75% of all proteins belong to one of three categories: cytoskeletal proteins, nucleic acid binding or structural proteins. albumin, cluster or keratin (keratin type i-iii) and cluster of pyruvate kinase were most highly overexpressed. expression of 14 proteins was decreased to o1 to 50%, belonging to 12 different protein classes. half of them belong to defense/immunity proteins, enzyme modulators, hydrolases, nucleic acid binding and carrier proteins. expression of lactotransferrin, proline-rich protein and prolactin-inducible protein was most profoundly decreased. compared to healthy controls, a high number of protein is found to be differentially expressed in tears in ocular cgvhd. among them high expressions are observed for proteins that may indicate disturbed integrity of ocular surface and leakage of conjunctival capillaries. most profoundly decreased proteins include proteins with important functions in host defense and immunomodulation. more detailed pathway analysis is necessary to identify biomarkers for ocular cgvhd. disclosure of conflict of interest: none. steroid-dependent chronic gvhd after allogeneic peripheral blood stem cell transplantation is a great problem. nonresponders to corticosteroid therapy are at high risk of mortality. we hypothesized that such patients could benefit from treatment strategy using in patients with primary severe autoimmune diseases like multiple sclerosis and crohn's disease. patient z., 4 y.o. was diagnosed in july 2011 with myelomonocytic leukemia (jmml). initially he was treated with low-dose of cytarabine and epigenetic agents. in september 2013, jmml progression was observed with leukocytosis, thrombocytopenia, splenomegaly. bone marrow aspirate showed 19.2% monocytes and 19.8% blast cells. splenectomy was performed in november 2013 due to refractoriness to blood components transfusions. in december 2014 unmanipulated haploidentical peripheral blood stem cell transplantation from mother with 3.2 × 10 6 /kg cd34+ and 3.5 × 10 8 /kg cd3+ was performed. the conditioning regimen was myeloablative including melphalan 100 mg/m 2 day − 5 and treosulfan 14 000 mg/m 2 days − 4, − 3, − 2. no organ toxicity 4grade 2 was observed. gvhd prophylaxis consisted of hatg 10 mg/kg on days − 5, − 3, − 1, +1, i.v. tacrolimus from d − 1 and mmf 25 mg/kg from d 9. engrafted was fast and prompt (100% donor) with wbc41.0 × 10 9 /l on d +8, plts420 × 10 9 /l on d +10. acute gvhd of stage ii was observed in early posttransplant period and treated with steroids and tnf-α inhibitor (infliximab). patient also received five procedures of ecp. all attempts of immunosupression tapering failed and the patient was staying on high dose of tacrolimus, mmf and courses of steroids till october 2015. in october 2015, gvhd stage ii flare with blood eosinophilia occurred after another attempt of steroids withdrawal. clinical examination showed that the patient was in complete remission with full donor chimerism. mild response of gvhd to steroids was observed. in april and may 2016 patient received two doses of rituximab 375 mg/m 2 with no significant response. in order to restore naive immune system first course of chemotherapy with cyclophosphamide 2000 mg/m 2 was performed in the end of may 2016. no toxicity 4grade 2 was observed. the patient recovered wbc41.0 × 10 9 /l on d +12, plts420 × 10 9 /l on d +11. in the phase of hematological recovery he was mobilized with g-csf and two leukaphereses of pbscs were performed. in june 2016 our patient was transplanted with previously collected 0.5 × 10 6 cd 34+/kg following nonmyeloablative regimen including cyclophosphamide 1500 mg/m 2 on day − 3 and fludarabine 10 mg/kg, on days − 3, − 2, − 1. second dose of cyclophosphamide was not administered because of severe hyponatriemia with seizures due to the cpm administration. no other significant toxicity was observed. the patient did not require either blood product or i.v. antibiotics. doses of tacrolimus and mmf were picked on 2 months late and no more steroids were given. the patient is well in cr with no signs of gvhd for 7 months. we speculate that pbsc collection from patients under massive immunosupression underwent allogeneic transplant is difficult but feasible. the nonmyeloablative regimens in such group of patients could be well tolerated and ensure the restoration of naive recipient immune system. this option could be discussed as an attractive alternative for treating resistant gvhd in steroid resistant patients. disclosure of conflict of interest: none. severe acute gi-gvhd is a serious early complication of allotransplants, and still remains a clinical diagnosis.(1) endoscopic biopsies provide the best supportive evidence, but are invasive and morbid in patients who are already medically compromised. 18 f-fdg pet/ct may be able to stratify patients who require endoscopy and biopsy. to evaluate the performance of 18 fdg pet/ct in differentiating moderate to severe gi-gvhd from no or mild disease in pediatric patients with suspected gi-gvhd. retrospective chart review of all paediatric allo-transplant patients referred for 18 f-fdg pet/ct with suspected gi-gvhd from 2009 to 2015. clinical follow-up, endoscopy and biopsy findings were correlated with 18 f-fdg pet/ct. regional suv parameters were extracted by placing rois around stomach, duodenum, distal ilium, caecum, ascending, transverse, descending colon, recto-sigmoid colon and rectum. regional, and average large and small bowel suv data were statistically compared between patients with no or mild git-gvhd vs moderate to severe disease. the clinical and biopsy-supported diagnosis of acute gi-gvhd was taken as the true positive diagnosis for acute gi-gvhd. roc curves were generated for whole bowel suvmax values. about 50 scans in 34 patients, median age of 9 years (6 mths to 18 y), were performed at a median of 71 days post bmt. there were 15 stage 1, 13 stage 2-4 and 22 with no acute gi-gvhd. transverse colon suvmax was significantly higher in the stage 2-4 gi-gvhd compared to no or stage 1 disease (mann-whitney-u-test p o0.05). there was a non-significant trend for average large bowel suvmax to be higher in the stage 2-4 group than the no or stage 1 disease group (mean suvmax 4.16 compared to 2.94, p = 0.07). a cut off whole bowel suvmax 2.74 had a sensitivity of 79% and specificity of 61% for detecting moderate to severe gi-gvhd. 18 f-fdg pet/ct is a feasible and potentially useful non-invasive tool in the diagnosis and monitoring of therapeutic efficacy in acute gi-gvhd (2) . large bowel suvmax may be higher in patients with stage 2-4 gi-gvhd, and transverse colon suvmax could have the ability to differentiate children with no or stage 1 gi-gvhd from those with stage 2-4 disease. a negative 18 fdg-pet/ct could serve as a criteria to avoid invasive endoscopic procedures and observe for the persistence of gastrointestinal symptoms before subjecting these patients to an imageguided biopsy. in patients too unwell for endoscopy, suvmax 44 (roc curve specificity 75%) and a high suvmax in the transverse colon could serve as supportive evidence for moderate to severe acute gvhd, in the absence of biopsy findings. a major advantage of a pre-endoscopic 18f-fdg pet/ ct is to guide the procedurals to sample areas with the best diagnostic yield. prospective controlled studies are needed. oral mucosal progenitor cells (omlp-pcs) possess immunomodulatory and antibacterial properties, suggestive of their in vivo function in healthy tissue and their potential contribution to scarless wound healing in the buccal mucosa (2, 3). our aim was to establish whether the function of oral stromal progenitors is impaired in chronic graft versus host disease (cgvhd) and restored with response to treatment. a patient with grade 3 oral cgvhd was treated with systemic thalidomide for 9 weeks (200 mg/day). punch biopsies of buccal mucosa were taken before and after treatment. oral progenitor cells were isolated and expanded in vitro. numbers of progenitors was assessed using colony forming unitfibroblast (cfu-f) assays. stem cell markers (cd90, cd105, cd73, cd29, cd34, cd45, hla i and ii) were evaluated by flow cytometry. wound healing and antibacterial potential were assessed using a collagen gel lattice assay and bacterial cocultures as previously described (2, 4) . secreted levels of relevant cyto-and chemokines associated with wound healing were assessed by elisa. significant clinical improvement with reduced inflammation in the oral mucosa and healing of ulcers was seen after 3 weeks of thalidomide treatment, with continued improvement after 9 weeks. cell surface expression of cd90 and cd105 on omlp-pcs was elevated postthalidomide; markers correlated with stemness and angiogenesis in mesenchymal stromal cells. this correlated with a restoration of wound healing potential and antibacterial function after thalidomide treatment ( figure 1 ). figure 1 : antibacterial testing demonstrated a loss of antibacterial function against (a) gram positive and (b) gram negative micro-organisms in the cgvhd omlp-pcs that could be completely or partially restored to levels comparable with healthy controls after thalidomide treatment. *p ⩽ 0.05, **p ⩽ 0.01, ***p ⩽ 0.001. we demonstrate, for the first time a correlation between clinical improvement of oral cgvhd with thalidomide treatment and restoration of endogenous progenitor cell function. this study highlights the importance of a dysfunctional oral mucosal stroma in the pathogenesis of cgvhd. further studies should focus on the role of the stroma in promoting cgvhd and the precise mechanisms by which thalidomide is able to restore its functions. chronic graft-versus-host disease (cgvhd) is a major cause of late morbidity and treatment-related mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (hsct). cgvhd is driven by a th2 biased t-cell mediated alloreactive immune response that leads to chronic inflammation and fibrosis in various organs. thymic stromal lymphopoietin (tslp) is an epithelial cell-derived cytokine that mainly affects myeloid cells. upon stimulation with tslp, dendritic cells are polarized towards a dc2 phenotype driving th2 biased immune response. we hypothesized that tslp is involved in the pathogenesis of cgvhd and that elevated levels of tslp post-transplant may lead to an increased risk of cgvhd. in the present study, we measured plasma tslp levels during hsct to study associations with clinical outcomes including cgvhd. about 38 adult patients undergoing myeloablative hsct at rigshospitalet, denmark, from 2011 to 2013 were included. diagnoses included aml (n = 15), all (n = 11), myelodysplastic syndrome (n = 6), other malignancies (n = 4) and anemias (n = 2). donors were either hla matching siblings (n = 11) or mud (n = 27). grafts were either bmsc (n = 24) or pbsc (n = 14). conditioning included tbi (n = 31) or high-dose chemotherapy alone (n = 7). plasma tslp was measured by elisa (abcam) before transplantation, at the day of transplantation and at day +7, +14, +21 and +90 post-hsct. monocytes were counted daily, and t, b and nk cells were measured at day +30 and +90 using flow cytometry. about 35 (92%) patients engrafted; acute gvhd grade 2-4 was seen in 20 (53%) patients, and 16 (42%) patients developed severe cgvhd. oas was 57.9 %, trm 26.3% and relapse rate 15.8%. median plasma tslp levels increased from before conditioning (101 pg/ml) to reach a peak at day +21 (313 pg/ ml, p = 0.03), followed by a gradual decline. the plasma levels of tslp at day +21 were positively correlated with same-day monocyte counts (ρ = 0.58, p = 0.006). approximately half of the patients (n = 14) experienced an overall rise in tslp from baseline (100 pg/ml) to day +90 (328 pg/ml). this increase in tslp was not significantly associated with any transplantrelated baseline characteristics. however, patients, who had an increase in tslp levels from baseline to day +90, had a significantly higher risk of extensive cgvhd compared to those in whom tslp levels at day +90 were similar or below baseline levels (cumulative incidence of cgvhd: 77% (increased tslp at day +90) vs 38% (normal/low tslp at day +90), p = 0.01). development of cgvhd was also associated with the nucleated cell dose infused (p = 0.04) and transplant using pbsc (p = 0.08). tslp plasma levels were not associated with acute gvhd, oas, trm, relapse rate or numbers of t cell, b cell or nk cells posttransplant. we have found that increased levels of tslp from baseline to day +90 were associated with an increased risk of extensive cgvhd. this association may be due to the ability of tslp to polarize the immune system toward a th2 response. importantly, the increase in plasma tslp levels was not associated with any transplant-related characteristics suggesting that tslp may be an independent predictor of cgvhd. these findings indicate that anti-tslp treatment may be a new approach to fight severe cgvhd. disclosure of conflict of interest: none. thymopoiesis following hct: a retrospective review comparing interventions for agvhd in a paediatric cohort c roberts 1 , am flinn 1 , ma slatter 1,2 , r skinner 2 , h robson 2 , j lawrence 2 , j guest 2 and ar gennery 1,2 1 institute of cellular medicine, newcastle university and 2 great north children's hospital, newcastle-upon-tyne, uk acute graft-versus-host disease (agvhd) is a life threatening complication of allogeneic haematopoietic cell transplantation (hct), treated with topical and/or systemic corticosteroids. in steroid-refractory agvhd extracorporeal photopheresis (ecp) can be effective. ecp exposes apheresed mononuclear cells to 8-methoxypsoralen and ultra-violet radiation. systemic corticosteroids and agvhd are damaging to thymic tissue. delayed immune reconstitution, especially of the t lymphocyte compartment, is associated with increased morbidity and mortality. 1 therefore, management strategies must be effective in treating agvhd but endeavour to minimise resulting thymic damage. we compare the effect of topical steroid therapy, corticosteroids and ecp on thymic reconstitution following hct in paediatric patients. statistical analysis was performed using the kruskal-wallis test. about 155 paediatric allogeneic hcts were performed between june 2010 and april 2016, at the great north children's hospital, newcastle for malignant and non-malignant disease. we reviewed computerised records to categorise patients into four groups: no agvhd, mild agvhd treated with topical steroid, agvhd treated with systemic steroid, agvhd treated with ecp. laboratory data were reviewed to provide values of naive (cd4+ and cd4 − )cd45ra+cd27+ t-lymphocytes at 3, 6, 9 and 12 months post-hct. values for thymic output for the ecp group were additionally recorded at 3, 6, 9 and 12 months during ecp. excluded were patients with no available data, those with o12 months follow-up, those with chronic gvhd, recipient of 41 hct or received dli post-hct. about 104 patients were included, 42 (40.4%) had no agvhd, 49 (47.1%) had agvhd treated topically or systemically, 13 (12.5%) had agvhd and received ecp. for analysis, the group treated with steroids were divided into those treated with topical therapy and those given systemic steroids. the median values of all groups at each time point (3, 6, 9 and 12 months) are shown ( figure 1 ). there was a significant difference between the rate of thymopoiesis (measured by the addition of cd4+ and cd4 − cd25+cd27+ cells) between all groups (no agvhd, agvhd treated with topical or systemic steroids, and agvhd treated with ecp) at 3, 6, 9 and 12 months post-transplant (p = 0.002, p o0.001, p o0.001, p = 0.001 respectively). further analysis excluded those treated with ecp (so including the no gvhd (n = 42), topical treatment (n = 23) and systemic steroid treatment group (n = 26)). at each time point p = 0.001, p = 0.019, p = 0.021and p = 0.019, respectively, demonstrating a statistically significant difference in time to thymopoiesis between those that had developed agvhd and those that had not. acute graft-versus-host disease (agvhd) is a significant cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. which despite first line treatment is well-established (esteroids), second line is not well defined. evaluate the results with different second line treatment used and the risk factors associated with of sr-agvhd. we review the clinical records of 281 consecutive patients undergoing allogenic hsct from 2005 to 2015 in our hospital. about 53% presented agvhd. sr-agvhd was defined as progression after 3 days, no clinical change in 5 days or incomplete response after 14 days of treatment. about 34 patients (25%) met s235 criteria for sr-agvhd. there were no significant differences between both groups (sr-agvhd vs no sr-agvhd) respect to age (recipient/donor), unrelated donor, prophylaxis of gvhd, cd3 lymphocyte and cd34 cell. the median time between transplantation and agvhd diagnosis was 25 days (7-123). patients who did not respond on fifth day of steroid treatment have an 80% rate mortality vs 33% on no sr-agvhd group (p = 0.03). sr-agvhd: 34 patients presented sr-agvhd and this was related to: hla mismatch (35% sr-agvhd vs 15% no sr-agvhd, p = 0.008), male recipient/female donor (38% sr-agvhd vs 17% no sr-agvhd, p = 0.02) and advanced underlying disease (56% sr-agvhd vs 22% no sr-agvhd, p = 0.001). second line: basiliximab (82.4%); extracorporeal photopheresis (ep) (2.9%), timoglobulin (8.8%) and others therapies (5.9%). two patients (6%) obtained complete response (cr) and 10 patients (29%) partial response (pr). global response (cr, pr) after second line (mainly basiliximab) showed better overall survival (p = 0.009). third line: basiliximab (8.3%); ep (41.7%), mesenchymal cells (msc) (8.3%), ruxolitinib (16,7%) and others (24.9%). ruxolitinib improve gvhd cutaneous and hepatic but not intestinal. the best results were achieved with ep (2 cr, 1 pr) and basiliximab/msc (1 pr, respectively). only patients who achieved cr survived. fourth line: msc (50%)/ ruxolitinib (50%) does not improve the prognosis. no serious adverse effects were observed with msc therapy, basiliximab and ep. about 14% of patients showed cmv reactivation with basiliximab. about 27 patients died (80%), 21 patients with early mortality ( o6 months) due to refractory agvhd (40%) or secondary infections (60%). overall survival at 6 months and 2 year was 28 ± 8% and 0%, respectively. in multivariate analysis the main factor for trm was the steroid-refractory vs steroidsensitive (hr 2.00, 95% ci 0.91-4.39; p = 0.083) and was unfavorable the association of hepatic and intestinal agvhd (hr 2.24, 95% ci 0.90-5.57; p = 0.082) no sr-agvhd: 115 patients. trm-100 was 18% (n = 7), mainly due to infection (71%). trm-1 year was 37% (n = 15), mainly by gvhd (40%) and infections (40%). median follow-up of 26 months, os-6 months and 2 year were 84 ± 3%/75 ± 4%, respectively. trm was associated with not obtained cr/pr after second line (p = 0.001), no cr after third line (p = 0.018) and relapse of gvhd despite achieving cr initially (p = 0.004). in our series only the patients that obtained cr/pr after second-line or cr after third-line improved os. the best results in sr-agvhd were obtained with basiliximab and extracorporeal photopheresis. trm was associated with relapse of gvhd and advanced disease to the transplant. randomized clinical trials are needed to assess different treatment modalities for sr-agvhd. [p226] disclosure of conflict of interest: none. extracorporeal photopheresis (ecp) is a therapy for steroidrefractory chronic graft versus host disease (cgvhd). therapeutic response to ecp has been linked with a progressive increase in circulating granulocytic myeloid-derived suppressor cells (g-mdsc) in acute gvhd, but not in cgvhd 1 . low density neutrophils (ldn) phenotypically resembling g-mdsc (putative g-mdsc) show marked flux in cgvhd patients receiving ecp, and a reduction in their frequency is associated with a sustained therapeutic response to ecp 2 . recent data has identified lectin-type oxidized ldl receptor-1 (lox-1) as a specific marker of ldn with t-cell (tc) suppressive activity 3 . using this marker we have conducted a cross-sectional study to assess whether putative g-mdscs in this patient cohort have suppressive activity. about 15 patients with steroid refractory or steroid-dependent cgvhd (mean treatment duration of 9 months) receiving ecp and 8 healthy controls were recruited. patients had gvhd affecting skin (15/15), liver (3/15) and gut (2/15). pbmc were isolated and immunophenotyped by flow cytometry for markers of g-mdscs (cd14 − ve , cd16, cd66b, hla-dr − ve , cd33 int ) and lox-1 expression. suppressive function was determined by measuring the inhibition of proliferation of anti-cd3/cd28-activated purified cd3 tc from healthy donors by 4-day co-culture with g-mdscs from patients. statistical analysis was conducted using graphpad 6. ecp patients had substantially greater frequencies of circulating putative g-mdsc than healthy controls (p o0.0001; median: 13% and iqr 2%-32% vs 0.2% and iqr 0.1%-0.6%, respectively). while there were substantially greater frequencies of circulating lox-1 + cells in pbmc from ecp patients than healthy controls (p o0.0001; median: 1.5% and iqr 0.39%-35% vs 0.053% and iqr 0.029%-0.062%, respectively), these were mainly the minority population within the putative g-mdsc fraction with no significant difference between ecp patients and healthy controls in the proportion of lox-1 + cells (29% ± 16% vs 21% ± 9%, respectively). ecp had no significant effect on circulating putative g-mdsc frequency measured before and the day after treatment (median: 8.4% and iqr 4%-44% vs 16% and iqr 6%-25%; n = 11, respectively) nor on lox-1 frequency (median: 1% and iqr 0.29%-12% vs 2.8% and iqr 0.88%-7.3%; n = 9, respectively). at a tc:g-mdsc ratio of 1:1, isolated g-mdscs from ecp patients suppressed cd3 tc proliferation (mean ± sd: 52% ± 23 %; n = 14). however, the potency of suppression was highly variable (min-max: 18-82%). the pattern of lox-1 expression suggests that only a subset of putative g-mdscs in ecp patients are suppressive and may explain why suppressive function in this cell fraction is so highly variable. however, the relatively high frequency of lox-1 cells in this patient cohort might contribute to immunosuppression resulting in increased susceptibility to opportunistic infections. according to the revised eortc/msg criterion, 29 patients were diagnosed with cns-ifd. among those patients without cns-ifd (3826 patients), cns-ifd were matched in a 1:3 ratio for analyzing the risk factors of cns-ifd. and among 594 (15.4%) patients who occurred pulmonary ifd without cns involvement, 87 patients were selected as control group for analyzing the risk factors associated with involvement of cns in pulmonary ifd. we selected the control group using a 1:3 ratio matched-pair method with the variates of (1) age; (2) sex; (3) underlying disease. we retrospectively reviewed 29 patients complicated with cns-ifd after hsct in our single center during a 10 years period. most patients received haploidentical stem cell transplantation. the median onset time of cns-ifd was 173 (24-972) after hsct, and most (82.7%) of them have prior pulmonary ifd. the most frequent pathogen was aspergillus, while no crypoccosis and candidas were found. the most common clinical presentation was space-occupying symptoms and signs. brain abscess were the most common imaging finding. prior pulmonary ifd (po 0.001, hr 62.746(95% ci, 14.28-275.27)) was the only risk factors associated with occurrence of cns-ifd. while poor response at 6 weeks (p = 0.045, hr 2.574 (95% confidence interval: 1.021-6.487)) was the only risk factor predicting the involvement of cns in pulmonary ifd. the response (complete and partial response) at 12 weeks and last follow-up was 27.6% and 20.6%, respectively. the overall survival was 24.2% at the last follow-up with a median 289 (27-3341) days after transplantation. in conclusion, patients with pulmonary ifd had higher risk of cns-ifd, especially in those with poor response after 6 weeks of treatment. and the prognosis of cns-ifd was very poor after hsct. disclosure of conflict of interest: none. adv may cause severe infections in hsct recipients, especially from unrelated donors or cord blood particularly in pediatrics. disseminated infections usually occur after digestive reactivations. at 3 mo.post-hsct, the incidence of adv digestive infection and viremia in pediatric hsct is about 30% and 15%, respectively. therapeutic strategies to control adv infections are limited to the use of infusion of cidofovir (cdv) or ex vivo anti-adv selected cytotoxic lymphocytes (ctl). however cdv is not labeled for adv treatment, presents a renal toxicity and has shown limited efficacy. specific-ctl remain difficult to produce. brincidofovir (cmx001, bcv, chimerix, usa) is an orally-available lipid conjugate of the nucleotide analog cdv that has demonstrated broad clinical antiviral activity against double-stranded dna viruses (that is, herpes-, adeno-, orthopox-and polyomaviruses. the drug has an increased bioavailability compared to cdv and has shown encouraging results. we report here the results obtained with this compound in 20 patients treated in six centers from january 2015. there were 20 pts (8m/12f), median age at hsct: 65 mo. (15-202). hsct indication was all in nine, pid in six, aml in two, fa in two and ibmf in one. donor was 9/10 or 10/10 mud in four and six pts, respectively; haplo-identical familial donor in 4; 5/6 or 6/6 unrelated cb in two and three pts, respectively; msd in one. stem cell source was bm for 11 pts, cb in 5 and pbsc in 4. two pts underwent a second hsct. cond' regimen were mac in 16 pts. all pts received either ex vivo or in vivo t-cell depletion. three pts presented with adv-disseminated disease, seven pts with blood + other site (throat, urine or stools) adv infection, three with adv-related gut disease, three with blood infection and three with gut infection. the remaining patient received bcv for jc viremia with fever. median time for virus infection diagnosis was d20 post-hsct (range: d-126 to d+300). about 13 pts experienced 24 other viral infection episodes after hsct (cmv: 8; ebv: 5; bk: 5; hsv: 3; hhv6: 2; influenzae: (1). about 12 pts received 1-6 injections of cdv prior to bcv treatment. one pt received specific-adv ctl before bcv without efficacy. the reason to switch from cdv to bcv was uncontrolled adv infection (n = 11) or cdvinduced renal failure (n = 1). two additional pts experienced renal impairment after cdv. about 14 pts received 1-4 lines of immunosuppressive therapies (including ecp) in addition to calcineurin inhibitor at time of bcv therapy due to grade iii and iv acute gvhd in seven and seven pts, respectively. median adv load at time of bcv initiation was 4.5log copies/ ml (range: 3-9) in blood and 5log copies/ml (3.3-7.5) in stools. median duration for bcv therapy was 3 weeks (range: 1-18). about seven pts with blood adv infection or disseminated disease experienced adv disappearance as well as four pts with gut disease or infection. three of them experienced adv infection relapse and received thereafter cdv, bcv or advspecific ctl. five pts presented with grade 2-4 diarrhea during bcv treatment. about 13 were alive at end point where seven died from septis (n = 2), multi-organ failure (n = 2), gvhd (n = 2) and adv disseminated infection (n = 1). adenovirus infections occur often in immunocompromized pts receiving concomitant nephrotoxic drugs that may avoid cdv use.bcv appears as efficient therapy against adenovirus infection in such pediatric pts since here 13 out of 20 pts where alive after adv infection and bcv treatment. in this study we retrospectively analyzed cmv reactivation determined by pcr and response to pre-emptive therapy in patients receiving an haplo (n = 68, 24%) comparing them with a control group of non haplohsct (110 mrd and 106 mud) (n = 216, 76%). median age was 52 years (range: 16-71), 56 for haplohsct and 52 for control group. conditioning regimen was myeloabaltive (mac) in 33.5% and reduced intensity (ric) in 66.5%. haplohsct characteristics: haplo conditioning was fludarabine (30 mg/m 2 or 50 mg/m 2 × 4 days in ric or ma regimen) and busulfan (3.2 mg/kg × 2 in ric or 3 days in ma) (19.1% mac, and 80.9% ric). cyclophosphamide-post was used for gvhd prophylaxis in 94%. median of days to reach more than 500 × 10 9 granulocytes and more than 20 × 10 9 platelets were 17 (13-31) and 22 (0-103), respectively. incidence of acute gvhd was 70% (grade i-ii 64.2%, and iii-iv 5.7%), with two steroid-refractory cases. cmv reactivation: 66.2% of haplohsct patients presented cmv reactivation, vs 29.8% in control group (p = 0.000). median number of cmv reactivation episodes was 1 in both groups. median time to cmv pcr detection was 35 days (4-70) and 40 (0-186) in haplohsct and control group respectively (p = 0.042). average maximum cmv iu by pcr was 17.499 in haplo vs 8.206 in the control group (p = 0.035). first antiviral pre-emptive therapy (valganciclovir in 65.7%) was effective in 82% in haplohsct vs 65% in control group (p = 0.064). main reason for antiviral treatment switch was failure in cmv iu reduction, and foscarnet was the most used therapy in refractory cases. twenty patients developed cmv disease (5 in haplo and 15 in control group) (gi disease 90% and pulmonary disease 10% in both groups). in a multivariate cox-regression model, receiving an haplohsct, serological cmv status (positive patient/negative donor), mac regimen and development of acute gvhd grade i/ii or grade ii/iv were variables associated with a higher risk of cmv reactivation. based on these results, haplohsct is associated with a higher cmv reactivation compared to non-haplohsct, despite a lower incidence of all other risk factors as agvhd or mac in the haplo group. although it is not statistically significant, response to pre-emptive therapy is higher in haplohcst and no differences in cmv disease were observed. disclosure of conflict of interest: none. although a number of patients with hiv infection and hematological disease have successfully undergone allogeneic hsct together with combination anti-retroviral therapy (cart), short and long-term outcomes remain not well known. we report the spanish experience treating hiv-infected adult patients with high-risk hematological malignancies with allogeneic hsct. we retrospectively reviewed 17 hiv-positive patients who received allogeneic hsct in three institutions in spain within geth (grupo español de trasplante hematopoyético y terapia cellular). seventeen patients have been transplanted between 1999 and 2015. median age was 44 (30-57), 82% male. diagnosis and transplant characteristics are summarized in table 1 . cumulative incidence of neutrophil and platelet engraftment were 88% at 30 days (median 15 days), and 76% at 60 days (median 13 days), respectively. with a median follow-up of 42 months (22-87), os and efs were 35%. trm was 17% at 12 months and 32% at 36 months. grade ii-iv agvhd rate was 41%, and moderate/severe cgvhd rate was 41%. all patients received cart. two patients showed severe toxicity related to interaction of immunosuppressive s238 drugs and protease inhibitors. about 76% of patients showed infectious complications. viral infections were the most frequent cause: cmv (9), bk (2), adv (1), hhv-1 (2), hcv (1), hhv-8 (1), parainfluenza (1). two patients had invasive aspergillosis and one patient presented disseminated tuberculosis. causes of death were: relapse (4), infection (3), gvhd (2) and toxicity (1). all surviving patients showed undetectable hiv load after hsct. allogeneic hsct is an effective therapy for high-risk haematological malignancies in patients with hiv infection, and long-term hiv suppression with cart is feasible. however, interactions between immunosuppressive agents and anti-retroviral drugs, high rates of significant gvhd, and frequent infectious complications account for a complex procedure in this population. selected hiv-infected patients with hematologic malignancies should be considered for allo-hsct when indicated, in experienced centers. disclosure of conflict of interest: none. clostridium difficile infection (cdi) is one of the most common causes of nosocomial infectious diarrhea in europe and usa, which results in high morbidity and mortality among hospitalized patients. allogenic hematopoietic stem cell transplant (hsct) recipients remain at high risk for cdi. incidence rate ranges from 2 to 27%. numerous risk factors including acute graft-versus-host disease (agvhd), hla matching status, conditioning-intensity, use of total body irradiation (tbi) may play an important role in the course of cdi in these patients. the aim of this study was to evaluate the prevalence of cdi in children, and to assess the influence of such factors as gender, age, diagnosis, hla matching status, conditioning-intensity, use tbi-containing regimen, source of graft (bone marrow/bm/ vs peripheral blood/pb/)or agvhd on course, duration of treatment and outcome in children undergoing hsct. between 2014 and 2015 a total of 342 hscts were performed in five polish pediatric transplant centers, including 267 allogeneic and 75 autologous. all patients were followed up to 6 months post hsct. we analyzed retrospectively 29 episodes of cdi infection in the group of 342 children. twenty-one of 29 children were diagnosed with hematological malignancies: acute lymphoblastic leukemia (all), acute myeloblastic leukemia (aml), myelodysplastic syndrome (mds), two were diagnosed with severe aplastic anemia (saa), one with chronic granulomatous disease (cgd) and 5 of them-with solid tumors. the median age was 9.3 years (range: 2.5-19.0 years). majority of patients underwent myeloablative conditioning protocol (24/29). in allogeneic setting 21/24 patients underwent mud-hsct, 2/24 pts msd-hsct and one patient was given a haploidentical pbsct. in this series, in 7 out of 24 cases bm was a transplant source, and pb in 17 out of 24. cdi was defined as having diarrhea that tested positive for c. difficile via pcr, cytotoxin assay, or dual enzyme immunoassays. kruskal-wallis test, wilcoxon test and χ 2 -test were used to estimate the influence of risk factors on severity of disease, duration of treatment and outcome. we observed 29 episodes of cdi (8.5 %) in hsct recipients: in 24 allotransplant recipients (8.9% of all transplants) and in 5 autotransplant recipients (6.7% of all auto-hsct). nine patients responded to therapy with metronidazole, seven patients responded to vancomycin alone, and in two patients rifaximine was administered. six children required adding second drug: vancomycin or metronidazole, five patients were not given any medications. there was no significant correlation between such factors as diagnosis, gender, age, conditioning regimen, hla matching, agvhd and severity of disease, and duration of treatment. recurrence rate was difficult to assess due to lack of data. we observed three deaths. one of them was connected with cdi. there was one 17-year-old boy with saa (mud-pbsct, hla 10/10) with no agvhd. the other two deaths were due to progression of s239 disease. cdi occurred in nearly 9% of pediatric patients undergoing hsct, surprisingly often in autologous hsct too (6.7%). almost all patients experienced mild cdi with adequate response to antibiotic therapy. cdi is a rare cause of death among transplant recipients. disclosure of conflict of interest: none. antifungal prophylaxis in high-risk paediatric patients with haematological malignancies: a monocentric experience k perruccio, i capolsini 1 , a carotti 2 , n albi 3 , l pitzurra 4 , a velardi and m caniglia 1 pediatric haematology oncology section; 2 haematology and clinical immunology section; 3 transfusion medicine and 4 the choice of antifungal prophylaxis in high risk paediatric haematological patients (according to the latest ecil6/seifem guidelines) remains an open question. a recent retrospective survey from associazione italiana ematologia oncologia pediatrica (aieop) showed that, in these patient categories, the only variable which significantly impacted on invasive fungal infection (ifi) occurrence was the presence or not of antifungal prophylaxis at the ifi onset (unpublished data). from january 2012, in our pediatric hematology oncology unit, 31 allogeneic hematopoietic stem cell transplantations (hsct) were performed (median age: 10 years; range: 6 months-23 years), mainly for acute leukaemia (median follow-up: 24 months; range: 4-50). patients received liposomal amphotericine b (n = 20), micafungin (n = 10), or fluconazole (n = 1) as primary antifungal prophylaxis until neutrophil recovery (41 × 109/l). seven patients developed acute gvhd (22%) which evolved in gvhd in 5 (16%). as outpatients, they continued with posaconazole (n = 17), voriconazole (n = 4), or micafungin (n = 10) until cd4+t-cell recovery (4200/cmm) or gvhd immune suppressive prophylaxis/treatment withdrawn. during the last year, according to ecil6/seifem guidelines1, we administered primary antifungal prophylaxis also to 10/12 high risk (hr) acute leukaemia patients. two patients with aml were treated with posaconazole, four patients with hr-all received micafungin, four relapsed all patients received micafungin (n = 2), or liposomal amphotericine b (n = 1), or posaconazole (n = 1). one aml patient was then transplanted; all relapsed patients are waiting for transplant. no differences were observed in terms of breakthrough proven/probable (pp)-ifi incidence, according to antifungal prophylaxis in the various patient groups. in particular, in the early phase, we observed a pp-ifi incidence of 10% in both treatment arms (micafungin vs liposomal amphotericine b, p = ns). in the late phase, we observed 1 case of pp-ifi who were receiving posaconazole as prophylaxis. overall survival (os) was 96%, with 4% mortality rate. in hr leukaemia patient group, we observed pp-ifi in the only two patients who were not receiving any antifungal prophylaxis at the ifi onset. antifungal prophylaxis is strongly recommended in paediatric patients with haematological malignancies who are at high risk of ifi. the choice of antifungal drug depends on the treatment phase, drug interactions (particularly for azoles), patient compliance and clinical conditions which interfere with intestinal absorption. in our experience, as no differences were observed in term of efficacy, micafungin resulted the best choice in terms of tolerability, toxicity, compliance and cost saving. antifungal prophylaxis with micafungin and bridging to inhaled liposomal amphotericin b after engraftment in patients undergoing allogeneic hematopoietic stem cell transplantation d rivera* ,1 , c de ramon 1 , a avendaño 1 , j carrillo 1 , d caballero 1 , l lopez 1 , i ormazabal 1 , a navarro 1 , a martin 1 micafungin is an effective antifungal for prophylaxis, active against candida spp. including those resistant to other antifungals (c. glabrata and c. krusei) and also active against aspergillus spp. guidelines focused on antifungal prophylaxis, recommend its use during preengraftment and early postengraftment period in allogeneic hematopoietic stem cell transplant (allo-hsct) recipients. moreover, its profile of low drug interactions and side effects, makes it a suitable alternative for patients who need concomitant treatments, present hepatic insufficiency and in those who do not tolerate oral drug administration. the addition of inhaled liposomal amphotericin b (lamb) after engraftment, provides an alternative way to effectively prevent mold infections, that are acquired mainly by inhalation. inhaled lamb has good tolerance with absence of drug interactions and low toxicity. the aim of this study was to describe the experience in the hsct unit of the university hospital of salamanca with micafungin and lamb as primary prophylaxis in patients undergoing allo-hsct with reduced intensity conditioning (ric) and graft-versus-host disease (gvhd) prophylaxis with tacrolimus and sirolimus. thus evaluating efficacy and tolerability in our population. retrospective observational study from january 2013 to august 2016, including all adult patients undergoing allo-hsct with ric and gvhd prophylaxis with tacrolimus and sirolimus, in whom an azole derivative is not indicated, due to drug interactions. therefore received prophylaxis with micafungin during the preengraftment period and bridging to lamb after engraftment at discharge, and continuing it during the first 100 days post-transplant. data from 106 patients from our hsct unit. ten (9.4%) patients who had invasive fungal infection before undergoing allo-hsct, and 16 (14.8%) patients who received prophylaxis with drugs other than micafungin-lamb were excluded. underlying disease was grouped by leukemia in 44 (41.5%) patients, lymphoma in 23 (21.7%), myelodysplastic syndromes in 18 (17%), multiple myeloma in 11 (10.4%) and other diseases in 10 (9.3%) patients. eighty patients underwent peripheral blood allo-hsct, of whom were related donor in 40 (50%) patients and unrelated donor in 40 (50%). prophylaxis with micafungin in 80 (75.5%) patients, dose of 50 mg per day, with a mean of 19 days (±6 days) with postengraftment bridging with lamb 24 mg weekly, continuing it during the 100 days posttransplant. days of neutropenia during preengraftment, o14 days in 24 (31.2%) patients, 14-28 days in 49 (63.7%), more than 28 days in 4 (5.2%). during follow-up there were three cases (3.8%), two catheter related candida infection, and one esophageal candidiasis. there were no reported aspergillosis cases (possible, probable or proven), according to the european organization for research and treatment of cancer (eortc) criteria. finally, prophylaxis with micafungin and inhaled lamb, was considered an effective and safe strategy in 77 (96.3%) patients, with no side effects reported. according to our experience with micafungin and the addition of inhaled liposomal amphotericin b, the results indicate that, this is an appropriate alternative for antifungal prophylaxis, in patients undergoing allo-hsct, because of their efficacy, few side effects and drug interactions. disclosure of conflict of interest: none. recipients of allogeneic hematopoietic cell transplantation (allohct) are at high risk of developing invasive fungal infections (ifi). in the early phase (o100 days) after allohct, the use of antimold prophylaxis has been generalized, although there is no consensus on the best therapeutic strategy. the use of nebulized liposomal amphotericin b and fluconazole has been shown to be effective, safe and associated with low economic costs in lung transplantation 1 . however, the use of this prophylactic strategy in the early phase of allohct setting has not been evaluated. we included all consecutive patients who received their first allohct in our center from january 2013 to august 2016 and who underwent antifungal prophylaxis according to the prospective ambineb protocol (nebulized liposomal amphotericin b 24 mg administered three times per week as loading dose and once per week and fluconazole 200 mg per day until day +90). patients with a previous ifi were excluded. patients with graft-versus-host disease (gvhd) receiving high dose corticosteroids were allowed to be changed to voriconazole or posaconazole at physician's discretion. the primary objective of the study was the incidence of ifi at day +180. the secondary objectives were to assess adherence and toxicity of the ambineb protocol. only cases with proven or probable ifi according to eortc-msg criteria were considered. a multidisciplinary team of experts in hematology, infectious diseases, microbiology and radiology prospectively evaluated and categorized each case. we included 102 patients with a median age of 50 years (range: 18-70) and a median follow-up for survivor of 14 months (range: 2-47). patients received allohct mainly for acute leukemia (55%), non-hodgkin lymphoma (16%) and myelodysplastic syndrome (12%) . patients received allohct from hla unrelated (59%) or related donors (28%) mostly using a reduced intensity conditioning (57%). graft-versushost disease (gvhd) prophylaxis was performed with calcineurin inhibitors mainly in combination with sirolimus (57%) or methotrexate (17%). after the comprehensive review, only one case of proven or probable ifi at day +180 was diagnosed. prophylaxis with ambineb was completed in 75 patients (74%) while 27 (26%) stopped the treatment. the most frequent causes of discontinuation were possible ifi (6%), gvhd (5%), admission in intensive care unit (5%) and toxicity (6%) (figure) . ninety-four patients (92%) did not have adverse advents associated with ambineb. eight patients presented organ toxicity which was at least partially attributed to ambineb, including gastrointestinal symptoms (n = 4), liver function test abnormalities related to fluconazole (n = 3) and cough (n = 1). of the 27 patients who discontinued ambineb, 22 (81%) were switched to other antifungal drugs including (echinocandins (12, 54%) posaconazole (4, 18%), voriconazole (2, 9%) or others (4, 18%)). overall survival and non-relapse mortality of all patients at median follow-up were 64% (95% ci 58-69) and 25% (95% ci 20-30), respectively. the combination of nebulized ambisome and fluconazole is effective in preventing ifi in the early phase of allo-hct and is associated with high adherence and low toxicity. neutropenia-related infections is a common complication of apsct in patients (pts) with mm and dlbcl. our study aims are to: (1) assess antibacterial susceptibility patterns of isolated organisms, to guide antibiotic prophylaxis (2) identify the epidemiology of bacteremia with susceptibility patterns to direct empiric therapy of febrile neutropenia (3) assess the interval between the occurrence of neutropenia and the isolation of resistant bacteremia to identify the best timing to start prophylaxis (4) identify contributing factors for the development of bacteremia and mortality. our retrospective study included 191 adult pts who underwent apsct for mm and dlbcl between 2005 and 2015. we recorded the following: age, gender, basic illness, comorbidities, number of cd34 + cells infused, a central venous catheter, duration of neutropenia, diarrhea and mucositis, mechanical ventilation, positive bacterial cultures with susceptibility profiles and history of broad-spectrum antibiotic intake for more than 72 h for the past 3 months on hospital setting, and mortality. statistical analysis was carried out using spss (version 19). about 102 isolates were obtained: urine (46.5%), blood (28.5%), sputum (9%), wound (7%), venous catheter (7%) and stool (2%). gram-negative (gn) species were predominant (73.5%) with e. coli (32.5%), klebsiella (k) (11%) and pseudomonas (pseudo) (11%). isolates sensitive to third generation cephalosporins (3gc) represented 83% of the enterobacteriaceae (entero) including 78% in e. coli and 73% in k. all entero isolated were susceptible to carbapenems (carba), pipercillin/ tazobactam (pip/taz), amikacin and ciprofloxacin (cipro). all pseudo (n = 11) and acinetobacter (n = 4) isolates were susceptible to carba, pip/taz, amikacin, cipro, colistin and tigecycline. as for gram-positive (gp) bacteria (26.5%), coagulase negative staphylococci (cns) were predominant (14%) . oxacillin susceptibility reached 29% and two isolates methicillin resistant s. aureus were identified. all gp were susceptible to glycopeptides. a total of 29 bacterial isolates were identified (29 episodes of bacteremia) from 24 pts. gn were predominant (72.5%) with e. coli being most common (24%). all gn were susceptible to 3gc, carba, pip/taz, amikacin and cipro. as for gp (27.5%), cns predominated (24%) including 28% oxacillin-susceptible causing seven episodes of bacteremia with six central line-associated. no glycopeptide resistance was identified. none of the clinical features and pts' characteristics reached statistical significance as risk factor for bacteremia. however, the need for mechanical ventilation and mortality were higher in bacteremic vs non-bacteremic pts (16.7% vs 3%, p = 0.004, and 12.5% vs 3%, p = 0.036, respectively). all bacteremic episodes occurred after developing neutropenia (median = 2.5 days, range: 1-9) except for one case of clabsi caused by e. coli occurring 1 day before neutropenia. pip/taz was prescribed in 21% of bacteremia episodes followed by quinolones (11%) and carbapenems (4%). no previous use of third and fourth generation cephalosporins was observed. we recommend quinolone prophylaxis in apsct pts. for empiric therapy, antibiotics recommended by international guidelines including, cefepime and pip/taz still fit. thus, we could spare the use of carba and other last-resort antibiotics to other conditions. we also recommend continuous surveillance of resistance. disclosure of conflict of interest: none. fever is an almost universal complication in patients undergoing autologous stem cell transplantation (asct), however, microbiological documentation is only achieved in 20-30% of such febrile episodes (fe). 1 this low diagnostic efficiency makes epidemiological assessment in transplant units difficult, and may lead to a suboptimal empirical treatment. we have studied the utility of strict blood culture (bc) extraction as a mechanism to improve microbiological documentation of fe in these patients. we conducted a retrospective study over 66 consecutive asct performed in our centre between june 2015 and may 2016 (1 year). about 33 patients were male and 27 female, with age between 27 and 72 years (mean 57.8). diagnosis was 4 hodgkin lymphoma, 27 non hodgkin lymphoma and 35 multiple myeloma. ascts were performed in reverse isolation conditions, in rooms equipped with hepa and pall filters. prophylaxis against herpes virus and p. jirovecii with acyclovir and pentamidine was used. no prophylaxis against gram negative bacteria or filamentous fungi was performed. bc were extracted at the beginning of every fe and every 48-72 h if fever persisted (or more frequently, following clinical criteria). blood samples from intravascular devices and peripheral blood were collected in two bactec bottles each (for aerobic and anaerobic microorganisms). complementary diagnostic techniques and empiric antibiotic therapy were performed following our institution's guidelines. fe were classified in microbiologically documented infections (mdi), clinically documented infection (cdi) and fever of unknown origin (fuo) following his criteria. 2 85 fe were studied (average 1.28 fe per patient, 6.04 days of fever duration per fe). about 28% of fe were classified as mdi, 26% as cdi and 46% as fuo. mdis were cause by gram positive bacteria (56%), gram negative bacteria (26%) and polymicrobial infections (17%). no viral or fungal infections were observed. an average of 7.2 bc per fe and 7.6 per patient were extracted. the proportion of fe classified as mdi was related to the number of blood cultures extracted during the episode. only 8% of fe with three or less bc extracted were classified as mdi, 21% if 4-6 bc were extracted, and 55% if 7-9 bc were extracted (p o0.05). no significant difference in proportion of mdi classified fe between the extraction of 10 or more bc and 7-9. all patients were discarded in good clinical conditions. according to our experience, a strict 7-9 blood culture extraction is related to a high rate of microbiological documentation of febrile episodes. moreover, we have not observed the rise in gram negative bacteria reported by other studies and gram positive cocci persist as the main infection cause in our centre. candida which has been traditionally related to duration of neutropenia, emerges as a pathogen beyond the aplastic period in allogeneic haematopoietic cell transplantation (allohct). in the setting of alternative transplants and aggressive immunosuppressive therapy, these infections are a challenging problem. there is scarcity of data regarding the significance of breakthrough candidaemia in allohct. to that end, we aimed to determine the incidence, clinical and microbiological characteristics and outcome of candidaemia in allohct recipients. we studied consecutive allohct recipients from january 2014 to june 2016. blood cultures were obtained from peripheral vein or central venous catheters (cvcs) routinely and on febrile patients. well-known risk factors for candidaemia were studied: neutropenia, type of transplant, moderate to severe graft-versus-host disease (gvhd) and coexisting infections. among 108 allohct recipients, we identified seven patients with candidaemia: five post matched unrelated (four myeloablative and one reduced intensity conditioning) and two post haploidentical transplant. in median time of 3.5(1.3-8) months, 20 episodes of candidaemia were noted, despite antifungal prophylaxis with echinocandins or azoles. infections with non-albicans candida spp. occurred more frequently (19/20) and c. parapsilosis was the predominant microorganism (11/19). other species were isolated: c. famata (5), c. krusei (2) and c. haemulonii (1). all candida spp. isolates were phenotypically susceptible to antifungal agents already administered to patients. there was no resistance to echinocandins indicated by minimum inhibitory concentrations (mics). all patients had severe acute or late-onset gvhd with intestinal involvement and cvcs prior to candidaemia. although cvcs were removed in 7/7 and patients were treated with echinocandins, new cvcs were re-contaminated in 4/7 with the same or other species. all patients presented well known risk factors for candidaemia (use of broad spectrum antibiotics due to severe bacterial infections, total parenteral nutrition due malnutrition, long-term high-dose corticosteroids and other immunosuppression), but no neutropenia. one patient survived, whereas five patients succumbed to gvhd and multi-organ failure and one patient to sepsis due to bacteremia. candidaemia was observed in non-neutropenic patients with agvhd and cvcs on antifungal prophylaxis, despite difficulties in diagnosis due to poor sensitivity of blood cultures. the epidemiology of candidaemia has changed in the last decade and its risk is more diverse and complex. the irreversible intestinal gvhd lesions might be the main source of candida in patients receiving antifungal prophylaxis. our data show that candidemia remains an important issue in profoundly immunosuppressed patients contributing to excessive morbidity. our aim was to compare the rate of neutropenic sepsis, defined as fever of 438 o c and a neutrophil count of o0.5 × 10 9 /l, before and after the introduction of ciprofloxacin prophylaxis. one hundred and eight adult patients, of which 60 had acute myeloid leukaemia, 17 had acute lymphoblastic leukaemia, 12 had lymphoma and 19 had other haematological malignancies, were identified through our admission database. of these 108 patients, 48 received oral ciprofloxacin during their neutropenic phase. the median duration of neutropenia was 13 days in both the no-prophylaxis and ciprofloxacin groups. there was a significant reduction in the rate of neutropenic sepsis from 88.3% (53/60) in the no-prophylaxis group to 68.8% (33/48) in the ciprofloxacin group (p = 0.012). prolonged infection, suggested by the use of broad-spectrum antibiotic treatment for more than 10 days, was more common in the group which did not receive prior ciprofloxacin prophylaxis (45.0% vs 20.8%, p = 0.009). rate of intensive care admission (15.0% vs 0.0%, p = 0.005) was also reduced by the use of ciprofloxacin. however, there was no significant difference in the length of stay (mean of 28 vs 25 days, p = 0.187), or in the 30-day infection-related readmission rate (17.9% vs 13.3%, p = 0.536) between the two groups. in terms of the cause of neutropenic sepsis, escherichia coli, klebsiella pneumoniae and pseudomonas aeruginosa were the most common bacteria isolated from cultures in the no-prophylaxis group. eighty percent of these organisms showed sensitivity to ciprofloxacin. in the ciprofloxacin group, staphylococcus epidermidis was the most frequently found bacteria. with regards to treatment related adverse effects, none of the patients who received ciprofloxacin prophylaxis developed clostridium difficile diarrhoea. in conclusion, ciprofloxacin is still an effective antibacterial prophylaxis during neutropenia following allogeneic stem cell transplantation. clinicians should have a high suspicion of a gram-positive infection in patients who develop neutropenic sepsis on ciprofloxacin prophylaxis. disclosure of conflict of interest: none. s243 hematopoietic stem cell transplantation (haplohsct) to cure leukemia, malignancy and some inherited diseases, different additional reasons interfere microbiota metabolism and integrity. among them are radiation and chemotherapy, mucositis, infection and graft versus host disease (gvhd). the curative mechanism of fmt is based on the ability of donor intestinal microbiota to substitute and to provide all necessary functions of altered patient's microbiota. three patients (3, 10 and 28 years old) after haplohsct, who observed pseudomembranous colitis (toxin b-positive) as gvhd of intestine outcome, were enrolled to the study and performed fmt. relatives (mother, father and brother) were used as microbiota donors. donor and patient examination have included routine clinical and biochemistry laboratory data, microbiota cultural methods, pcr of most common intestinal microorganisms. additional for patient-level of fecal calprotectin by elisa was tested, identification of drug resistant bacteria and histology of intestine were made. patient's preparation for fmt included-probiotic (inulin) administration 72 h prior procedure, discontinuation of all antibiotics 24 h prior procedure and antiemetics (5ht3agonist), prokinetics and proton pump inhibitor. delivery of donor's microbiota was performed in two consecutive steps under total intravenous anesthesia: with esophagogastroduodenoscopy-to the duodenum; with colonoscopy-to the caecum. all patients observed complete clinical response in 14-28 days after fmt (table 1 ). in 10 days we have revealed significant quantitative and qualitative changes in microbiota composition, which was matched to donor's microbiota. in 35 days after fmt we identify microbiota changes in oropharynx and urogenital tract similar to donor microbiota. this leads to substitution of multidrug resistant klebsiella pneumoniae strains by drug sensitive microorganisms and helps to treat severe infection complications after haplohsct. platelet aphereses were carried out in 83 donors (50 males and 33 females with median age 38.5 years) using haemonetics instrument with simultaneous leucoreduction. quantitative detection of cmv, ebv and hhv-6 dna was performed by multiplex real-time quantitative pcr kit (interlabservice, russia) in donors' whole pb, plasma and platelet aphereses at the time of platelet collections. viral load in hsct recipients was monitored weekly after hsct and 7 days before hsct by the same pcr kit. lower limit of detection (llod) of the applied kit for all viruses was 500 copies/ml. in specimens of platelet donors we additionally performed ultra-sensitive pcr with llod 100 copies/ml. only one patient (2.4%) was cmv-positive by pcr prior to hsct. cmv reactivation after hsct ⩾ 500 copies/ml was noted in whole pb of 9 patients (22.0%) with median time of 41 days (range: 0-71). donor source in cmv-reactivated patients was as follows (3 mud, 1 msd, 3 haplo). cmv viremia ⩾ 1000 copies per ml was detected in seven patients (17.1%). cmv disease was found in five cases (12.2%). none of patients were positive by pcr for ebv or hhv-6 prior to hsct. ebv reactivation ⩾ 500 copies/ml was found in six cases (14.6%), ⩾ 1000 copies/ml in 5 (12.2%) with median time of 27 days (range: 20-56). no signs of ptld or other ebv-dependent clinical symptoms were observed. hhv-6 level after hsct ⩾ 500 copies/ml was detected in 17 patients (41.5%), ⩾ 1000 copies/ml in 11 ones (26.8%) with median time of reactivation 19 days (range: 13-36). hhv-6 disease was observed in one patient. none of platelet donors were cmv-positive in plasma, whole blood or platelet aphereses products. ebv ⩾ 500 copies/ml was detected in whole pb specimens of five platelet donors (6.0%). application of ultra-sensitive pcr revealed low level of ebv-viremia in additional 11 pb cases with median ebv level 260 copies/ml (range: 115-410). none of platelet donors have any clinical signs of ebv disease. there is no any ebv-positive case among platelet concentrate specimens. in two cases low levels of hhv-6 was found in a whole pb (110 and 180 copies/ ml). none of hhv-6-positive case was observed among plasma and platelet concentrate specimens. despite high incidence of cmv, ebv and hhv-6 reactivation after hsct in pediatric patients we could not show that source of viral reactivation was contamination of platelet apheresis products by donorderived herpes viruses. disclosure of conflict of interest: none. conventional respiratory virus (crv) infections are known to be major causes of morbi-mortality after stem cell transplantation (sct) due to the increased risk of progression to lower respiratory tract infection (lrti) in this setting. risk of developing severe lrti is mostly related to factors specific to the patient and the underlying disease, although the intrinsic virulence of crvs may also determine their outcomes. we conducted a single-center retrospective study including all adult sct recipients who had crv disease (defined as patients with symptomatic respiratory disease and crv identification) during a 7-year period (2009-2016) with the main objective of evaluating epidemiological changes over time and their association with infection outcomes. during the study period 137 episodes of crv disease were diagnosed in 104 patients (median age: 48 years, 58% male, 49% aml or mds as baseline disease). 83 patients (80%) received an allogeneic-sct (allo-sct) (30% had a prior sct) and 21 (20%) an autologous sct. crv disease was diagnosed at a median of 343 days after sct (range: 1-4361), with 33 cases (24%) occurring before day +100. during the infectious episode 31 of 83 allo-sct recipients (37%) had active gvhd and 39 (47%) were under s244 prednisone (pdn)415 mg/kg. most of the patients (84%) had symptoms compatible with an upper respiratory tract infection (urti), with 15 of them (11%) progressing to a lrti, while 7 (5%) had a lrti only. hospital admission was required in 46 episodes (33%) with a median duration of hospitalization of 11 days (range: 1-30), 17% required supplemental oxygen, 6% were transferred to the intensive care unit and 4% required mechanical ventilation. the most commonly identified pathogens over time are shown in figure 1 . twenty-four cases (17%) had concomitant bacterial or fungal infections. influenza a virus was the most frequent crv detected (49 episodes, 36%) followed by human respiratory syncytial virus (37 episodes (27%) and human parainfluenza virus type 3 (18 episodes, 13%). during the 2009 flu pandemic, only 2 of the 13 crv infections diagnosed in sct recipients (15%) were associated to influenza a virus h1n1. antiviral treatment was started in 62 episodes (45%), antibiotics in 56% and combined therapy in 25% during a median of 7 days (range: 4-21). the rv resolved in 129 cases (94%) at a median of 12 days (2-72) from onset, with crv being considered the leading cause of death in only 3 patients (3% of all cases and 3/22 (14%) in those with a lrti). predictors of severe crv infection (including icu admission, need for supplemental oxygen, need for mechanical ventilation requirement or death) in multivariate analysis were lymphocyte count o 200 cells/μl (hr: 4.2, 95% ci: 4-26, p = 0.01) and co-infection with other pathogens (hr: 4.8, ci95%: 1.5-16, p = 0.00). no specific crv nor period post-sct of the infection influenced the risk of severe infection. our results confirm that crv infections are a frequent cause of morbidity after sct with a high need for hospital-based care. temporal changes in the principal circulating crvs has been identified during the 7-year study period, with influenza a virus being the most common. profound lymphocytopenia and presence of co-pathogens are associated with infection severity. [p246] disclosure of conflict of interest: none. the 127 consecutive hsct performed from 2012 to 2016 are being analysed retrospectively. out of them 60 (47%) performed in hepa filter room and 67 (53%) in non-hepa filter room, criterion was purely financial to make this decision. 96/127 (76%) were allogeneic and 31/127 (24%) were autologous hscts. blood cultures both bacterial and fungal were taken at onset of fever and with every change of antibiotics till patient became afebrile. chest x-ray and if required hrct chest was done for all patients who had respiratory complaints. we did not use antibacterial prophylaxis; however, antifungal prophylaxis was administered along with conditioning; and at the onset of fever systemic antibiotics were started. antifungal agents were added if fever persisted for 3 days pre empatively. extremely well trained nurses were looking after both the groups. all treatment protocols, antibiotic/antifungal policies were same in both the groups. median time for neutrophil engraftment was 12 days in hepa filter room and 13 days in non-hepa filter room. total 4/127 (3 %) patients did not engraft till 30 days. out of them 02/60 (3 %) were in hepa filter room and 2 /67 (2.9 %) in non hepa filter room. blood cultures were positive in total 20/127 (16%) patients, 17 were positive for bacterial and 3 for fungal organisms. in hepa filter hsct 11/60 (18%) were positive and in non-hepa filter hsct were 09/76 (13%) positive. total 34/127 (27%) patients developed pneumonia, out of them 14/60 (23%) were in hepa filter and 20/67 (30%) hsct were in non-hepa filter room. statistically not significant. no central venous access cather issues or infections were documented in any groups gr 2-4 agvhd : hepa rooms 11/60 (18.3%),non hepa rooms 14/67 (20.8%) :was not statistically significant the 30-day mortality was 25/127 (20%), 07/60 (12%) patients were from hepa filter rooms and 18/67 (27%) were from non-hepa filter rooms. cost : average cost of allogeneic hsct in hepa room : usd 22 000. average cost of allogeneic hsct in non hepa room: usd 13 700. average cost of auto hsct in hepa room: usd 12 000. average cost of auto hsct in non hepa room : usd 8500. incidence of blood culture positivity & incidence of pneumonia was not different. these are two very important issues in outcome of hsct. agvhd incidence did not depend on the room type. these are significant findings from this study. results were slightly better in hepa filter rooms compare to non-hepa filter rooms, which was statistically insignificant. our study had few confounding factors hence we could not be concluded that hepa-filtered rooms are not necessary. nevertheless, our experience suggests that availability of dedicated hepa units with special air-handling equipment should not be considered a critical and essential precondition for providing allogeneic hsct to patients even in developing world with financial constraints. these would otherwise succumb to potentially curable hematological illnesses with background of financial constraints and wait list of hepa filter rooms. early hsct in a clean patient in non hepa rooms is extremely cost effective with comparable outcomes. nursing care, experience of the team, experience in hsct program & well established protocols are more important in outcome of hscts. disclosure of conflict of interest: none. we present five cases of cytomegalovirus (cmv) pneumonitis occurring in patients after recent allogeneic stem cell transplantation (allohsct). these cases were complicated by an organising pneumonia (during the recovery period) with a predominantly central peribronchial pattern. all patients presented with evidence of active cmv pneumonitis which was treated successfully with anti-viral therapy but was followed by persistent severe dyspnoea, cough and hypoxia. high resolution computed tomography (hrct) imaging showed widespread central peribronchial consolidation with traction bronchiectasis. in most cases there was a marked clinical and physiological improvement after treatment with systemic corticosteroids. however, in all patients the lung function remained abnormal and in some cases imaging revealed a fibrosing lung disease. these cases represent a previously undescribed central peribronchial pattern of organising pneumonia complicating cmv pneumonitis that can result in chronic lung damage. disclosure of conflict of interest: none. cytomegalovirus reactivation in pediatric acute leukemia after stem cell transplantation has an effect on relapse and survival in aml but not in b-precursor all j-s kühl 1 , l sparkuhl 1 and s voigt 1 department of pediatric oncology/hematology/sct, charité universitätsmedizin berlin, berlin, germany several studies have indicated better survival after stem cell transplantation (sct) for acute leukemias, especially acute myeloid leukemia (aml), in case of cytomegalovirus (cmv) reactivation. here, we investigated if cmv reactivation had an impact on survival after sct for aml or acute lymphoid leukemia (all) in children. 177 pediatric allogeneic stem cell transplant recipients from our institution who received myeloablative conditioning were included. transplant indications included aml, t-all and b-precursor all. cmv reactivation was correlated with relapse, mortality as well as acute graft-versus-host disease (gvhd) and was analyzed by fisher's exact test or χ 2 -test (if n4100). from the 177 patients included, 42 were transplanted for aml (24%), 22 for t-all (12%), and 113 for b-precursor all (64%). mortality and relapse rates (27-37% and 18-26%, respectively), cmv reactivation rates (21-36%) as well as numbers of negative cmv serology status (19-32%) of donor and recipient were comparable between different acute leukemias. when patients were analyzed altogether, cmv reactivation had no effect on relapse rates or mortality. however, a tendency towards fewer relapses after cmv reactivation was observed in aml patients (no relapse (0%) with cmv reactivation vs 11 relapse cases (33%) without cmv reactivation; p = 0.083). in those 128 leukemia patients capable of reactivating cmv (that is, donor or recipient cmv seropositive prior to sct), cmv reactivation had a protective effect on relapse rates in aml (no relapse (0%) with cmv reactivation vs 11 relapse cases (44%) without cmv reactivation; p = 0.017). a similar tendency could be seen in t-all whereas no effect in patients with b-precursor all was documented. numbers of acute gvhd cases grade 4i between aml and t-all with or without cmv reactivation were similar. different effects of cmv on relapse rates and mortality in aml vs b-precursor all were noticed in 79 patients who were either not capable of cmv reactivation or who did reactivate cmv post sct. in 17 aml patients, there were no relapses (0%) and 2 deaths (12%) in contrast to 17 relapse cases (27%) and 24 deaths (39%) in 62 children with b-precursor all (p = 0.017 and p = 0.044, resp.). latently cmv infected aml patients without documented cmv reactivation after sct have a significant worse prognosis compared with all other aml patients. this is also likely to be the case in patients with t-all, however, patient numbers in our cohort were too few. the protective effect of cmv reactivation in aml and possibly t-all does not appear to be gvh-related since the rate of relevant acute gvhd cases was comparable. cmv reactivation after sct for b-precursor all lacks significance. disclosure of conflict of interest: none. infections are among the most frequent and relevant complications of hematopoietic stem cell transplantation (hsct). little is known about the role of dental foci for the prevalence of infections in hsct. dental status was prospectively evaluated in all patients at our center before undergoing hsct. a total of 132 different patients before undergoing 163 hsct (83 allogeneic and 80 autologous), with a median age of 52 years (range: 4-70 years) were evaluated. for evaluation a panoramic x-ray evaluation was performed. dental findings included the status of third molars and root fillings as well as caries, periodontitis, destructed teeth and apical bone loss. as non-dental parameters we used age, sex, type and status of central venous line, mucositis, and type of transplantation. these were correlated with neutropenic fever, bacteremia and pneumonia in a bivariate manner before a multivariate analysis was performed. no correlation of initial dental status to neutropenic fever, bacteremia or pneumonia was found. however, bacteremia and suspected infection of central venous lines was a significant predictor of neutropenic fever. in conclusion, dental surgery should only be performed prior to hsct if urgently required and limited to those individuals with overt infection. disclosure of conflict of interest: none. [p250] early experience with clinimacs prodigy ccs method in generation of virus-specific t-cells for pediatric patients with severe viral infections after hematopoietic stem cell transplantation k kallay 1 viral reactivation especially in children is a frequent complication of allogeneic hematopoietic stem cell transplantation. most of these episodes can effectively be controlled by an antiviral or antibody therapy; in refractory cases a novel virusspecific t-cell therapy could be a promising management option. in our pediatric cohort of 43 allogeneic transplantation during 1 year 9 patients fulfilled criteria for virus-specific t-cell therapy (5 boys, 4 girls, median age of 11 (1.5-16) years). six patients were transplanted because of hematological malignancies and 3 for inborn errors. donor distribution was the following: 7 matched unrelated, 1 sibling and 1 haploidentical donor. in 5 cases bone marrow, 3 cases peripheral blood and 1 case cord blood was used as a stem cell source. the underlying viral illness was cmv in 3, ebv in 2 and adenovirus in 1 case, while more than one virus was detected in 3 cases (cmv+adenovirus 2 cases, cmv+ebv 1 cases). viral diseases necessitating a t-cell therapy were cmv pneumonitis and colitis, adenovirus enteritis and cystitis and ptld. patients initially received cidofovir for adenovirus, rituximab for ebv and a combination of gancyclovir and foscarnet for cmv infections. the indication for t-cell therapy was progressive viral disease in 8 of the 9 cases and uncontrollable viral load in 1 case. the procedure was performed on a median of 63 (49-113) day post transplant. donors were 1st degree relatives in 5 cases, 2nd degree relatives in 3 cases and an unrelated person in 1 case, the best hla match was haploidentical. the median age of the donors was 47 (33-60) years. cells were produced by the clinimacs prodigy cytokine capture system (ccs) method after mononuclear leukapheresis. the system produced a median of 9.9 (6.7-25) times 103/kg cd4+ and a median of 32.6 (16-125.1) times 103/kg cd8+ interferon producing cells while the non-interferon producing cells were far below gvhd limit with a median of 3.6 (1.5-12.4) times 103/kg cd4+ and a median of 3.85 (1.4-4.5) times 103/kg cd8 + cells. the t-cell products were administered uneventfully in all but one case. we observed a manageable cytokine storm in one patient. glucocorticoid treatment was ongoing due to acute gvhd in 5 children; however we could manage to keep the steroid dose below 1 mg/kg in all cases. eight patients became completely asymptomatic, while 7 also cleared the virus. we experienced decreasing viral load in all cases, the first negative viral results were achieved on a median day of 13 (13-55). six patients are alive without viral illness or sequale, and complete viral dna clearance in peripheral blood with a median follow up of 329 (144-580) days. one patient with cmv pneumonitis improved during the first week but deteriorated on the second week and died of respiratory insufficiency despite of mechanical ventilation. in 2 cases the viral illness improved or cleared, but the patients died of invasive aspergillosis. no cases of gvhd, rejection, organ toxicity or recurrent infection were noticed. virus-specific t-cell therapy produced by the clinimacs prodigy ccs is a feasible, fast, safe and effective way to control resistant viral diseases after pediatric hematopoietic stem cell transplantation. this treatment can be implemented within a week in most cases. in order to define the appropriate place of this approach for patients with viral reactivations more data should be collected. disclosure of conflict of interest: none. central venous catheter (cvc) is essential for the treatment of recipients of stem-cell transplant. it is usually placed for the administration of conditioning regimen, stem cell infusion, intravenous antibiotics, immunoglobulins, electrolyte and nutritional support and blood concentrates. this patient group is at high risk for catheter-related bloodstream infections that can result in substantial morbidity and mortality. the neutropenia secondary to the conditioning regimen determines the risk of catheter-related infections, which may serve as an entry into the blood circulation, leading to bacteremia, fungemia, and consequently to septic shock and death. the risks of infection and the spectrum of infectious syndromes differ according to the type of transplant, conditioning regimen, type of implant of stem cells and therapies used after the procedure. gram-positive bacteria, particularly coagulase-negative staphylococcus spp, remain the leading cause of catheter-related bloodstream infection, although an increase in gram-negative bacteria as the causative agent has been noted. aim of the study: to evaluate the impact of the early cvc removal on the frequency of febrile episodes and infections in our group of patients. during a 15 years period we have treated 351 patients with hematologic neoplasm with high-dose chemotherapy and stem cells transplantation. patients were treated in sterile room conditioned with hepa filtration. in every patient was introduced double-lumen cvc (321 subclavia, 7 jugular, and 23 femoral). 44% were febrile (10% fuo), catheter-related infection was present in 9%, while positive culture from cvc was present in 28%. the most frequent isolated bacteria from cvc were gram positive-staphylococcus coagulasa negative. the catheter was removed on the day of discharge. trm is 2.4%. from 01 january 2016 to 01 november 2016 we have transplanted additional 40 patients. to aim to decrease infection related mortality we perform strategy to remove cvc on day +2 after stem cell transplantation. the febrile episodes decreased on 25% (10/40), there were no early post-transplant mortality due to infection. early removal of the cvc and adequate handling from the nursing staff is essential for outcome of this patient population in regard of infective complications efficient prevention, early diagnosis, and effective treatment of catheter related infection are essential to providing the best care to these patients and can minimized morbidity and mortality. disclosure of conflict of interest: none. fever in patients with agranulocytosis during autologous hematopoietic stem cell transplantation (autohsct) can be associated with non-infectious causes due to g-csf, vancomycin, engraftment syndrome. in this case biochemical markers, such as presepsin (psp), procalcitonin (pct) and c-reactive protein (c-rp), can help in differential diagnosis of fever of infectious and non-infectious genesis. psp, pct and c-rp were assessed on the day of admission to the hospital (da), on d+1, on d+3, on d+7 and on the day of discharge (dd). if patients developed neutropenic fever (nf), the markers were assessed at the beginning of the fever, 6 h after, then on the second, third, fourth days after. if patients developed nf immediate empirical antibiotic therapy (at) was implemented with meropenem. in cases of ineffective 1st line ab, 2nd line at was added or totally changed. there were 100 patients included in the study: 41 patients with hodgkin lymphoma, 27 with non hodgkin's lymphoma, 32 with multiple myeloma, out of 100 patients there were 51 women and 49 men. the median age was 41 years (18-66 years). the conditioning regimens were cbv, beeac or hd melphalan. 69 patients developed infectious complications (ic): 2 of them had sepsis and others-nf. the median of nf development was 5.5 days. depending on the efficiency of at therapy patients were divided into two groups: group 1: patients that have had effective at (they 've had fast clinical response and they haven't needed to change medicine (n = 45)); group 2: patients that have had ineffective 1st line at, they haven't had response to 1st line at and they've needed to change another at (n = 24)). there were significant differences in psp levels on the third day after ab had been admitted: 365.3 pg/ml in group 1 and 750.6 pg/ml in group 2 (p = 0.0019). similar differences between the analyzed groups were observed on the fourth day: 350.5 and 569.7 pg/ml, respectively (p = 0.024). pct and c-rp didn't show any significant changes between group 1 and 2 on each day of the study (table 1) . disclosure of conflict of interest: none. enterovirus related immune reconstitution inflammatory syndrome (iris) following haploidentical stem cell transplantation in an mhc class ii deficient child r shah 1 , s waugh 2 , k foong ng 1 , z nademi 1 , t flood 1 , m abinun 1 , s hambleton 1 , a gennery 1 , m slatter 1 and a cant 1 1 paediatric immunology and bmt, great north children's hospital, newcastle upon tyne, uk and 2 department of virology, great north children's hospital, newcastle upon tyne, uk immune reconstitution inflammatory syndrome (iris) has been described after hsct in association with fungal, viral and bcg infections. we describe a case of post-hsct iris associated with enterovirus infection. case: a girl with mhc ii deficiency (rfxp2c.362 mutation) underwent treosulfan/fludarabine/ thiotepa/atg conditioned tcrαβcd3+ depleted haploidentical hsct at 1.8 years of age. pre-transplant work up did not reveal any viral or fungal infections except norovirus in stool. cyclosporine (csa) was given as gvhd prophylaxis. neutrophil and platelet engraftment occurred on d+15 and d+9, respectively. on d+6, her stool was tested positive for enterovirus (taqman pcr), however; she was asymptomatic. the child started having fevers and irritability from d+21 which persisted despite the use of antimicrobials. no evidence of fungal or bacterial infection was found. enterovirus pcr in blood was found positive on d+31 (cycle threshold value, ct 32.4) and further typing showed it to be echovirus 13. at this time, symptoms progressed with diarrhoea, developmental regression and signs of radiculopathy. mri (brain and spine) was normal and csf showed pleocytosis (815 wbc/mcl-100% lymphocytes, protein 4.23 g/l) with positive enterovirus pcr (ct 17). subsequently, immunoglobulin prophylaxis was increased to 0.5 g/kg bi-weekly, and with supportive measures, the patient slowly recovered. blood enterovirus pcr remained positive. with no evidence of gvhd, csa was tapered off by day+105 and child was discharged on d+112 on a bi-weekly ivig replacement. she presented 5 days later with signs of raised intracranial pressure. mri showed hydrocephalus, and vp shunt was placed and broad spectrum antibiotics administered. csf showed wbc o1/mcl, protein 0.23 g/l and enterovirus positive. methylprednisolone 2 mg/kg/day was started suspecting iris. in subsequent csf testing 3 days later, enterovirus was negative. enterovirus pcr remained positive in blood during this period. patient's clinical deterioration correlated with a rise in cd4/cd8 counts and c reactive protein with clearance of enterovirus from csf, blood and stool ( figure 1 ). subsequently, the child showed gradual but marked improvement and discharged home. discussion: the clinical features of index case fits into criteria for iris 1 . markedly raised crp suggests high il-6 levels without any bacterial or fungal pathogens being isolated. in addition, iris occurs at the site of prior active infection (brain in index case) and viral clearance and clinical recovery demonstrated with the continuation of steroids. the incidence of enterovirus infection in hsct recipients is around 10% 2 . iris, in this case, had a temporal correlation with discontinuation of csa, and it has been shown that discontinuation of immunosuppression is associated with higher risk of iris. a high index of suspicion for iris is necessary during immune recovery post-hsct especially when immunosuppression is being tapered in a patient with pre-existing infection. aggressive antiviral treatment (when available) and judicious immunosuppression are the keys to managing iris complications. posttransplantation lymphoproliferative disease (ptld) is a significant cause of morbidity and mortality in allogeneic stem cell transplant patients. identifying high risk patients, routine pcr screening, early diagnosis and therapy are crucial for successful management. patients and methods primary objectives of this study were to describe epidemiology of ebv associated ptld and to assess risk factors in our paediatric cohort. additionally, role of immunoglobulin (ig) levels as a possible diagnostic/prognostic marker was analyzed. between 1 january 1 2011 and 30 june 2016, 140 allogeneic transplantations were performed in 118 pediatric patients (82 boys and 36 girls) at our center. median age was 7.68 years (0.03-18). underlying diseases were hematological malignancies (68%), nonmalignant hematological conditions (13%), immunodeficiencies (11%) and others (8%). stem cell source was bone marrow (62%), peripheral blood (19%) and cord blood (19%). donors were unrelated (75%), sibling (18%), haploidentical (4%) or other matched family donors (3%). routine ebv pcr screening and ig level detection were performed weekly. rituximab prophylaxis was given only in nine cases. results ebv dnaemia was found in 16/118 patients (13.6%), while ptld was diagnosed in 11/118 patients (9.3%). all ptld cases were related to ebv infection, median of highest viral load was 11 790 copies/ml (688-6 670 000). diagnosis was confirmed by biopsy in 6/11 cases, further five fulfilled criteria of probable ptld (positive pcr with appropriate clinical symptoms). ptlds occurred at a median of day +48 (19-85) after transplantation. all patients received rituximab treatment along with a reduction of immunosuppressive therapy. four patients died of ptld (mortality 36%), all confirmed by autopsy. a higher incidence of male gender (10/11; 90.9% vs 67.3%), bone marrow graft (10/11; 90.9% vs 59.8%), hematological malignancy (10/11; 90.9% vs 67.3%) and second transplantation (5/11; 45.5% vs 14%) could be detected among ptld patients when compared to the non-ptld group. elevated igg, iga or igm levels were observed in 13/118 patients. nine out of 13 had positive ebv pcr testing, eight of them developed ptld. five of the ptld patients had monoclonal or biclonal immunoglobulin elevation, two of them died. in 3 cases, elevated ig level preceded the positive ebv pcr results by at least 1 week. conclusion: at our centre incidence and mortality of ptld was similar to published data. we observed a tendency that a higher representation of male gender, hematological malignancy, bone marrow graft and second transplantation could be confirmed in the ptld group however due to small number of patients, a correlation and statistical significance could not be calculated. elevation of immunoglobulin levels do not seem to be specific for ptld but in selected cases it could predict ebv disease earlier than pcr testing. disclosure of conflict of interest: none. autologous peripheral hematopoietic stem-cell transplantation is a procedure of a stem cell rescue with patients' own previously collected hematopoietic stem cells, after myelotoxic therapy. the purpose of stem cell reinfusion is to ensure adequate recovery of hematopoiesis, shorten the period of profound neutropenia and to reduce the risk of infections. the transplantation itself carries a moderate risk for infection but some patients have higher risk due to the nature of underlying disease, earlier treatment and in case of severe mucositis. for these reasons, all treated patients are in isolated clean rooms and receive ciprofloxacin, fluconazole and acyclovir prophylaxis. in the 3.5-year period, 177 autologous transplantations were performed. the patients were 20-72 years old, with median of 55.18 years. of all transplanted patients, 106 or 59.88% had multiple myeloma, 66 or 37.3% had lymphoma and 5 or 2.82% had acute myeloid leukemia. all of the patients received pegfilgrastim 6 mg on the first or the second posttransplant day. febrile neutropenia (ne o0.5 × 10 9 /l) was reported if patient's temperature was above 38.3°c in one measurement or above 38°c in two consecutive measurements. these patients were treated empirically with piperacillin/tazobactam 4.5 g four times a day with the addition of vancomycin in the case of severe mucositis or pulmonary infiltrates. in all cases blood and urine cultures were performed, as well as testing for seasonal flu. time to neutrophil recovery (ne40.5 × 10 9 /l) was 7-20 days, with a median of 10 days, and average of 10.25 days. febrile neutropenia was reported in 76 patients (42.94%) and in 39 (51.31%) patient's samples pathogen was isolated. gramm negative bacteria caused sepsis in 54.29% of patients. we had to change empirical therapy according to antibiogram in 37.2% patients. in 1 month follow-up period, there were two (1.12%) infection related deaths. our data on incidence of infections is consistent with literature data but large number of papers show satisfactory results of safety of patients discharged from hospital immediately after the autologous stem cell transplantation and who were treated at home during the phase of profound neutropenia. there is still an ongoing debate whether it is possible to conduct this procedure in such manner in our health system. disclosure of conflict of interest: none. fluconazole was equal to mold-active drugs in preventing early invasive fungal disease after allogeneic stem cell transplantation regardless of transplantation type y sun, j hu, h huang, j chen, j-y li and x-j huang there are still controversies that whether mold-active drugs is better than fluconazole in preventing invasive fungal disease (ifd) after allogeneic stem cell transplantation (hsct). we hypothesis that the optimal prophylaxis might be different in patients with different risk profile, such as in different time period after hsct or received alternative donor transplantation. in the prospective china assessment of antifungal therapy in haematological disease (caesar) study database, 661 out of 1401 patients received primary antifungal prophylaxis were analyzed. the ifd incidence of different time period after transplantation (early, late and very late) and survival were compared among different drug groups. in patients with fluconazole, itraconazole, voriconazole or micafungin prophylaxis, the overall incidence of ifd after transplantation were 7.2%, 12.6%,1.4% and 5.2%, respectively (p = 0.0379). however, there is no difference in early ifd (o40 days post hsct) among 4 groups of patients. the risk factors associated with occurrence of ifd were neutropenia duration 414 days (po0.01, or 3.73 (1.66-8.36)), adult (p = 0.02, or 3.37 (1.23-9.18)) and alternative donor (unrelated donor or haploidentical donor) transplantation (p = 0.01, or 5.88 (1.48-23.32)). in the sub-group analysis with only alternative donor (unrelated donor and haploidentical donor), it also demonstrated that fluconazole is equal to other mold-active drugs in preventing early ifd. patients received fluconazole prophylaxis has even better overall survival. the overall survival in patients with fluconazole, itraconazole, voriconazole or micafungin prophylaxis were 88.3%, 83.5%,78.9% and 72.4%, respectively (p = 0.0047). our current [p259] study suggests that fluconazole is equal to mold-active drugs to prevent early ifd in hsct patients, even in high-risk patients received transplantation from alternative donors. however, further prospective randomized study was warranted to confirm this conclusion. disclosure of conflict of interest: none. (9.5%) received autologous hsct and 38 (90.5%) allogeneic hsct. sixty-five out of 107 patients (60.7%) were affected by different haematological diseases: 36 by lymphoma, 11 by multiple myeloma, 7 by chronic lymphocytic leukaemia and 11 by others diseases including mastocytosis, amyloidosis and essential thrombocythemia. hbv reactivation prophylaxis prescribed was entecavir for 4 hbsag+ inactive carrier patients and prolonged lamivudine (lmv) course for 103 (96%) patients. in 6 patients (5.6%) lmv prophylaxis was withdrawn 12-18 months after the end of immunosuppressive therapy. eight out of 107 patients (7.4%) experienced hbv reactivation: 4 of them during lmv treatment and then they were switched to entecavir or tenofovir therapy, 4 patients reactivate hbv after lmv interruption (3.7%). in these patients reactivation was observed after an average time of 4 months (range: 3-6) after discontinuation of lmv prophylaxis. median duration of prophylaxis was 49 months (range: 40-60) after the end of immuno-suppression. three out of 4 patients (75%) underwent allogeneic hsct and 3 patients (75%) received rituximab. one out of 4 (25%) seroreverted in hbsag positive and hbsab negative status, with hbv-dna42000 ui/ml (table 1) . two patients out of 4 (50%) experienced hbv-dna detection below 20 ui/ml. disclosure of conflict of interest: none. [p260] [p260] the severity is measured on grades (grade 1: microscopic hematuria to grado 4: clots cause urinary tract obstruction). the treatment is based on support measures: hyperhydration, continuous bladder irrigation, instillation of topical agents and in severe cases must be performed a cystoscopy for clot evacuation. in the case of the presence of poliomavirus virus (bk virus) the use of cidofovir had been demonstrated in vitro studies to have activity against bk virus. we performed 42 allogenic transplants of which 10 are haploidentical from 2010 to october 2016. we realized a retrospective case study to analyses the experienced in the management of hc. results: of a total of 42 allogenic transplants realized, developed a hc: 1 patient received an identical hla transplant and the 4 patients remaining haploidentical allotrasplant. all cases were male, with an age range of 18-42 years. the status of the disease was: 3 were in complete remission and 1 had visible disease. 3 of the 4 patients received cyclophosphamide as immunosuppressive therapy and all patients received cyclosporine and mofetil micofenolate also. the onset of the symptomatology was between day 9 and day 82 post transplant and the range of duration was from 14 to 45 days. the four patients precised continuous bladder irrigation but because of the poor response they received instillation of hialuronic acid (4 doses). two patients required the use of cidofovir (3 doses). one of the four patients required urinary tract catheterization because of hydronephrosis and renal impairment. in our review we confirmed that this entity is more frequent in the haploidentical transplant and bkv is the most prevalent cause in the late hc. -the three patients received 3 doses of cidofovir (1mg/kg) without probenecid and had a good response. -three patients present acute renal failure associated to hc. the four patients needed bladder instillations with saline but they had poor response and received at least 4 doses of hyaluronic acid. disclosure of conflict of interest: none. hsv infection in allo-hsct setting is mostly reactivation of latent virus. hsv disease commonly presents as mucocutaneous lesions of the oral cavity. however some patients develop serious fatal visceral dissemination. prophylactic use of acyclovir has markedly reduced the incidence of hsv disease during the period of neutropenia after allo-hsct. in this study, our aim is to demonstrate the incidence, clinical outcome and risk factors for hsv disease in adult allo-hsct. between 2015 and 2016, 89 patients who underwent allo-hsct in our center were included to the study. all hsct candidates and donors were tested for hsv-1/2 immunoglobulin g (igg) antibodies prior to transplantation. all patients received acyclovir prophylaxis (related transplants 400mg tid, unrelated transplants 800mg tid) during conditioning and after allo-hsct up to 3 months. chlorhexidine oral solution as well as bioadherent oral protective gels was used for oral hygiene. all patients were followed for symptoms of reactivation. hsv1/2 igg seropositivity was detected in 66 recipients (74%) and 48 donors (54%). the distribution of hsv status was as follows: recipient and donor seropositive in 34 (38%), recipient and donor seronegative in 9 (10%), recipient seropositive and donor seronegative in 32 (36%), recipient seronegative and donor seropositive in 14 (16%) transplants. the median age of the patients was 41 (range: 18-67), 48 patients were male (54%) and 79 (89%) had malign disease. the stem cell source was peripheral blood in 73 (82%) patients and 48 (54%) received grafts from related donors. sixty four patients (72%) received myeloablative conditioning regimen. the most common graft-vs-host disease (gvhd) prophylaxis administered was cyclosporine (csa) and methotrexate (mtx) in 60 patients (67%). acute graft vs host disease was detected in 29 patients (33%).four patients from seropositive 66 patients (6%) had hsv reactivation, the patient characteristics are given in the table. all patients had hsv reactivation within 1 month of allo-hsct except one patient had symptoms at sixth month posttransplant when he suffered from oral gvhd. all patients s252 and donors were seropositive prior to allo-hsct and responded well to antiviral treatment. the incidence of hsv reactivation in allo-hsct was detected as 6% which is lower to previous studies. successful primary prophylaxis and oral hygiene might reduce the incidence. all patients were responded to antiviral treatment and no visceral dissemination was detected. disclosure of conflict of interest: none. patients who have received hematopoietic stem cell transplantation (hsct) may suffer, to some extent, losses in humoral and cell immunity against antigens to which they had been previously exposed naturally (infection caused by wild microorganisms) or artificially (through vaccination). the conditioning regimen for hsct replaces the patient's immune system and involves the loss of previous immunity. this study analyzed patients included in the vaccination program for hsct recipients in the salamanca health care complex during the period 2010-2016. we assessed the serological status prior to hsct for the following immunopreventable diseases (hepatitis b, hepatitis a, varicella), and the study after hsct also included measles, rubella and parotitis, prior to their inclusion in the hsct vaccination program. the study included 302 patients, 53.8% of which (n = 168) were men. 83.3% of the patients (n = 260) were allogeneic hsct recipients with an average age of 47 ± 16 years, and 16.7% (52) were autologous hsct recipients with an average age of 43 ± 16 years. prior to hsct, 40% of the patients showed immunity against hepatitis b (hbv antibodies 410 ui/l), 82.6% against hepatitis a (positive for hav igg) and 85% against varicella (positive for varicella igg). no statistically significant differences were observed regarding this variable hepatitis b anti-hbs 410 ui/l, hepatitis a, igg positive, varicella igg positive, measles igg positive, rubella igg positive, parotitis igg positive. table 1 compares the serological status before and after transplantation. in the pre-transplant serological study we observed that less than half of the patients are protected against hepatitis b, while over 80% of them are protected against hepatitis a and varicella. regarding the diseases in which we know the serological status before and after transplantation (hepatitis a, hepatitis b and varicella), we observed that most patients maintain immunity. in the case of rubella, measles and parotitis we only have access to the serological status after transplantation, and we observed that parotitis is the disease with the lowest seroprotection. therefore, vaccination would be indicated, just as in the case of hepatitis b. the clinical results support the need to adapt the vaccination schedule to the immunological status of the patients after hsct individually. disclosure of conflict of interest: none. impact of cumulative steroid dose on infectious diseases after allogeneic hematopoietic cell transplantation m watanabe 1 , j kanda, t kitano, t kondou, k yamashita and a takaori-kondo 1 after allogenic hematopoietic cell transplantation (hct), highdose steroids are used to treat transplantation-related complications such as graft-versus-host disease (gvhd). however, the use of high-dose steroids is associated with an elevated risk of infectious diseases. information on the association between cumulative steroid dose and infectious diseases after hct is scarce. a total of 238 patients who underwent their first hct in kyoto university hospital from 2005 to 2015 and survived at least 30 days after transplantation were included in this study. we analyzed the association between cumulative steroid dose used within 30 days after transplantation and the occurrence of infectious diseases, including invasive fungal infection (possible/probable/proven cases), cytomegalovirus (cmv) antigenemia, and bacteremia through 180 days after transplantation. sixty-three patients received transplantation from a related donor, 114 received unrelated bone marrow grafts, and 61 received unrelated cord blood units. their median age was 51 (range: 17-66) years and median day of neutrophil engraftment after transplantation was 21. patients were categorized into 3 groups according to their cumulative steroid dose within 30 days: no steroid administration (n = 183), low-dose cumulative steroid administration under 500 mg of prednisolone in total (n = 27), and high-dose cumulative steroid administration over 500 mg of prednisolone in total (n = 28). reasons for steroid administration were treatment for gvhd in 35 patients, engraftment syndrome in 11, and other reasons including lung complications in 9. the rate of invasive fungal infection was 6% (12 possible cases with pneumonia and 1 proven case of candida blood stream infection) and we found no apparent association between fungal infection and steroid use regardless of dose. cmv antigenemia was diagnosed in 41%, 67% and 60% of patients in the 3 groups respectively, and both low-dose and high-dose steroid groups were significantly associated with a high risk of cmv antigenemia (low-dose group, adjusted hazard ratio (ahr), 2.37, p = 0.004; high-dose group, ahr 1.84, p = 0.01). bacteremia was diagnosed in 9.8%, 11% and 21% of patients in the 3 groups, respectively. high-dose steroid use was a risk factor for bacteremia (ahr 1.70, p = 0.027). seven patients died from infection (fungal, 2; viral, 2; bacterial, 3). two of three bacterial infection-related deaths occurred in the highdose steroid group, although the number of events was too small to analyze. our data confirmed that steroid administration is itself a risk factor for cmv antigenemia and close observation to detect cmv antigenemia is mandatory for patients using steroids regardless of its cumulative dose. high-dose cumulative steroid use is a risk factor for bacteremia. contrary to our expectations, steroid administration showed no apparent association with invasive fungal infection in our study, perhaps because of its generally low incidence in our hospital. disclosure of conflict of interest: none. impact of different t-cell depletion techniques on the incidence of infectious complications after allogenic hematopoietic stem cell transplantation k aikaterini 1 , s federico 1 , d-l vu 2 , e boely 2 , c dantin 1 , a pradier 1 , y tirefort 1 , a-c mamez 1 , o tsopra 1 , c stephan 1 , y beauverd 1 , e roosnek 1 , s masouridi-levrat 1 , c van delden 2 , y chalandon 1 1 department of oncology, hematology unit, university hospital of geneva and 2 department of medicine specializations, infectious diseases, university hospital of geneva t-cell depletion (tcd), obtained by either in vivo antithymocyte globulin (atg) administration or ex vivo depletion, is a well-established strategy for graft-versus-host-disease (gvhd) prevention after allogeneic hematopoietic stem cell transplantation (hsct)1-4. however, the prolonged lymphopenia associated with tcd can result in increased incidence of disease relapse1 and infections. although many studies investigated the impact of tcd on disease relapse 1-4, little is known about the impact of tcd strategies on the incidence of infectious complications after allogeneic hsct. we retrospectively evaluated the incidence of infectious complications in 236 consecutive patients who underwent allogeneic hsct at our center from september 2010 to december 2015. 100 patients received tcd grafts obtained by in vivo atg administration as part of the conditioning regimen (atg group). 17 patients received partially tcd grafts obtained through incubation with alemtuzumab in vitro washed before infusion followed on day +1 by an add-back of donor t cd3+ cells5 (ptcd group). 60 patients received grafts tcd by both methods combined. 59 patients did not receive any form of tcd (no-tcd group). cumulative incidence estimates of infectious complications were calculated and compared using the gray test. given the increased risk of infection associated with gvhd and its treatments, gvhd or death from other causes were defined as competitive events in the analysis. we didn't observe any significant difference in the 1-year cumulative incidence of bacterial infections in patients receiving tcd by atg (45% (95% ci 35-54.5%)) ptcd (58.8% (95% ci 31.2-78.5%)) or both (55% (95% ci 41.4-66.7%)) compared with patients receiving no tcd (52.5% (95% ci 38.9-64.5%)). similarly, the 1-year cumulative incidence of viral infections or reactivations was comparable in patients receiving no-tcd grafts (80.3% (95% ci 66.8-88.8%)) compared with patients receiving tcd grafts (atg: 82.2% (95% ci 72.9-88.6%); ptcd: 76.5% (95% ci 45.7-91.2%); atg+ptcd: 81.7% (95% ci 68.9-89.6%)). finally, no significant impact of tcd was observed on 1-year cumulative incidence of fungal (no-tcd: 6.8% (95% ci 2.2-15.2%); atg: 18.1% (95% ci 11.2-26.3%); ptcd: 11.8% (95% ci 1.8-31.9%); atg+ptcd: 18.3% (95% ci 9.7-29.1%)) and parasitic (no-tcd: 1.7% (95% ci 0.1-8%); atg: 1% (95% ci 0.1-4.9%); ptcd: 5.9% (95% ci 0.3-24.2%); atg +ptcd: 1.7% (0.1-8.3%)) infections. image/graph: 1-year cumulative incidence estimates of infectious complications depending on the tcd strategy employed. the results of our retrospective analysis indicate that the cumulative incidence of bacterial, viral, fungal and parasitic infectious diseases are similar in patients receiving tcd grafts compared to those receiving no-tcd graft, suggesting a favorable toxicity profile of different tcd strategy in respect of infections. these results should be confirmed by similar analysis in large scale, prospective clinical trials assessing the potential benefits of tcd on transplantation outcomes. 57 patients with aml were considered eligible for hsct, 7 died before transplantation. 13 patients (26%) underwent transplantation from hla-identical sibling, 13 (26%) from haploidentical family donor and 19 (38%) from matched unrelated donor, while 5 patients (10%) received unrelated cord blood cells. twenty (35%) out of 57 eligible patients have had an ifi episode before transplant: 6 were proven, 4 probable and 10 possible; (9 (47%) pneumonia, 4 (19%) gastroenteritis, 3 (15%) sinusitis, 2 (10%) candida sepsis, 1(5%) meningitis and 1(5%) cutaneous abscess were registered). five (25%) out of 20 patients with a previous ifi and 2 (5%) out of 37 without previous ifi did not receive hsct (or 5.83 95% ci 1.02-33.96, fisher test p: 0.04). the majority (55%) of patients with a previous ifi waited hsct more than 6 months from the date of eligibility in comparison with those without a previous ifi (55% vs 30%; or 0.37, 95% ci 0.12-1.13, p-value 0.08 fisher test) overall a post transplant ifi episode was diagnosed in 13 (26%) of transplanted patient; 4 (26%) had a relapse of a past ifi vs 9 (25%) of the patients without a previous ifi who had a new episode. (or 1.53, 95% ci 0.39-5.90, p-value 0.4 yates test).a higher number of patients with ifi (10 out of 15, 66%) respect to those without a previous ifi (10 out of 35, 30%) died in a median time of 160 days(range: 22-480 ) after hsct. furthermore, those who had a previous ifi had a lower median survival (317 days (range: 22-1095)) compared to patients without a previous ifi (530 days period (range: 20-1490)) (student's t-test p: 0.014)). a previous ifi episode in the pre transplant period slows and limits the accessibility to hsct, and is significantly associated with an increased mortality. disclosure of conflict of interest: none. s254 delayed immune reconstitution has been described for haploidentical hematopoietic stem cell transplantation (hsct) compared to conventional hsct, nevertheless the incidence of invasive aspergillosis infections (iai) in haploidentical sct and the efficacy of primary prophylaxis are not well defined. our objective is to describe the incidence, risk factors and mortality of iai in our patients, using as prophylaxis micafungin during the conditioning and neutropenia period, switched to posaconazole or voriconazole when oral intake is feasible. we retrospectively analyzed 40 consecutive patients from 2014 to 2016 who received haploidentical grafts: unmanipulated for 20 adults, tcrab depleted in 6 children and cd45ra depleted in 14 children. the stem cell source was peripheral blood in all cases. adults (22-70 yo) were treated for aml/mds (n = 8), all (n = 2) and lymphoma (n = 10). children (6 mo-15 yo) were treated for aml (n = 6), all (n = 9), aplastic anemia (n = 2) and immunodeficiencies (n = 3). conditioning regimen was bu-flu-cy (n = 18, adults), thio-bu-flu (n = 2, adults), flu-mel-thio for all pediatric patients; atg was used in 6 children and tli in 14 children. median follow up was 12 months (1-30) for adults and 7 months (1-28) for children. we used eortc criteria for iai and analyzed probable or definite as cases. there were 6 events of iai, with a bimodal presentation: 3 events (7.5%) during neutropenia period and 3 (7.5%) after 6 months of hsct ( figure 1 ). five of them were probable and one definite (aspergillus niger). site of infection was mainly pulmonar; cns was suspected in two adult patients and skin was proven in one adult patient. all 3 patients at the late period had chronic gvhd at diagnosis. one patient had primary graft failure. severe cmv disease (hepatitis and colitis) was present in one adult. mortality related to iai was high (5/6), patients died at a median of 35 days. figure 1 . iai patients characteristics the global incidence of iai in haploidentical hsct is similar to conventional hcst. primary prophylaxis with micafungin switching to oral triazole is successful (92.5%) during the early period. late cases (7.5%) had clearly known risk factors (chronic gvhd, steroids and cmv), and primary prophylaxis had been modified due to toxicity or interactions. iai mortality in our patients is very high (84%) despite effort in prophylaxis, diagnosis and treatment. visceral intractable abdominal pain prior to skin lesions from herpes zoster can be misdiagnosed as gvhd post stem cell transplantation which may lead to initial increase in immunosuppression and hence high mortality if we don't suspect. case report and literature review through pubmed results: 13year-old male with relapsed all post mud pbsct (10/10) transplant in following cy tbi atg conditioning presented at day +117 with intractable diffuse abdominal pain with constipation. no history of nausea, vomiting or skin rash. on physical examination his abdomen was soft, diffuse tenderness but no rigidity, muscle guarding and rebound tenderness. laboratory tests including liver function test, amylase, lipase were normal. usg abdomen and mri abdomen showed no abnormalities, except for presence of fecolith. during the stay his pain worsened needing morphine infusion, pca and later ketamine. he had previous history of acute gut gvhd controlled on budesonide and cyclosporine which was later being weaned once his symptoms were controlled. in view of previous history of gvhd, gi consultation was sought and he underwent ugi endoscopy and biopsy which was non-significant. on day 7 of his admission he developed a pustular skin lesion on thigh and scrapping from that showed vzv and his blood pcr was also positive, he was started on intravenous acyclovir. his lesions improved and crusted and his abdominal pain subsided after 72 h of acyclovir and was discharged on oral acyclovir after 14 days of intravenous therapy. review of literature illustrated in table 1 . severe abdominal pain in patients who received an allogeneic stem cell transplant has a broad differential. here we describe a case of vzv presenting with intractable abdominal pain needing opioids. because of the poor prognosis and life-threatening nature of disseminated vzv disease, it should be considered and included in the patient's workup. intravesical cidofovir (5 mg/kg, diluted in 90 ml sterile water) was once weekly applied until symptom control for 60 min. via a transurethral catheter, i.v. cidofovir was initiated if no symptom control was achieved after 3 local applications. in patients with hc 3 or 4 a lavage catheter was added. bkv cystitis (dysuria (n = 8) or dysuria combined with hematuria (n = 10)) developed in 18 out of 152 transplants (12%). median age was 54 years, 89% were female and 50% received a mismatch transplantation after 1 mac or 17 ric conditioning regimens. in 67% of bkv cystitis cases also cmv reactivation within the first 180 days could be detected. 67% had acute gvhd ii°-iv°at the onset of bkv cystitis and 83% received steroid medication. the median time to symptom occurrence was day +40 after hsct (iqr 25-75: 31-136). 5 patients (3 with dysuria and one either hc 1°and 2°) didn´t require therapy due to self limiting symptoms. 3 (23%) of 13 treated patients showed only dysuria, 3 (23%) hc 1°, 5 (38%) hc 2°, 1 (8%) hc 3°and 1 (8%) hc 4°. the first patient was treated with i.v. cidofovir twice and symptoms relieved. all the following 12 patients were exposed to intravesical cidofovir as 1st line therapy. 8 patients (67%) achieved a complete remission with a median of 2 intravesical procedures (range: 1-3). 1 patient showed symptom improvement and all 9 patients didn´t require further therapy. 3 patients had to be switched to i.v. application due to bladder spasms during intravesical application (n = 1) or to insufficient symptom control (n = 2). 2 out of these 3 responded to i.v. treatment, whereas 1 patient receiving 2nd transplant didn´t respond at all. in patients with spontaneous symptom relieve the median bkv concentration at the time of symptom onset was 2 log lower compared to those requiring antiviral therapy. local therapy reduced bkv viruria by 2 log. pain during cidofovir instillation in 50% of patients was the only significant side effect of local therapy compared to creatinine increases by 450% in 66.6% of i.v. treated patients. intravesical treatment of symptomatic bkv cystitis with cidofovir (5 mg/kg) is safe and effective with an 75% symptom improvement rate and no systemic side effects. in patients without sufficient symptom or bleeding control i.v. cidofovir is still an option, which however induces significant renal toxicity. we therefore recommend intravesical cidofovir as 1st line therapy in case of dysuria or hematuria induced by bkv after hsct. disclosure of conflict of interest: none. haemorrhagic cystitis is a recognised complication of stem cell transplant (sct), with a reported incidence of 5-25% of cases (1) . the majority of cases are associated with bk polyomavirus (bkv), and less often adenovirus and cytomegalovirus. there are a lack of high quality studies on the optimal prevention and management of haemorrhagic cystitis. treatment options are restricted by conditioning toxicity, immunosuppression and other co-morbidities such as renal impairment. cidofovir has an inhibitory effect on bkv replication and has been used extensively in the treatment of haemorrhagic cystitis. however, severe nephrotoxicity limits routine intravenous use in sct patients. alternative options include using low dose intravenous cidofovir or intravesical administration. we conducted a retrospective case review of post sct patients presenting with bk virus associated haemorrhagic cystitis in our institution between january 2010 and november 2016. we identified 5 patients in total (4 male,1 female). the indications for stem cell transplant were as follows: severe aplastic anaemia 1 high risk aml 1 relapsed aml 1 relapsed all 2 onset of symptoms (haematuria and painful micturition) ranged from day − 4 to day +27, and the time to resolution of symptoms varied from 16 days to 68 days. four of the patients were treated with intravesical cidofovir only, with the number of doses required varying from 3 to 7. one patient received combination treatment with both intravenous (7 doses), and intravesical cidofovir (5 doses). all patients had a good clinical response with complete resolution of symptoms and no major complications. however, the level of bk virus in the urine did not always correlate with clinical response. some of the patients did not tolerate urethral catheterisation and required a general anaesthetic for the placement of the urethral catheter; 1 patient required a supra-pubic catheter. currently 3 out of 5 patients are alive and well; 2 patients died from causes not related to bk virus associated haemorrhagic cystitis. our experience shows that intravesical administration of cidofovir is a safe and effective option for the treatment of bk virus associated haemorrhagic cystitis. an allogeneic stem cell graft from a cytomegalovirus (cmv) seronegative donor puts recipients at high risk of cmv reactivation which can lead to cmv disease and mortality. based on the immunogenicity of cmv phosphoprotein 65 (cmvpp65) we initiated a clinical phase i trial with a novel vaccine designed by our group: a cmvpp65-derived peptide in water-in-oil emulsion (montanide) plus administration of granulocyte-macrophage colony stimulating factor. ten patients received four vaccines s.c. at a biweekly interval after allogeneic stem cell transplantation. we monitored the patients for their clinical outcome and cmvpp65 antigenemia. multi-color flow cytometry test were performed to assess cmvspecific cd8+ and gamma-delta t cells. novel neutralizing anti-cmv antibody assays were established and correlated to clinical parameters. findings: in general, patients tolerated the peptide vaccination well, no drug-related adverse events others than rash or induration at the site of injection were detected. seven of nine patients with cmvpp65 antigenemia cleared the cmv after four vaccinations and were hitherto free from antigenemia. two patients with cmv reactivation showed persisting cmv antigenemia. one of these two refractory patients received additional four injections and remained hitherto free from cmv antigen. another patient obtained a prophylactic vaccination and did not develop antigenemia. an up to six-fold increase in frequency of both cmv-specific cd8+ t cells or vdelta2-gamma-delta t cells was detected in five patients. moreover, titers of neutralizing antibodies increased in four patients up to 10-fold over the time of vaccination. humoral and cellular immune responses correlated with clearance of the cmv load. cmvpp65 peptide vaccination was safe and well tolerated in patients after allogeneic stem cell transplantation at high risk for cmv reactivation. the vaccine showed encouraging immunological and clinical results. a prophylaxis study using the vaccine in solid-organ transplant patients is ongoing. disclosure of conflict of interest: none. sporopachydermia cereana is a rare yeast found in necrotic cactus tissue, predominantly in the americas. infection in humans has only been reported in 4 neutropenic patients with fatal course, either directly from the pathogen or other complications of immunosuppression. treatment is complicated by difficulties in pathogen-identification with conventional diagnostic techniques and by resistance to echinocandins. here we present a patient with acute myeloid leukemia (aml) and s. cereana infection. this is the first patient who was successfully treated with antifungal therapy and who survived s. cereana infection. case presentation we present the case of a 50-year-old female patient who was diagnosed with normal karyotype aml with dnmt3a and idh2 mutations in december 2015. she achieved complete remission after two cycles induction chemotherapy. during the 2 nd induction cycle the patient developed persistent fever in neutropenia despite broad-spectrum antibiotics and the replacement of prophylactic fluconazole to caspofungin. blood cultures showed growth of s. cereana, shown to be sensitive to azoles (mic fluconazole o1 mg/l, mic voriconazole o0.12 mg/l) as well as amphotericin b (mic o0.25 mg/l), but resistant to caspofungin (mic44 mg/l). following the susceptibility profile the treatment was changed first to liposomal amphotericin b, and with the availability of mic results to voriconazole. metastatic fungal infection (that is, endocarditis, endophthalmitis, hepatosplenic candidiasis) was excluded. after regeneration of peripheral blood values the treatment was switched to oral voriconazole. a ct scan of the chest and abdomen prior to allo-hsct after 6 weeks of treatment with voriconazole revealed new multiple necrotic mesenteric lymph nodes. an ultrasound-guided biopsy of a node revealed no growth on fungal cultures, a grocott stain revealed no hyphae or spores. a panfungal pcr of an its (internal transcribed spacer) fragment revealed fungal dna, which could be confirmed as s. cereana. at this time the level of voriconazole in serum was found to be sub-therapeutic (0.4 mg/l), and the dosage was increased accordingly. subsequent ct scans 4 and 6 weeks later revealed a regression of the affected abdominal lymph nodes. in the further course non-myeloablative conditioning with fludarabine and busulfan prior to allo-hsct using pbsc from her hla-matched brother was performed. under prophylaxis with cyclosporine, methotrexate and antithymocyte globulins (atg) graft-versus-host disease (gvhd) remained absent. the allo-hsct was performed under voriconazole treatment with no further complications and the patient engrafted at day 20. the treatment was changed to fluconazole 400 mg daily before discharge. due to the complete radiological regression of the infection in follow-up scans and excellent general condition of the patient 5 months after hsct, fluconazole was discontinued. the patient remains in morphological complete remission 6 months after hsct and has a 100% donor chimerism. the first published case of survival of infection with s. cereana exemplifies the continual progress made in treating infections in the severely immunocompromised patient. diagnosis via its sequence-analysis seems reliable but a high index of suspicion is required for neutropenic patients who do not respond well to standard antimycotic therapy. the increased availability of the technology may lead to more frequent diagnoses in the future. disclosure of conflict of interest: none. neutropenic enterocolitis (ne) is a clinical syndrome characterized by fever and abdominal pain in patients who received chemotherapy for hematological malignancies and who treated with stem cell transplantation (sct). the aim of this study was to determine the incidence, risk factors and outcome of ne after autologous sct (auto-sct). we retrospectively evaluated 226 patients with non-hodgkin lymphoma (nhl), hodgkin lymphoma (hl) and multiple myeloma (mm) who underwent auto-sct between january 2013 and december 2016 in our center. patients with lymphoma were conditioned with carmustine, etoposide, cytarabine, melphalan (beam) or thiotepa, etoposide, cytarabine, cyclophosphamide, melphalan (tecam). patients with multiple myeloma were treated with melphalan as conditioning. diagnosis of ne was established in case of neutropenic fever, abdominal pain or diarrhea, and bowel wall thickening 44 mm on abdominal ultrasonography. febrile neutropenia was seen in 199 (88%) patients of all. the median time from transplantation to neutropenia was 4.5 days (range: 0-9 days). ne occurred in 22 (9.7%) in all neutropenic patients. the median time to ne after auto-sct was 7 days (range: 2-9 days). the median neutrophil engraftment time was 12.5 days (range: 9-18 days). abdominal pain was seen in all patients with ne. twenty one patients (95%) had diarrhea. ileus was seen in 1 (4.5%) patient and septic shock was developed in 3 (13.6%) patients. five (22.7%) of 22 patients had bloodstream infection. klebsiella pneumoniae in 1, pseudomonas aeruginosa in 1, escherichia coli in 1, staphylococcus aureus in 1 and coagulasenegative staphylococcus in 1 patient were documented in patient's blood stream. early diagnosis was made by abdominal ultrasonography in all patients at a day of median 7 days (range: 2-9). twenty (91%) patients were resolved completely with good supportive care and proper antibiotherapy. two (9%) patients died of septic shock and ileus. ne is a rare but serious complication in patients underwent high dose chemotherapy followed by auto-sct. gramnegative bacteria are the main causative pathogens. abdominal ultrasonography is the simple, cheap, fast diagnostic and noninvasive procedure that allows the early diagnosis and effective treatment. disclosure of conflict of interest: none. [p274] neutrophil transfusions in the treatment of neutropenic patients submitted to allogeneic hsct: possible role on graft failure s giammarco, p chiusolo 1 , l laurenti 1 , f sorà 2 , n piccirillo 2 , l teofili 2 and s sica 2 1 hematology department, università cattolica del sacro cuore and 2 hematology departement, università cattolica del sacro cuore granulocyte transfusions (gtx) from g-csf-stimulated donors have been shown to increase the absolute neutrophil count (anc) before expected haematopoietic recovery in neutropenic patients after chemotherapy or haemopoietic sct. thus gt offers a therapeutic option along with antimicrobial agents and growth factors to improve clinical outcome of neutropenic patients with severe infections. the primary limitations of gt include low component cell dose and leukocyte incompatibility. the transfusion of g-csf-mobilized, hla-matched granulocyte components resulted in sustained anc increments, but the efficacy of this procedure has not been established by convincing randomized control trials. aim: we focused our attention on gt in the setting of allogeneic hsct, in particular on the feasibility and safety of this procedure on the rate of engraftment. between 2006 and 2016 our centre performed 211 allogeneic hsct. we analyze data from 59 transplanted patients receiving gt at some point during their disease. indication for gt was severe sepsis mainly due to mdr gram-bacteria. patients received a median of 4 gt (1-34), in different phase: 24 patients during induction therapy, 18 during hsct, 9 at diagnosis and during hsct and 8 after hsct. patients' characteristics are summarized in table 1 . median cd34+ cells dose was 6.4 × 10 6 /kg (range: 1.2-24). donor source was in 49 patients g-csf mobilized peripheral blood, 6 bone marrow and 4 cord blood. median neutrophil recovery (4500/mmc) was 16 days and platelet recovery (420 000/ mmc) was 14 days. sepsis were documented in 36 pts and 45 pts developed fuo. relapse was documented in 21 pts (35%). twenty-two pts are still alive and in complete remission (37%), death occurred in 37 pts: 19 due to trm and the remaining 18 for disease relapse. graft failure occurred in 16 of the 211 pts submitted to hsct. among the 11 patients (18%) who experienced graft failure, six (54%) received gt before hsct, because of sepsis during the induction therapy, and the remaining 5 after hsct, during aplasia period. in the remaining group (152 pts) not receiving gt, only 5 (3%) graft failure were observed. thus a statistically difference (p = 0.0005 fisher's exact test) increase in the rate of graft failure was detected in patients receiving gt. the role of gt in the treatment of infections in neutropenic patients remain still unclear for several reasons including the lack of clinical trials convincingly and consistently demonstrating efficacy, by availability of gt donors and by center's experience. gt has been successfully used in our center in patients with severe sepsis from mdr gram-bacteria during severe neutropenia but an increase number of graft failure has been registered in patients subsequently receiving hsct. alloimmunization to hla antigens in patients receiving gt might lead to an excess of graft failure requiring hla antibodies detection and attempt to reduce titer prior to hcst and maximizing stem cell dose. disclosure of conflict of interest: none. viridans streptococci are microorganisms frequently isolated from blood cultures of patients undergoing myeloablative allogeneic hematopoietic cell transplantation (allohct). poor dentition status has been associated with an increased risk of streptococcal bacteremia in the immediate post-allohct neutropenic period. the objective of this study was to evaluate the impact of oral health status on bacteremia risk in a cohort of patients undergoing therapy for acute myeloid leukemia (aml). a retrospective study was conducted in patients with aml treated at dana-farber/brigham and women's cancer center (df/bwcc) from 2007 to 2011. there was no formal dental assessment prior to aml induction therapy. all patients underwent protocol directed pre-allohct dental evaluation that included a standardized examination, comprehensive dental radiographs, and detailed treatment planning guidelines. poor oral health status was defined as presence of acute or chronic odontogenic infection, and it was assumed that oral health status at the time of induction therapy was the same as the pre-allohct evaluation findings. oral health status at the time of allohct was determined by the completion of required dental treatment. positive blood cultures were recorded from aml induction to day +60 post allohct. organisms that caused bacteremia were classified as 'of possible oral source' by a blinded microbiologist. two-sided fisher's exact test was used to compare the oral health status of the entire cohort to patients with blood cultures of potential oral source. from january 2007 to january 2011, 181 patients with aml underwent myeloablative allohct at df/bwcc and were s260 followed through today +60, and of these, 92 patients met the inclusion criteria and were included in the cohort. the median age was 48 years (range: 24-66) and there was similar distribution of genders. the most common aml induction regimen was daunorubicin and cytarabine (63/92; 68%) and of those that received consolidation therapy (49/92; 53%), almost all patients were treated with cytarabine. nearly all patients (90/92; 98%) received cyclophosphamide and total body irradiation for allohct conditioning and the majority of patients (83/92, 90%) received tacrolimus/methotrexate (n = 51) or tacrolimus/sirolimus (n = 32) for gvhd prophylaxis. over half of patients (51/92, 54%) experienced mucositis during their course of therapy for aml. pre-allohct dental evaluations were completed in 91/92 (99%) of patients. of the 13/91 (14%) patients identified as having poor oral health status, 13/13 (100%) completed all required dental treatment prior to allohct. bacteremias occurred in 63/92 (68%) patients, and 12/63 (19%) had positive blood cultures of potential oral source. of the 12 patients with positive blood cultures of potential oral source, 1/12 (8%) patient developed bacteremia during induction and 11/12 (92%) patients developed bacteremia during allohct. of the 13/91 (14%) patients identified as having poor oral health status, one patient (1/13; 8%) had a positive blood culture with a bacteria of potential oral source during induction/consolidation (p = 0.68). oral health status was not associated with risk of bacteremia of potential oral source at either aml induction/consolidation or allohct. risk of such bacteremia in the setting of myeloablative allohct may be related more to overall gastrointestinal translocation. disclosure of conflict of interest: none. is one of the main alternatives to trimethoprimsulfamethoxazole (tmp-smx) for prophylaxis of pneumocystis pneumonia (pcp)(maertens et al. jac 2016). ato is less effective than tmp-smx to prevent pcp1 but the reasons of this lower efficacy are not well understood. ato acts on pneumocystis, plasmodia and toxoplasma species by inhibiting mitochondrial pyrimidine biosynthesis. ato is highly lipophilic and its absorption in volunteers is improved by a fatty meal. there is a wide inter-individual variability in bioavailability and many drug interferences. the aim of this study was to assess the plasma concentrations of ato in patients under pcp prophylaxis with ato oral suspension and explore the factors which might impact its bioavailability. all adult patients receiving ato for pcp prophylaxis in the hematology and clinical immunology wards between may and september 2016 were included in the study. the prescribed dose was 750 mg of oral suspension twice a day. blood samples were collected around 12 h after the evening dose (cmin) and 1-5 h after the morning dose (cmax). plasma was immediately separated after each sample and frozen at − 20°c until proceeding to the assay. ato plasma levels were measured by uv-high-performance liquid chromatography. clinical and biological data, exact timing and modalities of intake (during a meal or not), and concomitant medications were collected. cmin and cmax results are presented as median (iqr 25-75%) and compared by mann-whitney u-test or signed rank test when appropriate. patients: a total of 85 measurements were performed in 33 patients (allogeneic hsct patients: 19; hematology non-transplanted patients: 6; hiv-infected patients: 7). the mean age (range) was 53 years (33-75), the m/f ratio was 21/12. only two patients were neutropenic. the median cmin was 11.3 μg/ml (6.2-27.8) and the median cmax was 13.4 μg/ml (6.0-28.3). thirteen of the 33 (39%) patients had a cmin. disclosure of conflict of interest: none. presepsin as a marker of infectious complications during high-dose chemotherapy following autologous hematopoietic stem cell transplantation in lymphoma patients y dubinina, v sarzhevskiy and v melnichenko national pirogov medical surgical center lymphoma patients, who undergo high-dose chemotherapy following autologous hematopoietic stem cell transplantation (autohsct), are at high risk of developing infectious complications (ic). mortality from ic during the transplantation, according to various data ranges from 12 to 42%. thus the development of models of early prognosis of ic during autohsct has become more urgent. it's reasonable to include the dynamics of biochemical markers of inflammation in these models. presepsin (psp), procalcitonin (pct) and c-reactive protein (c-rp) were assessed on the day of admission to the hospital (da), on d+1, d+3, d+7 and on the day of discharge (dd). if patients developed neutropenic fever (nf), the markers were assessed at the beginning of the fever, 6 h after, then on the second, third, fourth days after. there were 100 patients included in the study: 41 patients with hodgkin lymphoma, 27with non-hodgkin's lymphoma, 32with multiple myeloma, out of 100 patients there were 51 women and 49. the median age was 41 years (18-66). the conditioning regimens were cbv, beeac or hd melphalan. depending on the presence of ic, the patients were divided into 2 groups: group 1patients without infectious complications (n = 31), group 2patients with the development of infectious complications (n = 69). the median of the nf development was 5.5 days. 53 patients from group 2 had no microorganism growth in blood stream, either in repeated studies. gram+ flora was detected in 12 patients, 1 patient had gram-, 2 patients had mixed flora and 1 patient had pneumocystis jirovecii infection with respiratory insufficiency grade 3. significant differences in psp level between groups 1 and 2 were determined on d+3, on d+7 and the dd after autohsct. considering the median day of the nf appearance (5.5 days), it's supposed both the prognostic value (differences on d+3, that is, 2 days before the clinical manifestation of infection) and the diagnostic value of psp (differences on d+7 and on the dd) ( table 1 , graph 1). [p278] disclosure of conflict of interest: none. hc is often a serious complication and occurs in 70% of allo-hsct recipients. early bleeding is usually the result of chemotherapy toxicity however late occurring hc is multifactorial. bk virus infection has been shown to be related with hc. most studies demonstrate bk virus at the time of bleeding therefore not allowing the risk imposed by asymptomatic infection to be estimated. in this study, our aim is to show the effect of risk factors as well as pre-transplant bk viral load in asymptomatic recipients on development of hc in allo-hsct. between 2014 and 2016, we prospectively evaluated 59 allo-hsct. in order to detect the bk viral load, we performed quantitative bk virus pcr (altona diagnostics, germany) from blood samples at days 0, 30, 60 and 90 after allo-hsct. informed consents were obtained from all participants. bk virus pcr was considered positive if any number of copies were detected above the analytical sensitivity of the tests. the patients were monitored for signs and symptoms of hs. the risk factors for the development of hs were evaluated by univariate and multivariate analysis. p o0.05 was considered statistically significant. the median age of the group was 41 (range: 22-71), 18 of the patients (31%) were aged 450. male to female ratio was 1.36 (34/25). fifty two patients (88%) had diagnosis of malign hematological disease. stem cell source was peripheral blood in 51 (86%), bone marrow in 8 (14%) allo-hsct. patients received stem cells from 26 related donors (44%) vs 33 (56%) unrelated or haplo donors. myeloablative conditioning was administered in 47 patients (80%). forty-four of the conditioning regimens (75%) included cyclophosphamide. hc was diagnosed in 22 patients (37%) at a mean of 100 days (range: 0-367), early hc was detected in 4 of 22 patients (18%). the frequency of bk viremia and number of viral copies are given in detail in table. the frequency of bk viremia increases during transplantation in relation to clinical hc (66%, 66%, 87%, 100%; p = 0.007). acute graft vs host disease (agvhd) was diagnosed in 37 patients (63%) at a median time of posttransplant day 67: grade i-ii gastrointestinal/skin/liver in 31 (84%), grade iii-iv gastrointestinal/ skin/liver in 6 patients (16%). the most common gvhd prophylaxis preferred was cyclosporine and methotrexate in 50 patients (85%). in univariate and multivariate analysis (age450, sex, diagnosis, stem cell source, donor type, conditioning regimen, agvhd, cy administration, bk virus pcr at days 0, 30, 60) bk virus titer positivity at day 0, 30, 60 (p = 0.008, p o0.001, p o0.001), myeloablative conditioning (p = 0.018), the presence of agvhd after day 30 (p = 0.018) and conditioning regimen that includes cyclophosphamide (p = 0.024) are found to be related with increased risk of hs. patients with hc and clots were treated with continuous bladder irrigation as well as 8 of patients with bk viremia received cidofovir and six of them responded to treatment (75%). our study showed that, bk titer positivity, myeloablative conditioning, presence of agvhd, cyclophosphamide containing conditioning are associated with hc. detection of bk viremia in later transplant period is more sensitive for clinically proven hc. prophylactic treatment might be considered in patients with asymptomatic bk viremia in pretransplant period. [p279] disclosure of conflict of interest: none. this project has been granted by ankara university scientific research committee numbered as 15b0230007. high-dose chemotherapy with peripheral blood progenitor cell (pbpc) collection followed by a myeloablative conditioning and autologous stem cell transplantation (asct) is considered the standard of care of relapsed/refractory non hodgkin/hodgkin lymphoma (nhl/hl). a widely adopted conditioning regimen is the combination of carmustine etoposide cytarabine and melphalan (beam), whose feasibility and efficacy has been largely demonstrated. high dose fotemustine plus etoposide, cytarabine and melphalan (feam) has in some cases replaced beam conditioning. neutropenic enterocolitis (nec) is a life threatening complication of patients (pts) treated with chemotherapy (cht) with mortality rate up to 50%. it's a clinical syndrome in neutropenic patients (pts) characterized by abdominal pain (ap), fever (f) and diarrhoea (d). ultrasound (us) was used to evaluate bowel-wall thickening (bwt), and 44 mm is considered diagnostic of nec. early diagnosis is crucial to start conservative medical management (cmm), which appears the optimal strategy for most cases. objective: 1. to evaluate if nec incidence and outcome differs in beam vs feam and 2. to evaluate prospectively if bed-side-us (bus) can detect early signs of nec and guide a prompt treatment (cmm or surgical) in order to reduce mortality. in the last 5 years all pts with nhl/hl admitted in our bmt unit wards at university of pisa (italy), undergoing asct were prospectively enrolled. abdominal us was performed, baseline before treatment, and as only one symptom (or a combination) appeared within 12 h from onset: f and/or d and/or ap in cht-related neutropenic pts. 95 pts were conditioned with beam and 52 pts with feam. nec was diagnosed in n = 19/52 feam and in n = 25/95 beam patients. incidence was 36% and 25% respectively, without a statistically significant difference (p = 0.234). two pts died/19 in feam arm (10.5%) and 2pts/24 in beam arm (8.3%), without a statistically significant difference (p = 0.778). at time of diagnosis (dx) symptoms were: f+ap+d 45%, f+d 4%, f+ap 3%, ap+d 35%,d 3%,ap 10%. f alone was never present at diagnosis of nec. at dx, f was absent in 18/44 nec episodes (40%). all pts were treated promptly as bus allowed diagnosis with cmm except one 1 pts who underwent surgery, guided by us features, during neutropenia. the likelihood of nec dx in a discriminant st model (bayes theorem) for pts with bwt and ap = 98.8%, ap+d = 99.9%, ap+d+f = 100%, ap+f = 99.9%, d+f = 5%. bus allowed to detect early signs of nec and to start prompt treatment in this life threatening complication, of nhl/hl pts undergoing asct. this is a prospective study thus the true incidence of nec in nhl/hl undergoing asct should not be underestimated. there is not a statistically significant difference in incidence and outcome of nec in pts conditioned with beam in respect to feam. with bus pts do not live the isolation room. fever is not a condition sine qua non for nec diagnosis. early diagnosis allows most of pts to be treated with cmm. images of bus and ct were superimposable with lower costs, and less radiation exposure. a low mortality rate in pts with a 25-36% chance of developing this life threatening complication suggests that a prompt bus in neutropenic patients as just one symptom presents allows to make early diagnosis of this life threatening complication and guide prompt treatment (conservative or surgical), reducing mortality. disclosure of conflict of interest: none. quantiferon-cmv in the evaluation of cmv-specific immunity after autologous and allogeneic hsct j moreno 1 , ltesta 1 , l zanetti 1 , l serra 1 , b pereira 2 , m souza 1 , a carolina souza 2 , mp souza 1 , vr colturato 1 and cm machado 1,2 1 hsct program, amaral carvalho foundation and 2 virology laboratory, institute of tropical medicine, university of são paulo cytomegalovirus (cmv) is a major cause of morbidity and mortality after allogeneic hsct. the same is not observed in autologous hsct recipients who do not need to receive immunosuppression after transplantation. in the present study, we compared the reconstitution of cmv-specific immunity in autologous and allogeneic hsct recipients. patients were invited to participate in the study and signed the informed consent. cmv surveillance with the antigenemia (ag) test (cmv brite, biotest, germany) was done weekly in the first 3 months of transplant in allogeneic hsct recipients. preemptive ganciclovir therapy was initiated whenever a positive antigenemia was detected. the presence or absence of cmvimmunity was determined by a commercial interferon (inf) gamma release assay (quantiferon cmv, qiagen) before hsct and monthly thereafter up to d+90. from january to october 2016, 106 hsct recipients (29 auto and 77 allo) were included in the study. ag was positive in 54 (70%) of the allohsct recipients at a median of 39 (range: 14-146) days. ag recurrences occurred at a median of 81.5 (38-249) days, in 15 of the 54 pts (27.8%) who had at least one episode of positive ag. 103 hsct recipients were included in the analysis of qtf-cmv. in the pre-hsct sample, qtf-cmv was reactive in 60 of the 85 allohsct (70.6%) and in 25 of the 28 autohsct (89.3%). significantly less allo hsct recipients recovered cmvimmunity at day +30 (31.8%) and day+60 (59.3%) in comparison with autohsct (95% and 100%, respectively, p o0.01). up to day +90, all autohsct have recovered cmvimmunity, in comparison to 68% of the allohsct recipients (p = 0.096, figure 1 ). the qtf-cmv test performed at d+30, d +60 and d+90 did not predict the risk of cmv reactivation in the following month. similarly, the test did not anticipate the risk of ag recurrences: 80% of the hsct recipients with undetermined or non-reactive qtf-cmv test at d+60 had ag recurrence after this period, in comparison with 70% of the patients with a reactive result (p = 0.56). in the present study, the qtf-cmv test alone could not predict the risk of cmv reactivation or recurrences. [p281] disclosure of conflict of interest: qiagen. recovery of vδ2+ γδ t cells is critical to epstein-barr virus reactivation after haploidentical hematopoietic stem cell transplantation j liu, z bian, q fu, l xu, x zhang, y wang and x-j huang peking university people's hospital, peking university institute of hematology, beijing, china epstein-barr virus (ebv) reactivation and its related disease are life-threatening complications in patients undergone haploidentical hematopoietic stem cell transplantation (haplohsct). our previous studies found that impaired cd4 − cd8 − t-cell recovery correlated to the increased occurrence of ebv infection after haplohsct. γδt cells make up 50-90% of cd4 − cd8 − t cells in the peripheral blood of healthy donors. expansion of vδ1+ γδt t cells after hsct has been reported and this subset could respond against autologous ebv-lcl in vitro. selective activation and expansion of vγ9vδ2-t cell could inhibit ebv-lpd development in humanized mice. however, the association of γδ t-cell recovery with ebv reactivation after allohsct remains unknown. this is a prospective cohort study including 110 consecutive patients who were diagnosed as hematological malignancy and underwent haplohsct. recovery of t lymphocyte and a panel of subsets, including cd3+, cd4+, cd8+, cd4-cd8-, tcrαβ+, tcrγδ+, vδ1+, and vδ2+ t cells, were determined by flowcytometry at 30, 60, 90, 180 days after haplohsct. all recipients and donors were tested negative for ebv dna in the peripheral blood before transplantation. recipients were monitored weekly for ebv dna load until day 100 after transplantation. recipients with peripheral blood plasma ebv dna load 41000 copies/ml at least on two consecutive occasions were diagnosed as ebv reactivation (ebv +). ebv − cohort generally represents patients whose ebv dna loado1000 copies/ml in peripheral blood. within 100 days after haplohsct, 17 of 110 (15.5%) recipients were diagnosed as ebv reactivation. compared to recipients with negative ebv dna load, the counts of cd3+, cd8+, and tcrαβ+ t cells were not statistically different in the ebv+ cohort from 30 to 180 days after haplohsct. in contrast, recoveries of cd4+ and cd4-cd8-t cells in ebv+ patients were significantly hampered at 30 days after transplantation (p = 0.021 and p = 0.046, respectively). although the tcrγδ+ t-cell counts were also decreased at 30 and 60 days in the ebv+ cohort, the comparisons did not reach the statistical significance (p = 0.072 and p = 0.082, respectively). notably, recoveries of vδ2+ γδ t cells at 30, 60 and 90 days were continuously delayed in recipients with ebv reactivation (p = 0.029, p = 0.001 and p = 0.046, respectively). whereas the counts of vδ1+ γδ t cells were similar between the two groups from 30 to 180 days in this context. in this prospective and large cohort study, we showed that the occurrence of epstein-barr virus (ebv) reactivation was associated with the hampered recovery of vδ2+ rather than vδ1+ γδ t cells after haplohsct. our findings will help explore γδt subset-dependent therapeutic strategies to control the serious complications due to ebv infection post transplantation and improve the overall survival of haplohsct recipients. disclosure of conflict of interest: none. in particular, bloodstream infection (bsi)is a frequent complication in the pre-engraftment phase with an impact on the morbidity and mortality of these patients. objectives: to analyze the incidence of bsi in patients undergoing hsct in our center, and to identify predisposing factors for the development of bsi in pre-engraftment phase patients after hsct. fifty-one consecutive patients undergoing hsct were analyzed retrospectively in our center during the period of 1 july 2015 and 30 june 2016. the characteristics of the sample are shown in table 1 . we have reported all the bsi between day 0 and day 30 after stem cell infusion. 70.5% (36 patients) received antibacterial prophylaxis with ciprofloxacin, five patients with broad spectrum antibiotics and five did not received any drug. the average days of fever have been 3.30 days (0-12 days). a total of 184 blood cultures has been collected (3.6 per patient). there have been 15 bsi (21.5% of the patients) with 8 (53.4 %) of cases caused by gram-negative organism (4 escherichia coli, klebsiella pneumoniae, acinetobacter baumanii, proteus vulgaris and delftia acidovorans) and 7 (46.6%) by gram-positives (1 enterococcus faecium, 1 enterococcus faecalis, 3 staphylococcus epidermidis, streptococcus mitis and streptococcus viridians group). one patient presented 3 different episodes of bsi, two patients 2 independent episodes and the rest eight, only one microorganism isolated. we have identified two bsi by extended-spectrum betalactamases (esbl-producing organism) and one isolation of carbapenem-resistant gram-negative bacteria. the rate of quinolone-resistant is 80% in all the sample. in univariate analysis, several factors like presence of comorbidities, presence of severe mucosits, type of catheter and antibacterial prophylaxis modality don't increased the risk to develop bsi (p40.05). the place where the procedure is performed does not influence the development of bsi. although the presence of previous infections is not a risk factor, hospitalization for infection in the 90 days before hsct does influence the development of bsi with statistical significance (po0.05). the crude mortality rate of the sample has been very low (2%), with only one death related to bloodstream infection. bsi are a common relative complication in the patient undergoing hsct but with an extremely low mortality in our sample. hospitalization for infection in the 90 days before hsct does influence the development of bsi. it is important to note that outpatient model and conventional rooms don't increased the incidence of bsi. although the use of quinolones in prophylaxis does not result in an increase in infections caused by multiresistant micro-organisms (esbl and carbapenemias) with acceptable resistance rates (80%), it also does not reduce the incidence of bsi in our sample. according to our analysis, his routine employment still throws light and shadows. [p283] disclosure of conflict of interest: none. septic episodes with multiple bacterial strains during antithymocyte globulin (atg) therapy for conditioning for allogeneic stem cell transplantation under rifaximin gut decontamination d markel 1 , c schultze-florey 1 , t brockmeyer 1 , v panagiota 1 , c lück 1 , a schwarzer 1 , m beck 1 , e dammann 1 , a ganser 1 , g beutel and m eder 1 recent evidence demonstrates the importance of the enteric microbiome for the development of gastrointestinal graftversus-host disease (gvhd) and mortality after allogeneic stem cell transplantation (sct) (1, 2) . accordingly, the usage of the non-absorbed rifamycin derivate rifaximin for gut decontamination has been reported to preserve the intestinal microbiota composition with a positive effect on overall survival in a single centre retrospective analysis (3). we here report severe septicaemia requiring therapy at the intensive care unit (icu) during atg application for conditioning in three patients with rifaximin used as single agent for gut decontamination within 6 months. after changing our gut decontamination from a chinolon-metronidazole regimen to rifaximin, three cases of severe septicaemia by gram-negative and gram-positive bacteria during atg treatment occurred within 6 months. patient #1 was a 52-year-old woman with tmds/aml after breast cancer conditioned according to the flamsa-bu protocol. the second (#2) and third (#3) patient were 55and 37-year-old males with a complex karyotype secondary aml after omf and relapsed inv(16) aml with meningeosis leucaemica, respectively. patients #2 and #3 were treated with flamsa-bu and flamsa-tbi, respectively. all patients received rabbit atg (atg fresenius/grafalon) at a dose of 3 × 10 mg/kg body weight and rifaximin (2 × 200 mg) for gut decontamination. patient #1 developed severe escherichia coli and pseudomonas aeruginosa septicaemia on day − 3 of the conditioning regimen and had to be transferred to the icu with septic cardiomyopathy for therapy with vasopressants and levosimendan. in patient #2 escherichia coli, klebsiella oxytoca, staphylococcus hemolyticus and staphylococcus epidermidis were simultaneously detected in blood cultures at day − 2. the patient was transferred to the icu and treated with vasopressants for septic shock. patient #3 developed septic shock due to klebsiella pneumoniae and enterobacter cloacae on day − 4 under atg therapy. mechanical ventilation and vasopressor therapy were required. fortunately, all three patients survived and completely recovered without any sepsis related disabilities under escalated anti-infective and intensive care therapy. all were discharged from the hospital in the outpatient clinics. interestingly, all isolated gram-negative pathogens were found to be sensible for a chinolon based gut decontamination. the reasons for these septic complications under atg therapy are not exactly understood but raise a note of caution on the use of rifaximin as single agent gut decontaminant during atg application in conditioning for allogeneic sct. infections with mycobacterium genavense were described for the first time in 1990. since then, several cases have been reported, but almost exclusively in patients with aids. most patients who underwent hsct have insufficient cellular immunity. here we report a mycobacterium genavense infection in a patient mimicking a lymphoma-relapse after hsct. a 58year-old female patient was diagnosed in july 2013 with stage ivb alk-negative anaplastic t-cell-lymphoma with cervical, retro-/supraclavicular, mediastinal, axillary and retroperitoneal lymphadenopathy as well as pulmonary manifestation. two chemotherapy treatment lines and autologous stem cell transplantation resulted in a partial remission. to improve remission prior to hsct the patient received 2 courses of brentuximab-vedotin. after conditioning therapy with fludarabine, busulfan, cyclophosphamide and atg, hsct from a hla compatible unrelated donor was performed in april 2014. a pet-ct-scan in november confirmed complete remission. after hsct the patient remained lymphocytopenic with cell count of cd4+ cellso100/μl. after acute stage iii gastrointestinal graft-versus-host disease (gvhd) low dose immunosuppressive therapy was maintained due to mild chronic gvhd of the liver and the upper gastrointestinal tract. beginning in june 2015 the patient experienced increasing fatigue, general weakness, loss of appetite, nausea, night sweating and fever. abdominal ultrasound, urine and blood culture as well as ct scans revealed no focus of infection. different lines of empirical antibiotic therapy resulted only in short term improvement. several blood culture tests remained sterile. a fdg-pet-ct scan showed a paraaortal and parailiacal lymphadenopathy with a high fdg uptake (suv between 19.7 and 20.1), highly suggestive of lymphoma relapse. endoscopic evaluations revealed two polypoid lesions in the bulbus duodeni. histology of duodenal biopsies revealed a massive accumulation of weakly pas-positive bacilli. pcr analysis confirmed an infection with mycobacterium genavense. despite several attempts mycobacteria were not recoverable on solid media even by long term culture. treatment was started with rifampicin, ethambutol, ciprofloxacin and clarithromycin. lymph node manifestation responded to therapy with decreasing fdg-uptake (suv 8.2) in a control fdg-pet-ct scan 3 months later. after 9 months treatment was terminated due to therapy refractory nausea. lymphocytopenia was persisting with cd4+ cellso100/μl. six weeks after stopping the antibiotic therapy, symptoms as fever and weakness reappeared. duodenal biopsy could not confirm persistent mycobacterial infection. fdg-positive intraabdominal lymph nodes (suv 11.2) and spleen (suv 8.2) were detected in a control fdg-pet-ct-scan. five lymphnodes were surgically removed. immunohistology detected histiocytic cell proliferation with no sign of lymphoma relapse. pcr confirmed the presence of mycobacteria-dna. consequently, antibiotic treatment was resumed. mycobacterium genavense can present with all the symptoms of a lymphoma relapse and should be considered in immune compromised patients. reliable diagnosis can only be obtained from lymph node biopsies and/or endoscopic evaluation. treatment has to be accompanied by restoring cellular immunity and should only be stopped after pcr-negative biopsies. disclosure of conflict of interest: none. stratification of patients with multiple myeloma and lymphoma undergoing autologous hematopoietic stem cell transplantation in term of antifungal prophylaxis r moghnieh, s khaldieh, l awad, d abdallah, n droubi, a youssef, a mougharbel, t jisr and a ibrahiim makassed university hospital, beirut, lebanon autologous hematopoietic stem cell transplantation (ahsct) is at intermediate risk for invasive fungal infections (ifi). the recommendations of international scientific societies are not homologous regarding prophylaxis against ifi in patients (pts) undergoing ahsct. the primary end point was to assess risk factors for the need of empiric/preemptive antifungal therapy in ahsct recipients, and to extrapolate to the subgroup of pts that requires antifungal prophylaxis in our population of ahsct pts. the secondary endpoint was to determine the fungal species distribution infecting or colonizing the pts. our study included adult pts (418 yo) who underwent ahsct for lymphoma and multiple myeloma (mm) between 2005 and 2015. all febrile neutropenic pts are being managed according to the 2010 infectious diseases society of america (idsa) guidelines regarding the use of antimicrobial agents in neutropenic pts with cancer. eligible pts were divided into two groups: those who received empirical antifungal therapy and those who did not need it. we recorded demographic and baseline clinical characteristics including: age, gender, comorbidities, stage, disease status at ahsct, high-dose therapy regimen, the presence of mucositis and its grade, the number of cd34 + cells transfused, the presence of central line or portacath, the need for mechanical ventilation, the presence of diarrhea, the duration of neutropenia, and the presence of bloodstream infections. pts who had lung infiltrates suggestive of ifi were analyzed separately. the causative fungal pathogens and colonizers were analyzed. univariate and multivariate analysis of potential risk factors to assess further significance was performed using spss. 190 patients were included.106 pts (56%) had lymphoma and 84 pts (44%) had mm. the need of empiric antifungal therapy was statistically more significant in lymphoma than mm pts (po0.01).the presence of mucositis grade ⩾ 3 showed a statistical significance for the need of antifungal therapy (p = 0.02). in the lymphoma group, remission status (pr vs cr) was not a significant factor for the need of empiric antifungal therapy (p = 0.49).the presence of mucositis grade ⩾ 3 was at the limit of significance ( p = 0.05). in the mm group, remission status (pr vs cr) did not affect the need of empiric antifungal therapy (p = 1). however, mucositis grade ⩾ 3 was found to be a significant risk factor for the need of empiric antifungal therapy (p = 0.02). following factors: the number of cd34 + cells transfused, the presence of central line and portacath, the need for mechanical ventilation, the presence of diarrhea, the duration of neutropenia, and bloodstream infections did not show any significance for the need of antifungal prophylaxis in both groups. all recovered fungal isolates (n = 14) were not from deep seated tissues biopsies or blood, and were identified as candida albicans in 7 with lymphoma, and in 7 with mm. they reflected the candida ecology in this pts series rather than deep seated fungal infections. we suggest to give antifungal prophylaxis to all lymphoma pts because of the higher need of empirical antifungal therapy, and give antifungal prophylaxis to mm pts having a predisposition for severe mucositis. fluconazole is the antifungal of choice for prophylaxis since all the fungal isolates were candida albicans. keywords: autologous hematopoietic stem cell transplantation, antifungal prophylaxis. disclosure of conflict of interest: none. a 48-year-old previously fit woman from a rural area of eastern europe was admitted to the hospital for severe aplastic anemia. steroids, csa, antinfective prophylaxis and supportive therapy were administered without response; therefore rabbit atg was then administered, with minor response; the year later, she underwent allogeneic-hsct (mud 9/10, ric: tbi, cyclophosphamide and fludarabine; gvhd prophylaxis: atg, csa, mtx). several days after transplantation she developed left migraine with ipsilateral back-eye pain. brain mri and ct showed a diffuse opacification of paranasal sinuses, mainly in the sphenoid sinus. the symptoms gradually improved with a specific treatment. the patient achieved a quick and complete haematological recovery and she was discharged. at follow-up visits she complained a flare of the migraine, with a left-sided headache that did not improve with nsaids. the headache gradually intensified until vision in the left eye became blurred with conjunctival injection. after consultation with ophthalmologist, for suspected toxoplasma retinitis, administration of intravitreal steroids and clindamicine was begun with partial benefit. however 15 days after (d +90) she was admitted in hospital because of worsening headache, irradiated in the occipital area, and weakness in the right hemibody. tests on csf were negative for neurotropic pathogens. an mri showed a complete occlusion of the intracranial tract of left internal carotid artery, with likely infectious material localized in the left lateral cerebral fissure. a chest tc showed a nodule with initial excavation in the right superior pulmonary lobe. for suspected tuberculosis she started antitubercular therapy. despite a second lumbar puncture confirmed pleocytosis compatible with acute purulent meningitis, microbiological research for bacteria, fungi and bk were negative. so antitubercular and antitoxoplasma therapy were stopped and the patient underwent surgical biopsy within the sphenoid sinus. pathological examination of the biopsy specimens showed acute and chronic inflammation of the respiratory mucosa, periodic acid. schiff and grocott staining ( figure 1 ) highlighted several septate fungal hyphae. cultural analysis revealed colonies of scedosporium apiospermum so the patient started targeted voriconazole intravenous therapy. nevertheless, 10 days later, she developed aphasia and right hemiparesis. a brain angio-mri confirmed the appearance of new lesions compatible with infectious localizations associated to an increased defect of left internal carotid artery vascularisation and complete left choroid detachment. after 2 weeks of voriconazole a significant clinical improvement have been observed and she was discharged, continuing oral antifungal therapy with voriconazole. at the last follow-up she achieved a complete resolution of neurologic symptoms, with permanent left eye blindness. 8 months later (d +370) she was asymptomatic, with normal haematological and neurological conditions and was able to stop the antifungal therapy. this case-report confirms that the risk of invasive fungal infection (ifi) is relevant in patients receiving hsct for aa, probably due to the prolonged neutropenia and association of other risk factors such as the immunosuppressive therapy and the iron overload. in this very poor prognosis infection, the early diagnosis of cns ifi remains challenging, but the administration of voriconazole was extremely effective. disclosure of conflict of interest: none. in this study, we aim to present the seroprevalence of ebv and incidence of posttranplant lymphoproliferative disease as well as to evaluate the relation with gvhd. between 2006 and 2015, the ebv serology of 364 patients that underwent allogeneic hematopoietic stem cell transplantation and their donors were evaluated in the study. ebv ig g (vca-igg, ebna ig g, ea-igg) and igm (vca-igm) antibodies were detected by chemolluminesance method (abbott, abd). all patients were followed for reactivation. ebv igg seropositivity was detected in 338 patients (93%) and 238 donors (77.7%). there was no statistically difference in related vs unrelated transplants in seropositivity. the median age of the patients was 37 (range: , 217 patients were male (60%) and 295 (81%) had malign disease. the stem cell source was peripheral blood in 299 (82%) patients and 258 (71%) received grafts from related donors. myeloablative conditioning regimen was received by 273 of patients (75%) (table) . all patients received acyclovir prophylaxis (related transplants 400mg tid, unrelated transplants 800 mg tid) during and after allo-hsct up to 3 months. twenty six-yearold pretransplant ebv seropositive aplastic anemia patient had ebv ig m positivity after 3 months of allo-hsct and developed lymphoproliferative disease. he was in complete remission after 4 courses of rituximab and methylprednisolone. three patients were ebv igm seropositive in 4th, 9th and 24th months of allo-hsct and received symptomatic treatment. acute gvhd was detected in 223 patients (61%) whereas 285 patients (78%) had chronic gvhd. acute gvhd and chronic gvhd incidences were similar in comparison of donor ebv seropositive vs seronegative status (78% vs 22%, p = 0.72; 80% vs 20%, p = 0.199). ebv seropositivity was detected in 92.8% of patients. the donor ebv serology was not related with acute or chronic gvhd. [p290] disclosure of conflict of interest: none. the umc utrecht pediatric experience with brincidofovir after allo hsct ca lindemans, m bierings and jj boelens pediatric blood and marrow program, dept. of pediatrics, university medical center utrecht, the netherlands viral reactivation with dna viruses form a considerable complication of allogeneic hematopoietic stem cell transplantation (hsct). there are little effective antiviral therapies and most have considerable toxicity. especially for adenovirus, there is no satisfactory therapeutic option. recently a new oral antiviral agent, the cidofovir prodrug brincidofovir became available to european patients only on the basis of urgent medical need and after a case by case approval by the health authorities. the aim was to describe our single center experience with brincidofovir in the pediatric allogeneic hsct setting. in the umc utrecht, pediatric patients receive t-replete bone marrow or unrelated cord blood (ucb) as the donor source after mostly myeloablative conditioning regimens (+ serotherapy in unrelated-hct). as gvhd prophylaxis patients receive cyclosporine a (csa) and mtx for bone marrow, csa and prednisone for ucb. patients are by standard weekly monitored for the presence of adenovirus, ebv, cmv en hhv6 viremia by rt pcrs in the plasma. extensive immune reconstitution measurements are performed every 2 weeks. since 2015, patients that developed viral reactivation with adenovirus, or a combination of other dna viruses (cmv, bk or hhv6) were offered brincidofovir if the viremia was progressive or in the context of poor immune reconstitution. brincidofovir was given in suspension (10 mg/ml) at the dose of 2 mg/kg biw, or 100 mg biw for larger children. de drug was discontinued when the viral load was below detection level. in total, six pediatric patients (age range: 0-18) received brincidofovir (2 patients tablets, 4 the suspension). four received it for adenovirus reactivation, a 5th patient for cmv and bk and a 6th patient for cmv en hhv6. the median day post-hsct of the first administration was 29 days post hsct (range: − 4 to 101), the median day post detection of viral reactivation 14 days . the median duration of administration was 36 days (10-98) with two patients being discontinued because of death. in no patient the drug was discontinued due to toxicity issues. the patients that died had multi-organ failure due to a combination of severe agvhd and multiple infectious issues. the patients were discontinued when the viral load was low and when they had cd4 counts of at least 50/μl. none of the four alive patients reactivated after the drug was discontinued. urgent medical need administration of brincidofovir is feasible. in our limited series we found the drug was well tolerated. disclosure of conflict of interest: i am a medical consultant for brincidofovir (chimerix). reactivation of herpes simplex virus 1 (hsv-1) or varicellazoster virus (vzv) occurs frequently after allogeneic stem cell transplantation (asct). here, we report three unusual cases, two with reactivation of hsv-1 and one with vzv. patients and methods: patient (pt) 1 (50-year-old, male) was allografted for high risk acute lymphoblastic leukemia in first complete remission after conditioning with total body irradiation (12 gy) and etoposide (60 mg/kg). graft-versus-host disease (gvhd) prophylaxis was performed using cyclosporine a, short course methotrexate and anti t-lymphocyte globulin (atg). pts 2 (44year-old, female) and 3 (67-year-old, male) were allografted for acute myeloid leukemia in second and first complete remission, respectively. conditioning regimens used were flamsa-ric in pt 2 and fludarabine/busulfan in pt 3. in both cases, gvhd prophylaxis consisted of cyclosporine a, mycophenolate mofetil, and atg. pts 1 and 2 had already experienced hsv-1-positive oral mucositis following induction chemotherapy and had successfully been treated with acyclovir. both developed hsv-1-positive oral mucositis again after asct. in both cases, initial therapy with acyclovir i.v. at a dose of up to 10 mg/kg t.i.d. was ineffective. to explore the mechanism leading to clinical acyclovir resistance, the thymidine kinase genes of both viral strains were sequenced. pt 3 presented with severe abdominal pain and nausea 11 months after asct. in this case, acyclovir prophylaxis post asct had been stopped 2 months before due to side effects. moreover, low dose prednisolone therapy was necessary for chronic gvhd. the hsv-1-strain from pt 1 showed a single base pair deletion in the region from nucleotide position 430 to 436 of the thymidine kinase gene (which consists of a guanosine repeat). in pt 2 a single base pair insertion in the same region was found. both genetic alterations lead to a loss of enzyme activity and acyclovir resistance. in both pts treatment was changed to foscarnet which led to rapid improvement. in the case of pt 3, multiple mucosal erosions were found on endoscopy of the esophagus. in these vzv dna was detected by polymerase chain reaction (pcr). only 4 days later, a vesicular skin eruption developed, which did not follow a dermatomal distribution. again, in the vesicular fluid vzv dna was detected by pcr. in this patient, acyclovir (10 mg/kg i.v., t.i.d.) resulted in rapid improvement. reactivation of hsv-1 and vzv after asct is a frequent finding. usually, hsv-1 strains respond well to acyclovir. in some cases, resistance can develop, especially in patients that had been treated with acyclovir before. acyclovir resistance of hsv-1 caused by mutations in the thymidine kinase gene can be overcome by treatment with foscarnet which directly inhibits the viral dna polymerase. disseminated vzv reactivations after asct have been described. clinical presentation can be misleading, for example, beginning with severe abdominal pain that precedes the vesicular eruption by several days. disclosure of conflict of interest: none. toxoplasmosis is a rare but severe complication after hematopoietic stem cell transplantation (hsct) (1) . it can involve the central nervous system alone or can manifest as a disseminated disease. in the paediatric population the mortality rate is high and sequelae are often severe. new diagnostic tools, such as the pcr assay, may allow for rapid diagnosis and preemptive therapy (2, 3) . we retrospectively analysed all children who underwent allogeneic hsct in our centre between january 2011 and december 2015. patients lost to follow up before day +100 were excluded. patients and donors were tested before transplant in order to assess their immunological status against t. gondii. a total of 187 allo-hsct were analysed. before transplant, 28.8% of recipients (r) were toxo-igg positive and 71.2% were toxo-igg negative. among donors (d), serology was available only for 152/187: 23% were toxo-igg positive, 77% were toxo-igg negative. we found a high number of not tested donors (18.7%, 35/187) which included, in most cases, mud from foreign registries. the group at higher risk for toxoplasmosis, d − /r+, included 21.7% pairs, whereas d − /r − were 55.2%, d+/r-were 15.1% and d +/r+ were 7.9%. in our series the cumulative incidence of toxoplasmosis disease was 2.1%, with 4 cases out of 187 transplants. two of them (case 1 and 3) had cerebral toxoplasmosis, one (case 2) had disseminated toxoplasmosis and case 4 had toxoplasmic chorioretinitis. mortality rate was 50%: two patients died because of multiorgan failure and disseminated toxoplasmosis respectively. in no case localized cerebral toxoplasmosis was the main cause of death. no complications were seen in surviving patients. all patients who developed toxoplasmosis were toxo-igg positive before hsct and three of them were transplanted from a toxoplasma igg negative donor (fourth donor not tested). in the two fatal cases the interferon-gamma releasing assay (igra) never became positive, confirming the absence of specific cellular immunity. toxoplasmosis disease can affect hsct outcome in paediatric recipients and pre-hsct seropositivity is the most important risk factor for toxoplasma disease in the post transplant period. in our cohort seroprevalence was higher than expected, probably due to the high number of patients coming from eastern europe. in order to reduce the burden of toxoplasmosis disease in our population we decided to implement a real-time pcr screening protocol for d − /r+ pairs, to provide rapid diagnosis and early therapy. all positive recipients with a seronegative donor will undergo real-time pcr screening starting on the day of stem cells infusion, and regularly until cd4+ t cell recovery. in the future we will analyse the impact of this strategy in this particular subset of immunocompromised patients. treatment with brincidofovir for adenovirus disease in pediatric hematopoietic transplants introduction adenovirus may cause serious morbidity and mortality after allogeneic hematopoietic transplants in children. severe lymphopenia is the main risk factor associated with progression to disseminated and often fatal disease. treatment with unlicensed cidofovir is based on monitoring of plasma viral load by pcr. however, cidofovir is only moderately effective at controlling adenovirus and it is associated with significant renal toxicity. brincidofovir is a lipid conjugate of cidofovir. it has a good oral bioavailability and achieves higher intracellular levels of active drug than cidofovir with a better safety profile. it is a potent inhibitor of viral dna synthesis so it could be indicated in immunocompromised patients with adenovirus disease. patients and methods we present three children of 3, 5 and 9 years old diagnosed of acute lymphoblastic leukemia (all) in 2nd complete remission (the first two patients) and severe aplastic anemia the last one. there were 2 girls and 1 boy. they underwent a peripheral blood hematopoietic stem cell transplantation using αβ/cd19 depletion with a haploidentical donor in the two patients with all and cd45ra depletion with a matched unrelated donor in the other patient. patients that underwent haploidentical transplants developed early acute graft versus host disease grade iii with gut and skin involvement so immunosuppressive treatment with corticoids was started. they developed severe lymphopenia ( o300/ mm 3 ). in the first month after transplant an adenovirus disease was diagnosed in the three patients from the weekly monitoring of plasma viral load by pcr. adenovirus was also tested in stools, urine and respiratory sample. in all patients adenovirus was also detected in urine sample. in one of them adenovirus was detected in nasal exudate too and in the other the virus was isolated in stools and in a skin biopsy. results: all of them were initially treated with cidofovir with poor results. foscarnet and gancyclovir was also used without improvement. finally they started a treatment by compassionate use with oral brincidofovir twice a week. with the first dose of brincidofovir plasma viral load started to go down until its complete disappearance. brincidofovir tolerance was good with only mild and limited diarrhea in two cases in the day they were taking brincidofovir. two of the three patients were alive without signs of adenovirus disease. in the other patient blood adenovirus load by pcr decreased below 1000/ml, but remain high in urine. she died of respiratory failure due to pulmonary graft versus host disease. conclusion brincidofovir may be a promising therapeutic option for the treatment of severe adenovirus disease in immunocompromised patients with a good toxicity profile. disclosure of conflict of interest: none. table 1 . all patients were transplanted with pbsc for haematological malignancy, and s269 received reduced intensity conditioning (ric) regimens with in vivo t-cell depletion. the proportion of patients with baseline and post-vaccination hi titres ⩾ 1:40 were 28.6 and 25% for a(h1n1)pdm09, 14.3% at both time points for a (h3n2), and 32.1 and 25% for b/phuket. pre and postvaccination geometric mean titres gmt) were higher by mn than hi for a(h1n1)pdm09 and a(h3n2), but lower for b/ phuket (p = 0.05). no post-vaccination seroconversions were detected by hi, while a single seroconversion to a(h1n1) pdm09 was detected by mn in a patient vaccinated at 0-3 months. the mn assay did not detect any additional low-titre seroresponses (negative to detectable titre) below hi threshold. none of patient age, lymphocyte count, days from transplant to vaccination, donor type, and gvhd or ist at vaccination correlated with baseline or post-vaccination titres by either assay. response to iiv was virtually absent throughout the first year post-hsct, with a single seroconversion to a(h1n1)pdm09 detected by mn but not hi, although the sample size was small and half of patients were vaccinated at 0-3 months. there is a clear need for a novel, immunogenic seasonal iiv and/or novel vaccination regimens in this population. vaccination of recipients' relatives and close contacts, and hsct healthcare workers should be strongly encouraged. pre-and post-transplant iron overload (io) has been associated with considerable long-term morbidity and mortality in pts undergoing transplantation. classically, management of io in the post-allo-hsct setting has been based in the performance of therapeutic phlebotomies (tp), which are inconvenient for the patient and are often not feasible due to ongoing anemia. we recently published the first prospective study of deferasirox in adult allo-hsct pts with io (vallejo, et al. haematologica 2014). in this retrospective analysis, we analyzed the real-life management of io in the post-allotransplant setting. this study includes the last 113 pts with a minimum follow-up of 6 weeks, who underwent allo-hsct in our center (october 2014-october 2016). 63 pts were male (55.8%) and 44 female (44.2%). median age was 53 years (range: 7-69). baseline diseases were: aml (44.2%), lymphoproliferative disorders (16.8%), mds (12.4%), all (8.8%), chronic myeloproliferative diseases (7.1%), mm (5.3%), and bm failures (5.3%). donor was unrelated in 61 cases (54%; 14 of them hla mismatched), and related in 52 (46%; 21 of them haplo-identical). conditioning regimen was: busulphan-based (68.1%), melphalan-based (13.3%), tbi-based (7.1%), and others (11.5%). progenitors source was pb in 102 (90.3%), and bm in 12 (9.7%). pre-hsct: pts had been transfused with a median of 23 prbc (range: 0-147), and their median serum ferritin (sf) was 1359 ng/ml (range: 22-5116). day +180 post-hsct: 15 pts had died, and 24 pts had not reached that day yet, so 74 pts were evaluable. they had been transfused with a median of 31 prbc (range: 0-157), and their median serum ferritin (sf) was 1127 ng/ml (range: 56-7993). 55% pts had sf superior to 1000 ng/ml. liver mri (by sir method) to assess liver iron concentration (lic) was performed in 44 pts at day +180. seven pts (15.9%) had no io (lic 0-2 mg/g), 12 pts (27.3%) had moderate io (lic 2.1-4.4 mg/g), and 25 pts (56.8%) had severe io (lic superior to 4.5 mg/g). median lic was 4.66 mg/g (range: 0.6-11.34). among the 29 cases with history of more than 20 prbc transfused and sf higher than 1000 ng/ml at day +180, 28 (96.6%) were proved to have liver io by mri; the other pt had io in spleen. 30 pts started some kind of therapy to treat the io: 6 pts with severe io initiated a tp program and 24 pts (6 out of 12 with moderate io, and 18 out of 25 with severe io) initiated chelation therapy with deferasirox. the drug was started at low dose (2.5-5 mg/kg/ day), and was increased if tolerated up to a maximum of 20 mg/kg/day. of note, the majority of pts were also taken a number of medications (immunosuppressants, statins, antimicrobials, etc). 3 of those 24 pts (12.5%) did not tolerate the drug, and were changed to tp. for more details, see the table. (1) the combination of the history of prbc transfusions and serum ferritin levels was, in the majority of cases, enough to assess the io in the post-allo-hsct setting. (2) liver mri (by sir method) helped to assess io in doubtful cases. (3) deferasirox, initiated at low doses and increased if tolerated, was safe and its use helped to avoid the need of therapeutic phlebotomies for the majority of patients. this study reproduces, in a real-life setting, our previous findings in a prospective clinical assay. [p297] disclosure of conflict of interest: none. a case-control study of risk factors of primary graft failure with a focus on associated early-onset severe infections v alcazer 1 , a conrad 2 , f-e nicolini 1 , s ducastelle-lepretre 1 , f barraco 1 , x thomas 1 graft failure (gf) is a rare but devastating event after allogeneic haematopoietic stem cell transplantation (ahsct), exposing the recipient to disease relapse, drawbacks of marrow aplasia, infections and death. the aim of this study was to analyse the risk factors associated with graft failure after ahsct, with a specific focus on early-onset severe infections (esi). we conducted a retrospective, observational, single-centre, matched case-control (1:2) study among adult s270 ahsct recipients transplanted at the haematology department of our institution between 2008 and 2015, with a subsequent follow-up of 12 months. engraftment was assessed at day+42 post-ahsct. gf cases were classified as primary gf (pgf), defined as failure to achieve donor-derived absolute neutrophil count (anc) ⩾ 0.5 × 109/l or lasting more than 3 consecutive days without evidence of disease relapse and early-secondary gf (esgf), referring to the loss by day 42 post-ahsct of a previously functioning graft associated without evidence of disease relapse. each case was matched with two controls according to underlying haematological disease, hla matching, stem cell source, intensity of conditioning and temporal proximity of ahsct. demographics, haematological and graft characteristics as well as esi report were retrieved. esi were classified in invasive fungal infections, viral infections (cmv, ebv, hhv-6, other viruses), toxoplasmosis and severe sepsis of bacterial origin. during the study period, 598 ahsct were performed at our center. seventeen (3.1%) gf cases were identified, of which 15 pgf and 2 esgf, and were matched with 34 controls. in the descriptive analysis, gf and control populations did not significantly differ when considering demographics, haematological characteristics and hematopoietic stem cell source. regarding pretransplantation status and graft characteristics, only disease status (progressive disease) and cell dose (both cd34+ and cd3+ cells number/ kg) were associated with graft failure. the proportion of patients with ⩾ 1 esi before day 42 was significantly higher in cases than in controls (11/17 vs 11/34, p = 0.038), with an overall number of esi events of 19 and 12 among cases and controls, respectively. five cases had ⩾ 2 concurrent esi. the median time from ahsct to the first esi event for gf cases was 17 days (interquartile range (iqr), 11-24) vs 15 (iqr, 8-34) days for controls (p = 0.779). in the gf setting, the most prevalent infections were herpesviridae infections (n = 7 including hhv-6 n = 4, ebv n = 2, cmv n = 1), probable ifi (n = 4), severe sepsis of documented bacterial origin (n = 3), toxoplasmosis (n = 2) among whom one patient developed haemophagocytic syndrome. when further analysing subsets of esi using logistic regression, only toxoplasmosis was a significant risk factor for gf (p = 0.018). death related to an infection was proven for 8 gf patients vs 5 control patients (p = 0.012). the overall survival probability at 12 months was significantly lower in the gf setting than in control patients (hr = 2.59 (95% ci 1.25 − 5.36), p = 0.01). the survival rates at 12 months were 35.3% and 57.7% for gf and control patients, respectively. at our center, graft failure is statistically associated with early-onset severe infections, and already known graft characteristics such as cell dose and disease status. however, our study would need more power to increase its significance. disclosure of conflict of interest: none. allogeneic stem cell transplantation (asct) is a curative option for hematological disorders, especially malignancies. in immunosuppressed women after asct, the progression from cervical dysplasia to invasive carcinoma is accelerated, and cervical cancer is likely a more aggressive disease. therefore, follow-up protocols after asct should include regular gynecologic evaluation with papanicolaou (pap) smears. we retrospectively evaluated 32 pap smears in 20 women who underwent asct and searched the risk factors for abnormal cervical cytology. the median age at transplantation was 44.5 years (range: 22-65 years). the most frequent indication for asct was leukemia (70%), and 85% of the patients received a transplant from a sibling hla-matched donor. stem cell source was peripheral blood in all patients. myeloablative conditioning regimen was used in 50% of patients. cyclophosphamide, busulfan and fludarabin were used in 20 (100%), 18 (90%) and 10 (50%) patients, respectively. acute graft versus host disease (gvhd) occurred in 7 patients (35%) and chronic gvhd in 4 patients (20%). secondary cancer (1 breast cancer) was reported in only one patient at 40 months after asct. the follow-up time was 23 months (range: 3-104 months). after asct, benign and abnormal pap smears were found in 12 (60%) and 8 (40%) women, respectively. the median time between asct and development of abnormal cytology was 2 months (range: 1-11 months). four (20%) women had at least one smear with atypical squamous cells of unknown significance (asc-us), one (5%) had a low-grade squamous intraepithelial lesion (lsil), one (5%) had atypical squamous cells/high-grade lesion (asc-h) and one (5%) had asc-us and asc-h. one (5%) patient had malign smear. two patients with asc-h showed high-grade atypia mimicking cancer but had a negative follow-up. patient who had malign smear died because of aorta dissection. cervical biopsy showed cervical intraepithelial neoplasia (cin) i in 3 (15%) women who had asc-us or asc-h. one patient was hpv-positive. we did not find any relationship between cervical cytological abnormality and clinical factors. after asct, patients are high risk for abnormal cervical cytology and secondary gynecological cancer. regular surveillance of patients is the most important factor for decreasing the risk of developing cervical and other secondary cancers. gynecologic examinations and cervical cytological testing after asct allows early diagnosis and effective management of cervical abnormalities. disclosure of conflict of interest: none. kidney dysfunction is a frequent complication of allogeneic stem cell transplantation (sct) and contributes to the morbidity and mortality of the procedure. incidence of severe acute kidney injury (aki) in patients undergoing nonmyeloablative allogeneic sct for malignant diseases ranges from 14 to 47%. lymphoma patients are often heavily pretreated through both chemotherapy and autologous sct and may be at increased risk of developing kidney injury. we performed a retrospective analysis of 108 consecutive patients with lymphoma undergoing nonmyeloablative allogeneic sct between 2004 and 2016 (table 1) . acute kidney injury (aki) within 100 days of allogeneic sct was diagnosed and staged according to rifle-criteria, and severe aki was defined as rifle stage i-e (4doubling of creatinine or 450% decrease of egfr). chronic kidney disease was defined as an estimated glomerular filtration rate (egfr) o60 ml/min/1.73 m 2 1 year after allogeneic sct. we performed multivariate logistic regression to evaluate potential risk factors for severe aki. severe aki developed in 75 patients (69.4%). reduced overall survival was observed in these patients, although not statistically significant. no significant associations were seen with age at transplantation, baseline kidney function or prior autologous sct. severe aki was associated with acute graft versus host disease (gvhd) (or 2.8, p = 0.026) and the use of an unrelated donor (or 2.8, p = 0.025). chronic kidney disease was observed in 20 (18.5%) of patients alive after 1 year. we report a substantially higher incidence of severe aki after nonmyeloablative allogeneic sct for lymphoma than has been reported for other malignancies. acute gvhd and unrelated donor stem cell s271 source were associated with severe aki, while prior autologous sct, age and baseline kidney function were not. [p301] disclosure of conflict of interest: none. patients with acute myeloid leukemia who are treated with conventional chemotherapy still have a substantial risk of relapse. we, therefore, retrospectively analyzed data to investigate the effects and some risk factors of allogeneic hematopoietic stem cell transplantation in relapsed and refractory acute myeloid leukemia patients, and to provide some suggestion for the clinical treatment. a total of 84 refractory and relapsed acute myeloid leukemia patients receiving allogeneic hematopoietic stem cell transplantation in our center between february 2005 and december 2014 were retrospectively analyzed, including 23 patients in no-remission (nr) and 61 patients in second complete remission (cr2) at the time of transplant. the median age was 35 years (range: 9-55). conditioning was myeloablative using cyclophosphamide, busulfan and total-body irradiation (bu/cy, n = 44; tbi/cy, n = 3), and others were underwent nonmyeloablative stem cell transplantation. 81 patients had successful engraftment. acute-gvhd and chronic-gvhd appeared in 47and 37 patients. the 3year overall survival (os), relapse rate and disease-free survival (dfs) of the cases was 50 ± 6.0%, 44.8 ± 5.8% and 37.1 ± 0.3%, respectively. the 3-year dfs were higher for patients in cr patients (52.2 ± 6.7%) than in nr patients (21.3 ± 10.1%), and the relapse rate in nr group and cr group were 60.4 ± 1.6% and 29.7 ± 0.4% respectively. there was no significant difference in treatment-related mortality compared cr group with nr group. sex, age, related-donor graft were not independent factors affecting os, dfs and relapse rate. it is concluded that allo-hsct is an effective salvage therapy for patients with refractory and relapsed aml. non-remission before transplant and severe agvhd are high risk factors of poor prognosis for allo-hsct. patients in cr group who accept reinduction chemotherapy before transplantation have better prognosis than those in nr. the overall outcome seems related to the disease status. hsct during refractory and relapsed can achieve long-term survival in selected patients with individual therapy. disclosure of conflict of interest: none. the incidence of most hematologic malignancies increases with age. aging is related with a greater prevalence of impaired functional status and comorbidities. although cure of malignant and non-malignant hematological diseases is potentially possible with allo-hsct, it could lead to significant transplant-related mortality. decision making about referral to allo-hsct in older adults is a challenging task. in this study we aim to present our geriatric allo-hscts. from 2007 to 2016, 33 [p303] patients (age 3 60) underwent allo-hsct in our center included to this retrospective study. pre-transplant status as well as posttransplant toxicities, complications and outcomes were determined. the age distribution of the group: 27 patients was aged 3 60 and o65, 5 patients was aged 465 and o70, 1 patient was 71 years old. the median age of donors was 49 (range: 21-73). the pre-transplant patients' characteristics are given in the table. remission was achieved in twenty-three (70%) patients. twenty-six patients (79%) had neutrophil engraftment (40.5 × 10 9 /l) at a median day of 19 (range: 10-41) and platelet engraftment (20 × 10 9 /l) at a median day of 20 (range: 14-54). post-transplant complications are detailed in the table. acute graft vs host disease (gvhd) was occurred in 10 patients (31%) and chronic gvhd in 12 patients (36%). eight patients (24%) were diagnosed with a relapse and 1 year relapse-free survival was 15%. the 1-year and 2-year os were detected as 30% and 12%. the most common reason for mortality was sepsis. the 1-year os was higher in patients who had reduced intensity conditioning regimen and remission status pre-transplant however they were not statistically significant (30% vs 21%, p = 0.6; 31% vs 25%, p = 0.9) (figure) . since increasing number of older patients being diagnosed with hematologic malignancies, this trend of increasing number of allo-hsct will continue. tolerability and effectiveness are lesser, toxicity is higher in older adults. although study population is relatively small, reduced-intensity conditioning and pre-transplant remission status may be related to better survival. comprehensive geriatric assessment may be considered prior to allo-hsct for global evaluation. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (asct) is a procedure with high morbidity and mortality (10-20%) requiring a complex hospital infrastructure. improved support measures and development of homecare units has allowed that asct at-home programs may be possible. our center has launched a pioneering program in our country in patients with asct to perform at home the following of aplasia, control of immunosuppressive therapy (ist) and intravenous support from the d+1 of asct until the engraftment and independent ambulatory patient. to evaluate the patient safety, we compared the group of patients at-home (asct-op) with a cohort of asct 'in patient' with similar characteristics (asct-ip). 26 asct patients between january 2014 and october 2016 at the hospital clinic of barcelona. 13 patients performed asct-op and 13 had an asct-ip. all patients received conditioning (myeloablative-mac-or reduce intensity-ric-) in the hospital with fludarabine 40 mg/m 2 (d1-4) and busulphan 3.2 mg/kg (2-4 doses), prophylaxis of gvhd was performed with tacrolimus/mycophenolate (mmf) in asct-op group and cyclosporine(csa) and methotrexate (mtx) or mmf in asct-ip group. in all patients, the infectious prophylaxis was conventional (levofloxacin, fluconazole and acyclovir). moreover, the asct-op group received prophylaxis with ceftriaxone 1 g intravenous (iv) once daily and liposomal amphotericin b inhaled 25 mg twice a week during neutropenia. the asct-op group from d+1 received a nurse visit once daily and physician visits twice a week in the hospital. baseline characteristics were analyzed those related to toxicity and patient outcomes. the median age (range) was 56 years (23-69), male/female 16/10; (62% male). the source of the progenitors was peripheral blood in all cases and analysis of the results detailed in the table: disclosure of conflict of interest: none. an increase in rdw-sd after allogeneic hematopoietic transplantation is associated with a poor prognosis s leotta, a cupri, a di marco, a spadaro, l scalise, g sapienza, mg camuglia, g avola, g moschetti 1 and g milone 1 istituto oncologico del mediterraneo red cell distribution width (rdw), is an erythrocyte index influenced by stress erythropoiesis, inflammation and antioxidants. rdw predict mortality in sepsis, chronic kidney diseases and in cardiovascular disease. no data are available on rdw after hematopoietic transplantation. in a retrospective study we collected data on changes of rdw-sd in a group of 81 patients who received allogeneic hematopoietic transplantation. fortyeight patients were affected by acute leukemia, 13 by lymphoma, 9 by mm, and 11 by other diagnosis. rdw was studied at baseline and monthly for the first 3 months. a subset of 34 patients were studied prospectively for clinical and laboratory signs of microangiopathy. at baseline before the transplant a rdw-sd higher than normal upper limit was observed in 43% of allogeneic candidates. a high co-morbidity score (htc-ci score 2-5) at the pre-transplant screening was a factor associated to high rdw-sd (χ 2 p = 0.01). a value of rdw-sd higher than normal range, at baseline, was not associated to any other factors, such as age, diagnosis, phase of the disease, previous transplantation, c-reactive protein, bilirubin, creatinine and arterial hypertension. early after allogeneic transplant we noticed at day +30 a significant reduction of rdw-sd but subsequently (at day +60) the proportion of patients showing an abnormal rdw-sd increased to 63%. an abnormal rdw-sd at s274 day +60 was registered in 70% of allogeneic transplant patients who presented an acute gvhd while in only 30% of patients who did not presented during the first 3 months an acute gvhd (χ 2 p = 0.02). in allogeneic transplantation group, patient who, at day +60, had a rdw-sd higher than normal value had a inferior outcome in respect to patients having a rdw-sd within normal ranges (os was 70% vs 30%; logrank: p = 0.04;), (ci of trm: 45% vs 18%).these two groups were not significantly different for pretransplant features in the subset of patients studied prospectively, abnormal rdw-sd was associated to presence of schystocytes in pb (chi test: 0.004) and patients having ⩾ 2% schystocytes had a median rdw-sd of 71 (iqr 31) vs a median rdw-sd of 46 (iqr 12.2) in patients who did not show schystocytes in pb (mann-whitney u-test p = 0.004). rdw-sd was significantly correlated also to serum triglycerides (r = +0.4, p = 0.0004) and to red blood cell mean corpuscular volume (r = +0.32, p = 0.02). abnormal rdw-sd is frequent after allogeneic transplantation. abnormal rdw-sd is associated to acute gvhd and its value obtained at day +60 marks a group of patients with poor prognosis because of high trm. this simple parameter warrant further studies to determine its clinical usefulness in monitoring of patients suffering acute-gvhd and in diagnosis and monitoring transplant associated microangiopathy. [p305] disclosure of conflict of interest: none. sickle cell disease (scd) poses a lot of psychological burden for the patient and the caregiver. it also poses a significant financial burden over the family. ohaeri et al. developed a 16 point questionnaire to asses sickle cell disease burden called as sickle cell disease burden index (scdbi) and its impact on caregiver's quality of life (qol). we used this questionnaire to assess the impact of hematopoietic stem cell transplant (hsct) on caregiver's qol. 16 point questionnaire was sent to 15 set of parents whose child underwent hsct between january 2016 and june 2016. scdbi contained 16 questions in various domains (3:family finances, 3:family interactions, 5:routine family activity and 5:parental coping ability). answers were graded on a score of 0-3 (0:never occurred and 3:occurred regularly or had a severe impact on the family). the results were interpreted in two headings a. family finances and interactions (0: no impact; 1-3: insignificant impact; 4-6: moderate impact; 7-9: severe impact) and b. routine family activity and parental coping ability (0: no impact; 1-5: insignificant impact; 6-10: moderate impact; 11-15: severe impact). all these domains were assessed before and after hsct. ten parents replied with duly filled questionnaire. mean age at hsct was 8.1 years (range: 1-14), m/f:7/3. all were symptomatic for 46 months before hsct with 90% having more than 2 hospital admissions. majority of parents were from middle class with median family income of 30 000 usd per annum (range 16 000-200 000 usd). median score for family finances and interactions (a) before hsct was 6 (range: [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] which decreased to 0 (range: 0-3) after hsct. median score for routine family activities and parental coping ability (b) before hsct was 13 (range: which decreased to 0 (range: 0-6) after hsct. our results suggest that before hsct there was a moderate impact on family finances and interactions which reduced to no impact after hsct. similarly there was severe impact on family activities and parental coping ability before hsct which changed to no impact after hsct. our study suggests that hsct not only improves the qol of the child but also of the caregivers. chronic graft versus host disease (cgvhd) is a late complication of allogenic hematopoietic stem cell transplantation (hsct) that affects many tissues and organs and manifests with polymorphic clinical features similar to autoimmune diseases. poorly understood pathophysiological mechanisms are implicated in inflammation and tissue fibrosis which is a hallmark of cgvhd. the affection of lachrymal glands is frequent and contributes to ocular manifestations presenting as dry eye syndrome. autologous serum eye drops (aesds) are used topically to facilitate tissue healing and ease the symptoms in a variety of ocular diagnosis. it is unclear if the serum of a patient with cgvhd is suitable for remedy preparation and if the transplanted patient himself can meet the criteria for autologous donation. aim is to show the safety, feasibility and efficacy of autologous serum preparations in ocular lesions after allogenic hsct. donors should meet criteria for autologous blood donation (infectious disease status, complete blood count hgb4110 g/l, hct433%, adequate venous access). aesds are prepared from 150 ml of autologous blood left to clot, irradiated and centrifuged to separate serum which is diluted with saline in 1:5 ratio or 1:1 if requested. product is dispensed into 1.5 ml ampules, stored at − 20°c and a 3-month supply is released to the patient after receiving negative results of sterility testing. in period from 2005 to 2016. in the aesds program 12 patients (4 female, 8 male) with ocular symptoms were included. all met required predonation criteria. of 29 collections performed, one failed due to venous access problem and one product had to be discarded due to hemolysis. cgvdh global nih score of the patients at start of the program was: 4 severe, 6 moderate, 1 mild and 1 not scored. all patients presented with moderate to severe dry eye symptoms. in 6 (50%) patients aesds alleviated dry eye symptoms. in 3 (25%) out of 12 patients referred to aesd program, more than 3 autologous blood collections were performed (range: 4-10) and aesds were used regularly through period of 10-36 months, which points to the beneficial effect of the long-term use of the serum. three patients dropped out because aesds showed no advantage compared to commercial lubricant eye drops preparations. one patient dropped out because of a venous access problem, 4 patients had disease s275 progression and needed other therapies: 3 cases of amniotic membrane application of which 2 continued with aesds to facilitate the healing effect. one patient was recently included and the effect of aesd is still evaluated. autologous donations in cgvhd patients are feasible, safe and autologous serum preparations can help relieve symptoms of dry eyes. it needs to be further elucidated specifically in which patients and at what point of the disease course the effect of the aesds is the most beneficial to make optimal use of these preparations. disclosure of conflict of interest: none. idiopathic pneumonia syndrome (ips) is a non-infectious pulmonary complication with diffuse lung injury that develops in 5-10% of patients who undergo hematopoietic cell transplantation (hct) and the mortality rate remains high at 80%1. the major aim of this study was to identify prognostic biomarkers for ips and establish positive and negative predictive values (ppv and npv) of ips. in a case-control study, we compared 41 patients with ips with available samples (transplanted between 1988 and 2014 at fhcrc) with 162 hct control recipients who did not require bronchoscopic examination and who did not grow any bacterial or fungal blood cultures. for each subject, plasma samples at day 7 post hct and onset of ips or matched time points for controls were analyzed. the 'onset sample' for controls was the sample closest to day 24 (median day of onset for patients with ips). we measured six proteins by elisa: suppressor of tumorigenicity 2 (st2), tumor necrosis factor receptor 1 (tnfr1), interleukin-6 (il-6), lymphocyte vessel endothelial receptor (lyve)-1, endothelial protein c receptor (epcr), and herpes virus entry mediator (hvem). multivariable logistic regression models were used to evaluate the association of each protein with ips vs controls. cytokine cutoff values that maximized discrimination between ips and controls were identified using receiver operating characteristic (roc) analysis. ppv and npv of ips were calculated using the identified cytokine cutoffs across a range of hypothetical ips prevalence values (0-15%) day 200 weighted kaplan-meier survival curves were estimated for high/low cytokine subgroups. similarly, a weighted log-rank test was used to evaluate p-values. a multivariable logistic regression model including six cytokines showed that st2 and il-6 were significantly important markers to identify ips at the onset (table 1) . st2 value at day 7 post hct was significantly associated with occurrence of ips and il-6 had a marginal association. predictive values for ips by a plausible percentage of the actual hct population (up to 15%) are shown in figure 1 . of the six proteins, st2 showed the highest ppv both at onset and day 7 post hct followed by tnfr1, and il-6. npv were high in all the markers. to analyze whether st2 and il-6 at day 7 after hct can predict survival following ips, we dichotomized the patients into cytokine high and low groups (cutoff level: st2, 19 ng/ml; il-6, 35 pg/ml) and compared survival after downweighting the observations to represent a plausible percentage of the actual population (ips prevalence, 5%). day 200 survival rate were significantly lower in st2 high value group than in st2 low value group (80% vs 88%, p = 0.015). similarly, il-6 high value was associated with high mortality (day 200 survival rate, 78% vs 88%, p = 0.003). st2, il-6, and tnfr1 were good prognostic markers for occurrence ips. especially, st2 and il-6 at day 7 after hct can be a predictor for both ips occurrence and survival following ips. these results require validation in an independent prospective hct population. body composition parameters are sensitive nutritional indicators that influence response to treatment and mortality in cancer patients. research is not conclusive on the changes in muscle attenuation and adipose tissue areas in the stem cell transplantation (sct) phases. objective is to assess the changes in adipose tissues, skeletal muscle index (smi) and waist circumference (wc) among stem cell recipients in the peri-transplantation phase. study design: institutional review board approved this retrospective study with 61 adult patients (age416 years) having b and t lymphoma who underwent sct. each patient was imaged by pet/ct scan pre-sct and 3 months post transplantation. a cross sectional image was analyzed at the level of the l3 to calculate total adipose tissue (tat), visceral adipose tissue (vat), intra-muscular fat (imf), smi and wc. data was analyzed by gender since body composition parameters differed significantly between the two categories in the literature. the study sample consisted of 61 patients (mean age: 38.2 ± 13.7 years, 35 (57%) males, 51(83.6%) autologous sct, median overall survival in months: 39.8 in males and 40.5 in females). death was observed in 6 (17.1%) males and 1(3.8%) female. patient characteristics were similar for males and females except for weights (kg) and body mass index (kg/m 2 ): 86.7 and 28.6 vs 63.5 and 24.1 in males and females respectively. changes from pre-sct to 3 months post sct revealed that tat, vat, smi and wc decreased with mean differences of 42 ± 61.2 cm 2 , 18.28 ± 37.6, 3.3 ± 7.5 cm 2 /m 2 and 4.58 ± 5.4 cm, respectively in males (po0.01). in females, tat and wc significantly decreased with mean differences of 18.4 ± 37 cm 2 and 3.1 ± 4.3 cm, respectively (po0.01). in females, vat and smi decreased clinically but did not reach clinical significance. in multivariate analysis, no significant associations were shown with mortality and progression rates. this study fills a research gap by providing data on the evolution of body composition parameters in the peri-transplantation phase. tat, vat, smi and wc decrease 3 months post transplantation. future studies should evaluate the associations of these parameters with major outcomes on larger sample sizes. [p310] disclosure of conflict of interest: none. . 5 patients received tbicontaining preparative regimen. all these patients were exposed to calcineurin inhibitors for prevention and treatment of gvhd. 16 patients suffered from cgvhd-grade moderate or severe. all patients required systemic corticosteroids, because of gvhd (16pts) or during basic treatment of lymphoma (2 pts). all patients had deficient states of vitamin d initially and required replacement. all of them, except for 2 patients, had balanced adrenal insufficiencies and 2 patients had balanced hypothyroidism. all women had premature ovarian failure (2 received hrt). according to measurements of bone mineral density (bmd), low bone mass was detected in 15 patients; osteopenia (11pts), osteoporosis (4pts). bone loss of femoral neck (8-osteopenia, 3-osteoporosis) occurred more often than lumbar vertebral ( 6-osteopenia, 2-osteoporosis) or radius (3osteopenia, 1-osteoporosis). presence of avascular necrosis of bone (avn), confirmed by mri, was detected in 12 patients and the most common site of involvement was the femoral head(all patients), knee(3 pts) and shoulder(1 pts). one of the first symptoms of avn was pain and functional limitation. all patients required intensive analgesic treatment, usually nsaids and 4 patientsfentanyl. fractures occurred in 12 patients. the femoral neck (7 pts) and thoracic or lumbar vertebral (3 pts) were two most common fracture sites. all patients were qualified for surgery; 6 patients required hip replacement, 6patients still awaited to perform surgery or were disqualified because of severe, skin cgvhd. bone complications may occur in about 17% of allo-hct survivors (including 30% patients with gvhd, and up to 60% patients with severe or moderate cgvhd) within first 4 years after allotransplantation. bone loss, particularly at the femoral head, is the most common complication. avascular necrosis usually requires surgical intervention because of fractures. exposure to higher doses of corticosteroids (during treatment of gvhd) increases risk of bone complications. early diagnosis by mri and dxa may help to detect bone complications. (41%) and (e) 3 (21%). a total of 41 (30%) pts failed to meet these criteria but remained alive on day 5 and 39 (28%) died before day 5. the overall survival (figure 1 ) for the 58 pts was 28% at 3 years with an overall mortality in icu of 33% (19/58) compared to 71% (29/41) for those who did not meet our criteria. the overall survival for pts that met our criteria at fifth day and were discharged to the haematology ward (n = 39), was 49% at 3 years. in this study, 50% of patients survived their icu admission. patients could be stratified according to the reason for admission and given an individualized 5-day trial: those who met our criteria for successful icu trial (42%) had a low icu mortality (33%) and those who were subsequently discharged home had a overall survival of 49% at 3 years. this study raises the possibility of offering a short-term icu stay to oncohematologic patients and perhaps allows for the ceiling of intensive care for those who fail these criteria. [p312] disclosure of conflict of interest: none. in contrast, only 2.98% of patients without cgvhd showed a thromboembolic complication in the later time course, with one patient showing an additional thrombotic risk factor. in multivariate analysis cgvhd was an independent risk factor for thromboembolic complications after hsct. 1.8% of patients with thrombosis before hsct showed one afterwards. thrombosis before hsct was not found as risk factor for thromboembolic complication after hsct. our retrospective analysis showed an increased risk for thromboembolic complications after allogeneic hsct, with substantial higher risk in patients with chronic gvhd (8.9%). in ongoing studies we currently investigate a vascular screening procedure with additional biomarkers according to inflammation and endothelial damage in patients with cgvhd prospectively. we hope to identify patients at risk for thromboembolism and prevent future complications on an individualized basis. disclosure of conflict of interest: none. (1). pts with cns involvement received intrathecal therapy with cytarabine and in one case additional cns irradiation was applied. 10/11 pts died after a median time of 9 mts (range: 1-25 mts) due to resistant systemic relapse, infectious complications or extensive graft-versus-host disease following allohsct. 1 patient remains alive and disease-free at 88+ mts following secondary allohsct. conclusions: our data indicate that em disease following allohsct affects a significant proportion of pts with aml. sites of em relapses vary widely among the pts with skin and cns being frequently involved. an aggressive approach combined of local and systemic therapy including secondary allohsct may produce favorable response in a small proportion of pts, however, overall prognosis for pts with isolated em relapses still remains poor. due to the lack of effective treatment strategies, there is a need for novel approaches to manage isolated em relapses after allohsct. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct)-associated thrombotic microangiopathy (tma) is a multifactorial complication, and has variable incidence in study populations due to different diagnostic criteria. aim: our aim was to identify pediatric patients with hsct associated tma using 4 different diagnostic tma criteria published in literature and to compare the various groups for tma parameters and outcomes. we enrolled 33 pediatric patients who underwent allogeneic hsct using treosulfan based or reduced intensity conditioning therapy. 4 different tma diagnostic criteria, the bmt ctn toxicity committee consensus definition (1), the overall thrombotic microangiopathy grouping (2), the diagnostic criteria created by city of hope (3) and the criteria proposed by jodele et al. (4) were used to startify the patients. we determined and registered the following tma activity markers: presence or development of increased ldh and decreased haptoglobin levels, new onset anemia, thrombocytopenia, fragmentocytes, coombs test, kidney function, proteinuria, hypertension and terminal complement complex (sc5b-9). complement pathway activities, components and sc5b-9 were measured during early hsct period. two/33 (1), 7/33 (2), 3/33 (3) and 10/33 (4) subjects met the different tma diagnostic criteria according to the four different systems on day 12 and 34 (1) and on median 44 (2), 43 (3), 61 (4) post-hsct days. all of the 6/10 patients who were defined with the first three criteria, met the forth definition. due to normal haptoglobin levels and kidney function, 4/10 patients fulfilled only the forth criteria. tma coexisted with acute graft-versus-host disease in 7/10 cases (7/10 vs 4/23; p o0.01). patients who met any of the different tma diagnostic criteria had higher sc5b-9 level on day 28 (411 vs 201 ng/ml; p = 0.003) compared to those without. all of the 10/33 subjects defined with tma had elevated sc5b-9 (4250 ng/ml) level during the early hsct period. two patient died before day 100 after hsct, out of which one patient met all of the four tma diagnostic criteria. after a median 2.29 (1.2-3.1) year follow-up time, overall survival was 24/33. 8/10 patients with tma survived, compared to 16/23 patients without tma. relapse related mortality was the most common cause of death (n = 7/9, po0.05), while tma was not a significant cause of mortality after reduced toxicity conditioning therapy. hsct-associated tma has a variable and complex pathophysiology. using the different diagnostic criteria may influence the incidence and the time of diagnosis of this transplant-related complication. monitoring all of the published tma activity parameters, including complement terminal pathway activation marker, may help to guide physicians to recognise tma after hsct. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) is associated with a risk of non-relapse mortality (nrm). it's important to assess the risk of complications and mortality before the hsct. some indexes quantify the impact of patients' comorbidities on hsct outcome. the most frequency used is the hct comorbidity index (hct-ci) and the european group for blood and marrow transplantation score (ebmts). this study tried to determine which of the two indexes best predicts the outcome in a series of patients submitted to hsct in a single center. between 2011 and 2015, 259 hsct were performed in our center. a total of 215 hsct have been analyzed (we excluded patients o18 years (yr), 2 nd hsct, haploidentical donors and hsct for specific diseases with very low number ( o3%) of hsct performed: aplastic anemia, cll, prolymphocytic leukemia, mycosis fungoides, sezary syndrome, dendritic cell neoplasia, plasma cell leukemia and poems syndrome). the hct-ci and ebmts were calculated retrospectively (yr 2011-2013) and prospectively (yr 2014-2015). overall survival (os), relapse incidence (ri) and nrm were analyzed in the overall series and separately according to the type of hsct: autologous hsct (auto-hsct) or allogeneic hsct (allo-hsct). male: 89 (59%) patients. median age: 54 yr (range: 18-71). diseases: aml 54 (25%), all 19 (9%), mm 67 (31%), nhl 41 (19%), hl 12 (5%), mds 17 (8%), cmpd 5 (3%). disease status: 1 st complete remission (cr) or 1 st chronic phase 102 (48%), ⩾ second cr 30 (14%), first partial remission (pr) 55 (26%), ⩾ second pr 12 (5%), no response 11 (5%) and without previous treatment 5 (2%). auto-hsct in 120 patients (56%) and allo-hsct in 95 (44%) patients. related and unrelated donor were 52 (55%) and 43 (45%), respectively. the conditioning regimen was standard in 57 (60%) cases and reduced intensity in 38 (40%). hct-ci and ebmts grouped 0-2, 3 and ⩾ 4 were 57 (48%), 29 (24%), 34 (28%) and 38 (32%), 51 (42%), 31 (26%) in auto-hcct and 61 (64%), 18 (19%), 16 (17%) and 34 (36%), 32 (34%), 29 (30%) in allo-hsct, respectively. median follow-up was 3.15 yr (0.66; 5.73) for the overall series, 3.18 yr (0.66; 5.73) for auto-hsct and 2.60 yr (0.97; 5.70) for allo-hsct. significant differences in os and nrm were found according to the ebmts in patients submitted to auto-hsct. one-yr-os and 3-yr-os were 91% (95% ci: 85%; 97%) and 68% (95% ci: 57%; 79%), respectively, in patients with ebmts 0-3, vs 73% (95% ci: 57%; 87%) and 56% (95% ci: 37%; 75%), respectively, in patients with ebmts ⩾ 4 (p = 0.033). one-yr-nrm and 3-yr-nrm were 2% (95% ci: 0%; 7%) in patients with ebmts 0-3, vs 13% (95% ci: 4%; 28%) in patients with ebmts ⩾ 4 (p = 0.015). no significant differences were observed for ri according to ebmts in patients submitted to auto-hsct. no significant differences in os, ri and nrm were observed according to ebmts in patients submitted to allo-hsct. no significant differences regarding os, ri or nrm were found when the hct-ci was assessed. in our series, only the ebmts was predictive of os and nrm in patients submitted to auto-hsct. failure to find statistically significant differences for the hct-ci and for ebmts in allo-hsct recipients could be due to an insufficient number of patients or to a partial retrospective collection of data. infertility is common after hct predominantly as a result of the chemoradiotherapy used in conditioning. nonetheless, some patients do retain or recover fertility. newer reduced intensity regimens may be less gonadatoxic. in addition, patients are increasingly encouraged to store gametes, or embryos before transplant. we sent questionnaires to 602 ebmt centers requesting retrospective details of number of pregnancies and pregnancy outcome for all patients treated between 1995-2015. 27 centers responded from 13 countries detailing 234 patients who became pregnant/partners conceived. the most frequent underlying diagnoses were acquired bone marrow failure (n = 44, 25f) aml (n = 42, 15f), hd (n = 26, 15f), cml (n = 25, 7f), all (n = 19, 4f) and b nhl (n = 18, 10f). other diagnoses included mds, mps, solid tumours, autoimmune disease, cll, t-nhl, haemoglobinopathy. of 110 females (f), 35 (32%) involved assisted reproductive techniques (art). 30f had tbi ( seven o4 gy) of which 16 (50%) had art. 25f had reduced intensity conditioning of whom 6 (24%) had art. 70f were specified as having standard conditioning of whom 24 (34%) had art. 73f had allogeneic (26 art, 36%) and 37f had autologous transplants (9 art, 24%). of 124 men (m) whose partners conceived, 61 (49%) had art. 54m received tbi of which 36 (67%) had art. where specified, 19 had reduced intensity hct (3 art, 16%) and 94 had standard conditioning (48 art, 51%) .93 had had allogeneic hct (43 art) and 31 autologous (12 art). 19 men had reduced intensity transplants. 53 men received tbi (two o4 gy) of whom 36 (68%) had art compared to 69 men without tbi, 16 (23%)of whom had art. data on return of menstruation was available for 84. 64 indicated yes and 12 (19%) had art. 20 indicated amenorrhoea of whom 14 (70%) had art. 224 specifying number of children had 324 live births (lb) and 87 (39%) patients had more than one child after hct. 146 lb occurred in female patients (41 art, 28%) and 178 lb were in partners of male patients (88 art, 49%). the median gestational age for 61 female patients was 39 weeks (range: 22-42) and the median birth weight was 3 kg (range: 0.3-4.19). there were 3/80 congenital anomalies. the median follow up of the offspring was 5 y (range: 0-15). developmental problems were indicated for 1/71 (fine motor skills) and learning difficulties in 1/70 (adhd). in partners of male patients the median gestational age for 62 offspring was 39 weeks (range: 26-43). the median birth weight for 56 offspring was 3 kg (range: 0.87-4.16). congenital malformations occurred in 4/89. one infant died of pulmonary infection. in women, several methods of assisted conception were used including hormone stimulation, ivf, cryopreserved embryos, donor embryos and cryopreserved ovarian tissue. the most frequent method was use of donor embryos (22/35) in which a minimum of 30 attempts led to 21 lb. the median number of attempts was 1 (range: [1] [2] [3] [4] [5] . art were frequently used in this group of posttransplant patients particularly in male patients vs female, tbi vs non-tbi, amenorrhoeic vs menstruating women, standard conditioning vs ric. in patients who conceive after hct, successful pregnancy leading to healthy offspring is the likely outcome. disclosure of conflict of interest: none. reduction of trm after sct was observed over the transplant periods and supportive care with danaparoid was found to be significantly effective to reduce trm. therefore, prophylactic administration of danaparoid is considered to be a reasonable option to improve the transplant outcomes for children. [p320] disclosure of conflict of interest: none. the attainment of transfusion independence after transplant is sometimes hampered by a combination of factors, ranging from infections to the need of combined therapy for clinical complications, as well as control of gvhd. iron overload is frequently observed in hematological patients before and after hematopoietic stem cell transplantation (hsct). whereas several reports have focused on iron overload before transplant, up to now, this is the only report that show full recovery of hematopoiesis and correlate this to deferasirox chelation performed on this particular subset of patients. we report on 19 patients, transplanted for hematological diseases (17 acute leukemia, 1 aplastic anemia, 1 multiple myeloma) heavily transfused before transplant that, considering the iron overload, were treated with deferasirox after hsct. before starting deferasirox, the patients were fully engrafted and in complete remission, although transfusion dependent, and with incomplete hematological reconstitution after allogeneic hsct. patients were selected according to the following inclusion criteria: (1) transfused pre-transplant with more than 20 rbc units; (2) incomplete hematological recovery; (3) transfusion-dependence; (4) serum ferritin 41000 ng/ml; (5) normal creatinine value. the workup for other aetiologies resulted negative. all patients received an initial dose of deferasirox 10 mg/kg/day, later adjusted according to side effects. all patients experienced an increase in hemoglobin levels, with a reduction in the frequency of rbc transfusions, followed by transfusion independence (median time: 24 days from the first dose of deferasirox). in addition, it was promptly (median time: 27 days) associated with hematological improvement, with sustained values and no further platelet support or growth factors administration. no relevant modifications with immunosuppressive or myelosuppressive drugs were made during deferasirox treatment. deferasirox was well tolerated. basing on our results, we think that deferasirox determined stimulatory, and/or depressive effects on hematopoiesis after allo-hsct. this clinical experience raises the possibility of a potential additive benefit on hematopoiesis after transplant following iron chelation therapy with oral deferasirox. further long term studies, in larger cohorts of patients are needed to confirm these data and to design an efficient strategy to reduce iron loading after transplant. disclosure of conflict of interest: none. supported in part by ail pesaro onlus. (4) hypertension (n = 2). all five patients had normal adamts13 levels and negative testing for shiga toxin. complement mutation genetic studies were obtained for four patients including 10 genes (n = 2) and 12 genes (n = 2) and were all negative. testing for complement pathway including c5b-9 were obtained for 2 patients and were normal. all five patients were treated with eculizumab with induction treatment at 900 mg weekly × 4 doses, followed by one dose of 1200 mg on the fifth week, and 1200 mg every 2 weeks thereafter. patients had a recovery of hemoglobin and platelets and a rise in haptoglobin and a normalization of ldh within 4-6 weeks from the start of eculizumab. eculizumab was discontinued for 3 of the 5 patients without recurrence of their tma; they are now 18-24 months since the discontinuation of eculizumab. in summary, there is a subacute syndrome of thrombotic s282 microangiopathy that can occur late post transplant. this syndrome appears to be complement mediated as shown by its response to a terminal complement inhibitor. it also appears to be transient without recurrence following treatment discontinuation. disclosure of conflict of interest: none. transplant associated microangiopathy (tam) is a very severe complication occurring after allogeneic bone marrow transplantation (bmt), burdened by a high case-fatality rate. it is characterized by abnormal complement activation, triggered by various agents (calcineurin inhibitors, acute gvhd, infections) with subsequent endothelial damage. in the literature, 6 cases of mutations in recipient complement genes are described, but none in donor dna. here we describe for the first time 3 patients affected by tam, carrying mutations in donor complement genes. in our lab, we studied 6 patients affected by tam; they were screened for cfh autoantibodies, adamts13 function and variants and macro-rearrangements in cfh (and related), cfi, cfb, cd46, c3, dgke, thbd genes and at-risk haplotype (cfh-h3 and mcpggaac) by means of next-generation sequencing (ngs) and multiplex ligationdependent probe amplification analysis (salsa mlpa p236 armd mix-1; mrc holland). ngs was used to sequence dna by haloplex kit (agilent) on a miseq (illumina) platform with 450-fold coverage of every target base. the bioinformatic analysis was performed using sophia genetics and the pathogenicity was assessed by means of in silico predictions (polyphen2, sift, mutationtaster, aligngvgd). all of the predicted pathogenetic variants were confirmed using sanger sequencing. the same genetic screening was extended also to donor dna in all 6 cases. the screening for known causes of tam revealed mutations in recipient complement genes in one case; no mutations were found neither in recipient nor in donor dna in two cases; instead, donor genetic alterations were found in 3 patients whose characteristics are summarized in table s283 donor hematopoietic cells. in the three cases presented, tam was relatively delayed with respect to hsct, in particular in two cases (6 months) and this timing is compatible with the concept of reticulo-endothelial 're-population' by donor cells of monocytic lineage, responsible for the production of regulatory proteins of the alternative pathway of the complement. we also underline the response to anti-c5 inhibition in the 2 patients who were treated with eculizumab; this fact further supports the hypothesis that the disease was related to complement dysregulation. we therefore suggest that both the recipient and the donor should be screened for complement gene mutations, so that more cases could be identified and the pathogenesis of tam could be further clarified. among these we observed one autologous engraftment, one death due to septic shock before engraftment and two primary gf. we used a desensitization treatment based on 4 plasma exchange procedures, intravenous immunoglobulin (1 g/kg) and rituximab (375 mg/sm) in 2 patients. one of these patients (aml, haploidentical donor) had dsa against hla-b50 (mfi 900). she experienced primary gf with increasing titles of dsa (maximum mfi 10 500); so, on day 38, a second transplant from the same donor was performed after a desensitization treatment. a progressive decrease in dsa was documented (up to mfi ⩽ 200). on day 12 patient achieved neutrophil count over 500/μl and on day 23 platelet count over 20 000/μl. the second patient (mds, haploidentical donor), instead, received a desensitization procedure before the first transplant. she had dsa against hla-a24 (mfi 3700), and after desensitization dsa levels decreased and reached 0. on day 20 patient achieved neutrophil count over 500/μl and on day 38 platelet count over 20 000/μl. dsa were detected in 1/9 of usct candidates (11%) and 4/17 of haplosct candidates (24%) and they were associated with failure to obtain allogeneic engrafment in 3 cases. desensitization treatment achieved dsa clearance and engraftment in the 2 patients in which it was performed, underlining the potential benefit of this procedure in the setting of hsct with dsa that has to be validated by prospective and controlled studies. disclosure of conflict of interest: none. early complications and late effects and quality of life at myeloma multiplex patients z trajkovska-anchevska, a pivkova, s genadieva-stavrich, l chadievski, z stojanoski, l chevreska and b georgievski university hematology clinic, skopje, macedonia the subject of this research is the quality of life at patients with myeloma multiplex at diagnosis and during therapy within 6-12 months. the research aims to analyze patients to be able to continue activities which will contribute for improving their quality of life as a priority task placed before the patient, his family, health institutions and social environment. this research was conducted at the university clinic for hematology skopje in the period from june 2009 to march 2012. it covers patients infected with multiple myeloma, diagnosed and treated during this period. a total of 80 patients analyzed, using the eortc qlq c30 ver. 3.0 standardized questionnaires for hr quality of life that analyzed the physical, cognitive, emotional, personal and social functions related to the patients. it also analyzed and general health and quality of life. analysis of physical functioning at diagnosis is 27.5 during treatment 59.5, significantly improved. personal functioning at patients at the diagnosis is 17.9, during therapy − 36.4. analyzing emotional functioning in patients at diagnosis is 39.9, during the therapy over 73.3 significantly improved. in examining the cognitive functioning is also a significant difference at diagnosis 55.2, during treatment 72.5. social functioning of the patients was 26.2 at the diagnosis; during the treatment grow to 50.8. significant improvement was notices in these patients' symptoms like fatigue, nausea and vomiting, pain, dyspnea, insomnia, loss of appetite, constipation and diarrhea. the analysis of the financial difficulties of patients at diagnosis is 76.2 and 72.5 during treatment, meaning no significant difference in the time given. the analysis of the overall health and quality of life at patient has a value of 23.9, and during therapy 58.8. quality of life at patients with myeloma multiplex that makes the research group was significantly improved as a result of on time diagnosis and treatment with modern medicaments and the role of social worker with the application of certain social skills, continuous counseling, guidance and education for their reintegration in the community. installing the quality of life as a separate category and investigating the factors that affect its expression in the daily functioning of the patients within the changed framework of action, as like this example for malignant disease. the needs of clearly defined interactions patient illness and treatment, quality of life and specifying the segments where it can effectively act and improve in order to achieve positive progression towards improving the qualitative features of this category is a clear and primary objective that must be inserted into the current approaches to monitoring patients with malignant hematological diseases. acute graft-versus-host disease (agvhd) is a common and severe complication after allogeneic stem cell transplantation. since the current first-line treatment is based on treatment with systemic glucocorticoids (gc), steroid-induced hyperglycaemia develops frequently in patients with (agvhd) potentially impacting on their outcome. we performed a retrospective analysis on 104 patients who received systemic gc for agvhd and thoroughly investigated the consequences of aberrant glucose metabolism. in particular, we focused on glucose parameters early after initiation of gc. with a median of 50 (range: 4-513) blood glucose measurements during gc treatment, increasing mean, median and maximum glucose levels as well as the need for insulin treatment were associated with decreased overall survival (os) in simple and multiple survival analysis. early hyperglycaemia, as defined by mean blood glucose levels 4125 mg/dl during the first 3 days of gc therapy, was also found to be highly associated with adverse outcome: in multivariate analysis, the hazard ratio (hr) for death was 2.5 (95% ci 1.32-4.87, p = 0.005) in patients with early hyperglycaemia. while the risk of death due to relapse was not increased, the hr for death due to non-relapse mortality was 3.26 (95% ci 1.53-6.92, p = 0.0021) in a competing risk analysis. a score based on early hyperglycaemia and non-response to gc within 7 days allowed the identification of three risk groups: patients with both risk factors had an inferior os at 5 years of 4.1% as compared to 75.4% in patients with none. patients with one risk factor had a 5-year os rate of 32.0% (p = 0.0002 for trend). in this retrospective study, we identified early hyperglycaemia after gc initiation as a prominent factor predicting increased nonrelapse mortality in agvhd patients. in addition, a score based on early hyperglycaemia and lack of response to gc was highly predictive for overall survival in these patients. disclosure of conflict of interest: none. early toxicity because of infectious complications not relapse is the main cause of death after allogeneic transplantation in aplasia for patients with refractory or relapsed acute myeloid leukemia high-dose cytarabin was given in 13/25 pts with induction failure. the search for a stem cell donor was started immediately after results of high-risk cytogenetic, no achievement of bone marrow aplasia on day 14 of induction therapy, or immediately after diagnosis of relapse. four patients had a related 10/10 donor, for 17 patients a 10/10 matched unrelated donor was identified and 10 patients received a transplant from a 9/10 unrelated donor. the interval between diagnosis of primary disease or relapse and tx was 3 (1-7) months (mo) for both groups . in 24 patients melphalan (100-140 mg/m 2 ) was used to induce an aplasia before starting conditioning therapy. the interval between melphalan and conditioning therapy was 13 (9-21) days. three pts started the conditioning therapy while in aplasia after previous chemotherapy. the conditioning therapy was of reduced intensity in all pts. and consisted of treosulfan (30 g/m 2 )/fludarabin(flu) in 19 pts, tbi(8gy)/flu in 7 pts and busulfan(8 m/(kg)/flu in 5pts, respectively.atg was given to all pts with an unrelated donor. most pts (21/31) had a severe neutropenia with a median of 0.3/nl ( 0.1-5.2) before starting melphalan because of refractory leukemia. after a median follow-up of 21 (4-68) mo 11 pts (35%) were alive without relapse. 6 (19%) pts died because of a relapse after a median of 6 (3-25) mo. the nonrelapse mortality was 45% (14/31 pts). most of these pts (10/14, 71%) died because of infectious complications early after transplantation (med 1; 0-19mo). in 4 pts graft versus host disease was the main cause of mortality. in this retrospective 'real-life' analysis, we showed that an early allogeneic transplantation is feasible for patients with primary refractory or relapsed aml. a reduced intensity conditioning after induction of aplasia with melphalan offers a chance of long-term relapse-free survival for about 30% of patients with an otherwise dismal prognosis. nrm is high, especially because of infectious complications early after transplantation, probably related to the long period of severe neutropenia. therefore, the focus has to be set on early recognition and intervention of infectious complications. disclosure of conflict of interest: none. recent evidence supports the effector role of complement activation in several types of thrombotic microangiopathythe atypical hemolytic uremic syndrome (ahus) as well as the transplantation-associated thrombotic microangiopathy (ta-tma). the blockade of the terminal complement complex formation by anti-c5 monoclonal antibody eculizumab provides an effective treatment option in severe and devastating cases of ta-tma. the experience with the use of eculizumab in this indication is slowly accumulating in the hsct community, however the published data originate from small case series or uncontrolled trials and sharing of emerging real-life observations may be valued. on case reports of two pediatric patients treated with eculizumab for ta-tma with very detailed followup of multiple complement parameters, including terminal complex sc5b-9 and eculizumab drug levels we would like to demonstrate: (1) achieving therapeutic levels of eculizumab (499 μg/ml) may be unsuccessful even with initially intensified dosing interval. furthermore, we documented rapid eculizumab clearance from circulation which allowed only for short periods ( o48 h) of efficient drug levels during the weekly dosing. (2) we did not observe tightly correlated sc5b-9 and eculizumab levels within the dosing intervals; however the long-term sc5b-9 formation suppression was achieved concomitantly with improved eculizumab levels and slowed drug clearance. (3) classical complement pathway activity assay (ch50) may not reliably substitute for therapeutic efficiency monitoring in case of hypocomplementaemia due to protein losses (profound diarrhea, proteinuria, gi bleeding, catabolism). this holds true also for the alternative pathway activity which remained low during treatment in both patients. (4) mycotic infections may represent serious therapy related risks in eculizumab treatment after hsct (both patients achieved control of complement activation after multiple doses of eculizumab, however suffered fatal infections subsequently). besides, we observed a significant increase in c3a concentrations correlated with clinical onset of infection which invites for further investigation of this complement cascade product as early indicator of mycotic infection. in conclusion, we would like to highlight the great added value of timely available complement assay results, including sc5b-9 and especially eculizumab drug level values-to be used together with detailed clinical parameters for directing effectively these highly personalized (and also costly) treatments. [p329] disclosure of conflict of interest: none. table 1 . no difference in terms of drug-related adverse events was observed in the three patient cohorts with no reported serious adverse events. similar results were obtained performing two separate sub-analysis only for lymphoma or myeloma patients. despite the limitations due to the non-randomized nature of the study, from our data on a large cohort of patients s286 with a long-term follow-up biosimilar filgrastim (zarzio®) could be considered substantially equivalent in terms of efficacy and safety to lenograstim (myelostim®) and peg-filgrastim (neulasta®), when used for hematological recovery and febrile neutropenia prophylaxis after asct in adult patients with hematologic malignancies. disclosure of conflict of interest: none. we studied all 107 adults who underwent allo-hsct during a 23-month period (01 january 2015 to 22 november 2016) in our center. a total of 6 pts (5.6%) received epag for pfg with thrombocytopenia. three pts were male, and three female. median age was 59 years (24-67). the baseline diagnoses were: alm (2), mds-raeb (1), idiopathic myelofibrosis (1), aa (1), and cll (1). three transplants were from family donor (all of them haplo-identical), and 3 from unrelated donor (the three of them hla 9/10). sc source was pb in 5 cases, and bm in 1. epag was started at 50 mg/day and escalated each 2 weeks to 75, 125 and 150 mg if platelet count was o50 × 10 9 /l. we analysed the platelets, anc, and hgb at epag initiation and 90 days after being with the maximum dose. median time between allo-hsct and eltrombopag initiation was 120 days (17-155). median maximum dose used of epag was 150 mg/day (125-150). median platelets, anc and hgb before starting treatment were 13 × 10 9 /l (5-28), 1 × 10 9 /l (0.07-11.2) and 8.6 g/dl (7.6-12.1), respectively. five patients (83%) were severely thrombocytopenic (platelet count ⩽ 20 × 10 9 /l), 4 (67%) were anemic (hbg o 10 g/dl), and 3 (50%) were neutropenic (anc o1.0 × 10 9 /l). median platelets, anc and hgb at day +90 of maximum dose were: 37 × 10 9 /l (8-108), 2.4 × 10 9 /l (0.93-9.62) and 11.8 g/dl (7.9-14.5), respectively. the 5 thrombocytopenic pts (100%) responded to epag, with increases of 120 00, 25 000, 28 000, 39 000 and 96 000 × 10 9 /l in the platelet count. three anemic pts (75%) responded and achieved increases of hgb of 1.1, 4.7 and 6.8 g/dl. finally, the 2 neutropenic pts (66.6%) responded and achieved increases of anc of 4080 and 9550 × 10 9 /l. at the moment of the analysis close, pts are at a median of +11.5 months post-hsct (8) (9) (10) (11) (12) (13) (14) (15) (16) , and all but one (who died from a septic shock) are alive and outpatient. this survival is striking for subjects who develop a complication with such a high expected mortality as pfg. pgf is a life-threatening complication, relatively frequent after alternative donor hsct, whose treatment has been very disappointed. we report our experience in pts who developed pgf during the last 2 years. epag induced responses in platelets in all pts of the studied group. bilineal and trilineal responses were also seen. in our opinion, prospective studies are warranted in order to confirm epag as a new efficient treatment of post-hsct poor graft function. disclosure of conflict of interest: none. s287 ing 19 patients who developed es with an equal number of patients who did not between january 2013 and november 2016. we analyzed variables such as cd34+ cells per kg infused, use of granulocyte colony-stimulating factor (csf-g) and engraftment day. analytical data, including baseline and maximum determination of serum glutamic oxaloacetic transaminase (got) and glutamic pyruvic transaminase (gpt), c-reactive protein (crp) and procalcitonin (pct), as well as clinical data fever, weight gain, digestive and respiratory symptoms, pulmonary infiltrates were analyzed. sixty-eight patients were women. median age was 59 years old (range: 39-73). patients were conditioned with beam (52%), melphalan 200 mg/m 2 (42%) and bcnu-tt (5%). nineteen patients developed es in our series, which correspond to eight percent of all asct. case and control groups were matched according to age, sex, diagnosis and conditioning regimen. the most prevalent baseline disease in the group with es was myeloma (42.1%), followed by mantle cell lymphoma (26.3%). all patients who developed es had fever, 79% skin rash, 37% respiratory symptoms, 16% pulmonary infiltrate an 9% digestive symptoms. a summary of the comparison of data analyzed in subgroups is shown in table 1 . we found significant difference in the percentage of weight gain (p = 0.007), increase of tgo (p = 0.0001), increase of tgp (p = 0.001) and increase the number of cd34+ cells per kg infused (p = 0.043), we found an inverse correlation between the number of cd34+ cells per kg infused and incidence of es. however, in terms of post-transplant csf therapy (p = 0.075) and crp and pct valor (p = 0.85 and p = 0.25, respectively) we did not find significantly difference to develop es. in our series, weight gain and tgo and tgp rise were risk factors for es development. therefore, we should be aware of es in patients who develop fever, elevated liver enzymes and weight gain during graft phase. we did not find a significant difference in crp and pct suggested in other studies. further studies are required to better characterize risk factors of es development. busulphan-based (155), melphalan-based (122), beam (58), tbi-based (22), and others (43). weight at hospital discharge was significantly lower than at admission (5.6% in allo-hsct, and 5.4% in auto-hsct). weight at day +100 was also significantly decreased compared with the admission (8.6% in allo-hsct, and 6.7% in auto-hsct). weight at day +100 was lower than the ideal for their sex and height in the allo-hsct setting. contrarily, among the patients undergoing auto-hsct, the weight at day +100 remained higher than the ideal for their sex and height in a high proportion of cases. regarding serum albumin, it was significantly decreased at discharge (9% in allo-hsct, and 17.5% in auto-hsct), but recovered values similar to admission at day +100. in the auto-hsct setting, prealbumin levels were significantly reduced at discharge (39%), and in lower proportion at day +100 (8%), compared with admission values. in the allo-hsct patients, prealbumin levels were significantly reduced at discharge (5%), but had been recovered at day +100, compared with admission values. disclosure of conflict of interest: none. recently, blood and marrow transplant clinical trials network has proposed a composite endpoint: gvhd-free, relapse-free survival (grfs) for hsct outcomes. this endpoint includes as event: iii-iv acute gvhd (agvhd), relapse, death or chronic gvhd (cgvhd) requiring systemic treatment. in the last embt annual meeting a redefinition of this endpoint was proposed changing cgvhd event from those patients with cgvhd requiring systemic treatment (the original one) to those with just severe cgvhd (the redefined one). we retrospectively analysed 603 patients consecutively transplanted (1995-2014) excluding non-malignant diseases, second allo-sct and those o16 years old age. we had generated two composite endpoints: in both iii-iv agvhd, relapse or death were considerated events but we defined grfs1 as the one with cgvhd event including those who required systemic treatment (as the original one) and in grfs2 just those with severe cgvhd (the ebmt redefined one). the median age was 49 years (16-69) and 59% (362) were males. other characteristics of patients are resumed in table 1 . with a median follow up for patients alive of 39 months , the median estimated survival in months and the % at +1 year and +2 years was: 114 months, 71% and 62% overall survival (os); 24 months, 58% and 50% event free survival (efs); 6 months, 35% and 26% grfs1; 11 months, 46% and 38% grfs2. 138 (23%) and 210 (35%) hadn't any event in grfs1 and in grfs2, respectively. in grfs1, event's incidence was: 90 (15%) for iii-iv agvhd, 170 (28%) for cgvhd, 151 (25%) for relapse and 54 (9%) for death; in grfs2 was 90 (15%), 65 (11%), 173 (28%) and 65 (11%), respectively. considering those patients with cgvhd as event in grfs1, 105 of them hadn't the event as cgvhd at the same time in grfs2 (since they had cgvhd requiring systemic treatment but not severe cgvhd). for these patients, the alternative event in grfs2 was: 72 without any event, 22 relapsed and 11 died. in the multivariate, the factors associated with better outcomes were: in grfs1 early ebmt stage (p o0.001 with early as reference; intermediate p factor with more impact in both, but it is interesting to point it out that haploidentical donor had an advantage in grfs1. these results are being validated in a large series and the definitive results will be available at the moment of the meeting congress. [p334] disclosure of conflict of interest: none. steroid refractory acute graft-versus-host disease (gvhd) remains a major complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). affected patients have a very poor prognosis. gvhd has been associated with transplant-associated thrombotic microangiopathy (ta-tam). endothelial damage mediated by radiation, viral reactivation, drug exposure or alloreactivity results in exposure of subendothelial collagen, activation of coagulation and small vessel occlusion to a degree that results in organ failure. complement is thought to be a major mediator of endothelial damage. although a consensus exists about the exceedingly high morbidity and mortality of ta-tam and diagnostic criteria have been converging to a consensus, no biomarkers to diagnose tam and predict outcome have been established. we hypothesize that a ta-tma, related to dysregulation of the alternative complement pathway correlates with organ damage. a retrospective analysis of 660 consecutive patients with hematological malignancies receiving an allo-hsct at the university hospital basel in the period from 2003 to 2013 was performed. data on the occurrence, risk factors and outcome of patients with ta-tma and the correlation with acute gvhd was collected. available biopsies of organs suspected to be affected by tam and/or gvhd will be performed. routine bone marrow biopsies for histological, immunohistochemical signs of ta-tam and complement activation will be analyzed. serum samples will be used to characterize markers of complement activation using plasma levels of c5b-9 and c5b-9 deposition in tissues biopsies. 660 patients (aml n = 260; all n = 152; mds/mpn n = 93; lymphoid neoplasm n = 85; plasma cell disorder n = 53; bone marrow failure n = 17) underwent myeloablative (n = 432) and non-myeloablative (n = 228) allo-hsct at a median age of 47 years (range: 19-71 years). forty-eight (7.3%) patients matched the established diagnostic criteria for tam (increased ldh, platelet count o50 g/l or o 50% of normal baseline, schistocytes 42 per high power field, creatinine increase). the median time to onset of tam was 36 days post-transplant (range: 22-67 days). subjects with ta-tam had significantly higher 3-year nonrelapse mortality compared to those without (47.8% vs 18.2%, p o0.001). grades 2-4 agvhd and cytomegalovirus viremia were independent risk factors for ta-tam, and serum ldh level 4500 u/l as well as arterial hypertension were early signs of ta-tma occurrence. patients with clinically relevant agvhd (⩾ grade 2) had more ta-tam than patients without agvhd (45% vs 24%; po 0.001). tam correlated with agvhd severity; the higher the agvhd grade, the more the patients who suffered from tam. allo-hsct recipients with grades 2-4 agvhd or cytomegalovirus viremia should be closely monitored for the presence of ta-tma. at the meeting first results of histological, immunohistochemical and complement activation analyses will be presented. disclosure of conflict of interest: none. hemorrhagic cystitis (hc) after stem cell transplantation (sct) can cause significant morbidity and prolonged hospitalization. early bleeding occurs almost exclusively when using cyclophosphamide (cy) (5-25% of cases), while late onset hc are classically attributed to bkv infection, and occurs up to 58% of patients (pts) receiving myeloablative haplo-sct who had positive bk viruria (1, 2). we retrospectively studied hc cases among pts submitted to haplo-hsct in our department. thirty-eight pts receiving an haplo-sct with post-transplant cy (pt-cy) were included (table 1) . prophylaxis for cy included hyperhydratation (3 l/m 2 of 0.9% saline) and mesna administration (200 mg for each 1000 mg of cy/daily divided into three doses). hematuria was graded as follows: grade i, microscopic; grade ii, macroscopic; grade iii, with clots; and grade iv, leading to urinary retention or requiring surgical intervention (1). pts with hc and clots were treated with continuous bladder irrigation. twenty-three pts (60.5%) developed hc at a median of 9.5 days post-sct (range: 1-57). clinical severity was grade i in 6 cases (26.1%), grade ii in 13 cases (56.5%), grade iii in 2 cases (8.7%) and grade iv in 2 cases (8.7%). at the onset of hc diagnosis, bk viruria was investigated in 13/23 pts. five pts (38.5%) had bkv negative (bkv − ) hc and 8 pts (61.5%) bkv positive (bkv+) hc. bkv-hc occurred after a median of 5 days (range: 5-52) while bkv+ hc after 14.5 days (range: 5-57), respectively (p = 0.06). among bkv+ pts, 4 received iv cidofovir 5 mg/kg once a week for 2 weeks and then once every 2 weeks. median number of administrations was 3 (range: 2-4). oral probenecid was given at the dose of 2 g 3 h before and 1 g 2 and 8 h after cidofovir administration. two pts obtained a complete response (cr) after 70 and 110 days, respectively, one patient reached a partial response after 31 days and one pt failed to obtain a response. no pts developed renal toxicity during treatment. one pt received ganciclovir for concurrent cmv viremia and bkv+ hc resolved in 55 days. three patients did not receive any treatment for mild or asymptomatic cystitis. all of them achieved remission after a median of 10 days from the onset (range: 5-57.) among bkv-hc, 3 pts obtained spontaneous resolution after a median of 4 days (range: 1-52), while two pts died early after sct. finally, among pts for whom bk viruria was not available, a remission was reached in 6 of them after a median of 28.5 days (range: 12-43), while 4 pts died early after sct. in our cohort of pts, hc occurrence was of 60.5% and bkv was responsible for the 61.5% of cases. contrary to its high incidence, hc showed a relative benign course, with an overall remission rate of 87.5%, regardless of treatment. finally, we found a trend for a longer interval between sct and hc onset in pts with bkv+ hc, as compared to cy-related hc (p = 0.06). sos is a rare and serious complication of hematopoietic stem cell transplantation (hsct). it is diagnosed using the modified baltimore criteria of hyperbilirubinemia, weight gain or ascites 45% over baseline, hepatomegaly or right upper quadrant pain of liver origin. only defibrotide has been approved for the treatment of veno-oclusive disease. hdmp has been described as effective sos therapy in a few case series (1, 2). we describe our experience of treating adult sos using hdmp. objective is to retrospectively analyze the treatment efficacy and overall survival of patients diagnosed with sos after hsct and treated with hdmp. we used vilnius university hospital data base to identify patients diagnosed with sos under baltimore criteria and treated with hdmp over 2007-2016 period. patient demographics, transplant and clinical data, response, survival (kaplan-meier survival analysis) and hdmp infusion related complications were analyzed. we identified 11 patients (9 males) of whom 10 had had allogeneic hsct (6 reduced intensity conditioning) and one had received a double autologous hsct. sos was diagnosed on the median day +22 (+7 to +81 days). the median bilirubin value was 61.7 μmol/l (11.1-137 μmol/l). all patients had liver enlargement of median 210 mm (160-235 mm) on ultrasound. two patients had normal bilirubin values but displayed the remaining signs and symptoms of sos at diagnosis. patients received intravenous methylprednisolone 500 mg/m 2 every 12 h for 3 days. none received defibrotide. seven (64%) patients responded on median day +12 (+3 to +20 days) after the start of hdmp. four responded by decrease in serum bilirubin by 50% and resolution of symptoms without the need of further treatment. the remaining three responders received maintenance treatment after one course of hdmp with reduced doses of methylprednisolone until resolution of symptoms. four patients failed to respond and died of multiorgan failure on median day +12 (+5 to +41). the median observation time was 6 months (0-44 months). the median overall survival for the sos group was 8 months (range: 0-18) and it was 27 months among the responders. no adverse reactions related to hdmp infusion were observed. hdmp therapy in adult sos results in clinically relevant response rate. further prospective trials are required to assess hdmp efficacy in comparison to defibrotide or as add on therapy. prevalence of hypertension (ht) in general pediatric population is~4%, while in children treated with hematopoietic stem cell transplantation (hsct) it is up to 30%. we assessed factors contributing to the development of ht in children treated with hsct and usefulness of ambulatory blood pressure monitoring (abpm) in this population of patients. the study included 30 children (21 boys, 9 girls; mean age 10.9 years) treated with hsct for neoplasms (n = 22; 73%) or non-neoplastic disorders (n = 8; 27%). control group included 19 children (8 boys, 11 girls; mean age 12 years). abpm measurements (spacelab device) were performed before hsct and after a mean of 7 months after hsct (in 16 of the 30 children). blood samples were collected from 10 children treated with hsct and all controls. total rna extraction was performed and microarray analysis was conducted using genechip human gene 1.0 st arrays (affymetrix). in patients after hsct no antihypertensive treatment was used. mean systolic blood pressures (sbp) before and after hsct did not differ significantly from the control group. mean diastolic blood pressures (dbp) before and after hsct were 68.7 ± 12.2 mm hg and 65.4 ± 9.42 mm hg, respectively, and mean dbp percentiles were 77.1 ± 7.9 and 78.3 ± 6.7, respectively; the differences between the study group and the control group were significantly higher before hsct. mean 24-hour arterial pressure (map) percentiles were 83.3 ± 11.1 and 79.2 ± 8.7, respectively; the differences between the study group and the control group were significantly higher before hsct. before hsct and after the procedure, the european society of hypertension criteria for high normal blood pressure (bp) and ht were fulfilled in 16%/12% patients and 20%/0% patients, respectively. nocturnal bp decrease o 10% was found in 46%/ 53% patients and 420% nocturnal bp decrease in 3%/7% patients, respectively. in the control group o 10% nocturnal bp decrease was found in 10% of children and 420% nocturnal bp decrease in 5% of children. when the groups of patients before and after hsct were compared, highly significant differences were found in gene expression levels for mthfr ( in children referred for hsct a trend towards higher bp values was seen. in children assessed 6 months after hsct more abnormalities in nocturnal bp measurements were seen, which may be a predictor of ht. in children treated with hsct significant differences in the expression of ht-related genes were found. abpm was useful in bp monitoring in children treated with hsct. hypothyroidism may complicate of allogeneic hematopoietic stem cell transplantation (allo-hsct); risk factors are analysed. we studied 229 patients with aml who underwent an allo-hsct between 2003 and 2013 with different conditioning regimens (myeloablative, reduced-intensity, chemotherapybased, total body irradiation-based). thyroid stimulating hormone (tsh) and free thyroxin levels (ft4) were available in 104 patients before and after allo-hsct. median age at transplantation (n = 104) was 47 years (iqr 40-59), 37 (35.6%) were female and overall mortality was 34.6% (n = 36) ( table 1) ursodeoxycholic acid (udca) has been shown to have a protective effect in the liver complications after allogeneic stem cell transplantation (allo-sct), but it also has other immunomodulatory effects; it has been described also a potential benefice as graft-versus-host disease (gvhd) protection. we retrospectively analysed 618 patients consecutively transplanted between 1995-2014 excluding second allo-sct and those o16 years old. we analysed the differences between those with and without prophylactic udca using spps v20. results: the median age was 49 years (16-69) and 59% (362) were males. other patient characteristics are resumed in table 1 objective of study was to evaluate the impact of disease status on the outcomes of allogeneic hematopoietic stem cell transplantation (allo-hsct) in the treatment of patients with refractory and relapsed acute lymphoblastic leukemia(all). 52 patients with refractory and relapsed all, including 19 cases in advanced stage (nonremission, nr) and 33 cases in more than or equal to second complete remission(⩾ cr2), received allo-hsct after myeloablative conditioning regimen in our department. results: 51 patients engrafted successfully. the transplantation-related mortality (trm) rate of nr and ⩾ cr2 was 10.5% vs 12.1% (p = 0.815). the incidence of agvhd was 52.6% vs 57.6% (p = 0.730), including 42.1% vs 33.3% (p = 0.527) with mild (grade i-ii) and 10.5% vs 24.3% (p = 0.399) with severe (grade iii-iv) agvhd. the incidence of cgvhd was similar also(41.6% vs 57.9%, p = 0.660). with a median follow-up of 12(1.8-44.5) months, the cumulative relapse rate of nr and ⩾ cr2 was 47% vs 34.3%(p = 0.425), respectively. the estimated 2 year overall survival (os) and 2 year leukemia-free survival (lfs) rate were 42.6% vs 45.7% (p = 0.487) and 46.3% vs 46.2% (p = 0.571), respectively. multivariate analysis results showed that cgvhd was independent favorable risk factor for os and lfs of r/r all. for relapsed patients, os was significantly better with first cr duration 46 months and time to transplant ⩽2 months. alio-hsct is an effective salvage treatment option for patients with refractory and relapsed all. our retrospective analysis showed that r/r all with different status prior transplant had similar outcome post transplantation. disclosure of conflict of interest: none. the deleterious effect of intensive care unit (icu) admission during hematopoietic stem cell transplantation (sct) on patient survival is well established. however, it is unknown whether admission into the icu during the chemotherapy for the underlying disease has any impact on survival after sct. we reviewed patients who had received a first sct between the years 2000 and 2016 in our institution, and we compared the overall survival (os), relapse incidence (ri) and non-relapse mortality (nrm) between patients who required icu admission during the chemotherapy prior to the sct (icu group) with matched patients (1:2) who did not need it (no-icu group). sixty-six patients were included, 22 of them in the icu group and 44 in the no-icu group. as shown in table 1 , the main clinic-biologic variables and the sct procedure were comparable between the patient groups. the causes of icu admission for the icu group patients were: 11 (50%) respiratory failure, 4 (18%) septic shock, 4 (18%) neurological disturbance, 2 (9%) post-surgery and 1 (5%) tumor lysis syndrome. seventeen patients (77%) needed mechanical ventilation. the median time between icu admission and the sct procedure was 144 days (range: 106-1097), and the median days of icu stay were 12.5 . with a median follow-up after sct of 5.47 years (0.50-16.22) for the icu group and 4.52 (0.73-15.85) for the no-icu group, the 5 year os (ic 95%) probabilities were 49% (28-70%) and 45% (29-61%) in the icu and no-icu patients (p = 0.353), the 5-yr probabilities of relapse were 34% (14-56%) and 42% (25-58%)(p = 0.755) and the 5-yr probabilities of nrm were 32% (14-52%) and 24% (12-38%)(p = 0.333), respectively. there were no differences in either os, ri or nrm between icu and no-icu in the allogeneic or autologous subgroups considered separately. at the moment of the study, s295 12 (54%) of icu and 22 (50%) of no-icu group had died. the causes of death in the icu group were: relapse in 5 (42%), infection in 4 (33%), gvhd in 1 (8%) and gvhd plus infection in 2 (17%). the causes of death in the no-icu group were: relapse in 8 (36.4%), infection in 4 (18.2%), gvhd in 3 (13.6%), gvhd plus infection in 5 (22.7%) and veno-occlusive disease and secondary malignancy, one each (4.55%). in this series, admission to the icu before sct did not have an impact on outcomes after sct. these results suggest that these patients benefit from this treatment as much as the other patients, without expecting worse outcomes as a result of a previous icu admission. supported in part by grants rd12/0036/0029 (rticc, fejer), pi14/01971, instituto carlos iii, and sgr225 (gre), spain. disclosure of conflict of interest: none. autologous stem cell transplant (asct) is a well established treatment for several haematologic and non haematologic malignancies, either as front-line or rescue therapy. however it is associated with toxicity and complications which might lead to treatment-related mortality (trm). although decrease in trm has been reported, data about the precise reduction and detailed analysis of causes of mortality throughout years are scanty. the aim of this study was to evaluate early trm and its causes in patients who underwent an asct in a single center along the last three decades. data of all consecutive adults (415 years old) asct recipients were prospectively collected at a single center from december 1988 to august 2016 and then retrospectively analysed. trm was defined as mortality happened into the 100 days post asct or during conditioning treatment due to any cause except relapse or progression of main diagnosis. demographic characteristics, diagnosis, conditioning regimen and cause of death were analysed. data were compared for two periods: from december 1988 to december 2000 and from january 2001 to august 2016. a total of 849 patients were included, median age was 45 years (16-71) and 50.3% were male. diagnoses were: lymphoma (n = 391), multiple myeloma (mm) (n = 216), acute myeloid leukaemia (aml) (n = 93), amyloidosis (al) (n = 15), acute lymphoblastic leukaemia (all) (n = 39), solid tumours (including breast cancer and germ-cell tumours) (n = 89), chronic myeloid leukemia (cml) (n = 3), thrombotic thrombocytopenic purpura (ttp) (n = 2) and autoimmune disease (n = 1). the most frequent indication for asct was lymphoma (46.1%) and mm (25.5%). twenty patients died within 100 d from asct (trm). demographic characteristics and causes of death for this patients are shown in table1. the cumulative incidence of trm at day +100 was 2.8% (95% ci 1.9-4.1). comparing both periods, trm cumulative incidence was 7.9% (95% ci 4.9-11.8) in first period (1988-2000) vs 0.8% (95% ci 0.3-1.8) in last period (2001-2016) po 0.001. (figure 1 ). according to main diagnosis trm cumulative incidence was higher in patients diagnosed with solid tumour 6.7% (95% ci 2.7-13.2) and al 6.7% (95% ci 0.4-26.9) followed by acute leukaemia (aml/all) 4.5% (95% ci 1.9-9.1), mm 2.8% (95% ci 1.1-5.6) and lymphoma 1.3% (95% ci 0.5-2.8) p o0.03 (figure 2 ). sepsis (65%) was the main cause of death in both periods of time, and the only one cause of death in the last period. the second cause was sinusoidal obstruction syndrome (sos/vod) (20%), which only appeared in the first period. this study shows a low incidence of trm in asct recipients, with a significant decrease in the last period (2001-2016), despite the higher risk in some groups of patients such as those with amyloidosis and solid tumours. in our experience, infection is the main cause of early death in asct recipients and sos/vod has disappeared in last years as a cause of early transplant related mortality. disclosure of conflict of interest: none. incidence and risk factors for hepatic sinusoidal obstruction syndrome after allogeneic hematopoietic stem cell transplantation: a retrospective multicenter study of turkish hematology research and education group (threg) hepatic sinusoidal obstruction syndrome (hsos) is a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-hsct). the mean incidence of hsos was found to be 13.7% (0-40%) in the literature. we examined the incidence and risk factors for hsos after allo-sct. eight centers from turkey were enrolled in the study. we retrospectively evaluated the medical records of patients who were treated with allo-sct between january 2012 and december 2015. a baltimore criterion was used for assessment of hsos. two hundred eighty three (96%) of 295 patients who were treated with prophylaxis with defibrotide alone or one or more of the n-acetylcysteine, diuretics and heparin used defibrotide (10-25 mg/kg/day). the study included 889 patients (514 males/348 females) with median age of 37 (15-71) years. the demographic and clinical characteristics of patients were summarized in table 1 . the incidence of hsos was 9.3% (83). prophylaxis for hsos was used in 40 (48.1%) of patients, who developed hsos. defibrotide as prophylaxis was received by 32 of 40 (80%) of patients. hsos developed in a median of 13 (0-34) days after stem cell infusion. seventy-five (90.3%) of patients who developed hsos had infection at the time of diagnosis. forty-five of them had ascites, 63 had hepatomegaly and, 74 had weight gain. seventy-two (86.7%) of patients with hsos were treated with defibrotide after diagnosis. the median time of starting defibrotide in these patients was 14 (2-29) days. thirty-six (43.3%) of patients with hsos recovered completely and forty-seven (56.7%) of them died as a result of multi organ failure. the incidence of hsosrelated mortality in allo-hsct cohort was found to be 5.3%. in univariate analysis, statistically significant associations were not found between hsos incidence and age/sex of recipient, type of conditioning regimen, stem cell source and type of gvhd prophylaxis. on the other hand primary diagnosis of myelofibrosis, donor type, engraftment status and prophylaxis for hsos were significantly associated with hsos development. hsos prophylaxis was significantly decreased hsosassociated mortality (p = 0.006). hsos still remains a serious life-threatening complication of allo-sct. although the incidence is low, hsos is associated with increased 100-day nonrelapse mortality. hsos prophylaxis especially with defibrotide, seems to reduce hsos associated mortality in high risk patients. [p346] disclosure of conflict of interest: none. hemorrhagic cystitis (hc) is a serious complication occurring after allogenic hematopoietic stem cell transplantation (hsct) more frequent on haploidentical (haplo) hsct, with an incidence of 10-70% 1 associated mainly with the effect of cytotoxic agents such as cyclophosphamyde (cy). the conditioning regimen, bkpyv infection and graft versus host disease have an implication in the incidence. other authors related the reactivation of cmv and a previous transplantation as risk factors to hc development2. with this study we aim to describe the hc incidence and risk factors in all haplo-hsct performed in the canary islands. we analyzed all consecutive haplo-hsct from family donors performed at our hospital between 2013 and 2016. the conditioning regimen used for the transplant was the hopkins haplo protocol with high dose cy (50 mg/kg on days 3 and 4) posttransplantation (ptcy). we used as hc prophylaxis intense hydratation on the cy administration day and the following 24 h (using bladder wash only in 1 patient with cardiac dysfunction) and perfused mesna at 100% of cy dose beginning 15 min before the cy administration on 16 pts and at 20% of the last dose at 0, 4 and 8 h on all pts. we used spss v.23 to determine the cumulative incidence (ci) of hc. we performed 20 haplo-hsct, of which 10 were males (1 was transplanted 3 times) and 8 were women. the mean age was 40 (range: 16-64). the pts presented the following diagnosis: aml (10), all (1), eh (5), nhl (3), am (1). 45% of pts received the haplo-hsct in remission, 50% with refractory disease and 5% of pts did not receive previous treatment. 6 pts developed hc (36.5% ci at day +80) (figure 1a ) with a median time from haplo-hsct to onset of 23 days (range: 3-42), 1 (17%) was grade i, 4 (66%) grade ii and 1 (17%) grade iv. the grade i case did not received the mesna infusion like most of the other pts. no pts died due to hc and all cases resolved without sequelae. 12 pts received cy pre-and post-transplant and only 8 pts received ptcy. the ci at day +80 for the pts with ptcy was 33.3% and for cy preand post-transplant 38.3% (figure 1b) . we found no statistically significant difference on the ci of hc between these two groups. the development of hc was related to cy in 1 patient, who suffered from this complication on the second and third haplo-hsct. for the rest of the pts (after day +30) the hc was related to bkpyv infection, as a consequence of the immunosuppression state of the patient, we also observed all these pts had positive serum viral load for cmv. the incidence of hc associated to post-hsct high cy dose in our series is 15% lower than other ones. most of them on grade 1 or 2 and without mortality associated. the risk of hc is high, particularly in the setting of highly pre-treated patients (especially those undergoing a 2nd transplant). the development of hc after day +30 is evidently associated to bkpyv as a contributing factor for continuous inflammation and cmv reactivation (as an immunosuppression marker). in our study, hc did not have an impact on mortality of high-risk patients after haplo-hsct. the hc remains frequent with a high morbidity in particular when it is severe, often causing prolonged hospitalization and resource use. we need further studies to recognize the at-risk population early. [p347] ta-tam is not a rare post-transplant complication and it is potentially fatal. in survivors, it was often associated with longterm morbidity and chronic organ damage, mostly to the kidney with poor renal prognosis. our retrospective study showed ta-tam associated risk factors included t reg haplo hsct as the incidence was highest in this group, tbi-based conditioning or tbi based conditioning plus cyclophosphamide. although acute gvhd and infection were associated with ta-tam in retrospective studies, no association emerged between acute gvhd or infection preceding diagnosis in our series of patients. in order to prevent ta-tam we need to understand its underlying biological mechanism so we are investigating its pathogenesis by means of cytokine assays, histology and murine models. disclosure of conflict of interest: none. mortality in children requiring invasive mechanical ventilation (imv) after allogeneic hematopoietic stem cell transplantation (hsct) is known to be high. little is known about the longterm outcome of those who survive imv. we therefore reviewed the medical records of 55 children who survived s298 imv after they had received a hsct between 2000 and 2012 in the two pediatric hsct centers in the netherlands. retrospective multi-center cohort study in two university hospitals that perform all pediatric hscts in the netherlands. long-term survival of hsct recipients who had received imv was assessed. health status was reviewed more in detail for those who were still alive 2 years after discharge from the pediatric intensive care unit (picu). in the absence of standardized use of quality of life questionnaires, health status was expressed as the number of affected domains (cardio-respiratory, motor and miscellaneous, regardless of the degree of dysfunction) and level of education. health status was categorized as follows: no health problems when all four domains were normal; mild health problem when there was an abnormal score in one of the four domains; moderate health problems when there was an abnormal score in two domains; severe health problem when there were abnormal scores in three or all four domains. between january 2000 and december 2012, 641 patients underwent a hsct in the two study centers together. a total of 89 hsct recipients received imv within 1 year after hsct (14% of all hsct recipients). median time between hsct and picu admission was 59 days (iqr 17-102 days). the most common indication to start imv was respiratory failure (73%). median duration of imv was 10 days (iqr 5-18 days). 34 patients (38%) died during their picu admission. of the 55 patients who were discharged alive from picu, 27 patients were still alive 2 years after picu discharge (49% of those who survived picu admission). health status of these long-term survivors was assessed in december 2014 by hospital database review, using the most recent hospital contact. follow-up time varied from 2 to 11 years (median 6.5 years) after picu discharge. two patients (8%) had no health impairment, eight patients (33%) had mild health problems, five patients (21%) had moderate health problems, and nine patients (38%) had severe health related problems. very little is known about long-term mortality and morbidity of hsct recipients who survived imv. survival of picu treatment in pediatric hsct recipients is limited. however, long-term outcome of those who survive picu treatment seems promising: a considerable proportion of them still is alive 2 years later without obvious sequelae. this is the first study which assessed long-term outcome of imv after hsct. further studies in this population are urgently required to counsel parents and to optimize quality of life outcomes in these children. disclosure of conflict of interest: none. long-term surveillance data support lack of increase in mortality or cancer risk in brincidofovir clinical trial participants m morrison, k fitzgerald 1 , t brundage, a harrison and wg nichols 1 chimerix brincidofovir (bcv) is an orally bioavailable lipid conjugate of cidofovir (cdv), with broad-spectrum activity against doublestranded dna viruses, including cytomegalovirus (cmv), adenoviruses (adv), polyomaviruses (bk and jc viruses), and orthopoxviruses. bcv is being evaluated for prevention of cmv and other dna viruses in high-risk hematopoietic cell transplant (hct) recipients, and for the treatment of serious adv infection. bcv is also being developed for the treatment of smallpox under the us fda's animal efficacy rule. because bcv, cdv, and other marketed nucleoside analogs are reported to be carcinogenic in rodents, a registry was established to evaluate the long-term safety of bcv in subjects who have participated in bcv clinical studies. to date, the registry includes prior participants from study 301 (a placebo (pbo)-controlled study of bcv for cmv prevention) and study 304 (a single-arm study of bcv for adv treatment). subjects are encouraged to consent for long-term follow-up in the registry following participation in bcv clinical studies. registry participants are followed at 6-month intervals for a minimum of 3 years from the time of completion of the parent study. development of malignancies (new or relapsed), lifethreatening or fatal adverse events (aes) assessed as potentially related to bcv, and subjects' vital status are collected. a total of 649 subjects were enrolled in the parent studies (302 bcv and 148 pbo from study 301, 199 bcv from study 304). of these, 291 are enrolled in the registry as of 24 october 2016 (155 bcv and 80 pbo from study 301, 56 bcv from study 304). bcv recipients in the registry are 60% male, 84% white, with a median age of 47 ( o1-76) years, similar to the bcv recipients in the parent studies. the median duration of follow-up is 12 (0-30) months, with 80% of subjects continuing in follow-up at the time of analysis. all-cause mortality from the time of first dose in the parent study through current registry follow-up is 25% for bcv vs 22% for pbo (p = 0.559) in study 301, and 51% for bcv in study 304. all-cause mortality in the registry since completion of the parent study is 21% bcv vs 24% pbo for subjects from study 301 (p = 0.618) and 14% bcv for subjects from study 304 (figure 1) . the incidence of a new malignancy in registry subjects from study 301 is 17% bcv vs 23% pbo (p = 0.295), and the incidence of relapsed primary malignancy is 12% bcv vs 21% pbo (p = 0.055). in registry subjects from study 304, 7% developed a new malignancy and 4% had a relapse of the primary malignancy. no bcv-related life-threatening or fatal aes have been reported to date in the registry. registry data collected to date support no increase in late mortality or increased risk for carcinogenicity in patients treated with bcv. long-term surveillance for cancer risk and other drivers of mortality is important for novel compounds undergoing development in hct and other immunocompromised patient populations, with high background risk for these outcomes. [p351] disclosure of conflict of interest: all authors of this abstract are employees and stockholders of chimerix. hematopoietic stem cell transplantation (hsct) is a medical procedure that allows the cure of many paediatric diseases. it has been described an increased risk of new malignancies in this population and it represents an important cause of late mortality. we analyzed the late evolution of 371 patients submitted at pediatric age to hematopoietic transplantation (hsct) (allogeneic or autologous) in santa creu i sant pau hospital between 1984 and 2013. a total of 434 hsct was analyzed. it has been calculated the cumulative incidence of secondary malignancies at 30 years of follow-up. it has been done univariate and multivariate analysis of risk factors through χ 2 -test and binary logistic regression method (odds s299 ratio, or). it has been studied the relative risk (rr) for new malignancies through comparison of observed cases in our cohort with the expected cases in the general population. we observed 19 cases of secondary malignancies with a cumulative incidence of 6% at 15 years, 12% at 20 years and 36% at 30 years of follow-up. the risk was higher of expected in general population for each tumor type and in the different range of age, being the rr for malignancies in our cohort of 51.4 at 30 years of follow-up. solid tumors were the most prevalent malignancies (16 out of 19 cases). the median time of latency from hsct to diagnosis of malignancy was 16 years (1-31 years) . the thyroid tumors were the later ones and hematologic malignancies the earliest to be developed. chronic graft versus host disease was a statistically significant risk factor in univariate (or = 16; p = 0.006) and multivariate analysis (or = 15.4; p = 0.000). total body irradiation of conditioning was a significant risk factor only in multivariate analysis (or = 4.3; p = 0.03). previous radiotherapy was a significant risk factor only in univariate analysis (or = 3.1; p = 0.04). mortality was 42% (8 out of 19) between patients with a new malignancy and it was the cause of death for all the cases. we observed an incidence of secondary malignancies after hsct of 5.1% that is significantly higher compared to the expected in the general population (p = 0.000). the factors that have been related to an increased risk were chronic gvhd, tbi and previous radiotherapy. microalbuminuria defined, as urinary albumin: creatinine ratio (acr) of 30-300 mg albumin/g creatinine is a marker of endothelial dysfunction and inflammation. in general populations albuminuria predicts development of chronic kidney disease (ckd) and cardiovascular disease (1). in the general population microalbuminuria is associated with the metabolic syndrome (2) . in patients with hypertension, diabetes and the critically ill, it is a marker of adverse events and poor outcomes. following hematopoietic cell transplant (hct), microalbuminuria at day 100 was associated with a four-fold increased risk of chronic kidney disease (ckd) at 1 year in a single centre study; macroalbuminuria at day 100 was associated with a 6.8-fold increased risk of non-relapse mortality (3) . international guidelines for adult and children survivors of hct recommend that proteinuria is assessed at least annually (4, 5) . there is a paucity of data on the prevalence and implications of micro and macroalbuminuria in long-term survivors (410 years) of adult hct, however. this was a single-centre retrospective study conducted in patients attending a dedicated clinic for long-term (minimum 10 years) survivors of hct. we investigated prevalence of albuminuria and its association with renal disease, cardiac disease and the metabolic syndrome. of 55 patients, 8 were treated for acute leukemia, 2 for aplastic anaemia and 46 for cml. the median follow up time was 24 years (range: 13-37 years) and the median age at follow up was 55 years (range: 24-81 years). for 36/55 urinalysis was normal (group a) and 19 (34%) had microalbuminuria (group b). none had macroalbuminuria. group b were significantly more likely to have ckd grade 2-4 (egfr o 60) compared to those in group a (p = 0.001). group b patients were significantly more likely to have diabetes or impaired glucose tolerance 7/19 (37%) vs 2/35 (6%) in group a (p = 0.005). group b patients were also significantly more likely to have dyslipidaemia (p = 0.019 ) with 14/19 (70%) affected vs 23/35 (37%) in group a. cardiac disease and hypertension were more frequent in group b, 4/19 (21%) and 7/19 (37%), respectively vs group a 3/35 (9%) and 8/35 (22%) but these data were not statistically significant. the more features of the metabolic syndrome present, however, (elevated hba1c, /glucose, dyslipidaemia, hypertension) the more likely a patient was to have microalbuminuria (p = 0.007). our data demonstrates that microalbuminuria is a frequent finding in long term survivors of hct. patients with microalbuminuria are more likely to have ckd grade 2 or below. they are also more likely to have diabetes and dyslipidaemia. as this was a retrospective study we are not in a position to comment on whether microalbuminuria is predictive of the development of renal disease, metabolic syndrome or cardiovascular disease in this group of patients. this warrants further study as intervention, for example with ace inhibitors, may have the potential to reduce morbidity. the purpose of the study is the improvement of transplantation techniques and supportive care lead to an increasing number of long-term survivors after allogeneic hematopoietic stem cell transplantation (ahsct). recipients of ahsct have a higher prevalence of cardiovascular risk factors. ambulatory blood pressure measurement (abpm) is the 'gold standard' to diagnose arterial hypertension (ht). the prevalence and treatment control of ht by abpm is unknown in ahsct patients (pts). this prospective single center study at university hospital basel included all pts ⩾ 1 year after ahsct in complete hematological remission during annual follow-up consultation. office blood pressure (obp) was measured on both arms after 5 minutes rest. abpm by noninvasive continuous bp monitoring (pulse transit time method) was performed on the same day. ht was defined as obp ⩾ 140/90 mm hg, mean systolic bp ⩾ 130mm hg on abpm s300 (bp 24 ) and/or current use of antihypertensive drugs. 175 pts (39% female) were included with median age of 53 years (range: 19-75) and 9 years (range: 1-33) after transplantation. 108 (62%) pts received total body irradiation-based conditioning, 70 (40%) pts had chronic graft-versus-host disease, and 39 (22%) required immunosuppression. mean bmi (kg/m 2 ) (± sd) was 25 ±5, with 22 (13%) pts 430. twenty-seven (15.4%) pts were current smokers. fourty-three (25%) pts had chronic kidney disease (egfr o60 ml/min/1.73 m 2 ) and 17 (10%) diabetes. 82 (47%) pts were on antihypertensive drugs consisting of ace/at-ii-inhibitors in 55 (31%), calciumchannel blockers in 18 (10%), beta-blockers in 32 (18%) and diuretics in 24 (14%) pts. thirty-nine (22%) pts were on ⩾ 2 drugs. among our cohort 47 (27%) pts were normotensive without antihypertensive treatment (mean age 46 ± 13 years, 62% female and mean bp 24 (systolic/ diastolic bp) 113 ± 8/76 ± 8 mm hg). 128 (73%) pts were hypertensive and/or on antihypertensive treatment. untreated ht was diagnosed in 46 (26%) pts (mean age 52 ± 13 years, 41% female and mean bp 24 of 147 ± 22/91 ± 12 mm hg), including 14 (8%) with white-coat hypertension and 9 (5%) masked hypertension (normal obp, high abpm). in the group of pts with current antihypertensive medication 32/82 (39%) were controlled (mean age 55 ± 13 years, 25% female, and mean bp 24 119 ± 9/77 ± 7 mm hg) whereas 50/82 (61%) were hypertensive on abpm (mean age 55 ± 11 years, 24% female, mean bp 24 146 ± 13/90 ± 10 mm hg). thirty-four (68%) pts with uncontrolled ht were already hypertensive at obp. although long-term survivors after ahsct are known to be at elevated cardiovascular risk, diagnosis of arterial hypertension was missed in every fifth patient. the proportion of controlled hypertension is poor with only 39%. disclosure of conflict of interest: none. myasthenia gravis (mg) is a rare complication of allogeneic stem cell transplantation (sct) and is often associated with graft-versus-host disease (gvhd). we report a 49-year-old man who presented with oculobulbar and neck weakness 30 months after an unrelated donor, allogeneic sct for chronic myeloid leukaemia (cml). he was diagnosed in 2009 with chronic phase cml. this responded poorly to tyrosine kinase inhibitors (tkis) and he was found to carry the t315i mutation with additional monosomy 7. he underwent a fully hla matched unrelated donor sct with y 90 -anti cd66 targeted radiotherapy, fludarabine, melphalan and alemtuzumab conditioning. he had grade 1 cutaneous gvhd on ciclosporin withdrawal but no other significant gvhd. he has an immune mediated neutropenia since 4 months post sct and has reduced immune reconstitution as demonstrated by a sub-normal absolute cd4 level. he remains on pneumocystis prophylaxis and has not experienced increased infection. chemotherapeutic agents have a cytotoxic effect on the oral mucosa and is a major problem following cancer treatment. cooling the oral mucosa in conjunction with chemotherapy infusion, using ice chips, is known to reduce the severity of oral mucositis (1, 2) . although effective, ice chips are perceived as uncomfortable. the aim of the present study was to determine the optimal cooling temperature to prevent adverse effect of chemotherapeutic agents using tissue engineered oral mucosal models (teom). teom were incubated at 35°c, 30°c, 25°c or 20°c for 30 min followed by exposure to 162 μg/ml of 5-fu for 2 h (control models were incubated at 35°c). teom were then washed and further incubated for 48 h at 37°c co 2 . cell viability and inflammatory cytokine production (il-6 and tnf-α) were measured using (prestoblue) and (elisa), respectively this study demonstrates an increased capacity to restore cell viability with decreasing temperature (figure 1a ). teom treated with 5-fu further showed an increased secretion of the pro-inflammatory cytokines tnf-α and il-6 at all temperatures compared to un-treated controls. for il-6, secretion increased markedly when cells were incubated with 162 μg/ml 5-fu at 35°c and 30°c compared to cells incubated with medium alone at 35°c (figure 1b) . for tnf-α, secretion was significantly higher (p o0.05) in cells treated with 162 μg/ml 5fu at 35°c compared to untreated mucosal models and mucosal models treated with162 μg/ml 5fu but incubated at 20°c (figure 1c ). teom models incubated at 20°c has an increased capacity to restore cell viability following exposure to 5-fu. incubation at 20°c further reduces the release of pro-inflammatory cytokine compared to those incubated at 35°c. (2) and one received fludarabine and cyclophosphamide. all patients received campath-1h as part of the conditioning regimen. stem cell source: peripheral blood stem cells 73 patients and 3 bm. comorbidity was assessed using the haematopoietic cell transplantation co-morbidity index (hct-ci), with 16 patients (21%) having no co-morbidities, 35 (46%) a co-morbidity index of 1-3 and 25 (33%) had a score ⩾ 4. follow up of survivors ranged from 1 to 148 months (median: 32 months). at the specified end point 29 patients had relapsed (38%) with an actuarial 3-year relapse rate of 55%. there were 34 deaths (44%). relapse (23) was the main cause of death with transplant related mortality of 5% (4) at day 100, 8% (6) at 6 months and 13% (10) at 1 year. the actuarial os at 3 years was 48%, with a 3-year dfs of 39%. of the surviving relapsed patients all received chemotherapy and donor lymphocyte infusions resulting in effective recovery of remission, showing the utility of this approach. in terms of co-morbidity, actuarial survival rates were 60% in those with an hct-ci index of 0, 39% with an index of 1-3 and 50% with an index ⩾ 4. the results of this retrospective study indicate that allosct using reduced intensity conditioning regimens can be an effective treatment strategy for older patients with high risk myeloid malignancies including those with significant co-morbidities. relapse remains the main cause of treatment failure and strategies to reduce relapse risk are required. patients that relapse post allosct may respond to further treatment such as azacytidine or intensive chemotherapy and donor lymphocyte infusions. (3) whether patient-related variables were associated with disagreement. this is a secondary analysis of a cross-sectional multicenter study where patients and clinicians completed an identical qol questionnaire (fact-bmt) at day 90. clinical and demographic variables as well as anxiety and depression (hads) were collected. agreement was analyzed with the intraclass coefficient correlation (icc). rates of under-and over-estimation were calculated. logistic regression models identified predictors of disagreement. we analyzed 96 pairs of questionnaires, filled in by 96 patients and 11 clinicians. patients' median age was 54 years, 50 (52%) were men, and 50 (52%) received an allogeneic hct. clinicians' median age was 42 years, 7 were men and had worked on the transplant field for a median of 12 years (range: 3-23). agreement on qol was moderate (icc = 436). exploratory analyses revealed that agreement for emotional (icc = 092) and social (icc = 270) wellbeing was poor, whereas it was moderate for physical (icc = 457), functional (icc = 451) and bmt concerns (icc = 445). patients' wellbeing was overestimated in 41-59% of the categories of wellbeing parameters, and underestimated in 10-24%. patient-related variables explained 12-17% of the variance on disagreement across scales. specifically, anxiety contributed to disagreement in all subscales, except in social wellbeing, where non-significant univariate associations were observed (p40.05). type of transplant (allogeneic vs autologous), performance status, and graft-versus-host disease were not associated with disagreement (p40.05). patients and clinicians agreement on qol is suboptimal, particularly on emotional and social wellbeing. patients' wellbeing cannot be estimated from other sources than themselves. these results highlight the unmet needs of hct recipients with respect to qol-related issues; an outcome that must be addressed by hct programs since their wellbeing is as important as survival endpoints. disclosure of conflict of interest: none. . we wanted to test the function and the safety of picc device as alternative to standard cvc in patients submitted to autologous stem cell transplantation (abmt). the primary end point of the study was to individuate the cause leading to the failure of picc (its removal or the needing of another cvc during the abmt procedure). secondary end points were the correct function of the device and its praticity. twenty patients submitted to abmt for multiple myeloma (18) or lymphoma (2) experienced a double lumen picc device (17) or a single lumen (3) if the patient already carried a permanent single lumen cvc such as hickman or port-a-cath. we excluded from this experience patients with high risk of life-threatening situation or high risk of intensive care already before abmt. picc devices were placed from a specialistic nurse team by ultrasound identification of a deep venous vessel in upper arms. melphalan 200 or ceam were the standard conditioning regimens employed in myeloma and lymphoma abmt respectively. we considered a failure all the causes leading to the removal of picc or requiring another cvc before the end of the transplant procedure. at last we collected nurses and clinicians opinions about the picc functionality. no complication has been recorded in positioning phase. 19/20 patients maintained the picc device for all the time of transplant procedure. only one patient needed to remove the device for infection. the opinion of nurses and clinicians about the picc device was a significatively slower speed of infusion and resistance to the flow; in fact, 11/20 patients needed an infusional pump. the idraulic resistance of the catheter was particular evident against cellular fluids (stem cells suspension, transfusions of blood and platelets). for this reason picc seems to be less indicated in patients requiring many endovenous infusions (nurses' opinion). the rate of infection of picc devices seems to be lower compared to cvc, but the number of cases tested in this experience is too limited for definitive conclusions about it. for other aspects picc is similar to other cvcs. picc seems to be a valid alternative to standard cvc in patients who do not require intensive care, and in particular in patients with low intensity abmt who do not present a high number of endovenous infusions. maybe picc is less burdened of infections respect to normal cvc. this fact, summed to the lower risk during the positioning of the device, leads to consider the use of this device in abmt setting for standard risk patients. disclosure of conflict of interest: none. there are only few algorithms for the selection of hlamismatched unrelated donors, when no fully matched donor is available. indirect recognition of hla-mismatches can be predicted using the model of 'predicted indirectly recognizable hla epitopes' (pirche). the pirche model is a recently developed computer-based strategy, which classifies hladerived epitopes that are potentially presented by patientdonor shared hla-molecules. we performed a multicenter retrospective study evaluating the impact of pirche on outcome after allogeneic stem cell transplantation from hla 9/10 matched unrelated donors. the study cohort included 1997 adult patients who had undergone allogeneic stem cell transplantation for aml or mds. pirche scores were computed for 424 recipients of hla 9/10 matched unrelated donor transplants (9/10mud) using a web-based tool. primary endpoint was overall survival at 2 years. patients with a 9/10 mud were divided into 2 groups according to the sum of pirche i+ii values (pirche score). eighty-five (85) patients had a pirche score of 0 (no pirche detected), 339 a pirche score 40. km estimate of 2 year os was higher for 9/10 mud with pirche score = 0 compared to pirche score 40: 57% (95% ci: 51-63%) vs 47% (95% ci 41-53%), p = 0.04. os was similar for 9/10 mud with pirche score = 0 and 10/10 mud (57% vs 55%). cox regression analysis revealed poorer os for pirche scores 40 (rr 1.5, 95% ci: 1.0-2.1, p = 0.03). cumulative incidence of nrm at 2 years was lower for 9/10 mud with pirche score = 0 compared to pirche score40 (20% vs 32%, p = 0.05). multivariate cox regression analysis revealed poorer nrm for pirche score40 (rr 1.7, 95% ci: 1.0-2.9, p = 0.03). cumulative incidence of agvhd grade 2-4 at 6 months was not significantly different for 9/10 mud with pirche score 0 compared to pirche score40 (23% vs 30%, p = 0.2). cumulative incidence of cgvhd at 2 years was lower for 9/10 mud with pirche score 0 compared to pirche score40 (31% vs 49%, p = 0.04. our findings require confirmation, ideally in a large prospective cohort study. if validated, the pirche model would allow selection of permissible hla-mismatches that may be associated with an improved transplant outcome in terms of reduced nrm and better os. [p364] disclosure of conflict of interest: none. this study was supported by a research grant from pirche-ag to the university medical center, hamburg-eppendorf. pretransplant liver dysfunction has been recognized as a risk factor for complications and mortality after allogeneic hematopoietic cell transplantation (allo-hct). however, there is no consensus on the optimal way to evaluate liver function in hct candidates. transient elastography (te) is a noninvasive method for diagnosing liver damage and cirrhosis. while elastography is widely used in the setting of viral hepatitis, its possible role in allo-hct recipients has not been deeply evaluated. patients receiving allo-hct in our center from may 2014 are scheduled to receive pretransplant evaluation by a hepatologist under a prospective protocol. the evaluation includes a hepatologist consultation, liver function and infectious serology tests and te. all patients receive ursodiol from hct admission to day +30. this study constitutes the first evaluation of the ongoing protocol for patients receiving their first allo-hct from may 2014 to august 2016. sixty patients received a first allo-hct during the study period. sixteen patients did not undergo hepatologist evaluation due to timing issues (n = 6), unstable medical condition (n = 4) or other reasons (n = 6). finally, 44 patients received pretransplant evaluation by a hepatologist under the current protocol and constitute the study population. median age at transplantation was 51 years (range: 21-69). most patients received a transplant for acute leukemia (n = 23, 52%) or non-hodgkin's lymphoma (n = 10, 23%) mainly from hla matched unrelated donors (n = 21, 48%). thirty-two patients received reduced-toxicity regimens (73%). graft-versus-host disease (gvhd) prophylaxis consisted of tacrolimus in combination with another agent. median follow-up for survivors of 14 months (range: 3-29). median elastography was 5.6 kpa (range: 2.9-13.7). considering the hct-ci categories on hepatic dysfunction, 38, 6 and 0 patients scored 0, 1 and 3 points, respectively. there were two cases of veno-oclusive disease (vod). overall survival and non-relapse mortality of all patients at median follow-up were 76% (95% ci 69-83) and 22% (95% ci 14-30), respectively. in the univariate analysis, median elastography was not associated with a higher risk of nrm (p-value = 0.13), os (p-value = 0.11) or hepatic chronic gvhd (p-value = 0.32). the two patients with vod had normal pre-hct transaminase levels and te. this first analysis of an ongoing protocol with universal pre-hct evaluation of hepatic function indicates that increased values of transient elastography are not associated with higher nrm or lower os after the procedure. further studies including a larger number of patients are needed in order to clarify the possible role of elastography in the hct setting. disclosure of conflict of interest: none. allogeneic hematopoetic stem cell transplantation (hsct) remains associated with a high morbidity and mortality in spite of advances in hsct management. specifically, pulmonary complications account for a substantial proportion of deaths within the first 100 days after hsct. therefore, identification of lung dysfunction and additional comorbidities are crucial for preventive strategies in hsct. given the inconsistent association of pretransplant lung function s305 parameters on mortality after hsct and the significant changes in hsct care over the last decades, the aim of our study was to assess the effect of pulmonary function and comorbid conditions on mortality in patients undergoing hsct for hematological disorders. we retrieved relevant clinical data of all consecutive patients at the hematology division of the basel university hospital with a transplant for hematological disorders between 2008 and 2015. we examined the lung function at baseline and 3, 6 and 12 months after hsct-including the 1 s forced expiratory volume (fev1% of predicted), fev1/vcmax and diffusing capacity for carbon monoxide (dlco, adjusted for hemoglobin concentration). in addition, we assessed pretransplant conditions such as age, sex, karnofsky performance status (kps), donor type and various risk scores in hsct (hematopoietic cell transplantation comorbidity index (hct-ci), european society for blood and marrow transplantation (ebmt), revised pretransplant assessment of mortality score (pam)). using uni-and multivariate cox proportional-hazards regression analysis, we evaluated patient-and transplant-related risk factors for all-cause mortality by including the following categorical candidate variables: fev1 (⩾80% vs 50-79% vs o50% of predicted), kps ( o90% vs ⩾ 90%), age ( o54 vs ⩾ 54 years), conditioning intensity and donor type (matched-related vs mismatchedrelated vs matched-unrelated vs mismatched-unrelated). within the study period, 429 patients with predominantly acute leukemia (64%) or lymphoproliferative disorders (28%) underwent myeloablative (n = 330) and non-myeloablative hepatic veno-occlusive disease/sinusoidal obstruction syndrome (vod/sos) is a potentially life-threatening complication of conditioning for hematopoietic stem cell transplantations (hsct). recombinant thrombomodulin (rtm) is a new drug for treating disseminated intravascular coagulation (dic) and is an endothelial anticoagulant cofactor that promotes the thrombin-mediated formation of activated protein c (apc). rtm has been used to treat vod/sos, but its ability to prevent vod/sos has not been established. we evaluated the cases of 19 pediatric hematology and oncology patients (8 (43%) acute myeloid leukemia, 3 (16%) acute lymphoblastic leukemia, and 4 (21%) neuroblastoma patients, and 1 (5%) patient each with myelodysplastic syndrome, rhabdomyosarcoma, hemophagocytic syndrome (hlh), and wiskott-aldrich syndrome) who underwent hsct at our institution between 2007 and 2014 and had ≧ 1 risk factors for vod/sos. these risk factors included previous treatment with gemtuzumab ozogamicin (go), receiving 42 hsct, undergoing conditioning with busulfan (bu), and being diagnosed with hlh. the patients who received hsct after 2012 (n = 8; rtm group) were treated with rtm as a prophylaxis against vod/sos (380 u/kg per day for 7 days; from days 7 to 13) together with ursodeoxycholic acid (urso) and low-molecular-weight heparin (lmwh), and the others (n = 11; control group) were only treated with urso and lmwh. the incidence of vod/sos was evaluated, and various coagulation parameters and markers of endothelial injury (plasminogen activator inhibitor type (pai-1) and apc) were measured in both groups. the patients' median age was 2 (range: 0-18) years, and 11 (58%) were male. clinical characteristics, including vod/sos risk factors, were wellmatched in both groups. the risk factors possessed by the patients included receiving 42 hsct (9/19, 47%), previous go treatment (6/19, 32%), conditioning with bu (3/19, 16%), and a diagnosis of hlh (1/19, 5%). although vod/sos occurred by post-hsct day +35 in 3 (27%) patients in the control group, vod/sos was not seen in the rtm group. two of the former 3 patients (2: previous treatment with go, 1: a diagnosis of hlh) suffered severe vod/sos, and 1 (a diagnosis of hlh) died of the condition. no grade 3/4 adverse events involving bleeding or severe organ damage were reported in the rtm group. interestingly, the mean peak value of pai-1 and apc (markers of endothelial injury) were significantly lower in the rtm group (table 1) . [p367] disclosure of conflict of interest: none. protective effect of early human cytomegalovirus reactivation on relapse of myeloproliferative disorders after allogeneic hematopoietic stem cell transplantation z peric 1,2 , j wilson 2 , n durakovic 1,2 , l desnica 2 , a ostojic 2 , vv rezo 2 , v marekovic 1,2 , r serventi-seiwerth 2 and r vrhovac 1,2 1 school of medicine, university of zagreb, zagreb, croatia and 2 university hospital centre, zagreb, zagreb, croatia there have been conflicting results regarding the association between early cytomegalovirus (cmv) reactivation and decreased incidence of relapse after allogeneic hematopoietic stem cell transplantation (allo-hsct). this prompted us to retrospectively evaluate the potential impact of cmv reactivation on transplantation outcomes in a study population of 161 consecutive adult patients who underwent allo-hsct in our institution and were treated and followed in a homogenous manner. patients were monitored for cmv reactivation once weekly for the first 100 days after allo-hsct. monitoring was done with a real time qpcr with lower limit of detection of 150 genome copies per ml of blood. when cmv viremia was detected, all patients were treated with intravenous ganciclovir or oral valganciclovir untill two consecutive negative qpcr assays. univariate and multivariable proportional hazards models using the fine and gray approach were considered to evaluate the variables for relapse, treating death as competing event. between 2011 and 2014, 97 male and 64 female patients underwent allo-hsct at a median age of 46 years (range: 18-64). among them, most patients were treated for myeloid malignancies (74 aml, 11 mds and 24 mpn with 11 cml, 11 mf and 2 cmml), while the rest had lymphoproliferative disorders (24 all, 10 nhl, 6 mm, 5 mh and 6 cll) and one patient had aplastic anemia. the donors were unrelated in 79 cases, related in 77 patients and haploidentical in 5 patients. most of the patients (70%) received peripheral blood stem cells after a reduced-intensity conditioning regimen (56%). with a median follow-up of 23 months, early cmv reactivation occured in 62% patients at a median of 27 days after transplantation and did not affect relapse incidence in patients with lymphoproliferative disorders. on the contrary, the cumulative incidence (ci) of hematologic relapse in patients with myeloproliferative disorders (aml and mpn) at 20 months after allo-hsct was 36% (95% ci, 21-52%) in patients without, opposed to 18% (95% ci, 10-29%) in patients with cmv reactivation (p = 0.04). however, cmv reactivation did not significantly affect (p = 0.21) overall survival between patients with (64%; 95% ci 53-77%) and without cmv reactivation (48%, 95% ci 34-68%). a striking and previously unreported correlation between cmv reactivation and relapse was found in patients with mpn; the ci of relapse was 50% (95% ci, 12-80%) in patients without, opposed to only 6% (95% ci, 25-100%) in patients with cmv reactivation (p = 0.01). a substantial and independent reduction of the relapse risk in myeloproliferative disorders (aml+mpn) associated with early cmv reactivation was confirmed by multivariate analysis using time-dependent covariate functions for high-risk disease, use of atg, chronic graft-versus-host disease (hazard ratio 3.33; 95% ci, 1.09-10.09, p = 0.03), and cmv reactivation (hazard ratio 2.37; 95% ci, 1.05-5.37, p = 0.04). in summary, this report supports an independent role of cmv reactivation on relapse in patients with myeloproliferative disorders. to our knowledge, we are the first to show a significant reduction of relapse incidence in patients with mpn, even though our findings are based on a relatively small number of patients. however, this putative virus-versus-myeloproliferation effect definitely warrants further research. [p368] disclosure of conflict of interest: none. final result of fact-bmt is score ranged 0-148 point (the higher the score, the better qol). for qualitative assessment of donor-recipient relationship, the adult sibling relationship questionnaire (asqr) in polish version was used. the asrq-s consists of 47 items which are spread over eight scales designed to investigate three factors: warmth, conflict and rivalry. the questionnaires were given to both subgroups, donors and recipients of msd-hsct and the results were compared to each other. the overall result of the fact-bmt questionnaire was 109.0 ± 7.5 points, which means that the examined group generally described their qol as 'quite good'. the best results were found in functional well-being (25.6 ± 0.9), while the worst in emotional well-being (20.7 ± 0.5) dimension. statistically, the qol score was not influenced by age at hsct (p = 0.256), current age (p = 0.378) or gender (p = 0.117) of the respondents. the recipients scored highest on warm factor (62.6 ± 7.8), while donor respondents scored slightly higher rivarly (60.3 ± 6.0) than warm (45.7 ± 5.4). the second dimension scored by recipients was rivarly (40.7 ± 6.8). conflict scores were lowest, although donor respondents scored higher on these than recipient respondents (38.6 ± 5.5 in donors vs 32.9 ± 3.6 in recipients). statistical analysis revealed that the being a donor or recipient of msd-hsct determines the level of rivarly in the sibling relationship (p = 0.007) with no impact on warm and conflict dimension. health-related qol in transplanted patients is quite good. sibling donor-recipient relationship is unbalanced with recipient respondents being more likely to assess a warm relationship, while rivalry was more likely to be present among donor. further multicenter studies based on larger cohort of patients are necessary to assess sibling relationship after transplantation life experience. disclosure of conflict of interest: the authors have nothing to disclose. this work was supported by grant from poznan university of medical sciences (502-14-01104119-10398). rate of re-admission in patients undergoing allogeneic transplants from identical siblings, unrelated donors or haploidentical donors f sora 1 , s sica, l laurenti, p chiusolo, s giammarco, i innocenti, e metafuni, a corbingi and a bacigalupo 1 department of hematology, catholic university of rome hla identical siblings (sib), unrelated (ud) and family hla haploidentical donors (haplo) are currently being used for patients undergoing an allogeneic transplant (hsct) for hematologic disorders. gvhd prophylaxis is usually different, and is commonly based on a calcineurin inhibitor (cni) and methotrexate (mtx) with or without atg for sibs and uds, wheres post-transplant cyclophosphamide (pt-cy)+a cni and mycophenolate (mmf) is used for haplos. we will refer as sib, ud, haplo platform, the combination of a given donor and a given gvhd prophylaxis. the outcome of these three different platforms is usually measured in terms of gvhd, non relapse mortality (nrm) and survival. days of admission and readmissions are important in terms of morbidity, but also of costs, and are usually not reported. aim of the study: assess the duration of the first admission and the incidence of a new re-admissions, in the first 100 days after the transplant. we retrospectively analyzed 151 patients from 2012 to2016. sixtyone received peripheral blood stem cell graft from an ud, and gvhd prophylaxis with cya+mtx+atg; 54 received a peripheral stem cell graft from a sib and gvhd prophylaxis with cya +mtx; 36 patients received bone marrow hsct from haplorelated donor and pt-cy+cya+mtmf. patients characteristics are shown in table 1 . relapses were excluded from the readmission analysis. the median time from the transplantation to discharge was 25 days for ud, 27 for haplo and 21 days for sib: there was no significant difference between haplo vs ud (p = 0.6), whereas the admission of both haplo and ud was longer than sibs (po 0.01). first readmission. fiftyone patient out of 151 required of a new admission for complications after tranplant (28 out of 61 after mud (46%), 13 out of 54 (24%) using a sibling donor and 10 out of 36 using an haploidentical donor (28%)). there were significantly more re-admissions in the ud vs sib group (0.01) and a trend for more ud readmissions vs haplo (p = 0.08); siblings had the lowest number of readmissions. time to neutrophil engraftment was comparable in haplo vs ud patients (p = 0.1) and in sib vs ud (p = 0.1); the time was longer in haplo vs sibs (p o0.01). the reason to re-admitted the patients in the hospital after tranplantation was fever in 14 out of 28 (50%) new admissions in ud setting,11 out of 13 (85%) in sib and 7 out of 10 (70%) in haplo; acute gvhd was the cause for re-admission in 5 out of 28 (18%) ud, 1 out of 13 (8%) sib and none in haplo. the other causes for re-admission in the hospital were hemorragic cistitis, thoracic or abdominal pain. second re-admission. of hospitalization is registered in 10 out of 61 patients in ud (7 for aghvd and 3 fever), 2 out of 54 (4%) in sib (2 episodes of fever) and 1 out of 36 (3%) patients in haplo (1 for fever and 1 progressive disease). also for second episodes, ud grafts had significantly more admissions compared to haplo and sibs. third re-admission was recorded only in ud patients (5 out of 61-8%). this study shows a comparable duration of admission for transplant for haplo and ud patients, both significantly longer than sib grafts. the number of re-admissions is comparable in haplo vs sibs and there is a trend for lower number of re-admission as compared to uds. we interpret this outcomes with caution given the relatively small sample size and heterogeneous disease population included. future studies need to confirme our results. disclosure of conflict of interest: none. prolonged thrombocytopenia (pt) is frequent event after allogeneic haematopoietic stem cell transplantation (hsct), especically in haploidentical transplantation, which could be up to 15% according to our previous report. pt has significant negative impact on long-term outcomes, mainly due to increased non-relapse mortality. however, there are no efficious treatment. in this study, we report the preliminary results of recombinant human thrombopoietin (rhtpo) in treating this kind of patients. from 2016.7 to 2016.10, 16 patients were enrolled under the following inclusion criterion: (1) diagnosed with dpe or sfpr after allogeneic stem cell transplantion; (2) no sign of minimal residual disease or recurrence of hematological malignancy; (3) not using other tpo receptor agonist or il-11 within 1 month of enrollment. pt include delayled platelet engraftment (dpe) and secondary failure of platelet recovery (sfpr). the former was defined as failure to achieve platelet counts ⩾ 20 000/μl for 7 consecutive without transfusion until 35 days after transplantation, while the latter was defined as a decline in platelet counts below 20 000/μl for 7 consecutive days, or requiring transfusion support after achieving sustained counts without transfusions for 7 consecutive days after hsct. the prescription of rhtpo was 15 000 iu once daily for 28 days, or if patients achieve platelet ⩾ 50 000/μl for 3 consecutive days with a duration o28 days. response was defined as success of achieve platelet counts ⩾ 20 000/μl for 7 consecutive days. the response time was defined as the first day achieve response from the start of prescription. the primary end point was response rate, and the secondary end point was reponse time. a total of 16 patients were enrolled, including 7 males and 9 females. the median age was 30 (18-50) years. all patients received haploidentical transplantation. among these patients, 10 patients were dpe and 6 were sfpr. all patients received a 28-day prescription. the overall response rate was 50% (8 out of 16) in the overall population, while 60% (6 out of 10) in dpe and 33.3% (2 out of 6) in sfpr, respectively. among the 10 patients with response, the median response time was 21 (10-28) days from the first dose of rhtpo. after 4 weeks of the last dose of rhtpo, none of the responsed patient lose response. since the followup time is too short, the impact of relapse, gvhd were not reported. this single-arm preliminary result suggest that rhtpo could be a efficious method to manage pt after stem cell transplantation. however, these result need further confirmation. disclosure of conflict of interest: none. reproductive health in long-term female survivors after allogeneic hematopoietic stem cell transplantation z peric 1,2 , a samardzic 2 , n durakovic 1,2 , d tina 1,2 , l desnica 2 , r serventi-seiwerth 2 and r vrhovac 1,2 1 school of medicine, university of zagreb, zagreb, croatia and 2 university hospital centre zagreb, zagreb, croatia most female recipients of allogeneic hematopoietic stem cell transplantation (allo-hsct) suffer from premature menopause, infertility and endocrine imbalance owing to gonadal damage from myeloablative conditioning. in order to evaluate ovarian recovery and long-term endocrine complications in our institution, we performed a retrospective study of female patients who received a myeloablative allo-hsct during their reproductive age. we identified 50 female patients who underwent myeloablative allo-hsct in our institution between 1983 and 2009 and were still alive with available follow-up at the time of this study. among them, 37 patients accepted to participate and responded to a query designed for this s308 purpose. the median age of our patients at transplantation was 32 years (range: 12-47 years). they were interviewed at a median of 20 years (range: 7-33 years) post allo-hsct. the majority of patients were transplanted for a myeloid malignancy (14 acute myeloid leukemia, 7 chronic myeloid leukemia, 3 myelodysplastic syndromes and 1 chronic myelofibrosis), while 7 patients had aplastic anemia and 5 had acute lymphoblastic leukemia. all patients received bone marrow transplant from a hla-matched related donor after a myeloablative conditioning. conditioning regimen consisted of cyclophosphamide with or without total body irradiation (tbi) or in combination with busulfan. only 6 patients (16%) resumed a normal menstrual cycle after allo-hsct, without the need for hormonal replacement therapy (hrt). all these patients were transplanted for aplastic anemia and none of them received tbi in the conditioning regimen. eight patients (22%) remained amenorrheic indefinitely and never started hrt, even though most of these women were transplanted under the age of 40 years. 25% of these patients were diagnosed with osteoporosis later in life. the remaining 23 patients (62%) started hrt at a median of 11 months after allo-hsct (range: 3-27 months). however, only seven patients on hrt (30%) resumed regular menstrual cycle. a median duration of hrt therapy was 6 years (range: 3-20 years). none of the women receiving long-term hrt had severe cardiovascular complications or breast cancer. finally, five women gave birth to eight healthy children in our study population. three unassisted pregnancies were observed in two female patients after spontaneous recovery of ovarian function (both patients with aplastic anemia). the remaining two patients restored ovarian function with the use of hrt and gave birth after an assisted pregnancy (one woman gave birth to triplets after an in vitro fertilization (ivf), while other became pregnant with a donated oocyte). in spite of the fact that almost all women who undergo allo-hsct develop an ovarian failure, spontaneous recovery is sometimes possible, particularly following conditioning regimen without tbi. in patients without spontaneous recovery, hrt should be initiated promptly to prevent the early and late unwanted effects related to estrogen deficiency. moreover, recovery of normal ovarian function and even a viable pregnancy is a realistic possibility in patients placed on hrt, particularly with the use of potential therapeutic interventions as ivf or oocyte cryopreservation. it is therefore crucial to provide adapted pre-transplant counselling and recommendations for regular post-transplant follow-up in female patients who undergo allo-hsct. disclosure of conflict of interest: none. transplant-associated thrombotic microangiopathy (ta-tma) is a multifactorial disorder caused by systemic vascular endothelial injury leading to end-organ damage often involving the kidney. ta-tma occurs in up to 30% of patients undergoing hsct, and may be associated with poor outcome. although pathogenesis has not been fully clarified, activation of the complement system has been suggested to play a central role, and eculizumab, a monoclonal antibody (mab) that mediates terminal complement blockade, has shown therapeutic benefit in cases unresponsive to immunosuppression modulation. we report the case of a pediatric allogeneic hsct recipient with severe ta-tma, who did not tolerate treatment with eculizumab, now successfully treated with oms721, a novel human mab targeted to the mannan-binding lectin-associated serine protease-2 (masp-2), a molecule central to the activation of the lectin pathway of complement. a 14-year-old girl received an allogeneic hsct from a hla-compatible unrelated donor for the treatment of diamond-blackfan anemia. at month +5 of the posttransplant course, she developed progressive deterioration of renal function, microhematuria and serositis, that prompted the cyclosporine discontinuation. from month +7, the patient experienced progressive trilinear cytopenia, elevated ldh, schistocytes, undetectable haptoglobin, hypertension, increased serum creatinine, nephrotic range proteinuria, and serositis, and a diagnosis of ta-tma was established. laboratory investigations documented no abnormalities in the patient but identified a stop-codon heterozygous 43 variant in cfhr5 c.485_489dupaa (p.glu163lysfs*10) in the donor's dna. the patient was initially treated with eculizumab, but she developed acute pulmonary edema soon after eculizumab administration as the consequence of a possible reaction to the drug which had to be discontinued. the patient was subsequently treated with plasma exchange, with only limited benefit. upon ta-tma relapse at month +11, eculizumab was re-administered at lower doses, but she developed a new episode of acute pulmonary edema, preventing further eculizumab continuation. renal function progressively deteriorated and she was started on hemodialysis, reaching a 3 times weekly regimen. the patient received oms721, kindly provided on a compassionate use basis by omeros corporation, seattle, usa, starting with an iv dosing schedule. she did not experience any adverse events, and was able to tolerate the treatment well. at 2 months from oms721 initiation, she has shown improvement in ldh and haptoglobin levels, and, more importantly, her creatinine levels have normalized, allowing for complete discontinuation of hemodialysis and partial outpatient management. anti-masp-2 mab oms721 is a promising new option for the treatment of ta-tma occurring after hsct, and seems to have a safe profile also in the pediatric/adolescent setting. disclosure of conflict of interest: none. severe cytokine release syndrome after t-cell replete haploidentical transplantation with post-transplant cyclophosphamide is associated with increased death rate d taurino 1 , j mariotti 1 , b sarina 1 , l morabito 1 , s bramanti 1 , c carlo-stella 2 , a santoro 2 and l castagna 1 1 bone marrow unit, humanitas cancer center, istituto clinico humanitas, rozzano, italy and 2 hematology department, humanitas cancer center, istituto clinico humanitas, rozzano, italy haploidentical stem cell transplant (haplo-sct) represents a potential curative strategy for several hematological malignancies. haplo-sct may represent an alternative option when a hla matched-identical sibling (hlaid) or a matched unrelated donor (mud) is not available. the syndrome of systemic inflammation, characterized by fevers, vascular leak, hypotension, and respiratory and renal insufficiency, in the context of elevated inflammatory markers and cytokine levels was previously described as cytokine-release syndrome (crs)1. recent publications have elicited the occurrence of crs after haploidentical transplant, especially after peripheral blood stem cell graft, and its high-related mortality 2-4. here we report the experience of our institution with crs after haplo-sct. between march 2014 and october 2016, we treated 29 patients with haplo-sct with a graft source represented by peripheral blood stem cells. we monitored the occurrence of crs symptoms and utilize a previously described grading system 1, 4 starting from day 0, up to day 14 after transplant. severe crs is defined as grade 3 or higher because it requires aggressive interventions and is characterized by oxygen requirement ⩾ 40%, 43 l nasal cannula, hypotension requiring high dose or multiple vasopressors, grade 3 renal toxicity or grade 4 transaminitis. other characteristics comprise newonset altered mental status without other explanation and new cardiomyopathy without wall motion abnormality. results: 27 out of 29 patients experienced fever between day 0 and day 14 post transplant with most episodes (24 patients) occurring between day 0 and day 4. on day 7 after transplant, 3 patients had grade 3, 6 grade 2 and 19 grade 0 crs, respectively. by day 14 post haplo-sct, 5 patients had crs grade 43, 5 grade 2 and 1 grade 1. overall, the incidence of crs any grade was 43% (95% ci 21-55%). 1 year after transplant 8 patients died because of non-relapse related side effects. with a median follow-up for alive subjects of 10 months, 1-year overall survival (os) was 64% (95% ci: 42-80%). 1-year os was 73% for patients with a crs 3 on day 7 (p = 0.007). conclusions: crs represent an important complication after haplo-sct. crs score 43 on day 7 after hst apparently correlates with long-term survival. better strategies need to be implemented for an early detection of severe crs in order to develop effective treatments, such as tocilzumab, for this important side effect. further studies are ongoing at our institution in order to correlate post-haplo crs with graft composition, laboratory parameters and immunereconstitution. hematopoietic cell transplantation (hct) is associated with significant morbidity that impairs survivor's sexual functioning. however, few studies have specifically addressed it. thus, we examined (1) sexual functioning during the first year post hct, (2) differences between allogeneic and autologous hct, and (3) whether demographic, clinical and psychological variables were associated with sexual functioning. this is a prospective multicenter study assessing patients before hct, at day 90, 180 and 360. sexual functioning was assessed with the changes in sexual functioning questionnaire, which yields a total score, along with scores for the dimensions of frequency, pleasure, orgasm, desire and arousal. anxiety and depression (hads) were also collected. we included 159 consecutive hct recipients: 91 (53%) were men, with a median age of 51 years (range: 18-71), 93 (58%) received an allogeneic hct and 66 (42%) an autologous hct. sexual functioning was significantly affected: 86% of the sample reported impairment at pre-hct, 91% at day 90, 87% at day 180 and 86% at day 360. mixed model analysis indicated that sexual functioning was not associated with time from hct (p = 0.802) or hct type (p = 0.538). however, there was an interaction between these two variables (p = 0.022), particularly at day 90, since sexual functioning had improved among autologous survivors and worsened among allogeneic survivors leading to nonsignificant differences between hct type (p = 0.082). frequency of sexual functioning improved during the study period (po 0.001), and no differences were observed between hct type (p = 0.111). again, there was a borderline interaction between post-hct time and hct type (p = 0.059), since autologous survivors reached higher frequencies than allogeneic survivors, with significant differences at day 90 (p = 0.003). pleasure significantly improved during the study period (p = 0.035), without observing differences between hct groups (p = 0.121). again, however, autologous survivors reported significant improvements in pleasure at day 90 (p o0.001) and a trend at day 180 (p = .093) when compared with allogeneic survivors. orgasm did not improve during the study period (p = 0.837), and no differences were obtained between hct groups (p = 0.413). allogeneic survivors had higher orgasm scores at pre-hct (p = 0.020), which worsened during the study period, particularly at day 90 (p = 0.028). in contrast, autologous survivors reported improvements in orgasm by day 90. non significant results were obtained in the sphere of sexual desire and arousal (p40.1). bivariate analyses indicated that women, older age and depression were associated with impaired sexual functioning at all assessed time-points (p o0.05). chronic graft-versus-host disease (gvhd) was associated with worse sexual functioning at day 180 (p = 0.045) and 360 (p = 0.020). no differences were obtained when considering diagnosis, having received previous hct, intensity of the conditioning regimen and whether patients lived with a partner (p40.05). stepwise multivariate regression analyses indicated that gender (p = 0.001) and extensive chronic gvhd (p = 0.012) predicted for worse sexual functioning at day 360. sexual functioning should be routinely assessed and considered for eventual targeted intervention in both hct populations, particularly during the first year post transplant. additional clinical efforts should focus on patients more vulnerable to impaired sexual functioning. disclosure of conflict of interest: none. significant improvement of qol by using atg as part of the conditioning regimen followed by hla-identical peripheral stem cell transplantation in acute leukemia patients. results from a prospective, randomized phase iii study (atg family study) b francesca 1 , s carlos 2 , w christine 3 , s mariarosaria 1 , p massimo 4 , s carmine 5 , m giuseppe 6 , b wolfgang 7 , cm angelo 8 , p francesca 9 , m nicola 10 cgvhd is a major complication after allogeneic sct. we previously demonstrated that the addition of anti-tlymphocyte globulin (atlg neovii, formerly atg-fresenius) to a myeloablative preparing regimen followed by peripheralblood sct from an hla-identical sibling for pts with acute leukemia resulted in a significant reduction of cgvhd, without increasing the risk of relapse or infection. 1 the study protocol included quality of life (qol) questionnaires (eortc qlq-30 and hdc29) before and after sct (day+ 100, 6, 12 and 24 mos). the qlq-c30 includes a global qol scale, five functional scales (physical, role, emotional, cognitive and social function) and nine symptom scales (fatigue, nausea-vomiting, pain, dyspnea, insomnia, appetite loss, constipation, diarrhea and financial problems). the qlq-hdc29 includes six multi-item scales and eight single items that describe impairment through highdose treatment. mixed models for repeated measures (mmrm) and linear mixed models (lmm) were used to analyze the time courses and the slopes of the outcomes depending on treatment arm (atg vs non atg), age, country, sex, and cgvhd. (clinicaltrials.gov: nct00678275). pts with a qol form returned decreased by visit (70% pre-sct, 45% at 100 days and 29% at 24 mos after sct). forty-nine percent in the atg and 60% in non atg arm provided any qol forms after sct. return of any post-sct qol forms by country was 68% for germany, 62% for italy and 25% for spain. pts with cgvhd were more likely to return qol questionnaires (66% vs 45% w/out cgvhd) while neither age nor sex were closely associated with qol form return. the majority of subscales of the qlq-30 indicated an average improvement of qol and reduction of symptoms over time, notably in the atg group. in an mmrm model controlling for country, age, sex and cgvhd, pts treated with atg showed significantly more pronounced improvement of global health status/qol over time compared to non-atlg (p = 0.02), with a treatment group difference of 2.8 ± 3.9 points (marginal mean ± sem) at day 100 and increasing to 10.5 ± 5.3 points at month 24 favoring atg. significant superiority of atg (po0.05) was also observed for four of the five functional scales as well as for several symptom scales scores including appetite loss, insomnia, nausea-vomiting and dyspnea. for the qlq-hdc29, significant treatment effects favoring atg were observed for gi side effects and impact on family. lmm analyses of qol by country indicate that patients from italy generally gave more favorable ratings for all functional scales and lower scores for most symptom scales than those from germany while the time courses and slopes were similar for most scales. these results underline the importance of the habits and cultural environment which are distinctive of each country. males and females showed similar qol ratings at pre-and post-sct. patients up to 34 years tended to provide more favorable functional ratings and less severe symptom scores than older patients and also showed more pronounced improvements of qol. pts receiving atg in a randomized study have significantly less cgvhd and improved grfs, resulting in an improved qol regarding global health status and most functional scales. notably, we also observed a significant difference in qol assessment between pts from germany and italy. oral mucositis (om) is a well-known side effect of high-dose chemotherapy and radiotherapy in hematological patients, which influences the health-related quality of life (hrqol) of the affected patients. the purpose of this study is to demonstrate the impact of om on hrqol in stem cell transplanted patients in routine care. prospective, noninterventional single-center observational study was performed at a german university hospital. inpatient allogenic and autologous stem cell transplant patients ⩾ 18 years with high-dose chemotherapy. om was assessed with the who oral toxicity scale, pain using the numeric rating scale (nrs) and the performance status with the ecog score. hrqol was captured with the eortc qlq-c30 and the qlq-oh15 questionnaires (3 days before hematopoietic stem cell transplantation (hsct); 7 days after hsct; 14 days after hsct). statistical significance was assumed p o0.05. a total of 20 patients (11 autologous and 9 allogenic) was included from august to december 2016. a total of 11 (55%) patients developed om. of these 11 patients, 3 suffered from grade 1, 3 from grade 2, 4 from grade 3 and 1 from grade 4 om. three days before hsct, the mean qol of all 20 patients was 45%, the mean qlq-c30 summary score 60.3% and the mean oral health related quality of life 82.3%. most of the patients suffered from om around day 7 after hsct. after 7 days, quality of life (qol) was higher in patients with no om (32.3%) than in patients with om (30.0%). the qlq-c30 summary score was significantly (p = 0.004) lower in patients affected by om (43.1%) than in patients who did not develop an om (65.8%). om affected patients had significantly more limitations in emotional (no om 84.4%; om 56.7%; p = 0.038) and cognitive functioning (no om 93.8%; om 51.7%; p = 0.002) and in fatigue (no om 58.3%; om 80%; p = 0.045), pain (no om 14.6%; om 55%; p = 0.005) and insomnia (no om 12.5%; om 60%; p = 0.001), they had a significantly higher rate of problems. oral health-related quality of life was significantly (p = 0.003) lower in patients who were affected by om (57.9%) compared to patients who did not develop an om (83.9%) and patients with an om had significantly more problems with a sore mouth (no om 8.3%; om 46.7%; p = 0.028), sticky saliva (no om 29.2%; om 63.3%; p = 0.045) and sensitive mouth (no om 8.3%; om 56.7%; p = 0.003). after 14 days, qol was higher in patients with no om (47.9%) compared to patients with om (46.7%). patients with no development of om had a higher but not significant physical functioning, cognitive functioning and social functioning. patients affected by om had higher levels of fatigue and pain and more often suffered from a sore mouth. oral health-related quality of life was higher in patients without om (75%) compared to patients with om (68.3%). comparing all assessed days patients with om had higher scores on the nrs increasing with a higher grade of om (mean nrs score grade 1; 2-2.3, grade 3; 4-4.5), the ecog index was higher in om affected patients during episodes with om (mean ecog score-2.3) compared to episodes without om (mean ecog score-1.9). om has a major impact on the hrqol, health-related symptoms and functionality. in the future, there has to be a higher awareness from clinicians and patients of the prevention, assessment und causes of om. more research has to be initiated to ease the symptomatology and to improve patients' quality of life. disclosure of conflict of interest: none. according to ebmt data, chronic gvhd (cgvhd) occurs in 40-70% of all patients after allogenic hematopoietic stem cell transplantation (allo-hsct). pulmonary cgvhd is the most severe form. but it is very unpredictable to use due to the fact that many factors can affect it (breath-dependent; need experience not only from physician but from patients also and so on). here we show that routine software-based image analysis algorithm can provide data that highly correlated with pft results and have excellent sensitivity and specificity in pulmonary cgvhd diagnosis. we blindly analyzed 120 ct scans (made without additional expiration) in 24 allo-hsct patients at different time points. all scans were performed on ct scanner aquilion 64, toshiba, japan. according to hounsfield units (hu) definition, − 1000 hu ('air') have approximate density at 0 g/ml; 0 hu ('water') have approximate density at 1 g/ml. the analysis of ct scans (heart, vessels and bronchi were excluded from analysis) was based on automated software conversion (image-analysis algorithm providing by multivox software, msu, moscow, russia) of each ct-image pixel from hu to density units (g/ml). pft were performed using standard procedures at same as ct scans time points (spirolab iii, italy). all patients with hematological malignancies (acute leukemia-17, aplastic anemia-1, chronic myeloid leukemia-1, t-cell lymphoma-1, chronic myeloproliferative disorder-1, myelodysplastic syndrome-3) were transplanted in national research center for hematology between 2012 and 2015. median of age was 41.5 years (range: 19-60 years). eight patients were males, 16-females. seventeen received reduced-intensity and 7-myeloablative conditioning regimen. graft from match unrelated donor (mud) were used in 17 cases, 'mismatch' mud-2, match related donor (mrd)-9, 'mismatch' mrd-1. median follow-up is 44.8 months. we analyzed lung tissue experimental density in patients before and after allo-hsct at different time points. median of lung tissue experimental density were 0.178 (interquartile range (iqr), 0.148-0.186), 0.17 (iqr, 0.153-0.185) and 0.147 (iqr, 0.131-0.172) for patients before allo-hsct, after allo-hsct with cgvhd (except pulmonary cgvhd) and with pulmonary cgvhd, respectively. mann-whitney u test was used to reveal significant differences between these groups (see figure 1 ). also, we found strong correlation between pft and experimental density (spearman's correlation coefficient r = 0.537) (see figure 2 ). forty-five ct scans of patients with pulmonary cgvhd and 59 ct scans of patients without pulmonary cgvhd at the time of ct scan as control subjects were included in roc analysis to assess the clinical values of our model. we generated an roc curve and found that the area under the curve (auc) was 0.77 (95% ci, 0.67-0. 86) (p o0.0001) (figure 3 ). standard ct scan is presented as easy to perform, breath-independent, standardized and wide spread method for every patient after allo-hsct. it can be performed many times during all their post-hsct life. ct scan with a simple software analysis allows to select a group with high probability of pulmonary cgvhd and who can be suspected of cgvhd development by this method with sensitivity-60% and specificity-81.36%. disclosure of conflict of interest: none. the choice of effectiveness criteria affects conclusions of economic evaluation of newer allogeneic bone marrow transplantation modalities :example based on a randomised multicenter trial comparing two reduced intensity conditioning regimen (flu-bu-atg) vs (flu-tbi) for matched related allo-sct s le corroller*, anne-gaelle 1 , c siani 2,1 , r tabrizi a re-evaluation of the per-diem hospitalization cost was performed in 2016 and included the utilization of hospital technical facilities and a more precise estimation of overheads costs. we performed three separated cost-effectiveness analysis, using, respectively, pfs, os and qaly as end point. when using pfs as effectiveness, relapse costs were not included. weighting coefficients for the cost per qaly analysis came from the literature. at 5 years, os and pfs were 41% and 29%, respectively, and did not statistically differ between groups. the mean total cost per patient was not statistically different between groups (111725€ for fba vs 98316€ for ftbi, ns). using pfs as end point, the icer of fba compared to ftbi is 35 034€ per year of pfs gained. using os, the icer became non-statistically significant, signifying that when handling uncertainty, no difference in term of cost-effectiveness was observed between fba and ftbi with os as end point. using s312 qaly, the icer was statistically ns again, showing no advantage in terms of cost per qaly of one conditioning regimen over the other. this result was obtained both considering three weighted health states (dfs, progression and death) and four weighted health states (dfs without gvhd, dfs with gvhd, progression and death) for the qaly calculation. using os and qaly, the two conditioning regimens were not different in terms of cost-effectiveness, while fba may be considered as more cost-effective using pfs as effectiveness criterion. using intermediary end points allows economic evaluation to be available earlier in the life cycle of an innovation. however, it implies strong hypotheses about the predictive value of the pfs over the os. longer period evaluation and qaly may reverse preliminary results. this situation is likely to exist in the hematology setting where alternatives between chances of cure and toxicities of treatment are often observed. research about allogeneic sct modalities is archetypical of such situations and decisions makers should be aware of the necessity of further economic re-evaluation along the development and diffusion process of innovative treatments. disclosure of conflict of interest: none. the impact of corticosteroids prophylaxis for the engraftment syndrome incidence during autologous stem cell transplantation in multiple myeloma and amyloidosis the es is a complication of asct characterized by an inflammatory response during peripheral blood recovery. the standard treatment is based on corticosteroid therapy. the incidence of es after asct increases in chemotherapy lowtreated patients such as those with multiple myeloma (mm) and amyloidosis (al).moreover, the es is associated with the use of g-csf after infusion of stem cells. therefore, our bmt team does not use g-csf since 2009 in this population reducing the incidence and severity of es. therefore, it makes sense to use low-dose prednisone to prevent this complication. in this study, we compared two consecutive cohorts of patients with mm/al that performed an asct while evaluating the corticosteroids prophylaxis (cp) in the es incidence and its effect on other clinical variables. we included 120 patients with mm (n = 96; 80%) and al (n = 24; 20%) that performed an asct between january 2011 and november 2016 in a single institution. the median age (range) was 56.7 (33.8-71.7) years. during the procedure, all patients received melphalan as conditioning chemotherapy and none received g-csf. fortyseven patients (39%) received intravenous methylprednisolone or oral prednisone 0.5 mg/kg/day from day +7 until reaching a neutrophil count ⩾ 500 per mm 3 for 3 consecutive days (cs group), and 73 (61%) patients did not receive corticosteroids (noncs group). the characteristics of patients in both groups (age, gender, status performance and previous treatment were similar (p40.05)). the cs group, received higher doses of cd34 + than the noncs group (3.68 × 10 6 /kg vs 2.92 × 10 6 /kg, respectively, p = 0.006). the median (range) days of neutropenia ( o 500 per mm 3 ) was 9 (4-25) days. es was diagnosed in 43 (36%) patients. fifty-seven (48%) patients had fever, showing infectious focus or microbiological isolation in 24 (20%) cases, whereas the incidence of grade iii-iv oral mucositis and relevant gastrointestinal toxicity was 8% and 2.2%, respectively. the complete analysis between groups (cs versus noncs) for the whole series and in the mm/al subgroups is detailed in table 1 . the administration of corticosteroids as prophylaxis seems to reduce the incidence of es in the overall series or in the analysis for the subgroups (mm and al) without increasing infection. [p381] disclosure of conflict of interest: none. chronic gvhd is a condition that might occur after allo-hsct and has been proved to impair long-term survival and quality of life of patients. graft failure is also a major potential complication for patients undergoing transplant for an aplastic anemia/bone marrow failure (bmf). partial in vivo t-cell depletion, employing anti-thymocyte globulin (atg) during conditioning, has been proved to successfully prevent the mentioned potentially life-threatening complications in highrisk patients. however, the possibility of developing epstein-barr virus (ebv)-induced post-transplant lymphoproliferative disorders (ptlp) has been a limiting factor to use atg. this study includes the last 100 pts with a minimum follow-up of 100 days, who underwent allo-hsct in our center (november 2014-august 2016). a total of 56 pts were male and 44 female. median age was 53 years (range: 7-69). baseline diseases were: acute leukemias (54), lymphoproliferative disorders (17), myelodysplastic syndromes (12), chronic myeloproliferative diseases (7), multiple myeloma (5) and bone marrow failures (5) . donor was unrelated in 57 cases, and related in 43 (including 18 haplo-identical). conditioning regimen was: busulphan-based (70), melphalan-based (13), tbi-based (8) and others (9). progenitors source was pb in 89 and bm in 11. patient/donor ebv pre-transplant serology was: +/+ in 94 cases, +/ − in 5 and − /+ in 1. rabbit atg (thymoglobuline) was employed in 58 cases: 54 at 4.5-6 mg/kg (urd transplants) (low dose), and 4 cases at 7.5 mg/kg (all of them pts with bmf) (intermediate dose). family donor (including haplo-identical) transplants of those pts with diagnosis different from bmf (42 cases) did not receive atg. systematic monitoring of ebv using quantitative pcr was employed. ebv reactivation was considered when dnaemia was superior to 1000 copies per ml. a total of 5 pts presented ebv reactivation: 0/42 (0%) in cases without atg, 4/54 (7.4%) in cases with low-dose atg and 1/4 (25%) in cases with intermediate-dose atg. median time of reactivation was the day +34 (range: +32 to +151). there was one single case of ebv-induced ptld which belonged to the intermediate-dose atg group. all cases (including the one with ptlp) were successfully treated with rituximab at 375 mg/m 2 /week. median number of doses employed were 3 (range: [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . mortality due to ebv was 0% in our series. limited donor availability in the form of either matchedrelated or unrelated donors drew attention to haplo-hct. donors of haplo-hct shares an exact haplotype with the recipient but is mismatched for hla genes on the unshared haplotype. most studies have shown promising results in terms of graft success and survival. in this study our aim is to present the early and late outcome of our haplo-hct patients. between 2012 and 2016, we retrospectively evaluated 16 haplo-hct in terms of post-transplant outcome, survival and complications who diagnosed and followed in our center. the median age of patients was 37 (range: 19-61), 10 (63%) of them were male recipients. the patient characteristics were given in table 1 . thirteen patients (81%) had pre-transplant active disease. neutrophil and platelet engraftment was achieved in 7 patients (44%) at a median day of 21 (range: 16-40) and 34 (range: 13-116). eight of 16 patients (50%) died within 1 month after transplant because of sepsis without achieving engraftment. haplo-hct is the second transplant in four of 16 patients (25%): 1 patient relapsed after full-matched related transplant, 1 patient relapsed after 9/10 matched unrelated transplant, 1 patient had engraftment failure after full-matched unrelated transplant, 1 patient underwent haplo-hct in another center, followed in remission for 2 years and relapsed. acute graft vs host disase (agvhd) was diagnosed in 6 patients (37%), whereas chronic gvhd in 4 patients (25%). four patients were relapsed (25%) during follow-up with median rfs of 6 months. three patient had bk virus-positive hemorrhagic cystitis (18%). the distribution of infections is shown in figure, viral infections were detected later than fungal and bacterial infections. previous history of invasive pulmoner aspergillosis was detected in 5 of the patients (31%) (2 of them were re-transplanted) and received secondary prophylaxis. overall survival (os) of 6 months and 1 year were 25% and 18%, respectively. the choice between alternative graft sources depends on the urgency of the transplant on each institutional preference. higher complication and infection rates in addition to decreased survival compared with previous studies since our patient population consisted of refractory patients with comorbidities. preferable patient profiles undergoing haplo-hct may have better outcomes. disclosure of conflict of interest: none. the third month risk factor score: detection of disease at day +100 of allogeneic stem cell transplantation is the most important risk factor of worse prognosis m celis 1 , c fernández 1 , l yáñez 1,2 , a bermúdez 1,2 , a insunza 1 , m colorado 1 , m lópez-duarte 1 , i romón 1 , s garcía-ávila 1 , a cabero 1 , a casado 1 , m sánchez-escamilla 1 , c richard 1 and e conde 1,2 1 hematology department, hospital universitario marqués de valdecilla and 2 university of cantabria before allogeneic stem cell transplant (sct), several index can provide prognostic information (ebmt risk score and hcti score). however, there is scarce data for the impact of the procedure during the first 100 days of transplant, in which opportunistic infections and the acute graft versus host disease (gvhd) can induce harmful effects. our purpose is to create a risk factor score, measured at day +100 post sct, to give information about the prognosis of the patient. we retrospectively analyzed seven clinical (disease, fungal and cmv infection, acute gvhd, treatment with corticosteroids, karnosfsky status and length of hospitalization) and eight analytical (related to immune status, liver and lung function, nutritional status, iron overload and platelet count) risk factors in 131 patients who underwent sct in our center between 2011 and 2015 and were alive at day +100. data were collected as categorical variables and compared by χ 2 -test. significant variables (p o0.05) were evaluated in a multivariate logistic regression model. those who maintained statistical significance were then assigned a point value calculated with their β-coefficient. summation of the points resulted in a weighted risk score. median age was 51 years (range: 4-73) and 80 were males (61.1%). the most frequent disease was aml, 49 patients (37.4%). the conditioning regimen was myeloablative in 97 patients (74%) and bone marrow was the principal stem cell source (71%). donor was mrd in 39 (29.8%), mud in 51 (38.9%) and mmd in 41 (31.3%). the median followup was 26 months (range: 3-66). the univariant model identified five prognostic variables: detection of disease by molecular, cytogenetic or flow cytometry asses in leukemias, myelodisplastic syndrome and multiple myeloma or image (ct scan ± pet) in lymphoma, dose of corticosteroids ⩾ 0.5 mg/kg/ day, ferritin 42500 ng/ml, albumin o3.0 g/dl and platelet o100 000 per mm 3 . table 1 shows variables evaluated. in the multivariate model, the detection of disease (hr 3.80, 95% ci 1.90-7.61, p 2500 ng/ml (hr 2.39, 95% ci 1.30-4.42, p = 0.005), and platelet o100 000 per mm 3 (hr 2.06, 95% ci 1.11-3.89, p = 0.022) were associated with higher risk of death and according with their-coefficient 4, 2 and 2 points were, respectively, assigned. the third month risk score (tmrs) was calculated in all patients and they were stratified into three groups: low risk of death (a, 0-2 points), intermediate risk (b, 4 points) and high risk (c, ⩾ 6 points). at 2 years post sct, the estimated overall survival according with the tmrs was 78.9% ± 4.3 in group a, 31.0% ± 8.6 in group b and 36.4% ± 14.5 in group c, po 0.001. although the harmful effect of the first 3 months of transplant can impact in the survival, the detection of disease at day +100 is the most determinant risk factor of death. this fact gives us the need of transplant in the best response and, in those who cannot, to plan promptly rescue strategies. the next objective is to confirm our risk score in a validation group. disclosure of conflict of interest: none. recombinant human soluble thrombomoduline alpha (rhtm) is a novel anticoagulant agent and approved for disseminated intravascular coagulation in japan. the aim of the study is to evaluate the therapeutic potential of rhtm for sinusoidal obstructive syndrome/hepatic veno-occlusive disease (sos/ vod). we retrospectively studied 878 times of allogeneic hematopoietic cell transplantation in toranomon hospital from june 2008 to june 2015. we extracted the patients who used rhtm for dic and satisfied the diagnostic criteria of sos/ vod around the same time, because the use of rhtm for sos/ vod alone is off-label. data on the patients who used rhtm for 43 days within 100 days after transplantation were analyzed. the patients who were already treated with rhtm before the emergence of the first symptom or sign of sos/vod, and who started rhtm over 30 days after the emergence of the first symptom or sign of sos/vod, were excluded from the [p383] analysis. to diagnose classical sos/vod (⩽21 days after transplantation), we used two classical criteria of the modified seattle and the baltimore. for late-onset sos/vod (4day 22 of transplantation), we used the criteria of ebmt. we defined as severe sos/vod, if the patients had renal (cr ⩾ 2 times of baseline), respiratory (spo2 ⩽ 90% or the need for positive pressure) or central nervous system failure until 2 weeks after the diagnosis of sos/vod. complete response (cr) was defined as the resolution of all the symptoms and the signs in sos/vod diagnostic criteria. a total of 39 patients were extracted. the median age was 60 years (range: 27-72) and 27 patients (69%) was male. donor cell sources were ucb (n = 34) and ubm (n = 5). most of the prophylaxis regimen was the combination of ursodeoxycholic acid and dalteparin in 36 patients (92%). classical sos/vod was diagnosed in 3 (8%) and 8 patients (21%) by the criteria of the modified seattle and the baltimore at the median day of 14 (range: 11-14) and 16 (range: 11-20), respectively. twenty-eight patients (72%) were diagnosed as late-onset sos/vod at the median day of 44 (range: 22-89). severe sos/vod developed in 33 patients (85%) (renal, n = 32; respiratory, n = 7; central nervous system, n = 15). the elevation of transaminase was observed in 18 patients (46%). the median interval from the emergence of the first symptom or signs of sos/vod to rhtm administration was 7 days (range: 0-23). the median duration of rhtm use was 11 days (range: 3-63). rhtm was used alone in 20 patients (51%), in combination with dalteparin in 7 (18%), with atiii in 5 (13%), with dalteparin and atiii in 3 (8%), with atiii and pge1 in 2 (5%), and with pge1 in 2 (5%). corticosteroid was used concomitantly in 32 patients (82%). finally, 13 patients achieved cr of sos/vod. the cumulative incidence of cr of sos/vod was 33.3 % at 1 year after the administration of rhtm (95% confidence interval, 18.5-48.9%). the median interval from the administration of rhtm to cr of sos/vod was 51 days (range: 6-141). at 1 year after transplantation, overall survival was 25.6% (95% confidence interval, 13.3-69.9%). from the administration of rhtm to 2 weeks after the cessation of rhtm, 23 hemorrhagic adverse events were observed. seven out of 23 events were at grade 3-5, and 5 out of 7 events were fatal (intra-abdominal in 2, gastrointestinal in 1, lung in 1 and brain in 1). we concluded that rhtm had a therapeutic potential for sos/vod. disclosure of conflict of interest: none. thrombopoietin receptor agonists for delayed and prolonged clinically-relevant severe thrombocytopenia after allogeneic hematopoietic stem cell transplantation v bosch vilaseca 1 , i garcía cadenas 1 , e roldán 2 , s novelli 1 , r martino 1 , p barba 2 , a esquirol, l díaz polo 1 , g orti 2 , d valcárcel 2 and j sierra 1 1 hematology department, hospital de sant pau, barcelona, spain and 2 hematology department, hospital de la vall d'hebron, barcelona, spain persistent thrombocytopenia is a common complication after allogeneic stem cell transplantation (allosct), which dramatically increases the patients' dependence on hospital-based healthcare. thrombopoietin receptor agonists (tpoa) increase platelet counts in other clinical settings; however, the experience regarding their use after allosct is limited. we retrospectively evaluated tpoa efficacy in 15 consecutive adult allosct recipients who received tpoa as a compassionate use for severe thrombocytopenia post-engraftment. five patients (33%) had primary and prolonged failure of platelet recovery, while 10 had secondary thrombocytopenia: in seven of these cases, gvhd and/or a viral infection were the 'attributed' cause, while three were classified as post-allosct itp. all 15 patients were dependent on platelet transfusions (median: 2 times per week, range 1-5), with severe bleeding episodes in nine cases (60%) before tpoa onset. tpoa was started at a median of 160 days after allosct (range: 65-1041). romiplostim was used in 13 (87%) cases. the median starting dose was 2 μg/kg once a week (range: 1-5 μg/kg), while the final dose identified as most beneficial was 4 μg/kg (range 1-10 μg/kg). eltrombopag was used in 2 cases (13%), with an initial dose of 50 mg daily; while the final doses were 100 and 150 mg daily. overall, 9/15 patients responded to tpoa therapy (defined as a stable platelet recovery to ⩾ 30 000/μl without transfusion support). the 180-day cumulative incidence of successful platelet recovery to ⩾ 30 000/μl and ⩾ 50 000/μl was 70% (95% ci, 67-73%) and 56% (95% ci, 42-69%), respectively, which were reached at a median of 21 and 48 days from start of therapy. five of the 9 patients (56%) with severe bleeding at onset responded to tpoa (4 of them without further hemorrhages) at a median of 93 days (range: 1-299). at a median follow-up of 511 days from start of therapy, three patients who responded continue tpoa treatment, while four other responders were able to discontinue it without recurrence of thrombocytopenia. among these 4 patients, s317 the median total duration of treatment was 201 days (range: 113-300). one patient lost his response within 5 months after tpoa onset when he developed thrombotic microangiopathy associated with progressive gvhd. the remaining responder experienced disease relapse on day +88 after allosct. among the 6 non-responders, 1 had leukemia relapse during tpoa treatment, 1 switched from romiplostim to eltrombopag without success and the remaining cases had active severe infections at tpoa onset (2 hemorrhagic cystitis and 1 cmv colitis) or non-controllable intestinal bleeding due to progressive gvhd. tpoa were well tolerated, with only 2 patients showing adverse events (grade 3 liver toxicity and grade 3 fatigue), which did not lead to any change in therapy. six patients (40%) underwent follow-up bone marrow biopsies that did not display any increase in marrow fibrosis, including the 1 patient who had myelofibrosis prior to allosct. although six patients in the study had active gvhd when tpoa was started, no patients showed worsening of gvhd. our results support the safety and efficacy of tpoa for the treatment of persistent thrombocytopenia in allosct recipients. further studies should compare the efficacy of romiplostim and eltrombopag and identify surrogate clinical and laboratory variables that are predictive of response to one (or both) of these tpoa. disclosure of conflict of interest: none. . clinical response in both groups was defined as improvement of organ function (no neurological residues; normalization of kidney function) and independence of red blood cell and platelet transfusions. results: the median time of ta-tma onset was 8.2 months (0.5-32.8) after hsct. thirty-five of 37 patients (95%) were under treatment with calcineurin-inhibitors or sirolimus at the time the ta-tma occurred. in all cases, the immunosuppressive drug was stopped promptly. in 28 patients, classical treatment was the primary therapy with a response rate of 52% (including four patients who switched to ec), whereas the response rate to ec treatment was significantly higher with 92% (p = 0.017). all patients receiving ec showed sufficient blockade of the terminal complement pathway after the second ec application (ch50 o 10%). despite the increased response rate for ec therapy, there was no difference seen between these two groups according to overall survival in weeks: classical treatment 9 (95% ci 0-19.3) vs ec treatment 14.9 (95% ci 6.9-22.7) p = 0.84. the main cause of death differed significantly between this two treatment approaches with a therapy-related mortality due to infection with 70% in the ec group during tma therapy and none seen in the classical treatment group (p = 0.001). progressive gvhd was identified as an adverse prognostic factor in both groups (p = 0.048 and p = 0.005). conclusion: in our analysis, we show that ec shows a significant higher response rate in severe ta-tma patients compared to the classical treatment approach. however, in both groups the outcome remained very poor. since most patients presented with advanced, severe ta-tma, especially in the ec group, we hypothesize that earlier diagnosis and treatment of ta-tma and more effective prevention and treatment of infections will improve the outcome of patients with this complication. however, randomized studies are essential for comparison of these two treatment strategies to identify patient groups that benefit from a treatment with ec. disclosure of conflict of interest: none. tocilizumab as an effective treatment in cytokine release syndrome as an early peri-transplant complications in patients subjected to allogeneic stem cell transplantationproinfammatory/autoimmune patient/donor hla haplotype life-threatening early allogeneic hsct complication risk factor hypothesis m-g patrycja 1,2 , p-j beata 1,2 , s marcin 1 , k ksenia 1 , s-k agnieszka 1 and sb aleksander 1,2 1 bone marrow transplantation unit, department of haematology, krakow university hospital and 2 jagiellonian university collegium medicum cytokine release syndrome (crs) is classical complication of car t cells therapy, but also can be connected with early peritransplant complications in patients subjected to allogeneic stem cell transplantation. it can be connected with atg infusion, but also with inflammatory response during periengraftmetnt period (pre-engraftment syndrome and engraftment syndrome) and septic infections. severity of these complication can differ depending on patient's performance status and therapeutic options from just observation and vigilance to mechanical ventilation need. we would like to present small patient series (n = 5) subjected to msd (n = 1) and mud (n = 4) with early transplant-related complications treated with combination of steroids (dexamethason) and tocilizumab. in two of them, tocilizumab was used after second dose of atg. both patients present hypotonia with decreased urine output, prompt increase of creatinine level and presence of acute inflammatory parameters crp, beta2microglonulin and procalcitonin level, fluid retention and decreased oxygen saturation. in another one patient, these symptoms were connected with pbsc infusion from unrelated donor. in later two patients, we observe almost the same clinical presentation in preengraftment phase. in every of patients infection was ruled out-blood cultures were negative. all these patients were treated with tocilizumab in a single dose of 8 mg/kg. in all patients, we observed prompt response-normalization of clinical state, renal function, oxygen saturation and decrease of inflammatory factors-crp, procalcitonin and beta2microglobuline. discussion: crs is a rare complication connected with early phase of allogeneic stem cell transplantation. there were no results of treatment with steroids, reduction of a dose of cyclosporine a according to decreased renal function, but all patient completely/fully recovered after single dose tocilizumab treatment. all our patients were subjected to reduced intensity protocols, what might be a risk factor to develop crs because non complete depletion of the patient origin monocytes/macrophages active population. we also analyzed other factor connected with crs in early peritransplant period finding possible connection with s318 proinflammatory hla phenotype. it was obvious in the patient one our patients with peri-engraftment phase crs-he was diagnosed previously with rheumatoid arthritis b27 pos, dr4. in three of five, we have found sle predisposition in hla phenotype (drb1*1501/dqb1*0602 or drb1*0301/ dqb1*0201), in later one-ra associated hla antigen drb1 0101.these patients were analyzed correlating with historical cohort of additional five patients with mortal and another three with very severe early peri-transplant complications and in all we have found the same 'sle or ra hla phenotype'. because small number of analyzed patients and documented high frequency of these haplotype in population, this is still an opened question is proinflammatory/autoimmune hla phenotype connected pathogenically with predisposition to develop severe transplant complications and are we able to treat all these patients with combination of steroids with tocilizumab. further analysis is needed. disclosure of conflict of interest: none. transplant-associated thrombotic microangiopathy (ta-tma) is a severe complication post haematopoietic cell transplantation (hct) leading to high mortality rates. however, outstanding questions regarding its diagnosis, pathophysiology and treatment remain in the literature. recent studies suggest evidence of complement activation, implicating that complement inhibition may be an effective alternative treatment strategy in refractory patients. therefore, we hypothesized that increased complement activation can be detected in ta-tma patients using a functional assay, the modified ham test. we enrolled consecutive patients with ta-tma according to the international working group criteria from january 2015 to june 2016. as controls, we studied patients with graft-versushost-disease (gvhd). complement activation was detected using the modified ham test, a cell proliferation assay based on the susceptibility of a pnh-like cell line to complement activated serum. normal human serum was used as a negative control and lipopolysaccharides(lps)-incubated normal serum as a positive control. all samples were tested in triplicates and twice. we studied 10 ta-tma patients transplanted from unrelated 8/8 matched (4) or 7/8 mis-matched (3) donors, identical (2) and haploidentical (1) siblings. all patients presented severe acute and/or chronic gvhd. ta-tma presented at median +109 (9-930) day post-transplant. in the control group, we studied two patients with steroidsensitive grii and two with steroid-refractory griv acute gvhd. we were able to detect significantly increased complement activation in the serum of ta-tma compared to gvhd patients (p = 0.039). based on previous studies and present controls, percentage of non-viable cells higher than 20% was considered a positive modified ham test, indicating increased complement activation in four ta-tma patients. regarding treatment outcomes, two patients with a negative modified ham test responded to cyclosporine cessation and steroid administration. plasma infusion with/without plasma exchange was initiated in seven patients. however, only three of them responded to second-line treatment. the modified ham test result was significantly increased in refractory patients (p = 0.048). the terminal complement inhibitor eculizumab was administered in one refractory patient with a positive modified ham test and renal failure at presentation. despite delayed initiation (28 days post ta-tma diagnosis), response was observed after three doses of eculizumab including evidence of reduced hemolysis, schistocytosis and transfusion needs. however, the patient succumbed to complications of end-stage renal disease (54 days post ta-tma diagnosis). among 10 ta-tma patients, 8 succumbed at a median +215 (73-430) day to transplant-associated complications, related to gvhd and infections from multi-resistant pathogens. ta-tma is associated with increased morbidity, mortality and severe complications, including gvhd. unlike gvhd, increased complement activation was observed in a significant portion of ta-tma patients. complement inhibition seems an encouraging therapeutic option in these patients. given the lack of robust functional assays for complement activation, the modified ham test may be useful for early recognition of patients that would benefit from complement inhibition. . this proposal includes, along with the 'classical sos' (cases diagnosed before day +21), the new type 'late onset sos' (cases diagnosed afterwards). new ebmt criteria for severy grading classify cases of sos into four grades (mild, moderate, severe, and very severe). the aim of this retrospective study is to analyze the cases of severe/very severe, both classical and late onset sos, occurred in our unit during the most recent period of time. we studied the last 100 pts, with a minimum follow-up of 100 days, who underwent allo-hsct in our center (november 2014-august 2016). 56 pts were male and 44 female. median age was 53 years (range: 7-69). baseline diseases were: acute leukemias (54), lymphoproliferative disorders (17), myelodysplastic syndromes (12), chronic myeloproliferative diseases (7), multiple myeloma (5), and bone marrow failures (5) . donor was unrelated in 57 cases, and related in 43 (including 18 haplo-identical). conditioning regimen was: busulphan-based (70), melphalan-based (13), tbi-based (8) , and others (9). all patient received prophylactic [p390] s320 ursodeoxycholic acid. progenitors source was pb in 89, and bm in 11. five patients developed severe/very severe sos (5% incidence); 3 were classical (at days +8, +11 and +20), and 2 were late onset (at days +34 and +44) (see table 1 ). four cases had received conditioning with a busulphan (iv)-based regimen (doses from 6.4 to 12.8 mg/kg), and one case with tbi plus cyclofosfamide at high doses. all cases presented with right upper quadrant pain, jaundice, ascites, weight gain, hiperbilirrubinemia, and renal function impairment. all but one had increased transaminases. the five cases were treated with defibrotide, in spite of which all of them died. considering that overall day +100 mortality was 9%, severe/ very severe sos was the most important cause of death of the series. [p391] although milder forms of sos might resolves within weeks, the most severe forms are still associated with a very high mortality rate. prophylaxis with defibrotide (the drug currently licensed for treatment) for high-risk patients has not been sufficiently studied yet. therefore, a high index of suspicion, early detection and early therapy are the only ways to try to reduce mortality due to sos in the hsct setting. disclosure of conflict of interest: this research has been performed entirely with public financial support. the royal marsden hospital, sutton, uk; 2 anthony nolan research institute, london, uk and 3 university college london, london, uk secondary poor graft function (spgf) complicates up to 15% allogeneic hcts, and is associated with increased mortality and poor quality of life due to recurrent infections and the need for ongoing blood product support. potential interventions include a second allograft using further conditioning, however many patients with spgf have a reduced performance status and are at an increased risk of complications from this procedure. unconditioned haematopoeitic progenitor cell (hpc) top-ups are associated with a high risk of gvhd if unmanipulated cellular products are used. cd34+ selection offers an attractive alternative, but incurs a loss of up to 20% hpcs and is an expensive procedure, unavailable to many centers internationally. alemtuzumab, a monoclonal anti-cd52 antibody, is routinely used in allogeneic transplant conditioning in the uk to prevent gvhd. we report the results of a retrospective study examining the efficacy of alemtuzumab conditioned hpc top-ups for spgf. data pertaining to patients who had undergone a second infusion of hpcs from their original donor were identified from our hospital-specific promise database. those who met the criteria of spgf defined as ⩾ 2 of hemoglobin 20 × 10 9 /l without support. 10 patients (4 pediatric, 6 adult) who underwent initial allogeneic transplants for malignancy (8) or bone marrow failure (2) received an alemtuzumab conditioned hpc top-up for spgf at our center 2005-2016. the diagnosis of spgf was made at a median 2.6 months post allograft (range 2-60) with trilineage cytopenias in 7 patients and bilineage cytopenias in 3 patients. all patients had received transplants from 10/10 (7 patients) or 9/10 (3 patients) matched unrelated donors. the median interval between initial transplant and top-up was 187 days (range 87-1853), and a median cd34 dose of 2.7 × 10 6 /kg recipient weight (range 0.3-7.63) was infused. 90% patients achieved haematological improvement (hi) at a median 20 days post-top-up (range 13-179), with the only failure to achieve hi seen in the patient who had received the lowest cd34 dose (0.3 × 10 6 /kg). one patient developed grade i agvhd post top-up but no grade ii-iv agvhd was observed. 1 year os was 80% and 2 year os 60% following hpc top-up. 3 deaths occurred due to infection at 1, 5 and 23 months post top-up, and one due to relapse of a prior non-haematological malignancy. 9 patients had an aplastic or hypocellular bm trephine pre-top up, which was repeated at 100 days post topup in 6 patients, of whom 5 had a normocellular bm trephine, while 1 remained hypocellular. alemtuzumab conditioned hpc top-up appears an effective intervention for spgf with results comparable to those of cd34 selected top-ups, and therefore represents a feasible alternative. larger studies are needed to exclude complications including viral reactivation and to investigate immune reconstitution following this procedure. disclosure of conflict of interest: none. high dose chemotherapy (hdt) followed by autologous stem cell transplantation (asct) has shown to improve outcome in patients with relapsed/refractory diffuse large b cell lymphoma (dlbcl). in the rituximab era, the benefit of asct has been debatable as prior study (coral study) has shown that patients who received r-chop as induction chemotherapy & responded to salvage chemotherapy had a poorer outcome following asct compared with those who received chop alone. in addition, it remains unclear whether addition of rituximab to standard high dose beam regimen provides any additional benefit. we retrospectively analyzed 63 dlbcl patients receiving high dose beam (n = 27) or rituximab +beam (r-beam) (n = 36) followed by asct for relapsed/ refractory dlbcl since 2002. all patients who received chop (n = 15) ± rituximab (n = 48) as first line therapy and who received ⩽ 2 lines of salvage chemotherapy before asct were analyzed. rituximab was given at the dose of 375 mg/m 2 on day +1 and +8 of asct. twenty-two (81%) patients in beam group and all the patients (100%) in r-beam group received rituximab-based salvage chemotherapy prior to asct. the 10year overall survival (os) was 71% and event-free survival (os) was 67% for the whole cohort. r-chop induced patients did not fare any worst after asct than chop induced patients (10 year os 73 vs 68 %; p = 0.91). there was a trend towards better survival in patients with pre-transplant disease free interval (dfi) 412 months compared to those with dfi 500/μl) time was 10 days and 11 days, respectively. median platelet recovery (420 000/μl) time was 17 days and 11 days, respectively (p = 0.11). ten year os (67% r-beam vs 77% s321 beam, p = 0.38) and efs (65% r-beam vs 73% beam, p = 0.28) were also comparable between both groups. hdt with beam and asct remains beneficial for patients with relapsed/ refractory dlbcl. it should be offered to all patients who respond to salvage chemotherapy with the expectation that they fare no worse than patients who do not receive rituximab in the induction chemotherapy. addition of rituximab following the standard beam for hdt and asct does not compromise haematopoietic recovery, but does not result in improved outcome in our study. prior use of rituximab during first-line or salvage therapy in most of the patients of r-beam group might have negated the beneficial effect of r-beam over beam. (1) . in this study, we aimed to develop a cns targeted chemotherapy regimen, which has lower toxicity and higher complete remission rates, in combination therapy. eight patients with secondary cns lymphoma (scnsl) and two with primary cns lymphoma (pcnsl), followed between the years 2010 and 2015, were included in the study, retrospectively. the patients were histologically diagnosed with biopsy and underwent autologous stem cell transplantation (apkht). all patients were treated with r-idaram/ rt (radiotherapy)/subsequently autologous stem cell transplantation (apsct) with r-beam protocol. the r-idaram regime consists of the following substances: rituximab 375 mg/m 2 , 50 cc/h infusion, day 1; cytosine arabinoside 1.0 gr/m 2 i.v., 1 h infusion, days 2 and 3; dexamethasone 100 mg, 12 h infusion, days 2, 3 and 4; idarubicin 10 mg/m 2 i.v.,15 min infusion, days 2 and 3; methotrexate 3 gr/m 2 (2 gr/m 2 at 445 years old-patients), 6 h infusion, day 4; and cytosine arabinoside 70 mg plus methotrexate 12 mg, intrathecally, days 2 and 8. the patients included seven males and three females. the median age was 44 years (range: 23-67). six scnsl patients were diagnosed in the application and two of them were diagnosed during r-chop chemotherapy (ct) protocol. five patients (57%) were stage ivb, and the others (43%) were stage iiib at diagnosis. after two or three chemotherapy cycles, patients were mobilized with growth factor support and median 5.510 6 cells per kg (range: 4-8) stem cells were collected. then, at a dose of 3600-4140 cgy cranial rt was administered for 14 days. after the third cycle of r/idaram, the state of remission was evaluated by cranial mri and lumbar puncture (lp). all patients achieved complete remission. neutrophil engraftment occurred at a median of 12 days (range: 10-18) and platelet engraftment occurred at a median 16 days (range: 11-21). after apkht, three patients relapsed and died at the fourth, ninth, and thirteenth months. grade i-ii manageable neurological toxicity occurred in two patients. the median follow-up time was 24 (range: 2-74) months. the five-year overallsurvival (os) was 63%. serious signs of infection were not observed in patients during transplantation. in pcnsl and scnsl, a standard treatment regimen has not yet been found. apsct with r-beam following modified r/idaram/rt is a curative and applicable therapeutic regimen with low toxicity, which can provide high rates of long-term survival and disease-free survival. despite the advent of novel therapies, autologous hematopoietic stem cell transplantation (ahsct) following melphalan (m)-based conditioning remains the standard of care for patients with multiple myeloma who are eligible. still, the majority of patients experience disease progression and ultimately succumb to their disease. we hypothesize that integrating novel agents in the conditioning is feasible and safe and may increase complete remission rates and overall survival. we completed a phase i, dose escalation study of carfilzomib (c) added to a backbone of bendamustine (b) and melphalan. all patients received a fixed dose (20 mg/m 2 ) of c on days (d) − 29, − 28, − 22, − 21, − 15 and − 14. in addition, patients were conditioned as described in table1. due to dose-limiting toxicity in cohort 2, the study was amended after the first 6 patients. subsequently, the dose of m was reduced to 140 mg/m 2 and the d +6 dose of c was omitted, per oversight of a data safety monitoring board. fifteen patients were enrolled, 9 males and 6 females. median age was 56 years (39-68). performance status was ⩾ 80% (kps) in all patients. per the international staging system (iss), 3 patients had stage i disease, 5 had stage ii, 6 had stage iii, and 1 had unknown staging. three patients had high-risk cytogenetics: 2 with t(4;14) and 1 with deletion 17p. four patients had undergone a prior ahsct. disease status at enrollment was stable disease (sd) (n = 3), partial response (pr) (n = 8), or very good partial response (vgpr) (n = 4). median cd34+ cell dose infused was 3.11 × 10 6 /kg (2.23 − 6.92 × 106). median follow-up was 18.2 months (1.4-28.7). all fifteen patients are evaluable s322 for engraftment. median time to neutrophil engraftment was 12 d (11-15). one patient died before achieving platelet engraftment. for the remaining patients, median time to platelet engraftment was 16 d (12-20) . non-hematologic toxicities included grade 3 acute mucositis (n = 1), lower gi complications (n = 7), electrolyte disturbances (n = 7), transaminase elevation (n = 1) renal insufficiency (n = 1), atrial fibrillation (n = 1), hypoxia (n = 1), prolongation of the qtc interval (n = 1), and grade 4 acute sepsis (n = 2), including 1 death (cohort 2) on d +44. eight patients went on to receive maintenance therapy: 3 with bortezomib, 3 with lenalidomide, and 2 with lenalidomide, dexamethasone, and c. posttransplant disease status was assessed per protocol by spep, spif, serum free light chains, and light chain ratio. twelve patients were evaluable on d +100. two patients had sd, 7 had vgpr, and 3 had complete response (cr). eight patients were evaluable on d +365. two patients had progressive disease, 1 had pr, 3 had vgpr, and 2 had cr. the combination of cbm prior to ahsct appears feasible, with manageable toxicities, at the doses described in cohort 3b. a prolonged follow-up and a phase ii study are warranted to determine response rates and long-term outcomes. disclosure of conflict of interest: none. beam (carmustine, etoposide, cytarabine, melphalan) is the most frequently used high-dose chemotherapy regimen for patients with lymphoma referred for autologous hematopoietic cell transplantation (autohct). in recent years a novel conditioning protocol containing bendamustine instead of carmustine (beeam) has been proposed in order to potentially increase the efficacy. so far, however data on its safety are limited. the aim of this study was to retrospectively compare the safety profile of beam and beeam based on single center experience. 174 consecutive patients with lymphoma treated with beam and 63 patients treated with beeam between year 2011 and 2016 were included in the analysis. the median age was 47 (19-69) years and 46 (22-73) years, respectively (p = ns). clinical characteristics of both groups were comparable. patients with hodgkin's lymphoma constituted 49% in the beam group and 40% in the beeam. among those with non-hodgkin lymphoma the diagnosis of dlbcl predominated. beam treatment consisted of carmustine 300 mg/m 2 on day − 6, etoposide 400 mg/m 2 /d on days − 5 to − 2, cytarabine 400 mg/m 2 /d on days − 5 to − 2, and melphalan 140 mg/m 2 on day − 1. in the beeam regimen carmustine was substituted by bendamustine administered on days − 7, − 6 at the total dose of 400 mg/m 2 i.v. peripheral blood was used as a source of stem cells. cd34+ cell dose was 5.1 (1.5-42.6) × 10 6 /kg in the beam group and 4.1 (2 − 16.8) × 10 6 /kg in the beeam group (p = ns). time to engraftment and the rates of adverse events up to day +100 after autohct were the study endpoints. all patients engrafted in both study groups. median time to neutrophil 40.5 × 10 9 recovery was 11 (7-37) days after beam and 10 (7-12) days after beeam (p = 0.13). median time to achieve platelet count 450 × 10 9 was 13 (7-44) days and 14 (7-33) days, respectively (p = 0.29). two patients died without progression before day +100 in the beam group, both due to bacterial infections. no early deaths were reported in the beeam group. the rates of grade 3 or 4 adverse events were comparable (see: table 1 ). administration of bendamustine instead of carmustin as part of conditioning does not affect engraftment as well as toxicity profile of the regimen. therefore beeam may be safely used in patients with lymphoma undergoing autohct. its efficacy requires evaluation in prospective studies focused on homogenous patient populations. [p398] disclosure of conflict of interest: none. the baltimore group reported a low dose tbi-based nonmyeloablative conditioning regimen followed by t cell replete bone marrow, with post-transplantation cyclophosphamide (pt-cy) to control gvhd and graft rejection. based on the fact that in our facility conventional low dose tbi was not available, we wanted to explore whether tmi/tli could be a potential substitute the aims of our study was to explore if tmi/tli can be considered an effective substitute of tbi in terms of os, pfs and nrm. retrospective analysis was applied in 159 cases of haploidentical hsct from april 2009 to october 2016. all patients underwent baltimore conditioning associating fludarabine (30 mg/m 2 /day) day − 6 to − 2, cy (14.5 mg/kg/day) on days − 6 and − 5, and tbi 2 gy in 135 patients and tmi/tli 2 gy in 24 patient at day − 1. unmanipulated bone marrow graft was infused at day 0. postgrafting immunosuppression consisted of cy (50 mg/kg/day) on day +3 and +4, and mycophenolate mofetil for 30 days, and tacrolimus or cyclosporine. no differences between the two groups was observed in term of age, gender diagnosis, disease status and donor type. 93% of patients engrafted in both arm (23/24 and 125/135). in tbi cohort vs tmi/tli cohort, the median time to anc 4500/μl and platelet recovery 420 000/μl was not different (22 and 27 days vs 20 and 27.5 days, p = 0.14 and 0.32, respectively). in all tmi/tli evaluable patients, full chimerism was observed at days +30. after a median followup of 17 months in tmi/tli cohort and 34 months in tbi arm, 1-year nrm was 25.9% and 13.9% (p = 0.16), respectively. the 1 years os and pfs were not statistically different in the two groups 70% vs 57.1%, p = 0.12 and 62.5% vs 48.1%, p 0.09, respectively). the 1-year relapse incidence was 26% in tmi/tli group and 23.6% in tbi group, p = 0.61. no difference in incidence of both agvhd and cgvhd was observed between the two groups. this retrospective analysis suggests that tmi/ tli could be considered an effective substitute of low dose tbi, with a sufficient degree of immunesuppression of recipient, allowing engraftment and full chimerism. the gvhd both acute and chronic as well as the 1-y nrm were not different. disclosure of conflict of interest: none. comparison of the beeam conditioning regimen and the beam conditioning regimen in the autologous transplantation for hl and nhl s lozenov 1 , p ganeva 1 , y petrov 1 , g arnaudov 1 and g mihaylov 1 1 the beam has established itself as a standard of care conditioning regimen in the autologous lymphoma hsct setting for most transplant centres in europe. yet however various other regimens are being compared with it in order to achieved better safety profile, better os and dfs, in order to improve results with chemoresistant and unfavourable patients. one such regimen is beeam (bendamustine, etoposide, cytarabine, melphalan).we aimed to compare the efficacy of the beam and beeam conditioning regimens and to compare their myelotoxicity profile. we evaluated retrospectively 114 adult patients (mean age 41.1197 with sd 11.12404), receiving auto-hsct at the national specialized hospital for active treatment of hematological diseases in sofia, bulgaria for relapsed/refractory hl or nhl (of them mh -57, dlbcl -26, pmbcl -15, fl -3, lbl -3, ptcl-nos -3, aitl -2, alcl -2, mcl -2, mzl -1) for the period from 1.01.2013 to 1.07.2016 with a follow-up of patients up to 1.11.2016. ninety-two of the patients received the beam (as previously described -bcnu 300 mg/m 2 i.v. day − 6, etoposide 200 mg/m 2 i.v. days − 5 to − 2, cytarabine 400 mg/m 2 i.v. days − 5 to − 2, and melphalan 140 mg/m 2 i.v. day − 1) regimen and 22 received beeam regimen (bendamustine on days − 7 and − 6 (160 mg/m 2 ); cytarabine, 400 mg/m 2 intravenously daily, from day − 5 to day − 2; etoposide, 200 mg/m 2 intravenously daily, from day − 5 to day − 2; and melphalan, 140 mg/m 2 intravenously on day − 1). the overall survival at the second and third years of follow-up (os-2, os-3) and dfs at the third year, the cr rates and the average time periods to hematological recovery, were compared. the os at 2 and 3 years, respectively, was 86.1% and 86.1%, for beeam and 78.8 % and 71% for beam, the dfs at 3 years was 76.4% for beeam and 73.2% for beam, provided that the differences did not have statistical significance (p 0.851 for os and p 0.890 for the dfs). the cr rate was 63.63% in the beeam group versus 50% in the beam group. from the patients who received autologous hsct in stable disease or progression pre-transplant status (chemoresistnat patients), 22.72% of the patients receiving beeam achieved cr at the first post-transplant evaluation versus 10.86% respectively for the beam group. the mean time to hematological recovery for neutrophils was 11.1765 ± 4.91471 days (beeam) versus 10.2469 ± 3.56216 days (beam) and 12.6471 ± 6.04091 days (beeam) versus 11.1235 ± 2.52677 days (beam) for platelets. beeam appears to be a non-inferior alternative conditioning regimen to the standard beam, it shows a trend towards higher myelotoxicity, but also a trend towards better response rates in chemoresistant patients. [p400] disclosure of conflict of interest: none. autologous hematopoietic stem cell transplantation (asct) is widely used as a consolidation therapy in aggressive non-hodgkin's lymphoma (nhl) and recurrent or refractory classic hodgkin's lymphoma (hl). in mexico, the use of carmustine (bcnu) in the conditioning regimen of these patients is limited due to the lack of access to the drug and its high costs. this study aims to compare results in terms of toxicity, disease-free and overall survival between a group of patients treated with the standard regimen beam and another group treated with a scheme in which carmustine was replaced by cisplatin (peam regimen). a comparison of two groups with lymphoma was performed and the clinical aspects of cisplatin 100 mg/m 2 d . the characteristics were well balanced between the two groups. the mean time for neutrophil grafting (4500 per mm 3 ) was significantly slower with beam than with peam (12 vs 11 days, p = 0.001), hospitalization time was longer with beam compared to peam (25 vs 22, p = 0.015). on the other hand, proportion of patients who require red blood cell s324 transfusion was significantly higher in beam group (58%) versus peam group (20%) (po 0.001), but total amount of platelet transfusion did not differ between groups. about the toxicity, beam patients had significantly more frequent incidence and severity of nausea/vomiting (95% vs 53.8%) and diarrhea (61.5% vs 90%) compared to peam (p o 0.01). no significantly differences were observed in incidence of mucositis (p = 0.65). at the moment of the analyses, 75% of patient of the peam group were in complete response versus 59% of the patients treated with beam, but it did not represent a significant difference. disease-free survival and 5-year overall survival in the peam vs beam scheme were similar with 64% vs 59% (p = 0.69) and 84% vs 76% (p = 0.35) respectively but with less toxicity using the peam scheme. peam regiment is not inferior scheme compared with beam, because it shows similar outcomes in disease-free survival and overall survival. additionally, peam is a well-tolerated regime and beam scheme was associated with greater gastrointestinal toxicity such as nausea, vomiting and diarrhea, also greater hematology toxicity such as more requirement of red blood cell transfusion. [p401] disclosure of conflict of interest: none. cumulative busulfan exposure is associated with relapse following busulfan and cyclophosphamide myeloablative allogeneic stem cell transplantation for acute myeloid leukaemia e wong, d kliman 1 , m chau 2 , j szer 2 , c nath 1 , p shaw 1 , d ritchie 2 , d gottlieb 1 and a bajel 2 1 westmead hospital, new south wales, australia and 2 royal melbourne hospital, victoria, australia the optimal busulfan exposure to reduce disease relapse in adult patients with acute myeloid leukaemia (aml) undergoing busulfan/cyclophosphamide myeloablative allogeneic stem cell transplant (allosct) is poorly defined. we retrospectively analysed busulphan pharmacokinetics (pk) and outcomes of patients who underwent busulfan/cyclophosphamide conditioned allosct for aml from 2010 to 2016. busulfan was administered intravenously over 5 days (1.6 mg/ kg/d for 2 days followed by 3.2 mg/kg for 3 days). peripheral blood was obtained for busulfan pk after the first dose. subsequent doses of busulfan were decreased if daily busulfan exposure (area under the curve; auc) was anticipated to exceed 5000 μm per min/day. cyclophosphamide was dosed at 120 mg/kg. the primary outcome was the cumulative incidence of relapse (cir) accounting for non-relapse mortality (nrm) as a competing risk. independent variables analysed included age, sex, cytogenetic risk group, disease risk index (dri), donor type, stem cell source, t-cell depletion, and cumulative busulfan auc (cumauc) calculated as previously described. 1 (figure 1) . t-cell depletion was also associated with increased cir (hr 3.3; p = 0.0049). patient age, sex, cytogenetic risk, dri and graft type were not significantly associated with cir. on multivariate analysis, cumauc 415 000 μm per min remained significantly and independently associated with lower cir (hr 0.38; p = 0.036). cumauc was not associated with nrm, rfs, os, or the incidence of acute or chronic gvhd. figure 1 . cumulative incidence of relapse in patients stratified by total busulfan exposure. [p402] cumulative busulfan exposure 415 000 μm per min is independently associated with reduced relapse following busulfan/cyclophosphamide allosct for adults with aml. these findings support further evaluation of the optimal busulfan exposure to reduce aml relapse in a prospective clinical trial, whereby patients could be randomised to target cumauc 415 000 μmol per min versus standard practice. hematopoietic stem cell transplant with busulfan and cyclophosphamide (bucy) based conditioning has a relatively high incidence of liver toxicity and sinusoidal obstruction syndrome (sos). busulfan and cyclophosphamide metabolites share the same glutathione conjugation in the liver metabolism. a small number of studies addressed different sequence of both drugs bucy vs cybu during conditioning. differences in liver toxicity, sos, transplant related mortality (trm), relapse incidence (ri) and overall survival (os) were reported favoring cybu conditioning. we decided to address the above issues at the umc ljubljana, slovenia. this was a retrospective study following patients with myeloid malignancies (aml, mds, mpn) with bucy (n = 30) and cybu (n = 14) conditioning through a three year period in a single institution. primary endpoint was detecting difference in liver toxicity by measuring levels of liver enzymes. secondary endpoints were incidence of sos, difference in trm, ri and os. patients characteristics between groups at the time of the transplant did not differ significantly. we observed significantly higher liver toxicity through elevated bilirubin and alt in the bucy 73.3% than cybu 64.3% patient group (picture 1). the highest probability of liver toxicity was around d0 in the bucy group and in the second week after the transplant in the cybu group. the incidence of sos, trm and ri were comparable between the groups. there was no difference in os between the patient groups during the 40-month follow-up. bucy conditioning for hematopoietic stem cell transplant causes higher incidence of liver toxicity compared to cybu conditioning. there is no difference in sos frequency, trm, ri and os between bucy and cybu conditioning. prospective controlled comparison would be needed for further study of the subject. disclosure of conflict of interest: none. early monocyte recovery is associated with better overall survival after busulfan containing myeloablative conditioning allogeneic hematopoietic cell transplantation in patients with acute myeloid leukemia a lojko-dankowska 1 the outcomes of allogeneic hematopoietic cell transplantation (allohct) in acute myeloid leukemia (aml) depend on different patient-, disease-and transplant related factors, including the dose and combination of agents used for conditioning. the aim of the study was to analyze the outcomes of allohct in patients with intermediate or high risk aml according to disease risk index (dri) who received myeloablative conditioning consisted of intravenous busulfan (9.6-12.8 mg/kg) combined with cyclophosphamide (120 mg/ kg) or fludarabine (150 mg/m 2 ) between 2010 and 2016 in our institution. the published data indicate that the combination of busulfan (bu) and fludarabine (flu) seems to have more favorable toxicity profile than combination of bu and cyclophosphamide (cy), so bucy regimen has been substituted with buflu as the myeloablative conditioning for aml patients in our institution practice since 2014. we evaluated the influence of type of regimen on transplant outcomes along with the impact of other potential prognostic factors, including age of patient, dri, donor type, hla and gender mismatches, stem cells source, and lymphocyte and monocyte recovery. the study group consisted of 71 aml patients, median age 37 years (range: 19-59), classified as intermediate (n = 57) or high (n = 14) risk according to the dri, who were conditioned with bucy (n = 38) or buflu (n = 33) followed by allohct from hla identical sibling (n = 23) or 9-10/10 matched unrelated donor (n = 48). the stem cell were collected from peripheral blood (n = 44) or bone marrow (n = 27). gvhd prophylaxis consisted of calcineurin inhibitor combined with mtx plus atg in allohct from unrelated donors. engraftment was observed in all patients. the median time to neutrophil count (0.5 g/l) and platelet count (20 g/l) recovery was shorter after buflu in comparison with bucy (18 days vs 22 days; p = 0.045 and 13 days vs 20 days; p o0.001), however peripheral blood stem cells were used more often after buflu regimen than after bucy (88% vs 40%, p340/mm 3 on+21 day after transplant (2-year os 77% vs 55%, p = 0.034) and intermediate vs high dri (2-year os 78% vs 53%, p = 0.068). in multivariate analysis higher amc after allohct remained the only independent favorable prognostic factor for os (rr 0.35 (95% ci 0.13-0.97), p = 0.04). our results suggest that early monocyte recovery after myeloablative bu containing conditioning allohct is significant favorable predictor of outcome. in our experience both bucy and buflu myeloablative regimens result in similar long-term survival after allohct in aml patients. [p403] disclosure of conflict of interest: none. the use of t-cell depletion as part of the conditioning protocol has the potential to improve the tolerability of allogeneic stem cell transplantation (hsct) through the reduction in graft versus host disease (gvhd). despite the wide spread adoption of this practice in many parts of the uk and europe, definitive recommendations regarding the most appropriate dose remain elusive. previous experience by our group with 100 mg of alemtuzumab combined with fludarabine and busulfan based conditioning demonstrated good long-term outcomes with low rates of gvhd. however, due to concerns of high relapse risk especially in patients with high-risk myelodsypastic syndrome and acute myeloid leukaemia, we instituted a policy change in 2013 to reduce the dose of alemtuzumab in the conditioning protocol from a total of 100-60 mg. we conducted a retrospective analysis of all consecutive patients undergoing reduced intensity unrelated allogenic stem cell transplantation with fludarabine (150 mg/m 2 ), busulfan (6.4 mg/kg iv or 12.8 mg/kg iv) and alemtuzumab (fb2c or fb4c, respectively) conditioning for neoplastic myeloid disorders between 2010 and 2016. patients were subsequently analysed in two cohorts; those receiving 60 mg of alemtuzumab (n = 84) and those receiving 100 mg of alemtuzumab (n = 95). apart from a decreased proportion of females in the 60 mg alemtuzumab group, the cohort was balanced across the different dose levels ( table 1 ). the longterm overall survival (os) of the entire cohort was good with a 5 year os of 51%. no significant differences in overall outcomes across the two groups were observed with a 5 year os of 51% in the 60 mg group vs 49% in the 100 mg group (p = 0.39). cumulative incidence of relapse (cir) and nonrelapse mortality (nrm) was 42% and 21% and 39% and 24% in the 60 mg and 100 mg groups, respectively. interestingly, age had a significant effect on nrm in the 100 mg (7% age o50, 28% age 50-65 and 41% age 465 p = 0.04), but not in the 60 mg group (11% age o50, 19% age 50-65 and 24% age 465 p = 0.8). the effect on relapse rate was not significant in either group (p = 0.6 and p = 0.11, respectively). this retrospective analysis did not demonstrate an overall improvement in transplant outcomes with dose de-escalation of alemtuzumab from 100 to 60 mg. in particular, we did not see the anticipated improvement in relapse rate in this cohort. notably older patients seem to tolerate the 60 mg dose better due to the lower nrm. prospective trials with accompanying translational work are required to determine the optimal dosing and schedule for this group of patients. disclosure of conflict of interest: none. bendamustine was given at 200 mg/m 2 /d for the first 4 pts then 100 mg/m 2 /d for the 4 subsequent pts and finally at 120 mg/m 2 /d for the remaining pts (22 pts). among the beam group, 68% had non-hodgkin's lymphoma (nhl) and 32% hodgkin's lymphoma (hl) compared to 87% and 13%, respectively, in the beeam group (p = 0.014). hhv-6 detection was performed by pcr for symptomatic pts (fever, rash or prolonged cytopenia). patients were housed in single bedrooms with air filtration and received the same supportive care. median age was 50 (18-66) and 56 (20-67) in the beam and beeam groups respectively and median of previous chemotherapy regimens was 2 (range: 1-5). fifty two out of 90 patients were male (37/60 in the beam group and 15/30 in the beeam group). pts were in cr (46.7% vs 56.7%) or pr (53.3% vs 43.3%) at time of transplant. there was no difference in terms of hematologic recovery (median = 11 days (range: 7-22)), blood and platelets transfusion, mucositis toxicity. there was no statistical difference in the incidence of acute renal failure when comparing the two groups. however, there was a very striking difference when considering the highest dose of bendamustine when compared as well to the two others doses of bendamustine (po 0.00001) as to the beam group (p = 0.005). additionally, we also observed a high incidence of symptomatic hhv-6 infections (53.3% vs 8.3%, p o0.00001), digestive toxicity (36.6% vs 15%, p = 0.03) and a longer hospitalization duration (25 days (range: 18-59) vs 21 days (range: 18-32), p = 0.001) for patients in the beeam group overall. with a median follow up of 18.3 and 9.7 months for beam and beeam respectively, overall survival (93% vs 86%), transplant related mortality (0% vs 3%) and event free survival (83% vs 78%) were comparable. overall, beeam regimen was associated with longer duration of hospitalization, higher rate of digestive toxicity and increased risk of symptomatic hhv-6 infection as compared to the beam regimen. in addition, higher doses of bendamustine (200 mg/m 2 /d for two consecutive days) were associated with unacceptable high rate of acute renal toxicity. with a still short follow-up, the absence of benefit on disease control together with higher short term toxicity does not allow to recommend the use of beam instead of classical beam. should it be used, we suggest that pts should be carefully monitored for renal toxicity and for hhv-6 infection in case of symptoms. disclosure of conflict of interest: none. high-dose treosulfan and melphalan for consolidation therapy in high-risk ewing sarcoma me abate, a paioli, a longhi, m cesari, e palmerini and s ferrari musculoskeletal department, rizzoli orthopaedic institutes, bologna, italy common toxicities observed after high dose chemotherapy with busulfan and melphalan for high risk ewing sarcoma (es) are generally well managed by current supportive care but some patients can develop severe complications. treosulfan is an alkylating agent that has recently been used as a substitute of busulfan to prevent potential serious complications related to busulfan. medical records of 7 es patients undergoing autologous peripheral blood stem cell (apbsc) transplantation after intravenous treosulfan (treo) and melphalan (mel) from 2011/6/1 to 2016/2/29 were analyzed with regard to toxicity and outcome. patients were included into the study if they were eligible for the protocols activated in our institution for es and presented reasons that did predict potential complications related to busulfan, such as previous radiotherapy on axial skeleton/pelvis or coexistence of high risk of epilepsy. as consolidation treatment patients received intravenous treo 12 g/m 2 over 3 days and mel 140 mg/sqm with support of apbsc transplant and use of granulocyte colony stimulating factor. in those patients with lung metastases total lung irradiation was performed at least 2 months after treomel. frequency of toxicity for treomel was recorded with at least 6 months of follow-up and was evaluated according to nci ctg common toxicity criteria. the median age at diagnosis of patients receiving treomel was 16 years (range 13-25 years), 3 males and 4 females. 5 patients had localized disease at diagnosis with poor radiological or histological response to standard chemotherapy; one patient had lung metastases at diagnosis and one patient had relapsed disease with lung metastases. before receiving treomel the primitive tumour underwent radiation therapy in 5 cases (3 pelvis, 1 cervical vertebra, 1 sacrum), surgical resection in one case(tibia) and surgical resection plus radiation therapy in one case (fibula). 2 patients showed eeg abnormalities at high risk of developing epilepsy. the median number of infused cd34+ cells was 5.9 × 10 6 /kg (range 3.4-15.9). febrile neutropenia occurred in 5/7 patients and lasted one day in 3 patients and 2 days in 2 patients. median time to granulocyte engraftment was 10 days (range 10-12 days); median time to platelet engraftment 420 000 was 10 days (range 9-16 days). only one patient needed 2 red blood cells transfusions; 5 patients needed 1 platelet transfusion and 2 patients needed 2 platelet transfusions. none developed grade 3-4 stomatitis or grade 3-4 [p406] hematuria or grade 3-4 liver toxicity. surprisingly, a patient became pregnant after 1 year and 10 months from transplantation. with a median follow-up of 24 months (range 8-65 months) 5 patients are alive in complete remission, one patient is alive with relapsed disease and one patient died for disease progression. these results, related to a limited cohort of patients, confirm the lower toxicity observed for treosulfan with respect to busulfan. although more data are needed to clarify the role of treosulfan in es, the impact of potential severe complications observed with busulfan, including infertility, should suggest its replacement with treosulfan in selected cases. disclosure of conflict of interest: none. immunoadsorption procedures prior to haploidentical allogeneic pbsct could prevent graft failure in patients with hematological malignancies displaying anti-donorspecific hla antibodies donor-specific anti-hla antibodies (dsa) have been shown to be associated with a high risk of rejection in solid organ transplantation and with graft failure (gf) in allogeneic hematopoietic stem cell transplantation. a combination of anti-cd20 (rituximab), plasma exchange (pe), and ivig in 12 patients with additional buffy coat infusion in 5 among them prevented graft loss in all patients that became c1q negative before sct. we addressed the question whether immunoadsorption in combination with rituximab can also be applied in patients with dsa to prevent graft failure in haploidentical pbsct. four patients with acute myelocytic leukemia or myeloma in second complete remission were enrolled. the presence of dsa was determined by luminex at pre bmt checking. immunoadsorption was performed with polyclonal sheep anti-human igg adsorbers (miltenyi biotec gmbh, germany) on life 18 apheresis system. in addition all patients received rituximab 375 mg/kg bw in a single dose. patients were conditioned with a reduced intensity regimen comprising tbi 2 gy, cyclophosphamide 29 mg/kg, and fludarabin 150 mg/m 2 . all patients received cyclophosphamide post bmt (ptcy) 100 mg/kg. non-t-cell depleted pbsct were transfused in a sequential manner in 2 doses each. the data of the patients and treatments is summarized in table 1 . two patients had a normal hematopoietic reconstitution and are alive at +21 and +15 months post-transplantation, one with hepatic gvhd; chimerism was 100% in peripheral blood on last follow up. one patient died following a graft failure. by a combination of rituximab and repeated immunoadsorption prior to allogeneic pbsct the titer of dsa could be lowered sufficiently to enable engraftment. ia turned out to be a safe procedure without relevant clinical side effects. hematopoietic reconstitution was in the normal range in 2 of 3 evaluable patients. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for patients with beta thalassemia major. however, the availability of hla-matched related donor remains the main obstacle for allogenic hsct. although, a few studies have been reported, experience with hla matched unrelated donors is limited. we present the result of 35 children with beta thalassemia major who received allogeneic hsct from hlamatched unrelated donors with using a novel conditioning regimen. we retrospectively assessed 35 unrelated hsct in children with beta thalassemia major. all patients received busulphan (bu) based myeloablative conditioning regimen. busulphan was used according to weight adjusted doses. in addition, all patients received fludarabine 150 mg/m 2 in 5 days, cylophosphamide 120 mg/kg in 3 days, thiotepa 10 mg/kg in one day and atg 30 mg/kg in 3 days. cyclosporin-a and mtx were used for graft versus host disease (gvhd) prophylaxis. donor chimerism was evaluated in the peripheral blood on days +30, +100 and +180. the median age of the patients was 6.9 years (range 14 month-15 year). two of the patients were grouped in class i and rest of them were class ii. the median serum ferritin level was 1.255 ng/ml (range, 585-5832). all of s329 the donors were matched 10/10 with high-resolution hla typing in gvhd direction but three of them 9/10 with graft failure direction. twenty-three of them received bm (median tnc: 6.2x108/kg) and 12 pbsc (median mnc:7.3x108/kg) with median cd34+ cell number 7.20x106/kg. the median neutrophil and platelet engraftment days were 13 and 14 days in pbsc and 17 and 20 days in bm group, respectively. grade i-iv acute gvhd was observed in 7 patients (26%) and only one experienced limited chronic gvhd with only skin involvement. mild to moderate vod was seen in 13 patients (37%) and treated with defibrotide successfully. all patients except one are alive with full donor chimerism (between 95-100 %) with a median 12 months (range 3-49 months) follow-up. one patient died because of cmv pneumonia. these data show that the results of hsct from unrelated donors in selected low risk thalassemia patients may be comparable to hsct of matched sibling donors. however, it needs further studies with long term follow up and larger study population. disclosure of conflict of interest: none. table 1 . data about cytogenetic risk of group 1 patients were available only in 9 individuals. differences between groups were analyzed by t-student and chi square tests. survival was analyzed by kaplan-meier method and differences in survival between groups were evaluated by log rank test. no differences were found between groups regarding gender, sc source, disease status at sct, type of donor and number of cd34+ cells infused. patients in group 2 were significantly older (median age for groups 1 and 2: 32 vs 38, p = 0.021). gvhd prophylaxis protocols included atg in a higher frequency in group 2. no differences between groups 1 and 2 were observed in neutrophils recovery (median days to anc4500/μl: 9 vs 8 respectively, p = 0.78) and platelets recovery (median days to platelets 420 000/μl: 8 vs 5 respectively, p = 0.51). patients in group 1 required more red cell transfusions (median packed rbc: 3 vs 1, p = 0.048). no differences were observed regarding platelets transfusion requirements or length of hospitalization. post-sct os was significantly better in group 2 (3 years-os group 1: 23%; group 2: 61%; p = 0.029) (figure 1). there were no significantly differences between groups regarding frequency of mucositis, diffuse alveolar haemorrage, sepsis, acute and chronic gvhd. vod was more frequent in group 1 (4/21 vs 0/20, p = 0.03). trm mortality was higher in group 1 (6/21 vs 3/20), being this difference no statistically significant (p = 0.29). as it was reported by others, the use of fludarabine-based conditioning regimen was associated with a significantly better post-sct os and a reduced frequency of vod in aml patients. reduction in trm and differences in the frequency of described complications are not statistically significant probably due to the small size of this sample. since march 2016, given the limited availability of melphalan, we administer the beac regimen (carmustine, etoposide, cytarabine, and cyclophosphamide), instead of the gold standard conditioning regimen beam, followed by autologous haematopoietic cell transplantation (ahct) in relapsed or refractory hodgkin (hl) and non-hodgkin (nhl) lymphoma patients. the primary goal of this analysis was to assess the immediate related toxicity of this alternative regimen. we used beac (carmustine 300 mg/m 2 , etoposide 800 mg/m 2 , cytarabine 800 mg/m 2 , and cyclophosphamide 140 mg/kg) in 22 consecutive lymphoma patients (13 hl, 9 nhl) who underwent ahct for relapsed or refractory disease. the median age of the patients was 42.5 years (17-64). they all received peripheral stem cell grafts with a median cd34+ cell dose of 5.13 × 10 6 /kg cells (2.07-15.78). disease status post salvage treatment (at ahct) was complete remission (cr) in 6, partial remission (pr) in 11 and progressive disease in 5. the disease was chemosensitive to salvage therapy in 17/21 patients. median follow up was 112 days (31-224). toxicity was assessed according to the who toxicity scale grading. all patients engrafted successfully. median time for engraftment was day +10 (d+10) for neutrophils (4500/mm 3 ) and d+11 for platelets (420 000/mm 3 , without transfusion within the previous 3 days). patients were hospitalized for a median of 19 days (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) . no treatment-related mortality occurred. two patients died due to disease progression (both nhl patients, on d+143 and d+63). toxicity assessment until d+30 is presented in table 1 : moreover, no hemorrhagic cystitis or macroscopic hematuria, and no cardiac events were encountered. febrile neutropenia was recorded in 4 and bacteremia in 9 patients (7 gram+, 2 gram-, 2/9 related to central venous catheter), with fever ≤ grade 2 in all cases. during d+30-100 two patients presented fever of unknown origin, and 3 patients had upper or lower respiratory infections, with no other adverse events being recorded. in terms of disease best response within 3 months post ahct (18/22 patients evaluated), 11 patients achieved or sustained cr, 5 pr (1 of these patients eventually died due to disease progression), 1 relapsed and 1 succumbed due to disease progression (no response). according to our preliminary results, the early toxicity profile of beac is very low, the regimen is easily tolerable for the patients, and without any treatment-related mortality. its use as an alternative conditioning regimen in ahct for lymphoma patients seems feasible. further investigation including more patients and comparative analysis to other conditioning regimens are warranted for reliable conclusions on the toxicity and efficacy of beac. disclosure of conflict of interest: none. veino-occlusive disease (vod) is a potentially fatal adverse event caused by intravenous (iv) busulfan used in bone marrow transplantation (bmt) conditioning. the objective of this study was to identify determinants of vod in children treated by iv busulfan. this was a retrospective analysis of data collected in 293 children from two bmt centers over 10 years. vod was diagnosed according to modified seattle criteria. individual pharmacokinetic data, including busulfan area under the concentration-time curve (auc) and maximal concentration (cmax) were estimated in all children by using a validated bayesian approach. 1 we examined the relationships between the occurrence of vod and available data in a learning (n = 243 patients) and validation set (n = 50 patients) obtained by random splitting. logistic regression was used as a continuous statistical model. in addition, we used classification and regression tree (cart) analysis, a machine learning and binary partitioning technique, to identify determinants of vod and their optimal cut-off values. the predictive performance of variables within both models was assessed by these results are compared with historical data from our service using beam as conditioning followed by auto-sct in lymphoma patients. nine patients were enrolled to receive neam: mitoxantrone 6 mg/m 2 day -6 to -4, etoposide 100 mg/m 2 every 12 hours and cytarabine 100 mg/m 2 every 12 hours day -6 to -3, and melphalan 140 mg/m 2 day -2, followed by auto-sct. the median age was 51 years (19-63); five non-hodgkin lymphomas (nhl) and four hodgkin lymphomas (lh). six patients were in partial remission (pr), two in complete remission (cr), and one with progressive disease at time of auto-sct. neam patients were compared with a historical control group of patients receiving beam regimen (n = 167). differences between groups were analyzed by t-student and χ 2 -tests. median cd 34+ cells infused in neam and beam groups was 3.48 × 10⁶/kg (3.09-8.72) and 4.14 × 10⁶/ kg (1.02 -25.6), respectively (p = 0.42). the median time to neutrophil recovery (4500/μl) was 16 days (11-22) and 10 days (3-30) (p = 0.000017) and median time for platelets recovery (420 000/μl) was and 15 days (9-30) and 8 days (1-49) (p = 0.018) respectively, for neam and beam patients. median duration of hospitalization was 34 days (24-55) with neam and 27 days (15-59) with beam (p = 0.002). among neam patients, 88% had one or more febrile episodes during neutropenia. no case of grade iii or iv mucositis was described. there was no transplant-related mortality (trm: 0%) associated with the use of neam regimen. at the present, all neam patients are alive, two of them in relapse (22%). due the difficulties in obtaining carmustine in our region, neam can be considered as a feasible alternative to beam. however, despite the sample was small enough to draw conclusions, we find that neam presents prolonged aplasia of significant value, we are currently exploring conditioning regimens followed by auto-sct in hodgkin's and non-hodgkin's lymphomas based on bendamustine, etoposide, cytarabine and melphalan. disclosure of conflict of interest: none. at present, decision-making about conditioning regimens for allogeneic hsct is based on patient's and donor's features, and disease characteristics. during the last years, terms as 'myeloablative/non-myeloablative/reduced-intensity' have been frequently employed in a confusing and unequal way among the different centers. knowing the expected intensity and myeloablative effect from each regimen is very useful and constitutes the aim of this analysis. we have analysed the severe neutropenia (anc o500 per mcl), and thrombocytopenia durations (platelets o20 000 per mcl), the need for platelet concentrates transfusion and the duration of the inpatient period of the 223 allo-hsct carried out during the last four years in our centre. these data are reported according to the conditioning regimen used and to the type of transplant performed. then, they are compared among them in order to stablish intensity ranks. results: population characteristics are described in table 1 : conventional intensive regimens (bu-cy2, cy-bu2, tbi-cy) reported more days of severe neutropenia and greater need of platelet concentrates transfusion. the regimens with less days of severe neutropenia, less need of platelet concentrates transfusion and fewer days of admission were flu-bu3 and flu-bu2. allotransplants carried out with stem cells from bm presented more days of severe neutropenia and longer hospital stay. similar platelet transfusion need was reported. haplo-identical allotransplants reported more days of severe thrombocytopenia, but were not asociated to longer neutropenia or longer hospital stay than the others. these data are described in detail in table 2 . flu-bu: the number (4, 3 or 2) expresses the doses of busulphan administered at 3.2 mg/kg/day. pc: platelet concretates data is expressed in medians. within the analysed conditioning regimens, an intensity rank is stablished regarding the myelosupression induced (in descending order): conventional intensive regimens (cy-bu2, bu-cy2, tbi-cy), flu-mel, flu-bu4, flu-bu3 and flu-bu2. all of them induced severe neutropenia and thrombocytopenia, and for that reason they must be considered myeloablative regimens. disclosure of conflict of interest: none. myeloablative allogeneic stem cell transplant for aml and mds: the impact of advanced age in the outcome m sánchez-escamilla* 1,2 , s garcía-ávila 1 , l yáñez san segundo 1,2 , ma bermudez rodriguez 3,2 , mm colorado araujo 1,2 , a casado diez 1 , m celis alvarez 1 , a cabero martinez 1 , c fernandez martinez 1 , a insunza garminde 1,2 , c richard espiga 1,2 and e conde garcía 1,2 1 allogeneic hematopoietic stem cell transplantation (allo-hsct) is the only curative option in high risk myeloid hematological malignancies. myeloablative conditioning (mac) regimen has been proven to be effective in the control of high risk diseases in advanced age patients. objective: the aim of this study was to analyze the efficacy of myeloablative allo-hsct in two cohorts of patients considering their age at transplant. we also analyzed the incidence of acute and chronic graft versus host disease (gvhd) and procedure related outcomes who underwent to myeloablative allo-hsct were retrospectively analyzed. the median age was 49 years (iqr 36-57). both groups were divided regarding their age at allo-hsct [group 1, age ⩾ 55 years (n = 41) and group 2, age o55 years (n = 93)]. patient´s characteristics are shown in picture 1. data were collected as either continuous data and compared by two-tailed unpaired t-test or mann-whitney test, or as categorical variables and compared by chi-square. the procedure related outcomes were analyzed with the kaplan-meier test. the incidence of acute gvhd grade ii-iv was similar in both groups (43.9% in group 1 and 42.2% in group 2, p = 0.761 ). the mean day to acute gvhd (grade ii-iv) development was 38 days in group 1 and 40 days in group 2. the most involved organs in both groups were skin (group 1: 94.4% and group 2: 72.1% [p = 0.258]) and gut (group 1: 55.6% and group 2: 62.8% [p = 0.598]). at day +100 post-transplant 123 patients were alive and evaluable for chronic gvhd. the incidence of cgvhd development was similar between group 1 and 2 (61.1% versus 68.4%, respectively, p = 0.140). however, severe grade s333 of cgvhd was high in group 1 patients (25.0% versus 18.4%). with a median follow up of 43 months (iqr, 9-70) the probability of os was significantly low (p = 0.004) in group 1 (46.3% +-7.8) compared with group 2 (67.0% ± 4.9). pfs was also significantly low (p = 0.003) in group 1 (41.5% +-7.7) compared with group 2 (66.3% ± 4.9). trm at 43 months was higher in group 1 compared with group 2 (34.1% versus 17.2%). mortality due to relapses was also higher in group 1 (17.0% versus 12.9%). most of the patients died during the first 24 month. comparing both groups at this time (24 months post-transplant), trm was higher in group 1 compared with group 2 (26.8% versus 14.0%). deaths due to relapse were also higher in this group (17.1% versus 10.7%). in our series, myeloablative conditioning regimen provides good survival rates and disease control in high risk hematopoietic diseases, however in patients aged ⩾ 55 years confers high toxicity. it may be necessary to evaluate other strategies in this group of patients. disclosure of conflict of interest: none. allogenic hematopoietic cell transplantation (hct) is reserved for a group of high risk multiple myeloma (mm) patients having relapsed after high-dose melphalan and autologous transplantation. in general, reduced-intensity conditioning (ric) regimen are applied in this patient group with the aim of reducing transplant related mortality (trm). however, relapse of disease remains a major challenge after allogeneic hct. to address this issue, we added radioimmunotherapy (rit) to a conventional ric regimen. we have used a 188 rhenium anti cd66 antibody in combination with a ric conditioning regimen. this ß-emitter leads to a so called 'cross-fire' effect allowing for bone marrow doses of 20 gy and in parallel may target cd66 on myeloma cells. we hypothesized that this strategy may decrease the incidence of relapse. so far, we have treated nine patients with high risk relapsed multiple myeloma. all patients had received one (n = 7), two (n = 1) or three (n = 1) high-dose regimens and autologous hct. conditioning therapy was flu/mel (n = 5), flu/ bu (n = 2) or flu/treo (n = 1). flu/cy and 2 gy tbi were applied before haploidentical transplantation. patients received g-csf mobilized pbsc from unrelated (n = 8) or haploidentical (n = 1) s334 donors. either tacrolimus/ methotrexate/ bortezomib or cyclosporine a/ methotrexate were used for gvhd prophylaxis. early extramedullary toxicity was limited. neutrophil and platelet engraftment was timely and complete in time in seven of nine cases. all patients achieved full donor chimerism around day fifteen after hct. severe acute graft-versus-host-disease (gvhd grade iii-iv) occurred in two patients and was lethal in both cases. two patients have experienced extramedullary relapse, one of them in the central nervous system and the other in the soft tissue. in two patients, a transplantation-associated thrombotic microangiopathy (ta-tma) was diagnosed. four patients are alive and in complete remission. we conclude that the combination of a ric regimen with a 188 rhenium anti-cd66 radioimmunotherapy is save and feasible. the incidence of gvhd, ta-tma and extramedullary relapse will be monitored closely and will be presented in a larger patient cohort. disclosure of conflict of interest: none. the sirolimus/tacrolimus (sir/tac) combination has been associated with a better outcome after allogeneic hematopoietic stem cell transplantation (allo-hsct) when compared with conventional prophylaxis for graft vs host disease (gvhd) as cyclosporine/methotrexate in the true reduced-intensity conditioning (ric) setting but not in the myeloablative setting. in moderate-intensity regimens as thiotepa/busulphan/fludarabine (tbf), the sir/tac combination has not been evaluated. from january 2009 to december 2015, all consecutive ric-allo-hsct recipients who received sir/tac combination to prevent gvhd in three spanish institutions were included in the study. the reduced-toxicity regimens used in this study where: (a) intravenous busulphan (6.4 mg/kg) and fludarabine 150 mg/ m 2 (bf), or (b) thiotepa days -4 and -3 and 10 mg/kg if 455 yrs old or 5 mg/kg if o55 yrs old) on days -6 and -5 added to the bf regimen (tbf). the gvhd prophylaxis with sir/tac was given as detailed elsewhere (cutler c blood 2014) and was consistent within the 3 center. the outcomes of the procedure according to the conditioning regimen were analyzed. overall, 125 patients were included: 66 tbf and 59 patients in the bf group, with a median follow-up of 22 months (range 3-83) and no difference in the median age (56 vs 58 years old). there were more males (71% vs 47%, p = 0.007) and more female donors to male recipients (35% vs 13%, p = 0.006) and more patients with lymphoid diseases and previous asct in the tbf group (27% vs 12%, p = 0.03), whereas there were more unrelated donors in the bf group (56% vs 76%, p = 0.02). other baseline characteristics were balanced between the 2 groups (table 1) . sir/tac prophylaxis had to be discontinued in 48% and 65% patients in the tbf and bf groups, respectively. toxicity was the main reason for discontinuation in the tbf group. the most frequent toxicities were renal injury (tbf 30% and bf 10%) and neurologic impairments (tbf 6%, bf 5%). in the bf group, the main reason of discontinuation was relapse or a mixed chimera. patients who received tbf presented higher incidence of extensive chronic gvhd (65% vs 39%, p = 0.02), higher nrm at 100 days (21% vs 4%) and at 2 years (42% vs 13%, p = 0.001). there were no differences in os (2 years) between both groups (49 ± 6.9% vs 80 ± 5.5%, p = 0.001) (figure) . there were no differences regarding to acute gvhd 2-4 (39% vs 36%, p = 0.31), acute gvhd 3-4 (23% vs 13%, p = 0.08), or relapse (up to 2 years, 22% vs 14%, p = 0.3) between the 2 groups, either. the combination of sir/tac as gvhd prophylaxis was associated with higher incidence of chronic gvhd and nrm in patients receiving conditioning regimen with tbf compared to those receiving bf. there were no differences in os between both groups. [p418] cr complete remission, pr partial remission, nr non remission, hct-ci hematopoyetic cell transplant comorbility index. disclosure of conflict of interest: none. reduced-intensity conditioning regimen with fractionated total body irradiation of 6 gy and cyclophosphamide 60 mg/kg for allogeneic hematopoietic stem cell transplant is well tolerated and offers a potential disease control as treatment of acute leukemia and lymphoproliferative disorders m adler, t girinsky 1 , s koscielny, g ferini, s wittnebel, s mayeur, c chahine, m vanghele, s pilorge, c castilla-llorente and j-h bourhis 1 the use of reduced-intensity conditioning regimens (ric) before allo-hsct is widely extended since it preserves the graft-versus-leukemia effect but reduces treatment related mortality. however, there exist different ric regimens with diverse outcomes and the choice of the ric regimen relies on the type of disease treated, experience of the center and previous therapies. this is a retrospective study of patients treated in our institution within 01/2000 and 12/2015. the ric regimen consisted of fractionated total body irradiation (ftbi) of 6 gy administered in 3 consecutive days (2 gy/day) and cyclophosphamide 60 mg/kg given in 2 days (30 mg/kg/day). post-transplant immunosuppression consisted of csa started the day before allo-hsct and short mtx on days 1, 3 and 6 after transplantation. for patients receiving transplant from unrelated donors, anti-thymocyte globulin at a dose of 5 mg/ kg (2.5 mg/kg/day for 2 days at day -2 and -1) was used as part of the immunosuppressant therapy. 78 patients (median age: 54 years: range: 36-64 years) were included. the median hct-ci was 0.5 (range: 0-4). primary disease was multiple myeloma (mm) in 45 (58%), al/mds in 14 (18%), cll in 10 (13%), nhl in 9 cases (12%) . 51 patients (65%) received transplant from matched related donors, 22 (28%) from matched unrelated donors and 5 (6%) from mismatched unrelated donors. female to male mismatch incidence was 23% (n = 18). most of the patients (n = 77) received a peripheral blood graft. 1 patient received a second allogeneic transplant. mm patients were transplanted in a "tandem" autologous-allogeneic hsct program in 42 cases. the median number of chemotherapy lines prior to transplant was 3.5 in cll, 2.8 in mm and 2.5 in nhl. 62 patients (91%) engrafted by day 28 post transplant. neutrophil engraftment occured at a median of 19 days (range: 14-35 days) and platelet engraftment at a median of 18.5 days (range: 9-103 days). full donor chimerism was observed in 62 out of 67 patients (92%) having survived by day 180. primary graft rejection was observed in 4 patients. treatment related toxicities consisted of grade 3/4 mucositis in 53 patients (68%), grade 3 (range: 2-4) cardiac toxicity in 6 patients (8%), grade 3 (range: 3-4) hemorrhagic complications in 4 patients (6%) including 3 cases of hemorrhagic cystitis and secondary malignancies in 4 patients, this within a median follow-up of 6.6 years. infectious complications during aplasia included fever of unknown origin (n = 52), bacteremia (n = 17) with 3 cases of bacteremia with severe sepsis and 8 cases of infections defined by bacterial foci. incidence of agvhd was 33% with 3 cases of grade 3/4 refractory agvhd. cgvhd occurred in 30 pts (39%). the non-relapse mortality (nrm) at 100 days was 5% including 2 cases of septic shock, 1 case of acute cardiac toxicity and 1 case of agvhd. the nrm at 1 year was 10%. 1-year survival rates were 60% in al, 80% in cll and 88% in nhl with extended survival benefit. in al patients, the relapse incidence was 36% comprising 2 patients who progressed during conditioning. the 1-year survival rate in mm patients was 77%. in mm patients who were in complete response prior to transplant, median overall survival was 4.6 years. the used ric regimen resulted in durable donor engraftment with an acceptable toxicity profile permitting efficient disease control in the described cohort. disclosure of conflict of interest: none. graft manipulation using selective depletion of αβ-t cells provides a source for haploidentical hematopoietic stem cell transplantation (haplo-hsct) enriched in effector cells. initial reports demonstrated safety and rapid immune reconstitution using this method, in malignant and non-malignant disorders using several proposed conditioning regimens. no specific considerations were given to hematologic malignancies. we reviewed a total of twenty seven pediatric patients who underwent haplo-hsct using αβ-t cell depletion between 2013-2016 in a single tertiary referral center. we report the results of 22 procedures performed in eighteen patients transplanted for malignancies. twenty two haplo-hsct were performed in eighteen patients. the indication for hsct was acute leukemia in sixteen (all = 11, aml = 5) and neuroblastoma in two. median age at hsct was 5.6 years. six patients had failed a prior hsct, and the remainder had no matched donor. the initial reduced-toxicity conditioning regimen consisted of melphalan, fludarabine, thiotepa and atg, and resulted in a high rate of graft rejections (7 of 9). thus, a totalbody irradiation (tbi)-based regimen was implemented, with prompt engraftment in all the patients. we observed rapid neutrophil and platelet engraftment kinetics (median time to engraft, 10 days and 11.5 days, respectively). significant treatment related complications were all due to graft failure in patients receiving reduced-toxicity conditioning, with two infection-related mortalities in the presence of prolonged neutropenia. none of the patients developed hepatic sinusoidal-obstruction syndrome, and no grade 3-4 acute graft-versus-host disease (gvhd) or chronic gvhd were observed with either regimen. importantly, the majority of patients with acute leukemia were free of immunosuppression in the first 100 days post hsct. the 2-year actuarial event-free and overall survival of the entire cohort were 34% and 55% respectively, with results for tbi-based conditioned patients being 58% and 88%. overall, we demonstrated that a tbibased conditioning for haplo-hsct using αβ-t cell depletion for malignant diseases resulted in acceptable outcomes in these high-risk patients without increased toxicity. disclosure of conflict of interest: none. high-dose chemotherapy conditioning regimens followed by autologous stem cell transplantation (auto-sct) generally provide good results in relapsed and refractory lymphomas. we evaluated the efficacy and safety of tecam regimen as conditioning with autologous stem cell support in patients with relapsed/refractory lymphomas. thirty-two (16 patients were refractory, 15 patients were relapse and one frontline treated) patients (21 m, 11 f) with lymphoma at various stages (stage ii, 19%; stage iii, 22%; stage iv, 59%) who underwent asct were included in this retrospective study. the median age at transplantation was 52.5 years (range, 28-69 years). the diagnosis were as follows: 9 diffuse large b-cell non-hodgkin lyphoma (nhl), 9 hodgkin lymphoma (hl), 5 mantle cell lymphoma, 3 follicular lymphoma, 3 marginal zone lymphoma and 3 t-cell nhl. all patients received tecam as conditioning regimen that consist of thiotepa (40 mg/m 2 × 4 days), etoposide (200 mg/m 2 × 4 days), cyclophosphamide (60 mg/ kg × 1 day), ara-c (200 mg/m 2 × 4 days) and melphalan (60 mg/m 2 × 2 days). median cd34(+) cells were 6.7 × 10 6 /kg (range; 1.9-19.3 × 106/kg) which were infused at day 0, followed by recombinant human granulocyte colonystimulating factor (rhug-csf) at a dose of 5 μg/kg/day. the median time between mobilization and auto-sct was 2 months (range; 1-13 months). the median time to recovery of absolute neutrophil and platelet counts independent of transfusion were 11 (range; 9-19) and 14 (range; 10-41) days, respectively. the median stay in hospital was 28 days (range, 11-108 days). bacterial, sitomegalovirus and invasive fungal infection were detected in 11 (34%), 4 (13%) and 2 (6%) patients, respectively in first 100 days of auto-sct. three s336 patients (9.3%) died from transplant-related complications. the overall response rate was 75% (22 cr, 68.8%; 2 pr, 6.2%) after auto-sct. relapse developed in 7 patients during median follow-up period of 6.5 months (range; 1-21 months) after auto-sct. the 1-year estimated dfs ( figure 1 ) and os were 70% and 45%, respectively. no statistical significance was observed for os and pfs in terms of gender, patient age ( o60 and ⩾ 60 years) and nhl and hl lymphoma group (p ⩾ 0.05). the tecam regimen for auto-sct in lymphoma seems to provide encouraging results in terms of response and its good tolerance with acceptable toxicity. [p421] disclosure of conflict of interest: none. allogeneic hematopoietic cell transplantation (allosct) is the only curative treatment for myelofibrosis. however, its widespread use is limited by early non-relapse mortality (nrm). the optimal modalities of the conditioning regimen are a major unmet clinical need. in an attempt to reduce early nrm, we used a tbf conditioning regimen (thiotepa, busulfan (bu), fludarabine (flu) and antithymocyte globulin (atg)). our aim was to reduce nrm and improve engraftment by using such tbf conditioning. thirty consecutive patients with a median age of 56 years (range, 32-69) who underwent allosct for primary (n = 18) or secondary (n = 12) myelofibrosis were included. according to the refined dynamic international prognostic scoring system (dipss-plus), patients were stratified as intermediate-1 (n = 3), intermediate-2 (n = 6), and high (n = 16) risk. five patients had blast transformation. ruxolitinib was given to 14 patients (47%) prior to allosct. graft source was pbscs in 26 patients (87%) and bm in 4 patients (13%). donors were matched related (mrd, n = 6), unrelated (n = 19) and haploidentical (n = 5). conditioning regimen was tbf in 18 patients (60%). in our historical cohort 8 patients (27%) received fb (flu, bu, atg). in addition, 4 patients received a 'tec-ric' sequential conditioning (thiotepa, etoposide, cyclophosphamide, and after 3 days rest, flu, bu and atg) for blast transformation (n = 2) or refractory proliferative myelofibrosis (n = 2). gvhd prophylaxis consisted of cyclosporine (csa) and mycophenolate mofetil in 26 patients (87%), csa and short course methotrexate in 3 patients (10%) with abo mismatch and csa alone in 1 patient (3%) with mrd. high dose posttransplant cy (pt-cy) was added in haplo cases. no significant difference was observed between tbf, fb and tec ric patients in terms of age, gender, karnofsky score, comorbidity index, number of previous treatment line, history of ruxolitinib administration and source of stem cells. median follow-up was 20 months (range, 3-75). two tbf patients died of septic shock before engraftment at day +12 and +19 after allosct, respectively. one fb patient died of graft failure at day +108 post allosct. median time to neutrophils and platelets (420 g/ l) recovery was 15 days (range, 9-28) and 20 days (range, 1-55) with tbf, 17 days (range, 14-53) and 18 days (range, 7-50) with fb, and 19 days (range, 15-24) and 14 days (range, 14-58) with tec ric. grade ii-iv acute gvhd occurred in 27.8% of tbf patients, 37.5% of fb patients, and 25% of tec ric patients (p = 0.90). moderate chronic gvhd developed in 1/13 evaluable tbf, 2/7 fb and 0/4 tec ric patients. no severe forms of chronic gvhd were observed. at last follow-up, 1 patient relapsed, 12 died and 18 are still alive. main causes of death were disease progression (n = 1), infection (n = 9) and gvhd (n = 2). nrm at 2 years was 33.3% in tbf patients, 50% in fb patients, and 25% in tec ric patients. the 2-year os were 66.7% in tbf patients, 37.5% in fb patients, 75% in tec ric patients, respectively. cd34+-selected stem cell boost without further conditioning allowed to 4 patients for poor graft function, with significant hematological improvement in 3 patients. tbf conditioning regimen seems to be efficient in allosct for patients with myelofibrosis and compares favorably with previously published fb regimens. these preliminary results give a rationale to support a prospective evaluation of this platform. disclosure of conflict of interest: none. we proposed here to compare the outcome of patients receiving either thymoglobuline (atg), a rabbit anti-human thymocyte immunoglobulin or campath, a recombinant dna-derived humanized monoclonal antibody directed against cd52. campath and atg are both commonly used as in vivo tcd before hsct, respectively in united kingdom and france but very few comparing data are available. all consecutive patients with acute myeloblastic leukemia (aml), myelodysplastic syndrome (mds) or myeloproliferative neoplasia (mpn) who received a reduced intensity hsct from an unrelated donor transplanted between 2006 and 2015 were included in this study. a propensity score was used to identify and control potential confounding to relate the treatment group to the outcomes. in the matched sample, cox regression model was used to describe the association between treatment and outcomes. 322 patients have been included. all patients received fludarabine and busulfan with either atg (n = 153) or campath (n = 169). patients treated by atg received cyclosporine plus mycophenolate mofetil or methotrexate and patients treated by campath received cyclosporine alone as gvhd prophylaxis. comparing patient and transplant characteristics, atg patients were older (62 vs 60 years), had less often aml (52 vs 69%), had higher disease risk (adverse dri: 14 vs 9%; poor cytogenetics: 25 vs 11%; high cibmtr score: 41 vs 28%), were less often in complete remission at time of transplant (62 vs 76%) and were transplanted less often from a mismatched hla donor (16 vs 26%). cumulative incidence of sustained engraftment was in 98% and 99% campath and atg patients. time to neutrophil engraftment was longer in atg patients (19 vs 13 days). acute gvhd ii to iv rate were higher after atg (44% vs 19%) as well as chronic extensive gvhd (26% vs 13%). relapse rate was higher after campath (44% vs 27%). disease-free survival (dfs) was higher after atg (53 vs 37%) and the gvhd-free relapse free survival (grfs) was similar (35% vs 32%). according to the prognostic factors for outcome, a propensity score was developed selecting 234 patients from the original cohort. the estimation of tcd effect was than studied. relapse risk was higher in patients treated by campath while there is a non-significant advantage for atg in dfs (table 1) . [p423] tcd with atg or campath gives similar os, dfs and grfs. severe acute or chronic gvhd is lowered by campath but the higher relapse risk counterbalances the potential benefit of campath finally given similar os. nevertheless, lower risk disease patient might benefit from campath while higher risk patients might benefit from atg. disclosure of conflict of interest: none. high-dose chemotherapy (hdt) with autologous stem cell transplantation is the standard of care for relapsed/refractory (rr) or high grade non-hodgkin-lymphoma (nhl) and hodgkin-lymphoma (hl)2. the standard hdt in autologous stem cell transplantation (asct) for lymphoma is carmustinebased hdt using a combination of carmustine, etoposide, cytarabine and melphalan (beam); this standard conditioning programme is used by most groups worldwide1. we have designed novels hdt regimens in which carmustine was substituited by an equal dose of fotemustine (feam)3 or thiotepa (team) and we compared these two hdt regimens in terms of engraftment times, toxicity, tolerability and frequency of relapse after asct. from february 2011 to september 2016 we consider a total of 67 relapsed/refractory patients affected by hl and nhl respectively 18 hl and 49 nhl with different grade of initial disease (grade i-iv) and different response to prior treatments. the all other drugs were administered according to a standard beam regimen1. after a day of rest, autologous peripheral blood progenitor cells were infused on day 0, followed by s.c. g-csf (5 mg/kg) from day 1 of asct until 2 consecutive days when the ancs were 4500 × 109/l3. the primary objectives of the study were to assess the feasibility and safety of the feam and team regimens in terms of acute toxicity, grade of mucositis, hemopoietic engraftment and relapse after asct. acute toxicity include chemotherapy-induced nausea and vomiting, diarrhea, hepatotoxicity, nephrotoxicity and infection complication. in all 67 patients cd34+ cells were collected from peripheral blood and the median number of infused cells per patient was 5.79 × 10e6/kg. the median time of engraftment was 9 days for neutrophil recovery (n4500 × 10 9 /l) and 11 days for plt recovery (420 000 × 10 9 /l). acute toxicity occurred in 14 total patients (20.8%), mucositis grade 3-4 occurred in 34 patients (50% of cases). frequency of relapse in all 67 cases was 43.2%. feam conditioning regimen was used in 41 cases showing a median time of neytrophil recovery of 10 days and a median time of plt recovery of 11 days. acute toxicity occurred in 4 of these cases (9.7%), mucositis grade 3-4 occurred in 18 patients (43.9% of cases). frequency of relapse in feam group of patients was 41.4%. team conditioning regimen was used in 26 cases showing a median time of neytrophil recovery of 9 days and a median time of plt recovery of 11 days. acute toxicity occurred in 10 of these cases (38.4%), mucositis grade 3-4 occurred in 16 patients (61.5% of cases). frequency of relapse in team group of patients was 50%. relapse/progression of lymphoma and conditioning regimen toxicities remain limitations to treatment success. the two novels hdt regimens feam and team are safe and feasible and show similar engraftment times, tolerability and frequency of relapse. maybe the team regimen shows toxicity slightly higher than feam regimen but longer follow-up is needed to evaluate fully its efficacy and long-term safety. disclosure of conflict of interest: none. treosulfan is a prodrug of a bifunctional alkylating cytotoxic agent. there are few reports regarding toxicological side effects of treosulfan-based conditioning prior to hsct. here we report on incidence of early potential treosulfan-related toxicity in 118 patients treated with treosulfan-based conditioning before hsct. treosulfan was given at a dose of 14 g/m 2 /d for 3 days in combination with fludarabin 30 mg/m 2 /d for 5 days prior to hsct. most patients (n = 93) had a haematological malignancy, while 25 patients had a non-malignant disorder as hsct indication. an hla-a, -b and -dr matched unrelated donor (mud) was used in 80 cases, 33 patients had a hla-identical sibling donor and 5 received an hla-a, -b or -dr allele mismatched unrelated donor. as graft versus host-disease (gvhd) prophylaxis, most patients (n = 93) received cyclosporine and methotrexate. patients medical records were scrutinized retrospectively to collect laboratory tests (aspartate aminotransferase (ast), alanine aminotransferase (alt), creatinine) before hsct and then weekly until 5 weeks after hsct. levels of ast and alt were significantly increased 1 week after hsct compared to before hsct. however, only a few patients had transaminase levels over 2 or 3 times the upper normal level (unl) levels decreased sharply after the first week. most of the cases with high levels of ast/alt at one week had normal or close to normal levels before hsct. creatinine levels increased after week 2 but no patient had levels ⩾ 2 × unl. clinical features of all oral mucositis (om) were recorded using the world health organization (who) scoring system. most patients (68%) had no or very limited (grade i) om, 22% had grade ii and 10% had grade iii or iv of om. according to our toxicological results this is low-toxic protocol. however, all patients became neutropenic, 61% already at the time of graft infusion, indicating that the protocol has a myelo-toxic effect comparable to conventional mac protocols. all patients engrafted, except three patients who died very early. median time to neutrophil and platelet engraftment was 18 (range 10-31) and 15 days (9-55), retrospectively, which is significantly later when compared to engraftment data for other ric protocols used at our centre (data not shown). median duration of neutropenia (o0.5 × 10 9 /l) was 17 days , comparable to what is expected after conventional mac conditioning. secondary graft failure (gf) occurred in 8 (6.8%) patients, all having a nonmalignant disorder and 6/8 having a urd. non-relapse mortality (nrm) was 7.5% (95% ci 3.7-13.3%) at 100 days and 11.9% (6.8-18.5%) at one year after hsct. causes of death within one year after hsct were: relapse 7, epstein-barr virus associated posttransplant lymphoproliferative disease (ptld) 4, other infections 4, organ failure 2, gvhd 2, hemophagocytic lymphohistiocytosis (hlh) 1. other infections occurring within 100 days after hsct were cytomegalovirus (cmv) reactivation 46 (39%), invasive fungal infection 6 (5.1%) and blood stream infection 47 (40%). veno-occlusive disease of the liver or sinusoidal obstruction syndrome (vod/sos) occurred in one patient and haemorrhagic cystitis in two patients. this study shows that early regimen-related toxicity after hsct was low despite similar marrow toxicities compared to mac regimens. disclosure of conflict of interest: none. allogeneic stem cell transplantation from haploidentical donors (haplosct) is an increasingly adopted option for patients (pts) with high-risk hematological malignancies. in our institution, we previously described a platform for unmanipulated peripheral blood stem cell (pbsc) haplosct using a calcineurin-free gvhd prophylaxis with sirolimus, micophenolate and anti-human t-lymphocyte immunoglobulin (atg) after conditioning with treosulfan and fludarabine (trramm; peccatori et al., leukemia 2015) . as an attempt to decrease relapse rate, especially in advanced-phase diseases, we designed a new phase ii prospective clinical trial intensifying conditioning regimen with the addition of 4gy total-body irradiation (tbi) (trramm4gy; eudract#2011-001534-42). we report results on 75 pts. 75 pts affected by aml (n = 49), other myeloid (n = 8) and lymphoid (n = 18) malignancies were prospectively enrolled from may 2010 to june 2015. median pts age was 45 y (range 17-67). revised disease risk index (r-dri) was low or intermediate in 31 pts, high in 34 pts and very-high in 10 pts. twenty-five pts had previously received an allogeneic stem-cell transplantation with a median time from 1st to 2nd sct of 17 months . median hct-comorbidity index by sorror et al. was 1 (0) (1) (2) (3) (4) (5) . pts received a myeloablative conditioning regimen consisting of treosulfan (14 g/m 2 /d from -6 to -4), fludarabine (30 mg/m 2 /d from − 6 to − 2) and tbi 4gy (fractionated in 2 doses, from − 1 to 0). source of stem cells were unmanipulated g-csf-mobilized pbsc from haploidentical donors. gvhd prophylaxis consisted of atg-fresenius (grafalon, neovii) 10 mg/kg/d from − 4 to − 2, rituximab 200 mg/m 2 on − 1, mycophenolate mofetil 10 mg/ kg from − 1 to +30 and sirolimus (target concentration 8-15 ng/ ml) from − 7. median infused cd34+ and cd3+ cell doses were 6.9 × 10⁶/kg and 2.15 × 10⁸/kg, respectively. median follow-up for survivors was 48 months (5-74). neutrophil engraftment occurred in 95% of pts with a median of 17 d (9-47), platelet engraftment was reached in 76% of pts with a median of 17 d (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) . the 100-d cumulative incidence (ci) of grade ⩾ 2 acute gvhd (agvhd) was 31 ± 10% and of grade ⩾ 3 agvhd 27 ± 10%; the 2 years ci of chronic gvhd was 34 ± 11%. the ci of transplant-related mortality (trm) at 1 y and 4 y were 31 ± 10% and 34 ± 11%, respectively. the ci of relapse at 1 y and 4 y were 27 ± 10% and 34 ± 11%, respectively, with a median time to relapse of 90 d . interestingly, we did not observe any extramedullary relapse; loss of mismatched hla-haplotype occurred in 33% of relapses. among 36 pts who were in active disease at time of haplosct and who were evaluable, 94% achieved complete remission (cr) and full donor chimerism at day+30. the 1y and 4y probabilities of disease-free survival (dfs) were 42 ± 11% and 33 ± 11%, respectively. at 4 y, 27% of pts are alive, disease-free and immunosuppression-free minimal residual disease, tolerance, chimerism and immune reconstitution the number of human leukocyte antigen (hla)-mismatched hematopoietic cell transplantation (hct), including cord blood transplantation, has been increasing. hla-flow method can discriminate mismatched hla antigens between donor and recipient by using flow cytometry, and can evaluate minimal residual disease (mrd) or chimerism after hla-mismatched hct. by developing more simple methodology, hla-flow might be more widely applicable. we have developed modified 6-colorbased hla-flow method. the aim of this study is to evaluate the utility of the 6-color-based hla-flow for monitoring of mrd and chimerism after hla-mismatched hct in children. from june 2013 to november 2016, serial monitoring of mrd or chimerism by the 6-color-based hla-flow was performed in twelve patients undergoing hla-mismatched hct (46 tests). nucleated cells obtained from bone marrow were stained by immunofluorescent antibodies against hla antigens mismatched between donors and recipients. these cells were also stained by immunofluorescent antibodies against surface antigens such as cd3, cd19, cd33, cd34 and cd16/cd56 for determining lineage of the cells. these surface antigens were also used as a marker of leukemic blasts in the mrd study. we used 6-color-based flow cytometry (facs-navious) and the data were analyzed with flow jo. erythroblasts and dead cells were excluded from the analysis. in each study, at least 105 cells were analyzed. for mrd analysis, we concurrently tested real-time quantitative polymerase chain reaction (pcr) of peripheral wt1 mrna or leukemia-specific fusion genes. pcr of polymorphic short tandem repeats or fluorescent in situ hybridization of x/y chromosomes was concurrently tested for chimerism study. age of patients ranged from 0 to 16 years. donor sources included bone marrow (n = 9) and cord blood (n = 3). for mrd monitoring of acute leukemia (n = 9), the 6-color-based hla-flow could detect mrd in three patients. five patients have not experienced relapse. no discordance with other mrd markers was observed in these patients. hla-flow could not separate donor-derived cells from recipient-derived ones in one patient receiving bone marrow transplantation. as for chimerism testing (n = 3), the 6-colorbased hla-flow could successfully evaluate quantitative lineagespecific chimerism in all patients. there is no discrepancy between hla-flow and other methods. we could complete evaluation of the 6-color-based hla-flow within two days in all tests. the 6-color-based hla-flow is a simple, quick and useful method for the quantitative evaluation of mrd and lineagespecific chimerism after hla-mismatched hct in children, irrespective of donor sources. it is thought that our method is applicable in all institutions owing 6-color-based flow cytometry. acknowledgement: we thank drs. nobukazu watanabe, eri watanabe and natsuko sato (university of tokyo) for their technical advices. we also thank drs. tomoko okunushi (chiba university), hidefumi hiramatsu and katsutsugu umeda (kyoto university) for their care of patients involved in this study. disclosure of conflict of interest: none. survival (pfs), cumulative incidence of relapse (cir) and acute/ chronic gvhd incidence in aml patients (pts) submitted to allo-hsct at our institution between january 2003 and december 2014. this retrospective study evaluated 122 aml pts submitted to allo-hsct from 56 matched sibling donors (msd) and 66 matched unrelated donors (mud) who provided bone marrow (bm) or peripheral blood as stem cell grafts.ir was evaluated at 100, 180 and 365 days post-transplant in all 122 pts. cmv-dna copies were determined in peripheral blood by quantitative pcr twice weekly in the first 100 days post-transplant and subsequently once weekly. cmvi/r was analyzed as a timedependent covariate.effect of cmvi/r on os and pfs was estimated by cox proportional hazard model. cir and gvhd incidence were analized with a competing risk approach, considering death from any cause as a competing event. effect of cmvi/r on cir and gvhd were evaluated by fine & gray model. median age at allo-hsct was 50.7 years (19.1-68.9 ). in our population 68% of donors were seropositive for a previous cmv infection and pts transplanted from these donors showed a significantly lower cumulative incidence of cmvi/r than pts transplanted with seronegative ones (shr = 0.56, 95%,ci:0. our study demonstrates that cmvi/r influences the success of allo-hsct by determining a better ir characterized by a higher cd8+ cell number that might exert an immune protective control on disease outcome by improving os,pfs and cir with no effect on gvhd. another factor of utmost importance to achieve this same goal might be constituted by the significantly increased nk cell number six months after allo-hsct. to assess the dynamics of molecular response to treatment in aml adult patients with concomitant flt3 and npm1 mutations. this retrospective single center studystudy was approved by the institutional review board of american university of beirut medical center. twelve consecutive newly diagnosed (n = 11) or relapsed (n = 1) aml patients received idarubicin/cytarabine induction and one or two consolidation (s) ( table 1) . seven patients received allogeneic stem cell transplant (allo-sct) and 3 had haploidentical-sct (hap-lo_sct); all followed by post-transplant sorafenib maintenance. median follow-up was 11.5 (6-27) months. all transplanted patients remain alive and disease free.flt3 mutation was tested on dna using a qualitative method with a sensitivity of 0.01%. npm-1 mutation was tested on cdna using a qualitative or a quantitative rt-pcr with a sensitivity of 0.01% and 0.008 ncn respectively. patients were tested at diagnosis, after induction, after each consolidation, before and s341 at days 30, 60 and 100 after allo-sct for kinetics of npm1 and flt3 molecular response. after induction, flt3 became negative in all tested patients (n = 10). after first consolidation, flt-3 was not tested in 3 patients who had a negative result after induction, was negative in 8 patients including the 2 patients who were not tested after induction, whereas a molecular relapse was noted in one patient who developed a hematological relapse and rapidly died. another patient died after the third consolidation, in complete remission, due to septic shock. no molecular positivity for flt-3 was noted later on, whether after second consolidation or post-transplant. conversely, npm-1 mutation became negative in 2 out of 12 tested patients after induction, in 1 additional patient after first consolidation and in 7 additional patients after sct, mostly after starting sorafenib. npm-1 mrd value remained elevated in 3 out of 4 patients with quantitative assessment at diagnosis and post induction (figure1). flt3 become negative early after induction while npm1 negativity lags behind. persistent npm-1 mrd does not seem to predict post-transplant outcome and may indeed become negative after sorafenib. these results need confirmation in larger studies. disclosure of conflict of interest: none. in allogeneic stem cell transplantation (allo-sct), an early detection of the transplant outcomes such as overall survival (os), event-free survival (efs), cumulative incidence of relapse (cir) and non-relapse mortality (nrm) is fundamental regarding the use in time of additional therapy after sct. therefore, we investigated the association between early immune reconstitution (ir) on day +30 after allo-sct and outcomes in children suffering from acute leukemia or myelodysplastic syndrome (mds). this study collected data from 188 allo-sct from january 2005 until december 2014 in our institution. the median survival follow-up was 38 months. indications of allo-sct were all (n = 113, 60%), aml (n = 44, 23%) and mds (n = 31, 17%). the median age was 10 years (range, 0.6 -18). patients were transplanted in cr (n = 131, 70%) and pr/nr (n = 57, 30%). patients included in the study received 1st sct (n = 170, 90%), 2nd sct (n = 15, 8%) or 42 sct (n = 3, 2%). grafts were from sibling (msd; n = 42, 22%), matched unrelated (mud; n = 95, 51%), haploidentical (n = 45, 24%) or mismatched unrelated (mmud; n = 6, 3%). conditioning regimens were tbi-based (n = 87, 46%) or chemo-based (n = 101, 54%). stem cells were from bone marrow (n = 118, 63%) or peripheral blood (n = 70, 37%). we analyzed the absolute count of lymphocytes (alc), monocytes, cd3+ t cells, cd3 +cd4+ t-helper cells, cd3+cd8+ cytotoxic t-cells, cd3-cd56+ natural killer (nk) cells and cd19+ b cells assessed on day 30 ± 5 after sct. we used the percentiles of the lymphocyte subsets of the same cohort to categorize the samples throughout the study. patients with alc over the 50th percentile of alc (alc o 327 cells/μl) had a 1.97-fold increased hazard ratio (hr) to develop relapse (p = 0.017). nk cell counts on day 30 after sct were strong associated with os, efs, cir and nrm. patients with nk cell count over the 75th percentile (nk 4 268.8 cells/μl) had increased hr for os (hr = 1.8, p = 0.01) and efs (hr = 2.01, p = 0.017) compared to patients with nk count under the 75th percentile. patients with nk cells over the 25th percentile (nk o52.5 cells/μl) had a hr = 3.3 (p = 0.009) for relapse and hr = 0.364 (p = 0.016) for nrm compared to patients with nk cell count under the 25th percentile. monocyte cell count on day 30 correlated with os, efs and cir. patients with cd14+ cells count under the 15th percentile of cd14+ (cd14+ o242 cells/μl) has an increased hazard ratio for os, efs and relapse compared to patients with cd14+ cell counts over the 15th percentile. no association between absolute cell count of cd3+, cd4+, cd8+ and cd19+ on day +30 after allo-sct and any outcomes either os, efs, cir or nrm was found. the study confirms the strong association between early ir and outcomes after allo-sct in children. our study suggests that especially nk cell and monocyte cell count on day +30 may have significant prognostic implications. our findings suggest that the cells count of alc, nk cells and monocytes on day +30 after allo-sct could be useful to predict outcomes after allo-sct and should be taken into account in considering alternative treatment. disclosure of conflict of interest: none. early immune reconstitution (eir) has proven to be a significant determinant for the outcome of allogeneic hematopoietic stem cell transplantation. in the setting of unmanipulated haploidentical transplantation (haplo-hsct), some groups have identified the absolute leukocyte count on day +30 (alc30) as an independent prognostic factor in terms of overall survival (os), disease free survival (dfs) and infectious mortality (im). the aim of this study was to evaluate the impact of eir on os, dfs and im among patients who underwent haplo-hsct with postransplant cyclophosphamide (ptcy) at our institution. from july 2011 to april 2016, 83 haplo-sct were performed at our institution. threedied before day 30 after haplo-sct, and 13 patients had missing data. conditioning regimen consisted of fludarabine, cyclophosphamide and busulfan. twenty-nine patients received a reduced intensity conditioning regimen (1-2 days of busulfan) while 37 a myeloablative regimen (3-4 days of busulfan). gvhd prophylaxis comprised ptcy, cyclosporine and mycophenolate mofetil. patients were assessed for eir by means of alc30, cd3+ t lymphocyte count on +30 (cd3), nk lymphocyte count on +30 (nk) and nk cd56 bright percentage on +30(cd56 br ). we analyzed 66 pts, with a median follow-up of 21 months (9-36). the median age of the pts was 43 (range 30-57), 76%men. diagnosis were: aml(32%), hl (23%)non-hl (17%), all (8%),mds(8%), cml(6%), others(6%). 55% were in complete remission at the time of transplant, 21% in partial remission and 24% had overt disease. in terms of infectious complications, cmv reactivation was documented in 76% of the pts, 1% developed a proven invasive fungal infection and 26% suffered from bk+hemorrhagic cystitis. median os and dfs were 21 (9-36) and 17 months (7-31), respectively. im rate was 21% at the end of follow up. median follow-up was 21 months (9-36). roc curves were used to determine the optimal cut-off values for each of the studied parameters: 300 cells/μl for alc30, 120 cells/μl for cd3, 41 cells/μl for nk and 83% for cd56 br were chosen. pts with alc30 ⩾ 300/μl had better os (p = 0.005) and dfs (p = 0.05), than those with alc30 o 300/μl. median os and dfs were 25 months vs not reached (nr) and 22 months vs nr, respectively. pts with cd56 br ⩾ 83% had better os (p = 0.04) than those with lower values. median os was 25 months vs nr; however no difference was seen in terms of dfs. we didn´t observe statistically significant differences in os or dfs, among pts with different levels of cd3 and nk on +30. cumulative incidence of im was significantly lower in pts with an alc30 ⩾ 300 (p = 0.04), pts with cd3 ⩾ 120/μl (p = 0.006) and pts with nk ⩾ 41 (p = 0.04); patients with cd56 br ⩾ 83% showed tendency to have lower cumulative incidence of infectious mortality (p = 0.24, non-significant). cumulative incidence of relapse was not affected by alc30, cd3, nk or cd56 br . our study supports the independent prognostic value of early immune reconstitution after unmanipulated haploidentical transplantation with ptcy, especially in terms of lower infectious mortality. os and dfs were better among patients with alc30 ⩾ 300 cells/μl. pts with cd56 br ⩾ 83% also showed better os. no correlation was found between cd3 or nk on +30 with os or dfs. cumulative incidence of infectious mortality was affected by alc30, cd3 and nk on +30; while cd56 br seems to have less impact. [p431] disclosure of conflict of interest: none. an early absolute lymphocyte count (alc) recovery after autologous stem cell transplantation (asct) for hematologic malignancies has been related with an improved transplant outcome due to a faster autologous immune restoration. in this retrospective study we analyze post-transplant survival of non hodgkin lymphoma (nhl) patients and its relation with alc at day +15 post-asct. we analyzed 53 consecutive adult nhl patients who underwent asct at the hematology and sct department of hospital maciel (montevideo, uruguay). only individuals with at least 6 months post-transplant follow up were included. all patients received beam (bcnu, etoposide, cytarabine and melphalan) conditioning regimen followed by peripheral blood stem cells previously collected by apheresis. median cd34+ cell dose was 4.13 × 10e6/kg (1.62-12.58). median alc at day +15 was 500/μl. patients were divided into two groups: alc at day +15 inferior than 500/ul (group 1) and alc at day +15 superior or equal than 500/ul (group 2). differences between groups were analyzed using t-student and chi-square tests, with statistical significance determined at p o0.05. disease free survival (dfs) and overall survival (os) were analyzed by kaplan meier method. differences in survival between groups were determined by log-rank test. no differences were observed between groups regarding gender, histology, disease status at transplant and cell dose. patients in group 1 were older and more heavily pretreated. neutrophils and platelets engraftment were significantly faster in group 2 (table 1) . after a median follow up of 36 months, disease-free survival (dfs) and overall survival (os) were superior in group 2. median dfs was 47 months and not reached (p = 0.019) and os was 51 months and not reached (p = 0.016) in groups 1 and 2 respectively (figure 1 ). an early alc recovery after asct was associated with a superior dfs and os in nhl patients. individuals with alc major or equal than 500/ul had also a shorter time to neutrophils and platelets recovery and a shorter hospital stay. in this study, cd34+ cell dose does not seems to be a determinant factor for lymphocyte recovery. the load of previous treatment may influence lymphocyte recovery after asct. these results support the association between early post-asct lymphocyte recovery and improved survival in nlh patients. [p432] disclosure of conflict of interest: none. t cell depletion (tcd) reduces the risk of graft versus host disease (gvhd) but also the graft versus leukaemia (gvl) effect, thus increasing the risk of relapse. donor lymphocyte infusions (dli) can be given to boost donor chimerism, with the intention of enhancing the gvl effect. 1 it is not currently known whether giving dli based on bone marrow chimerism (bmc) influences survival, or whether certain groups of patients benefit more from dli than other groups. in addition, it is not known whether the overall aim of achieving 100% bmc associates with improved survival. we investigated whether day 100 (d100) bmc was predictive of survival, and whether giving dli based on this result was associated with improved overall survival. data were retrospectively collected from case notes and laboratory reports for patients who underwent allogeneic stem cell transplant (allosct) for aml or mds at the northern centre for bone marrow transplantation between 2010 and 2015. patients who died prior to d100 were excluded from the analysis. of the 147 patients analysed (aml 117, mds 30), 68% were male and 38% female. the median age was 59 years (range 22-74). conditioning was with flu/bu/ alemtuzumab (63), flu/mel/alemtuzumab (45), cy/tbi alemtuzumab (7), flamsa tbi/bu atg/alemtuzumab (27), other (5) . 103 (70%) received a graft from an unrelated donor, 42 (29%) a matched sibling donor and 2 (1%) another source. 143 (97%) received mobilised pbscs, 3 (2%) bone marrow and 1 (1%) cord blood. statistics were performed using graphpad prism. p values were calculated using the chi square test and taking po0.05 to determine significance. bmc was divided into 3 groups 100%, 90-99% and o 90%. 100% bmc at d100 was associated with a significant increase in 2 year overall survival (os) (74.6% vs 57.3% and 33.4% for 90-99% and o90%, respectively, p o0.0012). patients with a d100 bmc o80% had a 2 year os of o10% (with relapse the cause of death in 90%). in patients whose d100 bmc was o 90%, there was a significant improvement in 2 year os seen in those who received dli (61.7% survival at 2 years vs 0% with no dli, po0.0026) (figure 1 : os by d100 bm chimerism (with and without dli). attainment of 100% bmc at a subsequent time point also significantly improved survival in those with a d100 bmc of 90-99% (79.4% 2 year survival vs 33.6% who never attained 100%, p o0.001) and o90% (100% 2 year survival vs 14.7%, p o0.006). we found d100 bmc to be predictive of os in this population. in addition, dli was associated with an improvement in os, especially in patients whose bmc at d100 was o90%. there was also a statistically significant improvement in os seen in patients who subsequently attained a 100% bmc, where it was o100% at d100. the objectives of this analysis were to examine the optimal alc recovery cutoff utilizing receiver operator characteristics (roc) analysis and to examine infused allograft characteristics associated with early alc recovery. after due irb approval, patients (pts) with aml and all who underwent hct at our institution between 2010-2015 were identified. pts with t-cell depletion or maintenance post hct were excluded. data were collected retrospectively from the patient's records. cellular contents of infused products (cd34, cd3, tnc, mnc, alc and amc) in addition to alc post hct were analyzed and optimal cutoff, if present, was established using roc analysis for the end point of relapse. time to end point analysis was computed using the kaplan-meier with log ranks. for competing events, cumulative incidence was computed using grey's model. univariable and multivariable analyses were performed using cox proportional hazard regression. a total of 72 pts met the inclusion criteria and were analyzed. optimal alc cutoff by roc analysis was established to be on day +14 (d14) with alc 40.3 × 10 9 /l and was subsequently defined as early lymphocyte recovery (erl). pts with alc ⩽ 0.3 × 10 9 /l were deemed to have delayed lymphocyte recovery (dlr). patients were subsequently stratified accordingly and patient, disease and transplant related factors were well balanced between the groups. median follow up of the entire cohort was 17 (2-64.8) months. graft characteristics: roc analysis established optimal cellular cutoff, if present to predict elr. pts in the elr group were more likely to receive cd 34 × 10 6 /kg o6 (0.018), cd3 4 24 × 10 7 /kg (0.017) and alc 41.3 × 10 8 /kg (p = 0.015). we did not find a significant threshold for other allograft variables i.e. (tnc, mnc or amc). post hct outcomes: at 2 years, corresponding cumulative incidence of relapse (cir) and non-relapse mortality (nrm) was16.9% vs 46.9% (p = 0.025) and 23.2% vs 14.2% (p = 0.51), for elr and dlr cohorts, respectively. there was a trend towards improved progression free survival (pfs) and overall survival (os) in favor of elr vs dlr at 61.9% vs 40.1% (p = 0.09) and 70.1% vs 53.9% (p = 0.12), respectively. median time to neutrophil and platelet engraftment was 17 and 24 days, respectively for both groups. incidence of acute graft vs host disease (agvhd) was similar (p = 0.4); however, chronic gvhd (cgvhd) was more prevalent in the elr group at 70% vs 27%, respectively (p = 0.0006). on s344 multivariable analysis for relapse, elr retained its prognostic significance with hr 0.27 (0.05-0.94; p = 0.038). cgvhd and first complete remission (cr1) at the time of hct were also protective factors from relapse in multivariable analysis. we observed that elr is an independent predictor for relapse in patients receiving allogeneic hct for acute leukemia with a trend towards improved os. this is possibly related to higher incidence of cgvhd. elr was influenced by infused allograft characteristics particularly cd34 count. given the sample size and retrospective nature of the analysis, these important observations should be examined prospectively. disclosure of conflict of interest: none. allosct is the only curative option for the treatment of hematological disorders with depression of hematopoiesis and primary immunodeficiencies.non-myeloablative conditioning (mac) regimens lead to long persistence of mixed chimaerism (mc) in the majority of patients. purpose: to estimate the relationship between type of hematopoietic chimaerism and appearance of gvhd in patients with non-malignant diseases after allosct eleven patients (8 boys and 3 girls) with median age of 9 years (range 3-17) were included in the current study. among them there were 7 patients with severe aplastic anemia (saa), 2 with fancony anemia (fa), 1 with thalassemia, 1 with nijmengen syndrome, treated in our center from 2008 to 2016. donors' sources were as follows: siblings in 7 cases, mud (10/10) in 4 ones. in 5 cases bone marrow aspirate were used, in 6 mobilized peripheral blood hematopoietic stem cells. conditioning regimens included fludarabin, cyclophosphamide and horse atg for saa patients, in fa and nijmengen syndrome patients this scheme was augmented by low-dose busulfan. in thalassemia patient we used mac with busulfan, fludarabin and horse atg. in majority of case gvhd prophylaxis consisted of tacrolimus and methotrexate combination. when allosct was performed form mud patients were additionally administered with mycophenolate mofetil. median of follow-up period was 32 mo (range 7-93). quantitative evaluation of chimerism was done by multiplex amplification of str loci with subsequent fragment analysis using «cordis plus» kit («gordiz llc», russia). we analyzed whole bone marrow and peripheral blood together with cd3+ and cd19+ lymphocyte subpopulations. presence of ⩾ 99% donors' hematopoietic cells was considered as complete donor chimerism (cc), less than 99% was considered as mc. all patients engrafted in time and all of them are alive at the time of current analysis. there were no severe life-threatening complications, infections or graft rejections. only 2 patients achieved cc at day +28. at day +100 only these 2 patients stayed in cc. at this time point mc was mainly revealed in cd3 + lymphocytes. in 1 year after hsct proportion of cc patients enlarged to 82% (2 patients did not achieved this time point). there is no any correlation between time of engraftment and chimerism value at day +28, either between the dose of transplanted cd34+ cells and chimerism level ((p40.05 in both cases). severe gvhd was noted only in 2 female patients with cc at day +28. in the first case it was acute gvhd grade iii after hsct from mud, in the second case extensive form of chronic gvhd in 1 year after hsct from sibling was observed. there are no other cases of grade iii-iv acute gvhd in the observed cohort of patients. localized form of chronic gvhd [p434] was revealed in 4 (36%) patients. in other patients there were no signs of chronic gvhd. despite limited number of observations we assumed that fast achievement of cc corresponds to severe gvhd. and vice versa, long persistence of mc prevented emergence of gvhd. however our findings need to be confirmed in a larger group of patients and preferably in a multicenter setting. disclosure of conflict of interest: none. interest of quantitative assessment of hematopoietic chimerism by real-time quantitative polymerase chain reaction after hematopoietic cell transplantation for hematological malignancies: a retrospective analysis on 347 adult patients from rennes university hospital g laure 1 , c mathilde 2 , b marc 1 , n stanilsas 1 , d charles 1 , l christine 2 , a mehdi 2 , lb laetitia, s gilbert 3 , l thierry 1 and r virginie 2 1 department of hematology, chu pontchaillou, rennes; 2 immunogenetics and histocompatibility laboratory, etablissement français du sang, rennes and 3 etablissement français du sang-bretagne chimerism (percentage of recipient versus donor-derived blood cells) is used to document engraftment after hematopoietic stem cells transplantation (hsct). detection of persistant host cells, 1-2 as well as an increase in recipient cells chimerism has been associated with impaired dfs and os. 3 quantitative real time pcr (qrt-pcr) 4 is a highly sensitive, reproducible method, which can detect very low levels of recipient cells. the aim of this study was to evaluate the prognostic impact of early chimerism 30 days (d30) and 100 days (d100)) after hsct and the meaning of detection of an increase of chimerism, even at low levels, during follow-up. 347 adult patients who underwent hsct in rennes between 2002 and 2013 were included in this retrospective study. all chimerism analyses were done with qrt-pcr using whole blood sample. complete chimerism (cc) was defined by less than 0.1% recipient cells detected. with a median follow-up was 653 days, 101 patients relapsed with a median time of 116.5 days after hsct. both d30 and d100 mixed chimerism (mc) (40.1 % recipient cells detected) were associated with an increased relapse risk (p = 0.0003 and p o0.00001 respectively) compared to patients with cc in univariate analysis. however, when looking at subgroups analysis, d30 and d100 mc vs cc was significantly associated with increased relapse risk in this cohort for myeloid diseases (p = 0.0049 and p o0.0001) but not for lymphoid diseases (p = 0.506 and p = 0.059). no difference in os was observed (p = 0.32 and p = 0.34). more important, detection of an increased of mc (imc) was associated with an increased relapse risk in univariate and multivariate analysis (or = 9.69 [5.42; 17 .34], or = 10.05 [5.35; 18 .90]), (po0.0001), as well as impaired os (p = 0.0043) and dfs (p o0.0001). among the 103 patients with aml and at least 2 chimerism analysis available, only 3 relapsed without imc detected but the patients' last available chimerism analysis was 75, 84 and 138 days before relapse respectively. median levels of recipient cells detected was 1.2 %. altogether, these results indicate that serial analyses of chimerism with qrt-pcr are a useful tool for post-transplant monitoring and might help identify patients at highest risk to relapse after transplant, especially in myeloid disease. 3 monitoring frequency is critical in order to obtain the highest clinical impact, and the timing of monitoring as well as the safety and type of pre-emptive interventions still need to be explored. considering the kinetics of the disease, frequent analysis in myeloid pathology might improve the detection of impending relapse. although an approximately 1-log higher sensitivity of quantitative pcr (qpcr) compared to short tandem repeat (str) has been documented in different studies, the latter remains the standard procedure for hc assessment to date. we hypothesized that qpcr could be superior to str for monitoring the molecular kinetics of donor cell engraftment, response to donor lymphocyte infusions (dli) and development of relapse post-hct. we analyzed 30 patients (pts) who underwent mainly 10/10 hla-matched unrelated hct mostly for acute myeloid or lymphatic leukemia at the university hospital essen between 2006 and 2013. transplant conditioning was mostly myeloablative and gvhd prophylaxis was by cyclosporin a and methotrexate without anti-thymocyte globulin (atg). median follow-up of pts was 1504 days (d) (317-2891). cytomegalovirus (cmv) reactivation in the first 100d posttransplant was measured by pp65 (n = 22) or pcr (n = 8). a total of 459 retrospective genomic dna samples from peripheral blood (pb; n = 364) or bone marrow (n = 95) collected between d21 and d2302 post-transplant were available for hc analysis in parallel by str (mentype chimera, biotype) and qpcr (alleleseq, abbott). threshold for hc positivity in qpcr was set at 0.1% following published protocols. concordance in hc analysis between qpcr and str was found in 365/459 (79.5%) samples, with all 94 discordant cases positive in qpcr but negative in str. engraftment could be assessed in 110 samples drawn at d21-d213 from 18 pts without relapse in the first 6 months post-hct. these samples showed concordant negative or positive qpcr and str results reflective of full donor engraftment or persistent mixed chimerism (pmc) in 5 and 3 pts, respectively. in 2 pts, qpcr but not str documented delayed conversion to full donor chimerism until d200. in the remaining 8 pts, positive results in qpcr but not str during early engraftment were observed exclusively in bm, in particular those drawn before d35 post-hct. qpcr but not str was also able to document the kinetics of conversion to full donor chimerism which took 126d and 196d in 2 pts receiving dli for treatment of pmc and relapse, respectively. 8 informative relapses could be assessed in 33 samples drawn at least 192d before onset. 6/6 bm and 23/26 pb were positive in qpcr, compared to 2/6 bm and 2/23 pb in str, with a sensitivity of 8/8 (100%) and 3/8 (37.5%) relapses, respectively. consistent with previous reports on a protective effect of early cmv reactivation on relapse in gvhd prophylaxis regimens without atg, relapse occurred in 1/10 (10%) pts who experienced cmv reactivation in the first 100d post-transplant, compared to 10/20 (50%) pts who did not. no apparent associations were observed between early cmv reactivation and engraftment kinetics post-hct. hc assessment by qpcr is highly concordant with str, but markedly superior for molecular monitoring of engraftment kinetics and relapse. positive qpcr results in bm should be interpreted with caution during early engraftment, while both bm and pb were highly informative for relapse in our series. these results advocate the feasibility and clinical utility of qpcr for post-hct hc monitoring in routine use. disclosure of conflict of interest: the commercial assays for qpcr chimerism analysis were provided by abbott molecular free of charge. tyrosine kinase inhibitor (tki) has become the standard of care in patients (pts) with chronic myeloid leukemia (cml) and an unavoidable tool in the combined therapy for pts with philadelphia chromosome positive (ph+) acute lymphoblastic leukemia (all). nevertheless, allogeneic stem cell transplantation (hsct) remains the standard therapy of all ph+ and of cml pts failing 1st line therapy with tki, with failure or insufficient response or intolerance or mutations resistant to 2nd generation tki, or in the advanced phase at diagnosis. in the past decade the feasibility and safety of post-hsct imatinib administration as prophylactic or therapeutic strategy was confirmed. second and 3rd generation tki administration after hsct is under investigation. here we are reporting our experience in post-hsct treatment with the 3rd generation tki ponatinib in 5 pts treated between 2011 and 2016 at our institution. pts data and information were collected from institutional database and chapters revision. a written consent was given by pts allowing the use of medical records for research in accordance with the declaration of helsinki. pts and diseases features are reported in table 1 . pre-transplant treatment for the all ph+ patient consisted of chemotherapy combined with dasatinib, followed by a 1st mud hsct and dasatinib in maintenance. the patient relapsed 1 year after hsct with documentation of mutation v299l. ponatinib was introduced as salvage treatment to bridge 2nd haplo hsct. pre-transplant treatment for the cml patients consisted of tki therapy with combination of chemotherapy in case of uncontrolled progression of disease. two pts received a 1st mud hsct but relapsed respectively 5 months and 4 years later. ponatinib was introduced as salvage treatment to bridge 2nd haplo hsct. four pts received ponatinib 45 mg daily before the last hsct: one patient achieved sustained major molecular response, 4 pts obtained transient response. all pts were presenting 2nd generation tki resistant mutations (table 1) . ponatinib was started at a median of 157 days after hsct (range, 117-583) as salvage treatment in overt relapse (3 cases), prophylaxis (1 case) or preemptive therapy (1 case). acute gvhd was diagnosed in 4 pts before ponatinib administration, 2 of them also experienced chronic gvhd. no new cases of gvhd were observed after initiation of ponatinib. immunosuppressive treatment and azoles treatment were discontinued before ponatinib in all but one patient who was under combined treatment for chronic gvhd: therapeutic drug monitoring was closely performed without evidence of drug-drug interaction. pts were regularly evaluated for toxicities and monitored for cardiovascular events. no serious adverse events were reported in our experience: we administered ponatinib at a median maximum dosage of 30 mg daily (range, 15-45 mg), for a median of 24 weeks (range, 4-132 weeks). at last evaluation one patient maintained the status of molecularly undetectable leukemia (follow-up post hsct: 34 months) and one major molecular response (follow-up post hsct 29 months). three patients who received therapeutic ponatinib in overt relapse did not respond and died for progressive disease. ponatinib is safe and well tolerated as bridge to hsct and to maintain disease control after transplant. prophylaxis targeted therapy and preemptive therapy with ponatinib may lead to the reduction of disease relapse for high-risk ph+ leukemia. disclosure of conflict of interest: none. at nuh singapore we have adopted the cd45 ra depletion to ameliorate graft versus host disease. materials and methods: we have transplanted 14 leukemia patients with cd3 depleted hsct followed by cd45ra depleted donor lymphocyte infusion. no additional gvhd prophylaxis or gcsf was used. results: 100% patients achieved primary engraftment. median time for neutrophil engraftment (4500/μl without gcsf) was 14 days (range 7-17 days), platelet engraftment (450 000) was 13 days (range 10 to 29 days). immune reconstitution was rapid with median cd4 and cd8 cell counts 4200/μl at day 30. by day 200 median cd4 count was 390/μl (range 312-817/μl). no patient developed grade iv acute gvhd. there was a significantly reduced incidence of invasive viral infections as compared to conventional transplants. importantly, all patients achieved complete remission (cr) on day +21 and remained in cr for longer time as compared to conventional transplants. conclusion: our preliminary data suggests that rapid immune-reconstitution of nk cells and t cells with this strategy correlates with reduced infection related mortality without loss of graft versus leukemia effects. disclosure of conflict of interest: none. the use of post-transplantation high-dose cyclophosphamide (pt-cy) has overcome the need for extensive depletion of t lymphocytes from haploidentical donor grafts, which traditionally resulted in severe and prolonged immunosuppression. it is therefore relevant to investigate the degree and the tempo of immune reconstitution after t cell replete haploidentical stem cell transplantation (haplo-sct) by use of pt-cy. we prospectively monitored cellular immunity in 15 consecutive adult patients (male/female: 9/6), who underwent haplo-sct with pt-cy for myeloid (n = 12) or lymphoid (n = 3) malignancies. the median age at transplant was 56 (range, 27-68) years. the conditioning regimen was myeloablative in 10, reduced-intensity in 4, and non-myeloablative in 1 case. the source of the graft was peripheral blood (n = 7) or bone marrow (n = 8). in addition to pt-cy, graft-versus-host disease (gvhd) prophylaxis included tacrolimus and mycophenolate mofetil. absolute counts of cd3+cd4+, cd3+cd8+, cd19+ and cd16+cd56+ cells were measured by flow cytometry at 1, 3, 6, 9, 12, 18 and 24 months post transplant. the median doses of infused cd34+ and cd3+ cells were 4.13 (range, 1.16-9.61) × 10 6 /kg and 4.45 (range, 2.2-38) × 10 7 /kg, respectively. neutrophil engraftment (4500/ul) was achieved at a median of 18 (range, 16-35) days, whereas platelet engraftment (420 000/ul) was observed at a median of 23 (range, 13-54) days. seven patients developed acute gvhd (grade i/ii: 6, grade iii: 1). chronic gvhd occurred in 3 patients, and was extensive in the 2 of them. cytomegalovirus infection was detected in 9/15 cases at a median interval of 42 (range, 30-89) days post transplant. two patients were administered rituximab for epstein-barr virus reactivation at 10 months, whereas one patient developed bk virus-associated hemorrhagic cystitis at 2.5 months following haplo-sct. there was 1 death due to gvhd and infection at 7 months post transplant. at a median follow-up of 12 (range, 3-33) months, 14/15 patients remain alive and disease-free. the absolute counts of t and b cells were extremely low early post transplant, while nk cells recovered from the first month (mean count, 254/μl). the number of cd8+ t cells started to increase beyond the first month, and exceeded lower normal limit at 3 months (mean count, 296/μl). cd4+ t cells remained in general low ( o100/μl) for the first 3 months, increased moderately by 6 months (mean count, 186/μl), and approached lower normal values at 9 months (mean count, 325/μl) [ figure 1 ]. of note, cd4+cd45ra+ naïve t cells remained significantly impaired (absolute count range, 4-52/μl) in all 7 patients in which they were assessed beyond the 1st year from transplant. cd19+ b cells were suppressed for the entire first trimester (mean count at 3 months, 68/μl), but increased rapidly between 6 and 9 months (mean counts, 165/μl and 366/μl, respectively). in haplo-sct with pt-cy, reconstitution of cellular immunity can be achieved at adequate levels by 6-9 months following transplant. the observed deficit in the recovery of naïve t-helper cells may be related to a possible effect of pt-cy on thymopoiesis and warrants further investigation. [p440] disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) is a curative therapy for patients with sickle cell disease (scd). hemoglobin a in scd ameliorates the manifestations of the disease and this could be achieved with stable mixed chimerism after a reduced intensity hsct. this study aims to estimate the proportion of patients who develop mixed chimerism after hsct for scd and to characterize its progression in patients who develop it. this is a retrospective cohort study conducted at sultan qaboos university hospital (squh) bone marrow transplant unit in oman. we included all patients with scd who received hsct over the course of 10 years between may 2007 to may 2016. patients who received second hsct were excluded. short tandem repeat polymerase chain reaction was used for chimerism assessment. mixed chimerism was defined as 5 − 95% chimerism at 6 months from hsct. the data was analyzed by r program 3.1.2. χ 2 or student t-test were used to assess the impact of acute graft versus host disease (agvhd) prophylaxis, age at transplantation, gender, red blood cell antigen alloimmunization, preparative regimen, and ferritin on the development of mixed chimerism. we included 56 eligible patients. the median follow-up time after hsct was 26 months (interquartile range: 17.3 − 50.3 months). the mean age at transplant was 19.9 years (standard deviation: 8.44). fifty-nine percent of patients were male. most patients had s/s genotype (77%), followed by s/beta-thalassemia mutation (20%). the indications for bmt were: stroke in 7%, acute chest syndrome (acs) in 9%, recurrent vaso-occlusive crisis (voc) in 38%, stroke and acs in 7%, acs and voc in 31%, orbital compression syndrome in 2%, stroke and moyamoya disease in 4%, and moyamoya disease in 2%. the two most frequently used preparative regimens were busulfan/fludarabine/atg in 49% and thiotepa/treosulfan/fludarabine in 42%. twenty-five percent of patients developed mixed chimerism at six months after hsct. on follow up of patients with mixed chimerism, 10% rejected the graft, 20% developed complete chimerism, and 70% continued to be in mixed chimerism. preparative regimen and the development of agvhd were statistically significant predictors of mixed chimerism at 6 months (p values: 0.00079 and 0.01817 respectively). age at transplant, gender, red blood cell antigen alloimmunization, and ferritin were not statistically significant predictors of the mixed chimerism (p40.05). the study confirmed that mixed chimerism can commonly be achieved in patients with scd after hsct and in majority, it remains stable on long-term follow-up. reduced intensity preparative regimen and lack of agvhd predicts the development of mixed chimerism. larger prospective studies are needed to confirm these results. disclosure of conflict of interest: none. there was a majority of male patients (69%). the median hbf level was 15% (0-77), median monocyte count was 5.109/l (range: 0-74.8), median platelet count was 64.109/l (4-377), median marrow blasts was 5% (0-37) above 96 patients who were explored by marrow karyotype, 27% of them had a monosomy 7. mutations in ptpn11 were detected in 33 patients. fifty patients (43%) were treated with the bu/cy/mel regimen, whereas 22 patients (19%) received the bu/flu/mel regimen. at 6 years, the overall survival (os) was 62% (95% ci: 53-72). nineteen and 9 patients developed vod after bu/cy/ mel and bu/flu/mel conditioning regimen, respectively. the cumulative incidence of agvhd 3-4 was 26% (95% ci: 19-35). the 6-year cumulative incidence of relapse and non-relapse mortality was 30% (95% ci: 22-39) and 18% (95% ci: 11-25), respectively. the median delay of relapse was 90 days (range 15-1330). among relapsing patients, 16 were transplanted twice and one underwent 3 hsct. in multivariate analysis, female donor to male recipient sex-mismatch, cmv status, total body irradiation and ras-double mutation/other additional mutation predicted poorer outcomes. the bu/flu/mel conditioning regimen was associated with a decreased risk of relapse. however, there was no statistical difference for os between the two main preparative regimens, bu/cy/mel vs bu/ flu/mel. our results show that allogeneic hsct may cure approximately 60% of patients with jmml and are similar to the best results published by other groups. relapse represents the main cause of treatment failure and a second hsct should be proposed. despite a decreased risk of relapse with the bu/ flu/mel regimen, there was no statistical difference in terms of os between the two main conditioning regimens, bu/flu/mel vs bu/cy/mel. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (ahsct) is the only curative treatment modality for the majority of pediatric patients with myelodysplastic syndrome (mds) and myeloproliferative neoplasms (mpn). the purpose of this study is to evaluate overall (os) and failure (relapse or death from any cause) free survivals (ffs), non-relapse mortality (nrm) and relapse incidence in children who underwent ahsct for mds or mpn in a single center from turkey. we retrospectively analyzed 45 ahsct carried out in 43 patients (median age: 9.2 years; range: 0.4-18; 24 males). thirty four had primary mds and 9 had secondary mds. according to the modified who mds and mpn classification, 18 had refractory cytopenia (rc), 12, refractory anemia with excess blast (raeb), 1, refractory anemia with excess blast in transformation (raeb-t) and 12, juvenile myelomonocytic leukemia (jmml). amongst patients with secondary mds, 4 had been treated for acute myeloid leukemia, 2 had been treated for non-hodgkin's lymphoma and 1 had been treated for acute lymphoblastic leukemia, retinoblastoma and osteosarcoma, each, previously. donors were related in 18 transplantation (5 haploidentical transplantation) and the stem cell resources were bone marrow (n = 27), peripheral blood (n = 14), cord blood (n = 2) and bone marrow +peripheral blood (n = 2). three-year ffs and os for patients with mds were 55% and 57.0%, respectively; and for patients with jmml, 50% and 64%, respectively. crude incidence of nrm and relapse for entire group were 33% and 22%, respectively. ahsct offers durable ffs and os for a significant group of pediatric patients with mds and mpn. less toxic conditioning regimens could result in better results in some patients. disclosure of conflict of interest: none. allogeneic stem cell transplantation in children with autism z antonella, g alessandra, ma beatriz and r vanderson bone marrow transplantation unit, hospital sirio-libanes, são paulo, brazil autism spectrum disorders (asd) are severe heterogeneous neurodevelopmental abnormalities characterized by dysfunctions in social interactions and communication skills, restricted interests, repetitive, and stereotypic verbal and non-verbal behaviors. the etiology of asd remains unknown, but recent studies suggest a possible association with altered immune responses and asd. inflammation in the brain and central nervous system has been reported with microglia activation and increased cytokine production in postmortem brain specimens of individuals with asd. other studies have established a correlation between asd and family history of autoimmune diseases, associations with mhc complex haplotypes, and abnormal levels of various inflammatory cytokines and immunological markers in the blood. the paracrine, secretome, and immunomodulatory effects of stem cells would appear to be the likely mechanisms of application for asd therapeutics. we describe two cases of patients with asd who underwent hsct for acute lymphoblastic leukemia (all) and whose symptoms were markedly decreased like an improvement of social interaction, communication, and behaviors. the first patient is an 11-year-old girl with asd who was diagnosis with ph-positive all in october 2011 (at the end of treatment, bcr-abl remained positive). she underwent a matched sibling hsct in march 2015. the conditioning regimen was total body irradiation (tbi) and cyclophosphamide. during the 20-month follow-up period, we observed improvement in social interaction, communication, and behaviors (according to the childhood autism rating scale-cars). the second case is a 7-year-old boy with asd, asperger syndrome, who was diagnosis with all in september 2012. he presented with bone marrow and testicular relapse in may 2015 and underwent a matched unrelated hsct in november 2015. the conditioning regimen used was etoposide, atg and tbi. during the 12-month follow-up period, we observed improvement in social interaction, communication, and behaviors (according to cars). there is no treatment for asd thus every effort to minimize the symptoms are valuable. in both cases, social interaction was significantly increased, and the aggressive behaviors decreased. clinical cases have reported responses in autistic children receiving cord blood cd34+ cells. several incurable neurological disorders have shown benefits with cellular therapy. thus, autism should be explored as an indication. clinical studies are an immediate need to fully explore its potential in autism. disclosure of conflict of interest: none. conditioning is the initial phase of hematopoietic stem cell transplantation, based on high dose chemotherapy, combined or not with total body irradiation, aiming to eradicate the disease and prepare the environment of the bone marrow for the new cells. conditioning regimens can be characterized as myeloablative or non-myeloablative. during the period of conditioning and immunological reconstitution, signs and symptoms of the gastrointestinal tract are frequent, negatively influencing oral food intake, and may require the use of complementary nutritional therapies, aiming at an adequate caloric intake with the objective of avoiding decreasing in the nutritional status. the study aims to describe the association between the regiment intensity and the nutritional aspects during hospitalization of children and adolescents undergoing allogeneic hematopoietic stem cell transplantation (hsct) at a tertiary hospital. a retrospective study with medical records of patients undergoing allogeneic hsct, aged between 0 and 19 years of age (incomplete) between january 2009 and december 2014. data were collected (regimen intensity, clinical signs of mucositis and nutritional therapies used) during the hospitalization and analyzed by the relative risk (rr). sixty-three patients were evaluated, being 56% male, with a median age of 10 years. nineteen types of conditioning protocols were used. of these, 64% were high intensive regimen and 36% were low intensive regimen. the four most applied (59% of cases) were bucy (busulfan + cyclophosphamide) with and without atg (thymoglobulin), as well as cytb (cyclophosphamide+total body irradiation), also with and without atg. mucositis were observed in 83% of patients, being 50% grade 3 and grade 4. the association was positive when analyzed the regimen intensity (myeloablative) with mucositis (rr = 1.51 (1.10-2.08)) as well with the use of parenteral nutrition (rr = 2.49 (1.17-5.13)). patients showed high prevalence of mucositis during hospitalization decreasing food intake, being necessary to use the parenteral nutrition. myeloablative regimen needed more nutritional therapy intervention when compared to non-mieloablative regiment. results demonstrate that an appropriate nutritional screening tool considering these aspects could help to intervene earlier maintaining an adequate nutritional status. autoimmune cytopenia (aic) is a potentially serious complication of hematopoietic stem cell transplantation (sct). autoimmune hemolytic anemia (aiha) is the most common aic, followed by immune thrombocytopenic purpura and autoimmune neutropenia. aic after sct is considered difficult to treat and associated with high morbidity and mortality. the aim of this cohort study is to evaluate incidence, outcome, potential risk factors and current treatment strategies and to explore the immune dysregulation predisposing to aic. the ebmt-promise database was accessed to identify all pediatric scts between 2000 and 2016 complicated by aic at our center. potential risk factors (i.e., age, gender, diagnosis, donor type, stem cell source, conditioning regimen) for aic after sct were assessed using univariate and multivariate cox regression analysis. in addition, we summarized treatment decisions of all aic patients. a nested matched case-control study was performed to search for possible biomarkers for aic. of 531 consecutive scts, 27 were complicated by the development of aic (cumulative incidence 5.2%) at a median of 5 months post-sct (figure) . aiha was the most common aic (48%), followed by combinations of two or more aics (evans syndrome, 33%). non-malignant disease, young age, alemtuzumab serotherapy pre-sct, non-tbi based conditioning regimen and cmv reactivation were associated with aic in univariate analyses. using multivariate cox regression analysis, non-malignant disease (hr 3.6, p = 0.028), alemtuzumab use (hr 2.4, p = 0.035) and cmv reactivation (hr 3.7, p = 0.013) were independently associated with aic (figure) . for patients with cmv reactivation, diagnosis of aic was made at a median of 4 months (iqr [1] [2] [3] [4] [5] [6] [7] [8] ) after detection of maximum viral load. in our nested case-control analysis, serum levels of individual anti-and proinflammatory, and regulatory cytokines did not differ significantly between patients and controls. however, the cytokine profile of aic patients appeared to skew towards a more pronounced th2 response, compared to controls. firstline treatment, usually with prednisone and/or ivig, or a waitand-see approach led to resolution of aic in 9 (33%) cases. second and subsequent-line therapies, often in combination with continuation of other treatments, consisted of rituximab (n = 16), bortezomib (n = 7) or sirolimus (n = 3) and eventually led to resolution of aic in 44%, 57% and 100% of cases, respectively. overall survival of aic patients was 78%. in this retrospective cohort study, we identified cmv reactivation post-sct, alemtuzumab use and non-malignant disease as independently associated clinical risk factors for the development of aic. treatment with first-line therapy was mostly insufficient. for patients with severe aic, rituximab, bortezomib or sirolimus can be regarded as promising step-up therapies. figure (bkv) may cause polyomavirus-associated nephropathy or polyoma virus-associated hemorrhagic cystitis in bone marrow-transplant patients.we present 19 patients with bk polyoma virus (bkv) ascociated hemorrhagic cystitis and 2 patients with bk polyoma virus associated hemorrhagic cystitis and nephritis. between 2013 and 2016, 124 patients received an allogeneic bmt at acıbadem adana hospital pediatric bone marrow transplantation unit. 21 patients occurred bkv associated hemorrhagic cystitis and nephritis. bkv was detected in the urine analysis and blood by pcr (polymerase chain reaction) in all patients. we presented 21 patients with bkv infection, age ranging from 3 to 20 with a average of 13.1 years. they underwent allogeneic bmt due to thalassemia major (13 patients), aplastic anemia (4 patients) and acute lymphoblastic leukemia (4 patients). the patients were treated with hydration, continuous bladder irrigation, ciprofloxacin, and weekly intravesical hyaluronic acid instillation for four weeks, and cidofovir. fourteen patients showed complete resolution of hematuria. one patient with refractory above these therapy also received hyperbaric oxygen and recombinant factor viia (rfviia, novoseven; novo nordisk,bagsvaerd, denmark). hemodialysis was performed in two patients who developed renal failure due to nephritis. bkv is ubiquitously present in the general population. 1 reactivation of infection occurs under conditions of immunosuppression, particularly hsct or renal transplantation, and causes late-onset hc. bkv the management of bkv cystitis and nephritis sometimes may be very difficult and refractory all treatments, we presented our experience of bkv infection and management in transplanted patients in our center. patients with high-risk hematologic malignancies (hrhm) are among those in the highest risk group for developing invasive fungal disease (ifd), especially mold infections. allogenic hematopoietic stem cell transplantation (alsct), acute myeloid leukemia (aml), refractory and relapsed acute lymphoblastic leukemia (all), myelodysplastic syndromes and chronic extensive graft-versus-host disease are considered hrhm. ifd are a major cause of morbidity and mortality in these patients, however, the optimal strategy for antifungal p448 s352 prophylaxis in this population is not well defined yet. we performed a retrospective, observational study to investigate documented ifd during antifungal prophylaxis in children with hrhm who were admitted in our unit between 2010 and 2016. demographic and clinical data were collected from patient's electronic medical records. all patients were treated with prophylactic voriconazole (vcz) according to our local practice. oral administration was preferred when available. vcz therapeutic drug monitoring (tdm) was not available in our center until june 2016. breakthrough ifd was defined as occurrence of a proven or probable ifd according to eortc/ msg criteria while on vcz prophylaxis (⩾7 days of treatment) or within 15 days after discontinuation of prophylaxis. during the study period, 75 hrhm patients were treated with prophylactic vcz in our unit. 4 patients out of 75 developed a breakthrough ifd. patient's demographic characteristics, main diagnosis and treatment are collected in table 1 . initial and maintenance vcz doses are adjusted by weight in all patients except in patient-4 (adjusted according to vcz plasma level). adherence and tolerance to treatment was excellent in all patients. disclosure of conflict of interest: none. (3) stable mixed chimerism (smc) when fluctuations of ac were o5%; and (4) mixed progressive chimerism (pmc) when ac were ⩾ 15%. 2-3 101 hscts performed in 85 patients (pts) were included: 72 children with a median age of 2.01 yrs (iqr 0.62-7.35 yrs) at diagnosis and 6.2 yrs (iqr 2-11 yrs) at hsct received one hsct (84.7%),10 pts two hsct (11.8%), and 3 pts three hscts. primary diagnosis were bone marrow failures in 37 pts (43.5%), primary immunodeficiencies in 25 (29.4%), inborn errors of metabolism in 15 (17.5%) and haemoglobinopathies in 8 (9.41%). the donor was match related in 23 (23%) procedures, match unrelated in 63 (62%), and haploidentical in 15 (15%); stem cell source was bone marrow in 55 (54%), peripheral blood in (26%) and cord blood in 20 (20%). conditioning regimen (cr) included busulfan in 19 hscts (18.8%), treosulfan in 33 (32.7%), while 48 hscts (47.5%) were conditioned with reduced intensity crs (including low dose of tbi in 9); 1 pt did not received cr. gvhd prophylaxis was based on csa/mtx (or mmf) in association with atg (69) or alentuzumab (16) ; recipients of tcrαβ/cd19 depleted haploidentical graft did not received post transplant immunosuppression. engraftment was observed in 87 hscts (79 after 1 st , 7 after 2 nd and 1 after 3 rd hsct) after a median of 18 day (iqr 14-23 days). acute gvhd occurred in 45 hscts at risk (52%), and it was severe (gr. iii-iv) in 20 (23%), chronic gvhd in 31 (31%). at last follow-up (median 4.35 yrs), 75 (88%) pts were alive, while 10 pts are dead for infections (n = 5), vod (n = 1), c-gvhd (n = 3) and vascular event (n = 1). figure 1 reported the evolution of chimerism over time. in our experience in children with non malignant disease, cc increased from 36% to 67% at subsequent analyses. 60% of pts with mc at 1 st evaluation became cc, 16% remained smc, 5% evolved in pmc, and 19% rejected. only 2 pts with cc at first time point rejected the graft. this study highlight the extreme variability of chimerism in the early post transplant course of children with non malignant disease and confirmed the relevance of performing serial analysis to monitor and, if necessary, improve graft function. naive t-cells identified by cd45ra expression are believed to cause graft-versus-host-disease (gvhd), while cd45ra-t-cells are memory cells that provide anti-infection and anti-tumoral effects. depleting cd45ra+ naïve cells and retaining memory t-cells in the graft is a novel approach to haploidentical hsct for children. 18 children with high risk leukemia (6 aml, 12 all) received cd45ra-depleted haploidentical hsct following non-myeloablative conditioning. cell-selection performed on g-csf-mobilized peripheral blood. two cellular products obtained using clinimacs device, infused to each patient: a cd34 selection and a cd45ra depletion from the cd34negative fraction. product infused contained a median of 6.04 (range 4.04-9.93) x106/kg cd34+ cells and a median of 6.5 (range 1.3-490) × 10 3 /kg of cd3+ cells in the cd34-selected s353 graft. the second product was the cd45ra depletion, cd45ra +/kg was a median of 6.15 × 10 3 /kg (range 0-498 × 10 3 /kg) and a median 4.70 (range 2.21-6.37) depletion log of cd45ra + cells. median dose of cd45ro+ cells (memory t-cells) infused was 8 (range 3.8-102) × 10 7 /kg. seventeen patients achieved neutrophil engraftment at median of 10 days (range 8-12) post-transplant. one patient could not achieve engraftment, died at day +8 due to sepsis. two patients presented secondary graft failure (day +18 and +20), both received a second hsct. three patients developed agvhd 4grade ii with gastrointestinal tract involvement, all steroids responsive. three patients presented clinical features of cgvhd. patients have an extensive skin involvement, with hepatic findings in one and pulmonary affection in other, at day +315, +130 and +330 post. ten of 18 patients (55.5%) remain alive in remission with median follow-up 156 (range 8-597) days post-transplant. eight patients died, 3 due progression at day +128, +117, +162 (2 presented positive minimal residual disease at hsct), 4 due to infectious complications (days +8, +44, +50, +55) and 1 due to cardiogenic shock at day +253. four patients relapsed, 3 of them died afterward with progressive disease. t-cells led immune recovery, achieved values higher than 500, 600, 1500 and 2400 cells/mcl at day 30, 60, 90 and 210 respectively. most of t cells were cd8+cd45ra-(median of 288, 370 and 2334 × 10 6 /mm 3 respectively on day +30, +60 and +90) and cd4+cd45ra-t cells (median of 129, 161 and 767 × 10 6 /mm 3 respectively on day +30, +60 and +90), while cd8+45ra+ and cd4+45ra+ cells remained low recapitulating the cd45ra depleted graft composition. six patients presented cytomegalovirus reactivation, one progressed to cmv disease. five patients with hhv-6 encephalitis. probable aspergillosis in 1 patient (aml-m7 with secondary graft failure) at day +16 after second hcst. two cases of toxoplasmosis (1 cns, 1 pulmonary). cd45ra-depleted haplo-hct showed acceptable tolerability with rapid and sustained engraftment as well as a full donor chimerism, minimal risk of acute gvhd and accelerated inmunologic reconstitution. to note the high incidence of hhv-6 encephalitis seen. disclosure of conflict of interest: none. collection of peripheral blood stem cells in teenager sibling donors: a single center experience c carvalho 1 , f amado, f bordalo, s ferreira, s lopes, c pinho and s roncon 1 serviço de terapia celular, instituto português de oncologia do porto francisco gentil, epe human leukocyte antigen (hla) compatibility is important in allogeneic haematopoietic stem cell transplantation in order to reduce post-transplant complications; however, siblings only present a 25% chance of hla-match with the patient. the well-known advantages and the low risk of complications associated to peripheral blood stem cells (pbsc) collected by apheresis made this procedure the first option in teenagers. the aim of this retrospective study was to analyse and characterize the paediatric sibling pbsc donor population assuring safety during the collection procedure, providing a high-quality product and accomplishing patient needs. we consulted the clinical files of donors under 18 years old since 1995-2015; a database in excel ® was created to register population characteristics, collection parameters and graft requirements. the informed consent was obtained from parents before procedure. the leukapheresis were performed with a cobe spectra system; since 2009, we use a spectra optia apheresis system. the donor/patient weight ratio (proposed by styczynski et al.) was determined for each pair. the collection was programed based on clinical and analytical donor's features as well as transplant requirements. the analytical assays were done by a certified laboratory. we performed 29 pbsc apheresis in 23 healthy donors, 10 females and 13 males ( table 1) . all of them started on the 5 th day after mobilization with granulocyte colony-stimulating factor (g-csf) administered subcutaneously, bidaily. the weight ratio was o1 in eight situations. most of donations were performed by peripheral vein; a central venous catheter (cvc) was placed into a femoral vein in six adolescents. a median of 4 (3) (4) (5) blood volemias were processed during 174(115-318) minutes; the anticoagulation used was citrate+heparin (ratio 25:1). in general, one-collection day was enough to obtain the number of cd34+ cells required; six donors had to perform a 2 nd collection. in 19 cases, we cryopreserved the exceeding cells after graft infusion. the procedure was well tolerated, with only 2 adverse reactions registered (one hematoma in the puncture local; one paraesthesia due to hypocalcaemia induced by citrate). no blood products were used after the procedure or needed for the priming of the extracorporeal circuit. so far, no serious long-term adverse events were observed. table 1 . median (range) of donors and leukapheresis products data. our long lasting experience shows that pbsc collection in the teenage population is safe and feasible, allowing us to obtain a high-quality product for the patients. there were no adverse events associated with the g-csf mobilization or cvc placement which is different from the experience of other groups. we recognize that leukapheresis by peripheral vein is a lengthy procedure but no complaint was reported to the collection team. [p451] disclosure of conflict of interest: none. correspondence between clinical and hystological grading of gastro-intestinal grading acute graft versus host disease in children m faraci 1 , a rizzo 2 , p gandullia 3 , s arrigo 4,3 , a barabino 3 , e lanino 1 , s giardino 1 and c coccia 5 1 hematopoetic stem cell transplant unit, institute g gaslini, genoa, italy; 2 pediatric department, institute g gaslini, genoa, italy; 3 gastroenterology and digestive endoscopy unit, institute g gaslini, genoa, italy; 4 gastroenterology and digestive endoscopy unit, institute g gaslini, genova and 5 department of pathology, institute g gaslini, genoa, italy diagnosis of gastro-intestinal acute graft versus host disease (gi-agvhd) is frequently confirmed by apoptosis findings on mucosal biopsies. 1 aims of this single center retrospective study is to evaluate the correlation between clinical and histological grading of gi-agvhd in children undergoing allogeneic haematopoietic stem cell transplantation (allo-hsct), and to describe histological findings obtained by gi endoscopies in order to evaluate usefulness in the diagnosis of gi-agvhd. 348 allo-hscts were performed in our department between january 2000 and december 2015. gi biopsies were performed in 26 pts (7.4%) because of suspected gi-agvhd. 14 pts were transplanted for malignant (53.8%) and 12 for not malignant diseases. the median age at hsct was 9.5 years (0.5-16.9).14 pts (54%) received myeloablative and 12 (46%) reduced intensity conditioning regimen. 21 pts (80.7%) received an unrelated donor (ud), 4 pts a related donor (rd) (15.3%), and 1 an haploidentical donor (3.8%). at onset of diarrhea, microbiological examinations of stool were performed and pcr research for cmv, adenovirus, hhv6, ebv were evaluated in blood and in mucosal biopsies. mucosal biopsies were obtained with esophago-gastro-duodenoscopy in 4 pts (15.3%),esophago-gastro-duodeno-colonscopy in 3 (11.5%), pancolonscopy in 11 (42.3%), flexible sigmoidoscopy in 3 (11.5%), and rectal suction biopsy in 5 pts (19.2%). all mucosal biopsies, except in case of rectal suction, were obtained under sedation. the interval between mucosal biopsies and onset of gi acute symptoms was 23 days (from − 66 to 103 days). biopsies were taken from different sites in the gi tract, were stained using hematoxylin-eosin and evaluated using histological grading of agvhd. 1 in these 26 pts the maximum grade of agvhd was: grade 2 in one (4%), grade 3 in 14 (54%), and grade 4 in 11 pts (42%). at time of histological evaluation, diarrhea was the most common gi symptom (84.6%); 2 children had also cutaneous agvhd and 5 hepatic agvhd. pcr-cmv was positive in 2 mucosal biopsies obtained with pancolonscopy, pcr-adenovirus in other 2 obtained with upper and pancolonscopy, pcr-hhv6 in 2 rectal biopsies, and pcr-ebv in one with upper and pancoloscopy. the comparison between clinical and histology grading of gi-agvhd is shown in table 1 . mucosal biopsies were positive in 1/4 pts evaluated with esophago-gastroduodenoscopy (25%) (grade 1 agvhd), in 3/3 pts undergone esophago-gastro-duodeno-colonscopy (grade 1 in 2 and grade 3 in 1), in 8/11 (73%) who received a pancolonscopy (grade 1 in 5, grade 2 in 1, grade 4 in 2), and 7/8 (87%) of rectal biopsy obtained by sigmoidoscopy or rectal suction biopsy (grade 1 in 3, grade 2 in 1, grade 3 in 1, and grade 4 in 2). one patient developed duodenal intraparietal hematoma after upper endoscopy. in our experience, we did not demonstrated a overall correlation between clinical and histological grading of agvhd showing that hystological examinations underestimated the grade mild or moderate of agvhd. we confirmed 2,3 that rectal biopsies represent to be more effective and safe diagnostic method for the confirm of diagnosis of gi-agi. during the past few decades, hematopoietic cell transplantation (hct) as a treatment modality for primary immunodeficiencies (pid) has undergone remarkable advancement mainly due to better availability of alternate donors resulting in increase in not just matched unrelated donor (mud) but also increased haploidentical (haplo) and cord blood transplants (cbt). additionally, refinement of the conditioning regimens and better graft versus host prophylaxis have presumably led to better survival outcomes. however, a literature gap is identified in evaluation of these outcomes in general with respect to donor and conditioning regimens. we conducted a systematic review by performing a comprehensive search of the pubmed and ovid library from its inception to august 2016. mesh terms included 'primary immunodeficiency (immunodeficiencies)', 'stem cell transplant', 'bone marrow transplant' and 'hematopoietic cell transplant'. all pid studies which used hct as a treatment modality were included. experimental cellular therapies were excluded. both cellular immunodeficiencies (e.g. scid, was, a-t), and innate immunity disorders (e.g. ifngr, cgd) were included in the search. reviews, case reports, meta-analysis and non-english language articles were excluded from our electronic search. publication bias was excluded by performing a methodological search of unpublished conference abstracts from the annual meetings of cis, aspho, asbmt, ebmt, and siop from 2000 to 2016. the data were analyzed considering the outcomes -overall survival and gvhd. 21 studies fulfilled the strict selection criteria for the electronic search comprising of 1010 pid patients. in majority of the hcts, a myeloablative conditioning regimen (mac) was utilized (47% of the evaluable) but a shift towards more reduced intensity conditioning (ric) was observed in the later years. 120 cbts were identified. 27% of patients developed some degree of acute gvhd, whereas chronic gvhd was identified in 15% of the patients. total number of haplos was 317. overall survival was found to be 71% post-hct. a meta-analysis could not be performed due to the heterogeneity of both the predictor variable data (combined stem cell sources were also used for hct) and due to the extremely small number of the patients when categorized in subgroups (e.g. for omenn syndrome, rag deficiencies). this is the largest study of hcts in pid, and we observe that alternate donor hcts have increased significantly over the past decade for the treatment of pid. while the incidence of chronic gvhd was low, acute gvhd still remains a problem in about a third of the pid patients transplanted. disclosure of conflict of interest: none. hepatic veno-occlusive disease (vod) is a common and serious complication of hemotopoietic stem cell transplantation (hsct) in children. we aimed to assess prospectively the use of prophylactic defibrotide in pediatric patients undergoing hsct. in this study, 113 patients who underwent hsct were given defibrotide prophylaxis as 25 mg/kg per day in four divided intravenous infusions over 2 h, starting on the same day as the pretransplantation conditioning regimen. the mean duration of use of defibrotide is 20 days as a prophylaxis. in this study, 113 patients were recruited, 66 male patients and 47 female patients, with the average of 9.1 years, range 1-20; 8% infants, 55% children and 37% adolescent. there were 50 patients with thalassemia major, 41 patients with leukemia, 11 patients with aplastic anemia, one patient with diamond blackfan anemia, two patients with congenitale dyserythropoetic anemia, one patient with osteopetrosis, four patients with famial hemophagocytic lymphohistiocytosis, two patienrs with severe immune deficiency and one patient with kostman syndrome. all transplants were allogeneic. no serious side effects were seen. in eight patients developed clinical vod (seattle criteria). in these patients, defibrotide dose was increased to a treatment dose of 40-60 mg/kg per day. one infant patient with kostman syndrome died due to hepatic and pulmonary veno-occlusive disease. after 36 months of follow up, 7 patients who developed vod are being well and no patient have transplant related complications. hepatic veno-occlusive disease, which is caused by hepatocyte and sinusoidal vessel endothelium damage, can ocur early after hsct, and in its severe form, may lead to liver faillure, hepatorenal syndrome, portal hypertension, and eventually death from multiorgan faillure. in this prospective study, we demonstrated that the use of defibrotide is safe and effective in preventing and treating vod in pediatric patients at high risk. immune reconstitution (ir) is critical for clinical outcome after allogeneic hematopoietic stem cell transplantation (hsct). host and proceeding-related factors affect the ir dynamics and survival. isolated ir parameters are commonly correlated and proposed to predict clinical outcomes after hsct, but these approaches only confer prognostic value at single time points or for single markers. we aim to demonstrate an appropriate methodology to assess the capability of combined serial measurements of lymphocyte subsets to reflect the impact of infections on ir after paediatric hsct. retrospective data of patients receiving a first hsct for any indication with any cell source in the paediatric hsct program from 2006 to 2015 were included. to characterize the kinetics of immune reconstitution, cd3+, cd4+, cd8 + t-cells, b-cells, nk-cells and their naive and memory subsets were measured and analysed at various time points at 2 years post-hsct to stablish a joint model for the evolution of cell subpopulations. slope per month (cellular increase or decrease) of each lymphocyte subsets were calculated and compared with clinical outcomes and cumulative risk of infections. a total of 88 children (range from 0-15 y.o. median 5 y.o.) were included, with cb (n = 19) pb (n = 22) and bm (n = 47) as cell sources. the cumulative incidences after early period were 45% for viral infections (ebv 27%, cmv 22%, bk 11%, adv 4%) and 30% for bacterial infections. data on ir were available for 77%, of the diseasefree survivors. in a exploratory multivariate analysis we detected mainly differences in cd8+, cd8+cd45ro+ memory and nk cells at 1 year after hsct, with dependent tendency according on the cell source and hla compatibility. analysis of the slope tendency patterns were stablished for the analysis of the impact of infections in the ir. delay in cd8+ and cd8+ra+ appearance (mean slope/m = − 7.1% and − 1.8% respectively) remarks the ir profile for bacterial infections, and delayed in nk, cd8 and cd8ro+ (−8.4%, − 5.8%, − 26% respectively) for overall viral infections. additional correlations allow differences in ebv (cd8+ra+ high mean slope/m = 15.9%), cmv (delayed in cd8ro+ slope/m = 10%), and bk infection (cd8+ra + plus cd4ro+ and nk high mean slope/m = 24.9%, 36% 22%). understanding the dynamics of reconstitution by integrating information from the monitoring of lymphocyte subpopulations allows the establishment of kinetic profiles that may help to evaluate the risk of infections and adjust infection prophylaxis in the follow-up of transplanted patients. mortality rate in hsct patients admitted to intensive care unit (icu) is still as high as 20% to 70%. this rate increases when respiratory complications progress to acute respiratory failure (arf) requiring mechanical ventilation (mv). 1 the aim of this study was to determine the feasibility and effectiveness of early continuous positive airway pressure (cpap) delivered in a pediatric hematology-oncology ward to prevent occurrence of arf requiring mv. we retrospectively analysed children treated with cpap in our pediatric hematology-oncology ward between october 2011 and october 2016. thirty-two patients received cpap delivered with helmet during the study period. data were available for 26 patients, 15 males and 11 females, median age 12 years [range 2-20]. eighteen patients underwent allogenic hsct: 1 from sibling donor, 11 from matched unrelated donor, 4 from haploidentical family donor, 2 from cord blood unit. seven patients had a malignant disease: 5 all, 1 aml,1 ewing sarcoma. infectious pneumonia was the main cause of arf in 16/26 patients (61.5%): 9 viral pneumonitis (4 rhinovirus, 3 parainfluenzae virus, 1 respiratory syncitial virus and 1 cmv). five patients had proven/ probable invasive fungal infection according to eortc criteria (3 aspergillosis and 2 mucormycoses). other causative agents were pneumocystis jiroveci (1), bacillus of calmette and guerein (1), toxoplasma gondii (1) and st. mitis (1) . non infective causes of arf were acute transfusion related lung injury (2), hemorragic alveolitis (2), cryptogenic organizing pneumonia (3), tumor lysis syndrome (1), and alveolar oedema due to renal failure (1). according to chest imaging, 13/26 patients (50%) presented with pulmonary consolidations, while 31% had both interstitial infiltrates and pulmonary consolidations. at baseline median neutrophil count was 2.05 × 10 3 /μl (range 0-21.0 × 10 3 /μl), mean heart rate 128 bpm, pulsiossimetry saturation in room air 86%. h-cpap was applied in 19/26 patient with a curative aim, in 7/26 patients as palliative support to reduce respiratory distress. median positive pressure delivered was 10 cmh 2 o (7-12 cmh 2 o), median fio 2 was 40% (30-100%). h-cpap was applied for a median of 11 days . no patient failed h-cpap because of agitation or adverse events (skin breakdown, conjunctivitis, gastric distension or epistaxis). ten patients were transferred to icu (34.6%), 8/10 because of hsct complications. median icu stay was 8.7 days (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) . only 3 patients required mechanical ventilation, in 2 cases associated to ecmo. nether psao 2 in room air (p 0.98 ci 95%) nor cpap level (p 0.76 ci 95%) correlated with the need of icu admission. patients requiring higher fio 2 during cpap demonstrated a not statistically significant trend to higher icu admission rate (p = 0.149).there was a higher rate of mv in patients with higher cpap fio 2 level (p = 0.008). mv prolonged icu stay (p 0.0049). cumulative mortality was 34.6% (9/26); only 1 patient died in icu (10%), because of post hsct parainfluenza virus pneumonitis requiring ecmo. helmet cpap delivery in pediatric hsct ward is feasible and safe, both for curative and palliative aim. if applied early, cpap could reduce icu admission rate for mv and icu mortality. veno occlusive disease (vod) and graft versus host disease (gvhd) are both dreadful complications of hematopoietic stem cell transplantation (hsct). although they have different clinical signs, it is suggested that they share similar pathophysiological pathway. defibrotide is used in the treatment of vod for a long time but it is very less known about its effect on gvhd. in this study, we analyzed a 'high risk' patient population in pediatric hsct to show the effect of defibrotide on acute gvhd. between june 2014-august 2016 totally 75 'high risk' pediatric allogenic hsct procedures were enrolled in this study. 'high risk' definition involves busulphan/ melphalan usage in conditioning regimen, second myeloablative hsct, pre-existing liver disease, allogenic hsct for leukemia with second relapse and diagnosis of hemophagocytic lymphohistiocytosis (hlh) or osteopetrosis. defibrotide prophylaxis group (n = 22) received 25 mg/kg/day per day and continued for at least 14 days after transplantation. the control group (n = 53) received only continuous infusion of low-dose heparin until 30 days after transplantation. for the comparison between groups, the fisher's exact test was used. all analyses were performed using spss 20 and p-value of 0.05 was considered statistically significant. we analyzed totally 75 hsct procedures with different diagnosis; 17 beta thalassemia major, 14 leukemia, 9 hlh, 18 primary immunodeficiencies, 3 osteopetrosis, 4 fanconi aplastic anemia (faa), 2 myelodysplastic syndrome, 2 neuroblastoma, 1 congenital amegakaryocytic thrombocytopenia, 1 krabbe disease, 1 aplastic anemia, 1 hypereosinophilic syndrome and 1 sickle cell disease. all the procedures meet the 'high risk' definition; most of them (n = 50) have busulphan for conditioning, also there are 9 hlh and 3 osteopetrosis patients, 2 neuroblastoma patients had the second myeloablative regimen, faa and aplastic anemia patients had pre-existing liver disease, and 2 of the leukemias had beyond second relapse. the mean age was 6.7 years old (0.25-19.6), 27 hsct performed from match sibling donor (msd) (36%), 3 hsct from match family donor (mfd) (4%), 43 hsct from match unrelated donor (mud) (57%) and 2 hsct from haploidentical mother (3%). we especially focused on gvhd and vod. totally 13 vod cases (17%) in these 75 hsct procedures were detected. only two of them detected in the prophlaxis group (9%) and 11 cases in the control group (20%). there were 32 cmv reactivation cases detected in 75 hsct procedures (42%). in the prophlaxis group there were 11 cases (50%) and in the control group 21 cases (39%). we detected 36 acute grade i-iv gvhd cases in 75 hsct procedures (48%). only 4 of them were in the prophlaxis group (18%) and 32 cases were in control group (60%). the prophlaxis group's agvhd ratio was significantly lower than the control group (p = 0.001). defibrotide for vod prophylaxis is confirmed by several studies, but its benefits for agvhd is not clear. in this study, we show the significant effect of defibrotide on agvhd. we speculate that the protective effect of defibrotide on both vod and agvhd could be explained by the similar pathophysiology of these complications. we need larger studies on the pathophysiological pathways, then we could invent more effective interventions. disclosure of conflict of interest: none. conventional extracorporeal photopheresis (ecp) has proven efficient for the treatment of graft-versus-host-disease (gvhd) but is limited to patients with sufficient body weight. a mini buffy coat ecp (mini-ecp) 'off line' technique that allows treatment of small children has been developed, using various methods for mononuclear cell (mnc) separation from whole blood. we present treatment of low body weight child with mini-ecp 'off line' technique using sepax system for mononuclear cell (mnc) selection and macogenic irradiator. a toddler with juvenile myelomonocytic leukemia (jmml) developed acute gvhd, after a matched unrelated stem cell transplantation (sct) performed at the age of 12 months. acute gvhd of the skin occurred three months after sct and responded to high dose steroids, but recurred six months after sct (biopsy of the skin confirmed acute gvhd) together with gvhd of the liver. because of the resistance to steroids and cyclosporine, mini-ecp was introduced as therapy. the patient weighed 8 kilograms. blood was collected from tunneled central venous catheter, and collected volume was replaced with saline infusion. the cord blood collection bag (macopharma, france), which contains 21 ml citrate phosphate dextrose (cpd) anticoagulant solution was used for whole blood collection. whole blood was processed using sepax system separator (biosafe, switzerland), and final volume of buffy coat was set on 25 ml. extracted buffy coat was transferred into the uv-a illumination eva bag (macopharma, france) and diluted with saline solution up to 200 ml. 8methoxypsoralen (gerot, austria) was injected directly into the uv-a illumination bag, and cells were irradiated by the uv-a illumination device macogenic 2 (macopharma, france). irradiated cells and autologous residual blood were reinfused back to the patient. during the whole procedure patient's vital signs were monitored. ecp procedures were performed 3 times per week for 4 weeks, followed by 2 times per week at 2 weeks intervals for 2 months. in 3 month period 28 mini-ecp procedures were performed. median of collected whole blood was 92 ml (range 52-100). median of total nucleated cell (tnc), and mononuclear cell recovery after sepax separation were 88.3% (range 66.7-104), and 90.8% (range 63-102), respectively. median of hematocrit in final irradiated product was 4% (range 3.8-6%). patient was reinfused with median of 1.0 (range 0.66-1.8) tnc × 10 8 /kg bw, and median of 4.95 (range 3.62-13.72) lymphocyte × 10 8 /kg bw. after one month of ecp together with steroids and cyclosporine, gvhd of the skin improved, and the steroids were gradually weaned, with no recurrence. gvhd of the liver showed no improvement, and other therapies had to be introduced, but without steroids. for the first time in croatia, mini-ecp was performed in a child with gvhd, in whom conventional ecp could not be used because of insufficient body weight. mnc separation using automated closed system sepax separator has proven efficient and safe. mini-ecp treatment was continued for three months, without technical difficulties. positive effect was experienced concerning the skin gvhd, but not the liver gvhd. after the first experience in our country, in future we plan to use this technique for low-weight patients or patients with contraindications for apheresis, which are in need of second-or third-line therapy for gvhd. disclosure of conflict of interest: none. gonadal failure represents one of the late effects of haematopoietic cell transplantation (hsct) with a negative impact on quality of life in young patients (pts) undergoing hsct1,2. the aim of this retrospective multicentre ebmt study was to assess gonadal function in untreated pts undergoing allogeneic hsct between 5 to 20 years (yrs) of age, after a preparative regimen with busulphan (bu) or treosulfan (treo). eighty-seven pts (32 females, 55 males) were reported from 17 out of 123 contacted ebmt centers: 26/87 (30%) received allogeneic hsct during pre-pubertal and 61/87 (70%) in pubertal phase. of the 87 pts, 76 (87.4%) received bu in myeloablative dose [25 pre-pubertal, (median age of 6.7 yrs) and 51 pubertal, (median age of 13.4 yrs)] and 11 pts (12.6%) received treo (1 in pre-pubertal and 10 in pubertal period). underlying diseases were primary immunodeficiency (34.5%), chronic myeloid leukemia (33.3%), myelodisplastic syndrome (24.1%), familial haemophagocytic lymphohistiocytosis (6.9%) and shwachman-diamond syndrome (1.1%). 17/26 of prepubertal pts (71%) developed spontaneous puberty (69.5% in the bu group and 100% in treo group). 21/28 (75%) females undergoing hsct during puberty completed their pubertal development (71.4% in bu group and 100% in treo group). none of females (4/4) with bu during pre-pubertal phase developed spontaneous menarche (sm), while 33.3 %(7/21) of females who received bu in pubertal period had sm. all females (n = 5) treated with treo during pubertal phase had sm (100%). for both conditioning regimens, the 42.8% (12/28) s358 of females treated during the puberty experienced sm. among the remaining 14 females (for 2 pts the information is missing) who did not developed sm, 13 received hrt 2.5 yrs after hsct and 5 of them had ovarian recovery after a median of 2.3 yrs from hsct (1.43-6.72). the median age at last follow up was 15.8 and 13.2 yrs in bu and treo pre-pubertal group, and 22.2 and 19.9 yrs in bu and treo pubertal group respectively. in the pubertal group, 18 females (69.5%) are still receiving hormonal replacement therapy (hrt) (16 in the bu group and 2 in treo group). 2 pts (7.4%) had spontaneous pregnancy. no problems in newborns are reported. sperm analysis was performed in 18.2% of pubertal pts (6/33) of males, and 66% (n = 4/6 treated with bu) were azoospermic (data regarding 2 pts were missing). the sperm analysis was repeated in half of the males. until now no paternity was reported. in this experience, the pubertal development in pts who received treo (n = 6) was normal, and in the bu group the majority of females (70%) had normal puberty. the rate of sm is higher (100%) in females after treo than bu (28%). the hrt is ongoing at last follow-up in 76% of females treated with bu and in 40% of those who received treo. our data suggests that treo may have a better outcome than bu in young girls receiving allogeneic hsct and larger studies are warranted. male patients require longer follow-up. prevention in patients transplanted from partially matched donors. we report the single centre experience in haploidentical sct. in years 1999-2016 in the department of pediatric bmt, oncology and hematology at wroclaw medical university, 72 children underwent sct from partially matched, haploidentical parental donors. graft manipulation in 38 patients consisted of cd34sel, in 22 patients the cd3 immunomagnetic depletion (tcd-cd3) was performed, and in 12 -tcr alpha-beta depletion (tcd-ab). we analysed the impact of type of manipulation procedure, conditioning regimen, demographic factors, and kir genotype on survival and probability of neutrophil recovery. the probability of engraftment and neutrophil recovery was 86% vs 77% in cd34sel group (p = ns). probability of 5 year overall survival in the tcd group was similar to the cd34sel group (45% vs 34%, p = ns). in the tcd patients, neither use of busulfan vs treosulfan, nor kir genotype, nor donor sex had noticeable impact on sct result and survival. patients transplanted after tcd due to non-malignant disease had higher survival probability, than those with malignancies (69% vs 28%, p = 0.04). the trm in tcd patients was reduced in comparison to cd34sel (24 vs 58%, p = 0.003). the trm after tcd resulted mostly from severe viral infections in tcd-cd3 patients. in 4/34 tcd patients spontaneous acute, skin (stage 2) gvhd was diagnosed and successfully treated. two patients received unmanipulated donor lymphocyte infusions (dli) and developed severe acute steroid-resistant (grade 4) gvhd, in one of them with fatal outcome. tcd methods are superior to cd34sel due to significant reduction in treatment related mortality. haploidentical sct after tcd can result in durable engraftment, but warrants intensive post-transplant monitoring for infectious complications and cautious approach to dli therapy. disclosure of conflict of interest: none. median of 15 days for neutrophils in both groups, 17 for platelets (23 in ptcy,12 in αβ+cd3+/cd19+depleted, p 0.005). donor chimerism was complete in 22 patients (84.6%). in αβ +cd3+/cd19+depleted group, 4 patient rejected (33.3%:1 primary and 3 secondary reject, 22, 28, 55 and 195 days after haplo, respectively) and were rescued with a second transplant. seven patients (50%) developed acute (a-) gvhd in ptcy group (grade 1-2 in 4; grade 3-4 in 3), compared to one (8.3%: grade 4) in αβ+cd19+depleted group (p 0.02). among patients at risk, 3 out of 9 in ptcy group developed chronic (c-) gvhd (33.3%:1 score-3, 1 overlap, 1 score-1), compared to 0/9 patients in αβ+cd3+/cd19+depleted group (p 0.08). the cumulative risk of cmv-reactivation was 72% and 58% in ptcy and αβ+cd3+/cd19+depleted groups, respectively (p 0.63). t-cell reconstitution was significantly different in the two groups,with a median absolute number of cd16+56 +cd3-and γδ+cd3+ higher in αβ+cd3+/cd19+depleted group on day +120 (p 0.03) and a median number of cd3 +cd8+ higher in ptcy group on day+180 (p 0.04). length of hospitalization was shorter in the αβ+cd3+/cd19+depleted group, with a median time from haplo to discharge of 23 days compared to 31 days in the ptcy group (p 0.003). some children have not donor and an urgent need to proceed to transplantation because of disease status. we reviewed the role of haploidentical transplantation in children and report our single center experience. ten children were transplanted from haploidentical family members donors (median age:12.5 years). we performed alfa beta t cell depleted transplantation in three patients and unmanipulated bone marrow transplantation with posttransplant cyclophosphamide in seven patients. the diagnosis were eight high risk leukemias (three all and five aml) and two severe aplastic anemia. patients were myeloablative conditioned with cyclophosphamide, fludarabine and total body irradiation in aplastic anemia received alfa beta t cell depleted grafts with a median cd34 cell dose of 2.9 × 10 6 /kg (range:2.6-3.2) and busulphan, cyclophosphamide in high risk leukemias received unmanipulated bone marrow grafts with posttransplant cyclophosphamide in 3rd and 5th day of posttransplant with a median cd34 cell dose of 7.4 × 10 6 /kg (range:2.38-16.1). median follow up of our patients 10 months. six of 10 patients are alive and in disease free follow up. four patients were relapsed and dead median 5.5 months of transplantation. the rate of relapse was 50 % for leukemia patients in remission and 50% for patients with active disease. myeloablative conditioning regimen followed by haploidentical bone marrow transplantation with posttransplant cyclophosphamide may be an option in high risk leukemia patients need urgent transplantation because of desease status who have not donor. table 1 . all patients received hd-cy 50 mg/kg on d+3 and d +4. cyclosporine a 3 mg/kg/d i.v., then 6 mg/kg/d p.o. adjusting for blood levels 200-400 ng/ml and mycophenolate mofetil 15 mg/kg 2 times daily po were started on d+5. mmf was discontinuated on d+35, csa-after d+100. all pts received anti-microbial prophylaxis for bacteria, fungal, herpes infection and pneumocystis according to institutional practices. analysis for donor chimerism and mrd were performed at d+30, +60, +100, +180. pts, donors and stem-cell harvest characteristics are described in table 1 . 4 pts had high risk hematological malignancies, and 1 relapsed after auto-sct neuroblastoma (hr-nb). 1 pt was transplanted in 1st cr (aml m7) and others in 2nd cr. 3 pts had full engraftment (neutrophil engraftment at 17,18 and 22 days). 1 pt (hr-nb) was concerned as a primary failure for achieving neutrophil and platelet engraftment by d+30, despite of complete donor chimerism in bone marrow. he was transplanted additionally with the same donor at d+49 after 1st transplant. 1 pt died before engraftment at d+20 (fulminant ps. aeruginosa-sepsis). 2 pts remain alive in cr (2ndcr-aml and 1st cr-m7 aml) with follow-up of 375 and 164 days (05/12/2016) without cgvhd with complete donor chimerism. 2 pts relapsed after d+100 ( were transplanted in 2nd cr-flt3 aml and 2nd cr-nb) and died. 1 pt died because of infectious complication at d+20 (transplanted in 3d cr-all). 4/5 pts had grade 2 acute gvhd. hla-haploidentical hsct with post-transplant t-cell in vivo repletion grafts by using hd-cy is feasible and effective in children with hr-haematological malignancies. [p463] who were match unrelated donor. thalassemic reconstitution occurred in three patients. acute graft-versus-host disease (gvhd) of grade ii-iv occurred in 17 % and chronic gvhd in 12%. acute and chronic gvhd were seen more frequently in patients with class 2-3 compared to class 1. mortality rate was also higher in these groups. seven patients died. one patient died on post-transplant day 26 due to intracranial bleeding. the other 6 patients with chronic gvhd died between 182 and 257 days, on average 219 days post-transplant. these data suggest that allogeneic bmt remains an important treatment option for children with beta-thalassaemia major, particularly when compliance with iron chelation is poor. the society to support children suffering from cancer, also known as mahak, was set up in 1991 as a non-governmental and non-profit organization. in the past two decades, the organization has attracted a vast public support and fulfilled a great part of its mission which is to support children with cancer, reduce the child mortality rate and create an appropriate environment that empowers families who have children with cancer. pediatric stem cell transplant also is used to treat many types of conditions affecting children and adolescent, including cancer and certain hematologic, immune reconstitution inflammatory syndrome (iris) is a clinical condition emerging after immune recovery of an immunocompromised status, mostly after the initiation antiretroviral therapy (art) in human immunodeficiency virus (hiv) infected patients, but also in several other settings, such as the recovery from the severe combined immunodeficiency (scid) status after hematopoietic stem cell transplantation (hsct). herein, we report a patient transplanted for scid who developed iris for two times, namely shortly after transplantation and after donor lymphocyte infusion (dli) ( table 1 ) in our patient, t cells passing from the donor probably contributed to the immediate post-transplant increase in the size of granulomas. this inflammatory response waned after the institution of immunosuppressive and methylprednisolone therapy. however, immunosuppressives were stopped due to lowered chimerism at follow-up, and the inflammatory response re-appeared after administering stem cell support containing a large amount of t cell from the donor for dli purpose. although the mechanism by which dli results in clinical responses is unclear, it is presumed to be a t cellmediated process. several studies have been performed to strengthen our understanding of the immunopathogenesis of iris. while some of those studies put forth t cell-associated causes, others implicated cytokines and non-t cells. the reaction that developed in our patient is suggestive of t cellassociated causes. immune reconstitution inflammatory syndrome remains a poorly understood entity. the dli procedure in our case provides a unique clue supporting a t cell mediated process. pediatric transplant teams need to be s364 aware of the previous iris phenomenon of bcg-adenitis while making the decision of dlis. [p469] disclosure of conflict of interest: none. pediatric patients treated with a hematopoietic stem cell transplantation (hsct) often suffer from late side effects caused by the treatment. the aim of this study is to investigate the late effects of a hsct on dental development. in addition, patients and parents awareness on this topic was investigated. 42 young adults treated and followed at the ghent university hospital who were under the age of 12 y at the time of hsct were examined clinically and radiographically (planmeca promax 2d). transplants (11 autologous/31 allogeneic) were done for malignant disease in 34 pts. eight patients received a hsct for a non-malignant disease. twelve patients underwent a conditioning regimen with total body irradiation (tbi), 21 patients with busulfan and 9 patients with other chemotherapeutic agents. 16 patients were o3 y, 9 patients were 3-6 years and 17 patients were 4 6 years at hsct. every patient was evaluated on dental agenesis, microdontia and rootcrown ratio. patients and their parents were asked about their knowledge and interest for dental screening at the follow-up clinic using a questionnaire. overall, the prevalence of agenesis and microdontia of one or more dental elements is respectively 51.3% and 46.2% in our study population. 76.3% of patients have a strongly aberrant root-crown ratio of at least one element. patients treated o3 years of age show significantly more microdontia (76.9%; po0.001) as well as agenesis (92.3%; p o0.001) compared to patients treated at an older age. the first premolar of the mandible is the most vulnerable element for agenesis as well as for microdontia. more microdontia is found in patients treated with a busulfan conditioning regimen compared to the other conditioning regimens (68.4% versus 25%). patients older than 6 years, treated with busulfan have statististically more microdontia compared to patients 46 y treated with tbi conditioning regimen (p = 0.044). there was no difference of the conditioning regimens on agenesis nor on root-crown ratio. almost all patients/parents find it important to receive information about the dental late effects of a hsct and are interested in dental screening at the follow-up clinic. treatment with hsct has an explicit negative impact on dental development. the degree of this effect depends on age at hsct and used conditioningregimen. dental follow-up of these patients is essential and should be incorporated in the follow-up program. disclosure of conflict of interest: none. importance of body composition in the outcome of hematopoietic stem cell transplantation in elderly patients l koch 1 , n hamerschlak 1 , r garcia 1 , c prado 1 , c silva 1 and a pereira 1 hospital israelita albert einstein the loss of muscular mass is a well recognized cause of the decline in muscle strength and functionality that accompany the aging process. in 1989, irwin rosenberg proposed the term 'sarcopenia' to describe the decline in muscle mass associated with aging. changes in body composition after hematopoietic stem cell transplantation (hsct) have been the subject of previous studies. immunosuppressive therapy and corticosteroids are known to alter skeletal muscle metabolism. infections and graft-versus-host disease (gvhd) that can occur after hsct may also affect body weight and composition. therefore, both the treatment and complications after hsct exert large negative effects on lean muscle mass, especially in elderly patients. patients with hematologic malignancies are usually well nourished before undergoing hsct. objective: the aim of this study is to determine in an elderly population whether parameters of body composition could be correlated to outcomes after hsct. we performed a retrospective longitudinal study through review of medical records of 48 patients ⩾ 60 years old undergoing hsct at hospital israelita albert s365 einstein, from 2013 to 2015, that were subject to tomography scans (cts) in a period ranging from 60 days before and 15 days after hsct. table 1 . there were no differences between groups with respect to age, gender, diagnosis, stage of disease, and source of stem cells. in ly patients, the quantity of peripheral cd34+ cell dose (×10 6 /kg) infused was different between groups (group ly-ct the incidence of nf was significantly higher in group mm-g (19 (59.4%) vs 19 (37.3%); p = 0.049). no differences were observed in the incidence and severity of mucositis, first day and duration of fever, documented bacterial infections or readmission rate between mm patients groups. this study suggests that in at home asct, the use of piperacillintazobactam prophylaxis significantly reduces the incidence of neutropenic fever and hospital readmission in patients with ly, and also that no administration of g-csf in mm patients reduces significantly the incidence of neutropenic fever. disclosure of conflict of interest: none. [p472] allogeneic stem cell transplantation (sct) has been recognized as a curative treatment for patients with wiskott-aldrich syndrome (was). in sct for was, myeloaberative conditioning (mac) has been indicated to avoid a mixed chimera. however, risk factors for a mixed chimera in patients with was who have received sct have not been evaluated. here, we analyzed the outcomes of sct and risk factors for a mixed chimera in 108 patients with was who underwent sct in japan since 1985. we reviewed medical records of 108 consecutive was patients who received sct since january 1985 who were registered with the japan society for hematopoietic cell transplantation. the age of the patients at transplantation ranged from 3 months to 23 years, and the mean age was 3.81 years. the origin of the stem cells was related bone marrow (bm) or peripheral blood stem cells (pbsc), unrelated bm or pbsc, and unrelated cord blood (cb) for 36, 41 and 27 patients, respectively. a preparative conditioning regimen consisting of mac was provided to 76 patients, and reduced-intensity conditioning was provided to 30 patients. fifty-one patients received prophylaxis against graft-versus-host disease (gvhd) with cyclosporine in combination with methotrexate (mtx) or a steroid, and 51 patients received tacrolimus (tac) with mtx or a steroid. chimerism analysis had been performed in 91 patients. neutrophil engraftment was achieved in 91 patients (82.7%). the engraftment rate was significantly higher in patients who received tac for gvhd prophylaxis, (p = 0.0001) overall survival rate was significantly higher in patients with complete chimerism than in patients with mixed chimerism (88.2 ± 6.1% and 66.7 ± 9.9%, respectively, p = 0.003), though there was no significant difference in stem cell sources. using multivariate analysis, the rate of complete chimerism in patients who received mac including cyclophosphamide (cy) at more than 200 mg/kg was significantly higher (p = 0.02) than the other conditioning. not only patients with mixed chimerism but also patients with complete chimerism were complicated with auto-immune diseases. in this study, achievement of complete chimerism after sct was important for survival in patients with was. we found that patients who underwent mac including cy at more than 200 mg/kg had a higher rate of complete chimerism. we also found a higher neutrophil engraftment rate in patients who received tac for gvhd prophylaxis. thus, mac including cy at more than 200 mg/kg and tac for gvhd prophylaxis are optimal conditions of sct for patients with was. disclosure of conflict of interest: none. adenosine deaminase (ada) deficiency is an inherited autosomal recessive immunodeficiency which represents about 10-15% of scid. since 1992 we diagnosed 29 patients affected by ada-scid: 10 underwent hematopoietic stem cell transplantation (hsct), 10 were treated with replacement therapy with peg-ada and 4 received gene therapy; 5 patients died before or after treatment. maternal t lymphocyte engraftment is frequently detected in scid patients, but this is never been found in ada deficient patients. a 3-months-old italian girl, from non-consanguineous parents, presented to our hospital with a history of frequent bronchiolitis associated with dermatitis, mycosis, hypogammaglobulinaemia, marked lymphopenia (t cells cd3, 171/mmc; cd3/cd4, 158/mmc; cd3/ cd8, 8/mmc, b cells 15.2/mmc, and nk cells, 110/mmc) and in vitro absence of proliferative response to pha. level of immunoglobulins was almost normal (igg 439 mg/dl, iga 87 mg/dl, igm 57 mg/dl). high levels of toxic metabolites were found: axp, 1.573 micromol/ml rbc; daxp, 0.629 micromol/ml rbc; %daxp, 28.5. ada activity in rbc lysates was abnormally high for scid-ada (0.54 u/g hb). molecular analysis confirmed diagnosis: the sequencing of exon 10 revealed two mutations: a missense mutation previously reported called p.ser291leu (c. 872c4t) and a new missense mutation defined p. leu298pro (c.893t4c). t-cells str analysis of patient showed 54.1% maternal t lymphocytes engraftment never reported before in ada-scid patients. the girl was transferred to the isolated bmt unit and the respiratory symptoms progressively improvement. replacement therapy with peg-ada was started immediately at a dose of 30 u/kg/twice per week. ig therapy was started at a dose of 200 mg/kg every two weeks. after three months of treatment patient showed an increase in t cells count (cd3, 411/mmc), and a decrease of toxic metabolites: axp, 1.652 micromol/ml rbc; daxp, 0.011 micromol/ml rbc; %daxp, 0.7 maternal t-cell engraftment persists, despite a good response to the peg-ada therapy. the last examination before hsct reveals maternal t-cell engraftment of 9.2%. patient underwent hsct from mud hlaidentical donor after a myelo-ablative reduced intensity conditioning regimen protocol d ebmt/esid guidelines. the number of infused cd34+ cells was 14.29 × 10 6 /kg and 69.47 × 10 6 cd3/kg. she is actually at day+108 post hsct, is doing well and shows 100% engraftment of donor cells. disclosure of conflict of interest: none. graft versus host disease (gvhd) is a frequent complication in patients undergoing haematopoietic stem cell transplantation. while the exact pathophysiology of gvhd is not known, the gut microbiome has been implicated in its development since it was shown that total gut decontamination (tgd) decreases the incidence of gvhd. with this study we aim to get insight into the diversity of the gut microbiota before, during and after total gut decontamination in comparison with selective gut decontamination (sgd). secondly, we want to identify changes in microbiota composition that relate to the occurrence of graft-versus-host disease. for this prospective cohort study we recruited 22 children (o 18y) that were eligible for a stem cell transplantation at the leiden university medical center between january and december 2015. of these, 64% (n = 14) received tgd (consisting of piperacillin/ tazobactam and oral amphotericin b), whereas the other 36% (n = 8) received selective gut decontamination with polymyxin /neomycin and oral amphotericin b. in total, 129 fecal samples were collected, weekly during admission for the stem cell transplantation and monthly thereafter up to 6 months after transplantation. also samples were collected from family stem cell donors as healthy controls. samples were processed within 24 hours and stored in the -80 freezer, after which 16s v4 amplicon sequencing (illumina hiseq, rapid mode, 250 bp read length) was applied. data analysis (taxonomy and shannon diversity) was primarily done using qiime (ref). compared to microbiota diversity in stem cell donors (mean shannon index (si) 3.43), we observed an overall lower mean si during tgd (1.90) and slightly higher mean si during sgd (2.43). microbiota diversity months after sgd (2.45) was similar to diversity during sgd (2.43), while diversity months after tgd (2.63) was higher than during tgd (1.90). further analysis of repopulation dynamics demonstrated no differences in repopulation duration after both decontamination strategies. however, we did observe differences in the type of bacteria that repopulated, with bacteroidales being more prominent in sgd and lactobacillales more prominent in tgd patients. actinomycetales (genus rothia) was exclusively present in tgd patients during decontamination. also, the clostridiales (blautia, lachnospiraceae and peptostreptococcaceae) were bacteria that appeared after the decontamination period. four patients (18%) in this cohort developed gvhd grade 1 or more. in these patients we did observe individual compositional changes of the gut microbiota at the time of ghvd diagnosis, e.g very low diversity or dominance of enterobacteriales. considerable microbiota diversity is observed in patients that received tgd. different repopulation dynamics were observed between tgd and sgd. no common pattern was found in the gvhd cases. disclosure of conflict of interest: none. minimal residual disease (mrd) pre-and post-hct for children with aml is highly predictive of event-free survival: a pediatric blood and marrow transplant consortium study d jacobsohn 1 johns hopkins all children's hospital, 16 children's hospital los angeles, usc keck school of medicine multicenter data regarding the significance of mrd in children with aml pre-and early post-hct are lacking. we hypothesized that pre-and post-hct mrd assessments using wt1 pcr combined with multi-dimensional flow cytometry (mdf) would be predictive of disease relapse and event-free survival (efs) at 2-yrs post-hct. subjects were o21 yrs with aml in morphologic cr undergoing ma allogeneic hct. stem cell sources included bm, pbsc, or cb. bm and pb samples were collected at 3 time points: baseline ( o3 weeks prior to preparative regimen); day+42 (±14 days); and day+100 (±20 days). bm samples were analyzed for both wt1 expression and mdf mrd (single reference lab using a 'difference from normal' approach without access to diagnostic phenotype); pb samples were analyzed for wt1 only. mdf detection limit was 0.02%; however, we required that 2 independent analysts certify that the abnormal population was aml. in addition, sorted mrd+ cells were tested for chimerism. wt1 positivity was defined as ⩾ 1300 copies for bm and ⩾ 200 copies for pb. results were not available to the treating clinician. 150 subjects were enrolled at 34 centers in us and canada. 20 enrolled subjects did not undergo hct and 6 were excluded for progression prior to hct or other ineligibility. in 124 eligible subjects, 2-yr efs and os were 52% and 61%, respectively. the 2-yr ci of relapse and trm were 36% and 13%, respectively. mdf identified 7 subjects pre-hct having 0.2-14% residual disease. the 2-yr relapse rate in subjects with +mrd by mdf pre-hct was 100% vs 32% (23-40%) in those who were negative. 2-yr dfs and os were 0% and 29% (4-65%) for positive mdf pre-hct, and 54% (45-63%) and 62% (53-71%) for negative mdf. pre-hct mdf sensitivity for 2-yr dfs was 10%; specificity was 100%. mdf mrd at days 42 and 100 were similarly predictive of outcome. sorted mrd+ cells from 19 post-hct samples were all noted to be of recipient origin. pb wt1 had no correlation with dfs or relapse; bm wt1 at day+100 correlated with 2-yr os (79% (68-88%) low/neg vs 57% (39-75%) high). other wt1 cutoffs studied demonstrated no correlation with outcomes. figure 1 : relapse probability by flow cytometry mrd at 3 time points. mdf mrd pre-hct and at days +42 and +100 was significantly associated with lower efs and os in children with aml undergoing hct. mdf is specific but not sensitive, as many negative mdf patients relapsed. our goal was to define a reproducible assay that did not depend on having the initial aml profile. this would facilitate multi-institutional studies aimed at decreasing relapse. given that constraint, we were able to detect clear mdf mrd in a small percentage of patients that was highly predictive and can be used in trials. wt1 level was not predictive in this multi-institutional trial. the sensitivity of flow was significantly affected by not having the initial flow available. future attempts to improve sensitivity should include initial flow and/or test higher channel flow or molecular pcr techniques. in addition, we confirmed that mrd + cells obtained by cell sorting post-hct were of recipient origin. future testing of 'suspicious' sorted cells by fish, molecular, or comparative genomic hybridization could possibly increase mfd sensitivity. novel cellular or targeted therapies should be tested in clinical trials to improve outcomes in patients with mfd mrd noted either pre-or post-hct. [p480] disclosure of conflict of interest: none. novel mutations were identified with ngs and low intensity of conditional regimen succeeded in children with fanconi anemia who received allo-hsct s hu 1 , h hou, j lu, p xiao, x bian, h liu, y hu, j ling, l li, l kong, z zhai and y yao 1 children's hospital of soochow university to explore the possiblity of applying next-generation sequencing (ngs) to diagnose the disease of fanconi anemia (fa) and evaluate the efficiency and safety of low intensity conditional regimen on children with fa receiving allogenic hematopoietic stem cells transplantation (allo-hsct). five patients initially suspected as severe aplastic anemia were diagnosed as fa by the method of next-generation sequencing (ngs)-based genetic diagnosis panel. one patient received hla-identical sibling donor hematopoietic stem cell transplantation (mrd), three patients underwent unrelated donor matched (ud) hsct, and one patient received unrelated cord blood transplantation (ucb). the conditional regimen consisted of either 300 cgy tbi or 3.2-3.6 mg/kg of busulfan with 20-40 mg/kg of cyclophosphamide. meanwhile, atg at 10 mg/kg and fludarabin at 140-180 mg/m 2 were included as well. cyclosporin or tacrolimus as well as mycophenolate mofetil (mmf) were used for the prophylaxis of graft versus host disease (gvhd). engraftment of neutrophil and platelet and complications followed transplantation such as infection, gvhd, and hemorrhagic cystitis (hc) were observed. of 5 cases diagnosed as fa by ngs, only 1 case showed the abnormality of chromosome fragility test which has been regarded as golden criteria in the diagnosis of fa. meanwhile, we found 5 novel mutations in 3 cases of fa which enriched chinese national database with data of rare diseases by ngs. the counts of mononuclear cells (mnc) were (3.87-18.57) × 10 8 /kg for non-ucb and 9.83 × 10 7 /kg for ucb. the counts of cd34+ were (4.01-9.57) × 10 6 /kg for non-ucb and 2.56 × 10 5 /kg for ucb. all 5 cases succeeded in allo-hsct with the low intensity of conditional regimen. the median time for neutrophils engraftment was 11 days (range 9~15 days), median time to platelets (plt) engraftment was 14 days (range 8~28days). one case occurred with grade i of agvhd, 2 cases with hemorrhagic cystitis. after transplantation, all patients were monitored the copies of ebv-dna and cmv-dna of whole blood, and five case with ebv positive and 3 cases with cmv positive. no patient suffered of ebv or cmv disease. the hepatic veno-occlusive disease (vod) and hc were observed in 5 fa patients after transplantation. ngs showed much more specific and facilitated for the diagnosis of fa. low intensity of conditional regimen is efficient and safe which should be recommended for the treatment of fa patients. disclosure of conflict of interest: none. outcome of alternate donor stem cell transplantation in children: an indian experience sp yadav 1,2 , n rastogi 1,2 , d thakkar 1,2 , s kohli 1,2 , s nivargi 1,2 , r misra 1 and s katewa 2 1 in india due to lack of donor registries and cord blood banks very few alternate donor stem cell transplants (sct) are performed. haploidentical sct has become feasible with availability of post-transplant cyclophosphamide (ptcy) technique. here we present our experience of setting up alternate donor program for sct for children in india and report the outcomes of the same. we collected data retrospectively of all children who underwent alternate donor sct during jan 2013 to dec 2016 in two centres. a total of 47 sct were performed for 43 children; median age 6 years (1-18 years) and 37 were boys and 6 girls. of these, 41 underwent haploidentical (35 ptcy and 6 tcr alpha-beta/cd19 depleted), 4 matched unrelated donors (mud) and 2 unrelated cord blood (ucb) sct. the diagnosis was: primary immunodeficiency-10, thalassemia major-14, sickle cell disease-3, inherited bone marrow failure-4, acquired aplastic anemia-3, acute lymphoblastic leukemia-3, acute myeloid leukemia-4, neuroblastoma-4, ewings sarcoma-1 & leukodystrophy-1. the conditioning was with fludarabine, cyclophosphamide and total body irradiation backbone in 36 children (thiotepa added in 24), fludarabine and treosulfan in 5, fludarabine and busulfan in 2, busulfan and cyclophosphamide in 4. serotherapy was part of conditioning, rabbit anti-thymoglobulin 4.5 mg/kg in 38 and campath 1 mg/kg in 9. graft vs host disease (gvhd) prophylaxis was ptcy along with tacrolimus and mycophenolate mofetil in 37 patients (35 haploidentical, 1 mud & 1 ucb) and ex-vivo tcr alpha-beta depletion in 6 and cyclosporine and methotrexate in 4. all were transplanted after a signed informed consent. a median of 8 million of cd34cells/kg was infused (range 5-24 million/kg).graft source was peripheral blood in 39 and bone marrow in 6 and ucb in 2. five children died before engraftment. the remaining 42 had neutrophil engraftment by median of 14 days (range 8-35) and platelet engraftment by median of 14 days (range 9-48). chimerism at day+100 was available in 34 cases; 31 of them had full donor hematopoiesis. one had mixed chimerism and 3 fully recipient. four children underwent a second haploidentical sct after rejection of which 2 are alive and disease free. the median follow-up of remaining patients is 9 months (range 1-40); the cumulative incidence of graft versus host disease (gvhd) acute and chronic extensive was 26% and 20% respectively. grade-iii-iv acute gvhd was seen in 3 patients. a total of 15 patients have died (sepsis-4, stroke-1, gvhd-3, vod-3, encephalitis-3 and progressive disease-1). among encephalitis deaths, one child had undergone ucb with ptcy and another tcr alpha-beta depleted second sct.; both had bk virus in the csf.there were 11/41 deaths in haploidentical (ptcy-10/35 & tcr alpha-beta-1/6), 3/4 in mud and 1/2 in ucb sct. overall survival is 68% and disease free survival is 66% at last follow up. alternate donor sct is an acceptable curative option for children lacking a matched sibling donor. haploidentical donor sct is more feasible in the setting of lack of donor registries having indian ethnicity donor. disclosure of conflict of interest: none. hematopoietic stem cell transplantation (hsct) from an unrelated donor (ud) is largely used for pediatric patients with all in second complete remission (cr) lacking an hlaidentical sibling. in this study, we retrospectively analyzed outcome of patients (pts) given ud-hsct in centers affiliated to the associazione italiana di ematologia ed oncologia pediatrica (aieop) network between 2000 and 2013. three hundred fifty-six pts with all in second cr experiencing either bone marrow (bm), isolated extramedullary or combined relapse were included in the study; 139 were males and 217 females, median age at hsct being 4.8 years (range 0.5-19). bm, peripheral blood (pb) and cord blood (cb) were the stem cell source in 64%, 16% and 20% pts, respectively. all children received a myeloablative conditioning regimen, either tbi-(293 pts) or chemotherapy-based (63 pts). as gvhd prophylaxis, the combination of cyclosporine a, short-term mtx and atg was employed in most pts. according to the berlin-frankfurt-munster (bfm) classification of first leukemia recurrence, 59% and 42% of pts were assigned to the s1+s2 and s3 +s4 groups, respectively. level of pre-hsct minimal residual disease (mrd), measured within 30 days before hsct through flow cytometry (fcm) in the laboratory of padova, is available in 37 children; more data will be presented during the s372 meeting. with a medium follow-up of 6.5 years (range 0.5-15), the overall survival (os) was 52%, while the event-free survival (efs) was 50%. the cumulative incidence of transplant-related mortality (trm) and leukemia recurrence were 24% and 25%, respectively. the efs probability for children transplanted in the time period [2000] [2001] [2002] [2003] [2004] [2005] [2006] [2007] [2008] [2009] and 2010-2013 was 45%, 56% and 52%, respectively (p = ns). patients who received a tbi-based conditioning regimen had a significantly better outcome in comparison to children who received chemotherapy-based treatment, efs being 53% and 36%, respectively (p = 0.01). efs of pts belonging to s1+s2 and s3 +s4 groups was 66% and 35% respectively (p = 0.0001). the difference in efs is largely explained by an increased incidence of leukemia recurrence in s3+s4 (34%) compared to s1+s2 pts (23%) (p = 0.0002). efs of pts who experienced grade ii acute gvhd was 68%, while that of pts with either absent/grade i acute gvhd or grade iii-iv acute gvhd was 52% and 11%, respectively (p = 0.0001). pts with limited chronic gvhd had a better efs as compared to those with either extensive or absent chronic gvhd (77%, 35% and 61%, respectively; p = 0.039). the choice of stem cell source (bm, pbsc, cb) did not influence the probability of efs, which was 56%, 46%, 40% respectively (p = ns). importantly, among pts with evaluable mrd before hsct (n 37), the group with detectable levels 0.001% (n 9), respectively 54% and 17% (p = 0.038). conclusions. outcome of children with 2nd cr all who underwent transplant from an ud is significantly influenced by the presence of tbi in the conditioning regimen, limited severity of acute and chronic gvhd and bfm classification at time of 1st relapse. notably, mrd level before transplant, namely with a cut-off of 0.001%, influences efs. disclosure of conflict of interest: none. the median mononuclear cell dose was 5.5 × 10 8 /kg. the median time to reach absolute neutrophil count 40.5 × 10 9 /l was 13 days, and the median time to platelet count 420 × 10 9 was 16 days. grade 2 and grade 3 mucositis was seen in 61% of our patients. transplant-related mortality at 100 days not occurred. only three patients relapsed 15, 18 and 30 months after transplant (mean 21.5 m.). with a median follow-up of 39 months (4-48 months) after transplant the event free survival were 84%. only one patient had death, two years after transplantation. no significant different between cbv group vs ceam group in engraftment day. high-dose therapy with stem cell rescue can lead to durable remissions in children and adolescents with advanced hd. future investigations should focus on strategies designed to decrease relapse after auto-transplantation, particularly in patients at high risk for relapse. our analysis suggests that these regimens (ceam, cbv) are feasible in pediatric patients with acceptable engraftment and toxicity. pbsc collection may be difficult in small children owing to the large volume apheresis compared to the child's weight. various problems, such as metabolic or haemodynamic disorders may be were seen. peripheral stem cell harvest can be performed in lowweight children under safe and effective conditions even when systematic priming by blood is avoided. processing with increase of blood volume may to increase in the yield by recruiting progenitor cells. disclosure of conflict of interest: none. outcomes of children with hemophagocytic lymphohistiocytosis given allogeneic hematopoietic stem cell transplantation in italy allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for patients with familial hemophagocytic lymphohistiocytiosis (hlh) or relapsed/ refractory hlh. we analyzed outcomes of a cohort of 109 patients (65 m, 44 f) with hlh given hsct between 2000 and 2014. median age at hsct was 2 years (range 0.4-20). genetic testing was performed for 94/109 patients (86%). mutation of prf1 was found in 31 patients (32%), of unc13d in 32 (33%), of stxbp2 in 2 (2%), of rab27a in 6 (6%), of sh2d1a in 5 (5%), of birc4 in 2 (2%) and of lyst in 1 patient (1%). no known gene abnormality was found in 15 patients who had recurrent/ refractory hlh. central nervous system (cns) involvement at diagnosis was recorded for 79 patients (72%) and was present in 30 of them (38%). the primary endpoint was event-free survival (efs), defined as the probability of being alive and in continuous complete remission (cr) at last follow-up. in order to determine efs, death from any cause, relapse or graft failure were considered events. ninety-five patients received one transplant, while 14 received more than one hsct, because of rejection in 8 patients or disease relapse in 6 (preceded by rejection in 1 case): 2 hsct were performed in 12 cases, while 3 and 4 hsct were performed in 1 case each. donor for first transplant was an hla-matched sibling for 25 patients (23%), an unrelated donor for 73 patients (67%) and a partially matched family donor for 11 patients (10%). conditioning regimen was busulfan-based for 61 patients (56%), treosulfan-based for 21 patients (20%) and fludarabine-based for 26 patients (24%). the 5-year probability of overall and efs were 71% and 60% respectively (fig. 1) . twenty-six (24%) patients died due to transplant-related causes, while 14 (13%) and 10 (9%) patients experienced graft rejection and/or relapse, respectively (see also fig. 1 ). twelve out of 14 children (86%) given a 2nd hsct after graft failure/relapse are alive and disease-free. active disease at hsct was not statistically associated with adverse outcomes, while patients had a trend for a worse outcome if the interval between diagnosis and hsct was 46 months. patients transplanted from partiallymatched family donors (pmfd) had a significantly worse efs (9%) than recipients of a matched family donor transplant (73%) or a matched unrelated donor allograft (63%, po 0.001). the main reason for the dismal efs of pmfd recipients was graft rejection, which, however, was largely rescued by a 2nd hsct. patients given peripheral blood stem cell transplantation had a lower efs probability (39%) as compared to bone marrow (60%) or cord blood recipients (76%, p = 0.0185). children given hsct o o/u46 months from diagnosis had a better efs as compared to those transplanted 46 months from diagnosis (69% vs 50%, p = 0.069). in multivariate analysis, only the use of a pmfd predicted a worse efs probability (relative risk:12.26, p = 0.0008). these data suggest that in patients with hlh allogeneic hsct is able to cure 2/3 of patients. haploidentical hsct in patients with hlh is currently associated with unsatisfactory rate of engraftment, new approaches being needed to ameliorate this outcome. active disease does not preclude the chance of benefiting from transplantation, which should be ideally performed within 6 months from diagnosis. [p485] defibrotide shows efficacy in the prevention of sinusoidal obstruction syndrome (sos) after allogeneic hematopoietic stem cell transplantation: a retrospective study on 237 patients. disclosure of conflict of interest: none. standard gvhd prophylaxis regimens impair the graft-versustumor (gvt) effect, delay immune reconstitution and are associated with high rate of infections. high-dose posttransplantation cyclophosphamide (ptcy) targets alloreactive donor t cells proliferating early after bmt, promotes regulatory t cell and permits rapid immune reconstitution. in this pilot trial we evaluate the safety and effects of ptcy in unmanipulated haploidentical and matched unrelated transplantation (mud) in pediatric patients with all. fifteen pediatric patients with high risk all underwent unmanipulated allogeneic bone marrow (bm) (n = 11) or peripheral blood stem cell (pbsc) (n = 4) transplantation followed by ptcy between april 2014 and march 2016 with a median follow-up of 24 months (7-27). eight patients were transplanted from haploidentical donors and 7 from mud. the median age was 9.5 years (range 1.9-17) and were in complete remission (cr) at the moment of bmt. in 2 patients this was a second bmt. all pts. received myeloablative conditioning regimen (treosulfan-based n = 14, tbi based n = 1) and ptcy on day +3, +4, posttransplant prophylaxis consisted of tacrolimus from day +5 (n = 5), tacrolimus/mmf (n = 8), atg (rabbit, thymoglobuline) at 5 mg/kg without other posttransplant prophylaxis(n = 2, both from mud). primary engraftment was achieved in 100% of pts., the median time to neutrophil recovery was 20 days and to platelet recovery was 22 (7-69) days. all pts. had full donor chimerism on day +30. causes of death included viral infections (n = 1); gvhd and viral infection (n = 1). cumulative incidence (ci) of acute gvhd grade ⩾ ii was 33% (95% ci: 16-68), grade iii-iv-13.3% (95% ci: 3.7-48) and chronic gvhd-21.2% (95% ci: 7.7-58.4). two-year event-free survival (efs) and overall survival (os) were 86.7% (95% ci: 69.5-100) and were equal. median time of follow-up for survivors is 2 years (range 0.7-2.3). we demonstrate that unmanipulated hsct and posttransplantation cyclophosphamide allows for high rate of engraftment with acceptable transplant-related mortality in pediatric patients with all. all major outcomes were equivalent between transplantation from unrelated and haploidentical donor. gvhd prophylaxis including ptcy was effective. event-free survival was high despite chemotherapybased conditioning in most patients. disclosure of conflict of interest: none. serotherapy with atg is frequently used in allogeneic hsct to prevent gvhd and rejection. however, the choice of the two most frequently used rabbit atg brands depends on country, disease protocol, national recommendations and/or physician's preference. atg-genzyme (atg-g, thymoglobulin) is prepared by immunizing rabbits with human thymocytes, whereas rabbits are immunized with a jurkat cell line for production of atg-fresenius (atg-f, recently named as antihuman t-lymphocyte immunoglobulin atlg, grafalon, noveii biotech). the recommended dose of both brands differs a factor 4-5. we have previously reported the pharmacokinetics/ pharmacodynamics (pkpd) of atg-g in a large cohort of pediatric hsct recipients and concluded that the clearance of the active component of atg, which is the portion of atg binding to lymphocytes, had a major impact on immune recovery post-hsct, while total atg did not. 1 both atg brands have frequently been compared according to disease outcome, without detailed analysis of composition and clearance of the active components. in the present study, we compared clearance of the active component and immune recovery after atg-g and atg-f, respectively. the serum concentrations of total and active atg were measured longitudinally after hsct in 56 children (40 atg-g, 16 atg-f), transplanted with bm or pbsc of unrelated donors for all or aml between january 2010 and june 2016 in leiden (n = 36) or copenhagen (n = 20). atg-g treated patients received a total dose of 6-10 mg/kg and atg-f was given at a total dose of 30-60 mg/kg in both cohorts administration was divided over 3-4 days. serum samples (pre-conditioning, day of hsct, +1; +2; +3; +4 and +6 weeks and +2 and +3 months after hsct) were analyzed by elisa for total atg and by quantitative flow cytometry on hut78 cells for active (lymphocyte binding) atg. lymphocyte (sub-)populations were analyzed at +1, +2 and +3 months post-hsct by flow cytometry. as reference group for immune recovery, 22 children transplanted for all or aml with an hla-identical donor and not receiving serotherapy were included. the median serum concentration of total atg at the day of hsct was 6 times higher for atg-f (atg-g 67 μg/ml, atg-f 403 μg/ ml; figure a) as the result of the higher dose of atg-f given. the active atg concentration was twice as high for atg-f (atg-g 11.1 au/ml, atg-f 23.4 au/ml figure b ). three weeks later at the expected time of engraftment, the total atg concentration was decreased with the same factor for both atg brands (atg-g from 67 to 25 μg/ml, factor 2.7; atg-f from 403 to 139 μg/ml, factor 2.9). however, the active atg concentration showed a much faster decline for atg-f (atg-g from 11.1 to 1.03 iu/ml, factor 10.8; atg-f from 23.4 to 0.23 iu/ml, factor 100). correspondingly, the number of cd3 t-cells at 1 month post-hsct was higher after atg-f than after atg-g (atg-g, atg-f and no-serotherapy 41, 169 and 386 cells/μl, respectively. figure c) . this is the first study to compare the pkpd of total and active atg-genzyme and atg-fresenius. active atg-f showed a much faster clearance than atg-g, which was associated with a significantly faster cd3 t-cell recovery at 1 month post hsct. thus, atg-f is not only quantitatively but also qualitatively very different from atg-g, which will clearly impact hsct outcomes. reduced toxicity myeloablative conditioning regimen in pediatric hematologic malignancies not associated with improved outcomes s chaudhury 1,2 , i helenowski 2 , r duerst 1,2 , wt tse 1,2 , m kletzel 1,2 , j schneiderman 1,2 and d jacobsohn 3 1 ann and robert h. lurie children's hospital of chicago; 2 northwestern university feinberg school of medicine, chicago and 3 children's national health system, washington dc allogeneic (allo) hematopoietic cell transplantation (hct) is the only curative potential therapy in refractory and relapsed pediatric leukemias. poor outcomes in allo hct are associated with treatment-related mortality (trm), mostly due to regimen-related toxicities (rrt) and graft-versus-host disease (gvhd) after myeloablative conditionings (mac), but high relapse rate with reduced-intensity or nonmyeloablative regimens. 1 to improve trm, without compromising conditioning intensity, we prospectively explored the feasibility and efficacy of a mac but reduced-toxicity conditioning (rtc) regimen, consisting of fludarabine 30 mg/m 2 /d (given first) × 5d, daily busulfan dosed to target an auc of 4000 microm*min/d × 4, ratg 1.5 mg/kg/d × 3 and 400cgy of total body irradiation in 30 patients (table 1) with hematologic malignancies. gvhd prophylaxis was cyclosporine and mmf. all patients tolerated the rtc well, with no graft failures. rrt included moderate mucositis (67%), infections (bacterial 33%, viral reactivation 63%, fungal 20%) and 3 cases of venoocclusive disease (vod). cumulative incidence d100 ⩾ gr 3 acute gvhd was 32% (95% confidence interval [ci], 16-50), extensive chronic gvhd was 3.5% (95% ci, 0.2-16). mortality at 100 days was 10.7% (95% ci 3-25), 2 due to infections with agvhd and 1 vod. with a median follow-up of 1.5 y (range, 0.6-5), the cumulative incidences of relapse at 1 years was 29% (95% ci, 14-47). mortality due to severe agvhd was 90%. overall survival (os) and progression-free survivals (pfs) for 1year was 63% (95% ci, 42-78), and 56 % (95% ci, 36-72) respectively. on univariate analysis there was no association of outcomes with donor type, graft source, disease or busulfan exposure except significantly higher cgvhd in unrelated donors, agvhd severity with peripheral blood. in summary, the use of the myeloablative rtc resulted in comparable trm, with high relapse rate was high, including in those developing chronic gvhd. this suggested a less robust graft-versusleukemia effect resulting in poor pfs and os. nonetheless, this regimen may be used as a lower-trm platform to combine with other strategies, intensive disease monitoring pre and post hct, addition of post hct maintenance therapy in combination with marrow as the stem cell source to decrease relapse or gvhd. specific immune response to vaccinations decline after hematopoetic stem cell transplantion (hsct). re-vaccination of all hsct recipients is recommended in all guidelines but bcg vaccination is not recommended due to safety concerns after hsct. mycobacterium tuberculosis can cause severe disease in children including meningitis and milliary tuberculosis (tb). the bacille-carmette-guerin (bcg) is a liveattenuated vaccine with documented efficacy against milliary disease and meningitis. routine vaccination of all infants residing in countries with high tb incidence is recommended by world health organization. however, there is no data in literature regarding its safety in post hsct setting. here, we report 34 children who underwent matched related allogeneic hsct at ankara university pediatric bone marrow transplant (bmt) unit and received bcg 24-months post-transplant. all patients were free of graft versus host disease (gvhd) and immunosuppressive therapy (ist) and had negative ppd skin test prior to vaccination. none of the recipients developed local or disseminated tuberculosis as a complication of bcg with a median follow up of 10 years. we conclude that the bcg vaccine is safe in the post hsct period when administered at least 24 months out of transplant to a selected group of patients who are free of gvhd and ist. disclosure of conflict of interest: none. single centre experience of harvesting bone marrow from donors o3 years of age r raj, r uppuluri 1 , d subburaj 1 , d jayaraman 1 , k mullanfiroze 1 , v swaminathan and l vaidhyanathan 1 1 department of paediatric blood and marrow transplantation, apollo speciality hospital harvesting bone marrow for allogeneic marrow transplantation from donors o15 kg presents special challenges. we present data on 67 sibling donors from our institution between 2006 and 2016. the mean age was 23 months with a range between 8 months to 48 months. children less than one year accounted for 25% of our donors with the youngest being 8 months of age and the smallest donor weighed 5.5 kg. all aspirations were performed from iliac crests and all donors were given general anaesthesia by a paediatric anaesthetist. irradiated blood was transfused in 97 % of the donors during the procedure. the volume of marrow obtained ranged from 5 to a maximum of 20 ml/kg donor weight. the product contained an average cd34 count of 5.5 × 10 6 /kg recipient weight with a range from 1.6 to 12 × 10 6 /kg. only on one occasion was a second harvest needed, where the donor weighed 12 kg and recipient 42 kg with major blood group incompatibility requiring red cell reduction. the yield of cd34 cells per ml of bone marrow was on average 22% higher than children above 3 years of age. all recipients showed brisk engraftment in 2 weeks. none of these donors experienced major difficulties following the aspiration procedure. thus, very young children may safely donate marrow for allogeneic transplantation and the yield of stem cells obtained is substantial. this data is particularly relevant in transplantation for haemoglobinopathies like thalassaemia major and sickle cell anaemia, where families are being counselled about a target of 15 kg for the donor in order to plan transplantation. disclosure of conflict of interest: none. sinusoidal obstruction syndrome-veno-occlusive disease in pediatric patients given either autologous or allogeneic hematopoietic stem cell transplantation (hsct). a retrospective study of the aieop-sct (italian haematology-oncology association-stem cell transplantation) group m faraci 1 , r luksch, e calore 2 , f saglio 3 , a prete 4 , mc menconi 5 , v trevisan 6 , g de simone 7 , v tintori 8 , s cesaro 9 , s santarone 10 , mg orofino 11 , e lanino 1 , m zecca 12 and a bertaina 13 sinusoidal obstruction syndrome (sos), known as venoocclusive disease (vod), is a potentially life threatening complication that can develop after hsct. although sos progressively resolves within few weeks in most patients, the severe forms result associated with multi-organ dysfunction and high mortality rate (480%). aim of this survey is to evaluate incidence and management of sos in a large cohort of children receiving either allogeneic or autologous hsct. we retrospectively reviewed pediatric hscts performed in 12 (46%) out of 26 aieop affiliated centers, between january 2000 and april 2016. new ebmt criteria have been used for the diagnosis of sos (serum total bilirubin ⩾ 2 mg/dl and 2 of the following criteria: painful hepatomegaly, weight gain 45%, and ascites) and for the classification of severity grading. 1,2 among a total number of 6208 hsct procedures (2980 autologous and 3228 allogeneic), we identified 94 (1.5%) patients with sos. this complication occurred in 37 and 55 cases after autologous and allogeneic hsct, respectively. fiftytwo pts (55%) received iv busulphan (bu) at myeloablative dose, 28 (30%) oral bu, while 14 (15%) were treated with different conditioning regimen. the median time of sos occurrence was 16 days after hsct. details about prophylaxis and therapy are reported in figure 1 . out of the 92 children, 54 (59%) fulfilled all sos-ebmt criteria. bilirubin ⩾ 2 mg/dl, gain of weight 45%, ascites, and painful hepatomegaly did not occurred in 16, 3, 4 and 2 patients, respectively. thrombocytopenia was present in 85 pts (92%), thickening of gallbladder in 63 (68%) and abnormalities of coagulation parameters in 78 (84%). according to sos ebmt severity grading, levels of transaminases were mild in 18 pts (19%), moderate in 21 (22%), severe in 13 (14.1%), and very severe in 42 (45.6%). notably, creatinine was mild in 67 pts (71%), while 5 (5.4%), 10 (10.8%), and 12 (13%) children showed moderate, severe and very severe grade of renal failure. thirty-three pts (36%) had respiratory failure, and 28 (85%) of them experienced right pleural effusion. six out of the 25 patients who developed acute kidney injury, required dialysis. severe encephalopathy occurred in 8 pts (8.6%) and 22 (24%) out of the 92 pts evaluated, were admitted in intensive care unit. as therapy of sos, 69 pts received defibrotideâ (df); the dosage was 25 mg/ kg/day in 63% of them. the median duration of df treatment was 15.5 days (range 4-104). thirty-three (35%) pts received methylprednisolone (median dose of 2 mg/kg). fifteen pts (16.3%) died due to mof (2 in moderate, 6 in severe, and 7 in very severe group) at a median time of 6 days from sos diagnosis (range 3-75 gg). our multicenter survey showed that, at least in our experience, there is a significant variability in the management approaches to sos/vod in children, while, diagnostic evaluations are more homogeneous. interestingly, in our cohort, the increase of bilirubin may be an absent criteria, while thrombocytopenia and abnormalities of coagulation parameters are more frequent. as expected, mof occurred mostly in patients experiencing severe sos. df represents first strategy to treat sos in the majority of patients, even if steroids and ursodeoxycholic acid are still used. the hyper-ige syndromes are characterized by marked elevations in plasma ige levels and eosinophilia with impairment in t cells which clinically results in combined immune deficiency. dock8 deficiency, the autosomal recessive form, brings about allergic/atopic manifestations and unusual susceptibility to infections with herpesvirus family members (herpes simplex virus, human papilloma virus) and molluscum contagiosum. symptoms in patients with dock8 deficiency typically emerge during childhood, and the majority results in death because of infections and malignancy by the third decade. hematopoietic stem cell transplantation (hsct) is now considered a standard of care for dock8 deficiency when an appropriate donor is available. in this study, we present the 5 unrelated hsct results of 4 children with dock8 mutation. the demographic and clinical data of the 4 patients with 5 transplantations studied are shown in table 1 . hsct was administered between august 2013 and august 2015 at bahçeşehir university medical park antalya hospital and the clinical data of the hscts are presented in table 2 . all patients were transplanted from unrelated donors with bone marrow, except one with cord blood. the cord blood transplantation´s regimen was non-myeloablative which resulted with rejection. despite existence of serious morbid problems before transplantation, all the patients engrafted successfully. majority of the complications mentioned in the table 2 were improved and they are in the follow-up in an outpatient basis. discussion dock8 deficiency has high mortality, and hsct should be considered as early as possible before development of significant organ damage. despite myeloablative conditioning and high morbidity before the transplantation, survival was very good in our patients. myeloablative and nonmyeloablative transplants have been performed from related and unrelated donors and have reported successful results even without the preparative regimen. in our center, all transplants performed from unrelated donors by myeloablative regimen have been successful but have resulted in transplant rejection with cord blood transplantation after nonmyeloablative regimen. in all of our patients, stable full chimerism has been detected, however mixed chimerism have also been shown to be useful in several reports. whether hsct also cures the autoimmune complications and reduces the risk of cancers is as yet undetermined. however, a myeloablative conditioning regimen followed by allogeneic hematopoietic stem cell transplantation from unrelated donors in dock8 deficiency results in improvement of the clinical phenotype with a low incidence of regimen-related toxicity. disclosure of conflict of interest: none. successful bone marrow transplantation after myeloablative conditioning in a child with ipex syndrome b kuşkonmaz 1 , d ayvaz 2 , mh abur 3 , fv okur 1 , g karagüzel 4 , f orhan 5 , i̇ tezcan 2 and du çetinkaya 1 immune dysregulation, polyendocrinopathy, enteropathy, x-linked (ipex) syndrome is a rare disorder. although most patients present in infancy with a clinical triad of intractable diarrhea, insulin-dependent diabetes, and eczematous dermatitis, some patients present with severe food allergies and other autoimmune manifestations. the disease is caused by mutations in the forkhead box p3(foxp3) gene, a transcription factor that is essential for the development and function of regulatory t (treg) cells. this cells plays an essential role in controlling immune responses and preventing autoimmunity. patients usually die in the first years of life without treatment. the only effective cure is hematopoietic stem cell transplantation (hsct). here we report a patient with ipex syndrome who underwent hsct after myeloablative conditioning. 9 months of age boy with the history of diarrhea, insulin-dependent diabetes, eczematous dermatitis, pneumonia, coombs positive hemolytic anemia, referred to our hospital for investigation of immunodeficiency. on admission physical examination showed eczematous skin rash, submandibular lymphadenopathy, hepatosplenomegaly. before hsct the patients treated with immunosuppressive agents including methylprednisolone, mycophenolate mofetil and monthly intravenous immunoglobulin. complete blood count revealed anemia (hb: 7.7 g/dl), and eosinophilia (1900/mm 3 ). serum immunoglobulins were: ig g: 1550 mg/dl (463-1006), igm: 172 mg/dl (46-159), iga: 60.9 mg/dl (17-69), ige :1538 iu/ml. lymphocyte subset analysis showed cd3 64%, cd4 22%, cd8 40%, cd16+56 13%, cd19 19%. foxp3 gene analysis showed c.748_750delaag mutation. at the age of 1 year, patient underwent hsct from his hla matched sibling. myeloablative conditioning regimen including busulfan (20.4 mg/kg) and fludarabine (160 mg/m 2 ) was given to the patient. cyclosporine a and methotrexate (day +1, day +3, day +6) were used as graft versus host disease prophylaxis. bone marrow was used as the stem cell source and the number of cd34+ cells was 4.5 × 10 6 /kg. neutrophil and platelet engraftment were achieved on day +13 and +35 [p493] s378 respectively. acute and chronic gvhd were not observed, but patient developed veno-occlusive disease treated with defibrotide, sepsis treated with broad spectrum antibiotics. chimerism analysis showed %98 donor profile at the third month of hsct. after hsct, autoimmune hemolytic anemia, eczematous dermatitis, food allergies, diarrhea and type 1 diabetes resolved completely within two months after hsct. now the patient is in good clinical condition without any symptoms 5 months after hsct. early hsct provides better outcome in patients with ipex, before the organ damage due to autoimmunity and/or adverse effects of immunosuppressive therapy. myeloablative conditioning is associated with substantial transplantation-related mortality whereas nonmyeloablative conditioning carries an increased risk of rejection because of dysregulated effector t-cell function. in this patients, myeloablative conditioning was preferred because of the risk of rejection. although the required levels of donor chimerism and conditioning intensity are unknown, engraftment of donor treg cells seems to be sufficient to control the disease. the patient is well without any symptoms of ipex after hsct with full donor chimerism. disclosure of conflict of interest: none. interferon gamma receptor 1 deficiency (ifnr1) is a rare autosomal recessive immune deficiency disorder associated with very poor outcome secondary to severe and disseminated mycobacterial infections. hematopoietic stem cell transplantation (hsct) has been proposed as a curative option. however, hsct for these patients is particularly difficult owing to a high rate of graft rejection. the use of a non t-cell depleted transplant from an hla-identical sibling and fully myeloablative conditioning regimen has been shown to have improved outcomes. we report the first successful hsct with a t-depleted haplo-identical donor, performed in a girl with severe ifnr1 deficiency. we reviewed the medical chart of a 2-year-old hispanic girl with ifnr1 deficiency who was diagnosed at birth, since her brother had previously been diagnosed with the same complete ifnr1 deficiency. they were found to have a novel mutation variant detected at c.201-1g4t. as expected with this disorder, she developed disseminated infection with mycobacterium abscessus infection at 2 months of age and was subsequently found to have mycobacterium abscessus osteomyelitis. she was treated with multiple antibiotics including: amikacin, linezolid, meropenem and clarithromycin while tigecycline was added a few weeks prior to admission for hsct. she was continued on this therapy until day + 30 following which antimicrobials were gradually weaned off. she was enrolled on the bp-004 trial, a multicenter, prospective phase i-ii trial (enrolling both malignant and non-malignant diseases) evaluating αβtcr +/cd19+ depleted haplo-transplantation followed by administration of bpx-501 t cells containing the ic9 suicide gene, (clinicaltrials.gov nct02065869). her conditioning regimen included busulfan (4 mg/kg/day for 4 days) and cyclophosphamide (50 mg/kg/day for 4 days). fludaragbne, tli (900 cgy) . gvh prophylaxis with atg/rituximab. the patient received a graft with: tnc-9.98 × 10 8 cells/kg, cd34+ cells-16 × 10 6 cells/kg, and αβtcr+ t cell content of 4.78 × 10 4 cells/kg. as per protocol, since the αβ tcr+ t cells in the product was below threshold of 1 × 10 5 cells/kg, she did not receive any post-transplant immune suppression. bone marrow recovery occurred at day +10 with anc 4500/mm 3 and platelet recovery at day +18. full engraftment with 100% donor chimerism based on cytogenetic analysis was observed at day +28 after transplantation and has remained stable. she is currently 14 months post-transplant, and has done well without major complications and or signs of mycobacterial infection. there is limited data in patients receiving hsct for ifnr1 deficiency with very poor outcomes either relating to graft failure, transplant complications and progressive mycobacterial infection. to our knowledge, this is the first patient with ifnr1 deficiency transplanted successfully with a haploidentical donor and alive without any active mycobacterial infection. this report suggests that using a highly immunopotent graft depleted of only αβtcr+ t cells while retaining other immune effectors might offer a potential strategy to engraft these high risk patients using haplo-identical donors thereby allowing access to virtually all patients in need. disclosure of conflict of interest: none. tandem autologous stem cell transplantations for high risk pediatric embryonal central nervous system tumors: a single center experience k rosenfeld 1 , r dvir 1 , s constantini, j roth 2 , s edelman 1 , a tal 1 , d levin 1 , m manisterski 1 , s achituv 1 and r elhasid 1,3 1 department of pediatric hematology-oncology, tel aviv medical center; 2 department of pediatric neurosurgery, tel aviv medical center and 3 sackler faculty of medicine, tel aviv university pediatric embryonal central nervous system tumors are highly malignant tumors, which tend to disseminate through the cerebrospinal fluid to the brain and spinal cord and include: medulloblastoma, pinealoblastoma and primitive neuroectodermal tumors (pnets). the recommended treatment for these tumors is a complete surgical excision, craniospinal radiation and chemotherapy. the use of high dose chemotherapy with tandem autologous hematopoietic stem cell transplantation (hsct) has been advocated for high risk patients, and infants who could not be irradiated. between july 2010 and november 2016, 16 pediatric patients (11 males, 5 females) suffering from high risk medulloblastoma, pnet or pinealoblastoma underwent tandem autologous hsct. they were treated according to two protocols: group a consisted of ten patients with median age of 8.2 years (range 3.9-15.5 years) received the st jude sjmb03 protocol, while group b consisted of six patients with median age of 2.1 years (range 1.4-3.5 years) who received the children's oncology group -acns0334 protocol. all patients engrafted with median time for neutrophil engraftment of 11 days (range 7-14 days) and for platelets engraftment (420 000) of 13 days (range 13-24 days). median follow-up was 3.5 years (range 1 week-6 years). neurological toxicity: two group a patients had convulsions episodes, one occurred during infusion of cryopreserved stem cells, and the other was a result of progressive disease during the last course of hsct. gastrointestinal toxicity: seven patients required total parenteral nutrition due to mucositis. diarrhea occurred in seven patients, two of them were diagnosed with rota virus and two with clostridium difficile. infectious complications: all patients suffered from at least one episode of neutropenic fever which was treated with broad spectrum antibiotics. there were 7 documented bacteremia in 6 patients. (1 klebsiella pneumonia, 1 proteus mirabilis, 3 staphylococcus aureus, 1 streptococcus viridans and 1 staphylococcus epidermidis). metabolic complications: four patients in group a developed reversible syndrome of inappropriate anti-diuretic hormone secretion (siadh) during chemotherapy, and all group a patients developed hypomagnezemia. four patients died, one due to progressive disease, one due to early relapse 3 months post treatment, one due to late relapse 5 years post treatment and one due to sepsis 4 months post treatment. another patient relapsed 1.5 years s379 post treatment, underwent surgery and radiotherapy and is now 3 years post therapy. late effects: four group a patients developed endocrinological sequelae at a median of 20 months (range 17-21 months) and require hormone replacement therapy. tandem autologous hsct is a feasible treatment for pediatric high risk embryonal tumors, with good engraftment and acceptable toxicities using sjmb03 and acns0334 protocols, with overall survival of 75%. long follow-up is needed in order to diagnose and treat late effects. disclosure of conflict of interest: none. the diagnostic role of liver stiffness measurement in predicting hepatic veno-occlusive disease (vod) in pediatric hematopoietic stem cell transplantation (hsct) k kleinschmidt 1 , f ravaioli 2 , r rondelli 1 , g marasco 2 , r masetti 1 , a prete 1 , a colecchia 2 , d festi 2 and a pession 1 1 pediatric oncology and hematology unit, department of pediatrics, university of bologna, sant 'orsola-malpighi hospital and 2 department of medical and surgical sciences, university of bologna vod is a potentially life-threatening complication associated with hsct in which immediate therapeutic action is crucial for patients' outcome. liver stiffness measurement (lsm) using fibroscan represents a non-invasive method to detect the grade of liver fibrosis and portal hypertension as in case of vod. to evaluate the predictive potential of lsm in pediatric patients (pts) at risk for developing vod, a prospective, ongoing, single-center study has been performed at the university hospital of bologna. lsm was performed by using the fibroscan device, which consists of a 3.5 mhz ultrasound transducer probe that transmits low-frequency vibrations (50 hz) to the liver tissue. the propagation velocity is proportional to the stiffness (elasticity) of tissue. lsm will obtain pathological high values (47.5 kpa) when the tissue is altered like in liver fibrosis, or post-sinusoidal portal hypertension. from november 2014 -september 2016, 25 pediatric pts (18 male, 7 female), aged 3-20 years (mean 11.7), affected by hemato-oncologic disease, eligible to allogeneic (22) or autologous (3) sct conditioned with busulfan-based chemotherapy, were enrolled. pts were scheduled for 4 study examinations with lsm: at t0 (baseline) before chemotherapy, t1 (day 7-10 after sct), t2 (day 17-20) and t3 (day 27-30). the diagnosis of vod was defined according to modified seattle/baltimore criteria. twenty-five pts were enrolled in the protocol, 22 of which were evaluable for the study (pts characteristics table 1 ). 4 out of 22 pts (18%) developed vod. the cumulative incidence (se) of vod in our setting was 19 % (8.6). baseline lsm values on t0 of all pts were normal (7.5 kpa at t2 (p = 0.002) and t3 (p = 0.004). from our observations, an anticipating pattern of pathological lsm in presence of clinical and laboratory parameters within normal ranges in patients who develop vod can be derived. preliminary data indicate a high predictive potential of lsm in the diagnosis of vod, however the number of cases is not sufficiently representative to draw definitive conclusions. to optimize the predictive potential of the method, more frequent (daily) measurement in the critical time frame are currently investigated. [p497] all= acute lymphoblastic leukemia, aml= acute myeloid leukemia, bu= busulfan, treo=treosulfan, fluda= fludarabine. disclosure of conflict of interest: none. [p497] the exact role of extra-corporeal photopheresis in children with gvhd: an unanswered question ss anak, h bilgen 1,2,3,4,5,6,7 , y yaman, et saribeyoglu, k ozdilli, v hazar, m elli, am kokrek, h hizli and k payalan ecp continues to be a controversial treatment, probably due to the mechanism of action not being identified, the varying photopheresis procedures and treatment schedules, and the difficulty of conducting trials on relatively rare diseases with involvement of clinically heterogeneous organs. ecp was performed in our pediatric transplant center to 8 patients mean age of 12 years ( 4-18) diagnosed to have all ( 3 pts), thalassemia ( 2 pts), aplastic anemia (1), blacfan diamond (1), refractory hodgkin disease (1) following our internal protocol for 152 ecp sessions. five of the patients had mud, 3 had hla id sibling transplants. chronic gvhd was diagnosed in 2 of the patients 6 had acute gvhd. skin was involved in all the patients, liver in 6 of the patients, lung in 3, gut in 6 and mucous membranes in 7 patients. the ecp treatment consisted essentially of three steps: (1) collection of mncs from the patient, (2) processing of mnc buffy coat, and (3) return of mncs to the patient. collection was performed using a cell separator (haemonetics mcs plus), processing two blood volumes. our protocol provides for a maximum final mnc volume to be collected at 150 ml, with a hematocrit (hct) value below 5%. the maximum procedure time was set at 180 min. the mncs collected were adjusted to a constant volume of 300 ml by the addition of saline and 3 ml of 8-mop in aqueous solution, to always obtain a final concentration of the drug of 200 ng/ml. the diluted buffy coat was transferred into a special uv-a-permeable bag (pit-kit medtech solutions), and uv-a radiation at 2 j/cm 2 was performed (uva-pit irradiator). the photoactivated mncs were returned to the patient within 30 minutes using a blood transfusion set. during ecp procedure, patients' vital signs were monitored. anticoagulation consisted in acidcitrate-dextrose formula a set at a variable ratio (1:14-1:20) according to the patient's characteristics (clinical conditions, body weight, coagulation values) and platelet (plt) count. prophylaxis of hypocalcemia consisted of the administration of calcium gluconate (5 ml diluted in 5-10 ml saline) every 30 to 45 minutes. all procedure related side effects were recorded. during the reinfusion and postreinfusion phases, the patients were monitored for fever, chills, headache, rash, erythema, urticaria, itching and edema. no serious complication was detected. all the patients had also steroids, 4 had concurrent mesenchimal stem cells. ecp was applied on 2 consecutive days every 2-4 weeks which is continued for approximately 6 months followed by a maintenance schedule tapered to an every 2-to 4-weeks. the mean session cycle was 19 ( 6-51) between february 2015 to november 2016 . the most commonly involved organ was the skin which demonstrated a response rate of 75%, followed by liver (66%), lung (25%), gut (18%) and mucous membranes (68%) the concurrent immunosuppression could be reduced during ecp therapy, and no increase in opportunistic infections was detected. 1/8 patient died after a relapse, 7/8 are alive with chronic mild gvhd. however, despite our good response rates, our understanding of ecp remains limited. patients who suffer from acute and chronic gvhd have limited treatment options. ecp remains an important therapeutic option. future basic, translational, and clinical research studies will provide a better understanding of its mechanism of action and optimize its therapeutic potential. disclosure of conflict of interest: none. tolerability and responses to ex vivo il2 activated nk cells from haploidentical parental donors in paediatric patients with refractory leukaemia/lymphoma pl tan 1 prognosis for patients with refractory leukaemia/lymphoma ineligible for transplants and those who relapse posttransplant is poor. in adult settings, adoptive transfers of ex vivo il2 activated natural killer ('ank') cells from nk alloreactive donors, especially for nk sensitive cancers, has been successful in bridging patients to curative transplants.(1) this approach has not been reported in paediatric patients. we report our experience in 8 consecutive patients, of median age 9 (range, 1-15) years, with refractory leukaemia/ lymphoma (aml, 2; all, 2; mixed phenotype acute leukaemia, 2; lymphoma, 2) who received 9 treatments with 'ank' from haploidentical parental donors on institutional protocol, between aug 2012 and 2016. parents/legal guardians/patients provided informed consents as per institutional guidelines for donors and patients procedures. donor lymphocytes harvested at steady state were cd3 depleted followed by overnight culture in il2 before being infused into patients lymphodepleted with fludarabine and cyclophosphamide. additional rituximab were given to 3 patients and another received tbi 2 gy. subcutaneous il2 injections at doses 1-3 mu/m 2 /dose started on d-1 and were planned for 6 doses, as tolerated. nk alloreactive donors (kir-ligand mismatch) and kir b/x genotype were available to all except 2 patients. two patients were treated for post-transplant relapse; 1 of whom also received 'ank' pre-transplant; other 6 patients had failed best conventional therapy including cd19/cd3 bispecific t cell engager (blinatumomab) in 1. lymphodepletion was well tolerated. a median tnc and cd56+ dose of 9.8 (range, 2.9 to 38) × 10 7 /kg and 1.8 (range, 1.2-18) × 107/kg, respectively were administered. cytokine release syndrome (crs) was observed in 8 of 9 treatments (6 grade 1, 1 grade 3, 1 grade 4). the patient with dock8 deficiency, disseminated ebv+ cerebral lymphoma had grade 4 crs and robust tumour lysis syndrome but succumbed to neurotoxicity. of the 9 treatments, there were 7 responses, including the 2 given posttransplant. excluding the 2 treatments given post-transplant and 2 non-responders, median peak donor chimerism was 93% (range, 7-100%) occurring at a median of 12 (range, 7-22) days. five patients (4 responders, 1 non-responder) proceeded to transplants at a median of 43 (range, 35-96) days after 'ank.' responders had longer survival time compared to nonresponders (median 314 vs 88 days). two responders (25%) achieved sustained minimal residual disease (mrd) remission after transplants and are alive 138 and 380 days from 'ank.' five eventually died of their primary leukaemia/lymphoma; 1 from crs. our preliminary experience in a small cohort of 8 paediatric patients with refractory leukaemia/ lymphoma showed that adoptive transfers of ex vivo il2 activated nk cells from haploidentical parental donors were tolerable; with responses seen in 75% of patients; and 25% achieving prolonged mrd remissions after transplants. patients with cerebral diseases might be at increased risks of neurotoxicity with this approach; and care must be taken in patient selection and the design of the lymphodepletion therapy. alternative donor choices are limited in multi-racial, multiethnic societies with small families such as singapore. unrelated cord blood transplant provides a feasible alternative to patients lacking adult stem cell donors in children with primary immunodeficiency diseases. method: we describe our experience using unrelated cord blood transplant (ucbt) for 9 children with pid from august 2005 to november 2014. during this period we performed hsct for 12 children with pid: 9 with unrelated cord blood (75%); 2 with msd and i mud. out of 9 cases of ucbt there were 5 severe combined immunodeficency (scid), 2 chronic granulomatous disease (gcd), 1 hyperigm syndrome and 1 wiskott aldrich syndrome (was). the median age of transplant was 13.7 months (range 1.3 to 83.3 months). all presented with multiple infections ranging from disseminated bcg infection to parainfluenza /rsv /rotavirus infection to pseudomonas sepsis, staphylococcal endocarditis to pulmonary aspergillosis for scid. hyper igm presented with pnemocystitis carini pneumonia while cgd conditions presented with perianal abscess and fungal pneumonia. the child with was had life threatening git bleeding and a hemorrrhage trachaebronchial cast removed after a failed initial extubation for gastroscopy. conditioning regimes consisted of reduced intensive (fludarabine based) conditioning regime for scid and myeloablative regime for the rest. the median tnc dose was 12.7 × 10(7)/kg (range 4.2 to22.5) and median cd34+ cells dose was 3.68 × 10(5)/kg (range 1 to 8.9). results: all engrafted well except for one graft failure in cgd. he refused 2nd transplant and died 1.5 years post transplant from fungal pneumonia. median engraftment time for neutrophil was 21 days (range 13 to 33) and platelet was 30 days (range 18 to 65 days). grade 1 skin aghvd occurred in one patient while another patient died of agvhd of liver and lungs. chronic gvhd was found skin and liver in one patient. trm was 11 % (due to agvhd). median follow up was 1255 days (ranged 327 to 4109). overall 5 years survival was 78%. post-transplant complication with life threatening puemonitis was not uncommon. one patient developed biopsy -proven idiopathic interstitial pneumonitis and required ecmo for one month. he received immunosupressive drugs including methylprednisolone, infliximab, oral imatinib (tk inhibitor), azithromycin and nebulised becotide. he was weaned off oxygen after 2-3 months. conclusion: our limited experience showed unrelated cord blood is good source of stem cell for transplant in pid in a multiracial population. one case of graft failure was likely due too low cell dose cd34 +cells dose 1 × 10(5)/kg. the expertise in icu has enabled us to support several patients who presented with infective pneumonia pre-transplant and post -transplant. with better technology like alpha/beta depletion haploidentical transplant may be a better option to achieve engraftment earlier so as to avoid stormy post-transplant infections seen in unrelated cord blood setting. disclosure of conflict of interest: none. in spite of these recommendations, literature from developing countries suggest that pbscs are used more and more frequently without compromising the transplant results, as they seem to be preferred graft source for donors in many countries incl. poland. therefore we analyzed the efficacy of mud-hsct in children with saa transplanted in our centre. clinical data of 44 saa and pnh children and adolescents (27 boys and 17 girls), who underwent mud-hsct between october 2000 and july 2016 were retrospectively analyzed. the median age was 11.2 years (range 0.7-20 years) according to the graft source, the patients were divided into pbsct group (37 patients) and bm group (7 patients). four patients required second mud transplant due to graft rejection. overall survival for all patients was 66 %. estimated 5-year overall survival (os) was not statistically different between pbsct group and bm group [(68% vs 54% ) p = 0.42]. there was no significant difference in os between group who had ist before transplant and the group, who had an upfront transplant as a first line of therapy [65% vs 62%, p = 0.79].the time to neutrophil and platelet engraftment was statistically longer in bm group than in pbsc group [(anc 16 vs 15 days, plt 29 vs 16 days, respectively) p = 0.017]. the incidence of grade iii-iv acute graft-versus-host disease (gvhd) in pbsct group was similar to that in bm group [37% (14/37) vs 29% (2/7)]. the incidence of chronic gvhd in pbsct group was similar to that in bmt group [8% (3/37 ) vs 14% (1/7)]. other transplantrelated complications like heart failure, central nervous bleeding, incidence of infections were comparable within the two regimens. there were 15 deaths in the whole group. the main reason of death were infectious complications or multiorgan failure (mof) in severely pretransfused patients in this historical cohort of patients. unrelated donor pbsct in children and adolescents with saa seems to be not inferior to unrelated donor bmt. the incidence of chronic gvhd was surprisingly low in saa recipients of mud pbsc. increased morbidity and mortality due to infections was due to individual poor clinical situation of patients before transplant (i.e. fungal infections, contamination with resistant bacteria, prolonged neutropenia). disclosure of conflict of interest: none. dyskeratosis congenita (dc) is characterized by the clinical triad of reticular skin pigmentation, nail dystrophy, and oral leukoplakia. the majority of patients with dc develop bone marrow failure (bmf), which is the main cause of death in dc patients. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative treatment for bmf associated with dc. transplant-related morbidity/mortality is common, especially after myeloablative conditioning regimens. hsct has been introduced into the management of dc, which has had remarkable clinical results. we report our experience in 4 children with dc who underwent allogeneic transplantation at a single medical center. patients received a fludarabine-based reduced intensity conditioning (ric), and the graft source was unrelated peripheral blood stem cells. median age at the time of hsct was 5.5 years (range, 4-13 years). the numbers of infused mononuclear cells and cd34+ cells were 15.62 ± 3.04 × 10 8 /kg and 5.80 ± 3.37 × 10 6 /kg, respectively. the median time of neutrophil and platelet recovery were 13.5 days (range, 12-17 days) and 21.5 days (range, 19-26 days). two patients experienced grade ii-iii acute graftversus-host disease (gvhd), and chronic gvhd was only observed in one patient. all four patients remained alive and transfusion independent at the median follow-up of 18.5 months (range, 9-31 months). correction of previously existing physical defects was observed in two patients. unrelated peripheral blood hsct can be a curative option for dc. ric based on the type of disease is important to s382 achieve successful hsct. a larger sample size and extended follow-up of this rare patient population are needed to determine whether the changes in therapy will improve longterm survival. disclosure of conflict of interest: none. autosomal recessive hyper-ige syndrome due to dock8 mutation is a combined primary immunodeficiency, characterized by severe eczema, recurrent infections, and susceptibility to autoimmunity, malignancy, and multiple allergies, in addition to unusual high serum ige level. dock8 patients tend to have a progressive severe clinical course with mostly fatal outcome during second to third decade of life without hematopoietic stem cell transplantation (hsct). in our center we have a large number of dock8 patients. during a period of 11 years (2006-2016), we transplanted 14 patients with documented dock-8 mutation confirmed by molecular genetics. one patient did not receive any conditioning because of poor clinical condition and he died from severe cutaneous and gut gvhd and another patient received cbt with bu/flu with zero engraftment. the rest of the patients received hsct from hla full matched donor with chemoablation with bu/cy for all with 100% lymphoid and myeloid engraftment (str). among those patients who received chemoablation, gvhd developed in 6 patients mostly grade i and ii. in addition 3 patients died: one died of severe gvhd and the other two died of sepsis. for dock8 patients we highly recommend early hsct if fully matched donor is available to prevent the high mortality associated with the disease. alloreactivity triggered by interactions between killer cell immunoglobulin-like receptors (kir) and natural killer (nk) cells plays a role in graft-versus-tumor (gvt) effects after hematopoietic stem cell transplantations (sct). in particular, kir-ligand mismatching between the donor and recipient might promote nk cell alloreactivity after unrelated cord blood transplantations (ucbt) in adult patients with acute myeloid leukemia (aml). recently, it has been suggested that allogeneic nk cells could be the effector cells that mediate gvt effects after mismatched allogeneic transplants for refractory childhood solid tumors. however, there are few reports about the efficacy of kir-ligand mismatched sct in pediatric cases. here, we report the excellent outcomes of kirligand mismatched cbt (kir-cbt) in pediatric patients with refractory malignant disease. we evaluated the cases of 9 pediatric hematology and oncology patients [4 (44%) aml, 1 (12%) myelodysplastic syndrome [mds] , and 4 (44%) neuroblastoma [nbl] patients] who underwent kir-cbt between 2010 and 2016 at our institution. among the 4 aml cases, one involved refractory disease (induction failure), and the other three involved relapsed aml (one patient relapsed after the 1st sct because of 5q-). all 4 nbl patients underwent kir-cbt followed by auto-peripheral blood stem cell transplantation (pbsct) because of stage 4 disease. the mds patient underwent kir-cbt because of refractory anemia with excess blasts. kir mismatching was defined as incompatibility between the donor kir and recipient kir ligand, and only inhibitory kir that interacted with human leukocyte antigen (hla)-bw4, -c1, or -c2 group ligands were considered. the median age of the patients was 4 (range 2-18) years. all 4 of the aml patients were in complete remission (cr) at the time of the hsct (cr1 = one case, cr2 = 3 cases). the mds patient was in a non-cr state, and all of the nbl patients were in their 1st cr at the time of the hsct. the aml patients received total body irradiation (tbi)-based conditioning (12 gy tbi and 120 mg/kg cyclophosphamide [cy]), and the mds patient received busulfan (bu)-based conditioning (19.2 mg/kg bu and 120 mg/kg cy). the nbl patients received reducedintensity conditioning regimens (125 mg/m 2 fludarabine, 140 mg/m 2 l-pam, and 2 gy tbi). the cb exhibited hla 2-4 locus mismatches (dna typing), including at least one inhibitory kir gene mismatch. the prophylaxis for graftversus-host disease (gvhd) consisted of tacrolimus and shortterm methotrexate. anti-thymocyte globulin (atg) was not used as a gvhd prophylaxis in any case. after the median follow-up period of 25 months (range: 4 -54 months), all 9 patients were alive, and none of them had relapsed after the kir-cbt. although grade ii-iv gvhd was observed in 6 patients (67%), it was controlled with prednisolone. chronic gvhd was not seen in any case. the present findings suggested that nk cell alloreactivity plays a role in preventing childhood myeloid leukemia and nbl relapse after kir-cbt. although our results are limited, this report provides novel data to support further investigations into the use of kir-cbt for the treatment of pediatric refractory malignant disease. disclosure of conflict of interest: none. impact of fcm-based minimal residual disease on transplant outcomes in patients with aml in hematological complete remission t oka, j kanda 1 , k ohmori 2 , m hishizawa 3 , t kitano 4 , t kondo 5 , k yamashita 6 it is reported that the presence of minimal residual disease (mrd) before hematopoietic stem cell transplantation (hsct) is associated with poor overall survival in patients with acute myelogenous leukemia (aml) in hematological complete remission (cr). we retrospectively analyzed the association between flowcytometry (fcm)-based detection of mrd and transplant outcomes. we included 56 adult patients with aml in hematological cr, who underwent their first allogeneic hsct between april 2005 and may 2015 at kyoto university hospital. mrd of bone marrow before hsct was measured using fcm. to search for target antigens to detect mrd, threecolor fcm analyses were performed using a differential panel for every disease and patient, which allowed us to detect ⩾ 0.1% of mrd. of the 56 patients (median age: 48.5, range: 18-66), 41 patients were included in the mrd-negative group (mrd o0.1%), whereas 15 were included in the mrd-positive group (mrd ⩾ 0.1%). in the latter group, 6 patients were included in the mrd-low group (mrd o0.6%), and 9 were included in the mrd-high group (mrd ⩾ 0.6%). there was no significant difference in the patient background between the mrd-negative and mrd-positive groups. the 3-year overall survival rates for the mrd-negative, mrd-low, and mrd-high groups were 82%, 63%, and 30%, respectively (p = 0.007, figure 1 ). in a multiple regression analysis, the mrd-high group was significantly associated with higher overall mortality than the mrd-negative group (mrd-low vs mrd-negative, hazard ration [hr] 1.62, p = 0.554; mrd-high vs mrd-negative, hr 8.47, po 0.001). the 3-year relapse rates for the mrdnegative, mrd-low, and mrd-high groups were 15%, 0, and 67%, respectively (p o0.001). there were no significant differences in non-relapse mortality among the three groups. the analysis of fcm-based detection of mrd revealed that an mrd positivity of ⩾ 0.6% was significantly associated with high risk of relapse and death even in patients with aml with hematological cr. the stronger consolidation or conditioning therapy before hsct based on mrd could improve transplant outcomes in these patients. [p506] disclosure of conflict of interest: none. post-transplant cyclophosphamide (ptcy) and megadose t cell depleted (tcd) haplohsct for tolerance induction f aversa 1 , e bachar-lustig 2 , n or-geva 3 , y zlotnikov klionsky 2 , l prezioso 1 , s bonomini 1 , a monti 1 , i manfra 1 , c schifano 1 , s pratissoli 4 , f lohr 5 , r lamanna 6 , v sgobba 6 , n giuliani 7 and y reisner 2 1 hematology and bmt unit, university hospital of parma, italy; 2 department of immunology, weizmann institute of science, rehovot, israel; 3 neurology department, stanford school of medicine, stanford, california; 4 radiotherapy unit, university hospital of modena, italy; 5 radiotherapy unit, university hospital of modena, italy; 6 genetic unit, university hospital of parma, italy and 7 hematology and bmt unit, university hospital of parma, italy the use of ptcy is associated with reduced risk for gvhd in t cell replete nma haplo-hsct; however, this intervention is still not sufficiently safe to justify treatment of non-malignant diseases or as a platform for organ transplantation. experimental data: in a total of 66 mice, we showed that combining the power of megadose tcd hsct with high dose ptcy (fig.1a) , enables marked and durable chimerism following nma conditioning, while each modality alone was ineffective (figure 1a) . chimerism included all myeloid and lymphoid lineages, and lda analysis of alloreactive t cells revealed specific immune tolerance towards donor stimulators (fig.1b) , also associated with acceptance of donor but not 3 rd party skin. clinical trial: a similar protocol was developed for clinical use. the first patient, a 54 yr old male with high-risk multiple myeloma in cr after autohsct, received megadose (15.4 x10 6 cd34+ cells/kg) cd3/cd19 depleted (1.17 x10 5 cd3+t cells/kg) haploidentical pbpcs after atg, fludarabine and 2 gy single frcation tbi. ptcy was given to control both hvg and gvh reactions (fig. 1c) . hematopoietic engraftment was achieved at day +15 with over 97% donor type chimerism during the first 6 months in the myeloid and b cell lineages. t cells during this period were predominantly of host type (10-23% donor type), gradually increasing to 63-72% at 9-12 months post transplant (fig. 1d) . the patient overcame cmv and subsequently ebv reactivation without any treatment (fig. 1e-1g) . dextramer facs analysis revealed that cmv and ebv specific cd8 t cells were exclusively of host origin (fig. 1f-1h) . at +18 months, cr and normal free light chain ratio were confirmed. the second patient, a 50 year-old male with high risk heavily pretreated multiple myeloma (tandem auto-hsct, 3 yr maintenance with lenalidomide, salvage therapy with vd) received a similar hsct (10.8 x10 6 cd34+ cells/kg, 1.2x10 5 cd3 +t cells/kg). despite transient engraftment (50% donor cell on day +17), graft failure with autologous recovery (0.04% donortype chimerism) was documented on day +30. this may be due to the extended treatment (3 yrs) with lenalidomide, but rejection cannot be excluded. after 5 months, this patient tolerated a second haplo-hsct (different donor) after myeloablative conditioning (atg, treosulfan, thiotepa and fludarabine) and alfa/beta tcr/cd19-depleted pbpcs. at 8 month follow up, he shows no sign of gvhd, good immunological reconstitution, excellent quality of life, and remains in complete remission. collectively, our murine proof of concept data supported by clinical experience in the first high risk mm patient. the marked level of host t cells persisting over the first year after hsct can provide anti-viral immune protection until thymus-derived donor t cells are generated. avoiding additional post transplant immune suppression ensures a robust anti-viral immunity and a graft vs tumor effect. the rejection experienced by the 2 nd patient, although corrected by a 2 nd myeloablative tcd hsct, indicates that the conditioning must be fine-tuned to optimize engraftment in every patient. we are therefore testing, increasing tbi from 2 gy to 3 gy. further studies will determine the efficacy of this approach in elderly mm patients, in non-malignant hematopoietic diseases, or as a prelude for organ transplantation and cell therapy. over the last decade the addition of alemtuzumab to fludarabine-based reduced intensity conditioning regimen is common practice in the unrelated donor allograft setting. in recent years, however, its use has extended to reduced intensity hla-identical sibling donor allografts with the aim of providing an additional prophylaxis against gvhd. it is difficult to assess though whether this practice has any negative influence in the relapse rate or whether it has any net benefit or disadvantage in terms of overall survival. in this retrospective study we have analysed a historical cohort of 65 patients [41 males, 24 females, mean age 59.05 (40-73)] who s385 received a ric fully matched unrelated donor (49 patients) or sibling donor (16) hsct as consolidation treatment for hr aml in 2 transplant centres in uk and greece. the conditioning regimen included fludarabine in all cases, together with melphalan and alemtuzumab(29 patients), busulphan and campath (21 patients), busulphan and thiotepa (1 patient), melphalan (3 patients), busulphan with and without atg (9 patients) total body irradiation (200 cgy, 2 patients). in total, 50 patients received alemtuzumab (35 mud 50 mg alemtuzumab and 15 sibling donor hsct recipients 30 mg alemtuzumab) and 9 patients (7 mud and 2 sibling donor hsct recipient) received atg with 5 patients receiving t replete allografts. gvhd prophylaxis was ciclosporin for patients receiving alemtuzumab based or atg based regimen and ciclosporin with low dose methotrexate for t-replete allografts. the median follow up was 32.3 months (range 3-154 months).all but four patients were transplanted in cr1 overall, patients receiving conditioning without alemtuzumab suffered more frequent (po0.0001) and more severe (po0.0001) acute gvhd. this group, however, had a significantly (po 0.05) lower relapse rate. the overall survival remained unaffected. the subgroup of patients receiving allografts from mud had a clear benefit in terms of a lower incidence (p o0.0001) and severity (p o0.0001) of acute gvhd: none of the patientsreceiving alemtuzumab experienced grade iv agvhd, but up to 5/14 patients not receiving alemtuzumab suffered severe grade iv gvhd. however, the use of campath was associated with a significantly higher rate of relapse or progression of the aml (po 0.02), so that none of the 6 mud recipients not having campath relapsed, while 9/26 patients having alemtuzumab relapsed. although none of these factors had a net impact on survival, there was a nonsignificant (p = 0.07) trend towards a higher survival in patients who received alemtuzumab. in the sibling donor allograft setting, alemtuzumab had no significant impact on the incidence of acute gvhd, relapse or survival. finally, in diseases where cytogenetic or molecular markers of high risk were available, our results showed a better overall survival (po0.06) in ric alemtuzumab conditioning undergoing fully matched unrelated donor hsct, probably as a result of the protection against graft versus host disease while maintaining graft versus leukaemia effect. overall, alemtuzumab is a highly protective agent against agvhd in mud hsct recipients while it maintains the graft versus leukaemia effect.however it did not show any clear benefit of its use in the identical sibling donor setting. larger prospective studies are required in order to determine the need for this agent in this particular setting. disclosure of conflict of interest: none. blastic plasmacytoid dendritic cell neoplasm (bpdcn) is a rare disease which constitutes o1% of all hematologic neoplasms annually. majority of bpdcn present with diverse skin involvement prior to leukemic dissemination, whereas a minority (~10%) have systemic involvement at diagnosis. there are no established therapies for bpdcn and most pts receive acute leukemia, myeloid or lymphoblastic, induction regimens; but responses are short-lived and prognosis is poor upon relapse. allogeneic hematopoietic cell transplantation (allo-hct) is offered to bpdcn cases based on small retrospective or registry case series. we retrospectively analyzed outcomes of bpdcn pts who received an allo-hct at 5 transplant centers in the usa. a total of 20 pts were eligible for analysis ( table 1 ). the primary endpoint was overall survival (os). twenty patients (m = 18, 90%), median age of 51 (14-71) yrs, received an allo-hct from a matched related (n = 9, 45%), matched unrelated (n = 7, 35%), mismatched-unrelated (n = 2, 10%), umbilical cord (n = 1, 5%) or haploidentical (n = 1, 5%) donor using myeloablative (mac) (n = 13, 65%) or reducedintensity (ric) (n = 7, 35%) conditioning. fifteen pts received hyper-cvad as pre-allograft therapy (front-line = 14, salvage = 1). the majority (n = 17, 85%) were allografted in cr1. median f/u for survivors was 26.3 (3.9-128.8) months. median time-to-neutrophil and platelet engraftments were 16 (12-26) days and 15 (7-45) days, respectively. five pts never dropped s386 platelet counts below 20 000/μl. three pts (mac = 2, ric = 1) relapsed at 6, 7, and 99 months, respectively. all 3 relapsed with marrow involvement (1 had also skin involved). mean os was 81.9 (53.1-110.8) months. one-year and 3-year os were 85% (95% ci = 64-95%) and 70% (95% ci = 48-85%), respectively. there was no difference in 3-year os when comparing mac versus. ric (hr = 1.68 (95% ci = 0.34, 8.4), p = 0.53). median time to onset of acute gvhd was 35 (10-117) days; grade ii-iv acute gvhd occured in 6 cases. chronic gvhd was seen in 5 cases (mild = 2, mod/severe = 3). allo-hct is an effective therapy for bpdcn resulting in durable remissions. encouraging outcomes observed in this analysis may be explained by offering allo-hct early in the disease course and in the setting of complete remission. larger studies are needed to better understand risk factors for relapse to develop post-transplant strategies to improve outcomes. disclosure of conflict of interest: none. a risk-factor analysis for overall survival in patients with acute leukemia that relapse following t-replete haploidentical transplantation: on behalf of the acute leukemia working party of the european society for blood and marrow transplantation s piemontese 1,2 , m labopin 2,3 , f ciceri 1,2 , c schmid 2,4 , a ruggeri 2,3 , w arcese 5 , z gulbas 6 , y koc 7 , j tischer 8 , b bruno 9 , w depei 10 , d blaise 11 , d beelen 12 , g ehninger 13 , a boumendil 2,3 , m houhou 2,3 , m mohty 2,3 and a nagler 2, 14 relapse of acute leukemia is the leading cause of transplantation failure with devastating results. relapse post t-replete haploidentical transplantations (haplo-sct) is not well characterized. the objective of this study was to identify riskfactors for overall survival in patients with al that relapsed after a haplo-sct. from 2007 to 2014, 1660 haplo-sct were performed in 186 ebmt centers as first allogeneic transplantations for adults with acute leukemia. out of 657 patients for whom we were able to receive updated data, 208 relapsed and were included in this analysis. median follow-up among survivors was 25 months after haplo-sct (2-97) and 7.2 months (1-71) after relapse. median time from haplo-sct to relapse was 5 months (11 d-36m). diagnosis was acute myeloid leukemia (aml) in 72% and acute lymphoblastic leukemia (all) in 28% of the patients, respectively .fifty-two (25%) patients were transplanted in first complete remission (cr1), 42 (20%) in cr2 or cr3, while 114 (55%) were transplanted in active disease. ric regimen was used in 116 (57%) patients and 85 (41%) received bone marrow as stem cell source. post-transplant cyclophosphamide (pt-cy) was used for graft-versus-host disease (gvhd) prophylaxis in 121 patients (58%). fifty-two (25%) of the patients who relapsed post haplo-sct experienced previously acute gvhd and 42 (21%) chronic gvhd post transplantation. treatment of relapse varied and included: none in 42 (21%), ist withdrawal only in 15 (8%), chemotherapy (ct) only in 59 (30%), tyrosine-kinase inhibitor (tki) only in'(2%), tki and ct in 6 (3%), dli only in 9 (4%), subsequent transplant in 12 (6%), ct and dli in 37 (19%), ct and subsequent transplant in 8 (4%), tki ct and subsequent transplant in 1 (0.5%), dli and subsequent transplant in 5 (2.5%) patients. donors for second allogeneic transplant were unrelated (n = 1), haploidentical (n = 23) and cord blood (n = 2). second transplant was performed in cr for 8 patients and in relapse for 18 patients. only 2 patients who received a second haplo were alive at 47 and 69 months post second transplant. the majority of patients who received dli were in relapse at time of dli (81%), and 26% achieved cr after dli. os 1y after dli was 27%, 7 patients being alive at a median time of 18 mo (4-55) post dli. overall, the one-year overall survival (os) following relapse was 17% (95% ci: 11.6-22.3). in univariate analysis disease status at haplo-sct (cr vs active disease), cytogenetics (good/intermediate vs poor) and median time from haplo-sct to relapse (4 or o 5.03 months) were associated to a higher os at one year after relapse: 27% (p = 0.003), 27% (p = 0.03) and 25% (p o10 -4 ), respectively. in multivariate analysis complete remission at haplo-sct (p = 0.004; hr 0.64; ci:0.47-0.87) and time from haplo-sct to relapse higher than 5.03 months (p = 0.001; hr 0.58; ci: 0.41-0.81) were risk factors for a higher os after relapse. in the 58 patients transplanted in cr and relapsing more than 5 month after haplo, 1 and 2 y os were respectively 33% and 20%. these findings suggest that similar to other transplantation setting os for acute leukemia that relapse post haplo-sct is dismal. disease status at transplant and time from transplant to relapse are the two important prognostic factors that can predict somewhat better survival. indication for second transplant should be carefully evaluated. integrations with novel therapies are in unmet need to prevent and treat relapse post haplo-sct. disclosure of conflict of interest: none. patients (pts) with aml who relapse after autologous stem cell transplantation (asct) have a dismal outcome but some can be rescued with an allogeneic transplantation (allohsct). yet, available evidence presently stems from analyses of limited patient numbers. we decided to analyze the ebmt registry to evaluate the outcome and determine the prognostic factors in a large series of such pts. the ebmt registry was screened for adult pts with de novo aml (non-apl) who received an allograft in cr2 or first relapse (2000-2015) after being autografted in cr1. pts receiving ex vivo t cell depletion (tcd) were included only if they received a haploidentical allohsct. inclusion criteria were met by 537 pts (48% female, median age 45 [range 18-78] years). median time from asct to relapse was 10 (range 0.6-176, iqr 5.8-19.1) months. at allohsct, pts were in 1 st relapse (25%) or cr2 (75%). donors were matched sibling (18%), unrelated (57%), haploidentical (13%), or cord blood (12%), respectively. conditioning was myeloablative in 46% and reduced intensity in 54% of the pts, respectively. the median follow up was 52 months (range o1-167 months). at 3 years post allograft (figure), leukemia free survival ( 4 -18] of the pts. all factors significantly associated with ⩾ 1 endpoint in univariate analysis were entered in a multivariate cox regression model (table 1) . ri was lower in pts transplanted in cr2 rather than in relapse (31.1% vs 44.7%; hr 1.76, p = 0.004) and in pts who relapsed later (410 months, median value) as opposed to those who relapsed early post asct (25.5% vs 43.2%; hr (per month) 0.97, p o10 -3 ). ri was lower in pts transplanted with an unrelated donor (ud) in comparison to those transplanted from a matched sibling donor (29.1% vs 50.5%; msd, hr: 0.49, p o10 -3 ). patient age, poor cytogenetics, transplantation in relapse, previous tbi for asct, myeloablative conditioning (mac) vs reduced intensity (ric) and ud, haplo or cbt vs msd all significantly increased nrm. lfs was significantly better in pts with good risk (47.3%) than in pts with intermediate risk or poor risk cytogenetics (29%; hr 0.62, p = 0.007) or in pts who relapsed late (per month: hr 0.99, p = 0.003) post asct. lfs was worse in pts who previously had received tbi (20% vs 45%; hr = 1.82; po10 -3 ). the same prognostic factors were significant for os. haploidentical (hr 2.25, p = 10 -3 ) and cord blood (hr 2.09, p = 0.003) transplants resulted in lower os than those from msd. finally, date of transplant significantly influenced os which was higher in pts transplanted after january 2008 vs those allografted before; 48.2% vs 31.7%, hr (per year) 0.96, p = 0.02). about one third of adult patients with aml who relapse post asct can be rescued with an allogeneic transplantation, especially if the duration of persisting cr post asct is long and no tbi was received in the past. transplantation from an msd while in cr2 rather than at relapse offers the best outcome. disclosure of conflict of interest: none. high incidences of graft-versus-host disease (gvhd) and relapse have seriously impeded the widespread application of haploidentical hematopoietic stem cell transplantation (haplo-hsct) for high-risk acute leukemia lacking conventional hla-matched donors. one hundred and ten high-risk acute leukemia patients underwent haplo-hsct with idarubicin (ida) intensified conditioning regimen (ida intensified bucy2 for acute myelocytic leukemia (aml) and ida intensified tbi-cy for acute lymphoblastic leukemia (all)). for donor-recipient hla 3/6 or 4/6 transplant, we separately administered a total of 9 mg/kg or 6 mg/kg antithymocyte globulin (atg) and basiliximab for gvhd prophylaxis. all enrolled patients were observed longitudinally until death or lost to follow-up. the 100-day cumulative incidences of ⅱ-ⅳ and ⅲ-ⅳ agvhd for all patients were 30.3%, 14.7%, respectively. the 2-year cumulative incidence of extensive cgvhd was 12.2%. the relapse rate was 18.2%. the 3-year probability of overall survival (os) reached 63.1%. the patients in non-complete remission (nr) showed significantly higher relapse and worse survival than complete remission (cr) minimal residual disease (mrd) (-) and cr mrd (+) patients. however, the relapse, 3y-os and disease-free survival (dfs) of cr mrd (-) did not differ from cr mrd (+) patients, indicating our intensified transplant technique could overcome the poor prognosis of mrd. for whatever aml or all patients, the relapse rates, agvhd, cgvhd and the estimated 3-year os and dfs between two atg group were equivalent, except that all patients in atg 9 mg/kg experienced higher relapse (33.0% vs 19.2%, p = 0.226). although the incidence of cytomegalovirus (cmv) reactivation in atg 9 mg/kg and 6 mg/kg was 77.4%, 72.9%, the average episodes of cmv reactivation were remarkably [p512] higher in 9 mg/kg. our ida intensified haplo-hsct technique could improve the outcome of high-risk acute leukemia and could be recommended as a good alternate for patients lacking hla-matched sibling donors. diagnosed secondary aml were randomized 1:1 to cpx-351 or standard 7+3 therapy. cpx-351 induction was 100 units/m 2 on days 1, 3, 5 (first induction) and days 1, 3 (reinduction); 7+3 first induction was cytarabine 100 mg/m 2 /day × 7 days and daunorubicin 60 mg/m 2 on days 1, 2, 3, and reinduction was cytarabine 100 mg/m 2 /day × 5 days and daunorubicin 60 mg/ m 2 on days 1, 2. a dynamic allocation procedure stratified patients by age group (60-69 or 70-75 years) for each study arm. patients with complete response (cr) or cr with incomplete platelet or neutrophil recovery were considered for allogeneic hct, based on institutional criteria. overall survival (os) landmarked at the time of hct was assessed. a total of 309 patients were enrolled on the induction trial. . patient and aml characteristics in the hct age subgroups were generally similar between arms. in both age subgroups of patients receiving hct, median os was longer in the cpx-351 arm than in the 7+3 arm (table 1 ). in the 60-69 group, serious adverse events (saes) prior to hct in the cpx-351 and 7+3 arms occurred in 28% and 22% of patients, respectively; in the 70-75 group, in 13% and 67%, respectively. the most common sae was febrile neutropenia (cpx-351, 11.5%; 7+3, 5.3%), occurring in all age groups. relapse after allogeneic haematopoietic stem cell transplant (allo-hsct) for acute myeloid leukaemia (aml) and myelodysplastic syndrome (mds) remains the main cause of treatment failure. it is associated with dismal prognosis and short survival. proposed salvage strategies are tapering of immunosuppressive therapy, re-induction with chemotherapy and consolidation with donor lymphocyte infusion (dli) or second allo-hsct, although, results remain disappointing. azacitidine (aza) and dli has proved to be an effective and well-tolerated outpatient approach in this setting, and results in at least temporary disease control in the majority of patients, thus, representing a valuable alternative to current treatments. between january 2010 and november 2016, 16 patients with relapsed aml or mds after allo-hsct were treated with subcutaneous aza 100 mg/m 2 days 1-5 every 28 days and escalating doses of dli if feasible at manchester royal infirmary, uk. aza was continued until cr or disease progression. patients characteristics: median age 60 (range 45-69) years, 56% males, diagnoses were aml (n = 12) and mds (n = 4). five (31%) patients had either monosomal or complex karyotype. fifty percent of patients were in cr1 before transplant, 12.5% in cr2, 12.5% had a partial response and 25% did not receive any chemotherapy before the transplant. fifteen out of 16 received fludarabine-base reduced intensity conditioning regimen and all but one had a t-cell depleted graft. at relapse 88% had mixed donor chimerism. median time to relapse was 9.5 (range 2-21) months after allo-hsct. with a median follow up of 6.5 (range 1-25) months a median of 5 (range 1-16) courses of aza were administered and median of 2 (range 1-6) dli were infused. doses of dli were administered starting at 0.1 x107/kg and escalating by log5. aza and dli infusions were well tolerated; only two patients withdrew due to intolerance. seven patients were admitted at least once due to infections (86%) or progressive disease. only two patients developed mild gvhd grade 1. complete remission was achieved in 12.5% patients and stable disease in 56%. patients in cr had full donor chimerism. median overall survival for patients in cr was 25 months compared to 9 months for those who did not respond (p = 0.031). patients with more than 20% blasts on bone marrow at time of relapse after allo-hsct had a worse outcome than those with less than 20% blasts (11 months and 25 months respectively, p = 0.09). no differences were seen when compared time to relapse ( o 6 months vs ⩾ 6 months) s390 and outcome, or disease and overall response, although numbers in this series are small. image/graph: overall survival following azacitidine and dli, patients in complete remission, stable disease and disease progression. azacitidine and dli can provide long term remissions in patients with relapsed aml/mds post allo-hsct with low toxicity. lower disease burden at relapse carries better outcomes. low rates of gvhd are seen following azacitidine and dli most likely showing the immunomodulatory effect of azacitidine described by other groups. acute myeloid leukemia (aml) is a frequent complication in patients affected by telomere maintenance disorders ('telomeropathies') such as dyskeratosis congenita (dkc). treatment of aml in dkc patients by chemotherapy and hematopoietic stem cell transplantation is characterized by frequent remission failure, high organ toxicity and poor outcome. a 27-yearold patient with aml was admitted to our hospital in december 2014. he had been treated with 6 cycles beacopp for hodgkin´s lymphoma (hl) in 2009. on admission, the patient presented clinical signs of premature aging with hair greying and lack of fully recovered hair growth after chemotherapy (cx) for hl. flow-fish analysis revealed tl below the 1% percentile within leucocytes in line with the suspected diagnosis of telomeropathy. retrospective tl analysis by confocal q-fish from bm at hl diagnosis confirmed short tl before the start of any chemotherapy. he received standard aml induction cx (3+7), but follow-up revealed persistence of aml. salvage cx with flag-ida was applied resulting in partial remission with only weak regeneration of normal hematopoiesis. the patient received an allogeneic stem cell transplantation (asct) after conditioning with 140 mg/m 2 melphalan and fludarabin from his hlamatched brother whose tl was found to be normal. after asct, he developed sinusoidal obstructive syndrome and progressive liver failure treated with defibrotide and he was admitted to icu for sepsis. leucocyte count showed sufficient engraftment on day 14; however, liver function recovered only partially. during critical care treatment, the patient showed cardiomyopathy, renal failure and extensive wound healing problems without epithelial proliferation indicative of severe replicative exhaustion. finally, he died due to sepsis with acute liver failure on day 91 after asct. aml arising from dkc is a rare event with substantial impact on patients´prognosis. therapy remains challenging due to poor bm function and high risk of organ toxicity, especially liver failure and lung fibrosis. dose reduction of alkylating agents and avoidance of total body irradiation are necessary in conditioning prior to asct in patients with dkc and aml, however no clear data or recommendations exist for the management of these patients. tl screening can help to identify patients with suspected dkc related bm failure or aml and to identify family donors without telomeropathy. physicians should be aware of possible dkc related aml, especially in familial cases of aml or bone marrow failure, impaired or prolonged recovery following cytoreductive treatment or coincidence of solid (e.g. oral cavity carcinomas) and hematological malignancies. disclosure of conflict of interest: none. chronic graft-versus-host disease and donor lymphocyte infusions in patients with non-de novo acute myeloid leukemia or advanced myelodysplastic syndromes after allogeneic stem cell transplantation pg hemmati 1 , k pfeifer 1 , lg vuong 1 , cf jehn 1 , p le coutre 1 , b dörken 1 and r arnold 1 1 medizinische klinik mit schwerpunkt hämatologie, onkologie und tumorimmunologie, charité-universitätsmedizin berlin, campus virchow-klinikum, berlin, deutschland aml with myelodysplasia-related changes and therapy-related aml (taml), collectively termed secondary aml (saml) in daily clinical routine, represent distinct subgroups in the 2016 revised who classification of myeloid neoplasm and leukemias. as compared to de novo-aml, saml is associated with a poor survival when using conventional chemotherapy approaches. this is mainly due to unfavorable cytogenetics, older age and/or the presence of comorbidities as well as poor response to induction therapy. furthermore, cumulative organ toxicity resulting from treatment of the antecedent solid malignancy in patients with therapy-related disease has to be taken into account. allogeneic stem cell transplantation (allosct) represents the only option to achieve long-term disease control and definitive cure. we retrospectively analyzed 204 patients with saml or advanced mds (eb-2 according to who) transplanted at our center between 1995 and 2015. at the time of allosct, 98 patients (48%) were in complete hematologic remission (chr), whereas 106 patients (52%) had active disease. cytogenetic risk was categorized according to the swog/ecog classification and was favorable (n = 3; 2%), intermediate (n = 94; 46%), unfavorable (n = 84; 41%), or unknown/undetermined (n = 23; 11%). standard myeloablative conditioning (mac) using 12 gy total body irradiation (tbi) and cyclophosphamide was used in 41 patients (20%), whereas fludarabin/busulfan/atg-based reduced intensity conditioning (ric) was applied in 163 patients (80%). grafts were from related (n = 51; 25%) or unrelated (matched: n = 112; 55% or mismatched: n = 41; 20%) donors. the median follow-up of the surviving patients was 46 (5-24) months. a graft failure occurred in 5/204 patients (3%). at last day of follow-up 72/204 patients (35%) were alive and in chr. relapse occurred in 77/204 patients (38%) after a median interval of 4.6 (range: 0.1-135) months. cause of death were either relapse or nrm (gvhd and/or infections) in 69/204 patients (34%) or 56/204 patients (28%). at 1, 3, 5, and 10 years after allosct overall survival (os) or disease-free survival (dfs) of the entire cohort was 56%, 46%, 38%, and 29% or 50%, 38%, 36%, and 27 %, respectively. at the same time points, the cumulative incidence of relapse (ci-r) or non-relapse mortality (ci-nrm) was 30%, 37%, 37%, and 40% or 20%, 25%, 27%, and 33%, respectively. extensive uni-und multivariate analyses revealed a number of factors associated with inferior outcome, e.g. poor-risk cytogenetics, the presence of taml, advanced age, reduced physical performance, and comorbidities, whereas donor type (unrelated versus unrelated), and remission status had no significant impact on overall outcome. furthermore, the development of gvhd, especially the presence of cgvhd, and the use of donor-lymphocyte infusions (dli), either in a prophylactic or pre-emptive setting, were identified as independent predictors for a reduced relapse incidence, which in turn, led to an improved os and dfs. our results indicate that allosct represents an important treatment option for patients with saml. however, a relapse rate of 30% at 12 months prompts the development of novel approaches to prevent early disease recurrence. strategies to augment the graft-versus-leukemia (gvl) effect of allosct may help to improve the results. disclosure of conflict of interest: none. myeloid sarcoma (ms) is a rare hematologic myeloid neoplasm that can involve any site of the body. it can occur as an exclusively extramedullary form or it can be associated with an acute myeloid leukemia (aml), a chronic myeloproliferative neoplasm (mpn) or a myelodysplastic syndrome (mds) at onset or at relapse. the rarity of ms does not enable prospective clinical trials and therefore a specific multicenter register can be useful for the clinical and biological studies of this rare disease. we report the clinical characteristics and outcome of 48 histologically confirmed ms, diagnosed and treated in 9 italian hematological centers in the last 10 years. the patient's median age was 46 years. there were 9/48 de novo extramedullary ms, 24/48 de novo aml-related ms and 15/48 were secondary aml-related ms. the most common extramedullary anatomic sites of disease were: skin, lymph nodes and soft tissues. forty-three patients (90%) underwent a program of intensive chemotherapy including flai, hdac-ida, hypercvad and mec schemes, with a cr rate of 44% (19/43). twenty-two (46%) patients underwent allogeneic sct, 13 from a mud, 8 from an hla-identical sibling donor and 1 from an haploidentical donor. the median os of the whole population (48 pts) was 16.7 months. the os probability at 1, 2 and 5 years was 64%, 39% and 33%, respectively. the os was better in patients that underwent an intensive therapeutic program (median os: 18 months vs 5 months). among the intensively treated patients, in univariate analysis, the os was better in young patients (p = 0.008), in patients that underwent allo-sct (p = 0.009) and in patients that achieved a cr during treatment (p = 0.001), and was worse in pts with secondary aml-related ms (p = 0.007). age, response to intensive chemotherapy and allo-sct were the only three variables that significantly influenced dfs (p = 0.02, p = 0.01 and p = 0.04, respectively). in multivariable analysis, allo-sct and response to intensive chemotherapy remained significant in predicting a better os (p = 0.04 and p = 0.001, respectively), and response to intensive chemotherapy was the only significant variable in predicting dfs (p = 0.01). after allo-sct we observe a survival advantage in patients who achieved a pre-transplant cr (p = 0.008) and in those who developed a chronic gvhd (p = 0.05). patients with ms, both with de novo and secondary forms, still have a very unfavorable outcome and require an intensive therapeutic program, that includes allo-sct, whenever possible. the outcome after allo-sct is positively influenced by the development of chronic gvhd suggesting a graft versus ms effect. disclosure of conflict of interest: none. relapse of acute lymphoblastic leukemia (all) after allogeneic stem cell transplantation (sct) is associated with poor prognosis. blinatumomab may enhance the efficacy of donor lymphocyte infusions (dli) in this specific situation but data on the concurrent use of dli and blinatumomab are sparse. the patient presented here was diagnosed with standard risk pre-b-all (presence of t(3;9); bcr-abl and cd20 negative) at the age of 23. during treatment according to the german multicenter all-study group (gmall) protocol he presented with molecular relapse and 8 months after initial diagnosis he received a tbi-based myeloablative sct from an unrelated hla-identical (10/10) donor. post sct he was negative for minimal residual disease (mrd) with 100% donor engraftment. given the high relapse risk he received 3 prophylactic dli without occurrence of graft-versus-host disease (gvhd). one year after 1st sct he presented with an extramedullary (testes) and molecular relapse. after remission induction resulting in negative mrd he received a 2nd sct from an alternative, hlaidentical (10/10) donor after reduced intensity conditioning. this again resulted in negative mrd with 100% donor chimerism without any gvhd. six months after 2nd sct he presented with bone marrow relapse. we decided on the concurrent use of blinatumomab and dli. the first cycle of blinatumomab was initiated at standard dose including dose escalation without relevant toxicities. on day 40 of the 2nd cycle, i. e. in the infusion-free interval before the 3rd cycle the patient received the first dli at 1x107 cd3/kg. no toxicities or gvhd occurred. the 3rd cycle of blinatumomab was initiated and a second dli at 2.5x107 cd3/kg was applied on day 3 of the 3rd cycle. on day 32 of the 3rd cycle, i. e. day 29 after 2nd dli the patient presented with signs of overlap gvhd (mouth, skin) and topical steroids were started. upon progression of clinical gvhd systemic steroids were initiated with immediate response. steroids were rapidly tapered and a 4th cycle of blinatumomab was started. gvhd did not recur. current staging after the 4th cycle blinatumomab, i.e. on day +413 after 2nd sct and 7 months after initiation of blinatumomab treatment revealed complete remission with negative mrd, 100% donor chimerism and no signs of extramedullary relapse. counts of cd4-cells at that time point were 147/μl. no relevant infections or relevant blinatumomab-associated toxicities were present during the entire course after the 2nd sct. in this case concurrent treatment of blinatumomab and dli resulted in the longest disease-free interval for our patient compared to preceding chemotherapy or dli alone. together with the small number of reported cases (ueda et al.) this supports the concept of concurrent blinatumomab and dli as an effective post sct treatment. the objective of the study is to evaluate the clinical efficacy and safety of decitabine (dac) in combination with haag regimen [homoharringtonine (hht), cytarabine (ara-c), doxorubicin (acla) and recombinant human granulocyte colony stimulating factor (g-csf)] for advanced patients with acute myeloid leukemia (aml). thirty-six patients with advanced aml receiving dac combined with haag chemotherapy in our center from december 2012 to august 2015 were enrolled in this study. eighteen of them were refractory or relapsed aml, and another 18 patients were those who didn't achieve complete remission (cr) after a course of induction chemotherapy. the therapeutic responses, side effects and longtime survival were retrospectively analyzed. after a course of treatment, the rate of cr and partial response (pr) was 58.3% (21/36) and 22.2% (8/36) respectively, while the overall response rate (orr) was 80.6% (29/36) in the cohort. for the patients with refractory or relapse aml, cr was 61.0% (11/18), pr was 22.2% (4/18), and orr was 83.3% (15/18). while for the other not getting cr after a course of induction chemotherapy, cr was 55.6% (10/18), pr was 22.2% (4/18), and orr was 77.8% (14/18). grade 4 hematological toxicities were observed in all patients, and 72.2% cases experienced infection. and all non hematological side effects were mild and well-tolerated. with a median follow-up of 7.5 (0.5~33.3) months, the 1-year overall survival (os) rate was 43.3%, 24.2% for the refractory or relapsed aml patients, and 61.6% for those not achieving cr after a course of induction chemotherapy. the difference was significantly (p = 0.01). conclusion dac combined with haag regimen is safe and effective salvage treatment for advanced stage aml patients. disclosure of conflict of interest: none. aml patients harboring flt3-itd mutation are associated with decreased survival compared to patients without flt3-itd mutation. nevertheless, whether flt-itd mutation also has negative impact on the post-transplant survival is less clear. for flt3-itd mutated aml, a decreased leukemia-free survival (lfs) after allogeneic hsct was observed in ebmt analysis but not cibmtr. in this study, unlike studies of ebmt or cibmtr which only pre-specified populations of patients were analyzed (cr1 in ebmt, cr1+cr2 in cibmtr), we examined the prognostic impact of flt3-itd mutation on post-transplant outcome of "all" the adult aml patients reported to taiwan bone marrow transplant registry (tbmtr). tbmtr is a research collaboration affiliated to the taiwan society of blood and bone marrow transplantation. it comprises all the 14 transplantation centers in taiwan that contribute detailed data on hsct. adults aged ⩾ 18 years with a diagnosis of aml and with known flt-itd mutation status in the registry were included. patient characteristics and transplant outcome following allogeneic hsct for flt3-itd mutated and nonmutated aml were compared. kaplan-meier estimates were used to calculate the probability of lfs and overall survival (os). multivariable analyses for lfs and os were performed using cox proportional hazards model. 365 patients who met the eligibility criteria were enrolled for analysis. the median follow-up of survivors was 21 months. of the 365 patients, 94 (25.8%) were positive and 271 (74.2%) were negative for flt3-itd mutation. flt3-mutated patients had significantly more transplantation at cr1 (57.4%), shorter time interval between diagnosis and hsct (5.6 months), and higher wbc count at diagnosis (51.7 × 10 9 /l) comparing to patients without flt3 mutation (43.5% at cr1, 6.5 months from diagnosis to hsct, and 11.8 × 10 9 /l wbc count at diagnosis). significant more flt3 mutated patients had intermediate-risk (80.9%) and normal (64.9%) karyotype at diagnosis. the age, donor type, stem cell source, conditioning regimen, and atg use were not significant different between flt3-mutated and non-mutated patients. of the whole population, flt3 mutation status did not negatively impact the transplant outcome (2 years os for flt3 mutated and non-mutated patients: 45.2% vs 50%, log rank p = 0.624; 2 years lfs for flt3 mutated and non-mutated patients: 40.2% vs 32.4%, log rank p = 0.192). when different pre-transplant conditions (cr1, subsequent cr, and no cr) were analyzed separately, flt3-itd mutation status is still not a significant prognostic factor of os and lfs for patients in cr1 (equally good) and no cr (equally bad). however, for patients in subsequent cr, flt3-itd mutation is the only significant factor predicting poor os and lfs in multi-variable analysis (median os and lfs for flt3 mutated and nonmutated patients: 378 vs 1252 days, log rank p = 0.005; 204 vs 1049 days, log rank po 0.001 respectively). the incidence of non-relapse mortality, grade 3/4 acute gvhd and extensive chronic gvhd is comparable between flt3-mutated and nonmutated patients. flt3-itd mutation is a significant and strong predictor of poor survival for aml patients in subsequent cr at hsct. for flt3-itd non-mutated aml, a sizable portion of patients can have disease free survival after allogeneic hsct at subsequent cr. however, allogeneic hsct at cr1 should be strongly recommended for flt3-itd mutated aml. [p523] disclosure of conflict of interest: none. allogeneic stem cell transplantation (asct) is a curative strategy in acute myeloblastic leukemia (aml) and myelodysplastic syndrome (mds). however, relapse keeps being the main cause of treatment failure. extramedullary relapse (er) is a rare event and its management is not well standardized. we retrospectively analyzed patients who received asct from 2006 to 2016 and developed er in our centre. we performed a descriptive study to analyze characteristic of these patients, post-relapse treatment and survival. statistic analysis was performed using spss v.22. we found a total of 18 patients with er, one of them with 2 er after 2 consecutive asct, so we analyzed 19 cases of er. patient and transplant characteristics are summarized in table 1 . at day +100, 95% of patients were in complete response (cr). er occurred after a median of 13 (2-98) months post-asct. eleven patients (58%) presented with a bone marrow relapse concomitant with the er. er affected central nervous system (cns) in 7 patients (36.8%), bone in 4 patients (21%), skin or soft tissue in 3 patients (15.8%), mama in 2 patients (10.5%), ocular globe in 2 patients (10.5%) and teste in 2 patients (10.5%). two of them presented with multiple sites affected. between the 7 patients who developed cns relapse, 2 of them had received intrathecal prophylaxis. regarding post-er management, immune modulation was conducted in 16 patients (immunosupression tapering in 9, donor lymphocyte infusions in 4 and both strategies in 3). all patients except one received systemic treatment (salvage chemotherapy in 11, azacitidine in 5, low dose arac in 1 and atra in 1 patient with a promyelocytic leukemia). together with systemic treatment, 12 received radiotherapy and intrathecal therapy was used in all 7 patients with cns involvement. response: 12 out 18 patients treated, 12 (63.2%) achieved cr and 6 (31.6%) progressed. two responding patients received a 2 nd asct. after a median follow-up of 67 months (15-123), 8 patients are alive and disease free, with an estimated overall survival of 45% at 5 years. patients receiving salvage chemotherapy followed or not by a 2 nd asct experienced a significantly better os than those receiving other therapies (median os 92 vs 10 months; p = 0.005). patients with bone marrow involvement at relapse show a worse prognosis (median os 39 vs 54 months; p = 0.23) although not statistically significant due to small number of patients (image 1). ten patients died due to disease progression. er must be considered in patients receiving an asct in case of organ symptoms. patients can be rescued with salvage chemotherapy followed or not by a 2 nd asct achieving good results in terms of long term os. it seems that involvement of bone marrow at relapse confers a worse prognosis, what should be confirmed in a larger series of patients. [p524] disclosure of conflict of interest: none. flag-ida regimen as bridge therapy to allotransplant in refractory/relapsed aml patients: a single-center experience c pasciolla, m delia, d pastore, p carluccio, a ricco, a russo rossi, a mestice, f albano and g specchia university of bari, italy although treatment outcome in acute myeloid leukemia (aml) adult patient has improved over the past decade, relapse still occurs in up to 50-70% of cases. furthermore, 15-30% of patients fail to achieve complete remission (cr) because of treatment-resistance. the management of primary refractory and/or relapsed disease remainschallenging for clinicians. in our study, we reviewed the outcome of 116 refractory and/or relapsed aml patients who underwent salvage therapy with the flag-ida regimen between 2005 and 2015 at our institution. the study aim was to determine the efficacy of the flag-ida regimen in order to clarify which variables (who ps, ldh, bone marrow, peripheral blood blasts and platelets counts, white blood cells (wbc), pmn, molecular-cytogentic risk, duration of response and relapsed or refractory disease), present before starting flag-ida treatment, might have an impact both on cr and on os. we analyzed 116 consecutive adult patients (56 males, 60 females; median age 48 years, range 17-72) with newly diagnosed acute myeloid leukemia refractory to standard induction regimens or relapsed after cr, who received the flag-ida protocol as salvage therapy between january 2005 and december 2015. sixty-eight of the 116 patients (58%) were in first relapse, forty-seven patients (42%) were refractory to conventional chemotherapy. median wbc count before salvage therapy was 10.1 x109/l (range 0.56-88). median bone marrow and peripheral blasts counts were 52 and 20%, respectively; median platelets count was 91x10e3/μl. according to the fab classification, 14 patients had m0, 5 m1, 53 m2, 16 m4, 22 m5, 4 m6, 2 had biphenotype acute leukemia. according to molecular-cytogenetic risk stratification 51 (44%), 44 (38%) and 21 (18%) patients belonged to poor, intermediate and good risk group, respectively. sixty-nine of 116 patients (59%) achieved complete remission (cr); forty-seven 41%) patients were refractory to the salvage therapy. in multivariable analysis, variables with positive impact on response rate were lower wbc counts (o10e3/μl, p = 0.0047), higher platlets counts (450x10e3/μl, p = 0.046), molecular-cytogenetic risk (p = 0.032), duration of response in relapsed aml (p = 0.006) and relapsed rather than primary refractory disease (p = 0.042), respectively. median os was 17 months (m). cox regression analysis confirmed that both higher platlets counts, p = 0.002 (17 (450x10e3/μl) vs 11 m (o50x10e37ul), log rank, p = 0.05) and relapsed disease, p = 0.041 (23 (relapsed) vs 17 m (refractory), gehan-breslow, p = 0.021) correlated with better survival. of note, molecular-cytogenetic risk evaluated before starting treatment was associated with cr, while no correlation was found with os. our data seem to confirm the value of flag-ida in relapsed amland may suggest its best usage as bridge-therapy in patients awaiting allotransplantation. disclosure of conflict of interest: none. s395 leukemia relapse is the major cause of death in patients received allogeneic hematopoietic stem cell transplantation (allo-hsct). the precise etiological mechanisms of leukemia relapse remain unclear. both leukemia cells themselves and hematogenesis micro-environment are involved in the relapse event. in our previous study, we reported a case of donor derived relapse of acute myeloid leukemia (aml) after allo-hsct. the patient and his donor-sister both harbored a germline mutation(c.584-589dup) in cebpa gene. donor hematopoietic cells transformed to aml by developing two somatic cebpa mutations (247dupc and 914-916dup) in the patient's microenvironment. hence we suspect that 584-589dup mutation of cebpa gene may altered hematopoiesis microenvironment and increased the survival of aml cells. to conform our hypothesis, we transfected mesenchyme stem cells (mscs) with cebpa 584-589dup or wide type and took vector as control. aml cell line hl60 cells were co-cultured with transfected mscs and then treated with 40ng/ml doxorubicin. apoptosis and cell cycle were detected at day 3. mscs protected hl60 cells from toxicity of doxorubicin. this protection was enhanced by overexpression of cebpa 584-589dup . apoptosis rates of hl60 cells in group of msc-vector and msc-cebpa 584-589dup were 61.7 ± 5.8% vs 30.9 ± 5.6% (p＜0.05). a larger part of hl60 cells remains quiescent with s396 higher rate of g0/g1 phase in msc-cebpa 584-589dup group, which may reduce the sensibility of hl60 cells to doxorubicin. to explore mechanisms involved in the alteration of microenvironment, we performed rna sequence with each group of mscs. we found that col1a1, col1a2 and col3a1 were upregulated in msc-cebpa 584-589dup group compared with msc-cebpa wt group (col1a1:cebpa wt vs cebpa 584-589dup was 1713.65 vs 2317.88, p = 4.70e-19; col1a2:cebpa wt vs cebpa 584-589dup was 2260.02 vs 2755.81, p = 2.76e-10; col3a1: cebpa wt vs cebpa 584-589dup was 746.20 vs 964.82, p = 1.06e-06). furthermore, we found that ddit3 and herpud1 genes, which were important factors in cellular unfolded protein response(upr) and to topologically incorrect protein, failed to augment in cebpa 584-589dup group (ddit3 : vector vs cebpa wt the cure rate of childhood acute lymphoblastic leukemia (all) has improved considerably and approaches 80% today. however, the outcomes of patients who suffer from leukemic relapse remain unsatisfactory. despite the high cure rate of children and adolescents with all a subgroup of patients benefit from allogeneic hsct. allo hsct remains the standard treatment for intermediate/high risk aml patients. 59 patients, all = 42 and aml = 17 age 1 to 20 years with median age 11 years, m/f = 33/26(m/f all = 15/18, aml = 8/9) underwent sct in our hospital (from 2012 to 2016). fifty-eight patients transplanted allo hsct and 1pt aml auto hsct. conditioning regimens consisted of busulfan (iv) +cyclophosphamide for allo and cyclophosphamide + vp16 +cytarabine for auto hsct. peripheral blood (pb) was the source of progenitor cells in 47 patients, bone marrow (bm) in 11 patients and cord blood in one patient. in allo hsct, 50 patient transplanted 6/6 matched and 8 patients 5/6 matched. gvhd prophylaxis regimen was cyclosporine + mtx. all patients engrafted. in allogeneic pbsct all patients' median time to absolute neutrophil count (anc) 40.5 × 109/l was 12 days, and the median time to platelet count 420 × 109 was 15 days vs 17 and 21 days in allo bm all patients. in allogeneic pbsct aml patients median time to anc 40.5 × 10 9 /l was 12 days, and the median time to platelet count 420 × 109 was 14 days. (all patients with aml transplanted with pb). at present 47 pts are alive (36 all, 11 aml) and 12 pts died due to ards, vod, hemorrhagic stroke, sepsis and relapse. trm was 9% at 100 days. median time of death after transplantation was 195 days in all and 153 in aml. in allo pbsct all patients hospitalization period were 36 days vs 45 in allo bm all patients. acute gvhd appeared in 78% pts. chronic gvhd appeared in 55% pts. with a median follow-up of 35 months (3-51 months) after transplant the event-free survival were 73% and four years overall survival 75% in all patients. a median follow-up of 32.5months (4-48months) after transplant the event-free survival were 68% and three years overall survival 63% in aml patients. hematopoietic stem cell transplantation can lead to durable remissions in children and adolescents with leukemia and increase in survival of children. pbsct in childhood all was consistent with significant faster anc and platelet recovery in allogeneic pbsct, hospitalization was shorter. longer follow-up is required to evaluate fully efficacy and long-term results. disclosure of conflict of interest: none. group hla-c1/c2 subtypes were defined as previously practiced. median age of patients was 41, 47% of them were male. allo-hscts were performed from 60% unrelated donors vs 40% related donors. remission status was detected in 67% of patients whereas 12% had active disease pre-transplant. stem cell sources were as follows: 88% peripheral blood, 7% bone marrow, 3% cord blood, 2% bone marrow plus peripheral blood. the most frequent fab subtype was aml-m4. patients were grouped by hla-c status: 23% c1/c1 homozygote, 57% c1/c2 heterozygote and 20% c2/c2 homozygote. the frequency of hla c donor/recipient mismatch allo-hscts was 19%. relapse was detected in 26% of patients. the relapse risk was significantly lower in c1/c1 homozygote patients compared to c2/c2 homozygotes (13% vs 36%, p = 0.02). lfs was similar between c1/c1 homozygote group and c2/c2 homozygote group (p = 0.324) (figure 1 ). in multivariate analysis (age, sex, remission status, related/ unrelated transplant, aml subtype), lfs was increased by pre-transplant remission status (p1 year). there was no difference detected between 2-years os in c1/c1 homozygote group and other groups (57% vs 50%, p = 0.51) (figure 2) . when similar analysis were repeated with donor hla type results were not significant. our results confirm two earlier published reports on aml and all. even in the absence of kir genotyping, hla group c1 has a protective effect. if hla matched donor is not possible a donor-recipient hla-c mismatch favoring c1 to c2 may be preferable. disclosure of conflict of interest: none. immunomodulatory kits do not induce aml-blasts' proliferation ex vivo: ipo-38 is an appropriate and reliable marker to detect and quantify proliferating blasts c plett, dc amberger, a rabe, d deen, z stankova, a hirn, y vokac, j-o werner, j schmohl, d krämer, a rank, c schmid and h schmetzer aml-blasts can be converted to dcleu by immunomodulatory 'kits' (ex vivo). t-cells' energy can be overcome after stimulation with dc/dcleu and results in antileukemic reactivity. a potential induction of blast-proliferation (e.g. by immunomodulatory kit-application in vivo) in aml-pts has to be excluded. 8 kits containing combinations of gm-csf with 1-2 additional factors (pge-1/2, picibanil, ifnα, tnfα, calciumionophore) were studied with respect to the generation of dc/dcleu from blasts, mediation of antileukemic reactivity (after dc/dcleu-stimulation) and with respect to their potential to induce blast-proliferation in a whole blood (wb) culture-system. we studied 3 different markers (ipo-38, ki-67, cd71) and quantified blast proliferation before/after culture. we correlated findings with (ex vivo) antileukemic functionality, with disease-entities and the course of the disease. dc-generation: we could generate dc/dcleu regularly from wb culture from 36 aml-pts. detection of blast proliferation: ø65.6 % (range46-82%) of uncultured blasts expressed ipo-38, 33.1% (16-50%) cd71, 25.4% (8-43%) ki-67. induction of blast proliferation: pooling all results we found lowest amounts of proliferating blasts after culture with kit i (gm-csf+picibanil, 10% ± 13.32), kit k (gm-csf+pge2, 9.14% ± 12.01), kit m (gm-csf+pge1, 7.67% ± 11.79). amounts of proliferating blasts were lower compared to uncultured cells. highest expression of proliferating blasts was found with ipo-38 followed by cd71 and ki-67.we found few individual aml-samples with increased blast-proliferation after ex vivo kit-culture. antileukemic activity: t-cells stimulated with dcleu (generated with kits) improved antileukemic activity. correlations between blast-proliferation and antileukemic activity will be presented. clinical correlations: pts with bad (vs good) cytogenetic risk were characterized by higher proportions of proliferating blasts in uncultured blasts; in some pts with iron-deficiency anemia (ida) proportions of cd71+unculured blasts were lower than of ipo-38/ki-67+ blasts. ipo-38 is a stable marker to be used to quantify proliferating blasts in aml-pts. cd71 is also a good marker, although not suitable for some pts with ida, ki-67 is no reliable marker for every given pt. subtypes of pts correlated with proportions of proliferating blasts. in general kit treatment of blasts did only exceptionally induce blast proliferation ex vivo. in general lowest risk for blast proliferation was seen after culture with kit i, k and m. t-cellstimulation with dc/dcleu generated after kit-treatment resulted regularly in antileukemic reactivity. we conclude that an in vivo treatment of aml-pts with kits i, k or m might be safe (no induction of blast proliferation). disclosure of conflict of interest: none. the occurrence of additional cytogenetic abnormalities (acas) is common in philadelphia chromosome-positive acute lymphoblastic leukemia (ph+ all), but is of unknown significance in the tyrosine kinase inhibitor era. recent study [aldoss et al., 2015] has revealed the acas appear to have a significant deleterious effect on outcomes post-hsct in adult ph+ all patients only. we retrospectively analyzed data from adult and pediatric patients with ph+ all who had undergone allogeneic hematopoietic stem cell transplantation (allo-hsct) at our university between 2008 and 2015. among 65 patients with ph+ all, 53 patients had available data on conventional cytogenetics before allo-hsct. all patients and transplant characteristics are listed in table i . thirty-three of 53 patients (51%) had isolated t(9;22). acas were revealed in 20/53 (31%) pts, including 13/53 (20%) pts with ⩾ 3 cytogenetic abnormalities (with complex karyotype, ck). the median follow-up was 645 (26-2461) days. overall survival (os) and event free survival (efs) were 48% (95% ci 7-33) and 30% (95% ci 17-44) at 4 years, respectively. in univariate analysis, prognostic factors associated with increased os and efs were donor type (match related/match unrelated vs haploidentical; p = 0.02 for both), the disease status at transplant (cr1 vs beyond cr1; p = 0.01, only for efs), acas (aca-vs aca+; p = 0.04, only for os) and, especially, the complex karyotype (ck-vs ck+; p = 0.01, only for os) (figure 1 ). multivariate analysis showed that the independent prognostic factors for os and efs remained the complex karyotype (hr-2.79, 95% ci, 1.23-6.34; p = 0.01) and disease status at transplant (hr-2.15, 95% ci, 1.13-4.09; p = 0.01), respectively. the study demonstrates the acas and disease status at allo-hsct to be independent prognostic factors not only for adult, but for pediatric ph+ all patients too. up to 20% of newly diagnosed acute myeloid leukemia (aml) patients (pts) present initially with hyperleukocytosis consequently placing them at increased risk for morbidity and mortality during induction. 1,2 whereas early publications 3 have indicated that hyperleukocytosis is an adverse prognostic factor associated with poor long term outcome, it is currently unknown whether hyperleukocytosis still retains prognostic value for aml patients undergoing allogeneic stem cell transplantation. furthermore, it is unknown whether hyperleukocytosis retains prognostic value when modern molecular markers such as flt3 and npm1 are accounted for. we hypothesized that hyperleukocytosis at initial diagnosis is still an independent adverse prognostic factor influencing long term outcome in aml pts undergoing allogeneic stem cell transplantation. we performed a retrospective analysis using the multicenter registry of the acute leukemia working party (alwp) of the european society for blood and marrow transplantation (ebmt). pts included in the analysis were over 18 years of age, with de-novo non-m3 aml, a presenting white blood cell count of over 100k, with an hla matched related or unrelated donor, transplanted between 2005 and 2014. clinical outcome indices of hyperleukocyotosis pts namely, non-relapse mortality (nrm), graft versus host disease (gvhd), relapse incidence (ri), leukemia free survival (lfs), overall survival (os) and gvhd-free/relapse-free survival (grfs) were compared to a cohort of pts without presenting leukocytosis. multivariate analyses were used to assess whether hyperleukocytosis was independently associated with ri, nrm, os, lfs, and grfs. age, gender, number of chemotherapy inductions, cytogenetics, donor type, fms-like tyrosine kinase-3 (flt3) status, nucleophosmin (npm1) status, and conditioning intensity were covariates for regression modeling. a cohort of 357 pts with hyperleukocytosis (159 patients with wbc over 50k and less than 100k, and 198 patients with wbc over 100k) was compared to 918 pts without hyperleukocytosis. pts with hyperleukocytosis were younger, had an increased rate of favorable risk cytogenetics, were more likely to be flt3 and npm1 mutated, and had an increased rate of myeloablative conditioning. on univariate analysis pts with hyperleukocytosis had an increased rate of ri (30% vs 22.7%, p = 0.013), and decreased incidence of grfs (36.6% vs 45.3%, p = 0.022). in multivariate regression analysis, hyperleukocytosis was significantly associated with increased ri (hazard ratio [hr] of 1.55, 95% confidence interval [ci], 1.145-2.124; p = 0.004), s399 poorer lfs (hr of 1.38, 95% ci, 1.071-1.785; p = 0.013), decreased grfs (hr of 1.38, 95% ci, 1.117-1.71; p = 0.002), and poorer os (figure 1) (hr of 1.4, 95% ci, 1.073-1.846; p = 0.013). image/graph hyperleukocytosis at initial presentation retains a significant prognostic role for aml patients undergoing allogeneic stem cell transplantation even in the current era of advanced molecular prognostication. outcome after hematopoietic stem cell transplantation for philadelphia-positive aml: relatively favorable outcome in patients allografted in first complete response; a survey from the acute leukemia working party of the european society for blood and marrow transplantation (ebmt) v lazarevic 1 , m labopin 2 , w deipei 3 , i yakoub-agha 4 , a huynh 5 , p ljungman 6 , n schaap 7 , d blaise 8 , jj cornelissen 9 , n maillard 10 , p pioltelli 11 , t gedde-dahl 12 , s lenhoff 1 , m houhou 2 , j esteve 13 , m mohty 2 and a nagler 2,14 aml with t(9;22) and bcr-abl rearrangement (ph-pos aml) is a very rare aml subtype, recognized as a new provisional entity in the recent who 2016 classification. the role of stem cell transplantation (sct) in the era of abl tyrosine-kinase inhibitors (tkis) is mostly unknown. we analyzed long-term outcome in patients ⩾ 18 years after allogeneic or autologous sct performed between 2000-2013 in ebmt centers responding to a designated survey. patients with blast crisis cml and philadelphia-positive all were excluded. primary end-point was os. secondary end-points were nrm, acute gvhd, chronic gvhd, lfs, ri, and the effect of tki on outcome. 65 patients (median age, 48 years, range: 19-67; 31 males and 34 females) with ph-pos aml undergoing sct (allogeneic, 57; autologous, 8) were identified. median wbc count at diagnosis was 57x10 9 /l (1.2-366) and 25% had splenomegaly (data missing on 10 patients). translocation t(9;22) was the sole abnormality in 36 patients (55.5%). the majority of the patients received one or two courses of chemotherapy before transplant and 79% attained cr after one course. the majority (70%) received a tki (mostly imatinib, 34/43) before transplant, with a median period of exposition of 86 days (iqr 60-173), while 21 (34%) received tki after the transplant either as maintenance (n = 11) or treatment for relapse (n = 9). at time of transplant, 56 patients were in complete response (cr1-53, including all autosct; cr2-3) and the remaining 9 patients were allografted in advanced phase. among 40 patients with available information, 14 achieved a mrd negative status at transplant (ratio bcr-abl/abl o10 4 ).regarding allosct, conditioning regimen was myeloablative (mac) in 29/30 (96%) patients o50 years, while in patients 450 years 15 received a reduced intensity regimen (ric) and 9 mac. cell source was peripheral blood stem cells in 39 and bone marrow in 18 allogeneic transplants. the donor was a hla matched sibling (msd) in 32 cases and unrelated (ud) in 25, amongst whom 18 were 10/10 and 7 were 9/10 hla matched, respectively. in the 8 patients undergoing autologous sct the majority received busulfanbased conditioning (n = 6) and peripheral stem cells (n = 7). median follow-up was 6. 36.4%, 46.5%, 59.4%, and 34.9%, respectively. by the univariate analysis, age ( o50 vs ⩾ 50) was associated with ri (5-yr: 22.7 vs 50%), lfs (5-yr: 61.9 vs 31.8%, and grfs (5-yr: 52.4 vs 18.2%), whereas mrd-negative status before allosct was associated with an improved grfs (38.9 vs 16.7%). in the 8 patients autografted, ri, nrm, lfs and os at 5-year was 50% (12.5-79.4), 0%, 50% (15.4-84.6), and 62.5% (29-96), respectively. outcome of patients with ph-pos aml who received allosct in cr in recent years was relatively favorable, especially among younger patients, probably reflecting the beneficial effect of tki. disclosure of conflict of interest: none. outcome of allografting for aml-cr2 is equivalent across the bsbmt and ebmt and is associated with encouraging os and dfs across all age groups j byrne, j perry 1 , k kirkland 1 , r pearce 1 and g jackson 2 1 bsbmt and 2 newcastle university and freeman hospital, newcastle relapsed aml has a very poor prognosis with a high mortality, even if a second cr2 is achieved. the only curative treatment is with an allogeneic hsct but allografts for aml in cr2 are considered to have a worse outcome compared to those performed in cr1, especially in older patients for whom this therapy may not be considered. the bsbmt undertook a bench-marking study analysing the outcomes for all patients allografted for aml-cr2 from 2006 to 2011. the uk outcomes from 534 paediatric and adult patients were compared to 3070 non-uk patients transplanted for the same indication reported to the ebmt during the same period. allogeneic transplants for aml-cr2 represent an important part of any allograft program and numbers referred for allograft were stable between 2006-2011 in both programs. the median age of patients was 45.8 yrs and 43.3 years in the bsbmt and ebmt cohorts respectively, with 14% and 15% of patients aged o 18 years and 19% and 16% aged 4 60 years in the 2 groups. the length of first remission was missing in many of the ebmt registrations so time from diagnosis to transplant was used as a surrogate for this and was similar in both cohorts (18 m and 19 m respectively). similarly the presence of comorbidities was poorly reported in both databases but was similar. the bsbmt cohort included fewer patients undergoing ric conditioning protocols (55% vs 66%), fewer sibling transplants (25% vs 38%) and more pbsc allografts (79% vs 20%). transplant related morbidity and mortality were similar across the two cohorts (bsbmt v ebmt) with rates of severe acute gvhd (grade iii and iv) 6% v 10%, limited chronic gvhd 35% v 28%, relapse at 1 year 22% v 20% and death in cr at 1 year 20% v 18%. chronic gvhd (both limited and extensive) appeared more common in the bsbmt cohort (50% v 36%) although reporting of cgvhd was more comprehensive and complete within the bsbmt registry and may be under-reported in the ebmt registry. outcomes were excellent in both cohorts with outstanding rates of leukaemia free survival in this high risk cohort at day 100 (bsbmt v ebmt) 86% v 83%, at 1 year 63% v 67%, at 3 years 49% v 53% and at 5 years 41% v 43%. although os and lfs was significantly shorter in patients aged 4 60 years, at 37% and 30% the results in this high risk age group are acceptable and warrant its continued use. multivariate analysis of the combined cohorts showed that age at transplant ( o 18 yrs/18-60/460 yrs), time from diagnosis to transplant and the presence of agvhd were important factors affecting dfs. risk factors for relapse included the type of conditioning used, presence of agvhd and time from diagnosis to transplant, whereas those for trm included age, agvhd, source of stem cells and time to transplant. there was no significant difference in outcomes between the bsbmt and ebmt for this indication. outcomes for patients allografted for aml-cr2 are excellent and appear superior to those reported for patients not undergoing an allograft in both the bsbmt and ebmt cohorts. the os and dfs observed are comparable to those reported for allografts in aml-cr1 and, although this study has not considered outcomes for patients who did not achieve a 2 nd cr, it nevertheless supports the practice of risk stratification of aml patients such that only high risk patients are offered an allograft in cr1 with the remainder being offered an allograft as salvage after relapse. disclosure of conflict of interest: none. to evaluate the efficacy and safety of intensive chemotherapy followed by allogeneic hematopoietic stem cell transplantation (allo-hsct) for atl. a total of 21 evaluable atl patients treated at our center from 1995 to 2015 are retrospectively analyzed. the htlv-i proviral dna load in peripheral blood mononuclear cells using pcr assay was developed, in comparison with htlv-1 tax protein expression measured by western-blot, to confirm the diagnoses and monitor the disease control. 13 patients were male and 8 patients were female. median age was 50.8 (range 28-66) years. all obtained patient samples were subjected to flow cytometric examination and karyotype analysis. 19 patients received chemotherapy as the induction therapy while 2 quit at the time of diagnosis, 5 with da-edoch regimen while 14 with other regimens such as chop, vcap and amp. da-edoch regimen is a variation of dose-adjusted epoch regimen with the replacement of prednisone (60 mg/m 2 per day) by dexamethasone (15 mg/m 2 per day). before the conventional regimens bucy followed by prophylaxis donor lymphocyte infusion, both received a course of salvage chemotherapy including fludarabine and cytarabine for 5 days registered on http://clinicaltrials.gov (nct02328950). the gvhd prophylaxis consisted of atg, csa and mtx. the patient characteristics, therapeutic effect and survival data were collected. all patients came from the coastal area in the south-east of china. subtype classification of these 21 atll were 18 acute, 2 lymphoma and 1 chronic type. the main manifestations were characterized with cutaneous and respiratory involvement, hepatosplenomegaly, lymphadenopathy and the laboratory abnormalities as leukocytosis with atl cells, hypercalcemia and elevated serum ldh. typical morphological characterisitic of "flower cells" were observed in 85.7% cases and most of the atll cells are cd4+cd8-. chromosomal abnormalities were detected in 4 cases. all 14 patients who didn't receive da-edoch regimen died of disease progress, while among 5 patients with da-edoch regimen, 2 achieved cr, 2 pr and 1 died. with a median follow-up of 18.6 (10-24.1) months, 3 patients respond to da-edoch are still alive. 2 patients in cr achieved successful engraftment with complete donor chimerism in one month post haplo-identical transplant. both were received prophylaxis donor lymphocyte infusion and the immunosuppressive agents were abruptly discontinued for induction of a graft-verus-atl (gvl) effect. they keep remain alive and disease free longer than 2 years so far without severe graftversus-host (gvhd), and the htlv-1 proviral dna became undetectable after allo-hsct. conclusion: it shows great promise of da-edoch regimen followed by allo-hsct to the long-term cure of atl with apparent clearance of the virus. haplo-identical transplantation can be an alternative option for the atl patients without increasing non-relapse mortality. [p537] disclosure of conflict of interest: none. in the absence of an hla-matched related donor or a good matched unrelated donor in time, haploidentical stem cell transplantation (haplo-sct) is an option for patients requiring an allogeneic hematopoietic stem cell transplant. substantial progress has been made in the last two decades with a dramatic improvement in patient outcomes, with some groups reporting preliminary beneficial effects similar to the ones in hla matched unrelated donor and cord blood transplant. several strategies have been adopted through the years for the procedure. the two strategies used in haplo-sct are ex vivo t-cell depletion and t-cell replete transplantation. the latter can be done with a combination of immunosupressive drugs ( beijing approach) or with post-transplantation highdose cyclophosphamide (post-cy). due to of its lower cost, feasibility and practicality, post-cy has become the most often used platform for haplo-sct in the majority of allogeneic transplantation units worldwide. we analyzed our experience in haplo-sct, since the first one in march 2104 to the last one that has just been done in october 2016. we collected all complications reported, also mortality related to treatment and to the disease. we analyzed the overall survival (os). 9 transplants were treated, with different sct indications, the most common being acute myeloblastic leukemia (n = 5, 55%), the rest of indications are exposed in figure 1. all our patients, independent of the conditioning receive post-cy as t cell depletion measure and stem cells were collected from peripheral blood. age at the time of transplant was 40.6 years, 77% were males, 23% females, the rest of patient characteristics are listed in table 1 . in our series the treatment related mortality (trm) was low with only 1 patient (11%) that died before the day +100. as complications, we reported 33% of hemorrhagic cystitis, 22% of sinusoidal obstruction syndrome, 22% reports systemic inflammatory response syndrome, 55% of citomegalovirus (cmv) reactivation. neutrophils graft is 18.2 days (r = 14-23) and the platelets graft is 24.2 days (r = 13-34). in our series we haven't reported any case of graft failure, one of the transplantes the patient had antihla antibodies, this was treated with a plasmapheresis previous the stem cell infusion, and was infused with a high number of cd34+ cells (8 × 10 6 /kg), no graft problems, and has had no complications since then with the graft. the os for the whole group is 22 months, with a median not reach at 36 months, with 2 patient's dead at time of analysis. two patients had a relapse after the haplo-sct (22%), both of them received lymphocyte donor infusion, sadly, neither of them responded. the haplo-sct procedure is been adopted by many centers for high risk hematology malignancies, mainly because the fast availability of donors, and because of the preliminary results that have been reported place it as good as the unrelated donor or cord unit transplant. our center is getting experience in these types of transplants, and our results reflect similar outcomes as larger studies. with longer follow ups we will be able to keep the trend of good results both in procedure safety and disease efficacy. os, toxicity and trm are expected for these high risk malignancies. in overall, the haplo-sct seems a reasonable technique that is reflecting in our short series, the results being reported in studies worldwide at bigger scale. disclosure of conflict of interest: none. complex karyotype, the presence of flt-3 itd and losses of genetic material in chromosome 7, are all considered high risk markers in aml. patients bearing these abnormalities could undergo a myeloablative allogeneic transplantation in cr1 whenever this is possible, although this could significatly reduce the chances for cure in older patients due to increased transplant related mortality. ric allografts could be performed in older patients in order to overcome the deleterious effects of these individual abnormalities but its effect still remains controversial in the high risk group. between 2005 and 2015, a total 65 patients [41 males, 24 females, mean age 59.05 (40-73)] received a ric allograft (49 from fully matched unrelated donors, and 16 from an identical sibling) for high risk aml in 2 transplant centres. high risk disease was classified according to their response to treatment, the presence of complex karyotype, the presence of individual cytogenetic/molecular abnormalities or a combination of these. in particular, 24 patients presented one single karyotype abnormality and 17 presented three or more. ten patients presented alterations of chromosome 7 and 7 patients presented flt-3 itr. the conditioning regimes included fludarabine in all cases, together with melphalan and campath (29 patients), busulphan and campath (21 patients), busulphan and thiotepa (1 patient), melphalan (3 patients), busulphan with and without atg (9 patients) total body irradiation (200 cgy, 2 patients). graft versus host disease prophylaxis was ciclosporin for patients receiving alemtuzumab or atg but for patients who had a t-replete allograft ciclosporin and low dose methotrexate was the preferred prophylaxis. all but four patients were transplanted in cr1 patients were followed-up for a mean 32.3months (range 3-150). thirty-one (47%) patients remain alive. the causes of death (34 cases) include relapse or progression of the original disease (13), transplant-related causes (17) and unknown in 4 cases. the influence of the genetic abnormalities on survival was analysed, showing there were no significant differences between patients with normal karyotype, single chromosomal abnormalities and two or more abnormalities. likewise, the kaplan-meier survival analysis of patients bearing flt-3 itd was not significantly different to the rest of the cohort (p = 0.4; 3/7 died, with only one case being related to relapse). patients bearing chromosome 7 abnormalities (with or without other chromosomal aberrations) had a comparable, not significantly (p = 0.6) different survival to the rest of the cohort: 6/10 patients bearing this abnormality died although, most interestingly, none of these deaths were related to relapse. our results indicate ric allografts can provide an adequate consolidating effect in hr aml with complex karyotype, alteration of chromosome 7 or flt-3 itr, yielding clinical results that are comparable to those obtained in patients with aml allografted for other indications. this is particularly important as these alterations are more frequent in patients whose age prevents them from having myeloablative grafts. disclosure of conflict of interest: none. early detection of inapparent replicative human cytomegalovirus (hcmv) infection together with its preemptive antiviral treatment has led to a marked reduction of life-threatening hcmv disease after allogeneic hematopoietic stem cell transplantation (allosct). we first reported in a retrospectively performed study that hcmv reactivation is associated with a reduced risk for relapse in patients with aml after transplant. now, we evaluate the impact of early hcmv replication on the risk for leukemic relapse in patients with aml after t cell depleted transplantation in a prospectively performed observational study (registration trial drks00004300). between january 2012 and march 2015 we enrolled 251 patients with aml in this trial who were consecutively transplanted at the university hospital of essen. 239 patients received a myeloablative regimen (tbi based conditioning n = 83, chemotherapy based conditioning n = 156) and 12 patients a ric (n = 12 chemotherapy based regimen). patients were transplanted in 1.cr (n = 123), 2.cr (n = 51) or more progressive disease stages (n = 77) from hla-identical sibling donors (n = 60) or hlaidentical unrelated donors (urd) (n = 126) or mismatched unrelated donors (n = 65). patients who received a second transplant were excluded from the study. the median age of patients was 54 years (range 18-72) and that of the donors 35 years (range 14-69). gvhd prophylaxis was performed with mtx and csa, or csa and mmf with (n = 182) or without atg (n = 69) (30-60 mg total dose). the incidence of acute gvhd grade 2-4 was statistically not different in both groups (46% versus 40%). hcmv-reactivation (hcmv-r) detected by pcr occurred between 23 and 87 days (median 45 days) after allo sct. only patients surviving day 60 after transplant were included in the study for estimation of relapse incidence (cir) or overall survival (os). hcmv status of recipients (r) or donors (d) were in 29% r-/d-, 12% r-/ d+; 28% d+/r-and 31% r+/d+. patients with a documented hcmv-r had a cir at 4-year after transplant of only 30% as compared to 47% in patients without a hcmv-r (p = 0.016). estimates for 4-year os were in favor for patients with hcmv-r (61% for patients with hcmv-r versus only 48% for patients without hcmv-r), but this did not achieve statistical significance. non-relapse mortality was greater in patients with hcmv reactivation (23% versus 12%, ns) a substantial and independent reduction of relapse risk associated with early hcmv replication was confirmed by multivariate analysis including competitive factors as unfavorable cytogenetics according to eln, advanced disease stages of aml, hla-identical donor versus mismatched donor, sibling versus unrelated donor, presence of acute gvhd grades ii-iv, chronic gvhd, and hcmv-r [(hazard ratio: 0.61, 95% ci: 0.38-0.98, p o0.042) for occurrence of hcmv-r]. the final result of this first prospective performed study confirms an independent advantageous effect of early hcmv replication on the leukemic relapse risk in patients with aml after transplant. disclosure of conflict of interest: none. . primary engraftment of wbc and platelets was achieved in 64 pts, one patient (pt) died at day +3 after hsct, and one had a secondary graft failure. a median time to wbc engraftment was 13 days (9-27), to platelets -14 days (9-32). cumulative incidence (ci) of acute gvhd (agvhd) grade ⩾ ii was 25% (95% ci:16-38): from haplo-23%(12-45), from mud 26% (15-45),p = 0.7. ci of agvhd4iii 8%(95% ci:3-18): haplo vs mud -7% (95% ci:2-25) vs 9%(95% ci:3-25), respectively,p = 0.7. ci of chronic gvhd (cgvhd)-16% (95% ci:9-28). ci of agvhd was significantly lower in a group with regimen 2 (2014-2016) of gvhd prophylaxis: 10% (95% ci:4-25) vs 32%(95% ci:18-57), po 0.0001. regimen 2 was also effective in prevention of cgvhd: ci at 2 year after hsct was 10% vs 24%, p = 0.14. сi of trm was 12%(95% ci:6-24): haplo-7%(2-25), mud -14% (6-32). early mortality (before +100-day) was relatively low (n = 3): 1 pt died from bacterial sepsis; two pts died due to adv infection. thirteen pts died after 100 days: 9 pts relapsed and died due to complications of second hsct; bacterial sepsis in 1 pt and viral infection (adv and cmv) in 3 pts (2 with extensive chronic gvhd). ci of relapse was 24% (95% ci:15-38) at 2 year: from haplo-12% (95% ci:4-36), from mud-32% (95% ci:20-52),p = 0.086. event-free survival (efs) at 2 years was 64% (95% ci: 52-77): haplo -81%(95% ci: 66-97), mud -54% (95% ci:37-70), p = 0.06. os was 70% (95% ci: 58-82) at 2 years: haplo-77% (95% ci: 57-77), mud -65% (95% ci: 48-81), p = 0.3. relapse-gvhd-free survival at 2 years was different among recipients of haplo and mud hsct: 51%(95% ci: 28-75) vs 32% (95% ci: , p = 0.3. we confirm that the depletion of tcrαβ+/cd19+ depletion from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric aml pts. there is a trend towards better efs for haploidentical transplantation. gvhd prophylaxis including ratg/rituximab/bortezomib improves gvhd control s404 in recipients of tcrαβ+/cd19+depleted grafts in comparison to hatg/tacro/mtx apparently without loss of anti-leukemic activity. disclosure of conflict of interest: none. results of t-cell depleted haploidentical stem cell transplantation in adults with acute leukemia improve with time: a study from the acute leukemia working party of the european society of blood and marrow transplantation (ebmt) s simona 1,2 , m labopin 3 , a ruggeri 1,3,4 , a velardi 5 , f ciceri 6 , j maertens 7 , l kanz 8 , f aversa 9 , d bron 10 , d bunjes 11 , m mohty 1,3 and a nagler 3, 12 t cell-depleted (tcd) transplants from haploidentical donors are increasingly used in the absence of a hla full-matched donor for patients (pts) with high risk acute leukemia (al). progress has been made in optimization of conditioning regimens and post-transplant cellular therapy to potentiate the graft-versus-leukemia effect with no graft-versus-host disease (gvhd). however, relapse incidence (ri) and non relapse mortality (nrm) remain the main obstacles for pts outcomes. we report 308 adults with de novo al, given a tcd haplo from 2005 to 2015. to analyze the effect of transplant period on tcd haplo outcomes, pts were analyzed in two separate groups: 2005-2011 (n = 191) and 2012-2015 (n = 117). tcd haplo were performed in 66 ebmt centers. median follow-up was 11 months with no difference according to transplant periods. median age was different between groups, being 41 and 46 years respectively (p = 0.044). the majority of pts had acute myeloid leukemia (aml) (75% vs 69%, p = 0.261) and disease status at haplo was first complete remission (cr1) in 55% and 64% of pts respectively (p = 0.115). pts transplanted before 2012 had more frequently a karnofsky performance status o90% (19% vs 10%, p = 0.041). conditioning was myeloablative in 76% and 77% of tcd haplo before and after 2012 (p = 0.935), mainly based on fludarabine(flu)-tbi, flu-melphalan-thiotepa or cyclophosphamide-tbi. as for ric it was flu-melphalan-thiotepa, flu-tbi or cyclophosphamide-tbi. the cumulative incidence (ci) of neutrophil engraftment, grade ii-iv acute gvhd and chronic gvhd were not different according to transplant period, being 93% and 90%, p = 0.302; 20% and 22%, p = 0.667; 19% and 11%, p = 0.119, respectively. in the whole population 2-year ri and nrm were 20% and 48%, with no difference before and after 2012 (21% vs 19%, p = 0.722; 54% vs 38%, p = 0.109, respectively). ri was 20% before 2012 versus 15% after 2012. the main cause of nrm was infection, with no difference over time (46% vs 50%, p = 0.316). in multivariate analysis, disease status was the only factor associated with ri (hr 2.05, 95% ci 1.09-3.84, p = 0.025). tcd haplo after 2012 (hr 0.57, 95% ci 0.38-0.86, p = 0.008), younger age (hr 0.82, 95% ci 0.63-0.98, p = 0.023) and ric (hr 0.53, 95% ci 0.32-0.88, p = 0.014) were independently associated with lower nrm. 2-year os was 36% with a marked improvement for tcd haplo performed after 2012 (47% vs 29%, p = 0.024), while lfs and grfs were 31% and 24%, respectively. according to disease status, 2-year lfs, os and grfs were higher for pts transplanted in cr1 (38% vs 22%, p o0.001; 41% vs 29%, p = 0.006; 30% vs 17%, respectively p = 0.004). in multivariate analysis, tcd to further establish the role of haplosct in high-risk aml, we performed a retrospective matched-pair comparison of hlamatched sct vs haplosct/ptcy in two german centers. highrisk aml was defined by any of the following criteria: refractory or relapsed aml, secondary aml, or genetic aberrations classified as intermediate-high or adverse accordingly to the eln classification. all consecutive adults, who fulfilled ⩾ 1 of these criteria before either hla-matched or haplosct/ptcy were included (n = 200). recipients of haplosct were pair-matched with patients receiving a matched donor sct. matching variables were (1) stage at sct, (2) genetic subgroups accordingly to eln, and (3) age (±5y). 39 patients (pts) undergoing haplosct/ptcy could be successfully pairmatched (p = 1.0 for stage and genetic subgroup, 0.9 for age) with 39 recipients of matched sct (12 family, 27 unrelated sct). within the entire cohort, median patient age was 57y (24-70). at start of conditioning, 20% of patients were in cr, 18% had refractory, 52% had relapsed, and 10% had untreated disease. genetics were favorable (16%), intermediate i (51%), intermediate ii (12%) and unfavorable (21%). hla-matched sct was uniformly performed following flamsa-ric. 34 recipients of haplosct/ptcy (87%) received cytoreductive chemotherapy with flamsa (n = 16) or clofarabine (n = 18) before ric was started. median follow-up among survivors was 33 months. overall cr rate at d+30 was 95%, 4 patients suffered from refractory disease or early death, (n = 2 each). overall-survival (os) for the entire cohort was 74%/53.9% at 1y/3y from sct. the corresponding 1y/3y leukemia-free survival (lfs) was 63.5%/46.6%. median time to engraftment was 18.0 and 17.5 days after matched and haplosct, respectively (p = 0.8). with respect to outcome, no difference was observed between the two groups: os at 1y/3y was 78.5%/54.5% after matched sct, and 61.5%/55.2% after haplosct/ptcy (p = 0,71, figure 1 ). lfs at 1y/3y was 76.2%/ 42.6% within the hla-matched group and 56.4%/50.8% within the haploidentical group (p = 0,99). recipients of haplosct showed a higher incidence of agvhd ⩾ ii°(46% vs 18%, p = 0.014), as well as a trend towards increased 1y-nrm (18% vs 5%, p = 0.08), whereas 1y-relapse rates were comparable (23% after haplosct/ptcy vs 26% after matched sct, p = 0.5). relapse was the most frequent cause of death in both cohorts, main causes of nrm were gvhd and infections (no difference between the two groups). allogeneic sct following sequential conditioning can achieve excellent results in high-risk aml. in our study, results after haploidentical transplantation were comparable to those obtained after hla-matched sct. hence, haplosct/ptcy following sequential conditioning can be considered as a reasonable option for patients with high-riaml. [p544] disclosure of conflict of interest: none. a significant proportion of patients with acute myeloid leukemia ( aml) will either be refractory to initial chemotherapy or will suffer refractory relapse. the role of allogeneic transplantation (hct) in active disease is contentious. there is a growing body of literature that sequential chemotherapy, pioneered by the german flamsa regimen, followed by ric hct is a safe and efficacious modality in these patients, and there have been numerous modifications of this regimen, especially as amsacrine is not widely available. fludarabine, cytarabine and etoposide (vp16) (flav) have been reported as an an effective salvage regimen. here we report on single center outcomes of a variation of the flamsa regimen, substituting amsacrine for etoposide with mainly myeloablative conditioning. patients were consented for a clinical protocol if they had aml that was refractory to 2 cycles of chemotherapy, or 1 cycle and considered to be at risk of complications of a second cycle, and if they had a matched related donor. patients with myelodysplasia received flav if they had high or very high risk cytogenetics. cytoreductive chemotherapy consisted of fludarabine 30 mg/m 2 / day × 4 days, cytarabine 2g/m 2 /day × 4 days, etoposide 100 mg/m 2 /day × 3days, commenced simultaneously. after 3 days of rest, conditioning chemotherapy consisted of fludarabine 30 mg/m 2 × 2 days and and iv busulfan 0.8 mg/ m 2 q 6 hours; the number of busulfan doses varied between 8-12, depending on patient comorbidity. ten patients (76%) received myeloablative doses of busulfan. patient received 2 doses of atg at 2.5 mg/m 2 /day on day -3 and -2. patients received gcsf mobilized peripheral blood hematopoietic cells. post-transplant gvhd prophylaxis was csa and mmf. csa was tapered from day+60 and stopped at day +90 in the absence of gvhd. mmf was discontinued between day +30 and day +40. donor lymphocyte infusions were collected for planned prophylactic dli. thirteen patients received a flav-sct between march 2014 and october 2016. the median age was 39(15-57); male:female ratio was(7: 6). 10 patients (77%) had aml and 3 (23%) pts had mds. all patients had detectable disease prior to flav. the median time for plt engraftment was 19 days (9-50). the median time for anc engraftment was15 days (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) . cytogenetic cr rate on a day 28-30 bone marrow was 46%, and morphological cr was 60%.3 patients (23 %) developed veno-occlusive disease. acute gvhd grade ii-iv occured in 4 pts ( 30%). 3 (23%) patients developed chronic gvhd. death was due to disease relapse in 5 (38%) and nrm in 1(7.7%) patients, resulting from h1n1 pneumonia. 5 patients (38%) received dli for post transplant relapse, and one of these is in molecular remission. at a median follow up of 4.8 months post transplant (1.6-29m), 1 year dfs was 23%. the 1 year and 2 year os was 51% (±14%) (figure 1 ). our experience, consistent with published data, demonstrates that for patients with active aml refractory to chemotherapy, transplantation is an effective modality of disease control and may be the only curative therapy in a significant proportion. etoposide appears to be a suitable alternative to amsacrine. our patients tolerated busulfan at myeloablative doses, and this may be required for adequate disease control. our report is limited by small numbers and relatively short follow-up, but is encouraging enough for us to continue offering flav hct for these high-risk patients. [p545] disclosure of conflict of interest: none. sequential high-dose chemotherapy reinduction followed by haploidentical transplantation in acute leukemias l brunello 1,2 , cm dellacasa 1 , l giaccone 1,2 , e audisio 3 , d ferrero 3,2 , s d'ardia 3 , b allione 3 , s aydin 3 outcomes after t-cell replete haploidentical stem cell transplantation (haplo hsct) have been encouraging and haplo hsct has become an alternative option for patients without a hla-identical related or unrelated donor. 1,2 as previously published, the sequential use of intensive chemotherapy and allogeneic transplantation represents a possible approach to the treatment of high-risk acute myeloid leukemia (aml). 3, 4 between 2010 and 2015, 19 acute leukemia (al) patients received sequential therapy (s.t.) consisting of high-dose chemotherapy reinduction and haplo hsct during the chemotherapy-induced neutropenia. median age at transplant was 50 years (range: 21-62); median number of previous therapy lines was 2 (range: 1-3) and 5/19 (26%) patients had received a previous allogeneic hsct. twelve and 5 out of 19 patients had de-novo aml and secondary aml, respectively; furthermore two patients presented blastic crises of chronic myeloid leukemia. all patients had active disease at the time of s.t. and median marrow blast count before reinduction was 25% (range: 6-88%). hematopoietic cell transplantation comorbidity index was ≥ 3 in 12/19 patients (63%). all patients received high-dose cytarabine (≳1 g/sqm) containing regimens as reinduction therapy. the conditioning regimen was started after a median of 9 days from the end of reinduction (range: 4-15). sixty-eight percent of patients (13/19) were conditioned with a myeloablative regimen (thiotepa tot. 10 mg/kg, busulfan tot. 9.6 mg/kg, fludarabine tot. 150 mg/ sqm), while 6/19 (32%) patients received a non-myeloablative conditioning. bone marrow was used as stem cell source in 18/19 (94%) patients. graft-versus-host disease (gvhd) prophylaxis consisted of post-transplant cyclophosphamide with calcineurine inhibitors and mycophenolic acid. all patients engrafted but one, who was rescued with a second haplo-hsct with peripheral blood stem cells from the same donor. median day of neutrophil recovery was day +18 (range: 14-24). median follow-up of survivors was 4.2 years (range: 1.6-5.4); 1-year overall and event-free survivals were 37% and 32%, while 1-year relapse incidence and non relapse mortality were 42% and 26%, respectively. overall cumulative incidences of acute and chronic gvhd were 39% and 38% at day +100 and +400. among patients who developed gvhd, 2 grade iii-iv acute gvhd and 2 moderate-severe chronic gvhd were observed. at 4.2 years post haplo-hsct, 20% of patients are alive and disease free. in our cohort of heavily pre-treated and high-risk patients, s.t. with a myeloablative conditioning was safely used to reduce leukemic burden pre-transplant and enhance graft-versus-leukemia effects. only the prompt availability of a haploidentical donor allowed to implement this treatment modality. though small the patient cohort, our findings suggest that transplant-related toxicity was acceptable and early relapse was the major treatment-failure. however, long-term survival and disease-free rates of 20% in these very poor prognosis patients are highly encouraging. in elderly patients with acute lymphoblastic leukemia (all) and kinase activating lesions allogeneic stem cell transplantation (allo-sct) is considered to be the only curative option. 1 however, high risk of relapse and non-relapse mortality (nrm) often withholds elderly patients with existing comorbidities from definitive therapy. 2 even though, age alone as the most important eligibility criterion for allo-sct has become less important. 3 a 61-year old patient with a medical history of adipositas, hypothyreosis, arterial hypertension, hypercholesterolemia and coronary artery disease with stent implantation was diagnosed with common-b-cell-all based on immunophenotype. cytogenetic analysis showed two coexisting clones with an abnormal karyotype of 47,xx; t(1;9)(q22;q34), +17 [3] and 46,xx; t(1;9)(q22;q34),der(6)(6;17)(p23;q21) [2] and no evidence of bcr-abl1 positive disease using fluorescence in situ hybridization (fish) technique. rt-pcr and sequencing of the fusion transcript was performed to validate the rcsd1-abl1 t(1;9)(q22;q34) fusion between exon 3 of rcsd1 and exon 4 of abl1. western analysis of phosphorylated abl1 and its downstream target crkl was performed to investigate the in vivo activity of dasatinib. clinical monitoring of minimal residual disease (mrd) levels has been performed via rt-pcr of the rcsd1-abl1 fusion transcript followed by nested pcr of the amplicon to detect early relapse or mrd. as positive control the plasmid pcr2.1-topo_rcsd1-abl1(626bp) was synthesized encoding rcsd1-abl1 amplicon with the fusion site. our patient was enrolled on gmall elderly (01/2003) and treated accordingly. thereby, no sustained remission could be achieved. flag-ida re-induction and study treatment with an oral pi3k/mtor inhibitor remained futile. cytogenetics and verification of the rcsd1-abl1 fusion gene prompted salvage treatment with the tyrosine kinase inhibitor (tki) dasatinib as single agent. the in vivo activity of dasatinib was highlighted by a decrease of rcsd1-abl1 amplicon and inhibition of phosphorylated abl1 and its downstream target crkl was shown. clofarabine and cyclophosphamide complemented treatment and mrd negative remission was achieved due to administration of the bi-specific t-cell engager blinatumomab. consolidation with allo-sct was performed. ongoing remission has been achieved for more than 30 months now. we demonstrate that monotherapy with tki like dasatinib is effective in refractory rcsd1-abl1 positive all. to the best of our knowledge, this is the first elderly patient with rcsd1-abl1 positive all with a sustained and ongoing complete remission. thereby, we suggest allo-sct after successful tki even for elderly patients with existing comorbidities and uncommon cytogenetics. relapse is the most important cause of failure of allogeneic hematopoietic stem cell transplantation (hsct) for flt3-itdpositive acute myeloid leukemia (aml). in those cases, treatment with flt3 tyrosine kinase inhibitors (tki) constitutes a promising clinical approach to induce remission without conventional chemotherapy. forty-eight-year-old woman was diagnosed of aml secondary to myelodisplastic syndrome with npm1 mutation and internal tandem duplications of the flt3 gene (flt3-itd). after achieving complete remission (cr), she received a sibling allogenic-hsct. four months later, aml relapsed at the medullary level, without central nervous system (cns) involvement, treated with conventional chemotherapy and donor lymphocytes infusions (dli). she achieved second cr and developed chronic graft-versus-host disease (cgvhd). nine months later, she suffered the first extramedullary relapse, at the mammary, cutaneous and probably pericardial levels. there was not medullary involvement. disappearance of the lesions at all levels was achieved with conventional chemotherapy and radiotherapy, and full donor chimerism. eight months later, she referred atypical precordial pain irradiated to the back. cardiac mri was performed in which several masses were visualized in a pericardial sac up to 5 cm in diameter (dec-15). bm was maintained in cr. in study of pericardial fluid, infiltration by leukemic flt3 positive cells was observed. the patient was not considered candidate for further systemic chemotherapy nor radiotherapy treatment. then, treatment with the flt3 sorafenib inhibitor was started, by compassionate use, at dosage of 200 mg/12 h, which maintains after one year. after first month with sorafenib, pericardial lesions decreased considerably, ranging from 5 cm in diameter to 1.7 cm (jan-16). in the subsequent ct controls, progressive decrease of the lesions has been observed and no new lesions have appeared in other locations. in the last ct (oct-16) pericardial thickening is almost non-existent, without new lesions. after one year of treatment, she maintains cr at medullary and extramedullary levels images. in our patient, treatment with sorafenib has achieved sustained control of extramedullary disease, which had escaped the mechanisms of action of conventional chemotherapy, allotransplant and dli. further studies are needed to corroborate the efficacy of flt3 inhibitors in the control of aml extramedullary disease and in the treatment of relapses after allo-hsct. all pts with tbi-based regimen received rabbit atg. tcrαβ+/cd19+ depletion of hsct with clinimacs technology was implemented in all cases according to manufacturer's recommendations. the median dose of cd34+ cells in transplant was 10 × 10 6 /kg (range: 3.9-18.8), tcrα/β-23 × 10 3 /kg (range: 0.2-300). primary engraftment was achieved in 68 of 71 pts. (2 pts died before engraftment, one received second hsct), the median time to neutrophil and platelet recovery was 13 and 14 days, respectively. early (100 day) mortality was 7%(95% ci: 3-17), 2-year ptrm-17% (95% ci: 10-30). the three early deaths were due to bacterial sepsis (n = 2) and viral infections(n = 1), seven late: viral infection in 4 pts (adv = 2, adv+cmv = 1, cmv = 1), bacterial sepsis in 2 pts and rhinocerebral mucormycosis in 1 pt, all late deaths were associated with cgvhd and prolonged corticosteroid therapy. ci of acute gvhd grades ii-iv and iii-iv was 22.5% (95% ci: 9.6-53), and 7% (95% ci: 1.3-37), respectively. ci of cgvhd was 17.6% (95% ci: 6.4-48). regimen 2 was more effective in prevention of agvhd ii-iv: ci at 2 year after hsct was 14% vs 35,7% in regimen 1, p = 0.033 and in cgvhd 7% vs 35.7%, p = 0.006. ci of relapse at 2 years was 32% (95%ci:15.7-64.5). two-year pefs(event = death or relapse) was 49.9% (95% ci: 37-62), 2-year pos-54% (95% ci:41-67). in patients, who received tbi-based conditioning pefs was 59% (95% ci: 38-81), as compared with treosulfan-based 47% (95% ci: 32-61), p = 0.65. median time of follow-up for survivors was 2.2 years (range: 0.3-4.4). we confirm that the depletion of tcr-alpha/ beta and cd19 lymphocytes from the graft ensures high engraftment rate and acceptable transplant-related mortality in pediatric all patients. viral infections and leukemia relapse await further improvement of control. all major outcomes were equivalent between transplantation from unrelated and haplo donor. disclosure of conflict of interest: none. the prognosis of patients (pts) with relapsed/refractory acute lymphoblastic leukemia (all), especially after allogeneic hematopoietic stem cell transplantation (allo-hsct), is very poor. therapeutic options for these pts are limited. blinatumomab is a bispecific t-cell engager (bite) antibody construct with dual specificity for cd19 and cd3. bite therapy may help to overcome the resistance to chemotherapy (ct) with minimal toxicity, and may be a bridge to allo-hsct. we analyzed data of 34 pts from 4 hematologic centers in russian federation with relapsed cd19 positive all, who received bite. the median age was 22 (range: 1-62) years, 9 (26%) pts o18 yrs, 25 (74%) pts ≥ 18 yrs. the diagnosis was all b-i (egil) subtype in 11 pts, b-ii-in 16, b-iii-in 5 pts, and 1 patient had mixed phenotype leukemia (m5 (fab) and b-all). three (9%) pts had philadelphia positive (ph+) all. in 11 pts it was the first relapse of all, in 13-second, in 10-third. thirty pts had isolated bone marrow relapse, 4 pts-combined relapse (bone marrow and extramedullary sites). the bone marrow blast infiltration was o 50% in 15 pts, 450% in 19 pts. disease relapse was revealed after ct in 19 pts (7 (37%) pts received allo-hsct after the therapy of relapse), after allo-hsct (3-from related, 9-from unrelated, 3-from haploidentical donors)-in 15 pts (4 pts received second allo-hsct after the therapy). in 8 pts with posttransplant relapse donor lymphocyte infusion (dli) was used in combination with bite. every patient received from 1 to 7 cycles (median 2) of bite. complete remission (cr) was achieved in 18 (53%) pts (in 14 (41%) pts it was minimal residual negative remission): in 8 (42%) pts with all relapse after ct, in 10 (67%) pts-after allo-hsct. pts with less than 50% blasts in bone-marrow at baseline experienced substantially higher response rates compared with patients with 50% blasts or higher (67% (10/15) vs 52% (10/19)). response rates were similar although the number of relapse-45% (5/11) in first relapse, 61% (8/13) in second relapse and 70% (7/10) in third relapse. pts with posttransplant all relapse who received bite in combination with dli had higher response rate than pts, who received bite as monotherapy: 87.5% (7/8) vs 43% (3/7), respectively. one-year os was 50% (95% ci 23-77%). one-year dfs was 48% (95% ci 21-75%). grade ≥ 3 hematological toxicity (neutropenia, thrombocytopenia) was observed in 13 (38%) pts, grade ≥ 3 liver toxicityin 4 (12%) pts. five patients (15%) developed toxic neuropathy during the therapy. cytokine release syndrome occurred in 3 (9%) pts. one patient after allo-hsct (but not after dli) developed grade i agvhd. there were no fatal treatment related toxicity. tree (17%) pts who responded to bite had relapse. eighteen (53%) pts died: 14 pts-of disease relapse/ progression. the treatment of relapsed/refractory all with bite is effective and has acceptable toxicity. we demonstrated high efficacy in therapy of posttransplant all relapses, especially when bite was combined with dli, perhaps, due to additional immunological effect of the transplant. disclosure of conflict of interest: none. the mechanism of sorafenib anti-leukaemic effect seen in aml patients relapsing post allohsct involves the augmentation of alloreactivity which includes infiltration of the affected marrow by cd8+ cells having pd-1 receptor which presence characterize lymphocytes with antitumour potential multikinase inhibitor (mki) sorafenib is clinically active in acute myeloid leukaemia (aml) patients, especially in those with flt3 itd who receive allohsct as a part of their primary treatment. to throw some light on the mechanism of this antileukemic post-transplant sorafenib effect we studied the fate of two patients (flt3 itd-positive, npm1-positive) who relapsed 56 (53-year-old male) and 256 (50-year-old female) days post-transplant and their salvage treatment included sorafenib. the multikinase inhibitor (400 mg twice daily) was given either together with flag or da 2+5. the response was prompt. the patients became, after completion of the chemotherapy, leukaemia free. both patients continued the sorafenib (200 mg twice daily) treatment together with the aml standard maintenance chemotherapy (female case) or without any chemotherapy (male patient, substantial comorbidity and liver toxicity). (1) . the patients responded well to the therapy and were free of leukaemia for 16 and 32+ months after initiation of the mki treatment (flt3 itd negative, 100% chimerism documented in the blood and in the marrow). (2) . in both patients, 3 and 25 months on sorafenib, skin lesions appeared either in the context of cgvhd, which progressed to a life-threatening level in a male patient or as a photodermatitis-like cheek eruption. histopathology revealed the presence of severe cd3+ cells infiltration in affected tissues in both patients. (3) . cd8 positive lymphocytes colonized the marrow of both patients. these marrows infiltrating cells co-expressed cd279 (pd-1 receptor) in proportions which were higher than those seen in the blood (14.72% ± 1.45% vs. 3.63% ± 1.21%, p = 0.002). a similar observation was made for cd8+cd69+ cells (37.26% ± 3.50% vs. 1.58 ± 0.43%, po 0.002). 5. transcriptome analysis of the marrow cells, which addressed the genes involved in lymphocyte activation, revealed the presence of proinflammatory profile which included a higher expression of tlr9 and il-12. (1) sorafenib given with or without moderate chemotherapy was effective in two patients in maintaining the anti-leukaemic effect of salvage chemotherapy. (2) this was associated with the presence of alloreactivity (affected tissues infiltration with cd3+ cells) clinically seen as a severe fatal cgvhd aggravated by sorafenib treatment associated unwanted effects in one cases and with rather mild skin lesions appearing later during the treatment. the outcome of elder patients with acute myeloid leukemia or high risk myelodysplastic syndrome treated with allogeneic hematopoietic stem cell transplantation ch tsai 1,2,3 1 division of hematology, department of internal medicine, national taiwan university hospital; 2 tai-cheng stem cell therapy center, national taiwan university and 3 genome and systems biology degree program, national taiwan university allogeneic hematopoietic stem cell transplantation (hsct) is a curative-intent treatment for patients with high-risk hematologic diseases, including acute myeloid leukemia (aml) and myelodysplastic syndrome (mds). both the incidences of aml and mds increase with age and patients elder than 60 years of age were traditionally excluded from hsct because of high possibilities of therapy related morbidity/mortality. recently, with the introduction of reduced intensity conditioning regimens and the improvement of hsct care, more and more elder patients could undergo hsct for consolidation or salvage purposes. however, literature regarding the treatment outcome of elder patients receiving hsct is scarce. patients diagnosed as aml or high risk mds aged equal or more than 60 years were recruited consecutively at national taiwan university hospital. the high risk mds was defined to include myelodysplastic syndrome with excess of blasts-1 and 2 according to the 2016 world health organization (who) criteria. the cytogenetic risk stratification was based on original medical research council classification. from 2008 to 2016, a total of 51 patients were enrolled consecutively. the median age was 63.1 (range: 60-73) years and the gender distribution was even. among them, 11 (21.6%) patients had high risk mds, 27 (53%) had de novo aml, 10 (19.6%) had secondary aml, and three (5.9%) had therapy related aml. at diagnosis, four (7.8%) patients had extramedullary disease. nine (17.6%) had unfavorable-risk cytogenetics, 12 (23.5%) had either unfavorable-risk cytogenetics or intermediate-risk cytogenetics but with flt3-itd mutations. regarding the pre-hsct disease status, nine patients had the first complete remission (cr), 11 had the second cr, and 31 patients were treatment naive or had refractory disease. the graft-versushost-disease(gvhd)-free/relapse-free survival (grfs) in which events include grade 3-4 acute gvhd, chronic gvhd with moderate severity according to cibmtr criteria, relapse, or death of any cause. with median follow-up of 38 months (range: 0.8-85.2), the median overall survival (os) for all patients was 13.9 months, the relapse-free survival (rfs) was 11.3 months, and the grfs was 9.0 months. in univariate analysis for os and rfs, high-risk mds was a favorable prognostic factor but secondary or therapy related disease (p = 0.013 for os and 0.007 for rfs, respectively), unfavorablerisk cytogenetics or intermediate-risk cytogenetics but with s410 flt3-itd mutations (p = 0.005 and 0.002, respectively), pre-hsct refractory disease (p = 0.018 and 0.037, respectively), and grade 3-4 acute gvhd (p = 0.001 and 0.002, respectively) were unfavorable prognostic factors. however, for grfs, only unfavorable-risk cytogenetics or intermediate-risk cytogenetics but with flt3-itd (p = 0.020) and pre-hsct refractory disease (p = 0.018) were unfavorable prognostic factors. in multivariate cox proportional hazards regression analysis for os and rfs, grade 3-4 acute gvhd was a significant unfavorable risk factor; for gfrs, pre-hsct refractory disease status was a significant unfavorable risk factor. our results showed that the choice of hsct should not solely based on the age factor and pre-hsct disease status. incorporating cytogenetics and genetic mutation status could risk-stratify elder patients with hsct. further prospective trials are warranted to validate these findings. disclosure of conflict of interest: none. children affected with acute lymphoblastic t cell leukaemia (t-all) and relapse after allogeneic stem cell transplantation (sct) have limited treatment options and a poor prognosis. immune checkpoint inhibitors targeting the programmed death (pd-1) receptor pathway may enhance the graftversus-leukaemia (gvl) effect by blockade of inhibitory signals to t cells mediated by its ligand pd-l1. we report a 9-year old girl with refractory t-all after allogeneic sct, who was treated off-label with the pd-1 inhibitor pembrolizumab. the girl was diagnosed with t-all (8.2 g/l wbc, 82% bone marrow infiltration, cns negative, t (6;11)) and underwent hlahaploidentical bone marrow transplantation from her mother with post-transplant cyclophosphamide since she failed to achieve molecular remission despite an intensified chemotherapeutic regimen. on day 100 post sct, she had a 100% donor chimerism and decreasing minimal residual disease (mrd) marker (minimal 1 × 10 − 6 ). 140 days post sct she had a molecular relapse with an mrd of 5 × 10 − 3 and a subsequent morphological relapse as well as mixed donor chimerism. further treatment regimens included chemotherapy, intrathecal therapy and four donor lymphocyte infusions (dlis). initially, she displayed a good morphological response to dlis but the leukaemic burden eventually remained stable with an mrd of 2 × 10 − 2 . considering 54.2% pd-1 expression on cd3+ t cells in the patient's bone marrow and the encouraging data in other hematologic malignancies an off-label therapy with the pd-1 inhibitor pembrolizumab1-4 was initiated. the patient and her parents gave informed consent and she received a single dose of pembrolizumab at 3.3 mg/kg 343 days after sct. one week after administration of pembrolizumab, the patient developed acute gvhd grade iv of the skin, mucosa, liver, lung, central nervous system and eyes. she had a severe generalized inflammatory reaction with high inflammatory markers, increased hepatic transaminases and lymphocytic infiltration of the liver, cerebrospinal fluid and bronchoalveolar lavage fluid. magnetic resonance imaging (mri) of the brain revealed periventricular white matter lesions and hyperintensities of basal ganglia and bilateral temporal lobe consistent with autoimmune encephalitis. treatment with high-dose corticosteroids, cyclosporine and the anti-interleukin 6 receptor antibody tocilizumab slightly improved her clinical condition. her mrd value significantly decreased to 4 × 10 − 4 two weeks after administration and she achieved a 100% donor chimerism in bone marrow. despite this promising response her medical condition deteriorated and the severe inflammatory reaction caused fatal multi-organ failure. this is to our knowledge the first report on a remarkable and fast response to pd-1 inhibition in a patient with pediatric t-all refractory to multiple lines of therapy including allogeneic sct. this case illustrates the potential risk of checkpoint inhibitors to trigger severe gvhd that is not responsive to steroids. induction of inflammatory gvl responses without causing severe gvhd by therapeutic checkpoint inhibition needs to be addressed in future clinical trials. in recent years, there is a remarkable trend in the use of haploidentical-related hematopoietic cell transplantations (haplo-hct) in patients who do not have a hla matched related or unrelated donor. here, we report our single-center experience, in patients who underwent haplo-hct for acute leukemia. between 2011 and 2016 seventeen consecutive adult patients, seven males and ten females, median age 42 years (range: 18-61 years) with high-risk acute leukemia underwent unmanipulated, bm or pbsc transplantation from an haploidentical family donor. eleven patients transplanted for acute myeloid leukemia (5 in cr1, 1 in cr2, 1 in minimal active disease after cr1, 1 second trasplant in cr2, 1 transformed mds in cr1, 2 aml secondary to myelofibrosis in cr1), 5 for acute lymphoblastic leukemia (2 in cr1, 3 in active disease) and 1 mastcell leukemia (secondary to aml) in active disease. sixteen patients received myeloablative conditioning, and 1 reduced intensity, respectively. in five patients stem cells source was bm, in 12 were g-csf mobilized pbsc. the median infused cd34+ cell dose was 4.47 × 10 6 (range: 1.0 × 10 6 -8.2 × 10 6 ). conditioning regimens were: bu-flu-mac (n = 9), tbf-mac (n = 7), tbf-ric (n = 1) the regimens for gvhd prophylaxis were: ptcy as sole gvhd prophylaxis (n = 1), mtx-csa-atg (n = 9), methylpred-atg-tacrolimus (n = 6), atg-csa-mtx-mmf (n = 1). sustained trilineage engraftment occurred in 15 patients (88%), two patients died of transplantation-related complications before day 21 after transplantation without myeloid recovery. for patients receiving bm or pbsc grafts, the median time to 4500 neutrophils recovery was 16 days (range: 10-36), and 420,000 platelets recovery was 16 days (range: 13-37). 7/15 patients (46.6%) and 2/15 (13.3%) had ii-iv and iii-iv grade of acute gvhd, respectively.the incidence of grade ii-iv cgvhd was 27%. after a median follow-up of 11 months, 4/17 patients (23.5%), 4 out 5 patients transplanted in cr1, are alive and disease free at 50, 28, 19, 16 months (inclusing the patient transplanted for aml after imf). the 2-year probability of overall and progression-free survival was 40% (95% ci, 4.0-58.0%) and 28.6% (95% ci, 2.0-20.0%), respectively. causes of death were: sepsis (n = 1 ), fatal agvhd (n = 1), pneumonia (n = 1), toxicity (n = 2), progression (n = 4) and relapse (n = 4). in our experience unmanipulated bm or pbsc transplantation from haploidentical family donor is feasible approach with high engraftment rates and acceptable trm (23%) and rate of grade iii-iv agvhd, associated with durable remission in a proportion of patients with high-risk acute leukemia, specially in patients with aml transplanted in first remission. it is generally recognized that allogeneic hematopoietic stem cell transplantation (allo-hsct) should not be administrated to patients with severe aplastic anemia (saa) or very severe aplastic anemia (vsaa), when they got active infection. however, without neutrophil, severe infection is usually difficult to control and even fatal. under these circumstances, rapid recovery of neutrophil by allo-hsct might be an alternative to control infection. from january 2002 to december 2015, there were 175 young patients received allo-hsct for saa or vsaa at shanghai children's medical center in china. among them, 22 patients (11 males and 11 females) with a median age of 7.0 years (range: 3.0-14.0 years) received allo-hsct with refractory active infections. refractory active infection was defined as persistent neutropenic fever with nonresponse to standard doses of broad-spectrum antibacterial agents and antifungal agents for more than three weeks, with or without definite focus of infection. prior to allo-hsct, four patients had persistent fever of unknown origin, 11 patients with singlesite infection, and 7 patients with multiple-site infections. sites of infection included lung, sinus, cellular tissue, peritoneum, liver, spleen and skin. the conditioning regimen consisted of fludarabine, cyclophosphamide and rabbit-antithymocyte globulin with or without total body irradiation (tbi) (2-3 gy). twelve patients were transplanted from mismatched unrelated donors, 3 from matched sibling donors, and 7 from haploidentical donors. sixteen patients received g-csf mobilized peripheral blood stem cells, three patients g-csf mobilized peripheral blood stem cells plus g-csf primed bone marrow stem cells, two patients bone marrow stem cells, and 1 patient umbilical cord blood stem cells. a median of 11.4 × 10 8 /kg mononuclear cells with 4.6 × 10 6 /kg cd34+ cells were transfused, except the patient who underwent ucbt with a total of 1.3 × 10 8 /kg mononuclear cells and 1.5 × 10 6 /kg cd34+ cells transfused. eighteen patients achieved recovery of neutrophil and finally control of infections, including one patient who suffered primary graft failure and had autologous marrow recovery. three patients died of infection and one patients died of acute renal failure before recovery of neutrophil. one patient died of pneumonia 8 months after allo-hsct. one patient become thrombocytopenia after allo-hsct. the other 16 patients are all disease-free. there were five patients developing grade i-ii acute gvhd, and 4 patient grade iii-iv acute gvhd. all were cured at last. three patients had localized chronic gvhd and one patient had extensive chronic gvhd. with a median of 2 years follow-up, the overall survival rate and disease-free survival rate are 77.3% ± 8.9% and 71.3% ± 10%, respectively. allo-hsct could be a feasible way to control infection for children with saa or vsaa in the present of refractory active infections. disclosure of conflict of interest: none. paroxysmal nocturnal hemoglobinuria (pnh) may present hemolysis isolated (classical pnh) or associated with aplastic anemia (aa; aa/pnh syndrome). while classical pnh patients require anti-complement treatment (eculizumab), the treatment of aa/pnh patients should target their underlying aa by immunosuppression (ist), or even bone marrow transplantation (bmt). however, in a few patients clinically meaningful aa and hemolysis may be concomitant, eventually justifying both ist and eculizumab. to date there is no standard treatment for s413 this rare condition. amongst a large cohort of 145 pnh patients (between 2007 and 2016) at our reference centers, st. louis hospital (paris) and federico ii university (naples), we retrospectively assessed characteristics and outcomes of patients diagnosed with aa/pnh who received intensive ist during or immediately before (3-6 months) eculizumab treatment. nine patients were identified. eight patients fulfilled the criteria of severe aa, and one had an immunemediated isolated agranulocytosis. since no patient had a hla-matched related donor for bmt, all patients received intensive ist according to institutional guidelines. six out of 9 patients were already on eculizumab treatment at the moment of starting intensive ist (concomitant treatment) whereas 3 patients received ist in the 3-6 months (median time of 3 months) before the introduction of anti-complement therapy (sequential treatment). for all patients already on treatment, eculizumab was not discontinued to minimize the risk of rebound intravascular hemolysis and thrombotic complications. eculizumab was administered at the standard dose of 900 mg fortnightly in all but one patient, who needed an increased dose (1200 mg) because of pharmacokinetic breakthrough. six patients (5 aa and 1 agranulocytosis), including the three undergoing a sequential treatment, received standard ist with horse-antithymocyte globulin (h-atg, 40 mg/kg for four consecutive days) combined with cyclosporine a (csa). the remaining three aa patients received alemtuzumab (3-10-30-30 mg subcutaneously in four consecutive days) and csa within the prospective trial nct00895739; one of these patients a few months later also received a second ist course with rabbit-atg (3.5 mg/kg for five consecutive days) and csa. all the patients completed the scheduled treatment without any side effect, including infusion-related reactions. lymphocyte depletion (o 100/μl) was observed in all patients irrespective of sustained therapeutic complement blockade. all the patients were available for response assessment at 6 months. among the six patients receiving a concomitant treatment we observed one partial response (pr) and two complete responses (cr), whereas the three remain patients were non-responders (nr). of them one was rescued with an unrelated bmt, while two remained on eculizumab treatment. one of the cr relapsed at 3 years showing clonal evolution and finally died. all the other patients are alive, keeping their hematological response. patients receiving a sequential therapy were one in pr and two in cr 6 months after introduction of eculizumab. in conclusion, for patients diagnosed with severe aa/pnh syndrome intensive ist and eculizumab treatment, can be safely delivered either concurrently or sequentially, with an overall response rate of nearly 70%. this is the first systematic description of the management of severe aa in hemolytic pnh patients receiving eculizumab treatment. disclosure of conflict of interest: none. (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) in aa, 12 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) in rcc, and 8 (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) years in rcmd. sixty-five patients underwent bmt from hla-matched (related 41, unrelated 24) and 23 from hla-mismatched (related 6, unrelated 17) donors. conditioning regimens were used as follows; cyclophosphamide (cy)+antithymocyte globulin (atg) ± total body irradiation (tbi) (n = 29), fludarabine (flu)+cy ± atg ± tbi (n = 42), and flu +melphalan (mel) ± atg ± tbi (n = 17). all patients got engraftment after bmt. however, late graft failure was found in 6 patients with rcc, and 7 with rcmd, but none with aa. out of 13 patients who developed late graft failure, 8 patients used flu+cy ± atg ± tbi, 3 used cy ± atg ± tbi, and 2 used flu +mel ± atg ± tbi for conditioning regimens. five-year cumulative incidence (ci) of graft failure was higher in rcmd (36 ± 6.0%) than in aa (0%) and rcc (20 ± 1.8%), significantly (po0.01). five-year ci of graft failure tended to be higher in flu regimen (23 ± 2.2%) than in cy+atg ± tbi regimen (10 ± 0.65%), but not significant (p = 0.20). five-year ci of graft failure did not differ between with (21 ± 2.1%) or without tbi (29 ± 6.3%) (p = 0.57). multivariate analysis revealed that the morphological classification was a significant risk factor for graft failure (po0.01). five-year failure free survival rate (63 ± 11%) in rcmd was significantly lower than in aa (96 ± 3.8%) and rcc (74 ± 7.9%) (p = 0.02). graft failure, second malignancy, and death were considered as failure events. one patient with aa died of infection, four with rcc died of infection (n = 2), bleeding (n = 1) and myocarditis (n = 1), and one with rcmd died of infection. five-year overall survival rates were not different among 3 groups (aa, 96 ± 3.9%; rcc, 89 ± 5.2%; rcmd, 95 ± 4.7%) (p = 0.53). high incidence of graft failure in rcmd may be due to higher bm cellularity than in aa and rcc. the optimal conditioning regimen of bmt should be established for children with abmf based on the bm cellularity and morphological classification. disclosure of conflict of interest: none. recent studies have suggested inferior outcome of patients treated with rabbit atg (thymoglobulin, sanofi) as compared to horse atg (atgam,pfizer or lymphoglobulin, genzyme), and a higher early mortality with rabbit atg has been suggested to explain this difference. aim: to assess early mortality, response rates at 3,6 and 12 months and long term outcome, in a large cohort of aa pts, treated in europe or asia with rabbit atg and cyclosporin, as first line treatment. eligible for this study were pts with aa, treated with thymoglobulin between 2001 and 2012 in europe (n = 519) or asia (n = 457). median year of treatment, was 2008: characteristics were comparable : median age 20 and 21 years, interval diagnosis treatment (23 and 25 days) and severity of the disease (46% and 48% with vsaa). early mortality was analyzed for all 976 patients.long term outcome was also analyzed for 800 pts for who response data (no, pr, cr) were available. mortality o90 days was 5.5% and 2.1%, respectively, in the time period 2001-2008 and 2009-2012 (p = 0.007). in these 2 time periods, early mortality for patients aged 0-60, was reduced from 3.5% to 1.4% and for patients over 60, from 22% to 9%. overall response was recorded in 800 patients. at 6 months the cumulative incidence of response was comparable in the 2 time periods: 62% vs 66%, and at 1 year, 73% vs 75% (p = 0.8). response rates at 6 months were age dependent: 68%, 66%, 62%, 40% respectively in patients aged 0-20, 21-40, 41-60, 460 (p = 0.0006). when non responders at 3 months were reevaluated at 1 year, 59% had responded, 26% were non responders, 5% had died, and 10% had received other treatment. responses at 6 months, were 60%, 66%, 74%, in pts with very severe, severe and non severe aa (p = 0.0001). the actuarial 10 year survival for the entire population was 71%, and 70%, when pts were censored as surviving at transplant. actuarial 10 year survival in univariate analysis was as follows: 89% vs 61% for day 90 responders vs non responders (p o0.01), 68% vs 80% for males versus females (p = 0.07); 82%, 72%, 66%, 27% in pts aged 0-20, 21-40, 41-60, 460 years (p o0.001); 67%, 78%, 76% in pts with neutrophils o0.2 × 10 9 /l, 02-05 × 10 9 /l and 40.5 × 10 9 /l (p o0.001); 77%, 75%, 68% for pts with an interval diagnosis-treatment of o30 days, 31-60 days or 4 60 days (p = 0.002). finally pts treated 42008 had a 5 year survival superior to pts treated before 2008 (84% vs 77%, p = 0.01). survival at 5 years, in the recent period (2009-2012), was 83% for pts aged 1-60 and 60% for pts over 60 years. in multivariate cox analysis the following variables remained independent predictors of survival: patient age, year of treatment, severity of the disease, interval diagnosis treatment, and gender. thymoglobulin +csa is effective and safe in patients with aa. the outcome is mainly age dependent. the inrerval between diagnosis and treatment remains a strong predictor: the earlier the better. for pts o60 years old current early mortality. 4. for pts 460 years of age, current early mortality is higher (9%), response rate (40%) and 5 year survival (70%) are lower. 5. the actuarial 10 year survival for the enire population was 71%. survival at 5 years has improved from 77% (o o/u42008) to 84% (2009-2012), especially for pts over 60 years (37% vs 70%, p = 0.006). [p560] s415 disclosure of conflict of interest: we thank centers for providing up date follow up of their patients . this study was supported by a grant of sanofi-genzyme. both of the patients have extremely low anc o0.05 × 10 9 /l, reto0.5%, plt o20 × 10 9 /l. both was given antibiotic treatment with carbapenem, vancomycin/linezolid, voriconazole or amphotericin b liposome and got no response. no pathogenic bacteria or fungus was found from either of the patients. both of them had no full sibling or matched unrelated donor and had their father as their haploidentical donor. bone marrow combined with peripheral blood stem cell (pbsc) was adopted. conditioning: fludarabine days − 5 through − 2, (40 mg/m 2 × 4), intravenous busulfan (1.1 mg/kg q6h) on days − 4 to − 2. gvhd prophylaxis: high-dose cyclophosphamide 40 mg/kg on days +3 and +4, mmf and tacrolimus since days +5. rabbit anti-thymocyte globulin (thymoglobulin) 2.5 mg/kg on days − 4 to − 2. stable neutrophil engraftment (anc 40.5 × 10 9 /l) occurred on day +13 and day+19 respectively. platelet achieved 20 × 10 9 /l on day +11 and day +55, respectively. both transplant course was complicated by febrile neutropenia without detected etiology, while both children have no fever since the first day anc 40.5 × 10 9 /l.the facial swelling was resolved in both patients except for palatal fistula and fistula of maxillary sinus as the sequela of severe nasosinusitis. no acute or chronic gvhd. case1 had hemorrhagic cystitis on day +30 which last for about 30 days, and suspected thrombotic microangiopathy (tma) with hypertension, thrombocytopenia, elevated ldh and creatine on day +51 which was resolved soon after discontinue of tacrolimus. case2 had delayed engraftment of platelets and herpes simplex virus 6 encephalitis on days +40 which was cured by ganciclovir and high dose intravenous immunoglobulin. now they are 9 and 7months post-hsct respectively and are doing well with 100% chimerism and no gvhd. alternative donor hsct may be considered as the first line salvage therapy for patients of vsaa with extremely low anc and active infection. fast reconstruction of neutrophil helped to control the infection. hallo-identical hsct make sure nearly every patients can find a donor. ptcy is proved to be efficient and safety in gvhd prophylaxis and facilitating engraftment in these two challenging cases. disclosure of conflict of interest: none. long-term outcome of patients with severe aplastic anemia receiving allogeneic hematopoietic cell transplantation using nonmyeloablative conditioning with fludarabine and low dose total-body irradiation l cheryl xiu qi 1 , l yeh ching 2 , p michelle li mei 1 , t lip kun 1 , h william ying khee 2 , g yeow tee 2 , t patrick huat chye 2 and k liang piu 1 1 department of haematology-oncology, national university cancer institute, singapore and 2 department of haematology, singapore general hospital, singapore allogenic haematopoeitic stem cell transplant (ahct) offers the best prospect of cure in patients with severe aplastic anaemia (saa). the use of myeloablative hct is however limited by the toxicity of preparative regimens, the lack of matched sibling donors, transplant related mortality and graft rejection. the introduction of non-myeloablative (nm) conditioning offers the possibility of extending this potentially curative treatment to patients in whom ahct was previously contradindicated. in 2006, we reported the outcome of 8 patients with saa who have received ahct using nonmyeloablative conditioning comprising of 3 days of fludarabine at 25mg/m 2 and total body irradiation at 2 gy (flu + tbi 2gy). here, we report a longer follow-up, with 6 additional patients who had recevied ahct with this regimen. fourteen patients with a median age of 37 years old (range: 17-48 years old) received filgastrim-mobilised peripheral blood stem cell transplant from either hla identical sibilings (n = 12) or matched unrelated donor (n = 2) after receiving nm conditioning consisting of flu + tbi 2gy. the first two patients received cyclosporine (cya) and mycophenolate mofetil (mmf) for the post-transplant immunosuppessive therapy. the remainining 12 patients received cya, mmf and a short course of methotrexate (mtx) for additional graft-versus-host dsease (gvhd) prophylaxis. results all patients achieved prompt engraftment. the median time for engraftment of neutrophils (40.5 × 10 9 /l) and platelets (420 × 10 9 ) were 16 days (range: 13-20 days) and 13 days (range: 8-25 days), respectively. chimerism analysis on day 28 and subsequently showed 495% donor cells in all patients except 1, who developed secondary graft failure at 3 months and required salvage hct. none of the patients experienced grade 3 and above regimenrelated toxicity. five patients developed grade ii-iv acute gvhd and 2 patients developed limited chronic gvhd. with a median follow-up of 8.8 years (range: 0.54-14.52 years), the estimated overall survival and event free survival were 86% and 79% respectively. the two patients who did not receive mtx developed acute gvhd of the liver and succumbed to infective complications. the remaining 12 patients who had received triple immunosuppressive therapy were well, with limited chronic gvhd seen only in 2. our results suggest that the nm conditioning regimen comprising of flu + tbi 2gy provides sufficient immunosuppression to allow prompt and stable engraftment with minimal regimen-related toxicity. it is an attractive option for patients with saa who require ahct but are at increased risk of regimen-related complications from more intensive cyclophosphamide-based regimens. disclosure of conflict of interest: none. paroxysmal nocturnal hemoglobinuria (pnh) is a rare acquired clonal disorder of hematopoietic cells characterized by the triad of hemolytic anemia, cytopenias and high risk of venous thrombosis. due to the rarity of the disease, most reported data derive from multicenter studies. we describe the natural history of the disease in a 30-year (yrs) long single center series of pnh patients (pts). we performed a retrospective analysis of 42 consecutive pts followed at our center from 1985 to 2016. since 1985, the diagnosis was made by ham test; starting from 2000, flow cytometry (fc) analysis was used to diagnose new pts and to confirm pnh in pts previously diagnosed by the ham test. at diagnosis, 26 pts had classic pnh, 9 aplastic pnh and 7 intermediate form. the cumulative incidences of thrombosis, cytopenia and clonal neoplasm were 39%, 18% and 10%, respectively. except for 1 pt with aplasia, no severe infections were diagnosed, nor renal failures or pulmonary hypertention. the 30 yrs overall survival (os) was 84%. a nonsignificant better os was associated to the absence of thrombotic events (96% vs 80%) and to a diagnosis made during the last decade (100% vs 90% vs 75%).up to 2005 the treatment options were supportive care or allogenic bone marrow transplantation. since 2005, eculizumab was used in transfusion-dependent patients and/or in case of a thrombotic history. overall, 14 pts were transfusion-independent for the entire period of the illness, 28 were transfusion-dependent and/or had thrombotic events(8pts). six of the latter pts never received eculizumab but only transfusion support (3 pts) or allogeneic bone marrow transplant (3 pts), while 22 pts received eculizumab (the first 4 pts were included in the phase iii triumph and shepherd trials).considering the increased risk of meningococcus infection for pts on eculizumab, vaccination with conjugated anti-meningococcus serotypes acwy was employed and, since 2016, conjugated antimeningococcus serotype b was added. overall, 18 pts treated with eculizumab became transfusion-independent and four remained transfusion-dependent. no thrombotic event was observed after eculizumab, even if 8 pts had recurrent thromboembolisms prior to receiving the drug. no severe infection was documented. one patient developed extravascular hemolysis and receive a successfully selective splenic artery embolization. the 10 yrs os in the eculizumab group was 92%.no pnh-associated death occurred. our study confirms that thrombosis is a major complication in pnh pts not receiving eculizumab, influencing os. the better os in the last decade is probably due to the use of eculizumab and to lack of thrombotic events. in particular, for 22 pts on eculizumab the 10 year os was 92%, even though half of the pts had thromboembolism and diagnosis made prior to the last decade. although kidney failure and lung hypertension have been reported, we did not observe these complications in our long follow-up case series. we can assume that the availability of a dedicated emergency room at our center allows to perform, promptly, hyper-hydration or transfusion support in case of hemoglobinuria crisis, reducing the risk or organ damage. no infections have been observed after eculizumab, probably due to the vaccination program schedule recommended in the literature, plus the addition of conjugated anti-meningococcus serotype b. however, shared guidelines are needed. disclosure of conflict of interest: none. mortality following hsct in saa pts over the age of 40 is reported to be in the order of 50%, without taking in to account long term sequelae such as chronic gvhd, known to be more frequent in older patients. this has prompted international guidelines to recommend first line immunosuppressive therapy above 40 years of age. the question is whether this is still true in 2017. the aim of the study is to assess whether trm in saa patients grafted 2010-2015 is reduced,as compared to the era 2001-2009. we used the wpsaa ebmt registry, and identified 748 pts aged 40 years or more, with acquired saa, grafted between 2001 and 2015. we divided pts in 2 transplant eras:2001-2009 (n = 327) and 2010-2015 (n = 407). in the more recent period (2010-2015) pts were older (53 vs 49 year, p o0.01), were more often grafted from alternative donors (alt) (64% vs 43%, po0.01), with a greater use of bm (54% vs 41%, p o0.01), and with a longer interval dx-tx (317 vs 258 days, p0.01), and more often received a fludarabine containing regimen (55% vs 42%, p o0.01). the os 5 year of pts grafted in 2001-2009 was 57%, compared with 55% for pts grafted 2010-2015 (p = 0.7). in multivariate analysis, including the interval diagnosis transplant, patient's age, donor type, stem cell source and conditioning regimen, the lack of improved survival in 2010-2015 was confirmed (p = 0.3). a very strong age effect was shown both in univariate and multivariate analysis: survival of pts aged 40-50 years, 51-60 years and 461 years, was respectively 64%, 54%, 41% (p o0.0001) and this was confirmed in multivariate analysis. the conditioning regimen, also proved to be a significant predictor, with improved survival for alt transplants receiving flu containing regimens (56% vs 46%, po0.001). in general pts receiving either cy200 or a flu containing regimen, did significantly better than pts receiving other preparative regimens (58% vs 50%, p = 0.02). the use of a sibling donor (sib) did not prove to predict survival in multivariate analysis. pts receiving campath in the conditioning,did significantly better than pts not receiving campath (65% vs 54% po0.01); similarly survival of patients with atg was superior 59% vs 41% compared to patients not receiving atg (p o0.01). when pts receiving either campath or atg (n = 564) were compared to patients not receiving either (n = 161), the difference in survival was 61% vs 41% (p o0.0001), and this was significant also in multivariate analysis. combined primary and secondary graft failure was reduced from 16% to 12% in the two time periods (p = 0.02), acute gvhd grade ii-iv was reduced from 15% to 11% (p = 0.1) and chronic gvhd was also reduced from 32% to 26% (p = 0.04) infections remain the leading cause of death in both transplant eras (18% and 22% respectively), followed by gvhd (5% and 4%) and graft failure (5% and 2%), whereas ptld have been reduced from 3% to 0.5%. hsct in pts with acquired saa aged 40 and over, continues to carry a significant risk of trm also in 2010-2015, ranging from 36% in younger pts (40-50) to 59% in older pts (460 years). survival is predicted in multivariate analysis, by two crucial predictors: patients' age and the use of either campath or atg,the latter giving a 20% survival advantage over no campath/atg. alt and sib donors produce similar survival. this study gives further support to current guidelines, suggesting first line therapy with atg+csa, in pts over the age of 40. [p565] disclosure of conflict of interest: none. autoimmune diseases p566 allogeneic haematopoetic stem cell transplantation as curative therapy for early-onset, refractory crohn's disease e groene 1 , p bufler 1 , k krohn 1 , s immler 1 , g marckmann 2 , t feuchtinger 1 , s koletzko 1 and m albert 1 1 dr. von hauner university children's hospital and 2 institute of ethics, history and theory of medicine, lmu results of a recent randomized trial suggest that autologous hsct is an option in adult patients with severe, therapyrefractory crohn's disease (cd) with an associated mortality risk of 4%. however, relapse of the disease is frequent (1). in contrast allogeneic hsct has resulted in long-term cure of cd in affected patients transplanted because of haematological malignancy (2). we report a 17 year old girl who was diagnosed with severe cd at age seven (paris classification l3, l4a, b1). neither next generation sequencing nor immunological work up identified a monogenetic cause of cd. progressive chronic inflammation manifesting ubiquitously in the gastrointestinal tract resulted in severe complications, such as perianal fistulas with rectal stenosis, intestinal abscesses, dysphagia, severe weight loss, failure to thrive, delayed puberty and the need for ileostomy and long-term exclusive enteral nutrition via tube feeding. despite multiple lines of therapy, including repeated nutritional therapy, steroids, immunosuppressants (methotrexate, azathioprine) and biologicals (infliximab, adalimumab, certolizumab) a lasting remission could not be achieved resulting in poor quality of life. after careful risk/benefit assessment including ethical counselling allogeneic hsct was offered. she underwent allogeneic hsct from a matched (10/10) unrelated bone marrow donor (4.3 × 10 8 /kg total nuclear cells). conditioning was performed according to a protocol successfully applied in adolescents with chronic granulomatous disease (3) with alemtuzumab (3 × 0.2 mg/kg/d), targeted busulfan (tauc 53770 ng × h/ml) and fludarabine (6 × 30 mg/m 2 ). cyclosporine a and mycophenolate mofetil were used as gvhd prophylaxis. neutrophil and platelet engraftment were observed on days +20 and +24, respectively. the post hsct course was complicated by grade i acute skin gvhd treated with topical steroids and toxic megacolon secondary to scarring stenosis on both ends of the unused colon on day +130 requiring surgery and a colostomy. at 12 months post hsct the patient is well, off immunosuppressive medication, without gvhd and exhibiting 495% donor chimerism. the cd is in complete clinical and histological remission as proven by endoscopy and biopsies. stoma reversal with restitution of intestinal continuity is planned for the next 12 months. refractory cd can lead to life-threatening complications and severely reduced quality of life. although long-term outcome in our patient will need to be carefully assessed, allogeneic hsct may offer a curative therapy in children and young adults with severe cd, even in the absence of an identified monogenetic cause. current ebmt recommendations include consideration of ahsct in exceptional circumstances for patients with severe refractory cd. the only randomised trial of ahsct in cd (astic) confirmed substantial short-term benefits but failed to meet its primary 1 year endpoint. to further clarify the longterm safety and efficacy of ahsct in cd we performed a retrospective analysis of patients undergoing ahsct for cd outside the astic trial using the ebmt registry. patients were identified from the ebmt registry. all adult patients undergoing ahsct for a primary diagnosis of cd from 1997 to 2015 were eligible for inclusion. patients who were enrolled in the astic trial were excluded. from a total of 99 patients (across 27 centres) on the ebmt registry, data were obtained from 76 patients transplanted in 14 centres in 7 countries. median patient age was 30 yrs (range: 20-51) and 63% were female. median age at first diagnosis of cd was 18yrs (range: 2-48). patients were heavily pre-treated, having failed or been intolerant to a median of 6 previous lines of therapy (range: 3-10). 55% had received experimental therapy prior to auto-hsct. 80% of patients had undergone at least 1 operation. the median time from first diagnosis of cd to auto-hsct was 12.3 years (range: 1.3-25.8). all patients received peripheral blood stem cells following conditioning with cyclophosphamide 200 mg/kg and 84% received anti-thymocyte globulin (atg). the median cd34+ dose infused was 5.5 (range: 2.4-40.6) × 10 6 /kg. twelve percent of patients underwent cd34+ selection. neutrophil and platelet engraftment occurred at a median of day 10 (range: 6-18) and day 9 (range: 1-44), respectively. sixety-one percent received post transplant g-csf. median length of follow-up following auto-hsct was 42 months (range: 6-174). at 100 days post auto-hsct, 64% of patients were in clinical remission (cr), defined as no abdominal pain and normal stool frequency. a further 27% experienced significant improvement, defined as improvement in abdominal pain and stool frequency. for 5% there was no appreciable change in disease and in 4% the disease worsened compared to baseline. at 1 year post auto-hsct, 39% were in cr, 19% were improved, 20% were unchanged and 22% had worsened. at last follow-up, 37% were in cr, 23% were improved, 25% were unchanged and 15% had worsened. overall 74% restarted medical therapy post auto-hsct and 38% required further surgery. overall 26% developed an infection requiring treatment post auto-hsct (11% bacterial, 12% viral). ebv and cmv reactivation occurred in 7% and 4% respectively and herpes zoster occurred in 4%. a secondary autoimmune disease developed in 13%, most commonly thyroid disease (63%). malignancy developed in 5%, of which skin cancer accounted for 75% of cases. one patient died at 56 days post auto-hsct due to cmv infection, sepsis and multiorgan failure. this large retrospective series further supports the safety and efficacy of ahsct in a population with severe and treatment-refractory crohn's disease, 60% of patients experienced complete remission or significant improvement in cd symptoms with long-term follow-up. trm observed was similar to ahsct for other indications. in summary, ahsct appears to be an extremely promising therapy for severe refractory cd. further follow up of astic patients and future randomised trials are warranted. disclosure of conflict of interest: none. memory stem t cells (tscm) are long living self-renewing memory t cells with long-term persistence capacity, which play a relevant role in immunological memory and protection against infectious diseases and cancer1,2,3,4,5,6. the aim of this work is to investigate the potential role of tscm as a reservoir of arthritogenic t cells in rheumatoid arthritis (ra). we analysed the dynamics of circulating tscm (here identified as cd45ra+ cd62l+ cd95+ t cells) and other memory t-cell subpopulations by multiparametric 11-color flow cytometry in 27 patients with active ra and in 14 of them also during treatment with anti-tnfα biological agents (etanercept). to analyse cytokine productions, functional assays were performed stimulating peripheral blood mononuclear cells (pbmcs) with pma/ionomycin and brefeldin a. after the stimulation, cells were stained for surface markers, fixed and stained for intracellular cytokines. we traced circulating antigen specific cd4+ t cells for the vimentine-derived citrullinated peptide (vimcit) 65savracitssvpgvr77 7,8 in hla-drb1 × 04:01 ra patients before and during the anti-tnfα treatment using custom mhc class ii tetramers. viral antigen specific cd8+ t cells were traced using mhc class i dextramers. age-matched healthy donors (hds) were used as control for all the experiments. we found a significant expansion of cd4+ tscm in patients with active ra both in terms of frequency and absolute counts. notably, cd4+ tscm significantly contracted upon anti-tnfα treatment, suggesting a role of tnfα in tscm accumulation. in contrast to cd4 +t cells, cd8 compartment did not show significative alterations compared to (hds). furthermore, cd4+tscm in ra patients displayed an enrichment in the th17 phenotype, largely implied in autoimmune disorders, while the other t cell subpopulation were not enriched in the th17 phenotype. at the antigen specific level, we were able to trace in hla-drb1 × 04:01 patients antigen specific cd4+ t cells, comprising tscm, specific for the vimentin-derived citrullinated peptide. of notice, citrullinated vimentin specific cd4+ t cells, including tscm, contracted during anti-tnfα administration, while viral-specific cd4+ t cells (ebvbhrf-1) and antiviral cd8 specificities (cmvpp65, flump, ebvbmlf-1) were not affected by etanercept administration, thus suggesting a possible role of cd4+ tscm as reservoir of arthritogenic autoreactive t cells. overall, our results suggest that tscm, by representing a long-term reservoir of undesired specificities, might play a non redundant role in sustaining ra and possibly other t cell mediated disorders, thus representing novel biomarkers as well as therapeutic targets. ongoing experiments will characterize the tcr repertoire on sorted tscm and cd4+ memory subsets in order to identify a possible oligoclonality in tscm repertoire. in conclusion, the analysis of tscm dynamics in autoimmune disorders could have relevant clinical implications as new biomarkers and for devising innovative therapeutic strategies. ebv and cmv reactivation following autologous haematopoietic stem cell transplantation (hsct) for autoimmune neurological diseases resolves spontaneously and rarely requires treatment c mapplebeck 1 , b sharrack 1 , h kaur 1 , y ezaydi 1 , h jessop 1 , l pickersgill 1 , l scott 1 , m raza 1 and ja snowden 1 1 departments of haematology, neurology and virology, sheffield teaching hospitals nhs foundation trust, sheffield, uk autologous haematopoietic stem cell transplantation (hsct) for severe autoimmune diseases involves immunosuppressive conditioning regimens and current guidelines recommend monitoring for viral reactivation of cytomegalovirus (cmv) and epstein barr virus (ebv) (snowden et al 2012) . however, the incidence, degree and management of viral reactivation are not established. we performed a retrospective observational service evaluation study of all patients receiving cyclophosphamide 200 mg/kg + rabbit anti-thymocyte globulin 6 mg/kg (atg, thymoglobulin) followed by autologous hsct for various autoimmune neurological diseases between 2011 and 2016 at our centre. data collected included the baseline serological status of the patient prior to transplant and serial blood pcr quantitation (copies/ml). if ebv and cmv reactivation occurred details of further management was collected and descriptive statistics were used to summarise outcomes. twenty-three patients received autologous hsct between january 2011 and october 2016; 21 patients with multiple sclerosis (ms), 1 with chronic inflammatory demyelinating polyneuropathy (cidp) and 1 with stiff person syndrome. twenty-two patients had positive ebv igg serology prior to transplant and 1 patient had an equivocal result. seventeen patients had evidence of ebv reactivation and a further patient had ebv dna detected post-transplant but with less than 250 copies/ml. the average time to peak ebv pcr was 26.5 (range: 12-44) days post-transplant and a range: in ebv pcr peak level from 623 to 577 000 copies/ml. the 4 patients who had ebv pcr results of over 100 000 copies/ml had ct scans of chest, abdomen and pelvis performed which did not demonstrate significant lymphadenopathy or hepatosplenomegaly. in all patients monitored for a detectable ebv reactivation, the ebv pcr spontaneously began to fall within 2 months (average 36 days, range: 18-60 days) post-transplant and no specific treatment was required. one patient had late ebv reactivation of 3480 copies/ml at 6 months post-hsct associated with chronic tonsillitis and tonsillectomy specimens showed follicular hyperplasia without evidence of post-transplant lymphoproliferative disorder (ptld) and ebv pcr levels normalised without other treatment. 8 (35%) patients had positive cmv igg serology prior to transplant and one patient had an equivocal result. only 1 of 23 patients had a significant reactivation of cmv with 51 300 copies/ml at 21 days post-transplant, successfully treated with intravenous immunoglobulins and valganciclovir. two other patients had low level cmv reactivation with 94 and 476 copies/ml, respectively which resolved spontaneously without treatment. ebv reactivation in patients with neurological autoimmune disease undergoing autologous hsct is common and usually resolves spontaneously without treatment. asymptomatic cmv reactivation occurs in approximately 13% of patients in this setting and may require treatment. autologous hematopoietic stem cell transplantation (hsct) has been utilised for the treatment of severe multiple sclerosis (ms). it results in significant improvement of neurological function, although patients can experience exacerbations of ms-related symptoms during the procedure. we reviewed 17 patients with ms who underwent stem cell mobilisation and collection from march to november 2016. the median age was 40 years (24-55). nine patients (53%) were male. the interval from diagnosis to hsct was 114.3 months (range: 11.6-128.3). 9 patients (53%) had relapsing-remitting (rrms), six patients (35%) secondaryprogressive (spms) and two patients (12%) primary-progressive (ppms) multiple sclerosis. only 2 patients (12%) had not received any prior treatment, whereas 10 patients (59%) received two prior treatments, three patients (17%) received three treatments and two patients (12%) received four treatments. the median expanded disability status scale (edss) score was 6 (range: 2-6). peripheral blood stem cells were mobilised with cyclophosphamide (cy) 2 g/m 2 on day +1 and daily gcsf (5 μg/kg subcutaneously) from day +3 until the completion of the harvest. hsct was performed at a median of 33 days after mobilisation (range: 25-59). the conditioning regimen consisted of cy (50 mg/kg/day from day − 5 to − 2) and atg (2 mg/kg/day from day − 4 to − 2). exacerbation of ms symptoms was defined as the appearance of new or worsening of old symptoms for at least 24 h duration in a previously stable (4 weeks) patient. of the total cohort, 13 patients (76%) underwent mobilisation with cy+gcsf uneventfully. only two patients (12%) had an exacerbation of ms requiring hospital admission after collection (one with fatigue and increase of spasticity, other with worsening weakness). no patient required hospital admission during the mobilisation procedure. the median cd34+ cell dose was 8.39 × 10 6 /kg (range: 2.2-24). the median number of apheresis was 1(1-2). a total of 14 patients have undergone hsct at the time of this analysis. during transplant a total of 11 patients (78%) experienced an exacerbation of ms. of these, 54% (n = 6) before day 0 and 46% (n = 5) between day +4 and +7. symptoms of exacerbation were: muscle spasms in 4 patients (36%), weakness and reduced power of limbs in 4 patients (36%), increase instability and tremor in two patients (18%) and one patient (10%) with worsening of neuropathic pain. only three patients (28%) received treatment with methylprednisolone for ms exacerbation and symptoms had fully resolved by discharge in all patients. other transplant complications included neutropenic fever in all, invasive fungal infection in 1, fluid overload in 9 (64%) and atg related complications in 11 (78%) such as fever (n = 10) and pericarditis/serositis (n = 1). the median time to neutrophil engraftment was 10 days (10-14) and the median duration of hospital admission was 20 days (15-25). exacerbation of ms symptoms is common during hsct and can also occur during mobilisation. in our hands, after cy and gcsf mobilisation only two patients (11%) developed an exacerbation of ms symptoms compared with 11 patients (78%) after ct and atg conditioned hsct. it is possible that the addition of atg to cy triggers an immunological response involved in this transient deterioration of the ms symptoms. further studies are required to confirm this hypothesis. disclosure of conflict of interest: none. inflammatory immune response syndrome (iris) is a noninfectious worsening of neurological condition during immune recovery and has been documented to occur in hiv and in multiple sclerosis following alemtuzumab. the manifestation of iris includes headache, nausea, weakness, neurologic deficits, and mri enhancing lesion. we report three cases of iris after autologous non-myeloablative hematopoietic stem cell transplantation (hsct) in patients for which the transplant indication was an inflammatory neurologic disease: neuromyelitis optica (nmo), chronic relapsing inflammatory optic neuritis (crion), and multiple sclerosis (ms). mobilization was with cyclophosphamide 2 gr/m 2 and gcsf. conditioned regimen was 200 mg/kg cyclophosphamide (50 mg/kg/d) and 6.0 mg/kg ratg (thymoglobulin). the conditioning regimen for nmo and crion also included 1000 mg rituximab. case 1. a 22 years old african-american female with systemic lupus erythematosus (sle) and nmo was discharged day 10 and readmitted on day 14 for fever, headache, progressive altered mental status with dysarthria and legs. brain mri had numerous t2/flair hyperintense and enhancing lesions in the subcortical and periventricular white matter. a lumbar puncture was negative for infection including jcv. complete recovery occurred after treatment included high dose of steroids and plasmapheresis. case2. a 48 years old female with crion experienced blindness, weakness and slurring of speech three months post hsct. mri showed a large enhancing brain stem lesion. lumbar puncture was jcv negative. complete recovery occurred after solumedrol and rituximab. mri 6 months later demonstrated complete resolution of the enhancement with return of vision to baseline. case3. a ten year-old boy diagnosed with paediatric ms developed hemichorea seven days after hsc reinfusion. brain mri revealed a gadolinium-enhancing lesion in the contralateral basal ganglia. lumbar puncture was negative for infection including jc virus. symptoms resolved spontaneously after seventeen days. the appearance of new neurologic symptoms and mri enhancing lesions early after autologous hsct is unexpected and may be related to lymphocytes in the graft, immune recovery post engraftment, and or persistent auto-antibodies. it is mandatory to perform a lumbar puncture to exclude the possibility of infections including progressive multifocal leukoencelopathy (pml) due to jcv. the timing of presentation, the negativity of jc viral load, and the complete recovery with or without immune suppression suggest the hypothesis of iris, as an epiphenomenon of the immune reconstitution following autologous hsct for neurologic diseases disclosure of conflict of interest: none. hematopoietic stem cell transplantation is the effective method of therapy for cns autoimmune disorders in children. long-term outcomes and late effects estimation required. the aim of the study is to estimate long-term outcomes and late effects at children underwent auto-hsct for multiple sclerosis (ms) and allo-hsct for neuromyelitis optica (nmo). twelve pts. with ms and 3 pts. with nmo were included to the analysis. ms pts. gender: female -75% (n = 9), male -25% (n = 3 allogeneic haematopoietic stem cell transplantation (hsct) remains the sole curative option for patients with myelofibrosis (mf). although a spectrum of conditioning regimens has been used, the optimal preparative treatment before hsct remains to be defined. we did a phase ii randomized study at 21 transplant centers in italy with the aim of comparing the reduced-intensity conditioning (ric) fludarabine-busulfan (fb) (conventional arm), that had been already tested in the prospective ebmt study (1) with the ric fludarabine-thiotepa (ft) (experimental arm), that has been widely used in italy in the last two decades (2) . eligible to this study were patients with primary mf or a mf subsequent to a previous essential thrombocytemia or polycyhemia vera, an age ≥ 18 ≤ 70 years, a karnofsky performance status 4 60, a comorbidity index o o/u4 5 and with at least one of the following unfavorable prognostic factors: anemia (hb o 10 g/dl), leukocitosis (25 × 10 9 /l), circulating blasts 41% or constitutional symptoms. patients were randomized to receive intravenous busulfan 0.8 mg/kg for 10 doses or thiotepa 6 mg/kg for two doses associated to fludarabine 30 mg/m 2 for impact of pre-transplant ruxolitinib in myelofibrosis patients on outcome after allogeneic stem cell transplantation syed abd kadir, sharifah shahnaz, author 1,2 , zabelina, tatjana, co-author 1 , christopeit, maximilian, co-author 1 , wulf, gerald, co-author 3 , wagner, eva, co-author 4 , bornhaeuser, martin, co-author 5 , schroeder, thomas, co-author 6 , crysandt, martina, co-author 7 , mayer, karin tina, co-author 8 , stelljes, matthias, co-author 9 , badbaran, anita, co-author 1 , wolschke, christine, co-author 1 , ayuk ayuketang, francis, co-author 1 , triviai, ioanna, co-author 1 , wolf, dominik, co-author 8 ruxolitinib (rux) is the first approved drug for treatment of myelofibrosis. because spleen size and constitutional symptoms may influence outcome after allogeneic stem cell transplantation (asct), rux is recommended before stem cell transplantation in order to reduce therapy-related morbidity and mortality and improve outcome (ebmt/eln recommendation, leukemia 2015) the aim of this retrospective study was to evaluate the impact of pretreatment with rux in comparison to transplantation of rux-naïve mf patients with regard to outcome after asct. we included 149 myelofibrosis patients (pts) with a median age of 59 years (range: 28-74) who received asct between 2000 and 2015 from related (n = 23), matched (n = 86) or mismatched (n = 50) unrelated donor. all patients received busulfan-based reduced intensity conditioning. while 113 pts (66%) did not receive rux, 46 pts (34%) received rux at any time point prior to asct. the median daily dose of rux was 30 mg (range: 10-40 mg) and the median duration of treatment was 28 days (range: 12-159 days). in 11 pts rux was stopped before stem cell transplantation because of no response or loss of response, while in 35 pts rux was given until start of conditioning. gvhd prophylaxis consisted of cni plus short course mtx or mmf and anti-lymphocyte globulin. according to dynamic ipss (dipss) (n = 170) the patients were either low (n = 2), intermediate-1 (n = 36), intermediat-2 (n = 72), or high risk (n = 36). as the median follow up was shorter for patients treated with rux (15 vs 73 months, po0.001). primary graft failure was seen in 2 pts in the rux and three in the non-rux group. the median leukocyte engraftment was 13 days (range: 9-32) in the ruxolitinib and 14 days (range: 7-34) in the non rux group (p = 0.7). the incidence of acute gvhd grade i to iv was significantly lower in the rux group (49% vs 64%, p = 0.05), while agvhd grade ii-iv (33% vs 44%, p = 0.14) and grade iii/iv (23% vs 25%, p = 0.48), did not differ significantly. the ci of nrm at 1 year was 18% (95% ci: 6-30%) for the rux group and 22% (95% ci: 14-30%) for the non-rux group (p = 0.58), and the ci of relapse at 2 years was 8% (95% ci: 0-16%) vs 20% (95%ci: 12-28%, p = 0.25). the 2 years rfs and os was 66% (95%ci: 50-82%) and 69% (95%ci: 51-87) for the rux group and 59% (95% ci: 49-69%) and 70% (95% ci:62-78%) for the non-rux group (p = 0.29 and p = 0.45, respectively). within the rux group (n = 53), 24 pts responded to rux (more than 25% spleen size reduction), while 29 pts did not respond or lost response prior to stem cell transplantation. here, no significant difference could be seen between the responding and non-responding group for nrm (19% vs 17%, p = 0.69), relapse (4% vs 13%, p = 0.62), rfs (61% vs 72%, p = 0.81) and os (63% vs 75%, p = 0.89). in a multivariate analysis including rux treatment as variable there was a non-significant trend in favor for in the tyrosine kinase inhibitor (tki) era, allogeneic haemopoietic stem cell transplantation (allo-hsct) has become the later-line therapy but still remains the only known curative treatment for chronic myeloid leukemia (cml). since the introduction of tki in our centre in 2004, the trend of allo-hsct among our cml cohort has changed over time. the purpose of this study is to examine hsct outcomes of our cml cohort who was either tki naïve or has received tki therapy prior hsct. between may 1999 and december 2015, 98 cml patients in our center received allo-hsct with 39% were tki naïve. the time of diagnosis to transplant was significant shorter among the tki naive group as compared to those received tki prior hsct (17.29 ± 7.29 months versus 42.33 ± 31.92 months, respectively). there were no gender different (60% males) but the median age at hsct was younger among tki naïve group, 29.50 years (range: 14-44 years) versus 33.50 years (range: 16-59 years) respectively. malays remained majority ethnic group but the percentage was reduced among patients received tki prior hsct (60.5% versus 46.7% respectively). the disease phase at hsct was significant different whereby majority of tki naïve group was in first chronic phase (cp1) (60.5%) as compare to patients with prior tki exposure (35.0%). all the patients in the tki naïve group received hla-matched related siblings donor (mrd) with 81.6% marrow stem cell source whereas only 88.8% of patients who have prior tki exposure received mrd with 93.3% were from peripheral blood stem cell (pbsc). all patients in the tki naïve group but only 73.3% among patients who have prior tki exposure received full myeloablative conditioning regimen. there was slower neutrophil and platelet engraftment (19.97 ± 4.50 days versus 15.02 ± 3.55 days and 20.03 ± 6.72 days versus 13.93 ± 4.70 days respectively) among tki naive group. at 30 june 2016, the 1-year overall survival (os) of cml at all disease status was 50% in tki naive group versus 32% for patients who have prior tki exposure and transplanted in more advance disease stage. in general, patients in cp1 have the best os. there was higher incident of grade 2 to 4 acute graft-versus-host-disease (gvhd) among the tki naïve group (48.6% versus 16.7%, respectively) likely due to intensity of conditioning regimen with no significant different in chronic gvhd incident. similarly, there was higher relapse rate among tki naive patients (44.7% versus 16.7%, respectively) as upfront post transplant tki was not routinely given to this group of patients in the past. further multivariate analysis to ascertain predictors of transplant outcome among this cohort of patients included disease status, donor-recipient gender combination, ethnic difference will be presented. in conclusion, despite emergent of effective and potent next generation tki, hsct still has it role as curative modality for patients who failed tki. as showed in our data, the transplant outcome is excellent for patients who remain in cp1 at the time of hsct and it is important to identify patients earlier, before disease progression, especially young patients, in order to optimize transplantation outcomes. disclosure of conflict of interest: none. the purpose of this analysis was to provide 10-year follow-up of the gcllsg cll3x trial which aimed at evaluating reducedintensity conditioning (ric) hsct in patients with poor-risk cll. the cll3x trial included 100 patients (median age 53 (27-65) years), of whom 90 patients were allografted with blood stem cells from related (40%) or unrelated donors (60%) using fludarabine-alkylator-based ric regimens. 24% had refractory cll at hsct, and 35% had a tp53 deletion and/or mutation. the 6-year follow-up of the trial including the observation that genetic risk factors such as tp53 lesions and sf3b1 and notch1 mutations had no prognostic impact has been previously reported. survival and relapse information was requested for all patients who underwent hsct within the cll3x trial in 9 german centres (the canadian centre was unavailable for follow-up) and were alive at the 6-year followup. results: follow-up information was received for 37/44 patients (84%) alive at the 6-year follow-up. of these, 5 patients had died (3 cll, 1 chronic gvhd, 1 secondary cancer), and 3 had experienced disease recurrence. with a median follow-up of survivors of 9.7 (0.6-15.2) years, 10-year nrm, relapse incidence (rel), event-free survival (efs), and overall survival (os) of all 90 patients allografted was 25%, 55%, 31% and 51%, respectively, without significant effects of tp53 lesions on outcome. absence of minimal residual disease (mrd) at the 12-month landmark post hsct was highly predictive for a reduced relapse risk, in particular if mrd eradication occurred only after immunosuppression withdrawal, suggesting of effective graft-versus-leukemia activity (gvl; 10-year rel 12%). in the 32 patients who were alive and event-free 6 years post allohct, nrm, rel, efs, and os 4 years after this landmark (or 10 years after transplant) was 3.4%, 18%, 79%, and 94% with a median follow-up of 4.3 years (1.2-9.2) after the 6-year landmark. notably, no relapse event occurred beyond 10 years post hsct. of those who remained event-free beyond 10 years, all 8 patients who were available for mrd assessment at their most recent follow-up were mrdnegative. altogether 39 of the 90 allografted patients had cll recurrence after transplant; 34 between 2003 and 2010, and 5 from 2011 onwards. whilst the median survival of those patients who relapsed during the earlier period was 19 months, all 5 patients with late relapse are currently alive 4-62 (median 28) months after the event. conclusions: long-term observation of patients allografted in the cll3x trial confirms that ric hsct can provide gvl-mediated sustained disease control in a sizable proportion of patients with poor-risk cll independent of the tp53 status. patients who are in mrdnegative remission one year after hsct have an 87% probability of remaining disease-free at least for 10 years. however, late relapses do occur but may benefit from strategies involving innovative pathway inhibitors. hallek: consultancy and speakers bureau for pharmacyclics, llc and an abbvie company; speakers bureau for janssen; m. kneba: consultancy, honoraria, travel grants and research funding from gilead and roche; consultancy, honoraria and travel grants from abbvie and janssen-cilag; research funding from amgen; travel grants from glaxo-smithkline; p.dreger: consultancy for roche and janssen; consultancy and speakers bureau for novartis and gilead. no evidence for an increased gvhd risk associated with post-transplant idelalisib given for relapse of chronic lymphocytic leukemia or lymphoma: first results of a survey by the ebmt chronic malignancy and lymphoma working parties p dreger 1,2 , a boumendil, l koster 2 , c scheid 3 idelalisib is a kinase inhibitor (ki) approved for the treatment of cll and follicular lymphoma (fl). idelalisib has a specific adverse effect profile including immune-mediated inflammatory conditions such as colitis and pneumonitis, raising concern about the safety of this ki if administered for treatment of malignancy recurrence after allogeneic hematopoietic cell transplantation (allohct). the purpose of this ongoing study is to provide information on the safety and efficacy of idelalisib in this setting. we included in this study adult patients who had been registered with the ebmt for an allohct for cll or lymphoma and who received idelalisib for treating disease relapse or persistence at any time after transplant as indicated by participating investigators upon request by the ebmt study office in leiden. baseline patient, disease, and transplant data were collected from med-a forms. centers were requested to provide additional treatment and follow-up information. as of november 29, 2016, a total of 19 patients have been registered, of whom a full dataset as required for this analysis was available for 14 patients (cll 9, fl 2, diffuse large b-cell lymphoma (dlbcl) 1, peripheral t-cell lymphoma 1, unspecified 1) who had undergone allohct between july 2009 and april 2015. all patients except one were male. median age at transplantation was 52 (36-63) years and the median interval from diagnosis to allohct was 3.5 (0.8-12.2) years. prior to allohct, 3 patients (1 cll and 2 lymphoma) had received an autohct and two other patients had been exposed to ki (idelalsib 1, ibrutinib 1). disease status at allohct was sensitive in 71% of the patients. conditioning was reduced-intensity in 71% of the transplants and included in vivo t cell depletion in the majority of cases (71%). donors were identical siblings in 43% with pbsc being the stem cell source in all cases. the interval between hct and idelalisib commencement was 18 (2-68) months in the cll group but only 3 (1-57) months in the lymphoma group. prior to idelalisib, grade ii-iv acute gvhd and chronic gvhd had been observed in 7% and 36% of the patients, but was still active at the time of idelalisib commencement in only two cases (14%) . four patients with cll had already failed ibrutinib given for post-hct relapse prior to idelalisib. the median time on idelalisib until documented withdrawal or event (progression, retreatment, death) was 237 (9-569) days. after start of idelalisib, one patient developed grade 2 acute gvhd and subsequently chronic gvhd, however, in this patient idelalisib was started as early as 30 days after transplant. efficacy of idelalisib in this high-risk patient sample was limited with only one pr in the cll group (stable disease 4, progressive disease 1, not available 3; lymphoma not available), translating into a median event-free survival after start of idelalisib of 240 days. five patients with cll underwent a subsequent treatment with an alternate ki (ibrutinib 3, venetoclax 2). altogether, there were five deaths, all due to diease progression (cll 2, lymphoma 3). median overall survival was 360 days for the whole sample and not reached for cll. this preliminary data does not support concerns about the safety of idelalisib in the post allohct setting. updated results of this ongoing study will be presented at the meeting. tested patients (67%) achieved a ccyr and at least a mmolr, respectively. the response to transplant by day 30 assessment correlated significantly with the disease status before transplant. a higher percentage of patients who experienced cytogenetic response before transplant experienced molecular response post-transplant (77%) compared with those who did not (61%; p = 0.027). for the entire group, the 1-year cumulative incidence (ci) of acute gvhd grade ii-iv and grade iii-iv were 41% and 15%, respectively; 5-year ci of extensive chronic gvhd was 31%. there was no significant difference in the ci of severe acute or chronic gvhd between donor types. the ci of nrm at 100 days and 1 year was 14% and 30%, respectively. the ci of cytogenetic and molecular relapse at 5 years was 22% and 31%, respectively. overall the 5-year os, pfs and gvhd-free, relapse-free survival (grfs) were 49%, 34%, and 22%, respectively. in multivariable analysis for grfs, transplant in cp2 and the use of haploidentical donor significantly associated with better grfs. the 5-year grfs of patients in cp2, ap and bp before transplant was 24%, 16% and 14%, respectively (p = 0.013). ( figure 1a ) patients receiving a haploidentical donor had a better 5-year grfs when compared with hla matched transplants (53% vs 21%, p = 0.019). ( figure 1b ) for pfs, transplantation in cp2, using a haploidentical donor and mac regimen associated with better pfs while age, cytogenetic and molecular response before transplant did not predict survival. ahsct is curative for a proportion of patients with advanced cml. pfs and grfs are favorably influenced by percentage of bm blasts and donor type, with haploidentical donor having at least as good outcomes as hla matched donors, while molecular and cytogenetic response before transplant do not appear to correlate with survival posttransplant disclosure of conflict of interest: none. allogeneic stem cell transplantation (sct) has been considered as the treatment of choice for younger patients (pts) with high-risk chronic lymphocytic leukemia (cll). role of allogeneic sct in era of novel drugs is widely discussed. here we present our results after sequential use of chemotherapy and reduced-intensity conditioning (ric) in cohort of 25 high-risk cll pts. high-risk cll was defined by one of the following: disease refractory to purine analogs, short response or early relapse (within 24 months) after purine analog combination treatment, and/or progressive disease with unfavorable genetic abnormalities (del [17p]/tp53 mutation). we analyzed 25 pts with high-risk cll undergoing chemotherapy and ric sct in our centre from august 2007 to june 2016. the median of pretransplant lines were 3 (range: 2-4), novel drugs (idelalisib, ibrutinib) were used in 20% of pts (5/25). fludarabine (30 mg/m 2 ) and cytarabine (2 g/m 2 ) for 4 days (fc) were used for cytoreduction in all pts. after 3 days of rest, ric consisting of 4 gy tbi, anti-thymocyte globulin 10-20 mg/ kg/day for 3 days, and cyclophosphamide 40-60 mg/kg/day for 2 days followed. median age of pts was 53 years (range: five-year overall (os) and relapse-free survival (rfs) was 59% and 57%. ci for cgvhd in pts surviving more than 3 months post-hct was 35% after 5 years and 49% after 10 years. in a multivariate cox-regression model the occurrence of cgvhd independently improved os (p = 0.001, hr 0.27; 95% ci 0.12-0.59%) as well as rfs (po 0.001, hr 0.19; 95% ci 0.08-0.46). high risk dipss plus score demonstrated significant inferior survival compared to intermediate-2 (os p = 0.006; rfs p = 0.009), int-1 (os p = 0.037; rfs p = 0.042) and low risk (os p = 0.021; rfs p = 0.014) which could be confirmed in multivariate analysis for os (p = 0.001, hr 3.13; 95% ci 1.56-6.3) and rfs (p o0.001, hr 4.84; 95% ci 2.05-11.43). rfs additionally was improved by splenomegaly (n = 60) vs. normal spleen size (n = 11) at time of hct (p = 0.01, hr 0.29; 95% ci 0.1-0.75). ruxolitinib (n = 20) or none (n = 45) pre-treatment compared to other drug therapy (n = 19) resulted in improved os (p = 0.013) and rfs (p = 0.031) and was an independent factor for rfs in multivariate analysis (p = 0.046, hr 0.39; 95% ci 0.16-0.99). non-relapse mortality (nmr) was significantly determined by high-risk dipss plus score (p = 0.001) or dipss high and int-2 (p = 0.009). relapse incidence was significantly lower in pts with splenomegaly compared to asplenic or normalspleensized pts prior to hct (p = 0.027). our data point out that pre-therapy and dipss or dipss plus score are relevant pre-transplant outcome factors while chronic gvhd is the most important independent hct-related factor. furthermore, splenomegaly at hct reduces risk of relapse and therefore improves rfs. [p582] disclosure of conflict of interest: none. thalassaemia major affects 10 000 new babies in india each year and haematopoietic stem cell transplantation offers the only chance of cure. we present data on 179 children with thalassaemia major aged between 9 months and 17 years using a uniform conditioning regimen consisting of thiotepa 8 mg/kg, treosulphan 42 gm/m 2 and fludarabine 160 mg/m 2 . equine antithymocyte globulin at a dose of 45 mg/kg was added to children who were undergoing transplantation from an unrelated donor source. there were eight deaths before engraftment due to sepsis or bleeding and two related to graft versus host disease. all patients showed complete chimerism on day 28. however, in 21 children there was an acute drop in donor chimerism between day 60 and 120 post transplantation. immunosuppression was abruptly stopped when donor chimerism dropped below 95% in all children. seven children responded well and re-established complete chimerism with this measure. seven children progressed to develop complete graft loss. donor lymphocyte infusion (dli) in the form of small aliquots of peripheral whole blood from the donor was administered in seven children. dli was used in a graded fashion every 2 weeks starting from 1 × 10 4 /kg of cd3, followed by 1 × 10 5 /kg and 5 × 10 5 /kg. all of them continued to maintain their graft with these interventions. drop in chimerism was seen particularly in children less than 3 years at the time of transplantation comprising 14 out of 21 children. older children with lucarelli class iii were also prone to rejection in our earlier series and this complication has now been eliminated with pre-transplant immunosuppression and hypertransfusion. children above the age of 7 years were more prone to graft versus host disease and required on average 18 months of immunosuppression. treosulphan based protocol has been equally well tolerated by all age groups, all lucarelli classes of children with thalassaemia major and different donor sources. the transplant related mortality and graft rejection rates have been low at 5.5% and 3.9%, respectively. however, children less than 3 years need to be monitored carefully during the first 4 months of transplantation as early withdrawal of immunosuppression can prevent graft rejection resulting in excellent outcomes. disclosure of conflict of interest: none. 16 institute of cellular medicine, newcastle university, newcastle-upon-tyne, uk hemophagocytic lymphohistiocytosis (hlh; hemophagocytic syndrome) is a rare syndrome of potentially fatal, uncontrolled hyperinflammation. allogeneic stem cell transplantation (allosct) is indicated in familial, recurrent or progressive hlh. additional recommendations include central nervous system involvement and unknown triggering factor. while data for allosct outcome are available for the pediatric setting, information for adults is very limited. the aim of this study was to retrospectively analyze the information from the ebmt databases about adult hlh patients who underwent allogeneic stem cell transplantation. we obtained data of 67 adult (≥18 years of age) patients transplanted due to hlh. additionally, an hlh-oriented questionnaire was sent to the clinical centers, with 23 responses received so far. median age at transplantation was 28 (range: 18-65). there was a slight male predominance 43/67 (64%). the majority of patients were reported with secondary hlh 33/67 (49%), the familial disease was reported in 29/67 (43%) patients. in two patients triggering factor was attributed to malignancy. the majority of patients received stem cells obtained from the peripheral blood (52/67; 78%) while for the remaining ones it was bone marrow. reduced intensity conditioning was used since 2004 in 23/63 (37%) of patients. thirteen (19%) patients received tbi. donor choice was: 33 matched unrelated (50%), 7 mismatched unrelated (11%), 26 identical sibling (39%). engraftment was observed in 55/61 (77%). the cumulative incidence of acute graft versus host disease (gvhd) at 100 days was 26% (95% ci 15-37%). the cumulative incidence of chronic gvhd at 1 year after allosct was 13% (95% ci 2-23%) and increased to 31% at 3 years (95% ci 16-47%). the 3-year probability of overall survival is shown in fig.1 . the median survival time was 8 months. the 3-year os was 41% (95% ci 28-55%). for patients who survived until 3 months, this proportion was more favorable with an os of 62% (95% ci 45-78%) at 3 years after transplantation. among 19 patients with observation times longer than 15 months, only one patient died (in the 48th month after allosct due to relapse which occurred in the 12th month. after 12 months no more relapses of hlh were recorded-the cumulative incidence reached 19%. the non-relapse mortality reached 42% after 15 months. the familial disease was associated with a better prognosis than secondary hlh (p = 0.04). unlike the pediatric population, where reduced intensity conditioning (ric) was associated with higher survival, in adult patients there was no difference between the conditioning types. data form the 23 questionnaires confirm clinical picture typical for hlh at the diagnosis: fever in 21/22 (95%), splenomegaly in 19/20 (95%), hemophagocytosis in 18/20 (90%) and hyperferritinemia with median concentration of 4215 ng/ml (range: 63-260 160). image fig.1 overall survival after allogeneic stem cell transplantation for adult hlh patients until 36 months (95% confidence intervals are shown in grey). the number of patients at risk is indicated below the time axis at the corresponding time points. to our knowledge, this is the largest group of adult patients with hlh who underwent allogeneic stem cell transplantation. relatively low relapse incidence shows that allosct can effectively cure hlh. patients who survive the first period after this procedure can expect a long disease-free survival. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) is the only curative option for children suffering from various life-threatening inherited non-malignant diseases with best results using hla-identical family donor. hsct from unrelated or mismatched family donors is associated with increased risk of agvhd and graft rejection.use of post-transplantation cyclophosphamide (ptcy) with or without additional immunosuppression has been shown to be effective prophylaxis against gvhd in patients with hematological malignancies. there are limited reports of hsct using pt cy for patients with non-malignant disorders. we retrospectively analyzed results of 18 hsct in patients with life-threatening non-malignant diseases using ptcy-based gvhd prophylaxis. patients characteristics are presented in table 1 . thirteen patients (72.2%) were transplanted upfront, 5 patients (27,8%) were rescued after primary or secondary graft failure after first hsct. donors were hla-matched (n = 8) or mismatched (8-9/10) (n = 4) unrelated, haploidentical (n = 5) or hla-identical family (n = 1). bone marrow was used as graft source in 13 (72.2%) patients and peripheral blood stem cell in 5 (27.8%). median cd34+/kg recipient weight-7.25 × 10 6 (3.0-13.5), cd3+/kg-9.785 × 10 7 (0.78-90.6). the conditioning regimen was myeloablative in 12 patients (conventional-3, reduced toxicity-9), reduced intensity-6. the gvhd prophylaxis consisted of a combination of ptcy at dose of 50 mg/kg on days +3 and +4 with calcineurin inhibitors (tacrolimus-6 pts, cyclosporine a-10 pts) or sirolimus (1 pt) and mmf (15 pts) starting on day +5. all but one patients received also serotherapy with rabbit (12 pts) or horse atg (5 pst) and rituximab (4 pts). with a median follow-up of 8 months (range: 1-33), the kaplan-meier estimates of os − 81.5%. one patient with thalassemia died before engraftment on day+11 from severe vod. 15/17 pts (88%) achieved engraftment. the median time for neutrophil and platelet engraftment was 23 (16-28) and 19 days (12-32), respectively. primary graft failure was observed in 2 patients (1 was successfully retransplanted from another haploidenticle donor, 1 was not eligible for a second transplantation, but alive). at last follow up, 10 (67%) patients had full donor chimerism, 4 (27%) had stable mixed chimerism without signs of disease progression. one patien with wiscott-aldrich syndrome had secondary graft failure with progressive loss of donor chimerism and were successfully rescued with second haploidentical transplant from the same donor. of 15 engrafted patients, agvhd ii-iv was seen in 4 (26.7%) patients. one patient developed grade ii (gut stage ii) agvhd, which resolved with systemic steroids. severe (griii-iv) agvhd was observed in 2 pts after second hsct, both had calcineurin and mtor-inhibitors induced toxicity leading to discontinuation of this drugs, but responded on combined (steroids and ruxolitinib) therapy. one patient with was developed grade iii gvhd (gut stage 4) after severe cmv-colitis and died on day from multiple organ failure (suspected tma). one patient developed extensive chronic gvhd of kidney (minimal change [p587] disease) after tapering of immunosupression. one patient with hurler syndrome had seizures, died on day+29 from multiple organ dysfunction syndrome. conclusion: ptcy is a promising option for agvhd prophylaxis in patient with non-malignant disease, lacking an hla-matched family donor. disclosure of conflict of interest: none. an exploratory, open-label study to evaluate the safety and feasibility of atir201, a t-lymphocyte enriched leukocyte preparation depleted ex vivo of host alloreactive t-cells (using photodynamic treatment), as adjuvant treatment to a t-cell depleted haploidentical hematopoietic stem cell transplantation in patients with beta-thalassemia major c selim 2 , w rob 3 , l sarah 4 , f josu de la 5 previous studies demonstrated that donor lymphocytes, selectively depleted of alloreactive t-cells (atir), could be given safely in adult patients receiving a haploidentical hsct. in 42 patients a single dose of atir, at doses up to 2 × 10 6 viable t-cells/kg, was given and no grade iii/iv acute gvhd has been reported. this confirms the efficacy of the elimination method of allo-reactive t-cells and attributes to its beneficial safety profile. in an ongoing phase 2 study, cr-air-007 (nct01794299), infusion of atir101 at 28 days post-hsct results in a reduction of transplant-related mortality (trm) and improvement of overall survival and event-free survival. adjunctive treatment with donor lymphocytes in patients receiving a t-cell depleted, haploidentical hsct for nonmalignant diseases such as beta thalassemia major, could provide early immunological support and better immune reconstitution in the absence of gvhd. in an open-label, multicenter phase 2 study (cr-bd-001; eudract 2016-002959-17), 10 patients age ≥ 2 years and ≤ 25 years with beta thalassemia major will undergo a haploidentical hsct with adjunctive administration of atir201. patients will receive a t-cell depleted graft (cd34-selected, or cd3/cd19 depleted, or tcr-αβ depleted, depending on the experience of the study center) from a related, haploidentical donor, patient conditioning will be myeloablative following standard practices at the study center. atir201 infusion at a dose of 2 × 10 6 viable t-cells/kg is given between 28 and 32 days after the hsct. to assess safety, patients will be evaluated for the occurrence of dose limiting toxicity (dlt), defined as acute gvhd grade iii/iv within 180 days post hsct. efficacy will be primarily evaluated by transfusion-free survival (tfs), occurrence of severe infections, and time to t-cell reconstitution, taking into account hematologic and sustained engraftment. all patients will be closely monitored for cmv, ebv and adenovirus titers, with initiation of pre-emptive treatment upon rising blood titers. regulatory authorities in the united kingdom and germany have approved this clinical study protocol. enrolment of the study is expected to continue during 2017, with first report of safety of atir201 to be expected first half 2018. disclosure of conflict of interest: j. rovers is employee of kiadis pharma, sponsor of the study. sickle cell disease (scd) can be cured with haematopoietic cell transplantation (hct), yet progress in the practice and research of hct for scd has not come without risks and uncertainty. the information and decisions that families and physicians encounter in this field are complex and hanging. in this hermeneutic study, we analyze the case of one family who advocated for hcts for two of their four children knowing the potential risks. these experiences have had a profound impact on both the family and the medical team. this study was conducted through the research method of hermeneutic phenomenology. hermeneutic inquiry is described as the practice and theory of interpretation and understanding in human contexts and aims to make sense of the particulars of these contexts and arrive at deeper understandings. data collection: in-depth interviews were conducted with the mother of the family, the hct nurse coordinator, and the hct physician. the interviews were audiotaped and transcribed verbatim. the transcribed interviews were later reviewed by the physician, who then wrote an additional reflection. this work culminated in approximately 70 pages of single spaced data in textual form. in hermeneutics, interpretation takes place through a careful reading and re-reading of the data, looking for statements and instances that resonate with the researcher. initial individual interpretations of researchers are then raised to another level of interpretive analysis in the research team's communal attention to the data. particular criteria guide the analysis: agreement, coherence, comprehensiveness, potential, and penetration. the following excerpts and interpretations are provided as examples of the analysis, with names changed for confidentiality. "being heard" arose repeatedly in this family's experience, including at the time of their request for a transplant without meeting the traditional criteria for hct. they persisted in their belief that their children would benefit from hct. "they gave us options to see if there was a chance for a transplant...how life would look…. and then we figured…a transplant for him was better at the time…worth the risks…. and you wouldn't even know. he plays basketball now, he plays sports, he's active and he can exercise and run. i never had any regrets because i felt it was better and the most important thing is his organs were really intact; none of the organs were destroyed…so i think it's the right decision we made" (mother). "this family has changed my career, and my life as a result. they challenged my practice and way of thinking. they did so in a considerate way, out of a duty to advocate for their children. we worked through the tension of different viewpoints with respect and all of us grew in the process. at least i can say our team did. i certainly did... i am humbled by their trust and respect…i am grateful to them" (physician). patients and providers are deeply impacted by their interactions. dr. robert buckman stated that it was the individual case that changed his practice always. he claimed he could not walk into a new patient's room without his practice being forever changed. in presenting this hermaneutic analysis, we aim to remind ourselves of the opportunity for growth that can result from reflection on this sacred patientprovider relationship. disclosure of conflict of interest: none. defibrotide (df) prophylaxis and adjustment of busulfan schedule to prevent veno-occlusive disease and thrombotic microangiopathy in an infant with a membrane cofactor protein (mcp) gene mutation and metachromatic leukodystrophy undergoing hematopoietic stem cell gene therapy (hsc-gt) v calbi 1,2 , f fumagalli 1,3,4 , r penati 3 , g consiglieri 3 , m migliavacca 3,1 , d redaelli 1 , s acquati 1 , v attanasio 1 , r chiesa 5 , f ferrua 1,3 , f barzaghi 1,3 , m cicalese 3,1 , a assanelli 3,6 , p silvani 7 , s tedeschi, r arora 8 , a soman 8 , f ciotti 3 , m sarzana 3 , g antonioli 3,1 , c baldoli 9 , s martino 10 , gl ardissino 11 , mg natali sora 4 , l naldini 1,12 , f ciceri 12,13 , a aiuti 1,12,3 and me bernardo hepatic veno-occlusive disease (vod) and thrombotic microangiopathy (tma) are life-threatening complications that can occur after hsc transplantation. expert consensus guidelines support use of df for treatment and prophylaxis of vod due to its ability to restore thrombo-fibrinolytic balance and protect endothelial cells. presymptomatic monozygous twins affected by late infantile metachromatic leukodystrophy (mld) underwent investigational hsc-gt after conditioning with busulfan. no comorbidities were evident at baseline. the dose of transduced cd34+ cells was similar in both patients (18.2 × 10 6 cd34+/kg for patient 1 and 14.1 × 10 6 cd34+/kg for patient 2). patient 1 (p1): at 8 months of age, received conditioning with iv busulfan 80 mg/m 2 /dose for 4 doses (target auc 85 mg × h/l). on day (d) +18 after gt, he developed severe vod and was treated with diuretics, fresh frozen plasma, paracentesis and df. on d+39 schistocytes in peripheral blood, marked proteinuria, complement factor consumption, and increases ldh and bilirubin were observed. the patient's condition worsened, with reduced urine output and generalized oedema with pleural effusion. stool, urine and blood cultures were negative and adamts13 activity was 35%; anti-complement factor h (cfh) antibodies (ab) were positive (1371 ui/ml). these findings led to the diagnosis of atypical hemolytic uremic syndrome (ahus; a form of tma) and eculizumab (300 mg/weekly dose) was started on d +40. patient subsequently developed pulmonary oedema and needed non-invasive ventilation. molecular analysis revealed a heterozygous deletion of cfhr3-r1 and ala353val mutation in the mcp gene, a defect previously shown to be associated with ahus. due to the presence of ab anti-cfh and antiplatelet, 4 weekly doses of rituximab (375 mg/m 2 ) were administered. after treatment, p1 progressively improved although he showed prolonged severe anaemia and thrombocytopenia and bone marrow (bm) hypoplasia, secondary bleeding which required reinfusion of unmanipulated autologous bm cells on d +66. nine months after hsct-gt p1 has shown good hematopoietic recovery, stable engraftment of the transduced hscs, no signs of renal damage or complement activation, albeit with neurodevelopmental delay. patient 2 (p2): given his twin history and genetics, this 9 month old infant was considered at increased risk of vod, so prophylaxis with df (25 mg/kg/d) was administered from d-4 to d+30 and the busulfan conditioning was modified by adjusting the auc to a lower target (1 mg/kg/dose for 14 doses; target auc 67.2 mg × h/l). the child had a good clinical recovery and didn't develop signs of vod or tma after hsc-gt. on d+12 and +14, respectively, anti-cfh and anti-platelet ab were positive. considering the history of the twin, 4 weekly doses of rituximab were administered. p2 is currently 8 months after gt with persistent engraftment of transduced hscs and no signs of tma. data from this case-control report of monozygous twins diagnosed with mld, and subsequently shown to also harbor mutations in complement regulator gene, suggest that df prophylaxis and busulfan adjustment may have helped prevent systemic microangiopathic damage in the second twin. patients with rare disease may have mutations in genes in addition to those that cause their disease. patients at risk of post-transplant tma following hsc-gt for genetic diseases may require tailored df prophylaxis and treatment. disclosure of conflict of interest: a. aiuti is the principal investigator of the tiget-mld clinical trial of gene therapy. the mld gene therapy was licensed to glaxosmithkline (gsk) in 2014 and gsk became the financial sponsor of the trial. all authors declare no other competing interests. hematopoietic stem cell transplantation (hsct) using an optimized conditioning regimen is essential for the longterm survival of patients with inherited bone marrow failure syndromes (ibmfs). we report hsct in 25 children with fanconi anemia (fa, n = 12), diamond-blackfan anemia (dba, n = 7), dyskeratosis congenita (dc, n = 5) and shwachman-diamond syndrome (sds, n = 1) from a single hsct center. the graft source was peripheral blood stem cells (n = 20) or cord blood stem cells (n = 5). fa, dc and sds patients received reduced-intensity conditioning, while dba patients had myeloablative conditioning. the median numbers of infused mononuclear cells and cd34+ cells were 14.4 × 10 8 /kg and 4.5 × 10 6 /kg, respectively. the median time for neutrophil and platelet recovery was 12 and 17 days, respectively. there was one primary graft failure. after median follow up 3 years the overall survival was 96%. the incidence of grade ii-iii acute and chronic graft versus host disease (gvhd) was 32% and 16% respectively. in a multivariate analysis, the type of conditioning regimen was the only factor identified as significantly associated with grade ii-iii acute gvhd (p = 0.01). we conclude that hsct can be a curative option for patients with ibmfs. disease specific conditioning regimen was important to disease the transplant-related mortality. [p591] disclosure of conflict of interest: none. homozygous sickle cell disease (scd) patients suffering from end-stage renal disease (esrd) show a variable outcome after kidney transplantation as underlying disease can cause poor allograft survival and disease-specific problems. we present a case of a 27-year old patient with severe scd and esrd who underwent haploidentical bone marrow transplantation (bmt) with consecutive living kidney transplantion (lkt). the patient suffered from multiple complications of scd including stroke with secondary hemorrhage, symptomatic epilepsy, esrd and uncontrolled hypertension. the patient had been on hydroxyurea without success and required regular blood transfusion due to severe renal anemia. the rationale for bmt was uncontrollable iron overload. a reduced intensity conditioning regimen was used with (fludarabine, cyclophosphamide and 2gy of tbi, dose-adjusted to esrd). graft-versus-host disease (gvhd) prophylaxis consisted of post-transplant high-dose cyclophosphamide, cyclosporine a (cya) and mycophenolate mofetil (mmf). the donor was her 56-year old mother with hbs trait, the stem cell source was bone marrow, the cell dose 4.74 × 10 8 nucleated cells/kg. during conditioning daily hemodialysis was performed to keep drug levels stable. neutrophil engraftment occurred on day +26, chimerism at day +19 was 98%. hbs increased from 1.3% pre-hsct to 40.0% 6 months after hsct. hemoglobin values increased from 70 g/l pre-hsct to 110 g/l post-hsct and reticulocytes from 16 g/l to 124 g/l. erythropoietin levels increased from 2.3 iu/l pre-hsct to 178 iu/l 6 months after hsct. during the follow-up, the patient did not show any sign of acute gvhd or vaso-occlusive crisis, hemolysis or sickling. relevant complications were disease-related (therapy resistant hypertension and epileptic seizure due to former brain damage). on day +151 a lkt from the same donor was performed. the initial immunosuppressive treatment with mmf was continued, cya was switched to tacrolimus and steroids were added for 3 months. the post-transplant period was uneventful. currently, 12 months after haploidentical bmt and 6 months after lkt there are no signs of gvhd, the blood chimerism is 100%, the kidney allograft function is very good (gfr 73 ml/min/1.73 m 2 ) and immunosuppression is withdrawn. iron overload is being corrected by regular phlebotomies. the patient no longer requires antihypertensive medication and there is evidence of vascular remodeling. this is the first report of a successful haploidentical bmt followed by kidney transplantation from the same donor in a patient with scd. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (hsct) can cure non-malignant diseases, such as primary immune deficiency (pid), severe aplastic anemia (saa) and osteopetrosis (op). in the absence of a well-matched donor, transplantation from a haplo-identical donor maybe considered. post-transplant cyclophosphamide (ptcy) is a new strategy derived from the treatment of malignant diseases in adults that has been little studied in high-risk pediatric nonmalignant diseases. fifteen children (2.2 years, range: 0.19-10.88) underwent hsct in the pediatric immunology and hematology unit of necker hospital, paris, between december 2014 and september 2016. these children were suffering from op (n = 3), saa (n = 2), hemophagocytic lymphohistiocytosis (hlh) (n = 3), immunodysregulation polyendocrinopathy enteropathy x-linked (ipex) syndrome (n = 2), combined immune deficiency (n = 4) and leukocyte adhesion molecule deficiency (n = 1). three patients received a low-intensity conditioning regimen (cr) (based on fludarabine, cyclophosphamide, and total body irradiation) whereas the other 12 received myeloablative cr (based on busulfan auc targeted and fludarabine). fourteen patients received serotherapy before hsct. post-transplant cyclophosphamide (50 mg/kg/ day) was given on d3 and d4 and graft versus host disease (gvhd) prophylaxis with cyclosporine and mycophenolate mofetil was initiated on d5. the transplanted stem cells were obtained from bone marrow in all cases. engraftment with full donor chimerism was observed in 13 patients. the median cd34+ cell dose was 14.8 × 10 6 cells/kg body weight (range: 7.4-35.9 × 10 6 ). neutrophils recovered after a median of 18 days (range: 14-32), and overall survival (os) was 80% after a median follow-up of 1 year (range: 0.18-1.98). three patients died due to graft failure (n = 2) or infectious complications related to gvhd (n = 1). grade ii acute gvhd occurred in 8 of the 13 patients displaying engraftment (62%), and chronic gvhd and/or autoimmune complications were observed in four patients (31%). viral complications were frequent, occurring in 10 patients (77%) with cmv infection (n = 8) /disease (n = 1), adenovirus disease (n = 1) and bk virus cystitis (n = 4). haploidentical transplant with ptcy is feasible in high-risk patients with non-malignant diseases. chronic gvhd and autoimmunity were more frequent than for more conventional approaches in such patients. infection rates were high. disclosure of conflict of interest: none. sickle cell disease (scd) remains associated with high risks of morbidity and early death. even best of supportive care fails to improve quality of life. hematopoietic stem cell transplant (hsct) can be considered for selected group of patients. in long run it is not just economical but also substantially improves quality of life (qol). we report our experience with hsct for scd from india. seventy three consecutive patients suffering from scd who underwent hsct between january 2006 and november 2016 were included in the study. fifty two underwent matched sibling donor (msd), 2 matched family donor (mfd), 3 matched unrelated (91/0 or 10/10), 2 cord blood transplant cbt (1 matched sibling cord blood and 1 matched unrelated) and 15 patient underwent haploidentical transplant. different conditioning regimens were used and so was the graft versus host disease prophylaxis depending on institutional protocols as depicted in table 1. a total of 73 patients underwent sct. the median age was 9 years (10 months-29 years). m/f ratio was 45/28. majority of patients were either from african union or oman. all patients suffered from one or other severe symptoms making them eligible for sct. graft source was bone marrow (bm) in 30 with median cd34 count of 5.3x106/kg (0.92-10.7), peripheral blood (pb) in 36 with median cd34 count of 8.5x106/kg (3.9-20.18), cord blood in 2 with median cd34 count of 1.27x105/kg (0.44-2.1) and combined bm & pb in 5 with median cd34 count of 6.37x106/kg (1.5-23.3). of the 73 patients, 61 are alive and disease free with lansky/karnofsky scores of 100. there were 8 deaths (4 msd/mfd/mud; 3 haploidentical and 1 matched unrelated cbt). four patients rejected the graft (2 haploidentical and 2 msd/mfd/mud). at the last follow up, the probabilities of survival, scd-free survival, and transplantrelated mortality were 89%, 83.5%, and 11%, respectively. outcome of hsct in scd has improved significantly. with better conditioning regimens, improved supportive care, the outcome of alternative donor transplant and adult scd has improved and matches sibling donor transplant. hsct should be strongly considered as a curative modality for selected patients suffering from scd. disclosure of conflict of interest: none. s434 staff jointly defined more than 50 local standard operating procedures. patients with low-risk characteristics (age ≤ 7 years, liver size ≤ 2 cm below costal margin) and a hla matched sibling donor were considered eligible in this initial phase of activity. a downstaging protocol with hydroxyurea and deferoxamine or deferasirox was adopted. conditioning regimen included iv busulfan and cyclophosphamide. gvhd and rejection prophylaxis included atg from day − 12 to − 10 and csa, mtx and methylprednisolone. gcs-f primed bone marrow was chosen as stem cell source. the first allogeneic hsct of the whole iraq was performed in a child with thalassemia at hiwa hospital in october 2016. up to now, 3 patients (2 females, 1 male) underwent hsct; median age at transplantation was 2 years; median infused tnc 18.5 × 10 8 /kg, cd34+ 10.1 × 10 6 /kg. all of them engrafted. no major complication were observed. one of them developed grade ii agvhd (skin only) which resolved after increasing the dose of steroids. a huge number of patients with low-risk thalassemia are now in the waiting list and some of them have already started downstaging having planned hsct in a short time. a matched sibling transplant program in children with thalassemia is feasible and safe in kurdistan. such a program can provide many advantages: far less psychosocial and financial burden for the families and significant saving for the government. the estimated costs of performing locally hsct are much less than in the countries where patients were previously referred. the continuation of cooperation is of paramount relevance for further implementing the activity and extending the transplant accessibility to patients with other hemato-oncological disorders of childhood. disclosure of conflict of interest: none. long term follow-up after reduced-intensity conditioning and stem cell transplantation for thalassemia major r rihani, a natsheh, sm abu, e khattab, r najjar, f sheab, s sharma, n hussein, a tbakhi and m sarhan bone marrow and stem transplantation program-king hussein cancer center, amman, jordan hematopoietic stem cell transplantation (hsct) is the only curative treatment for thalassemia major (tm). reducedintensity conditioning (ric) before hsct for high risk tm patients results in fewer complications, when compared with myeloablative regimens. one hundred and three tm patients received hscts from an hla-identical related donor at king hussein cancer center, between january 2002 and november 2016. of those, 62 were high risk tm (60%) who received ric hscts. in this report, we describe follow-up beyond 2 years (median, 42; 24.6-126 months) post ric hscts. forty-four class ii-iii patients (58%) were identified (25% with hepatitis c); with a median age of 14 (13.4-28) years. females accounted for 59% (n = 26). conditioning regimen consisted of oral busulfan 8 mg/kg, fludarabine 175mg/m 2 ,tli 500cgy and atg followed by pbsct. gvhd prophylaxis consisted of mmf and csa. median infused stem cell dose was 5.4 × 10 6 /kg. all patients attained neutrophil and platelet engraftment (median, 15.3 and 21.3 days, respectively). persistent mixed donor or full donor chimerism were observed in 95.5% (n = 42) and 4.5% (n = 2), respectively. immune-suppressive therapy for gvhd treatment was required in 16 (36.4%) patients (agvhd, n = 8; cgvhd, n = 8). moreover, veno-occlusive disease occurred in 4 patients (9%) that resolved completely. secondary graft failure was noted in 11 (25%) patients. the 5-year overall survival was 100%, while the 5-year probability of thalassemia-free survival was 72.2%. other factors evaluated include: growth parameters, endocrine and other organ functions, in addition to functional status. this report confirms the safety and efficacy of ric regimens in hscts for high risk tm patients. those regimens are associated with excellent engraftment and sustained mixed donor chimerism; and lead to excellent thalassemia-free and overall survival rates. [p596] disclosure of conflict of interest: none. in-time hsct for pts. with hurler syndrome (hs) can significantly improve the results. long-term follow-up and late effects estimation required to prepare a special observation and rehabilitation programs. aim. to analyze our experience with hsct for hs in the field of special observation and rehabilitation programs. forty hsct during the 2004-2016 were performed for 38 pts. with hs. median age at the diagnosis was 15 months (3-38 months), at hsct-24 months (9-48 months). bm used in 62.5% (n = 25), pbsc-32.5% (n = 13), cb-5.0% (n = 2). mac conditioning was used for 29 hsct, ric-for 11. ric regimen: flu+mel+atg, mac: bu/treo +flu+thio/mel (bu was used in early 2000) and atg +rituximab (in case of mud hsct). all pts. with ric received mud hsct, pts. with mac mud-18 pts., mrd-5 pts. pts. received csa/tacro-based gvhd prophylaxis. mmf/mtx was additionally added in all cases. in 3 ric hsct immunomagnetic сd3/сd19+ depletion of pbsc (by clinimacs) was used. a special observation protocol including somatic and neurocognitive estimation was developed. all pts. engrafted with full donor chimerism on d+30. median of engraftment day-20 (11-29 days). thirty three pts. survived. reasons of death-mac: infections-3 pts., ric: trali-1 pt., agvhd-1 pt. trm improved, over the years, with improving of supportive care and donor selection as well as pre-transplant screening. no early severe toxicity revealed. pulmonary infection episodes was registered in 30% of pts. in our study. gvhd: grade iideveloped 14 pts., grade iii-iv-2 pts. (after ric), local cgvhd-3 pts. (ric). no extensive cgvhd. 5 pts. rejected (mac and ric rejection rate was same). at median follow up of 60 months (8-160 months), the estimated 5 years pos was 81%. best response correlated with early hsct (and better status before hsct) and higher level of aidu after. late effects estimation showed that 47.4% (n = 18) of patients experienced late effects: cardio-vascular-16 pts., skeletal-15 pts., endocrine-3 pts. all pts. with cardio-vascular effects received mac. skeletal effects affected patients of older age, pts. transplanted in younger age do not have such effects. median period of late effects arising after hsct was 15 month (3-27 months). only 3 pts. experienced serious pulmonary late effects (infections), all episodes was before 2010. no pts. in our study have progressive retinal degeneration. 80% of pts. improved in the neurosensory component and all pts. improved in neurocognitive status and development after hsct. best response correlated with neurocognitive rehabilitation based on unique computer model used by our group in russian national rehabilitation center "russkoe pole." in-time hsct is an effective and safe way to stop neurodegenerative process for pts. with hs. both mac and ric regimens can be used with the same effectiveness. mac regimens associated with bigger number of cardiovascular late effects. long-time follow-up showed that these patients require the special observational protocol including estimation of cardio-vascular, skeletal, endocrine and neurocognitive risks. better neurocognitive response correlated with intensive rehabilitation using computer model. russian joint study showed effective cooperation for treatment pts. with hs in the national setting. disclosure of conflict of interest: none. little is known about pathogenesis of solid tumors after hsct but, intensive cytotoxic conditioning therapy with defective dna repair of persisting stem cells/stromal cells, viral infection, and immunosuppression may play a role. 3/6 patients with solid tumors had a melphalan-based conditioning. melphalan was linked to sarcoma and lung cancer in animal model. there are few data linking parotid mec to infection by cmv and hhv6 which can remain dormant in the salivary glands. both affected patients had hhv6 during the transplant period. p8 and p11 had a family history of solid tumor pointing to a possible genetic factor. whilst secondary malignancy post-hsct for patients with malignant disorders is well recognised, non-ptld malignancy post-hsct for pid has not previously been reported. a larger study is needed to evaluate incidence and risks. allogeneic hsct is a treatment of choice for the bone marrow failure in patients with sds. hsct from unrelated or mismatched family donors is associated with higher morbidity and mortality compared with matched sibling. combined pgd and hla antigen testing is a possible option to preselect a compatible donor for an affected sibling requiring hsct. we describe a case report demonstrating first successful hsct for 6 years girl with sds by using preimplantation genetic diagnosis and hla matching. diagnosis of sds was suspected at 5 m.o., based on clinical features, family history, laboratory studies. at 6 m.o., bone marrow (bm) aspiration revealed hypocellular marrow with signs of dysplasia and expansion of blasts (15.6% blasts). the sanger sequencing of sbds gene showed c.183-184ta4ct and c.258+2t4c mutations. the patient had recurrent infections, including bilateral pneumonia caused by phaeohyphomyces, bloodstream infection, cmvdisease. due to the lack of matched related or unrelated donors, hsct with ric (flu, mel, atg) from haploidentical father was performed at 16 months of age. after the 1st allo-hsct, engraftment was achieved on d+13, initial str study showed full donor chimerism. post-transplant period was complicated with severe cmv-infection and signs of secondary hlh. at d+135, graft rejection was registered. the girl became dependent on regular rbc and platelet transfusions, bm examination revealed hypocellularity with moderate signs of myelodysplasia without elevated blast count. due to lack of available hla-compatible donors, an option of in vitro fertilization (ivf) with preimplantation selection of a normal hla-matched embryo was considered. after controlled ovarian hyperstimulation 2 embryos were hla-compatible and healthy (first, wild-type; second, heterozygous for sbds gene mutation c.183-184ta4ct). hence, the only unaffected hla-identical embryo was transferred resulting into full-term pregnancy. at the age of 5.5 years after 1st hsct, the 2nd s437 transplant was performed with a combination of cb and bm as a source of hematopoietic stem cells. the donors' age was 2 years a reduced toxicity conditioning regimen (rtc) based on flu150 mg/m 2 , treo42 g/m 2 , thiotepa 10 mg/kg with serotherapy (thymoglobuline 7.5 mg/kg) was used. because of neurotoxicity, arterial hypertension, since d+1 csa was changed to sirolimus +mmf for gvhd prophylaxis. the total number of infused nc was 2.5 × 10 8 /kg; cd34+, 3.85 × 10 6 /kg; cd3+, 2.4 × 10 7 /kg. engraftment was achieved on d+28. any signs of gvhd, severe infectious or toxic complications were not observed. eight months later, the patient is alive, has full donor chimerism in bm and is not transfusion-dependent. in the absence of hla-identical donor, ivf with preliminary pgd and hla-typing could be a chance for matched donor to cure patients with non-malignant genetic diseases. in case of low cord blood cellularity, a combination of cb and bm from the same sibling could be used. our experience showed a successful engraftment of sds patient and stable donor chimerism after second hsct of cb and bm from pgd-selected sibling with rtc. disclosure of conflict of interest: none. the safety and efficacy of familial haploidentical (fhi) stem cell transplantation utilizing cd34 enrichment and cd3 addback in patients with high risk sickle cell disease (scd) ( figure 1a ). probability of 1yr efs is 87.4% (ci95: 58-97%) ( figure 1b ). immune cell reconstitution has been robust and similar to rtc and msd allosct in scd (table 1 ). there have been 3 deaths, vod, steroid refractory agvhd and cgvhd. mac followed by fhi utilizing cd34 enrichment and t-cell addback in patients with high-risk scd is safe, tolerable and results in long-term donor chimerism and absence of scd symptoms or complications. a larger cohort and follow-up will be required to confirm these preliminary findings. disclosure of conflict of interest: none. supported by r01fd004090-01a1. [p601] s438 lymphoma p602 a clinical prognostic index for assessing patients aged 460 being considered for high-dose therapy and autologous stem-cell transplant in relapsed or refractory high-grade non-hodgkin lymphoma d edwards 1 , k kirkland 1 , r pearce, s robinson 1 and g cook 1 bsbmt patients with relapsed high-grade nhl or disease refractory to first-line therapy can still be cured with high-dose therapy and autologous stem cell transplant if they respond to salvage chemotherapy. this aggressive algorithm is accepted in younger patients but is less well established in the elderly. age 460 has a negative predictive score in the international prognostic index (ipi) and there are concerns that the outcomes of hdt in these patients are significantly worse. deciding which older patients will benefit from hdt is challenging and there are no established predictive tools to guide physicians. we present a clinical prognostic index derived from information readily available at the time a patient is being assessed for asct the bsbmt audited the outcomes for uk patients aged 460 transplanted between 2004-2009 (n = 371) and benchmarked against the european bone marrow transplant (ebmt) database for the same period (n = 2695). the primary outcome was progression-free survival (pfs) but data was also analysed for overall survival (os), relapse rate (rr) and non-relapse mortality (nrm). we included all patients with a diagnosis of high grade nhl and the following demographic features were also analysed: age at diagnosis; age at transplant; m/f; year of transplant; cr/not cr at transplant; no. of prior therapies; no. of cells infused; clinical staging; karnofsky status at transplant; histology; ipi at diagnosis; mobilising regime and conditioning regime. candidate prognostic indices were factors achieving significance in univariate and multivariate analyses of the main outcomes by regression analysis. the best prognostic index was selected based on the bsbmt dataset and then applied to the rest of the ebmt dataset (the validation dataset). there were no significant differences in patient characteristics between the uk and non-uk groups nor in outcomes of pfs, os, rr or nrm. (figure 1 ). in both univariate and multivariate analysis the following features were associated with a significantly worse outcome for pfs, os, rr and nrm : age466, karnofsky score. disclosure of conflict of interest: none. underwent an allo-sct at our center after a treosulfan-based conditioning regimen. eleven pts received a mrd, 19 pts a mud, and 11 pts a haplo unmanipulated pbsc allo-sct. at allo-sct 17 pts were in cr, 10 pts were in pr, and 14 pts had sd/pd. hct-ci was evaluable for 34 pts, 18 had a score ≥ 3. the backbone conditioning regimen consisted of treosulfan 14g/m 2 from day − 6 to − 4, and fludarabine 30 mg/m 2 from day − 6 to − 2; twenty-five pts were treated with this reduced toxicity conditioning (rtc) regimen. intensification with other alkylating agent (melphalan, thiotepa, or cyclophosphamide) or radiotherapy (4gy total dose) was applied on the remaining 16 pts (myeloablative conditioning, mac). gvhd prophylaxis was based on cyclosporine a and methotrexate (17 pts) or rapamycin and mycophenolate mofetil (24 pts), plus anti-thymocyte globulin or post-transplant cyclophosphamide accordingly to donor type. median numbers of infused cd34 +/kg and cd3+/kg were 6.66 × 10 6 (range: 2.72-12.24) and 2.34 × 10 8 (range: 0.03-6.89), respectively. median follow-up was 61 months (range: 18-139). thirty-nine pts were evaluable for engraftment; median time to neutrophil ≥ 0.5 × 10 9 /l was 16 days (range: 10-30), and 16 days (range: 10-59) to platelet ≥ 20 × 10 9 /l. treosulfan conditioning provided a cr in 3 and 6 pts respectively in pr and sd/pd at transplant. no graft failure was observed. one and 5 years overall survival (os) was 58.5% and 52.6%, respectively. progression free survival (pfs) and gvhd-free/relapse-free survival (grfs) were respectively 51.2% and 41.5% at 1 year, 44.3% and 23% at 5 years. one and 5 years relapse/progression incidence (ri) was 29.3% and 36.1%, respectively. transplant related mortality (trm) was 14.6% at 100 days, 19.5% at 1 year and for the entire follow-up. the 100-day cumulative incidence (ci) of agvhd grade ≥ 2 was 4.9%; ci of moderate to severe cgvhd was 24.8% at 2 years. the outcome of pts in cr at 5 years was significantly better compared to that of pts with active disease in terms of both os (82.4% vs 33.3%, p o0.005), pfs (68.6% vs 25%, p o0.005), grfs (36.3% vs 12.5%, p o0.005), and ri (19.6% vs 50%, p o0.05). no statistical differences in os, pfs, and ri were found when pts were stratified according to donor type and [p602] the use of rtc or mac regimen. at last follow-up, 22 patients are alive and disease free; 3 of them obtained a durable cr using chemotherapy and/or dli for disease progression after allo-sct. treosulfan-based conditioning regimen is effective and well-tolerated in patients with advanced b-nhl undetgoing allo-sct. disclosure of conflict of interest: none. systemic anaplastic large cell lymphoma (salcl) is a very infrequent well-defined histological entity that comprises around 11% of all t-cell non-hodgkin lymphoma. in the absence of prospective clinical trials, autologous stem cell transplantation (autosct) is considered the standard of care as consolidation therapy after first line therapy for those patients not expressing the alk protein (alk neg salcl) and for patients with relapsed disease. the objective of this retrospective analysis was to analyse the long-term outcome of patients diagnosed with salcl and being treated with autosct during the course of the disease, making special emphasis on the potential impact of the administration of brentuximab vedotin (bv). eligible for this study were patients 18 years or above with salcl who underwent autosct between 2010 to 2014 and were reported to the ebmt. baseline patient, disease, and transplant data were collected from ebmt med-a standard forms. centers with potentially eligible patients were contacted to provide additional treatment and follow-up information including a written histopathology report for central review. seventy-nine patients (48 males) with a median age at diagnosis of 43 years (range: 14-70) and at transplantation of 45 years were included in the final analysis. thirty-nine patients were alk negative, 38 alk positive and in 2 patients expression of alk protein was unknown. at diagnosis, 60 patients (76%) presented with advanced stage and 48 (61%), with b symptoms. sixty-three patients (80%) received 1-2 lines of therapy before autosct. ten patients were treated with bv at some point before autosct; two patients as second line therapy, three as third line, one as fourth line and four as fifth line therapy. the median number of bv doses was 5 (range: [3] [4] [5] [6] [7] [8] . the median time between diagnosis and transplantation was 12 months (range: . most patients had chemosensitive disease at autosct [65 patients (82%)] and in all but 2 patients peripheral blood was used as the source of stem cells. conditioning regimen consisted on beam / beam-like protocols in 72 patients (91%). all patients engrafted. with a median follow up for surviving patients of 34 months (range: 2-71), 57 patients are alive (72%), 20 patients died (25%) and 2 patients (3%) are lost for follow up. disease relapse after transplantation was the most frequent cause of death after the procedure. cumulative incidence of non-relapse mortality for the whole series was 3% (95% ci, 0.5-9) at 100 days, 1 year and 3 years. cumulative incidence of relapse was 27% (95% ci 17-27) and 32% (95%ci 21-44) at 1 and 3 years, respectively. 1 and 3years progression free survival (pfs) was 70% (95% ci 60-82) and 65% (95% ci 54-78), respectively and 1 and 3-years overall survival (os) was 82% (95% ci 73-91) and 71% (95% ci 61-83), respectively. there were no significant differences in any of the outcomes between bv treated and non-treated patients. autosct results in a promising pfs and os in patients with salcl. the potential impact of the administration of bv as salvage strategy before the procedure needs to be further elucidated. disclosure of conflict of interest: none. coeliac disease (cd) is a t-cell immune-mediated enteropathy to dietary gluten, characterized by small bowel villous atrophy resulting in malabsortion. the enteropathy is reversible with a gluten-free diet (gfd), however symptoms and signs which persist 41 year are defined as refractory coeliac disease (rcd). rcd is divided into type i and ii, depending on absence/ presence respectively of clonal intra-epithelial t-lymphocytes (iels) with an aberrant phenotype (cytcd3 pos, membranous cd3, cd4 and cd8 neg). rcdii patients have a 5 year survival of 1.0, plts 420) was successful at a median of 11.5 (range: 10-15) days and no transplant-related mortality occurred. all patients achieved a clinical complete remission, with normalization of nutritional indices at 100 days, but persistently abnormal iels and clonal t-cells on duodenal biopsy. with a median follow-up of 42.5 (range: 22-56) months, 5 patients remain in clinical remission, 1 patient relapsed with rcd and no patient progressed to eatl. chemotherapy and asct is a safe and effective strategy for the treatment of rcd offering the possibility of sustained clinical responses. clonal tcr in duodenal biopsy/blood and iel flow cytometry form part of the patient evaluation prior to the chemotherapy/asct program. most patients with hodgkin lymphoma (hl) are cured with conventional chemotherapy. however, approximately 20% of patients relapse after primary treatment. for those, high-dose chemotherapy (hdc) followed by autologous stem cell transplantation (asct) is the standard of care. fifty seven adult patients with relapsed or refractory hl submitted to asct between 2000 and 2015 were reviewed. variables examined were sex, age, ann arbor stage (i-ii vs iii-iv), b symptoms, bulky disease, extranodal involvement, nodal areas involved (≥3vs12vs ≤ 12 months) and response to the treatment prior to asct. log-rank test was used to compare differences in survival for each factor. patients median age was 31 (17-64) years at diagnosis. ann arbor stage iii-iv in 35 (61%) patients, b symptoms in 25 (44%), extranodal involvement in 20 (35%) and bulky disease in 13 (23%). all patients were treated according to the abvd protocol in first line. indications for asct were relapsed disease (n = 30, 52.6%) and lack of complete response (cr) or progressive disease with 1st line treatment (n = 26, 45.6%). there were a median of 2 (2-5) treatment-lines before asct (protocols eshap, ice, beacoop, gvd and others). the disease was chemosensitive in 86% cases: cr in 24 and partial response (pr) in 25 patients prior to asct. refractory disease (rd) in 14% (n = 9). in 84.2% patients, the hematopoietic cells mobilization was performed under stimulation with granulocyte-colony stimulating factor in hematologic recovery after the cycle of 2nd line chemotherapy, and most of which required 1 (1-4) apheresis. conditioning regimens were beam (93%) and gmb (7%). the median time to hematologic recovery was 11 days (8-14) for neutrophils4500/ul and 13 days (9-25) for platelets420,000/ ul. three months after asct, thirty-nine (68.4%) patients had cr, one (1.8%) patient maintained pr and 6 (10.5%) patients had disease progression. status unknown in 7 patients and four (7%) patients died. relapse rate 32% (n = 15/47). with a median follow-up time after asct of 52 (0-169) months, median disease-free survival (dfs) was 26 (0-169) months and overall survival (os) was 52 (0-169) months. there were 19 deaths (33.3%), four (7%) related to early infectious complications of asct, two (2.6%) due to late infectious complications, eleven (21.1%) due to disease progression and 1 (1.8%) in context of secondary acute myeloid leukemia. response to the treatment prior to asct was the only factor with survival influence. the dfs and os differed significantly in chemosensitive disease compared with rd (dfs mean: 51 vs 21 months,p = 0.025, os mean: 64 vs 22 months, p = 0.007). the response to salvage treatment prior to asct is the main prognostic factor for survival after asct. prognosis remains poor in patients with rd or early and disseminated relapses. for these patients, the therapeutic approach should include intensive treatment with tandem hdc and stem cell transplantation, allogeneic transplant or early consolidation with brentuximab-vedotin after asct. hodgkin's lymphoma (hl), although considered a curable neoplasm in adults, could be associated with a very poor prognosis when refractory to primary induction therapy or when it relapses within 12 months from an autologous stem cells transplant (auto-sct). the optimal treatment of patients with heavily pretreated/refractory hl is controversial. brentuximab vedotin (bv) is an active single agent in this context; unfortunately, there are no well established therapies when patients fail to respond or progress after bv. encouraging results were recently described with checkpoint inhibitors. similarly, data pertaining to efficacy of bendamustine (benda) shows encouraging activity in various refractory lymphomas. we included in this study adult patients with hl who relapsed post auto-sct and were refractory to or progressed after salvage bv and were treated with benda as salvage therapy with an intention to proceed with an allo-sct. this study was [p606] conducted in two major centers in lebanon, the american university of beirut medical center (aubmc) and makassed university hospital. we identified 12 eligible cases. the primary study endpoint was objective response rate (orr). the secondary endpoint evaluated successful rate of bridging into an allo-sct. the median follow-up times from auto-sct and from benda salvage were 35 (14-59) and 10 (4-35) months, respectively. the median age of patients was 27 years (17-54). all patients had bv as salvage therapy post auto-sct, and all of them progressed after a median of 4 (3-6) cycles. clinical characteristics are outlined in table 1 . patients received a median of 6 cycles (2-8) of benda. the treatment was well tolerated, with rather infrequent adverse events and transient and manageable toxicities. the orr was 75%, in 9 of 12 patients, with 43% obtaining a complete response. eventually, 6 of 9 proceeded to allo-sct using a matched related donor, and the remaining 3 patients are planned for allo-sct. only one patient died from disease progression after 24 months post allo-sct. two of 3 patients who progressed following benda received salvage therapy with nivolumab and are being planned for haplo-identical transplant while the third one is being planned for therapy with nivolumab. from the initiation of benda, the median duration of response for the 9 patients was 10 months (4-29); all these patients had maintained a continuous response at the last follow-up examination. conclusion: notwithstanding the limitations associated with our analysis, namely a small sample size and its retrospective nature, these results suggest a role for bendamustine in post bv failures. these findings also provide the basis to evaluate the concept of benda as a bridge to allo-sct in a large prospective study. [p607] disclosure of conflict of interest: none. brentuximab vedotin for relapsed or refractory hodgkin lymphoma, single center experience king faisal specialist hospital and research center, riyadh, kingdom of saudi arabia ms rauf 1 , i maghfoor 1 , a badran 1 , mn zahir 1 and s akhtar 1 1 hodgkin lymphoma (hl) patients with relapsed or progressive disease after high dose chemotherapy (hdc) and auto-sct have limited curative options. fda granted approval of brentuximab vedotin (bv) for the treatment of hl and anaplastic large cell lymphoma (alcl) patients who fail auto-sct or have had at least 2 prior multiagent chemotherapy regimens and are not candidates for auto-sct. we are reporting single center experience of bv usage in this "approved" setting. medical records were reviewed to collect required data. kaplan-meier (km) method was used to calculate overall survival (os) and progression free survival (pfs) from date of first dose of bv. from 2013-2015, 25 patients received bv. 24/25 had hl (22 classic hl-nodular sclerosis, 2 hl-mixed cellularity) and 1 alcl. 19/25 (76%) pts were primary refractory or had early relapse after initial treatment. 15/25 (60%) pts received bv were refractory to the last treatment. all the baseline characteristics of patients are mentioned in table 1 . median bv cycles administered were 6 (2-16). overall response rate (orr) was 40% (10 patients): cr in 6 (24%), pr in 4 (16%) (5/10 were primary refractory or early relapsed). median pfs for whole group was 5 months (95% ci, 3.6-6.3). km estimated 1-year os was 74% and 2 year was 68%, median os has not been reached yet. for 10 patients who responded, pfs at 12 months was 68% (95% ci, 38%-98%), median pfs not reached. for 15/25 patients with progressive disease (pd) or non responders after bv, median pfs was only 3 months (95% ci, 1.1-4.8). there was no difference in os between patients with responders and non responders. median os has not yet been reached in either group as mentioned in survival curves. at the median follow up of 19 months (range: 4-55 months) 18 patients are alive, 10 patients are alive without disease, 4 patients received consolidation bone morrow transplant (2 auto-sct and 2 allo-sct). 2 patients completed 16 courses and achieved cr. rest of 4 patients who are alive without disease; they had pd on bv but achieved cr with other treatments. 8 patients are alive with disease; 1 patient is on bv and 7 are on another treatment. 7 patients have died, 2 because of pneumonia while being on bv and 5 due to pd. 3/15 patients who received bv, achieved cr after failing all previous treatments and are in cr. peripheral sensory neuropathy developed in 3 patients; one required dose reduction. 1 patient stopped treatment due to pulmonary toxicity. we are reporting largest single center data from middle east which confirms that bv as a single agent is effective and safe. overall response rate is lower as compare to pivotal trial but cr rate is comparable to other reported case series. this analysis also concludes that bv can be used as bridge to transplant in patients who don't respond salvage chemotherapy. disclosure of conflict of interest: none. was used to diagnose hiv infections. cox proportional hazards models were used to evaluate risk factors of overall mortality. fifty-six patients with nhl (1.4%) and 23 patients with mm (0.8%) were positive for hiv antibody. in patients with nhl, overall survival was significantly lower in the hiv-infected patients than in the hiv-negative patients [5year overall survival: hiv-infected patients, 44% (95% confidence interval, 29%-58%) vs. hiv-negative patients, 65% (95% confidence interval, 63%-67%), p o0.001)]. in a multivariate analysis, hiv infection was significantly associated with an increased risk of mortality (hazard ratio 2.39, p o0.001), and this effect was consistent regardless of transplant year. on the other hand, overall survival in patients with mm was similar between the 2 groups [61% (95% confidence interval, 31%-82%) vs. 63% (95% confidence interval, 63%-67%), p = 0.988]. previous studies in europe and the united states showed comparable survival rates between hiv-infected and hiv-negative patients with nhl. however, our study showed that hiv infection was associated with a higher risk of mortality in patients with nhl in japan. suppression of t cell-mediated immunity or hiv related diseases might affect transplant outcomes in japanese patients. [p609] disclosure of conflict of interest: none. while beam and beac regimens (bcnu, etoposide, cytosar in both regimens and melphalan or cytoxan, respectively) are commonly used as conditioning high-dose therapy (hdt) in patients with non-hodgkin lymphoma (nhl), there have been few reports comparing these regimens. a retrospective analysis found the superiority of beam over beac in terms of overall survival (os) and event-free survival (efs). toxicities were similar, except that beam was associated with more frequent lower gastrointestinal (gi) mucositis. other studies reported that these regimens had similar efficacy and outcome. recently, a concern regarding cardiotoxicity of beac has risen. the current study aimed to compare efficacy and toxicity of beac and beam as consolidation hdt in young patients with mantle cell lymphoma (mcl) undergoing autologous stem cell transplantation (asct). this is a retrospective analysis of outcomes in mcl patients who received hdt with beam or beac followed by asct at 3 bone marrow transplant centers in israel. os, disease-(dfs) and progressionfree survival (pfs) and regimen toxicity were compared. seventy seven mcl patients who were diagnosed between 1995-1/2016 and received consolidation with beac or beam were included in the analysis. forty nine patients were treated with beam and 28 patients-with beac. no significant differences between the groups were revealed in terms of age, sex, the mantle cell lymphoma international prognostic index (mipi) risk score, induction protocol and% of patients transplanted in first complete response (cr1) (mean age 57 yrs in beam vs 59 yrs in beac group; 69% of patients in beam group had mipi risk score 2-3 vs 62% in beac group; 68% of patients in beam group were transplanted in cr1 vs 71% in beac group). the amount of infused cd34 cells was significantly higher in the beam group (median cd34 cells/ kg: 8.2 in beam vs 4.6 in beac groups; p = .001); the number of days to platelet engraftment was significantly greater in the beac group (median 12 days in beam vs 14 days in beac group; p = .02). there were no differences in the number of blood transfusions or hospitalization days between the groups. the rate of grade 3-4 upper mucositis was significantly higher in the beam group (41% in beam vs 18% in beac group; p = .046); no other differences in toxicity (grade 3-4 lower mucositis, pulmonary congestion, infections) were observed between the regimens. non-relapse mortality by day 30 posttransplant was 0% in both groups. a median follow-up was 29 (range: 1-119) months. the 3-yr dfs in beam and beac groups was 58% and 64%, respectively (p = .65). there was no difference in the 3-yr os between the groups (70% in beam and 84% in beac group; p = .51). there was a trend to improved dfs and os in patients transplanted in cr1 receiving beam (p = .09, figure) . in multivariate analysis, low-to-intermediate mipi and transplant in cr1 were found to significantly increase pfs (p = .04 and.01, respectively), while the hdt regimen did not affect pfs. beac and beam hdt regimens followed by asct had similar efficacy in mcl patients. there was a trend to improved dfs and os in patients transplanted in cr1 and treated with beam vs beac. the toxicity profile was similar in both groups, except a significantly higher rate of grade 3-4 upper gi mucositis. [p610] disclosure of conflict of interest: none. early or refractory relapsed ( o1 year) diffuse large b-cell lymphoma has a very poor prognosis especially for those not responding to salvage chemotherapy. allogeneic stem cell transplantation is potentially curative. even though this is less likely in those not responding or having frank progression pretransplantation. methods: at our institution we identified all patients with aggressive b-cell lymphoma (diffuse-large b-cell lymphoma and blastoid mantle cell lymphoma) who were refractory or progressive to salvage chemotherapy with r-dhap and who had peripheral blood stem cells (42 × 10 6 cd34+/kg body weight) collected after the 1st or 2nd cycle. after high-dose melphalane and autologous stem cell transplantation 13 patients had a partial and 6 a complete remission. 1 patient died due to neutropenic infection, 2 patients died due to progressive disease leading to a transplant related mortality of 3.5%. median progression-free survival after autologous transplantation was 4.6 months. 24 proceeded to allogeneic stem cell transplantation. 8 patients had a matched related sibling, 9 had a matched unrelated donor and 7 had a mismatched unrelated donor. transplant related mortality was 42% in this heavily pretreated population. 2-year overall survival of all patients intended for treatment is 21%. one of these patients with relapsed mediastinal lymphoma after allogeneic transplantation was cured by salvage radiotherapy and is in long-term remission (42 years). conclusions: salvage high-dose melphalane and autologous peripheral blood stem cell transplantation for diffuse large b-cell lymphoma as a bridge to allogeneic transplantation is potentially curative for a minor fraction of these patients. however, the remission rate of 79% (46% pr, 21% cr) and progression-free survival of 4.6 months after high-dose melphalane and autologous stem cell transplantation provides a window of opportunity to use new drugs and cellular therapies in these poor prognosis patients. high dose chemotherapy and autologous stem cell transplantation is the treatment of choice for patients with relapsed refractory hodgkin lymphoma. several factors including number of chemotherapy lines received before conditioning, time of relapse and remission status before transplantation can predict survival and pfs in patients undergoing autologous stem cell transplantation. in 2012, we reported on a 63 patients who underwent high dose chemotherapy followed by autologous stem cell transplantation from 2003 to 2008. all patients with relapsed or refractory hodgkin lymphoma in the period of 2009-2013, who underwent high dose chemotherapy followed by autologous transplantation were retrospectively analyzed. the main outcomes of the study were complete remission (cr) at day 100, overall survival (os) and relapse-free survival (rfs). the impact of the following variables on os and rfs: (a) disease status at the time of transplant, (b) number of chemotherapy lines prior to conditioning and (c) time of relapse 12 months and (d) age. a total of 78 patients were identified. the median age was 31 year. there were 50.6% females and 49.4% males. complete remission (cr) was achieved in 48.7% of patients and 49.9% with chemotherapy sensitive disease at the time of transplantation. prior to conditioning regimen, 43.2% received two chemotherapy lines, and 56.8% received more than two lines. 41% relapsed in less than 12 months and 57% relapsed more than 12 months after completion of therapy cr at day 100 was 69.2%. the median os for the whole group was 62.0 months; the median rfs was 10,6 months. the number of chemotherapy lines significantly impacted os and efs. cr status before conditioning, favorably influenced os and efs with a trend toward better os in favor of those who underwent abmt while in complete remission. the time of relapse and the age did not affect survival outcomes. [p614] the outcome of patients with relapsed or refractory hodgkin lymphoma is favorable and the number of chemotherapy lines received before conditioning is the only factor that had a statistically significant impact on os and efs. since the identification of human immunodeficiency virus (hiv), a clear association between hiv and specific malignancies has been recognized. high-grade b cell lymphomas are the most common malignancy complicating hiv infection and one of three aids defining malignancies. diffuse large b cell lymphoma (dlbcl) accounts for 80% of cases. before 1996, lymphomas were the cause of 16% of all deaths attributable to aids. after the introduction of highly active antiretroviral therapy (haart) overall incidence of adm declined, however longer survival and exposure to environmental risk factors have increased the incidence of non adm (adm) such as hodgkin's lymphoma (hl). since haart has improved overall survival substantially, the aim of chemotherapy should be complete remission rather than palliation with careful consideration of drug interactions and side of haart. between 2011 and 2016 a total of 510 patients were detected hiv positive. twenty-one of these patients were diagnosed with a malignancy and 8 patients referred to our department with a hematologic malignancy were evaluated retrospectively. diagnosis, stage, treatment, survival data were recorded. haart during chemotherapy, nadir cd4 count and cd4 count at diagnosis of malignancy was evaluated. four patients were diagnosed with high grade b cell lymphoma, 2 patients with primary central nervous system lymphoma (pcnsl), 1 patient with hl and 1 patient with multiple myeloma (mm). all patients were male and median age at diagnosis was 40.5 (24-63). hiv seropositivity was identified during evaluation of malignancy in both pcnsl patients. median duration of hiv seropositivity before diagnosis of malignancy was 18 months for the remaining patients. patient characteristics, treatment modification and cd4 counts are summarized in tables 1 and 2 . lymphoma was fatal in 5 and the cause of death was identified as lymphoma progression in all patients including one patient diagnosed with hodgkin's lymphoma. a patient presented with multiple plazmositomas was diagnosed with multiple myeloma is currently receiving induction treatment together with haart. hiv related lymphoma patients frequently present with extra nodal disease, incidence of central nervous system involvement is also higher and prognostic score tends to be in the intermediate or high-risk groups. prognosis is also worse than hiv negative population. degree of immunosuppression is implicated and the duration of immunosuppression is directly correlated with the risk of developing lymphoma rather than hiv itself. haart allowed the use of aggressive chemotherapy since it improved immune system and decreased infectious complications. multiple myeloma is a rare neoplasm observed in hiv infection and the treatment is based on data obtained from hiv negative patients. treatment of such patients as well as lymphomas should take into consideration the toxic effects of haart combined with chemotherapy. since hiv positive [p615] patients are excluded from most studies, there are no guidelines to direct treatment and avoid toxicities. drug interactions should be monitored closely and modifications should be made accordingly. interruption of haart may not be mandatory since studies have shown safety of continuation of haart during chemotherapy. for newly diagnosed hiv and malignancy, careful clinical and laboratory evaluation should be made before postponing haart until after chemotherapy. disclosure of conflict of interest: none. the outcome of hdct and asct in refractory hodgkin lymphoma (r-hl) is not as encouraging as in relapsed hl. ten years ago we analyzed and reported outcomes of asct in our r-hl patients, however the follow-up was short. now we a reporting long term outcomes in r-hl after asct in one of the largest numbers reported to date. between 1996 and 2014, patients with hl who underwent hdc and asct for r-hl in adult medical oncology (age 4 14 years) were identified. r-hl is defined as partial response (pr), no response (nr), stable disease (sd), progressive disease (pd), relapsing within 3 months (relapse o3 m) of finishing the planned (chemotherapy + radiation therapy (xrt)) treatment or refractory to salvage chemotherapy. kaplan-meier (km) method was used to estimate progression free survival (pfs) and overall survival (os) from the day 0 of asct while progression is defined as progression of disease, relapse and death from any cause. all percentages are rounded to nearest. 307 patients underwent hdc and asct during 1996-2014 and 177 of them met the criteria of r-hl. male 97 (55%), female 80 (45%), median age at diagnosis was 22.2 years (8-61 years) and at asct was 24 years (14-62 years). initial therapy was abvd in 153 (86.4%), mopp/copp alternating with abv or abvd in 11 (6%) and others in 13 (7%). 49 (28%) had xrt after initial chemotherapy. response to initial chemo + xrt was pr in 80 (45%), pd in 51 (29%), cr in 38 (21%) (28/38 relapsed within 3 months and others have refractory relapse) and no response in 4 (2%) and others in 4 (2%). prior to salvage chemotherapy, 119 (67%) had stage iii-iv, 90 (51%) extra-nodal involvement, 43(24%) bulky disease and 31 (18%) had b symptoms, spleen involvement in 43(24%), performance status 0, 1 in 155 (88%). eshap was used as first line salvage in 153 (86%) or 3 rd line 13 (7%). post salvage / prior to hdc and asct disease status was pr in 112 (63%), cr in 53 (30%) and nr/sd in 12 (7%). 111 (63%) patients had a fdg-pet scan prior to asct, 45 (25%) were in cr. beam was used as conditioning regimen. median follow-up for all alive patients is 81 months (15-224) from asct. response rate post asct: cr in 105 (59.3%), pr in 16 (9%), nr/sd in 1 (0.6%) and pd in 45 (25.4%) patients, others /unknown in 10 (5.6%). 61 (35%) patients had xrt post auto-sct. type of first post hdc auto-sct event was no event in 81 (46%), persistent disease in 17 (10%), pd in 45 (25%), relapsed disease in 23 (13%), treatment related mortality in 6 (3%) and died of other cause 5 (3%). at last follow-up in november 2016, 94 patients (53%) are alive with no disease, 6 (3%) alive with disease, 64 (36%) died of disease and 13 (7%) died of treatment related mortality or other causes. for entire group, km estimated median os is 129 months, 1,2,3,5 and 7 year survival is 83%:73%:64%:59%:55% respectively. median pfs is 48.5 month, 1,2,3,5 and 7 year pfs is 57.6%:53.6%:51%:50%:47% respectively. we are reporting a very high risk group of patients with a very long follow-up. in patients with r-hl, eshap + beam combination resulted in high response rate (68.3%). these remissions are durable. a 5 year os survival of greater than 50% in our population is higher than most reports with similar numbers. although our cohort has a 7 year os survival of 55%, 45% patients have either relapsed or died underscoring need for improvements in the management refractory hl. [p616] disclosure of conflict of interest: none. here we update the previously reported results of our reduced-intensity conditioning (ric) allo-hsct experimental program, initiated in 2002. as of november 2016, in our centre 34 patients underwent ric allo-hsct. donors were hla-identical sibling in 16, fullymatched unrelated in 7, 1 or 2-mismatch-unrelated in 9 and haploidentical relative in 2. median age was 53 years (range: 19-66). all patients (22 m and 12 f) had stage iib/iv refractory mf (n = 22) or refractory ss (n = 12). median number of previous treatment lines was 6 (range: 2-12). source of stem cells was peripheral blood in 31 patients and bone marrow in 3. median time from diagnosis to hsct was 46 months (range: 13-264). conditioning included flu/ctx/tbi200, pentostatin +tbi200 and flu/mel in case of hla-identical or unrelated donor, whereas the tt/flu/ctx/tbi200 regimen was used in the haplo setting. gvhd prophylaxis included csa/mmf in all patients, with the addition of atg in cases with unrelated donor and post-transplant ctx (50 mg/kg giorni +3 e +4) in cases with haploidentical donor. full donor chimerism was obtained in 28/33 of the evaluable patients, in a median time of 2 months (range: 1-12). grade ii-iv acute gvhd occurred in 16 patients (57%), while grade iii-iv was observed in 8 patients (28%). chronic gvhd occurred in 10 patients (36%), being extensive in 4 (14%), all transplanted from hla-identical sibling (no atg). following transplantation, a complete remission (cr) was achieved in 22 out of the 33 evaluable patients (67%), of whom 2 experienced relapse at +25 and +35 months, respectively. transplant-related death occurred in 6 patients (17%), of whom 4 were in cr. out of the 11 patients who did not achieve cr, 9 died from progressive disease (median follow-up of 12 months, range: 3-31), 1 from a secondary malignancy and 1 is still alive with disease 41 months after transplant. of note, all pts who died in progression had chemoresistant disease at time of transplant. at the last follow-up, 18 patients were alive and 16 (89%) maintained cr after a median time of 66 months (range: 4-189). in the whole population, the 5-year overall survival was 52% (95% ci 34-70) and the 5-year disease-free survival (dfs) was 44% (95% ci 27-62). however, when mf and ss were analysed separately, 5-yrs dfs were 32% (95% ci 12-51) and 69% (95% ci 38-99), respectively (figure) . apart from diagnosis, outcome appeared to be primarily associated with the disease status at transplantation, with a 5-yr dfs of 100% in the group of patients (n = 8) who were in cr before starting the conditioning. after a median follow-up longer than 5 years, we confirm the efficacy of ric allo-hsct as a powerful therapeutic strategy in inducing and maintaining remission in selected patients with chemosensitive advanced-stage ctcl, with results particularly encouraging in ss. [p617] disclosure of conflict of interest: none. outcomes of allogeneic hematopoietic stem cell transplantation for hodgkin lymphomas: a retrospective multicenter experience of the rete ematologica pugliese (rep) f gaudio 1 , p mazza 2 , am carella 3 , d pastore 1 , g pisapia 2 , a mele 4 , p galieni 5 , n cascavilla 3 , g specchia 1 and v pavone 4 1 hematology, university of bari, bari, italy; 2 hematology, ospedale "san giuseppe moscati", taranto, italy; 3 hematology, ospedale "casa sollievo della sofferenza", san giovanni rotondo, fg, italy and 4 hematology, ospedale "cardinale panico", tricase, le, italy; 5 hematology, ospedale "c. g. mazzoni", ascoli piceno, italy hodgkin's lymphoma (hl) is a potentially curable disease, and modern therapy is expected to successfully cure more than 80% of the patients. second-line salvage high-dose chemotherapy and autologous stem cell transplantation (sct) have an established role in the management of refractory and relapsed hl, leading to long-lasting responses in approximately 50% of relapsed patients and a minority of refractory patients. patients progressing after intensive treatments, such as autologous sct, have a very poor outcome. allogeneic sct represents the only strategy with a curative potential for these patients; this study reports a retrospective multicenter experience of the rete ematologica pugliese (rep) over the past 16 years aiming to define the impact of patient, disease, and transplant-related characteristics on outcomes. 67 patients with histologically confirmed diagnosis of hl who received allogeneic sct from 2000 to 2016 were retrospectively studied. the median age was 34 years (range: 16-57 years) and 36 (54%) were male. the majority of patients (84%) had had a prior autologous sct. at the time of allogeneic sct, 28 (42%) patients had a chemosensitive disease and 39 (58%) were chemorefractory. most (93%) patients received reduced-intensity conditioning, 54% received matched sibling donor and 46% matched-unrelated donor grafts. the disease status at day 100 post-transplant was reported in 62 out of 67 evaluable patients. of the 26 patients with chemosensitive disease, 18 (70%) achieved a cr, 7 (27%) had a pr or stable disease and 1 (3%) had progressive disease. of the 36 patients with chemorefractory disease 7 achieved a cr (20%), 26 had a pr or stable disease (72%) and 3 (8%) had progressive disease. although the overall survival has improved significantly in mantle cell lymphoma (mcl) according to advanced treatment options, relapsed or refractory disease remains a challenge. recently, lots of targeted agents actively have been tried clinical studies and adapted to clinical practice in indolent lymphoma. however, the role of frontline autologous hematopoietic stem cell transplantation (auto-hsct) has not been fully understood in patients with mcl, compared with a few impressive published data about auto-hsct as salvage treatment option for patients with relapsed mcl. so, we retrospectively evaluated consecutive patients diagnosed mcl, and compared the clinical outcomes of high-dose chemotherapy followed by auto-hsct and conventional chemotherapy alone. between january 2003 and december 2014, consecutive patients with newly diagnosed with mcl at catholic blood and marrow transplantation center in south korea were included in this study. all of the patients received high-dose cytarabine-containing regimen or chop with/without rituximab regimen for induction therapy regardless of transplant eligibility. the treatment approach in our institution for patients was based on the physician discretion for transplant eligibility or ineligibility that depend on patient age, comorbidities, and disease status. seventy patients were included in the analysis. initial chemotherapy regimens were consisted of chop (n = 12, 17%), r-chop (n = 44, 63%), r-hypercvad (n = 10,14%), and hypercavd (n = 4, 6%). demographics and disease characteristics of both groups are shown in table1. patients received auto-hsct were superior s449 overall survival (os; p = 0.015) and progression-free survival (pfs; po0.001). the subgroup analysis according to high-risk of mcl international prognostic index (mipi) or bone marrow involvement was performed. between the two treatment arms among the high-risk mcl group, the clinical parameters were not different. the high-risk mcl patients with frontline auto-hsct showed superior os (p = 0.0216) and pfs (po0.001) compared with conventional chemotherapy alone. although mcl is classified within indolent lymphoma, frontline auto-hsct can be considered for patients diagnosed with mcl in the group of high-risk mipi or bm involvement with the favorable survival outcomes. disclosure of conflict of interest: none. nasal type extranodal nk/t-cell lymphoma (enktl) is a very rare and agressive malignancy characterized by a poor outcome. current standard therapy is not yet established. the role of high dose therapy followed by haematopoietic stem cell transplantation (hsct) is still controversial. we evaluated the outcomes of all the enktl patients undergoing hsct in a multicenter analysis on patients registered by the société francophone de greffe de moelle et de thérapie cellulaire (sfgm-tc) and compared them with a population of french patients who received chemotherapy alone. sixty four enktl (48 males and 16 females) received hsct, including 19 allogeneic (allosct) and 45 autologous transplantations (autosct). median age at the time of hsct was 43 years (range: 17 to 70 years). overall, 57% of the patients presented with disseminated disease (64% and 55% in the allosct and autosct, respectively), 61% were in complete response (cr) at the time of hsct (74% and 61% in allosct and autosct groups, respectively) and 82% had received l-asparaginase regimen prior to hsct (73% and 84% in allosct and autosct groups, respectively). five (26%) and 20 (44%) patients of the allosct and autosct groups underwent upfront hsct therapy, respectively. four patients received tandem autologous/ allogeneic transplants. in allosct, stem cell source was a matched related donor in 13 patients, an unrelated donor in 3 patients and an umbilical cord blood in 3 patients. reduced intensity conditioning regimens (based on fludarabine-busulfan combination) and beam regimen were used in 42% and 84% of patients from the allosct and autosct groups, respectively. median overall survival for the whole cohort was 17.1 months (range: 1 to 131 months). the 3-year non-relapse mortality was 16.1% and 22.7% in the allosct and autosct groups, respectively (p = 0.58). the 3-year overall survival (os) and progression free survival (pfs) were 47.5% and 40.8% in the autosct and 43.4% and 47.4% in the allosct group, s450 respectively ( figure 1a) . the absence of cr prior to hsct was associated with a poor prognosis (p = 0.008). as compared to allosct, autosct resulted in a better outcome in patients who didn't achieve cr before transplant (p = 0.002) and tended to have better outcome in high pink risk score (figure 1 b-c) . finally, at 3 years pfs and os of patients who have been treated by chemotherapy alone (ct) (n = 55) or followed by allosct (n = 12) or autosct (n = 17) in cr1 were 55%, 81% and 50%, 54% and 44%,50%, respectively ( figure 1d ). in this french cohort, more patients received autologous hsct in upfront therapy than allogeneic hsct. in cr1, there is no evidence suggesting that transplantation is associated to a better outcome than chemotherapy alone. however, a precise matching based on the pink score will be evaluated to ensure that patients who were intensified were not of worst prognosis. in refractory patients there is also no clear advantage to perform allosct when compared to autosct. however, in relapsing disease after ct or autosct allosct, allowed to obtained durable control of the disease. disclosure of conflict of interest: none. high relapse rate is one of concerns for allo-sct in pts with relapsed/refractory aggressive lymphoma. an optimal conditioning regimen designed for aggressive lymphoma may reduce relapse, especially during early post-transplantation period. however, it is not established yet. results of a german phase 2 study of allo-sct with conditioning regimen of fludarabine, busulfan (12 mg/kg po or 9.6 mg/kg iv), and cyclophosphamide with or without post-transplantation rituximab for relapsed/refractory aggressive lymphoma suggested the role of myeloablative busulfan-containing regimen in reducing relapse rate in pts with aggressive lymphoma. based on these results, we conducted a single institution prospective study to explore feasibility of the bmf regimen consisted of full-dose busulfan, melphalan, and fludarabine in pts with relapsed/refractory aggressive lymphoma (umin000013940). patients with aggressive lymphoma who achieved at least sd with salvage chemotherapy after experiencing either pd during first-line therapy, early relapse ( o12 mo) after firstline therapy, late relapse (≥12 mo) but refractory to salvage therapy, relapse after auto-sct,; age 20-65; ecog ps of 0-2; and without severe organ dysfunction were eligible. donor source could be 6/6 matched related or unrelated donor pb/bm or cb with ≤ 2 antigen mismatch; the bfm regimen was consisted of busulfan 12.8 mg/kg iv, fludarabine 180 mg/m 2 , and melphalan 80 mg/m 2 (yamamoto h. bbmt 2016). gvhd prophylaxis was csa + mtx (related pb), tac + mtx (unrelated bm), and tac + mmf (cb). primary end point of the study was survival with engraftment at day 60, and secondary end points were engraftment rate at day 100; nrm and relapse rate at day 100 and 1 y; progression free survival (pfs), overall survival (os), and gvhd at 1 y. protocol was approved by irb and written ic was obtained from all pts. twelve pts (male 10, female 2) with a median age of 55 y (33-63) were enrolled. ps was 0-1 in 11 pts. diagnosis were dlbcl (n = 4), transformed fl (n = 3), enktcl (n = 3), ptcl (n = 1), and aitl (n = 1). median number of previous line of therapy was 3.5 (2-5) and 5 pts had failed previous auto-sct. diseases status at transplantation was cr (n = 6), pr (n = 4), and sd (n = 2). donor source was cb (n = 6), unrelated bm (n = 5), and related pb (n = 1). survival with engraftment at day 60, primary endpoint of the study, was achieved in 100%. neutrophil engraftment was achieved at a median of day 18 (13-32). full donor chimerism at day 30 was achieved in all of the 11 pts evaluated. two pts developed vod which was manageable. with a median follow-up of 20 mo, 3 pts had progression of lymphoma at 2, 5, 6 mo. five pts died and cause of death were progression of lymphoma in 3, interstitial pneumonitis in 1 (at 5 mo), systemic adenovirus infection in 1 (at 5 mo), and agvhd in 1 (at 4 mo). os and pfs at 1y were 54% and 46%, respectively. relapse and nrm rates were 8% and 0% (day 100), and 27% and 27% (1y), respectively. agvhd of grade ii-iv was observed in 7/12 pts and 2 pts developed limited cgvhd. this prospective study shows that allo-sct using myeloablative conditioning regimen with full-dose busulfan, melphalan, and fludarabine for relapsed/refractory aggressive lymphoma is feasible and deserves further evaluation. disclosure of conflict of interest: none. for patients with advanced ctcl, the allogeneic hsct seems to be curative with graft versus lymphoma effect playing a major therapeutical role. in this retrospective study, 16 patients with a median age of 52 years (range: 24-67) affected by ctcl underwent allogeneic hsct after a median of 4 (range: 1-8) lines of chemotherapy, including autologous transplant for 2 of them. the median time from diagnosis to hsct was 46 months (range: 9-309). the diagnoses were: sezary syndrome (ss, n = 8). mycosis fungoides (mf, n = 2), primary cutaneous cd30+ lymphoma (n = 4), panniculitis-like t-cell lymphoma (n = 1), nk t cell lymphoma (n = 1). at time of hsct, 2 patients (12.5%) were in complete remission (cr), 9 (56.3%) in partial remission (pr) and 5 (31.2%) had active disease. the patients were transplanted from an hla-identical (n = 7), mismatched (n = 1) or haploidentical (n = 1) sibling, from matched unrelated donor (n = 5) or from a single cord blood unit (n = 2). different pre-transplant regimens were used as myeloablative (mac) in 6 (th-bu-flu, n = 4; bu-cy, n = 2) or as reduced intensity (ric) in 10 (th-flu-cy, n = 7; th-bu-flu, n = 3). al patients engrafted for neutrophils at a median of 17 days (range: 12-46) and 14 patients engrafted for platelets at a median of 14 days (range: 12-77). acute gvhd was of grade 0-i in 9 patients and ii-iv in 7 (40.7%). skin was the most common organ involved. five of 11 evaluable patients experienced chronic gvhd which was mild in 3 and severe in 2. at a median of 3 months (range: 1-11), 7 patients died (4 mac and 3 ric) because of gvhd (n = 4), vod (n = 1), pneumonia (n = 1) or multiorgan failure (n = 1). all 12 patients surviving at 3 months from transplant were in cr. only patients prepared with a ric (n = 4) relapsed respectively at 3, 9, 10 and 11 months from hsct. these patients received dli associated or not to chemotherapy. three achieved cr, which remained stable in 2, while one patient died in cr from post dli acute gvhd. one patient (nk-t cell) not achieving cr is still alive with active disease. for all 16 patients the median survival was 10 months (range1-130). with a median follow up of 76 months (range: 4-130), 9 patients (2 mac, 7 ric) are alive, 8 in cr and 1 with active disease. at 10 years, the os was 54 ± 13%; at 5 years dfs was 34 ± 12%. according with the median time (46 months) from diagnosis to transplant, the 10-year os was 88 ± 12% for patients transplanted early and 25 ± 15% for the others (p o0.04), while dfs was respectively s451 58 ± 19% and 13 ± 12% (p o0.05). despite the small number of patients, our results confirm the high susceptibility of ctcl to the graft versus lymphoma effect and point out the time to transplant as a crucial prognostic factors for the outcome. finally, the long-term follow up of our series strongly supports hsct for the cure of ctcl. disclosure of conflict of interest: none. recently, a new prognostic score, the nccn-international prognostic index (ipi) has been developed 1 to stratify patients affected by diffuse large b cell lymphoma (dlbcl), and in high-intermediate and high risk groups the survival was equal or less than 50%. the aim of this analysis was to evaluate the outcome of a cohort of dlbcl patients undergoing high dose chemotherapy (hdc) as consolidation following first line chemo-immunotherapy, after their re-classification according to the nccn-ipi. we performed a retrospective study on 221 patients diagnosed with dlbcl, with a high/intermediate or high-risk disease according to the ipi (2-5), who received upfront hdc with asct, in 2 institutions. the patients were then re-stratified according to the nccn-ipi and arbitrarily classified in 2 groups: low risk (nccn-ipi ≤ 3) and high risk (nccn-ipi ≥ 4). the pre-transplantation disease status was assessed by positron emission tomography (pet) or computed tomography (ct). the primary endpoints were non-relapse mortality, progression-free survival (pfs), overall survival (os) and relapse risk. the estimated 3-year pfs for all patients was 80.1% (95% confidence interval [ci] 74.2-86.0) and the 3-year os was 91.0% (95% ci 86.7-95.3). of these patients, 93 had a low risk ipi score (ipi = 2) and 128 were considered high risk (ipi ≥ 3). subsequently, the whole cohort was re-stratified according to the nccn-ipi: 133 patients were allocated to the high-risk (nccn-ipi ≥ 4) group, and 88 to the low-risk group (nccn-ipi ≤ 3). the analyses were then carried out for both groups. the 3-year pfs was 92.0% (95% ci 85.7-98.3) in the low-risk group and 71.2% (95% ci, 62.2-80.2) in the highrisk group (po 0.01), whereas the 3-year os was 98.8% (95% ci 96.4-100) in the low-risk group and 85.0% (95% ci 77.9-92.1) in the high-risk group (p = 0.02). the significant difference in os and pfs between the two groups was mainly due to the cumulative incidence of relapse at 3 years (graph 1): 8.0% (95% ci 3.2-15.7) in the low-risk group and 27.1% (95% ci 18.7-36.2) in the high-risk group (po 0.01). non-relapse mortality was comparable in both cohorts: 1% (95% ci 0.2-3.3) for all patients. figure 1 : cumulative incidence of relapse following hdc and according to nccn-ipi. patients affected by high-risk dlbcl still have an unsatisfactory prognosis after treatment with conventional therapy regimens, even in the rituximab era. the 5-year os and pfs in patients with nccn-ipi score ≥ 4 range: from 33% to 64% and from 30% to 51% respectively 1 . although this is a retrospective analysis subject to all related biases, our results suggest that upfront intensive therapy with autologous stem cell transplantation may significantly improve the outcome of these patients compared to conventional chemotherapy. the role of hdc in the treatment of dlbcl is controversial. however, new entities or new risk stratifications, as the one reported here, could allow to identify high risk subpopulations that could benefit from this approach. enteropathy-associated t-cell lymphoma (eatl) is an exceedingly rare and often rapidly fatal subtype of peripheral t-cell lymphoma, arising from intraepithelial lymphocytes. eatl type i is associated with celiac disease; type ii occurs in patients without inflammatory pre-conditions (according to who 2016 classification now called monomorphic epitheliotropic intestinal t-cell lymphoma (meitl)). surgical debulking and anthracyclinebased chemotherapy (ctx) followed by high-dose chemotherapy (hdctx) and autologous cell rescue (asct) are pursued when possible in this often malnourished and frail patient cohort. yet, even with intensive consolidation relapse occurs in 40-70% of patients. the value of allogeneic hematopoietic cell transplantation (hct) is not clarified as of today due to limited reports. here, we report on a patient with meitl who was rescued with an allo-hct for his 2nd relapse following prior asct. moreover, we summarize the available literature on the use and outcomes of allo-hct for eatl and meitl. a 48y old man with spontaneous intestinal perforation was diagnosed with meitl following emergency partial resection of the small intestine. histology revealed infiltration by monotonous medium-sized lymphocytes with abnormal immunophenotype (cd3+, cd56+, cd8+, cd5-, cd30-, tia-1+) consistent with type ii eatl. post-surgical 18f-fdg pet-ct scan showed abnormal uptake in gastric antrum and pyloric region but no other manifestations. ctx with cho(e)p (6 × ) followed by beam hdctx and asct was performed and achieved a complete remission (cr1). however, 9m post asct disease relapsed and was treated with 2 × dhap, and 4 × dhaox. cr2 was achieved after the 3rd cycle of salvage therapy. due to anthracyclineinduced cardiopathy allo-hct could not be performed at that time. 5m after completion of salvage therapy, disease relapsed again, and was progressive under pralatrexat treatment (1 cycle, 6 infusions). by then cardiac function had recovered and therapy was switched to dose-reduced mini-beam (2 × ). in cr3 reduced intensity conditioning (ric; fludarabine, busulfan, atg) and allogeneic hct from a matched sibling donor was performed. ciclosporin a (csa) and mycophenolate mofetil (mmf) were given as gvhd prophylaxis. prompt engraftment in blood (day+14) and full donor chimerism in the marrow (d+100) were achieved. immunosuppression was tapered and discontinued on d+55 (mmf) and d+193 (csa), respectively. 18f-fdg pet-ct scan at 3m post-hct showed cr, but at 6m relapse was suspected (under work-up). only few cases of patients with eatl/meitl treated with allo-hct are reported in the literature (n = 9, table 1 ), and the value of this highly aggressive therapy is not clear at this point. of note, the patients listed in table 1 were given allo-hct instead of asct. long-term complete remission (cr) could be achieved in 4/9 patients, while 5 patients suffered from early relapse and died of the disease (n = 4 before d+100 post allo-hct). asct following surgery and ctx appears to cure 33-60% of patients in available series. no treatment concept is available for relapse following asct, and no published data are available for allo-hct for relapse post asct. the disease is exceedingly rare and is afflicted with very poor outcomes. therefore, patients given this aggressive treatment should be reported, even when treatment outcomes are not positive. disclosure of conflict of interest: none. strong graft versus lymphoma effects with low toxicicty of haploidentical hematopoietic stem cell transplantation comparing with hla-identical in t cell lymphomas: a retrospective multicenter study s bramanti, r devillier, s fuerst, b reda, a granata, s harbi, c faucher, i legrand, a santoro, d blaise and l castagna istituto clinico humanitas rozzano consolidation treatment of relapsed/ refractory t-nhl with allogeneic stem cell transplant (sct) is considered a curative options but few patients manage to undergo this procedure, due to the highly refractory nature of the disease. the primary aim of this work is to evaluate the gvl effects among t-nhl with both hla identical and haploidentical donors. we have retrospectively analized the long term outcome of 43 consecutive patients affected by t-nhl, received hlaidentical or t-cell replete haplo-sct with pt-cy, in 2 european centers, between february 2010 and october 2015. the patients received nonmyeloablative (nmac) or reduced intensity (ric) conditioning regimen. gvhd prophylaxis consisted of 50 mg/kg of pt-cy (day +3 and +4) in haplo setting and atg plus cyclosporine a in the hla identical setting. patients characteristics were reported in the table 1 . no differences were founded in the two groups . most of the patients were transplanted in complete remissions but only 8 as consolidation of first line. no graft failure occurred. the cumulative incidence of acute gvhd grade 3-4 was 5% in the haplo setting vs 11% in the hla id .extensive chronic gvhd was seen in 7% of haplo, and in 28% in the hla id . 9 patients had cmv reactivation, 3 hemorrhagic cystitis, and 1 ebv reactivation. after a median follow-up of 3 years os was 83% and 71% and pfs was 77% and 64% in the haplo vs hla id group see figure 1 . nrm was 5% in haplo setting and 11% in hla identical one. the 3 years cir is 16% and 23% in haplo and hla id setting respectively. this study confirm a strong anti-lymphoma effect of allo hsct without prohibitive toxicities. haplo-hsct with pt-cy shows low rate of cgvhd in a contest of poor prognosis t-nhl patients. [p625] disclosure of conflict of interest: none. ibrutinib is the first-in class bruton tyrosine kinase inhibitor that has been approved for the treatment of relapsed mantle cell lymphoma. however, despite the high response rate of 68% including 21% of complete response, the median duration of response is relatively short with an overall survival of 58% at 18 months (wang ml, et al, n engl j. med 2013). we report a single experience of 3 patients with relapsed mcl who underwent allogeneic stem cell transplant (allosct) after ibrutinib monotherapy salvage. all 3 patients had previous autologous stem-cell transplantation (asct) before and were given ibrutinib at a dose of 560 mg daily after the second or subsequent relapse. all patients had to be at least in pr according to cheson 2014 criteria before allosct. patients had an unrelated 10/10 (2) or 9/10 (1) allo-sct from peripheral hematopoietic stem cells after a reduced conditioning regimen with busilvex, fludarabine and antithymocyte globulin in association with zevalin according to our recent published phase 2 study protocol (bouabdallah k, et al. ann oncol 2015). graft versus host disease (gvhd) prophylaxis consisted on ciclosporine and methotrexate. patients (2m/1f) were aged from 62 to 65 years and received between 2 and 3 previous chemotherapy regimens including asct in their last treatment strategy before introduction of ibrutinib. all patients had extensive disease with gastric involvement in 2 patients and pulmonary localization in 1 patient. median time between diagnosis and ibrutinib introduction was 5 years (3-7) and the median time between asct and allosct was 4 years (4) (5) . median duration of ibrutinib treatment was 5 months (5-6) and it was stopped one week before proceeding to allo-sct. it was not planned to restart btk inhibitor after transplant. patients were assessed for response after at least 3 months of treatment with ibrutinib. at time of evaluation, all patients were in complete (2) or very good partial response (1) before allosct. the patient in partial response had 92% tumor reduction with persistent gastric ulcer where histology examination shows cd5+ but negative cycline d1 lymphoid cells. all patients engrafted (median duration of pnn o500g/l = 11 days (10-15) and median duration of platelets o20g/l = 2 days (0-14)) with fulldonor chimerism at 1 month. one patient had a grade ii cutaneous chronic gvhd (cgvhd) with favorable outcome and developed 8 months later a bronchopulmonary obstruction syndrome related to cgvhd. with a median follow-up of 9 months (9-23) after allo-sct, all 3 patients are alive in cr. one patient, in complete metabolic response before transplant had a gastric relapse 3 months later but achieved again a cr 3 months after reintroduction of ibrutinib. after the first case reported by furtado m et al (leuk lymphoma 2016), we report here 3 additional cases with longer follow-up after allogeneic transplantation. the excellent tumor control after treatment with ibrutinib together with a very good outcome after allosct should drive to consider this approach in young patients with mcl relapse after asct. disclosure of conflict of interest: none. autologous hematopoietic stem cell transplantation (autosct) is considered the standard approach for high risk or relapsed/ refractory non-hodgkin and hodgkin lymphoma. although a large variety of conditioning regimens are available, including the widely used beam (carmustine, etoposide, cytarabine, melphalan), there is no consensus regarding a standard approach. in the context of carmustine shortage, we have chosen to replace it by thiotepa. however, clinical data about thiotepa-based autosct conditioning are still sparse, except some retrospective data for primary central nervous system lymphoma. thus, we designed a multicenter prospective study (nct02504190) to assess the efficacy and toxicity of a team (thiotepa, etoposide, cytarabine, melphalan) conditioning regimen. team regimen consisted in total dose thiotepa of 8 mg/kg on day-6; etoposide 100 mg/m 2 /12 h and cytarabine 200 mg/m 2 /12 h (day-5 to -2); melphalan 200 mg/m 2 on day-1. patients underwent autosct with team conditioning, and were included in this analysis if they have fullfilled the following criteria: age older than 18 years, biopsy-proven hodgkin or non-hodgkin lymphoma, hiv seronegative, and first autosct. thirty-three male and nine female with a median age of 59 years (range: 19-72) were analyzed thus far. karnofsky score was 20g/l was 13 days (range: 8-48). of note, 5 patients received thrombopoietic agents after engraftment because of persisting thrombocytopenia. the most significant regimen-related toxicities were mucositis in 100% of patients (median grade = 3, range: 2-4) and diarrhea in 98% of patients (median grade = 1, range: 0-3). other non-hematologic grade 3 adverse events occurred in 13 patients (31%) and no grade 4 adverse events were observed. central line-associated bloodstream infection occurred in 11 patients (26%). surprisingly, 16/36 evaluable patients (44%) developed human herpesvirus 6 reactivation. only 2 patients required intensive care unit transfer. the median duration of hospital stay was 29 days (range: 20-62). after a median follow-up of 8 months (range: 2-20), the non-relapse mortality (nrm) was 0%. only one patient relapsed of refractory aitl 2 months after autosct and died 1 month after. the estimated 1-year overall survival and progression-free survival were 98% and 98%, respectively. a team conditioning regimen seems to be a safe and valid platform in autosct for patients with high-risk or relapsed/ refractory lymphoma. although mucositis and diarrhea were frequent, there were no grade 4 adverse events and no deaths related to the treatment. updated results with updated followup will be presented. disclosure of conflict of interest: none. the cell of origin has no prognostic impact on high-dose chemotherapy with r-beam and autologous stem cell transplant for diffuse large b cell lymphoma s lozano cerrada, r saliba, s srour, s ahmed, c hosing, r champlin and y nieto university of texas, md anderson cancer center, department of stem cell transplantation and cellular therapy diffuse large b-cell lymphoma (dlbcl) is a biologically heterogeneous disease that can be classified according to its cell-of-origin (coo). the germinal center b-cell (gcb) subtype has better outcome with frontline r-chop than the activated b cell (abc) subtype. however, the prognosis of these two types of dlbcl after high-dose chemotherapy and autologous stem cell transplant (asct) is less clear. the purpose of our study was to evaluate progression-free survival (pfs), event-free survival (efs) and overall survival (os) in a cohort of dlbcl patients treated with r-beam (rituximab, carmustine, etoposide, cytarabine, melphalan) and asct according to coo. we have the dicep regimen effectively reverses the poor outcome for lymphoma patients with suboptimal response or failure post 1st salvage treatment p kaloyannidis 1 the outcome of patients (pts) with refractory hodgkin's (hl) and non-hodgkin lymphomas (nhl) post 1st salvage treatment (salv1) is considered poor. the published data, have shown extremely low survival rates (15-20%) even after 2nd salvage treatment (salv2) followed by autologous stem cell transplantation (asct), due to the low response rates post salv2 and the high relapse rates post asct, confirming that the management of these pts remains a major challenge. we herein evaluated the dicep regimen [dose intesified cyclophoshamide (1750 gr/m 2 ), etoposide (350 mg/m 2 ) and cisplatin (35 mg/m 2 ), days 1-3] as a salv2 treatment, in terms of safety and efficacy regarding disease response and stem cell mobilization/collection. moreover, we evaluated pts' long term outcome post asct. we retrospectively analyzed the data of 27 (11 hl, 16 nhl) pts, with a median age of 32 (16-61) yrs. twenty-one had suboptimal response (75% reduction): 4 and minor response (≤50% reduction): 2). three pts had stable disease while 3 experienced progression. overall 23/27 pts underwent asct after a median of 44 days (range: 22-70) post dicep. no pt was considered ineligible for the asct due to unacceptable toxicity post dicep; 4 did not undergo asct because of progressive (n = 3) or stable (n = 1) disease. the 5-yr overall survival (os) was 70% for the whole cohort of pts (71% for hl and 66% for nhl, p = ns) while the 4-yr progression free survival (pfs) from dicep administration (± asct) was 62% (60% for hl and 64% for nhl p = ns). in particular, for the 23 autografted pts, the 5-yr os was 70% (80% for hl, 70% nhl p = ns) and the 4-yr pfs was similar, 70% (65% for hl, 72% for nhl, p = ns) our data demonstrate that dicep is an effective salvage regimen with acceptable toxicity and no negative impact on the cd34+ collection. the promising response rates post dicep in combination with the very encouraging pfs rates achieved post asct, in this unfavorable and heavily pretreated group of patients, strongly support the rationale for using dicep as 1st line salvage regimen in selected pts in order to proceed to a successful asct. for the treatment of aggressive lymphoma, high dose chemotherapy followed by autologous stem cell transplant (asct) is an important component. however, the role of upfront asct in patients with diffuse large b cell lymphoma (dlbcl) is still controversial. furthermore, there is currently no consensus on a single best conditioning regimen for asct in patients with dlbcl. we retrospectively analyzed the records of 184 patients with dlbcl who underwent upfront asct in state of complete remission (cr) or partial remission (pr) from 17 institutions in korea. we evaluated the outcomes and prognostic factors of upfront asct in patients with dlbcl. we compared the outcomes of most widely used two conditioning regimens for asct; carmustine based regimens and busulfan containing regimens. total 141 patients (81.0%) achieved cr after asct and overall response rate (orr) was 86.7%. with median follow up of 34 months, 65 patients (35.3%) had progression or relapse. the 3-year overall survival (os) rates and progression free survival (pfs) rates were 75% and 65%, respectively. infection events were found in 99 patients (54.5%) and treatment related mortality was 3.8%. these outcomes were comparable with the results of previous other studies. cox multivariate analysis for os showed that eastern cooperative oncology group performance status (ecog ps) ≥ 2 (p = 0.003) and rituximab based induction therapy (p = 0.062) were significant prognostic factors. in addition, the following factors were significantly associated with pfs in multivariate analysis; female (p = 0.031), ps ≥ 2 (p = 0.018) elevated β2-microglobulin (p = 0.008), failure to achieve cr with induction chemotherapy (p = 0.004), carmustine based conditioning regimen (p = 0.012) and melphalan based conditioning regimen (p = 0.031). there were no significant differences in os and pfs according to stage, b symptom, bulky disease, high lactate dehydrogenase, bone marrow involvement, high or high-intermediate international prognostic index (ipi), absolute lymphocyte count and absolute monocyte count. therefore, it is considered that upfront asct can overcome the poor prognosis in patients with advanced stage or high risk ipi. in the analysis with conditioning regimen, neutrophil and platelet engraftment were slower in the carmustine group compared to the busulfan group. there were no significant differences in os between busulfan group and carmustine groups with 3-year os rates of 74.4% and 77.9%, respectively (p = 0.797). pfs at 3years was 66.0% in busulfan group versus 59.9% in carmustine group (p = 0.241). however, carmustine based conditioning regimen was poor prognostic factors for pfs in multivariate [p631] s457 analysis (p = 0.004). in subgroup analysis, busulfan group had significantly higher pfs compared to the carmustine group especially in female patients (67.9 months vs. 7 months, p = 0.002), with b symptom (67.9 months vs. 7.4 months, p = 0.033) and abnormal serum ldh level (70.2 months vs. 25 .9 months, p = 0.045). the outcomes of upfront asct in patients with dlbcl after induction therapy were acceptable. it is considerable in selected high risk patients who achieve cr with induction treatment, and have good performance status at diagnosis. in cases of conditioning regimen, busulfan based regimen resulted in improved outcomes compared with carmustine based regimen especially in patients with disseminated disease or female patients. disclosure of conflict of interest: none. no heavy chain was present in 25%). the predominant light chain was kappa (60%). 80 patients had bence-jones positive myeloma. 109 received bortezomib as induction therapy before transplant. we analyzed overall survival (os) and progression-free survival (pfs) in 5 groups of patients. we separated the groups according to improvement in grade of response from preasct to postasct. the post-asct grade of response was measured 3 months after asct. the os and pfs were estimated by the kaplan-meier method. pfs was measured from diagnosis to disease relapse and os was measured from diagnosis to death by any cause. results by subgroups of patients are detailed in table 1 . median os and pfs of the whole group was 8 years and 54 months, respectively. if we analyze groups only by their grade of response before asct we find the following results: rc (5years os rate 78.9%, median pfs 95 months); pr/vgpr (median os 7.31 years and pfs 40 months); sd/progression (median os 6.46 years and pfs 21 months). according grade of response after asct, instead: rc (5 years os rate 78%, median pfs: 66 months); pr/vgpr (median os 7,31 years, and pfs 40 months); sd/progression (median os 1.05 years and pfs 11 months). in our experience, the grade of response before asct is a capital predicting factor for patients os and pfs. patient in cr before asct that preserve it after transplant, have a median pfs of 95 months, the 5 years os rate being 80.4%. patients in situation of progression after asct have a very dismal prognosis (median os 1.05 years, pfs: 11 months), however, patients who change from sd/progression to pr after asct have a median pfs of 29 months and a os of 6.4 years. comparing these results we observe that this second group is particularly benefited by transplant. autologous peripheral blood hematopoietic stem cell transplantation in elderly patients with multiple myeloma as a standard therapeutic procedure. is it feasible? a single-center experience l cadievski, s genadieva stavric, z stojanoski, a pivkova veljanovska, d miloska, b kocoski, l cevreska and b georgievski university clinic of hematology, department for hematopoietic stem cell transplantation, university ss. cyril and methodius, skoje, republic of macedonia autologous peripheral blood stem cell transplantation (pbsct) represents a standard therapeutic approach in the treatment protocol of myeloma patients. it is known that multiple myeloma is a hematological disease that is a characteristic for the older population. autologous pbsct ideally should be performed in every myeloma patient, but with the elderly myeloma patients the procedure might be risky if know the possible comorbidities, or the possibility of the body to fully compensate the side effects of the conditioning regimen, the procedure or its possible complications. we present our experience in using high dose conditioning with melphalan 200 mg/m 2 followed by autologous pbsct for elderly myeloma patients, using the age limit od 65 years. our retrospective analysis of our data during 16 years of experience, shows that we have performed autologous pbsct on 12 patients with myeloma at the age of 65 or older. 11 males (91.6%), and 1 female (8.4%). 6 patients (50%), were diagnosed with igg type myeloma, 5 patients (41%) with iga myeloma, and 1 patient (9%) with light chain myeloma. median age of the patients was 66.9 years (65-73). all patients were initially treated with cy-thal-dex regimen. in 5 (41%) patients complete response (cr) was achieved, in 5 (41%) very good partial response (vgpr), and in 2 (18%), partial response (pr). in all patients the mobilisation of hematopoietic stem cells was performed with g-csf, and a median of 2 apheresis procedures were performed, and the average number of collected cells was 3.11 × 10 8 /kg tt mononuclear cells (range: 6.0-2.1). days to confirmed engraftment in our group of patients was 11.5 (range: 9-15). the number of blood transfusions was on average 2.8 (range: 0-6), and the number of transfusion of thrombocytes 38.1 units (range: 10-74). in the majority of patients, mainly after the year 2012 (that represents 10 patients of the whole group), we used noncryopreserved hematopoietic stem cells, kept under the temperature of 40c, for median of 2 days, thus avoiding the toxicity of dmso. additionally, we used central venous catheter inserted in the femoral vein for apheresis and application of the stem cells afterwards. the day after, the catheter was removed, thus avoiding catheter associated infections. all patients received standard infectious prophylaxis with fluconasole 200 mg/daily, ciprofloxacin 500 mg/ two times daily, acyclovir 200mg/ three times daily, cefixime 400 mg/once daily, and ursodeoxycholic acid for vod prevention. no serious infectious complications were reported. our transplant related mortality was 0%. in the group with noncryopreserved stem cells no graft failure was reported. in two patients we even performed tandem autologous pbsct with no major complications. of the group of 12 patients, the majority, 8 patients (66%), had hta as comorbidity, 1 (8%) with cardiomyopathy, and 1 (8%) with inserted prosthetic aortic valves. three patients (24%) have died because of relapse of the disease. our oldest patents were 72 and 73 years old, and are still alive 1 year posttransplant, in cr. we can conclude the performing autologous pbsct in elderly myeloma patients can be safe and effective therapeutic option, but with careful selection of the patients, balancing the risk profile of the patient and the benefit, or the risk of the procedure. affective supportive care, monitoring and reducing the risk of complications is an imperative to a good result. disclosure of conflict of interest: none. autologous stem cell transplantation program for patients with multiple myeloma in an outpatient setting k lisenko 1 , s sauer 1 , g egerer 1 , j schmier 1 , m witzens-harig 1 , a schmitt 1 , ad ho 1 , h goldschmidt 1,2 , j hillengass 1 and p wuchter 1,3 1 department of medicine v, heidelberg university hospital, heidelberg, germany; 2 national center for tumor diseases heidelberg (nct), heidelberg university, heidelberg, germany and 3 institute of transfusion medicine and immunology, mannheim, german red cross blood service baden-württemberg-hessen, medical faculty mannheim, heidelberg university, germany the first and second authors contributed equally. high-dose chemotherapy with melphalan and autologous blood stem cell transplantation (absct) for treatment of symptomatic multiple myeloma (mm) is performed in the usa and canada mostly on an outpatient basis, whereas in germany and western europe an inpatient setting is the standard. we report on a german single-centre program to offer the procedure on an outpatient basis to selected patients. major inclusion and exclusion criteria for eligibility were defined as follows: patients had to be able to reach the hospital within 45 minutes, had reliable support from their family at home, had an ecog performance score of 0-1 and were willing and able to comply with the demands of the program. patients with severe co-morbidities were not included. all patients were treated on our outpatients' clinic and examined on daily visits by a team of physicians. feedback from patients was obtained by means of a questionnaire. from september 2012 to september 2016, 26 patients with mm stage iiia were enrolled. all engrafted within the expected time range: (median time to leukocyte 41,000 /μl and neutrophil recovery 4500 μ/l: 14 days; median time to platelet recovery 420/nl: 10 days, 450 /nl: 14 days). twenty patients (77%) had an episode of neutropenic fever but only in 5 patients (19%) blood cultures were found to be positive. there occurred no cases of infection with multiresistent bacteria. although rather liberal criteria for hospital admission were applied, 18 of 26 patients (69%) could be treated entirely on an outpatient basis. eight patients (31%) were temporarily admitted for inpatient treatment with a median duration of 4.5 days (range: 2-18 days), mainly because of neutropenic fever. no severe adverse events occurred. feedback from patients revealed a high level of satisfaction with the outpatient setting. high-dose chemotherapy and absct on an outpatient basis is safe and feasible if conducted in a comprehensive surveillance program. the feedback from patients was very positive, thus encouraging further continuation and expansion of the program. disclosure of conflict of interest: none. high dose of melphalan (bor-mel). we retrospectively analyzed 69 patients with mm who underwent asct between january 2014 and march 2016. in these patients, conditioning regimen consisted of a high dose of melphalan (140-200 mg/ m 2 ) intravenously on day -2 and two doses of intravenous bortezomib at 1.3 mg/m 2 administered on days − 1 and +2. this cohort was compared with patients underwent asct between 2003 and 2013, conditioned with high dose of melphalan alone. response rate was evaluated according to imwg criteria. all patients were evaluated after induction therapy and 3 months after asct. all patients were followed until death or reference date (november, 2016). results: patients' demographics and baseline disease-related characteristics are shown in table 1 . [p636] no difference was found in terms of neutrophil and platelet engrafment, hospitalization days (p=0.723) and use of mechanical invasive ventilation (p=0.415). bor-mel regimen did not enhance severity of preexisting peripheral neuropathy (pn) in any patients, and only one presented de novo grade 2 pn. non relapsed mortality was 1.4% and 1% in the bor-mel and mel cohorts, respectively (p=0.673). complete response rate after transplant was significantly better in the bor-mel cohort than in the mel cohort (65.2% vs. 40.7%; p=0.001) ( figure 1b) . when the analysis was restricted to patients who received bortezomib-based therapy, this difference was also statistically significant (64.1% vs. 44.4%; p=0.024) ( figure 1d ). median of follow-up was 12 months in the bor-mel vs. 42 months in the mel cohort. no difference was found in terms of overall survival (os) and progression free survival (pfs) between both groups. for all patients, a post-transplant deeper response was associated with better os and pfs (p=0.008 and p o0.001, respectively). our results are in line with previous studies demonstrating that bortezomib combined with melphalan is a well tolerated conditioning regimen and may enhance the response rate after transplant, even in patients receiving bortezomib in the induction therapy. these results should be confirmed in a randomized trial. for newly diagnosed patients (pts) with multiple myeloma (mm), the triple-agent induction treatment based on bortezomib plus dexamethasone in combination with cyclophosphamide (vcd) or lenalidomide (vrd) represent extremely reliable regimens, which in combination with early autologous stem cell transplantation (asct) result in high response rates and prolonged long-term outcomes. however, though both regimens are widely used, there are extremely limited studies that compare the vrd vs. vcd in terms of safety and efficacy. in the present study we compared the outcomes of 19 newly diagnosed mm pts who received induction treatment vrd (n = 10) or vcd (n = 9) and proceeded early to asct. the vrd and vcd pts groups were similar regarding age at diagnosis (52 vs.54 ys, p = ns), interval between diagnosis-asct (5,8 vs. 5,7 months, p = ns) and maintenance treatment post asct (8 vs. 6 pts, p = ns). per revised international scoring system (riss), the vrd-group had slightly more advanced disease (stage i: 1, stage ii: 7 and stage iii: 2), compared to vcd-group (stage i: 3, stage ii: 5 and stage iii: 1), however this difference was not statistical significant. the conditioning regimen consisted of single agent melphalan: 200mg/m 2 . the t-test, kaplan-meir and cox regression were utilized for the statistical analysis. following a median of 4 cycles of treatment (range: 4-6 for vrd vs. 2-6 for vcd, p = ns), in the vrd-group 4 pts achieved complete remission (cr), 5 pts very good partial remission (vgpr ≥ 75% reduction of m-band) and 1 pt partial remission (pr: 50-75% reduction of m-band) while in the vcd-group cr:3, vgpr:2 and pr:3 pts (p = ns). the toxicities in terms of peripheral neuropathy, myelosuppression, liver and renal function were well tolerated and no patient discontinued treatment due to severe side effects. the 5-yr overall survival (os) was 100% for the vrdgroup vs. 75% for the vcd-group; nevertheless, the difference was not significant due to the size sample of the pt groups. the stage at diagnosis, the disease status pre-asct and the maintenance post-asct did not influence the os. interestingly, the 4-yr progression free survival (pfs) was significantly superior for patients who had been induced with the vrd regimen (75% vs. 36% p = 0.05) and for patients who achieved cr or vgpr before asct (pfs: 50%) while no pts with pr pre-asct was progression-free 2 yrs post asct (p = 0.001). in multivariate analysis, only the cr or vgpr status before asct favorably affected the long term pfs. our results are in line with the limited published data from other studies with larger series of patients. in our study, very low disease burden before asct proven to be an independent factor for prolonged pfs. taking into consideration that vrd resulted in more cr or vgpr status, it is reasonable to conclude that vrd is a highly effective regimen and could be first treatment choice for newly diagnosed mm patients who are fit for early asct post induction. lenalidomide cohort and 64 in the maintenance bortezomib cohort. baseline characteristics and outcome data were obtained via chart review. the primary outcome was pfs. the secondary outcomes were overall survival (os) and treatment-related toxicities. the median follow-up time was 33 months. median time to death (4.28 years vs 5.77, p = 0.47) and median time to progression (1.71 years vs 1.74, p = 0.77) were not significantly different in the maintenance lenalidomide cohort compared to the maintenance bortezomib cohort. in the multivariate analysis, pfs was worse in patients at international staging system (iss) stage 3 at diagnosis compared to those at iss stages 1 and 2 (hr, 1.86; 95% ci, 1.11 to 3.12; p = 0.02) and worse in patients with less than very good partial response (vgpr) to last prior therapy compared to those with a response to prior therapy of at least vgpr (hr, 2.05; 95% ci, 1.14 to 3.69; p = 0.02) [see figure 1 ]. pfs was improved in patients with more than two years of maintenance therapy compared to those with less than two s464 years of maintenance therapy (hr, 0.40; 95% ci, 0.22-0.70; po0.01), but this result does not account for patients who ended maintenance therapy due to disease progression. os was worse in patients at iss stage 3 at diagnosis compared to those at iss stages 1 and 2 (hr, 3.87; 95% ci, 1.44 to 10.39; p = 0.01). peripheral neuropathy was more common in the bortezomib cohort (39% vs 9%, p o0.01), while cytopenias were more common in the lenalidomide cohort (30% vs 3%, po0.01). figure 1 kaplan-meier curve for pfs for the maintenance lenalidomide group versus the maintenance bortezomib group by log-rank test (p = 0.16). lenalidomide and bortezomib maintenance after transplantation have equal efficacy in prolonging progression-free and overall survival in patients with multiple myeloma. iss stage significantly affects time to progression and overall survival, and response to last prior therapy affects time to progression. length of maintenance therapy may be a significant predictor and warrants further analysis. these findings suggest that both lenalidomide and bortezomib are acceptable maintenance therapy options for post-transplantation multiple myeloma patients. autologous stem cells transplantation (auto-hct) is an accepted method in multiple myeloma (mm) patients, but usually it is not curative. the issue of allogeneic hematopoietic stem cells transplantation (allo-hct) is challenging yet for myeloma. we investigated allo-hct in mm and compared with auto-hct. in this retrospective study, we recruited 272 patients from january 2011 to january 2015 (218 (80.15%) patients in autologous group and 54 (19.85%) in allogeneic group). we performed allogeneic hct with peripheral blood stem cells source in our center for patients who are relatively young (less than 55 years old) with good performance, have match sibling donor and accepted allogeneic hct. the conditioning regimens in autologous group was melphalan 200 mg/m 2 only and in allogeneic groups was fludarabine 30 mg/m 2 plus melphalan 140 mg/m 2 in 5 consequent days. gvhd prophylaxis consisted of methotrexate and cyclosporine. the outcomes then compared between two groups using log-rank and gray tests and cox proportional hazard regression. the median follow-up in the autologous and allogeneic group was 17.02 months. three years disease-free survival of auto-hct was 38.61% (ci: 27.37%, 49.72%) and 68.88% (ci: 50.74%, 81.47%) for allo-hct patients (p value = 0.0363). three years overall survival of auto-hct was 77.26% (ci: 66.08%, 85.16%) and 82.15% (ci: 64.92%, 91.44%) for allo-hct patients (p value = 0.6363) showing no significant statistical difference between two groups. mortality rate was 11.01% for auto-hct and for allo-hct was 12.96%. the most common cause of death between two groups was relapse of primary disease. three year relapse incidence was 20.83% (ci: 9.04%, 35.30%) for allo-hct and 54.33% (ci: 42.02%, 65.09%) for auto-hct (gray's test p value = 0.018). the three year trm incidence was 10.36% (ci: 2.92%, 23.33%) and 7.01% (ci: 3.14%, 12.98%) in allogeneic and autologous patients respectively (gray's test p value = 0.42). despite there was no statistically significant difference between two groups in terms of os but dfs and relapse incidence was meaningfully better in allogeneic group. so, perhaps the reason of non-significant os improvement in allogeneic group is higher early death due to higher trm. we suggest that this study needs longer follow up to see whether allo-hct resulted in os improvement. disclosure of conflict of interest: none. myeloablative allogeneic hematopoietic stem cell transplantation from unrelated donors for patients with relapsed or refractory multiple myeloma n tsukada 1 , s shingaki, m ikeda, t ishida and k suzuki 1 division of hematology, japanese red cross medical center allogeneic hematopoietic stem cell transplantation (allo-sct) for patients with multiple myeloma (mm) is increasing in number despite in the era of novel agents, especially as a second line treatment and beyond. it has been reported that allo-sct for patients with mm resulted in high incidence of treatment related mortality (trm). high incidence of disease relapse is also a major problem especially after reducedintensity stem cell transplantation (rist). it is an important issue to reduce the incidence of trm while preventing disease relapse. the use of stem cells from unrelated donors is required for those without hla-matched sibling donors. the purpose of this study is to evaluate the feasibility of an intensified conditioning regimen incorporating both 140 mg/ m 2 of melphalan and 8 gy of total body irradiation (tbi), followed by allo-sct from unrelated donors for patients with relapsed or refractory mm. we retrospectively analyzed eight consecutive patients who received allo-sct from unrelated donors with the conditioning regimen including 8gy of tbi, fludarabine 25 mg/m 2 for five days, and melphalan 140 mg/m 2 between april 2013 and july 2015 at the japanese red cross medical center. six patients received unrelated bone marrow transplantation (bmt) and two patients received cord blood transplantation (cbt). graft-versus-host disease (gvhd) prophylaxis was consisted of tacrolimus and short term methotrexate. the median age at allo-sct, the time from diagnosis of myeloma to allo-sct, and the numbers of prior treatment lines were 48.5 years (range: 31-60 years), 38.5 months (range: 8-64 months), and 3.5 lines (range: 1-7 lines), respectively. five patients are female. no episode of either grade ≥ iii toxicity or non-relapsed mortality was documented during the median follow-up period of over two years. cumulative incidence of grade ≥ ii acute and severe chronic graftversus-host disease were 37.5% (95% confidence interval [ci] 7.2%-69.4%) at 100 days and 25.0% (95% ci 2.9%-58.1%) at 180 days, respectively. probabilities of progression-free survival and overall survival were 22.5% (95% ci 0.0%-58.7%) and 58.3% (95% ci 22.0%-94.7%), at 3 years, respectively. the results suggest that allo-sct conditioned with this intensified regimen may be tolerable for patients with relapsed or refractory mm. disclosure of conflict of interest: none. the role of allogeneic stem cell transplantation (allosct) in the era of novel myeloma drugs remains controversial. it is the only curative treatment option but non-relapse mortality makes the decision making difficult as opposed to achievements with autologous sct and new mm drugs by which the median survival is nowadays nearing 10 years. aim of this study was retrospectively evaluate the outcome of allosct for mm performed at our institute, including evaluation of factors affecting survival. all 66 consecutive patients allotransplanted for mm between 1986 and 2014 were included. the data were collected from our transplant registry. frequencies and medians were produced as appropriate. kaplan-meier method was used to calculate os and pfs and log rank test for comparisons. univariate analysis for factors affecting survival was performed with cox proportional hazard model. median age of all 66 patients was 55 (36-66) years. half of the patients had igg myeloma, 23% had iss score 3 (score available for 30 patients), and 33% had high-risk cytogenetics (data available for 39 patients). response to treatment at sct was at least vgpr in 80% of patients, and transplant timing was early (within 15 months from dg) in 58% of patients. sibling donors were used in 58% and muds in 42% of transplants, and conditioning was ma for 50% and ric for another 50% of patients. acgvhd grade 0-2 occurred in 78% and grade 3-4 in 22% of patients; 32% of patients had extensive chrgvhd. posttransplant cr rate was 83%. 45% of pattients have relapsed after allosct, and 42% are alive with the median follow-up of 5,6 years. non-relapse mortality has been 30% (35% until 2005, 27% since then). the median survival of patients up to age of 60 years is 4.9 years vs 3.0 years for patients 460 years (n = 8) with survival plateau after 6 years at 40% level. transplant period, cytogenetics, donor type, conditioning intensity or occurrence of chrgvhd had no statistical impact on survival. significant differences in os were observed between disease status at scr 4 vgpr vs o15 months from dg vs later), grade of acgvhd 0-2 vs 3-4, and best resonse post-transplant cr vs not less than cr. the respective differences for pfs were in sct timing, grade of chrgvhd, and best post-transplant response. in univariate cox regression analysis the only significant factors for os were severity of acgvhd and cr vs other responses after sct, and for pfs allosct timing, severity of chrgvhd, and best response to sct. with allosct ca. 40% of mm patients can be cured but at the cost of high non-relapse mortality. the occurrence of grade 3-4 acgvhd and less than cr response to sct predict poor survival. considering the increasing survival expectations with modern standard therapy for mm, allosct may be recommended for younger patients with high-risk features, and allosct should be done early in the disease course. disclosure of conflict of interest: none. angiogenesis plays an important role in the pathophysiology of hematological malignancies including plasma cell myeloma (pcm). microrna-21 (mir-21) is overexpressed and displays oncogenic activity in cancers. however, little is known about the role of mir-21 in pcm. the aim of the present study is to examine the expression level of peripheral mir-21 in pcm patients and to determine its role in angiogenesis. vegf serum levels and mir-21 in pbmcs was measured in 93 patients with pcm directly before melphalan 200 mg/m 2 followed by autologous hematopoietic stem cell transplantation (auto-hsct) and 2 months after hsct; and 35 healthy controls. the study population was divided into two groups after therapy: responders (stringent complete response, complete response, very good partial response, partial response) and nonresponders (stable disease, progressive disease). gene expression of mir-21 was quantified by sybr green real-time fluorescent quantitative pcr. further tube formation of huvecs and vegf secretion was measured in mir-21 mimic or inhibitor transfected human plasma cell myeloma cell lines h929 and rpmi-8226. the expression level of mir-21 was significantly increased (2.7 ± 0.55 versus 0.78 ± 0.22; p o0.01) in pbmcs of pcm patients compared with healthy controls. further, serum vegf levels were increased in pcm patients (477 ± 145 pg/ml versus 178 ± 78 pg/ml in normal controls; p o0.01). after auto-hsct, the expression level of mir-21 was significantly different in responders compared to nonresponders. responders had a lower expression of mir-21 compared to non-responders. further, serum vegf levels decreased in responders to auto-hsct compared to nonresponders. vegf expression was increased in the supernatant from mir-21 mimic transfected human pcm cell lines h929 and rpmi-8226 compared with the negative control, while s467 vegf was decreased in the mir-21 inhibitor transfected cell lines. the angiogenic ability of huvecs was increased under pretreatment with the supernatant from h929 and rpmi-8226 cells transfected with mir-21 mimic compared with negative controls and decreased when pretreated with mir-21 inhibitor transfected cells (fig. 1) . this study demonstrated that mir-21 was upregulated in pcm patients. responders to auto-hsct had a decrease of mir-21 expression and vegf levels. further, mir-21 regulated angiogenesis. therefore inactivation of mir-21 or activation of its target gene may be a potential therapeutic approach in pcm. fig. 1 : in vitro matrigel tube formation assay. (i), normal control (ii, iii), mir-21 mimic transfected h929 cells (iv), mir-21 mimic transfected rpmi-8226 cells (v), mir-21 inhibitor transfected h929 cells. original magnification × 100. disclosure of conflict of interest: none. [p647] pilot study of busulfan/thiotepa as conditioning regimen followed by allografting and post transplantation cyclophosphamide in advanced relapsed myeloma patients c wolschke, e klyuchnikov, d janson, m heinzelmann, m christopeit, f ayuk and n kröger university medical center hamburg-eppendorf despite the significant improvement in outcomes has been observed for myeloma patients, the disease still remains incurable. due to limitations, such as trm and gvhd, the role of allogeneic stem cell transplantation as salvage therapy in this setting remains unclear. in present pilot study we provide data on the use of post cyclophosphamide (ptcy) as gvhd prophylaxis after a busulfan/thiotepa based conditioning regimen in patients who relapsed after autologous stem cell transplantation. between 11/2014 and 08/16 17 myeloma patients (male n = 10, female n = 7) with a median age of 55 years (range: 45-66) (pts), who relapsed after autologous stem cell transplantation received allogeneic stem transplantation with with ptcy as gvhd prophylaxis after busulfan (9.6 mg/kg for age 460y and 6.4 mg/kg for age 460years) and thiotepa (10 mg/ m 2 )and for haploidentical and mmud additional fludarabin (90 mg/m 2 ). all pts. were relapsed after one or two autologous stem cell transplantations. donors were haploidentical (n = 1), mmud (n = 4), mud (n = 6) and hla-identical sibling (n = 6). stem cell source was pbsc (n = 15) or bm (n = 2). all patients received cyclophosphamide 50 mg/kg of body weight on day +3 and +4, which was in 10 pts (n = 5 mrd, n = 5 mud) the only gvhd prophylaxis, while patients with mmud and haploidentical donor received also cyclosporine a from day +5 and mmf (until day 35) and 2 patients (mrd and mud) received additional cyclosporine. we observed no primary or secondary graft failure. the median time for neutrophil and platelet engraftment was 19 (range: 15-24) and 51 days (range: 22-279), respectively. major toxicities grade 3 and 4 were: renal (n = 1) and mucositis (n = 1). major infectious complications were: cmv: n = 10 cmv-reactivations (n = 10), sepsis (n = 5), pneumonia (n = 3) rsv-(n = 2) and hsv (n = 1). acute gvhd grade ii to iv and ii/iv was noted in 29% and 24%, respectively and mainly seen in patients with cyclophosphamide as single gvhd prophylaxis. remission rate were n = 8 complete remission, n = 7 vgpr, n = 1 partial remission, n = 1 n.a. after a median follow up of 12 months 3 pts progressed and 7 patients (n = 1 relapse, n = 6 trm) died. the 1 year pfs was 18% (n = 3). busulfan/thiotepa is an active conditioning regimen for advanced relapsed myeloma patients. post cyclophosphamide might increase anti-myeloma activity, but as single gvhd prophylaxis it causes significant agvhd in mrd and mud and additional immunosuppressive agents such as cyclosporine should be added. disclosure of conflict of interest: none. magnetic resonance imaging (mri) for multiple myeloma (mm) is a sensitive, non-invasive and non-toxic method for detecting myeloma lesions. the goal of the study was to assess whether quantitative mri metrics can detect treatment response and replacement of neoplastic cells by fat marrow. the study was hipaa-compliant and irb-approved. we retrospectively identified all patients who achieved a complete response (cr) after induction therapy between 2000 and 2014. inclusion criteria for the study was total spine mri imaging at diagnosis and after achieving cr. cr was determined using the imwg criteria. spinal vertebrae t12 through l5 were outlined with imagej software. fractures and lesions were excluded. images were analyzed using histogram-based (entropy, skewness, kurtosis) and texture-based statistics. a two-sided t-test was used to compare quantitative mri metrics from before therapy and after achieving cr. cox regression was used to explore the association between progression free survival (pfs) and change in each quantitative mri metric based on a median split. pfs was defined as the time from the second mri to death or progression of disease. nineteen patients met the above criteria. median age was 61.5 years (range: 37.5-72.2). majority of patients (68%) were male. majority of patients had iss stage 1 disease (57.9%) and standard-risk cytogenetics (89.5%). an induction regimen containing an imid and/or a proteasome inhibitor was commonly used (73.7%). all patients received an autologous stem cell transplant (asct) consisting of high dose melphalan followed by autologous stem cell rescue. three patients received a planned second asct. seven patients (36.8%) were in cr before asct. nine patients (47.4%) were treated with imid maintenance after planned initial therapy. median time to repeat mri imaging after cr was 10 months (range: 4.4-19.8). mean change in measurements of kurtosis, skewness, entropy and 6 texture analyses are shown in table 1 . no significant change was detected between preand post-cr mri. furthermore, no significant association was seen between the change in any quantitative metric and pfs. [p649] despite promising results by other groups, we could not find a significant association between quantitative t1 image analysis and cr or pfs. there was heterogeneity in the time of repeat mri imaging which may have limited our ability to study interval change. although no definitive conclusions can be made from this small sample, correlation between pfs and kurtosis or texture d may be promising and should be investigated in a larger group prospectively. multiple myeloma (mm) treatment (tx) has evolved in recent years. solid data on the impact of new tx on patient (pt) outcomes outside clinical trials, however, is lacking. this study aimed at investigating tx practices, pt journeys, and outcomes in the real-world in countries with different access to new tx. the study was conducted between 04/2015 and 06/2016 in bulgaria, croatia, czech republic, poland, romania, and slovakia. it consisted of a cross-sectional (x) and a retrospective (r) phase. for the x-phase, investigators included all symptomatic mm pts seen during a 3-week counting phase to provide a snapshot of where in the pathway pts were at a given moment. for r-phase, investigators collected data on current and past tx, including symptoms, dosages, administration schedule, tx durations, tx interruption, reasons for change/discontinuation, and tx response. pts were selected in reverse chronological order with a quota of a maximum of 3 pts who completed first-line (1l) tx within the past 3 months (mo), 4 pts in second-line (2l) and 7 pts in third or higher lines (3+l). pts included in the x-phase could also be included in the r-phase, if they met the respective inclusion criteria. in total, 39 physicians included 522 pts in the x-and 35 physicians included 277 pts in the r-phase. in the x-phase, 52% of pts were o 65, 36% were 65-75, and 12% were 475 years; the median time since diagnosis was 27 mo. 57% of pts were currently undergoing tx, 41% were previously treated and 2% had never been treated. of currently-treated pts, 37% received 1l, 30% 2l, 19% 3l and 14% 4+l. in the r-phase, 47% of pts were o65 years. of pts receiving 1l, 59% continued to 2l, 33% to 3l, 15% to 4l and 8% to 5l. of the 38% of pts eligible for stem cell transplantation (sct), 55% ( = 21% of all pts) received sct at 1l; these proportions were similar across countries. the most frequently-used regimens in 1l and 2l were bortezomib-based (57% and 53%, respectively), in 3l and 4+l lenalidomide-based (47% and 35%, respectively). median duration of 1l was 6 mo, followed by a median disease-free interval (dfi) of 4 mo. median dfi was longer in pts with sct than in those without (6.5 mo vs 1.5 mo). time to progression (ttp) decreased with later tx lines, from median 9 mo at 1l to 4 mo at 3l. depth of response, as assessed by the treating physician, decreased with each additional line of tx: 50% of pts achieved at least very good partial response (≥ vgpr) in 1l, while only 25% achieved ≥ vgpr at 3+l. ttp was longer in pts with better response levels: in 1l, median ttp for pts with ≥ vgpr was 22 mo versus 6 mo for pts with 6 mo for pts with ovgpr. the most common ( ≥20%, all grades) adverse events (aes) and co-morbidities in 1l were anemia (42%), thrombocytopenia (29%), neutropenia (25%), neuropathy (25%), and fatigue (22%). these aes disrupted treatment in 57% in 1l, 41% in 2l and 55% in 3+l. the study found that of sct eligible pts, only slightly more than half were transplanted. poorer outcomes and increasing ae incidence with each tx line highlight the challenges of mm tx. information on real-world pt management may be valuable for physicians to plan their tx strategies and can provide input for health economic evaluations of existing and new tx. disclosure of conflict of interest: daniel coriu declares to have received consulting fees or other remuneration (payment) from novartis, amgen, pfizer, takeda, janssen. ivan spicka declares to have received research grants from celgene, consulting fees or other remuneration (payment) from bms, takeda, celgene, janssen-cilag, and amgen, and to be a member of the speakers bureau of celgene, janssen-cilag, amgen, and bms. zdenka stefanikova declares to have received consulting fees or other remuneration (payment) from amgen, celgene, and takeda and be a member of the speakers bureau of amgen, celgene, and takeda. daniela niepel, krisztian szabolcs toka, and paul schoen are amgen employees and hold amgen stock. dominik dytfeld, and georgi mihaylov have nothing to declare. safety and efficacy of autologous stem cell transplantation in elderly patients with multiple myeloma t maia 1 , c marini 1 , p medeiros 1 , e aguiar 1 , j cancela pires 1 , r bergantim 1 , f trigo 1 and je guimarães 1,2 1 hematology department, centro hospitalar de são joão and 2 faculty of medicine, university of porto autologous stem cell transplantation (asct) is considered standard treatment for multiple myeloma (mm) patients under the age of 65 years, but its safety and efficacy still uncertain for patients over this age. retrospective analysis from one single centre concerning mm patients under, equal or over 65 years who underwent asct between january/2010 and july/2016. it was also compared to 65-70 years old mm patients diagnosed in this period of time who were not transplanted. we analysed a total of 160 patients, 135 of which underwent asct. onehundred-and-six of the transplanted patients were aged 65 years or less (median 56, iqr 10 years), 29 patients were aged more than 65 years (median 67, iqr 2 years) and 25 patients were non transplanted (median 68, iqr 4). the conditioning regimen for younger patients who underwent asct consisted mainly of melphalan 200mg/m 2 (mel200) while half of the elder patients received melphalan 140mg/m 2 (mel140). regarding transplant-related myelotoxicity there were no statistical differences between patients aged 65 years or less and over 65 years old, however the first group needed less days of g-csf (p = 0.04). non-hematopoietic toxicity measured by infections and mucositis was not influenced by age. patients 465 years conditioned with mel200 had more days of aplasia (p = 0.05), greater need of g-csf (p = 0.01) and transfusional support (p = 0.04) than patients ≤ 65 years. there were no differences on non-hematopoietic toxicity. in the elderly group, patients conditioned with mel200 presented more aplasia days (po0.01), higher grade of mucositis (p = 0.03) and more days of iv antibiotics (p = 0.02) than those transplanted with reduced dose of melphalan. comorbidities had no effect on transplant-related toxicity, either by age or by dose of melphalan. days of hospitalization and post-transplant complications did not differ according to age group. transplant related mortality was 2% at day 100 posttransplant. survival after transplant in patients 65 years old or under vs older patients (median follow-up time, 30 months), was not influenced by age (os, 83mo vs 59mo, p = 0.17; pfs, 38mo vs 37mo, p = 0.59). regarding the non-transplanted elderly group, these are patients with more renal disease (p = 0.02) and poorer performance status (p = 0.04) than the transplanted cohort. there is also higher cytogenetic risk (p = 0.01). induction regimens were similar in transplanted group and non-transplanted group 465 years old, and response to first line therapy (before asct of transplanted group) revealed no differences. infections were the most common complication in both groups. transplanted patients needed less days of hospitalization (p = 0.04). comparing the long term outcome of these two groups, survival curves of the elderly patients transplanted were clearly superior to the nontransplanted (os, 62mo vs 21mo, p o0.01; pfs, 45mo vs 20mo, p o0.01) although one has to consider that the non-transplant group has worse features than the elderly transplant group. transplantation in the elderly still debatable but this study shows that it might bring benefit. globally, transplant related toxicity is not influenced by age. regarding dose of melphalan, higher dose in elderly patients has higher toxicity, without apparent benefit in survival. therefore, age should not restrict the access to asct, but instead selection must be based on individual clinical and functional status. disclosure of conflict of interest: none. second autologous stem cell transplantation for relapse after allografting in multiple myeloma using cd 34+ selected donor cells without immunosuppression p novak 1 number of patients receiving a second allogeneic stem cell transplantation (sct) in europe is increasing despite high treatment related mortality (trm). in multiple myeloma only very few reports of second allogeneic sct exist with limited number of patients and substantial mortality. while in most hematological malignancies, the donor cell chimerism is dropping down if patients are relapsing, in myeloma donor cell chimerism remains complete despite relapse. to reduce trm we thought that full donor cell chimerism may allow us to perform a second high dose busulfan based chemotherapy followed by "autologous transplantation" after stem cell mobilization and collection. however, because two consecutive patients failed to collect sufficient cd34+ cells for an autologous transplantation even with plerixafor, we used donor t cell depleted cd34+ selected cells and transplanted those patients in an "autologous" fashion without any immunosuppression. to enhance graft-versus-myeloma effect, we added donor lymphocyte infusion (dli) at day 100. we report here on 11 myeloma patients with a median age of 58 years (range: 48-68) who relapsed after allogeneic sct and underwent a second "autologous" sct with cd34+ selected donor cells. all patients had received one (n = 8) or two (n = 3) autologous sct before 1. allografting. 6 patients received an upfront auto-allo protocol and 5 patients received 1. allogeneic sct as a salvage therapy. 73% of patients received a reduced intensity melphalan based conditioning regimen for 1. allogeneic sct and the median pfs was 39 months (range: 22-56). before 2. allograft patients had received overall a median of 5 (range: 3-7) treatment lines. at the time of 2. allogeneic sct all patients had a full or nearly full donor cell chimerism and remission status was very good partial remission (n = 1), partial remission (n = 5), stable disease (n = 4), progressive disease (n = 1). 82% of patients received a myeloablative busulfan based conditioning regimen and all received cd 34+ selected stem cells with a median number of 5.3 × 10 6 /kg cd34+ cells (range: 1.4-7.5) and 5 × 10 3 /kg cd3+ cells (range: 1.6-6). engraftment was noted in 100% at a median of 10 days (range: 9-14). no further graft-versus-host disease (gvhd) prophylaxis was performed and no acute gvhd (agvhd) was observed. according to treatment plan, 9 patients received escalating dli around day +100, starting with a median dose of 2 × 10 6 /kg (range: 0.5-5) in combination with lenalidomide maintenance in 6 patients. 4 patients experienced agvhd ii-iv after dli. two patients had a severe gvhd (grade iii) which resolved completely after steroid therapy. no nonrelapse mortality after sct and dli was observed. after a median follow up of 43 months (range: 6-49) the median pfs was 16 months (range: 8-24) which translates into a pfs for all patients of 61% at 1 year and 13% at 2 years. median os was 31 months (range: 13-48) and an os of 69% at 2 years and 27% at 3 years was observed. for patients with advanced multiple myeloma relapsing after allografting, a second "autologous" sct with cd34+ selected donor cells without immunosuppression followed by dli is an encouraging treatment option with low toxicity. disclosure of conflict of interest: none. second autologous stem cell transplantation as treatment option for relapsed patients with multiple myeloma: a single center experience (cic 859) p ganeva, y petrov 1 , m mincheff 2 , i tonev 3 , m guenova, l gartcheva 4 , a michova 5 , g arnaudov 6 and g mihaylov 7 1 ya. petrov; 2 m. mincheff; 3 i. tonev; 4 l. garcheva; 5 a. michova; 6 g. arnaudov and 7 g. mihaylov the use of modern therapies such as bortezomib, lenalidomide, thalidomide coupled with upfront high-dose therapy and autologous stem cell transplantation (asct) has resulted in improved survival in patients with newly diagnosed multiple myeloma (mm). the role of second asct as salvage therapy for relapse is unclear because of the availability of new agents to treat progression in multiple myeloma (mm). as the treatment options for management of patients with relapsed mm has become increasingly complex, physicians must consider both disease-and patient-related factors when choosing the appropriate therapeutic approach, with the goal of improving efficacy while minimizing toxicity. we retrospectively reviewed all mm patients who received a second asct as salvage therapy at our center from 2009 to december 2015. for this period we performed 211 transplants for mm patients. twenty five (11.8%) patients received second asct (18 patients were relapsed) and for 7 patients asct was performed as tandem transplant. we analyzed only second asct for relapse. the median time to relapse after first transplant was 18.8 months (range: 8-50 months). all patients received reinduction therapy before the second asct. conditioning was performed with melphalan with two different doses (200 mg/m 2 and 140 mg/m 2 ). the median age at second transplant was 51.8 years (range: 40-67 years), and female/man ratio was 4/14. median interval between first and second asct was 28.3 months (range: 12-60 months). we have no observed early deaths. until now 8 (45%) patients are dead because of progression disease. response rate was assessed three months after asct, nine (50%) patients achieved vgpr, three (16.6%) patients achieved at least a partial response, three (16.6%) had sd and three (16.6%) progressed despite salvage asct.median overall survival (os) was 35.6 months ( relapse ≥ 24months = 47.7; ≤ 24 months = 20). second asct is a feasible and safe option for salvage therapy in mm, especially in bulgaria where the possibility of using novel agents such as carfilzomib, lenalidomide, daratumomab for relapsed patients is limited to clinical trials, because of no reimbursement. the best results were observed in patients whose time to progression was more than 24 months after first asct. advances in treatment of multiple myeloma (mm) has improved overall survival in these patients (pts). a steady increase in the risk of secondary malignancies has been reported over the last decades in mm survivors. estimated incidence of secondary acute myelogenous leukemia or myelodysplastic syndrome (t-mds/aml) after treatment with alkylating agents is 1%-1.5% per year 2-10 years after primary chemotherapy. no specific risk factor has been recognized, but genetic instability, natural history of the disease as well as induction therapy and autologous stem cell transplantation (hct) have been implicated. recently, novel anti-myeloma treatments have been linked with an increase in secondary malignancies, but no solid relationship has been established yet. in a retrospective study, we analyzed the incidence of secondary malignancies (t-aml/mds and solid tumors) in patients suffering from mm who had undergone autologous hct using high-dose melphalan conditioning regimen in our bmt unit. study population consisted of 192 consecutive pts with median age of 55 years (29-70), 56.5% of them being male, who were transplanted during a period of 28 years . type of myeloma was igg/a/d in 56%, 18.8% and 0.5% respectively, while 17.2% was light chain and 7% nonsecretory. the majority of pts presented with k light chain myeloma (62.8%). there was almost equal distribution between iss stages i and ii (45%/38.5%) and only 16.6% were diagnosed with advanced stage myeloma. most pts received two lines of chemotherapy (60%) and all of them more than one. treatment regimens before autologous hct included vad (63pts), bortezomib-based (133pts), dcep (8pts) and rd (29pts) and 34 pts received radiotherapy. chemotherapy administration for mobilization was used in 18 pts (9.3%). conditioning regimen before autologous hct consisted of high-dose melphalan (200 mg/m 2 ) and in case of renal insufficiency 140 mg/m 2 . incidence of a secondary malignancy was 5.7% after a median follow up period of 46 months. t-aml /mds was diagnosed in 9 (4.68%) pts and 2 (1.02%) were diagnosed with breast and lung cancer respectively. pts diagnosed with secondary malignancy were previously exposed in induction therapy to melphalan (6), vad (3), bortezomib (3), high-dose cyclophosphamide as mobilization treatment (4) and radiotherapy (4) . cytogenetic analysis was available in 6 patients diagnosed with t-mds/aml and the majority (4/6) presented complex karyotype. abnormalities mainly observed were deletions and insertions in chromosomes 5,7,17. pts with secondary malignancies had an overall survival of 68 months (26-178), however, after malignancy diagnosis, survival was very poor, four months only . secondary malignancies in pts with multiple myeloma after autologous hct occur with a substantial frequency and have a dismal prognosis. the role of novel treatment agents has to be elucidated. further studies are needed to identify new risk factors and develop better surveillance strategies. [p654] disclosure of conflict of interest: none. survival analysis after allogeneic hematopoietic stem cell transplantation in patients diagnosed with multiple myeloma: a single center experience p patricia hernandez* 1 , r maria calbacho 2 , a laura posada 3 , g fabio augusto ruiz 2 , r anabelle chinea 2 1 hospital universitario nuestra señora de candelaria, santa cruz de tenerife (spain); 2 hospital universitario ramón y cajal, madrid (spain) and 3 hospital universitario cruces, barakaldo (spain) allogeneic hematopoietic stem cell transplantation (allohsct) may provide long term remission cures for patients diagnosed with high-risk multiple myeloma. however, its use is limited since it has a high rate of treatment-related mortality (trm), and because its efficacy compared to autologous hsct is not fully established. we studied 16 patients that underwent allohsct between 2002-2015. population characteristics are in table 1 . all patients were treated at least with one prior therapy lines (1) (2) (3) (4) (5) , all including autohsct (43.75% underwent 2 prior autohsct). 83% had 2 or 3 prior therapy lines. 11 of them received bortezomib as part of treatment regimens. donor characteristics: 2 non-related; 14 hla-identical. gvhd prophylaxis: methotrexate plus a calcineurine-inhibitor: 12 cyclosporine and 4 tacrolimus. median follow-up 15.5 months (1.3-174.5), average was 35.9 months. seven patients died (43.75%); 2 because of progression (12.5%), and 5 (31.25%) due to trm, including infections and immediate complications of transplantation, such as toxicities, icu admission and agvhd: infections: 7 cmv reactivations, 3 invasive fungal and 7 bacterial infections. disease status: 6 patients were in cr prior to allohsct. 3 of them maintained it after. remaining patients died before disease was evaluated. seven patients were in pr prior to transplant, and 4 reached cr after allohsct. one had progressive disease and reached cr after the procedure. two had stable disease and progressed after allo; one of them is in cr after additional therapy lines, and the other one died 4 months after due to it. donor characteristics: hla-identical sibling donors: 87.5% (1 hla-mismatch, passed away 2.7 months after allo due to trm). one of the nonrelated donors, had an hla-mismatch, and died 4 months after allohsct due to trm, the other one is alive after 21 months. gvhd: 10 (62.5%) developed agvhd and 3 of them maintained it chronically. two suffer from cgvhd, plus 3 that initiated it as agvhd. 9 were refractory to steroids. longterm survivors: 3 patients had overcome three years after allohsct. they were among 39 and 50 years old at the time transplant was performed. none of them received bortezomib as part of therapy protocols for the disease. all had 2 therapy lines prior allograft. 2 were submitted to 2 prior autologous hsct. relapse: 4 patients relapsed after allohsct (25%, median time to relapse 6.2 months), being alive 50% at the end of the study. allogeneic hsct is associated with a high incidence of nrm and a low incidence of relapse. rates of acute and chronic gvhd are high. in our cohort, besides that more than 50% are alive until now, they suffer from extensive chronic gvhd and are in need of treatment. long-term survival may be related with patient factors such as young age, but also low tumor burden, or less prior therapy lines; in our group there are no differences in this aspect. studies including high-risk abnormal cytogenetics should help to define which patients are best candidates to allohsct. high-dose melphalan followed by autologous haematopoietic cell transplantation (ahct) remains the standard of care for eligible multiple myeloma (mm) patients. the majority of patients in clinical practice and trials receive a melphalan dose of 200 mg/m 2 (mel200), but a reduced dose of 140 mg/m 2 (mel140) is often used in patients perceived to be unable to tolerate mel200. it remains to be determined whether this considerable dose difference results in different clinical outcomes. we therefore analysed 1978 patients with mm who underwent a first single mel140 or mel200-conditioned ahct between january 2008 and december 2012. all patients were included in the calm study, an analysis of a prospectively defined cohort of patients with data reported retrospectively to the ebmt, covering ahcts for mm and lymphoma. patients in the mel140 group were older than patients in the mel200 group at the time of ahct (median 64 years [range: 28-73] vs 59 years ; p o.001). compared to the mel200 group (n = 1733, 87.6%), fewer patients in the mel140 group (n = 245, 12.4%) were overweight or obese (49.5% vs 63.9%; p = .003). compared to the mel200 group, more patients in the mel140 group had received proteasome inhibitor-containing induction therapy (71.7% vs 57.5%; p = .001), had a karnofsky score of ≤ 80 (38.2% vs 28.1%; p = .002), and were transplanted in less than pr (13.0% vs 7.8%; p = .025). overall survival (os) from the time of ahct was not significantly different between the mel140 and mel200 group (6significantly different between mel140 and mel200 (12 days in both groups for neutrophil recovery; 16 vs 15 days for platelet recovery). however, late neutrophil recovery was noted in a small proportion of patients in the mel200 group. neutrophil recovery 421 days post ahct was not observed in any engrafting patient in the mel140 group, but occurred in 37 (2.2%) engrafting patients in the mel200 group (p = .011). a cox proportional hazards model that included melphalan dose, age, and remission status at ahct showed that melphalan dose had no effect on os, pfs and relapse risk. the findings suggest that mel140 is not inferior to mel200 in younger and older mm patients and may reduce the risk of delayed haematological recovery in some patients. further analyses in relevant subgroups such as patients with high-risk features or renal impairment are required. disclosure of conflict of interest: none. high-dose therapy (hdt) followed by autologous stem cell transplantation (asct) remains the standard of care for patients younger than 65 years of age with multiple myeloma (mm). different agents are being used to control the disease before asct, including the older thalidomide based combination or the newer bortezomib and lenalidomide based combination. the relation between the initial induction regimen and outcomes after asct is not completely clear. to evaluate the effect of different induction regimens on asct outcome, we retropsectively evaluated the outcomes of a low cost older regimen of thalidomide based combination in doublets or triplets with newer novel agents like bortizomib or lenalidomide based combination in a low resources country in transplant-elegible patients with multiple myeloma who underwent autologous stem cell transplantation at king hussein cancer center bmt program we retrospectively reviewed the files of patients diagnosed with mm from january 2008 till december 2015, who received induction treatment followed by hdt and asct and followed up in a single institution. we compared the effects of different induction regimens, disease stage, and remission status before transplantation on over-all survival (os), event free survival (efs) and progression free survival (pfs) using kaplan meier curves. a total of 94 patients were included, 54 (57.4%) of them received thalidomide based induction (group 1) and 40 (42.6%) received bortezomib and lenalidomide based induction (group 2). patients also offered no consolidation nor maintenance therapy. 35 (37.2%) patients were stage i, 34 (36.2%) stage ii and 15 (16%) were stage iii. stage was not documented for 10 (10,6%) of cases. 58 (61.7%) were in complete remission (cr) and 36 (38.3%) were in partial remission (pr). the estimated 5-year os for the whole group was 57.7%. there was no statistically significant difference between both groups in regards to initial iss stage of disease (p = 0.332) or cr status before asct. 32 patients (59.3%) in group 1 achieved complete remission ( cr ) or very good partial response (vgpr), while 25 (62.5%) patient in group 2 achieved cr or vgpr. there was no statistically significant difference between group 1 and group 2 in 5-years os ( 5-year os was 60% vs 57%, p = 0.5007), efs (39.6% vs 52.6%, p = 0.8029) or pfs (45.2% vs 57.8, p = 0.8033). the use of an old, low-cost, thalidomide-based regimen in a low-resources country achieved a favorable transplantation outcomes in patients with multiple myeloma who received hdt and asct. double autologous stem cell transplantation (asct) is a useful treatment for multiple myeloma (mm) patients. we can make the second asct (2asct) without reinduction treatment (tandem regimen) or after a reinduction treatment after first asct (1asct) relapse (salvage regimen). we have conducted a retrospective study over 61 mm patients undergoing a double asct performed in our centre from 1996 to 2016. we have compared the different conditioning regimens used, and if there are any difference between tandem or salvage asct. we do not use maintenance treatment systematically. characteristics of patients and conditioning regimens in table 1 . the overall survivals (os) of our patients are 139 months (m) from treatment start till last control. the most important prognostic factors are the duration of the progression free survival (pfs) after 1asct (hr: 0.96 (0.94-0.99); p = 0.006), and the use of bumel like conditioning regimen at the 1asct or at the 2asct vs another conditioning regimens (hr: 3.43 (1.4-8.39); p = 0.007). today there are 27 patients alive (43%), but only 10 (37%) are free of mm now. the 25 patients who were treated with tandem have a little better os than salvage patients (166m vs 103 m; p = 0.55. not significative). patients at tandem group who received different conditioning regimen at the 1asct and at the 2asct live more time than patients treated only with melphalan 200 (mel200) at both asct. at salvage group the duration of pfs after 1asct is better than the pfs after 2asct (28 m vs 13 m). the use of the same conditioning regimen at the both asct has worse results than if we use different treatment. patients who were treated with s474 bumel at the 1asct or 2asct have better os than patients treated with cbv or mel200. patients who not responded to reinduction treatment before 2asct have worst pfs after 2asct (rc:29 m, response; 19m and not response; only 10 m). attention is drawn to the fact that patients who received bumel at 1asct have large os, but they are very few (8) patients. only one patient has died during the 2asct, and was a patient of salvage group treated with bumel. double autologous transplantation continues to be a useful treatment despite the new mm treatments, and allows to prolonged the os. tandem asct probably is a useful treatment in high risk mm patients. salvage treatment is most useful in patients with a large pfs after 1asct, and good response to reinduction treatment. although mel200 continue to be the standard conditioning regimen for asct in mm patients, we have observed that patients treated with different conditioning regimen at 1asct and 2asct have better prognostic, and bumel has the best results in our serie. disclosure of conflict of interest: none. allogeneic haematopoietic stem cell transplantation (allo-hsct) is an effective treatment for myelodysplastic syndrome (mds) and acute myeloid leukemia (aml). the prognosis of elderly patients with mds and aml after chemotherapy is poor. allo-hsct is feasible in these patients; however the management of elderly patients with mds and aml for allo-hsct is difficult. we performed a retrospective survey of allo-hsct for elderly patients with mds and aml in our institution. we retrospectively analyzed the records of elderly patients ≥ 60 years with mds and aml who underwent allo-hsct in our hospital between january 2011 and december 2015. in this study, we assessed the ipss-r (revised international prognosis scoring system) cytogenetic score and the ipss-r score against the outcome of elderly mds and aml patients who treated with allo-hsct. fifty-one elderly patients with mds and aml were treated with allo-hsct in our institution, 47 patients with mds (29 with mds overt aml) and 4 with de novo aml. ages ranged from 60 to 71 years (median 64), 18 patients were female and 33 were male. there was a history of malignant disease in 14 patients. according to the ipss-r cytogenetic scores of mds patients, 10 patients fell in the good risk group, 7 were in the intermediate risk group, 7 were in the poor risk group, and 23 were in the very poor risk group. regarding the ipss-r score, 1 patient fell in the low risk group, 5 in the intermediate risk group, 6 in the high risk group, and 35 in the very high risk group. sixteen patients were in 1st complete remission (cr), 1 patient was in 2nd cr, 9 patients were in partial remission, and 25 patients were not in remission (nr) upon administration of allo-hsct. all patients received a reduced intensity conditioning regimen. 45 patients [p658] were treated with fludarabine (flu), melphalan and low dose tbi-containing regimens; 5 patients were treated with flu, intravenous busulfan and low dose tbi; and one patient was treated with flu, cyclophosphamide and low dose tli. graftversus-host disease (gvhd) prophylaxis consisted of tacrolimus plus methotrexate in 46 patients, and tacrolimus, methotrexate and mycophenolate mofetil in 5 patients. thirty-four patients received anti-thymocyte globulin (atg). the donor source was sibling bone marrow (bm) in 1 patient, sibling peripheral blood stem cell in 7, unrelated bm in 36 and unrelated cord blood in 7. relapse-free survival (rfs) and overall survival (os) were 40.7% (95% confidence interval (ci): 27.2-53.8%) and 49.7% (95% ci: 35.1-62.7%) at 1 year, 31.4% (95% ci: 18.2-45.5%) and 33.6% (95% ci: 19.2-48.5%) at 3 years, respectively (figure 1.) . in this study, 4 patients died before engraftment. non-relapse mortality (nrm) was 19.6% at day 100. twenty-five patients developed chronic gvhd (3 patients limited and 22 extensive). the causes of death were disease progression (10 patients), treatment-related mortality (13 patients), infection (4 patients) and other causes (3 patients). we suggest that many elderly allo-hsct patients with mds and aml were in the very poor risk group when the ipss-r cytogenetics score was assigned, in the very high risk group when the ipss-r score was assigned and nr upon administration of allo-hsct. rfs and os were 31.4% and 33.6% at 3 years, respectively. there is a need for novel treatment strategies to manage elderly mds and aml patients for allo-hsct. [p659] disclosure of conflict of interest: none. counting bone marrow blasts as a percentage of nonerythroid cells provides superior risk stratification for mds patients with erythroid predominance a sun 1 , y yu 1 , t zhang 1 , q wang 1 , d liu 1 and s chen 1 1 the first affiliated hospital of soochow university, jiangsu institute of hematology, suzhou, china patients with erythroid predominance (≥50% erythroblasts, mds-erythroid) compose a significant proportion of patients with mds. the erythroid/myeloid subtype was divided from the aml category into mds-erythroid by the 2016 who classification of myeloid neoplasms. at that time, there was no consensus on a more appropriate way of enumerating bone marrow (bm) blasts from tncs or necs in mds-erythroid patients. to clarify these questions, 1283 mds patients were retrospectively analyzed in our center. mds-erythoid was observed in 27.0% of patients (346/1283), and these patients had similar clinico-pathological features and overall survival, with 937 cases of mds with o50% encs. by calculating the percentage of bm blasts from necs, 73 of 200 patients (36.5%) with mds-erythroid who were diagnosed within who subtypes without excess blasts (eb) were moved into higherrisk categories and showed shorter os than those who remained in the initial categories (p = 0.041). recalculating the international prognostic scoring system-revised (ipss-r) by enumerating blasts from necs, 40 of 168 (23.8%) mdserythroid patients with relatively lower risk were re-classified as higher-risk and had significantly poorer survival than those who remained in the lower-risk category (p = 0.030). this was especially true for the intermediate risk group that was stratified by ipss-r (unchanged patients vs. shifted patients, p = 0.007). however, the impact of enumerating bm blasts from necs on classification and prognostication was not evident in all mds patients. in conclusion, our results suggested that enumerating the percentage of bm blasts from necs significantly improved the prognostic assessment of mds-erythroid, especially for patients within the intermediate risk group stratified by ipss-r. disclosure of conflict of interest: none. myelodisplastic syndrome (mds) is a group of clonal and heterogeneous diseases, characterized by ineffective hematopoesis. the incidence of mds is about 5% of all blood disorders in children, approximately 40% of them develops acute leukemia. allogeneic hematopoietic stem cell transplantation (allo-hsct) is effective curative treatment of mds in children, but depends on disease status, type of clonal chromosomal abnormalities presented at the time of allo-hsct and graft quality. the aim of this study: to analyze the influence of graft quality on the outcome of childhood mds after allo-hsct. allo-hsct were performed in 58 patients (pts) p662 hypomethylating agents vs. allogeneic sct in elderly patients with advanced myelodysplastic syndromes: a single center study j cermak, a vitek, m markova-šťastná, j soukupova-maaloufova and p cetkovsky institute of hematology and blood transfusion, prague, czech republic a group of 26 patients older than 50 years of age with myelodysplastic syndrome (mds) raeb ii or with acute myeloid leukemia with multilineage dysplasia with less than 30% of bone marrow blasts (mds raeb-t according to the fab classification) was treated with hypomethylating agents (hma) and the results were compared to those obtained in an age and diagnosis matched group of 16 patients who underwent allogeneic stem cell transplantation (sct). in the hma group, 22 patients received azacytidine (vidaza) in the dose of 75 mg/ m 2 × 7 every 28 days and 4 patients were treated with decitabine (dacogen) in the dose of 20 mg/m 2 × 5 every 28 days. median number of cycles administered was 10.8 (range: 3-31). in the transplanted group, 8 patients were transplanted upfront and 10 patients were pretretated with combination chemotherapy, 8 patients received myeloablative conditioning and 8 patients were transplanted after reduced conditioning regimen. a hematologic response to hma (cr,pr, hematologic improvement) was observed in 15 patients (61.5%), cr was achieved in 8 patients (31.8%). in sct group, engraftment was achieved in 14 out of 16 patients, 8 patients died after sct ( 5 on complications related to sct, 3 patients relapsed). no difference in 1 year survival was observed between both the groups (65.6% for hma vs. 62.5% for sct), however, median overall survival (os) was 19.0 months in hma treated group compared to 47.6 months in sct group (p = 0.03). in a recent analysis performed at 48 months after starting the treatment, 2 patients treated with hma (7.7%) and 6 transplanted patients (37.5%) were alive, 16 patients in hma group and 3 patients in sct group relapsed. estimated 5 years survival was 31.3% in sct group and only 3.8% in hma group (p = 0.001). no significant differences in results and adverse effects of treatment were observed between patients aged 51-60 years and those older than 60 years in both hma and sct groups. our results confirm previous observations showing that despite a promising effect of hma resulting in hematologic response in more than 50% of elderly patients with advanced mds, allogeneic sct still represents the only potentially curative treatment connected with long-term survival in a significant number of patients even in this mds patients subgroup. disclosure of conflict of interest: none. immunophenotypic assessment of erythroid dysplasia by a simplified cocktail in myelodysplastic syndromes in taiwan c-c li 1 , p-f weng 1 , c-t lin 1 , j-l tang hypomethylating agent (hma) is commonly used as a bridge therapy to prevent leukemic transformation prior to selection of a donor for allogeneic stem cell transplantation (sct) in patients with myelodysplastic syndrome (mds), and showed low toxicity. although its roles are known, the underlying genetics and clonal dynamics upon hma treatment has not been systematically examined using serial samples, especially in allogeneic stem cell transplantation (sct) setting. in this study, we performed targeted serial sequencing on 66 bone marrow samples from 22 mds patients treated with hma for bridging of allogeneic sct. to perform targeted deep sequencing, bm mononuclear cells before hma treatment and, and fractionated t-cell samples (cd3+ fraction) as controls were taken before hma treatment. analysis of genetic mutations were performed using targeted resequencing by illumina hiseq 2000 (sureselect custom probe set targeting [p664] entire exon regions of a myeloid panel consisting of 84 genes). all 22 patients received hma (decitabine: 15, azacitidine: 7), and the median number of cycles was four (range: 2-12). the overall response rate for hma pre-treatment was 55%: there were four cases of complete remission (cr) (18%), six cases of marrow cr (27%), and two cases of hematologic improvement (9%). targeted sequencing revealed 37 mutations in 16 patients (16/22, 73%) with median of 2 mutations per patient (range: 2-5). mutated genes were then grouped into 8 biological pathways, defined in the cancer genome atlas (tcga) aml study. the most frequent biological pathway at diagnosis was dna methylation (32%), followed by activated signaling (27%), chromatin modifiers (18%), tumor suppressors (18%), spliceosome (14%), cohesin complex (9%), npm1 (4%), and myeloid transcription factors (tfs) (4%). when assessing the difference in pattern of variant allele frequency (vaf), we found the significant reduction of vafs in four (25%) patients after hma. with a median follow-up of 63.4 months, 5-year overall survival (os) were 69.6% (95% ci, 49.0-90.2). there was no significant difference in os according to the presence of mutations in each biological mutational pathway (all, p40.05). to identify prognostic value of mutational dynamics, we subclassified the change of variant allele frequencies (vafs) after median fourth cycles of hma [no mutated or reduction of vafs (11 patients) vs. stable or increased (11 patients)]. however, there was no significant association between the dynamic of mutation and os (p = 0.374). these data show that hma using as bridge therapy for allogeneic sct in mds patients is insufficient to achieve the sufficient molecular responses and, mutational pathway and dynamics may not prognostic in this clinical setting. to clearly demonstrate the role of hma pre-treatment in mds, systematic assessment on a larger cohort is necessary. disclosure of conflict of interest: none. any role of high-dose chemotherapy in mediastinal nonseminoma germ cell tumors? p pedrazzoli, s secondino, a necchi 1 , f lanza 2 and g rosti 1 istituto nazionale tumori, milano and 2 ospedale santa maria delle croci, ravenna among germ cell tumors, primary mediastinal nonseminoma germ-cell tumors (pmnsgcts) have the poorest outcome with 5-year overall survival ranging from 40 to 45%. indeed, the presence of mediastinal location defines per se a "poor prognosis" category according to the igcccg classification. this clinically and biologically distinct disease entity is associated with lower complete response rates to chemotherapy (ct), high rates of relapse and disappointing results from salvage ct. current standard first line treatment for patients with mediastinal primary location is still four cycles of peb, as for all igcccg poor-prognosis patients. we have reviewed available data present in the literature, including recommendations and expert opinions, on the use of high-dose chemotherapy (hdc) with autologous stem cell support in pmnsgcts. the use of hdc as both early intensification (that is, first-line setting) and at disease recurrence (salvage setting) have been reported in small cohorts of patients. according to the largest retrospective comparison, it has been suggested that hdc, given up-front, may produce a 15% to 20% absolute improvement in survival compared with standard dose ct. studies of the ebmt suggest that responsive disease after induction therapy may have a better outcome. mediastinal primary had salvage rates by hdct of less than 15% based on an international multicenter analysis and an ebmt study. the use of hdct in pmnsgcts warrants further investigation, preferably with the use of modern hdct strategies (that is, multiple carboplatin and etoposide courses). while hdc cannot be routinely recommended in pmnsgcts, selected patients with chemosensitive disease may benefit from early intensification. a retrospective analysis evaluating the large ebmt database is ongoing; results will be presented at the meeting. disclosure of conflict of interest: none. high dose therapy and autologous stem cell transplantation in gynaecological malignancies: a monocentric retrospective study m nderlita 1 , i vergote 1 and d dierickx 2 1 university hospitals leuven, department of gynaecology; university hospitals leuven and 2 department of hematology high-dose chemotherapy (hdt) followed by autologous stem cell transplantation (asct) has been established as a treatment option in many relapsed hematologic malignancies. however, in spite of many small trials, there still is no proven role for this treatment in solid tumors including most gynaecological epithelial carcinomas. however, in some recurrent non-epithelial ovarian cancers, such as sex cord stromal tumors, germ cell tumors, neuroendocrine gynaecological tumors and gestational trophoblastic disease, some studies suggest a possible role for hdt followed by asct. we performed a monocentric retrospective descriptive analysis of all patients diagnosed with gynaecological malignancies and treated with hdct followed by asct in our center. clinical, laboratory, pathological and imaging data were collected and analysed, together with information on treatment and outcome. eleven patients were included in this analysis, with a median age of 29 years (range: 14-56) at time of diagnosis. eight patients suffered from ovarian neoplasia. at time of diagnoses 6 patients showed metastatic disease. first line therapy consisted of surgery (n = 4), chemotherapy (n = 2) or a combination of both (n = 5). median time to progression after first line therapy was 39.8 months (range: 0-192) with a median time between primary diagnosis and start hdt of 54.7 months (range: 4-306). three patients underwent single ast, whereas the other 8 patients had a tandem ast, with a median time of 2 months between first and second hdt (range: 1-4). treatment related toxicity was manageable, although there was 1 treatment-related death. at last follow up 5 patients (45%) were still alive with a median follow up of 3.9 years (range: 0.25-15.1) after last asct for all patients. of the 6 deceased patients 5 died with progressive disease. although the number of patients is very small, this retrospective study shows that hdt and asct is feasible in heavily pretreated patients with relapsed/refractory gynecological malignancies, although further studies are mandatory for optimal selection of patients, histological subtype and timing of hdt during the disease course. disclosure of conflict of interest: none. the human endogenous retroviruses (hervs) are remnants of ancient exogenous retroviral infections of the humans: they represent about 8% of the human genome 1 . the basic genes of hervs are group-specific antigen (gag), polymerase (pol) and envelope (env); there are also two regulatory regions, long terminal repeat (ltr), located at 5' and 3' ends. several reports have shown that hervs may play a role in the development of autoimmune diseases, such as multiple sclerosis 2 . additionally the existence of a strong relationship between hervs expression and cancer, based on the mrna expression profile of hervs in normal and cancer tissues has been suggested 2 . the increased level of expression level of herv-h in colorectal cancer (crc), a major cause of cancer death worldwide has been already shown. the aim of the study was to analyse the expressions of env genes of herv-r, herv-h, herv-k and herv-p in the peripheral blood mononuclear cells (pbmcs), in the tumor and in the adjacent normal tissues of 20 colorectal cancer patients. a group of control composed by pbmcs from 46 healthy subjects was also included. rna was isolated from the biological samples and a reverse transcription assay was conducted. quantitative real time pcr was performed to evaluate the expression of the hervs env gene. all the env genes were related to the expression of an housekeeping gene, gapdh. the quantification was carried out using comparative ct method and the difference between the levels of env gene expression in pbmcs, cancer and adjacent normal tissue was given by fold difference. fold difference values were relative to a calibrator: first the pbmcs of patients and then pbmcs of control healthy group. δct values were analysed using the paired sample t-test, followed by a bonferroni correction. higher levels of expression of herv-h, herv-k and in particular herv-p were found in tumor tissues, as compared to pbmcs and to adjacent normal tissues of patients, with an increase of 24-, 10-and 78folds, respectively. the δct distribution of herv-h, herv-k and herv-p in cancer tissues were statistically significant (po0.05) ( table 1 ). the expression of herv-h, herv-k and herv-p env gene resulted increased in the colorectal tumor tissues also when compared with the pbmcs of the healthy controls (5-, 15-and 26-folds, respectively). the δct distribution of herv-h, herv-k and herv-p in tumor tissues were statistically significant ( ρ < 0.05). no difference of expression was observed between pbmcs of healthy controls, pbmcs and normal adjacent tissues of patients (figure 1 ). hervs env gene expression cannot be used as a diagnostic biomarker, but it is conceivable that hervs are directly involved in the pathogenic process of cell transformation and, if the protein expression will be demonstrated, the protein of hervs env gene could be the target for new immunotherapy strategies against colorectal cancer. [p668] disclosure of conflict of interest: none. a biosimiliar g-csf filgrastim is as effective as a reference drug however itis not as cost effectiveas it supposed to be and by the way no impact on the health care system m kurt yüksel, g pekcan, u sahin, s bozdag, s toprak, p topcuoglu, o arslan, g gurman and m beksac ankara university school of medicine biosimiliars are up to 1000 times the size of small molecule generic drugs and far more structurally complex. additionally biosimiliars are manufactured in living cell lines using processes that cannot be exactly replicated from one manufacturer to the next. a biosimiliar cannot be identical to its reference biologic drug. with 67billion dollars in global sales of biologic medicines anticipated to go off patent by 2020.this lead to fast production of biosimiliar drugs. besides, it is expected that biosimiliar drugs will be more cost effective than the reference drugs and will have a meaningful impact on health care systems around the world. aim: to compare biosimiliarfilgrastim (leucostim) with two reference g-csf filgrastim (neupogen) and lenograstim(granocyte) in the context of safety, efficacy and cost effectivity. records of patients with multiplmyeloma(mm) whom underwent autologous stem cell transplantation(asct) and received g-csf sc5mikrogram/kg/day from +day 5 until engraftment were [p668] retrospectively evaluated 60 mm patients were treated with high dose melphalan and asct at the ankara university school of medicine bone marrow transplantation unit between 2013 and 2016. the median age was 59 (38-75 years) with 55% male. patients were divided into three groups (n = 20) whom received reference filgrastim (neupogen), lenograstim (granocyte) and biosimiliarfilgrastim (leucostim): groups a, b and c respectively. the total cost of each g-csf in dollars was calculated by one package of g-csf multiplied by total used days . chi-square, mann-whitney u and kruskal-wallis tests were used for analyses of variance. the percentage of patients who received melphalan 200 mg/m 2 were%80, 85, 80in groups a, b, c respectively (p = 0.9).there was no statistically significant difference between the engraftment day of neutrophil 500 and 1000; platelet 20 000 and 50 000 in the groups. (p = 0.07, p = 0.55, p = 0.33, p = 0.81 respectively) themedian numbers of g-csf administered days were 7(5-18), 8 (5-12), 7 (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) in groups a, b, c, respectively .eventhough there was no statistical difference between the numbers of days( p = 0.23), the total cost in dollars was statistically difference between a vs b and c vs b (both p o0.0001) and there was no statistical difference between a vs c (p = 0.89), total cost in dollars as follows: 155$ (112-288$), 416$(260-624$) and 166$(81-250$) for the group a, b and c respectively. our results demonstrate that biosimiliar gcsf leucostim is highly similar to existing licenced biologic products in turkey with no clinically meaningful difference interms of safety and efficacy. on the other hand it as a biosimiliardoes not have a meaningful impact on the cost savings to the health care system as expected when compared with reference filgrastim. disclosure of conflict of interest: none. in this study, we investigated the roles of prx ii, one of 3 critical peroxidases besides catalase and gpxs, in cml primary cells at diagnosis and remission while patients were treated with sti (signal transduction inhibitor) and tested the same roles in imatinib(im) sensitive ph+ cell lines and resistant cell lines as well. newly diagnosed cml cells, im resistant k562 cells and parental k562 cells were treated stis and analyzed western blot assay to detect bcr-abl, phosphorylated bcr-abl and prx2 protein expression level. we added n-acetylcysteine (0-5mm, 6hr) to k562 cells to show antioxidant effect of imatinib and analyzed dcf-da detection for intracellular ros level and western blot for prx2 protein level. mtt assay was performed to detect cell death by nac time-dependent treatment of 5mm nac(0, 24, 40, 48hr). imatinib resistant k562 cells were established by treatment of gradual increment of imatinib. we also repeatedly investigated the effects of im therapy using prxii overexpressed k562 cells by transfection. at diagnosis of cml, ros level was elevated and prx ii was either absent or significantly suppressed. as ph chromosomes were decreased with stis, suppressed or absent prxs levels were restored to the level of normal individuals. these findings were also inversely correlated with the level of ph chromosomes in the cases of disease progression and re-remission with further treatment. when sti were treated in ph positive cell line, we found deceased cell survival and ros level by mtt assay and dcf-da methods respectively, but elevated prx ii by western blot. by the treatment of nac into ph+ cell lines, the level of dcf-da was decreased and mtt level was down, but prx ii level was elevated. interestingly, the level of bcr-abl oncogene were decreased in prx ii tranfected cells. meanwhile, we observed that prx ii restoration was mild or weak in imatinib resistant k-562, which we established in our lab. the importance of the roles of ros and its prx ii, antioxydant enzymes in cml is further established by our work. our finding may contribute to find a new pathway on which tkis are working besides the mechanisms of atp binding competitively, blocking the binding of abl-bcr kinase to the substrate resulting apoptosis of ph+ cells. furthermore, our finding may be useful to overcome the stis resistant cml in the clinics in the future. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation for the treatment of mucopolysaccharidosis f shunqiao, s xiaodong and l junhui department of hematology, capital institute of pediatrics, china mucopolysaccharidosis (mps) is a lysosomal storage disorder caused by deficient activity of the iduronate-sulfatase.this leads to accumulation of glycosaminoglycans(gags) in the lysosomes of various cells,which causes progressive multisystem involvement with ensuing death.the aim of this study was to exploit the effect of treatment with allogenic hematopoietic stem cell transplantation and administration of high doses of cyclophosphamide early after haplobmt in these cases. we retrospectively reviewed data from 3 mps patients (2 cases mps ii, and 1 case mps i). the two mps ii patients were 44-month-old and 35-month-old boy and the mps i patient is a 84-month-old girl at the time of transplantation. the reduced-intensity of bu+flu conditioning regimen in allo-hsct for these patients was as follows: busulfan 4 mg/kg at 5-2 days before transplantion,fludarabine 40 mg/m 2 at 6-3 days before transplantion.graft-versus-host disease(gvhd) prophylaxis:rabbit antithymocyte globulin 2.5 mg/kg daily at 5-3 days before transplantation,shortcourse methotrexate,posttransplantation high-dose cyclophosphamide on days +3 and +4 was followed by mycophenolate mofetil and cyclosporine.the donors all were their hlahaploidentical father. these three patients' neutrophil engraftment occurred on +14d, +12d and +15d after transplantation respectively, platelet engraftment occurred on day +14d, +10d and +15d after transplantation respectively.complete donor type engraftment was confirmed by short tandem repeat-polymerase chain reaction(str-pcr) on day 14 after transplantation. no regimen-related toxicity occurred,gvhd and graft failure were not observed. 1 month after transplantation, the activity of the iduronate-sulfatase was increased to normal. the motion of metacarpophalangeal joints ameliorated, regression of hepatosplenomegaly, the neurocognitive function improved. allogeneic hematopoietic stem cell transplantation is an effective measure to treat patient with mps at least mps ii and mps i. the reduced-intensity conditioning regimen was helpful to decrease the regimen-related toxicity. posttransplant cyclophosphamide approach successfully used and reduced the incidence of gvhd. this study aimed to evaluate the feasibility of alternative donor hematopoietic stem cell transplantation (hsct) using busulfan, fludarabine, and thymoglobulin conditioning for patients with chronic granulomatous disease (cgd) who lack an hla-matched familial donor. medical records of 11 consecutive patients who received alternative donor hsct between may 2010 and may 2016 were reviewed, and the transplant-related outcome measures were analyzed retrospectively. the donor source was unrelated peripheral blood (pb) in 4, unrelated cord blood (cb) in 4, and haploidentical father in 3 patients. only 2 transplants (8/8 allele-matched unrelated pb) were hla-matched according to current standards relevant to the donor type. the conditioning regimen was uniform; fludarabine 40 mg/m 2 on days -8 to -4, busulfan 3.2 mg/kg/d (or 120 mg/m 2 /d) on days -6 to -3, and thymoglobulin 2.5 mg/kg/d on days -3 to -1 (or on days -8 to -6 in cb recipients). all but one patient were male and their median age at transplantation was 6.5 y (range: 1.1-26.3). one patient who received a cord blood graft suffered from primary engraftment failure, while the other 10 patients were successfully engrafted with their chimerism levels ranging from 66% to 100% (median 100%) at 1 month post-transplant. the median days to neutrophil and platelet engraftment were 12.5 (range: 11-22) and 27 (range: 11-47), respectively. among the 10 patients engrafted, one patient experienced secondary graft failure which was rescued by a second transplantation. the remaining one patient who failed to engraft was also rescued with a haploidentical graft from his mother. eight patients (73%) developed cmv antigenemia, and one of those patients developed cmv hepatitis. three patients developed grade 3 acute gvhd which were manageable. one patient who developed grade 4 hepatic gvhd eventually died. two patients developed extensive chronic gvhd, but became free of immunosuppressants after a complete resolution in one and with remaining stable mild joint contractures in the other. including 2 patients who were rescued by additional transplantation, 10 patients are alive with their latest chimerism levels ranging from 86.8% to 100% (median 100%). the estimated 5-y overall survival rate was 85.7% with a median follow-up of 49 months (range: 6-72). even though the majority of our cohort underwent a mismatched transplantation, the survival rate was excellent. while conditioning with busulfan, fludarabine, and thymoglobulin seems feasible for alternative donor hsct in patients with cgd, special attention needs to be payed on cmv infection and severe gvhd which might offset the high survival rate. disclosure of conflict of interest: none. diarrhea is a common infectious complication in patients who had hematopoetic stem cell transplantation 2 so, we aimed to detect entamoeba histolytica ratio before engraftment, amoung 375 patients who had diarrhea after periferic hematopoetic stem cell transplantation (phsct) in our clinic. allogenic phsct patients had a median age of 29 (range: 15-63) and autolog phsct patients had a median age of 54 (range: 18-74). diarrhea is described as an abnormal increase in the frequency (3 or more times per day), volume or liquidity of stools. we based upon this description in this study. we made stool examination in the first day of diarrhea. as stool examination, we used direct microscopic evaluation and adhesin antigen test specific for e.hystolytica with enzyme linked immunosorbent assay (elisa), e. histolytica ii, techlab, blacksburg, usa). we accepted e.hystolytica positivity as detecting cyst or/and trophosoit in stool and antigen test positivity at the same time. in our study, 185 of 375 patients had diarrhea in the first 28 days of phsct. diarrhea was found in 139 of 242 in autologous phsct patients (%57), 21 of 63 patients in allogenic phsct with non-myeloablative conditioning regiment (%33) and 25 of 70 patients in allo phsct with myeloablative conditioning regiment (%36). diarrhea occured at +8th day of transplantation and the median duration of diarrhea was 3 days. e. histolytica positivity was found 46 of 185 patients (25%) who underwent phsct within first 28 days of transplantation. infection is an important mortality and morbidity factor for patients who had hematopoetic stem cell transplantation, when especially before engrafment (between 0-30 days). 1 autologous phsct patients were elderly, with poor self-care and low socioeconomic status individuals. e. hystolytica is a frequent pathogen in posttransplant diarrhea at endemic regions. prophylactic metronidasole treatment should be used routinely for autologous phsct as in allogenic phsct. 3 patients and companions sholud be tested for e.hystolytica before autologus/allogenic phsct in endemic regions. prophylactic treatment for amebiasis and scanning patient/companions could be a part of solution for post phsct diarrhea. despite the emergence of disease modifying therapies (dmts) for multiple sclerosis (ms) a cohort of patients with aggressive disease have ongoing progression/relapse, associated with progressive disability. autologous haematopoietic stem cell transplantation (ahsct) has been used worldwide for aggressive ms with inflammatory changes on mri. we update on a uk single centre experience of ahsct in ms. a retrospective audit of ahsct performed for ms from 2012 to 2016 at 1 uk centre (king's college hospital) was undertaken. patients were selected for transplantation based on persistent clinical relapses (relapsing-remitting ms) or secondary progressive neurological disability with mri lesion activity despite use of at least 1 dmt. primary progressive patients were also eligible if new/active mri lesions were demonstrable. followup included clinical evaluation, edss assessment and mri scanning. we report our preliminary findings. as of november 2016, 30 patients (16 female, 14 male, 18 rrms, 10 spms, 2 ppms) had received ahsct. mean age at transplant was 40.6 years (range: 22-57). the mean baseline edss was 5.3 (range: 2.5-8.0). 29 patients underwent cyclophosphamide/atg conditioning, while 1 received beam/atg. whilst conditioning and stem cell infusion were well tolerated there was a high rate of infections, with 23/30 patients developing a culture confirmed infection. reactivation of ebv and cmv were observed in a number of patients (21 and 8, respectively) while a number of delayed herpes zoster infections were also seen (4 cases of shingles and 2 of disseminated varicella infection in patients who had previously experienced it in childhood). median follow-up was for 361 days (63-1479). of patients with a formal 6 month assessment (n = 16), 4 had a stable edss, 6 had an improved score (median improvement 0.5, range: 0.5-2.5) and 6 had a deterioration in their score (median 0.5, range: 0.5-1.0). at 12 months (n = 11), 1 had a stable edss, 4 had an improved score (median 0.75, range: 0.5-1) and 6 had a deterioration in score (median 0.5, range: 0.5-1.5). at 24 months, two patients assessed both had improvements in edss scores (median 1, range: 0.5-1.5). for patients who underwent mri at 6 month follow-up (n = 14), 10 had a stable lesion load, 2 demonstrated improvement in lesions and 1 had a new lesion (the remaining mri was difficult to read due to a high baseline lesion load in this patient). 4 patients had mri's at 12 months; 3 were stable and 1 demonstrated a reduction in lesion load. to date, no patients have developed secondary malignancies or autoimmune diseases. of patients with followup data, 4/18 rrms patients experienced suspected clinical relapses following hsct-only one had a new lesion on mri (with no gadolinium enhancement). 3 of the 4 received steroids to treat these relapses (it is unclear if the remaining patient received treatment). 1 patient tried a new disease modifying therapy (1 dose of rituximab) following hsct. ahsct in this cohort was feasible with universal mobilisation and harvest. whilst conditioning and stem cell infusion were well tolerated, there was a high rate of infectious complications in the neutropenic phase. however, the transplant related mortality was 0% despite significant levels of disability amongst this patient cohort. ahsct remains a treatment option to be further investigated in this difficult cohort of patients. disclosure of conflict of interest: none. peripheral blood (pb) stem cells (scs) mobilized with g-csf are the first-choice source for allogeneic transplantation. we carried out a prospective study on healthy donors (hds), to identify donor characteristics that could influence the effectiveness of mobilization with special focus on the value of the basal cd34+ cell count. sibling hds were analyzed in a prospective study. we tested somatic variables (sex, age, weight, height, volemia) and, basal blood counts (white blood cell, peripheral blood mononuclear cell, platelets, hematocrit, hemoglobin, cd34+ cell). hds received g-csf subcutaneously at a dose of 10 μg/kg day. two different determinations of cd34+ cells were done in each donor: baseline (before g-csf administration) and in pb on the morning of the fifth day (after g-csf administration). 128 consecutive hds (65 males) with a median age of 43 years were enrolled. the mean value of cd34+on day 5 was 90.8 cells/μl, while the median value was 75.5 cells/μl. we performed two multivariate analyses either by using median regression (to predict the median value of cd34+on day 5) according to the values of cd34+ at baseline, the first adjusted by gender, age and blood volume and the second by gender, age and bmi. results of both models indicate that from basal cd34+ values o = 1 to values ranging between 3 and 4 cells/μl, predicted median values of cd34+ on day 5 significantly increase, from 54.6 to 92.8 cells/μl for model adjusted by blood volume, and from 49.9 to 92 cells/μl for model adjusted by bmi. baseline, pb cd34+ cell count correlated with the effectiveness of allogeneic pbscs mobilization and could be useful to plan the collection. disclosure of conflict of interest: none. comparison of efficacy between chemotherapy plus granulocyte colony stimulating factor (g-csf) and chemotherapy plus g-csf and granulocyte-macrophage colony stimulating factor (gm-csf) for mobilization of peripheral blood stem cells (pbsc) and hematological recovery post-transplantation in patients with multiple myeloma (mm). a retrospective study of autologous peripheral blood stem cell (apbsc) mobilization data of 56 mm patients who treated with chemotherapy plus g-csf or chemotherapy plus g-csf and gm-csf from may 2008 to july 2016. the mobilization efficacy and hematopoietic recovery were analyzed. a total of 65 stem cell mobilizations were performed in 56 mm patients. in the univariate analysis, successful collection rate of single harvest in female and in patients at iss stage iii, r-iss ii/iii and chemotherapy plus g-csf was lower(po0.05). however, age(≦60 yrs vs 460 yrs), subtype, d-s staging (i+ii vs iii), cycles of chemotherapy before mobilization (≦6 cycles vs 46 cycles), disease phase before mobilization (pr vs cr) and interval diagnosismobilization (≦18 months vs 418 months) were not correlated with the cd34+ cell collection and successful mobilization rate(p＞0.05). in the multivariate model, rate of successful mobilization in patients who received chemotherapy plus g-csf+gm-csf mobilization regimen was high (or = 12.009, 95%ci 1.961-73.537). the effect of mobilization regimen remained significantly (p = 0.007). all patients successfully underwent hematopoietic reconstruction without transplantation-related mortality. chemotherapy plus g-csf +gm-csf mobilization regimen can significantly increase the effect of apbsc mobilization and ensure the reconstruction of hematopoietic function after transplantation. this mobilization regimen is a safe and effective method of mobilizing apbsc. disclosure of conflict of interest: none. clinical efficacy of bk virus specific t-cells in treatment of severe refractory hemorrhagic cystitis after hla haploidentical transplantation om pello 1 , a bradshaw 2 , a innes 2 , s-a finn 2 , s uddin, e bray 2 , e olavarria, jf apperley and j pavlu 1 centre for haematology, imperial college at hammersmith hospital, london, uk and 2 department of clinical haematology, hammersmith hospital, imperial college healthcare nhs trust, london, united kingdom hemorrhagic cystitis caused by bk virus (bkv) is a significant complication of allogeneic hematopoietic cell transplantation (hct). it is particularly common in the setting of hla haploidentical transplantation and can be challenging to manage. here we present a post haploidentical hct patient who developed severe bkv haemorrhagic cystitis resistant to standard therapy and who responded to adoptive transfer of donor t cells enriched with anti-bkv specific cells. a 40 year old man underwent hct for acute myeloid leukaemia with inversion of chromosome 3 and monosomy of chromosome 7 while in first complete remission. as he had no related or unrelated hla identical donor, cells from his hla haploidentical sister were used. on day +32 of this procedure he developed haemorrhagic cystitis. supportive treatment was initiated and cystoscopy showed diffuse bleeding from his urinary bladder with blood clots. urine pcr for bkv showed 5.2 billion copies/ml. despite bladder irrigation, local therapy to s483 bladder mucosa and intravenous hydration, he failed to improve, so treatment with weekly intravenous cidofovir was initiated on day +38. his symptoms improved, but on day +72 he again deteriorated on weekly infusions of cidofovir. his immunosuppression was slowly tapered off without any graft versus host disease (gvhd) but without any significant effect on his hemorrhagic cystitis. he underwent bladder diathermy, was treated with intravesicular hyaluronate and with intravenous cidofovir, but continued to have frank haematuria with blood clots and significant lower abdominal pain. although there was no evidence of obstruction his renal function deteriorated on cidofovir therapy. hence we elected to trial adoptive anti bkv therapy. a leukoapheretic lymphocyte collection was used to prepare an anti-bkv t cell enriched product using the clinimacs prodigy and the cytokine capture system from miltenyi biotec. the eluted product contained 50% and 5% of cd4+ and cd8+ lymphocytes expressing ifng+ respectively and the cd4+/cd8+ dose adoptively transferred on day +86 of transplantation was 0.34 × 10 4 /kg. in vivo expansion of anti-bkv t cells in the patient was analysed weekly for the first month using the research grade peptivators bkv lt and bkv vp1 and the rapid cytokine inspector (cd4/cd8 t cell) kit. bk viral load was monitored by pcr in urine samples twice weekly. ifng+ anti-bkv reactive t cells were undetectable in the patient for the first two weeks after adoptively transfer of donor t cells. twenty days after the adoptive transfer an increase in the cd4+ ifng+ population was observed, in response to the bkv vp1 peptivator. this observation correlated in time with a substantial decrease of the urine bkv viremia from 3.3 million copies/ml to 1360 copies/ml and a complete resolution of patient's symptoms. no gvhd, recurrence of urinary symptoms or any other problems have been observed to date (day +260 of transplantation, +174 days after the adoptive transfer). we are not aware of any other reports of successful adoptive anti bkv cellular therapy. our experience suggests safety and efficiency of the use of anti-bkv t cell enriched products using the clinimacs prodigy and the ifng capture system in hla haploidentical hct where bkv cystitis constitutes a significant complication. this opens the possibility of further clinical trials. disclosure of conflict of interest: none. haploidentical donor (hd) has been used as an alternative stem cell source when patients do not have a hla-matched related or unrelated donor. to overcome the hla barrier, haploidentical stem cell transplantation (haplosct) using post-transplantation cyclophosphamide (ptcy) has been conducted. here, we compared the clinical outcomes of haplosct using ptcy with those of unrelated donor transplantation. eighty-two patients (28 from hd and 54 from unrelated donor [ud]) who underwent allogeneic hematopoietic stem cell transplantation (hsct) in seoul national university children's hospital from january 2013 to june 2016, were analyzed. there were no significant differences between hd and ud patients with respect to median age of patients, sex distribution, and diagnosis [42. 6%], p = 0.081). the conditioning regimen of haplosct included targeted busulfan, fludarabine and cyclophosphamide using ptcy, tacrolimus and mycophenolate mofetil for graftversus-host disease (gvhd) prophylaxis. all patients showed engraftment except for a patient who underwent unrelated hsct. neutrophil engraftment of ud was faster than hd (median 11 days versus 15.5 days, respectively, po0.001). however, there was no significant difference of platelet engraftment. incidences of complications, such as hepatic venoocclusive disease, cmv infection, and hepatic dysfunction, between both groups, were comparable, except hemorrhagic cystitis (hd: 32.1%, ud: 7.4%, p = 0.004). moreover, cumulative incidence of acute gvhd (hd: 32.3%, ud: 44.7%, p = 0.260), severe chronic gvhd (hd: 8.4%, ud: 26.7%, p = 0.059), relapse (hd: 28.6%, ud: 25.1%, 7p = 0.323) and non-relapse mortality (hd: 0%, ud: 9.7%, p = 0.151) were not significantly different. the overall and event-free survival of hd and ud were 85.4% vs 86.2% (p = 0.703) and 75.0% vs 75.9% (p = 0.509), respectively. the clinical outcomes of haplosct using ptcy were comparable with those of ud, and a trend of lower cumulative incidence of severe chronic gvhd and non-relapse mortality was encouraging. it could be a promising alternative therapeutic option in pediatric hsct. disclosure of conflict of interest: none. , median number of apheresis procedures was 2,15 (1-6), median amount od dmso infused 20 ml (7-60). time to engraftment was median 11 days (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) . statistical comparison between cryopreserved pbsc grafts and bm showed benefit for pbsc in the terms (po0.01) of faster engraftment, less infective complications, less transfusion support and less hospital stay. in 253 patients (86%) dmso related events were not registered during graft administration. in 41 patients (14%) mild to moderate dmso related events were registered, as nausea in 34 patients (83.3%), vomitus in 20 patients (50%), tachycardia in 8 (20.8%), hematuria in 6 patients (16%) and 2 patients (4.16%) with bradycardia, hypotension, fever and high temperature during graft infusion. cryopreservation of stem cells is a feasible procedure at our institution. there are some issues that have to be improved. the process is standardized with achieved engraftment in all transplanted patients. disclosure of conflict of interest: none. effectivity of a fludarabine based conditioning regimen in allogenic hematopoietic stem cell tranplantation for patients with severe aplastic anemia and over twenty years old p mustafa 2,3 , k melya pelin 1 , s handan haydaroglu 2 and g ilknur 1 1 gaziantep university, faculty of medicine, department of internal medicine; 2 hematology and 3 bone marrow tranplantation unit, gaziantep, turkey severe aplastic anemia (saa) is an anemia with bone marrow hypocellularity and caused by hematopoietic stem cell failure (1) . allogenic periferic hematopoietic stem cell transplantation (aphsct) is a curative treatment choice (2) . although cyclophosphamide (cyc) and anti thymocyte globulin(atg) is accepted as standart conditioning regimen, especially for patients with high rejection risk, using fludarabin (fu) based regimens show increased successful engraftment ratio with minimal toxic side effects (3) . to the study, 20 saa patiens who were transplanted from hla matched sibling donors between the years 2010-2015 were included. the patients comprised of 13 male (%65) and 7 female (%35). median age was 22 (range: 20-42). the median time from diagnosis to transplantation was 3 (range: 2-108) months. conditioning regimen consisted of cyc (1200 mg/m 2 ), fu (120 mg/m 2 ), atg (fresenius rabbit, 15 mg/kg). the median dose of stem cells was 7 × 10 6 stem cell/kg (range: 5-12). methotrexate (10 mg/ m 2 given four days) and cyclosporine (cyca) (3-5 mg/kg given 18 months) were applied for graft versus host disease (gvhd) prophylaxis. all 20 patients ecog performance status were good (0-1). prior to transplantation only one of the patients received atg-csa, the others received only supportive treatment. after aphsct, neutrophil engraftment was occured at a median of 16 days (range: 11-20) and thrombocyte engraftment was occured at a median of 14 days (range: 11-20). posttransplant graft failure was observed in only one patient at tenth month and this patient had aphsct again from the same donor with the same conditioning regimen. acute gvhd didnot occur in any patient. the 5 (%25) of patients had common chronic skin/oral mucosa gvhd. these 5 patients received methylprednisolone (mp) and/or mycophenolate mofetil (mmf) in addition to the cyclosporine treatment. extracorporal photopheresis was applied to the two patients with chronic gvhd. all chronic gvhd patients had complete response to the immunsupresive treatment with a median follow up time 46 months (range: 1-64). one patient died from sepsis. at 5 year overall-survival rate was 90%. fu based conditioning regimen in aphsct with young saa patients has favorable results. fu based regimen might be a gold-standard treatment in the future. cgvhd; tgfb-induced factor homeobox 1, interleukin 2 receptor gamma, tetra trico peptide repeat domain 37, carbonic anhydrase i, serpin peptidase inhibitor clade a and myod family inhibitor. we established a 3-gene model (myod family inhibitor, tgfb-induced factor homeobox 1, tetra trico peptide repeat domain 37) to diagnose cgvhd. our 3-gene model showed 81.00% sensitivity, 90.40% specificity, 80.8% precision, 81.03% accuracy and 86.90% roc area in diagnosing cgvhd. tgfb-induced factor homeobox 1 increased in expression after rituximab treatment in responders. myod family inhibitor was found to be able to predict rituximab responses in steroid-refractory cgvhd patients. we could demonstrate that gene expression studies were useful in the diagnosis of cgvhd after allo-hsct. we developed a 3-gene model to diagnose cgvhd. hematopoietic stem cell transplantation (sct) is physically and psychosocially demanding. however, exercise interventions may have positive impact on sentiment and psychological well-being in patients undergoing sct. we report on a prospective, randomized study comparing the influence of a multimedia sensor-based practice with classical physiotherapeutic treatment (pt) on psychological aspects and quality of life (qol). patients undergoing sct were randomized into the control group (n = 23) receiving pt or the experimental group exercising on the nintendo-wii (n = 19). patients of both groups performed the exercises under the supervision of a physiotherapist and completed the functional assessment of cancer therapy -bone marrow transplantation (fact-bmt), hospital anxiety and depression scale (hads-d) and distress thermometer at the date of hospital admission (t1) and on day 14 (t2), 28 (t3) and 100 (t4) after sct. questionnaires were completed by the participants independently and without supervision. groups were compared using the mann-whitney u-test. a p value o0.05 was considered statistically significant. the median age of patients was 59 years in the control group and 57 years in the experimental group. results of fact-bmt generally showed a decline of the qol domains measured on t2 and t3 and a raise at t4 in both groups. physical well-being (pwb) showed the strongest fluctuation of all domains. it declined significantly between t1-t2 in both groups (pt p = 0.015, wii p = 0.019), followed by a significant increase between t2-t4 (both groups p = 0.001). however, only in wii-group results of pwb at t4 ranked significantly above t1 (p = 0.028). highest scores were proved for social and emotional well-being (swb/ewb) in both groups. in wii-group ewb increased significantly between t1-t4 (p = 0.015) and ranked above pt-group at all times. functional well-being (fwb) scored lowest in both groups at all times. the score of bone marrow transplant scale (bmts), the second lowest score in both groups, was always higher in wii-group. the level of distress was comparable between both groups. however, at t2 distress increased above the cut-off level of 5 in both groups (wii-group p = 0.006, pt-group p = 0.276). this was accompanied by an increase of anxiety (p = 0.705) and depression (p = 0.006) in the pt-group, while both parameters decreased in the wii-group (p = 0.087 and p = 0.220), respectively. anxiety in intervention group 5,8/4,4/5,0/,4 at t1/t2/t3/t4 stayed below standard group 5,9/6,4/5,9/6,4 at all times. depression averaged out at 4,9/6,5/5,4/5,7 in physiotherapy group and 5,5/4,3/5,9/3,8 in wii-group. to the best of our knowledge, this is the first study to show that exergaming using the nintendo-wii is feasible in the immediate phase after hsct. exergaming may be regarded as beneficial since our data indicate less psychological distress and higher qol in sct recipients exercising with nintendo-wii. therefore, it may be used in addition to pt. disclosure of conflict of interest: none. acute graft versus host disease (agvhd) is the most frequent and serious complication following haematopoietic stem cell transplantation (hsct), with a high mortality rate. a clearer understanding of the molecular pathogenesis may allow for improved therapeutic options or guide personalised prophylactic protocols. circulating micrornas are expressed in body fluids and have recently been associated with the etiology of agvhd, but global expression profiling in a hsct setting is lacking. this study profiled expression of n = 799 mature micrornas in patient serum, using the nanostring platform, to identify micrornas that were dysregulated at agvhd diagnosis. selected micrornas (n = 10) were replicated in independent cohorts of serum samples taken at agvhd diagnosis (n = 42) and prior to disease onset (day 14 post-hsct, n = 47) to assess their prognostic potential. sera from patients without agvhd were used as controls. dysregulated micrornas were investigated in silico for predicted networks and mrna targets. profiling identified 61 micrornas that were differentially expressed at agvhd diagnosis. mir-146a (p = 0.03), mir-30b-5p (p = 0.007), mir-374-5p (p = 0.02), mir-181a (p = 0.03), mir-20a (p = 0.03) and mir-15a (p = 0.03) were significantly verified in an independent cohort (n = 42). mir-146a (p = 0.01), mir-20a (p = 0.03), mir-18 (p = 0.03), mir-19a (p = 003), mir-19b (0.02) and mir-451 (p = 0.01) were differentially expressed 14 days post-hsct in patients who later developed agvhd (n = 47). high mir-19b expression was associated with improved overall survival (os) (p = 0.008), while high mir-20a and mir-30b-5p were associated with lower rates of non-relapse mortality (p = 0.05 and p = 0.008) and improved os (p = 0.016 and p = 0.021). pathway analysis associated the candidate micrornas with haematological and inflammatory disease. circulating biofluid micrornas are dysregulated at agvhd onset and have the capacity to act as prognostic and diagnostic biomarkers. their differential expression in serum suggests a role for circulatory micrornas in agvhd pathology, which warrants further investigation. disclosure of conflict of interest: none. factors associated with medication adherence amongst allogeneic hematopoietic stem cell transplantation recipients j lehrer 1 , e brissot 2,3 , a ruggeri 2,4 , r dulery 2 , a vekhoff 2 , g battipaglia 2 , f giannotti 2 , c fernandez 1,5,6 , m mohty 2,3 and m antignac 1 1 ap-hp, hôpital saint-antoine, service de pharmacie, paris, f-75012; 2 service d'hématologie et de thérapie cellulaire, hôpital saint antoine, assistance publique-hôpitaux de paris; 3 sorbonne patients with median ages of 23 years (range: 11-54 years) between december 2006 and march 2016, which including 7 case of primary hlh (homozygous missense mutation in unc13d: n = 3; homozygous missense mutation in prf1: n = 1; heterozygous missense mutation in prf1 in the combination with hemizygous missense mutation in sh2d1a: n = 1; mutation in rab27a: n =1; mutation in itk: n = 1). 5 cases of unknown causes hlh, 10 cases of lympgoma -hlh (nk/t-cell lymphoma: n = 6, primary γδt cell lymphoma in skin: n = 1; subcutaneous panniculitis-like t cell lymphoma: n = 2; primary t cell lymphoma in skin: n = 1) and 39 cases of ebv associated hlh. 41 patients achieved cr+pr before hsct, and 20 patients nr. 47 patients were transplanted from hla-haploidentical family donors, 13 from hla-identical sibling donors, and 1 from a matched unrelated donor. conditioning regimen include tbi and non-tbi. the median overall survival rate was 65.6% with a median survival time of 38 months (range: 5-119 months). os of primary hlh is 85.7%, os of unknown causes hlh is 60%, os of lymphoma-associated hlh is 60%, os of ebv-hlh is 64.1%. os of cr+pr is 80.5%, os of nr is 35.0% 6 patients without engraftment died because of 2 graft failure and 4 toxicity of conditioning regimen. 15 patients with engraftment died. of those, 1 patient died of hsct-associated tma, 3 patient died of grade iv agvdh, 5 patients died of relapsed hlh or organ failure as results from unsuccessful treatment of the progressively elevated ebv-dna load. 2 patient died of tumor relapse, and 4 patient died of infection. acute gvdh occurred in 42 patients with grade i-ii agvdh in25 patients and grade iii-iv agvdh in 17 patients; chronic gvdh occurred in 19 patients. 46 patients achieved completed chimerism, 9 patients appeared with mixed chimerism,and2 patient presented with graft failure. of 34 ebv-hlh with engraftment, reactivated ebv infection was found in 33 (97%) with the whole blood ebv-dna load at 103-107 copy numbers per ml. ptld occurred in 3 patients confirmed by pathology. after reduced immunosuppressors, negative result of ebv infection was obtained while patients developed gvdh. for 39 ebv-hlh, patients who carry with ebv loading ebvdna ≤ 105 copies/ml before transplantation, overall survival rate was significantly higher than that of ebvdna4105 copies/ml (po0.05); who achieved cr+pr os was significantly higher than that of nr (po0.05); who range: from diagnosis to transplantion ≤ 6 months os was significantly higher than that of 46 months (po0.05). allogeneic hematopoietic stem cell is an effective method for primary hlh and lymphoma-hlh, ebv-hlh,even haploid transplantation. the remission status before transplantation is decisive for the prognosis. disclosure of conflict of interest: none. hepatic veno-occlusive disease after allogeneic hematopoietic stem cell transplantation in a single centre: revised diagnosis and incidence according to new ebmt classification s santarone, a natale, p olioso, g papalinetti, t bonfini, p accorsi, s angelini, g iannetti, m di ianni and p di bartolomeo dipartimento di ematologia, medicina trasfusionale e biotecnologie, bmt center, ospedale civile, pescara, italy sinusoidal obstruction syndrome, also known as venoocclusive disease (sos/vod), is a potentially life threatening complication that can develop early after hematopoietic cell transplantation (hct). in this study we retrospectively investigated the incidence, risk factors and outcomes of sos/vod in 978 transplants, performed in 896 patients between march 1982 and may 2016, on the basis of the new diagnostic criteria and classification of the ebmt. the patient's median age was 31 years (1 to 71). of them, 536 were males and 442 females. 896 patients received one transplant and 82 two transplants. a diagnosis of hematological malignant and nonmalignant disease was present in 784 and 194 cases, respectively. the disease risk at hct was standard in 397 cases, intermediate in 237 and high in 344. an hla identical sibling donor was used in in 691 cases, an unrelated donor in 166 and a haploidentical family donor in 121. conditioning was myeloablative in 813 transplants and at reduced intensity in 165. source of hematopoietic stem cells was bone marrow in 680 transplants and peripheral blood in 298. we did not limit the diagnosis of sos/vod to the classical 21 days after hct, but all suspicious cases appearing in the first 100 days were evaluated. sos/vod was diagnosed in 56 cases, of which 47 in the first 21 days after transplant and 9 between day 22 and 50 (median day 9). their main clinical characteristics are shown in table 1 . the severity of sos/vod was mild in 5 patients (9%), moderate in 6 (11%), severe in 8 (14%) and very severe in 37 (66%). the cumulative incidence (ci) of sos/vod was 5.7+0.005%. among the most relevant variables studied in univariate analysis (recipient age and gender, ferritin level at hct, type of hematological disease, disease risk at hct, type of donor, number of transplants, time of transplant, drugs used in the conditioning regimen, intensity of the conditioning regimen, source of stem cells), there was no factor with an adverse impact on sos/vod incidence. of 56 patients with diagnosis of sos/vod, 41 (73%) died. sos/vod was the main cause of death in 9 patients and a relevant contributing cause of death in 10. of relevance, 6 of 8 patients (75%) with severe sos/vod and 34 of 37 patients (92%) with very severe sos/vod died, whereas only one patient with moderate sos/vod died and no patient with mild sos/vod died. among 56 patients with sos/vod, 19 received defibrotide therapy and 37 the best supportive available therapy. defibrotide was given for a median of 20 consecutive days (range: 5 to 87), starting at day 18 post-hct (range: 3 to 49) with a median total bilirubin level of 3,16 mg/dl (range: 1.4-20.7). the 1-year overall survival (os) of patients treated with defibrotide was better as compared to that of patients who received the supportive therapy (47% versus 27%) although the difference doesn't reach the significance (p = 0.25). the occurrence of sos/vod does influence significantly the 1-yr os considering that it was 72 +1.5% for patients without sos/vod and 33+6% for patients with sos/vod (p = 0.0001). in conclusion, the new ebmt diagnostic and severity criteria for sos/vod has been very useful in identifying patients with severe and very severe forms of this complication. if validated in prospective studies, these criteria will allow an earlier selection of patients requiring immediate therapeutic intervention. [p692] disclosure of conflict of interest: none. the prognosis of patients with newly diagnosed ewing's sarcoma family of tumors (esft) has improved significantly over the last few decades. nonetheless, the long-term survival is still below 35% patients with high risk features.the role of s490 high dose chemotherapy and autologous stem cell transplantation (hdct and asct) for high risk and relapsed esft was analyzed. a retrospective medical chart review was done on patients with efst who underwent hdct and asct between september 1998 and january 2015 at seoul national university children's hospital. indications for hdct and asct included metastasis at diagnosis, bulky primary tumor (4100 ml), axial/ central primary site, and relapsed disease. single hdct and asct was performed in the earlier period, and the regimen was changed from mec (melphalan, etoposide, carboplatin), to topothiocarbo (topotecan, thiotepa, carboplatin), and to bumel (busulfan, melphalan). tandem hdct and asct was performed in the recent period, 1st hdct with bumel and 2nd hdct with modified mec (melphalan, etoposide, carboplatin). twenty-one patients who were diagnosed with esft at a median age of 8.7 years old underwent conventional chemotherapy, radiation therapy and/or surgery and received hdct and asct in complete response (n = 14) or partial response (n = 7). the overall survival of the patients was 70.0% at median 3.6 years and the event free survival (efs) of the patients was 55.0% at median 2.9 years from the last asct. the efs of the patients who underwent single hdct and asct with mec (n = 11), topothiocarbo (n = 1), and bumel (n = 4) was 54.5%, 0.0% and 75% respectively. the efs of the patients who underwent tandem hdct and asct (n = 5) was 50.0%. seven patients relapsed at median 6.6 months from the last asct. despite further treatment, 5 patients died of disease and 2 patients are currently alive without disease. one patient developed acute myeloid leukemia at 17.8 months from the last asct and is currently alive without disease after additional chemotherapy, hla-haploidentical stem cell transplantation and donor lymphocyte infusions. one patient died of transplantation-related mortality due to septic shock and lung infection. hdct and asct may be a promising treatment option for patients with high risk or relapsed esft. further refinements may be needed to identify the optimal regimen and number of hdct and asct. disclosure of conflict of interest: none. post transplant cyclophosphamide (pt-cy) has expanded the use of unmanipulated haploidentical grafts which have a high hla disparity between host and donor. one of the consequences of hla disparity is the development of engraftment syndrome (es). this is an immunological reaction characterized by non-infectious fever and skin erythema that develops after neutrophil engraftment. es resembles an infectious process but treatment involves the use of high dose steroids. our hypothesis is that pts undergoing haploidentical transplants (haplo) with pt-cy should have a high rate of es given the high hla disparity between donor and recipient. objectives: to determine the incidence, symptoms, morbidity and mortality of es in patients undergoing haplo with pt-cy at our institution. retrospective analysis of 22 patients with highrisk hematological diseases undergoing haplo with pt-cy at clinica santa maria between november 2012 and august 2016. es was diagnosed using the spitzer criteria (1). es was diagnosed if pts met 3 major criteria or 2 major plus 1 minor criterion. symptoms could occur prior to or after neutrophil engraftment (neutrophils over 500 cells / ul). all patients signed informed consent and the study was reviewed by our institutional review board. 22 patients received haploidentical grafts ( table 1) . all patients had neutrophil engraftment at a median of 18 days. 9/22 patients (41%) had symptoms that met criteria for es ( table 2 ). 2/9 were transferred to icu due to hypoxemia and 1 patient died after diagnosis of es. 5/9 pts were treated steroids. all patients received broad spectrum antibiotics during the febrile period and neutropenia. blood cultures, ebv and cmv pcr were negative in all es pts. there were no significant differences in hospital stay or one-year overall survival (os) between patients who developed and pts who did not develop es (median 37 vs. 35 days respectively, p = 0.68; one-year os 56% vs. 57%, p = 0.86, respectively). es is a frequent complication in patients undergoing hsct haplo with pt-cy. the incidence of es in our study was higher when compared to historical full match related donors series and lower when compared to cord blood transplant studies (2) there was no increased morbidity and mortality associated with es diagnosis. prompt institution of steroids is recommended in es patients after ruling out an underlying infectious process to avoid further complications. haploidentical allogenic hematopoietic stem cell transplants (haplo-hsct) is an alternative transplant procedure for patients with hematologic malignancies that are in need of transplant and do not have a compatible donor. due to the broad hla disparity, the haplo-hsct can be performed with t-cell depletion and megadose of cd34+. alternatively haplo-hsct can be performed with non t-cell depleted transplants (t-replete) either in combination with anti-thymoglobuline serum (atg) or post-transplant cyclophosphamide (pt-cy) as gvhd prophylaxis strategy. center effect is a known risk factor for outcomes of haplo-hsct in both t-cell depleted (tcd) and t-replete settings. however, many centers tend to specialize in one gvhd prophylaxis strategy making it difficult to differentiate the treatment effect from the center effect. the objective was to investigate the role of center effects in gvhd prevention strategy, on leukemia-free survival (lfs) and overall survival (os) in a population of adult patients with acute leukemia receiving haplo-hsct. a retrospective multicenter study was conducted on patients reported to the ebmt registry. inclusion criteria were: age 418 years, lymphoblastic or myeloid acute leukemia (all or aml) in first or second complete remission (cr1 or cr2), receiving a haplo-hsct between 2005 and 2014. in this population (n = 606), in order to assess the interaction between center and gvhd prevention treatment, we then included in the study selected patients from the centers that had performed more than 20% of both tcd and t-replete haplo-hsct during the study period. center effects on the outcomes consisted of 1) center effect on the baseline risk of event and 2) interaction between center and strategy of gvhd prevention. these center effects were estimated using cox mixed-effects models and tested using permutation tests. all models were adjusted on age, cmv statuses, disease (all or aml), secondary leukemia, previous autologous transplant, disease status (cr1 or cr2), peripheral blood vs. bone marrow transplant, conditioning regimen. after selection, 226 patients were available across 29 centers in europe. one hundred and one (45%) patients received tcd, 125 t-replete haplo-hsct (62 (27%) using atg and 63 (28%) using pt-cy). overall, 175 (77%) patients had aml. there were 86 (69%) peripheral blood transplants in the tcd group and 92 (91%) in t-replete. median follow-up was 2.7 years. in adjusted analyses, without accounting for center effect, t-replete tended to be associated with better lfs (hazard ratio (hr): 0.70 (95%ci 0.45-1.07), p = 0.10) and os (hr = 0.67 (95%ci 0.43-1.04), p = 0.076). when center effects were included, there was significant heterogeneity across centers on the baseline risk of both outcomes (lfs: p = 0.036 and os: p = 0.048). when accounting for interaction between center by strategy for gvhd prevention, the effect of t-replete vs. tcd on the outcomes did vary across centers (p = 0.065 and p = 0.031 for interactions in lfs and os, respectively) ( figure 1 ). we found an interaction between center and strategy for gvhd prevention on outcomes of patients who received a haplo-hsct. the difference between the 2 strategies (tcd or t-replete) varied across centers, in size and direction. this could be in part related to the increase in expertise with each technique in some centers and with the different management of complications, such as infections-related and relapse. disclosure of conflict of interest: none. adherence included recognition of spuriously high levels (typically from contaminated lines) and delayed dose adjustment due to late reporting of levels by the laboratory. the most common cause of unjustifiable non-adherence was failure to increase the dose in response to a low level. inadequate or excessive dose adjustments may be due to lack of experience or unfamiliarity with the sop. two interventions were launched with the aim of improving adherence to the sop for therapeutic tacrolimus dosing. firstly, to provide a rapid and user-friendly calculation method, we developed a mobile phone application (tacrocalc, a dose calculator based upon the sop algorithm) for android and ios devices using python and swift, respectively. secondly, to reduce the number of spuriously high levels, all nurses responsible for specimen collection participated in an educational module delivered by medical and senior nursing staff. key messages included the need to: use only the dedicated colour-coded tacrolimus lumen to infuse iv tacrolimus; avoid sampling from this lumen; sample peripherally when other lumens are known to be contaminated (reasons for this are being explored); suspend infusion of iv tacrolimus 15 minutes before taking a level; send only immediately pre-dose levels for oral tacrolimus. initial re-audit of 16 episodes post intervention (data collection is ongoing) demonstrated a 40% increase in sop adherence (p = 0.03; fisher's exact test), with no cases of unjustifiable non-adherence and a significant reduction in spuriously high levels. in conclusion, the use of tacrocalc by doctors and the implementation of targeted teaching for nurses dramatically improved adherence to the tacrolimus sop. this should ultimately improve therapeutic dosing whilst avoiding toxicity, which may result in better transplant outcomes. tacrocalc is now being adapted to include an option for paediatric dosing, with the potential to dose related medications such as cyclosporine. disclosure of conflict of interest: none. king's college hospital, 2 imperial healthcare, charing cross hospital and 3 imperial healthcare, hammersmith hospital managed with calcium and vitamin d alone in 28/43 cases (65%) and together with bisphosphonates in 9/43 (21%). osteoporosis was managed with bisphosphonates ± calcium/ vit d in 19/36 and with calcium/vit d alone in 10/36. 8 /34 indicated that they would give bisphosphonates in the absence of osteoporosis, if a patient with osteopenia was receiving long term steroids. dissemination and implementation of existing guidance on investigation and managing low bmd post hct appeared to be poor amongst respondents to our survey. routine dxa scanning was underused; the trigger for dxa in the context of steroids is inappropriately high at many centres at 1 mg/kg daily for 3 months; in established osteoporosis, bisphosphonates were used less frequently than would be anticipated. these findings may reflect the limited data on which current recommendations have been made, or the large number of non-transplant guidelines for investigating and managing low bmd which confound management of this post-hct patient group. hematopoietic stem cell transplantation (hsct) still remains as the most efficient therapy for adult patients with acute myeloid leukemia. for older patients and those lacking a hlacompatible donor, autologous hematopoietic stem cell transplantation (auto-hsct) is a valid therapeutic option. 1 authors aimed for determining the effect of auto-hsct for acute myeloid leukemia patients and analyze group of patients who underwent auto-hsct. the study has been set as a retrospective single center study. clinical information included age, gender, aml type and cytogenetic risk. pretransplantation treatment, mobilization and conditioning were analyzed and thus subsequently authors used kaplan and meier method to calculate the actuarial overall survival rate. table 1 describes patients' characteristics. majority of patients received similar induction therapy based on combination of cytarabine and anthracycline. timespan from the diagnosis to auto-hsct varied from 74 days to 1791 days, median was 175 days. seventy (88,6%) patients received a preparative regimen consisting of busulfan at 1mg/kg orally, four times daily for 4 days for a total dose of 16 mg/kg administered on day -6 through day -3 and melphalan 100-150 mg/m 2 intravenously for over 4 hours on day -2. patients achieved an absolute neutrophile count (anc) of ≥ 0.5 × 109/l in between 10 to 40 days; median was 14 days. patients achieved not transfused platelet count ≥ 20 × 109/l in between 10 to 209 days; median was 19 days. median of patients' discharge from hospital was 19 days (range: from 13 to 44 days) since auto-hsct. hundred day mortality after autologous transplant was at 6.32% (5/79). on the date of our evaluation (april 30, 2016), 48 patients were alive and in continued cr. the relapse rate was 39.5% (32 patients) and 7 patients (8.6%) were lost from follow-up. the 5-year overall survival (os) was 60.8%, so the target median of overall survival has not been reached. [p701] the development of dyslipidaemia is commonly observed after haematopoietic stem cell transplantation (hsct). few data are available concerning lipid profiles over a long followup period or with regard to the different transplantation types (autologous vs. allogeneic) or the effect of multiple transplantations on the development of dyslipidaemia. a retrospective, single center cohort study including 1239 adult patients (416 years) who underwent hsct at the university hospital basel s496 1973-2013 and who survived ≥ 100 days was performed. patients with at least a baseline lipid measurement were included (n = 1096) and grouped according to the type of their first hsct (autologous or allogeneic). for the examination of the effect of subsequent hscts, patients with consecutive transplantations of the same type were included and other patients were censored when a different transplantation type was performed. serial lipid profiles (total-, ldl-and hdl-cholesterol and triglycerides) before and after transplantation were examined. of the 1096 patients, 407 underwent a first, and 89 of these at least one subsequent autologous hsct. 689 underwent a first, and 85 of these at least one subsequent allogeneic hsct. median age of patients at autologous hsct was 52y (iqr 39-61) and 43y (32-53) at allogeneic hsct. 62% and 58% were males, median bmi pre-transplant was 25 (22-28) and 24 (22-27). the majority of patients underwent s497 intensive conditioning before hsct. median follow-up time was 3.0 years in the autologous and 4.8 years in the allogeneic group, with a maximum follow up time of 26.1 and 34.3 years, respectively. table 1 shows the number and percentage of patients with dyslipidaemia (1st autologous and allogeneic transplants). the distribution of exact total cholesterol values along with comparisons with baseline measurements according to group are presented in the figure 1 . *% based on number of measurements available total, ldl-and hdl-cholesterol and tg increased within 3 months of transplantation, regardless whether autologous or allogenic transplantation or a first or a subsequent transplantation was performed. the percentage of patients with dyslipidaemia accordingly rose significantly within 3 months of transplantation and persisted throughout follow-up. although patients undergoing an autologous hsct presented with higher baseline values of total cholesterol, a significantly greater increase post-transplant was observed after allogeneic hsct. first and subsequent transplantations seem to behave similarly with respect to changes in lipid profiles. disclosure of conflict of interest: none. nuremberg, erlangen, germany; 4 department of cancer immunology, institute for cancer research, oslo university hospital, radiumhospital, oslo, norway; 5 kg jebsen center for cancer immunotherapy, institute of clinical medicine, university of oslo, oslo, norway; 6 department of haematology and oncology, university hospital of the goethe university, frankfurt, germany and 7 childrens hospital, goethe university, frankfurt, germany natural killer (nk) cells are lymphocytes of the innate immunity with a potent anti-tumor capacity. in tumor patients, such as multiple myeloma (mm) patients, an elevated number of nk cells correlates with a higher overall survival (os) rate. our study adressed nk cells characteristics and anti-tumor ability in mm patients. especially cytotoxicity of patientderived, cytokine-stimulated nk cells against mm cells has been analyzed at various time points (at diagnosis, before/ after chemotherapy and/or auto-sct). nk cells from patients were analyzed by facs after pbmcs isolation via ficoll separation at different time points: tp1, before the start of high dose chemotherapy (hdc)/auto-sct; tp2, after early leukocyte recovery (leukocytes 41000/μl) and tp3: at least 2 weeks after tp2. for testing nk cell cytotoxicity against mm cells, nk cells were purified via negative selection and expanded in vitro for 1-2 weeks in low doses il-2 and il-15. nk cells were divided into the cd56 ++ cd16 − or cd16 + and cd56 + cd16 ++ subsets. while the major nk cell subset at tp1 was the cd56 + cd16 ++ nk cell subpopulation (71.86%), after leukocyte recovery at tp2 cd56 ++ cd16 − /+ nk cells were the main subsets (cd16 − : 22.85%; cd16 + : 36.51%). we further evaluated the nk cell function upon tumor interaction at the defined time points. cd56 ++ cd16 − nk cells were the main subset to produce ifn-γ upon interaction with k562 cells at all different time points. the percentage of ifn-γ-positive cd56 + + cd16 − nk cells was slightly decreased at tp2 compared to tp1 but significantly increased from tp2 to tp3 (p-value: 0.0008). similarly, mip-1β-and cd107a-positive cd56 ++ cd16 − cells remained constant between tp1 and tp2, whereas their percentages increased from tp2 to tp3 [p-values: 0.0056 (mip 1β) and 0.0232 (cd107a)]. moreover, in a small group of mm patients, we isolated nk cells and expanded them for 1-2 weeks prior to the functional assays. as expected, the expansion rate was reduced after chemotherapy compared to nk cells from healthy controls, but the patients nk cells increased their ability to kill mm cells due to the ex vivo cytokine expansion. conclusion: our data demonstrate that nk cells have an altered phenotype and function after hdc/auto-sct. remarkably, these nk cells were able to secrete cytokines and still displayed cytotoxic capacity against different types of tumor cells. however, as the proliferative capacity of nk cells seemed to be reduced following chemotherapy, innovative nk cell therapeutic approaches further improve the patients nk cell activity by an ex vivo cytokine stimulation procedure. finally, we suggest that an additive cell therapy with cytokinestimulated autologeous nk cells might improve the outcome of mm patients. lymphoid and myeloid acute leukemia are the most frequent type of cancer and the most frequent cause of cancer related death in children. relapse and refractory disease are the main clinical problems that current therapies are still unable to solve. one of the main nk cell activating receptors is nk cell group 2d (nkg2d). nkg2d receptor recognizes human mica/ulbp1-6 ligands. these nkg2d ligands are expressed in leukemia cells and constitute suitable targets for immunotherapy. the expression of nkg2d ligands was analyzed in peripheral blood mononuclear cells from 61 pediatric patients suffering from acute leukemia (21 acute myeloid leukemia, 25 b cell acute lymphoid leukemia and 15 t cell acute lymphoid leukemia), as well as in 7 leukemia cell lines (k562, rs4-11, jurkat, nalm-6, molt-3, reh and cem), by flow cytometry using specific monoclonal antibodies directed against mica, micab, ulbp-1, ulbp-2, ulbp-3 and ulbp-4, and by quantitative pcr using taqman probes. peripheral blood mononuclear cells from healthy donors were labeled with cd45ra microbeads and depleted using automacs device. the hl20i4r-mndanticd19bbz lentiviral vector was derived from the clinical vector cl20i4r-ef1a-hgcopt27 but contained the extracellular domain of nkg2d, the hinge region of cd8a and the signaling domains of 4-1bb and cd3-z. the cassette was driven by mnd promoter. viral supernatant was produced by transient transfection of hek293t cells with the vector genome plasmid and lentiviral packaging helper plasmids pcagg-hivgpco, pcagg-vsvg and pcag4-rtr2. cytogenetic studies and array comparative genomic hybridization were performed to analyze the genetic stability of lentiviral-transduced memory t cells. the in vitro cytotoxicity of cd45ra − t cells against leukemia cells, healthy pbmc and mesenchymal stem cells (msc) was evaluated by performing conventional 4-hour europium-tda release assays or by flow cytometry using cfse and 7aad labeling of target cells. nkg2dl were heterogeneously expressed in leukemia primary cells and cell lines. for b cell all primary samples, we found expression of mica/b, mica and ulbp1 decreased in refractory disease compared to remission (p = 0.01, p = 0.03 and p = 0.02, respectively). lentiviral transduction of nkg2d-4-1bb-cd3z markedly increased nkg2d surface expression in cd45ra − memory t cells, which became consistently more cytotoxic than untransduced cells against leukemia cells. additionally, no chromosomal aberrations nor cytotoxic activity against healthy pbmc or mesenchymal stem cells was observed in nkg2d car expressing t cells. our results demonstrate nkg2d-car redirected cd45ramemory t cells target nkg2dl expressing leukemia cells in vitro and could be a promising and safe immunotherapeutic approach for acute leukemia patients. peripheral blood stem cell mobilization and collection from elderly patients (≥65 years) with multiple myeloma: a single center experience g cengiz seval 1 , sk toprak 2 , s civriz bozdag 2 , m kurt yuksel 2 , p topcuoglu 2 , o arslan 2 , m ozcan 2 , t demirer 2 , g gurman 2 , h akan 2 , m beksac 2 and o ilhan 2 1 clinic of hematology, yildirim beyazit university yenimahalle education and research hospital and 2 department of hematology, ankara university school of medicine high-dose melphalan followed by autologous hematopoietic cell transplantation (auto hsct) has become the standard procedure for patients with symptomatic multiple myeloma (mm). the ability to mobilize stem cells from healthy donors shows little deterioration with age, the influence of patients' age on auto hsct is uncertain and studies in patients' ≥ 65 years are scarce. severe studies specific to mm have failed to show an independent effect of patient age on cd34+ mobilization. we retrospectively compared myeloma patients below the age of 65 with patients above 65 years of age, analyzing cd34 mobilization into peripheral blood and the number of leukapheresis needed to collect at least one single stem cell graft. material and methods: from february 1999 through april 2016, data from 501 myeloma patients below the age of 65 were compared to 52 myeloma patients above 65 years of age. all these data were obtained from the ankara university faculty of medicine center for therapeutic apheresis and written informed consent was signed according to our institution regulations. most of the patients received only gcsf at a dose of 5 μg/kg bw twice-daily s.c. until stem cell procurement. patients underwent further pbsc collections until we obtained the target dose 420 cd34+ cells/μl blood. a maximum of 3 collections were performed in the first mobilization; if the cell dose was not achieved, we submitted patients to a second mobilization. fifty two of 553 patients were above 65 years of age (median age 66, range: 65-73) and 501 patients were below the age of 65 (median age 54, range: 29-64). baseline characteristics of the older and younger patient cohorts are summarized in table 1 . mobilization regimens for the younger patient population were cyclophosphamide based (n: 122), g-csf only (n: 372) and +plerixafor (n: 7). mobilization in the older population was with cyclophosphamide based (n: 10), g-csf only (n: 41) and +plerixafor (n = 1). the chemotherapy regimens were not statistically different between both age groups. there were no significant statistical differences in time from diagnosis to mobilization, number of prior therapies or disease status between both patient groups. the number of cd34+ circulating cells before scheduled leukapheresis was mean 69.28 cells/μl (median 49 cells/ μl, range: 2-397; sem ± 46.875) in all patients (including patients who failed mobilization). our data support the observation that after a standard mobilization regimen with anti-myeloma chemotherapy and once-daily growth factor support, patients above 65 years of age show an impaired cd34 mobilization into peripheral blood compared to a younger population. this can be overcome by an increased number of leukaphereses. still the number of progenitor cells in the actual graft is inferior compared to the younger population. [p712] disclosure of conflict of interest: none. donor and/or recipient citomegalovirus (cmv) seropositivity has been associated with a poor overall survival (os) in patients who have received an allogeneic hematopoietic stem cell transplantation (allohsct). in comparison with seronegative donors, hsct from seropositive donors has been associated with decreased disease-free survival (dfs) and increased non-relapse-related mortality (nrm). we analyzed the prognostic impact of cmv serology status (donor/ recipient) in patients diagnosed with acute leukemia (al) [p713] s503 who had received an allohsct in our institution. retrospective unicentric study of patients diagnosed with al between 2001 and 2015 who received allohsct.the following outcomes were studies: os, dfs, and cumulated incidences of relapse (ri), nrm, acute graft-versus host disease (agvhd) and chronic gvhd (cgvhd). the series included 163 patients (86 males, 77 females), median age of 44 years . al type: 42 (26%) all, 121 (74%) aml. type of transplant: 88 (54%) related donor, 42 (26%) unrelated donor and 33 (20%) unrelated umbilical cord blood. the majority, 111 (68%), received myeloablative conditioning. stem cells source: peripheral blood 124 (76%), cord blood 33 (20%) and bone marrow 6 (4%). cmv serology status: positive receptor 124 (76%), negative receptor 39 cases (24%); positive donor 100 (61%), negative donor 63 (39%). serology status combinations (d/r): +/+ 84 (52%), +/ − 40 (24%), − / − 23 (14%), − /+ 16 (10%). 56 patients developed agvhd and 14 (9%) cgvhd. the impact of donor/recipient cmv serology status on os, dfs, ri, nrm and incidence of agvhd and cgvhd for the overall series is reported in table 1 . no statistically significant differences were detected in any of the analyzed variables. in this study, donor/recipient cmv serology showed no influence on the analyzed variables os, dfs, al relapse, nrm, acute and chronic gvhd. however, the sample size limits the validity of the results. disclosure of conflict of interest: none. supported in part with the grants pi10/01417 from fondo de investigaciones sanitarias and rd12/0036/0029 from rticc, instituto carlos iii and 2014sgr225(gre), generalitat de catalunya, spain. petersburg, russia during the last two decades ahsct has been used as a treatment option for ms with promising outcomes. qol is an important outcome of ms treatment. its assessment gives the patient's perspective on the overall effect of treatment. we aimed to study qol in ms patients before and after ahsct and search the value of the data obtained for decision-making. a total of 135 patients with different types of ms were enrolled in the study: mean age-34 (range-17-54) years old; male/ female-53/82; mean edss-3.5 (range: 1.5-8.5). all patients were treated by ahsct. reduced-intensity beam-like conditioning was used (bcnu 300 mg/m 2 , etoposide 100 mg/m 2 , ara-c 100 mg/m 2 and melphalan 100 mg/m 2 ). mean follow-up was 24 months (range: 12-53 months). qol was assessed using generic questionnaire sf-36. for comparisons t-test for independent samples or mann-whitney test was used. qol parameters in ms patients at 12 months after ahsct improved in comparison to base-line: physical functioning-66.3 vs 52.6, role-physical functioning-62. 8 6 . further qol improvement was registered at long-term follow-up: integral qol index exhibited 0.50 at long-term follow-up as compared to 0.32 at base-line. qol improvement was more dramatic in relapsing-remitting ms than in progressive ms. we found a significant increase of all eight sf-36 scales in a year posttransplant as compared with base-line in relapsing-remitting ms patients (po 0.05). in progressive ms patients statistically significant improvement was registered for six out of eight sf-36 scales (except bodily pain and role-emotional functioning) (p o0.05). improved qol parameters were preserved over the entire study period in all the patients who did not have disease progression or relapse. in conclusion, qol monitoring in ms patients after ahsct provides clinicians with the unique information regarding the changes in physical, psychological and social well-being of patients who have been treated with this new treatment modality. it allows to evaluate risks/ benefits of ms patients undergoing ahsct and might influence decision-making. further studies are needed to examine the trajectory of qol changes in this patient population to better define treatment outcomes after ahsct. disclosure of conflict of interest: none. pediatric patients with leukocyte adhesion deficiency type-i (lad-i), a rare autosomal recessive primary immunodeficiency disorder, experience severe and recurrent lifethreatening bacterial infections. allogeneic haematopoietic stem cell transplantation (hsct) offers the possibility of curative therapy although the conditioning regimen used for hsct in lad-i is still a controversial issue. this study provides evaluation of outcome of the lad-i pediatric patients who underwent reduced-intensity conditioning (ric) hsct. twenty four patients (14 female) with severe lad-i who received 26 hscts between februay 2007 and september 2016 at our center were enrolled. the median age at hsct was 30 months (range:4 months-14 years). patients received bone marrow (n = 9), peripheral blood progenitor cells (n = 14) or umbilical cord blood grafts (n = 3) from hla-matched related donors (n = 18), mismatched related or unrelated donors (n = 4), unrelated fully matched donors (n = 1) and haploidentical relative donors (n = 1). ric regimen was provided with fludarabine, melphalan and anti-thymocyte immunoglobulin. cyclosporine a and prednisolon were used as graft-versus-host disease (gvhd) prophylaxis. engraftment occurred in 23/26, of which one patient experienced graft rejection.the median times to neutrophil and platelet engraftments were 12 days (range: 10-23days) and 15 days (range: 10-32days), respectively. with a median follow-up of 43 months (range: 2-95months), overall survival (os) was 70.8%.the main causes of death were gvhd and infection. acute gvhd occurred in ten patients (4 grade i-ii, 6 grade iii-iv) and 3 patients also developed chronic gvhd. there were no significant differences in acute gvhd occurence and also os regarding to the stem cell sources. at this time,10 patients with full chimerism and 6 patients with mixed chimerism are alive and disease free. conclusion: hsct offers long term benefit in lad-1 and should be considered as an early therapeutic option if a suitable hla-matched stem cell donation is available. as pretransplant infections in primary immunodeficient patients especially those affected by lad-1 lead to rise in mortality rate, ric regimen is found to be safe and mixed donor chimersim appears sufficient to prevent significant symptoms. disclosure of conflict of interest: none. tregs based immunotherapy may be beneficial in several immune mediated diseases including graft versus host disease (gvhd). the possibility of cryopreserving tregs might lead to the administration of multiple doses, thus potentially increasing their efficacy in chronic diseases. however, there are few and controversial data on the functionality of tregs after cryopreservation. here, we evaluated the phenotype and the inhibitory capacity of thawed tregs. tregs were purified from leukapheresis of normal donors (n = 3) by double immunomagnetic depletion (cd8 and cd19) followed by cd25 enrichment using the clinimacs system (miltenyi biotec) under gmp condition. the cells were cryopreserved in saline solution containing 10% human serum albumin (hsa) and 10% dmso with a controlled-rate freezing. cell viability was assessed by 7-aad staining. number/phenotype and function were evaluated on fresh and thawed tregs. cryopreserved autologous t effector (teff) cells were used in mlr assays. before cryopreservation the tregs enriched product mean viability was 95 ± 4% and the mean percentage of cd45+cd4 +cd25+cd127low and cd45+cd4+cd25+cd127lowfoxp3+ cells was 74 ± 13% and 66 ± 10%, respectively. we then analysed the tregs enriched product after thawing. mean viability of thawed tregs, by 7-aad staining, was 85 ± 7%. the viable tregs were almost totally cd4+cd25+ (97 ± 2%). the mean percentage of cd4+cd25+cd127low and cd4+cd25 +cd127lowfoxp3+ thawed cells was 73 ± 14% and 71 ± 20% respectively. the contaminant cells present in the treg enriched product were mostly cd4+cd25+cd127+ (around 18%). we further characterized the phenotype of the cd4 +cd25+cd127low population. this population was almost totally foxp3+ (93 ± 6%) and expressed selected markers at various degree (cd62l (50 ± 2%), cd15s (6 ± 2%), cd45ra+ (19 ± 3%), hla-dr+ (15 ± 10%), ccr7+ (74 ± 5%), cd49d (52 ± 14%), cd26+ (1 ± 0.4%), cd196+cd161+ (4 ± 1%). notably, viable thawed tregs were able to induce inhibition of autologous teff cells in a 1:2 tregs:teff ratio as freshly isolated tregs: 44 ± 16% (thawed) vs 55 ± 24% (fresh) of inhibiton (p4 0.1). in conclusion, here we demonstrated that thawed tregs from healthy donors mantain a stable phenotype. in addition, in our hands tregs show good suppressive ability after thawing despite lower expression of cd62l and cd15s relapsed and refractory malignant b cell diseases: evidence for therapeutic efficacy via subcutaneous administration of anti-cd20 × anti-cd3 antibody lymphomun r buhmann, p ruf, j hess, h lindhofer, u jacob 1 and m dreyling 1 the trifunctional antibody anti-cd20 × anti-cd3 lymphomun represents a chimeric immunoglobulin scaffold (mouse igg2a/ rat igg2b) with promising treatment outcome in patients suffering from malignant b cell diseases. by changing the lymphomun administration route from intravenous (i.v.) to subcutaneous (s.c.) the proinflammatory cytokine-mediated side effects were considerably slighter and generally welltolerated. most importantly, s.c. lymphomun showed outstanding responses in b cell depletion even in the absence of elevated cytokine levels (e.g. il-6) that are required for cytotoxic t cell activation. in summary, the clinical tolerability of s.c. lymphomun may result in a considerable improvement of the subjective well-being and in enhanced mobility due to decreased pain symptomatology. intestinal microbiota disruption is associated with acute gastrointestinal (gi) gvhd and inferior outcome in patients after allogeneic stem cell transplantation (asct). the wide use of systemic broad spectrum antibiotics adds a further risk factor contributing to major microbiota shifts. here, in a retrospective analysis of 200 patients undergoing asct at the regensburg university medical center we assessed the relative expression of paneth cell antimicrobial peptides (amps) in 292 human intestinal biopsies in relation to acute gi gvhd and systemic antibiotic treatment. the relative expression of paneth cell amps was significantly higher in biopsies of the upper gi tract than in the lower gi tract for reg3α (p ≤ 0.001), human defensin (hd) 5 (p ≤ 0.001) and hd6 (p ≤ 0.001). regarding the distribution of paneth cell amps in the gi tract we observed significantly higher expressions of all three paneth cell amps in the duodenum, jejunum and ileum compared to the stomach, colon and rectum (po0.001, figure 1 ). in the presence of acute gi gvhd, paneth cell amps reacted contrarily in the upper and lower gi tract: we observed a decrease of hd5, hd6 and reg3α in the upper gi tract (p ≤ 0.01), similarly paneth cell count dropped in case of severe gi gvhd stage 2-4 (po0.001). however in the lower gi tract severe acute gi gvhd was associated with an increase of paneth cell amps (p ≤ 0.03). initiation of additional systemic antibiotic treatment prior to day 10 after asct correlated with a significantly higher expression of hd5 (p = 0.002) and reg3α (p = 0.01) in intestinal biopsies compared to patients without or with initiation of systemic antibiosis after day 10. however, no significant differences were found in terms of hd6 expression in intestinal biopsies and start of systemic antibiotic therapy. the expressions of hd5, hd6 and reg3α in intestinal biopsies seem to respond to major microbiota disruptions caused by acute gi gvhd or systemic antibiotic treatment. while observations in the upper gi tract seem to reflect paneth cell damage, the relative increase in the lower gi tract may indicate inflammatory induction of amps in colonic epithelial cells in the course of gvhd. [p718] disclosure of conflict of interest: none. patients (55%) were in complete remission at the time of pcy haplo-sct. hematopoietic cell transplantation-comorbidity index was ≥ 3 in 20 patients (74%). thirteen patients (48%) received non-myeloablative conditioning regimen (as baltimore schema, luznik et al. bbmt 2008) prior to haplosct while remaining patients received busulfan-based regimen. all patients were given pcy and both csa and mmf as gvhd prophylaxis. day+100 cumulative incidence of grade 2 to 4 and 3 to 4 acute gvhd was 15% and 7%. 2-year cumulative incidence of chronic gvhd was 12%. the cumulative incidence of non-relapse mortality and relapse at 2 years were 38% and 27%, respectively. with a median follow up of 25 months (range: 4-63), 2-year progression-free and overall survivals were 36% and 39%, respectively. disease status at the time of haplosct was a major determinant for outcome. indeed, 2year nrm and os were 58% and 25% in patients transplanted with active disease, respectively, while corresponding values in patients transplanted in cr were 21% (p = 0.036) and 49% (p = 0.041), respectively ( figure 1a and 1b) . we can conclude that in selected patients who could be candidate for second transplantation, haplosct is feasible and may represent a curative option. the overall incidence of relapse of 27% is promising in this situation for which no alternative for cure is available, with relatively good survival in patients transplanted in cr. however, the very high nrm (58%) in refractory patients should make us consider second transplant with caution in this setting. for these patients, specific developments are needed to avoid procedure-related toxicity. [p722] disclosure of conflict of interest: none. secondary solid tumors following hematopoietic cell transplantation for thalassemia major a natale, s santarone, a meloni, a pepe, m di ianni, s angelini, p di bartolomeo 1 1 dipartimento di ematologia, medicina trasfusionale e biotecnologie-ospedale civile, pescara, italy secondary solid tumors (sst) have been described after hct, in particular for patients affected by hematologic malignancies. there is limited information about the incidence of sst following hct for thalassemia major (tm). the aim of this study was to determine the incidence of sst in 134 patients with tm who received hct in our center between 1983 and 2013. 117 patients survived more than 3 years after hct and were enrolled in the study. of them, 57 were males and 60 females. their median age at time of hct was 10 years (1-29). as conditioning regimen, they received busulfan (14 mg/kg) and cyclophosphamide (200 mg/kg). the gvhd prophylaxis included cyclosporine and methotrexate. all patients received bone marrow cells from an hla identical donor. at time of this report, 112 patients were cured, whereas 5 patients rejected their graft and are now under regular transfusion treatment. overall, the median follow-up after hct was 24 years (3-34). seven patients developed a malignancy 3.2 to 28 years (median 16.4 years) after hct including 2 carcinomas of the tongue, 1 oral squamous cell carcinoma, 1 colorectal cancer, 1 thyroid carcinoma, 1 carcinoma of the uterine cervix, and 1 parotid carcinoma. the 30-yr cumulative incidence (ci) of developing sst was 10+0.17%. all patients underwent surgical resection of the tumor and in addition 4 of them received chemotherapy and/or radiotherapy. of relevance, the 3 patients with cancer of the oral cavity were affected by severe chronic gvhd with buccal cavity involvement. 2 patients (1 with parotid and 1 with tongue carcinoma) died of tumor progression and 5 are living. we compared these results with 2 case control populations. first of all, we investigated the occurrence of solid tumors in the 117 individuals (64 males, median age 10 years at time of marrow donation), who served as stem cell donors for hct. one donor developed breast cancer 29 years after marrow donation at age of 38. the 30-yr ci of developing solid tumor for donors was 4.5+0.21% with a statistically significant difference (p = 0.03) as compared to that of transplanted patients. the second case control population consisted of 117 patients affected by tm treated with transfusions and iron chelation. the matching technique applied was based on the variables age and sex. one control per case (transplanted patient) was randomly selected from the miot (myocardial iron overload in thalassemia) registry and matched by sex and age with the transplanted patient population. 2 patients developed an hepatocellular carcinoma (hcc) at age of 39 and 44 years, respectively. one patient died and one is living. using the event rate measure, we observed an event rate of 0.102 at 30 years for the transplant group and 0.041 for the nontransplant group (p = 0.106). this study shows that the magnitude of increased risk of sst is twofold to threefold for patients treated with hct as compared with an age-and sex matched nontransplant tm patients or with stem cell donors. notably, among the transplanted patients we didn't observe any case of hcc, which is one of the most frequent solid tumor in nontransplant tm patients, whereas we observed 4 cases of head/neck cancers. in our series, cgvhd seems to be a strong risk factor in the development of new solid tumors. patients with cgvhd, especially those with involvement of the oral cavity, must receive a very long careful monitoring and surveillance in order to prevent the development of secondary cancers. disclosure of conflict of interest: none. sequential treatment with bortezomib plus thalidomide plus dexamethasone followed by autologous hematopoietic stem cell transplantation (hsct); consolidation and maintenance therapy in patients with multiple myeloma a bachiri 1 , ma bekadja 2 , s talhi 2 , s abderrahmani 1 , h ouldjeriouat 3 and r bouhass 2 1 department of hematology, hmru oran, oran, algeria; 2 department of hematology and cell therapy, ehu1st november, oran, algeria and 3 department of hematology and cell therapy, oran, algeria the management of multiple myeloma (mm) has been significantly improved in recent years in young patients, where ahsct and advent of new molecules was introduced as first line treatment. the sequential treatment (induction followed by autologous hematopoietic stem cell transplantation; consolidation and maintenance therapy) has increased rates response (cr and vgpr) as well as the overall survival. our purpose was to assess the efficacy and adverse effects of sequential treatment with vtd chemotherapy and autologous hsct followed by consolidation and maintenance therapy. in a prospective multicenter study, we evaluated this mm management strategy at oran, in two hematology centers. patients aged under 65 years with de novo mm, were treated with induction included: bortezomib (1.3 mg/m 2 , d1-d4-d8-d11), thalidomide (100 mg/ m 2 d1-d21) and dexamethasone (40 mg, d1-d4; d8-d11). a total of 3 to 4 cycles where delivered every 21 days. autologous stem cell was mobilized using g-csf alone (15 μg/kg/day for 5 days). leukapheresis to harvest stem cells were performed on day -2 and -1. the conditioning regimen consisted of melphalan 200 mg/m 2 . a consolidation phase was initiated two months later with the same protocol (vtd), followed by a maintenance treatment with thalidomide 50 mg/day given orally for 12 months. this study was done over a 6-years period (january 2010-december 2015). fifty patients were included. they include 19 women and 31 men (sex ratio = 1.63). the median age at diagnosis was 53 years (32-64). according to durie salmon staging, 80% of patients were in stage iii, while 38% were in stages iii according to iss staging. the monoclonal component was igg in 56% of patients. postinduction overall response rate in the 50 eligible patients was 100%, including 52% vgpr and 38% cr/ and 10% pr rates. the median of cd34 + rate was 3.88x10 6 /kg (1.41 to 11). all patients had engraftment on the median of day 10 (range; 7 to 14) and platelet transfusion independence on the median of day 13 (range; 9 to 19). there was no graft failure. one patient died following the procedure (trm). posttransplantation on day 100, cr and at least vgpr remained significantly higher (98%). in the 49 evaluable patients, the estimated os at 79 months was 82%, the estimated dfs at 43 months was 66% and the pfs at 78 months was 66.5%. at the 30/09/2016, 43 (86%) patients are alive and 39 (80%) without disease activity after a median follow-up of 33 months (range; 3-79). the main hematological toxicities post transpland (grade 3/4) were thrombocytopenia (49%), neutropenia (50%), and anemia (8%). the most frequently observed nonhematological toxicities (all grades) included peripheral neuropathy (66%). our experience suggests that the sequential protocol used in first line produce a better outcome with fewer adverse events and is an interesting therapeutic option in term of efficacy and tolerance. disclosure of conflict of interest: none. micrornas are small, non-coding single-stranded rnas and regulate approximately 50% of all genes by repressing translation. they are present in bodily fluids, where they are protected from rnase-mediated degradation by encapsulation into extracellular vesicles (evs) and demonstrate a novel capacity to regulate the cellular differentiation of blood cells and immune function. candidate micrornas mir-377, mir--199, mir-93* and mir-423 have previously been associated with acute graft versus host disease (agvhd) in posthematopoietic stem cell transplant (hsct) patient plasma. however, validation in independent cohorts is necessary, and their presence within extracellular vesicles (evs) has not been explored. microrna expression was evaluated in a prognostic cohort (n = 81) of day 14 (d14) post-hsct patient serum samples by taqman qrt-pcr. further assessment in an independent cohort of serum samples taken at the time of agvhd diagnosis was also performed. expression was also assessed in serum evs at sequential time points (pre-hsct, d0, d7 and d14) and an independent verification cohort of d14 serum samples by ev isolation, rna extraction and taqman qrt-pcr analysis. this study replicated elevated serum expression of mir-423 (po 0.001), mir-199 (p = 0.04), mir-93* (p o0.001) and mir-377 (p = 0.03) in agvhd, in a prognostic cohort of d14 post-hsct patient samples (n = 81). expression was also associated with disease severity. further analysis at agvhd diagnosis in an independent cohort (n = 65) confirmed high expression of mir-423 (p = 0.02), mir-199 (p = 0.007) and mir-93* (p = 0.004) at disease onset. investigation of microrna expression patterns during early hsct at sequential time points (pre-hsct to d28) identified elevated micrornas at d7 post-hsct in all transplant patients. in a novel investigation of microrna expression in serum evs (n = 15), mir-423 (p = 0.09), mir-199 (p = 0.008) and mir-93* (p = 0.001) levels were lower at d14 in patients who later developed agvhd, and this was replicated for mir-423 (p = 0.02) and mir-199 (p = 0.04) (n = 47). comparing serum to circulating evs, at d14 patients remaining agvhd-free had significantly higher expression of mir-423 (p = 0.03), mir-199 (p o0.001) and mir-93* (p = 0.001) in the ev fraction. results validate the capacity for circulating serum mir-423, mir-199 and mir-93* to act as diagnostic and prognostic biomarkers for agvhd. novel findings of differential expression between whole serum and the ev compartment prior to disease onset suggest a role for ev micrornas in the biology of agvhd, which warrants further investigation. disclosure of conflict of interest: none. prior data indicate similar outcomes after transplants from hla-haplotype-matched relatives, hla-idntical siblings and hla-matched unrelated donors. we used our dataset to answer a clinically important question: who is the best donor for a person with acute leukemia. we analyzed data from persons with acute leukaemia in 1 st complete remission treated in a prospective, multi-centre study. patients were randomly divided into training (n = 611) and validation (n = 588) sets. 1199 consecutive subjects received a transplant from an hla-haplotype-matched relative (n = 685) or an hlaidentical sibling (n = 514). 3-year leukaemia-free survivals (lfss) were 75% (95% confidence interval [ci], 72, 78%) and 74% (70, 78%; p = 0.95). the multivariate model identified 3 major risk factors for transplant-related-mortality (trm): older donor/recipient age (donor440years/recipient430 years; hazard ratio [hr] = 1.88; [1.05, 3.35]; p = 0.03), female-to-male transplants (hr = 2.11; [1.50, 2.98; p = 0.01) and donor-recipient abo major-mismatch transplants (hr = 1.55 [1.08, 2.23; p = 0.02). a risk score was developed based on these three features. trms were 8% (5, 10%), 15% (12, 18%) and 31% (19, 43%) for subjects with scores of 0-1, 2 and 3 (p o0.001). 3 year lfs were 78% (75, 81%), 74% (70, 78%), and 58% (45, 71%; p = 0.003). the risk score was validated in an independent cohort. in recipients 450years, lfss were 69% and 86% (p = 0.08) after transplants from identical-sibling or children. our data confirm donor source or degree of hla-disparity is not significantly correlated with transplant outcomes. selection of the best donor needs to consider donor-recipient age, sex-matching and abo-incompatibility amongst persons with acute leukemia receiving transplants from family members. [p726] disclosure of conflict of interest: none. synergistic effect of kir ligands missing and cytomegalovirus reactivation in improving outcomes of haematopoietic stem cell transplantation for treatment of myeloid malignancies d cardozo, a marangon, r da silva, fj aranha, j visentainer, s bonon, s costa, e miranda, c souza and f guimarães. the lack of one or more hla class i alleles, whose protein products are the ligands for kir receptors, has been exploited as a prognostic factor for the outcome of patients with haematological malignancies treated by haematopoietic stem cell transplantation (hsct). although it has been accepted that kir-hla interactions may influence the outcome of the hlamismatched hsct, there is no consensus regarding the settings of hla-matched transplantation. there are studies that have reported either benefits, or no effects, under the influence of inhibitory kir-hla interactions. additionally, certain activating kirs and/or reactivation of cytomegalovirus (cmv) infection have been reported to affect the outcome of hla-matched transplantation. the goal of this study was to evaluate the influence of kir-hla genotypes on the outcome of patients undergoing treatment for haematological malignancies by non-t-depleted lymphocyte haematopoietic stem cell transplantation (hsct) from hla-matched sibling donors. the prospective study was conducted at the center of hematology, university of campinas, and 50 patients and their donors were followed up from 2008 to 2014. kir and hla class i genes were genotyped and patients grouped based on the presence of kir ligands combined with kir genotype of their respective donors. patients with all kir ligands present (n = 13) had a significantly higher (p = 0.04) incidence of acute graft-versus-host-disease (gvhd) than patients with one or more kir ligands missing (n = 37). the overall survival following transplantation of patients with myeloid malignancies (n = 27) was significantly higher (p = 0.035) in the group with one or more kir ligands missing (n = 18) than in the group with all ligands present (n = 9). presence of kir2ds2 was associated with a worsening of hsct outcome while reactivation of cytomegalovirus (cmv) infection improved the outcome of patients with one or more kir ligands missing. our results indicate that kir-hla interactions affect the outcome of the hla-matched transplantation, particularly in patients with myeloid malignancies. disclosure of conflict of interest: none. p = 0.015), lower disease-free survival (p = 0.015 and p = 0.015) and lower overall survival (p = 0.01 and p = 0.014). one-year cir of the above two groups were 5.6 ± 5.4% vs. 57.1 ± 18.7% in mrd negative and positive patients, respectively (p = 0.001). in addition, those who had consistent positive mrd prior to hlamatched sibling hsct showed even worse outcomes compared to patients without pre-mrd. unmanipulated haploidentical hsct might have the stronger graft-versus-leukemia effect compared to hla-matched sibling hsct. it suggested that those who received unmanipulated haploidentical hsct with pre-mrd might not need more intensive relapse intervention after transplantation. disclosure of conflict of interest: none. the retrospective study of allogeneic hematopoietic cell transplantation for 36 patients with mixed-phenotype acute leukemia in toranomon hospital, japan in the real clinical setting, however, there are substantial number of patients who can not achieve cr after chemotherapy. we conducted a retrospective study including such patients to elucidate the outcome of allogeneic hct in toranomon hospital, japan. we studied the patients with mpal diagnosed from july 2008 to september 2015. mpal was diagnosed according to who classification in 2008. from june 2013, we examined cytoplasmic myelo-peroxydase (cmpo) routinely for flowcytometric analysis in all the patients, to distinguish mpal from acute lymphoblastic leukemia (all). we included the patients who were diagnosed as mpal in toranomon hospital, regardless of their diagnosis or clinical course in the previous hospitals. a total of 36 mpal patients underwent their first allogeneic hct with related bone marrow or peripheral blood stem cells (r-bm/pb) (n = 9), unrelated bone marrow (u-bm) (n = 6), and unrelated umbilical cord blood (u-cb)(n = 21). the median patient age was 41 years (range:17-69). the immunophenotype of leukemia cells included 23 cases of b and myeloid (b/my) (64%) and 13 cases of t and myeloid (t/my) cell lineage(36%).eleven patients(31%) harbored philadelphia chromosome. the remission induction chemotherapy was performed with all-type regimens in 31 patients, and acute myeloid leukemia (aml)type regimens in 5 of 36 patients, 18 patients(50%) were not in cr at the time of transplantation. myeloablative conditioning (mac) regimens were used in 30 pantients(83%). the 2-year overall survival (os) rate was 43.1% (95% confidence interval (ci), 25.7-59.4%). to identify the factors that influenced os, we performed univariate analysis and compared the following pre-transplantation factors: age at the time of transplantation ( o41 vs.4 = 41 years), committed immunophenotype (b/my vs.t/my), karyotype (philadelphia chromosome (ph vs.non-ph), disease status at the time of transplantation (cr vs.non-cr), donor cell source (r-bm/pb vs.u-bm vs.u-cb, cb vs.non-cb), and conditioning regimen (mac vs.reduced intensity conditioning). cr at the time of transplantation was extracted as a significant predictive factor for the better os(2-year os; cr vs. non-cr, 63.0% (95% ci, 32.1-82.8%) vs.22.2% (95% ci, 6.9-42.9%), p = 0.009). the cumulative incidence of relapse rate (rr) at 2 years after transplantation was 53.3% (95% ci, 31.8-70.8%). to identify the factors that influenced relapse rate, we performed univariate analysis and compared pretransplantation factors same as above. harboring philadelphia chromosome was extracted as a significant predictive factor for lower relapse rate (2-year rr; ph vs.non-ph, 27.3%(95% ci, 0.1-77.3%) vs. 66.5%(95% ci, 41.4-82.8%), p = 0.003). the older patients(p = 0.07) and the patients in cr (p = 0.05) also showed a trend towards lower relapse rate. allogeneic hct provided 63.0% of 2-year os for mpal patients in cr at the time of transplantation. on the other hand, for patients not in cr, 2year os was approximately 20%. the use of tyrosine kinase inhibitors along with chemotherapy before transplantation might prevent relapse after transplantation in mpal patients with ph chromosome. disclosure of conflict of interest: none. allogeneic hematopoietic stem cell transplantation (allo-hsct) is a standard of treatment for many patients with hematological malignancies. however, the disease relapse and graft failure after first allo-hsct (1 st allo-hsct) lead to poor outcomes almost in all cases. second allo-hsct (2 nd allo-hsct) is one of primary options that can decrease the mortality in this group of patients. here we report our experience of 15 patients who underwent 2 nd allo-hsct. the aim of the study was to estimate a clinical efficiency and practicability of 2 nd allo-hsct. we included 15 patients (9 males/6 females) with acute myeloid leukemia (aml, n = 10), acute lymphoblastic leukemia (all, n = 3) and myeloproliferative disease (mpd, n = 2) who underwent 2 nd allo-hsct for relapse (66,7%) or graft failure (33,3%) from the same (n = 8) or another donor (n = 7) between november 2012 and october 2016. median age was 31 years (range: 18-42 years). three (20%) patients had a matched related donor (mrd), nine (60%) patients had a matched unrelated donor (mud) and three (20%) patients had a mismatched unrelated (mmud) at the second transplant. to evaluate time gap affecting outcomes all patients were divided into two groups: who underwent 2 nd allo-hsct in more/less than 6 months after 1 st allo-hsct. in "less than 6 months" group three patients were re-transplanted for relapse and one-for graft failure, in other group there were seven patients who received 2 nd allo-hsct for disease relapse and four-for graft failure. fisher's exact test were performed to exclude probability of imbalance between groups (p40.05). median of overall survival (os) and disease-free survival (dfs) after 2 nd allo-hsct was 13.5 months and 10.59 months respectively. (see figure 1a ,1c) two patients (13.3%) developed graft failure and three relapsed (20%). acute graft-versus host disease (agvhd) incidence was extremely low as 13.3% (n = 2) even despite use of mud/mmud in 80% of cases. mortality rate were 53.3% in a group of 2 nd allo-hsct. it should be noted that only 3 (20%) patients died because of disease progression. five patients (33.3%) died in complete remission due to severe infections or previous toxicity (e.g. heart failure). the effect of donor change on dfs was not significant (p = 0,88). our statistical analysis reveal significantly differences in os in patient with long-term interval (46 months) between 1 st and 2 nd allo-hsct. median of os in patients who underwent 2 nd allo-hsct in more/less than 6 months after 1 st allo-hsct was 20,59 vs 7,02 months respectively. (see figure 1b ,1d) for hazard ratio (hr) estimation mantel-haneszel approach were used hr for group who were transplanted in less than 6 months from 1 st allo-hsct was 8.36, (95% ci, 1.054 s511 to 66.38, p = 0.04). as for dfs difference was not significant (p = 0.07). according to our analysis, performing 2 nd allo-hsct in a period less than 6 months after 1 st allo-hsct seemed not very reasonable due to extremely high mortality even in young patients (hr-8.36, p = 0.04). as for "more than 6 months" group it can be considered even despite hla-disparity between donor-recipient pair due to extremely low agvhd rate (13.3%). donor change was not associated with better outcome (p = 0.88) disclosure of conflict of interest: none. hemopoietic stem cell transplantation (hsct) is an effective treatment for many hematologic disorders, and globally over 70 000 procedures/year are performed in more than 70 countries. however, not all the countries have enough resources and expertise to establish an hsct program, and patients are often forced to emigrate for transplantation, with heavy social and economic consequences. in the year 2015 the iuc (an italian ngo) identified the hiwa cancer hospital (hch) in sulaymaniya (iraqi kurdistan) as a possible site for the establishment of a new hsct transplant center. a hsct expert from italy (mi) following a visit to the hch, reported a positive conclusion on the feasibility of an hsct project. this was mainly due to the fact that many of the required technologies were already available at hch, including a 6-bed positivepressure, hepa-filtered-air clinical unit, 2 last-generation cell separators and a well equipped hla laboratory. following this preliminary survey, a capacity building project was rapidly made and submitted to the italian agency for development cooperation, that approved and funded it in march 2016 with the specific aim to cure thalassemia patients either of kurdistan and of the refugees population from syria and other parts of iraq. in april 2016, the joint italian and kurdish team started the project. a first autologous transplant was done in june 2016 followed by 8 more autografts (overall, 5 myeloma and 4 lymphoma patients). in october, following appropriate downstaging, a first low-risk thalassemia patient was allografted from her hla-id sibling, followed by 2 more patients. all the patients engrafted promptly, with one death occurring on day +23 with acute cardiac failure and a major toxicity recorded in a single patient (nhl, severe enterocolitis with perforation) that was successfully treated. the full process for the start-up included the following activities developed during 8-month time: (1) s512 of transplants, the hch group also submitted to ebmt an application for full membership, that was promptly approved. in all this project, the italian counterpart provided over 30 highly-experienced volunteer specialists (physicians, nurses, technicians and one physicist), each with a specific mission plan. despite the many difficulties and obstacles encountered, the clinical results obtained so far appear encouraging, though there is still need to furtherly support the hch in order to make it totally independent. following this intervention, the hch is the only one center performing both auto and allo hsct not only in the iraqi kurdistan region, but also in all the iraqi nation. we conclude that international cooperation may be fruitful also in the field of high-technology medicine, and may contribute to improve the capabilities of centers even in critical geographic areas, representing a valuable instrument also to implement nation-to-nation scientific exchanges. disclosure of conflict of interest: none. the use of plerixafor with g-csf in conditioning regimen for hematopoietic stem cell transplantations with tcr alpha/beta and cd19 depletion of graft in wiscott-aldrich syndrome patients: a single-center experience b dmitry 1 , l alexandra 2 , s larisa 1 , g elena 1 , s irina 1 , t pavel 1 , k rimma 1 , n galina 3 , m michael 1 and m alexei 1 grade 2 acute gvhd (agvhd) was 13% (2 pts). no pt experienced a grade 42 agvhd. three patients presented a limited form of chronic gvhd (21%). incidence of oral mucositis and gastrointestinal/liver toxicity has been extremely low in this population of patients, even in those with active disease and heavily treated at the time of transplant. eight out of fifteen pts (53%) are alive with a median follow-up of 31 months (range: 12 -48 m). seven (47%) are in cytogenetic/molecular remission. six out the eight patients who were transplanted in cr1 or cr2 are alive (75%), while two out the seven patients who were transplanted in advanced phase are alive (29%). in this preliminary clinical experience, we find that unmanipulated haploidentical transplants with post-transplant cyclophosphamide are a valid alternative and have outcome comparable to unrelated and match sibling transplants, in pts with hematologic malignancies. advanced disease is the only adverse factor for diseasefree survival. we therefore consider this therapeutic option when a match sibling or a 10/10 ag mud donor is not immediately available. disclosure of conflict of interest: none. autism spectrum disorder (asd) is a group of neurodevelopmental disorders characterized by impaired social communication and interactions with restricted and repetitive behaviors. although asd is suspected to have either heritable or sporadic genetic basis, its fundamental etiology and pathogenesis are poorly understood. recently researchers have suggested that stem cells have therapeutic potential for asd. wharton's jelly-derived msc (wj-msc) from third-party donors (tpd) have high proliferation and differentiation potential. this cell population has also non-immunogenic and immunomodulatory properties, thus seem to be a promising treatment stem cell source. the polish stem cell bank (pbkm) has provided wj-msc for clinical application in a medical therapeutic experiment for children with asd. twenty-three patients (pts) with asd aged from 3 to 18. 10/12 (median age: 7 years and 7 months), after bioethical committee approval, received intravenous injections of wj-msc, obtained from tpd. the cells were previously collected from healthy newborns, then processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapour. wj-msc immunophenotype was confirmed using flow cytometry assay. the pts received from 1 to 5 injections in intervals from 4 to 12 weeks. the average cell dose per infusion was 1.01 × 10^6/kg of body weight (bw). each pt was examined by the same neurologist at the day of infusion. comorbidities present in some patients: unspecified speech disturbances, flaccid paralysis, flaccid tetraplegia, unspecified encephalopathy, epilepsy, sensorineural hearing loss. one patient was diagnosed with 2 comorbidities: conductive hearing loss and intellectual disability. almost 80% of pts, after their treatment with wj-msc, revealed positive changes in neurological examination. an improvement in speech was observed in 10 pts and improvement of cognitive functions ensued in 7 pts. what is more, 26% of children showed progress in self-reliance, social interactions and improved their ability to concentrate. there was a reduction of aggressive behavior in 4 pts and 2 pts have experienced better quality of sleep. there was only one adverse event after wj-msc infusions -psychomotor agitation occurred in 24 hours after the administration. five follow-ups have not yet been completed. the administration of thirdparty donor wj-msc seems to be safe and efficient procedure with promising preliminary results in patients with asd. hematopoietic stem cell transplantation between red cell incompatible donor-recipient pairs red blood cell depletion from bone marrow and peripheral blood buffy coat: a comparison of two new and three established technologies human bone marrow processing using a new continuous-flow cell separation device disclosure of conflict of interest: none. references 1. zama d, et al. gut microbiota and hematopoietic stem cell transplantation: where do we stand? bmt the kyoto encyclopedia of genes and genomes-kegg metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation disclosure of conflict of interest: none antifungal prophylaxis in hematopoietic stem cell transplant recipients: the unfinished tale of imperfect success guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective differences in aspergillus-specific immune recovery between t-cell-replete and t-cell-depleted hematopoietic transplants toxoplasmosis following allogeneic hematopoietic stem cell transplantation diagnosis of toxoplasmosis after allogeneic stem cell transplantation: results of dna detection and serological techniques implementation of molecular surveillance after a cluster of fatal toxoplasmosis at 2 neighboring transplant centers management of high blood pressure genes for blood pressure a prospective studyon the predictive value of plasma bk virus-dna load for hemorrhagic cystitis in pediatric patients after stem cell translantation cidofovir for bk virusassociated hemorrhagic cystitis:a retrospective study hemorrhagic cystitis after bone marrow transplantation bcsh/bsbmt guideline: diagnosis and management of veno-occlusive disease (sinusoidal obstruction syndrome) following haematopoietic stem cell transplantation drug safety evaluation of defibrotide defibrotide for prophylaxis of hepatic veno-occlusive disease in paediatric haemopoietic stem-cell transplantation: an open-label, phase 3, randomised controlled trial safety and effects of prophylactic defibrotide for sinusoidal obstruction syndrome in hematopoietic stem cell transplantation disclosure of conflict of interest: none university children's hospital basel, division of paediatric oncology/haematology late complications subcommittee of translated related complications and quality of life wp; 5 clinic of paediatric haemato-oncology, department of women's and children's health, university of padova, italy; 6 department of surgery, division of transplantation division of blood and marrow transplantation, the children's hospital at westmead ovarian function after bone marrow transplantation during childhood pregnancies following high-dose cyclophosphamide with or without high-dose busulfan or total-body irradiation and bone marrow transplantation unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning haploidentical hematopoietic transplantation:current status and future perspectives t-cell replete haploidentical donor transplantation using post-transplant cy: an emerging standard-of-care option for patients who lack an hla-identical sibling donor hematopoietic stem cell transplantation in thalassemia major and sickle cell disease: indications and management recommendations from an international expert panel allogeneic stem cell transplantation for thalassemia major killer-cell immunoglobulin-like receptors reactivity and outcome of stem cell transplant kir b haplotype donors confer a reduced risk for relapse after haploidentical transplantation in children with all kir/hla interactions negatively affect rituximab-but not ga101 (obinutuzumab)-induced antibody-dependent cellular cytotoxicity reduction of minimal residual disease in pediatric b-lineage acute lymphoblastic leukemia by an fcoptimized cd19 antibody diagnoses: hodgkin's lymphoma(hl)-38 pts (refractory 22; relapsed 16); non-hodgkin's lymphoma(nhl disease status before asct: 1st (after refractority prior radiotherapy to the mediastinum -19/48 (39.6%); heavily pretreated patients with advanced disease (x 3 lines previous treatment) 18/48 (37.5%). grafts: pbsc -43/48 pts with median of cd34+cells-3 ccnu dose: 47/48 pts 300mg/m2; 1pt 400 mg/m2. engraftment: anc>500 cells/mkl: median=d+11(8÷24), 47/48pts. plt>50 000 cells/ mkl: median=d+ 28(13÷122), 43/48pts. full engrafted 43/ 48pts /8 pt required a short-term mechanical ventilation (2 of them died because of lung infection ad d+68 and d+82) aeruginosa associated sepsis on a background of graft failure); 2pts (4.2%)-d+68 and d+82 (pulmonary toxicities +infection; both had prior mediastinal radiotherapy). relapse/ progression after asct-8/48 pts (16.6%), 6 of them died. 1 pt achieved secondary mds (diagnosed 4.5 mo after asct). for this group of pts with relapsed/refractory lymphomas (n=48) 11-year os=0 for nhl(n=10) efs=0.88 (se ± 0.2) lomustine-containing conditioning regimen cem (lomustine, etoposide, melphalan) is effective and feasible in autologous stem cell transplant efficacy and toxicity of a ccnu-containing high-dose chemotherapy regimen followed by autologous hematopoietic cell transplantation in relapsed or refractory hodgkin's disease champlin re reduced-toxicity conditioning therapy with allogeneic stem cell transplantation for acute leukemia idarubicin-intensified bucy2 conditioning regimen improved survival in high-risk acute myeloid, but not lymphocytic leukemia patients undergoing allogeneic hematopoietic stem cell transplantation: a retrospective comparative study comparison of outcomes of idarubicin intensified tbi-cy and traditional tbi-cy conditioning regimen for high-risk acute lymphoblastic leukemia undergoing allogeneic hematopoietic stem cell transplantation: a single center experience inhibition of cd25 (il-2r alpha) expression and t-cell proliferation by polyclonal anti-thymocyte globulins csf-primed bone marrow transplantation for patients with high-risk hematologic malignancies in an exploratory analysis, os after hct appeared to be longer in the cpx-351 arm in both age groups. these results suggest that cpx-351 may provide an effective bridge to successful transplant for a high-risk subgroup of aml patients. support: celator pharmaceuticals, inc., a subsidiary of jazz pharmaceuticals plc consulting ambit biosciences, amphivena therapeutics, ariad, astellas pharma sunesis, tolero; institutional research funding abbvie chiarella and louie: employment celator/jazz; stock jazz pharmaceuticals plc. hoering disclosure of conflict of interest: none. inkt-/nk-/cik-cell (subsets) are important for immunesurveillance. antibody 6b11 targets the vα24-jα18-invariant-t-cell-receptor (tcr) in the cdr3-region, which is semiinvariantly rearranged in inkt-cells. we characterized: i.) inkt-/nk-/cik-subsets in pb-samples from healthy donors (n = 9 subsets under stimulation with dendritic-cells of leukemic origin (dc leu ), generated from aml-blasts in mononuclear cells(mnc) and whole-blood (wb, containing soluble/cellular components of pts' pb) with 'cocktails' (dc-generating-methods/kits). 1.1) compared to healthy mnc (significantly) lower proportions of inkt-cells comparable correlations were seen in adultall-and cll-pts. 2.1) we quantified inkt-/nk-/cik-subsets before/after mixed-lymphocytecultures (mlc) of t-cell-enriched immune-reactive cells stimulated with mnc/wb (with or without pretreatment 'cocktails' inducing blasts' conversion to dc leu ) from aml-pts. our findings show, that 1)inkt-/nk-/cik-cells increase after mlc independent of the stimulator-cells-suspension (under the influence of il-2); 2) pretreatment of mnc/wb-blasts with 'cocktails' increases inkt-counts and induces a shift in the composition of inkt-/nk-/cik-subsets after mlc, that might correlate with an improved antileukemic potential; 3) individual samples showed varying, however higher inkt-, cik-cell-counts after pretreatment with different (especially prostaglandin-containing) 'cocktails'; 4) dc-/inkt-/nk-/cikcells-values after mlc were comparable in physiological hypoxia vs normoxia; 5) in cases with antileukemic blast-lytic activity after mlc t-/inkt-/nk-/cik-cells were significantly increased-pointing to an involvement of these cells in antileukemic reactions. in summary: (1) healthy mnc present with significantly higher inkt-/nk-/cik-cells compared to aml/all/cll-leukemic mnc. (2) subtypes of inkt-cells differ in healthy vs leukemic samples, resembling a shift in the composition of inkt-cells. (3) amounts of inkt-/ nk-/cik-cells in aml/all/cll-mnc-samples correlate with prognosis. (4) 'cocktail'-treated aml-blasts (resulting in dc leu ) lead to a shift in t-,inkt-/nk-/cik-cell-counts/compositions, what correlates with improved antileukemic activity against aml-blastspointing to a cross-talk of these cells. proportions of inkt-/ nk-/cik-cells management of philadelphia chromosome-positive acute lymphoblastic leukemia (ph+ all) outcome of allogeneic stem cell transplantation for aml and myelodysplastic syndrome in elderly patients (⩾60 years) comorbidity-age index: a clinical measure of biologic age before allogeneic hematopoietic cell transplantation high rate of hematological responses to sorafenib in flt3-itd acute myeloid leukemia relapsed after allogeneic hematopoietic stem cell transplantation phase i trial of maintenance sorafenib after allogeneic hematopoietic stem cell transplantation for fms-like tyrosine kinase 3 internal tandem duplication acute myeloid leukemia haematopoietic cell transplantation with and without sorafenib maintenance for patients with flt3-itd acute myeloid leukaemia in first complete remission quantitative monitoring of minimal residual disease (mrd) after sct was performed by four-colour flow cytometry and/or real time pcr. the median time of neutrophil engraftment (above 0.5 × 10e9/l) was 16 days, 96% of pts (24/25) engrafted, one patient died in aplazia. non-relapse mortality (nrm) after 1 year and 2 years was 8% (2/25) and 13% (3/25). causes of death were refractory gvhd (n = 1), infection (n = 1) and multiorgan failure (n = 1). incidence of acute gvhd was evaluated in 23 pts: 52% (12/23) of pts had gvhd (grade i+ii in 7 pts, grade iii in 5 pts). incidence of chronic gvhd was evaluated in 22 pts, 41% (9/22) of pts had gvhd with median follow-up from sct 37 months (range: 4-90), 64% of all pts (16/25) were alive (14 in remission of cll with mrd negativity, 2 with relapse), 9 pts died (3 from nrm, 6 from cll relapse/progression), 8 relapses (32%; 8/25) occurred. sequential use of chemotherapy and ric regimen with allogeneic sct is safety and effective treatment of high-risk cll with reponse rate 86% and low nrm. progression-free survival and overall survival at 3 years from sct were 56% and 64% 1 department of hematology, hemostasis, oncology and stem cell transplantation hannover deutsche klinik für diagnostik helios klinik wiesbaden, germany; 19 imperial college london at hammersmith hospital du cane road centre for haematology london disclosure of conflict of interest: none. references 1. sibon d, brice p. optimal treatment for relapsing patients with at our institution, pr-hl is defined as partial response (pr), no response (nr), stable disease (sd), progressive disease (pd), relapsing within 3 months of finishing the planned treatment. progression free (pfs) and overall survival (os) from the day 0 of auto-sct was estimated by kaplan-meier (km) method. from 1996 to 2014, 234 patients with aethera trial criteria were identified. male 121 (52%), female 113 (48%), median age at diagnosis:22 yrs (12-61), at auto-sct: 24.3 yrs (13.8-63)(90% o40 yrs). initial chemo: abvd in 194 (83%). 70 (30%) had radiation therapy (xrt) after initial chemo. response to initial chemo + xrt was refractory disease:152 (65%), relapse between 3-12 months:49 (21%) and relapse after 12 months:33 (14%) aethera had 28% stable disease before sct vs we have only 6%. aethera 24 months pfs (45% control arm, 65% brentuximab arm, investigator assessment) and our 56.4% is not much different. despite having similar selection criteria, our median pfs is higher than both aethera trial placebo and experimental arm. clinically, rate of progression in both studies are very high and comparable at 24 months. given the very high cost of this drug and while waiting for survival fifty-nine (82%) and 13 (18%) patients had relapsed and primary refractory chemosensitive dlbcl, respectively. secondary ipi was 0-1 in 23 (44%) patients, 2 in 13 (25%) patients and 3-4 in 16 (31%) patients. fifty-one (71%) and 21 (29% patients had gcb and abc tumors, respectively. abc patients received more prior lines of chemotherapy than gcb patients (76% vs 48% received 4 2 lines of chemotherapy, p = 0.03). the rest of characteristics were equally distributed between both groups (table 1) disclosure of conflict of interest: none disclosure of conflict of interest: none. p631 upfront autologous stem cell transplantation in patients with diffuse large b cell lymphoma: focused on risk factors for survival and conditioning regimens ds kim 1 association between complete response and outcomes in transplant-eligible myeloma patients in the era of novel agents e jantunen 1 and v varmavuo 12 1 department of medicine disclosure of conflict of interest: none. and 6 hematology department lenalidomide after stem-cell transplantation for multiple myeloma bortezomib induction and maintenance treatment in patients with newly diagnosed multiple myeloma: results of the randomized phase iii hovon-65/gmmg-hd4 trial disclosure of conflict of interest: none we performed a retrospective study to investigate survival outcomes and toxicities of l maintenance therapy compared with b maintenance in mm patients post-ahct. this study included 156 patients who received ahct for mm between 2008 and 2015 after induction with l-or b-based therapy. all patients received ahct within 12 months of mm diagnosis and received melphalan 200 mg/m 2 conditioning. patients who received tandem transplantations (autologous or allogeneic) were excluded. only patients initiating maintenance therapy within 6 months post-ahct were included. maintenance therapy was defined as monotherapy with either l or b. the primary outcome was pfs. secondary outcomes were overall survival (os) and treatment-related toxicities. 92 patients received l maintenance and 64 b maintenance post-ahct. at baseline there were no differences in iss stage, ds stage or cytogenetic risk between maintenance cohorts. at time of analysis, 49% (n = 45) receiving l maintenance and 52% (n = 33) on b maintenance experienced disease progression. median time to progression (1.71 vs 1.74 yrs, p = 0.77) was not significantly different between cohorts. by multivariable analysis, choice of maintenance (l vs b) was not significant for pfs or os. variables significant for improved pfs were iss stage i disease response improved while on maintenance in 38% (n = 24) with l and 34% (n = 31) with b. median os was not statistically different between maintenance cohorts (4.28 vs 5.77 yrs, p = 0.47). iss stage i/ii vs iii while cytopenias were more common in the l cohort (30% vs 3%, p o 0.01). the median follow-up time for survivors was 33 months. these findings suggest that both lenalidomide and bortezomib are equivocal maintenance therapy options for post-transplantation mm patients. choice of maintenance therapy post-ahct for mm did not demonstrate a difference in survival outcomes. based on these data, maintenance choice should be guided by patient specific anticipated tolerance rather than drug type alone. iss stage and post-ahct disease response continue to be significant predictors for outcomes. toxicities recorded on maintenance were as anticipated. length of maintenance therapy may be a significant predictor and warrants further analysis. the analysis was underpowered to disclosure of conflict of interest: none. p650 real-world multiple myeloma management practice patterns and outcomes in six central and eastern european countries d coriu 1 , d dytfeld 2 , d niepel 3 , i spicka 4 second autologous stem cell transplantation as salvage therapy for multiple myeloma: impact on progression free survival and overall survival second autologous stem cell transplantation: an effective therapy for relapsed multiple myeloma second auto asct for treatment of relapsed multiple myeloma the role of second autografts in the management of myeloma at first relapse moving beyond autologous transplantation in multiple myeloma ebmt data office bortezomib-based versus nonbortezomib-based induction treatment before autologous stem-cell transplantation in patients with previously untreated multiple myeloma: a meta-analysis of phase iii randomized, controlled trials first-line therapy with thalidomide and dexamethasone in preparation for autologous stem cell transplantation for multiple myeloma mechanism of action of bortezomib and the new proteasome inhibitors on myeloma cells and the bone microenvironment: impact on myeloma-induced alterations of bone remodeling boys; 19 girls) with following mds types: refractory cytopenia of childhood-11 (19%), refractory anemia with excess blasts -14 pts (24%), refractory anemia with excess blasts in transformation-22 pts (38%), juvenile myelomonocytic leukemia in 11 pts (19%). the median of age was 7 years (1-19 years) mac consisted busulfan (bu) 16 mg/kg + cyclophosphamide 120 mg/kg. ric included fludarabine (flu) 150 mg/m 2 + melphalan (mel) 140 mg/m 2 , flu 150-180 mg/m 2 + bu 8mg/ kg. the bone marrow (bm) was used in 38 pts (66%), peripheral blood stem cells (pbsc) in 13 pts (22%), combination of bm and pbsc in 7 pts (12%). 5-years overall survival (os) was 45% os was in pbsc group -28%; bm group-50%, combination of bm and pbsc-25% there were two cases of mds, eb-2, although erythroid aberrancy can not be found, fc did disclose significant aberrancy on myelomonocytic lineages. on the other hand, all the normal control bm samples revealed no any erythoid phenotypic abnormality. our study suggests this simplified cocktail of 2-tube, 4-color, fc is very sensitive and useful in the assessment of erythroid phenotypic abnormalities in mds we analyzed 62 consecutive patients (44% were female, median age of 48 (range: 18-70) allografted for mds (median ebmt risk score of 3, median disease risk index of intermediate risk) over a 19-year period (1998-2016) with mac conditioning for 66% and ric for 34% pfs 40 ± 14%, grfs 26 ± 12%, ri 37 ± 13% and trm similarly, there was not difference between tdep and non tdep patients for 3-years pfs (48 ± 18% and 28 ± 20%, p value 0.321), 3-years gfrs (32 ± 17 vs 19 ± 17, p value 0.111) (graph), 3-years ri (36 ± 18% and 37 ± 20%, p value 0.622) and 3-years trm (26 ± 16% and 23 ± 18%, p value 0.933). finally, tdep had no significant impact on 3-years grade 2-4 agvhd when compared to the non tdep (26 ± 18% and 31 ± 16%, p value 0.656). it had not either on 3-years cgvhd (26 ± 18% and 28 ± 34%, p value 0.637). our study shows that tdep is feasible on patients undergoing hsct for mds disclosure of conflict of interest: none. p665 mutational pathway and dynamics may not be prognostic in patients with myelodysplastic syndrome receiving hypomethylating agent pre-treatment for allogeneic stem cell transplantation republic of korea; 3 department of computer science; 4 the donnelly center for cellular and biomolecular research amebiazis after bone marrow transplantation use of a five-agent gvhd prevention regimen in recipients of unrelated donor marrow impact of age on outcomes after bone marrow transplantation for acquired aplastic anemia using hla-matched sibling donors treatment of acquired severe aplastic anemia: bone marrow transplantation compared with immunosuppressive therapy-the european group for blood and marrow transplantation experience disclosure of conflict of interest: none. leukemia, myelodisplastic syndrome, juvenile myelomonocytic leukemia and chronic myelomonocytic leukemia s bondarenko hla-mismatched unrelated (n = 5, 10%), and haploidentical (n = 8, 15%) donors. response was achieved in 30% (n = 16) of pts after 1-5 (median 3) courses of hma therapy: complete remission (cr) in 2 (4%), partial remission (pr) in 14(26%) of pts. stabilization (s) was documented in 30 (56%) pts, in 8 (14%) pts there was disease progression (p) after beginning of hma therapy mismanaging the gift of life: noncompliance in the context of adult stem cell transplantation l'adhésion thérapeutique et at. des lieux en allogreffe de cellules souches hématopoïétiques (csh) dans des services de pédiatrie et d'adulte. rapport de la sfgm-tc predictive validity of a medication adherence measure in an outpatient setting data is limited to small case series, transplant registries and a single prospective multicenter observational study. here we report our institutional experience with auto-hct in patients with hrl. twenty patients with hrl [non-hodgkin = 14 (70%), hodgkin = 6 (30%)] and treatable hiv infection underwent hdt consisting of carmustine, etoposide, cytarabine and melphalan (beam) followed by peripheral blood auto-hct from 04/2006 to 07/2015. in 2 cases rituximab was administered as part of the preparative regimen. patient-, disease-, and transplant-related characteristics are summarized in table 1. median age was 48 years (range: 35-61). the median follow-up for surviving patients was 42 months (range: 6-110) abbreviation: n: number of patients; m: male gender; auto-hct: autologous hematopoietic cell transplant; nos: not otherwise specified; dlbc: diffuse large b-cell lymphoma ara-c), melphalan; cr1: first complete remission; cr2 :second complete remission disclosure of conflict of interest: none. p696 incidence of secondary primary malignancies (spm) in patients with multiple myeloma m curly 22 , g laurent 23 and k nicolaus 24 1 city of hope igm 21 (0.6%), lines of induction regimens prior to hsct one in 2003 pts (53%), two in 724 pts ( 19.3%), 4 2 in 348 pts (9.3%), and missing in 682 pts (18%). induction regimens included imids and proteasome inhibitor (pi)s with alkylating agents in 1266 pts (33.7%), imids and pis with no alkylating agents in 1328 (35.5%), and alkylating agents with no imids or pis in 478 (12.7%) and missing data in 685 (18%). radiotherapy was used pre hsct in 614 pts (16.3%), no radiation in 2461 pts (66%) and missing data in 682 (18.2%). plerixafor (p) was administered mostly for poor hsc mobilization as defined by the centers number of hsc collected o3 × 10 6 in 239 pts (6.4%), 3-5 in 397 pts ( 10.6%), 45 × 10 6 in 1394 pts (37%), and data missing in 1727 (46%). the number of cd 34+ hsc infused o3 × 10 6 in 760 pts (20%), 3-5 × 10 6 in 1055 pts (28%0, 45 × 10 6 in 799 pts (21%), and missing in 1143 (30%). a total of 141 pts developed spm with cumulative incidence of 5.4% (95%ci 4.4,6.3) at 72 mo. data are missing in 414 pts (11%) use of radiotherapy, type of induction, hsc cell dose did not influence the cumulative conflict of interest: f. sahebi, none declared, s. iacobelli, none declared, l. koster none declared l. gardaret none declared, n. kroger received research fund from sanofi, curly morris, none declared p697 interaction between center effect and strategy for gvhd prophylaxis on outcome of t-cell depleted and t-cell replete haploidentical transplant inserm u1153 ecstra team expanding transplant options to patients over 50 years-improved outcome after reduced intensity conditioning mismatched-unrelated donor transplantation for patients with acute myeloid leukemia: a report from the acute leukemia working party of the ebmt nkg2d ligands in tumor immunity comprehensive analysis of nkg2d ligand expression and release in leukemia: implications for nkg2d-mediated nk cell responses nkg2d cars as therapy for cancer russian federation high incidence of mixed chimerism with impaired graft function remains a significant issue in patients with wiskott-aldrich syndrome (was) after hsct. simultaneous use of plerixafor with g-scf is efficient in inducing stem cell release and opening of bone marrow niches. the use of plerixafor/g-csf in conditioning demonstrates better levels of donor chimerism in patients with acute myeloid leukemia. we report our experience of plerixafor/g-csf usage in patients with was as an addition to myeloablateive conditioning to improve stem cell engraftment p = 0,85. events were considered: death in 2 patients, graft rejection in 5 patients, mixed myeloid chimerism (less than 20% donor) in 2 patients. median time of event was 3,2 months after hsct (1.23-8.6) all patients are alive, median fu is 3 months, range: 0.43-6.3. 2 patients had acute gvhd: 1-grade 2 (gut), 1-grade 1 (skin), in both cases resolved after a short course of steroids. all patients had more than 95% donor chimerism monthly till the time of last fu. the comparison of peripheral blood chimerism (% of donor cells) in was patients transplanted with and without plerixafor/g-csf in conditioning is shown (figure 1). the additional use of plerixafor with g-csf references 1. moratto et al disclosure of conflict of interest: none. is undesirable. fifteen pts (3 males, 12 females, median age 48, range: 17 -65 years) with high risk hematologic malignancies ( acute myeloid leukemia n.10, 66%; acute lymphoblastic leukemia n 2, 13% pretransplant conditioning regimen consisted of thiotepa 10 mg/kg in two days, busulfan 9.6 mg/kg in three days, and fludarabine. source of stem cells was g-csf stimulated bone marrow in all. dose of marrow nucleated cells and cd34+ were 5.4 (range: 3.4-6.7) × 10 8 /kg and 3.5 (range: 2.1-5.8) × 10 6 /kg respectively. post-transplant cyclophosphamide at 50 mg/kg/ day was given on days 3 and 5 after transplantation, together with cyclosporine (starting at day − 1 until day 180 posttransplant) and mycofenolate (from day +1 to day +20) modeling autism spectrum disorders with human neurons autism spectrum disorders neurobiology and genetics of autism: a developmental perspective. the development of autism: perspectives from theory and research wharton's jelly-derived mesenchymal stem cells treatment in children with cerebral palsy: our second preliminary results of the clinical application in poland a mucha 1 , k kosterna 1 , m chroscinska-krawczyk 2 , m kotarska 2 , k mitosek-szewczyk 2 , m murzyn 3 the polish stem cell bank cases application potential of bone marrow mesenchymal stem cell (bmscs) based tissueengineering for spinal cord defect repair in rat fetuses with spina bifida aperta sensory neuron differentiation potential of in utero mesenchymal stem cell transplantation in rat fetuses with spina bifida aperta: sensory neuron differentiation of in utero mscs analysis of post allo-hct relapse in acute leukaemia patients, a comparative on second allo-hct and donor lymphocyte infusions g orti 1 , j sanz 2 , i garcia-cadenas 3 , i sanchez-ortega 4 , mj jimenez 5 , p barba 1 , c ferra 6 , r parody 4 , j sierra 3 , ma sanz 2 , s querol 7 and d valcarcel 1 1 hospital universitari vall d´hebron; 2 hospital universitario la fe; 3 hospital de sant pau i la santa creu; 4 hospital duran i reynals ico, 5 hospital germans trias i pujol ico; 6 hospital germans trias i pujol and 7 banc de sang i teixits acute leukaemia relapse after allogeneic hematopoietic cell transplantation (allo-hct) associates poor prognosis. in this scenario, lowering the tumour burden prior to a second allo-hct (2 nd allo-hct) or donor lymphocyte infusions (dli) is essential to improve survival. thus, patients that respond to chemotherapy and subsequently receive a dli or 2 nd allo-hct appear to associate better outcomes compared to patients receiving only chemotherapy, but data regarding this particular group of patients is lacking. we retrospectively analysed a cohort of post allo-hct relapsed acute leukaemia patients, who, after tumour reduction, were treated with either a 2 nd allo-hct or dli. data was collected from 5 centers, 42 patients were consecutively included from 1995 to 2016. patients were treated to reduce the tumour burden and received the 2 nd allo-hct or dli on morphological remission or postchemotherapy aplasia. 26 patients (62%) were diagnosed with aml and 16 (38%) with all. 23 patients (55%) underwent 2 nd allo-hct and 19 (45%) received dli. median patient age was 38 (4-66) years. the median follow-up was 674 (9-5823) days. since data regarding time from first allo-hct to relapse was unavailable, we calculated the time from allo-hct to 2 nd allo-hct or dli (time to 2 nd allo-hct or dli). median time to 2 nd allo-hct/dli was 336 (9-8823) days, and was 674 days and 336 days for 2nd allo-hct and dli respectively (p = 0.004). regarding the dli group, the median dli dose was 1.1x10 7 / cd3+ (0.01-10x10 7 ) cells and the mean number of infused dli was 1.4/patient. one-year os was 51% (se ± 8%). in os univariate analysis, longer time to 2 nd allo-hct/dli associated better survival rates (p = 0.003). the 1-year dfs was 39% (se ± 8%). a longer time to 2 nd allo-hct/dli (p = 0.006) and 2 nd allo-hct compared to dli (p = 0.047) (figure 3 ) associated better dfs. the 1-year nrm was 24% (se ± 8%). univariate analysis identified pb as stem cell source as linked to better nrm (p = 0.086). the 1-year relapse incidence (ri) was 35% (se ± 9%). ri univariate analysis related longer time to 2 nd allo-hct/dli (p = 0.013) to lower ri. on os multivariate analysis, longer time to 2 nd allo-hct/dli was associated to better survival (p = 0.042). this association was also observed on dfs multivariate analysis (p = 0.017). table 1 summarizes 2 nd allo-hct and dli univariate analysis. grade ii-iv acute gvhd was diagnosed in 8 (35%) and 5 (26%) patients post 2 nd allo-hct and dli, respectively. chronic gvhd was diagnosed in 8 (4 extensive) and 3 patients after a 2 nd allo-hct and dli, respectively. in this study, longer time to 2 nd allo-hct/dli associated better dfs. 2 nd allo-hct (compared to dli) associated better dfs on univariate analysis, but this association was not observed on multivariate analysis. of note, the 2 nd allo-hct group included more patients with longer time to 2 nd allo-hct/dli. this might be explained by 2 nd allo-hct patients relapsing later or by the fact that the preparation of a 2 nd allo-hct might require longer time than dli. results of this analysis warrant further study with larger number of patients.advancing age is associated with worse prognosis in acute myeloid leukemia (aml). intensive induction chemotherapy in patients aged ⩾ 60 years results in lower aml remission rates with increased induction mortality vs younger patients. cpx-351 is a liposomal formulation of cytarabine and daunorubicin encapsulated at a 5:1 molar ratio. a phase iii, randomized, open-label study of cpx-351 vs 7+3 (cytarabine and daunorubicin) in newly diagnosed older patients with high-risk secondary aml showed superior survival in the cpx-351 arm (hazard ratio 0.69; p = 0.005). in that trial, eligible patients went on to allogeneic hematopoietic cell transplantation (hct). an exploratory analysis of those patients by age strata is reported here. patients aged 60 to 75 years with newly p534 number, composition and/or antileukemic activity of (dc-stimulated) invariant nkt-, nk-and cik-cells is predictive for outcome of patients with aml, all and cll cl boeck 1# , dc amberger 1# , f doraneh-gard 1 , w sutanto 1 , t guenther 1 , j schmohl 2 , f schuster 3 , h salih 2 , f babor 3 , a borkhardt 3 myelofibrosis (mf) is a hematolgic malignancy which is characterised by extramedullary hematopoiesis due to bone marrow fibrosis resulting in spleno-and/or hepatomegaly. allogeneic stem cell transplantation (allo-hsct) is the only curative treatment for mf but is associated with therapy related morbidity and mortality. retrospective studies suggested an increase of liver toxcicity in mf patients in comparison to other diseases following allo-hsct. the aim of this prospective study was to evaluate the impact of liver stiffness measured by transient elastography (fibroscan) on liver toxicity after allo-hsct. between 2013 and 2015 we included 39 patients (male 64%, female 36%) who underwent allo-hsct due to primary mf(72%), postpv/et-mf (23%) or mf in transformation (5%). the median age of the patients was 62 y@@@ears (range: 35-74). conditioning regimen was mainly busulfan based reduced intensity. all patients received atg. gvhd prophylaxis was csa/mmf in all patients. stem cell source was peripheral blood in 95% and bone marrow in 5% of the patients. donor sources were as follows: mrd (18%), mud (77%) and haploidentical relative (5%). fibroscan was performed prior to conditioning. elevated liver enzymes, bilirubin above the normal value or the onset of veno-occlusive disesae (vod) from the time of conditioning start and within the first 100 post-transplant days were considered as indicators for liver toxicity. the median stiffness of the liver measured by fibroscan on the day before conditioning treatment start was 7.6 kpa (range: 4.4-39.7). six patients (15%) had prior liver diseases such as cirrhosis (n = 1), viral hepatitis (n = 3), steatosis (n = 1), or vod (n = 1). the median onset of liver toxicity was day 0 (range: − 2 until +92). the median bilirubin level of all 39 patients was 4 mg/dl (range: 0-17). the median ap level was 153 u/l (range: 80-833), the median ggt level was 343 u/l (range: 88-1647), the median alt level was 108 u/l (range: and the median ast level was 67 u/l (range: 20-12292). the pearson-test revealed a positive correlation between liver stiffness and the elevation of the ap (r = 0.55, p = 0.001) and ggt levels (r = 0.54, p = 0.008). the comparison of the median maximum enzyme and bilirubin levels is shown in table 1 . in two patients who developed severe vod requiering defibrotide, the liver stiffness level was 6.9 kpa and 13.8 kpa, respectively. the patient with the highest stiffness level (39.7 kpa) developed acute gvhd of the liver, which completely resolved after steroid treatment. only one of those five patients who had stiffness levels 413 kpa died due to liver toxcity and concurrent septic shock, he suffered from viral hepatitis prior to transplantation. liver stiffness measured by transient elastography (fibroscan) positively correlates with the elevation of the cholestatic enzymes ap and ggt in myelofibrosis patients after allo-hsct and may predict liver toxicity. disclosure of conflict of interest: none.[p574]in the era of tyrosine kinase inhibitors (tki) as superior first line treatment in the therapy of cml, the concept of allogeneic hsct has been pushed to the role of salvage therapy. to date, data on allogeneic hsct after tki-therapy are scarcely available. in this study, we report single center data on the outcome of 52 cml patients, for the most part pretreated with tki, who underwent allogeneic hsct between 1999 and 2015 with a follow-up of 12 months to 17 years. upon obtaining written informed consent 48 patients diagnosed with bcr-abl-positive cml and 4 patients with bcr-abl-negative atypical cml were included in this analysis. the majority of patients underwent myeloablative conditioning regimen. the median age at time of hsct was 47 years with a range: from 19 to 67 years. twenty-one patients were transplanted from a matched related donor, and 31 received stem cell grafts from an unrelated hla-compatible donor. 36/48 patients received tki-therapy before transplantation, 23 patients received more than 1 tki prior to hsct. 10/48 patients were treated with interferon prior to hsct. twenty-two patients were transplanted due to acceleration or blast crisis. twenty-six patients received an allogeneic hsct in chronic phase (cp, n = 16) or complete hematologic (chr, n = 7) or cytogenetic remission (ccyr, n = 3). kinase domain mutations could be identified in seven patients including t315i-mutation in four patients. seven patients showed "major route" cytogenetic aberrations. next to advanced disease status, tki intolerance (n = 4) and tki resistance (n = 11) were the main indications for hsct after 2001. after a median follow up of 5 years and 3 months, those 26 patients transplanted in cp, chr or ccyr showed an overall survival (os) of 79%. 3/27 patients died in remission and two patients died after cml relapse. after 2004 none of the 15 patients transplanted in cp, chr or ccyr died or relapsed so far, with a median follow-up of 1672 days. all of these patients received tki therapy prior to transplant. twenty-two patients transplanted in advanced stage cml (bc and ap) had after a median follow up of 5 years an os of 44%. the difference between survival curves is significant (log rank test p = 0.041; hr 0.3407, 95% ci of ratio 0.1211-0.9585). prior to transplantation 19 of these patients received a tki-therapy. in this group, four patients died due to cml relapse, one died after development of donor cell leukemia and five patients died in remission. one patient with atypical cml was transplanted in bc and died of progressive disease shortly after transplantation. the other three patients with atypical cml were transplanted in cp-phase. with a median follow-up of 648 days these patients are in ongoing remission. even in times of tkitherapy allogeneic hsct remains a successful and safe therapy option for cml patients with tki intolerance or resistance. patients transplanted in cp or complete remission had an excellent long-term outcome. allogeneic hsct should be considered in tki resistance or intolerance before the development of blast crisis. despite tki therapy, overall survival deteriorates in patients with advanced disease. however, this treatment modality can improve survival rates substantially compared to other available therapies. tkimaintenance therapy could be a possible strategy to prevent cml relapse, although randomized data on tki-maintenance therapy after allogeneic hsct are still lacking.[p583]disclosure of conflict of interest: none. use of first or second generation tki for cml after allogeneic hematopoietic stem cell transplantation: a study by the cmwp of the ebmt y chalandon, s iacobelli 1 , j hoek 2 , l koster 2 , l volin 3 , j finke 4 , jj cornelissen 5 , i yakoub-agha 6 patients (pts) relapsing with cml after allogeneic hematopoietic stem cell transplantation (allohsct) may be treated with tki and/or dli. as nowadays the majority of cml pts would have received at least imatinib prior to transplantation, we were interested in analizing (a) the type of tki used after allohsct, (b) the indication for tki treatment, (c) the outcome of this treatment and d) the temporal relationship with dli if given. 435 pts received tki after first allogeneic hsct for cml. transplants had been performed in cp1, n = 194, ap, n = 60 or for more advanced disease (bc/4 cp1, n = 177) from hla identical siblings (n = 231) or ud (n = 204) between 2000 and 2013. tki given prior to transplant was imatinib (n = 268), dasatinib (n = 162), nilotinib (n = 88), bosutinib (n = 4) and ponatinib (n = 7). median age at transplant was 44 (18.5-68) years, 274 pts (63%) were male. tki post allohsct were given between 2000 and 2015. first tki given was either imatinib (n = 223), dasatinib (n = 131), nilotinib (n = 67), bosutinib (n = 2) or ponatinib (12). the indications for tki therapy were the same as for transplantation (n = 25), for relapse/progression/ persistent disease (n = 246), for prophylaxis/pre-emptive (n = 147), planned (n = 5), others (n = 8) and missing (n = 4). median follow-up from start of tki was 55 (1-171) months. the median time interval from transplant to tki was 6 (0.2-165) months. it was longer for tki given for relapse/progression with 15 (1-89) months and shorter for tki given for prophylaxis/pre-emptive with 1.6 (0. haematopoietic cell transplant (hct) is the only curative approach for scd. due to concerns regarding the toxicities associated with myeloablative conditioning regimens in adults, a non-myeloablative protocol was developed by hsieh et al. (national institutes of health, nih protocol). the use of this novel regimen was able to achieve a curative degree of mixed donor chimerisms with minimal transplant-related complications. the alberta children's hospital (ach) has adopted this conditioning regimen in children due to the efficacy and low rates of toxicities published by the nih group. with generally lower rates of gvhd in younger recipients, our group had no reason to believe rates of toxicities would be greater in a younger population with fewer comorbidities secondary to scd than those described in the nih cohort. to our knowledge, there is no published literature describing the utilization of the nih protocol in a paediatric population. we describe our experience in children with scd who underwent matched sibling donor (msd) peripheral blood hct using nih protocol. this retrospective cohort describes outcomes of msd hct in children with scd who underwent hct with the nih conditioning regimen between 2013-2017. a total of 13 potential subjects were identified. eight subjects have consented to the analysis to date. msds with either normal haemoglobin or sickle cell trait were considered appropriate for donation. the transplant procedure: the conditioning regimen consisted of alemtuzumab 0.2 mg/kg/dose administered subcutaneously daily for five days (days − 7 to day − 3). patients received a tbi dose of 300 cgy on day -2, with testicular shielding for male recipients. gvhd prophylaxis consisted of a sirolimus load of 3 mg/ m 2 /dose (po) on day − 1, followed by 1 mg/m 2 /dose once a day starting on day 0. unmanipulated peripheral blood stem cells were infused on day 0. sirolimus was used for gvhd prophylaxis post-hct and continued until at least one year. weaning of sirolimus was initiated no earlier than 1 year post-hct and if donor t-cell chimerisms were greater than 50%. institutional supportive care protocols for scd hct were followed. patients were eligible for early discharge post-hct even prior to neutrophil engraftment. eight patients (5 f, 3 m) have been registered on this retrospective study. follow-up ranges from 1 to 52 months post-hct. there were no failed stem cell mobilizations. all patients had donor neutrophil engraftment at a median of 21 days. all patients are currently alive. there have been no cases of graft failure to date and no sickling crises post-hct. one patient has dropping myeloid chimerisms but still 4 20% donor. no cases of veno-occlusive disease, idiopathic pneumonia syndrome, cerebral hemorrhage, pres, or posttransplant lymphoproliferative disease were observed. three cases of cytomegalovirus (cmv) reactivation required pre-emptive therapy. only one patient did not initiate sirolimus weaning at 1 year post-hct due to donor t-cell chimerisms of 42%; this patient is 52 months post-hct and is likely to start weaning sirolimus soon. there have been no cases of acute or chronic graft-versus-host disease. nonmyeloablative conditioning regimen is safe and effective as curative therapy for scd. long-term follow-up of these children to assess organ function post-hct is underway. disclosure of conflict of interest: none.the number of new hiv/aids cases has been declining in developed countries, whereas it is still increasing in japan, with the cumulative number reaching 26,607 as of june 28, 2016. hiv infection is associated with an increased risk of hematological malignancies such as non-hodgkin lymphoma (nhl). autologous hematopoietic cell transplantation (auto-hct) is a treatment option for hiv-infected patients with nhl and multiple myeloma (mm). however, the prognosis after auto-hct in hiv-infected japanese patients remains unclear. the aim of this study is to evaluate the effect of hiv infection on transplant outcomes after auto-hct in japan. using the national database of the japan society for hematopoietic cell transplantation, we retrospectively evaluated patients with nhl (n = 3862) and mm (n = 2670) who underwent their first auto-hct between 2001 and 2014. presence of hiv antibodyperipheral t-cell lymphomas (ptcl) comprise a heterogeneous group of diseases among which ptcl-not otherwise specified (ptcl-nos) represents the most common histology. patients with ptcl are typically offered high-dose chemotherapy followed by autologous hematopoietic cell transplantation (auto-hct) as front-line consolidation. allogeneic hct (allo-hct) is generally offered in the relapsed setting; however, in selected cases it is also offered as front-line consolidation. no randomized controlled trial (rct) have been performed to date comparing offering an allo-hct versus other treatment modalities either in the front-line or in the relapsed setting. thus, we performed this systematic review/meta-analysis to assess the totality of evidence pertaining to the role of allo-hct in ptcl. search of the literature was undertaken via pubmed and web of science from inception until september 6, 2016. no search limits were applied but studies presented only in abstract form were excluded. data were collected on treatment benefits (complete remission (cr), progression-free survival (pfs), overall survival (os)) and harms (non-relapse mortality (nrm), grade ii-iv acute graft-versus-host disease (gvhd), and chronic gvhd). the search identified 1271 references; however, only 17 studies (6 in front-line (n = 132 pts), 11 in relapsed/refractory setting (n = 330 pts)) were eligible based on our inclusion criteria and had extractable data. three studies included both frontline and relapsed/ refractory cases but data for certain outcomes were reported separately. the median follow-up time for studies evaluating allo-hct in the front-line or relapsed/refractory setting ranged from 30-45 months and 12-85 months, respectively. in the front-line setting, allo-hct resulted in cr rates of 64% ((95%ci = 50-77%), 2 studies, n = 49 pts), pfs rate of 64% ((95% ci = 49-78%), 5 studies, n = 100 pts), and os rate of 72% ((95% ci = 62-81%), 5 studies, n = 95 pts). nrm rate was 6% ((95% ci = 0-15%), 3 studies, n = 68 pts). acute (grade ii-iv) and chronic gvhd rates were 39% (95% ci = 24-56%), 2 studies, n = 38 pts) and 33% (95% ci = 16-53%), 3 studies, n = 64 pts), respectively. in the relapsed/refractory setting, allo-hct resulted in cr rates of 68% ((95% ci = 70-97%), 5 studies, n = 75 pts), pfs rate of 39% ((95% ci = 31-47%), 6 studies, n = 203 pts), and os rate of 52% ((95% ci = 43-60%), 6 studies, n = 307 pts). nrm rate was 21% ((95% ci = 13-31%), 7 studies, n = 194 pts). acute (grade ii-iv) and chronic gvhd rates were 34% (95% ci = 23-46%), 7 studies, n = 197 pts) and 37% (95% ci = 30-44%), 8 studies, n = 199 pts), respectively. notwithstanding the need to perform a rct to compare the efficacy of allo-hct versus auto-hct as front-line consolidation in ptcl, the results of this systematic review/meta-analysis show very encouraging os rates of 72% following allo-hct. moreover, allo-hct also offers an encouraging os rate of 52% in patients with ptcl in the relapsed/refractory setting. the higher nrm rate in the relapsed/refractory setting probably reflects the adverse effect of a higher number of prior prescribed therapies. one of the limitations of our analysis is the inability to analyze outcomes for individual histologic subtypes due to the aggregate nature of the published data. disclosure of conflict of interest: none. high-risk patients with relapse or refractory hodgkin lymphoma do significantly better after hdc auto-sct compared to control arm of aethera trial. mature results from a cohort of 234 patients s akhtar, s rauf, tam elhassan and i maghfoor king faisal specialist hospital and research center, riyadh, kingdom of saudi arabia brentuximab vedotin use in hodgkin lymphoma (hl) patients who had hdc auto-sct has been reported to improve progression free survival (pfs) but not the overall survival (os) in a phase 3 trial (lancet 2015;385:1853-62). in this trial, after hdc auto-sct, 329 high risk hl patients were randomized to receive placebo (control gp) vs brentuximab (experimental gp) as consolidation therapy. we are reporting our experience of patients with similar selection criteria as control gp. hl patients z14 yrs who received hdc auto-sct with similar selection criteria as defined in aethera trial were identified that is, patients had at least one of the following risk advanced lymphomas still represent a therapeutic challenge and allo-hsct is among treatment options. between march 2007 and august 2016, seventy-three patients (pts) affected by r/r lymphomas (34 nhl and 39 hl) underwent an allo-hsct after a treosulfan-based conditioning regimen and sirolimus as calcineurin-inhibitor-free prophylaxis of gvhd. six pts received a mrd, 18 pts a mud, and 49 pts a haplo unmanipulated pbsc. at allo-sct 30 pts were in cr, 13 pts were in pr, and 30 pts had sd/pd; sixty patients underwent autologous sct before allo-hsct. hct-ci was evaluable for 64 pts, 33 had a score ≥3. thirty-three pts received treosulfan and fludarabine reduced toxicity conditioning regimen (rtc) and intensification with other alkylating agent or with 4 gy total body irradiation was added on the remaining 40 pts (myeloablative conditioning, mac). all pts received a backbone gvhd prophylaxis with sirolimus and mycophenolate mofetil; atg or pt-cy or both were added in 41, 25, and 3 pts respectively. median numbers of infused cd34+/kg and cd3 +/kg were 6.01 × 10 6 (range: 2.72-9.06) and 2.39 × 10 8 (range: 0.3-6.89), respectively. median follow-up was 44 months (range: 3-111); median time to neutrophil ≥ 0.5 × 10 9 /l was 17 days, and 20 days to platelet ≥ 20 × 10 9 /l. sixteen out of 43 patients with pre-transplant active disease obtained a cr after treosulfan conditioning; nine of them (6 hl and 3 b-nhl) achieved durable cr without post transplant treatment. oneand 3-years os was 62% and 48%, pfs was 47% and 37% at 1 and 3 years respectively; cumulative incidence of relapse/ progression was 32% and 42% at 1 and 3 years. grfs was 31% and 19% at 1 and 3 years, respectively. transplant related mortality (trm) was 15% at 100 days, 21% at 1 year and for the entire follow-up. the 100-day cumulative incidence (ci) of agvhd grade ≥ 2 was 19% and ci of agvhd grade ≥ 3 was 10%; ci of moderate to severe cgvhd was 23% at 2 years and for the entire follow-up. no differences in ci of agvhd or cgvhd were found if pts were stratified according to donor type, but ci of moderate-severe cgvhd was significantly higher in pts after mac regimens (p o0.0005). as expected, the outcome of pts in cr was significantly better compared with active disease, in terms of os (p = 0.0061), pfs (p = 0.00022), ri (p = 0.0028). in multivariate analysis, intensity of conditioning regimen (rtc vs mac), gvhd prophylaxis (use of atg, pt-cy or none), donor sex and age at allo-sct did not impact the transplant outcomes; both os and pfs were reduced by active disease at allo-hsct (hr = 4.37, ci 95% 1.76-10.86, p = 0.01 and hr = 4.37, ci 95% 1.97-9.7, p = 0.00, respectively) and by nhl histology (hr = 3.88, ci 95% 1.65-9.19, p = 0.02 and hr = 2.43, ci 95% 1.09-5.42, p = 0.03, respectively); grfs and ri were impacted only by active disease (hr = 2.25, ci95% 1.19-4.25 and hr = 4.89, ci 95% 1.87-12.62, p = 0.01, respectively). allo-hsct after treosulfan conditioning and sirolimus gvhd prophylaxis is feasible even in heavily pretreated pts affected by lymphomas. complete remission status before transplant remains crucial for better outcomes and in the era of new targeted treatments should be pursued. disclosure of conflict of interest: none.university of eastern finland, kuopio, finland and 12 department of medicine, kymenlaakso central hospital, kotka, finland autologous stem cell transplantation continues to have an important role in the treatment of patients with multiple myeloma (mm). in mm patients the most commonly used mobilization method is granulocyte-colony stimulating factor (g-csf) ± cyclophosphamide (cy). generally, up to 10-20% patients mobilize poorly with these methods and plerixafor may be used to enhance mobilization. the most important parameter of graft quality has usually been the number of cd34+ cells, but there are also significant numbers of other cell subsets in the grafts and they may also be of special interest in regard to post-transplant recovery and outcome. for example, a higher number of lymphocytes and nk cells in the grafts has been associated with improved lymphocyte as well as nk cell recovery, respectively. the mobilization methods used seem to affect the graft composition. however, there is currently no prospective data on the effects of plerixafor on the graft composition, post-transplant hematological and immune recovery or outcome in patients with mm. altogether eighty-seven patients with mm were included into this prospective study. seventy-seven patients were mobilized with g-csf ± cy (control group) and ten patients received also plerixafor due to poor mobilization (plerixafor group). in the control group 57/77 (74%) and in the plerixafor group 3/10 (30%) of patients were mobilized with g-csf+cy (p = 0.009). there were no statistically significant differences between the groups according to age, gender, paraprotein type, initial iss, induction therapy used or disease status at the time of mobilization. by imwg risk stratification, there were more high risk patients in the plerixafor group (5/10 vs. 13/77, p = 0.066). cryopreserved graft samples were analyzed with flow cytometry for t and b cells (cd3/cd8/cd45/cd19) as well as for nk cells (cd3/cd16+cd56). also, cd34+ cell subclasses were analyzed (cd34/cd38/cd133). complete blood counts were evaluated at +15 days, 1, 3, 6 and 12 months posttransplant. to evaluate immune reconstitution, flow cytometry of lymphocyte subsets (t, b, nk) was performed in a subset of patients at 1, 3 and 6 months after the graft infusion using the same antibody panel as for graft analysis. there were no significant differences between the groups in the number of cd34+ cells in the grafts. also, the median number of aphereses was two in the both groups (p = 0.086). the proportion of the more primitive cd34+ cells (cd34 + cd133 + cd38 -) was significantly higher in the plerixafor group (p = 0.001). in addition, the number of various lymphocyte subsets analysed was significantly higher in plerixafor group table 1 ). there were no statistically significant differences in the course of hematological recovery. the recovery of blood cd3+cd4+ t cells was significantly faster in the plerixafor group at one at three moths post-transplant. there was no significant difference in the progression-free survival (pfs) (log rank, p = 0.408) with the median follow-up time of 703 days in the plerixafor group and 882 days in the control group (0.099). in the present study plerixafor added to g-csf ± cy seemed to significantly alter the cellular composition of autologous blood grafts in poorly mobilizing mm patients. hematological recovery was comparable but the cd3+cd4+ t lymphocyte recovery was faster in the plerixafor group. the pfs was comparable between the groups. disclosure of conflict of interest: dr. valtola has received honoraria from sanofi and jansen-cilag. dr. silvennoinen has received a research grant from celgene and janssen, honoraria from genzyme and sanofi and participated in advisory board organized by amgen, janssen and takeda. dr. siitonen has received honoraria from amgen and celgene. dr. jantunen has received honoraria from genzyme, amgen and sanofi and has participated in eu leadership meeting organized by genzyme as well as medical advisory board meeting organized by genzyme and amgen. dr. varmavuo has received consultancy fees from abbvie, roche, celgene, amgen and sanofi. the other authors declare no conflicts of interest. bortezomib after high-dose melphalan as conditioning regimen before autologous stem cell transplantation in patients with multiple myeloma: a comparison with the historical conditioning regimen with melphalan alone ga ferini; ja arbelbide; al basquiera; e nucifora; n schutz; v otero; d fantl hospital italiano d buenos aires, buenos aires, argentinahigh dose of melphalan followed by autologous stem cell transplantation (asct) is the standard of care for younger patients with multiple myeloma (mm). to enhance the efficacy of the conditioning regimen, the intergroupe francophone du myelome added bortezomib to melphalan showing improved response rates, without significant toxicity. bortezomib has shown synergistic effects with melphalan, mainly if the bortezomib is administered 24 hours after the melphalan. since 2014, we have changed our conditioning regimen for patients with mm undergoing asct by adding bortezomib toallogeneic stem cell transplantation (allosct) is a potencially curative option for patients with multiple myeloma (mm). despite the improvement of reduced-intensity-conditioning (ric), transplant-related mortality (trm) remains high. there is no consensus on which graft versus host disease (gvhd) prophylaxis regimen is superior. some studies have suggested that tacrolimus-based prophylaxis is more effective than cyclosporine (csa) in terms of lower incidence of severe acute gvhd (agvhd), with no impact on overall survival (os). herein, efficacy and toxicity between two gvhd prophylaxis regimens is analyzed. we retrospectively analyzed 14 patients (pts) with relapsed mm who received allosct ric in the period from 2003 to 2015 in a single centre (table 1) . population: age, 51 years (40-73); median follow-up: 19 months (1-175). conditioning regimen: allo-ric (fludarabine + busulfan or melphalan regimens) and 100% was bortezomib-based in the tacrolimus group. donor: matched related (11 pts), unrelated (1), mismatch unrelated (1) and haploidentical (1) donor. gvhd prophylaxis: all patients received a short course of methotrexate + csa (9 pts, 64%) or tacrolimus (5 pts, 36%). complete response at transplant was 33% at csa group and 60% at tacrolimus group. all pts underwent toxicity related to chemotherapy (mainly mucositis and neutropenic fever) with organ impairment (renal or liver) in 100% tacrolimus arm as well as 4 pts in csa group. the incidence of agvhd was 80% and 77.8% in tacrolimus and cyclosporine groups, respectively (p = 0.99). grade iii-iv agvhd were reported in 2 pts (40%,tacrolimus) and 4 pts (44%, cyclosporine), with severe gastrointestinal and liver involvement. glucocorticoid resistance was observed in 75% in both groups. patients with refractory agvhd received other immunosuppressive therapies: more than 3 second-lines agents (3) (4) (5) (6) were necessary in fifty percent of pts in both groups to control gvhd. two patients had to interrupt tacrolimus due to neurological toxicity and suspected thrombotic thrombocytopenic purpura. no patients had to discontinue treatment in the csa arm because of toxicity. the 12-months os was 78.6% (80% in tacrolimus vs 66.7% in csa (p = 0.78)) and the 24-months was 68.8%. a total of 4 pts died because of gvhd. during follow-up, only 2 patients relapsed (10 and 126 months after allosct, respectively) in csa group. no relapse were seen in tacro group. in our experience, no significant differences were observed between both calcineurin inhibitor in terms of os, toxicity and gvhd incidence. an explanation could be our small number of patients. allosct is an effective therapy for selected patients but it is associated with high rates of gvhd and trm. a long-termsafety and effective prophylactic regimen is necessary as main objective.[p638]disclosure of conflict of interest: none. several parameters, including early lymphocyte, neutrophil, platelet recovery, and infused dose of cd34+ cells, have been associated with clinical outcome of patients with haematological malignancies. however, their prognostic significance remains uncertain. the aim of current study was to evaluate prognostic significance of clinical and laboratory parameters that might influence survival after autologous stem cell transplantation (asct) in hodgkin lymphoma (hl) and multiple myeloma (mm). this retrospective study included a total of 90 with hl and 114 mm patients (median age 32 years, 55 years, respectively) who underwent asct between november 2005 and june 2016. hl patients were conditioned with beam (84.4%) and cbv (15.6%) regimen, while mm patients received conditioning with high dose of melphalan. high ips (international prognostic score) at diagnosis had 68.9% hl patients and high iss (international scoring system) had 27.2% of mm patients, of which 8.8% had renal impairment. the average of transplanted cd34+ cells in hl patients was 7.15 × 10 6 /kg (range: 2-25.0 × 10 6 /kg), and 6.6 × 10 6 /kg (range: 2-15.51 × 10 6 / kg) in mm patients. after asct, favourable treatment response (partial/complete remission) achieved 83.3% hl patients, of whom 22.7% had infused o5 × 10 6 /kg cd34+ cells. median time to recovery of absolute lymphocyte count 500 × 10 6 /l or greater (alc500) was 16 days (range: 9-31 days), recovery of absolute neutrophil count ≥ 500 × 10 6 /l (anc500) was 12 (range: 6-26 days), and platelet recovery ≥ 20 × 10 6 /l (plt20) was 12 days (range: 5-44 days). after asct, 93.6% mm patients achieved favourable treatment response, of whom 26.5% had infused cd34+ cell dose48.7 × 10 6 /kg. median time to alc500 was 15 days (range: 9-23 days), anc500 was 13 (range: 9-24 days), and plt20 was 11 days (range: 5-26 days). median follow up of patients with hl was 67 months, while after asct, median event free survival (efs) was 20 months, and overall survival (os) was 38 months. treatment response after asct strongly influenced both efs and os after asct (po0.0001). in patients who achieved favourable treatment response, os and efs after asct were influenced by infused cd34+ cell dose (o5 × 10 6 /kg vs. ≥ 5 × 10 6 /kg), prolonged recovery of alc500 by day+20, plt by day +13, and achieving of anc500 by day +11(po0.05). multivariate analysis among significant variables showed that infused cd34+ cell dose was the most important parameter that influenced os and efs (po0.05). median follow up of mm patients was 50 months, while after asct, median efs was 26 months and os was 34 months. regarding patients who achieved favourable treatment response, os and efs after asct were influenced by the presence of renal impairment, infused cd34+ cell dose (≤8.7 × 10 6 /kg vs. 48.7 × 10 6 /kg) and plt20 recovery by day +13 (po0.05). among these significant parameters, multivariate analysis pointed out infused cd34+ cell dose as the most important parameter that influenced both os and efs (po0.05). these data suggest that number of infused cd34+ cells is an independent factor that may contribute to outcome of patients with hl and mm. disclosure of conflict of interest: none. high-dose therapy with autologous stem cell transplantation (asct) has become the treatment of choice for symptomatic eligible patients with multiple myeloma (mm). we studied an induction regimen of cyclophosphamide, bortezomib and dexamethasone (cybord) and showed rapid and deep responses after 4 cycles in patients with newly diagnosed mm and we subsequently done asct with melphalan (mel) conditioning. cost is the major limiting factor in developing world.all the drugs used are generic brands manufactured in india. a total of 25 mm patients (median age: 54.5 years, 76% male and 24% female) were transplanted between 2012 and 2016. in all, patients had igg kappa-12 (48%), igg lambda-03 (12%), iga lambda-05 (20%), iga kappa-02 (08%), kappa light chain 02 (08%), lambda light chain 01 (04%) patients. prior to autograft, all cases had received cybord with generic medicines. median time diagnosis to asct was 7.5 months (5 to 21 months). stem cell mobilization was done with g-csf alone in 17 (68%), g-csf plus plerixafor in 07 (28%) and chemo mobilization in 01 (04%) patients. all patients received asct support after conditioning with 200 mg/m 2 generic melphalan alone (dose adjustment was done according to renal status). all patients received thalidomide maintenance from march 2012. bortezomib used was manufactured by dr. reddy's lab, hyderabad and melphalan used was manufactured by emcure pharmaceuticals, pune, india. 68 patients from 1999 to 2011 received cyclophosphamide, vincristine, adriamycin and dexamethasone (cvad) protocol of originator medicines followed by originator melphalan conditioning and asct (cvad-mel-asct). at the time of autograft, 21 (84%) of patients were in complete remission, 02 (08%) in partial remission, 02 (08%) very good pr. median day of engraftment was 10 for neutrophils and 14 for platelet. transplant related mortality was 16% (4/25) out of which 2 died of infection and 2 deaths of cardiac events. the pfs and os rates were 80% and 84% at median follow up of 18.6 months. patients who were treated with cvad-mel-asct had efs of 74% at 2yrs and 52% at 5yrs. cost of bortezomib showed significant difference, generic was 4000usd where as for originator drug was 20000usd for 4cycles of chemotherapy. cost of melphalan also showed difference with 450usd for generic and 2000usd for originator drug. generic cybord showed excellent response rate and allows excellent stem cell collection and transplantation which can further consolidate response and improve outcome. cybord induction and melphalan conditioning with generic medicines can be considered a standard regimen for transplant-eligible patients with newly diagnosed mm in resource constraint situation. generic cybord-mel-asct is more cost effective than originator cvad-mel-asct. generic medicines produced in india are of good quality and cost effective. this study needs long term follow up to assess survival parameters at a median. disclosure of conflict of interest: none. and an extra copy of one or more odd-numbered chromosomes and as intermediate risk(ir) if they had t(4;14) or del(13) (q).overall survival (os) and relapse-free survival (rfs) were calculated from the time of allo hsct and auto hsct on day 0, from diagnosis to death or disease progression. the median age at presentation was 53.86 (range: 20-80) years, and 72 (63.7%) were men. at a median follow-up time of 18 months, 73% were alive.45 of the 113 patients with available fish samples underwent auto hsct. 24 patients (53.3%) achieved cr and 21 patients (46.7%) relapsed. of the 13 patients who had received allo hsct, five patients (38.5%) achieved cr and five patients (38.5%) remained alive. in patients who received auto hsct, the risk of relapse was 56% less than those never transplanted (p = 0.02), but the difference was not significant in patients who received allo hsct. the relapse-free survival in hr patients was 6 months (po 0.001), in ir was 11 months (p o0.001) and in sr was 37.67 months (p o0.001). in transplant patients, rfs in hr patients was 5.73 times more than sr group (po0.001) and in ir group was 3.35 times more than sr (p o0.001). the survival time in transplant patients was significantly better than non-transplanted patients (p o0.001). the median overall survival (os) in hr patients was 25.45 months, in standard risk group 30 months and in sr patients was 31 months. cytogenetic abnormalities detected by fish are of significant value in classification, risk stratification and management of patients with mm. we can use cytogenetic data to provide prognostic information and also to guide management and clinical practice. these data indicate that autologous stem cell transplantation could potentially be of benefit to myeloma patients. disclosure of conflict of interest: none. chronic graft-vs-host disease (cgvhd) is the most troublesome complication developing after allogeneic hematopoietic stem cell transplantation (allo-hsct). diagnosis of cgvhd has largely been based on clinical features only. we previously reported gene expression profiles in patients with cgvhd after allo-hsct. we extended our study to develop a molecular diagnostic method of cgvhd. we selected six most commonly expressed genes from the former dna expression study. and, a home-made 6-gene pcr array were used to evaluate gene expression profiles in the peripheral blood mononuclear cells of 39 patients given allo-hsct (20 cgvhd patients, 19 non-cgvhd patients) and 19 normal controls. the gene expressions of the allo-transplanted patients were compared to those of the stem cell donors. sybr green qpcr and multiplexqpcr were performed to confirm the usefulness of the selected genes in the diagnosis of cgvhd. infogainattributeeal and ranker were used to develop a gene model to diagnose cgvhd. k-nearest neighbor model and weka classifiers lazy ibk module were applied to evaluate the performance of the gene model. in another 21 steroid-refractory cgvhd patients (14 responders, 7 non-responders), the gene expression changes were analysed using our 6-gene pcr array before and 57 days after rituximab treatment. we identified six genes most accurately delineating cgvhd patients from those without treatments after allogeneic hematopoietic stem cell transplantation (hsct) are long and constraining for patients. medical adherence in hsct patients is of major concern in daily practice but it has been not yet described. 1, 2 the aims of our study were to evaluate treatment adherence and to identify factors associated with adherence behaviors. an observational single-center study was based on self-reported questionnaires completed by patients in a hematology day hospital between november 2015 and july 2016. the patientreported adherence was evaluated using the eight-item morisky medication adherence scale (mmas-8). 3, 4 individual item scores were summed: patients with a score of 8/8 were considered as good adherents to medication whereas a poor adherence referred to a score under 8. among the latter, medium adherence ranged to a score of 6-8, while a score of o6 was considered low adherence. socio-demographic and medical characteristics were collected by health records. a univariate model was used to evaluate if some of patients' characteristics were associated with adherence. statistical analysis was performed using r software (version 0.98.1103 -2009-2014 rstudio, inc). fifty-six patients were included in the current study. median age at transplantation was 55 years (range: 16-72 years). diagnosis were aml (n = 28), all (n = 10), myelofibrosis (n = 8) and other hematological diseases (n = 10). 24 patients received a hsct from a related donor (13 haploidentical). myeloablative conditioning was used in 18 patients and reduced intensity regimen in 38 patients. a total of 64.3% (36/56) of the patients were poor adherent according to mmas-8. among these patients, 6/36 were low adherent and 30/36 were medium adherent. the results of univariate analysis showed that a poor adherence was associated with a longer time since hsct and discharge at home. however elderly patients, patients treated with cyclosporine and patients with daily hydration at home were associated with a better adherence (po 0.05). our study presents the first data on adherence among patients undergoing hsct. risk factors associated with a poor adherence have been identified in order to determine patients' profiles that will benefit more from interventions to improve adherence. particular attention has to be paid to younger patients. efforts to establish a regular follow-up of these patients are needed in order to sustain patients in the treatment adherence to prevent the occurrence of severe complications. we studied the effect of basic fibroblast growth factor (fgfb) and dexamethason on expansion and immune modulation of mscs in patients with lymphomas. mscs were generated from bone marrow aspirates obtained from the patients with hodgkin's lymphoma (hl; n = 8) and non-hodgkin's lymphoma (nhl; n = 4). the adherent fraction of marrow aspirate was cultivated with/without the basic fibroblast growth factor (fgf-b, 10 ng/ml) or dexamethason (10 − 5 м or 10 − 8 м) to reach 80-90% confluence. then mscs were passaged with accutase and used for experiments after 1-2 passages. the number of msc precursors (cfu-f) in bone marrow of lymphoma patients was found to be significantly decreased both in patients with nhl (17 ± 5, p o0.01) or hl (26 ± 5, p o0.05). the time until 80-90% confluence was significantly increased and took on average 26 ± 2 days (vs 15.4 ± 0.6 in donors). finally, the immunosuppressive ability of patient msc was significantly lowered and was only registered at the high concentrations of mscs (1:1 and 1:2). the expansion of patient mscs was significantly promoted with fgfb resulting in a significant decrease of primary cell cultivation (from 25.4 ± 1.52 to 18.6 ± 1.21 days; p = 0.041) and a statistically significant twofold increase in the number of cells received at the first passaging. in addition, in cultures with fgfb there was a decrease in the relative amount of resting mscs and a threefold increase of cycled cells in cd73+ mscs. dexamethasone has also provided a moderate stimulating effect on the msc growth. in fact, the use of 10 − 5 м of dexamethasone resulted in the increase of the cell yield by 1.6 times and of 10 −8 м-by 1.9 times. however, fgfb and dexamethasone differed in their effect on the msc ability to inhibit the proliferative response of t lymphocytes upon stimulation with mitogens or alloantigens. indeed, fgfb failed to correct the impaired immunosuppressive activity of patient mscs, and median percentage of suppression still remained lowered-17% vs 16% without fgfb. in contrast to fgfb, dexamethason could increase the immunosuppressive activity of patient mscs by 1.5 times (in dose of 10 − 5 м) and by 2.1 times (for 10 − 8 м). our data indicate that fgfb and dexamethasone used during the generation of mscs exert a stimulating effect on the msc expansion. in contrast to fgfb, dexamethasone, in the broad range: of doses, was able to enhance the suppressive properties of mscs that are initially reduced in patients with lymphoma. these findings suggest the existence of at least two mechanisms of impairments in immunoregulatory function of mscs in lymphomas-dependent and independent of the msc proliferation. disclosure of conflict of interest: none. free nonabsorbable antibacterial digestive decontamination is associated with a low incidence of gastrointestinal acute gvhd and better gvhd-free/ relapse-free survival (grfs) in the atg-based conditioning regimens nabil yafour 1 commonly antibacterial prophylaxis based of oral no absorbable antibiotic such as (neomycin colistin, gentamicin, vancomycin) used before and after engraftment, other fluoroquinolone such as levofloxacine were recently used to prevent invasive infection. however the exact interaction with gastrointestinal acute graft versus host disease (gi-agvhd) remains unclear. the objective of this study was to evaluate a novel composite endpoint of gvhd-free/relapse-free survival (grfs), in which events include grades 3-4 gi agvhd, chronic gvhd requiring systemic therapy, relapse, or death in atg based-conditioning regimens, with free no absorbable antibacterial digestive decontamination prophylaxis. a total of 39 evaluable consecutive patients with hematological disease were included in period of february 2013 to mai 2016. patients with malignancies disease (n = 33) received myeloablative conditioning regimens plus atg (5 mg/kg); including once daily busulfan (130 mg/m 2 ,-6d to -3d, iv ) + fludarabine (40 mg/m 2 /d, -6d to -3d, iv) (aml = 26, all = 3, cml = 1) or melphalan (140 mg/m 2 , d-1, iv) (all = 3). six patients received cy/atg for saa. gvh prophylaxis consisted to; ciclosporine a (csa) + mtx. csa was maintain levels between 150-400 ng/ml and tapered at the discretion of the treating physician. all patients were received peripheral blood stem cells (pbsc) graft from a matched related donor. since december 2015 levofloxacine and voriconazole was administered as antibacterial and antifungal prophylaxis. diagnostic, clinical grading and treatment of gi-agvhd and gi-cgvhd were performed according to established criteria and nih recommendations. probability of grfs was estimate by kaplan-meier method. median age was 35 years (range: 6-60). median dose of cd34+ and cd3+ cell doses were 4.9 × 10 6 (range: 2.5-7) and 1.84 × 10 8 (range: 0,036-5,28). the median time to neutrophil and platelet recovery were13 days (range: 6-33) and 14 days (range: 10-43) respectively. at time of transplant 12/39 (31%) had an intestinal colonization with extended-spectrum betalactamase (esbl) producing bacteria. only 5/39 (13%) developed infectious diarrhea during the period of transplant. incidence of grade iii/iv gi-agvhd and gi-cgvhd requiring systemic therapy were 5% and 5% respectively. for patients with malignancies diseases (n = 33), 23 (70%) were alive at a median follow up of 12 months (range: 5-43). incidence of relapse, disease free survival rates were 30%, 67% respectively. the grfs rate as defined previously was 48% at 30 months. these results confirm that free no absorbable antibacterial digestive decontamination and atg-based conditioning regimens were associated with very low incidence of gi-gvhd and better grfs in patients with malignancies diseases. diverse bacterial populations of the gastrointestinal tract remain important factors to promote immune tolerance after allogeneic sct. disclosure of conflict of interest: none. g-csf primed hla haploidentical transplantation from maternal or collateral donor using atg plus reduced dose of posttransplantation cyclophosphamide: results of a phase ii prospective trial y wang 1 , x-j huang 1 1 peking university people's hospital, peking university institute of hematologythe transplantation milieu using granulocyte colony-stimulating factor (g-csf), and anti-thymocyte globulin (atg) for hlahaplotype-mismatched transplants from related donors has resulted in favourable outcomes with low transplant-related mortality (trm), without increased relapse rate. however, in this transplant modality, the poorer outcome owing to high incidence of graft-versus host disease (gvhd) related to maternal donor or collateral donor remains a concern. meanwhile the use of post-transplant cyclophosphamide (pt/cy) in recent years appears to be protective against severe acute and chronic gvhd. we performed a prospective pilot study of hla haploidentical stem cell transplantation (sct) from maternal or collateral donors with intensified conditioning including g-csf and atg, followed by two lower doses of pt/cy (14.5 mg/kg × 2 doses). outcomes were compared with those of 160 controls from matched-pair analysis who undergone haploidentical sct from other donors than mother or collateral relatives at the same time period. a total of 40 patients with myelodysplastic syndrome (mds) or leukaemia undergoing haploidenticla sct from maternal or collateral donors were enrolled in the study. incidence of grade ii-iv and grade iii-iv acute gvhd at day 100 were comparable between the study group and the control group (17.5% vs. 33.3%, p = 0.07; 5.0% vs. 12.5%, p = 0.24). incidence of cmv and ebv reactivation at day 100 were also comparable between the study group and the control group (75.0% vs. 85.0%, p = 0.16; 15.0% vs. 29.2%, p = 0.09). after a median follow-up of 303 days and 341 days, the incidence of trm and relapse at 1 year were comparable between the study group and the control group (5.0% vs. 13.3%, p = 0.16; 10.0% vs. 6.7%, p = 0.54); the probability of overall survival and lfs at 1 year were comparable between the study group and the control group (84.2% vs. 79.8%; p = 0.24; 83.0% vs. 77.7%, p = 0.48). in conclusion, conditioning with atg and low-dose pt/ cy might be a feasible option for patients undergoing hla haploidentical, t-cell replete sct from maternal or collateral donors. trial registration: the study is registered at www. clinicaltrial.gov as nct02412423. disclosure of conflict of interest: none. hematopoetic stem cell transplantation (hct) is a lifesaving treatment option for eligible patients with hematological malignancies. hct is inherently associated with a risk of nonrelapse mortality that varies greatly depending on transplant and patient characteristics. the assessment of the risk of complications and mortality before the procedure is extremely important. the hct comorbidity index (hct-ci) introduced by sorror m. is one of the tools proved to predict hct outcomes and was shown to be significant in various disease and hct settings. the objective is to evaluate hct-ci index of hct recipients, determine impact of different variables on ci score, particularly those, showing pulmonary and cardiac function. data of hct-ci of autologous (auto) and allogeneic (allo) hct recipients, transplanted during period january 2015-october 2016 were analyzed. impact of pulmonary and cardiac function values on ci score was evaluated: dlco (diffusing capacity of the lung for carbon monoxide), fev1 (forced expiratory volume) and ef (cardiac ejection fraction) are parameters, reflecting pulmonary and cardiac function, which values are included into hct-ci score. the statistical data analysis was conducted using spss program. the differences were considered statistically significant at p ≤ 0.05. records of 100 allo and 220 auto hct recipients, transplanted during 01.2015 -10.2016 in vilnius university hospital were revised. median age of allo hct and auto hsc recipients was 50 (19-75) and 58 (19-74) years respectively. main indication for allo hct was acute myeloid leukemia 48 (48%) patients and for auto hct -multiple myeloma 133 (60.5%) patients. hct-ci was completely calculated (no values missing) in 64 allo and 102 auto hct recipients. only patients with available complete hct-ci data were further analyzed. hct-ci in hct recipients was as shown in table 1 . hct-ci score o3 was calculated in 37 (57.8%) and ≥ 3 in 27 (42.2%) allo hct recipients. hct-ci score o3 was calculated in 45 (44.1%) and ≥ 3 in 57 (55.9%) auto hct recipients. hct-ci score did not differ statistically significant between male and female recipients in both hct categories as well as in different age groups of patients (below and above 40 years in allo and below and above 60 years in auto hct). dlco was found to be below normal values (o80%) in 43 (67.19%) allo hct and in 67 (65.7%) auto hct recipients. fev1 was less affected and found to be lower 80% in 6 (9.3%) allo hct and in 17 (16.7%) auto hct recipients. ef below 50% detected in 1 (1,6%) allo hct and in 6 (5.9%) auto hct recipients. low dlco was found to cause the greatest impact on hct-ci score and was statistically significantly associated with higher hct-ci (po0.001). the most common hct-ci in both hct groups was score 3. dlco was found to be below normal ranges in relatively large patient group and had the greatest impact on hct-ci score. further studies on reasons of pulmonary function impairment and it's impact on hct outcomes are warranted.[p690]disclosure of conflict of interest: none. haemophagocytic lymphohistiocytosis (hlh), a life-threatening hyper-inflammation syndrome, is classified into primary and secondary forms. primary hlh is caused by gene mutations resulting in impaired cytotoxicity of natural killer (nk) cells and cytotoxic t lymphocytes (ctls). secondary hlh arises in the setting of autoimmunity, infection, malignancy, or less commonly, may be idiopathic. treatment of hlh has two major goals: halting the triggering event and controlling the overactive immune system. however, patients with primary or recurrent secondary hlh should subsequently undergo allogeneic hct for long lasting disease remission. we retrospectively evaluated 61 hematopoietic stem cell transplantation (hsct) might be a valid treatment option for adults suffering from aggressive t-cell malignancies providing long term disease control. since a suitable hla-matched donor cannot be identified for all patients (pts) in need for transplantation, alternative donors graft sources such as related hla-haploidentical donors are considered. through introduction of t-cell-replete (tcr) hlahaploidentical transplantation (haplo-hsct) using post transplantation cyclophosphamide (ptcy) successful treatment with low non-relapse mortality rate (nrm) has been observed in lymphoma patients (luznik et al., bmt, 2008) . however, less data are available on the outcome of this haplo-approach in the treatment of t-cell malignancies, in particular when disease is refractory. we retrospectively evaluated the outcome of haplo-hsct using tcr grafts and ptcy in 8 pts with peripheral t-cell lymphoma treated between 2010 and 2015 at our institution (t-nhl = 6, t-all = 2; male n = 5; median age: 37 years). disease was refractory/active at time of transplantation in 7 pts, while one had achieved second cr. all patients received at least 2 prior treatment lines and one patient failed previous allogeneic transplantation. while fludarabine and cyclophosphamide served as backbone for conditioning, 3 pts received a tbi-based and 5 a drug-based conditioning regimen which was myeloablative in 50%. if disease was active at time of haplo-hsct, a sequential therapeutic concept was performed involving intensive chemotherapy (clofarabine n = 6) shortly preceding conditioning (zoellner ak et al., bmt, 2015) . post-grafting immunosuppression consisted of cyclophosphamide, tacrolimus and mycophenolate mofetil in all patients. graft source was bone marrow in 3 pts. no primary graft rejection occurred; 7/8 pts engrafted, one died early in aplasia. neutrophil/platelet engraftment was achieved at a median of 20 (range: 14-36) and 42 (range: 17-117) days, respectively. acute gvhd grade ii-iii was observed in 4 pts, whereas no patient developed grade iv agvhd. mild chronic gvhd occurred in one patient. 50% of the pts developed grade ii-iii treatment-related toxicities most commonly diarrhea (33%) and mucositis (25%); grade iv toxicity (mucositis) was observed in one patient only. no vod occurred. cmv reactivated in 4/5 pts at risk, whereas no ptld was seen. proven invasive aspergillosis was diagnosed in one patient. at day +30 seven pts achieved cr. 3 pts relapsed and 3 died (relapse n = 1, infection n = 2). 1-year nrm was 25%. at a median follow up of 46 months (range: 15-76) the estimated 1-year and 3-year overall survival (os) and progression-free survival (pfs) were 63%/63% and 50%/33%, respectively. three pts received haploidentical dlt pre-emptively (n = 2) and therapeutic (n = 1), leading to sustained cr in two, while no severe gvhd occurred. sequential therapy in the setting of tcr haplo-hsct using ptcy as gvhd prophylaxis is feasible, well tolerated and shows low rates of gvhd and acceptable nrm in patients with relapsed/refractory t-cell lymphoma/ leukemia providing an effective anti-lymphoma/leukemic activity. thus, we suggest that intensified tcr hapo-hsct using ptcy should be considered as an alternative for patients suffering from aggressive t-cell malignancies, lacking hlamatched donors. disclosure of conflict of interest: none. tacrolimus is a calcineurin inhibitor increasingly used as immunosuppression following allogeneic stem cell transplantation; maintenance of therapeutic serum levels is essential to reduce the risk of graft rejection and graft versus host disease. however, tacrolimus can be associated with serious side effects and potential drug interactions. regular monitoring of serum levels and appropriate dose adjustment is essential to ensure therapeutic levels and to avoid toxicity. in our adult bmt unit, an established standard operating procedure (sop) provides a prescriptive dosing algorithm for: (i) initiation of tacrolimus therapy; (ii) conversion between iv and oral routes; (iii) dose adjustment based upon tacrolimus serum level and interacting medications. we performed an audit assessing adherence to the sop dosing algorithm. 97 inpatient tacrolimus dosing episodes from five consecutive haploidentical transplants were retrospectively analysed. 17 episodes were excluded due to insufficient records. for the remaining 80 episodes, tacrolimus serum levels and corresponding doses were identified. the response of the medical team to each serum level was compared with the sop dosing recommendation. to account for sensible rounding of doses, a margin of error of ± 10% was permitted. adherence to sop dosing was 54%. non-adherence to the sop (46%) was subcategorised as justifiable (21%) or unjustifiable (25%). justifiable non-autologous hsct is currently being explored for its efficacy and safety in the treatment of multiple sclerosis (ms). as more experience is gained in treating this cohort, treatment related mortality has steadily improved although the procedure still carries a degree of risk. ebv reactivation is well described in allogenic stem cell transplants although less so in autologous transplantation. we investigated the frequency of ebv reactivation in patients with ms undergoing autologous hsct at a single uk site. 30 patients underwent autologous hsct for treatment of ms at king's college hospital between feb 2012 and aug 2016. all were mobilised with cyclophosphamide 4g/ m 2 and g-csf. 29 were conditioned with cyclophosphamide and atg, and one with beam/atg. previous exposure to ebv (ebv igg) was assessed prior to transplant and local posttransplant ebv monitoring was performed on whole blood samples by means of quantitative pcr in 26 patients. data was collected retrospectively. all 26 (100%) patients were positive for ebv igg pre-transplant. overall, 194 samples were tested for monitoring post-transplant. 20 (76.9%) patients demonstrated positive pcr post-transplant on local testing with one further patient being negative on local tests but later becoming positive on testing in their parent hospital (full results unavailable). of these 20, the median time to positive testing post-transplant was 24 days (7-91). maximal ebv dna titre was reached at a median time of 40 days post-transplant (7-101) with a mean maximum titre of 5.17 log (3.3-8.2). 2 patients experienced symptomatic reactivation with an associated large paraproteinemia. one of these developed hyper-viscosity requiring plasma exchange and developed neurological symptoms mimicking an ms relapse (max ebv titre of 8.2 log). this patient received rituximab and ebv level is declining, the other was observed carefully but developed right leg weakness which is slowly improving. the patient with raised ebv at their parent hospital also received rituximab (unclear if this reactivation was symptomatic). we have developed a protocol to pre-emptively treat ebv reactivation with rituximab once a 6 log titre is reached and one patient has so far been treated according to this. of the 18 patients with locally confirmed reactivation who did not receive rituximab, 9 (50%) self-resolved at a median time of 98 days (44-182), 5 (33.3%) have ongoing re-activation (4 with improving, 1 with stable titres) and 4 (22.2%) have not had any local bloods performed ≥ 6 months. 1 patient with selflimiting reactivation later had a further positive titre (370 days post-transplant and 182 days post initial resolution). ebv reactivation appears to be common in patients with ms in the first 3 months post autologous hsct. unlike in other patient groups such as aplastic anaemia patients receiving allogeneic transplants it can cause significant neurological symptoms which may be confused with ms relapse. the mechanism of this reactivation is probably related to atg administration but may be exacerbated by prior immune suppression in this heavily pre-treated group, the majority of whom have received highly active disease modifying therapies in the past. these results demonstrate the importance of monitoring for ebv reactivation following autologous hsct and the consideration of pre-emptive therapy. disclosure of conflict of interest: none. reduced bone mineral density (bmd) is a well recognised complication of hct. guidelines recommend scanning by dual energy x-ray absorptiometry (dxa) one year after transplant in all hct patients 1 or else specific groups of high risk patients. 2, 3 it is recognised that both dose and duration of steroids are risk factors for low bmd and it is recommended that prednisolone doses greater than or equal to 5mg/day for more than 3 months should prompt a dxa scan. for patients with osteopenia it is recommended that calcium/vitamin d supplements are given together with lifestyle advice including diet, smoking cessation and weight bearing exercise. 1 in this survey we have investigated the current practise in investigating and managing bone health in the context of hct. a survey was sent to all 453 centres including 45 countries registered with ebmt as of november 2016. 63 centres replied from 14 countries. response numbers to each question were variable and are indicated by the denominators. 5/36 used a national guideline to guide their practise, and 3/34 used an international guideline. no single guideline was quoted more than once. 25 low testosterone has been demonstrated to be an independent determinant of endothelial (dys)function in men. graftversus-host disease (gvhd) is a major contributor to nonrelapse mortality (nrm) after allogeneic stem cell transplantation (allosct). vulnerability of the recipients' endothelial cell system is a novel concept to explain why a proportion of patients with acute gvhd fail to respond to escalating immunosuppressive therapy and ultimately succumb to gvhd and related complications. this retrospective study investigated the prognostic impact of pre-transplant testosterone levels on nrm after allosct in male patients. between 2002 and 2014, a total of 277 male patients undergoing allosct at heidelberg university (median age 55 years) provided informed consent to participate in this observational study (training cohort). a total of 71 patients (26%) received transplants from related donors (rd). diagnoses were aml (48%), mds (29%), lymphoid malignancies (33%) and multiple myeloma (12%) . a total of 176 patients (78%) received statin treatment post allosct as per institutional standard policy. for validation, an independent patient cohort of 205 men allografted for aml and mds (median age 57 years, 18% rd, no statin treatment) at essen university was analysed. pretransplant serum samples were collected between 0 and 2 months before allosct and cryopreserved at − 80°c. testosterone and suppressor of tumorigenicity-2 (st2) levels were measured by radioimmunoassay and elisa, respectively. median pre-transplant testosterone level in the training and validation cohort was 13.6 nmol/l (range: 0.3-41.7 nmol/l) and 16.0 nmol/l (0.8-38.1 nmol/l), respectively. in the training cohort, lower pre-transplant testosterone as continuous variable was associated with shorter os (p = 0.009). lower testosterone levels showed a trend towards higher nrm (p = 0.09) and a significant association with nrm after onset of acute gvhd (p = 0.02). multivariate analysis confirmed lower pre-transplant testosterone levels as a significant predictor of an increased nrm risk after gvhd onset (p = 0.03). in the subgroup of patients not receiving statins post-transplant, lower testosterone levels were associated with increased incidence of transplant-associated microangiopathy (p = 0.01), and, in addition, with higher pre-transplant st2 levels indicating endothelial vulnerability. in the validation cohort, similar results with regard to overall survival (os, p = 0.02), nrm (p = 0.04), nrm after acute gvhd onset (p = 0.03) in univariate analysis, and to nrm after gvhd onset (p = 0.02) in multivariable analysis could be observed. the association of pre-transplant testosterone levels (in quartiles) and incidence of nrm after gvhd onset in the training and validation cohort is depicted in figure 1a and 1b, respectively. our study suggests that low pre-transplant testosterone is associated with serological and clinical evidence for endothelial damage and is an independent risk factor for a fatal outcome of gvhd. prospective studies in the allosct setting investigating testosterone and testosterone supplementation in deficient patients are highly warranted. disclosure of conflict of interest: none. nk cells anti-tumor ability in multiple myeloma patients s tognarelli 1,2 , b jacobs 3,4,5 , i von metzler 6 , h serve 6 , p bader, t klingebiel 7 , a mackensen 3 and e ullrich 1,2 1 department of pediatric stem cell transplantation and immunology, childrens hospital, goethe university, frankfurt, germany; 2 cellular immunology, loewe centre for cell and gene therapy, goethe university, frankfurt, germany; 3 department of hematology and oncology, university hospital erlangen-busulfan is one of essential drugs for hematopoietic stem cell transplantation (hsct). because of its narrow therapeutic range: targeted busulfan using therapeutic drug monitoring (tdm) has been used. generally, the initial dose of busulfan is determined by patients' body surface area as 120 mg/m 2 except for infants (80 mg/m 2 ). however, pharmacokinetic evidence of these initial doses is scarce. therefore, we investigated the full pharmacokinetics of busulfan in infant and child, and attempted to validate that these initial doses are acceptable. one hundred ninety-five pediatric patients undergoing hsct using four-day targeted busulfan were enrolled. of them, 6 patients received hsct when their age was ≤ 1 year old (infant group [ig]), and 19 patients received when 1-2 years old (toddler group [tg]). the remaining 170 patients were defined as a child group (cg). busulfan was administered intravenously once daily for 4 consecutive days. tg and cg received 120 mg/m 2 as the first dose, and ig received 80 mg/m 2 . using daily tdm, we adjusted the next dose of busulfan. target daily and total area under the curve (auc) were 18 750 μg*h/l/day and 74 000-76 000 μg × h/l, respectively. median first-day busulfan auc of ig, tg, and cg were 18 416, 22 529 and 20 410 μg × h/l, respectively, which was significantly different (p = 0.031). however, there was no significant difference in median total busulfan auc (ig; 74 180, tg; 73 406, and cg; 74 482 μg × h/l, respectively, p = 0.089). the coefficient of variance (cv) of four-day busulfan aucs in ig and cg was similar (median cv: 22.1% and 24.7%, respectively), whereas cv of tg was 40.4%. in sub-analysis of tg and cg who received equally 120 mg/m 2 as the first dose, there was an inverse correlation between age and first-day busulfan auc (r = − 0.148, p = 0.042), as well as between age and cv of four-day busulfan aucs (r = − 0.210, p = 0.004). initial busulfan dose as 80 mg/m 2 for infant could be acceptable in aspect of first-day auc and cv of four-day busulfan aucs. however, higher first-day auc and cv were shown in tg. although target total busulfan auc could be achieved safely by tdm, we suggest that reduction of initial dose less than 120 mg/m 2 is also necessary to patients with 1-2 years old to lower the relatively higher first-day auc. taken together, tdm is highly recommended to reduce busulfan toxicity, especially in younger children. disclosure of conflict of interest: none. post-induction treatment strategy of acute myeloid leukemia (aml) is currently driven by european leukemia net (eln) risk assessment at diagnosis. if it is well established that patients belonging to favourable-risk group can be treated with chemotherapy and/or autologous stem cell transplantation (sct) and that those belonging to the unfavourable-risk group should be addressed to allogeneic (allo) sct, for patients included in the intermediate-risk groups the best post-induction treatment has not been established yet. we report here a 6years (2010-2015) allo-sct single center experience in 78 aml patients. median age was 53 years (range: 20 -68), 17%, 23%, 9% and 51% were grouped in the eln favourable, intermediate-i, intermediate-ii and unfavourable risk category, respectively and 47% of the patients were allotransplanted in advanced disease-phase (2nd complete remission). half of the patients received a sibling hla compatible donor, 76% of the cases received peripheral blood stem cells and half of the patients received a myeloablative conditioning regimen. graft versus host disease prophylaxis was conventionally based on cyclosporine and shor-course methotrexate, with the addition of antilymphocyte immunoglobulin in case of matched unrelated donor. the clinical and transplant characteristics of the patients according to the eln-risk group were well balanced. with a median follow up of 20 months (range: 8-58 months), the projected 2 years overall survival (os) and disease free survival (dfs) is 45% (95% ci: 32-57%) and 43% (95% ci: 30-54%). the median os and dfs in favourable/intermediate-i vs intermediate-ii/unfavourable is 21.8 and 14.8 months ( figure 1a ; p = 0,67) vs 18 and 14,8 months ( figure 1b ; p = 0.66). the relapse rate (rr) and the non relapse mortality (nrm) at two years are 38% (95% ci: 26-50%), and 15% (95% ci: 8-26%), respectively. non differences were observed comparing the 2 years rr and the 2 years nrm of patients in the favourable/intermediate-i vs intermediate-ii/unfavourable eln risk group (36% vs 40%; p = 0.66 and 16% vs 18%; p = 0.95). interestingly, the percentage of patients allotransplanted in advanced phase of the disease was higher in those included in low/intermediate-i with respect to intermediate-ii/unfavourable eln-risk group (73% vs 43%; p = 0.001). our data suggest that allo-sct can cure approximately 40-50% of aml patients, with no difference within the eln risk groups. disease recurrence remains the major problem and this is highly correlated to the percentage of patients in advanced phase of the disease at transplant, particularly in eln favourable/intermediate-i patients. we are currently collecting the data on minimal residual disease (mrd) status of these patients during chemotherapy and before transplant using moelcular biology on target genes and/or multiparametric flow cytometry on leukemia associated immunophenotype, in order to assess if the prognosis of these patients may be refine by the prospective application of mrd data.[p707]disclosure of conflict of interest: none. outcome of allogeneic stem cell transplantation for patients with high-risk acute leukemia according to donor type and graft-versus-host disease prophylaxis s lindner, t berg 1 , j riemann 1 , s ajib 1 , z jedlickova 1 , s gueller 1 , f lang 1 , a sackmann 1,2 , n goekbuget 1,2 , h martin 1 , a bacigalupo 3 , h serve 1,2 and g bug 1 1 department of medicine ii, hematology and oncology, university hospital frankfurt, goethe university, germany; s500 2 german cancer consortium (dktk), german cancer research center, heidelberg, germany and 3 università cattolica del sacro cuore, fondazione policlinico universitario gemelli, roma, italyin high-risk acute leukemia (hr-al), allogeneic hematopoietic stem cell transplantation (hsct) is the only potentially curative treatment. increasingly, hsct is being performed utilizing alternative donors. we retrospectively analyzed the outcome of 148 consecutive patients (pts) with hr-al (aml/all, n = 118/30) undergoing first allogeneic hsct in our transplant unit between 1/2011 and 6/2015 according to donor type and graft-versus-host disease (gvhd) prophylaxis: in the matched related donor group (mrd, n = 26), hsct was performed with standard immunosuppression (is), that is, calcineurin inhibitor (cni) plus methotrexate or mycophenolate mofetil (mmf). for 10/10 hla-allele matched unrelated donors (10/10 mud, n = 84) or 9/10 hla-allele mud (9/10 mud, n = 24) we used is and anti-thymocyte globulin (atg fresenius/neovii). hsct with a haploidentical family donor or an 8/10 hla-allele mismatched unrelated donor was performed using is with cni plus mmf and post-transplant cyclophosphamide (pt-cy, n = 14). a myeloablative (n = 75) or reduced-intensity (n = 73) conditioning regimen was applied in complete remission (cr, n = 106) or active disease (n = 42). pts had a median age of 52 years (range: 19-72) and hematopoietic cell transplantation comorbidity index of 3 (range: 0-11). patient and treatment characteristics were well balanced between the groups except for a higher percentage of pts transplanted in cr in the pt-cy group (93% vs. 54-76%, p = 0.03). peripheral blood stem cells were preferred for mrd, 10/10 mud and 9/10 mud (81%, 95% and 96%, respectively) and bone marrow for 93% of pt-cy based hsct. all pts engrafted. with a median follow-up of 28 months (range: 1-60), probability of overall survival (os) at 3 years was 54 ± 10% for the mrd, 65 ± 6% for the 10/10 mud, 41 ± 11% for the 9/10 mud and 93 ± 7% for the pt-cy group, without significant differences (p = 0.05). however, the probability of achieving the combined endpoint gvhd-and relapse-free survival (grfs) at 3 years varied significantly between the groups (mrd 8 ± 5%, 10/10 mud 43 ± 6%, 9/10 mud 29 ± 10% and pt-cy 57 ± 13%, po0.01), reflecting the high cumulative incidence (ci) of chronic moderate and severe gvhd at 1 year in the mrd (58 ± 11%) as opposed to the other groups (10/10 mud 12 ± 4%, 9/10 mud 12 ± 8% and pt-cy 33 ± 14%, p o0.01). of note, donor type had no impact on ci of transplant-related mortality (trm) at 3 years (12 ± 3%), acute gvhd g3-4 at day +100 (10 ± 3%) or leukemic relapse at 3 years (34 ± 4%). overall, aml pts 460 years of age had a significantly inferior relapse-free survival compared to younger pts (50 ± 9% vs. 71 ± 6%, respectively, p o0.01) without a higher ci of trm (p = 0.37). median time to aml relapse was 6 months. our results suggest that pt-cy-based alternative donor hsct is safe in hr-al pts and provides a solid basis for a randomized clinical trial comparing hsct from haploidentical family donors and 9/10 mud, currently in preparation. while os did not vary between groups, grfs was dismal after mrd transplants without atg, due to high rates of severe chronic gvhd, consistent with published data. as leukemic relapse remains the major cause for treatment failure especially in elderly pts, maintenance strategies using novel drugs or cellular therapies are warranted. disclosure of conflict of interest: none. relapse following hematopoietic stem cell transplant (hsct) is the leading indication for a second transplant in patients with malignant disease. hsct has been shown to be superior to chemotherapy alone or palliative measures in these patients. for non-malignant disease a second transplant may be considered for graft failure after first transplant. data regarding the outcome of a second hsct for non-malignant disease is scarce. we retrospectively analyzed 29 patients who underwent a second hsct, for survival and toxicity data. twentynine patients (age 0-19 years) who received a second hsct at our institution during 1998-2015 were included in the analysis. thirteen patients had an underlying malignancy and 16 patients were transplanted for non-malignant indications, including inborn errors of metabolism, non-malignant hematologic diseases and immune deficiency. median follow up was 14 months (range: 1-180). there were 10 deaths (77%) in the malignant group, 7 (53%) were due to disease relapse and 3 (23%) were transplant related. fifty percent of deaths occurred within the first year following the second hsct. in the non-malignant group there were 5 deaths (31%), of which 2 (12%) were attributed to the underlying disease and 3 (18%) were transplant related. all deaths but one occurred within the first year post hsct. treatment related mortality following second hsct is higher compared to first transplant. the higher survival rate in the non-malignant group suggests that transplant following graft failure should be considered ins501 patients with otherwise incurable underlying disease. though the outcome for patients with relapse of malignant disease following hsct is poor, a second transplant may benefit a subset of these patients. attempts to achieve complete remission prior to transplant should be made to improve outcome. due to the small number of patients in our cohort, further multi-center trials are needed. disclosure of conflict of interest: none. disseminated bcg infection (bcg-osis) is a rare but most serious complication in vaccinized especially immunocompromised children. severe combined immunodeficiency disorder (scid) is probably the commonest primary immunodeficiency associated with bcg-osis, though there is no such definitive data as most of the cases described in literature are in the form of reports. hematopoietic stem cell transplantation (hsct) is a life-saving treatment for patients with scid, especially if therapy is instituted early, prior to onset of infections.as bcg vaccine is routinely given to all iranian children at birth, the likelihood of having an active infection at the time of transplant would be significantly high. the main objective of this study was to evaluate the outcomes of hsct in scid patients with disseminated bcg infection . sixteen scid patients underwent hsct in our center since 2007 to 2016, of which nine patients (7 male, 2 female) were enrolled in this analysis. all the 9 patients had received bcg vaccination according to the national vaccination protocol, and had undergone anti-tuberculosis (tb) treatment prior to transplant due to disseminated bcg infection. the mean age at hsct was 9.3 months (range: 6-13 months). patients received bone marrow (n = 1), peripheral blood progenitor cells (n = 6) or umbilical cord blood grafts (n = 2) from hla-matched related donors (n = 7) and mismatched unrelated donors (n = 2). three patients received unconditioned matched sibling donor transplants and ric regimen was provided with fludarabine, melphalan and rabbit anti-thymocyte immunoglobulin (thymoglobulin) in others . cyclosporine a and prednisolon were used as graft-versus-host disease (gvhd) prophylaxis. they also continued to receive anti-tb treatment. all patients but one engrafted. the median times to neutrophil and platelet engraftments were 12 days (range: 11-39), and 18 days (range: 17-90), respectively. engraftment with full chimerism (495%) occurred in 5 patients and the other 3 patients had mixed chimerism. with a median follow-up of 24 months (range: 3 -48 months), overall survival was 66.7%. the main cause of death was disseminated bcg infection.three out of 8 patients who achieved engraftment, developed acute gvhd (grade i-ii), while one patient developed extensive chronic gvhd. although anti-tb treatment continued, tuberculous dactylitis occurred in 3 patients post-hsct that were successfully treated. on last post-hsct follow-up, 4 patients with full chimerism and 2 with mixed chimerism are alive and disease free. scid is called as a pediatric emergency as it invariably leads to fatality in infancy without early aggressive therapy and hsct. in hsct recipients, the impaired cellular immunity renders these patients more susceptible to infection. as previous reports suggest, our study demonstrates that with appropriate anti-tb cover, immunological reconstitution with complete recovery from bcg infection can be achieved by early hsct. disclosure of conflict of interest: none. paraproteinemia occurrence after allogeneic hematopoietic stem cell transplant as a possible marker for chronic gvhd onset f monaco 1 , s tamiazzo 1 , f dallavalle 1 , l calcagno 2 , m pini 1 and m ladetto 1 1 hematology and 2 transfusion medicine, azienda ospedaliera ss. antonio e biagio e cesare arrigo, alessandria, italy transient monoclonal gammopathy is commonly reported after solid organ or stem cells transplant (sct) for hematologic malignancies. however the clinical significance of a paraproteinemia appearance is not fully understood, because the attempts to correlate its effect on survival rates, graft versus host disease (gvhd) occurrence and viral reactivations have led to controversial results. starting from these reports we decided to evaluate among our allogeneic transplanted patients the incidence of m-component and its possible relationship with chronic gvhd. one-hundred and one patients undergoing allosct at the hematology unit of alessandria (italy) between 2006 and 2015 were evaluated. 55% of patients were male and 45% were females. pretransplantation diagnosis included: 62 acute myeloid leukaemia/ high-risk myelodisplastic sindromes (62%), 14 acute lymphoblastic leukaemia (14%), 13 lymphoproliferative disorders (13%) and 12 other less common malignancies (12%) . patients with multiple myeloma were excluded from the study. all patients had, at least, two pre-transplantation serum electrophoresis with no evidence of pre-existing monoclonal component. serum electrophoresis was scheduled to be performed at 90, 180 and 360 days and 2 years after transplantation. forty-nine patients were submitted to allo-sct from a sibling donor and 52 from a matched related donor (mud); in vivo t-cell depletion with anti-thymocyte globulin was used in 63 patients. thirty-four patients relapsed after allosct, 52 (52%) developed chronic gvhd and 56 patients (56%) are currently alive at the last follow-up. posttransplantation follow up ranged from 81 to 2695 days with a median of 496 days. paraproteins were detected in 52 out of 101 patients (52%), being monoclonal in 28 patients, and bi or tri-clonal in the remaining cases; the immunoglobulin subclass most commonly observed was igg. ten-year overall survival of the whole population was 50%; splitting the population in two cohorts (with or without paraproteinemia) we did not detect any statistical differences in overall survival, gvhd development and relapse incidence at +90 and +180 days posttransplant; viceversa, after 360 days, a statistically significant difference was observed in chronic gvhd occurrence in patients with or without paraproteinemia (85% vs 42%, respectively, po 0.001). ten-year overall survival curves were significantly better in patients with paraproteinemia as compared with the paraprotein-free group (59% vs 45%, p = 0.04), and an even more evident significance was seen in ten-year relapse free survival curves (66% for patients with paraprotein vs 48% for patients without paraprotein, p = 0.009). monoclonal gammopathy, also in our experience, is frequent following allo-sct. we observed a strong correlation between the occurrence of paraproteinemia, chronic gvhd and a significantly better overall and relapse-free survival. recently many evidences showed that b cells are involved in the pathogenesis of chronic gvhd (cgvhd) and anti-b-cell therapy has been suggested for the treatment of cgvhd. we speculate that the presence of a monoclonal gammopathy after allogeneic transplant is expression of the activation of the b-cell compartment. a prospective study with a larger population should be considered, in order to confirm our results and assay post-transplantation monoclonal gammopathy as an early marker for gvhd development. disclosure of conflict of interest: none.inherited bone marrow failure (ibmf) syndromes are rare pediatric disorders that characteristically associate physical abnormalities, progressive bone marrow failure and predisposition to cancer. the most common of these disorders is fanconi anemia (fa). stem cell transplantation (sct) using related or unrelated donors are the only curative therapeutically approach when severe marrow failure is established. the aim of the study was to analyze the results of sct for patients with ibmfs in a single center. we performed a retrospective study in pediatric patients with ibmf admitted in pediatric hematology and bone marrow transplant department, fundeni clinical institute between january 2000 and september 2016. diagnosis and severityof ibmfs were established based on hematological results, bone marrow biopsy and clinical findings. genetic testing for ibmfs is not currently available in our country. indication for sct was established when patients developed moderate/severe aplastic anemia and became transfusion dependent.in case of dba, sct indication was established for steroid resistant disease. the donors were selected from family members or unrelated donors, 10/10 matched.the conditioning regimens used were reduced intensity (fludarabine 120-150 mg/m 2 , cy 20 mg/kg, f-atg 40 mg/kg) for af, dc and myeloablative (busulfan i.v., fludarabine 150 mg/m 2 , thiotepa 20 mg/kg, f-atg 30 mg/kg) for dba. gvhd prophylaxis consisted of standard methotrexate and csa/tacrolimus. all parents signed informed consent forms. in our center, between 2000 and 2016, 20 patients with ibmf were diagnosed: 10 (50%) patients with fa, 6 (30%) patients with diamond blackfan anemia (dba), 2 (10%) patients with diskeratosis congenita (dc), and 2(10%) patients with not classifiable ibmfs. the patient data is available in table 1 . seven out of 20 patients (35%) performed sct procedures: sibling 3 patients (2 patients with af, 1 patient with dc), mud 4patients (3 patients with af, 1 patient with dba). all patients (100%) engrafted for pmn (median = 17, range: 12-29 days) and platelet (median 21, range: 13-46 days).2/7 (42%) presented reactivation of cmv and received valganciclovir, 1/7 developed cmv disease (encephalitis and pneumonia), 2/7 (28%) developed bkv cystitis and required extensive hydration and levofloxacin. 4/7 (57%) developed grade i-ii skin acute gvhd day +100, which responded to topical treatment and low dose of corticosteroids. 1/7 (14%) developed grade iii intestinal acute gvhd, which responded to high-dose corticosteroids.1 /7 (14%) developed grade iv intestinal chronic gvhd (day +160), without response to high-dose corticosteroids, mmf and later died on day +221, due to infectious complications (severe pulmonary and cerebral aspergillosis). 6/7 patients (85%) are alive, with 100% donor chimerism 4/6(66%) or stable mixed chimerism 2/6 (33%). median follow-up for sct patients was 515 days (26 days-5y 6mo). conclusions in our study we observed a low incidence of severe complications associated with low mortality rate (14%) . sct is a procedure that associates multiple risk situations, but it remains the only curative cytomegalovirus (cmv) infections remain a significant cause of morbidity and mortality in patients whose immune systems are compromised, including hematopoietic stem cell transplant (hsct) recipients. although the adoptive transfer of third party cmv-specific t cells has proven both safe and clinically beneficial in treating even drug-refractory infections/disease, broader implementation and commercialization of this strategy has been hampered by (i) the postulated need for extensive cell banks generated from donors representing diverse hla profiles, and (ii) lack of large scale t cell manufacturing processes. to address these limitations we have developed a proprietary decision tool (cytomatch™) to identify a small panel of healthy donors who should provide almost universal hla coverage; and optimized a simple, scalable manufacturing process to generate large numbers of cmv-specific t cells. to assess the robustness of our strategy we generated a bank of cmv-specific t cells (viralym-c™) from 8 carefully selected healthy donors. the lines were polyclonal, comprising both cd4 + (21.3 ± 6.7%) and cd8 + (74.8 ± 6.9%) t cells, expressed central cd45ro+/cd62l+ (58.5 ± 4.2%) and effector memory markers cd45ro+/cd62l-(35.3 ± 12.2%), and were specific for the immunodominant cmv antigens ie1 and pp65 (ie1: 419 ± 100; pp65 1070 ± 31 sfc/ 2 × 10 5 , n = 8). a fixed-dose (2 × 10 7 cells/m 2 ) phase i clinical trial was subsequently initiated to test the safety and efficacy of these "ready to administer" t cells in pediatric and adult hsct recipients with drug-refractory cmv infections. using our bank of just 8 lines, we have identified a suitable line for 21 of 22 patients screened. of these, 7 patients have been treated with viralym-c cells; 6 received a single infusion and 1 patient required 2 infusions for sustained benefit. there were no immediate infusion-related toxicities; and despite the hla disparity between the viralym-c™ lines and the patients infused, there were no cases of de novo or recurrent graft versus host disease (gvhd). based on viral load (measured by quantitative pcr) and/or symptom resolution, viralym-c cells controlled infections in all patients with 5 complete (cr) and 2 partial responses (pr) achieved within 4 weeks of infusion. one patient with cmv retinitis had complete resolution of symptoms following viralym-c™ infusion. our results demonstrate the feasibility, preliminary safety and efficacy of "ready to administer" viralym-c™ cells that have been generated from a small panel of healthy, eligible cmv seropositive donors identified by our decision support tool. these data suggest that cost-effective, broadly applicable t cell anti-viral therapy may be feasible for patients following hsct and potentially other conditions. disclosure of conflict of interest: drs. juan vera, ann leen and brett giroir hold equity and drs. ifigeneia tzannou, sunitha kakarla are employed by viracyte. haploidentical stem cell transplantation (hsct) protocols utilizing ex vivo t-cell depleted grafts have been proven efficient in preventing graft versus host disease (gvhd), but cause a delay in early t-cell recovery that increases the risk of graft rejection, leukemia relapse and viral infections. conventional donor lymphocyte infusion (dli) after hsct transplantation is conditioned because of the high prevalence of gvhd even with low dose of t cells. here we present preliminary data of escalating cd45ro+ memory t cells as dli in three patients that received a selective graft depleted of naïve (cd45ra+) t-cells. three children that were transplanted following nonmyeloablative conditioning regimen with a graft consisting of cd34+ and cd45ra-cells, with mixed chimerism, lymphopenic and viral/opportunistic infections and minimal residual disease positive before hsct received dose scalating cryopreserved haploidentical cd45ra-memory t cell starting with a initial dose of 1 × 10 5 /kg, until a maximal dose of 1 × 10 8 /kg with a 21 days interval. we infused 10 products with a naïve (45ra+) t-cell dose less than 1 × 10 4 /kg with 499.9% purity of cd3 + cd45ro+ memory t-cells in all cases. all infusions were well tolerated without any side effect during infusions neither gvhd. following the dli, a progressive increase in t cell counts was observed. our preliminary data suggest that dose escalating of haploidentical memory t cells (45ro+) as dli provides a safety platform, even with high dose of t cells (1 × 10 8 /kg), for adoptive immunotherapy in haploidentical 45ra+ depleted grafts with no gvhd complications, and allows an increase in t cell reconstitution. however, efficacy of this strategy requires longer studies. relapse after allogeneic hematopoietic stem cell transplantation (allohsct) remains a major therapeutic challenge: outcome is very poor, without curative option in most cases. second allohsct may be considered in few selected patients because of anticipated limitations: (1) donor availability; (2) high toxicity due to previous treatments; (3) low efficacy considering the very advanced disease situation. we hypothesized that the use of post transplantation cyclophosphamide (pcy) haplo-sct after relapse following allohsct may deal in part with these limitations. in particular, the presence of full haplotype hla mismatch could provide a decisive antileukemic effect relative to alloreactivity. in absence of large series in this setting, we report here the outcome after haplosct for patients who relapse after a first allohsct. we retrospectively studied adult patients, who received a second pcy haplo-sct for hematological malignancies. patients were treated between 2009 and 2016. the objective was to assess both the feasibility and the efficacy of haplosct in this setting. twenty seven patients were included: median time between first allohsct and relapse was 11 months (range: 1-82). median age at second transplantation was 49 years old (range: 21-61). most of patients had acute myeloid leukemia (n = 12, 44%) or hodgkin lymphoma (n = 6 patients, 22%). fifteen the impact of minimal residual disease and its kinetics prior to different types of allogeneic hematopoietic stem cell transplantation on clinical outcomes in patients with acute myeloid leukemia z xiao-su 1,2 , l yan-rong 1 , yan-hong 1 , p xu-ying 1 , qian-jiang 1 , hao-jiang 1 , lan-ping, xu 1 , xiao-hui zhang 1,2 , yu-wang 1,2 , h xiao-jun 1,2 and c ying-jun 1 this study investigated the impact of minimal residual disease (mrd) and its kinetics prior to different types of allogeneic hematopoietic stem cell transplantation (hsct) on clinical outcomes in patients with acute myeloid leukemia (aml) in complete remission (n = 132). 107 patients who received unmanipulated haploidentical hsct and 25 patients who received hla-matched sibling hsct were enrolled. mrd measured using 8-color flow cytometry (fcm) at fixed time points before transplantation was retrospectively analyzed. the patients were divided into four groups based on mrd kinetics before transplantation: consistent negative, positive to negative, negative to positive and consistent positive. during the follow-up, total twenty (15.2%) patients underwent relapse. through unique variate analysis, none of mrd status at various time points before unmaipulated haploidentical transplantation was associated with clinical outcomes, as well as the dynamic change of mrd before hsct (p40.05), although the patients with consistent positive mrd before hsct seemed to have a relatively higher incidence of relapse (p = 0.151). one-year cumulative incidence of relapse (cir) were 11.2 ± 4.7% vs. 31.1 ± 13.8% in mrd consistent negative and consistent positive groups, respectively (p = 0.202). however, patients with positive mrd after the second chemotherapy or pre-mrd before hla-matched sibling hsct showed a significant poor outcomes including higher cir (p = 0.015 and both neuroblastoma (nrb) and rhabdomyosarcoma (rms) in childhood are the aggressive malignant disease with higher mortality. this paper aims to study the efficacy of autologous peripheral blood stem cell transplantation (apbsct) in the treatment of high risk advanced nrb and rms. 34 patients with high-risk stage iv nrb and 9 patients with advanced childhood rms were treated by apbsct in our hospital from october of 2010 to may of 2016. in the subgroup of nrb patients, 16 patients got complete remission (cr) and 1 patient got cru while 17 patients had tumor residual disease after intensive induction therapy before asct. the median age was 5.55 (1-9) years old. primary sites of the tumors included submaxilla (n = 2), cervical (n = 5), adrenal gland (n = 16)and retroperitoneal (n = 11). the conditioning regimen consisted of busulfan and melphalan (busulfan 1 mg/kg × 4d, melphalan 140mg/m 2 x1d) or cem regimen (carboplatin 600 mg/m 2 × 3d, etoposide 500 mg/m 2 × 3d, cyclophosphamide 1800 mg/ m 2 × 2d); the pathology of 9 stage iii childhood rms patients was embryonal rhabdomyosarcoma. there were 8 cases in cr and 1 case in partial remission (pr). the median age was 6.56 (3-13) years old. primary sites of the tumors included bladder (n = 1), left forearm (n = 3), retroperitoneal (n = 1), pelvic (n = 3) s513 and talus (n = 1). the conditioning regimen consisted of melphalan, cyclophosphamide and dactinomycin (melphalan 60 mg/m 2 × 3d, cyclophosphamide 1800 mg/m 2 × 2d, dactinomycin 0.0 13 mg/kg × 3d).there were 13 double apbsct cases (nrb n = 12, rms n = 1). all the relapse patients were treated with chemotherapy and radiation therapy. all the patients successfully underwent mobilization, collection and reinfusion. the time of hematopoietic reconstitution was (11. 0 ± 3) days, no severe toxicity was observed, no transplant-related death was found. with a median follow-up of 25.35(2-60) months, one of the patients was lost to follow-up. in the subgroup of nrb patients (n = 33): the 2-year event-free survival and total survival rate of all patients were 66.2% and 79.4%, respectively. the survival time of no recurrence was significantly different between the double transplantation group and single transplantation group (p o0.05). in the subgroup of rms patients (n = 9), 1 patient died, 6 patients live without pd(1 patients had double apbsct), 2 patients suffered recurrence but still alive. apbsct achieved good outcome in patients with high risk advanced nrb and rms. transplantation-related toxicities were tolerable. double apbsct significantly improved the depth of remission. disclosure of conflict of interest: none. transplantation outcomes of a once-daily intravenous busulfan and fludarabine conditioning for allogeneic hematopoietic stem cell transplantation in pediatric aml and high risk mds: single center experience in korea y-t lim, e-j yang and k-m park department of pediatrics, pusan national university children's hospital, yangsan, koreathere have recently been some reports suggesting that oncedaily intravenous busulfan as a conditioning regimen for hematopoietic stem cell transplantation (hsct) possibly reduces the toxicities without influencing the clinical outcome as compared with the traditional 4 times daily dosage schedule. but until recently there has been little research and limited data available on the safety and efficacy of oncedaily intravenous busulfan and fludarabine in pediatric allogeneic hsct. we report the outcomes for allogeneic hsc recipients, evaluating engraftment status, regimen related toxicities (rrt), and event free survivals (efs) after use of oncedaily intravenous busulfan and fludarabine conditioning for allogeneic hsct in children with aml and high risk mds in a single pediatric center of korea. from january 2005 to december 2015, 22 aml and 2 high risk mds children who received once daily iv busulfan/fludarabine based conditioning regimen for allogeneic hsct were reviewed, bu/flu ± atg consist of intravenous fludarabine (40 mg/m 2 ) and busulfan (110~130 mg/m 2 , once daily iv) on days -6 to -3, and antithymocyte globulin (atg) (3 mg/kg) on days -3 to -1. all patients received tacrolimus and mini-dose methotrexate (5 mg/m(2)) for graft versus host disease (gvhd) prophylaxis. 17 boys and 9 girls were enrolled with median age of 10.1 years (range: 0.6-17.9 years). the median period from diagnosis to transplantation was 7 months (range: 5-49 months). more than half of the patients had a matched sibling donor (n = 16, 62%), 27% patients (n = 7) had a matched unrelated donor, 8% patients (n = 2) had a mismatched unrelated donor, and the remaining 1 patient had a mismatched family donor. as a stem cell source, peripheral blood stem cells (pbsc) were 22 cases (85%), bone marrow and cord blood were 2 cases in each. the median follow-up for patients was 40 months. the median number of infused total nucleated cells and cd 34+ cells except cord blood transplantation were 9.2 × 10(8)/kg and 7.8 × 10(6)/kg. all patients including who received cord blood were successfully engrafted. the median time to absolute neutrophil count (anc) recovery (anc 4 500 × 10(6)/l) and platelet recovery (platelet4 20,000 × 10(6)/l) were 13 days, 18 days in each. the incidence of acute gvhd was 19.2%, while severe grade iii/iv gvhd was observed in only 2 patient (7.7%). there were only two cases (8.7%) of extensive chronic gvhd in this study. transplant-related toxicities were acceptable, there was no case with cns toxicity, eleven patients (42.3%) developed grade ii,iii mucositis and grade i-iii hepatic toxicity in twenty four (92.3%), but transient. there was 3 clinically diagnosed veno-occlusive disease (vod), but most recovered by fluid restriction and diuretics. nine patients (36%) showed positive cytomegalovirus (cmv) antigen/pcr but only one patient developed cmv colitis. eight patients died: 7 due to relapse/disease progression, 1 due to extensive chronic gvhd. the 5-year efs and overall survival were 62.2% and 66.1% respectively. at 3 year, the cumulative incidence of relapse was 19.2%. overall, once-daily intravenous busulfan and fludarabine was less toxic and effective as conditioning regimen in aml and high risk mds patients undergoing allogeneic transplantation in children. disclosure of conflict of interest: none. haploidentical stem cell transplantation from unmanipulated graft has becoming a practiced option for high risk hematological malignancies who lack a matched related or unrelated donor. lack of a matched sibling or unrelated donor (mud) can be a significant barrier to allogeneic transplantation in patients who stand to benefit from this procedure. hlahaploidentical donors are readily available for nearly all such patients. haplo transplantation has inherent advantages over mud transplantation including the lower cost of graft acquisition, greater availability of donors for ethnic minorities, and immediate access to the donor in patients in whom delay cerebral palsy (cp) is a heterogeneous group of conditions that result in permanent motor disability. it may occur due to perinatal hypoxic insults, developmental brain abnormalities, genetic diseases, traumatic or infectious causes. in general the condition is non-progressive, but improvement over time is rarely seen. various treatment methods have been used for the management of this disorder. however, there has been no absolute cure for cp. the ultimate goal of stem cells therapy is to use the regenerative capacity of the stem cells causing a formation of new tissues to replace the damaged tissue. the polish stem cell bank (pbkm) has provided wharton's jellyderived msc (wj-msc) for medical therapeutic experiment application in children with cp. wj-msc from third party donors were administered to 27 patients (pts) with cp aged from 1.6/12 to 16.9/12 (median age: 6 years and 1 month). twenty two pts have received infusions intravenously (i.v.), 1 pt intrathecally (i.t.), and 4 pts via both routes (first i.v., next i.t.). the cells were previously collected from healthy newborns, processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapour. msc immunophenotype was confirmed using flow cytometry assay. the pts have received from 1 to 6 infusions in intervals from 4 weeks to 6 months. median i.t. dose was 15 × 10 6 cells per infusion, while median i.v. dose was 1 × 10 6 cells/kg of body weight per infusion. each patient has been examined by the same neurologist at the day of each infusion and the result of examination has been described in a follow-up. twelve patients were diagnosed with epilepsy as comorbidity. eighteen pts (67%) showed positive changes in neurological examination after their treatment with wj-msc. almost half of the children experienced improvement of cognitive functions (12 out of 27 pts). muscle tension was reduced in 6 pts. improvement in the ability to concentrate, better contact with others and improved social interactions were observed in 19% of pts. correction of motility was noticed in 5 pts, 2 pts have experienced better quality of sleep. in 3 cases there has been a reduction in the number of epileptic seizures (1 pt even discontinued some of his medicines). there were no s515 noticeable changes in neurological examination of 2 patients. seven follow-up forms have been not received yet. the experiment data provide evidence that third-party donor wj-msc are suitable and efficient stem cells for treatment in patients with cp. however further and more extensive examination, with a greater number of patients is needed, which will be beneficial for far-reaching results. spina bifida (sb) is a congenital malformation resulting from failure of fusion in the spinal neural tube during embryogenesis. despite surgical repair of the defect, most patients who survive with spina bifida have multiple system damage due to neuron deficiency in the spinal cord. it has been confirmed that the mesenchymal stem cells (mscs) have the ability to survive, migrate and differentiate into cells of a neural lineage. wharton's jelly-derived mscs (wj-mscs) from third-party donors have high proliferation and differentiation potential along with non-immunogenic features, thus seem to be a promising stem cell source. the polish stem cell bank (pbkm) has provided wj-msc for clinical application in a medical therapeutic experiment for children with sb. eleven patients (pts) were qualified for administration of wj-mscs. three pts have been waiting so far for their therapy after bioethical committee approval. seven pts were in the middle of stem cell therapy (after 1 or 2 injections), 1 pt had finished one cycle of stem cell therapy (5 injections -ijs) and resumed therapy by administering a first dose of wj-mscs. the cells were previously collected from healthy newborns, processed, screened for bacterial contamination as well as endotoxin content, and frozen in liquid nitrogen vapors. six pts have received infusions intravenously (median dose: 1.01 × 10 6 /kg body weight per infusion), and 1pt was given 1 injection of 40 × 10 6 cells intrathecally. each patient has been examined by the same neurologist at the day of each infusion and the result of examination has been written in a follow-up. there were 6 pts, who received at least 2 doses of wj-msc, and all of them showed positive changes in neurological examination. the important improvement, declared by pts, was in areas: pronunciation and/or self-reliance (3pts), movement of arms and/or legs (4pts), quality of life (3pts), core stabilization (1pt). only one adverse event occurred after third injection of wj-msc: 1 pt had nausea and a fever. in case of other pts it was too early to provide reliable feedback. the transplantation of wj-mscs could stimulate the mscs to differentiate towards sensory neurons. this could be one of the reasons of observed improvement of many vital functions in patients, after mscs treatment. this approach might have value in the experimental treatment of sensory neuron deficiency in spina bifida.